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THE VITAMINS
Chemistry, Physiology, Pathology

VOLUME II

THE
VITAMINS

Chemistry, Physiology, Pathology

VOLUME II

EDITED
BY
W. H. SEBRELL, JR. Ropert S. Harris
Director, National Institutes of Health Professor of Biochemistry of Nutrition
Bethesda, Maryland Massachusetts Institute of Technology

Cambridge, Massachusetts

ACADEMIC PRESS INC., PUBLISHERS
New York + 1954

Copyright, 1954, by
ACADEMIC PRESS INC.
125 East 23rd Street
New York 10, N. Y.

All Rights Reserved

NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM,
OR ANY OTHER MEANS WITHOUT WRITTEN PERMISSION
FROM THE PUBLISHERS

Library of Congress Catalog Card Number: 54-7612

PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME II

H. J. Atmauisr (389-399, 400-419, 444-
447)*
Vice President and Research Director
The Grange Company
Modesto, California
CuHARLEs H. Best (104-128, 129-130)
University of Toronto
Toronto, Canada
CHARLES EI. Brus (132-223)
6403 Murray Hill Road
Baltimore, Maryland
Grorce M. Briaes (628-630, 633-669,
682-687)
National Institute of Arthritis and
Metabolic Diseases
National Institutes of Health
Bethesda, Maryland
ERWIN CHARGAFF (329-338)
College of Physicians and Surgeons
630 West 168th Street
New York,N.Y. |
T. J. Cunua (367-371, 381-382)
Agricultural Experiment Station
University of Florida
Gainesville, Florida
Fioyp 8. Darr (628-630, 633-669, 682-
687)
National Institute of Arthritis and
Metabolic Diseases
National Institutes of Health
Bethesda, Maryland
Grorce A. Emerson (311-317)
Department of Pediatrics
University of Texas Medical Branch
Galveston, Texas
WENDELL H. Grirritu (2-103, 128-129)
Department of Physiological Chem-

istry

School of Medicine

University of California Medical
Center

Los Angeles, California

ARILD EK. HANSEN (300-317)
Department of Pediatrics
University of Texas Medical Branch
Galveston, Texas
RosBert 8. Harris (2, 132, 268, 322, 388,
399-400, 452, 591)
Department of Food Technology
Massachusetts Institute of Technology
Cambridge, Massachusetts
W. Sranuey Hartrort (104-128, 129-130)
University of Toronto
Toronto, Canada
Raupu T. Hotman (268-300, 318-319)
The Hormel Institute
University of Minnesota
Austin, Minnesota
J. M. Hunpiey (452-538, 540-550, 551-
587)
National Institute of Arthritis and
Metabolic Diseases
National Institutes of Health
Bethesda, Maryland
James H. Jones (223-232, 253-256)
School of Veterinary Medicine
University of Pennsylvania
Philadelphia, Pennsylvania
ABRAM KANoF (232-253, 257-266)
Department of Pediatrics
The Jewish Hospital of Brooklyn
Brooklyn, New York
BENJAMIN KRAMER (232-253, 257-266)
Department of Pediatrics
The Jewish Hospital of Brooklyn
Brooklyn, New York
Henry A. Larpy (323-329, 342-351)
Institute for Vitamin Research
University of Wisconsin
Madison, Wisconsin
SaMUEL LepKovsky (591-598, 626-627,
680-681 )
Agricultural Experimental Station
University of California
Berkeley, California

« Numbers in parentheses indicate the pages on which each author’s contribution or contributions may

be found.

LIVRARY}]S

»

vi CONTRIBUTORS

Fritz LirpMANN (598-625)
Biochemical Research Laboratory
Massachusetts General Hospital
Harvard Medical School
Boston, Massachusetts
ArtTHUR H. LiveRMoRE (351-361, 363-
366)
Department of Chemistry
Reed College
Portland, Oregon
Coun C. Lucas (104-128, 129-130)
University of Toronto
Toronto, Canada
A. T. MinHorat (371-381, 382-386)
Payne Whitney Psychiatric Clinic
The New York Hospital
New York, N. Y.
JosePH F. Nyc (2-103, 128-129)
Department: of Physiological Chem-
istry
School of Medicine
University of California
Center
Los Angeles, California

Medical

Cuar.es A. OWEN, JR. (419-444, 447-448)
Mayo Clinic
Rochester, Minnesota
ELAIngE P. Rawr (669-678, 687-694)
Department of Medicine
New York University
477 First Avenue
New York, N. Y.
E. E. SNELL (3861-362, 366-367, 538-540,
550-551, 630-633, 678-680)
Department of Chemistry
University of Texas
Austin, Texas
E. R. WEIDLEIN, JR. (839-342)
Mellon Institute of Industrial Re-
search
University of Pittsburgh
Pittsburgh, Pennsylvania
Hitpa F. Wise (300-311)
Department of Pediatrics
University of Texas Medical Branch
Galveston, Texas

CONTENTS OF VOLUME II

Page
CON MRE UIPORS LEO, VOI UNE Te vr.c rs, sionals oA avs 2 1s a aoe a EE oe te ne Vv
CHAPTER 5. CHOLINE
leeNomenclaturevand Bormulay.. 0 «.ccie occ. 5 anos Rone Se ee ae 2
Ropert 8. Harris
IMEC HeMmiStiver ets sae ks oc Aare Sas Sic ee ee ye 2
WENDELL H. GRIFFITH AND JOSEPH F. Nyc
iitimeindustrialsbreparation ys. aoc. ces oct yice ne cee ny ne 14
WENDELL H. GRIFFITH AND JOSEPH F. Nyc
iWerBbiochemical’ Systems... .3-))..5.0..5--2. 0000560 Bee aN Sir oe RE CRT eee 15
WENDELL H. GRIFFITH AND JOSEPH F. Nyc
Wire SPEGiniCitysOl ACTON fr sc2 ccitis steed eisielt melt Greene ees 2 6c ee a 45
WENDELL H. GRIFFITH AND JOSEPH F. Nyc
re INST S re ts ee hin eo wry atte ata > cia, Gite ee alw a tcnye cus Sw dese Meas 49
WENDELL H. GRIFFITH AND JOSEPH F. Nyc
NV AMS ES SUIT ALO Eley te rene oy reps Aes oe earthen ete ae ie aati Rae eee 52
WENDELL H. GRIFFITH AND JOSEPH F. Nyc
Winer Standardization Of ACHVITY.< .a.c. 0.08400 Sedbea. cs od mdiew sdkin i cule ean euen ase 58
WENDELL H. GRIFFITH AND JOSEPH F. Nyc
IX. Occurrence...... Nas Fe PPS oe ats OP ee edt re ee SE REE, ee 58
WENDELL H. GRIFFITH AND JOSEPH F. Nyc
NOME ITECtSsOLeD ETICLEN CY) ry. oe aarti cnn ols IeVals Os Ee oe Re: 62
ea GeneraleVianitestatiOns ses ' sm be toh eis ate oa ee ose er es 62
WENDELL H. GRIFFITH AND JoSEPH F. Nyc
1B). TRISH he) See Oey Men te oc eens Reet ene Oe Ae Om: So mm RE Lg! Lene 63
WENDELL H. GRIFFITH AND JosEpH F. Nyc
CR Atviains SWE CIOS 47. seal. echt os cet tes RESIS ee re, 93
WENDELL H. GRIFFITH AND JosEpPH F. Nyc
ID) IDYOyits, Sacto Oe OS Seno Oe. Siero e Re EEE ho Can OL Gn OO a eect Or at pete 97
WENDELL H. GRIFFITH AND JosEPH F. Nyc
BOP GHETUSDERIES 10 sce ha. vikiiaavd' 4G Eels AAO a eT ES ee 101
WENDELL H. GRIFFITH AND JosEpH F. Nyc =
TES AY [Ee pee ected oe ga Oe) be Aa te 104
W. Stanuey Harrrort, Coiin C. Lucas AND CHARLES H. Best
PNGB AT ITN COLO PY, at hcp ess) or ess s/ Soctsrad teu Fe isl rand uk ISIS I erect otatt eee ee tones 123
W. Sranuey Harrrort, Coiin C. Lucas, AND CHARLES H. Brst
PCPA ITCREETIES? 25th 32 Gate OUND ea ee ane Oe OARS SO OS eee Sek Be 128
ENS LOSE TNT DOTY I) Siac Ce ILO Cee UL RCT REAPER MERCI a POPPE ALE dh Oo eae 128
WENDELL H. GRIFFITH AND JOSEPH F. Nyc
Bye re ELUNE DUI chee lee sat oti Oi aa Se earn See e ee Ce ee 129
W. Sranuey Harrrort, Coitin C. Lucas anp CHarLeEs H. Brest
CHAPTER 6. VITAMIN D GROUP
IPeNomenclatirevandsHormnulascnics) (eee sacle cece cote oreo ete ce ee ees 132

Rogpert §. Harris

vill CONTENTS
Page
ICH emis thy aes jakcila Rc SAR ee OS ee cee, oon ee 132
CHARLES E. BILus
be Industrial Preparation: 2.2 2:69 crt eags 2 ce site i ee 210
CHARLES E. BILLs
Ves Bs timvationic i): co... ss chisel aetererne oe ae 215
CuHaRLEs E. BILis
Vio Standardization of Activity-55..-060 02) ae ee eee 221
CHARLES E. BILus
Vii effects of Deficiency...< cease toe oe eo nee Oe eee 223
A. Tn vAniimalvn sc. Ae aeeebeea cece ks od cnmee Re eee Ce OO 223
JamEs H. JoNES
Be invPatholory<of FuumangBein gsi ernest Serer ee 232
BENJAMIN KRAMER AND ABRAM KANOF
Vil. ChemicalsPatholovysandtkharmacologya- oe as45 4 eae eee 248
BENJAMIN KRAMER AND ABRAM KANOF
VIET. Reqginenients: 3.050 0c4.. 3 tae cone bode eee: bleak eee eee 253
ASO fecA I ase ce hae 5, os eles Soeee ata ee ne a ae, Oe 253
JAMES H. JONES
Bs (Of Human. Beings. Ai... Ss ceorte cacti s ea a een Oe eee 257
BENJAMIN KRAMER AND A. KANOF
CHAPTER 7. ESSENTIAL FATTY ACIDS
i. Nomenclature:and: Formulas: + . Ao ondansetron ee 268
Ropert §. Harris
DE Chemistry nuncio. Wed enact Gee de Soe BO Lalick: See ene 2 a oo 268
RaueH T. HOLMAN
tik: BiochemicalSystemisin) 3.8 .26.0- ae Oe ae Re 8 eee 277
Rautpeu T. HommMan
LY: Specificityvof Actlome 57-922 ao ees ae oe ER oc 278
RaupyH T. HoLMAN
V. Biogenesis, and Metabolism. . ...02¢...2hcnei hates hs, ooo: 280
Raupey T. Hotman
WL. (BStimva tion netic eal: s bagsciac cs an. cin a OO ite Jc 2 blo Aon 286
RaupH T. HotMan
WIL. Occurrence in Hoods: «5... 22%. bbc cdo Geen Os a ee 291
Rate T. HOLMAN
VEL. Eécts*of Deficiency... 2. ach. oSaeS. Sane ey oe ne ee eee 292
‘A... In, Amima] ssanditinsects: ns): 23s Habe ctr nae eee ee 292
Raurpu T. HOLMAN
B. AnsHumankB eines #9 caey: ih ate cs Woe ias oe aa eee ee ee 300
ArILpD E. HANSEN AND HiLpa F. WIESE
Px Pharmacology...) kas ons ao cee oats Sy aia. Gia So nee baeeents 4ee See 311
Arttp E. HANSEN AND GEORGE A. EMERSON
Xi; Requirements: |... 2. ....ch oy Sacto. B+ 2h 7G6 vo ee oeeoet eees ote eee 318

Rate T. HOLMAN

CHAPTER 8. INOSITOLS

le

Nomenclaturesand! Formula 3.0 js 57 co ee oe ee oe eee 322
Rosert 8. Harris

CONTENTS 1x

Page
mepenscussion af. Lerminology: .. Ss. «22... .>-- = aeassarest Accs et -t 323
Henry A. Larpy
TIL, (CLG TaN reas le eens Pe eee ey ec ce no CADE a omens DERE 329
ERWIN CHARGAFF
Beeodustiial Preparauons «oh Avballh.. i aos ead ween nenes kp: 339
E. R. WEIDLEIN, JR.
Weeriognemical Systems) 42.50.56) fas. s: «5 <5) dee ae papre tat 342
Henry A. LARDY
Mem S NeCuicityOlACtiON: 726.0 .s 6.6 cone eos 4 oe Re ee tates © 351
ArtTHUR H. LIVERMORE
WIL. LORS TGS tte ae te eee Ra a Sp oc cen Sor Heeoies 354
ARTHUR H. LIVERMORE
WA StiMatiONn Of TNOSICOL. «.¢.2....6 ose. ¢ 6 y os vi anwre Sow a becca orks ae Roe eee ate 358
ORES MUSGLATIONS: 2's. oR ties 4's a stek did ee Hae DAR ON oaks oe eR ee eee 358
ARTHUR H. LIVERMORE
BeebyaChemircal: Methods’. irc. oes odes ds he scene + © ors cheered cee son 359
ARTHUR H. LIVERMORE
Pee ab amen hrormidGOeraplye. . Jaci cscs cine. oe. gue aeage wih 2 ls Aa a ee 361
ARTHUR H. LIVERMORE
DD Pa B yal VIO USE SASSAi Vin. sic. 4 seni etochees Dae sats GRY ae Eee os iene o oats 6 sie 361
ARTHUR H. LIVERMORE
HPeebvaniicropiolopical Agaay.< 2 “250 eaencat eeee toe oe eens fone ee ee 361
E. E. SNELL
Wa MAEMO) COUTTEETIC Cite co otk Mamepht we ise Shai y Se vstrieai as bain Bade RRC See GEERT ate 363
ARTHUR H. LIVERMORE
lON@mEeCtSeO he D CL CIEM Cyr rete. weais.0, be wich we erertimere Oe Sete OR Henn A pe 366
Atel nie ViNCROOLCANISIMG em frayar sks Mrsctbeae > € Ses AR eto Aa oe EES oe 366
E. E. SNELL
SEPT ASTRAL ees esac Aeapeie Saw ocusy ek ee AN ns he Ee a CEL 367
T. J. CUNHA
Cmweatholopgy in. umean: Beings. «...20,<.++ asim «cA NO. ants 2 2 371
A. T. MinHorat
ROME NATIMNACOLODY 2 ser nck ts. es ea cieew ee knee ee Fe IS Oe eos apa ke Rech cece 377
A. T. MrtHoRat
PXSIPRPRR CQ ULIT EIN CLUES 50 piieccrg eas Ses hd oie iaile SAVE tu AE ap ak FAQ Am He ele IR an a 381
FANON) fe CATARINA, Sr. Sec re stearic cos ails <i -apeaivdn ute ecto, cobra ROR SRE ELA cate nte 381
T. J. CUNHA ?
TB (OFA [7c POR a an nn aE OE ee Pes El ere ie chats eee <A 382
A. T. MinHorat
Celnivuenceron Other: Constituents of Diectheu-ni.-cekeeieet en iane sects = 383
A. T. Minyorat
Wmv Menesis in the BOUY., 2 24 ark oes ssi oaks = aa. BO ORR aa ae.) 385
A. T. Mityorat
CHAPTER 9. VITAMIN K GROUP
ieeNonenclatune and Kormulas):«-.060.042¢1cGshois eet ee ee 388
Rosert 8. Harris
PORES SECU th em ty eke ia scder i Se aslecrala cs anise eV me ale mika alt 389

H. J. AtmMquistT

Oe CONTENTS
Page
Litsindustrials:Preparation.: ». 004 o4:eac rata eee ee ee eee 399
Ropert §. Harris
IVieBiochemical. Systems: ... 2 ocehcs a bacon canoe Oe eee eee 400
H. J. ALmMQuist
VesEstimation: ... ...65 ts ios ee ee on Eo be ee ee 402
H. J. ALMQUIST
VilwOccurrence in Natirescs.53252 sons he ee eee 415
H. J. ALMQUIST
WAL Bfiectsiof: Deficiency: 52652 42.32)5. nso t aon shad oe ee ee 419
As Tn Animals 553.27 shee coin oreevcittens 0 Lace ne 419
H. J. ALMQUIST
B: In Human Beings sie. -2c neh. Ghee ees d oad oe Sees 1 One eee 419
CHARLES A. OwEN, JR.
WILE: ‘Phammnacologys sii cccibet ntccaninn tet 2 he eeedk es os be eee 435
CHARLES A. OWEN, JR.
EX. Reqgiremetits:...2..02<. i040 24. anc sack be St bee eee ee 444
(A XO feoAtnaN all S oy. oh se si wa Sco ahr bcs Seer oe 444
H. J. ALMqQuistT
iB; OfsMuman: Beings iis... 05/82 nae anced son eee eee oe sO oe eee 447
CHARLES A. OWEN, JR.
CHAPTER 10. NIACIN
i Nomenclaturesand :Kormulas=.— >). 5. oee oe ee eee ee eee eee 452
Ropert §. Harris
TL. Chemistry sac feed tency waa adlconte dc Seaon ara Hee Oy Reeaetiche Oe Ge eae 452
J. M. HunDLEY
Lil. Industrial) Preparation::...s..2 0.6 ¢s05. cad asin > oo seine Re eee Ore 478
J. M. HunDLEY
IV... BiochemicalsSystemSic0: «ke hs cies Dass se eationss fae eee ee 480
J. M. HunDLEY
V. Specificity of Action. ... .. ....<.4- i... See eee 0. eee 517
J. M. HunDLEY
Wil. BIO BENESIS): 5 coc ciste-o sc leuele sae aie Drage oe aoe ets eseoe ie: she 521
J. M. HunDLEY
WATS Estimation. 320524 6: wok eS ned corad whew s oa eae sale eee oe ds eee 534
A. Biological Assay so). sc ic.s coc ia +s o8 de veoes essa. 2 ee 534
J. M. HunDLEY
B(Chemical Methods: 2s .c.5.0 sacs soci noes. hee oe 534
J. M. HunpLEY
C.Microbiological, Methods 4% 2200.5. 4. sie ese ee eee eee 538
E. E. SNELL
Vill .Standardization: of VActivity...5 osc ..0.c.4 cs..0..)2 see rae ire te ele ee 540
J. M. HunpDLEy
WX TO CCULTENCE! cricncc tis coisa eo ot casac ete Cee Ios eT eae ani aT 541
J. M. HunpDLEy
X. Effects of Deficiency: s...6 264 ic. 5 ocak): maps OR totem ee bee eee 550
Ae Tr MirGroOrganisms :...ise6% ietacs 4 ofa eveos otto ey bro Sus ehh ei be es order oe eee 550
E. E. SNELL
B? In' Man and Animals!) 34/9 < -ccee nse ee ei oh SE eae =a ee 551

J. M. HunpDLEY

CONTENTS x1

Page
MEDAL OLO Dyer ten are ter cs ie ere cles onetey tase ss «snl w Sah GH RO ERE R os clsust auereke os Basra h seston ie 565
J. M. HunpiLEy
eaITeMBE NTR ETIN AC OLOG. Vip cetera sass ie once SES 3 GF 205-3 ek eR Sos Gees SESE oe te 571
J. M. HunDLEY
XIII. Requirements and Factors Influencing Them........................... 578
J. M. HUNDLEY
CHAPTER 11. PANTOTHENIC ACID
IMINOINGNClACUTE Ms waters cle a Sea cl cece ete shea hl ee cee eras 591
Rosert 8. Harris
ILL. (QUNGT CUT eee es te ene ain nA in ee ee nr tO nn BREE RRS he ruchinin ctor Res 591
SAMUEL LEPKOVSKY
See DERM CAl SV SLOM © 5.0855 cece acs siinc rhe he ose etree a hd > AERO SOON 598
Fritz LIpMANN
IWMemSOpeciiicrtyeOf Action irate: Bot cin: Dates dooeuens ata oo deme ase eee 626
SAMUEL LEPKOVSKY
WW, LBIGPaS TSENG. atta Fee ol ore rie Eee rc eon nn, 9 Sr arses PReRERCLG NAcc 627
SAMUEL LEPKOVSKY
WAL, TRESS TERRE ok Sag, oO rie Un Oe, eames eC E el an ma EME WA oA aa ene Re een ferme, 628
PMeaneonemical-and Physical Methods. ...0.0 7.4.4.5 fan si eee a eeuyenieaoe mes 628
GrEorRGE M. Brices AND Fuoyp 8. Darr
Eee Ss tOLopI Gar IVet BOGS 2.52.0 oes. 1. oo te we es se are oa ees Some cae Oa ele 628
GEORGE M. Briecs AND Fioyp 8S. Darr
CAViicroplologicaleNlethods src eictorie cae cei tocar) oe nee 630
E. E. SNELL
Danita standaLai zation Of ACtVIEY: 5.25)... cigs afscp noone tints ole ols we Pane ns wearin 633
GrorcE M. Briccs AND Fioyp 8S. Darr
VAT BINIPRN@.G CUTE NICE rt sent aed tee cacsit oo au tesvatees oieus autiard ay vote ydutay caine See we au apoasmnaperet che teks See eIe 634
GEORGE M. Briges AND FLoyp 8. Dart
DXeeEHeCtsHOfsDeficlenCys we eit. ona cutie erie a ea me elena ea 649
PAC Lite ATID) Sieh eae ee Nene wi 5 3 ee feo, er a A POM 0 ected chara ame 649
GEORGE M. Brices AND Fioyp 8. Darr
Va}y. lity MLS iiss ars cyte REARS ae PARE RRMA Ro a 0a At nee Per tetu s eR a Dele ne ee Res 669
ELAINE P. Raut
Ore ltr VNCrOONPANISIIS 5 ou cs oeai> ch. ly.< CROP RN eo cw Madani Deere 678
E. KE. SNELL
ToL LAVA pan Tete) (2) a Orage i a A AeA Rene an Are 680
SAMUEL LEPKOVSKY
XI. Requirements and Factors Influencing Them........................... 682
Ate O PATINA Siento te tat hus a eatin eet et, nc eR Meee aR Sopa ran 682
GEORGE M. BriccGs AND Fuioyp 8. Darr
134 CORN IEY Die ees ok ene Ae AR tare oe Pat oe SRE me AEE AN fa we ne ae 687
ELAINE P. RAL
RAY UETO TET CX oe ee Nr A ee, rr hs ne oes Aer een. nS pee re TA Oa GE hd da on 695

iy
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“45
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j 7

CONTENTS OF VOLUME I AND III

VOLUME I

CHAPTER 1. VITAMINS A AND
CAROTENES

Robert S. Harris, H. H. Inhoffen,
Karl E. Mason, F. H. Mattson, Nicho-
las A. Milas, H. Pommer, George Wald,
S. Burt Wolbach

CHAPTER 2. ASCORBIC ACID
Robert S. Harris, L. W. Mapson,

Mamie Olliver, Mary Elizabeth Reid,
Fred Smith, Richard W. Vilter

CHAPTER 3. VITAMIN Bie

Frank H. Bethell, Karl E. Folkers,
Robert S. Harris, Thomas H. Jukes,
William L. Williams, Donald E. Wolf

CHAPTER 4. BIOTIN

Paul Gyorgy, Robert S. Harris, Esmond
E. Snell

Author Index—Subject Index

VOLUME III
CHAPTER 12.
ACID

Robert S. Harris, Charles C. Scott,
Peter A . Tavormina, Lemuel D. Wright,
H. M. Wuest

CHAPTER 13. PTEROYLGLUTAMIC

ACID

Frank H. Bethell, Robert S. Harris,
EL. L. R. Stokstad

CHAPTER 14. PYRIDOXINE
Paul Gyérgy, Robert S. Harris, Charles
E. Keevil, Jr., John C. Keresztesy,
Henry Sherman, Esmond E. Snell, W.
W. Umbreit, Klaus R. Unna

CHAPTER 15. RIBOFLAVIN
Robert S. Harris, D. M. Hegsted, Max
K. Horwiit, Esmond E. Snell, Theodor
Wagner-Jauregg

CHAPTER 16. THIAMINE
Robert S. Harris, B. C. P. Jansen, W.
H. Sebrell, Jr., Klaus R. Unna, H. M.
Wuest

CHAPTER 17. THE TOCOPHEROLS
Philip L. Harris, Robert S. Harris,
Karl E. Mason, Henry A. Mattill

CHAPTER 18. NEW AND UNIDEN-

TIFIED GROWTH FACTORS

Vernon B. Cheldelin

Author Index—Subject Index

p-AMINOBENZOIC

Xili

\ ¢
j 7.
i
4 +
AL (i
é 4e + e
b ae
= 4 oe) Jn || ores
YY eee oi Oa ie ee
vai 1’ Al -aae wa ee ee ~
ry ad ' tiges) ‘ : ws i pariee
Ghent] Uae inline Op ee 18>) 8 re Tere
ee Se

ui ae Ging © ete) ee Ce ee

een ae ey ee ee rc.
MA) if tee 1 Sra ‘. Mie cn om,

CHAPTER 5

CHOLINE

Page

meomenciaturerand Pormulai 2. a ke ow ee em re an ne 2

PEO HCOUSUR VAD ALS Eo Pie Te oe See ee 5, & ss 2

A} Isolation . . Si al bec are Re | ee ae ee ee 2

B. Physical and Chemical erenerties SUL. Ok See SL Sg ceeicen nO

Ca Constitution and Synthesis. ~ . s : « « & 2 A + ee eeey ee) 0

III. Industrial Preparation . . se) ee gq- GRO TE Bee
(included in part in Section Tt C)

BPE MOE HeMmiGal OVStCINS. (5005. 5.55 8 ve cl hay Sc satel) cee tery ohye eee oP cat

A. Enzymes and Coenzymes . . Sees eyes ~ a, sgh ot eal

B. Choline Acetylase and Mec ichalimostornse ee obs yea eee LO

CaMechanisny of Action. of Choline, ..- 2 « 6 <is06 2 2 Ys

i Derivatives;or Phosphoryicholine . 2.5 « . « « »« » «= «6 . J8

2. Choline and Transmethylation . . Sse eens, Meee AY)

3. Nature of Methyl Donors in Tiranawnechylation ae 24

4. Methyl Acceptors, Transmethylation, and Formate-to “Methyl! Syn.

chesis:.*. = 26

5. Choline Gease na pie @noline: Bernine: @ipeme Relntioneeine 33

6. Oxidation of Methyl Carbons . . SBE Sect One is ee

7. Other Aspects of Methyl Transfer ae Gradetian Se Tee ee

MES CCIICTUN OfeNChION® GW as Se. ga ls cee So IU A a ee ie 346

Solr ta Pencaisn, fie. tig os os orca eaeey sees VR ee ae swe SD

VII. Estimation. . . base UN ooh ee te OP EOUS MGC Gaon

A. Chemical eeaeedures Sg PS Ba Pode ok Nort teens 0h) jet teh' Sr pees

EeeNitcrobiologiesal Procedures. \ Js. << ce Goh Se wo | oss aw) lene ee OD

MEE IOUIDICALSASSAV Sate) Go gcweshe tile yed tae dale, ahh eae ade eee tl Ol

DSEvsicwl) Bstimacion-.. .s 9.2 0’. «se. eos 4 oS & Oe be. 6 OU

Poe cvandaraization of Activity 0 . 5 « 2 «foes 2 oe «6 ee SS

IX. Occurrence. . . Ee yt RM ER Mees Ds 8 ark Bes, he toers . efeigis bagi |

X. Effects of Menueney Dyce oo Os ba oi te Crack AM MAUI) wats. AP A ES TING?

pemGeneral Manifestationgy. i. ic... eles. agrini Pe fd La Stealce BL So G62

BEC Ais: soees leiy be Dees Ee ner ed Bane Seo co ae Ronen Fey aan

1. Renal i eston ia ee Bale, rele sieve tn) Bich eee A line ek ered 1 Ons

2. Fatty Liver... etek. hie eee Wee Sua hig iae te io tee mie th Pod OU
3. Effect of Sulfur win’ Reiita Re Ee ee Re ee ee Lo
AP petect OMOther Nutrients 24 3: Las elise See OS. OTe 8O
HeMOLMGESHINEG EME 7 ISL IFALROl! cian ceitecs vipa iy ahhuast ile Paoax Ga } 88
ie Le tore Olive) Wet ee er ee ie ke a or eee Lemme © >)
pret CIC RMON ie Aion FORE: Go me: pS te Sh ge el cee toe ale Ske Oe
DORs sc RS. EE Te oy 9ei Ly! ba cx Is Saal Pilg: a? ier oh geeia Rall OG
. Other pecs SE ee a ote as Pal Moe as i ee eae el ee ee oer OI
Ph es Ge eee beet a. tes eee em Me bing Oe. Vi a ee

maimoo

2 CHOLINE

daStamaple Fat. 50 Vee ae Ge ARO oe

Pee C1506 | re Pet a le

3. Portal versus Non- Portal Choe a duke ee oo) 5 ee

KP SRP MarmMecolory 2 ks ae cal we lal a iy ce etm a ou et
XII. Requirements. .. . ste oe bo ca a gh ok er
A. Requirements of iene SER Sa yRIT SS les Neel locks 2 ee

B: Requirements of Human Beings . 5... « . = 7. 3) = seen

I. Nomenclature and Formula
ROBERT 8. HARRIS

Accepted name: Choline

Obsolete name: Bilineurine

Empirical formula: C;sH;;02N

Chemical name: 6-Hydroxyethyltrimethylammonium hydroxide
Structure:

|

OH

II. Chemistry
WENDELL H. GRIFFITH and JOSEPH F. NYC

A. ISOLATION

Choline is widely distributed in biological materials as free choline, as
acetylcholine, and as more complex phospholipids and their metabolic in-
termediates. It is an integral part of the lecithins, which accounts for its
occurrence, in combination at least, in all plant and animal cells. It is also
one of the bases of the sphingomyelins of animal tissues. Phosphorylcholine,
glycerylphosphorylcholine, and the ester of phosphorylcholine with sphingo-
sine have been reported to occur, but it is uncertain to what extent these
components of phospholipids normally exist free in tissues. Choline is char-
acterized by a trimethyl quaternary nitrogen. Substances related to choline
in this respect, include glycine betaine, carnitine, and ergothionine. From
the viewpoint of lability of methyl groups, related compounds are methio-
nine, dimethyl-8-propiothetin, and dimethylthetin. The latter is of con-
siderable importance in laboratory studies but is not known to occur
naturally.

II. CHEMISTRY 3

CH.— CH; N=(CHs3)s O==C—CH,— Y=(CHs)s
| + | | +
OH OZ
Choline Betaine
CH.—CH.—N=(CHs)s O==C—CH,—N=(CHs)s
| I+ | [+
O H
O—=C—CH;
Acetylcholine Betaine aldehyde
CH.—CH, N= (CH); O
| | + |
O CHC
| |
O= P—O_ =
| |
OH (CHs)2
Phosphorylcholine Dimethylthetin
O O
| |
eich. —Ch_-C_0H. @Hs-CH:—C 02
| | |
S NH, SS
SPs
CH; (CHs)2
Methionine Dimethy1-8-propiothetin

Surprisingly, the first isolations of choline were not from materials rich
in the complex lipids but from hog bile by Strecker! in 1849 and from an
alkaloid of white mustard seed (Sinapis alba) by Babo and Hirschbrunn?
in 1852. The latter workers named their product sinkaline, whereas Strecker?
in 1862 applied the name choline to the substance obtained from bile.
Subsequently, Liebreich* separated a fraction from hydrolyzed crude brain
lecithin (protagon), which he named neurine. Dybkowsky°® soon found that
Liebrich’s base was choline and not the vinyl compound known as neurine
at the present time, and Claus and Keesé® demonstrated the identity of
sinkaline and choline.

Choline has been obtained from a great variety of tissues and fluids

1A. Strecker, Ann. 70, 149 (1849).

2L. von Babo and M. Hirschbrunn, Ann. 84, 10 (1852).
3A. Strecker, Ann. 123, 353 (1862).

40. Liebreich, Ann. 134, 29 (1865).

5 W. Dybkowsky, J. prakt. Chem. 100, 153 (1867).

6 A. Claus and C. Keesé, J. prakt. Chem. 102, 24 (1867).

4 CHOLINE

since these original isolations. Wrede and Bruch’ extracted various tissues
with hot acidulated water, and the choline in these extracts was isolated
and weighed as the chloroaurate. Bischoff et al.,5 using a reineckate precipi-
tation, reported finding up to 45 mg. of free choline (calculated as the
chloride) per kilogram of muscle. Heesch® prepared extracts of blood serum
which had been treated with trichloroacetic acid and found in these ex-
tracts 2.5 to 10 mg. of choline per liter of blood. Strack e¢ al.'° have pre-
sented data which suggest that much of the evidence for the presence of
free choline in biological materials is unreliable, owing to delay in the
preparation of extracts, with resulting release of choline by autolysis. They
found that dog’s liver contained 0 to 43 mg. of choline if extracted imme-
diately after death of the animal and 136 to 164 mg. of choline per kilo-
gram of liver if extracts were made 5 hours after death. A similar slow
release of free choline occurred in experiments in which the fresh tissue
was suspended in alcohol. Strack et al.!! did not find free choline in rabbit,
dog, or beef muscle. On careful investigation the substance in muscle which
was precipitated as the reineckate and reported as choline by Bischoff et
al.8 was found to be carnitine.

Many solvents have been tested with respect to the thoroughness with
which total choline, combined and free, is extracted from natural products.
Among these are benzene, petroleum ether, ethyl ether, ethanol, methanol,
acetone, chloroform, and mixtures thereof. None has proved to have any
special advantage over methanol itself.” Engel” employed multiple ex-
tractions of samples with methanol in a Bailey-Walker extractor. The more
convenient method of extraction with the Soxhlet apparatus is generally
preferred. Glick has recommended the mixing of powdered samples with
No. 2 pulverized pumice, after weighing, to prevent caking of the sample
and the resultant channeling of the extracting solvent.

The residue of the methanol extracts of samples must be hydrolyzed if
the total choline content is to be determined. Barium hydroxide has been
favored as the alkali for the digestion process because there is no loss of
choline when pure choline solutions are used.!°!8 Gulewitsch™ studied the

7F. Wrede and E. Bruch, Hoppe-Seyler’s Z. physiol. Chem. 195, 255 (1981).

8 G. Bischoff, W. Grab, and J. Kapfhammer, Hoppe-Seyler’s Z. phystol. Chem. 207,
57 (1932).

90. Heesch, Arch. ges. Physiol. 209, 779 (1925).

10}, Strack, E. Neubaur, and H. Geissendiérfer, Hoppe-Seyler’s Z. physiol. Chem.
220, 217 (1933).

11, Strack, P. Wérdehoff, E. Neubaur, and H. Geissendérfer, Hoppe-Seyler’s Z.
physiol. Chem. 233, 189 (1935).

122 R. W. Engel, J. Biol. Chem. 144, 701 (1942).

13 M. Rhian, R. J. Evans, and J. L. St. John, J. Nutrition 25, 1 (1943).

14D). Glick, J. Biol. Chem. 156, 643 (1944).

15 W. Gulewitsch, Hoppe-Seyler’s Z. physiol. Chem. 24, 513 (1898).

II. CHEMISTRY 5

effect of heating choline in aqueous baryta as well as in alcoholic solutions
of sodium ethylate and found only a negligible breakdown of choline after
boiling in baryta solution for 6 hours or after heating in a 5% sodium
alcoholate solution for 24 hours.

Beattie!’ studied the acid hydrolysis of a lecithin emulsion prepared from
a commercial egg lecithin preparation and hydrolyzed in 7.8 % hydrochloric
acid at 110°. The maximum value of free choline was obtained after hy-
drolysis for 21 hours. Acid hydrolysis has been used also in the liberation
of choline from bound forms in tissues.2°-? Ducet and Kahane” refluxed
animal and vegetable tissues with 30% nitric acid until a clear solution
was obtained. After neutralization of the solution with powdered calcium
carbonate and dilution with several volumes of water, 10 ml. of 50 % ferric
sulfate and 5 g. of calcium carbonate were added for each gram of dry
tissue originally taken. The mixture was heated to boiling and filtered.
The filtrate and washings containing the choline were concentrated to a
small volume, and the choline was precipitated by one of the reagents
generally employed for this purpose. These workers found that no choline
was destroyed during this procedure.

The earliest methods which were employed for isolating choline from
biological extracts were dependent on the use of various sensitive though
non-specific precipitants. Choline may be precipitated from alcoholic solu-
tions as the double salt of platinum, gold, or mercury chlorides.!*: 23> 4
Precipitation as the reineckate or the periodide has been employed most
extensively for the removal of choline from aqueous solution.

Beattie!® observed that a quantitative precipitation of free choline as the
reineckate can be obtained in solutions containing as little as 0.03 mg. of
choline chloride per milliliter and that the choline in about 7 to 10 ml. of
a solution of this concentration can be quantitatively determined. The
slight extent to which other substances interfere with the reineckate pre-
cipitation and estimation of choline in animal tissues and fluids was demon-
strated by Beattie by analysis of tissue extracts, a tryptic digest, and urine
before and after the addition of known amounts of choline chloride (Ta-
ble I).

16 B. N. Erickson, I. Avrin, D. M. Teague, and H. H. Williams, J. Biol. Chem. 135,
671 (1940).

uw H. P. Jacobi, C. A. Baumann, and W. J. Meek, J. Biol. Chem. 188, 571 (1941).

18 J. Kapfhammer and C. Bischoff, Hoppe-Seyler’s Z. physiol. Chem. 191, 179 (1930).

oF. J. R. Beattie, Biochem. J. 30, 1554 (1936).

20 J. D. Fletcher, C. H. Best, and O. M. Solandt, Biochem. J. 29, 2278 (1935).

21 R. W. Luecke and P. B. Pearson, J. Biol. Chem. 158, 259 (1944); 155, 507 (1944).

22 G. Ducet and E. Kahane, Bull. soc. chim. biol. 28, 794 (1946).

*3 A. Lohmann, Arch. ges. Physiol. 122, 203 (1908).

*4C, T. Morner, Hoppe-Seyler’s Z. physiol. Chem. 22, 514 (1896).

6 CHOLINE

On the basis of a careful study of the reineckate method, as originally
modified by Jacobi et al.!’ and Engel,” Glick“ has proposed the following
procedure for the isolation of choline from natural materials:

A weighed sample, containing the equivalent of 2 to 5 mg. of choline
chloride, is placed in an alundum thimble of medium porosity (80 mm. long
and 22 mm. in diameter) for extraction in a Soxhlet apparatus fitted with
a 125-ml. boiling flask. About 100 ml. of methanol is used as the solvent,
and the extraction is allowed to proceed for 24 hours. With some finely
divided materials such as flour, the tendency to form a hard cake makes it
desirable to mix the sample intimately with No. 2 pulverized pumice to
facilitate the extraction. The boiling flask containing the methanol extract
is placed on a steam bath and, when only a few milliliters of solvent remains,
30 ml. of a saturated solution of barium hydroxide is added and the heating
is continued for 90 minutes. After the mixture is cooled, a drop of 1% al-
coholic thymolphthalein is added to the hydrolyzate and glacial acetic is

TABLE I
RECOVERY OF ADDED CHOLINE BY THE REINECKATE PROCEDURE!?

Choline chloride, mg./ml.

Originally Amount Total Amount

present added amount recovered
Kidney extract 0.13 0.69 0.85 0.72
Liver extract 0.18 Ona 0.94 0.76
Tryptic digest None 1.50 1.56 1.56
Urine None 1.50 1.56 1.56

introduced until the blue color is just discharged by one drop. The liquid
is then filtered by suction through a sintered glass filter tube of medium
porosity (15 to 30 ml. capacity) into a 125-ml. suction flask. The boiling
flask is rinsed with small portions of distilled water, and the rinsings are
used to wash the filter, a total of about 15 ml. of water being used. To the
combined filtrate and washings is added 6 ml. of a 2 % solution of reineckate
salt in methanol, and the flask is placed in a refrigerator at about 5° for 2
hours. The choline reineckate precipitate is filtered with suction into a
30-ml. sintered glass filter tube of medium porosity. The dried precipitate
is washed three times with 2.5-ml. portions of n-propanol and again dried
by means of the suction.

The above procedure avoids the precipitation of betaine reineckate which
is insoluble in acid solutions but soluble in slightly alkaline solutions. How-
ever, it has been noted®® that dimethylaminoethanol appears to be carried
down in part in the choline reineckate precipitate when a solution contain-
ing the two bases is treated with reineckate at a slightly alkaline pH.

26 T. H. Jukes, A. C. Dornbush, and J. J. Oleson, Federation Proc. 4, 157 (1945).

Il. CHEMISTRY "4

According to Coujard”* treatment of tissue sections with reineckate pre-
cipitates choline reineckate as birefringent crystals which are readily seen
with a polarizing microscope. Keenan” has described microscopic proce-
dures for the quantitative detection of traces of choline as the reineckate
and as the chloroplatinate.

The periodide separation is generally considered to be one of the most
sensitive methods of precipitating choline. Griess and Harrow” had utilized
the insolubility of the periodide to isolate choline as early as 1885. In 1896
Florence?’ described a medico-legal test for semen stains based upon the
typical crystals formed when this material was treated with iodine in po-
tassium iodide solution. Bocarius*® isolated the typical Florence crystals
and proved by chemical identification that choline was the substance which
gave the insoluble periodide. Booth*! estimated that in aqueous solutions
potassium triiodide gives a precipitate with choline at a dilution of about
1:50,000. Stanék®? studied the chemical composition of the choline perio-
dide precipitate and the conditions under which it is formed. A detailed
study of the periodide procedure for the isolation and subsequent estima-
tion of choline was made by Kiesel.**

Choline may also be precipitated from water with phosphotungstic,
silicotungstic, and phosphomolybdic acids.1®, #4 Ackerman®® used dipicryl-
amine as a precipitant, the choline salt being only slightly soluble in water
(0.02 g. in 100 ml. of water at 20°). The low solubility of the salt permitted
the separation of choline from betaine and aminoethanol. Schoorl’® pub-
lished descriptions and enlarged micrographs of the double salts of choline
hydrochloride with the following reagents: platinum chloride, sodium gold
chloride, mercuric chloride, mercuric iodide, potassium bismuth iodide,
picric acid, and picrolonic acid.

Several combined water-soluble forms of choline have been isolated from
biological materials. In 1929 Dale and Dudley*’ succeeded in isolating
acetylcholine from an extract of horse spleen in sufficient quantities for
chemical identification. Since that time the acetylcholine in tissues has

26 R. Coujard, Compt. rend. soc. biol. 142, 15 (1948).

27 G. L. Keenan, J. Assoc. Offic. Agr. Chemists 26, 96 (1943).

28 P. Griess and G. Harrow, Ber. 18, 717 (1885).

29 A. Florence, Arch. Anthropol. II, 11 (1896).

30 N. Bocarius, Hoppe-Seyler’s Z. physiol. Chem. 34, 339 (1901).

31 F. J. Booth, Biochem. J. 29, 2064 (1935).

2 V. Stanék, Hoppe-Seyler’s Z. physiol. Chem. 46, 280 (1905); 47, 83 (1906); 48, 334
(1906).

33 A. Kiesel, Hoppe-Seyler’s Z. physiol. Chem. 58, 215 (1907).

34 T,, Lematte, G. Bionot, E. Kahane, and M. Kahane, Compt. rend. 191, 1130 (1930).

35 TD), Ackermann, Hoppe-Seyler’s Z. physiol. Chem. 281, 197 (1944).

86 N. Schoorl, Pharm. Weekblad. 55, 363 (1918).

37 H. H. Dale and H. W. Dudley, J. Physiol. (London) 68, 97 (1929).

8 CHOLINE

been widely studied. A summary of the early work on acetylcholine has been
published by Gaddum.**

Hunt? described a biological test for choline based on its conversion to
acetylcholine and the demonstration of the effect of the ester in lowering
blood pressure in cats or rabbits or in decreasing the amplitude of the beat
of the frog’s heart. Ackermann and Mauer“ prepared the acetylcholine salt
of dipicrylamine, insoluble red crystals yielding a red solution with acetone
suitable for colorimetric estimation. Rossi et al.“! compared various deriva-
tives and found that the formation of the crystalline silicotungstate was a
useful method of distinguishing choline and acetylcholine.

Inukai and Nakahara® isolated phosphorylcholine from beef liver. A
yield of 0.3 g. of the crystalline picrate was obtained from 200 kg. of fresh
beef liver. The crystals, which were an addition compound of 1 mole of picric
acid and 2 moles of the ester, softened at 225° and melted at 228°. Both the
synthesis and hydrolysis of phosphorylcholine by intestinal phosphatase
have been reported. “ The enzymatic cleavage of phosphorylcholine has
been studied extensively by Baccari.**: *°

Isolation from animal tissues of a water-soluble substance believed to be
the phosphorylcholine ester of sphingosine has been reported by a number
of investigators.” 47-5! King and Aloisi® isolated glycerylphosphoric acid
from an acid extract of beef pancreas and assumed that it resulted from
hydrolysis of glycerylphosphorylcholine (GPC). Schmidt et al. isolated
the latter compound from incubated pancreas and, on the basis of diffi-
culties in the isolation of homogeneous products of this type, questioned
the identity of the product previously isolated by King and Small* and
believed by them to be sphingosylphosphorylcholine. Kahane and Lévy**°

38 J. H. Gaddum, Ann. Rev. Biochem. 4, 311 (1935).

39R. Hunt, J. Pharmacol. Exptl. Therap. 7, 301 (1915).

40, Ackermann and H. Mauer, Hoppe-Seyler’s Z. physiol. Chem. 279, 114 (1943).

41 T,, Rossi, A. D. Marenzi, and R. Lobo, Anales. farm. y bioquim. (Buenos Aires) 18,
31 (1942).

4. F. Inukai and W. Nakahara, Proc. Imp. Acad. (Tokyo) 11, 260 (1935).

43 §. Bouchilloux and A. Tissieres, Bull. soc. chim. biol. 29, 955 (1947).

44 J. Roche and S. Bouchilloux, Arch. sci. physiol. 2, 283 (1948).

45 V. Baccari, Boll. soc. ital. biol. sper. 21, 48 (1946).

46 V. Baccari and G. Auricchio, Boll. soc. ital. biol. sper. 22, 559 (1946).

47 &. Strack, E. Neubaur and H. Geissendérfer, Hoppe-Seyler’s Z. physiol. Chem. 229,
25 (1934).

48 F. J. Booth, Biochem. J. 29, 2071 (1935).

49. J. Booth and T. H. Milroy, J. Physiol. (London) 84, 32P (1935).

50D. H. Smyth, Biochem. J. 29, 2067 (1935).

51 B, J. King and C. W. Small, Biochem. J. 33, 1135 (1939).

82. J. King and M. Aloisi, Biochem. J. 39, 470 (1945).

63 G. Schmidt, B. Hershman, and 8. J. Thannhauser, J. Biol. Chem. 161, 523 (1945).

54. Kahane and J. Lévy, Compt. rend. 219, 431 (1944).

II. CHEMISTRY 9

presented evidence for the hydrolysis of lecithin to GPC by rat intestine,
and Shapiro** has demonstrated the formation of GPC from lecithin, by
a cell-free extract of pancreas.

Cyclic choline sulfate was isolated from Aspergillus sydowt by Woolley
and Peterson,” who obtained it both from an autolyzate and from an
acetone extract of defatted mycelium. The yield was 0.26% based on the
weight of the dry mycelium. The isolated cyclic sulfate was believed to be
identical with a synthetic product of the formula CsHis;04NS which had
been previously synthesized from choline chloride and sulfuric acid by
Schmidt and Wagner.®** Its use as a source of sulfur by Aspergillus oryzae
has been reported.°*®

As yet no methods exist for the accurate isolation and determination of
the different forms of choline in biological materials, particularly free
choline and combined water-soluble choline. Kahane and Lévy® defined
water-soluble choline as the total found in aqueous extracts of tissues after
suitable precipitation and filtration. Ferric hydroxide formed within the
mixture by addition of ferric sulfate and calcium carbonate was recom-
mended as the best precipitating agent. The choline of lecithin would not
be included in the total water-soluble choline.

Several attempts have been made to devise procedures for the separation
of free choline from combined water-soluble choline as well as from other
water-soluble substances which may interfere with its isolation and quanti-
tative determination. Gebauer-Fuelnegg and Kendall®™ applied electro-
dialysis to the separation of histidine from histamine or choline, and also
to the separation of an artificial mixture of protein or gelatin from histamine
or choline. This is reported to be a suitable method for the separation of
relatively strong, crystalloidal bases from mixtures with amphoteric or
more weakly basic substances.

Horowitz and Beadle® used Permutit columns to separate choline from
non-basic interfering substances. They found that a Permutit column meas-
uring 110 X 0.6 mm., containing approximately 1 g.-of Permutit, com-
pletely removes the choline from 5 ml. of a solution containing up to 0.5
mg. of choline per milliliter. Repeated tests showed that the absorbed
choline is quantitatively eluted with 10 ml. of 5% sodium chloride.

55}. Kahane and J. Lévy, Helv. Chim. Acta 29, 1322 (1946).

56 B. Shapiro, Nature 169, 29 (1952).

57D. W. Woolley and W. H. Peterson, J. Biol. Chem. 122, 213 (1937).
58 1. Schmidt and W. Wagner, Ann. 337, 51 (1904).

59, Egami and M. Itabashi, Igaku to Seibutsugaku 19, 292 (1951).
60, Kahane and J. Lévy, Bull. soc. chim. biol. 21, 223 (1939).

61). Gebauer-Fuelnegg and A. I. Kendall, Ber. 64B, 1067 (1931).

62 N. H. Horowitz and G. W. Beadle, J. Biol. Chem. 150, 325 (1943).

10 CHOLINE

Ducet® observed that free choline can be quantitatively adsorbed on
silica gel whereas the combined water-soluble choline remains in the solvent.

The isolation of choline by paper chromatography was investigated by
Munier and Macheboeuf.® These workers report that non-alkaloidal sub-
stances such as choline and betaine are readily separated from alkaloids
because their partition coefficients in various solvent systems are different.
Choline is detected on the paper strips by the blue color formed when the
chromatograms are treated with solutions containing phosphomolybdic
acid, acetic acid and stannous chloride. R; values are given by these workers
for choline when it is chromatogrammed with various solvent mixtures
containing n-butanol, acetic acid, and water.

B. PHYSICAL AND CHEMICAL PROPERTIES

Choline, hydroxyethyltrimethylammonium hydroxide, can be obtained
with difficulty as a colorless crystalline mass by drying under high vacuum
over P.O;.2%' © It is a strong base, decomposes ammonium salts, and has
a marked tendency to absorb water and carbon dioxide from the air.
Choline has no well-defined melting or boiling point but breaks down when
heated into trimethylamine and glycol. Dimethylaminoethanol and di-
methylvinylamine are also formed in lesser amounts by thermal decompo-
sition of the base.®® Dilute water solutions of the base are stable to heat,
but concentrated solutions give off trimethylamine when boiled.*

Choline is soluble in water, in formaldehyde, and in absolute methyl and
ethyl alcohols. It is sparingly soluble in amyl alcohol, chloroform, dry
acetone, and wet ether. Choline is insoluble in dry ether, carbon tetra-
chloride, carbon sulfide, toluene, benzene, and petroleum ether.®:

Edsall®® reported the Raman spectrum for choline chloride, and the
ultraviolet absorption spectrum of the base is described by Castille and
Ruppal”° and by Graubner.”

C. CONSTITUTION AND SYNTHESIS

The correct structure of choline was determined by Baeyer”? and by
Wurtz,” who carried out the first syntheses, using the reaction of tri-

63 G, Ducet, Compt. rend. 226, 1045 (1948).

64 R. Munier and M. Macheboeuf, Bull. soc. chim. biol. 31, 1144 (1949).
65 K. H. Meyer and H. Hopff, Ber. 54, 2274 (1921).

66 A. Wurtz, Compt. rend. 66, 772 (1868).

67 W. Roman, Biochem. Z. 219, 218 (1930).

68 . Klien and H. Linser, Biochem. Z. 250, 220 (1932).

69 J, T. Edsall, J. Am. Chem. Soc. 65, 1767 (1943).

70 A. Castille and M. Ruppal, Bull acad. roy. méd. Belg. 56, 263 (1926).
71 W. Graubner, Z. ges. exptl. Med. 63, 527 (1928).

72 A. Baeyer, Ann. 140, 306 (1866) ; 142, 322 (1867).

73 A. Wurtz, Compt. rend. 65, 1015 (1867).

II. CHEMISTRY i

methylamine either on ethylene chlorohydrin or on ethylene oxide with the
formation of the chloride or the free base, respectively.

Several of the early synthetic methods for choline were based on (2-bro-
moethyl)trimethylammonium bromide as the starting compound. This
substance is easily prepared by reacting trimethylamine with ethylene
bromide according to the following equation:

(CH3) 3N Le BrCH,CH,Br—Br(CH:;) 3NCH.CH.Br

Bode” converted the brominated product into choline by heating it in a
solution of silver nitrate. Kriiger and Bergell’® effected the same conversion
by heating its aqueous solution for 4 hours at 160° in a sealed tube. Lucius’®
heated the compound for 1 hour in an alcoholic solution of potassium
hydroxide at 120° and obtained a mixture of choline and neurine (vinyl-
trimethylammonium hydroxide).

Choline has been synthesized also by the exhaustive methylation of
aminoethanol with methyl iodide in a methanolic solution of potassium
hydroxide,” by modifications of the original methods of Wurtz’? using
trimethylamine,’*” by preparation of dimethylaminoethanol and its con-
version to choline through the methiodide,® and by hydrolysis of 2-(ethoxy-
methoxy )ethyl-trimethylammonium formate formed from the correspond-
ing dimethylamine derivative and methyl formate.*!

The general problem involving the synthesis of hydroxy bases and of
homologs of choline was studied by von Braun.*® This worker has shown
that, by means of the compounds Br(CH2),OBz and NHMez, bases of the
type Me.H(CH:),OBz can be prepared. These are quantitatively converted
to the hydroxy bases, Me2N(CH:2),OH, by alkaline hydrolysis. The methi-
odide of the product can then be treated with silver chloride to give
Me;sNCI(CH2),OH.

The synthesis of choline with the hydrogens of the methyl groups re-
placed by deuterium was first undertaken by du Vigneaud® and his co-
workers. Deuteriomethyl alcohol was converted with phosphorus and
iodine to deuteriomethyliodide. The iodide with aminoethanol yielded
choline with an overall yield of 64% based on deuteriomethyl alcohol.

74 J. Bode, Ann. 267, 268 (1891).

75M. Kriiger and P. Bergell, Ber. 36, 290 (1903).

76 R. Lucius, Arch. Pharm. 245, 248 (1907).

7 G. Trier, Hoppe-Seyler’s Z. physiol. Chem. 80, 409 (1912).

78 R. R. Renshaw, J. Am. Chem. Soc. 32, 128 (1910).

79F, Korner, French Pat. 736,107 (April 29, 1932).

79a H. Hopff and K. Vierling, German Pat. 801,210 (December 28, 1950).

80 J. von Braun, Ber. 49, 966 (1916).

81 W. F. Gresham, U.S. Pat. 2,457,226 (December 28, 1948).

8 V. du Vigneaud, J. P. Chandler, M. Cohn, and G. B. Brown, J. Biol. Chem. 134,
787 (1940).

1 CHOLINE

Walz et al. synthesized choline and acetylcholine labeled in the ethylene
chain with isotopic carbon-14. Acetylene-C™“, obtained from active car-
bonate in the usual manner,** was reduced to ethylene by reaction with
chromous chloride according to the method of Arrol and Glascock.® The
labeled ethylene was converted to ethylene bromohydrin-1,2-C™ with
N-bromoacetamide. The bromohydrin with excess trimethylamine in ether
yielded choline bromide with an 83% yield based on the bromohydrin.
Dauben and Gee* have published an alternative procedure starting with
carboxyl-labeled sodium acetate. This was converted to chloroacetic acid
which was esterified with diazoethane. The resulting chloroacetate was
allowed to react with dimethylamine, and the product was reduced to
N,N-dimethylaminoethanol with lithium aluminum hydride. The substi-
tuted ethanol was further methylated with methyl iodide, and the choline
iodide converted into choline chloride with C“ in the alcoholic carbon.

An improved synthesis of phosphorylcholine has been described by
Baer.*”: The compound was prepared by the catalytic hydrogenation of
diphenylphosphorylcholine produced by the reaction of diphenylphosphoryl
chloride and choline chloride in pyridine.

Glycerylphosphoric acid esters of choline have been prepared by Ravaz-
zoni and Fenaroli®® and by Aloisi and Buffa®® from bromocholine picrate
and the silver a- and 8-glycerylphosphates. These authors suggest that
previous workers may have confused the choline salts of the glycerylphos-
phates with the choline esters. The choline salts form readily and block
esterification. Baer and Kates*!: * prepared and studied the hydrolysis of
L-a-glycerylphosphorylcholine and noted a reversible shifting of the phos-
phoric acid between the a- and 8-carbons.

Salts of choline and of the common acids, including acetic, carbonic,
hydrochloric, nitric, oxalic, picric, picrolonic, and sulfuric acids, are soluble
in water and in ethanol, whereas the acid tartrate, chloroplatinate, mono-
phosphate, and ruffinate are insoluble in ethanol. Double salts with cad-
mium chloride and with zine chloride are also soluble in water and insoluble
in ethanol. Double salts with gold chloride and with mercuric chloride are
insoluble in water. Other water-insoluble salts include the hexaiodide,

83 PD. E. Walz, M. Fields, and J. A. Gibbs, J. Am. Chem. Soc. 78, 2968 (1951).
84 W. J. Arrol and R. Glascock, Nature 159, 810 (1947).

85 W. J. Arrol and R. Glascock, J. Chem. Soc. 1950 Suppl., Issue 2, § 335.

86 W. G. Dauben and M. Gee, J. Am. Chem. Soc. 74, 1078 (1952).

87 H}. Baer and C. 8. McArthur, J. Biol. Chem. 154, 451 (1944).

88 1]. Baer, J. Am. Chem. Soc. 69, 1253 (1947).

89 C. Ravazzoni and A. Fenaroli, Ann. chim. applicata 30, 318 (1940).

90 M. Aloisi and P. Buffa, Biochem. J. 48, 157 (1948).

1. Baer and M. Kates, J. Am. Chem. Soc. 70, 1394 (1948).

2 H. Baer and M. Kates, J. Biol. Chem. 175, 79 (1948).

II. CHEMISTRY Ls

periodate, enneaiodide, phosphotungstate, phosphomolybdate, reineckate,
and salts with Mayer’s reagent (potassium mercuric iodide) and with
Kraut’s reagent (potassium bismuth iodide). The chloroplatinate is mod-
erately soluble in water but very insoluble in ethanol. The flavianate is
sparingly soluble in ethanol and is insoluble in N-butanol.

The properties of some of the more important salts are listed below:

Chloride (CsHuONCI): Soluble in water, methanol, ethanol, and formal-
dehyde; less soluble in carbon tetrachloride, chloroform, and acetone;
insoluble in carbon disulfide, benzene, toluene, ether, and petroleum ether;
deliquescent; stable up to 180°, decomposing on heating to give dimethyl-
aminoethanol and methyl chloride.

Reineckate (CisH\sON -CaHzNSuCr): Melts above 250°;% soluble in
water at 18° up to 0.02 %, in 10% hydrochloric acid up to 0.03 %; in the
presence of excess ammonium reineckate the solubility in water is greatly
depressed ;*! insoluble in dilute ammonia, 0.1 N sodium hydroxide, ethanol,
benzene, and ether, but has an appreciable solubility in acetone. %

Periodides: Periodides of choline are precipitated by iodine in potassium
iodide solution, either as an insoluble oil or as a crystalline material, de-
pending upon the conditions.?*:

Hexatodide (CsHyON -I-I;): Black greenish iridescent oil obtained when
potassium triiodide solution is added to an excess of choline chloride; very
insoluble in water and soluble in ethanol; converted to the enneaiodide by
treatment with KI; solution or powdered iodine.

_ Enneaiodide (CsH\,ON -I-Is): Green needles, soluble in alcohol but very
insoluble in water; loses iodine rapidly in air and goes over to the hexa-
iodide.

Mercurie chloride double salt (CsHisON-Cl-6 HgCl,): Melts at 249 to
251°) 242 to 243°; insoluble in cold water and very insoluble in alcohol.

Chloroaurate (CsHuyON -Cl-AuCl;): Melts at 243 to 244° (slow heating),
259° (rapid heating),%” 257°% 267 to 270°; deep yellow needles from hot
alcohol or octahedra and cubes from dilute alcohol; sparingly soluble in
water, and very insoluble in alcohol.

Chloroplatinate (CsHuON -Cl)2PtCl:: Quickly decomposes on heating at
241 to 242°;° dimorphous; crystallizes in cubes and octahedra from hot
alcohol and water (1:1), but in six-sided pyramids or monoclinic rhombic

% G. Bischoff, W. Grab, and J. Kapfhammer, Hoppe-Seyler’s Z. physiol. Chem. 200,
153 (1931).

* EK. Strack and H. Schwaneberg, Hoppe-Seyler’s Z. physiol. Chem. 245, 11 (1936).

% H. Paal, Biochem. Z. 211, 244 (1929).

96 F,. H. Shaw, Biochem. J. 32, 1002 (1938).

7 J. Smorodinzew, Hoppe-Seyler’s Z. physiol. Chem. 80, 218 (1912).

% C. Reuter, Hoppe-Seyler’s Z. physiol. Chem. 78, 167 (1912).

14 CHOLINE

crystals from water; both forms of crystals are orange-red in color; very
insoluble in alcohol but moderately soluble in water.

Bromoplatinate (CsHuON Br)2PtBri: Melts at 240° (decomp.); large
dark-red prisms or octahedra; sparingly soluble in water.°®

Picrate (CsHyON -CsH207N3): Melts at 240°; readily soluble in water
and alcohol.!°°

Complex with uranium [CsHwON -UO2(NO3)el2: Yellow, non-hygroscopic
crystals insoluble in ethanol and ether and sparingly soluble in water;
aqueous solution fluoresces in ultraviolet light.1”

III. Industrial Preparation
WENDELL H. GRIFFITH and JOSEPH F. NYC

The reaction between trimethylamine and either ethylene chlorohydrin! ?
or ethylene oxide’ is used commonly in the manufacture of choline.

(CH;)3N + CICH.CH.OH — (CH;);N*+CH.,CH.OH + Cl
(CH;);N + CH.—CH, + H.O0 — (CH;:);N*CH,CH.OH + [OH}!
pss.
O

In Hopf and Vierling’s modification of the first reaction? gaseous trimethyl-
amine is passed through ethylene chlorohydrin at 80°. In K6rner’s pro-
cedure’ trimethylamine and ethylene oxide react in the presence of water
and carbon dioxide and the resulting choline is transformed to other salts
by treatment with various acids.

Choline has been prepared more recently by a two-step synthesis.4 The
quarternary salt, 2-(ethoxymethoxy)ethyltrimethylammonium formate, is
formed by heating 2-(ethoxymethoxy)ethyldimethylamine with an excess
of methyl formate at 140 to 150° under a pressure of 250 p.s.1. The quar-
ternary salt is then refluxed in a mixture of ethyl alcohol and hydrochloric
acid, and the reaction mixture is taken to dryness at a reduced pressure.
The crude choline chloride remaining in the residue is purified by crystal-
lization from isobutyl alcohol.

99 A. B. Weinhagen, Hoppe-Seyler’s Z. physiol. Chem. 105, 249 (1919).
100 YU. Suzuki, T. Shimamura, and 8. Odake, Biochem. Z. 48, 89 (1912).
101 C, Soye, Compt. rend. 228, 1228 (1949).

1R. R. Renshaw, J. Am. Chem. Soc. 32, 128 (1910).

2 H. Hopff and K. Vierling, German Pat. 801,210 (December 28, 1950).

3 F. Korner, French Pat. 736,107 (April 29, 1932).

4W.F. Gresham, U.S. Pat. 2,457,226 (December 28, 1948).

IV. BIOCHEMICAL SYSTEMS 5

IV. Biochemical Systems
WENDELL H. GRIFFITH and JOSEPH F. NYC

A. ENZYMES AND COENZYMES

Studies on choline and its derivatives have emphasized the biochemical
importance of these compounds as structural components of tissues, as
intermediates in vital metabolic reactions, and as specific chemical reactants
of marked biological potency. On the other hand, evidence for the partici-
pation of choline or of its derivatives in a specific manner as cofactors in
enzymatic systems is meager, although a few reports have linked it or its
phosphoric acid ester with phosphatases. Caution is needed in questioning
the importance of choline as a component of coenzymes, because relatively
little definite information is at hand regarding the functions and properties
of the lipoproteins that contain choline phospholipids.

Kielley and Myerhof! believe that a magnesium-activated adenosine-
triphosphatase (ATPase) of muscle may consist of a lipoprotein with a
choline-containing phospholipid as a constituent. The compound was de-
void of myosin and actomyosin, and there was no indication that it was
another form of myosin ATPase. Its pH optimum was 6.8, and it was
strongly inhibited by calcium. Inactivation of the enzyme and hydrolysis
of the phospholipid portion by lecithinase of Clostridium welchii paralleled
each other. The occurrence of the pyrophosphoric acid ester of choline in
the prosthetic groups of acid and alkaline phosphatases has been reported.”
Other workers* have noted that this ester contains a labile phosphate group,
hydrolyzable by crude and not by purified muscle pyrophosphatase, but
they are not of the opinion that it is a coenzyme of a phosphatase. Alkyl
nitrogen-substituted derivatives of aminoethanol and of choline activate
alkaline phosphomonoesterases.* Activation of an ATPase system in rat
submaxillary gland by acetylcholine in vitro has been reported.®

B. CHOLINE ACETYLASE AND ACETYLCHOLINESTERASE

Coenzyme A (CoA) appears to be a common coenzyme for most, if not
all, of the acetyl-transferring systems, including the acetylation of choline.
The constituents of one form, at least, of this important thermostable com-
pound include adenosine-2’(or 3’)-phosphate,®: 7 pyrophosphate,® panto-

1W. W. Kielley and O. Myerhof, J. Biol. Chem. 176, 591 (1948); 183, 391 (1950).

2 W. Kutscher and H. Sieg, Naturwissenschaften 87, 451 (1950).

3 J. Roche, N.-V. Thoai, and N.-V. Thiem, Compt. rend. soc. biol. 145, 168 (1951).

4R. Granger and J. Fraux, Trav. soc. pharm. Montpellier 5, 48 (1945-1946); 6, 93
93 (1946-1947).

5K. P. DuBois and V. R. Potter, J. Biol. Chem. 148, 451 (1943).

6 F. Lipmann, N. O. Kaplan, G. D. Novelli, and L. C. Tuttle, J. Biol. Chem. 186,
235 (1950).

16 CHOLINE

thenic acid,® and 6-mercaptoethylamine.®: ®: ° It is probable that the nucleo-
side is joined to the terminal alcoholic hydroxy] of the pantothenate by a
pyrophosphate bridge and that the sulfur component forms an acid amide
linkage with the carboxyl of the 6-alanine moiety of the pantothenate.
The pantothenic acid-mercaptoethylamine complex can be obtained from
CoA by hydrolysis by intestinal phosphatase!’ and is identical with
Snell’s Lactobacillus bulgaricus growth factor (LBF or pantetheine).°> !

The free-SH group is the principal site of reactivity in the CoA molecule,
and it is readily acetylated to acetyl CoA (CoA—S—CO—CHs) in the
presence of ATP. A partially purified preparation of acetyl CoA has been
obtained from baker’s yeast by Lynen et al. This molecule, formerly
designated “active acetate,” serves as a donor of acetyl groups in the
presence of specific apoenzymes. Energy in the form of ATP is required
for its formation, and it is of considerable interest that its acyl-mercaptide
linkage is an energy-rich bond.!*-!> CoA may exist in the disulfide form
(CoA—S—S—CoA), and mixed disulfides with other sulfhydryl compounds
have complicated its isolation.

The application of these findings to the metabolism of choline is illus-
trated by the system which transfers acetyl from citrate to choline. Ochoa
et al.1* have isolated a condensing enzyme from heart muscle which cata-
lyzes reversibly the reaction between acetyl CoA and oxalacetate to give
CoA and citrate. If choline and a second acetyl-transferring enzyme (acetyl-
ase) are present in addition to Ochoa’s condensing enzyme plus citrate and
CoA, acetylcholine is formed."

The probable identity of the factor (presumably CoA) required in the
acetylation of sulfanilamide and of choline was indicated in 1946.” Subse-
quently, Nachmansohn obtained a fraction termed acetylkinase from pigeon
liver and demonstrated that both sulfanilamide and choline were acetylated

7 J. Baddiley and E. M. Thain, J. Chem. Soc. 1951, 3421.

8 W. H. DeVries, W. M. Govier, J. S. Evans, J. D. Gregory, G. D. Novelli, M.
Soodak, and F. Lipmann, J. Am. Chem. Soc. 72, 4838 (1950).

9. E. Snell, G. M. Brown, V. J. Peters, J. A. Craig, E. L. Wittle, J. A. Moore,
V.M. McGlohon, and O. D. Bird, J. Am. Chem. Soc. 72, 5349 (1950).

10G. D. Novelli, N. O. Kaplan, and F. Lipmann, Federation Proc. 9, 209 (1950).

11 G. M. Brown, J. A. Craig, and E. E. Snell, Arch. Biochem. 27, 473 (1950).

12 F, Lynen, E. Reichert, and L. Rueff, Ann. 574, 1 (1951).

13 ¥, Lynen, E. Reichert, Angew. Chem. 68, 47 (1951).

14 J. R. Stern, B. Shapiro, E. R. Stadtman, and 8. Ochoa, J. Biol. Chem. 193, 703
(1951).

15H, R. Stadtman, J. Biol. Chem. 196, 535 (1952).

16 §. Ochoa, J. R. Stern, and M. C. Schneider, J. Biol. Chem. 198, 691 (1951).

17 ¥, Lipmann and N. O. Kaplan, J. Biol. Chem. 162, 743 (1946).

IV. BIOCHEMICAL SYSTEMS LG

in the presence of the enzyme, ATP, and acetate.!8 It may be assumed that
the fraction contained CoA as well as acetylkinase. Choline acetylase was
inhibited or inactivated by iodoacetate.!® Thiolacetate would replace the
ATP and acetate in this system and in acetylating systems prepared from
brain and from Lscherichia coli extracts, but not in the system obtained
from ganglia from the head of the squid.?° Surprisingly, dimethylamino-
ethanol was esterified at the same rate as was choline whereas neither
aminoethanol nor monomethylaminoethanol was active. It is of interest
that the acetic acid ester of dimethylaminoethanol bears no resemblance
in biological activity to the corresponding trimethyl compound, acetyl-
choline. Although revisions may be necessary in the present understanding
of phosphorylations during the acetylation of choline, it is reasonable to
assume that a failure to find changes in concentrations of either inorganic
phosphate or in ATP?! may be ascribed to the presence of acetyl CoA or,
possibly, of a substance such as thiolacetate.

The recognition of the indispensable role of a derivative of pantothenic
acid in the metabolism of acetate involves this vitamin in the over-all
synthesis of fatty acids from carbohydrate and, hence, in the production of
fatty livers. The same possibility is recognized in the case of thiamine
because of the role of diphosphothiamine in pyruvate metabolism.” 2 The
role of the vitamins of the B complex in choline-deficient animals is dis-
cussed in Section IV, p: 33.

The extreme physiological activity of acetylcholine makes it understand-
able that rapid inactivation is essential as part of the mechanism controlling
its concentration in tissues. Acetylcholine is hydrolyzed at various rates
by miscellaneous tissue esterases and at a rapid rate by pseudocholinester-
ase and by cholinesterase. An attempt at differentiation on the basis of
substrate specificity has been made*t and reports have appeared on the
localization in tissues? and on inhibitors?®: *’ of a specific acetylcholinester-
ase. A more satisfactory definition of a specific acetylcholinesterase and its
description must await the isolation and characterization of such an en-
zyme.

18 D. Nachmansohn, J. B. Wilson, 8S. R. Korey, and R. Berman, J. Biol. Chem. 195,
25 (1952).

19D. Nachmansohn and A. L. Machado, J. Neurophysiol. 6, 397 (1943).

20§. R. Korey, B. de Braganza, and D. Nachmansohn, J. Biol. Chem. 189, 705 (1951).

21 N.-V. Thoai, L. Chevillard, and 8. Mayer, Compt. rend. 229, 254 (1949).

2 R.A. Peters and J. R. O’Brien, Ann. Rev. Biochem. 7, 305 (1988).

23 E.§8. G. Barron, C. M. Lyman, M. A. Lipton, and J. M. Goldinger, J. Biol. Chem.
141, 957 (1941).

*4 K. Augustinsson and D. Nachmansohn, Science 110, 98 (1949).

2° G. B. Koelle, J. Pharmacol. Exptl. Therap. 108, 153 (1951).

26 M. G. Ord and R. H. 8. Thompson, Biochem. J. 46, 346 (1950).

*7 W.N. Aldridge, Biochem. J. 46, 451 (1950).

18 CHOLINE

C. MECHANISM OF ACTION OF CHOLINE
1. DERIVATIVES OF PHOSPHORYLCHOLINE

In so far as participation in the intermediary metabolism of phospho-
lipids is concerned, it is important to determine the relative activities of
choline and its phosphorylated derivatives. Of these phosphorylcholine and
glycerylphosphorylcholine (GPC) have received special attention. Riley
determined the rate of utilization of radioactive phosphorylcholine (P**) in
rats and found no evidence of its preferential use in phospholipid metabo-
lism? or in bone metabolism.?’ Severe choline deficiency did not alter the
uptake of P*? by the femur to any marked degree.*® Schmidt e¢ al.*! have

TABLE II

AMOUNTS OF FREE CHOLINE AND OF GLYCERYLPHOSPHORYLCHOLINE (GPC) 1n SoME
MamMALIAN TIssuEs*!

Choline, mg./g. moist
tissue

As choline As GPC
Beef heart, skeletal muscle, and brain, fresh Negligible Negligible
Beef spleen, fresh Negligible 34
Beef pancreas, fresh 22 53
Beef pancreas, incubated 32 169
Lamb testicle, fresh 5.3 6.4
Lamb kidney, fresh 22 48
Lamb liver, fresh frozen Negligible 75, 123, 126
Rabbit liver, fresh Negligible 35
Rat liver, adult, fresh frozen Negligible Negligible
Rat liver, weanling, fresh frozen Negligible Negligible
Human bile, fresh Negligible Negligible

analyzed tissues for free choline and GPC and have made the surprising
observation that GPC may make up a relatively large fraction of the total
free and combined tissue choline. This was true particularly in lamb liver
whereas beef and rat livers contained only negligible quantities of either
free choline or GPC (Table II). The incubation of ground beef pancreas
increased free choline slightly and GPC markedly. Neither compound oc-
curred in rat intestine in more than traces, but incubation of ground intes-
tine at pH 5.2 resulted in liberation of GPC whereas incubation at pH 8.2
yielded free choline. Schmidt e¢ al. believe that their findings suggest the

2 R.F. Riley, J. Biol. Chem. 153, 535 (1944).

29R. F. Riley, B. McCleary, and R. E. Johnson, Am. J. Physiol. 143, 677 (1945).

30 W. F. Neuman and R. F. Riley, J. Biol. Chem. 168, 545 (1947).

31 G. Schmidt, L. Hecht, P. Fallot, L. Greenbaum, and S. J. Thannhauser, J. Biol.
Chem. 197, 601 (1952).

IV. BIOCHEMICAL SYSTEMS 19

general importance of GPC as an intermediary in the phospholipid metabo-
lism of mammals.

Baccari*” has compared the hydrolysis of phosphorylcholine and of glyc-
erophosphoric acid by phosphatases in pancreas, intestinal mucosa, and
brain. Phlorizin and fluoride inhibited both hydrolyses. Hydrolysis of the
glycerophosphate but not of phosphorylcholine was greatly accelerated by
magnesium chloride.

The direct phosphorylation of choline by adenosine triphosphate in the
presence of extracts from brewer’s yeast and preparations from liver, brain,
intestine, and kidney of several species has been reported by Wittenberg
and Kornberg.*? The enzyme, choline phosphokinase, also catalyzed the
phosphorylation of aminoethanol and of the mono- and dimethyl (and
ethyl) aminoethanols. It was suggested that these phosphorylated deriva-
tives may be activated precursors of the respective phosphatides even
though previous attempts to demonstrate such a conclusion have been
unsuccessful.

Reference has been made previously to the occurrence of sphingosylphos-
phorylcholine. It is oxidized more rapidly by minced liver, kidney, and
brain than by other tissues of the guinea pig.**

2. CHOLINE AND TRANSMETHYLATION

The first intimation that the dietary supply of choline might have nutri-
tional significance resulted from survival studies on depancreatized dogs
subsequent to the discovery of insulin by Banting and Best. Both Fisher*
and Allan e¢ al.*° observed fatty and severely degenerated livers in the
animals deprived of the pancreas but supplied with insulin. In the latter
study, survival was reported in the case of one animal that received raw
pancreas in its diet. Six years passed before it was reported that the pro-
tective action of raw pancreas in depancreatized dogs was duplicated by
the feeding of lecithin.**-*§ At this time Best et al.*® observed that fatty
livers resulted from feeding rats mixed grains and fat and that dietary
lecithin was lipotropic, i.e., it prevented the accumulation of hepatic fat

8 V. Baccari, Boll. soc. ital. biol. sper. 20, 397, 398 (1945).

32a J. Wittenberg and A. Kornberg, J. Biol. Chem. 202, 430 (1953).

33 M. Aloisi, Atti accad. nazl. Lincet, Rend. Classe sct. fis. mat. e nat. 2, 98 (1947).

34 N. F. Fisher, Am. J. Physiol. 67, 634 (1924).

35 FN. Allan, D. J. Bowie, J. J. R. Macleod, and W. L. Robinson, Brit. J. Expil.
Pathol. 5, 75 (1924).

36 J. M. Hershey, Am. J. Physiol. 93, 657 P (1930).

37 J. M. Hershey and 8. Soskin, Am. J. Physiol. 98, 74 (1931).

38 C, H. Best and J. M. Hershey, J. Physiol. (London) 75, 49 (1932).

39C. H. Best, J. M. Hershey, and M. E. Huntsman, J. Physiol. (London) 76, 56
(1932).

20 CHOLINE

under these conditions. Best and coworkers soon noted that the effective
component in lecithin was choline.*°“” Betaine was also found to have
lipotropic activity in rats.

The use of the rat as a test animal in place of the depancreatized dog
facilitated greatly the extension of the investigations that comprised the
first phase of the study of the role of choline as a dietary essential. During
the next few years the study of the relation of dietary factors to choline-
preventable fatty livers in rats was pursued vigorously. Best et al. noted
the protective action of choline in diets containing added cholesterol*: “
and the protective effect of protein.*°-** Channon and coworkers examined
the effect of protein and of amino acids.**- Following the demonstration
of the antilipotropic effect of dietary cystine by Beeston and Channon,*!
Tucker and Eckstein® noted that supplements of methionine had an oppo-
site effect and were lipotropic. This similarity in the anti-fatty liver action
of choline and of methionine was the first observation in the second phase
of studies of the nutritional importance of choline, a phase that was to
place choline in a unique position in metabolism as a source of labile methyl
groups as well as a component of biochemically important tissue constitu-
ents.

In 1932 Jackson and Block™ presented the first evidence of a mammalian
requirement of methionine in experiments in which growth was improved
in rats by the addition of this amino acid to a diet low in sulfur amino acids.
In the same year Butz and du Vigneaud®® prepared homocystine, a de-
methylated product of methionine, by the action of strong sulfuric acid on
methionine, and du Vigneaud et al.°® showed that homocystine supported
the growth of rats on a cystine-poor diet. Later, Womack et al.*” found that

40 C. H. Best, J. M. Hershey, and M. E. Huntsman, Am. J. Physiol. 101, 7P (1932).
41 C. H. Best and M. E. Huntsman, J. Phusiol. (London) 75, 405 (1932).

42 C. H. Best, G. C. Furguson, and J. M. Hershey, J. Physiol. (London) 79, 94 (1933).
43 C. H. Best and J. H. Ridout, J. Physiol. (London) 78, 415 (1933).

44 C. H. Best, H. J. Channon, and J. H. Ridout, J. Physiol. (London) 81, 409 (1934).
45 C. H. Best and M. E. Huntsman, J. Physiol. (London) 88, 255 (1935).

46 C. H. Best and H. J. Channon, Biochem. J. 29, 2651 (1935).

47 C. H. Best, R. Grant, and J. H. Ridout, J. Physiol. (London) 86, 337 (1936).

48 C. H. Best and J. H. Ridout, J. Physiol. (London) 87, 55P (1936).

49H. J. Channon and H. Wilkinson, Biochem. J. 29, 350 (1935).

50 A. W. Beeston, H. J. Channon, and H. Wilkinson, Biochem. J. 29, 2659 (1935).

51 A.W. Beeston and H. J. Channon, Biochem. J. 30, 280 (1936).

52 A. W. Beeston, H. J. Channon, J. V. Loach, and H. Wilkinson, Biochem. J. 80,
1040 (1936).

53 H. F. Tucker and H. C. Eckstein, J. Biol. Chem. 121, 479 (1937).

54 RW. Jackson and R. J. Block, J. Biol. Chem. 98, 465 (1932).

55 T,. W. Butz and V. du Vigneaud, J. Biol. Chem. $9, 135 (1932).

56 V. du Vigneaud, H. M. Dyer, and J. Harman, J. Biol. Chem. 101, 719 (1933).

57 M. Womack, K. 8. Kemmerer, and W. C. Rose, J. Biol. Chem. 121, 403 (1937).

IV. BIOCHEMICAL SYSTEMS Pa

cystine was not an indispensable amino acid as had been believed since the
evidence of its supplementary potency presented by Osborne and Mendel
over twenty years earlier.*’ The experiments in Rose’s laboratory showed
the essential character of the methionine requirement” and that this amino
acid was a precursor of cystine in vivo.*® It was evident also that cystine
could spare the methionine requirement in so far as cystine was needed
by the animal.®°

The demonstration of growth-stimulating activity of homocystine in rats
on a methionine-poor diet was difficult to understand, inasmuch as cystine
was ineffective.* The explanation of the methionine-like action of homo-
cystine was soon found to depend upon the presence of choline in the diet.
Homocystine replaced methionine as a growth factor in young rats if the
water-soluble vitamins were provided in the form of concentrates of milk
and rice bran but not if purified vitamins were used. In the latter instance
markedly fatty livers were observed and the addition of choline to the
mixture of purified vitamins permitted homocystine to function as a source
of methionine. Choline was isolated from the concentrates of milk and
rice bran vitamins.

The concept of transmethylation or transfer of intact methyl groups was
established by du Vigneaud and his collaborators in a series of experiments
in which isotopically labeled compounds were employed. Choline containing
deuterium-labeled methyl was isolated from the carcasses of rats fed a
choline-deficient diet supplemented with methionine containing deuterium
in the sulfur methyl. By the same procedure the methyl of creatine was
shown to come from methionine.® In addition, the transfer of methyl from
choline to creatine® and to methionine®® occurred if labeled choline and
homocystine replaced methionine in the diet. The transfer of methyl to
guanidoacetic acid to form creatine, however, was irreversible. °* This im-
pressive evidence of transfer of intact methyls was given additional support
by the experiment in which choline was isolated after feeding rats doubly

588 T. B. Osborne and L. B. Mendel, J. Biol. Chem. 20, 351 (1915) .~

59 W.C. Rose and T. R. Wood, J. Biol. Chem. 141, 381 (1941).

60 M. Womack and W. C. Rose, J. Biol. Chem. 141, 375 (1941).

61 A. White and E. F. Beach, J. Biol. Chem. 122, 219 (1937).

62 V. du Vigneaud, H. M. Dyer, and M. W. Kies, J. Biol. Chem. 130, 325 (1939).

63 V. du Vigneaud, J. P. Chandler, A. W. Moyer, and D. M. Keppel, J. Biol. Chem.
131, 57 (1939).

64 V. du Vigneaud, M. Cohn, J. P. Chandler, J. R. Schenck, and 8. Simmonds, J.
Biol. Chem. 140, 625 (1941).

65 V. du Vigneaud, Biol. Symposia 5, 234 (1941).

66S. Simmonds, M. Cohn, J. P. Chandler, and V. du Vigneaud, J. Biol. Chem. 149,
519 (1943).

67 V. du Vigneaud, J. P. Chandler, and A. W. Moyer, J. Biol. Chem. 139, 917 (1941).

67a §. Simmonds and V. du Vigneaud, Proc. Soc. Exptl. Biol. Med. 59, 293 (1945).

22 CHOLINE

labeled methionine. Within experimental error the same ratio of C“ to
deuterium was found in choline and creatine methy]s as in the sulfur methyl
of the administered methionine.®

At the time these studies were in progress there was no reason to doubt
the supposed inability of the animal organism to synthesize so-called labile
methyl, the methyl of choline and of methionine. The ease with which
either choline or methionine deficiency was produced and the ease of pre-
vention of these deficiencies by methionine or by choline and homocystine,
respectively, made the concept of a dietary deficiency of labile methyl very
plausible. More recent findings, however, have made it necessary to revise
this concept. Discussions to follow indicate that the animal organism does
have the ability to synthesize the methyls found in choline or methionine
but only if the diet is otherwise adequate. Furthermore, under the best
circumstances the rate of the synthetic process may be limited.

This conclusion has been reached in three types of experiments: (1)
diets containing homocystine but devoid of methionine and of other known
sources of labile methyl have supported the growth of rats;%> 6» 70 (2)
deuterium-labeled choline methyl was found in the tissues of germ-free rats
supplied deuterium-labeled water ;”: ” and (3) C-labeled choline and me-
thionine methyls have been found after the incubation of tissue preparations
with C-labeled formate.” Although there is nothing in the present evi-
dence that questions the reality of metabolic transfer of intact methyl
groups under certain conditions, it is necessary to distinguish carefully
between methylation due to transmethylation and methylation due to
synthesis of a methyl from formate. The term transmethylation will be
used for the reactions in which intact methyls participate. The general
designation, formate transfer, and the more descriptive terms, formate-to-
methyl and methyl-to-formate transfer, will be employed for those reactions
in which there is synthesis or degradation of a methyl group. In these
instances a carbon-oxygen linkage replaces the carbon-hydrogen bonding,
or the reverse, in addition to transfer of the carbon.

In many instances the experimental data are insufficient to permit an
unequivocal differentiation between the two types of methylation. Particu-
larly disturbing is the difficulty in describing the series of reactions in which
a formate carbon becomes a labile methyl carbon. It is not possible to state

88 1). B. Keller, J. R. Rachele, and V. du Vigneaud, J. Biol. Chem. 177, 733 (1949).

69 M. A. Bennett, G. Medes, and G. Toennies, Growth 8, 59 (1944).

70M. A. Bennett, J. Biol. Chem. 168, 247 (1946) ; 187, 751 (1950).

7 V. du Vigneaud, C. Ressler, and J. R. Rachele. Science 112, 267 (1950).

72 V. du Vigneaud, C. Ressler, J. R. Rachele, J. A. Reyniers, and T. D. Luckey,
J. Nutrition 45, 361 (1951).

73 V. du Vigneaud and W. G. Verly, J. Am. Chem. Soc. 72, 1049 (1950).

IV. BIOCHEMICAL SYSTEMS 233

with certainty whether the first appearance of the newly synthesized methyl
is in methionine, thetin, or choline. The bulk of evidence supports the
hypothesis that three successive methylations convert aminoethanol into
choline. If this is the pathway of reactions for formate-to-methyl synthesis,
an alternative reaction must exist for the direct formation of choline by
transmethylation. Otherwise, an explanation of the appearance of doubly
labeled methyl (C™ and deuterium) in tissue choline following the adminis-
tration in rats of similarly labeled methionine is obscure.® In general, the
quantitative aspects of formate-to-methyl synthesis have been difficult to
assess and the isotopic data may be misleading in this respect. In vivo
experiments in which growth is the criterion show only that some methyla-
tion other than transmethylation has occurred. In no instance has optimum
growth of rats or chicks been demonstrated in the complete absence of
recognized dietary sources of labile methyl. For this reason, it will be
assumed in the following paragraphs that the requirements for growth or
for tissue repair may create demands for methyl that, in the absence of
adequate dietary sources, exceed the rate of production by one or more of
the reactions in the formate-to-methy] transfer with resulting evidences of
deficiency. Other nutrients, possibly folacin and By, appear to influence
the formate-to-methy] transfer and, possibly, transmethylation also.

Clarification of the roles of choline, betaine, methionine, and thetin must
await the recognition and isolation of the numerous enzymes and cofactors
concerned with the fascinating mechanisms of methyl synthesis and methyl
transfer. Although current findings are subject to different interpretations
and present a most complex picture, they are discussed below on the basis
of a relatively simple hypothesis in which certain arbitrary and provisional
assumptions are made. This possible oversimplification of the problem is
justified by the emphasis placed on phases that particularly require further
investigation. The tentative scheme distinguishes between transmethyla-
tion and formate-to-methyl] transfer as follows: :

(a) Methionine in its active form is the principal methyl donor for trans-
methylations resulting in the synthesis of creatine from guanidoacetic acid,
of choline from dimethylaminoethanol, of trigonelline and N‘!-methylnico-
tinamide from nicotinic acid and its amide, respectively, and of many other
methyl-containing metabolites.

(b) Homocysteine is converted to methionine by accepting labile methyl
from betaine, only one of the three methyls of which is labile.

(c) Homocysteine is also converted to methionine by a reaction involving
formate-to-methyl synthesis, possibly with a thetin as an intermediate.
These and other relationships are illustrated in Fig. 1.

24 CHOLINE

3. NATURE OF Metuyt Donors IN TRANSMETHYLATION

The process of transmethylation involves demethylation of a methyl
Donors of intact methyl

donor and methylation of a methyl acceptor.
groups appear to be limited to methylated quaternary nitrogen and sulfo-

Acetylcholine
thi
Lecithins (yabereine

Se a . uldehyde ce

(1) Choline (3) Betaine

(12) Methionine _\M

(13) Active (11) Thetin
methionine A
(4) Dimethylglycine

(10) Dimethyl-
aminoethanol

(15) | i
—\I

'
CH; | Labile methyl | CII ‘
| CH | Cees
1! (14) Histidine, cte.
ahs 1
I A
I
|
(16) Formate (5) Monomethylglycine

\
(17) Homocysteine
—M

+ Mor F?

(9) Monomethy1-
aminoethanol

(18) Homo-
cystine

ie Aminoethanol ‘
pers ae Serine

Ser ine ei ear
Cystathionine

Ba (20) Wwilcmamide (22) Creatine

Cystine <——> Cysteine and other acceptors

Fia. 1. Schematic representation of pathways of transmethylation, of formate-
to-methyl synthesis, and of methyl-to-formate oxidation. Letters M and F repre-
sent labile methyl and active formate, respectively. Directions of reactions are
indicated, but reacting compounds are not shown with the exception of the reaction
of homocysteine with methyl to yield methionine and with serine to yield eystathi-
onine. A distinction is indicated between the methyls of methionine and of active
methionine. The use of formate in the synthesis of thetin, shown by the dash line,

+Mor F?
(6) Glycine

Arginine

a
(21) Guanidoacetie acid

‘
N
‘

is hypothetical.

nium compounds. It is reasonable to relate lability of a methyl to its
attachment to a nitrogen or sulfur atom which has acquired an additional
covalent bond and positive charge. The main nitrogen compounds in this

IV. BIOCHEMICAL SYSTEMS 25

category, in addition to choline and choline-containing compounds, are
betaine aldehyde and betaine. Betaine was observed to be lipotropic in the
early experiments of Best,*! and du Vigneaud®: 7: 7® showed that it yielded
methyl to homocysteine to form methionine. Welch found betaine aldehyde
as well as betaine lipotropic in rats’® and mice.”

The known sulfur compounds that are methyl donors in transmethyla-
tion are dimethylthetin, propio-6-dimethylthetin, and active methionine.
The first, an analog of betaine (sulfobetaine), was first shown to be lipo-
tropic by Welch (cited by du Vigneaud ef al.7*: 78), and its part in transmeth-
ylation was demonstrated by du Vigneaud and coworkers.’”* Propio-6-di-
methylthetin has been isolated from a marine alga, Polystphonia fastigiata,
by Challenger and Simpson.7? It supports rat growth with homocystine
and is, therefore, a source of methyl.8°: §! The activity of methionine as a
methyl donor appeared to be an exception until it was shown by Cantoni
that it probably is converted to a sulfonium compound prior to its de-
methylation. Barrenscheen® had concluded previously that the sulfoxide
of methionine was the first intermediate in transmethylation. The formula
provisionally assigned to active methionine, S-adenosylmethionine, by Can-
toni, is as follows:*

Neenine=—=vihore—_6—_ CH — CH. — Gh-——C==0

CHs NH, O-

Presumably, substrate specificity is also involved because certain beta-
ines®: & and the sulfur analog of choline, sulfocholine,*® do not donate
methyl to homocysteine.

It cannot be assumed that choline serves directly as a methyl donor.
Present evidence suggests strongly that it must be oxidized to betaine
first and that dimethylglycine rather than dimethylaminoethanol is a prod-

74 V. du Vigneaud, J. P. Chandler, and A. W. Moyer, J. Nutrition 19, 11 (1940).

75 V. du Vigneaud, S. Simmonds, J. P. Chandler, and M. Cohn, J: Biol Chem. 165,
639 (1946).

76 A.D. Welch, J. Nutrition 40, 113 (1950).

77 A.D. Welch and M. S. Welch, Proc. Soc. Exptl. Biol. Med. 39, 7 (1938).

77a A.W. Moyer and V. du Vigneaud, J. Biol. Chem. 148, 373 (1942).

78 V. du Vigneaud, A. W. Moyer, and J. P. Chandler, J. Biol. Chem. 174, 477 (1948).

79 F. Challenger and M. I. Simpson, Biochem. J. 41, x1 (1947).

80 G. A. Maw and V. du Vigneaud, J. Biol. Chem. 174, 381 (1948).

81 G. A. Maw and V. du Vigneaud, J. Biol. Chem. 176, 1037 (1948).

82 H. K. Barrenscheen and T. von Valy-Nagi, Hoppe-Seyler’s Z. physiol. Chem. 283,
91 (1948).

8 G. L. Cantoni, J. Am. Chem. Soc. 74, 2942 (1952).

84H. E. Carter and D. B. Melville, J. Biol. Chem. 133, 109 (1940).

85 G. A. Maw and V. du Vigneaud, J. Biol. Chem. 176, 1029 (1948).

26 CHOLINE

uct of the demethylation. The role of choline oxidase in this regard is
described below.

The significance of the thetins in methyl metabolism is not clear. Neither
has been detected as a naturally occurring intermediate in animal tissues.
The very active dimethylthetin is the most potent known donor of methyl
to homocysteine in 7 vitro experiments. However, evidence that it supplies
methyl for creatine synthesis has not been presented. Clarification of the
function of methylated sulfonium compounds of this type can be confi-
dently expected because of the presence in tissues of a dimethylthetin
transmethylase.

4. Mretruyt Accreptrors, TRANSMETHYLATION, AND FORMATE-TO-
Merny SYNTHESIS

The early studies on labile methyl quickly brought to light three prime
examples of methyl acceptors—homocysteine, guanidoacetic acid, and ni-
acinamide. Each of these has been widely used experimentally, and the
first two, at least, give rise by methylation to highly important metabolites,
methionine and creatine, respectively. It may well be that N'methyl-
niacinamide also is more than a urinary end product and that it represents
some unrecognized function of the antipellagric vitamin.

The formation of choline by the methylation of a carbon-nitrogen pre-
cursor is obviously an indispensable reaction, but the exact nature of the
precursor has not been as evident as in the case of the three acceptors
named above. Whether the methylation of the precursor is an obligatory
reaction is also uncertain. Presumably, the substance in question is amino-
ethanol, a molecule readily synthesized by animal tissues.

The synthesis of choline in animals by transmethylation of the methyl
of methionine has been mentioned previously. ®» § Production of choline
by methylation involving formate-to-methyl synthesis is also well-estab-
lished, but these observations do not exclude the possibility that the forma-
tion of choline is secondary to the appearance of the new methyl] in methio-
nine or in a thetin.

The fact of synthesis of choline de novo has been established by
du Vigneaud et al.”:7 in experiments in which labeled choline was iso-
lated from tissues of germ-free rats fed heavy water. Other evidence for
this conclusion has been reported by Bennett*®*:7° and by Stekol and
Weiss*¢ on the basis of rat growth on diets lacking obvious sources of labile
methyl and containing homocystine and a suitable vitamin supplement.

Production of choline by methylation of a carbon-nitrogen precursor by
means of formate-to-methyl synthesis is well established. The amount of

86 J. A. Stekol and K. W. Weiss, J. Biol. Chem. 186, 343 (1950).

IV. BIOCHEMICAL SYSTEMS 27

radioactivity in the methyl groups of choline or of methionine isolated from
tissues of rats after the administration of C™ precursors indicates that an
appreciable transfer of carbon from methanol,’ 7: formaldehyde,*: 89: 9°
formic acid,*7-89, %!, * acetone,* and serine”: °* occurs. Formate is not the
only precursor of the 6-carbon of serine, according to Kruhgffer.°> In his
studies rat liver homogenates transferred glycine but not formate to serine,
although the conversion of formate to serine did occur with liver slices.
Although no evidence has been presented for the transmethylation of the
intact methyl group of methanol,” the administration of sodium deuterio-
C'-formate subcutaneously in the rat was followed by the isolation of tissue
choline containing C'*-labeled methy] with no detectable loss of deuteritum.%®
This finding suggests that in the formate-to-methyl synthesis one hydrogen,
at least, remains attached to carbon in the intermediate forms. The in-
corporation of C into the 8-carbon of serine occurs more rapidly from form-
aldehyde than from formate or methanol,*°® but this is not necessarily evi-
dence that formaldehyde is the precursor of the methyl group. Jonsson and
Mosher found the C™ label in both the carbon chain and in the methyl
of liver choline after the administration of 8-C'-labeled serine in the rat.

Reid and Landefeld” and Toporek et al.%*: 9° have presented evidence
that histidine is an important dietary source of the carbon of methyl groups
of choline and creatine. L-Histidine-2-C™ was fed to rats, and the labeled
carbon was found not only in tissue choline and creatine methyls but also
in the aminoethanol portion of choline. In the work of Toporek et al.,°® the
transfer to choline was enhanced in choline deficiency both in the intact
animal and in isolated perfused cirrhotic livers of rats with chronic choline
deficiency. Interestingly, these authors have calculated the histidine con-
tent of diets used by Griffith and Mulford!° and by Rose e¢ al.!° and have
concluded that this amino acid played an important role in their experi-

87 V. du Vigneaud, W. G. Verly, J. E. Wilson, J. R. Rachele, C. Ressler, and J. M.
Kinney, J. Am. Chem. Soc. 73, 2782 (1951).

88 H.R. V. Arnstein, Biochem. J. 48, 27 (1951).

89 V. du Vigneaud, W. G. Verly, and J. E. Wilson, J. Am. Chem. Soc. 72, 2819 (1950).

9 L. Siegel and J. Lafaye, Proc. Soc. Exptl. Biol. Med. 74, 620 (1950).

%1W. Sakami and A. D. Welch, J. Biol. Chem. 187, 379 (1950).

2 P. Siekevitz and D. M. Greenberg, J. Biol. Chem. 186, 275 (1950).

%W. Sakami, J. Biol. Chem. 187, 369 (1950).

% S$. Jonsson and W. A. Mosher, J. Am. Chem. Soc. 72, 3316 (1950).

% P,. Kruh@ffer, Biochem. J. 48, 604 (1951).

96 C. Ressler, J. R. Rachele, and V. du Vigneaud, J. Biol. Chem. 197, 1 (1952).

% J. C. Reid and M. O. Landefeld, Arch. Biochem. and Biophys. 34, 219 (1951).

% M. Toporek, Federation Proc. 11, 299 (1952).

99M. Toporek, L. L. Miller, and W. F. Bale, J. Biol. Chem. 198, 839 (1952).

100 W.H. Griffith and D. J. Mulford, J. Nutrition 21, 633 (1941).

101 R. H. McCoy and W. C. Rose, J. Biol. Chem. 117, 581 (1937).

28 CHOLINE

ments. The conversion of carbon 2 of histidine into choline methyls, pre-
sumably by way of formate, is supported by the finding of Soucy and
Bouthillier'!” that approximately one-fourth of the radioactivity of an hy-
drolyzate of liver protein was in the form of serine after feeding rats labeled
histidine. The reverse reaction, the synthesis of histidine from precursors
that include formate, has been demonstrated by Coon and Levy.1%> 1%
These observations are not unexpected in view of the earlier discovery by
Edlbacher!®>: 1°° of the enzyme, histidase, in liver and its disruption of the
imidazole ring to give a product yielding formate on acid hydrolysis.

It is significant that satisfactory proof is lacking for a methyl donor
function for mono- and dimethylaminoethanol.®* As in the case of the re-
ported transmethylation of methyl from sarcosine,!” unequivocal proof
would require double labeling of the methyl to rule out intermediary
formate formation, 1.e., oxidation and reduction of the carbon in question.
Du Vigneaud!® has shown that deutertum-methyl-labeled dimethylamino-
ethanol is converted in the rat into choline. This final methylation, yield-
ing choline, is apparently an irreversible reaction, Inasmuch as Muntz!
found dimethylglycine rather than dimethylaminoethanol after the incu-
bation of rat liver homogenate with added choline and homocysteine. Sub-
stantial support for the conclusion that oxidation of choline to betaine
is necessary before transmethylation occurs is provided by the observa-
tions of Dubnoff!!® and others on choline oxidase (Section IV, p. 33).

The methylation zm vivo of homocysteine, following its administration
as such or as homocystine, is discussed in Section IV, p. 19. Similarly, the
requirement of methionine in chicks is satisfied by homocystine plus
choline"! or betaine."? These metabolic reactions undoubtedly spare the
requirement of methionine, and there is no reason to doubt that the methio-
nine-homocysteine reaction may account for repeated transfers of intact
methyl. Nevertheless, it is pertinent to emphasize the fact that homo-
cysteine and its disulfide form are not known to occur naturally and that
their presence in the body is dependent on the methionine intake except in

102 R. Soucy and L. P. Bouthillier, Rev. can. biol. 10, 290 (1951).

103 M. J. Coon and L. Levy, Federation Proc. 10, 174 (1951).

104 T,, Levy and M. J. Coon, J. Biol. Chem. 192, 807 (1951).

105 S, Edlbacher and J. Kraus, Hoppe-Seyler’s Z. physiol. Chem. 191, 225 (1930); 195,
267 (1931).

106 §, Edlbacher, Hrgeb. Enzymforsch. 9, 131 (1948).

107 V. du Vigneaud, 8. Simmonds, and M. Cohn, J. Biol. Chem. 166, 47 (1946).

108 V. du Vigneaud, J. P. Chandler, 8. Simmonds, A. W. Moyer, and M. Cohn, J. Biol.
Chem. 164, 603 (1946).

109 J. A. Muntz, J. Biol. Chem. 182, 489 (1950).

110 J. W. Dubnoff, Arch. Biochem. 24, 251 (1949).

11 A, A. Klose and H. J. Almquist, J. Biol. Chem. 138, 467 (1941).

12H. J. Almquist and C. R. Grau, J. Nutrition 27, 263 (1944).

IV. BIOCHEMICAL SYSTEMS 29

so far as either is added to the diet as a synthetic product. It is not known
definitely if any one molecule of methionine can serve the dual function of
supplying both methyl for transmethylation and sulfur for cysteine forma-
tion.!8

Borsook and Dubnoff'" first demonstrated the 7n vitro synthesis of methi-
onine from homocysteine and choline by rat liver slices, homogenates, and
lyophilized preparations. Betaine was superior to choline as a methy! donor,
and homocysteine to homocysteinethiolactone or homocystine as a methyl
acceptor. The reaction was independent of oxygen and of inhibitors of
cellular oxidative reactions. These workers'"® subsequently compared the
methyl-donating capacities of choline, betaine, dimethylthetin, and propio-
6-dimethylthetin in rat liver slice preparations under anaerobic conditions
and in the presence of added homocysteine. Both sulfur derivatives were
more effective than betaine, and dimethylthetin was ten to twenty times as
effective. An enzyme was isolated in a partially purified state which trans-
ferred one methyl from the thetins. The enzyme was present in the liver
and kidney of rat, guinea pig, and hog but was absent from muscle,
pancreas, and spleen.

It appears quite reasonable to believe that methylation of homocysteine
may involve the direct transfer of methyl from the betaine formed by the
oxidation of choline. However, there is no certainty that homocysteine is
methylated directly by formate-to-methyl synthesis, although this is a
plausible conclusion, Using inhibition and dilution studies in which a labeled
methionine methyl appeared after incubation of guinea pig liver slices with
homocysteine and C'*-formate, Berg concluded that neither choline nor
betaine methyl was an intermediate.!'® In subsequent studies with a cell-
free extract of pigeon liver the chromatographic separation of the incubation
mixture yielded evidence for an unidentified product formed from C14-
labeled formate and homocysteine.!!” Reference has already been made to
active methionine studied by Cantoni.*® It is again emphasized that such
a compound as adenosylmethionine may represent the methyl donor struc-
ture of methionine!®: '!® without necessarily being the molecule in which
methyl first appears as a result of formate-to-methyl synthesis. Methionine
sulfoxide has been reported to be the first intermediate in transmethyl-
ation.

Stetten fed N'°-labeled aminoethanol to rats and determined the N® in

13 ZPD. J. Mulford and W. H. Griffith, J. Nutrition 28, 91 (1942).
14 H. Borsook and J. W. Dubnoff, J. Biol. Chem. 169, 247 (1947).
15 J. W. Dubnoff and H. Borsook, J. Biol. Chem. 176, 789 (1948).
u6 P. Berg, J. Biol. Chem. 190, 31 (1951).

u7 P. Berg, Federation Proc. 11, 186 (1952).

18 G. L. Cantoni, J. Biol. Chem. 189, 745 (1951).

9G. L. Cantoni, Federation Proc. 11, 330 (1952).

30 CHOLINE

the aminoethanol and choline isolated from the respective phospholipids.!2°
The data indicated that choline was formed from the precursor without
hindrance even though the diet was sufficiently low in sources of methyl
to cause deposition of extra liver fat. It is reasonable to accept this finding
as evidence of the conversion of aminoethanol to choline, but it does not
distinguish between methylation by transmethylation and methylation
by formate-to-methyl synthesis. The latter type of synthesis of choline
has been reported by Steensholt!':!* and by SBarrenscheen and
Skudrzyk”” in experiments in which various tissue preparations were
incubated with aminoethanol and either methionine or methionine sulf-
oxide. Steensholt also concluded that p-methionine is a more efficient
methyl! donor than the natural isomer.”"* According to Veitch and Zweig,!?"¢
caution is needed in the interpretation of tissue experiments in which
evidence of the synthesis of choline by transmethylation consists of a
decrease in the methionine content of the medium or in the appearance of
an otherwise unidentified reineckate. These workers noted that a p-amino
acid oxidase in the tissues used by Steensholt and by Barrenscheen de-
creased the level of methionine during the incubation period without
formation of choline. They showed, in addition that a reineckate of amino-
ethanol was actually formed under the conditions!» in which choline
reineckate was presumed to have been isolated.

The methylation in vivo of guanidoacetic acid (GA) by transmethylation
from choline and from methionine was demonstrated in rats by du Vigneaud
and his associates.» ®° The irreversibility of the reaction has been confirmed
by a number of investigators.*7: § 12-124 The apparent inability of the ani-
mal organism to avoid methylation of a part of a dietary supplement of
GA makes it possible to use this means of decreasing available methyl
groups in the whole animal.'®

Borsook and Dubnoff reported the zn vitro formation of creatine from GA
and methionine by rat liver slices and noted that choline was ineffective
as a donor of methyl unless homocystine was provided.'?*"8 Adenosine-

120 T). Stetten, Jr., J. Biol. Chem. 142, 629 (1942).

121 G, Steensholt, Acta Physiol. Scand. 10, 333 (1945); 11, 294 (1946); 14, 340 (1947).

11a G, Steensholt, Acta Physiol. Scand. 17, 276 (1949).

121» H, K. Barrenscheen and I. Skudrzyk, Hoppe-Seyler’s Z. physiol. Chem. 284, 228
(1949).

zie HP, Veitch and G. Zweig, J. Am. Chem. Soc. 74, 1921 (1952).

122K. Bloch, R. Schoenheimer, and D. Rittenberg, J. Biol. Chem. 138, 155 (1941).

123 K. Bloch and R. Schoenheimer, J. Biol. Chem. 181, 111 (1939).

124 WH. Griffith and D. J. Mulford, J. Am. Chem. Soc. 63, 929 (1941).

125 DT), Stetten, Jr., and G. F. Grail, J. Biol. Chem. 144, 175 (1942).

126 FH. Borsook and J. W. Dubnoff, J. Biol. Chem. 182, 559 (1940).

127 H. Borsook, J. Biol. Chem. 134, 635 (1940).

128 H, Borsook and J. W. Dubnoff, J. Biol. Chem. 160, 635 (1945).

Eee

IV. BIOCHEMICAL SYSTEMS 31

triphosphate was indispensable for the methylation of GA by methionine
if homogenized liver preparations were employed.!*: '° In this case, oxygen
was also essential, and the reaction was affected by inhibitors of oxidative
mechanisms. Cohen"!: 2 showed that homogenized liver preparations of
many species, including cattle, sheep, swine, chickens, hamsters, and
rabbits, formed little or no creatine in the presence of GA, methionine,
ATP, and oxygen. Similar preparations from adult guinea pigs were active,
but those obtained from embryonic specimens were active only if a member
of the Kreb’s cycle, such as fumarate, was added to the medium. Homogen-
ized rat liver required a pteridine derivative in addition to fumarate.
Surprisingly, 10-formylfolic acid, aminopterin, and A-methopterin were
almost as potent as folic acid. Attempts to replace methionine as the
methyl donor by choline, betaine, sarcosine, formate, and other com-
pounds were unsuccessful. Cohen prepared soluble enzyme systems by
centrifugation of homogenized rat and guinea pig liver and observed that
the creatine-forming activity of these preparations in the presence of GA,
ATP, methionine, and either oxygen or nitrogen was not enhanced by
additions of folic acid or of fumarate. It was evident, therefore, that the
latter were not required under these conditions for the methylation of GA.
Inasmuch as appropriate additions of mitochondria to the soluble enzyme
system restored the need of folic acid, fumarate, and oxygen, it appeared
that the maintenance of a high level of ATP in the presence of mitochondria
was dependent on an aerobic process involving folic acid or another pteri-
dine.

Menne*: 134 studied the formation of creatine in pulped muscle. Muscle
extracts were found to contain a myosin-like apoenzyme and a heat-stable,
water-soluble coenzyme. Atrophic and dystrophic muscles were unable to
synthesize creatine. Conditions for creatine synthesis in muscle were also
examined by Barrenscheen.™*: 86 According to this author creatine is formed
in muscle by methylation directly from methionine and indirectly from
choline with trimethylamine oxide as an intermediate. The pherase acting
on methionine was cyanide-sensitive whereas the cholinepherase was cy-
anide-insensitive. Vignos and Cantoni!*® prepared partially purified frac-
tions of a guanidoacetic acid methylpherase which were free of the enzyme
necessary for the synthesis of active methionine.

29 H. Borsook and J. W. Dubnoff, J. Biol. Chem. 171, 363 (1947).

130 J. Vignos, Jr., and G. L. Cantoni, Federation Proc. 11, 399 (1952).

131 §. Cohen, J. Biol. Chem. 193, 851 (1951).

132 §. Cohen, Federation Proc. 11, 197 (1952); J. Biol. Chem. 201, 93 (1953).

133 FW. Menne, Hoppe-Seyler’s Z. physiol. Chem. 273, 269 (1942).

134 F, Menne, Z. ges. exptl. Med. 112, 38 (1943).

135 A. K. Barrenscheen and J. Pany, Hoppe-Seyler’s Z. physiol. Chem. 283, 78 (1948).

186 H. K. Barrenscheen and M. Pantlitschko, Hoppe-Seyler’s Z. physiol. Chem. 284,
250 (1949).

32 CHOLINE

Binkley and Watson’ observed that methylphosphate was active in the
formation of creatine from GA by liver homogenates and extracts but in-
active in so far as growth of rats was concerned on diets containing homo-
cystine in place of methionine. To what extent this ambiguous finding is
related to the toxicity of the methylphosphate is not known.

Trigonelline and N!-methylniacinamide may be excreted in the urine
following the administration of niacin and niacinamide, respectively. Con-
siderable species variability exists in so far as the formation and elimination
of these substances are concerned, and the significance of the methylations
is not known. Rats, but not rabbits or guinea pigs, excrete N'-methylniacin-
amide after administration of niacinamide.'* Growth in rats on protein-low
and methyl-poor diets is affected by the amide if a sufficient quantity is fed,
a result doubtless explained in part by loss of methyl from the body.!9
Perlzweig et a!.4° demonstrated the synthesis of N'-methylniacinamide by
rat liver slices and noted that the process was strictly aerobic, that intact
cells were required, and that niacin was unaffected by the system in which
the amide served as a methyl acceptor. These findings were confirmed by
Ellinger,! who noted in addition that niacin was amidated by kidney and
brain tissue and by liver tissue if supplementary glutamine was provided.

This work was extended by Cantoni,’“” who was able to show that.

the methylation of niacinamide does occur in cell-free preparations of rat
liver under anaerobic conditions in the presence of methionine, magnesium
ions, and a source of energy-rich phosphate such as ATP. The enzyme sys-
tem, for which the name nicotinamide methylkinase was suggested, was
partially purified.

Epinephrine represents a product of transmethylation, according to
Keller e¢ al.“ Following the administration of C'-methyl-labeled methio-
nine in rats, this substance, formed presumably by the methylation of
norepinephrine, was isolated from the adrenals.

The methylations of dimethylaminoethanol, of homocysteine, of guanido-
acetic acid, and of niacinamide by transmethylation appear to be of two
types which differ on the basis of the methyl donor and according to the
need of energy-rich phosphate bond compounds. Apparently, the presence
of methyl-containing quaternary nitrogen or ternary sulfur groups is es-
sential, and, for such substances, transmethylation proceeds in the absence

137 F, Binkley and J. Watson, J. Biol. Chem. 180, 971 (1949).

138 P. Handler, J. Biol. Chem. 154, 203 (1944).

139 P. Handler and W. J. Dann, J. Biol. Chem. 146, 357 (1942).

140 W. A. Perlzweig, F. Bernheim, and M. L. C. Bernheim, J. Biol. Chem. 150, 401
(1943).

141 P, Ellinger, Biochem. J. 40, Proc. xxxi (1946); 42, 175 (1948).

142 G. L. Cantoni, J. Biol. Chem. 189, 203 (1951).

143 HY, B. Keller, R. A. Boissonnas, and V. du Vigneaud, J Biol. Chem. 183, 627 (1950)

IV. BIOCHEMICAL SYSTEMS 318)

of ATP. On the other hand, methionine must also be converted to a form
having ternary sulfur in order to serve as a methyl! donor. For its formation
ATP is necessary, and the reaction consists in the addition of adenosine
to methionine with the splitting out of 3 moles of H;PO,.** Active methio-
nine, or S-adenosylmethionine, is believed to be the donor of methyl to
guanidoacetic acid, to niacinamide, and, probably, to aminoethanol. Ac-
cording to the hypothesis presented earlier, homocysteine is remethylated
with methyl from betaine, the oxidation product of choline, or with methyl
from thetin.

Little is known of the mechanisms and of the energetics of formate-to-
methyl synthesis. Because of the original concept of labile methyl as an
indispensable dietary factor, emphasis has been largely on the proof of a
metabolic origin of methyl groups. The evidence for this is satisfactory from
a qualitative viewpoint. However, whether the intermediate is formate,
formaldehyde, a phosphorylated derivative of a one-carbon molecule, or,
possibly, folinic acid, is unknown. Of utmost importance is the identi-
fication of the formate acceptor, for instance, in the reaction that yields
monomethylaminoethanol. Does reduction of the carbon occur before or
after its addition to the amino nitrogen atom? If before, is thetin the formate
acceptor? Answers to these questions will mark a significant milestone in
the progress of intermediary metabolism.

5. CHOLINE OXIDASE AND THE CHOLINE-BETAINE-GLYCINE RELATIONSHIP
a. Choline Oxidase

The demonstrated transfer of labeled methyl 7n vivo from choline to homo-
cysteine gives no clue to the intermediate steps between the original donor
and the final acceptor. Several lines of investigation have supported the
hypothesis that the methyl of choline becomes a labile methyl only after
the oxidation of the alcohol, choline, to the acid, betaine. The presence of
an oxidizing enzyme, a choline oxidase, in tissues was first suggested by
Bernheim and Bernheim,'* who observed an increase in oxygen uptake when
acetylcholine was added to liver and kidney extracts of rat and cat. Guinea
pig liver was found to be inactive in this respect. The fact that choline was
oxidized by tissues was confirmed by Trowell,!> using liver slices. Mann
and Quastel'*® isolated the oxidation product of choline as the reineckate
and identified it as betaine aldehyde. Some evidence was obtained that
betaine aldehyde was also oxidized by rat liver slices and rat liver or kidney
extracts, though at a much slower rate than choline. It was suggested that
the oxidation product in this case was betaine.

144 F Bernheim and M. L. C. Bernheim, Am. J. Physiol. 104, 488 (1933) ; 121, 55 (1938)

45 Q A. Trowell, J. Physiol. (London) 85, 356 (1935).
46 P.J.G. Mann and J. H. Quastel, Biochem. J. 31, 869 (1937).

34 CHOLINE

Bernheim and Bernheim™ prepared a choline oxidase extract of rat liver
and showed that it was distinct from the enzyme which oxidized ethyl
alcohol. The oxygen uptake and the properties of the end products indi-
cated that at pH 6.7 choline was oxidized to betaine aldehyde with the up-
take of one atom of oxygen. At this pH betaine aldehyde was oxidized fur-
ther at a very slow rate, whereas at pH 7.8 the aldehyde was oxidized
rapidly to betaine, presumably by another enzyme system. The above
workers found the oxidase system in the liver and kidney of the rat but
failed to find it in blood, brain, or muscle.

Klein and Handler’ were able to show that the oxidation of betaine
aldehyde required diphosphopyridine nucleotide, but that of choline did
not. In the presence of the nucleotide, betaine aldehyde was also oxidized
by preparations of rat kidney, brain, and muscle, the activities of these
tissues compared to liver being 0.33, 0.18, and 0.10, respectively.

Mann and his coworkers"® observed that arsenocholine, like choline, is
oxidized to the corresponding aldehyde by the choline oxidase of rat liver.
The substitution of nitrogen by arsenic reduces the affinity of the enzyme
for the substrate. According to these authors the choline oxidase system
consists partly of a dehydrogenase. This was shown by the fact that rat
liver extracts, in the presence of choline or arsenocholine, rapidly reduce
sodium ferricyanide under anaerobic conditions. It was also observed that
choline oxidase in the presence of choline or arsenocholine reduces cyto-
chrome ¢ at room temperature.

An apparent phosphate requirement for choline oxidase activity was ob-
served by Angel and Miller.'*° When rat liver homogenates were prepared
with phosphate, veronal, and citrate buffers at pH 7.8, the choline oxidase ~
activities of the suspensions in citrate and veronal buffers were 18% and
50%, respectively, of that in the phosphate buffer. Addition of phosphate
to citrate and veronal suspensions stimulated the oxidase activity by 25
to 50%, respectively. The phosphate continued to stimulate the choline
oxidase activity in the presence of dinitrophenol (107? M).

The requirements of the rat liver choline oxidase system in whole liver
homogenates have been studied by Williams et al.,!°° who investigated the
effect of pH on the oxidation of choline by homogenates of rat liver. Only
small differences were observed in the activities at pH 6.8, 7.8, and 7.8.
The oxidation of choline at pH 6.8 and 7.8 gave sigmoidal oxygen consump-
tion curves if allowed to run for 2 hours, while the pH 7.3 homogenate gave
maximum activity during the first 10-minute period, with a gradual decrease

147 J, R. Klein and P. Handler, J. Biol. Chem. 144, 537 (1942).
148 P, J. G. Mann, H. E. Woodward, and J. H. Quastel, Biochem. J. 32, 1024 (1938).
1449 ©, Angel and O. N. Miller, Federation Proc. 11, 4385 (1952).
150 J. N. Williams, Jr., G. Litwack, and C. A. Elvehjem, J. Biol. Chem. 192, 73 (1951).

IV. BIOCHEMICAL SYSTEMS ae

in activity thereafter. From data obtained by these workers on 10-minute
incubations it appeared that, if two enzyme systems with different pH
optima for their activities were involved, the over-all activity in whole
rat liver homogenates was the same for the three pH values employed.
Since Williams and his coworkers! were interested in developing an assay
system for total rat liver homogenates, they also studied the product in-
hibition of the choline oxidase system. It was noted that the oxidation of
betaine aldehyde to betaine occurred at a much slower rate than the oxi-
dation of choline alone and that betaine aldehyde apparently inhibited the
oxidation of choline. Although betaine aldehyde was a potent inhibitor of
choline oxidase, betaine had no effect upon the oxidation of choline. Further
experiments showed that for the first 10 to 12 minutes oxidation of choline
proceeded linearly and that, as betaine aldehyde concentrations increased,
the rate of choline oxidation gradually decreased. Therefore, the first 10-
minute interval can be used as a measure of choline oxidase activity without
interference from the inhibitory action of betaine aldehyde or its slower
rate of oxidation. The requirements of the system for added cytochrome ¢
and for diphosphopyridine nucleotide were investigated also, and it was
concluded that for adequate assay no additional cofactors were necessary.
On the basis of the above findings Williams et al.1®° described an assay system
for measuring choline oxidase activity in total liver extracts. Their data
showed that rat liver choline oxidase is remarkably constant for comparable
animals. Niacinamide strongly inhibited the activity of the oxidase system
under all conditions. Both niacinamide and diphosphopyridine nucleotide
inhibited the activity of the oxidase system in total liver extracts.

Further studies by Williams !*! on the mechanism of the metabolism of
choline and betaine aldehyde have shown that the latter metabolite can be
converted to betaine either aerobically or anaerobically, whereas choline
oxidase appears to be an oxygen-requiring system.

Early studies on the intracellular distribution of the components of the
choline oxidase system were made by Lan,!®: 153 who failed to find any ap-
preciable amount of the oxidase activity in isolated nuclei of rat liver cells.
Kensler and Langemann found 78 % of the oxidase activity in the mitochon-
drial fraction of rat liver preparations and also demonstrated that the addi-
tion of succinate greatly reduced the oxidation of choline by tissue homoge-
nates, presumably because of saturation of proton and electron acceptors.1*:
155 Concentration of choline oxidase activity in mitochondria was confirmed

151 J. N. Williams, Jr. Proc. Soc. Exptl. Biol. Med. 78, 202 (1951).

162 'T. H. Lan, J. Biol. Chem. 151, 171 (1943).

153 T. H. Lan, Cancer Research 4, 37, 42 (1944).

154 ©, J. Kensler and H. Langemann, J. Biol. Chem. 192, 551 (1951).
155 H, Langemann and C. J. Kensler, Federation Proc. 11, 366 (1952).

36 CHOLINE

in extensive studies by Williams.!°* A potent heat labile inhibitor of choline
oxidase was found in the supernatant fraction. These studies also showed
that the betaine aldehyde oxidase activity of the supernatant fraction can
be considerably increased if diphosphopyridine nucleotide is added. The
nuclei and microsomes are unaffected by addition of this nucleotide.

Although choline oxidase activity has been found in the livers and kidneys
of rats, chicks, rabbits, dogs, and monkeys,!*” most of the in vitro studies
on this enzyme system have used tissues of the rat. It has been shown that
choline oxidase in the rat liver is cyanide-sensitive and that it is readily
inhibited by copper and phenylhydrazine. Barron and Singer’? have
classified it in the group of sulfhydryl enzymes. Choline oxidase loses its
activity at a fairly rapid rate if partially purified. The rate of loss, which is
that of a first-order reaction, is more rapid at pH 6.7 than at 7.8. Cystine
and semicarbazide increase the rate of inactivation both in presence and
absence of choline. Nickel, cobalt, and iron salts not only increase the in-
activation rate of the enzyme but also combine reversibly with a group on
the enzyme to inhibit it.!®° Halogenated alkylamines are powerful inhibitors
for choline oxidase.!® Among these is the nitrogen mustard, methylbis-
(6-chloroethyl)amine, which irreversibly inhibits choline oxidase. Colter
and Quastel! observed that benzedrine, ephedrine, tyramine, methylamine,
aminoethanol, and histamine not only reversibly inhibit choline oxidase
but also prevent the irreversible inhibition by the nitrogen mustard.

The enzyme in rat liver which oxidizes choline was named choline de-
hydrogenase by Bargoni and Di Bella.'® Its activity was ascribed to free
amino and carbonyl rather than to mercapto groups. The enzyme shows
no color with nitroprusside nor with Grote’s reagent and is inhibited by
cyanide but not by malonate. The dehydrogenation system involves the
oxidation of reduced cytochrome c.

At pH values below 7.0, stearic, palmitic, and oleic acids at 0.004 M
concentrations inhibit choline oxidase activity 61%, 41 %, and 29 %, respec-
tively. Fats and mixtures of cephaline and lecithin are without effect. It
has been suggested! that the fatty acids in liver may determine to some
degree how much free choline can be oxidized and how much can be used
for synthesis of phospholipids.

156 J. N. Williams, Jr., J. Biol. Chem. 194, 139 (1952) ; 195, 37 (1952); 197, 709 (1952).
157 J. 8. Dinning, C. K. Keith, and P. L. Day, Arch. Biochem. 24, 463 (1949).

158 F. Bernheim, J. Biol. Chem. 188, 485 (1940).

159). §. G. Barron and T. P. Singer, Science 97, 356 (1943).

160 G, §. Eadie and F. Bernheim, J. Biol. Chem. 185, 731 (1950).

161 &. S. G. Barron, G. R. Bartlett, and Z. B. Miller, J. Exptl. Med. 87, 489 (1948).
162 J. §. Colter and J. H. Quastel, Nature 166, 773 (1950).

163 N. Bargoni and 8S. Di Bella, Atti accad. sct. Torino 84, 149 (1949-1950).

164 F’. Bernheim, J. Biol. Chem. 133, 291 (1940).

IV. BIOCHEMICAL SYSTEMS 37

Evidence was presented by Williams'®> demonstrating a positive con-
nection between the choline oxidase system of rat liver and the probable
precursors of the Leuconostoc citrovorum factor (LCF) as well as LCF itself.
It was shown that when ascorbic acid and folic acid were incubated with
rat liver homogenate small but significant stimulation of the choline oxidase
system was observed. This effect was more pronounced in rats fed
aminopterin. Since aminopterin is believed to inhibit the conversion of folic
acid to LCF,!** Williams!® postulated that the stimulatory effect of the
folic acid and ascorbic acid added in vitro was due to the enzymatic con-
version of these substances to LCF, which was actually responsible for
stimulation of the enzyme system. In work that followed, Williams!” studied
various combinations of factors related to LCF, e.g., folic acid, ascorbic
acid, vitamin By, and synthetic LCF itself in relation to choline oxidase.
This work was done with a modified enzyme system containing homocys-
teine which amplified the effects of the above factors. The results of this
investigation showed that folic acid, Leuconostoc citrovorum factor, vitamin
By, and ascorbic acid individually or in combination markedly stimulate
choline oxidation in the liver homogenates of rats fed aminopterin. Only
ascorbic acid and folic acid were found to stimulate choline oxidation in
normal rat liver homogenates. The stimulation by these factors was ob-
served to be much more marked in aminopterin-fed rats.

Williams et al.!** subsequently noted that folic and folinic acids in whole
liver and in isolated mitochondria were markedly decreased in folic acid
deficiency in rats. Aminopterin-fed animals showed a similar decrease in
folinic acid, but there was no e‘fect on the folic acid content of the liver or
of the mitochondrial fraction. The loss in choline oxidase activity in the
latter fraction paralleled the disappearance of folinic acid, and it was con-
cluded that this substance rather than folic acid was involved in the main-
tenance of choline oxidase activity.

The influence of several dietary factors on tissue choline oxidase has been
reported. The removal of folic acid from the diet of the monkey or the
administration of aminopterin to this animal eliminated most of the choline
oxidase activity from the liver and kidney.!*” Further studies disclosed
that aminopterin-treated chickens did not exhibit the choline oxidase ac-
tivity normally found in bone marrow,!® and that livers from folic acid-
deficient chicks oxidized choline at a reduced rate.!®® Chick kidney choline
oxidase was not found to be significantly reduced in folic acid deficiency.
165 J. N. Williams, Jr., J. Biol. Chem. 191, 123 (1951).

166 ©, A. Nichol and A. D. Welch, Proc. Soc. Exptl. Biol. Med. 74, 52, 403 (1950).
167 J, N. Williams, Jr., J. Biol. Chem. 192, 81 (1951).
1672 J. N. Williams, Jr., A. Sreenivasan, Shan-Ching Sung, and C. A. Elvehjem, J.

Biol. Chem. 202, 233 (1953).

168 J. §. Dinning, C. K. Keith, P. L. Davis, and P. L. Day, Arch. Biochem. 27, 89
(1950).

38 CHOLINE

Williams!”° observed that rats fed aminopterin exhibit a concentration
of liver ascorbic acid which is less than 50% normal, suggesting that folic
acid is probably involved in ascorbic acid synthesis in the rat. His work
indicated that, whereas high levels of ascorbic acid in liver extracts stimu-
late the choline oxidase system, low levels of this vitamin added in vitro
actually inhibit the oxidation of choline. On the basis of these observations
Williams postulated that the low ascorbic acid concentration in the liver
of rats fed aminopterin may help to explain some of the previous observa-
tions on the low choline oxidase activity of livers of those rats.

Choline oxidase activity was greatly depressed in the fatty liver of rats
on a low methionine diet.!”! It was suggested that the oxidase inhibition may
be due to the increased lipid content of the liver, since fatty acids have been
shown to be inhibitory to the system.'* The importance of the choline oxi-
dase system in the development of fatty livers caused by choline deficiency
is suggested by the observations that guinea pigs which lack the enzyme
system cannot be made to develop fatty livers very readily’? and that
hamsters which have some enzyme but much less than rats do not accumu-
late as much liver fat on a deficient diet as do rats. A lack of the enzyme
system can be considered an advantage when minimal amounts of choline
are present for fat transport and metabolism, and the suggestion has been
advanced that it is the diminished choline oxidase activity which permits
the existence of a normal hepatic choline concentration (as phospholipid)
despite a dietary choline deficiency.!: 1 The work of Dubnoff!!® and
Muntz!? showed that the choline oxidase system also plays an important
role in transmethylation reactions. However, a mechanism must exist in
the guinea pig for the catabolism of choline. Dubnoff injected C'-methyl-
labeled choline and betaine intraperitoneally into this animal and found
that both contributed to labeled expired carbon dioxide as well as to labeled
tissues but the ratio of utilization of betaine methyl to choline methyl was
4.6.175

Liver choline oxidase was decreased by a riboflavin deficiency in
rats.176 177 'The B.-deficient rat livers showed a reduced activity of about
20 to 30% of the control levels.!” Atabrine, which has been known to in-
hibit flavin enzyme systems, 7n vitro was found to bring about an inhibition

169 J. S. Dinning, C. K. Keith, and P. L. Day, J. Biol. Chem. 189, 515 (1951).
170 J, N. Williams, Jr., Proc. Soc. Exptl. Biol. Med. 77, 315 (1951).

1 P, Handler and F. Bernheim, J. Biol. Chem. 144, 401 (1942).

12 P, Handler, Proc. Soc. Exptl. Biol. Med. 70, 70 (1949).

173 P, Handler and F. Bernheim, Proc. Soc. Exptl. Biol. Med. 72, 569 (1949).
174 H. P. Jacobi and C. A. Baumann, J. Biol. Chem. 142, 65 (1942).

175 J. W. Dubnoff, Arch. Biochem. 22, 474 (1949).

176 W. Hess and G. Voillier, Helv. Chim. Acta 31, 381 (1948).

177 B, Kelley, Federation Proc. 11, 238 (1952).

IV. BIOCHEMICAL SYSTEMS 39

of choline oxidase activity.1” The oxidase system was not influenced by a
deficiency of vitamin By. in the chick"* or by thiamine,!”® vitamin E,!*° or
histidine!*! deficiency in the rat. Intraperitoneal injection of urethane as
well as x-irradiation of mature hens reduced the bone marrow choline oxi-
dase.!® Whole body x-irradiation of adult male rats with a dosage of 200
r. appeared to decrease liver choline oxidase activity, but the effect was
uncertain in view of the marked increase in endogenous respiration.!® The
enzyme was poisoned by high oxygen tensions, probably due to its depen-
dence on its sulfhydryl groups. The injection of thyroxine had no effect
on the oxidase activity of the rat.1%°

Swendseid et al.'*** showed that the feeding of ethionine (S-ethylhomo-
cysteine) to rats decreased the choline and sarcosine oxidases but not the
succinoxidase of the liver. This possible antagonist of methionine also
inhibited choline and sarcosine oxidases in vitro in homogenized prepara-
tions, but the effect was absent if the preparation was dialyzed before its
addition. Neither methoxinine nor methionine sulfoximine inhibited choline
oxidase in the in vitro studies.

In summarizing the discussion of ‘“‘choline oxidase,” it is pertinent to
emphasize that, with few exceptions, the term has not referred specifically
to either of the dehydrogenases that convert choline to betaine aldehyde
and the aldehyde to betaine but has included the system responsible for the
transport of protons and electrons through the cytochromes to oxygen.
The two dehydrogenases appear to be different enzymes, although both
occur in liver mitochondria. They may be separated on the basis of the
greater solubility of the betaine aldehyde dehydrogenase.'**> Each is esti-
mated by the anaerobic reduction of ferricyanide in the presence of the
respective substrate, choline or betaine aldehyde. Strength et al.!8*> believe
that both enzymes are DPN-linked, although the necessity of DPN for
the action of choline dehydrogenase has been questioned.'®® Folinic acid!”°
and riboflavin'* 7 appear essential for the dehydrogenation of choline.
Ebisuzaki and Williams'*** demonstrated the importance of flavin adenine
dinucleotide (FAD) for liver choline oxidase activity, especially in prepara-

)

178 M. B. Gillis and R. J. Young, Poultry Sci. 30, 468 (1951).

179}, Egana, Publs. lab. med. exptl. clin. med. Univ. Chile 1, 127 (1946).

180}, L. Hove and J. O. Hardin, Proc. Soc. Exptl. Biol. Med. 78, 858 (1951).

181 J. W. Bothwell and J. N. Williams, Jr., J. Biol. Chem. 191, 129 (1951).

182 J. §. Dinning, I. Meschan, C. K. Keith, and P. L. Day, Proc. Soc. Exptl. Biol.
Med. 74, 776 (1950).

183 H. O. Kunkel and P. H. Phillips, Arch. Biochem. and Biophys. 87, 366 (1952).

184 F. Dickens, Biochem. J. 40, 171 (1946).

185 R. L. Smith and H. G. Williams-Ashman, Nature 164, 457 (1949).

185a MT. E. Swendseid, A. L. Swanson, and F. H. Bethell, J. Biol. Chem. 201, 803 (1953).

185b T). R. Strength, J. R. Christensen, and L. J. Daniel, J. Biol. Chem. 203, 63 (1953).

185e KK. Ebisuzaki and J. N. Williams, Jr., J. Biol. Chem. 200, 297 (1953).

40 CHOLINE

tions from riboflavin-deficient rats. These workers believe that FAD func-
tions in the hydrogen transport system prior to the participation of cyto-
chrome c.

b. The Choline-Betaine-Glycine Relationship

The evidence appears indisputable that choline serves as a source of
labile methyl by irreversible oxidation to either betaine aldehyde or to
betaine. Whether the aldehyde or the acid is the methyl donor is unknown,
although in Fig. 1 it is assumed that it is betaine which is demethylated
initially. If it is not, its reduction to the aldehyde must occur with great
rapidity. In any event, glycine is the product of the complete demethylation
of the choline molecule, and the di- and monomethy] derivatives of glycine
are logical intermediates in addition to glycine.

Stetten administered N!*-labeled ethanolamine, choline, glycine, and
betaine singly to rats and later isolated the first three compounds from
tissues. On the basis of the extent of labeling in the isolated materials he
concluded that glycine and ethanolamine were intermediates between be-
taine and choline!®* and that the major route of demethylation is the con-
version of betaine to glycine, a precursor of ethanolamine.'® Definite
evidence that choline is not demethylated was the finding of methionine
and labeled dimethylglycine, not dimethylaminoethanol, after incubation
of a rat liver homogenate containing homocysteine and N!*-labeled
choline.!°

The proof of the transfer of one intact methyl group, at least, from the
product of oxidation of choline, either betaine aldehyde or betaine, is im-
pressive. Following the feeding of deuteriomethyl-labeled choline to rats,
methionine and creatine containing deuteriomethyl groups were isolated
from tissues.*° This transmethylation occurred regardless of a dietary need
of methionine, indicating that the reaction was one of those characterized
by Schoenheimer as an ‘‘automatic and non-interruptable biochemical pro-
cess.’’!88 That betaine is a source of labile methyl in the sense commonly as-
cribed to the choline methyl has long been known.*!: 77: 174; 189, 199 Signifi-
cantly, betaine is not a complete replacement for choline in the chick which
is unable to form monomethylaminoethanol from aminoethanol. It is not
antiperotic, for instance, a function which appears to depend upon the in-
corporation of choline or of a similar molecule, such as arsenocholine, into
phospholipids.!*

186 T). Stetten, Jr., J. Biol. Chem. 138, 487 (1941).

187 TD). Stetten, Jr., J. Biol. Chem. 140, 143 (1941).

188 AR. Moss and R. Schoenheimer, J. Biol. Chem. 135, 415 (1940).
189 A. P. Platt, Biochem. J. 38, 505 (1939).

19 J. P. Chandler and V. du Vigneaud, J. Biol. Chem. 185, 223 (1940).
191 TH. Jukes, J. Nutrition 20, 445 (1940).

IV. BIOCHEMICAL SYSTEMS 4]

The series of reactions pictured in Fig. 1 indicates that only one methy]
of betaine takes part in true transmethylation, the remaining two being con-
verted to formate. Needless to say, the nitrogen is no longer quaternary
after the first demethylation. High radioactivity in the 6-carbon was found
in serine isolated from livers of rats after the administration of choline with
C-labeled methyl groups, thus demonstrating the conversion of one
methyl, at least, to formate.!% The zn vitro production of formate from the
methyls of choline has been demonstrated by Siekevitz and Greenberg in
experiments in which rat liver slices were incubated with similarly labeled
choline.*2 These authors concluded that the nitrogen-methyl carbons of
choline and the a-carbon of glycine are specific formate donors in the syn-
thesis of serine from glycine.

The contrast in the roles of betaine and of dimethylglycine in trans-
methylation is clearly illustrated in the experiments of du Vigneaud et al.
in which N1*-deuteriomethyl-labeled betaine and deuteriodimethylglycine
were fed to rats.7® Negligible labeling of tissue choline and creatine occurred
in the latter instance, whereas significant labeling with deuterium did oc-
cur after the administration of betaine. Little of the nitrogen of betaine
appeared in choline and creatine, showing that there had been no direct
reduction of betaine to choline. Oginsky!™ found more methionine formed
from homocystine and betaine by a rat liver homogenate than from homo-
cystine and choline.

The degradation of monomethylglycine, or sarcosine, has been studied
in several laboratories. Conversion to glycine was noted by Abbot and
Lewis in rabbits which excreted increased amounts of hippuric acid follow-
ing feeding of sarcosine and benzoic acid.!% The failure to observe a similar
effect of dimethylglycine and of betaine may be due possibly to a slower
rate of demethylation of these substances to the monomethyl] derivative.
Bloch and Schoenheimer observed glycine formation in rats fed N!*-labeled
sarcosine,!®® and Handler et al. demonstrated the conversion of dimethyl-
glycine and of sarcosine to glycine and formaldehyde in rat liver homog-
enates.19° Mackenzie isolated C'-formaldehyde as the dimedon derivative
from preparations containing rat liver homogenates or slices and labeled
sarcosine.!” Labeled formic acid and carbon dioxide were also identified.
According to this worker formaldehyde from methyl carbons of betaine
is a normally occurring metabolite, as is sarcosine.

The formation of serine by the addition of formate to glycine, with the

12 W. Sakami, J. Biol. Chem. 179, 495 (1949).

193 KH, L. Oginsky, Arch. Biochem. 26, 327 (1950).

194 T,, D. Abbot, Jr. and H. B. Lewis, J. Biol. Chem. 131, 479 (1939).

195 K, Bloch and R. Schoenheimer, J. Biol. Chem. 135, 99 (1940).

196 P. Handler, M. L. C. Bernheim, and J. R. Klein, J. Biol. Chem. 138. 211 (1941).
197 ©. G. Mackenzie, J. Biol. Chem. 186, 351 (1950).

42 CHOLINE

formate becoming the 6-carbon of the new serine molecule, appears defi-
nitely established. In zn vitro experiments with rat liver homogenates,
Winnick ef al.!% isolated carboxyl-labeled serine after addition of C'-car-
boxyl-labeled glycine and serine labeled in the a- and 6-carbons after addi-
tion of C'4-a-carbon-labeled glycine. Following the feeding of C'*-carboxyl-
labeled glycine and radioformate (C') to rats, Sakami!% isolated serine
from liver tissue and found C® in the serine carboxyl group and C* in the
8 position. Confirmatory evidence for the formation of serine from glycine
was supplied by Greenberg and his coworkers as a result of in vivo studies
in rats?°° and zn vitro experiments with rat liver slices.2" Ehrensvard e¢ al.2”
noted a rapid conversion of C'’-carboxyl-labeled glycine to serine by yeast.

Serine is also a source of formate, possibly by reversal of the reaction in
which formate is added to glycine. Shemin?® isolated hippuric acid after
the administration of benzoic acid and C-carboxyl-labeled, N?-labeled
serine in rats and guinea pigs and found the same N!*°:C® ratio in the gly-
cine component as in the serine. C'-formate is produced from C-6-carbon-
labeled serine by rat liver in in vitro experiments.!”

The mechanisms of the glycine-serine reactions are not known. Shemin?"
suggested the possibility of a removal of two hydrogens from the 8-position
of serine with the formation of a-amino-8-ketopropionic acid as the immedi-
ate precursor of glycine and formic acid. On the other hand, a serine dehy-
drase has been reported in mouse?" and rat liver.?°° In the latter instance
a-aminoacrylic acid was postulated as the product of the removal of water
from the 6-carbon of serine. Ratner et al.?°* have prepared a glycine oxidase
from liver and kidney of several species. Its prosthetic group is
flavin adenine dinucleotide, and the enzyme converts glycine to glyoxylic
acid plus ammonia and sarcosine to glyoxylic acid plus methylamine.
Paretsky and Werkman?” concluded that formaldehyde is an intermediate
in the dissimiliation of glycine by Achromobacter.

The evidence is clear-cut also for the decarboxylation of serine to amino-

198 T. Winnick, I. Moring-Claesson, and D. M. Greenberg, J. Biol. Chem. 175, 127
(1948).

199 W. Sakami, J. Biol. Chem. 176, 995 (1948).

200 P, D. Goldsworthy, T. Winnick, and D. M. Greenberg, J. Biol. Chem. 180, 341
(1949).

201 P, Siekevitz and D. M. Greenberg, J. Biol. Chem. 180, 845 (1949).

202 G. Ehrensvard, E. Sperber, E. Saluste, L. Reio, and R. Stjernholm, J. Biol. Chem.
169, 759 (1947).

203 T), Shemin, J. Biol. Chem. 162, 297 (1946).

204 F. Binkley, J. Biol. Chem. 150, 261 (1943).

205 HK), Chargaff and D. B. Sprinson, J. Biol. Chem. 161, 273 (1943).

206 §, Ratner, V. Nocito, and D. E. Green, J. Biol. Chem. 152, 119 (1944).

207 TD). Paretsky and C. H. Werkman, Arch. Biochem. 25, 288 (1950).

IV. BIOCHEMICAL SYSTEMS 43

ethanol. Stetten?% fed N!*-labeled serine to rats and found the N! in tissue
phospholipids and proteins, in cystine, and in aminoethanol. Levene and
Tarver?’ observed a rapid uptake of C!4-6-carbon-labeled serine in tissue
proteins and the appearance of the C™ in aminoethanol in rats fed the
labeled serine. Similiar evidence has been presented by other workers.”: *
%4, 210 Tn the experiments of Weissbach et al.,2!° rats fed serine, doubly labeled
by C™ in the B-carbon and by N!, converted the amino acid into choline-
containing C" in the alcoholic carbon and N™. This would be expected by
the decarboxylation of serine to aminoethanol and by the methylation of
the latter.

In so far as the carbon chain of choline is concerned, the evidence is
strong for a cycle as described in Fig. 1. In theory at least, the nitrogen-
carbon combination of choline might remain in the cycle indefinitely, but
with each complete series of reactions a new second carbon would enter the
cycle, entering as the 6-carbon and leaving as carbon dioxide from the car-
boxyl carbon of serine. As far as is known the glycine-serine reaction is the
only reversible reaction of consequence among those concerned with the
carbon chain of choline (compounds 1 to 10, Fig.1).

6. OXIDATION OF Mretuyt CARBONS

The rapid appearance of radioactive carbon dioxide in the expired air
of rats after the feeding of C'-methyl-labeled methionine clearly demon-
strated the oxidizability of the labile methyl group." A similar result fol-
lowed the intraperitoneal injection of C-labeled choline, betaine,
dimethylthetin, and dimethylpropiothetin.2”~"4 In each of the latter experi-
ments the highest level of radioactivity of carbon dioxide was found in the
first 4-hour period. Less choline was oxidized, but this may only have been
a reflection of its normal use in lecithins and in other metabolites. Reference
has already been made to the appearance of the labeled 6-carbon of serine
after administration of methyl-labeled choline or methionine. For this rea-
son, it is reasonable to assume, as indicated in Fig. 1, that formate is an
intermediate in the catabolism of a labile methyl and that the formate car-

208 T). Stetten, Jr., J. Biol. Chem. 144, 501 (1942).

209 M. Levine and H. Tarver, J. Biol. Chem. 184, 427 (1950).

210 A. Weissbach, D. Elwyn, and D. B. Sprinson, J. Am. Chem. Soc. 72, 3316 (1950).

211 CO. G. Mackenzie, J. P. Chandler, E. B. Keller, J. R. Rachele, N. Cross, D. B.
Melville, and V. du Vigneaud, J. Biol. Chem. 169, 757 (1947).

212 ©, G. Mackenzie, J. P. Chandler, E. B. Keller, J. R. Rachele, N. Cross, and V.
du Vigneaud, J. Biol. Chem. 180, 99 (1949).

213 ©, G. Mackenzie, J. R. Rachele, N. Cross, J. P. Chandler, and V. du Vigneaud,
J. Biol. Chem. 183, 617 (1950).

214M. F. Ferger and V. du Vigneaud, J. Biol. Chem. 185, 53 (1950).

44 CHOLINE

bon may be completely oxidized or built into serine or any other formate-
accepting anabolic product depending on a diversity of metabolic factors.
The demonstration of the loss of labile methyl groups by oxidation makes
understandable the continuous need of the organism for replacement meth-
yls.

7. OrneR Aspects oF Metruyt TRANSFER AND OXIDATION

Dimethylsulfone has been isolated from beef blood?!® and adrenal cortex”'®
and from the plants Equisetum palustre, EF. arvenue, and EF. hyemale?": *'8
Inasmuch as so little is known about the intermediate steps in the oxida-
tion of sulfur, it cannot be said that this compound is not a normal inter-
mediate. However, it appears most unlikely that it is important in trans-
methylation. Smythe demonstrated the formation of hydrogen sulfide from
cysteine by liver slices and suggested that it might be a precursor of the
sulfone.”!®

The chemical mobility of one methyl group in betaine, at least, was ob-
served by Willstatter at the beginning of the century.”° If heated to 300°
it is converted in part to the methyl ester of dimethylaminoacetic acid.
Challenger ef al. extended this observation to include the transfer of methyl
from betaine under the influence of heat with the formation of dimethyl
derivatives of selenium, tellurium, and sulfur from selenite, tellurite, and
sulfite, respectively.22: 22 It was also demonstrated that the heating of
betaine with aromatic primary amines such as aniline, p-toluidine, or
8-naphthylamine resulted in a transfer of methyl and the formation of the
corresponding monomethyl derivatives. Dimethylthetin also yielded the
methyl ester of thiomethylacetic acid on heating.’

Methylation of selenides and tellurides in the animal organism has been
the subject of much controversy since the original report by Hofmeister in
1894.24 The excretion in expired air of a volatile selenide or telluride has
not been questioned, but proof that the substance in question was a methyl
derivative has been unsatisfactory. However, McConnell and Portman have

25 J. J. Pfiffner and H. B. North, J. Biol. Chem. 134, 781 (1940).

216 T,, Ruzicka, M. W. Goldberg, and H. Meister, Helv. Chim. Acta 23, 559 (1940).
217 P. Karrer and C. H. Eugster, Helv. Chim. Acta 32, 957 (1949).

218 P. Karrer, C. H. Eugster, and D. K. Patel, Helv. Chim. Acta 32, 2397 (1949).
29 ©. V. Smythe, J. Biol. Chem. 142, 387 (1942).

220 R. Willstitter, Ber. 35, 584 (1902).

221 f. Challenger and C. Higginbottom, Biochem. J. 29, 1757 (1935).

222 f Challenger, P. Taylor, and B. Taylor, J. Chem. Soc. 1942, 48.

223 F Challenger and P. Fothergill, Biochem. J. 45, xxvii (1949).

224 F. Hofmeister, Arch. exptl. Pathol. Pharmakol. 33, 198 (1894).

V. SPECIFICITY OF ACTION 45

identified dimethylselenide and measured its excretion following the ad-
ministration in rats of sodium selenate containing radioselenium.*”°

V. Specificity of Action
WENDELL H. GRIFFITH and JOSEPH F. NYC

The following discussion deals with specificity of action of choline in the
animal organism. Certain aspects of this problem in microorganisms are
included in the section on biogenesis (Section VI).

Inasmuch as choline can be synthesized in vivo, the recognition of a
choline-like action in another compound depends in large part on whether
or not the compound in question is used with greater facility than choline
can be synthesized. The characteristic effects of choline are its role as a
potential source of labile methyl and its complex function as a constituent
of phospholipids. These properties are measured, respectively, by the rate
of growth of rats on diets containing homocystine but devoid of methionine
and by the prevention of increases of hepatic lipids in rats deprived of
choline. Presumably this latter property may be ascribed to phospholipids
such as lecithin which may also be responsible for the prevention of renal
lesions in young rats and of perosis in chicks and turkey poults and for
stimulation of growth in the young fowl. Although the specificity of choline
as a source of labile methyl or of phospholipid is high, it shares these proper-
ties to a limited extent. Pertinent data are shown in Tables III to VI which
are expanded from the material reported by Moyer and du Vigneaud.!

As indicated earlier (Section IV, p. 19), the lability of a methy] is related to
its attachment to a nitrogen or sulfur atom which has acquired an addi-
tional covalent bond and positive charge. In addition to compounds con-
taining preformed choline that may be liberated by hydrolysis (Table IT)
and to compounds with sulfur methyls and “onium”’ sulfur (Table IV), the
only substances known to be in this category are betaine and dimethyl-
ethyl-8-hydroxyethylammonium hydroxide or monoethylcholine (Tables
V and VI). This represents a high degree of specificity, indeed, and it
appears that conversion to glycine betaine or to a very similar structure is
a prerequisite for choline activity in substances of this type. It is clearly
evident, however, that the presence of a methylated quaternary nitrogen
in betaine is not the sole factor governing specificity because betaines of
225 K.P. McConnell and O. W. Portman, J. Biol. Chem. 195, 277 (1952).

1 A.W. Moyer and V. du Vigneaud, J. Biol. Chem. 143, 373 (1942).

46 CHOLINE

TABLE III
BroLoGicaL ACTIVITY OF CHOLINE AND OF SOME OF ITs DERIVATIVES

Prevention of

Renal Fatty Growth with
Compound Perosis lesion liver Fowl growth homocystine

Choline : +2, 3 4 +5 2, 3, 6 +7
Phosphorylcholine +8 +41
Lecithin +9 +5 +9 +1
Choline sulfate —10
Arsenocholine +2, 3 10, 11 Se 3 seat Se
Phosphocholine +10 +8
Sulfocholine +16 +16 16
Monoethylcholine +3, 14 10, 11 +1 +3, 14 sii
Diethylcholine 3, 14 +10, 11 +11 Ec ir§ =" 5
Triethylcholine Bese +10, 11 +17, 18 Sif 14 rand
Tripropylcholine —10 —10, 18
Homocholine +18 a!
Trimethyl-6-hydroxypropyl- —10 —i9

ammonium hydroxide
Diethylmethyl-6-y-dihy- —1

droxypropylammonium

chloride
Choline methyl ether —20
B-Methylcholine se! 257 = = an
B-Methylcholine ethyl ether =
a,a-Dimethylcholine —3 —3 —21
a-Methyl-6-phenylcholine —8

2T. H. Jukes, J. Nutrition 20, 445 (1940).

3T. H. Jukes and A. D. Welch, J. Biol. Chem. 146, 19 (1942).

4W.H. Griffith and N. J. Wade, Proc. Soc. Exptl. Biol. Med. 41, 188 (1939).

5>C. H. Best, J. M. Hershey, and M. E. Huntsman, Am. J. Physiol. 101, 7P (1932).

6 A. A. Klose and H. J. Almquist, J. Biol. Chem. 138, 467 (1941).

7V. du Vigneaud, J. P. Chandler, A. W. Moyer, and D. M. Keppel, J. Biol. Chem.
131, 57 (1939).

8 A. D. Welch and M.S. Welch, Proc. Soc. Exptl. Biol. Med. 39, 7 (1938).
°T. H. Jukes, Poultry Sct. 20, 251 (1941).

0 A.D. Welch, J. Nutrition 40, 113 (1950).

11 A.D. Welch, cited by T. H. Jukes, ref. 14.

122A. TD. Welch and R. L. Landau, J. Biol. Chem. 144, 581 (1942).

13 A.D. Welch, Proc. Soc. Exptl. Biol. Med. 35, 107 (1936).

14TH. Jukes, Proc. Am. Inst. Nutrition, J. Nutrition 21, Suppl. 13 (1941).
15 A.D. Welch, J. Biol. Chem. 137, 173 (1941).

16G. A. Maw and V. du Vigneaud, J. Biol. Chem. 176, 1029 (1948).

17H. J. Channon and J. A. B. Smith, Biochem. J. 30, 115 (1986).

18H. J. Channon, A. P. Platt, and J. A. B. Smith, Biochem. J. 31, 1736 (1987).
19C. H. Best and J. H. Ridout, Can. Med. Assoc. J. 39, 188 (1938).

20 A. P. Platt, Biochem. J. 33, 505 (1939).

21 A.D. Welch, cited by A. W. Moyer and V. du Vigneaud, ref. 1.

V. SPECIFICITY OF ACTION 47

TABLE IV

BroutocicaAL Activity OF METHIONINE, OF DIMETHYLTHETIN, AND OF SOME OTHER
S-ALKYL CoMPouUNDS

Prevention of

Renal Fatty Growth with

Compound Perosis lesion liver Fowl growth homocystine
Methionine —2 +4 23 =r
Methionine sulfoxide 24

‘ Homomethionine =
Ethionine
Dimethylthetin +10 10, 21 125
Methylethylthetin +10, 25 +25 +25
Diethylthetin —10, 25 —10, 25 oF
Dimethylpropiothetin +25 +25 o25
S-Methyleysteine 24, 26 Sue KE
S-Ethyleysteine 424
S-Methylthioglycolic acid —25 25
TABLE V

BroLtoGicaL AcTIvVITY OF BETAINE AND OF SOME OF [ts DERIVATIVES

Prevention of

Fatty Fowl Growth with

Compound Perosis Renal lesions liver growth homocystine

Betaine SE S10 YL 27 4.28 = a
+ 3

Arsenobetaine 10 2E8 ei
Phosphobetaine Silt =8
Triethylbetaine —22
Betaine aldehyde —3 10 +8 3, 29
Betaine aldehyde acetol +8
a-Alanine betaine =w23 10 =
B-Alanine betaine Toxic!
Cystine betaine 24 mat
Glutamic betaine == 21
Serine betaine TE 30 rant
Threonine betaine —30
Allothreonine betaine —30
Ergothioneine —31 Di]
Stachydrine aa
Trigonelline = ai

2 T. H. Jukes, J. Nutrition 22, 315 (1941).

223A. F. Tucker and H. C. Eckstein, J. Biol. Chem. 121, 479 (1937).

248. A. Singal and H. C. Eckstein, J. Biol. Chem. 140, 27 (1941).

25 G. A. Maw and V. du Vigneaud, J. Biol. Chem. 176, 1037 (1948).

26H. J. Channon, M. C. Manifold, and A. P. Platt, Biochem. J. 34, 866 (1940).
27 W. H. Griffith and D. J. Mulford, J. Am. Chem. Soc. 68, 929 (1941).

48 CHOLINE

other amino acids appear unable to serve as methyl donors (Table V).
Furthermore, sulfocholine is also negative in this respect, despite the great
activity of similar sulfur methyl groups in the thetins.

As far as is known, the attribute of being a methyl donor also confers
on a molecule the function of serving as a precursor of the methyl portion

TABLE VI
BroLogicaL AcTIVITY OF Vartous N-ALKYL AND RELATED CoMPOUNDS

Prevention of

: Fatty Fowl Growth with
Compound Perosis Renal lesions liver growth homocystine

Trimethylamine oxide —8
Trimethylammonium —32

chloride
Trimethylethylammonium Toxic’?

chloride
Tetramethylammonium Toxic®

chloride
Trimethylphenylammonium Toxic?

chloride
Tetra-6-hydroxyethylam- —20

monium chloride
Monomethylaminoethanol <4, 33 a, 33
Dimethylaminoethanol 5, 33 +10, 34 +10, 34 b. 334
Monomethylglycine — 35 =
Dimethylglycine — 34 — 34 =!
Dimethylglycine methyl all

ester
Creatine —22 —20 422 — 35
Creatinine — 35 =i
Caffeine —1 ="
Methanol —1 ="
Neurine Toxic!

* In presence of betaine and Bn.
In presence of Bis.

of choline. On the other hand, derivatives of choline are known that may
be used in the synthesis of lecithin but lack the methyl donor characteristic

28 C. H. Best and M. E. Huntsman, J. Physiol. (Lundon) 75, 405 (1932).

29'T. H. Jukes, cited by A. W. Moyer and V. du Vigneaud, ref. 1.

30 H. E. Carter and D. B. Melville, J. Biol. Chem. 188, 109 (1940).

31C. H. Best and J. H. Ridout, Ann. Rev. Biochem. 8, 349 (1939).

32M. E. H. Mawson and A. D. Welch, Biochem. J. 30, 417 (1936).

33 A. E. Schaefer, W. D. Salmon, and D. R. Strength, J. Nutrition 44, 305 (1951).

34.V. du Vigneaud, 8. Simmonds, J. P. Chandler, and M. Cohn, J. Biol. Chem. 165,
639 (1946).

35 V. du Vigneaud, J. P. Chandler, and A. W. Moyer, J. Biol. Chem. 189, 917 (1941).

a i

VI. BIOGENESIS 49

completely. In this category are arsenocholine, phosphocholine, and tri-
ethylcholine. Following the administration of the arsenic and phosphorus
analogs of choline, Welch!® noted lipotropic activity and, in the case of
arsenocholine, demonstrated its presence in tissue phospholipid.” ™ Similar
findings for triethylcholine were reported by McArthur.*®: * It is natural to
ascribe the lipotropic effect of these compounds to their corresponding
phospholipid forms, but the certainty of such an assumption remains to be
proved. Caution in this respect is required because of the possibility of
unrecognized biological properties of acetycholine or of other derivatives
of choline. That triethylcholine, for instance, may exhibit activity as an
antagonist of choline has been reported.*

VI. Biogenesis
WENDELL H. GRIFFITH and JOSEPH F. NYC

The biogenesis of choline in the animal organism, discussed in a. pre-
ceding section, depends on dietary sources of vitamins such as folic acid
and Biz: which may be required as cofactors in its enzymatic formation.
Limited synthesis occurs under advantageous dietary conditions in the rat
and chick, the two species used most commonly in choline studies. Severe
deficiencies result on diets low in labile methyl and in the vitamins in
question even though the diet may appear quite adequate in other respects.
On the other hand, unlimited synthesis characterizes the growth of most
microorganisms and plants.

Pneumococci': * and artificially produced mutant strains of Neurospora’: 4
are the only forms known to require external supplies of choline for normal
growth. Choline and homocystine can replace methionine for the growth
of L. casei? but not of H. colz.6 In preliminary experiments designed to
develop a microbiological assay for choline, Badger? found that amino-
ethanol would support the growth of a strain of pneumococcus in the
absence of choline. Other compounds which are related to choline and
aminoethanol were then tested for their activity in promoting growth of

36 ©. §. McArthur, Science 104, 222 (1946).

37 ©. §. McArthur and C. C. Lucas, Biochem. J. 46, 226 (1950).

38 A.S Keston and S. B. Wortis, Proc. Soc. Exptl. Biol. Med. 61, 439 (1946).

1. Rane and Y. SubbaRow, J. Biol. Chem. 134, 455 (1940).

2K. Badger, J. Bacteriol. 47, 509 (1944); J. Biol. Chem. 153, 183 (1944).

3N. H. Horowitz and G. W. Beadle, J. Biol. Chem. 150, 325 (1943).

4N.H. Horowitz, D. Bonner, and M. B. Houlahan, J. Biol. Chem. 159, 145 (1945).
5R. J. Evans, Arch. Biochem. 16, 357 (1948).

6M. N. Green and M. G. Sevag, Arch. Biochem. 9, 129 (1946).

50 CHOLINE

this organism. It was found that the active compounds contained N—C—
C—OH and N—C—C—C—OH linkages. Substitution of, or through, the
hydroxyl group resulted in complete inactivation of the molecule. Amino-
ethanol, monomethylaminoethanol, and dimethylaminoethanol were among
these substances supporting the growth of the organism. The activity of
triethylcholine, diethylaminoethanol, and similar compounds plus the in-
activity of betaine, methionine, and other compounds containing labile
methyl groups indicated that the role of choline in pneumococcal metabo-
lism is not that of transmethylation. The fact that amimoethanol is required
in ten times the concentration of choline for equivalent growth suggested
that choline is not demethylated to give ethanolamine.

In contrast to the pneumococcus, two genetically different strains of
Neurospora crassa are unable to utilize aminoethanol in place of choline.‘
These mutants are designated as strain 34486, or cholineless-1, and strain
47904, or cholineless-2. Each strain is believed to carry a mutation of a
different single gene concerned with the synthesis of choline,* and each
strain attains a growth rate comparable to that of the wild type in choline-
supplemented media. The following compounds show some activity for both
mutants: choline, dimethylaminoethanol, monomethylaminoethanol, ace-
tylcholine, arsenocholine, phosphorylcholine, dimethylethylhydroxyethyl-
ammonium chloride, diethylmethylhydroxyethylammonium chloride, tri-
ethylcholine, and methionine. The following compounds are inactive for
both mutants: aminoethanol, betaine, creatine, sarcosine, neurine, diethyl-
aminoethanol, dimethylamine, trimethylamine, and tetramethylammonium
chloride.*: 4:7: § The work of Horowitz? showed that an inherent difference
exists between the two cholineless mutants of Neurospora in their ability
to utilize monomethylaminoethanol and dimethylaminoethanol. The results
suggested that the gene-controlled deficiency in cholineless-2 may be con-
cerned with the methylation of a mono- or dimethylated precursor of
choline, whereas that in cholineless-1 blocks a prior step in the synthesis.
It was observed that cholineless-2 produces a substance which is imactive
for itself but which promotes the growth of cholineless-1. The substance
has been isolated from the cultures of cholineless-2 and identified as mono-
methylaminoethanol. This substance is therefore a normal intermediate in
the synthesis of choline in Neurospora. According to Horowitz, in choline-
less-1 the genetic block precedes monomethylaminoethanol, so that the
mutant cannot synthesize this intermediate but can utilize it for choline
synthesis if an exogenous supply is available. In cholineless-2 a partial
block exists between monomethylaminoethanol and choline. This mutant

7T. H. Jukes, A. C. Dornbush, and J. J. Oleson, Federation Proc. 4, 157 (1945).
8 T. H. Jukes and A. C. Dornbush, Proc. Soc. Exptl. Biol. Med. 58, 142 (1945).
9N. H. Horowitz, J. Biol. Chem. 162, 413 (1946).

VI. BIOGENESIS 51

can synthesize monomethylaminoethanol but is unable to convert it to
choline at the normal rate; as a result, the intermediate accumulates in the
culture. Hypothetical intermediates are bracketed, and vertical dotted
lines indicate points of blocking in the following scheme proposed by
Horowitz.

| : | |

ex. |= bee - a (GSI 5 : — | CH, — Choline
| | : | |

CH, CH, CH,

| cholineless-1 | cholineless-2 |

OH. OH OH

Challenger and coworkers!’-” showed that trimethylarsine is one of the
volatile odorous products evolved from mold cultures containing arsenious
acid. The mold Scopulariopsis brevicaulis not only methylates arsenite but
also converts selenite™ and tellurite“ to the respective dimethyl derivatives.
Challenger and Higginbottom!® suggested that choline or betaine might
supply methyl for these methylations. A means of separation and identifi-
cation of aminoethanol, devised by Simons,'° failed to show an accumula-
tion of this possible demethylation product of choline in a culture of S.
brevicaulis containing added choline and arsenite. No evidence was ob-
tained for the methylation of sulfur compounds by this organism except
by fission of dialkyl disulfides.!* !7 However, dimethyl sulfide was isolated
as a product of the fungus Schizophyllum commune Fr.!8 Smith and Schlenk
observed that thiomethyladenosine accumulated in yeast grown in a me-
dium containing an excess of methionine but not of sulfate, homocystine,
cysteine, or glutathione. The nucleoside was used by the yeast as a source
of sulfur if added to a deficient medium.'®

Although the Neurospora studies suggest similarity in the biogenesis of
choline between animals and microorganisms, there is little evidence that
degradation of choline proceeds along the same pathways. Trimethylamine
is the usual product, and its formation has been noted in choline-containing

10 F. Challenger and C. Higginbottom, Biochem. J. 29, 1757 (1935).

11 Ff. Challenger, C. Higginbottom, and L. Ellis, J. Chem. Soc. 1933, 95.

122. Challenger and A. A. Rawlings, J. Chem. Soc. 1936, 264.

13 F, Challenger and H. E. North, J. Chem. Soc. 1934, 68.

14M. L. Bird and F. Challenger, J. Chem. Soc. 1939, 163; 1942, 571, 574.

15 C, Simons, Biochem. J. 35, 749 (1941).

16. Challenger and 8. Blackburn, J. Chem. Soc. 1938, 1872.

17 F. Challenger and P. T. Charlton, J. Chem. Soc. 1947, 424.

18 J. H. Birkinshaw, W. P. K. Findlay, and R. A. Webb, Biochem. J. 36, 526 (1942).

19R. L. Smith and F. Schlenk, Federation Proc. 11, 289 (1952); Arch. Biochem. and
Biophys. 38, 159, 167 (1952).

Sy CHOLINE

cultures of Bacillus prodigiosus,?® Shigella alkalescens,! Proteus vulgaris,”
and many other organisms.

There is a wide distribution of choline phospholipids throughout the
plant world. Ducet”*: *4 examined seedlings of soybean, barley, and pea and
other young plants and concluded that synthesis of choline occurs in
rapidly growing tissues, such as leaf buds. He believed that glycerylphos-
phorylcholine is an intermediate in the synthesis of lecithin. The occur-
rence of small amounts of free choline was ascribed to the presence of
lecithinase.

VII. Estimation
WENDELL H. GRIFFITH and JOSEPH F. NYC

A. CHEMICAL PROCEDURES

Chemical procedures for the quantitative determination of choline in
biological materials have been extensively treated in various publications.!~4
The method based on the precipitation of choline as the periodide has been
one of the preferred chemical procedures. Earliest studies on the precipita-
tion of choline periodide were made by Stanék,°® who proposed the use of
potassium triiodide as the reagent for the quantitative precipitation of
choline. In 1923, Sharpe® published a quantitative chemical method, based
on the original work of Stanék, in which choline was precipitated with
iodine as the periodide which was washed free of excess reagent with ice-
cold water and decomposed with dilute nitric acid. The liberated iodine
was extracted with chloroform and estimated using a standard solution of
sodium thiosulfate.

Roman’ and Maxim’ investigated the original Stanék method and, as a

20D. Ackermann and H. Schiitze, Zentr. Physiol. 24, 210 (1910).

21 A. J. Wood and F. E. Keeping, J. Bacteriol. 47, 309 (1944).

22 G. N. Cohen, B. Nisman, and M. Raynaud, Compt. rend. 225, 647 (1947).

23 G. Ducet, Compt. rend. 227, 871 (1948).

24G. Ducet, Ann. agron. 19, 184 (1949).

1C. H. Best and C. C. Lucas, Vitamins and Hormones 1, 1 (1948).

2 P. Handler, Biol. Symposia 12, 361 (1947).

3 P. Gyorgy and 8. H. Rubin, in Vitamin Methods, Vol. 1, p. 243. Academic Press,
New York, 1950.

4 Association of Vitamin Chemists, Inc., Methods of Vitamin Assay, 2nd ed., p.
287. Interscience Publishers, New York, 1951.

5V. Stanék, Hoppe-Seyler’s Z. physiol. Chem. 46, 280 (1905); 47, 83 (1906); 48, 334
(1906).

6 J. S. Sharpe, Biochem. J. 17, 41 (1923).

7™W. Roman, Biochem. Z. 219, 218 (1930).

8M. Maxim, Biochem. Z. 239, 138 (1931).

VII. ESTIMATION 53

result of their findings, described the first microchemical method for the
estimation of choline. Roman obtained reproducible results within a range
of 5 y to 5 mg. of choline with a maximum error of 5%.

Erickson and her collaborators? improved the periodide micromethod of
Roman by using immersion filter sticks which facilitated washing the labile
choline iodide precipitate with minimum disturbance. The introduction of
this step in the procedure was desirable because the greatest source of
error in the periodide method was the volatile and unstable nature of the
choline precipitate. A further improvement was the introduction of bromine
oxidation to convert the iodine to iodate preceding titration with standard
sodium thiosulfate. The latter modification offered the advantages of ready
solubility of the precipitate in the bromine solution and a sixfold increase
in the final titration value.

Potassium periodide yields precipitates with dimethylamine, trimethyl-
amine, betaines, certain cyclic bases, and many alkaloids.!? The presence
of these substances in plants makes it impossible to relate the results ob-
tained by the periodide method to a definite constituent in a plant extract.
Reifer!® claimed that the greatest part, if not all, of the periodide-precipi-
table substances in plant extracts, with the exception of choline, were
removed by lead acetate and Norit.

At the present time the most widely used method for the quantitative
determination of choline appears to be by precipitation as reineckate.
Among the first to employ the reineckate method for the determination of
choline were Kapfhammer and Bischoff,!! who washed the choline reineck-
ate with ether and determined it gravimetrically. This method required
fairly large amounts of choline and, therefore, was not easily adaptable
for general use. A more sensitive method described by Beattie” consisted
of the precipitation of choline with a freshly prepared solution of ammo-
nium reineckate, solution of the precipitate in acetone, and comparison of
the bright red color imparted to the solvent with that of a standard solu-
tion. Using a visual colorimeter, quantities of the order of 0.3 mg. of choline
in a concentration of 0.003 % were estimated by this procedure with an
error of not more than 3%. The use of photoelectric colorimetry greatly
increased the sensitivity of the method.!*-!® According to the procedure of
Jacobi eé al.,!° the ground sample was extracted with boiling 1:1 alcohol-

2B. N. Erickson, I. Avrin, D. M. Teague, and H. H. Williams, J. Biol. Chem. 135,
671 (1940).

10 T. Reifer, New Zealand J. Sci. Tech. 22B, iii (1941).

11 J. Kapfhammer and C. Bischoff, Hoppe-Seyler’s Z. physiol. Chem. 191, 179 (1930).

2 EJ. R. Beattie, Biochem. J. 30, 1554 (1936).

13. W. Engel, J. Biol. Chem. 144, 701 (1942).

1447). Glick, J. Biol. Chem. 156, 643 (1944).

15H. P. Jacobi, C. A. Baumann, and W. J. Meek, J. Biol. Chem. 138, 571 (1941).

16M. H. Thornton and F. K. Broome, Ind. Eng. Chem. Anal. Ed. 14, 39 (1942).

54 CHOLINE

ether mixture and the residue of this extract was saponified for 2 hours at
80° with baryta. The choline, precipitated as the reineckate, was dissolved
in acetone and the color intensity was measured, using a light filter which
transmits at 520 mu.

Engel advised more exhaustive extraction of biological samples with
methanol and hydrolysis of extracts with baryta for 2 hours at 100° rather
than at 80°, as recommended by Jacobi et al. Glick" further improved the
reineckate method by showing that interference due to many compounds
which form insoluble reineckates is circumvented by precipitating the
choline reineckate in an alkaline medium. Propanol was found to be the
most suitable solvent for washing the reineckate. The maximum light
absorption for choline reineckate, as determined spectrophotometrically by
Glick, is near 526 mu. Glick’s reineckate method and the more recently
published modification by Willstaedt et al.” are in wide use today.

The development of a brown color, by adding an iodine reagent to an
aqueous solution of choline reineckate, was used as the basis of a method
by Shaw.'8 Still another modification of the reineckate method was proposed
by Marenzi and Cardini.!* It is based on the oxidation of the chromium of
choline reineckate to the chromic state by means of alkaline hydrogen
peroxide, followed by colorimetric determination of the chromate by means
of the violet-red color produced in an acid solution with diphenylearbazide
(Cazeneuve’s reaction). The authors claim that this method will allow
the estimation of as little as 15 y of choline. Winzler and Meserve?? in-
creased the sensitivity of the reineckate method by making use of the very
great absorption of acetone solutions of choline reineckate at 327 my. This
method has an accuracy of +5 % with samples containing 50 to 400 y of
choline hydrochloride.

Lintzell and Fomin?! introduced a new micromethod for choline estima-
tion based on the oxidative degradation of choline to trimethylamine and
titration of the latter. Neither the original method nor the modifications
of it??» % appears to have gained general acceptance.

Less well-known methods for the determination of choline are those of
Ambo and Aoki’ and of Eagle,?® who based their procedures on the volu-
metric determination of mercury in the mercuric salt of choline. A simple

17H. Willstaedt, M. Borggard, and H. Lieck, Z. Vitaminforsch. 18, 25 (1946).
18 F. H. Shaw, Biochem. J. 32, 1002 (1938).

19 A.D. Marenzi and C. FE. Cardini, J. Biol. Chem. 147, 363 (1943).

20R. J. Winzler and E. R. Meserve, J. Biol. Chem. 159, 395 (1945).

21W. Lintzel and S. Fomin, Biochem. Z. 238, 438 (1931).

22 G. Klein and H. Linser, Biochem. Z. 250, 220 (1932).

23 W. Lintzel and G. Monasterio, Biochem. Z. 241, 273 (1931).

24H. Ambo and T. Aoki, Trans. Japan. Pathol. Soc. 21, 171 (1931).

5K. Eagle, J. Lab. Clin. Med. 27, 103 (1941).

VII. ESTIMATION 59

gravimetric procedure for the assay of choline chloride in pharmaceutical
products, based on the precipitation of choline from absolute ethanol with
phosphotungstic acid, has been described by Gakenheimer and Reguera.?®
A recent gravimetric method” consists of precipitating the cadmium chlo-
ride complex of choline from an alcoholic solution. This method can be
used for the assay of pharmaceutical products but has not been studied
with biological] samples.

B. MICROBIOLOGICAL PROCEDURES

A microbiological method for the determination of choline by the use of
a mutant of Newrospora crassa was described by Horowitz and Beadle.?*
The mutant (34486) had been produced by ultraviolet irradiation of a
normal “wild type” strain. As a result of the induced mutation this organ-
ism fails to grow on an unsupplemented basal medium but grows if choline
is supplied. Combined choline, as in lecithin, is also utilized by the test
organism, but Jess efficiently than free choline. For this reason the choline
in samples must be liberated by hydrolysis prior to assay. Methionine has
a sparing action on choline and, when present in appreciable amounts,
must be removed from the sample. Jukes and Dornbush?’ have shown that
dimethylaminoethanol is also active for the mutant.

A second cholineless strain (47904) which differs genetically from strain
34486, was described by Horowitz et al.,*°: *! and this mutant may also be
used in the bioassay of choline. The growth of both cholineless mutants is
stimulated by such compounds as acetylcholine, phosphorylcholine, mono-
methylaminoethanol and dimethylaminoethanol. The following compounds
were found to be inactive for both mutants: betaine, creatine, sarcosine,
aminoethanol, neurine, diethylaminoethanol, dimethylamine, trimethyl-
amine, and tetramethylammonium chloride.

The procedure for the Neurospora assay has been described in several
texts.2: * According to the original method?’ the sample for assay is
autoclaved with 3% sulfuric acid for 2 hours at 15 pounds pressure to
liberate choline from its combined forms, and the solution is neutralized
with barium hydroxide. The solution is passed through a column containing
Permutit in order to separate the choline from methionine, and the ab-

26 W. C. Gakenheimer and R. M. Reguera, J. Am. Pharm. Assoc. Sct. Ed. 35, 311
(1946).

27 W. Seaman, J. J. Hugonet, and W. Leibmann, Anal. Chem. 21, 411 (1949).

28 N,. H. Horowitz and G. W. Beadle, J. Biol. Chem. 150, 325 (1943).

29'T. H. Jukes and A. C. Dornbush, Proc. Soc. Exptl. Biol. Med. 58, 142 (1945).

30 N. H. Horowitz, D. Bonner, and M. B. Houlahan, J. Biol. Chem. 189, 145 (1945).

31 N. H. Horowitz, J. Biol. Chem. 162, 413 (1946).

#2 B.C. Johnson, Methods of Vitamin Determination, p. 95. Burgess Publishing Co.,
Minneapolis, 1948.

56 CHOLINE

sorbed choline is then eluted from the column with 5% sodium chloride.
Aliquots of the solution and of standard choline solutions are added to
flasks containing the basal medium, and these are inoculated and incubated
for about 3 days at 25°. The response of the mold to the added supplements
is determined by weighing the dried mycelium. Siegel** has recommended
the removal of the mycelium from the culture fluid by filtration through
a tared, sintered glass filter of medium porosity which is then dried and
weighed. Dry weights from duplicate flasks agree within about 5%, and
choline values determined on different amounts of the same solution agree
within 10%. Standard curves must be run with each assay.

Luecke and Pearson*! have used the method for the determination of
free choline in plasma and urine and for the estimation of free choline in
animal tissues. Comparative determinations on the same samples showed
that the microbiological values were in excellent agreement with those
obtained by chemical methods. Hodson** used a modification of the above
procedure in the assay of milk products. The results obtained with the
Neurospora procedure indicate that this method is considerably more sensi-
tive and more specific than the chemical methods investigated to date.**®
The various analogs that support growth of the organism do not appear in
appreciable quantities in most biological materials or, as in the case of
methionine, can be separated by use of adsorbents.

Special attention has been given the determination of choline-containing
phospholipids in blood, liver, and other tissues by workers in the labora-
tories of Thannhauser,*7-9 Chaikoff,*°-*? Chargaff,*: “4 and McKibbin.**: 4

3 T,. Siegel, Sczence 101, 674 (1945).

84 R. W. Luecke and P. B. Pearson, J. Biol. Chem. 153, 259 (1944); 155, 507 (1944).

35 A. Z. Hodson, J. Biol. Chem. 157, 383 (1945).

36 Ff. J. Bandelin, J. Am. Pharm. Assoc. 38, 304 (1949).

37 G. Schmidt, L. Hecht, P. Fallot, L. Greenbaum, and S. J. Thannhauser, J. Biol.
Chem. 197, 601 (1952).

38§. J. Thannhauser, J. Benotti, and H. Reinstein, J. Biol. Chem. 129, 709 (1939).

39§. J. Thannhauser, J. Benotti, A. Walcott, and H. Reinstein, J. Biol. Chem. 129,
717, (1939).

40 C, Entenman, A. Taurog, and I. L. Chaikoff, J. Biol. Chem. 155, 13 (1944).

41 A. Taurog, C. Entenman, B. A. Fries, and I. L. Chaikoff, J. Biol. Chem. 155, 19
(1944).

42 ©, Entenman and I. L. Chaikoff, J. Biol. Chem. 160, 377 (1945).

43}. Chargaff, M. Ziff, and D. Rittenberg, J. Biol. Chem. 138, 439 (1941); 144, 343
(1942).

44 F. Chargaff, C. Levine, and C. Green, J. Biol. Chem. 175, 67 (1948).

45 J. M. McKibbin and W. E. Taylor, J. Biol. Chem. 178, 17, 29 (1949).

46 W. E. Taylor and J. M. McKibbin, J. Biol. Chem. 188, 677 (1951).

~J

VII. ESTIMATION

Kahane and Lévy worked out procedures for the estimation of free choline,
total water-soluble choline, and total choline.*7-*9

C. BIOLOGICAL ASSAYS

The first determination of choline by biological assay was based on its
acetylation to acetylcholine and on the estimation of the latter by virtue of
its pharmacological action on tissues. An extensive discussion of this pro-
cedure has been published by Chang and Gaddum.°*? The most satisfactory
tissues for the test are the rectis abdominis muscle of the frog, the isolated
intestine of the rabbit, and the longitudinal muscle of the leech. The
method was used by Abdon®*! for the determination of acetylcholine and
its precursor in tissues and by Fletcher e¢ al.®? in the analysis of a number
of foodstuffs for total choline. Accuracy is difficult because of interfering
substances such as histamine, potassium salts, and many other physio-
logically active constituents of tissues.

Attempts have been made to develop a biological assay procedure for
the determination of the total choline in foods and in other biological
materials by comparison of the degree of prevention of renal pathology by
the test substance and by choline in young rats on a choline-deficient
diet." *§ The method is not particularly sensitive and the interpretation
of results is complicated by variations in food consumption and in the
composition of the ration.

D. PHYSICAL ESTIMATION

“The ultraviolet absorption spectrum of choline*!: ®° is not useful as an
analytical tool although advantage is taken of the spectrum of choline
reineckate. It has not been possible to utilize other physical properties of
choline in its estimation.

47 G. Ducet and E. Kahane, Bull. soc. chim. biol. 28, 794 (1946).

48H. Kahane and J. Lévy, Compt. rend. 207, 642 (1938).

49 EF, Kahane, J. Lévy, and O. Libert, Bull. soc. chim. biol. 27, 65 (1945).

60 H. C. Chang and J. H. Gaddum, J. Physiol. (London) 79, 255 (1933).

51N. O. Abdon and K. Ljungdahl-Ostberg, Acta Physiol. Scand. 8, 103 (1944).
52 J. D. Fletcher, C. H. Best, and O. M. Solandt, Biochem. J. 29, 2278 (1935).
53 W. H. Griffith and D. J. Mulford, J. Am. Chem. Soc. 68, 929 (1941).

54 A. Castille and M. Ruppal, Bull. acad. roy. méd. Belg. 56, 263 (1926).

56 W. Graubner, Z. ges. exptl. Med. 63, 527 (1928).

58 CHOLINE

VIII. Standardization of Activity

WENDELL H. GRIFFITH and JOSEPH F. NYC

Choline is a fairly stable chemical that participates directly and in-
directly in metabolic reactions. The factors that control its synthesis by
transmethylation and by formate-to-methyl synthesis are not known defi-
nitely. Standardization of activity in the usual sense is, therefore, not
feasible.

IX. Occurrence
WENDELL H. GRIFFITH and JOSEPH F. NYC

The total choline present in typical foods (Tables VII and VIII) has
been determined by Engel,! and the amount in common meat cuts (Table
IX) by MeIntire ef al.2 Grains and milled wheat products were also ana-
lyzed by Glick*® (Table X). The examination of foodstuffs in India* and in
Sweden® has given values similar to those in the tables. Egg yolk, glandular
meats, and brain are the richest animal sources, and the germ of cereals,
legumes, and seed-oil meals are the best plant sources. Of interest is the
relatively high level in patent flour.

The choline content of fresh and of processed milk has been studied in
several laboratories.°* According to Hodson® little loss occurs in the prepa-
ration of dried milk, although Marquez’ reported that in both raw and
pasteurized milk no choline was present after standing 48 hours at room
temperature. Most of the choline of milk is found in the aqueous rather
than the cream layer.®

Sadhu’ noted a slight positive correlation between the choline content
of the brain and the lactose content of the milk of nine species of mammals
whereas a high correlation was found between the lactose level and the
brain cerebrosides. He suggested that sphingosine can react reversibly

1R. W. Engel, J. Nutrition 25, 441 (1948).

2 J. M. McIntire, B.S. Schweigert, and C. A. Elvehjem, J. Nutrition 28, 219 (1944).

3D. Glick, Cereal Chem. 22, 95 (1945).

4H. Chattopadhyay and 8. Banerjee, Food Research 16, 230 (1591).

5 N. E. Borglin, Acta Pharmakol. Toxicol. 3, Swppl. 1 (1947).

6 A. Z. Hodson, J. Nutrition 29, 137 (1945).

7V.M. Marquez, School Hyg. and Publ. Health, Biochem. Dept., Johns Hopkins
University (Separate), March 1942.

8K. Kahane and J. Lévy, Bull. soc. chim. biol. 27, 72 (1945); Compt. rend. 220, 97
(1945).

9D. P. Sadhu, J. Dairy Sct. 31, 347 (1948).

59

NCE

=,
wy)

OCCURRE

IX.

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IX. OCCURRENCE 61

with galactose or choline phosphoric acid to form either cerebrosides or
sphingomyelins.
The choline content of various biological fluids has been reported by

TABLE Ix
ToraL CHOLINE CONTENT OF Meats?

Choline, mg./100 g.

Fresh Dry
Sample Range Avg. Range Avg.
Veal
Leg 95-108 102 366-4382 389
Roast leg 125-141 132 388-392 360
Shoulder 83-100 93 268-373 337
Roast shoulder 133-1438 139 310-376 343
Sirloin chop 87-105 96 242-404 317
Braised chop 128-157 140 242-342 285
Shoulder chop 92-101 97 307-422 376
Braised chop 149-156 154 317-400 366
Stew meat 94-100 96 336-400 367
Cooked stew 137-149 142 360-3878 370
Lamb
Leg 75— 92 84 262-317 290
Roast 122-124 123 284-295 290
Sirloin chop (ie 76 179-198 189
Broiled chop 100-126 113 204-252 228
Stew meat 76-— 82 79 222-230 226
Cooked stew 116-128 122 247-291 269
Pork
Ham 101-129 120
Cured ham 98-129 122
Beef
Liver 470-570 510
Round ; 65-— 70 68
Tongue 108 108
Heart 170 170
Braised heart 200-275 238
Kidney 240-284 262
Brain 399-420 410
Miscellaneous
Bologna 60 60
Frankfurters 57 57
Pork links 48 48

Canadian bacon 80 80

62 CHOLINE

Eagle! (Table XI), and Schmidt et al.!! have recorded data on the occur-
rence of glycerylphosphorylcholine in tissues (Table IT).

TABI) xX
TotraL CHOLINE CONTENT OF GRAINS AND OF GRAIN PRODUCTS
(expressed as milligrams of choline chloride per 100 g. on a moisture-free basis)*

Variety Milled fractions

Hard spring wheat 91 Whole wheat 102
Hard winter wheat 79 Germ 354
Soft winter wheat 88 Bran 153
Soybeans Dern Low-grade flour 69
Oats 114 Bleached low-grade flour 69
Barley 110 Clear flour 61
Flax 107 Bleached clear flour 62

Patent flour 70

Bleached patent flour 69

TABLE Xi
FREE CHOLINE IN BroLoGicaL Fuurps?!®

Choline, mg./l.

Material No. of samples Average Range
Human saliva, male 12 11.6 8.6— 14.4
Human saliva, male? il Guo 4.7— 9.9
Human saliva, female? 75 16.9 6.2— 36.4
Human blood, male¢ 8 35.2 10.3- 17.1
Human blood, male? 10 13.5 8.5— 22.8
Human blood, female? 19 14.6 9.4 21.8
Human blood, pregnancy 62 13.1 9.9- 19.9
Human semen 17 341.0 234 .0-477 .0
Bladder bile, dog it 250.0 78 .0-380.0
Lymph, dog 3 18.2 14.2— 23.5

Collected while chewing paraffin.
Collected by use of dental siphon.
Normal.

Outpatients.

aoaewa

X. Effects of Deficiency
A. GENERAL MANIFESTATIONS
WENDELL H. GRIFFITH and JOSEPH F. NYC

The specific recognition of sources of labile methyl is accomplished by
the zn vivo administration of a suitably labeled, possible methyl donor or
10K. Eagle, J. Lab. Clin. Med. 27, 103 (1941).

1G, Schmidt, L. Hecht, P. Fallot, L. Greenbaum, and S. J. Thannhauser, J. Biol.
Chem. 197, 601 (1952).

X. EFFECTS OF DEFICIENCY 63

by its addition to an in vitro system followed by the isolation of methyl-
containing products and estimation of the isotopic carbon and hydrogen.
More general procedures include measurements of effectiveness in the
prevention of fatty livers and of renal lesions in rats, of growth of rats on
homocystine-containing, methionine-poor diets, of stimulation of growth
and prevention of perosis in chicks or turkey poults, and of extent of con-
version of homocysteine and of guanidoacetic acid to methionine and
creatine, respectively, in in vitro tissue systems. Choline, with at least one
labile methyl group after oxidation to betaine and with one or more carbons
that may participate in formate-to-methyl synthesis under the proper
conditions, reacts positively in each of these procedures. It is particularly
effective in reactions in which it serves directly, presumably as a constitu-
ent of phospholipids or of acetylcholine, rather than as a source of methyl.
This appears to be the case in so far as its lipotropie activity is concerned
and, possibly, its antihemorrhagic and antiperotic activities also. Obvi-
ously, it is not always possible to distinguish with certainty between evi-
dences of a deficiency of methyl and of choline in experiments on animals.
It is important, nevertheless, that deficiency symptoms, prevented by
choline, may be produced without difficulty in several animal species.

Be eA

WENDELL.H. GRIFFITH and JOSEPH F. NYC
1. RENAL LESION

The rat has been the principal test animal in investigations of choline.
The recognition of the lipotropic action of choline in adult rats by Best
has been described earlier (Section IV C 2). The importance of this sub-
stance in the nutrition of the young rat was demonstrated by Griffith c¢ al.,
who showed that choline was essential in weanling rats not only for the
prevention of the fatty liver but also for the maintenance of tissue structure
and even for survival.!4

The feeding of low choline diets to male rats, 20 to 26 days of age, results
in a spectacular series of effects which reach a crisis within 6 to 8 days.
This critical period may terminate fatally, or it may be followed by :
rapid, partial recovery. Recovery on the same diet that produces severe
degeneration of tissues is highly interesting and as yet an unexplained
phenomenon. A marked increase in liver fat occurs within 48 hours and is
maximal after 4 to 6 days. Renal lesions, named hemorrhagic degeneration,
develop between the sixth and eighth days; the animals become noticeably

1W. H. Griffith and N. J. Wade, Proc. Soc. Exptl. Biol. Med. 41, 188 (1939).
2W.H. Griffith and N. J. Wade, J. Biol. Chem. 181, 567 (1939).

3W.H. Griffith, J. Nutrition 19, 437 (1940).

‘W.H. Griffith, Biol. Symposia 5, 193 (1941).

64 CHOLINE

sick, and there is a marked elevation of blood non-protein nitrogen (Fig. 2).
Severely affected rats show an extensive regression of the thymus, and

Fic. 2. Effects of choline deficiency in weanling rats after an 8-day period with
choline (center) and without choline. The photograph illustrates the enlarged yellow-
ish fatty liver, the enlarged kidneys with hemorrhagic degeneration, and the partial
regression of the thymus. The sectioned kidneys above the deficient animals em-
phasize the cortical congestion which is evident in Fig. 3 and which, with or without
subcapsular hemorrhage, is responsible for the dark-red appearance of the kidneys.

ocular hemorrhage is frequently observed. The renal changes develop over
a 24- to 48-hour period and are characterized grossly by an increase in size
and weight and by hemorrhagic discoloration. Proteinuria but not hemat-
uria occurs. Recovery is noteworthy because of the rapidity with which
the renal function improves, evident also in the gross appearance of the
kidneys. The recovered kidneys are enlarged and pale brown in color and

X. EFFECTS OF DEFICIENCY 65

are frequently rough and scarred with a white incrustation, the so-called
frosted kidney. hes

Fic. 3. Section from cortex of kidney of a choline-deficient rat similar to those
illustrated in Fig. 2. Prominent changes include vascular congestion and tubular
degeneration.

Christensen has described the course of the involution of the thymus?
and the renal pathology® which is summarized as follows (Fig. 3). The

5K. Christensen and W. H. Griffith, Endocrinology 30, 574 (1942).
6K. Christensen, Arch. Pathol. 34, 633 (1942).

66 CHOLINE

primary vascular change during the acute stage is congestion of the periph-
eral cortical capillaries and capsular blood vessels and, sometimes, of the
glomerular blood vessels; hemorrhage, if present, occurs only in the capsule
and at the periphery of the cortex; peripheral cortical tubules undergo
necrosis or granular or hyaline droplet degeneration; hyaline casts are
always abundant in tubules of the inner zone of the cortex and of the outer
zone of the medulla; and calcification of degenerated peripheral tubules
occurs in the most severely affected kidneys.

Hartroft and Best7-!° have emphasized the role in this syndrome of small
droplets of stainable fat which appear in the epithelial cells of the proximal
convoluted tubules of the cortex. Concomitantly, generalized swelling of
the affected nephrons occurs, with obstruction of the intervening capillary
plexus in the cortex. Tubular ischemia and necrosis are followed by en-
gorgement of the proximal cortical vessels which may rupture into and
beneath the capsule. Choline feeding has relatively little effect on the total
renal lipids.!! That the acute renal lesion is an effect of a deficiency of choline
has been repeatedly confirmed’!®. Conditions for producing a uniformly
fatal outcome were described by Engel'*: ”. Handler showed that the lesion
occurred in adult rats after unilateral nephrectomy if the animals were
placed immediately on the deficient diet. The observations of Olsen and
Deane!® included changes in the adrenal cortex which is enlarged during the
acute phase with definite evidence of hyperactivity of the zona fasciculata
and zona glomerulosa, especially the latter.

The prevention of hemorrhagic degeneration is relatively specific for
choline, although methyl-sparing compounds, such as methionine or beta-
ine, and certain other compounds are effective. Substances which resemble
choline sufficiently to be built by anabolic processes into lecithin-like mole-
cules without obviously serving as inhibitors, such as arsenocholine or
triethylcholine, have more or less activity.8?° It may be that certain of
these compounds will be found later not to be antihemorrhagic. The onset

7™W.S. Hartroft and C. H. Best, Sczence 105, 315 (1947).
8W.S. Hartroft, Brit. J. Exptl. Pathol. 29, 483 (1948).
9C. H. Best and W. 8S. Hartroft, Federation Proc. 8, 610 (1949).
10W.S. Hartroft, Proc. 1949 Milbank Mem. Fund p. 144 (1950).
1G. Gualandi and 8. M. Tamburello, Boll. soc. ital. biol. sper. 26, 945 (1950).
122 P. Gyorgy and H. Goldblatt, J. Exptl. Med. 72, 1 (1940).
13R. W. Engel and W. D. Salmon, J. Nutrition 22, 109 (1941).
144P. Handler, J. Nutrition 31, 621 (1946).
15 F. IT. Dessau and J. J. Oleson, Proc. Soc. Exptl. Biol. Med. 64, 278 (1947).
16 R. E. Olson and H. W. Deane, J. Nutrition 39, 31 (1949).
17R. W. Engel, Proc. Soc. Exptl. Biol. Med. 50, 193 (1942).
-18 A.D. Welch, J. Nutrition 40, 113 (1950).
19 A. D. Welch, cited by T. H. Jukes, Proc. Am. Inst. Nutrition, J. Nutrition 21,
Suppl. 13 (1941).
20 J. M. Patterson and E. W. McHenry, J. Biol. Chem. 145, 207 (1942).

X. EFFECTS OF DEFICIENCY 67

of this syndrome, as is the magnitude of fatty infiltration, is influenced by
the intake of food and, in particular, by the adequacy of the caloric intake.”!
MeArthur and Lucas emphasized the inhibitory effect of triethylcholine on
appetite,” and Strength et al. suggested that this compound was not only
not a substitute for choline but that it functioned as an antimetabolite.”
No obvious explanation can be given for the protective action of atabrine
against hemorrhagic degeneration,”* unless it spares choline by inhibition
of choline oxidase.”*

Results do not permit an unequivocal conclusion regarding the mech-
anism of this choline effect in the kidney of the rat. Jacobi et al. were not
able to demonstrate any lack of choline in affected kidneys,”*: 7 but Pat-
terson and McHenry found a significant decrease in renal phospholipids.?8: 2°
The lesion has been reported to be associated with a low activity of alkaline
and acid phosphatase in the renal tubules.*°

Hemorrhagic manifestations in weanling rats on low choline diets have
been described in the eyes,” 4’ *- *! heart muscle,’ liver,®? adrenals,” and
brain,®* as well as in the kidneys. The observation by Jervis® that hemor-
rhagic lesions of the cerebellum occurred in young rats of mothers fed a
low choline diet after the eighth day of pregnancy followed the report of
Gyorgy and Goldblatt that the young of mothers on choline-deficient diets
developed a fatal paralysis.” This finding cannot be ascribed to changes in
the brain phospholipids, according to Foa et al.

2. Farry LIver

MacLean and Best described the histological picture of the rat liver
loaded with fat because of choline deficiency,** and this has been amplified
by Hartroft*®: *7 with particular reference to the development of experi-

212—. J. Mulford and W. H. Griffith, J. Nutrition 28, 91 (1942).

22 C.S. McArthur and C. C. Lucas, Biochem. J. 46, 226 (1950).

2312). R. Strength, E. A. Schaefer, and W. D. Salmon, J. Nutrition 45, 329 (1951).

241). M. Hegsted, J. M. McKibbin, and F. J. Stare, J. Nutrition 27, 149 (1944).

25 B. Kelley, Federation Proc. 11, 238 (1952).

26 H. P. Jacobi, C. A. Baumann, and W. J. Meek, J. Biol. Chem. 188, 571 (1941).

27H. P. Jacobi and C. A. Baumann, J. Biol. Chem. 142, 65 (1942).

28 J. M. Patterson, N. B. Keevil, and EK. W. McHenry, J. Biol. Chem. 158, 489 (1944).

29 J. M. Patterson and BE. W. McHenry, J. Biol. Chem. 156, 265 (1944).

30 M. Wachstein, Arch. Pathol. 38, 297 (1944).

31 J. G. Bellows and H. Chinn, Arch. Ophthalmol. (Chicago) 30, 105 (1943).

32 P. Gyérgy, Ann. N.Y. Acad. Sci. 49, 525 (1948).

33 G. A. Jervis, Proc. Soc. Exptl. Biol. Med. 51, 193 (1942).

34 P. P. Foa, H. R. Weinstein, and B. Kleppel, Arch. Biochem. 19, 209 (1948).

35D. L. MacLean and C. H. Best, Brit. J. Exptl. Pathol. 15, 193 (1934).

36 W. S. Hartroft, Rept. Ann. Meeting and Proc. Roy. College Phys. Surg. (Canada)
p. 120 (Nov. 25-26, 1949).

31 W. 8S. Hartroft, Anat. Record 106, 61 (1950).

68 CHOLINE

mental cirrhosis. Droplets of stainable lipid appear in the hepatic cells
surrounding the central veins within 24 hours after rats are placed on a diet
low in choline and in its precursors. Large intracytoplasmic masses of fat
are formed from smaller droplets during the first week, and the paren-
chymal cells are expanded to twice the original size. The fat content cf the
liver in this stage may be increased ten times, but it rapidly disappears if
choline is administered to the rat. If the choline-deficient diet is continued
for 1 to 2 months, distended cell walls rupture and large fatty masses,
called lipodiastemata, form by fusion of several cells. These and the fatty
liver cells compress the sinusoids in the center of lobules where the intra-
vascular pressure is lowest, with resulting interference with the oxygen
supply. The partial anoxia is believed by Hartroft to be an important factor
in the ensuing atrophy and regression of liver cells which are followed by
disappearance of the fat and by the growth of fibrous tissue.

The rapidity and extent of the increase in liver lipids in choline deficiency
as well as their removal following administration of choline suggest strongly
that the effectiveness of choline as a lipotropic agent depends on some func-
tion of choline-containing phospholipids in fat metabolism. Definite evi-
dence of an influence of choline on phospholipid activity in the form of
data on the turnover of P® has been presented, but the correlation of the
appearance or disappearance of liver fat with changes in lecithin levels is
not at all satisfactory. The accelerating effect of aminoethanol and of its
mono- and dimethy] derivatives in certain aspects of phospholipid activity
together with the lipotropic activity of betaine and methionine indicate
that the maintenance of choline in one or another of its combined forms is
a complex function of considerable importance. Actually, the choline re-
quirement in weanling male rats given an adequate supply of protein was
found by Griffith and Mulford** to be 2 and 5.5 mg. of choline chloride per
day for the prevention of renal lesions and of fatty livers, respectively.
The nature of the compounds sharing the lipotropic activity of choline is
indicated in Section V, p. 45.

The quantity and nature of the dietary fat influences the fatty liver but
is not the determining factor because accumulation of liver lipids occurs on
low choline diets that are low in fat.*?: “° Barrett ed al.4! fed deutertum-labeled
fatty acids and were unable to demonstrate the presence of the isotope in
the liver fat. There appears little question but that the bulk of the excess
fat in a fatty liver of this type is formed in the liver from carbohydrate.
Nevertheless, Channon and Wilkinson® found variations due to the nature

33 W. H. Griffith and D. J. Mulford, J. Am. Chem. Soc. 68, 929 (1941).

39 CO. H. Best and M. E. Huntsman, J. Physiol. (London) 88, 255 (1935).

40 W. H. Griffith, J. Biol. Chem. 132, 639 (1940).

41H. M. Barrett, C. H. Best, and J. H. Ridout, J. Physiol. (London) 93, 367 (1938).
42 H. J. Channon and H. Wilkinson, Biochem. J. 30, 1033 (1936).

X. EFFECTS OF DEFICIENCY 69

of the dietary fat, as follows, in the liver fat of adult rats fed a low choline
diet containing 5 % casein and 40 % fat: butterfat, 30.7 %; beef fat, 27.1%;
olive oil, 15.6%; and cod liver oil, 7.2%. The proportion of saturated fatty
acids containing 14 to 18 carbons in the diet appeared to increase the
deposition of fat more so than unsaturated fatty acids, including the solid
unsaturated acid, elaidic acid.** Stetten and Salcedo “+: * fed ethyl esters of
fatty acids in low choline diets and found the degree of the fatty liver to
increase as the chain length decreased from 18 to 14. No severe fatty livers
occurred if the dietary fatty acid contained less than 12 carbons. Ethyl
laurate, however, was extremely toxic and caused death within a few days
with symptoms resembling those of an acute deficiency of potassium. The
fatal myocarditis was not related to a lack of tissue potassium, however,
and the syndrome was prevented by choline.

Raman* compared the liver and carcass lipids resulting from feeding a
fat-free and a high fat diet with and without choline. The dietary fat was
highly saturated and fed at a 30% level. The proportion of saturated fatty
acids and the degree of unsaturation of the unsaturated fatty acids of liver
phospholipids increased in choline-deficient rats on both diets. The carcass
fat of animals on the high fat ration was increased by choline from 1.5 to
2.8% of carcass weight. Carcass fat on the fat-free diet was much higher
(12% of carcass weight) and was unaffected by choline. It was evident
that choline had little, if any, effect on the character of depot fat synthe-
sized and stored in rats fed the fat-free ration. The usual lipotropic action
of choline on liver lipids was observed in these experiments.

The feeding of cholesterol brings about the deposition of neutral fat and
of cholesterol esters in livers of rats fed low choline diets containing added
cholesterol. Choline prevents the fatty liver and partly reduces the level of
the sterol esters.”: 4* Similar results were obtained by supplements of ca-
sein‘: °°, 1 and of methionine. Choline deficiency was aggravated by
dietary cholesterol in weanling rats.*? Choline was protective over the
eritical 8-day period but not over a 30-day period in surviving animals.
Inositol was not more effective than choline upon cholesterol esters.™4

48H. J. Channon, 8. W. F. Hanson, and P. A. Loizides, Biochem. J. 36, 214 (1942).
44D. Stetten, Jr., and J. Salcedo, Jr., J. Nutrition 29, 167 (1945).

45H. D. Kesten, J. Salcedo, Jr., and D. Stetten, Jr., J. Nutrition 29, 171 (1945).
46C.S. Raman, Biochem. J. 62, 320 (1952).

47C. H. Best and J. H. Ridout, J. Physiol. (London) 78, 415 (1933).

48 C. H. Best, H. J. Channon, and J. H. Ridout, J. Physiol. (London) 81, 409 (1934).
49 C. H. Best and J. H. Ridout, J. Phustol. (London) 87, 55P (1936).

50 A. W. Beeston, H. J. Channon, and H. Wilkinson, Biochem. J. 29, 2659 (1935).
51 A. W. Beeston, H. J. Channon, and A. P. Platt, J. Soc. Chem. Ind. 66, 292 (1937).
52H. J. Channon, M. C. Manifold, and A. P. Platt, Biochem. J. 32, 969 (1938).

53 W. H. Griffith and D. J. Mulford, J. Nutrition 21, 633 (1941).

54 C. H. Best, C. C. Lucas, J. M. Patterson, and J. H. Ridout, Biochem. J. 48, 452
(1951).

70 CHOLINE

Little is known of the details of the synthesis of lecithin, for instance,
in the animal organism. In an attempt to clarify certain aspects of this
problem, Tolbert and Okey** injected P**-labeled phosphate and C'-methyl-
labeled choline simultaneously in rats and determined the specific activity
of various liver fractions after time intervals ranging from 3 to 96 hours.
Similar renewal rates for phospholipid choline and phospholipid phosphate
seemed likely because of the similarity in the rates of decrease in the spe-
cific activities of the two labeled fractions after reaching their initial maxi-
mum concentrations. Glycerophosphate satisfied the criteria for an imme-
diate precursor of phospholipid.

Artom has investigated thoroughly the effect of choline and of related
compounds on the phospholipid content of livers of rats 2 to 3 months old
and fed diets low in protein and in sources of labile methyl. Significant
decreases were found in the total and in the choline-containing phospho-
lipids.** These decreases were only partly prevented by supplements of
choline administered at the start of the experiment and not prevented at
all if fatty infiltration was allowed to exist for 7 or more days prior to the
addition of choline to the diet.*” The absence of an effect of choline in the
latter experiments was not prevented by simultaneous administration of
various vitamins and amino acids, including methionine. In the case of
diets varying in fat content, the addition of choline after the development
of the fatty liver did restore the phospholipid level if 20% or more of fat
was present in the ration.®** Choline was more effective also if lactose re-
placed dietary sucrose.®® The phospholipid turnover in intestinal mucosa
after the injection of P® and the feeding of choline with and without fat
was less if the choline and fat were administered separately.® The conclu-
sion seemed clear that choline increased phospholipid formation in the
intestine and did so especially during the digestion and absorption of fat.
Liver phospholipids were affected similarly.*t Aminoethanol and mono-
methyl- and dimethylaminoethanol stimulated the formation of total
phospholipids in both intestine and liver as much as did choline. The
increase with choline was limited to the choline-containing phospholipids,
whereas both choline-containing and non-choline-containing phospholipids
were increased by aminoethanol and by its methylated forms.”

Artom’s demonstration of the inadequacy of choline for the complete

55 M. EB. Tolbert and R. Okey, J. Biol. Chem. 194, 755 (1952).

56C, Artom and W. H. Fishman, J. Biol. Chem. 148, 405, 415, 423 (1948).
57 W. H. Fishman and C. Artom, J. Biol. Chem. 154, 117 (1941).

58 W. H. Fishman and C. Artom, J. Biol. Chem. 164, 307 (1946).

59C, Artom and W. H. Fishman, J. Biol. Chem. 170, 587 (1947).

60 ©, Artom and W. E. Cornatzer, J. Biol. Chem. 165, 393 (1946).

61 C, Artom and W. E. Cornatzer, J. Biol. Chem. 171, 779 (1947).

62 ©, Artom and W. E. Cornatzer, J. Biol. Chem. 176, 949 (1948).

X. EFFECTS OF DEFICIENCY fil

restoration of the decrease in choline-containing phospholipids caused by
choline deficiency in 2- to 3-month-old rats is particularly interesting be-
cause he was able to show that choline was largely effective in weanling
rats®* and that this effect was not duplicated by aminoethanol, methionine,
serine, or glycine.® He has interpreted these data as evidence for the par-
ticipation with choline of a factor essential in phospholipid syntheses and
present in weanling rats to a greater extent than in older animals. Artom
and Fishman*® have emphasized the fact that older rats were used in pre-
vious experiments which failed to demonstrate an effect of choline on total
liver phospholipids**: ®: ® or an effect on the choline content of livers,?’: 4%
whereas weanling rats were used in studies in which total phospholipids”: °°
or total choline-containing phospholipids***8 were reported to be increased
by administration of choline.

Brante®® found a significant increase in liver cephalin in rats maintained
for several months on a choline-deficient, high fat diet. The level of
choline-containing phospholipids in the liver was the same in rats with and
without choline. No difference in the total choline content of liver and
kidneys of rats on control and low choline diets was noted by Kahane et
al.,”° but the amount in the rest of the body was somewhat less in the
deficient group. Muscle creatine levels were not affected by an existing
deficiency of choline that permitted development of fatty livers.”

Kensler e¢ al.” have made the very significant observation that the choline
oxidase activity of the liver and kidneys of young rats increases sharply
during the age period in which they are particularly susceptible to acute
choline deficiency. Of interest in this connection is the finding that the
severe renal lesions of choline deficiency were less frequently fatal in 20-
day-old rats than in those 21 to 27 days of age.?

Chaikoff investigated the rate of phospholipid metabolism in rat liver
as measured by phosphorus turnover studies following the administration
of radioactive phosphorus (P®). Choline and betaine” and cystine and
methionine” accelerated the turnover of phosphorus, the effect of a single

68 W. H. Fishman and C. Artom, J. Biol. Chem. 154, 109 (1944).

64 J. H. Channon and H. W. Wilkerson, Biochem. J. 28, 2026 (1934).

65 F. Cedrangolo and V. Baccari, Arch. sct. biol. (Italy) 24, 311 (1938).

66 R. W. Engel, J. Biol. Chem. 144, 701 (1942).

67 T). Stetten, Jr., and G. F. Grail, J. Biol. Chem. 144, 175 (1942).

68 P, Handler and W. J. Dann, J. Biol. Chem. 146, 357 (1942).

69 G. Brante, Acta Physiol. Scand. 6, 291 (1943).

70H. Kahane, J. Lévy, and O. Tanguy, Arch. sct. physiol. 4, 185 (1950).

71K. Roberts and H. C. Eckstein, J. Biol. Chem. 164, 377 (1944).

72 C, J. Kensler, M. Rudden, E. Shapiro, and H. Langemann, Proc. Soc. Exptl. Biol.
Med. 79, 39 (1952).

73J. Perlman and I. L. Chaikoff, J. Biol. Chem. 127, 211 (1939); 180, 593 (1939).

4 T. Perlman, N. Stillman, and I. L. Chaikoff, J. Biol. Chem. 133, 651 (1940).

12 CHOLINE

supplement lasting 6 or more hours. Betaine was less effective than choline.
The latter substance was particularly active in rats fed cholesterol.7®> There
was little indication in these experiments of variations in the total phospho-
lipid content of the liver. A slight increase in this fraction of liver lipids
following the administration of choline or methionine was noted by Horn-
ing and Eckstein, but this was not correlated necessarily with lipotropic
activity.”6

Artom and Swanson observed that slices from livers of rats previously
fed a low choline, low protein diet incorporated P*®? into phospholipids at a
slower rate than slices from rats fed high protein diet or a stock diet. Prior
administration of choline in vivo reversed the fatty infiltration but did not
restore the ability of the isolated liver to synthesize phospholipids. In fact,
the addition of choline zn vitro inhibited phospholipid formation.” Boxer
and Stetten fed N'-labeled choline to rats and found the rate of replace-
ment of phospholipid choline in the liver to be 3.9 mg. per rat per day.
With cessation of the labeled choline supplement, new, non-isotopic choline
replaced the isotopic fraction at a rate of 1.3 mg. per day while fatty livers
developed. They concluded that the fatty livers were due to a decrease in
the rate of incorporation of choline into tissue phospholipids.7’ The admin-
istration of choline and of aminoethanol increased the rates of formation
of lecithin and of cephalin, respectively, in fasted and fed rats previously
given radioactive phosphate.7? Partial hepatectomy impaired the formation
of intestinal and hepatic phospholipids,*” *! but choline was ineffective in
preventing the resulting fatty liver.®

Deposition of excess lipids in the liver may result from many and diverse
causes, and, in certain instances, choline is without effect as a lipotropic
agent. Best has summarized the pertinent facts in this field. Of particular
significance is the distribution of accumulating fat, in the center of liver
lobules if due to hypolipotropism and in the periphery if due to the activity
of the anterior pituitary or to extracts of this gland. The administration of
anterior pituitary extract in fasting guinea pigs, rats, and mice mobilized
body fat in the liver.*: ® Inasmuch as this result did not occur if the ad-
renals were removed, MacKay concluded that the effect was mediated

75 J, Perlman and I. L. Chaikoff, J. Biol. Chem. 128, 735 (1939).

76M. G. Horning and H. C. Eckstein, J. Biol. Chem. 166, 711 (1946).

77 C. Artom and M. A. Swanson, J. Biol. Chem. 198, 473 (1951).

7G. E. Boxer and D. Stetten, Jr., J. Biol. Chem. 153, 617 (1944).

779A. P. Platt and R. R. Porter, Nature 160, 905 (1947).

80}, Chargaff, J. Biol. Chem. 128, 587 (1939).

81). Chargaff, K. B. Olson, and P. F. Partington, J. Biol. Chem. 134, 505 (1940).
82. M. MacKay and H. O. Carne, Proc. Soc. Exptl. Biol. Med. 38, 131 (1938).

83 ©. H. Best, Federation Proc. 9, 506 (1950).

84 C,H. Best and J. Campbell, J. Physiol. (London) 86, 190 (1936) ; 92, 91 (1938).
85 J. Campbell and C. C. Lucas, Biochem. J. 48, 241 (1951).

X. EFFECTS OF DEFICIENCY 73

through the adrenals.*® That depot fat was the probable source of the
mobilized fat was indicated by the use of deuterium as a means of dis-
tinguishing newly synthesized triglycerides.*!: *’ Liver fat was increased by
both growth hormone and by ACTH,*: * presumably the active materials
in the original pituitary extract employed by Best and Campbell.*! On the
other hand, the administration of cortisone in rats fed a low choline diet
failed to prevent the deposition of liver fat, although considerable protec-
tion against hemorrhagic degeneration was afforded.*° Olson and Deane!®
described changes in the adrenal cortex during the acute phase of hemor-
rhagic degeneration but believed the alterations to be secondary to the
renal lesions. Neither desoxycorticosterone acetate nor whole adrenal ex-
tract influenced the course of the acute phase of choline deficiency. The
marked hyperactivity of the zona glomerulosa at the time of the renal
lesion was ascribed to altered electrolyte balance.

The feeding of choline-deficient diets to adult rats removed the elevated
blood pressure resulting from partial nephrectomy in the experiments of
Handler and Bernheim. ACTH restored temporarily the hypertensive state
in these animals although it was without effect in normal controls, and it
was suggested that secretion of ACTH is impaired in choline-deficient rats.”!
Adrenalectomy partially prevented fatty livers in male rats on a choline-
deficient diet, but there was little effect in females.°?

Shipley ef al. noted increased liver fat in female rats but not in males
after castration.*? Testosterone propionate aggravated both renal and he-
patic lesions in female rats and hepatic lesions in male rats fed a low choline,
high fat diet.°* Male rats had been found more susceptible than females to
severe, acute choline deficiency.? Castration of male rats caused a marked
reduction in blood lipid phosphorus in rats on a hypolipotropic diet.”

Shipley reported that thyroidectomy prevented fatty livers in male and
female rats on a choline-deficient diet.°2 Handler, on the other hand, found
that removal of the thyroid or the feeding of thiouracil caused a small
increase in neutral fat and a marked increase of cholesterol in livers of both
control and choline-deficient rats.

86H. M. MacKay and R. H. Barnes, Am. J. Physiol. 118, 525 (1937) ; 120, 361 (1937).

87 TD). Stetten, Jr. and J. Salcedo, Jr., J. Biol. Chem. 156, 27 (1944).

8 L. L. Bennett, R. E. Kreiss, C. H. Li, and H. M. Evans, Am. J. Physiol. 152, 210
(1948).

89 C, H. Li, M. E. Simpson, and H. M. Evans, Arch. Biochem. 28, 51 (1949).

9, A. Sellers, R. W. You, J. H. Ridout, and C. H. Best, Nature 166, 514 (1950).

91 P, Handler and F. Bernheim, Am. J. Physiol. 162, 375 (1950).

2 R, A. Shipley, E. B. Chudzik, and P. Gyérgy, Arch. Biochem. 16, 301 (1948).

9% W. J. Emerson, P. C. Zamecnik, and I. T. Nathanson, Endocrinology 48, 548
(1951).

9% R. Honorato and M. Modak, Bol. soc. biol. Santiago Chile 4, 24 (1947).

95 P, Handler, J. Biol. Chem. 178, 295 (1948).

74 CHOLINE

The lipotropic effect in rats of exposure to cold constitutes a striking
exception to the usual correlation between severity of choline deficiency
and the metabolic level. Sellars and You found an average liver fat of 7.2
+ 1.24% in animals maintained at 2.5° whereas controls fed the same low
choline diet and maintained at 25° had an average liver fat of 24.8 + 4.90 %.
The ‘‘cold”’ animals consumed 50 % more food.** The choline-like effect of
cold was not a temporary phenomenon and continued through a 15-week
experimental period; it occurred in animals acclimatized to cold prior to
the feeding of the hypolipotropic diet; and it was not prevented by thyroid-
ectomy and subsequent administration of a daily maintenance dose of
thyroxin.*” However, exposure to cold was only partially lipotropic if the
diet contained 50% fat. Stimulation of the adrenals was not believed a
primary factor because cortisone failed to prevent the development of the
fatty liver in rats on a low choline diet.%° The study of the increased metabo-
lism in “cold” rats under various conditions has not permitted a satisfactory
explanation of the lipotropism due to the lowered environmental tempera-
ture.°* Although both thyroid and adrenal hormones were believed necessary
for the mechanism involved, hyperactivity of either gland was not con-
sidered an essential factor.°?: 1°

The administration of low molecular weight halogenated hydrocarbons
by injection or by inhalation in rats on low choline, low protein diets causes
severe fatty infiltration and degeneration of the liver. Heppel et al. found
methionine considerably more protective than choline, although the latter
with cystine increased the resistance of animals exposed to ethylene and
propylene dichloride.!": 1°? Calder concluded that choline plus a thermo-
labile factor in yeast, which was not thiamine, decreased the liver damage
caused by chloroform in rats.!°? Carbon tetrachloride caused anemia as well
as hepatic pathology in rats.!°* The former responded to methionine, but
neither methionine nor choline prevented the fatty degeneration. Coulson
and Brazda tested pyridine, quinoline, and a number of pyridine deriva-
tives and found choline relatively ineffective as a protective agent.!

Reference has been made previously to the degenerative changes in the

96H}, A. Sellers and R. W. You, Science 110, 713 (1949).

97. A. Sellers and R. W. You, Biochem. J., 51, 573 (1952).

98K. A. Sellers and 8. S. You, Am. J. Physiol. 168, 81 (1950).

99 f. A. Sellers, S.S. You, and N. Thomas, Am. J. Physiol. 165, 481 (1951).

100 H}. A. Sellers and R. W. You, J. Nutrition 44, 513 (1951).

101 |, A. Heppel, P. A. Neal, F.S. Daft, K. M. Endicott, M. L. Orr, and V. T. Porter-
field, J. Ind. Hyg. Toxicol. 27, 15 (1945).

102 |, A. Heppel, B. Highman, and V. T. Porterfield, J. Pharmacol. 87, 11 (1946).

103 R. M. Calder, J. Pathol. Bacteriol. 64, 355 (1942).

104 Hf. Benard and M. Gajdos-Torok, Compt. rend. soc. biol. 141, 122 (1947).

1065 R, A. Coulson and F. G. Brazda, Proc. Soc. Exptl. Biol. Med. 65, 1 (1947); 69,
480 (1948).

X. EFFECTS OF DEFICIENCY (5

liver that predispose to experimental cirrhosis in rats and to the fact that
hypolipotropic diets usually result in hepatic fibrosis that starts in the
centrolobular region. This type of liver pathology is prevented by lipo-
tropic agents, particularly by choline and methionine. Extent of curative
effects depends on the degree of damage that has occurred. This experi-
mental cirrhosis must be distinguished from the hemorrhage and necrosis
or necrotic degeneration that occurs in rats if fed certain deficient diets,
especially diets low in cystine and vitamin E, and from the pathology
caused by an excess of dietary cystine. These various conditions have been
described by many workers.!06!8

A characteristic feature of cirrhotic livers due to a deficiency of lipotropic
factors is an acid-fast and sudanophilic pigment named “‘ceroid”’ by Lillie
et al." Victor and Pappenheimer reported that ceroid was associated with
vitamin E deficiency,!! a conclusion supported by Hartroft,?® who sug-
gested the possibility that the deposit was a mixture of lipids and some
component of erythrocytes.!!9

Liver damage caused by the excessive administration of ethanol in rats
affords an excellent illustration of the protective role of lipotropic substances
such as choline.!°%: ”° Ashworth reported, however, that alcohol exerted
an effect unrelated to lipotropic factors.!”! Best et al.!? and Hartroft®* found
increased fibrous tissue formation in nearly 50% of rats fed a low protein,
low choline diet and given 15 % alcohol as drinking water. Pair-fed controls
developed the same degree of damage only if the total caloric intake was

106 A.C. Curtis and L. H. Newburgh, Arch. Internal Med. 39, 828 (1927).

107 P. Gyorgy and H. Goldblatt, J. Exptl. Med. 70, 185 (1939); 75, 355 (1942); 90,
73 (1949).

108 H. Blumberg and E. V. McCollum, Science 93, 598 (1941).

109 Ff, §. Daft, W. H. Sebrell, and R. D. Lillie, Proc. Soc. Exptl. Biol. Med. 48, 228
(1941); 50, 1 (1942).

110 JT), P. Earle and J. Victor, J. Exptl. Med. 73, 161 (1941); 75, 179 (1942).

111 R. D. Lillie, L. L. Ashburn, W. H. Sebrell, F. §. Daft, and J. V. Lowry, Publ.
Health Repts. (U.S.) 57, 502 (1942).

112 J, Victor and A. M. Pappenheimer, J. Exptl. Med. 82, 375 (1945).

13 J, Green and A. Brunschwig, Proc. Soc. Exptl. Biol. Med. 61, 348 (1946).

u4 P, Handler and I. N. Dubin, J. Nutrition 31, 141 (1946).

15 P, Handler and R. H. Follis, Jr., J. Nutrition 35, 669 (1948).

16 C, A. Hall and V. A. Drill, Proc. Soc. Exptl. Biol. Med. 69, 3 (1948).

17 |, EK. Glynn, H. P. Himsworth, and O. Lindan, Brit. J. Exptl. Pathol. 29, 1 (1948).

18 K. Schwarz, Proc. Soc. Exptl. Biol. Med. 77, 818 (1951).

19 W.S8. Hartroft, Science 113, 673 (1951).

120 J. V. Lowry, L. L. Ashburn, and W. H. Sebrell, Jr., Quart. J. Studies Alc. 6, 271
(1945).

121 C, T. Ashworth, Proc. Soc. Exptl. Biol. Med. 66, 382 (1947).

122 C.H. Best, W.S. Hartroft, C. C. Lucas, and J. H. Ridout, Brit. Med. J. II, 1001
(1949).

76 CHOLINE

made equivalent to the alcohol intake by isocaloric amounts of sucrose. It
was concluded that the effect of aleohol on the livers of rats in experiments
of this type was due to a deficiency of lpotropic factors, especially of
choline.

Kaufman et al. were unable to produce experimental hemochromatosis in
rats by feeding a diet low in lipotropic factors and containing a moderately
toxic level of copper.!”? The combination of copper and choline in this diet
decreased the body weight of the animals.

3. EFFECT OF SULFUR AMINO ACIDS

In 1935, Best and Huntsman reported that casein exhibited lipotropic
activity,®® a result confirmed by Channon and Wilkinson™! and extended
to egg white and beef protein, but not to gelatin, by Best et al.!® In a sur-
vey of the effect of various amino acids, Beeston and Channon found one,
cystine, with a marked antilipotropic activity.'’® In subsequent experiments,
negative results for most of the other amino acids were confirmed,!”7; 8 and
cysteine and homocystine were found to resemble cystine in augmenting
the deposition of fat in the livers of rats fed the low choline, 5% protein,
and 40 % fat diets that were commonly used.”°: The explanation of the
puzzling lipotropic character of many proteins was indicated by the im-
portant observation of Tucker and Eckstein that a supplement of methio-
nine reduced markedly the liver fat of rats on the experimental diets and,
in this respect, acted oppositely to cystine.'! The choline-like property of
methionine has been confirmed repeatedly.*”: !-!8> It is pertinent that Best
called attention to the significant fact that methionine rarely reduced the
level of liver lipids to normal values even though it was highly effective
in preventing excessively fatty livers.’ The participation of methionine in
transmethylation as a methyl donor with synthesis or sparing, at least, of
choline offers a reasonable explanation of the lipotropic property (Section

123 N. Kaufman, J. A. Cartaya, P. L. White, D. M. Hegsted, and T. D. Kinney, J.
Nutrition 46, 4383 (1952).

24 AH. J. Channon and H. Wilkinson, Biochem. J. 29, 350 (1935).

125 C, H. Best, R. Grant, and J. H. Ridout, J. Physiol. (London) 86, 337 (1936).

126 A.W. Beeston and H. J. Channon, Biochem. J. 30, 280 (19386).

127 §, A. Singal and H. C. Eckstein, J. Biol. Chem. 140, 27 (1941).

128 A.W. Beeston and A. P. Platt, J. Soc. Chem. Ind. 56, 292 (1987).

129 §. A. Singal and H. C. Eckstein, Proc. Soc. Exptl. Biol. Med. 41, 512 (1939).

130 H. J. Channon, M. C. Manifold, and A. P. Platt, J. Soc. Chem. Ind. 57, 600 (1938).

131 H. F. Tucker and H. C. Eckstein, J. Biol. Chem. 121, 479 (1937).

1322 C,H. Best and J. H. Ridout, J. Physiol. (London) 97, 489 (1940).

133 W. H. Griffith and N. J. Wade, J. Biol. Chem. 132, 627 (1940).

134 C, R. Treadwell, J. Biol. Chem. 176, 1141 (1948).

135 J. M. R. Beveridge, C. C. Lucas, and M. K. O’Grady, J. Biol. Chem. 160, 505
(1945).

X. EFFECTS OF DEFICIENCY 77

IV). The similar behavior of most proteins depends on their content of
this amino acid inasmuch as it is the only one of these units commonly
present in proteins for which significant lipotropic activity has been demen-
strated. 156-139

The effect of cystine and its apparent antagonism to methionine have
intrigued many investigators. Beeston and Channon noted that the cystine
effect was not proportional to the level of the cystine supplement,!2® and
this may explain the minimal influence of additions of the amino acid to
low choline, low protein diets in which the protein was gliadin’ or albu-
min,'*° both relatively richer in cystine than casein. Attempts to compare
the lipotropic activity of free methionine with equivalent amounts in vari-
ous proteins have not yielded uniformly consistent results.!32) 185, 187, 140-143
This is not surprising in view of differences in strains and in the age of
experimental rats, of changes in the nutritional state of the animals in
those instances in which a deficiency of protein was superimposed on a
deficiency of methyl, of variations in the supply of cystine, and of the
unrecognized presence in some diets of nutrients, such as Bis, which are
now believed to influence favorably the utilization of methionine.

Somewhat more consistent results have been obtained in studies of the
cystine effect in weanling rats. Griffith and Wade noted clear evidence of
the aggravating effect of added cystine on both renal lesions and deposition
of liver fat on diets ordinarily considered adequate in total protein.1** Cox
et al. had observed this result before choline was recognized as a dietary
factor. In line with data obtained on older rats,!® the maximum cystine
' effect in weanling rats fed a diet containing 0.05 % choline chloride and 18 %
casein resulted if 0.1 to 0.2% additional cystine was added. Cystine sup-
plements up to 2% had no additional effect.2! These data emphasized the
physiological role of a small supplement of cystine and removed any basis
for an explanation of the cystine effect because of toxicity. Mulford and
Griffith demonstrated that the addition of extra cystine to the 18 % casein
diet supplemented with sufficient choline to prevent fatty livers resulted in
improved growth of the rats as measured by increased body weight and
length and efficiency of utilization of food. Accordingly, it was concluded

136 H. J. Channon, J. V. Loach, P. A. Loizides, M. C. Manifold, and G. Soliman, Bio-
chem. J. 32, 976 (1938).

137 H. F. Tucker and H. C. Eckstein, J. Biol. Chem. 126, 117 (1938).

138 W. H. Griffith, J. Nutrition 21, 291 (1941).

139 M.G. Horning and H. C. Eckstein, J. Biol. Chem. 155, 49 (1944).

140 H. J. Channon, M. C. Manifold, and A. P. Platt, Biochem. J. 84, 866 (1940).

41 CR. Treadwell, M. Groothuis, and H. C. Eckstein, J. Biol. Chem. 142, 653 (1942).

142 J. M.R. Beveridge, C. C. Lucas, and M. K. O’Grady, J. Biol. Chem. 154, 9 (1944).

43 A. F. Tucker, C. R. Treadwell, and H. C. Eckstein, J. Biol. Chem. 185, 85 1940).

144 G. J. Cox, C. V. Smythe, and C. F. Fishback, J. Biol. Chem. 82, 95 (1929).

78 CHOLINE

that the phenomenon involving cystine was related to its improvement of
the nutritive value of the diet, 1.e., the removal of the existing sulfur defi-
ciency. The resulting increased metabolic level raised the choline require-
ment and exaggerated the deficiency signs in the absence of a supply of the
required extra choline. This explanation made unnecessary any hypothesis
based on a specific antagonism or competition between the sulfur amino
acids, methionine and cystine.

The fact that weanling rats develop severe evidences of deficiency on low
choline diets, even on diets adequate in protein, made it possible to study
protein effects without the risk of protein deficiency. Griffith fed mixtures
of casein, lactalbumin, fibrin, edestin, and gelatin with and without sup-
plements of methionine and cystine and found that dietary choline was not
required for the prevention of hemorrhagic degeneration if the food mixture
provided 0.8% or more methionine, regardless of whether this level was
attained by use of proteins or by methionine supplements.'** Renal lesions
and fatty livers occurred on low choline diets providing 10 to 25 % casein,!**
and 30% casein was required to prevent the cystine effect in the absence
of added choline.”! The latter observation was the basis for the conclusion
that a single molecule of methionine is not simultaneously a source of both
labile methyl and cystine sulfur, inasmuch as 22 to 24% casein supplies
the optimum level of sulfur if dietary choline is provided.”

The relation between the general adequacy of the diet for growth, except
for its choline content, and the deposition of liver fat was emphasized in
the first report on choline deficiency in weanling rats. With diets only
moderately deficient in choline, the deposition of liver fat was intensified in
those permitting the better rates of growth.? The concept that nutritionally
adequate diets involve increased needs of choline, and the converse, that
choline requirements are less if the food mixture or the food intake does not
allow growth, were supported by the results of caloric restriction in wean-
ling rats fed a choline-deficient diet. The development of renal lesions and of
fatty livers was completely prevented by removal of part of the carbo-
hydrate and fat of the diet.” Similar conclusions regarding the nature of
the diet and the requirement of choline have been reached by Beveridge
et al.'85» 142 Salmon,'45 Handler,'4* and Best.12?» 147

Handler prevented growth in young rats by feeding them a diet deficient
in choline and in the mineral component of the ration. After 4 weeks these
animals had not grown and the values for liver lipids were nearly normal
whereas rats restricted to the same amount of food containing the mineral
component grew slowly and had markedly fatty livers.16

45 W. D. Salmon, J. Nutrition 33, 155 (1947).

146 P, Handler, J. Biol. Chem. 149, 291 (19438).

147 C, H. Best, C. C. Lucas, J. H. Ridout, and J. M. Patterson, J. Biol. Chem. 186,
317 (1950).

X. EFFECTS OF DEFICIENCY 79

Salmon concluded that weanling rats on diets containing 18% or less
casein were subject to methyl deficiency unless supplemental choline or
methionine was supplied.® This deficiency was intensified by cystine and
by fat. Such animals also required supplementary nicotinic acid or addi-
tional tryptophan unless the diet was high in fat. It was not possible to
demonstrate a deficiency of cystine unless the methyl and nicotinic acid
deficiencies were remedied. In contrast is the surprising observation of
Tyner et al. that the cystine effect was abolished if generously adequate
levels of niacin or of tryptophan were provided."*

Treadwell and coworkers have also studied the cystine effect in both
young and adult rats.!*4; 49-15! The intensification of renal lesions in wean-
ling rats by supplements of cystine was confirmed, but the explanation
based on the increased demand for choline because of the otherwise im-
proved food mixture was questioned.!* Treadwell suggested that the me-
tabolism of cystine may require methyl groups or that extra cystine may
depress the demethylation of methionine by a mass action effect.!4° Neither
possibility explains the similarity of lipotropism in diets supplemented with
0.1 and 2.0 % cystine.”! The failure of cystine to induce growth on low casein
diets, as noted by Treadwell'*? and by Salmon, is not surprising. Griffith
and Nawrocki found that threonine increased the need of choline in cystine-
supplemented 8% casein diets and concluded that at this casein level
cystine (or methionine) and threonine were the first and second limiting
amino acids, respectively.!*?

The synthesis of choline in rats fed diets containing methionine as the
~ main methyl donor requires a suitable carbon-nitrogen moiety to serve as
the methyl acceptor and whatever cofactors are necessary for the enzy-
matic systems involved. There appears to be little difficulty in the provision
of the dietary essentials for these mechanisms. Rose et al. found no statis-
tically significant effect on the growth of weanling rats as a result of the
omission of glycine, serine, cystine, and most of the choline from a standard
diet containing a mixture of highly purified amino acids as the source of
nitrogen.!®? The same omissions caused a slight but significant retardation
of growth in similar diets in which the methionine level was decreased from
0.8 to 0.5% (as pL-methionine) and the threonine level from 0.7 to 0.4%
(as L-threonine) and in which the liver extract, a possible source of By,
was also omitted.

Ethionine (S-ethyl-pL-homocysteine) is a homolog of methionine and is,

48H}, P. Tyner, H. B. Lewis, and H. C. Eckstein, J. Biol. Chem. 187, 651 (1950).
49 ©, R. Treadwell, J. Biol. Chem. 176, 1149 (1948).

150 ©, R. Treadwell, H. C. Tidwell, and J. H. Gast, J. Biol. Chem. 156, 237 (1944).
161 C, R. Treadwell, J. Biol. Chem. 160, 601 (1945).

12 W. H. Griffith and M. F. Nawrocki, Federation Proc. 7, 288 (1948).

153 W. C. Rose, W. W. Burr, Jr., and H. J. Sallach, J. Biol. Chem. 194, 321 (1952).

80 CHOLINE

therefore, a potential antagonist of the natural amino acid. It was found to
inhibit methyl transfer from methionine to choline but not to creatine.!*4
Fatty livers occurred rapidly after its injection into female rats but not in
male rats, and this antilipotropic effect was opposed by methionine.!®®
Evidence has been provided for its incorporation in vive into an abnormal
type of tissue protein.1°® Stekol showed that ethionine was deethylated in
the rat!*” and that its inhibitory action on rat growth was prevented by the
administration of either choline or methionine.!®* In similar experiments
with triethylcholine, choline was more efficient than methionine in the pre-
vention of a deleterious effect on growth.!®® These data suggest the partici-
pation and interference of ethionine in transmethylation.

4. Errect oF OTHER NUTRIENTS

A lipotropic action of inositol was first reported by Gavin and McHenry!
and studied further by MacFarland and McHenry.'®* Rats, 90 to 100 g. in
weight, were depleted of fat and of B vitamins for 3 weeks on a fat-free,
high carbohydrate diet; during a fourth week test substances were adminis-
tered and the livers were analyzed for lipids. In such experiments a crude
beef liver fraction, a liver extract, or a biotin plus folic acid supplement with
extra thiamine, riboflavin, pantothenate, and nicotinic acid produced fatty
livers resistant to choline and responsive to inositol. McHenry emphasized
particularly the effectiveness of inositol in reducing the level of liver choles-
terol. A supplementary action of inositol with choline was described by
Engel!” and confirmed by Forbes.'* Beveridge noted that the inositol effect
was absent if the diet contained corn oil.!* 1° The favorable influence of
inositol was also observed by Handler,'®* but both Handler“ and Best ed
al.1*" reported that in the absence of choline renal lesions were intensified

154 §. Simmonds, E. B. Keller, J. P. Chandler, and V. du Vigneaud, J. Biol. Chem.
183, 191 (1950).

155 J). Jensen, I. L. Chaikoff, and H. Tarver, J. Biol. Chem. 192, 395 (1951).

156 M. Levine and H. Tarver, J. Biol. Chem. 192, 835 (1951).

157 J. A. Stekol, K. W. Weiss, and S. Weiss, J. Am. Chem. Soc. 72, 2309 (1950).

158 J. A. Stekol and K. W. Weiss, Proc. Soc. Exptl. Biol. Med. 17, 213 (1951); J. Biol.
Chem. 179, 1049 (1949); 185, 577 (1950).

159 J. A. Stekol and K. W. Weiss, J. Biol. Chem. 185, 585 (1950).

160 G, Gavin and E. W. McHenry, J. Biol. Chem. 189, 485 (1941) ; 141, 619 (1941).

161 M. L. MacFarland and E. W. McHenry, J. Biol. Chem. 159, 605 (1945); 176, 429
(1948).

1622 R. W. Engel, J. Nutrition 24, 175 (1942).

163 J. C. Forbes, Proc. Soc. Exptl. Biol. Med. 54, 89 (1943).

164 J. M. R. Beveridge, Science 99, 539 (1944).

165 J. M. R. Beveridge and C. C. Lucas, J. Biol. Chem. 157, 311 (1945).

166 P. Handler, J. Biol. Chem. 162, 77 (1946).

167 C. H. Best, C. C. Lucas, J. M. Patterson, and J. H. Ridout, Science 103, 12 (1946).

X. EFFECTS OF DEFICIENCY 81

by inositol. Best ef al. have reinvestigated the lipotropic activity of inositol
and have found that the analysis for liver lipids at the end of the 7-day test
subsequent to the depletion period may not be representative of values
existing earlier or later.°* They concluded that inositol has no unique lipo-
tropic properties and that it is inferior to choline in the reduction of either
liver fat or cholesterol esters. Its supplementary action with choline in fat-
free diets was confirmed. No explanation has been brought forward for the
influence of inositol on liver lipids other than its possible role in an inositol-
containing phospholipid.

In 1933 Blatherwick et al. reported the occurrence of fatty livers in rats
fed large amounts of whole liver.'® This result was confirmed by Beeston
and Wilkinson, who were unable to demonstrate a lipotropic action of
choline.'®® McHenry, using the depletion procedure referred to above, found
marked increases in liver fat and cholesterol following the feeding of a liver
fraction!” and biotin.!®: 16! The term “biotin fatty liver” was applied, and
it was believed that inositol was more effective than choline in this condi-
tion. The concept of a special type of fatty liver due to biotin was attacked
by Best et al.7! and by Handler.!** These workers were unable to find satis-
factory evidence for an unusual effect of inositol or for a special role of biotin
other than the effect to be expected as a result of changes in food intake and
utilization.

McHenry noted a marked increase in liver fat in rats following the addi-
tion of thiamine to a low choline, thiamine-deficient diet.!” Choline, but
not inositol, was found to be lipotropic for this so-called ‘thiamine fatty
- liver.”? Somewhat similar results were observed by Engel and Phillips, who
concluded that thiamine therapy caused a temporary pathological state as
a result of disruption of cells by excessive production of fat.!”? The hydropic
degeneration and fatty changes were not prevented by choline, but des-
iccated thyroid was effective. McHenry had suggested earlier that thiamine
might be essential for the synthesis of fat from carbohydrate.!”4 The proba-
bility of such an origin of the extra liver lipids in choline deficiency was
supported by the exclusion of increased absorption from the intestine!”® and
of increased mobilization of tissue fat*! as possible sources. Regardless of

168 N.R. Blatherwick, E. M. Medlar, P. J. Bradshaw, A. L. Post, and 8. D. Sawyer,
J. Biol. Chem. 103, 93 (1933).

169 A.W. Beeston and H. Wilkinson, Biochem. J. 30, 121 (1936).

170. W. McHenry and G. Gavin, Sriegae 91, 171 (1940).

l7inG: He Best; ©. C.-Lueas,- dz M. Patterson, and J. H. Ridout, Biochem. J. 40, 368
(1946).

1722 H.W. McHenry, J. Physiol. (London) 89, 287 (1937).

173. R, W. Engel and P. H. Phillips, J. Nutrition 18, 329 (1939).

174 HY. W. McHenry, Science 86, 200 (1937).

176 H. KE. Longenecker, G. Gavin, and E. W. McHenry, J. Biol. Chem. 134, 693 (1940).

82 CHOLINE

the relation of thiamine to the synthesis of fat, it is doubtful if the deposi-
tion of extra fat resulting from the supplementation of the deficient diet
has any different explanation from that which explains satisfactorily the
effects of biotin,” cystine,‘ and minerals.'4* This conclusion is given solid
support by the findings of Boxer and Stetten, who caused labeling of newly
synthesized fat by the administration of D.O in rats, produced fatty livers
on a low choline diet with and without dietary thiamine, and noted that
the decrease in the synthesis of fat in the thiamine-deficient animals was
paralleled by that in the animals supplied thiamine but restricted to the
same food intake as the thiamine-deficient group.!7°

By way of contrast to thiamine, it is of interest that pyridoxine has been
reported to be necessary for the prevention of fatty livers. Rats deficient
im vitamin B, developed fatty livers to a greater extent than controls,!”
and necrosis of the kidney cortex was noted more frequently as a symptom
of choline deficiency.!”8 The feeding of a very high casein diet after depletion
of fat and of B vitamins resulted in an additional weight loss and in the
development of fatty livers unless pyridoxine was supplied.’”® According to
Engel, pyridoxine and essential fatty acids must be present for the normal
functioning of choline as a lipotropic agent in rats.!®

Niacin and its derivatives have been linked with lipotropism in rats in a
variety of experiments. Niacinamide and, under certain conditions, the di-
phosphopyridine nucleotide inhibited choline oxidase in zn vitro studies.1*
Growth of rats on low protein, methyl-poor diets containing niacinamide
was affected adversely, presumably because of further depletion of methyl
due to the formation and excretion of N!-methylniacinamide.® The cystine
effect was absent in young rats unless nicotinic acid was present in the
diet,'*° and the cystine effect was abolished in older animals in the presence
of generously adequate levels of this vitamin.“8 Forbes found that the addi-
tion of niacin to a low choline diet increased the cholesterol content of
fatty livers and decreased the responsiveness of the animals to the lipotropic
effect of choline.*! It is not possible to conclude that any of these effects
represent direct relations between the metabolism of choline and of niacin
in vivo except the changes introduced by the methylation of the vitamin.
Many of these results may be explicable on the basis of non-specific effects
on food intake and utilizatione Handler showed that dietary niacinamide
(2%) resulted in a greater loss of weight in partially hepatectomized rats

176 G. EK. Boxer and D. Stetten, Jr., J. Biol. Chem. 158, 607 (1944).

177 N. Halliday, J. Nutrition 16, 285 (1938).

178 P, Gyorgy and R. E. Eckardt, Biochem. J. 34, 1148 (1940).

179, W. McHenry and G. Gavin, J. Biol. Chem. 188, 471 (1941).

180 J. N. Williams, Jr., G. Litwack, and C. A. Elvehjem, J. Biol. Chem. 192, 73 (1951).
181 J, C. Forbes, J. Nutrition 22, 359 (1941).

X. EFFECTS OF DEFICIENCY 83

than in controls.'® Fatty livers on low choline diets were prevented by ni-
acinamide or by a deficiency of thiamine. These results were believed due
to an impairment of the metabolism of the rat rather than to a specific
defect involving niacin or thiamine.

It is not possible to discuss the many investigations that indicate impor-
tant relationships of folic acid, folinic acid, and By, or their derivatives,
to choline and to methyl metabolism. That certain of these factors may be
involved in transmethylation, in formate-to-methyl synthesis, or in the
betaine-glycine-aminoethanol system appears highly probable. In so far as
the rat is concerned, observations up to the present time have demonstrated
that its ability to synthesize the methyl and the aminoethanol portions of
choline depends on one or more of these newer vitamins. Schaefer
et al.?3+ 183-185 have demonstrated that the requirement of choline for the
prevention of renal lesions and for the maintenance of normal liver lipids
in young rats on a diet low in choline and in methionine may be decreased
as much as one-half by the addition of Bi, and folic acid. These nutrients
appeared to increase the utilization of betaine and methionine in the methyl-
ation of aminoethanol to choline and of betaine in the methylation of homo-
cystine to methionine. In general, Bip was effective alone if present in ade-
quate amounts. If present at a subnormal level, its value was improved by
folic acid. The latter was believed ineffective in the absence of By. Hale
and Schaefer noted that weanling rats of the Sprague-Dawley strain had a
lower choline requirement for protection against renal hemorrhage and a
higher requirement for prevention of fatty livers than young of the Alabama
Experiment Station strain.!8* Lipotropic activity was ascribed to By. in the
experiments of Gyérgy'®’: 188 and to Bi. and folic acid in the studies of
Drill!!®: 189 and of Fischer.!%° Growth of rats on diets devoid of labile methyl
and containing By, and folic acid was reported by Bennett,!%* by Stekol,!°"
and by du Vigneaud.1%«

182 P. Handler and F. Bernheim, J. Biol. Chem. 148, 649 (1943).

183 A. E. Schaefer, W. D. Salmon, and D. R. Strength, Proc. Soc. Exptl. Biol. Med.
71, 193 (1949). 4

184 AE. Schaefer, W. D. Salmon, D. R. Strength, and D. H. Copeland, J. Nutrition
40, 95 (1950).

185 AE. Schaefer and J. L. Knowles, Proc. Soc. Exptl. Biol. Med. 17, 655 (1951).

186 Q, M. Hale and A. E. Schaefer, Proc. Soc. Exptl. Biol. Med. 77, 633 (1951).

187 P, Gyorgy ahd C. S. Rose, Proc. Soc. Exptl. Biol. Med. 78, 372 (1950).

188 ©, §. Rose, T. E. Machella, and P. Gyérgy, Proc. Soc. Exptl. Biol. Med. 64, 352
(1947); 67, 198 (1948).

189 H. M. McCormick and V. A. Drill, Proc. Soc. Exptl. Biol. Med. 74, 626 (1950).

199 Vf. A. Fischer and G. D. Hall, Federation Proc. 11, 211 (1952).

19a M. A. Bennett, J. Biol. Chem. 168, 247 (1946); 187, 751 (1950).

190% J. A. Stekol and K. W. Weiss, J. Biol. Chem. 186, 3438 (1950).

190¢e V. du Vigneaud, C. Ressler, J. R. Rachele, J. A. Reyniers, and T. D. Luckey,
J. Nutrition 45, 361 (1951).

84 CHOLINE

Many observations support the view that folic acid or a derivative in-
fluences formate metabolism in the rat and thereby affects the formation
of aminoethanol from serine and glycine and the formate-to-methyl syn-
thesis which may be involved in the conversion of aminoethanol to choline.
Totter et al. noted a favorable effect of folic acid on the metabolism of
glycine which was manifested by an increased excretion of porphyrins, be-
lieved to indicate improved synthesis of hemoglobin, and by a neutralization
of the symptoms of glycine deficiency produced by the administration of
sodium benzoate.!%! Elwyn and Sprinson found that the conversion of N!-
L-serine to glycine and the subsequent excretion of N!°-benzoylglycine were
reduced markedly in folic acid-deficient rats.!*? Stekol ef al. reported that
the deficiency impaired the use of the 6-carbon of serine and, to a lesser
extent, of the y-carbon of glycine in choline formation.» !%* The incorpora-
tion of C-formate into serine of liver proteins was greatly increased by the
treatment of deficient rats with folic acid,!® and the synthesis of the choline
methy] from methanol was improved by either folic acid or leucovorin.19® 1%
In confirmation of his own results!**: 19° and of those of others,?°° Williams
found in in vitro studies that symptoms of folic acid deficiency in rats caused
by feeding the antagonist, aminopterin, included an inhibition of the liver
oxidases that convert choline and betaine aldehyde to betaine and of the
enzymes that methylate homocystine.?°! In contrast to the slight influence
of a synthetic citrovorum factor, leucovorin, folic acid was necessary for
the optimal formation of N'-methylnicotinamide from administered nic-
otinamide in rats.?°

As in the case of folic acid, By. has been involved in both methyl and
formate metabolism. Oginsky found By»-deficient rats less able to form
methionine from homocystine and either choline or betaine.” The de-
ficiency had no appreciable effect on the formation of choline methyl from
methanol!®* or from the 6-carbon of serine.2“ Arnstein and Neuberger," how-

191 J. R. Totter, E.S. Amos, and C. K. Keith, J. Biol. Chem. 178, 847 (1949).

1922 JT), Elwyn and D. B. Sprinson, J. Biol. Chem. 184, 475 (1950).

193 J. A. Stekol, S. Weiss, and K. W. Weiss, Arch. Biochem. and Biophus. 36, 5 (1952).

194 J. A. Stekol, S. Weiss, B. Hsu, and P. Smith, Federation Proc. 11, 292 (1952).

195 G, W. E. Plaut, J. J. Betheil, and H. A. Lardy, J. Biol. Chem. 184, 795 (1950).

196 W. G. Verly, J. E. Wilson, J. M. Kinney, and J. R. Rachele, Federation Proc. 10,
264 (1951).

197 W. G. Verly, J. M. Kinney, and V. du Vigneaud, J. Biol. Chem. 196, 19 (1952).

198 J. N. Williams, Jr., J. Biol. Chem. 191, 123 (1951).

199 J. N. Williams, Jr., J. Biol. Chem. 192, 81 (1951).

200 J. §. Dinning, C. K. Keith, P. L. Davis, and P. L. Day, Arch. Biochem. 27, 89
(1950).

201 J, N. Williams, Jr., Proc. Soc. Exptl. Biol. Med. 78, 206 (1951).

202 T,, §. Dietrich, W. J. Monson, and C. A. Elvehjem, J. Biol. Chem. 199, 2 (1952).

203 H. L. Oginsky, Arch. Biochem. 26, 327 (1950).

204 H.R. V. Arnstein and A. Neuberger, Biochem. J. 48, ii (1951).

X. EFFECTS OF DEFICIENCY 85

ever, considered their data evidence for a role of By, in the reactions which
result in the formation of serine from glycine, and Stekol et al. arrived at
the same conclusion.!*?: !%* Choline prevented depletion of By. in the livers
of rats, but not of mice, on choline-deficient diets containing sulfasuxidine
and iodinated casein.?°° The decrease in the epinephrine content of the
adrenals of rats on a low Biz diet containing desiccated thyroid or iodinated
casein was partially prevented by Bis, and it was suggested that Bi. may
function in the formation of the methyl group of epinephrine.”°® The sar-
cosine oxidase content of livers of hyperthyroid rats deficient in folic acid
was unaffected but was reduced in By-deficient animals.2” Bis, however,
was ineffective in restoring the enzyme content.

