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Marine Biological Laboratory Library
Woods Hole, Mass.
Presented by
Academic Press, Ine.
New York City
SS
‘aa
Lb h9b02e00 TOEO O
WNNUNAN A
IOHM/18lW
THE VITAMINS
Chemistry, Physiology, Pathology
VOLUME III
THE
VITAMINS
Chemistry, Physiology, Pathology
VOLUME Ill
EDITED
BY
W. H. SEsBRELL, Jr. Rosert S. Harris
Director, National Institutes of Health Professor of Biochemistry of Nutrition
Bethesda, Maryland Massachusetts Institute of Technology
Cambridge, Massachusetts
AGADEMIC PRESS. ING.» 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
FraNK H. Betrue.u (202-217)*
University of Michigan
The Thomas Henry Simpson Memorial
Institute for Medical Research
Ann Arbor, Michigan
VeRNON H. CHELDELIN (575-600)
Professor of Chemistry
Oregon State College
School of Science
Corvallis, Oregon
P. Gyérey (280-290, 298)
University of Pennsylvania Hospital
3400 Spruce Street
Philadelphia 4, Pennsylvania
Puitie L. Harris (495-501)
Biochemistry Department
Distillation Products Industries
Rochester 3, New York
Rosert 8S. Harris (2, 121-124, 220-221,
300-301, 404, 482-483)
Department of Food Technology
Massachusetts Institute of Technology
Cambridge 39, Massachusetts
D. M. Heestep (361-371)
Department of Nutrition
School of Public Health
Harvard University
Boston 15, Massachusetts
M. K. Horwirt (334-361, 373-377, 379-
402)
Elgin State Hospital
Director, Biochemical Research Labo-
ratory
Elgin, Illinois
B. C. P. JANSEN (404-409, 425-466, 472-
478)
Nederlands Institut voor Volksvoeding
J. D. Meyeroldin 3
Amsterdam, Holland
* Numbers in parentheses indicate the pages on which each author’s contribution or contributio
be found
TO VOLUME III
CHARLES 8S. KereEvit, JR. (255-263)
Department of Chemistry
University of Texas
Austin 12, Texas
JouHN C. KEeREZzTESY (222-233)
National Institutes of Health
Public Health Service
Bethesda 14, Maryland
Karu E. Mason (514-570, 572-573)
Department of Anatomy
School of Medicine and Dentistry
University of Rochester
Rochester, New York
Henry A. Martiti (4838-495, 501-514,
570-572)
Department of Biochemistry
College of Medicine
State University of lowa
Towa City, Iowa
CHARLES C. Scorr (52-62)
Director of Pharmacology
Chilcott Laboratories
The Maltine Company
Morris Plains, New Jersey
W.H. SEBRELL, JR. (478-480)
Director
National Institutes of Health
Bethesda, Maryland
HENRY SHERMAN (248-253, 255, 264-276,
293-298)
Department of Food Technology
Massachusetts Institute of Technology
Cambridge 39, Massachusetts
Esmonp E. SNELL (253-263, 276-280, 37 2-
373, 377-379)
Department of Chemistry
University of Texas
Austin 12, Texas
Vi CONTRIBUTORS
K. L. R. Stoxstap (89-121, 124-202) LEMUEL D. Wricut (2-12, 13-52, 62-85)
Lederle Laboratories Director of Research in Microbiolog-
Pearl River, New York ical Chemistry
Perer A. TAVoRMINA (2-12, 13-52, 62-85) Research Division
Sharp and Dohme Sharp and Dohme
West Point, Pennsylvania West Point, Pennsylvania
W. W. UmsBreit (239-242)
Merck Therapeutic Institute
Rahway, New Jersey
THEODOR WAGNER-JAUREGG (301-333)
Medical Division
Army Medical Center
Kuaus R. Unna (290-298, 466-471) Edgewood, Maryland
Department of Pharmacology
Medical School H. M. Wusst (12-13, 409-425)
University of Illinois 137 Summit Avenue
Chicago, Illinois Montclair, New Jersey
CONTENTS OF VOLUME III
Page
ANTATRUPORSETO™ VOLUME se DLL me ce. hey. su ietin «aetna TA Liao inne be aMise Vv
RONAN TEROR TO DUMERY LAND Ls tacts iat Ours ea. sw een eters aici s wisieieaheye vite: xili
CHAPTER 12. p-AMINOBENZOIC ACID
HEINGMeNC atures srt Seco ere ae raed = Ad EN a enmersee, Nolet ohs aeasneie = 2
Ropert 8. Harris
WINS EPIRA TSG U ti eo Wee sah vt pec Ices Mien 5. w= te SRY MO ae RF gateT Siac Gad erat SR 2
LEMUEL D. WriIGHT AND PetTEerR A. TAVORMINA
PMPeMITIG HET IGE PENALAUOL:. .Ak 0 ceri iorc oe a oreo meters severe ciel eles tole oie: ste cer meas 12
H. M. Wurst
MUM OCHEMUNCAl OVStEME os ci ces hok wha sch bx Loaves Ae cou ces aie oR es cer eas tars 13
LEMUEL D. WriGHut AND PETER A. TAVORMINA
MP MERTOROTICSIS) rt ae Sra ore << Sepa cgere Se hveiie «Ry ort eyneaar tere Ube goer AE ete eee ener 16
LEMUEL D. Wricut AND PETER A. TAVORMINA
WAIL, TEVSU HDS na vig, Seeley Ce teers ee Rn nA ca eR fo li On cy ee one RR 19
LEMUEL D. WrIGHT AND PETER A. TAVORMINA
Miller OGCITTENCE NTE OOUSS «<6 nc. 5 5 eerste areal yet 2
LEMUEL D. WRIGHT AND PETER A. TAVORMINA
Wiltilemrmects Of DeEfClenGy 52.0 we cee cot OE: ae 2 hat, Abts =) ic eee ere 32
LEMUEL D. WRIGHT AND PETER A. TAVORMINA
EOI TAD OLLSIie. ee ee ec oe eed hors te oe eres, 1 43
LEMUEL D. WriGHT AND PETER A. TAVORMINA
DORIC T Gi! 400M. coc cee rite Se ee oo a DIE ia roe ellos Se ane aa 49
LEMUEL D. Wricut AND PETER A. TAVORMINA
PURE DATINACOLO RY sorcte Miers oe Pape oale sas Eee se ok ee ee 52
CHARLES C. ScotT
Kelme Detoxication. oro Arsenicals 3 wsc5. ocho aa eels ORI ee oe 62
LEMUEL D. WRIGHT AND PETER A. TAVORMINA
PellieSalfonamidesReversale tse eo: cc ee es ee te Se 66
LEMUEL D. WriGHT AND PETER A. TAVORMINA
PM PERITeTaDENUICSACHWItY 6. Fes ate arn Stes Sr keendekeeee 2 ob ORE e309 79
“Lemvuet D. Wricut AND Prerer A. TAVORMINA
CHAPTER 13. PTEROYLGLUTAMIC ACID
ipemomenciature and Formula nc. fire ets OR Serele lates. bh 89
E. L. R. Stoxstap
ULL) GLICS TEES ye ae ee SERS tanta eee me Je eR ee oo a 91
E. L. R. Stoxstap
MENT IG NEUTER PLE UAGALLON Ts shed «5.0 ee oe Ae espe a eo eed one gta ae at 121
Rospert 8S. Harris
MEO CHPEUMEAIM SURLENIS Mt air aise iad aes ota: Sidhe oS viva nese A Anen he vis aoa 124
KE. L. R. Stoxstap
MOE SHECINCIEV OT ACTION. we teers Haake ao dani nage oeys fot eee 142
E. L. R. Sroxstap
Wi. Estimation; ...:. Te Sean, Aa OMeck Wee ee; 3 pe ede RA eae 161
E. L. R. Sroxstap
. 71411
Vili CONTENTS
Page
VilleeStandardization.of Activity; eee aero neeer eae ee: eee 168
E. L. R. StoxstTap
VillieOccurrencé:in Foods... ...cgee cree teen een Cn ee 168
E. L. R. StoksTap
EX, *Eiects of Deficiency... .:..:.. cern see ae oe Pe ee 17
AS li Amiri alist io 5) &i-a.a «kc Se ee ge 171
E. L. R. Stoxstap
1B. In Microorganisins:. ...... 424.0 coche 2 eee i 199
E. L. R. StoxstTap
Gens Miatie« 6e4 ohsdis ncn thas bok SA OR el Oe 202
FraNK H. BETHELL
XK. (Pharmacology:....:..5 0.2... h20hahtae «apse oars ce ed gies eee 216
FrANK H. BETHELL
XI. Requirements and'Factors Influencing Them: |.......:.-) 4-2 ---eeeeee 217
FRANK H. BETHELL
CHAPTER 14. PYRIDOXINE AND RELATED COMPOUNDS
(Vitamin Bs Group)
i; Nomenclatures:, Sec <2 32 i les © a a ee 220
Rosert 8. Harris
DE. Chemistry 3 cosh eens SO a a ng So 222
Joun C. KEREZTESY
TI. Industrial Preparation: 2.2...00: Ses, eee 8. Sela ee ee 233
JOHN C. KEREZTESY
IV... Biochemical’ Systems)... 30 e eon e e e eee 234
W. W. UmBREIT
Vi." Specificity: of Action: ..9.(4 7. Oe, lee ee eee 239
W. W. UMBREIT
VI Biogenesis! 2 oncca 5, . eh eS ieidacc oe oe BOR De acs Oo 242
W. W. UMBREIT
Wall “Estimation: $5.05 4500 face ete Sete ae) ee 242
AS Physical Methodstix.c.:hcc a. seeded «+= Se tee Ore eee ... 243
HENRY SHERMAN
B. (Chemical Methods: «c......5 . select at oe ae ae 244
Henry SHERMAN
©. BiologiealMethods 1:22. 2.:ccq, «salt bos eae ee eee ee eee 249
HENRY SHERMAN
DE Macrobiologicall Methods\ty.. cece eerie tee 253
Esmonp EK. SNELL
Vill: Standardization of Activity........-42.2-: OS ed Rg i | ota ey 255
HENRY SHERMAN
EX Occurrence in FOOdS: «cc ck cc.cssaccis-cdoe ere ee es 255
Esmonp E. SNELL AND CHARLES S. KEEvIL, JR.
X. Effects of Deficiency..... Ce: ca nah : jae Sorin tee 264
i\s tho leaenouics aoe gem an: bis bk Soci Fe 264
Henry SHERMAN
B. In Animals........ ae ae oi ayle eee 265
HENRY SHERMAN
CONTENTS ix
Page
Rae Bi I OPOGMEENINIDS ©.) hia Vialc car hoy 035 ke LRM WD cutra potas MAES 3 276
Esmonp Ef. SNELL
1D ee GLY IV i ei ok ee PER car te are 280
P. GyéOrey
Oo ya tS UG eh ns ee ie Ai i te nh ee tr ae ee 290
Kuaus R. UNNA
XII. Requirements and Factors Influencing Them........................... 293
eae CDE A TYITIVAL ES OP ee inhi cs Perc date CRS co 3509, iyard ein RE Rl enka 293
Henry SHERMAN
Tek. AOA GNU IES sae Oe Aree eae A ee, fe Ase so geek glee ae 298
P. Gyérey
CHAPTER 15. RIBOFLAVIN
Pee MRITESTIENETIT ES. ALS, 5 Fe ks Sohva okie ola’) aps lo'é ae athe A ee Me A ee 300
Rosert 8. Harris
MMENSCENISUOY cst es Stine say law rere doa -5 ois o Be slad De eee sae eee 301
THEODOR WAGNER-JAUREGG
Seema usttinl PTOPATAtON.....\. 5.05. ..yucu's 22 x 244 eas aS ono RE 332
THEODOR WAGNER-JAUREGG
BPMPRINENEMICAl SYSCEMIG. 05 osc sises 02's. oo oe age eee ee NO RE © 334
M. K. Horwirtr
Mee HGeiuCtty Of ACTION. <5... ..0 52.0406 + ach» «cae mines oe 5 eeU nee eee 358
M. K. Horwitr
EOE S108 2 ei, oss Be Ride a he, a ORs Oe ek 359
M. K. Horwitt
VOULL TINS] AGG Yr a 00) 1 Rta oe ne ane a Re Ae ce! HG ee eM es LD 361
Pak aysicaL and Chemical Methods?) 22a we os ee eee 361
D. M. Heastrep
Bee IbOPICAl NWIOLNOUS:...: .:42.2Ghe cs \s,c RoR een ee EA. ere 369
D. M. HegstEep
eeantiorspiolorical Methods:a\s icons. . ss ee ee ole aloe A ee eee 372
Esmonp E. SNELL
ReneS LANCATOIZALION: ord neat, ee. Sos. ES ee 373
M. K. Horwitr
Pe OCcurrence inl) FOOGs 56 ots few le sh oe eee Oe A ss 374
M. K. Horwitrr
Bement areOL POUCICHOY. rer cs. re. ait AO ite ee Ia aE 377
Pee mETy CLT GT OOR OO MIRINA cr x wishes <; 6s Recents OR Somerton eens NE A eee ae, 377
Esmonp E. SNELL
Edad Gatgl 2d Fhe te een AO eR. ce i a. i cas 6 eset ae 379
M. K. Horwirr
(Ops) bee eV (oY 611 en OR, Bo ie aI AR ry RN oe On ne Wee 2 379
M. K. Horwirr
DUPE CATHITIAIA Ices eect cee ne Sion Mere ok ail ay ak dale s/. 380
M. K. Horwirr
Eyeme Len VU surat rem ari cats neg Pte i x, eS Ace ats wlohe seem olds ch. oj Chai hepa res 387
M. K. Horwitr
Col LELETEEER S700 "ca le ear esa eaten ees ee ore eT ee Sty 391
M. K. Horwirr
x CONTENTS
Page
XII. Requirements and Factors Influencing Them.....5.°.°2).. °F). ae 394
M. K. Horwirr
CHAPTER 16. THIAMINE
[ewNomenclature:.:.... 2:6 -:shhen aee tenaenoh sos Sek kee 404
Ropert 8S. Harris
PTE CHEmMIStLY «0.05.5 03.6 os ns os A So ee ee ee 404
B. C. P. JANSEN
Me Industrial Preparation... 29 qc. eh Gara ene a scaen t ee Oo One ee 409
H. M. WusEst
IVE Biochemical'Systems:...:. caer: oe es oer ee 425
B. C. P. JANSEN
Vi -specificity of “Action. 2%..: 264.5526. e eee ote hae ba 442
B. C. P. JANSEN
VI- Biogenesis: «...,.2c0 «Shes meas ees bin tee Oe 445
B. C. P. JANSEN
WEE Bestia tin ijco sic od olds eM eu oa AE 2 RR ee 448
B. C. P. JANSEN
Vili StandardizationlofActivity...0) 4.0). eee a ae ee 453
B. C. P. JANSEN
EX SOCCULFENCE INGE OO Gk. 5, co ask oak eae oe WOR Ia ey. ee oe ne ee 454
B. C. P. JANSEN
X. Effects ofDeficiency: <3... o.4.iias oes on dors Pee ee Oe ee 460
B. C. P. JANSEN
MI Pharmacology: fac ooh coh hlen otteet cach Saittle tb + ae pee eee ee 466
Kuaus R. UNNA
XII. Requirements and Factors Influencing Them............................ 472
A OP Amimal sg aisern iikdiides ase 2 30 scene cetes Oe coe 472
B. C. P. JANSEN
BS OF Miata ok ashe hte as Wat pe hEGl os 0G doz rereye Gel pe 478
W.H. SEBRELL, JR.
CHAPTER 17. THE TOCOPHEROLS
TE WNomenclatunewy.rck ojos osha os See boo Matas ae ee Ae Oe 482
RoseErt 8. Harris
Li mi@hemistrya.cc cc soa eons nes Wide See oe OE bah ae ee eee 483
Henry A. MattTitu
II. Industrial’Preparationy ...5.. 2265 424 «c:. Jae eee ee ee 495
Puitip L. Harris
TY;. ‘Biochemical’ Systems) oc 5..5 5 os crac aoc Ser s cod oasseere a AG ene ce Oe eee 501
Henry A. Martin
Wit ES tintatl Onis feos oc cea, Ce fs OT CI ge 506
Henry A. MatrTiuu
WI. Occurrence in ROOd icon ed oe ce ns ee Se eee 512
Henry A. Martinu
Vil-vEfiects of Deficiency. ...- 4. cc. cee eee div afersie tie se or 514
Karu EK. Mason
VILL. Pharmacology isci.aj deus \/ Pa eek @aetee he Hin be ee 562
Karu E. Mason
CONTENTS xi
Page
IX. Requirements and Factors Influencing Them.............. Mens 7 See 570
PAW COLMATININISU LR Stee Veen cp Per taro tatin 54 'o'in co-is cle Si ian SPRRM RMU Ac! NW octonele telsre a veaten od 570
Henry A. Marriun
Teh TOTEM USA CEe 48a or bcs Ginger ae ee aCe on iets © 0 0. So Geel Sige enn eran Fe 572
Karu EK. Mason
CHAPTER 18. NEW AND UNIDENTIFIED GROWTH FACTORS
VERNON H. CHELDELIN
I. Lipoic Acid (Thioctic Acid, Pyruvate Oxidation Factor, Protogen, Acetate
MGMIACeMOENt HF ACEOM) c.)ecc-'s < «<.5. 5 aeeters yaaa eerie a wot crtye yess la neo 575
PETC e (Vitamin! Bir) mee eke 50 ks ite is ae eee ere nee eae orks 583
ee Pentides:: =: 2.28.5 2). ars MI ERM Pi essetatis & on cath Bh cut Ue 585
JIN. TaFo.€0) a 2) ph Rie ioe eae oe Ratceee nee ae Pe ere cate arr is aii ree ee acae 588
STI AVENE RTS fol 8 Rs 8G, CER Oa Torco. > Sak vt a PRR de Oe ee ENS romeo 589
VI. Factors Required in Unheated Growth Media........................... 589
Miteoninea Pir Antistifiness Factor:... . oss s ths oe 2 Ol ae mes tts ete 591
PPE TSC EATICOUS PACCOLS i> c(i coo. otis Widens k corhareece eoeuare opoaroke aimee aerate mates 593
STIPE i ae ret en a ne eeu eR Mee Scans Oh of 601
SEDER IAUUSTUM ONE) HERS ef oi3< horney cate, bic Saute al cactamen ttten a Eryn fol ee nen eee 643
et a ae
ie Y me f4)ss ewe
ies LAY Ue ad 3 ee a ed w fre ofe) fi
(Egeend is aie
7
ss
? Nate AW: P pilin i
‘ mo > 65> ow oh,
Ae) | d
CONTENTS OF VOLUME I AND II
VOLUME I
CHAPTER 1. VITAMINS A AND
CAROTENES
Robert S. Harris, H. H. Inhoffen, Karl
E. Mason, F. H. Mattson, Nicholas A.
Milas, H. Pommer, Wald,
S. Burt Wolbach
George
CHAPTER 2. ASCORBIC ACID
Robert S. Harris, L. W. Mapson,
Mamie Olliver, Mary Elizabeth Reid,
Fred Smith, Richard W. Vilter
CHAPTER 3. VITAMIN By
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 II
CHAPTER 5. CHOLINE
Charles H. Best, Wendell H. Griffith,
Robert S. Harris, W. Stanley Hartroft,
Colin C. Lucas, Joseph F. Nyc
CHAPTER 6. VITAMIN D GROUP
Charles E. Bills, Robert S. Harris,
James H. Jones, Abram Kanof, Benja-
min Kramer
CHAPTER 7. ESSENTIAL FATTY
ACIDS
George A. Emerson, Arild E. Hansen,
Robert S. Harris, Ralph T. Holman,
Hilda F. Wiese
CHAPTER 8. INOSITOLS
Erwin Chargaff, T. J. Cunha, Robert
S. Harris, Henry A. Lardy, Arthur H.
Livermore, A. T'. Milhorat, Esmond E.
Snell, EZ. R. Weidlein, Jr.
CHAPTER 9. VITAMIN K GROUP
H. J. Almquist, Robert S. Harris,
Charles A. Owen, Jr.
CHAPTER 10. NIACIN
Robert S. Harris, James M. Hundley,
Esmond E. Snell
CHAPTER 11. PANTOTHENIC ACID
George M. Briggs, Floyd S. Daft, Robert
S. Harris, Samuel Lepkovsky, Fritz
Lipmann, Elaine P. Ralli, Esmond E.
Snell
Author Index—Subject Index
CHAPTER 12
p-AMINOBENZOIC ACID
I. Nomenclature . ee es... ame me D ocr eae Oe Weare ae ah a
MPEPEPEISUE YT Sages. 85 GN Or US OR cs ok ow eg eee ow oe a ee ae
A. Preparation ; 2
B. Physical Properties. A oe Sy he AN Re
PeMGneM CLA TOPETGIES! 5), fa, o-. 6. esp ok ee ee ek Gee tO
PREPCHEVALEVEBm. sc loee LPs. a) co Soe ee Pee ees poe er
BOOLOTANCRCHIONS +9 fro SC as’ cs d Ee ae te ee Ba, ve
MePMTTNGRLTI AE PReONALYUION. <<. hats, 3.8 | o's wake con es ee eee Unt eee
MeePEHOCHeMICHl SyhteMsS |... = pe a9 d » Fo Oe ees Go eo ee
SPENOPEHEDIS oc cs fatter Eh. was 5. ae ee Rey Sy ee
VI. Estimation. . . Rn res Mee Ce epee IR
A. Chemical Methods CF ORES Oe ae ol.) «res eee
Bevicrobiologieal. Methods: "2. . .._..-go cane = so eee tee oe ce
mieereccurrence an Foods. . ....1. Sj. «a teen ce} so ee ee cee
MmMenb eats GF Deticioncy. . .° /... 8” av...) =“ lease ae © ee ee
RemineBactarin 6: gee. 2 ses a eR Se Sy cc Me CS
MeUbTItON. Ss ee 65 ak ee es es. 6” eee ee
Per HORIG, Sh" te de, ys. Sates SR es, Op eee
fs SNAG IN ALS 25%, wi hed ts& a NE ath ees. <<... Rae egod
[CEN ah i de ie mee PO e a MMR ee Yah 0)
Miceas: & G2-b25), | ky ck: a rE gad (es) eee
RT OTS os) oh: Mle cl eas wv SERIES ty cl ok ot meine
hteksr sae ee Oe Le eee
Nae! SW oe so tn Regen Foe Le ee =" ee i ear
Geamignien te Fe he ena oa ey. 3 ht, 5) Se SR mente
IX. Metabolism. ... Pees oe Pet en Pe tc | Pc eS
A. Excretion in Lower iecaale ee ee Se ee
Be rxcretion.im: Human bemps. <p. ) a eee hs oo 2 Pee ie os Se
ReeEOxiciny . |.) ©: i Ne Gt: CE OE , Ssw oN hy ot Ee al ee
A. In iearntory erred Lg Tes OMe etee ta ie bere OM oe cheat Ms beet
Beelneblamanei einige Ss icne = 2 ce tet, Eee a, ky oe Oe Oe) COs bap rie ec OO
MPR RACOLOy To Te eee” A NE eh an oe
AP Absorption, Hate, and Pxeretion =...) is 2 2 a he ps. 2
Boxe... 2: ee ee Oe ee ee en 5. vO
C. Pharmacological eetonah Fs), LS 2 Sh ihc ho i
uc ireuiabary spate =”. Pee ee ede Go cel es | «OD
MBLOOGRin wea Le APS e iain awl. od mapleigd i>. 20 £99
. Gastrointestinal Tract ee eS eee ne a nr ee, 2:
SeNCE OUEST SR ALOMion Mrs ee A eo) Ge hes) a Ae OW
ONC ee RE OM ee gk et te os
CUE tnt! Sp gat ctl Pe ope balerrg oak OD Ae Rn AS a a a
pl Ge Tine Se ster se ee Le eee on SOR Sk ye yn od
Siiar One ti CTE Ma DME Y bn ewics Lipkl ts! Pe od n'y «Sq, spe eee
Core Ww bo
“IQ Ore W bo
~
2 P-AMINOBENZOIC ACID
Page
1D, Therapeutic: Uses: .. @ al ae ae ee ty ae ae2 ee
1. "Gray Hairs ...° . (92 epee a eee ie. ee ee
2° Dermatology =. . Warten eee ee ae A
3: Rickettsial Diseases!) <i. 4 a tre er ee
4° Neoplastic Diseases ..- 0.75%." Bos tele. 3), ee
b: Other Diseases: \. say 2 ak 8 Se he hee oh
XI, Detoxication of Arsenicals™/s\.: .¢5. “0.2 = Se ee
XI. ‘Sulfomamide; Reversal: <3 3.) . 1 see ee see an = cee
XIV; ‘Therapeutic Activity. . 9.9. “2 0x 2 2 Sons ats) eS eee ee
A. Rickettsial Diseases: . 2 5 1 a a: .s <se dp ae cel) oe euros en
B. Rheumatoid Diseases. «3 . <: « = « . “1 «otk Aenea
C. Achromotrichia, 2% 4. 3. os oe eS on
D. Thyrotoxicosis’ . 2. 3 228 2). 2% 05 % 2.5608 ees
FE. Miscellaneous Diseases: <0. . « «| 6). o < -s. ae BeOS!
I. Nomenclature
ROBERT 8S. HARRIS
Accepted name: p-Aminobenzoice acid
Obsolete names: Vitamin By, Anti-gray hair factor
B, factor Trichochromogenic factor
Chromotrichia factor Bacterial growth factor P
Anticanitie vitamin
Bacterial vitamin H
Empirical formula: C;H;NO»
Chemical name: 4-Aminobenzoic acid
Structure:
wn—€ _ S—coon
II. Chemistry
LEMUEL D. WRIGHT and PETER A. TAVORMINA
A. PREPARATION
Both reductive and oxidative methods have been used in the synthesis
of p-aminobenzoic acid (PABA).
The chemical reduction of p-nitrobenzoic acid has been effected with
ammonium sulfide,! tin and hydrochloric acid,?:* ferrous sulfate and am-
1G. Fischer, Ann. 127, 137 (1863).
2 J. Wilbrand and F. Beilstein, Ann. 128, 257 (1863).
31. Widnmann, Ann. 198, 202 (1878).
Il. CHEMISTRY 3
monia,‘ or sodium hydrosulfide.® Catalytic hydrogenation® has been carried
out with platinum oxide as catalyst. More recently, Ravenscroft and his
associates’ employed electrolytic reduction of the nitro compound to pre-
pare PABA in very high yields.
The oxidation of p-acetotoluide offers an alternative route for the prepa-
ration of PABA. Potassium® or calcium’ permanganate has been used for
this purpose. Hydrolysis of the p-acetamidobenzoic acid with mineral acid
yields the amino compound.
Of special interest are the syntheses leading to isotope-labeled PABA.
Murray and his collaborators!? have described a reaction whereby C" is
introduced in the carboxyl group:
1. C4O2
H.N—< pee _n-CaHoLi , [ain—C >] 2) H:0 .
PABA labeled with N has been synthesized by Lustig et al." by heating
a solution of p-bromobenzoic acid in a sealed tube with N?!*-labeled am-
monia.
BC coon a HN€ >—COOH
B. PHYSICAL PROPERTIES
H.N
PABA crystallizes in the form of monoclinic prisms which have a melting
point of 186 to 187°.2.*. ” Andrews and his coworkers reported a melting
point of 188 to 188.5°. The molal heat capacity of the solid was determined
to be 42.5 calories at 25° and 62.2 calories at the melting point, with a heat
of crystallization of 5000 calories per mole.”
PABA is soluble in water, aqueous alcohol, methanol, isopropanol, bu-
tanol, ether, glacial acetic acid, chloroform, and ethyl acetate; sparingly
4W.L. Lewis and H. C. Cheetham, J. Am. Chem. Soc. 48, 2117 (1921).
5 J. Hirata, Japanese Pat. 109,708 (Feb. 21, 1935) [C.A. 29, 4776 (1935)].
6 R. Adams, F. L. Cohen, and O. W. Rees, J. Am. Chem. Soc. 49, 1093 (1927).
7P. H. Ravenscroft, R. W. Lesis, and O. W. Brown, Trans. Electrochem. Soc. 84,
145 (1943).
8 A. W. Hofmann, Ber. 9, 1299 (1876).
9F. Ullmann and J. B. Uzbachian, Ber. 36, 1797 (1903).
10 A. Murray, III, W. W. Foreman, and W. Langham, J. Am. Chem. Soc. 70, 1037
(1948).
11 B. Lustig, A. R. Goldfarb, and G. Gerstl, Arch. Biochem. 5, 59 (1944).
122 C, L. Lazzell and J. Johnston, J. Phys. Chem. 32, 1331 (1928).
18 TF). H. Andrews, G. Lynn, and J. Johnston, J. Am. Chem. Soc. 48, 1274 (1926).
4 P-AMINOBENZOIC ACID
soluble in benzene and carbon disulfide; and insoluble in petroleum
ether.'4; 1%
The pH of a water solution of PABA at 25° has been reported!® to be
3.79 with the hydrogen electrode, and 3.63 with the antimony electrode.
The latter value agrees more closely with the calculated value of 3.60.
The acid and base dissociation constants of PABA have been determined
by a number of investigators’ to be of the order of Kg = 1.2 X 10~° and
KG lo ~ LO:
PABA in its isoelectric state in aqueous solution exists in the neutral
rather than the zwitterionic form. Evidence for this behavior was first
presented by Harris*: *4 in his classic papers on the formol titration of
various amino acids. A different approach to the problem was made by
Klotz and Gruen.”® By calculating the ionization constants of PABA and
of its methyl and ethyl esters from the ultraviolet absorption spectra of the
compounds, these authors reached the same conclusion as did Harris. It is
interesting to note that sulfanilamide, too, exhibits this behavior at its
isoelectric point.
The ultraviolet spectrum of PABA is characterized by two bands, one
of high extinction at long wavelengths, the other of low extinction at short
wavelengths. For the primary band Kumler?® reports a molar extinction
coefficient, in 95 % alcohol, of 17,400 at the wavelength of maximum ab-
sorption, 288 mu, and an extinction coefficient of 8220, for the secondary
band, at 220 mu. Kern and his associates!® found the wavelength of peak
absorption to be 288 my in 95 % alcohol or 99 % isopropanol, with extinction
coefficients ranging, for several experiments, from 15,760 to 16,250 and
from 18,400 to 18,900 for the respective solvents. In aqueous solution,
values for Amax of 266 my,!> 271 muy,” and 284 mys have been reported,
with extinction coefficients of the order of 14,000.
14 Beilstein’s Handbuch der organischen Chemie, 4th ed., Vol. 14, p. 418. Springer,
Berlin, 1931.
15 C, J. Kern, T. Antoshkiw, and M. R. Maiese, Anal. Chem. 20, 919 (1948).
16 fF. Fenwick and E. Gilman, J. Biol. Chem. 84, 605 (1929).
17 J. N. Pring, Trans. Faraday Soc. 19, 705 (1924).
18. Arnall, J. Chem. Soc. 117, 835 (1920).
19 A. Albert and R. Goldacre, Nature 149, 245 (1942).
20 L,. Michaelis and M. Mizutani, Z. physik. Chem. 116, 135 (1925).
21 Ke. Winkelblech, Z. physitk. Chem. 36, 546 (1901).
22 B. Holmberg, Z. physik. Chem. 62, 726 (1908).
23 L.. J. Harris, Biochem. J. 24, 1080 (19380).
244'T. W. Birch and L. J. Harris, Biochem. J. 24, 1086 (1980).
257. M. Klotz and D. M. Gruen, J. Am. Chem. Soc. 67, 848 (1945).
26W. D. Kumler, J. Am. Chem. Soc. 68, 1184 (1946).
27 KH. R. Riegel and K. W. Buchwald, J. Am. Chem. Soc. 51, 484 (1929).
28 T,, Doub and J. M. Vandenbelt, J. Am. Chem. Soc. 69, 2714 (1947).
Il. CHEMISTRY 5
Sodium p-aminobenzoate exhibits maximum absorption at 266 my with
an extinction coefficient of 14,700 to 14,900.'%: °8
The hydrochloride of PABA has an extinction coefficient of only 970 at
the wavelength of maximum absorption, 270 mu.Ӥ In this connection may
be mentioned the rule proposed by Kumler and Strait®® that an aromatic
amine, in a solution sufficiently acid to produce the salt, will have an ab-
sorption spectrum similar to the compound which one would have if the
amino (or alkylamino) group were replaced by hydrogen (or an alkyl group).
Kumler and Strait found that the spectrum of PABA hydrochloride was
remarkably similar to that of benzoic acid® which exhibits an absorption
maximum at 273 my with an extinction coefficient of 980.
Riegel and Buchwald” proposed that the high degree of absorption
which is characteristic of neutral PABA-type molecules (« = ca. 15,000)
ean be attributed to the hemiquinoid form in which such compounds can
exist.
The low degree of absorption of aniline in the vicinity of 270 mu was
accounted for on the basis that this substance cannot assume such a struc-
ture.
Kumler,?* however, demonstrated that the absorption of p-dimethyl-
aminobenzoic acid is actually greater than that of PABA but occurs in a
region of longer wavelength (e€ = 25,400 at 308 my). Kumler was able to
show that, with every increase in basicity of the amino group, peak ab-
sorption is shifted to a longer wavelength, and the extinction coefficient
becomes greater. These phenomena were explained?®: ?° on the basis of the
resonant forms of this type of molecule, a high degree of absorption being
attributable to a structure such as
OH
——— ve
LX > =c
a SS
OF
or, in the case of the dimethylamino derivative,
oe ee
a
% = = -
29 W. D. Kumler and L. A. Strait, J. Am. Chem. Soc. 65, 2349 (1943).
6 P-AMINOBENZOIC ACID
This theory can be extended to account for the low absorption of the
hydrochloride of PABA, since in the salt form the amino group can no
longer resonate with the benzene nucleus or with any group on the ring,
so that only the resonance within the ring itself, or between the ring and
the carboxyl group can function in absorption.
The high degree of absorption exhibited by the N-alkyl analogs of
PABA and their esters occurs at those wavelengths of light which are
responsible for sunburn. The investigation of these compounds as potential
sun-screening agents*’-*’ has received considerable attention in recent years.
C. CHEMICAL PROPERTIES
PABA is decomposed by prolonged boiling with water to give aniline
and carbon dioxide.** Concentrated hydriodic acid at elevated tempera-
tures degrades PABA to benzoic acid.*® Potasstum chlorate and hydro-
chloric acid convert the amino acid to chloranil.* Mild oxidation of PABA
with sodium hypobromite yields p-azobenzoic acid.*®
In those reactions involving the amino or carboxyl group PABA exhibits
the behavior usually associated with arylamines or benzoic acid-type mole-
cules. Alkylation of the amine function has been carried out with methyl,
ethyl, and allyl iodides and a base*’: *8 as well as with dimethyl] sulfate and
base.*® By the proper choice of conditions the reaction can be controlled
to yield the mono- or dialkylated amine*? or the trialkyl-p-benzobetaine.*”: *°
PABA shows no exception to the ease with which aromatic amines and
phenols, which are not sterically hindered, can be brominated. Beilstein
and Geitner*? used bromine water to produce a mixture of 3 ,5-dibromo-4-
aminobenzoic acid and 2,4,6-tribromoaniline. Francis and Hill,*! using a
mixture of potassium bromide and potassium bromate in acid medium to
generate bromine, found that at 0° formation of the dibromo compound is
favored, whereas above 40° the tribromoaniline is obtained. The use of
iodine monochloride in cold acid solution leads to the formation of 3-10do-
30$. Rothman and J. Rubin, J. Invest. Dermatol. 5, 445 (1942).
31 W. D. Kumler and T. C. Daniels, J. Am. Pharm. Assoc. Sci. Ed. 37, 474 (1948).
82 A.C. Giese, E. Christensen, and J. Jeppson, J. Am. Pharm. Assoc. 39, 30 (1950).
33 H}, Christensen and A. C. Giese, J. Am. Pharm. Assoc. 39, 223 (1950).
34 T,. McMaster and R. L. Shriner, J. Am. Chem. Soc. 45, 751 (1923).
35 A. Kwisda, Monatsh. 12, 419 (1891).
36 W. Meigen and EK. Nottebohm, Ber. 39, 744 (1906).
37 A. Michael and J. F. Wing, Am. Chem. J. 7, 195 (1885-1886).
38M. Jaffé, Ber. 38, 1208 (1905).
39 J. Johnston, Proc. Roy. Soc. (London) AT8, 82 (1906).
40F, Beilstein and P. Geitner, Ann. 189, 1 (1866).
41 A.W. Francis and A. J. Hill, J. Am. Chem. Soc. 46, 2498 (1924).
Il. CHEMISTRY 7
and 3 ,5-diiodo-4-aminobenzoic acid, whereas iodine and the potassium salt
of PABA react to produce p-iodoaniline.*: “
Diazotization of PABA has been used in the estimation of the vitamin
(see section on determination). Saunders“ recommends use of the ‘‘in-
verted”? method for the diazotization of monoamino sulfonic or carboxylic
acids which otherwise are not completely transformed to the diazonium
compound because of their low solubility. The “inverted’’ method consists
in adding the nitrite to the alkaline solution of the amino acid and then
running this mixture steadily into the chilled mineral acid. Diazotization
takes place instantly, and the insoluble material which forms is the diazo
compound.
The formation of such PABA salts as the hydroxylamine salt,*° the tri-
ethyl lead salt,4® and the chlorostannate*’ has been described. The acid
chloride can be prepared in the usual manner using thionyl chloride.®
Esterification can be carried out in the conventional manner, using an-
hydrous alcohols and dry hydrogen chloride.*® In practice, the preparation
of PABA esters is usually accomplished by reduction of the previously
esterified nitro compound.*9-*9
Esters of PABA have long been known to possess valuable properties
as local anesthetics.®° Although it is beyond the scope of this review to
discuss in great detail this aspect of the properties of PABA, it seems
desirable to summarize some of the more important considerations in this
# H.L. Wheeler and L. M. Liddle, Am. Chem. J. 42, 441 (1909).
43H. L. Wheeler and L. M. Liddle, Am. Chem. J. 42, 498 (1909).
“4K. H. Saunders, The Aromatic Diazo-Compounds and Their Technical Applica-
tions, p. 9, Edward Arnold and Co., London, 1936.
45 R. E. Oesper and M. P. Ballard, J. Am. Chem. Soc. 47, 2424 (1925).
46 H. Gilman and J. D. Robinson, Rec. trav. chim. 49, 766 (1930).
47 J. G. F. Druce, Chem. News 119, 72 (1919).
48 L.. McMaster and F. F. Ahmann, J. Am. Chem. Soc. 50, 145 (1928).
49S. D. Goldberg and W. F. Whitmore, J. Am. Chem. Soc. 59, 2280 (1937).
°° §. D. Goldberg, W. F. Ringk, and P. E. Spoerri, J. Am. Chem. Soc. 61, 3562 (1939)
51 J. R. Reasenberg and G. B. L. Smith, J. Am. Chem. Soc. 66, 991 (1944).
52 A.C. Cope and E. M. Hancock, J. Am. Chem. Soc. 66, 1448 (1944).
53 FE. M. Hancock and A. C. Cope, J. Am. Chem. Soc. 66, 1738 (1944).
54. M. Hancock, E. M. Hardy, D. Heyl, M. E. Wright, and A. C. Cope, J. Am.
Chem. Soc. 66, 1747 (1944).
55 R. O. Clinton, U. J. Salvador, S. C. Laskowski, and J. S. Buck, J. Am. Chem.
Soc. 72, 1331 (1950).
56D. Curtis, U.S. Pat. 2,518,525 (Aug. 15, 1950).
571). Curtis, U.S. Pat. 2,395,538 (Feb. 26, 1946).
588 J. L. Regnier, British Pats. 477,822-3 (Jan. 3, 1938).
59S. Avakian, U.S. Pat. 2,430,268 (Nov. 4, 1947).
60 A, Einhorn, Ann. 871, 125 (1909).
8 P-AMINOBENZOIC ACID
field. Most local anesthetics derived from PABA have the structure
Ri
H; — \ J ——— X \
ey
R»
Table I lists some of the more common anesthetics of this type. The
specific substituents for the variables in the general formula are indicated.
TABLE I
Common Loca ANESTHETICS DERIVED FROM PABA
Compound X= Ri = R2 =
Benzocaine (anesthesine) —C:2H; a —-
Propesin -n-C3H7 = =
Cycloform —CH(CHs3)2 a _-
Butesin -n-C4H — aa
CH;
Monocecaine —CH.CH,— —H —CH
x
CoH;
Amyleaine —CH.CH,— —H -n-C5sHi4
Novocaine (procaine) —CH.CH.— —C2H; —C2H;
Tutocaine CH—CH—CH, CH; —CH;
CH; CH;
CH;
Larocaine —CH,C—CH,— —C:H; —C.H;
CH;
Butyn CH,—CH.—CH, SAO ales SpAGnlalh
Panthesine —CH,—CH— —C.H; —CoH;
CH:
CH
Vis
CH; CH;
Pontocuine (pantocaine, —CH,.CH.— —CH; —CH;
tetracaine) (a p-(N-butyl-
amino) benzoate)
The nature of the substituents X, Ri, Re. determines toa large extent the
degree of toxicity as well as of anesthetic efficiency of the particular ester.
In general, the longer the group X (up to n-butyl), the greater is the anes-
II. CHEMISTRY 9
thetic value, a normal carbon chain being more effective than a branched
chain. Again, increasing the size of the R; , Re groups produces compounds
of enhanced toxicity and activity, the anesthetic value, however, increasing
more rapidly than does the toxic effect.
Hansen and Fosdick® have described a sulfur analog of novocaine,
“thiocaine”’ :5, 64
<_ D—coscn.cHn(cstty 2
This substance has an activity several times that of procaine, and only
one-half the toxicity of cocaine.®
Fellows®® has tested some substituted phenyl p-aminobenzoates. The
hydrochloride of 2-(piperidinomethyl)-4 ,6-dimethylphenyl-p-aminoben-
H.N
HN
00 2 Se2cirr
an
3
zoate was found to be more active topically than cocaine, without produc-
ing as high a toxic reaction as does the natural alkaloid.
The preparation and pharmacology of many other anesthetic esters of
PABA have been described in the chemical and patent literatures,**-*9; . ©
to which the reader is referred for more detailed information on this subject.
In recent years a number of derivatives of PABA of interest as potential
biochemical intermediates have been synthesized. Auhagen®™ has prepared
and described the properties of the N-(p-aminobenzoyl) derivatives of
D-, L-, and pui-leucine, of D-, L-, and pi-glutamic acid, of L-aspartic acid,
of glycine, and of glycylglycine.
King and Spensley® have synthesized the N-(p-aminobenzoyl) deriva-
tives of pL-isoglutamine, of pLt-glutamine, and of L-glutamine. Lampen and
61 R. Adams, E. K. Rideal, W. B. Burnett, R. L. Jenkins, and E. E. Dreger, J. Am.
Chem. Soc. 48, 1758 (1926).
6 W. B. Burnett, R. L. Jenkins, C. H. Peet, E. E. Dreger, and R. Adams, J. Am.
Chem. Soc. 59, 2248 (1937).
68 H. L. Hansen and L. S. Fosdick, J. Am. Chem. Soc. 55, 2872 (1933).
64 N. F. Albertson and R. O. Clinton, J. Am. Chem. Soc. 67, 1222 (1945).
6° L,. S. Fosdick and H. L. Hansen, J. Pharmacol. Exptl. Therap. 50, 323 (1934).
66H. J. Fellows, Proc. Soc. Exptl. Biol. Med. 58, 7 (1943).
67. Auhagen, Z. physiol. Chem. 277, 197 (1943).
68 F. E. King and P. C. Spensley, J. Chem. Soc. 3159 (1950).
10 P-AMINOBENZOIC ACID
Peterson®® have obtained the N-p-ribosido- and N-.-arabinosido-p-amino-
benzoic acids. N-Glucosido-p-aminobenzoic acid has also been reported.”
Cherbuliez and Mori™ mercurated PABA by means of mercuric acetate.
The reaction results in the formation of an inner salt in which the mercury
HgOH
HN >—cooH ao? | HN—< ss} —Co0H | >
atom is linked to the ring in the 2 position. Support for this structure is
to be found in the fact that when the internal salt is made to react with
iodine the mercury is displaced, and 2-iodo-4-aminobenzoic acid is obtained.
D. DERIVATIVES
A number of solid derivatives useful for the identification of PABA are
listed in Table IT.
TABLE II
DERIVATIVES FOR THE IDENTIFICATION OF PABA
Melting Melting Ref-
Compound point Reference Compound point erence
Acetyl 250-251 8, 72 3,5-Dinitrobenzoyl > 290 76
Benzoyl 278 73 3,5-Dinitrobenzoate salt 195 76
Benzylamide 89-90 74 Pieryl 287-288 77
m-Nitrophenylthiourea 221-222 75 s-Trinitrobenzene addi- 151 78
tion epd.
s-Trinitrobenzene add’n 1l11(d) 78
epd. (K salt)
69 J. O. Lampen and W. H. Peterson, Arch. Biochem. 2, 443 (1943).
70 A. Dansi, Farm. sci. e tec. (Pavia) 2, 195 (1947) [C.A. 42, 639 (1948)].
71}. Cherbuliez and M. Mori, Helv. Chim. Acta 28, 17 (1945).
72 A. Kaiser, Ber. 18, 2942 (1885).
7 A. Bruckner, Ann. 205, 113 (1880).
™ OQ. C. Derner and J. King, J. Org. Chem. 8, 168 (1948).
7° W.L. Tung, C. H. Kao, C. H. Kao, and P. P. T. Sah, Science Repts. Natl. Tsinghua
Univ. A8, 285 (1935) [C.A. 80, 2875 (1936)].
76 B. C. Saunders, J. Chem. Soc. 1988, 1397.
78. P. Mulliken, Identification of Pure Organic Compounds, Vol. 2, p. 68. John
Wiley and Sons, New York, 1916.
7% J. 8S. Sudborough and S. H. Beard, J. Chem. Soc. 97, 773 (1910)
Il. CHEMISTRY ll
E. COLOR REACTIONS
A number of color reactions have been described with reference to
PABA. One such reaction, which may be used as a qualitative test for the
acid, is to be found in Mulliken.” The addition of one drop of a saturated
solution of bleaching powder to an aqueous solution of the acid produces a
red to violet coloration which changes to an opaque orange after 5 minutes.
The colorimetric method for the determination of sulfanilamide developed
by Marshall and his colleagues’*-*! and subsequently modified by Bratton
and Marshall® has since been applied to the estimation of PABA. The
basis for the method is the formation of a red to purple dye which results
when diazotized PABA is made to couple with a suitable arylamine.
When PABA in glacial acetic acid solution is made to react with a 1%
solution of p-dimethylaminobenzaldehyde in the same solvent, a deep-
yellow color is produced. Tauber and Laufer* have described a quantitative
CH;
Se ; — ;
ae D—CHO + HNC >—cooHn aa
CH;
CH;
NS —
a
CH;
assay for PABA based on this reaction. The test is not given by the ali-
phatic amino acids or by tyrosine, phenylalanine, glutathione, urea, panto-
thenic acid, niacin, niacinamide, or thiamine. Substances which were found
to give rise to a similar coloration (e.g., the o- and m- aminobenzoic acids
and their esters, aniline and some of its derivatives) are not normally
present in biological materials.
Kirch and Bergeim®®: ** observed that diazotized thiamine will couple
with PABA to yield a red-colored dye.
Nakahara and his associates” have devised a scheme based on color tests
79 E. K. Marshall, Jr., K. Emerson, Jr., and W. C. Cutting, J. Am. Med. Assoc. 108,
953 (1937).
80}. K. Marshall, Jr., Proc. Soc. Exptl. Biol. Med. 36, 422 (1937).
81. K. Marshall, Jr., J. Biol. Chem. 122, 263 (1937-1938).
82 A.C. Bratton and E. K. Marshall, Jr., J. Biol. Chem. 128, 537 (1939).
83). Strauss, F. C. Lowell, and M. Finland, J. Clin. Invest. 20, 189 (1941).
84H. Tauber and S. Laufer, J. Am. Chem. Soc. 68, 1488 (1941).
85 fF). R. Kirch and O. Bergeim, J. Biol. Chem. 148, 445 (1948).
86 E. R. Kirch and O. Bergeim, J. Biol. Chem. 148, 575 (1942).
87 W. Nakahara, 8S. Ugami, and Y. Nagata, Sci. Papers Inst. Phys. Chem. Research
(Tokyo) 42, 45 (1945) [C.A. 42, 1531 (1948)].
12 P-AMINOBENZOIC ACID
for the differentiation of the isomeric aminobenzoic acids. Three samples
of the solution to be examined are treated individually with potassium
ferrocyanide, potassium ferricyanide, and sodium _ nitrosylopentacyano-
ferrate (II), and the three mixtures are exposed to sunlight. Table III
indicates the colors formed in this test. These colored complexes can be
extracted with butanol from the acidified aqueous solutions.
Other reactions in which the production of color has been attributed to
the presence of PABA have appeared in the literature. Mayer*’ found that
Myobacterium tuberculosis (human strain) when grown on a medium con-
taining PABA elaborates a yellow pigment. The process is enzymatic and
requires ions of magnesium and iron. Glucose, ascorbic acid, or sodium
cyanide represses formation of the pigment. Little is known concerning the
nature of this material, other than that it appears to be some oxidation
product of PABA.
TABLE II
Cotor TrEst FOR THE DIFFERENTIATION OF THE ISOMBRIC AMINOBENZOIC ACIDS
Acid KaFe(CN)e6 K3Fe(CN)e NazFe(CN)sNO
o-Aminobenzoic Brown Blue-green Brown-yellow
m-Aminobenzoic Violet (Colorless) Brown-yellow
p-Aminobenzoic (Colorless) (Colorless) Brown-red
III. Industrial Preparation
H. M. WUEST
The role of p-aminobenzoic acid in human nutrition has not been estab-
lished, nor is a deficiency disease in higher animals known that is caused
by its absence in the feed. The industrial importance of the compound as
a vitamin is therefore very limited. PABA and its salts, however, are recom-
mended in rickettsial diseases, including Rocky Mountain spotted fever
and scrub typhus (4 to 6 g. daily).
The U.S. Pharmacopeia describes PABA as a reagent (not as a pharma-
ceutical) and gives criteria for its purity.2 The industrial preparation starts
from p-nitrotoluene, which is oxidized (e.g., with dilute nitrie acid) to
p-nitrobenzoic acid; the reduction to the amino acid is done with tin or
iron and hydrochloric acid or by catalytic hydrogenation.
88 R. L. Mayer, J. Bacteriol. 48, 337 (1944).
1M. Ek. Howard, Modern Drug Encyclopedia, p. 694. Drug Publishers, New York
1952.
2U. 8S. Pharmacopeia, 14th revision, p. 735, 1947.
IV. BIOCHEMICAL SYSTEMS 13
Production, sales, and value of sales have decreased considerably in 1952
over 1951, as the following figures show:
p-AMINOBENzoIC AcID AND ITs SALTS
1951 1952
Production, Ib. 191,000 122,000
Sales, Ib. 169,000 90 ,000
Value of sales $595 , 000 $276, 000
The price of the acid (U.S.P.) in September, 1953, was $2.50, and $1.77
for 1 lb. technical grade.‘
The important role of p-aminobenzoie acid in the production of folic
acid (pteroylglutamic acid) is mentioned on p. 33.
IV. Biochemical Systems
LEMUEL D. WRIGHT and PETER A. TAVORMINA
The demonstration that a substance is an essential component of a sys-
tem which functions as a biological catalyst is perhaps one of the most
useful criteria for a vitamin. Such a relationship has been shown for many
of the B vitamins. The relative difficulty of freeing PABA from some of its
conjugate forms suggested to Miller and his associates! that PABA may
exist in nature in combination with a protein, that is, that it may function
as the prosthetic group of some enzyme. A number of crystalline enzymes
were examined for their PABA content, which was found to range from 13
y per gram of phosphorylase to 130 y per gram of yeast polypeptidase. It is
quite apparent from a consideration of the molecular weights usually as-
signed to these proteins that the vitamin was present as an impurity,
rather than as an integral part of the enzymes studied.
Williams and his coworkers? investigated a wide variety of proteins,
hormones, and viruses and found that in all cases their PABA content was
too low to be a constituent part of the molecule.
Despite the negative results obtained thus far in the search for a specific
enzyme with which PABA is associated, a number of interesting publica-
tions have appeared concerning enzymatic systems which involve PABA
or its conjugates. Cohen and McGilvery*® investigated the enzymatic con-
3U.8. Tariff Comm. Repts. 175, 36 (1951); 190, 33 (1952).
4Oil, Paint and Drug Reptr. September 14, 1953.
1D. R. Miller, J.O. Lampen, and W. H. Peterson, J. Am. Chem. Soc. 65, 2369 (1943).
2R. J. Williams, F. Schlenk, and M. A. Eppright, J. Am. Chem. Soc. 66, 896 (1944).
3P. P. Cohen and R. W. McGilvery, J. Biol. Chem. 166, 261 (1946).
4P, P. Cohen and R. W. McGilvery, /. Biol. Chem. 169, 119 (1947).
5 P,P. Cohen and R. W. McGilvery, J. Biol. Chem. 171, 121 (1947).
14 P-AMINOBENZOIC ACID
jugation of PABA and glycine as a model system for the study of peptide
bond formation. Rat liver and kidney cortex slices (but not heart, testis,
muscle, brain, or spleen) were found to carry out the synthesis of p-amino-
hippuric acid (PAHA). The reaction is aerobic and will not proceed in the
presence of cyanide, arsenite, iodoacetate, azide, fluoride, or malonate.’
Adenosinetriphosphate, however, will support the synthesis anaerobically.
Potassium and magnesium ions stimulate the conjugation, phosphate is
without effect, and calcium is inhibitory.* In order to maintain the reaction
at low tissue concentrations the addition of members of the citric acid cycle
was found necessary. Under anaerobic conditions the addition of diphospho-
pyridine nucleotide (DPN) inhibits the synthesis, presumably by diverting
to other reactions the energy furnished by ATP. The enzyme system re-
sponsible for PAHA synthesis was found associated with the large insoluble
particles of the liver cells and is unstable to freezing, acetone-drying, and
non-isotonic concentrations of salt. Cohen and McGilvery suggest the
name ‘‘synthetase”’ for enzymes involved in the formation of amides and
esters exclusive of phosphate esters.
The findings of Cohen and McGilvery regarding PAHA formation were
confirmed by Beyer and his associates.* The latter workers, employing
guinea pig liver and renal cortex, observed that conjugation is essentially
complete in | hour. Beyer e¢ al.® report that ‘“benemid” produces 70 % in-
evo
“‘Benemid’’
CH;CH2CH:2
s
NSO,
Va
CH;CH.CH:
hibition of the synthesis at a concentration of 25 X 10-4 WM, without, how-
ever, affecting the oxygen uptake in the aerobic system. PAHA formation
in the ATP-anaerobic system is also inhibited by ‘“‘benemid.” The com-
pound has no effect, however, on the zn vitro phosphorylation of glucose.
The authors interpret the data to mean that ‘“‘benemid”’ exerts its inhibi-
tory action specifically by obstructing the utilization of energy by the con-
jugase, rather than by affecting the production of high energy phosphates.
The inhibitory action of a nitrogen mustard (methylbis-8-chloroethyl-
amine hydrochloride) on PAHA formation in rat liver slices is similarly
interpreted by McKinney’ as being directed specifically toward the ‘‘syn-
thetase’”’ rather than involving the reactions which produce or utilize en-
ergy.
6K. H. Beyer, V. D. Wiebelhaus, E. K. Tillson, H. F. Russo, and K. M. Wilhoyte,
Proc. Soc. Exptl. Biol. Med. 74, 772 (1950).
7G. R. McKinney, J. Pharmacol. Exptl. Therap. 96, 188 (1949).
IV. BIOCHEMICAL SYSTEMS 15
Recently, Woolley’ reported that the thiaminase found in an extract of
carp viscera when incubated with synthetic 2-amino-4-hydroxy-6-pteridy]-
methyl-(4’-methyl-5’-hydroxyethylthiazolium) bromide and PABA will
yield pteroic acid. Substitution of p-aminobenzoylglutamic acid for PABA
OH HBr CH,.CH,0H COOH
ee N JN ‘s | carp
N _ CHs—< | ee Fuaminace.”
| ; uA
=»
J \N/N\N? CH; \
H.N |
NH.
OH ——
5 ; CH,.NH—< fp COOH
NW JNy WA ;
J NZ NY
HoN
gives rise to folic acid. Woolley points out that the specificity of the enzyme
appears to be for the thiazolium group, and that this is not necessarily the
mechanism whereby PABA is transformed into folic acid. The experiment
is especially worthy of note in that a new biosynthetic mechanism is pre-
sented, i.e., one in which the energy for the reaction is derived from a
quaternary ammonium ion rather than from a high-energy phosphate bond.
Baur and Rif’ were able to demonstrate that low concentrations of
PABA, or of sulfanilic acid, sulfathiazole, sulfanilamide, and sulfapyri-
dine, inhibit to a considerable extent the air oxidation of hydroquinone as
well as the degradation of tyrosine by potato tyrosinase.
Another indication that PABA exerts some influence on an enzyme
system is to be found in the work of Mayer,'° who observed that the growth
of Mycobacterium tuberculosis in a medium containing PABA is attended
by the formation of a yellow pigment. The pigment appears to consist, in
part at least, of some oxidation product of PABA, produced by a specific
oxidase which requires ions of magnesium and iron for activity. As might
well be anticipated, glucose, ascorbic acid and cyanide repress the enzy-
matie action, and pigment is not produced.
According to Makino and Yamamoto! rabbit and cattle livers contain
an enzyme which cleaves folic acid. The authors, using the methylene blue
8D. W. Woolley, J. Am. Chem. Soc. 73, 1898 (1951).
9. Baur and H. Rif, Helv. Chim. Acta 25, 523 (1942).
10R. L. Mayer, J. Bacteriol. 48, 337 (1944).
1K. Makino and K. Yamamoto, Sczence 1138, 212 (1951).
16 P-AMINOBENZOIC ACID
technique, found that the enzyme can oxidize L-glutamic acid or p-amino-
benzoylglutamic acid in essentially the same length of time as is required
for pteroylglutamic acid oxidation, which occurs without any detectable
formation of either PABA or p-aminobenzoylglutamic acid. They interpret
this to mean that folic acid is not oxidized as such but is hydrolyzed to
pteroic acid and glutamic acid, the latter being the agent responsible for the
reduction of the methylene blue. Accordingly, the enzyme must attack the
amide bond of folic acid. It would appear interesting, when one considers
the specificity of enzyme action, to investigate the behavior of this enzyme
toward other PABA conjugates, such as PAHA or the bound PABA re-
ported by Ratner.”
V. Biogenesis
LEMUEL D. WRIGHT and PETER A. TAVORMINA
The biogenesis of PABA is only one aspect of a much larger subject,
that of the biogenesis of aromatic compounds in general.
Quinic acid has been suggested as a precursor of aromatic compounds.
By the loss of three molecules of water this compound could yield p-hy-
droxybenzoice acid. Quinic acid when administered to man does indeed yield
urinary hippuric acid.'!:? Phenolic compounds are produced from quinic
acid by a variety of microorganisms including molds, yeast, and bacteria.’
Quinie acid will promote growth of a Neurospora mutant witha requirement
for an aromatic compound, although it is only about 10 % as active for this
purpose as tryptophan.* On the other hand, quinic acid is inactive in pro-
moting growth of any one of a variety of Escherichia coli mutants with
nutritive requirements for two to five aromatic compounds.*
meso-Inositol has been suggested by Fischer® as a precursor of the aroma-
tic ring. Aromatization conceivably could result from a loss of three mole-
cules of water. No actual experimental evidence indicating that PABA
originates from inositol has been published.
12 §. Ratner, M. Blanchard, A. D. Coburn, and D. E. Green, J. Biol. Chem. 155,
689 (1944).
13 §. Ratner, M. Blanchard, and D. E. Green, J. Biol. Chem. 164, 691 (1946).
1k). Lautemann, Ann. 125, 9 (1863).
2A. J. Quick, J. Biol. Chem. 92, 65 (1931).
$B. D. Davis, J. Biol. Chem. 191, 315 (1951).
4M. Gordon, F. A. Haskins, and H. K. Mitchell, Proc. Natl. Acad. Sci. U. S. 36,
427 (1950).
5H. O. L. Fischer, Harvey Lectures 40, 156 (1944-1945).
V. BIOGENESIS lz
Phloroglucinol has been implicated by Shive® as a possible intermediate
in the biogenesis of the aromatic ring. These studies involved a system where
a strain of Leuconostoc mesenteroides that requires pantothenic acid and
either acetate or aromatic amino acids for growth is inhibited by pL-N-
pantoyl-n-butylamine. Pantothenic acid reverses the inhibitor with an
index of about 300 in the presence of acetate. In the presence of phenylala-
nine, tryptophan, or tyrosine the inhibition index is increased to 3000.
Phloroglucinol, particularly in the presence of increased phosphate, is just
as effective as the aromatic amino acids. These data were interpreted to
indicate that pantothenic acid functions in the conversion of acetate to an
intermediate, such as phloroglucinol, common to the biogenesis of the aro-
HO, COOH
COOH
H H
H H —3H.0
H OH
HO H
HOH oe
Quinic acid p-Hydroxybenzoic acid
(1,3,4,5-tetrahydroxy-
cyclohexanecarboxylic acid)
OH OH
HO
H OH sto
> OH
HO H /
HO
H OH
Inositol Phloroglucinol
matic amino acids and presumably of PABA. Phloroglucinol is inactive in
promoting growth of certain Escherichia coli mutants*® with a nutritive re-
quirement for aromatic amino acids.
A scheme for the synthesis of aromatic compounds, including PABA,
has been advanced by Davis’ as a result of the study of a variety of mutants
of Escherichia coli selected by the penicillin method.7-? Mutants were ob-
tained in this manner with requirements for two or more of the aromatic
compounds tyrosine, phenylalanine, tryptophan, and PABA. For certain
mutants with a requirement for the above four aromatic compounds, shi-
kimiec acid was found to duplicate the growth-promoting effect of a combina-
6 W. Shive, Ann. N. Y. Acad. Sci. 52, 1212 (1950); M. Gordon, Ph.D. Thesis,
versity of Texas, 1948.
7B. D. Davis, J. Am. Chem. Soc. 70, 4267 (1948).
®§B. D. Davis, Proc. Natl. Acad. Sci. U.S. 35, 1 (1949).
9 J. Lederberg and N. Zinder, J. Am. Chem. Soc. 70, 4267 (1948).
18 P-AMINOBENZOIC ACID
tion of phenylalanine, tyrosine, tryptophan, and PABA. For rapid growth
one such strain requires, in addition, p-hydroxybenzoic acid (POB).!°
Certain other mutants, blocked further along in the pathway of aromatic
synthesis so that they require tyrosine and phenylalanine for growth, ac-
cumulate shikimie acid and another compound (compound X) in the me-
Gime: 20 4*
Compound X has been isolated in crystalline form and is characterized
as follows: m.p. 150 to 152°; [a}® — 52.1 + 1.0°; Amax (in ethanol) 233 my;
Phenylalanine
Tryptophan
OH
GE COOH een ae Unknown a :
ee (coenzyme A?) intermediates ec
PABA
OH
log. 4.0; pK 3.2; C, 48.83; H, 4.85; molecular weight 162; neutralization
equivalent 162, 179; reduces Fehling’s and Tollen’s reagents; forms crystal-
line semicarbazone, phenylosazone, methyl ester, and acetate. Compound
X has recently been identified as a 5-dehydroshikimie acid."
A tentative and necessarily incomplete outline of aromatic biosynthesis
in Escherichia coli may be summarized according to Davis as follows:
Aromatic
amino acids
COOH COOH JA
known Bee
ane (s) fee mediates
SS Shikimie feat
(5-dehydro- (3,4,5-trihydroxy-
shikimic acid) cyclohexene-1- COOH COOH
carboxylic acid)
wa (slow)
|e
Ss
|
OH NH,
POB PABA
Evidence has been presented by Davis” indicating that PABA partici-
pates in the synthesis of vitamin Bi., since PABA has a sparing effect on
the By requirement of certain Escherichia coli mutants. Although a eata-
10 B.D. Davis, Nature 166, 1120 (1950).
11 T, T, Salamon and B. D. Davis, Proc. 119th Meeting, Am. Chem. Soc. Boston (1981).
2B. D. Davis, J. Bacteriol. 62, 221 (1951).