Williams et al.?°: 2%> compared the effect of a deficiency of Bi. on liver
xanthine oxidase and betaine-homocysteine transmethylase levels and
concluded that the decreased xanthine oxidase activity was an indirect
metabolic effect, whereas the data indicated that the transmethylase may
require By. as a cofactor or as a precursor of a cofactor. Hawk and Elve-
jem?" found no evidence of a choline-sparing action of By»; , a biologically
different form of By. recently described by Lewis et al2%¢ Stekol et al.?°
observed no effect of Bi. deficiency on the transmethylation that yields
choline from methionine although the incorporation of methyl into choline
was diminished in both folic acid-deficient and pyridoxine-deficient animals.
It was suggested that By» , folic acid, and pyridoxine may be more concerned
with the synthesis of methyl acceptors than with transmethylation itself.

Dubnoff has made the interesting suggestion that one of the functions of
a Bie derivative is the reduction of homocystine to homocysteine, believed
to be the actual acceptor of methyl.?°° A By. concentrate increased the sulf-
hydryl content of rat liver slices, and this effect was particularly noticeable
in experiments on Byo-deficient rats. The activity was associated with the
By color after paper chromatography, although crystalline By. and Bio, were
negative. In subsequent observations Dubnoff demonstrated that homo-
cysteine or homocystine plus a reducing agent replaced B,. or methionine

205 J. J. Travers and L. R. Cerecedo, Federation Proc. 11, 457 (1952).

206 A. D’Jorio and G. W. E. Plaut, Arch. Biochem. and Biophys. 41, 153 (1952).

207 M. E. Swendseid, A. L. Swanson, and F. H. Bethell, Arch. Biochem. and Biophys.
41, 138 (1952).

207a J. N. Williams, Jr., W. J. Monson, A. Sreenivasan, L. 8S. Dietrich, A. E. Harper,
and C. A. Elvehjem, J. Biol. Chem. 202, 151 (1953).

207» J. N. Williams, Jr., W. J. Monson, A. E. Harper, and C. A. Elvehjem, J. Biol.
Chem. 202, 607 (1953).

207e H. A. Hawk and C. A. Elvehjem, J. Nutrition 49, 495 (1953).

207d U. J. Lewis, D. V. Tappan, and C. A. Elvehjem, J. Biol. Chem. 194, 539 (1952);
199, 517 (1952).

207e J. A. Stekol, S. Weiss, P. Smith, and K. W. Weiss, J. Biol. Chem. 201, 1 (1953).

208 J. W. Dubnoff, Arch. Biochem. 27, 466 (1950).

86 CHOLINE

for the growth of an Escherichia coli mutant (No. 113-3), a form which
requires either By. or methionine.”® The growth-promoting value of homo-
cysteine was enhanced by a trace of Bis, by a methyl donor such
as dimethyl-6-propiothetin, or by catalytic amounts of p-aminobenzoic
acid. Dubnoff suggested that Bis is necessary for the maintenance of homo-
cysteine in the reduced state and that it may also be required for the syn-
thesis of p-aminobenzoic acid which may be involved in the synthesis of
methyl groups. According to Shive, p-aminobenzoic acid, folic acid, and Bie
are involved in the metabolism of formate in microorganisms.” Ling and
Chow reported that Bi. corrected the decreased sulfhydryl content of blood
of Bie-deficient rats.?!!

Although the dog has been used in most of the investigations on lipocaic,
believed by Dragstedt to bea pancreatic hormonal anti-fatty liver factor,?”
studies on rats also have been conducted and with varying results. Data
have been presented which support the view that the lipotropic activity
of preparations of lipocaic is explained completely by their content of cho-
line and of protein.”!*-!6 In contrast to these results are findings which indi-
cate that the potency of lipocaic cannot be explained on the basis of its
content of choline, methionine, or inositol.?!7-2! More recently, successful
ligation of the pancreatic duct has been reported to result in fatty livers
in rats on a choline-deficient diet.??? As in the experiments on dogs to be
discussed later, this result appears due to the lack of a proteolytic factor
in the external secretion of the pancreas that normally releases a choline
precursor, presumably methionine, from dietary protein. Choline and the
anti-fatty liver factor of the pancreas were reported to have opposing effects
on the excretion of urinary ketones.

Rats fed a protein-free diet showed an accumulation of liver glycogen
and fat after one month, and the increase in lipid was only partially pre-

209 J. W. Dubnoff, Arch. Biochem. and Biophys. 37, 37 (1952).

210 W. Shive, Ann. N. Y. Acad. Sct. 52, 1272 (1950).

211 C.-T. Ling and B. F. Chow, Federation Proc. 11, 249 (1952).

212 T,, R. Dragstedt, J. Am. Med. Assoc. 114, 29 (1940); 115, 454 (1940).

213 F, X. Aylward and L. E. Holt, Jr., J. Biol. Chem. 121, 61 (1937).

214K}, M. MacKay and R. H. Barnes, Proc. Soc. Exptl. Biol. Med. 38, 410 (1938).

215 ©, H. Best and J. H. Ridout, Am. J. Physiol. 122, 67 (1938).

216 A. N. Wick and E. Laurence, Arch. Biochem. 20, 113 (1949).

217 H. J. Channon, J. V. Loach, and G. R. Tristram, Biochem. J. 32, 1332 (1938).

218 G. Gavin, J. M. Patterson, and E. W. McHenry, J. Biol. Chem. 148, 275 (1948).
219 T), E. Clark, M. L. Eilert, and L. R. Dragstedt, Am. J. Physiol. 144, 620 (1945).
220 M.L. Hilert and L. R. Dragstedt, Am. J. Physiol. 147, 346 (1946).

221 M. J. Raymond and C. R. Treadwell, Proc. Soc. Exptl. Biol. Med. 70, 43 (1949).
22 G. H. A. Clowes, Jr. and L. B. Macpherson, Am. J. Physiol. 165, 628 (1951).

223 V. Baccari and A. Guerritore, Boll. soc. ital. biol. sper. 24, 842 (1948).

X. EFFECTS OF DEFICIENCY 87

vented by choline.?** Extremely fatty livers resulted in choline-deficient
rats fed at 20 % protein diet for one week following 7 days of fasting or 21
days of protein depletion, although choline was effective in these experi-
ments.22> Prolonged feeding of diets low in protein and in choline resulted
in severe nutritional anemia and edema in rats.?**: 227 Both were prevented
by choline, and Engel suggested that choline was a more important factor
than dietary protein in the development of these abnormalities. The edema-
tous condition was associated with hepatic fatty infiltration and subsequent
cirrhosis. Anemia was also observed by Diaz et al. in rats fed a choline-de-
ficient ration.?*8

Weanling rats fed diets low in protein or in riboflavin or containing 1o-
dinated casein consistently showed decreased levels of liver and kidney
choline oxidase.22 Ebisuzaki and Williams observed a parallelism between
the flavin adenine dinucleotide and the choline oxidase levels of riboflavin-
deficient rats. The latter workers concluded that the riboflavin-containing
enzyme system was a part of the hydrogen transport mechanism involved
in the oxidation of choline by choline oxidase.?*

Singal et al. produced fatty livers in young rats fed low protein or low
amino acid rations deficient in essential amino acids such as lysine and
threonine but containing 0.2 % choline.*! Normal liver lipid levels resulted
from supplementation with the missing amino acid. High levels of choline
were lipotropic in the animals on the deficient diets but only partly so in
those fed the inadequate amino acid mixture. Bi. had no lipotropic action
in these experiments. As observed by Eckstein, threonine was without
effect in the absence of dietary choline. The rate of turnover of liver phos-
pholipids was increased by the administration of threonine to threonine-
deficient animals, although no such effect was noted in the turnover rate in
the kidneys or in the small intestine. These results are of unusual interest
in view of the observation that supplements of threonine in a low protein,
choline-deficient diet aggravated symptoms in young rats during the period
in which the acute phase of the deficiency occurred.!*?

24 C.F. Wang, D. M. Hegsted, A. Lapi, N. Zamcheck, and M. B. Black, J. Lab.
Clin. Med. 34, 953 (1949).

225 Q. M. Hale and A. E. Schaefer, J. Nutrition 46, 479 (1952).

226 R. W. Engel, J. Nutrition 36, 739 (1948).

227 H. D. Alexander and R. W. Engel, J. Nutrition 47, 361 (1952).

28 ©, J. Diaz, H. Castro-Mendoza, G. Paniagua, and F. Vivanco, Bull. Inst. Med.
Research, Univ. Madrid 1, 101 (1948).

229 T), A. Richert and W. W. Westerfeld, J. Biol. Chem. 199, 2 (1952).

230 K. Ebisuzaki and J. N. Williams, Jr., J. Biol. Chem. 200, 297 (1953).

231 §. A. Singal, S. J. Hazan, V. P. Sydenstricker, and J. M. Littlejohn, J. Brol.
Chem. 200, 867 (1952).

232 H. C. Eckstein, J. Biol. Chem. 195, 167 (1952).

88 CHOLINE

Morgan and Lewis noted the absence of fatty livers in rats on diets lack-
ing both choline and pantothenic acid.?%? They concluded that fat metabo-
lism was seriously deranged in these animals because of a depression of
adrenal activity resulting from the lack of pantothenic acid.

Honorato and his associates suggested that choline plays a role in the
antihemorrhagic activity of 2-methyl-1,4-naphthoquinone and reported
that the latter showed lipotropic activity.2*4 An antagonism between heparin
and choline with inactivation of the former?*® was not confirmed.” Lecoq
elt al. observed that the effect of dietary cystine on chronaxie of rats was
corrected by vitamin K plus choline.?*

5. OTHER EFFECTS

In the preceding discussion, emphasis has been placed on the renal and
hepatic changes that appear in choline-deficient rats. Many other effects
of the deficiency syndrome have been recognized, some traceable directly
to previous abnormalities in the kidneys and liver. Of unusual interest has
been the observation of Best and Hartroft,®: ?°* confirmed by others,”%%: 77°
that hypertension developed in rats 4 to 7 months after the onset of acute
choline deficiency even though fed a normal diet subsequent to the acute
episode. It had been demonstrated previously by Sobin and Landis that
hypertension did not result from a prolonged chronic deficiency of choline,”*!
and Handler ef al. noted that hypertension in partially nephrectomized
rats disappeared if a choline-deficient diet was fed.*! A partial solution of
this anomalous situation in which damage occurred as a result of restora-
tion of a normal diet was afforded by the demonstration that the hyperten-
sion following acute deficiency was prevented by decapsulation of the
kidneys.2” This suggested that the damaged capsule restricted the increase
in size of the recovering kidney as it developed on the normal diet and that
hypertension was the ultimate result as in the case of animals in which
hypertension was induced by coating the kidney with cellophane or other
restrictive materials.

In studies on the carcinogenic action of p-dimethylaminoazobenzene

233 A. F. Morgan and E. M. Lewis, J. B7ol. Chem. 200, 839 (1952).

234 C, R. Honorato and H. Molina, Rev. soc. argentina biol. 18, 481 (1942).

235 V. A. Cabezas and C. R. Honorato, Bol. soc. biol. Santiago Chile 2, 26 (1944).

236 G. W. Howe and C. L. Spurr, Proc. Soc. Exptl. Biol. Med. 71, 429 (1949).

237 R. Lecoq, P. Chauchard, and H. Mazoué, Compt. rend. soc. biol. 141, 220 (1947).

233 W.S. Hartroft and C. H. Best, Brit. Med. J. I, 423 (1949).

239 C,. Moses, G. M. Longabaugh, and R. 8S. George, Proc. Soc. Exptl. Biol. Med. 75,
660 (1950).

240 P. Handler and F. Bernheim, Am. J. Physiol. 162, 189 (1950).

241 §.§. Sobin and E. M. Landis, Am. J. Physiol. 148, 557 (1947).

242 P. Handler and F. Bernheim, Proc. Soc. Exptl. Biol. Med. 76, 338 (1951).

X. EFFECTS OF DEFICIENCY 89

(DAB), or butter yellow, demethylation prior to fission of the azo
linkage“*: “4 and methylation of the resulting p-monomethylaminoazoben-
zene2** were demonstrated in rats. This observation appeared to support
the conclusion of Jacobi and Baumann that DAB was a methyl donor be-
cause it prevented the development of renal lesions in young rats on a
choline-deficient diet.24° Later, Baumann was of the opinion that the pro-
tection against renal lesions must have had another explanation™*® in view
of the failure of choline or of choline deficiency to affect the incidence of
tumors following DAB administration.?*”-?>° Subsequently, it was shown by
the feeding of C-'-methyl-labeled DAB that the tagged carbon appeared
quickly as carbon dioxide in the expired air and that none was found in the
methyls of tissue choline or creatine.?*! Choline in drinking water did not
affect the induction of tumors in rats following the injection of 1,2,5,6-
dibenzanthracene?® or the lesions of bone marrow produced by nitrogen
mustards.”>?

Using a strain of rats with a high choline requirement,**: °° Engel et al.
observed that over one-half of chronically deficient animals developed neo-
plasms of various types whereas no lesions appeared in control animals.26 27
A similar finding was reported by Viollier.?°* Aloisi and Bonetti described
lesions in the muscles of rats suffering from a prolonged deficiency of methyl
donors.2°° The abnormality differed from the experimental dystrophy due
to a deficiency of vitamin E, but it was not possible to separate the direct
effect of a lack of methyl from the secondary influence of the hepatic
pathology.

Malignant tumors of the human brain contained elevated levels of free

243 KS. Stevenson, K. Dobriner, and C. P. Rhoads, Cancer Research 2, 160 (1942).

244 J. A. Miller, E. C. Miller, and C. A. Baumann, Cancer Research 5, 162 (1945).

245 H. P. Jacobi and C. A. Baumann, Cancer Research 2, 175 (1942).

246 ©, A, Baumann, Trans. 2nd Conf. on Biol. Antiox dants, New York, p. 114 (Oct.
9-10, 1947).

247 HC. Miller and C. A. Baumann, Cancer Research 6, 289 (1946).

248 PN. Harris, M. E. Krahl, and G. H. A. Clowes, Cancer Research 7, 162 (1947).

249 J. White and J. E. Edwards, J. Natl. Cancer Inst. 3, 43 (1942).

250 H. M. Dyer, J. Natl. Cancer Inst. 11, 1073 (1951).

251 R, A. Boissonnas, R. A. Turner, and V. du Vigneaud, J. Biol. Chem. 180, 1053
(1949).

252 J. W. Cook and R. Schoental, Brit. J. Cancer 3, 557 (1949).

253 J. Domokos and E. Kelemen, Intern. Z. Vitaminforsch. 21, 443 (1950).

254 R. W. Engel, Proc. Soc. Exptl. Biol. Med. 52, 281 (1943).

255 T). H. Copeland, Proc. Soc. Exptl. Biol. Med. 57, 33 (1944).

256 1), H. Copeland and W. D. Salmon, Am. J. Pathol. 22, 1059 (1946).

257 R. W. Engel, D. H. Copeland, and W. D. Salmon, Ann. N. Y. Acad. Sct. 49, 49
(1947).

258 H. Staub, G. Viollier, and A. Werthemann, Experientia 4, 233 (1948).

259 M. Aloisi and E. Bonetti, Arch. sct. biol. (Italy) 36, 206 (1952).

90 CHOLINE

and combined choline,?® and increased concentrations were also found in
transplanted epidermal carcinomas in mice.?*! In the latter studies the in-
crease appeared to be due to phospholipids.

The passage of fat through the intestinal mucosa during absorption was
accelerated by feeding choline with the fat, according to Frazer.?® Tidwell
presented evidence for the view that choline played a chemical role and did
not influence absorption because of an effect on emulsification.’ This view
is in line with the favorable influence of choline on phospholipid
synthesis.2*: ®» 264 Directly or indirectly, choline affected the intestinal ab-
sorption and distribution of vitamin A. Rats on a choline-deficient diet
containing liberal amounts of carotene developed fatty livers poor in vi-
tamin A although the kidneys were rich in the vitamin.?® The addition of
choline to solutions of vitamin A in olive oil yielded higher levels of liver
vitamin A after oral administration in normal rats.?°

Choline and pyridoxine were protective against the hyperplasia and ul-
cerations that appeared in the forestomach of rats fed a diet containing
white flour and cystine as the sources of nitrogen.?*? Sharpless suggested
that regurgitated bile might be an important cause of the lesions and that
choline prevented regurgitation by stimulation of the smooth muscle of
the intestine.?°

Abdon and Borglin reported that an impairment of oxidative metabolism
was an early symptom of choline deficiency in rats. The 7m vitro oxygen con-
sumption of minced muscle tissue from deficient rats was not increased by
choline or methionine but was returned to a normal value by an acetone-
insoluble preparation of splenic tissue.?&? Olson and Deane noted decreased
respiration of kidney slices during the acute phase of hemorrhagic degener-
ation.!6

Dinning et al. demonstrated that leucopenia was present in rats main-
tained for 65 days on a diet low in methionine (0.27 %) and in By and con-
taining a moderate level of choline (0.1%). The interference with leucocyte
formation was prevented by supplemental methionine with or without folic

260 J), Vincent, S. Daum, and M. Bouchet, Trav. membres soc. chim. biol. 23, 18638
(1941).

261 M. G. Ritchey, L. F. Wicks, and E. L. Tatum, J. Biol. Chem. 171, 51 (1947).

262 A.C. Frazer, Nature 157, 414 (1946).

263 H.C. Tidwell, J. Biol. Chem. 182, 405 (1950).

264 H. D. Friedlander, I. L. Chaikoff, and C. Entenman, J. Biol. Chem. 158, 231 (1945).

265 H. Popper and H. Chinn, Proc. Soc. Exptl. Biol. Med. 49, 202 (1942).

266 A Pederzini, Boll. soc. ital. biol. sper. 24, 1146 (1948).

267 G. R. Sharpless, Proc. Soc. Exptl. Biol. Med. 45, 487 (1940).

268 QR. Sharpless and M. Sabol, J. Nutrition 25, 113 (1943).

269 N. QO. Abdon and N. E. Borglin, Nature 158, 793 (1946) ; 159, 272 (1947) ; Acta Phar-
macol. Toxicol. 8, 73 (1947).

X. EFFECTS OF DEFICIENCY 91

acid and By, or by additional choline or betaine with supplements of folic
acid and By.” Later it was shown that leukemic mice excreted larger
quantities of creatine and allantoin than normal animals” and that this was
the result of increased synthesis of creatine.?”? Kelley et al. caused marked
leucocytosis in rats by feeding excessive levels of glycine (10 %) and choline
(2%). Riboflavin and folic acid were essential for this effect.?7

Chévremont concluded that choline was responsible for spontaneous trans-
formation of cells of cultures of skeletal muscle or of subcutaneous connec-
tive tissue into histiocytes.2? The change of muscle cells into histiocytes,
believed to be caused by high concentration of choline, was observed also
by Firket and Cornil.?”*

Abdon and Borglin observed bradycardia in choline-deficient rats, which
disappeared quickly following intravenous administrations of choline.?7®
Choline restored to normal an increased level of pseudocholinesterase in
male rats and a decreased level in female rats.?”

Guggenheim and Olson determined the specific activity of tissue fatty
acids after administration of carboxyl-labeled acetate and were unable to
find evidence of impaired lipogenesis in choline-deficient rats.?7* Deuel e¢ al.
showed that the lipotropic activity of choline was not due to an increased
rate of oxidation of fat.27? The deposition of vitamin A in the livers of de-
pleted rats was not affected by choline deficiency or by the presence of a
fatty liver.2®° Impaired metabolism of carbohydrate and of fat, as well as
functional damage to liver cells, decreased the hepatic synthesis of choline.?*!
Elevated environmental temperature increased the choline requirement of
~ rats.28 The report that choline deficiency resulted in a loss of contractility of
the uterus and partial atrophy of both uterus and ovaries in rats? has not

270 J. S$. Dinning, L. D. Payne, and P. L. Day, Arch. Biochem. 27, 467 (1950); J. Nutri-
tion 48, 525 (1951).

271 J. S. Dinning and L. D. Seager, Sctence 114, 2967 (1951).

272 J. S. Dinning, L. D. Seager, L. D. Payne, and J. R. Totter, Science 116, 121 (1952).

273 B. Kelley, M. Northrup, and P. D. Hurley, Proc. Soc. Exptl. Biol. Med. 76, 804
(1951).

274 M. Chévremont, J. Morphol. 76, 139 (1945).

275 J. Firket and A. Cornil, Compt. rend. soc. biol. 189, 51 (1945).

276 N. O. Abdon and N. E. Borglin, Acta Phormacol. Toxicol. 2, 247 (1946).

277 R. D. Hawkins and M. T. Nishikawara, Biochem. J. 48, 276 (1951).

278 K. Guggenheim and R. E. Olson, J. Nutrition 48, 345 (1952).

279 H. J. Deuel, Jr., S. Murray, L. F. Hallman, and D. B. Tyler, J. Biol. Chem. 120,
277 (1937).

280 T,. S$. Bentley and A. F. Morgan, J. Nutrition 31, 333 (1946).

281 H. K. Barrenscheen and D. Papadopoulou, Hople-Seyler’s Z. physiol. Chem. 284,
236 (1949).

282 ©. A. Mills, Proc. Soc. Exptl. Biol. Med. 54, 265 (1943).

283 M. Peet and M. M. Sampson, Scvence 107, 548 (1948).

92 CHOLINE

been confirmed.2* Sure concluded that choline was indispensable for lacta-
tion and growth in rats.?8> Rustiness of the hair was produced by omission
of either choline or pantothenic acid from the diet.2*° On the other hand,
so-called bronzing of the hair in young rats was ascribed to the choline
intake and was prevented by dietary yeast or by the presence of dextrin
in the ration.2%’ Dietary choline prevented toxic effects of excess
methionine.?**
6. FatE oF CHOLINE

The data of Luecke and Pearson may be cited as confirmatory evidence
of the conclusion of earlier investigators?’?: 2% that administered choline
and betaine disappear rapidly.2*! Of 10 to 20 g. of choline administered to
sheep, only 1 % was recovered in the urine during a 48-hour period, an equal
amount was found in the feces, and no accumulation was detected in liver,
kidney, or blood. Similar results were found in dogs. No excretion of betaine
followed the ingestion of 20 g. of this substance in a sheep. Davies showed
that 14 to 43 % of the nitrogen of choline and of betaine in the diet of cows
was excreted as trimethylamine oxide.?® Small amounts of trimethylamine
and traces of the di- and monomethylamines were found. Davies noted
that cattle fed sugar beet by-products consumed as much as 100 g. of betaine
daily without the excretion of detectable amounts. It is probable that tri-
methylamine is the result of the action of intestinal microorganisms on
choline or betaine,?** inasmuch as many organisms are known to have this
effect.2%295 Whether or not animal tissues also form trimethylamine needs
further study. This substance was identified as a product of in vitro me-
tabolism in liver slices, but the sterility of the system was not dem-
onstrated.2*%” The rapid disappearance of repeated sublethal doses of choline
in rats and mice was reported by Kahane et al.?°*

284 CO, P. Kraatz and C. M. Gruber, Sczence 109, 310 (1949).

235 B. Sure, J. Nutrition 19, 71 (1940).

286 H. S§. Owens, M. Trautman, and E. Woods, Science 93, 406 (1941).

287 G. M. Higgins, O. R. Joneson, and F. C. Mann, Proc. Staff Meetings Mayo Clinic
20, 320 (1945).

288 J. S. Roth and J. B. Allison, J. Biol. Chem. 183, 173 (1950).

289 M. Guggenheim and W. Léffler, Biochem. Z. 74, 208 (1916).

290 H. Fuchs, Z. Biol. 98, 473 (1938).

291 R. W. Luecke and P. B. Pearson, J. Biol. Chem. 158, 561 (1944).

2922 W.L. Davies, J. Dairy Sct. 7, 14 (1936).

293 H.R. Norris and G. J. Benoit, Jr., J. Biol. Chem. 158, 443 (1945).

294 JT), Ackermann and H. Schiitze, Zentr. Physiol. 24, 210 (1910).

295 A. J. Wood and F. E. Keeping, J. Bacteriol. 47, 309 (1944).

296 G. N. Cohen, B. Nisman, and M. Raynaud, Compt. rend. 225, 647 (1947).

297 CO, Artom and M. Crowder, Arch. Biochem. 29, 227 (1950).

2098 B. Kahane, J. Lévy, R. Bourgeois, and O. Tanguy, Arch. sci. physiol. (Italy) 4,
173 (1950).

X. EFFECTS OF DEFICIENCY 93

A most extensive study of the urinary excretion of choline in rats and
in man has been carried out by Borglin.2*? The output in the urine of rats
varied with the intake and amounted to 0.27 to 0.44% of the choline in
the food. The dietary choline was the principal factor governing excretion,
the presence or absence of other sources of methyl, of protein, and of fat
having relatively little effect.

The oxidation of methyl groups of choline to carbon dioxide has been
noted previously, and it is reasonable to assume that the nitrogen of the
demethylated choline or betaine is excreted as urea. There is some question
regarding the fate of administered trimethylamine. Davies reported the
quantitative excretion of this compound and of its oxide as the oxide in the
cow.2” Langley, however, recovered only 20 % of the trimethylamine given
to rabbits; the remainder of the nitrogen was believed to be excreted as
urea.*%

C. AVIAN SPECIES

WENDELL H. GRIFFITH and JOSEPH F. NYC

The primary effects of a deficiency of choline in chicks are interference
with growth and the appearance of perosis or ‘‘slipped tendon disease.”
This characteristic abnormality is influenced by multiple factors but, if
caused by a lack of choline, is prevented readily by supplements of choline
or of compounds with specific choline activity. There is gross enlargement
_ of the tibial-metatarsal joint, twisting or bending of the distal portion of
the tibia and of the proximal end of the metatarsus, and slipping of the
gastrocnemius tendon from its condyles. If this occurs in severe form, the
chick is incapacitated. Until 1940 a lack of manganese was considered the
principal cause of perosis.* In this year Jukes demonstrated that in cer-
tain diets, adequate in manganese, choline was an effective antiperotic and
erowth-stimulating agent.?” This unexpected finding was soon confirmed
in a number of laboratories.3-3%

An understanding of the relation of choline to the prevention of perosis
is complicated by the fact that the abnormality is aggravated by creatine,*”°

2099 N. E. Borglin, Acto Pharmacol. Toxicol. 3 Suppl. 1 (1947).

300 W. D. Langley, J. Biol. Chem. 84, 561 (1929).

301 H. §. Wilgus, L. C. Norris, and G. F. Heuser, J. Nutrition 14, 155 (1937).

302 T. H. Jukes, J. Nutrition 20, 445 (1940).

303 AG. Hogan, L. R. Richardson, H. Patrick, and H. L. Kempster, J. Nutrition 21,
327 (1941).

304 TD), M. Hegsted, R. C. Mills, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 188,
459 (1941).

305 P. R. Record and R. M. Bethke, Poultry Sci. 21, 271 (1942).

306 TH. Jukes, Proc. Soc. Exptl. Biol. Med. 46, 155 (1941).

94 CHOLINE

by glycine,*”: *°8 and by gelatin. *°7: 99 Briges showed that 10% gelatin
in chick diets increased the need of niacin and might cause niacin defi-
ciency.*!” This effect was counteracted by either niacin or tryptophan. Per-
osis caused by egg white injury*" was prevented by biotin.*!? In so far as
choline is concerned, its antiperotic effect does not necessarily depend on
the same deficiency state in which its growth-stimulating action is evident.
In choline-deficient chicks neither methionine*®”: *” nor betaine*?: 3% was
antiperotic unless the diet contained mono- or dimethylaminoethanol. This
is due to the fact that the chick is practically unable to convert
aminoethanol into monomethylaminoethanol.*!*: *44 On the other hand, ar-
senocholine is strongly antiperotic and growth-promoting,*”: #° even
though it does not cause the methylation of homocysteine.*!® This analog
of choline is known to be incorporated into the lecithin molecule,” and
it is a fair assumption that this occurs in the chick also. From the data in
Tables III to VI it is evident that antiperosis in choline-deficient chicks is
a function of choline, possibly in phospholipid form, and not a general func-
tion of sources of labile methyl. In this connection it should be noted that
4 and 9%, respectively, of the tagged methyl of C'-methyl-labeled methio-
nine were found in tissue choline isolated from 2 chicks 48 hours after the
feeding of amino acid.*!8 This is not conclusive evidence of transmethyla-
tion, even at a relatively slow rate, because the labeled choline may have
resulted from the methylation of dietary dimethylaminoethanol through
C-formate resulting from the partial oxidation of the labeled methionine
methyl.

For normal growth chick rations must contain 1.1% of methionine, one-
half of which may be replaced by cystine.*!® Homocystine can be substituted
for cystine and for methionine also if the supply of methyl donors is ade-
quate. Choline and betaine are suitable donors.

The requirement of chicks for choline has been reported as 150 and 100
mg.%, respectively, for a diet of natural foodstuffs*® and for a purified
diet.*** The allowance recommended by the Committee on Animal Nutri-

307 T. H. Jukes, J. Nutrition 22, 315 (1941).

308 TH. Jukes, and E. L. R. Stokstad, J. Nutrition 48, 459 (1951) ; 48, 209 (1952).
309 H. L. Lucas, L. C. Norris, and G. F. Heuser, Poultry Sct. 25, 93 (1946).

310 G, M. Briggs, J. Biol. Chem. 161, 749 (1945).

311 T,, W. McElroy and T. H. Jukes, Proc. Soc. Exptl. Biol. Med. 45, 296 (1940).
312 T, H. Jukes and F, H. Bird, Proc. Soc. Exptl. Biol. Med. 49, 231 (1942).

313 A. EH. Schaefer, W. D. Salmon, and D. R. Strength, J. Nutrition 44, 305 (1951).
314TH. Jukes, J. J. Oleson, and A. C. Dornbush, J. Nutrition 30, 219 (1945).

315 T, H. Jukes and A. D. Welch, J. Biol. Chem. 146, 19 (1942).

316 H. J. Almquist and T. H. Jukes, Proc. Soc. Exptl. Biol. Med. 51, 243 (1942).

317 A, D. Welch and R. L. Landau, J. Biol. Chem. 144, 581 (1942).

318 K. A. Burke, R. F. Nystrom, and B. C. Johnson, J. Biol. Chem. 188, 723 (1951).
319 C.R. Grau and H. J. Almquist, J. Nutrition 26, 631 (1943).

X. EFFECTS OF DEFICIENCY 95

tion of the National Research Council is 130 mg. %.**° For diets containing
homocystine in place of methionine, 60 mg. % of betaine-irreplaceable and
140 mg. % of betaine-replaceable choline are required, according to Alm-
quist.#2! Minimum levels of methionine and of irreplaceable choline were
reported as 500 and 100 mg. %, respectively, by McKittrick.*? West e¢ al.
found 100 mg. % of choline adequate if ample methionine was provided.*”
The growth-depressing action of excess methionine in chick diets was
counteracted by guanidoacetic acid and by serine but not by decreasing
dietary cystine. Excess homocystine did not depress growth unless choline
was also present. McKittrick also noted that betaine was a satisfactory
replacement of part but not of all the choline requirement.

Although the significance of betaine-replaceable choline in the chick is
not readily apparent in view of the reported failure of betaine to show anti-
perotic activity, the distinction between the two parts of the choline re-
quirement has emphasized the role of Bi, in this phase of avian metabolism.
Patton et al. noted a beneficial growth effect of fish meal in chick diets com-
posed mainly of corn and soybean oil meal.**4 Choline, betaine, and methio-
nine were also reported to have an interchangeable supplementary effect
in the improvement of the corn-soybean ration.*”*: #6 Gillis and Norris found
liver paste superior to supplements of choline or betaine and later obtained
similar results with By» in place of liver paste.®”? Schaefer et al. reported that
both folic acid and By». decreased the choline requirement for growth.1**: **
Jukes and Stokstad demonstrated that Bi, spared the choline requirement
for growth but not for prevention of perosis.*°* The response to homocystine
in the presence of By. was augmented by the addition of either choline or
betaine. However, a supplement of methionine has been reported to decrease
the By. requirement of chicks on a diet lacking animal protein.”® Methyla-

20 W. W. Cravens, H. J. Almquist, R. M. Bethke, L. C. Norris, and H. W. Titus,
Recommended Nutrient Allowances for Poultry, National Research Council
(U.S.), Washington, D. C., 1944, revised 1946.

321 H. J. Almquist, Science 103, 722 (1946). 7

322 TD). §. McKittrick, Arch. Biochem. 15, 133 (1947); 18, 437 (1948).

323 J. W. West, C. W. Carrick, 8S. M. Hauge, and E. T. Mertz, Poultry Sci. 30, 880
(1951).

324 AR. Patton, J. P. Marvel, H. G. Petering, and J. Waddell, J. Nutrition 31, 485
(1946).

325 J. A. Marvel, C. W. Carrick, R. E. Roberts, and S. M. Hauge, Poultry Sci. 28,
294 (1944).

326 TD). H. Mishler, C. W. Carrick, and 8. M. Hauge, Poultry Sct. 28, 24 (1949).

227 M. B. Gillis and L. C. Norris, J. Biol. Chem. 179, 487 (1949); Poultry Sct. 28, 749

(1949).

328 AE. Schaefer, W. D. Salmon, and D. R. Strength, Proc. Soc. Expil. Biol. Med. 71,
202 (1949).

#29 G, M. Briggs, E. G. Hill, and M. J, Giles, Poultry Sci. 29, 723 (1950).

96 CHOLINE

tion of mono-and dimethylaminoethanol in chicks fed betaine did not occur
unless Bis was present.?”

Contrary to the results of others, McGinnis et al. found methionine and
betaine effective antiperotic agents in certain diets, but this result was not
obtained if more highly purified diets were employed.*” In later experi-
ments, Gillis and Norris observed the same stimulation of growth by betaine
and by choline if the Bi. stores were normal but inferior growth with choline
if the By, stores were low.**! The absence of an effect of Bi, on the utilization
of betaine was also demonstrated by the equal stimulation of growth of
B,»-deficient chicks by methionine and by homocystine plus betaine.**?

Thus, the interrelationships of choline, betaine, and By. in the chick are
not clear. Gillis and Norris do not believe that Bi. is concerned with the
conversion of choline to betaine by choline oxidase because no evidence was
found of a decreased content of this enzyme in the livers of Bis-deficient
chicks.**! They concluded that at least one mechanism by which the chick
uses choline is impaired by a deficiency of Biz whereas betaine remains effec-
tive under the same conditions.

The depression of choline oxidase in the bone marrow?” and liver**? of
chicks deficient in folic acid and the reduction of this enzyme in the bone
marrow of hens exposed to x-irradiation*** have been mentioned previously.
Choline oxidase in the livers of chick embryos was decreased by folic acid
fed to the mother hen whereas much less reduction occurred if folic acid
was injected directly into the egg during or prior to incubation.**° Con-
flicting evidence has been presented regarding the need of dietary choline
by pullets and older hens. Abbott and DeMasters reported increased egg
production with dietary choline,*° but no significant differences in this
respect or in hatchability, fertility, or body weight were noted by Lucas
et al.**7 Hatchability was not improved in the study of Bethke et al.***
Ringrose and Davis likewise found no change in egg production with added
choline. It was noted in the latter experiments that twice as much choline
was present in the egg yolks as was present in the diet.**? Total blood choles-
terol and cholesterol ester and the levels of these components of aorta, heart
330 J, McGinnis, L. C. Norris, and G. F. Heuser, Proc. Soc. Exptl. Biol. Med. 56, 197

1944).
331 te = Gillis and L. C. Norris, J. Nutrition 48, 295 (1951).
332 M. B. Gillis and L. C. Norris, Proc. Soc. Exptl. Biol. Med. 77, 13 (1951).
333 J. §. Dinning, C. K. Keith, and P. L. Day, J. Biol. Chem. 189, 515 (1951).
334 J. §. Dinning, I. Meschan, C. K. Keith, and P. L. Day, Proc. Soc. Exptl. Biol.

Med. 74, 776 (1950).

335 J. N. Williams, Jr., M. L. Sunde, W. W. Cravens, and C. A. Elvehjem, J. Biol.

Chem. 185, 895 (1950).

336 O. D. Abbott and C. U. DeMasters, J. Nutrition 19, 47 (1940).

337 H. L. Lucas, L. C. Norris, and G. F. Heuser, Poultry Sci. 25, 373 (1946).

338 R. M. Bethke, D. C. Kennard, and V. D. Chamberlin, Poultry Sez. 25, 579 (1946).
339 R. C. Ringrose and H. A. Davis, Poultry Sct. 25, 646 (1946).

X. EFFECTS OF DEFICIENCY 97

muscle, and liver have been reported by Herrmann to be reduced by sup-
plements of 0.5 g. of choline chloride daily in the diet of old laying hens.*”

Ducklings require choline for growth and for prevention of perosis*™!: 3”
and of fatty infiltration of the liver.* Betaine was not found to be anti-
perotic or growth-stimulating, nor was methionine lipotropic in this
species.3”

Young turkeys require choline, and the effects of a deficiency are similar
to those in the chick. Perosis is produced more easily, severe symptoms ap-
pearing without the presence in the diet of aggravating materials such as
creatine, gelatin, or glycine. The antiperotic requirement of choline in poults
is greater than the requirement for growth.*?: *4%-347 Methionine has no ac-
tivity, although it is indispensable in the ration of the turkey.*S As in the
chick, arsenocholine is antiperotic, but betaine*? and creatine*® have been
reported ineffective. Scott*” and McGinnis,*** however, have observed that
betaine has a considerable degree of antiperosis if some choline is present
in the diet. Scott also found that creatine and sarcosine are as active as
betaine. This is an interesting finding in view of the demonstration that
choline was never completely effective as an antiperotic agent in diets lack-
ing animal protein unless either betaine or relatively high levels of glycine
and By were provided. According to Kratzer the effectiveness of choline
for growth was increased by Bi, but its antiperotic activity was decreased,
and the possibility was suggested that more choline was used in growth and
less in antiperosis in the presence of By.*°° Betaine caused only a small
- increase in growth and did not prevent perosis on a low choline diet. Biotin
and an unidentified organic substance®*! and niacin**? are believed to be
the contributing factors which with choline prevent perosis in the poult.

PD. DOG
WENDELL H. GRIFFITH and JOSEPH F. NYC

Choline is important in canine nutrition although it may be partly dis-
pensable in certain diets containing adequate levels of factors such as Bus,

340 G. R. Herrmann, Proc. Soc. Exptl. Biol. Med. 61, 302 (1946).

341 A. Roos, D. M. Hegsted, and F. J. Stare, J. Nutrition 32, 473 (1946).

342 R. Bernard and J. M. Demers, J. Cancer Research 27E, 281 (1949).

343 T. H. Jukes, J. Biol. Chem. 134, 789 (1940).

344 M. Rhian, Wash. Agr. Expt. Sta. Bull. 410, 33 (1941).

345 R. J. Evans, M. Rhian, and C. I. Draper, Poultry Sci. 22, 88 (1943).

346 R. J. Evans, Poultry Sct. 22, 266 (1943).

347 M. L. Scott, J. Nutrition 40, 611 (1950).

348 T. H. Jukes, Poultry Sci. 20, 251 (1941).

349 J. McGinnis, Poultry Sci. 25, 91 (1946).

350 F. H. Kratzer, J. Nutrition 48, 201 (1952).

351 H, Patrick, R. V. Boucher, R. A. Dutcher, and H. C. Knandel, J. Nutrition 26,
197 (1943).

382 G. M. Briggs, J. Nutrition 31, 79 (1946).

98 CHOLINE

as appears to be the case in the rat. Fouts produced a severe deficiency in
dogs which was characterized by loss of weight, anemia, dermal and peptic
ulcers, fatty cirrhotic livers, and death, and which responded to the com-
bined administration of choline and liver extract.**? It was also apparent
that choline is one of the nutrients essential for growth and for hemoglobin
production in young dogs.**!: > Hough et al. observed a decrease in hepatic
dye clearance and an elevation of serum phosphatase in puppies and in
adult dogs on choline-deficient diets.*°* Both manifestations of impaired
liver function were prevented or reversed by choline. McKibbin et al. de-
veloped a ration which produced fatal choline deficiency in 3 weeks or less
in puppies.**7 358 In addition to decreased bromosulfalein elimination and
increased plasma phosphatase, a fall in plasma cholesterol and in its ester,
and increase in prothrombin time, and decreases in hemoglobin, hemato-
crit, and plasma proteins were noted. Total lipids in the liver increased
markedly. In an extensive analysis of the tissues of normal and choline-
deficient puppies,**? little or no change was found in the lipids of the cere-
brum, spleen, pancreas, kidney, heart, and lungs, but a marked shift in
the pattern of the liver and plasma lipids was evident. In the liver this was
characterized by a decrease in choline and by an increase in sphingosine and
in other undetermined nitrogenous components of lipids, although the total
phospholipids were unchanged. In the plasma there was a marked decrease
in total phospholipids. The data suggested that sphingomyelins were re-
placing lecithins, but the percentage of choline phospholipids in the plasma
remained the same. Schaefer et al. were able to produce chronic choline de-
ficiency in dogs which was characterized by cirrhotic livers, edema, and
duodenal ulcers.*®° The protective level of choline was halved by adminis-
tration of By. Burns and McKibbin also observed this effect of Biz and
noted that liver impairment in some puppies was prevented completely
by Bie alone.3®
Chaikoff and coworkers have studied the mechanisms by which choline
exerts its lipotropic activity in dogs by means of determinations of the
363 P, J. Fouts, J. Nutrition 25, 217 (1943).
354 W. R. Ruegamer, L. Michaud, C. A. Elvehjem, and E. B. Hart, Am. J. Physiol.
145, 23 (1945).
355 A.B. Schaefer, J. M. McKibbin, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med.
47, 365 (1941).
356 V. H. Hough, E. P. Monahan, T. W. Li, and 8. Freeman, Am. J. Physiol. 189, 642
1943).
Sey : een ae S. Thayer, and F. J. Stare, J. Lab. Clin. Med. 29, 1109 (1944).
358 J. M. McKibbin, R. M. Ferry, Jr., S. Thayer, E. G. Patterson, and F. J. Stare,
J. Lab. Clin. Med. 30, 422 (1945).
359 J. M. McKibbin and W. E. Taylor, J. Biol. Chem. 185, 357 (1950).
360 A.B. Schaefer, D. H. Copeland, and W. D. Salmon, J. Nutrition 48, 201 (1951).
361 M. M. Burns and J. M. McKibbin, J. Nutrition 44, 487 (1951).

X. EFFECTS OF DEFICIENCY 99

turnover rate of phosphorus-containing compounds after the administration
of P*®®. Single feedings of choline increased the activity of the choline-contain-
ing phospholipids.*® Measurements on samples of blood and on liver biopsy
specimens after the administration of choline and of P® led to the conclusion
that the removal of liver fat under the influence of choline does not involve
increased transport of fat from liver to peripheral tissues via plasma phos-
pholipids.*® No increase in the calculated turnover rates of plasma lecithin
or sphingomyelin was found, contrary to a previous conclusion on this
point.2*! However, determinations of rates of disappearance of these two
phospholipids from plasma showed that the turnover rate of lecithin was
five times as great as that of sphingomyelin.** It was suggested that choline
acts on the utilization of fat within the liver itself and that glycerylphos-
phoric acid is a precursor of lecithin.*® The greater activity of liver lecithin
phosphorus than that of cephalin rules out the possibility of synthesis of
lecithin by the methylation of cephalin.

Liver lipids were unchanged after hypophysectomy even though the dogs
were observed as long as 32 months.*® Thyroidectomy, however, resulted
in fatty livers. The increase in lipids was more rapid and of greater magni-
tude in force-fed dogs if both thyroid and hypophysis were removed. The
hepatic changes were prevented for the most part by choline, but this sub-
stance did not prevent an increase in blood lipids.

Davis has reported a depression of cobalt-induced polycythemia in dogs
by choline*®* and has extended his findings to the involvement of choline in
an experimental anemia and in pathological changes in the nervous system
‘related to changes observed in pernicious anemia.**”: * This effect of cho-
line, ascribed by Davis to a vasodilator action on bone marrow arterioles
with a resulting decrease in hematopoiesis,*®® has not been confirmed.*”
Choline was ineffective in reducing arteriosclerotic lesions in dogs fed a
diet containing added cholesterol and thiouracil.*”! A limited beneficial ac-
tion of choline was observed in dogs with fat emboli produced by bone mar-
row curettage.” The latter study was prompted by the finding that the

362 ©. Entenman, I. L. Chaikoff, and H. D. Friedlander, J. Biol. Chem. 162, 111 (1946).

363 T). B. Zilversmit, C. Entenman, and I. L. Chaikoff, J. Biol. Chem. 176, 193 (1948).

364 T). B. Zilversmit, C. Entenman, and I. L. Chaikoff, J. Biol. Chem. 176, 209 (1948).

365 ©, Entenman, I. L. Chaikoff, and F. L. Reichert, Endocrinology 42, 210 (1948);
C. Entenman, I. L. Chaikoff, F. L. Reichert, and T. Gillman, zbzd. 42, 215 (1948).

366 J. EX. Davis, Am. J. Physiol. 127, 322 (1939); J. Pharmacol. 70, 408 (1940).

367 J. KH. Davis and D. E. Fletcher, J. Pharmacol. 88, 246 (1946).

368 J. KH. Davis, Science 105, 43 (1947).

369 J. KH. Davis, Am. J. Physiol. 142, 65 (1944).

370 M. F. Clarkson and C. H. Best, Science 105, 622 (1947).

371 J. D. Davidson, W. Meyer, and F. E. Kendall, Circulation 3, 332 (1951).

372. M. Monson and C. Dennis, Proc. Soc. Exptl. Biol. Med. 70, 330 (1949).

100 CHOLINE

fat content of human bone marrow was decreased markedly following the
intravenous administration of choline.*”*

Interest in choline as an anti-fatty liver factor originated with the first
experiments designed to test whether the administration of msulin was
sufficient to replace the internal secretion of the pancreas in depancreatized
dogs.*"*» 7° The manner in which these studies led to the recognition of the
lipotropic activity of choline has been described in Section IV, p. 19. In 1936
Dragstedt and coworkers published the first of a series of papers on lipocaic,
a factor believed to be a true hormonal product of the pancreas and in-
volved in lipid metabolism in the liver.?!: *7°879 Fatty infiltration and de-
generation, the hepatic changes in depancreatized dogs, were not observed
in animals after ligation of the pancreatic ducts or after preparation of total
pancreatic fistulae. Pancreatic juice was ineffective, if administered orally.
Choline was not believed to be the main protective agent because the po-
tency of raw pancreas was considered much greater than its content of
choline. For these reasons Dragstedt concluded that the external digestive
secretion of the pancreas was not an important factor. This latter finding
has not been confirmed. Ralli et al. were unable to find any difference be-
tween the fatty changes in the livers of dogs after pancreatectomy and
after ligation of the ducts,**° and Best and Ridout could find no satisfactory
evidence for lipocaic as a second pancreatic hormone.?!’ This problem was
reinvestigated by Chaikoff and coworkers, who arrived at the following
conclusions: (a) a fraction can be prepared from the pancreas which is more
protective than can be accounted for on the basis of its content of choline,
although sufficient choline is as effective as the pancreatic fraction ;**! (b)
ligation of the ducts does result in a fatty liver, and the liver changes are
prevented by the oral administration of pancreatic juice;**? (c) the fatty

373 F. B. Moosnick, E. M. Schleicher, and W. E. Peterson, J. Clin. Invest. 24, 228
(1945).

374 N. EF. Fisher, Am. J. Physiol. 67, 634 (1924).

375 FN. Allan, D. J. Bowie, J. J. R. Macleod, and W. L. Robinson, Brit. J. Hxpit.
Pathol. 5, 75 (1924).

376 J. Van Prohaska, L. R. Dragstedt, and H. P. Harms, Am. J. Physiol. 117, 166,
175 (1936).

377 W. C. Goodpasture, C. W. Vermeulen, P. B. Donovan, and L. R. Dragstedt, Am.
J. Physiol. 124, 642 (1938).

378 QO. C. Julian, D. E. Clark, J. Van Prohaska, C. Vermeulen, and L. R. Dragstedt,
Am. J. Physiol. 138, 264 (1942-43).

379 J. G. Allen, C. Vermeulen, F. M. Owens, Jr., and L. R. Dragstedt, Am. J. Physiol.
138, 352 (1942-43).

380 F}, P. Ralli, S. H. Rubin, and C. H. Present, Am. J. Physiol. 122, 43 (1938).

381 C, Entenman and I. L. Chaikoff, J. Biol. Chem. 188, 477 (1941).

382 T. L. Chaikoff, C. Entenman, and M. L. Montgomery, J. Biol. Chem. 160, 387
(1945).

X. EFFECTS OF DEFICIENCY 101

changes that occur in the livers of pancreatectomized or of duct-ligated
dogs can be prevented by the feeding of pancreas, pancreatic extracts,
methionine, choline, or merely by substituting hydrolyzed casein for the
casein of the basal diet;*** and, (d) trypsin is a possible factor that ensures
the liberation of methionine from dietary protein.**! In view of this solution
of the problem it is of interest that Ralli and Rubin had noted earlier that
pancreatectomized dogs fed extracted meat powder did not develop fatty
livers whereas those fed the untreated raw meat did so, a difference believed
due to the greater digestibility of the meat powder.*** Haanes and Gyoérgy
have extended the results of Chaikoff by showing that a highly lipotropic
pancreatic fraction without proteolytic activity actually contained trypsin
and trypsin inhibitor and that the inhibitor was overcome by enterokinase
in duodenal juice.**® Even though these data represent substantial support
for the enzymatic nature of lipocaic, it is possible that other lipotropic sub-
stances may occur in pancreas. Schilling et al. have reported the presence
in preparations of lipocaic of a non-enzymatic, heat-stable lipotropic ma-
terial 387 , 388

EK. OTHER SPECIES

WENDELL H. GRIFFITH and JOSEPH F. NYC

An increase in liver fat and an abnormality of gait without retardation
of growth were reported in swine after the feeding of a low choline ration.**®
Choline has been found necessary, in addition to pantothenic acid and py-
. ridoxine, for protection of pigs from locomotor incoordination resulting
from nerve degeneration.’ Poor reproduction, lactation, and survival of
young resulted from feeding sows a choline-deficient ration.*®! Baby pigs
fed a synthetic diet low in choline but containing 0.8 % methionine showed
gross symptoms of unthriftiness, were short-legged and pot-bellied, lacked
coordination of movements and the normal rigidity of joints, and exhibited
fatty infiltration of the liver, characteristic renal glomerular occlusion, and

383 T. L. Chaikoff, C. Entenman, and M. L. Montgomery, J. Biol. Chem. 168, 177
(1947).

384 M. L. Montgomery, C. Entenman, I. L. Chaikoff, and H. Feinberg, J. Biol. Chem.
185, 307 (1950).

385 }. P. Ralli and 8. H. Rubin, Am. J. Physiol. 188, 42 (1942-1943).

386 M. L. Haanes and P. Gyoérgy, Am. J. Physiol. 166, 441 (1951).

387 K. Schilling, /st Intern. Congr. Biochem. Abstr. Communs. (Cambridge, Engl.)
p. 15 (1949).

388 P. Bruun, H. Dam, and K. Schilling, Acta Physiol. Scand. 20, 319 (1950).

#9 M.M. Wintrobe, M. H. Miller, R. H. Follis, Jr., H. J. Stein, C. Mushatt, and S.
Humphreys, J. Nutrition 24, 345 (1942).

$90 N. R. Ellis, L. L. Madsen, and C. O. Miller, J. Animal. Sct. 2, 365 (1948).

39 M. E. Ensminger, J. P. Bowland, and T. J. Cunha, J. Animal Sci. 6, 409 (1947).

102 CHOLINE

some tubular epithelial necrosis.**?: *% No growth effect was noted if the
food intake was equalized. Doubling of the methionine content of the arti-
ficial milk was effective in the prevention of these symptoms,?% and it is
evident that the pig does not lack the ability to methylate aminoethanol
if labile methyl is available in diets otherwise adequate.

The need of choline by newborn calves fed a choline-deficient, artificial
milk was demonstrated by Johnson et al.*®> After 7 days an acute syndrome
developed which was characterized by marked weakness, labored or rapid
breathing, and anorexia. Certain of the calves showed evidences of renal
hemorrhage and fatty liver. These effects were prevented by choline, al-
though subsequent removal of the choline supplement was not injurious.
Karlier studies by Waugh ef al.*°® had emphasized the importance to the
calf of the relatively high concentration of choline in colostrum and the
relation of this fact to the level of choline in the blood of the calf. A decrease
in blood choline was noted in calves, 5 weeks of age, following the removal
of milk from the diet, although restoration of the choline level was not ob-
tained by dietary supplements of choline.

Evidence of choline deficiency in rabbits is lacking, but experiments on
the young have not yet been reported. Hypercholesteremia and aortic ather-
osclerosis were not prevented by choline in the study by Firstbrook,*%? but
others have concluded that choline does have a beneficial effect in the pre-
vention or removal of this induced abnormality in rabbits.*8“ Sprunt
concluded that subcutaneously administered choline inhibited the suscep-
tibility of the rabbit to infection with vaccinia,*! and a transitory decrease
in red cells has been observed following the administration of choline in
rabbits rendered polycythemic by cobalt.“ Transmethylation occurs in
this species as evidenced by the isolation of labeled choline, creatine,
anserine, and creatinine, after the feeding of deuteriomethionine.*%

A deficiency of choline or of methyl has not been demonstrated in the

89 B.C. Johnson and M. F. James, J. Nutrition 36, 339 (1948).

93 A. L. Neumann, J. L. Krider, M. F. James, and B. C. Johnson, J. Nutrition 38,
195 (1949).

394 R.O. Nesheim and B. C. Johnson, J. Nutrition 41, 149 (1950).

895 B. C. Johnson, H. H. Mitchell, J. A. Pinkos, and C. C. Morrill, J. Nutrition 48,
37 (1951).

396 R. K. Waugh, 8. M. Hauge, and W. A. King, J. Dairy Sci. 80, 457, 641 (1947).

397 J. B. Firstbrook, Proc. Soc. Exptl. Biol. Med. 74, 741 (1950).

398 TL. M. Morrison and A. Rossi, Proc. Soc. Exptl. Biol. Med. 69, 283 (1948).

399 A. Steiner, Arch. Pathol. 45, 327 (1948).

400 ©, Moses and G. M. Longabaugh, Arch. Pathol. 50, 179 (1950).

401 T). H. Sprunt, Proc. Soc. Exptl. Biol. Med. 51, 226 (1942).

402 M. Saviano and V. Baceari, Arch. fisiol. 48, 243 (1943).

403 J. R. Schneck, 8. Simmonds, M. Cohn, C. M. Stevens, and V. du Vigneaud, J.
Biol. Chem. 149, 355 (1943).

X. EFFECTS OF DEFICIENCY 103

guinea pig, and this is correlated with the observation that the turnover of
choline is relatively slow, owing presumably to the absence of choline ox1-
dase in the liver of this species.“°* Handler found no accumulation of fat in
the livers of guinea pigs fed seven different diets that did cause fatty livers
in rats, even though guanidoacetic acid was added to the guinea pig diets.*°*
Dubnoff and Borsook have reported the presence of an enzyme in the liver
and kidney of this animal, as well as in these organs of the rat and hog,
that transfers methyl from thetin to homocysteine.’ The metabolism of
choline in the guinea pig may differ only quantitatively from that in the
rat inasmuch as Dubnoff has noted some methyl lability in guinea pigs
following the administration of C'-methyl-labeled choline.*”

The mouse appears more resistant to choline deficiency than the rat, and
it has been used relatively little in studies of transmethylation. The pro-
duction of fatty livers was noted in Best’s laboratory,*! but the develop-
ment of renal lesions in weanling mice has been found to occur only if a
choline-deficient diet is eaten prior to weaning.*”* The presence or absence
of choline did not affect the rate of disappearance of deuterium from either
liver or depot fatty acids.”°°

Some evidence has been presented that choline decreases the acute liver
destruction that occurs in rhesus monkeys inoculated with yellow fever
virus.“” Choline was required for normal lactation in the hamster.*!! Trout
fed a choline-deficient diet showed renal degeneration and intestinal hemor-
rhage.” The omission of choline from an otherwise adequate synthetic
diet resulted in complete growth failure and death in the cockroach, Blatella
germanica 1.4") 444 Betaine was practically as effective as choline in the pre-
vention of the deficiency. Choline is stated to be essential for the develop-
ment of larvae of Aedes aegypti.*!”

454 F, Bernheim and M. C. L. Bernheim, Am. J. Physiol. 104, 438 (1933); 121, 55
(1938).

405 P. Handler, Proc. Soc. Exptl. Biol. Med. 70, 70 (1949).

406 J. W. Dubnoff and H. Borsook, J. Biol. Chem. 176, 789 (1948).

407 J. W. Dubnoff, Arch. Biochem. 22, 474 (1949). e

498 H}. A. White and L. R. Cerecedo, Proc. Am. Chem. Soc. 23B, (1946).

409 J). Stetten, Jr. and G. F. Grail, J. Biol. Chem. 148, 509 (1943).

410 A.W. Sellards and W.S. McCann, U.S. Naval Med. Bull. 43, 420 (1944).

411 J. W. Hamilton and A. G. Hogan, J. Nutrition 27, 213 (1944).

412 B. A. McLaren, E. B. Keller, D. J. O’Donnell, and C. A. Elvehjem, Arch. Bio-
chem. 15, 169 (1947).

13 J. L. Noland and C. A. Baumann, Proc. Soc. Exptl. Biol. Med. 70, 198 (1949).

414 J. L. Noland, J. H. Lilly, and C. A. Baumann, Ann. Entomol. Soc. Amer. 42, 154
(1949).

415 W. Trager, Proc. 29th Ann. Meeting New Jersey Mosquito Exterm. Assoc., p. 46
(1942).

104. CHOLINE

F. MAN

W. STANLEY HARTROFT, COLIN C. LUCAS, and CHARLES H. BEST ,

Direct evidence of disease in man due to choline deficiency is still lack-
ing, although two decades have elapsed since Best and Huntsman*!® dem-
onstrated the lipotropic action of choline in the experimental animal. It has
been suggested that, in such deficiency states as kwashiorkor, infantile
cirrhosis, and related conditions in parts of the world where the nutritional
status of much of the population is low, a deficiency of the lipotropic factors
as well as of other vitamins, coupled with a low intake of poor protein,
may be responsible in part for the lesions in liver, pancreas, and kidney.
There are many references to this question in the recent literature,” but
such a complex problem will not be discussed here. The clinician must await
more direct evidence based on controlled therapeutic trials before he can
properly assess the role of the lipotropic factors in these perplexing diseases.
Experimental research in this field has now progressed sufficiently to justify
attempts by physicians to provide clinical data to answer their own ques-
tions.

In contrast to our ignorance concerning the etiology of those hepatic
disorders which are endemic in countries with extremely low standards of
living, there is considerable evidence that the cirrhosis frequently encount-
ered in cases of chronic alcoholism is the direct result of choline deficiency
induced by the replacement of a large portion of the individual’s food intake
by alcohol—an alipotropic source of abundant calories. This conclusion is
based on data obtained from animal experiments and also from a few clini-
cal trials, which are understandably limited and incompletely controlled.
In experiments conducted in this laboratory”? an adequate supplement of
choline prevented the development of fatty and fibrotic hepatic changes
that occurred in rats given alcohol (15 %) in their drinking water. This
confirmed the earlier investigations of Sebrell and his associates in the U.S.
Public Health group.‘ In addition it was shown that in comparable paired-
fed animals given an amount of sugar isocaloric with the alcohol consumed,
identical lesions (Figs. 4 and 5) developed unless equal amounts of choline

416 C. H. Best and M. E. Huntsman, J. Physiol. (London) 88, 255-274 (1934-1935).

47 J. N. P. Davies, Trans. 9th Conf. Liver Injury, New York pp. 151-200 (1950) ; Lancet
I, 317-320 (1948).

418 J. C. Waterlow, Med. Research Council (Brit.) Spec. Rept. Ser. 263, 78 (1948).

“19 J, Gillman and T. Gillman, Lancet I, 169 (1948).

#0 V. Ramalingaswami, P.S. Menon, and P. 8. Venkatachalam, Indian Physician 7,
229-237 (1948).

#21 Kenneth R. Hill, Trans. 10th Conf. Liver Injury, New York pp. 263-820 (1951).

422 C. H. Best, W.S. Hartroft, C. C. Lucas, and J. H. Ridout, Brit. Med. J. II, 1001—
1006 (1949).

423 WS. Daft and W. H. Sebrell, Publ. Health Repts. (U.S.) 56, 1255-1258 (1941).

Fria. 4. Early fibrosis in the liver of a rat in which large amounts of fat had ac-
cumulated as a result of inducing a relative deficiency of choline by replacing the
drinking water with 15% alcohol (see text). Paraffin section; hematoxylin and
eosin stain; X 200. (Compare with Fig. 5.)

Fra. 5. Early fibrosis in the liver of a rat in which large amounts of fat had ac-
cumulated as a result of inducing a relative deficiency of choline by adding an amount
of sucrose to the basal diet, isocaloric with the aleohol consumed by the group of
rats illustrated in Fig. 4 (see text). Technical data as for Fig. 4.

105

106 CHOLINE

or its precursors were added to the diet to compensate for the additional
calories supplied by this amount of sucrose. These experiments indicate
that the ratio of lipotropic factors to the total caloric intake must be above
a certain critical level to protect the liver from harmful degrees of fat stor-
age and its almost inevitable successor, fibrosis. If the caloric intake be
raised by any means without a simultaneous increase in the lipotropes, the
resulting disturbance of liver function may eventually produce as grievous

\

|

|

|

Fria. 6. Gross appearance of the liver of rat which had been maintained on a
choline-deficient diet for approximately one year.

a result as the operation of an engine without adequate lubrication. Such
an imbalance has been produced in animals by choline-deficient caloric in-
crements supplied not only as carbohydrate and alcohol but also as fat or
methionine-poor protein. Persistent addiction to excessive amounts of
alcohol in man constitutes a fairly close clinical duplication of these experi-
mental conditions, and it is a reasonable working hypothesis that cirrhosis
of this type is due to choline deficiency. It is possible also that the fatty

424 Tt must be recognized, of course, that the alcoholic patient is probably suffering
from multiple dietary deficiencies, involving not only the vitamins but also min-
erals and particularly proteins.

X. EFFECTS OF DEFICIENCY 107

and fibrotic livers frequently associated with obesity*® should be included
in this category. Cases of gross obesity induced by chronic overindulgence
in candy, desserts, and starchy foods are possibly due to a lowered
choline/calorie ratio, but dietary histories are too inadequate to warrant
more than a suggestion at present. Investigators are hampered by the fact
that the glutton, like the drunkard, is frequently untruthful about his habit-
ual overindulgence.

The hypothesis that alcoholic cirrhosis in man is the clinical counterpart

Fic. 7. Gross appearance of the liver of a man aged 39 years who had been ad-
dicted to alcohol for several years before his death. If one allows for the differ-
ence in size of this liver and that illustrated in Fig. 6, the characteristics of the cir-
rhotic lesions are remarkably similar.

of cirrhosis in choline-deficient animals might be less tenable if the histo-
pathology of the two conditions were not essentially identical. The gross
appearances of the livers are certainly remarkably similar (Figs. 6 and 7),
but, although this is accepted by all investigators, there is lack of agreement
concerning the similarity of the microscopic characteristics. It has been
stated®”® that ‘‘This peculiar experimental hepatic cirrhosis of rats has no
counterpart in the usually described varieties of hepatic cirrhosis in man,”’

425 §. Sherlock, A. G. Bearn, and B. Billing, Trans. 10th Conf. Liver Injury, New York
pp. 205-262 (1951).

#6 R. D. Lillie, L. L. Ashburn, W. H. Sebrell, F. S. Daft, and J. V. Lowry, Publ.
Health Repts. (U.S.) 57, 502-508 (1942).

108 CHOLINE

and that ‘This experimental cirrhosis is not identifiable with any of the
previously described experimental toxogenic cirrhoses nor with any of the
usual varieties of hepatic cirrhosis in man.”’ If these statements were sup-
ported by the available facts, 1t would indeed be rash to assume that choline-
deficiency cirrhosis in rats bears any true relation to alcoholic cirrhosis in
man. But there is evidence that differences in the histopathology of the
two lesions are more apparent than real, and that in fact they can be re-
garded as minor variants of a fundamentally identical pattern. The impor-
tance of this question in any discussion of the pathology of choline deficiency
in man is self-evident, and it will therefore be considered in some detail.
Cirrhosis in choline-deficient rats has been said to differ from that in
human alcoholics in the following essentials: (a) Usually there are abundant
amounts of stainable fat in the cirrhotic rat’s liver, whereas frequently little
can be demonstrated in the alcoholic patient’s liver at autopsy. (b) Ceroid
is often deposited in the livers of choline-deficient rats,!2° whereas this pig-
ment is absent or present in only small amounts in alcoholic cirrhosis.*2°4”9
(c) The cirrhosis of alcoholism is described as ‘“‘portal’’; that in choline-
deficient rats as “non-portal.’”? These points will be considered in turn.

1. STAINABLE Fat

Staimable fat i cirrhotic livers of alcoholics, although a common finding,
is not constantly present at autopsy. This has been responsible for the con-
cept that this type of cirrhosis may not have been preceded by fatty paren-
chymal change. Although stainable fat may be absent from the liver of an
alcoholic at autopsy, there is much to suggest that abundant lipid might
have been demonstrable at an earlier, more actively progressive stage in
the development of the lesion. The fat content of cirrhotic livers at autopsy
and in the animal experiments has been shown to be inversely proportional
to the degree of fibrosis.°: “! This is true also for the experimental cir-
rhosis produced in rats by a dietary deficiency of choline.*”? In fact a stage
may be reached in the rat where, even though the animal has been main-
tained continuously on the low choline diet, microscopic examination fails
to reveal more than very small amounts of stainable fat in a liver that is
grossly cirrhotic (see Fig.10). This indicates that much of the abnormal lipid
that accumulates in the liver must eventually escape or disappear even
in the absence of choline. The pathways by which this occurs may be ab-
normal and involve the biliary and vascular systems.**8
427 A. M. Pappenheimer and J. Victor, Am. J. Pathol. 22, 395-412 (1946).

428 H. Popper, P. Gyorgy, and H. Goldblatt, Arch. Pathol. 37, 161-168 (1944).

#29 W.8. Hartroft, Proc. Roy. College Phys. Surg. (Canada) pp. 120-138 (1949).

430 C, L. Connor, Am. J. Pathol. 14, 347-364 (1938).

431 T. L. Chaikoff, C. L. Connor, and G. R. Biskind, Am. J. Pathol. 14, 101-110 (1938).

432 C, C. Lucas, J. H. Ridout, and W. 8S. Hartroft, Unpublished data (1952).
433 W. 8. Hartroft and J. H. Ridout, Am. J. Pathol. 27, 951-990 (1951).

X. EFFECTS OF DEFICIENCY 109

The accumulation of fat in non-portal regions of the livers of choline-
deficient rats may become so great that it can no longer be stored in intra-
cellular form but eventually bursts the cells, thus permitting the fat
to escape from the confines of the cell membrane. Single tenuous septa are
formed by the compression and stretching exerted on cell membranes of
adjacent cells distended by large fat spherules. These septa are frequently
the sites of ruptures when fat accumulation in the cells becomes excessive.

@; : Ger 7! SI

Fia. 8. A large fatty cyst in the liver of a male alcoholic (39 years old). In this
single, random section (5), the nuclei of nine parenchymal cells which form the wall
of the cyst may be seen. The greatest diameter of the cyst in this section is over 90 u.
If the cyst had been followed through serial sections, it is likely that the total num-
ber of cells in its wall would be found to total almost 50. Cells in walls of cysts are
usually more stretched and thinned than those illustrated here. Paraffin section;
hematoxylin and eosin stain; 800.

lo d : :
a AP an ah

This rupture permits the adjacent fat spherules from each cell to fuse and
so become enclosed by the conjoined parent cells. As a result, the latter
actually form a small two-celled epithelial cyst (Fig. 8), and these fatty
cysts*** #4 are the cytometaplastic links between the fatty and fibrotic
lesions of choline deficiency. Once formed, cysts may further increase in
size by fusing with other cysts or with fat-laden cells in the same manner
as the cysts were originally formed from individual cells. Eventually, large
cysts rupturing may cause damage to surrounding biliary or vascular
channels, and when this occurs fat may escape into the lumina of the latter
484 W.S. Hartroft, Anat. Record. 106, 61-87 (1950).

110 CHOLINE

and so leave the liver. Cell remnants of disrupted cysts may survive for
some time, as their nuclei are not often damaged. Condensation of these
atrophic parenchymal cells (along with their reticular stroma) produces
the so-called “fibrous” trabeculae of the cirrhotic lesion (Fig. 9). The con-
densation is responsible for the annular surface depressions, while paren-
chymal hyperplasia (compensatory in nature) in the portal regions produces
the nodular, raised areas.

htt

Fria. 9. The cells in the wall of this fatty cyst are thinned and stretched. The sur-
rounding stroma is condensed, and the cyst is enmeshed in a trabecula. A portion of
a radicle of an hepatic vein is shown in the upper left corner. Liver of a rat fed a
choline-deficient diet for 6 months; paraffin section; hematoxylin and eosin stain;
<s00.

The fact that the natural sequence of events is the formation, rupture,
and dissolution of fatty cysts with consequent escape of their contents
from the liver explains why the amount of fat in the liver decreases as the
fibrosis increases. For, with the rupture of each additional cyst, more fat
escapes from the liver and condensation of the cellular and stromal elements
of the torn cyst adds to the growth of trabeculae. The loss of parenchyma
incident to the eventual destruction of each cyst (a large one may con-
sist of as many as 60 cells) stimulates compensatory hyperplasia in portal
areas. This region commonly exhibits only a moderate degree of fat storage
which is almost entirely intracellular in nature. Intracellular fat is very
rapidly mobilized when the deficiency in lipotropic factors is corrected. This

X. EFFECTS OF DEFICIENCY Ase

may be effected in a variety of ways, as, for example, decreased growth of
the animal (thus sparing more methionine for lipotropic use), reduced food
intake, or the addition of choline to the diet. The result is a cirrhotic liver
without obvious fatty change (Fig. 10). This sequence has been observed
repeatedly in experimental animals. The clinical counterpart of this pre-
sumably occurs when a cirrhotic alcoholic in hospital is denied access to
his alcohol and is given a well-balanced diet (with or without therapeutic

rat has disappeared. That which remains is chiefly contained within persistent fatty
cysts. This demonstrates the small amount of abnormal fat which may be present in
a liver in which fibrosis has become severe. Liver of a rat fed a choline-deficient diet
for over a year. Paraffin section; hematoxylin and eosin stain; X 100.

adjuvants of choline or methionine). If, in addition, the patient’s caloric
intake drops sharply owing to terminal complications such as ascites or
ruptured esophageal varices, his death may be preceded by a significant
loss in weight (which may be masked by ascites or edema). This should mo-
bilize all intracellular fat from his liver and even much of the extracellular
lipid. Experiments with rats have demonstrated*®: 4° that most of the
extracellular fat in cysts will be resorbed eventually if the deficiency of
lipotropes is relieved. The only evidences of previous fatty change that can
then be found in liver sections are occasional remnants of cysts within the

485 W.S. Hartroft and E. A. Sellers, Am. J. Pathol. 28, 387-399 (1952).
486 C, H. Best, W. 8S. Hartroft, and E. A. Sellers, Gastroenterology 20, 375-384 (1952).

z SRE
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Ya Me

Fre. 11. Many of the fatty cysts illustrated in this field are undergoing involu-
tionary changes, since they are losing their contained lipid. The surrounding stroma
is condensed around the atrophic cysts. (Black material is ceroid pigment.) Liver of a
rat fed a choline-deficient diet for 6 months. Paraffin section; Ziehl-Neilson stain;
«400.

Fria. 12. Atrophic cysts in an area of reticular condensation in the liver of a human
alcoholic (male). A few of the cells in the walls of the cysts (center left) retain suffi-
cient characteristics so that they can still be identified as parenchymal. Compare
with Fig. 11. Paraffin section; hematoxylin and eosin stain. 400.

112

X. EFFECTS OF DEFICIENCY Liss

trabeculae (Fig. 11), and for these the pathologist may have to search as-
siduously. If such a search is successful, the clinical pathologist is well
rewarded in cases of cirrhosis in which the pathogenesis may be shrouded
in obscurity,*” for fatty cysts at any stage imply, by their very existence,
that at one time liver cells had been distended to the bursting point by fat
and this finding may provide an important clue to the etiology (Fig. 12).
The foregoing should serve to establish that the presence of large amounts
of stainable fat in cirrhotic livers is not a criterion for determining whether
or not the lesions were initiated by an excess accumulation of fat. Although
stainable fat is usually demonstrated with ease in experimental cirrhosis
of dietary origin, such animals are most frequently sacrificed before they
become moribund and while their food intake is still within normal limits.
Consequently the investigator commonly observes this type of cirrhosis at
the height of active progression of lesions when fat is abundant. This is in
sharp contrast to the clinical situation. Patients have usually been treated
by withdrawal of their alcohol, and terminal complications may have pro-
duced significant degrees of weight loss, both of which are potent factors
in clearing hepatic cells of stainable fat. These considerations may well
explain the frequent absence of stainable fat in livers of alcoholics at death.

2. CEROID

In the trabeculae of cirrhotic, choline-deficient rats, abundant amounts
of an orange-brown pigment may ke demonstrated. This material, which
was named ceroid by Lillie et al.,*2° is sudanophilic, acid-fast, and insoluble
~ in alcohol, xylol, and other common fat solvents. It is most easily and cer-
tainly identified by staining paraffin sections (from which all soluble lipids
have been removed by the dehydrating agents) with any of the dyes which
demonstrate fat (Sudan, Oil Red O, ete.). Ceroid in cases of cirrhosis inman
is absent or scanty ;*°°9 at most, only small amounts can be demonstrated
in alcoholics.

Deposition of ceroid in cirrhotic rats can be greatly reduced by either
supplementing their diet with large amounts of a-tocopherol, or replacing
unsaturated fats in the diet with hydrogenated vegetable oils such as Crisco
or Primex.***: #5449 Both zn vitro and in vivo experiments**! have now demon-
strated the possibility that ceroid is the product of the oxidation of unsatu-
rated fatty acids into an insoluble polymer, which, however, retains the
characteristic of sudanophilia. Tocopherol may act to inhibit this reaction
by virtue of its role as an antioxidant. Small hemorrhages frequently occur
437 W. S. Hartroft, Federation Proc. 11, 417 (1952).

438 J. Victor and A. M. Pappenheimer, J. Exptl. Med. 82, 375-383 (1945).
89 P. Gyorgy and H. Goldblatt, J. Exptl. Med. 89, 245-268 (1949).

40 P. Gyorgy and H. Goldblatt, J. Exptl. Med. 90, 73-84 (1949).

41 W.S. Hartroft, Science 118, 673-674 (1951).

114 CHOLINE

in ruptured fatty cysts,** with the result that red cells are bathed in liver
fat. Much of the ceroid that is found in experimental cirrhosis could be
explained as the polymerization of unsaturated fats which coat the surface
of erythrocytes in hemorrhagic fatty cysts. This explanation is supported
not only by direct observation of transition forms between red cells and
ceroid aggregates in sections and by the experiments cited above, but also
by the fact that, in livers that contain ceroid, hemosiderin is scanty or
absent. In cirrhotic livers of rats fed a diet designed to repress ceroid
formation, hemosiderin deposits were demonstrated.*” This finding sug-
gests that, if red cells become coated with ceroid, their disintegration, with
release of histochemically demonstrable iron, 1s prevented.

Hemosiderin pigment can usually be demonstrated in cirrhotic livers of
human alcoholics. The sequence of pathologic events responsible for this
finding may be fundamentally similar to those which produce ceroid in
choline-deficient rats, since manipulation of the diet of the latter will
diminish ceroid deposition in the animals’ livers and favor hemosiderin
release.“”? The chain of evidence will not be complete until alcoholics have
been encountered in which the converse has occurred—1.e., prevention of
hemosiderin formation by coating of red cells with ceroid. Although exam-
ples of such prevention have not been reported to date, there are sufficient
data at hand to suggest that it is unlikely that the presence of ceroid
deposits in experimental cirrhosis indicates any fundamental etiological
variant.

3. PorTAL versus Non-PortTAL CIRRHOSIS

The classical concept of cirrhosis associated with alcoholism in man is
that it is portal in distribution. Orginally this term was also applied to
cirrhosis in choline-deficient rats.“ It was only after careful and detailed
studies by the U. 8. Public Health group in Bethesda™ that the locus of
the initial sites of formation of the trabeculae in these animals was realized
to be, in fact, non-portal. This finding has now been independently con-
firmed.“* “4° It is in line with the lobular distribution of abnormal fat in
choline deficiency, which is also initially and primarily non-portal.** Thus
portal alcoholic cirrhosis and non-portal experimental cirrhosis appear to
differ fundamentally in their cytoarchitecture.

The initial observers of dietary cirrhosis in rats based their conclusion
regarding its portal distribution on careful inspection of microsections of

442 WS. Hartroft, Trans. 9th Conf. Liver Injury, New York pp. 109-150 (1950).

443 P, Gyérgy and H. Goldblatt, J. Hxptl. Med. 70, 185-192 (1939).

444 J, L. Asburn, K. M. Endicott, F. S. Daft, and R. D. Lillie, Am. J. Pathol. 23,
159-171 (1947).

445. E. Glynn, H. P. Himsworth, and O. Lindan, Brit. J. Exptl. Pathol. 29, 1-9
(1948).

X. EFFECTS OF DEFICIENCY WLS)

livers in an advanced stage of the lesion. These are more comparable to
those usually seen in human alcoholics at autopsy than are the earlier
stages of experimental cirrhosis. It is evident that conclusions concerning
the nature of the lesions in the two conditions should be based only on
comparisons of material at similar stages in their development. Karly cases
of alcoholic cirrhosis in man are not encountered at autopsy frequently

ex
Fia. 13. Thick (100 u) cleared slice of the liver of a rat injected with India ink at
the time of sacrifice. In the center of the field is a large conducting (see text) branch
of the portal vein, and to the right is a small branch of the hepatic artery. Note that
the surrounding sinusoids do not communicate with this large vein. The white arrow
in the lower left corner points to a small distributing (see text) branch of the portal
vein which is breaking up into sinusoids. X 250.

enough to permit careful comparison with beginning fibrosis in choline-defi-
cient rats. The tallacy of comparing early phases of cirrhosis in rats (non-
portal) with late phases in alcoholics (portal) is demonstrated by the fact
that late phases even in the rats were described as portal by expert patho-
logists when material from this stage only was available. Though it is well
established that the distribution of the initial lesions in rats is non-portal,
this should not be taken as evidence that experimental cirrhosis differs
fundamentally from that in man, until it is possible to conduct equally
complete and intensive studies of the early stages of cirrhosis in man.
Several questions arise from the foregoing. How does it happen that

116 CHOLINE

initially non-portal trabeculae in cirrhotic rats eventually assume charac-
teristics which are at least pseudoportal? Is it possible that the trabecular
distribution of advanced cases of alcoholic cirrhosis is also only pseudo-
portal? Is there in fact any essential difference in the histopathology of the

Fig. 14. Preparation (similar to that shown in Fig. 13), of a rat which had re-
ceived a single oral dose of carbon tetrachloride 24 hours before it was sacrificed.
Central veins (radicles of the hepatic vein) can be easily identified in such a specimen
as they are surrounded by partially ischemic zones in which liver cells have swollen
as a result of the toxin. A radicle of the hepatic vein occupies the center of the field.
The branch of the portal vein below and to the right is a terminal distributing vessel
(see text) and is breaking up into sinusoids. The surrounding parenchyma is func-
tionally periportal, whereas that illustrated around the conducting branch of the
portal vein in Fig. 13 is periportal only in a geographical sense. In livers of both
choline-deficient rats and alcoholic man, fibrosis develops around large branches of
the portal vein such as that in Fig. 13, but rarely and only in very advanced eases is
fibrosis found around these terminal branches illustrated here. * 250.

end stages of the two types of cirrhosis? An attempt to answer these ques-
tions will be made by surveying the general architecture of the hepatic
vasculature and relating it to the pathological anatomy of experimental
and alcoholic cirrhosis.

Large branches of the portal vein, like other large afferent vessels of the
body, rarely give off direct branches of sinusoidal or capillary dimensions.
Before supplying the bulk of the parenchyma, branches of this vessel, of

X. EFFECTS OF DEFICIENCY 7,

the order of veins, divide into small terminals of the order of venules, and
it is from the latter that blood enters the hepatic sinusoids (Figs. 13 and
14). These terminal venules (‘distributors’) of the portal vein should be
regarded as the most significant landmarks for orientation of lesions in
relation to the functional lobular units of the liver. Unhappily, in micro-

Fig. 15. Cleared and injected (India ink) slice (100 w) of liver of a rat fed a choline-
deficient diet for approximately one year. The white arrow (right center) points to
a large branch of the portal vein. Fibrosis in non-portal regions has destroyed the
sinusoidal patterns except for small trees at the ends of the terminal venules of the
portal vein. Note the manner in which the trabeculae (clear areas in illustration)
embrace the conducting portion of the portal vein as indicated by the position of the
arrow. Fibrosis at this site is periportal in an anatomical sense only. In functionally
periportal positions around the terminal portal venules, the sinusoidal pattern is
relatively intact. 100.

sections, these venules, with their accompanying bile radicles and hepatic
arterioles, are small and insignificant. Each venule supplies a parenchymal
unit which is many times its diameter in section (about 10 uw and several
hundred microns, respectively), and therefore thin random sections of the
unit frequently fail to include its small afferent vessels. In contrast, the
large, non-terminal veins (‘‘conductors’’) of the portal system readily attract
the attention of the morphologist by virtue of their large diameter and their
lengthy course as they pass through many parenchymal units to which
they are not directly related in a functional sense. The relation of the latter

118 CHOLINE

to such large vessels is analogous to that of houses at the edge of a highway
which must be approached circuitously by way of secondary sideroads and
streets. To reach such a house, the traveler would have to traverse as great
a distance after leaving the highway as if the building were not located
near any main route at all. In the liver, parenchymal cells beside large
conducting portal veins (in contrast to cells around terminal, distributing

te
-
Fa “s\

K

Fie. 16. Similar preparation to that shown in Fig. 15. A portion of a large conduct-
ing branch of the portal vein occupies the center of the field and is surrounded by
fibrosis (clear area). Branches from this extend toward the small trees of intact sinu-
soids in which terminal branches of the portal vein link with the sinusoids and are
free of encircling trabeculae (upper left corner). Although there is periportal fibrosis
around the large portal venous branches, the small distributing portal venules are
not directly involved and from a functional standpoint the distribution of the tra-
beculae is non-portal.

venules) are no more periportal in a physiological sense than cells in other,
more obvious, non-portal regions. Cells adjacent to non-terminal portal
canals are therefore periportal only in a limited regional sense. In terms of
blood supply, with reference to the entry of blood into a parenchymal unit,
these portions of the parenchyma are non-portal, since to reach them blood
must first traverse sinusoids for some distance. Lesions, such as fibrosis
around conducting portal triads, are therefore periportal in a geographical
sense only. To be periportal from a functional standpoint, the lesions should
involve parenchymal cells adjacent to the proximal end of the sinusoidal

X. EFFECTS OF DEFICIENCY 119

plexus. However, cells bordering Junctions between sinusoids and terminal
portal venules obviously have prior access to any essential food factors
(including choline). Actually it is cells more remote from these favored sites
that would first develop evidence of deficiency when the amount of an
essential substance reaching the liver is insufficient to supply all parts of
the organ. Cells adjacent to large conducting (but not distributing) portal

Fig. 17. This is a paraffin section of the liver of a rat comparable to the prepara-
tion illustrated in Figs. 15 and 16. The lower left portion of the field is occupied by a
large bile duct, branches of the hepatic artery, and a small portion of a large branch
of the portal vein. These structures are encircled by trabeculae, and to this extent
the fibrosis is periportal. But the small terminal branches of the portal vessels are
free of fibrosis, and in this functional sense the distribution of the trabeculae is non-
portal. The white arrow indicates a small terminal portal area which is enlarged in
Fig. 18. Hematoxylin and eosin stain. 200.

vessels are in a position which is no more favorable for obtaining metabo-
lites that may be in short supply than cells in other non-portal regions
equally remote from the site at which blood enters the parenchymal unit.

Both fatty and fibrotic lesions in choline-deficient rats make their initial
appearance and reach their most advanced stages in those portions of the
hepatic lobule which are farthest from the sites at which blood leaves
terminal portal venules to enter the sinusoids of each parenchymal unit.
The most non-portal areas of the liver are those which surround the radicles
of the hepatic vein, and these pericentral regions are the first sites of both

120 CHOLINE

fat storage and fibrosis. From here, trabeculae can extend only in directions
toward neighboring central veins or conducting portal veins; otherwise they
would encroach more directly on portal regions (Figs. 15 and 16). Thus,
extension of trabeculae in accordance with the non-portal principle eventu-
ally results in fibrotic replacement in regions around large portal triads
which are anatomically, but not functionally, periportal. By this process,
considerable areas along large portal veins become surrounded, often in

Fic. 18. The field immediately below the white arrow in Fig. 17 is enlarged in
this photomicrograph. A small bile duct is shown in cross section, and below it, but
less clearly shown, is a small terminal portal venule containing one or two red
cells. There is no fibrosis present, and with regard to these functionally periportal
regions, the fibrosis is non-portal. 800.

eccentric fashion (Fig. 16), by trabeculae of condensed hepatic stroma. But
even in relatively advanced stages of cirrhosis in rats, the terminal portal
venules at the points where they ramify into sinusoids remain almost com-
pletely free of fibrosis. This may not be readily appreciated in thin micro-
sections, for these venules are small and often missed by the plane of the
section. Furthermore, if the parenchyma surrounding them be fatty, they
are even more difficult to find under the lower powers of the microscope.
If the observer is not aware of their importance as landmarks in orienting
the lobular distribution of lesions, he may completely overlook these small
vessels. By contrast, he may be readily misled by the large conducting

X. EFFECTS OF DEFICIENCY 121

portal vessels which are always easily found and are rendered even more
conspicuous when partially surrounded by bands of fibrosis (Figs. 17 and
18). All these features have been followed in the rat through the various
stages of the development of cirrhotic lesions, and they offer a reasonable
explanation for the apparent disagreement between observers studying
different stages of the same condition. When cases of human alcoholic

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Fic. 19. Paraffin section of the cirrhotic liver of an alcoholic man. At the top of
the field a large branch of the hepatic artery (indicated by the upper white arrow)
is enmeshed by fibrous tissue. The portal area so identified is fibrotic and to this
extent the distribution of the lesion is periportal. But, as in the rat, there is no
fibrosis around terminal portal vessels (indicated by the white arrow at the lower
left). This region is enlarged in Fig. 20. Hematoxylin and eosin stain; X200.

cirrhosis are reexamined with these considerations in mind, similar features
are encountered.

In studying sections of livers from cases of so-called portal cirrhosis in
alcoholic men, we have been able to demonstrate repeatedly that fibrosis
is rarely present around the terminal portal venules, but of course is abun-
dant around many portions of the large conducting veins (Figs. 19 and 20).

In the latter sense only is this cirrhosis portal. But if this is accepted as
the definition of the term, then so must it be employed with reference to
the choline-deficient rat. By this standard, the cirrhosis in choline-deficient
rats is as much portal as that in human alcoholics. Conversely the cirrhosis

122 CHOLINE

of alcoholism is as much non-portal as that of choline deficiency. It must
therefore be conceded that the architectures of both lesions are essentially
similar. The distinction of portal from non-portal cirrhotic lesions has been
heightened by the anatomical nomenclature employed, but is apparently
only one of degree.

If the validity of these arguments is accepted, evidence based on dietary
studies would strongly suggest that alcoholic cirrhosis in man is indeed due

‘ . Rs NY @ Fes
Fia. 20. The field indicated by the white arrow in the lower left portion of Fig. 19
is enlarged in this photomicrograph. A small bile duct occupies the center of the
field, and at the bottom a terminal portal venule is seen in tangential section. There
is no fibrosis present. As in the rat, with regard to these functionally periportal areas,
the fibrosis is non-portal. 800.

in large part to the dietary deficiency of the lipotropic agents. Clinicians
might be richly rewarded were they to accept this as a working hypothesis
on which to base well-controlled studies designed to test its validity. The
results may well necessitate modification of the views presented in this
chapter, for it is quite possible that toxic substances, both organic and
inorganic, in commercial spirits may play an important role in the effects
of alcohol-containing drinks on man.

There is no evidence at present that extrahepatic lesions involving the
kidney, the eye, or the cardiovascular system of choline-deficient animals
have any clinical counterparts, despite some suggestions to the contrary.

XI. PHARMACOLOGY 125

There are strong anatomical and histological parallelisms between the vari-
ous forms of renal damage that develop in rats on low choline diets, and
lesions in some types of nephrotic conditions and in subacute and chronic
stages of Bright’s disease. But any assumption that choline deficiency may
play a part in the production of these conditions or that their treatment
with lipotropic factors might produce favorable results is completely with-
out either experimental or clinical foundation.

XI. Pharmacology
W. STANLEY HARTROFT, COLIN C. LUCAS, and CHARLES H. BEST

Choline given parenterally causes a fall in blood pressure (due to dila-
tation of peripheral vessels and slowing of the heart); it increases peristalsis
of stomach and intestines and increases salivation. Some variability of
response has been noted. Choline generally lowers the blood pressure,! but
sometimes there is, instead, a rise. It has been claimed that in cats a rise
generally follows the fall.2 The variable response does not depend upon
impurities,® as was suggested at one time. Choline acts powerfully upon
the autonomic (involuntary, vegetative, or visceral) nervous system. Cho-
line and most other quaternary ammonium compounds possess in varying
degrees three distinct types of pharmacological action: the so-called ‘“‘mus-
carinic,”’ the ‘‘nicotine-like,” and the ‘‘curariform’”’ effects.

Muscarine, a powerful drug occurring in the poisonous mushroom Ama-
nia muscaria, stimulates by direct action (not via the nerve endings)
smooth muscles and glands innervated by postganglionic parasympathetic
fibers. It causes cardiac slowing, peripheral vasodilation with consequent
fall in blood pressure, increased peristalsis (with vomiting and defecation),
bronchial constriction, salivation, sweating, and miosis. The similarity of
the responses of the heart, smooth muscle, and glands to choline (or acetyl-
choline) and muscarine led to the adoption of the term “‘muscarinic action”’
to describe these effects. Nicotine has long been known to stimulate auto-
nomic gangha before paralyzing them. Choline and acetylcholine produce
similar effects. Furthermore, both choline and nicotine have a similar
effect (stimulation, then inhibition) on skeletal muscle. For this reason the
stimulatory action of choline (and other quaternary ammonium com-
pounds) on ganglia and voluntary muscles has been called ‘“‘nzcotine-like.”’

1K. W. Mott and W. D. Halliburton, J. Physiol. (London) 21, xvii-xx (1897).

2 J. Pal, Z. exptl. Pathol. Therap. 9, 191-206 (1911).

®’R.R. Renshaw, F. P. Underhill, and L. B. Mendel, J. Pharmacol. Exptl. Therap.
3, 457-458 (1912).

124 CHOLINE

(This expression is to be preferred to the older term ‘‘nicotinic,”’ to avoid
confusion with the effects of nicotinic acid, now a product of therapeutic
importance.) Curare, a powerful alkaloid derived from several South Ameri-
can species of Strychnos, acts as blocking agent on skeletal muscles and
autonomic ganglia, causing muscular weakness ending eventually in paral-
ysis as the muscle becomes completely flaccid. Choline and other quater-
hary ammonium salts in higher concentrations have a “‘curariform’”’ effect.

Efforts to establish clearly the distinction between the muscarinic action
(peripheral effects on glands and smooth muscle cells) and the nzcotine-like
action (on ganglion cells and skeletal muscles) evoked much study of the
esters of choline and of related phosphonium, arsonium, stibonium, and
sulfonium bases.

Hunt and Taveau': * reported upon the circulatory effects and relative
toxicities of a large series of choline derivatives. Substitution of the hydro-
gen atom of the alcoholic OH group by acid radicals usually increases the
physiological activity, from 500 to many thousand times, depending upon
the substituent and the particular response observed.®: 7 Acetylcholine, for
example, is about 100,000 times more active than choline in causing a fall
in blood pressure. Replacing the hydroxyl group by a carboxyl (betaine)
leads to a product which is practically inert physiologically. Acetyl-6-
methylcholine (mecholyl) resembles acetylcholine except that it produces
practically no nicotine-like action. Carbaminoylcholine (carbachol, dory],
lentine), on the other hand, has a greater nicotine-like effect than acetyl-
choline.*

In comparison with many of its esters and many other quaternary am-
monium compounds the toxicity of choline is relatively low. Given as
chloride or citrate by mouth, choline has very low toxicity; it is consider-
ably more toxic by subcutaneous, intraperitoneal, or intravenous injection.
The relatively low (acute) toxicity of choline chloride may be illustrated
by a statement of Mott and Halliburton® in 1899 that ‘‘We have never
succeeded in killing an animal by injection of choline or choline hydro-
chloride.’”’ Others had succeeded, however. In 1885 Boehm!° found that
0.05 g. of choline chloride injected subcutaneously would kill a small frog,
and 0.1 g. a large one.

The minimum lethal dose, so-called, of choline chloride, for rabbits was

4R. Hunt and R. De M. Taveau, Brit. Med. J. II, 1788-1791 (1906).

5 R. Hunt and R. De M. Taveau, Hyg. Lab. Bull. (U. 8.) 73 (1911).

6H. H. Dale, J. Pharmacol. Exptl. Therap. 6, 147-190 (1914).

7G. A. Alles, Physiol. Revs. 14, 276-307 (1934).

8H. Molitor, J. Pharmacol. Exptl. Therap. 58, 337-3860 (1936).

9F. W. Mott and W. D. Halliburton, Trans. Roy. Soc. (London) B191, 211-267
(1899); see p. 223.

10 R. Boehm, Arch. exptl. Pathol. Pharmakol. 19, 87-100 (1885).

XI. PHARMACOLOGY ee

found by subcutaneous injection to be between 0.5 and 1.0 g. per kilo-
gram.'': © Dreyfus reported that rabbits are killed by 1 g. per kilogram
given rectally or subcutaneously but that only 0.11 g. per kilogram is
required intravenously. Cats are killed by 24 mg. of choline chloride per
kilogram given intravenously.!! The rate of administration is, of course,
important. Arai’? found that if the drug was injected slowly cats could
tolerate 15 mg. of choline chloride per kilogram given intravenously without
any observable toxic effect; 30 mg. given slowly produced a reversible arrest
of respiration but 35 mg., even if injected slowly, was lethal. As choline is
rapidly destroyed (see below), about 0.8 to 0.9 mg. per kilogram per minute
may be injected practically indefinitely into a cat if the concentration is
between 0.2 and 0.4%. The MLD for mice (subcutaneously) is 0.7 g. per
kilogram.!°

The acute toxicity of choline chloride for both mice and rats has been
determined in recent years by using larger numbers of animals and by
applying the technique of probit analysis to the data. This permits a more
accurate estimate of the dose needed to kill one-half of a given population,
1.e., the so-called LD5). Hodge and Goldstein!® injected mice (weighing 18
to 26 g.) intraperitoneally with different volumes of 2% solution of choline
chloride. The LD5) was 6.4 mg., or about 300 to 320 mg. per kilogram. The
importance of the strength of the solution was shown by Neumann and
Hodge” when they found the LD50 in rats given choline chloride by stomach
tube to ke of the order of 3.4 g. per kilogram when concentrated solutions
(670 mg. or 500 mg. per milliliter) were given, but 6.1 g. per kilogram when
’ weaker solutions (400 or 200 mg. per milliliter) were used. McArthur and
Lucas® obtained a value for the LDs5 (oral) of choline chloride in rats
weighing 150 g. of about 760 mg., 1.e., about 5.1 g. per kilogram when
the drug was administered in 2 ml. By intraperitoneal or intravenous
injection the LDs5) in rats is very much less (one-tenth to one-fiftieth,
according to preliminary tests, 1.e., of about the same order as was found
in mice).

Prolonged ingestion of choline has not revealed any chronic toxicity.
Many investigators of lipotropic phenomena have fed rations containing
0.1 up to 2 % of choline chloride to rats for periods of from several weeks to
1! Brieger, Ueber Ptomaine. Hirschwald, Berlin, 1885-1886.

2 Karl Vogt, Sitzber. Abhandl. naturforsch. Ges. (Rostock), New Ser. I, 1 (1909):

quoted by Arai in ref. 15.

13 L,. Dreyfus, Compl. rend. soc. biol. 88, 481-483 (1920).

144A. Lohmann, Pfliigers Arch. ges. Physiol. 118, 215-227 (1907).

15K. Arai, Pfliigers Arch. ges. Physiol. 198, 359-395 (1922).

16H. C. Hodge and M. R. Goldstein, Proc. Soc. Exptl. Biol. Med. 61, 281-282 (1942).

17 N. W. Neumann and H. C. Hodge, Proc. Soc. Exptl. Biol. Med. 58, 87-88 (1945).
18 C.S. McArthur and C. C. Lucas, Biochem. J. 46, 226-231 (1950).

126 CHOLINE

several months with no evidence of any toxic effects, other than some slight
decrease in food intake at the higher concentrations.

Hodge!’ administered relatively large amounts of choline chloride to rats
for a period of 4 months, to some in their food, to others in the drinking
water. Prolonged consumption of diets or drinking water containing 1%
choline chloride produced no evidence of toxicity. Food intake was low and
growth poor when the diet contained over 2.7%; no deaths occurred in
those given 5 % choline chloride in the ration. Those given 10 % lost weight,
and three out of five died, but no histological evidence of damage was
detectable. When the drinking water contained 2.7% choline chloride a
40 % reduction in growth occurred; when it was increased to 4% no growth
took place and all the rats (five out of five) died within 3 months.

Davis”? has claimed that administration of choline chloride to dogs by
stomach tube (10 mg. per kilogram of body weight, once or twice daily)
produces hyperchromic anemia. However, when Clarkson and Best”! main-
tained dogs on a good ration under controlled conditions for a period suffi-
cient to stabilize the blood picture (satisfactorily constant basal values for
red cell counts and hemoglobin concentrations) and then added to the diet
extra choline chloride (up to 30 mg. per kilogram of body weight per day),
no evidence whatever of macrocytic anemia could be detected. On the
contrary, the condition of the dogs given choline chloride, either in the diet
or by stomach tube, improved steadily, probably owing to the general
excellence of the diet rather than to the added choline.

Roth and Allison” fed rats of the Sherman strain a diet containing
1.35 % choline chloride for 20 days. The rats were fed the same weight of
food as was consumed by another group given an excess (4.8 %) of methio-
nine in the diet. The animals in both groups lost about 35 % of their body
weight in the 20-day period. Although the food intake is not given, it would
appear from the data for ingested nitrogen that the rats (250 g.) obtained
only 6.7 g. of the ration (12 % casein) per day. Restriction of food intake
rather than toxicity of choline chloride appears to explain the loss in weight
observed in this study.

The fate of choline in the organism has not been fully elucidated. Choline
is destroyed in the body with considerable speed.?* When injected it dis-

19 H. C. Hodge, Proc. Soc. Exptl. Biol. Med. 58, 212-215 (1945).

20 J. EK. Davis, Am. J. Physiol. 142, 213-215, 402-406 (1944); 147, 405-411 (1946);
Science 105, 43-44 (1947); J. E. Davis and D. E. Fletcher, J. Pharmacol. Exptl.
Therap. 88, 246-253 (1946); J. E. Davis and J. B. Gross, Am. J. Physiol. 144, 444-
446 (1945).

21M. F. Clarkson and C. H. Best, Sczence 105, 622-623 (1947).

2 J. 8S. Roth and J. B. Allison, J. Biol. Chem 188, 173-178 (1950).

23 H. von Hoesslin, Beitr. chem. Physiol. Pathol. 8, 27-37 (1906); queted by Fuchs,
ref. 31.

XI. PHARMACOLOGY 1 Payl

appears rapidly from the blood stream*™ but does not increase appreciably
the amount in the urie.?°-?7

When Luecke and Pearson’® fed large doses of choline chloride (10, 20
and 40 g. to sheep and 5 g. to dogs), no significant increase resulted in the
free or total choline in plasma, liver, or kidney and only a very small
fraction (0.5 to 2.5%) was recovered in the urine (24-hour collection).
Before treatment normal sheep excreted in the urine about 2 mg. of choline
per 24 hours; after 40 g. daily for a period of 6 days the output increased
significantly to between 300 and 500 mg. daily in one animal and to be-
tween 600 and 1050 mg. in the other (0.7 to 2.5% of the intake). Johnson
et al.” found that equally small proportions (0.7 to 1.5%) of the choline
intake were excreted as such in the urine by human subjects.

Choline is not destroyed by blood in vitro.® The destruction probably
occurs largely in the liver. In some species (rat and hamster, but not guinea
pig) a specific enzyme, choline oxidase, is found in the liver and kidney?: *°
which oxidizes the alcohol group to give betaine aldehyde. A second enzyme
continues the oxidation to betaine. Only. negligible amounts of choline,
trimethylamine, or its oxide could be found in the urine of a large dog
given 20 g. of choline chloride during a 6-day period.*! Possibly the methyl
groups are oxidized in some species by another enzyme via formic acid to
CO, and water. .

The methyl groups of choline appeared to be labile in certain physiologi-
cal environments, but recent studies suggest that they become labile only
_after conversion to compounds such as betaine aldehyde or betaine.*?-*4

Popper®*: * has reported that in normal human beings about two-thirds
of orally administered doses of choline chloride appear in the urine as
trimethylamine (mainly in the form of its oxide). Since the proportion so
excreted is greatly reduced after treatment of the patient with antibiotics
and is even smaller when the choline chloride is administered intravenously,

24R. Hunt, J. Pharmacol. Exptl. Therap. 7, 301-337 (1915).

25 M. Guggenheim and W. Loffler, Biochem. Z. 74, 208-218 (1916).

26 R. W. Luecke and P. B. Pearson, J. Biol. Chem. 158, 561-566 (1945).

27 B. C. Johnson, T. 8. Hamilton, and H. H. Mitchell, J. Biol. Chem. 159, 5-8 (1945).

28 F. Wrede, E. Strack, and E. Bornhofen, Hoppe-Seyler’s Z. physiol. Chem. 183,
123-132 (1929).

29 F. Bernheim and M. L. C. Bernheim, Am. J. Physiol. 104, 488-440 (1933).

30 F. Bernheim and M. L. C. Bernheim, Am. J. Physiol. 121, 55-60 (1938).

31H. Fuchs, Z. Biol. 98, 473-478 (1938).

32 J. W. Dubnoff, Pederation Proc. 8, 195 (1949)

33. J. W. Dubnoff, Arch. Biochem. 24, 251-262 (1949).

34 J. A. Muntz, J. Biol. Chem. 182, 489-499 (1950).

8° HW. Popper, in Discussion, Trans. 10th Conf. Liver Injury, New York (1951).

36 J. de la Huerga and H. Popper, J. Clin. Invest. 30, 463-470 (1951).

128 CHOLINE

it would appear that intestinal bacteria are responsible for the breakdown
observed. In rats about 40% of orally administered choline chloride is
excreted in the urine as trimethylamine or its oxide.*”

XII. Requirements

A. REQUIREMENTS OF ANIMALS

WENDELL H. GRIFFITH and JOSEPH F. NYC

Requirements of choline in the rat, chick, and turkey poult have been
stated for specific dietary conditions in the preceding discussion. Require-
ments cannot be described otherwise at the present time because of the
multiplicity of factors that govern the dietary need of this substance.
There is experimental support for the provisional conclusion that choline
is indispensable in the diet of chicks. This conclusion may need revision if
it is shown that mono- and dimethylaminoethanol can be methylated by
formate-to-methyl synthesis or by transmethylation at a rate sufficiently
rapid to meet the chick’s demand for choline. In the event that such a
finding is established, the effect would be merely the transfer of dietary
indispensability from choline to monomethylaminoethanol. There is no
evidence of wide and ample distribution of the latter compound in natural
foods. In so far as the rat and other animal species are concerned, there
appears little question concerning the ability to produce methyl groups.
Whether or not the rate of formation by this means is ever adequate for
growth, reproduction and lactation on diets completely devoid of choline
but otherwise of reasonable composition remains to be determined.

Among the dietary factors that influence the requirement of preformed
choline are (1) those that are involved in the enzymatic reactions of trans-
methylation and of formate-to-methyl synthesis, (2) those that provide
labile methyl, and (3) those that affect metabolism generally. In the first
category are folic acid and By. without which the choline requirement is
definitely increased. In the second category are methyl donors other than
choline, such as methionine, betaine; and the thetins. Of these, only methio-
nine occurs widely in nature, and the quantity and composition of dietary
protein are, therefore, important factors in determining how much choline
should be contained in the ingested food. In the third category are any
modifications of the food mixture, including a decrease in the amount
consumed, that may lower the requirement of choline by depression of

37 H. Popper, J. de la Huerga, and D. Koch-Weser, J. Lab. Clin. Med. 39, 725-736
(1952).

XII. REQUIREMENTS 129

the rate of growth or of the level or character of metabolism. In this group
are fasting and inhibition of appetite due to a deficiency of thiamine or of
other nutrients.

Undoubtedly there are many ways in which the metabolism of lipids is
affected with a concomitant change in the rate of turnover of choline-
containing phospholipids and in the rate of utilization of choline. Further-
more, the demand for methyl groups in the synthesis of creatine and of
other methylated metabolites may divert part of the dietary supply of
choline to such uses. On the other hand, choline may spare methionine as
a source of methyl and thus allow additional amounts of this amino acid
to be used in protein synthesis, directly or after conversion of its sulfur to
cystine. Certainly, the need of choline is increased during growth.

The position of choline among dietary essentials is unique and at the
same time anomalous. Clear-cut evidence for activity as a biocatalyst is
lacking, but there can be no question regarding its nutritional importance
in very fundamental biochemical systems in association with vitamins and
other indispensable nutrients.

B. REQUIREMENTS OF HUMAN BEINGS
W. STANLEY HARTROFT, COLIN C. LUCAS, and CHARLES H. BEST

Although some data are available which make possible a rough estimate
of the choline requirement of the rat, dog, and poultry, there are no com-
parable data for man. The daily choline intake of adults eating average
mixed diets was determined in Toronto! by actual analysis of representative
servings. The values found (ranging from 250 to 600 mg.) are in good agree-
ment with calculated values of 300 to 500 mg. reported from Sweden by
Borglin.? The choline intake of young men on a constant, adequate diet
was determined in the United States by Johnson ef al.* for a 5-day period;
it varied from 624 to 899 mg., with a mean value of 737 mg., or about 150
mg. per day.

Borglin? found the excretion of choline by adults to vary normally be-
tween 2 and 4 mg. per day, 1.e., 0.5% to 1% of the intake. He noted that,
when the intake was reduced (e.g., starvation, ulcer diet) the amount
excreted fell. Dietary factors (e.g., fat, methyl acceptors) which increase
the requirement for choline reduced the choline excretion. Conversely,
diets rich in lipotropic agents (protein, methionine, betaine) increased the
excretion of choline, obviously indicating a lessened requirement for choline.
These findings in human subjects paralleled those made during his investi-

1J.H. Ridout, C. C. Lucas, J. M. Patterson, and C. H. Best, Biochem. J. 52, 79-83
(1952).

2 .N.E. Borglin, Acta Pharmacol. Toxicol. 3 Suppl. 1, 1-123 (1947).

§ B.C. Johnson, T. 8S. Hamilton, and H. H. Mitchell, J. Biol. Chem. 159, 5-8 (1945).

130 CHOLINE

gations in animals. Negligible excretion of choline has also been reported
by others‘ even after giving extra choline.®* Thus ‘‘loading tests” are of no
value in determining the choline requirement.

4T. Z. Csiky, J. Moéllerstrém, and O. V. Sirek, Arch. Internal Med. 84, 730-737
(1949).

5 H. Castro Mendoza, C. Jiménez Diaz, and J. del Rio, Bull. Inst. Med. Research
(Madrid) 1, 7-17 (1948).

6 J. de la Huerga and H. Popper, J. Lab. Clin. Med. 36, 816 (1950).

7 J. dela Herga, H. Popper, and F. Steigmann, J. Lab. Clin. Med. 38, 904-910 (1951).

8 R. Busset, Rev. méd. (Liége) 6, 60-64 (1951).

CHAPTER 6

VITAMIN D GROUP

Page

ieenomenciavuire and Bormulas "; . 5 2 a. 1 . 2. « eee 2 oe ae Loe
PEON RMISOEY LSP Ge oe I OO COS 2
A. TheSterols . . ae res 1: OE i ee eld

B. The Provitamin Mheory aad Sian Mezes ie SS hs 5 de gr ate!

Be SLOTY 5 eres 8 ire. os) Rte sens Aap rams “ir eles eee LS

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3. Chemical Siruciire and ei vatability ian earner 6.8 ogee =. AG

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Cathe Provitsmine DrimiNature 2) f°. (sora ea. . SR eS
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2. Ergosterol. . . Lees a En re eek eS ey ee, SS?

Sve Mehydeachalesten ol. ne a ee a eee a mic obte la!

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OSE Rer Wibamninse ey OA oe, Ro C8 og) eae eee tt Oe eu, S68
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Peelnolanion from IN abural SOURCES): trl: ». 448 ie ew ee oe LTS

SI RICE Ole ACtVatlOn ns hewitt, aye Sekd al dene” a eh

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IV. Estimation. . . hy de Wilt Apt tes ad Ud Nee Male tial AD ie Adela 9
A. Physical Methods EET ae aE Saws eae fy eA.) ae ee

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Ze olLowtn.. 2 Te 3 a a a ae ar a ee

3. Calcium and Phosphorus a ie Sena me Oe . ee ta pda eee 25)

Ap ESOC eh ME RORDMAUAGO soo eo) Sa) covienlts un, Mer 2 be) ge Pa 226

5. X-Ray Examination. . . Sota aaa eee Md Be, Banh ah rrint 247 (

6. Calcium and Phosphorus BAlanos Ra Peay ee Rp oy ee ee

i, Memity of Intestinal’Contemtsyi.) 0. ke ke es BD
S.pone Ash. . . « Bere eto umee nem bane eRe)

9. Histological Cheneeas in ihe Tiieestie Bane A Fe i? ae ia ees a |

Bee eee OF Human Beige 2 oe sk ww a os BR
VII. Chemical Pathology and Rudi sided AOS pe Aiea) Ber eety Pe) Pe eee oe
VIII. hell cana ee A ee Ca eS mee zat eA ee eS
Pee Rm ee ee eke ee ee ce gas. allay %, Ge a athe Oe

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Dm livpervinsmainosis Dr's... ks. i.) a Ae te i re

131

SZ VITAMIN D GROUP

I. Nomenclature and Formulas
ROBERT 8S. HARRIS

Accepted names: Vitamin Dp», (or calciferol) from ergosterol by irradia-
tion; ergocalciferol
Vitamin Ds; (or activated 7-dehydrocholesterol) from 7-
dehydrocholesterol by irradiation; cholecalciferol
Obsolete names: Rachitamin
Rachitasterol
Antirachitie vitamin
Empirical formulas: Vitamin D2: CosHu0
Vitamin D3: Co;HysO
Chemical names: Vitamin D»
Vitamin D3;
Structure:

Vitamin D,;

II. Chemistry
CHARLES E. BILLS

The term vitamin D is applied to several antiricketic! substances derived
from, or associated with, the sterols. The existence of the vitamin was first

* The English word rickets, which probably derives from wrygates (crooked goings),

II. CHEMISTRY 133

indicated in experiments by Mellanby,? who found that rickets in puppies
is a deficiency disease, and that cod liver oil contains a factor which pre-
vents it. In 1922 the vitamin was recognized as a substance distinct from
vitamin A by McCollum et al. These workers bubbled air through heated
cod liver oil until the antixerophthalmic factor, vitamin A, was destroyed,
leaving the antiricketic factor which they later* termed vitamin D. Zucker
et al. found that vitamin D appears in the unsaponifiable, or sterol, fraction
of cod liver oil, and they suggested that “it may be a sterol related to
cholesterol or a cholesterol derivative.”

Proof of the relationship to sterols followed the discovery in 1924 of the
antiricketic activation of foods. It was found by Steenbock® and Hess and
Weinstock’ that numerous foods acquire vitamin D activity when exposed
to ultraviolet rays. In 1925 further experiments by Hess et al.,3 Steenbock
and Black,® and Rosenheim and Webster!® demonstrated that in foods it
is the sterol content which is activated. Extension of this work led in a
few years to the preparation of calciferol, or vitamin Ds, in the pure state,
and it is noteworthy that this was accomplished before any form of the
vitamin had been isolated from natural sources.

A. THE STEROLS

The literature of the sterols is voluminous, and the subject complex;
only the facts most pertinent to vitamin D will be given here. Bills’ review
of the physiology of the sterols" will be drawn upon for early material of
a biochemical nature. The organic chemistry of vitamin D, particularly in
its three-dimensional aspects, has been covered by Lettré and Inhoffen,!!*

antedates the unrelated classical coinage, rachitis (inflammation of the spine), and
is more accurately descriptive of the disease. Hence the terms rickets, ricketic and
antiricketic appear preferable to rachitis, rachitic, and antirachitic.

2 E. Mellanby, Med. Research Council (Brit.) Spec. Rept. Ser. 61, (1921).

3K. V. McCollum, N. Simmonds, J. E. Becker, and P. G. Shipley, J. Biol. Chem.
53, 293 (1922). “i

4E. V. McCollum, N. Simmonds, J. E. Becker, and P. G. Shipley, J. Biol. Chem.
65, 97 (1925).

5'T. F. Zucker, A. M. Pappenheimer, and M. Barnett, Proc. Soc. Exptl. Biol. Med.
19, 167 (1922).

6H. Steenbock, Science 60, 224 (1924); U.S. Pat. 1,680,818 (1928).

7A. F. Hess and M. Weinstock, J. Biol. Chem. 62, 301 (1924).

8 A. F. Hess, M. Weinstock, and F. D. Helman, J. Biol. Chem. 63, 305 (1925).

®°H. Steenbock and A. Black, J. Biol. Chem. 64, 263 (1925).

10Q. Rosenheim and T. A. Webster, Lancet I, 1025 (1925).

1C. EK. Bills, Physiol. Revs. 15, 1 (1935).

us A. Lettré and H. H. Inhoffen, Uber Sterine, Gallensiuren und verwandte Natur-
stoffe. Ferdinand Enke, Stuttgart, 1936.

134 VITAMIN D GROUP

Sobotka,” Strain,’ Rosenberg," Fieser and Fieser,!® Shoppee,!® and Deuel,”
and will not be repeated in detail. It is to be noted that the nomenclature
and symbolism of sterol chemistry are in a state of flux, the most recent
usage being that of Fieser and Fieser and of Shoppee. The book by Reed
et al.8 emphasizes the physiological and therapeutic aspects of vitamin D.

The sterol skeleton is shown in Fig. 1. This is a perhydro-1 ,2-cyclopen-
tenophenanthrene ring system, with methyl groups at positions 10 and 12
and a side chain at position 17. The conventional numbering of the carbon
atoms and the lettering of the rings are as indicated. The evidence leading
to this structural concept is briefly reviewed by Heilbron et al.!® Several
routes to the synthesis of the ring system have been described,” and recently
Woodward et al.2! have achieved the total synthesis of cholesterol.

The hydrocarbon skeleton common to the sterols is one of the largest
ring systems with which living cells have to deal. It occurs not only in the
common sterols, but also in the sex and cortical hormones, bile acids, car-
diac glucosides, toad poisons, and some carcinogens. But in vitamin D the
nucleus is broken in ring B, so that the structure is no longer a sterol in
the strictest sense. The several forms of vitamin D are sometimes classed
with the steroids in recognition of their origin, but properly they are not
sterols.

As an introductory convenience, the student of vitamin D may narrow
the field to three types of naturally occurring sterols, differing in the degree
of saturation in their ring system. In any of these types the side chain may
or may not be saturated, and always there are possibilities of structural
and steric isomerism.

1. The cholesterol type, characterized by just one double bond in the ring

12H. Sobotka, Chemistry of the Steroids. Williams and Wilkins Co., Baltimore,
1938.

13 W.H. Strain, in Organic Chemistry, 2nd ed., Vol. II, Chapter 19. John Wiley and
Sons, New York, 1943.

14H. R. Rosenberg, Chemistry and Physiology of the Vitamins. Interscience Pub-
lishers, New York, 1945.

16 ],, F. Fieser and M. Fieser, Natural Products Related to Phenanthrene, 3rd ed.
Reinhold Publishing Corp., New York, 1949.

16 C. W. Shoppee, Vitamins and Hormones 8, 255 (1950).

17H. J. Deuel, Jr., The Lipids. Interscience Publishers, New York, 1951.

18 C. I. Reed, H. C. Struck, and I. E. Steck, Vitamin D. University of Chicago Press,
1939.

19 J. M. Heilbron, J. C. E. Simpson, and F. 8. Spring, J. Chem. Soc. 1933, 626.

20 J. W. Cook and C. L. Hewett, J. Soc. Chem. Ind. 52, 451 (1933); J. Chem. Soc.
1933, 1098; L. Ruzicka, L. Ehmann, M. W. Goldberg, and H. Hoésli, Helv. Chim.
Acta 16, 833 (1933); G. A. R. Kon, J. Chem. Soc. 1988, 1081; A. Koebner and R.
Robinson, zbid. 1938, 1994; C. K. Chuang, C. M. Ma, Y. L. Tien, and Y. T. Huang,
Ber. 72, 949 (1939).

21. R. B. Woodward, F. Sondheimer, and D. Taub, J. Am. Chem. Soc. 78, 3548 (1951).

II. CHEMISTRY 135

system, always at C-5. Examples: cholesterol, sitosterol, stigmasterol,
brassicasterol, campesterol, fucosterol, and others (Fig. 2).

2. The saturated ring type, in which there is no double bond in the ring
system. Examples: dihydrocholesterol (cholestanol), dihydrositosterol (si-
tostanol), ete. (Fig. 3).

3. The ergosterol, or provitamin, type, characterized by two double
bonds in the ring system, always at C-5 and C-7. Examples: ergosterol
(provitamin D,), 7-dehydrocholesterol (provitamin D3), and the incom-
pletely identified provitamins D of mollusks (Fig. 4).

20 21 22 23 24
H.C CH(CH;):CH,:CH,:CH.:CH(CHs)»

Fra. 1. The sterol ring structure, with a typical side chain at C-17. Rings A, B, C,
and D are sometimes designated I, II, III, and IV.

EeCu
H;C
~ HO ;

Cholesterol R = —CH(CH;)-CH2-CH2-CH2-CH(CH3)2
B-Sitosterol R = —CH(CH;)-CH2:-CH2-CH(C2H;)-CH(CHs)2
Stigmasterol R = —CH(CH;)-CH=CH-CH(C:2H;)-CH(CHs;)2
Brassicasterol R = —CH(CH;)-CH—CH-CH(CH;)-CH(CHs)2
Campesterol R = —CH(CH;3)-CH2-CH2-CH(CH;)-CH(CHs)2

Fucosterol R = —CH/(CH;) -CH2-CH2-CH=C(CH3)-CH(CHs3)2
Fia. 2. Examples of cholesterol-type sterols.

Cholesterol (Fig. 2) is the principal sterol of the higher animals, or
vertebrates. It is abundant in nerve tissue, fats, and skin, and present to
some extent in all bodily structures. It is usually accompanied by traces of
other sterols, which in a few instances have been identified as dihydro-
cholesterol (cholestanol) and 7-dehydrocholesterol.

In the invertebrates cholesterol is partly or wholly replaced by mixtures
of other sterols, and in these mixtures 7-dehydrocholesterol and other pro-
vitamins D may be present in considerable amounts. The sterols of mollusks
are of particular interest in connection with vitamin D, and among these it
is to be noted that the sterols of the Pelecypoda (bivalves, such as clams
and oysters) differ from those of the Gastropoda (snails). Examples are

136 VITAMIN D GROUP

given in Table I, taken from Bergmann.” The bivalves may exhibit some
cholesterol, but chiefly their sterols are difficultly separable mixtures of
Cog and Cy sterols, with di-unsaturated C-24 epimers of Cs sterols pre-
dominating.” °° The snails, however, show chiefly cholesterol, like the
higher animals, but with small amounts of phytosterol-like sterols.

The sterols of plants, especially the higher plants, or phanerogams, are
collectively known as phytosterols. By far the commonest phytosterol is
sitosterol, a sterol which exists in three isomeric forms designated a, 8, and
y, which are so difficultly separable that they are usually considered as one.

H,c &

HO
Cholestanol R = —CH(CH;)-CH2-CH2-CH2:-CH(CH:3)2
B-Sitostanol R = —CH(CH;)-CH2-CH2-CH(C2H;)-CH(CHs)2
Fia. 3. Examples of saturated-ring sterols.

H,c B®

Ergosterol R = —CH(CH;)-CH—CH-CH(CH;)-CH(CHs3)e
7-Dehydrocholesterol R = —CH(CH;)-CHe2-CH2-CH2-CH(CH3).
Fig. 4. Ergosterol and 7-dehydrocholesterol, commonest examples of provita-
mins D.

Sitosterol is usually accompanied by traces of dihydrositosterol (sitostanol)
and a provitamin which might be expected to be 7-dehydrositosterol but
which is probably ergosterol. Other sterols occurring with sitosterol in the
higher plants are campesterol, brassicasterol, stigmasterol, and spinasterol.
In the lower plants, or cryptogams, sitosterol appears in the green algae,
but in the brown algae it is replaced by fucosterol. In the fungi the principal
sterol is ergosterol, accompanied by small amounts of 5-dihydroergosterol
and traces of several other sterols not of interest in connection with vita-
min D.

From the structural formulas it can be seen that the sterols which pre-
dominate in the higher forms of life differ only in their side chains. Their

22,W. Bergmann, J. Marine Research (Sears Foundation) 8, 137 (1949).
23 W. Bergmann and EH. M. Low, J. Org. Chem. 12, 67 (1947).

Il. CHEMISTRY 137

ring systems are identical and exhibit but one double bond, located at C-5.
These sterols and the dihydro derivatives which accompany them in traces
lack any system of conjugated double bonds and hence do not show ab-
sorption bands in the ultraviolet region when examined spectrographically.

TABLE I
Sterols of Mollusks (Bergmann??)

Class and species eee 2 | Principal sterol
Pelecypoda
Tapes phillippinarum 137 |
Corbicula leana | 126-127 Corbi-, brassicasterol
Cristaria plicata 137-138
Meretrix meretrix 137-138 Meretristerol
Ostrea gigas 136-137 Conchasterol
Ostrea virginica 134-135 Ostreasterol
Mya arenaria 131
Volsella modiolus 127-128
Tridacna gigas 156-157 Shakosterol
Venus mercenaria 131
Modiolus modiolus 131
Modiolus demissus 156-157 Brassicasterol
Gastropoda
Haliotis gigantea lly Cholesterol
Turbo cornutus 116 Cholesterol
Rapana thomasiana 120 Cholesterol
Cellana nigrolineata 115 Cholesterol
Tegula xanthostigma 118 Cholesterol
Fulgur (Sp.) Ile Cholesterol
Buccinum undatum Cholesterol
LIittorina littorea Cholesterol
Nerita pelerunta Cholesterol
Nassa obsoleta Cholesterol
Cephalopoda Y
Sepia officinalis Cholesterol
Octopus vulgaris Cholesterol

On the other hand, the sterols which are most characteristic of the lower
forms of life and which occur in traces with the sterols of the higher forms
(ergosterol, 7-dehydrocholesterol) have double bonds at C-5 and C-7. As
would be expected on theoretical grounds, these sterols with conjugated
double bonds exhibit ultraviolet absorption bands. With these principles in
mind, one can look back understandingly on the development of the pro-
vitamin theory.

138 VITAMIN D GROUP

B. THE PROVITAMIN THEORY AND SYNTHESES
1. History

When it became evident that in foodstuffs the sterol fraction contains the
acceptor of the activating rays, investigators tried to explain chemically the
changes induced in the sterols by irradiation. Hess and Weinstock” intro-
duced the use of the quartz spectrograph for investigating the chemistry
of activation. They found that ordinary cholesterol and wheat phytosterol
(sitosterol) were somewhat opaque to ultraviolet light, and that irradiation
decreased their opacity. On the other hand, dihydrocholesterol and dihydro-
sitosterol, which were not activatable, were practically transparent. Un-
fortunately, a mercury are was used as the light source for the spectrograms.
Since this gives a discontinuous spectrum, nothing could be learned of the
spectral structure in the region of absorption.

Schlutz and Morse,?> with better spectrographic technique, determined
that the absorption spectrum of ordinary cholesterol is banded. They rec-
ognized two maxima of absorption, at approximately 294 and 283 my and
were thus the first to record the provitamin absorption spectrum. They
noticed that after brief irradiation the inflections gave way to general ab-
sorption, and, considering Beer’s law, they postulated that either the cho-
lesterol had been at least half metamorphosed, or else “the substance in
which the absorption spectrum is changed may be a small amount of im-
purity in the cholesterol which is not removed by repeated crystallizations
from alcohol, and which is exceedingly absorptive.”

Rosenheim and Webster?* found that cholesterol which had been regen-
erated from cholesterol dibromide was so pure that it no longer showed the
characteristic absorption spectrum and was no longer activatable. Heilbron
et al.2” reported that fractional crystallization of cholesterol led to the ac-
cumulation, in the least soluble fraction, of the substance responsible for
the characteristic absorption spectrum. They recognized a third absorption
maximum, A 269 muy. Irradiation destroyed the three bands, leaving only
general absorption. Pohl,” by the technique of photoelectric photometry,
also detected three absorption bands in cholesterol, which faded upon ir-
radiation. With the knowledge that complete disappearance of the bands
corresponded to the destruction of only a trivial fraction of the cholesterol,
he concluded that the absorbing substance was present only in minute

24. A. F. Hess and M. Weinstock, J. Biol. Chem. 64, 181 (1925); 64, 193 (1925).

25 WF. W. Schlutz and M. Morse, Am. J. Diseases Children 30, 199 (1925).

26Q. Rosenheim and T. A. Webster, J. Soc. Chem. Ind. 45, 932 (1926); Biochem. J.
21, 127 (1927).

27 [. M. Heilbron, E. D. Kamm, and R. A. Morton, J. Soc. Chem. Ind. 45, 932 (1926);
Biochem. J. 21, 78 (1927).

28 R. Pohl, Nachr. Ges. Wiss. Gottingen, Math. physik. Kl. 142 (1926).

Il. CHEMISTRY 139

amounts. He found that cholesterol purified via the dibromide, or via allo-
cholesterol, and sitosterol purified via the dibromide, did not show the usual
absorption.

These studies made it plain that an impurity present in ordinary choles-
terol and sitosterol is responsible for at least the greater part of the anti-
ricketic activity conferred upon the sterols by irradiation. The impurity,
or provitamin, was presently identified as ergosterol, a sterol which
Rosenheim and Webster?’ had previously found to be activatable, but with
which they had made no quantitative studies. Pohl®® found that the three
known absorption bands of ordinary cholesterol are also exhibited by ergos-
terol, but in vastly greater intensity. Additional chemical studies and a
biological assay were made by Windaus and Hess,*! who found that, as
expected, the antiricketic potency of irradiated ergosterol was far greater
than that of irradiated cholesterol. Rosenheim and Webster® also conducted
chemical and biological studies and concluded that the precursor of vitamin
D “is ergosterol, or a highly unsaturated sterol of similar constitution.”

Bills et al.** confirmed these studies in a way which supported the view
that ergosterol was the contaminant provitamin. By the use of a continuous
light source they discovered that ordinary cholesterol exhibits a fourth
absorption band, \ 260 my. The same band was shown by ergosterol, making
the series \A 293.5, 282, 270, and 260 muy, with four points of identity in-
stead of three. Moreover, the spectra faded at the same rate when acetone
solutions of ordinary cholesterol and of provitamin-free cholesterol plus
added ergosterol were oxidized with permanganate.

In spite of all the evidence, it turned out that the identification of the
provitamin of cholesterol as ergosterol was incorrect, yet it was so con-
vincing that it was generally accepted from 1927 to 1934. However, there
was during that period a growing knowledge of the multiple nature of
vitamin D, which has been reviewed elsewhere in some detail. In par-
ticular, Massengale and Nussmeier®> observed that activated ergosterol is
much less effective than cod liver oil, rat unit for rat unit, in the prevention
of rickets in chicks. They offered no explanation of this phenomenon, but
their work provided an important tool, the chick-rat efficacy ratio, for
future studies on the D vitamins.

It remained for Waddell** to demonstrate that the provitamin D of

29 OQ. Rosenheim and T. A. Webster, Biochem. J. 20, 537 (1926).

80 R. Pohl, Nachr. Ges. Wiss. Gottingen, Math. physik. Kl. 185 (1927).

31 A. Windaus and A. Hess, Nachr. Ges. Wiss. Géttingen, Math. physik. Kl. 175 (1927).
8 O. Rosenheim and T. A. Webster, Lancet I, 306 (1927); Biochem. J. 21, 389 (1927).
83 C. E. Bills, E. M. Honeywell, and W. A. MacNair, J. Biol. Chem. 76, 251 (1928).
8 C. E. Bills, Cold Spring Harbor Symposia Quant. Biol. 3, 328 (1935).

35 QO. N. Massengale and M. Nussmeier, J. Biol. Chem. 87, 423 (1930).

36 J. Waddell, J. Biol. Chem. 105, 711 (1934).

140 VITAMIN D GROUP

cholesterol is not ergosterol. He compared irradiated cholesterol with ir-
radiated ergosterol on chicks and rats in a series of experiments designed
to be free from alternative interpretations. His findings proved beyond
question that the vitamin D from the provitamin of cholesterol is different
from the vitamin D from ergosterol, and therefore the provitamin of cho-
lesterol is not ergosterol.

CsHiz CsHiz
ey ae oxidation ohh Pew s
eee yl acetate cant yl acetate
CsHiz CsHiz
ota. benzoylation ohh “pyrolysis hee
OBz
7-Hydroxycholesterol 7- ee cholesteryl dibenzoate
C,H 17 CsHiz
ae saponification gh
f ae drocholesteryl benzoate 7-Dehydrocholesterol

Fig. 5. Synthesis of 7-dehydrocholesterol (method of Windaus et al.*8).

2. SYNTHESES

A few months before the appearance of Waddell’s paper, Callow*’ sug-
gested, on theoretical grounds, that there might exist in nature a provi-
tamin D which ‘‘is a cholesterol derivative with the double bonds in the
critical position, a demethyl-dihydro-ergosterol.’”’ This substance, now
usually known as 7-dehydrocholesterol, was synthesized by Windaus e¢ al.**
in the following manner (Fig. 5). Cholesteryl acetate was oxidized by chro-
mic acid to 7-ketocholesteryl acetate. This was reduced by aluminum
isopropylate to 7-hydroxycholesterol, the dibenzoate of which gave upon
strong heating in vacuo the benzoate of 7-dehydrocholesterol. The latter
was saponified to the free provitamin. 7-Dehydrocholesterol is highly acti-

37 R. K. Callow, Sci. J. Roy. Coll. Sct. 4, 41 (1984).
38 A. Windaus, H. Lettré, and F. Schenck, Ann. 520, 98 (1935).

Il. CHEMISTRY 141

vatable and exhibits ultraviolet absorption bands in practically the same
position and intensity as ergosterol. It also shares the property of being
somewhat unstable. These facts account for the original misidentification
of the provitamin in cholesterol.

By reactions analogous to the ones used in the synthesis of 7-dehydro-
cholesterol, the corresponding 7-dehydro derivatives of several other natural
sterols have been made, namely, 7-dehydrositosterol,*? 7-dehydrostigmas-

Hc B
ne CL
HO ea
Activat-

ability Sterol Side chain
+ Ergosterol
+ Epiergosterol 20) 21 22 PB pH!
+ Lumisterol R = —CH(CHs3)-CH—CH:CH(CHs;)-CH(CHs)2
— Pyrocalciferol ;

— Isopyrocalciferol
O

JES
—CH(CH:;)-CH-CH-CH(CHs)-CH(CHs)2
—CH(CHs)-CH2:CH2-CH(CHs)-CH(CHs)2

+ 22,23-Oxidoergosterol

+ 22-Dihydroergosterol

+ 7-Dehydrocampesterol

? 7-Dehydroclionasterol

? 7-Dehydrositosterol

? 7-Dehydrostigmasterol
+ 7-Dehydrocholesterol }

ll

—CH(CHs)-CH2:CH2-CH(C2Hs)-CH(CHs)2
—CH(CH:;):-CH—CH:CH(C2Hs)-CH(CHs)2
—CH(CHs:)-CH2:CH2-CH2:-CH(CHa)2
—CH(CHs3):-CH—CH:CH2:CH(CHsa)2
—CH(CHs)-CH2:CH2:CH2:CH»s:CHs;
—CH(CHs:)-CH2:CH2-COOH
—OH

Fic. 6. The provitamins D and related sterols with double bonds at C-5 and C-7.
The sign + means that the sterol becomes antiricketic upon irradiation, the sign —
means that the irradiation product exhibits no antiricketic action, and the sign ?
means that the sterol has not been adequately tested for activatability.

+ 7-Dehydroepicholesterol

? A5,7.22-Cholestatriene-3-ol

? A5.7~Norcholestadiene-3-ol

? 8-Hydroxy-A57-choladienic acid
? 3,17-Dihydroxyandrostadiene

DPXODDA DAH DD
ll

Io ow ol

terol,*° 7-dehydrocampesterol,*! and 7-dehydroclionasterol.” The structures
of these and other sterols unsaturated in the critical C-5 and C-7 positions
are shown in Fig. 6.

A provitamin D of special theoretical interest is 22-dihydroergosterol,
which is intermediate in structure between ergosterol and 7-dehydrocho-
lesterol, and epimeric on C-24 with 7-dehydrocampesterol. This provitamin
was prepared by Windaus and Langer* by saturating the double bond in

89 W. Wunderlich, Hoppe-Seyler’s Z. physiol. Chem. 241, 116 (1936).

40 Q. Linsert, Hoppe-Seyler’s Z. physiol. Chem. 241, 125 (1936).

41W.L. Ruigh, J. Am. Chem. Soc. 64, 1900 (1942); U.S. Pat. 2,378,435 (1945).
# W. Bergmann, A. M. Lyon, and M. J. McLean, J. Org. Chem. 9, 290 (1944).
48 A. Windaus and R. Langer, Ann. 508, 105 (1933).

142 VITAMIN D GROUP

the side chain of ergosterol. To do this, ergosteryl acetate was heated with
maleic anhydride to form an addition product in which the two double
bonds in ring B were saturated. The double bond at C-22 was then hydro-
genated, after which the maleic anhydride was thermally split from the
molecule, thus restoring the double unsaturation in ring B which is essential
for activation. The free provitamin was obtained by saponification of the
ester.

Ten other sterols with double bonds at C-5 and C-7 are also known.
Their structures are included in Fig. 6. Epiergosterol is prepared by the
reduction of ergosterone with aluminum isopropylate.“ It has not been
obtained pure, but has been made free from ergosterol. Lumisterol is the
initial irradiation product of ergosterol.**: 46 It has not been found in nature,
but it undoubtedly occurs in natural products where vitamin D is being
formed by insolation. Pyrocalciferol and isopyrocalciferol are thermal trans-
formation products of calciferol.** 22 ,23-Oxidoergosterol has been prepared,
but the details of its synthesis were not disclosed.” Rosenberg" surmises
that it was made by treating the maleic anhydride addition product of an
ergosterol ester with a mild oxidizing agent to form the 22 ,23-oxido com-
pound, which upon thermal decomposition would yield the 22 ,23-oxido-
ergosterol ester. 7-Dehydroepicholesterol has been made by applying the
reactions in Fig. 5 to epicholesterol,*8 and also, in poor yield, by reducing
dehydrocholestenone with aluminum isopropylate.*? A®:7.?"-Cholestatriene-
3-0] has been identified®® indirectly as one of the provitamins of Myizlus
edulis; it has not been prepared synthetically. A®:7-Norcholestadiene-3-ol
has been made by a series of reactions from the oxidation products of
sitosterol.®! 3-Hydroxy-A®7-choladienic acid is obtained by the pyrolysis of
the dibenzoate of methyl 3 ,7-dihydroxy-A*-cholenate in dimethylaniline.*®
3,17-Dihydroxyandrostadiene is made from androstenediol by reactions
analogous to those in Fig. 5.°%

Besides the chemically definite provitamins cited in the preceding para-
graphs, there are provitamins of unknown structure which will be consid-
ered under the heading, Provitamins D of Invertebrates. There are also

44 A. Windaus and K. Buchholz, Ber. 72, 597 (1939).

45 A. Windaus, A. Liittringhaus, and P. Busse, Nachr. Ges. Wiss. Géttingen, Math.
physik. Kl., Fachgruppe III, 150 (1932).

46 A. Windaus, K. Dithmar, and E. Fernholz, Ann. 493, 259 (1932).

47K. Dimroth and J. Paland, Ber. 72, 187 (1939).

48 A, Windaus and J. Naggatz, Ann. 542, 204 (1939).

49 A. Windaus and O. Kaufmann, Ann. 542, 218 (1939).

50 J. van der Vliet, Rec. trav. chim. 67, 246, 265 (1948).

51C, G. Alberti, B. Camerino, and L. Mamoli, Helv. Chim. Acta 32, 2038 (1949);
33, 229 (1950).

52 G. A. D. Haslewood, J. Chem. Soc. 1938, 224.

53 A. Butenandt, E. Hausmann, and J. Paland, Ber. 71, 1316 (1938).

II. CHEMISTRY 143

several vaguely recognized bodies from which antiricketic materials have
been obtained by irradiation or other treatment; these will be discussed
under Minor and Obscure Forms of Vitamin D.

3. CHEMICAL STRUCTURE AND ACTIVATABILITY
a. The Hydroxyl Group at C-3

It has long been established that certain esters of crude cholesterol, i.e.,
of 7-dehydrocholesterol, are made antiricketic by irradiation. These include
the acetate,” *4 *5 benzoate,®> isobutyrate,®® palmitate,?® and phosphate
(monocholesteryl) .°* Ergosterol esters undergo the chemical changes of acti-
vation as readily as the free sterol. However, the esters of calciferol which
are formed by the irradiation of the corresponding esters of ergosterol are
not uniformly effective when administered to rats. Irradiated ergosteryl
acetate,*”: 5 irradiated ergosteryl ethyl carbonate,*® and irradiated dier-
gosteryl phosphate? are as effective as irradiated ergosterol. The irradiated
benzoate was at first claimed to be as effective” as irradiated ergosterol but
later was found to be considerably less effective.®*: °9* The palmitate is also
less effective.®**: °° The allophanate, cinnamate, diphenylacetate, oxalate,
a-naphthylurethane, and phenylurethane are inactive at the usual curative
levels.°* The differences in antiricketic effectiveness are due merely to
differences in the assimilability of the esters, for by saponification the full
effectiveness of the vitamin D in any of the esters can be brought out. This
phenomenon has also been observed in the natural vitamin D esters of
fish oils.®°

When the hydroxy] group in 7-dehydrocholesterol is replaced by halogens,
the molecule is considerably stabilized. The chloride and bromide of this
provitamin D undergo changes upon exposure to ultraviolet rays which
indicate spectrographically that the corresponding halides of vitamin D3 are
formed. These halides, which are resistant to saponification, are devoid of
antiricketic action when fed to rats and chicks, even in large doses.®! Com-
pounds such as these, where the structure is present but the activity hidden,
raise the poser: ‘‘When is a vitamin not a vitamin?”

54 A. F. Hess, M. Weinstock, and E. Sherman, J. Biol. Chem. 67, 413 (1926).

55 C. EK. Bills and F. G. McDonald, J. Biol. Chem. 72, 13 (1927).

56 H. von Euler and A. Bernton, Ber. 60, 1720 (1927).

57 QO. Rosenheim and T. A. Webster, Lancet II, 622 (1927).

58 R. K. Callow, Biochem. J. 25, 79 (1931).

°° H. von Euler, A. Wolf, and H. Hellstrém, Ber. 62, 2451 (1929).

5% A. Windaus and O. Rygh, Nachr. Ges. Wiss. Géttingen, Math. physik. Kl. 202
(1928).

60 B. E. Bailey, J. Fisheries Research Board Can. 6, 103 (1943).

$1 §. Bernstein, J. J. Oleson, H. B. Ritter, and K. J. Sax, J. Am. Chem. Soc. 71, 2576
(1949).

144 VITAMIN D GROUP

By replacing the hydroxy] group with a thiol group, Strating and Backer®
and Bernstein and Sax® have recently prepared 7-dehydrothiocholesterol
(7-dehydrocholesteryl mercaptan or cholestadiene-5 ,7-thiol-3). This com-
pound exhibits an absorption spectrum similar to that of the provitamins
D and undergoes similar spectral changes upon irradiation, but the thiol
isostere of vitamin D3 which apparently resulted was not antiricketic for
chicks, even at generous doses.

Strating and Backer®™ have also prepared the compound, ‘‘7-dehydro-3-
homocholesterol,” in which the hydroxyl group is replaced by the HOCH:
group, i.e., 3-hydroxymethyl-cholestadiene-5 ,7. This exhibits the typical
provitamin D absorption spectrum, but no studies on its activatability have
been reported.

The C-3 position is a center of asymmetry. Sterols which differ from nor-
mal only in the steric arrangement of substitutions on C-3 are defined as
episterols.*8 They are not precipitable by digitonin. The epi configuration
is exhibited by epiergosterol and 7-dehydroepicholesterol. Both are highly
activatable, 7-dehydroepicholesterol acquiring about one-tenth as much
activity as is acquired by 7-dehydrocholesterol under similar exposure to
ultraviolet hight.“4: *

b. Orientations at C-9 and C-10

Isopyrocalciferol is the C-9 epimer of ergosterol, and except for this differ-
ence the two are identical in structure. The fact that isopyrocalciferol does
not become antiricketic upon irradiation is evidence that the normal con-
figuration at C-9 is essential for activation. Lumisterol differs from ergos-
terol only in the epimerization of its substituent methyl group at C-10.
The fact that it is fully activatable is evidence that the configuration at
C-10 is not a determinant of activatability. Pyrocalciferol differs from ergos-
terol in the spatial arrangements at both C-9 and C-10, and it is not activa-
table.

c. The Side Chain

The sex hormone, 3, 17-dihydroxyandrostadiene, differs structurally from
ergosterol and 7-dehydrocholesterol only in the absence of a side chain,
the substitution at C-17 being an hydroxyl group. This compound, when
irradiated, undergoes the spectral changes indicative of vitamin D forma-
tion (Fig. 7), but the product shows no antiricketic activity.”

The cholanic acid analog of ergosterol and 7-dehydrocholesterol, known
as 3-hydroxy-A®*7-choladienic acid, differs from the activatable provita-

62 J. Strating and H. J. Backer, Rec. trav. chim. 69, 909 (1950).

63 §. Bernstein and K. J. Sax, J. Org. Chem. 16, 685 (1951).
64 J, Strating and H. J. Backer, Rec. trav. chim. 70, 389 (1951).

II. CHEMISTRY 145

mins only in having a short side chain terminating in a carboxyl group. It
is nevertheless not activatable.®

The sterol, A®.7-norcholestadiene-3-ol, differs from 7-dehydrocholesterol
in that its side chain is unbranched and has one fewer CH: group. Although
it is described as ‘‘a new provitamin D,” no study of its antiricketic acti-
vatability has appeared.

The natural compound, A*7.?-cholestatriene-3-ol, or 22-dehydro-7-de-
hydrocholesterol, differs from 7-dehydrocholesterol only in having a double

Original 30 Sec. Irrad. 60 Sec. Irrad. 3 Min. Irrad. 6 Min. Irrad.

(a od a, Pe

WA
A WAL

Fic. 7. Spectral changes during the irradiation, under identical conditions, of
ergosterol (upper curves) and 3,17-dihydroxyandrostadiene (lower curves). (After
Dimroth and Paland.‘”)

15,000

10,000

Extinction Coefficient

5,000

0 280 260 280

bond at C-22. It has not been isolated in sufficient purity for direct studies
to be made on its activatability, but as a component of the provitamin D
mixture occurring in Mytilus edulis it probably contributes to the vitamin
D potency of the irradiated mixture.*® One would expect it to be activatable,
because the double bond at C-22 is known (in ergosterol) not to prevent the
formation of an effective vitamin D.

7-Dehydrocholesterol and 7-dehydroepicholesterol have identical side
chains, the shortest and simplest found in any provitamin D of unques-
tioned activatability. A double bond at C-22, as in ergosterol, is favorable
to activation, but not essential for it, because when it is saturated, as in
22-dihydroergosterol, activation can still occur, although the vitamin D

65 G, A. D. Haslewood, Biochem. J. 38, 454 (1939).

146 VITAMIN D GROUP

produced is of lesser potency.®*: ®” When the double bond is replaced by an
oxido group, as in 22,23-oxidoergosterol, the activatability is sharply re-
duced.“

7-Dehydrocampesterol is a C-24 epimer of 22-dihydroergosterol.®: ® It
is about one-tenth as activatable as ergosterol, being in this respect some-
what inferior to 22-dihydroergosterol. Thus it appears that the difference
in antiricketic activity due to stereoisomerism on C-24 is somewhat greater
than the difference caused by the double bond at C-22.

The presence of an ethyl group at C-24, as in 7-dehydrostigmasterol,
instead of a methyl group as in ergosterol, apparently destroys activata-
bility. An ethyl group at C-24 is present in 7-dehydrositosterol, which in
early experiments was found to be activatable.*®: ® Ruigh,®® however, has
presented evidence that the observed activatability is due to the presence
in the 7-dehydrositosterol of a quantity of 7-dehydrocampesterol or other
provitamin D as an impurity. 7-Dehydroclionasterol, the C-24 epimer of
7-dehydrositosterol, has not been tested for activatability, but in consider-
ation of the presence of the ethyl group, one would not expect it to acquire
marked antiricketic properties.

Several of the biological tests for the activatability of the sterols in Fig. 6
leave much to be desired. In most instances the irradiated products were
tested only on rats, and only by oral administration. The conclusions in
regard to the relation of chemical structure to antiricketic activatability
might have to be modified if the products were tested at higher dosage
levels, or by parenteral administration, or with a larger number of species.
One is reminded that, if it were customary to use only chicks in testing
vitamin D, irradiated ergosterol would be found inactive, or, at most,
feebly active, in comparison with irradiated 7-dehydrocholesterol. The ques-
tion mark is therefore applied to some sterols in Fig. 6, even though the
best reported tests have indicated that their irradiation products do not
cure rickets.

4. NEWER SynTHETIC METHODS

The provitamins D, especially 7-dehydrocholesterol, are so important
commercially that much effort has been applied to improving the original
synthesis*® and to devising new procedures for dehydrogenating common
sterols. Windaus and Schenck”? patented the original procedure as applied
to cholesterol, sitosterol, and stigmasterol and offered zirconium and mag-
nesium alcoholates as alternatives to aluminum isopropylate for the re-

66 F. G. McDonald, J. Biol. Chem. 114, Proc. Ixv (1936).

67 W. Grab, Hoppe-Seyler’s Z. physiol. Chem. 243, 63 (1936).

68 H. Fernholz and W. L. Ruigh, J. Am. Chem. Soc. 68, 1157 (1941).
69 W. L. Ruigh, J. Am. Chem. Soc. 64, 1900 (1942).

70 A. Windaus and F. Schenck, U.S. Pat. 2,098,984 (1937).

Il. CHEMISTRY 147

duction of the keto to the hydroxy sterols. They also obtained a product
patent” on 7-hydroxycholesterol. Barr et al.”? made 7-hydroxycholesterol
by oxidizing cholesteryl hydrogen phthalate with permanganate. Hasle-
wood”? found that better yields of the monobenzoate of the dehydro sterols
were obtained when the dibenzoate of the 7-hydroxy sterols was treated
with boiling dimethylaniline instead of being heated dry in vacuo. Rosen-
berg” patented the use of organic bases in general for this purpose. Winter-
steiner and Ruigh” discovered that the dibenzoate, when treated at room
temperature with sodium methylate in methanol, undergoes an unexpected
partial hydrolysis, the acyl group at position 3 being removed, while the
acyl group at position 7 remains. The resulting 7-monobenzoate of the
hydroxy sterol can then be heated in dimethylaniline to give the desired
7-dehydro sterol in superior yield and purity.

Wintersteiner’* made 7-hydroxy sterols by treating stabilized colloidal
solutions of the original sterol with molecular oxygen, and Wintersteiner
and Bergstrém” obtained good yields of 7-ketocholestero] and 7-hydroxy-
cholesterol by this means. Krémli’ prepared 7-ketocholesteryl acetate by
anodic oxidation, reduced it to 7-hydroxycholesterol with sodium methylate
in methanol-benzene, and dehydrated the product to 7-dehydrocholesterol
by boiling with concentrated oxalic acid solution. The dehydration pro-
cedure was admittedly open to improvement, and the product was not well
characterized. Horvath and Krdmli7? obtained, among other products, 7-
dehydrocholesterol from cholesterol by the action of Azotobacter and®° a
good yield of 7-hydroxycholesterol by the action of Proactinomyces roseus.

Milas and Heggie*! have patented the dehydrogenation of cholesterol and
similar sterols by treatment with a wide variety of agents. Their examples
were confined to cholesteryl acetate, which they dehydrogenated with qui-
none, chloranil, sulfur, diphenyl sulfide, benzaldehyde, methylene blue in
the presence of light, and the succinodehydrogenase of beef heart. Their
best example (with quinone) showed a yield of 20% of provitamin. Mazza

71 A, Windaus and F. Schenck, U.S. Pat. 2,098,985 (1937). -

72 T. Barr, I. M. Heilbron, E. G. Parry, and F. 8. Spring, J. Chem. Soc. 1936, 1437.

733 G. A. D. Haslewood, J. Chem. Soc. 1938, 224; Biochem. J. 33, 454 (1939).

7”. R. Rosenberg, U.S. Pat. 2,209,934 (1940).

75 O. Wintersteiner and W. L. Ruigh, J. Am. Chem. Soc. 64, 1177 (1942); U.S. Pat.
2,411,177 (1946).

76 Q. Wintersteiner, U. 8. Pat. 2,400,380 (1946).

77 Q. Wintersteiner and 8. Bergstrém, J. Biol. Chem. 187, 785 (1941); S. Bergstrém
and O. Wintersteiner, bid. 141, 597 (1941); 145, 309, 327 (1942).

78 A. Krdmli, Arch. Biol. Hung. 17, 337, 343 (1947).

79 J, Horvath and A. Krdmli, Nature 160, 639 (1947).

80 A. Krdmli and J. Horvath, Nature 162, 619 (1948); 163, 219 (1949).

81 N. A. Milas and R. Heggie, J. Am. Chem. Soc. 60, 984 (1938); U. S. Pat. 2,260,085
(1941).

148 VITAMIN D GROUP

and Migliardi*®? dehydrogenated cholesteryl acetate with quinone in boiling
acetic acid during exposure to filtered light (to activate the quinone) and
obtained a yield of 7-dehydrocholesterol amounting to 30 %. Sah** made the
provitamin by heating cholesteryl acetate with ordinary quinone and sev-
eral homologs and analogs thereof.

A new semisynthesis of provitamin D, based on the selective bromination
of sterols at position 7 by means of N-bromosuccinimide, has been described
by Henbest et al.,*4 Bide e¢ al.,5° and Buisman ed al.** Cholesteryl acetate gives
7-bromocholesteryl acetate,.and this, when heated in diethylaniline, splits
off hydrogen bromide, leaving 7-dehydrocholesteryl acetate, from which
the free provitamin is obtained by saponification. Yields up to 30 % have
been claimed. Schaaf’? has patented some technical details of the reaction,
claiming yields up to 40 %. Redel and Gauthier* bring about the dehydro-
bromination by heating with collidine, and Ruigh and Gould*® use quinal-
dine, which they claim gives 50 % better yields than diethylaniline. Schaaf*
effects a further improvement in yield by employing quinaldine diluted
with aromatic hydrocarbons.

C. THE PROVITAMINS D IN NATURE
1. ISOLATION

Only rarely does a provitamin D occur in such abundance, or so free
from other sterols, that it can be obtained in fair purity simply by the
recrystallization of unsaponifiable extracts. The exceptions are ergosterol
in certain fungi, and the new provitamin D, in the ribbed mussel,
Modiolus.*! Usually it is necessary to employ special techniques, such as
digitonin precipitation to concentrate the sterol fraction, esterification with
suitable agents for the double purpose of protecting the provitamin from
destruction and enhancing fractionation, and above all, chromatographic
separation of the provitamin or its esters. Esterification with 3 ,5-dini-
trobenzoyl chloride in pyridine is a characterization procedure borrowed

82 FP. Mazza and C. Migliardi, Quaderni nutriz. 8, 86 (1941).

83 P| P. T. Sah, Rec. trav. chim. 59, 454 (1940).

84 H. B. Henbest, E. R. H. Jones, A. E. Bide, R. W. Peevers, and P. A. Wilkinson,
Nature 158, 169 (1946); E. R. H. Jones and R. W. Peevers, British Pat. 574,432
(1946).

85 A. E. Bide, H. B. Henbest, E. R. H. Jones, R. W. Peevers, and P. A. Wilkinson,
J. Chem. Soc. 1948, 17838.

86 J. A. K. Buisman, W. Stevens, and J. van der Vliet, Rec. trav. chim. 66, 83 (1947).

87 K. H. Schaaf, U. S. Pat. 2,542,291 (1951).

88 J. Redel and B. Gauthier, Bull. soc. chim. France 15, 607 (1948).

89 W. L. Ruigh and D. H. Gould, U. 8. Pat. 2,546,787 (1951).

9K. H. Schaaf, U. S. Pat. 2,546,788 (1951).

21H. G. Petering and J. Waddell, J. Biol. Chem. 191, 765 (1951).

II. CHEMISTRY

149

TABLE II

OccURRENCE OF THE PROVITAMINS D IN PLANTS AND ANIMALS?

Source

Provitamin D, parts per
thousand of total sterol

Phanerogams
Cottonseed oil
Rye grass
Scopolia root
Spinach
Wheat germ oil
Cocksfoot grass
Horse chestnut
Rutabaga
Carrot
Bean
Cabbage
Cryptogams
Mold, Aspergillus niger
Mushroom, Cortinellus shiitake
Ergot, Claviceps purpurea
Yeast, Saccharomyces cerevisix
Mold, Penicillium puberulum
Alga, seaweed, Fucus vesiculosus
Vertebrates
Skin, pig
Skin, chicken feet
Skin, rat
Skin, wild pig
Skin, mouse
Skin, calf
Skin, human adult
Skin, human infant
Skin, cow
Skin, deer
Skin, eel
Skin, chicken trunk
Liver, tuna, Japanese
Liver, cod, Atlantic
Liver, shark
Liver, tuna
Liver, pig
Liver, cod, Japanese
Liver, halibut
Liver, tuna, bluefin
Liver, whale
Brain, rabbit
Brain, lumpfish
Brain, human fetus

bo oO © & bo

.06*

_

SOCCCOCO OHHH BH OPH HH BEA
SS ops

150

TABLE II—Continued

VITAMIN D GROUP

Source

Provitamin D, parts per
thousand of total sterol

Vertebrates—Continued
Brain, sheep

Brain, deer

Brain, human infant
Brain, horse

Brain, cow

Brain, human adult
Eggs, duck, Chinese
Eggs, duck, Dutch
Eggs, cod (roe)
Eggs, herring (roe)
Eggs, hen

Eggs, cormorant
Eggs, mire crow
Eggs, silver gull
Eggs, lumpfish (roe)
Body of frog

Venom of toad

Wool fat, sheep
Milk, cow

Placenta, cow
Pancreas, beef

Blood serum, cow
Spinal cord, beef
Mice, gutted carcasses
Colostrum, cow
Thymus, cow

Bile, ox

Herring oil

Spleen, cow

Milt, herring

Heart, calf

Lymph, dog

Rats, gutted carcasses
Gallstones, man
Lung, calf

Blood, dog

Sclerotic aortas, man

Invertebrates

Poriferans
Commercial species of sponges
Cliona celata, sponge
Spheciospongia vesparia, loggerhead sponge
Halychondria panicea, sponge

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II. CHEMISTRY 51

TABLE II—Continued

Provitamin D, parts per

Source thousand of total sterol

Invertebrates— Continued

Coelenterates
Metridium dianthus, sea clove 86
Actinoloba dianthus, sea anemone 52*
Actinia equina, sea anemone 50
Urticina crassicornis, sea anemone 45
Pennatula quadrangularis, sea pen 42
Alcyonium digitatum, sea finger 34
Anemonia sulcata, sea anemone 17
Commercial species of corals 10
Australian species of sea anemones 9
Unidentified species of sea anemones trace
Bryozoan
Flustra securifrons, sea mat 65*
Annelids
Tubifex (Sp.), waterworm 210*
Lumbricus terrestris, earthworm 170*
Arenicola marina, lugworm, sandworm 55*
Hirudo medicinalis, leech 39*
Nereis virius, ragworm 16*
Arthropods
Tenebrio molitor, mealworm 120*
Melolontha vulgaris, cockchafer grubs 89
Gyronomus (Sp.), gnat 61
Dytiscus marginalis, diving beetle 40
Carausius morosus, locust eggs 25
Aleurobius farinae, meal mite 25
Eriocheir sinensis, wool hand crab 23*
Blatta orientalis, cockroach 23
“‘Kieferspinnerraupe,”’ pine caterpillar 22
Cantharis vesicatoria, Spanish fly 22
Cancer pagurus, common crab 1B
Bombyx mori, silkworm eggs 8.9*
Daphnia (Sp.), waterflea 7.5
Musca domestica, housefly 7.0
Melolontha vulgaris, cockchafer, May beetle 5.0
Apis mellifica, honeybee 4.5
Crangon vulgaris, shrimp 3.8*
Munida bamffica, crustacean 3.5
Homarus vulgaris, lobster 2.5
Mollusks
Modiolus demissus, ribbed mussel 370*
Arion empiricorum, slug, red road snail 220*
Buccinum undatum, whelk, wave horn snail 180*
Littorina littorea, periwinkle 170*
Archidoris tuberculata, sea snail 150

a

152 VITAMIN D GROUP

TABLE II—Concluded

Provitamin D, parts per

Source thousand of total sterol

Invertebrates—Continued

Ostrea (Sp.), Australian oyster 130
Arion (Sp.), black road snail 120
Mytilus edulis, sea mussel 100*
Helix pomatia, edible snail, vineyard snail 97*
Anodonta cygnea, swan mussel 80
Ostrea virginica, oyster 80
Pecten (Sp.), Australian seallop 65
Mytilus planulatus, Australian mussel 62*
Cardium edule, cockle, sand shell 50*
Cardium tenuicostatum, Australian cockle 41*
Ostrea edulis, oyster 34*
Limaz agrestis, earth snail 32
Sepia (Sp.), cuttlefish, squid 2%
Echinoderms

Astropecten irregularis, little sea aster 4.5
Asterias rubens, starfish, big sea aster 3.8

* Data from Windaus,™ Gillam and Heilbron,®* van derVliet,®* and others. Figures
marked (*) are the average of two or more determinations.

from the organic chemists by Callow, who first applied it to vitamin D
work in the isolation of calciferol from irradiated ergosterol. It has proved
useful in the identification of both the vitamins and provitamins D, to which
latter purpose it was first applied by Boer et al.® Boer was also the first
to adapt chromatography to the isolation of a provitamin D, and he gave
a good working account of this procedure.

The unsaponifiable fractions of the fats of nearly all plant and animal
tissues (Table II) exhibit the provitamin D absorption spectrum, but the
quantity of provitamin D present is usually so small that its isolation is a
task of considerable magnitude.” Isolation has been achieved in only a few
instances, leaving great territories unexplored. The following examples con-
stitute most of the published work to date.

92 R. K. Callow, Chemistry at the Centenary (1931) Meeting of the British Associa-
tion for the Advancement of Science, p. 149. Heffer, Cambridge, 1932.

93 A.G. Boer, E. H. Reerink, A. van Wijk, and J. van Niekerk, Proc. Koninkl. Akad.
Wetenschap. Amsterdam 39, 622 (1936); A. G. Boer, J. van Niekerk, E. H. Reerink,
and A. van Wijk, Dutch Pat. 45,849 (1939).

% A. Windaus, Nachr. Ges. Wiss. Géttingen, Math. physik. Kl., Fachgruppe III
185 (1936). ’

95 A. E. Gillam and I. M. Heilbron, Biochem. J. 30, 1253 (1936).

96 J, van der Vliet, Chem. Weekblad 39, 271 (1942).

97 A. Windaus and O. Stange, Hoppe-Seyler’s Z. physiol. Chem. 244, 218 (1936).

II. CHEMISTRY 153

2. ERGOSTEROL

Tanret® discovered ergosterol in ergot, and Gérard? noted its occurrence
generally in the fungi. Heiduschka and Lindner!°® compared the ergosterol
content of ten fungi, including yeasts, finding from 0.29 to 1.17 % of ergos-
terol in the dry substance. The ergosterol content of twenty-nine species
and strains of yeast, grown under similar conditions, was determined spec-
trographically by Bills et al.!°' and found to vary from a trace to as high as
2.0% of the dry material. The ergosterol of commerce is obtained from
baker’s yeast, i.e., strains of Saccharomyces cerevisiae grown in aerated wort.
Part of the supply was formerly obtained from Aspergillus niger,’ but this
source has been abandoned in favor of yeast. Savard and Grant!” cited
previous studies on the occurrence of ergosterol in several species of Penicil-
lium and isolated some ergosterol from the mycelium of P. notatum grown
in submerged culture. Although available in quantity from the antibiotics
industry, Penicillium has not become a commercial source of ergosterol,
because the yield is comparatively low.

Windaus and Stange**: 7 isolated ergosterol from the sterol of hens’ eggs.
Its presence there, in association with cholesterol, was surprising but ex-
plainable on the basis that it had been transferred from the feed of the
hens (cf. grass, grains, and earthworms, below). It is known! that hens,
unlike mice, rats, rabbits, or dogs, absorb small amounts of fed ergosterol.
The finding of ergosterol in eggs does not preclude the possibility that other
provitamins D were also present but less readily isolated by the chromato-
graphic technique which was employed.

Bock and Wetter!®® studied the sterols of the vineyard snail, Helix
pomatia, the red road snail, or slug, Arion empiricorum, and the common
earthworm, Lumbricus terrestris. In these the major sterols were cholesterol
and some phytosterol-like substances, but the only provitamin isolated
from the mixtures was ergosterol. The coexistence of other provitamins was
not precluded, and almost certainly others were present, for Boer ed al.10% 1%7

9 C. Tanret, Ann. chim. et phys. Ser. 5, 17, 493 (1879); Compt. rend. 108, 98 (1889);
Ann. chim. et phys. Ser. 6, 20, 289 (1890); Ser. 8, 15, 313 (1908).

99}. Gérard, Compt. rend. 114, 1544 (1892) ; 121, 723 (1895) ; 126, 909 (1898); J. pharm.
chim. Ser. 6, 1, 601 (1895).

100 A, Heiduschka and H. Lindner, Hoppe-Seyler’s Z. physiol. Chem. 181, 15 (1929).

101 C, KE. Bills, O. N. Massengale, and P. 8. Prickett, J. Biol. Chem. 87, 259 (1930).

1022 A. Zimmerli, U. 8. Pat. 1,893,317 (1933).

103 K. Savard and G. A. Grant, Science 104, 459 (1946).

104 R. Schénheimer and H. Dam, Hoppe-Seyler’s Z. physiol. Chem. 211, 241 (1932);
W. Menschick and I. H. Page, bid. 211, 246 (1932).

106 F, Bock and F. Wetter, Hoppe-Seyler’s Z. physiol. Chem. 256, 33 (1938).

106 A. G. Boer, J. van Niekerk, E. H. Reerink, and A. van Wijk, U.S. Pat. 2,163,659
(1939).

107 A. G. Boer, J. van Niekerk, E. H. Reerink, and A. van Wijk, U. 8S. Pat. 2,266,674
(1941).

154 VITAMIN D GROUP

have ascribed to the ‘“‘spectroscopically pure’ provitamin of earthworms
physical constants different from those of ergosterol and have further de-
scribed the earthworm provitamins as a “chicken provitamin D.”’ Indeed,
van der Vliet®* ascribes to earthworm provitamin D, activated, an efficacy
ratio of 100.

Pollard!“ found ergosterol in cocksfoot grass, and Windaus and Bock
identified it in wheat germ oil,!°? cottonseed oil, and scopolia root sterols.!!°
The 7-dehydro derivatives of sitosterol, stigmasterol and campesterol, which
might have been expected in these sources, were not found and, in fact,
have never been found in nature.

The foregoing chemical studies are supported and extended by chick-rat
assays on irradiated vegetable materials. Thus Bethke e¢ al." found the low
efficacy ratio characteristic of irradiated ergosterol in irradiated specimens
of cottonseed oil, wheat middlings, and alfalfa leaf, as well as in yeast and
Aspergillus. Haman and Steenbock" found the low ratios in irradiated
coconut, peanut, and wheat germ oils, and Black and Sassaman!® got simi-
lar results with alfalfa unsaponifiables and maize oil phytosterol. Koch and
Koch," however, reported that irradiated maize oil phytosterol showed a
somewhat higher efficacy ratio than irradiated ergosterol. On the whole, it
appears that, with one exception, no provitamin D but ergosterol has yet
been identified in the vegetable kingdom. The exception is 22-dihydroergos-
terol, which Santos Ruiz!!® has separated chromatographically from the
minor sterols of ergot.

3. 7-DEHYDROCHOLESTEROL

In only three instances has 7-dehydrocholesterol been isolated from a
natural source. Boer et al.** obtained it from a sample of duck egg cholesterol
containing 4.5 % of provitamin D. That ducks’ eggs should contain a differ-
ent provitamin D from hens’ eggs is remarkable and doubtless reflects a
difference in diet, perhaps a predominantly fish diet in the case of the ducks.
Windaus and Bock"® isolated 7-dehydrocholesterol from pigskin, and Bock
and Wetter!™ obtained it from the wave horn snail, or whelk, Buccinum
undatum. It is noteworthy that this snail yielded 7-dehydrocholesterol,
whereas the two other snails studied by the same investigators gave ergos-
terol.

108 A. Pollard, Biochem. J. 30, 382 (1936).

109 A, Windaus and F. Bock, Hoppe-Seyler’s Z. physiol. Chem. 256, 47 (1988).

10 A, Windaus and F. Bock, Hoppe-Seyler’s Z. physiol. Chem. 250, 258 (1937).

111 R. M. Bethke, P. R. Record, and O. H. M. Wilder, J. Biol. Chem. 112, 231 (1935).
2 R, W. Haman and H. Steenbock, J. Biol. Chem. 114, 505 (1936).

113 A. Black and H. L. Sassaman, Am. J. Pharm. 108, 237 (1936).

4H}. M. Koch and F. C. Koch, J. Biol. Chem. 116, 757 (1936).

116 A, Santos Ruiz, Anales real acad. farm. 3, 201 (1941).

116 A, Windaus and F.. Bock, Hoppe-Seyler’s Z. physiol. Chem. 245, 168 (1937).

Il. CHEMISTRY 155

It is assumed, on the basis of chick-rat assays, that the provitamin D in
the cholesterol of all mammalian sources is 7-dehydrocholesterol. Com-
mercial cholesterol, such as was used in Waddell’s work,** comes from the
spinal cords of cattle. Bethke et al." found that activated samples of butter,
lard, and brain exhibit the same high efficacy ratio as cod liver oil or ir-
radiated ordinary cholesterol. Haman and Steenbock!” reported high
efficacy ratios for irradiated lard and chicken fat.

4, PROVITAMINS D or INVERTEBRATES

The phenomenal abundance of provitamin D in the sterols of the inverte-
brates has been demonstrated by a number of workers,®!: 4-96 105-107, 117-121
whose findings are included in Table II. In several instances provitamins
have been isolated, which, although “pure” by spectrographic tests, do
not correspond in other properties to any known form of provitamin D.
Some of these have considerable commercial, as well as theoretical, 1mpor-
tance.

Four provitamins were described in the patents of Boer ed al.10%: 1%, 117
The one from the earthworm, Lwmbricus terrestris, has already been dis-
cussed and shown to be a mixture of ergosterol with at least one other,
unidentified, provitamin. The one from the waterworm, Tubifex, has not
been further investigated chemically, but in view of the findings on Lumbri-
cus it seems unlikely that it is a single substance. In biological tests®® it has
been found to have an efficacy ratio of 100 when activated. The one from
_ the common sea mollusk, Mytilus edulis, has been reinvestigated by van
der Vliet,°° who considers it to be a mixture of three main components,
7-dehydrocholesterol, ergosterol, and a provitamin tentatively identified as
22-dehydro-7-dehydrocholesterol (i.e., A®*7:?-cholestatriene-3-ol). These
sterols were not isolated, but their presence indicated indirectly by fraction-
ations, degradations, and efficacy ratios. The presence of ergosterol could
account for the somewhat low efficacy ratio of 71% reported” for the
mixture. It has been suggested that the provitamin D of the periwinkle,
Littorina littorea, may be impure 7-dehydroclionasterol.’” After irradiation
it is said®® to exhibit an efficacy ratio of 100%. Such effectiveness seems
inconsistent with the presence of an ethyl group in the side chain.

Van der Vliet®® has determined the chick-rat efficacy ratios of several
other invertebrate provitamins D which have not been examined

17 A. G. Boer, J. van Niekerk, E. H. Reerink, and A. van Wijk, U.S. Pat. 2,216,719
(1940).

us P. Fantl, Australian J. Exptl. Biol. Med. Sci. 20, 55 (1942).

19 W.§S. Calcott, J. Waddell, and H. R. Rosenberg, U.S. Pat. 2,383,446 (1945).

20 H. R. Rosenberg and J. Waddell, J. Biol. Chem. 191, 757 (1951).

#21 H. Rosenberg, U. S. Pat. 2,475,917 (1949).

122 C. A. Kind and S. C. Herman, J. Org. Chem. 18, 867 (1948).

—

156 VITAMIN D GROUP

chemically. After irradiation, the provitamins of the mealworm, Tenebrio
molitor, the sea mussel, Mytilus edulis, the starfish, Astertas rubens, and
the sponge, Halychondria panicea, exhibited efficacy ratios of about 100%,
which indicates the absence of much ergosterol. But the activated pro-
vitamin of the leech, Hirudo medicinalis, showed only 65 % effectiveness,
and that of the crab, Cancer pagurus, only 10 %. These must have contained
ergosterol, or some other provitamin of low efficacy ratio.

The richest of all known animal sources of provitamin D is the ribbed
mussel, Modiolus demissus,*!: "°° in which the provitamin content
amounts to from 35% to 50% of the total sterol. The crude provitamin
appears to be a mixture of provitamins D, according to Rosenberg and
Waddell,!2° who noted differences in chick-rat efficacy ratios of specimens
made from mussels from different environments. From the crude sterols
Petering andWaddell ® isolated a provitamin which they tentatively called
provitamin D,, (m for Modiolus). This appears to be a single substance, with
29 carbon atoms, which in the activated form exhibits an efficacy ratio
10 % to 20 % higher than cod liver oil.!°

5. BIOGENESIS

Plants differ from animals in having no mechanism for the translocation
of lipoids. Therefore, in plants, the provitamin D, like all sterols, must origi-
nate and accumulate in the cells where it is found, whereas in animals its
origin may be remote from the site of accumulation.

In plants ergosterol is the predominant provitamin D. In some of the
lower species it comprises up to 95 % of the total sterol, and hence it would

_ seem to be a primary metabolic product. Sumi!” observed that the forma-

_ tion of ergosterol in the mushroom, Cortinellus shiitake, can occur at any

stage of the life cycle, and that the percentage of ergosterol steadily rises

cas the plant grows older.

It would seem that in plants ergosterol is synthesized from simple com-
pounds of carbon. Thus ergosterol has been produced in Penicillium glaucum
from sucrose and tartaric acid, in Mucor mucedo from lactose, in Pent-
cillium puberulum,!* Aspergillus fischeri,” and many other fungi’ from dex-
trose as the carbon sources.

123 M. Sumi, Sci. Papers Inst. Phys. Chem. Research Tokyo, 20, 254 (1933).

124 B. Gérard, Compt. rend. 114, 1544 (1892).

125 EB. Gérard, Compt. rend. 121, 723 (1895); J. pharm. chim. Ser. 6, 1, 601 (1895).

26 J, H. Birkinshaw, R. K. Callow, and C. F. Fischmann, Biochem. J. 25, 1977 (1931).

127 T,, M. Pruess, W. H. Peterson, and E. B. Fred, J. Biol. Chem. 97, 483 (1932).

128 T,, M. Preuss, W. H. Peterson, H. Steenbock, and E. B. Fred, J. Biol. Chem. 90,
369 (1931).

Il. CHEMISTRY rae

Massengale ef al.° demonstrated that the yeast Saccharomyces cerevisiae
elaborated widely different amounts of ergosterol, according to the sugar
which supplied the carbon to the basal medium. This work contributes one
of the few bits of information available on the mechanism of sterol forma-
tion. Seven fermentable sugars were used, with the finding that di- and
trihexoses occasioned the synthesis of greater amounts of ergosterol than
did the monohexoses (singly or in admixture) into which they are hydro-
lyzed. The amounts of ergosterol produced bore no relation to the protein
or fat content of the yeast cells. It is a reasonable inference, therefore,
that the monohexoses which are split from the higher sugars by the yeast
enzymes exist at the instant of cleavage in forms particularly suitable for
synthesis into sterol. According to this concept, ergosterol is primarily a
product of carbohydrate metabolism.

In this connection, an experiment by Maclean and Hoffert!*? is significant.
It was found that the presence of sulfite in the sugar solution in which the
yeast was incubated resulted in a marked decrease in ergosterol production
but in little or no interference with the elaboration of fatty acid. The
argument was advanced that in the formation of sterol, but not of fatty
acid, an aldehyde removable by sulfite comes into the picture between the
hexose and the final product. The action of the sulfite might have been
better explained as that of an oxygen remover. Actually, the production
of ergosterol is enhanced by the presence of oxygen carriers such as methy-
lene blue and various inorganic per-salts.!*!

_ Halden and his associates,“* who maintain that there is a biogenetic

relationship between fats and sterols, have succeeded in increasing the
ergosterol production of yeast ten- to fortyfold by keeping it in a state of
semidehydration and supplying alcohol and air. Under these conditions the
normal processes of budding and fermenting are repressed, while the pro-
duction of fat and sterol, especially the latter, is augmented to an extraor-
dinary degree. Halden’s papers contain a valuable review of ergosterol
production in yeast.

In the higher plants even less is known about the origin a the ubiquitous
ergosterol. Heilbron and Sexton! suggested that it arises from the simul-
taneous oxidation and reduction (dehydrogenation and hydrogenation) of
sitosterol, which at the time (1929) was believed to have 27 carbon atoms,
the same as ergosterol. This would relate the known wide occurrence of

1229 Q. N. Massengale, C. E. Bills, and P. 8. Prickett, J. Biol. Chem. 94, 213 (1931).

130 T. Smedley Maclean and D. Hoffert, Biochem. J. 20, 343 (1926).

131 W. G. Bennett, U. S. Pat. 2,059,980 (1936).

132 W. Halden, Hoppe-Seyler’s Z. physiol. Chem. 225, 249 (1934); Austrian Pat. 140,190
(1934); M. Sobotka, W. Halden, and F. Bilger, Hoppe-Seyler’s Z. physiol. Chem.
234, 1 (1935).

133 J. M. Heilbron and W. A. Sexton, Nature 123, 567 (1929).

158 VITAMIN D GROUP

dihydrositosterol and ergosterol to the commonest vegetable sterol, but,
unfortunately for this hypothesis, sitosterol and ergosterol were later found
to contain 29 and 28 carbon atoms, respectively .!*4 If ergosterol is the 7-de-
hydro derivative of anything, it would be of brassicasterol, but this sterol
is much less widely distributed than sitosterol. Also, against the hypothesis
of origin by oxidation-reduction is the fact that no provitamin except er-
gosterol (and 22-dihydroergosterol) has been found in the vegetable king-
dom. Until a better explanation is offered, it will have to suffice to regard
ergosterol as a primary metabolic product of the higher plants, as well as
of the lower.

In animals the provitamins D are partly absorbed from food and partly
formed within the body. The ergosterol in hens’ eggs is a clear example of
an exogenous provitamin. The ergosterol of worms, snails, and bivalves is
probably also derived from the food, but the 7-dehydrocholesterol of these
species may be endogenous. In carnivorous and omnivorous animals it is
probable that some of the 7-dehydrocholesterol is absorbed along with the
cholesterol of their prey. In a word, it appears that all ergosterol originates
in plants, and all 7-dehydrocholesterol in animals; almost nothing is known
about the origin of the still unidentified provitamins of the invertebrates.

Herbivorous mammals, which normally never ingest cholesterol after
weaning, and also the herbivorous lower animals, apparently must syn-
thesize their 7-dehydrocholesterol as well as their cholesterol, and it is
probable that all animals do this to some extent. It is not known whether
total synthesis from simple compounds of carbon or partial synthesis from
cholesterol takes place. There is no evidence of total synthesis other than
the analogy of the well-established fact that animals synthesize cholesterol
itself: 134a ri

There is considerable evidence that 7-dehydrocholesterol can be formed
in vivo from cholesterol. Schoenheimer™® in 1931 applied Wieland’s dehy-
drogenation theory to the explanation of the ubiquitous occurrence of de-
hydrocholesterol and dihydrocholesterol in cholesterol. According to this
view, dehydrogenation and hydrogenation of cholesterol occur simultane-
ously as a general biological process in which cholesterol functions as hydro-
gen acceptor. (It is immaterial that Schoenheimer spoke of ergosterol as
the provitamin in this system, for at that time ergosterol was thought to
have the same number of carbon atoms as cholesterol; his argument ap-
plies equally well to 7-dehydrocholesterol). In keeping with this theory
are the observations that dehydrogenation of cholesterol can be brought
about in vitro by an enzyme" and by bacterial action.” The constant forma-
134 A. Windaus, F. von Werder, and B. Gschaider, Ber. 65, 1006 (1932).

1348 K. Bloch and D. Rittenberg, J. Biol. Chem. 145, 625 (1942).
136 R. Schoenheimer, Science 74, 579 (1981).

II. CHEMISTRY 159

tion of traces of 7-dehydrocholesterol by some sort of oxidation-reduction
mechanism may also explain the reported presence of the unstable provi-
tamin D in roughly normal amount in the cholesterol from the brain of a
mummy 1400 years old.1*

It may be that the biosynthesis of 7-dehydrocholesterol occurs in steps,
rather than as a dehydrogenation-hydrogenation reaction. Bergstrém and
Wintersteiner’’ have shown that position 7 in cholesterol is extremely sus-
ceptible to attack by molecular oxygen. Haslewood!** and others have de-
tected 7-hydroxycholesterol in the animal body, and although this was
possibly an artifact, the ease with which it is formed from cholesterol in
stabilized colloidal solutions points to a possible route of synthesis, since
only the dehydration step is needed to convert it to 7-dehydrocholesterol.
7-Ketocholesterol, which precedes 7-hydroxycholesterol in the laboratory
synthesis, has also been found in the animal body.!*7> 139

Whatever the reactions involved, the fact of conversion of cholesterol
to 7-dehydrocholesterol appears to be established by the experiments of
Scott e¢ al. These workers found provitamin D in the lining of the small
intestine of guinea pig, rat, and ox in amounts as large or larger than in
the skin, usually regarded as the site of the greatest concentration. It per-
sisted in the gut wall despite fasting for 24 hours or the feeding of a low-
sterol diet. When spectroscopically pure cholesterol was fed to fasted guinea
pigs the amount of provitamin in the small intestine increased during the
period of absorption but returned to normal when absorption was
completed. Since during the same cycle the amount of provitamin in the
liver increased progressively, it follows that the provitamin was formed in
the gut wall and stored in the liver. The concentration of provitamin was
greatest in the duodenum and was associated largely with the mucosa and
lamina propria.

Partially supporting this work is a note by Rosenberg,'! who found in
clams feeding on algae a much higher concentration of provitamin in the
viscera than in the body. The kind of provitamin was not determined, but
the efficacy ratio was the same for the visceral as for the body material, and
it was much higher than would be expected of a vegetable source. It would
seem that in these clams provitamin D was being elaborated in the gut wall,
from nutrients in the process of absorption, but whether the synthesis was
a total one or merely a dehydrogenation is not clear.

136 H. King, O. Rosenheim, and T. A. Webster, Biochem. J. 23, 166 (1929).
137 §. Bergstrém and O. Wintersteiner, J. Biol. Chem. 141, 597 (1941).

1388 G. A. D. Haslewood, Biochem. J. 33, 709 (1939); Nature 154, 29 (1944).
139 V. Prelog, L. Ruzicka, and P. Stein, Helv. Chim. Acta 26, 2222 (1943).

140 M. Scott, J. Glover, and R. A. Morton, Nature 163, 530 (1949).

141 H. R. Rosenberg, Nature 164, 795 (1949).

160 VITAMIN D GROUP

D. THE VITAMINS DD
1. OccURRENCE AND ORIGIN

In contrast to the almost universal occurrence of provitamin D, vita-
min D itself is very limited in its distribution.“ In the vegetable kingdom
it is especially rare and never found in high concentration. Occasional
specimens of coconut oil show antiricketic activity, but the potency here
is not natural; it is man-made in the primitive process of drying the copra
under the sun. It is historically interesting to note that what was probably
the first therapeutic use of an irradiated material was made a century ago
by Thompson, who, writing on the anemia of tuberculosis, stated that
“the use of almond-oil and of olive-oil was not followed by any remedial
effect, but from cocoa-nut oil results were obtained almost as decided as
from the oil of the liver of the Cod...”

Yeast normally never contains vitamin D, but it can be activated to a
potency of 10,000 I.U. per gram by exposing it in powdered form to sun-
light under controlled conditions.“* Clover hay when cured in the dark is
inactive, but when cured in the sun it acquires a slight antiricketic activ-
ity.1*° Cocoa shell, as obtained from fermented beans dried in the sun, has
been reported to contain 28 I.U. of vitamin D per gram, part of which may
have originated in the surface fungi known to be present.'* The antiricketic
activity of ergot!’ is probably due to something other than vitamin D,
because the active substance cannot be extracted from the unsaponifiable
fraction with petroleum ether.

Marine vegetation being the ultimate food of fishes, much interest at-
taches to the possible occurrence of vitamin D in the plant life of the ocean.®
Leigh-Clare™® observed that the marine diatom, Nitzschia closierium, fails
to effect any genesis of vitamin D, when cultured under conditions of maxi-
mum insolation. Drummond and Gunther"? found little or no vitamin D
in mixed phytoplankton taken from the ocean. Johnson and Levring!”®

142, P. Daniel and H. E. Munsell, U. S. Dept. Agr. Misc. Publ. 275 (1937); L. E.
Booher, E. R. Hortzler, and E. M. Hewston, Vitamin Values of Foods. Chemical
Publishing Co., Brooklyn, 1942.

143 'T. Thompson, Proc. Roy. Soc. (London) 7, 41 (1854).

144 OQ. N. Massengale, C. E. Bills, and P. S. Prickett, J. Biol. Chem. 94, 213 (1931);
C. E. Bills, U. 8. Pat. 1,877,382 (1932).

145 H. Steenbock, E. B. Hart, C. A. Elvehjem, and 8S. W. F. Kletzien, J. Biol. Chem. 66,
425 (1925). i

146 A. W. Knapp and K. H. Coward, Analyst 59, 474 (1934).

147 F}. Mellanby, E. Surie, and D. C. Harrison, Biochem. J. 23, 710 (1929).

148 J, L. Leigh-Clare, Biochem. J. 21, 368 (1927).

1449 J. C. Drummond and E. R. Gunther, Nature 126, 398 (1930); J. Exptl. Biol. 11,
203 (1934).

150 N. G. Johnson and T. Levring, Svenska Hydrograf.-Biol. Komm. Skrifter, Ser. 3,
1, No. 3, 7 pp. (1947) [C. A. 45, 236 (1951)].

\

II. CHEMISTRY 161

found no vitamin D in two species of higher algae, Enteromorpha intesti-
nalis and Laminaria saccharina, but in Fucus vesiculosus they reported
slight activity. They found provitamin D, but no vitamin D, in phytoplank-
ton oil.

Darby and Clarke!®! investigated the floating brown alga, Sargassum, in
the area of its origin near the Tortugas Islands. Here in clear water under
the tropical sun enormous quantities of the weed develop. Masses of it
break away and drift into the Gulf Stream, in which it floats as far as Ice-
land. As it drifts along it becomes heavily infested with mollusks, shrimps,
and other invertebrates, and thus indirectly it is a major food of fish.
Sargassum free of foreign matter contains about 3 % of oil on the dry basis,
and this oil was reported to have an antiricketic potency of the same order
as some of the poorer fish liver oils.

An especially interesting example of the natural occurrence of vitamin
D in the vegetable kingdom is found in the work of Scheunert e¢ a/.1°? with
edible mushrooms. Four species were studied, and they showed a vitamin
D content of 0.21 to 1.25 I.U. per gram when grown in the absence of strong
light. One species, the common Agaricus campestris, exhibited 0.21 I.U. per
gram when grown in a cellar, and three times as much when grown in a
meadow where light presumably was a factor. In these ergosterol-rich
plants, a minute fraction of the provitamin apparently is activated with-
out the agency of light.

There is no evidence that vitamin D plays any role in plant physiology,

_and it is not difficult to understand why it is seldom found in live plant
tissues. The lower, non-pigmented, non-photosynthesizing plants, which
contain much ergosterol, thrive in dark places and perish in the light. The
higher plants, containing relatively little provitamin, possess pigments
which presumably filter out the activating rays at the short end of the
solar spectrum. Even if the vitamin could be formed in the superficial
layers, plants could not store it, unprovided as they are with any system
for the translocation of lipoids. f

In the animal kingdom vitamin D is less rare than in plants, but it is
abundant only in certain fishes. In mammals it is found principally in the
milk and liver, and in birds in the liver and egg yolks. These foods contain
from a fraction of a unit to severa] units per gram—only enough to make
them marginal sources of vitamin D for human nutrition. The amount can
be substantially increased by irradiating the animals or adding vitamin
D to their food.”

The fact that mammals and birds are subject to rickets is proof that they
cannot synthesize vitamin D. Except for occasional amounts gained by eating
151 W. H. Darby and H. T. Clarke, Science 85, 318 (1937).

182 A. Scheunert, M. Schieblich, and J. Reschke, Hoppe-Seyler’s Z. physiol. Chem. 235,
91 (1935).

—

162 VITAMIN D GROUP

such foods as fish and eggs, the vitamin must normally be obtained by ex-
posure of the body surface to the sun (the ‘‘sunshine vitamin’’). Since
sunshine is variable, both daily and seasonally, the amounts obtained
by insolation vary widely. The body has two mechanisms for adjustment
to the variable supply. Excess vitamin D is stored, principally in the liver,
where it may be held and released over long periods of time. This is the
basis of the ‘“‘Stosstherapie’’ method of preventing rickets in children,
which consists in administering massive doses of vitamin D at intervals of
several months.!*?: 154 Prolonged insolation results in tanning, a process of
temporary pigmentation which presumably has the effect of filtering out
the activating rays received in superabundance. It is well known that
Negroes and other dark-skinned people are more susceptible to rickets than
white people, and this difference appears to be due at least in part to pig-
mentation.!®*, 156 The picturesque suggestion has been made that in human
evolution vitamin D played a role in the appearance of blonde races in the
cold countries, where they have an advantage over dark races in the ab-
sorption of the limited sunlight.

That man and animals are well adapted to a variable supply of vitamin
D is clearly shown in the phenomenally wide margin which exists between
the therapeutic and the toxic doses of this substance.!! For rats, doses 1000
times the antiricketic dose, administered daily for months, are just per-
ceptibly harmful, and much larger single doses can be tolerated. For man,
the margin appears to be somewhat less, but still enormous. Probably no
other potent therapeutic agent exhibits such a wide margin of safety, but
if it were not wide, animal life could not have survived the fluctuations in
solar activity and vitamin D formation to which it has always been sub-
jected in nature.

The formation of vitamin D on the body surface has been thoroughly
studied. Sunshine, like fish oil, is an old remedy for rickets. In 1919 Huld-
schinsky!” clearly demonstrated, by means of radiographs, the healing ac-
tion of sunlight and of the light from the quartz mercury arc. Hess ei al.1*
determined that the effective wavelengths of light are the shorter ultra-
violet waves of the solar spectrum, or the still shorter waves of artificial

153 G. O. Harnapp, Wien. klin. Wochschr. 58, 698 (1940).

154 H. Uflacker, Z. Kinderheilk. 67, 350 (1949).

155 A. F. Hess, Rickets, Osteomalacia and Tetany, p. 92. Lea and Febiger, Phila-
delphia, 1929.

156M. M. Eliot and E. A. Park, in Brennemann’s Practice of Pediatrics, Vol. 1,
Chapter 36, pp. 3, 12. W. F. Prior Co., Hagerstown, Md., 1948.

157 K, Huldschinsky, Deut. med. Wochschr. 45, 712 (1919); Z. orthopdd. Chir. 38, 426
(1920).

168 A. F. Hess, A. M. Pappenheimer, and M. Weinstock, Proc. Soc. Exptl. Biol. Med.
20, 14 (1922).

Il. CHEMISTRY 163

sources. Goldblatt and Soames!’ discovered that the livers of irradiated
rats, when fed to non-irradiated rats, convey some of the virtue of irradia-
tion to the animals which eat them. Hess et al. found that lanolin, the fat
of wool, is activatable, and Hess and Weinstock" discovered that skin
itself becomes antiricketic upon irradiation and that irradiated cholesterol
is effective when administered subcutaneously.

Rekling!® found that irradiation did not protect rats from rickets when
they were prevented from licking their fur. Hou and Tso! found that the
skin of normal rabbits was slightly antiricketic, the dorsal skin more so than
the ventral, but that the skin of rickety rabbits or of normal rabbits reared
indoors was without protective action. Hou!” noted that the effectiveness
of ultraviolet irradiation for curing rickets in rabbits was almost lost when
the skin had been previously washed with ether. Rowan'!® observed that
birds of prey on a meat diet developed rickets, and that the addition of
feathers to the diet supplied protection. He suggested that the preen gland
is concerned with the formation of vitamin D. Hou!® made an elaborate
study of the formation of vitamin D in birds. In brief, his findings were as
follows. Birds differ from mammals in having only one gland of a sebaceous
nature. This is the glandula uropygialis, or preen gland. Preen gland oil
contains provitamin D, which birds, by preening, distribute over their
feathers and effectively expose to sunlight. The vitamin D is either in-
gested by swallowing the feathers, or absorbed by the skin from the
feathers. The feathers and skin of normal birds were shown to be anti-
ricketic, but in rickety birds, or birds whose preen glands had been removed,
the feathers and skin had little, if any, antiricketic action. Removal of the
preen gland made the birds susceptible to rickets, and rickety birds without
the gland were not benefited by exposure to ultraviolet radiation or sun-
shine.

Thus Hou!® was led to see that ‘‘ . . . vitamin D or its precursor is princi-
pally derived from the oil secretion rather than from the diet. This may be
more or less true for all birds. For although nocturnal birds, and the car-
nivorous animals which prey upon other forms of ‘feather and fur,’ may
derive their vitamin D supply mainly from their victims, yet the source of
the vitamin in the prey would appear to lie in the oil secretion. This may
explain the absence of oil gland in some species of birds . . . and the necessity
of adding rabbits or small birds with the fur or feather intact, to the diet

159 H. Goldblatt and K. M. Soames, Biochem. J.17, 446 (1923).

160 BH. Rekling, Strahlentherapie 25, 568 (1927).

161 H. C. Hou and E. Tso, Chinese J. Physiol. 4, 93 (1930).

1682 H.C. Hou, Chinese J. Physiol. 4, 345 (1930).

163 W. Rowan, Nature 121, 323 (1928).

164 H.C. Hou, Chinese J. Physiol. 2, 345 (1928) ; 3, 171 (1929) ; 4, 79 (1930); 5, 11 (1931).
1665 H. C. Hou, Chinese J. Physiol. 8, 171 (1929).

164 VITAMIN D GROUP

of young carnivora in captivity to ensure their successful development .. .
Further, it is commonly known that if herbivora, e.g., horses, are scrubbed
thoroughly with soap and water, they do not thrive. It may thus be inferred
that the sebaceous secretion of the preen gland of birds . . . is an important
source of vitamin D in the mammal.”’

Hou!®* found that rickety chickens with preen glands removed could be
cured by irradiating the feet, even though irradiation of the body or of the
head was ineffective. In this case the substance activated was obviously
neither preen gland oil nor circulating blood, but something in the tissues
of the feet which was directly absorbed.

Evidence enough has been presented to show that the higher animals ob-
tain vitamin D in at least three ways, the relative importance of which
must vary with the habits, requirements, and opportunities: (1) by eating
such foods as eggs, fish, whole furred or feathered animals, and insolated
dead vegetable tissues; (2) by ingesting insolated sebaceous matter in the
process of neatening the body—licking and preening; and (3) by directly
absorbing the products of insolation formed on or in the skin.

The occurrence of vitamin D in fishes is a phenomenon of great biochemi-
cal, as well as practical, interest. Nearly all fish oils contain vitamin D,
but the amount varies prodigiously with the species (Table IIT) and other
factors. Both free and esterified forms are present.®: 1°

The principal fat storage depots of fish are the muscle tissue and the
liver. Some fat is also found in the viscera and head. Species which store
much oil in the muscle, such as salmon, have small livers which contain
little oil. Species which store little oil in the muscle, such as cod, have large
livers which contain much oil. The amount of oil which is stored in muscle
or liver depends on the abundance of food and the demands of spawning

The occurrence of vitamin D in fish body oils was first noted by Bills!”°
in experiments with commercial menhaden oil. In most physicochemical
characteristics the liver and body oils of a given species are similar, but the
concentration of vitamin D is generally higher in the liver oil. In some
species, such as the tunas, the liver oil may contain several thousand times

166 AW. C. Hou, Chinese J. Physiol. 5, 11 (1931).

167 C. E. Bills, F. G. McDonald, O. N. Massengale, M. Imboden, H. Hall, W. D. Her-
gert, and J. C. Wallenmeyer, J. Biol. Chem. 109, Proc. vii (1935).

16 >. §. Jordan, B. W. Evermann, and H. W. Clark, Check List of the Fishes and
Fishlike Vertebrates of North and Middle America North of the Northern Bound-
ary of Venezuela and Columbia. Report of the U. 8. Commissioner of Fisheries
for the Fiscal Year 1928 with Appendixes, Part 2. Gov’t Printing Office, Washing-
ton, 1930.

169 K. C. D. Hickman, Ind. Eng. Chem. 29, 1107 (1937); K. C. D. Hickman and E. L.
Gray, zbid. 30, 796 (1938).

170 C, E. Bills, Studies on the Antiricketic Vitamin: Dissertation. Johns Hopkins
Press, Baltimore, 1924.

II. CHEMISTRY 165

TABLE III
DISTRIBUTION OF VITAMINS D aNp A IN THE LiveR Ors oF 100 Species oF Fis
Common name of fish Scientific name Zoological order van , Vian A,
Oriental tuna* Thunnus orientalis Percomorphi 45,000 170,000
Frigate mackerel* Auais thazard Percomorphi 44,000 30,000
California bluefin tuna* Thunnus saliens Percomorphi 42,000 65,000
Striped tuna* Katsuwonus pelamis Percomorphi 42,000 36,000
Meji tuna* Parathunnus sibi Percomorphi 38,000 22,000
Bonito* Sarda lineolata Percomorphi 35,000 57,000
Yellowtail* | Sericla dorsalis Percomorphi 25,000 67,000
Red snapper Lutianus campechanus | Percomorphi 22,000 61,000
Atlantic tuna Thunnus secundodorsalis Percomorphi 16,000 80,000
Albacore* Thunnus germo Percomorphi 13,000 12,000
Yellowfin tuna* Neothunnus macropterus Percomorphi 12,000 35,000
White sea-bass* Cynoscion nobilis Percomorphi 11,000 92,000
Jewfish* Stereolepis gigas Percomorphi 9,000 500,000
Ishinagi* Stereolepis ishinagi Percomorphi 7,000 500,000
Swordfish* Xiphius gladius Percomorphi 7,000 130,000
Black perch Embiotoca jacksont Holeonoti 7,000 6,000
Broadfin sole Lepidopsetta bilineata Heterosomata 6,800 13,000
Oriental mackerel* Scomber japonicus Perecomorphi 6,300 59,000
Corsair rockfish Sebastomus rosaceus Cataphracti 5,800 75,000
Mackerel seads Decapturus muroadsr Percomorphi 5,800 5,900
Grouper Epinephelus morio Percomorphi 4,800 24,000
Barracuda* Sphyrzna argentea Percomorphi 4,700 67,000
Starry rockfish Sebastomus constellatus Cataphracti 4,500 89,000
Yellowtail rockfish Sebastosomus flavidus Cataphracti 4,000 82,000
Red rockfish Rosicola miniatus Cataphracti 2,600 14,000
Totuava* Eriscion macdonaldi Percomorphi 2,500 190,000
Jack smelt Atherinopsis californiensis Percomorphi 2,400 96 , 000
Spearfish* Makaira mitsukurii Percomorphi 2,300 120,000
' Bastard halibut* Paralichthys californicus Heterosomata 2,300 69,000
Sardine (pilechard) Sardinia cerulea Tsospondyli 2,300 16,000
Rockfish Pteropodus vexillaris Cataphracti 2,200 49 ,000
Red rockfish Sebastopyr ruberrimus Cataphracti 2,100 100,000
Snoek* Thyristes atun Percomorphi 2,000 23,000
Bocaccio Sebastodes paucispinis Cataphracti 1,800 77,000
Yellowbacked rockfish Pteropodus maliger Cataphracti 1,800 32,000
Pacific hake Merluccius productus Anacanthini 1,500 50,000.
Black rockfish Sebastosomus mystinus Cataphracti 1,500 37,000 ©)
China rockfish Pteropedus nebulosus Cataphracti 1,400 110,000
Fringe sole Psettichthys melanostictus Heterosomata 1,400 10,000
Halibut* Hippoglossus hippoglossus Heterosomata 1,200 75,000
Shad Alosa sapidissima Isospondyli 1,200 17,000
Striped bass - Roccus sazatilis Percomorphi 1,200 500
Orange rockfish Rosicola pinniger Cataphracti 1,100 86, 000
Green spotted rockfish Sebastomus chlorostictus Cataphracti 1,100 47,000
Rockfish Acutomentum entomelas Cataphracti 1,100 8,000
Wall-eyed perch Hyperprospon argenteus Holconoti 1,100 3,500
Rabbitfish Cyclichthys schepfi (?) Plectonathi 1,100 2,200
Starry flounder Platichthys stellatus Heterosomata 1,000 8,200
Striped rockfish Hispaniscus elongatus Cataphracti 990 74,000
Ainame Hezagrammos otakaii Cataphracti 950 3,900
Ling cod* Ophiodon elongatus Cataphracti 920 160,000
Striped perch Txniotoca lateralis Holconoti 900 4,300
Rubberlip perch Rhacochilus toxotes Holeonoti 890 9,300
Rockfish black bass Sebastosomus melanops Cataphracti 830 49,000
Spanish flag rockfish Hispaniscus rubrivinctus Cataphracti 810 32,000

166

VITAMIN D GROUP

TABLE IlI—Concluded

Common name of fish Scientific name Zoological order View ae ve
Boston mackerel Scomber scombrus Percomorphi 750 31,000
California mackerel* Pneumatophorus diego Percomorphi | 730 45,000
Pufferfish Spheroides maculatus Plectonathi 570 1,500
Cabezon Scorpenichthys marmoratus Cataphracti 530 16,000
Round-nose sole* Eopsetta jordani Heterosomata 520 76,000
Nibe Sciena mitsukurit Percomorphi 500 5,400
Pacific white perch Phanerodon furcatus Holconoti 460 6,400
Fork-tail perch Damalichthys argyrosomus Holeonoti 410 2,700
Black cod* Anoplopoma fimbria Cataphracti 310 42,000
Chili-pepper Sebastodes goodei Cataphracti 270 150,000
Cabrilla* Epinephelus analogus Percomorphi 260 160,000
Newfoundland turbot Reinhardtius hippoglossoides Heterosomata 260 7,000
Pacific cod* Gadus macrocephalus Anacanthini 190 4,800
Atlantic salmon Salmo salar Isospondyli 180 12,000
Rock cod Sebastolobus alascanus Cataphracti 150 6,400
Rex sole Errex zachirus Heterosomata 140 8,200
Pointed sole Parophrys vetulus Heterosomata 140 6,100
Sculpin Scorpena guttata Cataphracti 140 3,000
Sand dab Citharichthys sordida Heterosomata 120 3,700
Atlantic hake* Urophycis (Sp.) Anacanthini 120 2,300
California turbot Pleuronichthys decurrens Heterosomata 110 8,200

. Menuke* Sebastodes baramenuke Cataphracti 100 120,000
Atlantic cod* Gadus morrhua Anacanthini 100 1,400
Yellow sole Pseudopleuronectes dignabilis | Heterosomata 87 17,000
Widowfish Acutomentum ovale Cataphracti 82 73,000
Tinker mackerel Pneumatophorus grex Percomorphi 77 9,300
Atlantic pollack* Pollachius virens Anacanthini 70 2,300
Pacific pollack* Theragra chalcogramma Anacanthini 67 8,300
Sheepshead Pimelometopon pulcher Pharyngognathi 62 6,600
California flying fish Cypselurus californicus Synentognathi 51 35,000
Corbina Menticirrhus undulatus Percomorphi 51 11,000
Rosefish* Sebastes marinus Cataphracti 33 26,000
Common skate of California | Raja inornata Batoidei 25 9,800
Abura karei Hippoglossoides dubius Heterosomata 25 5,100
Big skate of California* Raja binoculata Batoidei 24 4,100
Kichiji Sebastolobus macrochir Cataphracti 22 4,900
Wolffish Anarhichas lupus Jugulares 19 1,300
(pacific dogfish* Squalus suckleyt Tectospondyli 13 13,000
Same karei Clidoderma asperrimum Heterosomata 12 4,900
Thresher shark | Alopias vulpinus Euselachii 9 2,400
Basking shark Cetorhinus maximus Euselachii 6 <100
Atlantic dogfish Squalus acanthias Tectospondyli 3 1,700
Ratfish Hydrolagus colliet Chimzroidei 2 180
Gray sole Glyptocephalus cynoglossus Heterosomata <2 8,900
Sturgeon Acipenser fulvescens Glaniostomi <1 600

a From Bills ef al.16’ with revisions; nomenclature from Jordan et al.188 wherever possible. The species
marked (*) are represented by more than one assay.

as much vitamin D per gram as the body oil. Different species show dif-
ferences in the physicochemical constants of the oil, but these differences
yield no clue as to the amount of vitamin D in the oil or how it got there.

The vitamin D values given in Table III represent average figures for
the more important species, and occasional figures for the less common ones.

S 7

/

II. CHEMISTRY 167

Within any species, however, great variations in potency are noted when
the oils of individual livers are assayed. Hess ef al.‘”! found that the vitamin
D potency of the oils from individual cod livers varies inversely with the |
oil content, and as much as 1000 times. Similar inverse ratios were noted
for haddock and pollack.

Bills e¢ al.” investigated the commercial halibut catch of Seattle at
weekly intervals. In January the oil content of the pooled livers was at
the lowest point, 12%, and the vitamin D content of the liver oil
was highest, 1400 I.U. per gram. In August the oil content reached its
highest point, 25%, and the vitamin D potency was lowest, 900 I.U. per
gram. Thus, although the decline in vitamin D potency was a little less
than can be accounted for by dilution with inactive new oil, it was about
what one would expect from dilution with oil containing a little vitamin D.
Nevertheless this explanation is not wholly satisfactory, for it was noted
that the vitamin A content of the liver oil varied with the vitamin D con-
tent, but several times more widely. It declined from 240,000 I.U. per
gram in January to 35,000 in August, thus reaching a low point which was
much lower than could have resulted merely from dilution. It is possible,
of course, that the two vitamins are so different in origin and metabolism
that comparisons have no significance.

Pugsley!”* observed that the content of both vitamins D and A in the
liver oil and intestinal oil of the Alaska cod, Gadus macrocephalus, varied
inversely with the fatness of the livers. Pugsley ed al. found that the vi-
tamins D and A of the liver oil of the Atlantic cod, Gadus morrhua,

varied inversely with the oil content, but remained constant when figured

as units per gram of body weight for fish of a given age group. However,
the vitamin content of the liver oil increased with the age of the fish in years.
Bailey!”® reported that the vitamin D potency of commercial pilchard oil,
made from the whole fish including stomach contents, varied inversely
with the oil yield, and Pugsley!” noted that the inverse pelanotsy held
for both vitamins D and A in this species.

Hess et al.!”* reported that the milt and roe of cod and other species con-
tain vitamin D, but the fry were ‘“‘practically devoid of this factor, which
evidently had been used up in the course of the development of the larvae.”
Thus it appears that fish begin life with little vitamin D, and as they grow

171 A, F. Hess, C. E. Bills, and E. M. Honeywell, J. Am. Med. Assoc. 92, 226 (1929).

12 C, E. Bills, M. Imboden, and J. C. Wallenmeyer, J. Biol. Chem. 105, Proc. x (1934).

173 L. I. Pugsley, J. Fisheries Research Board Can. 4, 405 (1939).

74 L. I. Pugsley, C. A. Morrell, and J. T. Kelly, Can. J. Research F23, 243 (1945).

76 B. E. Bailey, Biol. Board Can. Pacific Progr. Repts. 23, 11 (1935).

176 A. F. Hess, C. E. Bills, M. Weinstock, E. Honeywell, and H. Rivkin, Proc. Soc.
Exptl. Biol. Med. 25, 652 (1928).

168 VITAMIN D GROUP

up they somehow accumulate it, at times losing part of their supply for
spawning, but on balance always gaining more.

The function of the vitamin D in fish is unknown. If it is related to bone
calcification, the questions remain why different species require widely
different amounts of it, and why most species store much greater quantities
than do the marine mammals, such as seals and whales, which consume
them. Several investigators!” have associated a low vitamin D content in
the liver oil with imperfect bone formation in the so-called cartilaginous
fishes—elasmobranchs and cyclostomes—including representative sharks,
dogfish, skates, rays, chimeras, and lampreys. It is well established that in
these species the liver oil seldom contains as much as 100 I.U. of vitamin
D per gram, and usually much less. Examples are given in Table III.
It is evident, however, that some cartilaginous fishes store more vitamin
D than some fishes with hard bones; also that sturgeons, which are usually
classified as true fishes in spite of their skeletal softness, have the lowest
vitamin D content of all.

The studies on the cartilaginous fishes marked the first correlation be-
tween zoological classification and vitamin D content. They were followed
by the work of Bills et al.,!”8 who made a taxonomic study of the distribution
of vitamins D and A in 100 species of fish (Table III). This work revealed
that several fishes of the order Percomorphi exhibit extraordinary concen-
trations of vitamin D in their livers (tunas, basses, and swordfish). On the
other hand, livers of fishes of the order Heterosomata, which includes the
halibuts, flounders, and other flatfish, were comparatively poor in vitamin
D, though rich in vitamin A. Livers of fishes of the order Cataphracti, the
rockfishes, were better than average sources of both vitamins.

After Massengale and Nussmeier*® in 1930 demonstrated that irradiated
ergosterol is less effective than cod liver oil, per rat unit for chicks, many
writers, especially in the medical field, were given to distinguishing the two
forms as ‘‘synthetic’’ and “natural.’”’ The implication was that the natural
vitamin D of fish oils was a single definite substance. That this is not so
was first shown in 1934 by Bills e¢ al.17° who found that a specimen of bluefin
tuna liver oil exhibited a chick-rat efficacy ratio of about 15, whereas the
ratio for cod liver oil was 100 and that for irradiated ergosterol was 1.
(The tuna species was described as Thunnus thynnus, the designation some- *

“17 KH. Poulsson, Strahlentherapie 34, 648 (1929); S. Schmidt-Nielsen and 8. Schmidt-
Nielsen, Hoppe-Seyler’s Z. physiol. Chem. 189, 229 (1930); R. K. Callow and C. F.
Fischmann, Biochem. J. 25, 1464 (1931); E. André and R. Lecoq, Compt. rend. 194,
912 (1932).

1728 C. E. Bills, F. G. McDonald, O. N. Massengale, M. Imboden, H. Hall, W. D.
Hergert, and J. C. Wallenmeyer, J. Biol. Chem. 109, Proc. vii (1935).

179 C. E. Bills, O. N. Massengale, and M. Imboden, Science 80, 596 (1934).

II. CHEMISTRY 169

times applied to all bluefins; this was later corrected'*® to T’.. saliens, the
California bluefin, in accordance with the Jordan classification.1%)

Confirmation of this work appeared in the studies of Haman and Steen-
bock!” and Black and Sassaman.'® It was shown!” that a commercial,
mixed-species tuna liver oil was ‘somewhat less effective’? on chickens,
per rat unit, than other oils which were compared. Repeated trials!’ with
three Japanese species of tuna (bluefin, striped, and yellowfin) showed that
the liver oils were only from 40 % to 63 % as effective, rat unit for rat unit,
as cod liver oil on chickens. Bills e¢ al.8° extended the work with the oils
of twenty-five species of fish. They found conspicuously low efficacy ratios
for albacore, striped tuna, totuava, and California bonito, as well as for
the original California bluefin tuna. Several other species, especially white
sea bass and Pacific dogfish, appeared to have efficacy ratios that were
distinctly above par.

It is true, nevertheless, that most fish oils have an efficacy ratio of around
100, the value characteristic of cod liver oil and activated 7-dehydrocholes-
terol (vitamin D3). Thus sardine (pilchard) body oil, which Bills 8° in
1927 found similar to cod liver oil in assays with rats, has for many years
been successfully used in poultry raising. Rygh!®! could recognize no signifi-
cant differences in the efficacy ratios of the liver oils of seventeen species of
fish. Haman and Steenbock!” found that sardine body oil, halibut liver oil,
and burbot liver oil had about the same efficacy ratio as cod liver oil. Black
and Sassaman!™ found that the liver oils of cod, halibut, swordfish, and
Japanese mackerel were about the same in efficacy ratios. In the work of
Rygh'®! and especially of Dols,!®? the vitamin D of tuna liver oil was found to
have the same efficacy ratio as cod liver oil; the tuna was the European blue-
fin, Thunnus thynnus, a species distinct from the California and Japanese
forms.

The fact that cod liver oil and many other fish oils exhibit the efficacy
ratio of vitamin D; is no proof that this is the one and only form of vitamin
D which these oils contain. It is possible that other forms are present which
are equally effective for rats and chicks, or that forms of higher and lower
efficacy ratio are present in admixture. Hickman and Gray'®® utilized the
technique of molecular distillation in an investigation of the vitamin D of
several fish oils. They found at the outset that about 70% of the vitamin
D of cod liver oil occurs as esters, and so it was necessary to conduct the
short path distillations with saponified material. Cod liver oil exhibited a
complex elimination curve indicative of two major, two minor, and two
180 C. FE. Bills, O. N. Massengale, M. Imboden, and H. Hall, J. Nwtrition 18, 485 (1937).
ne © 5). Bills, J. Biol. Chem. 72, 751 (1927).

181 Q. Rygh, Nature 136, 552 (1935).
182 M. J. L. Dols, Z. Vitaminforsch. 5, 161 (1936).

170 VITAMIN D GROUP

trace forms of vitamin D. The vitamin D of tuna liver oil was so unstable
that good curves could not be obtained. White sea bass liver oil gave an
unusually pure curve, indicative of a preponderance of one form of vitamin
D. Spearfish liver oil gave a fairly good curve, with a somewhat higher
elimination maximum, suggesting a vitamin D of higher molecular weight.
Albacore liver oil had chiefly one form of vitamin D, with lesser amounts of
higher and lower boiling forms. The most volatile vitamin D of cod liver
oil was one of the minor forms, and this, when assayed! with rats
and chicks, exhibited an efficacy ratio of between 25% and 50%.

In two instances the vitamin D of fish oils has been isolated. The vitamin
D from halibut liver oil!** was identified as vitamin D;, and that from tuna
liver oil®> was vitamin D3; with an admixture of vitamin Dz. It is therefore
established, by efficacy ratio studies, by molecular distillation, and by
actual isolation, that more than one kind of vitamin D occurs in fish oils, and
it seems probable that several kinds may be present. Whether or not the
oil of any given species always contains the same kind or mixture is not
known, but it would be reasonable to assume that some qualitative varia-
tion, as well as quantitative, occurs as a result of differences in food and
other environmental factors.

Little but conjecture can be written about the origin of vitamin D in
fish oils. Steenbock and Black,’ in one of their early papers on the activation
of foods, emphasized the suggestion that vitamin D is formed by the inso-
lation of plankton, which is ingested by little fish, and these in turn by
larger fish, and so on. So far as green phytoplankton is concerned, this
hypothesis has received no experimental support, but the discovery of vi-
tamin D in the brown alga, Sargassum, renews interest in it.4*! Furthermore,

since ergosterol is a vegetable sterol, it seems more likely than not that

the activated ergosterol (vitamin D,.) found in tuna liver oil originated in”
the vegetable kindgom, although it could be that the ergosterol was ab-
sorbed as such and activated in a mollusk or fish. On the other hand, the
activated 7-dehydrocholesterol (vitamin D3) which was the only form of
vitamin D identified in halibut liver oil and the predominant one in tuna
liver oil, almost certainly originated in an animal, because cholesterol is
never found in the vegetable kindgom.

Marine animal life other than fish does not exhibit much vitamin D.
Drummond and Gunther™? found a small amount in zooplankton oil. Belloc
et al.'8® found that zooplankton collected in the spring contained provita-

183 C, E. Bills, O. N. Massengale, K. C. D. Hickman, and E. L. Gray, J. Biol. Chem.
126, 241 (1938).

184 H, Brockmann, Hoppe-Seyler’s Z. physiol. Chem. 245, 96 (1937).

1865 H, Brockmann and A. Busse, Naturwissenschaften, 26, 122 (1938); Hoppe-Seyler’s
Z. physiol. Chem. 256, 252 (1938).

186 G. Belloc, R. Fabre, and H. Simonnet, Compt. rend. 191, 160 (1930).

Il. CHEMISTRY 171

min D but no vitamin D, whereas that taken in midsummer showed both
provitamin and vitamin. Their data are interesting in view of the July
change in vertical distribution of zooplankton described by Russell.!87 Cop-
ping!** demonstrated the presence of small amounts of vitamin D in cope-
pods collected in summer. Of the larger marine invertebrates, it may be
noted that the body oil of squid showed about 6 I.U. of vitamin D per gram
in a test reported by Bills.!8°

Clear sea water is sufficiently transparent for the activating rays of the
sun to reach a depth of about one meter.!8*: 19° It is therefore possible, and
in view of Belloc’s work even probable, that in the smaller zooplankton
where the body area is large in relation to the volume, some vitamin D is
formed by insolation. It is unlikely that any significant amount of vitamin
D is formed by the insolation of fish or of the larger invertebrates, for most
of these species do not inhabit the top meter of the sea. The basking shark,
Cetorhinus maximus, is an exception in that it basks for hours at the sur-
face, feeding on little fish and plankton; yet its liver oil is poor in vitamin
D, even for an elasmobranch.!*! The common goldfish, Carassius auratus,
which feeds in shallow pools, accumulates hardly a trace of vitamin D in
its body oil.!%* River catfish, [clalurus punctatus, when experimentally ir-
radiated, proved very sensitive to ultraviolet rays, but even after many ex-
posures did not accumulate more vitamin D than catfish not irradiated.'8%

The suggestion that vitamin D may be synthesized by fish was first made
by Bills!®* in 1927. He observed that during the first four weeks of the
summer fattening season the cod of the Newfoundland shore fisheries
gorged themselves on a little fish called capelin, Mallotus villosus, and that
during this time the oil content of their livers doubled without diminution
of vitamin D potency. It was roughly estimated that the capelin eaten
could not account for all the vitamin D accumulated, and the balance was
attributed to synthesis. In view of the later work of Pugsley and others,
the original premises may have been faulty, in that they did not contem-
plate the possibility that the cod population may have involved different

187 F, §. Russell, Natwre 126, 472 (1930).

188 A. M. Copping, Biochem. J. 28, 1516 (1934).

189 W. R. G. Atkins and H. H. Poole, Trans. Roy. Soc. (London) B222, 129 (1933);
H. H. Darby, E. R. F. Johnson, and G. W. Barnes, Carnegie Inst. Wash. Publ.
475, 191 (1937).

199 N. G. Jerlov, Nature 166, 111 (1950). Jerlov’s claim that effective radiations
penetrate to a depth of 20 meters was based on the use of light filters centering
on 310 my. One suspects that the radiations detected at this depth represented
leakage at the long wave side of the pass band.

191 '§. Schmidt-Nielsen and 8. Schmidt-Nielsen, Hoppe-Seyler’s Z. physiol. Chem.
189, 229 (1930).

1918 C, E. Bills, unpublished observation.

iP VITAMIN D GROUP

age groups. But the work did inspire further attempts to demonstrate the
synthesis of vitamin D under controlled conditions.

Bills'* kept young catfish in darkened aquaria for six months on a diet
of veal muscle, which contained no detectable amount of vitamin D. At
the end of this period the fish had doubled in weight, and their visceral oil
deposits had increased. The visceral oil of these fish contained at least as
much vitamin D per gram as the oil of fish assayed at the beginning. Al-
though this experiment was not conclusive, it strongly suggested that syn-
thesis had occurred. Hess et al.!™ tried to demonstrate synthesis by allowing
cod livers to digest with added ergosterol, but the findings were negative.
In another experiment,!” captive codfish were given ergosterol by mouth,
and also intramuscularly, but there was no indication that any of it was
converted to vitamin D.

Fish feed extensively on mollusks, such as whelk,!* and thereby ingest
quantities of various provitamins D, yet the chief sterol of fish is cholesterol
containing only the traces of provitamin which are characteristic of the
higher animals. To associate the ingestion of much provitamin D with the
accumulation of much vitamin D implies a synthesis, for which there is
considerable evidence but as yet no proof. An up-to-date version of the
experiments of Hess and Bills would comprise the digesting of 7-dehydro-
cholesterol with the intestinal tissue or with the enzyme-rich pyloric cecum
of species known to be good accumulators of vitamin D, such as the tunas.

2. ISOLATION FROM NATURAL SOURCES

Isolation of vitamin D is a task beset with difficulties. In the first place,
the amount of vitamin D present, even in such rich sources as fish oils, is
small. Assuming that the vitamin D of cod liver oil has the same potency
as vitamin Ds, it can be figured that a ton of oil contains only 2 or 3 g. of
vitamin. But this vitamin, according to the evidence of molecular distilla-
tions,!*® exists in several forms. Furthermore, when taken out of its protect-
ing esters and oily vehicle, it is unstable. It is thus understandable that
vitamin D has only once been obtained in the crystalline state from a
natural source.

The crystalline preparation was isolated from tuna liver oil by Brockman
and Busse.!*® The procedure, in brief, consisted in the following steps: (1)
saponification and separation of the unsaponifiable fraction of the oil, (2)
partial separation of vitamin D from vitamin A by partitioning between

192 A. F. Hess, C. E. Bills, M. Weinstock, and M. Imboden, Proc. Soc. Exptl. Biol.
Med. 29, 1227 (1982).

193 A. H. Cooke, in Cambridge Natural History, Vol. 3, p. 59. Macmillan, London,
1913.

194 H, Brockmann, Hoppe-Seyler’s Z. physiol. Chem. 241, 104 (1936); H. Brockmann
and A. Busse, zbid. 249, 176 (1937).

II. CHEMISTRY 173

hydrocarbons and alcohols, (3) chromatographic adsorption on alumina gel,
aided at certain stages by an indicator dye, (4) removal of sterols by crys-
tallization from methanol and precipitation with digitonin, (5) esterifica-
tion to the 3,5-dinitrobenzoate, (6) chromatographic purification of the
crude esters, (7) recrystallization of esters, (8) saponification of purified
ester to free vitamin, and (9) crystallization of the free vitamin.

The product thus obtained consisted of vitamin D3; with an admixture
of vitamin D» to the extent of about 10%. Previously Brockmann had
made from tuna liver oil and halibut liver oil'** preparations of vitamin
D which were crystallized as dinitrobenzoates but not as free vitamin. They
were regarded at the time as pure vitamin D; esters, but in the light of the
later work they must be considered as somewhat less than pure. However,
in view of the known high efficacy ratio of halibut liver oil, it seems unlikely
that any vitamin D, was present in this preparation. Zucker et al.19°» 196
obtained the vitamin D of tuna liver oil in the form of its 3 ,5-dinitroben-
zoate and allophanate, and that of cod liver oil as allophanate. Apparently
these products were about three-quarters pure. Haslewood and Drum-
mond!” also have worked with tuna liver oil, obtaining the allophanate of
the vitamin in semipure condition.

In the isolation techniques the importance of the exclusion of air is
emphasized, even of air dissolved in solvents. Oxygen-free nitrogen and
carbon dioxide gases are used to replace air in apparatus and liquids.
Numerous modifications of the procedure of isolation described above are
possible. Alternative methods of removing vitamin A from vitamin D in-
clude the reacting of the vitamin A with maleic anhydride.!* Presumably,
citraconic anhydride would react similarly.!%? An alternative method of
separating vitamin D from hydrocarbon oils is to esterify the vitamin D
with phthalic anhydride, leaving the hydrocarbons behind.!** An alternative
method of purifying vitamin D by adsorption consists in the use of tri-
calcium phosphate instead of alumina gel.?°°

3. Puysics oF ACTIVATION

Since the absorption spectra of the several provitamins D are almost
identical in shape and position and not much different in height, it will

195 E. J. H. Simons and T. F. Zucker, J. Am. Chem. Soc. 58, 2655 (1936).

196 T, F. Zucker, E. J. Simons, H. C. Colman, and B. Demarest, Naturwissenschaften
26, 11 (1938).

197 G. A. D. Haslewood and J. C. Drummond, J. Soc. Chem. Ind. (London) 55, 598
(1936).

1% OQ. Dalmer, F. von Werder, and T. Moll, Hoppe-Seyler’s Z. physiol. Chem. 224,
86 (1934).

199 A. Windaus, O. Linsert, A. Liittringhaus, and G. Weidlich, Ann. 492, 226 (1932).

200S. E. Miller, U. S. Pat. 2,179,560 (1939).

174 VITAMIN D GROUP

suffice to show the absorption spectrum of ergosterol (Fig. 8). The location
of the maxima at 262, 271, 282, and 293.5 mu are based on recent deter-
minations with the Beckman spectrophotometer, and they are probably
a bit more accurate than earlier data obtained photographically. The
absorption minima at 263, 277, and 289.5 my are also shown in the figure.

Optical Density

230 240 250 260 270 280 290 300 310 320

Wavelength, millimicrons

Fia. 8. Absorption spectrum of ergosterol in alcohol 1:50,000 w./v. Ergosterol
monohydrate recrystallized from alcohol-benzene 2:1. (Courtesy of the Sulphite
Pulp Manufacturers’ Research League.)

Other maxima, detectable in more concentrated solutions, will be discussed
in the section on the chemistry of activation.

The wavelengths which affect ergosterol must be among those which it
absorbs. Ergosterol absorbs strongly from 300 my to 230 my in the far
ultraviolet region, but it exhibits some absorption beyond these limits in
both directions. The shortest effective wavelength has not been ascertained,
but it is known that 230 my activates ergosterol readily.2" Wavelengths
as long as 313 my are known to activate ergosterol slightly °° and this

201 A. L. Marshall and A. Knudson, J. Am. Chem. Soc. 52, 2304 (1930).
202 R. W. Haman and H. Steenbock, Ind. Eng. Chem. Anal. Ed. 8, 291 (1936).

II. CHEMISTRY 175

wavelength also appears to be the upper limit for 7-dehydrocholesterol.?°-?"
There is reason to believe that still longer waves play a part in the decompo-
sition of ergosterol and/or of its irradiation products.?°* The predominance
of these longer waves may explain the relatively feeble effect of sunlight
in activation. White light in the presence of optical sensitizers produces
chemical changes in ergosterol, such as peroxide and pinacol formation, but
it does not produce vitamin D.2

Ergosterol in the form of vapor, spray, powder, or solution can be ac-
tivated by a high-frequency oscillating discharge, with or without elec-
trodes.?-?!° High-voltage direct current has also been claimed to be effec-
tive.2°? Some activation is induced by cathode rays”: 4% and by radium
emanation." X-rays apparently do not activate it.?”

In an early (1927) study of the disappearance of ergosterol during irra-
diation in dilute solution, Morton et al.? concluded that the time-disappear-
ance curve is a straight line. They measured the disappearing ergosterol
spectrographically and did not know at the time of the existence of the
intermediate products of irradiation which contribute irrelevant absorption
to the system. Bourdillon ei al.2* measured the disappearance by digitonide
precipitation and found that the rate slows down as the irradiation proceeds.
The problem was finally resolved by Dasler?!® in 1938. He found that the
time-disappearance curve had the shape of a first-order curve. The reaction
involving the disappearance was, however, apparently a zero-order reac-
tion, because the half-life of the ergosterol was directly proportional to the
initial ergosterol concentration. The first-order character of the curve was
explained by the fact that increasing amounts of light were filtered out by
the piling up of light-absorbing irradiation products.

Before the structure of vitamin D was known, Pohl” suggested that

203 J. W. M. Bunker, R.S. Harris, and L. M. Mosher, J. Am. Chem. Soc. 62, 508 (1940).

204 A. F,. Hess and W. T. Anderson, Jr. J. Am. Med. Assoc. 89, 1222 (1927).

205 C. Sonne and E. Rekling, Strahlentherapie 25, 552 (1927).

206 Lahousse and Gonnard, J. phys. radium, Ser. 6, 10, 1148 (1929).

207 A. Windaus, P. Borgeaud, and J. Brunken, Nachr. Ges. Wiss. Géttingen, Math.
phystk. Kl. 313 (1927); A. Windaus and P. Borgeaud, Ann. 460, 235 (1928); A. Win-
daus and J. Brunken, Ann. 460, 225 (1928).

208 N, A. Milas, U.S. Pat. (Reissue) 22,038 (1942).

209 J. G. Farbenindustrie, Austrian Pat. 119,210 (1930).

210 ©, C. Whittier, U. S. Pats. 2,106,779, 2,106,780, 2,106,781, 2,106,782 (1938); W.
Dasler and C. D. Bauer, J. Biol. Chem. 167, 581 (1947).

2108 A. Knudson and C. N. Moore, J. Biol. Chem. 81, 49 (1929).

211 R. B. Moore and T. DeVries, J. Am. Chem. Soc. 58, 2676 (1931).

212 H. Goldblatt, Ergebn. allg. Pathol. u. pathol. Anat. 2. Abt. 25, 58 (1931).

213 R.A. Morton, I. M. Heilbron, and E. D. Kamm, J. Chem. Soc. 1927, 2000.

214 R. B. Bourdillon, C. F. Fischmann, R. G. C. Jenkins, and T. A. Webster, Proc.
Roy. Soc. (London) B104, 561 (1929).

25 W. Dasler, Swmmaries of Doctoral Dissertations Univ. Wisconsin 3, 219 (1938).

176 VITAMIN D GROUP

activation may involve the addition of energy to the sterol molecule through
an electron displacement. This is an oversimplification of what happens,
although it is true that energy is required for the rupture of ring B with
the formation of a fourth double bond for the vitamin molecule.

Estimations of the energy requirements of activation have been made
by several investigators,”°!-*04, 216-221 most recently by Harris et al.””” The lat-
ter workers” found that 7.5 X 10% quanta were required to produce one
U.S.P. unit of vitamin D, from ergosterol. The U.S.P. unit official at the
time (1938) was nominally identical with the international unit, but ex-
perienced bioassayers were aware that the U.S.P. Reference Cod Liver Oil
was weak in terms of the international standard. Arnold?” found the U.S.P.
unit to be 20 % weak (49.7 U.S.P.U. = 40 1.U.), a conclusion with which
the present author closely agrees. Nelson®™ recognizes a deficiency of 6.6%,
but this probably refers to a later and improved batch of reference oil.
Applying Arnold’s correction, one figures that 9.3 X 10! quanta are re-
quired to produce one international unit of vitamin D, from ergosterol.

Bunker et al.??° found no significant differences in quantum efficiency for
the wavelengths within the region where ergosterol absorbs strongly, but
they got some slight evidence that wavelength 302.5 my, which is near the
edge of the region of absorption, is less efficient. Haman and Steenbock?”
had earlier found that line 313, where absorption is still less, is distinctly
less efficient.

In the activation of the provitamin D of cholesterol, Hess and Anderson?”
demonstrated as early as 1927 that the efficiency of line 313 is very low.
Bunker et al.,?* working with 7-dehydrocholesterol itself, observed no ac-
tivation by line 313. They reported that wavelengths 248.38, 253.7, 265.2,
280.4, and 302.5 my are substantially uniform per quantum of energy
applied. But they claimed?™: 22° that line 296.7 is a little more efficient
than the others, an observation which is difficult to reconcile with the fact
that the absorption spectra of 7-dehydrocholesterol and ergosterol are prac-
tically identical.

The production of vitamin D directly in animals by irradiation may be
regarded as a special instance of the activation of 7-dehydrocholesterol. It
is complicated by the presence in the skin of substances exhibiting general
absorption, a form of light-filtering which usually becomes more and more

216 §, K. Kon, F. Daniels, and H. Steenbock, J. Am. Chem. Soc. 50, 2573 (1928).

217 R.S. Harris, J. W.M. Bunker, and L. M. Mosher, J. Am. Chem. Soc. 60, 2579 (1938).
218 A. Knudson and F. Benford, J. Biol. Chem. 124, 287 (1938).

219 J. R. Owen and A. Sherman, J. Am. Chem. Soc. 59, 763 (1937).

220 J.W.M. Bunker, R.S. Harris, and L. M. Mosher, J. Am. Chem. Soc. 62, 1760 (1940).
221 J. W. M. Bunker and R.S. Harris, New Engl. J. Med. 216, 165 (1937).

222 A. Arnold, Proc. Soc. Exptl. Biol. Med. 68, 230 (1946).

223 H}. M. Nelson, J. Assoc. Offic. Agr. Chemists 32, 801 (1949).

II. CHEMISTRY Wied

manifest, the shorter the wavelength involved. Hence it is to be expected
that the longer wavelengths in the critical region will exhibit greater over-all
efficiency than the shorter ones. This is borne out experimentally. Bunker
and Harris”! found that line 296.7 was the most effective for producing
vitamin D in depilated rats. Knudson and Benford?® also used rats, but
under the conditions of their experiment line 280.4 was the most efficient.
The findings with animals represent a special situation, which has little
bearing on the physics of activation zn vitro.

The uniformity in quantum efficiencies for the production of vitamin D
is especially noteworthy, when it is considered that the conversion is not
a single step, but an over-all effect involving the production of several
substances in succession, of which vitamin D is not the last (Fig. 9). No

Ergosterol Lumisterol Tachysterol Precalciferol Calciferol Toxisterol Suprasterol

250 300 250 300 250 300 250 300 250 300 250 300 250 300
me

Fre. 9. Absorption spectra of ergosterol and its irradiation products. (After
Windaus et al.4® and Velluz et al.?23#)

studies have been reported on the energy requirements of the intermediate
reactions, but it is assumed that the requirements are less uniform than
for the over-all activation.

Before the energy relations in activation were understood, the suggestion
was occasionally advanced”? 748 that the yield of vitamin D might be
improved by the use of filtered light or of light sources predominating in
certain wavelengths. The goal was a light which would activate provitamin
D, but not destroy the vitamin D produced. Why this has never been
reached is evident from the spectra in Fig. 9, which show that ergosterol
and calciferol, as well as the intermediate products, absorb light over
essentially the same range, 230 my to 300 mu. There are, however, some
differences in the absorption maxima, which may account for the fact that

223a T,. Velluz, G. Amiard, and A. Petit, Bull. soc. chim. France 16, 501 (1949).

224 'T. A. Webster and R. B. Bourdillon, Biochem. J. 22, 1223 (1928).
224 T. M. Heilbron, E. D. Kamm, and R. A. Morton, Nature 120, 617 (1927).

178 VITAMIN D GROUP

the composition of the crude irradiation product varies with the wave-
length used.”° In particular, it has been demonstrated that long waves
favor the accumulation of lumisterol, and short waves of tachysterol, each
at the expense of the other.??°

The light sources used for irradiation are principally the quartz mercury
are and the cored carbon arc, carbons for the latter containing compounds
of iron, magnesium, or other metals. A favorite experimental source in the
early years was the magnesium spark.”*: 3°; 226-230 Tight filters have not come
into general use, but for special studies.the Corning’ or Jena??6: 228, 280
glasses or Vitaglass?4* have been employed, or filter cells containing ben-
zene,?”7 31 xylene,??6 731-83 diphenyl,”*! carbon disulfide,?** chlorine,?2*: 227
bromine,?™ chlorine-bromine,”*: 2» 2%? tartrazine,””* salicylic acid,” lead ace-
tate,”3> potassium nitrate,” nickel sulfate,’ or alcoholic cobalt chloride.?*4

The temperature coefficient of activation is small. Bills and Brickwedde?#6
found that cholesterol containing 1.2 parts of provitamin D per 1000 was
readily activated at —183°, although the product was somewhat less potent
than the product of similar irradiation at room temperature. Webster and
Bourdillon?** irradiated ergosterol at temperatures between —195° and
+78° and observed that the effect of temperature was only moderate.
Knudson and Moore” noted that a “less potent product”? was obtained
when ergosterol was exposed to cathode rays at liquid air temperature
than when the exposure was made at room temperature. These observations,
made before the structure of vitamin D was known, pointed to the fact
that activation is an intramolecular change, because bimolecular reactions
are generally inhibited at very low temperatures. More recently Dasler?!®
has stated that temperature has ‘‘no effect upon the time-disappearance
curve of ergosterol nor upon the time-activation curve of ergosterol.’’ The
fact that activation is best conducted in boiling solvents is not contra-

225 HW}. H. Reerink and A. van Wijk, Biochem. J. 28, 1294 (1929); A. Windaus, Nachr.
Ges. Wiss. Gottingen, Math. physik. Kl. 36 (1930); F. A. Askew, R. B. Bourdillon,
H. M. Bruce, R. G. C. Jenkins, and T. A. Webster, Proc. Roy. Soc. (London)
B107, 91 (1930).

226 P, Setz, Hoppe-Seyler’s Z. physiol. Chem. 215, 183 (1933).

227 WH}. H. Reerink and A. van Wijk, Biochem. J. 23, 1294 (1929).

228 A. Windaus, Nachr. Ges. Wiss. Géttingen, Math. physik. Kl. 36 (1930).

229 A. Smakula, Nachr. Ges. Wiss. Géttingen, Math. physik. Kl. 49 (1928).

230 A. Windaus, Deut. med. Wochschr. 57, 678 (1931).

231 T. G. Farbenindustrie, German Pat. 565,900 (1932).

232 FW. A. Askew, R. B. Bourdillon, H. M. Bruce, R. G. C. Jenkins, and T. A. Webster,
Proc. Roy. Soc. (London) B107, 91 (1930).

233 HW. H. Reerink and A. van Wijk, Biochem. J. 25, 1001 (1931).

234 N. V. Philips’ Gloeilampenfabrieken, British Pat. 385,626 (1932).

235 G. Sperti, R. J. Norris, R. B. Withrow, and H. Schneider, U. S. Pat. 1,982,029
(1934).

236 C, K. Bills and F. G. Brickwedde, Nature 121, 452 (1928).

Il. CHEMISTRY 179

dictory to the conclusion that the temperature coefficient of activation is
small; it merely reflects the better exposure of molecules in agitated solution
and perhaps also the driving of the precalciferol — calciferol equilibrium
to the right (see below).

Ergosterol can be activated in the solid state, in the vapor phase, or in
solution. When it is irradiated in the solid state the first irradiation products
which form on the exposed surfaces act as filters which prevent light from
reaching the under layers until the surface products are destroyed. The
degree of antiricketic potency attainable by the irradiation of solid ergos-
terol is therefore relatively low, and under practical conditions generally
not more than 10% of that attained in solution. Few studies have been
made of activation in the vapor phase, a procedure which is handicapped
by the fact that the melting point of ergosterol is higher than the tempera-
ture at which calciferol begins to undergo transformation into its inactive
pyro-isomers.

Irradiation in quiet solutions is less effective than in agitated solutions,”
because agitation breaks up the formation of strata of irradiation products
like those on the surface of crystals and promotes uniform exposure of all
portions of the liquid. Thus it is that in commercial practice the solutions
are always agitated by stirring, flowing, or boiling during exposure to the
activating rays.

Apart from any light-filtering action of solvents, and from any role which
they may play as carriers of dissolved oxygen, there appears to be what
Bills ed al.23* termed a specific solvent effect on activation. This was revealed
by parallel spectrographic and biologic examinations on the course of ac-
tivation in three transparent solvents—alcohol, cyclohexane, and ether.
The importance of the specific solvent effect was shown by the fact that
the time required for the attainment of maximum potency in ether was
longer than the time required for the entire sequence of activation and
destruction in alcohol (Fig. 10). The maximum potency reached in ether
was higher than in alcohol or cyclohexane, but certain spectral changes
(see below) were more conspicuous in alcohol. Dasler?!® reported that the
maximum potency obtained by the irradiation of ergosterol in alcohol
represented a 30% conversion of ergosterol to calciferol, while the maxi-
mum potency obtained in ether represented a 70 % conversion.

4. CHEMISTRY OF ACTIVATION

When a provitamin D is exposed to ultraviolet light of suitable wave-
length, the transformation into the corresponding vitamin D begins at once.

237 A. Windaus, K. Westphal, F. von Werder, and O. Rygh, Nachr. Ges. Wiss. Gét-
tingen, Math. physik. Kl. 45 (1929).
288 C. KE. Bills, E. M. Honeywell, and W. M. Cox, Jr., J. Biol. Chem. 92, 601 (1931).

180 VITAMIN D GROUP

Relative Potency

Cyclohexane

0 2 4 6 8 10 12 14 16 18
Hours

Fig. 10. Time-potency curves of the activation of ergosterol in alcohol, cyclo-
hexane, and ether. An example of the specific solvent effect. (After Bills et al.?%)

However, the provitamin, even under the most favorable conditions, never
gives a 100 % yield of vitamin. The transformation proceeds in overlapping
steps, with the formation of a series of products of which vitamin D is not
the last. The series has received special study by Windaus ei al.’> and
Setz,”2® whose conclusions, modified in the light of other work, can be
expressed in the following scheme. Here is illustrated the transformation
of ergosterol into calciferol (vitamin D2), but with other provitamins the
changes which take place are analogous.

Ergosterol

{

Lumisterol

{

Protachysterol

Tachysterol

|

Precalciferol

tT

Calciferol

YON
rated SS

Toxisterol — Suprasterols
(Substance 248) I and II

II. CHEMISTRY 181

The quantity of any one irradiation product present after a given period
varies with the conditions of exposure, and correspondingly there is a vari-
ation in the spectral picture of the total mixed products. As explained
above, the wavelength is a factor, particularly important in the accumula-
tion of lumisterol and tachysterol. Temperature appears to be important
in the accumulation of precalciferol. The specific solvent effect is a factor
in the accumulation of toxisterol.

The presence of dissolved oxygen in the solvent markedly affects the
spectral picture of activation.?** It does so by altering the by-products of

11000 Ergosterol

10000

7} minutes

9000

15 minutes

3 hours

3 hours
8000

22) minutes 2 hours
7000
30 minutes
6000 45 minutes

5000 1 hour

Extinction Coefficient

4000

3000

2060

1000

220 240 260 280 300 320 220 240 260 280 300 320 220 240 260 280 300
ne mye my
Fie. 11. Spectral changes during irradiation of ergosterol in alcohol by quartz
mercury lamp. Compare Fig. 12. (After Bills et al.?39)

activation, rather than by affecting to any large extent the formation or
destruction of vitamin D.”*° In one study,?4° extreme freedom from oxygen
during the irradiation of ergosterol did not occasion any enhancement of
antiricketic potency. The products of oxidation make difficult the crystal-
lization of vitamin D,™*! and are therefore especially to be avoided when

2388 A Smakula, Nachr. Ges. Wiss. Géttingen, Math. physik. Kl. 49 (1928); C. E. Bills,
E. M. Honeywell, and W. M. Cox, Jr., J. Biol. Chem. 80, 557 (1928); E. H. Reerink
and A. van Wijk, Biochem. J. 28, 1294 (1929).

239 C. KE. Bills, E. M. Honeywell, and W. M. Cox, Jr., J. Biol. Chem. 80, 557 (1928).

240 H. H. Beard, R. E. Burk, H. E. Thompson, and H. Goldblatt, J. Biol. Chem. 96,
307 (1932).

241 T. C. Angus, F. A. Askew, R. B. Bourdillon, H. M. Bruce, R. K. Callow, C. F.
Fischmann, J. St. L. Philpot, and T. A. Webster, Proc. Roy. Soc. (London) B108,
340 (1931).

182 VITAMIN D GROUP

the production of crystalline calciferol is desired. It has been shown?25: 241, 242
that vitamin D, either in crystalline form or as the crude resin, is decidedly
more stable to oxidation than some of the non-vitamin substances which
are formed with it in irradiation. It is, however, somewhat less stable than
ergosterol itself.

By following the course of activation both spectrographically and bio-
logically, curves can be constructed which illustrate the absorption spectra
of the mixed irradiation products from time to time, and the corresponding

Relative Potency

0 1 2 3
Hours

Fia. 12. Potency changes during irradiation of ergosterol in alcohol by quartz
mercury lamp. Compare Fig. 11. (After Bills et al.?39)

rise and fall of vitamin D potency (Figs. 11 and 12). The illustrations
depict the changes observed when an alcoholic solution of ergosterol at
room temperature is exposed to the unfiltered radiations of a water-cooled
quartz mercury vapor lamp. As explained above, different conditions give
different curves. Early expectations that the study of the crude absorption
spectra would yield useful information on the products of irradiation have
not materialized. It has been necessary to isolate the products and examine
them individually.

242 C. EH. Bills, F. G. McDonald, L. N. BeMiller, G. E. Steel, and M. Nussmeier,
J. Biol. Chem. 93, 775 (1931).
243 A. L. Bacharach, E. L. Smith, and S. G. Stevenson, Analyst 58, 128 (1933).

Il. CHEMISTRY 183

a. Ergosterol

Usual, or hydrated, form, C2sH.,0-H.O, molecular weight 414.648; an-
hydrous, C2sH4,0, molecular weight 396.632. Three double bonds. Melting
point about 165°, unsharp, varies with conditions. In vacuo, sublimation
occurs at 180°,** heavy vapors at 185°, and distillation with slight decom-
position at 198°.°’ Principal absorption maxima at 262, 271, 282, and 293.5
my (in alcohol). Absorption minima at 263, 277, and 289.5 mu. Molecular
extinction coefficient at 282 my = 11,500. [a]})’ = —135°, and [a]ite. =
—174° (anhydrous ergosterol in chloroform). Heat of combustion (an-
hydrous), 9,950 caloriesi; per gram.” Specific gravity (anhydrous), 1.040.244
Ratio of digitonide to sterol, 4.0:1.24°

Crude ergosterol as obtained from the unsaponifiable matter of the fat
of fungi contains other sterols in varying amounts. Tanret4* was the first
to prepare ergosterol of high purity. He made it from the crude sterols of
ergot by fractional crystallization in ether. The ergosterol of yeast is ac-
companied by a number of minor sterols to the extent of about 20 %.247: 248
Bills and Honeywell”? obtained from yeast a purified product identical
with Tanret’s by recrystallizing the crude sterols from 95 % alcohol-benzene
2:1. Alternatively, the same degree of purification was attained by convert-
ing the ergosterol into ergosteryl isobutyrate, which forms massive crystals
easy to purify. The ester was saponified, and the ergosterol recovered.
Callow** purified ergosterol via the benzoate, and by this means eliminated,
along with other impurities, an exceptionally persistent contaminant, 5-
dihydroergosterol, which may be present to the extent of about 5 %.25° This
ergosterol, hydrated, showed [a];’ = —128.7°, and anhydrous, [a]? =
—135° (in chloroform). Huber et al.2*! crystallized ergosterol from 95%
alcohol-benzene 1:2 (not 2:1, as used by Bills and Honeywell). They
obtained hydrated ergosterol which had [a], = —133° (in chloroform).
Although the ambient temperature was not stated, it would seem that this
value represented a sample of exceptional purity.

Ergosterol forms snow-white crystals, the gross appearance of which
varies with the solvent. From 95% alcohol it crystallizes in leaflets, and
from ether, chloroform, and acetone in fine needles.24* Larger crystals are
obtained from ether-acetone 1:3.749: 252 The presence of a little water in

244 C. Tanret, Compt. rend. 108, 98 (1889); Ann. chim. et phys. Ser. 6, 20, 289 (1890).
245 AW. Pénau and Z. Hardy, J. pharm. chim. Ser. 8, 9, 145 (1929).

246 C. Tanret, Ann. chim. et phys. Ser. 8, 15, 313 (1908).

247 A. Castille and E. Ruppol, Bull. acad. roy. med. Belg. Ser. 5, 18, 48 (1933).

248 W. Halden, Hoppe-Szyler’s Z. physiol. Chem. 225, 249 (1934).

249 C, E. Bills and E. M. Honeywell, J. Biol. Chem. 80, 15 (1928).

250 R. K. Callow, Biochem. J. 25, 87 (1931).

251 W. Huber, G. W. Ewing, and J. Kriger, J. Am. Chem. Soc. 67, 609 (1945).

252 C. E. Bills, U. S. Pat. 1,775,548 (1930).

184 VITAMIN D GROUP

ethyl acetate-alcohol or in benzene-alcohol mixtures promotes the forma-
tion of large crystals.?°* Under the microscope ergosterol crystals are found
to be elongated six-sided prisms, the angles adjacent to the long sides
measuring about 127°, and the angles between the short sides 106°. The
extinction is parallel, the sign positive, and the plane of the optical axes
transverse to the length. The needles differ from the leaflets only in exag-
geration of length. It is somewhat uncertain whether the crystals are mono-
clinic or orthorhombic, but examination in convergent light suggests that
they are monoclinic.”**: 749

Ergosterol usually contains one molecule of water of crystallization.®’> 24%
246, 251 Tt, retains this water so tenaciously that drying at room temperature
over sulfuric acid or calcium chloride removes only part of it.®°» 24° Tanret?46
apparently felt that ergosterol can be completely dehydrated without de-
composition by heating to 105° in a vacuum or in carbon dioxide (time not
specified, but presumably brief). The product thus obtained regains its
original weight after a few hours of exposure to air, but this is not the case
if it has become discolored. Callow®® warns that the water “is difficult to
remove completely without decomposition.’’ Bacharach et al.2* state that
“attempts to remove the water of crystallization by ordinary methods
involve some decomposition of the sterol.’

Tanret** 4 noted that ergosterol oxidizes readily, especially in solution
or under the influence of warmth and light; it becomes yellow and odorous
and its melting point and optical rotation are lowered. Callow®® observed
that anhydrous ergosterol oxidizes more readily than does the hydrated
form. However, hydrated ergosterol, kept over calcium chloride and ex-
posed to air occasionally, took up five atoms of oxygen in the course of a
year, arriving at practically constant weight. The oxygen uptake curve was
that of an autocatalytic oxidation. The destruction of ergosterol is very
noticeable in hot solutions,?**: 49, 25! and it is probable that some damage
occurs whenever an ergosterol solution is heated, unless extraordinary pre-
cautions are taken to exclude oxygen.

To preserve ergosterol, Tanret?4* recommended that it be kept in a vac-
uum or under carbon dioxide. Callow®® reported that in sealed tubes it
remains unchanged for long periods. Bills and Honeywell”? found that
ergosterol, kept in the dark at 0°, remained colorless for a year, even though
exposed to air. However, Bacharach et al.2*3 observed yellowing in a sample
kept in the dark at “‘a low temperature.” It is the experience of industrial
users that ergosterol can be kept indefinitely in well-sealed bottles, which
have been gassed and stored in a cool, dark place.

The melting point, or melting range, of ergosterol has never been satis-
factorily established. Callow®’ reported that the purest ergosterol, partially

253 W. A. Carlson, U. S. Pat. 2,167,272 (1939).

II. CHEMISTRY 185

dehydrated, melted in sealed capillaries at 160° to 163°. The circumstances
of heating were not stated, nor was it made clear whether any precautions
against oxidation were taken. Tanret*** found that purified hydrated ergos-
terol in sealed capillaries melted from 4° to 6° lower than in capillaries
filled with carbon dioxide or than it did on the Maquenne block,?* which
minimizes oxidation by rapid heating. He gave the melting point as 165°.
Bacharach ed al.?** emphasized the importance of rapid heating. They found
melting points between 162° and 164° for commercial hydrated ergosterol
in open capillaries. Huber et al.?*! reported the same range, 162° to 164°,
for a purified sample of hydrated ergosterol, but did not specify how the
determinations were made. Bills and Honeywell?’ observed that the point
at which molten purified ergosterol in sealed capillaries becomes clear de-
pends upon the water content. The clearing points (in distinction from the
liquefying points given by others) ranged from 166° to 183°, according to
the degree of hydration.

The turbidity associated with moisture may be related to the phenome-
non of mesomorphism described by Friedel**® and noted by Gaubert?5® 257
in ergosterol complexes (‘‘liquid crystals’’). Certainly it makes difficult the
observation of the true melting point, for it lengthens the apparent melting
range, thus more or less offsetting the depressing effects of oxidation. All
facts considered, one must conclude that the melting point of ergosterol
should be taken in the open to facilitate the escape of water, and rapidly
to minimize oxidation. Tanret’s value of 165° on the Maquenne block is
perhaps the best determination so far recorded.

The melting points of thirty ergosterol esters are given in Table IV.
Many of these esters, and others studied by Gaubert,”** exhibit melting
point anomalies associated with mesomorphism. It is probable, moreover,
that the single melting points ascribed to certain other esters in the table
represent only the final, or clearing, points, the authors having neglected
to note earlier phases. Thus, Tanret?*® gave the melting point of ergosteryl
butyrate simply as 129.5°, whereas Gaubert, working later with the same
specimen of this ester, recorded a spread of 28° between the liquefying and
clearing points (100° to 128°). Besides the esters, there are several loose
combinations of ergosterol with glycerol, orcinol, urea, and substituted
ureas, which exhibit more or less extended turbid phases in their melt-
ing.°6 27 Tt is well known that mesomorphic states are exhibited by numer-
ous esters and complexes of cholesterol and other sterols, as well as ergos-
terol. However, with the ergosterol compounds, the viscous, or smectic,

264 T,, Maquenne, Bull. soc. chim. France Ser. 2, 48, 771 (1887).
255 G, Friedel, Ann. phys. Ser. 9, 18, 273 (1922).

266 P. Gaubert, Bull. soc. frang. minéral. 32, 62 (1909).

257 P. Gaubert, Compt. rend. 149, 608 (1909).

186 VITAMIN D GROUP

TABLE IV
ERGOSTEROL HSTERS

Ester, reference

Melting point, °C.