VI. ESTIMATION 19
lytic function for PABA was not excluded, structural origin of the benzene
ring of vitamin By from PABA was the favored hypothesis.
VI. Estimation
LEMUEL D. WRIGHT and PETER A. TAVORMINA
A. CHEMICAL METHODS
PABA, being a primary aromatic amine, diazotizes with nitrous acid,
and the resulting diazo compound then couples with various other aromatic
compounds to yield colored products that may be determined spectro-
photometrically. The Bratton-Marshall method! for the determination of
sulfanilamide as applied to the determination of PABA entails diazotization,
removal of excess nitrous acid with ammonium sulfamate,? and coupling
of the diazo product with N-(1-naphthyl)ethylenediamine dihydrochloride.
This affords the basis of the preferred chemical method for PABA deter-
NaNO2 ‘sp =
<_>—coon Hol > HOOC—K_—_Y—N:*Cl
H
N—CH,CH.NH,
VAN
HOOC—{ Nir He =e
NZ
H
x
H.N
N—CH,CH,NH;
|
aN
NN
|
N=N—€ coon
mination. The colored compound formed by coupling diazotized sulfanila-
mide with N-(1-naphthyl)ethylenediamine has an absorption maximum at
545 mu.!
1 A.C. Bratton and E. K. Marshall, Jr., J. Biol. Chem. 128, 537 (1939).
2. K. Marshall, Jr., and J. T. Litchfield, Jr., Science 88, 85 (1938).
20 P-AMINOBENZOIC ACID
Several advantages derive from the use of this amine: ease of purification
of the diamine, rapid coupling, greater solubility of the dye, increased sensi-
tivity, elimination of the use of buffer, and stability of the color.
Prior to the use of N-(1-naphthyl)ethylenediamine as the coupling agent,
N, N-dimethy]-1-naphthylamine*~’ and N-ethyl-1-naphthylamine® had been
advocated as coupling agents. The determination of PABA in which N,N-
dimethyl-1-naphthylamine is the coupling agent has been described by
Kekert.’
CH; CH; H C.Hs
NY Bie
N N
YS ‘
SAF NF
N, N-Dimetby]-1-naphthylamine N-Ethyl-1-naphthylamine
In the original Marshall method in which the coupling agent is N ,N-
dimethyl-l-naphthylamine,® metals’ and thiocyanate interfere with color
production.’ Presumably these objections also apply to the improved
method where the coupling agent is N-(1-naphthyl)ethylenediamine.!
A disadvantage in the Bratton-Marshall method for the determination
of PABA is that the color reaction is not specific and a variety of biological
compounds all give essentially the same color reaction. Among the known
biological compounds that interfere are 5(4)-amino-4(5)-imidazolecarboxa-
mide,®: !° kynurenine," ? anthranilic acid," tryptophan,! and indole.®
The Bratton-Marshall method has been applied extensively in studies
of PABA and/or other arylamines formed by sulfonamide-sensitive and
-resistant strains of staphylococci.!: 1°
3. K. Marshall, Jr., K. Emerson, Jr., and W. C. Cutting, J. Am. Med. Assoc. 108,
953 (19387).
4H. K. Marshall, Jr., Proc. Soc. Exptl. Biol. Med. 36, 422 (1937).
5K. K. Marshall, Jr., J. Biol. Chem. 122, 263 (1937-1938).
6G. Hecht, Dermatol. Wochschr. 106, 261 (1938).
7H. W. Eckert, J. Biol. Chem. 148, 197 (1948).
8M. I. Gregersen and EK. E. Painter, Am. J. Physiol. 128, 83 (1988).
9W. Shive, W. W. Ackermann, M. Gordon, M. E. Getzendaner, and R. E. Eakin,
J. Am. Chem. Soc. 69, 725 (1947).
10M. R. Stetten and C. L. Fox, Jr., J. Biol. Chem. 161, 333 (1945).
1M. G. Sevag and M. N. Green, J. Bacteriol. 48, 615 (1944).
12 J. Tabone and C. Magis, Bull. soc. chim. biol. 28, 744 (1946).
13 A. W. Eckert, J. Biol. Chem. 148, 205 (1943).
i M. Landy, N. W. Larkum, E. J. Oswald, and F. Streightoff, Science 97, 265 (1943).
15 W. W. Spink, L. D. Wright, J. J. Vivino, and H. R. Skeggs, J. Exptl. Med. 79, 331
(1944).
VI. ESTIMATION 21
PABA also may be determined chemically!® by a procedure in which dia-
zotized thiamine couples with PABA to yield a colored product that is
measured quantitatively in the spectrophotometer after extraction into
isoamy! alcohol.
p-Dimethylaminobenzaldehyde reacts with aromatic amines to give a
yellow color. This reaction has been suggested as a method for the deter-
mination of sulfonamides and PABA.'7-*! The compound responsible for the
color with sulfanilamide is p-dimethylaminobenzylidene-p-aminobenzene-
sulfonamide. Presumably the compound responsible for the color with
PABA is the following:
CH,
Zs ie CH= N= COOH
NS —CH=N—K_
We
CH,
p-Dimethylaminobenzylidene-p-aminobenzoie acid
R; values for o-, m-, and p-aminobenzoic acid by paper chromatography
in the system butanol saturated with 5 N NH,OH were found to be 0.38,
0.19, and 0.12, respectively, by Lederer.” Ry values by paper chromatog-
raphy for PABA, PAHA (p-aminohippuric acid), and PASA (p-amino-
salicylic acid) in the system n-butanol-water have been published by Kele-
men ef al.2> These values may be of aid in the quantitative determination
of PABA and related compounds, particularly in urine.
B. MICROBIOLOGICAL METHODS
Prior to the discovery of microorganisms with a growth factor require-
ment for PABA, a number of investigators had examined natural material
microbiologically for sulfonamide inhibitor.*4?% A test for ‘sulfonamide
inhibitor” that depends upon the activity of natural material in reversing
the bacteriostatic activity of sulfapyridine for Escherichia coli was employed
16}. R. Kirch and O. Bergeim, /. Biol. Chem. 148, 445 (1943).
17H. Tauber and S. Laufer, J. Am. Chem. Soc. 68, 1488 (1941).
18 A. EK. A. Werner, Lancet I, 18 (1939).
19C. J. Morris, Biochem. J. 35, 952 (1941).
270A. T. Fuller, Lancet I, 760 (1942).
21W. V. LaRosa, Proc. Soc. Exptl. Biol. Med. 58, 98 (1943).
22M. Lederer, Australian J. Sci. 11, 208 (1949).
23. Kelemen, B. Tanos, and D. Halmadgyi, Biochem. J. 47, 138 (1950).
24, J. S. Lockwood, J. Immunol. 35, 155 (1938).
25T. C. Stamp, Lancet 287, 10 (1939).
26C. M. MacLeod, J. Exptl. Med. 72, 217 (1940).
27 J. S. Lockwood and H. M. Lynch, J. Am. Med. Assoc. 114, 935 (1940).
28 A. N. Green, Brit. J. Exptl. Pathol. 21, 38 (1940).
29 A. Fleming, J. Pathol. Bacteriol. 50, 69 (1940).
22 P-AMINOBENZOIC ACID
by MacLeod?* in a survey of a wide variety of substances of animal and
bacterial origin. “‘Sulfonamide inhibitor” is found in many tissue extracts.
The amount found is greatly increased by prior autolysis or acid hydrolysis.
In certain species of bacteria the inhibitor is found in the cells only and is
not demonstrable in the culture medium, whereas in other species the in-
hibitor is found in the culture supernatant, and the cells themselves are
relatively free. Sulfonamide resistance (fastness) in a strain of pneumococ-
cus is accompanied by greatly increased production of ‘‘sulfonamide in-
hibitor.”’
MacLeod was quite aware of the limitations of a microbiological assay
that depends upon the reversal of sulfonamides for the determination of
PABA (see section on sulfonamides). He found that the ‘‘sulfonamide in-
hibitor’’ present in some natural materials is not extractable by ether at
an acid pH as would be expected if it were PABA.
The first essentially specific microbiological method for the determina-
tion of PABA was proposed by Landy and Dicken.*® Their method was
based on the primary observations of Underkofler et al.*! and Lampen et al.*”
that Acetobacter suboxydans requires PABA as a growth factor. Their origi-
nal basal medium contained a purified acid hydrolyzate of casein, plus
tryptophan and cystine as nitrogen sources, glycerol as an energy source,
inorganic salts, and the only other growth factors required by the or-
ganism, namely, pantothenic and nicotinic acids. With this basal medium
the organism responds linearly to PABA over a concentration range of 0
to 0.03 y of PABA per 10 ml. of medium. A variety of compounds with some
relationship to PABA in structure were examined for activity, but no com-
pound examined had more than 2 % of the activity of PABA. Water extracts
or solutions of natural materials were examined for apparent PABA con-
tent. Yeast and yeast extract were by far the best sources of PABA studied.
Landy and Streightoff*? subsequently have shown that the incorporation
of purines (adenine alone in relatively large amounts or a mixture of ade-
nine, guanine, and xanthine) in the original basal medium greatly increases
the sensitivity of response of Acetobacter suboxydans to PABA. In the ab-
sence of purines (original medium) a response to PABA could be detected
at a level of 0.01 y per 10 ml. of culture. The inclusion of purines permits
a detectable response to PABA at a level of 0.001 y per 10 ml. Cheldelin
and Bennett*! have modified the Landy-Streightoff medium by the further
inclusion of glucose, Norit-treated peptone, Norit-treated liver, and addi-
30M. Landy and D. M. Dicken, J. Biol. Chem. 146, 109 (1942).
31 ,, A. Underkofler, A. C. Bantz, and W. H. Peterson, J. Bacteriol. 45, 183 (1943).
82 J. OQ. Lampen, L. A. Underkofler, and W. H. Peterson, J. Biol. Chem. 146, 277
(1942).
88 M. Landy and F. Streightoff, Proc. Soc. Exptl. Biol. Med. 62, 127 (1948).
34.V, H. Cheldelin and M. J. Bennett, J. Biol. Chem. 161, 751 (1945).
VI. ESTIMATION 23
tional Norit-treated hydrolyzed casein. These modifications were found to
promote more growth of Acelobacler suboxydans in response to PABA than
had previously been obtained.
The Acelobacler suboxydans method for the determination of PABA has
been applied by Landy et al." and by Spink ef al.!® to a study of PABA pro-
duction by sulfonamide-sensitive and -resistant bacteria.
A microbiological method for PABA determination was proposed by
Lewis® that depends upon the essential nature of PABA for Lactobacillus
arabinosus.*® The strain employed, the basal medium, and the cultural
details are quite similar to those outlined by Snell and Wright? for the
microbiological determination of nicotinic acid. It is essential that the acid-
hydrolyzed, vitamin-free casein used as the main source of nitrogen be
vigorously Norit-treated to remove traces of PABA or other contaminating,
microbiologically active material. The Lactobacillus arabinosus method is
an extremely sensitive method for the determination of PABA with a re-
sponse range to the compound of about 0 to 0.0005 y per 10 ml.
Considerable difficulty has been experienced in a number of laboratories
in the use of this method for the determination of PABA because of excess
growth in the ‘“‘blanks’”’ (no added PABA). Although contamination of the
glassware and the medium with PABA appears to be the most plausible
explanation for the difficulty, it is now the conclusion of a number of in-
vestigators,*® including also the reviewers, that spontaneous mutants of
Lactobacillus arabinosus are produced with such frequency that selection
of these mutants readily occurs. For this reason most laboratories have
abandoned the use of the Lactobacillus arabinosus method for the determina-
tion of PABA.
The Lactobacillus arabinosus method has been applied by Spink e¢ al.!°
to astudy of PABA synthesis by sulfonamide-sensitive and -resistant strains
of staphylococci.
A microbiological assay for PABA has been developed by Mitchell et al.**
and by Thompson et al.4° that depends upon the essential nature of PABA
for an x-ray induced mutant of Neurospora crassa discovered by Tatum
and Beadle.*':” The latter investigators determined that a variety of com-
pounds related in structure to PABA do not promote growth of the mutant,
that both the mutant and the normal parent strain are inhibited by sulfanil-
35 J. C. Lewis, J. Biol. Chem. 146, 441 (1942).
36H. Isbell, J. Biol. Chem. 144, 567 (1942).
37 . E. Snell and L. D. Wright, J. Biol. Chem. 1389, 675 (1941).
38 J). Pennington, Science 103, 397 (1946).
39 H. K. Mitchell, E. R. Isbell, and R. C. Thompson, J. Biol. Chem. 147, 485 (1943).
40 R.C. Thompson, E. R. Isbell, and H. K. Mitchell, J. Biol. Chem. 148, 281 (1943).
41 E. L. Tatum and G. W. Beadle, Proc. Natl. Acad. Sci. U.S. 28, 234 (1942).
4G. W. Beadle and E. L. Tatum, Proc. Natl. Acad. Sci. U.S. 27, 499 (1941).
24 P-AMINOBENZOIC ACID
amide, that PABA reverses sulfanilamide inhibition of both strains, and
that the presence of benzoic acid, p-hydroxybenzoic acid, or tyrosine does
not increase the amount of PABA synthesized by the parent strain.
In the development of an assay method for PABA with the Neurospora
crassa mutant it was observed by Thompson et al.**: 4° that water extraction,
autolysis, or enzymatic digestion of natural materials is not always sufficient
to release all the PABA contained in the sample. Autoclaving at 120° for 1
hour with 6 N H.SO, was the procedure finally recommended for release
of “bound”? PABA.
As originally described, the Neurospora crassa method involves the prepa-
ration of a number of petri plates, each containing basal medium supple-
mented with a definite amount of PABA solution or material to be assayed.
An inoculum block is then placed on each plate and, following a 20-hour
period of growth, the diameter of the mold growth surrounding the inoculum
block is measured with calipers and is dependent on the amount of PABA
in the culture plate. From a response curve obtained with PABA the po-
tency of unknown samples may be calculated. Although the authors claim
a number of advantages for this method of assay, including rapidity and
freedom from contamination due to the short incubation period employed,
it would appear that the method is subject to a number of possible sources
of error owing to the cumbersome method of inoculating the test plates.
Agarwala and Peterson® have described procedures for the determina-
tion of PABA with the PABA-less mutant of Neurospora crassa in which
the mold is grown in liquid culture. Following growth of the organism in
response to PABA or unknown material, the mycelial pads are removed
from the flasks in which they grew and are dried and weighed in the
conventional manner. A variety of compounds (see Table IV) related
to PABA in structure, including folic acid, were found to be devoid of
microbiological activity. Acid and alkaline hydrolysis were studied as
methods for the liberation of “bound PABA.” Although either method of
hydrolysis gives higher apparent PABA values than are obtained without
hydrolysis, the authors point out that the increments observed probably
represent destruction of folic acid. Since the structure of folic acid was
unknown at the time that most of the microbiological methods for the de-
termination of PABA were worked out, many of the data concerning the
distribution of “bound PABA” (see Table V, p. 30) obtained by micro-
biological methods of assay are subject to re-evaluation.
The essential nature of PABA (along with biotin) for Clostridium aceto-
butylicum forms the basis for the microbiological methods for the deter-
mination of PABA with this organism.*4-“
488. C. Agarwala and W. H. Peterson, Arch. Biochem. 27, 304 (1950).
44$.D. Rubbo, M. E. Maxwell, R. A. Fairbridge, and J. M. Gillespie, Australian J.
Exptl. Biol. Med. Sci. 19, 185 (1941).
VI. ESTIMATION 25
Lampen and Peterson*® have described a rapid turbidimetric method
involving a basal medium containing Norit-treated casein as the only
semipurified component. Anaerobic conditions for the growth of Clostridiwm
acetobutylicum were established by the use of sodium hydrosulfite (NaS.0,)
and reduced iron as components of the basal medium, and by incubating
the assays in an “oat jar,” where respiration of the oats establishes a par-
tial pressure of CO. The assay range is approximately 0.3 to 1.5 my of
PABA per 10-ml. culture tube. Lampen and Peterson, confirming other
investigators, found PABA to exist in many natural materials, including
water-soluble products in microbiologically unavailable forms. They re-
ported that combined PABA may be released by alkaline hydrolysis but
that strong acid hydrolysis destroys PABA.
Housewright and Koser*® utilized Clostridium acetobutylicum for PABA
determination with a basal medium containing all purified components.
Anaerobic conditions for the assay were established by alkaline pyrogallol.
A number of compounds related in structure were examined by House-
wright and Koser for microbiological activity. p-Nitrobenzoic acid, p-
aminobenzoylglycine and p-nitrobenzoylglycine are essentially as active as
PABA. Certain other related compounds are considerably less active. As
with Acetobacter suboaydans, less PABA is required for maximum growth
when the basal medium contains added purines (adenine, guanine, and
xanthine). The microbiological method was applied to a study of PABA
synthesis by sulfonamide-sensitive and sulfonamide-resistant strains of
Staphylococcus aureus. The findings of Landy et al.4 and Spink ef al.!® that
resistant strains produce considerably more PABA were confirmed.
Mirick*®® has proposed the use of a pseudomonas organism isolated from
soil for the highly specific determination of PABA. A specific adaptive en-
zyme is formed by this organism which oxidizes PABA presumably to COs: ,
H,0, and NHs3. Diazotizable amine is determined on samples before and
after the action of the enzyme. The difference obtained is attributed to
PABA. A few related compounds are attacked by the enzyme, but none
of the compounds thus oxidized undergoes the diazo reaction. The main
limitation of the method is primarily a matter of sensitivity of the diazo
45S. D. Rubbo and J. M. Gillespie, Nature 146, 838 (1940).
46 J. O. Lampen and W. H. Peterson, J. Am. Chem. Soc. 63, 2283 (1941).
47 ©. R. Park and W. B. Wood, Jr., Bull. Johns Hopkins Hosp. 70, 19 (1942).
48 J. O. Lampen and W. H. Peterson, J. Biol. Chem. 153, 193 (1944).
49. D. Housewright and 8. A. Koser, J. Infectious Diseases 76, 113 (1944).
60 G. S. Mirick, J. Exptl. Med. 78, 255 (1943).
518. D. Rubbo and J. M. Gillespie, Lancet, I, 36 (1942).
52. Wyss, M. Rubin, and F. B. Strandskov, Proc. Soc. Exptl. Biol. Med. 62, 155
(1943).
83 J. O. Lampen and W. H. Peterson, Arch Biochem. 2, 443 (1943).
26 P-AMINOBENZOIC ACID
TABLE IV
SpEcIFIcIry OF MicropioLoGicAL MrtrHops FOR THE DETERMINATION OF PABA
Per cent of PABA activity for the microorganisms indicated
Compounds Acetobac- F
Clostridium acetobutylicum ee a iat prac
p-Aminobenzoic acid (PABA) 100 (49) 100 (30) 100 (40) 100 (35)
o-Aminobenzoic acid 0.01-0.1 (49); 0.001 (51) 0 (30) 0 (41, 42)| 0.00005 (35)
m-Aminobenzoic acid 0.01-0.1 (49); 0.001 (51) 0 (80) 0 (41, 42) 0.009 (35)
p-Nitrobenzoiec acid 90-100 (49) 1.0-1.2 (41, 42)
0 (40)
p-Aminophenylacetie acid 0.01 (49)° 1.6 (30) 0.09 (52)
Sulfanilice acid 0 (49)
p-Nitroaniline 0 (49)
p-Nitrobenzaldehyde 1.0 (49)
p-Dimethylaminobenzalde- 0.1 (49)
hyde
pL-a-Aminophenylacetic acid 0.1 (49)
Ethyl-p-aminobenzoate 10 (49) 0.06 (30)
Methyl-p-aminobenzoate 8-10 (49)
Procaine 10 (49); 1 (51) 2.0 (30)
p-Aminobenzoylglycine 100 (49)
p-Nitrobenzoylglycine 100 (49) 0.35 (30)
p-Chloroacetylbenzoylglycine 10 (49) 0.2 (30)
p-Aminobenzaldehyde 100 (44, 51)
p-Aminobenzamide 0.001 (51) 3 (40)
p-Aminobenzyl aleohol 0 (51)
p-Aminophenol 0 (51)
Benzamide 0 (51)
Aniline 0 (51)
Benzoic acid 0 (51)
p-Acetamidobenzoic acid 0.04 (53) 0 (80) | 0 (40); 2.6 (41, 42)
Pantocaine 0.8 (53)
N-p-Ribosido-p-aminobenzoic 87 (53)
acid
p-Hydroxybenzoic acid 0 (44, 51) 0 (41, 42)
p-Benzamidobenzoic acid 10 (51)
2-Fluoro-4-aminobenzoie acid 38 (52) 38 (52)
2-Bromo-4-aminobenzoic acid 1.05 (52) 1.05 (52)
2-Iodo-4-aminobenzoic acid 0.10 (52) 0.19 (52)
4-Aminoisophthalie acid 0.13 (52) 0.13 (52)
p-Glyeylaminobenzoie acid 9.0 (30)
Folic acid and related com- | See text See text
pounds
* Bach of the following compounds also has been found to be inactive as a source for Neurospora crassa:
p-aminobenzoyl-6-alanine, p-aminobenzoyl-L-aspartie acid, p-aminobenzoyl-pL-aspartic acid, p-amino-
hippurie acid, p-aminobenzoylglutamic acid, p-aminobenzoyl-e-glutamyl-y-glutamylglutamie acid
tetraethyl ester (also the corresponding y,@ and a,@ compounds), p-nitrobenzoyl-a-glutamyl-y-glutamie
acid tetraethyl ester, p-nitrobenzoyl-L-aminosebacie acid, p-nitrobenzoyl-y-glutamyl-f-alanine, p-nitroben-
zoylethanolamine, p-nitrobenzoyldiethanolamine, p-nitrobenzoyl-pu-alanine, p-nitrobenzoylpiperidine,
p-nitrobenzoyl-e-glutamyl-pu-leucine, p-nitrobenzoyl-y-glutamylglycine diethyl ester, p-nitrobenzoy]-p1L-
a-aminopimelie acid, p-nitrobenzoyl-pL-a-aminoadipie acid, p-nitrobenzoyl-pi-threonine, p-nitrobenzoyl-
morpholine, p-nitrobenzoyl-e-glutamylglutamie acid, sodium pteroate, pteroyl-y-glutamyl-y-glutamylglu-
tamic acid, pteroyImonoglutamie acid.4%
’ Stated by Rubbo and Gillespie to be ten times as active as PABA. °
VII. OCCURRENCE IN FOODS 27
reaction which requires the presence of PABA to the extent of at least one
part per million.
VII. Occurrence in Foods
LEMUEL D. WRIGHT and PETER A. TAVORMINA
A variety of natural materials of both plant and animal origin have been
found to contain PABA (Table V). The vitamin occurs not only as such
but also in conjugated form. Thompson and his colleagues! have reported
that approximately 80% of the PABA content of animal tissues is in the
bound form, whereas in plants the bound form represents only 44 % of the
total PABA. |
Several procedures have been utilized for the liberation of the PABA
from the conjugate form. Autolysis,?“* enzymes,?:* and acid!*:° and alka-
line!: *» ®.7 hydrolysis all have been employed. Although it is generally ac-
cepted that autolytic and enzymatic methods free only part of the bound
PABA,?:*:® there is little agreement concerning which of the two alterna-
tive procedures is the more desirable. Thompson ef al.! reported that pure
PABA is destroyed in part by either acid or basic hydrolysis. Lampen and
Peterson? obtained maximum values for liver PABA, using 5 N alkali.
Pure PABA added to a liver sample prior to hydrolysis is recovered in 92
to 100 % yields. Lampen and Peterson suggest that part of the bound PABA
in natural materials is in a form which resists acid hydrolysis. On the other
hand, Pennington® reports that tissue PABA, but not pure PABA, is virtu-
ally completely destroyed by alkaline hydrolysis.
The first indication that not all of the PABA of yeast is in the free state
was the report by Loomis and his associates’ of an antisulfanilamide frac-
tion from yeast which was ether-insoluble, not diazotizable, and not inac-
tivated by acetylation. Blanchard‘ observed that yeast has a higher PABA
content after autolysis, and he attributes this to the presence in yeast of a
1R.C. Thompson, E. R. Isbell, and H. K. Mitchell, J. Biol. Chem. 148, 281 (1943).
2 H. K. Mitchell, E. R. Isbell, and R. C. Thompson, J. Biol. Chem. 147, 485 (1943).
3 J. O. Lampen and W. H. Peterson, J. Biol. Chem. 158, 193 (1944).
4K. C. Blanchard, J. Biol. Chem. 140, 919 (1941).
5D. Pennington, Science 108, 397 (1946).
6 J. C. Lewis, J. Biol. Chem. 146, 441 (1942).
7T. Ekstrand and B. Sjégren, Nature 156, 476 (1945).
8 A.J. Haagen-Smit, A. G. R. Strickland, C. E. P. Jeffreys, and J. G. Kirchner,
Food Research 11, 142 (1946).
9T. A. Loomis, R. 8. Hubbard, and E. Neter, Proc. Soc. Exptl. Biol. Med. 47, 159
(1941).
28 P-AMINOBENZOIC ACID
bound PABA. The conjugated form was thought to be a peptide involving
the arylamino group, since diazotization was found to occur only after
autolysis.
A second form of bound PABA was discovered by Ratner and her col-
laborators.!°: |! This conjugate is believed to consist of one PABA moiety
associated with ten or eleven glutamic acid residues and one other acidic
amino acid. In contrast to the complex reported by Loomis® and by Blan-
chard, the PABA in the peptide described by Ratner is linked to the rest
of the molecule through the carboxyl group. The amino group is free, as
shown by its positive response to the Bratton-Marshall test.
In recent years PABA has been shown to occur as a component of sev-
eral biologically important entities. These include the group of factors that
comprise the folic acid family, namely, folic acid (the liver L. casez factor),”
pteroyl-y-glutamyl-y-glutamylglutamic acid (the fermentation L. casei fac-
tor), pteroylhexaglutamylglutamic acid (Be conjugate),!® rhizopterin
(the S. faecalis R factor),” and folinic acid (the L. citrovorum factor).
COOH
O) MICH.
OH oo |
ey CHNH—€_>—CNHCHCOOH
| a
ye NZ
H.N
Folie acid
O COOH CH,
OH | | |
ke CHEK >—C NHCHCH.CH.CO/,NHCHCOOH
Z N:
N if
sm NY
H.N
Fermentation L. casei factor
10S. Ratner, M. Blanchard, A. D. Coburn, and D. E. Green, J. Biol. Chem. 155, 689
(1944).
11§. Ratner, M. Blanchard, and D. E. Green, J. Biol. Chem. 164, 691 (1946).
VII. OCCURRENCE IN FOODS 29
H.N
Rhizopterin
COOH
CH,
O COOH CH,
OH — ee |
De ca yaa NHCHCH.CH.CO/,NHCHCOOH
(fo
nS
Be conjugate
H,
0) CH:
OH CHO fa al
J CHAE >—CNHCHCOOH
RN VA
|
aS
Folinic acid
2 R. B. Angier, J. H. Boothe, B. L. Hutchings, J. H. Mowat, J. Semb, E. L. R.
Stokstad, Y. SubbaRow, C. W. Waller, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith
Jr., Science 103, 667 (1946).
13 E. L. R. Stokstad, B. L. Hutchings, J. H. Mowat, J. H. Boothe, C. W. Waller, R.
B. Angier, J. Semb, and Y. SubbaRow, J. Am. Chem. Soc. 70, 5 (1948).
4B. L. Hutchings, E. L. R. Stokstad, J. H. Mowat, J. H. Boothe, C. W. Waller, R.
B. Angier, J. Semb, and Y. SubbaRow, J. Am. Chem. Soc. 70, 10 (1948).
16 J. H. Boothe, J. H. Mowat, B. L. Hutchings, R. B. Angier, C. W. Waller, E. L. R.
Stokstad, J. Semb, A. L. Gazzola, and Y. SubbaRow, J. Am. Chem. Soc. 70, 1099
(1948).
16 J. J. Pfiffner, D. G. Calkins, E. S. Bloom, and B. L. O’Dell, J. Am. Chem. Soc.
68, 1392 (1946). 4
30
PABA Content oF Naturau MATERIALS
P-AMINOBENZOIC ACID
TABLE V
PABA, (y/g. wet weight)
Source Hine eer References
Free Total
Alfalfa meal 2.0 5 19
Asparagus juice concentrate (dry hilt) 2.00 Alkaline 2 6
weight)
Banana 0.43-0.46 | Acid ul 9P) 5
Cabbage, dried 9.7 13.9 Alkaline 2 6
Carrots 0.08 0.1-0.22 | Acid 1, 2, 4 1,5
Carrots, dried 0.178 0.43 Alkaline 2 6
Corn meal 0.3 5 19
Malt extract (Difco) 0.74 2.20 Alkaline 2 6
Molasses, y /ml. 0.01-0.2 0.32 Acid 4,5 i ak!)
Mushroom 0.5 i183 Acid 4 2
Oats, rolled 0.33 5 19
Oats, seed 0.13 0.5 Acid 4 2
Peanuts, raw 1.6-1.7 Acid Ibs 9) 5
Pineapple juice (solid portion) 0.17-0.22 | Acid 2 8
Potato, Irish 0.34 0.36-0.5 Acid 1, 2,4 aD
Potato, sweet 0.11 0.06-0.12 | Acid 1, 2,4 1,5
Rice bran concentrate, y/ml. ital 5 19
Rice bran concentrate (Galen 2.0 16.2 Alkaline 2 6
“B”)
Rice polish concentrate (Labco) 2.95 9.2 Alkaline 2 6
Spinach 0.12 0.6-1.3 Acid 12; 4 US OR
Wheat germ 0.5-1.0 1.7-1.8 Acid 1,2, 4,.5 1, 5, 19
Wheat middlings 0.52 5 19
Whole wheat 0.25 0.49-0.63 | Acid 1, 2,4 1,5
Yeast 4.9-5.5 Acid ih? 5
Yeast, baker’s pressed 3.6-4.7 4.0-7.9 Acid, autolysis 3,4 1 24
Yeast, brewer’s 102. 5 19
Yeast, brewer’s, dried (3 sam- 6.6-61 9.3-59. Alkaline 2 6
ples)
Yeast extract (Bacto) 40. 5 19
Yeast extract (Difco) 157. 156 Alkaline 2 6
Yeast, autolyzed, dried (Difco) 7.4 12.0 Alkaline 2 6
Yeast torula, autolyzed (fresh 1.74 2.04 Alkaline 2 6
basis)
Spent torula culture medium 0.162 0.235 Alkaline 2 6
(fresh basis)
Brain, rat 0.14 0.7 Acid 4 1
Heart, rat 0.15 1.35 Acid 4 1
Kidney, rat 0.13 1.8 Acid 4 1
Liver, beef <0.1-0.2 1.5-2.5 Acid 12; 4 1,:2;°5
Liver, calf OR 5 19
Liver extract, Lilly 343 5.0 5 19
Liver A (dried powdered sam- 126 0.8-11.0 | Acid, alkaline, 6 3
ple) enzymatic,
autolysis
Liver A, water extract, y/g. of 1.6 2.3-6.7 Acid, alkaline 6 3
original A
Kee 0.07 0.25-0.4 Acid 124 15
Egg, whole, dried (2 samples) 0.195-0. 247 0.238-0.30 Alkaline 2 6
Egg, whole, dried (Difeo) 0.36 2 6
Egg yolk, dried (Difco) 0.80 2 6
Egg albumin, dried, (Difco) 0.055 2 6
Epidermis, mouse, normal | 2.4 Acid 4 20, 21
Source
Epidermis, mouse, methyl
cholanthrene treated
Epidermis, mouse, transplanted
carcinoma
Muscle, rat
Muscle, beef
Meat extract
Beef extract (Difco)
Neopeptone (Difco)
Peptone
Peptone (Difco)
Proteose-peptone (Difco)
Protone (Difco)
Tryptone (Difco)
Gelatin (Difco)
Casein ‘‘PABA-free’”’
Pork
Pork chop
Nucleic acid (Eastman)
Milk, y/ml.
Milk, skim, fresh, y/ml.
Milk, skim, slightly sour, y/ml.
Milk, skim, sour, y/ml.
Blood, human, y/ml.
Blood, mouse, dried, y/g.
Blood, rat, y/ml.
Blood, ox, whole, y/ml.
Urine, human, y/ml.
Sweat, human, y/ml.
Spinal fluid, human, y/ml.
VII.
OCCURRENCE IN FOODS
TABLE V—Concluded
PABA, (y/g. wet weight)
Free
0.40
0.08-0.15
0.0046
0.011
0.0075-0.019
0.035
0.06
0.0004
0.004-0.021
0.0024
0.25
Total
3.09
iS ¢¢
0.04-0. 64
0.157
0.204
0.24-2.6
0.8
0.26-0.34
0.64
0.03-0.10
0.0043
0.0098
0.03-0.04
1.49
0.27
0.325-0.5
Method of
hydrolysis
Acid
Acid
Acid
Acid
Alkaline
Alkaline
Alkaline
Acid
Acid
Alkaline
Acid
Alkaline
Alkaline
Acid
Acid
Acid
Acid, alkaline
Method of
assay”
2, 4, 5, 6
5
5
dl
References
20, 21
20, 21
o
o
bo
to
StH DD HD OH WA>AAARADAAH AA oe
ao
_
©
1, 3, 6, 19
23
19
? Method of assay:
Leuconostoc mesenteroides
. Lactobacillus arabinosus
. Bratton-Marshall colorimetric analysis
. Acetobacter suboxydans
. Clostridium acetobutylicum
Ls
2
3
4. Neurospora crassa mutant
5
6
17 TZ). E. Wolf, R. C. Anderson, E. A. Kaczka, 8. A. Harris, G. E. Arth, P. L. South-
wick, R. Mozingo, and K. Folkers, J. Am. Chem. Soc. 69, 2753 (1947).
18 A. Pohland, E. H. Flynn, R. G. Jones, and W. Shive, J. Am. Chem. Soc. 73, 3247
(1951).
19 M. Landy and D. M. Dicken, J. Biol. Chem. 146, 109 (1942).
20K. L. Tatum, M. G. Ritchey, E. V. Cowdry, and L. F. Wicks, J. Biol. Chem. 163,
675 (1946).
21M. G. Ritchey, L. F. Wicks, and E. L. Tatum, J. Biol. Chem. 171, 51 (1947).
22 R. D. Housewright and 8. A. Koser, J. Infectious Diseases 75, 113 (1944).
23 B.C. Johnson, H. H. Mitchell, and T. 8. Hamilton, J. Biol. Chem. 161, 357 (1945).
32 P-AMINOBENZOIC ACID
VIII. Effects of Deficiency
LEMUEL D. WRIGHT and PETER A. TAVORMINA
A. IN BACT ERX
1. NUTRITION
Investigations in bacterial nutrition have uncovered a number of micro-
organisms with growth factor requirements for PABA. These species to-
gether with their approximate requirements for PABA are summarized in
Table VI. Note in particular the wide range in PABA requirement from
about 0.00003 y per milliliter for one-half maximum growth of Lactobacil-
lus arabinosus to about 0.001 y per milliliter for one-half maximum growth
of Acetobacter suboxydans.
The requirement of certain microorganisms for PABA forms the basis
for several microbiological methods for the determination of PABA. This
subject is discussed in considerable detail under that heading.
Wyss et al.! have shown that the growth factor requirement of a PABA-
less mutant of Neurospora crassa®:* is a function of the pH of the medium.
With increase in the pH of the medium from 4 to 7, larger amounts of
PABA are required for equivalent growth in a given length of time. Wyss
et al. point out that PABA has a dissociation constant of about 2 X 107°.
At pH 4.8 PABA exists in solution as equal amounts of molecules and ions.
At pH 5.8 the molecular form decreases from 50 to 10 %. The portion present
as the molecule drops almost tenfold with each unit rise in pH above 5.8.
They conclude on the basis of the above data that the efficiency of the
vitamin in the nutrition of Neurospora crassa is a function of the molecular
form rather than of the ion. Although the reviewers cannot subscribe to
this explanation as the only possible one, the establishment of the pH opti-
mum for the response of the organism to PABA is of significance in the use
of the PABA-less mutant for the microbiological determination of the fac-
tor.
The specificity of the growth factor requirement for PABA has been ex-
amined with a number of species. These data are summarized in Table IV.
As might be expected, the ortho and meta isomers of PABA are essentially
inactive for all species studied. Rubbo and Gillespie* have reported that
p-aminophenylacetic acid is ten times as active as PABA as a growth factor
for Clostridium acetobutylicum.
It is reasonable to assume from the structure of folic acid as well as other
10. Wyss, V. G. Lilly, and L. H. Leonian, Science 99, 18 (1944).
2.1L. Tatum and G. W. Beadle, Proc. Natl. Acad. Sci. U.S. 28, 234 (1942).
3G. W. Beadle and E. L. Tatum, Proc. Natl. Acad. Sci. U.S. 27, 499 (1941).
4S. D. Rubbo and J. M. Gillespie, Lancet I, 36 (1942).
VIII. EFFECTS OF DEFICIENCY 33
relationships (see section on sulfonamides) that PABA is a precursor of this
vitamin. Mayer® observed that Mycobacterium tuberculosis produces a yel-
low compound in the presence of high concentrations of PABA. Mills et al.®
showed that in the presence of high concentrations of PABA Mycobacterium
tuberculosis produces increased amounts of vitamins By) and By (folie acid
or its conjugates). Possibly these compounds are responsible for the yellow
color originally described by Mayer.
Sarett? demonstrated that Lactobacillus arabinosus synthesizes folic acid
in proportion to the PABA content of the basal medium. The bulk of the
folic acid synthesized occurs free in the medium, only a small fraction re-
maining in the bacterial cells. The synthesis of folic acid from PABA is
largely completed at the end of the log growth period (20 hours) and is
greater in acid-hydrolyzed casein medium than in enzymatically digested
casein or amino acid medium. Addition of L-glutamic acid does not increase
folic acid synthesis.
The extent to which folic acid can satisfy the PABA requirement of bac-
teria is a controversial subject. Sarett? found that folic acid or pteroic acid
has some activity in replacing PABA as a growth factor for Lactobacillus
arabinosus. Similarly, Lampen and coworkers®: * reported that folic acid,
pteroyltriglutamic acid, pteroic acid, or p-aminobenzoylglutamic acid is
less active on a molecular basis than is PABA as a growth factor for Lacto-
bacillus arabinosus. Koft et al.°:" demonstrated the unstable nature of
folic acid and related compounds in solution. It would appear from the
data of these investigators that the PABA activity of folic acid for Lacto-
bacillus arabinosus is due to the presence of PABA in the compound as an
impurity or to the unstable nature of folic acid in solution.
Folic acid was found by Lampen et al.” to be inactive as a source of PABA
for a PABA-less mutant of Escherichia coli. As might be expected (see
section on sulfonamides) the PABA requirement of this mutant may be
satisfied by a combination of amino acids, a purine, and thymine. Under
these conditions, however, growth is not optimal.
Zalokar® found that the PABA requirement of the PABA-less mutant of
Neurospora crassa described by Tatum and Beadle? cannot be satisfied by
5R. L. Mayer, Science 98, 203 (1943).
6R. C. Mills, G. M. Briggs, Jr.. T. D. Luckey, and C. A. Elvehjem, Proc. Soc.
Exptl. Biol. Med. 56, 240 (1944).
7H. P. Sarett, J. Biol. Chem. 171, 265 (1947).
8 J. O. Lampen, R. R. Roepke, and M. J. Jones, J. Biol. Chem. 164, 789 (1946).
9 J. O. Lampen and M. J. Jones, J. Biol. Chem. 170, 133 (1947).
10 B. W. Koft, M. G. Sevag, and E. Steers, J. Biol. Chem. 185, 9 (1950).
1B. W. Koft and M. G. Sevag, J. Am. Chem. Soc. 71, 3245 (1949).
12 J. O. Lampen, M. J. Jones, and R. R. Roepke, J. Biol. Chem. 180, 423 (1949).
13M. Zalokar, Proc. Natl. Acad. Sci. U.S. 34, 32 (1948).
34 P-AMINOBENZOIC ACID
folic acid, pteroic acid, pteroyltriglutamic acid, or p-aminobenzoylglutamic
acid.
More recent studies by Strehler™ have shown that methionine satisfies the
PABA requirement of a PABA-less mutant of Neurospora crassa produced
with nitrogen mustard. Apparently methionine has not been tested for
PABA activity with the x-ray induced mutant of Tatum and Beadle,?: 3
the mutant more commonly employed in microbiological assays. If active,
it would be a source of interference to be contended with in the conduct
or interpretation of assays carried out with Neurospora crassa.
2. SYNTHESIS
The synthesis of PABA by representatives of a great number of bacterial
species has been investigated by Landy et al.,!° using his Acetobacter suboxy-
dans assay.’ 18 Prior to PABA assay, half of each bacterial culture was
filtered through a Seitz filter while the other half was adjusted to pH 5.0
and hydrolyzed by autoclaving at 120° for one-half hour. The PABA
values for cells were calculated from the difference in assay between that
found for the whole culture and that found for the medium. A summary of
the data obtained is contained in Table VIT. Note that in most instances the
bulk of the PABA produced is found in the culture medium rather than
in the cells. The method used by Landy e¢ al.'® in preparing samples for
assay is not sufficient to break down folie acid or related compounds, so
that it would appear that the data obtained are reasonably valid.
PABA synthesis by Torula utilis is adversely affected by a deficiency of
iron according to Lewis.!® Normal yeast cells contain about 60 y of PABA
per gram, whereas yeast suffering from iron deficiency contains about 3 y
per gram. Lewis ef al.2° showed that immediate separation of yeast cells
from medium is essential for retention of PABA activity within the cells.
In one experiment 18.3 y per gram was found on immediate separation, and
12.0 y per gram after separation was delayed for 18 hours while the fermen-
tation mixture was held at 2°.
Jppwright and Williams?! found that the addition of thiamine, pyri-
doxine, alanine, and inositol to a synthetic medium is accompanied by a
14 B. L. Strehler, J. Bacteriol. 59, 105 (1950).
1 W.D. McElroy, J. KE. Cushing, and H. Miller, J. Cellular Comp. Physiol. 30, 331
(1947).
16M. Landy, N. W. Larkum, and E. J. Oswald, Proc. Soc. Exptl. Biol. Med. 52, 338
(1943).
17M. Landy and D. M. Dicken, J. Biol. Chem. 146, 109 (1942).
18 M. Landy and F. Streightoff, Proc. Soc. Haptl. Biol. Med. 52, 127 (1948).
19 J. C. Lewis, Arch. Biochem. 4, 217 (1944).
20 J. C. Lewis, J. J. Stubbs, and W. M. Noble, Arch. Biochem. 4, 389 (1944).
21M. A. Eppright and R. J. Williams, J. Gen. Physiol. 30, 61 (1946).
VIII. EFFECTS OF DEFICIENCY 35
decrease in PABA synthesis by Saccharomyces cerevisiae from 42 y per liter
to 0.83 y per liter. The specific vitamin concerned with this effect on
PABA synthesis was not established.
Agarwala and Peterson” found a depression of PABA synthesis with
four yeasts when the vitamins thiamine, pyridoxine, biotin, pantothenic
acid, nicotinic acid, and inositol were added to a synthetic medium. Thia-
mine appears to be the factor most concerned in influencing PABA synthe-
sis, since reductions from 124 y per gram to 41 y per gram and from 11.8 y
per gram to 2.7 y per gram were observed with Candida krusoides and Torula
utilis, respectively.
TABLE VI
MIcROORGANISMS WITH A GRowTH Factor REQUIREMENT FOR PABA
PABA requite-
ment,
Microorganism y/ml. References
Acetobacter suboxydans 0.001 17, 18, 23-25
Chlamydomonas Moewusii mutant a 26
Clostridium acetobutylicum 0.0001 27-33
Clostridium butylicum a 27
Clostridium felsineum a 27
Clostridium sporogenes 0.0001 34
Clostridium thermosaccharolyticum 0.001 35, 36
Corynebacterium diphtheriae gravis (Dundee) a 37
Escherichia coli mutants 0.00011 8, 12, 38, 39
Lactobacillus arabinosus 0.00003 7, 40, 41
Leuconostoc mesenteroides P-60 0.0001 42
Neurospora crassa mutant 0.004 1-3, 15, 43-46
Ophiostoma multiannulatum mutant a 47
Plasmodium knowles a 48
Propionibacterium sps. a 49
Rhizobium trifolir Stim. 50
Rhodopseudomonas palustris a 51
Rhodotorula aurantiaca 0.001 52
Saccharomyces cerevisiae 0.007 53-55
9 Not specified.
228. C. Agarwala and W. H. Peterson, Arch. Biochem. 27, 327 (1950).
23 J,. A. Underkofler, A. C. Bantz, and W. H. Peterson, J. Bacteriol. 45, 183 (1943).
24 J. O. Lampen, L. A. Underkofler, and W. H. Peterson, J. Biol. Chem. 146, 277
(1942).
25 J. H. Marshall and J. R. Postage, Proc. 1st. Int. ‘Congr. Biochem. p. 338 (1949).
26R. A. Lewin, Nature 166, 196 (1950).
27 J. O. Lampen and W. H. Peterson, Arch. Biochem. 2, 443 (1943).
28 R. D. Housewright and S. A. Koser, J. Infectious Diseases 75, 113 (1944).
29§$. D. Rubbo and J. M. Gillespie, Nature 146, 838 (1940).
ELABORATION OF PABA By Various ORGANISMS
Organism
Aerobacter aerogenes
Alkaligenes fecalis
Bacillus megatherium
Bacillus subtilis
Bacillus vulgatus
Brucella abortus
Clostridium botulinum
Clostridium sporogenes
Clostridium tetani
Corynebacterium diphtheriae
Diplococcus pneumoniae—I
Diplococcus pneumoniae—l,
sulfonamide-resistant
Diplococcus pneumoniae—II1
Diplococcus pneumoniae—II1
Eberthella typhosa
Escherichia coli
Klebsiella pneumoniae
Lactobacillus arabinosus
Lactobacillus caset
Mycobacterium smegma
Mycobacterium stercoris
Mycobacterium tuberculosis
Proteus vulgaris
Pseudomonas aeruginosa
Salmonella paratyphi
Salmonella schottmuellert
Serratia marcescens
Shigella dysenteriae
Shigella paradysenteriae
Shigella paradysenleriae Sonne
Shigella paradysenteriae Sonne,
sulfonamide-resistant
Staphylococcus albus
Staphylococcus aureus
Staphylococcus aureus, sulfon-
amide-resistant
Streptococcus hemolyticus
Streptococcus salivarius
Streptococcus scarlatinae
Tubercle bacillus, H387 (human)
Tubercle bacillus, Ravanel (bo-
vine)
Vibrio cholerae
Yeasts
TABLE VII
PABA, y/ml.
Supernatant Whole culture
0.028 0.045
0.005 0.006
0.007 0.007
0.019 0.019
0.030 0.071
0.013 0.013
= 0.004
== 0.008
= 0.008
0.171 0.241
0 .0027—0 .004 0 .0029-0 .005
= 0.0026
0.003 0.004
0.003 0.005
0.018 0.018
0.007 0.045
0.032 0.042
0 0
0.016 0.016
0.052 0.059
0.019 0.022
0.200 —
0.019 0.020
0.050 0.060
0.019 0.020
0.029 0.050
0.007 0.014
0.018 0.018
0.011 0.017
0.057 0.059
0.080 0.084
0.047 0.061
0.023-0 .063 0 .026-0 .067
0 .347-3 .40 0.361-2.40
0.004 0.012
0.005 0.010
0.030 0.050
0..0-0.083 0.0216
0.002 —
0.022 =
0.04-8.5
0.02-8.5
Reference and
method of assay®
16,
16,
16,
16,
S
ce PP PPP PS PB
2
2
_—
&
Torrperrnerererprereepeeeepeeee
16, a
28, 58, a, b
28, 58, a, b
16, a
16, a
16, a
59-61, a, ¢
61, a
16, a
16, 62, a, a
* Method of assay: a. Acetobacter suboxydans
b. Clostridium acetobutylicum
ce. Colorimetric assay of Tauber and Laufer®$
d. Neurospora crassa mutant
36
VIII. EFFECTS OF DEFICIENCY ot
B. IN ANIMALS
1. Rats
The discovery by Woods and Fildes™ that PABA is somehow concerned
in bacterial metabolism served as the impetus for studies on the significance,
if any, of this compound in animal nutrition. Ansbacher® in 1941 concluded
that PABA is a “vitamin,” since its oral administration at a level of 3 mg.
per day to black or piebald rats that had become gray on a purified basal
diet containing pantothenic acid was accompanied by a return of the fur
to normal pigmentation. Untreated controls continued to show typical
achromotrichia. These observations appeared to contradict much contempo-
rary evidence that pantothenic acid deficiency alone is responsible for nu-
% J. O. Lampen and W. H. Peterson, J. Am. Chem. Soc. 68, 2283 (1941).
31 C. R. Park and W. B. Wood, Jr., Bull. Johns Hopkins Hosp. 70, 19 (1942).
8 K.Shikata, T. Yamaki, and 8. Yamagulin, Acta Phytochim. (Japan) 15, 113 (1949).
33 A. E. Oxford, J. O. Lampen, and W. H. Peterson, Biochem. J. 34, 1588 (1940).
34 G. M. Shull and W. H. Peterson, Arch. Biochem. 18, 69 (1948).
35 F. M. Clark, T'rans. Illinois State Acad. Sci. 36, No. 2, 120 (1943).
36 F. M. Clark and W. R. Mitchell, Arch. Biochem. 3, 459 (1944).
37 F. W. Chattaway, F. C. Happold, B. Lythgoe, M. Sandford, and A. R. Todd,
Biochem. J. 36, vi (1942).
3% B. D. Davis, Nature 166, 1120 (1950).
39 B. D. Davis, Federation Proc. 10, 406 (1951).
40 J. C. Lewis, J. Biol. Chem. 146, 441 (1942).
41H. Isbell, J. Biol. Chem. 144, 567 (1942).
#2 TD). Pennington, Science 108, 397 (1946).
48H. K. Mitchell, E. R. Isbell, and R. C. Thompson, J. Biol. Chem. 147, 485 (1943).
* R.C. Thompson, E. R. Isbell, and H. K. Mitchell, J. Biol. Chem. 148, 281 (1943).
498. C. Agarwala and W. H. Peterson, Arch. Biochem. 27, 304 (1950).
46G. W. Beadle, Physiol. Revs. 25, 643 (1945).
47N. Fries, Nature 155, 757 (1945).
% C. B. Anfinsen, Q. M. Geiman, R. W. McKee, R. A. Ormsbee, and E. G. Ball,
J. Exptl. Med. 84, 607 (1946).
49R. C. Thompson, J. Bacteriol. 46, 99 (1943).
50 V. G. Lilly and L. H. Leonian, J. Bacteriol. 50, 383 (1945).
51S. H. Hutner, J. Bacteriol. 52, 213 (1946).
2 W. J. Robbins and R. Ma, Science 100, 85 (1944).
53N.S. Cutts and C. Rainbow, Nature 164, 234 (1949).
54C. Rainbow, Nature 162, 572 (1948).
56 N. 8. Cutts and C. Rainbow, J. Gen. Microbiol. 4, 150 (1950).
58M. Landy, N. W. Larkum, E. J. Oswald, and F. Streightoff, Science 97, 265 (1943).
59'T. Ekstrand and B. Sjégren, Nature 156, 476 (1945).
6 ©. D. Bird, Nature 159, 33 (1947).
61H. Pope and D. T. Smith, Am. Rev. Tuberc. 54, 559 (1946).
8S. C. Agarwala and W. H. Peterson, Arch. Biochem. 27, 316 (1950).
63H. Tauber and S. Laufer, J. Am. Chem. Soc. 68, 1488 (1941).
6D. D. Woods and P. Fildes, J. Soc. Chem. Ind. (London) 59, 133 (1940).
65S. Ansbacher, Science 93, 164 (1941).
38 P-AMINOBENZOIC ACID
tritional achromotrichia.**?° It should be mentioned, however, that Wil-
liams’! was unable to show any effect of pantothenic acid on gray hair in
rats, and Frost et al.” were able to protect only a small percentage of their
rats against achromotrichia with pantothenic acid alone, whereas liver con-
centrates were much more effective.
Martin” in further studies produced evidence that PABA is essential for
growth and prevention of achromotrichia in the rat only when inositol is
present in the diet. Conversely, the presence of PABA was reported to pro-
voke a need for inositol. Thus the six B vitamins—thiamine, riboflavin,
pyridoxine, choline, nicotinic acid, and caleitum pantothenate—or these six
plus PABA and inositol afford normal nutrition, but the omission of either
one of the last two factors induces a requirement for the other.
The forementioned findings of Ansbacher and Martin with respect to the
significance of PABA in animal nutrition instigated one of the most pro-
longed polemics in the nutrition field. Thus Unna ei al.,’7* Emerson,” and
Henderson et al.7® confirmed the activity of pantothenic acid as an achromo-
trichial factor and reported only negative results from their studies with
PABA. Ershoff” failed to confirm Martin’? by showing that no adverse
effects in rats are encountered when either PABA or inositol alone is ineor-
porated in their diet. On the other hand, a number of investigators have
reported beneficial effects from the feeding of PABA. Sure’: 7° reported
that, on a purified diet containing thiamine, riboflavin, pyridoxine, choline,
calcium pantothenate, nicotinic acid, and factor W concentrate from liver
extract, female rats are unable to nurse their young. When the diet is sup-
plemented further with PABA and inositol most of the young born to
mothers consuming the supplemented diet are raised successfully. Climenko
and McChesney®® confirmed the essential nature of inositol for lactation
66 P. Gyorgy and C. E. Poling, Science 92, 202 (1940).
67 K. Unna and W. L. Sampson, Proc. Soc. Exptl. Biol. Med. 45, 309 (1940).
68 G. A. Emerson and H. M. Evans, Proc. Soc. Exptl. Biol. Med. 46, 655 (1941).
69K. Unna, Am. J. Physiol. 188, 473 (1941).
70 C. A. Elvehjem, L. M. Henderson, 8. Black, and E. Nielsen, J Biol. Chem. 140,
xxxvl1 (1941).
71 R. R. Williams, Sczence 92, 561 (1940).
72 —D. V. Frost, R. C. Moore, and F. P. Dann, Proc. Soc. Exptl. Biol. Med. 46, 507
(1941).
73 G. J. Martin, Am. J. Physiol. 186, 124 (1942).
7™ K. Unna, G. V. Richards, and W. L. Sampson, J. Nutrition 22, 553 (1941).
75 G. A. Emerson, Proc. Soc. Exptl. Biol. Med. 47, 448 (1941).
76 T,. M. Henderson, J. M. McIntire, H. A. Waisman, and C. A. Elvehjem, J. Nutri-
tion 28, 47 (1942).
7 B. H. Ershoff, Proc. Soc. Exptl. Biol. Med. 56, 190 (1944).
78 B. Sure, Science 94, 167 (1941).
79B. Sure, J. Nutrition 26, 275 (1948).
80 PD. R. Climenko and KE. W. McChesney, Proc. Soc. Exptl. Biol. Med. 51, 157 (1942).
VIII. EFFECTS OF DEFICIENCY 39
in the rat and also demonstrated a favorable effect of PABA on the mor-
tality rate of the newborn.
The etiology of achromotrichia in the rat appeared to be further com-
plicated when Martin*!: * reported that black rats receiving a purified
sulfaguanidine-containing diet adequate in calcium pantothenate and
PABA become gray. The graying could be cured with folic acid concen-
trates. Similar findings were reported by Wright and Welch® with black
rats receiving succinylsulfathiazole. Accompanying the achromotrichia seen
in the latter studies were low hepatic stores of pantothenic acid. The achro-
motrichia responded favorably, and the hepatic stores of pantothenic acid
were restored to normal with the daily oral administration of folic acid and
biotin. It was concluded that folic acid and biotin are concerned with ‘‘utili-
zation of pantothenic acid in the rat.’’
Some clarification with respect to the role cf PABA in nutrition resulted
when it was announced by Angier and coworkers* that PABA is a structural
moiety of folic acid. There seems little doubt but that the beneficial effect
observed from the feeding of PABA, as, for example, in the studies of
Briggs and coworkers,®° may be directly attributable to an increased syn-
thesis of folic acid on the part of intestinal microorganisms (and possibly
even tissue cells**) in the presence of the structural component PABA.
A plausible explanation for the occurrence of achromotrichia in rats
receiving diets containing both pantothenic acid and PABA, and its cure
with folic acid and biotin, is possible as a result of three converging lines
of investigation. Firstly, it has been demonstrated by Lipmann and co-
workers” that pantothenic acid is present in tissues largely, if not en-
tirely, in the form of coenzyme A, a ‘‘modified”’ nucleotide containing ade-
nine as the purine component. Secondly, free pantothenic acid has been
found by Wright ef al.88 to have a high renal clearance so that pantothenic
acid probably does not remain long in the animal body without some mecha-
nism for retention by tissue cells. Thirdly, PABA in the form of folic acid,
81G. J. Martin, Proc. Soc. Exptl. Biol. Med. 51, 353 (1942).
82 G. J. Martin, Federation Proc. 1, 58 (1942).
83 T,. D. Wright and A. D. Welch, Science 97, 426 (1943).
84 R. B. Angier, J. H. Boothe, B. L. Hytchings, J. H. Mowat, J. Semb, E. L. R.
Stokstad, Y. SubbaRow, C. W. Waller, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith,
Jr., Science 103, 667 (1946).
85 G. M. Briggs, Jr., T. D. Luckey, R. C. Mills, C. A. Elvehjem, and E. B. Hart,
Proc. Soc. Exptl. Biol Med. 52, 7 (1943).
86 F.S. Daft, Public Health Repts. (U.S.) 62, 1785 (1947).
87 #. Lipmann, N. O. Kaplan, G. D. Novelli, L. C. Tuttle, and B. M. Guirard, J.
Biol. Chem. 186, 235 (1950).
8 L. D. Wright, K. H. Beyer, H. R. Skeggs, H. F. Russo, and E. A. Patch, Am. J.
Physiol. 145, 633 (1946).
40 P-AMINOBENZOIC ACID
or probably more correctly folinic acid, as recently reviewed by Shive,*
is concerned with purine synthesis. Thus it would appear that dietary PABA
is a precursor of folic or folinic acid which is essential for the synthesis of
the purine portion of coenzyme A. Coenzyme A is visualized as the form in
which pantothenic acid is retained. Pantothenic acid, then, rather than
PABA emerges as the factor most directly concerned with normal pigmenta-
tion of fur or hair. Obviously, any break in the postulated sequence of
events, such as dietary restriction of pantothenic acid, dietary restriction
of folic acid combined with the administration of a poorly absorbed sul-
fonamide, the feeding of hydroquinone, or other bacteriostatic agent, as
well as dietary imbalance of B vitamins (possibly by inositol), may be ex-
pected to cause achromotrichia. The condition then will respond to what-
ever measure will restore the normal sequence of events. Some suggestions
with respect to practical means by which ample tissue stores of pantothenic
acid (as coenzyme A or other combined form) might be achieved as a
possible means of combating achromotrichia involve (1) administration of
LBF (Lactobacillus bulgaricus factor,®°) a compound intermediate in struc-
ture between pantothenic acid and coenzyme A; (2) administration of large
amounts of pantothenic acid along with ‘‘caronamide’’ or ‘‘benemide’’*?
to decrease the renal elimination of the vitamin along the lines studied by
Roholt and Schmidt; and (3) administration of repository forms of panto-
thenic acid alone or as in (2).
In rats fed certain sulfonamides such as sulfaguanidine (but not sulfanilie
acid or guanine or succinylsulfathiazole) hyperemia and enlargement of
the thyroid occur, according to MacKenzie et al.** The thyroid hyperplasia
is accompanied by a decreased food intake, a lessened rate of body growth
and development, and a lessened basal oxygen consumption. This thyroid
effect of sulfaguanidine is not increased with PABA, but, on the contrary,
PABA actually enhances the effect.°® The effect of PABA in inhibiting
the thyroid was confirmed by Astwood,** who found that of the three iso-
meric aminobenzoic acids PABA, the only one known to occur naturally,
is the most active. Thiouracil and PABA were compared as antithyroid
89 W. Shive, in Vitamins and Hormones, 9, 76 (1951).
9}. HE. Snell, G. M. Brown, V. J. Peters,. J. A. Craig, E. L. Wittle, J. A. Moore,
V.M. MeGlohon, and O. D. Bird, J. Am. Chem. Soc. 72, 5349 (1950).
91K. H. Beyer, A. K. Miller, H. F. Russo, HE. A. Patch, and W. F. Verwey, Am. J.
Physiol. 149, 355 (1947)
92K. H. Beyer, H. F. Russo, E. K. Tillson, A. K. Miller, W. F. Verwey, and 8. R.
Gass, Am. J. Physiol. 166, 625 (1951).
931K. Roholt and V. Schmidt, Scand. J. Clin. Lab. Invest. 3, 108 (1951).
4 J. B. MacKenzie, C. G. MacKenzie, and KE. V. MeCollum, Science 94, 518 (1941).
95C. G. MacKenzie and J. B. MacKenzie, Endocrinology 32, 185 (1948).
96 1}. B. Astwood, J. Pharmacol. Exptl. Therap. 78, 79 (1943).
VII. EFFECTS OF DEFICIENCY 4]
agents by Gordon et al.’ Both thiouracil and PABA (0.2 % and 3.0 % of the
food, respectively) administerered for 19 to 45 days cause marked thyroid
hyperplasia, basophilia, and appearance of “thyroidectomy” cells in the
anterior hypophysis, and increased resistance of rats to lowered barometric
pressures (190 mm. Hg.). These effects, however, are more pronounced with
thiouracil than with PABA. Both drugs reduce the basal metabolic rate;
neither alters the resistance of the rats to the degree of lung hemorrhage
or extent of eye cataract encountered at the reduced pressures. Thiouracil
inhibits normal weight gains and, after 38 days, induces slight anemia and
granulocytopenia. PABA, on the other hand, does not retard body growth
and exerts no effect on the blood picture.
The effect of PABA on uptake of radioactive iodine by surviving slices
of sheep thyroid has been studied by Franklin et al.°8 This compound as
well as p-aminophenylacetic acid has a pronounced inhibitory effect on the
rate of conversion of inorganic iodide to thyroxine and diiodotyrosine.
2. Micr
Martin and Ansbacher®? have reported that PABA is essential for the
prevention of achromotrichia in the black mouse fed a purified diet adequate
with respect to pantothenic acid. In the black mouse the administration of
hydroquinone causes an achromotrichia that may be cured, according to
Martin and Ansbacher,! with PABA.
On the other hand, Fenton e¢ al.,!°! employing a number of purified diets
and working with two strains of mice (C57 and A) could find no evidence
for the essential nature of PABA in the diet of this species. Growth was
just as good in the absence of PABA and inositol asin their presence. Inosi-
tol did not provoke a requirement for PABA.
Keresztesy et al.1° reported that the inhibition of tumor growth by inosi-
tol previously noted by Laszlo and Leuchtenberger!® may be reversed with
PABA. Their chemotherapeutic experiments involved the use of groups
of seven or eight female Rockland mice bearing transplanted tumors of
similar size (sarcoma 180, 7 to 10 days after transplantation) and main-
tained on a normal diet. Each mouse of a group was injected intravenously
twice a day for 2 consecutive days with the substance or mixture of sub-
stances under test. Forty-eight hours after the first injection the animals
97 A.S. Gordon, E. D. Goldsmith, and H. A. Charipper, Endocrinology 87, 223 (1945).
9% A. L. Franklin, I. L. Chaikoff, and S. R. Lerner, J. Biol. Chem. 158, 151 (1944).
99 G. J. Martin and 8. Ansbacher, Proc. Soc. Exptl. Biol. Med. 48, 118 (1941).
100 G. J. Martin and S. Ansbacher, J. Biol. Chem. 188, 441 (1941).
101 P. F. Fenton, G. R. Cowgill, M. A. Stone, and D. H. Justice, J. Nutrition 42, 257
(1950).
102 J.C. Keresztesy, D. Laszlo, and C. Leuchtenberger, Cancer Research 6, 128 (1946).
103 T). Laszlo and C. Leuchtenberger, Science 97, 515 (1943).
42 P-AMINOBENZOIC ACID
were killed with ether and the tumors were removed, freed from surround-
ing tissue, and weighed. The reversal of the tumor-inhibiting effect of 100
y of inositol was found to be complete with 100 y of injected PABA. Fifty
micrograms of PABA was described as reversing only partially the 100
of inositol. o- and m-Aminobenzoic acids were described as much less effec-
tive antagonists of inositol than PABA.