Rotation (in CHCls)

Ergosteryl acetate?*®

Ergostery] allophanate®%4
Ergosteryl 6-anthraquinone car-
bonate?574
Ergosteryl benzoate’
Monoergostery! esters of n-butane-
1,2,3,4-tetracarboxylic acid:
the more soluble isomer?®°
the less soluble isomer?®
Ergosteryl butyrate?#®
Ergostery] 2-chloro-3 , 5-dinitroben-
zoatels4
Ergosteryl cinnamate®®*

Ergosteryl 3,5-dinitrobenzoate?!®

Ergosteryl 3,5-dinitrobenzoate?®!

Ergosteryl 3,5-dinitro-4-methyl-
benzoate!*4

Ergostery! diphenylacetate®%*

Ergosteryl ethyl carbonate®®

Ergosteryl formate?*®

Ergosteryl isobutyrate**®

Ergosteryl isovalerate?”

Ergosteryl 6-naphthoate?®"*
Ergosteryl a-naphthylurethane®9*
Ergosteryl 3-nitrobenzoate?®®
Ergosteryl 4-nitrobenzoate?®®
Ergosteryl 3-nitro-4-methylben-
zoate?>!
Diergosteryl oxalate®%*
Ergosteryl palmitate®*
Ergosteryl phenylurethane®%*
Diergosteryl [6-chloroethyl] phos-
phate®®
Diergosteryl phosphate®®
Monoergosteryl phosphite®?
Monoergosteryl phthalate?5’: 259
Diergosteryl propionate?‘®
Monoergosteryl succinate*®9

179 turbid
181 clear
250
195-200

169-171.5

168 decomp.
230 decomp.
100-129.5
203-204

175 turbid
190 clear
202
198-199
213-214

186

150-153.5

161.5

148 viscous, turbid
159 thin, turbid
162 clear

138 viscous, turbid
157 thin, turbid
160 clear

175

186

151

182

191-193

255
107-108
185
165-167

180-182
146

169
147.5
162

[a]5 = —90°

[a]ése, = —88.3°

[a]> = —73°
[e]p = —38°

[a];’ = —50.8°

la]> = —40.8°
[a]? = —49°
[e]lp = —60°
[a}éaen = —111.1°
la]> = —97.9°
[e]5 = —84°
[a]p = —82°
lalp = —55°
la]> = —71°
lalp = —49.5°
[a]> = —47.2°

[a]> = —51°
la]> = —77°

II. CHEMISTRY 187

phase is more pronounced, while the color play frequent with other sterols
is usually absent.

Ergosterol is insoluble in water,?4* but the presence of 0.2% of water in
ethyl acetate is claimed*** to increase its solubility in the latter solvent.
Weak aqueous colloidal solutions can be made by means of a mutual solvent
such as acetone, or by the action of supersonic waves, or with the aid of
dispersing agents. Better colloidal solutions can be made by dissolving the
sodium, potassium, or ammonium salts of ergosteryl acid esters, e.g., the
monoergosteryl esters of phthalic,?* » 2° succinic,?°® and especially n-butane-
1,2,3,4-tetracarboxylic?® acids.

Ergosterol is sparingly soluble in methyl formate and in methanol, which
makes these low-boiling liquids useful for washing ergosterol crystals in
analytical procedures, and for recovering unconverted ergosterol from crude
irradiation resin. Ergosterol is soluble in most organic solvents and in oils
and fats. Examples of its solubility in certain solvents are given in Table V.

The stability of ergosterol in different solvents varies. Alcoholic solutions
keep for several weeks if stored in a dark place. Pyridine favors decomposi-
tion and oxidation.®® Ethylene dichloride is unsuitable because of the dif-
ficulty of keeping it free from traces of hydrogen chloride,** to which er-
gosterol is very sensitive. Chloroform, which is so widely used for the
determination of the specific rotations of organic compounds that it may
be said to be the solvent of first choice, must be used with caution for ergos-
terol. Hydrated ergosterol dissociates in chloroform, giving the unstable
anhydrous sterol and a quantity of free water sometimes sufficient to create
turbidity and optical unfitness.24* Moreover, a solution of ergosterol in
chloroform discolors in a week, and if exposed to daylight it becomes brown
in a few hours.’ #46 Chloroform intended for optical test solutions should
be tested for traces of hydrogen chloride, which might induce isomeriza-
tion.?*! Any solvent subject to peroxide formation, such as acetone or ether,
should, on general principles, be removed from ergosterol crystals to pre-
vent the possible initiation of destructive changes.

Ergosterol is strongly levorotatory, more so, in fact, than any sterol
likely to be associated with it. Consequently its rotation is a reliable index
of purity. On this basis it would seem that the preparations of Callow®
and Huber et al.**' are the purest ever described. Ergosterol does not
exhibit mutarotation (in chloroform).*4° Its rotation is more influenced than
that of sugars by the wavelength employed, and it is also greatly influenced
by the solvent. Data on these factors, obtained by Bacharach ef al.?** with

57a M. Sumi, Sci. Papers Inst. Phys. Chem. Research Tokyo 30, 252 (1936).

268 H. Emerson and F. W. Heyl, J. Am. Chem. Soc. 52, 2015 (1930).

259 F. Hoffmann-La Roche and Co., German Pat. 495,450 (1930).

260 R. Schénheimer and F. Breusch, Hoppe-Seyler’s Z. physiol. Chem. 211, 19 (1932).

188 VITAMIN D GROUP

a commercial sample of hydrated ergosterol, are shown in Table VI. From
the table it appears that the ratio [a ]s5a91: [a] is 1.27 at 20° and is prac-
tically independent of the solvent. The specific rotations, however, vary

TABLE V
SOLUBILITIES OF ERGOSTEROL

A. Data from Tanret?4é

Solvent Temperature, °C. ie ee to
Acetone 20 200
Acetone Boiling } 32
Alcohol, 95% Cold 526
Alcohol, 95% Boiling 36
“Benzine”’ 16 94
Chloroform 18 50
Chloroform Hot Few
Ether, anhydrous 20 50
Ether, anhydrous Boiling 28
Ether, hydrated 20 112
Ether, hydrated Boiling 50
Water Insol.

B. Data from Honeywell and Bills?6°2

Solvent Temperature, °C. saute
Acetone 56 27
Alcohol, 96% 78 50
Benzene 80 4.6
Kther, U.S.P. 35 70
Ethyl acetate Hil 6.5
Hexane 65-70 24
Isopropyl alcohol 82 10
Methanol 65 280
Methyl acetate 54 35
Methyleyclohexane 101 <2
widely with the solvent, being highest in chloroform, [a]; = —125.25°,
and lowest in acetone, [a];’ = —92.0°. From the data of Bills and Honey-

well,*“° it appears that the ratio is only slightly influenced by temperature,
being 1.30 at 20° and 1.27 at 25°. The values themselves, however, showed
considerable temperature effect. From the data of Callow,®* it appears that

2600 H}. M. Honeywell and C. E. Bills, J. Biol. Chem. 99, 71 (1932).

II. CHEMISTRY 189

the ratio is essentially the same for hydrated ergosterol, 1.30, as for an-
hydrous ergosterol, 1.29.

The specific rotations of twenty-one ergosteryl esters are given in ‘Table
IV. In several instances the values seem anomalous, in that they are widely
different from what would be expected on the basis of differences in molecu-
lar weight. This does not necessarily indicate contamination or error, for
similar anomalies have been observed in cholesterol esters.?®

The absorption spectrum of ergosterol, Fig. 8, reveals the four commonly
recognized bands with maxima at 262, 271, 282 and 293.5 mu. In addition
to these, there are four other bands, or inflections, which can be seen clearly
only in more concentrated solutions. Smakula”® reported a band at 232
my, and Sumi?* noted an inflection at 250 my. Hogness et al.?** located the

TABLE VI
OpTicaAL ROTATION OF ERGOSTEROL?

Solvent [e261 [al Ratio
Chloroform —158.5° —125.25° 27
Benzene —156.0° —124.0° 126
Ethyl acetate —120.0° —95.0° 1.26
Ether —120.0° —94.0° 1 ge
Alcohol (absolute) —119.0° —93.0° 1.28
Acetone —118.0° —92.0° 1.28

Average value of ratio = 1.27

@ Data from Bacharach et al.243 on a commercial grade of ergosterol.

latter band at 252 my and found two more at 325 and 337.5 mu. The fact
that ergosterol absorbs, even slightly, these longer waves, probably accounts
for the otherwise unexplained destructive effect of subdued daylight.

Hogness et al.?® and Huber et al.2*! compared the absorption spectra of
ergosterol in several solvents. The wavelengths of the maxima were essen-
tially the same in alcohol, hexane, and isoéctane. The extinction coefficients
were highest in alcohol and hexane, and a little lower in isodctane. In
chloroform the extinction was considerably lower, and the position of the
maxima shifted 3 mu toward the visible region.

Sterol color reactions depend on the double bond systems and hence are

261 R. L. Shriner and L. Ko, J. Biol. Chem. 80, 1 (1928).

262 C, Liebermann, Ber. 18, 1804 (1885).

263 F. A. Askew, R. B. Bourdillon, H. M. Bruce, R. K. Callow, J. St. L. Philpot, and
T. A. Webster, Proc. Roy. Soc. (London) B109, 488 (1932).

264 M. Sumi, Biochem. Z. 204, 397 (1929).

265 T. R. Hogness, A. E. Sidwell, Jr., and F. P. Zscheile, Jr., J. Biol. Chem. 120, 239
(1937).

190 VITAMIN D GROUP

specific only as to classes. Reactions of interest in the study of ergosterol
and/or other provitamins D include the following: the Tanret or reversed
Salkowski reaction,”*: 74° the Liebermann-Burchard reaction,!: 746, 2& the
Tortelli-Jaffé reaction,?*3: 26, 267, 263 the Rosenheim reactions (trichloroacetic
acid or chloral hydrate),?*: 26° the Rosenheim-Callow reaction,” 2” the
Tschugajeff reaction,’ 2” and the antimony trichloride reaction.?#: 266 267
A modification of the trichloroacetic acid reaction, notable for its extreme
sensitivity, is described by Christiani and Anger.” Most of these reactions
distinguish the provitamins D from sterols of the cholesterol-phytosterol
class. Some distinguish provitamins from their esters,?® 2” and oxidized
from fresh specimens of provitamin.?%: 246, 250

For the quantitative estimation of ergosterol, three general methods are
available: (1) color reactions, (2) digitonide precipitation, and (3) spectro-
photometric measurement. No method is known which will distinguish
minute amounts of ergosterol from other provitamins D, but when sufficient
material is on hand, the esters and oxidation products can sometimes be
identified. The color reactions have the advantage of being applicable to
minute quantities, but they are subject to considerable error. Heiduschka
and Lindner! used the Liebermann-Burchard reaction for the determina-
tion of ergosterol in yeast, a questionable procedure because this reaction
is given not only by ergosterol but (more slowly) by sterols with a single
double bond.*** Bilger et al.2 used the Liebermann-Burchard reaction for
the total sterols of yeast, and a modification of the Rosenheim trichloro-
acetic acid reaction for the ergosterol. Page? applied a modification of the
Rosenheim reaction to the estimation of ‘‘ergosterol”’ in animal tissues; see,
however, the comments of Bilger. The more recent (1939) Christiani-
Anger?” modification, in which the chloroform solution of the unknown is
treated with trichloroacetic acid and lead tetraacetate, deserves a quanti-
tative development. In spot tests it detects as little as 0.1 y of ergosterol,
and it distinguishes esters from free sterols.

Ergosterol, like all sterols of normal steric configuration, gives with
digitonin a precipitate of low solubility which lends itself to gravimetric
estimation. The digitonin reaction is accurate but time-consuming. It does
not take place with esters, and thus when applied to tissue extracts before

266 H}, P. Haussler and E. Brauchli, Helv. Chim. Acta 12, 187 (1929).

267 J. M. Heilbron and F.§. Spring, Biochem. J. 24, 133 (1930).

268 U. Westphal, Ber. 72, 1243 (1939).

269. Rosenheim, Biochem. J. 28, 47 (1929).

270 O. Rosenheim and R. K. Callow, Biochem. J. 25, 74 (1931).

271 1. Tschugajeff, Chem. Z. 24, 542 (1900); Z. angew. Chem. 13, 618 (1900).
272 A. F,v. Christiani and V. Anger, Ber. 72, 1124, 1482 (1939).

273 FH, Bilger, W. Halden, and M. K. Zacherl, Mikrochemie 15, 119 (1934).

274 T. H. Page, Biochem. Z. 220, 420 (1930).

Il. CHEMISTRY 191

and after saponification, it provides a means of determining the percentages
of free and esterified total sterols. Digitonin does not give a precipitate
with any form of vitamin D. The critical details of conducting an analysis
with digitonin are given by Pénau and Hardy,”* Castille and Ruppol,?47
and Bilger et al.?”

With the availability of modern apparatus, the spectrophotometric
method of determining ergosterol and other provitamins D is usually the
method of choice. The characteristic spectrum (Fig. 8) is shown by the
free provitamins and also by their esters, provided that the acid radical
does not itself absorb in the critical region.®%*: 25! If the unknown is free of
“ultraviolet dirt,’’ it is only necessary to compare the optical density at
the 282 my maximum with that of pure provitamin D. To prove that the
unknown is not dirty, it is usually sufficient to measure the absorption at
the nearby minimum, 277 my; the relation of this to the maximum should
be 0.8. If irrelevant absorption cannot be avoided, one may resort to the
procedure employed by Castille and Ruppol*” in studies with yeast. This
consists in precipitating the total sterols with digitonin, dissolving the
washed and dried digitonides in absolute alcohol, and making the spectral
measurement on the resulting solution. In this case it is desirable to use
as the standard a preparation of pure ergosterol digitonide.

The accepted formula of ergosterol is the work of many chemists. Tan-
ret?## in 1889 recognized the single hydroxyl group and the molecule of
water of crystallization, and he presented analyses corresponding to the
- formula CosHw»O-H.0. In 1908 he™® revised the formula to C2;Hy.O-H,0,
which remained accepted until 1932, when Windaus ef al.,'%4: 27> by the
analysis of halogenated nitrobenzoic esters and other complex derivatives,
established the empirical formula as C2sHu0-H.O. The structural formula
(Fig. 4) was established by Windaus ei al.27* in 1934. Confirmation of de-
tails followed in papers by Fernholz and Chakravorty?”” and Dimroth and
Trautmann.”8 The complicated proofs of structure of ergosterol and related
sterols are reviewed by Rosenberg and Fieser and Fieser.2”9

b. Lumisterol

CosHyO. Three double bonds. Melting point 118°. Principal absorption
maxima at 265 and 279 mu. Molecular extinction coefficient at 279 my =
8500.28 [a]? = +192°, and [a]éis1 = +235° (in acetone).

275 A. Windaus and A. Liittringhaus, Nachr. Ges. Wiss. Géttingen, Math. physik K1.,
Fachgruppe III 4 (1932).

276 A. Windaus, H. H. Inhoffen, and 8. von Reichel, Ann. 510, 248 (1934).

277 BH}. Fernholz and P. N. Chakravorty, Ber. 67, 2021 (1934).

278 K,. Dimroth and G. Trautmann, Ber. 69, 669 (1936).

279 T,. F. Fieser and M. Fieser, Natural Products Related to Phenanthrene, 3rd ed.
Reinhold Publishing Corp., New York, 1949.

230 T. M. Heilbron, G. L. Moffet, and F. 8. Spring, J. Chem. Soc. 1987, 411.

192 VITAMIN D GROUP

Lumisterol, the initial phototransformation product of ergosterol, was
isolated by Windaus et al.**! and Askew et al.?® in 1932. From acetone-
methanol it crystallizes in fine needles. It is easily soluble in chloroform,
ether, and acetone, somewhat difficultly soluble in methanol. In air it
oxidizes ‘‘only slowly.” Like ergosterol, it is isomerized by hydrogen chlo-
ride. It gives the color reactions of ergosterol with minor differences. It
does not give a precipitate with digitonin. It is physiologically inert but
is activated by irradiation. It forms a molecular addition compound with
calciferol, this complex being the old vitamin D, of the German school.

The structure of lumisterol has been the subject of many studies, which
are critically reviewed by Rosenberg,!* with the conclusion that the only

R R
a)
HO HO
Ergosterol Lumisterol
R R
O ~
HO Ze HO
Tachysterol Calciferol

Fig. 13. Ergosterol irradiation products of known structure.

difference between lumisterol and ergosterol is an epimerization of the
methyl group at C-10. It is conventional to indicate such steric differences
by a dotted bond line, as shown in Fig. 13.

c. Protachysterol

In 1931, before much was known about the individual products of irra-
diation, Windaus and Auhagen?®? made a remarkable study of the stability
of irradiated ergosterol. A solution of ergosterol in dioxane-petroleum 1:9
was irradiated, the unconverted ergosterol frozen out and filtered off, and
the filtrate stored and examined polarimetrically and spectroscopically—all
in a closed system practically devoid of oxygen. It was found that, over a
period of 50 days at room temperature, the specific rotation changed from
—17° to a constant value at +7°, and the absorption spectrum at 280 mu

281 A. Windaus, K. Dithmar, and E. Fernholz, Ann. 498, 259 (1932).
282 A. Windaus and E. Auhagen, Hoppe-Seyler’s Z. physiol. Chem. 196, 108 (1931).

Il. CHEMISTRY 193

showed a substantial increase. These changes were not accompanied by
any significant loss of vitamin D potency.

To explain the observed spectral change, Windaus et al.*° postulated the
existence of an unstable ‘‘protachysterol”’ which changes into tachysterol,
the intermediate characterized by intense absorption at 280 my. This ex-
planation does not account for the change in specific rotation, which was
opposite to what would be expected if tachysterol were being formed.
Interpretation of this dark reaction is further complicated by the subse-
quent discovery of precalciferol, described below.

d. Tachysterol

CogsHsgO. Four double bonds. Melting point of 3 ,5-dinitro-4-methyl ben-
zoate 154° to 155°. Absorption maximum at 280 my, minor bands at 268
and 294 my. Molecular extinction coefficient at 280 mu = 24,000. [a]§ =
—70°, and [a]5161 = —86.3° (in ““Normalbenzin’’).

Tachysterol was isolated from activation resin by Windaus, Liittrimghaus
and Busse* in 1932, and its preparation and properties were described in
detail by Windaus, von Werder, and Liittringhaus.?® It got its name in
recognition of the speed (Gr. fachys) with which it reacts with maleic or
citraconic anhydride to form an adduct useful in its separation. Tachysterol
has not been obtained in crystalline form, although tachysteryl acetate-
citraconic anhydride and tachysteryl 3 ,5-dinitro-4-methyl benzoate form
good crystals. Tachysterol is a source of trouble in the separation of irra-
_diation products, because, besides failing to crystallize, it exhibits an ex-
ceptional affinity for oxygen, and the oxidation of calciferol is promoted by
its presence.*® It is insoluble in water, but easily soluble in the common
organic solvents, including methanol. It does not give a precipitate with
digitonin. It gives in modified form the color reactions of ergosterol.

The structure of tachysterol (Fig. 13) has been elucidated largely through
the work of Miiller*** and Grundmann,”** supported by von Werder.?*§ In
tachysterol ring B is ruptured, with the formation of a fourth double bond.
Thus, like calciferol, it does not, in the strict sense, possess the sterol ring
structure. Its similarity to calciferol is further shown by the fact that, upon
reduction with sodium in alcohol, both compounds yield the same dihydro
derivative.” Tachysterol is converted into calciferol by irradiation,?** and
calciferol is converted into tachysterol by the introduction and removal of
an atom of iodine.?3°

283 A. Windaus, F. von Werder, and A. Liittringhaus, Ann. 499, 188 (1932).
284 M. Miller, Hoppe-Seyler’s Z. physiol. Chem. 288, 223 (1935).

285 W. Grundmann, Hoppe-Seyler’s Z. physiol. Chem. 252, 151 (1938).

286 F. von Werder, Hoppe-Seyler’s Z. physiol. Chem. 260, 119 (1939).

286a P| Meunier and G. Thibaudet, Compt. rend. 223, 172 (1946).

194 VITAMIN D GROUP

Tachysterol exhibits considerable pharmacological activity. It is about
one-half as toxic as calciferol in producing kidney tubule calcification in
the mouse.”** For calcifying bone it is greatly inferior to calciferol although
still a potent agent. Windaus et al.*** observed no antiricketic activity from
doses of 1 y daily to rats, but higher doses were not explored. Meunier and
Thibaudet?*™ observed healing at the 10-y level. This is about 500 times
the requirement of calciferol. Probably the antiricketic action was not
due to contamination with calciferol, for it was the same in samples of
tachysterol prepared the usual way and by the deiodination of iodocal-
ciferol.

e. Precalciferol

CogsHyO. Probably four double bonds. Melting point of 3 ,5-dinitroben-
zoate 103° to 104°. [a], of 3,5-dinitrobenzoate = +45° in chloroform and
+30° in benzene, with mutarotation. Absorption maximum (of free form,
by graphic subtraction), at 265 my. Molecular extinction coefficient at
265 mp = 9600. In solution, transforms into calciferol.

In 1948-1949, when the irradiation series had come to be regarded as a
closed book, a renewed interest in the dark reaction came out of the studies
of Velluz and his associates.**7 Ergosterol in ether was irradiated under
nitrogen, the ether distilled off, the unconverted ergosterol frozen out of
alcohol, and the alcohol removed—all at a temperature below 25°. Without
heating, the resin was converted into 3 ,5-dinitrobenzoates, and the product
fractionated on alumina. The least strongly adsorbed fraction was crystal-
lized from ligroin, giving yields of precalciferyl 3 ,5-dinitrobenzoate which
amounted to as much as 50% of the resin taken. That such an abundant
product should have been so long overlooked is surprising but understand-
able because it transforms itself into calciferol in solution, especially when
warmed.

Precalciferyl 3 ,5-dinitrobenzoate crystallizes from ligroin in fine pale-
yellow needles, m.p. 103° to 104°, obviously different from the bright yellow
massive crystals of calciferyl 3 ,5-dinitrobenzoate, m.p. 158° to 159°. The
ester upon saponification gives a non-crystalline free form of precalciferol,
which does not precipitate with digitonin and which gives the calciferol
color reactions of Pesez”®* in reduced intensity. The absorption spectrum
of precalciferol is strikingly similar to that of calciferol, except that it is
only about half as intense. The curve (Fig. 9) shows no evidence of admix-
ture of other precursors of calciferol. When a benzene solution of precal-

287 L. Velluz, A. Petit, G. Michel, and G. Rousseau, Compt. rend. 226, 1287 (1948);
L. Velluz, A. Petit, and G. Amiard, Bull. soc. chim. France 15, 1115 (1948); L.
Velluz and G. Amiard, Compt. rend. 228, 692, 853 (1949); L. Velluz, G. Amiard, and
A. Petit, Bull. soc. chim. France 16, 501 (1949).

288 M. Pesez, Bull. soc. chim. France 16, 507 (1949).

II. CHEMISTRY 195

ciferol or precalciferyl 3 ,5-dinitrobenzoate is held at 60° for several hours
in the dark, the dextrorotation increases to almost that of calciferol or
calciferyl 3,5-dinitrobenzoate, and the latter products can be isolated.
Conversely, solutions of calciferol or calciferyl 3 ,5-dinitrobenzoate produce
some precalciferol or precalciferyl 3,5-dinitrobenzoate. In any of these
solutions, calciferol always predominates at equilibrium. The relationship of
the two isomers is presumed to be a phenomenon of transitory cyclization.
The increasing dextrorotation in the Windaus and Auhagen”’ experi-
ment, heretofore unexplained, was probably due to the precalciferol-cal-
ciferol transformation, which was overshadowed spectrally by the simul-
taneously occurring protachysterol-tachysterol transformation. Thus it
appears that there are two dark reactions in the activation series.

Mention should be made of the claim by Raoul e¢ al.”*° that, in ionizing
solutions, calciferol and tachysterol are interchangeable. It was found that
in an ionizing agent such as symmetrical dichloroethane, activated with
1 % glycerol dichlorohydrin, calciferol undergoes a change, its optical prop-
erties simulating those of tachysterol. If the ionization is checked before it
has continued too long, the product reverts to calciferol, but if it has been
prolonged, the change back to calciferol can be brought about only by
irradiation. That the product into which the calciferol changed was really
tachysterol may be questioned, since it was reported that an attempt to
prepare the 3,5-dinitro-4-methylbenzoate of tachysterol resulted only in
the formation of the corresponding ester of calciferol.

f. Caleiferol. Vitamin D2. Ergocalciferol

CosHuO. Four double bonds. Melting point 121°.?°° Distils, with de-
composition, at 150° in high vacuum.”!! Broad absorption band with maxi-
mum at 265 my; molecular extinction coefficient = 19,400 in alcohol
(E1% = 490).29°. 2900 [a]? = +4106°, [a}2s, = +125° (in absolute alco-
hol)?

In 1930-1931 the English and German teams of investigators succeeded
in isolating pure vitamin D from the irradiation resin of ergosterol. An
ingenious method for the distillation and fractional condensation of the
resin in a high vacuum was described by Askew ef al.?* Small amounts of
potent crystals were obtained. The method was improved, and a detailed
study of the active crystals was reported by Angus ef al.*4! The name,
calciferol, was given to the crystalline substance. A few months later, in
1931, Askew et al. discovered that the original calciferol was a mixture,

289 Y, Raoul, J. Chopin, P. Meunier, and N. Le Boulch, Compt. rend. 228, 1064 (1949).

290 H. Pénau and G. Hagemann, Helv. Chim. Acta 29, 1366 (1946).

2902 §. KK. Crews and E. L. Smith, Analyst 64, 568 (1939).

201 F. A. Askew, R. B. Bourdillon, H. M. Bruce, R. G. C. Jenkins, and T. A. Webster,
Proc. Roy. Soc. (London) B107, 76 (1930).

196 VITAMIN D GROUP

separable via the 3 ,5-dinitrobenzoate into pure calciferol and two inactive
sterols. One of these, pyrocalciferol, was merely a thermal transformation
product, produced during the distillation. The other, ‘‘sterol X’’ (lumis-
terol), had come over from the original irradiation product. By the new
esterification technique calciferol could be obtained directly from the resin,
without distillation.

While the isolation of calciferol was in progress in England, Windaus and
his associates in Germany were developing a different technique to the same
end. Windaus?”? and Windaus et al.*°? reviewed their preliminary studies on
activation and announced that a crystalline vitamin D was obtained by
removing the inactive components of the resin by means of maleic or citra-
conic anhydride. Presently Linsert?** in 1931 obtained another crystalline
preparation somewhat different in its properties from the first. For a few
weeks Windaus and Liittringhaus?® regarded these two preparations as
distinct forms of vitamin D, and called them vitamin D, and vitamin Dz,
respectively. Soon, however, it was realized by Windaus et al.!® that the
Linsert preparation was essentially identical with Bourdillon’s pure cal-
ciferol, while the first German preparation was an addition compound of
calciferol with lumisterol.

The name vitamin D, has been abandoned, but the name calciferol has
been retained, so that vitamin D» and calciferol are now synonyms for the
pure product. The Commission on Nomenclature of Biological Chemistry
of the International Union of Pure and Applied Chemistry?%* has adopted
the name ergocalciferol as another synonym, for the purpose of making
clearer the distinction from vitamin Ds, which they call cholecalciferol.

Callow’s” procedure of purifying calciferol via the 3 ,5-dinitrobenzoate
works so well that the properties and constants of calciferol are known
with better agreement than those of ergosterol.

Calciferol crystallizes from methanol in clusters of colorless needles,?®
and from acetone in long prisms.!*? It is very soluble in most organic sol-
vents, but less so in methanol than in acetone.?® The solubility in acetone
amounts to 1 g. in 14 ml. at 7°.1°° At 26° 100 ml. of acetone dissolves 25 g.,
100 ml. of absolute alcohol 28 g., and 100 ml. of ethyl acetate 31 g. of cal-
ciferol.?%°

The stability of calciferol has been reviewed and studied by Huber and
Barlow.” Calciferol is even more unstable than ergosterol. Samples kept
under ordinary laboratory conditions show signs of decomposition in 2 or
292 A Windaus, Proc. Roy. Soc. (London) B108, 568 (1931).

293 A. Windaus, A. Liittringhaus, and M. Deppe, Ann. 489, 252 (1931).
294 Q. Linsert, cited, in a footnote, by A. Windaus, A. Liittringhaus, and M. Deppe,

Ann. 489, 252 (1931); U. S. Pat. 1,902,785 (1933).

295 A. Windaus and A. Liittringhaus, Hoppe-Seyler’s Z. physiol. Chem. 203, 70 (1931).

296 A.M. Patterson, Chem. Eng. News 30, 104 (1952).
297 W. Huber and O. W. Barlow, J. Biol. Chem. 149, 125 (1943).

Il. CHEMISTRY 197

3 days. Inert gases or low temperatures greatly diminish, but do not pre-
vent, the decomposition. In the author’s experience, a sample stored for 6
years in the dark at 0° in a closed but not hermetically sealed bottle, re-
tained its crystalline form but became deep yellow, and shortly after
warming to room temperature it mildly exploded. Huber and Barlow found
that samples sealed under vacuum in amber ampules and kept in the re-
frigerator, showed no change up to 9 months. The esters of calciferol
(Table VIII, page 204) with various nitrobenzoic acids are remarkably
stable and provide a means of storage as well as of characterization. They
remain unchanged over a period of years if a slight photodecomposition is
avoided by keeping them in amber bottles.2% Fresh calciferol can then be
had at any time, merely by applying a simple saponification procedure
which avoids recrystallization and its attendant hazards to purity. Solu-
tions of calciferol in edible oils or in propylene glycol, and dispersions of it
in canned milk, have excellent keeping qualities, even under severe condi-
tions.?%7

The melting point data on commercial calciferol are reviewed by Ander-
son et al.2** The melting point is close to 116°, values between 115° and
117° being the usual range. Perhaps because of slight thermal decomposi-
tion, the actual melting point is unsharp. Highly purified calciferol melts
at 121°, according to Pénau and Hagemann.2%°

Calciferol is strongly dextrorotatory, although less so than lumisterol;
the presence of the latter as an impurity can be determined quantitatively
by a procedure described by Setz.?° The positive rotation decreases mark-
edly with increasing temperature; in alcohol this decrease amounts to
0.515° per degree of temperature rise.?°” The dextrorotation of calciferyl
3,5-dinitrobenzoate, on the other hand, increases with increasing tempera-
ture. The specific rotations of both calciferol and its 3 ,5-dinitrobenzoate
increase substantially with the concentration of the test solution. The
effects of solvents on the specific rotation are even greater than for ergos-
terol, but in a generally reversed order, as shown in Table VII in com-
parison with Table VI. Since the specific rotation of calciferol is highest in
absolute alcohol, and also because calciferol is extremely sensitive to traces
of hydrogen chloride,”*' it is customary to determine the specific rotation
in alcohol, rather than in chloroform.

The absorption spectra of calciferol and several of its substituted nitro-
benzoic acid esters have been studied by Huber et al.2*! The curve for
calciferol is smoother than some previously published and shows no evi-
dence of inflections other than the one broad band at 265 my. Identical
values were obtained in alcohol and hexane, and the curves were also
identical with those of vitamin D; in the same solvents. The molecular
extinction coefficient of 18,200 is in good agreement with previously re-

2974, W. Anderson, A. L. Bacharach, and E. L. Smith, Analyst 62, 430 (1937).

198 VITAMIN D GROUP

ported values, but inferior to the value 19,400 (H\%, = 490) found by
Pénau and Hagemann?’? and Crews and Smith.?°"

Calciferol gives no precipitate with digitonin. In the well-known color
reactions, it generally gives weaker and less well-defined colors than ergos-
terol. These responses are described by Askew et al.,?® Windaus et al.,!°°
and Bacharach et al.24* Color reactions of special interest in connection
with vitamin D are described by Pesez,?** Banchetti,?* Schaltegger,?®® Villar
Palasi,?°° and De Witt and Sullivan.?!

The structural formula of calciferol (Fig. 13) was established by Windaus
and Thiele? in 1935 and by Heilbron e¢ al.° in 1936. Their published work

TABLE VII
OpticaAL ROTATION OF CALCIFEROL?

Solvent a ee [ay Ratio

Alcohol (absolute) +125 .0° +106.25° 1.18
Ethyl acetate +113 .25° + 95.0° 1.19
Ether +105.5° + 88.75° 1.19
Benzene +102.12° + 87.5° ibsily/
Acetone + 99.5° + 83.5° 1.19
n-Hexane + 66.5° + 56.25° 1.18
Chloroform + 61.75° + 52.25° 1.18
1.18

Average value of ratio =

2 Data from Bacharach et al.243 on commercial lots of refined material.

was the culmination of a long series of attacks on the problems of structure
which are reviewed by Fieser and Fieser?”® and by Rosenberg.“ The cor-
rectness of the formula has been confirmed, and detailed stereochemical
relations of the atoms within the molecule established, by Crowfoot and
Dunitz*™ through a study of the X-ray diffraction pattern of calciferyl
4-iodo-5-nitrobenzoate.

g. Toxisterol. Substance 248

CosHy4O. Melting point about 50°.*°° Narrow absorption band with max-
imum at 248 mu; molecular extinction coefficient = 18,300." [a]? = —16°
(in chloroform) .*°°

298 A. Banchetti, Ann. chim. appl. 38, 394 (1948).

299 H. Schaltegger, Helv. Chim. Acta 29, 285 (1946).

300 V. Villar Palasi, Natwre 160, 88 (1947).

301 J. B. De Witt and M. X. Sullivan, Ind. Eng. Chem. Anal. Ed. 18, 117 (1946).

302 A. Windaus and W. Thiele, Ann. 521, 160 (1935).

303 T. M. Heilbron, R. N. Jones, K. M. Samant, and F. S. Spring, J. Chem. Soc. 1936,
905.

304 T). Crowfoot and J. D. Dunitz, Nature 162, 608 (1948).

305 Q. Linsert, U. S. Pat: 2,030,377 (1936).

II. CHEMISTRY 199

In 1927 Morton e¢ al.,?!° extending the work of Pohl,*®° showed that when
ergosterol is irradiated its characteristic absorption bands disappear, and
a single new band of great intensity develops at 247 or 248 my, which in
turn fades away to weak general absorption in the far ultraviolet region.
With inadequate biological evidence, the assumption was easily made that
this new band represented vitamin D. Smakula,”2* however, concluded that
substance 248 was not the vitamin. Bills et al.”° showed that the appear-
ance of the band at 248 my coincided not with the development, but with
the destruction, of antiricketic potency. They, and also van Wijk and
Reerink,*°* associated substance 248 with isoergosterol. Cox and Bills?
contributed further evidence of relationship to the isoergosterols, but noted
a point of difference, namely, that substance 248 does not precipitate with
digitonin.

Bills et al.#° found that irradiation products withdrawn for examination
at the moment when substance 248 was at its maximum concentration still
contained some vitamin D. Further irradiation totally eliminated anti-
ricketic activity, while only a small amount of substance 248 was destroyed.
Laquer and Linsert*® attributed a toxic quality to substance 248, and
proposed the name Toxisterin (toxisterol). The product which they in-
vestigated still contained some vitamin D, but not enough to account for
the relatively great toxicity. It was evidently a mixture similar to the one
which Hoyle®® obtained by limited overirradiation and which he reported
to have toxic-caleifying properties all out of proportion to the antiricketic
potency. Such a mixture, unfortunately, seems to have been the “vitamin
D” of the I. G. Farbenindustrie patent*!° of 1928 (the old Vigantol of toxic
repute). The claims of this patent, and of the corresponding American
patent,*" call for the irradiation of ergosterol to be continued just until
the absorption spectrum of what is now recognized as toxisterol attains its
maximum! The mistake was clearly the result of assuming, in the absence
of proper bioassays, that the spectrographically most conspicuous irradia-
tion product was the vitamin. There is considerable evidence, both spectro-
graphic and toxicologic, that toxisterol is formed most readily when alcohol
is the solvent in which the ergosterol is irradiated.28: 289, 306, 309, 312

The purest described preparation of toxisterol is that of the Linsert
(Farbenindustrie) patent*®® for isolating, from the irradiation products of

506 A. van Wijk and E. H. Reerink, Nature 122, 648 (1928).

507 W. M. Cox, Jr., and C. E. Bills, J. Biol. Chem. 88, 709 (1930).

$08 F. Laquer and O. Linsert, Klin. Wochschr. 12, 753 (1933).

309 J. C. Hoyle, J. Pharmacol. Exptl. Therap. 40, 351 (1930).

310 J. G. Farbenindustrie Aktiengesellschaft, British Pat. 296,093 (1928).

311 W. Zimmermann and W. Frankenburger, U.S. Pat. 1,896,191 (1933).

82 W. KE. Dixon and J. C. Hoyle, Brit. Med. J. 2, 832 (1928); L. J. Harris and T. Moore,
Biochem. J. 22, 1461 (1929); J. C. Hoyle and H. Buckland, ibid. 23, 558 (1929);
R. Kern, M. F. Montgomery, and E. U. Still, J. Biol. Chem. 98, 365 (1931).

200 VITAMIN D GROUP

vitamin D, a “product which is essentially characterized by its increasing
action on the level of blood calcium.’ According to the example, crystal-
line vitamin D, made from ergosterol, is irradiated in heptane in the ab-
sence of oxygen until its absorption maximum at 265 my substantially
disappears. The product is converted into the 3 ,5-dinitrobenzoate, which
is crystallized free of the suprasterols. Saponification of the dinitrobenzoate,
which is dextrorotatory, [a]?? = +33° in acetone, m.p. 130°, gives the free
drug as a whitish powder which is levorotatory, [a]? = —16° in chloroform.
(A similar anomalous difference in sign of rotation between dinitrobenzoate
and free sterol is noted in tachysterol;3!*).

The exceptionally low melting point, about 50°, casts some doubt on the
purity of the preparation, but the product is stated to be “practically
antirachitically inactive.” It is worth noting that the patent description of
this drug, which was one of a series of blood calcium raising substances
then under investigation by the I.G. (following the Vigantol episode),
makes no mention of the term toxisterol and no reference to the absorption
spectrum of the product by which it could be identified as substance 248.
Although the isolation of toxisterol has never been described elsewhere
than in this patent and its European counterparts, the German workers
appear to have been familiar with toxisterol to the extent of publishing its
absorption spectrum showing the extinction coefficient.”

h. Suprasterol I

CosH,4O. Three double bonds. General absorption below 250 mu. [a] =
—76° in chloroform. Melting point 104°.

Suprasterol II

C»sH,,O. Three double bonds. General absorption below 250 my. [a]? =
+63° in chloroform. Melting point 110°.

The products of extreme overirradiation, the end products of the series,
were designated Suprasterine by Windaus et al.* in 1930. They recognized
two isomers, suprasterol I and suprasterol II, which they separated by
taking advantage of differences in the solubility of the allophanic acid esters.
The designations I and II do not refer to sequence of formation, but to the
order in which the two allophanates crystallize out of solution. The su-
prasterols, especially suprasterol II, are much more soluble in organic sol-
vents than ergosterol. They are unaffected by irradiation. They can be
distilled unchanged at 190° 7n vacuo. They do not precipitate with digitonin.
Of their color reactions, the most conspicuous is the intense red violet which
they give with chloral hydrate. They show no antiricketic activity at dosage

313 A. Windaus, M. Deppe, and W. Wunderlich, Ann. 538, 118 (1937).
314 A, Windaus, J. Gaede, J. Késer, and G. Stein, Ann. 483, 17 (1930).

II. CHEMISTRY 201

levels several times higher than the usual dose for calciferol. They are only
slightly toxic.

Suprasterol I crystallizes in fine needles from acetone, and suprasterol II
in dense prisms from acetone or methanol. Suprasterol I is less sensitive to
oxidation than ergosterol or calciferol, but slowly yellows in contact with
air; suprasterol IT was not observed in this connection.

From the work of Windaus e¢ al.“ and other studies reviewed and ex-
tended by Miiller,* it appears that the suprasterols have three double
bonds and are therefore tetracyclic, but the ring structure may not be that
of the sterols. In other words, the ring closure suffered by calciferol in the
formation of the suprasterols may not be at the point of rupture of the
original ring B of ergosterol and lumisterol. Certainly the double bonds are
no longer in conjugated position, for the suprasterols show no absorption
bands.

t. 7-Dehydrocholesterol. Provitamin Ds

Cx,HyO-H.2O. Molecular weight 402.638. Two double bonds. Melting
point 149° to 150°.7° Principal absorption maxima at 262.5, 271, 281.5, and
293 mu.” 265 Molecular extinction coefficient at 281.5 mu = 10,920 (in
aleohol).25! [a]? = —124° (in chloroform).”°

The series of irradiation products which stem from 7-dehydrocholesterol
is in every respect analogous to the ergosterol series, but it has not been
studied in quite as much detail. Windaus e¢ al.33 have proposed a nomencla-
ture for these products, according to which the names of the ergosterol
series are retained, but a subscript number 3 is added; thus lumisterol; and
tachysterol; are the 7-dehydrocholesterol products corresponding to lumi-
sterol and tachysterol. Similarly, for the 22-dihydroergosterol series the
subscript 4 is used.

7-Dehydrocholesterol crystallizes in slender platelets from ether-meth-
anol.** It is difficultly soluble in methanol, and readily soluble in ether.”°
The crystals contain one mole of water?*! which cannot be completely re-
moved.** 7-Dehydrocholesterol forms a precipitate with digitonin.* It also
gives the color reactions of ergosterol with minor differences.** Its specific
rotation is a little less strongly levorotatory than that of ergosterol.’° Its
absorption spectrum is practically identical with that of ergosterol,*°!: °°
and bgsides the four major bands, minor inflections are exhibited at 252,
321, and 336 muy.?®

7-Dehydrocholesterol is much more unstable than ergosterol.*: 7°! *!° It
shows yellowing in less than 2 days when exposed to air at room tempera-
ture, even in the dark. However, when it is crystallized together with cho-
lesterol, a remarkable stabilization results, depending upon the amount of

316 H. R. Rosenberg and W. W. Woessner, U.S. Pat. 2,434,015 (1948).

202 VITAMIN D GROUP

cholesterol. Thus a sample containing 59% cholesterol showed no change
after 5 weeks under the above conditions.*!® The presence of much choles-
terol may be presumed to have been a factor in the survival of the provi-
tamin found in the brain of the mummy, mentioned above in another con-
nection. The substituted nitrobenzoates of 7-dehydrocholesterol are also
stable. Huber ef al.2°! have recommended converting the provitamin into
its 3,5-dinitrobenzoate, both as a means of purifying it and preserving it,
so that fresh samples of the free provitamin can be had at any time merely
by subjecting the ester to a simple saponification.

7. Lumisterols

C7H4O. Dimorphous: needles from acetone, melting point 87° to 88°;
mixed forms from methanol, melting point 63° to 64°. fa]® = +197° in
chloroform. Absorption spectrum almost the same as that of lumisterols.

The initial irradiation product of 7-dehydrocholesterol is lumisterol;, iso-
lated by Windaus ef al.*43 in 1937. It is distinguished from lumisterol. by
the fact that it does not form a molecular addition compound with its cor-
responding vitamin D.

k. Tachysterols

Co,HasO. Melting point of 3 ,5-dinitro-4-methyl benzoate 137°. Free form
has [a]? = —11.5° in ‘Normalbenzin,” but the ester is dextrorotatory.
Absorption spectrum like that of tachysterols, but somewhat less intense.

From the irradiation products of 7-dehydrocholesterol, tachysterol; was
isolated by Windaus e¢ al.*" in 1937. Like tachysterols, it does not crystal-
lize, but forms crystallizable esters.

l. Precalciferols

CwHwO. Melting point of 3,5-dinitrobenzoate 110° to 111° [a]!8 of
3,5-dinitrobenzoate = +52° in chloroform and +38.5° in benzene, with
mutarotation.

Velluz et al.*!® in 1949 obtained precalciferol; in the form of its 3 ,5-di-
nitrobenzoate, which crystallizes in pale yellow, thread-like needles not
exhibiting the dimorphism of the corresponding ester of vitamin D;. The
free form does not crystallize. The free (or ester) form exists in solution in
equilibrium with the corresponding free (or ester) form of vitamin Ds,
with the vitamin (or ester) predominating. of

m. Vitamin D3. Calciferol;. Cholecalciferol

Cx;HyO. Three double bonds. Melting point 84° to 85°.%!: 2°” Broad ab-
sorption band with maximum at 265 my; molecular extinction coefficient
= 18,200 in alcohol or hexane.?®! [a|?? = +84.8° in acetone.2%”

316 J,, Velluz and G. Amiard, Compt. rend. 228, 1037 (1949); L. Velluz, G. Amiard, and
A. Petit, Bull. soc. chim. France 16, 501 (1949).

II. CHEMISTRY 203

Vitamin D; was obtained in crystalline form by Schenck*” in 1937. The
isolation procedure was essentially the same as for calciferol, 1.e., purifica-
tion via the 3 ,5-dinitrobenzoate, followed by saponification. Vitamin D; is
more difficult to crystallize than De, and the yields are inferior.?97) 313, 318
There has been described a double compound of the vitamin with cholesterol
(or with cholestanol or coprosterol), which crystallizes in good yield and
represents the most economical way to obtain a preparation of uniform
purity.?*7 *19 The constants of the esters of vitamin D3; are shown in Table
VIII.

Pure crystalline vitamin D; is slightly more stable than D2, and much
more stable than its precursor, 7-dehydrocholesterol. Considerable deteri-
oration occurs in 72 hours when the vitamin is exposed to air at room tem-
perature, even in brown bottles. Deterioration is negligible after 12 months’
storage in evacuated amber ampules at refrigerator temperature.?*? The
complex with cholesterol is less stable than the pure vitamin.?” As with
calciferol, the various nitrobenzoic acid esters of vitamin D3 are remarkably
stable. They remain unchanged for at least 5 years with no special pre-
cautions other than the avoidance of light. Solutions of vitamin D; in edible
oils or in propylene glycol and dispersions of it in canned milk have excel-
lent keeping qualities, even under severe conditions.”

Vitamin Ds is sensitive to hydrogen chloride.?*! It does not give a pre-
cipitate with digitonin.*"” It gives a yellow color in the antimony trichloride
reaction.” In these respects, as well as in optical properties and chemical
reactions, it reveals its similarity to calciferol. The similarity in most
non-biological properties and reactions of these two forms of vitamin D
supports the quip that only a bird can tell them apart.

5. Minor AND OBSCURE FoRMS

The number of steroids which exhibit antiricketic action is considerable,
but many of them are so vaguely characterized that an exact count is im-
possible. In attempting to enumerate them, one is confronted with the in-
adequacy of many reported animal tests and with the prebability that, if
tests were conducted differently, the number of recognized vitamins D
would be larger. There is also the problem of contamination, which has
confused investigators ever since the first crude cholesterol was irradiated
and which now is involved in the so-called vitamin Ds, or activated 7-de-
hydrositosterol. Lastly, there is the factor of the occasional bad assay,
which, according to the statisticians, appears even in the best-regulated
laboratories at unpredictable times.

317 F, Schenck, Naturwissenschaften 25, 159 (1937); A. Windaus and F. Schenck,

U.S. Pat. 2,099,550 (1937).

318 A. Windaus, F. Schenck, and F. von Werder, Hoppe-Seyler’s Z. physiol. Chem.

241, 100 (1936).

319 Q, Linsert, U. S. Pat. 2,264,320 (1941).

204

VITAMIN D GROUP

TABLE VIII
VITAMIN D EsTERS

Ester, reference

Melting point, °C.

Specific rotation

Calciferyl (Vitamin D2) ace-
tate?

Calciferyl allophanate??®

Calciferyl anisate??®

Calciferyl benzoate?”

Monoealciferyl esters of n-bu-
tane-1,2,3,4-tetracarbox-
ylic acid (unseparated iso-
mers)269

Calciferyl chaulmoograte””

Calcifery] 2-chloro-3,5-dini-
trobenzoate!*4
Calciferyl 3,5-dinitrobenzo-

ate263, 2974

Calciferyl 3,5-dinitrobenzo-
ate?97

Calciferyl 3,5-dinitrobenzo-
ate?97

Calciferyl 3,5-dinitro-4-meth-
ylbenzoate?%”

Calciferyl B-naphthoate?®7*
Calciferyl 4-nitrobenzoate?”
Calciferyl 3-nitro-4-methyl-
benzoate?”
Calciferyl oleate”
Calciferyl phenylurethane??®
Calciferyl propionate?”
Vitamin D; anisate*!s
Vitamin D,; 3,5-dinitroben-
zoate (dimorphous)
From benzene-methanol?%”
From ether?*
Leaflets?1®
Needles?!§
Vitamin Ds; _ 3,5-dinitro-4-
methylbenzoate”*?
Vitamin D; 4-nitrobenzoate?%”
Vitamin D, 3,5-dinitrobenzo-
ate31
Vitamin D,, 3,5-dinitrobenzo-

atell9
\

86

194-195

99 .5-101

92

90-100 decomp.

53
132

147-149

146-147

116-117

132
94.5-95
119-120

Liquid
122

hE

114

132
141
142
150
128-129

125-126
127-128

128-128 .5

[a]s70 = +38° acetone
[a]?? = +50.4° chloroform

[a]$ = +120° chloroform
[a]: = +100°acetone

[a]s70 = +52° chloroform

[a]5 = +60° acetone
[a]}3?3. = +69° benzene
[a]? = +91.5° chloroform
[oe]? = +86.5° acetone
[a];’ = +95.8° chloroform
[a]? = +150° chloroform
[a]}?? = +105.2° chloroform
[a]; = +106.8° chloroform

{e]s70 = +18.7° chloroform
[a]3° = +49.2° chloroform
[a]s790 = +37.6° acetone
[a]> = +127° chloroform

[a];? = +97.9° chloroform
[a]?? = +97° chloroform
[a]? = +106.6° chloroform
[a]?? = +114.6° chloroform
[a]> = +93.2° acetone

[alo = +92° chloroform

II. CHEMISTRY 205

(1) Of the minor forms, the best known is vitamin D,, produced by the
irradiation of 22-dihydroergosterol. Windaus and Trautmann*° in 1937
prepared this vitamin in crystalline form. Windaus and Giintzel*”! repeated
the preparation, and also isolated other members of the irradiation series,
namely, lumisterols, tachysterols, and suprasterol II,. Vitamin D, crystal-
lizes out of acetone in long needles, m.p. 96° to 98°, [af?? = +85.7° in
acetone, spectrum similar to that of calciferol.*?! The potency of the crystal-
line vitamin amounts to 20,000 to 30,000 I.U. per milligram for the rat*2°
(50 % to 75 % of the value of Dz or D3). The chick-rat efficacy ratio of vi-
tamin D, may be estimated, from the findings of McDonald®* and Grab,
at about 20%, which places it between D2 (1%) and D; (100%), quite in
keeping with its structure. Vitamin D, ranks as a minor form only because
it has received no practical application.

(2) The vitamins D produced by the irradiation of mollusk provitamins
are obscure forms chemically but important commercially because they are
widely used in poultry feeding. As indicated in the discussion headed Pro-
vitamins D of Invertebrates, the provitamins of different mollusks, even
when “‘pure”’ spectrographically, differ from each other and from any chem-
ically known form of provitamin D. They are probably mixtures, perhaps
containing some 7-dehydrocholesterol and occasionally a little ergosterol,
but predominantly composed of other sterols, such as the provitamin D,, of
Petering and Waddell® which appears to have 29 carbon atoms. The vi-
tamin D obtained from provitamin D,, is notable for exhibiting a chick-
rat efficacy ratio somewhat above 100%. “°: ?° It has been crystallized in
the form of its 3 ,5-dinitrobenzoate, m.p. 128° to 128.5°, [a], = +92° in
chloroform."® Even though this may not be a pure substance, there is no
doubt but that it represents an obscure vitamin D characterized by high
efficacy and high molecular weight.

(3) In the discussion of vitamin D in fish oils, it was shown that several
forms are present, although only Dz and D; have been identified. One of
the minor forms detected in cod liver oil by Hickman and Gray'® exhibited
a boiling point in molecular distillation so low as to suggest the absence of
any side chain in the molecule at C-17. This form, in a chick-rat assay, was
found to have an efficacy ratio of between 25% and 50%. It must be re-
garded as a distinct form of vitamin D.

(4) It has been amply demonstrated by Waddell,** Bills e¢ al.,8° Remp
and Marshall,*”? and others that, rat unit for rat unit, vitamin D; and cod
liver oil are about equally effective for chicks. From this has come the un-

#20 A. Windaus and G. Trautmann, Hoppe-Seyler’s Z. physiol. Chem. 247, 185 (1937);
‘A. Windaus, U. S. Pat. 2,128,199 (1938).

321 A. Windaus and B. Giintzel, Ann. 588, 120 (1939).

322 T). G. Remp and I. H. Marshall, J. Nutrition 15, 525 (1938).

206 VITAMIN D GROUP

warranted deduction, occasionally seen in medical literature, that vitamin
D3; is the vitamin D of cod liver oil. It has been shown by Matterson e¢ al.5”8
and by Waddell and Kennedy** that, when vitamin D; and cod liver
oil are standardized with chicks kept on a diet in which most of the phos-
phorus is inorganic (e.g., the A.O.A.C. diet), and then again assayed with
chicks or turkey poults kept on a diet in which most of the phosphorus is
phytic, the vitamin D; is found to be much more effective than the vitamin
D of cod liver oil. This can mean only that vitamin D; is not the dominant
vitamin D of cod liver oil and that the dominant form (or forms) of vitamin
D in cod liver oil is something different from any recognized form.

(5) In determining the efficacy ratios of the liver oils of twenty-five species
of fish, Bills e¢ al.1®° found a few oils with a ratio much above 100. The ex-
treme example was the liver oil of the white sea bass, Cynoscion nobilis,
which had a ratio of about 300. This oil, when subjected to molecular dis-
tillation,!® showed an exceptionally pure elimination curve, indicative of a
preponderance of one form of vitamin D. If the assays can be confirmed,
it will be established that white sea bass liver oil contains a new form of
vitamin D characterized by the highest known efficacy ratio.

(6) Windaus and Trautmann*®° and Dimroth and Paland* briefly men-
tion the irradiation product of 22 ,23-oxidoergosterol. Described only as a
feebly active product, this is presumably 22 ,23-oxidocalciferol.

(7) Windaus and Buchholz*®> have prepared an oily ketone of vitamin D2
which was probably uncontaminated with traces of the vitamin, and which
exhibited about 1499 of the potency of the vitamin in tests with rats.

(8) McDonald ** has found that by the irradiation of 7-hydroxycholes-
terol (or an impurity associated therewith) a slight antiricketic potency is
developed. Conceivably, the irradiation causes dehydration, with the for-
mation of traces of 7-dehydrocholesterol, in which case the vitamin pro-
duced would not be a new form, but merely D3.

(9) Weinhouse and Kharasch*” claim to have produced a form of vitamin
D by irradiating the heated reaction product of 7-ketocholesteryl acetate
and isobutyl magnesium bromide.

(10) In the section headed Chemical Structure and Activatability, men-
tion was made of the vitamin D from 7-dehydrocampesterol. Corresponding
to the structure of its precursor, this vitamin can be regarded as the C-24
epimer of vitamin Dy.

(11) The vitamin D from epiergosterol is, presumably, epicalciferol.

323 TL. D. Matterson, H. M. Scott, and E. P. Singsen, J. Nutrition 31, 599 (1946).

324 J. Waddell and G. H. Kennedy, J. Assoc. Official Agr. Chemists 30, 190 (1947).

325 A. Windaus and K. Buchholz, Hoppe-Seyler’s Z. physiol. Chem. 256, 273 (1938).

326 KF. G. McDonald, cited by C. E. Bills, The Vitamins, Chapter xxiii. American
Medical Association, Chicago, 1939.

327 §. Weinhouse and M. 8. Kharasch, J. Org. Chem. 1, 490 (1936).

Il. CHEMISTRY 207

(12) The vitamin D from 7-dehydroepicholesterol is, by the same analogy,
epicalciferols.

(13) The compound dihydrotachysterol, better known under its trade
names A. T. 10 (anti-tetany compound No. 10) and Hytakerol, possesses
some antiricketic activity, in addition to its useful property of elevating
blood calcium in parathyroid tetany. It was obtained by von Werder*® in
1939 by the reduction of tachysteryl 3 ,5-dinitro-4-methylbenzoate with
sodium and alcohol, followed by saponification and chromatographic puri-
fication. This drug should not be confused with its isomer, 22-dihydro-
tachysterol (tachysterol,), which is a product of the irradiation of 22-dihy-
droergosterol or of the reduction of tachysteryl acetate adduct with
hydrogen. The side-chain double bond in dihydrotachysterol is intact, the
reduction being effected in ring A. With the reduction there occurs a migra-
tion of the remaining double bonds into the alignment characteristic of
calciferol. Thus, although the product is genetically dihydrotachysterol, it is
chemically a dihydrocalciferol, differing structurally from the latter only in
that the methylene group at C-19 is reduced. With this simple change, the
compound loses most of its antiricketic activity, while retaining a relatively
large proportion of its blood calcium-elevating activity.

Dihydrotachysterol, CosHisO, crystallizes in colorless needles from 90%
methanol. It is easily soluble in organic solvents. It has m.p. 125° to 127°,
[a]? = +97.5° in chloroform, strong absorption maxima at wavelengths
242, 251, and 261 muy.’ Somewhat varying reports on its antiricketic
effectiveness has been made by von Werder,*”8 Correll,*?? McChesney,**°
Motzok,**! and others. It appears to have a potency of about 90 I.U. per
milligram for rats, which is between 1499 and 99 of the potency of
calciferol and the same as the potency of tachysterol itself. When compared
with cod liver oil or vitamin D; with rats and chicks, it shows an efficacy
ratio of 400 to 750%. Willgeroth e¢ al.**? claim that it is somewhat more
effective on turkey poults than on chicks.

(14) In 1928 Bills et al.,3* repeating the work of the English and German
workers in destroying the provitamin D in ordinary cholesterol, found that
treatment with charcoal or bromine never resulted in a total, but only in
a relative (to about 149), reduction of activatability. Samples so treated,
when examined spectrographically in sufficiently high concentrations, still
showed the characteristic provitamin bands, and, also, two new bands at

88 F. von Werder, Hoppe-Seyler’s Z. physiol. Chem. 260, 119 (1939); U. S. Pat.
2,228,491 (1941).

89 J. T. Correll and E. C. Wise, J. Nutrition 23, 217 (1942).

330 H. W. McChesney, J. Nutrition 26, 81, 487 (1943).

381 T. Motzok, D. C. Hill, and H. D. Branion, Poultry Sci. 25, 644 (1946).

32 G. B. Willgeroth, J. L. Halpin, H. R. Halloran, and J. C. Fritz, J. Assoc. Official
Agr. Chemists 27, 289 (1944).

208 VITAMIN D GROUP

wavelengths 304 and 315 mu. Heilbron et al.*** suggested that the extra
bands might be those of cholesterilene, which is non-activatable and hence
irrelevant. Other studies with specially purified cholesterol are discussed in
the author’s review of the multiple nature of vitamin D.*4 It will suffice
here to note that Hathaway and Lobb performed chick-rat assays on the
irradiation product of cholesterol which had been purified via the dibro-
mide. They found that its efficacy ratio was very low, in a class with that
of irradiated ergosterol. Since no known cholesterol derivative has a low
efficacy ratio, and since ergosterol could not have survived bromination and
could not have been formed anew from a sterol of lower molecular weight,
it must be concluded that the irradiated purified cholesterol contained a
new form of vitamin D. Presumably the provitamin was the substance
which showed the absorption bands in the Bills preparation, and which orig-
inated during or after the debromination of the cholesterol dibromide.

Koch e¢ al.,**° in a series of papers summarized*** in 1935, found that by
strongly heating highly purified cholesterol in the presence of traces of
oxygen, the provitamin D activity could be enhanced 100 times. The typi-
cal provitamin spectrum was not observed, but it may have been present
and obscured by a strong general absorption. Waddell**” found that by
heating the cholesterol in the presence of water, the provitamin D activity
could be enhanced 100 to 150 times. The vitamin D from heat-treated cho-
lesterol was found by Hathaway and Lobb**4 and Waddell**’ to have a high
efficacy ratio in chick-rat assays. This fact, together with the later work
on the synthesis of provitamins D, makes it seem probable that the pro-
vitamin formed by heating cholesterol is 7-dehydrocholesterol. Unless this
assumption is disproved, one cannot regard the vitamin D from irradiated
heat-treated cholesterol as a new form.

(15) Meunier and Thibaudet#*® have prepared a monoiodocalciferol, m.p.
150°, which is at least as potent as the parent substance. Removal of the
iodine with hyposulfite yielded tachysterol.

(16) Bills and McDonald*® treated ergosterol with ethyl nitrite, and then
treated the product with isopropylamine, obtaining a substance which

showed a trivial antiricketic action with rats. Since the conventional form,

333 T. M. Heilbron, R. A. Morton, and W. A. Sexton, Nature 121, 452 (1928).

334 M. L. Hathaway and D. E. Lobb, J. Biol. Chem. 118, 105 (1936).

335 F.C. Koch, E. M. Koch, and I. K. Ragins, J. Biol. Chem. 85, 141 (1929); E. M.
Koch, F. C. Koch, and H. B. Lemon, ibid. 85, 159 (1929); M. L. Hathaway and
F.C. Koch, ibid. 108, 773 (1935).

336 H}. M. Koch and F. C. Koch, Science 82, 394 (1935).

337 J. Waddell, U. S. Pat. 2,028,364 (1936).

388 P, Meunier and G. Thibaudet, Compt. rend. 228, 172 (1946).

889 C, E. Bills and F. G. McDonald, cited by C. E. Bills, Cold Spring Harbor Symposia
Quant. Biol. 3, 328 (1935).

II. CHEMISTRY 209

of vitamin D are destroyed by nitrites, this active substance must be differ-
ent from any known form of vitamin D.

(17) The oldest of the minor forms is the antiricketic product which Bills
and McDonald*° made in 1926 by treating cholesterol with fuller’s earth.
Its potency was trivial, but interesting at the time in view of the irradiated
cholesterol which had been discovered the year before. The preparation was
repeated by Kon et al.?!* and later the reaction was investigated by Yoder,**
who identified the product as a sulfonated cholesteriline which resulted
from dehydration followed by sulfonation attributed to traces of sulfur
trioxide in the preheated acid clay. In a series of experiments Yoder and
his associates*” worked out the details of ‘‘antiricketic sulfonation” and
found that a considerable family of hydroxy steroids can be activated by
this means. One of the treatments was essentially the Liebermann reaction.
The potency attained was never high by irradiation standards, and the
healing action on rats was often accompanied by weight loss. Yoder and
Thomas** have reported that the product from cholesterol has an exception-
ally high chick-rat efficacy ratio. .

According to Raoul e¢ al.*“4 the action of sulfuric acid on cholesterol in the
Liebermann and Salkowski reactions results in the formation of an “ether-
oxide” of “‘tachysterol”” (meaning ditachysteryl; ether?), which, in an ioniz-
ing solvent, and without irradiation, transforms itself into ‘‘vitamin D.”
The French authors state, ‘““These experiments teach that it is not sulfo-
nation which is the cause of the transformation of cholesterol into an anti-
ricketic product. There is really formed a vitamin D by the intermediary
of tachysterol under the sole influence of ionizations of various origins
(acetyl chloride, acetic anhydride, sulfuric acid) as two of us envisaged in
1947.”’ The claim that vitamin D can be made from cholesterol by a simple
chemical treatment is most interesting, but the description of what happens
is unclear and lacks explanation of the required dehydrogenation step. In
a later paper, Raoul et al.*4° admit that the antiricketic compound formed
is not vitamin D;. It seems well established that Yoder’s active preparations
are monosulfonated unsaturated hydrocarbons, but it may be that products
of the type studied by Raoul are also formed in the same reactions.

340 C. E. Bills and F. G. McDonald, J. Biol. Chem. 67, 753 (1926).

441 J. Yoder, J. Biol. Chem. 116, 71 (1936).

32 J. C. Eck, B. H. Thomas, and L. Yoder, J. Biol. Chem. 117, 655 (1937); J. C. Eck
and B. H. Thomas, zbid. 119, 621, 631 (1937); 128, 257 (1939); L. Yoder and B. H.
Thomas, zbid. 178, 363 (1949); Ind. Eng. Chem. 41, 2286 (1949).

343 J,. Yoder and B. H. Thomas, Arch. Biochem. and Biophys. 82, 14 (1951).

344 Y. Raoul, J. Chopin, P. Meunier, A. Guérillot-Vinet, and N. Le Boulch, Compt.
rend. 229, 259 (1949); Y. Raoul, J. Chopin, P. Meunier, N. Le Boulch, and A. Gué-
rillot-Vinet, ibid. 282, 1154 (1951).

345 Y. Raoul, N. Le Boulch, P. Meunier, J. Chopin, and A. Guérillot-Vinet, Comot.
rend, 232, 1258 (1951).

210 VITAMIN D GROUP

III. Industrial Preparation
CHARLES E. BILLS

The patent index in Rosenberg’s! book bears witness to the effort and
ingenuity which have been applied to the industrial development of vitamin
D. The index includes 242 titles and short abstracts of the more important
United States and foreign patents through 1941.

The preparation of fish oils is described in Tressler and Lemon’s? “‘Marine
Products of Commerce”’ and especially in Brocklesby’s’ ‘‘Chemistry and
Technology of Marine Animal Oils.”’ The ancient method of rendering fish
liver oils by the natural disintegration of livers began to be replaced by
steam cooking about the middle of the nineteenth century.4 With technical
improvements, steaming sufficed for most vitamin oil production until
about 1933, when interest developed in the production of high-potency oils
from low-yield livers.

Nearly all high-potency fish liver oils are manufactured by a process
based on the liquefaction of the liver with alkali. This process was worked
out independently and at about the same time by Young and Robinson?
and Wallenmeyer.® Sufficient alkali is added to the livers so that, with
heating, they go into solution, but the amount of alkali is limited so that
substantial saponification of the oil does not occur. The oil is then separated
centrifugally from the aqueous liquor, washed, and refined as desired. The
vitamin D in liver oils is remarkably stable, but in consideration of the
less stable vitamin A, the oils are usually stored under inert gas or in tanks
with floating tops.

The patent literature contains many references to equipment designed
for the antiricketic activation of foods under the basic Steenbock’ patent.
The common aim in these inventions is the brief exposure of a shallow,
moving layer to the rays of a mercury or carbon arc. Milk, because of its
suitability for treatment, and its importance to infants and children, has
been most extensively activated. A representative apparatus for irradiating
milk is shown in Fig. 14. Direct activation of milk is practiced less now than
formerly; instead, fortification with vitamin D is accomplished by the

1H. R. Rosenberg, Chemistry and Physiology of the Vitamins. Interscience Pub-
lishers, New York, 1945.

2—D. K. Tressler and J. McW. Lemon, Marine Products of Commerce. Reinhold
Publishing Corp., New York, 1951.

3H. N. Brocklesby, Chemistry and Technology of Marine Animal Oils with Par-
ticular Reference to Those of Canada. Fisheries Research Board of Canada, Ot-
tawa, 1941.

4C. E. Bills, Chem. Revs. 3, 425 (1927).

5 F. H. Young and H. D. Robinson, U.S. Pat. 2,136,481 (1938).

6 J. C. Wallenmeyer, Mexican Pat. 35,140 (1934).

7H. Steenbock, Science 60, 224 (1924); U.S. Pat. 1,680,818 (1928).

III. INDUSTRIAL PREPARATION PA |

addition of concentrated fatty emulsions or propylene glycol solutions of
vitamin D» or Ds. Milk can also be fortified by irradiating the cow or feed-

5 SIO ELIE Clee sea ea

Fia. 14. Irradiating unit for milk and other aqueous fluids. From the regulating
chamber at the left, the milk enters the annular top trough, flows over the weir,
and cascades down the wall of the tank to the bottom trough. Carbon are lamp and
fume exit shown swung out of position. Forced air for ventilation enters at bottom.
(Courtesy of National Carbon Co.)

ing the cow irradiated yeast. The vitamin D in irradiated milk, or milk from
irradiated cows, presumably is vitamin D3; in milk from cows fed irradiated
yeast it is vitamin D».

Zi VITAMIN D GROUP

Concentrated preparations of vitamins D2, D3, and D,, are produced in
a much greater total unitage than fish oils for medicinal use and animal
feeding. The manufacture of fish oils has continued at a high level because
of their importance as a source of vitamin A, but since 1950, with the ad-
vent of synthetic vitamin A at low cost, the trend to replacement by syn-
thetics has been accelerated. The end use, as well as the source, of vitamin D
has undergone great change. In 1925 vitamin D, then almost solely in the
form of cod liver oil, was used principally to cure rickets in children. By
1950 florid rickets had become a rarity, thanks to the use of vitamin D in
many forms to prevent it. During the same period, the use of vitamin D in
poultry raising expanded from the experimental stage to the principal field
for this vitamin.

The first synthetic vitamin D manufactured in the United States was
produced by pumping a peanut oil solution of ergosterol through the cooling
jacket of a quartz mercury vapor lamp and then through a heat exchanger,
cyclically, until the desired conversion was effected.’ A more efficient unit,
employing the carbon arc, and designed to activate provitamins D in
transparent solvents, is shown in Fig. 15. This unit, still in use after more
than twenty years, has a daily capacity of the equivalent of 8 metric tons
of cod liver oil. In a different arrangement, shown in Fig. 16, the solution
flows through a series of activating units, each containing a water-cooled
Hanovia lamp and an outer quartz chamber for the provitamin D solution.

In commercial practice, ether is usually the preferred solvent. The irradia-
tion is continued until a certain percentage of the provitamin is transformed,
the exact amount being determined for optimum vitamin yield under the
conditions of exposure, particularly the type of light source and the kind
of provitamin. Usually not over 50% transformation is allowed. The un-
changed provitamin is recovered by evaporating the solvent, taking up the
resin in methanol, and freezing out. After the methanol is removed, the
resin is either dissolved without further treatment in an edible oil, or it is
converted into 3 ,5-dinitrobenzoate for the preparation of crystalline vita-
min D.

Some manufacturers use light filters, and others do not. The advantages
in yield gained by using light of about \ 280 mu are more or less offset by
the loss of light, but filtered light is probably advantageous when minimal
accumulation of tachysterol is desired, as in the production of crystalline
vitamin D. Some choice of wavelengths is available to users of carbon arcs,
for carbons can be purchased with different core materials, and large users
can even have them made to order. The hot mercury are differs spectrally
from the cold, but both are rich in the shorter waves, the cold especially so.

It is customary to exclude oxygen during irradiation, either by using

8 C. E. Bills, U.S. Pats. 1,808,760 (1931); 1,848,305 (1932).

Fig. 15. Apparatus for the activation of ergosterol. The light source is an alter-
nating current carbon are operating at 40 volts, 40 amperes across the terminals.
By the use of a constant current (floating secondary) transformer and a system of
relays sensitive to voltage changes, the automatic feeding of the carbons is closely
regulated. Equidistant from the are four fused quartz flasks are located, each of 1
liter bulb capacity. The neck of each is greatly extended, so as to form the inner tube
of a reflux condenser. Refrigerated water is circulated through the insulated jacket.
40 g. of ergosterol and 3600 cc. of purified ether constitute a charge. CO» is passed
through the solution to expel air, and immediately the open end of the condenser is
capped with a toy balloon. Thus in the “‘breathing”’ incident to irregular boiling,
the system is compelled to breathe its own breath, and ingress of air is prevented.
(After Bills et al.!*)

213

214 VITAMIN D GROUP

closed systems or by replacing the air over solutions with inert gas. Ether
used as a solvent should be peroxide-free. Stabilizers added to the solution
are claimed to greatly increase the yield of vitamin. Among these, various
sugar amines have been employed.’ The most effective stabilizers appear to
be the non-provitamin D sterols, such as cholesterol or the inactivatable
fraction of mollusk sterols. Waddell and Woessner’? claim that in the activa-

Fria. 16. Irradiating units in series for vitamin D production. Each unit consists of
a vertical quartz mercury lamp surrounded by two cylindrical chambers through
which water circulates for cooling. The solution of ergosterol or other provitamin D
is circulated through a third cylindrical chamber. (Courtesy of EK. R. Squibb and
Sons.)

tion of 7-dehydrocholesterol the presence of an equal amount of cholesterol
increases the vitamin yield almost 40%, besides enhancing the recovery of
unconverted provitamin. The highly unstable provitamins D of mollusks
are protected by the non-provitamin D sterols which naturally accompany
them. When, in the course of repeated irradiations, the protecting sterols
of the mollusk sterol mixture accumulate excessively, they can be removed
and converted into provitamins by reactions analogous to those employed
in the synthesis of 7-dehydrocholesterol.!! The stabilization of provitamins
°H. W. Elley and J. Waddell, U.S. Pat. 2,234,554 (1941).

10 J, Waddell and W. W. Woessner, U.S. Pat. 2,410,254 (1946).
1H. R. Rosenberg, U.S. Pat. 2,475,917 (1949).

IV. ESTIMATION 215

by other sterols occurs not only in activation by irradiation, but in the less
widely practiced activation in the vapor phase by a high-frequency elec-
trical discharge.”

IV. Estimation
CHARLES E. BILLS

Vitamin D is the only one of the better-known vitamins for which
physical or chemical methods of estimation have not largely replaced bi-
ological methods. The reason for this is plain: vitamin D is so potent that
even in the richer sources its concentration is usually minute. Conversely,
the substances which interfere with physical or chemical determination of
it are likely to be present in relatively overwhelming amounts. In biological
assays, however, rats or chicks show measurable responses to vitamin D
when it is present in their diet in amounts as little as 1 part in several
hundred million. Interference with the biological response occurs only when
comparatively gross quantities of materials such as phosphates are en-
countered.

With the publication of Gyérgy’s “Vitamin Methods,’ the art of esti-
mating vitamins is presented in two volumes. Therefore, the techniques will
be presented here only in outline, in order to guide the reader to the
more detailed sources of information.

A. PHYSICAL METHODS

In very concentrated forms, vitamin D can be estimated by purely
physical means. Thus Pirlot? recommends infrared spectrophotometry for
determining calciferol in the irradiation products of ergosterol. For calciferol
itself, or concentrated oily solutions thereof, Pénau and Hagemann*® em-
ployed ultraviolet spectrophotometry. The estimation, based on the ex-
tinction at \ 265 my, is straightforward, provided that the absorption curve
is normal. If there is displacement of the maximum, accentuated asym-
metry, or other abnormality, the presence of impurities or degradation
products is indicated. Concentrated oily solutions are diluted with choloro-
form and absolute alcohol to make a 0.0015 % solution of calciferol. The
extinction is determined, and correction is made for the oil, if known. If the
oil is unknown, the unsaponifiable fraction is separated, and allowance is
made for saponification loss, which usually amounts to about 7.5 %.

2C, E. Bills, F. G. McDonald, L. N- BeMiller, G. E. Steel, and M. Nussmeier,
J. Biol. Chem. 98, 775 (1931).

1P, Gyorgy, Vitamin Methods. Academic Press, New York, 1950-1951.

2G. Pirlot, Anal. Chim. Acta 2, 744 (1948) [C.A. 48, 7997 (1949)].

3H. Pénau and G. Hagemann, Helv. Chim. Acta 29, 1366 (1946).

216 VITAMIN D GROUP

B. CHEMICAL METHODS

A drawback to direct spectrophotometric estimation is the fact that the
absorption maximum of vitamin D lies at 265 mu, a region where ultra-
violet dirt is especially likely to show up. Accordingly, in the devising of
chemical methods of estimation, it has been sought to form strong and
stable colors in the visible range, less susceptible to irrelevant absorption,
and, of course, as nearly as possible specific to the vitamin.

The most widely used color reaction is that given by antimony trichloride
and first applied to pure vitamin D, by Askew ef al.4 A deep yellow color
is developed, with the absorption maximum at 500 muy. Nield et al.® in-
creased the sensitivity of the reaction to the point where they could measure
the color given by 0.2 y of calciferol, and they also increased the permanence
of the color. Milas et al.* applied modifications of the reaction to the assay
of fish liver oils. In one procedure they made interference corrections for
sterols and vitamin A, and in their preferred method they first removed the
interfering substances by coupling them with maleic anhydride and then
measured the color developed at 500 mu. They were able to get fairly good
agreement with bioassays in the case of fish oils containing 10,000 units or
more of vitamin D per gram.

Details of the antimony trichloride reaction were worked out by Vacher
et al.,” Shantz,® Nielsen,’ and others. The important step of chromatographic
purification was introduced and improved successively by Ewing et al.,!°
De Witt and Sullivan," Miiller,!? Ewing e¢ al.," and Fujita and Aoyama."
Under favorable conditions the pigment generated in this reaction from
vitamin D, or D; shows E}”,, = 1800 at 500 mu. This is an intense absorp-
tion, nearly four times as great as that of the vitamin itself at 265 my, and
comparable with that of strongly absorbing dyes such as fuchsin. Hence it
seems unlikely that efforts to find a better color reaction will substantially
improve upon this one with regard to intensity of color, although there is

4F. A. Askew, R. B. Bourdillon, H. M. Bruce, R. K. Callow, J. St. L. Philpot,
and T. A. Webster, Proc. Roy. Soc. (London) B109, 488 (1932).

5 C. H. Nield, W. C. Russell, and A. Zimmerli, J. Biol. Chem. 136, 73 (1940).

6N. A. Milas, R. Heggie, and J. A. Raynolds, Ind. Eng. Chem. Anal. Ed. 13, 227
(1941).

7M. Vacher, Y. Lortie, and H. Colson, Bull. soc. chim. biol. 26, 206 (1944).

8. M. Shantz, Ind. Eng. Chem. Anal. Ed. 16, 179 (1944).

9P.B. Nielsen, Nutrition Abstracts & Revs. 19, 57 (1949).

10T). T. Ewing, G. V. Kingsley, R. A. Brown, and A. D. Emmett, Ind. Eng. Chem.
Anal. Ed. 15, 301 (1948).

1 J.B. De Witt and M. X. Sullivan, Ind. Eng. Chem. Anal. Ed. 18, 117 (1946).

12 P, B. Miller, Helv. Chim. Acta 30, 1172 (1947).

13 —D. T. Ewing, M. J. Powell, R. A. Brown, and A. D. Emmett, Anal. Chem. 22, 317
(1948).

144A. Fujita and M. Aoyama, J. Biochem. (Japan) 37, 113 (1950).

IV. ESTIMATION 217

room for improvement with regard to permanence and specificity. In its
more highly developed forms, the antimony trichloride reaction and its
associated steps of purification are complicated procedures, which give
fairly reliable estimations with oily solutions containing 5000 or 10,000
I.U. per gram or more of vitamin D.

Another color reaction has been described by Sobel e¢ al.!® With a chloro-
form solution of dichlorohydrin and acetyl chloride, vitamin D gives a green
color with peak absorption at 625 my. The color is more stable and more
nearly specific than that of the antimony trichloride reaction. Rouir and
Pirlot,!® applying Sobel’s reaction to pharmaceuticals, stressed the impor-
tance of correcting for oil. Campbell!’ increased the sensitivity of the re-
action twentyfold, which makes it only five times less sensitive than the
antimony trichloride reaction. He found that by measuring the absorption
at 410 mu, instead of 625 mu, the interferences given by maize oil, fish oil,
ergosterol, and 7-dehydrocholesterol were minimized. The reaction deserves
further study with high-potency oils.

A third promising color reaction is that of Schaltegger.18 Carbenium salts
produced by treating vitamin D with perchloric acid are reacted with alde-
hydes, giving colors of improved specificity. Banchetti,!® using vanillin as
the aldehyde in the reaction, was able to detect 50 y of vitamin D in the
presence of two hundred times as much of other sterols. The possibilities
of this reaction have not been explored.

Investigators of color reactions should note the report of McMahon et
al.,?° in which is described the separation and identification of vitamin D
from other sterols by partition paper chromatography, the sterol spots
being developed by spraying with antimony pentachloride in chloroform.

A chemical method for estimating vitamin D, which is not a color reac-
tion in the usual sense, was described by Green”! in 1951. It is based on the
liberation of iodine in the reaction between vitamin D and iodine trichloride
in carbon tetrachloride. A comprehensive study was made of methods for
separating vitamin D from interfering substances by means of differential
solubility, digitonin precipitation, and chromatography on Floridin. Highly
efficient purification is claimed, and the purified product can be reacted
stoichiometrically with the reagent. The older techniques of condensation
with maleic or citraconic anhydride, and of selective ultraviolet irradiation

15 A. E. Sobel, A. M. Mayer, and B. Kramer, Ind. Eng. Chem. Anal. Ed. 17, 160 (1945)°

16. V. Rouir and G. Pirlot, Bull. soc. chim. biol. 29, 1005 (1947).

17 J. A. Campbell, Anal. Chem. 20, 766 (1948).

18H. Schaltegger, Helv. Chim. Acta 29, 285 (1946).

19 A, Banchetti, Ann. chim. appl. 38, 394 (1948).

20 J. M. McMahon, R. B. Davis, and G. Kalnitsky, Proc. Soc. Exptl. Biol. Med. 75,
799 (1950).

21 J. Green, Biochem. J. 49, 36, 45, 54 (1951).

218 VITAMIN D GROUP

for the separation of vitamin A from vitamin D, are not, in Green’s opinion,
good ones, for considerable losses of vitamin D may be encountered.

C. BIOLOGICAL METHODS

The author has elsewhere”? presented a chapter on the use of the rat in
the estimation of vitamin D, and Waddell and Kennedy* have published
a companion chapter on the use of the chick for this purpose. New workers
in the field of bioassay are advised first of all to absorb the wisdom in
Coward’s* “Biological Standardization of the Vitamins.’”’ The practical,
theoretical, and statistical aspects of this subject are also covered by
Gyorgy,! and in particularly fine detail.

The official method of estimating vitamin D in medicinal oils is described
in U.S. Pharmacopeia (14th revision). It is essentially a 7-day curative
technique, observations being made by the line test on rats previously
rendered ricketic. The official procedure for estimating vitamin D in milk
is given in the A.O.A.C. ‘‘Methods of Analysis.’’?> It is similar to the U.S.P.
method but requires the feeding of skim milk powder to the reference rats
in amounts equivalent to the milk given the test animals, thus to counter-
balance the interfering action of milk per se. The A.O.A.C. method for
estimating vitamin D intended for poultry use is a 21-day prophylactic
technique with baby chicks, observations being made on the percentage of
ash in the tibiae. These are all pass-or-fail tests, useful in quality control,
but not otherwise very informative as to the amount of vitamin present.
To gain quantitative information, one must either repeat the assays at
numerous levels or, better, interpret the findings by means of response
curves.

To illustrate the use of response curves, two are shown in Fig. 17. Let us
consider the cod liver oil curve, bearing in mind that it portrays the typical,
or ideal, response of chicks to graded doses of vitamin D under an established
set of conditions. (With a different basal diet, or with chicks of different age
or different breed, the curve would be different.) Under the prescribed con-
ditions, 10 I.U. of cod liver oil vitamin D will produce, on the average, a
bone ash of 43 %. Now suppose that an assay is conducted with two groups
of chicks. The reference group is given 10 I.U. of vitamin D per 100 g. of
diet in the form of a reference cod liver oil (the curve was made when cod

2 C. H. Bills, Biol. Symposia 12, 409 (1947).

23 J. Waddell and G. H. Kennedy, Biol. Symposia 12, 435 (1947).

24K. H. Coward, The Biological Standardization of the Vitamins, 2nd ed. Williams
and Wilkins, Baltimore, 1947.

25 Methods of Analysis, 7th ed. Association of Official Agricultural Chemists, Wash-
ington, 1950.

IV. ESTIMATION 219

liver oil was the official standard of reference, but vitamin Ds; gives re-
sponses not very different). At the end of the assay the reference group

1a is aseRe eee beet af SESE SeeeR gees ceed deans eee eeebae ae
saa = ages GRRenueneuGs +
Ht : ot SERED (ae ceeae as
48a SSESGGEEEEEE HH Cod liver oil
tS SEH EECA EEE EEE FECEEEEEEE EEE
+3 TAG r a =e .250 Ht Ts pomunensst 5 0 EB
47k a s/aeuune 5 ; oe ‘ Irradiated ergosterol °
3 seen
3 Fl peueaeses HH
46 is +
tf : Ff SetCGsshieettasttiE
: 4
| : H t - : t oh Th ceesue -
45 1 + 3 ~ + ~ t : : H wo
seats f HEHEHE f HF + seas
44} + t
43
42 A
41
t
40 +
+ : Ht
qe
39 te
38
37 Hed :
+
SoEBE = I. U. of vitamin D per 100 gm. of diet —
He EEE soueee Upper numbers for cod liver oil
36 7 aan Lower numbers for irradiated ergosterol
Is SUGGS GSG008 00000 8GcRqs0eGueaI ! SUUSUROEGUSEUSESUSSuSSc reso” SECcace
v0 10 20 30 40 50 60 70 80 90 100

9 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000

Fic. 17. Response of chicks to graded doses of cod liver oil and irradiated ergos-
terol assayed with rats, showing that efficacy ratios vary with the dose. (After Mas-
sengale and Bills.?°)

shows a bone ash of, say, 42.3 %. The assay group has been given an unknown
cod liver oil, the amount by weight being the same as that of the reference
oil. This group shows a bone ash of, say, 44 %. On the pass-or-fail basis, and
without reference to the curve, it can be said that the unknown oil contains

220 VITAMIN D GROUP

more vitamin D than the reference oil. But how much more? By the curve,
it is seen that 44% ash corresponds to 11.6 I.U. However, the reference
group in this assay showed only 42.3% ash, which corresponds to 9 I.U.
Apparently, then, the chicks, for some unknown reason, are responding a
little low, and a correction is called for: X:11.6::10:9, or X = 12.9. Thus
the unknown oil is found to be supplying 12.9 I.U. of vitamin D per 100
g. of the diet.

The example given above is a practical, everyday illustration of the use
of response curves, and it introduces the important subject of error. For a
discussion of error, the reader is referred to the paper by Massengale and
Bills?° from which Fig. 17 was taken, and especially to the more professional
discussions of statistical methods given by Coward™ and Bliss.?” Sugges-
tions on means of minimizing error are given in the technical articles
previously mentioned,22:”* and the chapter by Guerrant in the second
volume of Gyérgy’s book.!

In 1951 a method for the determination of vitamin D with radiophos-
phorus was described by Snyder e¢ al.28 Applied as yet only to the rat, it
would appear to be adaptable also to the chick. This method is the fifth
satisfactory means of measuring the response of animals to vitamin D, the
others being the line test, x-ray, bone ash, and growth methods. The new
method is claimed to give results comparable in accuracy to the other
methods, and it has the advantages of requiring distinctly less time and
labor, and covering an extraordinarily wide span of units (0.5 to 50 units)
in one test.

The procedure is as follows. Young (21-day) rats are placed on ricketo-
genic diet, and, after 16 days, are given by mouth a single dose of the test
solution of vitamin D. Forty-eight hours later a solution containing ap-
proximately 20 ue. of P*? is injected intraperitoneally. After another 10 days
without further treatment, the forepaws of the still-living rats are located
beneath the window of a Geiger counter. The observed radiation count is
interpreted by means of a response curve established from points at the
upper and lower limits of linearity at the time of the assay.

In the course of twelve series of experiments over a year it was noted
that the slope of the response curves varied considerably. This is another
way of saying that the response of animals, or even of groups of animals,
is variable, as is well known. It is because of this variability that it is

26Q. N. Massengale and C. E. Bills, J. Nutrition 12, 429 (1936).

27 C. I. Bliss, in Vitamin Methods, Vol. 2, p. 445. Academic Press, New York,
1951.

28 R. H. Snyder, H. J. Eisner, and H. Steenbock, J. Nutrition 45, 305 (1951).

V. STANDARDIZATION OF ACTIVITY Papal

necessary to check response curve interpretations by the use of reference
(positive control) groups, as described on the preceding page.

V. Standardization of Activity
CHARLES E. BILLS

The present international standard for vitamin D, adopted by the World
Health Organization! in 1949, is pure vitamin D;. The international unit
(1.U.) is defined as the vitamin D activity of 0.025 y of the standard. The
U.S. Pharmacopeial unit is also now defined in terms of vitamin Ds; and is
identical with the international unit. The former A.O.A.C. chick unit is
now replaced by the international chick unit, which also represents 0.025
y of vitamin D;. Four of the A.O.A.C. units equal three international chick
units. A review of the development of the international unit is given by
Coward,” and a brief account of the recent changes is given by Nelson.*

The original international unit of vitamin D was defined* ‘“‘as the vita-
min D activity of 1 mg. of the international standard solution of irradiated
ergosterol.” Parenthetically, and not as a part of the definition, it was noted
that ‘the international standard solution has been prepared to have such
potency that approximately 1 mg. thereof given daily to a rachitic rat for
eight successive days will produce a wide line of calcium deposits in the
metaphysis of the proximal ends of the tibiae and of the distal ends of the
* pao,

The original international standard solution was an olive oil solution of
ergosterol irradiation products, prepared under defined conditions and is-
sued from the National Institute for Medical Research, London. Solutions
of similar composition and potency had served, since 1927, as the Pharma-
ceutical Society’s standard, and, since 1930, as the official standard of the
Medical Research Council of Great Britain.?: > At the second international
conference on vitamin standardization, 1934, the definition of the unit was
broadened to read, ‘‘the Vitamin D activity of 1 milligram of the interna-
tional standard solution of irradiated ergosterol, which has been found equal
to that of 0.025 microgram of crystalline vitamin D.” Provision was made

1 World Health Organization: Expert Committee on Biological Standardization,
Chronicle World Health Org. 3, 147 (1949).

2K. H. Coward, J. Pharm. Pharmacol. 1, 737 (1949).

3. M. Nelson, J. Assoc. Offic. Agr. Chemists 32, 801 (1949).

4 Report of the Conference on Vitamin Standards, No. C.H. 1055 (1). League of
Nations, Geneva, 1931.

65K. H. Coward, F. J. Dyer, and B. G. E. Morgan, Analyst 57, 368 (1932).

222, VITAMIN D GROUP.

for future lots of the international standard solution to be made from crys-
talline vitamin Dz, of specified physical constants.® The 1949 decision to use
crystalline vitamin D;, rather than D2, was prompted by the knowledge
that D3, which exhibits approximately the same potency as Dz for the rat,
has the additional feature of being also effective for chicks.

The U.S.P. unit of vitamin D, officially adopted’ in 1934, was ‘‘equal, in
antirachitic potency for the rat, to one International Unit of Vitamin D as
defined and adopted by the Conference of (sic) Vitamin Standards of the
Permanent Commission on Biological Standardisation of the League of
Nations in June of 1931.’ An official reference cod liver oil of declared
potency was issued, and this remained the standard of reference in the

TABLE IX

VITAMIN D ContEntT oF AVERAGE Cop Liver O1t IN TERMS OF VARIOUS
Systems or Units. (ADAPTED FROM BILLs?®)

Unit system Potency
International, since 1931 (see text) | 100 units/g.
U.S.P., since 1934 (see text) 100 units/g.
Medical Research Council, 1930? 1° 100 units/c.c.
Steenbock, 1930"! 37 units/g.
American Drug Manufacturers’ Association, 1931!” 350 units/g.
Oslo, 1928 (Poulsson and Lévenskiold!%) 110 units/g.*
Oslo, 1933 (Poulsson and Ender") 160 units/g.¢
German, 1929 (rat unit)? 15 units/e.c.
German (clinical unit) 15 0.15 unit/e.c.
American Medical Association, 193116 2.8 ‘““D” potency

“ Vaguely defined.

United States until the new vitamin D reference standard of vitamin D3 in
oil became official in 1950.8

6 Quart. Bull. Health Organization League Nations 8, extract 15 (1934).

7U.S. Pharmacopeia, 10th revision, Interim Revision Announcement ud, 2, 1934.

8 U.S. Pharmacopeia, 14th revision, Easton, 1950.

°C. E. Bills, Physiol. Revs. 15, 1 (1935).

10 Medical Research Council, Lancet II, 503 (1930).

11H. Steenbock, EK. B. Hart, F. Hanning, and G. C. Humphrey, J. Biol. Chem. 88,
197 (1930).

2 A.D. Holmes, J. Am. Pharm. Assoc. 20, 588 (1981).

13H}. Poulsson and H. Lévenskiold, Biochem. J. 22, 135 (1928).

14. Poulsson and E. Ender, Skand. Arch. Physiol. 66, 92 (1933).

16 A. Scheunert and M. Schieblich, Biochem. Z. 209, 290 (1929); F. Holtz and E.
Schreiber, Hoppe-Seyler’s Z. physiol. Chem. 191, 1 (1930); F. Holtz, F. Laquer, H.
Kreitmair, and T. Moll, Biochem. Z. 237, 247 (1931).

16 New and Nonofficial Remedies, p. 414. Council on Pharmacy and Chemistry,
American Medical Association, 1931.

VI. EFFECTS OF DEFICIENCY Poa

The older literature contains many references to assays reported in terms
of vitamin D units no longer in use. The values of the various systems are
compared in Table IX.

VI. Effects of Deficiency
A. IN ANIMALS

JAMES H. JONES

The over-all effect of a deficiency of vitamin D in the higher animals is
known as rickets. According to Park,! Jost and Koch have stated that
rickets is a common disturbance among puppies, pigs, lambs, and kids but
occurs less frequently among colts, calves, and rabbits. However, from at
least four different Agricultural Experiment Stations in this country have
come reports of either spontaneous or experimental rickets in calves,?-> and
poultry should also be added to the above list. Rodents, such as the rat,
are not so susceptible to rickets as the higher mammals and poultry. Rickets
in this type of animal can be produced only if the diet is abnormal with
respect to calcium or phosphorus as well as deficient in vitamin D.

McCollum et al.,° Sherman and Pappenheimer,’ and Steenbock and
Black® have developed diets which will produce rickets in rats. All these
_ diets are composed principally of cereals and are high in calcium and
moderately low in phosphorus. Part of the phosphorus in these diets is in
the form of phytic acid, and the high calcium increases the unavailability
of this form of phosphorus.’ The above diets are all of the low phosphorus
—high calcium type, but if the phosphorus is sufficiently low, rickets in rats
can be produced without increasing the calcium to unusually high levels.!°

The low phosphorus type of rickets in rats simulates rickets in humans,

1. A. Park, Physiol. Revs. 3, 106 (19238).

2§. I. Bechdel, K. G. Landsburg, and O. J. Hill, Penna. Agr. Expt. Sta. Bull. 291

1933).

3 ‘5 W. Rupel, G. Bohstedt, and E. B. Hart, Wis. Agr. Expt. Sta. Research Bull.

115 (1933).

4H. E. Bechtel, E. T. Hallman, C. F. Huffman, and C. W. Duncan, Mich. Agr.

Expt. Sta. Tech. Bull. 150 (1936).

5 J. W. Hibbs, W. E. Krauss, C. F. Monroe, and W. D. Pounden, Bimonthly Bull.

Ohio Expt. Sta. 30, No. 232 (1945).

6 E.V. McCollum, N. Simmonds, P. G. Shipley, and E. A. Park, Bull. Johns Hopkins

Hosp. 33, 31 (1922).

7H. C. Sherman and A. M. Pappenheimer, J. Exptl. Med. 34, 189 (1921).

8H. Steenbock and A. Black, J. Biol. Chem. 64, 263 (1925).
| 9H.M. Bruce and R. K. Callow, Biochem. J. 28, 517 (1934).

10 J. H. Jones, J. Nutrition 28, 7 (1944).

224 VITAMIN D GROUP

but the low calcium type of rickets with either moderate or low levels of
phosphorus also has been studied in the rat.1' Experimental rickets has also
been produced in mice,!? hamsters,! foxes,’ and sheep.'® In the last two
cases the diets contained ample calcium and phosphorus.

One of the first investigators to study experimental rickets in animals
was Findlay,!® who worked with pups. Mellanby likewise used pups in his
early work on rickets.” From this work he concluded that rickets was due
to a deficiency of a specific dietary factor. These results aroused considerable
interest and led to a very large number of investigations in this field during
the following decade.

The signs of rickets in animals are not particularly different from those
in humans, and those of spontaneous rickets are essentially the same as in
experimental rickets. In the latter case, however, the condition may be
allowed to go to a more advanced state than in spontaneous rickets. Below
are discussed the various signs of rickets in animals. Most of the cases
discussed are experimental in type.

1. EXTERNAL APPEARANCE

Numerous investigators have described (either by photographs or words)
the outward signs of rickets in various animals. The signs in the calf have
been described very well by Bechtel et al.‘ as follows: “‘...the skeletal
changes included bowing of the forelegs either forward or to the side,
swelling of the knee and hock joints, straightening of the pasterns, occa-
sional ring-like swellings on the pasterns, and humping of the back. Poste-
rior paralysis occurred in cases of fractured vertebrae. Fractured femora
sometimes occurred. Other symptoms frequently observed were stiffness
of gait, dragging of the rear feet, standing with the rear legs crossed, ir-
ritability, tetany, rapid respiration, bloat, anorexia for grain and roughages
but not for milk, weakness and inability to stand for any length of time,
and finally the retardation or complete cessation of growth in body weight.”

Similar signs are seen in other types of animals but with some variations,
depending on the anatomy of the animal and on the severity of the disease.
Bowing of the forelegs, enlargement of the hock and the knee joints, and a
tendency to drag the hind legs are very characteristic of all animals suffering
from severe rickets. Another common sign is the enlargement of the costo-
chondral junctions or beading of the ribs. Deformities of the thorax are

1A. T. Shohl, J. Nutrition 11, 275 (19386).
2 C. Foster, J. H. Jones, W. Henle, and S. A. Brenner, J. Infectious Diseases 85,

173 (1949).

133 J. H. Jones, J. Nutrition 30, 143 (1945).
14 7,. EK. Harris, C. F. Bassett, and C. F. Wilke, J. Nutrition 48, 153 (1951).
15 J, Duckworth, W. Godden, and W. Thomson, J. Agr. Sci. 33, 190 (1943).

16],, Findlay, Brit. Med. J. II, 13 (1908).
17K. Mellanby, Lancet I, 407 (1919).

VI. EFFECTS OF DEFICIENCY 2a9

also common if the disease is in an advanced state. In the rat, enlargement
of the carpal joint appears to be rather characteristic.

2. GROWTH

As mentioned above, Bechtel et al.4 found decreased growth in ricketic
calves, and Steenbock and Black'® used the increase in weight of rats over
the ricketic controls as a criterion for the activity of antiricketic substances.
Hart et al.!® have demonstrated the marked effect of sunlight on the growth
of chicks on a synthetic diet which contained ample calcium and phosphorus
but was low in vitamin D. The vitamin A was supplied by fresh ground
clover. One group was given the basal ration without sunlight, and the
other received the same diet but was exposed to summer sunlight one-half
hour each day. At the end of 6 weeks on the experimental diets the two
remaining ricketic fowls weighed 80 and 90 g., respectively, and the two
controls weighed 145 and 180 g. The authors present radiographs of one
bird from each diet which show a pronounced difference in the degree of
calcification, especially of the long bones. Evidence that the lack of vitamin
D also markedly inhibits the growth of pups has been presented by Steen-
bock et al.2° Steenbock has repeatedly emphasized the essential requirement
of vitamin D for growth.

The relation of rickets to growth has also been discussed by Rosenberg.?!
He believes that this effect is of primary significance and not secondary
to other factors such as anorexia.

3. CALCIUM AND PHOSPHORUS OF THE SERUM

One of the most consistent changes in the composition of the blood during
rickets is a decrease in the level of inorganic phosphate, which was first
noted by Howland and Kramer”? while working with children. In Table X are
given the calcium and inorganic phosphorus in the serum of two ricketic
litter-mate pups as well as the calctum and phosphorus in the serum of three
other members of the same litter which were on the identical basal diet
but received vitamin D in addition.?° The level of these blood constituents is
given at 6 and 9 weeks after the pups were put on the experimental diet.
Both the calcium and inorganic phosphorus are considerably lower in the
serum of the ricketic than control animals. The calcium, although low, is
about the same at 6 weeks and 9 weeks on the diet, but the phosphorus is
considerably lower at the later period than it was earlier. Bechtel ef al.4
18 H. Steenbock and A. Black, J. Biol. Chem. 61, 405 (1924).

19 EK. B. Hart, H. Steenbock, 8. Lepkovsky, and J. G. Halpin, J. Biol. Chem. 58, 33

(1923-1924).

20 H. Steenbock, J. H. Jones, and E. B. Hart, J. Biol. Chem. 58, 383 (1923-1924).

21 H. R. Rosenberg, Chemistry and Physiology of the Vitamins. Interscience Pub-
lishers, New York, 1942.

22 J. Howland and B. Kramer, Am. J. Diseases Children 22, 105 (1921).

226 VITAMIN D GROUP

report that the first detectable signs of rickets in calves is a decrease in the
level of inorganic phosphorus in the serum. The same investigators also
found a low calcium content of the serum of their animals which was ac-
companied in some cases by tetany. Previously, Steenbock and associates”®
had observed tetany in ricketic pups. The same laboratory has also found
low calcium and low phosphorus in the serum of chicks?’ and low phosphorus
in swine” during rickets. In the later case, no calcium studies were reported,
but Loeffel e¢ al.2°> observed low calcium and low inorganic phosphorus in
the sera from their ricketic pigs.

When rickets is produced in rodents by means of low phosphorus diets,
the phosphorus of the serum is, as expected, considerably below normal. If

TABLE X

Errecr or Rickets IN Pups ON CALCIUM AND PHOSPHORUS IN SERUM
AND ON Femur AsH2°

Serum
Phosphorus,
Calcium, mg./100 ml. mg./100 ml. Femur ash

Dog / 6wk.on 9 wk. on 6wk.on 9 wk. on
No. Ration ration ration ration ration Grams %
35 Basal 1.99 7.99 4.96 2.23 1.69 28 .28
36 Basal 7.99 7.32 4.60 1.67 1.91 28 .86
37 Saponified cod 11.32 11.72 7.88 8.57 6.27 46.45

liver oil
38 Saponified cod 10.87 12.98 7.67 9.75 5.46 45.59

liver oil

39 Cod liver oil 12 12.25 7.07 9.00 5.75 47.61

the calcium in the diet is high,®*® the serum calcium is either normal or
slightly elevated. The rat, for instance, never develops tetany on this type
of diet.

4. Buoop PHOSPHATASE

Kay* and Bodansky and Jaffe?” have reported high serum phosphatase
(alkaline) during rickets in children, and Common’ found the same to be
true during rickets in chicks.

23 H. Steenbock, E. B. Hart, J. H. Jones, and A. Black, J. Biol. Chem. 58, 59 (1923-
1924). ;

24 H, Steenbock, E. B. Hart, and J. H. Jones, J. Biol. Chem. 61, 775 (1924).

25 W. J. Loeffel, R. R. Thalman, F. C. Olson, and F. A. Olson, Nebr. Agr. Expt. Sta.
Research Bull. 58 (1931).

26H. D. Kay, J. Biol. Chem. 89, 249 (1930).

27 A. Bodansky and H. L. Jaffe, Am. J. Diseases Children 48, 1268 (1934).

22 R. H. Common, J. Agr. Sci. 26, 492 (1936).

VI. EFFECTS OF DEFICIENCY DA

Sure et al..29 on the other hand, failed to observe a marked increase in
blood phosphatase during rickets in rats. Dikshit and Patwardhan*? not
only found no increase in the phosphatase of the blood of rats during rickets
but observed a pronounced fall as the disease progressed. Truhlar e¢ al.*!
found no change in the phosphatase content in the lung, liver, kidney, and
heart of ricketic rats.

In the above work on rats the diets were low in phosphorus and high in
calcium. Whether this accounts for the lower phosphatase in rickets in rats
as compared to the human or the chick, or whether it is a species difference,
apparently is not clear. Rupel eé al.’ have reported an increase in serum
phosphatase in ricketic calves. Here, again, the rise was not pronounced,
and the diet contained ample calcium and phosphorus.

5. X-Ray EXAMINATION

Owing to the failure to deposit calcium salts in the skeleton of ricketic
animals, the bone is less impervious to the roentgen ray. It is thus possible
to detect rickets by means of the x-ray.

Steenbock and others?’ have presented the roentgenograms of the right
rear leg of a ricketic pup and of two normal controls. The ricketic pup was
on a basal diet free from vitamin D but containing sufficient calcium and
phosphorus. The controls received the same diet, but, in addition, the
non-saponifiable fraction of cod liver oil was given to one control and whole
cod liver oil was supplied to the other. Calcification in general was consider-
ably less in the bones from the ricketic dog than in those from either of the
two controls. The patella and the condyles in the former are barely visible,
and the cortices of the tibia are thin and less opaque. The diameter of the
tibia is enlarged, and the distance between the diaphysis and the epiphysis
is increased. In the controls the line of demarcation between the diaphysis
and the epiphysis is distinct and sharp but narrow, whereas that in the
ricketic animal is indistinct and irregular.

Roentgenograms of the costochrondal junction of r ahaa calves have been
presented by Bechtel e¢ al.* Here again the junction of diaphysis and car-
tilage in the ricketic animals is irregular and indefinite and in places shows
areas of incomplete calcification.

Hart and associates! published radiographs of the complete bony struc-
ture of a ricketic chick and of a normal control. In the ricketic animal there
was very little differentiation between cortex and marrow cavity, and the
whole skeleton was almost devoid of any dense bone.

29 B. Sure, M. C. Kik, and K. 8. Buchanan, Proc. Soc. Exptl. Biol. Med. 35, 209
(1936-1937).

80 P. K. Dikshit and V. N. Patwardhan, Indian J. Med. Research 35, 91 (1947).

1 J. Truhlar, L. Drekter, G. MeGuire, and K. G. Falk, J. Biol. Chem. 127, 345 (1939).

228 VITAMIN D GROUP

Pappenheimer ef al.*? have presented roentgenograms of the rear leg of a
ricketic rat which shows the same lack of calcification and the wide area
of uncalcified cartilage at the junctions of the diaphyses and epiphyses.

6. CALCIUM AND PHOSPHORUS BALANCE

Some of the early work on the influence of the antiricketic factor on the
excretion or retention of calcium and phosphorus was reported in a series
of publications by Hart, Steenbock, and their associates.** They found that
lactating cows and goats on a diet of grains and timothy hay had a pro-
nounced negative calcium balance, and frequently the balance of phos-
phorus was also negative. It was possible to bring the milking goats into
a positive calcium and phosphorus balance by giving alfalfa hay or cod
liver oil or by irradiating the animals with ultraviolet ight. More difficulty
was encountered when an attempt was made to bring about a retention of
calcium and phosphorus in the lactating cow. The best results were ob-
tained with alfalfa hay, and the green plant was better than the dried hay.
The giving of as much as 10 ml. of cod liver oil per day caused the cows to
lose their appetite.

Nicolaysen* found that vitamin D decreased the amount of calcium ex-
creted in the feces of rats and increased the excretion in the urine. The ab-
sorption of calcium from an isolated intestinal loop was also augmented by
vitamin D.

Bergeim*®® has made a more detailed study of the absorption of calctum
and phosphorus from the intestinal tract. He found that in the normal
animal calcium is absorbed in the upper part of the intestines and a portion
of it is re-excreted in the lower part of the tract. In rickets the absorption
is normal, but a larger proportion is re-excreted.

Phosphorus, on the other hand, is excreted into the upper portion of the
intestine and reabsorbed from the lower part. In rickets this reabsorption
is below normal. The administration of cod liver oil resulted in a positive
balance of calcium and phosphorus. Cohen and Greenberg,*® also with the
rat, found only a slight increase in the intestinal absorption of radioactive
phosphorus, although there was a marked improvement in the deposition
of phosphorus in the bone when vitamin D was administered.

In contrast to phosphorus, Greenberg” found a definite increase in the

32 A. M. Pappenheimer, G. F. McCann, and T. F. Zucker, J. Exptl. Med. 35, 421
(1922).

33 &. B. Hart, H. Steenbock, E. C. Teut, and G. C. Humphrey, J. Biol. Chem. 84,
367 (1929).

34 R. Nicolaysen, Biochem. J. 31, 122, 323 (1937).

35. Bergeim, J. Biol. Chem. 70, 51 (1926).

36 W. E. Cohn and D. M. Greenberg, J. Biol. Chem. 130, 625 (1939).

37 ID. M. Greenberg, J. Biol. Chem. 157, 99 (1945).

VI. EFFECTS OF DEFICIENCY 229

intestinal absorption of radioactive calcium following the administration of
vitamin D to ricketic rats. Shohl and Bennett** observed a decrease in
calcium retention, but at no time did the balance become negative in
ricketic pups. Phosphorus was still less positive in respect to the control
animals than was the calcium, and in some cases the phosphorus balance
was negative. The authors state that even on a diet high in phosphorus and
low in calcium the most marked deficiency lies in the phosphorus retention.

7. Actpiry oF INTESTINAL CONTENTS

Zucker and Matzner*® were the first to show that the pH of the feces of
rats becomes higher as the animals develop rickets, and falls again when
cod liver oil is fed. Similar results have been obtained with rats by Jephcott
and Bacharach*® and Heller and Caskey.*!

Abrahamson and Miller,?? Yoder, and Redman et al.“* observed a higher
pH of the intestinal contents of ricketic rats than in non-ricketic controls.
This difference appeared to be constant through the small and large in-
testines. When cod liver oil was given to the ricketic animals the hydrogen
ion concentration of the intestinal contents increased. Grayzel and Miller
have made similar observations on the dog, and Kline and associates*® have
shown that irradiation of ricketic chicks decreases the pH of the proximal
part of the intestines but not of the distal portion.

The significance of this change in acidity of feces and intestinal contents
and its relation to the cure of rickets is not clear at the present. According
_to Shohl and Bing,” the change in acidity of feces did not occur when rats,
made ricketic on the Steenbock-Black diet, were cured by irradiation of the
food or by the addition of alkaline phosphates. Oser*® reports that the
effect of vitamin D on increasing the acidity of the intestinal contents and
feces of rats is inconsistent and non-specific, and Jones*® found that increas-
ing the acidity of the intestinal contents by other means did not cause a

88 A. T. Shohl and H. B. Bennett, J. Biol. Chem. 76, 633 (1928).

39'T. F. Zucker and M. J. Matzner, Proc. Soc. Exptl. Biol. Med. 21, 186 (1923-1924).

40H. Jepheott and A. L. Bacharach, Biochem. J. 20, 1351 (1926).

41V. G. Heller and C. Caskey, J. Nutrition 2, 59 (1929-1930).

42. M. Abrahamson and E.G .Miller, Jr., Proc. Soc. Exptl. Biol. Med. 22, 438 (1924—
1925).

43 J. Yoder, J. Biol. Chem. 74, 321 (1927).

44°T. Redman, 8. G. Willimott, and F. Wokes, Biochem. J. 21, 589 (1927).

45D. M. Grayzel and E. G. Miller, Jr., Proc. Soc. Exptl. Biol. Med. 24, 668 (1926-
1927).

46Q. L. Kline, J. A. Keenan, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 98,
121 (1932).

47 A. T. Shohl and F. C. Bing, J. Biol. Chem. 79, 269 (1928).

48 B. L. Oser, J. Biol. Chem. 80, 487 (1928).

49 J. H. Jones, J. Biol. Chem. 142, 557 (1942).

230 VITAMIN D GROUP

comparable increase in calcification. According to Friedman,*® the increase
in acidity during the healing of rickets is caused by a change in bacterial
flora of the intestinal tract, from non-acid-forming bacteria to acid formers.

8. Bone ASH

In his early work on rickets in pups Mellanby"” observed a decrease in
the calcium content of the skeleton of his animals. 'Telfer®! found a very low
bone ash in his ricketic pups. The percentage of ash in the dry limb bones
decreased from 44.9 in the normal controls to 17.7 in the deficient animals.
Steenbock and associates?’ made comparable observations. Table X gives
the percentages of ash in the dry, fat-free femurs of their ricketic and control
pups. The decrease in ash content as reported by Steenbock was somewhat
less than that given by Telfer.

McCollum and associates*? found a low ash in the bone of ricketic rats.
Bethke et al.>* as well as Dutcher et al.** have made detailed studies of the
changes of the bone ash of ricketic rats and correlated the percentages of
bone ash with the level of serum phosphorus. Dutcher and coworkers report
a bone ash of 62 % in the dry, fat-free bones from normal rats; in rickets it
fell as low as 24 % with an average percentage of 26.5.

A reduction in the percentage of ash in long bones or ricketic pigs has
been reported by Elliot e¢ al.*> and Loeffel e¢ al.?° In a like manner, there is
less ash in the bones of ricketic calves than in the normal controls,” * °
and the same is true for ricketic fowls.” The official method for determining
vitamin D in poultry feed*’ is based on the increase in the ash of bones of
chicks when vitamin D is supplied to animals previously made ricketic.
According to Chick et al.,° the best criterion of defective calcification is
given by the value of the ratio of the amount of ash to the amount of
organic material contained in the fat-extracted bone. This is essentially the
same as the percentage of ash in the dry, fat-free bone which has been used
more frequently.

50H. Friedman, J. Nutrition 12, 165 (1936).

5119. V. Telfer, Quart. J. Med. 16, 63 (1922-1923).

52 B®. V. McCollum, N. Simmonds, HE. M. Kinney, and C. J. Grieves, Bull. Johns
Hopkins Hosp. 33, 202 (1922).

53 R. M. Bethke, H. Steenbock, and M. T. Nelson, J. Biol. Chem. 58, 71 (1923-1924).

54R. A. Dutcher, M. Creighton, and H. A. Rothrock, J. Biol. Chem. 66, 401 (1925).

55 W. E. Elliot, A. Crichton, and J. B. Orr, Brit. J. Exptl. Pathol. 3, 10 (1922).

56 CO. F. Huffman and C. W. Dunean, J. Dairy Sci. 18, 511 (1935).

57 Official Methods of Analysis, 7th ed., p. 792. Association of Official Agricultural
Chemists, 1950.

68 H. Chick, V. Korenchevsky, and M. H. Roscoe, Biochem. J. 20, 622 (1926).

VI. EFFECTS OF DEFICIENCY 2Zo1

9. HISTOLOGICAL CHANGES IN THE RICKETIC BONE

The following description of the microscopic changes of the costochondral
junction and adjoining areas of the ricketic rat is condensed from the dis-
cussion by Pappenheimer.*®

The zone of proliferating cartilage does not differ greatly from the normal
in extent or in arrangement of its cells. It is difficult, however, to define the
boundaries of this zone, owing to the lack of calcification in severely ricketic
bones. When there is lateral swelling of the cartilage the columns of cells
are separated by an excessive amount of matrix.

In the zone of preparatory calcification are found the most pronounced
changes. There is a complete lack of calcification, and the depth of this
zone is greatly increased. In the normal rib there is little variation in the
depth, and it seldom exceeds four or five cells, whereas in the ricketic rib
the depth may be fifty cells or more. The extent to which this zone is
enlarged depends upon the length of time that the animals have been on the
ricketogenic diet and the extent of growth. If no growth has taken place
during the time on the experimental diet, there is but little enlargement of
this zone. In general, the more the animal grows, the greater the depth of
this zone, but usually this enlargement is one of the most dependable char-
acteristics of rickets. It has been frequently observed that ricketic lesions
are more severe in animals that do show definite growth during the period
on the ricketogenic diet. This is true in spite of the fact that vitamin D. is
necessary for continued growth, as previously discussed. The arrangement
~ of the cartilage cells in the columns is usually maintained in the basal
portion of the zone of preparatory calcification, but toward the diaphysis
this arrangement of the cells is entirely lost, and there is considerable
variation in the size and shape of the cells. The uncalcified matrix takes on
the appearance of osteoid tissue which forms a considerable portion of the
metaphysis, particularly surrounding the perforating vessels.

After 4 weeks on the ricketogenic diet there is formed an excessive amount
of calcium-free osteoid tissue in the region of the primary spongiosa. The
arrangement of the trabeculae is no longer orderly, but instead they are
broad, convoluted masses of osteoid several times thicker than normal
trabeculae. Their relation to the original trabeculae is completely obscured.
Some of the osteoid masses contain a core of calcified tissue, but many
others show no trace of calcification. The osteoid is usually homogeneous
and stains evenly and deeply with eosin. The demarcation between osteoid
and calcified tissue is always very sharp.

The perichondral osteoid of the ricketic rib is entirely free from calcifica-

59 A. M. Pappenheimer, J. Exptl. Med. 36, 335 (1922).

Zon VITAMIN D GROUP

tion, and it forms a large mass which contributes largely to the swelling of
the junction.

Both the endosteal and periosteal surfaces of the calcified cortex are
covered by osteoid tissue. It is most extreme near the epiphysis but extends
the entire length of the shaft. At times the osteoid may be so abundant as
to decrease the diameter of the marrow cavity. The calcified portion of the
cortex is considerably reduced in thickness, and in places calcium salts
appear to be entirely lacking.

The blood vessels are not dilated, and hyperemia is not pronounced.

Bechtel et al. have given a detailed description of the microscopic changes
in the bones of ricketic calves which for the most part agrees with the find-
ings of Pappenheimer.

B. IN PATHOLOGY OF HUMAN BEINGS
BENJAMIN KRAMER and ABRAM KANOF

The pathological changes in human beings resulting from deficiency of
vitamin D are almost entirely confined to the skeleton. Here there develops
a distortion of bone growth which gives rise to the clinical picture which we
designate as rickets. The primary disturbance responsible for this distor-
tion is a failure to mineralize newly formed osteoid tissue and cartilage
matrix. Hence, the unusual softness of the bone which under the stress and
strain of weight bearing and locomotion gives rise to the characteristic
deformities of the disease. To understand these changes it is important to
review the process of normal bone development and growth.®: *

From the point of view of embryologic development there are two types
of bone. The first is membranous bone, occurring in the vault of the skull,
the lower jawbone, and part of the clavicle. The first step in the formation
of these bones in the blastoderm is a condensation of the mesodermal cells
which soon develop into fibrous membrane. Between the cells of this mem-
brane a dense intercellular substance accumulates, and when the proper
stage of development of this substance is reached calcium is deposited into
it. The property of calcifiability is conferred upon the transformed connec-
tive tissue by the osteoblasts. Osteoblasts arise in the early embryo by
direct transformation from mesenchymal cells. In the adult they arise from
fibroblasts and reticular cells. It is through the activity of these osteoblasts
on the fibrous sheath that the bone attains increasing thickness, as addi-
tional layers of periosteum are laid down and ossified without the inter-

60 A. Robinson, Cunningham’s Textbook in Anatomy. William Wood and Co., New
York, 1923.

61 A. A. Maximow and W. Bloom, A Textbook of Hist: logy. W. B. Saunders and Co.,
Philadelphia, 1948.

VI. EFFECTS OF DEFICIENCY sys

mediate formation of cartilage.®? What is originally membrane thus becomes
cortex of the new bone.

The second type of bone, which includes all the bones not listed above, is
known as cartilage bone. In this type of bone, growth takes place with the
intermediation first of cartilage formation and then destruction of the
cartilage before bone can be formed. In the prenatal formation of these
bones there is first an aggregation or condensation of mesodermal] cells at
the location of the future bone. This aggregation of cells becomes demar-
cated from the surrounding mesoderm and forms a rudimentary model of
the future bone. In the area of the future shaft the peripheral cells become
fibroblasts and form the periosteum, while at the ends of the future bone
the cells form the periochondrium.

In the region of the shaft, where the membranous periosteum has formed,
bone formation proceeds in the manner described for membranous bone.
Elongation of the bone takes place at its ends, where the condensation of
mesenchymal cells has resulted in the formation of a cartilaginous plate
which during childhood remains distinct from the shaft, and is called the
epiphysis. The flattened cells in this cartilaginous plate are normally ar-
ranged in columns. Each cell in the column is separated from its fellow by
a thin bridge of matrix, while the adjacent columns are separated by wider,
parallel bands of matrix material. The epiphysis is attached to the diaphysis
by a number of calcified prongs remaining from those destroyed which form
a bridge between epiphysis and diaphysis.

As one proceeds from the relatively quiescent epiphysis toward the zone
of ossification, the appearance of the individual cells changes. The rows
of cells most distant from the shaft are composed of cells hardly differenti-
able from the ordinary cartilage, and they form the zone of resting cartilage.
As we proceed in the direction of the shaft or diaphysis toward the second
layer, the cells begin to show evidence of degeneration. The mitochondria
appear rod-like, and later shrink; vacuoles containing fat and glycogen
appear, and the cell nucleus shrinks. This zone, the zone of proliferating
cartilage, varies in depth, depending on the rate of bone growth. In the
third layer, the zone of preparatory calcification, the process of cell disinte-
gration is complete. Here we find clumps of degenerated cells, or only
lacunae from which the cells have disappeared. At the same time, if there
are adequate concentrations of calcium and phosphate in the blood plasma,
the matrix around the cells undergoes a notable change. There is a precipi-
tation of calcium salts which results in the matrix walls’ becoming calcified.
Immediately to the shaft side of this area of preparatory calcification, the
final stage of actual ossification takes place. This process is due to the
activity of the osteoblasts and blood vessels of the bone marrow. The cells

62. A. Park, Personal communication.

234 VITAMIN D GROUP

known as osteoblasts are fibroblast derivatives which possess the double
function of bone formation and bone destruction. Although it is in both
instances the same cell, the term osteoblast is applied to those cells which
are producing bone, and the term osteoclast is applied to those in the process
of bone resorption. As death of cartilaginous cells proceeds, there is an
invasion of the matrix by capillaries proceeding upward from the diaphysis.
These invade the empty lacunae. Some of the matrix trabeculae are resorbed
by activity of the osteoblasts, giving rise to an increasing size of the marrow
space and a reduction in the number but an increase in size of the trabecu-
lae. Into the enlarged lacunae the blood vessels grow, continuously carrying
with them connective tissue, osteoblasts, and osteoclasts. Upon the larger
trabeculae the osteoblasts lay down true bone. Between the osteoblasts and
the cartilage matrix, a new layer of tissue appears, gradually thickens, and
surrounds the contours of the cartilage projections. It is this tissue which
under favorable conditions begins to calcify as it is deposited and thus
becomes bone. Physiologically, there is a lag in calcification, resulting in
formation of an uncalcified osseous material known as osteoid. This appears
in a limited degree under physiological conditions but becomes enlarged
when there is local failure to supply calcium and phosphate. When this
failure is marked and generalized, there is an increase in the width of the
osteoid border. This is a picture typical of diaphyseal rickets and of oste-
omalacia. By this complex process (bone formation and bone resorption)
the bones become more hollow because of the increase in size of the marrow
spaces, and stronger because of the widening and ossification of the trabecu-
lae and the increased strengthening of the cortex.

Although the morphological changes associated with endochondral bone
formation have been carefully studied and adequately described, the chem-
ical changes related to this process are not so well understood. Mineraliza-
tion of bone matrix and osteoid consists of the deposition of calcium and
phosphate, plus, to a lesser degree, carbonates, fluorides, and perhaps other
anions combined with small amounts of calcium, sodium, magnesium, and
potassium. The nature of the mineral deposit has been the subject of much
investigation and controversy. The most acceptable concept has been that
the mineral matter of bone has a crystalline structure resembling that of
the apatite minerals, and that dissolved in this, perhaps as an adsorbate
or as a solid solution, are the calcium salts hydroxide, fluoride, carbonate,
etc. It seems clear that an adequate concentration of calcium and inorganic
phosphorus both in the bone matrix and the tissue fluid is essential for this
process, but the exact level at which calcification occurs is as yet poorly
defined, and the mechanism itself is not clear.

Studies on calcification of endochondral cartilage 7m vitro have added
much to our knowledge of this subject. Inhibition of this process may occur

VI. EFFECTS OF DEFICIENCY 235

in a variety of ways, and the explanation for such inhibition has been that
the inhibiting agent in some way affects an enzymic process, the ultimate
aim of which is the liberation of inorganic phosphorus from phosphoric
acid esters. The source of this inorganic phosphorus has been a stumbling
block in any theory of calcification. Shipley et al. produced in vitro calci-
fication with artificial serum ultrafiltrates containing an adequate concen-
tration of both calcium and inorganic phosphorus. Robison® demonstrated
the presence of alkaline phosphatase in the hypertrophic cartilage cells and
produced calcification im vitro using solution of calcium and hexosephos-
phoric acid. The latter was hydrolyzed by alkaline phosphatase, thus
liberating inorganic phosphorus. However, Shipley et al. pointed out that
plasma and presumably tissue fluid contain only traces of organically bound
phosphorus. Furthermore, preparatory cartilage cells contain only minute
amounts of organic phosphorus although rich in glycogen and phosphoryl-
ase. The problem of finding a source of organic phosphorus seemed. to
constitute an unsurmountable obstacle until Gutman®® suggested the proc-
ess of phosphylative glycogenolysis as a possible source of inorganic phos-
phorus and produced confirmatory evidence in the finding that substances
which interfered with this process inhibited 7n vitro calcification.

Glycogen, like alkaline phosphatase, may be demonstrated in the car-
tilage cells of the hypertrophic cartilage, just before and during calcifica-
tion, only to disappear as the process of calcification becomes complete.
That glycogen may play an important role in endochondral calcification is
indicated by the fact that zn vitro calcification will not take place if the
glycogen is removed with ptyaline.

The role of phosphatase in calcification has received the most intense
study.*® It is presumed to play an important role for the following reasons.

1. It is present in the preliminary stages wherever tissue is about to be
calcified, i.e., in cartilage matrix, in osteoid, and at the site of metastatic
calcification.

2. Substances that inhibit phosphatase activity inhibit 7m vztro calcifica-
tion. Some of these substances are potassium cyanide, hydrocyanic acid,
and fluoride.

3. In vitro calcification takes place in living cartilage in the presence of
phosphatase when phosphoric acid esters represent the only source of inor-
ganic phosphorus.

On the other hand, calcification 7m vitro can definitely occur in the ab-

68 P. G. Shipley, B. Kramer, and J. Howland, Biochem. J. 20, 379 (1926).

64 R. Robison, Biochem. J. 35, 304 (1924).

6° A. B. Gutman and T. F. Yu, Trans. 2nd Conf. Metabolic Interrelations p. 167,
1950.

66 R. H. Follis, Jr., Bull. Johns Hopkins Hospital 85, 360 (1949).

236 VITAMIN D GROUP

sence of phosphatase or in tissue heated to a temperature which destroys
it. Moreover, the optimal pH for phosphatase activity is much beyond the
pH of normal tissue fluids. Third, calcification 7m vitro will fail to occur
when cells are poisoned, although such substances may not affect phos-
phatase activity.

Endochondral calcification takes place in the presence of an adequate
concentration of plasma calcium and inorganic phosphorus. Deficiency of
inorganic phosphorus in rickets apparently cannot be made up by the local
action of alkaline phosphatase or phosphorylative glycogenolysis in the
hypertrophic cartilage cells under the same conditions.

Since all tissue fluids presumably have the same concentration of calcium
and inorganic phosphorus, there still remains the question of why certain
tissues can be mineralized whereas this occurs in others only under abnormal
conditions or not at all. Rubin and Howard®™ have demonstrated the pres-
ence of a mucopolysaccharide resembling chondroitin sulfate in tissues
potentially capable of undergoing calcification. This metachromic staining
material seems to form a combination with calcium and this may be the first
step in the process of calcium salt deposition. During the course of this
process the mucopolysaccharide seems to disappear and the tissue loses its
peculiar staining characteristics. This has been cited as a point against its
importance in primary calcification by Sobel,® but Rubin and Howard™
have shown that the polysaccharide does not disappear during calcification
but can be stained and its presence thus proved, if the section is decalcified
before staining.

Although much has been learned concerning the mechanism of mineral
salt deposition, little is known regarding the morphology, the chemical com-
position, including the enzyme content, and the metabolism of the organic
matter of bone, including the cartilage matrix and osteoid upon which the
calcium salts are deposited. Much has been added to our knowledge by the
use of special dyes and the ordinary light microscope, and this information
has been broadened and extended with the aid of the wide-angle diffraction
pattern and the electron microscope. The use of special dyes that react
specifically with certain constituents of the cell and the application of
enzymes that are specific cytoplasmic components afford further insight
into the functioning of osteoid and cartilage cells. Studies dealing with the
effects of vitamin C deprivation on osteoid structure and composition in
animals has thrown light on the mechanism of protein synthesis of cell
cytoplasm that may have far-reaching implications for cells in general.®

Cartilage matrix which under the ordinary light microscope and with the
67 P. S. Rubin and J. E. Howard, Trans. 2nd Conf. Metabolic Interrelations p. 155

(1950).
6 R. H. Follis, Jr., Trans. 4th Conf. Metabolic Interrelations p. 11 (1952).

VI. EFFECTS OF DEFICIENCY Oh

usual tissue fixation and staining appears quite homogeneous is found to
show a fibrillar network lying in a homogeneous matrix; this network is
made up chiefly of a protein, collagen, and the matrix itself contains a poly-
saccharide, probably chondroitic sulfuric acid. The collagen fibers show a
definite periodicity of bands in their structure with finer bands within the
periods. These collagen fibers resemble those found in skin, where the
chemical composition has been studied extensively and fascinating theories
concerning the arrangement of their chemical groups have been proposed
that may account for some of their physical properties.

The homogeneous matrix is also being studied and among other proper-
ties shows the property of metachromasia. This is present in the cartilage
matrix and seems concentrated about the hypertrophic cartilage cells; it 1s
probably due to the peculiar effect of the polysaccharides upon the specific
dye (polymerization).

The osteoblasts have been shown to contain lipids, glycogen, phospha-
tase, and lecithinase. Cytochromic oxidase has been demonstrated in the
osteoblasts in addition to ribonucleic acid and its enzyme, ribonuclease.
Less is known about the chemical composition of cartilage cells, but alkaline
phosphatase, phosphorylases, glycogen, lipoids, and lecithinase have been
demonstrated in these cells as well as some oxidases.

The reactivation of dormant osteoblasts in the scorbutic animal treated
with vitamin C has yielded valuable information concerning the mechanism
of synthesis of cell cytoplasm. Under the influence of vitamin C cell cyto-
plasm is synthesized: alkaline phosphatase reappears, protein synthesis is
accelerated, and cells become filled with ribonucleic acid and ribonuclease
and glycogen.

The electron microscope has revealed the location and orientation of
mineral crystals on the collagen fibers as well as their configuration.®

The mechanism of bone resorption remains as obscure as ever, although
the local application of parathormone can initiate and maintain such re-
sorption by stimulating osteoclastic activity. Osteoclasts can bring about
the resorption of both calcified and uncalcified cartilage matrix as well as
osteoid tissue. Just how the mineral matter is made to disappear during the
process of reabsorption of calcified trabeculae is not clear. Inorganic crystals
have been demonstrated in the osteoclasts. This is very rare, however, and
the mechanism of action of both osteoblasts as well as osteoclasts in the ab-
sorption of bone is quite obscure. Tension seems to affect enzyme activity,
a fact to bear in mind in explaining the effect of tension in molding bone.

The chief consequence of vitamin D deficiency is the disruption of the
orderly processes of bone formation which we have briefly described. In the

69 R. A. Robinson, Trans. 3rd Conf. Metabolic Interrelations pp. 271-289 (1951);
R. A. Robinson, Trans. 5th Conf. Metabolic Interrelations 1953 (In press).

238 VITAMIN D GROUP

shafts of the long bones, and in the membranous bones, there is produced
instead of endosteal and periosteal bone an excessive amount of uncalcified
bone, called osteoid. This osteoid is the framework of bone, without lime
salt addition, so that it is both soft and radiotranslucent. The amount of
this osteoid laid down varies at different points, increasing at areas of stress
and strain, but decreasing at points of tension. An example of the former,
is the excessive osteoid at the tendinous insertions into the bone, within
the angles of a fracture, and on the concave side of bone generally.

When this soft bone is subjected to various and changing stresses and
pressures such as those that occur in walking, sitting, etc., many types of
deformities result. The deformities depend on the severity and extent of
the disease process, its duration, the age of the child,’° and the stresses and
strains the bones are subject to. The age is important because it influences
other factors. The first of these is the rapidity of growth of the various
bones: the more active the growth, the greater the lability to damage.
Second, the child’s age and stage of development will determine which of
the bones are particularly subjected to strain and stress. For example, dur-
ing the first few months, the child les on its back, and the stresses on the
bones during these early months are those of gravity. During that period
also the head and chest are growing most rapidly. For these two reasons,
ricketic deformities encountered in the earliest period of life are most con-
spicuous in these two anatomic structures.

Changes in the skull are thus among the earliest manifestations of rickets.
First there is flattening of the occipital bones from the pull of gravity. The
lack of calcification in circumscribed areas of the skull (craniotabes) is due
to the failure of calcification in portions of this membranous bone. In the
areas where the bending of the bone is most pronounced, on the parietal
and frontal bones, there are accumulations of osteoid tissue which give rise
to the bossae which are typical of this disease.

Deformity of the chest and consequent physiological inadequacy of the
respiratory organs represent some of the most dramatic and serious conse-
quences of severe rickets.” In the advanced case, as the child lies in bed,
the front of the chest is seen as a blunt wedge protruding forward, with
the sternum and adjacent ends of the ribs corresponding to the prow of a
ship. The anterolateral portions of the ribs, corresponding to the costo-
chondral junctions, have sunk inward, producing the depressions running
the vertical length of the chest. The clavicles are exceedingly prominent
and bowed, giving undue prominence to the manubrium sternum. Often
there are multiple fractures of the ribs. When these fractures are anterior,
they form small mounds of callus; if posterior, they tend to eliminate the
70 M.M. Eliot and EK. A. Park, Brennemann’s Practise of Pediatrics. W. F. Prior Co.,

Hagerstown, Md., 1950.
71 E. A. Park and J. Howland, Bull. Johns Hopkins Hosp. 32, 101 (1921).

VI. EFFECTS OF DEFICIENCY 239

rounded character of the rib angle. Most important of all is what happens
at the costochondral junction. Normally, the cartilage and rib shaft are
accurately and rigidly joined end to end. In rickets, they are separated by
the soft metaphysis, which has little or no rigidity and permits of consider-
able movement during respiration. The metaphysis soon gives way to the
negative intrathoracic pressure, the ribs become more and more bent in-
ward, until finally the rib ends lie internal to the cartilage, and the enlarge-
ment of the costochondral junction is within the chest and compresses the
adjacent lung. Thus the lung is divided into an anterior emphysematous
portion and a posterior portion which is partly emphysematous and partly
atelectatic separated by a longitudinal zone of complete atelectasis.

As one watches such a child breathe, he observes that with inspiration
almost every one of these deformities becomes exaggerated. The moment
inspiration ends, the chest in early cases springs into the expiratory posi-
tion. Observation, or measurement, will reveal that the inspiration has
barely, if at all, increased the chest circumference. Moreover, it appears
that life is maintained not by the awkward and inefficient movement of
the rib cage, but by the exertions of the diaphragm. Later the elasticity of
the thoracic cage is lost and even the diaphragm is partly relaxed during
rest.

The essential difficulty physiologically is the loss of thoracic rigidity. As
this process progresses, the linear depressions which at first were present
only during inspiration tend to persist in expiration. As the efficiency of
each respiration is diminished by the chest collapse and the intrusion of
costochondral junctions and the rib ends into the chest cavity, there is an
attempt at compensation by increasing the frequency of respiration. But
the greater and more frequent the force applied, the greater is the collapse.
The diaphragm pulls its attachments further inward, and the accessory
muscles of respiration draw the bones out of position without stabilizing
the chest as a whole. As there is progressive decrease in chest capacity,
there develops increasing atelectasis, the pressure in the pulmonary circula-
tion rises, the right heart hypertrophies, and cyanosis may appear. The
dyspnoea is extreme, the respirations rising to 60 and 100 times a minute.
There may also appear dilatation of the nostrils, a grunt and a cough. If,
now, an additional burden, such as an infection, is superimposed, the mech-
anism and its compensations may fail, the vital capacity becomes equal to
or less than the tidal air, and permanent cyanosis is the result. Sudden
death may follow.

On the other hand, if the child does not die, he may improve. As the
rickets heals and lime salts are deposited in the bone, the chest regains its
rigidity. Despite the persistence of deformities, the efficiency of respiration
improves and then may gradually return to normal.

In neo-natal rickets, the pelvis becomes flattened, by virtue of the pull

240 VITAMIN D GROUP

of gravity on the soft structure. After a few months the child begins to
spend much of his time in the sitting position. The pelvis must now support
the weight of the head and trunk, and as a result of this new stress a dorsal
kyphosis appears. When the child stands up, and later when he begins to
walk, the strain on the spine changes again. Now the dorsal kyphosis
changes to a sharp lumbar lordosis, and the spine pushes forward at the
promontory of the sacrum, thus further decreasing the anterior-posterior
diameter of the pelvis. In the severest cases, the head of the femur may
push up the acetabulum, thus further encroaching upon the pelvic space.

In the period during which the child begins to sit up, various deformities
of the long bones may develop. The severely ricketic child sits cross-legged
and supports the weight of his body by extending his hands to the
table or floor. In this position the upper of the two crossed legs is bent by
gravity as it extends over the lower shin which acts as a fulerum. At the
same time the wrists against which the weight of the body rests may also
bend. This pressure against soft and rapidly growing bones results in bend-
ing deformities. Occasionally, the bending may progress to actual angula-
tion of the epiphyses which is then bent towards the diaphysis. As the
growth of the diaphysis in the original axis continues, however, the bend
is accentuated. The process of growth may carry the bend some distance
from the epiphysis so that its real nature may not be easily recognized.
Such deformities occur, particularly at the lower ends of the tibiae and
fibulae, and in the lower ends of the radius and ulna. They occur particu-
larly when the rickets is severe at about the age of 2 years, when growth
in these areas is particularly rapid. Bending deformities may also be due
to fractures which occur chiefly in the severer forms of the disease. In
addition to the actual bone involvement, there is great relaxation of the
tendons in rickets, and this looseness seems to initiate or accentuate many
of the deformities.

To summarize, curvatures of the shaft may result from bending of
softened bone, tilting and dislocation of the epiphysis, and as a result of
fractures. Posture may suffer from tendon relaxation. The typical deformi-
ties of the lower extremities which occur as a result of these forces are
bowlegs (genu varum), knock-knees (genu valgum), and saber shin de-
formity. The curvatures of the arms are much less marked than those in
the leg. Outward bowing of the humerus and exaggeration of the normal
curvatures of the radius and the ulna may be seen.

The type of rickets we have been describing is the moderately severe
type. Park has pointed out that rickets may vary in degree. Both he and
Follis have described cases in adults and in children in which the only
evidence of the disease is a slight to moderate increase in the osteoid seam
around the trabeculae. This may be limited even to one aspect or side of

VI. EFFECTS OF DEFICIENCY 241

the trabeculum, with or without minimal changes at the chondro-osseous
junction in the form of localized defects of cartilage matrix calcification.
Such cases show no change clinically or roentgenologically, and may or may
not show changes in the blood calcium or inorganic phosphorus. In contrast,
the process may be so severe that the bones are almost translucent and
difficult to differentiate on x-ray examination from the surrounding soft
parts. :
Since endochondral ossification ceases with the closure of the epiphysis,
deficiency of vitamin D in older people results only in shaft or diaphyseal
changes, and the disease characterized by this process is called osteomalacia.
Pathologically, we see only the superabundance of osteoid around the
trabeculae and a certain amount of osteoporosis. All the complications of
soft bones, which we have already described, can then occur. Severe osteo-
malacia occurs rarely in the western world except during exceptional times
of great stress. An endemic of this disease occurred in Germany during the
allied blockade in the first world war and in the subjugated countries
subjected to German genocide methods during the second world war. It is
sometimes seen following dietary restrictions either self-imposed or im-
posed as a therapeutic measure. It also occurs in India, in Japan, and in
northern China, among women, where prolonged lactation, with its drain
on the calcium reserves, may result in a negative calcium balance. It also
occurs among women of the higher classes, who practice purdah and are
therefore kept. confined indoors away from the sun.72

At the epiphyseal end of the bones where the process of endochondral
bone formation normally results in elongation of the bone, rickets mani-
fests itself by a disruption of the orderly processes which we have already
described for that area.” The progressive steps in that disruption may be
listed as follows:

1. Failure of calcium salt deposition in the cartilage matrix.

2. Failure of the cells to mature, making them impervious to invasion
and therefore leading to their accumulation rather than their destruction.

3. Compression of the proliferating cartilage cells. |

4. Elongation, swelling, and degeneration of the proliferative cartilage.

5. Abnormal pattern of invasion of the cartilage by tufts of capillaries.

There is some difference of opinion as to what is the very first change
from normal when the child is deprived of vitamin D. Park believes that
failure of calcium deposition in the matrix is the first deviation (Figs. 18
and 19). Because of this failure, the guiding influence of the calcified matrix
is absent when the invading blood vessels approach, and these vessels,
= A. F. Hess, Rickets Including Osteomalacia and Tetany. Lea and Febiger, Phila-

delphia, 1929.
73K. A. Park, Harvey Lectures 157 (1938-1939).

242 VITAMIN D GROUP

instead of taking a parallel course, are diverted and the cartilage is broken
up into uneven tongues. Park points out that in the earliest cases the his-
tologist will find only in the fastest growing bone some thinning of an
occasional spicule, and an area here and there where calcification is entirely
absent from one of the main partitions in the cartilage.”

As a result of the total or partial failure of the calcification in the matrix
substance of the cartilage, there is failure of support for the large cartilage
cells lying nearest the shaft. There results, therefore, the phenomenon of
compression of these cells which may be flattened out, or in severe cases

Fia. 18. Vertical section of the proximal end of tibia, showing early changes at
the chondro-osseus junction. Focal defects in the calcification of the zone of pre-
liminary calcification.

actually ruptured. Such compression is generally distributed in a spotty
manner and is severe and generalized only in bones where there is rapid
growth, and in a child with severe rickets. In a severe case the spicules
surrounding the cells are bent and buckled so that they may actually he
on their sides. Occasionally, the fractured spicules may be driven into the
cartilage.

The continued proliferation of cartilage cells, without concomitant min-
eralization and ossification, results in the lengthening of the cartilage plate
and in swelling of individual cells. The increase in size of the cartilaginous
plate is not due to an increased production of cartilage cells, as proposed
by Ziegler.” There is a normal production of these cells, but they fail to

™ EK. Ziegler, Lehrbuch der Allg. Pathol. Anat. 2, 179 (1898).

VI. EFFECTS OF DEFICIENCY 243

undergo normal senescence and they accumulate, thereby causing a widen-
ing of the cartilage plate in the proliferative zone (Fig. 20). If the rickets
persists, however, the rate of proliferation of these cells diminishes, so that
in extreme cases dwarfism may result. Besides this basic cause of dwarfism
in extreme rickets, shortness of stature may be due to a bending of the

+ Sidhe

Fre. 19. High power of Fig. 18, showing first maturation of cartilage cells with
disappearance of some of the cells at the chondro-osseus junction. Beginning irregu-
larity of the blood vessels. Deficient, almost absent calcification in the zone of pro-
visional calcification.

soft long bones, and accompanying fractures with shortening, and also to
squashing of the metaphysis.

The pattern of capillary invasion into the growing cartilage becomes
abnormal in two ways. In the normal bone, vessels invade the cartilage by
extension from the shaft only. In the ricketic bone, in addition to invasion
from the shaft, tiny blood vessels penetrate also from the epiphyseal end
and from the sides. The second abnormality is the type and formation of
the invading blood vessels. In the normal cartilage the capillaries are seen
extending up from the shaft and penetrating the matrix walls between the
cell columns, and by depositing lime salts on to these partitions they create

244 VITAMIN D GROUP

calcified fasicles. These calcified matrix columns in due time become trans-
formed into true bone. In rickets, the blood vessels attack several fasicles
together, and invade the very thick partitions. In severe cases an entire
bush of capillaries, branching from large arteries and attacking widespread
areas of the cartilage matrix, may cause gross defects in some parts of the
cartilage plate, while in other, perhaps adjoining areas, there may be a
mass of cartilage cells. Only the heaviest partitions remain, and the osteo-
blasts settle upon these and cover them with osteoid.

Wolbach® explained the occurrence of this abnormal calcification and

. ated Fa A

Fre. 20. Advanced rickets. Marked widening and irregularity of epiphyseal line
irregular vascular invasion, and defective calcification and ossification at chondro-
osseus junction. Osteoporotic appearance of metaphyseal region.

these changes at the chondro-osseus junction as follows: The capillaries
invade those regions which are uncalcified or soft, and after their penetra-
tion grow into large vessels, drawing away the circulation from the calcified
areas. Park believes, however, that the compressed cartilage cells we have
already described form the chief obstruction to the advancing capillaries.

The abnormal penetration of the blood vessels results in a variable blood
supply to the different parts of the cartilage, and the sections suffering from
insufficient blood may develop abnormally. Aside from the change of the
cartilage into osteoid rather than bone, there appear also bizarre, degenera-
tive forms of cartilage cells. The cells may vary greatly in size and shape,
with occasional appearance of double nuclei. At first these cells stain heavily

75 §. B. Wolbach, J. Am. Med. Assoc. 108, 7 (1937).

VI. EFFECTS OF DEFICIENCY 245

with hematoxylin, but later, as the degenerative process continues, they
lose their staining ability entirely.

The degeneration of cartilage cells may progress to the formation of
actual areas of necrosis. Although found most frequently in rats, such areas,
hemispherical and circumscribed, have also been described in humans.
Occasionally such degenerative and necrotic areas may dominate the patho-
logic picture.

Fig. 21. Large capillary tuft invading the cartilage. Severe rickets. Calcium
deficiency; large masses of uncalcified osteoid; connective tissue marrow.

We have already noted that during the ricketic process the proliferation
of new cartilage cells continues while the formation of bone is retarded. or
stopped. This, together with the invasion of this zone by large vascular
tufts (Fig. 21), results in the formation of a wide zone, soft and radiotrans-
lucent, between the shaft of the bone and the epiphysis. This zone, known
as the metaphysis, is an entirely abnormal area in which are scattered all
the elements of the pathologic picture of rickets, including large vascular
clumps, which enter the area from all sides, masses of cartilage broken up
into cartilaginous and osteoid trabeculae, bizarre cartilage cells, occasional
mineral salt deposits representing evanescent attempts at healing, and foci
of complete degeneration (Fig. 22). In this region we may note evidence

246 VITAMIN D GROUP

of the operation of mechanical forces on the newly and poorly formed
bone. Groups of cells may be compressed, a trabeculum may be bent
or doubled up on itself, the cartilage column at the periphery may fan
out to extreme angles, and there may even be a displacement of the up-
per metaphysis against the bone shaft so that the long axis of both
structures form an angle (Fig. 23). Moreover, since the human being seldom
suffers from complete absence of vitamin D, one finds evidence of transient
periods of healing, followed by recurrence of the ricketic process. To add

Fra. 22. Metaphyseal area. Irregular masses of cartilage cells, some swollen, some
compressed; compressed trabeculae of uncalcified osteoid tissue; irregular invasion
of cartilage by blood vascular marrow which shows early fibrous changes; some
degenerated cartilage and ghost cells; osteoid surrounding the invading marrow.

to the complexity of the picture, one may find evidence also of other vita-
min deficiency such as deficiency of vitamin C, or, less often, deficiency of
vitamin A and vitamins of the B complex. Deficiency of vitamin C may
lead to hemorrhage, osteoblastic degeneration, and fractures of newly calci-
fied trabeculae.

This variable and unpredictable histologic appearance is reflected in the
roentgenographs of the bones. Cupping and enlargement of the space be-
tween the epiphysis and the end of the shaft due to the transparency and
swelling of the metaphysis is most obvious. Cortical spurs or linear exten-
sions of the cortex which hug the proliferative cartilage may be an early
sign. Frayings, consisting of thread-like shadows extending from the end

VI. EFFECTS OF DEFICIENCY 247

of the shaft into the transparent cartilage, are seen. In early rickets the
individual threads are short, thin, and hard to see; in moderate and ad-
vanced cases the threads are long and coarse. In the shaft, the cortex may
appear thickened and composed of longitudinal, slightly curved interlacing
lamellae. The bone shadow may be slightly or moderately diminished. In
the severe forms, there is marked translucency of the entire shaft. If present,

Fic. 23. Cartilage mass which has been compressed projecting into metaphysis.
Predominantly connective tissue marrow.

the x-ray will always show these distortions and fractures as described,
especially after healing has begun.

Whether or not the condition of the teeth is influenced by the lack of
vitamin D is still an open question. It seems that primary dentition may
be delayed in rickets, and when the teeth do appear they may do so in an
abnormal order. The permanent teeth also show defects attributable to
rickets.7®: ” The teeth which are developing at the time when the rickets
is most active may, when they erupt, show a hypoplasia of the enamel.
76M. M. Eliot, S. P. Southner, B. A. Anderson, andS. Arnim, Am. .J. Diseases Chil-

dren 46, 458 (1933).
7 A. F. Hess and H. Abramson, Dental Cosmos 73, 849 (1931).

248 VITAMIN D GROUP

This condition is characterized by a symmetrical distribution of thinning
and pitting defects in the dental enamel. Although the relationship of
rickets to the incidence of caries has still not been established, it is generally
recognized that rickets results in enamel defects in the teeth which in turn
predispose to caries. This disabling disorder may be best prevented by the
ingestion of a calorically adequate and well-balanced diet, along with ade-
quate amounts of vitamin D."8: 7°

VII. Chemical Pathology and Pharmacology
BENJAMIN KRAMER and ABRAM KANOF

The chemical pathology of vitamin D deficiency is concerned with the
role which lack of this vitamin plays in preventing or delaying calcification.
In the last analysis rickets prevention depends upon the maintenance of a
normal concentration of calcium and inorganic phosphorus in the plasma
and presumably in the tissue fluids, thus in a large measure ensuring a
constant and adequate supply of these elements for mineralization of newly
formed cartilage matrix and osteoid. Exceptions to this rule are seen in the
very rapidly growing premature or newborn or in chronic nephritis or
hyperparathyroidism. An understanding of the mechanism of rickets pre-
vention therefore involves an understanding of the mechanism of calcium
and inorganic phosphorus, homeostasis. There are four facets to this prob-
lem: (1) the mechanism of absorption of calcium and inorganic phospho-
rus from the gastrointestinal tract; (2) the factors determining the level
of these elements in the blood; (3) the role of the kidneys in caletum and
phosphorus homeostasis; and (4) the factors which determine deposition
of calectum salts in the cartilage and osteoid, and the resolution of these
same materials (the local factor).

Just how calcium and inorganic phosphorus are absorbed from the gas-
trointestinal tract is unknown. We do know that soluble salts of caletum
are more readily absorbed than the less soluble combinations. A shift of
the pH of the intestinal contents to the acid side favors absorption, prob-
ably through conversion of the less soluble alkaline salts to the more soluble
acid forms. The presence of large amounts of fats, especially the higher
fatty acids, gives rise to highly insoluble calcium salts, while an excess of
carbohydrate, by increasing fermentation, shifts the pH of the intestinal
contents to the acid state and may give rise to the more volatile and more
soluble lower fatty acids.

78 M. C. Agnew, R. C. Agnew, and F. F. Tisdall, J. Am. Dent. Assoc. 20, 193 (1933).
72 A. F. Hess, H. Abramson, and J. M. Lewis, Am. J. Diseaces Children 47, 477 (1934).

VII. CHEMICAL PATHOLOGY AND PHARMACOLOGY 249

We have noted that the formation of insoluble salts of calcium such as
oxalate, tartrate, and alkaline phosphates, for example, tends to interfere
with absorption. Much of this interference with the absorption of calcium
can be overcome by inclusion of vitamin D in the diet, or by adequate ir-
radiation with ultraviolet rays of the correct wavelength. Thus, in the
absence of vitamin D, less than 20 % of ingested calcium is absorbed from
the gastrointestinal tract; if there is an adequate vitamin D intake, 50% to
80 % may be absorbed.

In the case of phosphate, which constitutes the most important anion
component of the calcium salts, the situation is rather more complicated,
for phosphorus plays an important role in innumerable enzymic processes
within the cells and is a constituent of many cellular components, e.g.,
nucleic acid, phosphatides, phosphoproteins, creatine phosphate, hexose
phosphates, ATP, ADP, etc. Phosphorus must be available at all times for
basic chemical processes.

It is therefore not surprising that the absorption of phosphorus from the
gut is in large measure independent of vitamin D intake, and that such
inefficiency of absorption as is observed in rickets is secondary to the failure
of calcium absorption. Moreover, such improvement of phosphorus absorp-
tion as is obtained by feeding vitamin D to the ricketic patient is a conse-
quence of improvement of calcium absorption.! The degree of phosphorus
absorption can be influenced by the pH of the intestinal tract. Adminis-
tration of cations, such as iron, aluminum, or beryllium which form insoluble
phosphates will interfere with the absorption of phosphate.’

The level of calcium and phosphorus in the blood is not entirely depend-
ent upon the amount of these elements absorbed from the gut. Physico-
chemical as well as endocrine factors play a part. The relationship between
the plasma calcium and inorganic phosphorus concentration is reciprocal,
as is that of the ions of any slightly soluble salt in a saturated solution, and,
presumably, the maximum concentration of either in the presence of the
other determines the magnitude of the solubility product. When the solu-
bility product falls below a certain critical value, lime salt deposition
becomes irregular, and if this is sufficient to interfere with the normal se-
quences of organic bone growth and mineralization of these tissues, rickets
develops. The magnitude of this solubility product in the human varies
with age, decreasing as the rate of bone growth diminishes. It can increase
to a higher level following fractures, or it may drop to a lower level with
infection. Both endocrine and physicochemical factors determine the satura-

1R. Nicolaysen, Biochem. J. 31, 122 (1937).

2 F. Albright, and E. C. Reifenstein, The Parathyroid Glands and Metabolic Bone
Disease, Williams & Wilkins Co., Baltimore, 1948, p. 38.

250 VITAMIN D GROUP

tion level. The attainment of the normal level in the otherwise normal
individual depends chiefly on an adequate intake of vitamin D.'

The availability of calcium to the plasma does not in itself determine its
level in the plasma, since introduction of calctum to the plasma in hypo-
parathyroidism results only in a temporary rise in the plasma calcium
concentration with a rapid re-establishment of the previous low calcium
level. In the ricketic child with hypocalcemia the administration of soluble
caleium salts in the absence of vitamin D will restore the normal serum
calcium level, but there will follow a simultaneous drop in inorganic phos-
phorus concentration. In the presence of adequate vitamin D intake,
normal calcium level is restored while normal inorganic phosphorus re-
mains unchanged, thus establishing a higher calctum phosphorus product.
Only toxic doses of vitamin D will raise this product still further. This
indicates the existence of a homeostatic mechanism which tends to keep
both ion concentrations at a level which is constant for the age group.
Vitamin D therefore influences the concentration of calcium in at least two
ways: by increasing absorption from the gastrointestinal tract, and by
raising the solubility product of calcium phosphate in the plasma, and
perhaps by regulating renal loss of calctum and phosphate either directly
or through the parathyroid glands. The mechanism seems to be as follows:
the vitamin D produces a rise in the serum calcium, which in turn depresses
parathyroid activity. Since the action of the parathyroids is to diminish
the renal threshold for phosphorus, their suppression raises the threshold
and elevates the serum phosphorus level.*

Under normal conditions bone acts as a reservoir for blood calcium. Hast-
ings’ has demonstrated a rapid restoration of plasma calcium after re-
moval of calcium from the blood and the return of such decalcified blood
to the circulation. Presumably this is accomplished by solublizing calcium
from bone. It is possible that the parathyroid glands play an important
role in this process. It is postulated that bone mineral forms the solid phase
of a bone-tissue fluid blood plasma system which is normally in equilibrium.
A reduction of plasma calcium concentration disturbs this equilibrium, and

3 For a given animal there would seem to be two products, one that is maintained
in the absence of vitamin D, and another, higher one when adequate vitamin D is
available. The former may be attained with a normal calcium level and a low
inorganic phosphorus concentration or a low calcium with a normal inorganic
phosphorus. The level of one ion is at the mercy of the amount of the other that
enters the plasma. With adequate vitamin D the total product as well as the
concentration of the individual components are stabilized, and if disturbed by
excessive ingestion of one, equilibrium is rapidly re-established at the normal level.

4F. Albright and E. C. Reifenstein, The Parathyroid Glands and Metabolic Bone
Disease, p. 127. Williams & Wilkins Co., Baltimore, 1948.

5 A.B. Hastings, New Engl. Med. J. 216, 377 (1937).

VII. CHEMICAL PATHOLOGY AND PHARMACOLOGY 251:

bone calcium is mobilized to restore the normal concentration of the liquid
phase. Just how this is accomplished is not clear, since calcium in bone is
presumably tied up with structural materials in bone, and resolution of
calcium salts usually goes on parallel with organic bone destruction.

The amount of phosphorus required is in excess of the amount needed
for bone mineralization alone, since phosphorus participates in most chemi-
cal processes in the cell. In addition it is an important buffer in maintaining
the normal reaction of blood plasma and in enabling the kidneys to excrete
large amounts of inorganic and organic acids within the normal span of
urinary pH. Although the mechanism of excretion of calcium and perhaps
the mechanism of its resorption by the kidney has not as yet received suffi-
cient attention, the problem of renal clearance of inorganic phosphorus has
received considerable study. These studies have concerned themselves not
only with normal subjects but also with individuals suffering from various
bone diseases, as well as other conditions such as acidosis, diabetes, and
parathyroid disturbances. When vitamin D is not available in the animal
with intact parathyroid, inorganic phosphate clearance seems to be in-
creased by virtue of a diminution in tubular resorption, a result of the un-
opposed activity of parathyroid hormone. This may explain the normal
plasma calcium level in most cases of rickets, in spite of defective calcium
absorption from the bowel. The hypophosphatemia in rickets is presumably
in part due to this defect. It is possible that the parathyroids play a part in
this process, since in experimental rickets these glands are found to be
enlarged. This may also explain the presence of a normal or only slightly

‘reduced calcium level in rickets due to vitamin D deficiency where absorp-

tion of calcium from the gastrointestinal tract is minimal. This increased
parathyroid activity has a twofold effect, the protection of the organism
against tetany by maintaining a normal calcium ion level in the face of
decreased calcium absorption from the gastrointestinal tract and an ex-
cessive inorganic phosphorus clearance by the kidney with resulting
hypophosphatemia. In this way the ricketic condition is aggravated while
the child is protected from the possibly fatal convulsions of tetany.

The action of vitamin D differs in the parathyroidectomized animal as
compared to animals with intact parathyroids. It is possible that with in-
tact parathyroids adequate intake of vitamin D depresses parathyroid ac-
tivity, resulting in a greater resorption of phosphorus by the renal tubules,
although the actual amount of phosphorus excreted in the urine exceeds
that found during active rickets (vitamin D deficiency). In parathyroidec-
tomized animals or human beings, vitamin D actually increases renal loss
of phosphorus.

The action of vitamin D in the ricketic animal is threefold: (1) It restores
the normal capacity of the gastrointestinal tract to absorb calcium. (2) It

Z5?, VITAMIN D GROUP

restores the normal phosphorus absorption which was secondarily impaired
by defective calcium absorption and the formation of shghtly soluble phos-
phates of calcium. (3) It increases urinary phosphorus excretion, although
phosphorus clearance actually is decreased because of increased tubular
resorption of phosphorus. This is accomplished presumably by the inhibi-
tory effect of increased plasma calcium upon parathyroid hormone secre-
tion. In the absence of the parathyroids phosphorus clearance is actually
increased by vitamin D intake.

Thus with more calcium and inorganic phosphorus available from exog-
enous sources and improved phosphorus resorption by the kidney, normal
levels of both elements are maintained, and normal minerglization of bone
is restored. Whether parathyroid hormone acts not only through the kidneys
but also directly on the bone itself is not known.

In the serum of normal children the concentration of calcium. 1s remark-
ably constant, at about 10 + 1.5 mg. %. The inorganic phosphorus content
of the serum is also fairly constant and in normal children is usually about
6 + 1mg. %. In the newborn infant the calcium content of the blood serum
may vary between 7.0 mg. and 11.0 mg. per 100 ml. The inorganic phos-
phorus content of the blood in premature and newborn full-term infants 1s
5.0 to 6.0 mg. %; it is slightly lower after 3 years of age, and still lower in
adults. It has been demonstrated that, whereas in tetany there is regularly
a marked reduction in the calcium of the serum, the drop in rickets is in-
frequent and may be caused by other factors than the vitamin D deficiency.
There is, however, a constant and sometimes marked decrease in the con-
centration of inorganic phosphorus with patients suffering from. rickets.
Howland and Kramer® and Iverson and Lenstrup’ independently made
observations with respect to inorganic and ecid-solub!e phosphorus in
patients with rickets and found that it varied from 0.6 to 3.2 mz. per
100 ml., the average being 2.0 mg., or less than 50% of the normal serum
content. Howland and Kramer showed that the administration of cod liver
oil in therapeutic doses had no effect on the calcium concentration but did
result in a marked increase of the phosphorus level.

We may say, therefore, that there is a fairly constant and a clinically
significant deficiency of the inorganic phosphorus of the plasma in rickets.
Howland and Kramer developed a mathematical formula which deter-
mines the chemical requirements for normal bone deposition.*® They found
that in most cases of uncomplicated rickets if the product of calcium con-
centration in milligrams per cent and the inorganic phosphorus concentra-

6 J. Howland and B. Kramer, Am. J. Diseases Children 22, 105 (1921).
7P. Iverson and E. Lenstrup, Porhandlingerne Ved. Forste Nordiske Kongres for
Pediatri. 1920.
. Howland and B. Kramer, T'rans. Am. Pediat. Soc. 34, 204 (1922).

VIII. REQUIREMENTS Dis

tion in milligrams per cent is less than 30, rickets exists; if it is above 40,
rickets is not present or is healing. Studies on zn vitro calcification indicate
that the blood of the normal child is slightly undersaturated with respect
to secondary calcium phosphate, and that equilibration experiments with
bone and artificial sera, as well as similar experiments using either tertiary
‘alcium phosphate or hydroxyapatite as the substrate, all point to the fact
that the determining factor in precipitation is the solublity product of this
compound (secondary calcium phosphate). Since both components enter-
ing into this product are represented by the first power, it follows that there
must be some relationship between the actual ion product and the product
expressed in this simple non-chemical way in terms of milligrams of each
component in 100 ml. of serum.

It must be emphasized, however, that the product is not the only factor
determining calcification. In rapidly growing premature infants osteoid
formation may proceed so rapidly that calcium salts deposition fails to
keep pace although the Ca & P product is normal.

Obviously the Ca X P product is only one basic essential for calcifica-
tion. Only certain tissues will calcify, although all are bathed presumably
with the same tissue fluid having its origin in the same plasma. Local pro-
cesses probably bring about a further increase of the product, presumably
by increasing the anion level. The many factors which may play a role in
this complicated process have already been discussed. Alkaline phosphatase
is the only one of the enzymes involved which can be readily measured.
The number of units reported in a particular case represents the number of
‘milligrams of inorganic phosphorus liberated by the amount of enzyme in
100 ml. of serum. The normal plasma values for children vary between 5
and 15 units. In active rickets this value is increased, and it is diminished
during healing. As the excess tissue either becomes calcified or resorbed, the
phosphatase concentration in the plasma tends to return to normal.

VIII. Requirements
A. OF ANIMALS
JAMES H. JONES

So many factors influence the requirements for vitamin D that it is diffi-
cult to express quantitatively the need of animals for this vitamin unless
the environment and diet are known. The most important of these factors
is sunlight. It is well recognized that exposure to sufficient summer sun-
shine makes the giving of vitamin D unnecessary. Consequently, the de-

254. VITAMIN D GROUP

termination of the requirements of any species for vitamin D is done under
conditions which exclude the actinic rays.

The amount and ratio of calcium and phosphorus in the diet also in-
fluence the need of the animal for vitamin D. As discussed previously, it
is impossible to produce rickets in rats and other rodents which have been
studied unless the diet is low in either phosphorus or calcium. However,
even with larger mammals and fowls which develop rickets on diets con-
taining ample amounts of caletum and phosphorus, the demand for vitamin
D is increased if the ratio of calcium to phosphorus is far removed from
that required by the animal, and/or the calcium or phosphorus in the diet
is deficient.

The availability of phosphorus (and also calcium) is another factor in
the requirements of animals for vitamin D. Most of the common ricketo-
genic diets contain approximately 0.3 to 0.4 % of phosphorus. If this were
all available, these diets would not produce rickets in rats, but they are
composed largely of cereals which contain considerable phytic acid. The
phosphorus of phytic acid is not available until the acid is hydrolyzed by
the phytase of the intestinal secretions. When a large amount of calcium
is added to the diet, the phytic acid is precipitated as the calcium salt.
The acid cannot be hydrolyzed, and the phosphorus cannot be made avail-
able. Mellanby! discussed the chemistry of phytic acid and its salts and their
relation to rickets. If the diet contains inorganic phosphates, 1t is probable
that here again the high calcium makes some of the phosphorus unavailable
by precipitating it as an insoluble calcium phosphate.

Still another factor which influences the requirements of animals for vi-
tamin D is the acidity of the diet. As the acidity is increased, the less rick-
etogenic the diet becomes.? The effect, in all probability, is due to an in-
creased solubility of the calectum and phosphorus. Shohl eé al.,> however,
found little difference among acid, neutral, and alkaline phosphates in
their effects on rickets in rats.

In the following discussion on the requirements of farm animals, it is
assumed that the animals are away from direct sunlight, but that the sup-
ply of calcium and phosphorus is adequate, and the ratio of these two ele-
ments to each other is not far from that demanded by the animals.

Recently the Committee on Animal Nutrition of the National Research
Council has issued several reports giving the recommended nutrient allow-

1. Mellanby, A Story of Nutritional Research. Williams and Wilkins Co., Balti-
more, 1950.

2T. F. Zucker, W. C. Johnson, and M. Barnett, Proc. Soc. Exptl. Biol. Med. 20,
20 (1922-1923).

3 A.T. Shohl, H. B. Bennett, and K. L. Weed, J. Biol Chem. 78, 181 (1928).

VIIL. REQUIREMENTS DSS

ances for domestic animals.* The following data dealing with the recom-
mendations for vitamin D are taken from these reports.

In Table XI are given the recommended daily amounts in international
units of vitamin D for the maintenance of chickens of various weights and
for laying and breeding. The recommended amounts of vitamin D per pound
of feed for starting chicks (0 to 8 weeks), growing chicks (8 to 18 weeks)
laying hens, and breeding hens are 180, 180, 450, and 450 A.O.A.C. units,
respectively. For turkey poults, growing turkeys, and breeding turkeys,
800 A.O.A.C. units of vitamin D per pound of feed is recommended.

The recommended daily amounts of vitamin D, in international units,
for various weights of market swine and for breeding stock are given in

TABLE XI¢
RECOMMENDED Datty ALLOWANCES? oF VITAMIN D ror CHICKENS

Maintenance
= | Laying | pees
Body weight, lb. Bop lied ges ps2: |e 2-Sia1 ts sOrlia On Hess) lis sspe {hse aly
| | |
White Leghorns /11.88} 18.5] 23.2) 26.1) 28.3) 31.1] 28.4 9207 92-7
and similar |
breeds |
Heavy breeds WAG. | US ocAl jee) aWcil 36.0 34.2/36.0? 111.6 111.6

* Adapted from Recommended Nutrient Allowances for Poultry: A Report of the Committee on
Animal Nutrition, National Research Council.
International units per animal.

Table XII The quantity of vitamin D per pound of feed recommended for
all types of swine is 50 I.U.

The daily allowances of vitamin D for dairy cattle are again based on
body weight. For heifers weighing 50, 100, 150, and 200 pounds, the rec-
ommended amounts are 200, 400, 600, and 800 I.U., respectively. The
allowances per pound of feed for dairy heifers weighing 50, 100, 150, and
200 pounds should be 220, 200, 150, and 130 I.U., respectively. No rec-
ommendations are made for heavier animals.

No general recommendations are made regarding the needs of beef cattle
for vitamin D. It is pointed out that usually these animals receive suffi-

4 Committee on Animal Nutrition, Recommended Nutrient Allowances for Domes-
tic Animals: No. I, Recommended Nutrient Allowances for Poultry (Revised
1950); No. II, Recommended Nutrient Allowances for Swine (Revised 1950);
No. III, Recommended Nutrient Allowances for Dairy Cattle (Revised 1950);
No. IV, Recommended Nutrient Allowances for Beef Cattle (Revised 1950);
No. V, Recommended Nutrient Allowances for Sheep (Revised 1949); No. VI,
Recommended Nutrient Allowances for Horses (1949).

256 VITAMIN D GROUP

cient sunlight to make a dietary source of vitamin D unnecessary. How-
ever, the report on the nutritive recommendations quote Bechtel e al.’ as
finding the vitamin D requirements of young beef calves to be about 300
I.U. per 100 pounds of live weight.

Sheep, like beef cattle, also spend considerable time on the range and
probably need no dietary source of vitamin D. Lambs away from sunlight
should be given this vitamin, and the Committee on Animal Nutrition
recommends a daily allowance of 250 to 300 I.U. of vitamin D per 100
pounds of live weight. In a similar manner, the working horse in all prob-
ability needs no dietary source of vitamin D because it is exposed to direct
sunlight for hours at a time. Where horses are confined away from the light
of the sun, it is recommended that the daily allowance be 300 I.U. per 100
pounds of live weight. There is no experimental information available on

TABLE XIfe¢
RECOMMENDED Datty ALLOWANCES? OF VITAMIN D FoR SWINE

Breeding stock

Pregnant females

and breeding boars Lactating females
Market stock Young Adults Gilts Adults
Live weight, Ib. 50 100 150 200 250 300 500 350 450

Vitamin D 150 265 340 375 415 300 375 550 625

* Adapted from Recommended Nutrient Allowances for Swine: A Report of the Committee on
Animal Nutrition, National Research Council.
International units per animal

the horse, but the recommendations are based on knowledge concerning
other animals.

It is to be remembered that these recommendations are not minimal
quantities, but a rather liberal allowance has been made for a margin of
safety.

Harris et al.6 found that for foxes on a basal diet which contained 0.82
1.U. of vitamin D per gram the addition of 200 I.U. per kilogram of body
weight “was not above the limit for best physiological efficiency.” They?
have also studied the requirements of minks for vitamin D, and they found
that 0.82 I.U. per gram of food was sufficient, provided that the calcium
to phosphorus ratio was between 0.75:1.00 and 1.70:1.00.

5H. E. Bechtel, E. T. Hallman, C. F. Huffman, and C. W. Duncan, Mich. Agr. Expt.
Sta. Tech. Bull. 150 (1936).

6 L,. E. Harris, C. F. Bassett, and C. F. Wilke, J. Nutrition 43, 153 (1951).

7C. F. Bassett, L. E. Harris, and C. F. Wilke, J. Nutrition 44, 433 (1951).

bo
t
“J

VIII. REQUIREMENTS

B. OF HUMAN BEINGS
BENJAMIN KRAMER and ABRAM KANOF

In general terms the amount of vitamin D required by any human being
is the amount needed to permit normal growth and mineralization of the
bones and teeth during infancy and childhood and to maintain these struc-
tures during later life, as well as to meet the increased demands of infection,
pregnancy, and lactation. Specifically, the determining factors for vitamin
D requirement are the varying capacity of people of various ages to absorb
and retain calcium, the rate of growth of the individual, and the adequacy
of the diet as regards not only the absolute amounts of calectum and inor-
ganic phosphorus but also the ratio of the elements to each other in the
diet. The nature of the compound of calcium or phosphorus may determine
the availability of the element for absorption by the intestine or utilization
by the tissues. Thus the less soluble calcium salts are more poorly absorbed
than are the more soluble ones. In phytin the phosphorus is almost com-
pletely unavailable. Inorganic phosphorus is better absorbed than organic
phosphorus from the gastrointestinal tract even in osteomalacia.

However, a high Ca/P ratio means poor phosphorus absorption, whereas
a very low ratio means poor calcium absorption, especially where the in-
testinal contents are alkaline in reaction. Similarly, any cation suchas alumi-
num or Fe which tends to form poorly soluble phosphorus compounds will
interfere with phosphorus absorption. The proportions of protein, fat, and
carbohydrate in the diet influence calctum and phosphorus absorption
through their effect on the reaction of the intestinal contents. Little is
known regarding the mechanism of absorption of calcium salts, but the less
favorable the condition for calcium and phosphorus absorption the more
vitamin D will be required to ensure optimal utilization of available ma-
terials.

Roughly, the daily need for calcium is 0.1 g. throughout infancy,® 0.3 g.
during childhood, and 0.5 g. during adolescence.’ The actual amount re-
quired by an individual will depend directly on his rate of growth, hence
the tendency of the rapidly growing infant to develop rickets under condi-
tions in which the adult will be free of disease. The prematurely born infant
is born with a minimal store of calcium and phosphorus, and in addition
under proper conditions his rate of growth is greater than that of the full-
term infant. He then suffers from a double handicap, namely, a greater
need of bone-forming minerals and vitamin D, with a decreased capacity
for absorption of these substance. The fetal need for calcium during the last

8’ White House Conference Reports: II Nutrition, p. 196. Century Co., New York,

1932.
°T. Leitch, Nutrition Abstr. & Revs. 6, 533 (1937).

258 VITAMIN D GROUP

month of gestation is 0.3 g. daily. A newborn premature infant simply can-
not ingest enough calcium to enable him to retain this amount plus the
extra amount needed for his rapid growth. Such an infant will need large
doses of vitamin D to prevent rickets.!°

The infant fed on the usual cow’s milk dilutions ingests an enormous
amount of calcium and inorganic phosphorus per square meter of body
surface as compared to the adult or the breast-fed infant. However, the
Ca/P ratio of cow’s milk is far from optimal and at times may be dangerous,
whereas the breast-fed infant, although receiving a much smaller amount
of bone-forming elements, ingests these materials in a more optimal ratio
and in amounts which do not tax the calcium and inorganic phosphorus
homeostatic mechanism of the kidney. It is perhaps this which explains
the lower incidence of rickets and ricketic tetany in breast-fed infants.
More than 95 % of ingested calcium goes to form bone. For this purpose an
amount of phosphorus equal to about 50% of retained calcium is needed.
Since phosphorus plays an important role in all intracellular chemical proc-
esses, an additional amount of phosphorus is needed by the cells. In addi-
tion to this factor, it seems also that the requirement of a breast-fed infant
for phosphorus is generally less than that of one fed on cow’s milk.

Infections may be associated with deficiency of blood calcium and in-
organic phosphorus, even when ordinarily adequate amounts of vitamin D
are ingested.

For a clearer understanding of the problem of vitamin D dosage in the
human being, it is important to differentiate between the prevention and
the cure of vitamin D deficiency (rickets) and also to determine whether
we are dealing with an organism that is initially normal or one suffering
from visceral or other disease or immaturity of vital organs. For practical
reasons, an adequate preventive dose of vitamin D is one that will prevent
all clinical and radiographic evidence of rickets whereas a therapeutic dose
is one that will re-establish normal calcification and bone growth and in due
time correct most of the deformities of the disease. Some telltale radio-
graphic evidence may persist in spite of adequate dosage, and occasionally
gross changes may require surgical intervention or other forms of therapy.
A dose of vitamin D that will cure rickets is also adequate to prevent the
disease.

For the prevention of rickets in the average normal infant 1000 units of
vitamin D given daily orally either as a concentrated fish oil or as vitamin
D dispersed in water will suffice. Vitamin D-enriched milk containing 400
U.S.P. units per liter is now widely used either with fresh cow’s milk, evapo-
rated, or dried milk. The advantage of vitamin D-enriched milk is that

10 P. C. Jeans and G. Stearns, The Vitamins, p. 493. American Medical Association,
Chicago, 1939.

VIII. REQUIREMENTS 259

calcium and phosphorus are ingested simultaneously and in a fixed ratio
to each other and to vitamin D. When concentrated forms of vitamin D are
used they should preferably be given in water-dispersed form in doses of
3000 units per day because of possible defective absorption of some fats
and fat-soluble vitamins. Occasionally, larger doses are required. The actual
amounts may have to be determined by the method of trial and error,
controlled with serial x-rays of the extremities and repeated determinations
of plasma, calcium, inorganic phosphorus, and phosphatase. Prematurely
born infants may require a somewhat larger dose.

This therapy should be continued throughout the year, rather than de-
pend on chance solar irradiation during the summer months. When mothers
cannot be depended upon to continue this medication or where conditions
exist that make adequate medical supervision impossible or in the presence
of prolonged infection, the large single dose of 600,000 units every 6 months
apparently may be used with favorable results. Clinical and pathological
studies show that there is little risk of hypervitaminosis with such therapy.

For the treatment of rickets in the otherwise normal child a similar dose

will suffice. Vitamin D highly dispersed in milk or in water seems to be
more effective than when administered as a concentrated fish oil. As little
as 400 units of vitamin D in milk will in due time cure the majority of rick-
etic children. When administered as vitamin D in oil, 1000 units daily is
usually required. It is important that the diet contain adequate amounts
of calcium and phosphorus, preferably in a 2:1 ration, as well as a mixture
of good proteins.
_ Where rickets persists in spite of adequate vitamin D dosage, an expla-
nation must be sought in a careful study of the organism itself. A number
of such refractory cases have been described, and many of these have been
subjected to exhaustive metabolic study. Defects of absorption (coeliac
disease, cystic fibrosis of the pancreas, sprue, idiopathic steatorrhea, in-
testinal shunts, intestinal anomalies, ulcerative colitis) may explain the
refractory state.

Where bile fails to enter the intestinal tract, as in acquired common duct
obstruction, congenital absence of bile ducts, or atresia of these structures
or biliary fistulae, absorption of fat-soluble vitamins, including vitamin D
is impaired and rickets may fail to respond to the usual doses of vitamin D.
Similarly, in cases of liver damage, as in cirrhosis, additional vitamin D
may be needed both to prevent as well as to cure rickets. For the child with
defective intestinal absorption the vitamin is preferably given in the form
of a water dispersion.

In some unknown manner infections may reduce the Ca X P product in
the blood plasma, giving rise to ricketic changes in the bones that are de-
monstrable histologically but not clinically or radiologically. Subsidence
of the infection results in a correction of the plasma calcium and inorganic

260 VITAMIN D GROUP

phosphorus concentration and healing of the ricketic lesion in the bones.
Increased dosage of vitamin D or its administration in highly dispersed
form in a watery dispersion or parenteral administration of the vitamin
may solve the difficulty in some of these cases. In other cases functional
disease of the kidney or diseases of the parathyroids may explain the persis-
tence of the ricketic state and its progress in spite of therapy. In some in-
stances much larger doses of vitamin D will suffice, whereas in others sup-
plementation of vitamin D with other substances is required. In one such
case, Albright, Butler, and Bloomberg found it necessary to increase the
daily dose of vitamin D to 1,500,000 units in order to initiate healing and to
give 150,000 units daily in order to maintain the normal state. However,
before one concludes that a patient has refractory rickets, one must be
certain that he is not dealing with some endocrine imbalance (hyperpara-
thyroidism) or some other abnormality such as a disturbance of acid-base
equilibrium, renal insufficiency, hypercalcemic rickets, liver damage, the
De Toni-Fanconi syndrome, the hyperplastic form of chondrodystrophy,
or rickets due to a disturbance of cysteine metabolism or due to biliary ob-
struction.

Refractoriness due to chronic acidosis should be treated with alkalies in
addition to large doses of vitamin D. Some cases of chronic acidosis are
due to a defect in the ammonia-producing mechanism in the renal tubules.
This is corrected by the administration of large amounts of base such as
sodium, potassium, calcium, or magnesium, an alkaline ash-yielding diet,
along with adequate doses of vitamin D, or simple sodium bicarbonate or
citrate. A form of rickets associated with chronic renal disease and hyper-
chloremia has been described. This responds to a mixture of sodium citrate
and citric acid, in addition to massive doses of vitamin D. In the De Toni-
Fanconi syndrome there is evidence of renal tubular dysfunction. There is
normal excretion of ammonia, but the blood shows a low bicarbonate, evi-
dence of compensated acidosis, marked and persistent hypophosphatemia,
occasional hypocalcemia, and, in the later stages, a decrease in the blood
sodium, potassium, and chlorides. Treatment is ineffective.

Although the practice is widespread of discontinuing prophylactic use of
the vitamin after the age of 2, when a mixed diet is ingested, Park’s studies
have demonstrated histological evidence of rickets in children even up to
14 years.!! This would indicate the need for the continuance of sup-
plementary vitamin D up to that age, in doses about half that for infants.
Normal adults may rely on sunshine and incidental ingestion of the vitamin,
but women during pregnancy and lactation require about 1000 units daily
to avoid the deleterious effects of vitamin D deficiency.

Single massive doses of vitamin D sometimes may be used. This method

1R. H. Follis, Jr.. D. Jackson, M. M. Eliot, and E. A. Park, Am. J. Diseases
Children 66, 1 (1943).

VIII. REQUIREMENTS 261

is advised when the mother cannot be trusted to give oral medication regu-
larly, or when rapid healing of rickets is indicated, as in thoracic rickets,
or in rickets complicated by pneumonia or whooping cough, when the per-
sistence of the ricketic state is itself a menace to the life of the child. It is
especially useful in tetany. For the single massive dose therapy 600,000
units may be given orally or by intramuscular injection. Post-mortem
studies have failed to reveal any detectable harm from such therapy.

Although the primary indication for the use of vitamin D is the preven-
tion and treatment of rickets and infantile tetany, there is some evidence
that the use of this vitamin may also help in the normal development of the
teeth.

In recent years vitamin D has been used extensively, usually in massive
doses, in the treatment of diseases having no obvious relationship to rickets,
tetany, or dental caries. Favorable results have been reported in intestinal,
bone, and lymph-node tuberculosis,” in arthritis," in acne vulgaris,“ in
hay fever,!® in asthma and urticaria,!® in psoriasis,” in the prevention of
anterior poliomyelitis;!® in the treatment of toxemia of pregnancy,!? and in
the amelioration of the bleeding tendency in obstructive jaundice.2° More
recently, Pascher has summarized the evidence in favor of vitamin D in
the treatment of lupus vulgaris.?! Sobel and his coworkers have studied the
role of vitamin D in experimental lead poisoning.” It should be emphasized
that some of these therapeutic reports have by no means been substantiated,
and that most of the instances of vitamin D poisoning have arisen in the
course of such treatment.

Table XIII summarizes the forms in which vitamin D may be given,
and the daily doses required for prevention and treatment of rickets.

2B. L. Wyatt, R. A. Hicks, and H. E. Thompson, Ann. Internal Med. 10, 534 (1936).

13 B. L. Wyatt, R. A. Hicks, and H. E. Thompson, Ann. Internal Med. 10, (1936);
I. Dreyer and C. I. Reed, Arch. Phys. Therapy 16, 537 (1935); E. G. Vritak and
R.S. Lang, J. Am. Med. Assoc. 106, 1162 (1936).

4 C. A. Simpson, F. A. Ellis, and H. Kirby-Smith, Arch. Dermatol. and Syphilol.
41, 835 (1940); M. B. Sulzberger, Year Book of Dermatology and Syphilology,
p. 14. Year Book Publishers, Chicago, 1949; F. C. Combes, Med. Times, Chicago
77, 473 (1949).

16 B. Z. Rappaport and C. I. Reed, J. Am. Med. Assoc. 101, 105 (1933).

16 B. Z. Rappaport, C. I. Reed, M. L. Hathaway, and H. C. Struck, J. Allergy 5,
541 (1934).

77. Olivetti and E. Ratto, Giorn ital. dermatol. e sifilol. 90, 187 (1949); H. Schmitz,
Prazis 36, 307 (1947); J. Krafka, Jr., J. Lab. Clin. Med. 21, 1147 (1936); E. T.
Cedar and L. Zon, Publ. Health Repts. (U. S.) 52, 1580 (1937).

18 J. A. Toomey, Am. J. Diseases Children 58, 1202 (1937).

19G. W. Theobold, Lancet I, 1397 (1937).

207,. B. Johnston, J. Med. 18, 235 (1937).

*1 F. Pascher, M. G. Silverberg, I. E. Marks, and J. Markel, J. Invest. Dermatol. 18,
89 (1949).

22 A. E. Sobel, H. Yuska, D. D. Peters, and B. Kramer, J. Biol. Chem. 182, 239 (1940).

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264 VITAMIN D GROUP

1. HypEerRvITAMINOsIS D

The pathologic effects of overdosage with vitamin D have been studied
chiefly in individuals who have received massive doses of calciferol in the
treatment of arthritis. In children there have been additional cases re-
ported as a side effect of vitamin D treatment for tuberculosis or after
receiving imperfectly irradiated ergosterol preparations containing large
amounts of toxic intermediates (vigantol). The basic pathologic effect is the
precipitation of calcium in various tissues. As a result of metastatic cal-
cification in the kidneys, kidney insufficiency may develop. Finally,
withdrawal of large amounts of calcium into these abnormal foci may re-
sult in demineralization of bone.

The gross and histologic findings have been frequently reported and are
similar in both children and adults, although fatalities seem to occur more
frequently in the young. There is diffuse calcinosis affecting the joints, syn-
ovial membranes, kidneys, myocardium, pulmonary alveoli, parathyroid
glands, pancreas, skin, lymph glands, large and medium-sized arteries, the
conjunctivae and cornea, and the acid-secreting portion of the stomach. The
abnormal calcification can be seen grossly as a whitish, chalky material.
The bones in the early stages may show accelerated calcification of the
provisional zone of calcification with thickening of the periosteum. In
more advanced cases, however, there is interference with cartilage growth,
and several authors’ have demonstrated diffuse demineralization of the
bones. Shelling and Asher pointed out that osteoporosis produced by hy-
pervitaminosis differs from that produced by parathormone in that the
resorbed areas are not replaced by fibrous tissue. Freeman e¢ al.*° have re-
ported an instance in a child in which doses of ertron resulted in a nega-
tive calcium balance. The most serious involvement is that of the kidneys,
and most of the fatal cases terminated in uremia. The best evidence seems
to indicate that the initial kidney damage is due to deposition of calcium in
the basement membranes of the cells of the distal tubules.?® There results
an inflammatory reaction, and later complete obstruction. As a result of
the obstruction, several nephrons at first dilate and then atrophy. As a
result of the inflammatory reaction, the lesion may spread rapidly through
the entire kidney. The kidney damage in turn is responsible for such path-
ology as hypertension, hypertensive retinopathy, and chemical evidence of
renal insufficiency.

23 D. H. Shelling, The Parathyroids in Health and Disease. C. V. Mosby Co., St.
Louis, 1935.

24 —D. H. Shelling, and D. Remsen, Bull. Johns Hopkins Hosp. 57, 158 (1935).

25 S$. Freeman, P. S. Rhoads, and L. B. Yeager, J. Am. Med. Assoc. 130,197 (1946).

26R. H. Freyberg and J. M. Bauer, Univ. Hosp. Bull. (Michigan) 11, 61 (1945);
T.S. Danowski, A. W. Winkler, and J. P. Peters, Ann. Internal Med. 28, 22 (1945).

VIII. REQUIREMENTS 265

The dosages administered before evidence of intoxication appeared varied
tremendously. As little as 400 units daily seemed to have produced fatal
pathology in one instance. The shortest length of time over which the vi-
tamin had been given before demonstrable calcification was produced was
14 days. The problem as to whether the toxic effects are due to the vitamin
D itself or to contaminating sterols cannot be answered. However, it has
been shown that pure, crystalline vitamin D, or any factor which can cause
an increase in the serum ionic calcium can produce toxic symptoms. Al-
bright and Reifenstein?’ ascribed all the manifestations of hypervitaminosis
D to an exaggeration of the normal action of the vitamin: (a) to an increase
in the absorption of calcium from the gastrointestinal tract, and (b) to an
increase in the urinary excretion of phosphorus.

Clinical symptoms in non-fatal cases are anorexia, nausea, vomiting,
diarrhea, polyuria, weakness, lassitude, headache, hyperesthesia, the ap-
pearance of areas of brown pigmentation over the skin, and evidence of renal
insufficiency.

There is no agreement as to the mechanism by which the abnormal calci-
fication takes place. Some believe that the first step is the appearance of
cellular damage followed by calcium deposition. On the basis of experimen-
tal data with rats it is believed that abnormal deposition of calcium is the
first step. The exact factors which are related to the precipitation of calcium
are not known. Abnormal calcification may occur in the presence of a normal
total serum clacium.

Conversely a high serum calcium may be present for a long time without
the occurrence of precipitation. It has been found that in rats, following ad-
ministration of large doses of calciferol, there was abnormal calcification
while the total serum calcium concentration was on the decline rather than
while the level was rising. The explanation seems to be that as the serum
calcium declined there was a rapid release of the part of the total calcium
held by the parathyroids and that this fraction was too great to be held in
solution. It would appear, therefore, that a fluctuating-serum calcium is
more apt to produce abnormal calcification than a high constant level. Calci-
fication is also aided by any factor which produces some degree of alkalosis,
as in continued vomiting. Reed et al.?8 have pointed out that patients with
gastrointestinal complaints are more susceptible to the toxic effect of vita-
min D. Other factors of importance are the dose of vitamin D ingested, the
vehicle, the degree of exposure to sunlight, the amount of dietary calcium
ingested, the susceptibility of the individual, the pathologic state for which

27 F. Albright and E. C. Reifenstein, Parathyroid Gland and Metabolic Bone Dis-
ease, p. 95. Williams & Wilkins Co., Baltimore, 1948.

22 C. I. Reed, I. E. Steck. H. C. Struck, and H. Deutsch, Ann. Internal Med. 10, 951
(1937).

266 VITAMIN D GROUP

the vitamin is administered, the status of the endocrine system, and the
age of the patient.

The story of vitamin D, its identification as a separate calcifying vitamin,
its isolation in pure form, and its application in eradicating what was a
serious and crippling disease, represents a brilliant chapter in sterol chem-
istry, in nutritional physiology, and in clinical medicine. However, little
is known as to its action. In some mysterious manner it facilitates the ab-
sorption of calcium from the intestinal tract and deposits it as a phosphate
or carbonate in osteoid and cartilage matrix. It controls in part the clear-
ance of inorganic phosphorus by the kidney, and still more remarkably it
molds the deformed tissue of the ricketic animal into a normal configura-
tion and pattern characteristic of the involved tissue. Little is known con-
cerning the mechanism of absorption of the vitamin itself, its manner of
transport, or its storage in the tissues. Much has been revealed, much still
remains to be discovered. Newer methods are needed for these studies.

Vir.
VIIl.

1X:

CHAPTER 7

ESSENTIAL FATTY ACIDS

. Nomenclature and Formulas
. Chemistry

. Isolation

. Synthesis

. Commercial Pr apncagane
. Chemical Properties

. Oxidation

. Physical Proper Pag

Jem oOoOWeS

. Biochemical Systems
. Specificity of Action
. Biogenesis and Metabolism

A. Dietary Factors Affecting Mictabelien

. Estimation

A. epesmontotamenitc Nas sis.
B. Chemical Methods
1. Thiocyanogen Value
2. Polybromide Methods
C. Enzymatic Method
D. Animal Assay.
Occurrence in Foods
Effects of Deficiency
A. In Animals and Taeeeie
. Rat
Dog ;
. Chicken
. Mouse
. Large Animals
. Insects
: Histousiholocy
B. In Human Beings
1. Low Fat Diets in isErcacie Beings
2. Indirect Evidences of Need for Essential Fatty hee
3. Data from Animal Studies with Possible Applications .
4. Spectroscopic Study of Serum Lipids in Human Subjects
5. Summary of Effects of pee: a Eada
Pharmacology Ste ek
A. Chemical Aspects
B. Biological Aspects 2
C. Vitamin Interrelationships
D
E

SIO oR wh

. Action on Physiologic Processes
}. Chemotherapy

267

Page
268
268
270
272
272
273
274
275
Pik
278
280
283
286
286
288
288
289
289
290
291
292
292
292
295
296
297
297
298
298
300

. 301

303
306
310
dll
311
311
313
315
316
317

268 ESSENTIAL FATTY ACIDS

X. Requirements: -. 6 0 se © Ge) epee ee ole cing Sella)
AMOfAntovals: p02, Ae gas Nay hice tee co, ee
B Of Human Beings’ 2 02) Sea we ss ee

I. Nomenclature and Formulas
ROBERT S. HARRIS

Accepted names: Essential fatty acids
Obsolete name: Vitamin F
Empirical formulas: Linoleic acid: CigH3202
Linolenic acid: CigH3 902
Arachidonic acid: CooH3202
Chemical names: Linoleic acid: A9 , 12-octadecadienoic acid
Linolenic acid: A9,12,15-octadecatrienoic acid
Arachidonic acid: A5,8,11,14-eicosatetraenoic acid
Structural formulas:

CH;3(CH:),CH=CHCH.CH—CH(CH.);COOH
Linoleic acid

CH; CE; Ch—CHCEH. Ch—CHCH CAi—CH(CHe), Coon
Linolenie acid

CH;(CH,),CH==CHCH,.CH=CHCH,.CH=CHCH,CH=CH(CH2);COOH

Arachidonic acid

II. Chemistry
RALPH T. HOLMAN

Rigid exclusion of fat from an otherwise adequate diet of a variety of
animals results in the delayed appearance of a deficiency syndrome first
described in 1929 by Burr and Burr.'* This deficiency has been shown to be
caused by the lack of certain fatty acids which have come to be called the
essential fatty acids. Incorporation of these acids in the diet either pre-
vents or cures the deficiency symptoms. Although the fat deficiency
phenomenon parallels vitamin deficiency phenomena, the term vitamin
has not been generally applied to the essential fatty acids, probably be-
cause they are required in larger quantity than are most vitamins.

Fatty acid deficiency and the essential fatty acids have been the subject

1a G. O. Burr and M. M. Burr, J. Biol. Chem. 82, 345 (1929).

269

CHEMISTRY

ite

270 ESSENTIAL FATTY ACIDS

of several previous reviews to which the reader is referred.!>-4 In the present
treatment, the discussion of the essential fatty acids and the deficiency
phenomenon caused by their absence from the diet of animals will be
general, reference being made only to selected papers from which the
reader can obtain more detailed reference.

The essential fatty acids area group of naturally occurring polyunsat-
urated fatty acids, some of whose structures are well established. They
possess the common structure of methylene-interrupted polyunsaturation.
The group is generally regarded as consisting of three members, linoleic
acid (9,12-octadecadienoic acid), linolenic acid (9,12 ,15-octadecatrienoic
acid), and arachidonic acid (probably 5,8,11,14-eicosatetraenoic acid).
However, the higher polyunsaturated acids, clupanodonic acid and docosa-
hexaenoic acid, are closely related to the essential fatty acids and should be
included with them in the more general sense of the classification. Molecu-
lar models and conventional structural formulas for the first three acids
are shown in Fig. 1. Linoleic and linolenic acids are of vegetable origin and
are probably the dietary precursors of the animal polyunsaturated acids.
Arachidonic acid is found only in animal lipids and is probably a functional
essential fatty acid. As will be mentioned later, linoleic and linolenic acids
are not entirely equivalent in action, and linoleic acid is the only vegetable
acid which meets all the requirements of the animal for essential fatty acid.
Thus, it is apparent that the term essential fatty acids, as ordinarily used,
is generic, including many members not equivalent in metabolic action.
As more information is gained regarding the roles of the various acids, best
usage will require their individual names.

A. ISOLATION

Linoleic acid is a major constituent of a variety of vegetable oils. Excel-
lent sources of the acid for preparative purposes are corn oil, cottonseed oil,
and safflower seed oil, in which little or no conjugated or more highly
unsaturated acids are found. The problem in isolation is the separation of
linoleic acid from saturated and unsaturated acids of various chain lengths.
The various acids present in such mixtures have such slight differences in
chemical and physical properties that separation is a difficult procedure,
and the product gained is rarely pure.

The classical procedure for linoleic acid preparation is the bromination-

1b G. O. Burr, Federation Proc. 1, 224 (1942).

2A. E. Hansen and G. O. Burr, J. Am. Med. Assoc. 182, 855 (1946).

3R. T. Holman, Proc. 8rd. Conf. on Research, Am. Meat Inst. Univ. Chicago p. 1
(1951); Fette wu. Seifen 58, 332 (1951).

4R. T. Holman, Symposium on Biol. Significance of Lipids, Robert Gould Research
Foundation, 1950.

II. CHEMISTRY 271

debromination method of Rollet.? Advantage is taken of the differences in
petroleum ether solubility of tetrabromostearic acid formed from linoleic
acid and the solubilities of the saturated acids and the dibromostearic acid
formed from oleic acid. This method has been carefully worked out and
standardized by McCutcheon.* Best practice requires constant protection
of the unsaturated material from oxygen. Thus during saponification, de-
bromination, washing, and transferring, the material should be kept under
a blanket of nitrogen. The resulting methyl ester after final distillation
through an efficient fractionation column should have an iodine number of
172 to 178, less than 0.1% conjugated acids, and consist of only Cis acid.
Preparations of linoleate or more highly unsaturated acids should always
be kept in evacuated or nitrogen-filled ampules at low temperature to
prevent oxidation of these very easily oxidizable esters or acids.

Rollet? and McCutcheon’ have developed similar bromination-debromi-
nation procedures for preparation of linolenic acid from the acids of linseed
oil. Arachidonic acid is prepared by bromination-debromination of the
acids from beef adrenal phospholipids® or from fresh hog liver fatty acids.
With these preparations the danger of oxidation is even greater and
extreme care must be taken to prevent entry of oxygen. The quality of the
preparation is roughly indicated by its color—the lighter the color, the
better the preparation. Spectral absorption should be low in the ultra-
violet, and iodine number should be close to theoretical. Hydrogen number
is a better characteristic constant for the highly unsaturated acids than is
iodine number.

Low-temperature crystallization methods developed by Brown and his
coworkers!’-? provide a means of preparing these acids without danger
of isomerization by chemical treatment. The low-temperature crystalliza-
tion methods yield natural isomers, but numerous recrystallizations are
required to approach purity.

Chromatographic procedures yield very pure natural products, but as yet
the method has not been used on a large scale. Swift et al. and Riemen-
schneider et al. have prepared linoleate’ and linolenate “ in a high degree of

5A. Rollet, Hoppe-Seyler’s Z. physiol. Chem. 62, 410 (1909).

6 J. W. McCutcheon Org. Syntheses 22, 75 (1942).

7A. Rollet, Hoppe-Seyler’s Z. physiol. Chem. 62, 422 (1909).

8 J. W. McCutcheon, Org. Syntheses 22, 82 (1942).

9W.C. Ault and J. B. Brown, J. Biol. Chem. 107, 615 (1934).

10 J. B. Brown and J. Frankel, J. Am. Chem. Soc. 60, 54 (1938).

1G. Y. Shinowara and J. B. Brown, J. Am. Chem. Soc. 60, 2734 (1938).

2D. T. Mowry, W. R. Brode, and J. B. Brown, J. Biol. Chem. 142, 671 (1942).

13C, EK. Swift, W. G. Rose, and G. 8. Jamieson, Oil & Soap 20, 249 (1943).

4 R. W. Riemenschneider, 8. F. Herb, and P. L. Nichols, J. Am. Oil Chemists’ Soc.
26, 371 (1949).

ie ESSENTIAL FATTY ACIDS

purity for use as analytical standards, and White and Brown! and Herb
et al.'® have isolated arachidonate by chromatography for the same purpose.
Of the methods available, chromatography yields the purest product, but
can handle only small amounts. Bromination-debromination is most con-
venient for preparing large quantities, but the product contains isomers of
the desired unsaturated acid.

B. SYNTHESIS

Only linoleic acid of the polyunsaturated acids has yielded to a total
synthesis. Noller and Girvin™ and Baudart!® have succeeded in the synthe-
sis of an unnatural isomer of 9,12-octadecadienoic acid by condensation
methods. More recently Walborsky et al.1® synthesized natural czs,cis-
linoleic acid through acetylenic compounds:

OCH:
C;H;,;C=CCH.Br + BrMgC=C (CH,),CH CuBr: .

OCH,
OCH,

C;H,,C=C—CH.C=C(CH.) 7CH

hydro]lysis OCH:

<AgNOs
C;Hi1C==C—CH.—C==C (CH.);CHO

He

cis, cis-9,12-octadecadienoic acid

This latter synthesis will be useful in the preparation of radioactively
labeled linoleic acid for use in pertinent metabolic studies.

C. COMMERCIAL PREPARATIONS

The investigator should be critical of the source of commercially available
linoleic, linolenic, or arachidonic acids. Products available commercially
under the name of linoleic acid are far from pure and in all likelihood are
merely corn oil or cottonseed oil fatty acids. One such preparation labeled
C.P. linoleic acid was analyzed in the author’s laboratory and found to

15M. F. White and J. B. Brown, J. Am. Chem. Soc. 70, 4269 (1948).

16S. F. Herb, R. W. Riemenschneider, and J. Donaldson, J. Am. Oil Chemists’ Soc.
28, 55 (1951).

17C. R. Noller and M. D. Girvin, J. Am. Chem. Soc. 59, 606 (1937).

18 P. Baudart, Bull. soc. chim. [5] 11, 336 (1944).

19 H. M. Walborsky, R. H. Davis, and R. Howton, J. Am. Chem. Soc. 73, 2590 (1951).

II. CHEMISTRY 273

contain less than 50% linoleic acid. It was packaged in an ordinary screw-
cap bottle and thus had access to oxygen of the air. The essential fatty
acids and their compounds are readily attacked by oxygen, and any
preparation which is not stored in an ampule or sealed under nitrogen or
under vacuum should not be accepted as pure. To the writer’s knowledge,
the only source of authentic bromination-debromination linoleic and lino-
lenic acids and their esters is the Hormel Foundation of Austin, Minnesota.
Arachidonate concentrate containing a maximum of 70 % arachidonic acid
was once available from the Nutritional Biochemicals Corporation.

D. CHEMICAL PROPERTIES

The polyunsaturated acids belonging to the essential fatty acid group are
similar in their properties. Chemically their reactivity is restricted to re-
actions of double bonds and of carboxyl groups, and segregation of these
acids on the basis of chemical reaction is difficult and only partial. For prac-
tical purposes, there is negligible difference between the carboxyl reactivi-
ties of the various members of the group, and all separative procedures
based upon chemical reactivity utilize the differences in the number of
double bonds of the various members of the group. Thus, the preparation
of linoleate is based upon the insolubility of tetrabromostearic acid in pe-
troleum ether, and the preparation of linolenic acid is based upon the in-
solubility of hexabromostearic acid and the solubility of tetrabromostearic
acid in ethyl ether. The more highly unsaturated acids form polybromides
which are all virtually insoluble in ether, and thus separation of the higher
members of the series is impossible by this method. For example, arachi-
donic acid prepared by bromination-debromination always contains a con-
siderable amount of pentaenoic acid.

All the essential fatty acids add halogens, and this reaction is the basis
of the commonest method for determination of total unsaturation. Various
halogenating reagents have been employed for the addition reaction, but
the results are expressed as grams of iodine absorbed per 100 g. of fat, and
the value is called the iodine value.

Thiocyanogen also adds to double bonds, but not to all double bonds, pre-
sumably because of steric hindrance. Thus oleic acid adds one mole of
thiocyanogen, linoleic acid adds one, and linolenic acid adds two. The thio-
cyanogen value, combined with the iodine value, can be used to calculate
the linoleic acid content of fats.

The unsaturated linkages in the essential fatty acids are readily hydro-
genated, and in hydrogenated vegetable oils the essential fatty acid content
is reduced. Partial hydrogenation disproportionately reduces the essential
fatty acid content of fats, for the polyunsaturated acids are preferentially
hydrogenated. Although hydrogenation of essential fatty acids is the basis

274 ESSENTIAL FATTY ACIDS

of preparation of shortenings from liquid vegetable oils, the resultant prod-
ucts contain essential fatty acids in quantities in the same order as that
found in other edible fats.”°

The essential fatty acids, which contain only cis double bonds, can be
catalytically converted by selenium isomerization to trans forms. The iso-
merization has been accomplished on both linoleic! and linolenic” acids.
The isomers thus formed have no biological value, although they are
metabolized by the animal.”

Treatment of the essential acids with alkali at high temperatures causes
the double bonds to shift, yielding conjugated isomers. Because such con-
jugated polyenes possess strong ultraviolet absorption, this reaction has
been made the basis of a spectrophotometric method for the analysis of
the essential acids. The conjugated acids themselves have no biological
potency.

The saponification and esterification reactions are the only important
carboxyl reactions of the essential fatty acids. However, the differences in
saponification or esterification rates of the different long-chain acids are
so small as to be of no practical importance. For discussion of these re-
actions, the reader is referred to Markley.”

EK. OXIDATION

The polyene acids are highly susceptible to oxidative rancidity. After a
variable induction period, the oxygen absorption increases autocatalyti-
cally. The maximum rates of oxidation of the various polyunsaturated acids
increase with each additional double bond in the molecule, so that with
arachidonic acid autoxidation is very rapid. In Fig. 2 the rates of oxidation
of the esters of essential fatty acids are compared with ethyl oleate. The
rates of oxidation are roughly 1:40:100:200 for oleate, linoleate, linolenate,
and arachidonate, respectively.?*

The autoxidation of the essential fatty acids is accompanied by radical
changes in the ultraviolet absorption spectrum.?® The major changes are
development of a strong absorption in the conjugated diene region (2340 A.)
and a smaller increase around 2700 A. The former has been shown to be
due to the conjugated diene hydroperoxide which is the primary product
of oxidation, and the latter is probably due to unsaturated ketonic sec-

20H. J. Deuel, Jr., S. M. Greenberg, L. Anisfeld, and D. Melnick, J. Nutrition 45,
5385 (1951).

21 J. P. Kass and G. O. Burr, J. Am. Chem. Soc. 61, 1062 (1939).

2 J. P. Kass, J. Nichols, and G. O. Burr, J. Am. Chem. Soc. 63, 1060(1941).

23 R. T. Holman, Proc. Soc. Exptl. Biol. Med. 76, 100 (1951).

24K. §. Markley, Fatty Acids. Interscience Publishers, New York, 1947.

25 R. T. Holman and O. Elmer, J. Am. Oil Chemists’ Soc. 24, 127 (1947).

26 R. T. Holman and G. O. Burr, J. Am. Chem. Soc. 68, 562 (1946).

II. CHEMISTRY 215

ondary products. The general mechanism of oxidation is given on the
following page.

For a more detailed discussion of autoxidation of the essential fatty acids,
the reader is referred to a recent review.”

Oxidative rancidity of edible oils is due largely to the oxidation of the
essential acids contained in them. The products of oxidation of these
acids, at least in large quantities, are probably harmful, for rancid fats are
toxic.

Moles Oxygen\\ Mole Ester
° o
> n

50 100 Hours

Fic. 2. Rates of oxidation of ethyl oleate (1), ethyl linoleate (2), ethyl linole-
nate (3), and methyl arachidonate.?® (4)

F. PHYSICAL PROPERTIES

The essential fatty acids are colorless oils at room temperature. Their
light absorption in the ultraviolet region is negligible but increases with
decrease in wavelength. These acids do exhibit strong maxima in the
vacuum ultraviolet, or Schumann region.?°

The acids themselves boil under high vacuum with some decomposition,
but their esters can conveniently be distilled at pressures of 2 to 4mm. Hg.
They have negligible solubility in water, but are highly soluble in organic
solvents such as ether, ethanol, acetone, and chloroform. They form col-
loidal soap solutions above pH 9. By virtue of their high unsaturation,
these compounds have high refractive indexes, and the refractive index

278. Bergstrom, and R. T. Holman, Advances in Enzymol. 8, 425 (1948).

28 I. I. Rusoff, R. T. Holman, and G. O. Burr, Oil & Soap 22, 290 (1945).

297. I. Rusoff, J. R. Platt, H. B. Klevens, and G. O. Burr, J. Am. Chem. Soc. 67,
673 (1945).

ESSENTIAL FATTY ACIDS

276

"OO

—HO—HO=HO—HO=HO—HO

hi

“OO

—HO—HO—HO=HO—HO=HO—HO—

|

—HO—HO=—HO—HO—HO=HO—‘HO—

i

a
SH) 0h =o — ooo
—HO—'HO—HO=HO—HO=HO*HO—

—HO—HO=HO—HO—HO=HO—HO—
iat €I al Il OL 6 8

prqay
sou BUOSOY

(a[]noe,our
9} 89[0UT|
zeqjoue

m101})

“H+

——————
-H-—

HOO

—HO—HO=HO—HO=HO—HO—*HO—

10

HOO

|

—HO—0—HO— 0 10H Ba
10
— —HO—B0=—50—B0—bD—20 no

HOO

—HO—HO=HO—HO—HO=HO—HO—

III. BIOCHEMICAL SYSTEMS PAT

of mixed acids or oils can be used as a measure of the unsaturation. A few
important physical and chemical properties are summarized in Table I.

TABLE I
PHYSICAL AND CHEMICAL PROPERTIES OF THE ESSENTIAL Fatty AcIps
Linoleic Linolenic Arachidonic
Refractive index, 50° 1.4588 1.4678 =
Specific gravity, 20°/4° C. 0.903 0.914 =
Melting point —65° —11° —50°
Boiling point 149.5/1 mm. — 160-165/1 mm.
182.4/4 mm. 184/4 mm.
Molecular weight 280.4 278.4 304.5
Iodine value 181.0 PD 333.5

III. Biochemical Systems
RALPH T. HOLMAN

The biological role of the essential fatty acids has not yet been elucidated,
and only a few pieces of isolated information bear on the matter. The poly-
unsaturated acids present in animal tissues are found in greater concentra-
tion in the phospholipids than in the depot fats,’ ? and the concentration of
_ these acids in the total fatty acids of the so-called vital tissues is greater
than in the remainder of the carcass or in skeletal muscle. Changes in phos-
pholipid unsaturated fatty acids are greater than corresponding changes in
neutral fat fatty acids caused by fat deficiency or supplementation. It
appears that these substances are associated with tissue structural elements
rather than depot lipids, for alkaline hydrolysis of the tissues gives greater
yield of these acids than does exhaustive extraction with solvents.

A study of the effects of fat deficiency upon enzyme systems should re-
veal some information about the metabolic role of the essential fatty acids.
Thus far the effects upon a few enzyme systems have been reported. Hess
and Violler® found that lipase activity in blood of rats decreases to about
half of normal on a fat-deficient diet, but that cholinesterase activity
remained unchanged. Kunkel and Williams? found that fat deficiency caused
an increase in liver cytochrome oxidase activity and an increase in endogen-
ous respiration, whereas succinoxidase activity remained normal. They

1T. G. Rieckehoff, R. T. Holman, and G. O. Burr, Arch. Biochem. 20, 331 (1949).
2R. Reiser, J. Nutrition 42, 325 (1950).

3 W. Hess and G. Viollier, Helv. Chim. Acta 31, 381 (1948).

4H. O. Kunkel and J. N. Williams, J. Biol. Chem. 189, 755 (1951).

278 ESSENTIAL FATTY ACIDS

reported that choline oxidase activity rose slightly in the deficient state.
With the exception of choline oxidase, all enzyme systems reverted toward
normal activities upon the administration of linoleate to the deficient ani-
mals. Thus it appears that essential fatty acid 1s involved in at least some
enzyme systems. The lipid in concentrated cytochrome oxidase prepara-
tions has been found to contain polyunsaturated fatty acids, and the lipo-
protein of blood is subject to autoxidation, as are the polyunsaturated
acids, indicating that these acids occur in important lipoprotein structures.

The essential fatty acids are themselves the substrates for a highly specific
enzyme in the plant kingdom. The occurrence of this enzyme, lipoxidase,
in animal tissues is not conclusively established. For more detailed informa-
tion concerning this enzyme and its action, the reader is referred to several
reviews.>” Lipoxidase attacks compounds in which there is multiple methyl-
ene-interrupted unsaturation of all czs configuration. The commonest and
simplest substrate is linoleate, containing the structure

— CH

|

CH

|

CH
This enzyme is of considerable importance in plant seeds where linoleic and
linolenic acids are storage materials, but it is doubtful that the lipoxidase is

of much metabolic importance in animal tissue if it exists there, for the
potential substrate is largely bound in structural lipid.

IV. Specificity of Action
RALPH T. HOLMAN

There has been considerable uncertainty over the comparative curative
values of the various fatty acids and over their minimum daily requirements.
Through the mass of collected information it is clear that the polyunsat-
urated fatty acids are divided into two classes: those that cure skin symp-
toms and restore growth, and those that restore growth but do not cure
skin symptoms.

Linoleic and arachidonic acids both restore growth and cure skin symp-
toms. Turpeinen! reported that arachidonic acid was three times as effective

°§. Bergstrém and R. T. Holman, Advances in Enzymol. 8, 425 (1948).

6R. T. Holman and S. Bergstrém, in The Enzymes, Vol. 2, Part 1, Chapter 60.
Academic Press, New York, 1951.

7W. Franke, Fette u. Seifen 52, 11 (1949).

10. Turpeinen, J. Nutrition 15, 531 (1938).

IV. SPECIFICITY OF ACTION 279

as linoleic acid, using the method of minimum dose for maximum response.
Hume et al.2 found arachidonic acid doubly as effective as linoleic acid when
fed at low levels. Tests by Burr et al.’ indicate a slightly greater response

TABLE II
BrotocicaL Activity oF UNSATURATED AcIpDS AND THEIR DERIVATIVES
Growth Skin Water
Compound effect effect intake References
cis ,cis-9,12-Octadecadienoic acid + + Normal 3
trans ,trans-9,12-Octadecadienoic acid 0 0 5, 6
Conjugated linoleic acid = _ 5-7
10,13-Nonadecadienoie acid 0 0 8
11,14-Eicosadienoiec acid 0 0 8
9 ,10-Octadecadienoic acid 0 0 8
9,11-Octadecadienoic acid 0 0 8
Oleic acid 0 0 9, 10
Elaidie acid 0 0 11
12-Octadecenoie acid 0 0 1
Linoley] alcohol “ + 1
Erucic acid 0 0 1
Ricinoleie acid 0 0 1
Linolenic acid a 0 High Cn Aik
Hexahydroxystearic acid + 0 12
Elaidolinolenic acid + 0 6, 7
Eleostearic acid 0 0 9
Dioxidostearic 0 0 128
Trihydroxystearic 0 C 128 °
Tetrahydroxystearic 0 C 12% *
’ Chaulmoogric acid 0 0 i,
Clupanodonic acid Toxic 13
Arachidonic acid + + Normal iB
Docosahexaenoie acid + 0 12
Cod liver oil esters + 0 High

2. M. Hume, L. C. A. Nunn, I. Smedley-Maclean, and H. H. Smith. Biochem. J.
34, 379, 384 (1940).

3G. O. Burr, J. B. Brown, J. P. Kass, and W. O. Lundberg. Proc. Soc. Exptl. Biol.
Med. 44, 242 (1940).

4S. M. Greenberg, C. E. Calbert, H. J. Deuel, Jr., and J. B. Brown, J. Nutrition
45, 521 (1951).

5G. O. Burr, Chemistry and Medicine, p. 101. University of Minnesota Press, 1940.

6R. T. Holman, Proc. Soc. Exptl. Biol. Med. 76, 100 (1951).

7R. T. Holman, Proc. 3rd Conf. on Research, Am. Meat Inst. Univ. Chicago p. 1
(1951); Fette wu. Setfen 53, 332 (1951).

8 P, Karrer and H. Koenig, Helv. Chim. Acta 26, 619 (1943).

9G. 0. Burr, M.M. Burr, and E.S. Miller, J. Biol. Chem. 97, 1 (1932).

10 Hf. M. Evans and S. Lepkovsky, J. Biol. Chem. 96, 148 (1932).

1R. G. Sinclair, J. Nutrition 19, 131 (1940).

12H. M. Hume, L. C. A. Nunn, I. Smedley-Maclean, and H. H. Smith, Biochem. J.
32, 2162 (1938).

13 U. Tange, Sci. Papers Inst. Phys. Chem. Research (Tokyo) 20, 13 (1933).

280 ESSENTIAL FATTY ACIDS

from arachidonic acid than from linoleic acid. Very recent tests in which
natural methyl arachidonate, prepared by physical means, was compared
with bromination-debromination linoleic acid, seem to indicate that the
arachidonate was six times as effective as the linoleic acid in promoting
growth (see Greenberg ef al.4). The proper comparison between comparable
natural linoleate and arachidonate esters has not yet been made.

Linolenic acid and docosahexaenoic acid restore growth but do not
improve the animal’s skin condition, indicating that these acids are not
able to meet all the metabolic requirements for polyunsaturated fatty acid.
Many unsaturated fatty acids and compounds related to them have been
tested for potency. The results of such tests are gathered in Table II.
From the table it is apparent that only linoleic and arachidonic acids are
fully effective in restoring fat-deficient rats to normal, and that linolenic
and docosahexaenoic acids allow growth without meeting the requirement
for normal skin condition. This points to the dual character of the essential
fatty acid deficiency.

V. Biogenesis and Metabolism
RALPH T. HOLMAN

The formation of linoleic and linolenic acids in the plant is generally
considered to be a conversion from carbohydrate. The mechanism has not
been worked out beyond the observation that in a ripening seed the fat
content increases whereas the carbohydrate decreases. The reverse is true
during germination,! a time when linoleic and linolenic acids are oxidized
preferentially, lipoxidase activity is initially high, and catalase activity is
temporarily high.

The synthesis of the essential fatty acids in the animal body appears to
be a slow and inadequate process. Although many reports indicate that
essential fatty acid is not synthesized by the animal,?: * more recent reports
indicate that synthesis can take place to a limited degree.*: > The polyun-
saturated fatty acids are dietary essentials because the animal cannot
synthesize them in sufficient quantity for its needs. The animal, however,
is able to make interconversions in the polyunsaturated fatty acids. Smed-

1R. T. Holman, Arch. Biochem. 17, 459 (1948).

2K. Bernhard, H. Steinhauser, and F. Bullet, Helv. Chim. Acta 25, 83 (1942).

3R. Schonheimer, The Dynamic State of Body Constituents, p. 18. Harvard Univer-
sity Press, 1949.

4G. Medes and D. C. Keller, Arch. Biochem. 15, 19 (1947).

6’ V.H. Barki, R. A. Collins, E. B. Hart, and C. A. Elvehjem, Proc. Soc. Expil.
Biol. Med. 71, 694 (1949).

V. BIOGENESIS AND METABOLISM 281

ley-Maclean® has shown by means of polybromide analysis that the poly-
unsaturated acids decrease in rats in a deficiency state and that curing the
deficiency with linoleate restores the polyunsaturated content of the animal.
By means of the spectrophotometric method of analysis, this has been
verified in greater detail, showing that, when supplements of different oils’
or pure fatty acids’ are made, changes take place in the polyunsaturated
fatty acids in the animal, depending on the substance fed. From these
studies it appears that the dominant interconversions in some tissues are
the synthesis of arachidonate from linoleate and hexaenoate from linolenate.
Increases of pentaenoate were observed in blood and kidney as the result
of linolenate supplementation in fat-deficient rats. Hearts of fat-deficient

2 N= NORMAL CONTROL
HEART 6 | D= FAT DEFICIENT

O = STEARATE (no IF)

1=OLEATE (1. F)

2= LINOLEATE (2 F)

3 = LINOLENATE (3 F)

4 = ARACHIDONATE (4 F)
KIDNEY KIDNEY KIDNEY
4F 5 F 6 F

Z

°

On

é
Z
Z
a
Z
iA
a2
A
a
A
=
a
=
zZ
Zz
Z

A

ESTIMATED CONTENT OF FATTY ACIDS - %
NAAN

QUINN

Ny

b\

Fig. 3. Changes in the polyunsaturated fatty acids of the heart and kidney as
the result of single fatty acid supplementation.®-!°

rats accumulate a high proportion of trienoic acid which is not normally
present.? The administration of arachidonate to fat-deficient rats results
in its slow deposition.’ During a period of 7 weeks daily supplementation
with 30 mg. of methyl arachidonate, the total arachidonate content of the
animals increased equivalent to only 10 % of that fed. The recovery in poly-
unsaturated acid content of the animal parallels the slow recovery from the
deficiency disease and emphasizes the low rate of turnover of these acids in
the tissue. The typical metabolic changes in polyunsaturated acids are il-

6 I. Smedley-Maclean, The Metabolism of Fat. Methuen & Co., London, 1943. .

71. G. Rieckehoff, R. T. Holman, and G. O. Burr, Arch. Biochem. 20, 331 (1949).

8 C. Widmer and R. T. Holman, Arch. Biochem. 25, 1 (1950).

9R. T. Holman and T. 8. Taylor, Arch. Biochem. 29, 295 (1950).

10R. T. Holman, Proc. 3rd Conf. on Research, Am. Meat. Inst. Univ. Chicago p. 1
(1951); Fette w. Seifen 58, 332 (1951).

282 ESSENTIAL FATTY ACIDS

lustrated in Fig. 3, for the fatty acids of heart and kidney, in which these
changes are most pronounced.

The metabolic conversions have been studied using some unnatural
isomers of the essential fatty acids.!! The responses to the natural and

WEIGHT GAIN g/RaT FATTY ACIDS
g /RAT
100 10
=
SS
=
=
g
t= pede ==
= iI< ~|< hand we
50 Jul SS]ul a2 5 <=] | =lu
Ye] OS] =} 8/9 =! 13is
S/S/S 5/812 SeREE
~ ~ ~
12} s 2 fal t]e e]e/ S/S} s| 2] 8
</=] & Fl=] je S| S]O] YS) =) =
[3 FS < ol Sle} sla] 12
Y/OIS] ¢}/O] ef <
{ Frei = ed eS ees
| SV ESS aml) aa

TETRAENOIC ACID HE XAENOIC ACID

200 mg /rat 200 mg /rat

wu

ww ~ wa

~
100 << ey 3S Fai
Sac =| 8/5
5 ome) = SSS
cole =] Seas a Ss] =</O16
ole]= =|=<}/SIa = 2|2)=
SIISSIR Ses Sipe ep a} Go] S| =
HPS SH S11) ~ Albal| all
ala oil~— <x (e) Oli
ie) OES hae «| = Z| ola
= [|= <x ws = Sar] jill is
= S| { a| =

Fia. 4. Weight response, total fatty acid synthesis, tetraenoic acid synthesis, and
hexaenoic acid synthesis as the result of supplementation of fat-deficient rats with
isomeric essential fatty acids.1% 1!

unnatural isomers are summarized in Fig. 4. Only the natural (cis) isomers
promote normal growth, but elaidolinolenic acid (trans ,trans ,trans-lino-
lenic) promotes a suboptimal weight gain. Total fatty acid synthesis is
stimulated by the natural essential fatty acids and by ¢rans-linolenic acid.
Tetraenoic acid deposition is stimulated only by the natural essential acids,
and hexaenoic acid deposition by the natural isomers plus ¢rans-linolenate.

11R. T. Holman, Proc. Soc. Exptl. Biol. Med. 76, 100 (1951).

V. BIOGENESIS AND METABOLISM 283

Linolelaidate (trans ,trans-9 , 12-octadecadienoic) is totally ineffective as an
essential acid, and conjugated linoleic acid is deleterious.

Similar interconversions have been observed to take place in the chicken
as evidenced by the analyses of eggs of a laying hen.” There appears to be
a difference between the hen and the rat in the conversions possible. Reiser
reported conversion of dienoic acid to pentaenoic and tetraenoic acids and
the conversion of trienoic acid to all acids having from two to six double
bonds. However, a word of caution should be spoken concerning the ex-
tensive interpretation of spectrophotometric data of the type used for
these studies when as yet no analytical standards exist for the determinations
of five and six double bonds. It is questionable whether calculations of
saturated, oleic, linoleic, and linolenic acid contents are valid when the
estimations of four, five, and six double-bonded acids are only relative and
proper corrections for their concentrations cannot be made (see p. 287).

Two studies have been made of the metabolic conversions of conjugated
unsaturated fatty acids. It was observed by Miller and Burr® that the eleo-
stearic acid (9,11,13-octadecatrienoic acid) of tung oil was converted to
conjugated dieneoic acid in the rat. Reiser“ has confirmed this finding and
studied the metabolism of conjugated dienoic acid in the laying hen. How-
ever, the established conversion of conjugated trienoic acid to conjugated
dienoic acid in the animal should not be confused with similar conversions
in the non-conjugated essential fatty acids. The two groups of acids differ
markedly in physical and chemical properties and in biological activity,
and in their metabolism and in mechanism of 7n vitro oxidations they bear
no relation one to the other.

A. DIETARY FACTORS AFFECTING METABOLISM

The first recognized relationship of essential fatty acids to other dietary
essentials was that pyridoxine deficiency produces deficiency symptoms in
rats very similar to those of fat deficiency.!*!7 A double deficiency of fat
and pyridoxine results in rapid development of more severe symptoms, and
supplementation with either linoleate or pyridoxine reduces the severity
of the symptoms (see Fig. 5). Medes et al.4 studied the relationship of these
two dietary essentials, and the results showed that relief from the double

12 R. Reiser, J. Nutrition 44, 159 (1951).

13K. S. Miller and G. O. Burr, Proc. Soc. Exptl. Biol. Med. 36, 726 (1937).

4 R. Reiser, Arch. Biochem. and Biophys. 32, 113 (1951).

18 L. R. Richardson, A. G. Hogan, and K. F. Itschner, Univ. Missouri Research Bull.
333.

16 F. W. Quackenbush, H. Steenbock, F. A. Kummerow, and B. R. Platz, J. Nutri-
tion 24, 225 (1942).

7 KR. Jiirgens, H. Pfaltz, and M. Reinert. Helv. Physiol. et Pharmacol. Acta 3, 41
(1945).

284 ESSENTIAL FATTY ACIDS

deficiency by supplementation with either pyridoxine or linoleate caused
an increase in body fat and an increase in polyunsaturated acids. These
results indicated that a fat-deficient rat could synthesize unsaturated acids
if given pyridoxine.

Kummerow et al.'® made a similar study with linolenate and pyridoxine
and observed that doubly deficient rats given linolenate showed lower gains
and worsened symptoms than did unsupplemented controls. When pyri-
doxine was present in the diet, more polyunsaturated acid was present in
the tissues.

Experiments in the author’s laboratory indicate that total arachidonate
and hexaenoate in rats remains low when weanling animals are kept on a
at-deficient diet or a diet deficient in fat and pyridoxine. Preliminary

Fia. 5. Severe acrodynia in the rat. (Jiirgens et al.)

experiments showed that supplementation with either pyridoxine or lino-
leate caused increases in arachidonate and hexaenoate and that, when
linoleate plus pyridoxine is administered to the animals, greater increases
in arachidonate and hexaenoate are observed.!° The same was true for
linolenate.

A more extensive study of the pyridoxine~essential fatty acid relationship
has recently been completed.!® Rats which had been on a doubly deficient
diet one month after weaning age were given supplements of pyridoxine,
linoleate, pyridoxine plus linoleate, linolenate, or pyridoxine plus linolenate
for an 8-week period. Deficiency symptoms disappeared with linoleate
feeding but disappeared more rapidly with linoleate plus pyridoxine. Ani-
mals fed linolenate developed even worse symptoms, a finding parallel to
Kummerow’s observations on weights. Linolenate plus pyridoxine caused
18 F. A. Kummerow, E. Otto, G. Jacobson and P. Randolph, Trans. 4th Conf. on Biol.

Antioxidants, New York (1949).
19P. W. Witten and R. T. Holman Arch. Biochem. and Biophys. 41, 266 (1952).

V. BIOGENESIS AND METABOLISM 285

no change in symptoms. Total fatty acids in the rats were highest in the
animals given linolenate plus pyridoxine, and these animals also contained
the greatest amounts of hexaenoic acid. The animals receiving linoleate
plus pyridoxine synthesized the largest amounts of arachidonic acid. These
results again point to the dual nature of the polyunsaturated acids: Lino-
leate is converted by the animal principally to arachidonic acid and symptoms
of deficiency disappear, whereas linolenate is converted principally to hex-
aenoalte and symptoms are not alleviated but growth and fat synthesis are
stimulated. Pyridoxine is involved in the conversion process, possibly as a
coenzyme in some enzyme system.

Hove and Harris”? demonstrated a relationship between tocopherol and
linoleate. Tocopherol increased the effectiveness of suboptimal doses of
linoleate in preventing or curing essential fatty acid deficiency. The effect
was not limited to antioxidant action of the tocopherol within the gastro-
intestinal tract, for the effect was observed when the linoleate and tocoph-
erol were fed separately on alternate days. However, tocopherol fed alone
aggravates fat-deficiency symptoms.

Anisfeld et al.2! were unable to show any relationship between linoleate
and tocopherol requirements in fat-deficient rats, using growth response
as a criterion. The metabolic relationship between tocopherol and essential
fatty acids is not clear, but suggestions have been made that tocopherol has
a sparing effect on the unsaturated acids by virtue of its antioxidant prop-
erties.

The Conferences on Biological Antioxidants”? produced much discussion
of possible 7m vivo antioxidant action. Tocopherol is a well-known effective
antioxidant zn vitro, and suggestion has been made that it may act as an
antioxidant in living organisms. Little information has been brought to
bear on the problem. The keeping time of rat fats is extended by inclusion
of tocopherol in the diet.2? Tocopherol has been shown to stimulate the
utilization of linolenate and oleate synthesis in rats,'* and other antioxidants
have been shown to have a similar action. Dam and coworkers” observed
that tocopherol-deficient rats deposit an oxidized polymerized fatty ma-
terial when fed highly unsaturated fats. This deposition of oxidized fat
contains peroxides and is presumably the result of abnormal oxidation of
the unsaturated acids. The effect is prevented by administration of cystine

20}. L. Hove and P. L. Harris. J. Nutrition 31, 699 (1946).
217, Anisfeld, S. M. Greenberg, and H. J. Deuel, Jr., J. Nutrition 45, 599 (1951).
22 1st-Sth Conferences on Biol. Antioxidants Josiah Macy, Jr. Foundation, New York
(1946-1950).
23 W. O. Lundberg, R. H. Barnes, M. Clausen, N. Larson, and G. O. Burr, J. Bvol.
Chem. 168, 379 (1947).
*4 Hf. Dam and H. Granados, Acta Physiol. Scand. 10, 162 (1945).

286 ESSENTIAL FATTY ACIDS

or nordihydroguaiaretic acid,?® substances that have antioxidant action.
Conversely, feeding oxidized fat to animals has toxic effects.

These various observations suggest a possible antioxidant action for
tocopherol in sparing metabolic unsaturated acids. Hickman” has suggested
that perhaps a balance of pro-oxidants and antioxidants is maintained in
tissue to insure the proper metabolism of such substances as essential fatty
acids, that a ‘‘pro-oxidant condition” would promote oxidation and that an
“antioxidant condition” would protect the acids against oxidation. In recent
experiments testing this hypothesis some unexpected results were ob-
tained.” The conversions of linoleic and linolenic acids to more unsaturated
acids by fat-deficient rats were used as measures of utilization of these
substances, when they were administered either alone or in combination
with benzoyl peroxide (pro-oxidant) or tocopherol. Best growth response
and greatest synthesis of body fat was obtained with supplements of benzoyl
peroxide plus linoleate, and least with benzoyl peroxide alone. The toxic
effect. of benzoyl peroxide was thus reversed when fed with linoleate. The
synthesis of arachidonate from linoleate or linolenate was unaffected by
either benzoyl peroxide or tocopherol. Benzoyl peroxide increased the
formation of hexaenoate from linoleate but not from linolenate. Thus it
will be seen that the matter of relationship between tocopherol and essential
fatty acids is at present rather confused.

The riboflavin requirement for normal growth of rats has been found
to be increased when cottonseed oil is incorporated in the diet at a mod-
erately high level.2”7 Although the effect of the oil on intestinal flora may
be a cause of the diminished growth, an alternate explanation may be that
riboflavin is involved in the metabolism of unsaturated acids. Riboflavin
is a constituent of the enzyme which causes the reduction of cytochrome c,”8
and cytochrome c is an essential component of the enzyme system which
oxidizes saturated fatty acids.2? Thus, an increased metabolism of fat may
require an increased amount of riboflavin.

VI. Estimation
RALPH T. HOLMAN
A. SPECTROPHOTOMETRIC ANALYSIS

Observations by Dann and Moore! that prolonged saponification of oils
increases their ultraviolet light absorption has led to a spectrophotometric

25 H. Granados, E. Aaes-Jorgensen, and H. Dam, Brit. J. Nutrition 3, 322 (1949).
26 P| W. Witten and R. T. Holman, Arch. Biochem. and Biophys. 87, 90 (1952).

27 R. Reiser and P. B. Pearson, J. Nutrition 38, 247 (1949).

22 A. L. Lehninger and E. P. Kennedy, J. Biol. Chem. 173, 753 (1948).

29 H. Haas, B. L. Horecker, and T. R. Hogness, J. Biol. Chem. 136, 747 (1940).
1W. J. Dann and T. Moore, Biochem. J. 27, 1166 (1933).

VI. ESTIMATION 287

method of analysis for the essential fatty acids. Mitchell et al.2 reduced the
phenomenon to a quantitative method using 7.3 % KOH in ethylene glycol
as reagent and a treatment of 30 minutes at 180°. This has been modified
and improved by Brice and Swain.’ They used 11% KOH in glycerol to
reduce the variable blank absorption, and they have introduced background
corrections in the calculations to improve the accuracy of the method.
Protection of the sample during isomerization by an atmosphere of nitrogen
has also been recommended. The method, even in its present highly
developed form, is still subject to some limitations. The 11% KOH reagent
is optimum for linoleic and linolenic acids, but gives only a low degree of
the specific conjugated isomers of the more highly unsaturated acids.‘

The isomerization treatment of a hexaenoic acid results in production of
a variety of hexaene isomers in which six, five, four, three, or two of the
double bonds are conjugated. Thus the spectrum of such an isomerized
sample shows principal maxima at 3750 A., 3475 A., 3000 A., 2700 A..,
and 2340 A. Treatment of a pentaenoic acid will give rise to similar maxima
showing pentaene, tetraene, triene, and diene conjugation. Arachidonic
acid will give rise to three types of conjugation, linolenic acid two, and lino-
leic acid only one. Thus it is apparent that, if linoleic acid is to be deter-
mined in the presence of the more highly unsaturated acids, appropriate
correction must be made for each of the others.

The ratios of the various kinds of conjugated isomers derived from a
single polyunsaturated acid can be varied by alkali concentration and time
of treatment. For example, the conjugated tetraene yield from arachidonate
treated with 23% KOH for 8 minutes is twice that from arachidonate
treated with 11% KOH for 30 minutes.* Thus, use of 11% KOH reagent
for “determination” of linoleic and linolenic acids in animal fats can lead
to error, because the tetraene, pentaene, and hexene acid content of the
sample is ordinarily overlooked and no correction is made for it. The polyun-
saturated acids of all types are thus unknowingly reported as linoleic and
linolenic acids. The confusion is compounded by the present lack of em-
pirical analytical standards for the five and six double-bond values. Thus
any analysis which pretends to give the contents of various polyunsaturated,
oleic, and saturated acids of animal fats by use of the 11% KOH alkaline
isomerization method and iodine value should be regarded only as an
approximation.

By use of 23 % KOH reagent the sensitivity of the method for the four,
five, and six double-bond acids is doubled. The author has used this reagent
in estimating the relative contents of these acids in animal fatty acids. To

2 J. H. Mitchell, Jr.. H. R. Kraybill, and F. P. Zscheile, Ind. Eng. Chem. Anal.
Ed 15, 1 (1948).

3B. A. Brice and M. L. Swain, J. Opt. Soc. Amer. 35, 532 (1945).

4R. T. Holman and G. O. Burr, Arch. Biochem. 19, 474 (1948).

288 ESSENTIAL FATTY ACIDS

express the results in a relative manner, standard values based on certain
assumptions® have been employed, but it should be understood that the
results, as expressed, are tentative relative approximations. No attempt has
been made to extend the calculations to include linoleic and linolenic acids.

Objection has been raised that the spectral methods are not specific for
the essential fatty acids but will detect unnatural isomers as well. This
apparently is the case, judging from the results of a study of metabolism
of unnatural isomers of the essential fatty acids in which spectrophoto-
metrically detectable polyunsaturates were found in animals suffering from
fat-deficiency symptoms.* Even so, this 1s not a serious criticism of the
method, for it is used largely on natural substances where unnatural isomers
are not often encountered.

The spectrophotometric method is the best current method for deter-
mination of linoleic and linolenic acids in natural vegetable oils. The method
as employed by Brice and Swain,* requiring 100-mg. samples, is excellent
for use on vegetable oils where linolenic acid is the most unsaturated acid
present. Some micro modifications of the method have been developed for
smaller samples,’’ ® but these are of more limited application. When pure
hexaene and pentaene acids have been isolated and their responses measured
under various conditions, a modified method will be available for the quanti-
tative measurement of more highly unsaturated members of the group.
However, it seems doubtful that a spectrophotometric method of precision
for the simultaneous determination of all members of the polyunsaturated
group will be forthcoming.

B. CHEMICAL METHODS
1. THIOCYANOGEN VALUE

The estimation of the content of oleic, linoleic, and linolenic acids in a
fat can be made if the thiocyanogen value, iodine value, and saturated fatty
acid content are known. Theoretically, thiocyanogen adds to the double
bond of oleic acid, one of the two double bonds of linoleic acid, and two
of the three in linolenic acid. The halogen in the iodine value reagent adds
to all double bonds. Correction for the total saturated acid content of the
fat then allows the setting up of a set of simultaneous equations for the
calculation of the contents of the three unsaturated fatty acids. Although

5 ©. Widmer, Master’s Thesis, Agricultural and Mechanical College of Texas, 1949.

6R. T. Holman, Proc. Soc. Exptl. Biol. Med. 76, 100 (1951).

7L. C. Berk, N. Kretchmer, R. T. Holman, and G. O. Burr. Anal. Chem. 22, 718

(1950).

8 P. W. O’Connell and B. F. Daubert, Arch. Biochem. 25, 444 (1950).

8a Recently S. F. Herb and R. W. Riemenschneider have made a detailed study of
the effect of alkali on the conjugation of polyunsaturated fatty acids, and have es-
tablished some constants for pentaene and tetraene acids. J. Am. Oil Chemists Soc.
29, 456 (1952).

VI. ESTIMATION 289

the method has been made official by the American Oil Chemists’ Society,*?
it is not popular because the preparation of thiocyanogen reagent is tedious,
and the method is not applicable to oils containing acids of higher unsatura-
tion. The method is empirical in nature, for pure acids do not give the
theoretical thiocyanogen uptake. The method has been largely superseded
by spectrophotometric analysis for routine work.

2. POLYBROMIDE METHODS

An empirical method has been worked out for the determination of
linoleic acid in mixed fatty acids by bromination in cold petroleum ether.®
The weight of the insoluble tetrabromides formed from linoleic acid can
be related to the linoleic acid content of the sample. The yield of tetra-
bromides is affected by sample size and by the presence of other acids.
However, if the empirical conditions are strictly followed, the analysis
can be made quite accurate for determination of linoleic acid in samples
containing no linolenic or more unsaturated acids.

The hexabromide number of fatty acids can be used to determine the
linolenic acid content, if linolenic acid is the most unsaturated acid present.!°
The bromination is done in cold ethyl ether, and the weight of insoluble
hexabromides precipitated is a measure of the amount of linolenic acid in
the sample. The yield is affected by size of sample and the presence of other
acids. Application of a similar bromination technique to methyl esters allows
the detection and measurement of arachidonate, subject to the same errors
mentioned above." The advantage claimed for these polybromide methods
is that only the natural isomers of polyunsaturated acids and thus only
the physiologically active forms are measured. These methods employ a
sample of the order of 1 g.

C. ENZYMATIC METHOD

Soybean lipoxidase has been shown to be specific in attacking only lino-
leic, linolenic, and arachidonic acids.» * The reaction can be carried out
under conditions in which the conjugated end product is proportional to the
amount of linoleic acid in the sample. The method is applicable to the meas-
urement of 10 to 100 y of linoleic acid. The method has been used to detect
and measure linoleic acid in chromatographic eluates. The presence of
oleic and saturated acids partially inhibits the enzyme, thus limiting its

8b Official and Tentative Methods, American Oil Chemists’ Society Cd 2-38 (1946).
9M. F. White and J. B. Brown, J. Am. Oil Chemists’ Soc. 26, 385 (1949).

10M. F. White and J. B. Brown, J. Am. Oil Chemists’ Soc. 26, 133 (1949).

uM. F. White and J. B. Brown, J. Am. Oil Chemists’ Soc. 26, 85 (1949).

128. Bergstrém and R. T. Holman, Advances in Enzymol. 8, 425 (1948).

13R.T. Holman and S. Bergstrém, The Enzymes, Vol. 2, Part 1, Chapter 60. Aca-

demic Press, New York, 1951.
144R. T. Holman and W. T. Williams, J. Am. Chem. Soc. 73, 5285 (1951).

290 ESSENTIAL FATTY ACIDS

use to samples in which linoleic acid is the major component. Empirical
conditions might be found to extend its usefulness as a micromethod.
Because of its specificity, the enzymatic method should be useful in meas-
urement of total essential fatty acids on a microscale.

D. ANIMAL ASSAY

Recently a method has been developed for the assay of essential fatty
acids by measurement of the growth response induced by supplementing
fat-deficient rats with the test fat.!°> Male rats maintained on a fat-deficient
diet until growth ceased resumed growth when they were given supplements
containing linoleate, and this growth has been shown to be proportional

o

15 WEEKS
L)

100 12 WEEKS

A 9 WEEKS

3.8 8

6 WEEKS

&

3 wEeKs

WEIGHT GAIN- GRAMS

LOG. DOSE - MILLIGRAMS

° O2 04 0.6 Or) v) 12 1.4 16 te 20

Fic. 6. Relationship between weight gain and logarithm of dose of linoleate in
male fat-deficient rats.1®

to the logarithm of the linoleate dose. This proportionality is shown in Fig.
6. This assay has been used to measure the essential fatty acid content
of butter and margarine.'®

The advantage of this assay is that it measures physiological response,
non-active isomeric forms which would be measured by other analyses not
affecting the assay. It suffers the disadvantages of the inaccuracy of bio-

logical assay methods, the large sample required, and the long time con-
sumed for the assay.

158. M. Greenberg, C. HE. Calbert, E. E. Savage, and H. J. Deuel, Jr., J. Nutrition
41, 473 (1950).

16H. J. Deuel, Jr.. 8S. M. Greenberg, L. Anisfeld, and D. Melnick, J. Nutrition 45,
535 (1951).

VII. OCCURRENCE IN FOODS 291

Thus far no microbiological assay methods have been developed for es-
sential fatty acids, although it would seem possible to find an organism
which requires essential fatty acids. The demonstration of essential fatty
acid deficiency in insects points to their possible use as assay animals."

VII. Occurrence in Foods
RALPH T. HOLMAN

The essential fatty acids are abundant in nature. Most common vegetable
oils contain linoleic acid or linolenic acid, and lard and other animal
lipids contain linoleic, arachidonic, and more highly unsaturated acids.
Although linoleic and linolenic acids occur as major constituents in numer-
ous vegetable oils, arachidonate is found only as a minor constituent in

TABLE III
EssEeNnTIAL Farry Actp CONTENTS OF SOME COMMON FATS AND OILS
Fat or oil Linoleic acid Linolenic acid Arachidonic acid

Corn oil 34-42% — —
Cottonseed oil 40-48% — —
Soybean oil 50-60% 2-8% —
Olive oil 0.5-9.0% — —
‘Lard 6.0% == 0.4%
Butterfat 14% = Trace
Beef tallow 1-3% = —

common animal fats. Its concentration in certain tissue lipids, however,
may be as high as a few per cent.!: ? The presence of linolenic acid in animal
fats is probable, but unequivocal evidence for its presence is not available
except in a few cases. Many of the reports of linoleic acid and linolenic acid
in animal fats are based upon analyses not specific for linoleic acid, such as
iodine plus thiocyanogen values, or upon spectrophotometric analyses in
which the higher polyunsaturated acids were overlooked (see p. 287).
However, the occasional very high values reported for linoleate and lino-
lenate, even when such methods of analysis are used, speak loudly for the
occurrence of these acids in animal lipids.

As an illustration of the amounts of the essential fatty acids available
in common fats and oils, a few examples are given in Table III. Similar
17 G. Fraenkel and M. Blewett, J. Exptl. Biol. 22, 172 (1946).

1C. Widmer and R. T. Holman, Arch. Biochem. 25, 1 (1950).
2R. T. Holman and T. 8. Taylor, Arch. Biochem. 29, 295 (1950).

292 ESSENTIAL FATTY ACIDS

data can be found in standard references on the composition of fats and
oils, and for more information the reader is referred to them.*-*

The highly unsaturated fatty acids are abundant in fish oils such as cod
and shark liver oils, but their value as essential fatty acids is doubtful. The
structures of some acids isolated from these sources differ from the essential
fatty acids in that they have ethylene-interrupted rather than methylene-
interrupted polyunsaturated systems. The literature contains a few hints
that fish oil acids are toxic to the animal.

The widespread occurrence of linoleate in vegetable oils and arachidonate
in animal tissues makes the possibility of spontaneous fat deficiency unlikely
n man or in animals if a natural selection of food is allowed.

VIII. Effects of Deficiency

A. IN ANIMALS AND INSECTS
RALPH T. HOLMAN

1. Rat

The discovery of a fat-deficiency syndrome arose through the attempts
of Evans and Burr to produce a synthetic diet free of vitamin E.! The syn-
drome was first recognized and described as fat deficiency by Burr and
Burr,’ and since then the deficiency has often been called the Burr and
Burr syndrome. Simultaneously McAmis ed al. reported a reduced growth
on fat-free diets? but did not recognize the essential nature of the fatty
acids responsible. These workers were able to produce the deficiency because
of their departure from past practice in substituting sucrose for starch
as carbohydrate energy source. (Starch contains an appreciable amount
of unsaturated fat as a component of the granule.) Using sucrose as energy
source and exhaustively extracted casein as protein component, they were
able to reduce the fat content of the ration enough to demonstrate fat de-
ficiency. Rats maintained on fat-deficient rations reach an early growth
plateau, their skin becomes scaly and their coat rough, usually within a

3G. 8. Jamieson, Vegetable Fats and Oils. Reinhold Publishing Co., New York,
1943.

4 A.E. Bailey, Industrial Oil and Fat Products. Interscience Publishers, New York,
1945.

6'T. P. Hilditch, The Chemical Constitution of Natural Fats, 2nd. ed. John Wiley
and Sons, New York, 1947.

1H. M. Evans and G. O. Burr, Proc. Soc. Exptl. Biol. Med. 24, 740 (1927).

2G. O. Burr and M. M. Burr, J. Biol. Chem. 82, 325 (1929).

3 A. J. McAmis, W. E. Anderson, and L. B. Mendel, J. Biol. Chem. 82, 247 (1929).

VIII. EFFECTS OF DEFICIENCY 293

period of 3 to 4 months. Necrosis of the tail often develops in late stages
of the deficiency, and hematuria is sometimes observed. These outward
manifestations of derangement are not exhibited regularly, but histological
changes are invariably found in kidneys of fat-deficient rats.

Subsequent studies by Burr and his coworkers indicated that the water
consumption! and basal metabolic rate® of the fat-deficient rat are higher
than those of the normal animal. The ability of animals to reproduce is
impaired by fat deprivation.‘ In the female rat, conception is followed by
resorption of all or some of the fetuses,!: ®7 the gestation period is pro-

f

-
sd
4.
a
3

Fic. 7. Schachtelhalmschwanz—exaggerated caudal symptoms of fat deficiency
in the young rat. (From Guggenheim and Jiirgens.®)

longed, and only a variable and low number of live young are produced.
These, however, do not live long after birth, and even at the age of a few
days show damage of the tail (Fig. 7). The male rat on a fat-free diet
becomes sterile and will not mate.‘: 8 The ability to reproduce returns when
the animals are given diets containing adequate essential fatty acid.
When weanling rats are placed upon a fat-free diet, fat-deficiency symp-
toms begin to appear within 3 months, and the animals can be maintained
in the fat-deficient condition well over a year with moderate mortality.

4G. O. Burr and M. M. Burr, J. Biol. Chem. 86, 587 (1930).

5G. O. Burr and A. J. Beber, Proc. Soc. Exptl. Biol. Med. 31, 911 (1934).

6K. C. Maeder, Anat. Record 70, 73 (1937).

7H. M. Evans, §. Lepkovsky, and E. A. Murphy, J. Biol. Chem. 106, 431 (1934).
8H. M. Evans, S. Lepkovsky, and E. A. Murphy, J. Biol. Chem. 106, 445 (1934).

294 ESSENTIAL FATTY ACIDS

If the dams are maintained on a fat-free diet from the time of conception,
the young which are born show symptoms within a few days and do not
live beyond a month.’ The accentuated caudal degeneration observed

oe Masini:

Fia. 8 (a) Dog maintained on low fat diet. Age 9 months.

under these conditions (Schachtelhalmschwanz) appears within a few days
and may lead to the complete necrosis of the tail (see Fig. 7).

Older animals placed on fat-free diet do not spontaneously develop
classical fat-deficiency symptoms. Only by superimposing a strain can adult
animals be brought to show fat-deficiency symptoms. Barki ef al.!° severely

9M. Guggenheim and R. Jiirgens, Helv. Physiol. et Pharmacol. Acta 2, 417 (1944).
10 V, H. Barki, H. Nath, E. B. Hart, and C. A. Elvehjem, Proc. Soc. Exptl. Biol.
Med. 66, 474 (1947).

VIII. EFFECTS OF DEFICIENCY 295

depleted adult rats by restricting food intake until the animals were half
their starting weight. When the animals were then fed fat-free diet ad
libitum, classical symptoms of fat deficiency appeared. These symptoms
could be prevented or cured by ethyl linoleate. The symptoms disappeared

Fia. 8. (b) Dog maintained on low fat diet for 7 months, then given diet contain-
ing 5% of total calories as fat for 3 months. At 7 months this animal showed sim-
ilar symptoms to those illustrated in (a). (From Hansen and Wiese."!)

spontaneously after prolonged periods of ad libitum feeding of fat-free diet,
indicating the possibility of synthesis of some essential fatty acid.

2. WoG

The fat-deficiency syndrome has been observed in a variety of animals.
Hansen and Wiese!'- have made extensive studies in fat deficiency of dogs.

11 A, E. Hansen and H. F. Wiese, Texas Repts. Biol. Med. 9, 491 (1951).
12H. F. Wiese and A. E. Hansen, Texas Repts. Biol. Med. 9, 516 (1951).
13H. F. Wiese and A. HE. Hansen, Jexas Repts. Biol. Med. 9, 545 (1951).
14 A. E. Hansen, 8. G. Holmes, and H. F. Wiese, Texas Repts. Biol. Med. 9, 555 (1951).

296 ESSENTIAL FATTY ACIDS

Young puppies develop dermal symptoms after 4 to 5 months on a low fat
diet. The flaky desquamation of the skin and loss of hair is usually followed
by secondary infections. Alopecia and emaciation accompanied by a greasy
skin condition develop at later stages of deficiency. Dogs first develop an
excitable temperament and later become tremulous. This syndrome in dogs
can be prevented or cured by inclusion of unsaturated fats such as lard,
bacon fat, butterfat, or Crisco at a level of 16 % of the calories. One per cent
of calories as linoleate or arachidonate caused definite improvement, but
did not provide a complete cure within 6 months. Dogs have been main-

=
Fic. 9. Four-week-old chicks on fat-free and 2% cottonseed oil diets. (From
Reiser.!6)

tained on fat-deficient diet for as long as 6 years. Figures 8a and 8b show
a fat-deficient dog compared with his normal control.

3. CHICKEN

Karly experiments by Russell and his coworkers,” in which natural diets
were solvent-extracted to reduce the fat content, indicated that fat was not
needed in quantity for the growth of the chicken. The more recent work of
Reiser, in which synthetic fat-free diets were used, indicates that total
exclusion of fat from the diet of chicks causes slow weight gain and high
mortality.!® Chicks which received supplements of lard or cottonseed oil

18 W.C. Russell, M. W. Taylor, and L. J. Polskin, J. Nutrition 19, 555 (1940).
16R. Reiser, J. Nutrition 42, 319 (1950).

VIII. EFFECTS OF DEFICIENCY 297

grew as rapidly as chicks on a mash diet and showed a much lower mortality.
In chicks, fat deficiency is sometimes associated with subcutaneous edema,
and in a few cases general edema in the body cavity was observed. The
deficient birds were unthrifty and had poor feathering (Fig. 9). Removal of
yolk sacs from the newly hatched chicks did not seem to affect the develop-
ment of the deficiency.

4. Mouse

Mice kept on a fat-deficient diet develop the same general symptoms as do
rats.” The first symptom to appear is a dandruff-like dermatitis on the scruff
of the neck, which spreads over the animal. The dermatitis is followed by
necrosis of the extremities, and hematuria is observed in a few cases.
Decker e¢ al.!8 have shown that adult mice can be brought to a state of
chronic essential fatty acid deficiency without showing any external symp-
toms, and that in these animals acute symptoms may be brought out by
stresses such as injuries, pregnancy, and x-irradiation. Although most
studies in the field of essential fatty acid nutrition and metabolism have
been carried out upon rats, it would seem that the mouse could effectively
replace the rat as an experimental animal because mice develop deficiency
symptoms more quickly, consume less food, and require less space.

5. LARGE ANIMALS

No conclusive work has been done on the essential fatty acid require-
ment of large animals because of the tremendous cost of such experimenta-
tion. Cows require a minimum of 4% extractable fat in the diet for milk
production,!*: 2° and calves on a low fat diet do not thrive.*! However, no
evidence has been brought out to relate these observations to essential
fatty acid requirements. Hogs on a low fat diet have a drastically reduced

22-25

linoleic acid content, but no deficiency symptoms have been observed.”

All the vertebrates in which fat-deficiency symptoms have been produced
experimentally have straight alimentary canals. In the author’s laboratory,
attempts to develop fat deficiency in guinza pigs failed, although many
animals were maintained on a fat-free diet for periods of time longer than

a White, J. R. Foy, and L. R. Cerecedo, Proc. Soc. Exptl. Biol. Med. 54, 301
(1943).

18 A.B. Decker, D. L. Fillerup, and J. F. Mead, J. Nutrition 41, 507 (1950).

19 G. Gibson and C. F. Huffman, Michigan Agr. Expt. Sta. Quart. Bull. 244, (1939).

20 L.A. Maynard, K. E. Gardner, and A. Z. Hodson, Cornell Univ. Agr. Expt. Sta.
Bull. 722 (1939).

1 T. W. Gullickson, F. C. Fountaine, and J. B. Fitch, J. Dairy Sci. 25, 117 (1942).

22, N.R. Ellis and O. G. Hankins, J. Biol. Chem. 66, 101 (1925).

23 .N. R. Ellis and H. S. Isbell, J. Biol. Chem. 69, 219 (1926).

*4N.R. Ellis and H. S. Isbell, J. Biol. Chem. 69, 239 (1926).

2° N.R. Ellis and J. H. Zeller, J. Biol. Chem. 89, 185 (1930).

298 ESSENTIAL FATTY ACIDS

are required to develop fat deficiency in rats. It seems possible that for
animals having a cecum, intestinal bacteria may play a role in the syn-
thesis of essential fatty acids as well as of many of the water-soluble
vitamins.

6. INSECTS

Fat-deficiency phenomena are not limited to vertebrates, for Fraenkel
and Blewett?® were able to develop fat-deficient moths of four species.
Feeding fat-free diets during the larval stage prevents proper wing develop-
ment in the adult (see Fig. 10). Linoleic and linolenic acids prevent this
condition, but docosahexaenoic acid does not. However, the latter does
benefit growth. Results with these lower animals again point to the differ-
ences between the members of the polyunsaturated fatty acid group. Emer-

No emergence

8

Fia. 10. Response of Ephestia keuhniella wing development to various levels of
linoleic acid in the larval diet. 1. Normal wing, 8. Fat-free diet. (From Fraenkel
and Blewett.?5)

gence from the pupal stage and scale development of the adult wing paral-
lels the linoleic acid content of the diet. It appears that these moths or
other insects might be useful and inexpensive animals for essential fatty acid
assay.

As yet studies of the requirements of microorganisms for essential fatty
acids have not been made, nor have mutants requiring these acids been
isolated. It seems likely, however, that mutants requiring the acids could
be produced and isolated for use in the assay of these essential fatty acids.

7. HISTOPATHOLOGY

In addition to the gross abnormalities of fat-deficient animals mentioned
in the previous section, a few observations have been made on the histo-
pathology accompanying fat deficiency. Histological examinations of the
kidneys of deficient rats?”: 28 indicate that the renal tubules are calcified

26 G. Fraenkel and M. Blewett, J. Exptl. Biol. 22, 172 (1946).

27 V. G. Borland and C. M. Jackson, Arch. Pathol. 11, 687 (1931).

22 R. H. Follis, The Pathology of Nutritional Disease. Charles C Thomas, Spring-
field, Ill., 1948.

VIII. EFFECTS OF DEFICIENCY 299

_ and necrotic areas of the renal medulla appear in some cases. The cells of
the tubular epithelium are filled with lipoid and the tubular lumena contain
fat droplets and albuminous material. In cured animals the gross appearance
of the kidneys was normal and histologically no difference could be demon-
strated between cured and normal animals. The implication that choline
deficiency may have been a contributing factor’ to the histopathology thus
does not seem to be justified.

Histological studies of the reproductive system of the female rat® indicate
underdevelopment of the uterine mucosa and failure of corpora lutea to
develop during pregnancy. Embryos are resorbed or remain in utero beyond
the normal gestation period.

Fria. 11. Liver sections from fat-deficient and linoleate-fed young rats. Sudan
stain. Dark areas indicate fat deposition. (From Guggenheim and Jiirgens.°)

In the male fat-deficient rat the testis is macroscopically atrophied.®
The tubular epithelium degenerates, sperm are absent, and giant cells
are present. Seminal vesicles are small and flaccid, and the prostate is
atropic. The epididymis shows no significant deviation from the normal, but
the penis and associated glands are atrophied. If male rats in this condition
are given gonadotropin, the accessory sex organs tend to revert to normal,
indicating that the fat-deficient animal can probably still synthesize testos-
terone.2? However, the testes remain degenerate, indicating that they have
lost the ability to respond to gonadotropin. Essential fatty acids are thus
required to maintain testicular structure and function.

Histological examination of the livers of young fat-deficient rats showed

29 P. EK. Barrios, Master’s Thesis, Agricultural and Mechanical College of Texas,
1949

300 ESSENTIAL FATTY ACIDS

a fatty infiltration of the liver (Fig. 11) which is prevented by fat in the
diet.° In this respect, essential fatty acids are lipotropic.

The best histological studies on the skin lesions of fat deficiency have
been made on the dog.'* The abnormalities include loss of hair and des-
quamation, and compactness of the stratum corneum. The epidermis
thickens and there is edema in the dermis. Hair follicles become hyperkera-
tinized, the hair shafts become plugged, and the sebaceous glands enlarge.
Illustrations of the epidermis of dogs on low fat and normal diets are shown
in Fig. 12:

The pathological conditions recognized as essential fatty acid deficiency
are generally produced by maintaining animals on a low-fat or fat-free diet.
Such diets are used because it is difficult to prepare a diet free of essential

pete

7a ay!

Fie: 12: Pree of dogs (a) on control diet ae (b) on low-fat diet. 120. (From
Hansen et al.1*)

fatty acids without removing all fat. These diets supplemented with
essential fatty acids in adequate amounts still do not allow optimum growth,
reproduction, and lactation, but the pathological states developed on such
a dietary regime are still poorly characterized. The studies of pathology of
fat-deficient animals have not differentiated between the two types of fat
deficiency, but the pathological changes described are prevented or cured by
linoleate, thus making it reasonably certain that the conditions described
are due to essential fatty acid deficiency.

B. IN HUMAN BEINGS
ARILD E. HANSEN and HILDA F. WIESE
Specific information regarding the essentiality of linoleic acid and arachi-

donic acid in relation to human nutrition is completely lacking. Hence, in
a discussion of the human requirement for these fatty acids, it is necessary

VIII. EFFECTS OF DEFICIENCY 301

to interpret some of the observations and experimental findings which have
been made with human subjects on various fat regimens in the light of
fundamental facts established from experimental work with animals. Con-
sideration will be given to observations from studies on the use of low fat
diets in human subjects and to indirect evidences of possible special need
for fat as gained from other clinical studies. Inasmuch as correlations have
been found between dietary fat and the composition of the tissue lipids
as well as the gross and histologic alterations in dogs maintained on diets
with and without fat,?°-* these data will serve as a chief source of material
for evaluating fat requirements for man. Reference also will be made to
the measurement of diene, triene, and tetraene fatty acids in blood serum
as a possible means for ascertaining the human requirement for these
fatty acids.

1. Low Fat Diets In Human BEINGS

Reports concerning the effect of comparatively low fat diets in human
subjects are meager and of a rather general nature. An attempt was made as
early as 1919 by von Gréer* to determine the need of infants for dietary
fat. Detailed observations were made on two infants by this worker, and
although gain in weight was not entirely satisfactory, no striking abnormali-
ties developed within a period of 9 months. The low fat diet was discontin-
ued in one infant because of anorexia and in the second infant because of
infection. Holt and coworkers*®> used a low fat diet in the study of three
infants. Although these studies were of short duration, one of the infants
‘repeatedly developed a skin eruption when fat was lacking in the diet.
In 1937, von Chwalibogowski** observed two infants on a low fat regimen
for periods of 13 to 15 months. No typical findings were evident, and this
led the author to conclude that fat in the diet was not essential for infants.
Four human subjects have been studied in our clinic and laboratories for
periods of 2 to 23 months. The first was one of twins who, when on a low fat
diet, was found to have a definitely lower iodine number of the serum fatty
acids than the twin infant who was maintained on a normal diet. Another
infant on the low fat diet developed a rather intractable impetigo but
showed no other clinical abnormalities. The third case concerned a rather
extensive study which was made on a child with chylous ascites. Beginning

80 A. E. Hansen and H. F. Wiese, Texas Repts. Biol. Med. 9, 491 (1951).

31 H. F. Wiese and A. E. Hansen, Texas Repts. Biol. Med. 9, 516 (1951).

32 H. F. Wiese and A. E. Hansen, Texas Repts. Biol. Med. 9, 545 (1951).

83 A. KE. Hansen, 8. G. Holmes, and H. F. Wiese, Texas Repts. Biol. Med. 9, 555 (1951).

34 F. von Groéer, Biochem. Z. 97, 311 (1919).

3° L. E. Holt, Jr. H. C. Tidwell, C. M. Kirk, D. M. Cross, and S. Neale, J. Pediat.
6, 427 (1935).

36 A. von Chwalibogowski, Acta paediat. 22, 110 (1938).

302 ESSENTIAL FATTY ACIDS

at the age of 3 weeks, this infant was fed on a diet composed mostly of skim
milk and added carbohydrate, with supplements of minerals and vitamins
so that the food intake was complete in so far as other dietary essentials
were known. Development of this child was satisfactory, and his appetite
remained good. Owing to marked abdominal enlargement, at various inter-
vals it was necessary to remove the chylous fluid from the peritoneal cavity,
thereby causing marked fluctuations in his weight curve. During the mid-
summer season when the child was about 7 months of age, he developed
marked prickly heat. The eruption later took on the appearance of a chronic
dermatitis which persisted for a period of about 3 months, long after other
infants in the same ward who also had suffered from prickly heat were free
from eruption. Later the infant developed impetigo along with several other
infants in the same ward. Again the eruption persisted and was much more
resistant to treatment than in the other infants. On several occasions small
eczematous patches developed, but these responded readily to local treat-
ment. There was no family history of eczema, asthma, or hay fever. The
vitamin A and #-carotene values in the blood were similar to those of
control subjects. The iodine number of the total fatty acids of the serum
was much lower than that of normal infants of a similar age. There was no
change in the serum fatty acids or their iodine number following a cream
meal. Observations were terminated when the child died during an operation
in which an attempt was made to establish a continuity between the venous
circulation and the peritoneal cavity by means of reflecting the mternal
saphenous vein superiorly. As reported by Brown and coworkers,*” the
fourth patient was an adult male subject who was maintained on low fat
diet for a period of 6 months. His total intake was not more than 1 g. of
fat daily. A study of the respiratory quotient during the low fat period
showed an increase similar to that observed in rats. The iodine number of
the total fatty acids of the serum was definitely less while on the low fat
diet. The linoleic and arachidonic acid content of the serum determined as
tetra- and polybromides at the end of the low fat period were 3.2 and 1.9%
of the total fatty acids, respectively. Six months after the normal diet was
resumed, these values were 5.7 and 3.2 %, respectively.

Summary. Although experimental observations are scanty and of rela-
tively short duration, the effects of a low fat diet in human subjects appear
to be the following: Highly unsaturated fatty acids are not synthesized;
respiratory quotients follow the pattern of those of experimental animals
on low fat diets; possibly there is increased susceptibility to infection
(skin and respiratory); there may be a tendency to develop a mild derma-
titis; growth and development are not greatly affected; and the appetite
in most instances remains fairly satisfactory.

37 W. R. Brown, A. E. Hansen, G. O. Burr, and I. McQuarrie, J. Nutrition 16, 511
(1938).

VIII. EFFECTS OF DEFICIENCY 303

2. INDIRECT EVIDENCES OF NEED FOR ESSENTIAL Farry ACIDS
a. Observations on Patients with Eczema

Inasmuch as one of the outstanding features of the low fat diet in experi-
mental animals results in a development of skin abnormalities, the rela-
tionships which have been found between dietary fat and eczematous con-
ditions of the skin may be more than incidental. Study**: *° of the iodine
number of the serum fatty acids of patients with eczema disclosed definitely
lower values than for those of control groups of patients. For example:
four out of five infants under 2 years of age suffering eczematous eruptions,
three out of four children from 2 to 15 years, and over half of the subjects
over 15 years had iodine values of the serum fatty acids definitely below the
normal range. In the study by Hansen e¢ al.,*° there were 171 patients with
eczema and 101 in the control group. This observation has been confirmed
by a number of investigators.*!“** The tetra- and polybromide numbers of
the total fatty acids from pooled samples of serum from patients with
eczema were found to be lower than in serum samples from control sub-
jects.*° Finnerud e¢ al.“ found no consistent differences in the polybromide
numbers of the fatty acids between control subjects and eczematous pa-
tients, and there was no correlation between the polybromide number and
the clinical condition of the patient.

A number of workers*®: 4°: 43: 44, 46-48 have reported that the addition
of fat rich in unsaturated fatty acid to the diet of patients with chronic
eczema favorably influences the condition of the skin. In studies in our own
‘clinics, before a change was made in therapeutic regimen the patients usually
were observed from periods of 3 weeks to 3 months. As a rule, fresh lard or
vegetable oil was added beginning with 1 teaspoonful two or three times a
day, and gradually increased so that by 2 weeks the patient was receiving
the equivalent of 1 tablespoonful two or three times a day. In most in-
stances no particular difficulty was encountered in consuming the extra fat;
however, in some instances the patients preferred to use fresh lard as a

38 A. EK. Hansen, Proc. Soc. Exptl. Biol. Med. 30, 1198 (1983).

39 A. E. Hansen, Am. J. Diseases Children 58, 933 (1987).

40 A. E. Hansen, E. M. Knott, H. F. Wiese, E. Shaperman, and I. McQuarrie, Am.
J. Diseases Children 78, 1 (1947).

41H. K. Faber and D. B. Roberts, J. Pediat. 6, 490 (1935).

42 7,. Schornstein, Z. Kinderheilk. 59, 52 (1937).

43.C. W. Finnerud, R. L. Kesler, and H. F. Wiese, Arch. Dermatol. and Syphilol.
44, 849 (1941).

44 A.V. Stoesser, J. Allergy 18, 29 (1947).

45 W. R. Brown and A. E. Hansen, Proc. Soc. Exptl. Biol. Med. 36, 113 (1937).

46 A. Kk. Hansen, Proc. Soc. Exptl. Biol. Med. 31, 160 (1933).

47'T. Cornbleet and E. R. Pace, Arch. Dermatol. and Syphilol. 31, 224 (1935).

48. Azerad and C. Grupper, Bull. mém. soc. méd. hép. Paris 64, 21 (1948); Bull.
soc. frang. dermatol. syphilig. 55, 24, 230 (1948); Semaine hép. Paris 25, 684 (1949).

304 ESSENTIAL FATTY ACIDS

spread on crackers or bread or mixed with cinnamon, jelly, or jam. When
favorable results occurred, usually the first evidence of improvement was
observed 2 to 4 weeks after the addition of fat to the diet. From the clinical
viewpoint it was felt that if improvement was to occur it would be evident
within 2 or 3 months. It must be emphasized that, although the attempt
was made to change only one factor, it was necessary to continue the same
local medication and dietary regimen as used previously and to prevent
injury from scratching. One continued to be on the alert for possible
allergens as playing a role. In order to control the clinical observations as
closely as possible, graphic records were used, as illustrated in a previous
report.*°

pa Babies Children Adults

Se ecs Eee

excellent 43 m4 a 5

Pie but P77 \2 VIA? WU?

Poor or none (13 eee |g a Nr
The entire gro 100 cases

Good or beens Buses) 52

excellent Ee aa ee

eeeaiter: but yt. 33

Poor or none | rao | (74

Fia. 13. Summary of results of Grupper and Azerad in a study of 100 patients with
eczema. (A. Burgos.48#)

Azerad and Grupper“’ of Paris summarized their experience with the ad-
dition of fat to the diet of 100 patients, as indicated in Fig. 13. These
results have been confirmed in a second series of 100 patients (personal
conversation, 1950). One of the most striking clinical results obtained by
Azerad and Grupper is illustrated in Fig. 14 through the courtesy of these
workers. The clinical results, in general, are similar to those reported earlier
by Hansen et al.4° It must be pointed out that not all workers*®-* report |
the addition of fat to the diet to be so helpful in altering the course of
patients with eczema.

48a A. Burgos, Contribution au traitment de ]’eczema par les acides gras non saturés.
R. Toulon, Paris, 1949.

49§. J. Taub and S. J. Zakon, J. Am. Med. Assoc. 105, 1675 (1935).

50 J. KE. Ginsberg, C. Bernstein, and L. V. Iob, Arch. Dermatol. and Syphilol. 36, 1033
(1947).

5tN. N. Epstein and D. Glick, Arch. Dermatol. and Syphilol. 35, 427 (1937).

VIII. EFFECTS OF DEFICIENCY 305

It may be of some significance that the incidence of eczema>? and skin
eruptions® is less in infants maintained on breast milk than on cow’s milk
mixtures. Admittedly many factors may be concerned with this difference;
however, it may be pointed out that the content of linoleic acid is much
greater in breast milk than in cow’s milk.*

Fra. 14. Photographs of a patient included in the study of Grupper and Azerad.
(A. Burgos.*8#)

b. Low Fat Intake Because of Necessity or Because of Derangement in Fat
Absorption a

It is recognized that a prolonged low fat diet leads to a craving for fat.
This is due probably to the satiety value of fat per se rather than to a need
for specific acids. It has been observed, however, that when people are
deprived of fat for long periods of time, an increase in the incidence of skin
abnormalities seems to appear. Freudenberg of Basel, Switzerland, in a
personal conversation, stated that he had observed this phenomena in
Central Europe in both World Wars I and II. It may well be that some of

8 C. G. Grulee and H. N. Sanford, J. Pediat. 9, 223 (1936).
53H. C. Robinson, Am. J. Diseases Children 59, 1002 (1940).
54°T. P. Hilditch and M. L. Meara, Biochem. J. 38, 29 (1944); 38, 437 (1944).

306 ESSENTIAL FATTY ACIDS

this reflects a need for certain fatty acids. Luzzatti and Hansen,** in study-
ing the serum lipids of patients suffering from the celiac syndrome, found
the iodine number of the serum fatty acids to be abnormally low. This
finding is in keeping with a relatively low intake of unsaturated fatty acids.
In addition, in reviewing the literature Luzzatti and Hansen found that a
number of authors®®-*8 reported a high incidence of abnormalities of the
skin in patients with poor fat absorption. This type of disorder is almost
always associated with malnutrition and recurrent respiratory infections,
two symptoms which were consistently observed in fat-deficient dogs.*°
To accentuate the situation, patients with inability to absorb fat adequately
usually are placed on a low fat diet, by their own choice or upon the recom-
mendation of the physician. If there is a need for specific unsaturated fatty
acids one might expect latent abnormalities to become manifest.

c. Summary

Study of the serum lipids in patients with eczema suggest that abnormally
low iodine values are common. The feeding of dietary fats rich in the un-
saturated fatty acids to patients suffering from eczema frequently appears
to improve the condition of the skin. Prolonged maintenance on a diet low
in fat because of faulty absorption regularly results in a low iodine value
of the serum fatty acids. Children suffering from faulty fat absorption quite
commonly develop chronic dermatitis, frequently suffer from respiratory
tract infections, and often become severely malnourished. The role of specific
fats in producing such conditions has not been ascertained.

3. DaTA FROM ANIMAL STUDIES WITH POSSIBLE APPLICATIONS

The work of Burr and Burr®?: °° regarding the essential nature of linoleic
and arachidonic acid for the rat is the basis of nutritional interest in these
fatty acids. Observations from studies with the rat as the experimental
animal which possibly may be applicable to the human subject are the
effect of fat deficiency on the condition of the skin and the loss in weight
of the animals. In an attempt to establish a means for ascertaining the
human need for special fatty acids, Hansen and Wiese* undertook detailed
studies with the dog in regard to the interrelationships of dietary fat, com-
position of serum, and tissue lipids, and the gross and microscopic altera-
tions of the tissues. Reference to some of their findings are presented.

55 T,, Luzzatti and A. E. Hansen, J. Pediat. 24, 417 (1944).

56 T. I. Bennett, D. Hunter, and J. M. Vaughan, Quart. J. Med. [N.S.] 1, 603 (1932).
57 ©. Konstam and H. Gordon, Proc. Roy. Soc. Med. 29, 629 (1936).

587. D. Riley and M. B. Leeds, Lancet I, 262 (1939).

59 G. O. Burr and M. M. Burr, J. Biol. Chem. 82, 345 (1929); 86, 587 (1930).

60 G. O. Burr, M. M. Burr, and E. 8. Miller, J. Biol. Chem. 97, 1 (1932).

61 A. E. Hansen and H. F. Wiese, Proc. Soc. Exptl. Biol. Med. 52, 205 (1943).

VIII. EFFECTS OF DEFICIENCY 307

a. Gross Changes

Maintenance of young dogs on diets practically devoid of fat (less than
1% of the total calories consumed) resulted in marked changes in the ap-
pearance of the animals.*? The distinctive abnormalities were dryness of
the skin and hair with desquamation, increased susceptibility to infection
and later tremulousness, drainage from the auditory canals, swelling and
redness of the paws, and emaciation. Animals placed on a low fat diet at
weaning began to show skin changes within a period of about 3 months.
Improvement varied both with the amount and type of fat fed. When fat
was fed to equal 5% of the total calories, there was little improvement in
the appearance of the skin; at the 10% level some improvement resulted;
but even at a 15% level healing was rather prolonged. Lard or bacon
drippings as the source of dietary fat were more effective in bringing about
restitution of the abnormalities than butter fat, both in relation to time
and degree. When the deficient animals were given fat at the 30% caloric
level, improvement was rapid (few weeks) and definite, being more rapid
with lard and bacon drippings than with butter fat. One animal was on the
low fat diet for as long as 6 years, and it is rather remarkable that, although
the skin and hair abnormality had been present for this length of time,
when sufficient fat again was added to the diet the skin and hair resumed a
healthy appearance. In only a few instances in the studies with dogs was it
possible to use linoleic or arachidonic acid as the source of fat. When fed at
a 1% caloric level, however, there was definite improvement in the skin
and renewed growth of hair. Nevertheless, it was found that this level was
not sufficient to effect complete and permanent cures within a 6-month
period. In control animals given the same diet from the time of weaning
except for the isocaloric substitution of sucrose with lard, bacon drippings,
or butter fat in amounts equal to approximately 30 % of the total calories,
no abnormalities developed. Illustrations of the gross appearance of dogs
suffering fat deficiency are presented in the discussion by Holman in
another portion of this volume.

b. Tissue Lipids

Rather extensive studies*! of the tissue lipids have demonstrated that
the degree of unsaturation of the fatty acids of the blood serum, skin, sub-
cutaneous fat, liver, kidney, and heart is definitely decreased in the animals
on the low fat regimen. In general, the differences in the iodine number of
the fatty acids between healthy control animals and fat-deficient animals
are much greater in the serum than in the skin or other tissues. The iodine
number of the serum fatty acids which are particularly involved have been
those present as cholesterol ester. Direct correlations have been observed
between the appearance of the skin and hair of the animals and the iodine

308 ESSENTIAL FATTY ACIDS

number of the serum fatty acids as well as the kind and amount of dietary
fat.
c. Spectral Analysis for Unsaturated Fatty Acids

The total fatty acids of the serum, skin, liver, and heart consistently
showed high percentages of diene fatty acid*? when adequate levels of fat

27

Fig. 15 (a)

containing linoleic acid were fed, and all animals receiving these diets
presented a normal appearance of the skin and hair. The total fatty acids
of the serum showed more tetraene fatty acid when diets containing linoleic
or arachidonic acids were fed than when the diet was extremely low in fat.
Dogs maintained on a low fat diet and exhibiting fat-deficiency symptoms
showed definitely lesser amounts of diene and tetraene acids in the liver,
kidney, skin, and heart than animals with healthy skin. At 2350 A., H 1%,
values of the serum ranged from 125 to 150, and at 3000 A. from 11 to 20,
whereas the serum from healthy dogs receiving 15 % or more of their caloric
intake as fat (lard or bacon drippings) varied from 250 to 315 for the

VIII. EFFECTS OF DEFICIENCY 309

former and from 40 to 90 for the latter. In animals receiving amounts of fat
inadequate to effect a completely normal skin, the serum diene and tetraene
values were intermediate between the normal and fat-deficient groups. The
correlations observed between the dietary history, the serum lipids, and the

Fira. 15 (6) ~
Fia. 15. Photomicrograph (magnification 1425X) section through the skin of a dog
(a) on a low fat diet and (b) after 6 months on a diet with 29% calories as fresh lard
substituted isocalorically for sucrose. (From Hansen and Wiese.**)

gross changes in the animals seem to be very significant and have been
valuable in interpreting the results of spectral analysis of serum fatty acids
in human subjects.

d. Histologic Features

Definite alterations occur in the histologic structures of the skin of dogs
which have been deprived of dietary fat.** Changes are discernible in the
stratum corneum, epidermis, and dermis (see Figs. 15a and 15b; see also

310 ESSENTIAL FATTY ACIDS

Figs. 12a and 12b, p. 300). Control animals having fat in the diet and grossly
exhibiting healthy skin and hair present an epidermis 2 to 3 cell layers in
thickness, a stratum corneum which is thin, wavy, and lacelike, the cell
outlines and nuclei being well delineated, a dermis with well-defined col-
lagen fibers, normal hair follicles, thin endothelial cells with large lumina
to the small vessels, and slight cellular infiltration. In the fat-deficient dog
presenting distinct abnormalities of the skin with marked desquamation
and loss of hair, the stratum corneum is smudgy and indistinct and often
there is parakeratosis, the epidermis is greatly thickened with palisade
formation at the basilar layer with intracellular bridges evident, and the
dermis shows evidence of irregular collagen bundles, extensive edema, pres-
ence of cellular material, swollen endothelial cells with small lumina of the
vessels, increase in the cells of the hair follicles, plugging of the hair shafts,
degeneration of the hair, and enlarged sebaceous glands. Later the seba-
ceous glands become shrunken. When fat is added to the diet there is good
correlation between the amount and kind of dietary fat and a reversal in
the microscopic appearance of the skin. Changes similar to these have been
found in many human subjects, but heretofore no attempt has been made to
relate such changes to the dietary history.

e. Summary

On the basis of data obtained from studies with laboratory animals in-
cluding the dietary history, serum lipid values, especially for the diene and
tetraene fatty acids, and gross and histologic observations regarding the
skin have given leads for study of the human requirements for essential
fatty acids.

4. Spectroscopic Stupy oF SERUM Lipips IN HUMAN SUBJECTS

In so far as is known, no sustained effort has been made to determine for
human subjects the highly unsaturated fatty acid levels of serum. Wiese,”
in work done at our laboratories under a contract sponsored by the Bureau
of Human Nutrition and Home Economies, U. 8. Department of Agricul-
ture, adapted the spectrographic technique to a semimicro basis which
makes it possible to study the values for diene, triene, and tetraene fatty
acids on samples of serum on a clinical basis. Under the same contract, and
with this method of analysis, the serum levels of these fatty acids were de-
termined for 75 human subjects. The individuals studied were either
healthy adult subjects or hospitalized children who were considered to be in
a good nutritional state clinically, or children markedly underweight and
appearing malnourished. The H}%n. values at 2350 A. (diene fatty acids)

62H. F. Wiese and A. E. Hansen, J. Biol. Chem. 202, 417 (1953).
63 A. EK. Hansen and H. F. Wiese, Federation Proc. 11, 446 (1952).

IX. PHARMACOLOGY ah

were between 250 and 325 in thirty-three subjects, and both children and
adults in this group were considered to be in a good state of nutrition. The
Et”. values at 3000 A. (tetraene fatty acids) ranged from 30 to 76. In con-
trast, in a group of eighteen infants and children who were markedly under-
weight and malnourished in appearance, the #;%,,. values at 2350 A. varied
from 90 to 170 and at 3000 A. from 15 to 42. These subjects were suffering
from cystic fibrosis of the pancreas or celiac disease, or had been on notor-
iously inadequate diets. In the remaining one-third of the subjects studied,
intermediate values both for serum diene and tetraene fatty acids were ob-
served. In general, the clinical appearance suggested quite a good state of
nutrition in all these children. The disorders among these included celiac
disease, eczema, cystic fibrosis of the pancreas, healed osteomyelitis, tuber-
culosis meningitis, laryngeal obstruction, mental retardation, lymphatic
leukemia, and rheumatic heart disease. The relative proportion of triene
fatty acids was extremely low in all three groups of subjects studied.

a. Summary

Spectrographic analysis of the serum fatty acids of human subjects indi-
cates that the content of diene and tetraene fatty acids varies with the
nutritional status of the individual. The proportion of two, three, and four
double-bond fatty acids of the total fatty acids in a group of thirty-three
well-nourished adults and children averaged 27.8, 1.3, and 12.7 %, respec-
tively. In a group of eighteen subjects in poor nutritional state, these values,
were 10.5, 2.5, and 7.5%, respectively.

5. SUMMARY OF EFFECTS OF DEFICIENCY

Human beings appear not to synthesize fatty acids with two and four
double bonds (linoleic and arachidonic acid) sufficient to maintain high
blood serum levels. There is a possibility that poor nutritional status and
susceptibility to dermatitis, skin infections, and respiratory infections may
be related to an inadequacy of dietary fat containing essential fatty acids.
No data whatsoever are available to indicate the magnitude of the human
requirement for linoleic and arachidonic acids.

IX. Pharmacology
ARILD E. HANSEN and GEORGE A. EMERSON
A. CHEMICAL ASPECTS

Progress in basic chemical knowledge has aided in clearer interpretation
of biological phenomena in relation to essential fatty acids. The linoleic

S12 ESSENTIAL FATTY ACIDS

acid of beef tallow has been identified as cis,cis-9 ,12-linoleic acid, and the
linolenic acid from the same source as cis,cis,cis-9,12,15-linolenic acid,
which implies that these should function as essential fatty acids.! Canadian
lard? has been found to contain, on the average, 8.7 % of linoleic, 0.6% of
linolenic, and 0.4% of arachidonic acid. Arachidonic acid has been identi-
fied? 4 in pig back fat. The liquid fatty acids of shark liver oil,’ which are
60 % of the total, contain 15 % linoleic and 2.6 % linolenic acid. It is realized®
that the fatty acid composition of oils such as linseed oil cannot be predicted
from the iodine value alone. Cardiolipin has been recognized’ as a complex
phosphatidic acid containing a large amount of linoleic acid.

Relative reactivities toward hydrogenation have been noted? for several
mono-, di- and triethenoic acids, and a saturase has been found® in pumpkin
seedlings. Oxidation of the essential fatty acids has been intensively studied,
both with soybean lipoxidase,!°-” which is specific for the essential fatty
acids, and with heme compounds.!*!> Both hemin and hemoglobin are de-
stroyed during catalyzed autoxidation of linoleic and linolenic acids, with
liberation of inorganic iron, under physiologic conditions.!® Crude linoleic
acid in the diet causes a progressive secondary anemia in rats.” Oxygenated
linoleic acid is very susceptible to further oxidation.'® Copper, in trace
amounts, greatly accelerates autoxidation of linoleic acid,!® and it has been
suggested that peroxides formed in the brain may be destroyed by linolenic
and linoleic acids in a reaction catalyzed by copper,”° since the catalase con-

1H. B. Knight, E. F. Jordan, Jr., and D. Swern, J. Biol. Chem. 164, 477 (1946).

2H. J. Lips and G. A. Grant, Can. J. Research F25, 63 (1947).

3. B. Shorland and P. B. D. de la Mare, Biochem. J. 39, 246 (1945).

4P. B.D. de la Mare and F. B. Shorland, New Zealand J. Sci. Technol. B27, 465
(1946).

5T. M. Gajjar, J. Sci. Ind. Research (India) 5, 18 (1946).

6. P. Painter and L. L. Nesbitt, O71 & Soap 20, 208 (1948).

7M. C. Pangborn, J. Biol. Chem. 168, 351 (1947).

8 A. E. Bailey and G. S. Fisher, Oil & Soap 23, 14 (1946).

9A. Zeller and F. Maschek, Biochem. Z. 312, 354 (1942).

10R. T. Holman and G. O. Burr, Arch. Biochem. 7, 47 (1945).

118. Bergstrom, Arkiv Kemi, Mineral. Geol. 21A, No. 15, 1 (1946).

122 R. T. Holman, Arch. Biochem. 10, 519 (1946).

13 F, Haurowitz, Compt. rend. ann. et arch. soc. turq. sci. phys. et nat. 8, 10 (1941-1942).

144, Haurowitz and P. Schwerin, Enzymologia 9, 193 (1941).

15 F. P. Simon, M. K. Horwitt, and R. W. Gerard, J. Biol. Chem. 164, 421 (1944).

16 F, Haurowitz, P. Schwerin, and M. M. Yenson, J. Biol. Chem. 140, 353 (1941).

17 P. Gyorgy, R. Tomarelli, R. P. Ostergard, and J. B. Brown, J. Exptl. Med. 76,
413 (1942).

18 H. von Euler, L. Ahlstrém, and B. Ek, Arkiv Kemi, Mineral. Geol. 24A, No. 17,
1 (1947).

19 F. G. Smith and E. Stotz, N. Y. State Agr. Expt. Sta. Tech. Bull. 276, 1 (1946).

207. Huszak, Orvosok Lapja 2, 1245 (1946).

IX. PHARMACOLOGY 313

tent of the brain is so low. The essential fatty acids are capable of destroying
the chemical carcinogens 3 ,4-benzopyrene”! and 20-methylcholanthrene by
a coupled oxidation, but 1,2 ,5,6-dibenzanthracene is destroyed much more
slowly.” Linoleic and arachidonic acids” also destroy N , N-dimethylamino-
azobenzene. Linseed oil and lard have no effect on actions of carcinogens
incorporated in these lipids.”

The RQ of isolated rat liver is increased to 1.23 by linolenic acid, and to
0.95 by a mixture of oleic and linoleic acids, although both the latter acids
decrease it when added singly.”4

B. BIOLOGICAL ASPECTS

Considerable progress has been made in the study of the normal role of
the essential fatty acids in microorganisms, plants, and animals. Linoleic
acid is a growth stimulant for some species of Lactobacillus,?>: 2° but not for
others.?8 Linoleic, linolenic, and arachidonic acids are essential to growth
of an unidentified Micrococcus,?? and enhance growth of others,*® whereas
the saturated acids are ineffective. Linoleic acid is essential for sustained
growth in vitro of Trichomonas vaginalis.*!

A possible relation of linolenic acid content to longevity and germination
of pine seeds is suggested® through parallel species variation.

The biological importance of variation in linoleic acid content of plants
used as food by insects is noted*® in regard to the sugar beet webworm,
Loxostege sticticalis L. Sterile females of this species are also abnormal in
that they contain no linoleic acid; of their host plants, lamb’s quarters con-
tain 20.5 % and sugar beets or sage less than 1 % of linoleic acid, in terms of
total fatty acids, suggesting the significance of diet in sterility in this species.
Ephestia spp., Tenebrio molitor, and various beetles and moths have been

21 G. C. Mueller and H. P. Rusch, Cancer Research 5, 480 (1945).

22 G. C. Mueller, J. A. Miller, and H. P. Rusch, Cancer Research 5, 401 (1945).

23 H. A. Davenport, J. L. Savage, M. J. Dirstine, and F. B. Queen, Cancer Research
1, 821 (1941). r

24H. Annau, A. Eperjessy, and Z. L. Zanthureczky, Hoppe-Seyler’s Z. physiol. Chem.
279, 66 (1943).

25 W.L. Williams, H. P. Broquist, and E. E. Snell, J. Biol. Chem. 170, 619 (1947).

26 B. M. Guirard, E. E. Snell, and R. J. Williams, Arch. Biochem. 9, 361 (1946).

27 EH. Kodicek and A. N. Worden, Biochem. J. 39, 78 (1945).

*8V. R. Williams and E. A. Fieger, J. Biol. Chem. 166, 335 (1946).

29R. J. Dubos, Proc. Soc. Exptl. Biol. Med. 68, 56 (1946).

30 R. J. Dubos, J. Exptl. Med. 85, 9 (1947).

31H. Sprince and A. B. Kupferberg, J. Bacteriol. 58, 441 (1947).

2N. T. Mirov, Nature 154, 218 (1944).

33 J. H. Pepper and E. Hastings, Montana Agr. Expt. Sta. Bull. 418, 1 (1943).

314 ESSENTIAL FATTY ACIDS

studied in regard to their requirements for essential fatty acids;*4*° it is
possible in /phestia to differentiate effects of linoleic and arachidonic acids.

Several studies have dealt with the requirements of mammals for essential
fatty acids under various conditions. Rats are most commonly used, since
the deficiency causes well-defined signs and symptoms. In dogs, fat defi-
ciency is characterized by skin changes and decreased resistance to infection.*”
Although nutritive value of dietary fats depends on their content of linoleic,
linolenic, and arachidonic acids, the relative proportions are also important,
and forced exercise can elicit deficiency symptoms in rats fed a diet con-
taining horse fat, even though this fat supports growth better than does
linseed oil.’8 In rapidly growing pigs, less linoleic acid is incorporated into
back fat than during slower growth.** In rats, there is no sex difference in
the composition of stored fat, but males are more susceptible to essential
fatty acid deficiency.*® Deficiency in essential fatty acids does not affect fat
absorption in rats.“! Effects of diet on distribution and composition of fat
in the rat have been intensively studied.” “* Synthetic margarine, contain-
ing no unsaturated fatty acids or phosphatides, is well absorbed but pro-
duces diarrhea, renal damage, and yellow atrophy of the liver.“ Rats main-
tained for long periods on a high carbohydrate diet with deficiency in
essential fatty acids show a markedly increased absorptive capacity for
glucose, whereas mature rats fed a high fat diet for relatively short periods
show a decreased absorption of glucose.*® Caffeine is reputed*® to increase
the unsaturated fatty acid content of the liver when it is given orally, but
not subcutaneously. If a fat-free diet is given to rats during pregnancy,
cis ,cis-9 , 12-linoleic acid may be found in the mother but not in the young.”
Offspring of rats fed a fat-poor diet develop Schachtelhalmschwanz, a rush-
like jointed tail.*8 If deutertum-tagged fatty acids are fed to pregnant rats,
they are found also in the fetuses.*® It is possible to produce an essential

34 G. Fraenkel and M. Blewett, Natwre 155, 392 (1945).

35 G. Fraenkel and M. Blewett, Biochem. J. 40, xii (1946).

36 G. Fraenkel and M. Blewett, Biochem. J. 41, 475 (1947).

37 A. 2. Hansen and H. F. Wiese, Proc. Soc. Exptl. Biol. Med. 52, 205 (1943).
38 A. von Beznak, M. von Beznak, and I. Hajdu, Erndhrung 8, 209 (1943).
39 F. B. Shorland and P. B. D. dela Mare, J. Agr. Sci. 35, 33 (1945).

40H. G. Loeb and G. O. Burr, J. Nutrition 33, 541 (1947).

41 R. H. Barnes, E. 8. Miller, and G. O. Burr, J. Biol. Chem. 140, 773 (1941).
42 T. Smedley-MacLean and EK. M. Hume, Biochem. J. 35, 990 (1941).

43 T, Rieckehoff, R. Holman, and G. O. Burr, Federation Proc. 6, 418 (1947).
44. H. Rein, Food Inds. 19, 1353, 1466 (1947).

45 R. G. Sinclair and R. J. Fassina, J. Biol. Chem. 141, 509 (1941).

46 H. Hindemith, Arch. exptl. Pathol. Pharmakol. 199, 167 (1942).

47 K. Bernhard and H. Bodur, Helv. Chim. Acta 29, 1782 (1946).

48K. Bernhard and H. Bodur, Helv. Physiol. et Pharmacol. Acta 4, C41 (1946).
49. W. H. Goldwater and D. Stetten, Jr., J. Biol. Chem. 168, 723 (1947).

IX. PHARMACOLOGY 315

fatty acid deficiency in mice,*° preventable or curable by lard, as in rats.
A deficiency may also be produced in the mature as well as the young rat,
and ethyl linoleate rapidly relieves the symptoms; recovery also follows a
sufficiently prolonged maintenance on a fat-free diet, suggesting that syn-
thesis of essential fatty acids can occur under these circumstances.*! On the
other hand, no synthesis of linoleic or linolenic acids is detected in rats given
D.O, as reflected by incorporation of deuterium into the fatty acid molecule,
although deuterium is found in palmitic, stearic, and even oleic acids of rats
so treated.*?: *> As mentioned above, plants*® and some insects** can syn-
thesize linoleic acid. Among the unsaturated fatty acids of hair of human
adults, the essential fatty acids may have a bactericidal role akin to the
fungicidal action postulated for the odd-number C;-:3; acids present, in
conferring resistance to ringworm in the adult.*

C. VITAMIN INTERRELATIONSHIPS

Supplementation of the diet of rats fed carotene with the unsaturated
fatty acids of butter results in accumulation of twice as much vitamin A
in the liver as when saturated fatty acids are given.®> Other work°® indicates
that the utilization of carotene from oils depends on the vitamin E rather
than linoleic acid content. Gossypol has been shown to increase the effects
of suboptimal supplements of vitamin A and lard or methyl linoleate in
diets deficient in both vitamin A and the essential fatty acids, acting as an
antoxidant.*” An antagonism between carotene and linoleic or linolenic
esters in rats deficient in vitamin A is said to be prevented by a-tocopherol,
probably by the same mechanism.*®® Among the vitamin B complex, the
essential fatty acids antagonize the lipotropic effect of inositol in rats but
have no effect on the action of choline.*? In rat dermatitis, ethyl linoleate
has a curative action whereas pyridoxine at best only alleviates, but pyri-
doxine will potentiate subcurative doses of ethyl linoleate.®° The anti-

50K. A. White, J. R. Foy, and L. R. Cereeedo, Proc. Soc. Exptl. Biol. Med. 54, 301
(1943). ce

51.V.H. Barki, H. Nath, E. B. Hart, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med.
66, 474 (1947).

52 K. Bernhard, H. Steinhauser, and F. Bullet, Helv. Chim. Acta 25, 1913 (1942).

53 F. Bullet and K. Bernhard, Helv. Physiol. et Pharmacol. Acta 1, C39 (1943).

54 A.W. Weitkamp, A. M. Smiljanic, and 8S. Rothman, J. Am. Chem. Soc. 69, 1936
(1947).

55. F. Brown and W. R. Bloor, J. Nutrition 29, 349 (1945).

56S. D. Rao, Nature 156, 234, 449 (1945).

57K. L. Hove, J. Biol. Chem. 156, 633 (1944).

58 W. C. Sherman, Proc. Soc. Exptl. Biol. Med. 47, 199 (1941).

59 J. M. R. Beveridge and C. C. Lucas, J. Biol. Chem. 157, 311 (1945).

60 fF. W. Quackenbush, H. Steenbock, F. A. Kummerow, and B. R. Platz, J. Nwtri-
tion 24, 225 (1942).

316 ESSENTIAL FATTY ACIDS

acrodynic potency of various seed oils is related to the linoleic acid content,
except when the linolenic acid content is high, in which case less effect than
expected is obtained.* Earlier studies®: ® indicated that ethyl linoleate has
no sparing action on pyridoxine, or that both are necessary for complete
cure. In rats on a diet deficient in both pyridoxine and essential fatty acids,
pyridoxine or linoleic acid supplements are equally effective in permitting
growth, and the content of highly unsaturated fatty acids increases in rats
regardless of which supplement is given. When chicks deficient in vitamin
E are fed different fractions of hog liver fatty acids, the most highly un-
saturated fraction is the most toxic,® and unsaturated fatty acids increase
symptoms of vitamin E deficiency in chicks.®* Vitamin E-deficient rats
given unsaturated fatty acids show a diffuse brown pigmentation of stored
fat, together with a lack of the normal pigment of the incisors which is due
to iron.” The pigmentation of lipids is taken to indicate®® a metabolic
interrelation of vitamin E and the essential fatty acids. Vitamin E increases
symptoms of essential fatty acid deficiency but potentiates strongly with
the effects of unsaturated fatty acids in counteracting this deficiency.®

D. ACTION ON PHYSIOLOGIC PROCESSES

The essential fatty acids are necessary for normal reproduction and lacta-
tion in the rat.7°.7 The comparative potencies of the actions of methyl
arachidonate and methyl] linoleate on growth are different from relative
curative effects on other disturbances in essential fatty acid deficiency in
rats.”» 73 Very small amounts of methyl linoleate greatly increase fat storage
in the rat.”

If linseed meal is added to an otherwise adequate diet, chicks show in-

61D. 8. Anthony, F. W. Quackenbush, A. J. Ihde, and H. Steenbock, J. Nutrition 26,
303 (1943).

62 G. A. Emerson, Proc. Soc. Exptl. Biol. Med. 47, 445 (1941).

63 P. Gross, J. Invest. Dermatol. 3, 505 (1940).

64 G, Medes and D. C. Keller, Arch. Biochem. 15, 19 (1947).

65H. Dam, J. Nutrition 28, 297 (1944).

66 H. Dam, J. Nutrition 27, 193 (1944).

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(1942).

71 J. K. Loosli, J. F. Lingenfelter, J. W. Thomas, and L. A. Maynard, J. Nutrition
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34, 879 (1940).

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™ T. Smedley-Maclean and L. C. A. Nunn, Biochem. J. 34, 884 (1940).

IX. PHARMACOLOGY Fs WP

hibition of growth; this can be prevented by prior soaking of the meal in
water, and drying before mixing with the diet.”*: 7° Since formation of HCN
was previously shown” to be the most toxic factor in use of the meal, it is
not likely that the essential fatty acids are involved. Linoleic acid decreases
the synthesis of acetylcholine.”® Linoleic and linolenic acid deficiency is
accompanied by a reduction in time of excitation of peripheral motor nerves
and a prolongation of muscular excitation ;79 these aberrances are corrected
by treatment with linoleic and linolenic acids. The incidence of ulcers of
the gizzard in chicks fed a diet containing cinchophen decreases if methyl
arachidonate or the methyl esters of highly unsaturated fatty acids from
the phosphatides of beef adrenals are fed.°°

EK. CHEMOTHERAPY

Antibiotics from yeasts and Torula utilis are a mixture of unsaturated
fatty acids;*'! that from the mycelia of Penicillium crustosum is linoleic
acid; and the antibiotic lipid from Tetrahymena geleii is a mixture of un-
saturated Co»-24 acids.®’ It is probable that other, unidentified antibiotics

may ultimately be found to be essential fatty acids. Linoleic and linolenic
acids inhibit oxygen uptake by Mycobacterium tuberculosis hominis.** These
acids also inhibit germination of spores of Clostridium botulinum at a con-
centration of 1 mg. % but have no effect on vegetative cells; addition of 1%
starch to the medium antagonizes this inhibition.’® These acids also inac-
tivate the neurotropic strain of yellow fever virus, while the virus retains
its antigenicity in mice.’® Fever appears to cause a flooding of unsaturated
fatty acids into the blood, and the temporarily beneficial effect of fever in
eczema may be related to this.*”

7 H. I. MacGregor and J. McGinnis, Poultry Sci. 27, 141 (1948).

76, H. Kratzer and D. E. Williams, Poultry Sct. 27, 236 (1948).

7 R. Vuillaume and J. Gillet, Compt. rend. soc. biol. 136, 614 (1942).

78 C. Torda and H. G. Wolff, Proc. Soc. Exptl. Biol. Med. 59, 246 (1945).

7 R. Lecoq, P. Chauchard, and H.Mazoué, Bull. soc. chim. biol. 29,717 (1947).

8H. Dam, Acta Physiol. Scand. 12, 189 (1946).

81K. Uroma and E. A. Virtanen, Ann. Med. Exptl. et Biol. Fenniae (Helsinki) 25,
36 (1947).

82 R. F. Riley and D. K. Miller, Arch. Biochem. 18, 13 (1948).

8 C. M. McKee, J. D. Dutcher, V. Groupé, and M. Moore, Proc. Soc. Expil. Biol.
Med. 65, 326 (1947).

8 §. Bergstrom, H. Theorell, and H. Davide, Nature 157, 306 (1946).

85 J. W. Foster and E. 8S. Wynne, J. Bacteriol. 55, 495 (1948).

8G. M. Findlay, Trans. Roy. Soc. Trop. Med. Hyg. 36, 247 (1943).

87 A.V. Stoesser, J. Allergy 18, 29 (1947).

318 ESSENTIAL FATTY ACIDS

X. Requirements

RALPH T. HOLMAN
A. OF ANIMALS

The daily linoleic acid requirement for maximum growth of rats has been
variously set at 20 to 100 mg.t-> The prophylactic dose of linoleic acid
seems to be of the order of 20 mg. per day. Using highly purified diet, Mac-
Kenzie ef al.6 found that 23 mg. of methyl linoleate per day was sufficient
to allow normal growth and reproduction. Greenberg ef al.’ found that the
daily dietary requirement for the male rat was in the range of 50 to 100
mg. of linoleic acid, whereas the female requires between 10 and 20 mg.
They also found that linolenic acid, if sparked by some linoleic acid, had
growth-promoting activity equal to that of lnoleic acid. However, Deuel
et al.8 found that 10 % cottonseed oil in the diet of rats receiving an optimum
level of linoleate gave added growth, indicating the possibility of a benefi-
cial effect of oil itself or of some constituent of the oil.

The linoleic acid requirement varies with the composition of the diet.
Rations including corn starch or rice starch fail to produce fat deficiency®
because of unsaturated fatty acids bound in the starch granules. Diets con-
taining saturated fats as the sole source of energy accentuate fat defi-
ciency.'!® When Sinclair fed trielaidin es dietary fat, the deficiency symp-
toms could not be cured by a daily dose of 20 mg. of corn oil." These
scattered observations suggest that either the requirement for linoleic acid
is Increased when non-essential fat is metabolized, or that some synthesis
of essential fatty acids is possible from sucrose.

Several early studies seemed to indicate that tissue linoleic acid parallels

1G. O. Burr and M. M. Burr, J. Biol. Chem. 86, 587 (1930).

20. Turpeinen, J. Nutrition 15, 531 (1938).

3G. O. Burr, J. B. Brown, J. P. Kass, and W. O. Lundberg, Proc. Soc. Exptl. Biol.
Med. 44, 242 (1940).

4. M. Hume, L. C. A. Nunn, I. Smedley-Maclean, and H. H. Smith, Biochem. J.
32, 2162 (1938).

5 EH. M. Hume, L. C. A. Nunn, I. Smedley-Maclean, and H. H. Smith, Biochem. J.
34, 879 (1940).

6C. G. MacKenzie, J. B. MacKenzie, and E. V. McCollum, Biochem. J. 33, 935
(1939).

78. M. Greenberg, C. E. Calbert, E. E. Savage, and H. J. Deuel, Jr. J. Nutrition
41, 473 (1950).

8 H. J. Deuel, S. M. Greenberg, C. E. Calbert, E. E. Savage, and T. Fukui, J. Nutrz-
tion 40, 351 (1950).

9H. M. Evans and 8. Lepkovsky, J. Biol. Chem. 96, 143 (1932).

10 H. M. Evans and 8. Lepkovsky, J. Brol. Chem. 96, 157 (1932).

1R. G. Sinclair. J. Nutrition 19, 131 (1940).

X. REQUIREMENTS 319

the dietary intake of this acid.’ Bernhard et al. and Schoenheimer!
found that tissue linoleic and linolenic acids incorporated deuterium from
the body water enriched with heavy water at a much lower rate than did
the other fatty acids, suggesting that the animal does not synthesize essen-
tial acids. On the other hand, studies using spectrophotometric techniques
for the measurement of the unsaturated acids seem to indicate synthesis of
small amounts of essential fatty acids by animals on a fat-deficient diet.
Medes and Keller” found that rats synthesized polyunsaturated fatty
acids when diets deficient in Bs and fat were supplemented with vitamin
B, . In their studies on fat deficiency in adult rats, Barki et al. found spon-
taneous cures and demonstrated that the essential fatty acid content of the
animals rose when they recovered spontaneously.!®’ The discrepancies
between the deuterium experiments and these latter observations were
probably due to the relatively short-term nature of the deuterium experi-
ments and the apparent slow rate of turnover of the polyunsaturated fatty
acids. It appears that the essential fatty acids can be synthesized by the
animal, but not in sufficient quantity to provide for the needs of the growing
animal.

B. HUMAN BEINGS

There is no specific information regarding the essentiality of linoleic acid,
or similar unsaturated fatty acids, in human nutrition. The evidence indi-
cates that these fatty acids may be essential, that the requirement is rela-
tively small, and that ordinary diets easily supply this requirement. The
Food and Nutrition Board of the National Research Council recommends
that an adequate diet should contain essential fatty acids to the extent of
1% of the total calories.

12 N.R. Ellis and O. G. Hankins, J. Biol. Chem. 66, 101 (1925).

13 A. Banks, T. P. Hilditch, and E. C. Jones, Biochem. J. 27, 1375 (1933).

14H. E. Longenecker, J. Biol. Chem. 128, 645 (1939).

15 K. Bernhard, H. Steinhauser, and F. Bullet, Helv. Chim. Acta 25, 83 (1942).

16 R. Schoenheimer, The Dynamic State of Body Constituents, p. 18. Harvard Uni-
versity Press, Cambridge, Mass., 1949. .

117 G. Medes and D. C. Keller, Arch. Biochem. 15, 19 (1947).

18V. H. Barki, R. A. Collins, E. B. Hart, and C. A. Elvehjem, Proc. Soc. Exptl.
Biol. Med. 71, 694 (1949).

a je

‘
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mn 4
t ; i al yr ws wf
‘
; P ‘J « ®
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TEE:

IV.

VII.

VET,

CHAPTER 8
INOSITOLS

Nomenclature and Formula.
A. Discussion of Terminology .
Chemistry .
A. Nomenclature.
B. Planar Projections .
C. Spatial Constellations.
D. Distribution .
1. meso-Inositol .
2. Seyllitol . :
3. d- and l-Inositol .
4, Other Cyclohexitols.
E. Properties .
Industrial Eeopenniiont
A. Presence in Plants . ;
B. Corn Steepwater as a Source of Tnosieal.
C. Precipitation of Phytate from Corn Steepwater
D. Hydrolysis of Phytate to Inositol sts
E. Isolation of Inositol from ee
Biochemical Systems
A. Bioecatalytie Functions of myo- Siasitel
1. Studies with Inhibitory Analogs .
2. Bound Forms of myo-Inositol .
3. Lipotropic Effect of Inositol
B. Metabolism of Inositol
1. In Bacteria
2. In Animals.

. Specificity of Action
. Biogenesis .

A. Inositol Seaihenis by Wiverabradeiamee

B. Inositol Synthesis by Higher Plants

C. Inositol Synthesis by Animals

Estimation of Inositol .

A. By Isolation . . .

B. By Chemical Methods . :
1. Reaction with Potassium Todemercuravel
2. Reaction with Periodate
3. Other Chemical Methods

C. By Paper Chromatography .

D. By Mouse Assay.

E. By Microbiological Assay

Occurrence . ape a

A. In Foods :

B. Natural Antivitamins ae Taste

321

. 322
. 323
. 329
. 329
. 330
. ddl
. d04
. dd4
. 300
. 300
. 306
. 336
. 339
. 339
. 340
. 340
. 341
. 341
. 342
. 342
. 343
. 345
. 347
. 347
. 347
. 349
. dol
. 304
. 354
. 000
. 356
. 008
. 308
. 359
. 359
. 309
. 360
. 361
. 361
. 361
. 363
. 363
. 363

BA INOSITOLS

IX. Effects of Deficiency
A. In Microorganisms .
B. In Animals. a
C. Pathology in Human Bein
X. Pharmacology
A. Heart d
B. Gastrointestinal Tract
C. Thyroid
D. Neuromuscular Sy stem
E. Miscellaneous.
XI. Requirements .
A. Of Animals.
B. Of Man .

1

». Influence of Other @anctituents a Diet 2
D. Synthesis in the Body

I. Nomenclature and Formula
ROBERT 8. HARRIS

Accepted name: Inositol
Obsolete names: meso-Inositol
Dambose
Meat sugar
2-Inositol
Inosite
Nucite
Phaseomannite
Empirical formula: Cs>H120¢
Chemical name: Hexahydroxycyclohexane
Structures of natural inositols:

OH OH OH OH
Hi HW H OH H H
OH HH
HO H HO OH
Jl OH H OH
myo-Inositol D-Inositol
OH Or: OE EL
HO OH HO H
OH 1 H OH

L-Inositol Scyllitol

. 366
. 366
. 367
. d71
sou
» Ord
. 379
. 380
. 380
. 381
. 381
. 381
. 382
. 383
. 385

I. NOMENCLATURE AND FORMULA 328

A. DISCUSSION OF TERMINOLOGY!
HENRY A. LARDY

There are nine possible stereoisomeric forms of hexahydroxycyclohexane,
commonly called inositols or cyclohexitols.* Several of these isomers occur
naturally, and a few others have been synthesized. Of the nine isomers,
seven are optically inactive or meso forms, and two are asymmetric enantio-
morphs which have been designated d and J. The isomer which is most
widely distributed in nature, and which is of importance for the nutrition
of animals and certain microorganisms, has been called meso-inositol or 7-
inositol (referring to the fact that it is optically inactive). It has been re-
named myo-inositol.2 The prefixes 7 or meso are not at all suitable for dis-
tinguishing this isomer from the other six optically inactive isomers, since
they too are meso forms. Furthermore, derivatives of myo-inositol, may
become optically active by asymmetric substitution, and to use 7 or meso in
the name of such derivatives is chemically incorrect and confusing. For
these reasons it seemed desirable, when proposing a systematic nomencla-
ture for the inositols and their derivatives,? to rename this isomer. Since
Scherer*® had originally isolated this compound from muscle and had in
fact called it ‘‘muscle sugar,” the name myo-inositol? (Greek, yuo, pvos,
muscle) was proposed? and soon adopted in the chemical literature.°

In Table I the structures of the nine stereoisomeric inositols are shown,
together with their trivial names and a designated? sequence for numbering
the carbon atoms. In the formulas the hydroxyl groups are indicated by
. vertical lines. Four of the isomers—myo-, D-, and L-inositols, and scyllitol—
occur naturally, either in the free form or as a wide variety of derivatives.®: 7
Three of the remaining isomers—ep?, allo, and muco—have been synthe-
sized, the first by Posternak® and the latter two by Dangschat.*:

1 (Editors’ note.) At the present time the nomenclature of the inositols is confusing.
This section was written by Dr. Lardy in an attempt to eliminate this confusion. The
systemic nomenclature suggested by Dr. Lardy has not yet been approved and
adopted by the American Association of Biological Chemists, or some similar sci-
entific body. For this reason the Editors have not insisted that all contributors to
this chapter use the proposed nomenclature. Instead, each author has been permitted
to use the nomenclature he prefers.

1a T,, Maquenne, Les Sucres et leurs Principaux Dérivés, p. 190, Carre et Naud,
Paris, 1900.

2H. G. Fletcher, Jr., L. Anderson, and H. A. Lardy, J. Org. Chem. 16, 1238 (1951).

3 J. Scherer, Ann. Chem. Justus Liebigs 73, 322 (1850).

4 Compare the analogous myoglobin, myoblast, myocyst, myosin.

5B. Magasanik, J. Am. Chem. Soc. 78, 5919 (1951).

6H. G. Fletcher, Jr., Advances in Carbohydrate Chem. 3, 45 (1948).

7W. W. Pigman and R. M. Goepp, Jr., Carbohydrate Chemistry. Academic Press,
New York, 1948.

8 T. Posternak, Helv. Chim. Acta 19, 1333 (1936).

9G. Dangschat, Naturwissenschaften 31, 146 (1942).

324 INOSITOLS

The systematic naming of derivatives of the inositols is complicated
by the fact that optically mactive parent iositols can yield optically
active derivatives by asymmetric substitution. Furthermore, monoketo-
inositols may be related to two different parent inositols. It can be readily
appreciated how this situation gave rise to inconsistencies of nomenclature
as more derivatives of known types were prepared and as new types of
derivatives were discovered.

TABLE I
THE NUMBERING OF THE NINE STEREOISOMERIC INOSITOLS

i= N=
—> 1 YA
I II Il

Unknown

IV
neo-Inositol

VII
Seyllitol

epi-Inositol

myo-Inositol

VIII
D-Inositol
(dextrorotatory
Inositol)

allo-Inositol

VI
muco-Inositol

Ix
L-Inositol
(levorotatory
Inositol)

To eliminate the confusion, a systematic nomenclature for this im-
portant class of compounds and its derivatives was developed.? The system
is based on the numbering sequence shown in Table I and on three rules.

The numbering sequence provides identical numbers for stereochemically
equivalent carbon atoms. For p- or L-inositols the numbering begins with
the carbon atom marked 1 and must proceed in the designated direction.
Isomer IV and epi-, allo-, myo-, and muco-inositols may be numbered
either clockwise or counterclockwise, depending on which results in the
lower numbers of substituents. In isomer I (all cis) and in scyllitol (all
trans) all carbon atoms are equivalent, and derivatives of these isomers

% Since this was written, an inositol corresponding to IV has been synthesized by
S. J. Angyal and N. K. Matheson (Abstr. 123rd Meeting, Am. Chem. Soc., p. 120, 1953)
and named neo-inositol.

I. NOMENCLATURE AND FORMULA 320

would be named to give the lowest possible number to the carbon atoms
bearing groups other than unsubstituted hydroxyl.

The explicit rules for naming inositol derivatives are presented below
with amplifications in which emphasis is given to naming derivatives of the
vitamin myo-inositol.

Rute 1. Jn accordance with established practice in the carbohydrate field
the trivial names of the parent inositols will be used.

This rule is self-explanatory from Table I and from what has been
said above concerning reasons for using the prefix myo rather than 7 or
meso to designate the nutritionally important isomer.

Rute 2. The structures of the optically active cyclitols and of all optically
active derwatives of the other cyclitols are designated as belonging to the D or L
series, based upon the configuration of the highest numbered carbon atom.

Some method is obviously needed to distinguish the name of a compound
from that of its enantiomorph. In the field of carbohydrate chemistry it
has been the general practice to designate the configuration of a sugar
molecule according to the configuration of its highest numbered asymmetric
carbon atom. The configuration of this carbon atom is established by
relating it to the configurations of the recognized standard compounds, pD-
and ut-glyceraldehyde. This practice may be extended to the inositols by
projecting the highest numbered carbon atom (i.e., number 6) onto a plane
with carbon atoms | and 5 projecting below the plane (away from the viewer)
and the H and OH above the plane (toward the viewer).

5 C 5 C
6 H—C—OH 6 HO—C—H
1c hee

D series L series

This rule would be applied to naming two enantiomorphic monophos-
phoric esters of myo-inositol, X and XI, in the following manner.

ORO ta,
x XI

By comparison with Table I it is apparent that both are myo-inositol-1-
phosphates. Their configurations are determined by projecting carbon
atom 6 onto a plane, as described above. Hudson’s'® projection formulas

10 C. $. Hudson, Advances in Carbohydrate Chem. 3, 1 (1948).

326 INOSITOLS

of the two structures in question are:

EL Ss ORO ia 1 O;PO Ne H

X=L XI=D
Thus X would be named t-myo-inositol-1-phosphate, and XI would be p-
myo-inositol-1-phosphate.

RuLE 3. When an inositol derivative contains less than six asymmetric
carbon atoms, the compound will be given the trivial name of one of the parent
mmositols to which it is related, the choice being that parent inositol which has
the maximum number of cis-hydroxyl groups. Where this offers more than one
possibility, as in the diketoinositols, the following also apply. Secondarily,
the parent inositol chosen is that which has the lowest possible number for its
substituents cis to the hydroxyl on carbon 1, or, finally, that which would
confer the lowest possible numbering on substituents other than hydroxyl.

This rule may be illustrated by its application to the ketoinositols
(named inososes by Posternak®) obtainable by oxidation of myo-inositol.

O O
O
XII XIII XIV
2-Keto-myo-inositol L-2-Keto-epi-inositol D-2-Keto-ep?-inositol
(myo-Inosose-2) (L-epi-Inosose-2) (D-epi-Inosose-2)

The bacterium Acetobacter suboxydans oxidizes myo-inositol" 2 to a mono-
ketoinositol which was shown by Posternak® to have the structure XII.
This compound (XII) has three hydroxyl] groups projecting upward and
only two projecting downward. Hence, according to rule 3 it receives the
trivial name of myo-inositol which has an hydroxyl group projecting upward
in position 2. The name of choice for XII is therefore 2-keto-myo-inositol
or myo-inosose-2. It had previously been named _ scyllo-meso-inosose by
11 A. J. Kluyver and A. G. J. Boezaardt, Rec. trav. chim. 58, 956 (1939).

2 H. E. Carter, C. Belinsky, R. K. Clark, Jr., E. H. Flynn, B. Lytle, G. E. McCas-
land, and M. Robbins, J. Biol. Chem. 174, 415 (1948).

I. NOMENCLATURE AND FORMULA 327

Posternak, since it could be selectively reduced to either scyllitol or
meso-inositol (myo-inositol).

Nitric acid oxidation of myo-inositol produces a monoketoinositol which
is a racemic mixture of XIII and XIV. Posternak™ resolved this mix-
ture and demonstrated that the levorotatory isomer had structure XIV.
Although this compound was obtained from myo-inositol, according to
rule 3 it must be assigned the trivial name of the inositol in which the
hydroxyl on carbons 1, 2, 3, 4, and 5 are cis, i.e., epi-inositol. By rule 2,
structure XIV is placed in the p configurational series, and the exact name
is therefore D-2-keto-epi-inositol or D-epi-inosose-2. The enantiomorphic
XIII is t-2-keto-epz-inositol or L-ep7-inosose-2.

The diketoinositol shown in formula XV is related to both myo-and muco-
inositols (but not to either p- or L-inositol, because these have fewer cis-
hydroxyl groups—see rule 3). Since the substituents cis to the hydroxyl on
carbon | in myo-inositol have a numbering sequence (1, 2, 3, 5) lower than
that in muco-inositol (1, 2, 4, 5), the compound is correctly named 1,3-
diketo-myo-inositol.

O Ou: O
XV XVI-a XVI-b
1,3-Diketo-myo- 1,5-Diketo-muco-
inositol inositol

Aminodesoxyinositols. Several derivatives of inositol in which amino
groups replace hydroxyl groups have been prepared synthetically,!°- and
at least one, streptamine, is known to occur naturally. Carter and his co-
workers!* have coined the term znosamine as a generic designation for this
class of substance, and other authors!” * have continued the usage. The
two monoaminodesoxyinositols derivable from 2-keto-myo-inositol (XII)
have been synthesized!*: 8 and their configurations established!®: 1°: 2° as
XVII and XVIII. Each of these meso structures may readily be named

13'T. Posternak, Helv. Chim. Acta 25, 746 (1942).

4 T. Posternak, Helv. Chim. Acta 29, 1991 (1946).

165 J. Grosheintz and H. O. L. Fischer, J. Am. Chem. Soc. 70, 1479 (1948).

16H. EK. Carter, R. K. Clark, Jr., B. Lytle, and G. E. McCasland, J. Biol. Chem. 175,
683 (1948).

17 E. May and E. Mosettig, J. Org. Chem. 14, 1137 (1949).

18 J,, Anderson and H. A. Lardy, J. Am. Chem. Soc. 72, 3141 (1950).

19 T. Posternak, Helv. Chim. Acta 38, 1597 (1950).

20 G. E. McCasland, J. Am. Chem. Soc. 78, 2295 (1951).

328 INOSITOLS

NH,
- =
XVII XVIII
2-Amino-2-desoxy-myo-inositol Aminodesoxyscyllitol
(myo-Inosamine-2) (scyllo-Inosamine)

through reference to the corresponding inositol, the myo-inositol relative,
XVII, being called 2-amino-2-desoxy-myo-inositol (or myo-inosamine-2),
while XVIII is simply aminodesoxyscyllitol (or scyllo-inosamine). The
reduction of nitrogenous derivatives of DL-2-keto-ep7-inositol (XIII, XIV)
gives two enantiomorphic pairs of inosamines: DL-2-amino-2-desoxy-ept-
inositol (pL-ep7-inosamine-2, XIX—XX) and pi-4-amino-4-desoxy-myo-in-
ositol (pL-myo-inosamine-4, XXI-X XII). One of these compounds was
made by Carter and his coworkers.!® The other has been synthesized by
Rieke et al.21 The methods of synthesis (cf. Anderson and Lardy") and
properties of the compounds indicate that the compound prepared by Carter
et al.6 (and designated EA) is DL-epi-inosamine-2 (XIX—XX), and that
prepared by Rieke et al.?! is pt-myo-inosamine-4 (XXI-XXII).

The antibiotic, streptomycin, contains a diamino didesoxyinositol, which
has been given the trivial name, streptamine. Previous evidence indicated
that streptamine has the configuration shewn in XXIIT.”? Wintersteiner

NH,
XIX XX
D-2-Amino-2-desoxy-ep?-inositol L-2- Amino-2-desoxy-ep?-inositol
(D-epi-Inosamine-2) (L-epi-Inosamine-2)
H Ks
NH,
XXI XXII
D-4-Amino-4-desoxy-myo-inositol L-4-Amino-4-desoxy-myo-inositol
(D-myo-Inosamine-4) (L-myo-Inosamine-4)

NH,
XXIII
1,3-Diamino-1,3-didesoxyscyllitol
21H. Rieke, H. A. Lardy, and L. Anderson, J. Am. Chem. Soc. 75, 694 (1953).
22M. L. Wolfrom, 8. M. Olin, and W. J. Polglase, J. Am. Chem. Soc. 72, 1724 (1950).

II. CHEMISTRY 329

et al. prepared a monoamino monodesoxyinositol by nitrous acid deamina-
tion of a derivative of streptamine in which one amino group was blocked.
Such deamination generally occurs with Walden inversion.!® Synthetic
DL-myo-inosamine-4 (XXI-XXIT) is identical with the compound pro-
duced from streptamine by Wintersteiner, thus confirming the all trans
structure (XXIII) for streptamine.”!

II. Chemistry

ERWIN CHARGAFF

The inositols! are a family of isomeric hexahydroxy derivatives of cyclo-
hexane. They are the ultimate representatives of the series of hydroxy-
cyclohexanes that can be subsumed under the general term cyclitol.? The
designation cyclohexitol to describe the inositols is also sometimes en-
countered in the literature, in contrast to the cyclopentitols or desoxy-
inositols (quercitols), the cyclotetritols, etc. The hexahydroxycyclohexanes
can exist in two optically active and seven inactive configurations, in addi-
tion to the racemic dl compound. Only a few of those, however, have so
far been found to occur in nature, and two of the isomers do not appear to
have been prepared as yet.

A. NOMENCLATURE

In a cyclic compound of the molecular formula (CHOH). it is sufficient
to indicate the positions of the hydroxyl groups; the corresponding steric
positions of the hydrogen atoms are thereby defined and need not be speci-
fied. In the following discussion all numbers and graphic representations
will, therefore, refer only to the hydroxyl groups.

The difficulties facing attempts at arriving at a numbering system that,
with a single convention, will permit an unequivocal description of all
inositol isomers and their derivatives are almost insuperable. Maquenne?
adopted the practice, at that time followed in carbohydrate chemistry,
of denoting the numbers of the carbon atoms carrying hydroxyl groups
situated above and below the plane of the ring respectively by way of a
cra or ieee or 2008 or pias if the

46 35 24 Zoe? |
23. Wintersteiner and A. Klingsberg, J. Am. Chem. Soc. 73, 2917 (1951).

1 P. Fleury and P. Balatre, Les Inositols—Chimie et Biochimie. Masson, Paris, 1947.
2 For a recent review on the chemistry of cyclitols, see H. G. Fletcher, Jr., Ad-

vances in Carbohydrate Chem. 3, 45 (1948).
’ T,. Maquenne, Les Sucres et leurs Principaux Dérivés, Carré et Naud, Paris, 1900.

fraction. meso-Inositol thus would be

330 INOSITOLS

numbering proceeds in a clockwise direction. The multitude of possibilities
points to the unsatisfactoriness of this system, especially if inositol deriva-
tives are to be described.

Magasanik and Chargaff* proposed a system, which they applied to the
cyclohexitols and their keto and desoxy derivatives, in which the carbon
atoms are numbered clockwise, positions 1 and 6 chosen with their hy-
droxyls in trans position. The structure is arranged in such a manner as to
have as many hydroxyl groups as possible cis to the one in position 1.
Where this scheme allows two possibilities, that arrangement is chosen in
which the carbon atoms with hydroxyls cis to the one in position 1 have the
lowest positional numbers. The planar projection of meso-inositol would,
consequently, be numbered in the following manner.

The inositol isomers are designated cyclohexanehexol, their desoxy deriv-
atives cyclohexanepentol, their monocarbonyl derivatives cyclohexane-
pentolone, the corresponding diketones cyclohexanetetroldione, etc. In the
case of the inositols it is sufficient to indicate the position of the cis hy-
droxyls. meso-Inositol is designated as cyclohexane-(1 ,2 ,3 ,5)cis-hexol.
Similarly, its 2-keto derivative is described as cyclohexane-(1 ,3 ,5)cis-
pentol-2-one. The corresponding 2-desoxyinositol is cyclohexane-(1 ,3 ,5)-
cis-4,6-pentol. In this case the numbers of all hydroxyl groups must be
given.

This system has the advantage that structures can be derived from
designations without reference to any visual aid. It leads, however, to
rather clumsy names, when sterically different polyfunctional derivatives
are to be described. Moreover, it requires enantiomorphs to be numbered
differently in equivalent positions.

A scheme which avoids the last-mentioned disadvantage has been pro-
posed by Fletcher et al.® (see the preceding section by Henry A. Lardy,
p. 323).

B. PLANAR PROJECTIONS

The planar projections of the cyclohexitols, including those not yet en-
countered or prepared, are shown in Table I. The structure of meso-inositol
(V) is based on the work of Dangschat and Fischer* and Posternak’. The

4B. Magasanik and E. Chargaff, J. Biol. Chem. 174, 173 (1948).

5H. G. Fletcher, Jr., L. Anderson, and H. A. Lardy, J. Org. Chem. 16, 1238 (1951).
6G. Dangschat, Naturwissenschaften 30, 146 (1942).

7™T. Posternak, Helv. Chim. Acta 25, 746 (1942).

II. CHEMISTRY oor

structures of II,® VII,’ and [X° were elucidated by Posternak, those of
III and VI by Dangschat and Fischer.!°: |! Table I also indicates the num-
bering devices and trivial names suggested by Fletcher et al.*

C. SPATIAL CONSTELLATIONS

Most textbooks and even review articles represent meso-inositol and
its isomers in the form of planar structures. This is, however, far from
acceptable, since it could lead to the assumption that the various hydroxyls
situated in what would be considered as the same plane are chemically
equivalent. Actually, it has been shown by Magasanik and Chargaff?: 4
that the several hydroxyl groups in czs position are treated differently in
a biological system, according to their position in space. These findings on
the selective oxidation of the inositol isomers by resting cells of Acetobacter
suboxydans, to yield mono- or dicarbonyl derivatives, were later extended
to cell-free enzyme preparations.}*: 1°

It is, of course, evident that the structural formulas I-IX in Table I,
representing the series of inositol isomers, do not describe the actual posi-
tions in space of the various atoms, but merely represent planar projections
based on the conventions introduced into stereochemistry by Emil Fischer."”
If the biological function and fate of meso-inositol and its isomers are to be
understood, the element of rigidity, i.e., the lack of free rotation around
carbon-to-carbon bonds, prevailing in these derivatives of cyclohexane,
cannot be ignored.

The well-known Sachse-Mohr concept postulates the existence of puck-
ered rings in cyclohexane; two strain-free forms are possible, the chair
configuration and the boat configuration. Recent work based on electron
diffraction studies'® and spectroscopic!® and thermodynamic”? properties
has led to the conclusion that at room temperature cyclohexane exists pre-
dominantly in the chair form. Similar considerations have been brought to
bear upon the structures of the mono- and dimethyl derivatives of cyclo-

8 T. Posternak, Helv. Chim. Acta 29, 1991 (1946).

9T. Posternak, Helv. Chim. Acta 19, 1007 (1936).

10 G. Dangschat and H. O. L. Fischer, Naturwissenschaften 27, 756 (1939).

1H. O.L. Fischer, Harvey Lectures 40, 156 (1945).

2H. Chargaff and B. Magasanik, J. Biol. Chem. 165, 379 (1946).

18 B. Magasanik and E. Chargaff, J. Biol. Chem. 175, 929 (1948).

144 B. Magasanik and E. Chargaff, J. Biol Chem. 175, 939 (1948).

18 R. E. Franzl and E. Chargaff, Federation Proc. 9, 173 (1950).

16 R. E. Franzl and E. Chargaff, Nature 168, 955 (1951).

17K. Freudenberg, Stereochemie, p. 662, Deuticke, Leipzig and Vienna, 1933.

18 QO. Hassel, Tidsskr. Kjemi, Bergvesen Met. 3, 32 (1943); Quart. Revs. (London)
7, 221 (1953).

19R.S. Rasmussen, J. Chem. Phys. 11, 249 (1943).

20 C. W. Beckett, K. S. Pitzer, and R. Spitzer, J. Am. Chem. Soc. 69, 2488 (1947).

332 INOSITOLS

hexane”? 2! and upon the various hexachlorocyclohexanes.” If the con-
ceptions applied to cyclohexane and its derivatives are extended to the
cyclohexitols, it will be seen that six of the substituents (hydroxyl or
hydrogen) surround the ring of carbon atoms in an equatorial belt; the six
others are perpendicular to the plane formed by the carbon ring: three
above (north polar), three below (south polar). Each carbon atom will

TABLE II
SPATIAL CONFIGURATIONS OF CYCLOHEXITOLS?
Compound Position of hydroxyls on carbon atoms
Planar : wre
eae Trivial name Designation Equatorial ona ae
I Cyclohexane- ao 2, 4, 6 —_
(1,2,3,4,5,6)-
cis-hexol
II epi-Inositol Cyclohexane- lo Bp Gp, (0 Ph dl —
(1,2,3,4,5)-cis-
hexol
Iil allo-Inositol Cyclohexane- We, 2,4 5
(1,2,3,4)-czs-hexol
IV Cyclohexane- Los 4 2 5
(1,2,3)-cts-hexol
V meso-Inosi- Cyclohexane- I pAb, a, 2 —
tol (1,2,3,5)-c7s-hexol
VI muco-Inosi- Cyclohexane- 1550 2, 4 3
tol (1,2,4,5)-cis-hexol
WAIL Seyllitol Cyclohexane- 1h 5 Bees BO =
(1,3,5)-cis-hexol
VIII d-Inositol Cyclohexane- 1, 4, 5, 6 2 3
(1,2,5)-cis-hexol
TX l-Inositol , Cyclohexane- 2, 3, 4, 5 1 6

(1, 2,4) -cis-hexol

* Based on the chair configuration and on the numbering system of Magasanik and Chargaff.4

therefore carry one polar and one equatorial substituent. A twist of the six
carbon atoms through a single plane to the opposite chair form renders the
equatorial substituents polar and vice versa.2! Two geometrically tautomeric
forms will, consequently, exist for each inositol isomer. By analogy to the
methyl-substituted cyclohexanes,?° it may, however, be assumed that the
tautomer possessing the smaller number of polar hydroxyls will predomi-
nate.

The constellations of the hydroxyl groups in the chair forms of the iso-

21 F, D. Rossini and K. 8. Pitzer, Science 105, 647 (1947).
22 QO. Bastiansen, @. Ellefsen, and O. Hassel, Research (London) 2, 248 (1949).

II. CHEMISTRY 333

meric cyclohexitols are listed in Table II, with the use of the designations
and the numbering system proposed by Magasanik and Chargaff.4 The
scheme of Fletcher et al.> cannot be followed for this purpose, since, as
may be seen in Table I, it employs two alternative routes of numbering for
five of the nine cyclohexitols. A system for the graphic presentation of the
constellations of the cyclitols has been proposed recently by Magasanik
et al.?® In this notation, which is employed for the depiction of the inositol
constellations in Table III, north polar hydroxyls are indicated by full

TABLE III

CoNSTELLATIONS OF CYCLOHEXITOLS?

ae,
ae
.

I III
Unknown ep?-Inositol allo-Inositol

a,
a2
iz.

IV VI
Unknown meso-Inositol muco-Inositol
Ash shesk
VII VIII Ix
Sceyllitol d-Inositol L-Inositol

* Based on the chair configuration. Graphic notation??: north polar hydroxyls, full circles; south
polar hydroxyls, open circles; equatorial hydroxyls, lines.

circles, south polar hydroxyls by open circles, equatorial hydroxyls by
radial lines; the hydrogen atoms are not shown.

The Peolbaieat significance of the spatial eSiemuEAtORS of the cyclo-
hexitols has been demonstrated by the selectivity, dependent upon the
stereochemistry of the different substrates, with which specific hydroxyl
groups are oxidized by Acetobacter suboxydans to mono- or dicarbonyl
derivatives. These studies have led to the following rules defining the steric
requirements for oxidation:*: ¥: .?3 (1) Only polar hydroxyl groups are
oxidized. (2) The carbon in meta position to the one carrying the polar
hydroxyl group (in counterclockwise direction, if north polar; clockwise,
if south polar) must carry an equatorial hydroxyl group.

°3 B. Magasanik, R. E. Franzl, and E. Chargaff, J. Am. Chem. Soc. 74, 2618 (1952).

334 INOSITOLS

D. DISTRIBUTION

1. meso-INOSITOL

meso-Inositol, discovered by J. Scherer™ in 1850, appears to be an almost
ubiquitous and possibly essential cellular component. A survey of its oc-
currence in plant and animal tissues has been given by Fleury and Balatre.1
The extent to which it is present in all microorganisms cannot yet be
stated. It is doubtless an important constituent of the lipids and other
fractions of acid-fast bacteria from which it has been isolated in quan-
tity.25 26

When meso-inositol is stated to occur in almost all types of cell, this
must not be taken to mean that it can always be isolated from them without
more or less extensive degradative action. There are obviously many types
of derivatives in which inositol is able, and often has been shown, to exist,
such as ethers, esters, lipids, and therefore also in lipoproteins, phospho-
proteins, etc. It has frequently been found in the free form; but this may
often have been due to enzymatic liberation taking place in the course of
the preparation or the assay.

Among the naturally occurring derivatives of meso-inositol, the following
may be mentioned. Bornesitol, a dextrorotatory monomethyl ether; *
sequoitol, an optically inactive monomethy] ether;?*: *° and dambonitol, an
inactive dimethyl ether.*!** One of the most widely distributed forms of
meso-inositol is phytin, the hexaphosphoric acid ester. The various phos-
phates of inositol have been reviewed recently by Courtois.* The occur-
rence of meso-inositol as a component of an entire series of lipids is of
great interest. It was first discovered, in this combination, in lipid prepara-
tions from tubercle bacilli and related acid-fast microorganisms,?*: 2° and
later it was shown to occur in soybean phosphatides**: ** and in brain
phosphatides.*” The structure of the soybean inositide, for which the name

24 J. Scherer, Ann. Chem. Justus Liebigs 78, 322 (1850).

25 R. J. Anderson, J. Am. Chem. Soc. 62, 1607 (1930).

26H}. Chargaff and R. J. Anderson, Hoppe-Seyler’s Z. physiol. Chem. 191, 172 (19380).
27 A. Girard, Compt. rend. 73, 426 (1871).

28H). R. Flint and B. Tollens, Ann. Chem. Justus Liebigs 272, 288 (1893).
29). C. Sherrard and E. F. Kurth, J. Am. Chem. Soc. 51, 3139 (1929).

30 N. V. Riggs, J. Chem. Soc. 1949, 3199.

31 A. Girard, Compt. rend. 67, 820 (1868).

32. C. O. Weber, Ber. deut. chem. Ges. 36, 3108 (1903).

33 A.W. K. De Jong, Rec. trav. chim. 27, 257 (1908).

34 J. E. Courtois, Bull. soc. chim. biol. 38, 1075 (1951).

35 EB. Klenk and R. Sakai, Hoppe-Seyler’s Z. physiol. Chem. 258, 33 (1939).
36D. W. Woolley, J. Biol. Chem. 147, 581 (1943).

37 J. Folch and D. W. Woolley, J. Biol. Chem. 142, 963 (1942).

II. CHEMISTRY 335

lipositol has been suggested,®® appears to be complex. The best charac-
terized inositol-containing lipid doubtless is the diphosphoinositide iso-
lated from brain by Folch,** which contains meso-inositol in the form of a
diphosphate. The structure of this diphosphoric acid ester is not yet known
except for the relative position of the two acid residues, which is meta.
There exist probably many other forms in which inositol occurs in tissues;
one could think of polyinositol phosphates and even of inositol polyphos-
phates. Indications of the existence of highly polymerized substances con-
taining inositol have been reported.**: 4? No more than brief reference can
be made here to streptamine, a component of streptomycin, which is a
meta-diaminocyclotetritol.*!

2. SCYLLITOL

This cyclohexitol (structure VII) has been found in the organs of several
fish varieties (dogfish, sharks, etc.),*?“* in acorns,** and in the leaves of the
coconut palm*é and of dogwood.” Its occurrence in human urine has been
reported recently.8 A C-methyl derivative*® of scyllitol, mytilitol, has been
isolated from the mussel Mytilus edulis.°°*

3. d- AND [-INOSITOL

The optically active cyclohexitols occur in nature in the form of mono-
methyl ethers. The dextrorotatory pinitol, which gives rise to d-inositol
(structure VIII), was first found in Pinus lambertiana,® but it occurs also
in a variety of other plants or plant products (Madagascar rubber, red-
wood, loco weed).!: 2 The following structure has recently been assigned to

38 J. Folch, J. Biol. Chem. 177, 505 (1949).

39D. W. Woolley, J. Biol. Chem. 139, 29 (1941).

40 J. Folch and F. N. Le Baron, Federation Proc. 10, 183 (1951).

41 R. U. Lemieux and M. L. Wolfrom, Advances in Carbohydrate Chem. 3, 337 (1948).

42 G. Staedeler and F. T. Frerichs, J. prakt. Chem. 73, 48 (1858). -

43 J). Ackermann and M. Mohr, Z. Biol. 98, 37 (1937).

44M. Mohr, Z. Biol. 98, 276 (1937).

45 ©, Vincent and Delachanal, Compt. rend. 104, 1855 (1887).

46H. Miiller, J. Chem. Soc. 91, 1767 (1907); 101, 2383 (1912).

47C. E. Sando, K. S. Markley, and M. B. Matlack, J. Biol. Chem. 114, 39 (1936).

48 P.F. Fleury, J. E. Courtois, and A. L. Jouannet, Abstr. 12th Intern. Congr. Pure
and Applied Chem. New York p. 82 (1951).

49 T. Posternak, Helv. Chim. Acta 27, 457 (1944).

50 B. C. P. Jansen, Hoppe-Seyler’s Z. physiol. Chem. 85, 231 (1913).

51 J). Ackermann, Ber. deut. chem. Ges. 64, 1938 (1921).

52 R. J. Daniel and W. Doran, Biochem. J. 20, 676 (1926).

53M. Berthelot, Compt. rend. 41, 392 (1855); Ann. chim. phys. 46, 66 (1856).

336 INOSITOLS

pinitol.*! According to the nomenclature of Fletcher et al.* it would conse-

quently be designated as 5-methyl-p-inositol.

The levorotatory quebrachitol is a monomethy] ether of J-inositol (struc-
ture [X). It was discovered by Tanret®> in the bark of the quebracho tree.
In addition to several other plants,!: 2 it occurs in the latex of Hevea brasi-
liensis,°® which appears to be the best source. The natural occurrence of
racemic inositol also has been reported.*”

4. OTHER CYCLOHEXITOLS

None of the other isomers of meso-inositol appears as yet to have been
found in nature. It is, however, perhaps suggestive that the y isomer of
hexachlorocyclohexane (‘‘gammexane”’), widely used as an insecticide, has
been shown to have the configuration of muco-inositol (structure VI).°* In
this connection, a brief mention of the structures of the other hexachloro-
cyclohexanes may be of interest: the a isomer corresponds to d,l-inositol
(structures VIII and IX), the 6 isomer to scyllitol (structure VII), the
6 isomer to meso-inositol (structure V), and the e isomer to the unknown
ceyclohexitol [V.”

EK. PROPERTIES

The cyclohexitols are white crystalline, non-reducing substances, soluble
in water, the most sparingly soluble being scyllitol (VIL). They are usually
recrystallized from aqueous ethanol. For additional characterization the
hexaacetates are often employed; and the distillation in vacuum of similar
hexa derivatives has been suggested as a procedure for final purification.”

Some of the characteristics of the various isomers are as follows: ep7-
inositol (II), m.p. 285° (with decomposition); hexaacetate, m.p. 188°;
allo-inositol (III), m.p. 270-275°; meso-inositol (V), m.p. 225-227°; hexa-
acetate, m.p. 216-217°; muco-inositol (VI), m.p. 285-290° (with decomposi-
tion); scyllitol (VII), m.p. 352° (with decomposition); hexaacetate, m.p.

54 A. B. Anderson, D. L. MacDonald, and H. O. L. Fischer, J. Am. Chem. Soc. 74,

1479 (1952).

55 C. Tanret, Compt. rend. 109, 908 (1889).
56 A.W. K. De Jong, Rev. trav. chim. 25, 48 (1906).
57 ©. Tanret, Compt. rend. 145, 1196 (1907).

88 G. W. van Vloten, C. A. Kruissink, B. Strijk, and J. M. Bijvoet, Nature 162,
771 (1948).

II. CHEMISTRY 337

296-297°: d-inositol (VIII), m.p. 249-250°; [a]2> = +65.0° (in water);
l-inositol (IX), m.p. 249-250°; [a]?? = —63.8° (in water).

The behavior of cyclohexitols toward oxidizing agents is not yet com-
pletely understood. Vigorous action of nitric acid, as it is employed in the
Scherer test for the qualitative demonstration of inositol, leads to aromati-
zation and a whole series of six- and five-membered polycarbonyl com-
pounds, such as tetrahydroxyquinone and rhodizonic, croconic, and leuconic
acids. Under milder conditions meso-inositol (V) is converted by nitric
acid to pL-2-keto-ep7-inositol (numbering as in Table I).°°

meso-Inositol (V) behaves in an anomalous fashion toward oxidation
by periodic acid.® Instead of the expected uptake of 6 equivalents of oxi-
dizing agent, only 4 equivalents are consumed in a first rapid phase, which
is followed by a second, much slower one. The reaction mechanism is not
yet understood completely. It is the opinion of this writer, however, that
the spatial constellation of the hydroxyl group in a given inositol isomer,
as discussed in Section II.C and in Tables IT and III, will probably be
found to influence the course of its oxidation by periodate. In this connection
it is noteworthy that the product of the oxidation of meso-inositol by
Acetobacter suboxydans,”: ® which carries a carbonyl group in position
2 instead of the polar hydroxyl,‘ is cleaved in a normal manner by periodic
acid,” and that, on the other hand, the tetraacetyl derivative of meso-
inositol containing a czs-glycol structure, which comprises a polar and an
adjacent equatorial hydroxyl, is not attacked by periodate.®

A number of mono- and dicarbony! derivatives of the different cyclo-
hexitols may be obtained through the action of Acetobacter suboxydans.
(Compare also Section II. C.) The inosose produced from meso-inositol®
has the following structure.’

ept-Inositol (II) yields a levoratory keto derivative with the following

structure.*: 8» 12
ous

5 T. Posternak, Helv. Chim. Acta 19, 1333 (1936).

© P. Fleury, G. Poirot, and J. Fievet, Compt. rend. 220, 664 (1945).

St A. J. Kluyver and A. G. J. Boezaardt, Rec. trav. chim. 58, 956 (1939).
® 1D). B. Sprinson and E. Chargaff, J. Biol. Chem. 164, 433 (1946).

338 INOSITOLS

Mono- and dicarbony] derivatives also have been prepared from the opti-
cally active isomers.*: ?: ¥

Conversions of one inositol isomer into another have frequently been
effected with the use of the keto derivatives as intermediaries. In this
manner, meso-inositol (V) has been converted to epi-inositol (II)>* and
to scyllitol (VII), and meso-inositol has been produced from d-inositol
(VIII)."* The preparation of allo-inosito] (III) and muco-inositol (VI) from
the naturally occurring tetritol conduritol!® should also be mentioned.
Another type of conversion is carried out through the drastic action of
halogen acids at elevated temperature.* Thus, meso-inositol (V) is con-
verted to dl-inositol (VIII + IX).*: ® Corresponding reactions have also
been described with quebrachitol and J-inositol (IX) as the starting pro-
ducts.*”

The synthesis of meso-inositol by the catalytic hydrogenation of hexa-
hydroxybenzene® appears to have been repeated recently,®® although other
workers”: 7 experienced difficulty in confirming it.

Addendum: Among other literature reports, the following may be
mentioned: a new total synthesis of meso-inositol (V) ;” the demonstration

OCH;

of the structure of quebrachitol, a monomethyl ether of /-inositol (IX);”
and a discussion of the nomenclature of the cyclitols and of some of their
isopropylidene derivatives.”

6% T. Posternak, Helv. Chim. Acta 24, 1045 (1941).

6 A. Miiller, J. Chem. Soc. 91, 1780 (1907); 101, 2383 (1912).

® T. Posternak, Helv. Chim. Acta 31, 2242 (1948).

6 Hf. G. Fletcher, Jr., and G. R. Findlay, J. Am. Chem. Soc. 70, 4050 (1948).
67 A. Contardi and B. Ciocea, Gazz. chim. ital. 79, 694 (1949).

6 H. Wieland and R.S. Wishart, Ber. deut. chem. Ges. 47, 2082 (1914).

*® R. Kuhn, G. Quadbeck, and E. Réhm, Ann. Chem. Justus Liebigs 565, 1 (1949).
7M. R. Stetten and D. Stetten, Jr., J. Biol. Chem. 164, 85 (1946).

1 R. C. Anderson and E. 8. Wallis, J. Am. Chem. Soc. 70, 2931 (1948).

2 'T. Posternak, Helv. Chim. Acta 33, 1597 (1950).

%'T. Posternak, Helv. Chim. Acta 35, 50 (1952).

™§. J. Angyal and C. G. Macdonald, J. Chem. Soc. 1952, 686.

III. INDUSTRIAL PREPARATION 339
III. Industrial Preparation

E. R. WEIDLEIN, JR.

In the absence of a practical chemical synthesis for inositol, the com-
pound has been obtained industrially by isolation of the natural vitamin
from plant sources.

A. PRESENCE IN PLANTS

Inositol is found widely distributed in many different types of plants.
It is most frequently encountered in combination with phosphoric acid,
although free inositol and monomethyl and dimethyl ethers of inositol
have also been isolated from some plant species. Phospholipids which con-
tain inositol, rather than glycerol, as the polyalcohol are an important
class of phosphoric acid derivatives in plants as well as in animals. How-
ever, the most common form for the occurrence of inositol in plants is as a
hexaphosphorie acid ester called phytic acid, a compound which is capable
of forming salts with potassium, calcium, magnesium, iron, manganese,
and other metals of importance in plant nutrition.

Phytic acid and its salts appear to be heavily concentrated in seeds and
cereal grains, in which phytate may account for as much as 86% of the
total phosphorus present.! In some instances the phytate has also been
shown to exist in combination as a protein complex.? Little is known of
the functions of phytates in plant metabolism, but they have been pre-
sumed to act as stores of phosphoric acid which may be utilized by the
plant during sprouting. This opinion is supported by the fact that the
concentration of free inositol has been found to increase in several different
types of seeds during germination.’ It also has been recognized that phytates
may function as carriers for trace metals needed to insure normal plant
growth.*

Approximately 80% of the phosphorus in corn exists as phytate, corn
having been reported to have a content of phytic acid phosphorus which
varies from 0.199 to 0.270 %, compared with a variation in total phosphorus
of 0.248 to 0.330% on the same basis.! Among the other plants in which
phytic acid has been found are wheat, rye, oats, peas, beans, barley, rice,
cottonseed, flaxseed, soybeans, and peanuts.

In extracting the phytate from plant materials, investigators have most

1H. Mollgaard, K. Lorenzen, I. G. Hansen, and P. E. Christensen, Biochem. J.
(London) 40, 589 (1946).

2T. D. Fontaine, W. A. Pons, Jr., and G. W. Irving, Jr., J. Biol. Chem. 164, 487
(1946).

3V. H. Cheldelin and R. L. Lane, Proc. Soc. Exptl. Biol. Med. 64, 53 (1943).

4E. R. Weidlein, Jr., Mellon Inst. Bibliographic Ser. Bull. 6 (1951).

340 INOSITOLS

often used dilute hydrochloric or sulfuric acids to treat the whole seed or
individual fractions of the seed, but other reagents have also been used.
From these extracts, various phytate salts have been precipitated and free
inositol obtained by hydrolysis of the phytate salt.

B. CORN STEEPWATER AS A SOURCE OF INOSITOL

Because of the relatively high concentration of contained phytate and
the large volume of raw material available, corn steepwater has proved to
be a highly satisfactory source of inositol. At least two commercial pro-
ducers of inositol are known to have developed processes utilizing corn
steepwater as a basis for the isolation of the natural vitamin.

Steepwater is obtained during the process for the industrial wet-milling
of corn. Shelled corn entering the wet-milling plant is first soaked in a
warm, dilute, aqueous sulfur dioxide solution. This operation softens the
kernel and facilitates the subsequent separation of hull and fiber, germ,
gluten, and starch. The presence of sulfur dioxide in the steepwater inhibits
the action of microorganisms on the grain, and the liquid medium com-
pletes the cleansing of the kernels before milling. Most important, the
effect. of the steeping process is to remove the water-soluble and acid-
soluble substances present in corn, phytates being included in this category.
Soluble protein, sugars, gums, and similar organic compounds, as well as
inorganic substances, are present in the steepwater along with phytate.

In the form of phytic acid and its salts, inositol ordinarily constitutes
approximately 2% of the steepwater total solids. Precipitation of the
phytate in steepwater, hydrolysis of the phytate, and isolation of inositol
from the hydrolyzate are the steps involved in current industrial processes
for the preparation of inositol.

C. PRECIPITATION OF PHYTATE FROM CORN STEEPWATER

The phytate in steepwater is effectively separated by the addition of a
slurry of slaked lime which reacts with the soluble phytate present in the
steepwater to precipitate an insoluble phytate salt. Steepwater itself con-
tains calcium, magnesium, iron, sodium, and potassium; consequently
free phytic acid as such undoubtedly is not present in steepwater. Rather,
it appears probable that the phytate is present as soluble partial salts of
calcium, magnesium, iron, sodium, and potassium. Thus, the phytate
product precipitated from corn steepwater by the addition of lime is not
a pure calcium salt of phytie acid but is a mixed salt which contains other
metal ions present in steepwater, as well as some protein.

In the precipitation step,° lime is usually added until the steepwater is
brought to a pH of 5 to 7. The resultant heavy slurry is then filtered, and

5 F. A. Hoglan and E. Bartow, Ind. Eng. Chem. 31, 749 (1939).

III. INDUSTRIAL PREPARATION 341

the filter cake is washed thoroughly with water to remove retained steep-
water. The crude product, essentially a mixed calcium-magnesium salt of
phytic acid which contains the impurities mentioned, has often been refer-
red to in the literature as “‘phytin” or as “calcium phytate.”’ This product
may be dried by conventional procedures or may be used directly for
hydrolysis to inositol.

Various procedures also may be employed to improve the quality of
crude phytate obtained from steepwater. Use of so-called “light steep-
water” for precipitation of the phytate, purification of the crude phytate
by treatment with alkali, and other modifications may help to reduce the
nitrogen content of the crude phytate and give a better raw material for
hydrolysis to inositol.*: 7

DD. HYDROLYSIS, OF PHYTATE TO INOSITOL

Inositol has been obtained from steepwater phytate by hydrolysis under
a variety of conditions.*!? Temperature, pressure, pH, and time of hy-
drolysis are variables which have been considered. Temperatures employed
have ranged from approximately 100° to 200°, pressures from atmospheric
to mild autoclaving conditions, and the medium for hydrolysis from a 25 %
calcium hydroxide slurry to a 60% sulfuric acid solution. The time re-
quired for completion of hydrolysis has usually been several hours.

According to Bartow and Walker® inositol can be obtained conveniently
by heating phytate under pressure with either water or acid. The quality
of the phytate used and the nature of the impurities present during hy-
drolysis appear to have as much influence on the yield of inositol as does
any other factor, including the nature of the conditions chosen for hy-
drolysis. The calculated yield of inositol based on pure dry calcium phytate
is 20.27 %, but the yields of inositol reported to be obtained from steepwater
_ phytate have generally been in the range of 7 to 12 %.

K. ISOLATION OF INOSITOL FROM HYDROLYZATE

Hydrolysis of phytate produces 6 moles of inorganic phosphate for each
mole of inositol. The inorganic phosphate produced by hydrolysis of phytate
may be removed by filtration if the hydrolyzate is first adjusted to a pH
that is slightly alkaline.

The phosphate filter cake is washed with hot water to remove dissolved
and adsorbed inositol, and the wash filtrate is combined with the original
filtrate. The combined filtrate, a crude aqueous solution of inositol, may be

6 F. A. Hoglan and E. Bartow, J. Am. Chem. Soc. 62, 2397 (1940).

7 Corn Products Refining Co., Brit. Pat. 601,273 (May 3, 1948).

8 KH. Bartow and W. W. Walker, Ind. Eng. Chem. 30, 300 (1938).

°K. Bartow and W. W. Walker, U.S. Pat. 2,112,553 (March 29, 1938).
10H. Elkin and C. M. Meadows, U.S. Pat. 2,414,365 (January 14, 1947).

342 INOSITOLS

decolorized with bone black or activated carbon, filtered to remove carbon,
and concentrated by vacuum-pan evaporation.

Crystallization of inositol from the mother liquor is generally accom-
plished by gradual cooling to as low a temperature as practical with con-
tinuous, moderate agitation. Crystals are separated from the mother
liquor by centrifugation and are then further washed and dried. Several
crops of crystals may be recovered by concentration of the mother liquor
and a repetition of the crystallization process.

The inositol obtained in this manner is an anhydrous, colorless, crystal-
line material having a melting point of approximately 225°, which is char-
acteristic of the pure myo isomer.

IV. Biochemical Systems

HENRY A. LARDY

The widespread occurrence and relatively great abundance of myo-
inositol make this compound unique among the vitamins in that it may
act both catalytically, like the other vitamins, and as an energy-yielding
foodstuff or metabolite. These two aspects of the biochemical systems in
which myo-inositol participates will therefore be discussed separately.

A. BIOCATALYTIC FUNCTIONS OF MYO-INOSITOL

myo-Inositol is an essential nutrient for several strains of yeast,’* for
fungi,’ > for a mutant of Neurospora crassa,® and for mice,’: * rats,’ cotton
rats,!°: ! hamsters,! and chicks under certain deitary regimens. In spite

1K. V. Easteott, J. Physiol. Chem. 32, 1094 (1928).

2R. J. Williams, R. E. Eakin, and E. E. Snell, J. Am. Chem. Soc. 62, 1204 (1940).

3 L. Atkin, A. 8. Schultz, W. L. Williams, and C. N. Frey, Ind. Eng. Chem. Anal. Ed.
15, 141 (1943).

4F. Koégl and N. Fries, Hoppe-Seyler’s Z. physiol. Chem. 249, 93 (1937).

5 W. J. Robbins, J. E. Mackinnon, and R. Ma, Bull. Torrey Botan. Club 69, 509
(1942).

6G. W. Beadle, J. Biol. Chem. 156, 683 (1944).

7D. W. Woolley, J. Biol. Chem. 136, 113 (1940).

8D. W. Woolley, J. Biol. Chem. 139, 20 (1941).

9T. J. Cunha, 8. Kirkwood, P. H. Phillips, and G. Bohstedt, Proc. Soc. Expil. Biol.
Med. 54, 236 (1943).

10 J. M. McIntire, B. S. Schweigert, and C. A. Elvehjem, J. Nutrition 27, 1 (1944).

1B. 8. Schweigert, Vitamins and Hormones 6, 55 (1948).

12 J. M. Cooperman, H. A. Waisman, and C. A. Elvehjem, Proc. Soc. Exptl. Biol.
Med. 52, 250 (1943).

13 —D, M. Hegsted, G. M. Briggs Jr., R. C. Mills, C. A. Elvehjem, and E. B. Hart,
Proc. Soc. Exptl. Biol. Med. 47, 376 (1941).

IV. BIOCHEMICAL SYSTEMS 343

of the great variety of organisms which require inositol, virtually nothing
is known of the function of the vitamin in terms of enzymes or coenzymes.
Since animals probably can synthesize myo-inositol (see Section VI), it
has been assumed that the dietary requirement of this compound reflects
a requirement of the intestinal flora. Woolley found that feeding panto-
thenic acid benefited mice deficient in inositol. White rats do not require
myo-inositol when fed purified diets,!®: 1® but Cunha et al.° found that rats
on diets containing soybean meal showed poor growth and hair loss. These
symptoms lessened after feeding of inositol. The fact that biotin or the
sulfur-containing amino acids can prevent the deficiency symptoms” lends
support to the hypothesis that the beneficial effect of inositol is indirect
and is probably exerted through intestinal microorganisms.

1. Stupries witH INHIBITORY ANALOGS

The possibility that the insecticide, hexachlorocyclohexane, exerts its
physiological action by antagonizing a normal function of myo-inositol
was first suggested by Slade.!® Evidence tending to support this possibility
has been obtained with several inositol-requiring microorganisms.!°*! For
example, Kirkwood and Phillips!® found the y isomer of hexachlorocyclo-
hexane to inhibit growth of an inositol-requiring strain of Saccharomyces
cerevisiae. The inhibitory effect was competitively, but incompletely, re-
versed by myo-inositol. The 6, a, and 6 isomers, in decreasing order, were
not as strongly inhibitory as the y isomer, and their effects were not in-
fluenced by increasing concentrations of myo-inositol. The relatively low
inhibition index,” indicating a close similarity of structure between the
inhibitor and metabolite, was taken by Kirkwood and Phillips!’ to indicate
that the y isomer of hexachlorocyclohexane had the same configuration as
myo-inositol. Bijvoet”? and coworkers” as well as Blekkingh*® conclude from
physical measurements that the y isomer does not have the configuration
of myo-inositol but appears to have that of muco-inositol. Dissimilarity of
configuration between the y isomer and myo-inositol introduces complica-

47). W. Woolley, J. Exptl. Med. 75, 277 (1942).

6 T. H. Jukes, Proc. Soc. Exptl. Biol. Med. 45, 625 (1940).

16 J,, R. Richardson, A. G. Hogan, B. Long, and K. I. Itschner, Proc. Soc. Expitl.
Biol. Med. 46, 530 (1941).

17R. R. Spitzer and P. H. Phillips, Proc. Soc. Exptl. Biol. Med. 68, 10 (1946).

18 R. E. Slade, Chemistry & Industry 64, 314 (1945).

19§. Kirkwood and P. H. Phillips, J. Biol. Chem. 163, 251 (1946).

20H. W. Buston, 8S. E. Jacobs, and A. Goldstein, Nature 158, 22 (1946).

21R. C. Fuller, R. W. Barratt, and E. L. Tatum, J. Biol. Chem. 186, 823 (1940).

22 A. MclIlwain, Brit. J. Exptl. Pathol. 23, 95 (1942).

23 J. M. Bijvoet, Rec. trav. chim. 67, 777 (1948).

24 G. W. Von Vioten, C. A. Kruissink, B. Strijk, and J. M. Bijvoet, Nature 162, 771
(1948).

26 J. J. A. Blekkingh, Rec. trav. chim. 68, 345 (1949).

344 INOSITOLS

tions of interpretation but should not lessen interest in the biological com-
petition between these two compounds.

Several workers have been unable to demonstrate a competitive rela-
tionship between myo-inositol and y-hexachlorocyclohexane.?**> However,
the negative results have, for the most part, been obtained with strains of
microorganisms which do not require preformed myo-inositol. From the
results of Kirkwood and Phillips,!® Schopfer e¢ al.2° and Fuller et al.” it
appears that the competitive relationship is demonstrable only in organisms
which are nutritionally dependent upon myo-inositol and that there is a
second, non-specific, inhibitory effect of hexachlorocyclohexanes which is
not influenced by myo-inositol.

myo-Inositol has also been reported by Chargaff and coworkers?’ to
counteract the arrest of nuclear division and the tumor formation induced
in onion root tips by either colchicine or y-hexachlorocyclohexane. pD-In-
ositol and sorbitol were ineffective. The effect of myo-inositol has been
confirmed,*°: *! although a number of sugars were found to have similar
activity.*° Other workers*: ** were unable to confirm the competitive
relationship between myo-inositol and colchicine in onion root cells. How-
ever, a more detailed study of this effect in fibroblast cultures from areolar
tissue of the adult rat has again shown myo-inositol to exert a distinct
inhibition of the effect of colchicine on mitosis.*** Other compounds which
were found not to influence the effect of colchicine were D-inositol, myo-
inosose-2 , DL-ep7-inosose-2,"4 sorbitol, ribose, sucrose, and glucose.

In higher animals the toxic effects of y-hexachlorocyclohexane appear
not to be counteracted by myo-inositol.**-*8 The convulsive effects of y-hex-
achlorocyclohexane appear to be prevented by previous administration
of the 6 or 6 isomers.” Another interesting finding is that the 6 isomer

26 W. H. Schopfer, T. Posternak, and M. L. Boss, Schweiz. Z. allgem. Pathol. u.
Bakteriol. 10, 443 (1947).

27 C. Fromageot and M. Confino, Biochim. et Biophys. Acta 2, 142 (1948).

28 P. Chaix, Bull. soc. chim. biol. 30, 835 (1948).

29 KH. Chargaff, R. N. Stewart, and B. Magasanik, Science 108, 556 (1948).

30 F. D’Amato, Carylogia 1, 223 (1949) [C. A. 48, 9170 (1949)].

31 F, D’Amato, Carylogia 1, 358 (1949) [C. A. 44, 4089 (1950)].

32 G. Deysson and M. Deysson, Bull. soc. chim. biol. 82, 276 (1950) [C. A. 44, 10052
(1950)].

33§. Carpentier and C. Fromageot, Biochim. et Biophys. Acta 5, 290 (1950).

33a M. R. Murray, H. H. de Lam, and E. Chargaff, Exptl. Cell Research 2, 165 (1951).

34 For clarification of the nomenclature of these inositol derivatives, see Section I
of this chapter.

35H. A. Doisy, Jr., and B. C. Bocklage, Proc. Soc. Exptl. Biol. Med. 71, 490 (1949).

36 H. A. Doisy, Jr., and B. C. Bocklage, Proc. Soc. Expil. Biol. Med. 74, 613 (1950).

87 B. P. McNamara and 8. Krop, J. Pharmacol. Exptl. Therap. 92, 140 (1948).

38 B. P. McNamara and 8. Krop, J. Pharmacol. Exptl. Therap. 92, 147 (1948).

IV. BIOCHEMICAL SYSTEMS 345

(which probably has the configuration of myo-inositol*’) stimulates the
respiration of rat brain homogenates.*? The hexamethyl ether of myo-
inositol produces convulsions in rabbits similar to those produced by
y-hexachlorocyclohexane; however, the ether is only 1/100 as active as the
chlorine-containing analog.*!

Several workers’: *® consider it likely that the biological antagonism
between hexachloroxycyclohexane and myo-inositol is actually exerted be-
tween the former and a bound or complex form of the latter. Since the
hexachlorocyclohexanes are much more soluble in fat solvents than in
aqueous systems, it is possible that they exert their effect on systems re-
acting with phospholipids containing myo-inositol.

Some years ago Williams and coworkers” found relatively high concen-
trations (0.4%) of inositol in a partially purified preparation of a-amylase
from pancreas. This led Lane and Williams® to test the effect of y-hexa-
chlorocyclohexane on the catalytic activity of the enzyme. Their report
that the chlorine-containing analog inhibited the enzyme and that myo-
inositol competitively reversed the inhibition could not be confirmed by
two groups“: 4° who used the more purified and crystalline enzyme.

2. Bounp Forms oF myo-INOSITOL

Although there are appreciable quantities of free myo-inositol in many
biological materials, by far the greater part of this substance exists in
various complex forms. The more common materials with which it com-
bines include phosphate, proteins, fatty acids, glycerol, and galactose.
Although these complex compounds may be considered “structural ma-
terial” of the cell, they may be expected to have specific biocatalytic
functions as well.

a. Inositol-Containing Phospholipids

The phosphatides of the tubercle bacillus,‘ “” the soybean,®*: 4° cotton
seed,°’ and brain®!: * and liver®® tissue contain inositol. Inositol mono-

39 Q. Bastiansen, @. Ellefson, and O. Hassel, Research 2, 248 (1949).

40 B. P. McNamara and 8. Krop, Science 109, 330 (1949).

‘tN. P. Buu-Hoi, E. Philippot, M. J. Dallemagne, and M. A. Gerebtzoff, Compt. rend.
soc. biol. 144, 1568 (1950).

# R. J. Williams, F. Schlenk, and M. A. Eppright, J. Am. Chem. Soc. 66, 896 (1944).

48R.L. Lane and R. J. Williams, Arch. Biochem. 19, 329 (1948).

44 E. H. Fischer and P. Bernfeld, Helv. Chim. Acta 82, 1146 (1949).

45S. Schwimmer and A. K. Balls, J. Biol. Chem. 179, 1063 (1949).

46 R. J. Anderson, J. Am. Chem. Soc. 62, 1607 (1930) [C. A. 24, 2490 (1930)]

47 J. Cason and R. J. Anderson, J. Biol. Chem. 126, 527 (1988).

48 E. Klenk and R. Sakai, Hoppe-Seyler’s Z. physiol. Chem. 258, 33 (1939).

49D. W. Woolley, J. Biol. Chem. 147, 581 (1943).

5° H.S. Oleott, Science 100, 226 (1944).

346 INOSITOLS

phosphate has been isolated as an hydrolysis product of the phosphatides
from the tubercle bacillus” and from soybean. However, inositol diphos-
phate was obtained by Folch® from brain phosphatide. Periodate oxidation
demonstrated that the phosphate groups were in meta position to one an-
other. The brain phosphatide contains, in addition to inositol diphosphate,
an equimolar proportion of fatty acid (of rather high equivalent weight)
and glycerol. Soybean “‘lipositol’’*® was found to contain myo-inositol,
galactose, oleic acid, a mixture of three saturated fatty acids—cerebronic,
palmitic, and stearic—phosphoric acid, L(+)-tartaric acid, and smaller
quantities of ethanolamine. Folch*** has obtained from a purified soybean
phosphatide: inositol 2 (moles), carbohydrate (as galactose) 2, phosphoric
acid 2, glycerol 2, fatty acids 3, and amine 1.

Claude has found that inositol is present in relatively high concentra-
tion in the mitochondria and submicroscopic particles (microsomes) of
the liver cell. Approximately 40 to 45 % of the dry weight of the microsomes
is lipid material, and 2% of this is inositol. Claude calculates that, if all
the inositol were bound as “‘lipositol,’’ this component would comprise
more than one-fourth of the total lipid and almost one-half of the phos-
pholipid.

b. Phytic Acid

In plant materials, and especially in the seeds of the grasses, a major
part of the inositol is present as the hexaphosphoric ester.**-*’ This com-
pound is called phytic acid, and its mixed calcium and magnesium salt is
called phytin. The phytic acid of corn is the source of commercial inositol
(Section III). Several possible formulas have been proposed for phytin or
phytic acid, including those in which adjacent phosphoric groups may
combine to form pyrophosphate linkages or in which hydroxyphosphoric
acid groups may be present.

Although a specific biological function for this compound has not been
established, it is known to act as a reservoir of phosphate which becomes
available to the young plant at germination. Complexes between phytic
acid and animal or plant proteins have been obtained by a number of
workers. However, it appears likely that these are merely non-specific
combinations, for the phytic acid may be separated by dialysis.**

51 J. Folch and D. W. Woolley, J. Biol. Chem. 142, 963 (1942).

52 J. Folch, J. Biol. Chem. 177, 505 (1949).

53 T,. B. Macpherson and C. C. Lucas, Federation Proc. 6, 273 (1947).
53a J. Folch, Federation Proc. 6, 252 (1947).

54 A. Claude, J. Exptl. Med. 84, 61 (1946).

55§. Posternak, Compt. rend. 137, 202 337, 439 (1903).

56 C. Neuberg, Biochem. Z. 9, 557 (1908).

57 R, J. Anderson, J. Biol. Chem. 44, 429 (1920).

58 J. Bourdillon, J. Biol. Chem. 189, 65 (1951).

IV. BIOCHEMICAL SYSTEMS 347

Phytin occurs also in animal tissues, for example, in the nucleated eryth-
rocytes of the chicken and turtle.*®

3. Lirpotropic Errect or INOSITOL

There are a number of dietary variables which can lead to deposition of
unusually large amounts of lipid in the liver. Certain nutrients favor this
deposition, whereas others (lipotropic agents®’) favor mobilization of the
lipids and a return to the normal fat content of the liver. Choline® and
other biological methyl donors are particularly well known as dietary
lipotropic agents, but inositol is also effective under certain conditions in
both experimental animals®-® and human subjects.®: ® The effect of
inositol is apparent even with diets containing an adequate supply of
methyl group donors.®: ®§ Although relatively little is known concerning
the mode of action of hpotropic agents, at least two of them—choline and
inositol—are essential constituents of phospholipids, and it seems likely
that their effectiveness in reducing liver fat may be a reflection of that
fact. Since all other vitamins eventually become structural units of proto-
plasm too, it seems arbitrary to separate the lipotropic activities of choline
and inositol from their respective ‘‘vitamin’’ activities.®®

B. METABOLISM OF INOSITOL
1. In BacTEeria

Although the pathways of inositol metabolism in bacteria are better
understood than those in animal tissues, knowledge in this field is still in a
relatively primitive state.

The oxidation of myo-inositol” to yield myo-inosose-2® can serve as the
major source of energy for the growth of Acetobacter suboxydans. The oxida-
tion system is not specific for myo-inositol; other polyhydric cyclohexanes
having polar hydroxyl groups are also attacked.®® Polar hydroxyl groups

59 J. Rapoport, J. Biol Chem. 135, 403 (1940).

60 ©. H. Best, M. E. Huntsman, and J. H. Ridout, Nature 135, 821 (1935).

61 G. Gavin and E. W. McHenry, J. Biol. Chem. 189, 485 (1941).

62 C. H. Best, C. C. Lucas, J. M. Patterson, and J. H. Ridout, Biochem. J. (London)
40, 368 (1946).

68 R. W. Engel, J. Nutrition 24, 175 (1942).

64 J. C. Abels, C. W. Kupel, G. T. Pack, and C. P. Rhoads, Proc. Soc. Exptl. Biol.
Med. 54, 157 (1943).

65]. M. Ariel, J. C. Abels, H. T. Murphy, G. T. Pack, and C. P. Rhoads, Ann. In-
ternal Med. 20, 570, 580 (1944).

66 ©. H. Best, C. C. Lucas, J. Patterson, and J. Ridout, Biochem. J. (London) 48,
452 (1951).

87 A. J. Kluyver and A. G. J. Boezaardt, Rec. trav. chim. 68, 956 (1939).

88 T. Posternak, Helv. Chim. Acta 25, 746 (1942).

69 B. Magasanik and E. Chargaff, J. Biol. Chem. 174, 173 (1948).

348 INOSITOLS

A. suborydans

are those oriented at approximately right angles to the thick plane of car-
bon atoms in the ‘‘chair” conformation of the cyclohexane ring. Equatorial
hydroxyl groups, i.e., those extending the thick plane formed by the carbon
atoms, are not oxidized by this organism. Franzl and Chargaff’?:™ have
obtained cell-free enzyme preparations from A. suboxydans which oxidize
myo-inositol and other inositols in the same manner as do the whole cells.
The enzyme is present in particulate material which is sedimented by
20,000g for 2 hours but which remains in the supernatant fluid after
centrifugation at 2000g for 30 minutes. A heat-stable cofactor, obtained
from whole cells, accelerates the oxidation of myo-inositol by the enzyme
but is not required for the oxidation of glucose. This factor may be replaced
in part by Ca++ or Mgt+. The cell-free preparations did not oxidize sorbitol,
which would seem to indicate that separate enzymes are involved in the
oxidation of polyhydric alcohols and cyclitols, respectively, by this
organism.

Aerobacter aerogenes may be grown under aerobic conditions with myo-
inositol as the sole source of carbon. Magasanik” has obtained consider-
able information about the pathway of inositol degradation by this
organism. Cells grown on myo-inositol under aerobic conditions fermented
myo-inositol to yield 0.68 mole of CO:, 0.72 mole of ethanol, and 1.54
equivalents of an unknown acid. Experiments with inhibitors led to the
conclusion that glucose was not an intermediate. 2-Keto-myo-inositol and

-1 ,2-diketo-myo-inositol were fermented by these cells two to three times
more rapidly than myo-inositol. L-1-Keto-myo-inositol was also fermented,
but no more rapidly than myo-inositol. The following metabolic pathway
was proposed by Magasanik.

Si Ss tha

myo-Incsitol y 2-Keto-myo- aNGEHO Ly wid L-1 ,2-diketo-myo- inositol ;

He 7 Tt i
H;C— va C—O- —s H;C— C—C—O-

H

OH O

] l
mr pai <_—:- H,C—C—0O-
H CO,

IV. BIOCHEMICAL SYSTEMS 349

It is to be expected that more work of this nature with bacterial prepara-
tions will help elucidate the pathway by which animals metabolize myo-
inositol. It is already apparent that, if the pathway in animal tissues is
similar to that shown above, the formation of pyruvate or lactate would
be an adequate explanation of the antiketogenic effect of inositol and of
the conversion of stably bound deuterium in inositol to stably bound
deuterium in urinary glucose (see below).

100

80

60

40

Per cent absorption

0 8 16 24 32
Hours after dosing
Fia. 1. Rate of inositol absorption in fasted rats.”

2. In ANIMALS
a. Absorption

myo-Inositol is absorbed from the digestive tract of animals but at a
relatively slow rate compared with glucose. Figure 1 (from Wiebelhaus
et al.) shows the rate of absorption of approximately 280-mg. doses of
myo-inositol administered by stomach tube to rats of approximately 200-¢.
body weight. The rate of absorption was measured by quantitative deter-
minations® of the inositol remaining in the gastrointestinal tract. A com-
parison of the assay values for the hydrolyzed and unhydrolyzed samples
indicated that no conversion of administered inositol to a bound form had
occurred in the intestine. However, about one-half of the 8 to 12 mg. of
inositol present in the tract of fasted animals (or of the experimental ani-

70 R. E. Franzl and E. Chargaff, Federation Proc. 9, 173 (1950).

mR. E. Franzl and E. Chargaff, Natwre 168, 955 (1951).

2B. Magasanik, J. Am. Chem. Soc. 78, 5919 (1951).

73°V.D. Wiebelhaus, J. J. Betheil, and H. A. Lardy, Arch. Biochem. 18, 379 (1947).

350 INOSITOLS

mals after absorption of the test dose was complete) was in a form not
available to the strain of yeast used in these assays.

The urinary excretion of inositol varied from 3 to 5 mg. per rat for a
24-hour period and was not significantly greater for the rats which had
received from 260 to 335 mg. of inositol per os than for the fasted control
rats. The limit of accuracy of the inositol assay permits the conclusion
that urinary excretion accounted for less than 1% of the administered
inositol.

The absorption of inositol monophosphate was also studied.” From 150
to 200 mg. of the monophosphate isolated from soybean lipositol was
administered to 200-g. rats. The rate of absorption appeared to be more
rapid than for inositol, especially during the first 2 hours. Approximately
80 % of the dose was absorbed in from 1 to 2 hours, and complete absorption
occurred in rats killed at 12 and 24 hours. In all animals killed later than
2 hours after administering inositol monophosphate, almost all the inositol
in the intestinal contents was found to be in a form available to the assay
organism. Since the original isolated inositol monophosphate gave a growth
response equivalent to only 8 to 15 % of its contained inositol, it appeared
that the phosphoric ester was rapidly hydrolyzed in the intestine. The
initial rapid disappearance of inositol might indicate an extremely rapid
absorption of the inositol monophosphate per se, with subsequent slow
absorption of the free inositol which was presumably liberated by intestinal
phosphatases.

Other bound forms of myo-inositol are also apparently readily hydrolyzed
in the intestinal tract of animals. For example, phytin can supply both the
inositol and inorganic phosphate requirements of rodents. Soybean ce-
phalin and even synthetic inositol hexaacetate can replace dietary inositol
for the mouse, indicating that they are cleaved to free inositol.”

Intraperitoneally administered myo-inositol appears to be absorbed,
since it is as effective in depressing ketone body excretion in fasted rats
as is the orally administered compound.”

b. Catabolism

Inositols are isomeric with the common 6-carbon sugars, and perhaps
for this reason the possible conversion of myo-inositol to carbohydrate or
carbohydrate breakdown products in animal tissues has been studied by
many investigators. Although earlier results were inconclusive, it is now
certain that inositol is metabolized, at least in part, like carbohydrate.
Stetten and Stetten” fed deuterium-labeled myo-inositol to a phlorhizin-
ized rat and found that at least 7% of the dose fed appeared in urinary

74D. W. Woolley, J. Biol. Chem. 140, 461 (1941).
75M. R. Stetten and D. W. Stetten, Jr., J. Biol. Chem. 164, 85 (1946).

V. SPECIFICITY OF ACTION 351

glucose. The slow rate of absorption of myo-inositol apparently precludes
the formation of any significant amount of liver glycogen when this com-
pound is fed to fasted rats.’* However, myo-inositol given either orally or
intraperitoneally does depress urinary ketone body excretion. This provides
additional evidence that myo-inositol is metabolized to carbohydrate or to
intermediates which are common to carbohydrate metabolic pathways.
Virtually nothing is known about the pathways by which the inositol
molecule is cleaved and converted to the carbohydrate-like products in
animal tissues.

V. Specificity of Action

ARTHUR H. LIVERMORE

There are nine theoretically possible isomers of inositol, differing only
in the steric arrangements of their hydroxyl groups. Determining the
specific steric configuration which is essential for vitamin activity in a
particular organism becomes, then, simply a matter of testing all the
available isomers of inositol on the organism in question. Only seven of the
nine possible isomers are known, but of those tested only one, myo-inositol,
has vitamin activity for all the inositol-requiring organisms which have
been found up to the present time. There are some organisms for which
-myo-inositol is the only isomer which serves as a vitamin, and other organ-
isms for which other isomers have slight vitamin activity.

The vitamin activity of myo-inositol for yeast was first demonstrated
in 1928 by Eastcott,! and for mice in 1940 by Woolley.2 Following this,
Woolley determined the comparative vitamin activities of various inositols
and inositol derivatives for mice and yeast. The results of this investiga-
tion are summarized in Table IV.* Alopecia in mice was cured not only by
myo-inositol but also by phytin, the hexaphosphate of myo-inositol, and
by mytilitol, which has since been shown to be C-methylscyllitol.4 Other
inositol isomers were completely inactive for mice. The yeast Saccharo-
myces cerevisiae, on the other hand, utilized the mono- and tetraphosphate
esters to a small extent only and did not utilize phytin at all. Mytilitol
was much less effective as a growth stimulant than was myo-inositol. As in
mice, the other isomers of inositol which were tested had no vitamin
activity.

1K. V. Eastcott, J. Phys. Chem. 32, 1094 (1928).

2D. W. Woolley, Science 92, 384 (1940).

’D. W. Woolley, J. Biol. Chem. 140, 461 (1941).
4T. Posternak, Bull. soc. chim. biol. 38, 1041 (1951).

352 INOSITOLS

More recent work with other organisms has demonstrated that L-inositol
has some vitamin activity for Hremothecitum ashbyii and for the inositol-
less mutant of Neurospora crassa, but that D-inositol is completely inactive
for these organisms.’ Two other isomers, ep2-inositol and scyllitol, were
also tested and found to have some vitamin activity for the former but not
for the latter organism. For Hremothecitum ashbyii myo-inositol monophos-
phate has from 50 to 100 % of the activity of the free form;> for Neurospora
crassa, the ester has little or no activity.*” For another organism, Rhizopus
Cohnii®: ° myo-inositol was the most active isomer, but mytilitol, isomytili-

TABLE IV
Activity oF ComMrouNps RELATED TO INOSITOL FoR MICE AND FOR YEAST?

Growth effect

Curative on yeast as

effect on compared to

Compound mice myo-inositol, %

e
S
So

myo-Inositol ae
Phytin +
Sodium phytate —
L-Inositol —
p-Inositol ade
Quercitol =
Quebrachitol (monomethyl ether of L-inositol) —
Pinitol (monomethyl ether of p-inositol) —
Mytilitol (monomethy! ether of myo-inositol) +
Quiniec acid (tetrahydroxyhexahydrobenzoic acid)

Soybean cephelin +
myo-Inositol hexaacetate ==
myo-Inositol monophosphate

myo-Inositol tetraphosphate

Inosose

—
Rm DD OTR RB BOD OR Rs i

4 Hansen No. 1 strain of Toronto yeast (Saccharomyces cerevisiae).

tol, hydroxyisomytilitol, and epz-inositol also had some activity. myo-
Inositol monophosphate was also quite well utilized by this organism.
The reversal of the inhibitory action of malonate on Clostridium sac-
charobutyricum by myo-inositol has been shown to be quite specific.!° The
Dp and L isomers did not reverse the inhibition.
It has been suggested® that, for certain microorganisms at least, three

5P. Chaix, Bull. soc. chim. biol. 30, 835 (1948).

6B. M. Iselin, J. Am. Chem. Soc. 71, 3822 (1949).

7W. H. Schopfer, T. Posternak, and M. L. Boss, Intern. Z. Vitaminforsch. 20, 121
(1948) [C. A. 48, 1457 (1949)].

8 W. H. Schopfer, Helv. Chim. Acta 27, 468 (1944).

9W.H. Schopfer, Bull. soc. chim. biol. 33, 1113 (1951).

10 A. J. Rosenberg, Compt. rend. soc. biol. 142, 443 (1948).

V. SPECIFICITY OF ACTION 353

adjacent c7s-hydroxyl groups, as in myo-inositol, are essential for activity.
The activity for some organisms of scyllitol and mytilitol (C-methylsecyl-
litol), which have no cis-hydroxyl groups, and L-inositol, which has two
pairs of czs-hydroxyl groups, one on each side of the ring, indicates that the
three adjacent cis-hydroxyls are not always essential.

A different kind of specificity is suggested by the work of Magasanik
and Chargaff!! on the oxidation of inositol isomers by Acetobacter suboxy-
dans. This organism, which can oxidize inositol to a cyclic ketone, shows a

N

N

Fig. 2.44
TABLE V
OXIDATION OF INosITOL BY Acetobacter suboxydans
Number of Position of OH groups in this isomer Positions of
Inositol isomer er eee tale ———E———— OH group(s)
of inositol Equatorial North South oxidized
myo-Inositol 1 il, B, 2 HC 2 = 2
L-Inositol 2 25°05 45,0 1 6 1G
p-Inositol 2 1, 4,5; 6 2, 3 2,3
Scyllitol 0 OOO == =
epi-Inositol 1 il, Bp Gy, & 2,4 = 2
p-Quercitol 2 iL, Gy, 2 3 (Not deter-
mined)

very interesting enzyme specificity for inositol isomers. In the isomers
which were oxidized by this organism, either one or two hydroxyl groups
in the molecule were oxidized depending upon the inositol isomer tested.
To explain this specificity Magasanik and Chargaff!! made two assump-
tions: first, that the inositols exist chiefly in the ‘‘chair” form, as cyclo-
hexane does; and, second, that the most stable configuration of an inositol
molecule has the maximum possible number of hydroxyl groups in the
equatorial plane (positions marked “Eq.” in Fig. 2) and the minimum num-
ber of hydroxyl groups in the pole regions (positions marked ‘‘N” and ‘S”
in Fig. 2). The results of these experiments are summarized in Table V."

1B, Magasanik and E. Chargaff, J. Biol. Chem. 174, 173 (1948).

354 INOSITOLS

It will be noted that in every case it was the “pole” hydroxyl groups which
were oxidized by Acetobacter suboxydans. Another organism, Aerobacter
aerogenes, has also been found to oxidize the polar hydroxyl group of myo-
inositol.”? It has not yet been determined whether the relationship between
the polar and the equatorial groups is also a factor in the vitamin activity
of inositol for various organisms.

VI. Biogenesis

ARTHUR H. LIVERMORE

That plant tissues can synthesize inositol is evident; that animal tissues
can do so has not yet been established. Increases in the inositol content of
animal tissues may represent synthesis by intestinal flora rather than
synthesis by the animal body itself. Increases in the inositol content of
plant tissues, on the other hand, can be accounted for only by synthesis
in the plant tissues. The mechanism of this synthesis has not yet been
demonstrated, but present evidence implicates the sugars, and particularly
glucose, as the precursors of inositol. Fischer! has suggested that

.. it could be deduced from chemical evidence . . . that inositol is most likely an
intermediate between carbohydrates and aromatic substances; moreover it very
likely serves as a reserve carbohydrate, storing away glucose in a form which could
be easily mobilized. Finally, there is the speculative possibility that inositol could
act as an intermediate enabling the easy transformation of one hexose into another.

Fischer also presented chemical evidence to support his suggestion that
inositol may be synthesized from glucose in biological tissues.

A. INOSITOL SYNTHESIS BY MICROORGANISMS

The presence of inositol in the phosphatides of the tubercle bacillus was
first reported in 1930.2 The ability of two strains of this bacillus to synthe-
size inositol* indicates that the tubercle bacillus need not be dependent upon
the host for a supply of this vitamin. The synthesis of inositol by other
bacteria, grown in vitamin-free media, has also been demonstrated.*: ®
The amounts synthesized were indicated by the observation’ that various

122 B. Magasanik, J. Am. Chem. Soc. 78, 5919 (1951).

1H. O. L. Fischer, Harvey Lectures 40, 156 (1945).

2 R. J. Anderson and E. G. Roberts, J. Biol. Chem. 89, 599, 611 (1980).

3H. Pope and D. T. Smith, Am. Rev. Tuberc. 54, 559 (1946) [C. A. 42, 3017 (1948)].
4R. C. Thompson, Univ. Texas Publ. 4237, 87 (1942).

5L. W. Jones and J. E. Greaves, Soil Sci. 55, 393 (1943) [C. A. 37, 6300 (1943)].

VI. BIOGENESIS 355

types of bacteria grown in an inositol-free medium for 24 hours contained
from 870 y (Clostridium butylicum) to 1700 y (Pseudomonas fluorescens) of
inositol per gram of dry cells.

The ability of various yeasts to synthesize inositol can be modified by
various procedures. It has been shown,® for example, that Saccharomyces
cerevisiae can be converted from a heterotrophic to an autotrophic habit by
successive changes in the medium. If inhibitors are included in the medium?
or if the cells are damaged by ultraviolet light,® the ability of the yeast to
synthesize inositol is also changed. Torulopsis utilis has been shown’ to
synthesize less inositol in an iron-deficient medium than in one containing
an adequate amount (0.1 to 3 p.p.m.) of iron.

The suggestion” that Aerobacter aerogenes catabolizes myo-inositol to
COz, acetate, and pyruvate through the intermediate formation of 2-keto-
and 1 ,2-diketo-myo-inositol, leads one to speculate on the possibility that
these metabolic products might in turn be precursors of inositol in this
organism. There is as yet no experimental evidence for this speculation.

B. INOSITOL SYNTHESIS BY HIGHER PLANTS

A number of studies of the changes in the inositol content of seeds, leaves,
and fruits have been made. Although the total inositol content of plants
increases during growth, the concentration in the plant tissues may de-
crease. For example, it was shown" that there was a higher concentration
of inositol in the growing tips of cucurbit and tomato plants than in the
mature leaves. In addition it was found” that the inositol concentration
in the fruits of these plants remained relatively constant or decreased
slightly during development.

The possibility that inositol is synthesized from glucose and glucose
derivatives in plants was strengthened somewhat by the observation™
that the leaves of the rubber plant, Lactuca virosa L., yielded an aqueous
extract which, when incubated with glucose for 3 days, gave rise to a sub-
stance which appeared to be inositol. It has also been reported" that apricot
leaves produced inositol when incubated with glucose and glucose phos-
phates.

6 L. H. Leonian and V. G. Lilly, Science 95, 658 (1942).

7M. A. Eppright and R. J. Williams, J. Gen. Physiol. 30, 61 (1946).

8 A. M. Webb and J. R. Loofbourow, Biochem. J. (London) 41, 114 (1947).

9 J. C. Lewis, Arch. Biochem. 4, 217 (1944).

10 B. Magasanik, J. Am. Chem. Soc. 78, 5919 (1951).

uC. L. Withner, Am. J. Botany 36, 355 (1949).

2C.L. Withner, Am. J. Botany 36, 517 (1949).

18 QO. Fernandez, G. Izquierdo, and E. Martinez, Farm. nueva (Madrid) 9, 563 (1944)
[C. A. 40, 4115 (1946)].

144Q. Fernandez, M. de Mingo, and E. Martinez, Farm. nueva (Madrid) 10, 541 (1945)
[C. A. 48, 4229 (1949)].

356 INOSITOLS

When fresh tea leaves were stored in the dark for 20 hours, the glucose
and sucrose contents decreased and the inositol content increased.’ In-
filtration of the leaves with glucose, fructose, sucrose, and mannose as
well as other glucosides also increased the inositol content of the leaves,
the glucosides being more effective than the free glucose.!® 7 Glucose-1-
phosphate likewise resulted in a more rapid synthesis of inositol than did
free glucose.'® All this work suggests, but in no way proves, that inositol
is synthesized from sugars in these plants.

The observation that the phloroglucinol content of tea ies increased
during the infiltration of the leaves with sugar solutions was interpreted to
mean that myo-inositol is a precursor of this aromatic compound in the
tea leaves. This is in agreement with Fischer’s suggestion! that inositol
is an intermediate between the sugars and aromatic substances. Apparently,
in the tea leaves the reverse reaction—conversion of phloroglucinol to
inositol—does not take place, for infiltration of the leaves with phloro-
glucinol did not increase their inositol content.!®

The possible interconversion of inositol and ascorbic acid has been sug-
gested,!® following the observations that these are the only two vitamins
present in significant amounts in citrus fruits. No experimental evidence
for such a conversion has yet been presented.

C. INOSITOL SYNTHESIS BY ANIMALS

Rosenberger,”’ in 1908, found that he could isolate inositol from rabbits
which had been allowed to stand for several days after being killed, al-
though he was unable to find inositol in freshly killed rabbits. He also
stated that he found an increase in the inositol content of beef muscle on
standing and postulated the presence in muscle of an “inositogenic sub-
stance.’’ Winter”! likewise reported an increase in the inositol content of
dog heart muscle on standing. Since both investigators used an alkaline
hydrolysis to liberate inositol, some doubt is cast on these observations, for
it is now known that inositol is somewhat alkali-labile.

Woolley” found that young mice which were fed on an inositol-deficient

1 A. L. Kursanov, N. N. Kryukova, and E. Vyskrebentseva, Biokhimiya 13, 530
(1948) [C. A. 48, 3070 (1949)].

16 A. L. Kursanov, M. Vorob’eva, and E. Vyskrebentseva, Doklady Akad. Nauk.
S.S.S.R. 68, 737 (1949) [C. A. 44, 1568 (1950)].

17 A. L. Kursanov, Izvest. Akad. Nauk. S.S.S.R., Ser. Biol. 2, 44 (1951) [C. A. 45,
7644 (1951)].

18 A. L. Kursanov, E. Vyskrebentseva, and M. Vorob’eva, Doklady Akad. Nauk.
S.S.S.R. 68, 893 (1949) [C. A. 44, 1172 (1950)].

19 W. A. Krehl and G. R. Cowgill, Food Research 15, 179 (1950).

20 F. Rosenberger, Z. Physiol. Chem. 56, 373 (1908).

217,, B. Winter, Biochem. J. (London) 28, 6 (1934).

22T). W. Woolley, J. Exptl. Med. 75, 277 (1942).

VI. BIOGENESIS S157

diet for 4 weeks after weaning showed an increase in the inositol content
of their tissues from a total of 3.4 mg. at weaning to 8.6 mg. after 4 weeks
on the experimental diet. However, when the experimental diet was de-
ficient in pantothenic acid as well as in inositol, there was no increase in
the total inositol content of the animals. Woolley compared the intestinal
flora of the mice which recovered spontaneously from the inositol deficiency
symptoms (alopecia) with the flora from mice which retained the symptoms
and found that in the first case the flora had the ability to synthesize ino-
sitol but in the latter case they did not. It would appear, therefore, that in
the mouse at least the intestinal flora can synthesize all the inositol re-
quired by the animal, provided that sufficient pantothenic acid is pro-
vided for normal growth of the intestinal organisms.

It was observed”’ that the inositol content of hen’s eggs increased from
73 to 456 y per gram of tissue during 23 days of incubation. This increase

TABLE VI

CHANGE IN INosIToL CoNTENT oF Haes

Inositol content, y/g.
Day of incubation

Free Total

0 46 220
20 190 240

is apparently not due to synthesis of inositol by the egg tissues but rather
to the liberation of inositol from a bound form. This liberation of bound
inositol was demonstrated by Woolley,** who determined both the free
and bound inositol in the incubated eggs. The results of this experiment
are shown in Table VI.

Although there has not yet been a clear-cut demonstration of the syn-
thesis of inositol by animal tissues, an interconversion of inositol and glu-
cose has been reported.”> myo-Inositol labeled with deuterium was fed to
phlorizinized rats, and the deuterium content of the urinary glucose was
determined. The high deuterium content of the glucose indicated its for-
mation from inositol, but since the location of the deuterium in the glucose
was not determined the significance of the observation has been ques-
tioned.?6: 27

23). E. Snell and E. Quarles, J. Nutrition 22, 483 (1941).

24D. W. Woolley, Proc. Soc. Exptl. Biol. Med. 49, 540 (1942).
25M. R. Stetten and D. Stetten, Jr., J. Biol. Chem. 164, 85 (1946).
26'T. Posternak, Bull. soc. chim. biol. 83, 1041 (1951).

27 W. H. Schopfer, Bull. soc. chim. biol. 38, 1113 (1951).

358 INOSITOLS

It is tempting to assume that inositol is synthesized from glucose in
plant tissues and that the reverse reaction can take place in the animal.
The fact remains, however, that up to the present no unequivocal experi-
ments to demonstrate either conversion have been reported.

VII. Estimation of Inositol

Although qualitative color tests for inositol (e.g., the Scherer test) have
been developed, none of them can be used for a quantitative determina-
tion of the vitamin. Neither is there a specific chemical reaction of any
sort by which inositol can be determined unequivocally in biological ma-
terial. This lack of specificity is due in part to the fact that there are a
number of biologically inactive isomers of inositol and in part to the simi-
larity of inositol to the sugars in structure and chemical reactivity. Any
chemical determination of inositol requires, therefore, a preliminary separa-
tion and purification of the vitamin to remove sugars. Such procedures
inevitably result in losses of material and are less desirable for a quantita-
tive determination of the vitamin than are the microbiological procedures
which require no separation of the vitamin from the tissues. The recently
developed paper chromatographic method, however, is a promising im-
provement in the chemical determination of inositol.

A. BY ISOLATION

ARTHUR H. LIVERMORE

The earliest methods for determination of inositol were developed before
it was recognized to be a vitamin. Scherer! in 1850 discovered inositol in
animal tissues and determined its presence after precipitating it with basic
lead acetate, a reagent used also by many subsequent investigators. Scherer
found? that, by treating the inositol first with nitric acid and then with
ammonia and calcium chloride and finally evaporating the solution, he
obtained a red-colored product. It has been shown that this colored com-
pound is a salt either of rhodizonic acid or of tetrahydroxyquinone.’: 4
This test is quite specific for the inositol structure and has been used as a
qualitative test for inositol by many investigators (see, for example, Hutt

1J. Scherer, Ann. Chem. Justus Liebigs 78, 322 (1850).

2 J. Scherer, Ann. Chem. Justus Liebigs 81, 375 (1852); Beilsteins Handbuch der
Organischen Chemie VI, 1196 (1923).

3 F, A. Hoglan and E. Bartow, J. Am. Chem. Soc. 62, 2397 (1940).

4P.W. Preisler and L. Berger, J. Am. Chem. Soc. 64, 67 (1942).

VII. ESTIMATION OF INOSITOL 359

et al.®). The Scherer test has recently been discussed in some detail by
Fleury.®

The introduction of the use of 70 % acetone for the extraction of inositol?
improved the isolation procedure. Estimation by isolation and weighing
remains, however, a very crude method for inositol determination.

B. BY CHEMICAL METHODS
ARTHUR H. LIVERMORE

The introduction of chemical methods for the estimation of inositol was
some improvement over the earlier procedures. However, considerable
purification of the inositol to remove interfering substances was still re-
quired.

1. REacTION witH PotTassiuM IODOMERCURATE®: 9

Potassium iodomercurate in alkaline solution is reduced to free mercury
by inositol and also by sugars. The mercury can then be dissolved in a
measured excess of a standard iodine solution and the unreacted iodine
determined by titration with thiosulfate.

2. REACTION WITH PERIODATE

The most satisfactory chemical method for determining inositol is to
oxidize it with periodate.® 1°! In the method of Platt and Glock,!® the
inositol must first be extracted from tissues with boiling water and im-
‘purities removed by precipitating some with 70% acetone and extracting
others with ether. Glucose can be destroyed by fermentation with yeast,
and then lactic acid and other interfering substances can be removed by
adsorption on carbonaceous zeolite and M.P.D. resin.

The inositol solution obtained in this fashion is treated with HIO. at
6° to8° for 48 hours, at which time the theoretical amount of HIO, required
by equation 1 is used up. The formic acid produced is then titrated with

C.-H.(OH). + 6HIO, — 6HIO; + 6HCOOH a)

0.01 N NaOH. Platt and Glock found that, when the theoretical amount
of HIO, had reacted, 90% of the theoretical amount of formic acid was
present in the solution.

5H. H. Hutt, T. Malkin, A. G. Poole, and P. R. Watt, Nature 165, 314 (1950)
6 P. Fleury, Bull. soc. chim. biol. 33, 1061 (1951).

7G. Momose, Biochem. J. (London) 10, 120 (1916).

8P. Fleury and J. Marque, J. pharm. chim. [8] 10, 241 (1929).

°L. Young, Biochem. J. (London) 28, 1435 (1934).

10 B.S. Platt and G. E. Glock, Biochem. J. (London) 37, 709 (1943).

1 P. Gyorgy, Vitamin Methods, p. 252, Academie Press, New York, 1950.
2 P. Fleury and M. Joly, J. pharm. chim. [8] 26, 341, 397 (1937).

360 INOSITOLS

In order to differentiate between inositol and glycerol which might be
present in the solution, a titration is carried out 90 minutes after the start
of the periodate oxidation. At that time 100% of the glycerol but only
2% of the inositol has been oxidized by the periodate.

In an earlier application” of the periodate oxidation method, the in-
ositol-periodate mixture was treated after 24 hours with 0.1 N As.O3 and
20 % KI, and 10 minutes later the excess As,O3 was titrated with standard
iodine solution.

More recently the CO, which was produced during the periodate oxi-
dation was determined in a Warburg apparatus. At 30°, between 1.1 and 1.3
moles of CO, were produced in 4 hours from 1 mole of inositol. Interfering
sugars were destroyed by heating the mixture with MgO at 100°. Lactic
acid was removed by alcohol and ether extractions.

It would appear that at the low temperatures used by Platt and Glock”
there is little formation of CO, (in 48 hours), whereas at the higher tem-
perature used by Fleury and Recoules® some COs, is produced. The source
of the CO, has been determined by Fleury, who has shown‘® that oxidation
of inositol by periodate at 30° takes place in two steps. The first is a rapid
one in which 2 moles of formic acid and 2 moles of glycolic acid are formed,
within a few minutes, from each mole of inositol. This is followed by a
slow phase in which the glycolic acid is oxidized by the periodate according
to equation 2. This reaction is still incomplete after several days.

2CH,OHCOOH + 40 — 2HCOOH + 2CO:2 + 2H:0 (2)

The possibility of estimating inositol by determining the amount of
glycolic acid formed during periodate oxidation led Fleury and his co-
workers to develop a semimicro method for determining this compound.®: 14
In this method the violet color obtained by heating glycolic acid with
chromotropic acid and H.SO, is measured.

3. OTHER CHEMICAL METHODS

The Hagedorn-Jensen method for determining sugars has been used for
inositol estimation.!®> Also, inositol in the form of its hexaphosphate ester
(phytin) has been estimated!® by determining the amount of organic phos-
phate in a partially purified tissue extract.

13 P. Fleury and A. Recoules, Compt. rend. 227, 691 (1948).

“4 P. Fleury, J. E. Courtois, and R. Perles, Mikrochemie ver. Mikrochim. Acta 36/37,
863 (1951) [C. A. 45, 5073 (1951)].

15 W.R. Todd, J. Vreeland, J. Myers, and E.S. West, J. Biol. Chem. 127, 269 (1939).

16 J, K. Courtois and C. Perez, Bull. soc. chim. biol. 30, 195 (1948).

VII. ESTIMATION OF INOSITOL 361

C. BY PAPER CHROMATOGRAPHY

ARTHUR H. LIVERMORE

A qualitative determination of inositol can be made readily with filter
paper chromatograms.'!® Although inositol is not readily oxidized in
solution by ammoniacal silver nitrate, this reagent can be used to detect
inositol on the filter paper chromatogram. The reduction of the silver by
the inositol spot proceeds more readily if a little NaOH is added to the
silver reagent.”

Quantitatively the inositol can be extracted from the filter paper, oxidized
with periodate, and the resultant formic acid titrated.!* No data have been
given from which the accuracy and precision of this method can be de-
termined.

D. BY MOUSE ASSAY

ARTHUR H. LIVERMORE

Woolley?’ demonstrated that inositol-deficient mice developed alopecia
and that administration of inositol to the deficient animals cured this
disorder. He used this method?!’ to determine the inositol activity of
various analogs of the vitamin. The mouse assay has not been developed
into a strictly quantitative method for determining inositol. Moreover, it
has now been supplanted by the more convenient and rapid microbiological
assays.

EK. BY MICROBIOLOGICAL ASSAY

K. E. SNELL

Inositol, the first component of ‘‘bios” to be identified, was first shown
to promote growth of yeast by Eastcott.24 Many, but not all, yeasts re-
quire this compound, together with one or more other vitamins,?> and the
response of suitable strains of yeast to it remains the basis for the most
convenient procedures for its estimation.

Of the several naturally occurring compounds related to inositol, only the

17§. M. Partridge and R. G. Westall, Biochem. J. (London) 42, 238 (1948).

18 T,, Hough, Nature 165, 400 (1950).

19 #}. L. Hirst and J. K. N. Jones, J. Chem. Soc. 1949, 1659.

20 C. L. Withner, Am. J. Botany 36, 355 (1949).

21D). W. Woolley, J. Biol. Chem. 140, 461 (1941).

2 7—D. W. Woolley, J. Biol. Chem. 139, 29 (1941).

23D. W. Woolley, J. Biol. Chem. 140, 453 (1941).

44K. V. Eastcott, J. Phys. Chem. 32, 1094 (1928).

25 P. R. Burkholder, I. MeVeigh, and D. Moyer, J. Bacteriol. 48, 385 (1944).

362 INOSITOLS

free vitamin, meso-inositol, has growth-promoting activity for yeast.”!: 7°
Bound forms, such as phytic acid and those occurring in lipids, are in-
active. For estimation of total inositol, therefore, hydrolysis of the sample
is essential. Treatment of the finely divided sample with 18 % HCl under
reflux for 6 hours, followed by evaporation of excess HCl and neutralization
of the dissolved residue, is the method of choice.?6

Of the several suggested yeast growth procedures, an unpublished modi-
fication of the procedure of Atkin et al.2® for determination of vitamin Bs
is simple and highly satisfactory. Inositol is omitted from the basal medium,
and an excess of pyridoxine is added. Increased growth of the test organ-
ism, Saccharomyces carlsbergensis 4228, in response to increased amounts
of inositol is obtained in the concentration range of 0 to 1 y of inositol per
milliliter. Incubation is for 18 to 24 hours, with shaking, at 30°. Other
details of the assay resemble those for vitamin Be assay with the same
organism,”* and have also been given in detail elsewhere.?® Good results are
also obtainable by the method of Woolley,?® which employs a strain of
Saccharomyces cerevisae as the test organism; the basal medium, however,
is much more troublesome to prepare, and the method does not appear to
possess compensating advantages.

A fundamentally different assay procedure for inositol is possible by use
of an inositol-less mutant of Newrospora crassa.?* 77, 8 The amount of my-
celium formed in response to added inositol is determined by direct weigh-
ing after 3 days of incubation. Although the procedure appears accurate,
it is more tedious than yeast assay and requires considerably longer time.

None of the lactic acid bacteria or artificially induced mutants of bac-
teria require inositol; indeed, it is not certain whether or not bacterial
cells contain any of this substance. Assay procedures that utilize such
organisms consequently are not available.

26D. W. Woolley, zn Biological Symposia, XII. Estimation of the Vitamins, p. 279,
Jaques Cattell Press, Lancaster, Pa., 1947.

26a T,, Atkin, A. 8. Schultz, W. L. Williams, and C. N. Frey, Ind. Eng. Chem. Anal. Ed.
15, 141 (1943).

26b H. EH. Snell, in Vitamin Methods, Vol. I, p. 327, Academic Press, New York, 1950.

27 G. W. Beadle, J. Biol. Chem. 146, 109 (1942).

2H. L. Tatum, M. G. Ritchie, E. V. Cowdry, and L. F. Wicks J. Biol. Chem. 163,
675 (1946).

VIII. OCCURRENCE 363
VIII. Occurrence

ARTHUR H. LIVERMORE

The naturally occurring inositol isomers are found in many plants and
in some animal tissues.!:? The two optically active isomers have been
found chiefly in various trees. p-Inositol occurs as the monomethyl ether,
pinitol, and L-inositol as the monomethyl ether, quebrachitol. The other
naturally occurring isomer of inositol is scyllitol. Scyllitol has been found
both in plant products and in elasmobranch fishes. It has also recently been
found, together with myo-inositol, in urine and in blood.’ In addition to
these compounds, other related substances have also been found in plant
products. Among these are the desoxyinositols, C-methylinositols, inososes
(pentahydroxycyclohexanones), and also the tri- and tetrahydroxy hexane
carboxylic acids (quinic acid and shikimic acid).!:?

A. IN FOODS

Since myo-inositol is the only inositol isomer having significant vitamin
activity, only this isomer has been determined extensively in food products.
Most of the determinations of the inositol content of foods have been made
by microbiological methods. Since the microorganisms which are used for
these determinations use myo-inositol specifically, the results of these
determinations undoubtedly give the myo-inositol content of the food
products. The same cannot be said of chemical methods since these pro-
cedures (e.g., periodate oxidation) will not distinguish between the various
isomers, and it is possible that scyllitol and p- or L-inositol might in some
cases be determined as well as myo-inositol.

Some of the results of analyses of various food products for inositol
have been collected in Table VII. This is by no means an exhaustive list
of the food products studied. The original literature cited should be con-
sulted for other foods.

B. NATURAL ANTIVITAMINS OF INOSITOL

The structure of the streptamine moiety of streptomycin is very similar
to the structure of inositol. Streptamine is a diaminotetrahydroxycyclo-

1H. G. Fletcher, Jr., Advances in Carbohydrate Chem. 3, 45 (1948).

2W. W. Pigman and R. M. Goepp, Jr., Chemistry of the Carbohydrates, p. 266,
Academic Press, New York, 1948.

’P. Fleury, Bull. soc. chim. biol. 33, 1061 (1951).

4V.H. Cheldelin and R. J. Williams, Univ. Texas Publ. 4237, 105, (1942).

Tue InosttoLt CoNTENT OF VARIOUS BioLoGicaL MATERIALS

TABLE VII

Vegetable Products

Inositol content, y/g. of fresh tissue

Reference

Cereals
Wheat germ 6,900 4
Whole wheat 1,700 4
1,900 5
Bread, whole wheat 644 6
1,030 4
Flour, white 830 4
Flour, whole wheat 1,105 6
Oats 3,160 5
1,000 if
Barley 3, 9202 5
Corn 500 7
Fruits
Oranges 2,100 4
Orange juice 1,040-1,700 (per ml.) 8
Grapefruit 1,500 4
Grapefruit juice 880-1,120 (per ml.) 8
Cantaloupe 1,200 4
Tomatoes 460 4
Apples 240 4
Vegetables
Peas, English, green 1,620 4
Cabbage 950 4
Potatoes, sweet 660 4
Potatoes, white 290 4
Lettuce 550 4
Carrots 480 4
Spinach 270 4
Miscellaneous
Peanuts, roasted 1,800 +
Molasses 1,500 4
3, 200° 9
Tea leaves, dry 10,000 8
Yeast, Torulopsis utilis 2,700 10
Yeast, brewer’s 500 7
Animal Products
Meats
Beef, muscle 115 4
2,640 i
Beef, heart 2,600 4
16,000 7
Beef, brain 2,000 4
6,000 if
Beef, liver 3,400 Uf
510 4
Pork, loin 360-450 4
Veal, chop 320-350 4

364

VIII. OCCURRENCE 365

TABLE VIIl—Continued

Animal Products

Inositol content, y/g. of fresh tissue Reference
Fowl and Fish
Chicken, breast 480 4
Oyster 440 4
Halibut 170 4
Salmon 170 4
Egg 220 7
Milk Products
Whole milk, cow 500 7
180 (per ml.) 4
Whole milk, human 330 (per ml.) 4
4

Cheese 250

? Determined as phytin phosphorus. These inositol weights were calculated from Courtois and Perez
values for phytin phosphorus, assuming a formula CasMg(CsHi202Ps6-3H2O)2 for phytin.
® This included 1340 y of free inositol and 1860 y of bound (phytin) inositol.

hexane which is an analog of either the scyllitol structure (1)" or the myo-
inositol structure (II). It has been observed! that lipositol from brain

I II

inhibited the action of streptomycin on Staphlococcus aureus, and a con-
firmation of this observation has been reported.* However, it has been
found'® that lipositol has no antistreptomycin effect on Escherichia colt.

Another relationship between inositol and streptomycin has been re-
ported.!® The yeast Torula utilis was adapted to grow on a medium con-
taining inositol in place of glucose. In contrast to the parent strain, this
yeast which had been adapted to inositol was sensitive to streptomycin.

> P. Gyorgy, Vitamin Methods, p. 252, Academic Press, New York, 1950.

5 R. R. Sealock and A. H. Livermore, J. Nutrition 25, 265 (1943).

7D. W. Woolley, Biol. Symposia 12, 279 (1947).

8 W. A. Krehl and G. W. Cowgill, Food Research 15, 179 (1950).

9W.W. Binkley, M. G. Blair, and M. L. Wolfrom, J. Am. Chem. Soc. 67, 1789 (1945).

10 J. J. Stubbs, W. M. Noble, and J. C. Lewis, Food Ind. 16, 9, 68, 125 (1944).

1M. L. Wolfrom, 8. M. Olin, and W. J. Polglase, J. Am. Chem. Soc. 72, 1724 (1950).

2T. Posternak, Bull. soc. chim. biol. 38, 1041 (1951).

137. Rhymer, G. I. Wallace, L. W. Byers, and H. E. Carter, J. Biol. Chem. 169, 457
(1947).

4, Sdderhjelm and B. Zetterberg, Upsala Lakareféren. Férh. 5/6, 235 (1948). As
quoted by W. H. Schopfer, Bull. soc. chim. biol. 33, 1113 (1951).

16'T, F. Paine, Jr. and F. Lipmann, J. Bacteriol. 58, 547 (1949).

16 Y. H. Loo, H. FE. Carter, N. Helm, and B. Anderlik, Arch. Biochem. 26, 144 (1950).

366 INOSITOLS

No other natural antivitamins of inositol have been found. myo-Inositol
has been reported” to reverse the inhibitory effect of malonate on Clos-
tridium saccharobutyricum, whereas the p- and L-inositols do not. However,
borate also reversed the malonate inhibition, so a specific inhibition of
inositol by malonate was probably not involved.

IX. Effects of Deficiency

A. IN MICROORGANISMS
KE. E. SNELL

The physiological activity of inositol was first discovered by Eastcott,!
who showed that the substance was required for growth of a strain of
yeast. Since these initial observations, many yeasts have been shown to be
stimulated by it; for 15 of 163 strains investigated by Burkholder e¢ al.?
inositol was essential for growth. Several fungi are known that require the
vitamin, e.g., Nematospora gossypit,?: * Lophodermium pinastr7,? and others.
Mutants of Neurospora crassa that require it have also been produced.®
In all such organisms, growth increases with the inositol concentration
over a definite range of concentrations below the optimum; microbiological
assay methods for inositol depend upon this fact.6 Although the vitamin
is required for growth of these microorganisms, nothing is known of the
essential metabolic role played by it; no distinctive metabolic aberrations
due to its lack have been reported. Presumably, in these organisms as in
higher plants and animals, inositol may be required for the formation of
essential lipid components of the cell;’: * this, however, is mere speculation.
It is a striking fact that no bacteria have so far been reported to require
inositol, and it is not certain whether inositol is present at all in some bac-
teria, e.g., Escherichia coli.? Other bacteria, e.g., the tubercle bacillus, are
known to contain it.!° Indeed, the occurrence of inositol in the phosphatide

17 A. J. Rosenberg, Compt. rend. soc. biol. 142, 443 (1948).

1K. V. Eastcott, J. Phys. Chem. 82, 1094 (1928).

2 P. R. Burkholder, I. McVeigh, and D. Moyer, J. Bacteriol. 48, 385 (1944).

3H. W. Buston and B. N. Pramanik, Biochem. J. (London) 25, 1656 (1931).

4F. Kégl and N. Fries, Hoppe-Seyler’s Z. physiol. Chem. 249, 93 (1937).

5G. W. Beadle and E. L. Tatum, Am. J. Botany 32, 678 (1945).

6H. E. Snell in P. Gyérgy, Vitamin Methods, Vol. I, p. 327, Academic Press, New
York, 1950.

77. Rhymer, G. I. Wallace, L. W. Byers, and H. E. Carter, J. Biol. Chem. 169, 457
(1947).

8D. W. Woolley, J. Biol. Chem. 147, 581 (1943).

9D. W. Woolley, J. Exptl. Med. 75, 277 (1942).

10R, J. Anderson, J. Am. Chem. Soc. 52, 1607 (1930).

IX. EFFECTS OF DEFICIENCY 367

fraction of the latter organism!® was the first evidence that inositol was
associated with lipid material in any organism. In some yeasts and fungi
that require inositol as a nutrient, the toxic action of y-hexachlorocyclo-
hexane is directed against inositol and can be overcome by it; the phenome-
non, however, does not throw additional light on the metabolic role played
by inositol. The insecticide also has a pronounced toxic action that is not
overcome by inositol; it appears both in inositol-requiring and in inositol-
synthesizing microorganisms.!!

B. IN ANIMALS
T. J. CUNHA

Woolley? showed that an inositol deficiency in the mouse resulted in
retarded growth and alopecia. Of considerable interest was the pattern of
the hair loss obtained. No hair loss occurred from the tail or head or from
the legs below the knees. The areas of loss of hair on most other parts of
the body were bilaterally symmetrical, and in most of these areas the
alopecia was nearly complete. Martin!® observed only slight alopecia in
mice fed an inositol-deficient diet. Woolley'®: " also observed that only
about 50% of the animals showed signs of an inositol deficiency and that
spontaneous cures of the deficiency occurred frequently. He also found
that, in the absence of pantothenic acid, alopecia developed even though
the ration contained enough inositol. With large amounts of pantothenic
acid and a lack of inositol, however, some animals still developed signs of
inositol deficiency and died unless inositol was administered. Woolley”
also showed that inositol is synthesized under certain conditions by the
intestinal flora of mice. Frequently, the amount of inositol synthesized was
equivalent to the minimum dose effective in the prevention of the alopecia.
Thus, it appears that inositol deficiency symptoms in mice are most likely
affected by intestinal synthesis.

Paveek and Baum® reported that inositol cured ‘‘spectacled eye’ in rats
fed a purified diet. Subsequently, Nielsen and Elvehjem!*® were able to
cure a similar spectacled eye condition in the rat with biotin. The fact
that both groups of investigators were able to cure the spectacled eye
condition with two different vitamins might well indicate that therapy

1R,. C. Fuller, R. W. Barratt, and E. L. Tatum, J. Biol. Chem. 186, 823 (1950).
2D. W. Woolley, Science 92, 384 (1940).

13D. W. Woolley, J. Biol. Chem. 186, 113 (1940).

4D. W. Woolley, J. Biol. Chem. 139, 29 (1941).

16G. J. Martin, Science 93, 422 (1940).

16 TD). W. Woolley, Proc. Soc. Exptl. Biol. Med. 46, 565 (1941).

7P—). W. Woolley, J. Exptl. Med. 75, 277 (1942).

18 P. L. Pavcek and H. M. Baum, Science 98, 502 (1941).

19 K. Nielsen and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 48, 349 (1941).

368 INOSITOLS

with one vitamin in one case may have stimulated the intestinal synthesis
of the other. This would mean that the spectacled eye condition could be
due to a biotin deficiency, but in the work of Paveek and Baum! the in-
ositol may have acted indirectly in stimulating the intestinal synthesis of
biotin. This postulation is strengthened by the finding of Lindley and
Cunha”? that inositol alleviated to a large extent the deficiency symptoms
prevented by biotin with the pig fed a purified diet. Cunha et al.?! reported
an alopecia in rats reared on a natural diet composed chiefly of corn and
soybean meal which could be prevented and cured by inositol. The hair
loss started in the dorsal part of the head and proceeded bilaterally along
the sides to the tail region and then downward to the hind legs. With inositol
therapy the hair returned inversely, proceeding from the caudal portions
forward. Of interest is the finding that the hair loss did not occur until
pyridoxine and a folic acid preparation were added to the control ration.
The addition of these two factors caused a decrease in growth and the
development of the alopecia. This may have been due to some imbalance,
some interrelationship of the vitamins, some change in intestinal synthesis,
or to other unknown causes. Spitzer and Phillips,” using a sucrose-soybean
oil meal ration, produced an alopecia in the rat similar to that observed by
Cunha et al.?! The hair loss was prevented by supplementation with inositol
or biotin or with both. However, the hair loss did not occur if the ration
contained added cystine or methionine. They also stated that, whereas
low levels (1 to 4 y) of biotin have been shown to prevent the loss of hair,
higher levels (12 y) of this vitamin may actually accentuate the condition.
The hair loss resulting from feeding the high level of biotin was prevented
by supplementation with adequate inositol. No explanatation is available
for their results, but, undoubtedly, the ultimate answer will be of con-
siderable interest. Nielsen and Black?’ also found that inositol prevents
the development of a symmetrical alopecia in the rat fed a purified ration
plus sulfasuxidine. The sulfonomide in some way brought out a need for
inositol. It must be pointed out that in many experiments rats are fed
purified diets apparently free of inositol and they do not develop spectacled
eye or alopecia. An example is the report of Ershoff,* who found that
inositol and PABA, in combination or separately, had no effect on growth
of rats fed a purified diet. Thus, it appears that under special conditions the
type of diet influences the need for inositol by the rat. Under ordinary
conditions it is of no benefit, but occasionally the diet is such that a need

20 D. C. Lindley, and T. J. Cunha, J. Nutrition 32, 47 (1946).

21°'T. J. Cunha, 8. Kirkwood, P. H. Phillips, and G. Bohstedt, Proc. Soc. Exptl. Biol.
Med. 54, 236 (1943).

22. R.R. Spitzer and P. H. Phillips, Proc. Soc. Exptl. Biol. Med. 68, 10 (1946).

23H. Nielsen and A. Black, Proc. Soc. Exptl. Biol. Med. 55, 14 (1944).

24 B. H. Ershoff, Proc. Soc. Exptl. Biol. Med. 56, 190 (1944).

IX. EFFECTS OF DEFICIENCY 369

for inositol can be shown. Whether the inositol is effective per se or whether
it acts indirectly by stimulating the synthesis of biotin or other factors is
not yet clear.

Sure?> reported that inositol and p-aminobenzoic acid improved the rate
of survival of newborn rats. Later, Sure?® found that p-aminobenzoic acid
was primarily responsible for this action. Climenko and McChesney”
reported that, on a purified diet, either with or without p-aminobenzoic
acid, the addition of inositol reduced the mortality rate of the young rats
and increased the milk yield of lactating rats. The inclusion of p-amino-
benzoic acid alone, however, appeared to have an adverse effect upon
lactation. The reverse of this finding was reported by Sure.?® Ershoff and
MeWilliams® reported reduced fertility when inositol was fed in a purified
ration containing both p-aminobenzoic acid and sulfaguanidine. However,
the reduced fertility did not occur if either sulfaguanidine or inositol was
omitted from the diet. Ershoff?‘ later found that massive doses of inositol
or p-aminobenzoic acid exerted no deleterious effects on growth or reproduc-
tion and that lactation may occur on diets containing | % p-aminobenzoic
acid or inositol. The divergent findings of these various investigators can-
not be definitely explained. It is possible that the differences are due to
the different diets used and their subsequent effect on the type of intestinal
flora present in the rats.

Lindley and Cunha found that inositol was of no benefit when added
to the ration of the pig fed a purified diet. They concluded that either the
pig synthesizes enough inositol or it does not need the vitamin added to the
ration. In that trial, however, it was found that, if a biotin deficiency was
produced by using sulfathalidine in the ration, inositol alleviated to a
large extent the deficiency symptoms prevented entirely by biotin. A pos-
sible explanation is that inositol acted indirectly by stimulating intestinal
synthesis of biotin. Data by Ross et al.2° and Cunha et al.*° have shown that
inositol was beneficial in lactation when added to a corn-soybean ration
for brood sows and rats. However, these studies need more confirmation
with other types of natural rations.

Hegsted et al.*! reported that inositol supplementation slightly increased
the growth rate of chicks fed a partially purified diet. Dam*® found that

25 B. Sure, Science 94, 167 (1941).

26 B. Sure, J. Nutrition 26, 275 (1943).

27 TD). R. Climenko and E. W. McChesney, Proc. Soc. Exptl. Biol. Med. 61, 157 (1942).

28 B. H. Ershoff and H. B. McWilliams, Proc. Soc. Exptl. Biol. Med. 54, 227 (1943).

29Q. B. Ross, P. H. Phillips, and G. Bohstedt, J. Animal Sci. 1, 353 (1942).

30 T. J. Cunha, O. B. Ross, P. H. Phillips, and G. Bohstedt, J. Animal Sct. 3, 415
(1944).

31 TD). M. Hegsted, G. M. Briggs, R. C. Mills, C. A. Elvehjem, and E. B. Hart, Proc.
Soc. Exptl. Biol. Med. 47, 376 (1941).

32 AH. Dam, J. Nutrition 27, 193 (1944).

370 INOSITOLS

encephalomalacia and exudative diathesis, two symptoms frequently en-
countered in chicks fed vitamin E-deficient diets, were prevented by adding
inositol to the diet.

Hogan and Hamilton* found that the rate of growth of guinea pigs fed a
partially purified diet was increased by inositol supplementation.

Cooperman et al.** reported an increased rate of growth of hamsters
when inositol was added to a purified diet. Hamilton and Hogan,** however,
found that inositol supplementation did not increase the growth rate of
hamsters; but the vitamin did counteract reproductive difficulties in ham-
sters in which the young were born dead or as shapeless bloody masses and
where the mothers frequently failed to survive parturition.

MclIntire** found that the addition of inositol to a purified diet almost
doubled the rate of growth of cotton rats. This increase in growth rate is
the greatest observed with any species fed a purified diet which was not
supplemented with a sulfonamide.

There appears to be general agreement among investigators that inositol
is a lipotropic factor. Gavin and McHenry®” found that the addition of
biotin to a purified diet caused a fatty liver in rats that could be prevented
by the further addition of inositol. The lipotropic action of inositol in
rats has been confirmed by Engel,®* Forbes,?? Handler,*? and McFarland
and McHenry.’ Gavin et al.” found that, when thiamine was the only
B-complex vitamin supplement added to a purified diet, the liver fat of
the rat could be maintained at a normal level by supplying one lipotropic
agent, choline. However, when other B vitamins were added to the ration,
the fatty livers responded to both inositol and choline. All the observations
of McFarland and McHenry“ indicate that a fatty liver, of the type pro-
duced by in vivo fat synthesis, is made resistant to choline and responsive
to inositol by increasing the intake of B vitamins, both in kind and in
quantity. Handler*® has suggested that a large increase in food intake, with
a surge in fatty acid synthesis, may be the factor causing choline resistance
and inositol responsiveness. However, McFarland and McHenry“ reported
on paired feeding tests which showed that food consumption is a contri-

33 A. G. Hogan and J. W. Hamilton, J. Nutrition 23, 533 (1942).

34 J. M. Cooperman, H. A. Waisman, and C. A. Elvehjem, Proc. Soc. Exptl. Biol.
Med. 52, 250 (1943).

35 J. W. Hamilton and A. G. Hogan, J. Nutrition 27, 213 (1944).

36 J. M. McIntire, B. S. Schweigert, and C. A. Elvehjem, J. Nutrition 27, 1 (1944).

37 G. Gavin and E. W. McHenry, J. Biol. Chem. 139, 485 (1941).

33 R. W. Engel, J. Nutrition 24, 175 (1942).

39 J. C. Forbes, Proc. Soc. Exptl. Biol. Med. 54, 89 (1943).

40 P. Handler, J. Biol. Chem. 162, 77 (1946).

41M. L. McFarland and E. W. McHenry, J. Biol. Chem. 176, 1 (1948).

42 G. Gavin, J. M. Patterson, and E. W. McHenry, J. Biol. Chem. 148, 275 (1948).

IX. EFFECTS OF DEFICIENCY ayia

buting factor, but there is also a specific effect from the B vitamin supple-
ments.

C. PATHOLOGY IN HUMAN BEINGS
A. T. MILHORAT

Evidence of a specific need for inositol by human beings has not been
presented, nor have symptoms of deficiency of inositol in humans been
described. However, the wide distribution of inositol in the body*** and
the data accumulated in investigations in animals make it reasonable to
predict that increased knowledge probably will demonstrate an important
role of inositol in the human organism. The observations of Best and
others” on the lipotropic action of inositol and choline were made in ani-
mals maintained on diets deficient in this substance. These observations
form the basis for the use of inositol in the management of fatty infiltra-
tion and cirrhosis of the liver in patients, but when one considers that
these abnormalities may be due to a variety of causes, of which inositol
deficiency probably is of great rarity and perhaps even non-existent, the
paucity of evidence on the therapeutic usefulness of inositol in liver disease
is not surprising.

Ariel et al.® found a high incidence of fatty infiltration of the liver in
patients with gastrointestinal cancer. In 28 fasted patients Abels et al.‘
observed an average concentration of 16.4 g. of fat in 100 g. of wet liver
tissue. The administration of 280 mg. of inositol to 10 patients and of
1200 mg. to 8 patients 10 hours before the operation appeared to reduce the
amounts of fat in the liver, since the average concentration in the first
group was 8.2 g. per 100 g. of wet liver tissue, and in the second group 6.9 g.,
representing, in the authors’ opinion, reductions of 50 and 58 %, respectively.
Lipocaic and choline similarly reduced the concentration of fat in the liver.
It was concluded, on the basis of comparative experiments, that the effect
of lipocaic could not be explained entirely by the choline content of the
lipocaic but may be due to the inositol content.*?: °° Echaurren and Jor-

48 F. Rosenberger, Hoppe-Seyler’s Z. physiol. Chem. 64, 341 (1910).

447. B. Winter, J. Physiol. 108, 27 P (1944).

45 J. Needham, Biochem. J. (London) 18, 891 (1924).

46 J. Foleh and D. W. Woolley, J. Biol. Chem. 142, 963 (1942).

47C. H. Best, C. C. Lucas, J. H. Ridout, and J. M. Patterson, J. Biol. Chem. 186,
317 (1950).

487. M. Ariel, J. C. Abels, H. T. Murphy, G. T. Pack, and C. P. Rhoads, Ann. In-
ternal Med. 20, 570 (1944).

49 J. C. Abels, C. W. Kupel, G. T. Pack, and C. P. Rhoads, Proc. Soc. Exptl. Biol.
Med. 64, 157 (1943).

60 J.C. Abels, I. M. Ariel, H. T. Murphy, G. T. Pack, and C. P. Rhoads, Ann. Internal
Med. 20, 580 (1944).

a2 INOSITOLS

quera*! treated 10 patients with cirrhosis of the liver with a regimen of a
high protein diet plus 600 mg. of inositol daily, and in 7 noted gain in body
weight, disappearance of gastrointestinal symptoms, increase in diuresis,
and definite subjective improvement. However, Patek and Post™ had pre-
viously demonstrated the beneficial effects of diets of high nutritive value
in 54 patients with cirrhosis of the liver. The diets employed by Patek were
of high protein content and were supplemented with yeast, liver extract,
and thiamine chloride. Goldstein and Rosahn*? and Broun™ considered
inositol to be of value in the treatment of cirrhosis of the liver, but definitive
clinical experiments in which the diet was controlled are lacking, and the
data at hand make it difficult to ascribe any observed effects to the ad-
ministered inositol and not to general composition of the diet. In this con-
nection, it should be noted that Sellers et al.°® could find no evidence that
inositol favorably influences the course of cirrhosis experimentally pro-
duced in rats by the administration of carbon tetrachloride. In contrast,
the addition of either choline or dl-methionine to the diet induced con-
siderable improvement in the cirrhotic livers. Felch and Dotti°® adminis-
tered 3 g. of inositol daily to 30 diabetic patients with hypercholesteremia
and concluded that inositol is an effective agent in lowering serum choles-
terol and lipid P, but neither Shay,” who gave 1.2 g. of inositol daily to
patients with diabetes, nor Gephart,®* who gave 3 g. daily to a patient
with xanthomatous biliary cirrhosis, observed any significant change in
the concentration of cholesterol in the blood. Similar negative results were
obtained by Lupten e¢ al.,°® who likewise found no change in the insulin
requirements of the patients. Gross and Kesten® noted significant reduc-
tions in the concentration of serum cholesterol of 55 out of a series of 64 |
patients with psoriasis whose blood levels of cholesterol were above normal,
when preparations obtained from soybeans were administered. The daily
dose of these preparations contained 0.6 g. inositol, but the presence of
other factors such as choline permit no interpretation regarding the effect
of the inositol or indeed of any single component of the preparations.

51 A. P. Echaurren and R. Jorquera, Rev. Medica de Chile 71, 755 (1943) [abstr. in
J. Am. Med. Assoc. 124, 66 (1944)].

22 A. J. Patek, Jr., and J. Post, J. Clin. Invest. 20, 481 (1941).

53 M. R. Goldstein and P. D. Rosahn, Connecticut State Med J. 9, 351 (1945).

54 G. O. Broun, Postgrad. Med. 4, 203 (1948).

55 BH. A. Sellers, C. C. Lucas and C. H. Best, Brit. Med. J. 1948, I, 1061.

56 W. C. Felch and L. B. Dotti, Proc. Soc. Exptl. Biol. Med. 72, 376 (1949); Bull. N.
Y. Acad. Med. 26, 261 (1950).

57H. Shay, Am. J. Digest. Diseases 10, 48 (1943).

58 M. C. Gephart, Ann. Internal Med. 26, 746 (1947).

59 A.M. Lupton, T. W. Battafarano, F. E. Murphy, and C. L. Brown, Ann. West.
Med. Surg. 3, 342 (1949).

60 P, Gross and B. Kesten, N. Y. State J. Med. 50, 2683 (1940).

IX. EFFECTS OF DEFICIENCY 373

However, observations indicating that factors other than the absolute
level of cholesterol in the blood may contribute to the production of
atherosclerosis leave open the possibility that inositol could function as a
therapeutic or prophylactic agent in man, not necessarily by its effect on
the concentration of cholesterol but in some other manner not yet defined,
for example, by regulating the cholesterol :phospholipid ratio in the blood.
Thus, Ladd et al.“ found that atherosclerosis experimentally produced in
rabbits by the feeding of cholesterol was accompanied by considerable in-
crease in the concentration of cholesterol and only a slight increase in
the phospholipid level in the blood. These investigators observed that the
intravenous administration of Tween 80 to animals fed cholesterol increased
the blood phospholipids to levels that were as high or higher than the cor-
responding cholesterol levels, and decreased both the incidence and the
severity of the atherosclerosis. Several other workers also have emphasized
the importance of the relative levels of phospholipids and cholesterol.®-®
Duff and Payne® in their formulation of the pathogenesis of experimental
cholesterol atherosclerosis in the rabbit have stated the opinion that
instability of cholesterol in the blood rather than hypercholesterolemia
per se is the general condition responsible for the deposition of cholesterol in
the arterial walls. Duff and Payne considered the interrelations of the
lipids to be more important for their stability than is their relation to the
serum protein. In their experiments on normal and alloxan-diabetic rabbits
that were fed cholesterol, they found, as had Ladd, Kellner and Correll,
that the elevation of phospholipids was the important factor for the
stability of serum cholesterol, with a minor effect being exerted by the
neutral fats. Observations along similar lines by Ahrens and Kunkel
indicate that the clarity of sera of high lipid content is related closely to
proportional elevation of the phospholipids; conversely, ‘‘milkiness”’ is
present in such sera when the relative concentrations of phospholipids are
low. Ahrens and Kunkel considered the concentration of serum phospho-
lipids available for complex formation with serum proteins to be an im-
portant factor in determining the size of the lipid particles in the serum.
More recently, Pollak® has emphasized the role of albumin in the pro-
tection of the blood vessels by its ability to stabilize cholesterol. Leinwand
and Moore® noted that the administration of a total of 3 g. of inositol daily

61 A. T. Ladd, A. Kellner, and J. W. Correll, Federation Proc. 8, 360 (1949).
62 J. P. Peters and E. B. Man, J. Clin. Invest. 22, 707 (1943).

68 T. Leary, Arch. Pathol. 47, 1 (1949).

64M. M. Gertler and 8. M. Garn, Science 112, 14 (1950).

65 A. Steiner, Geriatrics 6, 209 (1951).

66 G. L. Duff and T. P. B. Payne, J. Exptl. Med. 92, 299 (1950).

67 —. H. Ahrens and H. G. Kunkel, J. Exptl. Med. 90, 409 (1949).

68 Q. J. Pollak, Geriatrics 6, 183 (1951).

69 T, Leinwand and D. H. Moore, Am. Heart J. 38, 467 (1949).

374 INOSITOLS

to patients with disorders of lipid metabolism decreased the total lipids
and increased the lipid phosphorus and cholesterol levels of the blood
during the earlier periods of treatment, but subsequently lowered both
the lipid phosphorus and the cholesterol concentrations as the treatment
was continued. They concluded that inositol may have potential value in
the management of atherosclerosis in man.

Inositol has been employed in the management of several other condi-
tions in man. For example, on the basis of observations of Woolley” on
the relationship of inositol to the growth of hair in the mouse, Vorhaus
et al.” administered inositol to subjects with alopecia but observed no
beneficial effect. Milhorat and Bartels” in their observations on creatinuria
in patients with muscular dystrophy noted that the simultaneous adminis-
tration of inositol and a-tocopherol lowered the creatine output, although
the use of either compound alone was without effect. This effect was seen
only in patients in the early or moderately advanced stage of the facio-
scapulohumeral form of the disease and not in either the advanced stages
of this form or in the pseudohypertrophic type. Moreover, certain other
sugars such as galactose and mannose had similar effects, and the mecha-
nism of action of these substances is obscure. Beckmann’ has confirmed
these observations on inositol and believes that their application may be
of value in the management of muscular dystrophy. On the other hand,
John” noted no effect of inositol and vitamin E on creatinuria in this
condition.

In summary, it may be stated that, although on the basis of theoretical
considerations inositol would appear to be of therapeutic promise in cer-
tain pathologic states in humans, the usefulness of inositol in the manage- :
ment of any pathologic condition in man still remains to be established.

Inosituria. The occurrence of inosituria appears to have been noted
first by Cloetta,”> who in 1856 isolated inositol from the urine of a patient
with chronic nephritis. The observations that actually stimulated interest
in this subject, however, were those of Vohl,’° who, 2 years later, isolated
from 18 to 20 g. of inositol per day from the urine of a patient with diabetes
insipidus. Inosituria as an inconstant occurrence in diabetes insipidus was
soon confirmed by a number of investigators, (e.g., Strauss’? and v.d.

70D. W. Woolley, J. Biol. Chem. 189, 29 (1941).

71M. G. Vorhaus, M. L. Gompertz, and A. Feder, Am. J. Digest. Diseases 10, 45
(1943).

72 A. T. Milhorat and W. E. Bartels, Federation Proc. 6, 414 (1947).

73 R. Beckmann, Deut. Z. Nervenheilk. 167, 16 (1951).

748. John, Z. klin Med. 148, 245 (1951).

75 A. Cloetta, Ann. Chem. Justus Liebigs 99, 289 (1856).

76H. Vohl, Arch. physiol. Heilk. 17, 410 (1858).

77 Strauss, Dissertation, University of Tibingen, 1864.

IX. EFFECTS OF DEFICIENCY 375

Heyden”) and a number of hypotheses were proposed to account for the
urinary excretion of the inositol. Considering the type and limited amount
of data available at the time, these hypotheses are easily understandable.
For example, in one study, inositol was found in the urine of patients with
diabetes insipidus who likewise had lesions of the fourth ventricle of the
brain, whereas inositol was absent from the urine of two other patients
with diabetes insipidus in whom no lesion of the fourth ventricle was
present.” The fact that inosituria is not limited to cases of diabetes insipidus
was indicated not only by the original observation of Cloetta,’®> which seems
to have been neglected by the workers of that time, but also by the dis-
covery that the urine of adults and children with a wide variety of diseases
may contain inositol. An account of these early observations was pub-
lished by Kiilz.8° Reichardt* and Kiilz® made the important observation
that, following the ingestion of large amounts of water, inosituria could
occur simultaneously with the polyuria. Kiilz found inosituria in normal
students who had consumed large volumes of wine or beer, and, although
these beverages contain inositol (Perrin® and Meillére*’) the diuresis, and
not the inositol intake, was considered to be the cause of the inositol ex-
cretion. Evidence for this opinion was produced when Kiilz administered
from 6 to 1014 1. of water to six normal adults, who previously had excreted
no inositol. From the large quantities of urine obtained in each case, Kiilz
was able to isolate from 0.4 to 0.9 g. of inositol. Later he** showed that uri-
nary excretion of inositol could be elicited by the parenteral administration
of saline in rabbits. Meillére and Fleury*® attempted to extend the earlier
work of Gallois*’ and Meillére and Camus*® and to establish a correlation
between glycosuria and inosituria. Gallois had found inositol in the urine
of one out of every six patients with glycosuria, and Meillére and Camus
had previously produced both glycosuria and inosituria by means of punc-
ture of the floor of the fourth ventricle. Meillére and Fleury confirmed the

78 vy. d. Heyden, Dissertation, University of Leiden, 1875.

79 Schultzer, Klin. Wochschr. 35 (1875).

80 &. Kiilz, in C. Gerhardt, Handbuch der Kinderkrankheiten, Vol. 3, p. 285, Tiibin-
gen, 1878.

81 KE. Reichardt, in C. Gerhardt, Lehrbuch der Kinderkrankheiten, p. 540, Tiibingen,
1874.

82 B. Kiilz, Sitzber. Ges. Beférder. ges. Naturwiss. Marburg 7 (1875); Centr. med. Wiss.
550 (1876); Z. anal. Chem. 16, 135 (1877); Bettrdége Pathol. u. Therap. d. Diabetes
melitus u. tnsipidus 1, 2, Marburg (1877).

83 G. Perrin, Ann. chim. anal. 14, 182 (1909).

84 G. Meillére, J. pharm. chim. [6] 30, 247 (1909); Chem. Zentr. 2, 1776 (1909).

85 B. Kiilz, J. pharm. chim. 29, 187 (1879).

86 G. Meillére and P. Fleury, Compt. rend. soc. biol. 2, 343 (1909).

87 Gallois, Z. anal. Chem. 4, 264 (1865).

88 G, Meillére and L. Camus, Compt. rend. soc. biol. 2, 159 (1906).

376 INOSITOLS

observation of Gallois and were further able to demonstrate the existence
of inosituria in phlorhizin diabetes and in experimentally produced diabetes
after removal of the pancreas. They concluded that inosituria is intimately
linked with glycosuria,*® whatever the cause of the latter might be, and
that it accompanied polyuria only when this is associated with glycosuria.
The situation appeared to be resolved by the observations of Starkenstein”°
and Needham.* Starkenstein showed that inositol has no significant rela-
tion to diabetes mellitus except that, when the volume of urine is large,
inosituria may occur. The urine of diabetic patients, when of small volume,
contains no inositol. Needham induced polyuria and inosituria in rats by
feeding them a salt diet over a period of 110 days. Although the diet con-
tained no inositol and the inosituria was of considerable magnitude and
duration, no diminution in the inositol content of the tissues could be
demonstrated. These experiments are important, since they not only prove
that urinary excretion of inositol may be induced by measures that produce
polyuria but they demonstrate also that the animal body is able to syn-
thesize inositol. How profound an inosituria may accompany polyuria is
indicated by the early observation of Vohl’* and the later isolation by Hop-
kins*! of about 15 g. of inositol daily from the urine of a patient with diabetes
insipidus. Using a modification of the Saccharomyces cerevisiae G. M. method
of Williams e¢ al.,°? Johnson et al.*? studied the excretion of inositol in the
sweat and urine of four adult male subjects under constant environmental
and dietary conditions. The average excretion of inositol in the sweat during
an 8-hour period of exposure to “hot moist’? conditions was 0.118 mg. per
hour. Under the same conditions, the average urinary loss was 0.494 mg.

per hour. The corresponding average losses under ‘‘comfortable”’ conditions -

were 0.027 mg. in sweat and 0.626 mg. in the urine. Thus, with a greater
loss of inositol in the sweat under the hot moist conditions, there appeared
to be a compensatory decrease in the excretion of inositol in the urine.

89 G. Meillére and P. Fleury, Répert. pharm. [3]21, 498 (1909) [from the Tribune méd.
Sept. 4, 1909; C. A. 4, 934 (1910)].

90 EK. Starkenstein, Z. exptl. Pathol. 5, 378 (1908).

1 F. G. Hopkins, (1923), quoted by J. Needham, ref. 45.

9% R. J. Williams, A. K. Stout, H. K. Mitchell, and J. R. McMahan, Univ. Texas
Publ. 4137, 27 (1941).

8B. C. Johnson, H. H. Mitchell, and T.S. Hamiiton, J. Biol. Chem. 161, 357 (1945).

_

X. PHARMACOLOGY td

X. Pharmacology

A. T. MILHORAT
A. HEART

A physiologic role of inositol in cardiac function is suggested by the
demonstration of the cyclitol in the heart muscle of rabbit, dog, sheep,
pig, and ox.!* The ventricle of the ox heart was found by Winter* to con-
tain from 85.7 to 134.8 mg. of inositol per 100 g. of tissue, and the auricle
contained from 77.1 to 92.2 mg. Less inositol was found in the bundle of
His than in tissue from another part of the same ventricle. The inositol
content of the Purkinje fibers was 53 mg. per 100 g. of tissue. Winter?
found the survival changes in the heart muscle of the dog to be accom-
panied by an increase in the inositol content and, therefore, postulated the
existence of a combined from of inositol, in addition to the free substance.
Moreover, since a combined form of inositol could not be detected in the
hearts of herbivorous animals such as the sheep and ox or in the heart of
the carnivorous pig, it appeared unlikely that the combined form in the
heart of the carnivorous dog was a substance such as phytin and had been
derived from plant sources in the food. His evidence for the presence of a
combined form of inositol in heart muscle is reminiscent of the experi-
ments of Rosenberger,® who reported on the presence of both free and com-
bined inositol in the white mouse. Rosenberger’s determinations were made
on the entire carcass after removal of the stomach and intestines (see also
reference 6). Whether the function of inositol in heart muscle is similar to
that in striated muscle, as postulated by Portmann, cannot be stated in a
definitive way at the present time. Portmann’ found that the fin muscles
of fish, and especially of the shark, contain large amounts of inositol. Since
the liver of these fish contains no glycogen or other reserve carbohydrate,
Portmann believed that the inositol probably is a reserve carbohydrate
that is formed from glucose and is stored in the fins to serve as an available
source of glucose for the blood by the reopening of the inositol ring. Sug-
gestive support of this hypothesis is furnished by the success of Grosheintz
and Fischer’ in the cyclization of glucose to inositol by purely chemical

1J. Needham, Biochem. J. 17, 422 (1923).

2L. B. Winter, Biochem. J. 28, 6 (1934).

3L. B. Winter, Biochem. J. 34, 249 (1940).

4L. B. Winter, J. Phystol. (London) 108, 27P (1944).

5 F. Rosenberger, Z. physiol. Chem. 64, 341 (1910).

6 A. Taylor, M. A. Pollack, and R. J. Williams, Univ. Texas Publ. 4237, 41 (1942);
V.H. Cheldelin and R. J. Williams, Univ. Texas Publ. 4237, 105 (1942).

7A. Portmann, quoted by H. O. L. Fischer, Harvey Lectures 40, 156 (1944-1945).

8 J. M. Grosheintz and H. O. L. Fischer, Harvey Lectures 40, 156 (1944-1945).

378 INOSITOLS

means, and by the demonstration by others that inositol in the organism
can be converted into glucose. Although Mayer® had found no increase in
the amounts of liver glycogen of three rabbits given 10 g. of inositol, nor had
he observed an increasein urinary glucose of diabetic patients given as much
as 50 g., Greenwald and Weiss!° later found a small but unmistakable in-
crease in the urinary glucose: nitrogen ratio when inositol was administered
to dogs with phlorhizin diabetes. Perhaps pertinent to this problem are the
observations of Needham."' who found that the injection of glucose into
the fertilized unincubated egg causes a very large rise in the inositol con-
tent of the egg during its subsequent development. More recently, Stetten
and Stetten! demonstrated a transformation of meso-inositol into glucose
in the phlorhinized rat, when they found significant concentration of
deuterium in the urinary glucose after administration of deuterio-inos-
itol. The differences in the results and interpretations of the earlier
workers, using less definitive methods, are easily understood when one
considers that the data of Stetten and Stetten indicate a transformation
to glucose of only 7% of the administered meso-inositol. Moreover, in this
experiment the inositol was administered by intraperitoneal injection,
whereas the earlier investigators used the oral route by which absorption is
incomplete.

The original observations of Sachs" that inositol affects cardiac function
was confirmed by subsequent workers. Sachs perfused the frog’s heart
with Ringer’s solution and noted increased amplitude and rate of con-
traction when inositol in low concentrations was added to the perfusion
fluid. However, when an isotonic solution of inositol was substituted for
the Ringer’s solution containing inositol, the heart immediately was stopped .
in systole. Hewitt and de Souza“ employed approximately the same con-
centrations of inositol in their perfusion studies on the frog’s heart. The
heart responded rapidly to inositol by an increase in the force of its beat
and just as rapidly returned to its original state when the inositol was
replaced by Ringer’s solution alone. In six of the twenty-five experiments,
inositol had no apparent effect on the heart, and in two its perfusion led
to diminution in force of beat. The rate of the heart, as a rule, was not
altered by inositol. The action of inositol was considered to be on the
heart muscle, since the effects were noted also in the atropinized heart. A
concentration of 6%, such as was employed by Sachs, was found to be
strongly toxic and soon stopped the heart in systole. Brissemoret and

9P. Mayer, Biochem. Z. 2, 393 (1907).

107. Greenwald and M. L. Weiss, J. Biol. Chem. 31, 1 (1917).

11 J. Needham, Biochem. J. 18, 1371 (1924).

12 R, Stetten and D. Stetten, Jr., J. Biol. Chem. 164, 85 (1946).

13 F, Sachs, Pfltigers Arch. ges. Physiol. 115, 550 (1906).

14 J. A. Hewitt and D. de Souza, J. Physiol. (London) 64, CXIX (1921).

X. PHARMACOLOGY 379

Chevalier!® perfused the rabbit’s heart with inositol in concentrations of
0.05 and 0.1%. The lower concentration of inositol produced definite
acceleration and more forceful activity, but use of the larger amounts of
inositol was followed by gradual slowing of the heart and stoppage in
systole. Meyer!® observed that, in the perfused rabbit’s heart, inositol
concentrations of 0.01 and 0.02 % produced a decrease in amplitude and
arrythmias. However, since the concentrations of inositol employed in
these experiments are higher than those that occur normally in the blood,
no definitive conclusions can be drawn relative to the physiologic role of
inositol in cardiac activity. Sonne and Sobotka,” in their application of
the nephelometric micro bioassay with Saccharomyces carlsbergensis to the
determination of inositol, observed levels of from 0.37 to 0.76 mg. per 100
ml. in the blood plasma of fasting patients and normal persons. Pooled
samples from miscellaneous patients showed values that ranged from 0.54
to 1.87 mg. per 100 ml. Moderate increases in the plasma inositol levels
followed the ingestion of 1.5 g. of inositol daily. Waldstein and Steigmann™*
gave amounts up to 1 g. of inositol by intravenous injection to patients
and concluded that relatively large amounts are well tolerated and appear
to be without untoward effects.

B. GASTROINTESTINAL TRACT

The effect of inositol on the activity of the stomach and small intestine
was studied in dogs by radiographic means by Martin et al.,'8 who found
considerable increase in the peristalsis of these organs. No spastic state
was produced except a pylorospasm which the authors attributed in part
to the constipating diet and in part to a possible contracting effect of the
administered inositol on the pyloric sphincter. Other observations are of a
more precursory nature but, in general, suggest a stimulating action of
inositol on the gastrointestinal tract. For example, Anderson!’ and Bly
et al.2° noted diarrhea in dogs after administration of inositol, whereas
Vorhaus et al.2! and Shay”? administered from 1 to 2 g. of inositol daily to
patients without production of gastrointestinal symptoms. As indicated
in the discussion on the factors influencing its requirements, inositol prob-

15 A. Brissemoret and J. Chevalier, Compt. rend. 147, 217 (1908).

16 A. KE. Meyer, Proc. Soc. Exptl. Med. 62, 111 (1946).

17§. Sonne and H. Sobotka, Arch. Biochem. 14, 93 (1947).

17a §. $8. Waldstein and F. Steigmann, Am. J. Digest. Diseases 19, 323 (1952).

1G. J. Martin, M. R. Thompson, and J. de Carvajal-Forero, Am. J. Digest. Diseases
8, 290 (1941).

19 R, J. Anderson, J. Biol. Chem. 25, 391 (1916).

20 C.G. Bly, F. W. Heggeness, and E. S. Nasset, J. Nutrition 26, 161 (1943).

21M. G. Vorhaus, M. L. Gompertz, and A. Feder, Am. J. Digest. Diseases 10, 45
(1943).

22H. Shay, Am. J. Digest. Diseases 10, 48 (1943).

380 INOSITOLS

ably is relatively poorly absorbed, and it is probable that the effect on
motility of the gastrointestinal tract is due to an irritating action. In this
connection, the observations of Clark and Geissman*® that inositol does not
potentiate the action of epinephrine on an isolated smooth muscle prepara-
tion are of interest. Torda and Wolff found that inositol increases the
sensitivity of striated muscle of the frog to acetylcholine, but data on the
smooth muscle of the gastrointestinal tract are not available.

C2 LEME OLD

The presence of inositol in the thyroid gland was demonstrated by Tam-
bach” and Meyer.!® However, few data on the effect of inositol on thyroid
function are available. Abelin®® produced toxic symptoms in rats by the
administration of 100 mg. of thyroglobin daily for 1.5 months, with re-
duction in body weight and in muscle creatine and increase in rate of
respiration. The daily parenteral administration of 50 to 100 mg. of panto-
thenic acid prevented the occurrence of toxic symptoms. Inositol and py-
ridoxine had similar effects. Handler and Follis”’ observed that a decreased
level of thyroid activity induced by thyroidectomy or by thiouracil or p-
aminobenzoic acid feeding prevented or retarded the development of he-
patic necrosis or fibrosis associated with choline and cystine deficiencies
in the rat and that thyroid administration hastened the death of cystine-
deficient rats. Inositol, as well as sulfasuxidine and taurine, prevented the
development of hepatic cirrhosis in animals on a diet deficient in choline.
This effect of inositol, however, was not due to lipotropic action or anti-
thyroid activity, since inositol did not reduce the fat content of the fatty
livers, nor did it produce morphological changes in the thyroid.

D. NEUROMUSCULAR SYSTEM

Inositol is a constituent of striated muscle!: ?!: *4.?5 and of the spinal
cord and brain.?? Investigations on the pharmacologic action of inositol
on the brain apparently have not been done, but Lecoq et al.*° in their
studies on the effect of a number of compounds on nerve chronaxia in rats
observed that imositol caused an increase in nerve chronaxia. Moreover,
inositol had no effect on the increase in chronaxia after repeated adminis-

°3.W. G. Clark and T. A. Geissman, J. Pharmacol. Exptl. Therap. 95, 363 (1949).

*4C. Torda and H. G. Wolff, Am. J. Physiol. 145, 608 (1945-1946).

25 R. Tambach, Pharm. Zentralhalle 37, 167 (1896).

26 T. Abelin, Experientia 1, 231 (1945).

*7 P, Handler and R. H. Follis, Jr., J. Nutrition 35, 669 (1948).

28 J. Scherer, Ann. Chem. Justus Liebigs 78, 322 (1850).

29 J. Folch and D. W. Woolley, J. Biol. Chem. 142, 963 (1942).

30 R. Lecoq, P. Chauchard, and H. Mazoué, Bull. soc. chim. biol. 80, 296 (1948);
Compt. rend. 227, 307 (1948); Compt. rend. soc. biol. 142, 428 (1948).

X. PHARMACOLOGY 381

tration of epinephrine, and it abolished the reduction in chronaxia produced
by adrenochrome.*! Inositol does not potentiate the action of epinephrine
on a smooth muscle preparation, however, nor does it affect the autoxida-
tion of epinephrine.” Torda and Wolff*4 found that inositol increases the
sensitivity of the rectus abdominis muscle of the frog to acetylcholine.

EK. MISCELLANEOUS

Inositol was found by Williams and Watson® to activate slightly if at
all, transamination by the rat kidney.

Doisy and Bocklage® in their studies on hexachlorocyclohexane noted
that the toxicity of the a, 8, y, and 6 isomers was not affected by inositol.

The thromboplastin inhibitory action of inositol phosphatide observed
by Overman and Wright* is believed by Kay and Balla*® to be due to the
adsorption of the inositol phosphatide by the surface of the thromboplastin,
thereby preventing the typical reaction which normally occurs when
thromboplastin alone is employed. Inositol phosphatide was found by Kay
and Delancey*® to be without influence on the mortality rate of rats suffer-
ing experimental burns.

XI. Requirements

A. OF ANIMALS

T. J. CUNHA

No definite requirements have been worked out for the inositol needs
of animals. Many investigators have been able to obtain good growth of
rats, mice, chicks, pigs, and hamsters when these animals were fed purified
diets without inositol. On the other hand, other groups of workers, under
different conditions, have shown inositol to benefit the diet of rats, mice,
chicks, pigs, hamsters, guinea pigs, and cotton rats. It is apparent that
under certain conditions a need for inositol can be shown. The reason for
the need under those conditions is not exactly known. Since inositol has
been shown to be synthesized by rats! and mice,” it is logical to assume that

31 P. Chauchard, H. Mazoué, and R. Lecoq, Compt. rend. soc. biol. 142, 1346 (1948).
32 H. L. Williams and E. M. Watson, Rev. can. biol. 6, 43 (1947) [C. A. 41, 4182 (1947)].
83. A. Doisy, Jr., and B. C. Bocklage, Proc. Soc. Exptl. Biol. Med. 74, 613 (1950).
34 R.S. Overman and I. 8. Wright, J. Biol. Chem. 174, 759 (1948).

85 J. H. Kay and G. A. Balla, Proc. Soc. Exptl. Biol. Med. 73, 465 (1950).

36 J. H. Kay and H. Delancey, Surg. Forwm Proc. 1961, 514.

1J. Needham, Biochem. J. 18, 891 (1924).

2D. W. Woolley, J. Exptl. Med. 16, 277 (1942).

382 INOSITOLS

under many conditions intestinal synthesis of inositol is altered, and so
are the inositol requirements of the animal. There are also interrelationships
of nutrients, and thus the diet fed and its content of various nutrients may
influence inositol needs. It is also possible that there are substances, anti-
metabolites for example, in natural feeds which may have an effect on the
inositol needs of the animal. There undoubtedly are other possible explana-
tions. Regardless of which may be the right one, there is still no definite
postulate which can be regarded as established. Considerably more work
is needed to clear up the problem of the factors influencing inositol require-
ments of animals.

Many investigators have used levels of 0.05 to 0.3 % of inositol in their
experimental rations. However, none of these levels are regarded as definite
requirements.

Several investigators have studied the utilization of phytate P.**> Moll-
gaard and his associates have indicated the importance in the pig of the
intestinal pH and of the presence of phytase in the food. Woolley® investi-
gated the ability of several substances to replace meso-inositol in the nutri-
tion of the mouse. Inositol hexaacetate, phytin, and soybean cephalin were
effective in curing alopecia in this series. In contrast, the strain of yeast
used was incapable of utilizing esters of inositol. Wiebelhaus eé al.? found
the quantity of inositol absorbed from the intestinal tract of fasting rats
to be approximately a linear function of time and observed that the ab-
sorption of inositol monophosphate was more rapid than the absorption of
inositol.

B. OF MAN
A. T. MILHORAT

Williams® estimated that an average mixed diet of 2500 cal. contains
about 1 g. of inositol. Since evidences of inositol deficiency are not observed
on good mixed diets, the requirements of man cannot, under ordinary cir-
cumstances, exceed this amount. Williams concludes that 1 g. daily is a
“safe” level of intake but emphasizes that definite data are lacking. Ander-
son and Bosworth® found that of 0.5 g. of inositol per kilo of body weight,
given by mouth, only 9% was found in the urine, and none in the feces.
The inositol in the diet is predominantly from vegetable sources. How

3H. M. Bruce and R. K. Callow, Biochem. J. 28, 517 (1934).

4M. Gedroye and S. Otolski, Arch. Chem. i Farm. 68, 106 (1936).

5 H. Mollgaard, K. Lorenzen, I. G. Hansen, and P. E. Christensen, Biochem. J. 40,
589 (1946).

6D. W. Woolley, J. Biol. Chem. 140, 461 (1941).

TV. D. Wiebelhaus, J. J. Betheil, and H. A. Lardy, Arch. Biochem. 18, 379 (1947).

8R. J. Williams, J. Am. Med. Assoc. 119, 1 (1942).

®R. J. Anderson and A. W. Bosworth, J. Biol. Chem. 25, 399 (1916).

XI. REQUIREMENTS 383

efficiently man can utilize the inositol of phytic acid is not known, but on
the basis of investigations in various species of animals it is probable that
appreciable amounts of phytic acid may be hydrolyzed in the human
intestinal tract. The enzymic cleavage of phytic acid in the intestinal
tract would be determined, at least in some measure, by the type of grain
ingested, for certain grains such as wheat,!° rye, and barley contain phytase,
whereas oats and maize do not.® Cruickshank and his associates," in their
observations on four adult subjects, consuming a diet rich in oatmeal,
noted that the phytate P of oatmeal was almost completely digested when
the calcium intake was normal. Additional calcium decreased the digesti-
bility of phytate P if the supplementary calcium was taken together with
the oatmeal and not if it was taken separately. Sonne and Sobotka” noted
moderate increases in the concentration of the blood from average levels of
0.37 to 0.76 mg. per 100 ml. when 1.5 g. of inositol was administered orally.
Definite data on utilization and on possible synthesis of inositol in the
human organism as well as on the effect of other constituents of the diet
on the requirements for inositol are not available. For the present, these
questions can be answered only by inference based on data obtained in
animals.

Anderson" found inositol to be absorbed very slowly from the intestines
of the dog and was able to recover in the feces as much as 77 % of the ad-
ministered amount. He noted that the urine contained only minimal
amounts. It would appear that the differences between his findings in the
dog and in man’ might be explained by the fact that in the dog inositol
produced diarrhea, but his findings in the dog were corroborated by
Dubin." However, it should be noted that Bly et al.!® found inositol to
act as a cathartic. Greenwald and Weiss,!® who administered inositol to
dogs with phlorhizin diabetes, found increases in urinary glucose that sug-
gested most of the administered inositol had been utilized.

C. INFLUENCE OF OTHER CONSTITUENTS OF DIET
A. T. MILHORAT

Considerable evidence has accumulated indicating that the composition
of the diet influences the requirements for inositol. Thus, Best et al.”

10 R. J. Anderson, J. Biol. Chem. 20, 475 (1915).

1H. W. H. Cruickshank, J. Duckworth, H. W. Kosterlitz, and G. M. Warnock, J.
Physiol. (London) 104, 41 (1945).

1228. Sonne and H. Sobotka, Arch. Biochem. 14, 93 (1947).

13R. J. Anderson, J. Biol. Chem. 25, 391 (1916).

“4A. Dubin, J. Biol. Chem. 28, 429 (1916-1917).

15 C. G. Bly, F. W. Heggeness, and E. 8. Nasset, J. Nutrition 26, 161 (1943).

16 J. Greenwald angl M. L. Weiss, J. Biol. Chem. 31, 1 (1917).

7C. H. Best, C. C. Lucas, J. H. Ridout, and J. M. Patterson, J. Biol. Chem. 186,
317 (1950).

384. INOSITOLS

have pointed out the necessity not only of dose-response curves but also
of standardized dietary conditions for estimating requirements. These
investigators observed that the lipotropic effect exerted by inositol when
it is administered with fat-free diets is absent when the diet contains fat.
Beveridge et al.* in their studies with other lipotropic agents noted the
influence of protein on the effect of these agents. In another study, Beve-
ridge and Lucas!® stated the opinion that the antagonistic effect of corn oil
on the lipotropic action of inositol might be due to the glycerides of the
essential fatty acids. Pertinent to this problem are the investigations of
Dam and Glavind?? and Dam,?! who showed that the effect of cod liver oil
in increasing the exudative diathesis of vitamin E-deficient chicks may be
counteracted by inositol. Handler”? obtained suggestive evidence of a syner-
gistic activity of inositol and tocopherol in his studies on the inhibition of
the lipotropic action of inositol by unsaturated fatty acids.

The role of B vitamins in increasing the lipotropic action of choline has
been demonstrated by Gavin et al.?? and McFarland and McHenry.”
The observation of Sure?> that, in the albino rat, inositol has an injurious
influence on lactation which is counteracted by p-aminobenzoic acid is at
variance with that of Climenko and McChesney,”* who in the same species
found that the addition of inositol to a diet containing B vitamins resulted
in normal lactation. p-Aminobenzoie acid in these experiments had no sig-
nificant influence on the effect of the inositol. Martin and Ansbacher??
and Martin? had previously studied the effects of adding inositol and p-
aminobenzoic acid to a diet containing thiamine, riboflavin, pyridoxine,
choline, nicotinic acid, and calcium pantothenate and reported that the
addition of one produced a deficiency syndrome of the other. However,
Ershoff? was unable to confirm these observations. Pantothenic acid was
found by Woolley*® to influence alopecia in mice. Concomitant administra-
tion of magnesium was found by Muset*! to increase the effect of inositol

18 J. M. R. Beveridge, C. C. Lucas, and M. K. O’Grady, J. Biol. Chem. 154, 9 (1944);
ibid. 160, 505 (1945).

19 J. M. R. Beveridge and C. C. Lucas, J. Biol. Chem. 157, 311 (1945).

20 H. Dam and J. Glavind, Science 96, 235 (1942).

21H. Dam, J. Nutrition 27, 193 (1944); ibid. 28, 289 (1944).

22P. Handler, J. Biol. Chem. 162, 77 (1946).

23 G. Gavin, J. M. Patterson, and E. W. McHenry, J. Biol. Chem. 148, 275 (1943).

24M. L. MacFarland and E. W. McHenry, J. Biol. Chem. 176, 429 (1948).

25 B. Sure, J. Nutrition 26, 275 (1943).

26 PD. R. Climenko and E. W. McChesney, Proc. Soc. Exptl. Biol. Med. 51, 157 (1942).

27 G. J. Martin and S. Ansbacher, Proc. Soc. Exptl. Biol. Med. 48, 118 (1941).

2G. J. Martin, Am. J. Physiol. 136, 124 (1942).

29 B. H. Ershoff, Proc. Soc. Exptl. Biol. Med. 56, 190 (1944).

30D. W. Woolley, J. Biol. Chem. 140, 461 (1941); J. Nutrition 2b Suppl., 17 (1941).

31 P. Puig Muset, Acta Med. Hispanica 36, (1947).

XI. REQUIREMENTS 385

in decreasing the cholesterol content of guinea pigs given fats per os.
More recently, Waldstein and Steigmann*® noted that inositol had no
effect on either the degradation or excretion of choline when these sub-
stances were administered orally or by intravenous injection. Observations
somewhat along the same lines were made by Diognardi and Magnoni,**
who observed that 300 mg. of inositol daily did not alter the amounts of
trigonellin excreted in the urine by a normal subject.

D. SYNTHESIS IN THE BODY
A. T. MILHORAT

The demonstration of Needham! that rats could be maintained on
inositol-free diets for periods as long as 8 months without diminution in the
inositol content of the body gave evidence that inositol can be synthesized
in the organism. Even more convincing were his studies in which inosituria
was experimentally induced for a period of 110 days; although there was a
continuous and vigorous excretion of inositol, and the diet contained no
inositol, there was no change in the amount of inositol contained in the
tissues of the rat. Previously Vohl* had isolated more inositol from the
urine of a person with diabetes insipidus than could have been contained
in the diet.

Woolley? showed that the mouse can synthesize inositol when panto-
thenic acid is present in the diet, but it is unable to carry out the syn-
thesis when pantothenic acid is absent. He observed, further, that the
intestinal tract of animals which showed spontaneous cure of alopecia
contained microorganisms which could synthesize more inositol than could
the organisms from the intestines of mice that did not recover spontan-
eously. Mitchell and Isbell®® also have presented evidence of synthesis of
inositol in the intestinal tract of rats. Johansson and Sarles** have reviewed
the general problem of intraintestinal synthesis of the B vitamins. Fenton
et al.*” studied the cecal flora of mice and found that the presence or absence
of inositol in the diet appeared to have no influence on the number of or-
ganisms present. Seeler and Silber** were able to maintain dogs in apparent
good health for a period of as long as 4.5 years on a diet containing no sig-
nificant amounts of inositol. These findings might suggest a synthesis of
inositol in the body, but of course they do not offer definitive evidence.

32$. S. Waldstein and F. Steigmann, Am. J. Digest. Diseases 19, 323 (1952).

33 N. Diognardi and A. Magnoni, Acta Vitaminol. 5, 264 (1951) [C. A. 46, 5707 (1952)].

34H. Vohl, Arch. physiol. Heilk. 17, 410 (1858).

35 H. K. Mitchell and E. R. Isbell, Univ. Texas Publ. 4287, 125 (1942).

36 H. R. Johansson and W. R. Sarles, Bacteriol. Revs. 18, 25 (1949).

37 P. F. Fenton, G. R. Cowgill, M. A. Stone, and D. H. Justice, J. Nutrition 42, 257
(1950).

38 A. O. Seeler and R. H. Silber, Am. J. Med. Sci. 209, 692 (1945).

386 INOSITOLS

Starkenstein®® had early postulated that inositol may be destroyed in the
intestines by bacteria, but data to support such a view are not available.
The addition of sulfasuxidine to the diet was found by Nielsen and Black*
and Handler” to increase the need for inositol, presumably as a result of
inhibition of intraintestinal synthesis.

39 K). Starkenstein, Z. exptl. Path. 5, 378 (1908).
40 K}. Nielsen and A. Black, Proc. Soc. Exptl. Biol. Med. 55, 14 (1944).

BE:
. Biochemical Systems .
. Estimation.

VI.

VET:

Vii.

IX.

CHAPTER 9

VITAMIN K GROUP

. Nomenclature and Formulas
. Chemistry .

A. Isolation ‘
B. Chemical and Pay aical Braperties F
C. Constitution .

D. Synthesis .

E. Specificity

Industrial Preparation

A. Biological auemation

1. Animals

. Diet . ‘

. Administration a Supplements :

. Measurement of Supplement Effect.

. Computation of Results

. Standards of Potency ;

7. Comparison of Assays by Eeferent "Victhods :
B. Chemical and Physical Estimation
Occurrence in Nature ‘

A. In Plants .

B. In Microorganisms.

C. Natural Antagonists.
Effects of Deficiency .

A. In Animals

B. In Human Beings . 5

1. Hemorrhage Resulting Far a lipencioney, a Vitamin K

2. Induction of Vitamin K Deficiency.

3. Blood Coagulation Changes with Epes K Deficiency
Pharmacology Sf CER tn Ben js
A. Vitamin K EaSnenecace
B. Dosages ;

C. Mode of Adnictretion:
D. Action of Vitamin K .
E. Toxicity

Requirements.

A. Of Animals

1. Chick

Diemaitia ee

3. Dog - es
B. Of Human Beings .

Om orm W bo

387

388
389
389
390
393
395
396
399
400
402
402
403
404
405
406
408
409
410
413
415
415
416
418
419
419
419
419
421
432
435
435
435
436
437
442
444
444
444
446
447
447

388 VITAMIN K GROUP

I. Nomenclature and Formulas
ROBERT 8. HARRIS

Accepted name: Vitamin K
Obsolete names: Antihemorrhagic vitamin
Phylloquinones
Koagulations vitamin
Coagulation vitamin
Prothrombin factor
Empirical formulas: Vitamin Ky: Csi:H46O2
Vitamin KG; Ca,H; O02
Phthiocol: Cy,H;03
Menadione: CiH302
Chemical names: Vitamin K,: 2-methyl-3-phytyl-1 ,4-naphthoquinone
Vitamin K.: 2-methyl-3-difarnesyl-1 ,4-naphthoqui-
none
Phthiocol: 2-methyl-3-hydroxy-1 ,4-naphthoquinone
Menadione: 2-methyl-1 ,4-naphthoquinone

Structure:
O
aay
HZ ( ees
AY ss eo Oe ma
|
el I CH; CH; CH; CH;
Vitamin K,
O

| |

CH; CH;

0
Wain
O 0
sie | H |
HZ“ ie HZ a
H ye HY Ve
oa y
fe)

Phthiocol Menadione

II. CHEMISTRY 389

II. Chemistry
H. J. ALMQUIST

There had appeared, prior to 1935, several reports of modifications of
the diet!-? which had led to hemorrhagic symptoms or changes in the blood-
clotting power of the chicken. Although these reports may now be inter-
preted in terms of vitamin K, they did not contain sufficient information
which would clearly link the condition with a lack of a new vitamin-like
factor, as distinguished from possible deficiency effects of known dietary
requisites, or toxic substances, impaired absorption, etc.

In 1935, papers by Dam®: ° and by Almquist and Stokstad!”: ™ furnished
the first strong chemical evidence of a new vitamin which was shown to be
in the fat-soluble, non-saponifiable, non-sterol fraction. It was also shown
that the vitamin was formed in certain feedstuffs otherwise relatively free of
it if these were subjected to the action of microorganisms. Schgnheyder”: ¥
made studies of blood composition in this deficiency disease and found a
specific lowering of the prothrombin fraction, but no other abnormalities.
This evidence, collectively, was sufficient to permit recognition of a new
vitamin, distinct from the known fat-soluble vitamins.

A. ISOLATION

Green leafy material was found to be a relatively rich source of the vita-
min. The good heat stability of the vitamin!® made possible the use of
commercially dehydrated alfalfa meal as a starting source for isolation
studies.

Extraction of the vitamin was carried out by means of a solvent such as
ethyl ether, petroleum ether, or hexane. It was soon found that the vita-
min was too labile during alkaline saponification to permit use of this step
for the removal of green pigments and fats.» !® Chlorophyll and similar

1H. Dam, Biochem. Z. 215, 475 (1929).

2H. Dam, Nature 133, 909 (1934).

3H. Dam and F. Schgnheyder, Biochem. J. 28, 1355 (1934).

4W.F. Holst and E. R. Halbrook, Science 77, 354 (1933).

5A. A. Horvath, Am. J. Physiol. 94, 65 (1930).

6 W. D. McFarlane, W. R. Graham, Jr., and G. E. Hall, J. Nutrition 4, 331 (1931).

7W. D. McFarlane, W. R. Graham, Jr., and F. Richardson, Biochem. J. 25, 358
(1931).

8H. Dam, Nature 135, 652 (1935).

9H. Dam, Biochem. J. 29, 1273 (1935).

10H. J. Almquist and E. L. R. Stokstad, Nature 136, 31 (1935).

1H. J. Almquist and E. L. R. Stokstad, J. Biol. Chem. 111, 105 (1935).

2 F, Schgnheyder, Nature 135, 653 (1935).

13 Ff, Schgnheyder, Biochem. J. 30, 890 (1936).

4H. J. Almquist, J. Biol. Chem. 114, 241 (1936).

390 VITAMIN K GROUP

coloring matter could be removed by adsorption on carefully added amounts
of activated magnesium oxide or carbon. There followed a series of steps
which consisted typically in chilling the preparations in various solvents
to crystallize out impurities. By such means, for example, there was ob-
tained a reddish oil which protected chicks against the hemorrhagic disease
when fed at 2 mg. per kilogram of diet.'* The vitamin was further purified
in a molecular still, a fourfold concentration being effected.!® The hemor-
rhagic disease in chicks was then prevented by 0.5 mg. of the preparation
per kilogram of diet.

Dam and Schgnheyder” concentrated the vitamin by adsorption on cal-
cium carbonate or sucrose. Dam e¢ al.!8 announced isolation of the vitamin
in a highly purified form. Important details in the isolation steps were dis-
closed later by Karrer et al.!®

McKee ef al.*° used primarily chromatographic adsorption on Decalso
and Permutit on a large scale in their isolation of the vitamin from a petro-
Jeum ether extract of alfalfa meal and from putrified fish meal. Details of
the procedures were published, ultimately, by Binkley et al.?! Many other
adsorbents were found to be inefficient or destructive for the retention of
the vitamin. Repeated adsorptions yielded practically pure vitamin K,.

Tishler and Sampson” have isolated from Bacillus brevis a vitamin K
which proved to be identical with the vitamin K, previously obtained by
McKee et al.2° This isolation was accomplished by reducing the vitamin
by hydrosulfite, then extracting it from a petroleum ether layer by dilute
potassium hydroxide containing hydrosulfite, recovering the vitamin in
petroleum ether on dilution of the alkaline layer with water, and crystal-
lizing the product from chloroform and methanol.

B. CHEMICAL AND PHYSICAL PROPERTIES

Dam ef al.'® reported their yellowish oil preparation to have constant
composition and biological activity after repeated chromatographic ad-
sorption. It contained carbon 82.2%, hydrogen 10.7%, and 2 atoms of

15 A. J. Almquist, J. Biol. Chem. 117, 517 (1937).

16H. J. Almquist, J. Biol. Chem. 115, 589 (1936).

17H. Dam and F. Schgnheyder, Biochem. J. 30, 897 (1936).

18 H. Dam, A. Geiger, J. Glavind, P. Karrer, W. Karrer, E. E. Rothschild, and H.
Salomon, Helv. Chim. Acta 22, 310 (1939).

19P. Karrer, A. Geiger, R. Legler, A. Ruegger, and H. Salomon, Helv. Chim. Acta
22, 1464 (1939).

20 R. W. McKee, S. B. Binkley, D. W. MacCorquodale, S. A. Thayer, and E. A.
Doisy, J. Am. Chem. Soc. 61, 1295 (1939).

218. B. Binkley, D. W. MacCorquodale, S. A. Thayer, and E. A. Doisy, J. Biol.
Chem. 130, 219 (1939).

22M. Tishler and W. L. Sampson, Proc. Soc. Exptl. Biol. Med. 68, 136 (1948).

II. CHEMISTRY 391

oxygen per molecule. Karrer and Geiger?’ reported a provisional formula of
C30-32, Has. 48, or 50, Ov, and a molecular weight of 445 to 450. Doisy and
coworkers had obtained two final products, one from alfalfa and one from
putrified fish meal.2* 24 The first was a yellow oil and was called IX,; the
melting point was given as 53.5 to 54.5 and 52.5 to 53.5.2 The vitamin
from alfalfa contained carbon 82.76 and 82.54%, and hydrogen 10.65 and
10.66 %. Its molecular weight was determined as 443 to 464, and its probable
formula as C32H4s-5002. Almquist and Klose®* investigated the alkaline deg-
radation product of vitamin K,. This derivative is weakly acid, which
facilitates its purification. Analyses and molecular weight determination
indicated a provisional formula of C3:H50O.. This showed that no appre-
ciable fragment of the vitamin had been split off and that 2 oxygen atoms
and probably several hydrogen atoms had been added. At least one of the
oxygen atoms was phenolic. The empirical formula of vitamin Ky is now
known to be C3:H4.Q., and its molecular weight 450.37. Figure 1 shows the
structural formulas of the various forms of vitamin K.

The vitamin was found to be unstable to light.!®» 24 Phenyl isocyanate,
eyanic acid, and dinitrobenzoyl chloride, which are reagents for an alcoholic
hydroxyl group, did not form an isolatable derivative or affect the activity
of the vitamin. Bromine was readily absorbed. Oxidation destroyed activity.
A positive nitration test was obtained for an aromatic structure.” A di-
acetate was formed upon reductive acetylation from which the vitamin
could be regenerated.?!: *8

Almquist!® reported that the vitamin absorbed strongly in the ultraviolet
range with considerable destruction. Dam et al.!8 reported ultraviolet ab-
sorption maxima at 248, 261, 270, and 328 mu. The extinction coefficient,
E}”,,, for the 248 line was 280. McKee e¢ al.2° found the extinction co-
efficient at 248 to be 385 and suggested that their product was purer than
the one described by Dam et al. However, the biological activity of prepara-
tions from these laboratories was found to be equivalent in simultaneous
assays by another laboratory.”®

Ewing et al.*° investigated very carefully the fee aeration spec-

23 P, Karrer and A. Geiger, Helv. Chim. Acta 22, 945 (1939).

24D. W. MacCorquodale, S. B. Binkely, R. W. McKee, S. A. Thayer, and E. A.
Doisy, Proc. Soc. Exptl. Biol. Med. 40, 482 (1939).

25 R. W. McKee, S. B. Binkley, 8S. A. Thayer, D. W. MacCorquodale, and E. A.
Doisy, J. Biol. Chem. 131, 327 (1939).

26 H. J. Almquist and A. A. Klose, J. Biol. Chem. 1380, 791 (1939).

*7 A. A. Klose, H. J. Almquist, and E. Mecchi, J. Biol. Chem. 125, 681 (1938).

8S. B. Binkley, D. W. MacCorquodale, L. C. Cheney, S. A. Thayer, R. W. McKee,
and E. A. Doisy, J. Am. Chem. Soc. 61, 1612 (1939).

°9 H. J. Almquist and A. A. Klose, J. Biol. Chem. 130, 787 (1939).

80D. T. Ewing, J. M. Vandenbelt, and O. Kamm, J. Biol. Chem. 181, 345 (1939).

392 VITAMIN K GROUP

tra of vitamins K, and K, prepared by Doisy and coworkers. Each vitamin
showed maxima at 243, 249, 260, and 270 my and a broad absorption in
the region of 310 and 340. The H{%,, at 249 mp was 540 for Ki and 305
for Ko. These extinction coefficients were higher than those reported pre-
viously.'8 2° The purified vitamin Ky in hexane solution exposed to daylight
decreased in absorption so fast that in 15 minutes the extinction coefficient
at 249 was about 420 and at 1 hour about 350. The other maxima also

O O
Cer renee)
OH
O O
2-Methyl-3-hydroxy- 2-Methyl-1,4-naphthoquinone
1,4-naphthoquinone Menadione
Phthiocol
O
CH,
Beee eT ee ae
O (Csi 3 CH 3 CH; (lst

2-Methyl-3-phytyl-1,4-naphthoquinone
Vitamin K,

O
Ee eee ae
O CH; CH;

2-Methy]-3-difarnesy]-1,4-naphthoquinone
Vitamin K,

Fic. 1. Forms of vitamin K.

dropped similarly. Ewing e¢ al.*! have further studied the absorption spec-
trum of vitamin Ky, again finding evidence of rapid drops in absorption
as the vitamin in hexane solution was exposed to ultraviolet light. A new
value for the extinction coefficient at 249 mu was given at 435 + 5. The
literature was thoroughly discussed. More recently, the extinction co-
efficient for K. at 249 my has been given as 520, which is considerably
higher than the earlier values.” It seems evident, therefore, that unless
determined almost instantly after the vitamin was exposed to light the
absorption at 249 mu would have decreased by an indeterminate amount
and could hardly be used as a criterion of purity.

31D, T. Ewing, F. 8. Tomkins, and O. Kamm, J. Biol. Chem. 147, 283 (19438).

II, CHEMISTRY 393

At very low pressures, 10-4 to 10-> mm. of mercury, vitamin K, could
be distilled at 120 to 140° with little or no loss.'® #4 Vitamin K, was distilled
with some loss at 2 X 10-4 mm. and 200°.

C. CONSTITUTION

The characteristic yellow color of these purified vitamins, the loss of
color upon hydrogenation, and its reappearance upon exposure to oxygen
in air suggested that the vitamins might be quinones.”!: ® Among a num-
ber of bacteria tested Mycobacterium tuberculosis had been found to possess

O
Soe
CH ACEI Wen 2) CH (CH,) CH (CH 2) antes
O CH; CH; CH; CH;
Vitamin K,
I
ey CH; COOH
CH,COOH COOH
O.
2-Methyl-1,4-naphthoquinone- Phthalic acid

3-acetic acid

O—C(CH) CH (CH,),CH (CH;),CHCH,

CH 3 CH; H 3 CH;
2,6,10-Trimethylpentadecanone-14

Fia. 2. Oxidation products of vitamin K, .

appreciable vitamin K activity. The principal pigment in the lipids of
this organism had been isolated and synthesized by Anderson and coworkers
several years before.** The pigment, phthiocol, was 2-methyl-3-hydroxy-
1 ,4-naphthoquinone (Fig. 2). Certain features of the absorption curve and
other properties of this compound compared closely with those reported
for vitamin K,. Synthetic phthiocol tested with vitamin K-deficient chicks
was found to be distinctly active in restoring normal blood-clotting time
and thus became the first completely identified form of vitamin K.** The
next step was to test the functional importance of the methyl and hydroxy]

2H. J. Almquist, C. F. Pentler, and E. Mecchi, Proc. Soc. Exptl. Biol. Med. 38, 336
(1938).

33 R. J. Anderson and M.S. Newman, J. Biol. Chem. 101, 773 (1933).

34H. J. Almquist and A. A. Klose, J. Am. Chem. Soc. 61, 1611 (1939).

394 VITAMIN K GROUP

groups. Chick assays of the 2-methyl, the 2-hydroxy, and the 2-methyl-3-
hydroxynaphthoquinone showed that the 2-methyl compound (see Fig. 1)
was more active and the 2-hydroxy compound less active than phthiocol.
This indicated that the methyl group was functionally important whereas
the hydroxyl group seemed to reduce activity.*® Phthiocol was readily
brought into water solution which was effective by intramuscular or in-
travenous injection. It is probably the first synthetic form of the vitamin
to be employed in human cases.

Confirming reports of vitamin K activity of these and certain other syn-
thetic and natural substituted naphthoquinones immediately appeared.3*-39
It had also been noted that the absorption spectrum of 2 ,3-dimethyl-1 ,4-
naphthoquinone most closely resembled that of vitamins K, and Ko, and
it was suggested that the vitamins were derivatives of 1 ,4-naphthoquinone
with side chains at the 2 and 3 positions.

Since it had become plainly evident that the active nucleus of the natu-
rally occurring vitamin was represented by 2-methyl-1,4-naphthoquinone
with substituents at position 3, the remaining problem in the cases of vita-
mins K,; and K» was to determine the nature of the substituents. Mac-
Corquodale et al.4° have published the details of their studies on the struc-
ture of vitamin K;. Oxidation with chromic acid resulted in the formation
of a mixture from which two acids were isolated. One of these was phthalic
acid, and the other was ultimately identified with 2-methyl-1 ,4-napthoqui-
none-3-acetic acid by comparison of melting point and other properties
with those of products synthesized for this purpose. A comparable acid
was obtained from the oxidation of diacetyl dihydro vitamin K, (Fig. 2).

Also among the oxidation fragments was a ketone which could be sep-
arated by steam distillation. This was found to be identical with 2,6, 10-
trimethylpentadecanone-14, which was previously known to be formed in
the oxidation of phytol (Fig. 2). Phytol, if present as a side chain on the
vitamin, was evidently located so that its double bond was between the
second and third carbon atoms from the quinone ring, since this was the
point of scission upon oxidation. The formula of vitamin Ky was given as
2-methyl-3-phytyl-1 ,4-naphthoquinone (Fig. 2).

35 H. J. Almquist and A. A. Klose, J. Am. Chem. Soc. 61, 1923 (1939).
36S. Ansbacher and E. Fernholz, J. Am. Chem. Soc. 61, 1924 (1939).
37 LL. F. Fieser, D. M. Bowen, W. P. Campbell, M. Fieser, E. M. Fry, R. N. Jones,

B. Riegel, C. W. Schweitzer, and P. G. Smith, J. Am. Chem. Soc. 61, 1925 (1939).

38 J,, F. Fieser, D. M. Bowen, W. P. Campbell, E. M. Fry, and M. D. Gates, Jr.,

J. Am. Chem. Soc. 61, 1926 (1939).

39§. A. Thayer, L. C. Cheney, 8. B. Binkley, D. W. MacCorquodale, and E. A. Doisy,

J. Am. Chem. Soc. 61, 1932 (1939).

40D). W. MacCorquodale, L. C. Cheney, 8. B. Binkley, W. F. Holeomb, R. W. McKee,
S. A. Thayer, and E. A. Doisy, J. Biol. Chem. 181, 357 (1939).

Il. CHEMISTRY 395

McKee et al.”* stated that vitamin K, was a 2 ,3-substituted naphthoqui-
none with six double bonds in the side groups. It would take up 9 molecules
of hydrogen, 3 for the naphthoquinone portion. Iodine absorption indicated
6 molecules of halogen taken up by non-aromatic unsaturated bonds. Upon
oxidation vitamin K, yielded the same quinone acid (or aldehyde) and
phthalic acid that had been obtained from K, (Binkley eé aJ.“"). The vitamin
K. was, evidently, similar to Ki, but with a much longer side chain at posi-
tion 3. Fragments of the side chain were isolated and identified as acetone
and levulinaldehyde in a mole ratio of 1:5 per mole of K2. This aldehyde is
also obtainable from the oxidation of farnesol. A structural formula was
proposed having two farnesyl groups linked together to form a long side
chain at position 3 (Fig. 1). The empirical formula was given as Cy H5¢602.

D. SYNTHESIS

Synthesis of vitamin K,; was accomplished by simple procedures. It was
evident that a phytyl side chain would account for the difference in molec-
ular weight between the methylnaphthoquinone and vitamin Ky. 2-Methyl-
3-phytyl-1 ,4-naphthoquinone was synthesized by Almquist and Klose*® by
coupling methylnaphthoquinone with phytyl bromide in petroleum ether
and glacial acetic acid in contact with zinc dust. The product was refined
in a molecular still. It exhibited color, oily form, color reaction, carbon
and hydrogen analysis, and biological potency very similar to those of
vitamin K;}.

- Doisy and coworkers* submitted a report of synthesis of the same com-
pound by condensing phytyl bromide with the monosodium salt of 2-
methyl-1 ,4-naphthohydroquinone in benzene, and by direct coupling of
phytol and methylnaphthoquinone with zine chloride. Details of these
methods were given later.*® Fieser“ carried out the synthesis of this com-
pound by condensation of phytol with methylnaphthoquinone in dioxane
with oxalic acid as catalyst. These preliminary reports appeared in the
same issue of the same journal. Synthesis of vitamin K, has not yet been
accomplished.

The ultraviolet absorption of synthetic vitamin K; was identified with
that of the natural vitamin*! (Fig. 3).

41§. B. Binkley, R. W. McKee, S. A. Thayer, and E. A. Doisy, J. Biol. Chem. 138,
721 (1940).

4H. J. Almquist and A. A. Klose, J. Am. Chem. Soc. 61, 2557 (1939); J. Biol. Chem.
132, 469 (1940).

43§. B. Binkley, L. C. Cheney, W. F. Holcomb, R. W. McKee, S. A. Thayer, D. W.
MacCorquodale, and E. Doisy, J. Am. Chem. Soc. 61, 2558 (1939).

447, F. Fieser, J. Am. Chem. Soc. 61, 2559 (1939).

396 VITAMIN K GROUP

E. SPECIFICITY

In contrast to the history of most other vitamins, synthesis of vitamin Ky
did not prove to be the ultimate goal in the search for antihemorrhagic
vitamins. Immediately after the report on phthiocol,** a number of com-
munications appeared on tests of other naphthoquinones, mostly based
upon rapid curative assays which may be done in a few hours. Ansbacher
and Fernholz®* confirmed the activity of phthiocol and indicated an even

400

300

lcm.

E1%

200

100

240 280 320 360
Wavelength, mu

Fig. 3. Ultraviolet absorption spectrum of synthetic vitamin K; . (From Ewing
et al.*1)

higher potency for 2-methyl-1,4-naphthoquinone than reported by Alm-
quist and Klose*® or Thayer eé al.*® Ultimately, this compound was reported
to have a greater activity than vitamin Ki, by two to three times.?9: 4-®

Methylnaphthohydroquinone was equally as active as its oxidized

45H. J. Almquist and A. A. Klose, Proc. Soc. Exptl. Biol. Med. 46, 55 (1940).

46S. Ansbacher, J. Biol. Chem. 188, iii (1940).

47, P. Dann, Proc. Soc. Exptl. Biol. Med. 42, 663 (1939).

4A D. Emmett, R. A. Brown, and O. Kamm, J. Biol. Chem. 132, 467 (1940).

49}, Fernholz and 8. Ansbacher, Science 90, 215 (1939).

50S, A, Thayer, R. W. McKee, S. B. Binkley, and E. A. Doisy, Proc. Soc. Exptl.
Biol. Med. 44, 585 (1940).

51H, Dam, J. Glavind, and P. Karrer, Helv. Chim. Acta 33, 224 (1940).

82 T,. F. Fieser, M. Tishler, and W. L. Sampson, J. Biol. Chem. 137, 659 (1941).

II. CHEMISTRY 397

form.** 4° The diesters of this compound were also found to be active. The
bisulfite addition product of methylnaphthoquinone is water soluble and
highly active.** A number of other compounds easily convertible to methyl-
naphthoquinone in metabolism probably owe their activity to such con-
version. Among these are 1-hydroxy-2-methyl-, 1-hydroxy-3-methyl-, and
l-amino-2-methylnaphthalene, 3-methyl-1-tetralone, and 2-methyl-1-tetra-
lone.* The water-soluble compounds, 4-amino-2-methyl-1-naphthol hydro-
chloride and 4-amino-3-methyl-1-naphthol hydrochloride were found to be
approximately as potent as vitamin K,.°° Using the Craven’s ethyl cyano-
acetate color reaction for quinones as a quantitative method, Richert®®
demonstrated the biological conversion of 4-amino-2-methyl-1-naphthol,
2-methyl-1 ,4-naphthohydroquinone diphosphate, and 2-methy! tetralone
to methylnaphthoquinone. The excretion of the latter in the urine of the
rabbit and chicken increased significantly when any of the first three were
fed.

Several naturally occurring 3-hydroxynaphthoquinones having a larger
side chain in place of the methyl group of phthiocol were reported to show
some activity,” but this could not be confirmed.*®: 7 Among these were
lapachol, hydrolapachol, and lomatiol. 1-Methyl-2-hydroxy-, 2-methyl-3-
hydroxy-, and 1-methyl-4-hydroxynaphthalenes were not appreciably ac-
tive.*! A discussion of the relation of structure to activity has been pub-
lished by Fieser et al.

In view of the results with the above-mentioned compounds it was sur-
prising that the 2-methyl-1 ,4-naphthohydroquinone diphosphoric acid es-
ter was found to be more active on a molecular basis than the parent methyl-
naphthoquinone.** *> 8 The corresponding disulfate, however, was
markedly less potent.®?: °° These differences may be related to differences
in speed of absorption or of regeneration of the methylnaphthoquinone.
The interest in these and other water-soluble forms of vitamin K hinges
on their greater convenience for injection and their independence on bile
or bile salts for absorption from the alimentary tract.

Comparative five-day assays of vitamins K, and Ky have indicated that
the relative potencies are in the ratio of 1.25:1.4° This is the approximate
ratio of the amount of the active methylnaphthoquinone nucleus contained
in each. Assays by shorter procedures have indicated a potency ratio of

53 B. R. Baker, T. H. Davies, L. McElroy, and G. H. Carlson, J. Am. Chem. Soc. 64,
1096 (1942).

54M. Tishler, L. F. Fieser, and W. L. Sampson, J. Am. Chem. Soc. 62, 1881 (1940).

55 J). Richert, S. A. Thayer, R. W. McKee, 8S. B. Binkley, and E. A. Doisy, Proc. Soc.
Exptl. Biol. Med. 44, 601 (1940).

56D. A. Richert, J. Biol. Chem. 154, 1 (1944).

57 7,. F. Fieser, W. P. Campbell, and E. M. Fry, J. Am. Chem. Soc. 61, 2206 (1939).

588 R. H. K. Foster, J. Lee, and U. V. Solmssen, J. Am. Chem. Soc. 62, 453 (1940).

398 VITAMIN K GROUP

approximately 1.5,°9 which may mean that K; is more rapidly absorbed or
metabolized than K, during a short period of time. It is obvious, however,
that the long side chains on these vitamins are not specifically required
for activity.

An additional form of vitamin K has been reported in extracts of alfalfa.®°
This form did not give the typical strong color reaction in sodium ethylate
which has been reported!® for vitamin K,; yet it showed considerable po-
tency.®® The product was nearly colorless. Vitamin K,; has been converted
into a 2,3-oxido derivative, a nearly colorless oil, which gives no color re-
action with sodium ethylate and resembles in these respects the second
form of vitamin K reported to be in alfalfa.®! This second form may be one
which was reported as existing in the form of relatively colorless crystals
at low temperature after isolation from alfalfa.™

Antihemorrhagic activity in the anthraquinone series was investigated
briefly by Almquist and Klose,” who found no activity in anthraquinone
and 1 ,2-dihydroxyanthraquinone, and by Thayer e¢ al.*® who found none
in phenanthraquinone, anthraquinone sulfonic acid, and dihydroanthra-
quinone diacetate. Martin and Lischer® extended these studies and de-
tected very slight activity in 1,,2,4-trihydroxyanthraquinone and certain
other polyhydroxyanthraquinones having three hydroxyls on one ring.
These workers suggested that such polyhydroxy compounds might be more
labile to ring rupture and subsequent formation of 2 ,3-substituted naptho-
quinones.

A few distinct examples of activity are known where conversion to meth-
ylnaphthoquinone does not seem possible. 2 ,5-Dimethylbenzoquinone and
perhaps certain other substituted benzoquinones have very low but meas-
urable activity.®-® A degradation product of vitamin E, a-tocopherylqui-
none, appears to have slight antihemorrhagic activity.* It is a benzoqui-
none with three methyl groups and a long side chain derived from phytol.
This compound is also reported to be antagonistic to vitamin K.*s Both
of these reports may be correct if the a-tocopherylquinone displaces vita-
min K from an enzyme system forming another enzyme of very low but

59S. A. Thayer, R. W. McKee, S. B. Binkley, D. W. MacCorquodale, and E. A. Doisy,
Proc. Soc. Exptl. Biol. Med. 41, 194 (1939).

60 §. Ansbacher, E. Fernholz, and H. B. MacPhillamy, Proc. Soc. Exptl. Biol. Med.
42, 655 (1939).

61 ],, F. Fieser, M. Tishler, and W. L. Sampson, J. Am. Chem. Soc. 62, 1628 (1940).

62H. J. Almquist, Natwre 140, 25 (1937).

63 G. J. Martin and C. F. Lischer, J. Biol. Chem. 187, 169 (1941).

64S. Ansbacher and E. Fernholz, J. Biol. Chem. 131, 399 (1939).

66 H. J. Almquist, Physiol. Revs. 21, 194 (1941).

66 R. Kuhn, K. Wallenfels, F. Weggand, T. Moll, and L. Hepding, Naturwissenschaf-
ten 27, 518 (1939).

66a 1), W. Woolley, J. Biol. Chem. 159, 59 (1945).

III. INDUSTRIAL PREPARATION 399

positive antihemorrhagic efficiency (somewhat after the mechanism pro-
posed by Quick and Collentine®). 3-Methyl-4-hydroxycoumarin also ap-
pears to have activity. This compound may be regarded as analogous to
phthiocol in which the 4-carbonyl has been replaced by an oxygen atom.

Several reports have appeared of antihemorrhagic activity of phthalic
acid and its diesters.*® However, Dam7° was unable to detect any activity
in potassium acid phthalate or diethylphthalate. Blumberg and Arnold”
carried out further investigations on this question but were unable to de-
tect any activity in diethyl-, dipropyl-, diisopropyl-, or dibutylphthalate,
even at high levels per chick. The diethyl ester also showed no antagonistic
action to methylnaphthoquinone.

III. Industrial Preparation
ROBERT S. HARRIS

There is no important demand by the food industries for vitamins K
as concentrated from natural sources or as synthesized, because the amounts
present in the food supply and the amounts synthesized in the intestinal
tracts of most species, including man, are adequate for normal individuals.

The clinical demand for vitamins K is now met by synthetic processes.
Concentrates prepared! from natural sources are sometimes preferred be-
cause they contain vitamin K;. Synthetic vitamin K;, is also available,’ ?
but it is not widely used because of high cost of production. Synthetic 2-
methyl-1 ,4-naphthoquinone*-'° is now preferred in the clinic because it is
highly effective and least expensive to produce.

67 A. J. Quick and G. FE. Collentine, Am. J. Physiol. 164, 716 (1951); J. Lab. Clin.
Med. 36, 976 (1950).
68 P. Meunier and C. Mentzer, Bull. soc. chim. biol. 25, 80 (1943).
69 M. M. Shemiakin and L. A. Schukina, Nature 164, 513 (1944).
70H. Dam, Nature 152, 355 (1943).
1H. Blumberg and A. Arnold, Proc. Soc. Exptl. Biol. Med. 57, 255 (1944).
1§. Ansbacher, E. Fernholz, and M. L. Moore, U.S. Pat. 2,233,279 (1941).
2H. J. Almquist and A. A. Klose, J. Am. Chem. Soc. 61, 2557 (1939).
3... F. Fieser, J. Am. Chem. Soc. 61, 2559 (1939).
4L. F. Fieser, W. P. Campbell, E. M. Frey, and M. D. Gates, Jr., J. Am. Chem.
Soc. 61, 2559, 3216 (1939).
5§. B. Binkley, R. W. McKee, S. A. Thayer, and E. A. Doisy, J. Biol. Chem. 133,
721 (1940).
6 R. T. Arnold and R. Larson, J. Org. Chem. 5, 250 (1940).
7P.P.T. Sah, W. Brill, and H. Holzen, Ber. 73, 762 (1940).
8P. P. T. Sah, Rec. trav. chim. 59, 461 (1940).
9P,P.T. Sah and W. Briill, Ber. 73, 1430 (1940).
10}, A. Poulsson, J. Soc. Chem. Ind. (London) 60, 123 (1941).

400 VITAMIN K GROUP

Because vitamins K; and K, and also 2-methylnaphthoquinone are fat-
soluble, it is necessary that bile acids be present for their proper absorption
from the intestinal tract. Water-soluble forms of vitamin K can be ab-
sorbed as such and are more effective in cases where the flow of bile is im-
paired; furthermore they are useful for intravenous injection. Several
water-soluble forms have been introduced commercially: 2-methyl-1 ,4-
naphthohydroquinone-3-sodium sulfonate," 4-amino-2-methyl-1-naphthol
hydrochloride,2-"“ and sodium 2-methyl-1,4-naphthoquinone dphosi-
phate.”

IV. Biochemical Systems
H. J. ALMQUIST

Very little is known concerning the mechanism by which vitamin K-
active substances promote the formation of prothrombin. Vitamin K is not
found to any significant extent in blood. Large quantities of the prothrom-
bin fraction of normal chicken blood fed to small vitamin K-deficient chicks
failed to effect a cure.! Dried beef blood fed at 10% of the diet to defi-
cient chicks also showed no activity.? The vitamin in contact with pro-
thrombin-deficient chick blood 7n vitro does not accelerate clotting.’ * Even
the more water-soluble forms such as the methylnaphthoquinone, phthiocol,
and the diphosphoric acid ester have no direct effect on deficient chick
blood.2 When vitamin K, emulsion is given intravenously to deficient chicks,
the prothrombin does not rise immediately but requires at least 5 hours
to reach a normal level.! It is unlikely that the vitamin occurs in blood
except in transport. Therefore, the vitamin does not seem to act as a pros-
thetic group in combination with any blood elements.

That the principal site of prothrombin formation is the liver is indicated

1M. B. Moore, J. Am. Chem. Soc. 61, 2049 (1941).