Although the tumor-inhibiting effect of inositol against mammary adeno-
carcinoma of mice has been confirmed by Hesselbach and Burk,!™ Harris
et al.’°> found that neither inositol nor PABA has any effect on p-dimethyl-
aminoazobenzene carcinogenesis in rats. It may be pointed out that others
have reported that PABA has a retarding effect in chronic myelogenous
leukemia (see separate section) which is just the reverse of its reported
effect in antagonizing a tumor-inhibiting compound.
3. HAMSTERS
Cooperman et al.!°* reported that weanling hamsters fail to grow and die
when receiving purified diets containing the six B vitamins—thiamine,
riboflavin, calcium pantothenate, pyridoxine, nicotinic acid, and choline
plus biotin. When the diet is supplemented further with PABA and inositol,
there are fewer deaths. They conclude that PABA and/or inositol is re-
quired by the growing hamster. Here, again, PABA may be functioning as
a source stimulant for the synthesis of folie acid.
4. CHICKS
Ansbacher®® reported that chicks fed a ration consisting largely of wheat
middlings and yellow corn that had been heated at 110° for about one
week are stimulated in growth when the diet is supplemented with 300 7
of PABA per gram of diet. Similarly Briggs et al.8° found that PABA at
levels of 5 to 15 mg. per 10 g. of diet produces growth responses in chicks
receiving purified diets low in folic acid but complete in all other respects.
5. SWINE
Cunha ef al.” found that during a seven-week feeding experiment with
growing pigs no beneficial effect on external appearance, growth, or effici-
ency of feed utilization is obtained when either PABA or folic acid is added
alone or in combination with inositol and biotin to a purified basal ration
which contains the six B complex vitamins—thiamine, riboflavin, niacin,
104 M. L. Hesselbach and D. Burk, Record Chem. Prog. 5, 37 (1944).
106 P, N. Harris, M. E. Krahl, and G. H. A. Clowes, Cancer Research 7, 162 (1947).
106 J. M. Cooperman, H. A. Waisman, and C. A. Elvehjem, Proc. Soc. Exptl. Biol.
Med. 52, 250 (1943).
7 T. J. Cunha, L. K. Bustad, W. E. Ham, D. R. Cordy, E. C. McCulloch, I. FP.
Woods, G. H. Conner, and M. A. McGregor, J. Nutrition 34, 173 (1947).
IX. METABOLISM 43
pyridoxine, pantothenic acid, and choline. The addition of PABA or folic
acid alone to the basal purified ration did, however, stimulate hemoglobin
formation to a small extent.
Johnson and James!’ could find no definite evidence for a requirement
for PABA or inositol when their combined deficiencies were superimposed
on a choline deficiency, although their omission from the diet of growing
pigs appeared to accentuate the degree of fatty infiltration of the liver.
6. Fis
MeLaren ef al.1°® found that a deficiency of PABA in the diet of trout
results in fatty livers, so that PABA is considered to be a vitamin for this
species. The requirement of trout for PABA is placed at 10 to 20 mg. per
100 ¢. of ration.
IX. Metabolism
LEMUEL D. WRIGHT and PETER A. TAVORMINA
A. EXCRETION IN LOWER ANIMALS
The fate of intraperitoneally administered N'-labeled PABA has been
studied in the mouse by Lustig et al.1 A male mouse weighing 22 g. received
three subcutaneous doses of 10 mg. within a 24-hour period. Nineteen hours
after the last injection, however, only traces were found in the organs, but
227 y of N*, corresponding to 82% of that injected, was present in the
excreta.
Similar results with respect to the rapidity with which administered
PABA is excreted by mice were reported by Tabor et al.? One gram of PABA
per kilogram as the sodium salt was administered either orally or intra-
peritoneally to mice. At intervals after administration of the PABA, ani-
mals were homogenized in a Waring Blendor with water. Aliquots of each
homogenate were assayed for total amine (Bratton-Marshall method,
hydrolysis in 6 N HCl at 100° for 30 minutes). In mice killed immediately
after administration of the drug, all the PABA is recovered in the tissues.
After oral or intraperitoneal administration the total amine disappears
rapidly, so that only 50 % is present in the tissues after 3 hours, and little,
or none at all, after 8 hours.
108 B. C. Johnson and M. F. James, J. Nutrition 36, 339 (1948).
19 B. A. McLaren, E. Keller, D. J. O’Donnell, and C. A. Elvehjem, Arch. Biochem.
15, 169 (1947).
1B. Lustig, A. R. Goldfarb, and G. Gerstl, Arch. Biochem. 5, 59 (1944).
2C. W. Tabor, M. V. Freeman, J. Baily, and P. K. Smith, J. Pharmacol. Exptl.
Therap., 102, 98 (1951).
44 P-AMINOBENZOIC ACID
The distribution of PABA in the tissues of rats 2 hours after the oral
administration of the sodium salt at a level of 5 g. per kilogram of body
weight was studied by Tabor et al.? Free amine is found in greatest con-
centration in lung, liver, and kidney (ca. 1.8 mg. per gram), and in a lower
concentration in brain and muscle (ca. 0.4 mg. per gram). The per cent
conjugated is low and probably insignificant. The concentration in plasma
(1.35 mg. per gram) is twice that in red blood cells.
The acetylation of PABA as a method of ‘‘detoxification” prior to elimin-
ation in the urine appears to have been observed first in the rabbit by
Ellinger and Hensel* and subsequently in man by Muenzen et al.4
Increased amounts of acetylated PABA were found by Harrow et al.®
to occur in the urine of rabbits as a result of insulin injections. The same
group observed that acetylation of PABA by the rabbit is increased by
the simultaneous administration of a variety of compounds that presum-
ably function as a source of acetyl donors.*°
Increased acetylation of PABA (0.2 g. per kilogram per os) was observed
in rabbits by Venkataraman et al.!° when glycine or pL-malic acid was fed
along with the compound. The excretion of PABA as the glucuronide was
also observed. When excretion of acetylated PABA was increased by the
simultaneous administration of glycine or pi-malic acid, there was a de-
crease in that portion of PABA excreted in the acetylated form. Chara-
lampous and Hegsted! point out that the administration of PABA in
large amounts imposes a stress on the animal, so that increases in acetyla-
tion thus observed represent a measure of the effectiveness of such com-
pounds as acetyl donors.
Acetylation of PABA is impaired in the rat made diabetie with alloxan."
Acetylation in such animals is normal again after the administration of
insulin, dicarboxylic acids of the tricarboxylic acid cycle, ATP, acetyl
phosphate, or diacetyl. A return to normal is seen in the fasting animal as
well as in animals on a high fat diet. Such an effect is not apparent, how-
ever, when acetate, acetylmethyl acetate, pyruvate, lactate, glycerol
diacetate, acetoin, butylene glycol, phosphate salt, adenylic acid, or panto-
3 A. Ellinger and M. Hensel, Z. physiol. Chem. 91, 21 (1914).
4 J. B. Muenzen, L. R. Cerecedo, and C. P. Sherwin, J. Biol. Chem. 67, 469 (1926).
5 B. Harrow, A. Mazur, and C. P. Sherwin, J. Biol. Chem. 102, 35 (1933).
6B. Harrow, A. Mazur, E. Borek, and C. P. Sherwin, J. Biol. Chem. 105, xxxiv
(1934).
7B. Harrow, A. Mazur, E. Borek, and C. P. Sherwin, Biochem. Z. 298, 302 (1937).
8 KH. A. Doisy, Jr., and W. W. Westerfeld, J. Biol. Chem. 149, 229 (19438).
9B. Harrow, F. W. Power, and C. P. Sherwin, Proc. Soc. Exptl. Biol. Med. 24,
422 (1927).
10 P, R. Venkataraman, A. Venkataraman, and H. B. Lewis, Arch. Biochem. 26,
173 (1950).
11 F.C. Charalampous and D. M. Hegsted, J. Biol. Chem. 180, 623 (1949).
IX. METABOLISM 45
thenic acid is administered. Charalampous and Hegsted" note that of the
compounds effective in increasing acetylation of PABA by the alloxan-
treated rat only ATP, or compounds expected to stimulate ATP synthesis,
are effective. Reduced acetylation of PABA in the alloxan-treated rat,
then, is due to a relative deficiency of ATP, the immediate energy source
of the reaction.
As might be expected from the fact that pantothenic acid, as coenzyme
A, is concerned with the acetylation of such compounds as sulfanilamide™:
and choline,“ pantothenic acid deficiency is accompanied by impaired
acetylation of PABA in the rat. Riggs and Hegsted’® found that normal
rats acetylate 70% of the amount of PABA excreted in 24 hours after a
l-mg. or 2.5-mg. dose of PABA administered intraperitoneally. On the
other hand, pantothenic acid-deficient rats acetylate only 50% of a 1-mg.
dose or 37 % of a 2.5-mg. dose similarly administered. Acetylation of PABA
is less than normal in thiamine-deficient and riboflavin-deficient rats,
but the magnitude of the effect is not so great as in pantothenic acid de-
ficiency.'® Acetylation by riboflavin-deficient rats is not influenced by the
administration of pantothenic acid. Differences in acetylating ability as a
function of diet are only apparent following the administration of amounts
of PABA of the order of 1 to 2 mg. At a dose of 10 mg. or more, normal,
thiamine-, riboflavin-, or pantothenic acid-deficient rats all acetylate about
the same percentage of injected PABA, namely, about 40 %.
Dumm and Ralli” confirm the effect of pantothenic acid deficiency as a
factor in reducing the ability of the rat to acetylate PABA. Male rats on a
normal diet, a pantothenic acid-deficient diet, or a pantothenic acid-high
diet were found to acetylate a greater percentage of peritoneally injected
PABA than female rats on corresponding diets.
Riggs and Hegsted'® have studied the acetylation of PABA by normal
and pantothenic acid-deficient rats of various body weights after the intra-
peritoneal injection of 1 mg. of the amine. Rats weighing about 300 g.
acetylate a greater percentage of the PABA administered than do weanling
rats of about 40 g. body weight. Young rats show decreased acetylation
almost immediately upon withdrawal of pantothenate from the diet. Older
animals continue to acetylate PABA normally for at least two months.
These latter findings are correlated with the well-known difficulties en-
countered in producing vitamin deficiencies in older animals.”
2 F. Lipmann, J. Biol Chem. 160, 173 (1945).
13 F. Lipmann and N. O. Kaplan, Federation Proc. 5, 145 (1946).
4 F. Lipmann and N. O. Kaplan, J. Biol. Chem. 162, 743 (1946).
1s T. R. Riggs and D. M. Hegsted, J. Biol. Chem. 172, 539 (1948).
16 T. R. Riggs and D. M. Hegsted, J. Biol. Chem. 178, 669 (1949).
17M. E. Dumm and E. P. Ralli, J. Nutrition 44, 265 (1951).
8 T.R. Riggs and D. M. Hegsted, J. Biol. Chem. 193, 669 (1951).
19 FE. C. Miller and C. A. Baumann, J. Nutrition 27, 319 (1944).
46 P-AMINOBENZOIC ACID
Bray and his associates?’ have studied in detail the chemical nature of
the metabolites of PABA that occur in the urine of the rabbit. In the light
of their experiments it appears that PABA may be excreted by the rabbit
in a variety of forms: as free PABA, as the ester-type glucuronide, as the
elycine conjugate (PAHA), as acetylated PABA, as acetylated PAHA, and
as acetylated glucuronide. Although the various metabolites were not
estimated individually, Bray et al. found that only 0 to 21% of the ad-
ministered dose (0.25 to 1.0 g. per kilogram) is excreted as the acetylamino
derivatives (acetylated PABA, acetylated PAHA, and acetylated glu-
curonide of PABA), whereas 44 to 87% of the dose is excreted as “free
diazotizable material’? (PABA, PAHA, and glucuronide of PABA).
These findings render difficult the proper interpretation of any studies
on the zn vivo acetylation of PABA, either in humans or in lower species,
unless such studies have been conducted with reference to the specific
form(s) in which the PABA conjugate under investigation has been ex-
creted.
B. EXCRETION IN HUMAN BEINGS
PABA has been determined in urine by Lewis”! with his Lactobacillus
arabinosus microbiological method. Normal urine was found to contain
about 0.01 to 0.02 y of ‘free’ PABA per milliliter, and about 0.3 to 0.5 v
of “combined” PABA per milliliter. When these values are recalculated
in terms of probable daily output, about 10 to 20 y of “free”? PABA and
about 300 to 500 y of ““combined”’ PABA appear to be excreted.
Landy and Dicken,” using the Acetobacter suboxydans assay, found about
0.01 to 0.02 y of “free’? PABA per milliliter of normal urine or, on recal-
culating to daily output, about 10 to 20 y of “free”? PABA per day.
Thompson et al.?? with the Neurospora crassa p-aminobenzoic-less
mutant found 0.02 y of “free”? PABA or about 0.5 y of ‘‘“eombined’”’? PABA
per milliliter of normal urine. In agreement with Lewis and with Landy
and Dicken, this indicates a daily output of about 500 y of ‘‘combined”
PABA per day.
PABA excretion in normal adults has been studied by Denko eé al.,™4
using the Neurospora crassa p-aminobenzoic-less assay of Thompson et al.”
No mention is made in their paper of hydrolysis of samples prior to deter-
mination, so it may be presumed that the values reported represent ‘‘free”’
20H. G. Bray, H.S. Lake, F. C. Neale, W. V. Thorpe, and P. B. Wood, Biochem J.
42, 434 (1948).
21 J. C. Lewis, J. Biol. Chem. 146, 441 (1942).
22M. Landy and D. M. Dicken, J. Biol. Chem. 146, 109 (1942).
23. R. C. Thompson, E. R. Isbell, and H. K. Mitchell, J. Biol. Chem. 148, 281 (1943).
24 C, W. Denko, W. E. Grundy, J. W. Porter, G. H. Berryman, T. E. Friedemann, and
J. B. Youmans, Arch. Biochem. 10, 33 (1946).
IX. METABOLISM 47
PABA. Urinary excretion of PABA was found to be of the order of 150 y
per day. This value for ‘‘free’? PABA is much higher than that reported
by Lewis”! or by Landy and Dicken.” Fecal excretion of PABA, also deter-
mined without prior hydrolysis of the samples, was found by Denko ef al.
to average about 250 y per day.
PABA excretion by normal adults has been studied by Bloomberg,?°
using the microbiological assay of MacLeod*® that measures PABA in
terms of sulfonamide-reversing activity. Like others, Bloomberg found
PABA to occur in the urine both in the ‘free’? and in the ‘‘combined”
state. Less than 0.002 y of ‘free’? PABA per milliliter was found, while
the “combined”? PABA, determined after acid hydrolysis, was found to
be about 0.02 y per milliliter. From a study of the behavior of pure solu-
tions of derivatives of PABA to acid hydrolysis, Bloomberg concluded
that the “combined” form of PABA present in urine is mainly p-acetyl-
aminobenzoic acid. In the opinion of the reviewers, the approach of Bloom-
berg is subject to criticism in that the presence of other derivatives of
PABA of the same acid lability is not precluded. When the results reported
by Bloomberg are recalculated in terms of probably daily output, less than
2 7 of ‘free’ and about 20 y of ““combined’”’ PABA appear to be excreted.
These values are considerably lower than those found by Lewis,?! Landy
and Dicken,” Thompson et al.?? and Denko et al.?4
The investigations of Bray et al.?° (see Excretion in Lower Animals) on
the nature of the metabolic forms of PABA found in the urine of rabbits
have been extended by Tabor ef al.,> who made a similar study in normal
human beings. Using the countercurrent distribution technique, Tabor
and her collaborators concluded that very little of the administered PABA
(single 6.2-g. dose) is eliminated as such, and only a small fraction is ex-
creted as acetylated PABA. The bulk of the PABA is excreted either as
the glycine conjugate or as the glucuronide. Additional evidence for the
excretion of PABA in the form of the glucuronide is to be found in the
work of Zarafonetis and Chandler,’ who suggested that this was the re-
ducing substance’s: 2% found in the urine of patients receiving PABA, rather
than glucose as had been earlier presumed.*°
Gershberg and Kuhl*! report that 95 % of a small dose (100 mg.) of PABA
2° B. M. Bloomberg, S. African J. Med. Sci. 11, 51 (1946).
26 C. M. McLeod, J. Exptl. Med. 72, 217 (1940).
27 C. J. D. Zarafonetis and J. P. Chandler, J. Lab. Clin. Med. 37, 425 (1951).
28 R. H. Grekin and C. J. D. Zarafonetis, J. Invest. Dermatol. 12, 319 (1949).
29 C. J. D. Zarafonetis, R. H. Grekin, and A. C. Curtis, J. Invest. Dermatol. 11,
359 (1948).
30 C, J. D. Zarafonetis, G. A. Andrews, M. C. Meyers, and F. H. Bethell, Blood 3,
780 (1948).
31 Hf. Gershberg and W. J. Kuhl, Jr., J. Clin. Invest. 29, 1625 (1950).
48 P-AMINOBENZOIC ACID
is acetylated by the normal human. Although this high percentage may
appear at first sight to be at variance with the results of Tabor et al.,? two
points must be kept in mind: (1) the per cent of administered PABA ace-
tylated decreases with increase in the size of the dose (the small percentage
of acetylated PABA reported by Tabor et al. is associated with a dose of
6.2 g.); (2) Gershberg and Kuhl used the Bratton-Marshall reaction as
their method of assay, so that in actuality the ‘amount acetylated” repre-
sents not only acetyl PABA, but also acetyl PAHA, acetyl PABA glu-
curonide, and any other PABA derivatives in which the amino group is
involved.
Gershberg and Kuhl included in their studies the ‘‘acetylation” of PABA
in subjects suffering from various metabolic disorders. A standard dose of
500 mg. of PABA was used in most experiments. Normal (control) in-
dividuals were found to ‘“‘acetylate” 88 + 1.9% of the standard dose. In
patients with severe liver disease acetylation 1s normal. The authors infer
that acetylation does not occur exclusively or necessarily primarily in the
liver but that other tissues also participate in acetylation. Acetylation is
normal in patients with rheumatoid arthritis, diabetes mellitus, hypo-
thyroidism, sprue, and leukemia. Patients with hyperthyroidism have a
diminished capacity to acetylate. This usually returns to within normal
limits as the hyperthyroidism is controlled. In two patients with hyper-
thyroidism the administration of sodium acetate was found to increase the
capacity to acetylate. The authors suggest that in hyperthyroidism there
is a decrease in available acetate, owing to its rapid utilization, which may
contribute to the decreased ability to acetylate PABA.
Zilli and Di Ferrante” found very little difference between normal sub-
jects and patients with icterus and cirrhosis of the liver, in the rate at
which 1 g. of injected PABA is excreted, in the total excretion, or in the
proportions of free and acetylated PABA excreted.
Although PABA has been reported to inhibit the 7n vitro conversion of
tyrosine and melanine by tyrosinase,*? White ef al.°* found that PABA
(20 g. daily) is without effect on the excretion of homogentisic acid by an
aleaptonuric.
No data could be found in the literature concerning acetylation of PABA
in individuals in whom a pantothenic acid deficiency might be suspected
from a study of their nutritional history. It would appear to the present
reviewers that such a study could be instrumental in demonstrating the
essential nature of pantothenic acid in human nutrition.
32H}. Zilli and N. Di Ferrante, Acta Vitaminol. 2, 111 (1948).
33 K. EK. Paschkis, A. Cantarow, W. M. Hart, and A. E. Rakoff, Proc. Soc. Hzpil.
Biol. Med. 57, 37 (1944).
34 A. G. White, J. G. Parker, and F. Block, J. Clin. Invest. 28, 140 (1949).
X. TOXICITY 49
The fate of PABA in the kidney has been studied by Lundquist,*® using
the techniques of modern renal physiology. It was concluded that PABA
is secreted by the tubules in addition to being filtered by the glomerulus.
Unfortunately, here, too, PABA was determined by the Bratton-Marshall
colorimetric method which does not differentiate between PABA and
p-aminohippuric acid (PAHA). Since it is well known that PAHA is
secreted by the tubules, the significance of Lundquist’s conclusions may
be questioned. Similarly, Terp’s conclusions*® that PABA is eliminated by
tubular secretion in the dog and rabbit is open to question because the
method employed in the determination of PABA does not differentiate
PABA from PAHA.
Beyer ef al.*7 have studied PABA clearance in the dog, utilizing the
Cohen and McGilvery*® method involving ether extraction at pH 3.95 for
the separation of PABA from PAHA. Extremely low clearance ratios for
PABA were found, thus indicating tubular reabsorption. Although com-
parisons between the human being and the dog with respect to renal phys-
iology are not always warranted, it would be surprising, indeed, if clearance
studies of PABA in the human subject, utilizing a specific method for the
determination of PABA, did not show low renal clearance for PABA,
indicative of tubular reabsorption.
PABA is excreted dermally to the extent of about 0.24 y per 100 ml. of
sweat, according to Johnson ef al.*° Their results were based on microbio-
logical assays with Acetobacter suboxydans.
X. Toxicity
LEMUEL D. WRIGHT and PETER A. TAVORMINA
A. IN LABORATORY ANIMALS
The acute toxicity of PABA and its sodium salt has been determined
in animals by Scott and Robbins.! The results are summarized in Table
VIII.
Orally, acute toxic signs in mice consist at first only of weakness and
loss of normal posture, death occurring in several hours. Mild chronic
36 F. Lundquist, Acta Pharmacol Toxicol. 1, 307 (1945).
36 P. Terp, Acta Pharmacol. Toxicol. 7, 259 (1951).
37 K. H. Beyer, E. K. Tillson, H. F. Russo, G.S. Schuchardt, and A. A. Pitt, Federa-
tion Proc. 11, 13 (1952).
38 P. P. Cohen and R. W. McGilvery, J. Biol. Chem. 166, 261 (1946).
39 B. C. Johnson, H. H. Mitchell, and T. 8. Hamilton, J. Biol. Chem. 161, 357 (1945).
1C. C. Scott and E. B. Robbins, Proc. Soc. Exptl. Biol. Med. 49, 184 (1942).
50 P-AMINOBENZOIC ACID
convulsions with death in 5 to 10 minutes result from intravenous lethal
doses in mice and rats. In dogs, 1 to 2 days elapse before death occurs.
Toxic signs are tremor, listlessness, and weakness. Toxic and chronic
convulsions were observed in one dog receiving 3.0 g. per kilogram. Au-
topsies in the animals which received lethal doses show acute gastroenteritis
with hemorrhages apparently of capillary origin in the small intestine.
Acute necrosis of the liver occurred in the dogs given the two highest
dosages. In chronic toxicity trials of 28 days’ duration, growing rats re-
ceiving up to 1.4 g. of PABA per kilogram orally per day made as good
gains as the controls. At autopsy none of the rats receiving the drug showed
evidence of toxicity.
Richards? studied the toxicity of sodium p-aminobenzoate at pH 6.0 in
a number of species. Only 30% of a group of rats died after intravenous
injections of 4 g. per kilogram. The symptoms consisted of convulsions and
TABLE VIII
Acute Toxicities oF PABA anp Its Sopium SautT
Substance Animal Administration LDso + standard error, in g./kg.
Free acid Mouse Oral 2.85 + 0.4
Rat Oral >6.0
Dog Oral Between 1.0 and 3.0
Sodium salt Mouse Intravenous 4.60 + 0.21
Rat Intravenous 2.76 + 0.24
respiratory paralysis that might be due to the high osmotic pressure of
the solution. On a dose of 4 g. per kilogram given intraperitoneally, 40 %
died within a few hours. No deaths occurred after oral dosages unless at
least 6 g. per kilogram was fed daily for 3 successive days. The LDso in
rabbits is approximately 2 g. per kilogram, given intravenously. The blood
pressure of dogs and cats is raised slightly by doses of 100 mg. of PABA
per kilogram, but this rise was found to depend upon the rate of injection
of the drug. No change in respiration occurs.
Chronic intraperitoneal toxicity of PABA in growing rats at the single
level of 7.5 mg. per day has been studied by Sullivan and Archdeacon.’
After 48 days of treatment, rats receiving PABA show distinct symptoms
of toxicity. Prominent evidences of toxicity are depressed growth, bloated
abdomens, diarrhea, and increased weight of the adrenals.
Chronic oral toxicity of PABA in growing rats at a level of 3% of the
diet was studied by Gordon et al. PABA feeding causes a significant in-
2 R. K. Richards, Federation Proc. 1, 71 (1942).
’C. D. Sullivan and J. W. Archdeacon, Endocrinology 41, 325 (1947).
4 A.S. Gordon, Ef. D. Goldsmith, and H. A. Charipper, Endocrinology 37, 223 (1945).
X. TOXICITY 51
crease in the size of the thyroid gland and a reduced oxygen consumption.
It does not cause significant alterations in the blood picture.
The chronic oral toxicity of PABA fed in the diet in an amount cal-
culated to furnish 2 g. per kilogram of body weight has been studied in
rats by Upton and Zarafonetis.® This dose is approximately five times that
ordinarily administered as a therapeutic dose in man. After the animals
had received the PABA-containing diet for 6 months, complete autopsies
were done and tissue sections examined. No significant pathological changes
were observed with the exception of histological alterations in the thyroid.
These thyroid changes consisted of a reduction in the amount and eosino-
philia of intrafollicular colloid associated with epithelial hypertrophy and
hyperplasia. No hepatotoxic effects were observed.
Ershoff® has fed purified diets containing 1 or 2% of PABA or 1% of
PABA and 1 % of inositol to female rats. No adverse effects on growth or
reproduction were noted in the animals fed these diets for a number of
months. Lactation was poor, but this was observed on unsupplemented
control diets as well and could not be ascribed, therefore, to PABA.
B. IN HUMAN BEINGS
Studies by Strauss and Finland’ have shown that large amounts of
PABA may be administered orally to human beings without untoward
effects. One patient received 2 g. of PABA at 3-hour intervals for 12 hours.
Another patient was given PABA at 2-hour intervals over a 52-hour period
during which time 29 g. was ingested. Later the same patient received 33
g. of PABA over a 66-hour period. Thus in less than two weeks this patient
received over 62 g. of PABA.
With the advent of PABA for the treatment of rickettsial diseases,
amounts of this compound of the order of 25 to 30 g. per day have been
administered orally to hundreds of patients. Concerning the toxicity of
PABA in the treatment of rickettsial diseases, Cruickshank and Mitchell’
state: “No record of pathological changes caused by the drug in human
subjects has been found, although the pathology of two fatal cases of
typhus, treated with it, is said to have been studied’’.°
On the other hand, Cruickshank and Mitchell® were the first to note
toxicity when they observed two cases of acute rheumatic fever and one of
arthritis in children who were treated with PABA where the outcome was
> A.C. Upton and C. J. D. Zarafonetis, Proc. Soc. Exptl. Biol. Med. 76, 450 (1950).
6B. H. Ershoff, Proc. Soc. Exptl. Biol. Med., 63, 479 (1946).
7 E. Strauss and M. Finland, Am. J. Med. Sci. 201, 730 (1941).
8 A. H. Cruickshank and G. W. Mitchell, Jr., Bull. Johns Hopkins Hosp. 88, 211
(1951).
9A. Yeomans, J. C. Snyder, E. S. Murray, C. J. D. Zarafonetis, and R. 8. Ecke,
J. Am. Med. Assoc. 126, 349 (1944).
5p P-AMINOBENZOIC ACID
fatal in each instance. Each patient received about 20 g. per day for about
a week. At autopsy these cases showed marked to extreme deposits of fat
in the epithelial cells of the liver, kidney and myocardium. Similar fatty
changes in liver, kidney, and heart of rabbits were produced experimentally
with PABA.
XI. Pharmacology
CHARLES C. SCOTT
The literature reveals little of pharmacologic interest concerning PABA
prior to recognition of its vitamin activity. Early works dealt with acetyla-
tion processes by the body, toxicity, and relation to certain local anesthetics.
Obviously, PABA seemed relatively weak in ordinary pharmacologic
actions, which no doubt accounts for lack of interest in the compound by
early investigators.
Ansbacher in 1944! and Molitor and Emerson in 1948? have published
extensive reviews on the pharmacology of this substance.
A. ABSORPTION, FATE, AND EXCRETION
PABA is readily absorbed by usual routes of administration. For ex-
ample, oral and subcutaneous doses resulted in maximal blood concentra-
tions in | hour when given to mice and rabbits.’ Elimination of the substance
occurs mainly in the urine. Ellinger and Hensel‘ noted that PABA was
partially acetylated (approximately 30%) by rabbits before excretion.
Man and rabbit were found to acetylate the substance before excretion,
but acetylation did not occur in dogs.®: ® According to Harrow and co-
workers,’ rabbits acetylated 25% of PABA, and this process was greatly
increased by insulin. Conversely, acetylation of PABA was significantly
lower than normal in alloxan-diabetic rats.*
Recent work, however, has shown that in man, at least, PABA is ex-
creted predominantly in other forms. Smith ef al.? found PABA to be
18. Ansbacher, Vitamins and Hormones 2, 215 (1944).
2 H. Molitor and G. A. Emerson, Vitamins and Hormones 6, 69 (1948).
3 J. K. Miller, N. Y. State Dept. Health, Ann. Rept. Div. Labs. and Research 1940,
9. [C. A. 35, 7027 (1941)].
4A. Ellinger and M. Hensel, Z. physiol. Chem. 91, 21 (1914).
5 C. P. Sherwin, Proc. Soc. Exptl. Biol. Med. 22, 182 (1924).
6 J. B. Muenzen, L. R. Cerecedo, and C. P. Sherwin, J. Biol. Chem. 68, xvi (1925).
7B. Harrow, A. Mazur, and C. P. Sherwin, J. Biol. Chem. 102, 35 (1933).
8 F. C. Charalampous and D. M. Hegsted, J. Biol. Chem. 180, 623 (1949).
9H. W. Smith, N. Finkelstein, L. Aliminosa, B. Crawford, and M. Graber, J. Clin.
Invest. 24, 388 (1945).
XI. PHARMACOLOGY 53
converted to p-aminohippuric acid and excreted in this form in the urine.
Conversion of PABA occurred in the liver, normally being rapid, but was
depressed in primary liver disease.!° Apparently the p-aminohippurate is
the main excretory form, but there is still another. According to Tabor
and coworkers,!! man excreted very little PABA in either the tree or acety-
lated forms, excretion occurring mainly as a conjugation product with
glycine or glucuronic acid. Zarafonetis and Chandler” also noted the re-
ducing substance, glucuronic acid, in urine of patients receiving large doses
of PABA. Dogs were found to excrete p-aminohippuric acid when PABA
administration was prolonged.°
Acetylation of PABA in various species of cold-blooded animals is like-
wise variable. Chen and associates“ found that the spadefoot toad and the
nebulous toad were able to acetylate PABA, whereas acetylation did not
occur in the leopard frog or the turtle.
Renal clearance studies have shown that excretion of PABA occurs
partly through secretion by the renal tubules as well as glomerular filtra-
tion. | Secretion may be blocked by o-odohippurate.
Excretion is accomplished by routes other than the urinary tract. In
fact, Denko et al.!® have found that fecal excretion is greater than urinary,
but this is probably owing to synthesis of PABA by intestinal flora. Johnson
et al.” noted a concentration of 0.24 y per 100 ml. in the sweat of four
male humans.
Studies on the entire animal have shown that mice metabolize PABA
rapidly." No significant amount of the substance can be detected after 8
hours. It appears to penetrate freely into the cells of liver, lung, and kidney,
but only slowly into muscle, brain, and erythrocytes. N'° studies by Lustig
et al.8 likewise have revealed no measurable amount of PABA accumulating
in organs and tissues of injected mice.
Be TOXIC Y
Gibbs and Hare!’ in 1889 concluded that PABA was without effect on
the animal organism. Doses of 500 mg. to 2 g. in dogs weighing 5 to 7 kg.
10 W. P. Deiss and P. P. Cohen, J. Clin. Invest. 29, 1014 (1950).
11 C. W. Tabor, M. V. Freeman, J. Baily, and P. K. Smith, J. Pharmacol. Epil.
Therap. 102, 98 (1951).
22 C.J. D. Zarafonetis and J. P. Chandler, J. Lab. Clin. Med. 37, 425 (1951).
13. R. B. Failey, Jr., R. C. Anderson, F. G. Henderson, and K. K. Chen, J. Phar-
macol Exptl. Therap. 78, 366 (1943).
14 F. Lundquist, Acta. Pharmacol. Toxicol. 1, 307 (1945).
15 P. Terp, Acta Pharmacol. Tozicol. 7, 259 (1951).
16 GC. W. Denko, W. E. Grundy, J. W. Porter, G. H. Berryman, T. E. Friedemann, and
J.B. Youmans, Arch. Biochem. 10, 33 (1946).
17 B.C. Johnson, H. H. Mitchell, and T.S. Hamilton, /. Biol. Chem. 161, 357 (1945).
8 B. Lustig, A. R. Goldfarb, and B. Gerstl. Arch. Biochem. 5, 59 (1944).
19 W. Gibbs and H. A. Hare, Am. Chem. J. 11, 435 (1889).
54 P-AMINOBENZOIC ACID
caused no toxic reactions when the substance was administered as a single
dose by stomach tube. In mice, 50 mg. subcutaneously was without effect,
although larger amounts caused temporary excitation followed by pa-
ralysis.2” Ellinger and Hensel‘ noted that 1 g. subcutaneously produced
death in some rabbits, but Hensel?! subsequently found that doses up to
2 g. subcutaneously per day for 4 days were apparently non-toxic.
Tadpoles could withstand a 0.5% solution for 3 days, when death oe-
curred.”? A dose of 1 g. per kilogram was tolerated by dogs but 10 g. per
kilogram caused emesis. In humans, 5 g. produced nausea and vomiting,
although this dose is now commonly exceeded. Toxic symptoms in mam-
mals were similar to those following aniline, namely, fall of body tempera-
ture, marked muscular weakness, accelerated respirations, cardiac depres-
sion, tetanic cramps, and finally death from respiratory standstill.
According to Selbie,”* the oral lethal dose in mice was 100 to 200 mg.
Scott and Robbins™ found PABA to be more toxic to mice and dogs than
to rats. In dogs, single oral doses larger than 1 g. per kilogram caused death
in some instances, with acute gastroenteritis and hemorrhage into the
small bowel. Acute necrosis of the liver resulted from doses of 2 g. per kilo-
gram. Rats, however, tolerated a daily oral dose of 1.4 g. per kilogram for
4 weeks without inhibition of growth or appearance of pathologic changes.
Intravenous injection of 4 g. per kilogram was lethal to 30 % of rats, mani-
festations of toxicity being convulsions and respiratory paralysis.?° Given
intraperitoneally, this dose killed 40% of the animals within 4 hours.
Given orally, no deaths occurred unless at least 6 g. per kilogram was fed
daily for 3 consecutive days. The LDs intravenously in rabbits was ap-
proximately 2 g. per kilogram. Young rats injected with 200 to 500 mg.
per kilogram daily for 21 days grew slightly faster than controls. No ab-
normalities in the blood or urine were noted. After receiving 600 mg. per
kilogram intraperitoneally, rats showed a slight increase of oxygen con-
sumption.
On feeding 1% PABA in the diet of female rats, Ershoff?® could find no
adverse effect on growth, reproduction, or lactation. Adult male rats which
ate a diet containing 3% PABA gained weight normally, and the blood
picture did not change.’ On the other hand, Sullivan and Archdeacon’
20 H. Hildebrandt, Beztr. Chem. Physiol. Pathol. 3, 365 (1903).
21 M. Hensel, Z. physiol. Chem. 98, 401 (1915).
22 A. Oswald, Chemische Konstitution und Pharmakologische Wirkung. Gebriider
Borntrager, Berlin, 1924.
23 F. R. Selbie, Brit. J. Exptl. Pathol. 21, 90 (1940).
24 ©. C. Scott and E. B. Robbins, Proc. Soc. Exptl. Biol. Med. 49, 184 (1942).
25 R. K. Richards, Federation Proc. 1, 71 (1942).
26 B. H. Ershoff, Proc. Soc. Exptl. Biol. Med. 63, 479 (1946).
27 A.S. Gordon, E. D. Goldsmith, and H. A. Charipper, Endocrinology 37, 223 (1945).
23 C. D. Sullivan and J. W. Archdeacon, Endocrinology 41, 325 (1947).
ew
a |
oS
XI. PHARMACOLOGY
found depression of growth in young rats injected intraperitoneally with
only 7.5 mg. daily for 48 days. Marked accumulation of intestinal gas
occurred, along with diarrhea. Young human patients who received up to
148 g. of PABA over a period of days in treatment of acute rheumatic
fever on autopsy showed deposits of fat in epithelial cells of liver, kidney,
and myocardium, according to Cruickshank and Mitchell.?? These authors
noted similar changes in rabbits.
It is obvious from the above that the toxicity of PABA is relatively of
a very low grade, either in acute or chronic administration.
C. PHARMACOLOGICAL ACTIONS
1. CIRCULATORY SYSTEM
Like procaine, intravenous injection of PABA reduced the incidence of
ventricular fibrillation induced by epinephrine in dogs under cyclopropane
anesthesia.*°
According to Gibbs and Hare,!® PABA given intravenously in doses of
0.5 to 1 g. per 8 kg. had no effect on pulse rate or blood pressure of dogs.
This was confirmed by Richards,?° who noted only slight elevation of blood
pressure of dogs or cats injected intravenously with doses of 100 mg. per
kilogram.
2. BLoop
PABA was found to increase blood and tissue iron of liver and muscle
of rabbits treated simultaneously with reduced iron.*! Likewise, Annoni*
found that certain cases of anemia responded to PABA, with a rise of
hemoglobin level amounting to 8 to 12 % and a small increase of red blood
cells. PABA was capable of restoring prothrombin time to normal when
depressed by sodium salicylate.**
3. GASTROINTESTINAL TRACT
Baldini and Cei**: ** reported that PABA temporarily stimulated gastric
motility and gastric secretion but had no effect on gastric acidity of hu-
mans. Merlo and Ercoli** confirmed the gastric secretory effect except in
29 A. H. Cruickshank and G. W. Mitchell, Jr., Bull. Johns Hopkins Hosp. 88, 211
(1951).
30 B. A. Marangoni, C. L. Burstein, and E. A. Rovenstine, Proc. Soc. Exptl. Biol.
Med. 44, 594 (1940).
31 [,. Perosa and A. Tarantini, Boll. soc. ital. biol. sper. 23, 970 (1947).
82 G. Annoni, Acta Vitaminol. 1, 65 (1947).
33 F. Muratore and T. Putignano, Boll. soc. ital. biol. sper. 24, 270 (1948).
34M. Baldini and C. Cei, Boll. soc. ital. biol. sper. 22, 945 (1946).
35 M. Baldini and C. Cei, Boll. soc. ital. biol. sper. 22, 948 (1946).
36 P. Merlo and G. Ercoli, Vitaminologica 1946, 104. [C. A. 41, 5629 (1947)].
56 P-AMINOBENZOIC ACID
histamine-resistant patients. This effeet occurred following either oral or
parenteral administration of PABA.
4. NERVOUS SYSTEM
PABA may exert a mild sedative effect in certain autonomic nervous,
extrapyramidal, Parkinsonian, and choreic syndromes, tics, and epilepsy,
according to a recent report by Morpurgo and Fachini.*”
5. Muscie
Torda and Wolff*® observed no effect of PABA on acetylcholine sen-
sitivity of muscle. Survival of frog muscle and nerve after somatic death
was found by Sollman* to be prolonged by PABA.
6. ENZYME SYSTEMS
a. Cholinesterase
In physiologic concentrations, PABA inhibited serum cholinesterase by
10 to 30 %, according to Zeller.?° This may account for the acetylcholinergic
action of PABA reported by Danielopolu.*! The weak effect of PABA on
serum cholinesterase of humans was confirmed by Paget and Dhellemmes.”
However, Koelle* reported no protective action by PABA against Inactiva-
tion of brain cholinesterase by diisopropyl fluorophosphate.
b. Tyrosinase
The in vitro destruction of epinephrine by tyrosinase was shown by
Martin et al.“4 to be inhibited by PABA.
c. Phosphatase
PABA apparently depresses phosphatase in certain locations but stim-
ulates in others. Tamayo and Cuellar*® and Javier de Elio and Felix Sanz
Sanchez** reported inhibition of serum phosphatase, and Silver and Gold-
37 . Morpurgo and G. Fachini, Acta Vitaminol. 4, 210 (1950).
38 ©. Torda and H. G. Wolff, Am. J. Physiol. 145, 608 (1946).
39'T., Sollman, J. Pharmacol. Exptl. Therap. 90, 14 (1947).
10 HY. A. Zeller, Helv. Chim. Acta 25, 216 (1942).
41 T), Danielopolu, Acta Med. Scand. 118, 1 (1944) [C. A. 39, 2563 (1945)].
2M. Paget and G. Dhellemmes, Compt. rend. 224, 503 (1947).
43 G. B. Koelle, J. Pharmacol. Exptl. Therap. 88, 232 (1946).
44 G. J. Martin, C. T. Ichniowski, W. A. Wisansky, and8. Ansbacher, Am. J. Physiol.
136, 66 (1942).
46M. L. Tamayo and F. T. Cuellar, Anales fis. y. quim. (Madrid) 40, 1322 (1944).
[C. A. 42, 7448 (1948) ].
46 FB. Javier de Elio and F. Felix Sanz Sdnchez, Farmacoterap. actual (Madrid) 3,
468 (1946).
XI. PHARMACOLOGY 57
ing” noted depression of bone phosphatase by this substance. On the other
hand, alkaline phosphatase of renal distal tubuli of rats was increased by
PABA and glycosuria was said to result.*’ The significance of these findings
is not clear. Sulfonamides in general were not antagonistic but exerted the
same effect as PABA.
7. ENDOCRINE SYSTEM
a. Thyroid
The Mackenzies*® first noted that PABA caused enlargement of the
thyroid gland of rats, and this observation was confirmed almost simul-
taneously by Astwood.®® The substance likewise produced this effect in
mice and dogs.*! Jn vitro studies by Franklin et al.*? showed that PABA in a
concentration of 10~-* 17 depressed the rate of conversion of inorganic iodide
to thyroxine and diuodotyrosine. Berman** studied the effect of PABA in
six human patients with thyrotoxicosis and obtained a good antithyroid
response in each case. Dosage was | to 1.5 g. parenterally six times weekly
for 3 to 9 months.
Adult male rats fed large doses of PABA for 19 to 45 days? responded
with an increase in size of the thyroid gland from 17 mg. to 41 mg. and a
fall of oxygen consumption of 30%. However, the goiterogenic activity of
PABA was relatively weak.®*! According to McGinty and Bywater,®® the
activity of PABA is 0.3, with thiouracil taken as 100. Vanderlaan and
Bissell®* also noted the goiterogenic action of PABA to be slight but defi-
nite. In large doses (2 g.) to human subjects, Gualco ef al.*? claimed anti-
thyroid activity of PABA to be nearly comparable to that of thiouracil and
methylthiouracil. PABA was found to resemble thiouracil in its action
rather than sulfaguanidine in that both its goiterogenic and hyperplastic
actions were inhibited by iodide.*®® Both PABA and thiouracil inhibited the
in vitro formation of acetylthyroxine from acetyldiiodotyrosine.*®
47 P. H. Silver and J.S. R. Golding, Lancet I, 528 (1945).
4° M. Koltay, K. Kovdes, M. Majoros, D. Halmdgyi, and E. Kelemen, Orvos? Hetilap.
90, 382 (1949) [C. A. 48, 9245 (1949)].
°C. G. MacKenzie and J. B. MacKenzie, Endocrinology 32, 185 (1943).
50 BE. B. Astwood, J. Pharmacol. Exptl. Therap. 78, 79 (1948).
51 G. J. Martin, Arch. Biochem. 3, 61 (1943).
52 AL. Franklin, I. L. Chaikoff, and S. R. Lerner, J. Biol. Chem. 158, 151 (1944).
53 J,. Berman, Proc. Soc. Exptl. Biol. Med. 59, 70 (1945).
64K. A. Jensen and K. Kjerulf-Jensen, Acta Pharmacol Toxicol. 1, 280 (1945).
56D. A. McGinty and W. G. Bywater, J. Pharmacol. Exptl. Therap. 84, 342 (1945).
568 W. P. Vanderlaan and A. Bissell, Endocrinology 38, 308 (1946).
57 §. Gualeo and V. Patrono, Minerva med. 2, 353 (1947) [C. A. 42, 1657 (1948)].
55 C. G. MacKenzie, Endocrinology 40, 137 (1947).
59 R. Pitt-Rivers, Biochim. et Biophys. Acta 2, 311 (1948) [C. A. 48, 6298 (1949)].
58 P-AMINOBENZOIC ACID
b. Miscellaneous
In studies utilizing the Warburg technique and bioassay in ovariec-
tomized rats, Ansbacher et al.®° found that PABA inhibited the destruction
of stilbesterol. Possibly a similar effect was noted recently by Wiesel e¢ al.®!
in that PABA made possible a marked reduction in the cortisone require-
ments of rheumatoid arthritic patients.
8. DETOXICATING ACTION
In a series of papers, Sandground and Hamilton®-® reported that PABA
was highly effective in affording protection against high lethal doses of
certain pentavalent arsenicals and antimonials. This protective action of
PABA did not interfere with the trypanocidal activity of the metalloids.
If injected before the arsenical, PABA protection was maximal. The effect
was distinctly reduced when PABA was administered after the toxic agent,
and protection was further decreased with increasing time interval. This
would suggest a competitive effect for action on some receptor, but no
chemical similarity was noted. Protection was not afforded against the
trivalent arsenicals, Inorganic arsenious acid, mapharsen and arsphenamine,
or tartar emetic (trivalent antimonial). However, PABA did protect
against neoarsphenamine, and this was confirmed by McChesney et al.®
Peters® also found no detoxicant action by PABA against mapharsen. The
mechanism of protection was studied by Harris,** who showed a decrease
in extent and severity of renal damage of rats treated with PABA as com-
pared with animals which received only the toxic drug.
Inhibition of the convulsive action of procaine by PABA was reported
by Richards and Kueter.’°:” However, the peripheral local anesthetic
effect of procaine was not inhibited. The chemical similarity here is obvious.
Voss and Tatum” found a definite, although low-grade, protection by
PABA against certain organic bismuth preparations in rats.
60S, Ansbacher, W. A. Wisansky, and G. J. Martin, Federation Proc. 1, 98 (1942).
61 J,. L. Wiesel, A. S. Barritt, and W. M. Stumpe, Am. J. Med. Sci. 222, 243 (1951).
62 J. H. Sandground, Science 97, 73 (1948).
63 J. H. Sandground, Proc. Soc. Exptl. Biol. Med. 52, 188 (1943).
61 J. H. Sandground and C. R. Hamilton, J. Pharmacol Exptl. Therap. 78, 109, 203
(1943).
65 J. H. Sandground, J. Pharmacol. Exptl. Therap. 78, 209 (1948).
66 J. H. Sandground and C. R. Hamilton, J. Lab. Clin. Med. 28, 1821 (1948).
67}. W. McChesney, O. W. Barlow, and G. H. Klinck, Jr., J. Pharmacol. Exptl.
Therap. 80, 81 (1944).
68 [,, Peters, Proc. Soc. Exptl. Biol. Med. 58, 147 (1948).
69 P_ N. Harris, J. Pharmacol Exptl. Therap. 82, 254 (1944).
70R. K. Richards, J. Biol. Chem. 159, 241 (1945).
71 R. K. Richards and K. E. Kueter, J. Pharmacol. Exptl. Therap. 87, 42 (1946).
72 1). Voss and A. L. Tatum, J. Pharmacol. Exptl. Therap. 90, 161 (1947).
XI. PHARMACOLOGY 59
D. THERAPEUTIC USES
1. Gray Harr
Early in the work on PABA as a vitamin-like substance, Ansbacher™
reported the substance to be effective against nutritional achromotrichia
of rats. Subsequently, Martin and Ansbacher found that PABA cured the
gray hair of mice caused by hydroquinone” and nutritional deficiency.”
Emerson,’® however, could obtain no chromotrichial effect with PABA
in grayed rats, although pantothenic acid was effective. Her work was con-
firmed by Unna et al.,” Henderson et al.,’> and Brown and Sturtevant.”
Ershoff,°° who also was unable to confirm the work of Martin, stated that
the latter employed near-marginal levels of some of the B-complex vitamins.
Early clinical trials by Sieve®! and Eller and Diaz were said to show
evidence of darkening of gray hair in human beings, even though the gray
color was of many years’ duration. Friedgood* also noted a few patients
in whom PABA seemed to have chromotrichial activity. Photographs
showing darkening of hair were presented by DeVilbiss*! for a series of
sixteen patients who received the substance. Stimulation of libido was
noted, and this was also claimed by Sieve* in a series of 600 patients. Also
reported in this large series were darkening of gray hair and increased
pigmentation of normally pigmented areas as well as of areas lacking pig-
mentation. Nevi and freckles almost disappeared. Lactation was increased,
and amenorrhea was relieved in some cases. Of twenty-two women sterile
for over 5 years, PABA therapy was followed by conception in twelve
cases. Male impotency was said to be relieved and some cases of bronchial
asthma were improved. Confirmation of these extensive claims has been
lacking.
The effect on gray hair of human beings has not been verified by sub-
sequent work. Martino and Fabiano®*® found no effect on white hair when
73 §. Ansbacher, Science 98, 164 (1941).
74G. J. Martin and S. Ansbacher, J. Biol. Chem. 188, 441 (1941).
76 G. J. Martin and S. Ansbacher, Proc. Soc. Exptl. Biol. Med. 48, 118 (1941).
76 G. A. Emerson, Proc. Soc. Exptl. Biol. Med. 47, 448 (1941).
77K. Unna, G. V. Richards, and W. L. Sampson, J. Nutrition 22, 553 (1941).
78 L. M. Henderson, J. M. McIntire, H. A. Waisman, and C. A. Elvehjem, J. Nutri-
tion 23, 47 (1942).
79R. A. Brown and M. Sturtevant, Vitamins and Hormones 7, 171 (1949).
80 B. H. Ershoff, Proc. Soc. Exptl. Biol. Med. 56, 190 (1944).
81 B. F. Sieve, Science 94, 257 (1941).
82 J. J. Eller and L. A. Diaz, N. Y. State J. Med. 48, 1331 (1943).
83 H. B. Friedgood, New Engl. J. Med. 227, 788 (1942).
84 L. A. DeVilbiss, Med. Woman’s J. 49, 341 (1942).
8° B. F. Sieve, Southern Med. & Surg. 104, 135 (1942).
86 ],. Martino and A. Fabiano, Boll. soc. ital. biol. sper. 17, 702 (1942) [C. A. 40,
7389 (1946)].
60 P-AMINOBENZOIC ACID
PABA was given by intramuscular injection or by direct application to the
scalp. In carefully controlled observations, Brandaleone et al.’7: * found
PABA to be valueless in treatment of gray hair of human beings. These
authors pointed out the difficulty of ascertaining darkening of hair when
objective methods are not used. At present, the consensus is that vitamin
therapy is ineffective in treatment of ordinary gray hair of human beings.
2. DERMATOLOGY
According to Strauss et al.,°°: °° PABA in amounts sufficient to nullify
the bacteriostatic effects of sulfathiazole had no beneficial action on rash
or fever caused by the latter. Rothman et al.*' observed that PABA in an
ointment base protected the skin against sunburn. Irradiated solutions of
PABA caused erythema when injected intradermally, whereas non-ir-
radiated solutions did not. A 15% ointment of PABA protected against
fifty to one hundred times the usual erythema dose of ultraviolet light.”
These findings support the concept that ultraviolet erythema is due in
part to photochemical reaction of PABA in the skin. Costello® reported a
case of vitiligo successfully treated with PABA, which agreed with the
findings of Sieve.*®
3. RICKETTSIAL DISEASES
Snyder et al.% first noted that PABA in large doses was effective therapy
for experimental rickettsial infections. This was confirmed by Greiff e¢ al.*®
Human tests were first made on twenty cases of typhus fever by Yeomans
et al.,°® who found that when PABA was administered in large doses during
the first week of the disease the course was much less severe. Subsequently,
these findings have been substantiated in experimental rickettsial infections
by Murray et al.,°%7 Snyder and Zarafonetis,°* Hamilton,*? and Hamilton
87 H. Brandaleone, E. Main, and J. M. Steele, Proc. Soc. Exptl. Biol. Med. 53, 47
(1943).
88 H. Brandaleone, E. Main, and J. M. Steele, Am. J. Med. Sci. 208, 315 (1944).
89 H. Strauss, F. C. Lowell, and M. Finland, J. Clin. Invest. 20, 189 (1941).
90 #}. Strauss and M. Finland, Am. J. Med. Scz. 201, 730 (1941).
9 §. Rothman and J. Rubin, J. Invest. Dermatol. 5, 445 (1942).
2S. Rothman and A. B. Henningsen, J. Invest. Dermatol. 9, 307 (1947).
% M. Costello, Arch. Dermatol. and Syphilol. 47, 274 (1943).
%4 J. C. Snyder, J. Maier, and C. R. Anderson, Rept. National Research Council
Div. Med. Sci., (Dec. 26, 1942), quoted in ref. 2.
% J). Greiff, H. Pinkerton, and V. Moragues, J. Hxptl. Med. 80, 561 (1944).
9% A. Yeomans, J. C. Snyder, E. S. Murray, C. J. D. Zarafonetis, and R. 8. Ecke,
J. Am. Med. Assoc. 126, 349 (1944).
7.8. Murray, C. J. D. Zarafonetis, and J. C. Snyder, Proc. Soc. Exptl. Biol. Med.
60, 80 (1945).
9 J.C. Snyder and C. J. D. Zarafonetis, Proc. Soc. Exptl. Biol. Med. 60, 115 (1945).
9 H. L. Hamilton, Proc. Soc. Exptl. Biol. Med. 59, 220 (1945).
XI. PHARMACOLOGY 61
et al.!°° Clinical verification has been reported by Anigstein and Bader,'®!
Rose et al.,!° Tierney,!® Faust,!° Smith,!° Levy and Arnold,!°® Maroney
et al.,! Ravenel,!® Snyder et al.,!°? and Hendricks and Peters."
Faust states that PABA may be considered as relatively specific in the
treatment of the typhus group of diseases when given in the first week
following onset of infection. Dosage is very large. Ravenel specifies that
blood levels of 30 to 60 mg. per 100 ml. are desirable and that 60 to 80
mg. per 100 ml. may be necessary in some cases. Levels above 80 mg.
per 100 ml. of blood are dangerous. Alkalies also are necessary to prevent
acidosis. Under this regimen, a practical guarantee may be given that
fatality will not occur.
4, NEOPLASTIC DISEASES
Burk et al.!"! found that PABA exerted an anticarcinogenic action on the
production of hepatomas of rats fed p-dimethylaminoazobenzene and
excess biotin. Large doses of PABA produced a striking lowering of leuco-
cyte counts of six patients with chronic or subacute myelogenous leu-
kemia.!2 Less definite decreases in leucocyte counts occurred in cases of
chronic lymphatic leukemia. Prompt rise in counts followed discontinua-
tion of therapy. The use of PABA did not appear to be a practical form
of treatment.
5. OTHER DISEASES
Dry et al."'3: ‘4 reported the use of PABA to raise and maintain the blood
level of salicylates in patients with acute rheumatic fever. PABA alone
100 H. L. Hamilton, H. Plotz, and J. E. Smadel, Proc. Soc. Exptl. Biol. Med. 58, 255
(1945).
to. T,, Anigstein and M. N. Bader, Science 101, 591 (1945).
102 H. M. Rose, R. B. Duane, and E. E. Fischel, J. Am. Med. Assoc. 129, 1160 (1945).
103 N. A. Tierney, J. Am. Med. Assoc. 131, 280 (1946); Southern Med. J. 40, 81 (1947).
104 F.C. Faust, J. Am. Med. Assoc. 132, 965 (1946).
105 P| K. Smith, J. Am. Med. Assoc. 181, 1114 (1946).
106 M.D. Levy and W. T. Arnold, Texas State J. Med. 42, 314 (1946).
107 J. W. Maroney, H.C. Davis, and E.G. Scott, Delaware State Med. J.18, 104 (1946).
108 S. F. Ravenel, J. Am. Med. Assoc. 133, 989 (1947); Southern Med. J. 40, 801 (1947).
109 J.C. Snyder, A. Yeomans, D. H. Clement, E.S. Murray, C. J. D. Zarafonetis, and
N. A. Tierney, Ann. Internal Med. 27, 1 (1947).
110 W. J. Hendricks and M. Peters, J. Pediat. 30, 72 (1947).
11 J). Burk, J. M. Spangler, V. du Vigneaud, C. Kensler, K. Sugiura, and C. P.
Rhoads, Cancer Research 8, 130 (1943).
12-C, J. D. Zarafonetis, G. A. Andrews, M. C. Myers, and F. H. Bethell, Blood 3,
780 (1948).
us TJ. Dry, Proc. Central Soc. Clin. Research 19, 69 (1946).
14 -T. J. Dry, H. R. Butt, and C. H. Scheifley, Proc. Staff Meetings Mayo Clinic 21,
497 (1946).
62 P-AMINOBENZOIC ACID
seemed to be without therapeutic value. The mechanism by which PABA
elevated salicylate blood level was shown to be a depression of conjugation
of glycine with salicylate." After PABA, only small amounts of salicyluric
acid appeared in the urine. In addition, the urinary acidity was increased,
and this decreased the renal clearance of free salicylate. The above holds
true in man, but not in dog.
In experimental allergic encephalomyelitis of guinea pigs, Good et al.!!®
found that large doses of PABA plus salicylates had prophylactic value,
but no therapeutic action. Large doses of salicylates were less effective,
and PABA alone was actionless.
No therapeutic or prophylactic value could be demonstrated with PABA
against myxoma or vaccinia viruses in eggs.!7
Rosenblum and Fraser,"* in contrast to Dry, noted that PABA definitely
relieved both fever and joint pains in a series of nine patients with acute
rheumatic fever. Dosage was comparable to that used in rickettsial diseases.
Recently, Wiesel and associates® reported the use of PABA with cortisone
in treatment of fifteen patients with severe rheumatoid arthritis. A com-
pletely ineffective daily dose of cortisone (25 mg.) plus 12 g. of sodium
p-aminobenzoate daily brought about suboptimal control of the disease.
PABA alone was ineffective. The authors believed the action of PABA was
related to its ability to suppress hepatic inactivation of estrogens.
XII. Detoxication of Arsenicals
LEMUEL D. WRIGHT and PETER A. TAVORMINA
The concept of competitive inhibition stimulated a host of investiga-
tors to attempt a demonstration of this phenomenon in a variety of sys-
tems. In an experiment designed to explore the behavior of PABA as an
inhibitor of the trypanocidal activity of carbarsone, Sandground! made the
HNCONH—C_S—Asbll
observation that PABA confers an exceedingly high degree of protection
in rats infected with Trypanosoma equiperdum against the toxic properties
of the arsenical, without decreasing its trypanocidal effect. The adminis-
15 R. M. Salassa, J. L. Bollman, and T. J. Dry, J. Lab. Clin. Med. 33, 1393 (1948).
16 R. A. Good, B. Campbell, and T. A. Good, Proc. Soc. Exptl. Biol. Med. 72, 341
(1949).
17 ©. T. Lee, Proc. Soc. Exptl. Biol. Med. 75, 649 (1950).
18 H. Rosenblum and L. E. Fraser, Proc. Soc. Exptl. Biol. Med. 65, 178 (1947).
1 J. H. Sandground, Science 97, 73 (1943).
XII. DETOXICATION OF ARSENICALS 63
tration of PABA in conjunction with lethal doses of the pentavalent ar-
senicals tryparsamide, acetarsone, arsanilic acid, and benzenearsonic acid
was reported by Sandground!:? to be attended by similar protective ac-
tion, and this was found to be true regardless of the route of administration
of either the arsenical or the PABA. The protection is evident within 24
hours, and the usual symptoms of arsenic poisoning, such as tremor, di-
arrhea, paralysis, and emaciation, are minimized. Histologically, the most
significant manifestation of the beneficial action of PABA is in the con-
siderable reduction, both in extent and in severity, of the renal lesions pro-
duced by the arsenicals.®
It had long been known that the toxicity of trivalent arsenicals could be
substantially reduced by means of various agents. The injection of sulf-
hydryl-containing compounds (e.g., reduced glutathione) simultaneously
with, or prior to, the administration of the arsenical was used with success
by Voegtlin et al.‘ to offset to a large degree the toxic effects in the rat of
mapharside (3-amino-4-hydroxyphenylarsenoxide). Similarly, the sup-
pression of the toxic manifestations of neoarsphenamine was accomplished
by Durel® and by McChesney and his associates,® by means of ascorbic
acid. In contrast to these results with the trivalent arsenicals, no effective
means of counteracting the ill effects of the pentavalent compounds had
been demonstrated, and the elucidation by Sandground of this particular
function of PABA was attractive in its implication of potential clinical
significance.
In a series of papers devoted to a more detailed study of this detoxication
effect, Sandground and his associates reported that essentially all rats are
protected against a LDg of carbarsone (1000 mg. per kilogram) or of
arsanilic acid (400 mg. per kilogram) by the prior administration of 500
mg. per kilogram for the latter. The use of 750 mg. per kilogram of the
vitamin protects all animals against a dose of carbarsone of 1500 mg. per
kilogram, a dose which is not tolerated by a single unprotected rat. As
little as 15 mg. of PABA per kilogram protects 50% of animals subjected
to a LDgo of arsanilic acid.’
In addition to establishing the amount of the vitamin required for a
specific measure of protection, Sandground demonstrated*®:° the vital
2 J. H. Sandground and C. R. Hamilton, J. Pharmacol. Exptl. Therap. 78, 109 (1943).
3P.N. Harris, J. Pharmacol. Exptl. Therap. 82, 254 (1944).
4C. Voegtlin, H. A. Dyer, and C. S. Leonard, J. Pharmacol. Exptl. Therap. 25,
297 (1925).
5 P. Durel, Bull. soc. franc. dermatol. syphilig. 44, 1077 (1937) [C. A. 32, 3816 (1938)].
6B. W. McChesney, O. W. Barlow, and G. H. Klinck, Jr., J. Pharmacol. Expil.
Therap. 80, 81 (1944).
7 J. H. Sandground and C. R. Hamilton, J. Pharmacol. Exptl. Therap. 78, 203 (1943).
8 J. H. Sandground, J. Pharmacol. Exptl. Therap. 78, 209 (1943).
9 J. H. Sandground and C. R. Hamilton, J. Lab. Clin. Med. 28, 1821 (1943).
64 P-AMINOBENZOIC ACID
role of the time element in the detoxication of pentavalent arsenicals.
Injection of PABA up to 3 hours before the administration of the arsenical
affords protection for nearly all animals. On the other hand, when the
animals receive the vitamin subsequent to the administration of the ar-
senical, the detoxicating effect is less pronounced and continues to decrease
with increasing time interval. Sandground suggests’ that this time in-
terval coincides with the time required for the in vivo reduction of the
pentavalent arsenicals (or for the oxidation of the arsenobenzenes) to
the corresponding arsenoxides, the toxicity of which is not reduced by
PABA.®: '° In support of this theory the author points to the relatively
6 RAs = AsR lll RAsO ra RAsO;H:.
slow development of host toxicity and of parasiticidal activity of the pen-
tavalent compounds, as compared with the rapid appearance of both these
effects in the case of the arsenoxides. The fact that PABA does not de-
crease the trypanocidal effect of the pentavalent arsenicals is interpreted
by Sandground as indicating that the vitamin does not interfere in the
reduction of the arsonic acid to the arsenoxide.
The reviewers find some difficulty in subscribing to this view. If the
toxicity of arsonic acids can be ascribed to their conversion to the arsenoxide
(detoxication of which is not effected by PABA) and if PABA offers no
appreciable obstruction to the conversion, it would seem unlikely that
PABA could materially reduce the toxicity of the pentavalent arsenical.
In further studies Sandground"™ showed that the three isomeric amino-
benzoic acids, the hydroxy and nitro analogs of PABA, phenylacetic acid,
phenylpropionic acid, and benzoic acid, itself, all confer some degree of
protection. This observation led him to consider unlikely the direct com-
bination of arsenical and detoxicant to produce a non-toxic complex as a
mechanism of action. Similarly, the possibility of competition for a specific
enzyme site was excluded in view of the protective activity of compounds
so unrelated structurally to the arsonic acids as phenylglycine or benzyl
succinate. The only structural arrangement which appears to be a pre-
requisite for activity is a carbocyclic system of five or six members, as-
sociated with a free carboxyl group. Esterification or amidation of the acid
group reduces activity.
The apparent lack of specificity of PABA in this connection is supported
by the data of Martin,” who found that the administration of thyroxine
along with the vitamin nullifies to a great extent its detoxicating property.
The theory was advanced that PABA functions by virtue of its ability to
10 1,, Peters, Proc. Soc. Exptl. Biol. Med. 58, 147 (1948).
1 J, H. Sandground, J. Pharmacol. Exptl. Therap. 80, 393 (1944).
2G. J. Martin, Am. J. Pharm. 118, 394 (1946).
XII. DETOXICATION OF ARSENICALS 65
inhibit the thyroid, thus decreasing tissue metabolism and, concomitantly,
arsenoxide formation.
It has been mentioned that PABA does not influence the lethal action
of the more usual type of arsenicals such as mapharside, neoarsphenamine,
ete. In contrast to this, the trypanocidal action of y-(p-arsenosophenyl)-
butyric acid is completely inhibited by the vitamin. Williamson and
Lourie: ' demonstrated this interference both in vivo and in vitro. The
authors noted that lethal quantities of arsenic are absorbed by trypano-
some cells in a matter of seconds. Furthermore, the arsenical, when in-
jected, disappears from the blood stream in one-half hour, with or without
the prior administration of PABA. The rapidity with which the arsenical
takes effect is believed responsible for the inability of PABA to interfere
in the ‘‘cidal” activity when the vitamin is administered subsequently to
the arsenical.
In vitro, PABA immobilizes Trypanosoma rhodesiense reversibly and
innocuously and protects the organism for at least 24 hours from the ac-
tion of y-(p-arsenosophenyl)butyric acid. Williamson and Lourie express
the opinion that PABA immobilizes the parasite cell by absorption into,
or adsorption on the surface of, the organism. Presumably, this process
inhibits the lethal action of the arsenical by impeding its penetration into
the cell or its fixation on the surface of the cell.
The inhibition of the trypanocidal activity of y-(p-arsenosophenyl)-
butyric acid by PABA, a property which this vitamin does not exercise
against the usual arsenicals, is attributed by Williamson and Lourie to the
different process by which this arsenical permeates the cell. In support of
this view the authors cite the activity of this compound against trypars-
amide-resistant strains of the trypanosome, as well as the presence of the
acetic acid configuration, a structure which has been found to be common
to the group of arsenicals active against atoxyl-resistant trypanosomes.
These compounds are rendered more water-soluble by the presence of the
more polar groups and would be expected to penetrate the cell in a fashion
different from that of compounds devoid of hydrophilic groups. The latter
type of arsenical ostensibly enters via the lipid components of the organism.
It has been mentioned that except for its anomalous behavior in the
case of y-(p-arsenosophenyl)butyric acid PABA does not reduce the tryp-
anocidal action of any of the arsenicals. Schleyer and Schnitzer have
found, however, that the methyl! and ethyl esters and the amides of benzoic,
p-hydroxybenzoic, p-aminobenzoic, 2,4- and 2,5-dihydroxybenzoic, and
nicotinic acid do reduce the parasiticidal activity of mapharside or acriflavin
18, J. Williamson and E. M. Lourie, Ann. Trop. Med. Parasitol. 40, 255 (1946).
‘4 J. Williamson and E. M. Lourie, Nature 161, 103 (1948).
15 W.L. Schleyer and R. J. Schnitzer, J. Immunol. 60, 265 (1948).
66 P-AMINOBENZOIC ACID
on 7’. equiperdum. No such effect could be demonstrated with the corres-
ponding free acids. .
The observations reported by Sandground on the protective action of
PABA against the toxicity of arsenicals have been supplemented by anal-
ogous findings with respect to the detoxication of other organo-metallic
substances. Sandground"® extended his studies to the antimonial, “‘stibosan”’
CH,CONH—€_>—SbO.HINa
“Stibosan”’
and found PABA to afford a high order of protection against the toxic
action in rats of this drug, without a reduction of its trypanocidal potency.
Hardcastle and Foster!’ found that PABA appears to have promise as a
detoxicant for borax. Voss and Tatum! have reported that PABA exerts a
protective action in rats against lethal doses of sodium bismuthyl citrate,
mannonate, or tartrate. Similar findings have been published by Sala and
Borasi!® concerning the detoxication by PABA of both organic and in-
organic mercury compounds.
XIII. Sulfonamide Reversal
LEMUEL D. WRIGHT AND PETER A. TAVORMINA
The advent of the sulfonamides (1935-1940) stimulated considerable
research into the mechanism of action of these highly useful chemothera-
peutic agents. Lockwood in 1938 showed! that sulfonamide activity in
vitro can be reversed by peptone. Heat-killed bacterial cells or their extracts
were shown by Stamp? to reverse sulfonamide action. He showed also
that the sulfonamide-neutralizing activity of such bacterial extracts is
stable to heat, acid, and alkali. MacLeod* confirmed the antisulfonamide
activity of peptone and tissue extracts. He further demonstrated that the
production of sulfonamide-neutralizing material by the pneumococcus is
markedly increased as the organism becomes resistant to sulfonamides.
16 J, H. Sandground, Proc. Soc. Exptl. Biol. Med. 52, 188 (1943).
17 A. B. Hardcastle and A. O. Foster, Proc. Helminthol. Soc. Wash., D. C. 11, 60
(1944).
18H}. Voss and A. L. Tatum, J. Pharmacol. Exptl. Therap. 90, 161 (1947).
19G,. Sala and M. Borasi, Atti soc. lombarda sci. med. e biol. 3, 70 (1948) [C. A. 45,
3946 (1951)].
1J.S. Lockwood, J. Jmmunol. 35, 155 (1938).
2T. C. Stamp, Lancet II, 10 (1939).
$C. M. MacLeod, J. Exptl. Med. 72, 217 (1940).
XIII. SULFONAMIDE REVERSAL 67
Woods and Fildes,‘ and subsequently Woods® in more detail, showed that
the sulfonamide-reversing agent present in yeast in addition to being stable
to heat, acid, and alkali, as reported by Stamp, is inactivated by nitrous
acid, acetylation, or esterification. These data together with a considera-
tion of the solubility and acid-dissociation constant of the sulfonamide-
reversing agent led to a trial of synthetic PABA. This compound proved
highly active as a sulfonamide-neutralizing compound not only in vitro
but also zn vivo against sulfanilamide in the treatment of a hemolytic
streptococcal infection of mice.® Although the sulfonamide-neutralizing
activity of natural material was not isolated and identified as PABA by
Woods, this was accomplished subsequently by Rubbo and Gillespie?
and by Blanchard.§
Woods’ observation® that PABA is highly active in reversing the bac-
teriostatic action of the sulfonamides has been confirmed in many labora-
tories and against many strains of bacteria. PABA does not, however,
reverse sulfonamide action in all species, Bacteriwm tularensis being the
best-known exception.?°
Since PABA is an effective reversing agent for the sulfonamides, at least
in the case of the majority of microorganisms, it was apparent to Woods
that the development of sulfonamide resistance in bacteria may be as-
sociated with increased synthesis of PABA.® For a critical test of this hy-
pothesis, a specific test for PABA was essential. With the development by
Landy and Dicken!® of a microbiological assay for PABA based on the
essential nature of this compound for Acetobacter suboxydans, the synthesis
of PABA by sulfonamide-sensitive and sulfonamide-resistant bacteria
could be examined. Landy and co-workers!!! observed in culture filtrates
from sulfonamide-resistant staphylococci fifty to one hundred times as
much PABA as was encountered in culture filtrates from sulfonamide-
sensitive strains. Increased PABA synthesis occurs even when the strains
of staphylococci are grown for many generations in the absence of sulfon-
amides. Similar findings with respect to the synthesis of large amounts of
PABA by sulfonamide-resistant staphylococci were recorded by Spink
et al.,” using both the Acetobacter suboxydans" and the Lactobacillus arabino-
4D. D. Woods and P. Fildes, J. Soc. Chem. Ind. (London) 59, 133 (1940).
5D. D. Woods, Brit. J. Exptl. Pathol. 21, 74 (1940).
6 F.R. Selbie, Brit. J. Exptl. Pathol. 21, 90 (1940).
7§. D. Rubbo and J. M. Gillespie, Nature 146, 838 (1940).
8K. C. Blanchard, J. Biol. Chem. 140, 919 (1941).
9J.T. Tamura, J. Bacteriol. 47, 529 (1944).
10M. Landy and D. M. Dicken, J. Biol. Chem. 146, 109 (1942).
1M. Landy, N. W. Larkum, E. J. Oswald, and F. Streightoff, Sczence 97, 265 (1943).
12 W. W. Spink, L. D. Wright, J. J. Vivino, and H. R. Skeggs, J. Exptl. Med. 79, 331
(1944).
68 P-AMINOBENZOIC ACID
sus methods for PABA determination. Increased synthesis of PABA by
sulfonamide-resistant strains of staphylococci was noted by Housewright
and Koser, using the Clostridium acetobutylicum assay. Increased PABA
production was subsequently found by Landy and Gerstung!: !® to be
associated with the development of sulfonamide resistance in Nezsseria
gonorrhoeae. Less conclusive evidence that PABA synthesis is correlated
directly with sulfonamide resistance in Dziplococcus pneumoniae,*: 7: 38
strains of Clostridia,!® Brucella paramelitensis,?° and Shigella sonnei™ has
been presented. On the other hand, Landy et al." did not find increased
PABA synthesis associated with sulfonamide resistance in strains of Es-
cherichia coli, Vibrio cholerae, Shigella dysenteriae, or Diplococcus pneu-
moniae, and Housewright and Koser™ concluded as a result of microbio-
logical assays with Clostridium acetobutylicum that sulfonamide resistance in
Shigella paradysenteriae and Diplococcus pneumoniae is not associated with
increased PABA production. It must be concluded that with certain species
of bacteria, particularly the staphylococci, sulfonamide resistance is ex-
plainable in terms of increased PABA synthesis, but that in other species,
for example, Escherichia coli, sulfonamide resistance is not associated with
the same phenomenon.
Of equal interest with respect to the functions of PABA in metabolism
is a consideration of the reversal of sulfonamide action by compounds
unrelated to PABA in structure.
Soon after Woods observed that PABA reverses sulfonamide action, it
was observed that methionine exhibits a similar activity.2!*! Adenine or
hypoxanthine is reportedly as active as PABA in negating the effect of
sulfanilamide against Streptococcus hemolyticus infection in mice,?> and
various purines reverse the effect of sulfonamides on lactic acid bacteria.”®
More recently the sulfonamide-reversing activities of thymine,?’?? folie
13 J. C. Lewis, J. Biol. Chem. 146, 441 (1942).
14 R. D. Housewright and 8. A. Koser, J. Infectious Diseases 75, 113 (1944).
15M. Landy and R. B. Gerstung, J. Bacteriol. 47, 448 (1944).
16M. Landy and R. B. Gerstung, J. Immunol. 51, 269 (1945).
17 A. Zimmerman and R. M. Pike, J. Bacteriol. 45, 522 (1943).
18 G.S. Mirick, J. Bacteriol. 45, 66 (1943).
19G. B. Reed, J. H. Orr, and R. W. Reed, J. Bacteriol. 48, 233 (1944).
20 H. N. Green and F. Bielschowsky, Brit. J. Exptl. Pathol. 23, 1 (1942).
21, A. Bliss and P. H. Long, Bull. Johns Hopkins Hosp. 69, 14 (1941).
22 J.S. Harris, and H. I. Kohn J. Pharmacol. Exptl. Therap. 78, 383 (1941).
23 J.S. Harris and H. I. Kohn, J. Biol. Chem. 141, 989 (1941).
*4H. I. Kohn and J. 8. Harris, J. Pharmacol. Exptl. Therap. 77, 1 (1948).
25 Gq. J. Martin and C. V. Fisher, J. Biol. Chem. 144, 289 (1942).
26K. KE. Snell and H. K. Mitchell, Arch. Biochem. 1, 93 (1948).
27 J. O. Lampen and M. J. Jones, J. Biol. Chem. 170, 133 (1947).
28 J. O. Lampen and M. J. Jones, J. Biol. Chem. 166, 485 (1946).
29 K. C. Winkler and P. G. deHaan, Arch. Biochem. 18, 97 (1948).
XIII. SULFONAMIDE REVERSAL 69
acid,’ arginine,*® histidine,®® lysine,?*: *° glutamic acid,*®® aspartic acid,*°
isoleucine,’ tryptophan,*': * and valine®® have been reported.
Considerable clarification with respect to the activities of the above
apparently unrelated compounds in reversing the action of sulfonamides
has come about from the use of znhibition analysis as developed by Shive
and co-workers.**: *4 Inhibition analysis has been explained in detail a
number of times by Shive, and these references should be consulted for a
full appreciation of the possibilities inherent in the procedure as well as
the significance of the terms used to describe the results obtained.
Sulfonamides inhibit the functioning of PABA in Escherichia coli as
determined by Shive and coworkers in a synthetic salts-glucose medium
with an inhibition index of about 3000 (1 part of PABA will reverse the
effect of 3000 parts of sulfanilamide).** In the presence of methionine, this
inhibition ratio is increased to 10,000 (which indicates that more sulfanil-
amide is required for inhibition in the presence of methionine). Thus it
would appear that one function of PABA, not necessarily as such but in a
form whose synthesis is inhibited by sulfanilamide, is concerned with
methionine synthesis. The reaction affected is postulated by Shive and
coworkers as follows:
NH, eee NH,
inhibition
index, 3000
HSCH.CH,CHCOOH CH;—S—CH.CH.CHCOOH
Homocysteine (or related Methionine
precursor)
In the presence of methionine the inhibition index of sulfanilamide for
Escherichia colt may be raised from 10,000 to about 30,000 by purines.*
Purines and their related compounds effective in reversing sulfanilamide
under these conditions include adenosine, xanthine, guanine, and inosine.
Adenine not only is ineffective in reversing sulfanilamide but acts syn-
ergistically as an inhibitor.
An increase in the inhibition index from 10,000 to 30,000 with addition
of purines to the medium indicates that PABA functions in the synthesis
of purines. In this case the precursor of the reaction blocked by the failure
of PABA, in the presence of sulfanilamide, to be converted to a meta-
bolically active form has been elucidated. There accumulate in /scherichia
30 F. Nitti, J. Tabone, and H. Mousset, Ann. inst. Pasteur 70, 366, 379 (1944).
31M. G. Sevag and M. N. Green, J. Bacteriol. 48, 623 (1944).
32 M. G. Sevag and M. N. Green, J. Bacteriol. 48, 631 (1944).
33 W. Shive, Ann. N. Y. Acad. Sci. 52, 1212 (1950); M. Gordon, Ph.D. Thesis, Uni-
versity of Texas, 1948.
34 R. J. Williams, R. E. Eakin, E. Beerstecher, Jr., and W. Shive, The Biochemistry
of B Vitamins, p. 458. Reinhold Publishing Corp., New York, 1950.
35 W. Shive and E. C. Roberts, J. Biol. Chem. 162, 463 (1946).
70 P-AMINOBENZOIC ACID
coli cultures grown in the presence of just sub-bacteriostatic amounts of
sulfanilamide, and, preferably, increased amounts of glycine,*® an amine
that has been isolated*? and identified as 5(4)-amino-4(5)-imidazolecar-
boxamide.*® The reaction blocked indirectly by sulfanilamide in the syn-
thesis of purines is as follows:
H.N—C=O N—=C—OH
| |
C—NH cree HO—C C—NH
L 1
~ “index, 30,000 > ~
CH ae
Ye jo
H,.N—C—N N—C—N
5(4)-Amino-4(5)-imidazole- Hypoxanthine
carboxamide (or riboside) (or ribose)
An increase in the inhibition index from 30,000 to 50,000—100,000 with
addition to the Hscherichia coli medium now containing methionine and
purines may be obtained by addition of serine.?? These data lead to the
conclusion (considerably strengthened by unrelated experiments from other
sourees) that PABA functions indirectly in the synthesis of serine.
CH,.COOH Pelee CH.—CHCOOH
| inhibition |
index, 50,000-100,000
NH; OH NH,
Glycine Serine
An increase in the inhibition index from 50,000—100,000 to 200,000-
300,000 may now be obtained in the presence of methionine, purines, and
serine with either relatively large amounts of thymine or much smaller
amounts of folic acid.?? In this instance the precursor of thymine is un-
known.
Inhibition
Unknown precursor index, 200,000-300,000
Thymine (or derivative
such as thymidine)
Although a study of the various compounds that reverse sulfonamide
inhibition of microorganisms leads to considerable clarification of the
reactions carried out by a metabolically active derivative of PABA whose
36 J. M. Ravel, R. EK. Eakin, and W. Shive, J. Biol. Chem. 172, 67 (1948).
37 M. R. Stetten and C. L. Fox, Jr., J. Biol. Chem. 161, 333 (1945).
38 W. Shive, W. W. Ackermann, M. Gordon, M. E. Getzendaner, and R. E. Eakin,
J. Am. Chem. Soc. 69, 725 (1947).
XIII. SULFONAMIDE REVERSAL a
synthesis is inhibited by sulfonamides, these studies were not in them-
selves instrumental in describing the derivative.
The site of action of the sulfonamides has been elucidated from several
other lines of investigation that appeared at the time to be unrelated.
When certain sulfonamides are incorporated into highly purified diets
adequate with respect to thiamine, riboflavin, pyridoxine, nicotinic acid,
and pantothenic acid, typical signs of dietary deficiency are produced in
rats consuming such diets.**** Nielsen and Elvehjem*! were the first to
report that the deficiency thus induced could be alleviated with folic acid
concentrates and biotin. The effectiveness of folic acid was soon confirmed
in a number of laboratories.**-#8 These animal studies were the first to show
that folic acid could be a derivative of PABA whose synthesis, in this case
by intestinal bacteria, is inhibited by sulfonamides.
A remarkably specific effect of sulfonamides in inhibiting folic acid
synthesis 7n vitro was pointed out by Miller*® in tests with Escherichia colt.
In her studies folic acid synthesis was 5 to 10 % of that observed in controls,
while synthesis of other B vitamins was not significantly altered.
Further evidence that the primary mode of action of the sulfonamides is
in inhibiting synthesis of folic acid from PABA is contained in experiments
of Lampen and Jones?’ *8 with folic acid-dependent and -independent
strains of lactobacilli. These investigators observed that lactobacilli with a
growth factor requirement for folic acid are relatively non-susceptible to
inhibition by sulfonamides, whereas those strains that can make their own
folic acid are readily inhibited.
The structure of evidence that sulfonamides act primarily by inhibiting
the synthesis of folic acid from PABA was essentially complete when it
was finally announced by Angier e¢ al.*° that folic acid is a derivative of
PABA.
39. Black, J. M. McKibbin, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 47,
308 (1941).
40 J. B. MacKenzie, C. G. MacKenzie, and E. V. McCollum, Science 94, 518 (1941).
41F.S. Daft, L. L. Ashburn, and W. H. Sebrell, Science 96, 321 (1942).
4 F.S. Daft, L. L. Ashburn, S. 8. Spicer, and W. H. Sebrell, U. S. Public Health
Repts. 57, 217 (1942).
483A. D. Welch, Federation Proc. 1, 171 (1942).
44 EF. Nielsen and C. A. Elvehjem, J. Biol. Chem. 145, 713 (1942).
45 A. D. Welch and L. D. Wright, J. Nwtrition 25, 555 (1943).
46 FS. Daft and W. H. Sebrell, U.S. Public Health Repts. 58, 1542 (1943).
47G. J. Martin, Proc. Soc. Exptl. Biol. Med. 61, 353 (1942).
48 A. Kornberg, F. 8. Daft, and W. H. Sebrell, Proc. Soc. Exptl. Biol. Med. 58, 46
(1945).
49 A. K. Miller, Proc. Soc. Exptl. Biol. Med. 57, 151 (1944).
5oR. B. Angier, J. H. Boothe, B. L. Hutchings, J. H. Mowat, J. Semb, E. L. R.
Stokstad, Y. SubbaRow, C. W. Waller, D. B. Cosulich, M. J. Fahrenbach, M. E.
TABLE IX
SULFONAMIDE REVERSAL BY DERIVATIVES OF p-AMINOBENZOIC ACID
Activity,
of PABA,
Compound Species % Reference
p-Aminobenzoic acid 100
o- and m-Aminobenzoie acid Str. hemolyticus, Diplococcus pneumo- 0 5, 13,o1
niae, Staph. aureus, Lactobacillus ara-
binosus
Ethyl p-aminobenzoate Str. hemolyticus 0.033 5
Str. pyogenes + 52
E. coli 0 53
Diethylaminoethyl p-aminobenzoate Str. hemolyticus ca 21 5
(procaine) E. coli ca 1 53
Dimethylaminoethyl p-(n-butylamino)- E. coli >0.01 53
benzoic acid (pantocaine)
p-Aminobenzamide Str. hemolyticus 0.86 5
p-Aminobenzoylglycine E. coli <0.02 54
p-Nitrobenzoic acid Str. hemolyticus 0.0067 5
p-Hydroxylaminobenzoic acid Str. hemolyticus ca 21 5
p-Hydroxybenzoic acid (POB) Str. hemolyticus 0 5
p-Acetamidobenzoic acid Str. hemolyticus 0.0067 5
p-Aminophenylacetic acid Str. hemolyticus, Diplococcus pneumo- 0 51, 55
niae, E. coli, Staph. aureus
Clostridium acetobutylicum 0 53
2-Chloro-4-aminobenzoie acid Str. hemolyticus ca 0.1 56
Str. pyogenes + 52
Diplococcus pneumoniae + 56, 57
E. coli oa 56
2-Fluoro-4-aminobenzoic acid E. coli 38 55
2-Bromo-4-aminobenzoie acid EF. coli 0.79 55
Diplococcus pneumoniae + 57
2-Iodo-4-aminobenzoic acid E. coli 0.13 55
2-Hydroxy-4-aminobenzoic acid (PASA) E. coli 4-16 58
4-Aminoisophthalic acid Str. pyogenes a 52
E. coli 0.07 55
2-(p-Aminobenzamido) pyridine Str. hemolyticus 0 5
2-Aminopyrimidine-5-carboxylic acid Str. pyogenes 0.05 59
3-Methyl-4-aminobenzoie acid Str. pyogenes + 52
p-(p-Amidobenzamido) benzoic acid Str. pyogenes + 52
Streptobacterium plantarum 0.08 60
p-Aminocinnamice acid Str. hemolyticus + 52
Diplococcus pneumoniae + 52
E, coli _ 61
4-(p-Hydroxyphenyl)azobenzoic acid Str. hemolyticus, E. coli + 62
p-Aminobenzoic hydrazide Str. hemolyticus, E. coli a 62
N-Glucosido-p-aminobenzoic acid Str. hemolyticus, E. coli + 63
Ethyl p-(N‘-acetylsulfanilamido) ben- Str. hemolyticus, E. coli + 63
zoate
p-Aminophenylglycine Str. hemolyticus, Diplococcus pneumo- 0 51
niae, Staph. aureus
4-Aminocyclohexanecarboxylic acid Staph. aureus 77 64
p-Toluic acid Str. hemolyticus 0 5
p-Ethylaminobenzoic acid Str. hemolyticus 0.06 65
p-n-Butylaminobenzoic acid Str. hemolyticus <0.07 65
3-Dimethylamino-1, 2-dimethylpropyl- Streptobacterium plantarum 3.5 60
p-aminobenzoate (tutocaine)
5-(p-Aminobenzoxyethyl)-4-methyl Streptobacterium plantarum 0.48 60
thiazole
Ethyl N-(p-aminobenzoyl)alanine Streptobacterium plantarum 0.17 60
p-Aminobenzenestibonic acid Streptobacterium plantarum 0.003 60
Trimethylbetaine of p-aminobenzoic acid Streptobacterium plantarum 0.02 60
Trimethylbetaine of sulfanilie acid Streptobacterium plantarum 0 60
~J
wy)
XIII. SULFONAMIDE REVERSAL 73
The extent to which compounds related to PABA in structure reverse
sulfonamide inhibition of bacteria is summarized in Table LX.
With the information of the preceding paragraphs forming the essential
“framework” for a ‘“‘working hypothesis” of the fate of PABA within the
cell as revealed by studies employing sulfonamides, some of the more con-
troversial aspects of the subject as well as further details of the scheme
may be examined.
Although it has been almost universally agreed for some time that sul-
fonamides act by inhibiting the conversion of PABA to folie acid, the
exact locus of this effect has not been established.
OH COOH
| |
C N CHNH—€_>—CONHCHCH.CH.COOH
ZEN
fear A
Cc CH
Sh NF
HN N
Folie acid
A logical point for interference is the conjugation of PABA with glutamic
acid. If this condensation is interfered with by sulfonamides, then sul-
fonamides should be ineffective in the presence of p-aminobenzoylglutamic
acid. With the exception of the results of Auhagen,®® who reported that
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith,
Jr., Science 108, 667 (1946).
51M. Landy and J. Wyeno, Proc. Soc. Exptl. Biol. Med. 46, 59 (1941).
52 A. R. Martin and F. L. Rose, Biochem. J. 39, 91 (1945).
53 A. K. Keltch, L. A. Baker, M. E. Krahl, and G. H. A. Clowes, Proc. Soc. Expil.
Biol. Med. 47, 533 (1941).
54K. H. Beyer, P. A. Mattis, E. A. Patch, and H. F. Russo, J. Pharmacol. Expil.
Therap. 84, 136 (1945).
55 Q. Wyss, M. Rubin, and F. B. Strandskov, Proc. Soc. Exptl. Biol. Med. 52, 155
(1943).
56 Q. H. Johnson, D. E. Green, and R. Pauli, J. Biol. Chem. 153, 37 (1944).
57 J. L. Sirks, Antonie van Leeuwenhoek, J. Microbiol. Serol. 11, 171 (1946).
58 W. C. Tobie and M. J. Jones, J. Bacteriol. 57, 573 (1949).
59 A. R. Martin, F. L. Rose, and G. Swain, Nature 154, 639 (1944).
60 R. Kuhn, E. F. Mdller, G. Wendt, and H. Beinert, Ber. 75B, 711 (1942).
61 KF. L. Tatum and G. W. Beadle, Proc. Nail. Acad. Sci. U. S. 28, 234 (1942).
82 C. Levaditi, C. Mentzer, and R. Perault, Compt. rend. soc. biol. 186, 769 (1942).
63 A. Dansi, Farm. sci. e tec. (Pavia) 2, 195 (1947) [C. A. 42, 639 (1948)].
64H. von Euler and P. Karrer, Helv. Chim. Acta 27, 1697 (1944).
65 G. R. Goetchius and C. A. Lawrence, J. Bacteriol. 48, 683 (1944).
56 EK. Auhagen, Z. physiol. Chem. 277, 197 (1943).
14 P-AMINOBENZOIC ACID
COOH
|
H.N—€ > —CONHCHCH.CH.COOH
p-Aminobenzoylglutamic acid
p-aminobenzoylglutamic acid is eight to ten times as active as PABA in
reversing sulfonamide inhibition of Streptobacterium plantarum, p-amino-
benzoylglutamic acid otherwise has been found to be less active than PABA,
and the reversal obtained is competitive, indicating according to inhibi-
tion analysis that p-aminobenzoylglutamiec acid is not a product of the
reaction inhibited by sulfonamides.?’: *8» %
The synthesis of pteroic acid is a possible site for sulfonamide inhibition.
Pteroic acid
As with p-aminobenzoylglutamic acid, pteroic acid is less active than
PABA in reversing sulfonamides, and the reversal so obtained is com-
petitive, indicating that pteroic acid is not a product of the reaction in-
hibited by sulfonamides.”’: *8» ®” Utilization of pteroic acid by Streptococcus
faecalis whose “folic acid” requirement can be satisfied with pteroic acid
is not inhibited by sulfonamides.”* *
O’Meara et al.® have suggested that sulfonamides may prevent a com-
bination of PABA with reductone, a,6-dihydroxyacrolein, by combining
irreversibly with it instead. They picture reductone as being either toxic
to the cell or essential at only certain phases of development. It would
seem more plausible to consider reductone as an essential intermediate
in the synthesis of folic acid and that sulfonamides can interfere with this
process.
OH
INTE CH.COOH
ae HO—C=CHOH |
| CH;
| | O— er |
JANN. H.N—€_>—CoNHCHCOOH
H.N NH,
2,4,5-Triamino- Reductone p-Aminobenzoylglutamie acid
6-hydroxypyrimidine
67 J. O. Lampen and M. J. Jones, J. Biol. Chem. 164, 485 (1946).
68 R.A. Q. O'Meara, P. A. McNally, and H. G. Nelson, Nature 154, 796 (1944).
XIII. SULFONAMIDE REVERSAL 75
It has been suggested by Tschesche® that sulfonamides interfere with
the conjugation of 2-amino-4-hydroxypteridine-6-aldehyde with PABA,
but strong experimental evidence for the hypothesis is lacking.
OH
| CHO
/ a
N YY
/ *N“NNZ
H.N
2-Amino-4-hydroxy-
pteridine-6-aldehyde
Recent studies indicate that folinie acid, the Leuconostoc citrovorum
factor or 5-formyl-5 ,6,7 ,8-tetrahydropteroylglutamic acid, is nearer to
the form in which folie acid functions in metabolism. It is not impossible
that folic acid is an artifact that does not actually occur in the normal
sequence of PABA utilization.
That sulfonamides actually interfere with the uptake of PABA by bac-
terial cells has been shown by Noll et al.7° in studies with Escherichia coli
and $*-labeled sulfathiazole. Competitive interference as determined by
radioactivity counts was demonstrated between sulfathiazole and PABA.
A consideration of the deficiencies produced in animals by the feeding
of a poorly absorbed sulfonamide in conjunction with highly purified diets
containing only thiamine, riboflavin, pantothenic acid, vitamin Bg , nico-
tinic acid, and choline as a source of B vitamins is most logically presented
in the chapter on folic acid. Mention is made here of the extent to which
PABA has been found to reverse the growth-inhibiting effect of the sul-
fonamides.
Black et al.**:™ found that PABA reverses the growth-inhibiting effects
of sulfaguanidine (sulfanilyl guanidine) when this compound is fed to rats
to the extent of 0.5% of a highly purified diet, provided that the PABA
is given from the start of the experiment. If PABA is given after a nutri-
tional deficiency has been developed by the feeding of the drug, some lag
period is observed before the beneficial effects of the PABA become ap-
parent. The growth-promoting effect of PABA in purified diets containing
sulfaguanidine first observed by Black et al.**: ™ has been confirmed by Mar-
tin” and by MacKenzie et al.*°
Welch*® reported that PABA does not antagonize the growth inhibition
produced by sulfasuxidine (succinylsulfathiazole) when this compound is
6° R. Tschesche, Z. Naturforsch. 2b, 10 (1947).
70H. Noll, J. Bang, E. Sorkin, and H. Erlenmeyer, Helv. Chim. Acta 34, 340 (1951).
18. Black, R.S. Overman, C. A. Elvehjem, and K. P. Link, J. Biol. Chem. 145, 137
(1942).
76 P-AMINOBENZOIC ACID
included in a purified diet to the extent of 1%. Other more extended studies
from the same*: 74 and other laboratories’®: 7® have shown that PABA
does have a certain small effect in reversing sulfasuxidine, especially with
respect to reversing the incidence of the induced hypoprothrombinemia.
The failure to observe complete reversal with PABA of the growth in-
hibition produced by the incorporation of succinylsulfathiazole in highly
purified diets, as contrasted with the reversal of soluble sulfonamides 7m
vitro, probably is attributable to absorption of PABA early in the gut so
that the compound does not reach the cecum and large intestine in effec-
tive concentration.
Emerson and Cushing”: have described a unique mutant of Neuwro-
spora crassa that requires sulfanilamide or other similar sulfonamide for
growth at 35°. At 30° or lower, sulfonamides are not strictly essential, but
growth rates are lower in their absence. For this strain PABA is an in-
hibitor that 1s competitively antagonized by sulfanilamide with an in-
hibition ratio for one-half maximum growth of about 100. Another mutant
obtained by crossing the sulfonamide-requiring mutant with a PABA-
requiring mutant requires both sulfonamide and PABA for growth. The
molar ratio giving maximum growth at 35° is about 1000 of sulfanilamide
to 1 of PABA. The possibility that sulfanilamide is utilized as a metabolite
by the sulfonamide-requiring strain was entertained,’* but it has been ruled
out by Zalokar,”’: 8° who showed that the sulfonamide-requiring mutant
may be made to grow by any one of the following ways: (1) by the presence
of sulfonamide; (2) by reducing available PABA by genetic means, that is,
by crossing the sulfonamide-requiring mutant with a PABA-requiring
mutant to give a strain that does not synthesize PABA in large amounts;
(3) by reducing available methionine by genetic means, that is, by similarly
crossing the sulfonamide-requiring strain with a methionine-requiring
strain to give a new strain that does not synthesize methionine in large
amounts; (4) by the addition to the medium of threonine, which acts as an
inhibitor; or (5) by making free ammonia available as a nitrogen source.
As summarized by Zalokar*®® the data are best interpreted as follows: The
72. D. Wright and A. D. Welch, Science 97, 426 (1943).
73. D. Wright and A. D. Welch, J. Nutrition 27, 55 (1944).
“47. D. Wright, H. R. Skeggs, A. D. Welch, K. L. Sprague, and P. A. Mattis, J.
Nutrition 29, 289 (1945).
75, W. Neumann, M. M. Krider, and H. G. Day, Proc. Soc. Exptl. Biol. Med. 62,
257 (1943).
76H. G. Day, K. G. Wakim, M. M. Krider, and E. E. O’Banion, J. Nutrition 26,
585 (1943).
778. Emerson and J. EH. Cushing, Federation Proc. 5, 379 (1946).
78S. Emerson, J. Bacteriol. 64, 195 (1947).
79M. Zalokar, Proc. Natl. Acad. Sci. U. S. 34, 32 (1948).
80 M. Zalokar, J. Bacteriol. 60, 191 (1950).
XIII, SULFONAMIDE REVERSAL 77
gene that is altered in the sulfonamide-requiring strain produces a dele-
terious reaction in Neurospora crassa with the following characteristics:
(1) it requires a precursor of methionine as substrate, and the amount re-
quired is greater than that needed for essential reactions normally using
the same substrate; (2) it requires PABA as a catalyst, and in much larger
amounts than are required by other reactions using the same catalyst; (3)
at the same time sulfanilamide is a much stronger competitor of PABA in
this reaction than in other reactions requiring PABA as a catalyst; and (4)
the reaction, or a product of the deleterious reaction, interferes with the
normal utilization of threonine. That the step in threonine utilization
interfered with may involve amination is suggested by the growth-pro-
moting activity of free ammonia.
As demonstrated by the content of the foregoing paragraphs, most of the
studies that have involved relationships between PABA and the sulfon-
amides have been carried out on bacteria. That antagonism between PABA
and the sulfonamides may be demonstrated in a variety of plant and ani-
mal systems has been demonstrated. Terzian*! has reported that sulfanil-
amide or sulfadiazine in the diet of the mosquito Aedes aegypti will increase
the insect’s susceptibility to the malarial parasite Plasmodium gallinaceum.
PABA fed along with the sulfonamide nullifies its effect on susceptibility
of the host. Hindmarsh® has shown with onion rootlets that sulfanilamide
prevents the nuclei from coming into prophase and upsets the spindle
mechanism so that anaphase rarely occurs. These effects are reversed by
PABA. Schopfer and Anker* found that PABA, guanine, adenylic acid,
and riboflavin exert an antagonistic effect on sulfonamide inhibition of
the growth of pea roots in aseptic culture. The growth-inhibiting action of
the sulfonamides on growth of pepper grass (Lepidiwm sativum) roots was
found by Andus and Quastel® to be neutralized by PABA. Sulfonamide
inhibition of blood carbonic anhydrase is reversed with PABA according to
van Goor.®® PABA is an antagonist of the inhibiting action of sulfanil-
amide on germination and growth of flax seed in the experiments of Ha-
zard.®®> Maier and Riley*’ found that the therapeutic effect of the sulfon-
amides on Plasmodium gallinaceum infections of chicks is negated by
administration of PABA. Sulfonamide inhibition of yeast growth is re-
versed by PABA. Growth of isolated tomato roots is inhibited by sul-
8.1L. A. Terzian, J. Infectious Diseases 87, 285 (1950).
82 M. M. Hindmarsh, Nature 163, 610 (1949).
88 W. H. Schopfer and W. Anker, Experientia 5, 117 (1949).
54. J. Andus and J. H. Quastel, Ann. Botany 12, 27 (1948).
85 H. van Goor, Enzymologia 11, 174 (1944).
86 R. Hazard, Compt. rend. soc. biol. 188, 972 (1944).
87 J. Maier and E. Riley, Proc. Soc. Exptl. Biol. Med. 50, 152 (1942).
88 M. Landy and D. M. Dicken, Nature 149, 244 (1942).
78 P-AMINOBENZOIC ACID
fonamides, and the inhibition is reversed by PABA in the experiments of
Bonner.*® Sulfanilamide inhibition of the growth of onion (Alliwm capa)
rootlets is reversed with PABA, according to Stoll.°° Germination of rice
seeds is retarded by sulfanilamide, and the effect is reversed with PABA in
the experiments of Ribeiro.*! Chodat and Soloweitchik®” found that PABA
reduces the cytostatic influence of sulfanilamide on various species of
green algae. Growth of a fresh-water diatom (Nitzschia palea) is inhibited
by sulfonamides and the inhibition is reversed with PABA, according to
Wiedling.*: ** In experiments of Efimov,®® PABA was reported to counter-
act the depressant effect of sulfanilamide on isolated frog hearts. PABA
has been reported to reverse the effect of sulfanilamide in inhibiting the
adsorption of methylene blue by charcoal.’® The foregoing enumeration of
antagonisms between PABA and the sulfonamides that have been demon-
strated in non-bacterial systems is by no means exhaustive.
Although the aromatic sulfones are quite different from the sulfonamides
in structure and have a different spectrum of antibacterial activity, such as
might be expected if their mechanism of action were different, it has been
shown by Steenken and Heise” that PABA antagonizes in vitro the anti-
tubercular activity of promin.
p-Aminosalicylic acid, a compound resembling both sulfanilamide and
PABA in structure, was found by Lehmann: °° to be a remarkably specific
chemotherapeutic agent against Mycobacteriwm tuberculosis both in vitro
and in vivo.
SO.NH:2 COOH COOH
| OH
Yl vA
NS
NH, NH, NH,
Sulfanilamide PABA p-Aminosalicylie acid
. (PASA)
89 J. Bonner, Proc. Natl. Acad. Sci. U. S. 28, 321 (1942).
90 R. Stoll, Compt. rend. soc. biol. 187, 170 (1948).
1 F, Ribeiro, J. Biol. Chem. 152, 665 (1944).
2 F. Chodat and 8. Soloweitchik, Compt. rend. soc. phys. hist. nal. Geneve 59, 167
(1942) [C. A. 38, 5530 (1944)].
%S. Wiedling, Nature 150, 290 (1942).
S$. Wiedling, Science 94, 389 (1941).
9 N. I. Efimov, Farmakol. « Toksikol. 8, No. 5, 28 (1945) [C. A. 40, 7409 (1946) ].
9% H.C. Kyster, J. Cellular Comp. Physiol. 21, 191 (1948).
7 W. Steenken, Jr., and F. H. Heise, Proc. Soc. Exptl. Biol. Med. 52, 180 (1948).
% J. Lehmann, Lancet 250, 14, 15 (1946).
99 J. Lehmann, Svenska Ldkartidn. 48, 2029 (1946) [C. A. 41, 1834 (1947)].
XIV. THERAPEUTIC ACTIVITY 79
The compound does not have appreciable bacteriostatic activity against
microorganisms other than the tubercle bacillus.!°°!" The bacteriostatic
activity of PASA against Mycobacterium tuberculosis is reversed by PABA
and to a small extent by salicylic acid.!%!9 PASA replaces PABA as a
growth factor for two mutant strains of ’scherichia coli.°*
The antibacterial activities determined in vitro of thirty compounds re-
lated in structure to PABA have been summarized by Shive.!°°
XIV. Therapeutic Activity
LEMUEL D. WRIGHT and PETER A. TAVORMINA
A. RICKETTSIAL DISEASES
PABA was found to be an antirickettsial compound when Snyder e¢ al.!
observed that mortality in mice infected with yolk sac suspension of swine
typhus is markedly reduced by oral administration of PABA as a com-
ponent of the diet. Their findings were confirmed in mice by Andrews et al.’
Quite independently, Greiff ef al.’ found PABA to be rickettsiostatic in
typhus-infected yolk sacs and in swine typhus infection in mice.
The effectiveness of PABA as a practical chemotherapeutic agent was
established by Yeomans et al.,* who found the drug useful in the treatment
of louse-borne typhus in humans. PABA was subsequently shown to be of
value in the treatment not only of louse-borne typhus but of other rickett-
sial diseases as well, including tsutsugamuchi disease (scrub typhus) and
Rocky Mountain spotted fever.*4® PABA continued to be the drug of
100 Q. Sievers, Nord. Med. 33, 145 (1947).
101 J. Lehmann, Rev. gén. sci. 54, 222 (1947) [C. A. 42, 4641 (1948)].
1022 FE). Diezfalusy, Arkiv. Kemi, Mineral. Geol. 24B, No. 13, 1 (1947) [C. A. 42, 5506
(1948) ].
103 G. P. Youmans, G. W. Raleigh, and A. S. Youmans, J. Bacteriol. 54, 409 (1947).
104 G. Ivanovies, Proc. Soc. Exptl. Biol. Med. 70, 462 (1949).
105 T). Yegian and R. T. Long, J. Bacteriol. 61, 747 (1951). ;
106 R. J. Williams, R. E. Eakin, E. Beerstecher, Jr., and W. Shive, The Biochemistry
of B Vitamins, p. 481. Reinhold Publishing Corp., New York, 1950.
1J. C. Snyder, J. Maier, and C. R. Anderson, Rept. Natl. Research Council, Divis.
Med. Sci. (Dec. 26, 1942).
*C. H. Andrews, H. King, and G. Van den Ende, Biol. Rept. Natl. Inst. Med. Re-
search, London (1943).
3D. Greiff, H. Pinkerton, and V. Moragues, J. Exptl. Med. 80, 561 (1944).
4A. Yeomans, J. C. Snyder, E. S. Murray, C. J. D. Zarafonetis, and R. 8. Ecke,
J. Am. Med. Assoc. 126, 349 (1944).
5N. A. Tierney, J. Am. Med. Assoc. 131, 280 (1946).
6C. J. D. Zarafonetis, J. C. Snyder, and E. 8S. Murray, Proc. Soc. Exptl. Biol.
Med. 61, 240 (1946).
80 P-AMINOBENZOIC ACID
choice in the treatment of rickettsial diseases until the advent of chlor-
amphenicol (chloromycetin) and aureomycin. PABA was usually ad-
7 J. C. Snyder and C. J. D. Zarafonetis, Proc. Soc. Exptl. Biol. Med. 60, 115 (1945).
8 C.J. D. Zarafonetis, and J. C. Snyder, E.S. Murray, Proc. Soc. Exptl. Biol. Med.
61, 240 (1946).
9H. L. Hamilton, H. Plotz, and J. E. Smadel, Proc. Soc. Exptl. Biol. Med. 58, 255
(1945).
10 M. L. Robbins, A. R. Bourke, and P. K. Smith, J. Immunol. 64, 431 (1950).
11J,, Anigstein and M. N. Bader, Tezas Repts. Biol. Med. 3, 253 (1945).
12 7,, Anigstein and M. N. Bader, Science 101, 591 (1945).
13 J,. Anigstein and M. N. Bader, Texas Repts. Biol. Med. 4, 260 (1946).
147, Anigstein and D. M. Whitney, Texas Repts. Biol. Med. 4, 338 (1946).
15 M.M. Best, D. J. Wolfram, and J. O. Flora, J. Indiana State Med. Assoc. 40, 631
(1947).
16 R. L. Cash, Kentucky Med. J. 46, 19 (1948).
17H. L. Chung, Chinese Med. J. 67, 421 (1949).
18 R. 8. Diaz-Rivera, J. J. Santos, and E. Perez-Santiago, Biol. Assoc. Med. Puerto
Rico 38, 189 (1946).
19], B. Flinn, J. W. Howard, C. W. Todd, and E. G. Scott, J. Am. Med. Assoc. 132,
911 (1946).
20 T,. EK. Fraser, H. Rosenblum, and J. A. Danciger, Am. J. Diseases Children 75,
493 (1948).
2
eS)
F. G. Kirby, Ann. Western Med. and Surg. 1, 203 (1947).
M. D. Levy and W. T. Arnold, Texas State J. Med. 42, 314 (1946).
28 J. W. Maroney, H.C. Davis, and E.G. Scott, Delaware State Med. J. 18, 104 (1946).
29 AH. H. Muntz and I. Salkin, J. Indiana State Med. Assoc. 41, 591 (1948).
30 E. 8. Murray, C. J. D. Zarafonetis, and J. C. Snyder, Proc. Soc. Exptl. Biol. Med.
60, 80 (1945).
31 J. R. Newman, Rocky Mt. Med. J. 44, 718 (1947).
32 J. O. Niles and G. C. Bourne, New Engl. J. Med. 238, 769 (1948).
33 $. F. Ravenel, J. Am. Med. Assoc. 138, 989 (1947).
34S. F. Ravenel, Southern Med. J. 40, 801 (1947).
35 H. M. Rose, R. B. Duane, and E. HE. Fischel, J. Am. Med. Assoc. 129, 1160 (1945).
36S. Ross and P. K. Smith, Clin. Proc. Children’s Hosp. 2, 258 (1946).
37 §. Ross, P. A. McLendon, and H. J. Davis, J. Pediat. 2, 163 (1948).
38§. Ross, P. A. McLendon, and H. J. Davis, Clin. Proc. Children’s Hosp. 4, 247
(1948).
39 F. Sanchez Ruiz, Medicina mex. 30, 165 (1950).
40 PK. Smith, J. Am. Med. Assoc. 181, 1114 (1946).
41 J. C. Snyder, A. Yeomans, D. H. Clement, E. S. Murray, C. J. D. Zarafonetis,
and N. A. Tierney, Ann. Internal. Med. 27, 1 (1947).
©, J. Tichenor, S. Ross, and P. A. McLendon, J. Pediat. 81, 1 (1947).
'3.C, J. Tichenor, S. Ross, and P. A. McLendon, Clin. Proc. Children’s Hosp. 3, 203
(1947).
3
4
®>G. W. James, III, and H. Walker, Virginia Med. Monthly 74, 161 (1947).
6
7
i<y I<"
2
XIV. THERAPEUTIC ACTIVITY 81
ministered orally at 2- to 3-hour intervals for a total daily dose of 25 to
30 g.
The mechanism by which PABA acts to inhibit rickettsia is not known,
but recent work of Davis and colleagues has thrown some light on the sub-
ject. For the W strain of Escherichia coli Davis showed": * that PABA is a
bacteriostatic compound. The bacteriostatic activity of PABA is reversed
by p-hydroxybenzoic acid (POB). Separate studies by Davis showed pre-
viously*® that for certain mutants of Escherichia coli with a requirement for
five aromatic compounds (quintuple aromatic auxotrophs) POB is a bac-
terial vitamin essential for rapid growth. Shikimic acid and compound X,
previously shown by Davis et al. to be precursors of the aromatic ring in
Escherichia coli, similarly are active in the reversal of PABA inhibition.
Thus it would appear that POB is an essential metabolite for which PABA
is a naturally occurring antimetabolite. This concept is slightly compli-
cated but not necessarily vitiated by the fact that POB may be derived
from PABA at a slow rate. As summarized by Davis,** it would appear
that PABA exerts three metabolic effects on Escherichia coli: it acts as a
normal vitamin at low concentrations, as a source of another vitamin,
POB, at moderate concentrations, and as a growth inhibitor at high con-
centrations.
Snyder and Davis have published a preliminary note®® concerning the
extent to which the metabolite-antimetabolite relationship between POB
and PABA established in Escherichia coli can explain the rickettsiostatic
effect of PABA. POB was found to reverse PABA in two tests with the
Brein| strain of epidemic typhus in chick embryos, in one test with murine
typhus in white mice, and in one test with Rocky Mountain spotted fever
in chick embryos. For reversal of rickettsiostasis in chick embryos, the
effective ratio of POB to PABA (on the basis of the amounts inoculated
into each egg as a single dose at zero time) appears to be in the range of
Hobo lL: 10.
B. RHEUMATOID DISEASES
PABA has shown some promise in the treatment of acute rheumatic
fever. Dry ef al.®*' encountered a case of acute rheumatic fever in an adult
44N. A. Tierney, Southern Med. J. 40, 81 (1947).
45 N. A. Tierney, J. Am. Med. Assoc. 181, 280 (1946).
46 A. Yeomans, J. Am. Med. Assoc. 126, 782 (1944).
‘7B. D. Davis, Federation Proc. 10, 406 (1951).
4% B.D. Davis, J. Expil. Med. 94, 243 (1951).
49B. D. Davis, Nature 166, 1120 (1950).
50 J. C. Snyder and B. D. Davis, Federation Proc. 10, 419 (1951).
T. J. Dry, H. R. Butt, and C. H. Scheifley, Proc. Staff Meetings Mayo Clinic 21,
497 (1946).
82 P-AMINOBENZOIC ACID
man that did not respond to salicylate therapy (10 g. per day). On more or
less empiric grounds the simultaneous oral administration of PABA was
instituted (initial dose of 4 g. followed by 2 g. every 2 hours). Prior to the
use of PABA the salicylate blood level varied between 12.5 and 15 mg. per
100 ml. With PABA there was a steady increase in salicylate level from
12.5 to 34.5 mg. per 100 ml. There was a dramatic and complete clinical
response as the content of salicylate reached 37.5 mg. per 100 ml. of blood.
Separate experiments with healthy men indicated that salicylates and
PABA appear to have a reciprocal effect in increasing their separate con-
centrations in the blood when they are administered together. Increased
blood levels of salicylate attained by the simultaneous administration of
PABA are accompanied by less salicylate appearing in the urine.
The findings of Dry et al.*' have been confirmed by Hoaglund,*? who
reported that a case of rheumatic fever with refractoriness to 10 g. of
aspirin daily, manifested by an uninterrupted fever for 5 weeks, showed
dramatic improvement after the salicylate therapy was supplemented with
PABA in a daily dose of 24 g.
Smith*? has compared the combination PABA and salicylate with sali-
cylate alone as a treatment for a variety of rheumatic diseases exclusive of
acute rheumatic fever. The pain relief with the PABA-salicylate combina-
tion was found to be superior to that with sodium salicylate alone in 64
patients with rheumatoid arthritis and fibrositis. In 60 patients with osteo-
arthritis or with cervical osteoarthritis with radicular involvement and
chronic painful shoulder, the superiority was less marked. The combination
therapy gives longer pain relief than sodium salicylate alone. Significant
toxic reactions to the combinations were not observed. Sodium salicylate
alone, however, produced some toxic effects in 69 (55.2%) of the 125
patients.
The mechanism of action of PABA as an adjunct to salicylates has been
investigated by Salassa et al.,°>* who found that PABA produces the fol-
lowing effects. First, it alters the detoxication of salicylate by interrupting
or greatly depressing the conjugation of glycine with salicylate so that only
very small quantities of salicyluric acid appear in the urine after ingestion
of salicylate. Second, it tends to lower the pH of the urine and thus de-
creases the renal clearance of the free salicylate fraction. Third, it causes a
decrease in the urinary excretion of total salicylate and a rise in the plasma
salicylate level as a result of the foregoing effects. In dogs, the administra-
tion of PABA does not alter the excretion of salicylate and does not in-
crease the plasma salicylate levels.
52 R. J. Hoaglund, Am. J. Med. 9, 272 (1950).
53 R. T. Smith, J.-Lancet 70, 192 (1950).
54 R. M. Salassa, J. L. Bollman, and T. J. Dry, J. Lab. Clin. Med. 33, 1393 (1948).
XIV. THERAPEUTIC ACTIVITY 83
PABA was used alone by Rosenblum and Fraser®® in the treatment of
acute rheumatic fever in nine children. The dosage schedule involved an
immediate dose of 3 to 4 g. followed by a maintenance dose of 1 to 3 g.
given at 2- to 3-hour intervals. That PABA has some effect alone in the
treatment of acute rheumatic fever was indicated by a decrease in tem-
perature, a decrease in severity of joint pains, and the attainment of a sense
of well being.
PABA and cortisone have been found by Wiesel et al.°* to funetion syner-
gistically in the treatment of rheumatoid arthritis. A trial of the combina-
tion appeared warranted for two reasons. Firstly, PABA alone or cortisone
alone is active in rheumatoid arthritis. Secondly, the inactivation of es-
trogens by the liver is inhibited by PABA,* and it might be reasoned that
the inactivation of cortisone, a compound closely related to the estrogens
in structure, might be similarly prevented, or at least inhibited, by PABA.
Although the preliminary paper of Wiesel et al. was limited to a study of
only 15 patients, the results obtained indicated that a combination of the
two drugs at levels where neither is active alone produces satisfactory con-
trol of rheumatoid arthritis. The euphoria and subsequent depression
frequently observed in patients receiving cortisone alone were not observed
on the combination.
C. ACHROMOTRICHIA
The reported chromotrichial effect of PABA observed by Ansbacher®
in the rat and by Martin and Ansbacher*? in the mouse stimulated a number
of clinical investigators to try PABA against human achromotrichia.
Sieve®’ reported that, in 30 patients from a group ranging in age from
20 to 55 in which PABA was the sole therapy, marked darkening of the hair
occurred in all cases. The dose of PABA administered was 100 mg. twice a
day. Subsequently, Sieve® reported the beneficial effect of PABA in achro-
motrichia in 82 out of 460 patients. Another paper by Sieve® reports a
variety of favorable effects, including darkening of the hair, in a group of
800 patients.
Brandaleone et al.** have studied achromotrichia in a group of 19 elderly
°° H. Rosenblum and L. E. Fraser, Proc. Soc. Exptl. Biol. Med. 65, 178 (1947).
56 L. L. Wiesel, A. S. Barritt, and W. M. Stumpe, Am. J. Med. Sci. 222, 243 (1951).
57 S$. Ansbacher, W. A. Wisansky, and G. J. Martin, Federation Proc. 1, 98 (1942).
58S. Anshacher, Science 93, 164 (1941).
*° G. J. Martin and S. Ansbacher, J. Biol. Chem. 188, 441 (1941).
6” B. F. Sieve, Science 94, 257 (1941).
1B. F Sieve, 161st Ann. Meeting, Mass. Med. Soc., Boston (1942), quoted by 8. Ans-
bacher, Vitamins and Hormones 2, 215 (1944).
® B. F. Sieve, Southern Med. and Surg. 104, 135 (1942).
88H. Brandaleone, E. Main, and J. M. Steele, Proc. Soc. Exptl. Biol. Med. 68, 47
(1943).
$4 P-AMINOBENZOIC ACID
men and women hospitalized for various chronic diseases common to old
age. Seven received 100 mg. of caletum pantothenate, 200 mg. of PABA
and 50 g. of brewer’s yeast daily, five received yeast and PABA, and seven
received yeast and calcium pantothenate. The treatment was continued for
eight months. Significant improvement was observed in only two in-
dividuals, both of whom were men receiving the combination calcium
pantothenate, PABA, and yeast. Only questionable improvement occurred
in those patients receiving one or the other compound along with the
yeast.
Eller and Diaz administered PABA to 88 persons of different ages and
both sexes in various stages of achromotrichia. Tablets of 100 mg. were
given three or four times daily over periods ranging from three to five
months. A few of the patients showed definite changes in the color of their
hair.
Visual darkening of the hair was reported by DeVilbiss® in all of 16
patients treated with PABA at a recommended dose of 100 mg. four times
daily. Photos taken before and after treatment, in color as well as in
black and white, supported the patients’ own opinion that PABA is of
value in the treatment of premature graying.
Friedgood®*® observed a few cases of human achromotrichia that appeared
to respond to PABA, but he was inclined to minimize the extravagant
claims of Sieve.”
D. THYROTOXICOSIS
PABA has been used clinically with some success as an antithyroid
compound by a number of investigators. Berman claimed good results in
the treatment of six cases of hyperthyroidism with parenteral PABA.
Papp® gave PABA by mouth with success in four patients with hyper-
thyroidism. Williams®? was not enthusiastic about the use of PABA in
thyrotoxicosis, since he was able to reduce the basal metabolic rate in only
two out of eight patients by use of PABA. 3,5-Diiodo-p-aminobenzoic
acid showed some promise in his series, presumably because of the affinity
of the thyroid for iodine. Goodwin et al.’° treated ten thyrotoxic patients
with PABA and obtained full control in only one, and six did not respond
at all. They conclude that PABA has a slight but definite antithyroid action
in safe dosage but that, to obtain an action comparable to that of the
64 J. J. Eller and L. A. Diaz, N. Y. State J. Med. 48, 1331 (1948).
65 J,. A. DeVilbiss, Med. Woman’s J. 49, 341 (1942).
66 H. B. Friedgood, New Engl. J. Med. 227, 788 (1942).
67 L.. Berman, Proc. Soc. Exptl. Biol. Med. 59, 70 (1945).
68 A. Papp, Marquette Med. Rev. 18, 23 (1947).
69 R. H. Williams, Arch. Internal Med. 80, 11 (1947).
70 J. F. Goodwin, H. Miller, and E. J. Wayne, Lancet 257, 1211 (1949).
XIV. THERAPEUTIC ACTIVITY 85
thiouracil group, toxic doses would be necessary. It is concluded that
PABA is suitable only for the treatment of mild cases of thyroidism.
. MISCELLANEOUS DISEASES
The effectiveness of PABA in the treatment of rickettsial diseases,
rheumatic fever, and related rheumatic diseases is unequivocal. Results
that may be summarized as encouraging, at best, have been obtained in the
treatment of the following conditions with PABA: lymphoblastoma
cutis,” ” lupus erythematosus,” active dermatomyositis,” “: ™ sclero-
derma,” ” dermatitis herpetiformis,”: 7° chronic myelogenous leukemia,’*78
and experimental allergic encephalomyelitis.”’
PABA has been found by a number of investigators to have a low or-
der of fungistatic activity.5°4
1 C. J. D. Zarafonetis, J. Lab. Clin. Med. 38, 1462 (1948).
72 C.J. D. Zarafonetis, Ann. Internal Med. 30, 1188 (1949).
ISO Je Ds Peratocetia: R. H. Grekin, and A. C. Curtis, J. Invest. Dermatol. 11, 359
(1948).
74C. J. D. Zarafonetis, A. C. Curtis, and R. H. Grekin, Univ. Hosp. Bull., (Ann
Arbor) 18, 122 (1947).
75 C.J. D. Zarafonetis, Am. J. Med. 5, 625 (1948). bs
76C. J. D. Zarafonetis, G. A. Andrews, M. C. Meyers, and F. H. Bethell, Blood 3,
780 (1948).
77H. B. May and J. Vallanece-Owen, Lancet 255, 607 (1948).
73 J. Bichel, Le Sang 21, 360 (1950).
77R. A. Good, B. Campbell, and T. A. Good, Proc. Soc. Exptl. Biol. Med. 72, 341
(1949).
80 G. W. K. Cavill and J. M. Vincent, J. Soc. Chem. Ind. (London) 68, 189 (1949)
[C. A. 48, 8443 (1949)].
81G. W. K. Cavill and J. M. Vincent, J. Soc. Chem. Ind. (London) 67, 25 (1948)
[C. A. 42, 4701 (1948)].
82 F’. Serri, Acta Vitaminol. 2, 73 (1948) [C. A. 48, 1826 (1949)].
83 EF. Biocca and C. S. Lacaz, Arguiv. biol. (Sao Paulo) 29, 151 (1945) [C. A. 40,
5090 (1946)].
34M. Milner, C. M. Christensen, and W. F. Geddes, Cereal Chem. 24, 507 (1947)
[C. A. 42, 1671 (1948)].
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CHAPTER 13
PTEROYLGLUTAMIC ACID
I. Nomenclature and Formula.
II. Chemistry .
A. Isolation
ils
9
—
Of Pteroy eink amic Acid (PGA).
2. Of PGA Conjugates .
3. eS
3. Physical and Chemical Properties .
il.
2.
Degradation Reactions .
Hydrogenation .
Other Reactions
C. Constitution .
D. Synthesis .
1. Simultaneous Gondenenttan oe Tamincoy Sardine 3- Canbon ce
2.
3.
—
kd
We
2.
termediate, and Aromatic Amine :
Condensation of Preformed Pterin with emetic ‘Acaines
Synthesis of Pteroylpolyglutamie Acid Derivatives
. Specificity .
Chemistry of Ricnopteria (soungl Pierdic TED a
Chemistry of Citrovorum Factor (Leucovorin, Folinie Acid)
III. Industrial Preparation
IV. Biochemical Systems .
A. Coenzymes and Enzymes
i:
2. Sulfonamide, p-Aminobenzoic Acid, and Purine Relationships .
Function of PGA in Synthesis of Purine and Py Panidines :
B. Mechanism of Action.
. Role of 4-Amino-5-Imid: Ao cleebexsaniden in i parine i hesia :
. Inosinic Transformylase. : :
. Effect of Aminopterin on Purine Sy wien: in Mice.
. Relation of p-Aminobenzoic Acid to PGA
. Role of PGA in Synthesis of Amino Acids .
. PGA in Single-Carbon Transfer in Animals.
V. een of Action ae
A. Relation of Santhaptenn ie PGA :
B. PGA Antagonists .
i
Ze
. Pteridine Derivatives ’
. Modification of Pterin Rielens ; ;
. Pteroyl Derivatives of Different Amino Gde
. Alkyl Derivatives . é
. 4-Aminopteroylglutamic ad enue. :
8.
“IO Ot CO
Substituted Py pede
Purine Derivatives
Effect of Antagonists on Experimental Tumors
VI. Estimation.
87
Page
89
91
91
91
93
95
99
101
103
104
104
106
107
108
109
109
110
121
124
124
124
127
128
128
132
133
133
136
137
142
145
149
150
151
152
153
154
155
157
159
161
88
WADE
VTE:
IX.
PTEROYLGLUTAMIC ACID
A, Chemical Methods
B. Microbiological Methods
a
Extraction from Tissues and Eigdrely: sis os Gonjientes
2. Microbiological Procedures.
Sandi ration of Activity
Occurrence in Foods
Effects of Deficiency
A. In Animals
ile
(0/0)
“IED Ot HR Wb
Rats.
Chicks .
. Monkeys
Pigs .
Mice. ;
Guinea Pigs .
. Mink.
. Dogs.
. Foxes
. Insects . ;
. Relation benween PGA aad ecorbie Rede
. Endocrine Relationships of PGA
oe
Role of Citrovorum Factor in Animal Natition :
B. si Microorganisms.
Dye
Oo Ore © bo
. Metabolism of Raves
Microbiological Activity of Chimonortiin Paetor
. In Man.
il :
. Idiopathic Refractory Megaloblastic Rete or Aenreanie Wena
. Megaloblastic Anemia of Pregnancy and the Puerperium
. Megaloblastic Anemia of Infancy
. Sprue and Idiopathic Steatorrhea .
. Megaloblastic Anemias Believed To Be Due Primarie He Lack of
Nutritional Macrocy ie Aneta :
Vitamin Bi. .
. Induced PGA Deacionen in ONE
. Summary .
X. Pharmacology.
A. Actions and Uses .
B. Dose.
C. Toxicity
XI. Requirements and Ructore Tagen "Ther
A. Of Animals
B. Of Man.
Page
161
162
163
167
168
168
171
171
171
175
180
186
189
189
189
189
190
190
191
195
197
199
200
201
202
203
205
205
207
208
209
213
215
216
216
216
216
217
217
217
I. NOMENCLATURE AND FORMULA 89
I. Nomenclature and Formula
E. L. R. STOKSTAD*
There are few examples in biochemistry where a single compound has
provided the key to as many nutritional phenomena as has pteroylglutamic
acid. Deficiency states of this vitamin had been described in man, monkeys,
chicks, rats, guinea pigs, insects, and microorganisms before pteroylglu-
tamic acid was identified and its structure determined.