122. A. Doisy, D. W. MacCorquodale, S. A. Thayer, 8S. B. Burkley, and R. W.
McKee, Science 90, 407 (1939).

13D. Richert, S. A. Thayer, R. W. McKee, S. B. Binkley, and E. A. Doisy, Proc.
Soc. Expil. Biol. Med. 44, 601 (1940).

14H. J. Almquist and A. A. Klose, Proc. Soc. Exptl. Biol. Med. 45, 55 (1940).

15 7, F. Fieser and E. M. Frey, J. Am. Chem. Soc. 62, 228 (1940).

16R. H. K. Foster, J. Lee, and U. V. Solmssen, J. Am. Chem. Soc. 62, 453 (1940).

17§. Ansbacher, E. Fernholz, and M. A. Dolliver, Proc. Soc. Exptl. Biol. Med. 48,
652 (1940).

1H. Dam, J. Glavind, L. Lewis, and E. Tage-Hansen, Skand. Arch. Physiol. 79, 121
(1938).

2H. J. Almquist, Physiol. Revs. 21, 194 (1941).

3H. Dam, F. Schgnheyder, and E. Tage-Hansen, Biochem. J. 30, 1075 (1936).

IV. BIOCHEMICAL SYSTEMS 401

by much evidence. Andrus et al.4 and Warren and Rhoads? found that re-
moval of the liver from dogs was followed by a blood prothrombin drop
which could not be prevented by administration of vitamin K and bile
salts. Warner® reported that removal of two-thirds of the liver from rats
caused a decrease in blood prothrombin. Liver damage from such toxic
agents as chloroform also impairs prothrombin formation.’ ® Traumatic
injury to the liver can cause a marked loss of blood prothrombin.® Pro-
thrombin loss from these causes is not readily alleviated by vitamin K.
For other examples of liver damage and prothrombin deficiency, the reader
is referred to a review by Ferguson.!°

In marked contrast to the chick and other species, the dog is reported
to utilize methylnaphthoquinone less efficiently than K,.!! Quick and Col-
lentine™ have proposed that vitamin K, is a prosthetic group on an enzyme
in liver which takes part in the formation of prothrombin. This subject
is discussed more fully in Section VIII of this chapter.

The suggestion has been advanced that vitamin K-active quinones act
through their oxidation-reduction powers.’ The redox potential values
found were 328 for vitamin K,, 458 for 2-methyl-1 ,4-naphthoquinone,
and 256 for phthiocol, which are in same general order as the potencies
of these compounds. Trenner and Bacher™ have also reported some stand-
ard oxidation-reduction potentials which are of the same order for the
compounds mentioned. above. Within the same range are found some naph-
thoquinones which possess practically no vitamin K activity, so it is evi-
dent that potency is not a simple matter of redox activity, yet this is not
excluded as a factor in potency.

Nothing has yet been reported to link vitamin K and its homologs in
moderate doses to any body component other than prothrombin. Defi-
ciency of the vitamin has no effect on growth until animals become ill be-
cause of hemorrhages. Lesions or erosions of the chick gizzard lining were
observed": 1° but did not prove to be a characteristic symptom of vitamin

4W.D. Andrus, J. W. Lord, Jr., and R. A. Moore, Surgery 6, 899 (1939).

5 R. Warren and J. E. Rhoads, Am. J. Med. Sci. 198, 193 (1939).

6K. D. Warner, J. Exptl. Med. 68, 831 (1938).

7K. M. Brinkhous and E. D. Warner, Proc. Soc. Exptl. Biol. Med. 44, 609 (1940).

8 J. L. Bollman, H. R. Butt, and A. M. Snell, J. Am. Med. Assoc. 115, 1087 (1940).

9 J. W. Lord, Surgery 6, 896 (1939).

10 J. H. Ferguson, Ann. Rev. Physiol. 8, 231 (1946).

1A, J. Quick and G. E. Collentine, Am. J. Physiol. 164, 716 (1951); J. Lab. Clin. Med.
36, 976 (1950).

2 FE. L. MeCawley and C. Gurchot, Univ. Calif. (Berkeley) Publs. Pharmacol. 1, 325
(1940).

13.N.R.Trenner and F. A. Bacher, J. Biol. Chem. 187, 745 (1941).

4H. J. Almquist and E. L. R. Stokstad, J. Biol. Chem. 111, 105 (1935).

15H. Dam and F. Schgnheyder, Biochem. J. 28, 1355 (1934).

402 VITAMIN K GROUP

K deficiency. Although crude sources of the vitamin would protect against
gizzard erosion, more purified fractions did not, nor did any of the known
vitamins. The existence of a separate unidentified anti-gizzard erosion factor
was shown.!® Further evidence on the non-identity of these factors was
found when it was observed that pure bile acids, especially cholic acid,
in the diet would protect the gizzard lining irrespective of large or no in-
take of vitamin K or prolonged clotting time.’ #8 Arachidonic acid has
been reported as the gizzard erosion preventive agent in fats.

Hypoprothrombinemia induced in chicks by vitamin K deficiency or by
adding dicoumarol to a practical diet does not furnish any histological
evidence of liver damage.?°

V. Estimation
H. J. ALMQUIST
A. BIOLOGICAL ESTIMATION

The biological assay for vitamin K must be viewed in a somewhat differ-
ent light from that of certain other vitamins. General dietary deficiency
in humans is practically unknown, and only in a few pathological or emer-
gency conditions and in pregnancy is administration of the vitamin indi-
cated. Synthesis of the vitamin by microorganisms in the intestinal tract
is usually sufficient to supply the needs of most animals, except fowls. Ample
supplies of cheap synthetic forms and substitutes render dependence on
natural sources of minor importance.

The assay methods therefore, are still primarily for research and may
differ greatly, depending upon the nature of the investigation. If intended
for the study of comparative potencies of vitamin K-active substances, a
relatively simple and convenient assay with the chick may suffice. On the
other hand, a study of the metabolism of vitamin K or of some substance
similar or synergistic or antagonistic to vitamin K may require a more
specific measurement of blood components and excretion products. For
general purposes it would seem advisable to employ an assay method which
is as simple as possible and which alters the blood from its natural compo-
sition to the least extent compatible with a degree of accuracy. No assay

16H. J. Almquist and E. L. R. Stokstad, Nature 187, 581 (1936).

17H. J. Almquist, Science 87, 538 (1938).

18 H. J. Almquist and E. Mecchi, J. Biol. Chem. 126, 407 (1938); Proc. Soc. Exptl.
Biol. Med 46, 168 (1941).

19H, Dam, Acla Physiol. Scand. 12, 189 (1946).

20. V. M. Emmel and H. Dam, Proc. Soc. Exptl. Biol. Med. 56, 11 (1944).

V. ESTIMATION 403

method as yet devised is free from some theoretical objections; nevertheless,
there are several good procedures which may yield results as consistent and
reproducible as can generally be expected from a bioassay with animals.

The early development of knowledge on vitamin K was accompanied
by the usual mistakes and errors in bioassays which have not been uncom-
mon with other vitamins. More specifically, the sources of error included
the following.

1. The test animals were so few that individual variability was a large
factor in the accuracy of the data.

2. The assay time was too short (in some cases only a few hours), there-
by placing more emphasis upon speed of absorption than upon intrinsic
potency in an equilibrated animal.

3. The dose level was too high or too low, placing the response outside
the most sensitive range.

4. The vitamin was lost or destroyed during assay, as by volatility or
oxidation when dispersed over the large surface of a ground diet, or the
sharply increased sensitivity to light in purified preparations of the vita-
min.!

5. The test animals were exposed to the dust of green plants containing
vitamin K or other antihemorrhagic materials (cases are known in which
alfalfa dust and the manufacture of 2-methyl-1 ,4-naphthoquinone nearby
were sufficient to vitiate an assay).

6. Owing to bacterial contamination and synthesis of the vitamin, some
ingredient of the diet may unexpectedly contain vitamin K. Also, cop-
_ rophagy may be a significant source of the vitamin.

1. ANIMALS

The young chick is the most readily depleted animal and has been used
almost exclusively. The chick presents a further advantage in that the
platelets do not furnish thromboplastin which might affect the clotting
power of the blood sample.?

Chicks from good stock fed practical breeder rations are most desirable.
Although a carryover of the vitamin from the parent hen to the chick may
be demonstrated,’: ** this does not seriously interfere with depletion of the
chick. Rapid chick growth is most desirable since it leads to earlier deple-
tion of the chick in respect to vitamin K.

Attempts to use the rat for the study of vitamin K have not been suc-

1D. W. MacCorquodale, S. B. Binkley, R. W. McKee, S. A. Thayer, and E. A.
Doisy, Proc. Soc. Exptl. Biol. Med. 40, 482 (1939).

2H. Dam, Acta Physiol. Scand. 12, 189 (1946).

3H. J. Almquist and E. L. R. Stokstad, J. Nutrition 12, 329 (1936).

3a W. W. Cravens, 8S. B. Randle, C. A. Elvehjem, and J. G. Halpin, Poultry Sci. 20,
313 (1941).

404. VITAMIN K GROUP

cessful quantitatively except when the bile is diverted away from the gut
by ligation of the bile duct, thus interfering with absorption.*: * Bile-
fistula dogs have been used in studies of vitamin K and blood prothrom-
bin level.* Dogs in which the bile was diverted to the kidneys also became
vitamin K deficient and were used for estimating the requirement of this
species.’ Individually standardized rabbits were employed in the investiga-
tions of the hemorrhagic sweet-clover disease which also produces prothrom-
bin variations in the blood.®

2. Diet

The formulation of a successful diet involves the problem of avoiding
ingredients which may contain vitamin K, at the same time providing all
other nutrients for substantially normal maintenance and growth of the
animal during the period of the assay. For the chick assay, the basal test
ration® given in Table I, or minor variations of it, has been widely used.

The ration is principally ground polished rice, chosen because this type
of cereal is free of vitamin K or any extraneous material that may contain
the vitamin. As a protein supplement to the polished rice, the most satis-
factory single product is sardine fish meal that has been extracted contin-
uously with ethyl ether for 24 hours or more to remove small amounts of
vitamin K. The fish meal also provides most of the mineral supplements in
the form of bone and trace elements. Instead of the 17.5 parts of fish meal
a mixture of 12.0 parts of ether-extracted casein, 2.5 parts of ether-ex-
tracted gelatin, and 3.0 parts of bone ash may be used.

Dried brewer’s yeast is a convenient source of the water-soluble vitamins
required by the chicks. The yeast should be extracted with ethyl ether to
remove possible traces of vitamin K. The full amount specified is needed
to supplement the ration adequately in the well-known and less well-known
vitamins and dietary factors of the water-soluble class required by chicks.

Cod liver oil U.S.P. is recommended as a source of vitamins A and D.
Other types of animal feeding oils have been found to contain significant
amounts of vitamin K and should not be used in vitamin K assay. Puri-
fied B-carotene and synthetic vitamins A and D may also be used in an
oil such as refined cottonseed oil with an antioxidant, such as hydroquinone.

4J.E. Flynn and E. D. Warner, Proc. Soc. Exptl. Biol. Med. 43, 190 (1940).

5 J. D. Greaves and C. L. A. Schmidt, Proc. Soc. Exptl. Biol. Med. 37, 48 (1937).

6H. P. Smith, E. D. Warner, K. M. Brinkhous, and W. H. Seegers, J. Hxptl. Med.
67, 911 (1938).

7A. J. Quick and G. E. Collentine, Am. J. Physiol. 164, 716 (1951); J. Lab. Clin. Med.
36, 976 (1950).

8 R.S8. Overman, M. A. Stahmann, W. R. Sullivan, C. F. Huebner, H. A. Campbell,
and K. P. Link, J. Biol. Chem. 142, 941 (1942).

9H. J. Almquist and A. A. Klose, Biochem. J. 33, 1055 (1939).

V. ESTIMATION 405

The ration is probably deficient in vitamin E. However, the storage of
this vitamin in chicks from hens fed practical diets is sufficient to carry
the chicks over the short period required for a vitamin K assay. Addition
of vitamin E to the ration is not advisable, since some of the oxidation
products of vitamin E that may be formed possess antihemorrhagic activity.

A diet has been proposed in which the grain portion is heated for 1 week
at 120°.1° Chicks grow very poorly on such a diet, and it is doubtful if it
possesses any real advantage over the one described above. Multiple de-
ficiencies should be avoided in biological assays. Another proposed diet!
is extremely deficient in calcium for the chick, and it is difficult to under-
stand how chicks could survive and grow on the diet as described.

TABLE I
BasaL RatTION FOR CHICK VITAMIN K Assay

Ingredient Per cent
Sardine meal, ether extracted Wieo
Dried brewer’s yeast, ether extracted eo
Ground polished rice (2.5
Cod liver oil 1.0
Caleium carbonate 0.5
Salt, common (contains 0.5% Mn in form of the sulfate or carbonate) 1.0

3. ADMINISTRATION OF SUPPLEMENTS

Considerable influence on potency by the carrier of the vitamin supple-
ment has been reported. Dann” found 2-methyl-1 ,4-naphthoquinone to be
three times as potent when given orally in oil as compared to water, whereas
Ansbacher et al. reported the quinone to be twice as potent in water as in
oil. Almquist and Klose“ obtained the same potency for the quinone in
water and ethyl laurate solutions. Vitamin K, manifested different po-
tencies in reference to 2-methyl-1 ,4-naphthoquinone, depending upon the
volume of oil carrier in a very short (6-hour) assay.!? Whatever solvent is
chosen, it should be the same and in the same volume per dose for the as-
sayed substances as for the standard employed, so far as possible.

Methods for parenteral administration have also been used. Phthiocol

10S. Ansbacher, Proc. Soc. Exptl. Biol. Med. 44, 248 (1940).

1 A.J. Quick and M. Stefanini, J. Biol. Chem. 175, 945 (1948).

2 FP. Dann, Proc. Soc. Exptl. Biol. Med. 42, 663 (1939).

13 §. Ansbacher, E. Fernholz, and M. A. Dolliver, J. Am. Chem. Soc. 62, 155 (1940);
Proc. Soc. Exptl. Biol. Med. 48, 652 (1940).

14H. J. Almquist and A. A. Klose, Proc. Soc. Exptl. Biol. Med. 45, 55 (1940).

15S, A. Thayer, R. W. McKee, 8. B. Binkley, and E. A. Doisy, Proc. Soc. Expil.
Biol. Med. 44, 585 (1940).

406 VITAMIN K GROUP

showed the same potency whether given orally, intramuscularly, or intra-
venously.!® 2-Methyl-1 ,4-naphthoquinone appeared somewhat less potent
by the intravenous route.’?’ 7 An emulsion of vitamin K, was effective by
intramuscular but not by subcutaneous administration."

For most purposes the oral dosing for 4 to 5 days should lead to a satis-
factory assay. !° If a quantitative assay is desired, a definite level of in-
take should be maintained in the test animal for a sufficient period of time
so that potency-modifying factors such as solubility, absorption, and rate of
metabolism have been able to approach a balance or plateau of influence
on activity. A period of 3 to 4 days is required for the depleted chick to
become adjusted to intake levels of the vitamin in an assay range. If the
work of setting up an assay has been carried out properly, the extra 3 or
4 days is only a small addition to the task and may lend greater precision.
From the emergency standpoint there is perhaps some advantage in a
very short assay which will evaluate a compound as much from its speed
of action as its intrinsic potency. For example, the effective dose of vitamin
K, is greater for a 6-hour assay than for 18 hours.” ?! These are matters
to be adjusted to the particular problem and the purpose of the antihemor-
rhagic substances.

Dam and Sgndergaard have recently shown that in promoting prothrom-
bin restoration in depleted chicks vitamin K, is more rapidly effective than
menadione or the sodium salt of the corresponding hydroquinone diphos-
phoric acid ester.”!#

4. MEASUREMENT OF SUPPLEMENT EFFECT

The earliest quantitative measurement of supplement effect was the
simple clotting time of whole blood. If a sufficiently large number of chicks
is included in each group, the average blood-clotting time for the group has
a fairly close relation to the activity of the supplement, like that of the
prothrombin time as illustrated in Fig. 4.2? Some objections to simple whole
blood-clotting time may be mentioned.

1. Blood-clotting time may remain nearly normal when the blood pro-

16H. J. Almquist and A. A. Klose, J. Am. Chem. Soc. 61, 1923 (1939).

17T),. Richert, S. A. Thayer, R. W. McKee, S. B. Binkley, and E. A. Doisy, Proc.
Soc. Exptl. Biol. Med. 44, 601 (1940).

18 H. Dam, J. Glavind, L. Lewis, and E. Tage-Hansen, Skand. Arch. Physiol. 79, 121
(1938).

19H. J. Almquist and A. A. Klose, J. Biol. Chem. 180, 787 (1939).

20}, Fernholz, S. Ansbacher, and H. B. MacPhillamy, J. Am. Chem. Soc. 62, 430
(1940).

21 7,. F. Fieser, M. Tishler, and W. L. Sampson, J. Biol. Chem. 187, 659 (1941).

21a AH, Dam and E. Sgndergaard, Hxperientia 9, 26 (1953).

2H. J. Almquist, E. Mecchi, and A. A. Klose, Biochem. J. 32, 1897 (1938).

a

V. ESTIMATION 407

thrombin level is less than half the normal, and it is probably less sensitive
to moderate reductions in prothrombin.

2. Blood-clotting time may be influenced by tissue juice or extract. It is
nearly impossible to avoid contamination with tissue juice completely by
any method of withdrawing blood samples that is not almost hopelessly
laborious when applied to a large number of chicks.

ce
ce)
LP
a

Ly

A

1.0

0.8

SSE SaAN

Reciprocal prothrombin time, x 10

Bees:
aS

0.2 0.6 1.0 1.4 1.8
Log vitamin K dosage x 10
Fic. 4. The relation of the reciprocal prothrombin time to the logarithm of the
vitamin K intake. Numbers have been multiplied by 10 for convenience in plotting.

Curves 1 and 2 from data by Almquist and Klose.’ Curve 3 adapted from data by
Stamler et al.*8 Curves 4, 5, 6, and 7 from data by Quick and Stefanini."

3. Blood-clotting time may actually decrease in certain chicks after
severe loss of blood. For this reason it is not advisable to test all chicks for
blood-clotting time before administering supplements.

4. Blood-clotting time is measured in periods from 2 to 30 minutes or
longer. It is often impossible to obtain any distinct values from certain
chicks with prolonged clotting time.

To avoid the above objections, Almquist and Klose® employed for vita-
min K assay the ‘‘prothrombin-time” method of Quick,!!: 4 which depends

23 R. T. Tidrick, F. T. Joyce, and H. P. Smith, Proc. Soc. Exptl. Biol. Med. 42, 853
1939).
4 _ J. Grids Am. J. Physiol. 118, 260 (1937).

408 VITAMIN K GROUP

more specifically on the blood prothrombin level and, in turn, the effect
of vitamin K supplements on the prothrombin level. The improved method,
while requiring more preliminary preparation, has several advantages over
simple blood-clotting time, namely:

1. A larger sample of blood is used and is, therefore, more likely to be
an accurate sample from the chick.

2. Measurements are made in seconds rather than minutes, and the
gathering of the final data is expedited. Determinations can be made in
duplicate.

3. Conveniently measurable values can be obtained from every chick.

4. The variability of the results within a test group is much lower than
that of the simple blood-clotting times”: 7° (Table IT).

TABLE II

MEANS, STANDARD ERRORS, AND COEFFICIENTS OF VARIABILITY OF SIMPLE
BuLoop-CLoTTING TIMES, AND WHOLE BLOoD-PROTHROMBIN

TIMES
Reference Blood-clotting time Whole blood-prothrombin time
standard of
vitamin K per Coefficient Mean and Coefficient
kilogram of diet, No. of Mean and standard Co) standard 0

mg. chicks error, min. variability error, sec. variability

3 15 10.07 + 2.31 61.1 ol.2 = 1.9 e/a

6 15 3.42 + 0.55 60.2 37.3 + 1.0 9.6

12 15 Wy Se Uear/ 72.1 30.5 + 0.5 6.1

Blood samples may be taken simply by cutting off the head of the chick.®
Samples have also been taken from a carotid artery,” or from a jugular
vein,””: 8 or from a wing vein." 2 Prothrombin has been determined by an
elaborate two-stage method,?’: *° or by a method in which the ratio of the
strength of a clotting agent (thromboplastin) required to clot the plasma
in a specified time to the strength required to clot normal plasma?® is de-
termined. The reader should consult the original papers for details of these
methods.

5. CoMPUTATION OF RESULTS

It was found that over the sensitive assay range a plot of the reciprocal
mean prothrombin time against the logarithm of the vitamin K dosage

25 H. J. Almquist, Physiol. Revs. 21, 194 (1941).

26H, Dam and J. Glavind, Biochem. J. 32, 485 (1938).

27K, W. Stamler, R. T. Tidrick, and E. D. Warner, J. Nutrition 26, 95 (1943).

28H}. D. Warner, J. Exptl. Med. 68, 831 (1938).

29H. J. Almquist and E. L. R. Stokstad, J. Nutrition 14, 235 (1937).

30 H. P. Smith, E. D. Warner, and K. M. Brinkhous, J. Exptl. Med. 66, 801 (1937).

V. ESTIMATION 409

yields practically a straight line.’ This relation, although possibly only
empirical, proves to be applicable to data from different laboratories, using
different procedures for estimating prothrombin and different methods of
administering supplements (Fig. 4).

Stamler e¢ al.,27 employing this relation to compare calculated prothrom-
bin levels with observed levels, have stated that the agreement is not “‘very
exact.’’? Very exact results are rarely obtained in biological assays; never-
theless the data plotted by the direct method of Almquist and Klose® ap-
pear to be quite consistent (Fig. 4, curve 3). Quick and Stefanini” have
objected to the use of an extract of chicken muscle instead of chicken brain
as a clotting agent for prothrombin determination. Whether or not this
criticism is important, the assay method operates well with either prepara-
tion (Fig. 4, curves 1 and 2). Of course the linear relation no longer holds
after an optimal prothrombin level or time has been reached.

In any assay it is desirable to establish some such line by means of at
least two groups fed different levels of a standard preparation, such as
2-methyl-1 ,4-naphthoquinone. It is then possible to interpolate and express
the potency of an assayed supplement in terms of the reference standard.*!
Reproducibility of results by this method has been adequately demon-
strated.

Another method is to measure whole blood-clotting time to the nearest
minute and then to compare the percentages of the chicks in which the
clotting time is reduced to 10 minutes or less; or the quantity of material
which will cure 50% of the group by the above criteria may be estimated
from several dose levels.** This is called the CD5o and is compared with the
CD50 of some standard substance which was employed in the same assay.
Improved methods have been suggested from time to time. The reader is
referred to papers by Almquist,*! Quick and Stefanini," Stamler eé al.,””
and Dam et al.*4

6. STANDARDS OF POTENCY

It has been suggested** that the highly potent foioenad: 2-methyl-1 ,4-
naphthoquinone, can be employed as a standard of activity and chai 1
unit be defined as the antihemorrhagic activity of 1 y of this compound.
The name menadione has been adopted as a non-proprietary term for this

31H. J. Almquist, Biol. Symposia 12, 508 (1947).

8 §. Ansbacher, J. Nutrition 17, 303 (1939).

33§. A. Thayer, D. W. MacCorquodale, R. W. McKee, and E. A. Doisy, J. Bi-?
Chem. 123, cxx (1938).

34H, Dam, I. Kruse, and E. Sgndergaard, cited by H. Dam, Ann. Rev. Biochem. 20,
265 (1951).

35S. A. Thayer, S. B. Binkley, D. W. MacCorquodale, E. A. Doisy, A. D. Emmett,
R. A. Brown, and O. D. Bird, J. Am. Chem. Soc. 61, 2563 (1939).

410 VITAMIN K GROUP

substance by the American Medical Association.**® Although one-half as
potent as the methylnaphthoquinone, 2-methyl-1 ,4-naphthohydroquinone
diacetate might serve as a better standard.*” Both of these compounds are
cheaply and easily prepared in a high state of purity, which can be tested
conveniently by means of melting point. The melting point of methylnaph-
thoquinone should be 105 to 106°, that of the diacetate 112 to 113°. The di-
acetate has the advantage of greater stability when mixed in the diet;
this, however, is of little or no importance for oral administration. For
convenience the methylnaphthoquinone 1-y unit will be used for expressing
comparative activities, with the caution that this unit may not represent
any fixed proportion of activity to weight units of vitamins K, or K: (Table
NAO):

TABLE III

ACTIVITIES OF CERTAIN ANTIHEMORRHAGIC ComMpouNDS BasED ON CuiIckK 5-Day
ASSAYS AND ExprRESSED IN 2-Mrtuyt-l,4-NAPHTHOQUINONE UNITS PER

MILLIGRAM
Compound Units per milligram
2-Methyl-1,4-naphthoquinone (menadione) 1000
2-Methyl-1,4-naphthohydroquinone diacetate 450
2-Methyl-4-amino-1-naphthol hydrochloride 500

2-Methyl-1,4-naphthohydroquinone diphosphoric acid ester (tetraso- 500
dium salt, hexahydrate)

2,3-Dimethyl-1,4-naphthoquinone 25
2-Methy1-3-phytyl-1,4-naphthoquinone (vitamin K,) 500
2-Methyl-3-difarnesyl-1,4-naphthoquinone (vitamin Ke) 400
2-Methyl1-3-phytyl-1,4-naphthohydroquinone diacetate 170

During the phases of isolation, purification, and synthesis the different
laboratories employed different assay methods and different standards or
units of potency which served their temporary purposes. These methods
and standards or units have been reviewed by Ansbacher.*®

7. COMPARISON OF ASSAYS BY DIFFERENT METHODS

Several of the present methods when carefully adhered to will yield
equivalent results. Repeated assays of 2-methyl-1 ,4-naphthohydroquinone
diacetate have all indicated that the potency of this compound is close to
one-half that of the methylnaphthoquinone.*! An average of relative values
obtained in other assays, as reviewed by Dam,” leads to the same conclusion.

36 Council on Pharmacy and Chemistry, J. Am. Med. Assoc. 116, 1054 (1941).

37 PD. T. Ewing, J. M. Vandenbelt, and O. Kamm, J. Biol. Chem. 181, 345 (1939).
38 §. Ansbacher, J. Nutrition 21, 1 (1941).

V. ESTIMATION 411

Almquist and Klose, assaying the compound 2-methyl-1 ,4-naphtho-
hydroquinone diphosphoric acid ester in comparison to 2-methyl-1 ,4-naph-
thoquinone by 4-day oral feeding to depleted chicks and comparison of
average prothrombin time 24 hours from the last dose, found the phosphoric
ester to be 1.5 times as potent as the quinone on a molar basis (one-half
as potent on a weight basis.) Assay of the identical preparations was made
by Lee et al.*® by subcutaneous administration, measurement of whole
blood-clotting time 18 hours from dosage, and expression of results as the
single dose required to bring the blood-clotting time of 50% of the chicks
to 10 minutes or less. The phosphoric ester was found to be 50 % more po-
tent than the quinone on a molar basis in close agreement with the above
results.

The potency of vitamin K,; was found to be approximately one-third
that of methylnaphthoquinone (Almquist*!). This ratio was also found in
an 18-hour curative assay by Fieser et al.?! Emmett et al.,*° by the method
of Thayer et al.,*° obtained 450 methylnaphthoquinone units per milligram.
Assays by the Dam-Glavind technique, as reported by Dam,’ indicate an
average potency approximately 39 % of that of the methylnaphthoquinone
on a weight basis.

Quick and Stefanini! have provided further data on the relative po-
tencies of these compounds in a 12-day preventive assay with chicks. These
data, plotted by the method of Almquist and Klose,°® are represented by
curves 4, 5, 6, and 7 in Fig. 4. Curve 4, obtained with methylnaphthoqui-
none dosages, has a slope of 1.35. Curve 5, obtained with vitamin Ky, has
a slope of 1.00. These curves converge at lower dose levels. The difference
between the curves at any prothrombin value or reciprocal prothrombin
time is the logarithm of the potency ratio of the two compounds. This ratio
is not constant. Curve 6 represents results from methylnaphthoquinone
dosage applied to chicks also receiving dicoumarol in their diets (p. 405).
As would be expected, the curve is displaced toward higher dose levels of
vitamin K; the slope of this curve turns out to be 1.35, however, the same
as in the absence of dicoumarol (curve 4). Curve 7 represents data from simi-
larly dicoumarol-poisoned chicks which received vitamin K;. This curve is
also displaced toward higher vitamin K levels, but it has the same slope
as the vitamin K, curve (5) in the absence of dicoumarol. Such agreement
could hardly be accidental. The remarkable consistency of these data is
brought out clearly by the graphical method of expression.

The fact becomes evident that there is no constant ratio of activity for
methylnaphthoquinone and vitamin K,. This ratio varies over a range
39 J. Lee, U. V. Solmssen, A. Steyermark, and R. H. K. Foster, Proc. Soc. Exptl.

Biol. Med. 45, 407 (1940).
40 A.D. Emmett, R. A. Brown, and O. Kamm, J. Biol. Chem. 132, 467 (1940).

412 VITAMIN K GROUP

from 1, or less, to as high as 2, approximately. At the lower levels, on a
molar basis, vitamin K;, is clearly the more efficient form of the vitamin.
It is probable that these compounds are so dissimilar that they cannot be
evaluated in terms of each other except at stated dose levels. Therefore,
they cannot be used interchangeably for assay standards.

These experimental observations are in agreement with relations that
may be derived on the basis of certain assumptions.*! If the relation of
vitamin K to prothrombin formation is expressed as follows:

kee a Kee (1)

where K = any form of vitamin K.
A = proenzyme.
KA = enzyme which forms prothrombin.
C = constant.
E = efficiency of formation of prothrombin by KA.
P = prothrombin — KA xX ii:

Then
UE pe OKRA ee ee (2)
Gite KA ” B
which may be written
d log Kk H P
dt Al aC Ou

or integrated and simplified to eq. 3 as a practical expression of the rela-
tion of these factors, over a given time interval.

5 eae
AKC ial

It is recognized that within the assay range P is for practical purposes
equal to a constant times the reciprocal of the prothrombin-clotting time, 7’.

Lome (3)

Ge
Ee 4
P = (4)
Hence
logic K = G ae (5)

ABC »6B GPR 2388 EL

It appears that A, which is similar to the ‘‘apoenzyme”’ recently pro-
posed by Quick and Collentine,’ is relatively constant in respect to the above

41H. J. Almquist, Arch. Biochem. and Biophys. 35, 464 (1952).

V. ESTIMATION 413

variables. At any value of P which is maintained by two different forms
of vitamin K,
log Ke Cy X Be
lop kG “COC

As previously mentioned, in the data on the prothrombin times and
levels in chicks maintained by two forms of vitamin K, namely 2-methyl-
1 ,4-naphthoquinone (menadione) and vitamin Kj," the prothrombin values
tend to converge at very low vitamin K dosage and diverge by a constant
ratio toward higher dosages until, of course, the physiological limit of P is
approached (Fig. 4). This is in agreement with eq. 6. A further implication
of this equation is that the potency ratio K,/ Ky, with respect to maintenance
of different P levels is not a constant. This conclusion derived from the
stated assumptions agrees with the experimental facts. The agreement does
not, of course, prove that the assumptions are correct; however, it would
appear that they are concordant with the facts.

A further analysis of the dicoumarol data brings out some indications
of interest. The feeding of a constant amount of dicoumarol does not change
the slope of the regression lines of prothrombin on log vitamin Kx. This
means that the terms in eq. 3, such as A, C, and F, the net effect of which
is that of a constant, are unaffected by the dicoumarol. From this observa-
tion one may conclude that the effect of the dicoumarol is only upon the
vitamin K. Furthermore, the effect cannot be merely that of a quantitative
inactivation of vitamin K but must be applied uniformly to the entire
quota of K present. The effect is, evidently, that of a competitive displace-
ment, or some form of reversible inhibition, or reduction of activity, which
is applied prior to the reaction system as expressed in eq. 1. In dog blood
the effects of both avitaminosis K and of dicoumarol are due to a decrease
of prothrombin.” For another discussion of these relations the reader should
consult ref. 48.

The relative potency of vitamin K, with respect to K», was found to be
1.25 (Almquist and Klose"), 1.5 (Thayer et al.“4), 1.4 (Dam), and between
1 and 1.5 (Tishler and Sampson‘).

B. CHEMICAL AND PHYSICAL ESTIMATION

The absorption spectra of vitamins K; and K, have been studied in de-
tail by several investigators. A most careful measurement of the ultraviolet

= Constant (6)

4 A.J. Quick, C. V. Hussey, and G. E. Collentine, Proc. Soc. Exptl. Biol. Med. 79,
131 (1952).

43 G. E. Collentine and A. J. Quick, Am. J. Med. Sci. 222, 7 (1951).

448. A. Thayer, R. W. McKee, S. B. Binkley, D. W. MacCorquodale, and E. A.
Doisy, Proc. Soc. Exptl. Biol. Med. 41, 194 (1939).

45M. Tishler and W. L. Sampson, Proc. Soc. Exptl. Biol. Med. 68, 136 (1948).

414 VITAMIN K GROUP

absorption has been reported** (Fig. 3). As already mentioned, the absorp-
tion of these vitamins deteriorates rapidly on exposure to ultraviolet light.
Vitamin K can also be determined polarographically.‘”: 4? These physical
methods have been used only for relatively pure concentrates. Oxidation-
reduction methods have also been described,‘*: °° but they seem to be sub-
ject to much interference from other non-vitamin K substances.

Numerous color reactions would be expected from a compound of the
naphthoquinone structure. Vitamin K, gives a strong transient blue color
reaction with sodium ethylate, which fades to a more permanent red-
brown stage. This color reaction may apparently be expected only from
vitamins K, and K., which have a double bond between the - and y-carbon
atoms in the side chain, or from similar naphthoquinone derivatives.*':
The more stable red-brown end stage of the reaction has a roughly quanti-
tative relation to the activities of concentrates. It was stated that the
end-stage color was due to phthiocol.*! However, the principal pigment pro-
duced in this reaction possessed an analysis and molecular weight indicating
that it was a derivative of vitamin K,.** A colorimetric assay based upon
the reaction of 2,3-substituted 1 ,4-naphthoquinones with sodium diethyl
dithiocarbamate has been reported.®*°

Colorimetric methods for 2-methyl-1 ,4-naphthoquinone have excited in-
terest because of the wide use of this compound in vitamin K-active phar-
maceutical preparations, There have been used such color developers as
dichlorophenolindophenol,** dinitrophenylhydrazine,*”: ** and ethyl cyano-
acetate.5® A specific color test for 4-amino-2-methyl-1-naphthol was de-
scribed.®° The reaction with aniline has been used for determination of
methyl naphthoquinone in blood and tissues.®!

46.). T. Ewing, F. 8S. Tomkins, and O. Kamm, J. Biol. Chem. 147, 233 (1943).

47). B. Hershberg, J. K. Wolfe, and L. F. Fieser, J. Am. Chem. Soc. 62, 3516 (1940).

48 H. Onrust and B. Wéstman, Rec. trav. chim. 69, 1208 (1950).

49@. V. Scudi and R. P. Buhs, J. Biol. Chem. 141, 451 (1941); J. Biol. Chem. 143, 665
(1942).

50 N. R. Trenner and F. A. Bacher, J. Biol. Chem. 187, 745 (1941).

511, F. Fieser, W. P. Campbell, and E. M. Fry, J. Am. Chem. Soc. 61, 2206 (1939).

52 P. Karrer, Helv. Chim. Acta 22, 1146 (1939).

53H. J. Almquist and A. A. Klose, J. Am. Chem. Soc. 61, 1610 (1939).

54H. J. Almquist and A. A. Klose, J. Biol. Chem. 130, 791 (1939).

55 F. Irreverre and M. X. Sullivan, Science 94, 497 (1941).

56 W. Bosecke and W. Laves, Biochem. Z. 314, 285 (1943).

57 A. Novelli, Science 98, 358 (1941).

588 B. E. van Koetsveld, Rec. trav. chim. 69, 1217 (1950).

591). A. Richert, J. Biol. Chem. 154, 1 (1944).

60 A. R. Menotti, Ind. Eng. Chem. Anal. Ed. 14, 601 (1942).

61}, BE. Martinson and G. I. Meerovich, Biokhimiya 10, 258 (1945).

VI. OCCURRENCE IN NATURE 415

VI. Occurrence in Nature
H. J. ALMQUIST
A. IN PLANTS

Green leafy tissue is a rich natural source of the vitamin. One or two
per cent of commercially dehydrated alfalfa meal in the diet of the chick
meets normal requirements.':? The tops of carrots are a good source, but
the roots contain little or no vitamin.’ The vitamin is more abundant in
peas sprouted in the light than in those sprouted in the dark, and the inner
leaves of the cabbage have about one-fourth the activity of the outer
leaves.* Other sources of the vitamin are spinach, kale, cauliflower, nettle,
and chestnut leaves. The vitamin is present to some extent in tomato,
hempseed, seaweed,* and soybean oil.® Berries of the European mountain
ash are reported to be a good source.®

Dam, Glavind, and Gabrielsen’ have reviewed and extended earlier work
on the distribution of vitamin K in plants. Paits of plants which do not
normally form chlorophyll contain little vitamin K. Although conifers are
able to form chlorophyll in the dark, the amounts of chlorophyll and vita-
min K formed in the dark or hight remain approximately proportional.
However, the yellow spotted leaf areas of certain plants contained as much
vitamin K as did the green areas.

Leaves of various plants with markedly different natural green color
did not show a proportional amount of vitamin K activity by assay with
chicks. Neither was there any close relation to the amounts of carotene or
xanthophyll. Maize plants that were grown on an iron-deficient medium
showed marked deficiency of chlorophyll, xanthophyll, and carotene and
a reduction in vitamin K as compared to normal control plants. Natural
loss of chlorophyll, as in the fall yellowing of leaves, does not bring about
a corresponding change in vitamin K. Dam, Hjorth, and Kruse® have also
shown that the vitamin K in the press juice of spinach leaves does not de-
crease on standing overnight and nearly all the vitamin is present in the
chloroplasts. It seems evident, therefore, that any relation between the
concentrations of the chloroplast pigments and vitamin K in leaves is only
incidental to the general synthesis of all these substances in the leaves. It

1H. J. Almquist and E. L. R. Stokstad, J. Biol. Chem. 111, 105 (1935).

2 A.J. Quick, Am. J. Physiol. 118, 260 (1937).

3H. J. Almquist, Nature 140, 25 (1937).

4H. Dam and J. Glavind, Biochem. J. 32, 485 (1938).

5H. J. Almquist and E. L. R. Stokstad, J. Nutrition 14, 235 (1937).

6G. Y. Shinowara, J. C. DeLor, and J. W. Means, J. Lab. Clin. Med. 27, 897 (1942).
7H. Dam, J. Glavind, and E. K. Gabrielson, Acta Physiol. Scand. 18, 9 (1947).
8H. Dam, E. Hjorth, and I. Kruse, Physiologia Plantarum 1, 379 (1948).

416 VITAMIN K GROUP

should be recalled that vitamin K, and chlorophyll are both compounds of
phytol and may be dependent on the supply of this substance.

Vivino et al.* have made the interesting observation that honey contains
distinct antihemorrhagic activity equivalent to approximately 0.25 y of
2-methyl-1,4-naphthoquinone per gram as determined by the chick assay
procedure and interpretation method of Almquist.!°

B. IN MICROORGANISMS

Early observations on the development of antihemorrhagic activity in
foodstuffs during bacterial spoilage! and in the droppings of vitamin K-
deficient chicks," led to a further study of vitamin K production by micro-

TABLE IV

VITAMIN K AcTiviTIES OF PREPARATIONS OF CERTAIN MICROORGANISMS, EXPRESSED
IN 2-MrTHyt-1,4-NAPHTHOQUINONE UNITS PER GRAM®

Microorganism Units per gram
Bacillus cereus 115
Bacillus mycoides 155
Bacillus subtilis 190
Bacterium aerogenes 20
Bacterium flexneri 30
Bacterium proteus 75
Bacterium typhosum 15
Erythrobacillus prodigiosus 20
Escherichia coli 15
Mycobacterium tuberculosis 59
Sarcina lutea 100
Staphylococcus aureus 60

® Adapted from Almquist.4% For data on several other microorganisms, see this reference.

organisms. Various pure strains of bacteria were grown on vitamin K-
free media, killed by heat, dried, and assayed with the chick. Comparative
data on those which showed vitamin K activity are given in Table IV.
When receiving very little vitamin K in the diet, the cow is, nevertheless,
well supplied with the vitamin which is synthesized by microorganisms in
the rumen." In general, species of the mold, yeast, and fungus types were
inactive.

9K. E. Vivino, M. H. Haydak, L. 8. Palmer, and M. C. Tanquary, Proc. Soc. Expil.
Biol. Med. 53, 9 (1943).

10H. J. Almquist, Biol. Symposia 12, 508 (1947).

1H. J. Almquist and E. L. R. Stokstad, J. Nutrition 12, 329 (1936).

2H. J. Almquist, C. F. Pentler, and E. Mecchi, Proc. Soc. Exptl. Biol. Med. 38, 336
(1938).

13H. J. Almquist, Physiol. Revs. 21, 194 (1941).

471. W. McElroy and H. Goss, J. Nutrition 20, 527 (1940).

VI. OCCURRENCE IN NATURE ANG

Bacterial action on wet fish meal has been employed in the preparation
of vitamin K concentrates.! !® The vitamin Ky, isolated from putrified fish
meal'!® has also been obtained from a culture of Bacillus brevis.“ Experi-
mental animals for vitamin K studies must be prevented from obtaining
the vitamin from food in which bacterial action may have synthesized the
vitamin, and from coprophagy.

The remarkably high vitamin K content of certain microorganisms raised
the question of the role of antihemorrhagic quinones in their metabolism.
Woolley and McCarter found a microorganism, Johne’s bacillus, which
responded with increased growth when vitamin K, phthiocol, or methyl-
naphthoquinone was added to the synthetic medium. Using another strain
of this bacillus Glavind and Dam!’ were unable to demonstrate any growth
effect from methylnaphthoquinone or phthiocol. Hand?? investigated the
requirement of M ycobacteriwm tuberculosis for vitamin K and found evidence
of a growth-depressing action. Kimler* found bacteriostatic action against
this organism to be exhibited by methylnaphthohydroquinone diphosphate,
2-methyl-4-amino-1-naphthol and the bisulfite addition compound. The criti-
eal concentrations were 100, 1, and 2 y per milliliter, respectively. The
antihemorrhagic naphthoquinones can act as non-specific antibiotics in
some cases, as shown by Armstrong et al.” It is obvious from the above
facts that microbiological methods for estimating vitamin K are not very
feasible. For references to other recent reports on miscellaneous effects of
vitamin K on caries, tumors, and microorganisms, the reader is referred
to a review by Dam.”

Inhibition of bacterial action in the digestive tract by ingestion of sul-
fonamides causes a decrease in the synthesis of vitamin K and also of pro-
thrombin if the vitamin K intake is inadequate to meet the needs of the
animal. Black et al.*4 found that sulfaguanidine or sulfasuxidine added to a
synthetic vitamin K-free diet of rats caused a relatively rapid onset of

16 R.W.McKee,S. B. Binkley, D. W. MacCorquodale, 8. A. Thayer, and E. A. Doisy,
J. Am. Chem. Soc. 61, 1295 (1939).

16S. B. Binkley, D. W. MacCorquodale, 8S. A. Thayer, ee K. A. Doisy, J. Biol.
Chem. 180, 219 (1939).

17M. Tishler and W. L. Sampson, Proc. Soc. Exptl. Biol. Med. 68, 136 (1948).

18 PE). W. Woolley and J. R. McCarter, Proc. Soc. Exptl. Biol. Med. 45, 357 (1940).

19 J. Glavind and H. Dam, Physiol. Plantarum 1, 1 (1948).

20 C, N. Iland, Nature 161, 1010 (1948).

21 A. Kimler, J. Bacteriol. 60, 469 (1950).

2 W.D. Armstrong, W. W. Spink, and J. Kahnke, Proc. Soc. Exptl. Biol. Med. 53,
230 (1943).

23H. Dam, Ann. Rev. Biochem. 20, 265 (1951).

248. Black, R.S. Overman, C. A. Elvehjem, and K. P. Link, J. Biol. Chem. 145, 137
(1942).

418 VITAMIN K GROUP

prothrombin deficiency. This could be prevented by adding vitamin K to
the diet. Confirming observations have been reported.>: 6

C. NATURAL ANTAGONISTS

A hemorrhagic agent isolated from spoiled sweet clover hay has been
shown to be a dicoumarin by Link and coworkers.”” The compound isolated
is 3 ,3’-methylenebis(4-hydroxycoumarin). When given to various species
of animals this compound induces a decrease in prothrombin which is most
profound when associated with low vitamin K intake and can be reversed
by vitamin K administration.?’’ 8 The dicoumarin is easily degraded to
salicylic acid by chemical means, and this fact suggested that the acid
might be produced by the metabolism of the dicoumarin. Administration of
salicylic acid to rats, either in the vitamin K-low diet or intravenously,
caused a hypoprothrombinemia similar to that produced by the dicoumarin
or by vitamin K deficiency. The effect was reversed by vitamin K. Neither
the dicoumarin nor salicylic acid had any effect on the clotting power of
blood in vitro (Link et al.?®). This subject is more fully discussed in Section
VII B of this chapter (p. 482).

Woolley®® observed that a-tocopherolquinone administered to pregnant
mice caused hemorrhagic symptoms in the reproductive system and resorp-
tion. The action of the a-tocopherolquinone was not prevented by large
doses of vitamin E but was reversed by small doses of vitamin K. The struc-
tural similarity of a-tocopherolquinone and vitamin K, was pointed out as
an explanation of the antivitamin activity.

Excessive intake of vitamin A is known to cause a variety of toxic effects
among which is a hemorrhagic tendency and prolonged coagulation time of
blood. The clotting defect as produced in rats may be remedied by a gener-
ous intake of vitamin K.*! The condition has been produced by alcohol and
ester forms of vitamin A prepared from natural sources, but it has not yet
been reported as caused by synthetic vitamin A. It remains to be seen if
vitamin A itself or some associated substance is the cause of these hemor-
rhagic tendencies. In chicks, even large doses of vitamin A had no effect on
the prothrombin time although the chicks received only a minimal level of
vitamin K. The effects in rats, therefore, may have been due to interference
in the synthesis of vitamin K by intestinal microorganisms.”

25 A.D. Welch and L. D. Wright, J. Nutrition 25, 555 (19438).

26H. G. Day, K. G. Wakim, M. M. Krider, and E. E. O’Banion, J. Nutrition 26,
585 (1948).

27 H. A. Campbell and K. P. Link, J. Biol. Chem. 138, 21 (1941).

28 A.J. Quick and M. Stefanini, J. Biol. Chem. 175, 945 (1948).

29K. P. Link, R. S. Overman, W. R. Sullivan, C. F. Huebner, and L. D. Scheel,
J. Biol. Chem. 147, 463 (1943).

30 TP). W. Woolley, J. Biol. Chem. 159, 59 (1945).

31R. F. Light, R. P. Alscher, and C. N. Frey, Science 100, 225 (1944).

VII EFFECTS OF DEFICIENCY 419

VII. Effects of Deficiency

A. IN ANIMALS
H. J. ALMQUIST

The pathology of vitamin K deficiency is unique among the vitamins
because the only primary effect yet known on any tissue is that upon the
prothrombin level of the blood. The prothrombin content of blood is greatly
decreased, and the clotting time is markedly prolonged. Thus the occurrence
of subcutaneous and intramuscular hemorrhages is frequently observed.

B. IN HUMAN BEINGS
CHARLES A. OWEN, JR.

A deficiency of vitamin K is characterized by a bleeding tendency secon-
dary to an alteration of the coagulability of the blood. The deficiency may
result from ingestion of insufficient vitamin K, lack of bacterial synthesis
of the vitamin within the intestinal tract, nadeanate intestinal absorp-
tion, or hepatic inability to utilize the available vitamin K.

1. HEMORRHAGE RESULTING FROM A DEFICIENCY OF VITAMIN K

Serious abnormalities of the blood coagulation mechanism may be present
without any evidence of bleeding. However, it is frequently stated that
when the plasma prothrombin falls to about 30 % of normal! the danger of
bleeding exists; this is based on repeated observations that bleeding only
infrequently occurs in vitamin K deficiency when the prothrombin is above
this level.

Prior to the discovery of vitamin K and recognition of its deficiency in
obstructive jaundice, bleeding was most commonly seen after surgical cor-
rection of a biliary obstruction.” This is now rare because of the generous
use of vitamin K in biliary surgery. At the present time hemorrhage from
injudicious use of dicoumarol—an “‘antagonist”’ of vitamin K—is probably
commonest.*

The bleeding caused by vitamin K deficiency may be first recognized
by slow oozing from the mucous membranes of the nasopharynx, by the
appearance of ecchymoses associated with mild trauma, or in surgical cases
by hematomas and persistent bleeding at the site of operation. Massive
hemorrhage may occur beneath the skin and within muscles, particularly
of the extremities. Hemorrhagic disease of the newborn is often first sus-

1 Values as high as 35 to 40% [K. M. Brinkhous, H. P. Smith, and E. D. Warner,
Am. J. Med. Sci. 196, 50 (1938)] and as low as 5% [F. Koller and P. Frick, Helv.
Chim. Acta 82, Part 1, 717 (1949)] have been suggested.

2K. W. Boland, Proc. Staff Meetings Mayo Clinic. 18, 70 (1938).

37. F. Duff and W. H. Shull, J. Am. Med. Assoc. 139, 762 (1949).

420 VITAMIN K GROUP

pected on the basis of melena or hematemesis; although bleeding may occur
almost anywhere, areas of predilection are: umbilicus, skin, nose and mouth,
intestine, and cerebrum.?: > Eecchymoses and hematomas are usually re-
lated to trauma in those receiving too intensive dicoumarol therapy.

For an extensive study of the gross and microscopic pathology of experi-
mental vitamin Kx bleeding the work of Ferraro and Roizin‘® is referred to.
They concluded that three factors are important in the causation of bleed-
ing: the degree of hypoprothrombinemia, the age of the animal (the
younger, the more severe the hemorrhage), and trauma.

TABLE V

DIFFERENTIAL DIAGNOSIS OF THE BLEEDING CAUSED BY VITAMIN K DEFICIENCY

Vitamin K Thrombo-

deficiency Scurvy Hemophilia cytopenia

Clot time Long Ne Long N
Plasma prothrombin Low N? N N
Bleeding time N Long N Long
Capillary fragility N Iner. N Iner.
Response to vitamin Ix AF 0 0 0
Response to vitamin C 0 + 0 0
Response to ‘‘antihemophilic globu- 0 0 + 0

lhrua\?”
Response to transfusion of whole ar =f +

blood

* N = normal; Incr. = increased; + = positive response; 0 = no response

6’ Unless there is a coexisting deficiency of vitamin K.

Although the gross appearance is not in itself diagnostic, the bleeding
tendency in vitamin K deficiency differs to some extent from that of scurvy,
hemophilia, or thrombocytopenia (Table V). In scurvy there are swollen,
spongy, bleeding gums and subperiosteal and intramuscular hemorrhagic
extravasations. The hemophiliac tends to bleed in and about joints, with
resultant hemarthroses and ankyloses.? The petechial bleeding of thrombo-
cytopenia and scurvy may be confusing, but the platelets are severely
depressed only in the former.

41,. Salomonsen, Acta paediat. 27, Suppl. 1, 1 (1939).

5 R.B. Scott, Practitioner 165, 182 (1950); B. P. Clark, J. Med. Assoc. State Alabama
20, 130 (1950); G. Fanconi, Die Stérungen der Blutgerinnung beim Kinde mit
besonderer Beriicksichtigung des K-Vitamins und der Neugeborenenpathologie.
G. Thieme, Leipzig, 1941.

6 A. Ferraro and L. Roizin, Am. J. Pathol. 22, 1109 (1946).

7R. K. Ghormley and R.§8. Clegg, J. Bone Joint Surg. 830A, 589 (1948).

VII. EFFECTS OF DEFICIENCY 421

2. INDUCTION OF VITAMIN K DEFICIENCY
a. Dietary Lack of Vitamin K

It was first noted that on certain inadequate diets superficial and internal
bleeding developed in chicks.’ The ability of dietary means alone to induce
a deficiency of vitamin K is almost exclusively an avian characteristic ;° but
in the rat,!° rabbit,!! mouse,” and perhaps man!’ moderate hypoprothrombin-
emia may occasionally develop. The newborn of man,": ! sheep, goat,!®
and guinea pig" are critically lacking in vitamin K, as shown by the fact
that small doses of this vitamin will correct the blood-clotting abnormality.

The infant undergoes a potentially serious blood-clotting change during
its first week of life; this can be corrected or prevented by administration
of vitamin K either to the baby or, shortly before the baby’s birth, to the
mother. The cause of the vitamin K deficiency is not clear, but apparently
negligible amounts of the naphthoquinone have been stored by the infant
at the time of birth. Salomonsen‘ suggested that there is probably some
storage during the summer months, since the incidence of actual bleeding is
less during the summer and fall than at other seasons. Waddell and Lawson!8
are in agreement, adding that the peak month for hypoprothrombinemia is
March, at which time there is the greatest incidence of deaths from birth
injuries. On the basis of a very limited observation Thordarson!® was un-
able to find a seasonal trend in the prothrombin levels of infants.

Just as the cause of the hypoprothrombinemia, or rather the neonatal
lack of stored vitamin K, is unexplained, there is little agreement on the
spontaneous recovery which most infants undergo by about the seventh

8H. Dam, Biochem. Z. 215, 475 (1929); 220, 158 (1930); W. D. McFarlane, W. R.
Graham, Jr., and G. E. Hall, J. Nutrition 4, 331 (1931); W. D. McFarlane, W. R.
Graham, Jr., and F. Richardson, Biochem. J. 25, Part 1, 358 (1931); W. F. Holst
and E. R. Halbrook, Science [N. S.] 77, 354 (1933). An analysis of the distribution
of hemorrhages in the chick has been made by S. Ansbacher, J. Nutrition 17, 303
(1939). 2

9H. Dam, F. Schgnheyder, and L. Lewis, Biochem. J. 31, Part 1, 22 (1937).

10H. Dam and J. Glavind, Z. Vitaminforsch. 9, 71 (1939).

"1H. Dam and J. Glavind, Acta Med. Scand. 96, 108 (1938).

2 R, Murphy, Science [N. S.] 89, 203 (1939).

*13R. Kark and E. L. Lozner, Lancet II, 1162 (1939); C. M. Thompson and D. J.
Hilferty, Med. Clin. N. Amer. 33, 1685, (1949).

14K. M. Brinkhous, H. P. Smith, and E. D. Warner, Am. J. Med. Sci. 198, 475 (1937).

16W. W. Waddell, Jr.. D. Guerry, III, W. E. Bray, and O. R. Kelley, Proc. Soc.
Exptl. Biol. Med. 40, 432 (1939).

16 A van Vyve, Acta Brevia Neerl. Physiol. Pharmacol. Microbiol. 11, 101 (1941).

7, Widenbauer and U. Krebs, Monatsschr. Kinderheilk. 91, 223 (1942).

1 W.W. Waddell, Jr., and G. McL. Lawson, J. Am. Med. Assoc. 115, 1416 (1940).

19Q. Thordarson, Klin. Wochschr. 20, 645 (1941).

422 VITAMIN K GROUP

day of life. Adler?° attributes the entire process to a transient hepatic defect,
whereas Quick and Grossman?! believe that vitamin K becomes available
to the infant only after sufficient bacteria have developed in the bowel to
produce vitamin K. Sells and associates” found insufficient vitamin K in
human colostrum to maintain the baby’s blood coagulability; however,
minimally adequate vitamin K was present in cow’s milk. This might
explain the older observation” that the prelacteal feedings of cow’s milk
prevent hemorrhagic disease of the newborn. Sells’ group” further found
that coagulability of the blood remained impaired as long as food (other
than aqueous glucose solution) was withheld from the infants.

When vitamin K was found to prevent newborn hypoprothrombinemia,
it was anticipated that this hemorrhagic disease could be eliminated by
the simple expedient of routine prophylactic administration of vitamin K
to the baby or mother. Early, carefully controlled studies!®: *4 indicated
that this was overoptimistic but that some benefit was to be obtained.
Recently several reports”® have questioned whether the routine use of
vitamin K is of any benefit. Since few clotting studies were carried out,
judgment should be reserved. The problem merits further careful study.

b. Inhibition of Synthesis of Vitamin K by Intestinal Bacteria

The most important source of vitamin K for the mammal is its own
intestinal bacteria,?®: 7? in particular, Escherichia coli.?8 In the absence of
dietary vitamin K this source alone is usually sufficient to maintain a
normally coagulable blood. It is felt that the bacterial (farnesyl) vitamin?®

20 B. Adler, Monatschr. Geburtschiilfe wu. Gyndkol. 118, 225 (1944).

21 A, J. Quick and A. M. Grossman, Am. J. Med. Sci. 199, 1 (1940).

22R. L. Sells, S. A. Walker, and C. A. Owen, Proc. Soc. Exptl. Biol. Med. 47, 441
(1941).

23H. N. Sanford, H. J. Morrison, and L. Wyat, Am. J. Diseases Children 48, 569
(1932).

24 7,. M. Hellman, L. B. Shettles, and N. J. Eastman, Am. J. Obstet. Gynecol. 40, 844
(1940).

25H}. L. Potter, Am. J. Obstet. Gynecol. 50, 235 (1945); H. N. Sanford, M. Kostalik,
and B. Blackmore, Am. J. Diseases Children 78, 686 (1949); J. D. Hay, F. P. Hud-
son, and T. 8S. Rodgers, Lancet I, 423 (1951).

26H. J. Almquist, Physiol. Revs. 21, 194 (1941).

27H. J. Almquist, C. F. Pentler, and E. Mecchi, Proc. Soc. Exptl. Biol. Med. 38, 336
(1938); H. J. Almquist and E. L. R. Stokstad, J. Biol. Chem. 111, 105 (1935); H. R.
Butt and A. E. Osterberg, J. Nutrition 15, Swppl., 11 (1938).

8 §. Orla-Jensen, A. D. Orla-Jensen, H. Dam, and J. Glavind, Zentr. Bakt. Para-
sitenk. Abt. 2, 104, 202 (1941-1942); H. Dam and J. Glavind, Biochem. J. 32, Part 1,
1018 (1938).

28a Tt is generally stated that the intestinal bacteria produce the farnesyl vitamin
[H. Dam, Med. Welt. 20, 958 (1951)]; however, Taveira reported the recovery of the
phytyl vitamin from Escherichia coli [M. Taveira, Ann. pharm. frang. 9, 344 (1951)].

VII. EFFECTS OF DEFICIENCY 423

is less effective than the leafy plant (phytyl) one.2® Presumably, then, the
intestinally synthesized vitamin exceeds that obtained from food, for,
though less potent, it can maintain an efficient clotting system, whereas
the reverse does not seem to be true. Fowls have a short, large intestine
which is believed to be inadequate for absorption of the bacterially produced
vitamin K.*° Also, the mammalian lower colon is probably incapable of
absorbing the fat-soluble vitamins K even in the presence of bile, for re-
tention enemas of vitamin K have proved ineffective,*! and Greaves® was
able to produce a vitamin K deficiency in rats by means of bile fistulas into
the colon. However, the cecum may be an important site of bacterial syn-
thesis of the vitamin. More careful attention to prevention of coprophagy
—the feces contain large amounts of vitamin K even when the diet is
vitamin K-free!!: *°: *—may account for the greater than average success
that certain investigators*® * have had in inducing a dietary vitamin K
deficiency in mammals.

Reduction of intestinal synthesis of vitamin K is most simply accom-
plished by bacteriostatic or bactericidal agents such as the sulfonamides*®
and possibly by certain antibiotic agents.*® p-Aminobenzoic acid, whether
given orally or parenterally, concentrates in the bowel and prevents sulfon-
amide depression of the intestinal flora,*”? thus it is not possible to assume a
non-intestinal effect of a parenterally administered drug.

29H. J. Almquist and A. A. Klose, Proc. Soc. Exptl. Biol. Med. 45, 55 (1940).

30 H. J. Almquist and E. L. R. Stokstad, J. Nutrition 12, 329 (1936).

31H. P. Smith, 8. E. Ziffren, C. A. Owen, G. R. Hoffman, and J. E. Flynn, J. Jowa
State Med. Soc. 29, 377 (1939).

3 J. D. Greaves, Am. J. Physiol. 125, 429 (1939).

33H. G. Day, K. G. Wakim, M. M. Krider, and E. E. O’Banion, J. Nutrition 26, 585
(1943).

34 H. Dam, Advances in Enzymol. 2, 285 (1942).

35 The sulfonamides depress HL. coli [H. J. White, Bull. Johns Hopkins Hosp. 71, 213
(1942)] and inhibit intestinal synthesis of vitamin K in the same order of effective-
ness [W. H. Sebrell, Harvey Lectures 39, 288 (1943-1944) (from most to least effec-
tive)]: sulfapyrazine, sulfadiazine [A. Kornberg, F. 8. Daft, and W. H. Sebrell,
Publ. Health Repts. (U. S.) 59, Part 1, 832 (1944)], sulfathiazole [B. M. Braganca
and M. V. Radhakrishna Rao, Indian J. Med. Research 35, 15 (1947)], sulfasuxidine
[A. D. Welch and L. D. Wright, J. Nutrition 25, 555 (1948)], and sulfaguanidine
[S. Black, R. S. Overman, C. A. Elvehjem, and K. P. Link, J. Biol. Chem. 145, 137
(1942)].

36 Z. A. Lewitus and A. Aschireli, Harefuah 35, 13 (1948); W. K. Rieben, Helv. Med.
Acta 13, 295 (1946); C. M. Thompson and D. J. Hilferty, Med. Clin. N. Amer. 33,
1685 (1949). The coagulation changes resulting from prolonged treatment with anti-
biotic substances are to be distinguished from their clot-accelerating activities
during the first hour or two after administration [L. F. Moldavsky, W. B. Hassel-
brock, C. Cateno, and D. Goodwin, Science [N. 8.] 102, 38 (1945); D. I. Macht,
ibid. 105, 313 (1947); Editorial, J. Am. Med. Assoc. 141, 924 (1949)].

37 A. Kornberg, F. 8. Daft, and W. H. Sebrell, J. Biol. Chem. 155, 193 (1944).

424 VITAMIN K GROUP

Since small doses of vitamin K correct the hypoprothrombinemia accom-
panying intestinal “‘sterilization,” the possibility of prothrombin changes
secondary to hepatic damage would seem to be excluded. However, Bra-
ganca and Radhakrishna Rao* reported that infiltration with fat de-
veloped in the livers of rats after 2 weeks of a diet containing sulfathiazole;
this hepatic change was prevented by addition of vitamin K to the sulfon-
amide-treated diet. The data of Mushett and Seeler*®® suggest that in
sulfonamide hypoprothrombinemia vitamin K, is 100 to 250 times as
effective as menadione; by analogy with vitamin K suppression of dicou-
marol action, an hepatic site of action might be considered, for in simple
vitamin K deficiency states menadione is the more potent.

Rats develop hypoprothrombinemia when subjected to a diet high in
triglycerides containing dihydroxystearic acid.*** The clotting change is ac-
companied by disappearance of vitamin K from the intestinal contents and
is corrected by the administration of the vitamin (0.1 mg. per day). Since
the intestinal bacteria are qualitatively and quantitatively normal during
the period of vitamin K deficiency, it seems that the vitamin-synthesizing
systems of the bacteria are blocked.**»

c. Reduced Absorption of Vitamin K

(1) Absence of Bile. Quick et al.* first reported blood-clotting changes in
human obstructive jaundice comparable to those already demonstrated in
vitamin K-deficient chicks. Hawkins and Brinkhous*? found the same clot-
ting condition in experimental biliary fistulas in dogs. The lack of bile in
the intestine was common to both states and was soon recognized to be
etiologically important in vitamin K deficiency.*! Fant] and coworkers*!*
have recently questioned this concept.

In rapid succession Warner et al.,” Butt e¢ al., and Dam and Glavind*
reported the successful correction of the clotting changes and of the bleed-

88 C. W. Mushett and A. O. Seeler, J. Pharmacol. Exptl. Therap. 91, 84 (1947).

38a F), E. Lockhart, H. Sherman, and R. S. Harris, Science [N. S.] 96, 542 (1942).

8b G. Nightingale, E. E. Lockhart, and R. 8. Harris, Arch. Biochem. 12, 381 (1947).

39 A. J. Quick, M. Stanley-Brown, and F. W. Bancroft, Am. J. Med. Sci. 190, 501
(1935).

40 W. B. Hawkins and K. M. Brinkhous, J. Hxptl. Med. 63, 795 (1936).

41H. P. Smith, E. D. Warner, K. M. Brinkhous, and W. H. Seegers, J. Exptl. Med.
67, 911 (1938).

4a P. Fantl, J. F. Nelson, and G. J. Lincoln, Australian J. Exptl. Biol. Med. Sci. 29,
433 (1951). .

# WH}. D. Warner, K. M. Brinkhous, and H. P. Smith, Proc. Soc. Exptl. Biol. Med. 37,
628 (1988).

48 H.R. Butt, A. M. Snell, and A. E. Osterberg, Proc. Staff Meetings Mayo Clinic 18,
74 (1938).

44H. Dam and J. Glavind, Lancet I, 720 (1938).

VII. EFFECTS OF DEFICIENCY 425

ing in patients with obstructive Jaundice when crude vitamin K concen-
trates were administered orally, along with bile. Bile alone was slowly effec-
tive, but the vitamin alone was without any effect. Thus it became established
that, like the other fat-soluble vitamins, vitamin K requires bile for its
transport through the intestinal wall. Desoxycholic acid, which unites with
vitamin Ix to form vitamin K-choleic acid,** is claimed to be the principal
component of bile for absorption of the vitamin.*® Butt and Snell*” demon-
strated that human acholic stools are rich in vitamin K, so that for coagula-
tion all that seems to be lacking in obstructive jaundice is the bile. It is
therefore puzzling why bile alone, although free of vitamin K,** is not more
efficacious when one considers the smal] amount of vitamin K necessary to
correct the deficiency (1 mg. or less).*

As might be expected, water-soluble analogs of vitamin K may be given
orally, without supplementary bile, to animals or man lacking intestinal
bile.6° However, the water-soluble preparations are usually given parenter-
ally because of the prompter action and insured dosage.°!

(2) Intestinal Disease. Altered blood coagulation is at times found in a
variety of intestinal diseases and conditions: intestinal obstruction, gastro-
colic fistula, external enterostomy, chronic ulcerative colitis, regional ileitis,
intestinal polyposis,”’ and tuberculous enteritis.°* Apparently vitamin IK
is mechanically prevented from being absorbed in these conditions. The
vitamin deficiency which occurs in sprue, both tropical* and non-tropical,’”
and in prolonged diarrhea (such as in pellagra)®> likewise represents an
inability to absorb the vitamin K which is abundantly present; this state
may be duplicated experimentally by a high concentration of mineral oil
in the diet.°® Activated carbon, by firmly adsorbing vitamin K, also pre-

45H. J. Almquist and A. A. Klose, J. Am. Chem. Soc. 61, 745 (1939).

46. T. Cohn and C. L. A. Schmidt, Proc. Soc. Exptl. Biol. Med. 41, 443 (1939).

47H. R. Butt and A. M. Snell, Vitamin K. W. B. Saunders Company, Philadelphia,
(1941).

48H. J. Almquist, Science [N. S.] 87, 538 (1938).

49). Fernholz and 8S. Ansbacher, Science [N.S.] 90, 215 (1939).

50 —. D. Warner and J. E. Flynn, Proc. Soc. Exptl. Biol. Med. 44, 607 (1940); H. P.
Smith and C. A. Owen, J. Biol. Chem. 134, 783 (1940).

51H. P. Smith, 8. E. Ziffren, C. A. Owen, and G. R. Hoffman, J. Am. Med. Assoc.
113, 380 (1939); H. R. Butt, A. M. Snell, and A. E. Osterberg, Proc. Staff Meetings
Mayo Clinic 14, 497 (1939).

8 R. L. Clark, Jr., C. F. Dixon, H. R. Butt, and A. M. Snell, Proc. Staff Meetings
Mayo Clinic 14, 407 (1939); T. T. Mackie, N. Y. State J. Med. 40, Part 2, 987 (1940).

53}. Tanner and F. Suter, Schweiz. med. Wochschr. 74, 552 (1944).

54R.S. Diaz y Rivera, Puerto Rico J. Public Health Trop. Med. 17, 124 (1941).

55}. D. Warner, T. D. Spies, and C. A. Owen, Southern Med. J. 34, 161 (1941).

56 M.C. Elliott, B. Isaacs, and A. C. Ivy, Proc. Soc. Exptl. Biol. Med. 48, 240 (1940) ;
W.A. Barnes, zbid. 49, 15 (1942).

426 VITAMIN K GROUP

vents its absorption.2° Whether the hypoprothrombinemia and bleeding
reported to occur in animals receiving large doses of vitamin A‘ is second-
ary to reduced vitamin K synthesis or absorption, or to an alteration of
hepatic function, is not clear; addition of a vitamin K analog to the diet
of the hypervitaminotic A anima] prevents the clotting changes without,
however, any alteration in the vitamin A concentration in the liver.** The
status of quinine®? and certain rare earths® is likewise uncertain; hypo-
prothrombinemia, which is corrected by vitamin K, has been observed
following their administration. Quick™ has questioned the quinine effect
on clotting.

(3) Diversion of Intestinal Lymph. In the rat a profound incoagulability
of the blood may be produced by withdrawal of intestinal or thoracic duct
lymph;® correction with parenteral vitamin K is prompt.® The pathway
for vitamin K absorption would thus seem to be via the intestinal lympha-
tics, through the thoracic duct, to the vascular tree. The possibility of a
vitamin K deficiency in prolonged loss of lymph resulting from thoracic
duct avulsion might be considered; actually, hematuria often does accom-
pany lymphatic chyluria.

d. Hepatic Utilization of Vitamin K

(1) Liver Damage. It has long been known that severe liver damage™ or
hepatectomy® leads to a prolongation of the clotting time of whole blood.
With the development of methods for estimating plasma prothrombin it
was discovered that a lack of prothrombin in the blood was a prominent
part of these clotting changes.®* Furthermore, administration of vitamin K
to man or animals with liver damage has proved ineffectual in correcting

57R. F. Light, R. P. Alscher, and C. N. Frey, Science 100, 225 (1944).

58 $. E. Walker, E. Eylenburg, and T. Moore, Biochem. J. 41, 575 (1947).

59 J,. A. Pirk and R. Engelberg, J. Am. Med. Assoc. 128, 1093 (1945).

60). Vincke and E. Schmidt, Hoppe-Seyler’s Z. physiol. Chem. 273, 39 (1942).

61 A. J. Quick, J. Lab. Clin. Med. 31, 79 (1946).

6 ©, A. Owen, Jr., Studies on the Conversion of Prothrombin to Thrombin; Effect
of Conversion Variations on Prothrombin Tests. Thesis, Graduate School, Uni-
versity of Minnesota, 1950.

63 J. D. Mann, F. D. Mann, and J. L. Bollman, Am. J. Physiol. 158, 311 (1949).

64M. Doyon, Compt. rend. soc. biol. 58, 30 (1905); M. Doyon, A. Morel, and N. Kareff,
ibid. 58, 493 (1905).

65 M. Doyon and N. Kareff, Compt. rend. soc. biol. 56, 612 (1904); P. Nolf, Arch.
intern. physiol. 8, 1 (1905-1906).

66 H. P. Smith, E. D. Warner, and K. M. Brinkhous, J. Exptl. Med. 66, 801 (1937);
E. D. Warner, zbid. 68, 831 (1938); W. D. Andrus, J. W. Lord, Jr., and R. A. Moore,
Surgery 6, 899 (1939); K. M. Brinkhous and E. D. Warner, Proc. Soc. Exptl. Biol.
Med. 44, 609 (1940); J. L. Bollman, H. R. Butt, and A. M. Snell, J. Am. Med. Assoc.
115, 1087 (1940); B. Uvniis, Acta Physiol. Scand. 8, 97 (1941); D. J. Ingle, J. E.
Nezamis, and M. C. Prestrud, Am. J. Physiol. 161, 199 (1950).

VII. EFFECTS OF DEFICIENCY 427

the lack of prothrombin or the lengthening of clot time; however, large
doses of vitamin K before exposure of animals to chloroform are reported
to be hepatoprotective.® A practical application is the evaluation of hypo-
prothrombinemia of uncertain origin; if parenteral vitamin I< analogs are
not corrective, hepatic function is presumed to be subnormal (vitamin K
tolerance test).®® Clotting studies suggest that mild hepatic insufficiency
results from fever,’? anesthesia,” or simple manipulation of the liver.”
(2) Anticoagulant Drugs. In contradistinction to such substances as chlo-
roform, carbon tetrachloride, and phosphorus, which depress a variety of
hepatic activities, including the synthesis of prothrombin and fibrinogen,
a class of chemicals has been developed within the past decade, primarily
by Link’s group,” which has found wide clinical use: the anticoagulant
coumarins, which include dicoumarol,” hydrocoumarol,”> tromexan,’® phen-
ylindanedione,” and anticoagulant ‘63.’ These chemicals are adminis-

67 §. J. Wilson, Proc. Soc. Exptl. Biol. Med. 41, 559 (1939); F. J. Pohle and J. K. Stew-
art, J. Clin. Invest. 19, 365 (1940); R. Kark and A. W. Souter, Lancet I, 1149 (1940).

68 M. A. Pessagno Espora, Dia méd. 22, 1264 (1950).

69 P. N. Unger, S. Shapiro, and S. Schwalb, J. Clin. Invest. 27, 39 (1948); P. N. Unger,
M. Weiner, and 8. Shapiro, Am. J. Clin. Path. 18, 835 (1948).

70R. K. Richards, Science 97, 313 (1943).

71 §.C. Cullen, S. E. Ziffren, R. B. Gibson, and H. P. Smith, J. Am. Med. Assoc. 115,
991 (1940).

72 J. W. Lord, Jr., Surgery 6, 896 (1939).

73K. P. Link, Harvey Lectures 39, 162 (1943-1944).

74 3 3’-Methylenebis(4-hydroxycoumarin); dicumarin, dicoumarin, dikumarin, di-
cumarol, ‘“‘A. P.,’’? bishydroxycoumarin (Formula 10); R. 8. Overman, M. A.
Stahmann, W. R. Sullivan, C. F. Huebner, H. A. Campbell, and K. P. Link, J.
Biol. Chem. 142, 941 (1942); H. R. Butt, E. V. Allen, and J. L. Bollman, Proc. Staff
Meetings Mayo Clinic 16, 388 (1941); Council on Pharmacy and Chemistry, J. Am.
Med. Assoc. 187, 1533 (1948); 145, 644, (1951); H. Dyckerhoff, Biochem. Z. 316,
397 (1944).

75 3,3’-Methylenebis(3,4-hydro, 4-hydroxycoumarol) (Formula 11); F. Ericksen, E.
Jacobsen, and C. M. Plum, Acta Pharmacol. Toxicol. 1, 379 (1945).

76 3,3’-Carboxymethylenebis(4-hydroxycoumarin) ethyl ester or 4,4’-dioxydicou-
maryl ethyl acetate; pelentan, ““B.O.E.A.,’’ ethyl biscoumacetate (Formula 12);
K. N. von Kaulla and R. Pulver, Schweiz. med. Wochschr. 78, 806 (1948); R. Della
Santa, zbid. 79, 195 (1949); C. Solomon, H. J. MeNeile, and R. Lange, J. Lab. Clin.
Med. 36, 19 (1950); G. E. Burke and I. 8. Wright, Conf. on Blood Clotting and Allied
Problems, New York 3, 57 (1950).

77 Phenyl-2-indanedione-1,3; ‘‘P.I.D.’’; ‘‘danilone’’; ‘‘hedulin’”? (Formula 13); P.
Meunier, C. Mentzer, and D. Molho, Compt. rend. acad. sci. U.R.S.S. 224, 1666
(1947); J. P. Soulier and J. Guéguen, Rev. hématol. 3, 180 (1948); N. W. Barker,
J. E. Estes, Jr., and F. D. Mann, Proc. Staff Meetings Mayo Clinic 26, 162 (1951);
L. B. Jaques, E. Lepp, and E. Gordon, Conf. on Blood Clotting and Allied Problems,
New York 8, 11 (1950).

78 2-Methyl-2-methoxy-4-phenyl-5-oxodihydropyrano(3,2-C) (1)-benzopyran; cou-
mopyran; M. Ikawa, M. A. Stahmann, and K. P. Link, J. Am. Chem. Soc. 66, 902

428 VITAMIN K GROUP

tered clinically in order to reduce the ability of the blood to clot and thus
lessen the chances for the development or progression of thromboses. Since
the complications of hemorrhage may be as serious as those of thrombosis,
the margin of safety is rather narrow. The therapeutic goal is an amount of
anticoagulant which will depress prothrombin and associated clotting fac-
tors but not quite prolong the whole blood-clotting time. Clinical results
have been very encouraging and seem to bear out the empirical method of
evaluating therapy.”?

Even before the specific toxic agent (dicoumarol) of spoiled sweet clover
was known, Roderick*®® recognized that the incoagulability of the blood of
cattle that had eaten the moldy clover was associated with a prothrombin
deficiency. The hypoprothrombinemia requires at least 12 to 24 hours to
develop after ingestion of the anticoagulant, probably because an interval
of time is required for circulating prothrombin to be catabolized after the
production of new prothrombin has been halted.*!: * This conclusion seems
warranted from studies of prothrombin disappearance rates; Carter et al.
found no evidence of prothrombin stores.

Vitamin K concentrates (from alfalfa) or the pure analogs have been
found to counteract the prothrombin-depressing activity of dicoumarol.* ®
The problem remains unsettled as to whether dicoumarol injures the liver,
an action reversed by vitamin K or whether there is a competitive inhibi-
tion of vitamin K activity by dicoumarol. Glavind and Jansen®® found that
in vitamin K-depleted chicks the reaction to dicoumarol was counteracted
by an amount of vitamin K which followed no fixed ratio; they concluded
that dicoumarol injures the liver. In a comparable experiment Collentine

(1944); W. D. Battle, R. T. Capps, O.S. Orth, and O. O. Meyer, J. Lab. Clin. Med.
35, 8 (1950); R. Rotter and O. O. Meyer, zbid. 36, 981 (1950); H. H. Hanson, N. W.
Barker, and F. D. Mann, Circulation 4, 844 (1951).

79K. V. Allen, N. W. Barker, and J. M. Waugh, J. Am. Med. Assoc. 120, 1009 (1942) ;
H. R. Butt, E. V. Allen, and J. L. Bollman, Proc. Staff Meetings Mayo Clinic 16,
388 (1941); N. W. Barker, H. R. Butt, E. V. Allen, and J. L. Bollman, J. Am. Med.
Assoc. 118, 1003 (1942); N. W. Barker, H. E. Cromer, Jr.,M. Hurn, and J. M.
Waugh, Surgery 17, 207 (1945).

80 J,. M. Roderick, Am. J. Physiol. 96, 413 (19381).

81K. P. Link, R. 8. Overman, W. R. Sullivan, C. F. Huebner, and L. D. Scheel,
J. Biol. Chem. 147, 463 (19438).

82M. Stefanini and A. V. Pisciotta, Sczence [N. S.] 111, 364 (1950).

83 J. R. Carter, G. H. Chambers, and E. D. Warner, Proc. Soc. Exptl. Biol. Med. 72,
52 (1949).

84R.S. Overman, J. B. Field, C. A. Baumann, and K. P. Link, J. Nutrition 28, 589
(1942).

85 J. Lehmann, Science [N. S.] 96, 345 (1942); S. Shapiro, M. H. Redish, and H. A.
Campbell, Proc. Soc. Exptl. Biol. Med. 52, 12 (1948); H. E. Cromer, Jr., and N.W.
Barker, Proc. Staff Meetings Mayo Clinic 19, 217 (1944).

86 J, Glavind and K. F. Jansen, Acta Physiol. Scand. 8, 173 (1944).

87G. E. Collentine and A. J. Gur Am. J. Med. Sci. 222, 7 (1951).

VII. EFFECTS OF DEFICIENCY 429

and Quick*’ depleted dogs of vitamin K stores by cholecystnephrostomy;
smal] doses of vitamin K, (or analogs) and of dicoumarol counteracted
each other. Because of the minuteness of the doses, Collentine and Quick
feel that the only explanation is a mutual competition for prothrombino-
genic enzyme systems. Almquist** has presented an interesting theoretical
analysis of the action of dicoumarol.

Certain observations suggest a primary hepatotoxicity from dicoumaroli-
zation. Overdoses of dicoumarol produce fatty livers in rabbits’ and central
necrosis of the liver in rats.’ Bollman and Preston®® reported that in the
presence of an already damaged liver the effect of dicoumarol was accen-
tuated. Similarly, Roller and Mudrak* demonstrated that, if the liver had
been damaged, smaller doses of dicoumarol gave the same effect as large
doses when hepatic function was normal. Irish and Jaques noted that
dicoumarol, like known hepatotoxins, stimulated fibrinogen production if
given in small doses, and in larger doses depressed the fibrinogen of the
blood.

The concept of a competition between vitamin K and dicoumarol has
been explored extensively by Meunier and Mentzer; these investigators
have stressed the similarity between compounds with hemorrhagic and
antihemorrhagic activity. Menadione (Fig. 5) (1) is a potent vitamin K
analog; addition of an hydroxyl in the 3 position, phthiocol (2), reduces
the activity; replacement of the methyl by an ethyl! radical (3) further
weakens the antihemorrhagic power; combining two phthiocols through
their methyl! groups yields an antivitamin K™ (4), as does doubling menadi-
one®® (5). Replacing the 2-methyl group in menadione by methoxy (6) also
reverses the compound’s action,®® and if one substitutes for the methyl] in
phthiocol’s 2 position 3-cyclohexylpropyl (7), or 2-methyloctyl (8), or
3-(decahydro-2-naphthyl)propyl (9) radicals, a similar reversal occurs.”
Some of the more potent hemorrhagic agents tend to resemble (4) and (5),
that is, “double molecules.” Although most clinical anticoagulants are

87a H, J. Almquist, Arch. Biochem. and Biophys. 35, 463 (1952).

88 K. F. Jansen, Nord. med. 20, 1993 (1943).

89 C. L. Rose, P. N. Harris, and K. K. Chen, Proc. Soc. Exptl. Biol. Med. 50, 228
(1942).

90 J. L. Bollman and F. W. Preston, J. Am. Med. Assoc. 120, 1021 (1942).

% PD. Roller and O. Mudrak, Z. ges. exptl. Med. 114, 75 (1944).

2 U.D. Irish and L. B. Jaques, Am. J. Physiol. 148, 101 (1945).

93 Reviewed in several articles in the International Colloquy ‘‘Les Vitamins,’’ Bull.
soc. chim. biol. (1948).

94 P. Meunier, C. Mentzer, N.P. Buu-Hoi, and P. Cagniant, Bull. soc. chim. biol. 25,
384 (1943).

9 C, Mentzer, Bull. soc. chim. biol. 30, 872 (1948).

96 T). Molho, J. Moraux, and P. Meunier, Bull. soc. chim. biol. 30, 637 (1948).

7 C. C. Smith, R. Fradkin, and M. D. Lackey, Proc. Soc. Exptl. Biol. Med. 61, 398
(1946).

430 VITAMIN K GROUP
symmetrical ‘‘double molecules’’—dicoumarol (10), hydrocoumarol (11), and

tromexan (12) (Fig. 6)—they need not be, for the asymmetric phenylin-
danedione (13) and others (14 and 15)*8 are also active. Dithiocoumarol (16)

O O
CH; CH, C,H;
OH OH
O O
(1) (2)

O
(3)
O O Y O
CH CH;
OH HO
O O O O
(4) (5)
O O Ha He
/ we
OCH; (CH2);— Ci pl:
OH ‘C=C
I He He
O O
(6) (7)
O O H H
yous OL i
CH,CH (CH:),—CH i
OH (CH:);CHs OH HO. cee
2 2
(8) (9)
HOH Jal, (Oyc!
OH OH
5 Oo O 5 0 Oo O
(10) (11)

Bras 5:

is weakly hemorrhagic.** R in compound (17) determines the activity: if R
is CH;, the compound is vitamin K-like; if R is an halide or a pheny]
derivative, it is an antivitamin K compound.®%

* P. Meunier, C. Mentzer, and A. Vinet, Helv. Chim. Acta 29, Part 1, 1291 (1946).

°° P. Meunier, C. Mentzer, and D. Molho, Compt. rend. acad. sci. (U.R.S.S.) 224,
1666 (1947).

VII. EFFECTS OF DEFICIENCY 431

Woolley!” remarks that the dicoumarol-vitamin K antagonism differs
in several respects from the characteristic analog competitions: vitamin K
counteracts dicoumarol over only a limited dosage range; within this range
the inhibition index is not constant; and most unusual is an index of less
than 1.0 (for example, in the rat 10 mg. of dicoumarol is inhibited by 25
mg. of vitamin K; that is, an index of 0.4).

Regardless of the exact nature of the antagonism between vitamin K and
dicoumarol, the recent evidence of Jaques’ group'”! suggests that the hepatic
cell is involved. Dicoumaro] labeled with carbon" in the methylene bridge
was administered intravenously to mice and rabbits; significant radio-

OH OH OH
O
O *| O

COO:C.H;
(12) (13)
OH
0 O ei lea 0 CH;
(14) . (15)
OH OH OH
ees (ee
O
S OT S O
(16) (17)

Fie. 6.

activity was found only in the liver, gallbladder, feces, and urine (only
degradation products in the urine). Intravenous administration of vitamin
K expedited disappearance of radioactivity from the mouse liver. Measur-
ing plasma dicoumarol directly, Shapiro et al.!° were unable to demonstrate
more rapid disappearance of dicoumarol from the blood when vitamin K was
administered. Dicoumarol or its active products are apparently excreted by
the kidneys, for nephrectomy or experimental nephritis prolongs the anti-
coagulant activity.°° The suggestion! has been made that the action of

100 J), W. Woolley, Physiol. Revs. 27, 308 (1947); Advances in Enzymol. 6, 129 (1946).

101 ©, C. Lee, L. W. Trevoy, J. W. T. Spinks, and L. B. Jaques, Proc. Soc. Exptl. Biol.
Med. 74, 151 (1950); J. W. T. Spinks and L. B. Jaques, Natwre 166, 184 (1950).

102 §. Shapiro, M. Weiner, and G. Simson, New Engl. J. Med. 248, 775 (1950).

103 P. Gley and J. Delor, Bull. soc. chim. biol. 30, 891 (1948); J. Badin, C. Mentzer, J.
Moraux, and P. Meunier, Compt. rend. soc. biol. 144, 871 (1950).

432 VITAMIN K GROUP

phenylindanedione is relatively brief because this substance reduces glomer-
ular capillary resistance and is excreted rapidly; to counteract this phe-
nomenon, vitamin P has been used to increase the capillary resistance,
apparently successfully.

Link’s group!™ has synthesized dicoumarol from and degraded it to
salicylic acid. Since salicylates have a limited anticoagulant action, it has
been suggested that dicoumarol may act through its degradation prod-
ucts.8!» 1%, 106 The relative inefficiency of salicylates (1/500 the activity of
dicoumarol) makes this unlikely; however, one might speculate with Jaques
and Lepp!” that intestinal bacteria convert a small portion of ingested
salicylate into dicoumarol. Arguing along similar lines, Shemiakin and
coworkers'® believe that phthalates are biologic degradation products of
vitamin K and are responsible for the naphthoquinone’s coagulant activity.

For the effect of other drugs on blood clotting the excellent review of
Seegers!"* is referred to.

3, BLoop COAGULATION CHANGES WITH VITAMIN K DEFICIENCY

Hemorrhage associated with obstructive jaundice has been known and
reported for centuries; vascular changes, deficiency of various clotting com-
ponents, and the presence of clot-inhibiting agents have all been suggested.
Of the older concepts only the suggestion that antithrombin is excessive in
vitamin K deficiency persists. Dyckerhoff and Marx! claim that the
increased thrombin-destroying function of plasma is corrected by adminis-
tration of the vitamin.

Quick et al.,?® in 1935, announced that prothrombin was lacking in the
poorly coagulable blood of a patient with obstructive jaundice. In the
hemorrhagic chick disease of dietary origin Schgnheyder”® suspected a
deficiency of plasma prothrombin; Dam e¢ al." clearly demonstrated that
adding prothrombin from normal plasma corrected this deficiency; further,
they were unable to recover prothrombin, by acetone or isoelectric precipita-
tion, from the plasma of bleeding chicks. Quick!” convincingly confirmed
the prothrombin lack in the chick bleeding state. In the bleeding of dogs

104 M.A. Stahmann, C. F. Huebner, and K. P. Link, J. Biol. Chem. 138, 513 (1941).

105 §, Shapiro, J. Am. Med. Assoc. 125, 546 (1944).

106 Q. O. Meyer and B. Howard, Proc. Soc. Exptl. Biol. Med. 58, 234 (1943); S. Rapo-
port, M. Wing, and G. M. Guest, zbzd. 58, 40 (1943).

107 L. B. Jaques and E. Lepp, Proc. Soc. Exptl. Biol. Med. 66, 178 (1947).

108 M. M. Shemiakin, L. A. Schukina, and J. B. Shevezov, Nature 151, 585 (1943); M.
M. Shemiakin and L. A. Schukina, zbzd. 154, 513 (1944).

108a W. H. Seegers, Pharmacol. Revs. 3, 278 (1951).

109 H. Dyckerhoff and R. Marx, Biochem. Z. 311, 1 (1942).

10 F, Schgnheyder, Biochem. J. 30, Part 1, 890 (1986).

11H. Dam, F. Schgnheyder, and E. Tage-Hansen, Biochem. J. 30, Part 1, 1075 (1936).

u2 A. J. Quick, Am. J. Physiol. 118, 260 (1937).

VII. EFFECTS OF DEFICIENCY 433

with biliary fistulas, Hawkins and Brinkhous*® detected severe hypopro-
thrombinemia. In these various hemorrhagic states, all attributable to a
deficiency of vitamin K, and studied by a variety of methods, the common
denominator has been found to be a lack of the plasmatic precursor of
thrombin. When this clotting change was found to be readily corrected by
vitamin K it was felt!*—and is still generally believed—that the lack of
prothrombin is the only immediate result of vitamin K deficiency, and hence
the sole cause of bleeding in this deficiency state.

Such a simple, attractive concept—pure hypoprothrombinemia—over-
looks some disturbing facts. The newborn infant has a prothrombin value
only one-fourth to one-third that of adults. The prothrombin titer rises
slowly, reaching normal adult levels near the end of the first year of life.
Despite this rather pronounced hypoprothrombinemia, the newborn’s clot
time is normal or even shortened, and the ‘‘prothrombin time” of Quick is
usually normal; bleeding rarely occurs under these circumstances.*: 1° 71»
14, 15 Turing the first week of life drastic changes occur; generally between
the second and fifth days the “prothrombin time” is prolonged and clot
times may be lengthened; if these changes are extreme, hemorrhage may
ensue—clinically described as “‘hemorrhagic disease of the newborn.” Actual
bleeding is uncommon, and spontaneous return to normal takes place by
the end of the first week of life, if digestive disturbances have not inter-
vened.'6 It is well known that vitamin K and its analogs can prevent or
quickly correct these clotting and bleeding changes. Yet, throughout this
brief neonatal period the true prothrombin level of the blood is low and
fluctuates little.

Here, then, is one instance of hemorrhagic diathesis developing without
a significant alteration in the plasma prothrombin concentration, and what-
ever the abnormality may be, vitamin K is corrective.

Mann and Hurn"’ have described a new blood coagulation factor whose
activity appears to be related to the function of thromboplastin in the first
stage of clotting, hence ‘‘cothromboplastin.’’ When this factor is deficient,
prothrombin, regardless of how much is present, can convert to thrombin
only with difficulty; as a result of a deficiency of cothromboplastin the
“prothrombin time” of Quick is prolonged. It has been found that cothrom-

113 Reviewed by K. M. Brinkhous, Medicine 19, 329 (1940).

4 W.W. Waddell, Jr., and D. Guerry, III, J. Am. Med. Assoc. 112, 2259 (1939).

115 C, A. Owen, G. R. Hoffman, S. E. Ziffren, and H. P. Smith, Proc. Soc. Exptl. Biol.
Med. 41, 181 (1939); H. Dam, E. Tage-Hansen, and P. Plum, Lancet II, 1157 (1939);
E. Oehler, Monatsschr. Kinderheilk. 90, 394 (1942).

116 F, Widenbauer and U. Krebs, Monatsschr. Kinderheilk. 90, 173 (1942); S. Rapoport
and K. Dodd, Am. J. Diseases Children 71, 611 (1946).

17 FF, D. Mann, Am. J. Clin. Pathol. 19, 861 (1949); F. D. Mann and M. M. Hurn,
ibid. 20, 225 (1950).

434 VITAMIN K GROUP

boplastin diminishes in the vitamin K deficiency of obstructive jaundice
and returns to normal under the influence of vitamin K therapy.!!® Certain
characteristics of cothromboplastin are its presence in normal plasma or
serum and even in aged plasma [thus clearly differentiating it from both
prothrombin and Ac-globulin (factor V or labile factor)].

During the interval when the “prothrombin time” of the newborn is
prolonged, a prothrombin conversion factor was found lacking by Randall
and Randall; this factor is present in normal plasma, aged plasma, and
serum.

Among the clotting changes resulting from dicoumaro] therapy is a
diminution of stable prothrombin conversion factor;!° this precedes the
drop in prothrombin. The’ stable factor—so named to distinguish it from
the labile factor of Quick—is present in normal plasma, prothrombin-poor
serum, and aged plasma. Cothromboplastin, in these and other respects,
appears to be identical with the stable factor. Both resemble, at least
superficially, the factor of Randall and Randall.

Dam and coworkers”! have given their support to the opinion that there
are non-prothrombic clotting changes in both simple vitamin K deficiency
and dicoumarolization; however, they suggest that a “delta” factor is
lacking in the former, but a different (‘‘kappa’’) factor is reduced in the
latter (delta present in dicoumarol plasma and kappa in vitamin K-deficient
plasma). That a difference exists between dicoumarol plasma and plasma of
simple vitamin K deficiency has been both confirmed!* and denied.!'»

It becomes increasingly clear that the “hypoprothrombinemia”’ of the
newborn infant, of obstructive jaundice, and of dicoumarol therapy in-
volves, in addition to lowered prothrombin, one or more non-prothrombic
factors.!22 Since vitamin K overcomes the coagulation defect in all three
conditions, one is inclined to recall the statement of Boyd and Warner:

118 FD. Mann, E. §. Shonyo, and F. C. Mann, Am. J. Physiol. 164, 111 (1951).

119 A, Randall, IV, and J. P. Randall, Proc. Soc. Exptl. Biol. Med. 70, 215 (1949).

120 ©, A. Owen, Jr., and J. L. Bollman, Proc. Soc. Exptl. Biol. Med. 67, 367 (1948);
C. A. Owen, Jr., T. B. Magath, and J. L. Bollman, Am. J. Physiol. 166, 1 (1951).

121 H, Dam, Nature 161, 1010 (1948); H. Dam and E. Sgndergaard, Biochim. et Biophys.
Acta 2, 409 (1948); @. Sgrbye, I. Kruse, and H. Dam, Acta Chem. Scand. 4, 549,
831 (1950); Anonymous, Nutrition Revs. 9, 36 (1951).

21a M. Verstraete and R. Verwilghen, Acta clin. belg. 6, 269 (1951).

121b A. J. Quick, C. V. Hussey, and G. E. Collentine, Proc. Soc. Exptl. Biol. Med. 79,
131 (1952).

122 See also: F. Koller, A. Loeliger, and F. Duckert, Acta Haematol. 6, 1 (1951); R. F.
Jacox and R. F. Bays, Blood 5, 313 (1950); R. L. MacMillan. Science [N.8.] 108, 416
(1948); P. A. Owren, Rev. hématol. 6, 135 (1951); C. A. Mawson, J. Lab. Clin. Med.
34, Part 1, 458 (1949); H. E. Schultze, Arch. exptl. Pathol. Pharmakol. 207, 173
(1949); G. Y. Shinowara and W. B. Smith, Am. J. Clin. Pathol. 20, 341 (1950); L.
A. Sternberger, Blood 4, 1131 (1949).

23 E. J. Boyd and E. D. Warner, J. Lab. Clin. Med. 33, 1431 (1948).

VIII. PHARMACOLOGY 435

“Tt is possible that vitamin K affects factors which govern prothrombin
conversion as well as those which control its concentration.”

VIII. Pharmacology
CHARLES A. OWEN, JR.

A. VITAMIN K PREPARATIONS

Phytyl menadione (thylloquinone, vitamin K,, 2-methyl-3-phytyl-1 ,4-
naphthoquinone) has been synthesized and is commercially available, al-
though expensive. Concentrates of natura! vitamin K and synthetic
menadione (U.S.P.) (menaphthone B.P., 2-methyl]-1 ,4-naphthoquinone)
are relatively inexpensive. All these substances are fat-soluble, and when
given orally in the absence of intestinal bile they require the administration
of bile salts.

For the simple deficiencies of vitamin K the water-soluble analogs are
generally preferred, such as the following: menadione sodium bisulfite;
4-amino-2-methyl naphthol HCl; or 2-methyl-1 ,4-naphthohydroquinone
diphosphate tetra sodium (a menadiol derivative). In the uncomplicated
vitamin K deficiencies menadione has been found to be the strongest cor-
rective analog with approximately two to four times the activity of phytyl
menadione.!:? Despite an occasional objection,’ the weight of evidence is
that vitamin K, is more effective than menadione in counteracting the
effects of the anticoagulant dicoumarins.

B. DOSAGES

For the vitamin K deficiency of obstructive jaundice, biliary fistula, or
hemorrhagic disease of the newborn, daily doses of 1 to 5 mg. of any of the
vitamin K analogs are usually adequate. One milligram is-stated to be
sufficient to prevent the clotting changes produced by 1 g. of sodium acetyl-
salicylate.4 Ten to twenty milligrams has been recommended for adminis-
tration to the mother during the last few hours of labor to prevent hemor-
rhagic disease of the infant. These doses are so much greater than minimal
effective ones that the slight differences of potency of the various vitamin

1H. J. Almquist and A. A. Klose, Proc. Soc. Exptl. Biol. Med. 45, 55 (1940).

25. A. Thayer, R. W. McKee,S. B. Binkley, and E. A. Doisy, Proc. Soc. Exptl. Biol.
Med. 44, 585 (1940); S. A. Thayer, R. W. McKee, 8S. B. Binkley, D. W. MacCorquo-
dale, and E. A. Doisy, ibid, 41, 194 (1939); H. J. Almquist and A. A. Klose, J.
Biol. Chem. 130, 787 (1939).

8S. Shapiro, M. Weiner, and G. Simson, New Engl. J. Med. 248, 775 (1950).

48. Shapiro, J. Am. Med. Assoc. 125, 546 (1944).

436 VITAMIN K GROUP

analogs become inconsequential. Little has been done to investigate the
possibility of using smaller quantities of vitamin K, but from a practical
point of view there would seem to be little advantage.

Richards and Shapiro® stress that larger doses of vitamin K are needed
for the treatment of overdicoumarolization, in the presence of hepatic
damage, or simply in the performance of an evaluation of hepatic function.
Cromer and Barker, among others,® found that 60 mg. of anhydrous
menadione bisulfite (equivalent to 72 mg. of the hydrated ester) was
sufficient to correct the prolonged ‘‘prothrombin time” of most patients
who had responded to dicoumarol with unusual vigor. However, an occa-
sional dicoumarolized patient is unresponsive to the water-soluble analogs
even in doses of 600 mg. given intravenously. In such eases the administra-
tion of phytyl menadione (up to 1 g. orally or intravenously) has been re-
markably successful.? The more stable oxide of phytyl menadione may
prove to be practical.

C. MODE OF ADMINISTRATION

The water-soluble naphthoquinones may be given orally, subcutaneously,
intramuscularly, intraperitoneally, or intravenously with substantially the
same results; regardless of the route a lag of 1 to 2 hours occurs before any
clotting response is perceptible.’ Inunctions of water-soluble vitamin K
derivatives have proved effective,’ but they may be irritating.!° The fat-
soluble K-quinones are generally given orally, accompanied by bile salts in
the case of obstructive jaundice or biliary fistula but not in antidicoumarol
therapy where bile is available to the bowel. Davidson and MacDonald’
have described the suspension of fat-soluble preparations for intravenous
use: up to 450 mg. of vitamin K, was dissolved in 15 to 20 ml. of absolute

®R. K. Richards and 8. Shapiro, J. Pharmacol. Exptl. Therap. 84, 93 (1945).

6 J. Lehmann, Science [N. S.] 96, 345 (1942); S. Shapiro, M. H. Redish, and H. A.
Campbell, Proc. Soc. Exptl. Biol. Med. 52, 12 (1943); H. E. Cromer, Jr., and N. W.
Barker, Proc. Staff Meetings Mayo Clinic 19, 217 (1944).

7C.§8. Davidson and H. MacDonald, New Engl. J. Med. 229, 353 (1943); S. P. Lucia
and P. M. Aggeler, Proc. Soc. Exptl. Biol. Med. 56, 36 (1944); D. F. James, I. L.
Bennett, Jr., P. Scheinberg, and J. J. Butler, Arch. Internal Med. 88, 632 (1949);
R. Miller, W. P. Harvey, and C. A. Finch, New Engl. J. Med. 242, 211 (1950); E. T.
Phelps and 8. N. Jones, Bull. Georgetown Univ. Med. Center 4, 41 (1950) ; D.M. Watkin,
T.B. Van Itallie, W.B. Logan, R.P. Geyer,C.S. Davidson, andF. J. Stare, J. Lab.
Clin. Med. 37, 269 (1950); A. S. Douglas and A. Brown, Brit. Med. J. 1, 412 (1952);
A. Rehbein, A. Jaretzki, III, and D. V. Habif, Ann. Surg. 135, 454 (1952); R.
Stragnell, Am. Heart J. 44, 124 (1952); F. Koller, A. Loeliger, and P. Fliickiger,
Helv. Med. Acta 19, 411 (1952).

8 H. C. Willumsen, H. E. Stadler, and C. A. Owen, Proc. Soc. Exptl. Biol. Med. 417,
116 (1941).

9H. K. Russell and R. C. Page, Am. J. Med. Sci. 202, 355 (1941).

10R. C. Page and Z. Bercovitz, Am. J. Med. Sci. 203, 566 (1942).

~~ |e

VIII. PHARMACOLOGY 437

alcohol, which was then slowly mixed with | to 1.5 1. of 5 to 10% sterile
glucose solution (the alcohol may lead to transient euphoria). Phelps and
Jones’ employed a similar technique but increased the dose of phytyl
menadione or its oxide to | g. Blood transfusions were reported by Kinsey!
to be more effective in the treatment of the bleeding of hepatic failure if
the blood donor was pretreated with vitamin K; confirmation is lacking.”

D. ACTION OF VITAMIN K

As has already been suggested, prothrombin production is believed to
be intimately related to the liver, for a constant level of prothrombin in
the blood is found only if hepatic function is approximately normal. Assays
of prothrombin in portal lymph demonstrated a concentration near that of
the blood, whereas lymph from other sources had lesser concentrations."
These facts suggest either that the plasma prothrombin is synthesized in
the liver or that the finishing touches of proteins made elsewhere are ac-
complished by the liver. Furthermore, the action of vitamin K is demonstra-
ble only if hepatic function is adequate. The work of Jaques’ group™
indicates that vitamin K displaces dicoumarol from the liver and so ap-
parently acts on that organ. There is other evidence that may link vitamin
K with the liver: Nassi and Ragazzini'> found that relatively large doses of
vitamin K (60 to 500 mg.) depressed hepatic glycogen. Dogs on normal diets
when given cinchophen did not show clotting changes, according to Hue-
per,!® but did show changes when they were given a vitamin K-free diet.
Honorato’s group” has reported that on either a choline-free or vitamin
K-free diet rats developed fatty livers; administration of choline or vitamin
K in either case reduced the hepatic defect. Field and Dam were unable to
detect any change in hepatic lipids of vitamin K-deficient chicks, however,
and the results of the common clinical tests of hepatic function are normal
in uncomplicated vitamin K deficiency states.!

Whether vitamin K acts as a coenzyme for a prothrombin-synthesizing
enzyme in the liver or whether a part or all of the vitamin is incorporated
in the prothrombin molecule is not known. The latter might be suspected,

UR. E. Kinsey, Arch. Internal Med. 73, 131 (1944).

2 ALR. Butt, T. B. Magath, and T. H. Seldon, Arch. Internal Med. 81, 131 (1948).

18K. M. Brinkhous and 8S. A. Walker, Am. J. Physiol. 182, 666 (1941).

144C.C. Lee, L. W. Trevoy, J. W. T. Spinks, and L. B. Jaques, Proc. Soc. Exptl. Biol.
Med. 74, 151 (1950); J. W. T. Spinks and L. B. Jaques, Nature 166, 184 (1950).

167. Nassi and F. Ragazzini, Boll. soc. ztal. biol. sper. 24, 703 (1948).

16W.C. Hueper, Arch. Pathol. 41, 592 (1946).

17C. R. Honorato and H. Molina, Rev. soc. argentina biol. 18, 431 (1942);G. 8S. Topel-
berg and C. R. Honorato, ibid. 19, 409 (1948).

18 J. B. Field and H. Dam, Proc. Soc. Exptl. Biol. Med. 60, 146 (1945).

19M. A. Pessagno Espora, Dia méd. 22, 1264 (1950).

438 VITAMIN K GROUP

since Lyons?’ found evidence for a quinone structure in thrombin. The
consensus, however, is that vitamin K does not actually constitute a part
of the prothrombin molecule. Dam and associates?! were unable to find
any vitamin K activity in refined prothrombin preparations. Quick” has
calculated that the amount of vitamin K which will raise the plasma pro-
thrombin is significantly less than 1 mole for each mole of prothrombin
(assuming a prothrombin molecular weight of 140,00023 and concentration —
of 20 mg. per 100 ml. in the plasma).”4 Such calculations have no meaning
for non-prothrombic factors whose homeostatic level also seems to depend
on vitamin K, for molecular data and plasma concentrations are unknown.
The preparation of menadione with CH; in the 2 position, or C™ in either
of the rings, has been described;?*> preliminary biologic studies with such
preparations have been reported.®

What enzymes or types of enzyme are involved in the vitamin K reaction
is by no means clear. A variety of isolated experiments has been performed
in which naphthoquinones with vitamin K activity have had a stimulating
or inhibitory effect; in most cases the evidence is not clear whether the
phenomena are actually vitamin K-specific or are a general property of
naphthoquinones. Enzyme systems reported to be inhibited by 1 ,4-naph-
thoquinones include heart muscle succinoxidase,”” choline acetylase,”® and
lactic acid-producing bacterial enzymes.” The configuration of menadione
and phytyl menadione is theoretically and actually adequate for Stecker
degradation of a-amino acids.*°

McCawley and Gurchot*! reported that the redox potential of vitamin K
is characteristic of those substances inhibiting catheptic proteolysis. They

20 R. N. Lyons, Nature 155, 633 (1945).

21H. Dam, J. Glavind, L. Lewis, and E. Tage-Hansen, Skand. Arch. Physiol. 79,
Suppl. 121 (1938).

22 A. J. Quick, The Physiology and Pathology of Hemostasis. Lea and Febiger,
Philadelphia, 1951.

23 W. H. Seegers and A. G. Ware, Federation Proc. 7, 186 (1948).

24 W. H. Seegers, E. C. Loomis, and J. M. Vandenbelt, Proc. Soc. Exptl. Biol. Med.
56, 70 (1944).

25 PP. P. T. Sah, Z. Vitaminforsch. 3, 40 (1949-1950) ; C. J. Collins, J. Am. Chem. Soc.
73, 1038 (1951); A. Murray and A. R. Ronzio, ibid. 74, 2408 (1952); R. V. Phillips,
L. W. Trevoy, L. B. Jaques, and J. W. T. Spinks, Can. J. Chem. 30, 844 (1952).

26 P. F. Solvonuk, L. B. Jaques, J. E. Leddy, L. W. Trevoy, and J. W. T. Spinks,
Proc. Soc. Exptl. Biol. Med. 79, 597 (1952).

27H. G. Ball, C. B. Anfinsen, and O. Cooper, J. Biol. Chem. 168, 257 (1947).

*8C. Torda and H. G. Wolff, Proc. Soc. Exptl. Biol. Med. 57, 236 (1944); C. Torda
and H. G. Wolff, Science [N. S.] 103, 645 (1946).

29 W. D. Armstrong, W. W. Spink, and J. Kahnke, Proc. Soc. Exptl. Biol. Med. 53,
230 (1943); P. Atkins and J. L. Ward, Brit. J. Exptl. Pathol. 26, 120 (1945).

30 A. Schgnberg, R. Moubasher, and A. Said, Nature 164, 140 (1949).

31H. L. McCawley and C. Gurchot, Univ. California Publs. Pharmacol. 1, 325 (1940).

VIII. PHARMACOLOGY 439

fee] that certain naphthoquinones act as plant respiratory pigments. This
is of interest, since the plant leaves in which vitamin K is found are capable
of synthesizing the vitamin to any extent only if exposed to sunlight.* *
Wright* intimates that vitamin K may be an essential metabolite of all
forms of life.

Bacteria are known to produce vitamin K, and the possibility that the
vitamin is a growth factor has been entertained. According to Guérillot-
Vinet*®* phthiocol and menadionein concentrations of 10~* to 10-° are growth
factors for Mycobacterium paratuberculosis and Aspergillus niger, but at the
concentration at which vitamin K is produced by bacteria (8 to 152 y per
gram of dry weight) it is definitely bacteriostatic. The inhibition in vitro
of many bacteria by vitamin K analogs has been reported: Streptococcus,
pneumococcus, Salmonella typhosa and paratyphi, Brucella, Staphylococcus,*®
anthrax,” mycobacteria,®® corynebacteria;*? the mode of action on the vari-
ous bacteria may vary with the analog employed.*® Iland?® doubts that the
vitamins K are true growth factors but suspects they may play some
metabolic role in the bacteria. The quinone growth-stimulating properties
may be unrelated to antihemorrhagic activity, since the 4-aminonaphthol
analog has only an inhibitory action.*® Bacterial inhibition by dicoumarol
is not reversed by vitamin K, but is reversed by vitamin P.*! However, the
strong bacteriostatic action of salicylic acid is opposed by vitamin K
(weakly), and, in the case of the inhibition of Fusaria by 2-chloromenadione,
menadione is strongly competitive.*®> Inhibition of streptococcal growth by
the pigment iodinin® and of the growth of yeasts by 2,3-dichloro-1 ,4-
naphthoquinone* is counteracted by menadione.

Other inhibitory actions of menadione have been demonstrated, again
without information as to the specificity of the inhibition. Menadione and
derivatives shorten the life ot Daphnia magna;* inhibit the growth of roots
of Allium cepa, the onion roots becoming yellow and flaccid within a week;**

2H. J. Almquist and E. L. R. Stokstad, J. Nutrition 12, 329 (1936).

38 H. Dam and J. Glavind, Biochem. J. 32, 485 (1938); J. Erkama and N. Pettersson,
Acta Chem. Scand. 4, 922 (1950).

47. D. Wright, J. Am. Dietet. Assoc. 28, 289 (1947).

35 M. Guérillot-Vinet, Bull. soc. chim. biol. 30, 863 (1948).

36 F. Mulé, Jl Policlinico (Rome) sez. prat. 58, 653 (1946).

37 L. Donatelli and R. Davoli, Boll. soc. ital. biol. sper. 22, 134 (1946).

38 A. Kimler, J. Bacteriol. 60, 469 (1950).

39 LL. Nassi, Boll. soc. ital. biol. sper. 22, 141 (1946).

40C. N. land, Nature 161, 1010 (1948).

41 J. Naghski, M. J. Copley, and J. F. Couch, Science [N. 8.] 105, 125 (1947).

#” AH. Mellwain, Biochem. J. 37, 265 (19438).

DD. W. Woolley, Proc. Soc. Exptl. Biol. Med. 60, 225 (1945).

44V. Schecter, Proc. Soc. Exptl. Biol. Med. 74, 747 (1950).

495M. Levine and §. A. Rice, Proc. Soc. Exptl. Biol. Med. 74, 310 (1950).

440 VITAMIN K GROUP

gel the cytoplasm of Arbacia punctulata eggs, stimulating parthenogenesis ;4° —
and inhibit respiration of these sea urchin eggs.*” Respiration of Plasmodium
knowlesi and of yeast cells is also depressed.??

In mammals a variety of actions, which seem unrelated to its blood
coagulating function, has been attributed to vitamin K. For example:
Chamorro® administered 10 to 20 mg. of vitamin K daily to prepubertal
rabbits over a 2-week period; mammary, uterine, and vaginal changes were
of the estrogenic type. Somewhat similar estrogenic effects have been ob-
served in the guinea pig and rat.?? Vitamin K administered in conjunction
with follicular hormone is reported to elevate the prothrombin level of
normal tomeats.°® In one case of breast malignancy vitamin K seemed to
oppose the action of stilbestrol.*! Another reported correlation between
vitamin K and hormones is the apparently successful treatment of Rh-
sensitized pregnant women with the vitamin plus progesterone.” Woolley*?
was able to induce toxic changes in pregnant rats and mice with a-tocoph-
erolquinone; these were apparently a manifestation of the quinone, for
vitamin K but not a-tocopherol prevented the toxicity. Chauchard et al.**
noted peripheral nerve chronaxial alterations in the vitamin K-deficient
rat; changes were also induced by either menadione or dicoumarol treat-
ment, but not if both were given together. Muscle strip sensitivity to acetyl-
choline and potassium is increased by vitamin K according to Torda and
Wolff.°> Vitamin K is said to lower glutathione in the blood of man or the
dog,** elevate the blood sugar of children,*” reduce the cholesterolemia of
parkinsonian encephalitis,®® elevate the depressed concentration of platelets
produced by salicylate therapy,°*? alter the serologic reaction and erythro-
cyte sedimentation rates of syphilitic patients,®° raise plasma euglobulin

46 A. Halaban, Biol. Bull. 97, 240 (1949).

47C. B. Anfinsen, J. Cellular Comp. Physiol. 29, 323 (1947).

48 A. Chamorro, Compt. rend. soc. biol. 140, 498 (1946).

49 F, Vicari and M. Gaglio, Boll. soc. ital. biol. sper. 28, 1142 (1947); T. Paladino, zbid.
24, 303 (1948); V. Truglio and G. Arcidiacono, zbid. 24, 1059 (1948).

50 H). Szirmai, Gynaecologia 133, 163 (1952). i

51G.G. Binnie, Brit. J. Radiol. [N. 8.] 24, 691 (1951). *

82 P. B. Hoffman and D. E. Edwards, Am. J. Obstet. Gynecol. 59, 207 (1950); L. J.
Paquette and J. T. Schmitz, Wisconsin Med. J. 51, 473 (1952). |

8D. W. Woolley, J. Biol. Chem. 159, 59 (1945).

54 P, Chauchard, H. Mazoué, and R. Lecoq, Compt. rend. soc. biol. 140, 474 (1946); —
R. Lecoq, P. Chauchard, and H. Mazoué, ibid. 140, 743 (1946).

55 C. Torda and H. G. Wolff, Exptl. Med. Surg. 4, 50 (1946).

56 KH. Azerad, Seeman, and Obadia, Semaine hép. Paris 27, 110 (1951).

57 |. Nassi, Boll. soc. ital. biol. sper. 24, 706 (1948).

588 G. Fattovich, Acta Vitaminol. 1, 26 (1946); Abstr. Intern. Z. Vitaminforsch. 20,
328 (1948).

59M. Pellegrini and M. Ghirlanda, Jl Progr. med. 5, 434 (1948); Abstr. Intern. Z
Vitaminforsch. 21, 381 (1949-1950).

60 G, Buccellato, Boll. soc. ital. biol. sper. 24, 126, 127 (1948).

VIII. PHARMACOLOGY 441

and albumin values (not confirmed by Piacentini®), accentuate the tuber-
culin reaction,® raise the serum bilirubin level in cases of hepatitis,®! and
rectify increased fragility.®* Maltaner and Thompson®* quote Schgnheyder’s
dissertation as reporting elevated phosphorus and lowered calcium in the
blood of vitamin K-deficient chicks; their data do not substantiate the
claim. The anemia of vitamin K deficiency is apparently not a primary
effect, as suggested by Thayer and associates,” but secondary to hemor-
rhage.® According to Biising and Zuzak,*® reduction of complement accom-
panies the hypoprothrombinemia of vitamin K deficiency in chicks and
rises when the vitamin is administered; yet Felix and associates’ found no
clear depression of complement with dicoumarol treatment. The effect of
menadione derivatives on serum agglutinins is disputed.7! Hypoprothrombi-
nemia associated with pernicious anemia has been found responsive to liver
extracts but not to vitamin K.”

Clinical syndromes and diseases reported to be improved or cured by
vitamin K, other than hypoprothrombinemic bleeding states, include: an-
gioneurotic edema,” arterial hypertension™ (Taveira7®), bacillary dysen-
tery,’® brucellar meningitis,” chilblain,’ cirrhosis of the liver,’? dental
caries*® (questioned by Granados and associates*!), hemophilia® (contrary

61 R. Breda, Atti soc. lombarda sci. med. e biol. 1, 85 (1946); Abstr. Intern. Z. Vita-
minforsch. 20, 328 (1947-1948).

6 C. Piacentini, Boll. soc. ital. biol. sper. 24, 530 (1948).

68 C. Rossi and L. Brighenti, Boll. soc. ital. biol. sper. 28, 327 (1947).

64 G. Gottsegen, Z. Horn, and M. C. Haéry, Acta Med. Scand. 140, 127 (1951).

65 G. Leonardi, Acta Vitaminol. 1, 54 (1947).

66 F. Maltaner and W. R. Thompson, Arch. Biochem. 2, 49 (1943).

67S. A. Thayer, R. W. McKee, D. W. MacCorquodale, and E. A. Doisy, Proc. Soc.
Exptl. Biol. Med. 37, 417 (1937).

6 H. J. Almquist, E. Mecchi, and A. A. Klose, Biochem. J. 82, 1897 (1938).

69 K. H. Biising and H. Zuzak, Z. Immunitdtsforsch. 102, 401 (1943).

70K. Felix, I. Pendl, and L. Roka, Hoppe-Seyler’s Z. physiol. Chem. 284, 198 (1949).

a1 J.K.Narat, J. Am. Med. Assoc. 116, 1310 (1941); M. R. H. Stoppelman, Acta Med.
Scand. 111, 408 (1942); A. Gammelgaard, E. H. Larsen, and P. V. Marcussen, zbid.
116, 8 (1943); G. Trénnberg, Nord. Med. 21, 13 (1944).

7” EK. D. Warner and C. A. Owen, Am. J. Med. Sci. 208, 187 (1942).

73 P. Kallés, Gastroenterologia 71, 171 (1946).

4 E. Bellini, Minerva med. 39, Part 1, 56 (1948); M. Lippi, Acta Vitaminol. 1, 113
(1947); J. Nasello and P. B. Camponovo, Prensa méd. argent. 87, 2864 (1950).

79M. Taveira, Ann. pharm. frang. 9, 344 (1951).

76M. F. Krause, Hospital O (Rio de Janeiro) 39, 361 (1951).

7R. Virgili and V. Del Vecchio, Boll. soc. ital. biol. sper. 24, 748 (1948).

73 A. A. Cordero, Prensa méd. argent. 38, 1158 (1951).

79M. Kiiley, Schweiz. med. Wochschr. 79, 365 (1949).

80D. Y. Burrill, J. C. Calandra, E. B. Tilden, and L. S. Fosdick, J. Dental Researc
24, 273 (1945); L. S. Fosdick, ibid. 27, 235 (1948).

81H. Granados, A. Snog-Kjaer, J. Glavind, and H. Dam, Proc. Soc. Exptl. Biol.
Med. 72, 669 (1949).

442 VITAMIN K GROUP

to almost universal opinion), hepatitis,®? menometrorrhagia,*® ‘‘scours”’ in
calves,®* serofibrinous pleurisy,®° thrombocytopenia,®!: 8° (denied by Zuck-
er’), tinea kerion,®® and whooping cough.®® The hypoprothrombinemia of
tuberculosis may be related to gastroenteric or hepatic involvement and
thus may be amenable to vitamin K therapy.°°

K. TOXICITY

Before the advent of dicoumarol, vitamin K was given in doses of only
a few milligrams a day. Now, doses of several hundred milligrams are given
liberally to overcome dicoumarol’s anticoagulant activity; for the most
part no toxic effects have been observed. Phelps and Jones’ did note nausea
in patients receiving 150 mg. of a menadiol derivative intravenously; with
600 mg. doses more severe reactions at times occurred. Intravenous injec-
tions of 1 g. of phytyl menadione led to no reactions, but the oxide of this
compound in the same dose was followed by a febrile response in one
patient. From studies on animals it seems likely that man can tolerate
amounts of vitamin K and its analogs much larger than those now being
tentatively employed.

Table VI outlines the toxicity of various menadione preparations given
in single doses by various routes; the amount administered in milligrams
per kilogram of body weight of the animal is recorded as that causing death
in half the animals tested. The immediate cause of death may be circula-
tory” or respiratory failure.®: %

Molitor and Robinson” demonstrated that lethal doses of menadione and
phthiocol were 500 and 350 mg. per kilogram of body weight, respectively,

& P, Ggtzsche, Ugeskrift Laeger 112, 1753 (1950); H.-O. Mossberg, Brit. Med. J. I.
1382 (1952).

83 T. Baranowski, H. Beck, and 8. Liebhardt, Am. Rev. Soviet Med. 3, 173 (1945);
R. Gubner and H. E. Ungerleider, Southern Med. J. 37, 556 (1944).

84 G. W. Anderson, W. M. DuPré, and J. P. LaMaster, Am. J. Vet. Research 18, 5
(1952).

85M. A. Josserand, Lyon méd. 176, 168 (1946); P. Pichat and H. Boucher, ibid. 176,
169 (1946).

86 F, Groér, T. Baranowski, and J. Rosenbusch, Am. Rev. Soviet Med. 3, 173 (1945).

87 M. B. Zucker, Proc. Soc. Exptl. Biol. Med. 62, 245 (1946).

88 ],, Nékdm, Jr., and P. Polgér, Acta Dermato-Venereol. 31, 344 (1951); H. Grimmer ~

and S. Rust, Z. Haut- u. Geschlechtskrank. 12, 102 (1952).

89 J. C. Oyhenart, Dia méd. 23, 527 (1951); J. Nemirovsky, Prensa méd. argent. 38,

1955 (1951); W. B. Pereira da Silva, Rev. brasil. med. 8, 673 (1951).

90 R. F. Sheely, J. Am. Med. Assoc. 117, 1603 (1941); E. Tanner and F. Suter, Schweiz.
med. Wochschr. 74, 552 (1944).

91§. Ansbacher, W. C. Corwin, and B. G. H. Thomas, J. Pharmacol. Exptl. Therap.
75, 111 (1942).

2 R. H. K. Foster, Proc. Soc. Exptl. Biol. Med. 45, 412 (1940).

93M. B. Shimkin, J. Pharmacol. 71, 210 (1941).

4H. Molitor and H. J. Robinson, Proc. Soc. Exptl. Biol. Med. 48, 125 (1940).

ee Ts 2 re iS

VIII. PHARMACOLOGY 443

when given to rats daily for 30 days. The growth rate of rats was found by
Hatton and associates®* to be diminished if 0.3 % of the diet was menadione;
when the concentration was raised to 0.8 %, depression of growth was severe
and was accompanied by anemia and splenomegaly. Sublethal doses of
menadione derivatives (but not of phytyl menadione) have induced anemia
in dogs,°® rats,** and rabbits, but occasionally polycythemia has been

TABLE VI
LD;) oF MENADIONE DERIVATIVES ADMINISTERED IN SINGLE DosEs To ANIMALS

Route of administration

Intramuscular or

Intravenous subcutaneous Oral
Com-_ Refer- Com-_ Refer- Com-_ Refer-
Animal Dose* pound® ence Dose* pound®- ence Dose* pound®- ence

Mouse 250 3 5 138 2 91 200 1 94

450 5 92 250 2 93 500 2¢ 94

275 4 98 300 4 98 620 2 91

100 of 91 800 2 93

80 5 93 300 5 91

400 5 93

. 400 4 98

Chick 840 2 91
Dog 100+ 3 5
100 4 98

Rabbit 15-20 2 98 15-20 2 98 250 2 91
120 3 5
100 4 98

* Dose in mg. per kg. body weight.

5 No toxicity with 6 g. phytyl menadione per kg. body weight when administered subcutaneously or
with 25 g. per kg. given orally.

© No toxicity with 25 g. phytyl menadione per kg. body weight when administered orally.

d Hsters of menadiol were progressively less toxic, in order: diacetate, dipropionate, di-n-valerate, di-
n-butyrate, di-isovalerate; this last one was not lethal in doses of 18 g. per kg. when administered orally.

* Compounds administered: 1, phthiocol: 2, menadione; 3, menadione bisulfite; 4, menadiol disucci-
nate; 5, menadiol.

observed in the same animals,°! apparently the result of chronically de-
creased oxygen consumption.*” Excess menadione has been found to cause
methemoglobinemia;*® Cannava®® has duplicated the hemoglobin change

% KE. H. Hatton, A. Dodds, H. C. Hodge, and L. 8. Fosdick, J. Dental Research 24,
283 (1945).

96 F. Koller, Schweiz. med. Wochschr. 69, 1159 (1939).

7 A. Cannavé and L. Untersteiner Occhialini, Boll. soc. ital. biol. sper. 28, 1041
(1947).

% K. Fromherz, Z. Vitaminforsch. 11, 65 (1941).

99 A. Cannava, Boll. soc. ital. biol. sper. 24, 593 (1948).

444 VITAMIN K GROUP

in vitro. Other toxic manifestations in animals are prophyrinuria, albumin-
uria,” and hemorrhagic extravasations into the liver and kidneys.°®

It may be noted that the naturally occurring phytyl menadione is non-
toxic in amounts that would be lethal if comparable quantities of the
artificial vitamins had been given. Similarly, when the solubilizing ester
chains are lengthened, toxicity decreases (Table VI). As the toxicity de-
creases with increasing molecular size, so does the antihemorrhagic activity ;
the two functions seem not to be directly related, however, for the former
diminishes at a much greater rate than the latter. An even more striking
example of disparity between activity and toxicity is the effect of methyla-
tion of naphthoquinone: toxicity diminishes 75%, prothrombinogenic
activity increases 2000-fold.”

Potentially, one of the more serious toxic manifestations of vitamin K
would be overcorrection of the prothrombin deficiency, that is, ‘‘hyperpro-
thrombinemia.” This has actually been reported! !° and also denied.!0: 1%
Curiously, the excess prothrombin is detected by one method alone: ‘‘pro-
thrombin times” on diluted (1:8) plasma. It should be noted that with this
method the fibrinogen concentration of the tested plasma is suboptimal
and the test may reflect fibrinogen changes! as readily as it does other
clotting factors; elevation of plasma fibrinogen has been noted with large
doses of vitamin K.1°

IX. Requirements

A. OF ANIMALS
H. J. ALMQUIST

Pe Gi zareic

Factors influencing the incidence of dietary hemorrhagic disease in chicks
were discussed by Almquist and Stokstad.! It was found that the vitamin
was synthesized in the droppings of chicks and in the lower intestinal tract
where appreciable absorption did not take place. The vitamin K intake of

100 R. 8S. Overman, J. B. Field, C. A. Baumann, and K. P. Link, J. Nutrition 23, 589
(1942).

101 J. B. Field and K. P. Link, J. Biol. Chem. 156, 739 (1944); P. N. Unger and 8S.
Shapiro, Blood 3, 137 (1948).

102 A. J. Quick, J. Lab. Clin. Med. 31, 79 (1946).

103 ©, EH. Brambel and F. F. Loker, Proc. Soc. Exptl. Biol. Med. 58, 218 (1943).

104 FH{. F. Deutsch and H. W. Gerarde, J. Biol. Chem. 166, 381 (1946).

105 G, A. Nitsche, Jr., H. W. Gerarde, and H. F. Deutsch, J. Lab. Clin. Med. 32, Part
1, 410 (1947).

1H. J. Almquist and E. L. R. Stokstad, J. Nutrition 12, 329 (1936).

IX. REQUIREMENTS 445

the parent hen was important in the survival of the chick on a K-deficient
diet and in controlling the blood-clotting time of the day-old chick.’ These
observations showed a transfer of the vitamin from the hen diet to the chick.
Alfalfa meal in the hen diet at a level of 2.5% was sufficient for protection
of the chick. The vitamin was found in egg yolk but not in egg white.
Certain indications® that the prothrombin content of chick blood rises for
the first 3 or 4 days of life despite very low vitamin K intake may have
been due to assimilation of vitamin K remaining in the unabsorbed yolk
which normally is present in the chick for some time after hatching. Cravens
et al.‘ have also found that the blood-clotting time and prothrombin content
of day-old chicks is dependent upon the vitamin K content of the ration
fed the parent hen. One to two per cent of dried alfalfa or grasses was
required in the hen diet to maintain a normal prothrombin content in the
day-old chick. In these studies!’ 4 higher intake by the hen of vitamin K
in the form of dried greens had little or no further effect on the chick.

Tidrick e¢ al.* injected 2-methyl-1 ,4-naphthohydroquinone disulfate into
hatching eggs and obtained chicks with superior reserve stores of vitamin
K, as indicated by retention of prothrombin levels in the chicks when fed a
vitamin K-deficient diet.

On constant submarginal levels of vitamin K in the diet chicks tend to
show a progressive increase of blood clotting time up to approximately two
weeks of age and then a decrease.” > § At this age the hemoglobin level of
chicks, even on a normal diet, passes through a minimum phase and begins
to increase again. The riboflavin requirement is probably at a maximum.
After 2 weeks the gain in weight per unit weight of food consumed decreases,
and the food intake of vitamins in reference to the weight of the bird begins
to increase. The age of 2 weeks appears to be a sensitive, if not critical,
physiological stage of the chick. It is evident, therefore, that the vitamin
K intake required to maintain any particular clotting time would vary
with the age of the chick over the first 2 to 3 weeks of life. However, the
vitamin K requirement does not seem to be related to the body weight of
the chick.’

If the vitamin K-deficient chick is given a sufficiently large dose of the
vitamin the clotting time or prothrombin time can be restored in 4 to 6
ours.’-1°

2H. J. Almquist, E. Mecchi, and A. A. Klose, Biochem. J. 32, 1897 (1938).

3F.W. Stamler, R. T. Tidrick, and E. D. Warner, J. Nutrition 26, 95 (1943).

4W. W. Cravens, 8. B. Randle, C. A. Elvehjem, and J. G. Halpin, Poultry Sez. 20,
313 (1941).

5R. T. Tidrick, F. W. Stamler, F. T. Joyce, and E. D. Warner, Proc. Soc. Exptl.
Biol. and Med. 47, 438 (1941).

6H. J. Almquist and E. L. R. Stokstad, J. Nutrition 14, 235 (1937).

7H. Dam, J. Glavind, L. Lewis, and E. Tage-Hansen, Skand. Arch. Physiol. 79. 121
(1938).

446 VITAMIN K GROUP

Much of the data on which comparisons of time of action and kind of
vitamin K may be based were expressed in terms of the number or per-
centage of depleted chicks in which the blood-clotting time was reduced to
10 minutes or less in 18 hours after dosage. A clotting time of 10 minutes
probably represents a blood prothrombin level of only 20 to 30% of nor-
mal.!° Nevertheless, the data obtained in these assays have comparative
value. The amount of vitamin K, for a curative effect by these standards
was found to be approximately 1 y per chick by several groups of investi-
gators.’ |» ? The corresponding dose of methylnaphthoquinone was 0.3 y™
to 0.64 7.3 With the same criterion of effect the curative dose in a 6-hour
assay was 1.5 y of vitamin K, and 0.5 y of methylnaphthoquinone."!

Later assays based upon the prothrombin-clotting time have shown that
5 y of methylnaphthoquinone was sufficient to bring the blood prothrombin
content of a chick from 10% of normal to normal in 4 hours.’ In another
example, a 16 y dose of methylnaphthoquinone was required for complete
recovery of prothrombin in 6 hours, whereas 8 y gave about 50 % recovery.!°
In 18 hours the prothrombin was restored to normal by a dose of 6 to 8 y of
methylnaphthoquinone while, at the same time, the whole blood-clotting
time was reduced to 2 minutes by as little as 1 y.!°

In longer assays, 5 days or more, approximately 2 y of methylnaphtho-
quinone daily per chick is required for a normal range of prothrombin.®*: ®
4,15 Vitamin K, at a daily level of 2 y is insufficient,“ and the full require-
ment may be as high as 3.8 y° in the young chick.

Qualitative requirement for the vitamin has been shown to exist with
several other kinds of birds.®

2. Rat

Greaves and Schmidt!” found that the low prothrombin in bile fistula rats
could be corrected by administration of a vitamin K concentrate. It was
difficult to deplete rats of vitamin K by dietary means, but eventually this

8 A. J. Quick and M. Stefanini, J. Biol. Chem. 175, 945 (1948).

9. Fernholz, S. Ansbacher, and H. B. MacPhillamy, J. Am. Chem. Soc. 62, 430
(1940).

10 R. T. Tidrick, F. T. Joyce, and H. P. Smith, Proc. Soc. Exptl. Biol. Med. 42, 853
(1939).

1. F. Fieser, M. Tishler, and W. L. Sampson, J. Biol. Chem. 187, 659 (1941).

122 A.D. Emmett, R. A. Brown, and O. Kamm, J. Biol. Chem. 132, 467 (1940).

13 J. Lee, U. V. Solmssen, A. Steyermark, and R. H. K. Foster, Proc. Soc. Exptl.
Biol. Med. 45, 407 (1940).

14H. J. Almquist and A. A. Klose, J. Biol. Chem. 180, 787 (1939).

16H. J. Almquist and A. A. Klose, Proc. Soc. Exptl. Biol. Med. 45, 55 (1940).

16H. J. Almquist, Physiol. Revs. 21, 194 (1941).

17 J. D. Greaves and C. L. A. Schmidt, Proc. Soc. Exptl. Biol. Med. 37, 43 (1937).

IX. REQUIREMENTS 447

was done by Greaves’ and by Dam and Glavind!® and the curative effect
of vitamin K demonstrated. Some individual rats remained highly resistant
to depletion attempts. Flynn and Warner”? found that depleted bile fistula
rats required approximately 1000 y of phthiocol or 2 y of methylnaphtho-
quinone daily for moderate recovery of prothrombin level in 2 days. The
disulfuric acid ester of methylnaphthohydroquinone, which is much less
potent than the parent compound, was required at 8 y per day to cure
depleted rats.?!

3: Doe

Dogs rendered vitamin K-deficient by cholecystnephrostomy were found
by Collentine and Quick” to respond to 9 y of phyty! menadione per kilo-
gram of body weight when administered intravenously. With this dose the
“prothrombin time”’ returned to normal in 4 hours and persisted at that
level for 24 hours. From this rapid response it seems likely that much smaller
doses would have appreciable effects. It should be stated that changes in
“prothrombin time”’ and in the actual prothrombin concentration bear no
fixed relationship to each other, so that no direct comparison can be made
of minimal dosages determined by the various methods.

B. OF HUMAN BEINGS
CHARLES A. OWEN, JR.

Few studies have been conducted on human beings to ascertain the
minimal quantities of vitamin K derivatives necessary to correct the clot-
ting changes of the vitamin’s deficiency. Even in animals few reports have
been published, primarily because a severe deficiency of vitamin K which
will be uniform for a period sufficient to enable one to analyze various
dosages is difficult to obtain. Another problem which faces the investigator
is the one of vitamin K stores. Although only small amounts of vitamin K
can be recovered from the various organs of normal animals,”’: *4 it is
apparent that these stores are significant when the minuteness of daily
needs is realized. Direct evidence that these stores are important may be
deduced from the observation that a prolonged vitamin K-free diet scarcely
alters the blood coagulability of rats; if ligation of the common duct is now |

18 J. D. Greaves, Am. J. Physiol. 125, 429 (1939).

19H, Dam and J. Glavind, Z. Vitaminforsch. 9, 71 (1939).

20 J. E. Flynn and E. D. Warner, Proc. Soc. Exptl. Biol. Med. 48, 190 (1940).

21. D. Warner and J. E. Flynn, Proc. Soc. Exptl. Biol. Med. 44, 607 (1940).

22. G. KE. Collentine and A. J. Quick, Am. J. Med. Sci. 222, 7 (1951).

23 A. J. Almquist and E. L. R. Stokstad, J. Nutrition 12, 329 (1936).

24H. Dam, J. Glavind, L. Lewis, and E. Tage-Hansen, Skand. Arch. Physiol. 79,
Suppl. 121, (1938).

448 VITAMIN K GROUP

performed, the plasma prothrombin falls much more rapidly than that of

rats similarly operated on, which, however, had a normal diet.?>-?7

In the newborn infant it has been suggested*§ that a single intramuscular
dose of 10 y of 4-amino-2-methyl naphthol HCl given at birth will prevent
almost completely a prolongation of the ‘‘prothrombin time” during the
first week of life; daily doses of 1 y were equally effective. Once the ‘‘physi-
ologic hypoprothrombinemia”’ had developed, 1 y was sufficient to reduce
the “prothrombin time” to normal. Hardwicke,”* using a diphosphoric acid
ester of menadiol, reported comparable results when the vitamin was given
orally. Adler’s*®® results also tend to be confirmatory.

Taylor and coworkers*! have found that the hypoprothrombinemia of
sprue is rectified by administration of cortisone; they suggest that improved
intestinal absorption of fats and fat-soluble vitamins is the explanation.
An attempt was personally made to find the least daily dose of vitamin
K which would elevate the plasma prothrombin in such patients. Re-
peated determinations of the plasma prothrombin of an untreated patient
with non-tropical sprue yielded values ranging from 2 to 4% of normal, yet
with no signs of bleeding. 4-Amino-2-methyl naphthol HCl given intra-
muscularly on consecutive days induced no response in doses of 1 and 2 7;
the prothrombin level rose to 11 % with 5 y, to 21 % with 10 y, and to 34%
with 20 y. Although no further vitamin K was given, it seems likely that
much less than 1 mg. would have raised this patient’s prothrombin to
normal.

25 J. D. Greaves, Am. J. Physiol. 125, 429 (1939).

26 ©. A. Owen, Jr., Studies on the Conversion of Prothrombin to Thrombin; Effect
of Conversion Variations on Prothrombin Tests. Thesis, Graduate School, Uni-
versity of Minnesota, 1950.

27 J. HK. Flynn and E. D. Warner, Proc. Soc. Exptl. Biol. Med. 43, 190 (1940).

2 R. L. Sells, S. A. Walker, and C. A. Owen, Proc. Soc. Exptl. Biol. Med. 47, 441
(1941).

29S. H. Hardwicke, J. Pediat. 24, 259 (1944).

30 B. Adler, Monatschr. Geburtschiilfe u. Gyndkol. 118, 225 (1944).

31 A.B. Taylor, M. W. Comfort, E. E. Wollaeger, and M. H. Power, J. Clin. Invest.
30, 678 (1951).

CHAPTER 10

NIACIN

ie Nomenclature:and! Hormulasiy y's OL 2 eee Sg 52
eG hemistrys W458 sali ks a cs sk ct ee SP Ree ae A?
Avmls OlaitilOnpreeant aa ae Zee a CSA ee perenne 2
1. Isolation of Nicotinic Acid ET PI Ad 0 Oy ae Rian tO

2. Isolation of Nicotinamide. . . . ae CO tate ee oe oA:

3. Separation of Nicotinie Acid and Nicenmamide eee ee ote

4. Isolation of N'-Methylnicotinamide . . . . TA od ae 256

5. Isolation of 6-Pyridone of N! ayie shyluicatinamide vos Vee eon

6. Isolation of Other Nicotinic Acid Metabolites. . . . . . . . 4657
(eelsolationyors © coenzymes leand te ee eens ee es tos

Be Chemical and Physical’ Properties: = 24 - a9. . Ye SS sw « 6 6460
NicotnIC ACIG. REGAN SPR ies. et 19, MERE. Peers os 6 5 460

. Nicotinamide . . Per TAO! ie 5 os BAG

; Other Compounds Related: to ‘Micotinic Acta pS eS ee AS

C. COMEIAGM- OF WhreoinMinG AOC Aes 8 6 6 oo co oo peo o 0a)
D. Synthesis. . . Seta te 1 Seas Aa GE Ie Bh Pe eo AAA
1. Nicotinic cial. ia he rerwctengera Seer! ee es aM) EN LN AE Vit ook ht A

2. Nicotinamide . eae 410

3. Synthesis of Belered Genipomnds if Bislocieal Innpariancet SR ose LN

JE. SYST as phos Seaee Mite Ae Se A Me te a ee Seen:
HMMA ria ee Para Oly o. ys 05 anc ae les Ce, OA WS 2 4 AGS
Siieobiochentieslh Systems... :. pica we gee ee ws. 480
A. Introduction. . . ie Ne eR OSS ASO
B. Coenzymes Gontarane Nicotinic ‘Acid OY eek RPGR AES ok 5s CAST
Cmisalation and: Identifications y 9.2) uted) ae eS ew +. ASE
DmOccurrenceiol DEN andMNPNe ge) 9 2s es fe 484
E. Structure and Properties . . . eA e serene oe eA)
1. Diphosphopyridine Nucleotide (DPN) SET TO UR Mons AOA.

2. Triphosphopyridine Nucleotide (TPN)... ..... . « 488

He MechanismcomAction: = .. 0 <2. f+ <n yon 20 Lee 490
TPE Te ORD AVIICES, i. jak Goel els. = Late os <-giaed | Ct Rat MORALE AP 4 2 AOD)

2. The Apoenzymes. . . ee heat) ace AGU

3. The Coenzyme-Enzyme Gonplcs (ridinency i) BPTI, ie xo, we A492

A SE Ot OLE yOrOPEMAONMSA gi se) sas. oh a ten, UROAMI AOD

G. Specificity... Lik ser Ny ie OS GRAY te heel Set Meee bse! a ze AOS
1. DPN-TPN SnduiteiGy ate PA Baie ey, Oe SR ot ae 4 ADS
Zespecincity olthe Molecule: Ny Vaeig ee) ee Ey oS . - 498

5. Nicotinamide’ Mononucleotide Gi 29.2) 2 Pee GOS ee, 5 494

Pe aeckemica) REACtIONAS «at se duos diate a ee ee oe 40 4A.
leeSyvstems, = -. SURE a) ad 5 AO6
1. Synthesis of Fich nee Presphate Bonde Sp eae LANA ~ éndOG

2. DPN and TPN in Glyeolysis «2... . Be SN et ere. AOS,
Spi evrUvate: MetaDOleMiaies «ieee ct ee RL Oe | A098

449

450

VI.

WALL;

Vie

EX:

NIACIN

4. In Pentose Biosynthesis
5. In Lipid Metabolism
6. In Nitrogen Metabolism
7. In Photosynthesis . .
J. Biological Synthesis of DPN cond TPN .
1. Synthesis of Nicotinic Acid
2. Amidation of Nicotinic Acid .
3. Synthesis of Ribose . :
4. Synthesis of Nicotinamide Mononuclenude:
5. Biosynthesis of DPN
6. Biosynthesis of TPN
K. Destruction of DPN and TPN .
L. Inhibitors of DPN and TPN ‘
M. Assay Procedures of DPN and TPN .
1. General Principles.
2. DPN
5, UP :
N. Tissue Distribution and: Pate:
1. Excretion ‘
2. Tissue ineibeten And: Timorer Bate
3. Urinary Metabolites .
O. N}-Methylation of Nicarmamide

. Specificity of Action .

A. Modifications of the Nicotinic eid Micisenle ;
B. Tryptophan and Derivatives .
Biogenesis
A. Demonstration of eesti Nee Eioesmaeste
B. Mechanism of Biosynthesis
1. The Ornithine Scheme . ‘
2. Biosynthesis from pe Daa :
C. Site of Biosynthesis .
1. Synthesis in Tissues . ; :
2. Synthesis by Intestinal Minenaueeanaedts ;
3. Utilization of L- and p-Tryptophan as Bepcuncons fon Ree:
Acid
4, Tr pippheceNieounte INGE Relationanineae in one Qusadion
Estimation cee lastan (are
A. Biological hee
B. Chemical Methods.
1. Nicotinic Acid .
2. Differential Assay of Micotinie era Pinptanemncdee antl Related
Compounds .
ENE ‘Methyinicotinannde - oe
6-Pyridone of Nese vayinico nna: :
. Trigonelline (N!-Methyl Nicotinic Acid BeORNED,
. DPN and TPN saan aii T and IT)
; Other Methods > ..... ag
C. Microbiological Methods
Standardization of Activity
Occurrence. .
A. Occurrence in Food

Pi ctr ie

XI.

XII.

PELL.

cole amc]

mMmOo8moat

te

CONT & Ore SH tO

No)

NIACIN

. Forms of Occurrence
. Sites of Occurrence
. Stability in Food . :
. Influence of Soil, RATRORIAE RE. an Heredity!
Nicotinic Acid in U.S. Diets.
. Antivitamins in Food
. Occurrence in Various Tissues
iffeets of Deficiency
. In Microorganisms
. In Man and Animals ays :
Relation of Biochemical Tne fait ai Mieatinie Aa £0 BE Reones

States .

. The Deficiency States

. Nicotinic Acid and Coenzyme Tete evel al:

. Urinary Excretion of Nicotinic Acid and Other aabetances:
. Stomach

. Heart

. Brain

. Skin

. Eyes

10.
We

Blood
Miscellaneous

Pathology .
A. In Animals

ie
. Hogs.

5 IRENE : :

. Cotton Rats nnd Mice

_ Weoley 5 Se :
. Chicks, Turkeys. aul Ducks ;
le

OS Or em & LO

Dogs

Other Cerise

B. In Man.
Pharmacology
A. Toxicity

1.

Pe
Be
. Toxicity in Diets .
Circulation
. Blood Sugar .
. Ketosis ;
Gastric Seuretion ;
. Serum Bilirubin
. Antiallergie Effects
Other Effects

RK mOtroOo

Rats and Wee ;
Dogs.
Other Species

Requirements and Baetars Taaueneine
A. Animals
B. Man.

451

543
544
545
546
547
548
5950
550
550
551

5ol
502
554
556
560
561
561
562
563
563
564
565
565
565
567
568
569
569
569
570
570
571
571
571
572
572
573
573
574
575
575
576
576
577
578
578
586

452 NIACIN

I. Nomenclature and Formulas
ROBERT S. HARRIS

Accepted names: Niacin, niacinamide (United States)
Nicotinic acid, nicotinic acid amide (British)
Obsolete names: Vitamin PP
PP factor
Pellagra preventive factor
Pellagramine
Niamid
Empirical formulas: Niacin: CsH;0.N
Niacinamide: CsHsON2
Chemical names: Niacin: pyridine-3-carboxylic acid
Niacinamide: pyridine-3-carboxylic acid amide
Structural formulas:

H H
C C
TaN Yeo
HC; tor <i ieee
| 2
HC, »CH EC CH
: oe
N N
Niacin Niacinamide

II. Chemistry
J. M. HUNDLEY

A. ISOLATION

Nicotinic acid, so named because it was first discovered as an oxidation
product of nicotine, was first isolated from natural materials by Funk in
1911.1 Funk thought, at first, that the substance which he obtained from
yeast and rice polishings was the pigeon beriberi factor. Almost simultane-
ously Suzuki and associates, who also were searching for the antiberiberi
vitamin, isolated nicotinic acid from rice. Considerably later Vickery* iso-

1C. Funk, J. Physiol. 48, 395 (1911) [C. A. 6, 1923 (1912)]; J. Physiol. 46, 173 (1913) ;
Brit. Med. J. I, 814 (1913).

2 U. Suzuki, T. Shimamura, and S. Odake, Biochem. Z. 43, 89 (1912); U. Suzuki and
S. Matsunaga, J. Agr. Tokyo Imp. Univ. 5, 59 (1912).

3H. B. Vickery, J. Biol. Chem. 68, 585 (1926).

a i I i

II. CHEMISTRY 453

lated nicotinic acid from yeast. However, none of these investigators real-
ized the true biological significance of the substance they had isolated.

The first demonstration of a biochemical function for this substance
came from the work of Warburg and Christian‘: ® who isolated nicotinamide
from coenzyme II (TPN) and demonstrated its function as part of a hy-
drogen-transporting coenzyme.®7 Almost at the same time von Euler et
al.’ isolated a substance from coenzyme I which was identified as nicotina-
mide (see p. 482). Coenzyme I (DPN, cozymase) had long been known as
a substance necessary for the alcoholic fermentation of carbohydrates by
yeast. Kuhn and Vetter® further established the potential importance of
nicotinamide in metabolism by isolating it from heart muscle.

The true nutritional and biological significance of nicotinic acid became
evident in 1937 following the reports of Elvehjem ef al.!° that it would cure
blacktongue in dogs and that nicotinamide could be isolated from liver
concentrates which were active in curing canine blacktongue.!! The activity
of nicotinic acid in curing blacktongue was quickly confirmed by Street
and Cowgill.“* Late in 1937 Spies et al.,! Fouts e¢ al.,! and Smith e¢ al.,!
almost simultaneously announced that nicotinic acid would cure pellagra,
a fact confirmed by innumerable reports since that time. The rather uni-
versal biological significance of nicotinic acid was further confirmed by the
reports of Knight,!® Mueller,!® Koser et al.,” Fildes,!8 and Landy’ in 1937
and 1938, showing that this substance was an essential growth factor for
a variety of bacteria.

40. Warburg and W. Christian, Biochem. Z. 274, 112 (1934).

*>O. Warburg and W. Christian, Biochem. Z. 275, 112, 464 (1935).

8 Q. Warburg, W. Christian, and W. Griese, Biochem. Z. 282, 157, (1935).

70. Warburg and W. Christian, Biochem. Z. 287, 291 (1936)

8H. von Euler, H. Albers, and F. Schlenk, Hoppe-Seyler’s Z. physiol. Chem. 237,
180 I (1935); 240, 113 (1936).

9. Kuhn and H. Vetter, Ber. 68, 2374 (1935).

10C. A. Elvehjem, R. J. Madden, F. M. Strong, and D. W. Woolley, J. Am. Chem.
Soc. 59, 1767 (1937); J. Biol. Chem. 128, 137 (1938). ;

1C, J. Koehn, Jr., and C. A. Elvehjem, J. Nutrition 11, 67 (1936).

a H.R. Street and G. R. Cowgill, Proc. Soc. Exptl. Biol. Med. 37, 547 (1937).

2 T.D. Spies, C. Cooper, and M. A. Blankenhorn, Paper presented before the Cen-
tral Society for Clinical Research, Chicago, Ill., November 5, 1937; J. Am. Med.
Assoc. 110, 622 (1938).

13 P. J. Fouts, O. M. Helmer, 8. Lepkovsky, and T. H. Jukes, Proc. Soc. Exptl. Biol.
Med. 37, 405 (1937).

42). T. Smith, J. M. Ruffin, and 8. G. Smith, J. Am. Med. Assoc. 109, 2054 (1937).

16 B.C. J. G. Knight, Biochem. J. 31, 731 (1937).

16 J. H. Mueller, J. Biol. Chem. 120, 219 (1937).

7§. A. Koser, A. Dorfman, and F. Saunders, Proc. Soc. Exptl. Biol. Med. 38, 311
(1938).

1 P. Fildes, Brit. J. Exptl. Pathol. 19, 239 (1938).

1M. Landy, Proc. Soc. Exptl. Biol. Med. 38, 504 (1938).

454 NIACIN

1. IsoLATION oF Nicotinic AcID

Nicotinic acid is easily isolated from most natural materials, the exact
technique to be adopted depending on the nature of the material. In yeast,
nicotinic acid may be extracted directly without preliminary hydrolysis.*
With most materials, acid or alkaline hydrolysis is necessary to liberate
the free acid. Removal of fat with suitable solvents is a desirable preliminary
to hydrolysis, especially when dealing with animal tissue. Nicotinic acid
may be extracted directly from the acidified hydrolyzed material using
organic solvents (see p. 460). The free acid may be separated from this
extract as such, or in the form of an ester, or as the copper salt. The free
acid can be obtained from the copper salt using hydrogen sulfide. Nicotinic
acid can be purified by recrystallization from water or alcohol or by subli-
mation.

Nye et al.2° isolated nicotinic acid from Neurospora mycelium in the fol-
lowing fashion. The myceliwm was extracted with acetone. The acetone
extract was dried, redissolved in water, filtered, and treated with Ba(OH)>
and heat. After neutralization with H.SO, filtration and further adjustment
of the pH to 4.2 with HCl, the nicotinic acid was adsorbed on charcoal
(Norit A). The active material was eluted with hot 4% aniline in water,
the aniline removed with ether, and the watery eluate taken to dryness.
The dry residue was dissolved in hot absolute alcohol, the solution filtered,
again taken to dryness, and dissolved in water. Nicotinic acid was crystal-
lized from the concentrated aqueous solution and was recrystallized from a
1:4 mixture of acetic acid and benzene. In a later publication”! this proce-
dure was modified. A paper strip chromatogram developed with n-butanol

saturated with 0.2 N ammonium hydroxide was used to separate nicotinic

acid (labeled with C“) from unwanted materials. Nicotinic acid could be
eluted from the appropriate part of the paper strip and crystallized with
added ‘“‘carrier” nicotinic acid. The very small amount of nicotinic acid
(300 to 900 y) which was present in the extracts studied by these workers
made special techniques necessary. Yanofsky and Bonner” also have de-
scribed procedures for isolating small quantities of nicotinic acid using
paper strip chromatograms.

2. ISOLATION OF NICOTINAMIDE

This substance can be isolated usually by water extraction, followed by
partial hydrolysis with 0.1 N sulfuric acid to liberate free nicotinamide.

20 J. F. Nye, H. K. Mitchell, E. Leifer, and W. H. Langham, J. Biol. Chem. 179, 783
(1949).

21. Leifer, W. H. Langham, J. F. Nyc, andH. K. Mitchell, J. Biol. Chem. 184, 589
(1950).

22 C, Yanofsky and D. M. Bonner, J. Biol. Chem. 190, 211 (1951).

a

Il. CHEMISTRY 455

This solution can then be extracted with n-butanol or chloroform,” : 74 in
which nicotinamide is soluble. The chloroform solution may be subjected
to fractional distillation, nicotinamide distilling at 150 to 160° at 5 & 10-4
mm. Hg.’ The distillate may be recrystallized from organic solvents such
as chloroform, benzene, or ethylene glycol.

Warburg and Christian’ have described the procedure they used to ob-
tain nicotinamide from red blood cells. Coenzyme I (DPN) and coenzyme
II (TPN) were first isolated in a purified state (see p. 482 for method). The
coenzyme (DPN) could then be hydrolyzed with dilute sulfuric acid, lib-
erating free nicotinamide. Adenine could be precipitated as the silver salt.
The mother liquor and washings from this step were cleared of silver with
hydrogen sulfide and the nicotinamide extracted with amyl alcohol. After
filtration and concentration, nicotinamide was precipitated from the amyl
aleohol solution with picrolonic acid. The picrolonic acid salt could be
recrystallized from hot water. Treatment with hydrochloric acid yielded
the free nicotinamide hydrochloride,*® which was in turn recrystallized from
hot ethanol. .

The procedure used by the Wisconsin workers in isolating nicotinamide
from liver was somewhat involved but essentially as follows.!®: 1!) 25, 26
Liver paste was suspended and extracted with a 40 % ethanol, 50 % diethyl
ether, and 10% water mixture. The concentrate from this step was ex-
tracted with acetone. Several successive concentrations and extractions
with amyl alcohol, 95 % ethanol, acetone, and finally water were used. The
nicotinamide was adsorbed from the water extract using charcoal (Norit A)
and eluted with a mixture of 4 parts methanol and 1 part pyridine. Metha-
nol and pyridine were removed by evaporation. The dry residue was ex-
tracted with acetone and the extract evaporated to dryness. This dry resi-
due was placed in a molecular still and held at 160 to 165° and 1 & 10-4 mm.
pressure for 3 hours. The distillate was dissolved in alcohol and treated
with alcoholic mercuric chloride to yield a precipitate which was dissolved
in dilute HCl and decomposed with H.S. Crystalline nicotinamide appeared
in the concentrated filtrate.

Bovarnick”’ has described a procedure by which she isolated pure nico-
tinamide from heated asparagine-glutamic acid mixtures. Presumably, a
paper strip chromatogram technique similar to that described for nicotinic
acid could be used in the isolation of small quantities of nicotinamide,
although this has not yet been reported. It is known, however, that nico-

23 H. von Euler, F. Schlenk, L. Melzer, and B. Hégberg, Hoppe-Seyler’s Z. physiol.
Chem. 258, 212 (1939).

24P. Karrer and H. Keller, Helv. Chim. Acta 22, 1292 (1939).

25 C. A. Elvehjem and C. J. Koehn, Jr., J. Biol. Chem. 108, 709 (1935).

26 C, J. Koehn, Jr., and C. A. Elvehjem, J. Biol. Chem. 118, 693 (1937).

7M. R. Bovarnick, J. Biol. Chem. 153, 1 (1944).

456 NIACIN

tinic acid and nicotinamide, as well as related metabolites, can be separated
on paper strips with appropriate solvent systems.?8-”

3. SEPARATION OF Nicotinic ACID AND NICOTINAMIDE

This can be effected by extraction of a water solution with ether, chloro-
form, or benzene. The amide dissolves in the organic solvent while the acid
remains in the watery phase. Nitranilic acid may also be used for this pur-
pose, since it forms an alcohol-soluble compound with the acid but an al-
cohol-insoluble compound with the amide. Certain amines such as
piperidine, n-butylamine, morpholine, or diethylamine will react with a
suspension of nicotinic acid, but not with nicotinamide, in a non-aqueous
solvent such as benzene to give a soluble amine salt. The unreacted nico-
tinamide can be filtered and recovered almost 100 % pure.**

The acid and the amide may be separated using paper strip chromatog-
raphy as mentioned previously. This technique also separates free nico-
tinamide from that which is bound in coenzymes.”®: 2° Free nicotinamide
may also be separated from coenzyme-bound nicotinamide by acetone ex-
traction which dissolves free nicotinamide but not the coenzymes.”: %°: 38
This separation must be carried out promptly when extracting mammalian
tissues, since many of them contain very active nucleotidases which break
down the coenzymes unless inhibited by acetone or some other agent (see
p. 507).

4. ISoLATION OF N!-METHYLNICOTINAMIDE

This substance was isolated in pure form from urine by Huff and.

Perlzweig,*” using a rather simple procedure. The urine was acidified with
acetic acid, clarified with charcoal, partially evaporated, the residue ex-
tracted with 95% ethanol, this extract again evaporated, and the residue
extracted with 80 % ethanol. After evaporation of this extract, the residue
was taken up in water and adjusted to pH 4 with acetic acid and sodium

28 B. C. Johnson and Pei-Hsing Lin, Federation Proc. 10, 203 (1951).

2H. Leifer, L. J. Roth, D. S. Hogness, and M. H. Corson, J. Biol. Chem. 190,
595 (1951).

30 ©, F. Huebner, Nature 167, 119 (1951).

31 E}. Kodicek and K. K. Reddi, Nature 168, 475 (1951).

32 E.G. Wollish, M. Schmall, and E. G. Shafer, Anal. Chem. 28, 768 (1951).

33H}. Miller, Hoppe-Seyler’s Z. physiol. Chem. 268, 245 (1941).

34 J. R. Berg, U.S. Pat. 2,496,114 (January 31, 1950).

35 H. von Euler and G. Giinther, Hoppe-Seyler’s Z. physiol. Chem. 248, 1 (1936).

86 H. von Euler, H. Heiwinkel, and F. Schlenk, Hoppe-Seyler’s Z. physiol. Chem. 247,
IV (1937).

87 J. W. Huff and W. A. Perlzweig, J. Biol. Chem. 150, 395 (1943).

Il. CHEMISTRY 457

acetate. The N!-methylnicotinamide was adsorbed on a Decalso (Permutit)
column. After the column had been washed to remove impurities, the N!-
methylnicotinamide was eluted, using 25% potassium chloride solution.
The eluate was alternately evaporated and extracted with ethanol until
almost entirely free of potassium chloride. The characteristic picrate of
N?-methylnicotinamide was then formed, recrystallized, and decomposed
with hydrochloric acid to yield the free N!-methylnicotinamide which could
be crystallized from absolute ethanol. Other investigators have used meth-
ods differing but slightly from that described above.*®: *® Beher*® has re-
ferred briefly to a method for isolating semimicro amounts of N!-methy]l-
nicotinamide in which the compound is adsorbed on a cation exchange
resin and eluted with dilute hydrochloric acid which in turn is removed
with an anion exchange resin. The procedure was stated to give better
yields and was less time consuming than older methods.

5. ISOLATION OF 6-PYRIDONE oF N!-METHYLNICOTINAMIDE

Knox and Grossman"! isolated this substance from urine. The urine was
decolorized with basic lead acetate and the compound adsorbed on Lloyds’
reagent. After washing with 0.01 N hydrochloric acid, the compound was
eluted from the Lloyd’s reagent with absolute ethanol. After evaporation,
the residue was dissolved in water and the compound extracted into iso-
butanol. This extract was dried with calcium sulfate, evaporated, and the
residue extracted with acetone. Crystals were obtained from the concen-
trated acetone extract. Recrystallization could be accomplished from either
acetone or water.

6. ISOLATION OF OTHER Nicotinic Actp METABOLITES

The isolation of dinicotinylornithine from chicken droppings has been
described by Dann and Huff.*? Ackerman* was apparently the first to
isolate nicotinuric acid. He administered nicotinic acid and isolated both
nicotinuric acid and trigonelline from urine. The nutritional significance of
nicotinic acid was not known at that time. Linneweh and Reinwein“t also
isolated this substance from human urine.

38 M. Hochberg, D. Melnick, and B. L. Oser, J. Biol. Chem. 158, 265 (1945).

9% J. M. Hundley and H. W. Bond, J. Biol. Chem. 178, 513 (1948).

40W. T. Beher, Abstr. 118th Meeting, Am. Chem. Soc., Chicago, p. 73C (September
3-8, 1950).

41.W. E. Knox and W. I. Grossman, J. Biol. Chem. 166, 391 (1946) ; 168, 363 (1947).

#2 W.J. Dann and J. W. Huff, J. Biol. Chem. 168, 121 (1947).

48D. Ackerman, Z. Biol. 59, 17 (1912).

“*W. Linneweh and H. Reinwein, Hoppe-Seyler’s Z. physiol. Chem. 207, 48 (1932);
209, 110 (1932).

458 NIACIN

7. ISOLATION OF CoENzyMES I AND II
a. Coenzyme I (Diphosphopyridine nucleotide, DPN, cozymase)

This substance has not yet been isolated in an absolute chemically pure
form.*** It can, however, be prepared in 95% or better purity. Ohlmeyer*®
obtained a product of exceptional purity, as judged by extinction coefficients
at 340 my and other criteria, by isolating reduced DPN from a purified
oxidized DPN preparation (see p. 483). The early investigations of von
Euler and his group and Warburg and associates in isolating this coenzyme
have been cited elsewhere (p. 480 and 482). The methods applied by these
workers to purify DPN and to separate DPN from TPN and other impuri-
ties have been outlined in Section IV. It should be noted that none of
the procedures described to date for isolating ‘‘pure’’? DPN (or TPN) has
yielded crystalline products.

Improved and less cumbersome procedures have been devised in more
recent years which permit the isolation of DPN in a purity quite satis-
factory for most biochemical work. Williamson and Green*® and Sumner
el al.’ have devised methods which have found considerable use. These
and other methods have been reviewed by LePage.” Still more recently
Kornberg and Pricer*? have described a modification of Williamson and
Green’s method which yields DPN in about 95% purity. This method is
attaining increasingly wide acceptance and use. The coenzyme is extracted
from starch-free baker’s yeast with hot water. This extract is treated with
basic lead acetate, and DPN precipitated with silver nitrate. The precipi-
tate is decomposed with H.S and the DPN again precipitated with acetone.
The product at this stage has a purity of 76 to 83 %. Further purification
is achieved by adsorption on Dowex-1 resin, elution with 0.1 N formic acid,
and reprecipitation with acetone. Other investigators have also employed
ion exchange chromatography to purify DPN.°*°

b. Coenzyme II (Triphosphopyridine nucleotide, TPN)

TPN has been isolated in a form almost as pure as DPN, but it has not
been completely purified. Warburg et al.6:7 were the first to describe a
method for isolating purified TPN. The details of their procedure are de-

44a KK. Wallenfels and W. Christian [Angew. Chem. 64, 419 (1952)] have recently an-
nounced the crystallization of DPN as a quinine salt. This product crystallized as
fine, almost featherlike needles arranged in sheaves and gave a melting point be-
tween 162 and 170° with decomposition.

45 P. Ohlmeyer, Biochem. Z. 297, 66 (1938).

46S. Williamson and D. E. Green, J. Biol. Chem. 135, 347 (1940).

47 J. B. Sumner, P. 8S. Krishnan, and E. B. Sisler, Arch. Biochem. 12, 19 (1947).

48G. A. LePage, Biochem. Preparations 1, 28 (1949).

49 A. Kornberg and W. E. Pricer, Jr., Biochem. Preparations 2, (1952).

50 J. B. Neilands and A. Akeson, he Biot Chem. 188, 307 (1951).

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460 NIACIN

scribed in Section IV. LePage and Muller®! have described a procedure for
obtaining TPN from hog liver. More recently, Kornberg and Horecker™
have developed a method, part of which is a modification of the method
of Warburg and Christian, which can produce TPN of 92 % purity. TPN is
extracted from freshly killed sheep liver with hot water. Proteins and nu-
cleic acids are removed by precipitation with trichloroacetic acid. TPN is
precipitated from the filtrate with mercuric acetate, the salt decomposed
with H.S, and TPN precipitated with acetone. This product, which has a
purity of 8 to 13%, is further purified by ion exchange chromatography
using a Dowex-1 formate column, and reprecipitation with acetone.

B. CHEMICAL AND PHYSICAL PROPERTIES
1. Nicotinic Acip

Nicotinic acid is an odorless, white crystalline compound which melts
at 234 to 237° and sublimes without decomposition above this temperature.
It has a molecular weight of 123.11, and the composition CsH;O.N. It has
a tart taste. Table I summarizes many of the properties of nicotinic acid
and related compounds.

a. Solubility

One gram is soluble in 60 ml. of water or in 80 ml. of ethanol at 25°. It is
almost insoluble in ether but is freely soluble in boiling water, in boiling
ethanol, and in aqueous solutions of alkali hydroxides and carbonates.*
The solubility of nicotinic acid, its hydrochloride, and its sodium salt in
some common solvents is shown in Table IT.

b. Stability

Nicotinic acid is non-hygroscopic and very stable in the dry state. Solu-
tions may be autoclaved at 120° for 20 minutes without destruction. It is
stable on heating with 1 or 2 N mineral acid and alkali.

51 G. A. LePage and G. C. Mueller, J. Biol. Chem. 180, 775 (1949).

52 A. Kornberg and B. L. Horecker, Biochem. Preparations 2 (1952).

53 Vitamins Reviews, Niacin and Niacinamide. Merck & Co., Rahway, N. J., 1947.

54.N. A. Lange, Handbook of Chemistry, 6th ed. Handbook Publishers, Sandusky,
Ohio, 1946.

55 Y. Sendju, J. Biochem. (Japan) 6, 161 (1926).

56 ©, A. Elvehjem and L. J. Teply, Chem. Revs. 33, 185 (1943).

57 P. Karrer, G. Schwarzenbach, F. Benz, and U. Solmssen, Helv. Chim. Acta 19, 826
(1936).

58. H. Gaebler and W. T. Beher, J. Biol. Chem. 188, 348 (1951).

89 FM. Strong and 8S. M. McElvain, J. Am. Chem. Soc. 55, 816 (1933).

60 T,, M. Henderson, J. Biol. Chem. 178, 1005 (1949).

II. CHEMISTRY 461

c. Crystal Structure

Commercial nicotinic acid crystallized from water or ethanol occurs as
needle-like crystals or as a crystalline powder.®’ Keenan® and Wright and
King® have made extensive studies of the crystal structure of nicotinic
acid. When carefully crystallized from water, 95% ethanol, or 1:1 mix-
tures of water and 95% ethanol, the crystals appeared either as small
rods or as flat, colorless monoclinic plates with {010} as the predominant
form and a characteristic edge angle of 114°. All crystals were found to be
twinned either about the c axis or across the 100 plane to a greater or lesser
degree and exhibited a fibrous structure. Examination under the polarizing

TABLE II
SoLuBILITy* oF Nicotinic Actp, Sopium NiIcoTINATE (HEMIHYDRATE), AND
Nicotinic Actp HypROCHLORIDE AT VARIOUS TEMPERATURES (FROM
Y. M. Stopopin anp M. M. GoitpmMan®2)

Nicotinic
Nicotinic acid Na nicotinate acid: HCl
Saturated
Temperature, °C. H20 96% EtOH NaCl solution H.0 H20
0 0.86 0.57 0.8 9.5 We)
15 1.3 0.92 1.03 23). Li 8.5
38 2.47 2.1 1.61 31-16 (0°) 14.23
61 4.06 4.2 2.45 39.11 17.01
78 6.0 7.06 3.54 43.2 19.5
100 9.76 = 5.9 49.78 21.48

° Solubility as grams per 100 ml.

microscope gave the results shown in Table III. The density of nicotinic
acid crystals was 1.473 + 0.002 (g. cm.~*) as determined by a flotation
method.” Debye-Scherrer x-ray, oscillation, and Weissenberg photographs
were also taken and the results recorded.”

d. Absorption Spectrum

Solutions of nicotinic acid exhibit a characteristic absorption spectrum.
The absorption spectrum in aqueous solution has been studied by Hiinecke,®
who reported a maximum at 385 A. Hughes e¢ al.** have made an extensive

60a Y. M. Slobodin and M. M. Goldman, Zhur. Priklad. Khim. 21, 859 (1948) [C. A.
43, 6207 (1949)].

61G. L. Keenan, J. Assoc. Offic. Agr. Chemists 26, 514 (1948).

62 W.B. Wright and G.S. D. King, Acta Cryst. 3, 31 (1950).

63 H. Hiinecke, Ber. 60, 1451 (1927).

64 E. B. Hughes, H. H. G. Jellinek, and B. A. Ambrose, J. Phys. & Colloid Chem. 63,
414 (1949).

462 NIACIN

study of the ultraviolet absorption spectrum of nicotinic acid at pH values
from 1.28 to 13.0 (Fig. 1). The concentration of nicotinic acid used in de-
riving the curves shown in Fig. 1 was 0.0004 M, and the ionic strength
was the same at each pH. It will be noted that the molecular extinction
coefficient varied with pH, although the absorption maximum was at the
same wavelength, 2615 A., in each instance. There was a straight-line rela-
tionship between extinction coefficients and concentration of nicotinic acid
(Beer’s law, Fig. 2). The validity of Beer’s law was also confirmed using
nicotinic acid solutions at pH 1.28 and pH 13.

TABLE III

OrtTicaL PROPERTIES OF Nicotinic AcID ror SopiumM Lieut (From W. B. WriGHT
AND G.S. D. Kine®)

Wright and King Keenan
n1 1.424 + 0.002 1.428 + 0.003
Ne 1.717 + 0.005 Indeterminate
n3 >1.75 (~1.79) >1.734

Extinction

Both straight and inclined;

Parallel, although the sub-

stance showed many rod-
like fragments which did
not extinguish sharply
X, in a direction 1314° from --

the c axis in the obtuse

the maximum extinction
angle observed was 1314°

Acute bisectrix

angle 6
Optic plane Optic plane and X3 normal to =
[010]
Optic axial angle 2Vi = 46° =
Optic sign Negative =
Maximum bire- >0.32 oa
fringence
Monoclinic angle Balas =

e. Dissociation Constants

From the data depicted in Fig. 1, Hughes et al.** were able to calculate
dissociation constants for nicotinic acid. The spectra fell into two groups,
each having two isobestic points indicating two equilibria. The first two iso-
bestic points were at wavelengths of 2495 A. and 2695 A. and were associated
with pH values from 1.28 to 4.08. They belong to the dissociation equilib-
rium of nicotinic acid when acting as a base.

4 _coou “3 COOH
| | + H,0 = | | + OH-
\N 7 \N

Ht

i, Be

II. CHEMISTRY 463

Molecular extinction coefficient

2400 2500 2600 2700
Wavelength A

Fic. 1. Absorption spectra of nicotinic acid at different pH values. (From Hughes
et al.®4)

Extinction coefficient

0 1.605 3.21 4.82 x10-4
Concentration in moles per litre

Fic. 2. Demonstration of Beer’s law in buffer at 4.25. (From Hughes ef al.*)

464 NIACIN

The second group of isobestic points at 2465 A. and 2710 A. covering a
range of pH values from 4.4 to 13.0 is associated with the acid equilibrium
of nicotinic acid.

AN coon 4 _coo-
yr sno (PO a no
NZ

NN Z \}

The acid and base dissociation constants as calculated by two methods
are shown in Table IV. Ostwald® reported the acid dissociation constant
of micotimie acid as Kén— ee SOR.)

f. Chemical Properties

The fact that nicotinic acid is an amino acid is often overlooked. Al-
though not an a-amino acid, it is nevertheless at once both a carboxylic
acid and an amine and, accordingly, exhibits the characteristic properties

TABLE IV

THERMODYNAMIC DiIssocIATION CoNSTANTS FOR NIcoTINIC AcID AT AN AVERAGE
TEMPERATURE OF 22° + 2°

Isoelectric
pKa Ka pKp Kp point
Method 1 4.95 1 eal Ome 10.45 saa Se TORE 4.25
Method 2 4.91 sah 36 IS 10.45 35515) >< 10m 4.23

associated with the simultaneous presence of these two groups in a molecule
(although it does not form a zwitterion in solution®). It is soluble in both
acids and bases, forming quaternary salts with the former and carboxylic
acid salts with the latter. It possesses an isoelectric point (pH 4.23-4.25).
Its high melting point is characteristic of amino acids. Its strength as an
acid exceeds its strength as a base. A 1% aqueous solution has a pH of
about 3."

(1) Properties associated with its basic character

Nicotinic acid, like other compounds containing a basic amino group,
forms quaternary ammonium compounds. Thus, hydrogen chloride con-
verts it to nicotinic acid hydrochloride, a white crystalline solid readily
soluble in water (see Tables I and II) but insoluble in benzene and ether.
It has a melting point at 272° (with decomposition).’

4 _coou 4 _coon
| +H | |
\N/ \
ve
H

85 W. Ostwald, Z. physik. Chem. 3, 369 (1889).

II. CHEMISTRY 465

Similar reactions occur with other acids. Nicotinic acid nitrate? melts
at 184 to 185°. The picrate of nicotinic acid separates from absolute alcohol
in modular aggregates of short rhombic prisms which melt at 221 to 222°
to a red oil which begins to decompose at 250°. This compound contains

O-

65.5 % picric acid.®
OH |
| |

7 \—COOH 0.N—7% \—NO. pee 0-H NO;
iE eles Lule eal
NN 7 \ \N 7

| H

NO;

2 |
NO»

Nicotinic acid (and nicotinamide) react with methyl iodide and similar
alkyl iodides to yield compounds which are readily soluble in water (see

p. 492).
(7 \—cOoH _ 7 \—cooH

\N7Z SN
CH;

(2) Properties associated with its acidic character

Salt formation occurs when nicotinic acid is treated with alkali or an
‘alkaline earth hydroxide. The ammonium, sodium, potassium, calcium,
magnesium, etc., salts are readily formed as white crystalline solids by
titration of the acid with the hydroxides, viz.:

72 C00 4~_coona
| | + NaOH — | | + HO
NN 7 \

Heavy metal salts convert nicotinic acid to the metallic nicotinates.
This property has proved useful in recovering nicotinic acid from reaction
mixtures®® and from dilute nicotinic acid solutions, since salts such as
copper nicotinate are quite insoluble at certain pH values. Nicotinic acid
(and nicotinamide) reacts with HgCl, to form insoluble chloromercurates
which crystallize in characteristic forms. Gautier® has proposed the use of
this reaction as a means of microchemically characterizing nicotinic acid
and nicotinamide. Nicotinic acid will react with mercuric acetate as shown
in the following equation.

66 F. C. Huber, U.S. Pat. 2,487,874 (November 15, 1949).
67 J. A. Gautier, Ann. chim. anal. 26, 89 (1944).

466 NIACIN “

Y COOH 4000
2 | + Cu(C2H;02)2 — | Cu + HC;H;302
\ 1

\N \NZ 5

Esters of nicotinic acid are obtained by heating the acid in the appropriate
alcohol in the presence of a catalyst, usually sulfuric acid.® Esters are also
obtained when nicotinyl chloride is treated with an excess of the alcohol.

The esters of nicotinic acid possess characteristic aromatic odors. The
physical properties of a number of these esters are given in Table V.

Nicotinic esters, like other esters, are converted to the amides by treat-
ment with an excess of amines.

O O

|

C=0C2H; C—NH,
Y +NH— /\/ + C.H;OH

4
Z,
—

NZ

2. NICOTINAMIDE

This compound is a white crystalline powder, odorless or nearly so,
with a bitter taste. It crystallizes as needles from benzene and melts at
129 to 131°. It has a molecular weight of 122.12, and the composition
CeH_eN20.**: 6? A solution in water is neutral to litmus (1 % solution in wa-
ter = pH 6).

a. Solubility. One gram is soluble in 1 ml. of water, in 1.5 ml. of 95%
ethanol, and in 10 ml. of glycerol. It is soluble in acetone, amyl alcohol,
ethylene glycol, chloroform, and butanol. It is slightly soluble in ether and
benzene. Nicotinamide has a strong solvent effect on riboflavin.”

b. Stability. In the dry state nicotinamide is quite stable if kept below
50°. In aqueous solutions it may be autoclaved at 120° for 20 minutes with-
out appreciable destruction.” It distills at 150 to 160° and 5 X 10-* mm.
Hg.° It is converted to nicotinic acid by acid and by alkali.

6 C. O. Badgett, R. C. Provost, Jr., C. N. Ogg, and C. F. Woodward, J. Am. Chem.
Soc. 67, 11385 (1945).

6 U.S. Pharmacopeia, 14th Revision, p. 379, 1950.

70D. V. Frost, J. Am. Chem. Soc. 69, 1064 (1947).

~

ee

Il. CHEMISTRY 467

(c) Crystal Structure. The detailed crystal structure of nicotinamide has
been investigated by Keenan,® by McCrone and Cook,” and by Wright
and King.” Keenan reported that nicotinamide crystallized in colorless
small rods which exhibited both parallel and oblique extinction. He gives
the following values for the refractive indices: a equal to 1.485 + 0.002
and y greater than 1.734. McCrone and Cook encountered considerable
difficulty in preparing crystals suitable for microscopic examination but

TABLE V
Some Esrmers or ’ Nicotinic Acrp (ADAPTED FROM Bava TT et al. a5
Boiling point Solubility : :
R SN SE a AA em ee er:
| Hes | Mm. | asin 25 |

“a as a ! ————a — ==
Methyl | m.p. 38° | | =e
Ethyl | 88 3.5 1.1047] 1.5008} 5.60 | 147.5-148.0
n-Propyl 72-73, 0.2 1.0711) 1.4964) 0.950 | 129.5-130.0
n-Butyl | 75-76 0.15 | 1.0471] 1.4933] 0.261.) 113.5-114.0
n-Amy] | 93 0.22 | 1.0217) 1.4847; 0.081 98 5-99 .0
Isoamyl | 259 Atm. | =
n-Hexyl | 103-104 0.2 1.0133} 1.4897; 0.046 | 87.5-88.5
n-Heptyl | 116 0.19 | 0.9939] 1.4846} 0.040 | 92.5-93.5
n-Octyl | 116-117 0.2 0.9871) 1.4856} 0.019 | 89.5-90.0
n-Nonyl 133.5 | 0.28 | 0.9852) 1.4853) 0.020 | 99 .5-100.0
n-Decyl 140-141 | 0.25 | 0.9714] 1.4847] 0.017 94.0-94.5
n-Undecyl | 159-160 0.5 0.9606) 1.4829} 0.017 | 103.5-103.8
n-Dodecyl AE, PSE = 0.9356) 1.4750) 0.015 99 .5-100.5
n-Tetradecyl | m.p. 40.2-40.8 | — a — | 0.012 | 102.4-103.0
n-Hexadecyl m.p. 46.7—-47.0 = = = 0.013 | 103.0-103.5
ee | m.p. 55.3-55.8 = aa ee ORO, i M077 107-9

| |
an cone |
\N 7

finally obtained crystals from ethylene glycol on a microscopic slide, as
shown in Fig. 3. In addition they found any one of four polymorphic forms
when nicotinamide was crystallized from a melt, two of which are shown
in Fig. 3. Nicotinamide I recrystallized from common solvents appeared
as long, prismatic monoclinic rods showing well-developed prisms and
domes. McCrone and Cook present the various measurements of the crystal
morphology and crystal optics of nicotinamide I. The density was 1.401 +
0.002 (g. cm.~*) as determined by a buoyancy method using carbon tetra-
chloride and petroleum ether.

1 W.C.McCrone, Jr., and J. W. Cook, The Frontier 10, 12-23 (1947)

468 NIACIN

ae ay
¥ 2: Ry wie C
. Pe, x ; a7 3 _ ea 4 %
fag) wa ” he PED
ra « <A s) ? ay
ca) , , &
+) : 4£Q5

Zig ®%
any: 4 &s % = J o& ‘Ee
2 EE; oy +
&

| i in. ”
ee eee q “@&. @& une A

(a) Crystals of a commercial product of nicotinamide.

BT if rf has

(b) Characteristic sublimate of nicotinamide; crossed Nicols.
Fra. 3 (a-d). Various crystalline forms of nicotinamide. (From McCrone and Cook.”)

Il. CHEMISTRY 469

(ec) Crystals of nicotinamide from ethylene glycol on a microscopic slide; crossed
Nicols.

(d) Polymorphic transformation. Nicotinamide I growing into nicotinamide I];
crossed Nicols.

470 NIACIN

Wright and King® obtained nicotinamide crystals which differed in some
respects from those of McCrone and Cook. In their work nicotinamide
crystallized from ethylene glycol as monoclinic crystals of prismatic habit,
elongated parallel to the c¢ axis and showing {110} as the predominant
form. All crystals were twinned either about the ¢ axis or across the 100
plane. When crystallized from water, acetone, benzene, glycerol, or from
mixtures of ethylene glycol and water (50% or more), they appeared as
lath-shaped monoclinic crystals, elongated parallel to the ¢ axis and ex-
hibiting {010} as the predominant form with a characteristic edge angle
of 99°. The optical properties of nicotinamide for sodium light, and the

Log Io/I

0 4
220 240 260 280 300 320 340
Wavelength (micromoles)
Fig. 4. Ultraviolet absorption spectrum of nicotinamide (@) and N!-methyl-

nicotinamide (©). The concentration of both compounds was 2 & 10-4 M, in water,
at pH 7.0. (From Cantoni.7!»)

findings from Debye-Scherrer x-ray, oscillation and Weissenberg photo-
graphs of nicotinamide crystals are presented in Wright and King’s publica-
tion,
(d) Absorption Spectrum. The absorption spectrum of nicotinamide in
water was determined by Warburg et al.,° who found a maximum absorp-
tion of 2600 A. with a molecular extinction coefficient of 4.5 & 10*. Kuhn
and Vetter? reported a similar absorption maximum but with a slightly
higher extinction coefficient. Figure 4 shows the ultraviolet absorption
spectrum of nicotinamide in comparison to N!-methylnicotinamide.
Jellinek and Wayne” have studied the ultraviolet absorption spectrum
of 0.0003 M nicotinamide Se Mas at pH values of 5.72 to —1.06 with
the results depicted in Fig. 5. They found an absorption maximum at

nb G, L. Cantoni, J. Biol. Chem. 189, 203 (1951).
2H. H.G. Jellinek and M. G. Wayne, J. Phys. & Colloid Chem. 55, 173 (1951).

Il. CHEMISTRY 471

6.5, x10°
6.0 = OFF,
ron Eee
55 SS 03.
Se
5.0 SW 13

+ ww *

a5 Daa

Molecular extinction coefficient

SAWN ae ge

3.0 \\\ 4.85
\ 5.72

25

2.0

AS)

1.0 WN

03400 2500 2600 2700 2800
Wavelength A

Fia. 54 Ultraviolet absorption spectra of nicotinamide at various pH values.
(From Jellinek and. Wayne.”)

x 108

€2

6.1

57 pK ,- 10.60

pK,,;= 13.50
ahs.

ond
Tes

A9«, --Ndg-- Experimental points ©
; Calculated curve ——
4.5
4.1

=)

Molecular extinction coefficient

Sh3)

29

7412)

=e al 0 1 2 3 4 5 6

pH

Fic. 6. Dissociation curve for nicotinamide at \ = 2615 A. (From Jellinek and
Wayne.7?)

472 NIACIN

2615 A. (the same as for nicotinic acid), with the molecular extinctions of
the maxima decreasing as the pH values increased. The molecular extinction
coefficients for the peak values are plotted against the pH values in Fig. 6.
(e) Dissociation Constants. Jellinek and Gordon™ studied the hydrolysis
of nicotinamide in hydrochloric acid solutions and found that it differed
considerably from the hydrolysis of other amides such as benzamide in that
the first-order rate constants for the hydrolysis did not pass through a
maximum between 0.5 N and 8.6 N HCl. According to their results, two
reactions occur. An equilibrium is set up rapidly: Ht RCONH, + H*t =
HtRCONH;?* followed by a slow hydrolysis: H+RCONH;* + H;0t >
Ht+tRCOOH + NH,+ + Ht. The hydrolysis showed a positive salt effect
(NaCl). The effects of temperature and hydrochloric acid concentration on
the rate of hydrolysis were determined.

The dissociation constant due to the amido group and the thermodynamic

dissociation constant of the nitrogen in the ring were also evaluated spec-
troscopically by Jellinek and Wayne.” Their results indicated a constant
of 2.24 X 10~" for the nitrogen in the ring and 3.16 X 10~“ as the base
constant for the amido group, the latter agreeing well with the studies of
HCl hydrolysis.
(f) Chemical Properties. Many of the chemical properties of nicotinamide
have been referred to previously. Nicotinamide undergoes the Hoffman
degradation to 3-aminopyridine.” If nicotinamide is distilled with P.O; at
25 mm. pressure or SOCl, at 100°, 3-cyanopyridine is formed.”

\

Y; 3
4 CONH: : Co ==CN
| | ss SOCk _, | |
or P2O5

Nicotinamide and esters of nicotinic acid react with acids and alkyl
halides to form quaternary salts as already depicted for nicotinic acid.*”: 7878
The carbon in the 6 position seems to be the most reactive ring carbon
in nicotinamide.”® Karrer et al.8° have demonstrated that an amide group

73H. H. G. Jellinek and A. Gordon, J. Phys. & Colloid Chem. 58, 996 (1949).

™ A. Philips, Ann. 288, 253 (1895).

75 T. M. Heilbron and H. M. Bunbury, Dictionary of Organic Compounds, Vol. IIT.
Oxford University Press, New York, 1946.

76 W. Konig, J. prakt. Chem. 69, 105 (1904); 70, 19 (1904).

7 W. Ciusa and G. Nebbia, Gazz. chim. ital. 79, 521 (1949).

2M. EF. Zienty, J. Am. Pharm. Assoc. 87, 99 (1948).

79 W. E. Knox and W. I. Grossman, J. Am. Chem. Soc. 70, 2172 (1948).

80 P. Karrer, F. Kahnt, R. Epstein, W. Jaffe, and T. Ishii, Helv. Chim. Acta 21, 223
(1938).

II. CHEMISTRY 473

in the 8 position of the pyridine ring specifically facilitates the reversible
reduction of corresponding pyridinium salts.

3. OTHER COMPOUNDS RELATED TO NIcoTINIc ACID

The properties of several compounds related to nicotinic acid are listed
in Table I.

C. CONSTITUTION OF NICOTINIC ACID

The structure of nicotinic acid was determined shortly after it was first
isolated from the oxidation products of nicotine.*!» * The presence of a
pyridine ring can be established by boiling nicotinic acid with calcium hy-
droxide. Carbon dixoide is split off and free pyridine obtained. The car-
boxylic acid group can be identified by the formation of metallic salts and
by the formation of derivatives such as esters and the acid chloride (see
pp. 464-6). The existence of the basic ring nitrogen can be inferred by the
formation of salts such as the hydrochloride and the hydrobromide and
by the formation of quaternary ammonium compounds such as nicotinic
acid methochloride and methoiodide.

The fact that the carboxylic acid group was in the 3 (8) position was
proved by Skraup and Cobenzl*: ** by oxidation of 3-phenylpyridine the
structure of which was proven. Benzo|f|quinoline was oxidized to 3-phen-
ylpyridinecarboxylic acid, from which 3-phenylpyridine was formed by
decarboxylation. This in turn oxidized to nicotinic acid.**: 86 The 3 position
of the carboxyl group was further established by the formation of nicotinic
acid from synthetically prepared m-dipyridyl.*%”

Simpler proof of the constitution of nicotinic acid is cited by von Richter®®
from the behavior of 2,3-pyridinedicarboxylic acid (quinolinic acid) and
3,4-pyridinedicarboxylic acid (cinchomeronic acid). When the former is
heated, nicotinic acid is formed by elimination of one carbon dioxide group.
When the latter is heated, both nicotinic acid and isonicotinie acid (4-pyri-
dinecarboxylic acid) are formed. Therefore nicotinic acid is 3-pyridine-
carboxylic acid.

81C,. Huber, Ber. 3, 849 (1870); Ann. 141, 271 (1867).

82H. Weidel, Ann..165, 331, 346 (1873).

83 Z. H. Skraup, Monatsh. Chem. 1, 800 (1880).

84 Z%. H. Skraup and A. Cobenzl, Monatsh. Chem. 4, 486 (1883).

85 V. von Richter, The Chemistry of the Carbon Compounds, Vol. IV, p. 214. Elsevier
Publishing Co., New York, 1946.

86 H.R. Rosenberg, Chemistry and Physiology of the Vitamins, p. 228. Interscience
Publishers, New York, 1942.

877. H. Skraup and G. Vortmann, Monatsh Chem. 4, 594 (1883).

474 NIACIN

D. SYNTHESIS
1. Nicotinic. AcID
a. From Nicotine
Nicotinic acid can be obtained from nicotine by treatment with fuming
nitric acid,” with chromic acid,*! or by permanganate’ as indicated below.
When nicotine is treated with bromine water, dibromoticonine is formed

which decomposes in barium hydroxide solution, through the intermediate
indicated in parentheses, to nicotinic acid, methylamine, and malonic acid.*?

lao C18 i ’ O— Ca Chibn O=C— CHO
ey | | bromine Z bs Als eae 2 dics La
Wee orale as emo BC
os bu, “N CH; CH;

4 5 : 9
Nicotine Morin. Dibromoticonine Eyoy
Chron, © acig

lone
0. a
if Perp ed wo
[)

7~~COOH
Cor + CH;NH, + CH.(COOH).
N

Nicotinic acid
b. From 8-Picoline and Other 3-Substituted Pyridines

Nicotinic acid can be synthesized from substituted pyridines such as
B-picoline®’-* and 3-ethylpyridine®-** by suitable oxidation methods. For
example, 6-picoline is refluxed for 2 to 3 hours with H;PO4 and HNOs, the
HNO; and H.O distilled up to 200°, and the oxidation repeated with small
portions of HNOs. The final residue is diluted with water, treated with
NaOH, giving nicotinic acid.°® This oxidation may also be carried out with
oxygen-containing gases in the presence of catalysts such as V2O5 or V2Os5
containing small quantities of Fe2O3,%

G-Picoline can be prepared by heating glycerol with P2O; and substances
containing ammonia or, better, ammonium phosphate.* It may also be

88 R. Laiblin, Ber. 10, 2136 (1877); Ann. 135, (1879).

89 V. von Richter, The Chemistry of the Carbon Compounds, Vol. IV, p. 337. Elsevier
Publishing Co., New York, 1946.

90 H. Weidel, Ber. 12, 1992, 2004 (1879).

1H. Ost, J. prakt. Chem. [2]27, 286 (1883).

2K. Seyfferth, J. prakt. Chem. [2]84, 258 (1886).

°3 H. Weidel and K. Hazura, Monatsh. Chem. 3, 783 (1882).

%4 A. Ladenburg, Ann. 301, 152 (1898).

95 A. Wischnegradski, Ber. 12, 1480 (1879).

96 F. E. Cislak and W. R. Wheeler, U.S. Pat. 2,396,457 (March 12, 1945).

97 F. E. Cislak and W. R. Wheeler, U.S. Pat. 2,437,938 (March 16, 1948).

9 P. Schwarz, Ber. 24, 1676 (1891).

II. CHEMISTRY 475

prepared by condensing 8-oxocarboxylic acid esters and 6-diketones with
aldehydes and ammonia (Hantzsch’s method).°? 8-Picoline is commonly
recovered from coal tar.

c. From Quinoline

Quinoline can be oxidized to 2,3-pyridinedicarboxylic acid (quinolinic
acid) with heat? or with acids.®!: 1%, 105 For example, quinoline plus
H.S0O,4, HNOs, and HgO (as a catalyst) when heated to 300° gives an 88.4 %
yield of nicotinic acid which can be recovered as the copper salt.1°° SeOs» is
also an effective catalyst in converting quinoline to nicotinic acid in high
yield.1%: 108

NS JN

TOTS —COOH 7 \—COOH
heat or

| | 1, oe acid

WAY . eon \N/

Quinoline Quinolinic acid Nicotinic acid

d. From Pyridine

Pyridine may be sulfonated with fuming sulfuric acid, yielding the
3-sulfonic acid. When the sodium salt of this compound is distilled with
potassium cyanide, 3-cyanopyridine is formed.!°? This compound may then
be hydrolyzed to nicotinic acid. An alternative procedure is to brominate

pyridine in the 3 position and treat with cuprous cyanide to yield 3-cyano-
' pyridine.!°

+ Cu.,(CN)>2 ayes

\N 7 NN Z \N 7

‘
| HCl

99 V. von Richter, The Chemistry of the Carbon Compounds, Vol. IV, p. 198. Elsevier
Publishing Co., New York, 1946. :

100 S$. Hoogewerff and W. A. van Dorp, Ann. 204, 117 (1880); 207, 219, 226 (1881);
Rec. trav. chim. 1, 122 (1882).

101 R. Camps, Arch. Pharm. 240, 353, 359 (1902).

102 H. Weidel and J. Herzig, Monatsh. Chem. 1, 16 (1880).

103 F. B. Ahrens and R. Gorkow, Ber. 37, 2063 (1904).

104 S. Hoogewerff and W. A. van Dorp, Ber. 14, 974 (1881).

105 H. Weidel and J. Herzig, Monatsh. Chem. 6, 982 (1885).

106 M.S. Larrison, U.S. Pat. 2,475,969 (July 12, 1949).

107 M. B. Mueller, U.S. Pat. 2,486,660 (February, 1948).

108 KF. Porter, M. Bumpers, and J. N. Crosby, U.S. Pat. 2,513,251 (July 27, 1950).

109 Q, Fischer, Ber. 15, 63 (1882).

10 §.M. McElvain and M. A. Goese, J. Am. Chem. Soc. 63, 2283 (1941).

476 NIACIN

e. Other Methods

Trigonelline (the betaine of 3-pyridinecarboxylic acid) can be converted
to nicotinic acid by heating at 200° for 1 to 3 hours with pyridine hydro-
chloride or hydrobromide. Nicotinic acid can be separated as the copper
salt which can be decomposed with hydrogen sulfide in formic acid.!!°* Trig-
onelline may also be converted to nicotinic acid by heating with hydro-
chloric acid in a closed tube at 260°! or by heating in a mixture of potas-
sium hydroxide and ammonia.!”

f. Isotopic Nicotinic Acid

Murray et-al.!’ have prepared carboxyl-labeled nicotinic acid in the
following fashion with an over-all yield of 60 to 80%:

\ nN Y
Z, \—Br : / Ibi VANS *OOH
| Be Wa Or ary seen | | i) C0) 3 eee | |
NN? \NZ Av ;
3-Bromo- n-Butyl- 3-Pyridyl- Nicotinic
pyridine lithium lithium acid

Nicotinic acid may also be labeled with deuterium. Trenner e¢ al." have
described a procedure whereby deuteronicotinic acid can be prepared by
direct exchange of nicotinic acid with deuterosulfuric acid. The amount of
tracer in the product can be determined using infrared spectroscopy.

2. NICOTINAMIDE
a. From Nicotinic Acid

Nicotinamide can be formed from nicotinic acid by treating with ammo-
nia or molten urea at 230°,1°-"8 preferably with a catalyst such as NH,
molybdate.

b. From Esters of Nicotinic Acid

Ksters of nicotinic acid such as the methyl and ethyl esters can be reacted
with aqueous or alcoholic ammonia to yield nicotinamide.!®: 119-!1

10a J. Weijlard, J. P. Messerly, and M. Tischler, U.S. Pat. 2,381,794 (August 7, 1945).

111 fF, Jahns, Ber. 20, 2840 (1887).

12 J. W. Huff, J. Biol. Chem. 166, 581 (1946).

13 A. Murray, III, W. W. Foreman, and W. Langham, Science 106, 277 (1947).

114 N.R. Trenner, R. W. Walker, B. Arison, and C. Trumbauer, Anal. Chem. 28, 487
(1951).

15 S$, Keimatsu, K. Kokata, and I. Satoda, J. Pharm. Soc. Japan 58, 994 (1933).

16 K). Cherbuliez and F. Landolt, Helv. Chim. Acta 28, 1438 (1946).

17 HY. F, Pike and R.S. Shane, U.S. Pat. 2,412,749 (December 17, 1946).

18 P. W. Garbo, U.S. Pat. 2,419,813 (April 29, 1947).

119 C, Wingler, Ber. 27, 1787 (1894).

20 B. Pollak, Monatsh. Chem. 16, 53 (1895).

121. B. LaForge, J. Am. Chem. Soc. 50, 2377 (1928).

Il. CHEMISTRY Ae.

c. From 3-Cyanopyridine

Nicotinamide can be produced directly from the above compound by
partial hydrolysis in an aqueous alkaline solution containing a sufficient
quantity of alkali to produce a larger portion of nicotinamide than nicotinic
acid but insufficient to complete the hydrolysis of the cyanopyridine.!”*:
Nicotinamide can also be produced from this compound by treating with
H.O2 and NaOH™ or by heating the compound with water, NH3;, or an
amine.”

3. SYNTHESIS OF RELATED COMPOUNDS OF BIOLOGICAL IMPORTANCE
a. 3-Acetylpyridine

Ethylnicotinate undergoes normal Claisen-type condensations. With
ethyl acetate in the presence of sodium ethoxide, ethylnicotinoacetate
is formed in good yield.

O O O

y O : yee |

Las C—O—C.H; | “ “—C—CH,C—OC2H;
. GHC =O Cx, 20s |

\N 7 \NZ

The latter may be hydrolyzed to 3-acetylpyridine in excellent yield using
hydrochloric acid.** ?°

O O O

| l |

Cu 6— 0G. CCH,
A/ ~~
| | _— | SOs eG HE OH
YNZ NN Z

b. N'-Methylnicotinamide

Nicotinamide (and nicotinic acid) react readily with methyl iodide and
similar alkyl iodides to yield the corresponding N'!-substituted deriva-
tives.*7: 76 These may then be converted to the corresponding chloride (the
form in which this compound is ordinarily used) by treatment with silver
chloride.

122 British Pat. 563,184 (August 2, 1944) [C. A. 40, 2473 (1946)].

123 B. F. Duesel and H. L. Friedman, U.S. Pat. 2,471,518 (May 31, 1949).

124 J. F. Couch and C. F. Kreevson, U.S. Pat. 2,453,496 (November 9, 1948).
125 H.R. Rosenberg, U.S. Pat. 2,446,957 (August 10, 1948).

126 H. Gilman and H.S. Broadbent, J. Am. Chem. Soc. 70, 2755 (1948).

478 NIACIN

c. 6-Pyridone of N'-Methylnicotinamide

This compound has been prepared from coumalic acid by ring closure
with methylamine.!”” It may also be prepared by an alkaline ferricyanide
oxidation and subsequent treatment with SOCI, and ammonia, using either
trigonelline or N!-methylnicotinamide as starting material.

d. Pyridine 3-Sulfonic Acid

This substance may be prepared by the direct sulfonation of pyridine
with fuming sulfuric acid.1°% ?9, 8° Mercury is an effective catalyst in the
reaction.

EK. SPECIFICITY

The biological effect of various modifications in the nicotinic acid mole-
cule is discussed in Section V (p. 517).

III. Industrial Preparation
J. M. HUNDLEY

The current (1952) annual production of nicotinic acid is probably in
excess of 2 million pounds. The latest available data! indicate that about
144 million pounds of nicotinic acid and nicotinamide were produced in
the United States during 1950 (Table VI). This accounts, on a weight
basis, for about 41% of all vitamins produced. Since the period covered
by this report, two new plants for the production of nicotinic acid have
gone into operation.? One of these plants has an annual capacity of over
1 million pounds. This large production reflects a market demand stimu-
lated not only by the widespread use of nicotinic acid and derivatives in
vitamin preparations and other medicinal products but the large quantities
used in the enrichment of bread and cereals.

The details of the manufacturing processes being used in the new plants,
referred to above, have not been revealed. However, one will base its
production on coal tar raw materials, presumably quinoline or 6-picoline,

127 FH. von Pechman and W. Welsch, Ber. 17, 2384 (1884).

128 J. W. Huff, J. Biol. Chem. 171, 639 (1947).

129 A.J. P. van Gastel and J. P. Wibaut, Rec. trav. chim. 58, 1031 (1934).
130 G, Machek, Monatsh. Chem. 72, 77 (1938).

1 Synthetic Organic Chemicals. U. 8S. Production and Sales of Medicinals. U. 8.
Tariff Commission, Washington, D. C., 1950 (Preliminary Report published July,
1951).

2 Chem. Eng. News 29, 4686, 5136 (1951).

III. INDUSTRIAL PREPARATION 479

both of which can be obtained from coal tar distillation. Several of the
chemical reactions which could be applied using these starting materials
have already been described (see p. 474). The oxidation of bases from coal
tar or petroleum? or from bone tar oil* which distill between 135 and 142°
is a well-known procedure. The second plant will base its production on
aldehyde collidine. This process is, in principle, the same as the quinoline
process, 1.e., the raw material is oxidized with manganese dioxide and
sulfuric acid with or without a selenium catalyst, or with nitric acid, to
yield the pyridinedicarboxylic acid which in turn is heated to remove one
of the carboxyl groups (see p. 475). Details of a procedure which can be
used to produce nicotinic acid from nicotine have been described by S. M.
McElvain.®

TABLE VI

ANNUAL (1950) PropucTion oF Nicotinic ACID AND OTHER VITAMINS
IN THE UNITED STATES

Pounds

Nicotinic acid and nicotinamide 1,447 , 600
Ascorbic acid and salts 1, 227,500
Thiamine 237 , 400
Riboflavin 199 ,000
Pyridoxine 17,500
Caleium pantothenate ~ 155, 400
Choline 104,900
Vitamin A 95 , 300
. Vitamin D:, and D; 2,600

Other (including vitamin K) 72,900

Many processes are available by which nicotinic acid could be produced.
The choice of a commercial process depends not only on the efficiency of
the chemical reactions but on the current availability and cost of the
various starting materials and the market for secondary reaction products
(e.g., isonicotinic acid derivatives). Chemical reactions which could be ap-
plied in the industrial preparation of nicotinic acid and of nicotinamide, as
well as many of the pertinent patents and references have been listed in
Section IT.

3A. Pinner, Ber. 38, 1227 (1900).

4H. Weidel, Ber. 12, 1992, 2004 (1879).

°S. M. McElvain, in Organic Syntheses, Coll. Vol. I, 2nd ed., p. 385. John Wiley
and Sons, New York, 1941.

480 NIACIN

IV. Biochemical Systems
J. M. HUNDLEY

A. INTRODUCTION

Like most other B vitamins, nicotinic acid (or a derivative) is required
by all living cells. It is an essential part of certain coenzymes which cata-
lyze chemical reactions essential to cellular life. So far as is known, nico-
tinic acid has no metabolic function other than that exerted through the
coenzymes of which it is a part.

Actually, specific biochemical functions for nicotinic acid were discovered
before it was known that this substance was a vitamin. In 1934 Warburg
and Christian':? found that nicotinamide was part of the coenzyme II
molecule. The following year nicotinamide was isolated from cozymase
(coenzyme I) by von Euler and associates’ and later by Warburg and
Christian.4 It was not until 1937, when Elvehjem et al. discovered that
nicotinic acid would cure blacktongue in dogs and the subsequent finding
that it would cure pellagra in man, (see p. 453) that the full biological
significance of this simple organic molecule was realized. Since these pioneer
discoveries more than forty biochemical reactions have been identified
which are dependent on these two coenzymes. More reactions will likely be
discovered. Most of these reactions have been studied in great detail. Much
exact information is available on the mechanism and kinetics of the reac-
tions, on the effect of environmental influences, and on the interdependence
of these with other biochemical reactions.

However, it is important to remember that most of the detailed informa-
tion on coenzyme I and II catalyzed reactions has been obtained from
in vitro studies using simplified enzyme systems. It is well known that
these reactions are only steps in the very complex series of reactions which
occur in the intact cell. There are undoubtedly many influences in the
intact cell which modify the reactions observed in simplified systems. The
in vivo consequences of blocking or impeding specific biochemical steps, the
ability or inability to activate alternate reaction pathways, and effects due
to the accumulation of substrate or abnormal metabolites are only examples
of the complexities involved in evaluating the biochemical function of nico-
tinic acid in intact organisms. This field has hardly been explored.

1Q. Warburg and W. Christian, Biochem. Z. 274, 112 (1934).

2O. Warburg and W. Christian, Biochem. Z. 275, 112, 464 (1935).

3H. von Euler, H. Albers, and F. Schlenk, Hoppe-Seyler’s Z. physiol. Chem. 287,
180 I (1935); 240, 113 (1936).

4Q. Warburg and W. Christian, Biochem. Z. 287, 291 (1936).

'C. A. Elvehjem, R. J. Madden, F. M. Strong, and D. W. Woolley, J. Am. Chem.
Soc. 59, 1767 (1937); J. Biol. Chem. 128, 137 (1938).

IV. BIOCHEMICAL SYSTEMS 481

Nicotinamide containing coenzymes have significance not only in verte-
brate biochemistry but in the vegetable kingdom as well. This became clear
quite early not only from the studies on yeast fermentation® but especially
due to the studies of the Lwoffs with factor V, an essential growth factor
for Hemophilus parainfluenza.’ They established the identity of factor V
with the nicotinamide coenzymes and discovered that these substances
functioned biochemically in these microorganisms much as had already
been found in enzyme systems derived from mammalian sources (also see
p. 550).

B. COENZYMES CONTAINING NICOTINIC ACID

Two, and only two, coenzymes are known to contain nicotinic acid.®*
These have been described under various names in the literature.

1. Coenzyme I, codehydrogenase I, cozymase, Harden’s coferment, core-
ductase, factor V, codehydrase I, and diphosphopyridine nucleotide.

2. Coenzyme IT, codehydrogenase II, Warburg’s coferment, codehydrase
IT, and triphosphopyridine nucleotide. _

Since names which depict chemical structure are generally less confusing,
the terminology of diphosphopyridine nucleotide (DPN H, for the reduced
form) and triphosphopyridine nucleotide (TPN and TPNH2) will be used
in this chapter to refer to coenzymes I and IT, respectively. It is recognized
that this terminology is not quite correct chemically, since the term nucleo-
tide itself implies phosphoric acid content. Nevertheless this terminology
seems preferable by virtue of common usage and in view of the fact that
‘these were the names originally suggested by Warburg and Christian.

C. ISOLATION AND IDENTIFICATION

1. TPN was the third coenzyme to be discovered and the first found to
contain nicotinamide as part of its structure. In 1934 Warburg and Chris-
tian! isolated a nitrogen-containing base from their ‘“‘Co-ferment’’ (TPN)
which was quickly identified as nicotinamide.? These and later studies? 7
showed that their coenzyme contained 1 molecule each of adenine and
nicotinamide, 3 molecules of phosphoric acid, and 2 molecules of pentose.
However, there was some uncertainty that one of the sugar molecules

6 A. Lwoff and M. Lwoff, Proc. Roy. Soc. (London) B122, 352, 360 (1937).

6a A probable exception to this has been recorded recently by T. P. Singer and E.
B. Kearney [Abstr. 2nd Intern. Congr. Biochem. Paris p. 307 (1952)], who discovered
a pyridine nucleotide, which was not identical with any known coenzyme, in
Proteus vulgaris. This coenzyme was necessary for the oxidation of cysteinesulfinic
acid to eysteic acid and for the dehydrogenation of 6-sulfinylacetic acid. The
structure of this pyridine nucleotide is not yet known.

70. Warburg, W. Christian, and W. Griese, Biochem. Z. 282, 157 (1935).

482 NIACIN

might be a hexose* and the manner of linkage of the various molecules
was not clarified.

Warburg and Christian encountered their ‘‘Co-ferment” as a factor
necessary for the action of Zwischenferment, an enzyme which oxidized
glucose 6-phosphate.’ They isolated the coenzyme in a “‘pure” form from
hemolyzed horse erythrocytes by a rather involved procedure employing
acetone precipitation of the red cell stromata, fractionation with mercuric
acetate and barium hydroxide, precipitation from methanol-HCl with ethyl-
acetate, and further fractional precipitation with lead acetate and alcohol.
A fluorescent contamination which interfered with spectrophotometric
studies could be removed by bromine treatment.’

2. DPN was the first coenzyme to be discovered and the second found
to have nicotinamide as a part of its molecule. It was found by Harden
and Young? as a material which could be dialyzed from the complex zymase
system of yeast, the system which catalyzes the alcoholic fermentation of
carbohydrates. Actually, this soluble dialyzable component of the zymase
system contained, as later shown, two coenzymes, cozymase (DPN) and
phosphorylating coenzyme (adenosine phosphate).

By 1933 it was known, from the work of von Euler and Myrbick,?° that
cozymase was an adenine nucleotide. About a year after Warburg and
Christian identified nicotinamide as part of the TPN molecule, von Euler
et al.’ isolated a base from cozymase which they believed to be nicotinamide.
Warburg and Christian‘ also isolated a pyridine base from cozymase and
published convincing evidence of its identity as nicotinamide. From the
work of both groups, it was evident that cozymase was closely related to
TPN, since it contained the same structural elements differing only in that
it contained one less phosphate group.

DPN was originally isolated from brewer’s or baker’s yeast, although
animal tissues were used in much of the early work. The first ‘pure’
preparations were obtained from yeast*: ™ by a procedure which consisted
of hot water extraction, lead acetate precipitation of impurities, coenzyme
precipitation in consecutive steps as the phosphotungstate, Hg, Ag, and
Cu salts, followed by removal of contaminations by Ba and Pb treatment,
alcohol fractionation, and purification with aluminum oxide. In using ani-
mal tissue (erythrocytes) as a source material, Warburg and Christian‘
were able to isolate not only DPN but TPN and adenosine phosphate as
well. They separated the three nucleotides, utilizing the fact that the
barium salt of adenosine phosphate is only slightly soluble in water. The
barium salt of TPN is only slightly soluble in dilute alcohol, whereas the

8’ O. Warburg and W. Christian, Biochem. Z. 242, 206 (1931).

9A. Harden and W. J. Young, J. Physiol. 32 Proc. 1904 (1905); Proc. Roy. Soc.

(London) BT7, 405 (1906).
10K. Myrbick, Ergeb. Enzymforsch. II, 1389 (1933).

1 Af. von Euler and F. Schlenk, Svensk Kem. Tidskr. 48, 185 (1936).

IV. BIOCHEMICAL SYSTEMS 483

barium salt of DPN is easily soluble in dilute alcohol. According to Le-
Page,” when 3 volumes of alcohol are added to a water solution of DPN
and TPN at pH 9 in the cold, DPN is soluble to the extent of 5 mg. per
milliliter or greater while TPN is quantitatively precipitated. Ochoa!’ was
able to isolate DPN from rabbit muscle in good yield and in ‘‘pure” form
by modifying the procedure of Meyerhof and Ohlmeyer."

In the light of more recent information, it is doubtful if any of these
“pure” preparations of the early workers were actually pure in the chemical

TABLE VII

CONCENTRATION OF DPN anp TPN In Various MATERIALS (ADAPTED FROM
F. ScHLENK!*»)

(y/g. of fresh weight)

DPN TPN

Brewer’s yeast 1000-1500 <10
Baker’s yeast 1000-1500 <10
Muscle (striated rabbit) >600
Liver (rat) 600-1200 30
Kidney (rat) 400-1000 40
Muscle (rat) 300-600 80
Muscle (human) 400
Liver (cat) 430
Thigh (cat) 250
Oxyntie cells, stomach (cat) 2000
Retina (cattle) 1700-41002
Erythrocytes (human) 60-90

_ Erythrocytes (horse) 100 >12
Erythrocytes (rat) 100 40
Lobster, Homarus vulgaris, tail 450
Chilomonas paramecium 650
Pollen (Saliz, Populus) 700-1000

“ Per gram of fat-free and dry tissue.

sense, although they may have been pure from the biochemical standpoint.
A possible exception to this is the work of Ohlmeyer,’ who isolated DPN H2
from a purified DPN preparation. The high molecular extinction coeffi-

122G. A. LePage in Respiratory Enzymes, p. 87. Burgess Publishing Co., Minneapolis,
1949.

13§,. Ochoa, Biochem. Z. 292, 68 (1937).

14Q,. Meyerhof and P. Ohlmeyer, Pfltigers Arch. ges. Physiol. 188, 114 (1921).

15 P. Ohlmeyer, Biochem. Z. 297, 66 (1938).

15a K. Wallenfels and W. Christian [Angew. Chem. 64, 419 (1952)] have recently
announced the crystallization of DPN asa quinine salt. This product crystallized
as fine, almost featherlike needles arranged in sheaves and gave a melting point
between 162 and 170° with decomposition. Using pure DPN recovered from the
crystallized salt, they obtained an extinction coefficient of 9.43 em.? per milligram.

15b F. Schlenk, in The Enzymes, Vol. 2, Part 1, p. 255. Academie Press, New York,
1951.

484 NIACIN

cient he observed, 6.3 X 10? (3840 my), indicates a product of exceptional
purity.!°>4

The more recent methods which have been developed to isolate DPN
and TPN have been described in Section II (see p. 458).

D. OCCURRENCE OF DPN AND TPN

Pyridine nucleotides have been found in every living cell examined thus
far. Table VII lists a number of substances and the concentrations of
DPN and TPN found. Many of these figures must be regarded as approxi-
mations only because of analytical limitations. Many cells contain sub-
stances which rapidly destroy these coenzymes once their cellular structure
is disrupted. Furthermore, it is known that TPN and DPN are intercon-
vertible (see p. 493). Nevertheless it is clear that these coenzymes have a
rather universal distribution, although their concentration varies widely in
different substances. DPN and TPN tend to occur together, although the
amount of TPN seems always to be less than DPN. In animal tissues, a
fairly constant proportion (35 to 45 %) of the DPN is in the reduced state."

Yeast, red blood cells, liver, and skeletal and cardiac muscle are good
sources of DPN. Fresh yeast may contain as much as 0.5 g. per kilogram”
and rabbit cardiac muscle 0.4 g. per kilogram.'® Yeast contains very little
TPN. Animal tissues, especially red blood cells, liver, and muscle are good
sources of TPN, containing as much as 40 to 80 y per gram.’

E. STRUCTURE AND PROPERTIES

1. DipHosPpHOPYRIDINE NucLEOTIDE (DPN)
a. Structure

H
oC N=C—NH
|
CH C—CONH. HC. C—N
| | \
CH CH CH
ee ve
N, N—C—N
| |
H—C H—CcC
| |
H—C—OH H—C—OH
O | O
H—C—OH | H—C—OH
|
H=Cc (Ome H—cC J

IV. BIOCHEMICAL SYSTEMS A485

The structural formula listed above is now firmly established for the
oxidized form of DPN. This formula was established in the following way.

1. Acid hydrolysis splits the molecule to yield nicotinamide,’ * adenine,”°
and 2 molecules of ribose-5-phosphate.?!: 7

2. Hydrolysis by weak alkali at low temperature splits off nicotinamide
and leaves adenosinediphosphate ribose moiety.”

3. Hydrolysis with weak alkali at high temperature yields adenosine-
diphosphate.”*

4. Enzymatic hydrolysis under certain conditions yields nicotinamide
riboside and adenosine.”®

5. The fact that phosphate was linked to ribose in the 5 position was
shown by von Euler et al.,?° since the pentose phosphate from DPN gave
no formaldehyde when reacted with periodic acid.

6. D-Ribose was already known to be the carbohydrate of adenosine.”

Von Euler e¢ al.8 partially hydrolyzed DPN with acid, treated with
phosphatase, and isolated a pentose, the MSE SIGS aie of which was iden-
tical with p-ribose. As pointed out by Schlenk,” these workers did not
establish that the ribose came from the nicotinamide-linked portion of the
molecule and furthermore the phenylosazones of D-ribose and D-arabinose
are identical. He, however, reached the same conclusion by isolating the
pentose from nicotinamide nucleoside (step 4) and forming the p-bromo-
phenylhydrazone and studying its other properties. In addition, he con-
firmed the linkage of ribose and phosphate in the 5 position of ribose.

Thus the structural formula for DPN as proposed originally in 1936?9-*!
“seems well established. It should be noted, however, that the customary

16S. Ochoa and C. G. Ochoa, Nature 140, 1097 (1937).

17Q. Meyerhof and P. Ohlmeyer, Biochem. Z. 290, 334 (1937).

18 H. von Euler, Angew. Chem. 50, 831 (1937).

19H. von Euler, F. Schlenk, H. Heiwinkel, and B. Hégberg, Hoppe-Seyler’s Z.
yan? ystol. Chem. 256, 208 (1938).
°H. von Euler and K. Myrbick, Hoppe-Seyler’s Z. physiol. Chem. 177, 237 (1928);
Baie as 17, 291 (1929).

21 F. Schlenk, Arkiv Kemi, Mineral. Geol. 12B, 20 (1936).

22 F. Schlenk, J. Biol. Chem. 146, 619 (1942).

23 FP. Schlenk, H. von Euler, H. Heiwinkel, W. Gleim, and H. Nystrém, Hoppe-Sey-
ler’s Z. physiol. Chem. 247, 23 (1937).

24. Vestin, F. Schlenk, and H. von Euler, Ber. 70, 1369 (1937).

25 F. Schlenk, Natwrwissenschaften 28, 46 (1940); Arch. Biochem. 3, 93 (1948).

26 H. von Euler, P. Karrer and B. Becker, Helv. Chim. Acta 19, 1060 (1936).

27 P. A. Levene and L. W. Bass, Nucleie Acids. American Chemical Society Mono-
graph No. 56, New York, 1931.

28 H. von Euler, H. Karrer, and E. Usteri, Helv. Chim. Acta 25, 323 (1942).

29 F. Schlenk and H. von Euler, Naturwissenschaften 24, 794 (1936).

30 H. von Euler and F. Schlenk, Hoppe-Seyler’s Z. physiol. Chem. 246, 64 (1987).

31K, Myrbick, T’abulae Biol. 14, 110 (1937).

32 L. J. Haynes and A. R. Todd, J. Chem. Soc. 1950, 303.

486 NIACIN

final proof of the structure of an organic compound by chemical synthesis
has not yet been reported. Some progress in this direction has been made
since some portions of the molecule can now be synthesized.” The lability
of various portions of the DPN molecule, especially the nicotinamide-
ribose bond, in the procedures ordinarily used in organic syntheses, makes
this a most difficult problem.

b. Properties

DPN is a white amorphous powder which is colorless in water solution.
It is moderately soluble in phenol and in methanol-HCl. Some of the

TABLE VIII
Some PRopERTIES OF DPN anp TPN (From F. ScHLENK**)
DPN TPN
Empirical formula Cx H27014N7P2 C21 H2s017N7P3
Structural units Nicotinamide, adenine, 2 moles of Nicotinamide, adenine, 2 moles of
ribose, and 2 moles of phosphoric ribose, and 3 moles of phosphoric
acid acid
Base equivalent
Oxidized state 1 3
Reduced state 2 4
Stability
Oxidized state
In 0.1 N HCl at 100° 50% destroyed after 8 min. 50% destroyed after 7.3 min.
In 0.1 N NaOH 50% destroyed after 17 min. (20°) 50% destroyed after 12 min. (23°)
Reduced state
In 0.1 N HCl at 20° Activity disappears immediately Activity disappears immediately
In 0.1 N NaOH at 100° Slight decrease in activity
In 0.1 N NaOH at 20° Stable Stable
Absorption spectrum
Oxidized state Em = 16.5 X 103 at 260 my Em = 16.5 X 103 at 260 my
Reduced state Em = 14.5 X 103 at 260 my Em = 14.5 X 103 at 260 my
Reduced state Em = 4.5 to 6.3 X 103 at 340 my Em = 4.5 to 6.3 XK 103 at 340 my
Oxidation-reduction poten- Ey = —0.28 to 0.31 v

tial

important properties are listed in Table VIII. It is stable to oxidants such
as bromine, H.O2, and permanganate in acid solution unless catalysts such
as iron are present.!: 1°: *! Hypoiodide results in rapid destruction,** whereas
hyposulfite reduces DPN to DPNH:.! Alkaline ferricyanide converts DPN
to a pyridone derivative. The latter can be condensed to yield a fluorescent
product, which reaction has been used as the basis for assay methods.*#-#6

32a F. Schlenk, in The Enzymes, Vol. 2, Part 1, p. 263. Academic Press, New York
1951.

33 P, Karrer, F. Schlenk, and H. von Euler, Arkiv Kemi, Mineral. Geol. 12B, 26 (1936)

34 W. E. Knox and W. I. Grossman, J. Biol. Chem. 166, 391 (1946); 168, 363 (1947).

35 F. Rosen, W. A. Perlzweig, and I. G. Leder, J. Biol. Chem. 179, 157 (1949).

36K. J. Carpenter and E. Kodicek, Biochem. J. 46, 421 (1950).

IV. BIOCHEMICAL SYSTEMS 487

Treatment with bisulfite and cyanide give addition compounds*’ while
strong acids give pyridinium salts.

DPN is sensitive to ultraviolet light, being destroyed thereby.**-4° DPN
shows no fluorescence in ultraviolet light, but DPNH. exhibits a whitish
fluorescence. DPN is optically active, the specific rotation being —20° at
643.9 mu and —70° at 546 mu.”

DPN shows an absorption peak at 260 mu in the ultraviolet. DPNH,

103 cm.2/g. DPN

Absorption coefficient

hela lind
ee SS

Fig. 7. Ultraviolet absorption spectrum of diphosphopyridine nucleotide (pH
9.7); oxidized (O) and reduced (@) forms. (Adapted from Warburg and Christian.*)

also exhibits a peak at 260 my and, in addition, a peak at 340 my which
is entirely absent in the oxidized form (Fig. 7). This band at 340 mu has
been the basis for innumerable assay methods and has major biochemical
usefulness.

37 Q. Meyerhof, P. Ohlmeyer, and W. Mohle, Biochem. Z. 297, 113 (1938).

38 QO. Warburg and W. Christian, Biochem. Z. 282, 221 (1935).

39 J. Runnstrém and L. Michaelis, J. Gen. Physiol. 18, 717 (1935).

40 H. von Euler and F. Schlenk, Arkiv Kemi, Mineral. Geol. 12B, 19 (1936).

“1K. Myrbiick, H. von Euler, and H. Hellstrom, Hoppe-Seyler’s Z. physiol. Chem.
212, 7 (1932).

488 NIACIN

2. TRIPHOSPHOPYRIDINE NUCLEOTIDE (TPN)

a. Structure

CH N—=C—NH,
\ |
HC C—CONH, HC C—NH
| | NN
HC CH CH
NY, VA
Nt N—C—N
| |
HC H—C O
| Liters ea
H—C—oOH HC 0—r— or
| O | O |
H—C—OH | H—C—OH OH
| |
H—¢ lo- 0) -HE=6
| | | |
GH. —_ ©— 2 0—_P—0—

As depicted in the above formula, TPN differs from DPN only by having
an additional phosphate group. The close similarity of DPN and TPN
became evident in the early work of Warburg and Christian? and of von
Euler’s group,’ 2° not only from their chemical similarity but in the simi-
larity of their biochemical action. The fact that DPN can be reversibly
converted to TPN also strengthens this close relationship.”-*

Warburg and Christian?’ 4 and Karrer and associates**: 46-5! investigated
the mode of linkage of nicotinamide with the rest of the molecule. These
investigations firmly established the pyridinium linkage as shown above.

However, the exact linkage of the remainder of the components remained
uncertain, especially the position of the third phosphate. The rather cum-

42). Adler, S. Elliot, and L. Elliot, Enzymologia 8, 80 (1940).

43 A. Kornberg, J. Biol. Chem. 182, 805 (1950).

44H. von Euler and E. Adler, Hoppe-Seyler’s Z. physiol. Chem. 252, 41 (1938).

45 R. Vestin, Naturwissenschaften 25, 667 (1937).

46 P. Karrer, F. Kahnt, R. Epstein, W. Jaffe, and T. Ishii, Helv. Chim. Acta 21, 223
(1938).

47P, Karrer, T. Ishii, F. W. Kahnt, and I. von Bergen, Helv. Chim. Acta 21, 1174
(1938).

48 P. Karrer, B. H. Ringier, J. Biichi, H. Fritzsche, and U. Solmssen, Helv. Chim.
Acta 20, 55 (1937).

49 P. Karrer, G. Schwarzenbach, and G. E. Utzinger, Helv. Chim. Acta 20, 720 (1937).

50 P. Karrer, G. Schwarzenbach, F. Benz, and U. Solmssen, Helv. Chim. Acta 19,
811 (1936).

51 P. Karrer and F. J. Stare, Helv. Chim. Acta 20, 418 (1937).

IV. BIOCHEMICAL SYSTEMS 489

bersome isolation procedure used by Warburg (p. 482) and the small yields
inhibited research on this subject. However, as improved methods of pre-
paring TPN were developed, the subject was again attacked so that the
structure has now been clarified to some extent.

Two principal possibilities were considered. One that the three phos-
phates were linked in a chain® or, as later suggested by Schlenk e¢ al.**
that the third phosphate group was attached to the pentose of the adenosine
portion of the molecule. This matter was not definitely settled until Korn-
berg and associates ®> subjected TPN to the action of a nucleotide pyro-
phosphatase which split the coenzyme into two fragments, nicotinamide
ribose phosphate and a diphosphoadenosine fragment which was not adeno-
sinepyrophosphate. Kornberg and Pricer®® were then able to hydrolyze
specifically the phosphate esterified to carbon 5 of the diphosphoadenosine
fragment leaving an adenosinemonophosphate. They then compared this
monophosphate derivative to known samples of adenylic acid ‘“a’’ and
adenylic acid ‘“‘b.’’>7 8 Kornberg and Pricer could show that the mono-
phosphate compound from TPN was not adenylic acid ‘‘b” and was indis-
tinguishable from adenylic acid ‘‘a.’’ This finding has been confirmed
independently by Wang et al.°° Adenylic acids ‘“‘a” and “‘b” were thought
at first to be adenosine-2-phosphate and adenosine-3-phosphate, respec-
tively, but this is now uncertain.®: & The 2 and 3 positions of ribose seem
the only likely places where the third phosphate could be attached, since
carbon 1 is in a-glycosidic linkage with adenine, carbon 5 is esterified with
the second phosphate, and carbon 4 is part of a furanose ring. However,
' the formula as shown above must be regarded as tentative until the struc-
tures of adenylic acid ‘‘a”’ and ‘‘b” are proved.

b. Properties

Many of the properties of TPN have already been listed (see Table VITT).
In general its properties are similar to DPN. It has the same absorption
spectrum. This might be expected, since the phosphates contribute little
to absorption in the usual ultraviolet range. As with DPN, the absorption
at 260 my is due principally to the adenylic acid moiety.*: 7 Nicotinamide

82 H. von Euler and F. Schlenk, Hoppe-Seyler’s Z. physiol. Chem. 246, 64 (1937).

3 F. Schlenk, B. Hégberg and 8. Tingstam, Arkiv Kemi, Mineral. Geol. 13A, No. 11
(1939).

54 A. Kornberg, J. Biol. Chem. 174, 1051 (1948).

55 A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 182, 763 (1950).

56 A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 186, 557 (1950).

57 C. E. Carter, J. Am. Chem. Soc. 72, 1466 (1950).

58 W. E. Cohn, J. Am. Chem. Soc. 72, 1471 (1950).

59 T. P, Wang, L. Shuster, and N. O. Kaplan, J. Am. Chem. Soc. 74, 3204 (1952).

60 JT). M. Brown and A. R. Todd, J. Chem. Soc. 1952, 44.

61 TD). M. Brown, O. I. Magrath, and A. R. Todd, J. Chem. Soc. 1952, 2708.

490 NIACIN

contributes slightly to absorption intensity at 260 mu, and the slightly
reduced absorption intensity of reduced DPN and TPN at 260 mu is
probably due to a disturbance in the conjugated double bonds of the
pyridine ring. The absorption at 340 my of reduced DPN and TPN is a
function of the addition of hydrogen to the pyridine ring. Since both DPN
and TPN react to form the same type of reduced compounds, an identical
spectrum at this wavelength would be expected.

There has been little uniformity in the reported values for the extinction
coefficients of DPNH: and TPNH: at 340 my. The published values, re-
cently reviewed by Drabkin,® have varied from 4.78 X 10° to 6.28 x 10°
sq. em. X mole. More recent studies using DPN- or TPN-dependent
reactions which go to completion with pure substrates have shown identical
extinction coefficients for DPNH: and TPNH: at 340 mu of 6.22 « 10°
sq. cm. X mole.

TPN is destroyed by ultraviolet light, as is DPN. Electrophoresis de-

terminations show two different dissociation constants, pK, = 1.8 and
pK. = 6.1. TPN is optically active, [a]ss9m, = —24.6 and [a]si4¢mz =
—29.4.%

F. MECHANISM OF ACTION
1. THE CoENZYMES

DPN and TPN function in oxidation-reduction systems by virtue of
their ability to accept hydrogen atoms (dehydrogenation) from certain
substrates and transfer these hydrogen atoms to other hydrogen acceptors
such as the flavin enzymes. In other words, DPN and TPN function by
reversibly alternating between the oxidized (I) and the reduced state (II),
as depicted below.

H (dD) gE (U0)
C Cc
Vi a
HC C—CONH:2 H HC C—CONH:2
| | XN |
ENG. CH 2Ht + 2e Cc CH
ey = A erg
Nt O7- H N On Hs
| | |
Ribose—O—P—_O—R Ribose—O— P—O—R
O O

62 TD). L. Drabkin, J. Biol. Chem. 157, 563 (1945).

63 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948).

64H. R. Rosenberg, Chemistry and Physiology of the Vitamins, p. 236. Interscience
Publishers, New York, 1942.

IV. BIOCHEMICAL SYSTEMS 491

In the oxidized state, the nicotinamide nucleus exists as a quaternary
pyridinium ion which forms an inner salt with one of the ionizable acid
groups in the pyrophosphate bridge. Upon reduction the pyridine nitrogen
is converted to a weakly basic tertiary amine, hence changing the proper-
ties of the compound.

The hydrogen-transferring property of DPN had been postulated in
19346° on the basis of its function as coenzyme for a number of dehy-
drogenases. It remained, however, for Warburg and associates in their
classic work! 2:4 to clearly demonstrate this mechanism in their studies
with TPN and later with DPN.

This reaction probably proceeds in more than one step with the inter-
mediate formation of semiquinoid radicals. As shown by Warburg and
associates, when DPN or TPN are reduced by hydrosulfite, an intensely

H H
C c
Vi oO
HC aoe H HC C—CONH;
| | SI
HC CH + NaS.0.+2H,0—> C CH + 2NaHSO, + HX
\ <
N H N
eS |
Bax | R

yellow semiquinoid substance (monohydrocoenzyme) is observed as an
intermediate in the reaction. This intermediate substance can be stabilized
‘by carrying out the reaction in a strongly alkaline medium.*®*-®

2. Tur APOENZYMES

DPN and TPN function in biochemical reactions only when joined with»
or activated by, specific proteins (apocodehydrogenases). Although the
exact mechanism by which the protein apoenzyme unites with the coen-
zyme is very unsettled, it is clear that the coenzyme will not function
catalytically without the influence of the apoenzyme. It has been postu-
lated that the apoenzyme functions by facilitating formation of the semi-
quinoid intermediates referred to above.®: 7°

Although the coenzymes are quite non-specific in that they function in

65 H. von Euler, Chemie der Enzyme, Vol. II, Part 3. Bergman, Munich, 1934.

66 P. Karrer and F. Benz, Helv. Chim. Acta 19, 1028 (1936).

67 —. von Euler, H. Hellstrém, and H. von Euler, Hoppe-Seyler’s Z. physiol. Chem.
242, 225 (1936).

68 F, Schlenk and T. Schlenk, Arch. Biochem. 14, 131 (1947).

69 J,, Michaelis and C. V. Smythe, Ann. Rev. Biochem. 7, 1 (1938).

70 T,. Michaelis, Advances in Enzymol. 9, 1 (1949).

492 NIACIN

a number of systems involving hydrogen transfer, the apoenzymes are
generally specific, a different apoenzyme being required for each substrate
system. The apoenzymes contribute more to specificity than the prosthetic
group. In other words, these two coenzymes are capable of acting with a
number of apoenzymes, alternating from one to another as needed, hence
the term “mobile” coenzymes.” Some of the apoenzyme-coenzyme com-
plexes are capable of catalyzing reactions other than that of their specific
substrates, but the reaction rates are generally low. There are some excep-
tions to this, however, in which a given coenzyme-apoenzyme system can
catalyze the oxidation of a number of substrates with efficiency.”

3. Tae CoENzYME-ENzYME COMPLEX (HOLOENZYME)

DPN and TPN are usually readily dissociable from their apoenzymes.
Reduced DPN and TPN appear to have a lower affinity for their apo-
enzymes than the oxidized forms. This may be due to the fact that quater-
nary nitrogen of the pyridine ring is changed to a tertiary amine in the
reduced coenzyme. The elimination of this strong basic group increases
the acidic properties of the coenzyme and may increase its dissociability
from the apoenzyme, hence leaving the coenzyme free to migrate to an-
other enzyme system where it can donate its hydrogens to another sub-
strate or hydrogen acceptor.

Some exceptions to the ready dissociability of coenzymes from their
apoenzymes have been recorded. Cori e¢ al.” have been able to crystallize
phosphoglyceraldehyde dehydrogenase in combination with DPN. Others
have reported a very firm attachment of coenzymes in the particulate
matter of cells which catalyze reactions in the Krebs cycle.”

4. Point or HyDROGENATION

The point of reversible attachment of hydrogen to the pyridine nucleus
is not entirely certain. It is clear from the studies of Karrer and associ-
ates*’ 46-51 with N!-substituted model compounds that hydrogen is attached
to one of the carbons adjacent to the ring nitrogen. Available evidence,
especially from the studies of Knox and Grossman,**: 7° indicates the prob-
able point as the 6 position.

71 J. K. Parnas, Nature 151, 577 (1943).

72 A. Meister, J. Biol. Chem. 184, 117 (1950).

73 C. F. Cori, S. F. Velick, and G. T. Cori, Biochim. et Biophys. Acta 4, 160 (1950).
74, M. Huennekens and D. E. Green, Arch. Biochem. 27, 418, 428 (1950).

75 W. E. Knox and W. I. Grossman, J. Am. Chem. Soc. 70, 2172 (1948).

IV. BIOCHEMICAL SYSTEMS 493

G. SPECIFICITY
1. DPN-TPN SpeEcIrFiciry

Most DPN-TPN-linked enzyme systems exhibit a definite preference, if
not a specific requirement, for either DPN or TPN. Well-known exceptions
to this are liver glutamic dehydrogenase’® ” and glucose dehydrogenase’,
which react equally well with either TPN or DPN. Mehler e¢ al.” studied
the specificity of the DPN or TPN requirement for a number of enzyme
systems. Malic and lactic dehydrogenases, for instance, reacted with either
DPN or TPN, but the reaction rates were many times faster with DPN.
On the other hand, isocitric dehydrogenase is strictly TPN-specific,’”: 79: °°
whereas triosephosphate dehydrogenase is strictly DPN-specific.*!

Earlier reports which indicated a relatively non-specific DPN-TPN re-
quirement for certain dehydrogenases may have been complicated by the
fact that certain liver” and yeast® preparations can catalyze the conversion
of DPN to TPN if ATP is present. It is also well known that some tissues
contain phosphatases which convert TPN to DPN.®: ®

In addition Colowick and associates*: 8° have obtained evidence of an
enzyme in Pseudomonas fluorescens extracts which appears to catalyze the
reaction TPNH, + DPN — TPN + DPNH:. An enzyme which carries
out this reaction has been observed in animal tissue.*®

2. SPECIFICITY OF THE MOLECULE

Only a few modifications or derivatives of the DPN-TPN molecules
have been studied to determine the range within which these molecules
can be modified and still retain enzymatic activity. Desamino DPN can
be prepared by treating DPN with nitrous acid® or with an enzyme de-
rived from takadiastase,® the free amino group of the adenine moiety

76 H. von Euler, E. Adler, G. Giinther, and N. B. Das, Hoppe-Seyler’s Z. physiol.
Chem. 254, 61 (1938).

77 A. H. Mehler, A. Kornberg, 8. Grisolia, and 8. Ochoa, J. Biol. Chem. 174, 961
(1948). oe

78H. J. Strecker and 8S. Korkes, J. Biol. Chem. 196, 769 (1952).

79§. Ochoa, J. Biol. Chem. 174, 133 (1948).

80, Adler, H. von Euler, G. Giinther, and M. Plass, Biochem. J. 33, 1028 (1939).

81 G. T. Cori, M. W. Slein, and C. F. Cori, J. Biol. Chem. 159, 565 (1945).

82 H. von Euler, E. Adler, and T. 8. Eriksen, Hoppe-Seyler’s Z. physiol. Chem. 248,
227 (1937).

88 N. O. Kaplan, S. P. Colowick, and M. M. Ciotti, J. Biol. Chem. 194, 579 (1952).

84§. P. Colowick, N. O. Kaplan, E. F. Neufeld, and M. M. Ciotti, J. Biol. Chem.
195, 95 (1952).

85 N. O. Kaplan, 8. P. Colowick, and E. F. Neufeld, J. Biol. Chem. 195, 107 (1952).

86 J. Stern, quoted in Federation Proc. 11, 238 (1952).

87 F, Schlenk, H. Hellstrém, and H. von Euler, Ber. 71, 1471 (1938).

494 NIACIN

being removed in the process. In the pig heart lactic dehydrogenase system,
desamino DPNH is actually more active than DPNH; for pyruvate reduc-
tion but is less active for lactate oxidation. In other dehydrogenase systems
desamino DPN has activity sometimes equal to, but generally less than,
the parent material.88 Whether desamino DPN has any normal function,
or even exists in tissues, is not known. Desamino TPN cannot be formed
by the same procedures which yield desamino DPN apparently due to the
influence of the third phosphate attached to the ribose adenine group.

3. NICOTINAMIDE MONONUCLEOTIDE

It has been shown that nicotinamide mononucleotide (nicotinamide-
ribose-5-phosphate) accumulates in red blood cells incubated with nico-
tinamide and glucose.*? Furthermore, certain tissues have been shown to
contain a nucleotide pyrophosphatase which splits DPN to yield this com-
pound.® 9% Although this substance cannot function enzymatically as
DPN or TPN, it may have considerable significance as an intermediate in
the biosynthesis of DPN and TPN (p. 506).

H. BIOCHEMICAL REACTIONS

DPN and TPN function in oxidation-reduction systems (although no
oxygen is actually transferred). The redox potentials of the systems cata-
lyzed by these coenzymes are generally quite similar to the redox potentials
of the coenzymes themselves. Thus the reactions are generally reversible,
depending on the concentration of the reactants and the products as well
as changes in redox potential induced by other reactions in the cell.

Eakin” has classified the important reactions catalyzed by DPN and
TPN into five categories. These are listed below, with some of the more
important specific systems cited as examples. In addition, a miscellaneous
category may be added to include reactions less clearly characterized or
which are not included in this classification. Schlenk” has carefully de-
scribed the most important of the enzyme reaction systems listed below.

1. Aldehyde = Primary alcohol

Acetaldehyde—ethanol (DPN)
2. Ketone = Secondary alcohol
Pyruvic acid —lactic acid (DPN)
Pyruvic acid—malic acid (TPN)
88 M. E. Pullman, 8. P. Colowick, and N. O. Kaplan, J. Biol. Chem. 194, 593 (1952).
89 T. G. Leder and P. Handler, J. Biol. Chem. 189, 889 (1951).
90 A. Kornberg and O. Lindberg, J. Biol. Chem. 176, 665 (1948).
% R. E. Eakin in The Biochemistry of the B Vitamins, American Chemical Society

Monograph No. 110, p. 141. Reinhold Publishing Corp., New York, 1950.

2 F. Schlenk in The Enzymes, Vol. II, Part 1, pp. 278-315. Academic Press, New

York, 1951.

IV. BIOCHEMICAL SYSTEMS 495

Oxalacetic acid—malic acid (DPN)
Oxalosuccinic acid—isocitric acid (DPN)
6-Hydroxybutyric acid—acetoacetic acid (DPN)
a-Ketoglutarate—isocitric acid (TPN)
3. Acyl phosphate = Aldehyde-1-phosphate
1 ,38-Diphosphoglyceric acid—3-phosphoglyceraldehyde (DPN)
4. Acid = Aldehyde (hydrate)
Gluconic acid—glucose (DPN) (TPN)
6-Phosphogluconic acid—glucose-6-phosphate (TPN)
Phosphoglyceric acid—phosphoglyceraldehyde (DPN)
Phosphogluconic acid—ribulose phosphate (TPN)
Acetic acid—acetaldehyde (DPN)
5. Imine = Amine
Iminoglutaric acid—.-glutamic acid (DPN-TPN)°*4
6. Miscellaneous
Reduction of nitrate to nitrite
The oxidation of liciferin to produce bioluminescence”
Dehydration of formic acid: %
HCOOH + DPN — CO, + DPNH,
Conversion of choline to betaine (DPN)9*-%
Conversion of vitamin A, to rhodopsin (DPN)*?
Reduction of methemoglobin (DPN)!
Destruction of testosterone by liver mince (DPN)!
L-a-Glycerophosphate to dihydroxyacetone phosphate!”
Dismutation of pyruvate (in bacterial extracts)!%
Pyruvate + phosphate + DPN — Acetyl phosphate +
CO; + DPNH; Pyruvate + DPNH — Lactate + DPN

I. SYSTEMS

The biochemical reactions listed previously do not occur independently
but as a part of a series of reactions by which an organism carries out the

a The glutamic acid dehydrogenase system of yeast and bacteria must be coupled
with TPN, although the corresponding system in animal tissues is non-specific
in its DPN-TPN preference.

93 R. Adler and M. Srenwasaya, Hoppe-Seyler’s Z. physiol. Chem. 249, 24 (1937).

% F. H. Johnson and H. Eyring, J. Am. Chem. Soc. 66, 848 (1944).

9% J.C. Wirth and F. F. Nord, Arch. Biochem. 1, 143 (1942).

96 P. J. G. Mann and J. H. Quastel, Biochem. J. 31, 869 (1937).

7 J. R. Klein and P. Handler, J. Biol. Chem. 144, 537 (1942).

9% J. N. Williams, Jr., J. Biol. Chem. 195, 37 (1952).

99 R. Hubbard and G. Wald, Science 115, 60 (1952).

100 Hf. R. Gutman, B. J. Jandorf, and O. Bodansky, J. Biol. Chem. 169, 145 (1947).

101M. L. Sweat and L. T. Samuels, J. Biol. Chem. 178, 433 (1948).

102 'T. Baranowski, J. Biol. Chem.180, 535 (1949).

103 §. Korkes, J. R. Stern, I. C. Gunsalus, and S. Ochoa, Nature 166, 439 (1950).

496 NIACIN

complicated process of obtaining energy and building blocks from its nutri-
ents or, during anabolic phases of metabolism, of synthesizing appropriate
molecules for deposition or incorporation into other tissue substances. Only
certain of these steps require the participation of DPN and TPN. Yet,
these coenzymes are necessary for the catalysis of steps which are of such
importance that, should these reactions be blocked, the cell cannot function
and indeed the organism itself cannot survive.

1. SynTHEsIS oF HicH-ENERGY PHOSPHATE BoNnpDs

Phosphoric anhydride compounds such as adenosinetriphosphate occupy
a key role in metabolism. They constitute a means whereby the cell can
conserve and store energy in a readily transferable form. DPN and TPN
are involved in the processes by which these high-energy phosphate bonds
are synthesized. For example, if an aldehyde such as 1 ,3-diphosphoglycer-
aldehyde is dehydrogenated (i.e., oxidized) by DPN, one of the phosphate
groups is converted into a reactive acyl phosphate which can be transferred
to adenosinediphosphate (ADP), thus forming adenosinetriphosphate
(ATP) as depicted below.!%-10

H
) H H O
| Peter?)
Fl sabia AER DEN —
| ire |
O H O H 0
H H H
0 H H O
| Gara) |
DPNH, + HOE Osan
| |
O H O O
H H H
O H H fo)
| OP Aall |
HO—P—o— : aire diner iro ADB
|
O H O O
H H H
i H i O
|
ATP + Ho) Or Os ae
O H O
H H

108 QO, Warburg and W. Christian, Biochem. Z. 301, 221 (1939).
105 QO. Warburg and W. Christian, Biochem. Z. 303, 40 (1939).
106 H}. Negelein and H. Bromel, Biochem. Z. 303, 132 (1939).

IV. BIOCHEMICAL SYSTEMS 497

Thus, during catabolic phases of cellular activity when organic substrates
are being oxidized to provide energy, a part of the energy may be stored
in the form of ATP. During anabolism, the process can be reversed, the
net effect of which is to convert organic acids to aldehydes, liberating a
reactive phosphate group which can be used to form glycosidic, ester, and
perhaps other (peptide) bonds. This mechanism could be used, for instance,
to supply the energy necessary to form the acetal bond by which glucose
is polymerized into glycogen. Reactions such as these represent a general
biological mechanism by which cells can convert the latent energy of
organic substances into readily stored and utilized energy.

In addition to the type of reaction listed above, it is known that there
are aerobic processes which convert inorganic phosphate into energy-rich
pyrophosphates by reactions in which hydrogen atoms of DPNH»2 and
TPNH; are transported to oxygen via the riboflavin and cytochrome en-
zymes.!” Using a particulate (mitochondrial) fraction from rat liver, Fried-
kin and Lehninger'® and Lehninger'? have been able to demonstrate the
synthesis of esterified phosphate during oxidations which are DPN linked.
Adenosinediphosphate appeared to be the phosphate acceptor in their sys-
tem. Lehninger™® has recently published a careful analysis of the evidence
pertaining to oxidative phosphorylation in DPN-linked systems. Although
some of the evidence is conflicting, and many details are as yet unclear, it
does seem probable that phosphorylation coupled to electron transport
between DPNH, and oxygen constitutes a general biological mechanism
for the esterification of high-energy phosphate. Green and associates,"
using a different system derived from pig heart, have also obtained evidence
indicating oxidative phosphorylation in DPN-—TPN-linked systems.

DPN and TPN participate in phosphorylation reactions in yet another
way. It has long been known that inorganic orthophosphate accumulates
during the respiration of certain tissues. As inorganic phosphate accumu-
lates, the balance between hexose phosphate and glycogen is disturbed,
resulting in the breakdown of glycogen to glucose-1-phosphate. This sup-
plies fuel for the glycolysis cycle, the energy from which can be utilized to
resynthesize phosphoric acid anhydrides from inorganic phosphates. As the
concentration of inorganic phosphate decreases and that of the organic
phosphate increases, the equilibrium shifts toward the formation of glyco-

107 F, Lipmann, Currents in Biochemical Research, pp. 137-148. Interscience Pub-
lishers, New York, 1946.

108 M. Friedkin and A. L. Lehninger, J. Biol. Chem. 178, 611 (1949).

109 A. L. Lehninger, J. Biol. Chem. 178, 625 (1949).

10 A. L. Lehninger, in Phosphorus Metabolism, p. 344. The Johns Hopkins Press,
Baltimore, 1951.

11 —P). E. Green and H. Beinert, in Phosphorus Metabolism, p. 330. The Johns Hop-
kins Press, Baltimore, 1951.

498 NIACIN

gen. This process is thought to constitute one of the important regulatory
mechanisms in carbohydrate metabolism.

The source of the inorganic phosphate which accumulates has long been
uncertain. It was widely held that most of it resulted from the hydrolytic
cleavage of ATP. In certain systems, however, it could be clearly shown
that ATP was not the source of the inorganic phosphate.%: 1) 13 Korn-
berg: "> recently established at least one source of this inorganic phos-
phate when he discovered an enzyme in yeast and in liver which catalyzed
the reversible reaction:

Nicotinamide mononucleotide + ATP — DPN + inorganic pyrophosphate

This reaction plus the ubiquitous inorganic pyrophosphatase could yield
orthophosphate. Schrecker and Kornberg!!® have demonstrated a similar
phenomenon with flavin mononucleotide.

It should also be noted that this mechanism provides for a biosynthesis
of DPN from nicotinamide mononucleotide (p. 506). The reverse of this
reaction, i.e., the phosphorolysis of a dinucleotide by inorganic pyrophos-
phate, resembles the action of inorganic phosphate on the reversible split-
ting of polysaccharides referred to above (p. 497).

2. DPN anp TPN In GLyYcoLysis

Several pathways are known by which glucose can be utilized.'!” The
classical series of reactions which seem to prevail in most organisms in-
volves some eleven steps, with pyruvic acid as the end product.!!8 Glucose
is phosphorylated and converted through a series of steps to fructose-1 ,6-
diphosphate. The latter compound is then split into two trioses, dihydroxy-
acetone phosphate and 3-phosphoglyceraldehyde. The latter compound
forms an addition product with phosphate, 1 ,3-diphosphoglyceraldehyde,
which is dehydrogenated by DPN and converted to 3-phosphoglyceric acid
as depicted on p. 496. The latter compound is converted in three steps to
pyruvic acid. The pyridine nucleotides are required for only one of these
eleven steps as indicated above.

3. In Pyruvate METABOLISM

Pyruvic acid can be utilized in a number of ways." When the supply
of oxygen is limited, pyruvic acid can be converted to lactic acid.

112 C, F. Cori, A Symposium on Respiratory Enzymes. Madison, 1942.

13 R. J. Cross, J. V. Taggart, G. A. Covo, and D. E. Green, J. Biol. Chem. 177, 655
(1949).

4 A, Kornberg, J. Biol. Chem. 182, 779 (1950).

115 A. Kornberg and W. HE. Pricer, Jr., J. Biol. Chem. 191, 535 (1951).

16 A.W. Schrecker and A. Kornberg, J. Biol. Chem. 182, 795 (1950).

17. §. G. Barron, Advances in Enzymol. 3, 149 (1943).

u8 QO, Meyerhof, Biol. Symposia 5, 141 (1941).

19 Ff}, Stotz, Advances in Enzymol. 5, 129 (1945).

IV. BIOCHEMICAL SYSTEMS 499

H
O} 20

0
| |

|
H,;C—C—C—OH + DPNH, = DPN + H;C—C—C—OH

|

H

It should be noted that this reaction could provide a mechanism to re-
oxidize the DPN which was reduced in the conversion of phosphoglyceral-
dehyde to phosphoglyceric acid.