In view of the many phases of biological work already in progress by the
time pteroylglutamic acid was synthesized and made available, it is not
surprising that a large number of papers soon appeared on its role in clinical
medicine, animal nutrition, bacterial metabolism, enzyme chemistry, and
tumor research.
Since work on this vitamin has proceeded independently in many labora-
tories employing different species, the literature is replete with the various
names used to designate it. A summary of the names which have been
given to various biologically active materials whose activity can be ascribed
to pteroylglutamic acid is presented in Table I.
The nomenclature in this field is also complicated by the existence of
different chemical forms which were subsequently shown to be conjugates
of the vitamin with varying numbers of glutamic acid residues. Thus the
name “fermentation L. casei factor’? was given to a compound isolated
from a fermentation product and which later was shown to be pteroyltri-
glutamic acid. The name ‘‘vitamin B,. conjugate’? was used to designate
the compound isolated from yeast and which was shown to contain seven
glutamic acid residues. This will be referred to as pteroylheptaglutamic
acid. The name ‘folic acid”? has found the most universal acceptance,
largely because of its brevity. The term ‘‘folacin,’’ recommended by a joint
nomenclature committee of the American Institute of Nutrition and the
Society of Biological Chemists, has not as yet received wide usage. In this
chapter the name pteroylglutamic acid, abbreviated PGA, will be used
except in accounts of original literature where the investigator used a
special name to designate a biologically active factor, the exact chemical
identity of which cannot be established.
One of the first published observations describing a clinical syndrome
which was the result of PGA deficiency was made by Wills! in 1931. This
disease was found to have a blood picture similar to that in pernicious
anemia, except that some of the other symptoms, such as achlorhydria and
nervous lesions observed in pernicious anemia, were absent. This condition
*It is a pleasure to acknowledge the assistance of M. J. Fahrenbach and H. P.
Broquist in writing portions of this review and the services of Carol Gunderson in
the preparation of the manuscript.
90 PTEROYLGLUTAMIC ACID
was observed in women patients in India and was associated with preg-
nancy. The anemia was relieved by feeding large doses of Marmite, which
is a concentrated extract of autolyzed yeast.
A deficiency disease similar to this clinical syndrome was experimentally
induced in monkeys by Wills and Stewart"! by feeding diets similar to those
consumed by the women in Bombay who developed the tropical macrocytic
anemia. This anemia in monkeys could be cured by autolyzed yeast extract
TABLE I
NOMENCLATURE OF PREPARATIONS Havine BroLocicaL AcTIvITY PRESUMABLY DUE
TO PTEROYLGLUTAMIC AcID OR RELATED COMPOUNDS
Name Description Date Ref.
Wills factor Yeast extract, effective in the treatment 1931 1
of tropical macrocytic anemia
Vitamin M Yeast and liver extract, effective against 1938 2
nutritional cytopenia in monkeys
Factor U Growth factor for chicks, present in yeast 1938 3
extract
Vitamin B, Adsorbed on fuller’s earth, prevented nu- 1939 4
tritional anemia in chicks
Norit eluate factor Growth factor for L. casei from yeast and 1940 5
liver
Folic acid Active for S. lactis R; concentrates prepared 1941 6
from spinach
L. casei factor Isolated from liver and yeast 1943 7
SLR factor Growth factor for S. lactis R, inactive for 1943 8
(rhizopterin) L. casei; obtained from Rhizopus nigri-
cans fermentation product
Note: In addition to the above, less clearly defined fractions have received separate names, including
vitamins Bio and Bu and factors R and 8.10
and certain types of liver extracts which were also effective in the treatment
of tropical macrocytic anemia in human patients. However, two liver
17,. Wills, M. A. Contab, and B.S. Lond, Brit. Med. J.1, 1059 (1931).
2P.L. Day, W. C. Langston, and W. J. Darby, Proc. Soc. Exptl. Biol. Med. 38, 860
(1988).
3K. L. R. Stokstad and P. D. V. Manning, J. Biol. Chem. 125, 687 (1938).
4A.G. Hogan and I. M. Parrott, J. Biol. Chem. 128, xlvi (1939).
5}. K. Snell and W. H. Peterson, J. Bacteriol. 39, 273 (1940).
6 H. K. Mitchell, E. E. Snell, and R. J. Williams, J. Am. Chem. Soc. 68, 2284 (1941).
7H. L. R. Stokstad, J. Biol. Chem. 149, 573 (1948).
8 J. C. Keresztesy, EK. L. Rickes, and J. L. Stokes, Science 97, 465 (19438).
9G. M. Briggs, Jr., T. D. Luckey, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem.
148, 168 (1943).
10 A. i}. Schumacher, G. F. Heuser, and L. C. Norris, J. Biol. Chem. 185, 313 (1940).
11], Wills and A. Stewart, Brit. J. Exptl. Pathol. 16, 444 (1935).
Il. CHEMISTRY 91
preparations were found which were effective in the treatment of human
pernicious anemia but which produced no response in the anemic monkeys
on the experimental diets. These observations thus served to distinguish
the anti-pernicious anemia factor of liver from the factor effective against
tropical macrocytic anemia.
Day and coworkers? gave the name ‘“‘vitamin M”’ to a factor essential for
hematopoiesis in monkeys. This factor was later shown to be pteroylglu-
tamic acid by Day e/ al.,” who also found that this deficiency disease could
be corrected by pteroyltriglutamic acid. Stokstad and Manning’ observed
that chicks receiving a diet consisting essentially of washed fish meal,
polished rice, riboflavin, thiamine, and a pantothenic acid concentrate
responded to an unknown growth factor in yeast, alfalfa, and wheat bran.
This factor was designated ‘“‘factor U’’ and was later shown to be identical
with PGA. Hogan and Parrott? gave the name “vitamin B,.”’ to a factor
present in liver which prevented macrocytic anemia in chicks.
Shortly after the publication of these experiments with animals, Snell
and Peterson® reported that Lactobacillus casei required an unknown growth
factor which they termed the ‘‘Norit eluate factor.’”’ The term ‘‘folic acid”
was given by Mitchell et al.® to a substance obtained from spinach, which
stimulated the growth of Streptococcus faecalis R and was active for L.
caset. It appeared to have the same microbiological properties as the
Norit eluate factor.
II. Chemistry
E. L. R. STOKSTAD
A. ISOLATION
1. Or PreroyieLutamic Acip (PGA)
Many papers have appeared describing the concentration and chemical
properties of PGA. In the first publication by the Wisconsin workers, Snell
and Peterson! reported that this factor was adsorbed by activated charcoal
and Lloyd’s reagent, was stable to acid and alkali, could be precipitated by
basic lead acetate, and was extracted from aqueous acid solutions by bu-
tanol. A later publication by Hutchings ef al.? from this same laboratory
described additional methods of purification by adsorption on Super Filtrol
and formation of a zine salt. The isolation of a highly active folic acid
2 P. L. Day, V. Mims, J. R. Totter, E. L. R. Stokstad. B. L. Hutchings, and N. H.
Sloane, J. Biol. Chem. 157, 423 (1945).
1K. E. Snell and W. H. Peterson, J. Bacteriol. 39, 273 (1940).
2B. L. Hutchings, N. Bohonos, and W. H. Peterson, J. Biol. Chem. 141, 521 (1941).
92 PTEROYLGLUTAMIC ACID
preparation from spinach was described in a series of papers by Mitchell
and coworkers at Texas.*-° These workers employed repeated adsorption
and elution from activated charcoal, precipitation with heavy metals, and
chromatographic adsorption on alumina. Since folic acid from spinach has
never been crystallized, it is not possible to establish its identity with
PGA. However, comparison of samples of folic acid concentrates of known
potencies with pure PGA’: ® indicates that the most active preparation ob-
tained by the Texas workers had essentially the same biological potency as
pure PGA.
The isolation of PGA from liver has been reported from two laboratories.
Pfiffner et al. obtained this compound by starting with liver in which the
conjugates had been converted into free PGA by autolysis.!° The isolation
process involved extraction with boiling water, adsorption and elution
using first the ion exchange resin Amberlite 4R and later activated charcoal,
extraction of an aqueous solution of the free acid with butanol at pH 3 to 4,
formation of a barium salt and a zine salt, and finally crystallization of the
free acid from water.
Stokstad" reported the isolation of the methyl ester of PGA and later of
the free acid.” The starting material was an 80% alcohol precipitate of an
aqueous extract of liver. The method involved adsorption and elution, first
with Norit and then with Super Filtrol, precipitation of the barium salt
with methanol, esterification in acid methanol, extraction of the methyl
ester from aqueous solution with butanol, chromatographic adsorption of
the methyl] ester on Super Filtrol, and fractional precipitation of the ester
from water and methanol]. The free acid was obtained by saponification of
the ester and crystallization from hot aqueous dilute acetic acid solution.
Pteroylglutamic acid has also been isolated from yeast extracts by Pfiffner
et al.’ after treatment with hog kidney enzyme which hydrolyzed the con-
jugated vitamin to free PGA.
3H. K. Mitchell, E. E. Snell, and R. J. Williams, J. Am. Chem. Soc. 66, 267 (1944).
4). Frieden, H. K. Mitchell, and R. J. Williams, J. Am. Chem. Soc. 66, 269 (1944).
> H.K. Mitchell and R. J. Williams, J. Am. Chem. Soc. 66, 271 (1944).
6 H. K. Mitchell, J. Am. Chem. Soc. 66, 274 (1944).
7B. C. Johnson, J. Biol. Chem. 168, 255 (1946).
8 V. Mims and M. Laskowski, J. Biol. Chem. 160, 493 (1945).
9J. J. Pfiffner, S. B. Binkley, E. S. Bloom, R. A. Brown, O. D. Bird, A. D. Em-
mett, A. G. Hogan, and B. L. O’Dell, Sczence 97, 404 (1943).
10 J. J. Pfiffner, S. B. Binkley, E. 8. Bloom, and B. L. O’Dell, J. Am. Chem. Soc. 69,
1476 (1947).
1. L. R. Stokstad, J. Biol. Chem. 149, 573 (1948).
2b. LR. Stokstad, B. L. Hutchings, and Y. SubbaRow, J. Am. Chem. Soc. 70, 3
(1948).
Il. CHEMISTRY 93
a. Existence of an Acid-Labile Chick Anti-Anemic Factor in Liver
In the isolation of vitamin B, (PGA) from horse liver, Pfiffner ef al.!®
described certain fractions whose activity for chicks and L. casei was in-
activated by acid conditions which had no effect on pure PGA. Concentrates
containing this acid-labile factor contained twice as much PGA activity
by S. faecalis as by L. casei assay. After acid treatment in 1.5% methanol-
hydrogen chloride, the L. casei activity decreased to 20 to 30% of its
original value and the activity as measured by the two organisms became
the same. A similar treatment of pure PGA produces insignificant losses in
activity. The zine salt of the acid-labile factor is more soluble than that of
PGA. There has been some speculation that this acid-labile form may be
the citrovorum factor or a related compound. The acid lability properties
of the citrovorum factor correspond roughly with those of the acid-labile
form. However, the acid lability of citrovorum factor is less than that of
the acid-labile form of PGA obtained by Pfiffner. Until further evidence is
available, no definite conclusions can be drawn regarding the identity of
these two compounds.
2. OF PGA CoNndgJUGATES
Soon after it became apparent that the growth factor for L. casei was also
involved in animal nutrition, evidence was obtained by Welch and Wright!’
which showed that milk possessed more activity for animals than could be
accounted for on the basis of its microbiological activity. Similarly Mallory
et al.* noted that yeast concentrates were more active in promoting growth
and preventing leucopenia in rats fed sulfasuxidine (succinylsulfathiazole)
than liver preparations containing up to fifteen times as much ‘‘Strepto-
coccus lactis R-stimulating factor.”” These workers also pointed out the
parallelism between the activity of liver and yeast preparations for the
sulfonamide-fed rat and for the vitamin M-deficient monkey, and the lack
of parallelism with the microbiological activity. They also suggested that
these yeast preparations contained potential ‘‘Streptococcus lactis R-stimu-
lating factors” which could be enzymatically converted to microbiologically
active compounds.
A conjugated form of PGA which is much more active for L. casei than
for S. faecalis was described by Hutchings ef al.!® This compound, which was
13 A.D. Welch and L. D. Wright, Science 100, 153 (1944).
14M. E. Mallory, V. Mims, J. R. Totter, and P. L. Day, J. Biol. Chem. 156, 317
(1944).
16 B. L. Hutchings, E. L. R. Stokstad, N. Bohonos, and N. H. Slobodkin, Scvence
99, 371 (1944).
94 PTEROYLGLUTAMIC ACID
later shown to be pteroyltriglutamic acid, was isolated by Hutchings et al.'®
from a cell-free filtrate obtained from the aerobic fermentation of a Coryne-
bacterium. The method involved adsorption and elution, esterification of
the barium salt to give the methyl ester, and extraction of the ester from an
aqueous solution with butanol. The methyl ester at this stage could be
dissolved in hot methanol and reprecipitated on cooling in the presence of
0.05 N sodium chloride. In the absence of an electrolyte a gel was formed
instead. By repeated reprecipitation of this ester from methanol in the
presence of an electrolyte, it was obtained in a microcrystalline form. The
free acid was obtained by hydrolysis of the ester and crystallization at pH
2.8 in the presence of electrolytes such as sodium or calcium chloride.
Although pteroylglutamic acid can be precipitated from water and
methanol in the absence of electrolytes, pteroyltriglutamic acid and its
ester require the presence of electrolytes for their precipitation. The solu-
bility of pteroyltriglutamic acid in the presence of calcium chloride at pH
2.8 is 3.0 mg. per milliliter at 80° and 0.10 mg. per milliliter at 5°.
Binkley et al.!”: '§ isolated in crystalline form a microbiologically inactive
conjugate of PGA (vitamin B, conjugate) from yeast. These workers found
that a yeast extract rich in anti-anemic activity for the chick was relatively
inactive for L. casei or S. faecalis. Digestion of this yeast concentrate with
a suitable enzyme from hog kidney increased the microbiological activity.
This conjugate which crystallized from 5% sodium chloride solution was
0.4 % as active as PGA for L. casez and 0.2 % as active for S. faecalis assay.
This conjugate was later found by Pfiffner et al.!® to contain seven glutamic
acid residues. Enzymes which split this conjugate have no effect on its
methyl ester, showing that the conjugases may be classified as carboxypep-
tidases. Although PGA exists in a microbiologically inactive form in liver,
no conjugates have yet been isolated from this source. A more detailed dis-
cussion of the various types of PGA precursors occurring in liver will be
given ina subsequent portion of this review.
a. Pteroylglutamic Acid—Protein Complex in Yeast
Specific and reversible combinations of proteins and vitamins have been
noted in many instances in which the vitamin forms the basic part of the
coenzyme. This has been demonstrated for thiamine, pyridoxal, riboflavin,
and nicotinamide. Attempts have been made to find similar combinations
16 B. L. Hutchings, E. L. R. Stokstad, N. Bohonos, N. H. Sloane, and Y. SubbaRow,
J. Am. Chem. Soc. 70, 1 (1948).
178. B. Binkley, O. D. Bird, E. S. Bloom, R. A. Brown, D. G. Calkins, C. J. Camp-
bell, A. D. Emmett, and J. J. Pfiffner, Science 100, 36 (1944).
8 J. J. Pfiffner, D. G. Calkins, B. L. O’Dell, E. 8S. Bloom, R. A. Brown, C. J. Camp-
bell, and O. D. Bird, Sczence 102, 228 (1945).
'9 J. J. Pfiffner, D. G. Calkins, E. 8. Bloom, and B. L. O’Dell, J. Am. Chem. Soc. 68,
1392 (1946).
Il. CHEMISTRY 95
between PGA and proteins. Allfrey and King?® found that during fractional
precipitation of a yeast autolyzate with ammonium sulfate there was a
correlation between the amounts of PGA conjugate and protein that were
precipitated. The PGA conjugate was measured microbiologically after
appropriate enzyme digestion. The linkage between PGA conjugate and
protein is essentially salt-like or highly dissociable. The bond is broken by
the addition of organic solvents such as ethanol, acetone, or dioxane to
protein fractions prepared by salting-out procedures. Heating for 1 minute
at 80° or for 5 minutes at 60° also released the PGA conjugate from the
protein. During dialysis of freshly prepared yeast autolyzates in viscous
casing the PGA conjugate was rapidly released from the protein. By the
use of fractional precipitation with ammonium sulfate between 2.4 WM and
3.1 M at varying pH values, protein preparations were obtained which
contained 160 y of PGA per gram in the form of the microbiologically
inactive conjugate. Pteroylheptaglutamic acid by itself in low concentra-
tions is not precipitated under these conditions.
Six crystalline proteins were obtained from this ammonium sulfate pre-
cipitate. Most contained little or no PGA activity, the highest having a
value of 75 y of PGA per gram.
3. DEGRADATION REACTIONS
a. Degradation of Pteroyltriglutamic Acid
The relationship between pteroyltriglutamic acid isolated from a fer-
mentation product and PGA isolated from liver was established by Stok-
stad et al.’ by the use of anaerobic alkaline hydrolysis. The triglutamic
acid derivative, which is active for L. casei but only slightly active for
S. faecalis R, was rapidly inactivated for both organisms by aerobic alkaline
hydrolysis. Anaerobic hydrolysis, however, produced only a slight decrease
in the activity for L. casei and greatly increased the activity for S. faecalis
R. On anaerobic alkaline hydrolysis the ratio of the activity for these two
organisms approached that of pteroylglutamic acid isolated from liver.
Two moles of a-amino acid nitrogen were liberated during anaerobic
alkaline hydrolysis, and the active compound which was formed was ap-
proximately half as active as pteroylglutamic acid by both L. casei and
S. faecalis R assay. This compound was later identified as racemic pteroyl-
glutamic acid by comparison with the synthetic material. The pteroyl-
glutamic acid had apparently been racemized by the anaerobic alkaline
hydrolysis.
Stokstad et al.2! found that aerobic alkaline hydrolysis of pteroyltriglu-
tamic acid or of racemic PGA resulted in the formation of a fluorescent pig-
20 V. G. Allfrey and C. G. King, J. Biol. Chem. 182, 367 (1950).
"1K. L. R. Stokstad, B. L. Hutchings, J. H. Mowat, J. H. Boothe, C. W. Waller,
R. B. Angier, J. Semb, and Y. SubbaRow, J. Am. Chem. Soc. 70, 5 (1948).
96 PTEROYLGLUTAMIC ACID
ment and a diazotizable aromatic amine which could be estimated by the
method of Bratton and Marshall.” In the absence of oxygen no diazotizable
amine or fluorescent pigment was produced by alkaline hydrolysis. The
fluorescent pigment proved to be a dibasic acid having pk, values of 3.9
and 7.7. Klementary analysis suggested the empirical formula C;H;N;03.
Decarboxylation of the fluorescent dibasic pigment at 300° resulted in the
liberation of approximately 1 mole of carbon dioxide and the formation of
a fluorescent monobasic acid with a pK, of 8.0. Oxidation of the original
dibasic acid with chlorine water, followed by hydrolysis with 0.1 N HCl at
140°, yielded a compound which gave positive color tests for guanidine. The
formation of guanidine under such conditions constitutes evidence for a
pyrimidine ring with an amino group in the 2 position. The fluorescent
dibasic acid showed characteristic absorption spectra in 0.1 N sodium hy-
droxide with maxima at 253 and 365 mu. The empirical formula C7H;N;Os,
the titration data, and formation of guanidine suggested a 2-aminopurine
or a 2-aminopteridine. The absorption spectra, however, eliminated the
possibility of a purine, because purines do not have absorption maxima
above 300 my. Thus the available evidence pointed toward a 2-aminopteri-
dine with an enolic and a carboxy group. With this evidence available,
attempts were made to synthesize pteridines having these functional
groups. The compound was identified as 2-amino-4-hydroxypteridine-6-
carboxylic acid by comparison with the synthetic compound, and the
monobasic fluorescent pigment produced by decarboxylation was identified
as 2-amino-4-hydroxypteridine.
The structure of these two pteridines was established by Mowat e¢ al.”
by the following series of reactions. Diethylmesoxalate was condensed with
2 ,4,5-triamino-6-hydroxypyrimidine (IIL) to yield isoxanthopterincarboxy-
lic acid (III). The structure of isoxanthopterincarboxylic acid (III) is shown
by formula IIT, although it had not previously been definitely established
whether the carboxyl group occupied the 6 or 7 position. On chlorimation
of isoxanthopterincarboxylic acid (III) and subsequent reduction with hy-
drogen iodide, one of the hydroxyl groups was removed to give compound
V, which was identical with the dibasic fluorescent pigment. Presumably
either the 4- or the 7-hydroxyl could have been removed by this proce-
dure. The presence of the 4-hydroxyl in compound V was shown in two
ways. First decarboxylation of 2-amino-4-hydroxypteridine-6-carboxylic
acid (V) gave 2-amino-4-hydroxypteridine (VI), the structure of which was
established by its synthesis from glyoxal and 2,4 ,5-triamino-6-hydroxypyr-
imidine (II). Its formation by this method demands a hydroxyl group in
22 A.C. Bratton and E. K. Marshall, Jr., J. Biol. Chem. 128, 537 (1939).
23 J. H. Mowat, J. H. Boothe, B. L. Hutchings, E. L. R. Stokstad, C. W. Waller,
R. B. Angier, J. Semb, D. B. Cosulich, and Y. SubbaRow, J. Am. Chem. Soc. 70,
14 (1948).
II. CHEMISTRY 97
the 4 position. Second, the synthesis of 2-amino-4-hydroxypteridine-6-car-
boxylie acid (V) was accomplished by condensation of 2,4 ,5-triamino-6-
hydroxypyrimidine (IJ) and ethyl-8 ,8-diethoxy-a-bromopropionate. This
reaction also established the presence of a hydroxyl group in the 4 position
of the pteridine.
Unequivocal proof establishing the 6 position of the carboxyl group in
compound V was obtained by degrading the corresponding methyl deriva-
COOC;H;
7 — Ks T. N. PCls; T. Tv.
H,N | NS_ NH, : © N coos = HO 7: | Ny _NH; POC Cl a NS-NH;
H.N - N HOOC 2 N HOOC SN ZN
OH OH OH
II C:H,O. ul Ill IV
-€- COOC2H;
CHO CuH,0
|
CHO HI
e NN SNH, 275" 275° ( ye ee KMnOQ, i) eee
AS eel H.C
OH NaOH,
VI ao
|ssou
noe H,.SO;
fst
Jel det =(0) H Y: E ‘ E
eal al | ie SC NH: Tone
Hooc—C—N-C-{_)-N—CH, ae H.C,
ra OH VIII
HOOC—CH,
Pteroylglutamic acid
Fic. 1. Chemical reactions leading to the establishment of the structural formula
of the pteridine (V) obtained by aerobic alkaline hydrolysis of pteroylglutamic
acid.
tive, 2-amino-4-hydroxy-6-methylpteridine (VII) to give a compound iden-
tical with 2-amino-5-methylpyrazine (VIII). The corresponding 7-methyl-
pteridine would have yielded 2-amino-6-methylpyrazine instead. Oxidation
of 2-amino-4-hydroxy-6-methylpteridine (VII) by alkaline permanganate
gave the corresponding 2-amino-4-hydroxypteridine-6-carboxylic acid (V).
These reactions are outlined in Fig. 1.
Hydrolysis with 0.5 N sulfurous acid at room temperature was shown by
Hutchings et al.24 to rapidly inactivate pteroyltriglutamic acid and give an
4B. L. Hutchings, E. L. R. Stokstad, J. H. Mowat, J. H. Boothe, C. W. Waller,
R. B. Angier, J. Semb, and Y. SubbaRow, J. Am. Chem. Soc. 70, 10 (1948).
98 PTEROYLGLUTAMIC ACID
aromatic amine and a fluorescent pigment. This pigment reacted rapidly
with typical aldehyde reagents, indicating the presence of an aldehyde
group. This fluorescent pigment did not possess a carboxyl group, as evi-
denced by the fact that its distribution coefficient between water and
butanol was the same at pH 3.0 as at pH 7.0. When the pigment obtained
by sulfurous acid hydrolysis was treated anaerobically with dilute sodium
hydroxide, approximately equal amounts of 2-amino-4-hydroxypteridine
6-carboxylic acid (V) and 2-amino-4-hydroxy-6-methylpteridine (VII) were
formed. The formation of approximately equal molal quantities of carboxy
and methyl derivatives from what is apparently an aldehyde probably
involves a Cannizzaro type of reaction, although the mechanism of this
reaction 1s obscure.
Prolonged aqueous hydrolysis of pteroyltriglutamic acid at pH 4 yielded
1-pyrrolidonecarboxylic acid, which on hydrolysis with alkali yielded L(+)-
glutamic acid.”4
The aromatic amine which was produced during sulfurous acid hydrolysis
was isolated as the barium salt. This compound when diazotized and
coupled with N-(1-naphthyl)ethylenediamine dihydrochloride yielded a red
pigment,” which indicated a primary aromatic amine with a highly negative
substituent group. The aromatic amine nitrogen as measured by the method
of Bratton and Marshall” constituted approximately 25% of the total
nitrogen. The remaining 75% of the nitrogen could be converted into
a-amino acid nitrogen by alkaline hydrolysis. From such hydrolyzates the
aromatic amine was isolated and identified as p-aminobenzoic acid. Micro-
biological assay of the hydrolyzate indicated the presence of 3 moles of
glutamic acid. The peptide linkage of the glutamic acid to p-aminobenzoic
acid must involve the carboxyl group of the latter as a primary aromatic
amine is required for reaction in the Bratton and Marshall test.”
The diazotizable aromatic amine obtained by aerobic alkaline hydrolysis
of racemic pteroylglutamic acid was found to contain 2.1 atoms of nitrogen
for each atom of aromatic amino nitrogen. On hydrolysis with 2 N sulfuric
acid it yielded p-aminobenzoic acid and 45 % of the total nitrogen appeared
as a-amino acid nitrogen. This was later identified as glutamic acid, and
thus the diazotizable amine was identified as p-aminobenzoylglutamic acid.
Evidence regarding the mode of linkage is furnished by the results of
alkaline hydrolysis.?! The absence of fluorescence and of the free aromatic
amine in the original pteroylglutamic acid, and the simultaneous appearance
of these two during aerobic alkaline hydrolysis, suggested that the pteridine
is linked to the aromatic amine nitrogen. As hydrolysis proceeded, the
liberation. of pteridine and aromatic amine appeared at approximately the
same rate.
Xeduction in acid solution, either catalytically or with zine dust, yielded
Il. CHEMISTRY 99
the aromatic amine and a reduced pteridine.* After reoxidation with
manganese dioxide, the pteridine obtained by zine reduction was identified
as 2-amino-4-hydroxy-6-methylpteridine.
The foregoing evidence indicated the following conclusions regarding the
structure of pteroylglutamic acid.
1. Aerobic alkaline hydrolysis, sulfurous acid cleavage, and chemical or
catalytic reduction each yielded a pteridine and a primary aromatic amine.
This indicated the linkage of the pteridine to the nitrogen of the aromatic
amine.
2. The aromatic amine formed during sulfurous acid cleavage of pteroyl-
triglutamic acid was a tetrapeptide, p-aminobenzoyldiglutamylglutamic
acid. The aromatic amine from pteroylglutamic acid was p-aminobenzoy]-
glutamic acid.
3. There was a single-carbon atom linkage between the pteridine and the
aromatic amine. This was indicated by the fact that only pteridines with a
single-carbon atom side chain were obtained and that no other 2-carbon
fragments could be detected in the two degradation reaction products.
The evidence also indicated that this single-carbon atom is present in a
methylene link. If this carbon atom were present in an amide linkage, the
cleavage would be hydrolytic and would not require oxygen. The formation
of 2-amino-4-hydroxy-6-methylpteridine by reduction also constitutes evi-
dence for the methylene linkage.
b. Degradation of Vitamin B, (Pteroylglutamic Acid)
Wittle et al.2® described the oxidative degradation of vitamin B,. (pteroyl-
glutamic acid) which had been isolated from liver. Oxidation with alkaline
permanganate or chloric acid gave a fluorescing pigment with a charac-
teristic absorption spectrum which was identified as 2-amino-4-hydroxy-6-
carboxypteridine. The chloric acid oxidation mixture also yielded an ethy]
acetate-soluble crystalline material which proved to be 3,5-dichloro-4-
aminobenzoylglutamic acid. This compound decomposed at a temperature
slightly above the melting point to yield two lower molecular weight frag-
ments which were identified as 3 ,5-dichloro-4-aminobenzoie acid and d/-pyr-
rolidonecarboxylie acid.
B. PHYSICAL AND CHEMICAL PROPERTIES
Pteroylglutamic acid when crystallized from water forms yellow, spear-
shaped leaflets (Fig. 2). On heating it darkens, and it chars at around 250°
without melting (Pfiffner et al.!°). Its solubility in water as the free acid is
10 mg. per liter at 0° and over 500 mg. per liter at 100°. The disodium salt
25 E. L. Wittle, B. L. O’Dell, J. M. Vandenbelt, and J. J. Pfiffner, J. Am. Chem. Soc.
69, 1786 (1947).
100 PTEROYLGLUTAMIC ACID
has a solubility of over 15 g. per liter. It forms highly insoluble salts with
zinc, lead, and silver (Pfiffner et al.'°). The alkali metal salts are soluble in
water but can be precipitated by the addition of alcohol. Pteroylglutamic
Fra. 2. Crystals of pteroylglutamic acid.
A ] yig
acid is practically insoluble in most organic solvents but is slightly soluble
in acetic acid (Pfiffner et al.?°).
The optical rotation Ja},’ is +16° in 0.1 N sodium hydroxide solution at
a concentration of 7.6 g. per liter (Weygand et al.*®).
Pteroylglutamic acid dried at moderate temperatures and at atmospheric
26 FP. Weygand, A. Wacker, and V. Schmied-Kowarzik, Chem. Ber. 82, 25 (1949).
Il. CHEMISTRY 10]
pressure contains an amount of water corresponding roughly to a dihydrate.
Samples dried at 145° in high vacuum lose this water of crystallization and
become hygroscopic.
Pteroylglutamic acid has a characteristic ultraviolet absorption spectrum
(Waller et al.2*) which is markedly influenced by pH (Fig. 3). In 0.1 N
sodium hydroxide it exhibits maxima at 256, 282, and 365 mu with 2}?
values of 585, 570, and 206. The peaks at 256 and 365 mu are due to the
pteridine portion of the molecule: the peak at 282 is due to the p-amino-
benzoic acid moiety. The glutamic acid exerts very little effect on the ultra-
600
1.0%
1.0cm.
E
400
220 420
Fig. 3. Ultraviolet absorption spectra: (1) synthetic PGA in 0.1 N sodium hy-
droxide; (2) synthetic PGA in 0.1 N hydrochloric acid; (8) natural liver PGA in 0.1
N sodium hydroxide; (4) natural liver PGA in 0.1 N hydrochloric acid; (5) pteroic
acid in 0.1 N sodium hydroxide. Courtesy J. Am. Chem. Soc. (Waller ef al.?").
violet absorption spectrum. The absorption spectrum of pteroic acid (Fig.
3) is slightly different from that of PGA. However, addition of more than
one glutamic acid does not change the position of the absorption peaks, and
the extinction coefficients of PGA conjugates are in inverse proportion to
their molecular weights.
1. HypDROGENATION
Pteroylglutamic acid and related pterins are readily hydrogenated and
can be reoxidized by atmospheric oxygen. O’ Dell et al.?8 found that pteroyl-
27C. W. Waller, B. L. Hutchings, J. H. Mowat, E. L. R. Stokstad, J. H. Boothe,
R. B. Angier, J. Semb, Y. SubbaRow, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith,
Jr., J. Am. Chem. Soc. 70, 19 (1948).
102 PTEROYLGLUTAMIC ACID
glutamic acid in alkaline solution with platinum oxide as a catalyst takes
up 1 mole of hydrogen to form a colorless dihydro derivative with an ab-
sorption maxima at 284 my in alkaline solution. In glacial acetic acid over
platinum 2 moles of hydrogen are taken up to form the tetrahydro deriva-
tive which has an absorption spectrum similar to that of the dihydro form.
Alkaline solutions of the dihydro derivative readily absorb oxygen to yield
PGA, whereas in acid solution reoxidation is less rapid. The tetrahydro
derivative is reoxidized to PGA by oxygen over platinum in glacial acetic
240 280 320 360 400
A, Mu
Fig. 4. Ultraviolet absorption spectra: — PGA at pH 11.0; — — — di-
hydro PGA at pH 11.0; —-— dihydro PGA at pH 3.1; —- - - - - tetrahydro PGA at
DEL MIM AO Par ante ene oxidized dihydro PGA at pH 11.0. Courtesy J. Am. Chem. Soc.
(O'Dell et al 28).
acid. The ultraviolet absorption spectra of PGA, dihydro and tetrahydro
PGA, and reoxidized dihydro PGA are shown in Fig. 4+ (O’ Dell et al.’).
Reduction of 2-amino-4-hydroxypteridine-6-carboxylic acid in alkaline
solution with hydrogen over platinum oxide yields the tetrahydro form.
The reduced solutions rapidly absorb oxygen from the air to yield the
parent compound. It is interesting to note that the 365-my absorption
band of pteroylglutamic acid disappears during formation of the di- or
tetrahydro derivatives, whereas the same band is shifted to longer wave-
lengths in the tetrahydro derivatives of 2-amino-4-hydroxypteridine-6-car-
boxylie acid and 2-amino-4-hydroxy-7-methylpteridine (Fig. 5, O’Dell et
fallen)
22 B. L. O’Dell, J. M. Vandenhelt, E.S. Bloom, and J. J. Pfiffner, J. Am. Chem. Soc.
69, 250 (1947).
Il. CHEMISTRY 103
Hydrogenation of pteroyltriglutamic acid with a palladium catalyst at
pH 1 results in rapid biological inactivation and liberation of p-amino-
benzoyltriglutamic acid (Hutchings et al.**). Reduction of PGA with zinc
in acid solution also cleaves the bond between the nitrogen of the p-amino-
benzoic acid and the methylene carbon attached to the pteridine ring. Thus
the reduced form of PGA is unstable in acid solution and rapidly splits to
give the free aromatic amine.
240 280 320 360 400
x, My
Fie. 5. Ultraviolet absorption spectra of pterins: ————— 2-amino-4-hydroxy-6-
carboxypteridine at pH 11.0; — — — tetrahydro-2-amino-4-hydroxy-6-carboxy-
pteridine at pH 11.0; —-— tetrahydro-2-amino-4-hydroxy-6-carboxypteridine at pH
3.0;----- 2-amino-4-hydroxy-6-carboxy-7-methyltetrahydropteridine at pH 11.
Courtesy J. Am. Chem. Soc. (O’Dell e¢ al.?8).
2. OTHER REACTIONS
Cosulich and Smith?® observed that treatment of PGA with cold nitrous
acid yields N!°-nitrosopteroylglutamic acid with biological activity approxi-
mately equal to that of PGA for the chick and for S. faecalis R. It will be
recalled that nitrous acid has also been used to remove the 2-amino group
of N'°-formylpteroic acid (rhizopterin) (Wolf ef al.*°). Heating PGA with
formic acid and acetic anhydride gives N!°-formylpteroylglutamic acid
(Gordon et al.*!). Stokstad et al. found that irradiation of PGA with sun-
29D. B. Cosulich and J. M. Smith, Jr., J. Am. Chem. Soc. 71, 3574 (1949).
30D). E. Wolf, R. C. Anderson, E. A. Kaczka, 8. A. Harris, G. E. Arth, P. L. South-
wick, R. Mozingo, and K. Folkers, J. Am. Chem. Soc. 69, 2753 (1947).
31M. Gordon, J. M. Ravel, R. E. Eakin, and W. Shive, J. Am. Chem. Soc. 70, 878
(1948).
2 EB. L. R. Stokstad, D. Fordham, and A. deGrunigen, J. Biol. Chem. 167, 877 (1947).
104 PTEROYLGLUTAMIC ACID
light rapidly destroyed the biological activity. The liberation of p-amino-
benzoylglutamic acid paralleled that of biological inactivation which showed
that the first step in the reaction consists in a rupture in the bond between
the C°-methylene group and the N!° atom of aromatic amino group.
C. CONSTITUTION
Boa : ( Ny NE,
oe —NH—CH—\ . A
a OH
i
HOOC
Pteroylglutamic acid: N-[4-{[(2-amino-4-hydroxy-6-pteridyl)methyl]amino } -
benzoyl]glutamic acid
D. SYNTHESIS
The final proof of the structure of this compound was obtained by syn-
thesis of pteroylglutamic acid by different methods. The method of Waller
et al.”” utilizes the simultaneous condensation of 2,4 ,5-triamino-6-hydroxy-
pyrimidine (II), p-aminobenzoylglutamic acid (X), and a,8-dibromopro- —
pionaldehyde in aqueous solution. Pteroic acid was obtained by the cor-
responding reaction, except that p-aminobenzoic acid was used instead of
p-aminobenzoylglutamic acid. The procedure of Hultquist e¢ al.** involved
the reaction of a ,8-dibromopropionaldehyde with pyridine, 2 ,4 ,5-triamino-
6-hydroxypyrimidine (II), and potassium iodide to yield N-[(2-amino-4-
hydroxy-6-pteridyl)methyl] pyridinium iodide (XI). This was then treated
with p-aminobenzoylglutamic acid (X) in ethylene glycol to yield pteroyl-
glutamic acid. The position of the methylpyridinium group on the 6 posi-
tion of the pteridine was established by oxidation with alkaline perman-
ganate to yield 2-amino-4-hydroxypteridine-6-carboxylic acid (V). These
two methods of synthesis are schematically outlined in Fig. 6.
Angier et al.*4 condensed diethyl N-(p-aminobenzoyl)glutamate with re-
33 M. E. Hultquist, E. Kuh, D. B. Cosulich, M. J. Fahrenbach, HE. H. Northey, D. R.
Seeger, J. P. Sickels, J. M. Smith, Jr., R. B. Angier, J. H. Boothe, B. L. Hutchings,
J. H. Mowat, J. Semb, E. L. R. Stokstad, Y. SubbaRow, and C. W. Waller, J.
Am. Chem. Soc. 70, 23 (1948).
34. B. Angier, KE. L. R. Stokstad, J. H. Mowat, B. L. Hutchings, J. H. Boothe, C. W.
Waller, J. Semb, Y. SubbaRow, D. B. Cosulich, M. J. Fahrenbach, M. E. Hult-
quist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith, Jr.,
J. Am. Chem. Soc. 70, 25 (1948).
Il. CHEMISTRY 105
ductone (2,3-dihydroxyacrylaldehyde) to give diethyl N-[p-(2,3-dihy-
droxy-2-ene-propylideneamino)benzoyl| glutamate. This was then con-
densed with 2,4 ,5-triamino-6-hydroxypyrimidine (II) in hot ethylene glycol
to give pteroylglutamic acid diethyl ester in 5 to 20% yield. A number of
derivatives of pteroic acid, including the ethyl ester, amide, and pteroyl-
glycine, were prepared by this procedure.
A fourth method, described by Boothe et al.,*° consists in attaching a pre-
‘em CHO ‘3
wooc—c—n-C-{_\—nu, + Br-CH,—-G—Br + BN "NH:
| | H.N ZA
CH, H
| OH
HOOC—CH,
x Il
p-Aminobenzoylglutamic acid
no O
pooo-b tL) kul
CH, OH
HOOC—CH,
Pteroylglutamic acid X in ethylene glycol
Br-
CHO a
== ines | H.N | Ny Diag ahd a ou
NY ie oh a =F HN ew 2 Sv
H
OH
JL Tol
Fria. 6. Two syntheses of pteroylglutamic acid.
formed pteridine to a derivative of p-aminobenzoic acid. 2-Amino-4-hydrox-
y-6-methyl-pteridine (VII) was prepared by the reduction of 2-amino-4-hy-
droxy-6-pteridylmethylpyridinium iodide (XI) with zine in alkaline solution
and subsequent oxidation by iodine of the resulting dihydro-2-amino-4-hy-
droxy-6-methylpteridine. This was brominated at 150°, and the resulting
crude 2-amino-4-hydroxy-6-bromomethylpteridine was condensed with p-
aminobenzoylglutamic acid diethyl ester in ethylene glycol at 100°.
35 J. H. Boothe, C. W. Waller, E. L. R. Stokstad, B. L. Hutchings, J. H. Mowat,
R. B. Angier, J. Semb, Y. SubbaRow, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith,
Jr., J. Am. Chem. Soc. 70, 27 (1948).
106 PTEROYLGLUTAMIC ACID
Numerous synthetic methods have subsequently been developed, the
large number of which is reflected in the many publications and patents in
this field. These procedures may be divided into two main categories: first,
those in which 2,4,5-triamino-6-hydroxypyrimidine (II) is condensed
simultaneously with a 3-carbon intermediate and p-aminobenzoylglutamic
acid; second, those in which a substituted pterin is first formed and then
coupled with an aromatic amine to give PGA.
1. SIMULTANEOUS CONDENSATION OF TRIAMINOPYRIMIDINE, 3-CARBON
INTERMEDIATE, AND AROMATIC AMINE
The first method of synthesis, that of Waller et al.?7 in which 2 ,3-dibromo-
propionaldehyde is used as the 3-carbon fragment, has already been de-
scribed. Examples of other 3-carbon systems which have been used are
given below. (X in the formula denotes chlorine or bromine. Reference
numbers are given in parentheses. )
O H
|
CH; xX——C—— CHO} G6) O=C—C=C—COOH (37)
Xx ex
(followed by deearboxyl-
ation)
O
H OH |
HO—C=C—CHO (34, 38-40) X— CH.—C—_ Che (41-43)
X—CH:;—CX:—CHO (44) C1CH,—C—-CHO (45)
NOH
36 J. H. Mowat (to American Cyanamid Co.), U. S. Pat. 2,436,073 (Feb. 17, 1948).
37 C. W. Waller and J. H. Boothe (to American Cyanamid Co.), U.S. Pat. 2,442,867
(June 8, 1948).
33 R. B. Angier (to American Cyanamid Co.), U. 8S. Pat. 2,442,836 (June 8, 1948).
39 R. B. Angier (to American Cyanamid Co.), U. 8S. Pat. 2,442,837 (June 8, 1948).
40 R. B. Angier (to American Cyanamid Co.), U. 8S. Pat. 2,466,670 (Apr. 12, 1949).
41, Weygand and V. Schmied-Kowarzik, Chem. Ber. 82, 333 (1949).
“MM. EK. Hultquist and P. F. Dreisbach (to American Cyanamid Co.), U. 8. Pat.
2,443,165 (June 8, 1948).
438. Uyeo, S. Mizukami, T. Kubota, and S. Takagi, J. Am. Chem. Soc. 72, 5339
(1950).
44 J. H. Boothe (to American Cyanamid Co.), U. 8S. Pat. 2,444,002 (June 22, 1948).
49 T). B. Cosulich (to American Cyanamid Co.), U. 8. Pat. 2,444,005 (June 22, 1948).
Il. CHEMISTRY 107
O
nN
X,C—CH——CH:2 (46) al a (47)
x
O O
H
xX—(or H)—C—C—CH—OC—CH; (48) CH.—=C—C—H. (49)
|
| |
Xe Oe Eon) oe
H
CH.—CH—CHO + Nal (50, 51) C.H;0—CH.CH—C=0O (52)
|
Oo O x
mh
tosyl tosyl
Cl—CH.:—_C—CH.Cl (53)
O
2. CONDENSATION OF PREFORMED PTERIN WITH AROMATIC AMINE
These methods involve formation of a 2-amino-4-hydroxy-6-methyl-
pteridine with a suitable substituent in the 6-methyl group for reaction with
p-aminobenzoic acid. Examples of these are (R = 2-amino-4-hydroxy-6-
substituted pteridine) :
R—CH.Cl + p-aminobenzoylglutamic acid (PABG)**: *°
R—CHO + PABG in formic acid, followed by hydrolysis**®: 7
46M. E. Hultquist and P. F. Dreisbach (to American Cyanamid Co.), U.S. Pat.
2,472,482 (June 7, 1949).
47 J. H. Boothe (to American Cyanamid Co.), U.S. Pat. 2,472,462 (June 7, 1949).
48M. E. Hultquist and D. R. Seeger (to American Cyanamid Co.), U.S. Pat. 2,472,481
(June 7, 1949).
429. Doub and L. Bambas (to Parke, Davis & Co.), U. 8S. Pat. 2,476,360 (July 19,
1949).
50 P. Karrer and R. Schwyzer, Helv. Chim. Acta 31, 777 (1948).
51 P. Karrer (to Hoffman-La Roche, Inc.), U. S. Pat. 2,520,882 (August 29, 1950).
8 J. Geraci (to American Cyanamid Co.), U.S. Pat. 2,501,168 (March 21, 1950).
88 —. I. Weisblat and A. R. Hanze (to The Upjohn Company), U.S. Pat. 2,560,616
(July 17, 1951).
54 Roche Products, Ltd. Brit. Pat. 624,394 (June 7, 1949).
55 J. H. Boothe (to American Cyanamid Co.), U. 8. Pat. 2,547,519 (Apr. 3, 1951).
56 Roche Products, Ltd., Brit. Pat. 631,516 (Nov. 3, 1949).
57 H. Lindlar and H. Klaeui (to Hoffmann-La Roche, Inc.), U. 8. Pat. 2,520,156 (Aug.
29, 1950).
108 PTEROYLGLUTAMIC ACID
Dihydro R—CHO + PGA in formic acid, followed by hydrolysis*®
R—CHO + PABG with catalytic hydrogenation?®: 59
R—CH,OH + p-nitrobenzoylglutamic acid with catalytic hydrogen-
ation®®
R—CH.OH + p-aminobenzoylglutamic acid®
R—CH; brominated + p-aminobenzoylglutamic acid*
=
R—CH2—N » + p-aminobenzoylglutamie acid*: @
] fa
R—CH—COOH 4+ p-aminobenzoylglutamic acid
Br
3. SYNTHESIS OF PTEROYLPOLYGLUTAMIC ACID DERIVATIVES
The isolation of pteroyltriglutamic acid from a fermentation product and
the existence of pteroylheptaglutamic acid in yeast made the synthesis of
various polyglutamic acid derivatives of pteroic acid a problem of interest.
The findings of Lewisohn et al. that pteroyltriglutamic acid from a fer-
mentation product caused spontaneous regression of mammary tumors in
mice also attached a special interest to the synthesis of this type of com-
pound. Pteroyl-e-glutamylglutamic acid was prepared by Mowat et al.®
and found to be 0.5 % as active as pteroylglutamic acid by S. faecalis assay
and 0.8% by L. casez, and fully active for chicks. Pteroyl-a ,y-glutamyldi-
glutamic acid was 0.14% active by S. faecalis and 0.17% by L. caset.
Pteroyl-y-glutamylglutamic acid (Boothe et al.**) is 71% active by S.
faecalis and 63 % by L. casei assay. In both cases pteroylglutamic acid was
used as a standard. Pteroyl-y-glutamyl-y-glutamylglutamie acid had an
activity of 2.5% by S. faecalis and 71% by L. casei. These activities are
very similar to those observed for pteroyltriglutamic acid isolated from a
fermentation product (Hutchings ef al.!).
588. Weygand, V. Schmied-Kowarzik, A. Wacker, and W. Rupp, Chem. Ber. 88, 460
(1950).
59 Roche Products, Ltd., Brit. Pat. 628,305 (March 15, 1948).
60 H. Spiegelberg (to Hoffmann-LaRoche, Inc.), U. 8S. Pat. 2,487,393 (Nov. 8, 1949).
61 J. Semb (to American Cyanamid Co.), U.S. Pat. 2,491,285 (Dec. 13, 1949).
6&2 M.E. Hultquist (to American Cyanamid Co.), U.S. Pat. 2,473,796 (June 21, 1949).
63 R. Tschesche, F. Korte, and R. Petersen, Chem. Ber. 84, 579 (1951).
64 R. Lewisohn, C. Leuchtenberger, R. Leuchtenberger, and J. C. Keresztesy, Science
104, 436 (1946).
65 J. H. Mowat, B. L. Hutchings, R. B. Angier, E. L. R. Stokstad, J. H. Boothe,
C. W. Waller, J. Semb, and Y. SubbaRow, J. Am. Chem. Soc. 70, 1096 (1948).
66 J. H. Boothe, J. H. Mowat, B. L. Hutchings, R. B. Angier, C. W. Waller, H. L. R.
Stokstad, J. Semb, A. L. Gazzola, and Y. SubbaRow, J. Am. Chem. Soc. 70, 1099
(1948).
Il. CHEMISTRY 109
E. SPECIFICITY
1. CuemistRY OF RHIZOPTERIN (FORMYL PrERoIc AcrID)
Keresztesy et al.®» ® reported the isolation of a compound which was
highly active for S. faecalis but relatively inactive for L. casez. This ratio of
activity for these two organisms is in contrast to that of pteroyltriglutamic
acid which is more active for L. casei than for S. faecalis. Rhizopterin was
obtained from a charcoal adsorbate derived from a fumaric acid fermenta-
tion product of Rhizopus nigricans. It was isolated by adsorption on Norit
and fuller’s earth and by chromatographic adsorption on alumina.
Wolf et al.*° described a novel method for recrystallizing rhizopterin by
forming a double salt with luteoethylenediamine cobaltic chloride. This
double salt was recrystallized from hot water and reconverted back to
rhizopterin by treatment with acetic acid.
Alkaline treatment of rhizopterin was shown by Rickes et al.®* to decrease
its activity for S. faecalis, to have no effect on its L. casei activity, and to
change the adsorption spectrum. This alkaline degradation product was
shown by Wolf e¢ al.*° to be pteroic acid. Acid hydrolysis of rhizopterin
yielded formic acid. The position of the formyl group on the N?° position
of the p-aminobenzoic acid was established by the following reactions.
Benzoylrhizopterin was prepared which yielded benzoylguanidine on oxida-
tion with potassium chlorate and hydrochloric acid. This showed that the
2-amino group of rhizopterin was free and that benzoylation occurred on
this nitrogen atom. Oxidation of rhizopterin with potassium chlorate and
hydrochloric acid yielded tetrachloro-p-benzoquinone, an oxidation product
of p-aminobenzoic acid and oxaloguanidine which in turn yielded guanidine
on acid hydrolysis. p-Aminobenzoic acid was obtained by sublimation of
the alkaline hydrolysis product (pteroic acid) at 220 to 360° at very low
pressure and by acid or alkaline hydrolysis. A number of different acyl
derivatives were prepared by heating rhizopterin with the corresponding
acid anhydrides. These acyl groups were attached to the 2-amino group.
The 2-amino group could be removed by nitrous acid to give desimino-
rhizopterin.
Rhizopterin was synthesized by treatment of synthetic pteroic acid, ob-
tained by the method of Waller,” with 98 % formic acid on a steam bath
87 J. C. Keresztesy, E. L. Rickes, and J. L. Stokes, Science 97, 465 (1943).
6 E. L. Rickes, L. Chaiet, and J. C. Keresztesy, J. Am. Chem. Soc. 69, 2749 (1947).
69 E. L. Rickes, N. R. Trenner, J. B. Conn, and J. C. Keresztesy, J. Am. Chem. Soc.
69, 2751 (1947).
110 PTEROYLGLUTAMIC ACID
2. CHEMISTRY OF CrITROVORUM FAcToR (LEucOvoRIN, Fouinic ActIp)
a. Biochemical Relations between Citrovorum Factor and PGA
In 1948 Sauberlich and Baumann’? noted that the organism Leuconostoc
citrovorum failed to grow in a chemically defined medium containing all the
known amino acids and vitamins, but the addition of small amounts of
natural materials such as liver extract or yeast to the medium promoted
luxuriant growth. The substance(s) in these natural materials necessary for
growth of Le. citrovorum was termed the citrovorum factor (CF). It was
also noted by these workers that the organism responded to thymidine or
to massive doses of PGA, but because the response to these substances was
delayed and submaximum, it appeared that these substances could not be
the citrovorum factor. It soon became apparent, however, that a close
relationship existed between PGA and CF. Thus Sauberlich” noted that
rats on a PGA-deficient diet excreted very little CF in the urine, but the
administration of PGA resulted in large increases in the CF content of the
urine. Subsequently it was noted by Sauberlich” and confirmed by Broquist
et al.” that concentrates of CF can competitively overcome the toxicity of
aminopterin (4-aminopteroylglutamic acid), an inhibitory analog of PGA,
for Le. citrovorum. These latter workers also showed that CF had approxi-
mately the same distribution coefficient as PGA between butanol and water
at pH 2. It appeared then that PGA might be a biological precursor of CF
and that a close chemical relationship existed between the two substances.
In a seemingly unrelated series of investigations, Bond et al.™ provided
evidence from microbiological studies for the existence of an unrecognized
form of PGA in natural materials. These workers studied the toxic effect
of ‘x methyl PGA” for growth of L. casez and observed that liver extract
was approximately fifteen times more active than PGA in counteracting
the toxicity of ‘““x methyl PGA” than could be accounted for on the basis of
its PGA content when assayed in the absence of inhibitor. With the use of
this inhibition assay highly refined concentrates of the factor from liver,
termed folinic acid by the Texas group, were prepared and found to be
highly active for Leuconostoc citrovorum. It thus seems very likely that
folinic acid and CF are identical.
Gordon et al.*! had previously reported the preparation of 10-formyl-
pteroylglutamie acid and observed that it was more active than PGA in
counteracting the inhibitory action of “x methyl PGA” for S. faecalis R,
70 H. EK. Sauberlich and C. A. Baumann, J. Biol. Chem. 176, 165 (1948).
7H. KE. Sauberlich, J. Biol. Chem. 181, 467 (1949).
72 A. EK. Sauberlich, Arch. Biochem. 24, 224 (1949).
73H. P. Broquist, E L. R. Stokstad, and T. H. Jukes, J. Biol. Chem. 185, 399 (1950).
™4T. J. Bond, T. J. Bardos, M. Sibley, and W. Shive, J. Am. Chem. Soc. 71, 3852
(1949).
Il. CHEMISTRY ila lah
This observation suggested that CF might be a formyl derivative of PGA in
view of the similar behavior of CF in counteracting PGA antagonists. This
supposition was confirmed by the finding that catalytic hydrogenation of
formyl PGA produced a mixture of compounds with high growth-promoting
activity for CF.75-”
b. Synthesis and Purification
The synthesis of leucovorin (XII) appears to consist in two essential
steps (cf. Fig. 7):
1. Reduction of either PGA (1) or 10-formyl PGA (XV) in 87 to 100%
formic acid at 0° to 30° over a platinum catalyst, whereby 2 moles of hy-
drogen are absorbed to form 10-formyl]-5 6,7 ,8-tetrahydro PGA (XIV).
2. Rearrangement of the neutralized reduction mixture under anaerobic
conditions by long standing at room temperature or by heating the alkaline
solution at pH 10 to 12. This treatment results in the rearrangement of
10-formy1]-5 ,6,7 ,8-tetrahydro PGA to leucovorin in a 40 to 50% over-all
yield from either PGA or 10-formyl PGA. During this process the formyl]
group shifts from the N!° to the N® position of the molecule. The leucovorin
is isolated by chromatographic adsorption and purification of the neutral
calcium or barium salt. The free acid of leucovorin is crystallized as the
free acid after acidification of the calcium salt to pH 3.5.
c. Stage of Reduction and Location of Hydrogen Atoms
Elemental analyses of crystalline leucovorin (XII) are in agreement with
the formula Co9H»2;N;O,7, and the presence of one formyl group has been
demonstrated. Thus from this analytical data and the method of prepara-
tion described above, the conclusion seemed justified that leucovorin is a
tetrahydroformyl PGA.
Allen and coworkers® studied the stage of reduction of pteridines by
means of the potential at the dropping mercury electrode in the polaro-
graph. They demonstrated that it is possible to distinguish between PGA,
10-formyl PGA, and dihydro and tetrahydro PGA by means of the potential
at pH 9.0. Thus, with PGA (and pteridines in general) the polarograph
gives three inflection points in the curve corresponding to the tetrahydro,
dihydro, and aromatic forms; the quantity of a particular reduction state
75 W. Shive, T. J. Bardos, T. J. Bond, and L. L. Rogers, J. Am. Chem. Soc. 72, 2817
(1950).
76 J. A. Brockman, Jr., B. Roth, H. P. Broquist, M. E. Hultquist, J. M. Smith, Jr.,
M. J. Fahrenbach, D. B. Cosulich, R. P. Parker, E. L. R. Stokstad, and T. H.
Jukes, J. Am. Chem. Soc. 72, 4325 (1950).
7K. H. Flynn, T. J. Bond, T. J. Bardos, and W. Shive, J. Am. Chem. Soc. 78, 1979
(1951).
73 W. Allen, R. L. Pasternak, and W. Seaman, J. Am. Chem. Soc. 74, 3264 (1952).
12 PTEROYLGLUTAMIC ACID
1g as Ga = sa NSN
R—NH-—CH,— 13N : CH
R—N
RC= O
I XV
(PGA) (10-Formyl PGA)
=e Bi ener _HCOOH , ‘NH
—NH—CH, Tones Sth ae:
Nn ve
XVI XIV
(5,6,7,8-Tetrahydro PGA) (10-Formy]-5,6,7,8-tetrahydro PGA)
“a Hoot F
HH :
H—+N~ Nx _nu, “EME
Pe |
R Thee N ZN H, al
HCOOH H | \ stcbnly
He—@
He=or =
XVII XIII
(5,10-Diformy]-5,6,7,8-
tetrahydro PGA)
HCOOH
i Ac,O
ene OH- “aca || eos coat
Ro nutes Spe XIX en coo
(Anhydro-
leucovorin-B)
ee on”
QUE pH 4
(Synthetic citrovorum factor, OH- aoe
leucovorin, folinic acid-SF) H
H
NN
paer SNE,
HOOC ) O98 1 € wk
+
H
N cunu-c{_\-x c
cunu—c{_\- | Na OH
(Isoleucovorin chloride)
0.1.N
OH- || pH 4
HCl /1 toge
ie CH
Pao a ee XVIII
CH HOOC—CH, (Anhydroleucovorin-A)
HOOC
Fic. 7. Chemical transformations in the synthesis of leucovorin from pteroylglu-
tamic acid.
.
II. CHEMISTRY 113
ean be readily determined, since it is a function of the height of the wave
between inflection points. Control experiments showed that neither the
pyrimidine ring nor p-aminobenzoylglutamic acid is reduced under the
same polarographic conditions. In this manner it was demonstrated that
the crude product formed by the reduction of PGA or 10-formyl PGA
under the conditions used in the synthesis’®: 7° is a tetrahydropteridine
derivative (XIV) because it produces the typical tetrahydropteridine wave
in the polarograph at pH 9.0. Leucovorin was found to give no wave in the
polarograph at pH 9.0, but after the mild acid treatment which destroyed
activity for Le. citrovorum but not for S. faecalis R the typical tetrahy-
dropteridine wave was found. This observation, discussed in greater detail
below, not only adds to the proof that leucovorin is a tetrahydropteridine
derivative but serves as the basis for a polarographic assay of leucovorin.
Thus, determinations are made before and after mild acid treatment, and
the difference in height of the tetrahydro wave forms a measure of the
concentration of leucovorin.
Available evidence indicates that it is the pyrazine ring which undergoes
reduction during hydrogenation of pteridines. Hydrogen and platinum
readily reduce pyrazine derivatives to the corresponding piperazines,*°: *!
whereas the pyrimidine nucleus is more resistant to catalytic hydrogena-
tion.®
It has been shown by Waller et al. that treatment of PGA with sul-
furous acid results in cleavage to 2-amino-4-hydroxypteridine-6-carboxalde-
hyde, probably through the corresponding intermediate dihydropteridine.
Under similar conditions leucovorin is partially destroyed.*! Since unsatura-
tion of the pyrazine ring appears necessary for the occurrence of this re-
action, the stability of leucovorin is a further indication of the 5,6,7,8-
tetrahydro structure (XII).
Additional evidence that the four hydrogen atoms of leucovorin reside in
the 5, 6, 7, and 8 positions of the pyrazine ring and not the pyrimidine ring
is provided by electrometric titration data.’ Thus, leucovorin exhibits a
phenolic hydroxyl group with a pK, of 10.4 which is comparable to the
value of 8.2 found for the hydroxyl group in the 4 position of PGA. If reduc-
tion of the pyrimidine ring had occurred, no enolic group would be found.
77B. Roth, M. E. Hultquist, M. J. Fahrenbach, D. B. Cosulich, H. P. Broquist,
J. A. Brockman, Jr., J. M. Smith, Jr., R. P. Parker, E. L. R. Stokstad, and T. H.
Jukes, J. Am. Chem. Soc. 74, 3247 (1952).
80 F. B. Kipping, J. Chem. Soc. 1929, 2889.
81 F. B. Kipping, J. Chem. Soc. 1932, 1336.
82 F. B. Brown and T. B. Johnson, J. Am. Chem. Soc. 45, 2702 (1923).
83 C W. Waller, A. A. Goldman, R. B. Angier, J. H. Boothe, B. L. Hutchings, J. H.
Mowat, and J. Semb, J. Am. Chem. Soc. 72, 4630 (1950).
84 A. Pohland, b. H. Flynn, R. G. Jones, and W. Shive, J. Am. Chem. Soc. 73, 3247
(1951).
114 PTEROYLGLUTAMIC ACID
The ultraviolet spectra of a number of PGA derivatives,** pyrimi-
dines,*®: 8° and their reduction products provides further support for the
hypothesis that all three double bonds in the pyrimidine ring of PGA remain
intact in its conversion to leucovorin. Thus, dihydropyrimidines such as
dihydrouracil or thymine glycol demonstrate little if any absorption in the
ultraviolet region.*® On the other hand, the ultraviolet absorption of tetra-
hydro PGA (XVI), 10-formyltetrahydro PGA (XIV), leucovorin (XII),
and 5,10-diformyltetrahydro PGA (XVII) closely resembles those of cer-
tain model pyrimidines. Furthermore, it is highly improbable that the
benzene ring of the p-aminobenzoyl portion of PGA would be reduced
under the relatively mild conditions employed in the synthesis of leucovorin.
d. Location of the Formyl Group
Since the reduction of 10-formyl PGA (XV) to a tetrahydro derivative
(XIV) does not result in appreciable leucovorin activity until the reaction
mixture has been allowed to stand or is poured into dilute alkali and heated,
the development of activity appears to depend on a subsequent rearrange-
ment. The resulting leucovorin (XII) has several unique chemical proper-
ties. Thus, it is quite stable in dilute alkali but is quickly inactivated in
dilute acid;”: 4) 87-89 the acid inactivated product, however, retains activity
for S. faecalis R.
Many possibilities have been considered to explain the pronounced
changes in biological activity and stability exhibited by leucovorin and the
various intermediates between PGA and leucovorin which have been iso-
lated. These include the formation of a new ring linking the N!® position
by a l-carbon bridge to position 5, 7, or 8 in the tetrahydropyrazine ring;
a shift of the N!°-formyl group to the pyrazine ring; and the introduction
of an additional formyl group (or groups) in the tetrahydropyrazine ring
with subsequent removal of the N!°-formyl group, by hydrolysis in the
alkaline treatment.
The possibility that the formyl group in leucovorin exists as a bridge
linking the 10 position with the 7 position of the tetrahydropteridine ring
was investigated® by means of oxidation with alkaline permanganate. Such
treatment converts 2-amino-4-hydroxy-6- or 7-alkylpteridines, including
PGA and its analogs, to the corresponding 6- or 7-carboxypteridines.?*
85 ],. F. Cavalieri, A. Bendich, J. F. Tinker, and G. B. Brown, J. Am. Chem. Soc
70, 3875 (1948)
86 T,, F. Cavalieri and A. Bendich, J. Am. Chem. Soc. 72, 2587 (1950).
87 'T. H. Jukes, H. P. Broquist, and E. L. R. Stokstad, Arch. Biochem. 26, 157 (1950).
88 J. ©. Keresztesy and M. Silverman, J. Biol. Chem. 183, 473 (1950).
89 M. Silverman and J. C. Keresztesy, J. Am. Chem. Soc. 78, 1897 (1951).
90D. B. Cosulich, B. Roth, J. M. Smith, Jr.. M. E. Hultquist, and R. P. Parker,
J. Am. Chem. Soc. 74, 3252 (1952). ’
Il. CHEMISTRY LW 5
Treatment of leucovorin with mineral acid followed by oxidation with
alkaline permanganate gave 2-amino-4-hydroxypteridine-6-carboxylic acid
in 68 % yield. Since no 2-amino-4-hydroxypteridine-6 ,7-dicarboxylic acid
was found, the existence of a carbon-to-carbon linkage to the 7 position of
the tetrahydropteridine ring seems quite unlikely. This narrowed the in-
vestigation to the remaining possibilities of a shift of the formyl group, or
a new ring formation involving the 5 or 8 position. From inspection of
molecular models it seems more logical to choose the 5 position because of
its proximity to the 10-formyl! group.
Further evidence for the location of the formyl group on the 5 position
has been provided by the study of numerous model compounds. The
properties exhibited by 2,4-diamino-6-hydroxy-5-formamidopyrimidine*®:
1, are found®® to be strikingly similar to leucovorin and other mono-
formyltetrahydropteridines, some of which are discussed below in more
detail. Thus, the 5-formamidopyrimidine is stable to hydrolysis upon heat-
ing for 1 hour in 0.1 N sodium hydroxide solution. It does not react with
nitrous acid in the cold, but, after hydrolysis of the formyl group with
mineral acid at room temperature, reaction with 1 mole of nitrous acid
occurs. Since the 2- and 4-amino groups in this model pyrimidine are inert
to formylation and nitrosation, it is logical to assume that the 2 and 8
positions of the tetrahydropteridine are similarly unreactive; this again
points to the 5 position of leucovorin as the most probable site for the
formyl! group.
Both 2-amino-4-hydroxy-6-methylpteridine (XX)** and 2-amino-4-hy-
droxy-6 ,7-dimethylpteridine (X XI) can be reduced and formylated to give
the corresponding 5-formyl-5,6,7,8-tetrahydropteridines (XXII* and
XXIII). As in the case of 2 ,4-diamino-6-hydroxy-5-formamidopyrimidine,
XXII is stable in 0.1 N sodium hydroxide solution for 1 hour at 95° and
shows no wave in the polarograph at pH 9; however, after treatment with
mineral acid, the characteristic tetrahydropteridine wave is found. There is
also no reaction with nitrous acid until the formyl group has been removed
by acid hydrolysis, at which time 1 mole is consumed. Reaction of the
2-amino group in this instance appears unlikely, since it has been shown’®
that XX does not react with nitrous acid under similar conditions. The
ultraviolet absorption curves of both XXII and XXIII are also similar to
that of leucovorin, and, as in the conversion of 5,6,7,8-tetrahydro PGA to
leucovorin, XXII and XXIII exhibit a much higher intensity of absorp-
tion than the corresponding unformylated tetrahydropteridines.
Another experiment which presents almost unequivocal proof that the
formyl group is located in the 5 rather than the 8 position involves the
W. Traube and H. W. Dudley, Ber. 46, 3839 (1913).
# W. Wilson, J. Chem. Soc. 1948, 1157.
116 PTEROYLGLUTAMIC ACID
@Nv\/Nv NH,
oe | N N
CH; sles Che NNN
OH OH
XX XXI
(2-Amino-4-hydroxy-6- (2-Amino-4-hydroxy-6, 7-
methylpteridine) dimethy|pteridine)
H H
Elise bol H
HY eRe 6a, VINO Se
ue iia CHA at N
|
i Eh siheae
CHO OH CHO OH
XXII XXIII
(2-Amino-4-hydroxy-5-formy1-6- (2-Amino-4-hydroxyl-5-formy]-6,7-
methyl-5,6,7,8-tetrahydropteridine) dimethyl-5,6,7,8-tetrahydropteridine)
preparation of 2-amino-4-hydroxy-5-formy1-6 , 7-diphenyl-8-ethyl1-5 ,6 ,7 ,8-
tetrahydropteridine (X XV) by reduction and formylation of 2-amino-4-
hydroxy-6 , 7-dipheny1-8-ethyl-7 ,8-dihydropteridine (X XIV).°° The reduc-
C.H;
C.H; ec ||
ee . CY S\N _nu,
CSF NN NAO NGO s)
C,H; ..- N
C,H | | N : \NZ\ 4
oe NNN, H |
| OH
OH HC=
OHV) BOK,
(2-Amino-4-hydroxy-6,7-diphenyl- (2-Amino-4-hydroxy-5-formy1-6, 7-diphenyl-
8-ethyl-7,8-dihydropteridine) 8-ethyl-5,6,7,8-tetrahydropteridine)
tion in 98 to 100% formie acid and formylation in the presence of acetic
anhydride are carried out under the same conditions as those used to prepare
the 6-methylpteridine model (XXII). The resulting product (X XV) reacts
similarly to XXII; thus, it has the same polarographic behavior, the formyl
group is stable in 0.1 N sodium hydroxide, and 1 mole of nitrous acid is
consumed only after treatment with mineral acid at pH 2. In this case, the
original dihydropteridine (XXIV) was isolated after reaction with nitrous
9 H.S. Forrest, R. Hull, H. J. Rodda, and A. R. Todd, J. Chem. Soc. 1951, 3.
Il. CHEMISTRY iL te
acid, which indicates that the action of nitrite on tetrahydropteridines is
one of oxidation rather than nitrosation. Furthermore, since XXV_ is
blocked in the 8 position with an ethyl group and yet has properties which
are similar to those of leucovorin, the possibility of a 5,8-linked ring system
in leucovorin can also be eliminated.
Because of the unique stability of the formyl group in 5-formyltetrahy-
dropteridines to dilute alkali, it seemed probable that there might be hy-
drogen bonding or even actual linkage of the formyl to the 4-hydroxy]l
group. This question was investigated by the preparation of 2-amino-4-
methyl-6 ,7-diphenylpteridine (XXVI),°° followed by the usual hydro-
genation and formylation to give 2-amino-4-methyl-5-formyl1-6 , 7-diphenyl-
5,6,7 ,8-tetrahydropteridine (XX VIT). As with the reduced and formylated
Hl
Bee ee
C a NNN NE Owe Be:
CHW ALN C.Hs N4\Y
| H iV
GH CH;
HiC==O
XXVI XXVII
(2-Amino-4-methyl-6, 7-diphenylpteridine) (2-Amino-4-methyl-5-formy1-6, 7-
diphenyl-5,6,7,8-tetrahydropteridine)
4-hydroxypteridines, XX VII is stable to hot 0.1 N sodium hydroxide and
surprisingly stable to acid, requiring hot 2.5 N hydrochloric acid to remove
the formyl group. Such behavior virtually eliminates the possibility of
bonding between the 4-hydroxyl and the formyl group and also presents
evidence that the 4-oxygen of pteridines in acid solution exists mainly as
the ketone form, since the 5-formyl group is more labile in the 4-hydroxy-
pteridines than it is in XXVII.
As additional evidence that the formyl group of leucovorin is not in-
volved in a new ring linking the 5 and 10 positions, reduced 10-methy1
PGA has been found to formylate under the usual conditions in the
presence of acetic anhydride to give 2-amino-4-hydroxy-5-formy]-10-
methyl-5 ,6,7 ,8-tetrahydro PGA (XXVIII).°% This analog has the same
type of alkali-stable, acid-labile formyl group as leucovorin and exhibits
the same behavior in the polarograph. When titrated immediately with
nitrous acid, XX VIII does not react, but after removal of the formyl group
by standing in dilute acid, nitrous acid is readily absorbed. The preparation
% PD. B. Cosulich and J. M. Smith, Jr., J. Am. Chem. Soc. 70, 1922 (1948).
118 PTEROYLGLUTAMIC ACID
of 10-nitroso PGA by treatment of PGA with nitrous acid in cold mineral
acid has been reported.?® This is evidence that the nitrogen atom in the 10
position of PGA is capable of reacting as a secondary aromatic amine.
Under similar conditions, 10-formyl PGA (XV) does not react with nitrous
H
|
HOOC Eee 2 :
| Loi
CHNH— ie Neo E
wn Cy vadal
Ks le
|
CH: Ho=O
|
HOOC
XXVIII
(2-Amino-4-hydroxy-5-formyl-10-methyl-5,6,7,8-tetrahydro PGA)
acid, which is reasonable proof that the formyl group is in the 10 position
in this compound. Furthermore, immediate and rapid addition of 1 mole of
nitrous acid to leucovorin in cold dilute hydrochloric acid results in the
formation of the 10-nitroso derivative.®° This shows that the 10 position,
as in PGA, is free to act as a secondary amine, and thus the formyl group
in leucovorin must be attached only at the 5 position. However, when leu-
covorin is allowed to stand in dilute mineral acid for a short time at room
temperature, there is no reaction with nitrous acid in the cold. This indi-
cates that both the 5 and 10 positions had become blocked.
The above experiments suggest that under mild acid conditions the
formyl group of leucovorin forms a new ring linking the 5 and 10 positions.
This assumption was proved by the subsequent isolation of an imidazolin-
ium salt, isoleucovorin chloride (XIII),°°: °° obtained by treatment of
leucovorin with hydrochloric acid at pH 1.3 or below. Isoleucovorin chlo-
ride does not react with nitrous acid; furthermore, it contains ionic chlorine
and a potential tormyl group, since | mole of formic acid is formed during
the drastic acid hydrolysis employed in the usual formy] analysis.
Leucovorin can be considered a formyl derivative of a N ,N’-aromatic
disubstituted ethylene diamine. As a model of such a system, the mono-
formyl derivative of N,N’-diphenylethylenediamine, N-(2-anilinoethyl)-
formanilide (X XIX), was prepared®* in order to compare its behavior to
95D. B. Cosulich, B. Roth, J. M. Smith, Jr., M. E. Hultquist, and R. P. Parker,
J. Am. Chem. Soc. 78, 5006 (1951).
96 M. May, T. J. Bardos, F. L. Barger, M. Lansford, J. M. Ravel, G. L. Sutherland,
and W. Shive, J. Am. Chem. Soc. 78, 3067 (1951).
Il. CHEMISTRY 119
acid with that of leucovorin. A study of the ultraviolet absorption of XXLX
under acid and basie conditions shows that it behaves quite similarly to
O
|
\Cilal yas
ve a Ht
oC xO a
en X pa
CH.—CH:
XXIX
[N-(2-Anilinoethyl)formanilide]
CH.—CH:
XXX
leucovorin; thus, in acid the maximum shifts in a manner which suggests a
more highly conjugated system (XXX). Furthermore, a rapid change from
strongly acidic to strongly basic solutions results in an equally rapid re-
generation of XXIX. At intermediate pH values, however, the intercon-
version of the two forms occurs much more slowly, which shows that the
shift of the maximum is not an immediate effect of pH but represents a
more drastic change in the molecular structure.
It is readily apparent that opening the imidazolintum ring of XXX to
form X XIX can give a product with the formyl group on either nitrogen;
but, because the molecule is symmetrical, only one product is formed. How-
ever, treatment of isoleucovorin chloride with alkali may give either the
10- or 5-formyl] derivative. The major product appears to be 10-formyl-
5,6,7 ,8-tetrahydro PGA (XIV), although some regeneration to leucovorin
also occurs. Thus, when isoleucovorin chloride is treated with alkali
aerobically, one-half of a molecular equivalent of oxygen is rapidly con-
sumed to give a product which slowly oxidizes in acidic solution to 10-formy!]
PGA (XV). Since 10-formyl PGA can be hydrolyzed readily to PGA with
alkali, it is therefore possible to reconvert leucovorin to PGA.
As can be seen from Fig. 7, several other products have been isolated
during investigations on the chemistry of leucovorin. One of these is an-
hydroleucovorin-A (XVIII), % which is formed by dissolving isoleu-
covorin chloride in boiling water, then cooling slowly. This crystalline sub-
stance is thought to be an imidazolinium “‘betaine-type”’ derivative of
leucovorin. By treatment of anhydroleucovorin-A or isoleucovorin chloride
120 PTEROYLGLUTAMIC ACID
with boiling pH 4.0 buffer, anhydroleucovorin-B (XIX)°*°: °° is formed; the
exact structure of this derivative has not been completely elucidated. Both
anhydroleucovorin-A and anhydroleucovorin-B can be converted to iso-
leucovorin chloride with 0.1 N hydrochloric acid and to leucovorin with
alkali under anaerobic conditions. Another derivative is 5, 10-diformyl-
5,6,7,8-tetrahydro PGA (XVII),”°: * which can be formed from either
5,6,7,8-tetrahydro PGA (XVI), 10-formyl-5,6,7,8-tetrahydro PGA
(XIV), or leucovorin (XII) by formylation with 98 to 100 % formic acid in
the presence of acetic anhydride; this substance has an activity for Le.
citrovorum which is equivalent to 0.5% of pure leucovorin. Alkaline hy-
drolysis removes the 10-formyl group to give leucovorin.
It is conceivable that, during migration of the formyl group which occurs
in the synthesis of leucovorin and its transformation products, an inter-
mediate hydroxymethylene bridge might result. Two stereoisomeric¢ con-
figurations are possible for such a bridge. Although many have claimed*”-*°
that the formyl and other acyl derivatives of aromatic 6-secondary diamines
exist as ring structures with hydroxymethylene bridges, recent results ob-
tained by infra-red absorption studies of some acyl compounds of similar
structure indicate that the open amide form exists at least to some extent.!°°
e. Relation of Natural Citrovorum Factor to Leucovorin
Recently Keresztesy and Silverman?! and Sauberlich'” have succeeded
in isolating crystalline citrovorum factor from liver which has permitted
comparison of the properties of the naturally occurring vitamin with
5-formyl-5 ,6,7 ,8-tetrahydro PGA. The product obtained from horse liver
by Keresztesy and Silverman was found to be approximately twice as active
as synthetic 5-formyl-5,6,7,8-tetrahydro PGA for growth of Leuconostoc
citrovorum. In 0.1 N NaOH solution (at a concentration of 10 mg. per liter)
leucovorin exhibits a maximum at 282 mu (optical density = 0.631) and a
minimum at 243 my (optical density = 0.123). When the two compounds
are examined spectrophotometrically under identical conditions, they are
indistinguishable. Since the natural factor is twice as active microbio-
logically as the synthetic product, and since there appears to be no spectral
difference between the compounds, it is possible that the synthetic factor
is a mixture of diastereoisomers, one of which is the biologically active,
naturally occurring form. Examination of the structure of 5-formyl1-5 ,6,7 ,8-
tetrahydro PGA indicates the possibility of diastereoisomers. In the synthe-
97 Q. Fischer, Ber. 34, 930 (1901).
% St. Niementowski, Ber. 20, 1874 (1887).
99 O. Fischer and M. Rigaud, Ber. 34, 4203 (1901) ; 35, 1258 (1902).
100 C.W. Smith, R.S. Rasmussen, and S. A. Ballard, J. Am. Chem. Soc. 71, 1082 (1949).
lol J. C. Keresztesy and M. Silverman, J. Am. Chem. Soc. 78, 5510 (1951).
102 Hf. EK. Sauberlich, J. Biol. Chem. 195, 337 (1952).
III. INDUSTRIAL PREPARATION PAL
sis of this compound a new asymmetric center has been created at position
6 in the pyrazine ring. Another point of difference between the natural and
synthetic citrovorum factor is that, when the two substances are exposed
to the action of dilute acid, the natural material loses about one-third of its
potency as a source of folic acid for S. faecalis whereas the synthetic
material following acid treatment increases 11 % in activity for S. faecalis.*9
Synthetic leucovorin has been separated into its two diastereoisomers by
Cosulich et al.!°4 by utilizing the difference in solubility of the calcium salts.
The /t form is the less soluble form, and it has an [a], of —15.1 compared
with + 15.3 for the original mixture of the two diastereoisomers. The cal-
cium /L-leucovorin has biological activities for S. faecalis and Leuconostoc
citrovorum strictly comparable to those for the natural material and is de-
creased in activity by acid treatment. The more soluble calcium du-leuco-
vorin has not been obtained in a pure state.
The existence of different forms of the citrovorum factor in nature was
first clearly demonstrated by Winsten and Eigen.'°%* When various crude
materials such as yeast extract or liver were chromatographed on paper and
the strips placed on an agar medium deficient only in CF and seeded with
Leuconostoc citrovorum, several distinct zones of growth appeared. It was
suggested that the chromatographically slower factors represent conjugated
forms of the faster forms. Different forms of the synthetic factor have also
been prepared corresponding to the mono-, di-, and triglutamic acid deriva-
tives of PGA.
III. Industrial Preparation
ROBERT S. HARRIS
Because of the intense activity of commercial groups in the development
of inexpensive and effective methods for the synthesis of pteroylglutamic
acid, it is not possible at this time to describe the methods now being used
in the industrial preparation of this vitamin. Instead it is necessary to
describe some of the synthetic methods first reported in the scientific litera-
ture and to list a few of the patents which have been issued.
The chemical synthesis of PGA was first reported by Angier ef al.,1 who
10s W. A. Winsten and E. Eigen, J. Biol. Chem. 184, 155 (1950).
104 DP. B. Cosulich, J. M. Smith, Jr., and H. P. Broquist, J. Am. Chem, Soc. 74, 4215
(1952).
1R. B. Angier, J. H. Boothe, B. L. Hutchings, J. H. Mowat, J. Semb, E. L. R.
Stokstad, Y. SubbaRow, C. W. Waller, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith,
Jr., Science 103, 667 (1946); Ann. N. Y. Acad. Sci. 48, 283 (1946).
122 PTEROYLGLUTAMIC ACID
reported success by four different methods. Method [':? consisted in re-
acting equimolecular amounts of 2,4,5-triamino-6-hydroxypyrimidine,
p-aminobenzoyl-ut-glutamic acid, and 2 ,3-dibromopropionaldehyde in the
presence of an acetate buffer. The resulting product was 15% active. The
dihydro compound was formed first, and this oxidized to the aromatic
compound during the course of the reaction. The crude mixture was dis-
solved in dilute alkali and the impurities removed by precipitation with
barium chloride, adjusting to pH 7.0, filtering, and extracting three times
with 10 volumes of butanol. The aqueous phase was concentrated, acidified
to pH 3.0, and cooled to 0.5°. The precipitate was redissolved in dilute
alkali, the solution was treated with charcoal and acidified to pH 3.0, and
the active compound was recovered by crystallization from hot water.
In Method IT’: * 2 ,3-dibromopropionaldehyde was reacted with pyridine,
and the product was condensed with 2,4 ,5-triamino-6-hydroxypyrimidine
and KI to yield N-[(2-amino-4-hydroxy-6-pteridyl)methyl|-pyridinium
iodide. By heating with Na methoxide in ethylene glycol at 140°, this com-
pound was condensed and the product was found to contain 15% of the
active compound.
Method III‘ involved the reaction of reductone (2 ,3-dihydroxyacrylalde-
hyde) with p-aminobenzoylglutamic acid to form p-(2 ,3-dihydroxy-2-ene-
propylideneamino)benzoylglutamie acid, then esterification and condensa-
tion of the ester with 2,4 ,5-triamino-6-hydroxypyrimidine.
Method IV® involved the reduction of 2-amino-4-hydroxy-6-pteridyl-
methylpyridinium iodide to 2-amino-4-hydroxy-6-methylpteridine, bro-
mination or chlorination, then condensation of the product with the diethyl]
ester of p-aminobenzoylglutamie acid.
These and similar methods have been patented by the American Cyana-
mid Company.®
2C. W. Waller, B. L. Hutchings, J. H. Mowat, E. L. R. Stokstad, J. H. Boothe,
R. B. Angier, J. Semb, Y. SubbaRow, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith,
Jr., J. Am. Chem. Soc. 70, 19 (1948).
3M. E. Hultquist, E. Kuh, D. B. Cosulich, M. J. Fahrenbach, E. H. Northey, B. L.
Hutchings, J. H. Mowat, J. Semb, HE. L. R. Stokstad, Y. SubbaRow, and C. W.
Waller. J. Am. Chem. Soc. 70, 23 (1948).
4R. B. Angier, E. L. R. Stokstad, J. H. Mowat, B. L. Hutchings, J. H. Boothe, C.
W. Waller, J. Semb, Y. SubbaRow, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, E. H. Seeger, J. P. Sickels, and J. M. Smith,
Jr., J. Am. Chem. Soc. 70, 25 (1948).
5 J. H. Boothe, C. W. Waller, E. L. R. Stokstad, B. L. Hutchings, J. H. Mowat,
R. B. Angier, J. Semb, Y. SubbaRow, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith,
Jr., J. Am. Chem. Soc. 70, 27 (1948).
6 American Cyanamid Co., U. S. Pats. 2,442,836, 2,442,837, 2,442,867, 2,443,165,
=. th 3)
III. INDUSTRIAL PREPARATION 123
IKXarrer and Sechwyzer’ condensed 2,4,5-triamino-6-hydroxypyrimidine
with glyceraldehyde or dihydroxyacetone, giving a mixture of 2-amino-4-
hydroxy-6-hydroxymethylpteridine and 2-amino-4-hydroxy-7-hydroxy-
methylpteridine. Reacted with p-aminobenzoylglutamic acid, the former
compound yielded PGA.
Roche Products Lts.§ obtained a patent for PGA synthesis by reacting
2-amino-4-hydroxy-6-hydroxymethylpteridine with thionyl chloride and
then treating the product with p-aminobenzoylglutamie acid, or hydro-
genating a mixture of pteridine and p-nitrobenzoylglutamic acid. This
company subsequently obtained a series of patents on the synthesis of
PGA and intermediate compounds, a few of which are listed.°
Hoffman-LaRoche Co.'° condensed 2 ,4 ,5-triamino-6-hydroxypyrimidine
with a ketohexose and oxidized the 2-amino-4-hydroxy-6-tetrahydroxy-
butylpteridine which resulted with lead tetraacetate or another agent
capable of producing the glycol cleavage. The 2-amino-4-hydroxy-6-pter-
idylaldehyde that was formed was hydrogenated in an inert solvent or in
formic acid in the presence of p-aminobenzoylglutamic acid and a catalyst,
and PGA was obtained. Some formylpteroylglutamic acid was produced,
and this was converted to PGA by treatment with ammonia. Several of
the patents issued to this company are listed."
Forrest and Walker” reacted glucose and fructose with 2 ,4 ,5-triamino-6-
hydroxypyrimidine in the presence of hydrazine and obtained 2-amino-4-
hydroxy-6-p-arabotetrahydroxybutylpteridine; in the absence of hydrazine
he found 2-amino-4-hydroxy-7-p-arabotetrahydroxybutylpteridine.
Uyeo and Mizukami® reported better yields when mercuric acetate was
used as a dehydration agent in Waller’s method? of synthesis. In a more
recent paper Uyeo et al.‘ described a further modification of this method
of PGA synthesis.
2,444,002, 2,444,005, 2,472,520, 2,500,296, 2,517,530, 2,520,479, 2,537,006, 2,547,519,
2,547,520, 2,568,597, 2,570,391, 2,570,392; British Pats. 12,491, 14,216, 24,564, 25,001,
25,002 (1948) ; 3,413, 631,494 (1949) ; 638,411, 638,480, 640,092 (1950) ; 644,913, 648,896,
650,276, 654,512, 655,771, 656,403, 657,902 (1951) ; 258,538 (1952).
7P. Karrer and R. Schwyzer, Helv. Chim. Acta 31, 777 (1948).
8 Roche Products Ltd., British Pats. 264,394, 630,751.
® Roche Products Ltd., British Pats. 624,394, 628,305, 626,171 (1949); Belgium Pat.
481,230 (1948).
10 Hoffman-LaRoche and Co., British Pats. 626,171, 628,305; Roche Products Ltd.,
British Pat. 631,516.
1! Hoffman-LaRoche and Co., U.S. Pats. 2,487,393, 2,520,882; British Pats. 629,440
(1949) ; 657,254 (1951); 664,721 (1952); Swiss Pats. 253,838 (1948) ; 255,409, 258,141,
263,147 (1949); 263,281 (1950); 263,147 (1951); 268,328 (1952).
2 H.S. Forrest and J. Walker, J. Chem. Soc. 1949, 2077.
18S. Uyeo and S. Mizukami, Japan. J. Pharm. & Chem. 21, 237 (1949).
1448S. Uyeo, S. Mizukami, T. Kubota, and 8S. Takagi, J. Am. Chem. Soc. 72, 5339
(1950).
124 PTEROYLGLUTAMICG ACID
Weygand et al.'> described a new synthesis of PGA by the reaction of
the condensation products of p-tolyl-p-isoglucosamine and sugars, with
6-hydroxy-2 ,4,5-triaminopyrimidine. Later Weygand and Schmied-Kowar-
zik!® described a series of syntheses of PGA by other reactions.
Kirsanova and Trufanov™ described the synthesis of PGA by reaction of
p-aminobenzoyl-p(1)-glutamic acid, 2,3-dibromopropionaldehyde, and
2,4 ,5-triamino-6-hydroxypyrimidine. This synthesis is similar to that re-
ported by Angier’ and others. They described an improved purification
process by which there was no loss in activity, whereas the method of
Angier! involved a loss of 85% or more.
Hultquist and Dreisbach'® have patented a method for the synthesis of
PGA.
Haehner et al.!® reported PGA synthesis by condensing 2-amino-95,5-
dibromobarbituric acid with p-[2 ,8-diaminopropyl)amino] benzoy! glutamic
acid to give an unstable intermediate product which rearranged to dihydro
PGA. On oxidation it gave a good yield of relatively pure PGA.
Petering and Schmitt? have patented methods for synthesizing 2 ,4-
diamino-6-(3-carboxy-1 ,2 ,3-trihydroxypropyl)pteridine and 2-amino-4-
hydroxy-6-(3-carboxy-1 ,2 ,3-trihydroxypropyl)pterines.
Merck and Co.2! and Parke, Davis & Co.” have patented other methods
for synthesizing this vitamin.
IV. Biochemical Systems
E. L. R. STOKSTAD
A. COENZYMES AND ENZYMES
The primary biochemical role of PGA appears to be the synthesis of
compounds such as purines, pyrimidines, and certain amino acids which
involve the incorporation of a single carbon fragment.
1. Function or PGA 1N SYNTHESIS OF PURINES AND PYRIMIDINES
The relationship between PGA and purines in the nutrition of lactie acid
organisms was noted early in the work on this vitamin. Snell and Mitchell!
15. Weygand, A. Wacker, and V. Schmied-Kowarzik, Chem. Ber. 82, 25 (1949).
16 fF, Weygand and V. Schmied-Kowarzik, Chem. Ber. 82, 333 (1949).
17V. A. Kirsanova and A. V. Trufanov, Biokhimiya 14, 4138 (1949).
18 M. E. Hultquist and P. F. Dreisbach, British Pat. 657,831 (1951).
19 fH}, Haehner, H. Nafziger, and H. Ludes, Alin. Wochschr. 29, 571 (1951).
20 H. G. Petering and J. A. Schmitt, U.S. Pats. 2,568,482, 2,568,684 (1951).
21 Merck and Co., U.S. Pat. 2,540,274 (1951); British Pat. 653,068 (1951).
22 Parke, Davis & Co., U.S. Pat. 2,476,360 (1949); British Pat. 639,154 (1950).
1. E. Snell and H. K. Mitchell, Proc. Natl. Acad. Sci. U. S. 27, 1 (1941).
IV. BIOCHEMICAL SYSTEMS 125
O O
l
| \
A A
HN C—CH HN C—=—=N
fiers aii ee EN
O=C2 4CH | | sCH
BA HC. 4C—N
N NYA H
i N
Thymine Adenine
(5-methyluracil) (6-Aminopurine)
reported that adenine or guanine stimulated the growth of Streptococcus
faecalis R and that a further response was produced by thymine. Stokstad?
found that the PGA requirement of Lactobacillus casei can be partially re-
placed by a combination of 0.5 y of thymine and 5.0 y of guanine per
milliliter of media. However, the maximum growth obtained by a combina-
tion of purine and pyrimidines is only about half that produced by concen-
trates of L. casez factor (PGA). Thymine could not be replaced by other
pyrimidines such as uracil or cytosine. The purine requirements were less
specific and could be met by guanine, xanthine, adenine, or hypoxanthine.
A number of pyrimidines, purines, and pterins have been tested by Stokes?
as possible substitutes for thymine in the nutrition of S. faecalis R. Of
these, thymidine (thymine desoxyriboside) is the only substance which has
thymine-like activity equal to that of thymine on a molar basis. Thymus
nucleic acid is inactive, which demonstrates the inability of S. faecalis R to
hydrolyze this nucleic acid to the riboside form. No information is available
on the activity of thymidine desoxynucleotide. A study of pyrimidine
analogs reveals that a methyl group in the 5 position is essential for ac-
tivity. Shifting the methyl group to the 1, 3 or 4 position, or replacing it by
an ethyl, amino, or nitro group, results in complete loss in activity.
A possible role of thymine in serving as a substitute for PGA was sug-
gested on the basis of experiments with these two compounds in the nutri-
tion of S. faecalis R and L. casei.’ It was found that, whereas PGA alone
would give a response with S. faecalis R, the maximum effect was not ob-
tained unless a purine also was added. In the presence of a purine five
thousand times as much thymine as PGA is needed for maximum growth.
With S. faecalis R, thymine gives the same maximum growth rate as can
be obtained with PGA, although with ZL. casei only half-maximum growth
is achieved.
The possibility of formation of PGA by S. faecalis R cells during growth
2h. L. R. Stokstad, J. Biol. Chem. 189, 475 (1941).
3 J. L. Stokes, J. Bacteriol. 48, 201 (1944).
126 PTEROYLGLUTAMIC ACID
on thymine was tested by autoclaving the cells with dilute hydrochloric
acid and assaying with L. casez. A “plateauing”’ of the L. casei assay response
at approximately half-maximum growth was observed. This response is
characteristic of that obtained with thymine and suggests that the material
in the S. faecalis R cells giving the response is thymine and not PGA.
These facts led to the suggestion that PGA functioned directly or indirectly
as a coenzyme in the synthesis of thymine by S. faecalis R.
Hitchings et al.‘ studied the effect of a large number of pyrimidines on the
growth of L. casei and found that only those compounds which retain a
methyl group in the 5 position possess any activity. Substitution of an
imino group for one oxygen such as in 5-methyleytosine or 5-methylisocyto-
sine yields compounds one-tenth as active as thymine. Replacement of
both oxygens by imino groups as in 5-methyl-2 ,4-diaminopurine results in
a still further decrease of activity. Replacement of the 5-methyl group by
oxygen, amino groups, or halides produces inhibitory compounds. Of
special interest is 5-bromouracil, which inhibits completely the growth of
L. casei with thymine as the nutrient but which produces slight growth
stimulation when a ‘folic acid concentrate’? (PGA) is used as a nutrient.
Further evidence concerning the function of PGA in purine and pyrimi-
dine synthesis has been provided by the work of Rogers and Shive® using
the method of inhibition analysis. The antagonist employed was ‘“‘x methyl
PGA,” which was prepared from a,8-dibromobutyraldehyde, and which
had been shown by Franklin et al.® to function competitively as a PGA
antagonist for rats and microorganisms. The inhibition ratios (ratio of
inhibitor to PGA for complete inhibition) for this antagonist were deter-
mined in the presence of various purines and thymine. Thymine was found
to have no effect on the toxicity of ‘‘x methyl PGA” in the absence of a
purine but did counteract it when a purine was present. The inhibition ratio
in the absence of a purine was approximately 30. The addition of 10 y of
hypoxanthine per milliliter increased it to 100. The further addition of 3
of thymine per milliliter further increased the antibacterial ratio to about
1000. These results also show that the PGA requirement for purine synthe-
sis 1s larger than that for thymine synthesis, since the synthesis of purines
is blocked at a lower concentration of antagonist. Thus at certain critical
levels of antagonist it is possible effectually to block one enzyme function
of a vitamin without seriously impairing another. Inhibition of growth by
high levels of the antagonist in the presence of adenine and thymine shows
that PGA has still another function. This is to be expected, since with L.
casev a combination of thymine plus purine gives only half-maximum
4G. H. Hitchings, FE. A. Faleo, and M. B. Sherwood, Science 102, 251 (1945).
°T. L. Rogers and W. Shive, J. Biol. Chem. 172, 751 (1948).
6 A. L. Franklin, E. L. R. Stokstad, M. Belt, and T. H. Jukes, J. Biol. Chem. 169,
427 (1947).
IV. BIOCHEMICAL SYSTEMS 127
growth. Similar results were obtained with S. faecalis using both ‘‘x methyl
PGA” and N?!°-methylpteroic acid as antagonists (Stokstad ef al.’). In this
case the antagonist had no effect, even at high concentrations when both
adenine and thymine were supplied. These results with N!°-methylpteroic
acid are shown in Table II. This indicates that the only function of PGA in
S. faecalis is purine and pyrimidine synthesis and is in contrast to the situa-
tion in L. casei where PGA has a third additional role.
TABLE II
Errect OF ADENINE AND THYMINE ON INHIBITION Ratio or N-Metruyt PrEerRoic
Acip—Ore@anisM: S. faecalis R (Sroxstap et al.?)
Pteroylglutamic N-Methyl pteroic adenine 7/10 Thymine, Growth in 44 hr.,
ml.
acid, y/10 ml. acid, y/10 ml. ¥/10 ml. optical density Inhibition ratio
0.00 ? 0.07
100 0.10
30 0.02
100 30 0.45
0.01 0.59
0.01 100 0.64
0.01 0.03 Only 5
0.01 0.11 0.0
0.01 0.03 30 0.33 7
0.01 0.1 30 0.04
0.01 Orn 100 0.55 30
0.01 0.3 100 0.02
0.01 (Deal 1000 0.43 25
0.01 0.3 1000 0.01
300 100 30 0.44 No inhibition
2. SULFONAMIDE, p-AMINOBENZOIC ACID, AND PuRINE RELATIONSHIPS
An interesting relationship between p-aminobenzoic acid and the metab-
olism of purines, amino acids, and pyrimidines has been shown in #. coli
by inhibition studies with sulfanilamide.
Shive and his associates*: ° and Winkler and de Haan! have shown that
sulfanilamide, in progressively higher concentrations, inhibits successively
7E.L. R. Stokstad, M. Regan, A. L. Franklin, and T. H. Jukes, Federation Proc. 7,
193 (1948).
8 W. Shive and E. C. Roberts, J. Biol. Chem. 162, 463 (1946).
9W.Shive, Ann. N. Y. Acad. Sci. 62, 1212 (1950).
10 K. C. Winkler and P. G. de Haan, Arch. Biochem. 18, 97 (1948).
128 PTEROYLGLUTAMIC ACID
the synthesis of methionine, purine, serine, and thymidine by F. coli. The
inhibition ratio for sulfanilamide to p-aminobenzoic acid is 3000. In the
presence of methionine it becomes 10,000; in the presence of methionine
and purine it is 30,000; and in the presence of methionine, purine, and serine
it becomes 100,000. Further addition of thymine increases it to 200,000.
Thymine may be partially replaced by PGA. This indicates that the synthe-
sis of methionine, purine, serine, and thymine are mediated directly or
indirectly by p-aminobenzoic acid and that the synthesis of each of these
end products can be blocked in succession by progressively increasing the
amounts of sulfanilamide. In order for any end product to be effective, the
other end products, whose production is blocked at lower antagonist con-
centrations, must be present in the medium. In the presence of methionine,
xanthine, and serine and with 2 mg. of sulfanilamide per milliliter and
approximately 3 my of p-aminobenzoic acid per milliliter, growth is in-
hibited. Growth can be restored at this point by the addition of either 30
y of thymine, 0.3 y of PGA, or0.010 y of p-aminobenzoic acid per milliliter.’
The fact that such a large amount of PGA is required relative to the p-ami-
nobenzoic acid is difficult to explain if one assumes that the former is
synthesized from the latter. It is possible, however, that the conversion of
preformed PGA to a more active intermediate may be limiting.
Valine synthesis is the next limiting factor after thymine requirements
are met. The growth produced by thymine or PGA reaches the maximum
in 72 hours, whereas with p-aminobenzoic acid it is reached in 32 hours.
Addition of valine at this point permits early growth, showing that this
amino acid becomes the limiting factor after inhibition of thymine synthe-
Sisal?
A similar series of products reverse the toxicity of sulfanilamide for
Salmonella typhimurium, and methionine, xanthine, thymine, and valine
have been reported to function similarly in sulfonamide reversal in Staphylo-
coccus aureus."
The relationship of these metabolites to sulfonamide inhibition and the
possible precursors are shown in Fig. 8.
B. MECHANISM OF ACTION
1. Rove oF 4-AMINO-5-IMIDAZOLECARBOXAMIDE IN PURINE SYNTHESIS
If PGA blocks the synthesis of purine, one might expect an intermediate
product to accumulate in a system in which purine synthesis is blocked by
a minimum amount of antagonist and in which growth is permitted by the
addition of a purine. This has not been demonstrated with lactie acid or-
1K. C. Winkler, P. G. de Haan, and J. van de Langerijt, Antonie van Leewwenhoek
J. Microbiol. Serol. 15, 129 (1949), cited by W. Shive, Vitamins and Hormones 9,
75 (1951).
IV. BIOCHEMICAL SYSTEMS 129
ganisms either in cells grown on vitamin-deficient media or with PGA
antagonists. However, in studies with sulfanilamide inhibition of /. coli
a compound, 4-amino-5-imidazolecarboxamide, was obtained which ap-
pears to be an intermediate in purine synthesis. The existence of this
compound was first noted by Stetten and Fox,” who observed that /. coli
grown in a synthetic medium containing amino acids and a bacteriostatic
concentration of sulfadiazine produce a diazotizable amine. The formation
Hs NH,
HS—CH,—CH,-CH-COOH "5 CH,—S—CH,.—CH.—CH—COOH
Homocysteine Methionine
NH,.—C=0O HN—C=0 :
| [ol Hypoxanthine
CN He RCN Xanthine
I eCH “sd GIGGOLe | l pe) Adenine
NH:—C_—N iis Guanine
H L or derivatives
5(4)-Amino-4(5)-imidazolecarbox- Hypoxanthine
amide (or derivative)
| O Nee
I
NH.—CH,—C—OH AOI0O0 HOCH,—CH— COOH
Glycine ' Serine
OH NEL NH—C=0O
CH;—CH—CH—COOH Danae O=C C—CH;
Threonine precursor NH—CH
Thymine
PGA (or derivative)
Fic. 8. Inhibition analysis with sulfanilamide (E. coli). Courtesy Ann. N. Y.
Acad. Sci. (Shive’).
of the amine is prevented by addition of p-aminobenzoic acid in concentra-
tions sufficient to block the action of the sulfonamide. Other bacteriostatic
agents such as atebrin or penicillin do not produce this diazotizable amine.
The fact that this amine forms under conditions of sulfanilamide inhibi-
tion where purine synthesis is inhibited, together with the fact that the
empirical analysis corresponded to that of a purine minus one carbon atom,
suggested that it might well be an intermediate in purine synthesis. On
this basis Shive ef al. synthesized 4-amino-5-imidazolecarboxamide and
12M. R. Stetten and C. L. Fox, Jr., J. Biol. Chem. 161, 333 (1945).
18 W. Shive, W. W. Ackermann, M. Gordon, M. E. Getzendaner, and R. E. Eakin,
J. Am. Chem. Soc. 69, 725 (1947).
130 PTEROYLGLUTAMIC ACID
found that it had the same properties as the compound isolated by Stetten
and Fox.” Subsequently it was observed that the synthesis of this amine
by E. coli is increased by addition of glycine and to a lesser extent by thre-
onine but not by serine."
PGA has not been shown to be directly involved in the synthesis of
purines in the sulfonamide-inhibited H. coli system.'° Addition of PGA to
this system at the points where methionine, purine, or serine synthesis is
critical has no effect on the inhibition ratio..° The inactivity of added
PGA, however, does not exclude the possibility that a more highly active
metabolic form or conjugate of PGA may be formed from p-aminobenzoic
acid which cannot be replaced by PGA itself. Support for such a view comes
from the observations of Woolley and Pringle, who found that the amino-
imidazolecarboxamide accumulates in the medium when £. coli is grown in
the presence of sufficient 4-aminopteroylglutamic acid to inhibit growth
slightly. In the presence of larger amounts of 4-aminopteroylglutamic acid
(250 y per milliliter) the growth of F. colz is inhibited in a medium contain-
ing purines. Growth can be restored by the addition of thymidine but not
by thymine or the desoxyribosides of hypoxanthine or guanine (Franklin
et al.!®). This evidence favors the view that p-aminobenzoic acid acts in the
synthesis of purines and pyrimidines by way of an intermediate synthesis
through PGA. This is in accord with the hypothesis of Woods" that the
primary action of sulfonamides is the inhibition of PGA formation. The
only place where PGA has thus far been shown to be directly involved in
FE. coli metabolism is in the synthesis of thymine where either thymine or
PGA affects the inhibition index. However, the role of PGA in purine and
thymine synthesis in both L. casei and S. faecalis, the presence of p-amino-
benzoic acid in PGA, and the action of 4-aminopteroylglutamie acid in
stimulating production of the amine in E. coli certainly invite the view that
PGA may be an intermediary in the synthesis of purine by p-aminobenzoic
acid.
Aminoimidazolecarboxamide can replace purine in the nutrition of cer-
tain organisms. Shive® reported that the amine promotes growth of ZL.
arabinosus in @ manner similar to purines and disappears from the medium.
It also replaces hypoxanthine or adenine in stimulating growth of Ophi-
astoma multiannulatum if a large inoculum is employed or. if suboptimal
concentrations of these purines are added to the medium.'®
14 J. M. Ravel, R. E. Eakin, and W. Shive, J. Biol. Chem. 172, 67 (1948).
1657). W. Woolley and R. B. Pringle, J. Am. Chem. Soc. 72, 634 (1950).
16 A. LL. Franklin, EK. L. R. Stokstad, C. E. Hoffman, M. Belt, and T. H. Jukes,
J. Am. Chem. Soc. 71, 3549 (1949).
17 TD), D. Woods, Bull. soc. chim. biol. 80, 730 (1948), cited by D. W. Woolley and R.
B. Pringle, J. Am. Chem. Soc. 72, 634 (1950).
18 N. Fries, Physiol. Plantarum 2, 78 (1949), cited by W. Shive, Vitamins and Hor-
mones 9, 75 (1951).
IV. BIOCHEMICAL SYSTEMS 131
It is interesting, however, that no 4-amino-5-imidazolecarboxamide has
been detected in cultures of L. cased grown with inhibitory concentrations
of “x methyl PGA” in the presence of some purine.’ This is not at all
surprising when one considers that both the C? and the C® atoms of the
purine ring are introduced as 1-carbon fragments. If the introduction of the
C®-carbon happens to be the first limiting reaction, then the imidazole-
carboxamide would not be expected to accumulate. It is ineffective as a
growth promoter for nine purine-less mutants of H. coli but is effective for
one mutant at the high level of 100 to 200 y per milliliter, which is about
fifteen times as high as the level of purine required to give the same ef-
fect.1%» 2° Another mutant was found which accumulates the amine when
grown in the presence of a purine. With 5 y of xanthine per milliliter, growth
is approximately half of maximum, and 11.3 y of aminoimidazolecarboxam-
ide accumulates per milliliter of medium.!°
A synergistic effect was observed between purine and aminoimidazole-
carboxamide.”® In the presence of only 4 y of any purine per milliliter,
which by itself gives only 30 % growth, 30 y of aminoimidazolecarboxamide
per milliliter gives maximum growth. Formylaminoimidazolecarboxamide
which contains the “missing” carbon atom, but not in the form of the
closed pyrimidine ring, is more active than the parent amine. Vitamin By»
(0.2 my per milliliter) increased growth of the purine-less #4. coli mutant
and increased utilization of added aminoimidazolecarboxamide. p-Amino-
benzoic acid (0.1 y per milliliter) increased growth but did not increase utili-
zation of the imidazole derivative.?°
The activity of vitamin By» in increasing the utilization of amine is of
special interest. Shive has found”! that vitamin By» increases the inhibition
ratio of sulfanilamide with H. coli at the inhibition levels for methionine,
purine, and serine synthesis. The non-specific effect of vitamin By, in this
case was explained on the basis that By aids in the conversion of p-amino-
benzoic acid to the active coenzyme.
An alternate explanation for the activity of vitamin By is that it facili-
tates the conversion of the imidazole derivative to a desoxyriboside. Certain
lactic acid bacteria requiring vitamin By» are able to utilize thymine desoxy-
riboside or purine desoxyribosides but not free thymine. Under these cir-
cumstances the function of vitamin By», appears to be synthesis of a desoxy-
ribose derivative capable of attachment to thymine. The recent work of
Greenberg”: *§ has shown that ring closure in a pigeon liver system does not
19 J. S. Gots, Arch. Biochem. 29, 222 (1950).
20 Ruth Ben-Ishai, B. Voleani, and E. D. Bergmann, Arch. Biochem. and Biophys. 32,
229 (1951).
21W. Shive, Vitamins and Hormones 9, 75 (1951).
22G. R. Greenberg, Federation Proc. 9, 179 (1950).
*3G. R. Greenberg, J. Biol. Chem. 190, 611 (1951).
2, PTEROYLGLUTAMIC ACID
occur with the free imidazole derivative. The latter may represent a break-
down product of an intermediate which may be a ribotide. This ribotide is
converted into inosinic acid (hypoxanthine ribose-5-phosphate). Evidence
for this is the acceleration in rate of incorporation of radioactive formate
into inosinic acid by the addition of ribose phosphate.
This hypothesis fits the observations of Gots™ that in sulfonamide-in-
hibited EZ. colz the p-aminobenzoic acid added at intervals after inocula-
tion is able to prevent the accumulation of imidazole derivative and to
increase growth but is unable to decrease the amount already formed. This
shows that imidazole derivative lies outside the active metabolic pool and
that the enzyme system formed from p-aminobenzoie acid is unable to
reutilize the imidazole once it has been converted to the free state. Green-
berg has recently obtained evidence that the aminoimidazolecarboxamide
as formed by sulfonamide-inhibited EH. coli is originally formed as the
desoxyriboside.?®
Although aminoimidazolecarboxamide is not an intermediate per se in
the synthesis of hypoxanthine, Schulman e¢ al.?° found that there was ap-
preciable disappearance of C-labeled aminoimidazolecarboxamide when
incubated with pigeon liver homogenates and that radioactive hypoxanthine
was formed.
Buchanan” also found that the conversion of the imidazole derivative to
hypoxanthine is catalyzed by soluble proteins. The presence of insoluble
proteins appears unnecessary for optimal activity. When non-isotopic
inosinic acid and hypoxanthine are incubated with either isotopic glycine
or the isotopic imidazole derivative in pigeon liver homogenate, the inosini¢
acid contains a concentration of C™ three to five times greater than that of
hypoxanthine. These results were interpreted to mean that the element of
ribose is added to the imidazole derivative prior to ring closure with formic
acid.
2. INostinic TRANSFORMYLASE
Buchanan” has observed that incubation of formate with inosinate in a
pigeon liver system results in rapid exchange of the carbon in position 2 of
the hypoxanthine with the carbon of formic acid. This enzyme system was
termed ‘“‘inosinic transformylase’” and was found to be catalyzed by the
citrovorum factor. This represents the first partially purified enzyme sys-
tem in which the function of a PGA derivative can be clearly recognized.
24 J. 8S. Gots, Federation Proc. 9, 178 (1950).
2° G. R. Greenberg, Information presented at American Society of Biological Chem-
ists, New York, 1952. Federation Proc. (In press, 1952).
26 M. P. Schulman, J. M. Buchanan, and C. 8. Miller, Federation Proc. 9, 225 (1950).
27 J. M. Buchanan, Abstr. 119th Meeting, Am. Chem. Soc. p. 13C, 1951.
*8 J. M. Buchanan, cited by A. D. Welch, Pharmacol. Revs. 3, 345 (1951).
IV. BIOCHEMICAL SYSTEMS 136
3. Errect oF AMINOPTERIN ON PURINE SYNTHESIS IN MICE
Skipper ef al.*° found that administration of 4-aminopteroylglutamic acid
(A-aminopterin) or 4-amino-N?°-methylpteroylglutamie acid (A-methop-
terin) decreases the incorporation of radioactive formate into the nucleic
acids and purine fractions to one-fifteenth the normal rate. The antagonists
were administered for 7 days prior to the injection of formate.
4. RELATION OF p-AMINOBENZOIC AcID TO PGA
The presence of p-aminobenzoic acid in PGA and the similarity in func-
tion has fostered the attractive hypothesis that the p-aminobenzoic acid
functions as a precursor of PGA. There is no doubt that under certain con-
ditions this is true. Whether it is always the case remains a disputed point
in spite of the large mass of data brought to bear on this subject.
If the only function of p-aminobenzoic acid is for synthesis of PGA, then
the following conditions might be expected: (a) PGA should promote
growth in organisms which require p-aminobenzoie acid; (b) PGA should
reverse sulfonamide non-competitively in all susceptible organisms; (c)
organisms which require PGA should be resistant to sulfonamides.
There are organisms whose nutritional requirements for p-aminobenzoic
acid can be met by PGA; in other cases this is not true. Pteroylglutamic
acid counteracts the toxicity of sulfonamide in only a few organisms. How-
ever, those organisms which do require PGA are resistant to sulfonamide.
In considering the data pertinent to this subject it might be well to divide
them into two categories: first, those in which PGA and p-aminobenzoic
acid have a common function; second, those in which they are not inter-
changeable.
a. Biochemical Systems Where p-Aminobenzoic Acid Functions via PGA
1. Sulfanilamide inhibits the synthesis of PGA by both susceptible and
resistant strains of F. colz.*° Levels of sulfanilamide (0.005 17) which pro-
duce only partial inhibition of growth decrease the PGA synthesis to less
than | % of the control level. In resistant strains inhibition of PGA synthe-
sis is less marked, but no reduction in growth occurs. This effect is not due
to a general lowering of cellular activity, as no marked effect on biotin
synthesis occurs.
2. Increasing concentrations of p-aminobenzoic acid increase the produc-
tion of PGA by growing cultures of L. arabinosus, the yield being approxi-
mately 5% on a molar basis.*!
29 H. E. Skipper, J. H. Mitchell, Jr., and L. L. Bennett, Jr., Cancer Research 10, 510
(1950).
30 A. K. Miller, Proc. Soc. Exptl. Biol. Med. 57, 151 (1944).
31H. P. Sarett, J. Biol. Chem. 171, 265 (1947).
134 PTEROYLGLUTAMIC ACID
3. Resting cell suspensions of Streptobacterium plantarum synthesize
PGA (measured by L. casei assay) in a system containing only buffer, glu-
cose, glutamic acid, and p-aminobenzoic acid.” A quantitative relationship
exists between the amount of PGA synthesized and the concentration of
p-aminobenzoic acid used. It is also significant that synthesis of PGA
begins at that level at which p-aminobenzoic acid becomes limiting for the
growth of organism. Synthesis of PGA is inhibited by sulfonamide and
reversed in a competitive manner by p-aminobenzoic acid.
4. The p-aminobenzoic acid requirement (0.1 y per milliliter) of L.
arabinosus can be replaced by approximately thirty times as much PGA
(Lampen and Jones*) or by much larger quantities of thymine (50 y per
milliliter) in the presence of a purine. The amount of PGA needed is thus
approximately 3.0 my per milliliter or about five times the requirement of
S. faecalis for this vitamin. The ability of a combination of thymine plus a
purine to produce maximum growth with L. arabinosus in the absence of
PGA parallels that of S. faecalis. In L. arabinosus the function of p-amino-
benzoic acid seems solely that of PGA synthesis which in turn mediates
the synthesis of thymine plus purine. The low activity of PGA compared
to p-aminobenzoic acid suggests that PGA is not the active biological form
synthesized by the organism from p-aminobenzoic acid. Citrovorum factor
is no more active than PGA in this system.**4
Sulfapyridine inhibits the growth of L. arabinosus and is competitively
reversed by p-aminobenzoic acid and non-competitively by both PGA (10
my per milliliter) and thymine (30 y per milliliter).*? The amount of PGA
required to induce growth in the presence of a sulfonamide approximates
that which is effective in its absence. Similar sulfonamide—p-aminobenzoic—
PGA relationship results are obtained with Streptobacterium plantarum and
with a strain of L. arabinosus trained to grow in the absence of p-amino-
benzoic acid.
5. Lampen and Jones*®> observed that L. casei and S. faecalis R which
require preformed PGA are relatively insensitive to sulfonamides. S.
faecalis Raltson, however, which requires PGA for rapid early growth, is
sensitive to sulfonamides, and this inhibition is reversed non-competitively
by PGA or by thymine. p-Aminobenzoic acid at a level equal to that of
PGA has only a small effect on sulfonamide toxicity. The amount of PGA
required to reverse the effect. of sulfonamide corresponds to that required
for growth of S. faecalis R.
Three pairs of different strains of enterococci (S. faecalis, S. zymogenes,
32 R. H. Nimmo-Smith, J. Lascelles, and D. D. Woods, Brit. J. Exptl. Pathol. 29,
264 (1948).
33 J. O. Lampen and M. J. Jones, J. Biol. Chem. 170, 133 (1947).
34H. P. Sarett, Arch. Biochem. 34, 378 (1951).
35 J. O. Lampen and M. J. Jones, J. Biol. Chem. 166, 435 (1946).
IV. BIOCHEMICAL SYSTEMS 135
and S. durans), one member of which required PGA and the other did not,
were compared for their sulfonamide sensitivity by Lampen and Jones.*®
None of the cultures requiring preformed molecule were inhibited by 1280
y of sulfadiazine per milliliter. Those members of strains which were able to
synthesize their own PGA were inhibited by 0.3 ug. of sulfonamide per
milliliter but became insensitive when supplied with an amount of pteroyl-
triglutamic acid equal to that used to promote growth of PGA-requiring
members of the same strain.
Thus in the case of the lactic acid bacteria and the Streptococci the evi-
dence is in harmony with the thesis that p-aminobenzoic functions solely
in the synthesis of PGA and that sulfonamide inhibits this conversion.
6. PGA behaves as a non-competitive sulfonamide antagonist for Cl.
acetobutylicum (two strains) and Streptobacterium plantarum (three strains).
These organisms, which require p-aminobenzoic acid, will also respond to
ten to one hundred times as much PGA. The amount of PGA necessary to
reverse the sulfonamide is approximately the same as that required to pro-
mote growth in its absence and is independent of the amount of sulfonam-
ide.** This is similar to results obtained with Streptobacterium plantarum.®
7. The Le. citrovorum factor (folinic acid) is able to replace p-amino-
benzoic acid for growth of L. mesenteroides and overcomes sulfonamide in-
hibition in a non-competitive manner. Neither PGA nor N!-formyl PGA
is active in this respect. These observations of Lascelles et al.3? lend support
to the view that the inactivity of PGA in replacing p-aminobenzoic acid
may reflect the inability of the organism to convert preformed PGA to the
more metabolically active form.
b. Biochemical Systems Where PGA Cannot Replace p-Aminobenzoic Acid
1. A p-aminobenzoic acid-less mutant strain of H. coli was observed
which would not respond to PGA.*§ Growth could be induced either by low
concentrations of p-aminobenzoic acid (0.2 y per milliliter) or by a combina-
tion of amino acids, purine, and thymine. This mutant strain, when grown
in the presence of 0.01 y of p-aminobenzoic acid per milliliter, produces a
material giving a growth response with S. faecalis on a PGA-free medium.
With other organisms, such as Acetobacter suboxydans 621 and a Neuro-
spora mutant, which require p-aminobenzoic acid, PGA reverses the action
of sulfonamide in a non-competitive manner.*®
2. PGA is unable to affect the inhibition ratio of sulfanilamide for LZ.
coli at the levels where methionine, purine, or serine synthesis is affected.
At the level of thymine synthesis, PGA is effective.*: !° The lack of activity
36 R. H. Nimmo-Smith and D. D. Woods, J. Gen. Microbiol. 2, x (1948).
7 J. Lascelles, M. J. Cross, and D. D. Woods, Biochem. J. 49, Ixvi (1951).
8 J. O. Lampen, M. J. Jones, and R. R. Roepke, J. Biol. Chem. 180, 423 (1949).
136 PTEROYLGLUTAMIC ACID
of PGA in sulfanilamide-inhibited HF. coli is similar to that observed with
E. coli mutants (Lampen et al.**).
In summary, it can be said that in many organisms PGA can replace the
requirement for p-aminobenzoic acid. This especially seems true where the
function of p-aminobenzoic acid is concerned only with synthesis of purines
and pyrimidines. In /. coli, where p-aminobenzoic acid is concerned in the
synthesis of methionine, serine, purine, and pyrimidine, PGA has been
implicated directly only in the synthesis of thymine and indirectly in that
of adenine. How then does p-aminobenzoic acid function in the synthesis
of methionine and serine? Three hypotheses seem probable: (a) p-Amino-
benzoic acid is converted efficiently to a PGA-lke type of coenzyme, but
the conversion of preformed PGA to this coenzyme is very inefficient; (b)
p-aminobenzoic acid functions as a coenzyme without being incorporated
into a PGA-like type of molecule; (c) p-aminobenzoic acid functions via
vitamin By» in the synthesis of methionine and serine.
Unfortunately little direct experimental data can be brought to bear on
this point. However, it is significant that the synthesis of methionine,
serine, and adenine, which cannot be taken over by PGA in E. coli, can be
ascribed to PGA function in other types of organisms. This will be discussed
in greater detail in the next section.
5. Rote or PGA IN SyntHeEsis oF AMINO ACIDS
PGA promotes serine synthesis in the growth of S. faecalis R.**: *° It has
been observed* that pteroylglutamic acid is essential for the synthesis of
serine from glycine and formate by resting washed cells of S. faecalis R.
In this particular system N!°-formyl PGA is 30% more active than PGA.
Tetrahydro PGA and citrovorum factor are no more active than PGA
when added simultaneously with other components. However, when the
cells are first incubated with citrovorum factor in glucose-phosphate, and
the glycine and formate added subsequently, serine synthesis is more rapid
than when PGA is similarly used. Also, in the above systems formation of
citrovorum factor accompanies the synthesis of serine promoted by PGA.
These same workers*® also found that the serine requirement for growth by
Leuconostoc mesenteroides can be replaced by high concentrations of glycine
plus a high carbon dioxide tension and that the citrovorum factor greatly
reduces the carbon dioxide requirement. It thus appears that carbon dioxide
may serve as the source of the single-carbon fragment in this experimental
system. The very small amount of citrovorum factor used precludes the
possibility of its serving as a stoichiometric source of the single-carbon frag-
ment, and this factor must function instead as a more efficient catalyst
39 Hf. P. Broquist, Thesis, University of Wisconsin, 1949.
'0 B. R. Holland and W. W. Meinke, J. Biol. Chem. 178, 7 (1949).
IV. BIOCHEMICAL SYSTEMS 137
which reduces the need for mass action pressure of large amounts of carbon
dioxide.
Serine synthesis in the rat from isotopic formate is decreased in PGA
deficiency.*!
PGA has been similarly shown to be involved in methionine synthesis
from homocystine in animals,” and methionine synthesis in yeast can be
inhibited by 4-aminopteroylglutamic acid and the action of the antagonist
reversed by the citrovorum factor.”
6. PGA 1n SINGLE-CARBON TRANSFER IN ANIMALS
Pteroylglutamic acid has been shown to be involved in the synthesis of
serine, the conversion of serine to glycine, the synthesis of methionine, and
the synthesis of purines in the rat. Both vitamin By: and PGA are concerned
in the synthesis of choline and methionine.
a. Serine-Glycine Relationships
Incorporation of radioactive formate into serine is greatly depressed in
PGA deficiency induced by sulfonamide feeding (Plaut et al.*!). Treatment
with PGA for 4 days prior to formate administration increases C™ fixation
into liver protein tenfold. Serine synthesis is most markedly affected;
glutamic acid, arginine, and glycine are less affected. The carbon of formate
was predominantly incorporated into the 6-carbon of serine.
Heme from the blood of PGA-treated rats also contains an appreciable
quantity of C™, whereas that from deficient rats contains none. Biotin
deficiency has no influence on incorporation of C“ from formate into
8-carbon of serine, showing that this effect of PGA deficiency is a specific
one and is not the result of a general manifestation of vitamin deficiency
such as inanition.
Pteroylglutamic acid is also concerned in the conversion of serine to
glycine for hippuric acid synthesis in the rat (Elwyn and Sprinson“'). From
the lower utilization of serine and the greater utilization of glycine in the
PGA-deficient rat, it has been estimated that the rate of conversion of
serine to glycine is reduced to one-sixth of the normal value in PGA defi-
ciency.
A Comparison has also been made of the rates of incorporation of a-carbon
of glycine and 6-carbon of serine into choline.*® By. is concerned mainly
with the utilization of a-carbon of glycine but not the utilization of 6-car-
1G.W.E. Plaut, J. J. Betheil, and H. A. Lardy, J. Biol. Chem. 184, 795 (1950).
#2 J. S. Dinning, C. K. Keith, and P. L. Day, J. Biol. Chem. 189, 515 (1951).
48H. P. Broquist, Federation Proc. 11, 191 (1952).
** TD. Elwyn and D. B. Sprinson, J. Biol. Chem. 184, 475 (1950).
45 J. A. Stekol, S. Weiss, and K. W. Weiss, Arch. Biochem. and Biophys. 36, 5 (1952).
138 PTEROYLGLUTAMIC ACID
bon of serine. Conversely PGA deficiency reduces the utilization of 8-carbon
of serine and to a smaller extent that of the a-carbon of glycine. These re-
lationships are expressed schematically in Fig. 9.
The zn vitro incorporation of glycine, containing C™ in the carboxyl posi-
tion, by chick liver homogenates is decreased in PGA deficiency (Totter
et al.*°). This effect was observed both in the liver proteins and the phospho-
lipid fractions and was not influenced by in vitro addition of PGA to the
deficient liver homogenates.
Elwyn and Sprinson” found that the methyl group of thymine is derived
from the a-carbon of glycine and the 8-carbon of serine. This indicates
that the methyl carbon of thymine is derived from a one-carbon intermedi-
ate by methylation of a pyrimidine nucleus.
b. Counteraction of Glycine Toxicity by PGA
Feeding large amounts of glycine depresses the growth of chicks and rats.
The growth depression induced by 10 % glycine in rats can be prevented by
*
By
HO NH,
H,C—CH,—COOH
HCOOH
PGA
Fia. 9. Effect of Biz and PGA on conversion of glycine and serine to ‘formate
pool.”’
supplementation with PGA whereas a liver extract containing vitamin By»
is ineffective (Dinning e¢ al.*®). Similarly Machlin et al.49 found that 6%
glycine depresses the growth of chicks and that additional quantities of
PGA prevent the growth depression. It is thus apparent that the PGA re-
quirements are increased by high levels of glycine.
c. Effect on Purine Synthesis in Animals
Drysdale e¢ al.*° studied the effect of PGA depletion in rats on the in-
corporation of formate into purines. The rats were rendered deficient in
PGA by maintenance on a sulfasuxidine-containing diet until they exhibited
symptoms of leucopenia. The control animals were given PGA for 4 days,
which induced a remission of the leucopenia. This procedure of depleting
46 J. R. Totter, B. Kelley, P. L. Day, and R. R. Edwards, Federation Proc. 9, 238
(1950).
47 J). Elwyn and D. B. Sprinson, J. Am. Chem. Soc. 72, 3317 (1950).
48 J. S. Dinning, C. K. Keith, P. L. Day, and J. R. Totter, Proc. Soc. Expil. Biol.
Med. 72, 262 (1949).
49 L,. J. Machlin, C. A. Denton, and H. R. Bird, Federation Proc. 10, 388 (1951).
60 G. R. Drysdale, G. W. E. Plaut, and H. A. Lardy, J. Biol. Chem. 198, 533 (1951).
IV. BIOCHEMICAL SYSTEMS 139
both groups of animals and then administering PGA to one group for a
short time makes the experiment more specific for PGA and eliminates
general effects of inanition which might occur. Under these conditions, in-
corporation of formate into adenine and guanine in the liver is depressed
to approximately 30% of that in the PGA supplemented. No difference be-
tween deficient and repleted animals was noted in visceral purines. PGA
deficiency decreases the entrance of formate into the C-2 and C-8 positions
of liver purines equally, showing that PGA is involved in the incorporation
of both these atoms.
In the formation of 4-amino-5-imidazolecarboxamide during 4-amino-
pteroylglutamic acid inhibition of F. coli!® the incorporation of the 2-carbon
is the only one affected. Apparently in this microorganism the incorporation
of the 8-carbon is not inhibited, as this is a part of the imidazole molecule.