Also, under anaerobic conditions pyruvic acid may undergo 6-carboxyla-
tion to yield oxalacetic acid,”° which in turn can be converted by malic
dehydrogenase, plus DPNH, as coenzyme, to malic acid as depicted below.

H
HO O G- HMO O
|

0
Pal Selle |

noe. —_€-__ OH -) DPN; — DEN -H0O—C—C—_C—_C__On
|
H Jef dal
Pyruvic acid may be converted by reductive fixation of carbon dioxide
to malic acid”! in the presence of ‘‘malic’”? enzyme, TPNH», and Mn** as
follows.

| M
CO. + H,cC—C—C—OH + TPNH: —=— HO—C—C—C—C—OH

Hy

It should be noted that this reaction provides a mechanism for carbon
dioxide fixation (see p. 503).

A fourth type of anaerobic utilization of pyruvic acid (in yeast) involves
decarboxylation to acetaldehyde which can be converted to ethanol.

O H
H,C—C—H + DPNH, — DPN + H;C—-C—OH

H

Pyruvic acid may be split to yield other 2-carbon compounds, i1.e., acetic
acid, or other 2-carbon acetyl fragments (activated acetate!) which are
now known to be combined with coenzyme A (acetyl coenzyme A). Under
aerobic conditions pyruvate may undergo oxidative decarboxylation to
120 H. G. Wood and C. H. Werkman, Biochem. J. 32, 1262 (1938).

121 §, Ochoa, A. H. Mehler, and A. Kornberg, J. Biol. Chem. 174, 979 (1948).
12 F, Lipmann, Advances in Enzymol. 6, 231 (1946).

500 NIACIN

yield acetyl coenzyme A. DPN is known to be reduced in the processes
which lead to the formation of acetyl coenzyme A, although the exact bio-
chemical mechanism is not known. The acetyl groups transferred by co-
enzyme A can be converted to acetic acid or commonly oxidized to carbon
dioxide and water via the tricarboxylic acid cycle.’ !4 In this complicated
series of reactions DPN is required for the interconversion of malic and
oxalacetic acids as described previously, and TPN is required for the con-
version of isocitric to oxalosuccinic acid.

H H

O O

|
-—C—Olsl @ C=O O

eZ [Ze
BEC ==, = SPN =P Nie He—-¢
lbE==(C Jal Oal eS Gael (Chal

| |

C=O C=O

| |

O O

H H

4. In Pentose BIOSYNTHESIS

Because of the importance of ribose (and desoxyribose) in nucleotide
metabolism, the biological origin of this substance has considerable interest.
The exact mechanism of its synthesis has been obscure until recently. A
series of reactions requiring TPN as a coenzyme has now been demonstrated
which can account for the biosynthesis of pentose. The initial reaction:

Glucose-6-phosphate + TPN = TPNH: + 6-phosphogluconie acid

was known from the early work of Warburg and associates.”: * It was also
known from studies by Lipmann!”® and by Warburg and Christian”®
that yeast extracts were capable of further oxidizing 6-phosphogluconic
acid in TPN-linked reactions. Dickens'’’ 9 identified a pentose in similar
reaction mixtures and postulated the formation of ribose-5-phosphate.

123 FT. A. Krebs, Advances in Enzymol. 3, 247 (1948).

124 HW. A. Lardy and C. A. Elvehjem, Ann. Rev. Biochem. 14, 1 (1945).
125 F. Lipmann, Nature 138, 588 (1936).

126 Q, Warburg and W. Christian, Biochem. Z. 287, 440 1936).

127 QO. Warburg and W. Christian, Biochem. Z. 292, 287 (1937).

128 F. Dickens, Biochem. J. 32, 1626, 1636 (1938).

129 #. Dickens and G. E. Glock, Nature 166, 33 (1950).

IV. BIOCHEMICAL SYSTEMS 501

Cohen and Scott!*: ! confirmed the formation of ribose-5-phosphate in
these reactions.

Horecker and Smyrniotis'? have purified the enzyme from yeast which
catalyzes this reaction and have been able to characterize the reaction.

6-Phosphogluconate + TPN — TPNH, + CO: + ribulose-5-phosphate

1

ribose-5-phosphate

The significance of these reactions may be considerably greater than
just as a mechanism for ribose synthesis. They may consitute an important
alternate pathway for the oxidation of glucose, and as a source of other
biological compounds. All the steps in the further metabolism of these
pentoses have not yet been elucidated. However, Horecker and Smyrniotis!’
have demonstrated the formation of a 7-carbon sugar, sedoheptulose, which
is apparently formed by the condensation of ribulose phosphate and a 2-
carbon fragment. In addition it should be noted that these reactions are
reversible,!* thus constituting a mechanism for the fixation of carbon di-
oxide, and may be of importance in photosynthesis.

5. In Lipip METABOLISM

The dependence of fat metabolism on DPN-—TPN-linked reactions has
been studied less extensively than with carbohydrates. It is clear that DPN
is required for the synthesis and degradation of glycerol. Dihydroxyacetone
phosphate, derived from the splitting of fructose-1 ,6-diphosphate, can be
reversibly converted to L-a-glycerophosphate by an enzyme which requires
DPNHE: as a hydrogen donor.!”> 135

1 Ey Oy JEL

HO—E—.0-_C—_C—_C—08 + DPN: =

| | |
O H H
H 7 H
O BD +O
| lai
DPN + HO—P—O—C—C—C—OH
| |

Ey Sele

130 §. 8. Cohen and D. B. M. Scott, Science 111, 543 (1950).

131 TF). B. M. Scott and 8. 8. Cohen, J. Biol. Chem. 188, 509 (1951).

132 B. L. Horecker and P. Z. Smyrniotis, J. Biol. Chem. 193, 371, 383 (1951).
138 B, L. Horecker and P. Z. Smyrniotis, Federation Proc. 11, 232 (1952).

134 B, L. Horecker and P. Z. Smyrniotis, J. Biol. Chem. 196, 135 (1952).

135 H, Baer and H. O. L. Fisher, J. Biol. Chem. 128, 463 (1939).

502 NIACIN

It is also clear that the pyridine nucleotides are indirectly important in
fatty oxidation, since the 2-carbon activated acetyl compounds which re-
sult from the 6-oxidation of fatty acids are further oxidized via the tricar-
boxylic acid cycle. These substances enter the cycle principally by condens-
ing with oxalacetic acid to form citric acid. Furthermore, these acetyl
compounds seem to provide the common link between fat and carbohy-
drate metabolism. Since they can be formed from either carbohydrate or
fat, they provide a means of synthesis of fat from carbohydrates or, vice
versa, the oxidation of fat to provide energy.

Lehninger™* has shown that DPN is required for fatty acid oxidation in
crude rat liver homogenates. Green and associates!!! also have demonstrated
fatty acid oxidation in enzyme systems which require DPN or TPN. The
exact reactions and mechanisms involved in these complex systems and
the specific role of the pyridine nucleotides (other than as a means of gen-
erating high-energy phosphate) is completely unknown.

The DPN requirement for fatty acid synthesis is even less clear. How-
ever, it is thought that fatty acid synthesis and degradation proceed through
identical, albeit reverse, reaction pathways. On this basis, DPN-TPN
would undoubtedly be required. Eakin*! believes that the pyridine nucleo-
tides are required for the numerous dehydrogenations which are probably
involved and has proposed a scheme based on known reactions, some of
which are DPN-linked, which could account for the synthesis of fatty acids.

DPN is important in the metabolism of the ketone bodies, since the
interconversion of L-G-hydroxybutyric and acetoacetic acids is linked to
this coenzyme.}*7: 188

H
Oa 0 Oe it) BO

| lho ieee |

|
H;C—C—C—C—OH + DPN = H,C—C—C—C—OH + DPNH:
H H H

6. In NrrroGen METABOLISM

The pyridine nucleotides play a direct role in the metabolism of amino
acids in only one system. Either DPN or TPN is required for the reversible
oxidative deamination of L-glutamic acid. The a-amino group is converted
to an a-imino group which spontaneously hydrolyzes to yield NH; and
a-ketoglutaric acid. Since this reaction is reversible, it provides a link for
the biological synthesis of an amino acid utilizing inorganic ammonia and
a compound which can be derived from carbohydrate. Conversely it pro-
136 A. LL. Lehninger, J. Biol. Chem. 161, 487 (1945).

137 J). E. Green and J. Brosteaux, Biochem. J. 30, 1489 (1936).
138 TJ), EK. Green, J. G. Dewan, and L. F. Leloir, Biochem. J. 31, 934 (1937).

IV. BIOCHEMICAL SYSTEMS 503

H H NH, O

O
Bill Heike

OC C—O 4-- DPN —

pace

H H H
ist
Oo H HN O
I odecenelirae ech
DPNH, + HO—C—C—C—-C—-C—OH
Ractl
1S Oaael lawl
O HH
ei
HO—C—C—C—C—C—OH + NH;
[eae
H H

vides a means whereby the organism can derive energy from protein by
converting the protein to glycogenic fragments.’® This reaction probably
has considerable biological significance, since glutamic acid is currently
believed to constitute a central pool to receive or donate amino groups.

Indirectly, DPN and TPN are important in protein metabolism when
the organism breaks down amino acids to provide energy. The deaminated
derivatives of the glycogenic amino acids are further oxidized via conven-
tional carbohydrate oxidative pathways as already described.

Meister” has recently shown that crystalline lactic dehydrogenase and
- DPNH: are capable of reducing a number of a, y-diketo, and a-keto acids
to products as yet unidentified (but not lactic acid). TPNH: can substitute
for DPNHg, but the reaction rate is much slower. This reaction may have
biological significance in the utilization of the keto derivatives of amino
acids as well as other metabolites.

7. In PHOTOSYNTHESIS

The biochemical mechanism of photosynthesis has been a subject of
great interest but little information for many years. It is now known that
animals as well as plants and bacteria can incorporate atmospheric carbon
dioxide into organic compounds. Wood" has listed seventeen compounds
in which carbon dioxide can be fixed, although only a few of these provide
for primary fixation of carbon dioxide.

Two recent research findings have emphasized the fact that the pyridine
nucleotides may have an important role in photosynthesis. Horecker and
Symrniotis have proved the reversibility of the following reaction:

Ribulose-5-phosphate + CO, + TPNH + Ht — 6-Phosphogluconate + TPN
139 H. G. Wood, Physiol. Revs. 26, 198 (1946).

504 NIACIN

Since 6-phosphogluconate can be converted to glucose-6-phosphate in
a TPN-linked reaction (p. 500), it is evident that this could provide a
mechanism for the incorporation of carbon dioxide directly into glucose.

An even more pertinent development was the demonstration by Ochoa
et al. of the following TPN-linked reaction catalyzed by ‘‘malic’”’ enzyme:

Mnt+
CO, + pyruvate + TPNH, —— Lt-Malate + TPN

When this reaction was coupled with a glucose-6-phosphate-TPN system
to provide a steady supply of TPNH., a highly efficient means of fixing
carbon dioxide resulted.“ A similar reaction occurs in the formation of
isocitric acid.79

M 4+
GO; + eketoglutarate 4 TPNH. === D-Isociteate PN

Furthermore, evidence has been obtained that green grana from spinach
chloroplasts can effect a reduction of DPN and TPN, presumably by utiliz-
ing the energy of light to transfer hydrogens from water to the coenzymes,
thus supplying the reduced coenzymes needed for the above reactions."
Carbon dioxide fixation forming malate or isocitrate was shown to proceed
in these green grana suspensions when illuminated, but not in the dark.
Ochoa and Vishniac!® also demonstrated that the DPNHs: generated in
the green grana could be utilized to catalyze other DPN linked reactions
such as pyruvate — lactate, oxalacetate = malate, and a-ketoglutarate =
L-glutamate. It should also be noted that DPNH:2 and TPNH» produced
by the action of light could be used to generate high phosphate bond energy,
i.e., convert light energy to chemical energy (p. 496). Tolmach™ and
Arnon have confirmed and extended much of the work reported above
and have provided additional evidence for the view that photosynthesis in
plants may operate basically through a reversal of a respiratory cycle
which is powered by radiant energy rather than chemical energy.“® The
light-induced reduction of the pyridine nucleotides may be a key step in
these processes.

J. BIOLOGICAL SYNTHESIS OF DPN AND TPN

It is now possible to account rather completely for the biological synthe-
sis of these coenzymes. Reactions other than, or in addition to, the ones

140 §, Ochoa and W. Vishniac, Science 115, 297 (1952).

141 W. Vishniac, Federation Proc. 10, 265 (1951).

142, W. Vishniae and 8. Ochoa, Nature 167, 768 (1951).

143 W. Vishniac and S. Ochoa, J. Biol. Chem. 195, 75 (1952).

144 T,, J. Tolmach, Nature 167, 946 (1951).

145 T), J. Arnon, Nature 167, 1008 (1951).

146 M. Calvin, J. A. Bassham, and A. A. Benson, Federation Proc. 9, 524 (1950).

IV. BIOCHEMICAL SYSTEMS 505

listed below may exist, but at least these reactions make possible one route
of formation.

1. SyntueEsis oF Nicotinic AcIpD

The sources of nicotinic acid may be diet, the conversion of tryptophan
to nicotinic acid (p. 523), or possibly intestinal microorganisms (p. 530).

2. AMIDATION OF NIcoTINIC ACID

Ellinger“’ has shown that kidney and brain slices are capable of amidating
nicotinic acid. Liver slices can also accomplish this conversion if a source
of NH; such as glutamine is provided. Slices from several other tissues were
unable to accomplish this conversion. It was assumed from early studies
that erythrocytes (human) could also amidate nicotinic acid since, both
in vivo and in vitro, nicotinic acid but not nicotinamide produced an increase
within the erythrocytes of material which had activity for Hemophilus
parainfluenza, for Hemophilus influenza, or for DPN-linked enzyme sys-
tems.'48-152 Furthermore, Liefer e¢ al.!°* used carboxyl-C"™-labeled nicotinic
acid and nicotinamide to show a rapid uptake and fixation of nicotinic
acid within the erythrocyte (mice). Nicotinamide, on the other hand, was
taken up by the erythrocytes but could be washed out readily, indicating
that it had not been incorporated into fixed non-diffusible molecules.

More recently, Leder and Handler*® have found just the contrary, L.e.,
that nicotinamide but not nicotinic acid resulted in an increased synthesis
of coenzyme-active material in erythrocytes. A good explanation for these
_ contradictory results is not apparent.

3. SYNTHESIS OF RIBOSE

A mechanism which can account for the synthesis of ribose from glucose
has already been detailed (p. 500). Other mechanisms are possible.!™

4. Synruesis oF NICOTINAMIDE MONONUCLEOTIDE

This fragment of the molecule (nicotinamide-ribose-phosphate) can be
synthesized by erythrocytes incubated with nicotinamide and glucose.®® A

47 P, Hllinger, Biochem. J. 42, 175 (1948).

148 H, I. Kohn and J. R. Klein, J. Biol. Chem. 130, 1 (1939).

149 A. I. Kohn and J. R. Klein, J. Biol. Chem. 135, 685 (1940).

150 C, L. Hoagland and 8. M. Ward, J. Biol. Chem. 146, 115 (1942).

151 P| Handler and H. I. Kohn, J. Biol. Chem. 150, 447 (1943).

1682 ©, L. Hoagland, S. M. Ward, and R. E. Shank, J. Biol. Chem. 151, 369 (1943).

153 E. Leifer, J. R. Hogness, L. J. Roth, and W. H. Langham. J. Am. Chem. Soc. 70,
2908 (1948).

164 B. L. Horecker, in Phosphorus Metabolism, Chapter III. The Johns Hopkins
Press, Baltimore, 1951.

506 NIACIN

mechanism which can account for the synthesis of this fragment has been
described by Rowen and Kornberg.!®® A reversible reaction which is cata-
lyzed by an enzyme preparation from hog liver and which requires ortho-
phosphate was observed.

Nicotinamide-riboside + orthophosphate
— Nicotinamide + ribose-1-phosphate + Ht

The further reaction is tentative, but preliminary evidence indicates a
direct phosphorylation of nicotinamide riboside by ATP:

Nicotinamide-riboside + ATP — Nicotinamide-riboside-phosphate + ADP
5. BIosyNTHESIS oF DPN

The biosynthesis of DPN had been observed in erythrocytes as described
earlier and in yeast fermentation systems.!®® Kornberg and _associ-
ates®®: 114, 115 have observed the following reversible reaction in a purified
enzyme system:

Nicotinamide-ribose-phosphate + ATP — DPN + inorganic pyrophosphate

This reaction has been confirmed, with P*® as a tracer.!!® From the kinetics
of this reaction and the concentration of the enzyme in liver, Kornberg!
estimates that the entire DPN content of liver could be synthesized in
less than 5 minutes if the substrates were optimal. It can be seen that this
reaction not only completes DPN synthesis but provides an origin for the
adenine-ribose-phosphate portion of the molecule, i.e., from ATP. The
mechanism of adenine biosynthesis is obscure, except that it is known to be
synthesized by mammalian tissues.

6. BrosyNTHESIS oF TPN

The biosynthesis of TPN from nicotinamide plus ribose and adenosine-
triphosphate (ATP) in cell-free extracts has been reported.!*” This report
has never been confirmed, and subsequent experience leaves considerable
doubt as to its validity. It is known that TPN can be synthesized enzymat-
ically from DPN?**: 153 when DPN is incubated with ATP and a yeast
preparation. Mehler et al.” also observed the formation of TPN from DPN
and ATP in crude liver fractions. The mechanism of this reaction has now
been shown to be a direct phosphorylation of DPN by ATP which is cata-
lyzed by an enzyme which can be purified from ale yeast.”

DPN + ATP — TPN + adenosinediphosphate (ADP)

155 A. Kornberg, in Phosphorus Metabolism, Chapter VI. The Johns Hopkins Press,
Baltimore, 1951.

166 A. Lennerstrand, Arkiv Kemi, Mineral. Geol. 14A, No. 16, 1 (1941).

167 K. I. Altman and E. A. Evans, Jr., J. Biol. Chem. 169, 463 (1947).

468 HZ. von Euler and R. Vestin, Arkiv Kemi, Mineral. Geol. 12B, No. 44 (1938).

IV. BIOCHEMICAL SYSTEMS 507

TPN may be readily converted to DPN by phosphatases from a number
of sources,**: 15° by yeast,“ and by ‘“‘cyclophorase” preparations from rabbit
kidney and rabbit liver.!®

K. DESTRUCTION OF DPN AND TPN

Some of the ways in which these coenzymes can be degraded have al-
ready been described, since most of the reactions described under biosyn-
thesis (p. 506) are reversible. Actually, it has long been known that many
animal tissues, plants, and microorganisms can enzymatically split these
coenzymes.!® Ohlmeyer!® and Heiwinkel!® identified adenylic acid as a
breakdown product of DPN. Das and von Euler!™: !®° found inorganic
orthophosphate when DPN was destroyed in animal tissues. Handler and
Klein!** identified nicotinamide as a breakdown product when DPN was
destroyed by brain, liver, kidney, and muscle preparations from several
animal species. Mann and Quastel'® also called attention to the potent
destructive power of certain animal tissues, especially brain, and showed
that this rapid breakdown could be inhibited with rather high concentra-
tions of nicotinamide. McIlwain and associates! '6° have also studied the
destruction of DPN in brain preparations. Govier and Jetter'° have shown
that a-tocopherol phosphate has an inhibitory effect on DPN breakdown
by heart DPNase. From these and other studies it appears that the com-
mon DPNase of animal tissue splits DPN at the glycosidic linkage between
nicotinic acid and ribose. Nason e¢ al." have concentrated a DPNase from
Neurospora which acts in a fashion similar to animal DPNase. This enzyme
is not inhibited by excess nicotinamide, however. Animal tissues also con-
tain another type of DPNase which splits DPN at the pyrophosphate
linkage.°°

These DPN-destroying enzymes are particularly active when the cells

159 ED). R. Sanadi, J. J. Betheil, and B. J. Katchman, Abstr. Am. Chem. Soc. 118th Meet-
ing, p. 52C, 1950.

160 B. Katchman, J. J. Betheil, A. I. Schepartz, and D. R. Sanadi, Arch. Biochem. and
Biophys. 34, 437 (1951).

161 Ff. Schlenk, Advances in Enzymol. 5, 207 (1945).

162 P. Ohlmeyer, Biochem. Z. 287, 212 (1936).

163 H. Heiwinkel, Arkiv Kemi, Mineral. Geol. 183A, No. 19 (1939).

164 N. B. Das and H. von Euler, Nature 141, 604 (1938).

165 N. B. Das, Arkiv Kemi, Mineral. Geol. 183A, No. 7 (1939).

166 P. Handler and J. R. Klein, J. Biol. Chem. 148, 49 (1942).

167 P| J. G. Mann and J. H. Quastel, Biochem. J. 35, 502 (1941).

168 H. MeIlwain and D. E. Hughes, Bivchem. J. 48, 60 (1948).

169 H, McIlwain and R. Rodnight, Biochem. J. 44, 470 (1949); 45, 337 (1949).

170 W. M. Govier and N.S. Jetter, Science 107, 146 (1948).

171 A. Nason, Federation Proc. 10, 228 (1951); A. Nason, N. O. Kaplan, and 8. P. Colo-
wick, J. Biol. Chem. 188, 379 (1951).

508 NIACIN

are disrupted so that special precautions must be taken when making ho-
mogenates or extracts of tissue for DPN or TPN analysis.

The extent to which these ‘‘destroying”’ mechanisms may have physio-
logical significance in the normal wear-and-tear breakdown of the pyridine
nucleotides is unknown. It is of considerable interest, however, that Zat-
man!” has recently used isotopic nicotinamide to demonstrate that spleen
DPNase catalyzes an exchange between free nicotinamide and that bound
into DPN. This provides a mechanism whereby the nicotinamide can be
removed and replaced in DPN-TPN without destruction of the rest of the
molecule.

L. INHIBITORS OF DPN AND TPN

Inhibitors in intact animals are considered elsewhere (p. 548).

Inhibitors such as cyanide and iodoacetate are known to inhibit some
codehydrogenase enzyme systems in vitro. Of potentially greater biological
significance is the fact that at least some DPN-—TPN-linked systems can
be inhibited by the components of the DPN molecule. The inhibitory effect
of nicotinamide on DPN breakdown has already been mentioned (p. 507).
Feigelson et al.”° have shown that high levels of nicotinamide inhibit the
respiration of rat liver homogenates, an effect reversible with DPN. From
an analysis of the Michaelis constants they believe this to be due to a com-
petition between nicotinamide and DPN for the apoenzyme. However,
Adler et al. believe that this phenomenon is more complicated than just
enzyme displacement. They found that pyridine-3-sulfonic acid (a nicotinic
acid inhibitor in bacteria) would inhibit glucose and lactic dehydrogenase
systems, but so would salicylic acid, nicotinic acid, adenine, adenosine,
and adenine dinucleotide. Inhibition could be reversed with DPN but not
by adenine compounds. In addition, succinic dehydrase, which is not DPN-
linked, was also inhibited by the compounds listed above. Williams!” re-
cently reported that adenine, adenosine, and adenosinetriphosphate would
inhibit a malic dehydrogenase system in a manner competitive with DPN.
Sulfathiazole and sulfapyridine have been reported to inhibit nicotinamide-
stimulated respiration and growth in certain microorganisms.!7° However,
Anderson and associates'”®* found no effect of these sulfa drugs on DPN-

172 J,. J. Zatman, Federation Proc. 11, 315 (1952).

173 P. Feigelson, J. N. Williams, and C. A. Elvehjem, J. Biol. Chem. 189, 361 (1951).

174 W. Adler, H. von Euler, and B. Skarzynski, Arkiv Kemi, Mineral. Geol. 16A, No.
9, 1, (1943); 17, No. 2, 1 (1948).

175 J. N. Williams, Jr., J. Biol. Chem. 195, 629 (1952).

176 R. West and A. F. Coburn, J. Exptl. Med. 72, 91 (1940).

176, W. G. Anderson, F. J. Pilgrim, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med.
55, 39 (1944).

IV. BIOCHEMICAL SYSTEMS 509

linked enzyme systems or on the respiration of rat liver in the presence of
fumarate or pyruvate.

M. ASSAY PROCEDURES FOR DPN AND TPN

Chemical methods have been described elsewhere (p. 537).

1. GENERAL PRINCIPLES

A number of enzymatic methods have been described which permit a
quantitation of DPN or TPN. The most widely used are the spectrophoto-
metric techniques which depend upon the fact that oxidized DPN-TPN
exhibit no absorption at 340 mu whereas the reduced enzymes absorb
strongly and identically at this wavelength. Since the molecular extinction
coefficients are known (p. 490) and since absorption is proportional to con-
centration, these coenzymes can be readily quantitated. The methods are
fairly sensitive. There are few substances, either components or reaction
products of the enzyme systems, which have interfering light absorption
at 340 mu. Determinations may be made quickly with small quantities of
materials and are especially useful in following the progress of reactions.
The methods may be adapted to measure either the appearance or disap-
pearance of the reduced forms, depending on whether the particular enzyme
system donates or accepts hydrogen. They may also be used to measure
the concentration of the apoenzymes or the substrates when these are
limiting factors in the reaction mixtures. Specificity for DPN or TPN can
be attained when dealing with mixtures by using an apoenzyme and a
substrate which specifically requires one or the other coenzyme. These
systems also permit the measurement of reactions not amenable to direct
spectrophotometric determination if they can be coupled to reactions de-
pendent on these coenzymes.!77-179

2. DPN

As an example, DPN may be determined as described_by Kornberg,!“
using crystalline aleohol dehydrogenase and alcohol to reduce the DPN.
The components of the test system are ethanol 0.3 ml., glycine (1.5%) 0.2
ml., sodium pyrophosphate buffer (0.03 M, pH 8.5) 1.5 ml., alcohol de-
hydrogenase 5 to 10 y, and water or unknown qs. 3.0 ml. Figure 8 shows
the sensitivity and proportionality of the method with known amounts
of DPN. The reaction was complete in 5 minutes. The specificity of the
procedure could be verified by observing the complete disappearance of

177 Q. Warburg, Ergeb. Enzymforsch. T, 210 (1988).
178 E. Schlenk, in Methoden der Fermentforschung. Academic Press, New York, 1945.
179 T. R. Hogness and V. R. Potter, Ann. Rev. Biochem. 10, 509 (1941).

510 NIACIN

absorption at 840 mu when small amounts of pyruvate and lactic dehydro-
genase were added.

DPN may also be determined as described by Cori eé al.'®° utilizing a
reaction originally proposed by Warburg and Christian!®® in which p-glye-
eraldehyde-3-phosphate is oxidized to p-glyceric acid phosphate by the
appropriate crystalline dehydrogenase plus DPN as a hydrogen acceptor.
Arsenate is required to make this reaction go to completion (arsenolysis).
The DPNH, formed is measured spectrophotometrically at 340 my. This
system is more complicated than the one described above since fructose-

600
500
“I~ 400
2
2
2 300
oO
me}
is
= 200
(S)
100
0 20 ‘40 60 80

ml. DPN added

Fia. 8. Spectrophotometric assay for DPN; response to known amounts of DPN.
(From Kornberg.1!*)

1 ,6-diphosphate is used to produce pD- poe erle aye: 3-phosphate accord-
ing to the following reaction:

Fructose-1,6-diphosphate + aldolase

i)

p-Glyceraldehyde-3-phosphate —=————— Dihydroxyacetone phosphate

isomerase

+ dehydrogenase

aN

|

p-Glyceric acid 3-phosphate + DPNH»

This system, however, has the advantage not only of providing for the
quantitation of DPN, but of being a sensitive method for the determination

180 G. T. Cori, M. W. Slein, and C. F. Cori, J. Biol. Chem. 173, 605 (1948).

IV. BIOCHEMICAL SYSTEMS 511

of fructose diphosphate and the triose phosphates either singly or together,
depending on the order of addition of the three enzymes concerned. This
may be considered an example of the way in which substances other than
DPN may be quantitated by linking the reactions to DPN-dependent
enzyme systems.

DPN may also be converted chemically to DPNHg, using hydrosulfite
reduction (p. 491), and measured by absorption at 340 mu.!*! This procedure
does not differentiate between DPN and TPN. Cyanide also will react
with DPN (p. 487) to form a complex with an absorption maximum at
340 mu This reaction may be used analytically for DPN, as shown by
Colowick et al.

DPNH:2 may be quantitated by oxidation with pyruvic acid and lactic
dehydrogenase, the disappearance of absorption at 340 my being a measure
of the amount of reduced coenzyme present.)

3. TPN

TPN can be determined spectrophotometrically in an isocitric dehydro-
genase system which specifically requires TPN.*° This system, as described
by Ochoa,”? is dependent on two reactions, the net result of which is:

picts

M
p-Isocitric acid + TPN ——— a-Ketoglutaric acid + CO, + TPNH,

Oxalosuccinic acid is an intermediate in the reaction.

TPN may also be determined utilizing the glucose dehydrogenase reac-
tion originally described by Warburg and Christian,*: 7: '**: 18° which is also
TPN-dependent.

Glucose-6-phosphate +- TPN — 6-Phosphogluconic acid + TPNH»

This reaction can be coupled to a system in which cytochrome c is the
final hydrogen acceptor to form a very sensitive assay procedure.!®* It
may also be linked to the system

hexokinase

Glucose + ATP === Glucose-6-phosphate + ADP

to supply glucose-6-phosphate for the TPN-dependent reaction listed above.
If ATP is the limiting factor in the mixture, then the amount of TPN re-
duced in the combined reaction will have a proportionality to the amount
of ATP present, permitting a quantitative ATP assay.!*

181 G. A. LePage, J. Biol. Chem. 168, 623 (1947).

182 §. P. Colowick, N. O. Kaplan, and M. M. Ciotti, J. Biol. Chem. 191, 447 (1951).
183 F Kubowitz and P. Ott, Biochem. Z. 314, 94 (1943).

184 Q, Warburg and W. Christian, Biochem. Z. 242, 206 (1931).

185 I}, Negelein and W. Gerischer, Biochem. Z. 284, 289 (1936).

186 F). Haas, C. J. Harper, and T. R. Hogness, J. Biol. Chem. 142, 835 (1942).

S12 NIACIN

The conversion of 6-phosphogluconic acid to COs and pentose phosphate
is also TPN-dependent and can be used as an assay for either 6-phospho-
gluconate or TPN, as shown by Horecker and Smyrniotis.'

6-Phosphogluconate + TPN — TPNH: + CO, + pentose phosphate

These enzymatic methods have largely replaced the older manometric
or methylene blue reduction procedures.!”: 187-19!

The spectrophotometric methods referred to above are possible only
with purified or semipurified enzyme systems. Chemical and microbiolog-
ical methods are available for use in crude tissue extracts (p. 537). However,
Feigelson and associates!” have developed a spectrophotometric method
which measures both DPN and TPN and can be used in crude tissue ex-
tracts. The tissue is rapidly frozen, homogenized in 2% trichloroacetic
acid with H,O2, to oxidize and stabilize the coenzymes, and centrifuged.
The coenzymes in the supernatant are absorbed on charcoal (Nuchar C).
After washing with trichloroacetic acid, the coenzymes are eluted with
pyridine. The coenzyme concentration is determined by comparing the
absorption at 340 my of oxidized and hydrosulfite-reduced samples, fol-
lowing the method of Gutcho and Stewart.'% The concentration of DPN-
TPN was calculated on the basis of dilution factors and the fact that 100
y of reduced coenzymes per milliliter in a 1-cm. cell has an optical density
of 0.840, according to Gutcho and Stewart!® and Schlenk.” The correctness
of the 0.840 factor just cited seems open to question. From recent data
on the extinction coefficient of DPN-TPN (6.22 X 10*) and with a 3-ml.
cell and a 1-cm. light path, this factor should be about 0.939 if only DPN
were present. By virtue of its somewhat higher molecular weight, TPN, if
present, would reduce this factor slightly. Since the method of Feigelson
et al. does not distinguish TPN, it would be difficult to determine how much
the factor should be reduced, especially when working with a variety of
animal tissues. In view of these considerations, it seems possible that the
recent intensive studies of the Wisconsin group on DPN and TPN in ani-
mal tissue contain a small systematic error, which would, however, not
affect the validity of the changes in tissue concentration which they ob-
served under various conditions.

187 K, Myrbiick, Hoppe-Seyler’s Z. physiol. Chem. 177, 158 (1928).

188 H. von Euler, Ergeb. Physiol. 38, 1 (1936).

189 K, Myrbiack, Ergeb. Enzymforsch. 2, 139 (1933).

199 A. &. Axelrod and C. A. Elvehjem, J. Biol. Chem. 131, 77 (1939).

1911 P| §. Krishnan, Science 105, 295 (1947).

12 P, Feigelson, J. N. Williams, Jr., and C. A. Elvehjem, J. Biol. Chem. 185, 741
(1950).

193 §. Gutcho and E. D. Stewart. Anal. Chem. 20, 1185 (1948).

IV. BIOCHEMICAL SYSTEMS ayilics

N. TISSUE DISTRIBUTION AND FATE

The tissue distribution and intermediary metabolism of administered
nicotinic acid has been poorly understood. Some studies have been done
measuring changes in tissue concentration and urinary excretion following
administration of the vitamin. Although such studies are valuable, they
are limited by their inability to distinguish the distribution and fate of
administered vitamins in relation to pre-existing tissue stores.

1. EXCRETION

Roth and associates! have applied radioactive tracer techniques to this
problem with very interesting results. They administered C'-carboxyl-
labeled nicotinic acid and nicotinamide intraperitoneally to mice and de-
termined radioactivity in exhaled air, urine, feces, and tissues as a function
of time.

A single dose of nicotinic acid, 0.7 mg., which is considerably in excess
of the normal daily requirement, resulted in a large excretion of radioac-
tivity in urine (about 60 % of administered dose) and in pulmonary carbon
dioxide (about 3 % of administered dose) within the first 24 hours. The latter
finding is of especial interest, since it proved, for the first time, that animal
tissues are capable of decarboxylating nicotinic acid. The rather large
urinary excretion would be expected, since the animals were, presumably,
already well nourished with respect to nicotinic acid. Excretion was much
less after 24 hours, and minimal after 48 hours. Nicotinic acid remaining
in the animal at this point was assumed to be a part of the normal tissue
enzyme stores. By comparing the amount of radioactivity excreted in
urine and in pulmonary carbon dioxide after 48 hours, these investigators
estimated that the mouse normally disposes of 15 to 20% of the nicotinic
acid liberated from the tissues as carbon dioxide. Since only the carboxyl
group of the administered nicotinic acid was labeled, this does not neces-
sarily mean that the pyridine ring was also oxidized to carbon dioxide.
In later studies'®® these investigators showed that this phenomenon of
pulmonary carbon dioxide excretion from labeled nicotinic acid and nico-
tinamide also existed in hamsters, rats, and dogs. Hamsters and rats ex-
creted somewhat more in this fashion, and dogs much less than the mouse.
Very little, if any, of the radioactivity appeared in the feces, a fact of
significance in the problem of intestinal synthesis of nicotinic acid (p. 530).

194 [| J. Roth, E. Leifer, J. R. Hogness, and W. H. Langham, J. Biol. Chem. 176, 249
(1948).

195 H. Leifer, L. J. Roth, D. S. Hogness, and M. H. Corson, J. Biol. Chem. 190, 595
(1951).

514 NIACIN

2. TissuE DISTRIBUTION AND TURNOVER RATE

The uptake of radioactive nicotinic acid by various tissues and organs
was determined and the concentration followed at intervals for 15 days.
Uptake was highest in kidneys and lowest in erythrocytes. No radioac-
tivity was found in plasma after 24 hours. The excretion half-time (i.e.,
turnover rate) was about 4 days in liver, kidney, and spleen, 5 days in
cardiac muscle and erythrocytes, and 8 days in brain, sternum, and skele-
tal muscle. Since nicotinic acid exists in tissues almost entirely as coen-
zymes, it seems clear that coenzymes are broken down at an unexpectedly
rapid rate. It is also of significance that the mouse handled nicotinic acid
and nicotinamide identically in the above studies.

TABLE IX

RELATIVE PER CENT DISTRIBUTION OF RADIOACTIVITY AMONG URINARY METABOLITES
oF C!4-Nicotinic AcID AND C1!4-NICOTINAMIDE 12 To 24 Hours AFTER INJECTION

C4 nicotinic acid C\4-nicotinamide
Metabolites Dog Rat Hamster Mouse Dog Rat Hamster Mouse
N?}-Methylnicotina- 94 56 23.6 13,6 94 73.5 35 20
mide
Nicotinuric acid 1 10.5 2.8 24.8 0.5 0 0 0.5
Nicotinie acid 0.3 Ona 2475) “avez 1 ee Ny 36.2
Unknown? 0 11.9 13.0 7.0 1 3.6 13 9
N!-Methyl-6-pyridone- 2 11296 LOG P16 2 2 10 20
3-carboxylamide
Nicotinamide 2.4 Bao) 2,5 6.1 1 5.6 25 14.3

4 Unidentified but possibly 2-pyridone of N'methylnicotinamide.

The rather large excretion of carbon dioxide derived from nicotinic acid,
as observed in the above experiments, also explains a fact noted by many
investigators namely, that less nicotinic acid (including all known deriva-
tives) may be excreted than is consumed or administered (in some species).

3. Urtnary METABOLITES

The urinary excretion pattern following administration of nicotinic acid
and nicotinamide was generally similar, but it differed in some details.
There was, also, considerable variation among species. Table [X summarizes
the results of Leifer e¢ al.!®° in four species. Paper chromatographic meth-
ods were used to separate the various metabolites. Trigonelline contained
no C#, indicating that it is not derived from nicotinic acid, in confirmation
of many other workers.

Perlzweig and associates!®® have studied the urinary excretion pattern

196 W, A. Perlzweig, F. Rosen, and P. B. Pearson, J. Nutrition 40, 453 (1950).

IV. BIOCHEMICAL SYSTEMS 515

of nicotinic acid in man and eight other mammalian species and have re-
viewed much of the earlier work on this subject. Table X has been adapted
from their data to indicate the marked species variation. Apparently, man,
dog, rat, and swine dispose of nicotinic acid largely as methylated products,
whereas the herbivora, rabbits, guinea pigs, sheep, goats, and calves, dis-
pose of nicotinic acid in some other fashion. Johnson e¢ al. also found that
nicotinic acid was excreted principally in a methylated form in human
subjects. Ellinger and associates!®* have studied this question extensively
in man, dog, cat, rat, rabbit, and guinea pig. They obtained results es-
sentially the same as Perlzweig et al.!9° with reference to the ability of

TABLE X
URINARY ExcrEeTION oF Nicotinic AciIpD AND Its DERIVATIVES

Per cent of administered or ingested
nicotinic acid

6-Pyri- Unaccounted
Species Dose NMN? doneb N.A.¢ for
Man Normal diet?# 33 41 6 20
Man 500 mg. nicotinamide 38 43 1 18
Dog 10 mg./kg. nicotinamide 80 0 8 12
Rat 100-200 mg./kg. nicotinamide 50 3 15 32
Pig 75- 80 mg./kg. nicotinamide 7 10 9 74
Rabbit 90-100 mg./kg. nicotinamide 0.5¢ 3 43 Sard
Guinea pig 150-200 mg./kg. nicotinamide 0.5 5 8 86.5
Goat 70-100 mg./kg. nicotinamide 0.5 0.5 14 85.0
Sheep 30- 60 mg./kg. nicotinamide 0.5 0.3 30 69.0
Calf 30- 70 mg./kg. nicotinamide 0.6 0 35 64

* N-Methylnicotinamide.

6 §6-Pyridone of N'methylnicotinamide.

© Includes nicotinic acid, nicotinamide, and nicotinuric acid, if present.

° The significance of these values below 1% is questionable owing to analytical limitations.
4 Nicotinic acid content estimated at 17 mg.

various species to excrete methylated derivatives of nicotinic acid. The cat
resembles other carnivorous and omnivorous species in that it does ex-
crete N'-methylnicotinamide. Chickens excrete a still different type of
nicotinic acid derivative—dinicotinylornithine.!*? N!-Methylnicotinamide is
not a major urinary product in the horse,?°: 2° although it is in the cotton
rat?

The biochemical mechanisms by which the urinary derivatives of nico-

197 B. C. Johnson, T.S. Hamilton, and H. H. Mitchell, J. Biol. Chem. 159, 231 (1945).

198 P, Ellinger and M. M. Abdel Kader, Biochem. J. 44, 77, 627 (1949).

199 W. J. Dann and J. W. Huff, J. Biol. Chem. 168, 121 (1947).

200 J. W. Huff, P. B. Pearson, and W. A. Perlzweig, Arch. Biochem. $, 99 (1946).

201 B. §. Schweigert, P. B. Pearson, and M. C. Wilkening, Arch. Biochem. 12, 139
(1947).

516 NIACIN

tinic acid are formed are almost completely unknown except for the amide
(p. 505) and N!-methy! derivatives (vide infra).

O. NUMETHYLATION OF NICOTINAMIDE

Nicotinamide can be methylated in certain tissues by an aerobic process
analogous to the methylation of guanidoacetic acid to creatine; i.e., the
reaction is dependent on oxygen and is inhibited by oxidation inhibitors.?”
Perlzweig and associates?® showed that rat liver slices, but not kidney or
muscle, can methylate nicotinamide and that methionine enhances the
reaction. This finding has been amply confirmed by others.” Cantoni?™
has now obtained a soluble enzyme system (cell-free) from rat, pig, guinea
pig, and dog liver which catalyzes the methylation of nicotinamide in the
presence of methionine, Mgt*, and adenosinetriphosphate. Only L-methi-
onine could be used in the system. Betaine and dimethylthetin were inac-
tive in replacing methionine unless homocysteine was present. The system
would not methylate nicotinic acid.

The methylation reaction is apparently irreversible under biological con-
ditions, since N'-methylnicotinamide cannot replace nicotinamide in the
diet. Likewise this methylated compound has no lipotrophic activity as it
would if it could give up its methyl group.2% Keller et al.?°* used a tracer
technique to prove that N!-methyl groups attached to nicotinamide do
not participate in transmethylation reactions.

The liver can, however, degrade N!-methylnicotinamide to unknown com-
pounds." Tt is apparently a lack of this degradative reaction which accounts
for the paradoxical increase in urinary N!-methylnicotinamide in animals
and in human subjects whose livers have been severely damaged by carbon
tetrachloride?” or phosphorus.?°%

Beher et al.2°® have found that small doses of testosterone decrease the
urinary excretion of N!-methylnicotinamide, apparently because of an in-
creased storage of DPN and TPN in the tissues. Calvo et al.!° found that
thyroidectomy or thiouracil administration decreased urinary N!-methy]l-
nicotinamide excretion. Excretion of this compound was also decreased by
202 Nutrition Revs. 6, 28 (1948).

203 W. A. Perlzweig, M. L. C. Bernheim, and F. Bernheim, J. Biol. Chem. 150, 401
1943).

204 e sale J. Biol. Chem. 189, 203 (1951).

205 P, Handler and I. N. Dubin, J. Nutrition 31, 141 (1946).

206 H. B. Keller, J. L. Wood, and V. DuVigneaud, Proc. Soc. Exptl. Biol. Med. 67,

182 (1948).

207 W. A. Perlzweig, J. W. Huff, and F. Rosen, Federation Proc. 5, 149 (1946).
208 A. Bonsignore and L. Bevilacqua, Bull. soc. ital. biol. sper. 23, 1219 (1947).
209 W. T. Beher, E. M. Crigger, and O. H. Gaebler, J. Nutrition 47, 353 (1952).

210 J, M. Calvo, C. C. Boehme, and J. Goemine, Bol. soc. biol. Santiago Chile 6, 88
(1949).

IV. BIOCHEMICAL SYSTEMS 517

feeding thyroglobulin, but methionine or choline tended to bring the urinary
levels back to normal. Vitamin By. may also be involved in the methylation
of nicotinamide, probably through its influence on the synthesis and trans-
fer of labile methyl groups.”"

V. Specificity of Action
J. M. HUNDLEY

A. MODIFICATIONS OF THE NICOTINIC ACID MOLECULE

Many substances related chemically to nicotinic acid have been tested
for their ability to replace this vitamin in mammalian nutrition. Table XI
represents a partial compilation of such compounds. The following con-
clusions may be drawn concerning the range within which the molecule
may be modified and still retain biological activity.

1. The pyridine molecule must have a substituent group in the 3 position.

2. This substituent group must be a derivative of —-C—-R. Molecules
with other groups on the 3 position such as CH;, NH» or CN have no, or
much less, biological effectiveness, presumably because they must be con-

O
!

verted to the —-C— grouping.

3. The R group attached to the 3-carboxyl may be any one of a great
number of compounds and types such as esters and N-substituted amides
and still retain biological effectiveness.

O

4. Substitution of an acetyl (C—CH:3) for the 3-carboxyl group leads to a
compound (3-acetylpyridine) which is a nicotinic acid antagonist under
certain conditions** but can be converted to nicotinic acid under other con-
ditions.”

211 T, KE. Liener and M. O. Schultze, J. Nutrition 46, 223 (1952).

1P, J. Fouts, O. M. Helmer, 8. Lepkovsky, and T. H. Jukes, Proc. Soc. Exptl. Biol.
Med. 37, 405 (1937).

2 P. Ellinger, G. Fraenkel, and M. M. Abdel Kader, Biochem. J. 41, 559 (1947).

3V.A. Najjar and L. E. Holt, Jr., Proc. Soc. Exptl. Biol. Med. 48, 413 (1941).

4P. Ellinger and R. A. Coulson, Biochem. J. 38, 265 (1944).

5 W. A. Krehl, P. S. Sarma, L. J. Teply, and C. A. Elvehjem, J. Nutrition 31, 85
(1946).

6D. W. Woolley, F. M. Strong, R. J. Madden, and C. A. Elvehjem, J. Biol. Chem.
124, 715 (1938).

TABLE XI

SPECIFICITY oF CoMPOUNDS RELATED To Nicotinic AcIpD

Rat
Compounds Man Urinary excretion Growth Dog
Substitution on the Ring
Nicotinic acid (3-carboxypyridine) + (1% + (2-4) + (5) + (6)
Picolinic acid (2-carboxypyridine) 0 (6)
Isonicotinie acid (4-carboxy- 0 (6)
pyridine) :
Quinolinic acid (2,3-dicarboxy- + (7, 8) 0 (3) + (10-12) + (13)
pyridine) + (2, 9, 10) 0 (6, 14)
Dinicotinic acid (3,5-dicarboxy- + (15) 0 (3)
pyridine) + (16)
Isocinchomeronie acid (2,5-di- + (9) + (12)
carboxypyridine)
Nipecotic acid (hydrogenated nico- 0 (6, 17)
tinic acid)
2,6-Dimethylnicotinic acid + (16)
+ (15)
2,6-Dimethyl-3, 5-dicarboxy- + (16) 0 (8)
pyridine
6-Methylnicotinic acid 0 (6)
3-Methylpyridine + (16) + (2, 18) + (6)
3-Aminopyridine + (16) + (19)
0 (6)
3-Cyanopyridine 0 (2) 0 (11, 20) 0 (6)
N} Substitutions
N!-Methylnicotinamide + (2) 0 (5, 11, 21) 0 (6, 17)
+ (26)
Trigonelline (betaine of nicotinic 0 (16, 22) 0 (2) 0 (5) 0 (6, 23)
acid)
Nicotinamide glucosidoiodide + (6)
Modifications of Carboxyl Group
Nicotinamide + (22) + (2-4) + (5) + (6)
N-Methylnicotinamide + (6)
N-Ethylnicotinamide + (2) + (5) + (6)
N, N-Diethylnicotinamide + (22, 24) + (2-4) + (6, 25, 3)
N-Allylnicotinamide + (2)
N-Pheny|lnicotinamide + (2) 0 (5)
N, N-Diphenylnicotinamide 0 (2)
4”-Methoxyphenylnicotinamide + (2)
N-Benzyl + (2)
N, N-Dibenzyl 0 (2)
Nicotinuric acid 0 (26° 23)
+ (6, 3)
Ethyl nicotinate + (5) + (6)
3-Acetylpyridine + (27) 0 (20) 0 (6)
+ (28)
Pyridine-3-sulfonic acid 0 (20) 0 (6, 28)
Thionicotinamide
N-(2-Thiazolyl) nicotinamide + (29)
Substitutes for Pyridine Ring
Pyrazinemonocarboxylic acid + (80, 24) 0 (8, 31) 0 (13)
Pyrazinemonocarboxylamide 0 (31)
Pyrazine-2,3-dicarboxylic acid + (30) 0 (8) 0 (18)
Miscellaneous
Ornithine 0 (9, 32) + (12)
Pyridine 0 (6)
DPN (coenzyme I) 0 (33)

44 = active; 0 = no nicotinic acid activity; =: = questionable; urinary excretion
tion of nicotinic acid metabolites, usually N-methylnicotinamide, after administration of the compound.
b’ Numbers in parentheses refer to appropriate references.

increased excre-

V. SPECIFICITY OF ACTION 519

TABLE XII

CoMPpouNDS RELATED TO OR DERIVED FROM TRYPTOPHAN.
ABILITY TO SUBSTITUTE FOR Nicotinic AcID

Rat
Urinary nicotinic
Compounds acid excretion Growth

pu-Tryptophan + (9, 48-45)¢: > + (46)
Acetyl-L-tryptophan + (48)
Indole-3-pyruvie acid + (43)
Indole-3-proprionic acid 0 (43)
Indole-3-butyrie acid 0 (47)
Indole-3-acrylie acid 0 (47)
Indole-3-acetic acid 0 (48, 48) 0 (20, 47, 48)

+ (49) +; (49)
Indole 0 (12, 20)
Naphthylacetie acid 0 (49) + (49)
L-Kynurenine 0 (50) (0). (UL Gail)

a3) + (52)
N-Formylkynurenine 0 (1)
Acetylkynurenine 0 (11)
Kynurenic acid 0 (48, 50) 0 (11)
Xanthurenic acid 0 (48, 50)
Anthranilie acid 0 (9) 0 (20)

=tenuhz)

3-Hydroxyanthranilie acid + (53) + (10, 11, 52, 54)
3,4-Dihydroxyanthranilie acid 0 (55) 0 (55)
N-Formylanthranilie acid 0 (11)

44 = active in substituting for nicotinic acid; 0 = not active.
+ Numbers in parentheses refer to appropriate references.

7R. W. Vilter and T. D. Spies, Lancet II, 423 (1989).

8H. P. Sarett, J. Biol. Chem. 198, 627 (1951).

9L. V. Hankes and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 78, 550 (1950).

10 7,. M. Henderson, J. Biol. Chem. 178, 1005 (1949).

1 W. A. Krehl, D. Bonner, and C. Yanofsky, J. Nutrition 41, 159 (1950).

2. V. Hankes, R. L. Lyman, and C. A. Elvehjem, J. Biol. Chem. 187, 547 (1950).

13W. J. Dann, H. I. Kohn, and P. Handler, J. Nutrition 20, 477 (1940).

144. A. Waisman, O. Mickelsen, J. M. McKibbin, andC. A. Elvehjem, J. Nutrition
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158. P. Vilter, W. B. Bean, and T. D. Spies, Southern Med. J. 81, 901 (1938).

16T. D. Spies, H. M. Grant, and N. E. Huff, Southern Med. J. 31, 901 (1938).

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18H. N. De and P. Datta, Jr., Current Sci. (India) 19, 279 (1950).

19 Y, SubbaRow, W. J. Dann, and E. Meilman, J. Am. Chem. Soc. 60, 1510 (1938).

20 W. A. Krehl, L. M. Henderson, J. de la Huerga, and C. A. Elvehjem, J. Biol.
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218. A. Singal, V. P. Sydenstricker, and J. M. Littlejohn, J. Biol. Chem. 176, 1069
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2 'T. D. Spies, W. B. Bean, and R. E. Stone, J. Am. Med. Assoc. 111, 584 (1938).

520 NIACIN

5. Almost any modification in the 2, 4, 5, or 6 position of nicotinic acid
leads to inactive compounds. The exceptions to this are quinolinic acid,

23 J. W. Huff and W. A. Perlzweig, J. Biol. Chem. 142, 401 (1942).

24 A. #. Axelrod, T. D. Spies, and C. A. Elvehjem, J. Biol. Chem. 138, 667 (1941).

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27 W. T. Beher, W. M. Holliday, and O. H. Gaebler, Abstr. 12th Intern. Congr. Pure
and Appl. Chem., New York, p. 84, 1951.

28 OQ. H. Gaebler and W. T. Beher, J. Biol. Chem. 188, 343 (1951).

29 T,. Cote and J. J. Oleson, J. Bacteriol. 61, 463 (1951).

30 C. E. Bills, F. G. McDonald, and T. D. Spies, Southern Med. J. 32, 793 (1939).

31 T,. Cote, J. J. Oleson, and J. H. Williams, Proc. Soc. Exptl. Biol. Med. 80, 434 (1952).

32 P. Ellinger and M. M. Abdel Kader, Nature 160, 675 (1947).

33 FS. Daft, H. F. Frazer, W. H. Sebrell, and M. Pittman, Sczence 88, 128 (1938).

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35 B.C. J. G. Knight, Biochem. J. 31, 731 (1937).

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38 P. Fildes, Brit. J. Exptl. Pathol. 19, 239 (1938).

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51 W. A. Krehl and D. Bonner, Unpublished data quoted in Vitamins and Hormones
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VI. BIOGENESIS 521

which is known to be easily and preferentially decarboxylated in the 2
position, forming nicotinic acid, and compounds which have been tested
principally by treating human pellagra, a difficult means of testing which
is subject to considerable clinical interpretation. The reported activity of
ornithine may be in a special category (p. 522). The reported activity of
isocinchomeronic acid seems very questionable.

6. N-substituted derivatives of nicotinic acid or nicotinamide may be
active or inactive, depending on the nature of the substituent group. The
reported inactivity of DPN is puzzling, since this substance is known to be
active in enzyme systems and for bacterial growth. This substance should
be retested, using more sensitive systems than acute canine blacktongue.

7. Man, rat, and dog seem to have about the same ability to use various
types of nicotinic acid derivatives.

The effectiveness of nicotinic acid derivatives in microorganisms has been
reviewed, elsewhere.?9: #5-#

B. TRYPTOPHAN AND DERIVATIVES

The amino acid and some of its derivatives can replace nicotinic acid,
since many organisms possess the ability to convert tryptophan to nicotinic
acid. This subject is considered in Section VI. The principal compounds
of interest are listed in Table XII. Tryptophan can replace nicotinic acid
in human nutrition,**-*? in the dog,® and in many other species (p. 578), as
well as in the rat.

VI. Biogenesis

J. M. HUNDLEY
A. DEMONSTRATION OF NICOTINIC ACID BIOSYNTHESIS

It has long been known that certain mammals can synthesize nicotinic
acid. Several groups of investigators showed that rats thrive on diets which
produced pellagra in humans, blacktongue in dogs, and deficiency states
in pigs and monkeys.!-> Other investigators using various diets could dem-

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aac, NIACIN

onstrate no nicotinic acid requirement for the rat.*-8 Shourie and Swami-
nathan,® Dann and Kohn,!® and Dann" showed that rats accumulated in
their tissues and excreted more nicotinic acid than they ingested, unequiv-
ocal evidence of synthesis. Similar findings have been recorded for
calves,!- horses,!>: 16 sheep,!”: 18 and man.!9-2!

B. MECHANISM OF BIOSYNTHESIS

Although these data certainly established the fact that nicotinic acid
could be synthesized in vivo, the mechanism of this synthesis was obscure
for many years.

1. THE ORNITHINE SCHEME

Earlier workers suggested that nicotinic acid might be synthesized ac-
cording to the scheme shown in Fig. 9. However, the evidence that this
mechanism provides any substantial amount of nicotinic acid in mammals
is very shaky. Neither proline, glutamic acid, nor ornithine will substitute
for nicotinic acid in diets; indeed proline may, along with other amino
acids, increase nicotinic acid requirements.2”: “> However, Bovarnick: 2° has

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13 B.C. Johnson, Euclides (Madrid) 8, 161 (1948).

4G. C. Esh and T. S. Sutton, J. Dairy Sci. 31, 909 (1948).

16 J. W. Huff, P. B. Pearson, and W. A. Perlzweig, Arch. Biochem. 9, 99 (1946).

6 B. S. Schweigert, P. B. Pearson, and M. C. Wilkening, Arch. Biochem. 12, 139
(1947).

7 A.H. Winegar, P. B. Pearson, and H. Schmidt, Science 91, 508 (1940).

8 P. B. Pearson, A. H. Winegar, and H. Schmidt, J. Nutrition 20, 551 (1940).

19V. A. Najjar, L. E. Holt, Jr., G. A. Johns, G. C. Mediary, and G. Fleischmann,
Proc. Soc. Exptl. Biol. Med. 61, 371 (1946).

20 P. Ellinger, R. A. Coulson, and R. Benesch, Nature 154, 270 (1944).

"1 P. Hilinger, R. Benesch, and W. W. Kay, Lancet I, 432 (1945).

A. C. Groschke, J. O. Anderson, and G. M. Briggs, Proc. Soc. Exptl. Biol. Med.
68, 564 (1948).

°° L. V. Hankes, L. M. Henderson, W. L. Brickson, and C. A. Elvehjem, J. Biol.
Chem. 174, 873 (1948).

24M. R. Bovarnick, J. Biol. Chem. 158, 1 (1944).

2° M.R. Bovarnick, J. Biol. Chem. 148, 151 (1943); 151, 467 (1943).

VI. BIOGENESIS 523

shown that a mixture of glutamic acid and asparagine (or glutamine) heated
in the presence of manganese and iron gives rise to nicotinamide. Ellinger
and Abdel Kader?* have suggested that ornithine and glutamic acid may
be involved in nicotinic acid synthesis on the basis of experiments using
Escherichia coli. Guvacine will substitute for nicotinic acid in the nutrition
of Staphylococcus aureus and Proteus vulgaris.’ Nicotinic acid synthesis in
rats may be increased by DL-é-amino-n-valeric acid.?? Hankes ef al.?® have

eo
bun, wo
bt Se ~ nat CH_NH:
ieee H CH— COOH EEG COOH
CH—NH, ann SNH;
COOH
Glutamic acid Proline Ornithine
H.C7 “C—COOH hed “CH HiC™ ~ CH:
es ——E
HEC CH, 18LC. COOH Hee COOH
ae Swit “NH,
Guvacin A?-§-Amino- 6-Amino-n-

NS pentenic acid valeric acid

HO7 ~C—COOH
HCx_CH

Nicotinic acid

On

Fic. 9. The ornithine scheme for nicotinic acid biosynthesis. (Adapted from
Williams.?’)

reported that anthranilic acid and ornithine together can apparently sub-
stitute for nicotinic acid under certain dietary circumstances.

2. BIOSYNTHESIS FROM TRYPTOPHAN

The mechanism of mammalian nicotinic acid biosynthesis which is now
firmly established is conversion of the amino acid tryptophan to nicotinic
acid. A series of brilliant investigations beginning in 1945 have unraveled

26 P. Ellinger and M. M. Abdel Kader, Biochem. J. 44, 285 (1949).

27 R. J. Williams in The Biochemistry of the B Vitamins, American Chemical Society
Monograph No. 110, p. 84. Reinhold Publishing Corp., New York, 1950.

28 J. W. Huff and W. A. Perlzweig, J. Biol. Chem. 142, 401 (1942).

29L. V. Hankes, R. L. Lyman, and C. A. Elvehjem, J. Biol. Chem. 187, 547 (1950).

524 NIACIN

many of the details of this mechanism. The door to this fruitful chapter of
biochemistry was opened by the report of Krehl et al.*° that tryptophan
could substitute for nicotinic acid in counteracting the unfavorable growth
effects of corn in certain rat diets. Shortly thereafter it was shown that
tryptophan markedly increased the urinary excretion of N'-methylnicotina-
mide,*!-*8 the first clear indication that tryptophan could substitute for
nicotinic acid by virtue of the fact that it was transformed into nicotinic
acid. Subsequent reports have fully confirmed the fact that tryptophan
can substitute for nicotinic acid in the diet of rats, dogs, pigs, rabbits, chicks,
ducks, monkeys, and cotton rats (p. 578). Impressive but indirect evidence
that tryptophan is converted into nicotinic acid has been obtained in
man,*4-89 rats,16 26, 36, 40-48 dogs.49 mice,°° rabbits,®! horses and cotton rats,1®
pigs,*®: *? calves, sheep, and guinea pigs.** Most of these studies have shown
increased urinary excretion of nicotinic acid and its derivatives, or increased
tissue levels following tryptophan administration.

30 W. A. Krehl, L. J. Teply, P.S. Sarma, and C. A. Elvehjem, Sczence 101, 489 (1945).

31 F. Rosen, J. W. Huff, and W. A. Perlzweig, J. Biol. Chem. 163, 343 (1946).

2 §. A. Singal, A. P. Briggs, V. P. Sydenstricker, and J. M. Littlejohn, J. Biol.
Chem. 166, 573 (1946).

33§. A. Singal, A. P. Briggs, V. P. Sydenstricker, and J. Littlejohn, Federation Proc.
5, 144 (1946).

34H, P. Sarett and G. A. Goldsmith, J. Biol. Chem. 167, 293 (1947).

35 W. A. Perlzweig, F. Rosen, N. Levitas, and J. Robinson, J. Biol. Chem. 167, 511
(1947).

36 J,, M. Henderson, G. B. Ramasarma, and B. C. Johnson, J. Biol. Chem. 181, 731
(1949).

37 W. I. M. Holman and D. J. de Lange, Nature 165, 112 (1950).

38 H. P. Sarett and G. A. Goldsmith, J. Biol. Chem. 167, 293 (1947); 177, 461 (1949);
182, 679 (1950).

39§. E. Snyderman, K. C. Ketron, R. Carretero, and L. E. Holt, Jr., Proc. Soc.
Exptl. Biol. Med. T0, 569 (1949).

40 7,. V. Hankes and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 73, 550 (1950).

41R. EH. Kallio and C. P. Berg, J. Biol. Chem. 181, 333 (1949).

42 J. M. Hundley, J. Nutrition 34, 253 (1947).

43 J. M. Hundley and H. W. Bond, Arch. Biochem. 21, 313 (1949).

44P. B. Junqueira and B.S§. Schweigert, J. Biol. Chem. 175, 535 (1948).

45 Y. Raoul and C. Marnay, Bull. soc. chim. biol. 30, 450 (1948).

46 C.-T. Ling, D. M. Hegsted, and F. J. Stare, J. Biol. Chem. 174, 803 (1948).

47 J. N. Williams, Jr., P. Feigelson, and C. A. Elvehjem, J. Biol. Chem. 187, 597
(1950).

48 J. N. Williams, Jr., P. Feigelson, C. A. Elvehjem, and 8. 8S. Shahinian, J. Biol.
Chem. 189, 659 (1951).

495. A. Singal, V. P. Sydenstricker, and J. M. Littlejohn, J. Biol. Chem. 176, 1051
(1948).

50 DP). W. Woolley, J. Biol. Chem. 162, 179 (1946).

51 J. G. Wooley, Proc. Soc. Exptl. Biol. Med. 65, 315 (1947).

82 R. W. Luecke, W. N. McMillen, F. Thorp, Jr., and C. Tull, J. Nutrition 36, 417
(1948).

VI. BIOGENESIS 525

Subsequent investigations using isotope tracer techniques have clearly
proved that, at least in the rat, tryptophan increases nicotinic acid synthe-
sis by virtue of the fact that it is converted into nicotinic acid. Heidelberger
and associates®*-** showed that C™ from the 3 position in the indole nucleus
of tryptophan became the carboxyl carbon of nicotinic acid. Likewise,
Schayer and associates” found that N™ in the indole nucleus of tryptophan
appeared as the ring nitrogen of urinary nicotinic acid derivatives.

These findings certainly established the conversion of tryptophan to
nicotinic acid but did not elucidate the exact biochemical steps. Some addi-
tional information was gained from tracer experiments since Heidelberger
et al.*-® and Shayer et al.*” found that C™ in the @ position of the side
chain and Hundley and Bond* found that C¥ from the carboxyl group of
tryptophan did not appear in nicotinic acid. It was evident from these
findings that the side chain of tryptophan was not used to form nicotinic
acid.

a. Nicotinic Acid Biosynthesis in Neurospora

Much of our information on the exact mechanism by which tryptophan
is converted to nicotinic acid came from a series of productive investiga-
tions using various strains of Neurospora crassa. Some of these strains, as
has now been shown, synthesize nicotinic acid from tryptophan in a fashion
quite similar to the mechanism in animals, whereas other strains have
genétic blocks so that they cannot carry out certain of the biochemical
steps. Beadle and associates® first noted that a mutant strain of Neurospora
could use either nicotinic acid or tryptophan for growth and that kynurenine
was involved in this interchangeable relationship. Bonner and Beadle’? iso-
lated a substance which they believed to be a precursor of nicotinic acid
in this organism. Mitchell and Nyc® and Bonner® showed that this sub-
stance was 3-hydroxyanthranilic acid and that it could substitute for nico-
tinic acid. Later studies using tracer techniques have fully confirmed the
fact that 3-hydroxyanthranilic acid can be converted to nicotinic acid by

53 ©, Heidelberger, M. E. Gullberg, A. F. Morgan, and 8. Lepkovsky, J. Biol. Chem.
175, 473 (1948).

54 ©. Heidelberger, M. E. Gullberg, A. F. Morgan, and 8. Lepkovsky, J. Biol. Chem.
179, 148 (1949).

55 ©, Heidelberger, E. P. Abraham, and S. Lepkovsky, J. Biol. Chem. 176, 1461
(1948).

56 C. Heidelberger, E. P. Abraham, and S. Lepkovsky, J. Biol. Chem. 179, 151 (1949).

57 R. W. Schayer, G. L. Foster, and D. Shemin, Federation Proc. 8, 248 (1949).

58 G. W. Beadle, H. K. Mitchell, and J. F. Nye, Proc. Natl. Acad. Sci. U. 8. 33, 155
(1947).

59D. Bonner and G. W. Beadle, Arch. Biochem. 11, 319 (1946).

60H. K. Mitchell and J. F. Nye, Proc. Natl. Acad. Sci. U. S. 34, 1 (1948).

61D. Bonner, Proc. Natl. Acad. Sci. U. S. 34, 5 (1948).

526 NIACIN

Neurospora.®: ® It has also been shown by Yanofsky and Bonner®™ that
kynurenine is converted into nicotinic acid. 8-Hydroxykynurenine probably
is an intermediate between kynurenine and 3-hydroxyanthranilic acid.®, ®
The exact steps between 3-hydroxyanthranilic acid and nicotinic acid are
still uncertain. It has been suggested®®: * that the benzene ring of 3-hy-
droxyanthranilic acid is oxidatively cleaved in the 3-4 position to yield
an intermediate similar to that shown in Fig. 10. This compound might
then undergo ring closure with the amino nitrogen to give quinolinic acid
which is in turn decarboxylated to nicotinic acid,® or this hypothetical
intermediate might be decarboxylated prior to ring closure to yield nico-
tinic acid directly.” Leifer et al. have suggested that a symmetrical diamino
compound may be the intermediate between 3-hydroxyanthranilic acid and
nicotinic acid, although the evidence supporting this theory has been ques-
tioned.® The details of the evidence for the various steps in the conversion
of tryptophan to nicotinic acid in Neurospora have been carefully reviewed
by Bonner and Yanofsky.®°

b. Biosynthesis from Tryptophan in Mammals

The pathway by which tryptophan is converted to nicotinic acid in mam-
mals is not as fully known as in Neurospora. Current information suggests
that it may proceed substantially as shown in Fig. 10; however, the evi-
dence is faulty, incomplete, and contradictory in some respects. Tryptophan
is known to be converted into kynurenine and kynurenic acid in several
animal species as first shown by Kotake’® and later proved in isotope tracer
experiments by Heidelberger et al.*3-°> However, kynurenic acid is inactive
as a substitute for nicotinic acid in rats, and the reports on kynurenine are
contradictory (see Table XII). Rosen et al.7! and Krehl et al.” found kynu-
renine inactive when given in the diet or by injection, whereas Kallio and
Berg*! and Wiss et al.”* reported that it would increase the urinary excretion

62 H. Leifer, W. H. Langham, J. F. Nye, and H. K. Mitchell, J. Biol. Chem. 184, 589
(1950).

63 ©, Yanofsky and D. M. Bonner, J. Biol. Chem. 190, 211 (1951).

64 C, Yanofsky and D. M. Bonner, Proc. Natl. Acad. Sci. U. S. 36, 167 (1950).

65 WF. A. Haskins and H. K. Mitchell, Proc. Natl. Acad. Sci. U. S. 35, 500 (1949).

66 ],, M. Henderson, J. Biol. Chem. 178, 1005 (1949).

67D). M. Bonner and C. Yanofsky, Proc. Natl. Acad. Sci. U. S. 35, 576 (1949).

68 J. M. Henderson, J. Biol. Chem. 181, 677 (1949).

69 J). M. Bonner and C. Yanofsky, J. Nutrition 44, 603 (1951).

70 Y. Kotake and J. Iwao, Hoppe-Seyler’s Z. physiol. Chem. 195, 139 (1931); Y.
Kotake, J. Japan. Biochem. Soc. 21, 155 (1949).

™ F. Rosen, J. W. Huff, and W. A. Perlzweig, J. Nutrition 38, 561 (1947).

2 W.A. Krehl, D. Bonner, and C. Yanofsky, J. Nutrition 41, 159 (1950).

73O. Wiss, G. Viollier, and M. Miiller, Helv. Physiol. et Pharmacol. Acta 7, C64
(1949).

VI. BIOGENESIS 57

of nicotinic acid metabolites and would promote growth. Knox and
Mehler’: 7° have shown that the liver of several animal species possesses
an enzyme system which converts tryptophan to kynurenine through the
intermediate n-formylkynurenine. However, the latter compound also was
inactive in nicotinic acid-deficient rats.” Krehl’* and Bonner and Yanofsky*®
have suggested that the well-known instability of kynurenine might ac-
count for these conflicting results.

O
I
CH,—CH—COOH C—CH,—CH—COOH
| ae oe |
N NH, NH, NH
H 2 2
Tryptophan Kynurenine
i
C—CH,—CH—COOH COOH
| if
NH; NH, NH,
OH OH
3-Hydroxykynurenine 3-Hydroxyanthranilie acid

acres Fears
ee
OHC OHC COOH
NH, NH
? ?

ees ? Gikincees
Sy COOH

‘N
Nicotinic acid Quinolinie acid
Fie. 10. Summary of known and probable steps in the conversion of tryptophan
to nicotinic acid.

The situation with 3-hydroxyanthranilic acid is much clearer, since it
does support growth in nicotinic acid-deficient rats®*: 7: 73: and increases
the urinary excretion of nicotinic acid metabolites.” Schweigert7? reported
that rat liver slices would convert 3-hydroxyanthranilic acid to a compound

™4W. E. Knox and A. H. Mehler, J. Biol. Chem. 187, 419 (1950).

75 A. H. Mehler and W. E. Knox, J. Biol. Chem. 187, 431 (1950).

76W. A. Krehl, Unpublished data quoted in Vitamins and Hormones 7, 140 (1949).
7H. K. Mitchell, J. F. Nye, and R. D. Owen, J. Biol. Chem. 175, 433 (1948).

73 P.W. Albert, B. T. Scheer, H. J. Deuel, Jr., J. Biol. Chem. 175, 479 (1948).

79 B.S. Schweigert, J. Biol. Chem. 178, 707 (1949).

528 NIACIN

which had nicotinic acid activity after acid hydrolysis. Later studies using
liver homogenates and other enzyme preparations®®: *! showed that the
actual compound formed in this system was quinolinic acid and that no
free nicotinic acid was produced. Other investigators independently re-
ported similar findings.” * Neither tryptophan nor kynurenine would give
rise to quinolinic acid in these preparations, nor would quinolinic acid give
rise to nicotinic acid. However, Makino et al. reported that horse and
cattle livers possess an enzyme system which will convert 3-hydroxyan-
thranilic acid to nicotinic acid, although it is difficult to be positive that
the end product of the reaction may not have been quinolinic acid which
either spontaneously or from heat and chemical treatment decarboxylated
to nicotinic acid and so reacted in their chemical assay procedures. Hurt
et al.§* also reported that rat liver slices would convert tryptophan to nico-
tinic acid, although actually their procedure for determining nicotinic acid
would not permit them to distinguish between nicotinic acid and quinolinic
acid. Makino e/ al.®* also reported that their enzyme preparation converted
3 ,4-dihydroxyanthranilic acid to nicotinic acid just as effectively as it did
3-hydroxyanthranilic acid, indicating that this substance may be an inter-
mediate in the conversion. Their system also formed small amounts of
nicotinic acid from tryptophan and from quinolinic acid. Henderson et al.**
were unable to confirm these findings with 3 ,4-dihydroxyanthranilic acid
using rat and hog liver preparations.

The intermediate between 3-hydroxyanthranilic acid and nicotinic acid
is not entirely clear, although considerable evidence indicates that quino-
linic acid is either an intermediate or a by-product of the reaction. Singal
et al.** were the first to note that tryptophan administration in rats and in
dogs*? resulted in the urinary excretion of an unidentified substance which
had nicotinic acid activity after acid, but not after alkaline, hydrolysis.
This substance was identified by Henderson® as quinolinic acid. This com-
pound can substitute for nicotinic acid in the rat (see Table XI), although
it is far less effective than nicotinic acid, tryptophan, or 3-hydroxyanthra-
nilic acid.” It seems clear that quinolinic acid can be used as a precursor of
nicotinic acid by the rat. However, the relatively large amount required
in comparison to the effective amounts of tryptophan, 3-hydroxyanthranilic
acid, and nicotinic acid induces considerable doubt that it is a normal inter-
mediate between 3-hydroxyanthranilic acid and nicotinic acid, although it

80 B.S. Schweigert and M. M. Marquette, J. Biol. Chem. 181, 199 (1949).

81 A. H. Bokman and B.S. Schweigert, J. Biol. Chem. 186, 153 (1950).

82 J,, M. Henderson and G. B. Ramasarma, J. Biol. Chem. 181, 687 (1949).

83 K. Makino, F. Itoh, and K. Nishi, Nature 167, 115 (1951).

84 W. W. Hurt, B. T. Scheer, and H. J. Deuel, Jr., Arch. Biochem. 21, 87 (1949).

85 ||. M. Henderson, H. N. Hill, R. E. Koski, and I. M. Weinstock, Proc. Soc. Expil.
Biol. Med. 78, 441 (1951).

VI. BIOGENESIS 529

may well be a side product which is normally excreted into the urine rather
rapidly after it is formed.

The conversion of tryptophan to nicotinic acid is a rather inefficient
process at best. Krehl et al.” list the relative activities of tryptophan, 3-
hydroxyanthranilic acid, quinolinic acid, and nicotinic acid as 10, 10, 20
to 40, and 0.2 mg., respectively, in correcting nicotinic acid deficiency in
the rat. On an equimolar basis 3-hydroxyanthranilic acid is somewhat less
active than tryptophan, which might be explainable if the liver converted
a considerable portion of the 3-hydroxyanthranilic acid to quinolinic acid
which was then promptly excreted and thus lost to the nicotinic acid econ-
omy of the tissues.

Recent evidence by Schayer and Henderson®* using doubly labeled tryp-
tophan makes it very likely that the intermediate between 3-hydroxy-
anthranilic acid and quinolinic acid is a substance in which the benzene
ring is split in the 3-4 position similar to that depicted in Fig. 10.

C. SITE OF BIOSYNTHESIS

There have been two principal schools of thought on this subject, one
holding that the principal site of the tryptophan to nicotinic acid conver-
sion is in the tissues and the other holding that the intestinal bacteria are
the predominant factor in this conversion.

1. SYNTHESIS IN TISSUES

In the rat it has been clearly demonstrated that the conversion of tryp-
tophan to nicotinic acid can proceed with normal efficiency without the
intervention of intestinal microbes. This conclusion could be inferred from
studies previously cited showing (1) that enzyme systems exist in tissues
which are capable of carrying out many of the steps in the conversion; (2)
that tryptophan and some of the intermediates in the conversion are effec-
tively changed into nicotinic acid when given parenterally; (3) that tryp-
tophan given parenterally leads to an increase in the nicotinic acid-con-
taining coenzymes of erythrocytes within 4 hours,*® an effect too rapid to
be explainable if tryptophan had to be excreted into the intestinal tract,
converted to nicotinic acid, reabsorbed, and then picked up and incor-
porated into coenzymes by the erythrocytes; and (4) that a deficiency of
pyridoxine interfered in the conversion of tryptophan to nicotinic acid or
inhibited certain steps in this process in intact animals and in tissue prepa-
rations.*®: 71, 84, 87-9 Finally, it was shown by Henderson and Hankes® and

86 R. W. Schayer and L. M. Henderson, J. Biol. Chem. 195, 657 (1952).

87 B.S. Schweigert and P. B. Pearson, J. Biol. Chem. 168, 555 (1947).

88 G. H. Bell, B. T. Scheer, and H. J. Deuel, Jr., J. Nutrition 35, 239 (1948).
89 P. B. Junqueira and B.S. Schweigert, J. Biol. Chem. 174, 605 (1948).

530 NIACIN

by Hundley®* that the conversion of tryptophan to nicotinic acid proceeded
with normal efficiency in rats surgically deprived of their entire intestine.

2. SYNTHESIS BY INTESTINAL MICROORGANISMS

These findings, however, do not necessarily mean that under normal
conditions the intestinal bacteria may not convert some tryptophan to
nicotinic acid or, in addition perhaps, synthesize some nicotinic acid from
other precursors which may be utilized by the host. The entire question
of intestinal synthesis of vitamins in relation to the vitamin economy of
the host is a complicated question which is very difficult to approach by
direct, conclusive experimentation. Some aspects of this question have
been reviewed in the section on requirements (p. 583).

There is little direct evidence indicating how much influence intestinal
bacteria may have on the nicotinic acid requirement of the host by virtue
of the participation of such bacteria in the conversion of tryptophan to
nicotinic acid. There is little, if any, direct relationship between the nico-
tinic acid content of the cecum or of the feces and tryptophan in
the diet.2*: 3!» 82 Junqueira and Schweigert* found that sulfasuxidine, added
to rat diets for the purpose of suppressing intestinal microbial activity,
had no influence on the efficiency of conversion of tryptophan to nicotinic
acid. However, if the diets were deficient in folic acid, then sulfasuxidine
did reduce the amount of nicotinic acid formed from tryptophan. Further-
more, Ellinger and Abdel Kader?®: % found that oral tryptophan was two
to five times as effective as parenteral tryptophan in increasing urinary
N'!-methylnicotinamide whereas tryptophan given orally to rats receiving
sulfasuxidine produced little or no increase in N'!-methylnicotinamide ex-
cretion. Other investigators have not found such a wide discrepancy be-
tween the relative effectiveness of oral and parenteral tryptophan. For
instance, Rosen et al.*! found oral tryptophan about two times as effective
as parenteral tryptophan in increasing urinary nicotinic acid metabolites.
Snyderman et al.*® found oral and parenteral tryptophan equally effective
in increasing urinary N'-methylnicotinamide in infants. Sung and Tung®

90 T,. M. Henderson, I. M. Weinstock, and G. B. Ramasarma, J. Biol. Chem. 189, 19
(1951).

21 J. P. Kring, K. Ebisuzaki, J. N. Williams, Jr., and C. A. Elvehjem, J. Biol. Chem.
195, 591 (1952).

92 M. Mason and C. P. Berg, J. Biol. Chem. 195, 515 (1952).

9 A. EK. Braunstein, E. V. Goryachenkova, and T. 8S. Paskhina, Biokhimiya 14, 163
(1949).

9 C. E. Dalgliesh, W. E. Knox, and A. Neuberger, Natwre 168, 20 (1951).

95 J,, M. Henderson and L. V. Hankes, Proc. Soc. Exptl. Biol. Med. 70, 26 (1949).

96 J. M. Hundley, Proc. Soc. Exptl. Biol. Med. 70, 592 (1949).

97 P, Klinger and M. M. Abdel Kader, Nature 160, 675 (1947).

9S. Sung and T. Tung, J. Biol. Chem. 186, 637 (1950).

wytos

VI. BIOGENESIS 531

found monomethyltryptophan (presumably amino-N-methyltryptophan)
equally effective by mouth or parenterally in increasing the urinary excre-
tion of nicotinamide and its derivatives in rats.

3. UTILIZATION OF L- AND D-[RYPTOPHAN AS PRECURSORS FOR
Nicotinic Acip

a. In Rat

There is a very limited amount of information on this subject. Both p-
and L-tryptophan can be used by the rat for nicotinic acid synthesis, al-
though p-tryptophan is somewhat less efficiently utilized.*!: 4) 7» °° Acetyl-
p-tryptophan, however, was completely inactive, and acetyl-L-tryptophan
was only about one-half as active as L-tryptophan.*!

b. In Poultry

In the chick, Grau and Almquist!° found p-tryptophan to be completely
inactive, although later studies by Wilkening and Schweigert!™ indicated
that the chick could use p-tryptophan 17 to 40% as efficiently as L-tryp-
tophan. Kratzer et al.! found p-tryptophan to be 30 % as active as L-tryp-
tophan in turkey poults. These studies in poultry evaluated the utilization
of p-tryptophan for growth. Its effect on nicotinic acid requirements was
not studied. Utilization of the isomers would not necessarily be the same
in the conversion of tryptophan to nicotinic acid. Anderson e¢ al.!% studied
this question in chicks under conditions where they could measure the
nicotinic acid-replacing value of the isomers of tryptophan. These workers
could find no evidence that p-tryptophan was utilized as a precursor of
nicotinic acid when the diet contained glucose as the principal carbohydrate.
However, when the ration contained starch, p-tryptophan was utilized to a
limited extent. These facts, plus the finding that the utilization of p-tryp-
tophan was reduced when the starch ration contained sulfasuxidine, indi-
cate that intestinal microorganisms may play some role in the utilization
of p-tryptophan.

c. In Dogs

In dogs, Singal and associates‘? obtained evidence that only L-tryptophan
was used as a precursor for nicotinic acid in spite of the fact that pL-tryp-

99W. A. Krehl, P.S. Sarma, L. J. Teply, and C. A. Elvehjem, J. Nutrition 31, 85
(1946).

100 ©. R. Grau and H. J. Almquist, J. Nutrition 28, 263 (1944).

101M. C. Wilkening and B.S. Schweigert, J. Biol. Chem. 171, 209 (1947).

102 FH. Kratzer, D. E. Williams, and B. Marshall, J. Nutrition 48, 223 (1951).

103 J. O. Anderson, G. F. Combs, and G. M. Briggs, J. Nutrition 42, 463 (1950).

532 NIACIN

tophan is used as efficiently as L-tryptophan for maintenance of nitrogen
balance and for plasma protein synthesis.!™

d. In Man

p-Tryptophan is apparently not utilized to any extent! for ordinary
metabolic purposes in man. Sarett and Goldsmith*® could find no evidence
that D-tryptophan was used as a precursor of nicotinic acid in man.

4. TRYPTOPHAN-NICOTINIC ACID RELATIONSHIPS IN OTHER ORGANISMS
a. Eggs

It is well known that nicotinic acid is synthesized by the developing
chick!* and turkey!” embryo. There is also an increase in the total amount
of pyridine nucleotide as the embryo develops.!% The precursors of this
synthesized nicotinic acid are not known. However, Schweigert et al.!%°
found that tryptophan introduced into the embryo resulted in a moderately
increased synthesis of nicotinic acid. Kidder and associates? were unable
to confirm this finding, although Denton and associates" were able to do
so. Furthermore, Ackermann and Taylor! have found that both nico-
tinamide and tryptophan can prevent toxicity from 3-acetylpyridine in the
developing embryo, although tryptophan was considerably less potent than
nicotinamide. These findings seem to indicate a tryptophan—nicotinic acid
relationship in this organism afd have additional interest since this is a
situation in which tryptophan can apparently be converted to nicotinic
acid in the complete absence of any microorganisms.

b. Plants

The fact that plants can and do synthesize nicotinic acid during germi-
nation and growth from the simplest of inorganic elements is well known.
There is some evidence that tryptophan may serve as a precursor of nico-
tinic acid under certain conditions. Nason" found that L- or pt-tryptophan
and 3-hydroxyanthranilic acid increased nicotinic acid synthesis in the

104 S$. C. Madden, R. R. Woods, F. W. Shull, and G. H. Whipple, J. Exptl. Med. 79,
607 (1944).

105 A. A. Albanese and J. E. Frankston, J. Biol. Chem. 155, 101 (1944).

106 W. J. Dann and P. Handler, J. Biol. Chem. 140, 935 (1941).

107 C, Furman, E. E. Snell, and W. W. Cravens, Poultry Sci. 26, 307 (1947).

108 M. Levy and N. F. Young, J. Biol. Chem. 176, 185 (1948).

109 B. 8. Schweigert, H. L. German, and M. J. Garber, J. Biol. Chem. 174, 383 (1948).

110 G, W. Kidder, V. C. Dewey, M. B. Andrews, and R. R. Kidder, J. Nutrition 37,
521 (1949). :

11 C, A. Denton, W. L. Kellogg, W. E. Rowland, and H. R. Bird, Arch. Biochem. and
Biophys. 39, 1 (1952).

12 W. W. Ackermann and A. Taylor, Proc. Soc. Exptl. Biol. Med. 67, 449 (1948).

113 A, Nason, Science 109, 170 (1949); Am. J. Botany 8, 612 (1950).

ow

VI. BIOGENESIS 533

incubating corn embryo. Gustafson" found that tryptophan increased,
slightly, the nicotinic acid content of broccoli, cabbage, and tomato leaves.
Banerjee and Banerjee!!® found that tryptophan increased nicotinic acid
in germinating Phaseolus mungo seeds, although Terroine!* could demon-
strate no such relationship in Phaseolus multiflorus embryos.

c. Insects

Neither Tribolium confusum nor Tenebrio molitor can use tryptophan in
place of nicotinic acid.” “Germ-free”’ Drosophila also require preformed
nicotinic acid and cannot synthesize it from tryptophan."8

d. Microorganisms

Davis et al."° have reported that a tryptophan-nicotinic acid relation-
ship exists in Xanthomonas pruni very similar to that in Neurospora crassa-
Tryptophan can replace nicotinic acid for the growth of the fungus 7'r7-
chophyton equinum.”° Schopfer and Boss”! found that 2-methyl-1 ,4-naph-
thoquinone inhibits growth and nicotinic acid synthesis in Phycomyces
blakesleeanus, an effect reversible by tryptophan, kynurenine, anthranilic
acid, indole, nicotinic acid, and nicotinamide. Whether this indicates a
tryptophan-nicotinic acid conversion mechanism in this fungus is not yet
clear. Kidder e¢ al."° could demonstrate no tryptophan-nicotinic acid re-
lationship in the animal microorganism T'etrahymena.

It is well known that many bacteria require nicotinic acid, and others
require tryptophan, whereas some require both nicotinic acid and trypto-
phan for growth. Many bacteria can synthesize both nicotinic acid and
tryptophan from simple inorganic molecules. However, little is known about
the exact mechanism of nicotinic acid biogenesis in bacteria. A tryptophan-
nicotinic acid interrelation in the same sense as it exists in Neurospora and
in animals is not known to exist in bacteria.!22-!"4

14 FG. Gustafson, Science 110, 279 (1949).

115 §. Banerjee and R. Banerjee, Indian J. Med. Research 38, 153 (1950).

16 T. Terroine, Compt. rend. 226, 511 (1948).

7G, Frankel and H. R. Stern, Arch. Biochem. 30, 438 (1951).

18s J. Schultz and G. T. Rudkin, Federation Proc. 7, 185 (1948).

119 TJ), Davis, L. M. Henderson, and D. Powell, J. Biol. Chem. 189, 543 (1951).
120 J,, K. Georg, Proc. Soc. Exptl. Biol. Med. 72, 653 (1949).

21 W. H. Schopfer and M. L. Boss, Helv. Physiol. et Pharmacol. Acta 7, C20-2 (1949).
122 B. E. Volcani and E. E. Snell, Proc. Soc. Exptl. Biol. Med. 67, 511 (1948).
23 P, Ellinger and M. M. Abdel Kader, Nature 163, 799 (1949).

124 P, Ellinger and M. M. Abdel Kader, Biochem. J. 44, 506 (1949).

534 NIACIN

VII. Estimation

Biological, chemical, fluorometric, photometric, and microbiological tech-
niques have been employed for the estimation of nicotinic acid and related
substances.

A. BIOLOGICAL ASSAY
J. M. HUNDLEY

Animal assays offer little opportunity for the exact determination of
nicotinic acid unless the substances to be tested are completely free from
protein and amino acids, especially tryptophan. Biological tests might offer
possibilities in assessing the over-all pellagra-protective potency of foods if
a suitable way to quantitatively express and compare the results could be
devised. Young nicotinic acid-deficient animals do show a growth response
proportional, within a narrow range, to the amount of nicotinic acid admin-
istered. This has been demonstrated with pure, or relatively pure, solutions
of nicotinic acid or nicotinamide in puppies,! weanling rats,?~* and chicks.®
Some idea of the nicotinic acid content of test solutions may be gained by
attempting to cure blacktongue in adult dogs, but precise quantitative re-
sults are almost impossible. Test substances may also be evaluated by
their ability to prevent blacktongue in dogs on suitable diets, but this tech-
nique also has many variables.

B. CHEMICAL METHODS

J. M. HUNDLEY
1. Nicotinic AcIp

The chemical determination of nicotinic acid is, in theory, quite simple.
Utilizing the Konig reaction,® nicotinic acid is treated with cyanogen bro-
mide and then with an aromatic amine to yield a colored compound which
can be quantitated photometrically. The underlying reaction proceeds in
two steps: first, the intermediate production of a pyridinium derivative
by reaction of the nicotinic acid with cyanogen bromide, and, second, the
production of a colored glutaconic dialdehyde derivative by reaction with
the aromatic amine.®: 7

1H. A. Waisman, O. Mickelsen, J. M. McKibbin, and C. A. Elvehjem, J. Nutrition
19, 483 (1940).

2W. A. Krehl, P. 8. Sarma, L. J. Teply, and C. A. Elvehjem, J. Nutrition 31, 85
(1946).

3 J. M. Hundley, J. Biol. Chem. 181, 1 (1949).

4. J. Harris and E. Kodicek, Brit. J. Nutrition 4, xiii (1950).

5M. E. Coates, 8. K. Kon, and E. E. Shepheard, Brit. J. Nutrition 4, 203 (1950).

6 W. Konig, J. prakt. Chem. 69, 105 (1904); 70, 19 (1904).

7™T. Zincke, Ann. 330, 361 (1904).

VII. ESTIMATION 535

However, the large number of modifications of this basic method which
have appeared in the past twelve years attests to the fact that the proce-
dure is not so simple as it seems when applied to a variety of biological
materials. Difficulties are encountered in hydrolysis in obtaining complete
liberation and maximum conversion of nicotinamide to nicotinic acid with-
out formation of interfering chromogens and without conversion of tri-
gonelline. The choice of reaction temperatures and proper pH, the choice
of the proper aromatic amine to yield the maximum stable color, and proper
blank corrections are important factors. Many of these difficulties have been
discussed and examined in detail in other publications.’ 9 Gyérgy and
Rubin’ present detailed instructions for three methods: (1) a procedure
using metol, devised by Perlzweig et al.!° and improved by Dann and
Handler,'' which has been used extensively in examining animal tissues;
(2) the aniline procedure of Melnick,” which has been applied extensively
to cereals; and (3) a p-aminoacetophenone (or p-aminopropiophenone) pro-
cedure developed by a British group.": Dennis and Rees! have recently
proposed a method which uses procaine hydrochloride for color develop-
ment. The Association of Vitamin Chemists!® has adopted two carefully
checked chemical methods, one of which is essentially the method of Friede-
mann and Frazier.® A new method has recently been proposed for adoption
by the Association of Official Agricultural Chemists.” '8 The method uti-
lizes sulfanilic acid or Tobias acid (2-naphthylamine-1l-sulfonic acid) for
color development and permits both a quantitative and a qualitative dif-
ferentiation of nicotinic acid and nicotinamide.

2. DIFFERENTIAL Assay oF Nicotinic Acip, NICOTINAMIDE, AND RELATED
CoMPoUNDS

These compounds may be differentially quantitated either by micro-
biological (p. 538) or by chemical methods. In addition to the method of
Sweeney and Hall, mentioned above,": '8 paper chromatography has proved
useful in separating and determining small quantities of these sub-
stances.!9-?1

8 P. Gyérgy and 8. H. Rubin zn Vitamin Methods, Vol. I, pp. 223-239. Academic
Press, New York, 1950.

9T. E. Friedemann and E. I. Frazier, Arch. Biochem. 26, 361 (1950).

10W. A. Perlzweig, E. D. Levy, and H. P. Sarett, J. Biol. Chem. 136, 729 (1940).

uW.J. Dann and P. Handler, J. Biol. Chem. 140, 201 (1941).

22 T). Melnick, Cereal Chem. 19, 553 (1942).

13K. M. James, F. W. Norris, and F. Wokes, Analyst 72, 327 (1947).

™C, Klatzkin, F. W. Norris, and F. Wokes, Analyst 74, 447 (1949).

15 P.O. Dennis and H. G. Rees, Analyst 74, 481 (1949).

16 Association of Vitamin Chemists, Methods of Vitamin Assay, 2nd ed., pp. 184-203.
Interscience Publishers, New York, 1951.

7 J. P. Sweeney, J. Assoc. Offic. Agr. Chemists 34, 380 (1951).

18 J, P. Sweeney and W. L. Hall, Anal. Chem. 28, 983 (1951).

536 NIACIN

Ciusa” described a procedure which depends on the Konig reaction be-
fore and after the unknown solution is boiled with chlorobenzene. Since
chlorobenzene reacts with nicotinamide, but not with nicotinic acid, the
two forms of the vitamin can be determined by difference. Scudi”* described
a fluorometric procedure for the determination of nicotinamide which in-
volves the conversion of nicotinamide to a fluorescent N!-methy] derivative.
Chaudhuri and Kodicek™ have developed a somewhat related method which
measures only nicotinamide in biological materials. They used cyanogen
bromide and treatment with strong alkali to yield a fluorescent derivative
which could be quantitated in a photoelectric fluorometer. Kato and
Shimizu®> had difficulty with this method, because of interference from
kynurenine, 3-hydroxykynurenine, and other substances. These interfer-
ing substances could be removed by chromatographing on a carboxylic-
type cation exchange resin and a strong-base-type anion exchange resin.

Ellinger and Abdel Kader?® have developed chemical methods with which
they could differentially determine nicotinic acid, nicotinamide, nicotinuric
acid, N!-methylnicotinamide, and trigonelline. The basis of the procedure
was hydrolysis of sample aliquots in various strengths of acid, alkali, and
alkali plus urea, with subsequent application of the K6nig reaction. (N1-
methylnicotinamide was assayed fluorometrically.)

3. N!-METHYLNICOTINAMIDE

Several methods have been devised to measure this substance in urine.””~*!
Some of these measured trigonelline as well as N'!-methylnicotinamide. The
method in most general use at present is that devised by Huff et al.* This
method is sensitive, accurate, and rapid and has given excellent results
in many hands. It depends on the reaction of N'-methylnicotinamide and
acetone in the presence of alkali to yield a fluorescent derivative which can
be quantitated against an internal standard of synthetic N'methylnico-

19C. F. Heubner, Nature 167, 119 (1951).

20}. Kodicek and K. K. Reddi, Nature 168, 475 (1951).

21). G. Wollish, M. Schmall, and E. G. Schafer, Anal. Chem. 23, 768 (1951).

22 W. Ciusa, Ann. chim. appl. 39, 93 (1949).

23 J. V. Scudi, Sczence 103, 567 (1946).

24 PK. Chaudhuri and E. Kodicek, Biochem. J. 44, 343 (1949).

25 M. Kato and H. Shimizu, Science 114, 12 (1951).

26 P. Ellinger and M. M. Abdel Kader, Biochem. J. 44, 77, 627 (1949).

27 J. W. Huff and W. A. Perlzweig, J. Biol. Chem. 150, 395 (1948).

28 M. Hochberg, D. Melnick, and B. L. Oser, J. Biol. Chem. 158, 265 (1945).

29V. A. Najjar, Bull. Johns Hopkins Hosp. 74, 392 (1944).

30 R. A. Coulson, P. Ellinger, and M. Holden, Biochem. J. 38, 150 (1944).

31H. P. Sarett, J. Biol. Chem. 150, 159 (1943).

3 J. W. Huff, W. A. Perlzweig, and M. W. Tilden, Federation Proc. 4, 92 (1945);
J. Biol. Chem. 167, 157 (1947).

VII. ESTIMATION 537

tinamide using a fluorometer equipped with the same filters as for thio-
chrome. As little as 0.3 y of the compound per milliliter of diluted urine
can be detected easily. This usually permits a sufficient dilution to eliminate
interference from highly pigmented urines and other substances. Urines
giving high blank values can be decolorized with charcoal and acetic acid.

4. 6-PyRIDONE oF N!-METHYLNICOTINAMIDE

This compound* can be assayed directly by means of its intense ultra-
violet absorption which has maxima at 260 and 290 mu. As little as 1 y
per milliliter can be assayed in this way. Rosen et al.*4 found the spectro-
photometric technique somewhat inadequate in that the values were too
high largely because of inability to obtain a suitable blank. They devised
a fluorometric method which gave satisfactory results. This method, al-
though somewhat laborious, is useful and does not detect N'-methylnico-
tinamide.

5. TRIGONELLINE (N!-Meruyt Nicotinic Acip BETAINE)

In spite of the close structural similarity of this substance to N!-methyl-
nicotinamide, it is now well established that it is not a metabolic product
of nicotinic acid in mammals. Several methods have been devised to measure
it in urine.” 35. 35a

6. DPN anp TPN (Cornzymes I anp II)

Enzymatic spectrophotometric methods have been described elsewhere
(p. 509). Total pyridine nucleotides in red blood cells may be determined
by means of a fluorometric procedure*® based on the same principle as
Huff, Perlzweig, and Tilden’s fluorometric determination of N'-methyl-
nicotinamide in urine.” The blood is made protein-free with trichloroacetic
acid. Acetone condensation is then carried out in the presence of alkali,
yielding a fluorescent derivative which can be quantitated fluorometrically.
The procedure yields results very similar to those obtained by Hemophilus
parainfluenza assay.

Essentially the same procedure can be applied to assay the contents of
coenzymes I and II in the tissues.*’ In this instance the tissue to be assayed
must be immersed promptly in 2 % nicotinamide solution to inhibit break-

33 W. E. Knox and W. I. Grossman, J. Biol. Chem. 168, 121 (1947).

34’. Rosen, W. A. Perlzweig, and I. G. Leder, J. Biol. Chem. 179, 157 (1949).

35S. W. Fox, E. W. McNeil, and H. Field, Jr., J. Biol. Chem. 147, 645 (1943).

36a A. P. Sarett, W. A. Perlzweig, and KE. D. Levy, J. Biol. Chem. 135, 483 (1940).

36 N. Levitas, J. Robinson, F. Rosen, J. W. Huff, and W. A. Perlzweig, J. Biol. Chem.
167, 169 (1947). ;

37 J. Robinson, N. Levitas, F. Rosen, and W. A. Perlzweig, J. Biol. Chem. 170, 653
(1947).

538 NIACIN

down of the coenzymes. The reduced coenzymes are oxidized, after which
the same procedure is used as for determining coenzymes in blood.

Kaplan et al.** have recently proposed a chemical method based on the
facts that oxidized DPN and TPN are split by alkali and that treatment
with strong alkali produces a bluish fluorescence which can be quantitated.
When crude tissue extracts were used, the difference in fluorescence before
and after the coenzymes were destroyed with an enzyme (DPNase from
Neurospora) was a measure of the amount of DPN-—TPN originally present.
N!-Methylnicotinamide interfered slightly in the test. The method has not
yet received its baptism of fire by being tried in tissue studies by other
investigators.

7. OTHER METHODS

Feinstein®? has devised a rapid, simple test to determine whether flour
has been enriched with nicotinic acid. A few drops of aniline are dropped
into the center of a packed sample of the flour. A few drops of cyanogen
bromide are dropped on top of the spot wet from the aniline. A canary
yellow color appears promptly, and the depth of the color is roughly pro-
portional to the amount of nicotinic acid in the flour.

C. MICROBIOLOGICAL METHODS

KE. E. SNELL

Nicotinic acid and nicotinamide have been found essential for a great
variety of bacteria and yeasts since the nutritive importance of these com-
pounds for bacteria was first indicated by the work of Lwoff and Lwoff
with influenza bacilli,?? of Knight with staphylococci,*! of Mueller with
diphtheria organisms,” and of Snell and coworkers with lactic acid bac-
teria.*

A number of nicotinic acid derivatives occur naturally (e.g., nicotina-
mide, coenzymes I and II, N!-methylnicotinamide, trigonelline, quinolinic
acid, etc.), and selection of an assay organism for which the activity of
these compounds parallels their activity for animals is important. Nico-
tinic acid and nicotinamide have widely different activities for staphylo-
cocci, corynebacteria, dysentery organisms, pasteurella, and several other
bacteria, and hence these organisms are not entirely satisfactory for assay

38 N. O. Kaplan, S. P. Colowick, and C. C. Barnes, J. Biol. Chem. 191, 461 (1951).

39 L,, Feinstein, Sczence 101, 675 (1945).

40 A. Lwoff and M. Lwoff, Proc. Roy. Soc. (London) B122, 352 (1937).

1B. C.J. G. Knight, Biochem. J. 31, 731, 966 (1937).

42 J.H. Mueller, J. Biol. Chem. 120, 219 (1937).

48H}, E. Snell, F. M. Strong, and W. H. Peterson, J. Am. Chem. Soc. 60, 2825 (1938);
J. Bacteriol. 38, 293 (1939).

VII. ESTIMATION 539

purposes, quite apart from their pathogenic character.*4 Some nicotinic
acid-requiring yeasts utilize trigonelline,*® and special procedures to correct
for this substance, which has no activity for animals, are necessary when
they are used. A wide variety of lactic acid bacteria require nicotinic acid;
furthermore, the most widespread of the active forms of the vitamin (nico-
tinic acid, nicotinamide, coenzymes I and II) are equally active on the
molar basis. The method of Snell and Wright,‘® which uses Lactobacillus
arabinosus as the assay organism, has been widely studied and has proved
highly successful both in its original form and in any of several minor modi-
fications.

The assay procedure of choice is the modification adopted after wide
collaborative study by the U 8. Pharmacopeia* and the American Associ-
ation of Agricultural Chemists. The method has been treated in detail
with respect to procedure, specificity, and reliability in several readily
available treatises.“ 47-49 Growth of L. arabinosus in the niacin-free basal
medium used increases with the niacin concentration in the range from 0 to
about 0.40 y per 10 ml. of medium. Pure nicotinic acid and samples to
supply the vitamin at several levels within this range are added to indi-
vidual tubes containing 5 ml. of the double-strength basal medium. Each
tube is then diluted to 10 ml., capped, autoclaved, cooled, and inoculated.
Response of the test organism is usually determined by acid titration after
72 hours incubation at 30 to 37°; under favorable conditions, turbidimetric
estimations of growth can be made as early as 24 hours. The niacin con-
tent of the sample is then determined by interpolation of the response
obtained with known levels of the samples onto the standard curve obtained
by plotting the responses to known levels of nicotinic acid. The procedure
is usually considered accurate to within +10%, which compares very
favorably with the best of the chemical procedures.

The extraction of nicotinic acid from natural materials preparatory to
assay Offers little difficulty because of the looseness with which the vitamin
is bound, and because of the stability of nicotinic acid. Autoclaving of the
finely divided sample at 15 Ib. pressure for 15 minutes with 0.1 N sulfuric
acid is the preferred procedure.” “8 Few interfering materials are known.
In contrast to animals, the test organism is unable to utilize tryptophan or
the intermediates between tryptophan and niacin for growth in the absence

44. E. Snell, Biol. Symposia 12, 183 (1947).

45 W.1L. Williams, J. Biol. Chem. 166, 397 (1946).

46). EK. Snell and L. D. Wright, J. Biol. Chem. 139, 675 (1941).

47U.S. Pharmacopeia, 14th revision, p. 737, 1950.

48H. E. Snell 7m Vitamin Methods, Vol. I, pp. 860-370. Academic Press, New York,
1950.

49 Association of Vitamin Chemists, Methods of Vitamin Assay, 2nd ed., p. 245. In-
terscience Publishers, New York, 1951.

540 NIACIN

of niacin.®® Although the assay values thus give the true niacin content of
a foodstuff, they do not therefore necessarily reflect the effectiveness of a
foodstuff in preventing pellagra or blacktongue. Indeed, the low values for
niacin first found in eggs and milk by this procedure*® and confirmed by
improved chemical methods,*! as contrasted with the known value of these
foods in preventing pellagra, were among the first findings pointing to the
importance of factors other than niacin in the etiology of pellagra.

For special purposes, assay methods with different specificities may be
of value. A strain of Leuconostoc mesenteroides responds to nicotinic acid,
but not to nicotinamide, and has been used for estimation of the former in
the presence of the latter.**: * Following appropriate hydrolysis, total nia-
cin is obtained. By use of the yeast, Torula cremoris, together with a variety
of hydrolytic procedures, one may estimate either (1) total niacin, (2)
niacin plus trigonelline, or (3) niacin plus trigonelline plus N!-methylnico-
tinamide.*® Some organisms, e.g., Pasteurella suiseptica, utilize nicotinamide
but not nicotinic acid;** assay of the former in the presence of the latter
should thus be possible. None of these procedures has been widely used.

VIII. Standardization of Activity
J. M. HUNDLEY

Both nicotinic acid and nicotinamide are readily available from commer-
cial sources in quite pure form. There is little need for reference standards
for most purposes. The purity of commercial products can be checked
readily by melting point and other tests; they are readily recrystallized if
necessary. However, a U.S.P. reference standard of pure crystalline nico-
tinic acid is available for those who wish to use it.! Commercial nicotinic
acid, U.S.P. grade,? must be at least 99.5 % CsHsNO2. When dried at 105°
for 1 hour it must lose no more than 1% in weight. When ignited there
must be no more than 0.1 % residue. It must have no more than 200 p.p.m.
of chloride, 200 p.p.m. of sulfate, and 20 p.p.m. of heavy metals. Nico-
tinamide (U.S.P.)? must be at least 98.5 % CsHsN2O and must lose no more
than 0.5 % of its weight after drying over H:SO, for 4 hours. On ignition
there must be no more than 0.1 % of residue, and it must contain no more
than 30 p.p.m. of heavy metals.

50 B. EB. Voleani and E. E. Snell, Proc. Soc. Exptl. Biol. Med. 67, 511 (1948).
51 W.J. Dann and P. Handler, J. Biol. Chem. 140, 201 (1941).

52 B. C. Johnson, J. Biol. Chem. 159, 227 (1945).

53§. Berkman and 8. A. Koser, J. Bacteriol 41, 38 (1941).

1U.8. Pharmacopeia Reference Standards, 46 Park Ave., New York 16, N. Y.
2 U.S. Pharmacopeia, 14th revision, p. 379 (1950).

_-

IX. OCCURRENCE 541

IX. Occurrence
J. M. HUNDLEY

There is a tremendous volume of literature which presents in detail the
nicotinic acid content of practically every form of living matter or sub-
stances derived from living matter (Table XIII). Much of this information
is of but limited value, from the standpoint of practical nutrition, since we
know that there are many factors which influence the amount of nicotinic

TABLE XIII

OccURRENCE OF NICOTINIC ACID IN ORGANISMS OF VARIOUS BIOLOGICAL PHYLA
(rrom Woops et al.)

(y/g. of moist tissue)

Frog (Rana) 11.7. Brewer’s yeast 126.0
Horned toad 26.8 Rat (whole) 61.0
Snake (Thamnophis) 28.0 Lamb leg muscle 75.0
Red ant (Dolichederus) 20.5 Veal leg muscle t2e0
Cockroach (Periplaneta americanus) 33.0 Chicken leg muscle 38.0
Termites (Zootermposis) 32.0 Chick embryo 28.4
Drosophila virilis larvae NY 36.5 Salmon steak 64.0
Drosophila virilis larvae NO 37.5 Fish (Crypinidae) 23.9
Oyster (Mytilus) 11.7 Whole wheat (seed) 41.0
Earthworm (Lumbricus terrestris) 15.0 Lima beans (dry seed) 9.8
Protozoa (V'etrahymena geleit) 11.7 Cauliflower (flower) et
Aerobacter aerogenes, aerobic 49.1 Carrots (root) 2.6
Serratia marcescens 40.1 Blackeye peas (dry seed) 13.0
Pseudomonas fluorescens 44.0 Apples (fruit) 0.8
Clostridium butylicum, anaerobic 73.0 Watermelon (fruit) 2.4
Mushroom (Coprinus atranentarius) 68.5 Lettuce (leaf) Det)
Mold 12.2 Irish potato (tuber) 4.3

acid that can be derived from a given diet, factors which cannot be readily
assessed quantitatively except by actual experiment. The amount of tryp-
tophan in relation to the balance of other amino acids, the type and amount
of carbohydrate, the amount of fat, the relative availability of the nicotinic
acid in the food, and the possibility of intestinal microbial synthesis all
influence the pellagra-preventive potency of a given diet. For this reason
the data on the pellagra-preventive potency of foods, gathered by Gold-
berger and his associates by actual test on pellagrins and on blacktongue
dogs, still are reliable for practical use.!*-"7 The potencies (good, fair, and

1 A.M. Woods, J. Taylor, M. J. Hofer, G. A. Johnson, R. L. Lane, and J. R. McMa-
han, Univ. Texas Publ. 4237, 84 (1942).

1a J. Goldberger and W. F. Tanner, Public Health Repts. (U. S.) 39, 87 (1924).

2 J. Goldberger, G. A. Wheeler, R. D. Lillie, and L. M. Rogers, Public Health Repts.
(U. S.) 41, 297 (1926).

542 NIACIN

little or none) found by these workers are listed in Table XIV. The nico-
tinic acid content of these foods is also listed. It is interesting that, with
a few exceptions, the nicotinic acid values compare fairly well with the
pellagra-preventive potency found by these workers in empirical testing.

TABLE XIV
PELLAGRA-PREVENTIVE PoTENcy or Foopsturrs (GoLDBERGER RaTiINnGs)*
Nicotinic Nicotinic | Nicotinic
Good ene. Fair ane Little or none eee
mg./100 g. mg./100 g. mg./100 g.
Beef (fresh) 4.2 Haddock (canned) 2.4 Pork, salt 0.9
Corned beef (canned) 3.4 Egg yolk (dried) 0.1 Butter 0.1
Chicken 8.0 Skim milk (fresh) 0.1 Casein (leached) 0.0
Pork liver 10.5 Skim milk (dried) isa Cornmeal 1.0
Pork shoulder, lean 4.0 Evaporated milk 0.2 Cornstarch 0
Rabbit 8.8 Wheat, whole 4.3 Oats, rolled 1.0
Salmon (canned) 1.3 Kidney beans 2.5 Rye meal 1.6
Buttermilk 0.1 Soybeans 2.3 Cod liver oil 0
Kale 1.7, Cabbage, green 0.3 Cottonseed oil 0
Collards 2.0 Cowpeas 2.2 Lard 0
Green peas 2.7 Mustard greens (canned) 0.7 Green beans (canned) 0.3
Tomato juice (canned) 0.8 Green peas (dried) onl Navy beans ORD)
Turnip greens (canned) 0.6 Spinach (canned) O23 Carrots 0.5
Wheat germ 4.6 Lettuce 0.2
Peanut meal 16.2 Greenandmatureonions 0.2
Yeast, baker’s 28.2 Sweet potatoes 0.6
Yeast, brewer’s (dried) 36.2 Irish potatoes ee)
Rutabaga 0.9
Dried apples and prunes’. 1.7
Gelatin 0
Beets (canned) 0.1
Whole whey 0.8
American cheese Trace

2 Most of the nicotinic acid values are taken from ‘‘Composition of Foods,’ in U.S. Department of Agri-
culture Handbook No. 8.

A. OCCURRENCE IN FOOD

The nicotinic acid’* content of all common food items has been com-
piled by the Bureau of Human Nutrition and Home Economics of the

3 J. Goldberger, G. A. Wheeler, R. D. Lillie, and L. M. Rogers, Public Health Repts.
(U. 8S.) 48, 1385 (1928).

4 J. Goldberger and W. H. Sebrell, Public Health Repts. (U. S.) 45, 3064 (1930).

5 W.H. Sebrell, Public Health Repts. (U. 8.) 49, 754 (1934).

6 W. H. Sebrell, G. A. Wheeler, and D. J. Hunt, Public Health Repts. (U. S.) 50,
1333 (1935).

7W.H. Sebrell, R. H. Onstott, and D. J. Hunt, Public Health Repts. (U. S.) 58,
72 (1938).

7a Nicotinic acid in this instance and in many other places in this section is used in
its generic sense to mean total nicotinic acid, irrespective of whether the actual
form present is the acid, the amide, or the bound forms of either.

IX. OCCURRENCE 543

U. S. Department of Agriculture.’ The nicotinic acid content of various
animal tissues has been determined and compiled by several groups.?-™
Cereal products have been analyzed and the results compiled.!°-!8 The
nicotinic acid content of many fresh fruits and vegetables is listed by
Russell e¢ al.!® Rich sources of nicotinic acid are yeasts and meats, especially
organ meats such as liver, pancreas, heart, and kidneys. Wheat, barley,
and rye are better sources than corn, oats, or rice. Peanuts are a much
richer source than soy and other varieties of beans. Milk and related dairy
products are low in nicotinic acid. Wheat germ and brewer’s yeast are
excellent sources.

B. FORMS OF OCCURRENCE

In living animal tissues almost all the nicotinic acid is found as the amide
bound into nucleotides (DPN and TPN).?° Meats as purchased for human
consumption undoubtedly contain a considerable amount of free nicotina-
mide, since the pyridine nucleotides are rapidly inactivated and broken
down after cellular death, particularly if the cells are disrupted (p. 507).
Krehl and associates”! studied the relative distribution of nicotinic acid
and nicotinamide in a variety of cereals, vegetables, and other natural
products. From 7 (yellow corn) to 70% (white potatoes) of the total ac-
tivity was found as nicotinamide, the remainder as nicotinic acid or as
substances which yielded nicotinic acid on hydrolysis. Skim milk powder
and rat tissues, on the other hand, contained 91 to 99% in the amide
form 22 +22

8B. K. Watt, A. L. Merrill, M. L. Orr, W. Wu, and R. K. Pecot, U. S. Dept. Agr.,
Agr. Handbook 8, (1950).

9. Bandier, Biochem. J. 38, 1130 (1939).

10H. A. Waisman and C. A. Elvehjem, The Vitamin Content of Meat. Burgess Pub-
lishing Co., Minneapolis.

11 Studies on the Vitamin Content of Tissues, Vols. I and II, Univ. Texas Publs.
4137 (1941); 4237 (1942).

2W. J. Dann and P. Handler, J. Nutrition 24, 153 (1942).

13 J. M. McIntire, H. A. Waisman, L. M. Henderson, and C. A. Elvehjem, J. Nutri-
tion 22, 535 (1941).

144 B. de M. Braganca, Ann. Biochem. and Exptl. Med. (India) 4, 41 (1944).

167. J. Teply, F. M. Strong, and C. A. Elvehjem, J. Nutrition 28, 417 (1942) ; 24, 167
(1942).

16R, MeVicar and G. H. Berryman, J. Nutrition 24, 235 (1942).

17), I. Frazier, J. Am. Dietet. Assoc. 26, 264 (1950).

18}. B. Brown, J. M. Thomas, and F. F. Bina, J. Biol. Chem. 162, 221 (1946).

19W.C. Russell, M. W. Taylor, and J. F. Beuk, J. Nutrition 25, 275 (1943).

20 J. Robinson, N. Levitas, F. Rosen, and W. A. Perlzweig, J. Biol. Chem. 170, 653
(1947).

21. W. A. Krehl, J. de la Huerga, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 166,
53 (1946).

22 P. K. Chaudhuri and E. Kodicek, Biochem. J. 44, 343 (1949).

544 NIACIN

The exact form in which nicotinic acid and its amide are bound in plant
tissue is not known. Several groups of investigators”**> found evidence of
an alkali-labile precursor of nicotinic acid in wheat bran. The substance
was purified to some extent and found to have nicotinic acid activity for
the dog.2® The exact nature of this substance has never been determined,
although the latter workers suggested that it might be a simple ester of
nicotinic acid. Chaudhuri and Kodicek*’ obtained evidence of a precursor
of nicotinic acid in wheat, barley, rice brans and corn.?§ This precursor is
inactive for L. arabinosus and differs from Krehl and Strong’s material in
that it is not active in animals (rats) unless first hydrolyzed. This material
has been purified to some extent, and the suggestion has been made that
it may be nicotinic acid with a residual substance attached through the
carboxyl group.??

These findings may offer an explanation for the work of Chitre and
Desai.?° These investigators developed an animal method for estimating
the availability of nicotinic acid in foodstuffs. Using this method, which
has some inaccuracies and limitations, they found that the availability of
nicotinic acid was only 60% in rice, 81 % in wheat, 76% in gram, 70% in
tur, and 92 % in yeast. They also showed that 7n vitro digestion, using en-
zymes to simulate in vivo digestion, effected a release of nicotinic acid to a
degree consistent with the 7m vivo availability experiments. Cheldelin and
Williams” also noted the apparently incomplete release of nicotinic acid
from cereals when enzyme digestion was compared to acid digestion.

C. SITES OF OCCURRENCE

The distribution of nicotinic acid, and its derivatives, is not uniform
within a given food product. In most foods of vegetable origin nicotinic
acid tends to be concentrated in the outer coverings. For example, wheat
bran contained 330 y per gram, and patent flour 12 y per gram, whereas
the whole wheat from which it was derived assayed 70 y per gram; pol-
ished rice contained 0.9 y per gram, brown rice 6.9 y per gram, and rice
polishings 96.6 y per gram.!° Likewise, much of the nicotinic acid in corn
is found in the bran and, like wheat, in the germ.*! Apple peelings contain

23 V. H. Cheldelin and R. R. Williams, Ind. Eng. Chem. Anal. Ed. 14, 671 (1942).

24 J. S. Andrews, H. M. Boyd, and W. A. Gortner, Ind. Eng. Chem. Anal. Ed. 14, 663
(1942).

25 W. A. Krehl and F. M. Strong, J. Biol. Chem. 156, 1 (1944).

26 W. A. Krehl, C. A. Elvehjem, and F. M. Strong, J. Biol. Chem. 156, 13 (1944).

27 TD). K. Chaudhuri and E. Kodicek, Biochem. J. 47, xxxiv (1950).

28H}. Kodicek, Biochem. J. 48, viii (1951).

29D. K. Chaudhuri and E. Kodicek, Nature 165, 1022 (1950).

30 R. G. Chitre and D. B. Desai, Ind. J. Med. Research 8, 471, 479 (1949).

31 P. R. Burkholder, I. McVeigh, and D. Moyer, Yale J. Biol. Med. 16, 659 (1944).

IX. OCCURRENCE 545

more than twice the concentration of nicotinic acid as the rest of the apple;
pear peelings have three times the concentration of peeled pears. Similar
findings have been recorded for plums, sweet potatoes, tomatoes, and yams.
White potatoes seem to be an exception in that peelings and the peeled
potato have about the same nicotinic acid concentration. In inactive bean
seeds, the concentration of nicotinic acid was 40 y per gram in the sprout,
28 y per gram in the cotyledons, and 7 y per gram in the cuticle. In the
whole bean, 96 % of the nicotinic acid was in the cotyledon.*

Little information is available on the distribution of nicotinic acid within
cells. EK. R. Isbell and associates® studied the distribution of several vita-
mins between nuclei and the whole cell in beef heart and mouse mammary
carcinoma cells. In beef heart the nuclei had a concentration of nicotinic
acid nearly three times that of the entire cell. In mouse carcinoma the
reverse was true in that the entire cell had considerably higher nicotinic
acid concentration than the nuclei. On a weight basis, three-fourths of the
total cellular nicotinic acid was in the cytoplasm of these cancer cells. In
the hen’s egg, the whites have seven times the concentration of the yolks
(on a dry-weight basis) .§

D. STABILITY IN FOOD

Nicotinic acid is one of the most stable of the vitamins against the ordi-
nary conditions met in food processing, storage, and cooking. Some losses
in cooking are encountered, particularly where there is an opportunity for
leaching out. Nicotinic acid is quite stable in canning processes. In canned
foods stored 2 years and at storage temperatures up to 100°F., the losses
rarely exceed 15 %.**-4° There is little or no loss with frozen storage or dry
storage.“'-*8 Usual methods of curing and cooking result in losses of 15 to

8 'T. Terroine and J. Desveaux-Chabrol, Arch. sci. physiol. 1, 117 (1947).

88 E.R. Isbell, H. K. Mitchell, A. Taylor, and R. J. Williams, Studies on the Vitamin
Content of Tissues, Vol. II, Univ. Texas Publ. 4237, 81 (1942).

34 L,. E. Clifeorn Advances in Food Research, 1, 39-104 (1948).

35 F.C. Lamb, A. Pressley, and T. Zuch, Food Research 12, 273 (1947).

36 N. B. Guerrant, O. B. Fardig, M. G. Vavich, and H. A. Ellenberger, Ind. Eng.
Chem. 40, 2258 (1948).

37§. Brenner, V. O. Wodicka, and 8. G. Dunlop, Food Technol. 2, 207 (1948).

3 J. F. Feaster, M. D. Tompkins, and W. E. Pearce, Food Research 14, 25 (1949).

39 B. B. Sheft, R. M. Griswold, E. Tarlowsky, and E. G. Halliday, Ind. Eng. Chem.
41, 144 (1949).

40 R. Millares and C. R. Fellers, Food Research 14, 131 (1949).

41P. F. Sharpe, J. B. Shields, and A. P. Stewart, Jr., Proc. Inst. Food Technol. p. 54
(1945).

4 A. F. Morgan, L. E. Kidder, M. Hunner, B. K. Sharokh, and R. M. Chesbro, Food
Research 14, 439 (1949).

43B. B. Cook, A. F. Morgan, and M. B. Smith, Food Research 14, 449 (1949).

546 NIACIN

20%, although with some foods and cooking methods losses as high as
50% have been recorded.” “4-48 The references cited are intended to be
only representative and do not even begin to cover completely the volumi-
nous literature on this subject.

E. INFLUENCE OF SOIL, ENVIRONMENT, AND HEREDITY

Soil and its nutrients can play some role in the nicotinic acid content
of plants. McCoy and coworkers‘? found that a reduction in the major
ions in their nutrient solution reduced the nicotinic acid content of oats.
Hunt and associates®® reported that liming the soil, or adding nitrates,
increased the nicotinic acid content of wheat. Liming decreased the nicotinic
acid content of oats in a dry year. Gough and Lantz*! studied eight varieties
of beans grown in three widely different localities and found that environ-
ment influenced nicotinic acid content. Gustafson® found more nicotinic
acid in tomatoes, beans, and soybeans grown at 28 to 30° than at 10 to
15°; the reverse was true with broccoli, cabbage, spinach, clover, peas, and
wheat.

However, the magnitude of the effects produced by soil and environment
seems to be much less than the influence of heredity, within the limits of
reasonable production yields. McElroy and Simonson® found no correla-
tion between soil type and nicotinic acid content for oats, wheat, and barley
grown in three different parts of Alberta. Richey and Dawson™ found that
extreme differences in production practices, 1e., fertilizers, green manures,
etc., had no significant influence on the nicotinic acid content of corn, al-
though different inbred strains of corn varied from 13.9 to 53.3 y per gram
of nicotinic acid. Hunt et al.®° examined samples of nine corn hybrids grown
at five different locations for 2 years. There were differences in nicotinic
acid content for both years and at all locations. However, the variations
were small compared to the variations in nicotinic acid content characteris-

44R. M. Griswold, L. M. Jans, and E. G. Halliday, J. Am. Dietet. Assoc. 25, 866
(1949).

45}. Hartzler, W. Ross, and E. L. Willett, Food Research 14, 15 (1949).

46 A. Lopez-Matas and C. R. Fellers, Food Research 13, 387 (1948).

47 B.S. Schweigert, J. M. McIntyre, and C. A. Elvehjem, J. Nutrition 27, 419 (1944).

48K. Causey, E. G. Andreassen, M. E. Hausrath, C. Along, P. E. Ramstad, and F.
Fenton, Food Research 15, 237, 249, 256 (1950).

49T. A. McCoy, S. M. Free, R. G. Langston, and J. Q. Snyder, Sozl Scz. 68, 375
(1949).

50 C,H. Hunt, L. D. Rodriquez, and R. M. Bethke, Cereal Chem. 27, 79 (1950).

51H. W. Gough and E. M. Lantz, Food Research 15, 308 (1950).

52 F. G. Gustafson, Plant Physiol. 25, 150 (1950).

53 T,, W. McElroy and H. Simonson, Can. J.Research 26F, 201 (1948).

54 FD. Richey and R. F. Dawson, Plant Physiol. 23, 238 (1948).

55 ©, H. Hunt, L. Ditzler, and R. M. Bethke, Cereal Chem. 24, 355 (1947).

—~.

IX. OCCURRENCE BAT

tic for each hybrid. Carroll and Lee Peng®® analyzed two varieties of corn,
four of wheat, two of oats, and two of soybeans all grown under the same
environmental conditions and found the nicotinic acid content to be more
characteristic of the species than the soil. Futhermore, Leng ef al.*’ grew
corn which was a cross between a high nicotinic acid sugar line (48.3 y per
gram) and a low nicotinic acid waxy line (18.0 y per gram). From each ear
of the resultant corn they could pick kernels which were sugary in type
(33.8 y of nicotinic acid per gram), waxy (26.0 y per gram), and dent (21.4
y per gram). This certainly demonstrates the predominance of heredity
over environment in this regard.

Different strains of corn are known to vary greatly in their nicotinic acid
content (Table XV). Sugary corns are generally higher than starchy strains.

TABLE XV
Nicotinic Acip IN VARIOUS STRAINS OF CoRN (FROM BURKHOLDER et al. *)
(y/g. air-dried mature grain)

Type of corn Number of strains Average nicotinic acid Range
Yellow field 94 21.4 11.3-36.3
White field 86 20.1 12.7-29.3
Sweet 46 34.6 18.2-62.1
Popcorn 7 17.4 7.9-21.6

F,. NICOTINIC ACID IN U. 8. DIETS

Studies made by the U. 8. Department of Agriculture indicate that our
food in 1951 supplied 24% more nicotinic acid than in the 1935-1939
period.’ Much of this increase can be accounted for by the widespread
enrichment of white flour, the Federal Standard for which provides a mini-
mum of 10 mg. and a maximum of 15.0 mg. of nicotinic acid per pound.*®
Booher and Behan® have analyzed composite food samples representative
of the national dietary for nicotinic acid, as well as other nutrients. Ac-
cording to their results a 3050-calorie diet provides 20.2 mg. of nicotinic
acid, considerably in excess of the recommended allowances of the National
Research Council (15 mg.).®! Earlier studies making direct analyses on foods
representative of the national dietary in 1940 and 1941 indicated that the

56 J. C. Carroll and C. A. Lee Peng, Science 113, 211 (1951).

57 H.R. Leng, J. J. Curtis, and M. C. Shekleton, Science 111, 665 (1950).

58 ©. M. Coons, Chicago Med. Soc. Bull. 58, 1015 (1951).

59 Federal Register, Aug. 3, 1943.

60 J,, E. Booher and I. T. Behan, J. Nutrition 39, 495 (1949).

61 Recommended Dietary Allowances, Bull. Natl. Research Council, Reprint and
Circ. Ser. 129, (1948).

548 NIACIN

average American diet provided about 11 mg. of nicotinic acid daily (no al- -
lowance included for enriched bread).®> ® These data are in contrast to
the 4.25 to 10.49 mg. of nicotinic acid per day which Frazier and Friede-
mann, using Goldberger’s diet records, have calculated were consumed
by families in which pellagra was prevalent. These facts, plus the virtual dis-
appearance of pellagra, indicate that the average American diet is ade-
quate in pellagra-preventive factors.

G. ANTIVITAMINS IN FOOD

The existence of substances in food which have antinicotinic acid prop-
erties has not been proved. There are innumerable reports in the older
literature suggesting or citing evidence for the presence of pellagragenic
factors in corn, particularly spoiled corn. More recently, Woolley® ex-
tracted a substance from corn which caused a pellagra-like syndrome in
mice which could be prevented by nicotinamide. Borrow and associates*®
attempted to repeat Woolley’s work but were in doubt as to whether the
toxicity they obtained was due to residual chloroform in the extract. The
latter group did, however, find evidence of a pellagragenic factor for mice
in corn bran. No further reports on this finding have appeared thus far.
Raska® reported producing “pellagra”’ in dogs by feeding large amounts of
adenine and phosphate. Apparently no attempt was made to prevent or
treat this condition with nicotinic acid. Hence, there is no assurance that
the condition was actually a nicotinic acid deficiency. Kodicek et al. re-
ported that indole-3-acetic acid, which does occur in corn, was pellagragenic
in rats. However, this claim has been denied®*~7! and was later retracted.”

Woolley et al.” found that 3-acetylpyridine was toxic in nicotinic acid-
deficient dogs but, in the same dosage, harmless to normal dogs. Woolley
later reported”: 7 that 3-acetylpyridine caused a ‘‘pellagra-like’”’ condition

6 R. L. Lane, E. Johnson, and R. R. Williams, J. Nutrition 28, 613 (1942).

63 V. H. Cheldelin and R. R. Williams, J. Nutrition 26, 417 (1943).

64H}. I. Frazier and T. E. Friedemann, Quart. Bull. Northwestern Univ. Med. School
20, 24 (1946).

65 TD). W. Woolley, J. Biol. Chem. 168, 773 (1946).

66 A. Borrow, L. Fowden, M. M. Stedman, J. C. Waterlow, and R. A. Webb, Lancet
254, 752 (1948).

67S. B. Raska, J. Biol. Chem. 165, 748 (1946); Science 105, 126 (1947).

68 H. Kodicek, K. J. Carpenter, and L. J. Harris, Lancet 251, 491 (1946).

69 ],, M. Henderson, T. Deodhar, W. A. Krehl, and C. A. Elvehjem, J. Biol. Chem.
170, 261 (1947).

70 F, Rosen and W. A. Perlzweig, Arch. Biochem. 15, 111 (1947).

71 -Y, Raoul and C. Marnay, Compt. rend. 226, 1043 (1948).

2 EK. Kodicek, K. J. Carpenter, and L. J. Harris, Lancet 258, 616 (1947).

73D. W. Woolley, F. M. Strong, R. J. Madden, and C. A. Elvehjem, J. Biol. Chem.
124, 715 (19388).

™%4D. W. Woolley, J. Biol. Chem. 157, 455 (1945).

IX. OCCURRENCE 549

in mice which could be counteracted by nicotinic acid or tryptophan. These
findings have not been confirmed. Indeed, Gaebler and Beher?® found that
3-acetylpyridine caused large increases in urinary N!-methylnicotinamide
excretion in dogs and rats, indicating that this compound could serve as a
precursor of nicotinic acid. Beher et al.” later used an isotope tracer tech-
nique to prove the conversion of 3-acetylpyridine to nicotinic acid. How-
ever, Ackermann and Taylor?’ found that 3-acetylpyridine induced rapid

TABLE XVI
Nicotinic Actp CoNTENT OF VARIOUS ORGANS AND TISSUES
(y/g. of wet tissue)

Man?’ Human fetus8? Rat8l Rats? Pigs3 Ox83
Heart 34 50 125 114 53 59
Liver 60 49 180 157 118 122
Brain 19.5 23 64 AT — —
Lung 20 = 51 48 == =
Kidney 40 38 115 87 68 58
Spleen 26 = 69 58 40 44
Thyroid = 25 = = 16 30
Muscle, smooth 31 = = — = —
Muscle, skeletal 42 26 80 78 47 49
Adrenal 25 — — — — 57
Stomach 22 33 — — — —-
Tleum 21 31 — — — —
Colon, mucosa 16 — — — — --
Ovary 18 — — = 38 =
Testes seminiferous 16 —_— — — 47 —_

tubules

Seminal ducts 8.2 — = — —
Skin 8.6 20 — — — —
Cartilage = 9.7 = — a= —
Bone = 13.0 — = — —

death in embryonating eggs, an effect competitively reversed by nicotin-
amide but much less successfully reversed with nicotinic acid or trypto-
phan. Hull et al.”8 reported that 3-acetylpyridine and pyridine-3-sulfonic

75D. W. Woolley, J. Biol. Chem. 162, 179 (1946).

76 OQ. H. Gaebler and W. T. Beher, J. Biol. Chem. 188, 343 (1951).

77 W.'T. Beher, W. M. Holliday, and O. H. Gaebler, Abstr. 12th Intern. Congr. Pure
and Appl. Chem., New York, p. 84 (1951).

ma W. W. Ackermann and A. Taylor, Proc. Soc. Exptl. Biol. Med. 67, 449 (1948).

73W. Hull, J. C. Perrone, and P. L. Kirk, J. Gen. Physiol. 34, 75 (1950).

79 Averaged from the data of A. Taylor, M. A. Pollack, and R. J. Williams, Univ.
Texas Publ. 4237, 41-55 (1942).

80 A. Lwoff and L. Digonnet, Compt. rend. 218, 1030 (1941).

81H. K. Mitchell and E. R. Isbell, Univ. Texas Publ. 4237, 37-40 (1942).

550 NIACIN

acid produced deleterious effects in embryonic chick heart cultures, but
nicotinic acid would not reverse this effect. Available evidence seems to
support the view that 3-acetylpyridine is a nicotinic acid antagonist under
certain conditions. However, animals (rat and dog at least) possess the
ability to convert this compound to nicotinic acid. If the antagonist is
administered in doses large enough to exceed the animal’s capacity to con-
vert it to nicotinic acid, then it acts as an antagonist. If given in lower doses
it does not act as an antagonist but will actually substitute for nicotinic
acid. There is no evidence that either 3-acetylpyridine or pyridine-3-sulfonic
acid occurs in food.

H. OCCURRENCE IN VARIOUS TISSUES

The data in Table XVI are presented to give a picture of the relative
distribution of nicotinic acid in various mammalian tissues and organs.
The data are not necessarily exactly comparable from one species to an-
other, because different analytical methods were used. Gounelle and associ-
ates*! found considerably higher values for normal human liver (150 y per
gram) than that reported by Taylor et al.79 (Table XVI).

X. Effects of Deficiency

A. IN MICROORGANISMS

EK. E. SNELL

Subsequent to the discovery of diphospho- and triphosphopyridine nu-
cleotides and the demonstration that these nicotinamide-containing coen-
zymes were identical with the long-known factor V required for growth of
Hemophilus influenza, nicotinic acid itself was shown to be an essential
growth factor for Staphylococcus aureus! and for Corynebacterium diph-
thervae.? Since then, it has been shown to be essential for a great variety of
other bacteria (e.g., Proteus vulgaris, Acetobacter, all lactobacilli so far
examined, Clostridium tetani, and others’: *), and for some yeasts.®: § Natu-

8S. A. Singal, V. P. Sydenstricker, and J. M. Littlejohn, J. Biol. Chem. 176, 1069
(1948).

83H}. Bandier, Biochem. J. 33, 1130 (1939).

84H. Gounelle, Y. Raoul, and J. Marche, Compt. rend. 189, 30 (1945).

1B. C.J. G. Knight, Biochem. J. 31, 731, 966 (1937).

2 J. H. Mueller, J. Biol. Chem. 120, 219 (1937).

3B.C.J.G. Knight, Vitamins and Hormones 8, 105 (1945).

4W.H. Peterson and M.S. Peterson, Bacteriol. Revs. 9, 49 (1945).

5 A.S. Schultz, L. Atkin, and C. N. Frey, J. Am. Chem. Soc. 60, 1514 (1938).

6 P. R. Burkholder, I. McVeigh, and D. Moyer, J. Bacteriol. 48, 385 (1944).

X. EFFECTS OF DEFICIENCY Gok

ral populations of fungi other than yeast that require nicotinic acid have
not been observed; however, mutant cultures of Neurospora crassa that
require it are readily obtained.’ All organisms that do not require it pre-
formed in the medium appear to synthesize it; many intermediates in the
route of this synthesis, which proceeds from tryptophan to niacin through
a series of intermediates, were first indicated by use of mutant cultures of
N. crassa."

In all cultures that require a source of nicotinic acid (or its amide) in
the medium, a deficiency in supply is reflected in decreased growth of the
culture. Concomitant with such decreased growth there is, of course, a de-
creased production of the metabolic end products of the particular culture
(e.g., lactic acid production by lactic acid bacteria). Microbiological assays
for the vitamin depend upon these effects.* Aside from these over-all effects
upon growth, no effects of nicotinic acid deficiency upon specific phases of
metabolism of microorganisms have been reported. Because of the role of
the nicotinamide-coenzymes in metabolism, one might expect the ability
of resting cells to carry out certain dehydrogenation reactions to be im-
paired by deficiency of nicotinic acid. Few studies of a sufficiently quantita-
tive character to detect such alterations have been made. Hughes, however,
reported that in a nicotinic acid-low medium, L. arabinosus synthesized
DPN at a considerably higher rate (24 my moles per milligram of cells per
hour) than was true in the presence of an excess of nicotinic acid (8 my
moles per milligram per hour).°

B. IN MAN AND ANIMALS
| J. M. HUNDLEY

1. RELATION oF BiocHEMICAL FuNcTIONS oF NicoTINic AcID TO
DEFICIENCY STATES

A dietary deficiency of nicotinic acid produces deficiency states by virtue
of the fact that nicotinamide is an essential component of coenzymes I
and II. These coenzymes are required for normal metabolism, indeed, for
life itself. If the dietary supply of nicotinic acid is insufficient and the
organism is unable to synthesize sufficient amounts of the vitamin (see
Section VI), normal metabolism becomes impossible, a deficiency state
develops, and the organism eventually dies, if the deficiency is sufficiently
severe.

A great mass of precise information is available on the many biochemical

7G. W. Beadle, H. K. Mitchell, and J. F. Nyc, Proc. Natl. Acad. Sci. U. S. 38, 155
(1947).

8 KE. E. Snell in Vitamin Methods, Vol. 1, p. 327. Academic Press, New York, 1951.

9D. E. Hughes, Biochem. J. 45, xxxvi (1949).

552 NIACIN

reactions which are catalyzed by these coenzymes. The chemistry of the
reactions and the manner in which the coenzymes function are known. Yet
with all this precise information, it is still impossible to offer any compre-
hensive explanation for the origin of many of the characteristic pathological
manifestations of human nicotinic acid deficiency such as dermatitis, skin
sensitivity to solar radiation, glossitis, atrophy of tongue papillae, and
mental abnormalities. An extensive field, still largely unexplored, exists in
which the biochemical reactions catalyzed by these coenzymes must be
integrated into the normal physiology and biochemistry of the intact ani-
mal. This will be necessary before any real picture of the specific effects of
malfunction of the enzyme systems can be drawn. Such will be a difficult
task, but until it is done our explanations for the manifestations of nicotinic
acid deficiency will be mostly educated guesswork based on reasoning
rather than demonstrated facts.

2. Tuer DEFICIENCY STATE

Only two mammalian species, man and dog, develop nicotinic acid defi-
ciency states which are characteristic and which can be readily differen-
tiated by their external manifestations from other vitamin deficiencies and
other diseases. The four D’s of pellagra in man, dermatitis, diarrhea,
delirium, and death, form a characteristic picture which is readily diagnosed
by an experienced clinician. Milder and atypical syndromes are frequently
observed. Many excellent clinical descriptions of pellagra have been pub-
lished and need not be repeated here.!® Pellagra has been produced experi-
mentally in man on two occasions. The original demonstration of the
nutritional etiology of pellagra by Goldberger and associates in 1915"-! is
a classic and well-known story in nutrition. Their work represented the
first experimental production of pellagra in man and clearly demonstrated
the role of diet in the origin and prevention of the disease. It is of more
than passing interest, in view of the recently demonstrated tryptophan-
nicotinic acid relationship, that Goldberger suspected that an amino acid

10 T. D. Spies in Clinical Nutrition, p. 531. Paul B. Hoeber, New York, 1950.

1 J. Goldberger and G. A. Wheeler, Public Health Repts. (U. 8S.) 30, 3336 (1915).

22 J. Goldberger, Public Health Repts. (U.S.) 81, 3159 (1916).

13 J. Goldberger, C. H. Waring, and D. G. Willets, Public Health Repts. (U. S.) 30,
3117 (1915).

14 J. Goldberger and G. A. Wheeler, U. S. Public Health Service Hyg. Lab. Bull. 120
(1920).

16M. X. Sullivan and K. K. Jones, U. S. Public Health Service Hyg. Lab. Bull. 120,
117-126 (1920).

16 J. Goldberger and W. F. Tanner, Public Health Repts. (U. S.) 39, 87 (1924).

17 J. Goldberger, G. A. Wheeler, E. Sydenstricker, W. I. King, W. 8. Bean, R. E.
Dyer, J. D. Reichard, P. M. Stewart, M. C. Edmonds, R. E. Tarbett, D. Wiehl,
and J. C. Goddard, U. S. Public Health Service Hyg. Lab. Bull. 153, (1929).

X. EFFECTS OF DEFICIENCY 553

deficiency, as well as a vitamin deficiency, was involved in the etiology of
pellagra. He singled out cystine and tryptophan as the probable culprits
and with W. F. Tanner conducted therapeutic trials in three patients.'®
The results were cautiously evaluated as a marked improvement in the
dermal lesions of two patients given cystine and a steady gain in weight
with some improvement in diarrhea when both cystine and tryptophan
were administered to the third patient. Apparently, tests with tryptophan
alone were conducted but they were never reported. The author of this
review is in possession of a copy of a progress report to Goldberger from
Tanner, dated August 5, 1921, which has never been publicly reported. In
it, Tanner relates the course of one pellagrous patient who was given “‘one-
half dram” of tryptophan before each meal. There was no improvement in
the diarrhea at the time of the report, but prompt and marked improvement
in the patient’s extensive dermatitis was noted. After describing the progress
of the skin lesions, Tanner stated: ‘‘I might add that the improvement in
this patient’s skin condition has surpassed anything I have ever seen in a
case of pellagra in an equal period of time.” There is no record of the final
result.

The second instance of the production of pellagra in man by dietary
means was recently described by Goldsmith e¢ al.1®9 These experiments are
of special interest, since the importance of tryptophan was realized and
both the tryptophan and nicotinic acid intakes were controlled. These
workers also provided supplementary vitamins such as riboflavin and folic
acid. It has long been a debatable issue as to the extent to which the
pellagra syndrome, as commonly seen clinically, may be modified by con-
comitant deficiencies of other vitamins. It is, therefore, of interest that
essentially typical pellagrous lesions appeared in spite of added vitamins
(not including nicotinic acid). These workers were also able to cure pellagra
with tryptophan, a clear demonstration of the ability of this amino acid
to cure pellagra in man.'** Other interesting aspects of these investigations
are discussed in later sections.

Dogs also develop a very characteristic reaction to nicotinic acid defi-
ciency, the so-called blacktongue. The severe oral lesions, drooling of
saliva, the very foul and characteristic oral odor, diarrhea, often bloody,
the emaciated appearance, and the complete lack of appetite for food and
water form a typical picture. This syndrome is described in detail in Sec-
tion XT.

18 J. Goldberger and W. F. Tanner, Public Health Repts. (U. S.) 37, 462 (1922).

19G. A. Goldsmith, H. P. Sarett, U. D. Register, and J. Gibbens, J. Clin. Invest. 81,
533 (1952).

1% W. B. Bean, M. Franklin, and K. Daum, J. Lab. Clin. Med. 38, 167 (1951), also
reported that tryptophan produced rapid improvement in the tongue of two
patients with pellagrous glossitis.

554 NIACIN

In all other species, except possibly the cat (p. 570), a deficiency of
nicotinic acid is manifested by the same signs and symptoms as can be
produced by many other deficiencies. Actually, there are only six deficiency
phenomena which can be found in all species susceptible to the deficiency.
These are: (1) reduced growth in young animals, (2) weight loss in either
young or adult animals if the deficiency is severe, (3) loss of appetite, (4)
reduced concentration of nicotinic acid and coenzymes I and II in some
but not all tissues, (5) reduced excretion of nicotinic acid and its derivatives
in the urine, and (6) death, if the deficiency is severe or long continued..
Difficulties in reproduction and lactation could probably be added to this
list, but these subjects have received little attention. All these phenomena,
except possibly the reduced tissue and urine levels, can be produced by
other deficiencies.

The general signs and symptoms and the pathology observed in various
species is described in Section XI. The discussion in this section will deal
with some of the physiological and biochemical defects which have been
reported in various species as well as some of the interesting and fairly
characteristic defects which are seen in human pellagra.

3. Nicotinic AcID AND COENZYME TissuE LEVELS

Nicotinic acid-deficient chicks,?°: 2! dogs,”?-*4 pigs,?* 74 rats,>9 rabbits,?°
and men*! exhibit a lowered concentration of nicotinic acid (and nicotinic
acid-containing coenzymes) in tissues such as liver, muscle, and, in some
species, brain. Other tissues such as red blood cells, heart, lung, spleen, and
kidney may maintain normal concentrations.”~*°: *!-*4 The level of nicotinic

20K. G. Anderson, L. J. Teply, and C. A. Elvehjem, Arch. Biochem. 3, 357 (1944).

21G. M. Briggs, Jr., T. D. Luckey, L. J. Teply, C. A. Elvehjem, and E. B. Hart,
J. Biol. Chem. 148, 517 (1948).

22H. I. Kohn, J. R. Klein, and W. J. Dann, Biochem. J. 33, 1482 (1939).

23 A. KE. Axelrod, R. J. Madden, and C. A. Elvehjem, J. Biol. Chem. 131, 85 (1939).

24 A. EK. Axelrod and C. A. Elvehjem, Nature 148, 282 (1939).

25§. A. Singal, V. P. Sydenstricker, and J. M. Littlejohn, J. Biol. Chem. 176, 1069
(1948).

26 J. M. Hundley, J. Nutrition 34, 253 (1947).

27 J. N. Williams, Jr., P. Feigelson, and C. A. Elvehjem, J. Biol. Chem. 187, 597
(1950).

28 J. M. Hundley, J. Biol. Chem. 181, 1 (1949).

29§. A. Singal, V. P. Sydenstricker, and J. M. Littlejohn, J. Biol. Chem. 171, 203
(1947); 176, 1063 (1948).

30 J. G. Wooley, Proc. Soc. Exptl. Biol. Med. 65, 315 (1947).

31H. Gounelle, Y. Raoul, A. Vallette, and J. Marche, Bull. mém. soc. méd. hép.
Paris 21, 1225 (1945).

32 A. E. Axelrod, T. D. Spies, and C. A. Elvehjem, J. Biol. Chem. 138, 667 (1941).

33 A. KE. Axelrod, E.S. Gordon, and C. A. Elvehjem, Am. J. Med. Sci. 199, 697 (1940).

34H. I. Chu, P. T. Kuo, and K. P. Chang, Chinese Med. J. 61, 181 (1942).

Y

X. EFFECTS OF DEFICIENCY +55 y9)

acid (or coenzymes) in liver and muscle shows a much better correlation
with the degree of nicotinic acid deficiency than most other tissues. There
seems to be little or no correlation between nicotinic acid levels in blood
and the deficiency. Furthermore, blood nicotinic acid may be depressed in
several pathological states other than nicotinic acid deficiency.*!: *°-87 The
degree of depression in coenzyme levels in tissues such as liver and muscle
from deficient animals has varied greatly in different studies. In dogs with
acute blacktongue Kohn et al.” found coenzyme levels 70% below normal
in liver and 35 % below normal in striated muscle. Paradoxically, the liver,
which showed a decreased coenzyme level, exhibited an increase of 35 % in
O: consumption, whereas kidney which showed no lowering of coenzymes
showed a 50% decrease in lactate oxidation. More recent studies by Wil-
liams and Elvehjem*® with rat liver homogenates from deficient animals
have shown a low endogenous respiratory rate which could be restored to
normal by the addition of coenzyme I zn vitro. Other studies* in rats have
shown that survival and some growth is possible in animals having a con-
centration of nicotinic acid 70% of normal in liver and 50% of normal in
muscle. The type of carbohydrate in the diet appeared to influence growth
and survival in animals having these lowered nicotinic acid tissue levels.
Glucose and carbohydrates containing glucose exerted a favorable effect,
but fructose and sucrose had the opposite effect.”

Factors other than nicotinic acid deficiency have been reported to lower
nicotinic acid tissue levels. Although simple starvation does not have this
effect,?® a protein-free diet,*®: ' a low protein diet, and a vitamin B-free
ration® do have this effect in rats. Deficiencies of thiamine and riboflavin
also have been reported to lower liver nicotinic acid.**: 4° Cobalt deficiency
has been reported to lower blood nicotinic acid in sheep.*®

Administration of nicotinic acid or tryptophan to deficient or normal sub-
jects results in a rise in blood and tissue nicotinic acid sometimes, especially

35 H. I. Kohn, F. Bernheim, and A. V. Felsovanyi, J. Clin. Invest. 18, 585 (1939).

36§. P. Vilter, M. B. Koch, and T. D. Spies, J. Lab. Clin. Med. 26, 31 (1940).

37 C, W. Carter and J. R. P. O’Brien, Quart. J. Med. [N.8.] 14, 197 (1945).

38 J. N. Williams, Jr., and C. A. Elvehjem, J. Biol. Chem. 183, 539 (1950).

89 B. C. Flinn, F. J. Pilgrim, H.S. Gregg, and A. E. Axelrod, Proc. Soc. Exptl. Biol.
Med. 63, 523 (1946).

40 J. N. Williams, Jr., P. Feigelson, C. A. Elvehjem, and S. 8. Shahinian, J. Biol.
Chem. 189, 659 (1951).

41§. Seifter, D. M. Harkness, L. Rubin, and E. Muntwyler, J. Biol. Chem. 176, 1371
(1948).

“LL.D. Wright and H. R. Skeggs, Proc. Soc. Exptl. Biol. Med. 68, 327 (1946).

48H, von Euler, F. Schlenk, L. Melzer, and B. Hégberg, Hoppe-Seyler’s Z. physiol.
Chem. 258, 219 (1939).

44K, Bhagvat and P. Devi, Biochem. J. 45, 32 (1949).

45 Y, Raoul, Bull. soc. chim. biol. 27, 371 (1945).

46S. N. Ray, W. C. Weir, A. L. Pope, and P. H. Phillips, J. Nutrition 34, 595 (1947).

556 NIACIN

in non-deficient subjects, to levels temporarily above normal.?°: %° 88, 47-97
Wooley*® found that tryptophan was less effective than nicotinic acid in
increasing the nicotinic acid level in skeletal muscle of deficient rabbits,
although both substances were equally effective in increasing the liver
nicotinic acid level and in alleviating deficiency symptoms. On the other
hand Williams e¢ al.’ found that nicotinic acid was less effective than tryp-
tophan in maintaining levels of coenzyme I and II in the liver of rats. This
seemed to be particularly true when protein-free rations were fed.4° How-
ever, in later studies Feigelson et al.°* found that nicotinic acid and trypto-
phan were equally effective, on an equimolecular basis, as precursors for
liver pyridine nucleotides, except that administration of tryptophan at
levels considerably in excess of requirements seemed to increase liver pyri-
dine nucleotides above “normal.” This effect was not observed with nico-
tinic acid.

4. Urinary Excretion or Nicotinic ACID AND OTHER SUBSTANCES
a. Urinary Pigments

For the first two or three years following the discovery of nicotinic acid
as the pellagra-preventive vitamin, interest in using urine for diagnostic
purposes centered about the excretion of porphyrin and other pigments.
Beckh, Ellinger, and Spies®* devised a test (the B.E.S. test) which they
believed to measure urinary porphyrin and which seemed to have a diag-
nostic relationship to pellagra. However, further investigations by Watson
and associates>*-® showed that the B.E.S. test measured urorosein, which

47 J. G. Leder and P. Handler, J. Biol. Chem. 189, 889 (1951).

48C. L. Hoagland, S. M. Ward, and R. E. Shank, J. Biol. Chem. 151, 369 (1943).

49K. Leifer, J. R. Hogness, L. J. Roth, and W. Langham, J. Am. Chem. Soc. 70, 2908
(1948).

50 ],. J. Roth, E. Leifer, J. R. Hogness, and W. H. Langham, J. Biol. Chem. 176,
249 (1948).

51 A. Bonsignore and L. Bevilacqua, Bull. soc. ital. biol. sper. 28, 1219 (1947).

52 W.J. Dann and H. IJ. Kohn, J. Biol. Chem. 136, 435 (1940).

53 C, Ling, D. M. Hegsted, and F. J. Stare, J. Biol. Chem. 174, 803 (1948).

54H. I. Kohn, Biochem. J. 32, 2075 (1988).

55 H. I. Kohn and J. R. Klein, J. Biol. Chem. 1380, 1 (1939).

56 A. Bonsignore and C. Ricci, Boll. soc. ital. biol. sper. 24, 171 (1948).

57 M. Duncan and H. P. Sarett, J. Biol. Chem. 193, 317 (1951).

57a P, Feigelson, J. N. Williams, Jr., and C. A. Elvehjem, J. Biol. Chem. 193, 737
(1951).

58 W. Beckh, P. Ellinger, and T. D. Spies, Quart. J. Med. [N.8.] 6, 305 (1937).

59 C. J. Watson, Proc. Soc. Exptl. Biol. Med. 39, 514 (1938); 41, 591 (1939).

80 C¢. J. Watson and J. A. Layne, Ann. Internal Med. 19, 183 (1948).

61 J, A. Layne and C. J. Watson, Ann. Internal Med. 19, 200 (1948).

62S. Schwartz, J. F. Marvin, J. A. Layne, and C. J. Watson, Ann. Internal Med.
19, 206 (1943).

X. EFFECTS OF DEFICIENCY bbye

is a normal constituent of many urines. No relation between urorosein
excretion and nicotinic acid malnutrition could be observed. Furthermore,
results of the B.E.S. test could not be correlated with the amount of
urinary porphyrin, and the excretion of porphyrin had no constant relation
to nicotinic acid deficiency. A red pigment which was observed in urine
from patients with pellagra and from dogs with blacktongue was identified
as a mixture of several related pigments similar to but not identical with
synthetic indirubin. These pigments were found in normal animals as well
as in those with nicotinic acid deficiency. Thus, it seems clear that these
urinary pigments have no special relationship to nicotinic acid deficiency.

b. Urinary Fluorescent Substances

In 1940 Najjar and Wood*® noted a substance in urine which gave a
bluish fluorescence with ultraviolet light, the concentration of which seemed
to be increased when nicotinic acid was administered. Shortly thereafter
Najjar et al.*: © noted that this substance (F2) seemed to be absent in the
urine of patients with pellagra and of dogs with blacktongue. They also
noted another fluorescent substance (F,;) in the urine of patients with
pellagra and of dogs with blacktongue which they did not find in normal
urine. These investigators suggested that measurement of F, and Fy» in
urine would be a useful diagnostic tool in pellagra. Later, Field and asso-
ciates,®* Perlzweig et al.* Raoul,® and Ellinger and Coulson*® noted similar
fluorescent substances in urine. The latter workers found that N!-methyl-
nicotinamide (nicotinamide methochloride) gave a reaction similar to F»2
in urine. In 1943 Huff and Perlzweig”® isolated F2 from urine and identified
it as N!-methylnicotinamide. Considerable controversy developed as to
whether the substance they isolated was identical with F»2.7!-“ In any event,
it is now well established that N!-methylnicotinamide is one of the principal

68 V. A. Najjar and R. W. Wood, Proc. Soc. Exptl. Biol. Med. 44, 386 (1940).

64V. A. Najjar and L. E. Holt, Jr., Sczence 93, 20 (1941).

66V. A. Najjar, H. J. Stein, L. E. Holt, Jr., and C. A. Kabler, J. Clin. Invest. 21,
263 (1942).

66 H. Field, Jr., D. Melnick, W. D. Robinson, and C. F. Wilkinson, Jr., J. Clin.
Invest. 20, 379 (1941).

67 W. A. Perlzweig, H. P. Sarett, and L. H. Margolis, J. Am. Med. Assoc. 118, 28
(1942).

68 Y. Raoul, Bull. soc. chim. biol. 25, 266, 271, 279 (1943).

69 P. Ellinger and R. A. Coulson, Nature 152, 383 (1943).

70 J. W. Huff and W. A. Perlzweig, J. Biol. Chem. 150, 395 (1943).

1 J. W. Huff and W. A. Perlzweig, Science 97, 538 (1943); 100, 28 (1944).

72 J.W. Huff, J. Biol. Chem. 167, 151 (1947).

733V. A. Najjar and V. White, Science 99, 284 (1944); 100, 247 (1944).

mV. A. Najjar, V. White, and D. B. M. Scott, Bull. Johns Hopkins Hosp. 74, 378
(1944).

558 NIACIN

urinary nicotinic acid excretion products and that it reflects the state of
nicotinic acid nourishment to some extent. F; has never been identified
nor has it been studied in recent years.

c. Nicotinic Acid and Its Derivatives in Urine

It is now known that there are a number of forms in which nicotinic
acid is excreted (Fig. 11) and that different species vary markedly as to
which derivatives predominate in their urine (Tables [X and X). In man,
the principal derivatives are N!-methylnicotinamide and the 6-pyridone
of N'-methylnicotinamide. The dog, however, excretes none of the latter

(9) © .O © 20 O

(Oe CF C7 eK
CJ Son i
N N Ne O7 “Ne

|
CH; CH;

Niacin Niacinamide N’-Methyl- 6-Pyridone of
nicotinamide N!-Methyl-
nicotinamide
e10,
(CH. CH.—CH,— CH— COOH Cus
NH NH * NH—CH,—COOH
(ahaa ela Nicotinuric acid
N N
2,5-Dinicotinylornithine
(birds only)

Fig. 11. Derivatives of nicotinic acid found in urine. The symbol (©) refers to
steps which have been proved by means of isotope tracer techniques.

compound. There have been innumerable studies of the urinary excretion
of one or more of these nicotinic acid derivatives in relation to nicotinic
acid deficiency or nicotinic acid intake. Some of the early studies were
complicated, intentionally or unintentionally, by the fact that the assay
methods measured trigonelline as well as nicotinic acid derivatives. It is
now known that trigonelline is not a product of nicotinic acid metabolism
in mammals.

In man, dog, and rat, the most studied species, it seems clear that urinary
N'-methylnicotinamide and the 6-pyridone of this compound (in man) re-
flect the amount of nicotinic acid available to the host. It also seems clear,
especially from the recent carefully controlled studies of Goldsmith and
her associates,!® that the finding of low or even zero amounts of these
nicotinic acid metabolites in urine does not necessarily mean that the
patient is actually suffering from pellagra. This is true, since the excretion
of these substances drops rapidly to a low and constant level soon after

X. EFFECTS OF DEFICIENCY 559

nicotinic acid restriction is instituted and considerably before any symp-
toms of the deficiency become evident. This finding might be anticipated
from animal studies and in view of the fact that the tissues would undoubt-
edly attempt to conserve all available nicotinic acid if the supply were
limited, hence leaving little available for excretion.

It should be mentioned that there are several published reports which
have failed to find any significant correlation between nicotinic acid intake
and excretion of nicotinic acid metabolites and hence are in seeming contra-
diction to the conclusions stated above.’>- It is obvious, however, that if
the dietary circumstances are such that appreciable and undeterminable
amounts of nicotinic acid are synthesized from tryptophan, or possibly by
intestinal microorganisms or from any other source, then it would be almost
impossible to establish any correlation of intake to excretion or to the state
of nicotinic acid nourishment of the tissues. As pointed out by Ellinger
and Coulson” the urinary excretion of nicotinic acid derivatives is influ-
enced by the intake, the need in the body, the amount of methyl donors,
the efficiency of the methylating mechanisms, the amount of biosynthesis,
and other factors.

Consequently, it is not surprising that most studies in which an attempt
has been made to determine nutritional status with respect to nicotinic
acid by determining the amount of urinary nicotinic acid derivatives have
encountered much variability and overlapping of results between normal
and presumably deficient groups.7°-**> Studies in animals, where the dietary
and environmental circumstances can be controlled, have given much more
consistent results.?® 28, 85

d. Load Tests

Attempts have been made to increase the sensitivity and diagnostic use-
fulness of urinary excretion by employing the “load” principle. The theory

75 P, Ellinger and R. A. Coulson, Biochem. J. 38, 265 (1944).

7%6V.A. Najjar, L. E. Holt, Jr.,. G. A. Johns, G. C. Mediary, and G. Fleischmann,
Proc. Soc. Exptl. Biol. Med. 61, 371 (1946).

77 A. P. Briggs, 8S. A. Singal, and V. P. Sydenstricker, J. Nutrition 29, 331 (1945).

78 QO. Mickelsen and L. L. Erickson, Proc. Soc. Exptl. Biol. Med. 58, 33 (1945).

79G. A. Goldsmith, Arch. Internal Med. 78, 410 (1944).

80 G. A. Goldsmith, Proc. Soc. Exptl. Biol. Med. 61, 42 (1942).

81 H.C. Hou and M. Y. Dju, Chinese Med. J. 61, 192 (1942).

82 W. A. Perlzweig, J. M. Ruffin, and D. Cayer, Federation Proc. 4, No. 1 (1945).

83 J. J. Angulo, Rev. med. trop. parasitol. bacteriol. (Habana) 11, 79 (1945).

81 F. Sargent, P. Robinson, R. E. Johnson, and M. Castiglione, J. Clin. Invest.
23, 714 (1944).

84a H. Field, Jr., P. P. Foa, and N. L. Foa, Arch. Biochem. 9, 45 (1946).

8b ©. W. Denko, W. E. Grundy, J. W. Potter, and G. H. Berryman, Arch. Biochem.
10, 33 (1946).

85 P, Ellinger and M. M. Abdel Kader, Biochem. J. 44, 77, 627 (1949).

560 NIACIN

of this test is simple. If an organism is suffering from nicotinic acid defi-
ciency, its tissues are depleted of the vitamin and urinary excretion should
be low. A standard dose of nicotinic acid (usually as the amide) should
then go principally to replete the tissue stores; less should be excreted in
the urine than when the same amount is given to individuals whose tissues
are already normally saturated with the vitamin. This test undoubtedly
has some usefulness, but interpretation of individual tests is often difficult.
It has been useful as a research tool.!9: 75: 77, 79, 80, 82, 86-876 T)oses of 50 to
500 mg. of nicotinamide and urine collection periods of 2 to 24 hours have
been recommended. There seems to be no agreement on any “standard”
technique for doing the test.

5. STOMACH

About 60% of pellagrins have histamine-resistant achlorhydria.!? 8 A
return of normal gastric secretion frequently follows therapy with nico-
tinamide. It is of interest that one of the human subjects in whom Gold-
smith and associates!? induced pellagra developed a histamine-resistant
achlorhydria with return of acid secretion following therapy. Another pa-
tient developed a slight reduction in gastric acidity. It has also been re-
ported that swine on a Goldberger type of blacktongue-producing diet
develop achlorhydria, although nicotinic acid-deficient dogs*® and rats%
show essentially normal gastric secretion. It is also well known that nico-
tinic acid and the amide are capable of stimulating gastric secretion in
normal individuals as well as in patients with pellagra (p. 575). The acid-
secreting (oxyntic) cells of the stomach are known to have a very high
concentration of coenzyme I (DPN) (see Table VII).

These facts have led to considerable speculation that DPN and TPN
may play a vital role in the mechanism by which gastric hydrochloric acid
is secreted. Patterson and Stetten®! have evolved a theory, and supported
it with some experimental evidence, that DPN is an essential component
of a stratified sequence of enzyme systems which transport the hydrogen
necessary for gastric hydrochloric acid production. Hawk and Hundley®®
were unable to obtain supporting evidence in nicotinic acid-deficient rats,
but these experiments certainly do not rule out the possibility of such a
mechanism.

86 H. P. Sarett and G. A. Goldsmith, J. Biol. Chem. 167, 293 (1947); 177, 461 (1949);
182, 679 (1950).

87 W. A. Perlzweig, E. D. Levy, and H. P. Sarett, J. Biol. Chem. 136, 729 (1940).

87a JT). Melnick, W. D. Robinson, and H. Field, Jr., J. Biol. Chem. 136, 145 (1940).

smb P, Ellinger and R. Benesch, Lancet II, 197 (1945).

88 V. P. Sydenstricker and E. S. Armstrong, Arch. Internal Med. 59, 883 (1937).

89 J. A. Layne and J. B. Carey, Gastroenterology 2, 133 (1944).

90 #. A. Hawk and J. M. Hundley, Proc. Soc. Exptl. Biol. Med. 78, 318 (1951).

21 W.B. Patterson and D. Stetten, Jr., Science 109, 256 (1949).

X. EFFECTS OF DEFICIENCY 561

These and other facts have led some observers to speculate that the
stomach may have a special relationship to the etiology of pellagra.”
Sydenstricker and associates® treated six cases of pellagra with gastric
juice from normal individuals and obtained very favorable results. These
investigators proposed that there was an “intrinsic factor’? produced in the
stomach of normal individuals but absent in pellagrins which was essential
in curing pellagra, as well as an “extrinsic factor” present in food which
was also required. Diaz-Rubio and Lorente®™ treated thirty-six cases of
pellagra with gastric juice from normal individuals and failed to obtain
very dramatic results (nine of the thirty-six died). They did note an im-
provement in diarrhea in a few patients and a striking improvement in the
neurological symptoms of many patients. The medical literature has been
singularly free of any reference to an “intrinsic factor” in pellagra since the
discovery that nicotinic acid was the pellagra-preventive vitamin.

6. HEART

Nicotinic acid deficiency is not known to selectively damage the heart,
such as occurs frequently in beriberi. Pathological studies have shown no
gross and only minor microscopic lesions. There is no reason to believe
that the biochemical defects present in this deficiency affect the heart any
more or less than any other organ. Yet, several independent studies of
human pellagra have shown a very high incidence of abnormal electrocardio-
grams. These defects were mainly of the myocardial type rather than
conduction defects. Inverted T; and/or Ty, Pardee-type S-T intervals,
notched or low-voltage ventricular complexes, and other defects were noted.
Most of these abnormalities disappeared promptly following therapy with
nicotinic acid.%°-%

7. BRAIN

Man is apparently unique among mammals in that no other species
develops such a striking and profound mental disturbance in nicotinic acid
deficiency as occurs frequently in man. Deficient animals customarily de-
velop apathy and other mild disturbances which might be referable to the
central nervous system. Pathological lesions have been observed in the
spinal cord of nicotinic acid-deficient dogs (p. 566). The hind limb paralysis
and acute collapse syndrome sometimes seen in dogs on Goldberger-type

9 V. P. Sydenstricker and J. W. Thomas, Southern Med. J. 30, 14 (1937).

9 V. P. Sydenstricker, E. S. Armstrong, C. J. Derrick, and P. 8. Kemp, Am. J.
Med. Sci. 192, 1 (1986).

%M. Diaz-Rubio and L. Lorente, Rev. clin. espan. 16, 72 (1944); Author’s summary
in Rev. asoc. méd. argentina 59, 1178 (1945).

95H. Feil, Am. Heart J. 11, 173 (1936).

96 Ff, Mainzer and M. Krause, Brit. Heart J. 2, 85 (1940).

97 M. Rachmilewitz and K. Braun, Am. Heart J. 27, 203 (1944).

562 NIACIN

blacktongue-producing rations is due to riboflavin deficiency rather than
nicotinic acid. Man, however, frequently develops a marked ‘‘encephalop-
athy’’® with irritability, insomnia, headaches, dizziness, tremor, jerky
movements, and rigidity of the body. Altered tendon reflexes, numbness,
and later paralysis of the extremities may develop. Insanity may occur if
the patients are not treated.!° Alcoholism, with its mental disturbances,
and concomitant thiamine deficiency, with its effect on the nervous system,
frequently complicates the picture.

DPN and TPN are known to be required for the biochemical reactions
by which the brain obtains energy from fuels such as fructose. It is easy to
imagine that a deficiency of these vital coenzymes could impede essential
biochemical reactions and permit the accumulation of metabolites such as
lactic acid which could cause some of these mental symptoms. However,
this is speculation and not experimental fact. Actually, no adequate bio-
chemical explanation can be offered for the occurrence of these striking
mental abnormalities.

8. SKIN

One of the most puzzling lesions in human pellagra is the rather typical
dermatitis and the pigmentation which usually accompanies it. Man, again,
is unique, since these lesions are not seen in deficient animals. It has long
been known that the dermatitis occurs principally on those parts of the
body normally exposed to the sun and that sunlight frequently precipi-
tates the lesions. The suggestion has been made repeatedly that the photo-
sensitive skin of pellagrins may be due to a disturbance in porphyrin
metabolism.®®: °9-10 This has never been proved (p. 556). Alcoholic pel-
lagrins do appear to show an increased amount of coproporphyrin III in
their urine and stools, but this does not seem to be specifically related to
nicotinic acid.5%: 103-105

Again, no adequate biochemical explanation can be offered for these
interesting phenomena.

8 N. Jolliffe, K. M. Bowman, L. A. Rosenblum, and H. D. Fein, J. Am. Med. Assoc.
114, 307 (1940).

99U. Bassi, Clin. med. ital. 65, 241 (1934).

100 P, Ellinger and F. Dojmi, Chemistry & Industry 54, 507 (1935).

101M. Massa, Riforma med. 48, 1669 (1932).

102 T. D. Spies, Y. Sasaki, and E. 8. Gross, Southern Med. J. 81, 483 (1938).

103 K. Dobriner, W. H. Strain, and §. A. Localio, Proc. Soc. Exptl. Biol. Med. 38,
748 (1938).

104 R, Kark and A. P. Meikeljohn, Am. J. Med. Sci. 201, 380 (1941).

105 T,, A. Rosenblum and N. Jolliffe, Am. J. Med. Sci. 199, 853 (1940).

o-*,

X. EFFECTS OF DEFICIENCY 563

9. EYES

Hubbard and Wald! have shown that DPN is necessary for the con-
version of vitamin A, to rhodopsin, a process essential to vision. In this
instance, a ready-made biochemical explanation is available for a defect
which has not been noted in either human or animal nicotinic acid de-
ficiency.

Simonelli!” found that cataractous lens (produced by massage) in rabbits
have a nicotinic acid concentration one-fifth to one-half of normal. The
same was true for cataracts induced by naphthalene. The concentration of
nicotinic acid in the crystalline lens from two humans with cataract seemed
to be slightly reduced. Pike! found that nicotinic acid had an effect in
preventing congenital cataract in rats. The effect seemed to be due to the
“sparing” effect of nicotinic acid on tryptophan. Large amounts of nicotinic
acid were ineffective if dietary tryptophan was too low. Cataract from
tryptophan deficiency is weil known.

10. Bioop

Anemia is an almost constant finding in human pellagra. It is generally
mild and may be macrocytic or normocytic hypochromic in type. The white
cell count is generally normal.’’ There is no certainty that this anemia is
due specifically to nicotinic acid deficiency. It does generally improve fol-
lowing nicotinic acid therapy, but most often other B vitamins, yeast, or
wheat germ as well as a generous diet are used in therapy. This could
provide adequate amounts of other vitamins, such as folic acid, which
may be more directly concerned in the anemia. Complicating deficiencies
of vitamins other than nicotinic acid almost certainly were present in some
of the early experiments showing that dogs and swine on Goldberger diets
developed anemia as well as leucopenia and granulocytopenia.!°?-!" How-
ever, later studies in swine, where complicating deficiencies were minimized,
revealed a normocytic anemia.! Wooley* found a mild anemia, leucopenia,
and granulocytopenia in nicotinic acid-deficient rabbits which responded
to therapy with either nicotinic acid or tryptophan. Rats and dogs do not
usually develop anemia in nicotinic acid deficiency on diets otherwise com-

106 R, Hubbard and G. Wald, Science 115, 60 (1952).

107 M. Simonelli, Boll. soc. ital. biol. sper. 20, 692 (1945).

108 R, L. Pike, J. Nutrition 44, 191 (1951).

109 J). K. Miller and C. P. Rhoads, J. Clin. Invest. 14, 153 (1935).

110 C, P, Rhoads and D. K. Miller, J. Exptl. Med. 58, 585 (1933).

1 TZ). K. Miller and C. P. Rhoads, J. Exptl. Med. 61, 173 (1935).

12 T. Spies and A. Dowling, Am. J. Physiol. 114, 25 (1935).

13 G, E. Cartwright, B. Tatting, and M. M. Wintrobe, Arch. Biochem. 19, 109 (1948).

564 NIACIN

plete in known growth factors." ! However, Handler and associates have
shown that, if the life of their dogs was prolonged beyond the stage of
blacktongue by parenteral saline therapy, then a macrocytic or normo-
cytic anemia and leucopenia developed."®"® These abnormalities would
respond to nicotinic acid even though the food intake was restricted during
therapy. This subject has been very carefully reviewed by Handler.'!® It
should also be noted that nicotinic acid-deficient dogs are much more
susceptible to the hemolytic effect of indole than are normal dogs.”?: ™!
These results seem to indicate that nicotinic acid is necessary for the
formation of red and white blood cells but that this can be demonstrated
only if proper conditions are found. It should be noted that none of Gold-
smith’s experimental pellagra patients developed an anemia which could
be attributed to nicotinic acid deficiency.'®

There seems to be nothing characteristic about other constituents of
blood such as sugar, non-protein nitrogen, and lactate-pyruvate ratios in
human pellagra,.!9 88, 122, 128

11. MiscELLANEOUS
a. Nitrogen Balance

Very few data are available on the subject. The patients of Goldsmith
et al.!® maintained positive nitrogen balance. Mancini and Fabriani* stud-
ied three cases of pellagra and noted a positive balance in each. One patient
treated with nicotinic acid showed an improved nitrogen retention after
therapy. Sure and associates” used a paired-feeding technique to show
that nicotinic acid had a “specific’’ effect in improving food utilization.

b. Pregnancy and Lactation

Lojkin et al.’* found that pregnant women, as well as pregnant rats,
showed a progressive tendency to excrete more urinary nicotinic acid de-

114 W. A. Krehl, P. S. Sarma, L. J. Teply, and C. A. Elvehjem, J. Nutrition 31, 85
(1946).

15 W. A. Krehl and C. A. Elvehjem, J. Biol. Chem. 158, 173 (1945).

116 P, Handler and W. J. Dann, J. Biol. Chem. 145, 145 (1942).

117 P, Handler, Proc. Soc. Exptl. Biol. Med. 52, 263 (1943).

us P, Handler and W. P. Featherston, J. Biol. Chem. 151, 395 (1943).

19 P. Handler, Z. Vitaminforsch. 19, 393 (1948).

1220 C, P. Rhoads, W. H. Barker, and D. K. Miller, J. Exptl. Med. 67,299 (1938).

121 W.H. Sebrell and J. M. Hundley, J’rans. Assoc. Am. Physicians 61, 288 (1948).

122 G. A. Goldsmith, Federation Proc. 6, 408 (1947).

23 M. Diaz-Rubio, E. Monsalvez, and J. M. Masaguer, Rev. clin. espan. 21, 299 (1946).

24 FW. Mancini and G. Fabriani, Boll. soc. ital. biol. sper. 20, 342 (1945).

125 B. Sure, L. Easterling, and 8. Jeu, J. Nutrition 46, 55 (1952).

126 M. E. Lojkin, A. W. Wertz, and C. G. Dietz, J. Nutrition 46, 335 (1952).

ee ee ee

XI. PATHOLOGY 565

rivatives as pregnancy progressed. During the last month of pregnancy
women excreted considerably more of these derivatives in the urine than
they ingested. Coryell and associates!’ studied nicotinic acid intake in
relation to the nicotinic acid content of human milk at various intervals
postpartum. Nicotinic acid in the milk increased from the first to the tenth
day postpartum. During mature milk production, about 7 % of daily nico-
tinic acid intake appeared in the milk and 3% in the urine.

c. Corn and Alcohol in the Etiology of Pellagra

The consumption of maize and the excessive consumption of alcohol are
the two outstanding factors known to show a positive correlation with the
incidence of pellagra. The role of maize in pellagra has been covered very
competently in two recent reviews, one by Handler!!? and one by Chick.’
This subject has also been considered in other sections of this chapter
(p. 548).

Pellagra is known to be frequent in alcoholics. The effect of aleohol seems
to be only that it inhibits consumption of a normal diet and therefore leads
to a deficient intake of nicotinic acid. There is no evidence that alcohol
increases the requirement for nicotinic acid. The few metabolic studies
which have been done indicate that ingestion of alcohol increases the
urinary output of N!-methylnicotinamide, possibly due to a “‘sparing”’
action of alcohol on the nicotinic acid requirement.”*: !?°

XI. Pathology

J. M. HUNDLEY

A. IN ANIMALS
1. Does

Chittenden and Underhill' were the first to produce experimentally the
dietary deficiency in dogs which later became established as the canine
equivalent of pellagra. Wheeler ei al.? called attention to the similarity of
this syndrome to what was known to American veterinarians as black-

127 M. N. Coryell, C. E. Roderuck, M. E. Harris, S. Miller, M. M. Rutledge, H. H.
Williams, and I. G. Macy, J. Nutrition 34, 219 (1947).
28 H. Chick, Nutrition Abstr. and Revs. 20, 523 (1951).
29 R. E. Butler and H. P. Sarett, J. Nutrition 35, 539 (1948).
1R.H. Chittenden and F. P. Underhill, Am. J. Physiol 44, 13 (1917).
2G. A. Wheeler, J. Goldberger, and V. Blackstock, Public Health Repts. (U.S.) 37,
1063 (1922).

566 NIACIN

tongue. Goldberger et al.* studied a case of naturally occurring blacktongue
and later described a similar disease in dogs which was produced experi-
mentally. Dogs suffering from this deficiency show very characteristic
findings. The first abnormality is, usually, refusal to eat. The animals are
quiet and apathetic. After a day or two the inner surfaces of the lips,
cheeks, and gums become diffusely inflamed and develop superficial yellow-
ish necrotic patches. There is an intensely foul odor. Excessive drooling of
thick ropy saliva is characteristic. The tip and margins of the tongue be-
come red and later develop dark bluish patches. Ulceration frequently
appears on or under the tongue. A diarrhea, generally bloody, is present.
Scrotal dermatitis may be found. Vomiting usually develops. Body tem-
perature may reach 40 or 41°. At autopsy, the tongue is found generally
to have assumed a deep bluish-black color. The inflammatory changes in
the mouth may extend to affect the epiglottis, the soft palate, and even
into the esophagus. The stomach may show small areas of congestion,
although during life it is apparently normal to endoscopic examination.*®
Small and large intestine, and particularly the rectum usually show patches
of intense congestion, less commonly small hemorrhages. Duodenal or
colonic ulcers may be found.

Denton® studied the pathological changes in Goldberger’s dogs. He found
degenerative lesions in the mucous membranes of the mouth, pharynx,
esophagus, intestines, and epithelium of the scrotum. The lesions seemed
to originate in the supporting tissues of the membranes and terminated in
extensive necrotic and diphtheritic inflammation. Lillie? made a detailed
histologic study of the tissues of blacktongue dogs. In addition to pre-
viously described findings, he noted degenerative changes in the nerves
regional to the oral lesions, granular degeneration of heart muscle fibers,
moderate passive congestion, and relatively little fatty infiltration of the
liver. The spleen was fibrotic and contained atrophic follicles. Albuminous
degeneration of the epithelium of the convoluted tubules of the kidney,
injection of the meninges, tigrolysis of the brain stem ganglia, nerve cell
atrophy, and pericellular vacuolation in the cortex and basal ganglia were
found. Jensenius and Norgaard’ also found extensive histological changes
in the central nervous system, particularly the spinal cord.

The fatty livers (yellow liver) sometimes seen in dogs on blacktongue-

3 J. Goldberger, W. F. Tanner, and E. B. Saye, Public Health Repts. (U. S.) 38,
2711 (1923).

4 J. Goldberger and G. A. Wheeler, Public Health Repts. (U.S.) 48, 172 (1928).

6 J. A. Layne and J. B. Carey, Gastrventerology 2, 133 (1944).

6 J. Denton, Am. J. Pathol. 4, 341 (1928).

7™R. D. Lillie, Natl. Insts. Health Bull. 162, 13 (1933).

8 H. Jensenius and F. Norgaard, Acta Pathol. Microbiol. Scand. 19, 433 (1942).

XI. PATHOLOGY 567

producing diets’: !° were undoubtedly due to concomitant riboflavin defi-
ciency as shown by Sebrell and associates.!! Smith ef al.” were apparently
the first to note the large number of fuso-spirochetal organisms in the
mouths of blacktongue dogs. The almost universal occurrence of this
Vincent’s-type infection is one of the most puzzling features of canine
blacktongue.

Nicotinic acid deficiency may occur in dogs without blacktongue, espe-
cially in puppies and when purified diets are used." Handler and Dann"™
found that dogs could be cured of blacktongue by treatment with intra-
venous saline only to continue to lose weight and develop a severe anemia
and leucopenia (p. 564). The bone marrow of such dogs has not, apparently,
been studied.

2. Hogs

Miller and Rhoads" fed swine a modified Goldberger diet and observed
a syndrome similar but not identical to blacktongue in dogs, i.e., stomatitis,
achlorhydria, anemia, diarrhea, loss of appetite, and weakness. Both macro-
cytic and microcytic anemias were observed. These deficiency symptoms
could be relieved with oral or parenteral liver extract. Several other in-
vestigators gave similar diets and observed the beneficial effect of nicotinic
acid on the deficiency state.1®?° Braude et al.2! used Chick’s diet!® and made
a study of the gross pathology of pig pellagra. In their experiments, the
pigs had a rough, “staring” coat but showed no actual dermatitis. Several
pigs had small ulcerations of the buccal mucosa and most had a severe
enteritis with necrotic ulceration of the cecum and large bowel. Fibrinous
pleurisy, mild anemia, and, frequently, pneumonia were found in animals
which died of the deficiency.

Unfortunately, most of the experiments referred to above were undoubt-
edly complicated by deficiencies of nutrients other than nicotinic acid.

9W.H. Sebrell, Natl. Insts. Health Bull. 162, 23 (1933).

10 W. H. Sebrell and R. D. Lillie, Natl. Insts. Health Bull. 162, 37 (1933).

1W. H. Sebrell, R. H. Onstott, and D. J. Hunt, Public Health Repts. (U.S.) 52,
427 (1937).

12212). T. Smith, E. L. Persons, and H. I. Harvey, J. Nutrition 14, 373 (1937).

13 P. Handler, Z. Vitaminforsch, 19, 393 (1948).

144 P, Handler and W. J. Dann, J. Biol. Chem. 145, 145 (1942).

15 J). K. Miller and C. P. Rhoads, J. Clin. Invest. 14, 153 (1935).

16H. Chick, T. F. Macrae, A. J. P. Martin, and C. J. Martin, Biochem. J. 32, 10, 844
(1938).

47}. H. Hughes, Hilgardia 11, 595 (1938).

18 T,. C. Madison, R. C. Miller, and T. B. Keith, Science 89, 490 (1939).

19M. M. Wintrobe, Am. J. Physiol. 126, 375 (1939).

20 A. N. Worden and G. Slavin, J. Comp. Pathol. Therap. 54, 77 (1944).

21 R. Braude, 8S. K. Kon, and E. G. White, Biochem. J. 40, 843 (1947).

568 NIACIN

However, more recent studies using purified diets more adequately supplied
with vitamins have shown findings generally similar to the observations
already reported.??-?°

3. Rats

Most investigators have observed little pathology in nicotinic acid-
deficient rats except retarded growth and non-specific deficiency signs such
as rough hair coat, porphyrin-caked whiskers and, occasionally, alope-
cia.2 27 Animals allowed to eat a nicotinic acid-deficient ration ad lib
voluntarily restrict their food intake and may thereby protect themselves
to some extent. Spector and associates”: 7° force-fed a diet severely deficient
in both nicotinic acid and tryptophan to rats and observed striking pathol-
ogy. Alopecia, bloating, diarrhea, hunchback, jumping, screeching, convul-
sions, mild anemia, kidney enlargement, degeneration of the testes, fatty
infiltration of the liver, atrophy and degeneration of visceral and cardiac
muscle, and keratinization of the cornea were observed. Most of these
abnormalities were undoubtedly due primarily to tryptophan deficiency
rather than nicotinic acid. The addition of nicotinic acid in the absence of
added tryptophan was without any substantial effect on the deficiency
syndrome. Nicotinic acid can protect to some extent against congenital
cataract in rats when the diet contains marginal amounts of tryptophan
as shown by Pike.*® The stomach of deficient rats is normal to gross in-
spection, and acid-pepsin secretion is normal.*! However, Bourne” found
that the gastric mucosa was reduced in thickness, mainly at the expense of
the oxyntic cells. Only slight degenerative changes were noted in the rest
of the gastrointestinal tract. Johnston and Weitz,** on the other hand,
found marked degenerative changes in the Golgi apparatus in the columnar
absorbing cells of the duodenum in nicotinic acid-deficient rats. Bourne”
also noted loss of staining ability and decrease in number of acidophile cells

2 G. E. Cartwright, B. Tatting, and M. M. Wintrobe, Arch. Biochem. 19, 109 (1948).

23 M.M. Wintrobe, H. J. Stein, R. H. Follis, Jr., and S. Humphreys, J. Nutrition 30,
395 (1945).

24.W.C. Powick, N. R. Ellis, L. L. Madsen, and C. N. Dale, J. Animal Sez. 6, 310
(1947).

25 W. Burroughs, B. H. Edgington, W. L. Robinson, and R. M. Bethke, J. Nutrition
41, 51 (1950).

26 W. A. Krehl, P. S. Sarma, L. J. Teply, and C. A. Elvehjem, J. Nutrition 31, 85
(1946).

27, W. A. Krehl, L. J. Teply, and C. A. Elvehjem, Science 101, 283 (1945).

28 H. Spector, J. Biol. Chem. 178, 659 (1948).

29 H. Spector and F. B. Adamstone, J. Nutrition 40, 213 (1950).

30 R. L. Pike, J. Nutrition 44, 191 (1951).

31. A. Hawk and J. M. Hundley, Proc. Soc. Expil. Biol. Med. 78, 318 (1951).

32 G. H. Bourne, Brit. J. Nutrition 4, xvi (1950).

33 P.M. Johnston and E. M. Weitz, J. Morphol. 91, 79: (1952).

XI. PATHOLOGY 569

in the anterior pituitary, cessation of bone formation at the costochondral
junctions, cessation of spermatogenesis, and a reduction in size of the
adrenal cortex. The spleen was reduced in size and showed fibrosis and
reduction of the red pulp. The epithelium of the trachea showed degenera-
tion. The tubular epithelium of the kidneys was severely degenerated.
Bourne did not report the use of pair fed controls to rule out the nonspecific
effects of simple inanition in the etiology of these lesions.

4. Corton Rats AND MIcE

Only poor growth has been reported as a symptom of nicotinic acid defi-
ciency in the cotton rat.** *° As reported by Woolley,** mice given 3-acetyl-
pyridine, a “nicotinic acid antagonist’? (p. 548), developed difficulty in
controlling their hind legs and finally became almost completely paralyzed.
The hair coat became wet and unkempt. Weight loss and emaciation devel-
oped. The skin of the chest wall, the sides of the abdomen, and the legs
became very red and inflamed. Fiery red tongues developed late in about
half of the animals.

5. RABBITS

Subnormal weight gain, anemia, leucopenia, and a terminal diarrhea,
sometimes bloody, were observed in nicotinic acid-deficient rabbits by
Wooley and Sebrell.*”: *8 No oral lesions were found. Histological examina-
tion showed nothing that could be attributed to nicotinic acid deficiency
aside from a few animals showing superficial ulcerations in the colon.
Olcese et al.*® observed only subnormal weight gain in their nicotinic acid-
deficient rabbits.

6. Cuicks, TuRKbYS, AND Ducks

Growing chicks on nicotinic acid-deficient rations showed diminished
growth, increased mortality, and inflammation of the entire mouth cavity
as well as the esophagus and the crop (blacktongue) except for the tip of
the tongue, which remained white. Excessive mucus may be found in the
mouth. Diarrhea, perosis, and poor feathering have been described.*°-* The

34 J. M. McIntire, B.S. Schweigert, and C. A. Elvehjem, J. Nutrition 27, 1 (1944).

35 B.S. Schweigert and P. B. Pearson, J. Biol. Chem. 172, 485 (1948).

3D). W. Woolley, J. Biol. Chem. 157, 455 (1945).

37, J. G. Wooley, Proc. Soc. Exptl. Biol. Med. 65, 315 (1947).

3 J. G. Wooley and W. H. Sebrell, J. Nutrition 19, 191 (1945).

39 Q. Olcese, P. B. Pearson, and P. Sparks, J. Nutrition 39, 93 (1949).

40 G. M. Briggs, Jr., R. C. Mills, C. A. Elvehjem, and E. B. Hart, Proc. Soc. Exptl.
Biol. Med. 51, 59 (1942).

1G. M. Briggs, A. C. Groschke, and R. J. Lillie, J. Nutrition 32, 659 (1946).

4 7,. R. Richardson, A. G. Hogan, and H. L. Kempster, Missouri Agr. Expt. Sta.
Research Bull. 390, (1945).

49H. M. Scott, E. P. Singsen, and L. D. Matterson, Poultry Sci. (Research Notes)
25, 303 (1946).

570 NIACIN

rather marked dermatitis noted in some of the early work of Briggs and
associates“! has not been found in more recent studies where the diets were
more adequately supplemented with vitamins other than nicotinic acid.
Nicotinic acid-deficient turkey poults exhibited similar findings.*°-“7

Nicotinic acid-deficient ducks show diminished weight gain, weakness,
and diarrhea. In some of the birds there was an accumulation of food
under the tongue and necrotic tissue beneath this.*8

7. OTHER SPECIES

Cats have been reported to develop a disease in nature which resembles
canine blacktongue and which responds to nicotinic acid.*® Nicotinic acid-
deficient foxes showed cessation of weight gain and anorexia, and, if the
deficiency was severe, they developed symptoms similar to blacktongue.*°
Rainbow trout developed swollen gills when deprived of nicotinic acid.*

B. IN MAN

Sundwall® and Harris® have reviewed the European and early American
literature up to about 1915. These authors and, more recently, Denton,**
Moore et al.,®° and Follis®* have published their observations of the pathol-
ogy of pellagra in the United States.

The usual pathological features of pellagra in this country include
changes in the skin, oral cavity, esophagus, colon, nervous system, and
blood. Skin from clinically involved areas shows dilatation of blood vessels,
some rarefaction of tissue, hyperkeratosis, parakeratosis, acanthosis, and,
in severe cases, bullae. The sebaceous glands may be atrophic, although
the sweat glands appear normal. Abnormalities of keratinization may be
found in clinically normal skin. The mucous membrane of the mouth,
tongue, esophagus, vagina, and colon show somewhat similar changes with

44G. M. Briggs, Jr., T. D. Luckey, L. J. Teply, C. A. Elvehjem, and E. B. Hart,
J. Biol. Chem. 148, 517 (1948).

45 G.M. Briggs, J. Nutrition 31, 79 (1946).

46TH. Jukes, E. L. R. Stokstad, and M. Belt, J. Nutrition 38, 1 (1947).

47B. G. Lance and A. G. Hogan, J. Nutrition 36, 369 (1948).

48D. M. Hegsted, J. Nutrition 32, 467 (1946).

49M. K. Heath, J. W. MacQueen, and T. D. Spies, Science 92, 514 (1940).

50 A. E. Schaefer, C. K. Whitehair, and C. A. Elvehjem, J. Nutrition 34, 131 (1947).

51 B, A. McLaren, E. B. Keller, D. J. O’Donnell, and C. A. Elvehjem, Arch. Bio-
chem. 15, 169 (1947).

8 J. Sundwall, U. S. Public Health Service Hyg. Lab. Bull. 106, 5-74 (1917).

53H. F. Harris, Pellagra, pp. 188-180. The Macmillan Co., New York, 1919.

54 J. Denton, Am. J. Trop. Med. 5, 173 (1925).

55 R. A. Moore, T. D. Spies, and Z. K. Cooper, Arch. Dermatol and Syphilol. 46, 100
(1942).

66 R.H. Follis, Jr., The Pathology of Nutritional Disease, p. 169. Charles C Thomas,
Springfield, Ill., 1948.

XII. PHARMACOLOGY 571

considerable atrophy of the epithelium. The buccal mucous membrane may
disappear entirely in some areas with grayish ulcerations which are loaded
with microorganisms. Cysts filled with mucus and inflammatory material
and, in advanced cases, ulcers are found in the colon. Chromatolysis of
ganglion cells in the brain and, in some cases, myelin degeneration of
peripheral nerves can be observed. Peripheral blood may show either
microcytic or macrocytic anemia. Bone marrow appears to have been
studied but little.

The specificity of these pathological changes in relation to possible con-
comitant deficiencies of other essential nutrients has been discussed else-
where (p. 553). The “pellagra” of Africans, as characterized by the Gill-
mans,” almost certainly is complicated by other deficiencies, since it does
not respond to nicotinic acid but does respond to certain materials which
contain high-quality protein. The extensive pathology of this severe nutri-
tional disease has been described thoroughly by the Gillmans.”

XII. Pharmacology
J. M. HUNDLEY
AX LOX CRE Y,

Both nicotinamide and nicotinic acid are quite non-toxic, there being a
ratio of at least 1:1000 between the effective therapeutic dose and the
toxic dose.

1. Rats anp Mice

The LD,’s of nicotinic acid (sodium salt when given parenterally) and
nicotinamide range from 3.5 to 5 g. per kilogram subcutaneously and 5 to
7 g. orally. Nicotinamide is almost twice as toxic as nicotinic acid.!* The
methyl ester of nicotinic acid injected into mice was tolerated at a level of
1.0 g. per kilogram, whereas the methoiodide of this compound was fatal
at 0.6 g. per kilogram. The methyl ester of N'-methyltetrahydronicotinic
acid (arecoline) was fatal at a dosage of 0.065 g. per kilogram.* N'-Methyla-
tion of nicotinic acid and related compounds does not result in a consistent

97 J. Gillman and T. Gillman, Perspectives in Human Malnutrition. Grune &
Stratton, New York, 1951.

1K. K. Chen, C. L. Rose, and E. B. Robbins, Proc. Soc. Exptl. Biol. Med. 38, 241
(1938).

2K. Unna, J. Pharmacol. Exptl. Therap. 65, 95 (1939).

3F. G. Brazda and R. A. Coulson, Proc. Soc. Exptl. Biol. Med. 62, 19 (1946).

4R. Hunt and R. R. Renshaw, J. Pharmacol. Exptl. Therap. 35, 75 (1929).

ie NIACIN

effect on toxicity. Brazda and Coulson’ found the following LDs9’s in rats
given the following compounds subcutaneously (grams per kilogram) : nico-
tinic acid 5.0, trigonelline 5.0, nicotinamide 1.68, N'-methylnicotinamide
2.4, N,N-diethylnicotinamide (coramine) 0.24, coramine methochloride
1.90, pyridine 1.00, and pyridine methochloride 0.28. Nicotinamide, cora-
mine, and their N'-methyl derivatives caused paralysis of the respiratory
centers. Pyridine and its methyl derivative caused deep anesthesia. Karasek
and coworkers® reported considerably lower LDso’s for nicotinic acid in
mice (1.8 g. per kilogram) than those recorded above. They also found that
as little as 0.4 g. per kilogram caused preglomerular, corticomedullary, and
interstitial hemorrhages in rats. Unna,? on the other hand, fed 1.0 g. per
kilogram of sodium nicotinate daily for 40 days to rats and observed no
effect on growth or on tissue histology. Chen and associates! found no
abnormalities in mice injected with 0.5 g. per kilogram daily for four weeks.
McCrea‘ gave 0.05 to 0.2 g. of nicotinic acid to rats intraperitoneally for
24 days with no ill effects. Unna? suggested that the toxic effect of nico-
tinic acid and its amide may be due, at least in part, to simple osmotic
effects, since the same volumes of a solution of sodium chloride equimolec-
ular to the sodium nicotinate solutions caused some deaths in their animals.

2. Docs

Dogs tolerated up to 2 g. of sodium nicotinate per kilogram of body
weight orally for 35 to 63 days without toxicity.? Chen and associates! gave
dogs 1 g. of nicotinic acid per day orally for eight weeks with no toxic
symptoms, although 2 g. per dog per day produced marked toxic symptoms
in 11 to 20 days.

3. OTHER SPECIES

Chickens tolerated sodium nicotinate up to 2 g. per kilogram of body
weight orally each day for periods as long as two months.? The minimum
lethal dose of sodium nicotinate by injection into guinea pigs was 3.5 g.
per kilogram. Rabbits tolerated 0.02 to 0.06 g. per kilogram per day for
ten days.® As much as 5.4 g. of nicotinic acid per day has been given orally
to man with no harmful effects.? Hawker® reported a 20% increase in
ovary weight and a decrease in pituitary gland weight of virgin guinea
pigs receiving nicotinic acid in amounts up to 150 mg. daily. This report
must be accepted with reservations because of the very small number of
animals used.

5 F. Karasek, O. Poupa, and V. Jelinek, Minerva med. 39, 28 (1948) [C. A. 48, 6291
(1949)].

6 F. D. McCrea, J. Pharmacol. Exptl. Therap. 63, 25 (1938).

7C.M. Kurtz, O. S. Orth, and G. Sepulveda, Wisconsin Med. J. 44, 761 (1945).

8 R. W. Hawker, Med. J. Australia I, 872 (1946).

XII. PHARMACOLOGY 573

B. TOXICITY IN DIETS

Handler and Dann? found that inclusion of 1% nicotinamide in a low
protein diet almost completely inhibited growth in rats. This growth in-
hibition could be prevented by methionine or by choline plus homocystine
but not by choline, betaine, homocystine, or cystine alone. One per cent
nicotinic acid in the same diet did not inhibit growth but did induce fatty
liver, an effect preventable by methionine, choline, and betaine but aggra-
vated by cystine or homocystine.’ '° Randoin and Causeret"™ confirmed
the dietary toxicity of 0.5 to 2.0% nicotinamide but failed to obtain re-
versal with methionine.” They did obtain reversal of toxicity with liver
powder or folic acid plus calcium pantothenate. One or two per cent
nicotinamide in the diet of rabbits and guinea pigs did not depress growth
and did not cause fatty liver. It is of interest that the latter two species
do not methylate nicotinamide to any extent, whereas the rat does. Two
per cent, nicotinamide reduced the growth of chicks but did not produce
fatty liver; neither glycine nor choline restored growth. Two per cent
nicotinic acid in this diet had no effect on chick growth."

Coulson and Brazda!® fed rats a series of pyridine derivatives, including
the N!-methyl] derivatives of each, at a level of 1% in a high protein diet.
None of the N!-methy] derivatives affected liver fat. N'-Methylation had
no consistent effect when the growth depression obtained with methylated
compounds was compared with that obtained with non-methylated com-
pounds. y-Picoline, nicotinic acid, and nicotinamide increased liver fat.
B-Picoline and coramine produced larger increases in liver fat, but this
could be prevented by methionine or choline. Coramine produced a large
increase in fat-free liver weight which could not be prevented by choline
or methionine. These results suggest that the toxicity of pyridine deriva-
tives may be only partially explained by the fact that the tissues are robbed
of the methyl groups necessary for their methylation and excretion.

C. CIRCULATION

Nicotinic acid, 20 to 30 mg. given intravenously or 100 to 500 mg. orally,
produces a transient vasodilatation of the cutaneous vessels, particularly
of the ears, face, neck, upper extremities, and trunk in most human sub-
jects. The reaction is accompanied by itching, burning, and tingling of the

°P. Handler and W. J. Dann, J. Biol. Chem. 146, 357 (1942).
10 A. Aschkenasy and J. Mignot, Compt. rend. soc. biol. 140, 261 (1946).
17. Randoin and J. Causeret, Compt. rend. 227, 367 (1948).
27. Randoin and J. Causeret, Compt. rend. 228, 504 (1949) [C. A. 48, 6296 (1949) ].
13 T,. Randoin and J. Causeret, Compt. rend. 227, 399 (1948).
4 P. Handler, J. Biol. Chem. 154, 203 (1944).
15 R. A. Coulson and F. G. Brazda, Proc. Soc. Exptl. Biol. Med. 65, 1 (1947).

574 NIACIN

affected areas.!*° The skin temperature may'® or may not” be elevated. In
spite of this reaction, blood pressure, pulse, and body temperature are
little affected. There is an increased blood flow in the hand and the forearm
but little in the leg.” The increase in blood flow is largely in the skin and
subcutaneous tissue, there, apparently, being a compensatory decrease in
the muscles and the viscera!’ ?° so that there is little change in blood
pressure and perhaps only a slight increase in circulating blood volume.*!
Some dilatation of the arteries of the retina may occur,!® but there is no
increase in cerebral blood flow, according to Scheinberg.” The latter is
somewhat surprising, since flushing doses of nicotinic acid are reported to
have a beneficial effect in certain types of headaches.”: *4 Nicotinamide
and many other related compounds do not cause this flushing reaction,”®
although a few derivatives such as methyl nicotinate, other alkyl nico-
tinates, and the alcohol of nicotinamide (Roniacol) do.**: ” Prior dosage
with 30 to 60 g. of glycine is said to prevent or retard the flushing action
of nicotinic acid in man.!* 2° Coramine (N , N-diethylnicotinamide) has a
marked stimulant action on the central nervous system. Other nicotinic
acid derivatives such as tetrahydrofurfuryl nicotinate*® have a direct hy-
peremic action on the skin. The reported ability of nicotinic acid or nico-
tinamide to increase the resistance of rats to anoxia may be related to some
of these circulatory effects.?°

Although nicotinic acid itself is quite inactive in lowering blood pressure,
its methyl ester, the methyl ester of N'-methylnicotinamide, and the methyl
ester of N'-methyltetrahydronicotinic acid (arecoline) are, in the order
given, quite active in that respect.*

D. BLOOD SUGAR

The extensive and contradictory literature on this subject has been
reviewed by Diaz-Rubio and associates.*° Various investigators seem about

16'T. D. Spies, W. B. Bean, and R. E. Stone, J. Am. Med. Assoc. 111, 584 (1938).

“PD. J. Abramson, H. H. Katzenstein, and F. A. Senior, Am. J. Med. Scz. 200, 96
(1940).

18 R. A. Murphy, Jr., J. N. McClure, Jr., F. W. Cooper, Jr., and L. D. Crowley,
Surgery 27, 655 (1950).

198. Artunkal, Bull. fac. méd. Instanbul I, 4024 (1944).

20 M. Salvini, Boll. soc. ital. biol. sper. 23, 907 (1947).

"1M. Salvini and C. Dal Palu, Boll. soc. ital. biol. sper. 24, 986 (1948).

22 P. Scheinberg, Circulation 1, 1148 (1950).

23 M. Atkinson, Ann. Internal Med. 21, 990 (1944).

24 J. W. Goldzieher and G. L. Popkin, J. Am. Med. Assoc. 131, 103 (1946).

25 W. B. Bean and T. D. Spies, J. Am. Med. Assoc. 114, 439 (1940).

26 L,. Chevillard, R. Charonnat, and H. Giono, Compt. rend. soc. biol. 144, 194 (1950).

27 W. Huber, U.S. Pat. 2,431,558 (November 25, 1947).

*8 CIBA Ltd. Swiss Pat. 258,714 (June 1, 1949).

29 R. M. Calder, Proc. Soc. Exptl. Biol. Med. 68, 642 (1948).

30 M. Diaz-Rubio, E. Monsalvez, and J. M. Masaguer, Rev. clin. espaii. 21, 299 (1946).

XII. PHARMACOLOGY 575

equally divided among those who find a hyperglycemia, those who find no
effect, and those who find a hypoglycemia after intravenous nicotinic acid.
More recent reports concerning both animals and man have not clarified
this subject, although most reports indicate either no effect or a slight
hypoglycemic action.*!-*” Poppalardo* found a marked hypoglycemic action
when nicotinic acid or nicotinamide was given by subarachnoid injection
in man. Insulin injected into the developing egg embryo induced develop-
mental defects. Nicotinamide prevented this effect, although a-ketoglutaric
acid also had a similar action.*?

EK. KETOSIS

Janes and Meyers’? discovered that rather large amounts of nicotinic
acid would induce ketosis in alloxan diabetic rats. The alcohol of nicotinic
acid (Roniacol) was more active, and nicotinamide less active, than nico-
tinic acid in this action.*! Banerjee” found that nicotinic acid, and several
other compounds having a pyridine nucleus and a carboxyl group, would
inhibit the diabetogenic action of alloxan in rabbits. However, there is
nothing specific about the action of nicotinic acid in either the induction
or the progress of alloxan diabetes. Janes and Brady* found that excessive
doses of nicotinic acid induced ketosis even in normal rats. Banerjee and
Ghosh* could detect no effect of large doses of nicotinamide on blood
acetone bodies in either normal or diabetic subjects.

F. GASTRIC SECRETION

Both nicotinic acid and nicotinamide have the ability to increase gastric
secretion, including free and total acid, in normal individuals“-4* and in

31 FF. K. Permyakov, Klin. Med. (U.S.S.R.) 24, 65 (1946).

2 C. Pelosio, Fisiol. e med. (Rome) 15, 481 (1947).

33§. Eser, Bull. fac. méd. Instanbul 11, 82 (1948).

34. Banerjee, N. C. Ghosh, and G. Bhattacharya, Indian J. Med. Research 36, 341
(1948).

35 G. Benedetti, B. Tortori-Donati, and F. Ferri, Rass. fisiopatol. clin. e terap. (Pisa)
20, 233 (1948).

36 H. M. Alesker, Klin. Med. (U.S.S.R.) 27, 70 (1949).

37§. Banerjee and N. C. Ghosh, J. Biol. Chem. 177, 789 (1949).

38 P. Poppalardo, Acta. Neurol. (Naples) 1, 102 (1946).

39 W. Landauer, J. Exptl. Zool. 109, 283 (1948).

40 R. G. Janes and L. Meyers, Proc. Soc. Exptl. Biol. Med. 63, 410 (1946).

41R.G. Janes, Proc. Soc. Exptl. Biol. Med. 75, 246 (1950).

42S. Banerjee, Sczence 106, 128 (1947).

48R.G. Janes and J. Brady, Am. J. Physiol. 159, 547 (1949).

44C, Malaguzzi-Valeri and P. Paterno, Boll. soc. ital. biol. sper. 14, 377 (1939); Gazz.
ospedali e clin. 60, 925 (1939).

45 G. Gabor, Orvosi Hetilap 85, 629 (1941).

467.8. Snitzer, Klin. Med. (U.S.S.R.) 27, 77 (1949).

576 NIACIN

pellagrins,!* 47-®° but usually not in achylic individuals.“ Other investi-
gators®! confirmed this action of nicotinic acid and nicotinamide in normal
individuals but could find little or no increase in patients with pellagra.

G. SERUM BILIRUBIN

Intravenous sodium nicotinate (30 to 300 mg.) induces a rather striking
rise in indirect reading serum bilirubin in both normals and those with
hepatic disease.*?->> The increase is generally higher and more prolonged in
patients with hepatic diseases than in normal subjects. A copious excretion
of bile and increased urinary urobilin were also noted.**: °* Nicotinamide
given intravenously did not elevate serum bilirubin but did increase bile
flow to some extent.*®

H. ANTIALLERGIC EFFECTS

There are quite a number of reports in recent European medical litera-
ture attributing antihistaminic and smooth muscle pharmacological effects
to nicotinic acid and nicotinamide. These reports constitute an interesting
facet of the pharmacology of nicotinic acid which has been largely over-
looked in the American literature except for the flushing (smooth muscle?)
action of nicotinic acid.

In 1941 Sceaffidi and Molino®™ noted that intravenous sodium nicotinate
caused a transient increase in tone and amplitude of the rhythmic contrac-
tions of rabbit uteri. In 1944 Halpern and Dainow® discovered that nico-
tinamide would protect against anaphylactic shock and histamine aerosol
asthma but not against bronchospasm induced by acetylcholine in guinea
pigs. These investigators also noted an antagonism between nicotinamide
and histamine on the isolated intestine and uteri of guinea pigs. Nicotina-
mide diminished the stimulant action of acetylcholine and barium chloride

47G. A. Goldsmith, H. P. Sarett, U. D. Register, and J. Gibbens, J. Clin. Invest. 31,
533 (1952).

48V. Rao, Riv. patol. sper. 26, 49 (1941).

49 G. Gotgiu and L. Romano, Ott. soc. med. chir. Padova 7, 3 (1939), quoted in ref. 44.

50 G. A. Goldsmith, H. P. Sarett, U. D. Register, and J. Gibbens, Federation Proc.
10, 383 (1951).

51 —D). Zamfir, C. C. Dimitriu, P. Ionescu-Stoian, and N. Neculescu, Rev. sttirnt. med.
31, 420 (1942), abstract in Ber. ges. Physiol. 181, 585 (1943).

52 C, Mattei, Minerva med. 37, 308 (1946).

53 1,, Marfori, M. Stefanini, and P. Bramante, Jl Policlinico (Rome) Sez. med. 58,
243 (1946); Am. J. Med. Sci. 218, 150 (1947).

54M. Stefanini, J. Lab. Clin. Med. 34, 1039 (1949).

55 M. Salvini and F.§. Feruglio, Boll. soc. ital. biol. sper. 24, 979 (1948).

56M. Stefanini, Am. J. Digestive Diseases 17, 337 (1950).

57.V. Scaffidi and N. Molino, Boll. soc. ital. biol. sper. 16, 284 (1941).

58 B. N. Halpern and I. Dainow, Z. Vitaminforsch. 15, 217, 250, 260 (1944).

-

XII. PHARMACOLOGY 577

on the isolated intestine but seemed without effect on uterine contractions
induced by posterior pituitary extracts. Alechinsky®? found that 100 mg. of
nicotinamide would protect guinea pigs against one MLD of histamine.
Frommel and associates®® tested a number of nicotinamide derivatives
against histamine aerosol in guinea pigs. None of the compounds had
greater efficacy than nicotinamide itself, although others have found it less
effective than sodium nicotinate.*! Nicotinamide has also been reported to
modify the systemic effects of histamine and to decrease the local effect
of histamine on skin.”

Nicotinic acid and nicotinamide increased the force of contraction of the
isolated intestine of rabbits.* A number of esters of nicotinic acid have
been studied for their spasmolytic potency on smooth, cardiac and striped
muscle.** Both nicotinic acid and nicotinamide have been found to antago-
nize curare and to reinforce the action of acetylcholine on the gastrocnemius
and soleus muscles of anesthetized cats. Nicotinic acid antagonized whereas
nicotinamide reinforced the action of prostigmine in this type of prepara-
tion.®° Nicotinic acid has been reported to shorten the period of spasms in
mice given strychnine and to give some protection against metrazole shock
in rats, rabbits, and guinea pigs®® and in dogs.”

I. OTHER EFFECTS

There are a number of reports dealing with unexpected effects of nico-
tinic acid administration in man and animals which have not been studied
extensively enough for their significance to be determined. These are reduc-
tion in electrical excitability of the cerebral cortex in dogs®’; increased
absorption and increased blood levels of iron®®: 7°; prevention and treatment
of bromidism in man and dogs;7! changes in blood electrolytes;7?’ 7 counter-

69 A. Alechinsky, Compt. rend. soc. biol. 141, 524 (1947).

60 F. Frommel, A. Bischler, I. T. Beck, F. Vallette, and M. Favre, Intern. Z. Vitamin-
forsch. 19, 193 (1947).

31H. Ginoulhiac and F. Semenza, Acta Vitaminol. 3, 20 (1949).

2 U. Butturini, Giorn. clin. med. (Parma) 27, 681 (1946).

3R. Savini, Boll. soc. ital. biol. sper. 24, 819 (1948).

§4R, Charonnat, L. Chevillard, H. Giono, M. Harispe, and J. V. Harispe, Ann.
pharm. frang. 6, 489, 490 (1948).

65 F, Valenzuela and F. Huidobro, J. Pharmacol. Exptl. Therap. 92, 1 (1948).

66 N. V. Sapezhinskaya, Farmakol. i Toksikol. 10, 11 (1947) [C. A. 42, 3081 (1948)].

67 Q. Calabro and R. Savini, Boll. soc. ital. biol. sper. 24, 823 (1948).

68 R. Savini, Boll. soc. ital. biol. sper. 24, 822 (1948).

69 A. Lattanzi, Boll. soc. ital. biol. sper. 24, 561 (1948); Intern. Z. Vitaminforsch. 21,
307 (1949).

707. Marinelli and C. Tramontana, Giorn. clin. med. (Parma) 81, 152 (1950).

71 R.S. Harris and P.S. Derian, Southern Med. J. 42, 973 (1949).

24, Biana, Boll. soc. ctal. biol. sper. 15, 1051, 1054 (1940).
M. Sacca, Boll. soc. ital. biol. sper. 22, 477 (1946).

578 NIACIN

action of the toxicity of crystalline borrilidin in rats; decreased serum
cholinesterase;’> and an increase (with thiamine) of basal metabolic rate.7®

XIII. Requirements and Factors Influencing Them
J. M. HUNDLEY
A. ANIMALS

Table XVII presents a summary of published information on the nicotinic
acid requirement of various species. The figures for tryptophan listed in

TABLE XVII

Nicotinic Acip REQUIREMENT AND AMOUNT OF TRYPTOPHAN REQUIRED TO REPLACE
Nicotinic AcIp

Species Nicotinic acid, mg./100 g. of diet Tryptophan, mg./100 g. of diet Remarks
Rat 1e5e(in2)4 L or DL, 50 (1, 2) Corn diets
0.5-1.5 (3) Diets with vari-
ous carbohy-
drates
Mouse 1.0 (4) 6 —
Cotton rat <2.5 (5, 6) b —
Dog 5.0) (7) — Corn diets
0.130-0.225/kg. body — Adult dogs
wt./day (8-10)
0.250-0.360 kg. body pt, 100 or 1, 50 (11) Puppies
wt./day (9)
Swine 6.5-17.0/day/animal DL, 250 (15, 16) Young pigs
(12-14)
0.6-1.0/kg. body wt./ opt, 200/day (12, 14) Young pigs
day (15, 16)
Rabbit 60 (17) DL, 220 (17) Glucose diet
10/kg. body wt./day opu,400/kg. body wt./day Sucrose diet
(18) (19)
Monkey 10-35 wk. (20) pL, 1000-4000/wk. (20) —
Chicken 1.8-2.0 (21-23) — Young chicks
3.0 (24, 25) L or DL, 200 (24) Young chicks
4.0-5.0 (26-28) L, 100 or pu, 200 (27-29) Young chicks
Turkey 3.0-10.0 (80-32) c Poults
Duck 2.5 (83) DL, 400 (34) Ducklings
Cat 1.7/day (35) d ==

@ Figures in parentheses are references.
+ Tryptophan can replace nicotinic acid in the mouse and cotton rat, but this has not been deter-
mined quantitatively.
¢ The ability of tryptophan to replace nicotinic acid in the turkey has not been determined.
4 Trypotophan does not appear to replace nicotinic acid in cats.

74 J. M. Cooperman, 8. H. Rubin, and B. Tabenkin, Proc. Soc. Exptl. Biol. Med. 76,

18 (1951).

XIII. REQUIREMENTS AND FACTORS 579

Table XVII represent the approximate amount required to substitute for
the nicotinic acid requirement. These figures do not include tryptophan

7 P. Salvi and A. Morelli, Acta neurol. (Naples) 2, 986 (1947).
76 C. Malaguzzi-Valeri, G. Conese, and D. Angarano, Intern. Z. Vitaminforsch. 22,
174 (1950).
1W. A. Krehl, P.S. Sarma, L. J. Teply, and C. A. Elvehjem, J. Nutrition 31, 85
(1946).
2 L. V. Hankes, L. M. Henderson, W. L. Brickson, and C. A. Elvehjem, J. Biol.
Chem. 174, 873 (1948).
3 J.M. Hundley, J. Biol. Chem. 181, 1 (1949).
4B.8. Schweigert and P. B. Pearson, J. Biol. Chem. 172, 485 (1948).
6 J. M. McIntire, B. S. Schweigert, and C. A. Elvehjem, J. Nutrition 27, 1 (1944).
6 B.S. Schweigert, Proc. Soc. Exptl. Biol. Med. 68, 522 (1948).
7™W.A. Krehl, L. J. Teply, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 58, 334
(1945).
8 T. W. Birch, J. Nutrition 17, 281 (1939).
9A. E. Schaefer, J. M. McKibbin, and C. A. Elvehjem, J. Biol. Chem. 144, 679
(1942).
10 W. H. Sebrell, R. H. Onstott, H. F. Fraser, and F. 8S. Daft, J. Nutrition 16, 355
(1938).
1§, A. Singal, V. P. Sydenstricker, and J. M. Littlejohn, J. Biol. Chem. 176, 1051
(1948).
122. R. W. Luecke, W. N. McMillen, F. Thorp, Jr., and C. Tull, J. Nutrition 36, 417
(1948).
13 R. EK. Butler and H. P. Sarett, J. Nutrition 35, 539 (1948).
14 RR. W. Luecke, W. N. McMillen, F. Thorp, Jr., and C. Tull, J. Nutrition 33, 251
(1947).
16 W. C. Powick, N. R. Ellis, L. L. Madsen, and C. N. Dale, J. Animal Sci. 6, 310
(1947).
16 W.C. Powick, N. R. Ellis, and C. N. Dale, J. Animal Sci. 7, 228 (1948).
17Q. Olcese, P. B. Pearson, and P. Sparks, J. Nutrition 39, 93 (1949).
18 J. G. Wooley and W. H. Sebrell, J. Nutrition 19, 191 (1945).
19 J. G. Wooley, Proc. Soc. Exptl. Biol. Med. 65, 315 (1947).
20 D. V. Tappan, U. J. Lewis, U. D. Register, and C. A. Elvehjem, J. Nutrition 46,
75 (1952).
21 G. M. Briggs, R. C. Mills, C. A. Elvehjem, and E. B. Hart, Proc. Soc. Exptl. Biol.
Med. 61, 59 (1942).
2 H.M. Scott, E. P. Singsen, and L. D. Matterson, Poultry Sci. (Research Notes) 25,
303 (1946).
23 PS. Sarma and C. A. Elvehjem, Poultry Sci. 25, 39 (1946).
24,J. W. West, C. W. Carrick, 8. M. Hauge, and E. T. Mertz, Poultry Sci. 31, 479
(1952).
26 G. R. Childs, C. W. Carrick, and S. M. Hauge, Poultry Scz. 31, 551 (1952).
26M. E. Coates, 8. K. Kon, and E. E. Shepheard, Brit. J. Nutrition 4, 203 (1950).
27 G. M. Briggs, A. C. Groschke, and R. J. Lillie, J. Nutrition 32, 659 (1946).
3G. M. Briggs, J. Biol. Chem. 161, 749 (1945).
*7 J. O. Anderson, G. F. Combs, and G. M. Briggs, J. Nutrition 42, 463 (1950).
30 G. M. Briggs, J. Nutrition 81, 79 (1946).
3. T. H. Jukes, E. L. R. Stokstad, and M. Belt, J. Nutrition 38, 1 (1947).
32 B. G. Lance and A. G. Hogan, J. Nutrition 36, 369 (1948).
33D. M. Hegsted, J. Nutrition 32, 467 (1946).

580 NIACIN

in the basal diet and hence do not represent the absolute tryptophan re-
quirement. The figures listed, both for nicotinic acid and tryptophan, can
be regarded as valid only under the particular conditions of the experiments
cited, in view of the well-known conversion of tryptophan to nicotinic acid
and the many other factors which influence requirements.

As indicated in Table XVII, rats, mice, cotton rats, dogs, swine, rabbits,
monkeys, chickens, turkeys, ducks, and cats require a dietary source of
nicotinic acid except, in some species, when sufficient tryptophan is sup-
plied. Fox puppies require nicotinic acid (0.39 to 2.0 mg. per kilogram of
body weight per day).*® The activity of tryptophan has not been reported
in this species. Guinea pigs require nicotinic acid.*”: *’ Banerjee el al.*® could
demonstrate no activity with tryptophan, although Cannon and associates*”
obtained some evidence that tryptophan could substitute for nicotinic acid
and Henderson et al.*® found that tryptophan increased the urinary excre-
tion of nicotinic acid and related compounds in guinea pigs.

Calves,**-” horses,*: 44 and sheep*®: 4°: 46 do not require a dietary source
of nicotinic acid, although they can convert tryptophan to nicotinic acid.

a. Tryptophan

The conversion of tryptophan to nicotinic acid is described in detail in
Section VI. Tryptophan may be added to the diet either as the pure amino
acid or as protein. Thus, dogs will develop blacktongue on a 20% casein
ration but will not if the casein is increased to 42 %.!! Rats develop nicotinic
acid deficiency on 9% casein, cystine-supplemented rations but do not if
the casein level is increased to 15 to 25%.*: “-®! Swine develop nicotinic

34. Bernard and J. M. Demers, Rev. can. biol. 8, 504 (1949).

35 A. C. da Silva, Acta Physiol. Latinoamer. 1, 20 (1950); A. C. da Silva, R. Fried,
and R. C. de Angelis, J. Nutrition 46, 399 (1952).

36 A. }. Schaefer, C. K. Whitehair, and C. A. Elvehjem, J. Nutrition 34, 131 (1947).

37 M. D. Cannon, G. J. Mannering, C. A. Elvehjem, and E. B. Hart, Proc. Soc. Exptl.
Biol. Med. 68, 414 (1946).

38§. Banerjee, R. Banerjee, and C. C. Deb, Indian J. Med. Research 38, 161 (1950).

39 |, M. Henderson, G. B. Ramasarma, and B. C. Johnson, J. Biol. Chem. 181, 731
(1949).

40 B.C. Johnson, A. C. Wiese, H. H. Mitchell, and W. B. Nevens, J. Biol. Chem. 167,
729 (1947).

418. C. Johnson, Huclides (Madrid) 8, 161 (1948).

4G.C. Esh and T.S. Sutton, J. Dairy Sci. 31, 909 (1948).

43 J. W. Huff, P. B. Pearson, and W. A. Perlzweig, Arch. Biochem. 9, 99 (1946).

44B. S. Schweigert, P. B. Pearson, and M. C. Wilkening, Arch. Biochem. 12, 139
(1947).

45 A. H. Winegar, P. B. Pearson, and H. Schmidt, Science 91, 508 (1940).

46 P. B. Pearson, A. H. Winegar, and H. Schmidt, J. Nutrition 20, 551 (1940).

47 J. M. Hundley, J. Nutrition 34, 253 (1947).

48 W.A. Krehl, L. J. Teply, and C. A. Elvehjem, Science 101, 283 (1945).

XIII. REQUIREMENTS AND FACTORS 581

acid deficiency on low casein diets but not if the casein level is increased
to 20 or 26%." ©

There is considerable evidence that tryptophan added to the diet as the
free amino acid results in considerably greater synthesis of nicotinic acid
than when an equivalent amount of tryptophan is added as protein.®: °
This interesting phenomenon might be explained by assuming that trypto-
phan presented to the tissues along with a well-balanced mixture of other
amino acids is used primarily in association with other amino acids in
processes involved in protein synthesis and replacement. However, when
tryptophan is presented to the tissues so that it is in excess of the available
amount of other amino acids, the excess tryptophan is diverted into the
channels which lead to nicotinic acid synthesis.

b. Other Amino Acids

Several groups of investigators have shown that proteins lacking or low
in tryptophan, amino acid mixtures simulating these proteins, and certain
amino acids, singly or in combinations, have a marked ability to increase
nicotinic acid requirements when added to deficient rations in rats?» 47» 54-87
and in chicks.” *8-®° Anderson e¢ al.,°' using chicks on a nicotinic acid
deficient diet, have recently shown that amino acids may be divided into
two groups, those which cause a growth depression correctable by nicotinic
acid and those which cause a growth depression not influenced by the
vitamin.

In each of the experiments cited above, the basal ration to which the
various protein and amino acid supplements were added contained a limited
amount of tryptophan as well as being deficient in nicotinic acid. In general,

49W.D. Salmon, J. Nutrition 38, 169 (1947).

50 W. D. Salmon, J. Nutrition 38, 155 (1947).

51M. M. Wintrobe, H. J. Stein, R. H. Follis, Jr., and S. Humphreys, J. Nutrition 30,
395 (1945).

2S. A. Singal, A. P. Briggs, V. P. Sydenstricker, and J. M. Littlejohn, J. Biol.
Chem. 166, 573 (1946).

53G. H. Bell, B. T. Scheer, and H. J. Deuel, Jr., J. Nutrition 35, 239 (1948).

54W. A. Krehl, L. M. Henderson, J. de la Huerga, and C. A. Elvehjem, J. Biol.
Chem. 166, 531 (1946).

55 F. Rosen, J. W. Huff, and W. A. Perlzweig, J. Biol. Chem. 163, 343 (1946).

568. A. Singal, V. P. Sydenstricker, and J. M. Littlejohn, J. Biol. Chem. 171, 203
(1947); 176, 1063 (1948).

57 F. Rosen and W. A. Perlzweig, J. Biol. Chem. 177, 163 (1949).

® A.C. Groschke, J. O. Anderson, and G. M. Briggs, Proc. Soc. Exptl. Biol. Med.
68, 564 (1948).

59G.M. Briggs, J. Biol. Chem. 161, 749 (1945).

60 A. C. Groschke and G. M. Briggs, J. Biol. Chem. 165, 739 (1946).

61 J. O. Anderson, G. F. Combs, A. C. Groschke, and G. M. Briggs, J. Nutrition
45, 345 (1951).

582 NIACIN

the more of the supplements which were added, the more severe the appar-
ent nicotinic acid deficiency became. In most instances nicotinic acid and
tryptophan were equally effective in overcoming the growth depression
(within certain limits).

Most, if not all, of these observations can be explained according to the
concept of amino acid balance. When the diet contains more tryptophan,
in proportion to available amounts of other amino acids, than is required
for protein synthesis and other anabolic demands, then a portion of the
tryptophan is converted into nicotinic acid in an efficient fashion. When,
however, the supply of tryptophan is restricted so that it is the most limit-
ing amino acid, then the tissues seem to metabolize it in a different fashion.
Its utilization becomes quite inefficient and very little nicotinic acid is
formed, as shown by Krehl and associates.* The greater the deficit of
tryptophan in proportion to the other amino acids, the more severe the
nicotinic acid deficiency seems to be. If nicotinic acid is supplied in these
low-tryptophan rations, the tissues seem to be relieved of the necessity
for converting tryptophan to nicotinic acid and the metabolism of trypto-
phan becomes efficient once again; i.e., the added nicotinic acid seems to
“spare” tryptophan.

This concept, if valid, implies that the metabolic channels into which an
amino acid can be directed may vary according to the needs of the tissues
for essential substances. It also implies that the metabolic utilization of an
amino acid may become quite inefficient when it is the most limiting amino
acid. Further, it is evident that the absolute requirement of an organism
for an amino acid varies not only with the relative supply of other amino
acids but with the dietary supply of essential substances which can, in case
of need, be made from the amino acid.

The role of amino acid balance in nutrition has been reviewed by EIl-
vehjem and Krehl® and by Krehl.®

c. Influence of Carbohydrates

Krehl and associates! were the first to note that dextrin, starch, glucose,
and lactose had a nicotinic acid sparing action when compared to diets
containing sucrose in the rat. These findings have been confirmed and
extended.*: *4: 6 A similar phenomenon may exist in the rabbit,” 8 al-
though the reverse apparently obtains in chicks; 1.e., starch increases
nicotinic acid requirements.2® The explanation usually given these phenom-
ena is that different carbohydrates alter the intestinal flora and thereby

62 W. A. Krehl, J. de la Huerga and C. A. Elvehjem, J. Nutrition 164, 551 (1946).

63 C, A. Elvehjem and W. A. Krehl, J. Am. Med. Assoc. 135, 279 (1947).

64 W. A. Krehl, Vitamins and Hormones 7, 111 (1949).

65 T,. M. Henderson, T. Deodhar, W. A. Krehl, and C. A. Elvehjem, J. Biol. Chem.
170, 261 (1947).

XIII. REQUIREMENTS AND FACTORS 583

change the amount of nicotinic acid consumed or produced by the micro-
organisms, which in turn influences the amount of nicotinic acid available
to the host. This may be the true explanation, although Hundley* has
advanced the idea, with some supporting data, that fructose and sugars
containing fructose require more nicotinic acid in their metabolism than do
glucose and sugars containing glucose. According to this theory, fructose,
or a portion of it, may be metabolized in a different manner from glucose,
a metabolic pathway which necessitates a greater utilization of nicotinic
acid-containing coenzymes, and may thereby increase the dietary nicotinic
acid requirement.

d. Intestinal Synthesis of Nicotinic Acid

The exact explanation for the effect of various carbohydrates on nicotinic
acid requirement is still in doubt. Indeed, much has been written about
the role of intestinal microorganisms in supplying the host with essential
nutrients. Ellinger®® has reviewed the extensive and excellent work he and
his associates have done, as well as that of other workers, which supports
the position that intestinal microbes play an important role in providing
nicotinic acid for the host. There is no doubt whatsoever that bacteria,
such as are found in the intestine, can and do synthesize nicotinic acid, but
on the other hand they may also consume and destroy it. ® There is also
no doubt that dietary variations such as changing the type of carbohydrate
will alter the microbial population of the intestine and will change the
amount of free nicotinic acid found in the intestine, as shown by the nu-
merous studies on carbohydrates cited previously.

The essential question is whether any of these changes result in any
substantial difference in the nicotinic acid economy of the host. The evi-
dence is conflicting. Ellinger and associates have found that some sulfa
drugs, but not others, taken orally result in a substantial decrease in
urinary N!-methylnicotinamide excretion.®: 7° There are clinical reports of
nicotinic acid deficiency being induced by oral “sulfa” drugs or anti-
biotics.7! 72 On the other hand, numerous attempts to produce nicotinic
acid deficiency in animals by means of sulfa drugs have been unsuccessful.
Several investigators have found no decrease in the urinary excretion of
N'-methylnicotinamide or other nicotinic acid metabolites after sulfa drugs

66 Pp. Ellinger, Hxperentia 6, 144 (1950).

87S. A. Koser and G. R. Baird, J. Infectious Diseases 75, 250 (1944).

68 R. Benesch, Lancet I, 718 (1945); Prensa med. (Argentina) 33, 1335 (1946).
69 P, Ellinger, R. A. Coulson, and R. Benesch, Nature 154, 270 (1944).

70 P, Ellinger, R. Benesch, and W. W. Kay, Lancet I, 432 (1945).

1$. W. Hardwick, Lancet 250, 267 (1946).

72 P, Ellinger and F. M. Shattock, Brit. Med. J. II, 611 (1946).

584 NIACIN

were given orally in man and animals,”7> although some drugs seem to
produce an increased excretion of nicotinic acid metabolites.7° The final
solution to this knotty question will have to await the availability of tools
which will permit a more direct approach to the problem.

e. Influence of Fat

Salmon7? has shown that diets high in fat result in a lowered dietary
requirement for nicotinic acid, in a fashion similar to the well-known
thiamine-sparing action of fat. This has been interpreted as providing
additional evidence for the role of nicotinic acid in carbohydrate metabo-
lism, although the correctness of this interpretation has been questioned.™

f. Other Influences

(1) Corn. The fact that the occurrence of human pellagra is so often
associated with the consumption of diets high in corn, and the fact that
pellagra will develop when corn diets are fed whereas other diets with less
nicotinic acid do not cause pellagra,®*: *! have led to innumerable attempts
to find some substance in corn which increases nicotinic acid requirements
(pp. 548 and 565). Although it has been shown that corn, under certain
circumstances, will increase nicotinic acid requirements in several species
of animals,’ 48 ® there is no conclusive evidence that this effect of corn is
due to anything more than the effect of its low tryptophan protein.*®: *8: 8
The weight of evidence seems to favor the view that these effects of corn
can be adequately explained on this basis plus perhaps a relatively low
digestibility and a low availability of the nicotinic acid in corn.**: ®

(2) Vitamin Bs. A deficiency of pyridoxine in the rat, if sufficiently
severe, reduces the excretion of urinary nicotinic acid derivatives after

73V. A. Najjar, L. E. Holt, Jr., G. A. Johns, G. C. Mediary, and G. Fleischmann,
Proc. Soc. Exptl. Biol. Med. 61, 371 (1946).

™H. N. De, M. C. Malaker, and A. K. Paul, Indian Med. Gaz. 84, 542 (1949).

75 A. E. Teeri, M. Leavitt, D. Josselyn, N. F. Caloves, and H. A. Keener, J. Biol.
Chem. 182, 509 (1950).

76 H. Spector, J. Biol. Chem. 173, 659 (1948).

7 P. Ellinger and A. Emmanuelowa, Lancet 251, 716 (1946).

78 P. Ellinger, M. M. Abdel Kader, and A. Emmanuelowa, Brit. J. Exptl. Pathol. 28,
261 (1947).

79W.D. Salmon, J. Nutrition 33, 155 (1947).

80). I. Frazier and T. E. Friedemann, Quart. Bull. Northwestern Univ. Med. School
20, 24 (1946).

81 W.R. Aykroyd and M. Swaminathan, Indian J. Med. Research 27, 667 (1940).

8 P. Handler, Proc. Soc. Exptl. Biol. Med. 52, 263 (1943).

83. W. A. Krehl, P.S. Sarma, and C. A. Elvehjem, J. Biol. Chem. 162, 403 (1946).

84H}, Kodicek, Biochem. J. 48, viii (1951).

85 R. G. Chitre and D. B. Desai, Ind. J. Med. Research 8, 471, 479 (1949).

XIII. REQUIREMENTS AND FACTORS 585

tryptophan administration®: 8*-88 and prevents the normal increase of pyri-
dine nucleotides in red blood cells following tryptophan.*? In most instances
these defects were overcome promptly after pyridoxine therapy. Since
pyridoxine deficiency is known to result in abnormal tryptophan metabo-
lism %°-% it was logical to believe that pyridoxine deficiency might also
interfere with the synthesis of nicotinic acid from tryptophan.

However, several reports**-°* appeared indicating that the liver of pyri-
doxine-deficient rats could still transform tryptophan to nicotinic acid
derivatives. Furthermore, deficiencies of thiamine or riboflavin or simple
reduction in caloric intake also appeared to result in a reduced excretion
of urinary nicotinic acid derivatives after tryptophan.®® Spector?’ found no
interference with the tryptophan to nicotinic acid conversion in force-fed
pyridoxine-deficient rats. Heimberg ef al.” obtained evidence that the in-
volvement of pyridoxine in nicotinic acid synthesis was not a direct meta-
bolic effect. These seemingly contradictory reports may be explained by a
recent investigation by Kring and associates,*8 who also found that pyri-
doxine deficiency did not inhibit the formation of liver pyridine nucleotides
from tryptophan. However, if they intensified the deficiency state by using
the antagonist desoxypyridoxine, they could demonstrate such an inter-
ference. However, even this severe deficiency did not interfere with the
formation of liver pyridine nucleotides from nicotinic acid.

So far as is known, this interesting interrelationship between pyridoxine
and tryptophan has no practical significance in altering the nicotinic acid
requirements of either animals or man.

(3) Folic Acid. Krehl and associates?’ !°° found that folic acid improved

86 F. Rosen, J. W. Huff, and W. A. Perlzweig, J. Nutrition 38, 561 (1947).

87 B. §. Schweigert and P. B. Pearson, J. Biol. Chem. 168, 555 (1947).

88 P. B. Junqueira and B.S. Schweigert, J. Biol. Chem. 174, 605 (1948).

89 C. Ling, D. M. Hegsted, and F. J. Stare, J. Biol. Chem. 174, 803 (1948).

90 P. J. Fouts and 8. Lepkovsky, Proc. Soc. Exptl. Biol. Med. 50, 221 (1942).

1$. Lepkovsky and E. Nielsen, J. Biol. Chem. 144, 135 (1942).

%§. Lepkovsky, E. Roboz, and A. J. Haagen-Smit, J. Biol. Chem. 149, 195 (1943).

9%3C.C. Porter, I. Clark, and R. H. Silber, J. Biol. Chem. 167, 573 (1947).

% J. N. Williams, Jr., P. Feigelson, C. A. Elvehjem, and §. S. Shahinian, J. Biol.
Chem. 189, 659 (1951).

% W.W. Hurt, B. T. Scheer, and H. J. Deuel, Jr., Arch. Biochem. 21, 87 (1949).

96 J. N. Williams, Jr., P. Feigelson, S.S. Shahinian, and C. A. Elvehjem, Proc. Soc.
Exptl. Biol. Med. 76, 441 (1951).

7M. Heimberg, F. Rosen, I. G. Leder, and W. A. Perlzweig, Arch. Biochem. 28, 225
(1950).

% J. P. Kring, K. Ebisuzaki, J. N. Williams, Jr., and C. A. Elvehjem, J. Biol.
Chem. 195, 591 (1952).

99W. A. Krehl and C. A. Elvehjem, J. Biol. Chem. 158, 173 (1945).

100 W. A. Krehl, N. Torbet, J. dela Huerga, and C. A. Elvehjem, Arch. Biochem. 11,
363 (1946).

586 NIACIN

the response of nicotinic acid-deficient dogs to nicotinic acid and helped
to maintain a more normal blood picture. Ruegamer et al.1° reported that
both folic acid and nicotinic acid were required to obtain a response with
liver extract in an anemia which developed in dogs on rations which were
probably deficient in all three factors. Teply et al.!° found an interrelation
between folic acid and nicotinic acid in the ceca of rats under various dietary
circumstances. Junqueira and Schweigert!™ found that sulfasuxidine inter-
fered with the conversion of tryptophan to nicotinic acid if folic acid was
not in the diet, but had no effect if the diet was supplemented with folic
acid. The significance, if any, of these reports in relation to the nicotinic
acid requirement of animals is not clear.

(4) Age. Adult animals require less nicotinic acid than growing animals,
as might be expected.!: 2 1%

(5) Climate. Mills and associates! found no difference in nicotinic acid
requirement between chicks kept at temperate coolness and at tropical heat.

(6) Alcohol. Available evidence (p. 565) suggests that alcohol ‘“spares”’
nicotinic acid in man. However, in view of the high incidence of pellagra
in alcoholics, one might doubt the practical importance of this effect.

B. MAN

The recommended daily dietary allowance of nicotinic acid which is
generally applied in the United States is that of the Food and Nutrition
Board of the National Research Council.!°* According to this standard, the
daily diet of man should provide 12 to 18 mg. of nicotinic acid, depending
on activity, that for women 10 to 15 mg., depending on activity and whether
they are pregnant or lactating. Infants and children should receive from 4
to 17 mg. daily, depending on age up to 13 years, and on age and sex from
13 to 20 years of age.

These allowances were based primarily on animal experiments and on
analyses of diets which were known to be pellagra preventive.!°* They
contain a deliberate margin of safety to provide for those who may have
requirements above average or who by reason of illness or injury might
have a temporarily increased need for the vitamin. It should be emphasized
that the figures cited above are recommended allowances, not minimum
physiological requirements.

101 W. R. Ruegamer, W. L. Brickson, N. J. Torbet, and C. A. Elvehjem, J. Nutrition
36, 425 (1948).

1022 |. J. Teply, W. A. Krehl, and C. A. Elvehjem, Am. J. Physiol. 148, 91 (1947).

103 P. B. Junqueira and B. S. Schweigert, J. Biol. Chem. 175, 535 (1948).

104 R. Braude, 8S. K. Kon, and E. G. White, Biochem. J. 40, 843 (1947).

105 ©, A. Mills, E. Cottingham, and E. Taylor, Am. J. Physiol. 149, 376 (1947).

106 Recommended Dietary Allowances, Bull. Natl. Research Council (U. S.) Reprint
and Circ. Ser. 129, (1948).

XIII. REQUIREMENTS AND FACTORS 587

However, the validity of these recommended allowances has recently
received important confirmation from two sources. Frazier and Friede-
mann*? recalculated the nicotinic acid and other nutrients in diets known to
be pellagra producing or pellagra preventive, using dietary records gathered
by Goldberger and others. They concluded that the minimum daily intake
of nicotinic acid in a marginal diet containing corn products is about 7.5
mg. per day. More recently, Goldsmith and associates!” produced pellagra
experimentally in women with corn-containing diets which provided 4.7
mg. of nicotinic acid and 190 mg. of tryptophan daily. Other subjects who
received the same diet supplemented with 2 mg. of nicotinic acid, or
‘‘wheat”’ diets providing 5.7 mg. of nicotinic acid and 230 mg. of tryptophan,
failed to develop pellagra.

Much less precise information is available on factors which influence
requirements in man than in animals. Tryptophan can substitute, at least
in part, for nicotinic acid in human diets. The possible influence of corn
products (p. 548) and alcohol (p. 565) has been mentioned elsewhere.
Several factors are known which, clinically, seem to precipitate or predis-
pose man to attacks of pellagra. These are sunlight, heavy work, disturbed
gastrointestinal function, intestinal parasitism, and surgery. Whether these
increase requirements or act in an indirect manner is not established. Like-
wise, it is uncertain whether the peak incidence of pellagra in the early
spring months is due to the effect of sunlight, to increased exercise, or
simply to the development of a deficit of nicotinic acid during the winter
months.

107 G. A. Goldsmith, H. P. Sarett, U. D. Register, and J. Gibbens, J. Clin. Invest. 31,
533 (1952).

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CHAPTER 11

PANTOTHENIC ACID

I. Nomenclature.
II. Chemistry .
A. Isolation .. :
B. Chemical and Eneeical Properties :
C. Constitution . ane: ne:
D. Synthesis . .
III. Biochemical Systems .
A. Introduction .
B. Identification of Pantathenda iMeicle as Part of Gueaey me vt
1. CoA Assay.
2. Relation of the aay ewes ime tecmined CoA Iie 6 Its Banto-
thenic Acid Content .
3. Arsenolysis Assay . . .
4. Other CoA Assay Sy Site
C. The Chemistry of Coenzyme A :
1. Phosphate and Adenylic Acid Content
. The Sulfur-Containing Moiety.
. Acetyl-CoA . ones
. The Structure of CoA
5. Preparation of CoA .
D. Some Nutritional Aspects of thee Gon problem Kah tues
1. Assay of Pantothenic Acid Based on the ied hodalary oF Pair
thenie Acid Liberation from CoA : sah atte EBT
2. The Alkali Stability of ‘‘Natural enmnanhenic Nerd?
E. Split Products of CoA as Microbial Growth Factors
1. Pantothenic Acid Conjugate of Cheldelin
2. Pantethine (Lactobacillus bulgaricus Factor)
F. The Metabolic Function of CoA
1. Distribution of CoA. . .
2. Acetyl and Acyl Transfer .
3. Fat and Steroid ewe 3
G. Conclusion. : oD
IV. Specificity of Action .
V. Biogenesis .
VI. Estimation. ..
A. Chemical and Bhysieal Methods.
B. Biological Methods
1. Rat Growth Assays
2. Chick Growth Assays.
3. Assays for Coenzyme A .
C. Microbiological Methods
VII. Standardization of Activity.

Hm CO bh

589

Page
591
591
591
596
597
597
598
598
599
599

601
601
603
603
603
605
607
608
609
610

610
611
611
613
613
614
614
615
622
624
626
627
628
628
628
629
629
630
630
633

590

VIII.

PANTOTHENIC ACID

Page
Occurrence in Food Fit 634
A. Assay Values in Foods, F ae, Tiaenen: aad Mianeollameoue Materials 634
B. Effect of Various Maetons on Pantothenic Acid Distribution. 634
1. Effect of Variety, Species, and Breed . 5 635
2. Effect of Environment during Period of Growth . 635
3. Effect of Germination and Embryonic Development 636
4. Effect of Diet on Tissue Composition . 636
5. Effect of Storage ; eer 637
6. Effect of Heat and @oaking 637
7. Effect of Stage of Lactation . 638
C. Distribution of Coenzyme A . 638
. Effects of Deficiency . 649
A. In Animals 649
1 Rats: 650
2. Chicks . 658
3. Turkeys Pee EG ela, Ot 661
4. Ducks, Pigeons, and Other Fowl. 661
5. Dogs. Ao ae 663
6. Guinea Pigs . 664
7. Swine . . : 664
8. Cattle and Bneee , 666
9. Monkeys 667
10. Mice. 667
11. Other Byers 668
B. In Man. 669
1. Introduction . ‘ 669

2. Summary of Baaalamien! facture in ‘ata Fed iste Mafeient
in Pantothenic Acid . oe 669

3. Pathological Lesions in Human Beme isusceunine on a Belrienes oe
Pantothenic Acid. 670
C. In Microorganisms. 678
. Pharmacology. : 680
. Requirements and Racers Tine esiatete "Them 682
A. Of Animals 682
1. Minimal Levels . : 682
2. Factors Influencing Peniieements é 683
B. Of Man. 687
1. Pantothenic Roan Content bE Podds 687

2. Urinary Excretion and Blood Levels of Bantothenre leith in Bac
Beings . 688

3. Approximate Pica te ed Peapod a Man as Bae on
the Known Requirement of the Experimental Animal . 691
4. Toxicity of Pantothenic Acid. 692
5. Summary . 693

II. CHEMISTRY 591

I. Nomenclature
ROBERT S. HARRIS

Accepted name: Pantothenic acid!
Obsolete names: Liver factor 2?
Antidermatitis factor?
Chick antidermatitis factor*
Liver filtrate factor®
Yeast filtrate factor®
Chick antipellagra (A.P.) factor?
Empirical formula: CgHyO;N
Chemical name: a,y-Dihydroxy-@ ,$-dimethylbutyryl-6’-alanine
Structural formula:

CH;

HOCH:—_€—_CHOH—_CO_NH—_ CH: CH, —CO0on

CH;

II. Chemistry
SAMUEL LEPKOVSKY
A. ISOLATION

The isolation of pantothenic acid was accomplished during a period in
the history of nutrition that was characterized by confusion and mis-
understanding. This was caused by the mistaken notion that vitamin G
or Bs was a single factor instead of a multiplicity of factors which had
similar chemical properties and were associated together in such food-
stuffs as yeast, liver, and wheat germ. Goldberger and Lillie! predicted
this in what became a masterpiece of understatement. ‘“The possibility,
remote though it seems, is not excluded that there may be in yeast more

1R. J. Williams, C. M. Lyman, G. H. Goodyear, T. H. Truesdail, and D. Holaday,
J. Am. Chem. Soc. 55, 2012 (1932).

2S. Lepkovsky, T. H. Jukes, and M. E. Krause, J. Biol. Chem. 115, 557 (1936).

3 J. C. Bauernfeind, A. E. Schumacher, A. Z. Hodson, L. C. Norris, and G. F.
Heuser, Proc. Soc. Exptl. Biol. Med. 39, 108 (1938).

40. Mickelsen, H. A. Waisman, and C. A. Elvehjem, J. Biol. Chem. 124, 313 (1938).

5 T. H. Jukes and 8. Lepkovsky, J. Biol. Chem. 111, 119 (1935).

6 C, EK. Edgar and T. F. Macrae, Biochem. J. 31, 886, 893 (1937).

7C. J. Koehn, Jr., and C. A. Elvehjem, J. Biol. Chem. 118, 693 (1937).

1 J. Goldberger and R. D. Lillie, Public Health Repts. (U. S.) 41, 297 (1926).

592 PANTOTHENIC ACID

than one such thermostable factor which further study may succeed in
differentiating. Moreover, it is not clear that all essential factors, or neces-
sary relations among such factors, for the nutrition of the albino rat, have
as yet been determined.” The confusion was largely attributable to three
causes:

1. Different species of animals such as the chicken, the turkey, the rat,
and the human being showed superficially similar symptoms, such as
dermatitis, which were very frequently and uncritically referred to as
‘pellagra.”? This resulted in the mistaken implication that one vitamin
only was involved when in fact each so-called ‘“pellagrous”’ condition was
due to the deficiency of a different vitamin.

2. Not all species reacted similarly to all vitamin deficiencies. Man,
dog, and pig were sensitive to the deficiency of nicotinic acid, but the rat
and the chick were not.2 It took some time for investigators to realize that
a pig developed pellagra on a diet whereas a rat on the same diet did not.

3. Adsorbing agents such as fuller’s earth and charcoal which were used
in the fractionation of different vitamins were not so specific as the in-
vestigators using them believed, leading to considerable misunderstanding.

Slowly and laboriously the weight of accumulating evidence brought to
light the chemical nature of pantothenic acid. Two completely independent
lines of investigations, proceeding simultaneously, one with microorgan-
isms and the other with chicks, finally merged, and together they were
largely responsible for the isolation of pantothenic acid and its recognition
as a vitamin.

The pioneering investigations of Williams, an organic chemist using
microorganisms and microbiological methods, were in large part responsible
for the isolation and characterization of pantothenic acid. The term was
coined by Williams and his coworkers and it comes from the Greek mean-
ing “from everywhere’ “ because it was found in all of a considerable num-
ber of plant and animal tissues from widely divergent sources, and they
correctly felt that it must play a fundamentally important biological role.
The retention of the name pantothenic acid and the failure to use any
letter-number designation for it marks the early departure from the tra-
ditional alphabetical-number vitamin nomenclature. Williams e¢ al.” ** used
the following techniques in the isolation of pantothenic acid from liver.

28. Lepkovsky, Nutrition Abstr. & Revs. 11, 363 (1942). This article carries an ex-
tensive bibliography on the subject.

3R. J. Williams, C. M. Lyman, G. H. Goodyear, J. H. Truesdail, and D. Holaday,
J. Am. Chem. Soc. 55, 2912 (1933).

3¢R. J. Williams, Advances in Enzymol. 8, 253 (1943). This article carries an ex-
tensive bibliography on the subject.

4R.J. Williams, J. H. Truesdail, H. H. Weinstock, Jr., E. Rohrmann, C. M. Lyman,
and C. H. McBurney, J. Am. Chem. Soc. 60, 2719 (1938).

4a f. A. Robinson, The Vitamin B Complex. John Wiley and Sons, New York, 1951.

II. CHEMISTRY 593

1. Autolysis to obtain a clear filtrate.

2. Adsorption on fuller’s earth to remove bases.

3. Adsorption of the vitamin on charcoal and its subsequent elution.

4. Preparation of brucine derivatives by evaporation to dryness on
kieselguhr in the presence of brucine and brucine oxalate.

5. Extraction of brucine pantothenate with chloroform.

6. Fractional distribution of the brucine salts between chloroform and
water.

7. Conversion of the pantothenic acid to its calcium salt.

8. Fractionation of the calcium salts with various solvents and solvent
mixtures.

The success of this procedure depended upon a rapid and accurate
quantitative assay method which was made possible by the use of yeast as
test organisms.*: > Without such a rapid test, this procedure would have
been hopeless. This work foreshadowed the wide use of microorganisms
for the assay of vitamins and amino acids.

Pure pantothenic acid has never been isolated by this or any other pro-
cedure. Only a small quantity of pantothenic acid of 90% purity has been
prepared. It was a colorless ‘‘varnish,’’ which, when ground, yielded a
white amorphous hygroscopic powder which was about 11,000 times as
potent as their “standard” rice bran preparation.*”’ *

Long before pantothenic acid was isolated, Williams and his coworkers
obtained extensive preliminary information about it, made possible in
large part by the extensive use of a relatively new tool, 1.e., fractional elec-
trical transport.*”’ ° ‘

1. The compound was an acid with a molecular weight? of about 150 and
with an ionization constant® of about 3.9 xX 10~°.

2. It was unstable in hot acids and alkalies.

3. It had a nitrogen atom with barely detectable basic properties.°

4. It had in its structure no olefin double bond, aldehyde, ketone, sulf-
hydryl, basic nitrogen, aromatic, or sugar group.