If PGA is concerned equally with both C-2 and C-8, it is possible to under-
stand why the imidazole derivative has not been observed during growth
of any lactic acid organism on a medium devoid of PGA and supplemented
with purine.
d. Thymine and Purines in Nutrition of Animals
The ability of thymine to replace PGA partially or completely in the
nutrition of certain bacteria has prompted interest in whether thymine can
replace PGA in the nutrition of animals. In the rat (Daft*!) and in the chick
(Stokstad et al.7) thymine is inactive both in growth stimulation and
hematopoietically. Thymine is ineffective for rats both on a PGA-free diet
containing sulfasuxidine and on a diet containing crude ‘‘x-methyl PGA”’
antagonist. Adenine is also ineffective in promoting growth in these experi-
ments. More recently Daft®? has found that a combination of adenine and
thymine, each at a level of 1%, produces a marked leucocyte and granulo-
cyte response in rats rendered deficient in PGA by the feeding of sulfona-
mide. No growth response is obtained, however. The animals remain or be-
come anemic, and kidney damage develops. Adenine appears to account
for most of the activity of the mixture. It was further observed that the
agranulocytosis symptoms of PGA deficiency which had previously been
observed on low protein diets can be corrected by a mixture of methionine,
tryptophan, and threonine plus either adenine, adenosine, or yeast extract.
Previously, mixtures of the three amino acids had been found ineffective
unless supplemented with PGA. This suggests that PGA-deficient rats are
unable to synthesize amounts of adenine needed for white cell formation.
The apparent inactivity of thymine is in accord with the information gained
51 F.S. Daft, Ann. N. Y. Acad. Sci. 48, 299 (1946).
82 F.S. Daft, Federation Proc. 11, 200 (1952).
140 PTEROYLGLUTAMIC ACID
by isotope studies that adult rats cannot utilize free thymine for nucleic
acid synthesis.**
Pigs, however, have been observed to give a hematological response to
thymine in doses of 10 g. daily (Welch and Heinle‘).
Of special interest is the fact that patients with pernicious anemia respond
hematologically to thymine, uracil, and thymidine. Spies ef al.°® found that
15 g. of thymine is capable of producing a hematological response similar to
that induced by PGA. About five thousand times as much thymine as PGA
is required. This ratio is similar to the relative effectiveness of these two
compounds in promoting the growth of S. faecalis (Stokes). In view of the
ineffectiveness of uracil in replacing thymine in the nutrition of PGA-
requiring organisms, the observations of Vilter e¢ al.°® that uracil in doses
of 15 to 30 g. per day often causes hematological remission are of special
interest. Quite surprising also is the fact that thymine proved effective in a
pernicious anemia patient who had become refractory to PGA.
Thymidine, which is able to substitute for vitamin By in the nutrition
of certain lactic acid bacteria, has been reported by Hausmann*” to be
effective in total doses of 2.0 and 2.8 g., respectively, and to produce a
response comparable to that elicited by PGA or vitamin By in pernicious
anemia.
e. Influence of PGA and Vitamin By, on Methionine and Choline Synthesis
Both vitamin Biz and PGA have been shown to have a role in choline
and methionine metabolism in chicks and rats. This was first shown by the
work of Bennett,°® who found that rats with a certain previous dietary
history were able to utilize homocystine for growth in the absence of labile
methyl donors when supplemented with PGA and with purified liver extract
containing vitamin B,.. This requirement for PGA was observed both in
the absence and presence of sulfasuxidine. Since rats normally do not de-
velop PGA deficiency on a purified type of diet unless supplemented with
sulfonamide, the results of Bennett suggest that the PGA requirements of
the rat are increased on a “‘labile methyl’’-deficient diet. Both vitamin By
and PGA are required for synthesis of methionine from homocystine under
this regimen.
On a methionine-deficient diet (containing alcohol-extracted peanut meal
53 A. A. Plentl and R. Schoenheimer, J. Biol. Chem. 158, 203 (1944).
54 A.D. Welch and R. W. Heinle, Pharmacol. Revs. 3, 345 (1951).
55T. D. Spies, W. B. Frommeyer, Jr., C. F. Vilter, and A. English, Blood 1, 185
(1946).
56 R. W. Vilter, D. Horrigan, J. F. Mueller, T. Jarrold, C. F. Vilter, V. R. Hawkins,
and A. Seaman, Blood 5, 695 (1950).
57 K. Hausmann, Lancet I, 329 (1951).
53M. A. Bennett, Sczence 110, 589 (1949).
IV. BIOCHEMICAL SYSTEMS 141
and oxidized casein) supplemented with homocystine, there is a relation-
ship between homocystine, choline, PGA, and vitamin By». requirements
(Schaefer and Knowles*’). On sucha diet no growth takes place, and there
is a 100% incidence of renal hemorrhage. The effects of methyl donors,
vitamin By , and PGA are shown in Table III. The results clearly indicate
that choline alone or a combination of betaine plus aminoethanol will pro-
tect against renal hemorrhage in the absence of both vitamin By and PGA.
On diets marginal in choline or betaine neither By. nor PGA has any effect
on renal damage. Complete protection can be achieved with a combination
TABLE III
Errect OF VITAMIN By AND PGA ON THE UTILIZATION OF BETAINE, CHOLINE, AND
AMINOETHANOL
Gain in Incidence of
Choline, Betaine, Aminoethanol, Vitamin Bio, PGA, body wt. in renal
g./kg. g./kg. g./kg. y/kg. mg./kg. 2wk.,g. hemorrhage, %
gat 0 100
0.8 a “f 14 75
0.8 60 a 28 50
0.8 a 2 17 50
0.8 60 2 35 0
1.6 P ts 14 0
1.6 60 er: 56 0
1.6 — 2 19 0
eG 60 2 62 0
3.2 0.3 “fi a —] 100
3.2 0.3 60 oe 0 100
3.2 0.3 ae 2 0 100
3.2 0.3 60 2 40 0
6.0 0.6 Pe Re 33 0
6.0 0.6 60 2 43 0
of these two vitamins or by increasing the level of betaine or choline. This
clearly shows that the requirements for PGA are higher on diets marginal
in choline.
Although a combination of vitamin By, and PGA affords complete pro-
tection against renal damage on diets marginal in choline, it does not pro-
duce a maximum growth response. Maximum growth is given only by
higher levels of choline supplemented with vitamin By. , PGA having little
effect on growth at these levels of choline.
A similar effect of PGA and By on lipotropism has been noted in rats
(Schaefer et al.®°). Fatty livers were noted in rats receiving 0.1 % of choline
59 A. EK. Schaefer and J. L. Knowles, Proc. Soc. Exptl. Biol. Med. 77, 655 (1951).
60D). R. Strength, E. A. Schaefer, and W. D. Salmon, J. Nutrition 45, 329 (1951).
142 PTEROYLGLUTAMIC ACID
which prevented kidney hemorrhage. Protection against lipotropism could
not be obtained by vitamin By. or PGA alone but was achieved with a com-
bination of By. and PGA or with a higher level of choline.
The influence of PGA on utilization of methanol as a source of methyl
groups in choline has been demonstrated by Verly et al.*! C'4-Deuterio-
methanol was injected into rats deficient in PGA and into similar rats
treated with PGA prior to methanol administration. PGA caused a two-
to threefold increase in utilization of methanol as measured by the C™“ and
deuterium content of isolated choline. Vitamin By under similar conditions
was without effect.
f. In Vitro Studies on Methionine Formation
Livers from PGA-deficient chicks exhibit reduced ability to form methi-
onine from homocystine in the presence of choline or betaine (Dinning et
al.*”). Choline oxidase is also decreased by PGA deficiency as evidenced by
slower oxidation of choline. Jn vitro addition of PGA to the deficient liver
does not affect methionine synthesis but addition of PGA to repleted livers
does. This effect of added PGA in vitro is difficult to understand. The effect
of PGA on choline oxidase offers an explanation for the effect of this vita-
min on methionine synthesis as it has been suggested that choline is first
oxidized to betaine before transmethylation can occur.
V. Specificity of Action
HL Ry SrOKSTAD
A large number of PGA analogs have been made. Most of these behave
as antagonists and will be considered in another section of this chapter.
Certain modifications can be made in the molecule without nullifying the
biological activity, but they may modify the activity for different species.
This information, which can best be presented in tabular form, is shown in
Table IV.
It is apparent from these data that the higher animals, with the exception
of the fox, are capable of utilizing derivatives of PGA containing more than
one glutamic acid molecule. It is of special interest that the diglutamic acid
derivative containing an a linkage is utilized efficiently by the chick even
though it has low biological activity for L. casei and S. faecalis. Chicken
liver conjugase has been shown to split a-glutamic acid linkages more slowly
61 W. G. Verly, J. E. Wilson, J. M. Kinney, and J. R. Rachele, Federation Proc. 10,
264 (1951).
143
V. SPECIFICITY OF ACTION
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144 PTEROYLGLUTAMIC ACID
than the y linkages (Dabrowska et al.'). Both pteroic acid and its N?°-
formyl derivative are inactive for the higher animals, Tetrahymena geleti
and L. casei. They are active only for S. faecalis and other Enterococci.
1W. Dabrowska, A. Kazenko, and M. Laskowski, Science 110, 95 (1949).
2H. L. R. Stokstad, B. L. Hutchings, J. H. Mowat, J. H. Boothe, C. W. Waller,
R. B. Angier, J. Semb, and Y. SubbaRow, J. Am. Chem. Soc. 70, 5 (1948).
3 J. H. Mowat, B. L. Hutchings, R. B. Angier, E. L. R. Stokstad, J. H. Boothe,
C. W. Waller, J. Semb, and Y. SubbaRow, J. Am. Chem. Soc. 70, 1096 (1948).
4T. D. Spies, R. HE. Stone, and R. L. Toca, Southern Med. J. 40, 175 (1947).
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and Y. SubbaRow, J. Am. Chem. Soc. 71, 2310 (1949).
6 J. H. Boothe, J. H. Mowat, B. L. Hutchings, R. B. Angier, C. W. Waller, E. L. R.
Stokstad, J. Semb, A. L. Gazzola, and Y. SubbaRow, J. Am. Chem. Soc. 70, 1099
(1948).
7™W.B. Castle, J. B. Ross, C. S. Davidson, J. H. Burchenal, H. J. Fox, and TI) H:
Ham, Science 100, 81 (1944).
§P.L. Day, V. Mims, J. R. Totter, E. L. R. Stokstad, B. L. Hutchings, and N. H.
Sloane, J. Biol. Chem. 157, 423 (1945).
9F.S. Daft and W. H. Sebrell, Public Health Repts. (U.S.) 58, 1542 (1948).
10 B. L. Hutchings, J. J. Oleson, and E. L. R. Stokstad, J. Biol. Chem. 163, 447 (1946).
1G. W. Kidder and V. C. Dewey, Arch. Biochem. 21, 66 (1949).
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32, 23 (1947).
13M. E. Swendseid, R. A. Brown, O. D. Bird, and R. A. Heinrich, Arch. Biochem.
16, 367 (1948).
14 J.J. Pfiffner, D. G. Calkins, B. L. O’Dell, E.S. Bloom, R. A. Brown, C. J. Camp-=
bell, and O. D. Bird, Science 102, 228 (1945).
15 C, W. Waller, B. L. Hutchings, J. H. Mowat, E. L. R. Stokstad, J. H. Boothe,
R. B. Angier, J. Semb, Y. SubbaRow, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith,
Jr., J. Am. Chem. Soc. 70, 19 (1948).
16R. B. Angier, J. H. Boothe, B. L. Hutchings, J. H. Mowat, J. Semb, E. L. R.
Stokstad, Y. SubbaRow, C. W. Waller, D. B. Cosulich, M. J. Fahrenbach, M. E.
Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith,
Jr., Sczence 108, 667 (1946).
7 T. D. Spies, G. G. Lopez, R. E. Stone, F. Milanes, R. O. Brandenberg, and T.
Aramburu, Blood 8, 121 (1948).
18 G. W. Kidder and V. C. Dewey, Proc. Natl. Acad. Sci. U. S. 38, 95 (1947).
19TH. Jukes and E. L. R. Stokstad, Physiol. Revs. 28, 51 (1948).
20D. E. Wolf, R. C. Anderson, E. A. Kaczka, S. A. Harris, G. A. Arth, P. L. South-
wick, R. Mozingo, and K. Folkers, J. Am. Chem. Soc. 69, 2753 (1947).
21. L. Rickes, L. Chaiet, and J. C. Keresztesy, J. Am. Chem. Soc. 69, 2749 (1947).
2G. W. Kidder and R. C. Fuller, IIT, Sczence 104, 160 (1946).
23 J. C. Keresztesy, E. L. Rickes, and J. L. Stokes, Science 97, 465 (1948).
24M. Gordon, J. M. Ravel, R. E. Eakin, and W. Shive, J. Am. Chem. Soc. 70, 878
(1948).
25 R. J. Williams, R. E. Eakin, E. Beerstecher, Jr., and W. Shive, 7n Biochemistry
of B Vitamins, p. 567. Reinhold Publishing Corp., New York, 1950.
V. SPECIFICITY OF ACTION 145
A. RELATION OF XANTHOPTERIN TO PGA
The role of xanthopterin in nutrition is an enigma in the PGA problem.
From the beginning, xanthopterin has crossed and recrossed the trail, some-
times guiding, sometimes serving only to confuse those intent on learning
the nature and function of PGA. This yellow pigment, one of a class of com-
pounds found in the wings of butterflies, has a structure closely related to
the pteridine portion of PGA.
OH OH
ke |
Soin 20H 4~/N\_on
ae tena
Ce 4
HN \w/\N Z H.N \n /\n 7 OH
Xanthopterin Leucopterin
Although our knowledge of xanthopterin originated in 1891 when Hop-
kins began his investigations on the wing pigments of the English brim-
stone butterfly,?® it was not until 1941 that Wieland and his collaborators
completed the degradation work with xanthopterin and leucopterin, and
Purrman”’ finally accomplished their synthesis. The problem of obtaining
sufficient material for chemical degradation studies was an acute one, and
altogether over 1,000,000 butterflies of various types were used by differ-
ent workers in this field.*8
Xanthopterin has also been isolated from human and animal urine and
designated uropterin by Koschara.**: *° It occurs in insects other than the
lepidoptera; it has been detected in the crab but apparently not in plants.”®
Rominger ef al.*! found that rats 3 to 4 weeks old developed macrocytic
anemia when fed exclusively on goat’s milk. The erythrocyte count fell to
around | million, and the animals did not respond to supplementation with
iron. Typical reticulocyte responses were obtained with liver preparations.”
Tschesche and Wolf* found that injection of uropterin (xanthopterin) ob-
tained from Koschara produced a rise in the erythrocyte count. A daily
dose of 1.0 y produced an elevation of 2 to 3 million in the erythrocyte
count in 14 days. Ten micrograms produced no greater response than 1.0
26 F. G. Hopkins, Nature 45, 197 (1891), cited by M. Gates, Chem. Revs. 41, 63 (1947).
27 R. Purrman, Ann. 548, 284 (1941).
28 M. Gates, Chem. Revs. 41, 63 (1947).
29 W. Koschara, Z. physiol. Chem. 240, 127 (1936).
80 W. Koschara, Z. physiol. Chem. 277, 159 (1943).
31 E. Rominger, H. Meyer, and C. Bomskov, Z. ges. erptl. Med. 89, 786 (1933).
32 E. Rominger and C. Bomskov, Z. ges. exptl. Med. 89, 818 (1933).
83 R. Tschesche and H. J. Wolf, Z. physiol. Chem. 244, I, (1936).
146 PTEROYLGLUTAMIC ACID
y. In contrast to the preceding report*! concerning the ineffectiveness of
iron, the anemia was found to respond to iron and copper as well as to
uropterin. These results with xanthopterin were repeated by the same
authors** with other samples of xanthopterin. However, Koschara reported
that Rominger was unable to get any response with the same sample of
uropterin that Tschesche had used in his experiments.*°
Xanthopterin was also found to have a hemapoietic effect when injected
into fingerling salmon with nutritional anemia.*® The anemia was produced
by feeding the salmon a high protein diet containing yeast as a source of
the vitamin B complex. The erythrocyte counts of the control fish varied
between 501,000 and 916,000 per cubic millimeter, whereas in those in-
jected with 50 y of xanthopterin the counts varied between 944,000 and
1,305,000. In a subsequent study these same investigators*” observed a
similar anemia on salmon raised on diets of 20 % liver and 80 % spawned-out
salmon. The ability of salmon to become anemic on a diet containing 20 %
liver certainly indicates that PGA is ineffective in this blood dyscrasia. A
larger variability existed in the erythrocyte count of these fish, but by
selection a group of uniform experimental fish having an average count of
800,000 cells per cubic millimeter was obtained. Injection of a single dose
of xanthopterin at a level of 10 mg. per kilogram of body weight increased
the RBC to 1,300,000 per cubic millimeter; 20 and 40 mg. per kilogram of
body weight increased it to approximately 1,580,000, with the peak on the
third to fifth day. The erythrocyte count returned to its original level
within 10 to 14 days. The erythrocyte counts of normal wild salmon caught
in fresh or salt water were in the range of 1,500,000 to 1,600,000. Compari-
son of different species of salmon raised on the experimental diet and having
the same initial erythrocyte count showed that different species varied in
their response to xanthopterin.
Variable results have been reported with xanthopterin in sulfonamide-fed
rats. Totter and Day*®* observed that 20 y of xanthopterin administered
daily to rats receiving 1% sulfasuxidine produced an immediate weight
gain and increased the leucocytes to 9400 cells per cubic millimeter, com-
pared with 3420 for the controls. The growth rate and the white blood cells
were not completely restored to normal, which showed that the response
produced by xanthopterin was qualitatively different from that produced
by liver fractions. These results with xanthopterin have not been confirmed
34 R. Tschesche and H. J. Wolf, Z. physiol. Chem. 248, 34 (1987).
35 W. Koschara and A. Hrubesch, Z. physiol. Chem. 258, 39 (1989).
36 R. W. Simmons and E. R. Norris, J. Biol. Chem. 140, 679 (1941).
37H}. R. Norris and R. W. Simmons, J. Biol. Chem. 158, 449 (1945).
38 J. R. Totter and P. L. Day, J. Biol. Chem. 147, 257 (1948).
V. SPECIFICITY OF ACTION 147
by others,’: **: 4° or by the same workers.*® O’Dell and Hogan*! have stated
that xanthopterin is inactive in chicks fed on a diet low in PGA.
In vitamin M deficiency in the monkey, xanthopterin has produced more
consistent effects. Totter and coworkers® found that administration of 2.5
to 10 mg. of xanthopterin to vitamin M-deficient monkeys produced a
reticulocytosis in 3 to 6 days. The white and red cell counts made transient
increases to normal levels in 3 to 13 days. Xanthopterin alone given pro-
phylactically failed to protect completely but did delay the onset of nu-
tritional cytopenia. One animal given xanthopterin plus heated liver (shown
to be ineffective by itself) maintained a normal white and red cell count for
71 days. Removal of xanthopterin at this point resulted in prompt return
of cytopenia, and reinstatement of xanthopterin therapy produced a re-
sponse similar to the first. Cerecedo and his associates found that xanthop-
terin, like PGA, produced significant improvement in the lactation per-
formance of mice* and of rats.4* Definite but small hematological responses
have been noted by Heinle et al.*° in two PGA-deficient pigs given 10 mg.
of xanthopterin parenterally for 10 days. A third pig responded to a single
dose of xanthopterin.*® Cartwright et al.47 observed a reticulocyte response
of 9% in one similarly deficient pig given a single injection of 20 mg. of
xanthopterin.
The larva of the insect L'phestia kuehneola is able to utilize xanthopterin
in place of PGA but at levels one thousand times that of the vitamin.®
Another curious and still largely unexplained effect of xanthopterin was
noted early by Wright and Welch.*? When fresh rat liver is incubated with
xanthopterin a marked increase in the folic acid content of the preparation
is observed as compared with rat liver incubated either alone or with
leucopterin or certain purines or pyrimidines. In a more detailed investiga-
tion of this phenomena the same workers‘? found that the folic acid content
of rat liver and muscle was influenced during digestion by factors such as
39. D. Wright and A. D. Welch, Science 98, 179 (1948).
40 A. E. Axelrod, P. Gross, M. D. Bosse, and K. F. Swingle, J. Biol. Chem. 148, 721
(1943).
41B. L. O’Dell and A. G. Hogan, J. Biol. Chem. 149, 323 (1943).
42 J. R. Totter, C. F. Shukers, J. Kolson, V. Mims, and P. L. Day, J. Biol. Chem.
152, 147 (1944).
43 T,. Mirone and L. R. Cerecedo, Arch. Biochem. 15, 324 (1947).
44 A. J. Sica, A. M. Allgeier, and L. R. Cerecedo, Arch. Biochem. 18, 119 (1948).
45 R.W. Heinle, A. D. Welch, and H. L. Shorr, J. Lab. Clin. Med. 34, 1763 (1949).
46 A.D. Welch and R. W. Heinle, Pharmacol. Revs. 3, 345 (1951).
7G. E. Cartwright, J. Fay, B. Tatting, and M. M. Wintrobe, J. Lab. Clin. Med. 33,
397 (1948).
48G. Fraenkel and M. Blewett, Biochem. J. 41, 469 (1947).
49L. D. Wright, H. R. Skeggs, and A. D. Welch, Arch. Biochem. 6, 15 (1945).
148 PTEROYLGLUTAMIC ACID
xanthopterin, salts, cyanide, degree of dispersion of the tissue, pH of the
tissue, and the addition of taka-diastase. This observation was confirmed
by Totter et al.,°° and it was found that the addition of xanthopterin in-
creased the content of microbiologically active PGA during incubation of
liver from vitamin M-deficient monkeys but not of liver tissue from normal
chickens. This species difference in response of liver tissue to xanthopterin
is further emphasized by the fact that addition of this pterin to hog liver
produces tenfold increases of PGA activity®! in contrast with the earlier
reports of two- to threefold increases with rat liver.
As an explanation of this phenomenon, it was suggested that liver con-
tains a precursor of pteroylheptaglutamie acid which is susceptible to attack
competitively by two types of enzymes.*® One reaction at pH 7.0 leads to
the formation of a compound, presumably pteroylheptaglutamic acid,
which is susceptible to further enzymic conversion at pH 4.7 to give PGA.
A second reaction at pH 7.0 leads to inactivation of this precursor and may
be inhibited by xanthopterin. This hypothesis rests on the following facts:
1. Incubation of pig liver with xanthopterin at pH 7.0 increases the PGA
from an original of 0.25 to a final value of 3.0 y per gram.
2. Incubation at pH 4.7 gives 0.65 y with or without xanthopterin.
3. Incubation for 4 hours at pH 7.0 followed by 14 hours at pH 4.7 gives
18.5 y with xanthopterin.
4. At pH 7.0 pteroylheptaglutamic acid is only partially converted to
PGA; at pH 4.7 the conversion is nearly complete.
If this explanation of the effect of xanthopterin is correct, then the true
amount of PGA derivatives in liver is much higher than that observed with
any combination of autolysis and digestion on unheated liver tissue.
If one accepts this explanation of the effect of xanthopterin, one might
account for the transient and partial responses observed with xanthopterin
in rats. It may be that in PGA-deficient animals there are small amounts of
PGA conjugates in the liver whose major pathway of metabolism would
normally cross that of inactivation. Xanthopterin, by inhibiting this in-
activation, increases the utilizable quantity of the vitamin, and as soon
as the PGA precursor is completely exhausted, no further response can be
elicited by xanthopterin.
The possibility that xanthopterin may be converted to PGA has been
entertained, but recent work with xanthopterin labeled with C™ in the 6
and 7 positions makes such a view unlikely.4* Administration of this pterin
to a PGA-deficient pig did give a small hematological response, but no
radioactivity could be detected in PGA isolated from the liver by use of
the carrier vitamin. In addition, incubation of the liver zn vitro with isotopic
60 J. R. Totter, V. Mims, and P. L. Day, Science 100, 223 (1944).
‘1 A.D. Welch, E. M. Nelson, and M. F. Wilson, Federation Proc. 8, 346 (1949).
V. SPECIFICITY OF ACTION 149
xanthopterin did not lead to the appearance of any labeled carbon in the
PGA isolated from the homogenate.
B. PGA ANTAGONISTS
The study of PGA antagonists has proved to have both academic and
clinical interest. These compounds have facilitated the study of the mech-
TABLE V
Types oF PGA ANTAGONISTS
1. Pyrimidine derivatives
Example: 5-Nitrouracil (Hitchings et al.**)
2. Purine derivatives
Example: 2,4-Diaminopurine (Elion*)
3. Pteridine derivatives
Example: 2,4-Diamino-6,7-diphenylpteridine (Daniel et al.**)
4. Modification of pterin nucleus
Example: Quinoxaline-2-carboxyl-p-aminobenzoylglutamie ‘acid (Woolley and
Pringle®®)
5. Pteroyl derivatives of different amino acids
/' Example: Pteroylaspartic acid (Hutchings et al.*)
6. Alkyl derivatives of pteroylglutamic acid
Example: N!°-Methylpteroylglutamie acid (Cosulich and Smith®)
. Substitution on the pterin nucleus
Example: 4-Aminopteroylglutamic acid (Seeger et. al.5%)
~J
8. Sulfur analogs with a sulfonyl group in place of the carboxyl of p-aminobenzoic
acid
Example: Benzimidazole-2-methyl-p-aminobenzene sulfonylglutamic acid (Ed-
wards et. al.®°)
anism of PGA function in microorganisms and in tissues of higher animals.
They have also provided new tools for the study of leukemic processes and
have proved to have some clinical application in the therapy of leukemia.
A complete cataloguing of the antagonists which have been synthesized
is well outside the scope of this review. For more detailed information the
reader is referred to the excellent reviews by Shive®® and Martin.*? The
2G. J. Martin, Biological Antagonism. Blakiston & Co., New York, 1952.
53G. B. Hitchings, G. B. Elion, E. A. Falco, P. B. Russell, and H. Vander Werff,
Ann. N.Y. Acad. Sci. 52, 1318 (1950).
54 G. B. Elion and G. B. Hitchings, J. Biol. Chem. 187, 511 (1950).
150 PTEROYLGLUTAMIC ACID
discussion will be confined to representatives of the main types of antago-
nists and to a description of their more important biological effects.
The principle classes of compounds which function as PGA antagonists
or in some cases possess PGA-like activity are shown in Table V.
1. SUBSTITUTED PYRIMIDINES
Since pyrimidines are formed by PGA-catalyzed reactions, it is not sur-
prising that substituted pyrimidines function as PGA antagonists under
certain conditions. A large number of pyrimidines have been prepared and
their growth-promoting or growth-inhibiting actions determined. In many
cases the antagonistic action is not reversed by PGA and must therefore
be regarded primarily as an inhibition of pyrimidine or purine function.
Detailed information on pyrimidine and purine antagonists has been
compiled in the excellent reviews of Hitchings e¢ al.®! and Wright.”
Replacement of the 5-methyl group of thymine (5-methyluracil) by
hydroxy, bromine, amino, or nitro groups produces compounds which in-
hibit the growth of L. casez (Hitchings et al.*). Of these compounds, 5-nitro-
uracil was reversed competitively by PGA, 5-aminouracil and 5-bromo-
uracil were competitively reversed by thymine, and 5-hydroxyuracil by
uracil.
Of special interest are the 2,4-diaminopyrimidines and their substituted
ring systems which include the 2,4-diaminopurines and pteridines. Hitch-
ings et al. found that nearly all 2 ,4-diaminopyrimidines inhibited L. casez
in the presence of PGA and in the absence of a purine. A possible structural
similarity between the PGA antagonist 2 ,4-diamino-5-p-chlorophenoxy-
pyrimidine and the antimalarial paludrine suggested that the former com-
pound may have antimalarial properties and the latter may have anti-PGA
activity (Falco eé al.®).
55 L,. J. Daniel, L. C. Norris, M. L. Scott, and G. F. Heuser, J. Biol. Chem. 169, 689
(1947).
56 T). W. Woolley and A. Pringle, J. Biol. Chem. 174, 327 (1948).
57 B. L. Hutchings, J. H. Mowat, J. J. Oleson, E. L. R. Stokstad, J. H. Boothe, C. W.
Waller, R. B. Angier, J. Semb, and Y. SubbaRow, J. Biol. Chem. 170, 323 (1947).
58 D. B. Cosulich and J. M. Smith, Jr., J. Am. Chem. Soc. 70, 1922 (1948).
59 J). R. Seeger, J. M. Smith, Jr., and M. E. Hultquist, J. Am. Chem. Soc. 69, 2567
(1947).
60 P. C. Edwards, D. Starling, A. M. Mattocks, and H. E. Skipper, Science 107, 119
(1948).
61G. B. Hitchings, G. B. Elion, E. A. Falco, P. B. Russell, and H. Vander Werff,
Ann. N. Y. Acad. Sci. 62, 1318 (1950).
62), D. Wright, Vitamins and Hormones 9, 131 (1951).
6§ G. B. Hitchings, G. B. Elion, and E. A. Falco, J. Biol. Chem. 185, 643 (1950).
64 G. B. Hitchings, G. B. Elion, H. Vander Werff, and E. A. Falco, J. Biol. Chem.
174, 765 (1948).
65). A. Falco, G. B. Hitchings, P. B. Russell, and H. Vander Werff, Nature 164, 107
(1949).
V. SPECIFICITY OF ACTION Tb5L
; \
u bes NEC Do
; N CH;
2,4-Diamino-5-p-chlorophenoxy Paludrine
pyrimidine
The action of 2,4-diamino-5-p-chlorophenoxypyrimidine was found to
inhibit the growth of L. casei at levels of 0.5 y per milliliter and to be com-
petitively reversed by PGA. Paludrine produced partial growth inhibition
at levels of 100 y per milliliter which could be reversed by PGA. Higher
levels of paludrine which produced complete growth inhibition could not
be reversed by an excess of PGA. The 2,4-diamino-5-p-chlorophenoxy-
pyrimidine was found to be as effective as quinine against Plasmodium
gallinaceum.®*
Burchenal et al.*® found twenty-three pyrimidine analogs to be ineffective
against transmitted leukemia Ak4 in mice.
2. PurRINE DERIVATIVES
Purines with amino groups in the 2,6-positions have been found to be
potent PGA antagonists (Elion and Hitchings®). The 2,6-positions of the
purine correspond to the 2,4-positions of pyrimidines and pteridines.
N N
f \—NH, ‘ee.
x HC “=
REN ANZ aaah
re alee <4
| NH:
NH,
2,4-Diamino- 2,6-Diaminopurine
pyrimidine
2,6-Diaminopurine inhibits growth of L. casez at a level of 0.1 y per
milliliter. Pteroylglutamic acid is capable of reversing the growth inhibition
produced by low levels of this compound but not that produced by high
levels. Adenine is also capable of reversing the antagonist at low levels of
the inhibitor. In contrast, the inhibitory action of 2-aminopurine is reversed
by either PGA or purines over a considerable range of concentration.
56 J. R. Burchenal, 8. F. Johnston, J. R. Burchenal, M. N. Kushida, E. Robinson,
and C. C. Stock, Proc. Soc. Exptl. Biol. Med. 71, 381 (1949).
152 PTEROYLGLUTAMIC ACID
Burchenal ef al.®® studied the action of six purines against Ak4 leukemia in
mice and found 2,6-diaminopurine effective. The corresponding methyl
derivative, 2,6-diamino-7-methylpurine, was ineffective.
3. PTERIDINE DERIVATIVES
A large series of 2,4-diaminopteridines and 2-amino-4-hydroxypteridines
have been prepared™ and their biological effects studied.
TABLE VI
ANTAGONISTIC ACTION OF SUBSTITUTED PTERIDINES
Inhibition Index
Compound S. faecalis R L. casei Ref.
2,4-Diaminopteridine 3,000 1,000,000 55
2,4-Diamino-7-methylpteridine 20,000 1,500,000 55
2,4-Diamino-6,7-dimethylpteridine 8, 000 100,000 55, 68
2,4-Diamino-6,7-dicarboxypteridine 400 , 000 55
2,4-Diamino-6,7-diphenylpteridine 8 100,000 55, 68
2,4-Diamino-6,7-phenanthro-(9, 10-e)-pteri- 3 55
dine
2-Amino-4-hydroxy-6, 7-diphenylpteridine 40,000 200 , 000 68
N N INS Pe
Tsk Oy NE EA \ NH
2
he ye eWN | a
x 4 ~
Ose wr
NH, OH
2,4-Diaminopteridine
(related to 4- amino-
2-Amino-4-hydroxy-
pteridine (related
pteroylglutamic acid) to pteroylglutamie acid)
The microbiological effects of some of these are shown in Table VI. One
interesting fact which is apparent from this data is the much higher ac-
tivity of the inhibitors for S. faecalis than for L. casei. Thus 2 ,4-diamino-
6,7-diphenylpteridine has an inhibition index (ratio of antagonist to
metabolite) of 8 for S. faecalis and 100,000 for L. casez. However, when the
2,4-diaminopteridine is incorporated into the PGA molecule to give
4-aminopteroylglutamic acid, the inhibition index for the two organisms is
approximately the same (Swendseid®). The inhibitory activity of the
2 ,4-diaminopteridines increases as the size of the substitution groups in-
6&7 ©. K. Cain, M. F. Mallette, and E. C. Taylor, Jr., J. Am. Chem. Soc. 70, 3026
(1948).
6&8 M. EK. Swendseid, E. L. Wittle, G. W. Moersch, O. D. Bird, and R. A. Brown, J.
Biol. Chem. 179, 1175 (1949).
V. SPECIFICITY OF ACTION 153
creases; the 6,7-diphenyl and the 6,7-phenanthro derivatives are much
more active than those containing hydrogen, methyl, or carboxyl groups.
A synergism between 2,4-diaminopteridine and sulfathiazole has been
observed by Daniel and Norris®® with #. coli and Staphylococcus aureus. For
example, 150 y of 2,4-diamino-6 ,7-dimethylpteridine per milliliter or 100
y of sulfathiazole is required to prevent growth of Staphylococcus aureus,
but a combination of 2 y of each of these gives the same degree of inhibition.
With LF. coli 100 y of this same pteridine or 5 y of sulfathiazole is required
to give the same degree of inhibition that can be attained with 10 y of
pteridine plus 1.0 y of sulfathiazole. This synergistic effect suggests that the
two antagonists are acting on two enzyme systems functioning in series.
The biological effect of a large number of pteridines has been determined
for chicks (Daniel et al.7°) and rats (Swendseid et al.®). The 2 ,4-diamino-
6,7-diphenylpteridine which is highly inhibitory for S. faecalis has no
effect on chick growth and hemoglobin formation.” Swendseid ef al.,®
however, observed that this same compound at a level of 500 mg. per kilo-
gram of diet produces leucopenia and a marked granulocytopenia in the rat
but has no effect on hemoglobin formation. This leucopenia can be pre-
vented by simultaneous feeding of 500 mg. of PGA per kilogram. The
corresponding 2,4-diamino-6,7-dimethylpteridine also produces a_ leu-
kemia, but higher levels of antagonist are required. The 2-amino-4-hydroxy-
6 ,7-diphenylpteridine is less active than the corresponding 2 ,4-diamino
derivative in producing leucopenia and granulocytopenia in the rat® but
is more active in depressing growth and hemoglobin formation in the
chick.”°
4. MopIFIcaATION OF PTERIN NUCLEUS
Replacement of the pteridine ring by benzimidazole® and quinazoline”
has yielded growth-promoting compounds rather than inhibitors. The sub-
stitution of a sulfonyl group for the carboxyl of p-aminobenzoic acid changes
the weak growth-promoting activity of benzimidazole-2-methyl-p-amino-
benzoylglutamic acid to a weak antagonist.*
Quinoxaline-2-carboxylyl-p-aminobenzoylglutamic acid is a weak an-
tagonist for S. faecalis.6® This may, however, be partially due to the fact
that there is an amide linkage between the aromatic amine and the quinox-
aline group instead of the methylene group present in PGA. The biological
activity of these compounds shows that major modifications of the PGA
molecule frequently yield growth promoters rather than inhibitors. The
formulas of these compounds are shown in Fig. 10.
69 L,. J. Daniel and L. C. Norris, J. Biol. Chem. 170, 747 (1947).
7 L. J. Daniel, M. L. Scott, L. C. Norris, and G. F. Heuser, J. Biol. Chem. 173, 123
(1948).
1G. J. Martin, J. Moss, and 8. Avakian, J. Biol. Chem. 167, 737 (1947).
154 PTEROYLGLUTAMIC ACID
5. Preroyt DERIVATIVES OF DIFFERENT AMINO ACIDS
A large number of pteroyl derivatives of different amino acids have been
prepared; the only one that functions as an antagonist is the aspartic acid
analog.’ The inhibition index is 40 for S. faecalis and 2000 for L. casez and
is constant over relatively large ranges of concentration. The inhibition
ratio for S. faecalis in the presence of pteroic acid is decreased to 2. Pteroyl-
HOOC O N
A < of \-x Cc é
HCN i=C=—ee
oer
eg
HOOC
Benzimidazole-2-methyl-p-aminobenzoylglutamic acid®
Growth promoter 0.01% as active as PGA for S. faecalis R
HOOC O T.
HCE s( \-» c-é
ole tall jeeps, ore
HG 0
Bee
HOOC
Benzimidazole-2-methyl-p-aminobenzene sulfonylglutamic acid?
Growth inhibitor inhibition ratio for S. faecalis R : 8000
er
HOOC
Quinazoline-4-p-aminobenzoylglutamie acid ‘°
Growth promoter 1 to 10% as active as PGA
ek i eel
| tpl
a O
a
HOOC
Quinazoline-2-carboxylyl-p-aminobenzoylglutamic acid®®
Antagonist inhibition ratio for L. caset + 300,000
Fre. 10. Modification of the pteridine nucleus of pteroylglutamie acid.
V. SPECIFICITY OF ACTION 155
aspartic acid inhibits growth and hemoglobin formation in the chick, the
inhibition ratio being approximately 500, but has no effect on rats at a
level of 1.5 mg. per day.
Kirsanova and Trufanov” found that the corresponding 6-carbon analog,
pteroylaminoadipic acid, was 5% as active as PGA for the chick and 10 to
20 % as active as PGA for the rat. Similarly, the 7-carbon analog, pteroyl-
aminopimelie acid,” was about 10% as active as PGA for L. casei, 20% as
active for rats, and 50% as active for chicks.
Wright ef al.“ found that the pteroyl derivatives of alanine, ¢-amino-
caproic, cystine, phenylalanine, serine, and valine were neither stimulatory
nor inhibitory activity. The B-alanine, methionine, and sareasine derivatives
had slight growth-promoting activity for S. faecalis R.
6. ALKYL DERIVATIVES
Alkyl] derivatives of PGA have been prepared in which the methyl group
has been added to the pteridine ring,”: 7° to the C® position” and the N!°
position,®® and to the 2-amino group.’
a. Biological Experiments with ‘x Methyl PGA”’
Martin ef al.’® first described a PGA antagonist designated 7-methyl-
pteroyl-p-glutamic acid which had an inhibition index of 150 for S. faecalis
R. This was prepared by the condensation of 2 ,4,5-triamino-6-hydroxy-
pyrimidine with a,8-dibromobutyraldehyde and p-aminobenzoyl-p-glu-
tamic acid. The methyl group was ascribed to the 7 position of the pteridine
ring, although this fact has not yet been definitely established.
The biological properties of a similar compound have been described by
Franklin et al.?° in which the natural L isomer of p-aminobenzoylglutamic
acid was used. The inhibition index of the crude reaction product desig-
nated crude ‘‘x methyl PGA” is 30 for S. faecalis, 1000 for L. casez, and 3000
for rats. The inhibition indices for microorganisms are relatively constant
over large ranges of concentration, and the inhibition produced in rats is
completely reversible by PGA. The syndrome produced in rats by the
antagonist is similar to, but more acute, than that induced by feeding a pu-
rified diet plus sulfasuxidine and was accompanied by oral lesions. Reversal
72 V.A. Kirsanova and A. V. Trufanov, Biokhimya 15, 243 (1950).
73.V_A. Kirsanova and A. V. Trufanov, Biokhimya 16, 367 (1951).
™W.B. Wright, Jr., D. B. Cosulich, M. J. Fahrenbach, C. W. Waller, J. M. Smith,
Jr., and M. E. Hultquist, J. Am. Chem. Soc. 71, 3014 (1949).
7 A. L. Franklin, E. L. R. Stokstad, M. Belt, and T. H. Jukes, 169, 427 (1947).
76 G. J. Martin, L. Tolman, and J. Moss, Arch. Biochem. 12, 318 (1947).
7M. E. Hultquist, J. M. Smith, Jr., D. R. Seeger, D. B. Cosulich, and E. Kuh,
J. Am. Chem. Soc. 71, 619 (1949).
7. B. Roth, J. M. Smith, Jr., and M. E. Hultquist, J. Am. Chem. Soc. 73, 2864 (1951).
156 PTEROYLGLUTAMIC ACID
of the syndrome by PGA in the presence of the antagonist was accompanied
by temporarily overcompensatory increases in total white blood cell and
granulocyte counts.
This antagonist has produced symptoms of PGA deficiency in chicks,
mice,’®: °° pigs,*! and dogs. The dog does not develop deficiency symptoms
on a purified diet devoid of PGA even when a sulfonamide is added.® The
deficiency syndrome which develops with the antagonist consists of loss in
appetite, skin changes, uticarious dermatitis, ulceration of bony prom-
inences, mild macrocytic anemia, and slight leucopenia. These symptoms
develop much more slowly than those in the rat or chick, and no diarrhea
or gingivitis occurs, which is characteristic of the antagonist-induced syn-
drome in the rat. Complete and rapid reversal of the deficiency disease is
produced by feeding of PGA with the continued feeding of the antagonist.
The effects of ‘“‘x methyl PGA” in affecting the action of the sex hormones
have already been described.
Shive et al.*4 found thymidine to be more effective than PGA in reversing
the action of ‘‘*x methyl PGA” for Leuconostoc mesenteroides. Thymine is
inactive.
b. 9- and 10-Methyl PGA derivatives
The biological effects of a series of methyl derivatives of PGA are shown
in Table VII. It should be noted that the inhibition indices reported for
9-methyl PGA and for 9,10-dimethyl PGA are lower than those reported
elsewhere.” It should be borne in mind that these values are not absolute
but will vary from one experiment to another, depending on the time of
incubation, ete. Of special interest is the fact that 10-methyl PGA, which
is a very potent antagonist for S. faecalis, has slight pro-PGA activity for
chicks. The 9-methyl derivative is an antagonist for chicks and can be
completely reversed by PGA. The inhibitory effect of 4-amino PGA and the
9- and 10-methyl derivatives of 4-amino PGA on chicks is only partially
reversed by PGA over a narrow range of concentration. The inhibition
ratios of these compounds tend to decrease with increasing concentration,
79 A. L. Franklin, E. L. R. Stokstad, T. H. Jukes, Proc. Soc. Exptl. Biol. Med. 65,
368 (1947).
80D. R. Weir, R. W. Heinle, and A. D. Welch, Proc. Soc. Exptl. Biol. Med. 69, 211
(1948).
81 A. D. Welch, R. W. Heinle, G. Sharpe, W. L. George, and M. Epstein, Proc. Soc.
Exptl. Biol. Med. 65, 364 (1947).
82 A. L. Franklin, T. H. Jukes, E. L. R. Stokstad, and M. Belt, Federation Proc. 8,
199 (1949).
83 T,. Michaud, A. R. Maass, W. R. Ruegamer, and C. A. Elvehjem, Proc. Soc. Exptl.
Biol. Med. 56, 148 (1944).
84 W. Shive, R. E. Eakin, W. M. Harding, J. M. Ravel, and J. E. Sutherland, J. Am.
Chem. Soc. 70, 2299 (1948).
V. SPECIFICITY OF ACTION ys
which shows that PGA is becoming less effective in reversing the action of
the antagonist. This is characteristic of systems in which inhibition occurs
at more than one step in the reaction series.
7. 4-AMINOPTEROYLGLUTAMIC AcID DERIVATIVES
This class of compounds is unique among the PGA antagonists. They
are highly potent antagonists for both microorganisms and animals and
have proved to be the most effective of the PGA antagonists in treatment
of experimental and clinical leukemias.
TABLE VII
SomE DERIVATIVES OF PTEROYLGLUTAMIC ACID AND THEIR BrioLocicaL Errects?
Inhibition? of S. faecalis R
at three PGA levels, y/ml. Toxicity for animals, p.p.m. of purified
of culture medium PGA-deficient diet for LDioo
e PGA derivative 10 100 1000 Mice Rats Chicks Reverse®
Crude ‘‘x-methyl”’ 30 20 30 1000 1000 1000 =F
9-Methy] 300 400 400 30-1000 a7
10-Methy1 1 1 0.8 Pro?
9,10-Dimethyl 3 2 2 100 30 30
4-Amino 6 3 2 1 1-3 3 =
4-Amino-9-methyl 2 2 2 10
4-Amino-10-methy] 2 0.5 0.3 1 3 5 _
4-Amino-9, 10-dimethy] 0.4 0.2 0.2 3 3 3 _
* Courtesy Ann. N. Y. Acad. Sci. (Jukes et al.85).
6 Inhibition ratio to PGA for half-maximum growth.
© Reversible by PGA over a wide range.
4 Slight PGA-like effect.
A number of derivatives containing an amino group in place of the
4-hydroxy group of the pteridine ring have been prepared (Seeger et al.**).
The replacement of the glutamic acid by alanine in 4-amino PGA results
in almost complete loss of antagonist activity (Wright ef al.™). Introduction
of methyl groups in the 9 and 10 positions increases the activity for S. fae-
calis R.*8
In contrast to the effects of ‘x methyl PGA” and 9-methyl PGA, which
are readily reversible by PGA, the inhibitory action of 4-amino PGA and
related compounds cannot be completely counteracted by PGA. In S.
faecalis the ratio of 4-amino PGA to PGA for half-maximum inhibition is
266 at 0.003 y of PGA and 0.07 at 0.3 y per milliliter. This shows that PGA
becomes less effective at higher concentrations of the antagonist, a feature
85 T. H. Jukes, A. L. Franklin, and E. L. R. Stokstad, Ann. N. Y. Acad. Sci. 52, 1336
(1950).
86 T). Seeger, J. M. Smith, Jr., and M. E. Hultquist, J. Am. Chem. Soc. 69, 2567 (1947).
158 PTEROYLGLUTAMIC ACID
which is characteristic of systems in which the antagonist blocks the enzyme
system at more than one point. The growth of chicks is retarded by 5 mg.
of 4-amino PGA per kilogram of diet. The toxicity is partially counteracted
by 5.0 mg. of PGA per kilogram of diet, but complete reversal is not at-
tained by as much as 25 mg. (Oleson et al.’7). In the rat, 10 y of 4-amino
PGA per day or 1 mg. per kilogram of diet is lethal in about 3 to 4 days.*7
The symptoms produced in the rat are severe diarrhea, weight loss, and
porphyrin-stained whiskers. Large amounts of PGA (20 mg. per kilogram
of diet) produced partial reversal of toxic symptoms. One milligram of
4-amino PGA per kilogram of diet produces high mortality in the mouse
which cannot be reversed by one hundred times as much PGA (Franklin
et al.)*8. Guinea pigs given 0.5 to 5.0 mg. of 4-amino PGA subcutaneously
daily lose weight and die within 11 to 28 days. The symptoms of toxicity
include normocytic anemia, leucopenia, agranulocytosis, thrombocytopenia,
and a hypoplastic bone marrow. PGA at levels of twenty-five to one
hundred times that of the analog prevents the development of leucopenia
but not the anemia (Minnich and Moore*’).
Other animals which are susceptible to the action of 4-amino PGA are
the dog,®° the monkey,® and Drosophila.®! Early embryonic death has been
induced in the dog and the mouse and rat.*? The level of 4-amino PGA (0.1
mg. per kilogram of body weight) which was used to induce fetal death
produced a temporary depletion of the bone marrow and a transient loss
in body weight but did not make the animals seriously ill.
The chick egg embryo is inhibited by 3 to 5 y of 4-amino PGA. The
inhibitor becomes relatively less toxic as embryonic development pro-
gresses.** Although PGA is almost completely ineffective in reversing the
action of the antagonist,%’ %* a mixture of thymidine and hypoxanthine
desoxyriboside produced™ a partial reversal. Thymine plus hypoxanthine
desoxyriboside was ineffective. This provides a parallel to the action of
thymidine in reversing the action of 4-amino PGA in F. coli. and Le.
citrovorum and is the only example outside of microorganisms where thy-
midine is capable of reversing the action of a PGA antagonist. The citro-
87 J. J. Oleson, B. L. Hutchings, and Y. SubbaRow, J. Biol. Chem. 175, 359 (1948).
88 A. L. Franklin, E. L. R. Stokstad, and T. H. Jukes, Proc. Soc. Exptl. Biol. Med.
67, 398 (1948).
89 V. Minnich and C. V. Moore, Federation Proc. 7, 276 (1948).
90 J. B. Thiersch and F.S. Philips, Proc. Soc. Exptl. Biol. Med. 71, 484 (1949).
91K. D. Goldsmith, M. H. Harnly, and E. B. Tobias, Ann. N. Y. Acad. Sct. 52, 1342
(1950).
2 J. B. Thiersch and F. 8. Philips, Proc. Soc. Exptl. Biol. Med. 74, 204 (1950).
9 TD). A. Karnofsky, P. A. Patterson, and L. P. Ridgway, Proc. Soc. Exptl. Biol. Med.
71, 447 (1949).
94. E. Snell and W. W. Cravens, Proc. Soc. Exptl. Biol. Med. 74, 87 (1950).
V. SPECIFICITY OF ACTION 159
vorum factor is also effective in counteracting the toxicity of 4-amino PGA
for the egg embryo.**
Tetrahymena geleit is unique among the PGA-requiring organisms in
that it is not inhibited by either 4-amino PGA or N!°-methyl PGA. It has
been reported that 4-amino PGA functions as a growth promoter, but this
was later shown to be due to appreciable amounts of PGA which were
present in the sample of 4-amino PGA (Heinrich et al.°®). Although N?°-
methyl PGA functions as a growth promoter and 4-amino PGA neither
stimulates nor inhibits growth, 4-amino-10-methyl PGA, as well as 4-amino-
9-methyl PGA, functions as an antagonist (Dewey et al.°”).
The resistance of 7’. gelezt to 4-amino PGA is similar to that of a strain
of S. faecalis R which had become resistant to 4-amino-10-methyl PGA by
repeated culture in the presence of this antagonist (Burchenal ef al.**).
Broquist et al.°° has found that the resistance of this organism to 4-amino
PGA is due to an increased ability to convert PGA to the citrovorum fac-
tor.
8. Errect oF ANTAGONISTS ON EXPERIMENTAL TUMORS
The striking effect of PGA in stimulating growth of white cells in PGA-
deficient animals provided a basis for the suggestion that PGA antagonists
might be able to reduce the large number of white cell blood elements
which form in leukemia. Clinical work has borne out the validity of the
assumption.!°° and a large amount of data has accumulated on the effects
of these antagonists in experimentally induced tumors of various types.
Interesting in this connection is the observation that the PGA and citro-
vorum factor content of leucocytes is higher in leukemia than in normal
blood. In advanced stages of the disease, leucocytes may contain as high
as 300 my of citrovorum factor per milliliter as compared with normal
levels of 30 to 80 my per milliliter (Swendseid et al.'°').
The growth of Rous chicken sarcoma, which is a virus disease and thus
not necessarily directly related to other types of tumors, is inhibited by a
PGA deficiency. The deficiency can be induced either by feeding a diet
% W.W. Cravens and E. E. Snell, Proc. Soc. Exptl. Biol. Med. 75, 43 (1950).
96 M. R. Heinrich, V. C. Dewey and G. W. Kidder, J. Am. Chem. Soc. 75, 5425 (1953).
7 V.C. Dewey, G. W. Kidder, and R. E. Parks, Jr., Proc. Soc. Exptl. Biol. Med. 78,
91 (1951).
% J. H. Burchenal, G. B. Waring, and D. J. Hutchison, Proc. Soc. Exptl. Biol. Med.
78, 311 (1951).
99 H. P. Broquist, A. R. Kohler, D. J. Hutchison, and J. H. Burchenal, J. Biol. Chem.
202, 59 (1953).
100 S$. Farber, Blood 7, 97 (1952).
101 M. E. Swendseid, F. H. Bethell, O. D. Bird, Cancer Research 11, 864 (1951).
160 PTEROYLGLUTAMIC ACID
low in PGA or by the administration of 4-amino PGA (Little et al.!°). The
growth of this tumor is also reduced by deficiencies of other vitamins such
as nicotinic acid, pantothenic acid, riboflavin,'!” and vitamin By .!°* PGA
exerted the greatest effect and was the only vitamin, a deficiency of which
completely prevented tumor growth.
Similar to the effect of PGA antagonists on virus-induced tumors in
animals is their inhibiting action on growth of plant tumors. 4-amino PGA
and three other methylated derivatives of this antagonist were effective in
suppressing the growth of tumorous tissue induced on carrots by inoculation
with crown gall bacteria (de Ropp?”*).
The action of 4-amino PGA in normal and in leukemic animals has been
studied in great detail by the workers at Sloan-Kettering Institute.10% 1°
In a study of ninety compounds related to PGA only four showed definite
chemotherapeutic effect as measured by doubling the survival time of leu-
kemic Ak4 mice. These compounds were 4-amino-10-methyl PGA, 4-amino-
9-methyl PGA, 4-amino-9 ,10-methyl PGA, and 2,6-diaminopurine. 4-am-
ino-PGA and 4-aminopteroylaspartic acid were less effective (Burchenal
et al.®*). In each case the compounds were given at the maximum tolerated
dose. In the treatment of a solid tumor, sarcoma 180, 4-amino PGA and
4-amino-10-methyl PGA were the most effective, 4-aminopteroylaspartic
acid, pteroylaspartic, and pteroyltriglutamic acid being ineffective (Stock
et al.!°7), The narrow margin between toxic and therapeutic dose is reflected
in the fact that in the case of 4-amino-10-methyl PGA 1.0 mg. per kilogram
of body weight per day shows no activity, 2.0 mg. is markedly toxic, and
1.5 mg. gives marked inhibition of tumor activity with little evidence of
toxicity.
One of the problems associated with clinical use of the PGA antagonists
in the treatment of leukemia is the eventual failure of therapy due to de-
velopment of resistance to this agent. If this resistance could be overcome,
the leukemia could conceivably be held indefinitely in check by use of the
therapeutic agent. This problem of resistance has been studied by the
development of a resistant strain of lymphoid leukemia, Ak4, by repeated
passage through mice treated with 4-amino-10-methyl PGA (Burchenal
1022 P. A. Little, A. Sampath, V. Paganelli, E. Locke, and Y. SubbaRow, Trans. N.Y.
Acad. Sci. 10, 91 (1948).
103 P_ A. Little, J. J. Oleson, and Y. SubbaRow, J. Lab. Clin. Med. 33, 1139 (1948).
104 J, J. Oleson and P. A. Little, Proc. Soc. Exptl. Biol. Med. 71, 226 (1949).
105 R.S. de Ropp, Natwre 164, 954 (1949).
106 F. §. Philips, J. B. Thiersch, and F. C. Ferguson, Ann. N. Y. Acad. Sci. 52, 1349
(1950).
107 C. C. Stock, J. J. Biesele, J. H. Burchenal, D. A. Karnofsky, A. E. Moore, and K.
Sugiura, Ann. N. Y. Acad. Sci. 52, 1360 (1950).
VI. ESTIMATION 161
et al.'°8), This strain was also resistant to other 4-amino PGA derivatives
but was not resistant to 2,6-diaminopurine or to “x methyl PGA” with a
PGA-deficient diet containing a sulfonamide.!°’ This therapeutic effect of
“x methyl PGA” on a strain of transplanted mouse leukemia in Ak mice
had previously been demonstrated by Weir ef al.!°® An interesting metabolic
difference between the normal strain, Ak4, and the resistant strain, Ak4R,
lies in their relative ability to incorporate radioactive formate into the
nucleic acid purines. Both strains have the same ability in this respect in
the absence of antagonist. On the administration of 4-amino-10-methyl
PGA the incorporation of radioactive formate was reduced to 4% of its
original level in the normal Ak4 strain but was reduced to only 25 % in the
resistant Ak4R strain (Skipper and Burchenal!!”),
Pteroyltriglutamic acid has been reported by Leuchtenberger, Lewisohn,
and associates!!! 1" to produce complete regressions in spontaneous breast
tumors in mice. Intravenous injection of 5 y per day led to complete re-
gressions in 43 % of a group of eighty-nine animals. No regressions occurred
in sixty control animals. In contrast, PGA had no inhibitory effect, and it
was observed that primary tumors in mice receiving 100 y of PGA grew
more rapidly than the untreated controls. The effect of pteroyltriglutamic
acid on transplanted sarcoma 180 has not been confirmed in investigations
by Burchenal et al.®° and Schoenbach ef al."
VI. Estimation
E. L. R. STOKSTAD
A. CHEMICAL METHODS
Pteroylglutamic acid on reduction in acid solution is split to yield a
methylpteridine and p-aminobenzoylglutamic acid.! This reaction has been
made the basis of a quantitative procedure? in which the sample is reduced
108 J. Burchenal, 8. F. Johnston, and G. B. Waring, Proc. Soc. Exptl. Biol. Med. 78,
348 (1951).
109 J). R. Weir, A. D. Welch, and R. W. Heinle, Proc. Soc. Exptl. Biol. Med. 71, 107
(1949).
110 A. E. Skipper and J. H. Burchenal, Cancer Research 11, 229 (1951).
11 R. Lewisohn, C. Leuchtenberger, and J. C. Keresztesy, Science 104, 436 (1946).
12 R. Leuchtenberger, C. Leuchtenberger, D. Laszlo, and R. Lewisohn, Science 101,
46 (1945).
13. B. Schoenbach, A. Goldin, B. Goldberg, and L. G. Ortega, Cancer 2, 57 (1949).
1B. L. Hutchings, E. L. R. Stokstad, J. H. Mowat, J. H. Boothe, C. W. Waller,
R. B. Angier, J. Semb, and Y. SubbaRow, J. Am. Chem. Soc. 70, 10 (1948).
2B. L. Hutchings, E. L. R. Stokstad, J. H. Boothe, J. H. Mowat, C. W. Waller,
R. B. Angier, J. Semb, and Y. SubbaRow, J. Biol. Chem. 168, 705 (1947).
162 PTEROYLGLUTAMIC ACID
with zine in 0.5 N hydrochloric acid and the aromatic amine estimated by
the method of Bratton and Marshall.’ In crude reaction products contain-
ing free p-aminobenzoic acid or its derivatives, the aromatic amine is de-
termined before and after reduction with zinc. The increase resulting from
reduction is a measure of the amount of PGA. p-Aminobenzoic acid and
its peptides with other amino acids give the same amount of color on a
molar basis when diazotized and coupled with N-(1-naphthyl)ethylene-
diamine dihydrochloride by the method of Bratton and Marshall. Thus the
method can be used for pteroic acid, its amide, ester, or peptide with any
amino acid. Any derivative which contains a substitution on the nitrogen
of the p-aminobenzoic acid cannot be estimated by this method because
the presence of a primary amino group is necessary for the diazotization
and coupling reaction.
Titanous chloride has been used by Glazko and Wolf? to reduce pteroyl-
glutamic acid. They found that, in the assay of tissue homogenates by the
zine reduction methods, adenine and nucleic acid products interfered by
producing diazotizable substances on reduction with zine. The use of
titanous chloride eliminates this difficulty and permits the analysis of
PGA in the presence of adenine.
A micromethod has been proposed by Allfrey et al.° which is based on
the oxidation of pteroylglutamic acid by alkaline permanganate solution
to give 2-amino-4-hydroxypteridine-6-carboxylic acid. This pterin is then
estimated by fluorometric methods. In the presence of extraneous fluores-
cing pigments, such as xanthopterin, which are affected by permanganate,
the 2-amino-4-hydroxypteridine-6-carboxylic acid is adsorbed on Florisil,
eluted, and then measured fluorometrically. This procedure has been
adopted to use with natural products and gives results which are com-
parable to, although slightly higher than, those obtained by microbiological
methods. Such a chemical procedure has the advantage that it measures
total soluble pteroylglutamic acid in free and in conjugated form.
B. MICROBIOLOGICAL METHODS
Two main problems are involved in the microbiological estimation of
pteroylglutamic acid in natural products. The first involves extraction and
hydrolysis of pteroylglutamie acid conjugates which may be present; the
other, assay of the microbiologically active compounds. Detailed descrip-
tions of these methods in review form have been published by Stokstad
and Hutchings® and Snell.?
3 A.C. Bratton and E. K. Marshall, Jr., J. Biol. Chem. 128, 537 (1939).
4A.J.Glazko and L. M. Wolf, Arch. Biochem. 21, 241 (1949).
5V. Allfrey, L. J. Teply, C. Geffen, and C. G. King, J. Biol. Chem. 178, 465 (1949).
6. L. R. Stokstad and B. L. Hutchings 7n Biological Symposia, The Microbiolog-
VI. ESTIMATION 163
1. ExrrRacTION FROM TissuES AND HYDROLYSIS OF CONJUGATES
A number of experiments have shown that microbiologically imactive
conjugates exist in natural products. Vitamin B, conjugate (pteroylhexa-
glutamylglutamiec acid) found in yeast is inactive both for S. faecalis and
L. casei. Pteroyltriglutamic acid isolated from a fermentation material is
active for L. casez but only slightly active for S. faecalis. Pteroyl-y-glutamyl-
glutamic acid, which has been prepared synthetically but not yet observed
in natural products, is active for both S. faecalis and L. casez. It is thus
apparent that L. casei is capable of responding to larger conjugates than
S. faecalis. This offers a possible explanation for the fact that many natural
products give a higher result by L. casez assay than by S. faecalis assay.
Rhizopterin and pteroic acid are active for S. faecalis but inactive for L.
casei. Thus the presence of a high activity for S. faecalis compared to L.
casei might indicate the presence of rhizopterin or pteroic acid in the sample.
A number of methods have been proposed for the release of pteroylglu-
tamic acid from tissues. Cheldelin et al.’ investigated the effect of a number
of commercially available enzymes on the liberation of folic acid from tis-
sues. This hydrolysis presumably consisted in two parts: first, the release
of pteroylglutamic acid from the tissue to give a soluble form; second, the
hydrolysis of the soluble but inactive conjugates to yield active compounds.
These workers found taka-diastase to be more effective than papain, pepsin,
trypsin, pancreatin, malt diastase, or pancreatic amylase. Digestion of hog
heart with taka-diastase gave about ten times as much pteroylglutamic
acid activity as autolysis alone. However, subsequent work’: '° indicated
that, although taka-diastase may be effective in releasing the nutrilite from
the tissues, it does not hydrolyze all the conjugates present. Mims et al.°
demonstrated the existence of an enzyme capable of activating a microbio-
logically inactive conjugate in yeast. Cell-free extracts of plants and tissues
which had been previously digested with taka-diastase could be further
activated by this enzyme which was obtained from liver. This distinguished
the action of the conjugate-splitting enzyme from that of taka-diastase.
a. Conjugases
Shortly after the observation that certain natural materials possessed
more activity by animal assay than could be accounted for by microbio-
ical Assay of Lactobacillus casei Factor (Vitamin B, , Folic Acid), Volume XII,
p. 339. Jaques Cattell Press, Lancaster, Pa., 1947.
7. E. Snell in Vitamin Methods, Microbiological Methods in Vitamin Research,
p. 327. Academic Press, New York, 1950.
8V. H. Cheldelin, M. A. Eppright, E. E. Snell, and B. M. Guirard, Univ. Texas
Publ. No. 4237, 15 (1942).
9V. Mims, J. R. Totter and P. L. Day, J. Biol. Chem. 155, 401 (1944).
10 P. R. Burkholder, I. McVeigh and K. Wilson, Arch. Biochem. 7, 287 (1945).
164. PTEROYLGLUTAMIC ACID
logical test, it was observed that the activity for microorganisms could be
increased by appropriate enzymatic digestion. The name ‘‘vitamin B,
conjugase”’ was given by Bird et al.'! to this enzyme because it was capable
of splitting vitamin B, conjugate (pteroylheptaglutamic acid).