5. It possessed several hydroxyl groups.’

Snell et al.!° were independently studying an essential nutrient for lactic
acid bacteria. They concluded that it was an acid, was adsorbed on char-
coal, lacked basic properties, and was unstable in hot acid or alkaline
solution.

5R. J. Williams, E. D. McAlister, and R. R. Roehm, J. Biol. Chem. 88, 315 (1929).

6 R. J. Williams and J. H. Truesdail, J. Am. Chem. Soc. 58, 4171 (1931).

TR. J. Williams, J. Biol. Chem. 110, 589 (1935).

8 R. J. Williams and R. J. Moser, J. Am. Chem. Soc. 56, 169 (1934).

9R. J. Williams, H. H. Weinstock, Jr., E. Rohrmann, J. H. Truesdail, H. K. Mit-
chell, and C. E. Meyer, J. Am. Chem. Soc. 61, 454 (1939).

10. E. Snell, F. M. Strong, and W. H. Peterson, Biochem. J. 31, 1789 (1937).

594 PANTOTHENIC ACID

They recognized a rough similarity between their factor and pantothenic
acid but concluded that the available evidence was too limited to warrant
discussion of the relationship of the two factors.

A clue to the nature of pantothenic acid was found with the discovery
that B-alanine was a yeast growth stimulant.!! This discovery led Williams
and his coworkers to suspect that there was a structural relationship be-
tween 8-alanine and pantothenic acid. This suspicion was soon confirmed
with the discovery that 8-alanine was in fact a cleavage product of panto-
thenic acid,” and they succeeded in isolating it in the form of 6-naphthalene-
sulfo-G-alanine.” They also found that their yeasts (G.M.) were capable of
synthesizing physiologically active pantothenic acid when @-alanine was
supplied in the medium.”

Attention was now concentrated upon the other cleavage product. The
evidence indicated that it was an a-hydroxy-y-lactone.“ Elementary
analysis of the most potent pantothenic acid concentrates yielded the
misleading information that this lactone possessed five carbon atoms"
instead of the six actually present. A partial synthesis of physiologically
active pantothenic was effected by condensing 6-alanine ester with impure
lactone obtained from a pantothenic acid concentrate.» This amazing
information, obtained about a compound before it had been obtained in
pure form, proved to be decisive in the ultimate isolation and synthesis of
pantothenic acid.

In the meantime, Snell e¢ al.1® continued their investigations of the acidic
compound required by lactic acid bacteria. In general their information
paralleled that obtained by Williams and his coworkers. They” also suc-
ceeded in accomplishing a partial synthesis of physiologically active panto-
thenic acid and concluded that their factor was indeed identical with the
pantothenic acid of Williams and his coworkers. ** ”

Norris and Ringrose® described in chicks a “‘pellagrous’” dermatitis
which was destined to play a key role in the isolation and characterization
of pantothenic acid. The cure of the ‘‘pellagrous’’ condition was attributed

1 R.J. Williams and E. J. Rohrman, J. Am. Chem. Soc. 58, 695 (1936).

122. H. Weinstock, Jr., H. K. Mitchell, E. F. Pratt, and R. J. Williams, J. Am.
Chem. Soc. 61, 1421 (1939).

13H. K. Mitchell, H. H. Weinstock, Jr., E. E. Snell, S. R. Stanberry, and R. J.
Williams, J. Am. Chem. Soc. 62, 1776 (1940).

4R. J. Williams, H. H. Weinstock, Jr., E. Rohrmann, J. H. Truesdail, H. K. Mit-
chell, and C. E. Meyer, J. Am. Chem. Soc. 61, 454 (1939).

16R. J. Williams, H. K. Mitchell, H. H. Weinstock, Jr., and E. E. Snell, J. Am.
Chem. Soc. 62, 1784 (1940).

16H. E. Snell, F. M. Strong, and W. H. Peterson, J. Am. Chem. Soc. 60, 2825 (1938).

17). E. Snell, F. M. Strong, and W. H. Peterson, J. Bacteriol. 88, 293 (1939).

18 1. C. Norris and R. C. Ringrose, Science 71, 6438 (1930).

II. CHEMISTRY 595

by these workers to vitamin Bz or G. By the use of a heat-treated ration
composed largely of yellow corn, middlings, and casein, Kline e¢ al.!® de-
veloped a basal ration which regularly produced dermatitis in chicks and
was very useful in studies of a factor referred to as the chick antidermatitis
vitamin. Using this technique for the quantitative determination of this
vitamin, Woolley e¢ al.?° obtained evidence that it was a hydroxy acid.
Further study indicated a close similarity between the properties of the
chick antidermatitis vitamin and those of pantothenic acid. Like panto-
thenic acid, the chick antidermatitis vitamin contained @-alanine in its
molecule. Moreover, these workers succeeded in producing a partial syn-
thesis of physiologically active chick antidermatitis vitamin by coupling
B-alanine with the acidic part of alkali-inactivated concentrates of the
chick antidermatitis vitamin.) 7°

As a result of their investigations, Woolley et al.”!: * suggested that the
chick antidermatitis vitamin was identical with pantothenic acid for the
following reasons.

1. Both are acids composed of $-alanine with an hydroxy acid in amide
linkage. ;

2. Both are labile to hot acid and alkali.

3. Both form heat-stable acetyl derivatives which distill at approxi-
mately the same temperature and pressure.

4. The solubilities in various solvents of the free acids and salts of both
these vitamins are similar.

Jukes™ fed a crude preparation of pantothenic acid, which he obtained
from Williams, to chicks with dermatitis and cured them. Although his
results are of limited significance because of impurity of the preparation,
they did serve to strengthen the evidence of Woolley and his coworkers.
In a later communication with a highly potent concentrate of pantothenic
acid, also obtained from Williams, Jukes*4 was able to strengthen the evi-
dence still further for the identity of pantothenic acid and the chick anti-
dermatitis vitamin by the similarity in their dissociation constants and by
the constancy in the ration of chick activity to yeast growth activity of the
pantothenic acid preparations that he fed. Final proof of the identity of

19Q. L. Kline, J. A. Keenan, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 99, 295
(1932).

20D. W. Woolley, H. A. Waisman, O. Mickelsen, and C. A. Elvehjem, J. Biol. Chem.
125, 715 (1938).

21D. W. Wooley, H. A. Waisman, and C. A. Elvehjem, J. Am. Chem. Soc. 61, 977
(1939).

22 TD). W. Wooley, H. A. Waisman, and C. A. Elvehjem, J. Biol. Chem. 129, 673 (1939).

23'T. H. Jukes, J. Am. Chem. Soc. 61, 975 (1939).

24TH. Jukes, J. Biol. Chem. 129, 225 (1939).

596 PANTOTHENIC ACID

the chick antidermatitis vitamin with pantothenic acid came with the
synthesis of pantothenic acid.**: 4%» 25» 26

Williams and Major” announced the synthesis of pantothenic acid
which was made possible by the isolation from pantothenic acid concen-
trates of the lactone moiety in pure form. They named this compound
pantoic lactone. The lactone and 6-alanine were coupled to form pure
pantothenic acid. Pure pantothenic acid thus was first prepared as a syn-
thetic compound. Its preparation clarified its relation to the “‘filtrate fac-
tor’ of the English workers*: 2? who were also well along toward its isola-
tion. They suggested that their factor and pantothenic acid were identical.”°
Pantothenic acid also seemed to be identical with factor 2 of Lepkovsky
Cid

B. CHEMICAL AND PHYSICAL PROPERTIES

Pantothenic acid is a pale-yellow viscous oil with a molecular weight of
219. It has weak basic properties. It has never been prepared in crystalline
form, but its sodium, potassium, and calcium salts crystallize readily. It is
generally distributed commercially as its calcium salt. It is readily soluble
in water, ethyl alcohol, dioxane, and glacial acetic acid.*! It is somewhat
soluble in ether acetone and amyl alcohol and practically insoluble in
benzene, chloroform, and the hydrocarbon solvents. It is hydrophilic and
is adsorbed on charcoal but not on fuller’s earth.*! It is readily esterified.
Its methyl or ethyl esters are active for animals but have little activity for
microorganisms. The vitamin activity of pantothenic acid is lost when the
OH groups in the pantoic acid part of the molecule are esterified. The
activity is regenerated by careful saponification.*! Its acetyl derivative can
be distilled at approximately 10-° mm. Hg.?°

Panthothenic acid is quite stable in neutral solution, but its activity is
destroyed by hot acid or alkaline solutions. Under these conditions, it
decomposes largely into pantoic acid or its lactone and 6-alanine.

The vitamin is optically active, owing to an asymmetric carbon atom in
pantoic lactone. Free pantothenic acid has a rotatory power [a];’ + 37.5.

25. T. Stiller, J. C. Keresztesy, and J. Finkelstein, J. Am. Chem. Soc. 62, 1779
(1940).

26}. T. Stiller, S. A. Harris, J. Finkelstein, J. C. Keresztesy, and K. Folkers, J. Am.
Chem. Soc. 62, 1785 (1940).

27 R. J. Williams and R. T. Major, Science 91, 246 (1940).

22'T. F. Macrae, A. R. Todd, B. Lythgoe, C. E. Work, H. G. Hind, and M. M. El
Sadr, Biochem. J. 33, 1681 (1939).

29 B. Lythgoe, T. F. Macrae, R. H. Stanley, A. R. Todd, and C. E. Work, Biochem.
J. 84, 1335 (1940).

30 §. Lepkovsky, T. H. Jukes, and M. E. Krause, J. Biol. Chem. 115, 557 (1936).

31H. R. Rosenberg, Chemistry and Physiology of the Vitamins. Interscience Pub-
lishers, New York, 1945.

II. CHEMISTRY 597

Its calcium salt is also dextrorotatory [a]?? + 24.3. It has an ionization
constant of about 3.9 & 1075.34 #1

C. CONSTITUTION

Pantothenic acid has the empirical formula CyHi;O;N. It consists es-
sentially of two compounds, pantoic acid and 6-alanine, combined in acid-
amide linkage by the coupling of the carboxyl group of pantoic acid with
the amino group of 8-alanine. It has been assigned the following structure.

CH;
HOCH,—_©__CHOH _C © __NHi—CH..—_€H,— CO0H
CH;
a-y-Dihydroxy-8, B-dimethyl butyryl-§-alanine

Pantoic acid has two hydroxy groups, one of them in the a- and the other in
the y position. In acid solution, especially upon heating, it forms a lactone
which is represented by the following structure.**: 4%

a-Hydroxy-8, 8-dimethyl-y-butyrolactone®
D. SYNTHESIS

A partial synthesis of pantothenic acid was effected before its struc-
ture was known. The total synthesis of pantothenic acid involves essentially
the synthesis of pantoic acid or its lactone followed by its condensation
with 6-alanine. The synthesis of pantoic acid is carried out as follows.*!
Isobutyraldehyde is condensed with formaldehyde to give a,a-dimethyl-
B-hydroxy-propionaldehyde. This compound is condensed with hydro-
cyanic acid or potassium cyanide in the presence of calcium chloride to
form racemic a-hydroxy-@ ,6-dimethyl-y-butyrolactone. This lactone may
then be condensed with @-alanine to form racemic pantothenic acid. Both
the racemic pantothenic acid and the pantoic lactone can be resolved by
the fractional crystallization of their quinine salts.*! The salts of quinine
methohydroxide, quinidine methohydroxide, and cinchonine methohy-
droxide have also been used to resolve racemic mixtures of these com-
pounds.** #4

Many methods are available for the condensation of pantoic lactone with
B-alanine. A simple and effective method is the direct condensation of

598 PANTOTHENIC ACID

B-alanine with the lactone. An approximately theoretical yield may thus
be obtained. This type of condensation has also been carried out with the ©
dry sodium salt of 6-alanine and yields directly the sodium salt of panto-
thenic acid. Pantothenic acid has also been obtained by the condensation
of the lactone with 6-alanine benzyl ester followed by the catalytic hydro-
genation of the pantothenic benzyl ester to free pantothenic acid.3: 4% *

III. Biochemical Systems
FRITZ LIPMANN

A. INTRODUCTION

In 1943 Roger Williams wrote in Advances in Enzymology: “The pre-
sumption, in view of the known function of other vitamins, is that panto-
thenic acid fits into some enzyme system or systems which is essential to
metabolism. What this enzyme system is or what these systems are is not
known. There are some facts which suggest that pantothenic acid may be
concerned with carbohydrate metabolism, but it is not certain.”

At that time, other than a slight connection between carbohydrate
storage and pantothenic acid,? found in Dr. Williams’ laboratory, there
existed only one promising observation, namely, a demonstration of a
specific stimulation of pyruvic acid oxidation by pantothenic acid in Proteus
morganit by Dorfman eé al.’ in this country, and independently by Hills*
in England. However, this observation remained an isolated fact for several
years.

In 1946, during the study of the acetylation of sulfonamide in cell-free
systems of pigeon liver, Lipmann®: © observed that a coenzyme was in-
volved in this reaction, which could not be replaced by any of the then
known coenzymes. Independently, in the study of acetylcholine synthesis
in brain extracts, Feldberg and Mann’ and Nachmansohn and Behrman®
found that for acetylation of choline a stable cofactor seemed necessary.
In view of the indication that a novel factor may be involved in acetylation
reactions, an isolation of the coenzyme was started and after preliminary
purification it was shown, as illustrated by Fig. 1, that the cofactors for

1R. J. Williams, Advances in Enzymol. 3, 253 (1948).

2R. J. Williams, W. A. Mosher, and E. Rohrmann, Biochem. J. 30, 2036 (1936).
3 A. Dorfman, 8. Berlman, and 8. A. Koser, J. Biol. Chem. 144, 393 (1942).

4G. M. Hills, Biochem. J. 37, 418 (1948).

5 F. Lipmann, J. Biol. Chem. 160, 173 (1945).

6 F. Lipmann and N. O. Kaplan, Federation Proc. 5, 145 (1946).

7W. Feldberg and T. Mann, J. Physiol. 104, 411 (1946).

8 D. Nachmansohn and M. Behrman, J. Biol. Chem. 165, 551 (1946).

III. BIOCHEMICAL SYSTEM 599

A

8

= 2
5 =
= 2
= i: 4
= ¥

a
3 2000 |
oT ~
5 3
2

=
= 5
5 ¢
o 1000
by

0 10 20

50
7 COENZYME

Fig. 1. Coenzyme A, the common activator of sulfanilamide acetylation in liver
extract and choline acetylation in brain extract.®* A highly purified preparation of
CoA was used.

the acetylation of sulfonamide in liver and for the acetylation of choline in
brain were identical.® An effort was then made to purify the coenzyme more
rigorously, and preparations were obtained which appeared to be far
enough advanced in purity to justify a chemical study of the product.

B. IDENTIFICATION OF PANTOTHENIC ACID AS
PART OF COENZYME A

1. CoA%® Assay

As a convenient assay system for the coenzyme, the sulfanilamide ace-
tylation system in liver was used.!° In this enzyme system, sulfanilamide is
acetylated in the presence of ATP and acetate. After autolysis of the ace-
tone-pigeon liver extract for 3 hours at room temperature, the originally
present CoA is completely decomposed. The autolyzed extract serves as
apoenzyme for measuring CoA. As a unit of CoA, such an amount was
defined as giving a half reactivation of the apoenzyme system under spec-
ified conditions. A standard curve obtained with this system is shown in
Fig. 2. It may be seen that between 0.5 and 1.5 units the curve is nearly a

9F,. Lipmann and N. O. Kaplan, J. Biol. Chem. 162, 743 (1946).

9¢ F, Lipmann, Advances in Enzymol. 6, 231 (1946).

% The following abbreviations are used: AMP = adenosine monophosphate; ADP =
adenosine diphosphate; ATP = adenosine triphosphate; CoA = coenzyme A; P =
phosphate; PP = pyrophosphate; and Ac = acetate, acetyl.

10N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948).

600 PANTOTHENIC ACID

50

Xo

i =1 unit

25

Sulfanilamide acetylated, 7

Units coenzyme A

Fig. 2. Coenzyme A assay. Concentration-activity curves for coenzyme A prepa-
rations of different purity. The arrow indicates the half-reactivation point which
represents our unit. O = crude coenzyme, 0.25 unit per milligram; X = purified
coenzyme, 130 units per milligram.

straight line. A modified assay procedure with the same enzyme solution
using the color change caused by acetylation of 4-aminoazobenzene was
recently described by Handschumacher et al.!! This color change may be
measured directly. On the same principle, Bessman™? in our laboratory re-
cently worked out a more convenient procedure using the much more solu-
ble 4-aminoazobenzene sulfonate.

In view of the likelihood that a new coenzyme may contain one of the
vitamins not covered then by coenzyme activity, the preparation was
analyzed for its vitamin content. Fortunately, in this task we were helped
by the experience of Dr. Roger Williams’ laboratory with vitamin assay —
in general, and in particular with pantothenic acid assay. Dr. Beverly
Guirard, who carried out the analysis of our coenzyme preparations, found
little or no activity with Lactobacillus arabinosus. However, after incubation
for a prolonged period with clarase and papain—at that time considered
enzymes which liberated the “protein-bound” pantothenic acid of natural
sources—a small amount started to appear, still only of the order of a

1 R. E. Handschumacher, G. C. Mueller, and F. M. Strong, J. Biol. Chem. 189, 335
(1951).

la §. P. Bessman and F. Lipmann, Arch. Biochem. and Biophys. 46, 252 (1953);
ef. also M. E. Jones, S. Black, R. M. Flynn, and F. Lipmann, Biochim. et Biophys.
Acta 12, 143 (1953).

III. BIOCHEMICAL SYSTEM 601

fraction of a per cent. This was, however, enough to make Dr. Guirard
suspect that pantothenic acid might be present but might not be liberated
effectively by the procedure used. She therefore tested for the presence of
B-alanine in the acid hydrolyzate of the coenzyme preparations; indeed,
the yeast assay showed G-alanine to be present in amounts corresponding
to a pantothenic acid content of 11%. 8 In view of the rare occurrence of
G-alanine, it was then considered almost certain that pantothenic acid was
part of CoA.

Because of the instability of pantothenic acid to alkali or acid, chemical
hydrolysis of CoA could not be applied for its liberation. Eventually the
unhinging of pantothenic acid from its links in CoA was accomplished by
enzymatic hydrolysis with intestinal phosphatase in conjunction with a
particular peptidase present in bird liver," in pig kidney,'® and, to a lesser
extent, in other organs.

2. RELATION OF THE ENZYMATICALLY DETERMINED CoA UNIT
To Its PANTOTHENIC AcID CONTENT

A unit of CoA corresponds to 0.7 y of pantothenic acid per milligram.”
This ratio has been of great value for assessment of purity; | uM. of CoA
containing 1 uM. of pantothenic acid corresponds to 310 units of CoA.
The molecular weight of the coenzyme was earlier determined by the
Northrup diffusion method!* to be around 800. Therefore, pure CoA was
expected to contain approximately 400 units per milligram. Analytical
data obtained with our purest preparations,!” which will be discussed below
in detail, indicate a molecular weight of 767 and a content of 410 units
per milligram of pure CoA, in excellent agreement with earlier predictions.

3. ARSENOLYSIS ASSAY

During the work on isolation, we became aware that the assay system
with crude pigeon liver extract is not specific for intact CoA, but responds
to certain fragments.’ This is due to enzymatic resynthesis of CoA from
partially dephosphorylated coenzyme in the presence of ATP. Products
which may be resynthesized to CoA have been obtained by controlled

122, Lipmann, N. O. Kaplan, G. D. Novelli, L. C. Tuttle, and B. M. Guirard, J.
Biol. Chem. 167, 869 (1947).

13 F, Lipmann, N. O. Kaplan, G. D. Novelli, L. C. Tuttle, and B. M. Guirard, J.
Biol. Chem. 186, 235 (1950).

14G. D. Novelli, N. O. Kaplan, and F. Lipmann, J. Biol. Chem. 177, 97 (1949).

15 T,, Levintow and G. D. Novelli, Abstr. Papers, Meeting Am. Chem. Soc. Atlantic
City, p. 38C (Sept. 14-19, 1952).

16 G. D. Novelli, R. M. Flynn, and F. Lipmann, J. Biol. Chem. 177, 493 (1949).

17 J. D. Gregory, G. D. Novelli, and F. Lipmann, J. Am. Chem. Soc. 74, 854 (1952).

18 G. D. Novelli, N. O. Kaplan, and F. Lipmann, Federation Proc. 9, 209 (1950).

602 PANTOTHENIC ACID

ACETYLP

ie) 5 10 15 20 25
CoA UNITS ADDED

Fig. 3. Proportionality of arsenolysis with coenzyme A concentration. The acetyl
phosphate values represent micromoles of acetyl phosphate decomposed in 15 min-
utes. Coenzyme A values are recorded in units per milliliter. Conditions: 0.05 M
potassium arsenate; 0.1 tris(hydroxymethyl)aminomethane (pH 8.0); 22 uM. acetyl
phosphate; 0.01 M cysteine; 0.002 M MgCl. ; 0.5 mg. of enzyme (from Lot L); am-
monium sulfate fraction, between 70 and 90% saturation. Final volume 1 ml. (Drawn
from Table IV in Stadtman e¢ al.?!2)

degradation. This procedure has been useful in the study of the structure,
in particular, in conjunction with assay procedures more specific for intact
CoA. As such, the arsenolysis assay!® has been very important.

In many microorganisms, in particular, in Clostridium kluyveri, an
enzyme, transacetylase,!*: 2° is present which equilibrates acetyl phosphate
and CoA to acetyl-CoA and phosphate. If arsenate is added to this system,
it equilibrates with arsenate, yielding acetyl arsenate, which decomposes
immediately.?° The arsenolysis of acetyl phosphate is CoA-dependent and
may be measured easily by following acetyl phosphate decomposition
with the hydroxamic acid reaction. CoA can be removed from microbial
extracts by shaking with Dowex-1 chloride. 2! The Dowex-treated apo-
enzyme may be used for CoA assay. Over a wide range, the rate of acetyl
phosphate disappearance is proportional with CoA concentration, as shown

19K. R. Stadtman, G. D. Novelli, and F. Lipmann, J. Biol. Chem. 191, 365 (1951)
20 K. R. Stadtman and H. A. Barker, J. Biol. Chem. 184, 769 (1950).

21 WH. Chantrenne and F. Lipmann, J. Biol. Chem. 187, 757 (1950).

1a Fi, R. Stadtman, M. Doudoroff, and F. Lipmann, J. Biol. Chem. 191, 377 (1951).

III. BIOCHEMICAL SYSTEM 603

in Fig. 3. In contrast to the pigeon liver assay, the arsenolysis assay is
sharply specific for intact CoA.

4. OTHER CoA Assay SYSTEMS

Assay systems have recently been devised where CoA concentration may
be measured spectrophotometrically in CoA-dependent DPN-reducing
systems. Sanadi and Littlefield in Green’s laboratory”: * found that DPN
reduction by ketoglutarate in a muscle extract is CoA-dependent, and this
system may be used for assay. Stadtman*™ found that the acetaldehyde-
DPN reaction in extracts of Clostridium kluyveri is CoA-dependent, and
this system likewise represents a promising assay system.

C. THE CHEMISTRY OF COENZYME A
1. PHOSPHATE AND ADENYLIC AcID CONTENT

Nearly pure preparations of CoA contain 1 mole of adenosine and 3
moles of phosphate per mole of pantothenic acid.** Only 1 of the 3 moles
of phosphate is split off by phosphomonoesterases (prostate phosphatase) .!”
The partially dephosphorylated product is easily rephosphorylated by
ATP through a kinase present in pigeon liver extract (Novelli?®). This
fragment, therefore, assays in pigeon liver acetylation assay as CoA but
is inactive in the arsenolysis assay. It may be reactivated for arsenolysis
by incubation with ATP in pigeon liver extract. The monoester-linked
phosphate is located at the ribose of adenosine (Baddiley’’).

Kaplan discovered an enzyme which specifically attacks a nucleotides
(-phospho-2-), but not the b form (-phospho-3-).?8 With this enzyme he
studied the position of the monoester-linked phosphate in CoA and found
that it corresponded to the b nucleotide series. Interestingly enough, the
monoester phosphate in TPN corresponds to the a series, according to his
analysis.

If CoA is treated with an enzyme preparation from potato similar to
the nucleotide pyrophosphatase of Kornberg,?° it is inactivated for arseno-
lysis, but not for crude pigeon liver assay.2* The pH optimum for the

22H. Beinert, R. W. Van Korff, D. E. Green, D. A. Buyske, R. E. Handschumacher,
H. Higgins, and F. M. Strong, J. Am. Chem. Soc. 74, 854 (1952).

2372). R. Sanadi and J. W. Littlefield, J. Biol. Chem. 198, 683 (1951).

24R. M. Burton and E. R. Stadtman, J. Biol. Chem. 202, 873 (1953).

25 W. H. DeVries, W. M. Govier, J. S. Evans, J. D. Gregory, G. D. Novelli, M.
Soodak, and F. Lipmann, J. Am. Chem. Soc. 72, 4838 (1950).

26 G. D. Novelli, in Phosphorus Metabolism, Vol. I, p. 414. Johns Hopkins Press,
Baltimore, 1951.

27 J. Baddiley and E. M. Thain, J. Chem. Soc. 1951, 3421.

28 T,. Shuster and N. O. Kaplan, Federation Proc. 11, 286 (1952).

29 A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 182, 763 (1950).

604 PANTOTHENIC ACID

splitting of the pyrophosphate bridge in CoA, however, was found to be
different from that of the pyridine nucleotides, being 4.5 rather than 7.76
No phosphate, however, may be liberated with this enzyme. If the nucleo-
tidase-treated preparation is exposed, subsequently, to phosphomonoester-
ase, then all three phosphates are split off and almost complete inactivation
for all assay systems results. In analogy to the results of Kornberg on DPN
and, in particular, on TPN with a similar enzyme,” it is concluded that the
nucleotidase splits a pyrophosphate bridge between a phosphopantothenate
derivative and diphosphoadenosine. The phosphopantothenate derivative,
now identified as phosphopantothenylthioethanolamine,*® was found to be
resynthesized to CoA with ATP and pigeon liver enzyme.*!

Evidence for the pyrophosphate bridge is further offered by the complete
inactivation with intestinal phosphatase, which, by virtue of its phos-
phodiesterase activity, opens the bridge and dephosphorylates completely.
The same effect obtains as mentioned, by a combination of dinucleotidase
treatment and partial dephosphorylation.?’> *

The nature of the phosphate attachment to pantothenic acid has recently
been settled definitely after pantothenate-2’-phosphate, pantothenate-
4’-phosphate, the diphosphate, and the cyclic phosphate had been syn-
thesized and made available for comparison.”*: ** Baddiley chromatograph-
ically identified a fragment of CoA obtained by cautious alkaline hydrolysis
with pantothenate-4’-phosphate. This observation had already made it
most likely that the pyrophosphate bridge links between the 5’ position
on ribose and the 4’ position on pantothenic acid.?’: *: *> Recently, Bad-
diley and Thain** have synthesized pantethine-4’-phosphate. This was
established as a fragment of CoA by enzymatic synthesis to CoA in a
pigeon liver fraction isolated by Levintow and Novelli!® which acts specif-
ically on phosphorylated pantethine.*®

Final proof for the structure outlined was obtained recently by Hoagland
and Novelli,*® who synthesized dephospho-CoA from ATP and synthetic
pantetheine-4’-phosphate and showed equivalent liberation of pyrophos-
phate according to the following equation:

30 #}. E. Snell, G. M. Brown, V. J. Peters, J. A. Craig, E. L. Wittle, J. A. Moore,
V.M. MecGlohon, and O. D. Bird, J. Am. Chem. Soc. 72, 5349 (1950).

31. W. M. Govier and A. J. Gibbons, Arch. Biochem. and Biophys. 32, 347 (1951).

32 G. D. Novelli, J. D. Gregory, R. M. Flynn, and F. J. Schmetz, Jr., Federation Proc.
10, 229 (1951).

33'T. EH. King and F. M. Strong, J. Biol. Chem. 189, 315 (1951); 191, 515 (1951).

34 J. Baddiley and EK. M. Thain, J. Chem. Soc. 1951, 246.

35 J. Baddiley and E. M. Thain, J. Chem. Soc. 1951, 2253.

36 J. Baddiley, E. M. Thain, G. D. Novelli, and F. Lipmann, Nature 171, 76 (1953).

36¢ M. B. Hoagland and G. D. Novelli, J. Biol. Chem. 207, 767 (1954).

III. BIOCHEMICAL SYSTEM 605

Pantetheine-4’-P ++ adenosine-P-P-P — Pantetheine-

(1)
4’-P-P-5-adenosine (= dephospho-CoA) + PP
Dephospho-CoA was then converted to CoA with ATP.
Pantetheine-4’-P-P-adenosine + ATP pantetheine-4’- 2)

P-P-5-adenosine-3-P (= CoA)

The enzymatic synthetis has been reviewed expertly by Novelli in the
CoA Symposium at the Federation Meeting of 1953.°°

2. THE SuULFUR-CONTAINING MOIETY

It was early observed that CoA preparations contained sulfur in di-
sulfide linkage." 97 The preparations gave a typical purple nitroprusside
reaction for SH-compounds, but only after reduction, for example, by treat-
ment with cyanide. The preparation in which the presence of pantothenic
acid was discovered! contained 1.97% sulfur and 11% pantothenic
acid, corresponding to 1.2 moles of sulfur per mole of pantothenic acid.

Initially, however, so much attention was centered on the evaluation of
the presence of pantothenic acid in this coenzyme that little attention was
paid to the sulfur-containing part. Eventually, through a number of cir-
cumstances, information was gained on the nature of the compound as well
as on its linkage to pantothenic acid. Very stimulatory in this respect was
the discovery by Williams eé al.® of a new growth factor for Lactobacillus
bulgaricus (LBF). This factor was identified as a pantothenic acid derivative
and a fragment of CoA.*9 4° Brown ef al. showed that intact CoA was in-
active as their factor.*? However, CoA could be activated by dephosphoryla-
tion with intestinal phosphatase. Very significant was the further observa-
tion by Snell’s group that the bird liver enzyme, which we had found to be
necessary for complete liberation of pantothenic acid from CoA, destroyed
LBF activity. By following the lead of these observations on Snell’s new
factor, we obtained by degradation of CoA with intestinal phosphatase
and subsequent chromatography” a product containing the pantothenic
acid linked to a ninhydrin-reactive sulfur compound which, however, was
not cystine, as had previously been tentatively assumed. Chromatograph-
ically, this degradation product behaved like LBF. A chromatogram of
intestinal phosphatase-treated CoA is shown in Fig. 4.

366 G. D. Novelli, Federation Proc. 12, 675 (1953).

37 F. Lipmann, N. O. Kaplan, and G. D. Novelli, Federation Proc. 6, 272 (1947).

38 W. L. Williams, E. Hoff-Jgrgensen, and E. E. Snell, J. Biol. Chem. 177, 933 (1949).
39 G. M. Brown, J. A. Craig, and E. E. Snell, Arch. Biochem. 27, 473 (1950).

40 R. A. McRorie, P. M. Masley, and W. L. Williams, Arch. Biochem. 27, 471 (1950).

606 PANTOTHENIC ACID

Independently, in a brilliant piece of work, Snell and his associates
arrived,°*° by isolation and synthesis, at a final identification of the LBF
fragment of CoA. They found it to be N-pantothenylthioethanolamine;
this compound was named ‘‘pantetheine” for the sulfhydryl form and
“nantethine” for the disulfide form. A new and relatively simple method
of synthesis for pantethine was recently described by Baddiley.*! #

AZ <- PANTETHINE

| eA

NITROPRUSSIDE GROWTH OF
REACTION ! A, SUBOXIDANS

Fia. 4. Paper chromatogram of phosphatase-treated coenzyme A (cf. ref. 18)-
The right-hand side represents the assay for pantothenic acid in all bound forms,
using Acetobacter suboxydans.

The left-hand side represents the disulfide test by cyanide-nitroprusside spray.

By this identification at the same time, the sulfur compound in CoA
was identified as the decarboxylation product of cysteine, also known under
the name of “cysteamine”’, a short and handy name. The synthesis of
pantetheine has been studied by various workers and appears to involve a
condensation of pantothenic acid with cysteine. This is followed by de-
carboxylation of the pantothenyl-cysteine to yield pantethine eventually.

Some differences between the synthetic compounds and substances iso-
lated from natural materials have been explained through a cross-linking of
various SH- compounds to reduced pantethine by simultaneous oxidation

41 J. Baddiley and E. M. Thain, J. Chem. Soc. 1951, 3425.
42 J. Baddiley and E. M. Thain, J. Chem. Soc. 1952, 800.
43 G. Medes and N. Floyd, Biochem. J. 36, 2591 (1942).

——

1 ae ee ER ret

A.

III. BIOCHEMICAL SYSTEM 607

in the process of isolation.*4 Such S-S- cross-links also contaminate all
cruder CoA preparations.!”: 4°

Govier and Gibbons*! obtained an independent support for the presence
of pantethine in CoA. They found that aged pigeon liver extract could
synthesize small but appreciable amounts of CoA from synthetic pan-
tethine + ATP. Levintow and Novelli!® in this laboratory recently found
that such a synthesis occurs much more readily in very fresh liver extracts.

3. AcETYL-CoA

The analysis of various acetylation systems involving CoA had led to
the recognition of its acetyl carrier function, a shuttling back and forth
between acetyl donor and acceptor systems.!* 4% 47 Recently, Lynen and
Reichert*: 4° succeeded in isolating acetyl-CoA in substance from yeast.
They furthermore became aware of the possibility that the SH-group in
CoA}: ?> may be the point of attachment of the acetyl group. In confirma-
tion, they could show in a brilliant piece of work that acetyl-CoA, which is
nitroprusside negative, becomes SH-positive by such alkali treatment as
is known to split the mercaptoesters. The nature of the acetyl-S-CoA link
was further verified by the lability of the acetyl group in CoA to mercuric
salt, a rather specific property of mercaptoesters.®° It reminds one interest-
ingly enough of the similar behavior of enol phosphate, which likewise is
easily split catalytically by mercuric salts.

Lynen and Reichert also showed that, with the acceptor enzyme of liver
extract, acetyl-CoA donates acetyl to sulfanilamide, and that SH- is
liberated by this enzymatic acetyl transfer. Acetyl-CoA was furthermore
identified as acetyl donor in citric acid synthesis.*!

More recently, Stadtman,” in continuation of earlier experiments on
transacetylase, worked out a rather efficient enzymatic method for the
preparations of acetyl-CoA. He used acetyl phosphate with the trans-
acetylase, for example, of Clostridiwm kluyveri, as the acetyl donor system,
with which CoA can be preparatively acetylated. Acetyl phosphate is
destroyed by heating at a pH of 4.5, whereas acetyl-CoA is stable. M. E.
Jones, in our laboratory, found that the product of enzymatic acetylation,

44G.M. Brown and E. E. Snell, Proc. Soc. Exptl. Biol. Med. 77, 138 (1951).

45 J. D. Gregory and F. Lipmann, Abstr. Papers 12th Intern. Congr. Pure and Appl.
Chem. New York, p. 74 (1951).

46 T. C. Chou, G. D. Novelli, E. R. Stadtman, and F. Lipmann, Federation Proc. 9,
160 (1950).

47}. R. Stadtman, Federation Proc. 9, 233 (1950).

48 F, Lynen and E. Reichert, Angew. Chem. 68, 47 (1951).

49 F, Lynen, E. Reichert, and L. Rueff, Ann. 574, 1 (1951).

50 G. Sachs, Ber. 54, 1849 (1921).

518. Ochoa, J. R. Stern, and M. C. Schneider, J. Biol. Chem. 198, 691 (1951).

608 PANTOTHENIC ACID

TABLE [7

Calcd., %” Found, % Ratio

Pantothenic acid 28.6 26.8 (enzymatic assay) if
25.6 (microbiological)

Adenine AG 17.0 (spectrophotometric) 1.05
Phosphorus (total) 12.12 10.6 2.83
Monoester phosphorus? = 3.6 0.96
Sulfur 4.18 4.13 1.07

@ Pantothenic acid, 2-mercaptoethylamine, 3-phosphoric acid, adenosine, —5H20; molecular weight 767.
» Liberated by prostate phosphomonoesterase.

acetyl-CoA, may be purified easily by resin chromatography. She obtained
in this manner practically pure acetyl-CoA.

A very convenient method for a preparation of various CoA derivatives
was recently described by Simon and Shemin,°*!* using the great affinity of
SH-compound for acid anhydrides. For example, they synthesized, in
aqueous solution, succinylSCoA from CoA-SH and succinic anhydride in
excellent yield, using only a slight excess of anhydride. This reaction is very
generally applicable.

4. THe STRUCTURE OF CoA

Analytical data on a nearly pure CoA preparation” are represented in
Table I. The data agree with the composition of pantothenic acid, thio-
ethanolamine, adenosine, and three phosphates, minus 5H2O. In con-
junction with this analytical data, the results of structural analysis pre-
sented in the previous paragraphs lead to the formulation shown in Fig. 5.
(This structure is now definitely established by analysis as well as by en-
zymatic synthesis. )

To summarize, the biosynthesis of CoA from pantothenic acid involves
the following steps:

1. Pantetheine, by condensation of pantothenic acid and cysteine, with
subsequent decarboxylation.

2. Phosphorylation of pantetheine in the 4’-position by ATP. This reac-
tion is catalyzed by Kinase I, which was partially purified from pigeon liver
extract.

3. Condensation of pantetheine-4’-phosphate and ATP, with elimina-
tion of inorganic pyrophosphate, by a condensing enzyme in pigeon liver
extract. The resulting product is ‘‘dephospho-CoA” also obtained from
CoA by dephosphorylation with prostate phosphomonoesterase.

4. The final step is catalyzed by Kinase II, which phosphorylates de-
phospho-CoA in the 3-position on the ribose part of adenylic acid.

51a #}, J. Simon and D. Shemin, J. Am. Chem. Soc., 75, 2590 (1953).

III. BIOCHEMICAL SYSTEM 609

GH, OH |
GHe—G —GH-C. ,CHe-GHe.
j CH, Nay 07°- NH-GH,-CH,- SH
u

-O

Fic. 5. The structure of coenzyme A.

For further details, the reader is referred to Novelli’s review in the CoA
Symposium of the Federation Meeting, 1953.5?

5. PREPARATION OF CoA

The early preparations of CoA were obtained from pig liver.” #: 7 In
view of the easy destruction of CoA by autolysis, only very fresh pig liver
can be used for the isolation. A rather complicated procedure was originally
used, involving metal precipitation and acetone fractionation.’ More
recently, in collaboration with Dr. Evans, Dr. Govier, and Mr. DeVries
ot the Upjohn Research Laboratory, a simpler procedure was elaborated
for isolation of CoA from cultures of Streptomyces fradiae.?® The coenzyme
was repeatedly adsorbed from acid solution on charcoal and eluted with
acetone-ammonia.

Final purification was worked out by Gregory after it had been observed
that a reduction step had to be included in the purification procedure to
remove cross-linking from sulfur compounds.*® The best preparation de-
scribed in the previous paragraph was obtained by following up the adsorp-
tion procedure with reduction with zine and hydrochloric acid and mercury
precipitation.” After removal of mercury by hydrogen sulfide, the solution
was chromatographed over Duolite-CS 100. Most of the impurities were
removed by washing with hydrochloric acid, and the coenzyme was even-
tually eluted with water and freeze-dried.

More recently, Green, Strong,” and their group at the University of
Wisconsin described a relatively simple, promising isolation procedure.
Yeast was used as the starting material, and preliminary purification was

5b G. D. Novelli, Federation Proc. 12, 675 (1953).

610 PANTOTHENIC ACID

carried out by charcoal adsorption as described by Gregory e¢ al.!7: 2° The
prepurified material was then coprecipitated with reduced glutathione
by the Hopkins reagent, cuprous oxide. The glutathione was removed by
resin chromatography. In this manner, in a rather good yield, 75% pure
CoA was obtained.

A very good preparation of CoA, averaging 360 units per milligram (about
75 to 80% pure) is now commercially available from the Pabst Labor-
atories, 1037 McKinley Avenue, Milwaukee 3, Wisconsin.

D. SOME NUTRITIONAL ASPECTS OF THE CoA PROBLEM

1. Assay oF PANTOTHENIC AcID BASED ON THE METHODOLOGY
OF PANTOTHENIC AcID LIBERATION FROM CoA

The difficulties encountered with the liberation of pantothenic acid
from its coenzyme, which have been described above, eventually resulted
in the elaboration of the dual enzyme treatment." Since, as shown in Table
II, fresh tissues contain almost solely CoA and very little free pantothenic
acid, it is obvious that the methodology for liberation of pantothenic acid
from the coenzyme should be generally applicable for microbiological
determination of pantothenic acid in natural sources, including foodstuffs.
With the treatment by intestinal phosphatase and liver enzyme, the first
fully satisfactory method for a complete liberation of pantothenic acid
from natural sources was developed as an accidental result of our studies
on CoA. The data in Table III may serve as an example of the effective-
ness of the method for reaching all bound pantothenic acid.

The importance of this method for the evaluation of true pantothenic
acid content in food is amplified by the work of Neilands and Strong,”
who compared the old clarase-papain procedure with the phosphatase-liver
enzyme method (cf. Table III). They found that the new method yielded
in some cases up to four times as much pantothenic acid as the old one. A
somewhat troublesome high blank in the liver enzyme has recently been
eliminated by Novelli and Schmetz** through Dowex treatment of the liver
enzyme solution. Recent work in our laboratory by Levintow indicates
that pig kidney may even be a better and more convenient source of the
second enzyme than bird liver.

Earlier discrepancies of pantothenic acid values obtained with the chick
and microbiological assays* have now found their explanation in the dif-
ference in availability of CoA-bound pantothenic acid. Hegsted and Lip- —

52 J. B. Neilands and F. M. Strong, Arch. Biochem. 19, 287 (1948).
53 G. D. Novelli and F. J. Schmetz, Jr., J. Biol. Chem. 192, 181 (1951).
54'T. H. Jukes, Biol. Symposia 12, 253 (1947).

III. BIOCHEMICAL SYSTEM 611

TABLE II"
RELATION OF COENZYME A ACTIVITY AND PANTOTHENIC ACID CONTENT IN ORGANS
oF RABBIT
(All values are given per gram of wet weight of tissue)

Pantothenic acid, y

Bound
Coenzyme A, oo
units Free Total Found Calculated®
Liver 112 2 75 74 73
Heart 26.4 ono 20.7 17 17
Kidney 49.5 2.7 45 42 32
Brain (cortex) 40.5 3.0 18 15 26
Testes 25.6 6.0 20.4 14 ily
5.1 9.9 5 4

Muscle (skeletal) 6

2 Calculated by multiplication of the unit value by 0.65, the average pantothenic acid content in micro-
grams of a unit of coenzyme A.

mann® could show that the chick almost completely utilized CoA-bound
pantothenic acid. Therefore, in contrast to most microorganisms, the ani-
mal body is able to utilize the vitamin fully when bound in the coenzyme.

2. THe ALKALI STABILITY oF “NATURAL PANTOTHENIC ACID”

It has been observed by various workers that natural pantothenic acid
in tissues and other fresh material resists alkali treatment much better
than free pantothenic acid, where the peptide bond between the 6-alanine
and pantoic acid is very labile to alkali (Jukes). Strong and King,** and,
independently, Baddiley,*> have now explained this discrepancy. They
show that phosphorylation of pantothenic acid confers to it resistance
to alkali. Since pantothenic acid in CoA is phosphorylated, the alkali
resistance now is explained through the presence of the phosphorylated
pantothenic acid derivative coenzyme A.

EK. SPLIT PRODUCTS OF CoA AS MICROBIAL
GROWTH FACTORS

In two instances a growth factor has been identified as a fragment of
CoA. These are the Acetobacter suboxydans factor (PAC) of Cheldelin!®: °°
and the Lactobacillus bulgaricus factor (LBF, pantethine), of Snell and
Williams.*°: #9 49 In a stricter sense, both factors are rather to be termed
growth stimulants. Acetobacter will grow relatively easily with pantothenic
acid, although with a pronounced lag period. The Lactobacillus bulgaricus
family responds poorly or not at all to small amounts of pantothenic acid
but will grow if rather large quantities are supplied. Since both organisms

55 T). M. Hegsted and F. Lipmann, J. Biol. Chem. 174, 89 (1948).
56 T. EK. King, I. G. Fels, and V. H. Cheldelin, J. Am. Chem. Soc. 71, 131 (1949).

612 PANTOTHENIC ACID

TABLE III

PANTOTHENIC AcID RELEASED FROM BIOLOGICAL MATERIALS BY
Various TREATMENTS?

Liver enzyme plus phosphatase® Mylase P None
Enzyme treatment Pantothenic acids, y/g.
A B

Spinach 27.5 30.0 11.5 Coz
21.8 23.6 11.4 7.8
Alfalfa 41.0 65.0 43.8 29.8
46.8 58.7 37.9 26.0
Wheat germ meal 30.0 30.0 15.2 14.6
29.3 28.6 17.2 17.3
Beef, dried 33.3 46.6 16.7 6.9
45.6 64.6 17.3 8.9
Whole liver powder 365 400 70.0 50.4
343 386 67.5 53.7
Yeast, dried 400 400 106 57.0
443 486 100 58.8

Vitab 385 400 132 121

418 436 138 130
Egg, fresh 50.7 53.3 13.0 9.9
49.0 57.4 12.6 10.4
Sardines, Pacific® 15.0 1255 6.0 5.0
18.4 19.3 6.0 3.9
Tuna — — 4.2 ii
8.4 14.3 4.2 igi
Sardines, Atlantic 12.0 11.5 4.7 4.1
lod 19.0 4.7 4.0
Mackerel, Pacific 11.3 1150 Ae 2.7
15.9 16.8 4.7 2.5
Salmon Se — 5.8 4.8
20.9 26.4 5.8 4.8
Mackerel, Atlantic 11.3 — 3.1 2.7
12.0 19.3 3.1 2.4

* In column B the substrate concentration was half that in column A. Enzyme concentrations were
identical.
» All the fish samples assayed were canned products.

III. BIOCHEMICAL SYSTEM 613

convert pantothenic acid into its functional form, namely, CoA, the growth
effect of the CoA fragment indicates the lack of one of the enzymatic
reactions needed for the synthesis of CoA from free pantothenic acid.
The sluggish response of both Acetobacter suboxydans an Lactobacillus
bulgaricus to pantothenic acid indicates a certain capacity for adaptive
formation of the lacking enzymatic step.

1. PANTOTHENIC AcID CONJUGATE OF CHELDELIN

In the case of the Acetobacter suboxydans factor, Cheldelin showed that
it is destroyed by intestinal phosphatase.**® It was found, subsequently,
that intact CoA as well as some of its fragments are active, and it was con-
firmed that intestinal phosphate destroys activity or reduces it to a low
level. During studies on the constitution of CoA, as already mentioned,
Novelli studied the Acetobacter factor in detail.2* He found that, if the
organism had been grown on CoA, it would respond best to intact CoA.
But if it had been grown on partially decomposed CoA, it would respond
most actively to a phosphorylated fragment obtained by treatment with
potato dinucleotidase, by mild acid hydrolysis, or with an enzyme ob-
tained from snake venom. This Acetobacter factor has now been identified
as phospho-4-pantethine which was recently synthesized by Baddiley and
Thain and biologically tested in this laboratory.*®

Acetobacter is the only organism known so far which responds reasonably
well to all bound forms of pantothenic acid. This organism has frequently
been used in this laboratory for a qualitative detection of pantothenic
acid in all bound forms, e.g., in chromatograms of CoA fragments.”®

2. PANTETHINE (Lactobacillus bulgaricus Factor)

In 1947, Williams et al.** discovered a new growth factor for Lactobacillus
bulgaricus. Snell et al.?? and Williams et al.4° independently observed that
large amounts of pantothenic acid would replace the factor. In their con-
centrates, pantothenic acid was found to be present in a bound form. As
mentioned already, Snell and his group compared this factor with CoA.**
They found that the intact coenzyme was inactive. But dephosphorylation
with intestinal phosphatase liberated a very active compound comparable
to their factor. On the other hand, treatment with liver enzyme completely
destroyed the activity of the LBF. This group of organisms, therefore,
seemed to be lacking in the enzyme which synthesizes the link to panto-
thenic acid, specifically hydrolyzed by the liver enzyme. This is now known
to be a peptidic link between pantothenic acid carboxyl and the amino
group of cysteamine. In summary, the anomaly in Lactobacillus bulgaricus
appears to be the lack of or slow adaptation to the enzymatic synthesis of
the link between thioethanolamine (or rather cysteine; cf. above) and the
carboxyl group of pantothenic acid.

614 PANTOTHENIC ACID

In conclusion, it seems worth mentioning that the two fragments of CoA,
which are active as growth factors, mirror, in the direction of synthesis,
the two degrading steps essential for the liberation of pantothenic acid
from CoA. The Acetobacter factor is destroyed by intestinal phosphatase
and the Lactobacillus bulgaricus factor by bird liver “peptidase,” the com-
bination of which is necessary to liberate pantothenic acid fully for the assay
with Lactobacillus arabinosus or Proteus morgani.“

F. THE METABOLIC FUNCTION OF CoA
1. DISTRIBUTION OF CoA

All living material so far tested for CoA was found to contain this co-
enzyme in variable amounts.!®: 4 The highest content in animal tissues is
found in the liver. Rat liver assays from 100 to 250 units per gram wet
weight. In Table II, which appears on p. 611, the enzymatically assayed
CoA content of various animal tissues is compared with the total amount
of pantothenic acid obtained by Lactobacillus arabinosus assay after double
enzyme treatment. There is very little free pantothenic acid present in
fresh tissues.“ Practically all pantothenic acid was found to be bound as
CoA. This may be seen from the close correspondence of the amounts of
pantothenic acid determined directly by microbial assay and calculated
from CoA assay by multiplying with the pantothenic acid factor of 0.7 y
per unit of CoA. It appears, therefore, unlikely that there is another panto-
thenic acid-containing coenzyme in animal tissue.

It is noteworthy that the adrenal gland is next highest to liver in CoA
content. The well-known relationship between pantothenic acid and adrenal
cortex®”: §8 is further emphasized by the decline of CoA in adrenal gland in
pantothenic acid deficiency,®® as shown in Fig. 6. A similar depletion of
CoA in pantothenic acid deficiency in other organs is shown in the same
figure for liver, heart, and kidney. With Lactobacillus arabinosus, the
depletion of CoA in a pantothenic acid-deficient organism is shown in
Table IV.® It is compared with an increase in coenzyme of the same or-
ganism after incubation with pantothenic acid. Very large amounts of CoA
are present in microorganisms carrying out 4-carbon fermentations. Clo-
stridium butylicum contains 1000 units per gram dry weight. Clostridium
kluyvert contains 500 units per gram. Some examples of the CoA content
in plants are shown in Table V.

It was to a large extent the early recognized presence of CoA in all
living matter which made us expect that this coenzyme had to fulfill im-
portant functions in intermediary metabolism.

57 A. F. Morgan, Vitamins and Hormones. 9, 162 (1951).

58H. P: Ralli, Trans. 1st Conf. on Adrenal Cortex, New York, p. 159 (1949).
59 R. E. Olson and N. O. Kaplan, J. Biol. Chem. 175, 515 (1948).

60 G. D. Novelli and F. Lipmann, Arch. Biochem. 14, 23 (1947).

Ill. BIOCHEMICAL SYSTEM 615

LIVER
220 @ PA DEFICIENT
@ RIBOFLAVIN DEFICIENT
© NORMAL CONTROLS
180
ADRENAL
Mo 140
=
<
c
°o
—
Fe wo to 100
WwW
3 2
x E
g 3
= 60 z 60
= <
S w
= =
ae N 20
= 2
o Ww
= 8
5 2 are a Te ar ee Can)
= WEEKS WEEKS
<
2 hes KIDNEY HEART
:
=
=<
60 & 60
S.
wn
=
z
5
20 < 20
fo)
o
2 4 6 10 4 6 10
WEEKS WEEKS

Fia. 6. Changes in the coenzyme A content of liver, adrenal, heart, and kidney
in rats on normal, riboflavin-deficient, and pantothenic acid-deficient diets. Coen-
zyme A content in units per gram of fresh weight of tissue plotted against the time on
the diet in weeks. Each point represents the mean value for pooled tissues from two
rats.

2. ACETYL AND AcyL TRANSFER

CoA was discovered during a study which was essentially undertaken to
elucidate the nature of the active acetate.° Isotope work had indicated a
great variety of synthetic reactions with the mysteriously reactive acetyl
as starting material.*! This is illustrated by Fig. 7.”

Then a connection of CoA with a great variety of primary processes in-
volved in acetyl transfer soon indicated that the coenzyme may act as an
acetyl carrier.®*-° This view was substantiated through a separation of

61K. Bloch, Physiol. Revs. 27, 574 (1947).

62 F. Lipmann, Harvey Lectures, Ser. XLIV, 99 (1948-1949).

63 N. O. Kaplan and F. Lipmann, Federation Proc. 7, 163 (1948).

6* R. C. Millican, S. M. Rosenthal, and H. Tabor, J. Pharmacol. Exptl. Therap. 97,
4 (1949).

6° G. D. Novelli and F. Lipmann, J. Biol. Chem. 182, 213 (1950).

616 PANTOTHENIC ACID

TABLE IV®
CoENZYME SYNTHESIS IN L. arabinosus

(Pantothenic acid was determined microbiologically. Coenzyme was determined
enzymatically.)

Per gram dry weight of harvested cells

Pantothenic

Pantothenic acid added acid bound in Free Bound
to growth medium, Units coenzyme, Yield of | pantothenic pantothenic
No. 7/100 ml. coenzyme calculated coenzyme, % aci acid®

1 1 0 — -—
50 44 31 4.8
100 81 57 4.2
500 89 62 10

2, 30 54 38 7.8 1 33

75 94 66 5.8 a 51

2 This value is calculated by multiplying the number of coenzyme units with the constant factor of 0.7
7 of pantothenic acid per unit of coenzyme.

> Bound pantothenic acid is determined after liberation through incubation with a mixture of acetone
pigeon liver extract and intestinal phosphatase. This enzyme mixture was shown to liberate pantothenic acid
from coenzyme A. Intact coenzyme does not test as pantothenic acid microbiologically.

© Too little to be determined accurately.

TABLE V
CoENZYME A IN PLANT MATERIAL

Units per gram fresh weight

Spinach 0.74
Tomato 1.3
Frozen peas 4.5
Wheat germ (commercial sample) 30
Royal jelly (bee) 0

CoA-requiring reactions into two distinct classes, namely, acetyl donor
and acetyl acceptor enzyme systems. Particularly useful for the identifi-
cation of partial reactions in acetyl transfer was the separation of the
various donor and acceptor enzymes present in pigeon liver extract by
Chou in our laboratory. Very important, furthermore, was the recognition
of an activating system in bacterial extract for acetyl phosphate to act as
an acetyl donor.!® * This enzymatic process of activation was identified
with the transacetylase reaction of Stadtman and Barker.!*: 2° This re-
action has been described in a previous paragraph as an assay system for
intact CoA. The CoA dependence of the phosphate exchange of acetyl-
bound and inorganic phosphate, as well as of the arsenolysis of acetyl

66 M. Soodak and F. Lipmann, J. Biol. Chem. 175, 999 (1948).
67 T. C. Chou and F. Lipmann, J. Biol. Chem. 196, 89 (1952).

III. BIOCHEMICAL SYSTEM 617

Carbohydrate Fat
Pyruvate Acetoacetate
N Va

Two Carbon Fragment
mn

Cholesterol <<——— = Citric Acid
Steroid Hormones <— Glutamic Acid

Hemin <— i> Proline
Fatty Acids <-————- !————___> Arginine

Fig. 7. Synthetic products derived from the 2-carbon fragment.

phosphate, led to the formulation of the enzyme-catalyzed process as
essentially the conversion of acetyl phosphate to acetyl-CoA by an equi-
libration reaction; this observation furnished definite evidence for the
existence of acetyl-CoA and its identity with ‘‘active” acetate.*® This was
further substantiated by the interchangeability of a great variety of donor
systems to donate acetyl by way of CoA to the sulfanilamide acetyl-ac-
ceptor enzyme.!*: 7!) 7, & Tt was shown that acetyl phosphate, through
phosphotransacetylase,!® pyruvate,” °° and, furthermore, the ATP-CoA-
acetate reaction of animal tissues and of yeast could furnish a common
acetyl donor, acetyl-CoA, for the same acceptor system.!* 2!) 8&7

The just-mentioned ATP-CoA-acetate reaction represents one of the
most important acetyl donor reactions in animal tissues. Its mechanism
remained for a long time obscure. It recently was clarified through the
work of Lipmann et al.7° The reaction was found unexpectedly to yield in-
organic pyrophosphate according to an over-all reaction:

CoA + acetate + ATP = Acetyl CoA + AMP + pyrophosphate

The reaction is freely reversible.7°* The finer mechanism of the reaction was
recently further clarified through use of isotope exchange reactions by
Jones et al.7%

Integrating the reactions just discussed, we are led to the construction
of what may be called a general acetyl transfer field, separated into donor

6 J. R. Stern, B. Shapiro, E. R. Stadtman, and S. Ochoa, J. Biol. Chem. 198, 703
(1951).

69 §. Korkes, A. Del Campillo, I. C. Gunsalus, and 8. Ochoa, J. Biol. Chem. 1938,
721 (1951).

70 F, Lipmann, M. E. Jones, 8. Black, and R. M. Flynn, J. Am. Chem. Soc. 74, 2384
(1952).

70a M. K. Jones, 8. Black, R. M. Flynn, and F. Lipmann, Biochim. et Biophys. Acta
12, 141 (1953); M. E. Jones, Federation Proc. 12, 708 (1953).

706 M.E. Jones, F. Lipmann, H. Hilz, and F. Lynen, J. Am. Chem. Soc. 75, 3285 (1953).

618 PANTOTHENIC ACID

DONOR SYSTEMS ACCEPTOR SYSTEMS
TRANSACETYLASES: ACETOKINASES:
Ac~Ph Acetyl SAM, choline

Histamine
Glucosamine
Amino acids
Hydroxylamine

Acetylformate
(pyruvate) <—- £0
Pee \: y = Citrate
Acetoacetate +Oxaloacetate
AMP
PP
Acetate + CoA+ ATP \

Acetoacetate

Fia. 8. The acetyl transfer system.

and acceptor territories, which are bridged by the acetyl-CoA carrier
system. This is illustrated by Fig. 8.°

As mentioned, a final confirmation and amplification was obtained by
Lynen and Reichert*®: 4° when they isolated acetyl-CoA from yeast. Of
great importance is their finding that the SH- group of CoA is the functional
group, that is to say, when functioning, CoA shuttles back and forth be-
tween sulfhydryl- and thioacetyl-CoA. This explains the long-known need
of large amounts of SH-compounds like cysteine or glutathione to activate
all acetylation systems.!°:7! This activation occurs by conversion of in-
active CoA disulfide into the active sulfhydryl-CoA.

It is quite remarkable that in CoA-linked acetyl the methyl end—we
call this in our laboratory the “‘tail’’? end—as well as the carboxyl end or
“head” is activated. Accordingly, we speak of ‘‘tail’’ or ‘“‘head’’ condensa-
tions.” Thus citric acid synthesis is a tail condensation. Straightforward
acetylation reactions are, of course, head condensations. One of the most
interesting reactions in this respect is acetoacetate synthesis. Stadtman
et al. showed that acetoacetate is synthesized by interaction of two active
acetyls, i.e., two acetyl-CoA’s, the head of one reacting with the tail of
another.”
71D. Nachmansohn and A. L. Machado, Neurophysiol. 6, 397 (1943).

72 F, Lipmann, Cold Spring Harbor Symposia Quant. Biol. 18, 127 (1948).
73 EK. R. Stadtman, M. Doudoroff, and F. Lipmann, J. Biol. Chem. 191, 377 (1951).

Ill. BIOCHEMICAL SYSTEM 619

COO- O
; VA
ClOy re) EH - CH.-'C + H:0
AS
CH, S:CoA
COO;
{

COO-
HO-C-CH,-C00- - HssConwy oe is
CH.
C00-
Fig. 9. Citric acid synthesis from acetyl-CoA and oxalacetate.

This reaction has now been fully clarified by the work of Lynen and his
group”*, by parallel work in Green’s laboratory”? and by Stern and del
Campillo in Ochoa’s laboratory.’** The reaction is formulated as follows:

CH;-CO-S-CoA + CH;-CO-S:-CoA
= CH;-CO-CH::CO-S8:CoA + HS-CoA

The equilibrium favors greatly the split of acetoacetyl-S-CoA + CoA-SH
to two moles of acetyl-S-CoA, the ‘‘thioclastic split” of Lynen.”¢ In the
process of fatty acid synthesis the condensation is followed by DPN-linked
hydrogenation of acetoacetyl-CoA to $-hydroxybutyryl-CoA. The 6-hy-
droxybutyryl-CoA, unlike the free /-compound, belongs to the d-series.7*
It now is dehydrated to crotonyl-CoA, which then is hydrogenated by a
flavin enzyme to butyryl-CoA. The butyryl-CoA condenses further with a
new molecule of acetyl-CoA to the 6-carbon-6-keto acyl-CoA and DPN-
linked hydration reduction; flavin-linked reduction and condensation con-
tinues until the 16- or 18-carbon chain is completed. Then stearyl or
palmityl-CoA condenses with glycerophosphate to form the phospholipids’
and presumably with glycerol to yield neutral fat.

73a F, Lynen, Harvey Lectures, Ser. XLVIII, 210 (1954); F. Lynen, Federation Proc.
12, 683 (1953).

735 H. Beinert, R. M. Bock, D.S. Goldman, D. E. Green, H. R. Mahler, 8. Mii, P. G.
Stansly, and S. J. Wakil, J. Am. Chem. Soc. 75, 4111 (1953).

73¢ J. R. Stern and A. del Campillo, J. Am. Chem. Soc. 75, 2277 (1953).

734 F. Lynen, Federation Proc. 12, 683 (1953).

73e A. L. Lehninger and G. D. Greville, J. Am. Chem. Soc. 75, 1515 (1953).

73f A. Kornberg and W. E. Pricer, Jr., J. Am. Chem. Soc. 74, 1617 (1952).

620 PANTOTHENIC ACID

Fatty acid oxidation proceeds by the same reaction chain in an opposite

direction.

For further reference the reader is referred to Lynen’s Harvey Lecture™#

and to papers by Lynen,”” and by Mahler™* in the Symposium on CoA.

A list of CoA-linked reactions known at the present time is given in
Tables VI and VII. It is quite notable that CoA is by no means exclusively

TABLE VI
CoENZYME A-CaATALYZED ENzyMAtTiIc Reactions I
AcETYL TRANSFER

Type of reaction Enzyme systems, extracts References
Donor Systems
ATP-CoA-acetate:
ATP + CoA + Ac= Ac-CoA + 5, 67, 70, 74
AMP + PP Pigeon liver, yeast 75
Phosphotransacetylation:
Ac-P + CoA = Ac-CoA + P Cl. kluyveri, EB. cola 19, 20, 24
Formotransacetylation:
Pyruvate + CoA= Ac-CoA+ E.coli 21, 69
formate
Transacetylase:
Ac-CoA + butyrate = Butyryl- Cl. kluyveri 76
CoA + acetate
Acetoacetate Pigeon liver ital
Citrate in reverse Pigeon liver 68
Acetaldehyde + CoA + DPN — _ Glyceraldehyde dehydro- 78, 79, 80
Ac-CoA genase, E. coli, Cl. kluyveri
Pyruvate + CoA + DPN — Ac-_ E. coli, Strep. faecales, heart 81, 82
CoA + DPNH: + CO2z and muscle
Acceptor Systems
Acetokinases
Aromatic amines Pigeon liver 5
Choline Rat brain 71
Histamine Pigeon liver 64
Amino acids Cl. kluyveri 20
Glucosamine Pigeon liver 83
Condensation
Acetoacetate Pigeon liver 66, 73
Citric acid Pigeon liver, yeast 68, 68
Pyruvate E. colt 21, 84-86

739 KF. Lynen, Harvey Lectures, Ser. XLVIII, 210 (1954).

73h B, Lynen, Federation Proc. 12, 683 (1953).

731 H. R. Mahler, Federation Proc. 12, 694 (1953).

7™ F, Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945).
75. Lipmann and L. C. Tuttle, J. Biol. Chem. 161, 415 (1945).

76K. R. Stadtman, J. Cellular Comp. Physiol. 41, Suppl. 1, 89 (1953).

Ill. BIOCHEMICAL SYSTEM 621

TABLE VII
CoENZYME-CATALYZED Reactions II

Acyl Transfer
Type of reaction Enzyme systems, extracts References

Succinyl Transfer
Donor system

Ketoglutarate + DPN + CoA Heart muscle 23, 87, 88
—Succinate-CoA + DPNH: +
CoA
ATP-succinate EL. coli 89
Acceptor system
Heme synthesis Red cells hemolyzate 90

Benzoyl Transfer

Hippuric synthesis Rat liver 91, 92
Stearyl Transfer
Phospholipid synthesis Rat liver 93
Complex Reaction Systems
Type of reaction Enzyme systems References
Fatty acid synthesis Cl. kluyvert extracts 20, 94
Butyrate oxidation Cl. kluyvert extracts 95
Fatty acid oxidation Liver homogenate 96
Steroid and fat synthesis Resting yeast cells 97
Liver slices 98

77M. Soodak in Phosphorus Metabolism, Vol. I, p. 291. Johns Hopkins Press, Balti-
more, 1951.

73 J. Harting, Federation Proc. 10, 195 (1951).

79 G. B. Pinchot and E. Racker, Federation Proc. 10, 233 (1951).

80 R. M. Burton and E. R. Stadtman, ef. in Phosphorus Metabolism, Vol. I, p. 229.
Johns Hopkins Press, Baltimore, 1951.

81 §. Korkes zn Phosphorus Metabolism, Vol. I, p. 259. Johns Hopkins Press, Balti-
more, 1951.

82 R.S. Schweet, M. Fuld, K. Cheslock, and M. H. Paul in Phosphorus Metabolism,
Vol. I, p. 246. Johns Hopkins Press, Baltimore, 1951.

83'T. C. Chou and M. Soodak, J. Biol. Chem. 196, 105 (1952).

84 F,. Lipmann and L. C. Tuttle, J. Biol. Chem. 154, 725 (1944).

85 H. J. Strecker, H. G. Wood, and L. O. Krampitz, J. Biol. Chem. 182, 525 (1950).

86M. F. Utter, C. H. Werkman, and F. Lipmann, J. Biol. Chem. 154, 723 (1944).

87 I. KE. Green and H. Beinert, in Phosphorus Metabolism, Vol. I, p. 330. Johns Hop-
kins Press, Baltimore, 1951.

88S. Kaufman in Phosphorus Metabolism, Vol. I, p. 370. Johns Hopkins Press. Balti-
more, 1951.

89 G. D. Novelli, unpublished results.

90D. Shemin and R. Rittenberg, J. Biol. Chem. 192, 315 (1951).

1H. Chantrenne, J. Biol. Chem. 189, 227 (1951).

9% R. W. McGilvery and P. P. Cohen, J. Biol. Chem. 183, 179 (1950).

622 PANTOTHENIC ACID

COOH

|
COOH CH:

|
CH, CH,

CH.«-——CO-CoA

CO-CoA CH:—COOH

y
HN
Fic. 10. The mechanism of pyrrole synthesis from succinyl-CoA and glycine.
(From Shemin et al.9°)

.

an acetyl carrier but is more broadly used by the cell as an acyl transfer
system.

Of major importance is the succiny] transfer function of CoA now studied
by Green’s group® and in Ochoa’s laboratory.®* The principal background
of this reaction is the observation that ketoglutarate oxidation, e.g., in
heart muscle extracts, is CoA-dependent.”* An ATP-succinate reaction was
shown by Kaufman* and has recently been also found by Novelli®® to
occur in Escherichia coli. Of particular interest appear the indications ob-
tained by Shemin® that succinyl-CoA is likely to be a building stone of the
pyrrole ring in the heme molecule. The tentative scheme of condensation
of two suecinyl-CoA’s with glycine is reproduced here from Shemin’s paper
as an illustration. This scheme, if further substantiated, would confirm on
the enzymatic level a previously suspected relationship of pantothenic acid
to erythrocyte metabolism.*$

3. FAT AND STEROID SYNTHESIS

The identification of acetate by isotope analysis as a precursor of fatty
acid, cholesterol, and steroid hormones® indicated that the initial con-
densation reactions leading to these syntheses should belong to the acetyl

93 A. Kornberg and W. E. Pricer, Jr., Federation Proc. 11, 242 (1952).

94H. A. Barker in Phosphorus Metabolism, Vol. I, p. 204. Johns Hopkins Press,
Baltimore, 1951.

95. P. Kennedy, ef. in Phosphorus Metabolism, Vol. I, p. 240. Johns Hopkins
Press, Baltimore, 1951.

96 V. H. Cheldelin, A. P. Nygaard, O. M. Hale, and T. E. King, J. Am. Chem. Soe.
73, 5004 (1951).

97 H. P. Klein, Federation Proc. 10, 209 (1951).

9% H. P. Klein and F. Lipmann, J. Biol. Chem. 208, 95 (1953); H. P. Klein and F.
Lipmann, J. Biol. Chem. 203, 101 (1953).

99 R. J. Williams, R. E. Eakin, E. Beerstecher, Jr., and W. Shieve, The iar Mesa
of the B rama: p. 423. Rerhold Bubliahing Corp., New Van. 1950.

. eo

III. BIOCHEMICAL SYSTEM 623

TABLE VIII

Lirip SyNTHESIS witH CoA-Poor aNpD CoA-RicH RESTING CELLS
or Saccharomyces cerevisiae®

Pantothenic acid Net synthesis, Total lipid,

Experiment added, y/ml. CoA, units/g. cells steroid mg./g. cells

1 0 37 —0.2
0.02 67 7

2 0 47 OR2 2.4

10 280 200 WET,

3 0 60 0.4 le@

10 310 1.8 8.6

4 0 57 0.2 Nee,

10 300 1ba7/ 9.6
5 0 60 1.8
10 165 6.2

@ Tdentical pantothenic acid-deficient yeast samples were incubated with and without pantothenic acid
for 2 to 3 hours, washed, and assayed for CoA. Subsequently, the yeast samples were incubated for 16 hours
in an acetate-phosphate medium and analyzed for total lipid and steroid.

acceptor territory and derive acetyl from acetyl-CoA. In confirmation a
still rather general connection between CoA and fat and steroid synthesis
has evolved from studies in this laboratory with Klein.” By a comparison
of CoA-poor and CoA-rich yeast samples (Table VIII), he showed that
that fat and ergosterol synthesis runs parallel with CoA concentrations.
A similar parallel between fat and cholesterol synthesis and CoA levels
was obtained in experiments with pantothenic acid-deficient rats.*’ The
incorporation of isotopic acetate into fat and cholesterol in liver slices from
normal and pantothenic acid-deficient animals is shown in Table IX.

TABLE IX%8
CH;C“OOH INcoRPORATION INTO PANTOTHENIC ACID-DEFICIENT Rat LIVER SLICES

Incorporation

Pantothenic CoA in per gram Liver
Group Age, weeks acid liver, units/g. of fatty acids, % cholesterol, %

1 6 + 98 14.9 Yell

_ 55 6.6 2.0

2 10 ae 85 2.7 1.4
— 62 0.4 0.4

3 11 a 85 7.4 0.9
_ 65 0.3 0.8

624 PANTOTHENIC ACID

TABLE X°%

INHIBITION OF Liprip SYNTHESIS BY PANTOYL-TAURYL-ANISIDIDE (PTA); PARTIAL
RELEASE BY LBF

C.p.m./g. tissue

No. PTA LBF, mg./7.5 ml. Fatty acids Cholesterol

il — — 2450 315
— 0.5 2300 305

3.7 X 107? M — 550 1000

Dal OX MOP WL 0.5 825 245

2 — a 1650 25
— 1.0 2000 680

od Sk MO ye a 365 210

168 SK UGS A 0 800 525

In view of the sensitivity of fat and steroid synthesis to various nu-
tritional effects,9* results obtained by the use of a pantothenic acid anti-
metabolite are of significance. Klein found (cf. Table X) that pantoyl-
tauryl-anisidide suppresses fat and steroid synthesis without appreciably
influencing respiration. Under certain conditions he also could demonstrate
an antagonism between pantethine*® and pantoyl-tauryl-anisidide,® while
free pantothenic acid appeared inactive.

In view of the much-discussed, rather specific relationship of panto-
thenic acid to the adrenal cortex, recently reviewed by A. F. Morgan*’
(cf. also ref. 58), a participation of CoA in the synthesis of steroids assumes
special importance. As mentioned before, CoA is very abundant in the
adrenal gland,*? and it declines in pantothenic acid deficiency obviously in
parallel with a depletion of steroids. A confirmation of this view seems to
derive from the recent work of Cowgill and his associates,!°° who report
that adrenal degeneration in pantothenic acid-deficient rats may be pre-
vented by corticosterol.

G. CONCLUSION

Since the discovery of CoA, the function of pantothenic acid has been
relegated to the metabolic territory of acetyl transfer in particular, and to
that of acyl transfer in a more general sense. The relative abundance of
pantothenic acid as CoA in living cells, e.g., up to 400 mg. of CoA per kilo-
gram of liver is explained by the variety of the metabolic reactions medi-
ated by this coenzyme. It appears that CoA is involved in the primary

99a §. Gurin, private communication.
100 G. R. Cowgill, R. W. Winters, R. B. Schultz, and W. A. Krehl, International Congress
on Vitamins (Havana, Cuba), report in The New York Times, January 26, 1952.

III. BIOCHEMICAL SYSTEM 625

pathway of carbohydrate oxidation through citric acid synthesis and
through its function in ketoglutarate and pyruvate oxidation. Through its
function in citrate and ketoglutarate metabolism, furthermore, it is in-
volved in the synthesis of various amino acids, like glutamic acid,!™ proline,
and others from carbohydrate sources. Through succinyl transfer, it seems
to enter into synthesis of the pyrrole ring in the heme molecule. Its role in
acetoacetate synthesis and its well established role in fatty acid and choles-
terol synthesis illustrate the instrumental part of the coenzyme in fat and
steroid synthesis.

The unequivocal identification of acetyl-CoA with ‘‘active acetate”
makes it appear legitimate to presume that any incorporation of isotopically
marked acetate indicates the intermediacy of acetyl-CoA at some stage of
the process. In this manner, by aiming at a closer recognition of the role
of acetyl-CoA, an opening is found for an enzymatic understanding of
such complex reactions as steroid, rubber, carotenoid, and terpene bio-
synthesis where acetate incorporation has been shown to occur. It is pre-
sumed that such structures are built up from 2-carbon residues exclusively,
as was shown to be the case for cholesterol by Bloch’s investigations.!
James Bonner’s work on the synthesis of latex in certain plants!™ indicates
that the isoprene unit may be derived from 2-carbon residues.

It is, however, obvious that an incorporation of acetate into a biological
structure has to be viewed judiciously because, for example, by milling
through the citric acid cycle, acetate incorporates into a large number of
molecular species, as amino acid, carbohydrates, and others. These, in
their turn, represent building stones and will spread acetate secondarily
into many compounds. Although initially acetyl-CoA participates in a
derivation of such building stones, eventual synthetic mechanisms may
easily not involve this coenzyme.

A few concluding remarks on the role of pantothenic acid in CoA appear
in order. Comparing CoA structurally to the pyridine and flavin adenine
dinucleotide, one finds that pantothenic acid, through its terminal hydroxy]
on the one side and the carboxyl on the other, links the reactive base cyste-
amine through the pyrophosphate bridge to the adenyl moiety, the latter
a common feature in many coenzymes. The question as to why in the case
of CoA this ‘‘tie’’ molecule has to have such an unusual structure remains
open, and the essentiality of the “precious”? vitamin in this coenzyme still
presents a challenge to our biochemical imagination.

101 §,. Shive, W. W. Ackermann, and J. E. Sutherland, J. Am. Chem. Soc. 69, 2567
(1947).

102 K. Bloch, Recent Progr. Hormone Research 6, 111 (1951).

103 J. Bonner and B. Arreguin, Arch. Biochem. 21, 109 (1949).

626 PANTOTHENIC ACID

IV. Specificity of Action
SAMUEL LEPKOVSKY

The activity of pantothenic acid resides only in its dextrorotatory form
which has been indicated to be the p configuration by application of Hud-
son’s amide rule.’* i(—)-Pantothenic acid appears to be inactive for
organisms requiring the intact vitamin. Pantothenic acid appears to be
highly specific and is incapable of modification without loss of activity
unless the organisms metabolizing it are capable of regenerating it in vivo.
The following are examples which illustrate this.

1. The methyl and ethyl esters of pantothenic acid are comparable in
activity to the vitamin in promoting growth of rats,?:* but ethyl panto-
thenate is only about 6.8 % as effective as pantothenic acid for Lactobacillus
casei.*-> The rats apparently can hydrolyze the esters zn vivo, but the bac-
teria cannot.

2. The alcohol corresponding to pantothenic acid (pantothenyl alcohol)
has been found to be effective in animals,*: ® but it cannot replace panto-
thenic acid in the nutrition of lactic acid bacteria and it even inhibits its
utilization in these organisms.’? The utilization of pantothenyl alcohol by
animals depends upon its 7m vivo conversion to pantothenic acid. Lactic
acid bacteria apparently cannot make this conversion.

3. Acetylation of pantothenic acid concentrates from natural sources
destroys its activity for chicks® and for bacteria. However, synthetic ethyl
monoacetyl p-pantothenate is as active as pantothenic acid for rats and
chicks but is only 0.7 % as effective as the vitamin for Lactobacillus caset.*-*
These are impressive examples of the relationship between the potency of a
vitamin and the presence of enzyme systems in the organism capable of
modifying the vitamin so that it can be metabolized.

Many analogs of pantothenic acid have been prepared. The -alanine
has been replaced with other amino acids such as a-alanine, 6-aminobutyric
acid, aspartic acid, lysine, and leucine. All such preparations were devoid of

1H. C. Park and E. J. Lawson, J. Am. Chem. Soc. 68, 2869 (1941)

2C. 8. Hudson, J. Am. Chem. Soc. 39, 462 (1917).

3R.J. Williams, R. E. Eakin, E. Beerstecher, Jr., and W. Shive, The Biochemistry
of the B Vitamins. Reinhold Publishing Corp., New York, 1950. The chapter on
pantothenic acid carries an extensive bibliography.

4K. Unna and C. W. Muschett, Am. J. Physiol. 185, 267 (1942).

6S. A. Harris, G. A. Boyack, and K. Folkers, J. Am. Chem. Soc. 68, 2662 (1941).

68. H. Rubin, J. M. Cooperman, M. E. Moore, and J. Scheiner, J. Nutrition 35, 499
(1948).

7. E. Snell and W. Shive, J. Biol. Chem. 158, 551 (1945).

8D. W. Woolley, H. A. Waisman, O. Mickelsen, and C. A. Elvehjem, J. Biol. Chem.
125, 715 (1938).

91. E. Snell, F. M. Strong, and W. H. Peterson, Biochem. J. 31, 1789 (1937).

V. BIOGENESIS 627

activity.!° The pantoic lactone portion of the pantothenic acid molecule has
also been modified in many ways, all of them resulting in great loss of
activity. The only modifications of pantothenic acid which retain appre-
ciable, but by no means full, activity are hydroxypantothenic acid!’ and
methylpantothenic acid. 1° It is not known whether modifications in vivo
account for all or part of the activity of these analogs.

V. Biogenesis
SAMUEL LEPKOVSKY

Microorganisms differ in their ability to synthesize pantothenic acid.
Some are capable of its total synthesis. This has been demonstrated by the
rumen microorganisms in cattle and sheep.! Its synthesis also takes place
in the intestinal tract of rats, apparently by the bacteria in the cecum. Some
organisms can produce only the pantoic lactone fragment but not 6-
alanine,?» whereas others can synthesize §-alanine but not pantoic
lactone.?:

6-Alanine is apparently formed by the decarboxylation of aspartic acid.?

COOH
|
cue Co
| et ENERO EEO Hee OOE
CH—NH,
COOH
Aspartic acid B-Alanine

B-Alanine is then coupled with pantoic acid to form pantothenic acid.
The steps in the biosynthesis of pantoic acid remains to be worked out.

10 R. J. Williams, Advances in Enzymol. 3, 253 (1943).
1L. W. McElroy and H. Goss, J. Nutrition 21, 405 (1941).
2 R. J. Williams, R. E. Eakin, E. Beerstecher, Jr., and W. Shive, The Biochemistry
of the B Vitamins. Reinhold Publishing Corp., New York, 1950.
3 H.R. Rosenberg, Chemistry and Physiology of the Vitamins. Interscience Pub-
lishers New York, 1945.

628 PANTOTHENIC ACID

VI. Estimation

A. CHEMICAL AND PHYSICAL METHODS
GEORGE M. BRIGGS and FLOYD S. DAFT

Several chemical methods for estimation of pantothenic acid are avail-
able, although they seem especially suited only for relatively pure mixtures.
Two proposed methods are based on the liberation of 6-alanine by chemical
means and its subsequent determination by colorimetric tests.!: !* Recently
this has been extended to the determination of pantothenic acid in crude
materials (urine and wheat), and the values agree well with the results ob-
tained by biological assay.1® !¢ Further studies are necessary, however, be-
fore chemical methods can be routinely used for biological materials.

The estimation of pantothenic acid in pure vitamin mixtures is possible
by the chemical release and determination of pantoy] lactone or of pantoic
acid.!? 1¢ This method does not differentiate pantothenic acid from its in-
active lactone moiety, however.

Also, it is known that pantothenic acid is reduced at the dropping mer-
cury electrode? and may be measured by polarimetric analysis.’ These
methods are not specific enough, however, to be used to assay pantothenic
acid in natural materials.

B. BIOLOGICAL METHODS
GEORGE M. BRIGGS and FLOYD 8S. DAFT

Biological methods of assay are the only satisfactory methods thus far
suggested. Microbiological assays (discussed elsewhere) and animal assays
are both used with success. Microbiological methods are more rapid and
are the method of choice for routine purposes, whereas animal assays are
helpful chiefly for checking the accuracy of microbiological methods in the
determination of total pantothenic acid activity.

In general, an animal assay of crude material measures not only panto-
thenic acid but also any “‘sparing”’ factor which might be present.* This
gives a greater significance to animal assays than to microbiological assays
as far as animal “pantothenic acid activity” of a material is concerned.

1R. Crokaert, Bull. soc. chim. biol. 31, 903 (1949).

ta C. R. Szalkowski, W. J. Mader, and H. A. Frediani, Cereal Chem. 28, 218 (1951).
1>R. Crokaert, 8. Moore, and E. J. Bigwood, Bull. soc. chim. biol. 38, 1209 (1951).
tc KF. Y. Refai and B.S. Miller, Cereal Chem. 29, 469 (1952).

1d. G. Wollish and M. Schmall, Anal. Chem. 22, 1033 (1950).

te C. R. Szalkowski and J. H. Davidson, Anal. Chem. 25, 1192 (1953).

2 J. J. Lingane and O. L. Davis, J. Biol. Chem. 187, 567 (1941).

3D. V. Frost, Ind. Eng. Chem. Anal. Ed. 15, 306 (1943).

4F.§8. Daft and K. Schwarz, Federation Proc. 11, 201 (1952).

VI. ESTIMATION 629

However, such assays have less significance in terms of the content of
pantothenic acid itself.

1. Rat GrowtnH ASSAYS

Satisfactory assays for pantothenic acid activity may be made by re-
moving pantothenic acid from any of the good, highly purified diets for
rats in use today. The pantothenic acid activity of crude materials may
then be calculated by comparing the growth obtained with such materials
with standard growth curves obtained by feeding graded levels of crystalline
calcium pantothenate.

As an example of the use of this principle, a successful rat growth assay
was used by Wisconsin workers® in 1951 to determine the activity of bound
forms of pantothenic acid. Total pantothenic acid activity of certain sam-
ples of liver, yeast, wheat bran, and rice bran was greater than indicated
by microbiological assay. A few other laboratories have also used rat
assays,®’7 but older rat assays now need modification Decate of newer
vitamins which have since been discovered.

2. Cuick GrRowTH ASSAYS

Assays for pantothenic acid activity may be made with chicks by the
same general method as that used with rats. Chick assays have the ad-
vantage over rat assays of being somewhat less expensive and of shorter
duration. For instance, Hegsted and Lipmann,® in 1948, measured the
pantothenic acid content of coenzyme A by using a purified-type diet low
in pantothenic acid. Day-old chicks were fed a commercial chick mash for
4 days and then placed on the pantothenic acid-low diet. After a short
depletion period the chicks were divided into uniform groups for assay
purposes. The length of the actual assay period was only 8 to 10 days.

Animal assays, especially chick assays, have been very useful as a stand-
ard in the development of improved microbiological methods for panto-
thenic acid. This is true because the animal can utilize bound forms of
pantothenic acid which usually are not available to bacteria unless re-
leased by special techniques.*®

5H. Lih, T. E. King, H. Higgins, C. A. Baumann, and F. M. Strong, J. Nutrition
44, 361 (1951).

6 J.S. D. Bacon, G. N. Jenkins, and J. O. Irwin, Biochem. J. 37, 492 (1943).

7N. B. Guerrant, M. G. Vavich, and O. B. Fardig, Ind. Eng. Chem. Anal. Ed. 17,
710 (1945).

8D. M. Hegsted and F. Lipmann, J. Biol. Chem. 174, 89 (1948).

9D. Pennington, E. E. Snell, H. K. Mitchell, J. R. McMahan, and R. J. Williams,
Univ. Texas Publ. 4187, 14 (1941).

10 A. L. Neal and F. M. Strong, J. Am. Chem. Soc. 65, 1659 (1943).

uA. L. Neal and F. M. Strong, Ind. Eng. Chem. Anal. Ed. 15, 654 (1943).

630 PANTOTHENIC ACID

Several laboratories have used the chick assay to study the distribution
of pantothenic acid in a wide variety of foods, animal tissues, and feed-
stuffs.8» 1619 (See the review on this subject by Jukes.'!) However, some of
this work was done before the importance of the more recently discovered
vitamins was known, so that many of the values may be somewhat high.
Also, much of the older work was done with natural-type diets heated in
the dry state to destroy the pantothenic acid content. This heating process
undoubtedly affected other nutrients as well, some of which could not be
added back satisfactorily.

3. ASSAYS FOR COENZYME A

It is now possible to measure coenzyme A by zn vitro methods from which
pantothenic acid values may be computed. Kaplan and Lipmann proposed
a method depending upon the reactivation by coenzyme A of an acetylating
system derived from pigeon liver extract.?° Assay values could be obtained
in a few hours. Various modifications of this method have since been
suggested.

C. MICROBIOLOGICAL METHODS
EK. E. SNELL

Pantothenic acid was discovered independently as a growth factor for
yeast?! 2 and for lactic acid bacteria® before its role in animal nutrition was
recognized. Following demonstration of its importance for animal life, repre-
sentatives of these two groups of organisms were used widely for its deter-
mination.

The assay of this vitamin is complicated by its occurrence in bound forms,

12. H. Hoag, H. P. Sarett, and V. H. Cheldelin, Ind. Eng. Chem. Anal. Ed. 17, 60

(1945).

13H}, Willerton and H. W. Cromwell, Ind. Eng. Chem. Anal. Ed. 14, 603 (1942).

“4TH. Jukes, Biol. Symposia 12, 253 (1947).

15 M. E. Coates, J. E. Ford, G. F. Harrison, 8. K. Kon, E. E. Shepheard, and F. W.
Wilby, Brit. J. Nutrition 6, 75 (1952).

16 H. A. Waisman, O. Mickelsen, and C. A. Elvehjem, J. Nutrition 18, 247 (1939).

17'T. H. Jukes, J. Nutrition 21, 193 (1941).

18'T. H. Jukes and 8. Lepkovsky, J. Biol. Chem. 114, 117 (1936).

19H. A. Waisman and C. A. Elvehjem, Vitamin Content of Meat. Burgess, Min-
neapolis, 1941.

20 N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948).

21 R. J. Williams, C. M. Lyman, G. H. Goodyear, and J. H. Truesdail, J. Am. Chem.
Soc. 54, 3462 (1932).

22R. J. Williams, Advances in Enzymol. 3, 257 (1943).

23H}. EK. Snell, F. M. Strong, and W. H. Peterson, Biochem. J. 31, 1789 (1937); J. Am.
Chem. Soc. 60, 2825 (1938).

a

VI. ESTIMATION 631

NHCH:CH.SH or —S—SR

CH;

B linkage 2

Diagram giving suggested formula for coenzyme A.

of which coenzyme A and its various degradation products are quanti-
tatively the most important.’ Our present knowledge of the relationships
of these products is summarized in the diagram, which gives a suggested
formula for coenzyme A.”>: °6 This formula will serve to illustrate the types
of linkages that must be broken to liberate pantothenic acid.

A variety of different products containing pantothenic acid are possible,
depending upon whether one hydrolyzes linkages 1, 2, or 3, or various
combinations of these. Hydrolysis of linkage 1 yields one or more forms of
the growth factor, LBF, closely related to or identical with synthetic
pantetheine, A.””* This growth factor, and coenzyme A itself, exist in a
variety of forms, depending upon whether their —SH group is free or

24.N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948).

25 J. Baddiley and E. M. Thain, J. Chem. Soc. 1951, 2253.

26 T. P. Wang, L. Shuster, and N. O. Kaplan, J. Am. Chem. Soc. 74, 3204 (1952).

27 G. M. Brown, J. A. Craig, and E. E. Snell, Arch. Biochem. 27, 473 (1950).

8 Ki. E. Snell, G. M. Brown, V. J. Peters, J. A. Craig, E. 1. Wittle, J. A. Moore, V. M.
McGlohon, and O. D. Bird, J. Am. Chem. Soc. 72, 5349 (1950).

29 J. A. Craig and E. E. Snell, J. Bacteriol. 61, 283 (1951).

29a), EK. Snell and G. M. Brown, Advances in Enzymol. 14, 49 (1953).

632 PANTOTHENIC ACID

combined in a disulfide linkage with any of several naturally occurring
—SH compounds.*®: *! Linkage 1 is cleaved by intestinal phosphatase.”
Hydrolysis of linkage 2, accomplished by an enzyme preparation from
pigeon or chicken liver,” liberates a fragment, B. Hydrolysis of linkage 3,
accomplished by a potato pyrophosphatase,** should liberate 4-phospho-
pantetheine. Hydrolysis of both linkages 2 and 3, by the combined action
of pyrophosphatase and the liver enzyme, would yield a pantothenic acid
phosphate. Synthetic phosphates of pantothenic acid, in contrast to panto-
thenic acid, are stable to alkali;?®: ** an alkali-stable form of the vitamin
that occurs naturally and is converted to an alkali-labile form by treat-
ment with phosphatases** may be identical with this fragment. Liberation
of free pantothenic acid, C’, from coenzyme A requires the combined action
of the liver enzyme and intestinal phosphatase®: ** to hydrolyze linkages 1
and 2.

Of these compounds, only LBF (A) and free pantothenic acid (C) have
growth-promoting activity for lactic acid bacteria, and only free panto-
thenic acid has such activity for yeasts.2® The activity of LBF is much
greater than that of pantothenic acid for one group of lactic acid bacteria
but is equal to or less than that of pantothenic acid for Lactobacillus casei
and Lactobacillus arabinosus, the organisms commonly used for assay of
pantothenic acid. Whichever of the methods of assay is used, it is therefore
necessary to liberate pantothenic acid by the combined action of liver
enzyme and intestinal phosphatase to obtain true values. Procedures for
this purpose have been summarized elsewhere.*: #88 Since such procedures
were unknown until recently, many of the early figures for the pantothenic
acid content of natural materials obtained by microbiological procedures are
low, a fact that was indicated quite early by their failure to agree with
chick assay procedures.*?

The most widely used microbiological procedure for pantothenic acid
employs Lactobacillus arabinosus as the test organism.*” The procedure and
medium are very similar to those used for determination of biotin and nico-
tinic acid; they have been described in detail several times.*”-*? Growth
in the pantothenic acid-free medium increases with the pantothenic acid

30 G. M. Brown and E. E. Snell, Proc. Soc. Exptl. Biol. Med. T7, 138 (1951).

31 G. M. Brown and E. E. Snell, J. Biol. Chem. 198, 375 (1952).

32 G. D. Novelli, N. O. Kaplan, and F. Lipmann, J. Biol. Chem. 177, 97 (1949).

33 G. D. Novelli, N. O. Kaplan, and F. Lipmann, Federation Proc. 9, 209 (1950).

34'T. EK. King and F. M. Strong, Science 112, 562 (1950).

35 A. L. Neal and F. M. Strong, J. Am. Chem. Soc. 65, 1659 (1943).

36 J. B. Neilands and F. M. Strong, Arch. Biochem. 19, 287 (1948).

37 #}, E. Snell, 72 Vitamin Methods, Vol. I, p. 327. Academic Press, 1950.

38 Association of Vitamin Chemists, Methods of Vitamin Assay, 2nd ed., p. 290. In-
terscience Publishers, New York, 1951.

39 T. H. Jukes, Biol. Symposia 12, 253 (1947).

VII. STANDARDIZATION OF ACTIVITY 633

concentration in the range from 0 to about 0.2 y of calcium pantothenate
per 10 ml. The pure vitamin and samples which supply it at several levels
within this range are added to individual tubes containing 5 ml. of the
double-strength medium. Each tube is then diluted to 10 ml., capped, auto-
claved, cooled, and inoculated. Response of the test organism is customarily
determined by acid titration after 72 hours of incubation at 30 to 37°, or
turbidimetric estimations of growth can be made as early as 24 hours.
B-Alanine does not replace pantothenic acid; fatty acids may interfere
slightly, and should be removed by filtration of the samples at pH 4.5
before assay.

Lactobacillus casei was used in many of the earlier procedures,” but it was
more troublesome than L. arabinosus because of its more complex nutrition
and because fatty acids interfered much more markedly with its response
to pantothenic acid. With present knowledge, both disadvantages could be
overcome, and further work might lead to a simpler assay procedure with
this test organism, inasmuch as it is one of the few investigated for which
pantethine (LBF) and pantothenic acid have equimolar activity.”®

Early yeast assay methods for pantothenic acid were relatively non-
specific when applied to natural materials because of the presence of toxic
materials, because 6-alanine could be used in place of pantothenic acid,
and because other components of the sample stimulated growth in the
simple media originally used.*” None of these objections apply to a more
recently developed procedure utilizing Saccharomyces carlsbergensis.*’» *°
For suitably equipped laboratories, this method should be found equally
as suitable as that utilizing L. arabinosus.

VII. Standardization of Activity
GEORGE M. BRIGGS and FLOYD S. DAFT

Now that the crystalline calcium salt of pantothenic acid is readily
available, the older “units” are obsolete. Since the calcium salt is usually
used in biological studies, it is sometimes convenient to know that 1.087 g.
of calcium pantothenate equals 1.0 g. of pantothenic acid. Pure pantothenic
acid is not used as a standard, since it is an unstable viscous oil and ex-
tremely hygroscopic. The sodium salt is also hygroscopic, whereas calcium
pantothenate in the dry form is reasonably stable to air and light.

A unit of coenzyme A has been defined as the amount which will activate
the zn vitro system of Kaplan and Lipmann to half the maximum activity.!
It contains approximately 0.7 y of bound pantothenic acid.

40. Atkin, W. L. Williams, A. S. Schultz, and C. N. Frey, Ind. Eng. Chem. Anal.

Ed. 16, 67 (1944).
1N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948).

634 PANTOTHENIC ACID

VIII. Occurrence
GEORGE M. BRIGGS and FLOYD S. DAFT

There is somewhat of a scarcity of reliable figures in the literature on the
pantothenic acid content of natural materials. This is because only in very
recent years have methods become available by which the various bound
forms of pantothenic acid are released to make them available for the
growth of microorganisms. Older values obtained by microbiological assay
tend to be somewhat low, especially for materials rich in bound forms.

Pantothenic acid is universally distributed in all living cells and tissues.
In general, liver, kidney, yeast, egg yolk, royal jelly, and fresh vegetables
are the best sources of pantothenic acid, and milk, meat, grains, fruits, and
nuts are considered fair to good sources. Certain canned products, as well
as egg white, beets, corn, and rice, may be considered relatively poor
sources.

A. ASSAY VALUES IN FOODS, FEEDS, TISSUES, AND
MISCELLANEOUS MATERIALS

The tables, which appear at the end of this section give the approxi-
mate pantothenic acid content of various classes of materials of natural
origin. The values given were selected as much as possible from the most
recent and reliable values found in the literature. Not all values, however,
have been determined by the use of modern methods of enzymatic release of
pantothenic acid, and, hence, they tend to be low. In a few instances,
values obtained by several methods are given for the sake of comparison.

The tables are separated according to the following classification:

Table XI (p. 638): Foodstuffs.

Table XII (p. 642): Feedstuffs (grains, animal by-products, etc.).

Table XIII (p. 644): Various varieties of oats, wheat, and soybeans.

Table XIV (p. 645): Diet ingredients, enzymes, and miscellaneous.

Table XV (p. 646): Organisms of different biological phyla.

Table XVI (p. 647): Normal tissues of rat, mouse, beef, hog, chick, and

human being.

Table XVII (p. 648): Milk of various species.

Table XVIII (p. 648): Blood of various species.

Table XIX (p. 649): Coenzyme A distribution.

B. EFFECT OF VARIOUS FACTORS ON PANTOTHENIC
ACID DISTRIBUTION

Various factors are known to affect the pantothenic acid content of
natural materials as discussed below.

VIII. OCCURRENCE 635

1. Errect oF VARIETY, SPECIES, AND BREED

Table XIII gives the pantothenic acid content of various varieties of
grains.! It is obvious that considerable variation exists which might be
expected from genetic, environmental, and other considerations. This varia-
tion convincingly demonstrates the importance of directly assaying any
diet or food if a true pantothenic acid value is wanted rather than relying
too heavily on published ‘‘average”’ values.

Myint et al.4 have reported that highly significant differences were ob-
served in the pantothenic acid content of different breeds of chickens fed
similar diets (containing 10% alfalfa) as follows:

Pantothenic acid content

Breed of leg tissue, y/g.
White Jersey Giant 11.6
New Hampshire 14.7
White Plymouth Rock 10.7
White Leghorn 11.0

No significant differences could be found in the pantothenic acid content
of milk of various breeds of cattle.4* Individual variation was greater than
differences in breed in the few samples studied.

2. Errect oF ENVIRONMENT DURING PERIOD OF GROWTH

The pantothenic acid content of fresh plants, such as alfalfa and clover,
is surprisingly not influenced by the stage of growth according to Bondi
et al.® These authors reported, however, that there were various seasonal
effects on pantothenic acid content of plants; e.g., plants grown in a dry
season had more pantothenic acid than plants grown during a rainy season.

In this same connection, Tepley et al.2 have shown that the pantothenic
acid content of wheat samples of the same variety differed with the locality
in which they were grown. For instance, wheat samples from Ohio were
lower in pantothenic acid than the same variety from other areas. These
differences are probably explained by soil and seasonal effects. Also, it is
known that rain may cause a leaching effect on pantothenic acid during
the period of field curing® which may then also be a factor in the final
pantothenic acid content of hays or grains. The pantothenic acid content of

1K. J. Frey and G. I. Watson, Agron. J. 42, 434 (1950).

2L. J. Teply, F. M. Strong, and C. A. Elvehjem, J. Nutrition 24, 167 (1942).

2% FY. Refai and B.S. Miller, Cereal Chem. 29, 469 (1952).

3 P.R. Burkholder and I. MeVeigh, Plant Physiol. 20, 301 (1945).

4T. Myint, C. I. Draper, and D. A. Glenwood, Abstr. Meeting, Am. Chem. Soc.
p. 3A (Sept. 3-8, 1950).

4a J. M. Lawrence, B. L. Herrington, and L. A. Maynard, J. Nutrition 32, 73 (1946).

5 A. Bondi, R. Etinger, and H. Meyer, J. Agr. Sci. 39, 104 (1949).

6 L. G. Blaylock, L. R. Richardson, and P. B. Pearson, Poultry Sci. 29, 692 (1950).

636 PANTOTHENIC ACID

corn is known to be affected by environmental factors™ ® and is markedly
affected by stage of maturity (the pantothenic acid level becomes less as
the stage of maturity increases).®

It is known that the composition of the soil may affect the vitamin con-
tent of a plant. For instance, the pantothenic acid content of oat plants has
been reported to be lowered by soils low in phosphorus® or in caleium.*4
Also, a soil high in phosphorus® or high in nitrate® produces oat plants
with higher than normal amounts of pantothenic acid.

3. EFFECT OF GERMINATION AND EMBRYONIC DEVELOPMENT

Burkholder’ has reported that the pantothenic acid content of germinated
oats, wheat, barley, and corn seeds is approximately double the content of
ungerminated seeds. Similar increases due to germination have also been
seen in the pea, the soybean, and the mung bean.’ This increase in the
synthesis of pantothenic acid during germination is no doubt a reflection
of the increased rate of metabolism, and hence of the requirement for co-
enzyme A, in the germinating seed.

No change occurs in the pantothenic acid content of incubating hen eggs
over the course of the entire period of incubation according to Snell and
Quarles.’

4. Errect oF Diet on TisSurE COMPOSITION

The pantothenic acid content of tissues may be influenced by the dietary
level of pantothenic acid, as well as other substances, although more infor-
mation on this subject is needed. In studies with chicks it has been shown
that there is a direct correlation between the pantothenic acid content of
the diet and the pantothenic acid content of various tissues.’ Deficient
chicks had from only 10 to 40% as much pantothenic acid as normal
chicks. Likewise, in studies with hens, it has been determined that the
pantothenic acid content of blood,!®!! eggs,"-" and other tissues! is
directly related to that of the diet. A similar relationship between dietary

6¢ 7. Ditzler, C. H. Hunt, and R. M. Bethke, Cereal Chem. 25, 273 (1948).

6» C. H. Hunt, L. D. Rodriguez, 8. Taylor, and R. M. Bethke, Cereal Chem. 29, 142
(1952).

8¢ T. A. McCoy, D. G. Bostwick, and A. C. Devich, Plant Physiol. 26, 784 (1951).

6¢ TD). §. Bostwick and T. A. MeCoy, Proc. Oklahoma Acad. Sci. 81, 112 (1950).

Se R. Langston, Plant Physiol. 26, 115 (1951).

7™P.R. Burkholder, Science 97, 562 (19438).

8H. EK. Snell and E. Quarles, J. Nutrition 22, 483 (1941).

9H. E. Snell, D. Pennington, and R. J. Williams, J. Biol. Chem. 138, 559 (1940).

10 P. B. Pearson and VY. H. Melass, Federation Proc. 5, 237 (1946).

1M. B. Gillis, G. F. Heuser, and L. C. Norris, J. Nutrition 35, 351 (1948).

2K. E. Snell, E. Aline, J. R. Couch, and P. B. Pearson, J. Nutrition 21, 201 (1941).

13 P. B. Pearson, V. H. Melass, and R. M. Sherwood, Arch. Biochem. 7, 353 (1945).

—

VIII. OCCURRENCE 637

and tissue pantothenic acid content has been seen in swine and in other
animals” (see also section on deficiency symptoms).

It is also interesting that the pantothenic acid, or coenzyme A, content
of certain animal tissues is changed as a result of deficiencies of other vita-
mins, as shown in studies with chicks, * rats,’ and humans'® (with beri-
beri, pellagra, or riboflavin deficiency).

5. Errect oF STORAGE

Pantothenic acid is reasonably stable in foods and feedstuffs during long
periods of storage, provided that oxidation and high temperatures are
avoided. For instance, it has been shown that grains may be stored for
periods up to a year in the intact or ground state without appreciable loss
of pantothenic acid.” Also, it is known that the pantothenic acid content of
pork!”¢ and of eggs!* is reduced very little after 12 months’ cold storage, or
longer.

6. Errect oF HEAT AND COOKING

It is well known that heating or cooking of feeds or foods may cause some
destruction of pantothenic acid. In usual cooking or baking procedures only
relatively small amounts of pantothenic acid are lost.*% 7! Normal de-
hydration temperatures over very short periods do not cause any loss of
pantothenic acid, as shown with studies on dehydrated alfalfa meal.® High
temperatures (100° to 150°) over long periods of time (2 to 6 days), how-
ever, do cause considerable loss of pantothenic acid. In fact, this is the
method by which pantothenic acid-low diets were obtained in the early
studies with this vitamin in chick nutrition.2°: 7!

4B, D. Owen and J. P. Bowland, J. Nutrition 48, 317 (1952).

14a R. M. Melampy and L. C. Northrop, Arch. Biochem. 30, 180 (1951).

15H}. M. Popp and J. R. Totter, J. Biol. Chem. 199, 547 (1952).

16a R, J. Evans, A. C. Groschke, and H. A. Butts, Arch. Biochem. and Biophys. 31,
454 (1951).

15b 'T. Terroine and J. Adrian, Arch. sct. physiol. 4, 485 (1950).

16S. R. Stanberry, E. E. Snell, and T. D. Spies, J. Biol. Chem. 185, 353 (1940).

17C. C. Lardinois, C. A. Elvehjem, and E. B. Hart, J. Dairy Sci. 27, 875 (1944).

17a B, D. Westerman, G. E. Vail, J. Kalen, M. Stone, and D. L. Mackintosh, J. Am.
Dietet. Assoc. 28, 49 and 331 (1952).

18 R. J. Evans, J. A. Davidson, and H. A. Butts, Poultry Sci. 31, 777 (1952).

19 F. M. Strong, A. Earle, and B. Zeman, J. Biol. Chem. 140, exxviii (1941); V. H.
Cheldelin, A. M. Woods, and R. J. Williams, J. Nutrition 26, 477 (1943); B.S.
Schweigert, and B. T. Guthneck, J. Nutrition 61, 283 (1953); S. Cover and W. H.
Smith, Jr., Food Research 17, 148 (1952).

20 QO. L. Kline, J. A. Keenan, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 99, 295
(1932); T. H. Jukes, J. Biol. Chem. 117, 11 (1937).

21 A.M. Pearson, J. E. Burnside, H. M. Edwards, R.S. Glasscock. T. J. Cunha, and
A. F. Novak, Food Research 16, 85 (1951).

638 PANTOTHENIC ACID

A factor affecting the pantothenic acid content of meat, which may be-
come more important in the future, is the loss found in the drip from de-
frosting frozen meat. Pearson et al. report a 33 % loss of pantothenic acid in
this manner.”!

7. EFFECT OF STAGE OF LACTATION

The pantothenic acid content of colostrum milk is significantly lower
than in milk obtained at a later stage of lactation, as determined in studies
with milk of the cow,‘” 7! human,’ ewe,”/* and sow.”!* This is rather a
surprising fact, since colostrum milk is known to be richer than normal
milk in most nutrients.

C. DISTRIBUTION OF COENZYME A

Only a few values for the distribution of coenzyme A in natural materials
are available at this time and are given in Table XIX. Coenzyme A 1s pres-
ent in large amounts in certain dried microorganisms and is in relatively
high amounts in animal tissues.

TABLE XI
PANTOTHENIC AcID ConTENT OF FOODSTUFFS?22-3%
(Fresh basis unless otherwise noted)

Pantothenic Method of assay,

Food acid, y/g. if given Reference and year
Almonds 4.0 -- 30, 1943
Apples 0.6 Microb. 28, 1942
Apricots, canned 0.9 Microb. 29, 1945
Artichoke 4.0 Chick 24, 1941
Asparagus, green, canned 2.0 Microb. 29, 1945

21a P, B. Pearson and A. L. Darnell, J. Nutrition 31, 51 (1946).
216 M. N. Coryell, M. E. Harris, S. Miller, H. H. Williams, and I. G. Macy, Am. J.
Diseases Children 70, 150 (1945).
tle V. EK. Davis, A. A. Heidebrecht, R. W. MacVicar, O. B. Ross, and C. K. Whitehair,
| | J. Nutrition 44, 17 (1951).
22.N. G. Guerrant, M. G. Vavich, and O. B. Fardig, Ind. Eng. Chem. Anal, Ed. 17
710 (1945).
23 A. L. Neal and F. M. Strong, Ind. Eng. Chem. Anal. Ed. 15, 654 (1948).
44'T. H. Jukes, J. Nutrition 21, 193 (1941).
25 HW. A. Waisman and C. A. Elvehjem, Vitamin Content of Meat. Burgess, Min-
neapolis, 1941.
26 J. B. Neilands and F. M. Strong, Arch. Biochem. 19, 287 (1948).
27 M. Ives and F. M. Strong, Arch. Biochem. 9, 251 (1946).
28 V. H. Cheldelin and R. J. Williams, Univ. Texas Publ. 4237, 105 (1942).
29 M. Ives, M. Zepplin, S. R. Ames, F. M. Strong, and C. A. Elvehjem, J. Am. Diet.
Assoc. 21, 357 (1945).
30 W. H. Peterson, J. T. Skinner, and F. M. Strong, Elements of Food Biochemistry,
p. 272. Prentice-Hall, New York, 1943.
30a A. J. Ihde, and H. A. Schuette, J. Nutrition 22, 527 (1941).

~

VIII. OCCURRENCE 639

TABLE XI—Continued

Pantothenic Method of assay,

Food acid, y/g. if given Reference and year

Bacon, sample 1 ae Microb. 28, 1942
Bacon, sample 2 9.8 Microb. 28, 1942
Beans, green string, canned 0.7 Microb. 29, 1945
Beans, green string, fresh 1.5 — 30, 1943
Beans, kidney, dried 5D = 30, 1943
Beans, lima, canned ia! Microb. 29, 1945
Beans, lima, dried 8.3 Microb. 28, 1942
Beans, navy, dried 6.0 — 30, 1943
Beans, yellow wax 0.6 = 30, 1943
Beef, brain 18.0 Microb. 28, 1942
Beef, dried 55.6 Microb. 26, 1948
Beef, heart 20.0 Microb. 28, 1942
Beef, kidney 35.0 — 30, 1943
Beef, liver 76.0 Microb. . 28, 1942
Beef, round 4.9 Microb. 28, 1942
Beef, steak 6.5 — 30, 1943
Beef, tongue 11.0 Microb. 25, 1941
Beets 2.0 = 30, 1943
Beet greens 5.0 = 30, 1943
Blackberries, canned 0.8 Microb. 29, 1945
Blueberries, canned 0.7 Microb. 29, 1945
Brazil nuts 25 — 30, 1943
Bread, rye (40%) 5.0 — 30, 1943
Bread, white 4.0 = 30, 1943
Bread, white enriched 4.6 Microb. 28, 1942
Bread, whole wheat 8.0 == 30, 1943
Broccoli 14.0 Chick 24, 1941
Brussels sprouts 6.0 == 30, 1943
Buckwheat 26.0 = 30, 1943
Cabbage 1.8 Microb. 28, 1942
Cantaloupe 2.3 Microb. 28, 1942
Carrots, canned el Microb. 29, 1945
Carrot, fresh 2.5 Microb. 28, 1942
Cauliflower 9.2 Microb. 28, 1942
Celery, bleached 3.0 = 30, 1943
Cheese, American 2.8 — 30, 1943
Cheese, cottage Pt = 30, 1943
Cheese, Swiss 3.5 = 30, 1943
Cherries, red sour, canned 12 Microb. 29, 1945
Chicken, dark 13.0 Microb. 25, 1941
Chicken, light 8.0 Microb. 25, 1941
Chocolate 1.9 Microb. 28, 1942
Codfish 5.0 = 30, 1943
Corn, white, meal 3.1 Microb. 28, 1942
Corn, yellow, sweet, dried 8.0 == 30, 1943

2.2 Microb. 29, 1945

Corn, cream style, canned

TABLE X1I—Continued

Pantothenic Method of assay,

Food acid, y/g. if given Reference and year

Corn, sweet, canned 2.9 Rat 22, 1945
Cowpeas, dried 18.0 — 30, 1943
Cucumbers 3.9 — 30, 1943
Egg, hen’s, whole 55.4 Microb. 26, 1948
Egg, hen’s, white 1.3 ae 30, 1948
Egg, hen’s, yolk 63.0 Chick 24, 1941
Endive 2.3 — 30, 1943
Flour, rye (dark) 14.1 Microb. 30a, 1941
Flour, rye (white) Lo? Microb. 30a, 1941
Flour, white, patent 3.5 Microb. 28, 1942
Flour, whole wheat 10.0 — 30, 1943
Grapefruit 2.9 Microb. 28, 1942
Grapefruit juice, canned el Microb. 29, 1945
Halibut 15 Microb. 28, 1942
Hominy grits 1.0 Microb. 28, 1942
Kale 3.0 Chick 24, 1941
Kohlrabi 16 — 30, 1943
Lamb, leg of 6.0 Microb. 28, 1942
Lamb, liver 53.0 Microb. 25, 1941
Lettuce, head 1.3 — 30, 19438
Lettuce, leaf ou Microb. 28, 1942
Lima beans Wad Microb. 27, 1946
Mackerel 13.9 Microb. 26, 1948
Milk, pasteurized 3.0 — 30, 1943
Milk, powder, skim 36.0 Chick 24, 1941
Milk, powder, whole 24.0 Microb. 28, 1942
Milk, skim, fresh 3.6 Chick 24, 1941
Milk, whole, raw 2.9 Microb. 28, 1942
Molasses 25.0 — 30, 1943
Mushrooms, canned 9.4 Microb. 29, 1945
Mushrooms, fresh 17.0 Microb. 28, 1942
Mutton, shoulder 4.3 Microb. 28, 1942
Oats, rolled 11.0 Chick 24, 1941
Onions, dry 153 Microb. 28, 1942
Oranges 3.4 Microb. 28, 1942
Orange juice, canned hoe) Microb. 29, 1945
Oysters 4.9 Microb. 28, 1942
Parsley 6.0 — 30, 1943
Peaches, canned 0.6 Microb. 29, 1945
Peaches, fresh 17 Microb. 28, 1942
Pears, halves, canned 225 Microb. 29, 1945
Peanuts, roasted 25.0 Microb. 28, 1942
Peas, blackeyed 10.4 Microb. 28, 1942
Peas, dried 18.0 — 30, 1943
Pawehfteatvercen 1.8 Microb. 23, 1943

F 5.8 — 30, 1943
PeseMercencameed 1.3 Microb. 29, 1945

. 2.0 Rat 22, 1945

640

TABLE XI—Concluded

Pantothenic Method of assay,

Food acid, y/g. if given Reference and year
Pecans 14.0 — 30, 1943
Peppers, green 1.2 — 30, 1943
Peppers, red, canned 1.0 Microb. 29, 1945
Pineapple, canned 0.8 Microb. 23, 1943
3.4 Microb. 28, 1942
Pork, ham, fresh es ian 30, 1943
Pork, ham, smoked 9.0 Microb. 25, 1941
Pork, heart 21.0 Microb. 25, 1941
Pork, kidney 30.0 — 30, 1943
Baceailicat {16.0 Microb. 23, 1943
; \49.0 Microb. 25, 1941
Pork, loin 11.0 Microb. 28, 1942
Potatoes, sweet, canned 4.3 Microb. 29, 1945
Potatoes, sweet 9.4 Microb. 28, 1942
Potatoes, white 6.5 Chick 24, 1941
Prunes, Italian, canned 10) Microb. 29, 1945
Pumpkin, canned 4.0 Chick 24, 1941
Raisins 0.9 Microb. 28, 1942
Rice Krispies a4 Microb. 28, 1942
Rice, bran 22.0 Chick 24, 1941
Rice, white 8.0 — 30, 1943
Set sane’ (26.4 Microb. 26, 1948
: pet Rat 22, 1945
Sardines 15.9 Microb. 26, 1948
Sauerkraut, canned 0.9 Microb. 29, 1945
Spinach, canned 0.6 Microb. 29, 1945
Soe iiciicsh ee Microb. 26, 1948
3 Ibgte: Microb. 28, 1942
Squash, Zucchini 3.0 Chick 24, 1941
Strawberries 2.6 Microb. 28, 1942
Feed ca tcanned ee Microb. 28, 1942
; 3.8 Rat 22, 1945
Tomatoes, ripe Sig) Rat 28, 1942
Tomato juice, canned 30 Rat 29, 1945
Tunafish 14.3 Microb. 26, 1948
Turnips 0.37 Microb. 28, 1942
Turnip greens, canned 0.7 Microb. 29, 1945
Veal, chop tet Microb. 28, 1942
Veal, heart 31.0 Microb. 25, 1941
Veal, liver 52.0 Microb. 25, 1941
Veal, round 16.9 Microb. 27, 1946
Walnuts, English 8.0 Chick 24, 1941
Watercress 125 — 30, 1943
Watermelon Bia il Microb. 28, 1942
Wheat, bran 30.0 — 30, 1943
Wheat, germ meal 29.3 Microb. 26, 1948
Wheat, whole 12.0 Microb. 28, 1942
Yeast, brewer’s, dry 200.0 Chick 24, 1941

641

642 PANTOTHENIC ACID

TABLE XII

PANTOTHENIC AcID CONTENT OF FEEDSTUFFS®: 24, 31-34
(Air-dry basis unless otherwise noted)

Pantothenic Method of assay,
Feedstuff acid, y/g. if given Reference and year

Alfalfa meal, dehydrated

17% protein 38.4 Microb. 32, 1942
20% protein 41.0 = 33, 1947
ee a Chick 24, 1941
4.1 Microb. 5, 1949

Barley malt 8.6 — 33, 1947
Beet pulp, dried 1.8 — 33, 1947
Blood flour 5.5 — 33, 1947
Blood meal eal o= 33, 1947
Bone meal, raw 2.2 — 33, 1947
Bone meal, steamed 1.8 — 33, 1947
Brewer’s dried grains 14.6 — 33, 1947
Buckwheat 12.3 — 33, 1947
Buttermilk, dried 34.2 Microb. 32, 1942
Buttermilk, fresh 4.6 Chick 24, 1941
Citrus pulp, dried 13.2 — 33, 1947
Coconut meal Ua) Microb. 5, 1949
Corn, dent, white 3.5 — 33, 1947
Corn, dent, yellow 4.6 Microb. 5, 1949
17.2 — 33, 1947

Ponte lieu ice’ 2.1 Microb. 5, 1949
Corn gluten meal (41% protein) 13.8 Microb. 32, 1942
Corn oil meal 4.0 _ 33, 1947
Cottonseed meal (41% protein) 14.0 Chick 24, 1941
Cowpeas 17.6 — 30, 1947
Distiller’s dried corn grains WI 7 — 33, 1947
Distiller’s dried grains, wheat with solubles 13.2 — 33, 1947
Distiller’s solubles 25.0 — 34, 1951
Fish meal, herring be eee ig ae
Fish meal, salmon (ee = 33, 1947
Fish meal, sardine 15.8 Microb. 5, 1949
Fish solubles, condensed 40.1 — 33, 1947
Hominy feed 7.4 — 33, 1947
Kafir 12.6 — 33, 1947

31H. Lih, T. E. King, H. Higgens, C. A. Baumann, and F. M. Strong, J. Nutrition
44, 361 (1951).

32 J. C. Bauernfeind, L. C. Norris, and G. F. Heuser, Poultry Sci. 21, 136 (1942).

33 R. V. Boucher and Committee on Feed Composition, Rept. Natl. Research Council
(U. 8S.) 1, Suppl. (1947).

34K. L. Smiley, M. Sobolov, F. L. Austin, R. A. Rasmussen, M. B. Smith, J. M. Van
Lanen, L. Stone, and C. 8. Boruff, Ind. Eng. Chem. 43, 1380 (1951).

:

VIII. OCCURRENCE 643

TABLE XII—Concluded

Pantothenic Method of assay,

Feedstuff acid, y/g. if given Reference and year
p ‘ 16.5 — 33, 1947
Beees OF med 7.1. Microb. 5, 1949
Liver meal 105.0 Chick 24, 1941
Meat scraps, 52% protein 4.6 — 33, 1947
Meat scraps, 60% protein 5.1 — 33, 1947
Meat and bone scrap, 50% protein 3.3 = 33, 1947
Millet 7.9 Microb. 5, 1949
Milo 1352 — 33, 1947
Molasses, cane, Cuban 39.5 — 33, 1947
Molasses, cane, Hawaiian 57.5 Microb. 32, 1942
Oats 15.0 — 33, 1947
Oat middlings 22.9 — 33, 1947
Oats, rolled 11.0 a 33, 1947
Peanut kernels 30n ll — 33,. 1947
Peanut oil meal, 438% protein 53.0 = 33, 1947
Peanut oil meal, 51% protein 34.8 Microb. 5, 1949
Rice bran 22.7 — 33, 1947
Rice polishings 12.1 = 33, 1947
Rye 9.3 — 33, 1947
Rye germ 13.9 Microb. 30a, 1941
Rye middlings Zor Microb. 30a, 1941
Sesame oil cake 5:6 Microb. 5, 1949
F ; : 29.9 Microb. 5, 1949
Skim milk, dried ee is 33. 1947
Soybeans, yellow varieties, whole Ge — 33, 1947
Soybean oil meal, 43% protein 11.0 Microb. 5, 1949
Soybean oil meal, 44% protein 13.9 — 33, 1947
Soybean oil meal, solvent extracted 16.1 = 33, 1947
Sunflower seed oil meal 11.0 Microb. 5, 1949
Tankage, digester, 60% protein 2.4 — 33, 1947
Wheat, hard red, winter 10.6 — 33, 1947
Wheat, northern, spring 14.1 — 33, 1947
Wheat, soft, white AOS) = 33, 1947
48.0 Rat 31, 1951
ee A Meee 5, 1949
Wheat germ ililets) — 33, 1947
Wheat standard middlings 20.0 — 33, 1947
; 60.0 Chick 24, 1941
eect ae i ecae 5, 1949
Whey, fresh 4.0 Chick 24, 1941

Yeast, brewer’s, dried 62.8 Microb. 32, 1942

200.0 Chick 24, 1941

644 PANTOTHENIC ACID

TABLE XIII
PANTOTHENIC ACID CONTENT OF VARIETIES OF GRAIN
(Air-dry basis)

Panto- Panto-
thenic thenic
acid, acid,

Oats! v/g. Wheat? y/g.% Soybeans?
Andrew 6.3 Turkey 13.2 Peking
Beaver 8.6 Tenmarg 12.4 Manchu
Bonda Uo? Chiefkan 11.8 Mukden
Clinton 8.5 Blackhull 12.9 Lincoln
Eaton San Kawvale 15.1 Dunfield
Forvic 10.6 Fulcaster 10.4 T)lini
Huron 9.4 Wabash 9.3 Anwei
Mohawk Uo Purdue 9.4
Overland 8.9 American Banner 11.4 Average
Vicland 10.7 Hymar 11.4
Wolverine Wa Ceres 15.0
Worthy 9.0 Marquis 15.3
Colo 8.3 Thatcher 14.8
Michigan 44720 9.2 Garnet 10.4

— Blue Stem 15.2

Average 9.0 Burbank 15.0
Dicklow 11.9

Average 12.6

@ }xpressed as calcium pantothenate. Other values for wheat also given in refs. 2 and 2a.

VIII. OCCURRENCE

TABLE XIV

PANTOTHENIC AciD CONTENT OF EXPERIMENTAL Dint INGREDIENTS, AND
MIiscELLANEOUS MATERIALS*

(Air-dry basis unless otherwise noted)

645

Sample

Pantothenic
acid, y/g.

Method of assay Reference and year

Beef heart infusion

Casein, commercial

Casein, alcohol-extracted
Casein, acid-washed

Fermented solubles of A. gossypi
Gelatin

Lactoglobulin, crystalline

Liebig meat extract

Liver powder 1:20 (Wilson)
Liver powder, whole (Wilson)
Malt extract

Peptone

Phosphorylase, muscle (crystalline)
Rice brain extract

Royal jelly (honey bee)

Trypsin, crystalline

Tryptone

Vitab

Yeast, dried brewer’s, strain G

Yeast, dried

Yeast extract, Group A
Yeast extract, Group B

=I

lor)
se

(Jy) bo
[te} mal
WNONODOOGTCONS

WNOOHFOCONDONHAOUDY

bo
CoO
SON aN

ou ee

°

a

Microb.
Microb.
Microb.
Microb.

Rat

Rat

Microb.
Microb.
Microb.
Microb.
Microb.

Rat

Microb.
Microb.
Microb.

Microb.
Microb.
Microb.

Microb.
Microb.
Microb.
Microb.

35,
32,
36,
36,
34,
35,
37,
35,
31,
26,
35,
35,

1944
1942
1945
1945
1951
1944
1944
1944
1951
1948
1944
1944
1944
1951
1942
1944
1944
1948
1942
1951
1948
1944
1944

* See ref. 37 for assay values in other purified proteins and enzymes.

36 J, L. Stokes, M. Gunness, and J. W. Foster, J. Bacteriol. 47, 293 (1944).
86M. D. Cannon, R. K. Boutwell, and C. A. Elvehjem, Science 102, 529 (1945).
37 R. J. Williams, F. Schlenk, and M. A. Eppright, J. Am. Chem. Soc. 66, 896 (1944).

646 PANTOTHENIC ACID

TABLE XV

PANTOTHENIC ACID IN ORGANISMS OF DIFFERENT BIOLOGICAL PHYLA?
(y/g. of moist tissue)

Pantothenic acid, y/g.

Fish (Cryprinidae)
Frog (Rana)

Snake (Thamnophis)
Chick embryo

Li)

Red ant (Dolichederus) 1
Cockroach 1
Termites il
Dros. virilis larvae, N. Y. 2

Oyster (Mytilus)
Earthworm

Protozoa (Tetrahymena geleiz) 1
A. aerogenes 3
C. butylicum 2
Mushrooms 1
Yeast, brewer’s 4
Mold

WWNHNDOWWHOONNOOTWN
SCOonmnoveomonvooonnornN

Rat (whole)

a

2 Data from the University of Texas.®® Values determined by microbiological assay.

38 A.M. Woods, J. Taylor, M. J. Hofer, G. A. Johnson, R. L. Lane, and J. R. McMa-
han, Univ. Texas Publ. 4237, 84 (1942).

VIII. OCCURRENCE 647

TABLE XVI
PANTOTHENIC AciID CONTENT OF NORMAL TISSUES?
(Dry basis)

Embryo Embryo

rat chick Mature
Mature Mouse, Beef, Hog, (12-day), (12 day), Chicken, human,
Tissue rat, y/g. /g. /g. ¥/g. ¥/g. ¥/g. y/e. —-v/e.
Entire carcass 37 — — —_ — — — =
Liver 370 120 150 160 140 300 190 136
Spleen 59 #50 Bil 37 -— — — 25
Heart 200 150 63 83 140 400 180 78
Kidney 190 150 (hs 150 = — — 99
Muscle 32 25 28 28 — — —- 53
Lung 58 26 36 44 -— — = 26
Brain 71 82 66 55 240 380 310 68
Ovary 40 — 78 — — — — 18
Pancreas 52 — 44 — = = =e =
Thymus 150 = 25 = = = a =
Thyroid 80 = 7 = = == = —
Testes 150 — 70 _— —_— — — 32
Skin (with hair) 5e2y == _- — — — — 7.5
Intestinal tract 63 — — — — — — 24
Adrenal cortex —- —— 64 — — — = =
Anterior pituitary = = 31 = as = a aos
Posterior pituitary — — 31 = = — 7 ay
Adrenal gland (whole) _- _ — — — — ~ 20

2 Data from the University of Texas.39-41 Values determined by microbiological assay. Other values for
various tissues?> and for 13 different chicken muscles* are available.

39. D. Wright, J. R. McMahan, V. H. Cheldelin, A. Taylor, E. EK. Snell, and R. J.
Williams, Univ. Texas Publ. 4187, 38 (1941).

40 R. J. Williams, A. Taylor, and V. H. Cheldelin, Univ. Texas Publ. 4137, 61 (1941).

41 A. Taylor, M. A. Pollack, and R. J. Williams, Univ. Texas Publ. 4237, 41 (1942).

42. E. Rice, E. J. Strandine, E. M. Squires, and B. Lyddon, Arch. Biochem. 10, 251
(1946).

648 PANTOTHENIC ACID

TABLE XVII

PANTOTHENIC AcID CoNTENT OF MILK OF VARIOUS SPECIES#
Source of milk y/ml.

Human being, white 1.6

Mare, thoroughbred 2.9

Cow 2.9

Goat 2.4

Dog, English bull 4.9

Mouse, albino 23 .0

@ Data from the University of Texas.‘3 Values given in terms of y/ml. fresh milk determined by microbio-
logical assay. Additional values for the pantothenic acid content of milk cf the guinea pig,“¢ ewe,7/4 sow,?2I¢
mare,489 human,”2 and cow,4@, 214, 43¢ are available.

TABLE XVIII
PANTOTHENIC AcID CONTENT OF THE BLOOD oF VARIOUS SPECIES“: 45
(Microbiological assay, 1941 and 1946)¢

Pantothenic acid, 7/100 ml.

Species Cells, % Blood Plasma Cells
Dog 46.7 26.3 31.8 22.4
Horse 32.9 44.8 37.4 51.9
Human being 47.8 19.4 17.0 23.6
Pig 42.8 30.0 34.7 29.6
Rabbit 32.3 (ks 7 57.8 84.6
Sheep 30.2 26.6 24.1 29.0
Chick*® ee 43.6 51.6 21.9

2 Also see section on effect of diet on tissue composition for additional references.

48R. J. Williams, V. H. Cheldelin, and H. K. Mitchell, Univ. Texas Publ. 4237, 97
(1942).

43a W. L. Nelson, A. Kaye, M. Moore, H. H. Williams, and B. L. Herrington, J.
Nutrition 44, 585 (1951).

430 A.D. Holmes, B. V. McKey, A. W. Wertz, H. G. Lindquist, and L. R. Parkinson,
J. Dairy Sci. 29, 163 (1946).

43¢ A. Z. Hodson, J. Nutrition 29, 137 (1945).

44P. B. Pearson, J. Biol. Chem. 140, 423 (1941).

45 P. B. Pearson, V. H. Melass, and R. M. Sherwood, J. Nutrition 32, 187 (1946).

VIII. OCCURRENCE 649

TABLE XIX
CoENZYME A DISTRIBUTION?
(Fresh basis unless otherwise noted)

Coenzyme A, units/g.%

Plant materials

Peas, frozen 4.5
Royal jelly (bee) 0
Spinach 0.74
Tomato 1.3
Wheat germ 30.0
Clostridium butylicium, dried extract 2000.0
Escherichia coli, dried 320.0
Tissues, rat
Liver 132
Adrenal 91
Kadney 74
Brain 28
Heart 42
Intestine 26
Thymus 20
Human red blood cells 3-4
Human plasma 0

* A coenzyme A unit contains 0.7 y of bound pantothenic acid. Data from Kaplan and Lipmann.‘6

IX. Effects of Deficiency
A. IN ANIMALS

GEORGE M. BRIGGS and FLOYD S. DAFT

The effects of pantothenic acid deficiency vary greatly from species to
species. In one species, the rat, some of the deficiency signs have been ob-
tained in rather low incidence or even only sporadically. In addition, in
some laboratories, certain of the generally recognized deficiency signs have
either not been observed at all or their relationship to a lack of pantothenic
acid has appeared questionable. Despite these unexplained discrepancies,
many interesting lesions may be ascribed with confidence to a low level of
this vitamin in the diet. Among the earliest deficiency signs correctly as-
cribed to pantothenic acid deficiency were growth failure in the chick and
rat, dermatitis in the chick, achromotrichia (graying of the hair) in rats
and other animals, and adrenal necrosis and hemorrhage in the rat. These
and other pathological changes will be discussed in the following sections.

46 N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948).

650 PANTOTHENIC ACID

1. Rats

The signs of pantothenic acid deficiency observed in the rat are failure
of growth, achromotrichia (in black, hooded, or brown animals), dermatitis,
porphyrin staining of the fur and whiskers, ‘‘spectacled”’ eyes (circumocular
loss of hair), a characteristic spastic gait, closure of the eyes by a sticky
exudate, and, in some animals, an obvious anemia. At autopsy, the adrenals
may be enlarged, very dark red in color, and apparently engorged with
blood. This advanced stage of adrenal damage has not been observed in all
laboratories but when present, it presents a striking picture that cannot be
overlooked. There is a marked reduction of body fat. Histological examina-
tion has revealed lesions in skin, adrenal, thymus, bone marrow, testis,
intestine, bone, and kidney. In mature pantothenic acid-deficient rats,
reproduction is impaired.

a. Adrenal Necrosis and Hemorrhage

One of the most interesting and significant of these changes is a fatal
cortical necrosis and hemorrhage of the adrenal gland.!: ? Hyperemia and
hemorrhage in the adrenal cortex and medulla had been described earlier
as part of a panmyelophthisis syndrome due to an unknown deficiency,’
and atrophy of the adrenal without mention of necrosis or hemorrhage had
also been described and attributed to filtrate factor deficiency.* Daft et al.?
demonstrated the effectiveness of pantothenic acid in the prevention or cor-
rection of the adrenal necrosis, hemorrhage, and other changes which they
had observed, and these results have been repeatedly confirmed.®*” Gross
and histological examinations of the adrenals have been made in several
laboratories.8-! Grossly, the adrenals are swollen and dark. Ashburn® de-
scribed the microscopic findings as congestion, hemorrhage, atrophy, ne-
crosis, scarring, fibrosis, hemosiderin deposition, and cortical fat depletion.
Deane and McKibbin” studied the sequence of events in the development
of the adrenal lesions. The first changes noted were a disappearance of

1F.S. Daft and W. H. Sebrell, Public Health Repts. (U.S.) 54, 2247 (1939).

2F.S. Daft, W. H. Sebrell, S.H. Babcock, Jr., and T. H. Jukes, Public Health Repts.
(U.S.) 55, 1833 (1940).

3P. Gyorgy, H. Goldblatt, F. R. Miller, and R. P. Fulton, J. Exptl. Med. 66, 579
(1937).

4A.F. Morgan and H. D. Simms, Science 89, 565 (1939).

5R.C. Mills, J. H. Shaw, C. A. Elvehjem, and P. H. Phillips, Proc. Soc. Exptl. Biol.
Med. 45, 482 (1940).

6 W. D. Salmon and R. W. Engel, Proc. Soc. Exptl. Biol. Med. 45, 621 (1940).

7K. Unna, J. Nutrition 20, 565 (1940).

[3 A. A. Nelson, Public Health Repts. (U.S.) 54, 2250 (1939).

\9 L. L. Ashburn, Public Health Repts. (U.S.) 55, 1337 (1940).

10H. W. Deane and J. M. McKibbin, Endocrinology 38, 385 (1946).

IX. EFFECTS OF DEFICIENCY 651

ketosteroid from the inner cortical zones (reticularis and fasciculata), ac-
companied and followed by progressive depletion of sudanophilic droplets
from the same area. Shortly thereafter, foci of necrosis and hemorrhage
appeared in these zones. This progressed in severe cases to almost total
destruction of the cortex, only a thin layer of intact cells remaining in the
zona glomerulosa.

A number of laboratories have reported failure to observe the more ad-
vanced stages of adrenal damage, and some investigators have found no
pathological changes observable in gross or by microscopic examination.
Cowgill and coworkers!! have obtained consistent, rapid, and marked
changes by using weanling rats from mothers placed on the deficient diet
immediately after birth of the offspring.

Because of the fact that depletion of the lipoids of the adrenal cortex
preceded cortical necrosis and hemorrhage, it was proposed by Ashburn®
that the adrenals of pantothenic acid-deficient rats might be functionally
insufficient. It had earlier been suggested by Morgan and Simms? that the
graying and other physical changes of senescence, which they reported as
being associated with a deficiency of filtrate factor, might be mediated
through the adrenal. These investigators reported further that graying in
rats could be cured by relatively large doses of adrenal cortical extract*
or by thyroid extract.” These results have not been confirmed.!*: In fur-
ther support of the concept of adrenal insufficiency in pantothenic acid
deficiency in rats are the results of Gaunt and coworkers,!®> who demon-
strated a greater sensitivity of their animals to water intoxication.

In opposition to the suggestions that the adrenal of the pantothenic acid-
deficient rat might be functionally insufficient, Deane and McKibbin” pro-
posed that there might be overstimulation of the gland due to a stress re-
action. They presented cytological and physiological data in support of
this point of view. McQueeney et al.!® showed also that deficient animals on
drastically restricted sodium intake showed no defect in sodium conserva-
tion. Perry and coworkers” reported that pantothenic acid-deficient rats
showed no loss in ability to react to stress as judged by discharge of ascorbic
acid from the adrenal. Ershoff e¢ al.!® presented evidence that there was no

1G. R. Cowgill, R. W. Winters, R. B. Schultz, and W. A. Krehl, Intern. Rev. Vitamin
Research 28, 275 (1952).

122 A. F. Morgan and H. D. Simms, J. Nutrition 19, 233 (1940).

13 ©, W. Mushett and K. Unna, J. Nutrition 22, 565 (1941).

4 R. B. Schultz, Thesis, Yale University School of Medicine, 1952, quoted by Cow-
gill et al. in ref. 11.

15 R. Gaunt, M. Liling, and C. W. Mushett, Endocrinology 38, 127 (1946).

16 A. J. McQueeney, L. L. Ashburn, F. 8. Daft, and R. R. Faulkner, Endocrinology
41, 441 (1947).

17 W.F. Perry, W. W. Hawkins, and G. R. Cumming, Am. J. Physiol. 172, 259 (1953).

18 B. H. Ershoff, R. B. Alfin Slater, and J. G. Gaines, J. Nutrition 50, 299 (1953).

652 PANTOTHENIC ACID

impairment of pituitary-adrenal function in deficient animals as judged
by resistance to egg white intoxication or as judged by the extent of ad-
renal ascorbic acid depletion and peripheral lymphocyte response follow-
ing epinephrine or ACTH administration.

Morgan and associates!® have also investigated adrenal function in
their animals and have concluded that pantothenic acid deficiency im-
poses a stress on the adrenal cortex, resulting in exhaustion of the gland and
adrenal hypofunction. Dumm ef al.?° have presented evidence that the de-
ficient rat’s ability to resynthesize adrenal cholesterol, after stress, is de-
creased.

Cowgill and associates" and Winters vt al.?! have made a further study
of the physiological events leading to and accompanying adrenal damage.
Using weanling rats which had suckled mothers receiving a pantothenic
acid-deficient diet, they were able to secure striking alterations of the adre-
nals. Large doses of ACTH (4 mg. per rat per day) produced a marked in-
tensification of the adrenal lesion, while cortisone at a level of 2 mg. per
rat per day protected the rats completely from cortical necrosis and hemor-
rhage. The adrenals in the latter animals (cortisone-treated and pantothenic
acid-deficient) were indistinguishable from control animals given cortisone.
These investigators concluded that a depletion of coenzyme A in the adrenal
cortex leads to increased secretion of ACTH and consequent adrenal hyper-
trophy; that the adrenal becomes increasingly unable to produce and
secrete steroid hormone; and that the sustained high level of circulating
ACTH acting upon the enfeebled gland leads, directly or indirectly, to
“hemorrhagic necrosis” of the adrenal cortex.

It is interesting that, although all observers are agreed that in panto-
thenic acid deficiency there is a depletion of adrenal cholesterol and other
steroids, it has been reported by Guggenheim and Olson?!* that lipogenesis
per se does not appear to be altered. In their experiments cholesterol con-
centrations of the liver, heart and blood serum in deficient animals were
normal and C™ incorporation into cholesterol was not significantly re-

duced.

19.8. Hurley and A. F. Morgan, J. Biol. Chem. 195, 583 (1952); R. R. Guehring, L.
8. Hurley, and A. F. Morgan, zbid. 197, 485 (1952); A. F. Morgan and E. M. Lewis,
J. Biol. Chem. 200, 839 (1953).

20 M. E. Dumm, H. Gershberg, E. M. Beck, and E. P. Ralli, Proc. Soc. Exptl. Biol.
Med. 82, 659 (1953).

21 R. W. Winters, R. B. Schultz, and W. A. Krehl, Proc. Soc. Exptl. Biol. Med. 79,
695 (1952); R. W. Winters, R. B. Schultz, and W. A. Krehl, Endocrinology 50, 377
(1952); R. W. Winters, R. B. Schultz, and W. A. Krehl, zbid. 50, 388 (1952); R. B.
Schultz, R. W. Winters, and W. A. Krehl, zbid. 51, 336 (1952).

21a K. Guggenheim and R. E. Olson, J. Nutrition 48, 345 (1952).

IX. EFFECTS OF DEFICIENCY 653

b. Blood Dyscrasias

A second lesion, which has been observed in a few laboratories in panto-
thenic acid-deficient rats, is a fatal aplasia of the bone marrow with anemia,
leucopenia, and granulocytopenia.*: 22-4 In 1937, before the isolation of
pantothenic acid, Gyérgy and coworkers’? observed panmyelophthisis in
72 of 319 rats on various deficient diets. Twenty-four of the 72 animals had
hyperemic and hemorrhagic adrenals. The authors stated that the panmye-
lophthisis was not cured or prevented by the known B vitamins available
(thiamine, riboflavin, and pyridoxine) or by a supposedly active filtrate
factor preparation. In view of subsequent investigations it appears very
probable that pantothenic acid in sufficient amount would have prevented
the appearance of the described syndrome even though it may not be a sign
of an uncomplicated pantothenic acid deficiency. Attempts to duplicate
the hematopoietic findings in other laboratories were unsuccessful until
1945 at which time Daft et al.?? and Carter and coworkers™ described very
similar blood changes in rats receiving purified diets deficient in pantothenic
acid. Daft and coworkers reported that therapy with pantothenic acid was
usually unsuccessful unless folic acid also was administered. Adequate
amounts of pantothenic acid, but not of folic acid, prevented the develop-
ment of the blood dyscrasias. Carter et al. did not test folic acid but reported
that therapy with pantothenic acid was successful in only 25% of their
animals.

A surprising aspect of the situation is that this striking deficiency syn-
drome has been observed in so few of the laboratories which have studied
pantothenic acid deficiency in rats and that even in those few laboratories
its appearance apparently has been sporadic. It appears probable that
special conditions must be necessary, possibly the simultaneous deficiency
of other food essentials.

c. Achromotrichia

A third aspect of pantothenic acid deficiency in black, hooded, or brown
rats is achromotrichia or graying of the hair. Morgan and coworkers®® and
Lunde and Kringstad*® showed that this condition could be prevented or
corrected by the inclusion of filtrate factor concentrates in the diet. This

22 F.S. Daft, A. Kornberg, L. L. Ashburn, and W. H. Sebrell, Public Health Repts.
(U.S.) 60, 1201 (1945).

23 7,. L. Ashburn, F. 8S. Daft, and R. R. Faulkner, Blood 2, 451 (1947).

24C, W. Carter, R. G. Macfarlane, J. R. P. O’Brien, and A. H. T. Robb-Smith,
Biochem. J. 39, 339 (1945).

25 A. F. Morgan, B. B. Cook, and H. G. Davison, J. Nutrition 15, 27 (1938).

26 G. Lunde and H. Kringstad, Avhandl. Norske Videnskaps—Akad. Oslo. I Mat.
Naturv. Kl., No. 1 (1938).

654 PENTOTHENIC ACID

finding was confirmed by others using concentrates,””-*® impure pantothenic
acid,®° and finally the pure compound.*!-*” Considerable controversy de-
veloped, however, as to how completely the achromotrichia of filtrate factor
deficiency (i.e., factors not adsorbed on fuller’s earth and known to be differ-
ent from thiamine, riboflavin, and pyridoxine) could be prevented or cured
by pantothenic acid. Williams* reported completely negative results, and
Frost e¢ al.*® found only a slight effect of the pure vitamin in preventing
graying in their animals. Dimick and Lepp* reported that some measure
of graying persisted in rats given 50 y of synthetic pantothenic acid daily,
and Gyorgy and Poling* indicated that depigmentation of a less striking
nature reappeared in their animals which had been cured of graying even
though pantothenic acid supplementation was continued. Emerson and
Evans"! and Pavcek and Baum” stated that pantothenic acid, although
quite effective, did not restore the hair of rats to its original color but re-
sulted in stippling. Despite the occasional negative findings and the incom-
plete repigmentation frequently observed, it is generally conceded that
pantothenic acid is an important factor in the prevention or treatment of
dietary achromotrichia. It is also recognized that under appropriate experi-
mental conditions other dietary deficiencies may be involved. The effect
of a copper deficiency on graying of the hair in rats is well established.¥-**

27 J. J. Oleson, C. A. Elvehjem, and E. B. Hart, Proc. Soc. Exptl. Biol. Med. 42, 283
(1939).

23 A, Mohammad, O. H. Emerson, G. A. Emerson, and H. M. Evans, J. Biol. Chem.
133, 17 (1940).

29 H. Chick, T. F. Macrae, and A. N. Worden, Biochem. J. 34, 580 (1940).

30 P. Gyorgy, C. E. Poling, and Y. SubbaRow, J. Biol. Chem. 132, 789 (1940).

31 P, Gyorgy and C. E. Poling, Science 92, 202 (1940).

32 P. Gyorgy and C. E. Poling, Proc. Soc. Exptl. Biol. Med. 45, 773 (1940).

33 K. Unna and W. L. Sampson, Proc. Soc. Exptl. Biol. Med. 45, 309 (1940).

34K. Unna, Am. J. Physiol. 133, 473 (1941).

85 C, A. Elvehjem, L. M. Henderson, S. Black, and E. Nielsen, J. Biol. Chem. 140
xxxvi (1941).

36K. Unna, G. V. Richards, and W. L. Sampson, J. Nutrition 22, 553 (1941).

37 L.. M. Henderson, J. M. McIntire, H. A. Waisman, and C. A. Elvehjem, J. Nutri-
tion 28, 47 (1942).

33 R. R. Williams, Science 92, 561 (1940).

39 PD). V. Frost, R. C. Moore, and F. P. Dann, Proc. Soc. Exptl. Biol. Med. 46, 507
(1941).

40 M. K. Dimick and A. Lepp, J. Nutrition 20, 413 (1940).

41G. A. Emerson and H. M. Evans, Proc. Soc. Exptl. Biol. Med. 46, 655 (1941).

42 P. 1. Paveek and H. M. Baum, Proc. Soc. Exptl. Biol. Med. 47, 271 (1941).

43H. L. Keil and V. E. Nelson, J. Biol. Chem. 93, 49 (1931).

44. AH. Free, Proc. Soc. Exptl. Biol. Med. 44, 371 (1940).

45 J. M. Hundley, Proc. Soc. Exptl. Biol. Med. 74, 531 (1950).

46 J. M. Hundley and R. B. Ing, Endocrinology 48, 482 (1951); J. M. Hundley and R.
B. Ing, Federation Proc. 10, 385 (1951)

oe a

aiid

ee

IX. EFFECTS OF DEFICIENCY 655

A deficiency of zinc*” appears to produce a similar effect on the color of the
hair. Folic acid deficiency induced by sulfonamides has been shown to pro-
duce achromotrichia in rats*: 4° as well as in other species, and reports have
appeared on chromotrichial effects from cystine,” choline,®® biotin,®? and
sodium chloride.*! p-Aminobenzoic acid, also, was reported from one labo-
ratory to be a chromotrichial agent,®*?: ** but this finding was not confirmed
by other investigators.*® 97» 54, 58

An extremely interesting finding in regard to the graying in pantothenic
acid deficiency is that of Ralli and Graef®® that rapid and diffuse, though
somewhat transitory, repigmentation, observable first in the skin and then
in the hair, occurs following adrenalectomy. The pigmentation of the skin
was even more pronounced in adrenalectomized control animals receiving
adequate amounts of pantothenic acid. Injections of desoxycortisone in-
hibited the adrenalectomy-induced melanin deposition.*”: *8 Hypophysec-
tomy caused an effect similar to removal of the adrenal but less striking.*®
The significance of these results is not entirely clear. They appear to demon-
strate quite convincingly that the adrenal is in some way concerned with
melanin production or deposition. It is tempting to conclude further that
the achromotrichia of pantothenic acid deficiency is mediated through the
adrenal. However, since the initial pigment deposition is greater than nor-
mal, since it occurs in supplemented control rats as well as deficient animals,
and since the effect is transitory, such a conclusion does not, at this time,
appear to be justified.

d. Nasal Discharge of Porphyrin

Most of the earlier reports on pantothenic acid deficiency in rats, includ-
ing those on studies carried out on “filtrate factor deficiency’’ before the
actual isolation and identification of the vitamin, referred to the accumula-
tion of red material around the nose, staining the whiskers and fur, as
“nosebleed” or “‘blood-caked”’ whiskers. Chick et al.2° and McElroy and

47. BH. Stirn, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 109, 347 (1935).
48 G. J. Martin, Proc. Soc. Exptl. Biol. Med. 51, 353 (1942).

49L. D. Wright and A. D. Welch, J. Nutrition 27, 55 (1944).

50 H.S. Owens, M. Trautman, and E. Woods, Science 93, 502 (1941).

51H. P. Ralli, D. H. Clarke, and E. Kennedy, J. Biol. Chem. 141, 105 (1941).
52S. Ansbacher, Science 98, 164 (1941).

53 G. J. Martin and S. Ansbacher, J. Biol. Chem. 138, 441 (1941).

54G. A. Emerson, Proc. Soc. Exptl. Biol. Med. 47, 448 (1941).

55 F. P. Dann, R. C. Moore, and D. V. Frost, Federation Proc. 1, 107 (1942).
56K. P. Ralli and I. Graef, Endocrinology 32, 1 (19438).

57 H. J. Spoon and E. P. Ralli, Endocrinology 35, 325 (1944).

58H. P. Ralli and I. Graef, Endocrinology 37, 252 (1945).

59 K. P. Ralli, Trans. 1st Conf. on Adrenal Cortex, New York, p. 159 (1950).

656 PANTOTHENIC ACID

associates,®® however, showed that the material was porphyrin in nature.
Chick and coworkers? tentatively identified it as protoporphyrin, but Mc-
Elroy et al.®° presented evidence for its identity with synthetic copropor-
phyrin I. The latter workers showed, also, by ablation experiments that the
source of the porphyrin was the Harderian glands. Figge and Atkinson®
reported that limitation of the drinking water of pantothenic acid-sup-
plemented rats led to a similar porphyrin deposition and concluded that
pantothenic acid may be involved in the regulation of water metabolism.
It has been pointed out by Smith® that similar deposits had been observed
earlier in riboflavin deficiency and that they, also, had been attributed to
dehydration.

e. Growth Failure

The effect of pantothenic acid concentrates (factor 2 concentrates) on
rat growth was clearly shown by Lepkovsky and coworkers® in 1936. Their
sharp separation of factor 1 (pyridoxine) from factor 2 (pantothenic acid)
by adsorption of factor 1 on fuller’s earth contributed greatly to progress
in the field of the B vitamins. Their conclusion that both factors were needed
for growth of rats was repeatedly confirmed in the following years with
concentrates of pantothenic acid®-7° and with the pure compound.?**:
7, 81-42, 71-73 Tn large measure the curtailment of growth in pantothenic acid
deficiency is due to inanition, but it has been shown by paired feeding ex-
periments that the administration of this vitamin results in a specific
growth-promoting effect not related to appetite.”

f. Dermatitis

The first clear differentiation of the dermatitis of pantothenic acid de-
ficiency in the rat from that of other B-vitamin deficiencies appears to have

60 LL. W. McElroy, K. Salomon, F. H. J. Figge, and G. R. Cowgill, Science 94, 467
(1941).

61 F. H. J. Figge and W. B. Atkinson, Proc. Soc. Exptl. Biol. Med. 48, 112 (1941).

628. G. Smith, Proc. Soc. Exptl. Biol. Med. 49, 691 (1942).

68§. G. Smith and D. H. Sprunt, J. Nutrition 10, 481 (1935).

64S. Lepkovsky, T. H. Jukes, and M. E. Krause, J. Biol. Chem. 115, 557 (1936).

65 C, EK. Edgar and T. F. Macrae, Biochem. J. 31, 893 (1937).

66 Y. SubbaRow and G. H. Hitchings, J. Am. Chem. Soc. 61, 1615 (1939).

67M. M. El-Sadr, H. G. Hind, T. F. Macrae, C. E. Work, B. Lythgoe, and A. R.
Todd, Nature 144, 73 (1939).

68 G. H. Hitchings and Y. SubbaRow, J. Nutrition 18, 265 (1939).

69 J. J. Oleson, D. W. Woolley, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 42,
151 (1939). ;

70D. W. Woolley, Science 91, 245 (1940).

1K. Unna, Am. J. Med. Sct. 200, 848 (1940).

72K. Schwarz, Z. physiol. Chem. 275, 245 (1942).

73K. Unna and G. V. Richards, J. Nutrition 28, 545 (1942).

™L,. Voris, A. Black, R. W. Swift, and C. E. French, J. Nutrition 23, 555 (1942).

IX. EFFECTS OF DEFICIENCY 657

been made by Gyérgy and associates’>-” soon after pyridoxine first became
available. These investigators described the lesion as an exfoliative dermati-
tis. Scaliness was observed most frequently in the region of the abdomen,
head, groin, and acillae. In the following years, most other investigators in
the field observed some degree of dermatitis in animals receiving thiamine,
riboflavin, and pyridoxine but deficient in pantothenic acid. After pure
pantothenate became available, it was widely noted that these lesions re-
sponded to its administration.2->: #!-#, 7-73 Sullivan and Nicholls, in their
series of studies on nutritional dermatoses in the rat, investigated the effect
of this deficiency.”® They described the lesions of pantothenic acid deficiency
as generalized scaling, small eczematous crusted plaques, diffuse alopecia
of the venter and the preauricular regions, and alopecia with or without
inflammation in the circumocular regions. They reported that the histologi-
cal lesions consisted of milk hyperkeratosis, acanthosis, occasionally para-
keratosis, a small amount of edema and vesiculation, dilatation of the hair
follicles, and, in the late stages, disintegration of the sebaceous glands in
the areas of alopecia. To most observers, the dermatitis has not been a
striking feature of pantothenic acid deficiency in the rat.

Collins et al.7* have reported that rats kept under conditions of low rela-
tive humidity developed dermatitis, whereas those kept at 50% relative
humidity or above did not.

g. Other Pathological Changes

Other pathological changes reported as occurring in pantothenic acid-
deficient rats are hemorrhages into the gastrointestinal tract with abscesses
and macrophage accumulation in the submucosa;*: 7 reduction of spermato-
genesis and other signs of damage to the testes;’:° lesions of the
kidney,’ 1°79» liver,’: 1° and heart; 7? hemosiderin deposition in the
spleen;®: ° inhibition of skeletal growth (cartilage hypoplasia) ;°: 1®: °° atro-
phy of the thymus;!®: !* 73 and changes in the bone marrow, ranging from
impairment of hematopoiesis®® to the almost complete aplasia which has
been discussed in an earlier paragraph.*: 2-4 Although some degree of some
of the above changes can be brought about by inanition alone (e.g., reduc-

75 P. Gyorgy, J. Am. Chem. Soc. 60, 983 (1938).

76 P. Gyorgy and R. E. Eckardt, Nature 144, 512 (1939).

77 P, Gyorgy, C. E. Poling, and Y. SubbaRow, Proc. Soc. Exptl. Biol. Med. 42, 738
(1939).

78M. Sullivan and J. Nicholls, Arch. Dermatol. and Syphilol. 45, 917 (1942).

78a R. A. Collins, M. Schreiber, and C. A. Elvehjem, J. Nutrition 49, 589 (1953).

786 J. J. Vitale, D. M. Hegsted, J. Di Giorgio, and N. Zamcheck, Metabolism 2, 367
(1953).

79 G. C. Supplee, R. C. Bender, and O. J. Kahlenberg, Endocrinology 30, 355 (1942).

80M. M. Nelson, E. Sulon, H. Becks, W. W. Wainwright, and H. M. Evans, Proc.
Soc. Exptl. Biol. Med. 73, 31 (1950).

658 PANTOTHENIC ACID

tion of spermatogenesis and inhibition of skeletal growth), it does not ap-
pear that any of the changes reported can be accounted for entirely in this
way.

2. CHICKS

Deficiency signs, now known to be due chiefly to a lack of pantothenic
acid, were described in chicks fed heated diets by Kline et al.*! in 1932.
Even before this time, studies with chickens had indicated that there were
unidentified factors of the B complex yet to be discovered (for example,
vitamin B; of Eddy e¢ al.8? and the unidentified factor of Norris and co-
workers* 8), It is now evident that perhaps a part, but not all, of these
early deficiencies can be accounted for by a lack of pantothenic acid. (See
the review by Williams.®°) Since 1932, basal diets have been improved for
the study of this vitamin and deficiency signs have been more adequately
described by a number of workers.’*-!"! That the use of purified or synthetic
pantothenic acid in the diet would prevent the occurrence of certain de-
ficiency signs was first shown in 1939-1940 by Woolley et al.,10, 1%
Jukes,!: 1° Babcock and Jukes,!°° and Stiller et al.1”

810. L. Kline, J. A. Keenan, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 99, 295
(1932).

82 W. H. Eddy, S. Gurin, and J. C. Keresztesy, J. Biol. Chem. 87, 729 (1930).

83 7,. C. Norris and A. T. Ringrose, Science 71, 648 (1930).

84 A. T. Ringrose, L. C. Norris, and G. F. Heuser, Poultry Sci. 10, 166 (1931).

85 R. J. Williams, Advances in Enzymol. 3, 253 (1943).

86 T). M. Hegsted and F. Lipmann, J. Biol. Chem. 174, 89 (1948).

87 M. E. Coates, J. E. Ford, G. F. Harrison, 8. K. Kon, E. E. Shepheard, and F. W.
Wilby, Brit. J. Nutrition 6, 75 (1952).

88 H. A. Waisman, O. Mickelsen, and C. A. Elvehjem, J. Nutrition 18, 247 (1939).

89 T. H. Jukes, J. Nutrition 21, 193 (1941).

90'T. H. Jukes and 8. Lepkovsky, J. Biol. Chem. 114, 117 (1936).

1 J. G. Lease and H. T. Parsons, Biochem. J. 28, 2109 (1934).

9 C, A. Elvehjem and C. J. Koehn, Jr., J. Biol. Chem. 108, 709 (1935).

93 §. Lepkovsky and T. H. Jukes, J. Biol. Chem. 114, 109 (1936).

94 A. T. Ringrose and L. C. Norris, J. Nutrition 12, 553 (1936).

95 O. Mickelsen, H. A. Waisman, and C. A. Elvehjem, J. Biol. Chem. 124, 313 (1938).

96 M. K. Dimick and A. Lepp, J. Nutrition 20, 413 (1940).

7 H. A. Waisman, R. C. Mills, and C. A. Elvehjem, J. Nutrition 24, 187 (1942).

98 'T. H. Jukes and L. W. McElroy, Poultry Sci. 22, 4388 (1943).

99'T. Ram, Poultry Sci. 28, 425 (1949).

100 J. L. Milligan and G. M. Briggs, Poultry Sci. 28, 202 (1949).

101 M. EK. Coates, S. K. Kon, and E. E. Shepheard, Brit. J. Nutrition 4, 203 (1950).

102 T). W. Woolley, H. A. Waisman, and C. A. Elvehjem, J. Am. Chem. Soc. 61, 977
(1939).

103 T). W. Woolley, H. A. Waisman, and C. A. Elvehjem, J. Biol. Chem. 129, 673 (1939).

104 'T. H. Jukes, J. Am. Chem. Soc. 61, 975 (1939).

105 TH. Jukes, J. Biol. Chem. 129, 225 (19389).

106 S. H. Babcock, Jr., and T. H. Jukes, J. Am. Chem. Soc. 62, 1628 (1940).

IX. EFFECTS OF DEFICIENCY 659

A unique feature of acute pantothenic acid deficiency in the young chick
is the occurrence of a severe dermatitis in the corners of the mouth, often
extending under the lower mandible and to the nostrils. The dermatitis is
the first specific symptom seen, usually when the chicks are 14 to 21 days
old. The eyelids become granular and eventually stick together in severely
affected birds unless they are manually separated. Mild dermatitis between
the toes and on the upper surface of the foot may also occur, but, if so,
this is not generally seen until several days after appearance of the mouth
and eye symptoms. The foot symptoms may never appear in borderline
deficiencies.

The dermatitis symptoms closely resemble those of a biotin deficiency in
the chick. Early reports of Norris and coworkers*: * in 1930 and 1931 de-
scribed symptoms chiefly of a biotin dermatitis in chicks, sometimes mis-
takenly attributed in the literature since then to be due mainly to a panto-
thenic acid deficiency. The deficiency described by these investigators was
produced, in part, by diets containing unheated egg white, which is now
known to cause a biotin deficiency. Distinction between the dermatitis of
pantothenic acid and of biotin deficiencies has been since described by Lease
and Parsons,” Ringrose and Norris,’ !° Lepkovsky and Jukes,!°? Hegsted
et al.,"° and others.!" In true pantothenic acid deficiency the mouth and
eyelids are chiefly affected and the feet are involved to a minor extent,
whereas in a biotin deficiency the feet are severely affected before the oral
symptoms appear.

Other signs of pantothenic acid deficiency in the chick include poor feath-
ers (which may become brittle and drop off),!°° lowered efficiency of feed
utilization,!°° lowered pantothenic acid content (and no doubt coenzyme A)
of blood and tissues,!” slow growth rate or weight loss, and, in severe cases,
incoordination, paralysis, and death at as early as 3 to 4 weeks of age.%!-10!
Although graying of hair has been associated with pantothenic acid defi-
ciency in certain other animals, no loss of feather pigment has been noted
in colored breeds of chicks. The feather depigmentation reported by Groody
and Groody in 1942" attributed to a pantothenic acid deficiency, is un-
confirmed and was more likely due to inanition which itself may cause de-
pigmentation.1* The positive control birds fed pantothenic acid in the

107 H. T. Stiller, S. A. Harris, J. Finkelstein, J. C. Keresztesy, and K. Folkers, J.
Am. Chem. Soc. 62, 1785 (1940).

108 A. T. Ringrose and L. C. Norris, J. Nutrition 12, 535 (1936).

109 §. Lepkovsky and T. H. Jukes, J. Biol. Chem. 111, 119 (1935).

10 TD). M. Hegsted, J. J. Oleson, R. C. Mills, C. A. Elvehjem, and E. B. Hart, J.
Nutrition 20, 599 (1940).

11 'T. H. Jukes, Biol. Symposia 12, 253 (1947).

12 i. E. Snell, D. Pennington, and R. J. Williams, J. Biol. Chem. 133, 559 (1940).

u3 T. C. Groody and M. E. Groody, Science 95, 655 (1942).

4G. M. Briggs, Poultry Sci. 15, 41 (1946).

660 PANTOTHENIC ACID

studies of Groody and Groody gained only 151 g. in 66 days, showing that
other growth factors were lacking also.

Histological study of the tissue of pantothenic acid-deficient chicks, as
reported by Phillips and Engel!> and Shaw and Phillips,!!* showed lesions
in the spinal cord characterized by myelin degeneration of myelinated fibers
distributed widely throughout the white matter but chiefly in the lateral

i

and anterior columns and extending to all segments of the cord. Axon de-
generation also occurred to some extent. The spinal cord alone and not the
peripheral nerves seemed to be involved.® Thymus involution and, oc-
casionally, liver damage were also seen. Coates et al.!"' have also reported
liver damage (‘‘dark and patchy livers’) in pantothenic acid-deficient
chicks.

Ram? found myelin degeneration in the motor apparatus of the brain
in addition to the spinal cord. Ram also found eroded gizzard lining, slightly
thickened proventriculus, and distended gall bladder. It is questionable
whether or not these symptoms are specific, since the diet of Ram contained
only 10 y each of added biotin and folic acid per 100 g. of diet, and no
weight values of the positive control birds were given for proper evaluation
of the results.

That hens also require pantothenic acid was suggested by Bauernfeind
and Norris in 1939"7: "8 and later confirmed by Gillis et al. (1942),"9 who
first used crystalline pantothenic acid. Deficient hens, as demonstrated
by more recent work, may not have the typical dermatitis symptoms seen
in the chick, but in severe cases they show loss of weight, possible drop in
egg production, poor hatchability of eggs (due to death of the 18 to 21-
day-old embryo), poor qualtity of down on the embryo, and loss of via-
bility and growth of progeny, as indicated chiefly by the work of Gillis e¢
ql 119-121

A deficiency in the hen also results in a lowering of the pantothenic acid
content of the egg,}?°: 1? 123 the blood, and the tissues.!?°: 174: 125 Low panto-
thenic acid content of eggs is associated with lowered amounts in hatched
chicks.!?? Higher than normal pantothenic acid content of eggs may give

15 P. H. Phillips and R. W. Engel, J. Nutrition 18, 227 (1939).

16 J. H. Shaw and P. H. Phillips, J. Nutrition 29, 107 (1945).

17 J. C. Bauernfeind and L. C. Norris, Science 89, 416 (1939).

u8 J.C. Bauernfeind and L. C. Norris, J. Nutrition 18, 579 (1939).

19 M. B. Gillis, G. F. Heuser and L. C. Norris, J. Nutrition 23, 153 (1942).

120 M. B. Gillis, G. F. Heuser, and L. C. Norris, J. Nutrition 35, 351 (1948).

121 M. B. Gillis, G. F. Heuser, and L. C. Norris, J. Nutrition 26, 285 (1943).

122 HY, E. Snell, E. Aline, J. R. Couch, and P. B. Pearson, J. Nutrition 21, 201 (1941). ~
123 P, B. Pearson, V. H. Melass, and R. M. Sherwood, Arch. Biochem. 7, 353 (1945).
124 P, B. Pearson, V. H. Melass, and R. M. Sherwood, J. Nutrition 32, 187 (1946). :
125 P. B. Pearson and V. H. Melass, Federation Proc. 5, 237 (1946). f

IX. EFFECTS OF DEFICIENCY 661

rise to depressed size of embryonic heart and brain and higher concentra-
tion of hemoglobin as compared with the normal.'?®

It is interesting that there have recently been occasional reports showing
that pantothenic acid resulted in small (and often insignificant) growth
responses when added to practical poultry rations, chiefly of the corn-
soybean oil meal type.'?”"° As a result of these reports many feed manu-
facturers are today adding supplementary synthetic calcium pantothenate
to certain poultry rations, especially turkey starters. In spite of this, at
present there is no clear-cut evidence that supplements of this vitamin need
to be added routinely to any commercial poultry feed. For every report
showing a possible positive effect from pantothenic acid addition there have
been many more showing negative results. Obviously, future studies on
this aspect are needed.

3. TURKEYS

Symptoms of pantothenic acid deficiency in the turkey poult are similar
to those in chickens and include dermatitis of the mouth, adhesions of the
eyelids, weakness, lowered growth rate, and, in severe cases, death. The
deficiency was first studied by Jukes"! in 1938, before the availability of
pure pantothenic acid. More recent studies, with pure pantothenic acid,
have been made by Lepkovsky et al. (1945),!°? who showed that the require-
ment was higher than that of chickens, and by Kratzer and Williams
(1948)° who used an improved basal diet. No information is available on
pantothenic acid deficiency in the mature turkey, although it could be as-
sumed that it is necessary.

4. Ducks, PIGEONS, AND OTHER FowL

Probably the first report of a pantothenic acid deficiency in the duck was
made by Trager!** in 1943, who used a heated grain-casein diet. Deficient

26 A. Taylor, J. Thacker, and D. Pennington, Science 94, 542 (1941).

127 J, A. Marvel, C. W. Carrick, R. E. Roberts, and 8. M. Hauge, Poultry Sci. 24, 253
(1945) ; H. R. Bird and M. Rubin, zbid. 25, 87 (1946); D. H. Mishler, C. W. Carrick,
R. E. Roberts, and 8. M. Hauge, ibid. 25, 479 (1946).

28 A. R. Robblee and D. R. Clandinin, Poultry Sci. 28, 781 (1949) ; 29, 777 (1950) ; 32,
579 (1953).

129 §, J. Slinger, W. F. Pepper, D. C. Hill, and E.S. Snyder, Poultry Sci. 31, 193 (1952);
M. L. Sunde, J. R. Vedvik, H. W. Bruins, and W. W. Cravens, ibid. 31, 571 (1952).

130 F, H. Kratzer and D. Williams, Poultry Sci. 27, 518 (1948).

131 T, H. Jukes, Poultry Sci. 17, 227 (1938).

132 §, Lepkovsky, F. H. Bird, F. H. Kratzer, and V. S. Asmundson, Poultry Sci. 24,
335 (1945).

133 W, Trager, J. Exptl. Med. 77, 557 (1948).

662 PANTOTHENIC ACID

ducks became weak, grew poorly, had lowered pantothenic acid content
of the blood, and had sticky eyelids, while the control ducks fed pantothenic
acid appeared ‘entirely normal.” More extensive studies have been made
by Hegsted and Perry,'** who found that 3- to 4-day-old ducks very rapidly
develop acute pantothenic acid deficiency when placed on a synthetic diet
low in pantothenic acid. In these studies ducks failed to grow after only 2
or 3 days on the diet and usually died within 4 to 7 days, whereas positive
control ducks with pantothenic acid gained as much as an average of 35
g. per day for 10 days. It may be pointed out that no other animal has been
reported to show such marked effects in such a short period when fed panto-
thenic acid-low diets.

Because of the obvious importance of pantothenic acid for the duck, this
species has been useful in studying the effect of a deficiency of this vitamin
on the metabolism of tissues. Olson and Kaplan,!* in 1948, and Olson and
Stare,!* in 1951, reported that coenzyme A values of liver and heart tissues
of pantothenic acid-deficient ducks fell 50 to 70 % in only 5 days and that
there was a decreased ability of the tissues to metabolize pyruvic acid. In
these two studies, ducks 7 days old were used in order to obtain better sur-
vival. The authors reported the occurrence of anorexia, conjunctivitis, and
decreased feed efficiency in the deficient ducks. Dermatitis was noted in
these studies,!° although Hegsted and Perry" did not observe dermatitis
in their ducks.

Some of the earliest studies on what is now known as pantothenic acid
were made with the pigeon, although the diets were complicated with other
deficiencies.®?: 8°» 187-140 At least part of the activity of vitamin B3;,® first
discovered in 1928, may probably be explained by pantothenic acid. Lee
and Hogan first used pure pantothenic acid in the pigeon and reported, in
1942, that this vitamin was needed for growth and for prevention of ane-
mia.!4! Additional studies with the pigeon, with the use of nutritionally
complete diets, are needed.

No reports have appeared on pantothenic acid deficiency in pheasants,
quail, geese, guinea, and other fowl, although presumably these animals
also require pantothenic acid.

134 T), M. Hegsted and R. L. Perry, J. Nutrition 35, 411 (1948).

135 R, EK. Olson and N. O. Kaplan, J. Biol. Chem. 175, 515 (1948).

136 R,. E. Olson and F. J. Stare, J. Biol. Chem. 190, 149 (1951).

137 R. R. Williams and R. E. Waterman, J. Biol. Chem. 78, 311 (1928).

1388 C, W. Carter and J. R. O’Brien, Biochem. J. 30, 48 (1936).

139 C, W. Carter and J. R. O’Brien, Biochem. J. 33, 1810 (1939).

1440 C, W. Carter, H. W. Kinnersley, and R. A. Peters, Biochem. J. 24, 1832 (1930).
141 J. G. Lee and A. G. Hogan, Missouri Agr. Expt. Sta. Research Bull. 342 (1942).

IX. EFFECTS OF DEFICIENCY 663

Pe WaGe

Before the availability of pure pantothenic acid, several reports in 1939
indicated that this vitamin may be important in dog nutrition.” 4-44 These
studies were obviously complicated with other deficiencies, and it wasn’t
until Morgan (1941),!° Schaefer et al. (1942),"° Seudi and Hamlin (1942) ,17
and Silber (1944)"8 used pure pantothenic acid that convincing evidence
was obtained for its need. Even these diets were inadequate by today’s
standards, however.

Pantothenic acid-deficient dogs usually show the following symptoms:"°:
48 lowered growth rate, decreased appetite, irritability and sudden prostra-
tion or coma, rapid respiratory and heart rate, convulsions, gastrointestinal
symptoms, and, eventually, death. Spasticity may occur in the hind quar-
ters during the last week. The hair of deficient dogs may appear coarser,
but there has been no evidence of alopecia, graying, or other skin or hair
disorder. The corneal reflex is sluggish at the time of collapse, and there
may be excessive salivation.

Gross examination of tissues of pantothenic acid-deficient dogs revealed
fatty livers (as high as 55 % fat), evidence of hemorrhagic kidney degenera-
tion in the cortex and medulla, frequently gastritis or enteritis and intus-
susception, and possibly mottled thymuses, The occurrence of mottled
thymuses and hemorrhagic kidneys has been questioned.'#8 No histological
studies have been presented except preliminary liver pathology.!”

In addition to these signs, deficient dogs show lowered pantothenic acid
content of liver, muscle, brain, and blood,™* lowered blood sugar,'* a low-
ering of cholesterol, cholesterol esters, lipoid phosphorus, and total lipoids
in the blood (correlated with occurrence of liver damage),!7 and possible
decreased gastrointestinal motility and poor digestion and absorption of
carbohydrate and protein.’ Although hemorrhagic adrenals were not seen
in earlier studies,'4* “8 Morgan and Guehring?®® stated that they obtained
hemorrhagic adrenals in their pantothenic acid-deficient dogs unless 1 % of
cholesterol was added to the diet. Fatty livers occurred whether or not
cholesterol was fed.1®°

142, P, J. Fouts, O. M. Helmer, and S. Lepkovsky, J. Nutrition 19, 393 (1940).

143 J, M. McKibbin, R. J. Madden, 8. Black, and C. A. Elvehjem, Am. J. Physiol.
128, 102 (1939).

44 J. M. McKibbin, 8S. Black, and C. A. Elvehjem, Am. J. Physiol. 130, 365 (1940).

45 AF. Morgan, Science 93, 261 (1941).

46 AE. Schaefer, J. M. McKibbin, and C. A. Elvehjem, J. Biol. Chem. 148, 321 (1942).

47 J. V. Scudi and M. Hamlin, J. Nutrition 24, 273 (1942).

48 R. H. Silber, J. Nutrition 27, 425 (1944).

49 C, G. Bly, F. W. Heggeness, and E. S. Nasset, J. Nutrition 26, 161 (1943).

150 A. F. Morgan and R. R. Guehring, Federation Proc. 10, 226 (1951).

664 PANTOTHENIC ACID

The amount of pantothenic acid excreted in the urine is in proportion
to the amount in the diet (Silber and Unna!®! and Silber™’). Only a small
fraction of ingested pantothenic acid may appear in the urine, whereas
fecal excretion is rather constant (Silber!*?).

Adult dogs are more resistant to a deficiency than are puppies, apparently
requiring less dietary pantothenic acid.“°48: 153 Little is known about pan-
tothenic acid requirements for reproduction of the dog.

6. GuINEA Pics

Reid in 1952'*4 demonstrated that young guinea pigs require a dietary
source of pantothenic acid when fed a synthetic-type diet. Symptoms of
deficiency include decrease in growth rate and loss of weight, anorexia ac-
companied by inactivity and weakness, rough hair coat, cyanosis at the
margin of the ears, and diarrhea. The report of Reid is the first study of a
clear-cut pantothenic acid deficiency in the guinea pig. A previous study by
Morgan and Simms” showing gray hair in guinea pigs was inadequate to
prove that pantothenic acid was the limiting factor.

7. SWINE

It would be difficult to determine who first described signs of pantothenic
acid deficiency in swine, because abnormal gait, now known to be associated
with pantothenic acid deficiency, had been seen in pigs many years before
the discovery of pantothenic acid (see the review by Wintrobe, et al.1®*).
Between 1938 and 1941 deficiency symptoms were obtained in pigs by a
number of workers, but it is clear now that the diets used were deficient in
other factors as well as in pantothenic acid.15>-1%°

The first studies in which responses were obtained with pure pantothenic
acid were made independently in 1942 by Hughes!® and Wintrobe et al.1®
with the use of diets very similar to those used previously. Since 1942, a
number of workers have made careful studies of pantothenic acid-deficiency

151 R, H. Silber and K. Unna, J. Biol. Chem. 142, 623 (1942).

152 R. H. Silber, Arch. Biochem. 7, 329 (1945).

153 A. QO. Seeler and R. H. Silber, J. Nutrition 30, 111 (1945).

154 M. E. Reid, Federation Proc. 11, 453 (1952).

155 M. M. Wintrobe, J. L. Miller, Jr., and H. Lisco, Bull. Johns Hopkins Hosp. 67,
377 (1940).

156 H. Chick, T. F. Macrae, A. J. P. Martin, and C. J. Martin, Biochem. J. 32, 2207
(1938).

157 Hi, H. Hughes, J. Nutrition 17, 527 (1939).

1588 M. M. Wintrobe, Am. J. Physiol. 126, 375 (1939).

159 N. R. Ellis and L. L. Madsen, J. Agr. Research 62, 303 (1941).

160}, H. Hughes, J. Agr. Research 64, 185 (1942).

161 M. M. Wintrobe, M. H. Miller, R. H. Follis, Jr., H. J. Stein, C. Mushatt, and S. —

Humphreys, J. Nutrition 24, 345 (1942).

IX. EFFECTS OF DEFICIENCY 665

symptoms in swine, although again much of this work was complicated by
other vitamin deficiencies.!®-1%

As a result of the above-mentioned studies, further clarified by studies
made since 1947 with more complete diets,!®°!” it may be concluded that
the following symptoms of pantothenic acid deficiency may be seen in young
swine (given approximately in the order of appearance): excessive lachryma-
tion, coughing, decrease in appetite, dermatitis, incoordinated movements
of the hind legs and a spastic gait (‘‘goose-stepping’’—one of the most
characteristic symptoms), dull and roughened hair coat and skin and even-
tually some alopecia, brown exudate around the eyes, diarrhea, poor weight
gains, loss of sucking reflexes and control of tongue, rectal hemorrhages,
and low urinary excretion of pantothenic acid. Postmortem gross examina-
tion reveals loss of subcutaneous and internal fat, soft ribs and long bones,
and light bone marrow.!® Gastritis!® and diffuse hyperemia, formation of
small ulcers, and inflammatory changes of the bowel'® may also be seen.

Changes in the tissues may include development of a moderate normo-
cytic anemia, a fall in serum chlorides, increase in the carbon dioxide com-
bining power of the blood, a terminal rise in non-protein nitrogen, and some-
times hypoglycemia.!® A lowering of the pantothenic acid content of blood
has also been observed in deficient animals!* as well as a lowering of creati-
nine, total lipids, total cholesterol, cholesterol esters, and free cholesterol.!”

Histological examination!® may show atrophy of the cells lining the
glands of the mucosa, abscess formation, and ulceration of the large intes-
tine. Sensory nerve degeneration is also seen.!®° The dorsal root ganglion
cells exhibit chromatolysis. The Nissl bodies become finer and finally dis-
solve, leaving a homogeneous ground substance. Loss of myelin of the pe-
ripheral nerves and axis cylinder degeneration is also found, as well as cer-

162 H}, H. Hughes and N. R. Ittner, J. Animal Sci. 1, 116 (1942).

163 MI. M. Wintrobe, R. H. Follis, Jr., R. Aleayaga, M. Paulson, and 8S. Humphreys,
Bull. Johns Hopkins Hosp. 78, 313 (1948).

164 N.R. Ellis, L. L. Madsen, and C. O. Miller, J. Animal Sci. 2, 365 (1948).

165 R. H. Follis and M. M. Wintrobe, J. Exptl. Med. 81, 539 (1945).

166 R, W. Colby, T. J. Cunha, C. E. Lindley, D. R. Cordy, and M. E. Ensminger,
J. Am. Vet. Med. Assoc. 118, 589 (1948).

167 R. W. Luecke, F. Thorp, Jr., W. N. McMillen, and H. W. Dunne, J. Animal Sct.
8, 464 (1949).

168 R, W. Luecke, W. N. McMillen, and F. Thorp, Jr., J. Animal Scz. 9, 78 (1950).

168 G. J,. Sharma, R. L. Johnston, R. W. Luecke, J. A. Hoefer, M. L. Gray, and F.
Thorp, Jr., Am. J. Vet. Research 18, 298 (1952).

169 A. C. Wiese, W. P. Lehrer, P. R. Moore, O. F. Pahnish, and W. V. Hartwell, J.
Animal Sci. 10, 80 (1951).

170 R. W. Luecke, J. A. Hoefer, and F. Thorp, Jr., J. Animal Sci. 11, 238 (1952).

171 Committee on Animal Nutrition, Recommended Nutrient Allowances for Swine.
National Research Council, Washington, D. C., 1950.

172 W. C. Russell and A. E. Teeri, Federation Proc. 7, 297 (1948).

666 PANTOTHENIC ACID

tain other changes.!® No changes in the adrenal gland have been noted
in swine.1®

The heated diet, used to a great extent in pantothenic acid studies with
poultry, has not been used to any great extent with swine. However, Ellis
and Madsen?*? in 1941, showed that incoordination and myelin degeneration
developed in animals fed a heated diet.

In experiments with sows, Hodgskiss et al. in 1950'”? demonstrated for
the first time that pantothenic acid-deficiency signs may develop in the adult
animal. Deficient sows showed loss of appetite, reduced water intake,
‘“‘voose-stepping”’ with hind legs, diarrhea, and rectal hemorrhages. Repro-
duction was abnormal because of death of the fetus. Autopsy of pregnant
sows showed macerating fetuses in the uterine horns as well as ‘‘hemor-
rhagiconecrotic cecocolitis,”’ gastroenteritis, and catarrh of the stomach and
small intestine.

It is of interest that in practical swine feeding there is some evidence that
the usual amount of pantothenic acid in corn-soybean oil meal type rations
is often not enough to provide for optimal growth.!®: 1%: 174 This is espe-
cially true if the diet is low in protein (less than 15 %) since higher levels of
soybean oil meal may protect pigs from a deficiency of pantothenic acid!”°
owing either to increased levels of protein or to some other sparing factor.
Several reports to the contrary, showing no effect of pantothenic acid in
practical rations, have appeared.’ Whether this vitamin should be added
to all commercial swine rations of a corn-soybean meal nature can only
be answered by further research.

8. CATTLE AND SHEEP

Although it is well known that all the B vitamins, including pantothenic
acid, are synthesized by the microflora in the rumen of cattle and sheep,
it is becoming increasingly evident that dietary sources of the B vitamins
are needed in the very young animal before the rumen starts to function.
For instance, the newborn calf requires a dietary source of pantothenic
acid when fed ‘‘synthetic-type”’ diets, according to Johnson e¢ al. in 1947.17
Signs of pantothenic acid deficiency included diarrhea, cessation of growth,
weakness of the legs with inability to stand, and decreased urinary levels

173 A. W. Hodgskiss, M. E. Ensminger, R. W. Colby, and T. J. Cunha, J. Animal Sci. —
9, 619 (1950); M. E. Ensminger, R. W. Colby, and T. C. Cunha, Washington Agr. |

Expt. Sta. Circ. 184, 1951.

174 J. KE. Briggs and W. M. Beeson, J. Animal Sci. 10, 813 (1951); R. W. Luecke, J. A.
Hoefer, and F. Thorp, Jr., J. Animal Sci. 12, 605 (1953).

174a T). V. Catron, R. W. Bennison, H. M. Maddock, G. C. Ashton, and P. G. Homeyer,
J. Animal Sci. 12, 51 (1953).

178 B. C. Johnson, H. H. Mitchell, T. S. Hamilton, and W. B. Nevens, Federation
Proc. 6, 410 (1947).

er ats

IX. EFFECTS OF DEFICIENCY 667

of pantothenic acid. These results are only preliminary, and more work is
necessary with this vitamin in cattle. Pantothenic acid deficiency has not
been studied in the young lamb as yet, but it would be expected they would
need this vitamin.

9. MonxKEYS

Very little information is available concerning pantothenic acid in mon-
key nutrition. McCall e¢ al.!76 in 1946 reported that pantothenic acid de-
ficiency in rhesus monkeys was characterized by lack of growth, ataxia,
graying and thinning of the fur, anemia, diarrhea, and cachexia. Dermatitis
was noted in one animal. These studies were complicated by a concurrent
deficiency of a factor present in liver, and complete recovery was not ob-
tained with the administration of pantothenic acid. Additional studies in
monkeys are needed, especially because of the close relationship between
this animal and the human being.

The daily intake of 3 mg. of pantothenic acid appears to be fully adequate
to prevent the occurrence of deficiency symptoms.

10. Mice

In most, but not all, respects, pantothenic acid deficiency in the mouse
resembles its counterpart in the rat. The most striking differences are the
absence of necrotic and hemorrhagic adrenals in the mouse and the de-
velopment of paralysis of the hind legs with nerve tissue degeneration in
this species.

Achromotrichia and growth failure of C57 black mice and repigmenta-
tion and growth resumption following pantothenic acid therapy were de-
scribed by Gyorgy and Poling.*? Martin!” reported that graying in Rockland
strain black mice was partially, but not completely, reversed by similar
therapy. Norris and Hauschildt!”® observed the development of dermatitis
in mice receiving thiamine, riboflavin, niacin, pyridoxine, and ‘“‘filtrate
factor.’’ There is considerable question, however, as to whether their ani-
mals were receiving adequate amounts of filtrate factor, i.e., pantothenic
acid. Cerecedo and coworkers!”®: 8° and Martin!” noted a very similar
dermatitis, together with alopecia and growth failure, in mice which were
deficient in this vitamin. All these changes were prevented or reversed by
pantothenic acid administration.

76 K, B. McCall, H. A. Waisman, C. A. Elvehjem, and E. S. Jones, J. Nutrition 31,
685 (1946).

177 G, J. Martin, Science 93, 422 (1941).

178 E.R. Norris and J. Hauschildt, Science 92, 316 (1940).

179 J. G. Sandza and L. R. Cerecedo, J. Nutrition 21, 609 (1941).

180 J. R. Foy and L. R. Cerecedo, J. Nutrition 22, 439 (1941).

668 PANTOTHENIC ACID

Morris and Lippincott!*!-!83 made histopathological as well as other stud-
ies of pantothenic acid deficiency in C3H mice. They reported growth fail-
ure, followed by depilation and a dermatosis which presented a scaly
‘““chalklike” appearance. An additional deficiency sign was a partial paraly-
sis of the hind legs. Histologically, the dermatosis was described as hyper-
keratotic and desquamative; myelin degeneration of the spinal cord, pos-
terior roots, and sciatic nerves was noted. The adrenal cortex was found to
be normal. These observations were, in general, confirmed by Woolley,'*
who observed hyperirritability, lack of muscular control followed by paral-
ysis, and closure of the eyes by a sticky exudate. Jones et al.®° found no
signs of paralysis in their deficient albino mice but noted spasticity of the
extremities, arching of the spine, and an awkward gait as well as a striking
alopecia, scaly desquamation of the skin, and hyperemia and edema of the
eyelids. Melampy and coworkers? noted lipid depletion of the adrenal
in adult mice of CF No. 1 strain kept on a pantothenic acid-deficient diet
but reported an even more rapid depletion in starvation. A strain variation
in susceptibility to pantothenic acid deficiency has been reported.'®°?: 18%¢

11. OTHER SPECIES OF ANIMALS

Pantothenic acid has been studied in several other animal species, and
it has been shown to be required for fish (McLaren et al.'**), the fox (Lunde
and Kringstad!*’ and Morgan and Simms!*), the hamster (Routh and
Houchin'*’), various insects (see reviews on this subject!*®: !%), and the
toad (tadpole stage, Catolla-Cavalcanti!”). In this connection, Shock and
Sebrell!*? found that the work output of frog muscles was improved by the
addition of calcium pantothenate to the perfusion fluid.

181 H. P. Morris and S. W. Lippincott, J. Natl. Cancer Inst. 2, 29 (1941).

182 §. W. Lippincott and H. P. Morris, J. Natl. Cancer Inst. 2, 39 (1941).

183 §. W. Lippincott and H. P. Morris, Am. J. Pathol. 17, 588 (1941).

184 T). W. Woolley, Proc. Soc. Exptl. Biol. Med. 46, 565 (1941).

185 J. H. Jones, C. Foster, F. Dorfman, and G. L. Hunter, J. Nutrition 29, 127 (1945).

185a R. M. Melampy, D. W. Cheng, and L. C. Northrop, Proc. Soc. Exptl. Biol. Med.
76, 24 (1951).

185) P. F. Fenton, G. R. Cowgill, M. A. Stone, and D. H. Justice, J. Nutrition 42, 257
(1950).

185¢ J). R. Weir, J. Nutrition 49, 425 (1953).

186 B, A. McLaren, B. E. Keller, D. J. O’Donnell, and C. A. Elvehjem, Arch. Bio-
chem, 15, 169 (1947); L. E. Wolf, Progressive Fish Culturist, 18, No.1, 17 (1951).

187 G. Lunde and H. Kringstad, Naturwissenschaften 27, 755 (1939).

188 A. I’. Morgan and H. D. Simms, J. Nutrition 20, 627 (1940).

189 J. I. Routh and O. B. Houchin, Federation Proc. 1, 191 (1942).

190 F, A, Robinson, The Vitamin B Complex. John Wiley and Sons, New York, 1951.

191 R. J. Williams, R. EH. Eakin, KE. Beerstecher, Jr., and W. Shive, The Biochem-
istry of B Vitamins. Reinhold Publishing Corp., New York, 1950.

1922 A. Catolla-Cavaleanti, Acta Vitaminol. 5, 162 (1951).

193 N. W. Shock and W. H. Sebrell, Am. J. Physiol. 142, 274 (1944).

IX. EFFECTS OF DEFICIENCY 669

The pantothenic acid content of the urine of the horse is influenced by
the amount of pantothenic acid in the diet, although this vitamin does not
appear to be low in natural-type diets for this species.!** 1% As yet, there
is no proof that this vitamin is needed in the diet of the horse at any stage
of growth.

Studies with the growing rabbit show that dietary pantothenic acid may
not be needed (Olcese et al.!°*). The rabbit is known to consume a consider-
able amount of soft, or night, feces which contain a large amount of panto-
thenic acid (50 y/g.) produced by synthesis within the gastrointestinal
tract.!*7 The pantothenic acid content of the soft feces is approximately six
times as great as the content of hard feces.!*7 These facts probably account
for the apparent non-essentiality of dietary pantothenic acid for the rabbit.

B. IN MAN
ELAINE P. RALLI

1. INTRODUCTION

Up to the present, no definite pathological lesions due to a deficiency of
pantothenic acid have been described in the human being. This is in contrast
to the detailed description of the pathological effects of a deficiency of this
vitamin in experimental animals.!%8-?" In view of the paucity of pathological
data in man, the only approaches open to a reviewer are, first, to examine
those conditions in which pantothenic acid has been used therapeuti-
cally and, second, to examine the pathological findings reported in situa-
tions of gross deficiency in human subjects and see wherein these might
compare with the pathological lesions described in experimental animals
on pantothenate-deficient diets. For this reason the pathological findings
in pantothenic acid-deficient animals are listed briefly.

2. SUMMARY OF THE PATHOLOGICAL FINDINGS IN ANIMALS FED Di1erETs
DEFICIENT IN PANTOTHENIC ACID

a. Atrophy of the hair follicles and bulbs and loss of melanin with re-
sulting graying of the fur.!%® 2° Rusting of the fur and porphyrin-caked

194 P. B. Pearson and H. Schmidt, Federation Proc. 1, 191 (1945).

195 P. B. Pearson and H. Schmidt, J. Animal Sci. 7, 78 (1948).

196 QO. Olcese, P. B. Pearson, and B.S. Schweigert, J. Nutrition 35, 577 (1940).
197 R, Kulwich, L. Struglia, and P. B. Pearson, J. Nutrition 49, 639 (1953).

1% L.. L. Ashburn, Public Health Repts. (U.S.) 55, 13837 (1940).

199 A. F, Morgan and H. D. Simms, Science 89, 565 (1939).

200 W. D. Salmon and R. W. Engel, Proc. Soc. Exptl. Biol. Med. 45, 621 (1940).
201 F. 8. Daft and W. H. Sebrell, Public Health Repts. (U. S.) 54, 2247 (1939).
202 H. W. Deane and J. M. McKibbin, Endocrinology 38, 385 (1946).

203 HY. P. Ralli and I. Graef, Endocrinology 32, 1 (1943).

670 PANTOTHENIC ACID

whiskers have also been observed in white rats. These findings have been
observed in mice,?™ rats,?°° foxes,?°® dogs,?” and monkeys.?%

b. Corneal changes, consisting of vascularization, thickening, and opac-
ity of the cornea.?°%

c. A tendency for fatty infiltration of the liver.?”

d. Atrophy of the adrenal cortex with necrosis and hemorrhage in about
50% of the animals.!%° The hemorrhagic lesion is probably present in
animals on the deficient diet who are stressed.

e. Failure of spermatogenesis due to necrosis of the tubular cells of the
testes.?0°

f. Delay or absence of the estrus cycle.?!°

g. Neurological lesions. Deficiency of pantothenic acid in the chick causes
widespread myelin degeneration of the spinal cord?!!: 2 and in swine?!3-216
ataxia with sensory neuron damage.

h. Reproduction. Hatchability of eggs is decreased in hens on a deficient
diet and can be markedly increased when pantothenic acid is added to the
diet.246 In rats, fetal abnormalities?!” have been observed as a result of pan-
tothenic acid deficiency, and interestingly enough the lesions involved prin-
cipally the nervous system. Absence of the eyes occurred and the ocular
globe and optic nerve could not be found. Massive diencephalus and altera-
tion in the morphology of the brain were also observed.

3. PaTHOLOGICAL LESIONS IN HuMAN BEINGS SUGGESTIVE OF A
DEFICIENCY OF PANTOTHENIC ACID

a. Effect of Administration of Large Doses of Pantothenic Acid in Certain
Clinical Conditions

(1) Graying of the Hair. Human subjects with gray hair have been given
large amounts of calcium pantothenate orally, and its effect on the color of

204 J. H. Jones, C. Foster, F. Dorfman, and G. L. Hunter, J. Nutrition 29, 127 (1945).

205 A. F. Morgan and H. D. Simms, J. Nutrition 19, 233 (1940).

206 A. K. Schaefer, C. K. Whitehair, and C, A. Elvehjem, J. Nutrition 34, 131 (1947).

207 R. H. Silber, J. Nutrition 27, 425 (1944).

208 K. B. McCall, H. A. Waisman, C. A. Elvehjem, and E. S. Jones, J. Nutrition 31,
685 (1946).

209 T,. L. Bowles, W. K. Hall, V. P. Sydenstricker, and C. W. Hock, J. Nutrition 37,
9 (1949).

210 F. H. J. Figge and E. Allen, Endocrinology 30, 81028 (1942).

211 J. H. Shaw and P. H. Phillips, J. Nutrition 29, 107 (1945).

212 PH. Phillips and R. W. Engel, J. Nutrition 18, 227 (1939).

213 M.M. Wintrobe, M. H. Miller, R. H. Follis, Jr., H. J. Stein, C. Mushatt, and S.
Humphreys, J. Nutrition 24, 345 (1942).

214 R. H. Follis, Jr., and M. M. Wintrobe, J. Exptl. Med. 81, 539 (1945).

25 R. L. Swank and R. D. Adams, J. Neuropathol. Exptl. Neurol. 7, 274 (1948).

216 M.B. Gillis, G. F. Heuser, and L. C. Norris, J. Nutrition 35, 351 (1948).

217 J. Boisselot, Arch. franc. pédiat. 6, 225 (1949).

IX. EFFECTS OF DEFICIENCY 671

the hair has been observed. The majority of subjects have been people in
whom premature graying of the hair had occurred, but the vitamin was
also given to elderly individuals with gray hair. The dosage used was more
than adequate (from 20 to 100 mg. of calcium pantothenate daily), so that
had the condition been due to deficiency of pantothenic acid it should have
responded. Except in an occasional subject, investigators?!’ 24° did not find
that the administration of caletum pantothenate had any consistent effect
in overcoming graying of the hair. Frankly, there seems little reason to
infer that, because the fur of animals grayed as a result of pantothenate
deficiency, pantothenic acid would cure gray hair in man. In the animal,
the “skin” is composed of the hair follicles and bulbs with a thin overlying
layer of cutis. The various hair follicles and bulbs go through phases of
atrophy and regeneration, and the melanin content, which is responsible
for the pigmentation, varies according to the state of the hair follicles and
bulbs. In the animal, pantothenic acid is clearly responsible for maintaining
the integrity of the hair apparatus and in its absence atrophy of the hair
follicles and bulbs and loss of melanin occur. In man, obviously, the ques-
tion of graying of the hair depends on many factors.

(2) Therapeutic Effects of Calcium Pantothenate or Pantothenyl Alcohol
on Various Skin Lesions. Therapeutic use of pantothenic acid has been
reported in a variety of skin lesions. The vitamin has been given orally or
applied topically as an ointment.” 2! Goldman”® treated patients with
discoid erythematosus of the scalp, giving 400 mg. per day, and noted im-
provement in the subacute type. Combes and Zuckerman”! applied the
vitamin as an ointment to patients with ulcerations of the skin due to a
variety of causes, including ulcers due to radium therapy, traumatic ulcers,
arteriosclerotic ulcers, and ulcerations in patients with sickle cell anemia.
Pantothenic acid ointment has also been used locally in the treatment of
burns and infected wounds,” apparently with beneficial results.

These findings suggest that pantothenic acid may be important to the
nutrition of the skin, and this may offer a parallel to the skin lesions, such
as scaliness and crusting, seen in animals on a pantothenic acid-deficient
diet.

(3) Effect of Pantothenic Acid on the Hematopoietic System. Crisalli?* re-
ported that pantothenic acid has a stimulating action on the hematopoietic
system in infants. However, in this respect it should be borne in mind that

218 H. Brandaleone, E. Main, and J. M. Steele, Proc. Soc. Exptl. Biol. Med. 53, 47
(1943).

219 JT, Kerlan and R. P. Herwick, J. Am. Med. Assoc. 128, 391 (1948).

220 T,, Goldman, J. Invest. Dermatol. 11, 95 (1948).

221 F.C. Combes and R. Zuckerman, J. Invest. Dermatol. 16, 379 (1951).

222 FB. Sciclounoff and E. Naz, Schweiz. med. Wochschr. 75, 767 (1945).

228 M. Crisalli, Boll. soc. ital. biol. sper. 23, 1047 (1947).

672 PANTOTHENIC ACID

the intestinal synthesis of folic acid and biotin is affected in pantothenic acid
deficiency,“ and it may be that any effects on the hematopoietic system
are the result of an associated deficiency of folic acid.

(4) Pantothenic Acid Therapy in Metabolic Disease. In view of the fact
that an absence of pantothenic acid in animals was associated with atrophy
and, in many instances, necrosis and hemorrhage of the adrenal cortex, the
use of this vitamin in the treatment of patients with Addison’s disease
naturally has suggested itself. The author has personally given three pa-
tients with Addison’s disease massive doses of calcium pantothenate (10
to 20 g. daily). There was no evidence of any effect on the course of the
disease or on the hormone requirement of these patients. In two of them
the etiological cause of the Addison’s disease was presumably tuberculosis,
as other evidences of tuberculosis were present in the patients. It may be
that the different etiological factors involved, i.e., pantothenic acid defi-
ciency in the rat and a chronic infection in the human being, explain the
fact that no clinical effect was observed.

Disturbances of carbohydrate metabolism have been reported as associ-
ated with pantothenic acid deficiency in many species.”?°-?° Gershberg et
al.?° studied the ability of patients to acetylate PABA and sulfadiazine
after having been administered large amounts of this vitamin (from 10 to
20 g.). No depression of acetylation was observed in patients with liver dis-
ease or in patients with rheumatoid arthritis, diabetes mellitus, hypothy-
roidism, sprue, or leukemia. In patients with hyperthyroidism, a diminished
capacity to acetylate was observed, which was improved by the administra-
tion of sodium acetate, suggesting that in hyperthyroidism there is a de-
crease in available acetate due to its rapid utilization. One might infer that
patients with hyperthyroidism would be more susceptible to pantothenic
acid deficiency than normal subjects. In this respect it is interesting that
Drill and Overman”! found that the requirement for pantothenic acid in-
creased in experimental hyperthyroidism in rats.

(5) Pantothenic Acid in Other Clinical Situations. Jacques?” reported the
use of pantothenic acid in the treatment of surgical patients with severe

224 B. N. Berg, T. F. Zucker, and L. M. Zucker, Proc. Soc. Exptl. Biol. Med. 71, 374
(1949).

225 T,. D. Wright, J. Biol. Chem. 142, 445 (1942).

226 A. H. Schaefer, J. M. McKibbin, and C. A. Elvehjem, J. Biol. Chem. 143, 321
(1942).

227 A. Dorfman, 8. Berkman, and S. A. Koser, J. Biol. Chem. 144, 393 (1942).

228 H. MclIlwain and D. E. Hughes, Biochem. J. 38, 187 (1944).

229 R. HE. Olson and N. O. Kaplan, J. Biol. Chem. 175, 515 (1948).

230 H. Gershberg, 8. H. Rubin, and E. P. Ralli, J. Nutrition 39, 107 (1949).

231 'V. A. Drill and R. Overman, Am. J. Physiol. 135, 474 (1942).

232 J. UH. Jacques, Lancet II, 861 (1951).

IX. EFFECTS OF DEFICIENCY 673

postoperative ileus. He was stimulated to try pantothenic acid in this situ-
ation as a result of the observations of Jurgens and Pfaltz?** that rats kept
on a diet deficient in pantothenic acid showed atony and distension of the
gastrointestinal tract. Sixteen cases were studied. Pantothenic acid was
given intramuscularly in doses of 50 mg., and most of the patients received
two or three injections. Jacques reported a very encouraging response to
the use of pantothenic acid in that a return of bowel motility took place as
evidenced by the passage of flatus. Many of the patients did not receive
pantothenic acid until they had been under treatment with either pituitary
extract, neostigmine, or other drugs. The clinical observations should be
confirmed, but they are suggestive of a possible therapeutic role of panto-
thenic acid.

Ralli et al.2*4 have reported the effects of large amounts of calcium panto-
thenate by mouth in normal male adults stressed by immersing them in
water of 9° for 8 minutes. When this stress was repeated after 6 weeks of
pantothenate therapy, it was observed that the initial eosinophile count
was higher and that there was less of an eosinopenia. The whole blood
ascorbic acid was significantly elevated, and there was no decrease in the
blood ascorbic acid following the stress. In the urine the uric acid/creati-
nine ratio was decreased after therapy. The oral administration of vitamin
By was not associated with any changes of this type. The authors suggest
that pantothenic acid may possibly increase the capacity of the tissues to
withstand stress, and this might result from the fact that pantothenic acid
constitutes part of coenzyme A. This coenzyme?* functions catalytically in
the condensation of pyruvate with oxalacetate to form citrate. An inter-
mediary in this reaction is acetyl-CoA, which is believed to represent an
essential step in the oxidative metabolism of carbohydrate. Therefore pan-
tothenate is an essential part of one of the key reactions for the oxidative
metabolism of cells, and when excessive amounts of pantothenate are pro-
vided to the tissues the production of coenzyme A might be facilitated and
so influence the response to stress.

b. Pathological Lesions among Prisoners of War in the Far East, Suggestive
of a Deficiency of Pantothenic Acid

It is obvious that more than one nutritional factor was involved in the
cases of gross malnutrition that occurred among the prisoners of war in the
Japanese prison camps. The diets were deficient in all fractions of the vita-
min B complex, in protein, and in fat. It is interesting that scurvy was

233 R. Jurgens and H. Pfaltz, Z. Vitaminforsch. 14, 243 (1944).

234 H). P. Ralli, Nutrition Symposium Natl. Vitamin Found. Ser. 5, 78 (1952).

235 §. Korkes, A. Del Campillo, I. C. Gunsalus, and 8. Ochoa, J. Biol. Chem. 193, 721
(1951).

674 PANTOTHENIC ACID

rarely encountered among the prisoners. Among prisoners of war it was
noted by physicians, who themselves were in prison camps and were able
to make observations at first hand, and among investigators, who had an
opportunity of studying the prisoners when they were released from the
camps, that many of the pathological lesions present did not respond to
specific fractions of the B complex, such as thiamine, riboflavin, or nico-
tinic acid, but did respond to dried brewers’ yeast or to liver extract. The
lesions that did not respond to specific fractions of the B complex were in
some instances suggestive of pantothenic acid deficiency in experimental
animals, and it is these findings that will be discussed.

(1) Ocular Changes. Among prisoners of war visual disturbances ranging
from complete blindness to just blurring of vision were reported by many
investigators. Pallor of the optic discs and central scotoma were also ob-
served.”*° Improvement did not occur on high vitamin diets. The fact that
there was no improvement with therapy may well have been due to the
fact that the visual symptoms had developed two to three years prior to
any therapy. Scott?*” reported that in some of the British West Indies islands
the eye changes were associated with itching and burning of the eyes and
inflammatory lesions in the mouth and fissures. Pallor of the optic dise
was noted in children in Kingston, and treatment with brewers’ yeast and
cod liver oil improved the visual disturbances, but not if the disease had
existed for some time.?**: 78° Bell and O’Neill*“° observed 95 cases of partial
optic atrophy from malnutrition occurring in liberated prisoners of war from
Hong Kong, which was an incidence of 20 %. The main findings were scoto-
mata and contraction of the peripheral visual fields. In discussing the oph-
thalmology, they felt that the lack of vitamin B and the hypoproteinemia
were factors and that, in addition, there was possibly a toxic factor. In-
vasion of the cornea was also noted by Kark.*4! Some observers felt that this
was due to riboflavin deficiency.

The fact that many of the prisoners of war had had symptoms of beriberi
and pellagra, the fact that the response to these fractions of the B complex
were not uniformly satisfactory, and the fairly high incidence of eye lesions
obviously suggest that other factors were involved in the production of
these lesions. In view of the corneal vascularization that occurs in panto-
thenic acid deficiency in animals, and in view of the other eye lesions occur-
ring in animals, it seems reasonable to suggest that pantothenic acid may

236 W.L. Roberts and T. H. Willcockson, Am. J. Ophthalmol. 30, 165 (1947).
237 H. H. Scott, Ann. Trop. Med. Parasitol. 12, 109 (1918).

238 T), Whitebourne, Am. J. Ophthalmol. 30, 169 (1947).

239 FD. Carroll, Am. J. Ophthalmol. 30, 172 (1947).

240 P. G. Bell and J. C. O’Neill, Can. Med. Assoc. J. 56, 475 (1947).

241 R, Kark, H. F. Aiton, and E. D. Pease, Ann. Internal Med. 25, 266 (1946).

IX. EFFECTS OF DEFICIENCY 675

have been one of the factors responsible for some of the eye lesions, possibly
those involving the cornea.

(2) Burning Feet Syndrome. Probably the findings in humans most sug-
gestive of pantothenic acid deficiency are those related to the syndrome
known as “burning feet.’ “8 This has no exact counterpart in the experi-
mental animal, and there is no justification for concluding that it is due
soley to a deficiency of pantothenic acid. This condition, seen in so many
of the men who were in prisoner-of-war camps in Japan and Burma, had a
characteristic syndrome, was associated with neurological and mental dis-
turbances, did not respond satisfactorily to therapy with thiamine chloride,
riboflavin, or nicotinic acid, and was reported by Gopalan™ as responding
to calcium pantothenate. The other fractions of the vitamin B complex
improved the nutritional state of the individual although they were not
successful in completely correcting the feet symptoms or the mental dis-
turbance. Dried brewers’ yeast was apparently the most successful therapy,
and this, of course, contains all fractions of the B complex.

Glusman,”** who himself was a prisoner of the Japanese from the time of
the fall of Corregidor until the war was over, had an opportunity of observ-
ing prisoners of war in the Philippines and in Japan. He reports that evi-
dence of weight loss and malnutrition became apparent soon after the
American troops were taken prisoner in the Philippines, but that three or
four months elapsed before the first patients with burning feet began to
appear. The incidence then increased rapidly so that approximately 300 of
some 800 patients were complaining of this symptom. In other camps, such
as Cabanatuan, some 2000 more cases were observed. In these patients
evidences of pellagra were common, as well as corneal ulcerations, constric-
tion of the visual fields, central scotomata, scrotal dermatitis, glossitis, and
cheilosis. Diarrhea was almost universal. The burning feet syndrome oc-
curred in conjunction with evidences of these other deficiencies, but Glus-
man reports that ‘it could occur as a distinct symptom complex by itself.”
As described by all authors, the onset was gradual, with numbness and
tingling in the toes, usually bilateral. The paresthesias then gave way to
burning pains in the toes and soles of the feet, and then the shooting pains
began. These radiated from the dorsa of the feet up the legs. There seemed
to be a relationship in these pains to the time of day, and they were much
more severe at night. In the most advanced cases, pains in the palms of the
hands also occurred. Relief occurred to a certain extent when the feet were
immersed in cold water. Preservation of the reflexes was the rule, and motor

242 G. F. Harrison, Lancet I, 961 (1946).

243 HH}. K. Cruickshank, Lancet II, 369 (1946).
244 C, Gopalan, Indian Med. Gaz. 81, 22 (1946).
245 M. Glusman, Am. J. Med. 3, 211 (1947).

676 PANTOTHENIC ACID

power was preserved. No paralysis of the extremities occurred; there was
no evidence of foot drop. The gait is best described by quoting from Glus-
man’s report: ‘“The gait was peculiar but this appeared to be due to pain
and hypersensitivity of the soles of the feet rather than to disturbance of
motor power or equilibrium. The patient walked as if the ground beneath
the soles of his feet were hot. He walked cautiously, gingerly, and on a some-
what widened base. Because of his reluctance to use his oversensitive toes
to grip the ground, the gait had a characteristic flat-footed quality. In
standing, patients frequently shifted their weight from one foot to the other
in a restless and repetitious fashion. It seemed as if they could not endure
the discomfort of resting their weight on one foot for more than a few mo-
ments at a time. The same restlessness was frequently noted while the pa-
tient was in bed. Often when the pain was severe patients would sit cross
legged in bed, holding the distal portions of their feet in their hands and rock
rhythmically backward and forward with the pain. This last attitude was
not only pitiful but it was so pathognomonic that any observer walking into
a ward could pick out those with burning feet at a glance.”

Attempts at therapy were, according to Glusman, disappointing. Nico-
tinic acid, as reported by other observers also, relieved the associated signs
of pellagra “but was without effect on the symptoms of burning feet.’’ The
same was true of thiamine chloride.

Glusman also mentions discussing the matter with Professor Kinosita,
and the report was the same as published in Page’s article.24° Kinosita
claimed that the Japanese had been unfamiliar with the syndrome of burn-
ing feet before the war. However, they began to see patients with this con-
dition among the Japanese military personnel who had been cut off by
allied operations in the southern battle areas for variable periods of time
before rescue. Professor Kinosita autopsied a small number of these Japa-
nese soldiers. He observed that the spinal cords and peripheral nerves in
these patients were normal. He found changes in the small arteries ‘‘whose
walls were diffusely thickened, and he commented on the absence of new
vessels.”’

A clue to the etiology of the syndrome is provided by Gopalan’s report,”
which was done in the Nutrition Research Laboratories in South India.
This author very kindly provided me with a microfilm copy of his report.
He points out that the “differences between ‘burning feet’ and the peripheral
neuritis associated with thiamine deficiency are so fundamental that it is
surprising that the two conditions have been confused.”’ He found that the
incidence was greatest among the poorer classes and that the diet consumed
was mostly rice gruel and cheap vegetables, the rice being of the parboiled
type which contains enough thiamine to prevent beriberi. Again, the signs

246 J, A. Page, Brit. Med. J. II, 260 (1946).

= ry ey ict ee etre deh rite 3:6, ck:

IX. EFFECTS OF DEFICIENCY 677

of riboflavin deficiency and other deficiencies were present in many of the
subjects. The description of the symptoms was much the same as Glusman’s,
but probably the most significant finding was the result of treatment.
Gopalan found that 50 mg. of thiamine chloride daily, 300 mg. of nicotinic
acid, and 10 mg. of riboflavin resulted in the disappearance of the deficien-
cies such as glossitis, stomatitis, and some of the ocular symptoms but
had no effect on the burning feet. A striking improvement occurred follow-
ing the administration intramuscularly of 20 to 40 mg. of calcium panto-
thenate daily. The burning and hyperhidrosis disappeared first, and the
pins-and-needles sensation later. Up to 80 mg. of calcium pantothenate was
given in some cases and in fact was necessary in the more severe cases. No
untoward reactions occurred. Gopalan points out the resemblance to the
neurological lesions occurring in pantothenate-deficient swine. Spies has
reported that riboflavin or biotin injections increased the blood level of
pantothenic acid.**7 This may account for the moderate improvement re-
ported by some other authors after treatment with the other fractions of
the B complex. The probability is that the other fractions of the B vitamin
complex also are involved to a certain extent in the syndrome. However,
it would seem that the burning feet syndrome was associated with a de-
ficiency of pantothenic acid and suggests that in humans a deficiency of
this vitamin will affect the small arteries and may involve the central ner-
vous system as it does in the experimental animal.?!°?!® Major Bruce Hunt,
of the Australian Army Medical Corps,”* who was taken prisoner by the
Japanese at Singapore, told me that personality changes and mental dis-
turbances were also present in many of the men suffering from the burning
feet syndrome.

(3) There were no reports on pathological examination of fatty infiltra-
tion of the liver or atrophy of the adrenals in men dying as a result of star-
vation in the prison camps, but apparently complete pathological examina-
tions were not feasible.

(4) Apparently some decrease in the secretion of the male sex hormone
occurred, as a common observation among the prisoners was the fact that
they had to shave much less frequently than normally, and loss of libido
was frequently reported. Whether or not this was due to atrophy or changes
in the tubular cells of the testes is not known, but undoubtedly it was the
result of malnutrition.

c. Summary

Obviously, at the present time one can only surmise, on the basis of ani-
mal experimentation, what the possible pathological changes in humans

47 'T. D. Spies, 8S. R. Stanbery, R. J. Williams, T. H. Jukes, and 8. H. Babcock,
J. Am. Med. Assoc. 115, 523 (1940).
248 B, Hunt, personal communication, Perth, Australia.

678 PANTOTHENIC ACID

would be as a result of a deficiency of pantothenic acid. The pathological
effects of a deficiency of this vitamin have not been clearly defined in hu-
mans. In view of the ubiquitous occurrence of the vitamin in nature, it is
probable that pantothenic acid deficiency is a rare occurrence in humans,
except possibly for the burning feet syndrome seen in the prisoners of war
and among some malnourished peoples in the Far East. Apparently, the
most propitious place to pursue the study of the deficiency would be in that
part of the world where this syndrome is most often encountered. Glusman
feels that the syndrome is due to a nutritional deficiency and suggests calling
it “nutritional melalgia’”’ (nutritional limb pain); he apparently considers
pantothenic acid one of the major nutritional factors involved.

C. IN MICROORGANISMS
EK. E. SNELL

Pantothenic acid was discovered independently as a growth factor for
yeast”? °° and for lactic acid bacteria;?*! it was only after extensive work
on its properties, distribution, and purification had been carried out with
these microorganisms that the active compound present in the concentrates
was shown to be identical with a relatively uncharacterized substance re-
quired for growth of animals.?°°: 2°. 253 The vitamin is required for growth
by a large variety of microorganisms;*°: 754: 25> those that do not require it
preformed synthesize it. In the absence of the vitamin, organisms that do
not synthesize it fail to grow, and in the presence of suboptimal amounts of
it, growth increases with the vitamin concentration. Microbiological as-
says for the vitamin are based upon this fact.?°®

In addition to its effects on growth, several rather specific relationships
of pantothenic acid to the metabolism of microorganisms have been noted.
Glycogen storage by yeast is reported to increase definitely when panto-
thenic acid is supplied to the growing cultures.?*” The requirement of many
lactic acid bacteria for pantothenic acid is considerably decreased by the

249 R. J. Williams, C. M. Lyman, G. H. Goodyear, J. H. Truesdail, and D. Holaday,
J. Am. Chem. Soc. 55, 2912 (1933).

250 R. J. Williams, Advances in Enzymol. 3, 253 (1943).

251 H. . Snell, F. M. Strong, and W. H. Peterson, Biochem. J. 31, 1789 (1937); J. Am.
Chem. Soc. 60, 2825 (1938).

252 TD). W. Woolley, H. A. Waisman, and C. A. Elvehjem, J. Am. Chem. Soc. 61, 977
(1939).

253TH. Jukes, J. Am. Chem. Soc. 61, 975 (1939).

254 B.C. J. G. Knight, Vitamins and Hormones 8, 105 (1945).

255 W. H. Peterson and M.S. Peterson, Bacteriol. Revs. 9, 49 (1945).

256 H}. KE. Snell zn Vitamin Methods, Vol. I, p. 327. Academic Press, New York, 1950.

257 R. J. Williams, W. A. Mosher, and E. Rohrmann, Biochem. J. 30, 2036 (1936).

Se =

IX. EFFECTS OF DEFICIENCY 679

presence of fatty acids in the medium;?°*: 2° similarly, the inhibitory effect
on these organisms of antivitamins related to pantothenic acid in structure
is greatly decreased by addition of free or combined fatty acids to the me-
dium.?*? Pantothenic acid greatly increases the synthesis of acetylcholine
by Lactobacillus plantarum.?®°

Dorfman and coworkers?! found that the rate of pyruvate oxidation by
cells of Proteus morganii was depressed when the cells were grown with sub-
optimal amounts of pantothenic acid; the rate of oxidation was increased
by addition of pantothenic acid to the resting cell suspension. Similar obser-
vations were made by Hills.2 Both authors considered that the vitamin
takes part in the oxidation of pyruvic acid to acetic acid. Novelli and Lip-
mann2* showed that this increased oxidation was mediated by coenzyme A,
which is formed almost quantitatively by such organisms from the panto-
thenic acid which they take up. This coenzyme is concerned in the initial
reactions (condensation of acetate with oxalacetate) leading to the oxidation
of acetate.2* 265 This was shown in striking fashion by the demonstra-
tion that acetate accumulated from ethanol in the presence of a pantothenic
acid-deficient yeast suspension, but not in the presence of a similar sus-
pension rich in pantothenic acid. The latter preparation of cells also re-
moved acetate from solution (by oxidation) much more rapidly than the
former deficient cells.?

As a result principally of the work of Lipmann and his group, pantothenic
acid is now known to be essential for a whole series of reactions (and possi-
bly all reactions) involving the acetyl group. As a preliminary to participa-
tion in such reactions, it must be built into its coenzyme form, coenzyme
A263, 266 The latter coenzyme, together with various degradation products
of it, accounts for essentially all the ‘“‘bound” pantothenic acid present in
tissues. Knowledge of these facts permits a ready explanation of most of
the metabolic effects described above. Thus, coenzyme A is required for
formation of fatty acids from acetate, and hence the addition of preformed
fatty acids in a form directly utilizable for cell synthesis might be expected
to decrease the pantothenic acid requirement of organisms such as L.

258 H. P. Broquist, Ph.D. Thesis, University of Wisconsin, 1950.

259 W. Shive, W. W. Ackermann, J. M. Ravel, and J. E. Sutherland, J. Am. Chem. Soc.
69, 2567 (1947).

260 Ff}. Rowett, J. Gen. Microbiol. 2, 25 (1948).

261 A. Dorfman, S. Berkman, and 8. A. Koser, J. Biol. Chem. 144, 393 (1942).

262 G. M. Hills, Biochem. J. 37, 418 (1943).

263 G. D. Novelli and F. Lipmann, J. Bacteriol. 54, 19 (1947); Arch. Biochem. 14, 23
(1947).

264 G. D. Novelli and F. Lipmann, J. Biol. Chem. 171, 833 (1947).

265 J. R. Stern, B. Shapiro, and 8. Ochoa, Nature 166, 403 (1950).

266 G. D. Novelli, N. O. Kaplan, and F. Lipmann, J. Biol. Chem. 177, 97 (1949).

680 PANTOTHENIC ACID

arabinosus. Since coenzyme A is essential for acetylcholine formation, it
is readily seen why pantothenic acid-deficient cells of L. plantarum form
decreased amounts of this ester. Since oxidation of acetate (and hence of
pyruvate) in many organisms proceeds through the Krebs cycle, and since
coenzyme A is necessary for the initial formation of citric acid in this cycle,
the reason for the stimulation of oxidation of pyruvate by pantothenate in
deficient cells of Proteus becomes apparent. By way of contrast, the anaero-
bic fermentation of glucose by yeast juice was not decreased in pantothenic
acid deficiency.?%

The extensive studies of McIlwain on the mechanism of the inhibitory
action of pantoyltaurine for pantothenic acid-requiring bacteria may also
be explained on this basis. McIlwain showed that addition of pantoyltau-
rine increased the lag phase and decreased the rate of growth during the
first half of the logarithmic phase of Streptococcus hemolyticus, observations
consistent with the assumption that pantoyltaurine slowed incorporation
of the vitamin into a substance necessary for normal growth.?® Pantothenic
acid disappeared from cultures of this organism; this disappearance was
dependent upon the simultaneous occurrence of glycolysis, but was pre-
vented by pantoyltaurine even though glycolysis was not. It may now be
assumed that the pantothenic acid was being converted to coenzyme A, an
endergonic process that was dependent upon glycolysis,?°* and that this
conversion was the process inhibited by pantoyltaurine. Related antime-
tabolites of pantothenic acid probably inhibit growth by a similar mecha-
nism.?7°

The observation that ether pantothenic acid or tryptophan suffices to
permit growth of a strain of Staphylococcus aureus under specified condi-
tions”! indicates a relationship of the vitamin to metabolism of this amino
acid. The nature of this relationship has not been clarified.

X. Pharmacology
SAMUEL LEPKOVSKY

Little is known concerning the pharmacology of pantothenic acid. Its
toxicity is very low and varies with different animals.!* Acute toxicity was

267 P| C. Teague and R. J. Williams, J. Gen. Physiol. 25, 777 (1942).
268 H. MeclIlwain, Biochem. J. 36, 364 (1942).
269 HT. McIlwain and D. E. Hughes, Biochem. J. 38, 184 (1944).
270 H. McIlwain and D. E. Hughes, Biochem. J. 38, 133 (1945).
271 M. G. Sevag and M. N. Green, J. Biol. Chem. 154, 719 (1944); J. Bacteriol. 48, 631
(1944).
1K. Unna and J. G. Greslin, J. Pharmacol. Exptl. Therap. 78, 85 (1941).

CE ce A ct No Rt.

X. PHARMACOLOGY 681

studied in mice, rats, dogs, and monkeys. The LD5 after subcutaneous
injection is 2.7 g. per kilogram in mice and 3.4 g. per kilogram in rats. The
LDs5o after oral administration in mice is 10 g. per kilogram of body weight,
but rats fed the same amount of pantothenic acid survived without showing
toxic symptoms. After intraperitoneal and intravenous injections the LD 50
for mice is 0.92 and 0.91 g. per kilogram, respectively, and for rats it is 0.82
and 0.83.1 Lethal doses produced prostration and respiratory failure in rats
and mice. No toxic symptoms were observed in five dogs and one monkey
fed 1 g. of calcium pantothenate per kilogram of body weight.! Pantothenic
acid is essentially non-toxic to human beings. At least 100 mg. may be in-
jected intravenously in man without producing any toxic reactions.”

Chronic toxicity was studied in rats, dogs, and monkeys. Daily doses
of 50 and 200 mg., respectively, of calctum pantothenate were fed to young
male and female rats over a period of 190 days. Growth was normal, and
autopsies at the end of the feeding period did not reveal any gross or mi-
croscopic changes in the organs. Offsprings of the group of rats receiving
50 mg. of calcium pantothenate daily were given this dose of pantothenic
acid from the day that they were weaned. These second-generation animals
also developed normally.

Adult dogs were fed daily 50 mg. of calcium pantothenate per kilogram,
and monkeys 1.0 g. per kilogram over a period of 6 months. Neither the
dogs nor the monkeys showed any toxic symptoms or loss of weight. Histo-
pathological examination of these animals failed to reveal any changes.!

Local effects of caletum pantothenate were studied on rabbits by subcu-
taneous injections and instillation into the conjunctival sac. No irritation,
inflammation, or abscess formation was observed following the subcutan-
eous injection of 1.0 ml. of 1, 2, 5, and 10% solutions of the vitamin. The
infiltration of the subcutaneous tissue subsided about as rapidly as that
following an injection of 1.0 ml. of saline solution. Instillation of 0.5 ml. of
a 10 % solution into the conjunctival sae did not produce any irritation.

Calcium pantothenate did not influence the metabolism, circulatory and
respiratory systems, or the smooth muscle organs of normal animals.

2T. D. Spies, D. P. Hightower, and L. H. Hubbard, J. Am. Med. Assoc. 115, 292
(1940).

3 F. A. Robinson, The Vitamin B Complex. John Wiley and Sons, New York, 1951.

4H. Molitor and G. A. Emerson, Vitamins and Hormones 6, 69 (1948).

682 PANTOTHENIC ACID

XI. Requirements and Factors Influencing Them
A. OF ANIMALS
GEORGE M. BRIGGS and FLOYD 8S. DAFT

The pantothenic acid requirement of animals varies with species, age,
type of diet, period of depletion, various stresses, and other factors to be
considered. Many careful attempts have been made to determine the mini-
mal level required for optimum growth for many animals but considerably
more work is needed. Accepted values of today may need to be changed
as new nutritional developments unfold.

1. Minima LEVELS

The requirements for pantothenic acid which are available are given in
Table XX with key references. The values given are minimal levels, with

TABLE XX
MINIMAL CatcrumM PANTOTHENATE REQUIREMENTS FOR Optimum GROWTH,
PERFORMANCE, OR PRODUCTION

Other key
references to
Mg./100 g. Mg./kg. body studies on
Animal of diet weight per day Mg./day requirement
Rat
Weanling 0.081: 2
0.04-0.102> 3
0.104
0.08-0.105 6 8
0.05—-0.107
0.69
Young adult (10 wk. old) 0.0256
Breeding 2.05
Chicken
Chick 0.910 11-17
Laying hen 0.1518 19
Breeding hen 1.038 19
Turkey 1.2517 20, 21
Duck 1.1872
Swine 1.02.07 0.17-0.2674 10-2075
Dog 0.126
Mouse 0.0327
1.028 0.023-0 .02928

* 40 y daily for maximum growth; 100 y daily for prevention of graying.

1K. Unna, J. Nutrition 20, 565 (1940).
2K. Unna and W. L. Sampson, Proc. Soc. Exptl. Biol. Med. 45, 309 (1940).
3G. A. Emerson and H. M. Evans, Proc. Soc. Exptl. Biol. Med. 46, 655 (1941).

oe oe

XI. REQUIREMENTS AND FACTORS INFLUENCING THEM 683

no added ‘‘allowance,” and are based on the latest figures available. Many
of the older values were obtained with diets considered incomplete by to-
day’s standards or with depleted animals. Values for many animals have
not been determined as yet.

2. Factors INFLUENCING REQUIREMENTS

Many factors are known to influence the pantothenic acid requirement
of any one species, so actually it is difficult to give one figure as the ‘‘re-
quirement.”’ A discussion of some of these factors follows:

a. State of Maturity of the Animal

As has been seen in the former section, adult animals, in general, require
less dietary pantothenic acid than growing animals. Deficiency symptoms
are most readily obtained in the very young animal (see section on path-
ology and deficiency symptoms).

4+C. A. Elvehjem, L. M. Henderson, 8S. Black, and E. Nielsen, J. Biol. Chem. 140,
xxxvi (1941).
> L. M. Henderson, J. M. McIntire, H. A. Waisman, and C. A. Elvehjem, J. Nutri-
tion 28, 47 (1942).
6 K. Unna and G. V. Richards, J. Nutrition 28, 545 (1942).
7J.S. D. Bacon and G. N. Jenkins, Biochem. J. 37, 492 (1948).
8C. A. Slanetz, Am. J. Vet. Research 4, 182 (1943).
9C. A. Mills, Arch. Biochem. 1, 73 (1942).
107). M. Hegsted and T. R. Riggs, J. Nutrition 37, 361 (1949).
11H. A. Waisman, R. C. Mills, and C. A. Elvehjem, J. Nutrition 24, 187 (1942).
2TH. Jukes and L. W. McElroy, Poultry Sci. 22, 488 (1943).
13 J. L. Milligan and G. M. Briggs, Poultry Sci. 28, 202 (1949).
1448, Lepkovsky, F. H. Bird, F. H. Kratzer, and V. S. Asmundson, Poultry Sci. 24,
335 (1945).
15 G. F. Heuser, M. B. Gillis, and L. C. Norris, Rept. 8th World’s Poultry Congr. p.
114 (1948).
16 J. C. Bauernfeind, L. C. Norris, and G. F. Heuser, Poultry Sci. 21, 142 (1942).
17 A. G. Hogan, Nutrition Abstr. & Revs. 19, 751 (1949-1950).
18M. B. Gillis, G. F. Heuser, and L. C. Norris, J. Nutrition 35, 351 (1948).
19M. B. Gillis, G. F. Heuser, and L. C. Norris, J. Nutrition 26, 285 (1943).
20 F. H. Kratzer and D. Williams, Poultry Scz. 27, 518 (1948).
217, H. Jukes, Poultry Sct. 17, 227 (1938).
22.W. Trager, J. Exptl. Med. 77, 557 (1943).
23 §. C. Stothers, R. R. Johnston, J. A. Hoefer, and R. W. Luecke, J. Animal Sci.
11, 777 (1952).
24. H. Hughes and N. R. Ittner, J. Animal Sci. 1, 116 (1942).
25 A. C. Wiese, W. P. Lehrer, P. R. Moore, O. F. Pahnish, and W. V. Hartwell, J.
Animal Sci. 10, 80 (1951).
26 A. K. Schaefer, J. M. McKibbin, andC. A. Elvehjem, J. Biol. Chem. 148, 321 (1942).
27 J. G. Sandza and L. R. Cerecedo, J. Nutrition 21, 609 (1941).
28 H. P. Morris and 8. W. Lippincott, J. Natl. Cancer Inst. 2, 29 (1941).

684 PANTOTHENIC ACID

b. Environmental Effects

Spector eé al.?® reported that the urinary and dermal excretion of panto-
thenic acid was greater in human subjects under hot moist conditions than
under comfortable conditions. Mills,? however, found no difference in the
dietary concentration of this vitamin required for maximum growth of the
rat at 68° F. and at 91° F.

c. Effect of Other Nutrients

It has long been known that the type of carbohydrate in the diet may
affect profoundly the requirements of rats and other animals for the B
vitamins. It was reported in 1926 by Fridericia®® that some rats receiving
uncooked rice-starch as the source of carbohydrate developed a condition
designated as ‘‘refection”’ and required no ‘‘B vitamin” in their food. Ani-
mals on the rice-starch diet which did not become refected spontaneously
developed the condition after ingestion of feces from refected ani-
mals (Fridericia and coworkers*!). It was also shown by Roscoe” that in
order to continue in a refected state an animal must continue to ingest his
own or similar feces. Morgan and coworkers® showed that rats receiving
starch as the source of carbohydrate grew well without filtrate factor and
that supplementation of the diet of these animals with filtrate factor con-
centrates improved the growth rate only slightly.

The level of protein, also, has been shown by Nelson and coworkers to
affect the dietary requirement of the rat for pantothenic acid.*4** Animals
deficient in this vitamin receiving casein at a level of 64% grew better and
survived longer than littermates maintained on a 24 % casein diet.** When
intermediate levels of casein were used, the sparing effect was proportional
to the protein level.*® When washed beef blood fibrin, which was even lower
in its pantothenic acid content than the casein, was used as the protein
component of the diet, even more pronounced differences between 64 % and
24 % levels of protein were seen.** The pantothenic acid requirement of the
pig has also been shown to be similarly dependent on the level of protein
in the diet.*7

Yacowitz et al.*® reported that the presence or absence of vitamin By: in

29 H. Spector, T. S. Hamilton, and H. H. Mitchell, J. Biol. Chem. 161, 145 (1945).

30 [,. 8. Fridericia, Skand. Arch. Physiol. 2, 55 (1926).

31. 8. Fridericia, P. Freudenthal, 8S. Gudjonnsson, G. Johansen, and N. Schoubye,
J. Hyg. 27, 70 (1927).

32M. H. Roscoe, J. Hyg. 27, 103 (1927).

33 A. F, Morgan, B. B. Cook, and H. G. Davidson, J. Nutrition 15, 27 (1988).

34M. M. Nelson and H. M. Evans, Proc. Soc. Exptl. Biol. Med. 60, 319 (1945).

35 M. M. Nelson, F. van Nouhuys, and H. M. Evans, J. Nutrition 34, 189 (1947).

36 M.M. Nelson and H. M. Evans, Proc. Soc. Exptl. Biol. Med. 66, 299 (1947).

37 R. W. Luecke, J. A. Hoefer, and F. Thorp, Jr., J. Animal Sci. 11, 238 (1952).

388 H. Yacowitz, L. C. Norris, and G. F. Heuser, J. Biol. Chem. 192, 141 (1951).

La: i Yee eo Pe |

XI. REQUIREMENTS AND FACTORS INFLUENCING THEM 685

the diet of chicks influences the requirement of pantothenic acid; without
By it was reported as 2 mg. per 100 g. of diet, whereas with adequate Bi»
it was only 1 mg. per 100 g. of diet. Jukes, however, concluded from his
experiments*®® that the requirement of chicks for pantothenic acid is not
significantly affected by the lack of Bis if the diet is adequate in methionine
and choline. In young pigs fed practical rations with only 14% protein,
Catron e¢ al.,°°* in 1953, also found a sparing action of vitamin By on the
pantothenic acid requirement.

Wright and Welch*® reported that a relationship exists between defi-
ciencies of folic acid and biotin and the development of signs of pantothenic
acid deficiency. Following the administration of succinylsulfathiazole to
rats, changes appeared which were very similar to those seen in pantothenic
acid deficiency, and a low level of this vitamin in the liver was demonstrated.
Administration of additional pantothenic acid did not correct these condi-
tions in any way, but administration of folic acid and biotin corrected
them completely. These investigators interpreted their results as indicating
that deficiencies of folic acid and biotin prevent the proper utilization of
pantothenic acid.

Daft*! showed that the inclusion of ascorbic acid at a level of 2 % in diets
deficient in pantothenic acid prevented the development of the usual panto-
thenic acid deficiency syndrome in weanling rats. Daft and Schwarz” re-
ported on these and similar experiments after the animals had remained
on the deficient diet for one year. Some rats grew to a weight of over 500 g.
during this period, having received from weaning a highly purified diet
with no supplementary pantothenic acid. Hundley and Ing** showed that
glucuronolactone and other related compounds could successfully replace
ascorbic acid in similar experiments. Everson et al.*”® reported that the in-
clusion of 2% of ascorbic acid in a pantothenic acid-deficient diet had a
beneficial effect on reproduction.

d. Effect of Antibiotics

In 1951, Swick e¢ al.4? and Lih and Baumann* reported that penicillin,
aureomycin, and streptomycin stimulated the growth of rats receiving

39 T. H. Jukes, Federation Proc. 11, 447 (1952).

39 J). V. Catron, R. W. Bennison, H. M. Maddock, G. C. Ashton, and P. G. Homeyer,
J. Animal Sci. 12, 51 (1953).

407,, D. Wright and A. D. Welch, Science 97, 426 (1948).

41 FS. Daft, Federation Proc. 10, 380 (1951).

42. S. Daft and K. Schwarz, Federation Proc. 11, 201 (1952).

42a J. M. Hundley and R. B. Ing, Federation Proc. 12, 417 (1953).

4226 G, Everson, L. Northrop, N. Y. Chung, and R. Getty, Federation Proc. 12, 413
(1953).

43 R. W. Swick, H. Lih, and C. A. Baumann, Federation Proc. 10, 395 (1951).

44H. Lih and C. A. Baumann, J. Nutrition 45, 148 (1951).

686 PANTOTHENIC ACID

limited amounts of pantothenic acid. The increase was that to be expected
from doubling or tripling the pantothenic acid content of the diet. Aureo-
mycin and streptomycin were reported as superior to penicillin in these
experiments. A few months later Sauberlich*? showed that aureomycin or
penicillin increased the growth of pantothenic acid-deficient rats during a
four-week period and concluded that these antibiotics have a marked
influence upon the pantothenic acid requirement of the weanling rat. In
1952 Daft and Schwarz reported on experiments which had lasted for a
year” and showed that weanling rats grew at an almost normal rate during
this period on highly purified diets unsupplemented with pantothenic acid
but containing small amounts of aureomycin. Somewhat similar results
with a variety of antibiotics have been reported by other investigators.4°*~4
Luecke et al.‘® reported that in his experiments aureomycin did not spare
the pantothenic acid requirement of pigs, although Catron e¢ al.*% did find
a sparing effect in pigs when the protein level was low (14 %).

e. Effects of Hormones

Drill and Overman*’ have shown that the pantothenic acid requirement
of rats is increased during experimental hyperthyroidism. Haque et al.,*8
on the other hand, observed a partial counteraction of pantothenic acid
deficiency signs in chicks by thyroxine injections.

The effects of adrenal and pituitary hormones on the development of the
pantothenic acid deficiency syndrome in rats have been discussed in a
previous section.

f. Effects of Stress and Miscellaneous Factors

Dumm ef al.4° have reported that rats deficient in pantothenic acid,
which were subjected to the stress of swimming or given injections of
ACTH, failed to show the expected lymphopenia. Following therapy with

45 H. KE. Sauberlich, J. Nutrition 46, 99 (1952).

45a A. Fidanza, G. Giunchi, M. L. Rutigliano, L. A. Scuro, and F. Sorice, Boll. soc.
ital. biol. sper. 28, 1393 (1952).

456 G, Giunchi, A. Fidanza, L. A. Scuro, and F. Sorice, Acta Vitaminol. 7, 75 (1953).

45¢ G, Giunchi, A. Fidanza, L. A. Seuro, and F. Sorice, Boll. ist. sieroterap. milan. 32,
159 (1953).

464K, Guggenheim, 8. Halevy, I. Hartmann, and R. Zamir, J. Nutrition 50, 245
(1953).

46 R. W. Luecke, J. A. Hoefer, and F. Thorp, Jr., J. Animal Sci. 11, 769 (1952); 12,
605 (1953).

47V. A. Drill and R. Overman, Am. J. Physiol. 135, 474 (1942).

48M. E. Hague, R. J. Lillie, C. 8. Shaffner, and G. M. Briggs, Endocrinology 42, 273
(1948).

49M. E. Dumm, P. Ovando, P. Roth, and E. P. Ralli, Proc. Soc. Exptl. Biol. Med.
71, 368 (1949).

|
;

XI. REQUIREMENTS AND FACTORS INFLUENCING THEM 687

pantothenic acid, the animals showed the usual lymphopenia after swim-
ming or ACTH administration. Dumm and Ralli®® also stated that a
daily intake of at least 4 mg. of calcium pantothenate is necessary to obtain
prolonged survival in adrenalectomized rats. Ralli and coworkers*!: * have
also observed a decreased excretion of ascorbic acid during pantothenic
acid deficiency or following adrenalectomy or hypophysectomy.

Rats depleted in pantothenic acid are less able to withstand exposure to
cold than their pair-fed or normal controls, according to Ershoff.*

B. OF MAN
ELAINE P. RALLI

The widespread occurrence of pantothenic acid in foods precludes the
possibility that a deficiency state due to the absence of this vitamin would
be readily produced. There have been no experiments on human subjects
maintained for long periods on diets deficient in pantothenic acid. There-
fore, the question of the nutritional requirement of human beings for panto-
thenic acid will be approached on the basis of the approximate daily intake
of this vitamin provided in an average diet in this country, on the studies
of the excretion of pantothenic acid in human subjects, and, finally, by
estimating the human requirement on the basis of the known requirement
of animals of different species.

1. PanrotHEeNic Acip CoNTENT oF Foops

Numerous analyses have been made of the pantothenic acid content of
uncooked foods.**-*§ From the data in Table XI (see p. 638) the average
American diet, which would include two glasses of milk, two helpings of
fruit, two helpings of vegetables, a generous helping of potatoes, 5 oz. of
meat, 4 oz. of beans, two eggs, and several slices of bread, would contain
about 9 mg. of pantothenic acid. Even if the diet consisted mainly of vege-
tables and two glasses of milk daily, the pantothenic acid intake would

50 M. E. Dumm and E. P. Ralli, Endocrinology 48, 283 (1948); M. E. Dumm and E. P.
Ralli, Metabolism 2, 153 (1953).

51M. KE. Dumm and E. P. Ralli, Endocrinology 45, 188 (1949).

62M. E. Dumm, LE. P. Ralli, and I. Graef, quoted by E. P. Ralli and M. E. Dumm,
Endocrinology 51, 135 (1952).

52a B. H. Ershoff, J. Nutrition 49, 373 (1958).

53M. L. Thompson, E. Cunningham, and E. E. Snell, J. Nutrition 28, 123 (1944).

54 A. J. Ihde and H. A. Schuette, J. Nutrition 22, 527 (1941).

55 T,. J. Teply, F. M. Strong, and C. A. Elvehjem, J. Nutrition 24, 167 (1942).

56 J. M. Lawrence, B. L. Herrington, and L. A. Maynard, J. Nutrition 32, 73 (1946).

57 V. H. Cheldelin and R. R. Williams, J. Nutrition 26, 417 (1943).

58 T. H. Jukes, J. Nutrition 21, 193 (1941).

688 PANTOTHENIC ACID

still be approximately 6 to 7 mg. The widespread distribution of panto-
thenic acid in foods undoubtedly guards against a deficiency of this vitamin.
The absorption of the vitamin is apparently fairly complete and is not
impaired even in such a disease as pernicious anemia.°*?

On the basis of the dietary intake of pantothenic acid, the requirement
would seem to be about 7 mg. daily. This is lower than the 10 to 12 mg.
suggested by Williams.°®°

2. URINARY EXCRETION AND BLooD LEVELS OF PANTOTHENIC
AcID IN HuMAN BEINGS

The urinary excretion of pantothenic acid has been measured in man by
many investigators,®!-®* and Oldham et al.* have determined the fecal
excretion. The data from the various reports on urinary excretion are in
good agreement and show that on normal diets the excretion ranges from
1.3 to 5.8 mg. per 24 hours. The excretion of pantothenic acid is influenced
by the intake. The excretion is not altered in elderly people.*” Oldham et
al. have done a very complete study on the excretion of pantothenic acid
in urine and feces at different levels of intake, and the data in Table XXI
are taken from their report. Approximately 54 to 84% of the excreted
vitamin was found in the urine, and 25 to 38 % in the feces.

Rubin and his associates® found that when pantothenic acid was given
in the form of pantothenyl alcohol a greater portion of the pantothenic
acid was excreted than when administered in the form of calcium panto-
thenate. They gave doses of 100 mg. of calcium pantothenate or an equiva-
lent dose of pantothenyl alcohol. Their interpretation was that the panto-
thenyl alcohol was physiologically more available than calcium panto-
thenate. However, I would question this interpretation and would suggest
rather that the blood levels may have risen more rapidly following the
administration of the pantothenyl alcohol, and that this was associated
with an increased rate of excretion. In renal clearance studies, Wright et al.®°
have studied the relation of the excretion of pantothenic acid to the blood

59 C. EK. Meyer, I. F. Burton, and C. C. Sturgis, Proc. Soc. Exptl. Biol. Med. 49, 363
(1942).

60 R. J. Williams, J. Am. Med. Assoc. 119, 1 (1942).

61H. P. Sarett, J. Biol. Chem. 159, 321 (1945).

62 H. G. Oldham, M. V. Davis, and L. J. Roberts, J. Nutrition 32, 163 (1946).

68 L. D. Wright and E. Q. Wright, Proc. Soc. Exptl. Biol. Med. 49, 80 (1942).

64M. J. Pelezar, Jr., and J. R. Porter, Proc. Soc. Exptl. Biol. Med. 47, 3 (1941).

65 P. B. Pearson, Am. J. Physiol. 135, 59 (1941).

66 H. Krahnke and E. 8. Gordon, J. Am. Med. Assoc. 116, 2431 (1941).

67, V. Schmidt, J. Gerontol. 6, 132 (1951).

68 $. H. Rubin, J. M. Cooperman, M. E. Moore, and J. Scheiner, J. Nutrition 35,
499 (1948).

69. D. Wright, K. H. Beyer, H. R. Skeggs, H. F. Russo, and E. A. Patch, Am. J.
Physiol. 145, 633 (1946).

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IXX ATAV.L

689

690 PANTOTHENIC ACID

levels. They found that at ordinary normal plasma concentrations (0.10 to
0.32 y per milliliter) only a trace of pantothenic acid was excreted in the
urine and that the renal clearance was of the order of 0.2 to 0.5 milliliter
per minute. On increasing the plasma concentration by oral or intravenous
administration to about 0.5 y per milliliter, there was an abrupt rise in the
calculated clearance. When the plasma concentration was increased to 1.2
y per milliliter, the renal clearance approximated the glomerular filtration
rate and the further increase of the plasma concentration to or above 100
y per milliliter did not alter materially the clearance ratio. These experi-
ments were done with dogs.

Pelezar and Porter® determined the plasma concentration of pantothenic
acid in seventeen normal subjects and found that it ranged from 0.030 to
0.099 y per milliliter of blood and averaged 0.059 y per milliliter. These
levels are lower than those observed in 1946 by Wright e¢ al. at the time
they did renal clearances.*® Pearson® reported the blood levels as varying
from 0.020 to 0.28 y per milliliter. The discrepancy is probably due to the
organism that was used in the microbiological assays. Wright et al. used
Lactobacillus arabinosus. Pearson used Lactobacillus casei ¢«, and Pelezar
and Porter used Proteus morganii.

Spies et al.”° made the interesting observation that the pantothenic acid
content of the blood was decreased in patients with pellagra, beriberi, and
riboflavin deficiency. They found that the administration of 20 mg. of
pantothenate daily increased the blood pantothenic acid and riboflavin
levels. Siegel et al.” studied blood pantothenic acid in patients with multiple
sclerosis and did not observe any significant differences from the blood
levels in normal subjects.

Another group of workers studied the pantothenic acid content of can-
cerous tissue and analyzed malignancies of liver, myocardium, brain, lung,
spleen, kidney, and muscle.” The pantothenic acid content in micrograms
per gram of fresh tissue was highest in carcinoma of the liver and lowest
in carcinoma of the lung and spleen. The pantothenic acid content of liver
was 31 y per gram, of kidney 16 y per gram, and of lung and spleen 5 y
per gram. The figures were lower than determined in rat liver carcinoma.

In Oldham’s study, the dietary intake of pantothenic acid was lower
(2 to 5 mg.) than what would normally be consumed in an ordinary diet
(7 mg.). It seems obvious that the subjects in this study had adequate
supplies of pantothenic acid in their tissues because the excretion paralleled

70T. D. Spies, S. R. Stanbery, R. J. Williams, T. H. Jukes, and S. H. Babcock, J.
Am. Med. Assoc. 115, 523 (1940).

11, Siegel, T. J. Putnam, and J. G. Lynn, Proc. Soc. Exptl. Biol. Med. 47, 362 (1941).

7 A. Taylor, M. A. Pollack, M. J. Hofer, and R. J. Williams, Cancer Research 2, 752
(1942).

XI. REQUIREMENTS AND FACTORS INFLUENCING THEM 691

the intake, and as the intake was increased the total excretion increased.
The effect of test doses of various magnitudes confirms this observation,
and as was shown by Rubin e¢ al.®8 the dose response relationship is quite
constant—the greater the test dose, the greater the excretion.

The question arises as to what use one can make of the data on the
excretion of pantothenic acid in estimating the daily requirement. The
fecal excretion remains fairly constant on different intakes, and it is the
urinary excretion which reflects the daily intake. If one assumes that the
amount of the vitamin excreted in the urine represents an excess of the
bodily need, then subtraction of the amount excreted daily from the
amount ingested might be considered as the amount required daily in a
normal, well-nourished subject. The average daily excretion (3.7 mg. in the
urine and 1.0 mg. in the feces) subtracted from an approximate average
intake of 10.4 mg., would leave approximately 6.4 mg. as the daily require-
ment.

3. APPROXIMATE PANTOTHENIC ACID REQUIREMENT OF MAN AS BASED ON
THE KNOWN REQUIREMENT OF THE EXPERIMENTAL ANIMAL

In all experimental studies in animals, the physiological state of the
animal influences its requirement for pantothenic acid. The requirement
of the growing animal is much greater than that of the adult. Similarly,
stress increases the animal’s requirement for pantothenate. As an example
of this, it was reported by Lotspeich” that symptoms of pantothenic acid
deficiency, which are very slow to appear in the adult rat on a deficient
diet, can be induced fairly rapidly if the animal is given injections of an
anterior pituitary hormone. Other forms of stress, such as pregnancy and
hyperthyroidism, will also increase the requirement for this vitamin.”: 7°

The pantothenic acid requirement for the rat decreases from about 2.5
mg. per kilogram of body weight daily at weaning to approach 0.1 mg. per
kilogram for the adult rat.7* More than 3 mg. is required daily by the
weanling mouse,” whereas the young dog requires only 0.1 mg.7® Since
adult dogs survive much longer on diets deficient in pantothenic acid than
do pups, the adult requirement is thought to be less than 0.1 mg. per kilo-
gram. I am indebted to Dr. Mary E. Dumm for the graphic summary of
these data, shown in Fig. 11, in which the requirement of pantothenic acid

73. W.D. Lotspeich, Proc. Soc. Exptl. Biol. Med. 73, 85 (1950).

74 J. Boisselot, Arch. franc. pédiat. 6, 225 (1949).

75 V. A. Drill and R. Overman, Am. J. Physiol. 185, 474 (1942).

76K. Unna and G. V. Richards, J. Nutrition 28, 545 (1942).

77 J. G. Sandza and L. R. Cerecedo, J. Nutrition 21, 609 (1941).

73 A. E. Schaefer, J. M. McKibbin, and C. A. Elvehjem, J. Biol. Chem. 148, 321
(1942).

692 PANTOTHENIC ACID

mg
x
3.0
x Mouse
© Rat
° O Dog

Pantothenic acid requirement, mg/kg of body weight

-2 -1 0 api
Log of body weight, kilos

Fig. 11. Pantothenic acid requirement of the mouse, the rat, and the dog, in
milligrams per kilogram of body weight. (Prepared by M. E. Dumm.)

per kilogram of body weight (at the beginning of the experiment) is shown
with reference to the log of the body weight in kilograms for the three
species on which quantitative data are available. In general, the data sug-
gest that the requirement for pantothenic acid, expressed per kilogram of
body weight, is greater for small mammals than for large ones, and that
within a given species the requirement may be considerably greater for the
young animal than for the adult.

Although the estimation of the human requirement on the basis of in-
formation obtained from other species is notably uncertain, the data in Fig.
11 might be interpreted as suggesting that the human requirement is prob-
ably less than 0.1 mg. per kilogram, or no more than 6 to 8 mg. daily for
adults.

4. Toxiciry oF PANTOTHENIC ACID

In discussing pantothenic acid intake and requirement, it seems reason-
able to mention briefly what is known of the toxicity of this vitamin.

The toxicity of pantothenic acid was studied by Unna and Greslin?® in
mice, rats, dogs, and monkeys. Very large doses, up to 1 g. per kilogram
in monkeys and 50 mg. per kilogram in dogs, seemed to have no toxic
effect. The author and associates®®: §! have administered from 10 to 20 g.
79K. Unna and J. Greslin, Proc. Soc. Exptl. Biol. Med. 45, 311 (1940).

80 H. Gershberg, S. H. Rubin, and E. P. Ralli, J. Nutrition 39, 107 (1949).
81 H. Gershberg and W. J. Kuhl, Jr., J. Clin. Invest. 29, 1625 (1950).

XI. REQUIREMENTS AND FACTORS INFLUENCING THEM 693

of calcium pantothenate daily to human subjects—these are, of course,
excessively large doses. The evidences of toxicity were an occasional diar-
rhea and, in three subjects with rheumatoid arthritis, retention of water
with edema of the face, the feet, and the lower parts of the leg occurred,
but it disappeared when calcium pantothenate was discontinued. This
latter observation is interesting because of the report by Gaunt et al.”
concerning the disturbance in water tolerance tests in rats on diets deficient
in pantothenic acid, and also because of the effects of large doses of calcium
pantothenate on the survival of the completely adrenalectomized rat.®*> 4
In our own experiments on intact rats on high pantothenate intakes®®> we
observed a decrease in the water tolerance test. One would judge that cal-
cium pantothenate under certain circumstances was associated with the
retention of water.

5. SUMMARY

On the basis of the data reported, it would appear that the approximate
daily intake of pantothenic acid in human subjects on a diet in the United
States varies from 3 to 12 mg. Judging from this and from the amounts
excreted daily, the amount of pantothenic acid in the diet is more than
adequate and protects against a deficiency of this vitamin. Combined with
the data in Fig. 11, it would appear that the daily requirement of panto-
thenic acid for adults is approximately 3 to 5 mg. The intake of pantothenic
acid is reflected in the urinary excretion, and when the diet is excessively
high the excretion will increase. The highest excretion reported on normal
diets was 5.3 mg. per 24 hours. The constancy of the fecal excretion indi-
cates good absorption of the vitamin. Probably the infant and growing
child would need relatively more pantothenic acid than the adult, approxi-
mately 5 mg. daily. On the basis of data previously mentioned on the effects
of the administration of very large doses of pantothenic acid to individuals
with a variety of metabolic disturbances, there seems to be no indication
that the requirement is significantly altered in these diseases. The proof
that the average human, at least in this country, has an adequate supply
of pantothenic acid is provided by the acetylation studies done in patients
with such severe diseases as cirrhosis of the liver, Addison’s disease, and
diabetes mellitus. It is significant in this respect that patients with hyper-
thyroidism* did not acetylate PABA as well as did normal subjects, and
probably in conditions of stress in humans and in situations where the
metabolic rate is significantly increased the pantothenic acid requirement
would be increased.

82 R. Gaunt, M. Liling, and C. W. Mushett, Endocrinology 38, 127 (1946).
83H. P. Ralli, Endocrinology 39, 225 (1946).

84M. E. Dumm and E. P. Ralli, Endocrinology 48, 283 (1948).

85 M. E. Dumm and E. P. Ralli, unpublished data.

694 PANTOTHENIC ACID

The nutritional requirement of pantothenic acid is, in all probability,
influenced by the intake of some of the other fractions of the vitamin B
complex. There are experimental data to show an interrelation between
vitamin By and pantothenic acid in chicks.8* Apparently By» will spare the
pantothenic requirement for growth and survival and will prevent the
dermatosis. Pantothenic acid similarly showed a sparing effect on the vita-
min By, requirement for the growth of normal chicks. Other data indicate
an interrelationship between biotin and pantothenic acid*’ and also between
ascorbic acid and pantothenic acid.’ As mentioned in the previous section,
the development of the burning feet syndrome in the prisoners of war as a
rule was preceded by the development of symptoms of thiamine, riboflavin,
and nicotinic acid deficiency. Similarly, the administration of these frac-
tions in some instances decreased the severity of the burning feet syndrome
although it definitely was not successful in clearing up this syndrome. Ob-
viously, our knowledge of the requirement of pantothenic acid will depend
on further data concerning the interrelation of other vitamins and panto-
thenic acid.

The data for the human requirement are inferential, and the daily re-
quirement arrived at by these means should under no circumstances be
taken as the final word on the subject.

86 H. Yacowitz, L. C. Norris, and G. F. Heuser, J. Biol. Chem. 192, 141 (1951).

87 G. A. Emerson and E. Wurtz, Proc. Soc. Exptl. Biol. Med. 57, 47 (1944).
88 M. E. Dumm and E. P. Ralli, Endocrinology 45, 188 (1949).

Author Index

Numbers in parentheses are footnote numbers. They are inserted to indicate the reference when an
author’s name is cited but his name does not appear on the page.

A

Aaes-Jorgensen, E., 286

Abbott, L. D., Jr., 41

Abbott, O. D., 96

Abdel Kader, M. M., 515, 518 (2, 32), 523
524 (26), 530, 533, 536, 537 (26), 559,
584

Abdon, N. O., 57, 90, 91

Abelin, I., 380

Abels, J. C., 347, 371

Abraham, E. P., 525, 526 (55, 56)

Abrahamson, E. M., 229

Abramson, D. I., 574

Abramson, H., 247, 248

Ackermann, D., 7, 8, 52, 92, 335, 457, 459
(43)

Ackermann, W. W., 532, 549, 625, 679

Adams, R. D., 670, 677 (215)

Adamstone, F. B., 568

Adler, B., 422, 448

Adler, E., 488, 493 (42), 503 (76), 507 (44),
508, 511 (80)

Adler, R., 495

Adrian, J., 637

Aggeler, P. M., 436

Agnew, M. C., 248

Agnew, R. C., 248

Ahlstrém, L., 312

Ahrens, E. H., 373

Ahrens, F. B., 475

Aiton, H. F., 674

Akeson, he 458

Albanese, A. A., 532

Albers, H., 453, 458 (8), 480, 481 (3), 482
(3), 485 (3), 488 (3)

Albert, P. W., 519 (53), 527

Alberti, C. G., 142

Albright, F., 249, 250, 265

Alcayaga, R., 665, 666 (163)

Aldridge, W. N., 17

Alechinsky, A., 577

695

Alesker, E. M., 575

Alexander, H. D., 87

Alfin Slater, R. B., 651

Aline, E., 636, 660

Allan, F. N., 19, 100

Allen, E., 670

Allen, E. V., 427, 428

Allen, J. G., 100

Alles, G. A., 124

Allison, J. B., 92, 126

Almquist, H. J., 28, 46, 94, 95, 389 (15),
390 (14), 391 (15), 393 (16), 394, 395,
396 (29, 34), 397 (85, 45), 398, 399, 400,
401, 402, 403, 404, 405, 406 (14), 407,
408 (9), 409, 410 (31), 411, 412, 4138,
414, 415, 416, 417(1), 422, 423, 425,
426 (26), 429, 4385, 489, 441, 444, 445
(1), 446, 447, 531

Aloisi, M., 8, 12, 19, 89

Along, C., 546

Alscher, R. P., 418, 426

Altman, K. I., 506

Ambo, H., 54

Ambrose, B. A., 461, 462 (64), 463 (64),
464 (64)

Ames, S. R., 638, 639 (29), 640 (29), 641
(29)

Amiard, G., 177, 194, 202, 204 (316)

Amos, E. S., 84

Anderlik, B., 365

Anderson, A. B., 336

Anderson, B. A., 247

Anderson, E. G., 508, 554, 556 (20)

Anderson, F.. W., 197, 204 (297a)

Anderson, G. W., 442

Anderson, J. O., 522, 531, 578 (29), 581,
582 (29), 584 (58)

Anderson, L., 323, 324 (2), 327, 328, 329
(21), 330, 331 (5), 333 (5), 336 (5)

Anderson, R. C., 338

696

Anderson, R. J., 334, 345, 346 (47), 354,
366, 367 (10), 379, 382, 383 (9), 393

Anderson, W. E., 292

Anderson, W. T., Jr., 175, 176, 178 (204)

André, E., 168

Andreassen, E. G., 546

Andrews, J. S., 544

Andrews, M. B., 582, 533 (110)

Andrus, W. D., 401, 426

Anfinsen, C. B., 488, 440 (27)

Angarano, D., 578 (76)

Angel, C., 34

Anger, V., 190

Angulo, J. J., 559

Angus, T. C., 181, 182 (241), 195

Angyal, S. J., 323 (Qa), 338

Anisfeld, L., 274, 285, 290

Annau, E., 313

Ansbacher, S., 384, 394, 396, 399, 400, 405
406, 409, 410, 421, 425, 442, 443 (91),
444 (91), 445 (9), 446, 655

Anthony, D.8., 316

Aoki, T., 54

Aoyama, M., 216

Arai, Ke. 1125

Arcidiacono, G., 440

Ariel, I. M., 347, 371

Arison, B., 476

Armin, S., 247

Armstrong, E. 8., 560, 561, 563 (88), 564
(88)

Armstrong, W. D., 417, 488

Arnold, A., 176, 399

Arnold, R. T., 399

Arnon, D. I., 504

Arnstein, H. R. V., 27, 28 (88), 43 (88),
84

Arreguin, B., 625

Arrol, W. J., 12

Artom, C., 70, 71 (56), 72, 90 (60), 92

Artunkal, S., 574

Aschireli, A., 423

Aschkenasy, A., 573

Ashburn, L. L., 75, 107, 108 (426), 113
(426), 114, 650, 651, 653, 657 (9, 16, 22,
23), 669, 670 (198)

Ashton, G. C., 666, 685, 686 (39a)

Ashworth, C. T., 75

Askew, F. A., 178, 181, 182 (225, 241),
189, 192, 195 (241, 263), 196 (263),
198, 204 (263), 216

AUTHOR INDEX

Asmundson, V.58., 661, 682 (14)

Atkin, L., 342, 349 (3), 362, 550, 633

Atkins, P., 438

Atkins, W. R. G., 171

Atkinson, M., 574

Atkinson, W. B., 656

Augustinsson, K., 17

Auhagen, E., 192, 195

Ault, W. C., 271

Auricchio, G., 8

Austin, F. L., 642, 645 (34)

Avrin, I., 4 (16), 53

Axelrod, A. E., 512, 518 (24), 554, 555,
556 (33)

Aykroyd, W. R., 584

Aylward, F. X., 86

Azerad, E., 303, 304, 305, 440

B

Babcock, 8S. H., 677, 690

Babcock, 8. H., Jr., 650, 656 (2), 657 (2),
658

Baccari, V., 8, 19, 71, 86, 102

Bacharach, A. L., 182, 184, 185, 187, 189,
190 (243), 197, 198, 204 (297a), 229

Bacher, F. A. 401, 414

Backer, H. J., 144

Bacon, J. S. D., 629, 682 (7)

Baddiley, J., 16, 603, 604 (27), 606, 611,
613, 631, 632 (25)

Badger, E., 49

Badgett, C. O., 466, 467

Badin, J., 431

Baer, E., 12, 501

Baer, M., 12

Baeyer, A., 10

Bailey, A. E., 292, 312

Bailey, B. E., 148, 164 (60), 167

Baird, G. R., 583

Baker, B. R., 397

Balatre, P., 329, 334 (1), 335 (1), 336 (1)

Bale, W. F., 27

Ball, E. G., 488, 440 (27)

Balla, G. A., 381

Balls, A. K., 345

Banchetti, A., 198, 217

Bancroft, F. W., 424, 432 (39)

Bandelin, F. J., 56

Bandier, E., 5438, 549 (83)

Banerjee, R., 533, 580

Banerjee, 8., 58, 5383, 575, 580

AUTHOR INDEX

Banks, A., 319

Baranowski, T.,

Bargoni, N., 36

Barker, H. A., 602, 616, 620 (20), 621 (20,
94)

Barker, N. W., 427, 428, 436

Barker, W. H., 564

Barki, V. H., 280, 294, 315, 319

Barlow, O. W., 196, 197 (297), 203 (297),
204 (297)

Barnes, C. C., 538

Barnes, G. W., 171

Barnes, R. H., 73, 86, 285, 314

Barnes, W. A., 425

Barnett, M., 133, 254

Barr, T., 147

Barratt, R. W., 348, 344 (21), 367

Barrenscheen, H. K., 25, 29 (82), 30, 31,
91

Barrett, H. M., 68, 73 (41), 81 (41), 103
(41)

Barrios, P. E., 299

Barron, E. 8. G., 17, 36, 498

Bartels, W. E., 374

Bartlett, G. R., 36

Bartow, E., 340, 341, 358

Bass, L. W., 485

Bassett, C. F., 224, 256

Bassham, J. A., 504

Bassi, U., 562

Bastiansen, O., 332, 336 (22), 345

Battafarano, T. W., 372

Battle, W. D., 428

Baudart, P., 272

Bauer, C. D., 175

Bauer, J. M., 264

Bauernfeind, J. C., 591, 642, 643 (82),
645 (382), 660, 682 (16)

Baum, H. M., 367, 368 (18), 654, 655 (42),
656 (42), 657 (42)

Baumann, C. A., 4 (17), 6 (17), 38, 538,
54 (15), 67, 71 (27), 89, 103, 428, 444,
629, 642, 643 (31), 645 (31), 685

Bays, R. F., 434

Beach, E. F., 21

Beadle, G. W., 9, 49, 50 (3), 55, 342, 362,
366, 525, 551

Bean, W. B., 518 (15, 22), 553, 574, 576 (16)

Bean, W. S., 552

Beard, H. H., 181

Bearn, A. G., 107

442, 495, 501 (102)

697

Beattie, F. J. R., 5,6 (19), 53

Beber, A. J., 293

Bechdel, S. I., 223, 230 (2), 232

Bechtel, H. E., 223, 224, 225, 227, 256

Beck, E. M., 652

Beck, H., 442

Becko ly dee oda

Becker, B., 485

Becker, J. E., 133

Beckett, ©. W., 3381, 3382) (20)

Beckh, W., 556, 562 (58)

Beckmann, R., 374

Becks, H., 657

Beerstecher, E., Jr., 622, 626, 627 (3), 668

Beeson, W. M., 666

Beeston, A. W., 20, 69, 76, 77, 81

Behan, I. T., 547

Beher, W. T., 457, 459 (58), 516, 517 (27)
518 (27, 28), 549

Behrman, M., 598

Beinert, H., 497, 502 (111), 603, 609 (22),
619, 621, 622 (87)

Belinsky, C., 326

Bell, G. H., 529, 581, 585 (53)

Bell, P. G., 674

Bellini, E., 441

Belloc, G., 170

Bellows, J. G., 67

Belt, M., 570, 578 (81)

BeMiller, L. N., 182, 183 (242)

Benard, H., 74

Bender, R. C., 657

Benedetti, G., 575

Benesch, R., 522, 560, 583

Benford, F., 176, 177

Bennett, H. B., 229, 254

Bennett, I. L., Jr., 436

Bennett, L. L., 73

Bennett, M. A., 22, 26 (69, 70), 83

Bennett, T. I., 306

Bennett, W. G., 157

Bennison, R. W., 666, 685, 686 (39a)

Benoit, G. J., 92

Benotti, J., 56

Benson, A. A., 504

Bentley, L.S., 91

Benz, F., 459 (57), 472 (57), 477 (57), 488,
491, 492 (50)

Bercowitz, Z., 436

Berg, B. N., 672

698

Berg, C. P., 519 (48), 524, 526, 529 (92),
531 (41)

Berg, J. R., 456

Berg, P., 29

Bergeim, O., 228

Bergell, P., 11

Berger, L., 358

Bergmann, W., 186, 137, 141

Bergstrém, S., 147, 159, 275, 278, 289, 312,
317

Berk, L. C., 288

Berkman, 8., 540, 672, 679

Berlman, 8., 598

Berman, R., 17

Bernard, R., 97, 578 (84)

Bernfeld, P., 345

Bernhard, K., 280, 314, 315, 319

Bernheim, F., 32, 33, 34, 36, 38 (164), 73,
83, 88 (91), 108, 127, 516, 555

Bernheim, M. L. C., 32, 33, 34, 41, 103,
127, 516 .

Bernstein, C., 304

Bernstein, 8., 143, 144

Bernton, A., 143

Berryman, G. H., 548, 559

Berthelot, M., 335

Bessman, 8. P., 600

Best, C. H., 5, 19, 20, 25 (41), 40 (41), 46,
48, 52, 57, 66, 67, 68, 69, 71 (48), 72,
73 (41), 74 (90), 75, 76 (89), 77 (132),
78 (122), 80, 81 (41), 82 (171), 86, 88
(9), 99, 100 (215), 103, 104, 111, 126,
129, 347, 371, 372, 383

Betheil, J. J., 84, 349, 350 (73), 351 (73),
382, 507

Bethell, F. H., 39, 85

Bethke, R. M., 98, 94 (305), 95, 96, 154,

155, 230, 546, 568, 636

Beuk, J. F., 543

Beveridge, J. M. R., 76, 77 (135), 78 (142),
80, 315, 384

Bevilacqua, L., 516, 556

Beyer, K. H., 688, 690 (69)

Bhagvat, K., 555

Bhattacharya, G., 575

Biana, L., 577

Bide, A. E., 148

Bigwood, E. J., 628

Bijvoet, J. M., 336, 343

Bilger, F., 157, 190, 191

Billing, B., 107

AUTHOR INDEX

Bills, C. E., 183, 139, 148, 153, 157, 158
(11), 160, 162, (11), 164, 166 (167), 167,
168, 169, 170, 171, 172 (U71); Lieeige:
180, 181, 182, 188 (242), 184 (249)
185, 186 (249), 187 (249), 188, 199
205, 206, 207, 208 (34), 209, 210, 212,
213, 218, 219 (26), 220 (22), 222, 518
(30)

Bina, F. F., 543

Bing, F. ©., 229

Binkley, F., 32, 42

Binkley, 8S. B., 390, 391 (20, 21), 392 (20),
393 (21, 25) , 394, 395 (25, 40), 396 (89),
397 (43), 398 (39), 399, 400, 403, 405,
409, 418, 417, 435

Binkley, W. W., 364 (9)

Binnie, G. G., 440

Bionot, G., 7

Birch, T. W., 521, 578 (8)

Bird, F. H., 94, 682 (14)

Bird, H. R., 582, 661

Bird, M. L., 51

Bird, O. D., 16, 409, 604, 606 (30), 611
(80), 624 (380), 631

Birkinshaw, J. H., 51, 156

Bischler, A., 577

Bischoff, G., 4 (18), 18, 53

Biskind, G. R., 108

Black, A., 1383, 154, 160 (9), 169, 170, 228,
225, 226 (8) 230 (23), 368, 386, 656

Black, M. B., 87

Black, 8., 417, 423, 600, 617, 620 (70),
654, 656 (35), 657 (85), 663, 682 (4)

Blackburn, S., 51

Blackmore, B., 422

Blackstock, V., 565

Blair, M. G., 364 (9)

Blankenhorn, M. A., 453

Blatherwick, N. R., 81

Blaylock, L. G., 635, 637 (6)

Blekkingh, J. J. A., 343

Blewett, M., 291, 298, 314, 315 (84)

Bliss, C. I., 220

Bloch, K., 30, 41, 158, 615, 622 (61), 625

Block, R. J., 20

Bloom, W., 232

Bloor, W. R., 315

Blumberg, H., 75, 399

Bly, C. G., 379, 383, 663

Bocarius, N., 7

Bock, F., 158, 154, 155 (105), 186 (110)

AUTHOR INDEX

Bock, R. M., 619

Bocklage, B. C., 344, 345 (36), 381

Bodansky, A., 226

Bode, J., 11

Bodur, H., 314

Boehm, R., 124

Boehme, C. C., 516

Boer, A. G., 152, 153, 154, 155

Boezaardt, A. G. J., 326, 337, 347

Bohstedt, G., 223, 227 (3), 230 (8), 342,
343 (9), 368, 369

Boisselot, J., 670, 691

Boissonnas, R. A., 32, 89

Bokman, A. H., 528

Boland, E. W., 419

Bollman, J. L., 401, 426, 427, 428, 429,
431 (90), 4384

Bonetti, E., 89

Bond, H. W., 457, 459 (39), 524, 525

Bondi, A., 635, 642 (5), 643 (5)

Bonner, D. M., 49, 50 (4), 55, 453, 518
(11), 519 (51),1 525, 526, 527 (72),
528 (72), 529 (72), 531 (72)

Bonner, J., 625

Bonsignore, A., 516, 556

Booher, L. E., 160, 161 (142), 547

Booth, F. J.,7,8

Borgeaud, P., 175

Borggard, M., 54

Borglin, N. E., 58, 90, 91, 93, 129

Borland, V. G., 298

Bornhofen, E., 127

Borrow, A., 548

Borsook, H., 29, 30, 31, 103

Boruff, C. S., 642, 645 (34)

Bosecke, W., 414

Boss, M. L., 344, 352, 533

Bostwick, D. G., 636

Bosworth, A. W., 382, 383 (9)

Bothwell, J. W., 39

Boucher, H., 442

Boucher, R. V., 97, 642, 643 (33)

Bouchet, M., 90

Bouchilloux, 8., 8

Bourdillon, J., 346

Bourdillon, R. B., 175, 177, 178, 181, 182
(225, 241), 189, 192 (263), 195 (241
263), 196 (263) 198 (263), 204 (263),
216

Bourgeois, R., 92

Bourne, G. H., 568

699

Bouthillier, L. P., 28

Boutwell, R. K., 645

Bovarnick, M. R., 455, 522

Bowen, D. M., 394, 397 (37)

Bowie, D. J., 19, 100

Bowland, J. P., 101, 6387

Bowles, L. L., 670

Bowman, K. M., 562

Boxer, G. E., 72, 82

Boyack, G. A., 626

Boyd, E. J., 434

Boyd, H. M., 544

Bradshaw, P. J., 81

Brady, J., 575

Braganca, B. M., 428, 424

Bramante, P., 576

Brambel, C. E., 444

Brandaleone, H., 671

Branion, H. D., 207

Brante, G., 71

Brauchli, E., 190

Braude, R., 567, 586

Braun, K., 561

Braunstein, A. E., 529 (98)

Bray, W. E., 421, 483 (15)

Brazda, F. G., 74, 571, 572 (8), 573

Breda, R., 441, 442 (61)

Brenner, S., 545

Brenner, S. A., 224

Breusch, F., 186 (260), 187, 204 (260)

Brice, B. A., 287, 288

Brickson, W. L., 522, 5380 (23), 578 (2)
581 (2), 586

Brickwedde, F. G., 178

Brieger, 125

Briggs, A. P., 519 (45), 524, 528 (32), 529
(32), 531, 559, 560 (77), 581

Briggs, G. M., 94, 95, 97, 369, 522, 578
(21, 27, 28, 29, 30), 581 (27), 582 (29),
584 (58), 658, 659 (100), 686

Briggs, G. M., Jr., 342, 554, 569, 570, 682
(13)

Briggs, J. E., 666

Brighenti, L., 441

Brinkhous, K. M., 401, 404, 408, 419, 421,
423, 426, 481 (14), 483 (14), 437

Brissemoret, A., 379

Broadbent, H.S., 477

Brocklesby, H. N., 210

Brockmann, H., 170, 172, 173 (184)

Brode, W. R., 271

700

Bromel, H., 496

Broome, F. K., 53

Broquist, H. P., 313, 679

Brosteaux, J., 502

Broun, GaOysi2

Brown, A., 436

Brown, C. L., 372

Brown, D. M., 489

Brown, E. B., 5438

Brown, E. F., 315

Brown, G. B., 11

Brown, G. M., 16, 604, 605, 606 (30), 607,
611 (30, 39), 613 (39), 624 (380), 631, 632

Brown, J. B., 271, 272, 279, 280 (4), 289,
BPA Bl} (UZ) 5 alls:

Brown, R. A., 216, 396, 409, 411, 446

Brown, W. R., 302, 303

Bruce, H. M., 178, 181, 182 (225, 241), 189,
192 (263), 195 (241, 263), 196 (263), 198
(263), 204 (263), 216, 228, 382

Bruch, E., 4

Bruins, H. W., 661

Brill, W., 399

Brunken, J., 175

Brunschwig, A., 75

Bruun, P., 101

Buccellato, G., 440

Buchanan, K. §., 227

Buchholz, K., 142, 144 (44), 206

Biichi, J., 488, 492 (48)

Buffa, P., 12

Buhs, R. P., 414

Buisman, J. A. K., 148

Bullet, F., 280, 315, 319

Bumpers, M., 475

Bunbury, H. M., 472

Bunker, J. W. M., 175, 176, 177

Burgos, A., 304, 305

Burk, R. E., 181

Burke, G. E., 427

Burke, K. A., 94

Burkholder, P. R., 361, 366, 544, 547, 550,
635, 636 (3), 644 (3)

Burkley, 8. B., 400

Burns, M. M., 98

Burnside, J. E., 637

Burr, G. O., 268, 270, 274, 275; 277, 279,
281, 285, 287, 288, 292, 293, 302, 306,
312, 314, 318

Burr, M. M., 268, 279, 292, 293, 306, 318

Burr, We W..,. drs 79

AUTHOR INDEX

Burrill, D. Y., 441

Burroughs, W., 568

Burton, I. F., 687

Burton, R. M., 603, 607 (24), 620 (24, 80)

Biising, K. H., 441

Buston, H. W., 348, 366

Busse, P., 142, 172, 177 (45), 180 (45), 193,
198 (45), 200 (45)

Busset, R., 130

Butenandt, A., 142

Butler, J. J., 436

Butler, R. E., 565, 578 (13)

Butt, H. R., 401, 422, 424, 425, 426, 427,
428, 437

Butts, H. A., 637

Buttutrini, U., 577

Butz, L. W., 20

Buu-Hoi, N. P., 345, 429

Buyske, D. A., 603, 609 (22)

Byers, L. W., 365, 366

Cc

Cabezas, V. A., 88

Cagniant, P., 429

Calabro, @., 577

Calandra, J. C., 441

Calbert, C. E., 280 (4), 290, 318

Calecott, W. S., 155, 156 (119), 204 (119),
205 (119)

Calder, R. M., 74, 574

Callow, R. K., 140, 143, 152, 156, 168, 181,
182 (241), 183, 184, 186 (58), 187, 188,
189, 190 (250) 192 (263), 195 (241,
263), 196, (263), 198 (263), 204
(263), 216, 223, 382

Caloves, N. F., 584

Calvin, M., 504

Calvo, J. M., 516

Camerino, B., 142

Campbell, H. A., 404, 418, 427, 428, 436

Campbell, J., 72

Campbell, J. A., 217

Campbell, W. P., 394, 397 (87), 399, 414

Camponovo, P. B., 441

Camps, R., 475, 476 (101)

Camus, L., 375

Cannavé, A., 443

Cannon, M. D., 580, 645

Cantoni, G. L., 25, 29, 31, 32, 33.{8a);
470, 516

Capps, R. T., 428

PP il

AUTHOR INDEX

Cardini, C. E., 54

Carey, J. B., 560, 566

Carlson, G. H., 397

Carlson, W. A., 184, 187 (253)

Carne, H. O., 72

Carpenter, K. J., 486, 548

Carpentier, 8., 344

Carretero, R., 524, 530 (39)

Carrick, C. W., 95, 578 (24, 25), 661

Carroll, F. D., 674

Carroll Cs, 547

Cartaya, J. A., 76

Carter, C. E., 489

Carter, C. W., 555, 653, 657 (24), 662

Carter, H. E., 25, 48, 326, 327, 328, 365,
366

Carter, J. R., 428

Cartwright, G. E., 563, 568

Caskey, C., 229

Cason, J., 345, 346 (47)

Castiglione, M., 559

Castille, A., 10, 57, 183, 191

Castro Mendoza, H., 87, 130

Cateno C., 423

Catolla-Cavaleanti, A., 668

Catron, D. V., 666, 685, 686

Causeret, J., 573

Causey, K., 546

Cayer, D., 559, 560 (82)

Cedar, E. T., 261

Cedrangolo, F., 71

Cerecedo, L. R., 85, 103, 297, 315, 667,
682 (27), 691

Chaikoff, I. L., 56, 71, 72, 80, 90, 99 (264),

100, 101, 108

Chaix, P., 344, 352

Chakravorty, P. N., 191

Challenger, F., 25, 44, 51

Chamberlin, V. D., 96

Chambers, G. H., 428

Chamorro, A., 440

Chandler, J. P., 11, 21, 22 (63), 25: (63),
26 (64), 28, 30 (66, 67), 40 (66), 41
(75), 46, 47 (7), 48 (7), 80

Chang, H. C., 57

Chang, K. P., 554

Channon, H. J., 20, 46, 47, 68, 69, 71
(48), 76 (52), 77, 86

Chantrenne, H., 602, 617 (21), 620 (21),
621

Chargaff, E., 42, 56, 72, 330, 331, 332 (13,

701

14), 333 (13, 14), 334, 387 (4), 338 (4,
12, 13), 344, 345 (29), 347, 348, 353

Charlton, P. T., 51

Charonnat, R., 574, 577

Chattopadhyay, H., 58

Chauchard, P., 88, 317, 380, 381, 440

Chaudhuri, P. K., 536, 548, 544

Cheldelin, V. H., 339, 364 (4), 365 (4),
377, 544, 548, 611, 613 (56), 621 (96),
629 (12), 637, 638, 639 (28), 640 (28),
641 (28), 645 (28), 647, 648, 687

Chen, K. K., 429, 571, 572 (1)

Cheney, L. C., 391, 394, 395 (40), 396
(389), 897 (48), 398 (39)

Cheng, D. W., 668

Cherbuliez, E., 476

Chesbro, R. M., 545

Cheslock, K., 620 (82)

Chevalier, J., 379

Chevillard, L., 17, 574, 577

Chévremont, M., 91

Chick, H., 230, 521, 565, 567, 654, 655, 656,
664

Childs, G. R., 578 (25)

Chinn, H., 67, 90

Chitre, R. G., 544, 584

Chittenden, R. H., 565

Chou, T. C., 607, 616, 617 (67), 618 (67),
620 (67, 83)

Chow, B. F., 86

Chopin, J., 195, 209

Christensen, J. R., 39

Christensen, K., 65

Christensen, P. E., 339, 382, 383 (5)

Christian, W., 453, 455 (5, 7), 458 (6, 7)
460, 470 (6), 480, 481 (2, 4), 482, 484
(15a) 485 (4), 486 (4), 487, 488, 489
(4,7), 491 (1, 2, 4), 496, 500 (4, 7), 510,
511

Chu, Ho L,, 554

Chuang, C. K., 134

Chudzik, E. B., 73

Chung, N. Y., 685

von Chwalibogowski, A., 301

Ciocea, B., 338

Ciotti, M. M., 493, 511

Cislak, F. E., 474

Ciusa, W., 472, 536

Clandinin, D. R., 661

Clark, B. P., 420

Clark, D. E., 86

702

Clark, H. W., 166 (168), 169 (168)

Clark, 1,585

@lark, Re ii... Jr, 425

Clark, RK’ ali 326,327,325) Cl)

Clark, W. G., 380, 381 (23)

Clarke, D. H., 655

Clarke, H. T., 161, 170 (151)

Clarke, M. F., 522

Clarkson, M. F., 99, 126

Claude, A., 346

Claus, A., 3

Clausen, M., 285

Clegg, R. S., 420

Clifeorn, L. E., 545

Climenko, D. R., 369, 384

Cloetta, A., 374, 375

Clowes, G. H. A., Jr., 86, 89

Coates, M. E., 534, 578 (26), 629 (15) 658,
659 (101), 660

Cobenzl, A., 473

Coburn, A. F., 508

Cohen, G. N., 52, 92

Cohen, P. P., 621

Cohen, S., 31

Cohen, 8. S., 501

Cohn, E. T., 425

Cohn, M., 11, 21, 25, 26 (64), 28, 30 (66),
40 (66), 41 (75), 48, 102

Cohn, W. E., 228, 489

Colby, R. W., 665, 666

Collentine, G. E., 399, 401, 404, 406 (7),
412, 413, 428, 429, 434, 447

Collins, C. J., 438

Collins, R. A., 280, 319, 657

Colman, H. C., 173

Colowick, S. P., 493, 494, 507, 511, 538

Colson, H., 216

Colter, J. 8., 36

Combes, F. C., 261, 671

Combs, G. F., 531, 578 (29), 581, 582 (29)

Comfort, M. W., 448

Common, R. H., 226

Conese, G., 578 (76)

Confino, M., 344

Connor, C. L., 108

Contardi, A., 338

Cook, B. B., 545, 653, 684

Cook, C. A., 522

Cook, J. W., 89, 134, 467, 468 (71), 470

Cooke, A. H., 171

Coon, M. J., 28

AUTHOR INDEX

Coons, C. M., 547

Cooper, C., 453

Cooper, F. W., Jr., 574

Cooper, O., 488, 440 (27)

Cooper, Z. K., 570

Cooperman, J. M., 342, 370, 578, 626, 688,
691 (68)

Copeland, D. H., 83, 89, 95 (184), 98

Copley, M. J., 439

Copping, A. M., 171

Cordero, A. A., 441

Cordy, D. R., 665

Cori, C. F., 492, 493, 498, 510

Cori, G. T., 492, 493, 510

Corley, R. C., 316

Cornatzer, W. E., 70, 90 (60)

Cornbleet, T., 303

Cornil, A., 91

Correll, J..T., 207

Correll, J. W., 373

Corson, M. H., 456, 518

Corwin, W. C., 442, 448 (91), 444 (91)

Coryell, M. N., 565, 638, 648 (21b)

Cote, L., 518 (29, 31), 521 (29)

Cottingham, E., 586

Couch, J. F., 439, 477

Couch, J. R., 636, 660

Coujard, R., 7

Coulson, R. A., 74, 518 (4), 522, 536, 557,
559, 560 (75), 565 (75), 571, 572 (8),
573, 583

Courtois, J. E., 334, 335, 360, 365

Cover, S., 637

Covo, G. A., 498

Coward, K. H., 160, 218, 220 (24), 221,
222 (2)

Cowdry, E. V., 362

Cowgill, G. R., 356, 364 (8), 385, 453, 624,
651, 652, 656, 668

CoxiGruendn

Cox, W. M., Jr., 179, 180 (238), 181, 182
(239), 199 (238, 239)

Craig, J. A., 16, 604, 605, 606 (30), 611
(30, 39), 613 (39), 624 (30), 631, 632
(29), 633 (29)

Cravens, W. W., 95, 96, 403, 445, 532, 661

Creighton, M., 230

Crews, S. K., 195, 198

Crichton, A., 230

Crigger, E. M., 516

Crisalli, M., 671

AUTHOR INDEX

Crokaert, R., 628

Cromer, H. E., Jr., 428, 436

Cromwell, H. W., 629 (13)

Crosby, J. N., 475

Cross, D. M., 301

Cross, R. J., 498

Crowder, M., 92

Crowfoot, D., 198

Crowley, L. D., 574

Cruickshank, E. K., 675

Cruickshank, EK. W. H., 383

Cullen, S. C., 427

Cumming, G. R., 651

Cunha T. J., 101, 342, 348, 368, 369, 637,
665, 666

Cunningham, E., 687

CurtiswAS ©. 110

Curtis; J. J., 547

Czaky, T. Z., 130

D

Daft, F. 8., 74, 75, 104, 107, 108 (426), 113
(426), 114, 423, 518 (83), 578 (10), 628,
650, 651, 653, 656 (2), 657 (2, 16, 22
23), 669, 670 (201), 685, 686

Dainow, I., 576

Dal Palu, C., 574

Dale, C. N., 568, 578 (15, 16)

Dale. Ho EH... 7, 124

Dalgliesh, C. E., 529 (94)

Dallemagne, M. J., 345

Dalmer, O., 173

Dam, H., 101, 153, 285, 286, 316, 317, 369,
384, 389, 390, 391, 392 (18), 396, 398
(18), 399, 400, 401, 402, 408, 406, 408,
409, 410, 411, 413, 415, 417, 421, 422,
423 (11), 424, 432, 433, 434, 437, 488,
439, 441, 445, 447

D’Amato, F., 344

Dangschat, G., 323, 330, 331, 337 (6), 338
(10)

Daniel, E. P., 160, 161

Daniel, L. J., 39

Daniel, R. J., 335

Daniels, F., 176, 209 (216)

Dann, F. P., 396, 405, 654, 655, 656 (39)
657 (39)

Dann, W. J., 32, 71, 82 (68), 457, 459 (42)
515, 518 (18, 19, 26a), 522, 532, 535,
540, 543, 546 (12), 554, 555 (22), 556,
564, 567, 573

703

Danowski, T. 8., 264

Darby, H. H., 161, 170 (151), 171

Darnell, A. L., 638, 648 (21 a)

Das, N. B., 498, 503 (76), 507

da Silva, A. C., 578 (85)

Dasler, W., 175, 178, 179

Datta, P., Jr., 518 (18)

Dauben, W. G., 12

Daubert, B. F., 288

Daum, K., 553

Daum, 8., 90

Davenport, H. A., 313

Davide, H., 317

Davidson, C. 8., 436

Davidson H. G., 684

Davidson, J. A., 637

Davidson, J. D., 99

Davidson, J. H., 628

Davies, J. N. P., 104

Davies, T. H., 397

Davies, W. L., 92

Davis, D., 533

Davis, H. A., 96

Davis, J. E., 99, 126

Davis, M. V., 688, 689 (62)

Davis, O. L., 628

Davis, P. L., 37 (169), 84, 96 (200)

Davis, R. B., 217

Davis, R. H., 272

Davis, V. E., 638, 648 (21c)

Davison, H. G., 653

Davoli, R., 439

Dawson, R. F., 546

Day, H. G., 418, 423

Day, P. L., 36, 37, 39, 84, 91, 96 (200)

De, H. N., 518 (18), 584

Deal, C. C., 518 (26)

Deane, H. W., 66, 73, 90 (16), 650, 651,
657 (10), 669, 670 (202)

de Angelis, R. C., 578 (35)

Deb, C. C., 580

de Braganza, B., 17

de Carvajal-Forero, J., 379

Decker, A. B., 297

De Jong, A. W. K., 334, 336

Delachanal, 335

de la Huerga, J., 127, 128, 130, 518 (20),
519, 548, 581, 582 (54), 585

de Lam, H. H., 344

de la Mare, P. B. D., 312, 314

Delancey, H., 381

704

de Lange, D. J., 521, 524

Del Campillo, A., 617, 619, 620 (69), 673

Della Santa, R., 427

Delor, J., 481

DeLor, J. C., 415

del Rio, J., 180

Del Vecchio, V., 441

Demarest, B., 173

DeMasters, C. U., 96

de M. Braganca, B., 543

Demers, J. M., 97, 578 (34)

de Mingo, M., 355

De M. Taveau, R., 124

Denko, C. W., 559

Dennis, C., 99

Dennis, P. O., 535

Denton, C. A., 532

Denton, J., 566, 570

Deodhar, T., 519 (47), 548, 582

Deppe, M., 196, 200, 201 (813), 202 (313),
204 (298, 313)

Derian, P. 8. 577

Derrick, C. J., 561

Desai, D. B., 544, 584

de Souza, D., 378

Dessau, F. I., 66

Desveaux-Chabrol, J., 545

Deuel, H. J., Jr., 91, 184, 274, 279, 280
(4), 285, 290, 318, 519 (53), 527, 528,
529 (84), 581, 585 (53)

Deutsch, H., 265

Deutsch, H. F., 444

Devi, P., 555

Devich, A. C., 636

DeVries, T., 175

DeVries, W. H., 16, 603, 605 (25) , 607 (25),
609 (25), 610 (25), 613 (25)

Dewan, J. G., 502

Dewey, V. C., 532, 533 (110)

De Witt, J. B., 198, 216

Deysson, G., 344

Deysson, M., 344

Diaz, C..J., 87

Diaz-Rubio, M., 561, 564, 574

Diaz y Rivera, R.8., 425

Di Bella, S., 36

Dickens, F., 39, 500

Dietrich, L. 8., 84

Dietz, C. G., 564

Di Giorgio, J., 657

Digonnet, L., 549

AUTHOR INDEX

Dikehhat PKs 3227,

Dimroth, K., 142, 144 (47), 145, 146 (47),
191, 206

Dinning, J. S., 36, 37 (169), 39, 84, 91,
96 (200)

Diognardi, N., 385

D’Torio, A., 85

Dimick, M. K., 654, 656 (40), 657 (40),
658, 659 (96)

Dimitru, C. C., 576

Dirstine, M. J., 313

Dithmar, K., 142, 192

Ditzler, L., 546, 636

Dixon, C. F., 425

Dixon, W. E., 199

Dju, M. Y., 559

Dobriner, K., 89, 562

Dodd, K., 433

Dodds, A., 448

Doisy, E. A., 390, 391 (20, 21), 392, 393
(21, 25), 394, 395 (25, 40), 396 (39),
397 (43), 398 (39), 399, 400, 403, 405,
409, 411 (33), 413, 417, 435, 441

Doisy, E. A., Jr., 344, 345 (86), 381

Dojmi, F., 562

Dolliver, M. A., 400, 405

Dols, M. J. L., 169

Domokos, J., 89

Donaldson, J., 272

Donatelli, L., 439

Donovan, P. B., 100

Doran, W., 335

Dorfman, A., 453, 521 (37), 598, 668, 672,
679

Dorfman, F., 670

Dornbush, A. C., 6, 50, 55, 94

Dotti, L. B., 372

Doudoroff, M., 602, 618

Douglas, A. 8., 4386

Dowling, A., 563

Doyon, M., 426

Drabkin, D. L., 490

Dragstedt, L. R., 86, 100 (212)

Draper, C. I., 97, 635

Drefus, L., 125

Drekter, L., 227

Dreyer, I[., 261

Drill, V. A., 75, 83 (116), 672, 686, 691

Drummond, J. C., 160, 170, 173

Dubin, H., 383

Dubin, I. N., 75, 516

AUTHOR INDEX

Dubnoff, J. W., 28, 29, 30, 31, 38, 85, 86,
103, 127

DaBboiss kK. P.; 15

Dubos, R. J., 313

DucetyG., 5; 10, 13. (22), 52, 57

Duckert, F., 434

Duckworth, J., 224, 383

Dudley, H. W., 7

Duesel, B. F., 477

Duff, G. L., 373

Dui. F., 419

Dumm, M. E., 652, 686, 691, 692, 693, 694

Dunean, C. W., 223, 224 (4), 225 (4), 227
(4), 282 (4), 256

Dunean, M., 556

Dunitz, J. D., 198

Dunlop, 8. G., 545

Dunne, H. W., 665, 666 (167)

DuPré, W. M., 442

Dutcher, J. D., 317

Dutcher, R. A., 97, 230

du Vigneaud, V., 11, 20, 21, 22 (63), 23
(68), 25 (63), 26 (64, 65, 68, 71, 72),
27 (73), 28, 30 (65, 66, 67, 67a), 32, 40
(66), 41, 48 (71), 45, 46 (1), 47 (1, 7),
48 (1, 7), 80, 83, 84, 89, 102

DuVigneaud, V., 516

Dybkowsky, W., 3

Dyckerhoff, H., 427, 432

Dyer, F. J., 221

Dyer, H. M., 20, 21, 89

Dyer, R. E:, 552

E

Eadie, G. S8., 36

Eagle, E., 54, 62

Eakin, R. E., 342, 494, 502, 622, 626, 627
(3), 668

Earle, A., 637

Earle, D. P., 75

Eastcott, E. V., 342, 351, 361, 366

Easterling, L., 564

Eastman, N. J., 422

Eek, J. C., 209

Kekardt, R. E., 82, 657

Eckstein, H. C., 20, 47, 71, 72, 76, 77, 79,
82 (148), 87

Eddy, W. H., 658, 662 (82)

Edgar, C. E., 522, 591, 656

Edgington, B. H., 568

Edlbacher, S., 28

Edmonds, M. C., 552

Edsallee a, 10

Edwards, D. E., 440

Edwards, H. M., 637

Edwards, J. E., 89

Egami, F., 9

Egana, E., 39

Ehmann, L., 134

Ehrensviard, G., 42

Eilert, M. L., 86

HKisner, H. J., 220

BEAR, ale

Eliot, M. M., 162, 238, 247, 260

Elkin, E., 341

Hllefsen, @., 332, 336 (22)

Ellefson, @., 345

Ellenberger, H. A., 545

Elley, H. W., 214

Ellinger, P., 32, 505, 515, 516 (147), 518
(2, 4, 32), 522, 523, 524 (26), 530, 533,
536, 537 (26), 556, 557, 559, 560 (75),
562 (58), 565 (75), 588, 584

Elliot, L., 488, 493 (42)

Elliot, 8., 488, 493 (42)

Elliot, W. E., 230

Elliott, M. C., 425

Ellis, F. A., 261

Ellis, L., 51

Ellis, N. R., 101, 297, 319, 568, 578 (15,
16), 664, 665, 666

Elmer, O., 274, 275 (25)

El-Sadr, M. M., 596, 656

Elvehjem, C. A., 34, 35 (150), 37, 39 (150),
58, 61 (2), 82, 84, 85, 93, 94 (304), 96,
98, 103, 160, 229, 280, 294, 315, 319,
342, 367, 369, 370, 403, 417, 423, 445,
453, 455 (10, 11), 459 (56), 480, 500,
508, 512, 518 (5, 6, 9, 12, 14, 17, 20, 24),
519 (46, 47, 48), 521 (40), 522, 523, 524,
529 (91), 530 (23), 531, 534, 543, 544
(15), 545 (15), 546, 548, 554, 555, 556
(20, 27, 33, 40), 564, 568, 569, 570,
578 (1, 2, 5, 7, 9, 20, 21, 23), 580, 581
(2), 582 (1, 54), 584 (7, 48), 585, 586
(1), 591, 595, 596 (20), 626, 630, 635,
637, 638, 639 (25, 29), 640 (25, 29), 641
(25, 29), 644 (2), 645, 647 (25), 650,
654, 655 (37), 656 (5, 35, 37), 657 (5,
35, 37), 658, 659 (92, 95, 97), 663, 664
(146) , 667, 668, 670, 672, 678, 682 (4, 5,
11, 26), 687, 691

706

Elwyn, D., 84

Emerson, G. A., 316, 654, 655, 656 (41),
657 (41), 680 (4), 682, 694

Emerson, H., 186 (258), 187

Emerson, W. J., 73

Ebisuzaki, K., 39, 87, 529 (91), 585

Emmanuelowa, A., 584

Kchaurren, A. P., 372

Emmel, V. M., 402

Emerson, O. H., 654

Emmett, A. D., 216, 396, 409, 411, 446

Ender, E., 222

Endicott, K. M., 74, 114

Engel, R. W., 4, 6, 53, 54, 57 (13), 58, 59
(1), 60 (1), 66, 67 (13), 71, 80, 81, 82
(162), 87, 89, 347, 370, 650, 660, 669,
670 (200)

Engelberg, R., 426

Engler, C., 476

English, M. A., 518 (26)

Ensminger, M. E., 101, 665, 666

Entenman, C., 56, 90, 99 (264), 100, 101

Eperjessy, A., 313

Eppright, M. A., 345, 355, 645

Epstein, N. N., 304

Epstein, R., 472, 488, 492 (46)

Ericksen, F., 427

Eriksen, T. S., 493

Erickson, B. N., 4 (16), 53

Erickson, L. L., 559

Ershoff, B. H., 368, 369, 384, 651, 687

Erkama, J., 439

Eser, 8., 575

Esh, G. C., 522, 580

Estes, J. E., Jr., 427

Etinger, R., 635, 642 (5), 648 (5)

Eugster, C. H., 44

Evans, E. A., Jr., 506

Evans, H. M., 73, 279, 292, 293, 299 (8),
318, 654, 656 (41), 657 (41), 682, 684

Evans, J. 8., 16, 603, 605 (25), 607 (25),
609 (25), 610 (25), 613 (25)

Evans, R. J., 4, 49, 97, 637

Evermann, B. W., 166 (168), 169 (168)

Everson, G., 685

Ewing, D. T., 216, 391, 392, 395 (31), 396
410, 414

Ewing, G. W., 183, 184 (251), 185 (251),
186 (251), 187 (251), 189 (251), 191
(251), 197 (251), 201 (251), 203 (251),

AUTHOR INDEX

Eylenburg, E., 426
Eyring, H., 495

Faber, H. K., 303

Fabre, R., 170

Fabriani, G., 564

Falk, K. G., 227

Fallot, P., 18, 56, 62

Fanconi, G., 420

Fantl, P., 155, 424

Fardig, O. B., 545, 629, 638, 640 (22) 641
(22)

Fassina, R. J., 314

Fattovich, G., 440

Faulkner, R. R., 651, 653, 657 (16, 23)

Favre, M., 577

Feaster, J. F., 545

Featherstone, W. P., 564

Feder, A., 374, 379, 380 (21)

Feigelson, P., 507, 512, 524, 554, 555, 556
(27, 40), 585

Feil, H., 561

Fein, H. D., 562

Feinberg, H., 101

Feinstein, L., 538

Felch, W. C., 372

Feldberg, W., 598

Felix, K., 441

Fellers, C. R., 545, 546

Fels, I. G., 611, 618 (56)

Felsovanyi, A. V., 555

Fenaroli, A., 12

Fenton, F., 546

Fenton, P. F., 385, 668

Ferguson, J. H., 401

Fernandez, O., 355

Fernholz, E., 142, 146, 191, 192, 394, 396,
399, 400, 405, 406, 425, 445 (9), 446

Ferraro, A., 420

Ferri, F., 575

Ferry, R. M., Jr., 98

Feruglio, F. 8., 576

Fidanza, A., 686

Fieger, E. A., 313

Field, H., Jr., 537, 557, 559, 560

Field, J. B., 428, 437, 444

Fields, M., 12

Fieser, L. F., 184, 191, 198, 394, 395, 396,
397 (37), 399, 400, 406, 411, 414, 446

Fieser, M., 134, 191, 198, 394, 397 (37)

AUTHOR INDEX

Fievet, J., 337

Figge, F. H. J., 656, 670

Fildes, P., 453, 521 (88)

Filer, L. J., 316

Fillerup, D. L., 297

Finch, C. A., 486

Findlay, G. M., 317

Findlay, G. R., 338

Findlay, L., 224

Findlay, W. P. K., 51

Finkelstein, J., 596 597 (26), 658 (107)

Finnerud, C. W., 303

Firket, J., 91

Firstbrook, J. B., 102

Fischer, E. H., 345

Fischer, H. O. L., 327, 331, 336 (11), 338
(10), 354, 356, 377, 501

Fischer, M. A., 83

Fischer, O., 475, 478 (109)

Fishback, C. F., 77

Fisher, G.S., 312

Fisher, N. F., 19, 100

Fischmann, C. F., 156, 168, 175, 181, 182
(241), 195 (241)

Fishman, W. H., 70, 71 (56)

Fitch... B., 297

Fleischmann, G., 522, 584, 559

Fletcher, D. E., 99, 126

Fletcher, H. G., Jr., 323, 324 (2), 329, 330,
331, 333, 335 (2), 336 (2), 338, 363

Bletcher, Jn Dy, 5,57

Fleury, P., 329, 334 (1), 335 (1), 336 (1),
337, 359, 360 (12), 363, 375, 376

Flinn, B. C., 555

Flint, E. R., 334

Florence, A., 7

Floyd, N., 606

Fliickiger, P., 436

Flynn, E. H., 326

Flynn, J. E., 404, 423, 425, 447, 448

Flynn, R. M., 600, 601, 604, 611 (16), 617.
620 (70)

Foa, N. L., 559

Foa, P. P.,. 67,559

Folch, J., 334, 335, 345 (51, 52), 346, 371,
380

Folkers, K., 596, 597 (26), 626, 658 (107)

Follis, R. H., Jr., 75, 101, 235, 236, 260,
298, 299 (28), 380, 568, 570, 580 (51),
581, 664, 665, 666 (163, 165), 670, 677
(218, 214)

707

Fomin, S., 54

Fontaine, T. D., 339

Forbes, J. C., 80, 82, 370

Ford, J. E., 629 (15), 658

Foreman, W. W., 476

Fosdick, L. 8., 441, 448

Foster, C., 224, 668, 670

Foster, G. L., 525

Foster, J. W., 317, 645

Foster, R. H. K., 397, 400, 411, 442, 443
(92), 446

Fothergill, P., 44

Fountaine, F. C., 297

Fouts, P. J., 98, 453, 518 (1), 585, 663

Fowden, L., 548

Fox, S. W., 537

Foy, J. R., 297, 315, 667

Fradkin, R., 429

Fraenkel, G., 291, 298, 314, 315 (34), 518
(2)

Franke, W., 278

Frankel, G., 533

Frankel, J., 271

Frankenburger, W., 199

Franklin, M., 553

Frankston, J. E., 532

Franzl, R. E., 331, 333, 348

Fraser, H. F., 578 (10)

Hraux, J, Ld

Frazer, A. C., 90

Frazer, H. F., 518 (83)

Frazier, E. I., 535, 543, 548, 584

Fred, E. B., 156

Frediani, H. A., 628

Free, A. H., 654

Free, 8. M., 546

Freeman, S., 98, 264

French, C. E., 656

Frerichs, F. T., 335

Freudenberg, K., 331

Freudenthal, P., 684

Frey, C. N., 342, 349 (3), 362, 418, 426,
550, 633

Frey, K. J., 635

Freyberg, R. H., 264

Frick, P., 419

Fridericia, L. 8S. 684

Fried, R., 578 (35)

Friedel, G., 185

Friedemann, T. E., 535, 548, 584

Friedkin, M., 497

708 AUTHOR INDEX

Friedlander, H. D., 90, 99 (264)

Friedman, H. L., 477

Friedmann, H., 230

Fries, B. A., 56

Fritz, J. C., 207

Fries, N., 342, 366

Fritzsche, H., 488, 492 (48)

Fromageot, C., 344

Fromherz, K., 443

Frommel, E., 577

Frost, D. V., 466, 628, 654, 655, 656 (39),
657 (39)

Fry, E. M., 394, 397 (37), 399, 400, 414

Fuchs, H., 92, 127

Fujita, A., 216

Jiolkaym, 40, silts}

Fuld, M., 620 (82)

Fuller, R. C., 348, 344 (21), 367

Fulton, R. P., 650, 653 (3), 657 (3)

Funk, C., 452

Furguson, G. F., 20

Furman, C., 532

G

Gabor, G., 575
Gabrielson, EH. K., 415
Gaddum, J. H., 8, 57
Gaebler, O. H., 459 (58), 516, 517 (27), 518
(27, 28), 549
Gaede, J., 200, 201 (314)
Gaglio, M., 440
Gaines, J. G., 651
Gajdos-Torok, M., 74
Gajjar, I. M., 312
Gakenheimer, W. C., 55
Gallois, 375, 376
Gammelgaard, A., 441
Garber, M. J., 532
Gardner, K. E., 297
Garbo, P. W. 476
Garn, S. M., 373
Gast Je He, 79
Gates, M. D., Jr., 394, 399
Gaubert, P., 185
Gaunt, R., 651, 693
Gauthier, B., 148
Gautier, J. A., 465
Gavin, G., 80, 81 (160), 82, 86, 347, 370,
384
Gebauer-Fuelnegg, E., 9
Gedroye, M., 382

Gee, M., 12

Geiger, A., 390, 391 (18), 392 (18), 398 (18)

Geissendorfer, H., 4, 8 (10)

Geissman, T. A., 380, 381 (23)

Georg, L. K., 533

George, R.S., 88

Gephart, M. C., 372

Gérard, E., 153, 156

Gerard, Re W., 312

Gerarde, H. W., 444

Gerebtzoff, M. A., 345

Gerhardt, C., 375

Gerischer, W., 511

Germany ds diy 532

Gershberg, H., 652, 672, 692, 693 (81)

Gertler, M. M., 373

Getty, R., 685

Geyer, R. P., 436

Ghirlanda, M., 440

Ghormley, R. K., 420

Ghosh, N. C., 575

Gibbens, J., 552, 558 (19), 560 (19), 564
(19), 576, 587

Gibbons, A. J., 604, 607 (31)

Gibbs; Jz-A: S12

Gibson, G., 297

Gibson, R. B., 427

Giles, M. J., 95

Gillam, A. E., 152, 155 (95)

Gillet, J., 317

Gillis, M. B., 39, 95, 96, 636, 660, 670, 682
(15, 18, 19)

Gilman, H., 477

Gillman, J., 104, 571

Gillman, T., 99, 104, 571

Ginoulhiac, E., 577

Giono, H., 574, 577

Ginsberg, J. E., 304

Girard, A., 334

Girvin, M. D., 272

Giunchi, G., 686

Glascock, R., 12

Glasscock, R. 8., 637

Glavind, J., 384, 390, 391 (18), 392 (18),
396, 398 (18), 400, 406, 408, 415, 417,
421, 422, 423 (11), 424, 428, 438, 439,
441, 445, 447

Gleim, W., 485

Glenwood, D. A., 685

Gley, P., 431

Glick, D., 4, 6, 53, 54, 58, 62 (3), 304

a i i

AUTHOR INDEX

Glock, G. E., 359, 360, 500

Glover, J., 159

Glusman, M., 675, 676, 677, 678

Glynn, L. E., 75, 114

Goddard, J. C., 552

Godden, W., 224

Goemine, J., 516

Goepp, R. M., Jr., 3238, 363

Goese, M. A., 475

Goldberg, M. W., 44, 134

Goldberger, J., 541 (8, 4), 542, 548, 552,
553, 560, 563, 564, 565, 566, 567, 587,
591

Goldblatt, H., 66, 67 (12), 75, 108, 118
(428), 114, 163, 175, 181, 650, 653 (3),
657 (3)

Goldinger, J. M., 17

Goldman, D. 8., 619

Goldman, L., 671

Gol’dman, M. M., 460

Goldsmith, G. A., 521 (56), 524, 582, 553,
558, 559, 560 (79, 80), 564, 576, 587

Goldstein, A., 343

Goldstein, M. R., 125, 372

Goldsworthy, P. D., 42

Goldwater, W. H., 314

Goldzieher, J. W., 574

Gompertz, M. L., 374, 379, 380 (21)

Gonnard, 175

Goodpasture, W. C., 100

Goodwin, D., 423

Goodyear, G. H., 591, 592, 593 (3), 630
678

Gopalan, C., 675, 676, 677

Gordon, A., 472

Gordon, E., 427

Gordon, E. S8., 554, 556 (33), 688

Gordon, H., 306

Gorkow, R., 475

Gortner, W. A., 544

Goryachenkova, E. V., 529 (93)

Goss, H., 416, 627

Gotgiu, G., 576

Gottsegen, G., 441

Gétzsche, P., 442

Gough, H. W., 546

Gould, D. H., 148

Gounelle, H., 550, 554, 555 (31)

Govier, W. M., 16, 507, 603, 604, 605 (25),
607 (25, 31), 609 (25), 610 (25), 613
(25)

709

Grab, W., 4, 13, 146, 205

Graef, I., 655, 669, 687

Graham, W. R., Jr., 389, 421

Grail G. Fe 30s“, 103

Granados, H., 285, 286, 316, 441

Granger, R., 15

Grant, G. A., 153, 312

Grant, H. M., 518 (16)

Grant, R., 20, 76

Grau, C. R., 28, 94, 531

Graubner, W., 10, 57

Gray, E. L., 164, 169, 170, 172 (169), 205,
206 (169)

Gray, M. L., 665

Grayzel, D. M., 229

Greaves, J. D., 404, 423, 446, 447, 448

Greaves, J. E., 354

Green, C., 56

Green, D. E., 42, 458, 492, 497, 502, 603,
609 (22), 619, 621, 622 (87)

Green, J., 75, 217

Green, M. N., 49, 680

Greenbaum, L., 18, 56, 62

Greenberg, D. M.., 27, 41, 42, 228

Greenberg, S. M., 274, 280 (4), 285, 290,
318

Greenwald, I., 378, 383

Gregg, H. S., 555

Gregory, J. D., 16, 601, 603 (17), 604, 605
(25), 607 (17, 25) , 608 (17), 609 (17, 25,
45), 610 (17, 25), 613 (25)

Gresham, W. F., 11, 14

Greslin, J. G., 680, 681 (1), 692

Greville, G. D., 619

Griese, W., 453, 458 (6, 7), 470 (6), 481,
488 (7), 500 (7), 511 (7)

Griess, P., 7, 10 (28), 13 (28)

Grieves, C. J., 230

Griffith, W. H., 27, 29, 30, 46, 47 (4), 57,
63, 65, 67 (2, 4), 68, 69 (40), 71 (8), 73
@)nu%6; 7% (21) 978 Gey 2ie 1387 ss).
79, 82 (4), 87 (152)

Grimmer, H., 442

Grisolia, S., 493, 506 (77)

Griswold, R. M., 545, 546

Groer, F., 442

Groody, M. E., 659, 660

Groody, T. C., 659, 660

Groothuis, M., 77

Groschke, A. C., 522, 569, 578 (27), 581
(27), 584 (58), 637

710

Grosheintz, J., 327, 377

Gross, E.8., 562

Gross, J. B., 126

Gross, P., 316, 372

Grossman, A. M., 422, 433 (21)

Grossman, W. I., 457 459 (41), 472, 486,
492 (34), 537

Groupé, V., 317

Gruber, C. M., 92

Grulee, C. G., 305

Grundmann, W., 193

Grundy, W. E., 559

Grupper, C., 303, 304, 305

Gschaider, B., 158, 186 (134), 191 (134),
204 (134)

Gualandi, G., 66

Gubner, R., 442

Gudjonnsson, S., 684

Guéguen, J., 427

Guehring, R. R., 652, 663

Guérillot-Vinet, A., 209, 439

Guerrant, N. B., 545, 629, 638, 640 (22),
641 (22)

Guerritore, A., 86

Guerry, D., III, 421, 433 (15)

Guest, G. M., 482

Guggenheim, K., 91, 652, 686

Guggenheim, M., 92, 127, 293, 294, 299,
300 (9)

Guirard, B. M., 318, 600, 601, 605 (12, 13),
607 (13), 609 (12, 13)

Gulewitsch, W., 4,5 (15), 7 (15), 13 (15)

Gullberg, M. E., 525, 526 (53, 54)

Gullickson, T. W., 297

Gunness, M., 645

Gunsalus, I. C., 495, 617, 620 (69), 673

Gunther, E. R., 160, 170

Giinther, G., 456, 458 (35), 493, 503 (76),
511 (80)

Giintzel, B., 204 (321), 205

Gurchot, C., 401, 438

Gurin, S., 624, 658, 662 (82)

Gustafson, F. G., 533, 546

Gutcho, S., 512

Guthneck, B. T., 637

Gutman, A. B., 235

Gutman, H. R., 495

Gyorgy, P., 52,55 (8), 66, G7 (12), 73, 75,
82, 83, 101, 108, 113 (428), 114, 215, 218
(1), 220 (1), 312, 318 (17), 359, 364

AUTHOR INDEX

(5), 366, 535, 650, 653, 654, 655 (32),
656 (3, 31, 32), 657 (3, 31, 32), 667

H

Haagen-Smit, A. J., 585

Haanes, M. L., 101

Haas, E., 286, 511

Habif, D. V., 436

Hagemann, G., 195, 196 (290), 197, 198,
204 (290), 215

Hajdu, I., 314

Halaban, A., 440

Halbrook, E. R., 389, 421

Halden, W., 157, 183, 190, 191 (278)

Hale, O. M., 83, 87, 621 (96)

Hall, C. A., 75, 83 (116)

Hall, G. D., 88

Hall, G. E., 389, 421

Hall, H., 166 (167), 168, 169, 172 (180)
205 (180), 206 (180)

Hall, W. K., 670

Hall, We Tis<535

Halliburton, W. D., 128, 124

Halliday, E. G., 545, 546

Halliday, N., 82

Hallman, E. T., 228, 224 (4), 225 (4), 227
(4), 232 (4), 256

Hallman, L. F.; 91

Halloran, H. R., 207

Halpern, B. N., 576

Halpin, J. G., 225, 227 (19), 408, 445

Halpin, J.1., 207

Halvey, 8., 686

Haman, R. W., 154, 155, 169, 174, 176
(202)

Hamilton, J. W., 103, 370

Hamilton, T. S., 127, 129, 376, 515, 666,
684

Hamlin, M., 663, 664 (147)

Hammond, M. M., 518 (26)

Handler, P., 32, 34, 38, 41, 52, 55 (2), 66,
71, 73, 75, 78, 80 (14), 81 (166), 82 (68,
146), 83, 88 (91), 103, 370, 380, 384,
386, 494, 495, 505, 507, 516, 518 (138,
26a), 532, 535, 540, 548, 546 (12), 556,
564, 565 (119), 567, 573, 584

Handschumacher, R. E., 600, 603, 609
(22)

Hankes, L. V., 518 (9, 12), 519, 522, 523,
524, 529, 5380 (23), 578 (2), 581 (2)

Hankins, O. G., 297, 319

AUTHOR INDEX

Hanning, F., 222

Hansen, A. E., 270, 295, 300, 301, 302, 303,
304, 306 (380), 307 (80, 31), 308 (382)
309 (33), 310, 314

Hansen, I. G., 339, 382, 383 (5)

Hanson, H. H., 428

Hanson, 8. W. F., 69

Haque, M. E., 686

Harden, A., 481, 482

Hardin, J. O., 39

Hardwick, 8. W., 583

Hardwicke, 8. H., 448

Hardy, Z., 188, 191

Harispe, J. V., 577

Harispe, M., 577

Harkness, D. M., 555

Harms, H. P., 100

Harman, J., 20

Harnapp, G. O., 162

Harper, A. E., 85

Harper, C. J., 511

Harris, H. F., 570

Harris, L. E., 224, 256

Harris, L. J., 199, 521 (4, 5), 534, 548

Harris, M. E., 638, 648 (21b)

Harris, P: 1.., 285, 316

Harris, P. N., 89, 429

Harris, R.S., 175, 176 (203), 177, 424, 577

Harris, S. A., 596, 597 (26), 626, 658 (107)

Harrison, D. C., 160

Harrison, G. F., 629 (15), 658, 675

Harrow, G., 7, 10 (28), 13 (28)

Hart, E. B., 93, 94 (304), 98, 160, 222, 223,
225, 226 (20), 227 (8, 19, 20), 228, 229,
230 (3, 20, 23), 280, 294, 315, 319, 342,
369, 543, 569, 570, 578 (21), 580, 595,
637, 654, 655, 658, 659

Harting, J., 621 (78)

Hartmann, I., 686

Hartroft, W. S., 66, 67, 68, 75 (86), 78
(122), 88 (9), 104, 108, 109 (433), 110
(484), 111, 113 (429, 484), 114 (433,
434)

Hartwell, W. V., 665, 682 (25)

Hartzler, E., 546

Harvey, H. I., 567

Harvey, W. P., 436

Hary, M. C., 441

Haslewood, G. A. D., 142, 145, 146 (65),
147, 159, 173

Haskins, F. A., 526

711

Hassel, O., 331, 332, 336 (22), 345

Hasselbrock, W. B., 423

Hastings, A. B., 250

Hastings, E., 318, 315 (33)

Hathaway, M. L., 208, 261

Hatton, E. H., 443

Hauge, S. M., 95, 102, 578 (24, 25), 661

Haurowitz, F., 312

Hauschildt, J., 667

Hausmann, E., 142

Hausrath, M. E., 546

Haussler, E. P., 190

Hawk, E. A., 85, 560, 568

Hawker, R. W., 572

Hawkins, R. D., 91

Hawkins, W. B., 424, 433, 651

lalehy, di, JD). 2eP

Haydak, M. H., 416

Haynes, L. J., 485, 486 (82)

Hazan, 8S. J., 87

Hazura, K., 474

Heath, M. K., 570

Hecht, L., 18, 56, 62

Heesch, O., 4

Heggeness, F. W., 379, 383, 663

Heggie, R., 147, 158 (81), 216

Hegsted, D. M., 67, 76, 87, 93, 94 (304),
97, 342, 369, 524, 529 (46), 556, 570,
578 (33), 585, 610, 629, 630 (8), 657,
658, 659, 662, 682 (10)

Heidelberger, C., 525, 526

Heidebrecht, A. A., 638, 648 (21c)

Heiduschka, A., 153, 190

Heilbron, I. M., 134, 1388, 147, 152, 155
(5), 157, 175; 177, 190; 1915198) 199
(218), 208, 472

Heimberg, M., 585

Heiwinkel, H., 456, 484 (19), 485, 507

Heller, V. G., 229

Hellman, L. M., 422

Hellstrém, H., 143, 186 (59), 487, 491, 493

Helm N., 365

Helman, F. D., 133, 163 (8)

Helmer, O. M., 458, 518 (1), 663

Hembest, H. B., 148

Henderson, L. M., 459 (60), 518 (10, 20),
519 (47, 55), 521, 522, 524, 526, 527
(66), 528, 529 (90), 580 (23), 533, 548,
548, 578 (2), 580, 581 (2), 582 (54), 654,
655 (37), 656 (35, 37), 657 (35, 37),
682 (4, 5)

2 AUTHOR INDEX

Henle, W., 224

Heppel, L. A., 74

Herb, S. F., 271, 272, 288

Hergert, W. D., 166 (167), 168

Herman, 8S. C., 155

Herrington, B. L., 635, 6388 (4a), 648 (4a),
687

Herris, M. E., 565

Herrmann, G. R., 97

Hershberg, E. B., 414

Hershey, J. M., 19, 20, 46

Herzig, J., 475

Hershman, B., 8

Herwick, R. P., 671

Hess, A. F., 183, 188, 139; 148, 162, 163,
167, 172, 175, 176, 178 (24), 204, 241,
247, 248

Hess, W., 38, 39 (176), 277

Heubner, C. F., 535

Heuser, G. F., 96, 591, 636, 642, 648 (32),
645 (32), 658, 659 (84), 660, 670, 682
(15, 16, 18, 19), 684, 694

Hewett, C. L., 134

Hewitt, J. A., 378

Hewston, E. M., 160, 161 (142)

Heyl, F. W., 186 (258), 187

Hibbs, J. W., 223

Hickman, K. C. D., 164, 169, 170, 172
(169), 205, 206 (169), 286

Hicks, R. A., 261

Higginbottom, C., 44, 51

Higgins, G. M., 92

Higgins, H., 603, 609 (22), 629, 642, 643
(31), 645 (31)

Highman, B., 74

Hightower, D. P., 680 (2), 681

Hilditch, T. P., 292, 305, 319

Hilferty, D. J., 421, 423

JBIIUL, ID), (Cr, AO/, (al

ESE Ge 95

Hill, H. N., 519 (55), 528

Hill, Kenneth R., 104

Hal Or J, 223% 23082)

Hills, G. M., 598, 679

Holz, H., 617

Himsworth, H. P., 75, 114

Hind, H. G., 596, 656

Hindemith, H., 314

Hirsechbrunn, M., 3

Hirst, E. L., 361

Hitchings, G. H., 656

Hjorth, E., 415

Hoag, E. H., 629 (12)

Hoagland, C. L., 505, 512 (152), 556, 604

Hochberg, M., 457, 536

Hock, C. W., 670

Hodge, H. C., 125, 126, 443

Hodgskiss, H. W., 665

Hodson, A. Z., 56, 58, 297, 591, 648

Hoefer, J. A., 665, 666 (170), 682 (23), 684,
686

Hofer, M. J., 541, 646, 690

Hoff-Jgrgensen, E., 605, 613 (88)

Hoffert, D., 157

Hoffman, G. R., 423, 425, 483

Hoffman, P. B., 440

Hofmeister, F., 44

Hogan, A. G., 98, 103, 283, 348, 370, 569,
570, 578 (32), 662, 682 (17)

Hogberg, B., 455, 456 (238), 458 (28), 484
(19), 489, 555

Hoglan, F. A., 340, 341, 358

Hogness, D.8., 453, 518, 514 (195)

Hogness, J. R., 505, 556

Hogness T. R., 189, 201 (265), 286, 509, 511

Hojorato, R., 73

Holaday, D., 591, 592, 593 (8), 678

Holcomb, W. F., 394, 395 (40), 397 (43)

Holden, M., 536

Holliday, W. M., 517 (27), 518 (27), 549

Holman, R. T., 270, 274, 275 (25), 277, 278,
279, 280, 281, 282 (10), 284 (10), 286,
287, 288, 289, 291, 312, 314

Holman, W. I. M., 521, 524

Holmes, A. D., 222, 648

Holmes, 8. G., 295, 300 (14), 301, 309 (33)

Holst, W. F., 389, 421

Holt, L. E., Jr., 86, 301, 518 (8), 522, 524,
530 (39), 557, 559, 584

Holtz, F., 222

Holzen, H., 399

Homeyer, G. P., 666, 685, 686 (39a)

Honeywell, E. M., 139, 167, 172 (171), 179,
180 (238), 181, 182 (239), 183, 184
(249), 185, 186 (249), 187 (249), 188,
199 (238, 239), 207 (33)

Honorato, C. R., 88, 437

Hoogewerff, 8., 475

Hopff, H., 10, 11, 13 (65), 14

Hopkins, F. G., 376

AUTHOR INDEX

Horecker, B. L., 286, 460, 490, 501, 503,
505, 512

Horn, Z., 441

Horowitz, N. H., 9, 49, 50 (8, 4), 55

Horning, M. G., 72, 77

Hortzler, E. R., 160, 161 (142)

Horvath, A. A., 389

Horvath, J., 147, 158 (79)

Horwitt, M. K., 312

Hésli, H., 134

Hou, H. C., 163, 164, 559

Houchin, O. B., 668

Hough, L., 361

Hough, V. H., 98

Houlahan, M. B., 49, 50 (4), 55

Hove, E. L., 39, 285, 315, 316

Howard, B., 432

Floward, J. E., 236

Howe, G. W., 88

Howland, J., 225, 238, 252

Howton, R., 272

Hoyle, J. C., 199

Hsu, B., 84, 85 (194)

Huang, Y. T., 134

Hubbard, L. H., 680 (2), 681

Hubbard, R., 495, 563

Huber, C., 473, 474 (81)

Huber, F. C., 465

Huber, W., 183, 184 (251), 185, 186 (251),
187, 189, 191 (251), 196, 197 (297), 201
(251), 202, 203 (251, 297), 204 (297),
574

Hudson, C. 8., 325, 626

Hudson, F. P., 422

Huebner, C. F., 404, 418, 427, 428, 432
(39), 456

Huennekens, F. M., 492

Hueper, W. C., 437

Huff, J. W., 456, 457, 459 (37, 42), 476, 478,
515, 516, 518 (16, 23), 519 (44, 50),
522, 523, 524, 526, 529 (71), 530 (31),
536, 537 (382), 557, 580, 581, 585

Huffman, C. F., 223, 224 (4), 225 (4), 227
(4), 232 (4), 256, 297

Hughes, D. E., 507, 551, 672, 680

Hughes, E. B., 461, 462, 463, 464 (64)

Hughes, E. H., 567, 664, 665, 682 (24)

Hugonet, J. J., 55

Huidobro, F., 577

Huldschinsky, K., 162

Hull, W., 549

Hume, E. M., 279, 314, 316, 318

Humphrey, G. C., 222, 228

Humphreys, 8., 101, 568, 580 (51), 581,
664, 665, 666 (163), 670, 677 (213)

Hundley, J. M., 457, 459 (39) , 524, 525, 530,
534, 554, 555 (28), 559 (26, 28), 560,
564, 568, 578 (3), 580, 581 (47), 583,
654, 685

Hiinecke, H., 461

Hunner, M., 545

Hunt, B., 677

Hunt, C. H., 546, 636

Hunt, D. J., 541 (6, 7), 567

Hunt, R., 8, 124, 127, 571, 574 (4)

Hunter, D., 306

Hunter, G. L., 668, 670

Huntsman, M. E., 19, 20, 25 (41), 40 (41),
46, 48, 68, 76 (39), 104, 347

Hurley, L. 8., 652

Hurley, P. D., 91

Hurn, M., 428

Hurn, M. M., 433

Hurt, W. W., 528, 529 (84), 585

Hussey, C. V., 413, 434

Huszak, I., 312

Hutt, H. H., 358, 359

I

Ihde, A. J., 316, 638, 640 (30a), 648 (30a),
687

Ikawa, M., 427

Tland, C. N., 417, 439

Imboden, M., 166 (167), 167, 168, 169, 172,
205 (180), 206 (180)

Ing, R. B., 654, 685

Ingle, D. J., 426

Inhoffen, H. H., 133, 191

Jonescu-Stoian, P., 576

Inukai, F., 8

Tob, L. V., 304

Irish, U. D., 429

Irreverre, F., 414

Irvine, (Ga WiraJieoo9

Irwin, J. O., 629

Isaacs, B., 425

Isbell, E. R., 385, 545, 549

Isbell, H. S. 297

Iselin, B. M., 352

Ishii, T., 472, 488, 492 (46, 47)

714

Itabashi, M., 9

Itoh, F., 528

Itschner, K. F., 288, 343

Ittner, N. R., 665, 682 (24)

Iverson, P., 252

Ives, M., 638, 639 (29), 640 (27, 29), 641
(27, 29)

Ivy, A. C., 425

Iwao, J., 526

Izquierdo, G., 355

J

Jackson, C. M., 298

Jackson, D., 260

Jackson, R. W., 20

Jacobi, H. P., 4 (17), 6, 38, 58, 54, 67, 71
(27), 89

Jacobs, 8. E., 348

Jacobsen, E., 427

Jacobson, G., 284, 285 (18)

Jacox, R. F., 434

Jaffe, H. L., 226

Jaffe, W., 472, 488, 492 (46)

Jahns, E., 476

James, D. F., 436

James, E. M., 535

James, M. F., 102

Jamieson, G.S., 271, 292

Jandorf, B. J., 495

Janes, R. G., 575

Jans, L. M., 546

Jansen, B. C. P., 335

Jansen, K. F., 428, 429

Jaques, J. E., 672

Jaques, L. B., 427, 429, 431, 482, 487, 438

Jaretzki, A., 436

Jeans, P. C., 258

Jelinek, V., 572

Jellinek, H. H. G., 461, 462 (64), 463 (64),
464 (64), 470, 471, 472

Jenkins, G. N., 629, 682 (7)

Jenkins, R. G. C., 175, 178, 182 (225), 195

Jensen, D., 80

Jensenius, H., 566

Jephcott, H., 229

Jerlov, N. G., 171

Jervis, G. A., 67

Jetter, N.8., 507

Jeu, S., 564

Jimenez Diaz, C., 130

Johansen, G., 684

AUTHOR INDEX

Johansson, H. R., 385

John, J. L. St., 4

John, 8., 374

Johns, G. A., 584

Johnson, B. C., 55, 94, 102, 127, 129, 376,
456, 515, 521, 522, 524, 540, 580, 666

Johnson, E., 548

Johnson, E. R. F., 171

Johnson, F. H., 495

Johnson, G. A., 522, 541, 559, 646

Johnson, N. G., 160

Johnson, R. E., 18, 559

Johnson, W. C., 254

Johnston, L. B., 261

Johnston, P. M., 568

Johnston, R. L., 665

Johnston, R. R., 682 (23)

Joliliffe, N., 562

Joly, M., 359, 360 (12)

Jones, EH. C., 319

Jones, E. R. H., 148

Jones, E. 8., 667

Jones, J. H., 223, 224, 225, 226 (20), 227
(20), 229, 230 (20, 23), 668, 670

Jones, J. K. N., 361

Jones, K. K., 552

Jones, M. E., 600, 617, 620 (70)

Jones, L. W., 354

Jones, R. N., 198, 394, 397 (37)

Jones, 8. N., 486, 487, 442

Joneson, O. R., 92

Jonsson, S., 27, 48 (94)

Jordan, D. S., 166 (168), 169

Jordan, E. F., Jr., 312

Jorquera, R., 372

Josselyn, D., 584

Josserand, M. A., 442

Jouannet, A. L., 335

Joyce, F. T., 407, 408 (23), 445 (10), 446

Jukes, T. H., 6, 40, 46, 47 (2, 3, 11), 48
(1, 22), 50, 55, 66, 93, 94 (802), 95
(808), 97, 348, 453, 518 (1), 570, 578 |
(31), 591, 595, 596, 610, 611, 629 (14),
630, 632, 6388, 639 (24), 640 (24), 641 |
(24) , 642 (24), 643 (24), 650, 656 (2), .
657 (2), 658, 659 (93, 98) , 661, 677, 678,

682 (12, 21), 684, 687, 690 :

KOPF <x

Junqueira, P. B., 524, 529, 530, 531 (44),
585, 586

AUTHOR INDEX

Jiirgens, R., 288, 298, 294, 299, 300 (9),
673
Justice, D. H., 385, 668

K

Kabler, C. A., 557

Kahane, E., 5, 7,8 (55), 9, 13 (22), 57, 58,
71, 92

Kahane, M., 7

Kahlenberg, O. J., 657

Kahnke, J., 417, 4388

Kahnt, F., 472, 488, 492 (46, 47)

Kalen, J., 637

Kallio, R. E., 519 (48), 524, 526, 531 (41)

Kallés, P., 441

Kaltnitsky, G., 217

Kamm, E. D., 138, 175, 177, 199 (213)

Kamm, O., 391, 392, 395 (31), 396 (81),
410, 411, 414, 446

Kapfhammer, J., 4 (18), 18, 53

Kaplan, N. O., 15, 16 (6), 489, 498, 494,
507, 511, 538, 598, 599, 601, 603, 605
(12, 13), 607 (13), 609 (12, 13, 37) 610
(14), 611 (14), 614 (10, 14), 615, 616
(10), 618 (10), 624 (59), 630, 631, 632,
633, 649, 662, 672, 679

Karasek, F., 572

Kareff, N., 426

Kark, R., 421, 427, 562, 674

Karrer, P., 44, 279, 390, 391 (18), 392 (18),
396, 398 (18), 414, 455, 459 (57), 472
(57), 477 (57), 485, 486, 488, 491, 492
(46, 47, 48, 49, 50, 51)

Karrer, W., 390, 391 (18), 392 (18), 398
(18)

Kass, J. P., 274, 279, 318

Katchman, B. J., 507

Kates, M., 12

Kato, M., 536

Katzenstein, H. H., 574

Kaufman, N., 76

Kaufman, S., 621, 622 (88)

Kaufmann, O., 142

Kay, H. D., 226

Kay, J. H., 381

Kay, W. W., 522, 583

Kaye, A., 648

Kearney, B., 481

Keenan, G. L., 7, 461, 462, 467

Keenan, J. A., 229, 595, 637, 658

Keener, H. A., 584

715

Keeping, F. E., 52, 92

Keese, C., 3

Keevil, N. B., 67, 90 (28)

Keil, H. E., 654

Keimatsu, 8., 476

Keith, C. K., 36, 37 (169), 39, 84, 96 (200)

Keith, T. B., 567

Kelemen, E., 89

Keller, D. C., 316, 319

Keller, E. B., 22, 23 (68), 26 (68), 32, 80,
103, 280, 283 (4), 516, 570, 668

Keller, H., 455

Kellner, A., 373

Kelley, B., 38, 39 (177), 67, 91

Kelley, O. R., 421, 483 (15)

Kellog, W. L., 532

Kelly. (Ja Ds; 167

Kemmerer, K. 8., 20, 21 (57)

Kemp, P.8., 561

Kempster, H. L., 93, 569

Kendall, A. I., 9

Kendall, F. E., 99

Kennard, D. C., 96

Kennedy, E., 655

Kennedy, E. P., 286, 622 (95)

Kennedy, G. H., 206, 218, 220 (23)

Kensler, C. J., 35, 71

Keppel, D. M., 21, 22 (63), 25 (63), 46,
47 (7), 48 (7)

Keresztesey, J. C., 596, 597 (26), 658 (107),
662 (82)

Kerlan, I., 671

Kern, R., 199

Kesler, R. L., 303

Kesten, B., 372

Kesten, H. D., 69

Keston, A. §., 49

Ketron, K. C., 524, 530 (89)

Kharasch, M. 8., 206

Kidder, G. W., 532, 533

Kidder, L. E., 545

Kidder, R. R., 532, 5383 (110)

Kielley, W. W., 15

Kies, M. W., 21

Kiesel, A., 7

Kik, M. C., 227

Kimler, A., 417, 439

Kind, C. A., 155

King, E. J., 8

King, G.S. D., 461, 462, 467, 470

King, H., 159

716 AUTHOR INDEX

King, T. E., 604, 611, 613 (56), 621 (96),
629, 632, 642, 643 (31)

King, W. A., 102

King, W. I., 552

Kingsley, G. V., 216

Kinnersley, H. W., 661

Kinney, E. M., 230

Kinney, J. M., 27, 84

Kinney, T. D., 76

Kinosita, 676

Kinsey, R. E., 437

Kirby-Smith, H., 261

Kirk, C. M., 301

Kirk, P. L., 549

Kirkwood, S8., 342, 343 (9), 344 (19), 368

Klatzkin, C., 535

Klein, G., 54

Klein, H. P., 621 (97, 98), 622 (98), 624
(98)

Klein, J. R., 34, 41, 495, 505, 507, 554, 555
(22), 556

Klenk, E., 334, 345, 346 (48)

Kleppel, B., 67

Klevens, H. B., 275

Kletzien, S. W. F., 160

Klien, G., 10

Kline, O. L., 229, 595, 637, 658

Klingsberg, A., 329

Klose, A. A., 28, 46, 391, 393, 394, 395, 396
(29, 34), 497 (35, 45), 398, 399, 400,
404, 405, 406 (14), 407, 408 (9), 409,
411, 413, 414, 423, 425, 435, 441, 445, 446

Kluyver, A. J., 326, 337, 347

Knandel, H. C., 97

Knapp, A. W., 160

Knight, B. C. J. G., 453, 521 (35, 41), 588,
550, 678

Knight, H. B., 312

Knott, E. M., 308, 304 (40)

Knowles, J. L., 83

Knox, W. E., 457, 459 (41), 472, 486, 492
(84), 527, 529 (94), 537

Knudson, A., 174, 175, 176 (201), 177, 178

Ko, L., 189

Koch, E. M., 154, 208

Koch, F. C., 154, 208

Koch, M. B., 555

Koch-Weser, D., 128

Kodicek, E., 313, 456, 486, 534, 535 (20),
536, 548, 544, 548, 584

Koebner, A., 134

Kogl, F., 342, 366

Koehn, C. J., Jr., 453, 455 (11), 591, 658,
659 (92)

Koelle, G. B., 17

Koenig, H., 279

Kohn, H. [., 505, 518 (18), 522, 554, 555,
556

Kokata, K., 476

Koller, F., 419, 434, 436, 443

Kon, G. A. R., 134

Kon, 8S. K., 176, 209, 534, 567, 578 (26),
586, 629 (15), 658, 659 (101), 660 (101)

Konig, W., 472, 477 (76), 534, 536

Konstam, C., 306

Korenchevsky, V., 230

Korey, 8. R., 17

Korkes, 8., 493, 495, 617, 620 (69, 81), 673

Kornberg, A., 19, 428, 458, 460, 488, 489,
490, 4938, 494 (55), 498 (90), 499, 504
(121), 506 (48, 77, 115), 507 (55, 90),
510, 511 (114), 608, 604 (29), 619, 621
(93), 653, 657 (22)

Korner, F., 11, 14

Koser, J., 200, 201 (314)

Koser, S. A., 453, 521 (37), 540, 583, 598,
672, 679

Koski, R. E., 519 (55), 528

Kostalik, M., 422

Kosterlitz, H. W., 383

Kotake, Y., 526

Kraatz, C. P., 92

Ginter ding dies Ao!

Krahl, M. E., 89

Krahnke, H., 688

Kramer, B., 217, 225, 252, 261

Krdémli, A., 147, 158 (79)

Krampitz, L. O., 620 (85)

Kratzer, F. H., 97, 317, 531, 661, 682 (14,
20)

Kraus, J., 28

Krause, M., 561

Krause, M. E., 591, 596, 656

Krause, M. F., 441

Krauss, W. E., 223

Kraybill, H. R., 287

Krebs, H. A., 500

Krebs, U., 421, 433

Kreevson, C. F., 477

Krehl, W. A., 356, 364 (8), 518 (5, 11, 17,
20), 519 (46, 47, 51), 524, 526, 527 (72),
528 (72), 529, 531 (72), 534, 543, 544,

fea

ye

AUTHOR INDEX

548, 564, 568, 578 (1, 7), 580, 581, 582
(1, 54), 584 (7, 48, 64), 585, 586 (1),
624, 651, 652

Kreitmaier, H., 222

Kreiss, R. E., 73

Kretchmer, N., 288

Koider; J+ L.,102

Krider, M. M., 418, 423

Kriger, K., 183, 184 (251), 185 (251), 186
(251), 187 (251), 189 (251), 191 (251),
197 (251), 201 (251), 203 (251)

Kring, J. P., 529 (91), 585

Kringstad, H., 653, 668

Krishnan, P. S., 458, 512

Krop, 8., 344, 345

Kriiger, M., 11

Kruhgffer, P., 27

Kruissink, C. A., 336, 343

Kruse, I., 409, 415, 434

Kryukova, N. N., 356

Kubowitz, F., 511

Kuhl, W. J., Jr., 692, 693 (81)

Kuhn, R., 338, 453, 455 (9), 470

Kiley, M., 441

Kulwich, R., 669

Kiilz, E., 375

Kummerow, F. A., 283, 284, 285 (18), 315
316

Kunkel, H. G., 373

Kunkel, H. O., 39, 277

Kuo, P. T., 554

Kupel, C. W., 347, 371

Kupferberg, A. B., 313

Kursanov, A. L., 356

Kurth, E. F., 334

Kurtz, C. M., 572

Kutscher, W., 15

L

Lackey, M. D., 429

Ladd, A. T., 373
Ladenburg, A., 474
Lafaye, J., 27

LaForge, F. B., 476
Lahousse, 175

Laiblin, R., 474
LaMaster, J. P., 442
Lamb, F. C., 545

Lan, T. H., 35

Lance, B. G., 570, 578 (32)
Landau, R. L., 46, 49 (12), 94

flrs

Landauer, W., 575

Landefeld, M. O., 27

Landis, E. M., 88

Landolt, F., 476

Landsburg, K. G., 223, 230 (2)

Landy, M., 453, 521 (39)

Lane, R. L., 339, 345, 541, 548, 646

Lang, R.S., 261

Lange, N. A., 459 (54)

Lange, R., 427

Langemann, H., 35, 71

Langer, R., 141

Langham, W. H., 454, 476, 505, 513, 526,
556

Langley, W. D., 93

Langston, R., 636

Langston, R. G., 546

Lantz, E. M., 546

Lapi, A., 87

Laquer, F., 199, 222

Lardinois, C. C., 637

Lardy, H. A., 84, 328, 324 (2), 327, 328,
329 (21), 330, 331 (5), 333 (5), 336 (5),
349, 350 (73), 351 (73), 382, 500

Larrison, M. S., 475

Larsen, E. H., 441

Larson, N., 285

Larson, R., 399

Lattanzi, A., 577

Laurence, E., 86

Laves, W., 414

Lawrence, J. M., 635, 638 (4a), 648 (4a)
687

Lawson, E. J., 626

Lawson, G. MelL., 421, 422 (18)

Layne, J. A., 556, 560, 566

Leary, T., 373

Lease, J. G., 658, 659 (91)

Leavitt, M., 584

Le Baron, F. N., 335

Le Boulch, N., 195, 209

Lecogq, R., 88, 168, 317, 380, 381, 440

Leddy, J. E., 438

Leder, I. G., 486, 494, 505, 537, 556, 585

Lee, C. C., 431, 437

Lee, J., 397, 400, 411, 446

Lee, J. G., 662

Leeds, M. B., 306

LeePeng, C. A., 547

Legler, R., 390

Lehmann, J., 428, 436

718 AUTHOR INDEX

Lehniger, A. L., 286, 497, 502, 619

Lehrer, W. P., 665, 682 (25)

Leibman, W., 55

Leifer, E., 505, 513, 514, 526

Leigh-Clare, J. L., 160

Leinwand, I., 373

Leitch, I., 257

Leloir, L. F., 502

Lematte, L., 7

Lemieux, R. U., 335

Lemon, H. B., 208

Lemon, J. McW., 210

Lennerstrand, A., 506

Lenstrup, E., 252

Leifer, E., 453, 456, 556

Leng, E. R., 547

Leonardi, G., 441

Leonian, L. H., 355

LePage, G. A., 458, 460, 483, 511

Lepkovsky, S., 225, 227 (19), 279, 293, 299
(8), 318, 453, 518 (1), 525, 526 (53, 54,
55, 56), 585, 591, 592, 596, 630, 656,
658, 659 (92), 661, 663, 682 (14)

Lepp, A., 654, 656 (40), 657 (40), 658, 659
(96)

Lepp, E., 427, 432

Lettré, H., 133, 140, 146 (388), 201 (38)

Levene, P. A., 485

Levine, C., 56

Levine, M., 80, 439

Levintow, L., 601, 607 (15)

Levitas, N., 521 (57), 524, 537, 54

Levring, T., 160 °

Levy, E. D., 535, 537, 560

Lévy, J., 8 (55), 9, 57, 58, 71, 92

Levy, L., 28

Levy, M., 582

Lewis, E. M., 88, 652

Lewis, H. B., 41, 79, 82 (148)

Lewis, J. C., 355, 364 (10)

Lewis, J. M., 248

Lewis, L., 400, 406, 421, 488, 445, 447

Lewis, U. J., 85, 578 (20)

Lewitus, Z. A., 423

16s OM s eas}

Li, T. W., 98

Libert, O., 57

Liebermann, C., 190 (262)

Liebhardt, S., 442

Liebreich, O., 3

Lieck, H., 54

Liener, I. E., 517

Light, A. E., 522

Light, R. F., 418, 426

Lih, H., 629, 642, 643 (31), 645 (31), 685

Liling, M., 651, 693

Lillie, R. D., 75, 107, 108 (426), 113, 114,
541 (3), 566, 567, 591

Lillie, R. J., 569, 578 (27), 581 (27), 686

Lilly, G. V., 355

Tilly, J2H:, 103

Lin, P.-H., 455

Lincoln, G. J., 424

Lindan, O., 75, 114

Lindberg, O., 494, 498 (90), 507 (90)

Lindley, C. E., 665

Lindley, D. C., 368, 369

Lindner, H., 153, 190

Lindquist, H. G., 648

Ling, C.-T., 86, 524, 529 (46), 556, 585

Lingane, J. J., 628

Lingenfelter, J. F., 316

Link, K. P., 417, 418, 423, 427, 428, 432
(81), 444

Linneweh, W., 457

Linsert, O., 141, 173, 196 (199), 198 (199),
199, 203

Linser, H., 10, 54

Lintzel, W., 54

Lipmann, F., 15, 16 (6), 365, 497, 499, 500,
598, 599, 600, 601, 602, 603 (17), 604,
605 (12, 18, 25), 607 (13, 17, 19, 25),
608 (17), 609 (12, 18, 17, 25, 37, 45),
610 (14, 17, 25), 611 (14, 16), 613 (25,
36), 614 (10, 14), 615 (5, 66), 616 (10,
19, 60, 65), 617 (19, 21, 67), 618 (10,
67), 620 (5, 19, 21, 66, 67, 70, 84, 86),
621 (98), 622 (98), 624 (98), 629, 630
(8), 631, 632, 633, 649, 658, 679

Lippi, M., 441

Lippincott, S. W., 668, 682 (28)

Lips, J. J., 312

Lipton, M. A., 17

Lisco, H., 664

Littlefield, J. W., 603, 621 (23), 622 (23)

Littlejohn, J. M., 87, 518 (21), 519 (45),
524, 528 (32, 49), 5380 (82), 531 (49),
549 (82), 554, 578 (11), 580 (11), 581

Litwack, G., 34, 35 (150), 39 (150), 82

Livermore, A. H., 364 (6)

Ljungdahl-Ostberg, K., 57

Loach, J. V., 20, 77, 86

F
|

AUTHOR INDEX

Lobb, D. E., 208

Lobo, R., 8

Localio, S. A., 562

Lockhart, E. E., 424

Loeb, H. G., 314

Loeffel, W. J., 226, 230

Loeliger, A., 434, 436

Loffler, W., 92, 127

Logan, W. B., 436

Lohmann, A., 5

Loizides, P. A., 69, 77

Lojkin, M. E., 564

Loker, F. F., 444

Long, B., 343

Longabaugh, G. M., 88, 102

Longenecker, H. E., 81, 319

ge, Yo H:, 365

Loofbourow, J. R., 355

Loomis, E. C., 438

Loosli, J. K., 316

Lopez-Matas, A., 546

Lord, J. W., 401

Lord, J. W., Jr., 401, 426, 427

Lorente, L., 561

Lorenzen, K., 339, 382, 383 (5)

Lortie, Y., 216

Lotpeich, W. D., 691

Lévenskiold, H., 222

Low, E. M., 136

Lowry, J. V., 75, 107, 108 (426), 113 (426)

Lozner, E. L., 421

Lueas, C. C., 49, 52, 67, 69, 72, 75, 76, 77
(135), 78 (122, 135, 142), 80, 81 (54),
82 (171), 94, 96 104, 108 125, 129, 315,
345 (53), 347, 371, 372, 383, 384

Lucia, S. P., 436

Lucius, R., 11

Luckey, T. D., 22, 26 (72), 88, 554, 570

Luecke, R. W., 5, 13 (21), 56, 92, 127, 524,
578 (12, 14), 581 (12), 586 (12), 665,
666 (167, 168, 170), 682 (23), 684, 686

Lundberg, W. O., 279, 285, 318

Lunde, G., 653, 668

Lupton, A. M., 372

Liittringhaus, A., 142, 173, 177 (45), 180
(45), 191, 198, 194 (283), 196 (199),
198 (45, 199), 200 (45), 204 (298)

Luzzatti, L., 306

Lwoff, A., 481, 538, 549

Lwoff, M., 481, 538

Lyddon, B., 647

719

Lyman, C. M., 17, 591, 592, 593 (8, 4), 630,
678

Lyman, R. L., 518 (12), 519, 523

Lynen, F., 16, 607, 617, 618, 619, 620

Lynn, J. G., 690

Lyons, R. N., 488

Lythgoe, B., 596, 656

Lytle, B., 326, 327, 328 (16)

M

McAlister, E. D., 593

McAmis, A. J., 292

McArthur, C.S., 12, 49, 67, 125

McBurney, C. H., 592, 593 (4)

McCall, K. B., 667, 670

McCann, G. F., 228

McCann, W.S., 103

McCarter, J. R., 417

McCasland, G. E., 326, 327, 328 (16)

McCawley, E. L., 401, 438

McChesney, E. W., 207, 369, 384

McCollum, E. V., 75, 183, 223, 226 (6),
230, 318

MacCorquodale, D. W., 390, 391 (20, 21),
392 (20), 393 (21, 25), 394, 395 (25,
40), 396 (89), 397 (43), 398 (89), 400,
403, 409, 411 (33), 418, 417, 435, 441

McCleary, B., 18

McClohon, V. M., 16, 631

McClure, J. N., Jr., 574

McConnell, K. P., 44,45

McCormick, H. M., 83

McCoy, R. H., 27

McCoy, T. A., 546, 636

McCrea, F. D., 572

McCrone, W. C., Jr., 467, 468 (71), 470

McCutcheon, J. W., 271

Macdonald, C. G., 338

MacDonald, D. L., 336

McDonald, F. G., 148, 146, 166 (167), 168,
182, 183 (242), 205, 206, 208, 209, 213
(12), 518 (30)

MacDonald, H., 436

McElroy, L., 397

McElroy, L. W., 94, 416, 546, 627, 655 (60),

656, 658, 659 (98), 682 (12)

McElvain, 8. M., 459 (59), 475, 477 (59),

479

MacFarland, M. L., 80, 81 (161), 370, 384

Macfarlane, R. G., 653, 657 (24)

McFarlane, W. D., 389, 421

720

McGilvery, R. W., 621

McGinnis, J., 96, 97, 317

McGlohon, V. M., 604, 606 (30), 611 (30),
624 (30)

MacGregor, H. I., 317

McGuire, G., 227

McHenry, E. W., 66, 67, 71 (20), 80, 81
(160, 161), 82, 86, 90 (28), 347, 370,
384

Mellwain, H., 343, 439, 507, 672, 680

McIntire, J. M., 58, 61 (2), 342, 370, 543,
546, 569, 578 (5), 654, 655 (87), 656
(37), 657 (37), 682 (5)

MacKay, E. M., 72, 73, 86

McKee, C. M., 317

McKee, R. W.., 390,391,392 (20) , 393 (25),
394, 395 (25, 40), 396, 397 (48), 399
400, 403, 405, 406, 409, 411 (33), 413,
417, 435, 441

MacKenzie, C. G., 318

MacKenzie, J. B., 318

McKey, B. V., 648

McKibbin, J. M., 56, 67, 98, 518 (14), 534,
578 (9), 650, 651, 657 (10), 663, 664
(146), 669, 670 (202), 672, 682 (26),
691

McKittrick, D.S., 95

McLaren, B. A., 103, 570, 668

MacLean, D. L., 67

McLean, M. J., 141

Maclean Smedley, I., 157

Macleod, J. J. R., 19, 100

McMahan, J. R., 376, 541, 629, 646, 647

McMahon, J. M., 217

McMillen, W. N., 524, 578 (12, 14), 581
(12), 586 (12), 665 (167, 168)

MacMillan, R. L., 434

MacNair, W. A., 139, 207 (83)

McNamara, B. P., 344, 345

MeNeil, E. W., 537

MeNeile, H. J., 427

Macpherson, L. B., 86, 345 (53)

MacPhillamy, H. B., 406, 445 (9), 446

McQuarrie, I., 302, 303, 304 (40)

MacQueen, J. W., 570

McQueeney, A. J.. 651, 657 (16)

McRorie, R. A., 605, 611 (40), 613 (40)

MeVeigh, I., 361, 366, 544, 547 (31), 550
635, 636 (3), 644 (3)

MacVicar, R. W., 638, 648 (21c)

MeVicar, R., 543

AUTHOR INDEX

MeWilliams, H. B., 369

Ma, C. M., 134

Ma, R., 342

Machado, A. L., 17, 618, 620 (71)

Macheboeuf, M., 10

Machek, G., 478

Machella, T. E., 83

Macht, D. I., 423

Mackenzie, C. G., 41

Mackie, T. T., 425

Mackinnon, J. E., 342

Mackintosh, D. L., 637

Macrae, T. F., 522, 567, 591, 596, 654, 655

(29), 656 (29), 664

Macy, I. G., 565, 638, 648 (21b)

Madden, R. J., 453, 455 (10), 480, 518 (6),

548, 554, 663

Madden, S. C., 532

Maddock, H. M., 666, 685, 686 (39a)

Mader, W. J., 628

Maeder, E. C., 293, 299 (8)

Madsen, L. L., 101, 568, 578 (15), 664, 665,
666

Magasanik, B., 323, 330, 331, 332 (13, 14),
333, (13, 14), 337 (4), 338 (4, 12, 13),
344, 345 (29), 347, 348, 353, 354, 355

Magath, T. B., 434, 437

Magnoni, A., 385

Magrath, O. I., 489

Mahler, H. R., 619, 620

Main, E., 671

Mainzer, F., 561

Major, R. T., 596

Makino, K., 528

Malaguzzi-Valeri, C., 575, 576 (44), 578
(76)

Malaker, M. C., 584

Malkin, T., 359

Maltaner, F., 441

Mamoli, L., 142

Man, E. B., 373

Mancini, F., 564

Manifold, M. C., 47, 69, 76 (52), 77

Mann, F. C., 92, 434

Mann, F. D., 426, 427, 428, 433, 434

Mann, J. D., 426

Mann, P. J. G., 33, 34, 495, 507

Mann, T., 598

Mannering, G. J., 580

Maquenne, L., 185, 323, 329

Marche, J., 550, 554, 555 (31)

AUTHOR INDEX

Marcussen, P. V., 441

Marenzi, A. D., 8, 54

Marfori, L., 576

Margolis, G., 518 (25)

Margolis, L. H., 518 (25), 557

Marinelli, L., 577

Markel, J., 261

Markely, K.S., 274, 335

Marks, I. E., 261

Marnay, C., 519 (49), 524, 548

Marque, J., 359

Marquette, M. M., 528

Marquez, V. M., 58

Marshall, A. L., 174, 176 (201)

Marshall, B., 531

Marshall, I. H., 205

Martin, A. J. P., 567, 664

Martin, C. J., 521, 567, 664

Martin, G. J., 367, 379, 384, 655, 667

Martinez, E., 355

Martinson, E. E., 414

Marvel, J. A., 95, 661

Marvel, J. P., 95

Marvin, J. F., 556

Marx, R., 482

Masaguer, J. M., 564, 574

Mascheck, F., 312

Masley, P. M., 605, 611 (40), 613 (40)

Mason, K. E., 316

Mason, M., 529 (92)

Massa, M., 562

Massengale, O. N., 139, 153, 157, 160, 166
(167), 168, 169, 170, 205 (180), 206
(180), 219 (26)

Matheson, N. K., 323 (9a)

Matlack, M. B., 335

Matsunaga, 8., 452

Mattei, C., 576

Matterson, L. D., 206, 569, 578 (22)

Matzner, M. J., 229

Mauer, H., 8

Maw, G. A., 25, 46, 47

Mawson, C. A., 434

Mawson, M. E. H., 48

Maxim, M., 52

Maximow, A. A., 232

May, E., 327

Mayer, A. M., 217

Mayer, P., 378

Mayer, S., 17

721

Maynard, L. A., 297, 316, 635, 638 (4a),
648 (4a), 687

Mazoué, H., 88, 317, 380, 381, 440

Mazza, F. P., 147, 148

Mead, J. F., 297

Meadows, C. M., 341

Means, J. W., 415

Meara, M. L., 305

Mecchi, E., 391, 393, 402, 406, 416, 422,

441, 445

Meerovich, G. I., 414

Medes, G., 22, 26 (69), 280, 283 (4), 316
319, 606

Mediary, G. C., 522, 559, 584

Medlar, E. M., 81

Meek, W. J., 4 (17), 6 (17), 53, 54 (15), 67

Mehler, A. H., 493, 499, 504 (121), 506,
527

Meikeljohn, A. P., 562

Meillére, G., 375, 376

Meilman, E., 518 (19)

Meister, A., 492, 503

Meister, H., 44

Melampy, R. M., 637, 668

Mellanby, E., 133, 160, 223, 230, 254

Melass, V. H., 636, 648, 660

Melnick, D., 274, 290, 457, 535, 536, 557,
560

Melville, D. B., 25, 48

Melzer, L., 455, 456 (23), 458 (23), 555

Mendel, L. B., 21, 123, 292

Menne, F., 31

Menon, P.S., 104

Menotti, A. R., 414

Menschick, W., 153

Mentzer, C., 399, 427, 429, 430 (95), 431

Merrill, A. L., 548, 545 (8)

Mertz, E. T., 95, 578 (24)

Meschan, [., 39, 96

Meserve, E. R., 54

Messerly, J. P., 476

Meunier, P., 193, 194 (286a), 195, 208, 209,
399, 427, 429, 430, 431

Meyer, A. E., 379, 380

Meyer, C. E., 593, 594, 688

Meyer, H., 635, 642 (5), 648 (5)

Meyer, K. H., 10, 13 (65)

Meyer, O. O., 428, 482

Meyer, W., 99

Meyerhof, O., 483, 484 (17), 487, 498

Meyers, L., 575

722

Michaelis, L., 487, 491, 508

Michaud, L., 98

Michel, G., 194

Mickelsen, O., 518 (14), 534, 559, 591, 595
596 (20), 626, 630, 658, 659 (95)

Migliardi, C., 148

Mignot, J., 573

Mii, S., 619

Milas, N. A., 147, 158 (81), 175, 216

Milhorat, A. T., 374

Millares, R., 545

Miller, B.S., 628, 635

Miller, C. O., 101, 665

Miller, D. K., 317, 521, 563, 564, 567

Miller, E. C., 89

Miller, E. G., Jr., 229

Miller, E.S., 279, 306, 314

Miller, F. R., 650, 653 (3), 656 (8), 657 (3)

Miller, J. A., 89, 313

Miller, J. L., Jr., 664

Miller, L. L., 27

Miller, L. N., 213 (12)

Miller, M. H., 101, 664, 670, 677 (213)

Miller, O. N., 34

Miller, R., 436

Miller, R. C., 567

Miller, S., 565, 638, 648 (21b)

Miller, S. E., 173

Miller, Z. B., 36

Millican, R. C., 615, 620 (64)

Milligan, J. L., 658, 659 (100), 682 (13)

Mills, C. A., 91, 586, 682 (9), 684 (9)

Mills, R. C., 93, 94 (804), 342, 369, 569,
578 (21), 650, 656 (5), 657 (5), 658,
659 (97), 682 (11)

Milroy, T. H.,8

Mirov, N. T., 318

Mishler, D. H., 95, 661

Mitchell, H. H., 102, 127, 129, 515, 522,
580, 666, 684

Mitchell, H. K., 376, 385, 454, 519 (54),
525, 526 (60), 527, 545, 549, 551, 593,
594, 629, 648

Mitchell, J. H., Jr., 287

Modak, M., 73

Moffet, G. L., 191

Mohammad, A., 654

Mobr, M., 335

Moldavsky, L. F., 423

Molho, D., 427, 429, 430

Molina, H., 88

AUTHOR INDEX

Molino, N., 576

Molitor, H., 124, 442, 443 (94), 680 (4)

Moll, T., 178, 222

Mollerstrém, J., 130

Mollgaard, H., 339, 382, 383 (5)

Momose, G., 359

Monahan, E. P., 98

Monasterio, G., 54

Monsalvez, E., 564, 574

Monroe, C. F., 223

Monson, E. M., 99

Monson, W. J., 84, 85

Montgomery, M. F., 199

Montgomery, M. L., 100, 101

Moore, C. N., 175, 178

Moore, D. H., 373

Moore, J. A., 16, 604, 606 (80), 611 (30),
624 (30), 631

Moore, M., 317, 648

Moore, M. B., 400

Moore, M. E., 626, 688, 691 (68)

Moore, M. L., 399

Moore, P. R., 665, 682 (25)

Moore, R. A., 401, 426, 570

Moore, R. C., 654, 655, 656 (39), 657 (39)

Moore, R. B., 175

Moore, 8., 628

Moore, T., 199, 286, 426

Moosnick, F. B., 100

Moraux, J., 429, 431

Morel, A., 426

Morelli, A., 578 (75)

Morgan, A. F., 88, 91, 525, 526 (53, 54),
545, 614, 624, 650, 651, 652, 653, 656
(4), 657 (4), 663 (12), 664 (12), 668,
669, 670 (199), 684

Morgan, B. G. E., 221

Moring-Claesson, I., 42

Morner, C. T., 5

Morrell, C. A., 167

Morrill, C. C., 102

Morris, H. P., 668, 682 (28)

Morrison, H. J., 422

Morrison, L. M., 102

Morse, M., 138

Morton, R. A., 138, 159, 175, 177, 199, 208

Moser, R. J., 593

Moses, C., 88, 102

Mosettig, E., 327

Mosher, L. M., 175, 176 (203)

Mosher, W. A., 27, 43 (94), 598, 678

AUTHOR INDEX

Moss, A. R., 40

Mossberg, H. O., 442

Mott, F. W., 123, 124

Motzock, I., 207

Moubasher, R., 438

Mowry, D. T., 271

Moyer, A. W., 21, 22 (63), 25 (63), 28, 30
(67), 45, 46 (1), 47 (1, 7), 48 (1), 48 (7)

Moyer, D., 361, 366, 544, 547 (31), 550

Mudrak, O., 429

Mueller, G. C., 313, 460, 600

Mueller, J. H., 453, 521 (36), 538, 550

Mueller, M. B., 475

Mulé, F., 439

Mulford, D. J., 27, 29, 30, 47, 57, 67, 68,
69, 76 (21), 77 (21), 78 (21), 79 (21)

Miller, E., 456

Miller, H., 335, 338

Miller, M., 193, 519 (52), 526, 527 (73)

Miiller, P. B., 216

Munier, R., 10

Munsell, H. E., 160, 161 (142)

Muntwyler, E., 555

Muntz, J. A., 28, 38, 40 (109), 127

Murphy, E. A., 293, 299 (8)

Murphy, F. E., 372

Murphy, H. T., 347, 371

Murphy, R., 421

Murphy, R. A., Jr., 574

Murray, A., 438, 476

Murray, M. R., 344

Murray, S8., 91

Mushatt, C., 101, 664, 670, 677 (213)

Mushett, C. W., 424, 626, 651, 693

Myerhof, O., 15

Myers, J., 360

Myrbiack, K., 482, 485, 486 (10, 31), 487,
512

Myint, T., 635

N

Nachmansohn, D., 17, 598, 618, 620 (71)

Naggatz, J., 142, 144 (48)

Naghski, J., 439

Najjar, V. A., 518 (3, 26), 522, 536, 557,
559, 584

Nakahara, W.,8

Narat, J. K., 441

Nason, A., 507, 532

Nassello, J., 441

Nasset, E. 8., 379, 383, 663

723

Nassi, L., 487, 489, 440

Nath, H., 294, 315

Nathanson, I. T., 73

Nawrocki, M. F., 79, 87 (152)

Naz, E., 671

Neal, A. L., 629, 632, 638, 640 (23), 641
(23)

Neal, P. A., 74

Neale, S., 301

Nebbia, G., 472

Neculescu, N., 576

Needham, J., 371, 376, 377, 378, 381, 385

Negelein, E., 496, 511

Neilands, J. B., 458, 610, 632, 638, 639

(26), 640 (26), 641 (26), 645 (26)

Neilsen, E., 386

Nékam, L., Jr., 442

Nelson, A. A., 650, 657 (8)

Nelson, E. M., 176, 221

Nelson, J. F., 424

Nelson, M. M., 657, 684

Nelson, M. T., 230

Nelson, V. E., 654

Nelson, W. L., 648

Nemirovsky, J., 442

Nesbitt, L. L., 312

Nesheim, R. O., 102

Neubaur, E., 4, 8 (10)

Neuberg, C., 346

Neuberger, A., 84, 529 (94)

Neufeld, E. F., 493

Neuman, W. F., 18

Neumann, A. L., 102

Neumann, N. W., 125

Nevens, W. B., 522, 580, 666

Newburgh, L. H., 75

Newman, M. S., 393

Nezamis, J. E., 426

Nichol, C. A., 37

Nicholls, J., 657

Nichols, J., 274

Nichols, P. L., 271

Nicolaysen, R., 228, 249

Nield, C. H., 216

Nielsen, E., 367, 368, 585, 654, 656 (35),
657 (35), 682 (4)

Nielsen, P. B., 216

Nightingale, G., 424

Nishi, K., 528

Nisman, B., 52, 92

Nishikawara, M. T., 91

724

Nitsche, G. A., Jr., 444

Noble, W. M., 364 (10)

Nocito, V., 42

Noland, J. L., 103

Nolf, P., 426

Noller, C. R., 272

Nord, F. F., 495

Norgaard, F., 566

Norris, E. R., 92, 667

Norris, F. W., 535

Norris, L. C., 93, 94, 95, 96, 591, 594, 636,
642, 643 (382), 645 (32), 658, 659 (83,
84, 94), 660, 670, 682 (15, 16, 18, 19),
684, 694

Norris, R. J., 178

North, H. B., 44

North, H. E., 51

Northrop, L., 685

Northrop, L. C., 637, 668

Northrup, M., 91

Novak, A. F., 637

Novelli, A., 414

Novelli, G. D., 15, 16 (6), 601, 602, 603
(17), 604 (26), 605 (12, 18, 25), 607
(18, 15, 17, 19, 25), 608 (17), 609 (12,
18, 17, 25, 37), 610 (14, 17, 25), 611 (14,
16), 613 (25, 26, 36), 614 (14), 615, 616
(19, 60, 65), 617 (19), 620 (19), 621,
622 (89), 632, 679

Nunn, L. C. A., 279, 316, 318

Nussmeier, M., 139, 168, 182, 183 (242),
213 (12)

Nye, J. F., 454, 519 (54), 525, 526 (60), 527,
551

Nygaard, A. P., 621 (96)

Nystrém, H., 485

Nytrom, R. F., 94

O

Obadia, 440

O’Banion, E. E., 418, 423

O’Brien, J. R., 17, 662

O’Brien, J. R. P., 555, 653, 657 (24)

Ochoa, C. G., 484 (16)

Ochoa, 8., 16, 483, 484 (16), 492, 493, 495,
499, 504 (79), 506 (77), 511, 607, 617,
620 (68, 69), 673, 679

O’Connell, P. W., 288

Odake, 8., 14, 452

O’Donnell, D. J., 103, 570, 668

Oehler, E., 433

AUTHOR INDEX

Ogg, C. N., 466, 467 (68)

O’Grady, M. K., 76, 77 (135), 78"G@aay
142), 384

Oginsky, E. L., 41, 84

Ohlmeyer, P., 458, 483, 484 (17), 487, 507

Okey, R., 70

Olcese, O., 569, 578 (17), 582 (17) 669

Olcott, H. S., 345

Oldham, H. G., 688, 689, 690

Oleson, J. J., 6, 50, 66, 94, 148, 518 (29,
31), 521 (29), 654, 656, 659

Olin, S. M., 328, 365

Olivetti, L., 261

Olson, F. A., 226, 230 (25)

Olson, F. C., 226, 230 (25)

Olson, R. E., 66, 72, 73, 90 (16), 91, 614,
624, 652, 662, 672

O'Neill, J. C., 674

Onrust, H., 414

Onstott, R. H., 541 (7), 567, 578 (10)

Ord si Ge, 17

Orla-Jensen, A. D., 422

Orla-Jensen, S., 422

Orr, J. B., 230

Orr, M. L., 74, 548, 545 (8)

Orth, O.S., 428, 572

Osborne, T. B., 21

Oser, B. L., 229, 457, 536

Ost, H., 474, 475 (91)

Osterberg, A. E., 422, 424, 425

Ostergard, R. P., 312, 313 (17)

Ostwald, W., 464

Otolski, S., 382

Ott, P., 511

Otto, E., 284, 285 (18)

Ovando, P., 686

Overman, R., 672, 686, 691

Overman, R. S., 381, 404, 417, 418, 423,
427, 428, 432 (81), 444

Owen, B. D., 637

Owen, C. A., 422, 423, 425, 433, 441, 448

Owen, C. A., Jr., 426, 434, 436, 448

Owen, J. R., 176

Owen, R. D., 519 (54), 527

Owens, F. M., 100

Owens, H. S8., 92, 655

Owren, P. A., 434

Oyhenart, J. C., 442

=

Paal, H., 13
Pace, E. R., 303

ee

AUTHOR INDEX

Pack, Gi T., 347,371

Page, I. H., 153, 190

Page, J. A., 676

Page, R. C., 486

Pahnish, O. F., 665, 682 (25)

Paine, T. F., 365

Painter, E. P., 312

Pal, J.,.123

Paladino, T., 440

Paland, J., 142, 144 (47), 145, 146 (47),
206

Palmer, L. S., 416

Pangborn, M. C., 312

Paniagua, G., 87

Pantlitschko, M., 31

Pany, J.,.31

Papadopoulou, D., 91

Pappenheimer, A. M., 75, 108, 113 (427),
138, 162, 223, 226 (7), 228, 231, 232

Paquette, L. J., 440

Paretsky, D., 42

Park, E. A., 162, 223, 226 (6), 233, 238,
241, 242, 260

Park, H. C., 626

Parkinson, L. R., 648

Parnas, J. K., 492

Parry, E. G., 147

Parsons, H. T., 658, 659 (91)

Partington, P. F., 72

Partridge, 8S. M., 361

Pascher, F., 261

Paskhina, T. §., 529 (93)

Patch, E. A., 688, 690 (69)

Patek, A. J., Jr., 372

Patel, D. K., 44

Paterno, P., 575, 576 (44)

Patrick, H., 93, 97

Patterson, A. M., 196

Patterson, E. G., 98

Patterson, J. M., 66, 67, 69, 71 (20), 78,
80, 81 (54), 82 (171), 86, 90 (28), 129
347, 370, 371, 383, 384

Patterson, W. B., 560

Patton, A. R., 95

Patwardhan, V. N., 227

Paul, A. K., 584

Paul, M. H., 620 (82)

Paulson, M., 665, 666 (163)

Paveek, P. L., 367, 368 (18), 654, 655 (42),
656 (42), 657 (42)

Payne, L. D., 91

Payne, T. P. B., 373

725

Pearce, W. E., 545

Pearson, P: Bi, 5, 13) (21),2563 92, 127,
286, 514, 515, 522, 524 (16), 529, 569,
578 (4, 17), 580 (4), 582 (4, 17), 585,
635, 636, 637 (6), 638, 648 (21a), 660,
669, 688, 690

Pease, E. D., 674

Pecot, R. K., 548, 545 (8)

Pederzini, A., 90

Peet, M., 91

Peevers, R. W., 148

Pelezar, M. J., Jr., 688, 690

Pellegrini, M., 440

Pelosio, C., 575

Pénau, H., 183, 191, 195, 196 (290), 197,
198, 204 (290), 215

Pennington, D., 629, 636, 659, 660

Pentler, C. F., 393, 416, 422

Pepper, J. H., 313, 315 (33)

Pepper, W. F., 661

Pereira da Silva, W. B., 442

Perez, C., 360, 365

Perles, R., 360

Perlman, I[., 71, 72

Perlzweig, W. A., 32, 456, 459 (37), 486,
514, 515, 516, 518 (23), 519 (44, 48,
50), 521 (57), 522, 523, 524, 526, 529
(71), 580 (31), 535, 536, 537 (32), 543,
548, 557, 559, 560 (82), 580, 581, 585

Permyakov, F. K., 575

Perrin, G., 375

Perrone, J. C., 549

Perry, R. L., 662

Perry, W. F., 651

Persons, E. L., 567

Pesez, M., 194, 198 (288)

Pessagno Espora, M. A., 427, 437

Petering, H. G., 95, 148, 155 (91), 156 (91),
205

Peters, D. D., 261

Peters, J. P., 264, 373

Peters, R. A., 17, 662

Peters, V. J., 16, 604, 606 (80), 611 (80),
624 (30), 631

Peterson, M. S., 550, 678

Peterson, W. E., 100

Peterson, W. H., 9, 156, 538, 550, 593, 594,
626, 630, 638, 639 (80), 640 (80), 641
(30), 678

Petit, A., 177, 194, 202, 204 (316)

Pettersson, N., 439

Pfaltz, H., 283, 673

726

Pfiffner, J. J., 44

Phelps, E. T., 486, 487, 442

Philippot, E., 345

Philips, A., 472

Phillips, P. H., 39, 81, 342, 348 (9), 344
(19), 368, 369, 555, 650, 656 (5), 657
(5), 660, 670

Phillips, R. V., 488

Philpot, J. St. L., 181, 182 (241), 189, 192
(263), 195 (241, 263), 196 (263), 198
(263), 204 (263), 216

Piacentini, C., 441

Pichat, P., 442

Pigman, W. W., 323, 363

Pike, E. F., 476

Pike, R. L., 563, 568

Pilgrim, F. J., 508, 555

Pinchot, G. B., 620 (79)

Pinkos, J. A., 102

Pinner, A., 479

Pirk, L. A., 426

Pinlot. Gya2le, 27

Pisciotta, A. V., 428

Pittman, M., 518 (83)

Pitzer, K.S., 331, 332 (20)

Plass, M., 498, 511 (80)

Platt, A. P., 40, 46, 48 (20), 69, 72, 76 (52),
LG

Platt, B. S., 359, 360

Platt, J. R., 275

Platz. BR... 283, ol5

Plaut, G. W. E., 84, 85

Plum, C. M., 427

Plum, P., 433

Pohl, R., 188, 189, 175, 178 (28, 30), 199

Pohle, F. J., 427

Poirot, G., 337

Polgar, P., 442

Polglase, W. J., 328, 365

Poling, C. E., 654, 655 (82), 656 (31, 32),
654 (31, 32), 657, 667

Pollack, M. A., 377, 549, 550 (79), 647, 690

Pollak, F., 476

Pollak, O, J., 373

Pollard, A., 153

Polskin, L. J., 296

Pons, W. A., Jr, 339

Poole, A. G., 359

Poole, H. H., 171

Rope AG laapop

Pope, H., 354

AUTHOR INDEX

Popkin, G. L., 574

Popp, E. M., 6387

Poppalardo, P., 575

Popper, H., 90, 108, 113 (428), 127, 128,
130

Porter, C. C., 585

Porter, F., 475

Porter, J. R., 688, 690

Porter, R. R., 72

Porterfield, V. T., 74

Portman, O. W., 44, 45

Portmann, A., 377

Post, A. L., 81

IROSteenone

Posternak, T., 323, 326, 327, 329 (19), 330,
331, 335, 337 (7, 8), 338 (59), 344, 346,
347, 351, 352, 357, 365

Potter, E. L., 422

Potter, J. W., 559

Potter, V. R., 15, 509

Poulsson, E., 222

Poulsson, E. A., 399

Pounden, W. D., 223

Poupa, O., 572

Powell, D., 583

Powell, M. J., 216

Power, M. H., 448

Powick, W. C., 568, 578 (15, 16)

Pramanik, B. N., 366

Pratt, E. F., 594

Preisler, P. W., 358

Prelog, V., 159

Present, C. H., 100

Preston, F. W., 429, 431 (90)

Prestrud, M. C., 426

Pricer, W. E., Jr., 458, 489, 494 (55), 498,
506 (55, 115), 507 (55), 603, 604 (29),
619, 621 (93)

Prickett, P. S., 153, 157, 160

Provost, R. C., Jr., 466, 467 (68)

Pruess, L. M., 156

Pugsley, L. I., 167

Puig Muset, P., 384

Pullman, M. E., 494

Pulver, R., 427

Putnam, T. J., 690

Q

Quackenbush, F. W., 283, 315, 316
Quadbeck, G., 338
Quarles, E., 357, 636

AUTHOR INDEX

Quastel, J. H., 33, 34, 36, 495, 507

Queen, F. B., 313

Quick, A. J., 399, 401, 404, 405, 406 (7),
407, 408 (11), 409, 412, 413 (11), 415,
418, 422, 424, 426, 428, 429, 432, 433
(21), 434, 488, 444, 445 (8), 446, 447

R

Rachele, J. R., 22, 23 (68), 26 (68, 71, 72),
27, 43 (71), 83, 84

Rachmilewitz, M., 561

Racker, E., 620 (79)

Radhakrishna Rao, M. V., 423, 424

Ragazzini, F., 487

Ragins, I. K., 208

Ralli, E. P., 100, 101, 614, 624, 652, 655,
669, 672, 673, 686, 687, 692, 693, 694

Ram, T., 658, 659 (99), 660

Ramalingaswami, V., 104

Raman, C. §., 69

Ramasarma, G. B., 521, 524, 528, 529 (90),
580

Ramstad, P. E., 546

Randall, A., IV, 434

Randall, J. P., 434

Randle, 8. B., 408, 445

Randoin, L., 573

Randolph, P., 284, 285 (18)

Rane, L., 49

Rao, 8S. D., 315

Rao, V., 576

Raoul, Y., 195, 209, 519 (49), 524, 548,
550, 554, 555 (381), 557

Rapoport, J., 347

Rapoport, 8., 482, 433

Rappaport, B. Z., 261

Raska, 8. B., 548

Rasmussen, R. A., 642, 645 (34)

Rasmussen, R. 8., 331

Ratner, §., 42

Ratto, E., 261

Ravazzoni, C., 12

Ravel, J. M., 679

Rawlings, A. A., 51

Ray, 8S. N., 555

Raymond, M. J., 86

Raynaud, M.., 52, 92

Raynolds, J. A., 216

Reckling, E., 163, 175

Record, P. R., 93, 94 (805), 154, 155 (111)

Recoules, A., 360

727

Reddi, K. K., 458, 585 (20)

Redel, J., 148

Redish, M. H., 428, 436

Redman, T., 229

Reed, C. I., 184, 261, 265

Reerink, E. H., 152, 158, 154 (98), 155
(106, 107), 178, 181, 182 (225), 199

Rees, H. G., 585

Refai, F. Y., 628, 635

Register, U. D., 553, 558 (19), 560 (19),
564 (19), 576, 578 (20), 587

Reguera, R. M., 55

Rehbein, A., 486

Reichard, J. D., 552

Reichert, E., 16, 607, 618

Reichert, F. L., 99

Reid, J. C., 27

Reid, M. E., 664

Reifenstein, E. C., 249, 250, 265

Reifer, I., 58

Rein, F. H., 314

Reinert, M., 283

Reinstein, H., 56

Reinwein, H., 457

Reio, L., 42

Reiser, R., 277, 283, 286, 296

Remp, D. G., 205

Remsen, D., 264

Renshaw, R. R., 11, 14, 123, 571, 574 (4)

Reschke, J., 161

Ressler, C., 22, 26 (71, 72), 27, 43 (71), 83

Reuter, C., 13

Reyniers, J. A., 22, 26 (72), 83

Rhian, M., 4, 97

Rhoads, C. P., 89, 347, 371, 521, 563, 564,
567

Rhoads, J. E., 401

Rhoads, P. 8., 264

Rhymer, I[., 365, 366

Ricci, C., 556

Rice, E. E., 647

Rice, 8S. A., 439

Richards, G. V., 654, 655 (36), 656 (36),
657 (36, 73), 682 (6), 691

Richards, R. K., 427, 486, 443 (5)

Richardson, F., 389, 421

Richardson, L. R., 93, 288, 343, 569, 635,
637 (6)

Richert, D., 397, 406

Richert, D. A., 87,397, 414

Richey, F. D., 546

728

Richtert, D., 400

Ridout, J. H., 20, 46, 48, 68, 69, 71 (48),
73 (41), 74 (90), 75, 76, 77 (132), 78
(122), 80, 81 (41, 54), 82 (171), 86, 100
(215), 103 (41), 104, 108, 109 (433),
114 (483, 434), 129, 347, 371, 383

Rieben, W. K., 423

Rieckehoff, I. G., 275, 281, 314

Riegel, B., 394, 397 (87)

Rieke, H., 328, 329 (21)

Riemenschneider, R. W., 271, 272, 288

Riggs, N. V., 334

Riggs, T. R., 682 (10)

Riley, I. D., 306

Riley, R. F., 18, 317

Ringier, B. H., 488, 492 (48)

Ringrose, R. C., 96, 594, 658, 659 (83, 84,
94)

Ritchey, M. G., 90

Ritchie, M. G., 362

Rittenberg, D., 30, 56, 158

Rittenberg, R., 621, 622 (90)

Ritter, H. B., 143

Rivkin, H., 167

Robb-Smith, A. H. T., 653, 657 (24)

Robbins, E. B., 571, 572 (1)

Robbins, M., 326

Robbins, W. J., 342

Robblee, A. R., 661

Roberts, D. B., 303

Roberts, E., 71

Roberts, E. G., 354

Roberts, L. J., 688, 689 (62)

Roberts, R. E., 95, 661

Roberts, W. L., 674

Robinson, A., 232

Robinson, E. C., 305

Robinson, F. A., 592, 595 (4a), 597 (4a), 598
(4a), 668, 680 (3)

Robinson, H. D., 210

Robinson, H. J., 442, 443 (94)

Robinson, J., 521 (57), 524, 537, 543

Robinson, P., 559

Robinson, R., 134

Robinson, R. A., 237

Robinson, W. D., 557, 560

Robinson, W. L., 19, 100, 568

Robison, R., 235

Roboz, E., 585

Roche, J., 8, 15

Roderuck, C. E., 565

AUTHOR INDEX

Roderick, L. M., 428

Rodgers, T.8., 422

Rodnight, R., 507

Rodriquez, L. D., 546, 636

Roehm, R. R., 593

Rogers, L. M., 541 (8)

Rohm, E., 338

Rohrmann, E., 592, 593 (4), 594, 598, 678

Roizin, L., 420

Roka, L., 441

Roller, D., 429

Rollet, A., 271

Roman, W., 10, 52

Romano, L., 576

Ronzio, A. R., 488

Roos, A., 97

Rosahn, P. D., 372

Roscoe, M. H., 230, 684

Rose, C: L., 429, 571, 572 (1)

Rose, C.5S., 88

Rose, W. C., 20, 21 (57), 27, 79

Rose, W. G., 271

Rosen, F., 486, 514, 516, 519 (44, 48, 50),
521 (57), 524, 526, 529 (71), 530, 537,
543, 548, 581, 585

Rosenberg, A. J., 352, 366

Rosenberg, H. R., 134, 142, 147, 155, 156
(119, 120), 159, 190 (14), 191 (14), 192,
198, 201, 202 (315), 204 (119), 205 (119,
120), 210, 214, 225, 473, 477, 490, 596,
597 (31), 598 (81), 627

Rosenberger, F., 356, 371, 377

Rosenblum, L. A., 562

Rosenbusch, J., 442

Rosenheim, O., 133, 188, 189, 148 (29),
159, 190

Rosenthal, S. M., 615, 620 (64)

Ross, O. B., 369, 638, 648 (21c)

Ross, W., 546

Rossi, A., 102

Rossi, C., 441

Rossi, L., 8

Rossini, F. D., 332

Roth, J. 8., 92, 126

Roth, L. J., 456, 505, 513, 514 (195), 556

Roth, P., 686

Rothman, §S., 315

Rothrock, H. A., 230

Rothschild, E. E., 390, 391 (18), 392 (18),
398 (18)

Rotter, R., 428

:
|
:

AUTHOR INDEX

Rouir, E. V., 217
Rousseau, G., 194
Rowan, W., 163
Routh, J. I., 668
Rowett, E., 679
Rowland, W. E., 532
Rubin, L., 555
Rubin, M., 661
Rubin, P. 8., 236
Rubin, S. H., 52, 55 (3), 100, 101, 535, 578,
626, 672, 688, 691, 692
Rudden, M., 71
Rudkin, G. T., 533
Rueff, L., 16, 607
Ruegamer, W. R., 98, 586
Ruegger, A., 390
Ruffin, J. M., 458, 559, 560 (82)
Ruigh, W. L., 141, 146, 147, 148
Rumery, R. E., 316
Runnstrém, J., 487
Rupel, I. W., 223, 227, 230 (3)
Ruppal, M., 10, 57
Ruppol, E., 183, 191
Rusch, H. P., 313
Rusoff, I. I., 275
Russell, F.S., 171
Russell, H. K., 436
Russell, W. C., 216, 296, 543, 665
Russo, H. F., 688, 690 (69)
Rust, 8., 442
Rutigliano, M. L., 686
Rutledge, M. M., 565
Ruzicka, L., 44, 134, 159
Rygh, O., 148, 169, 179, 186 (59a), 191
(59a)
Ss

Sabol, M., 90

Sacca, M., 577

Sachs, F., 378

Sachs, G., 607

Sadhu, D. P., 58

Sah, P. P. T., 148, 399, 438

Said, A., 438

Sakai, R., 334, 345, 346 (48)

Sakami, W., 27, 41, 42 (192)

Salcedo, J., Jr., 69, 73

Sallach, H. J., 79

Salmon, W. D., 48, 66, 67 (13), 78, 79
(145), 81 (23), 88, 89, 94, 95 (184), 96
(313), 98, 580 (49, 50), 584 (49), 650,
669, 670 (200)

729

Salomon, H., 390, 391 (18), 392 (18), 398
(18)

Salomon, K., 656

Salomonsen, L., 420, 421, 433 (4)

Saluste, E., 42

Salvi, P., 578 (75)

Salvini, M., 574, 576

Samant, K. M., 198

Sampson, W. L., 390, 391 (22), 392 (22),
396, 397 (52), 406, 411 (21), 418, 417,
446, 654, 655 (36), 656 (383, 36), 657
(33, 36), 682

Samuels, L. T., 495

Sanadi, D. R., 506, 603, 621 (23), 622 (23)

Sando, C. E., 335

Sandza, J. G., 667, 682 (27), 691

Sanford, H. N., 305, 422

Santos Ruiz, A., 154

Sapezhinskaya, N. V., 577

Sarett, H. P., 518 (8), 521 (56), 524, 532,
535, 5386, 537, 553, 556, 557, 558 (19),
560 (19), 564 (19), 565, 576, 578 (13),
587, 629 (12), 688

Sargent, F., 559

Sarles, W. R., 385

Sarma, P. S., 518 (5), 519 (47), 524, 531,
534, 564, 568, 578 (1, 23), 582 (1), 584,
586 (1)

Sasaki, Y., 562

Sassaman, H. L., 154, 169

Satoda, I., 476

Sauberlich, H. E., 685

Saunders, F., 458, 521 (87)

Savage, E. E., 290, 318

Savage, J. L., 313

Savard, K., 153

Saviano, M., 102

Savini, R., 577

Sawyer, S. D., 81

Sax, K. J., 148, 144

Saye, E. B., 566

Seaffidi, V., 576

Schaaf, K. H., 148

Schaefer, A. E., 48, 67, 81 (23), 83, 87, 94,
95 (184), 96 (818), 98, 570, 578 (9),
580, 663, 664 (146), 670, 672, 682 (26),
691

Schaefer, E. G., 585 (21)

Schaltegger, H., 198, 217

Schayer, R. W., 525, 529

Schecter, V., 489

730

Scheel, L. D., 418, 428, 432 (39)

Scheer, B. T., 519 (53), 527, 528, 529 (84),
581, 585 (53)

Scheinberg, P., 436, 574

Scheiner, J., 626, 688, 691 (68)

Schenck, F., 140, 146 (88), 147, 201 (88,
70), 203

Schenck, J. R., 21, 26 (64)

Schepartz, A. I., 507

Scherer, J., 323, 334, 358, 359, 380

Scheunert, A., 161, 222

Schieblich, M., 161, 222

Schilling, K., 101

Schleicher, E. M., 100

Schlenk, E., 509

Schlenk, F., 51, 345, 458, 455, 456 (23),
458 (8, 23), 480, 481 (3), 482 (3), 483,
484 (19), 485 (3), 486, 487, 488 (8, 29,
33), 489, 491, 492 (33), 498, 494, 507,
512, 555, 645

Schlenk, T., 491

Schlutz, F. W., 138

Schmall, M., 456, 506 (82), 535 (21), 628

Schmetz, F. J., Jr., 604, 610

Schmidt, C. L. A., 404, 425, 446

Schmidt, E., 9, 426

Schmidt, G., 8, 18, 56, 62

Schmidt, H., 522, 580, 669

Schmidt-Nielsen, S., 168, 171

Schmidt, V., 688

Schmitz, H., 261

Schmitz, J. T., 440

Schneck, J. R., 102

Schneider, H., 178

Schneider, M. C., 16, 607

Schoenheimer, R., 30, 40, 41, 153, 158, 186
(260), 187, 204 (260), 319

Schoental, R., 89

Schgnberg, A., 438

Schonheimer, R., 280

Schgnheyder, F., 389, 390, 400, 401, 421,
432

Schoorl, N., 7

Schopfer, W. H., 344, 352, 357, 365, 533

Schornstein, L., 303

Schoubye, N., 684

Schrecker, A. W., 498

Schreiber, E., 222

Schreiber, M., 657

Schuette, H. A., 638, 640 (80a), 648 (30a),
687

AUTHOR INDEX

Schukina, L. A., 482

Schultz, A. S., 342, 349 (3), 362, 550, 633

Schultz, J., 533

Schultz, R. B., 624, 651, 652

Schultze, H. E., 434

Schultze, M. O., 517

Schultzer, 375

Schukina, L. A., 399

Schumacher, A. E., 591

Schiitze, H., 52, 92

Schwalb, 8., 427

Schwaneberg, H., 13

Schwartz, S., 556

Schwarz, K., 75, 628, 656, 657 (72), 685,
686

Schwarz, P., 474

Schwarzenbach, G., 459 (57), 472 (57), 477
(57), 488, 492 (49, 50)

Schweet, R.S., 620 (82)

Schweigert, B.S., 58, 61 (2), 342, 370, 515,
522, 524 (16), 527, 528, 529, 530, 531
(44), 582, 546, 569, 578 (4, 5, 6), 580
(4), 582 (4), 585, 586, 637, 669

Schweitzer, C. W., 394, 397 (87)

Schwerin, P., 312

Schwimmer, 8., 345

Sciclounoff, F., 671

Scott, D. B. M., 501, 557

Scott, H. H., 674

Scott, H. M., 206, 569, 578 (22)

Scott, M. L., 97, 159

Scott, R. B., 420

Scudi, G. V., 414

Scudi, J. V., 536, 663, 664 (147)

Scuro, L. A., 686

Seager, L. D., 91

Sealock, R. R., 364 (6)

Seaman, W., 55

Sebrell, W. H., 75, 104, 107, 108 (426), 113
(426), 423, 518 (83), 541 (4, 5, 6, 7),
564, 567, 569, 578 (10, 18), 582 (18),
650, 656 (2), 657 (2, 22), 653, 668, 669,
670 (201)

Seegers, W. H., 404, 424, 482, 438

Sampson, M. M., 91

Seeler, A. O., 385, 424, 664

Seeman, 440

Seifter, 8., 555

Seldon, T. H., 487

Sellards, A. W., 103

Sellers, E.A., 73, 74 (90), 111, 372

_.o-_ - =

;

4

4

§
”

AUTHOR INDEX

Sells, R. L., 422, 448
Semenza, F., 577
Sendju, Y., 459 (55)
Senior, F. A., 574
Sepulveda, G., 572
Setz, P., 178, 180, 197, 204 (226)
Sevag, M. G., 49, 680
Seyfferth, E., 474
Sexton, W. A., 157, 208
Shafer, E. G., 456, 506 (82), 535 (21)
Shaffner, C.8., 686
Shahinian, 8. 8., 524, 555, 556 (40), 585
Shane, R. 8., 476
Shank, R. E., 505, 512 (152), 556
Shantz, E. M., 216
Shaperman, E., 3038, 304 (40)
Shapiro, B., 9, 16, 617, 620 (68), 679
Shapiro, E., 71
Shapiro, S., 427, 428, 431, 432, 435, 436,
443 (5), 444
Sharma, G. L., 665
Sharokh, B. K., 545
Sharpe, J.S., 52
Sharpe, P. F., 545
Sharpless, G. R., 90
Shattock, F. M., 583
Shaw, F. H., 13, 54
Shaw, J. H., 650, 656 (5), 657 (5), 660, 670
Shay, H., 372, 379
Sheely, R. F., 442
Sheft, B. B., 545
Shekleton, M. C., 547
Shelling, D. H., 264
Shemiakin, M. M., 399, 482
Shemin, D., 42, 525, 608, 621, 622 (90)
Shepheard, E. E., 534, 578 (26), 629 (15),
658, 659 (101), 660 (101)
Sherlock, S., 107
Sherman, A., 176
Sherman, E., 143
Sherman, H., 424
Sherman, H. C., 228, 226 (7)
Sherman, W. C., 315
Sherrard, E. C., 334
Sherwood, R. M., 636, 648, 660
Shettles, L. B., 422
Shevezov, J. B., 482
Shields, J. B., 545
Shieve, W., 622
Shimamura, T., 14, 452
, Shimizu, H., 536

731

Shimkin, M. B., 442, 443 (93), 444 (93)

Shinowara, G. Y., 271, 415, 434

Shipley, P. G., 1383, 223, 226 (6), 235

Shipley, R. A., 73

Shive, S., 625

Shive, W., 86, 521 (42), 626, 627 (3), 668,
679

Shock, N. W., 668

Shohl, A. T., 224, 229, 254

Shonyo, E.S., 434

Shoppee, C. W., 134

Shorland, F. B., 312, 314

Shourie, K. L., 522

Shriner, R. L., 189

Shull, F. W., 532

Shull, W. H., 419

Shuster, L., 489, 603, 631

Sidwell, A. E., Jr., 189, 201 (265)

Sieg, H., 15

Siegel, L., 27, 56, 690

Siekewitz, P., 27, 41, 42

Silber, R. H., 385, 585, 663, 664 (148), 670

Silverberg, M. G., 261

Simmonds, N., 133, 223, 226 (6), 230

Simmonds, S., 21, 25, 26 (64), 28, 30 (66,
67a), 40 (66), 41 (75), 48, 80, 102

Simms, H. D., 650, 651, 656 (4), 657 (4),
663 (12), 664 (12), 668, 669, 670 (199)

Simon, E.J.,608 —

Simon, F. P., 312

Simonelli, M., 563

Simonnet, H., 170

Simons, C., 51

Simons, E. J. H., 178

Simonson, H., 546

Simpson, J. C. E., 134

Simpson, C. A., 261

Simpson, M. E., 73

Simpson, M. I., 25

Simson, G., 481, 485

Sinclair, R. G., 279, 314, 318

Singal, 8. A., 47, 76, 87, 518 (21), 519 (45),
524, 528 (49), 530 (32), 531 (49), 549
(82), 554, 559, 560 (77), 578 (11), 580
(11), 581

Singer, T. P., 36, 481

Singsen, E. P., 206, 569, 578 (22)

Sirek, O. V., 180

Sisler, E. B., 458

Skarzynski, B., 508

Skeggs, H. R., 555, 688, 690 (69)

732

Skinner, J. T., 638, 639 (30), 640 (30), 641
(30)

Skraup, Z. H., 473

Skudrzyk, I., 30

Slade, R. E., 348

Slanetz, C. A., 682 (8)

Slavin, G., 567

Slein, M. W., 493, 510

Slinger, S. J., 661

Slobodin, Y. M., 461

Smakula, A., 178, 181, 189, 199

Small, C. W., 8

Smedley-Maclean, I., 279, 281, 314, 316,
318

Smiley, K. L., 642, 645 (34)

Smiljanic, A. M., 315

Smith, C. C., 429

Smith, D. T., 354, 453, 518 (25), 567

Smith, E. L., 182, 184 (243), 185 (243),
187 (243), 189 (243), 190 (243), 195,
197, 198 (248), 204 (297a)

Smith, F. G., 312

Smith, H. H., 279, 316, 318

Smith, H. P., 404, 407, 408 (23), 419, 421,
493, 424, 425, 426, 427, 431 (14), 433
(14), 445 (10), 446

Smith, J. A. B., 46

Smith, M. B., 545, 642, 645 (34)

Smith, P., 84, 85 (194)

Smith, P. G., 394, 397 (37)

Smith, R. L., 39, 51

Smith, 8. G., 453, 656

Smith, W. B., 434

Smith, W. H., Jr., 637

Smorodinzew, J., 13

Smyrniotis, P. Z., 501, 503, 512

Smyth, D. H.,8

Smythe, C. V., 44, 77, 491

Snell, A. M., 401, 424, 425, 426

Snell, E. E., 16, 318, 342, 357, 362, 366,
532, 533, 538, 539, 540 (46, 48), 551,
593, 594, 604, 605, 606 (30), 607, 611
(30, 39), 613 (88, 39), 624 (380), 626,
629, 630, 631, 632 (29), 633 (29, 37),
636, 637, 647, 659, 660, 678, 679 (256),
687

Snitzer, I. 8., 575

Snog-Kjaer, A., 441

Snyder, E.S., 661

Snyder, J. Q., 546

Snyder, R. H., 220

AUTHOR INDEX

Snyderman, 8S. E., 524, 530

Soames, K., 163

Sobel, A. E., 217, 261

Sobin, 8. S., 88

Sobolov, M., 642, 645 (34)

Sobotka, H., 134, 379, 380 (17), 383

Séderhjelm, L., 365

Solandt, O. M., 5, 57

Soliman, G., 77

Solmssen, U., 459 (57), 472 (57), 477 (57),
488, 492 (48, 50)

Solmssen, U. V., 397, 400, 411, 446

Solomon, C., 427

Solvonuk, P. F., 488

Sgndergaard, E., 406, 409, 484

Sondheimer, F., 134

Sonne, C., 175

Sonne, S., 379, 380 (17), 383

Soodak, M., 16, 603, 607 (25), 609 (25),
610 (25), 613 (25) 615 (66), 616, 620
(66, 77, 83)

Sgrbye, @., 434

Sorice, F., 686

Soskin, 8., 19

Soucy, R., 28

Soulier, J. P., 427

Souter, A. W., 427

Southner, S. P., 247

Soye, C., 14

Sparks, P., 569, 578 (17), 582 (17)

Spector, H., 568, 584, 585 (76), 684

Sperber, E., 42

Sperti, G., 178

Spies, T. D., 425, 458, 518 (7, 15, 16, 22,
24, 30), 552, 554, 555, 556, 560 (10),
562 (10, 58), 563, 570, 573, 574, 576
(16), 637, 677, 680 (2), 681, 690

Spink, W. W., 417, 438

Spinks, J. W. T., 431, 487, 438

Spitzer, R., 331, 332 (20)

Spitzer, R. R., 343, 368

Spoon, H. J., 655

Sprince, H., 313

Spring, F.S., 134, 147, 190, 191, 198

Sprinson, D. B., 42, 84, 337

Sprunt, D. H., 102, 656

Spurr, C. L., 88

Squires, KE. M., 647

Sreenovasan, A., 37

Srenwasaya, M., 495

Stadler, H. E., 436

AUTHOR INDEX

Stadtman, E. R., 16, 602, 6038, 607 (19,
24), 616, 617 (19), 618, 620 (19, 20, 24,
68, 80), 621 (20)

Staedeler, G., 335

Stahmann, M. A., 404, 427, 482

Stamler, F. W., 408, 409, 445, 446 (38)

Stanbery, S. R., 594, 637, 677, 690

Stange, O., 152, 153

Stanley-Brown, M., 424, 432 (39)

Stanley, R. H., 596

Stanék, V., 7, 18 (82), 52

Stansly, P. G., 619

Stare, F. J., 67, 97, 98, 436, 488, 492 (51),
524, 529 (46), 556, 585, 662

Starkenstein, E., 376, 386

Staub, H., 89

Stearns, G., 258

Steck, I. E., 1384, 265

Stedman, M. M., 548

Steel, G. E., 182, 183 (242), 218 (12)

Steele, J. M., 671

Steenbock, H., 133, 154, 155, 160 (9), 169,
170, 174, 176 (202), 209 (216), 210, 220,
222, 223, 225, 226 (8), 227 (19, 20),
228, 230, 288, 315, 316

Steensholt, G., 30

Stefanini, M., 405, 407, 408 (11), 409, 413
(11), 418, 428, 445 (8), 446, 576

Steigmann, F., 130, 379, 385

Stein, G., 200, 201 (314)

Stein, H. J., 101, 557, 568, 580 (51), 581,
664, 670, 677 (213)

Stein, P., 159

Steiner, A., 102, 373

Steinhauser, H., 280, 315, 319

Stekol, J. A., 26, 80, 83, 84, 85 (198, 194)

Stern, H. R., 533

Stern, J., 493

Stern, J. R., 16, 495, 607, 617, 619, 620
(68) , 679

Sternberger, A., 434

Stetten, D., Jr., 30, 40, 43, 69, 71, 72, 73,
82, 103, 314, 338, 350, 357, 378, 560

Stetten, M. R., 338, 350, 357, 378

Stevens, C. M., 102

Stevens, W., 148

Stevenson, E. S., 89

Stevenson, 8S. G., 182, 184 (243), 185 (248),
187 (243), 189 (243), 190 (243), 198
(243)

Stewart, A. P., Jr., 545

733

Stewart, E. D., 512

Stewart, J. K., 427

Stewart, P. M., 552

Stewart, R. N., 344, 345 (29)

Steyermark, A., 411, 446

Still, E. U., 199

Stiller, E. T., 596, 597 (26), 658

Stillman, N., 71

Stirn, F. E., 655

Stjernholm, R., 42

Stoesser, A. V., 303, 317

Stokes, J. L., 645

Stokstad, E. L. R., 94, 95 (808), 389, 401,
402, 403, 408, 415, 416, 417 (1), 422,
423, 439, 444, 445 (1), 447, 570, 578
(31)

Stone, L., 642, 645 (34)

Stone, M., 637

Stone, M. A., 385, 668

Stone, R. E., 518 (22), 574, 576 (16)

Stoppelman, M. R. H., 441

Stothers, 8. C., 688

Stotz, E., 312, 498

Stout, A. K., 376

Strack, E., 4, 8 (10), 18, 127

Stragnell, R., 486

Strain, W. H., 134, 562

Strandine, E. J., 647

Strating, J., 144

Strauss, 374

Strecker, A., 3

Strecker, H. J., 493, 620 (85)

Street, H. R., 453

Strength, D. R., 39, 48, 67, 81 (23), 83,
94, 95 (184), 96 (313)

Strijk, B., 336, 348

Strong, F. M., 453, 455 (10), 459 (59), 477
(59), 480, 518 (6), 538, 548, 544 (15),
545 (15), 548, 593, 594, 600, 603, 604,
609 (22), 610, 611, 626, 629, 630, 632,
635, 637, 638, 639 (26, 29, 30), 640 (28,
26, 27, 29, 30), 641 (28, 26, 27, 29, 30),
642, 648 (381), 644 (2), 645 (26, 31),
678, 687

Struck, H. C., 134, 261, 265

Struglia, L., 669

Stubbs, J. J., 364 (10)

Sturgis, C. C., 688

SubbaRow, Y., 49, 518 (19), 522, 654, 656,
657

Sullivan, M., 657

734

Sullivan, M. X., 198, 216, 414, 552

Sullivan, W. R., 404, 418, 427, 428, 432
(39)

Sulon, E., 657

Sulzberger, M. B., 261

Sumi, M., 156, 186 (257a), 189, 204 (257a)

Sumner, J. B., 458

Sunde, M. L., 96, 661

Sundwall, J., 570

Sung, S., 530

Sung, Shan-Ching, 37

Supplee, G. C., 657

Sure, B., 92, 227, 369, 384, 564

Surie, E., 160

Suter, F., 425, 442

Sutherland, J. E., 625, 679

Sutton, T.S., 522, 580

Suzuki, U., 14, 452

Swain, M. L., 287, 288

Swaminathan, M., 522, 584

Swank, R. L., 670, 677 (215)

Swanson, A. L., 39

- Swanson, M. A. 72, 85

Sweat, M. L., 495

Sweeney, J. P., 535

Swendseid, M. E., 39, 85

Swern, D., 312

Swick, R. W., 685

Swift, C. E., 271

Swift, R. W., 656

Sydenstricker, E., 552

Sydenstricker, V. P., 87, 518 (21), 519
(45), 524, 528 (32, 49), 530 (32), 531
(49), 549 (82), 554, 559, 560 (77), 561,
563 (88), 564 (88), 578 (11), 580 (11),
581, 670

Szalkowski, C. R., 628

Szirmai, E., 440

r

Tabenkin, B., 578

Tage-Hansen, E., 400, 406, 432, 483, 438,
445, 447

Tabor, H., 615, 620 (64)

Taggart, J. V., 498

Tambach, R., 380

Tamburello, 8. M., 66

Tange, U., 279

Tanguy, O., 71, 92

Tanner, E., 425, 442

Tanner, W. F., 541, 552, 553, 566

AUTHOR INDEX

Tannhauser, 8. J., 8, 18, 56, 62

Tanret, C., 153, 183, 184 (244, 246), 185,
186 (246), 187 (246), 188, 190 (244,
246), 191 (244, 246), 336

Tanquary, M. C., 416

Tappan, D. V., 85, 578 (20)

Tarbett, R. E., 552

Tarlowsky, E., 545

Tarver, H., 80

Tatting, B., 563, 568

Tatum, E. L., 90, 348, 344 (21), 362, 366, |
367 |

Taub, D., 134

Taub, S. J., 304

Taurog, A., 56

Taveira, M., 422, 441

Taylor, A., 377, 582, 545, 549, 550, 647,
661, 690

Taylor, A. B., 448

Taylor, B., 44

Taylor, E., 586

Taylor, J., 541, 646

Taylor, M. W., 296, 543

Taylor, P., 44

Taylor, 8., 636

Taylor, T.S., 281, 291

Taylor, W. E., 56

Teague, D. M., 4 (16), 53

Teague, P. C., 680

Teeri, A. E., 584, 665

Telfer, S. V., 230

Teply, L. J., 459 (56), 518 (5, 17), 519 (46),
521 (40), 524, 531, 5384, 543, 544 (15),
545 (15), 554, 556 (20), 564, 568, 570,
578 (1, 7), 580, 582 (1), 584 (7, 48),
586 (1), 635, 644 (2), 687

Terroine, T., 533, 545, 637

Teut, E. C., 228

Thacker, J., 661

Thain, E. M., 16, 603, 604 (27), 606, 611
(35), 613, 631, 632 (25)

Thalman, R. R., 226, 230 (25)

Thayer, S., 98

Thayer, S. A., 390, 391 (20, 21), 392 (20),
393 (21, 25), 394, 395 (25, 40), 396,
397 (43), 398, 399, 400, 403, 405, 406,
409, 411, 413, 417, 435, 441

Theobold, G. W., 261

Theorell, H., 317

Thibaudet, G., 193, 194 (286a), 208

Thiele, W., 198

AUTHOR INDEX

Thiem, N.-V., 15

Thoai, N.-V., 15, 17

Thomas, B. G. H., 442, 448 (91), 444 (91)

Thomas, B. H., 209

Thomas, J. M., 548

Thomas, J. W., 316, 561

Thomas, N., 74

Thompson, C. M., 421, 423

Thompson, H. E., 181, 261

Thompson, M. L., 687

Thompson, M. R., 379

Thompson, R. C., 354

Thompson, R. H.S., 17

Thompson, T., 160, 161 (142)

Thompson, W. R., 441

Thomson, W., 224

Thordarson, O., 421

Thornton, M. H., 53

Thorp, F., Jr., 524, 578 (12, 14), 581 (12),
586 (12), 665, 666 (167, 168, 170), 684,
686

Tidrick, R. T., 407, 408 (23), 409 (27), 445
(10), 446 (3)

Tidwell, H. C., 79, 90, 301

Mien, ¥. L., 134

Tilden, E. B., 441

Tilden, M. W., 536

Tingstam, S., 489

Tischler, M., 476

Tisdall, F. F., 248

Tishler, M., 390, 391 (22), 392 (22), 396,
397 (52), 406, 411 (21), 418, 417, 446

Tissieres, A., 8

Titus, H. W., 95

Todd, A. R., 485, 486 (82), 489, 596, 656

Todd, W. R., 360

Toennis, G., 22, 26 (69)

Tolbert, M. E., 70

Tollens, B., 334

Tolmach, L. J., 504

Tomarelli, R., 312, 313 (17)

Tomkins, F. §., 392, 395 (81), 396 (31),
414

Tompkins, M. D., 545

Toomey, J. A., 261

Toporek, M., 27

Torbet, N., 585, 586

Torda, C., 317, 380, 381, 438, 440

Tortori-Donati, B., 575

Totter, J. R., 84, 91, 637

Trager, W., 103, 661, 682 (22)

Tramontana, C., 577

Trautman, M., 92, 191, 205, 206, 655

Travers, J. J., 85

Treadwell, C. R., 76, 77, 79 (134), 86

‘Trenner, N. R., 401, 414, 476

Tressler, D. K., 210

Trevoy, L. W., 431, 437, 438

Ter, .G ake

Tristram, G. R., 86

Tromberg, G., 441

Trowell, O. A., 33

Truesdail, T. H., 591, 592, 593 (8, 4), 594,
630, 678

Truglio, V., 440

Truhlar, J:, 227

Trumbauer, C., 476

Tschugajeff, L., 190

Tso, E., 163

Tucker, H. F., 20, 47, 76, 77

Tull, C., 524, 578 (12, 14), 581 (12), 586
(12)

Tung, T., 530

Turner, R. A., 89

Turpeinen, O., 278, 279 (1), 318

Tuttle, L. C., 15, 16 (6), 601, 605 (12, 13),
607 (18), 609 (12, 13), 620 (84)

Tyler, D. B., 91

Tyner, E. P., 79, 82 (148)

U

Uflacker, H., 162

Underhill, F. P., 123, 565

Unger, P. N., 427, 444

Ungerleider, H. E., 442

Unna, K., 571, 572 (2), 626, 650, 651, 654,
655 (36), 656 (7, 33, 34, 36), 657 (33,
34, 36, 71, 73), 664, 680, 681 (1), 682
(6), 691, 692

Untersteiner Occhialini, L., 448

Uroma, E., 317

Usteri, E., 485

Utter, M. F., 620 (86)

Utzinger, G. E., 488, 492 (49)

Uvnas, B., 426

Vv

Vacher, M., 216

Vail, G. E., 637
Valenzuela, F., 577
Vallette, A., 554, 555 (31)
Vallette, F., 577

736

Vandenbelt, J. M., 391, 410, 488

van der Vliet, J., 142, 145 (50), 148, 152,
154, 155

van Dorp, W. A., 475

van Gastel, A. J. P., 478

Van Itallie, T. B., 436

Van Korff, R. W., 603, 609 (22)

van Koetsveld, E. E., 414

Van Lanen, J. M., 642, 645 (34)

van Niekerk, J., 152, 153, 154 (93), 155
(106, 107)

van Nouhuys, F., 684

Van Prohaska, J., 100

van Vloten, G. W., 336

van Vyve, A., 421

van Wijk, A., 152, 153, 154 (93), 155 (106,
107), 178, 181, 182 (225), 199

Vaughan, J. M., 306

Vavich, M. G., 545, 629, 638, 640 (22) 641
(22)

Vedvick, J. R., 661

Veitch, F. P., 30

Velick, S. F., 492

Velluz, L., 177, 194, 202, 204 (316)

Venkatachalam, P. 8., 104

Verly, W. G., 22, 27 (73), 84

Vermeulen, C. W., 100

Verstraete, M., 434

Verwilghen, R., 434

Vestin, R., 485, 488, 506 (45)

Vetter, H., 453, 455 (9), 470

Vicari, F., 440

Victor, J., 75, 108, 113 (427)

Vickery, H. B., 452, 454 (8), 459 (8), 464
(3), 465 (3)

Vierling, K., 11, 14

Vignos, J., Jr., 31

Villar Palasi, V., 198

Vilter, R. W., 518 (7, 15)

Vilter, 8S. P., 555

Vincent, C., 335

Vincent, D., 90

Vincke, E., 426

Vinet, A., 430

Viollier, G., 89, 277, 519 (52), 526, 527 (73)

Virgili, R., 441

Virtanen, E. A., 317

Vishniac, W., 504

Visscher, F. E., 316

Vitale, J. J., 657

Vivanco, F., 87

AUTHOR INDEX

Vivino, E. E., 416

Vogt, Karl, 125

Vohl, H., 374, 376, 385

Voillier, G., 38, 39 (176)

Voleani, B. E., 533, 540

von Babo, L., 3

von Bergen, I., 488, 492 (47)

von Beznak, M., 314

von Braun, J., 11

v. Christiani, A. F., 190

v. d. Heyden, 375

von Euler, E., 491

von Euler, H., 143, 186 (59), 312, 453, 455,
456 (23), 458, 480, 481, 482, 484 (18,
19), 485 (3), 486, 487, 488 (29, 33),
489, 491, 492 (33), 493, 503 (76), 506,
507 (44), 508, 511 (80), 512, 555

von Gréer, F., 301

von Hoesslin, H., 126

von Kaulla, K. N., 427

von Pechman, H., 478

von Reichel, §., 191

von Richter, V., 473, 474, 475

von Valy-Nagi, T., 25, 29 (82)

Von Vloten, G. W., 343

von Werder, F., 158, 173, 179, 186 (134),
191 (134), 198, 194 (283), 208, 204
(134), 207

Vorhaus, M. G., 374, 379, 380 (21)

Voris, L., 656

Vorob’eva, M., 356

Vortmann, G., 473

Vreeland, J., 360

Vuillaume, R., 317

Vritak, E. G., 261

Vyskrebentseva, E., 356

Ww
Wachstein, M., 67

Waddell, J., 95, 139, 148, 155 (91), 156 (91,

119, 120), 204 (119), 205 (119, 120),
206, 208, 214, 215 (10), 218, 220 (23)

Waddell, W. W., Jr., 421, 422 (18), 433
(15)

Wade, N. J., 46, 47 (4), 63, 76, 77, 78 (2,
133)

Wagner, W.,9

Wainwright, W. W., 657

Waisman, H. A., 342, 370, 518 (14), 534,
543, 591, 595, 596 (20), 626, 630, 638
639 (25), 640 (25), 641 (25), 647 (25),

AUTHOR INDEX

654, 655 (37), 656 (37), 657 (87), 658
659 (95, 97), 667, 670, 678, 682 (5, 11)

Wakil, 8. J., 619

Wakim, K. G., 418, 423

Walborsky, H. M., 272

Walcott, A., 56

Wald, G., 495, 563

Waldstein, 8S. S., 379, 385

Walker, R. W., 476

Walker, S. A., 422, 437, 448

Walker, 8S. E., 426

Walker, W. W., 341

Wallace, G. I., 365, 366

Wallenfels, K., 458, 484 (15a)

Wallenmeyer, J. C., 166 (167), 167, 168,
210

Wallis, E. S., 338

Walz, D. E., 12

Wang, C. F., 87

Wang, T. P., 489, 631

Warburg, O., 453, 455 (5, 7), 458 (6, 7),
460, 470, 480, 481 (2, 4), 482, 485 (4),
486 (4), 487, 488, 489 (4, 7), 491 (2, 4),
496, 500, 509, 510, 511

Ward, J. L., 488

Ward, S. M., 505, 512 (152), 556

Ware, A. G., 488

’ Waring, C. H., 552

Warner, E. D., 401, 404, 408, 409 (27),
419, 421, 424, 425, 426, 428, 431 (14),
433 (14), 484, 441, 445, 446 (3), 447,
448

Warnock, G. M., 383

Warren, R., 401

Waterlow, J. C., 104, 548

Waterman, R. E., 661

Watkin, D. M., 436

Watson, C. J., 556, 562 (59)

Watson, E. M., 381

Watson, G. I., 635

Watson, J., 32

Watt, B. K., 548, 545 (8)

Watt, P. R., 359

Waugh, J. M., 428

Waugh, R. K., 102

Wayne, M. G., 470, 471, 472

Webb, A. M., 355

Webb, R. A., 51, 548

Weber, C. O., 334

Webster, T. A., 133, 138, 139, 143 (29),
159, 175, 177, 178, 181, 182 (225, 241),

737

189, 192 (263), 195 (241, 263), 196
(263), 198 (263), 204 (263), 216

Weed, K. L., 254

Weidel, H., 473, 474 (82), 475, 479

Weidlein, E. R., Jr., 339

Weidlich, G., 173, 196 (199), 198 (199)

Weijlard, J., 476

Weiner, M., 427, 431, 435

Weinhagen, A. B., 14

Weinhouse, 8., 206

Weinstein, H. R., 67

Weinstock, H. H., Jr., 592, 593 (4), 594

Weinstock, I. M., 519 (55), 528, 529 (90)

Weinstock, M., 133, 138, 148, 162, 163 (8),
167, 172, 178 (24)

Weir, W. C., 555, 668

Weiss, K. W., 26, 80, 83, 84, 85 (193)

Weiss, M. L., 378, 383

Weiss, 8., 80, 84, 85 (193, 194)

Weitkamp, A. W., 315

Weitz, E. M., 568

Welch, A. D., 25, 27, 37, 40 (77), 46, 47
(3, 8, 10, 11, 15), 48 (8, 10), 49 (10, 12,
13), 66, 94, 418, 423, 655, 685

Welch, M.S., 25, 40 (77), 46, 47 (8), 48 (8)

Welsch, W., 478

Werkman, C. H., 42, 499, 620 (86)

Werner, E. D., 425

Werthemann, A., 89

Wertz, A. W., 564, 648

West, E. S., 360

West, J. W., 95, 578 (24)

West, R., 508

Westerman, B. D., 637

Westall, R. G., 361

Westerfeld, W. W., 87

Westphal, K., 179

Westphal, U., 190

Wetter, F., 158, 154, 155 (105)

Wheeler, G. A., 541 (8, 6), 552, 565, 566

Wheeler, W. R., 474

Whipple, G. H., 532

White, A., 21

White, E. A., 103, 297, 315

White, E. G., 567, 586

White, H. J., 423

White, J., 89

White, M. F., 271, 289

White; P..L-, 76

White, V., 557

Whitebourne, D., 674

738

Whitehair, C. K., 570, 580, 638, 648 (21c),
670

Whittier, C. C., 175

Wibaut, J. P., 478

Wick, A. N., 86

Wicks, L. F., 90, 362

Widenbauer, F., 421, 483

Widmer, C., 281, 288, 291

Wiebelhaus, V. D., 349, 350.(73) , 351 (73),
382

Wiehl, D., 552

Wieland, H., 338

Wiese, A. C., 580, 665, 682 (25)

Wiese, H. F., 295, 300 (14), 301, 303, 304
(40), 806 (80), 307 (30, 31), 308 (32),
309 (83), 310, 314, 522

Wilby, F. W., 629 (15), 658

Wilder, O. H. M., 154, 155 (111)

Wilgus, H.S., 93

Wilke, C. F., 224, 256

Wilkening, M. C., 515, 522, 524 (16), 531,
580

Wilkerson, H. W., 71

Wilkinson, C. F., Jr., 557

Wilkinson, H., 20, 68, 69, 76, 81

Wilkinson, P. A., 148

Willcockson, T. H., 674

Willerton, E., 629 (13)

Willett, E. L., 546

Willets, D. G., 552

Willgeroth, G. B., 207

Williams, D., 661, 682 (20)

Williams, D. E., 317, 531

Williams-Ashman, H. G., 39

Williams, H. H., 4 (16), 53, 565, 638, 648
(216)

Williams, H. L., 381

Williams, J. H., 518 (81)

Williams, J. N., Jr., 34, 35 (150), 36, 37,
38, 39 (150), 82, 84, 85, 87, 96, 277,
495, 508, 512, 524, 529 (91), 554, 555,
556 (40), 585

Williams, R., 600

Williams, R. J., 342, 345, 355, 364 (4), 365
(4), 376, 377, 382, 523, 545, 549, 550
(79), 591, 592, 593 (8, 4), 594, 596 (8a),
597 (a), 598, 622, 626, 627 (3), 629,
630, 636, 637, 638, 639 (28), 640 (28),
641 (28), 645 (28), 647, 648, 658, 659,
662 (85), 668, 677, 678, 680, 688, 690

AUTHOR INDEX

Williams, R. R., 544, 548, 654, 656 (88),
657 (38), 662, 687

Williams, V. R., 313

Williams, W. L., 318, 342, 349 (8), 362,
539, 540 (45), 605, 611 (40), 613, 633

Williams, W. T., 289

Williamson, 8., 458

Willimott, S. G., 229

Willstaedt, H., 54

Willstatter, R., 44

Willumsen, H. C., 486

Wilson, J. B., 17

Wilson, J. E., 27, 84

Wilson, 8S. J., 427

Windaus, A., 139, 140, 141, 142, 143, 144
(44, 48), 146 (88), 147, 152, 153, 154,
155 (94), 158, 178, 175, 177;7Siebee
182 (225), 186 (59a, 110, 134), 191 (59a,
134), 192, 198, 194, 195, 196, 198 (45),
200 (45), 201 (88, 70), 202, 203, 204
(1384) (298, 313, 321), 205, 206

Winegar, A. H., 522, 580

Wing, M., 432

Winkler, A. W., 264

Winnick, T., 42

Winter, L. B., 356, 371, 377

Winters, R. W., 624, 651, 652

Wintersteiner, O., 147, 159, 328, 329

Wintrobe, M. M., 101, 568, 567, 568, 580
(51), 581, 664, 665, 666 (163, 165), 670,
677 (2138, 214)

Winzler, R. J., 54

Wirth, J. C., 495

Wischnegradski, A., 474

Wise, E. C., 207

Wishart, S., 338

Wiss, O., 419 (52), 526, 527 (73)

Withner, C. L., 355, 361

Withrow, R. B., 178

Witten, P. W., 284, 286

Wittenberg, J., 19

Wittle, E. L., 16, 604, 606 (30), 611 (80),
624 (30), 631

Wodicka, V. O., 545

Woessner, W. W., 201, 202 (315), 214, 215
(10)

Wokes, F., 229, 535

Wolbach, 8S. B., 244

Wolf, A., 143, 186 (59)

Wolf, L. E., 668

Wolfe, J. K., 414

AUTHOR INDEX

Wolff, H. G., 317, 380, 381, 438, 440
Wolfrom, M. L., 328, 335, 364 (9)
Wollaeger, E. E., 448

Wollish, E. G., 456, 506 (82), 535 (21), 628

Wommack, M., 20, 21 (57)

Wood, A. J., 52, 92

Wood, H. G., 499, 503, 620 (85)

Wood, J. L., 516

Wood, R. W.., 557

Wood, T. R., 21

Wooden, M. B., 518 (26)

Woods, A. M., 541, 637, 646

Woods, R. R., 532

Woodward, C. F., 466, 467 (68)

Woodward, H. E., 34

Woodward, R. B., 134

Woods, E., 92, 655

Wooley, J. G., 524, 569, 578 (18, 19), 582
(18)

Woolley, D. W., 9, 334, 335 (36), 342, 343,
345 (51), 346 (49), 350, 351, 356, 357,
361, 362 (21), 364 (7), 365, 366, 367,
371, 374, 380, 381, 382, 384, 385, 417,
418, 431, 489, 440, 453, 455 (10), 480,
517 (34), 518 (6), 524, 548 (75), 554,
556 (30), 563, 569, 595, 596 (20), 526,
656, 658, 668, 678

Wordehoff, P., 4

Worden, A. N., 313, 567, 654, 655 (29),
656 (29)

Work, C. E., 596, 656

Wortis, 8. B., 49

Wostman, B., 414

Wrede, F., 4, 127

Wright, E. Q., 688

Wright, I. 8., 381, 427

Wright, L. D., 418, 423, 439, 539, 540 (46),
555, 647, 655, 672, 685, 688, 690

Wright, W. B., 461, 462, 467, 470

Wu, W.., 548, 545 (8)

Wunderlich, W., 141, 146 (89), 200, 201
(313) , 202 (313), 204 (313)

Wurtz, A., 10, 11

Wurtz, E., 694

Wyat, L., 422

Wyatt, B. L., 261

Wynne, E. S., 317

739

yy,

Yacowitz, H., 684, 694

Yanofsky, C., 453, 518 (11), 519, 526, 527
(72), 528 (72), 529 (72), 531 (72)

Yeager, L. B., 264

Yenson, M. M., 312

Yoder, L., 209, 229

You, R. W., 73, 74 (90)

You, 8.58., 74

Young, F. H., 210

Young, L., 359

Young, N. F., 532

Young, R. J., 39

Young, W. J., 482

Vuh coo

Yuska, H., 261

Z

Zacherl, M. K., 190, 191 (273)

Zakon, 8. J., 304

Zamcheck, N., 87, 657

Zamecnik, P. C., 73

Zamfir, D., 576

Zamir, R., 686

Zanthureezky, Z. L., 313

Zatman, L. J., 508

Zeller, A., 312

Zeller, J. H., 297

Zeman, B., 637

Zepplin, M., 638, 639 (29), 640 (29), 641
(29)

Zetterberg, B., 365

Ziegler, A., 242

Zienty, M. F., 472

Ziff, M., 56

Ziffren, S. E., 423, 425, 427, 433

Zilversmit, D. B., 99

Zimmerli, A., 153, 216

Zimmermann, W., 199

Zincke, T., 534

Zon, L., 261

Zscheile, F. P., 287

Zscheile, F. P., Jr., 189, 201 (265)

Zucker, M. B., 442, 672

Zucker, T. F., 1383, 173, 228, 229, 254, 672

Zuckerman, R., 671

Zuzak, H., 441

Zweig, G., 30

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Subject Index

A

Acetaldehyde,
formation from pyruvate, 499
in coenzyme A assay, 603
Acetic acid, 91
as inositol precursor, 355
formation from pyruvie acid, 499
in coenzyme A assay, 599
in lipid synthesis, 622-624
pantothenic acid and, 679
Acetoacetie acid, 502, 625
synthesis of, 618-619
Acetobacter suboxydans, inositol oxida-
tion by, 326, 331, 333, 337, 347-
348, 353-354
Acetone, in methyl group synthesis, 27
Acetylase, 16
Acetylation, in disease, 672
Acetylcholine, 2, 49, 123, 380, 381
adenosine triphosphatase and, 15
comparative activity of, 124
fatty acids and, 317
inactivation of, 17
in choline estimation, 57
in growth of microorganisms, 50, 55
isolation of, 7-8
niacin and, 576-577
oxidation of, 33
pantothenic acid and, 679, 680
synthesis of , 598-599
vitamin K and, 440
Acetylcholinesterase, 17
Acetyl-coenzyme A, 16, 602
isolation of, 607, 618
Acetyl compounds, oxidation of, 502
Acetylkinase, 16-17
Acetyl-8-methyleholine, resemblance to
acetylcholine, 124
Acetyl phosphate, 607
arsenolysis of, 602
as acetyl donor, 616, 617
3-Acetylpyridine,
antiniacin effect of, 548-550, 569
niacin and, 517

741

synthesis of, 477
toxicity of, 5382
Acetyl transfer, coenzyme A and, 615-621
Acetyl tryptophan, 531
Achlorhydria, in pellagra, 560
Achromotrichia, in pantothenic
deficiency, 653-655, 667
Acidosis, effect on rickets, 260
“Active acetate,’’ 615, 617, 625, see also
Acetyl coenzyme A
Actomyosin, 15
Acyl transfer, coenzyme A and, 620-622
Addison’s disease, pantothenic acid, and,
672
Adenine, 455, 481, 482
antiniacin effect of, 548
as inhibitor, 508
in pyridine nucleotides, 485, 489
Adenine dinucleotide, as inhibitor, 508
Adenosine, as inhibitor, 508
Adenosine phosphate, 15
Adenosine triphosphatase, 15
Adenosine triphosphte, 16, 17, 33
as inhibitor, 508
estimation of, 511
in acetyl transfer, 617
in active methionine formation, 33
in choline phosphorylation, 19
in coenzyme A assay, 599
in coenzyme A synthesis, 603
in creatine formation, 30-31
in methylation of niacinamide, 32, 516
pyridine nucleotides and, 496-498, 506
S-Adenosylmethionine, 29
in transmethylation, 25, 33
Adenylic acid, 507
in coenzyme A, 603
Adrenal cortical hormones, coenzyme A
and, 624
Adrenal gland,
achromotrichia and, 655
coenzyme A in, 614
fatty liver and, 72-73
in choline deficiency, 66

acid

742 SUBJECT INDEX

in pantothenic acid deficiency, 650-
652, 663, 667, 672
Adrenochrome, 381
Adrenocorticotropic hormone,
effect in choline deficiency, 73
pantothenic acid and, 652, 686-687
Aedes aegypti, choline and, 103
Age, effect on pantothenic acid require-
ment, 683
B-Alanine, 16
as growth stimulant, 594
formation of, 627
in coenzyme A preparations, 601
in pantothenic acid estimation, 628,

633
Albumin, effect in choline deficiency,
76, 77

Alcohol, in sterol synthesis, 157
Alcohol dehydrogenase, in diphospho-
pyridine estimation, 509
Alcoholism,
choline and, 104-108
pellagra and, 562, 565
Aldehydes, in vitamin D estimation, 217
Allantoin, excretion in leucopenia, 91
Allocholesterol, 139
Alopecia, in inositol deficiency, 367, 374
A-methopterin, in creatine formation, 31
p-Aminoacetophenone, 535
p-Amino acid oxidase, 30
Amino acids,
effect on niacin requirement, 581-582
coenzyme A and, 625
sulfur, effect in choline deficiency,
76-80
vitamin K and, 438
a-Aminoacrylice acid, in glycine forma-
tion, 42
4-Aminoazobenzene, in
assay, 600
p-Aminobenzoic acid, 368-369, 380, 423
achromotrichia and, 655
inositol and, 384
vitamin By and, 86
Aminodesoxyinositols, naming of, 327-
329
Aminoethanol, 15, 26, 29-30 (Ethanol-
amine)
effect in choline deficiency, 68, 70-71, 72
effect on choline oxidase, 36
formation of, 27, 42-43, 83-84

coenzyme A

in choline isolation, 7
in choline synthesis, 11, 23, 30, 33
in growth of chick, 94
in growth of microorganisms, 49, 50,
51, 55
methylation of, 40
phosphorylation of, 19
a-Amino-§-ketopropionic acid, in glycine
formation, 42
1-Amino-2-methylnaphthalene, vitamin
K and, 397
4-Amino-2-methyl-1-naphthol hydro-
chloride,
vitamin K and, 397
Aminopterin,
choline oxidase and, 37-38, 84
in creatin formation, 31
3-Aminopyridine, formation of, 472
6-Amino-n-valeric acid, niacin and, 523
a-Amylase, inositol and, 345
Androstenediol, 142
Anemia,
in choline deficiency, 87, 98, 99
in pellagra, 563-564
vitamin K and, 441, 443
Aniline,
in niacin estimation, 535
in vitamin K estimation, 414
methylation of, 44
Anserine, formation in rabbit, 102
Anthranilic acid, in niacin synthesis,
528, 533
Anthraquinone, vitamin K and, 398
Antibiotics,
effect on trimethylamine excretion,
127-128
fatty acids as, 317
niacin and, 583
Anticoagulant ‘63’, 427-428
Antidermatitis factor, see Pantothenic
acid
Antihemorrhagic vitamin, see vitamin K
Antimony pentachloride, in vitamin D
detection, 217
Antimony trichloride, in vitamin D
estimation, 216-217
Antirachitie vitamin, 132
Antithrombin, in vitamin K deficiency,
432
Antivitamins K, production of, 429-432
Apatite, 234

SUBJECT INDEX

Arachidonic acid, 268, 274, 284
effect in deficiency, 278-280
effect on gizzard erosion, 402
formation from linoleate, 281
isolation of, 271-272
Arecoline, 574
toxicity of, 571
Arsenate, 510
Arsenite, methylation of, 51
Arsenocholine, 40
effect in choline deficiency, 66
in growth of microorganisms, 50
in lecithin synthesis, 49
in perosis, 94, 97
oxidation of, 34
Arsenolysis, in coenzyme A assay, 601-
602
Arteriosclerosis, choline and, 99
Arthritis, 261, 264
Ascorbic acid, 694
choline oxidase and, 37-38
inositol and, 356
in pantothenic acid deficiency, 651-
652, 673, 687
pantothenic acid requirement and, 685
Asparagine, niacin synthesis and, 523
Aspartic acid, B-alanine and, 627
Atabrine,
effect in choline deficiency, 67
effect on choline oxidase, 38-39
Atherosclerosis,
choline and, 102
inositol and, 373-374
ATP, see Adenosine triphosphate
Aureomycin, pantothenic acid require-
ment and, 685-686
Autonomic nervous system, effect of
choline on, 123

B

Benzedrine, effect on choline oxidase, 36
Benzoic acid, 41, 42

in glycine deficiency, 84
Benzoyl peroxide, fatty acids and, 286
Beri-beri, 674

pantothenic acid and, 637, 690
Betaine, 6, 20, 51, 83, 91, 516

as methyl donor, 25, 29, 33

demethylation of, 40

effect on choline requirement, 2, 41,

45-48, 66, 68, 71-72, 103, 128, 129

743

effect on perosis, 94, 97
fate of, 92-93
formate formation from, 41
formation of, 25, 33, 39
in choline estimation, 53
in choline isolation, 7
in creatine formation, 31
in growth of microorganisms, 50, 55
in hippuric acid formation, 41
in methionine formation, 23
in methylation, 23
niacin toxicity and, 573
oxidation of, 43
physiological activity of, 124
relation to choline and glycine, 40
requirements of chick, 94-95
separation of, 10
utilization by guinea pig, 38
vitamin By and, 84
Betaine aldehyde, 127
as methyl donor, 25
effect on choline oxidase, 35
oxidation of, 33-84, 35, 39
Betaine aldehyde oxidase, 127
localization of, 36
Betaine-homocysteine transmethylase,
vitamin By, and, 85
Bile, 134
effect on vitamin D absorption, 259
in vitamin K absorption, 423, 424-425
Bilineurine, see Choline
Biotin, 655, 694
burning feet syndrome and, 677
deficiency, distinction from panto-
thenie acid deficiency, 659
effect in inositol deficiency, 343, 367—
368, 369
fatty liver and, 80, 81
pantothenic acid and, 671-672, 685
perosis and, 94, 97
Bismuth salts, in choline isolation, 7
Blacktongue, 453, 480
pathological changes in, 565-567
symptoms of, 553
Blatella germanica, choline deficiency
in, 103
Blood, in pantothenic acid deficiency, 653
Blood-clotting time, in vitamin K assay,
406-407
Blood pressure, effect of choline on, 123
Bone development,

144. SUBJECT INDEX

cartilaginous, 233-237
membranous, 232-233
Bone ash, in vitamin D estimation, 218—
220
marrow, in
deficiency, 653
Bornesitol, 334
Bradyeardia, in choline deficiency, 91
Brain, in pellagra, 561-562
Brassicasterol, 135, 136
relation to ergosterol, 158
Bromides, of unsaturated fatty acid, 289
(2-Bromoethy])trimethylammonium
bromide, in choline synthesis, 11
Burning feet syndrome, in nutritional
deficiency, 675-677
Butyric acid, synthesis of, 619

Bone pantothenic acid

C

Caffeine, fatty acids and, 314
Calciferol, 132, 142, see also vitamin D»
absorption spectrum of, 177
isolation of, 148-152
properties of, 195-198
Calcification, in rickets, 231
Calcinosis, in hypervitaminosis D, 264
Calcium,
balance in rickets, 228-229
deposition in bone, 233-234
effect on vitamin D requirement, 254
factors affecting serum levels, 248-253
in rickets production, 223-224
pantothenic acid and, 636
requirements, 257-258
serum levels in rickets, 225-226
Calf, choline deficiency in, 102
Caloric intake, effect on choline de-
ficiency, 78, 106
Campesterol, 135, 136
Capillaries, in ricketiec bone, 243-245
Carbachol, see Carbaminoylcholine
Carbaminoylcholine, resemblance to
acetylcholine, 124
Carbenium salts, in vitamin D estima-
tion, 217
Carbohydrates, 17
coenzyme A and, 625
effect in fat deficiency, 292
effect on calcium absorption, 248
effect on niacin requirement, 582-583

effect on pantothenic acid require-
ment, 684
in fatty acid formation, 280
in inositol synthesis, 356
metabolism in choline deficiency, 91
in sterol synthesis, 156-157
Carbon dioxide, from choline, 93
Carbon tetrachloride, liver fat and, 74
Carcinogens, 134
fatty acids and, 313
Cardiolipin, 312
Carnitine, 3
relation to choline, 2
Carotene, 90
fatty acids and, 315
vitamin K and, 415
Carotenoids, biosynthesis of, 625
Cartilage,
in bone formation, 233
in rickets, 231
Casein,
iodinated, 85, 87
lipotropic effect of, 76, 78
Catalase, 312-313
Cataract, niacin and, 563
Cattle, pantothenic acid deficiency in,
666-667
Celiac disease, serum fatty acids in, 306
Cephalin, choline oxidase and, 36
in choline deficiency, 71, 72
relation to lecithin, 99
Cerebronic acid, 346
Cerebrosides, 58-61
Ceroid, in choline deficiency, 113-114
in cirrhotic livers, 75
Chest, deformation in rickets, 288-239
Chick,
fatty acid deficiency in, 296-297
in pantothenic acid assay, 629-630
in vitamin K assay, 403
niacin deficiency in, 569-570
pantothenic acid deficiency in, 658-661
antidermatitis factor, see Pantothenic
acid
antipellagra factor, see Pantothenic
acid
Chloroform, liver fat and, 74

Chlorophyll, relation to vitamin Kk,
415-416

Chloroplasts, pyridine nucleotides in,
504

eee

Se

ee sel a SS

SUBJECT INDEX 745

Cholecalciferol, 182, see Vitamin D;
Cholestanol, 135, 203
A‘, 7, 2_Cholestatriene-3-ol,
as provitamin D, 142, 145
in invertebrates, 155
Cholesterilene, 208 sulfonated, 209
Cholesterol, 135, 203, 307, 625
arteriosclerosis and, 99
as source of dehydrocholesterol, 159
choline and, 69, 72, 97, 98
dehydrogenation of, 146-148
destruction of provitamins in, 207-208
effect on 7-dehydrocholesterol, 201—
202
fatty livers and, 20, 82
inositol and, 372-373, 384-385
in pantothenic acid deficiency, 652,
663, 665
irradiation of, 138-139
occurrence of, 135-136
source in animals, 158
synthesis of, 622-623
vitamin D and, 183, 214-215
Cholesterol dibromide, 138-139
Cholic acid, effect on gizzard erosion,
402
Choline, 51, 299, 371, 380, 487, 517, 655
deficiency,
amino acids and, 76-80
biochemistry of, 15-45
biogenesis of , 26-28, 43, 49-52, &3
chemistry of, 2-14
constitution and synthesis, 10-14
creatine and, 21-22
cyclic sulfate of, 9
demethylation of, 40
effects of deficiency, 62-123
avian species, 93-97
dog, 97-101
general manifestations, 62-63
man, 104-123
other species, 101-103
rat, 63-93
effect on fat absorption, 90
effect on fatty liver, 19-20, 347
estimation of, 52-57
biological methods, 57
chemical methods, 52-55
microbiological methods, 55-57
physical methods, 57
excretion of, 127

fate of, 92-938, 126-128
fatty acids and, 315
glycerylphosphorie acid esters of, 12
industrial preparation of, 14
in growth of bacteria, 49
in methylation of guanidoacetic acid,
30, 31

inositol and, 370, 385
in serine formation, 4]
intake of humans, 129
isolation of, 2-10
isotopic, 11, 12, 40
leucocytes and, 90-91
mechanism of action, 18-45
niacin toxicity and, 573
nomenclature, 2
occurrence of, 58-62
oxidation of, 25, 43
pantothenic acid and, 685
pharmacology of, 123-128
phospholipids and, 45, 72
precipitation of, 5
properties of, 10
relation to ethanolamine, 40
requirements

of aminals, 128-129

of chicks, 94-95

of humans, 129-130
separation from methionine, 55-56
salts of, 6, 7, 12-14
separation of different forms, 9
specificity of action, 45-49
standardization of activity, 58
toxicity of, 124-126
transmethylation and, 19-24, 29, 33
utilization by guinea pig, 38, 103
vitamin By. and, 84-85
vitamin K and, 88

Choline acetylase,

coenzyme A and, 15-17
inhibition of, 17
vitamin K and, 488

Choline dehydrogenase, see Choline

oxidase

Cholinesterase, 578

acetylcholine and, 17
aoe

in fat deficiency, 277

Choline oxidase, 67, 82, 127

in fat deficiency, 278
in guinea pigs, 103
inhibitor of, 36

746 SUBJECT

in susceptibility to deficiency, 71
in transmethylation, 26, 28, 33-40
localization in cell, 35, 36
vitamin Bye and, 96
Cholinepherase, 31
Choline phosphokinase, substrates of, 19
Choline phosphoric acid, 61
Chondroitin sulfate, 236-237
Chromatography,
in choline isolation, 9-10
in isolation of fatty acids, 271-272
in niacin assay, 535, 536
Cinchomeronic acid, niacin formation
from, 473
Cinchophen, 437
fatty acids and, 317
Cirrhosis,
inositol and, 372
portal versus non-portal, 114-123
Citric acid, 502, 680
in acetylcholine formation, 16
synthesis of, 607, 618, 625
Citrovorum factor,
choline oxidase and, 37
in methyl group transfer, 33
relation to choline, 83-84
Clupanodonic acid, as essential fatty
acid, 270
Coagulation vitamin, see Vitamin K
Codehydrase I, see Diphosphopyridine
nucleotide
Codehydrase II, see Triphosphopyridine
nucleotide
Codehydrogenase I, see Diphosphopyri-
dine nucleotide
Codehydrogenase II, see Triphospho-
pyridine nucleotide
Cod liver oil, 133
vitamins D in, 205-206
Coenzyme A, 673, 679
assay of, 599-601, 630
assay of pantothenic acid in, 610-611
chemistry of, 603-610
definition of unit, 633
distribution of, 614-615, 638
functions of, 624-625
in choline acetylation, 15-17
in fatty acid synthesis, 619
metabolic functions of, 614-625
pantothenic acid in, 601
preparation of, 609-610
reaction with acid anhydrides, 608

INDEX

removal from enzymes, 602
split products as microbial growth
factors, 611-614
structure of, 608-609
synthesis of, 604-605
Coenzyme I, see Diphosphopyridine
nucleotide, DPN
Coenzyme II, see Triphosphopyridine
nucleotide, TPN
Colchicine, inositol and, 344
Cold, effect in choline deficiency, 74
Collagen, in cartilage matrix, 236-237
Collidine, in industrial niacin produc-
tion, 479
Colostrum, choline in, 102
Conduritol, 338
Copper,
choline and, 76
deficiency of, 654
Coprophagy, vitamin K and, 417, 423
Coproporphyrin I, in pantothenic acid
deficiency, 656
Coproporphyrin III, in pellagra, 562
Coprosterol, 203

Coramine, see N,N-Dimethylnicotin-

amide
Coreductase, see Diphosphopyridine
nucleotide

Corn,
as source of inositol, 340-842
effect on niacin requirement, 584
pellagra and, 565
tryptophan and, 524
Cortical hormones, 134
Corticosterol, in pantothenic acid de-
ficiency, 624
Cortisone,
in hypoprothrombinemia, 448
in pantothenic acid deficiency, 652
liver fat and, 73, 74
Cothromboplastin, vitamin K and, 433-
434
Coumaliec acid, 478
Cozymase, see Diphosphopyridine nuc-
leotide
Creatine, 380, 516
excretion in leucopenia, 91
formation of 26, 27, 30-31, 40, 41, 102
in choline deficiency, 71, 129
in growth of microorganisms, 50, 55
in perosis, 93, 97
methyl groups of, 21-22, 23, 80

SUBJECR INDEX

Creatinine, in pantothenic acid defi-
ciency, 665
Creatinuria, inositol and, 374
Crotonic acid, synthesis of, 619
Curare,
niacin and, 577
resemblance to choline, 124
Cyanide, effect on choline oxidase, 36
Cyanogen bromide, in niacin estimation,
534-535
3-Cyanopyridine, 475
formation of, 472
niacinamide synthesis from, 477
Cyclohexitols, see Inositols
Cyclopentitols, 329
Cyclotetritols, 329
Cysteamine, 613, 625
in coenzyme A, 606
Cysteine, 44, 51
in acetylation systems, 618
Cysteinesulfinic acid, coenzyme in oxi-
dation of, 481
Cystine, 129, 380, 605, 655
deficiency, 75
effect on choline oxidase, 36
effect on fatty liver, 20
fatty acids and, 285-286
in choline deficiency, 71, 74, 76-78,
79, 82
in diet of chicks, 94
in inositol deficiency, 368
in pellagra, 553
methionine toxicity and, 95
niacin toxicity and, 573
relation to methionine, 21
serine and, 43
Cytochrome, 497
fatty acids and, 286
Cytochrome c, 39
choline oxidase and, 34, 35, 36
in triphosphopyridine nucleotide esti-
mation, 511
Cytochrome oxidase,
in fat deficiency, 277
in osteoblasts, 237
lipid in, 278

D

Dambonitol, 334
Dambose, see Inositol
Deformities, in rickets, 238

747

7-Dehydrocampesterol, 141
as provitamin D, 146, 206
Dehydrocholestenone, 142
7-Dehydrocholesterol, (Provitamin Ds)
activation of, 214
formation from cholesterol, 208
halides, provitamin D activity of, 143
in invertebrates, 155
in vitamin D estimation, 217
occurrence of, 1382, 137, 154-155
properties of, 201-202
source in animals, 158
synthesis of, 140
7-Dehydroclionasterol, 141
as provitamin D, 146
in invertebrates, 155
7-Dehydroepicholesterol,
as provitamin D, 144, 207
synthesis of, 142
7-Dehydro-3-homocholesterol, 144
7-Dehydrositosterol, 136, 141, 203
as provitamin D, 146
7-Dehydrostigmasterol, 141
as provitamin D, 146
7-Dehydrothiocholesterol,
min D, 144
Dephospho-coenzyme A, synthesis of,
604-605, 608
Dermatitis, in pantothenic acid de-
ficiency, 656-657, 659, 667-668
Desaminodiphosphopyridine nucleo-
tide, activity of, 493-494
Desoxycholic acid, in vitamin K absorp-

as provita-

tion, 425

Desoxycorticosterone, in choline de-
ficiency, 73

Desoxycortisone, achromotrichia and,
655

Desoxypyridoxine, 585

Diaphysis, 233

1,2,5,6-Dibenzanthracene, choline and,
89

Dibromostearic acid, in linoleic acid
isolation, 271

Dichlorohydrin, in vitamin D estima-
tion, 217

Dichlorophenolindophenol, in vitamin
K estimation, 414

Dicoumarin, see Dicoumerol

Dicoumarol, 419

effect on microorganisms, 439

748 SUBJECT INDEX

in vitamin K estimation, 411, 413
mode of action, 428-432
prothrombin and, 418, 424, 427-428
Dienoic acid, conversion to other acids,
283
Diet,
effect on choline oxidase, 38-39
effect on choline requirement, 128-129
effect on inositol requirement, 383-384
effect on niacin requirement, 541
effect on pantothenic acid in tissues,
636-637
in vitamin K deficiency, 421-422
in vitamin K estimation, 404-405
Diethylaminoethanol, in growth of
microorganisms, 50, 55
N, N-Diethylnicotinamide,
central effect of, 574
toxicity of, 572, 573
Dihydrocholesterol,
in dehydrocholesterol formation, 158—
159
irradiation of, 1388
occurrence of, 135
5-Dihydroergosterol, 136, 183
22-Dihydroergosterol,
as provitamin D, 145-146
irradiation of, 205
occurrence of, 154
synthesis of, 141-142
Dihydrositosterol, 135
irradiation of, 138
occurrence of, 136
relation to ergosterol, 158
Dihydrotachysterol,
effect on blood calcium, 207
properties of, 207
Dihydroxyacetone phosphate, 498
pyridine nucleotides and, 501
3,7-Dihydroxyandrostadiene,
as provitamin D, 144
formation of, 142
3,4-Dihydroxyanthranilic acid, in niacin
synthesis, 528
1,2-Dihydroxyanthraquinone, vitamin K
and, 398
3,7-Dihydroxy-A*-cholenate, 142
Dihydroxystearic acid, in vitamin K
deficiency, 424
Dimethylamine,
formation of, 92

in choline estimation, 53
in growth of microorganisms, 50, 55
Dimethylaminoacetic acid, formation
from betaine, 44
p-Dimethylaminoazobenzene, DAB, But-
ter yellow, choline and, 88-89
Dimethylaminoethanol, 25, 40
as methyl donor, 28
effect in choline deficiency, 68, 70, 128
esterification of, 17
formation from choline, 10
in choline isolation, 6
in choline synthesis, 11
in growth of microorganisms, 50, 55
in perosis, 94
methylation of, 23
phosphorylation of, 19
Dimethylaminoethanol acetic ester, bi-
ological activity of, 17
2,5-Dimethylbenzoquinone, vitamin K
and, 398
Dimethylethylhydroxyethylammonium
chloride, in growth of microor-
ganisms, 50
choline replacement by, 45
Dimethylglycine,
formation from choline, 40
in choline formation, 41
in hippuric acid formation, 41
in transmethylation, 25, 28
Dimethylpropiothetin, 86
oxidation of, 48
Dimethyl-6-propiothetin,
choline, 2
Dimethylselenide, excretion of, 45
Dimethyl] sulfide, in methylation, 51
Dimethylsulfone, isolation of, 44
Dimethylthetin, 44, 516
as methyl donor, 25, 26, 29, 33
oxidation of, 43
relation to choline, 2
Dimethylthetin transmethylase, 26
Dimethylvinylamine, formation from
choline, 10
Dinicotinylornithine, 457
excretion of, 515
Dinitrophenylhydrazine, in vitamin K
estimation, 414
Diphenylphosphorylcholine, 12
Diphosphoadenosine, in coenzyme A,
604

relation to

7
‘
i

SUBJECT INDEX

Diphosphoesterase, effect on coenzyme
A, 604
1,3-Diphosphoglyceraldehyde, 498-499
oxidation of, 496-497
Diphosphopyridine nucleotide, 35, 39,
455, 604, see also Pyridine nucleo-
tides
biosynthesis of, 506
effect on choline oxidase, 82
estimation of, 509-511
in coenzyme A assay, 603
in fatty acid synthesis, 619
in oxidation of betaine aldehyde, 34, 36
isolation of, 458, 482-484
nicotinic acid in, 453, 480
properties, 486-487
structure 484-486
Diphosphothiamine, 17
Dipicrylamine,
in acetylcholine isolation, 8
in choline isolation, 7
Disease, vitamin D treatment of, 261
Docosahexaenoic acid,
as essential fatty acid, 270
effect in deficiency, 280
Dog,
fatty acid deficiency in, 295-296, 307,
314
niacin deficiency in, 553, 565-567
pantothenic acid deficiency in, 663-664
Doryl, see Carbaminoylcholine
DPN, see Diphosphopyridine nucleotide
Duck, pantothenic acid deficiency in,
661-662
Ducklings, choline deficiency in, 97
Dyspnoea, in rickets, 239

E

Eezema, serum fatty acids in, 303-305
Edema, in choline deficiency, 86
Edestin, effect in choline deficiency, 78
Egg,

inositol in, 357

niacin synthesis in, 532

vitamin K in, 445
A5,8,11,14-Eicosatetraenoic

Arachidonic acid

Elaidic acid, 69
Elaidolinolinoleic acid, 282
Electrodialysis, 9

acid, see

749

Eleostearic acid, conversion to other
acids, 283
‘‘Eneephalopathy,
Enterokinase, 101
Ephedrine, effect on choline oxidase, 36
Epicalciferol, 206, 207
Epicholesterol, 142
Epiergosterol, 206
as provitamin D, 144
synthesis of, 142
Epinephrine, 380, 381, 652
formation of, 32
vitamin By. and, 85
Epiphysis, in bone formation, 233
Ergocalciferol, see Calciferol, vitamin D2
Ergosterol, 132, 135, 136, 137
absorption spectrum of, 174
as provitamin D, 139-140
coenzyme A and, 623
color reactions of, 189-191
esters, as provitamin D, 143
energy of activation, 176
formation in plants, 156-158
in invertebrates, 155-156
in vitamin D estimation, 217
irradiation of , 139-140, 212
nitrite treatment of, 208-209
occurrence, 153-154
properties of, 183-189
purification of, 183-184
source in animals, 158
Ergosterone, 142
Ergothionine, relation to choline, 2
Eryocalciferol, 132
Essential fatty acids, 384
biochemical systems, 277-278
biogenesis and metabolism, 280-286
chemistry of, 268-277
commercial preparations of, 272-273
deficiency,
histopathology of, 298-300, 309-310
in animals and insects, 292-300
in humans, 300-311
estimation of,
animal assay, 290-291
chemical methods, 288-289
enzymatic methods, 289-290
spectrophotometric methods, 286-
288
isomers,

”)

in pellagra, 562

750

ultraviolet absorption of, 274

utilization of, 274, 282-283
nomenclature and formulas, 268
occurrence in foods, 291-292
oxidation of, 274-275
pharmacology of,

biological aspects, 313-315

chemical aspects, 311-313

chemotherapy, 317

vitamin interrelationships, 315-316
physical properties of, 275-277
pyridoxine and, 283-285
requirements,

of animals, 318-319

of humans, 319
riboflavin and, 286
specificity of action, 278-280
synthesis by animals, 280-283
tocopherol and, 285-286

Esterases, acetylcholine and, 17
Ethanol, 348, 499

liver damage and, 75-76
oxidation of, 679

Ethanolamine, 346, see also Amino-
ethanol, relation to betaine and
choline, 40

Ethionine,

effect on choline oxidase, 39
effect on transmethylation, 79-80
2-(Ethoxymethoxy)ethyldimethylamine,
in choline synthesis, 14
2-(Ethoxy-methoxy)ethyl-trimethylam-
monium formate, in choline syn-
thesis, 11, 14
Ethyl cyanoacetate, in vitamin K esti-
mation, 414
Ethylene bromide, in choline synthesis,
11
Ethylene chlorohydrin, in choline syn-
thesis, 11, 14
Ethylene dichloride, liver fat and, 74
Ethylene oxide, in choline synthesis,
11, 14
S-Ethylhomocysteine, see Ethionine
Ethyl laurate, 69
3-Ethylpyridine, niacin synthesis from,
474-475
Eye,
in nutritional deficiency, 674-675

SUBJECT INDEX

F

Factor V, see Diphosphopyridine nucleo-
tide
Farnesol, in vitamin K, 395
Fats, 233
choline oxidase and, 36
effect in choline deficiency, 79, 91, 129
effect on calcium absorption, 248
effect on niacin requirement, 584
inositol and, 384
stainable,
in cirrhotic livers, 108-113
Fatty acid, 17, 625
essential,
fatty liver and, 82
in pantothenic acid assay, 633
pantothenic acid and, 678-679
pyridine nucleotides and, 502
synthesis of, 619, 622-624
unsaturated,
in ceroid production, 113
Fatty cysts, in choline deficiency, 109-
1138
Fatty liver,
in choline deficiency, 98
inositol and, 371
in pantothenic acid deficiency, 663
vitamin K and, 437
Fibrin, effect in choline deficiency, 78
Fibrinogen, vitamin K and, 444
Fibrosis, of liver, 108-110
Fish, vitamin D in, 164-172
Fish liver oils, production of, 210
Flavin adenine dinucleotide, 625
choline oxidase and, 39-40, 87
glycine oxidase and, 42
Flavin enzymes, pyridine nucleotides,
and, 490
Flavin mononucleotide, 498
Fluoride, 19
Folacin, see Folic acid
Folie acid,
achromotrichia and, 655
choline oxidase and, 37-38, 96
effect on niacin requirement, 530, 585-
586
fatty liver and, 80
in creatine formation, 31
in inositol deficiency, 368

SUBJECT INDEX

in methyl group synthesis, 23, 84-85
in pantothenic acid deficiency, 653,
671-672, 685

in pellagra, 553, 563

leucocytes and, 90-91

niacin toxicity and, 573

relation to choline, 49, 88-84, 129
Folinic acid, see Citrovorum factor
Formaldehyde,

formation from glycine, 41, 42

in methyl group synthesis, 27, 33

formation from sarcosine, 41
Formic acid, 83

formation of, 41, 127

in creatine formation, 31

in histidine synthesis, 28

methyl groups and, 22-23, 27, 33, 43-44

in serine synthesis, 27, 41-42

in transmethylation, 29

metabolism of, 83-84, 86
10-Formylfolic acid, in creatine forma-

tion, 31
N-Formylkynurenine, in niacin’ syn-
thesis, 527
Fructose-1,6-diphosphate, 498, 501, 510—-
511

Fucosterol, 135, 136
Fumarie acid, 509
in creatin formation, 31

G

Galactose,
reaction with sphingosine, 58-61
Gammexane, 336, see also Hexachloro-
cyclohexane
Gastric ulcers,
choline and, 90, 98
pyridoxine and, 90
Gastrointestinal tract,
inositol and, 379-380
pantothenic acid and, 672-673
Gelatin,
effect in choline deficiency, 76, 78
effect on perosis, 93-94, 97
Gliadin, effect in choline deficiency, 77
Glucose, inositol and, 354, 355-356,
377-378
Glucose dehydrogenase,

751

coenzyme of, 493
inhibition of, 508
in triphosphopyridine nucleotide es-
timation, 511
Glucose-1-phosphate, pyridine nucleo-
tides and, 497-498
Glucose-6-phosphate, 511
oxidation of, 482
Glucosides, cardiac, 134
Glucuronolactone, pantothenic acid and
685
Glutamic acid, 625
niacin synthesis and, 522-523
pyridine nucleotides and, 502
Glutamic dehydrogenase, coenzyme of,
493
Glutamine, 32
in niacinamide synthesis, 505
niacin synthesis and, 523
Glutathione, 51
in acetylation systems, 618
vitamin K and, 440
Lt-Glyceraldehyde, 325
Glyceraldehyde-3-phosphate, in diphos-
phopyridine nucleotide estima-
tion, 510
Glycerol, 501, 619
in inositol estimation, 360
Glycerophosphate, 8, 510
hydrolysis of, 19
in lecithin synthesis, 99
phospholipid synthesis and, 70
pyridine nucleotides and, 501
Glycerylphosphorylcholine, 2, 8-9, 12, 62
in lecithin synthesis, 52
in tissues, 18-19
Glycine, 574
conversion to glyoxylate, 42
effect on perosis, 93-94, 97
formate formation from 41
formation from sarcosine, 41
in aminoethanol formation, 83-84
in heme synthesis, 622
in serine synthesis, 27, 41, 42, 85
leucocytosis and, 91
methyl derivatives of, 40
niacin toxicity and, 573
relation to betaine, 40
relation to choline, 2, 40, 71, 79

752

Glycine oxidase, 42
Glycogen, 86, 233
formation from inositol, 351
in bone formation, 235, 237
pantothenic acid and, 678
pyridine nucleotides and, 497-498
vitamin K and, 487
Glycol, formation from choline, 10
Glycolic acid, in inositol estimation, 360
Glycolysis, pyridine nucleotides and, 498
Glyoxylic acid, formation from glycine
and sarcosine, 42
Gold salts, in choline isolation, 7
Gonadotropin, in fatty acid deficiency,
299
Gossypol, in vitamin deficiencies, 315
Growth,
in pantothenic acid deficiency, 656
vitamin D and, 220, 225
Growth hormone, liver fat and, 73
Guanidoacetic acid, 516
effect on guinea pig, 103
methionine and, 95
methylation of, 21, 23, 26, 30, 33
Guanidoacetic acid methylpherase, 31
Guinea pig, pantothenic acid deficiency
in, 664
Guvacine, niacin and, 523

H

Hair, discoloration of, 92
Harden’s coferment, see
pyridine nucleotide
Hatchability,
choline and, 96
in pantothenic acid deficiency, 660
Heart,
inositol and, 377-879
in pellagra, 561
Hemaptopoiesis, pantothenic acid and,
671-672
Heme, 625
synthesis of, 622
Hemin, fatty acids and, 312
Hemoglobin, fatty acids and, 312
Hemophilia, differentiation from vita-
min K deficiency, 420
Hemorrhage, in vitamin K deficiency,
419-420
Hemosiderin, relation to ceroid, 114
Heparin, choline and, 88

Diphospho-

SUBJECT INDEX

Hexabromostearic acid, in arachidonic
acid isolation, 273
Hexachlorocyclohexane, 367
as inhibitor, 343-845
relation to inositol, 336
Hexaenoic acid, 284
deposition of, 282
formation from linoleate, 281
Hexahydroxycyclohexane, see Inositol
Hexose phosphoric acid, in bone for-
mation, 235
Hippuric acid,
formation from sarcosine, 41
formation from serine, 42
Histamine,
effect on choline oxidase, 36
in choline estimation, 57
niacin and, 576-577
separation from histidine, 9
Histidase, products of, 28
Histidine,
choline oxidase and, 39
in methyl group synthesis, 27-28
separation from histamine, 9
Histiocytes, choline and, 91
Homocysteine, 516
arsenocholine and, 94
choline oxidase and, 37
methylation of, 23, 26, 29, 33, 103
Homocysteinethiolactone, as methyl ac-
ceptor, 29
Homocystine, 26, 51
effect in choline deficiency, 76
in creatin formation, 30-31
in diets, 45, 94-95
in growth of bacteria, 49
methylation of, 25, 26, 28-29, 83
niacin toxicity and, 573
relation to cystine, 20-21
relation to methionine, 21
toxicity of, 95
Human,
choline deficiency in, 104-123
fatty acid deficiency in, 300-311
niacin deficiency in, 552-553
niacin requirement of, 586-587
pantothenic acid deficiency in, 669-678
pantothenic acid requirements of , 687—
694
pathology of pellagra, 570-571
Hydrochloric acid, secretion of, 560

SUBJECT INDEX

Hydrocoumarol, 427-428
Hydrogen, attachment to pyridine nu-
cleotides, 492
Hydrogenation, effect on essential fatty
acids, 273-274
Hydrolapachol, vitamin K and, 397
Hydrosulfite, in diphosphopyridine nu-
cleotide estimation, 511
Hydroxamiec acid reaction, 602
3-Hydroxyanthranilic acid, in niacin
synthesis, 525-526, 527-528, 532-
533
B-Hydroxybutyric acid,
pyridine nucleotides and, 502
synthesis of, 619
3-Hydroxy-A*:7-choladienic acid,
as provitamin D, 144-145
formation of, 142
7-Hydroxycholesterol, 140
formation in body, 159
irradiation of , 206
synthesis of, 147
a-Hydroxy-8, 6-dimethyl-y-butyrolac-
tone, synthesis of , 597
3-Hydroxykynurenine, in
thesis, 526
Hydroxyisomytilitol, vitamin activity
of, 352

niacin syn-

1-Hydroxy-2-methylnaphthalene, vita-
min K and, 397
1-Hydroxy-3-methylnaphthalene, vita-

min K and, 397
Hydroxypantothenic acid, 627
Hypercholesteremia, choline and, 102
‘“Hyperprothrombinemia”’, 444
Hypertension, choline deficiency and, 88
Hyperthyroidism, acetylation in, 672
Hypervitaminosis D,

factors affecting, 265-266

clinical symptoms, 265

pathology of, 264
Hypoparathyroidism, 250
Hypophysectomy, effect on liver fat, 99
Hypoprothrombinemia,

dicoumarol and, 428

of newborn, 421-422, 433, 448

I

Indirubin, niacin and, 557
Indole, 533
Indole-3-acetic acid, 548

753

Infantile cirrhosis, choline and, 104
Infection, effect on Vitamin D absorp-
tion, 259-260
Tnosamines, 327
Inosite, see Inositol
Inositol, 86
absorption of, 349-350
antiketogenic effect of, 349, 351
biochemical systems, 342-351
biogenesis, 385-386
by animals, 356-358
by microorganisms, 354-855
by plants, 355-356
chemistry of, 329-838
effects of deficiency,
in animals, 367-371
in humans, 371-376
in microorganisms, 366-367
effect on cholesterol esters, 69
effect on fatty liver, 80-81, 347, 370, 384
estimation of,
chemical methods, 359-360
isolation, 358-859
microbiological assay, 361-362
mouse assay, 361
paper chromatography, 361
fatty acids and, 315
glucose formation from, 357-858
hydrolysis of bound forms, 362
industrial preparation of, 339-342
metabolism,
in animals, 349-351
in bacteria, 347-349
natural antivitamins of, 363-366
nomenclature and formulas, 322-330
occurrence of, 334-336, 363-366
pharmacology of, 377-381
gastrointestinal tract, 379-380
heart, 377-379
neuromuscular system, 380-381
thyroid, 380
properties of, 336-338
spatial constellations of, 331-333
specificity of action, 351-354
requirements,
of animals, 381-382
of humans, 382-383
allo-Inositol, 328, 324, 338
p-Inositol, vitamin activity of, 352
ept-Inositol, 323, 324, 338
vitamin activity of, 352

754

i-Inositol, 322
L-Inositol, vitamin activity of, 352-353
meso-Inositol, 322, 338

occurrence of, 334-335

synthesis of, 338
muco-Inositol, 323, 324, 338
myo-Inositol, 322, 323, 324

bound forms of, 345-347

requirement for, 342
Inositol hexaacetate, utilization of, 350
Inositol monophosphate,

absorption of, 350

vitamin activity of, 352
Tnosituria, relation to polyuria, 374-876
Inososes, 363

naming of, 326
Insects,

fatty acids and, 291, 298, 313-314

niacin and, 533
Insulin,

in discovery of choline, 100

niacin and, 575

relation to choline, 19
Intestinal disease,

effect on vitamin D absorption, 259

in vitamin K deficiency, 425
Iodine trichloride, in vitamin D estima-

tion, 217

lodine value, 273

of unsaturated fatty acids, 288-289
Todinin, vitamin K and, 439
Todoacetic acid, 17
Isocinchomeronie acid, 521
Isocitric acid, 500

in carbon dioxide fixation, 504
Isocitric dehydrogenase,

coenzyme of, 493

in triphosphopyridine nucleotide es-

timation, 511

Isoergosterol, 199
Isomytilitol, vitamin activity of, 352
Isonicotinic acid, formation of, 473
Isoprene, derivation of, 625
Isopyrocalciferol,

as provitamin D, 144

formation of, 142

K

7-Ketocholesterol,
occurrence in body, 159
synthesis of, 147

SUBJECT INDEX

7-Ketocholesteryl acetate, 140, 206
a-Ketoglutaric acid,

in coenzyme A assay, 603

insulin and, 575

oxidation of, 622, 625

pyridine nucleotides and, 502
Ketones, excretion of, 86
Kidney,

in choline deficiency, 64-67

in fatty acid, deficiency, 298-299

in hypervitaminosis D, 264
Kwashiorkor, choline and, 104
Kynurenic acid, formation from tryp-

tophan, 526

Kynurenine, in niacin synthesis, 525,

526-527, 533
L
Lactalbumin, effect in choline deficiency,
78
Lactation,

choline and, 92, 103

niacin and, 565

vitamin D requirement in, 260
Lactic acid,

formation from inositol, 349

pyridine nucleotides and, 498-499
Lactic dehydrogenase, 510-511

coenzyme of, 493

inhibition of, 508

in keto acid oxidations, 503
Lactobacillus arabinosus, in niacin as-

say, 539

Lactobacillus bulgaricus factor, 605

pantothenic acid and, 613
Lactose, relation to choline, 58, 70
Lanolin, as provitamin D, 163
Lapachol, vitamin K and, 397
Latex, synthesis of, 625
LBF, 16, see also Panteth(e)ine, Lacto-

bacillus bulgaricus factor

LCF, see Citrovorum factor
Lecithin, 2, 3, 9, 43

arsenocholine and, 94

choline and, 45, 68

choline oxidase and, 36

hydrolysis of, 5

in growth of Neurospora, 55

lipotropic effect of, 19-20

synthesis of, 48-49, 52, 70, 72

turnover, 99

SUBJECT INDEX

Lecithinase, 52
effect on adenosine triphosphatase, 15
in bone-forming cells, 237
Lentine, see Carbaminoylcholine
Leuconostoc mesenteroides, in differential
niacin assay, 540
Leucopenia, choline and, 90-91
Line test, in vitamin D estimation, 220
Linoleic acid, 268, 274, 280, 281
conjugated, 283
conversion to arachidonate, 281
effect in deficiency, 278-280
effect in double deficiency, 284-285
isolation of, 270-272
synthesis of, 272
Linolelaidic acid, 283
Linolenic acid, 268, 274
effect in deficiency, 280
effect in double deficiency, 284—285
effect on enzymes, 278
isolation of, 271-272
trans-Linolenic acid, 282
Lipase, in fat deficient diet, 277
Lipids,
in bone-forming cells, 237
in choline deficiency, 68-76, 98
inositol in, 334, 366-367
tissue, in fatty acid deficiency, 307,
308-309 , 310-311
Lipocaic, 371
choline and, 100-101
composition of, 86
Lipogenesis,
in choline deficiency, 91
in pantothenic acid deficiency, 652
Lipoprotein, 15
Lipositol, 334-335
composition of, 346
Lipoxidase, 280
in estimation of fatty acids, 289
specificity of, 312
substrates of, 278
Liver,
coenzyme A in, 614
dicoumarol and, 428-429
in choline deficiency, 63, 67
in fatty acid deficiency, 299-300
in prothrombin production, 426-427
vitamin K and, 401, 437
Liver factor 2, see Pantothenic acid
Liver filtrate factor, see Pantothenic acid

755

Lomiatol, vitamin K and, 397
Long bones, deformation in rickets, 240
Lumisterol, 181, 196, 205

as provitamin D, 144

formation of, 142, 178

properties of , 191-192, 202
Lymph, in vitamin K absorption, 426
Lysine, fatty liver and, 87

M

Malic acid, formation of , 499, 500
Malic dehydrogenase, 499
coenzyme of, 493
inhibition of, 508
“Malic”? enzyme, 499
in carbon dioxide fixation, 504
Malonic acid,
choline oxidase and, 36
reversal of inhibition by, 366
Manganese, 93
Meat sugar, see Inositol
Mecholyl, see Acetyl-6-methylcholine
Melanin, formation of, 655
Menadione, see also Vitamin K, 2-
Methyl-1,4-naphthoquinone
inhibitory effects of, 439-440
Mercaptoesters, behavior of, 607
@-Mercaptoethylamine, 16
Metals, effect on choline oxidase, 36
Mercury salts, in choline isolation, 7
Metaphysis, in rickets, 245
Methanol, in methyl group synthesis, 27,
84
Methionine, 28, 26, 90, 101
active, 25, 29, 31
as source of cysteine, 29
choline oxidase and, 38
creatine and, 21-22, 30-31
effect on fatty liver, 20, 76, 78
effect on perosis, 94, 97
ethionine and, 80
formation, 29, 40, 41
in epinephrine formation, 32
in growth of microorganisms, 49, 50, 55
in inositol deficiency, 368
in methylation of nicotinamide, 32,
516, 517
in niacin toxicity, 573
in thiomethyladenosine formation, 51
in transmethylation, 21-23
oxidation of, 43

756

pantothenic acid and, 685
relation to choline 2, 26, 66, 68, 69, 71,
74, 102, 128, 129
requirement of chicks, 94
separation from choline, 55-56
toxicity of, 92, 95
p-Methionine, as methyl donor, 30
Methionine sulfoxide, 29, 30
in transmethylation, 25
Methionine sulfoximine, choline oxidase
and, 39
Methoxinine, choline oxidase and, 39
Methyl acceptors, 26-83
Methylamine,
effect on choline oxidase, 36
formation from sarcosine, 42
Methylbis-(8-chloroethyl)amine,
on choline oxidase, 36
2-Methy] -3 - difarnesyl-1,4-naphthoqui-
none, see vitamin Ky,
3,3’-Methylenebis(4- hydroxycoumarin),
see Dicoumarol
Methyl] formate, in choline synthesis, 11,
14
Methyl] groups,
oxidation of, 43-44
synthesis of, 22

effect

3-Methyl-4-hydroxycoumarin, vitamin
K and, 399
1-Methyl-2-hydroxynaphthalene, vita-

min K and, 397
1-Methyl-4-hydroxynaphthalene,
min K and, 397
2-Methyl-3-hydroxynaphthalenes, vita-
min K and, 397
2-Methyl -3 - hydroxy - 1,4 - naphthoqui -
none, see Vitamin K
Methyl] iodide, in choline synthesis, 11
2-Methyl-1,4-naphthohydroquinone,
activity of, 396-397
comparative potency of, 410
2-Methyl-1,4-naphthohydroquinone di-
phosphate, vitamin K and, 397
2-Methyl-1,4-naphthoquinone, see also
Vitamin K
activity of, 396
choline and, 88
comparative potency of, 410-411
niacin and, 533

vita-

SUBJECT INDEX

2-Methyl]-1,4-naphthoquinone
phoric acid ester,
comparative potency of, 411
N’-Methylnicotinamide, 26, 82, 549, 583
estimation of, 536-537
excretion of, 515
folic acid and, 84
formation of, 23, 32
in niacin deficiency, 557-558
isolation of, 456-457
synthesis of, 477
toxicity of, 572
tryptophan and, 524, 530-531
N’-MethyInicotinamide-6-pyridone,
differential assay of, 537
excretion of, 558
isolation of, 457
synthesis of, 478
Methylpantothenic acid, 627
Methylphosphate, in creatin formation,
32
2-Methyl-3-phytyl-1,4-naphthoquinone,
see Vitamin K
2-Methyl-1-tetralone, vitamin K and,
397
3-Methyl-1-tetralone, vitamin K and, 397
Metol, 535
Metrazole, niacin and, 577
Microorganisms,
choline requirements of, 49-52
essential fatty acids and, 297-298, 313
inositol and, 357
in pantothenic acid assay, 630-633
niacin synthesis by, 530-531, 533, 583-
584
pantothenic acid deficiency in, 678-680
vitamin K and, 393, 402, 416-418, 422—
424, 439
Microsomes, inositol in, 346
Milk,
choline in, 58
irradiation of, 210-211
pantothenic acid in, 638
Mitochondria, 233
choline oxidase in, 35
in creatine formation, 31
inositol in, 346
Modiolus demissus, provitamin D in, 156
Monkey, pantothenic acid deficiency in,
667
Monoaminoethanol, formation of, 33

diphos-

SUBJECT INDEX

Monoethylcholine, 45
Monoiodocalciferol, vitamin D activity
of, 208
Monomethylamine, formation of, 92
p-Monomethylaminoazobenzene, 89
Monomethylaminoethanol, 17
as methyl donor, 28
effect in choline deficiency, 68, 70, 128
formation by chick, 40
in growth of microorganisms, 50-51, 55
in perosis, 94
phosphorylation of, 19
Monomethylglycine, see also Sarcosine,
degradation of, 41
Monomethyltryptophan, 531
Mouse,
choline deficiency in, 103
fatty acid deficiency in, 297, 315
niacin deficiency in, 569
pantothenic acid deficiency in, 667-668
Mucopolysaccharides, in calcification,
236
Musearine, resemblance to choline, 123
Myosin, 15
Mytilitol, 335, 363
vitamin activity of, 351, 352-353

N

B-Naphthylamine, methylation of, 44
Nerves, in pantothenic acid deficiency,
665-666, 667-668
Neurine, 3
formation of, 11
in growth of microorganisms, 50, 55
Neuromuscular system, inositol and, 380
Neurospora,
in choline estimation, 55-57
in inositol assay, 362
niacin synthesis and, 525-526
Niacin, see also Nicotinic acid
absorption spectrum, 461-462
acidic character of, 465-466
antihistaminic effect of, 576-577
antivitamins in food, 548-550
as inhibitor, 508
basic character of, 464-465
biochemical systems, 480-517
biosynthesis of, 505, 521-533
chemistry, 452-478
constitution of, 473
crystal structure, 461

757

decarboxylation of, 513
differential assay of, 535-536
dissociation constants, 462
effects of deficiency,

in man and animals, 551-565

in microorganisms, 550-551
effect on bilirubin, 576
effect on blood sugar, 574-575
effect on circulation, 573-574
effect on gastric secretion, 575-576
estimation,

biological, 534

chemical, 534-538

microbiological, 538-540
excretion of, 513, 522, 556-560
industrial preparation, 478-47
in U. S. diets, 547-548
isolation, 452-454
isotopic, 476
ketosis and, 575
load tests of deficiency, 559-560
niacinamide synthesis from, 476, 505
nomenclature and formulas, 452
occurrence of, 541-550
pathology,

in animals, 565-570

in humans, 570-571
pharmacology, 571-578
requirements,

in animals, 578-586

in human, 586-587
separation from niacinamide, 456
solubility, 460
specificity of action, 517-521
stability, 460, 545-546
standardization of activity, 540
synthesis of, 474-478
tissue, 514, 554-556
toxicity,

in dogs, 572

in other species, 572

in rodents, 571-572
urinary metabolites of, 514-516

Niacinamide, see also Nicotinamide
absorption spectrum, 470-472
chemical properties, 472-473
erystal structure, 467-470
differential assay of, 540
dissociation constants, 472
in destruction of pyridine nucleotides,
507, 508

758

isolation of, 454-456
methylation of, 516-517
solubility, 466
stability, 466
synthesis, 476-478
toxicity of, 571-572
Niamid, see Niacin
Nicotinamide, see also Niacinamide
effect on choline oxidase, 35
methylation of, 23, 26, 32, 33
Nicotinamide methochloride,
methylnicotinamide
Nicotinamide methylkinase, 32
Nicotinamide mononucleotide,
biosynthesis of, 505-506
inorganic phosphate and, 498
significance of, 494
Nicotine,
niacin synthesis from, 474
resemblance to choline, 123
Nicotinic acid, 592, see also Niacin
burning feet syndrome and, 676, 677
effect in choline deficiency, 79
fatty liver and, 80, 82-88
gelatin and, 94
methylation of, 23, 32
perosis and, 94, 97
Nicotinuric acid, 457
differential assay of, 536
Nitrate, pantothenic acid and, 636
Nitrogen metabolism,
in pellagra, 564
pyridine nucleotides and, 502-503
Nitrogen mustards, 89, see also Methyl-
bis-(6-chloro-ethyl])-amine
A®.7_Norcholestadiene-3-ol, 142
as provitamin D, 145
Nordihydroguaiaretic acid, fatty acids
and, 285-286
Norepinephrine, methylation of, 32
Nucite, see Inositol
Nuclei, choline oxidase in, 35
Nucleotide pyrophosphatase, effect on
coenzyme A, 603-604
Nutritional disease, in war prisoners,
673-677

see N’-

oO

Obesity, choline and, 106-107
A9 ,12-Octadecadienoic acid, see Linoleic
acid

SUBJECT INDEX

A9 ,12,15-Octadecatrienoic acid, see Lino-
lenic acid

Oleic acid,

autoxidation of, 274

effect on choline oxidase, 36
Ornithine, niacin and, 521, 522-523
Osteoblasts, in bone formation, 232-234
Osteoclasts, 234
Osteoid,

in bone formation, 234, 237-238

in osteomalacia, 241

in rickets, 231-232
Osteomalacia, 234
Osteoporosis, 241

in hypervitaminosis D, 264
Oxalacetic acid, 16, 502, 679

pyridine nucleotides and, 499, 500
Oxalosuccinic acid, 511

pyridine nucleotides and, 500
Oxidation, in choline deficiency, 90
22 ,23-Oxidocalciferol, 206
22,23-Oxidoergosterol, 206

as provitamin D, 146

formation, 142
Oxygen, effect on ergosterol activation,

181

P

Palmitic acid,
effect on choline oxidase, 36
Pancreatectomy, fatty liver and, 100-101
Pantetheine, 16
in pantothenic acid assay, 632, 633
release from coenzyme A, 631-632
Pantethine, 624
as growth factors, 613
synthesis of, 606
Pantoic lactone, 627
isolation of, 596
Pantothenic acid, 15-16, 17, 92, 101, 380
acetyl, 626
alkali stability of, 611
amino acid analogs of, 626-627
biochemical systems, 598-625
biogenesis of, 627
burning feet syndrome and, 677
chemistry, 591-598
comparative requirements for, 691-692
constitution of, 597
deficiency symptoms, 669-670

SUBJECT INDEX

effect in inositol deficiency, 343, 357,
367, 384, 385
effect of storage on, 637
effects of deficiency,
in animals, 649-669
in humans, 669-678
in microorganisms, 678-680
esters of , 626
estimation of,
biological methods, 628-630
chemical and physical methods, 628
microbiological methods, 630-633
excretion of, 688-691
factors affecting distribution, 634-638
fatty liver and, 80, 88
hair-color and, 670-671
human intake of, 687-688
in coenzyme A, 599-601
in red cell metabolism, 622
isolation, 591-596
levels in blood, 690
losses in cooking, 637-638
niacin toxicity and, 573
nomenclature, 591
occurrence, 634-649
pharmacology of, 680-681
release from coenzyme A, 610-611
requirements,
of animals, 682-687
of humans, 687-694
specificity of action, 626-627
standardization of activity, 633
synthesis of, 597-598
toxicity of, 692-693
Pantothenic acid conjugate, as growth
factor, 613
Pantothenic acid phosphate, coenzyme
A and, 604, 632
Pantothenyl alcohol, biological activity
of , 626
N-Pantothenylthioethanolamine, 606
Pantoyl-tauryl-anisidide, as antimetabo-
lite, 624
Pantoyltaurine, effect on growth, 680
Parathormone, in bone resorption, 237
Parathyroid gland, effect on calcium
levels, 250-252, 260
Pellagra, 453, 480, 674, 675, see also
Niacin, deficiency
in vitamin deficiency, 592

759

pantothenic acid and, 637, 690
symptoms of, 552-553
Pellagramine, see Niacin
Pellagra preventive factor, see Niacin
Pelvis, deformation in rickets, 239-240
Penicillin, pantothenic acid require-
ment and, 685-686
Pentaenoic acid, 273, 283
formation from linoleate, 281
Pentose phosphate, 512
Peptidase, coenzyme A and, 601
Perchloric acid, in vitamin D estimation,
217
Perhydro-1,2-cyclopentenophenan-
threne, 134
Periodic acid, in inositol estimation, 359-
360
Periodide,
in choline estimation, 52-53
in choline isolation, 7
Peristalsis, effect of choline on, 123
Perosis, in choline deficiency, 93, 97
Phaseomannite, see Inositol
Phenanthraquinone, vitamin K and, 398
Phenylhydrazine, effect on choline ox-
idase, 36
Phenylindanedione, 427-428
Phlorizin, 19
Phloroglucinol, inositol and, 356
Phosphatase, 16
choline and, 15, 67, 98
coenzyme A and, 601, 632
in bone formation, 235-236, 237
in phosphorylcholine synthesis, 8
in rickets, 226-227, 253
effect on choline oxidase, 34
in coenzyme A, 603
Phosphatides, 19
Phosphocholine, in lecithin synthesis, 49
6-Phosphogluconic acid,
in pentose synthesis, 500
in photosynthesis, 503-504
in triphosphopyridine nucleotide es-
timation, 512
3-Phosphoglyceraldehyde, 498
Phosphoglyceraldehyde dehydrogenase,
492
3-Phosphoglyceric acid, 498-499
Phospholipids, 2, 15, 40, 90, 94, 347, 619
aminoethanol in, 30
distribution of, 52

760

estimation of choline in, 56
fatty acids of, 277
in atherosclerosis, 373
in choline deficiency, 67, 68, 70-71, 72,
98, 99, 129
inositol in, 339, 345-346
in threonine deficiency, 87
serine and, 48
synthesis of, 36
Phosphomolybdie acid, in choline isola-
tion, 7
Phosphomonoesterase, 15
effect on coenzyme A, 603, 604
4-Phosphopantetheine, coenzyme A and,
632
4’-Phosphopantethine, as growth factor,
613
Phosphopantothenylthioethanolamine,
in coenzyme A, 604
Phosphorus,
effect on vitamin D requirement, 254
excretion of, 251
factors affecting serum levels, 248-253
in bone formation, 235
pantothenic acid and, 636
rickets and, 223-224, 225, 226, 228-229
turnover, choline and, 98-99
Phosphorylase,
in bone formation, 235
in cartilage cells, 237
Phosphorylcholine, 2
derivatives of, 18-19
hydrolysis of, 19
in growth of microorganisms, 50, 55
isolation of, 8
synthesis of, 12
utilization of, 18
Phosphotransacetylase, in acetyl trans-

fer, 617

Phosphotungstic acid, in choline isola-
tion, 7

Photosynthesis, pyridine nucleotides
and, 503-504

Phthalic acid, vitamin K and, 399
Phthiocol, 388

isolation of, 393
Phylloquinones, see Vitamin K
Phytase, 382, 383
Phytic acid,

function of, 346

inositol in, 339

SUBJECT INDEX

in rickets production, 223
phosphorus of, 254, 257
utilization of, 382-383
Phytin, 334
utilization of, 350
vitamin activity of, 351
Phytol, in vitamin K, 394
Phytosterols, occurrence of, 136
Picoline, toxicity of, 573
B-Picoline, in industrial niacin produc-
tion, 478-479
Picric acid, in choline isolation, 7
Picrolonic acid, in choline isolation, 7
Pigeon, pantothenic acid deficiency in,
662
Pinitol, occurrence of, 335-336, 363
Pituitary gland, fatty liver and, 72
Plants,
factors affecting pantothenic acid con-
tent, 635-636
inositol in, 339-340
niacin synthesis in, 5382-533
vitamin D in, 160-161
vitamin K in, 415-416
Platinum salts, in choline isolation, 7
Polycythemia, choline and, 99, 102
Porphyrin, discharge in pantothenic acid
deficiency, 655-656
excretion in glycine deficiency, 84
niacin and, 556-557, 562
Potassium iodomercurate, in inositol
estimation, 359
PP factor, see Niacin
Precalciferol, 181
properties of , 194-195, 202
Preen gland, as source of vitamin D, 163-
164
Pregnancy, niacin in, 564-565
vitamin D requirement in, 260
Procaine hydrochloride, 535
Progesterone, vitamin K and, 440
Proline, 625
niacin synthesis and, 522
Propio-8-dimethylthetin, as
donor, 25, 29
Propylene dichloride, liver fat and, 74
Prostigmine, niacin and, 527
Protachysterol, 193
Protagon, 3
Protein,

methyl

Se Pe

— ois

fant eee ee ee ee ee

SUBJECT INDEX

effect on pantothenic acid require-
ment, 684
fatty liver and, 20, 87
in choline deficiency, 72, 129
Prothrombin, 418, 419
dicoumarol and, 413
in vitamin deficiency, 389, 432-435
site of formation, 400-401
vitamin K and, 400
Prothrombin factor, see Vitamin K
Prothrombin-time, in vitamin K assay,
407-408
Provitamins D, activatability of, 143-146
biogenesis, 156-159
chemistry of activation, 179-203
history, 138-140
irradiation of, 138-140
isolation of, 148-152
of invertebrates, 155-156
physics of activation, 173-179
syntheses, 140-148, 146, 148
Provitamin Dm, properties of, 205
Provitamin Dz , 135
Provitamin D;, 135
Pseudocholinesterase,
acetylcholine and, 17
choline and, 91
Pteroylglutamic acid, see Folic acid
Ptyaline, 235
Pyridine,
liver fat and, 74
niacin synthesis from, 475
toxicity of, 572

2,3-Pyridinedicarboxylic acid, niacin
formation from, 473
3,4-Pyridinedicarboxylic acid, niacin

formation from, 473
Pyridine-3-carboxylic acid, see Niacin
4-Pyridinecarboxylic acid, 473
Pyridine nucleotides, 625

apoenzymes of, 491-492
biochemical reactions of, 494-504
destruction of, 507-508
estimation of, 537-538
high-energy phosphate and, 496-498
holoenzymes of, 492

inhibition of, 508-509

in lipid metabolism, 501-502

in niacin deficiency, 555

in nitrogen metabolism, 502-503
in pentose biosynthesis, 500-501

761

in photosynthesis, 503-504

interconversion of, 493

mechanism of action, 490-491

occurrence of, 484

semiquinoid intermediates of, 491

specificity of, 4938-494
Pyridine-3-sulfonic acid,

as inhibitor, 508, 549-550

synthesis of, 475, 478
Pyridoxine, 101, 380, 653, 656, 657

deficiency, 283-285

effect on niacin requirement, 529, 584—

585

fatty acids and, 315-316

fatty liver and, 82, 90

in inositol deficiency, 368

in methyl synthesis, 85
Pyrocalciferol,

as provitamin D, 144

formation of, 142, 196
Pyrophosphatase, 15

coenzyme A and, 632
Pyrrole, 625

synthesis of, 622
Pyruvic acid, 17, 509, 510, 511

as inositol precursor, 355

formation from inositol, 349

in acetyl transfer, 617

in pantothenic acid deficiency, 662

oxidation of, 625

pantothenic acid and, 598, 679

pyridine nucleotides and, 498-500

Q

Quebrachitol, occurrence of , 336, 363
Quercitols, 329
Quinic acid, 363
Quinine, vitamin K and, 426
Quinoline,
in industrial niacin production, 478-479
liver fat and, 74
Quinolinice acid,
activity of, 521
in niacin synthesis, 473, 475, 526, 528
Quinone,
in dehydrogenation of cholesterol, 147-—
148

R

Rabbit, niacin deficiency in, 569
Rachitamin, 132

762

Rachitasterol, 132
Radiophosphorus, in vitamin D estima-

tion, 220
Rat,
fatty acid deficiency in, 292-295, 306,
314

in pantothenic acid assay, 629
niacin synthesis in, 526-529
pantothenic acid deficiency in, 650-658
pathology of niacin deficiency, 568-569
Reineckate, in choline estimation, 53-54
Reineckate procedure, in choline isola-
tion, 6
Renal disease, effect on rickets, 260
Reproduction, on fat-free diet, 293, 316
Reproductive organs, in fatty acid de-
ficiency, 299
Rhodopsin, 563
Riboflavin, 445, 497, 567, 653, 677
burning feet syndrome and, 677
choline oxidase and, 38, 87
fatty acids and, 286
fatty liver and, 80
in pellagra, 553
leucocytes and, 91
niacin and, 555, 562, 585
pantothenic acid and, 637, 690
porphyrin and, 656
Ribonuclease, in osteoblasts, 237
Ribonucleic acid, in osteoblasts, 237
Ribose, in diphosphopyridine nucleo-
tide, 485
Ribose-5-phosphate,
500-501
Ribs, in rickets, 238-239
Ribulose phosphate, 501
in photosynthesis, 503
Rickets, 132-133, see also Vitamin D de-
ficiency
acidity of intestinal contents in, 229-
230
bone ash in, 230
bone changes in, 227-228
external appearance, 224-225
histological changes in, 231-232, 241—
247
in adults, 241 (see Osteomalacia)
in infants, 238-240
production in rats, 223-224
serum calcium and phosphorus in, 225—
226

biosynthesis of,

SUBJECT INDEX

Raniacol, 574
Rubber, biosynthesis of , 625

Ss

Salicylic acid,
as inhibitor, 508
prothrombin and, 418, 432
vitamin K and, 4389
Sarcosine, see also Monomethylglycine
as methyl donor, 28
conversion to glyoxylate, 42
in creatine formation, 31
in growth of microorganisms, 50, 55
perosis and, 97
Sarcosine oxidase,
ethionine and, 39
vitamin By, and, 85
Scurvy, 673-674
differentiation from vitamin K de-
ficiency, 420
Scyllitol, 322, 323, 324, 338
occurrence of, 335, 363
vitamin activity of, 352-353
Sedoheptulose, 501
Seeds, pantothenic acid in, 636
Selenite, methylation of, 44, 51
Semicarbazide, effect on choline oxidase,
36
Sequoitol, 334
Serine,
as source of formate, 42
decarboxylation of, 42-43
in aminoethanol formation, 84
in choline formation, 43, 71, 79
in methyl carbon oxidation, 43-44
in methyl group synthesis, 27
methionine toxicity and, 95
synthesis of, 27, 28, 41
Serine dehydrase, in glycine formation,
42
Sex hormones, 134
Shikimie acid, 363
Silicotungstic acid,
in acetylcholine isolation, 8
in choline isolation, 7
Sinkaline, 3
Sitostanol, 135
Sitosterol, 135, 143
dehydrogenation of, 146-147
irradiation of, 138-139

SUBJECT INDEX

occurrence of, 136
relation to ergosterol, 157-158
Sitosterol dibromide, 139
Skin,
in fatty acid deficiency, 300, 301-302,
309-310
in pellagra, 562
pantothenic acid and, 671
Skull, deformation in rickets, 238
Sodium acety] salicylate, vitamin K and,
435
Sodium ethylate, in vitamin K estima-
tion, 414
Sorbitol, 344
oxidation of, 348
Sphingomyelins, 2
choline and, 58-61
turnover, 99
Sphingosine, 2
in choline deficiency, 98
reaction with choline, 58-61
Sphingosylphosphorylcholine, 8
oxidation of, 19
Spinal cord, in pantothenic acid defi-
ciency, 660
Spinasterol, 136
Sprue, vitamin K and, 425, 448
Stearic acid, effect on choline oxidase, 36
Sterols, chemistry of, 133-137
Stigmasterol, 135, 136
dehydrogenation of, 146-147
Stilbestrol, vitamin K and, 440
Streptamine, 327
relation to inositol, 363-365
structure of, 328-329

Streptomycin,
pantothenic acid requirement and
685-686
reversal of inhibition by, 365
Stress,

in fatty acid deficiency, 294-295, 297
pantothenic acid and, 673, 686-687
Stress reaction, in pantothenic acid de-

ficiency, 651
Strychnine, niacin and, 577
Succinie acid, effect on choline oxidation,

35

Succinic anhydride, reaction with coen-
zyme A, 608

Succinie dehydrogenase, inhibition of,
508

763

Succinoxidase,
ethionine and, 39
in fat deficiency, 277
vitamin K and, 438
Succinylsulfathiazole, pantothenic acid
and, 685
Succinyl transfer, coenzyme A and, 622
Sucrose, in choline deficiency, 70
Sulfaguanidine, 369
vitamin K and, 417
Sulfanilamide, 16
Sulfanilic acid, in niacin estimation, 535
Sulfapyridine, pyridine nucleotides and,
508-509
Sulfasuxidine, 380, 386
in inositol deficiency, 368
in niacin biosynthesis, 530
vitamin By, and, 85
vitamin K and, 417
Sulfate, 51
Sulfathalidine, 369
Sulfathiazole,
in vitamin K deficiency, 424
pyridine nucleotides and, 508-509
Sulfite, methylation of, 44
Sulfobetaine, see Dimethylthetin
Sulfocholine,
as methyl donor, 25
biological activity of, 48
Sulfonamide, 655
acetylation of, 598-599, 607
in niacin deficiency, 583
in vitamin K deficiency, 423-424
Sunlight, effect on vitamin D require-
ment, 253-254
Suprasterol I, 200
Suprasterol II, properties of, 200-201
Suprasterol II, , 205
Swine,
choline deficiency in, 101-102
niacin deficiency in, 567-568
pantothenic acid deficiency in, 664-666

Ay

Tachysterol, 181
formation of, 178, 208, 209
in vitamin D production, 212
properties of, 193-194, 202
Tachysterol, , 205
Taurine, 380
Teeth, vitamin D and, 247-248, 261

764

Tellurite, methylation of, 44, 51
Temperature, effect on choline require-
ment, 91
Terpenes, biosynthesis of, 625
Testosterone, 299
effect in choline deficiency, 73
N’-methylnicotinamide and, 516
Tetany, effect of vitamin D on, 261
Tetrabromostearic acid, in linoleic acid
isolation, 271, 273
Tetraenoic acid, deposition of, 282
Tetramethylammonium chloride, in
growth of microorganisms, 50, 55
Thetins, see also Dimethylthetin
effect on choline requirement, 128
in methionine formation, 23
methyl groups and, 23, 26, 48, 103
Thiamine, 17, 129, 653
burning feet syndrome and, 676, 677
choline oxidase and, 39
fatty liver and, 74, 80, 81-82, 83
niacin and, 555, 562, 585
inositol and, 370
Thiocyanogen value,
in linolenic acid determination, 273
of unsaturated fatty acids, 288-289
Thioethanolamine, see also Cysteamine,
in pantethine, 613
Thiomethylacetic acid, 44
Thiolacetic acid, in acetylcholine for-
mation, 17
Thiomethyladenosine, formation of, 51
Thiouracil, 380
arteriosclerosis and, 99
effect in choline deficiency, 73
N’-methylnicotinamide and, 516
Threonine,
effect in choline deficiency, 79
fatty liver and, 87
Tihrombeey tapeaia: Alfferenhiation from
vitamin K deficiency, 420
Thromboplastin, 403, 408
inositol phosphatide and, 381
vitamin K and, 4383-434
Thyroidectomy, effect on liver fat, 99
Thyroid gland, 85
in choline deficiency, 73, 74
inositol and, 380
Thyroxine,
choline oxidase and, 39
pantothenic acid and, 686

SUBJECT INDEX

a-Tocopherol, 374, see also Vitamin E
effect on ceroid, 118
essential fatty acids and, 285-286, 315-
316
inositol and, 384
a-Tocopherol phosphate,
cleotides and, 507
a-Tocopherylquinone, vitamin K and,
398, 418, 440
p-Toluidine, methylation of, 44
Torula cremoris, in niacin assay, 540
Toxisterol, 181
properties of, 198-200
Transacetylase,
in acetate activation, 616-617
in acetyl-coenzyme A preparation, 607
in coenzyme A assay, 602
pie pact choline and, 19-24
Trienoic acid, 2&3
accumulation in fat deficiency, 281
Triethylcholine, 80
effect in choline deficiency, 66-67
in growth of microorganisms, 50
in lecithin synthesis, 49
Trigonelline, 385, 457, 478
derivation of, 514
differential assay of, 5386, 537
formation of, 23, 32
in niacin assay, 539, 558
niacin formation from, 476
toxicity of, 572
1,2,4-Trihydroxyanthraquinone,
min K and, 398
Trimethylamine,
fate of, 93, 127-128
formation of, 10, 51-52, 92
in choline estimation, 53, 54
in choline synthesis, 10-11, 14
in growth of microorganisms, 50, 55
Trimethylamine oxide,
excretion of, 127-128
formation of, 92
in creatine formation, 31
Trimethylarsine, formation of, 51
Triosephosphate dehydrogenase,
zyme of, 493
Triphosphopyridine nucleotide, 455, 603,
604, see also Pyridine nucleotides
biosynthesis of, 506-507
estimation of, 511-512
isolation of, 453, 458-460, 480, 481-482

pyridine nu-

vita-

coen-

SUBJECT INDEX

properties of, 489-490
structure, 488-489
Tromexan, 427-428
Trout, choline deficiency in, 103
Trypsin, fatty liver and, 101
Trypsin inhibitor, 101
Tryptophan, deficiency in rats, 568
effect in choline deficiency, 79
gelatin and, 94
in niacin assay, 539-540
in pellagra, 553
isomers, in niacin synthesis, 531-532
niacin and, 505, 521, 523-529, 580-581
pantothenic acid and, 680
Tuberculosis, 261, 264
Tumors, choline and, 89-90
Turkey, pantothenic acid deficiency in,
661
Turkey poults, choline deficiency in, 97
Tween 80, effect on phospholipids, 373
Tyramine, effect on choline oxidase, 36

U

Ultraviolet rays, vitamin D and, 133
Urea, from choline, 93

Urethane, choline oxidase and, 39
Urorosein, niacin and, 556-557

Vv

Vanillin, in vitamin D estimation, 217
Vigantol, 264
Vinyltrimethylammonium hydroxide, see
Neurine
Vitamin A, 210, 212, 246
choline and, 90, 91
destruction of, 133
fatty acids and, 315
in fish liver oils, 167
in vitamin D estimation, 216
niacin and, 563
vitamin K and, 418, 426
Vitamin(s) B, 246
inositol and, 384
Vitamin B., 591
Vitamin B,2, 79, 517, 673
choline oxidase and, 37, 39
effect on pantothenic acid require-
ment, 684-685, 694
effect on sulfhydryl groups, 85-86
in canine nutrition, 97-98
in chick nutrition, 95-96

765

in leucopenia, 90-91
in methyl group synthesis, 23
perosis and, 97
relation to choline, 49, 83, 128
transmethylation and, 84
Vitamin C, 246
effect on osteoblasts, 237
osteoid and, 236
Vitamin(s) D,
absorption in disease, 259
chemistry, 132-209
effect on calcium absorption, 249-252
effect on phosphorus absorption, 249—
252
effects of deficiency, 223-248
in animals, 223-232
in humans, 232-248
estimation of, 215
biological methods, 218-221
chemical methods, 216-218
physical methods, 215
form of administration, 259
formation in animals, 161, 170-172
industrial preparation, 210-215
isolation of, 172-173
minor and obscure forms, 203-209
nomenclature, 132
occurrence, 160-172
pathology in humans, 232-248
requirements,
factors affecting, 257
of animals, 253-256
of humans, 257-266
standardization of activity, 221-223
unit, 221
Vitamin D,;, composition of, 196
Vitamin D2 , 132
Vitamin D;, 132, see also Calciferols
as standard, 222
properties of, 202-203
Vitamin D,,
production of, 205
Vitamin D;, 203
Vitamin E, 89, see also a-Tocopherol
choline oxidase and, 39
deficiency, 75
inositol and, 369-3870
in vitamin K assay, 405
Vitamin F, see Essential fatty acids
Vitamin G, 591
Vitamin(s) K,

766

absorption of, 400
biochemical systems, 400-402
chemical and physical properties, 390—
393
chemistry, 389-399
comparative potencies of, 410-413,
424, 446, 447
consitution, 393-395
dosages, 435-436
effect of deficiency,
in animals, 419
in humans, 419-435
effect of route of administration, 405-
406
effect on bacteria, 417
estimation of,
biological, 402-413
chemical and physical, 413-414
sources of error, 403
estrogens and, 440
forms used therapeutically, 435
industrial preparation of, 399-400
isolation of, 389-390
miscellaneous effects, 440-441
mode of action, 487-442
mode of administration, 436-437
natural antagonists of, 418
nomenclature and formulas, 388
occurrence of, 415-418
pharmacology of, 435-444
requirements,
chick, 444-446
dog, 447
human, 447-448
rat, 446-447

SUBJECT INDEX

specificity of, 396-399
standards of potency, 409-410
synthesis of, 395
toxicity of, 442-444
water soluble forms of, 400
Vitamin P,
antivitamins K and, 432
dicoumarol and, 439
Vitamin PP, see Niacin

WwW

Warburg’s coferment, see Triphospho- .
pyridine nucleotide

Water metabolism, pantothenic acid and,
693

x

Xanthine oxidase, vitamin By, and, 85
Xanthophyll, vitamin K and, 415
X-rays,

choline oxidase and, 39

in vitamin D estimation, 220

Ȏ

Yeast, in inositol assay, 362
Yeast filtrate factor, see Pantothenic
acid
Yellow fever,
choline and, 103

Z

Zinc, deficiency, 655 |
Zwischenferment, coenzyme of, 482

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