Two main types of conjugase have been described, one present in hog
liver and having a pH optimum of 4.5,"> ? the other in chicken pancreas
with a pH optimum of 7 to 8.% Chicken pancreas conjugase is inactivated
at temperatures above 45° and is relatively stable to the action of crystalline
trypsin. Autolysis of chicken pancreas tissue increases the amount of ex-
tractable enzyme activity a thousandfold. Crystalline chicken pancreas
conjugase has not been obtained, but concentrates 2800 times as active as
the crude starting material have been prepared by Mims and Laskowski"
using adsorption on tricaletum phosphate gel, precipitation with alcohol,
and fractional precipitation with sodium sulfate. Calcium ions were found
to be a component of the enzyme system, which accounted for the apparent
loss of activity which sometimes occurred during dialysis and the increase
in activity observed on purification by adsorption on calcium phosphate.
Hog kidney conjugase has a pH optimum of 4.5. This enzyme does not
attack the methyl ester of pteroylheptaglutamate, a fact which led to its
classification as a carboxypeptidase. However, neither of the conjugases is
identical with carboxypeptidase, since the purified carboxypeptidase has no
conjugase activity.
Studies on the specificity of chicken pancreas conjugase by Dabrowska
et al.'® show that y-peptides of pteroylglutamic acid are attacked much
more readily than a-peptides. In the action of the enzyme on pteroyl-y-
glutamyl-y-glutamylglutamic acid only one mole of glutamic acid is re-
moved. This presumably is the terminal glutamic acid group, since pteroyl-
-glutamylglutamic acid is not attacked by chicken pancreas conjugase.
Thus it appears that the final product resulting from the cleavage of pteroyl-
heptaglutamic acid by chicken pancreas conjugase is pteroyl-y-glutamyl-
glutamic acid rather than the monoglutamate.
Hog kidney conjugase is capable of splitting pteroylheptaglutamate all
the way down to the monoglutamate. This is shown by the fact that pteroyl-
glutamic acid has been isolated from a hog kidney conjugase digest of the
11Q.D. Bird, 8. B. Binkley, E. 8. Bloom, A. D. Emmett, and J. J. Pfiffner, J. Biol.
Chem. 157, 413 (1945).
122Q. D. Bird, M. Robbins, J. M. Vandenbelt, and J. J. Pfiffner, J. Biol. Chem. 1638,
649 (1946).
13M. Laskowski, V. Mims, and P. L. Day, J. Biol. Chem. 157, 731 (1945).
14.V. Mims and M. Laskowski, J. Biol. Chem. 160, 493 (1945).
15M. Laskowski, Ann. Rev. Biochem. XIX, 21 (1950).
16W. Dabrowska, A. Kazenko, and M. Laskowski, Science 110, 95 (1949).
VI. ESTIMATION 165
heptaglutamate derived from yeast (Pfiffner et al.”). Evidence that hog
kidney conjugase consists of two enzymes has been obtained by Mims and
Bird.'® The hydrolysis of pteroylheptaglutamic acid was found to require
both components, whereas the hydrolysis of pteroyltriglutamic acid could
be effected by only one. Calcium ion has not been found to be a part of the
hog kidney conjugase system.
Conjugases have been observed by Simpson and Schweigert'® in the blood
of several animal species, including man. When the conjugase in blood is
allowed to act on the conjugates in blood, the pH optimum is observed to be
7. However, when the substrate is either pteroylheptaglutamic acid or the
conjugates present in taka-diastase, equal activity is obtained with blood
conjugase at pH 4.5 and at pH 7. This suggests that the conjugases in blood
have different pH optima, depending on the substrate. These observations
point out the hazards associated with the use of crude enzyme preparations
in the hydrolysis of conjugates prior to microbiological assay. Incubation
of taka-diastase with blood produces an increase in pteroylglutamic acid
activity which has been interpreted as indicating the presence of conjugates
in the blood which are hydrolyzed by conjugases in the taka-diastase. Ac-
tually, the converse has proved to be true, and the conjugases in the blood
are really hydrolyzing the conjugates present in the blood and in the taka-
diastase.
Complete hydrolysis of the pteroylheptaglutamate cannot be .accom-
plished by either chicken pancreas or hog kidney conjugase alone. Thus
Sreenivasan ef al.?° found that complete hydrolysis of conjugates for assay
purposes could be attained only by the stepwise use of chicken pancreas
and hog kidney conjugase. Rat liver contains two enzymes with pH optima
at 4.5 and 7. Autolysis of liver or digestion of liver by hog kidney conjugase
at pH 4.5 liberates very little PGA. Since hog kidney conjugase produces
approximately 75 % hydrolysis of the pteroylheptaglutamate, it is apparent
that this conjugate is not present in liver. Autolysis or chicken pancreas
digestion of liver at pH 7.0 produces an increase in activity. Further evi-
dence for the differences in the behavior of these two conjugases lies in the
fact that pteroyltriglutamic acid is completely split by both chicken pan-
creas and hog kidney conjugases where microbiological activity is used as a
eriterion of hydrolysis. The pteroyldiglutamic acid formed by chicken pan-
J.J. Pfiffner, S. B. Binkley, E. S. Bloom, and B. L. O’Dell, J. Am. Chem. Soc. 69,
1476 (1947).
18'V. Mims and O. D. Bird in Vitamin Symposium, Conference of American Associa-
tion for the Advancement of Science, Colby College, 1948; cited by M. Laskowski,
Ann. Rev. Biochem. XIX, 21 (1950).
19R. E. Simpson and B.S. Schweigert, Arch. Biochem. 20, 32 (1949).
20 A. Sreenivasan, A. 2. Harper, and C. A. Elvehjem, J. Biol. Chem. 177, 117 (1949).
166 PTEROYLGLUTAMIC ACID
creas digestion apparently possesses the same activity for the assay orga-
nism as PGA. Pteroylheptaglutamate is only 30 % hydrolyzed by chicken
pancreas and 75 % by hog kidney conjugase. A stepwise combination of the
two enzymes accomplishes complete hydrolysis of heptaglutamate com-
pound.
Hog kidney conjugase is readily inhibited by thymus nucleic, yeast nu-
cleic acid, and extracts of yeast and molasses (Mims e¢ al.!). A number of
proteins such as casein, gelatin, egg albumin, and crystalline bovine plasma
albumin also inhibit hog kidney conjugase.”
Thymus and yeast nucleic acids which inhibit hog kidney conjugase have
no effect on chicken pancreas conjugase.2! This enzyme is, however, in-
hibited by a glutamic acid polypeptide of p-aminobenzoic acid derived from
yeast.”? p-Aminobenzoyl-y-glutamyl-y-glutamylglutamiec acid also inhibits
chicken pancreas conjugase.”* The tetraethyl ester of this peptide gives no
inhibition, and the p-nitro derivative of the acid peptide is half as active
as the peptide with an amino group.
b. Digestion Procedures for Assay Purposes
Bird et al.?> described four methods for the enzymatic hydrolysis of
pteroylglutamic acid conjugates and compared the results with those ob-
tained by assay with chicks. These results, presented in Table VIII show
that enzymic hydrolysis followed by microbiological assay gave essentially
the same results as assay with chicks except in two samples. One sample of
liver extract, which gave only half as much pteroylglutamic acid by diges-
tion with acetone-desiccated hog kidney and microbiological assay as it
did with chick assay, was found to give comparable results when digested
with an extract of unheated almond. Certain natural materials were found
to contain enzyme inhibitors, and in these cases very large amounts of
enzyme were necessary to secure maximum hydrolysis of the conjugates
present.
Digestion with acetone-dried or fresh chicken pancreas at pH 7.2 is the
recommended procedure of the Association of Official Agricultural Chem-
ists.2® Enzyme digestion is preceded by autoclaving the finely ground sample
at the same pH to extract the factor from the tissue.
21.V. Mims, M. E. Swendseid, and O. D. Bird, J. Biol. Chem. 170, 367 (1947).
2 A. Z. Hodson, Arch. Biochem. 16, 309 (1948).
23H. S. Sims and J. R. Totter, Federation Proc. 6, 291 (1947).
24H. L. R. Stokstad, J. Pierce, T. H. Jukes, and A. L. Franklin, Federation Proc. 7,
193 (1948).
25 QO. D. Bird, B. Bressler, R. A. Brown, C. J. Campbell, and A. D. Emmett, J. Biol.
Chem. 159, 631 (1945).
26 Methods of Analysis, Association of Official Agricultural Chemists, 7th ed., p.
784, 1950.
VI. ESTIMATION 167
It is apparent that no one procedure can be recommended for digestion
of the conjugates in all types of natural materials. In order to determine
whether complete hydrolysis has been obtained, it may be necessary to
employ different enzyme systems and to vary the amounts of enzyme.
2. MicrRoBIOLOGICAL PROCEDURES
Although a number of organisms have been found which will respond to
pteroylglutamic acid, only two organisms, L. casei and S. faecalis, have
TABLE VIII
CoMPARISON OF PGA Assays MapE BY ASSAYS WITH CHICKS AND witH Lactobacillus
casei BEFORE AND AFTER ENZYME TREATMENT?
PGA content y/g.
Microbiological assay
Enzyme After enzyme
Substance assayed method”? Initial treatment Chick assay
Yeast extract* 1 2:9 50 55
Concentrate from yeast 1 11.6 198 200
Yeast extract 2 2.0 52 50
Yeast extract 3 2.0 57 50
Bacto-yeast extract? 1 Onn 26 25
Concentrate from yeast 2 12.6 250 280
Liver extract 1 20.0 26 54
Liver extract 2 20.0 52 54
Concentrate from liver extract 2 34.0 89 98
Asparagus juice concentrate 4 5.0 7 12
Concentrate from yeast 4
48.0 7670 8135
“ Courtesy J. Biol. Chem. (Bird et al.).
: Enzyme method: 1. Acetone desiccated hog kidney, pH 4.2-4.5.
2. Extract of unheated almonds, pH 6.0-7.0.
3. Extract of commercial almond meal, pH 6.0-7.0.
4, Extract of autolyzed hog kidney, pH 4.5.
© An aqueous extract of plasmolyzed yeast.
’ Difco Laboratories, Detroit.
been used for the assay of this vitamin. L. casei has the advantage in that
it is the more sensitive of the two organisms. It gives half-maximum growth
with approximately 0.05 my of pteroylglutamic acid per milliliter of me-
dium, whereas S. faecalis requires 0.3 my. L. casei does not give maximum
growth with purines and thymine, whereas S. faecalis does. Thus L. casei
is better suited to the assay of low-potency materials and those products
which may contain thymine. Approximately five thousand times as much
thymine is required to produce the same response as PGA both for S. faecalis
and L. casei. L. casei is a slower growing organism than S. faecalis, and the
assays with the former organism are usually carried out 72 hours and the
168 PTEROYLGLUTAMIC ACID
growth measured by titration of the acid produced. Assays with S. faecalis
can be made in 16 hours, and the growth measured turbidimetrically or by
titration after 40 hours incubation. A method has been developed by
Roberts and Snell” by using L. casez with a 16 to 24-hour incubation period.
A trypsin digest of casein is used in the basal medium which serves as a
source of strepogenin and permits maximum growth of the organism to be
reached in a short time.
VII. Standardization of Activity
E. L. R. STOKSTAD
The ease of microbiological assay has prevented extensive use of animal
assays for the measurement of pteroylglutamic acid. Chicks and rats are
used on occasion to determine the total pteroylglutamic acid activity for
animals. This affords a partial answer to the problem of whether the micro-
biological methods are a true measure of the pteroylglutamic acid activity
for animals.
Two methods have been proposed for chick assay. One is a curative test!
in which the sample is administered orally in six doses given every other
day. The increase in hematocrit is used as a measure of the response. A 4-
week prophylactic test using a purified type of diet of chemically defined
ingredients has been also used.?: #
Rats cannot be made deficient in pteroylglutamic acid by the simple ex-
clusion of this factor from the diet. By the use of sulfonamides, such as
sulfaguanidine or succinylsulfathiazole, in the diet it is possible to produce
a pteroylglutamic deficiency in this species. An assay method based on this
type of diet has been proposed in which growth is used as the criterion of
response in rats.
VIII. Occurrence in Foods
E. L. R. STOKSTAD
The distribution of PGA in foods is shown in Table [X. The data present
a summary of information compiled by the Bureau of Human Nutrition
27H}. C. Roberts and E. EK. Snell, J. Biol. Chem. 163, 499 (1946).
1B. L. O’Dell and A. G. Hogan, J. Biol. Chem. 149, 323 (1943).
2C. J. Campbell, R. A. Brown, and A. D. Emmett, J. Biol. Chem. 154, 721 (1944).
3C. J. Campbell, M. M. McCabe, R. A. Brown, and A. D. Emmett, Am. J. Physiol.
144, 348 (1945).
VIII. OCCURRENCE IN FOODS
Food
TABLE IX
PreroyLuGLutTAmMic Acip CONTENT OF Foops
Moisture, %
169
Total PGA content,
fresh basis, y/g.
Meats and Eggs
Beef, round steak
Beef, heart
Beef, kidney
Beef, sweetbreads
Beef, liver
Lamb, stew meat
Lamb, liver
Pork, loin
Pork, liver
Pork, ham (smoked)
Chicken, dark meat
Chicken, white meat
Chicken, liver
Eggs, whole
Egg white
Egg yolk
Nuts
Peanuts
Almonds
Coconuts
Vegetables, fresh
Asparagus
Beans, lima
Beans, green snap
Beets
Broccoli
Brussels sprouts
Cabbage
Carrots
Cauliflower
Corn, sweet
Cucumbers
Greens, beet
Greens, spinach
Lettuce
Peas, fresh
Peppers, green
Potatoes, peeled
Potatoes, peel
Pumpkin
Sweet potatoes
Tomatoes
Vegetables, dried
Beans, kidney
70
76
76
fod
7
68
59
69
66
68
67
64-81
0.06-0.14
0.03
0.58
0.23
2.9
0.03
2.76
0.03
2.2
0.10
0.03
0.03
3.77
0.03-0.08
0.004
0.18
0.48-1.15
0.03-0.54
0.12-0.35
0.04-0.09
0.04-0.06
0.08-0.20
0.05-0.10
0.05-0.19
0.02-0.15
1.90
170 PTEROYLGLUTAMIC ACID
TABLE IX—Concluded
Total PGA content,
Food Moisture, % fresh basis, y/g.
Beans, navy 11 1.28
Beans, soybeans 8 2.08
Peas, green split 9 0.24
Peas, yellow split 0.24
Fruits, fresh
Apple, whole 84 0.04
Apricots 87 0.30
Bananas 73 0.35
Berries, blackberries 84 0.39-1.15
Berries, cranberries 86 0.13
Berries, strawberries 91 0.60
Cantaloupes 91 0.47-0.88
Cherries 80 0.30
Grapefruit 89 0.24
Grapes 80 0.20-0.29
Lemons 91 0.87
Oranges 86 0.28-0.40
Orange juice 87 0.36
Peaches 88 0.08-0.20
Pears 85 0.11-0.16
Fruits, dried
Apricots 27 0.06
Peaches 25 0.07
Prunes 27 0.07
Raisins, seedless Ne 0.13
Cereals and grains
Barley 11 0.45
Corn, yellow iil 0.26
Oats 11 0.26
Rice, brown 5 0.12-0.35
Rice, milled 5 0.15
Sorghum grain
White kafir 11 0.21
Yellow milo 10 0.24
Wheat 8.5-10.6 0.384-0.45
Dairy Products
Buttermilk 88 0.11
Evaporated 73 0.007
Non-fat, dry og 0.02
Cheese, Cheddar 34 0.19-0.29
Cheese, cottage 73-77 0.92
IX. EFFECTS OF DEFICIENCY 171
and Home Economics and the Texas Agricultural Experiment Station.!
In addition to values for total PGA content measured by the L. casei
assays, as listed in the table, assays were also made with S. faecalis for
both free and conjugated forms. Bound PGA was measured after digestion
with chicken pancreas enzyme.
IX. Effects of Deficiency
A. IN ANIMALS
EK. L. R. STOKSTAD
Various animal species differ markedly in their requirement for PGA. This
reflects in part their ability to utilize bacterial intestinal synthesis as a
source of the vitamin. The chick, the monkey, and the guinea pig develop
deficiencies on a purified diet low in the vitamin. The rat, on the other hand,
constitutes an excellent example of an animal that is capable of meeting
its dietary requirements for PGA by intestinal synthesis; consequently,
deficiencies of this vitamin were not recognized in the rat until intestinal
antiseptics such as sulfonamides were employed. Other species, such as the
dog, do not develop deficiencies on a purified diet even with the use of in-
testinal antiseptics, and PGA antagonists are necessary to produce a de-
ficiency syndrome.
1. Rats
a. Production of PGA Deficiency by Sulfonamide Feeding
Rats can be grown on a diet free of PGA without development of any
deficiency symptoms unless an intestinal antiseptic is added or a stress is
imposed which increases the requirement of the vitamin. Black ef al.!
showed that addition of a sulfonamide depressed the growth rate which
could then be restored in the presence of the sulfonamide by the feeding of
liver extracts. Shortly after this, Spicer and coworkers? at the National
Institutes of Health observed hematological changes including leucopenia,
agranulocytosis, hypocellularity of the bone marrow, and less frequently
anemia, on diets containing sulfaguanidine or sulfasuxidine (succinylsul-
fathiazole). These symptoms could be prevented or cured by the feeding of
1K. W. Toepfer, E. G. Zook, M. L. Orr, and L. R. Richardson, U.S. Dept. Agr.,
Agr. Handbook 29 (1951).
1§. Black, J. M. McKibbin, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 47,
308 (1941).
‘S. S. Spicer, F. S. Daft, W. H. Sebrell, and L. L. Ashburn, Public Health Repts.
(U. S.) 57, 1559 (1942).
a2, PTEROYLGLUTAMIC ACID
liver extracts. Ransone and Elvehjem? later showed that “folic acid con-
centrates’’ prepared from liver were effective in increasing growth on the
sulfonamide diet.
The activity of pure PGA in correcting the blood dyscrasia was reported
by Daft and Sebrell* and Wright and Welch.® The blood of normal rats
contains 10,000 to 15,000 total leucocytes and 2000 to 4000 granulocytes.
These counts were reduced, respectively, to 700 to 2000 leucocytes and 10
to 500 granulocytes after 30 to 90 days feeding on a diet containing sulfa-
guanidine. The daily feeding of 10 y of PGA or 20 y of pteroyltriglutamic
acid for 4 days increased the leucocytes and the granulocytes to normal.
In some cases there was a surge of leucocytes and especially the granulocytes
to higher than the normal levels.
Definite evidence that PGA is actually synthesized in the intestinal tract
comes from the observation that the depressed growth resulting from sul-
fonamide feeding can be restored by the feeding of rat feces as well as yeast
and yeast extract (Light et al.®).
A thorough investigation of the effects of sulfonamide feeding to rats has
been made by Daft, Sebrell, and coworkers at the National Institutes of
Health, and their work has been excellently reviewed in a Harvey Lecture.’
One of these symptoms which has been frequently observed in rats receiving
sulfonamides is hypoprothrombinemia. This accounts for the subcutaneous
hemorrhages observed on these types of diets.’? This hypoprothrombinemia
can be prevented or cured by the feeding of vitamin K, folic acid concen-
trates,’ and p-aminobenzoic acid.® The effect of p-aminobenzoic acid can
be readily accounted for by its ability to reverse the bacteriostatic effect
of the sulfonamide in the intestine and thus permit resumption of bacterial
synthesis of vitamin K. The effect of the folic acid concentrates cannot be
as readily accounted for. Their method used in preparation of the folic
acid concentrate precludes the presence of a fat-soluble form of vitamin K.
Unfortunately, the effect of pteroylglutamic acid on hypoprothrombinemia
under conditions employed by Welch and Wright® has not yet been re-
ported.
Rats receiving a sulfonamide have an increased susceptibility to anemia
induced by frequent bleeding.'° Administration of pteroylglutamic acid has
3B. Ransone and C. A. Elvehjem, J. Biol. Chem. 151, 109 (1943).
4F.S. Daft and W. H. Sebrell, Public Health Repts. (U. S.) 58, 1542 (1948).
57. D. Wright and A. D. Welch, Science 98, 179 (1948).
6R.F. Light, L. J. Cracas, C. T. Olcott, and C. N. Frey, J. Nutrition 24, 427 (1942)3
7™W.H. Sebrell, Harvey Lectures Ser. 39, 288 (1943-44).
8 A.D. Welch and L. D. Wright, J. Nutrition 25, 555 (1943).
9S. Black, R. S. Overman, C. A. Elvehjem, and K. P. Link, J. Biol. Chem. 145, 137
(1942).
10 A. Kornberg, H. Tabor, and W. H. Sebrell, Am. J. Physiol. 142, 604 (1944).
IX. EFFECTS OF DEFICIENCY 173
a corrective and preventive effect on this anemia as measured by hemo-
globin concentration, hematocrit reading, and white count. The occurrence
of hydrocephalus in about 2 % of the young offspring of rats on a purified
ration has been noted.'' This abnormality can be largely prevented by the
addition of pteroylglutamic acid to the maternal diet. A high incidence of
infarction of the spleen has been noted in PGA deficiency in young rats
but not in control rats receiving 5 y of this factor per day.”
b. Production of PGA Deficiency by Stress of Other Deficiencies
Deficiencies of PGA can be induced by the stress of other vitamin de-
ficiencies as well as by inhibition of intestinal synthesis by use of sulfonam-
ides. In a series of studies by Kornberg et al.,'*-!6 granulocytopenia was ob-
served in a small percentage of animals fed diets not containing
sulfonamides. This dyscrasia was also produced by feeding diets low in
pantothenic acid, and under these conditions the symptoms of anemia,
leucopenia, granulocytopenia, and hypoplasia of the bone marrow which
are characteristic of PGA deficiency could be corrected by pantothenic acid
alone. The similarity of the blood dyscrasia observed in pantothenic acid
deficiency with that found in pteroylglutamiec acid deficiency is evidence
of a relationship between these two factors.
Folic acid concentrates have been reported to be effective in restoring
the color of black rats which had turned grey on diets containing sulfaguan-
idine.” The well-established role of pantothenic acid in the control of hair
color in the rat suggests a possible relationship in the metabolism of PGA
and pantothenic acid. Addition of sulfasuxidine to a purified diet containing
adequate amounts of the vitamin B complex produces a reduction in the
hepatic storage of PGA and pantothenic acid. No change occurs in the
riboflavin and nicotinic acid contents of the liver. Increasing the dietary
intake of pantothenic acid failed to increase growth or to alleviate the typi-
cal pantothenic acid deficiency symptoms such as alopecia, spectacled
eye, porphyrin-caked whiskers, and achromotrichia. Administration of
crystalline biotin and a folic acid concentrate did produce resumption of
growth, alleviate the symptoms, and increase the pantothenic and PGA
contents of the liver.
4 L. R. Richardson and A. G. Hogan, J. Nutrition 32, 459 (1946).
2 C.F. Asenjo, J. Nutrition 36, 601 (1948).
8 A. Kornberg, F. S. Daft, and W. H. Sebrell, Proc. Soc. Exptl. Biol. Med. 58, 46
(1945).
“ A. Kornberg, F. S. Daft, and W. H. Sebrell, Public Health Repts. (U. S.) 60, 1201
(1945).
8 A. Kornberg, F. 8. Daft, and W. H. Sebrell, Arch. Biochem. 8, 431 (1945).
6 A. Kornberg, F. S. Daft, and W. H. Sebrell, Science 103, 646 (1946).
™G. J. Martin, Proc. Soc. Exptl. Biol. Med. 61, 353 (1942).
174 PTEROYLGLUTAMIC ACID
Riboflavin deficiency also produced hematological changes resembling
those of PGA insufficiency. The leucopenia but not the anemia could in
some cases be corrected by administration of PGA, or by riboflavin, or by
a combination of both.!® Restriction of intake of a diet containing riboflavin
also produced the typical blood dyscrasia which suggests that limitation of
other dietary essentials by low feed consumption may be an important fac-
tor.
Severe granulocytopenia and anemia were produced in rats fed protein-
free diets.!® The condition was prevented by casein alone but not by PGA.
The blood dyscrasia could not be cwred by either casein or pteroylglutamic
acid alone but did respond to a combination of the two. When weanling
rats were given a 4% casein diet, the animals again became anemic, leuco-
penic, and granulocytopenic. In this case the leucocyte count was increased
by PGA alone or by parenteral 15-unit liver extract. The activity of the
liver extract could not be accounted for on the basis of its PGA content as
determined by microbiological assay. The simultaneous administration of
methionine and threonine with the PGA increased the incidence and magni-
tude of the response. This observation foreshadowed subsequent work which
established the role of PGA in the metabolism of these compounds.
The interchangeability of pteroylglutamic acid and liver extract in cor-
recting the agranulocytosis has been confirmed both on a normal and on a
low protein diet.'* In these experiments it was impossible to account for
the activity of the liver extract on the basis of PGA or its conjugates as
measured by microbiological assay after enzymatic hydrolysis.
Anemia, leucopenia, and hemorrhage and necrosis of the adrenals were
observed by Daft et al. in rats fed thiourea in a purified diet.!? The blood
dyscrasia was not prevented by thyroid powder or thyroxine but was cor-
rected by administration of PGA. These results are in contrast to the re-
ported ineffectiveness of PGA in preventing the appearance of agranu-
locytosis in two patients receiving thiouracil.2° Higgins observed that
pteroylglutamic acid at a level of 18 y per day counteracted the hypo-
chromic anemia induced by feeding of promin and promizole to young rats
on a purified diet.
c. PGA and Reproduction in the Rat
Reproduction performance in rats during lactation is improved by the
addition of PGA to a purified diet. The maximum improvement is obtained
when the supplement is given to the parent animals from the time of wean-
18 C. W. Mushett and G. A. Emerson, Federation Proc. 7, 295 (1948).
19-F.S. Daft, A. Kornberg, L. L. Ashburn, and W. H. Sebrell, Proc. Soc. Exptl. Biol.
Med. 61, 154 (1946).
20H. V. Newman and B. F. Jones, J. Am. Med. Assoc. 132, 77 (1946).
"1G. M. Higgins, Proc. Staff Meetings Mayo Clinic 19, 329 (1944).
IX. EFFECTS OF DEFICIENCY 175
ing rather than during gestation and lactation only (Sica ed al.??). Under the
same conditions xanthopterin has only a small effect. “Lactation leuco-
penia”’ in rats on a purified diet was found by Nelson et al.’ to be partially
prevented by addition of brewer’s yeast or liver extract. Subsequently,
PGA at a level of 1 mg. per kilogram of diet was observed to increase
maternal weight and the total leucocytes and granulocytes although the
effect was not as complete as that produced by a liver fraction.*4 In view
of subsequent information it seems probable that the additional improve-
ment noted with liver extract may have been due to its vitamin By con-
tent. The severity of the lactation leucopenia was increased and occasional
anemia was induced by addition of sulfasuxidine to a purified diet. Pteroyl-
glutamic acid was effective in preventing these symptoms.?°
2. CHICKS
A dietary deficiency of PGA is readily produced in chicks on purified
diets. Chicks appear to derive very little of their water-soluble vitamins by
intestinal synthesis and, in marked contrast to the rat, readily develop a
PGA deficiency by simple exclusion of this vitamin from the diet.
Beginning in 1938 several lines of investigation were initiated which led
to the recognition of PGA deficiency in chicks. Stokstad and Manning?®
found that chicks grew slowly on a diet composed primarily of polished
rice and water-washed fish meal and supplemented with sources of thiamine,
riboflavin, and pantothenic acid. Growth was increased by the addition of
yeast or alfalfa or their corresponding water extracts. This growth factor,
which was designated factor U, was adsorbed by fuller’s earth and was stable
to autoclaving with acid and alkali.
A macrocytic anemia and reduction of growth rate on a semipurified
type of diet were noted by Hogan and Parrott.?’: * Growth was restored
and anemia prevented by inclusion of liver extract in the diet. Evidence
was presented to show that the anti-anemia principle was distinct from any
of the vitamins previously described, and the name vitamin B, was given
to this factor. A third group of investigators, Hutchings ef al.,°° presented
strong evidence that the ‘‘Norit eluate factor’? required by Lactobacillus
22 A.J. Sica, A. M. Allgeier, and L. R. Cerecedo, Arch. Biochem. 18, 119 (1948).
23M. M. Nelson, F. van Nouhuys, and H. M. Evans, Proc. Soc. Exptl. Biol. Med. 61,
74 (1946).
24M. M. Nelson and H. M. Evans, Arch. Biochem. 18, 265 (1947).
25M. M. Nelson and H. M. Evans, Arch. Biochem. 18, 153 (1948).
26}. L. R. Stokstad and P. D. V. Manning, J. Biol. Chem. 125, 687 (1938).
27 A. G. Hogan and E. M. Parrott, J. Biol. Chem. 128, xlvi (1939).
°° A. G. Hogan and E. M. Parrott, J. Biol. Chem. 132, 507 (1940).
29 B. L. Hutchings, N. Bohonos, D. M. Hegsted, C. A. Elvehjem, and W. H. Peterson,
J. Biol. Chem. 140, 681 (1941).
176 PTEROYLGLUTAMIC ACID
caset was essential for the chick. Concentrates of this factor were effective
at levels of 100 mg. per kilogram of diet. The ‘‘Norit eluate factor” is now
known to be PGA.
Vitamin B, from liver (PGA) was first reported to be active for the chick
by Pfiffner et al.,?° who found that addition of 2.5 mg. of vitamin B, per
kilogram of purified diet prevented anemia and permitted normal growth.
a. Feather Growth and Pigmentation
Pteroylglutamic acid is necessary for feather growth and pigmentation.
Mills et al.*' reported that a concentrate of the ‘‘Norit eluate factor” for
L. casei promoted feathering, growth, and hemoglobin formation in chicks.
Frost et al. raised black leghorn chicks on purified diets containing pure
forms of the water-soluble vitamins. The black breed of bird has the ad-
vantage that it readily shows the effect of PGA on pigmentation as well as
feather growth. Preliminary experiments with concentrates of PGA showed
that 0.400 mg. per kilogram of diet gave good feather growth and pigmen-
tation. It is interesting to note that chicks which received the supplement
for the first 2 weeks only showed normal feathering and pigmentation up
to 10 weeks even though they grew at a decreasing rate and finally began
to lose weight after 7 weeks. When pure PGA was injected at levels of 1,
2.5, 5, and 10 y per day, it was found that the highest level gave normal
feathering and pigmentation. The growth rate, however, was not as high
as that produced by 10 % of brewer’s yeast added to the diet. Most marked
depigmentation occurred at the 5-y level, presumably because feather
growth at the lower vitamin levels was so poor that true pigmentation was
difficult to observe.
Depigmentation became most pronounced in the proximal portions after
the feathers were fairly well developed. The wing and tail feathers were
most affected but nearly all feathers showed some depigmentation.
b. Requirements of the Chick
The requirements of this vitamin have been found to be between 0.25
and 2.0 mg. per kilogram of diet, depending on the criteria of adequacy. Camp-
bell et al.** noted that 0.4 mg. per kilogram of diet was sufficient for main-
tenance for normal hemoglobin, hematocrit, red cell count, and
thrombocyte values, but 4 mg. per kilogram was required for maintenance
of normal leucocyte levels.
30 J. J. Pfiffner, S. B. Binkley, E.S. Bloom, R. A. Brown, O. D. Bird, A. D. Emmett,
A. G. Hogan, and B. L. O’Dell, Science 97, 404 (1943).
31R.C. Mills, G. M. Briggs, Jr., C. A. Elvehjem, and E. B. Hart, Proc. Soc. Expil.
Biol .Med. 49, 186 (1942).
32 D—D. V. Frost, F. P. Dann, and F. C. McIntire, Proc. Soc. Exptl. Biol. Med. 61, 65
(1946).
33 C, J. Campbell, R. A. Brown, and A. D. Emmett, J. Biol. Chem. 152, 483 (1944).
--—
IX. EFFECTS OF DEFICIENCY 177
Comparison of different methods of administration®! showed that sub-
cutaneous injection was slightly more effective than feeding by pipette.
In further studies Pfiffner ef al.*® reported that either PGA or its conjugate
at a level of 0.25 mg. per kilogram of diet gave maximum growth but sub-
maximal hemoglobin response. Briggs and Lillie*® have shown that 1 mg.
per kilogram of diet is necessary for normal feather pigmentation and 2
mg. for maximum growth. Oleson eé al.’ reported that PGA at a level of
1 to 5 mg. per kilogram of diet gave normal growth and pigmentation of
feathers in chicks. The requirement was not appreciably altered by addition
of sulfamerazine or certain other intestinal antiseptics.
Oleson ef al.** found that the requirements for maximum growth up to
4 weeks were 0.4 mg. per kilogram of diet in one experiment and 1.0 mg.
in another. In both cases 1.0 mg. was required for optimum hemoglobin
response. Pteroyltriglutamic acid was found to be as effective on a molar
basis as the monoglutamate. The addition of 0.7 % of either sulfasuxidine,
sulfaguanidine, or carboxysulfathiazole did not affect the response on mar-
ginal levels of pteroyltriglutamic acid. Robertson ef al *° found the require-
ments to be 0.45 mg. per kilogram of diet for growth and hemoglobin up
to 4 weeks of age. The requirement at 6 weeks for optimum hemoglobin
was stated to be 0.35 mg. and 0.55 mg. for normal feathering. In these ex-
periments the PGA requirements were not affected by addition of 1 and
2% sulfasuxidine to a purified diet. It thus appears that sulfonamides do
not have as marked an effect on the PGA requirements in chicks as they
do in rats.
Perosis has been observed by Hill e¢ al.*° in chicks on a purified-type of
diet adequately substituted with choline, biotin, and manganese but de-
ficient in PGA. The incidence was increased by addition of 2% sulfasuxi-
dine. The perosis was prevented by addition of 0.2 mg. per kilogram of PGA
without the sulfonamide and by 0.3 mg. in the presence of the sulfonamide.
Pteroyltriglutamic acid has been shown to be effective for the
chick." It has also been reported by Scott ef al. that the effectiveness of
34 C, J. Campbell, R. A. Brown, and A. D. Emmett, J. Biol. Chem. 154, 721 (1944).
35 J. J. Pfiffner, D. G. Calkins, B. L. O’Dell, E.S. Bloom, R. A. Brown, C. J. Camp-
bell, and O. D. Bird, Sczence 102, 228 (1945).
36 G. M. Briggs and R. J. Lillie, Federation Proc. 5, 124 (1946).
37 J. J. Oleson, B. L. Hutchings, and N. H. Sloane, J. Biol. Chem. 165, 371 (1946).
38 B. L. Hutchings, J. J. Oleson, and E. L. R. Stokstad, J. Biol. Chem. 163, 447 (1946).
39}. I. Robertson, L. J. Daniel, F. A. Farmer, L. C. Norris, and G. F. Heuser,
Proc. Soc. Exptl. Biol. Med. 62, 97 (1946).
40 FW. Hill, L. C. Norris, and G. F. Heuser, J. Nutrition 28, 175 (1944).
1B. L. Hutchings, E. L. R. Stokstad, N. Bohonos, and N. H. Slobodkin, Science 99,
371 (1944).
“2M. L. Scott, L. C. Norris, G. F. Heuser, and W. F. Bruce, J. Biol. Chem. 158, 291
(1945).
178 PTEROYLGLUTAMIC ACID
pteroyltriglutamic acid is increased by the addition of 5-pyridoxic acid
(2-methyl-3-hydroxy-4-hydroxymethyl-5-carboxypyridine) or 4-pyridoxic
acid (2-methyl-3-hydroxy-4-carboxy-5-hydroxymethylpyridine). This sup-
plementary effect has been observed both for growth and hemoglobin
formation in the young chick” and for regeneration of hemoglobin in hens
rendered anemic by bleeding.**
Jukes and Stokstad“ failed to observe any effect of the pyridoxie acids
in increasing the effectiveness of pteroyltriglutamic acid. In a similar way
pteroylheptaglutamic acid was found by Binkley et al.4° to be as effective
on a molar basis as-the monoglutamate without the addition of pyridoxic
acid,
c. Requirements of Turkeys and Ducks
Richardson et al.** reported that a deficiency of this factor in the diet of
the turkey produces cervical paralysis (Fig. 11) in addition to a reduction
in growth rate. No marked anemia was observed. Cervical paralysis was
also observed on a practical diet containing corn, wheat bran, and wheat
middlings, which indicates that a deficiency of PGA can occur on diets
composed primarily of natural ingredients. Spontaneous remissions of cervi-
cal lesions were not observed. Complete recovery was obtained in 4 to 6
hours by injection of 100 y of PGA. Thus far cervical paralysis has not been
reported in chicks deficient in PGA. Jukes ef al.” found the requirement
of this factor for maximum growth of the turkey to be approximately 1.0
mg. per kilogram of diet.
Although large variations exist, the PGA requirement of the turkey for
growth appears to be higher than that of the chick. Schweigert* reported
PGA and pteroyltriglutamic acid to be equally effective in increasing PGA
blood levels and preventing the symptoms of mild anemia to severe ane-
mia, poor feather condition, and weakened legs. After 8 weeks of the un-
supplemented diet, the hematocrit had dropped to 28 %, compared to 40 %
for those receiving 2 mg. of PGA per kilogram of diet. Curative treatment
with the vitamin increased the hematocrit to normal in 2 weeks.
Ducks grown on a purified type of diet give a marked growth response
to the addition of 100 y per day of pteroyltriglutamic acid.*®
43M. L. Scott, L. C. Norris, and G. F. Heuser, Sczence 108, 303 (1946).
4TH. Jukes and E. L. R. Stokstad, J. Biol. Chem. 168, 563 (1947).
45 §. B. Binkley, O. D. Bird, E. S. Bloom, R. A. Brown, D. G. Calkins, C. J. Camp-
bell, A. D. Emmett, and J. J. Pfiffner, Sczence 100, 36 (1944).
46 LL. R. Richardson, A. G. Hogan, and H. L. Kempster, J. Nutrition 30, 151 (1945).
47'T. H. Jukes, KE. L. R. Stokstad, and M. Belt, J. Nutrition 33, 1 (1947).
48 B.S. Schweigert, Arch. Biochem. 20, 41 (1949).
19D. M. Hegsted and F. J. Stare, J. Nutrition 30, 37 (1945).
IX. EFFECTS OF DEFICIENCY 179
d. Requirements for Reproduction
The requirements for egg production are low. A diet composed primarily
of polished rice and sardine meal and containing 0.12 mg. of PGA per
kilogram of diet by microbiological assay was found by Taylor®® to be
adequate for egg production. Further supplementation with 1.0 mg. of
PGA per kilogram yielded no increase in egg production although the
hatchability of fertile eggs was increased. These results are in agreement
with those of Schweigert et al.,°' who reported that egg production and
hatchability on a basal diet containing 0.42 mg. of PGA per kilogram (de-
Fic. 11. Normal turkey (left) and PGA-deficient turkey (right), showing symp-
toms of cervical paralysis.
termined by microbiological assay) and composed primarily of corn and
casein were not improved by the addition of 2.0 mg. of PGA. This was ob-
served with both turkeys and hens. The average PGA content of the eggs
before and after supplementation of the hen diet was 0.12 and 0.21 y per
gram in the case of the turkey and 0.099 and 0.133 y, respectively, in the
case of the chicken. For poults from hens adequately supplemented with
PGA, 0.8 mg. of the vitamin per kilogram appeared to be adequate. The
requirement was higher for poults from hens which received no supplemen-
tation. Thus, although 0.45 mg. of PGA per kilogram of diet (measured by
50 LL. W. Taylor, Poultry Sct. 26, 372 (1947)
‘1 B.S. Schweigert, H. L. German, P. B. Pearson, and R. M. Sherwood, J. Nutrition
35, 89 (1948).
180 PTEROYLGLUTAMIC ACID
microbiological assay in the basal diet) appeared adequate for egg produc-
tion and hatchability, it was not adequate for optimum livability of the
poult and storage of this vitamin in the egg.
3. MoNnKEYS
The rhesus monkey is peculiarly susceptible to a deficiency of PGA, and
the symptoms are more similar to those of the human than are those of any
other species. They include anemia, leucopenia, oral lesions, diarrhea, ul-
cerations of the colon, and increased susceptibility to infections of the intes-
tinal tract. Re-examination of the literature shows that certain of these
symptoms had been described and credited to unknown nutritional factors
long before PGA was recognized as a vitamin. Day has termed this factor
vitamin M and in an excellent review on the early work in this field*” points
out that ‘this syndrome, or certain manifestations of it, at least, have been
rediscovered and described every few years since 1919. The work has been
approached from many angles; the diets used have been very different, but
what appears to be the same syndrome has been found in each.”’
Wills and coworkers*’ were able to distinguish between the anti-perni-
cious factor in liver and the anti-anemic principle for monkeys, the anti-
pernicious anemia factor being precipitated by ammonium sulfate and the
monkey anti-anemia factor remaining 1n solution.
A wide variety of diets have been used for the production of vitamin M
deficiency (PGA deficiency) in the monkey. That used by Day and his
collaborators at Arkansas was composed of Labco vitamin-free casein,
polished rice, wheat, salt mixture, and ascorbic acid, and the known water-
and fat-soluble vitamins.**
a. Blood Changes
The following symptoms have been described by Day*® and credited to
PGA deficiency. There is a progressive decrease in all types of peripheral
blood cells. The normal values for the rhesus monkey are 5.0 million RBC,
10,000 to 20,000 total leucocytes. A profound leucopenia involving all
white cell types is the most striking aspect of the deficiency. There is an
almost absolute neutropenia and lymphopenia. Total white cells drop to
2000 to 3000, and there is a marked agranulocytosis. This agranulocytosis
is thus similar to that observed in rats fed a purified diet containing sul-
fonamide. Limited data indicate a thrombocytopenia. Some animals show
a pronounced anemia, but others will develop a marked leucopenia which
is rapidly fatal without showing any severe anemia. Minimum counts of 2
2 PL. Day, Vitamins and Hormones 2, 71 (1944).
53 T,. Wills, P. W. Clutterbuck, and B. D. F. Evans, Biochem. J. 31, 2136 (1937).
54 J. R. Totter, C. F. Shukers, J. Kolson, V. Mims, and P. L. Day, J. Biol. Chem. 152,
147 (1944).
IX. EFFECTS OF DEFICIENCY IS]
and 3 million are common, and counts of 1 million or slightly less have been
observed. The anemia is slightly macrocytic, the volume index and mean
cell diameter being slightly increased.
The addition of PGA, or concentrates of this vitamin, effects a reticu-
locyte crisis which may be as high as 40 %, a dramatic increase in white cells,
16 2S SS
Hemoglobin
12 w 6 o
2° § mee Z
8 gE ee Os See ®
& € Sete ate a 50 es
= > co)
see, 40 5
wo S) Erythrocytes 3
e = 30 2
a Cy
a
Sa See) 20
Reticulocytes
10
0 0 o-J-—--L--L.— 0
L. Casei factor L. Casei factor
28
24
Total white cells
White cells, thousands
a
0
Days on experiment
Fig. 12. Typical responses of the cytopenic monkey to natural pteroyltriglutamic
acid (L. casei factor). In each of the two experiments on this monkey the total dose of
vitamin was approximately 3 mg., given intramuscularly. Courtesy J. Biol. Chem.
(Day et al.®5).
and a slower return of red cell and hemoglobin values.** A typical response
of a deficient monkey to administration of pteroyltriglutamic acid (fer-
mentation L. casei factor) is shown in Fig. 12.
A megaloblastic bone marrow is found in monkeys with nutritional eyto-
penia. Wilson®*® reported that there is a relative hypoplasia in the myeloid
elements in monkeys in the terminal stages of peripheral leucopenia.
5° P_L. Day, V. Mims, and J. R. Totter, J. Biol. Chem. 161, 45 (1945).
66 H. E. Wilson, S. Saslaw, J. L. Schwab, O. C. Woolpert, and C. A. Doan, J. Am.
Med. Assoc. 121, 1411 (1943).
182 PTEROYLGLUTAMIC ACID
Further details of the blood picture may be obtained from the papers of
Wills and Stewart,” Langston et al.,°° and Wilson et al.°?
b. Oral Lesions
Oral lesions, particularly of the margins of the gums, are one of the most
frequently observed signs of nutritional cytopenia in the rhesus monkey.
These have been described in detail by Day et al.,®° Langston et al.,°® Chap-
man and Harris,®! and Saslaw et al. The oral lesions develop shortly after
the onset of leucopenia and begin as a recession of the gums which develops
into a yellowish ulceration. The ulceration usually develops among the in-
Fia. 13. Gum lesions in vitamin M deficiency in monkey. Courtesy Dr. P. L. Day.
cisors and may become so severe that the bone is exposed or the teeth fall
out. The gums are pale, and there are no marked spontaneous hemorrhages.
A photograph of the gingivitis in the terminal stages of an animal with nu-
tritional cytopenia is shown in Fig. 13. It was noted by Saslaw et al. that
57 T,. Wills and A. Stewart, Brit. J. Haptl. Pathol. 16, 444 (1935).
5883 W. C. Langston, W. J. Darby, C. F. Shukers, and P. L. Day, J. Exptl. Med. 68,
923 (1938).
59 H. E. Wilson, C. A. Doan, 8S. Saslaw, and J. L. Schwab, Proc. Soc. Exptl. Biol.
Med. 50, 341 (1942).
60 P. L. Day, W. C. Langston, and C. F. Shukers, J. Nutrition 9, 637 (1935).
61 QO, D. Chapman and A. E. Harris, J. Infectious Diseases 69, 7 (1941).
62S. Saslaw, J. L. Schwab, O. C. Woolpert, and H. E. Wilson, Proc. Soc. Exptl. Biol.
Med. 61, 391 (1942).
IX. EFFECTS OF DEFICIENCY 183
lesions of the oral mucous membranes often become infected with Sdrep-
tococct, Slaphylococci, or the flora of Vincent’s angina. In monkeys on normal
diets these lesions do not occur spontaneously, and where accidental trauma
of the gum or buccal mucosa does occur no secondary infection appears.
This illustrates the increased susceptibility of the monkey to bacterial in-
fection during a deficiency of PGA.
c. Diarrhea and Susceptibility to Bacillary Dysentery
Diarrhea is one of the most frequent and striking characteristics of the
vitamin M deficiency syndrome. The stools become watery in a short time,
and in some cases blood and mucus appear. Administration of PGA has a
rapid and dramatic effect. In a few days formed stools appear which almost
suggest that this vitamin has an effect on water balance in the intestine.
The diarrhea in the monkey and the marked effect of PGA are very similar
to that observed in sprue in clinical patients. When blood and mucus ap-
pear, the monkey becomes ill with the clinical symptoms of bacillary dysen-
tery. Stool cultures usually reveal the presence of Shigella type of organism.
Janota and Dack® demonstrated convincingly the relation between vita-
min M deficiency and dysentery in the monkey. They found that monkeys
which have been kept in the laboratory on a stock diet do not develop bacil-
lary dysentery, even though animals are received with the disease from the
dealers. If the resistance of the animal is lowered by vitamin M deficiency,
some of the animals develop bacillary dysentery. This suggests that Bact.
dysenteriae (Flexner) may live in a saprophytic existence in the intestine.
“The fact that control monkeys do not develop dysentery when kept in
the same room with animals on a vitamin M-deficient diet indicates that
the disease occurs as a result of lowered resistance due to vitamin deficiency
and not to contact infection.’”? These workers also found “‘that monkeys
deficient in vitamins C, A and D in addition to vitamin M did not show
any increased incidence of lesions of the mouth or bowel over those monkeys
deficient in vitamin M alone.”’
Day,” in his excellent review of this subject, reported that the feeding
of large numbers of living dysentery bacilli to a monkey receiving a normal
diet (a vitamin M-deficient diet supplemented with liver extract) did not
produce diarrhea, clinical dysentery, or any changes in the blood picture.
A year later this monkey was made deficient in vitamin M and spontane-
ously developed an acute clinical dysentery and died.
Saslaw et al.” found that monkeys on a vitamin M-deficient diet mani-
fested a “markedly lowered clinical resistance to spontaneous infections
with high mortality. The susceptibility to experimental infections with
68M. Janota and G. M. Dack, J. Infectious Diseases 65, 219 (1939).
184 PTEROYLGLUTAMIC ACID
Streptococcus hemolyticus Group C and to influenza virus A administered
intranasally was likewise increased in contrast with controls on normal
diets.”
d. Requirements of the Monkey
Daily injections of 0.1 to 0.2 mg. of PGA produced a reticulocyte response
of about 7%.** This apparently represents the minimum amount that will
elicit a response. In a separate investigation Day et al.®° found that totals
of 4 and 4.5 mg. of pteroyltriglutamic acid given in divided doses over 4 to
5 days produced reticulocyte responses of 10 and 8 % with prompt elevation
of the total white cells and granulocytes.
Cooperman et al.®° reported that daily administration of 100 y of PGA
produced a rise in white cells and a gain in weight in deficient monkeys.
No marked rise in hemoglobin occurred. Two hundred micrograms of
vitamin B, conjugate (pteroylheptaglutamate) produced less of a response
in one animal than 100 y of PGA. The monkey is obviously able, however,
to utilize some of the conjugate in yeast, since a sample of yeast which was
found to be low in free PGA (measured by S. faecalis R) was a good source
of vitamin M for monkeys.*! This same sample of yeast was subsequently
shown to contain PGA conjugate because its activity for S. faecalis was
increased by digestion with rat liver enzyme. Day and Totter® have re-
ported the minimum daily protective dose of 100 y on their basal diet which
furnished an additional 20 y per day. However, with a purified type of diet
Smith and Elvehjem®® obtained normal growth and blood cell formation
with 100 y per day. The minimum protective dose was not established.
It is of interest to compare the PGA requirements of the monkey and the
human. In pernicious anemia the minimal effective curative dose is ap-
proximately 5 mg. per day. On a body weight basis this would correspond
to approximately 0.2 mg. per day for a 3-kg. monkey.
e. Monkey Anti-Anemia Factor
The existence of an anti-anemia factor for monkeys has been postulated
by Cooperman et al.*® The evidence is based largely on the failure of PGA
at levels of 100 y per day to completely restore the blood picture in mon-
keys which have been depleted on a PGA-deficient diet containing the
64 H. EK. Wilson, 8. Saslaw, and C. A. Doan, J. Lab. Clin. Med. 31, 631 (1946).
66 P| L. Day, V. Mims, J. R. Totter, E. L. R. Stokstad, B. L. Hutchings, and N. H.
Sloane, J. Biol. Chem. 157, 423 (1945).
66 J. M. Cooperman, C. A. Elvehjem, K. B. McCall, and W. R. Ruegamer, Proc. Soc.
Exptl. Biol. Med. 61, 92 (1946).
67 V. Mims, J. R. Totter, and P. L. Day, J. Biol. Chem. 155, 401 (1944).
68 P. L. Day and J. R. Totter, J. Nutrition 36, 803 (1948).
69 S$. C. Smith and C. A. Elvehjem, J. Nutrition 45, 47 (1951).
IX. EFFECTS OF DEFICIENCY 185
other known B vitamins and ascorbic acid. These levels of PGA produce
a rise in white cells and an increase in growth, but the hemoglobin does not
rise to normal. Feeding of whole liver produces complete restoration of
hemoglobin. One marked characteristic of this syndrome is a reversal of
the normal ratio of neutrophiles to lymphocytes. It seems possible that the
levels of PGA used in these experiments were near the marginal level and
that the response produced by the liver may have been due to its PGA
content. This is supported by the observations of Smith and Elvehjem®?
that 0.5 to 1.0 mg. of PGA per day completely eliminated the need for the
monkey anti-anemia factor. This was about five to ten times the amount
needed for maintenance. Once the animals had been ‘‘cured’’ by 1.0 mg.
of PGA per day, they could be maintained with 0.10 mg. per day. A metha-
nol extract of liver which furnished only 4 y of PGA per day produced a
transient response. This amount of liver extract was able to maintain the
blood picture when supplemented with 50 y of PGA. Although the assay-
able amounts of PGA in the methanol extract were low, the possible pres-
ence of PGA conjugates cannot be ruled out. Other liver extracts have been
reported which gave PGA-like responses in excess of their assayable PGA
content. Thus the chick factors vitamins Bi) and By were liver fractions
which contained only traces of assayable Bi.. However, it was reported
that in the presence of sufficient PGA vitamins By and Bu were no longer
required by the chick.’° Certain parenteral liver extracts have also been
found which have a greater effect on the granulocyte count of sulfonamide-
fed rats than can be accounted for by their PGA content.'®
It is apparent that the monkey does not need the monkey anti-anemia
factor for maintenance, since purified basal diet supplemented with 100 y
of PGA per day and without any additional source of the monkey anti-
anemia factor is adequate for growth and maintenance of a normal blood
picture. The requirement for this has been shown only during the stress of
a deficiency of PGA or of riboflavin.”!
The possibility exists that the monkey anti-anemia factor is the citro-
vorum factor, since the latter has been shown to be more active than PGA
for the monkey in the anemia induced by ascorbic acid deficiency.” Smith
and Elvehjem point out that it is unlikely that the monkey anti-anemia
factor is “identical with the citrovorum factor of Sauberlich and Baumann
since reticulogen (injectable liver extract) which is a good source of CF is
inactive in the monkey” as a source of the anti-anemia factor.’> However,
70 T. D. Luckey, P. R. Moore, C. A. Elvehjem, and E. B. Hart, Sczence 103, 682 (1946).
1 J. M. Cooperman, H. A. Waisman, K. B. McCall, and C. A. Elvehjem, J. Nutri-
tion 30, 45 (1945).
72 C.D. May, R. D. Sundberg, and F. Schaar, J. Lab. Clin. Med. 36, 963 (1950).
73 W.R. Ruegamer, E. M. Sporn, U. D. Register, and C. A. Elvehjem, J. Nutrition
36, 405 (1948).
186 PTEROYLGLUTAMIC ACID
although reticulogen is active for Le. citrovorum at very low concentrations,
the actual amount of citrovorum factor supplied by the 45 U.S.P. units of
reticulogen would correspond to only about 5 y of CF per day. It would
indeed be interesting to see whether the citrovorum factor (folinie acid)
would be more effective than PGA under the conditions of this test.
4. Pies
PGA deficiencies in swine have not been observed on either purified or
natural diets without the use of intestinal antiseptics. Baby pigs which had
been given colostrum for 4 days and then placed on a diet of synthetic milk
plus 2% sulfathalidine (calculated on solids content of the milk) did not
develop any signs of PGA deficiency (Johnson et al.™). The baby pig diet
contained 30 % of Labeo vitamin-free casein and 27 % lard. Since subsequent
work with swine and previous work with rats have shown that PGA re-
quirements are decreased at higher protein levels, it seems possible that the
failure to produce any PGA deficiency as measured by growth or blood pic-
ture may be a reflection of the high protein level used.
With adult pigs blood changes have been produced by feeding of purified
diets containing sulfonamides. Cartwright e¢ al.7> fed a diet containing 26 %
of Labco vitamin-free casein and 2 % of sulfasuxidine. After 120 days growth
almost stopped and marked alopecia developed, especially over the flanks
and rump. A normocytic anemia developed, and the hematocrit dropped to
21%. Administration of biotin produced no change in the skin condition.
Administration of 1 ml. of purified liver extract for 9 days produced a retic-
ulocyte response with a peak of 9.4%, a marked growth response of 30
kg. in 50 days, and restoration of a normal hair coat. Four animals which
received sulfasuxidine and crude casein in place of the vitamin-free casein
were normal in all respects. In this particular experiment it appears that
vitamin By» was producing a response on a diet supposedly deficient in PGA.
In a subsequent paper these same workers,’® using a larger number of
animals, found that pigs on this same diet containing Labco vitamin-free
casein and sulfasuxidine would respond only partially to refined liver extract
but completely to PGA. The reticulocyte peak with PGA varied between
4 and 16% and that with liver extract between 5 and 8 %. Substitution of
crude casein for vitamin-free casein reduced the severity of the anemia, and
spontaneous remissions with broad reticulocyte responses sometimes oc-
curred. Reduction of the crude casein level from 26 to 10 % made the ane-
mia more severe.
™ B.C. Johnson, M. F. James, and J. L. Krider, J. Animal Sci. 7, 486 (1948).
79 G. E. Cartwright, M. M. Wintrobe, and 8S. Humphreys, J. Lab. Clin. Med. 81, 423
(1946).
76G. E. Cartwright and M.M Wintrobe, Proc. Soc. Exptl. Biol. Med. 71, 54 (1949).
IX. EFFECTS OF DEFICIENCY 187
Cunha ef al.” found that on a diet containing 26% of “Vitamin-Test
Casein” and 2 % sulfasuxidine no effect of PGA could be observed on growth
rate. However, a normocytic anemia developed in which the hemoglobin
content was raised from 8.6 g. to 14.4 g. % by the addition of 0.5 mg. of
PGA per kilogram of diet. Refined liver extract containing the anti-perni-
cious anemia factor produced a smaller hemoglobin response. It seems
probable that the diets employed were deficient both in PGA and in vita-
min By . Crude casein in the amounts used in the diets constitutes a good
source of By. Since pigs have been shown to require By , it seems very
likely that these pigs which received vitamin-free casein were deficient
both in vitamin By and in PGA.
Pteroylglutamic acid deficiency has also been induced in the pig by the
feeding of a crude ‘“‘x methyl PGA” antagonist.”2 This antagonist has the
advantage that its action can be completely reversed by pteroylglutamic
acid.*® The feeding of antagonist accentuates the anemia observed on a
purified diet containing sulfasuxidine. The anemia produced with the sul-
fonamide ration is normocytic.” The hematological manifestations observed
with sulfasuxidine plus antagonist include (a) severe macrocytic anemia,
(b) leucopenia, with a greater proportional reduction of polymorphonu-
clear than of mononuclear cells, (¢) mild thrombocytopenia, and (d) hyper-
plastic bone marrow with an increase in immature nucleated red cells which
resemble the megloblasts observed in pernicious anemia.’? The blood and
bone marrow are rapidly restored to normal by the injection of 10 to 20
mg. of either PGA or the corresponding diglutamate, triglutamate, or
heptaglutamate. Thus, all the known conjugates appear active. A partial
response was given by injection of a single dose of 0.10 mg. of PGA which
produced a reticulocyte peak of 10% and an increase in hematocrit read-
ing of 10% in 9 days. With larger amounts of PGA, reticulocyte responses
varied between 11 and 42 % and there was a corresponding rapid increase
in the hematocrit value between 10 and 20%. When 1 unit (corresponding
to approximately 1 y of vitamin By) of U.S.P. injectable liver extract was
given per day, the blood and bone marrow changes were neither prevented
nor was their onset postponed. When used curatively, liver extract, 30 to
150 units total, and vitamin B,., 150 y total, produced some hemapoietic
response although less than that elicited by PGA.
Thymine, which produces a hematological response in pernicious anemia,
was inactive; xanthopterin, tyrosine, adenine, and uraci! were also ineffec-
tive.
7 'T. J. Cunha, R. W. Colby, L. K. Bustad, and J. F. Bone, J. Nutrition 36, 215 (1948).
7 A.L. Franklin, E. L. R. Stokstad, M. Belt, and T. H. Jukes, J. Biol. Chem. 169,
427 (1947).
79G. E. Cartwright, B. Tatting, H. Ashenbrucker, and M. M. Wintrobe, Blood 4,
301 (1949).
188 PTEROYLGLUTAMIC ACID
In confirmation with observations previously made by these same work-
ers,’® on a sulfasuxidine diet the severity of the anemia was greater on a 10 %
casein diet than on a 26 % casein diet even when the ‘“‘x methyl PGA” was
present in the ration.
The urinary excretion of tyrosyl derivatives (hydroxyphenol) was ab-
normal in the PGA-deficient pigs and was not altered by therapy with PGA
or liver extract. This is an interesting observation in view of the findings
that the failure of ascorbutic guinea pigs to metabolize tyrosyl compounds
ean be corrected by administration of PGA.*°
Heinle et al.8! have found that pigs made deficient on a diet containing
sulfasuxidine and ‘‘x methyl PGA” respond well to PGA initially, but as
the deficiency is maintained the ability to respond diminishes. At this point
purified liver extract gives a response. Conversely, they also observed that
when a pig kept on this PGA-deficient diet for several months was given
large amounts of liver extract and later vitamin By, there was an initial
partial response. The blood values were maintained at an improved level
but never at a normal level. Very marked macrocytosis developed and per-
sisted, but was promptly corrected by administration of PGA. A vitamin
Bie deficiency was produced by feeding a diet containing purified soybean
protein. On this regimen an anemia developed which was less macrocytic
than that observed in PGA deficiency and no megaloblasts were found in
the bone marrow. The animals responded to vitamin By». therapy.
Johnson e¢ al.® studied the effects of PGA and vitamin By deficiency
in the baby pig using synthetic milk diets containing purified soybean pro-
tein. This protein is lower in vitamin By. than casein and permitted the
simultaneous production of PGA and vitamin By» deficiencies. When pigs
were placed on a 20% protein diet without vitamin By. or PGA and with
0.6% of sulfathalidine, the growth rate decreased and a mild anemia de-
veloped. After a 7-week depletion period, administration of vitamin Bi»
produced an increase in growth and a 7 % reticulocyte peak on the fifth
day. After 4 weeks of continued injection of 2 y of vitamin By». daily, the
oral administration of PGA produced a second sharp reticulocyte peak of
7% on the second day.
The pigs were also given a diet deficient in PGA and vitamin By but
without sulfathalidine for 3 weeks which was followed by the addition of
‘x methyl PGA” for the next 2 weeks. At this point either PGA or vitamin
Bi. provoked the maximum hematological and bone marrow responses but
only Bis elicited a growth response.
80 C, W. Woodruff, M. E. Cherrington, A. K. Stockell, and W. J. Darby, J. Biol.
Chem. 178, 861 (1949).
81. W. Heinle, A. D. Welch, and H. L. Shorr, J. Lab. Clin. Med. 34, 1763 (1949).
82 B. C. Johnson, A. L. Neumann, R. O. Nesheim, M. F. James, J. L. Krider, A. S.
Dana, and J. B. Thiersch, J. Lab. Clin. Med. 36, 587 (1950).
IX. EFFECTS OF DEFICIENCY 189
These results show that the pig requires both vitamin Biz. and PGA. The
basic requirement for blood cell formation seems to be that of PGA, but
in some cases of double deficiency either one may provoke a response. Con-
tinued administration of vitamin By. does not give complete protection
against anemia whereas continued administration of PGA apparently does.
The growth-stimulating function of vitamin By, cannot be substituted by
PGA.
5. MicE
Evidence has been obtained that mice require PGA for growth and re-
production. Nielsen and Black** observed that mice grown on a purified
diet containing sulfasuxidine give a growth response to folic acid concen-
trates prepared from liver extract. The extent of the deficiency was less
when the sulfonamide was omitted from the diet. Cerecedo and Mirone*™
investigated the nutritive requirements of lactation and found that addition
of 10 mg. of PGA per kilogram of a purified diet doubled the average num-
ber of mice weaned and increased the weight gain of the lactating mother.
No significant effect on litter size was observed.
6. Guinea Pies
A series of studies with guinea pigs on purified diets indicated that three
unidentified dietary factors are required, two of which were found present
in linseed oil meal. It was later shown by Woolley and Sprince that PGA
could replace one of the factors.s° The deficiency was characterized by
lethargy, salivation, loss in weight, terminal convulsions, and death.
7. MInk
The effects of pterolyglutamic acid deficiency in mink have been studied
by Schaefer et al.8° This was characterized by loss of body weight, diarrhea,
anorexia and leucopenia. The deficiency symptoms were rapidly corrected
by administration of pteroylglutamic acid. Some evidence was obtained
which indicated that an unidentified factor in liver was also needed.*®
8. Dogs
Dogs are very refractory to pteroylglutamiec acid deficiency. Michaud
et al. found that a purified ration supplemented with the water-soluble
vitamins will support excellent growth even in the presence of 4% sulfa-
83}. Nielsen and A. Black, J. Nutrition 28, 203 (1944).
84 1,. R. Cerecedo and L. Mirone, Arch. Biochem. 12, 154 (1947).
8® D. W. Woolley and H. Sprince, J. Biol. Chem. 157, 447 (1945).
86 A. E. Schaefer, C. K. Whitehair, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med.
62, 169 (1946).
37 L.. Michaud, A. R. Maass, W. R. Ruegamer, and C. A. Elvehjem, Proc. Soc. Expil.
Biol. Med. 56, 148 (1944).
190 PTEROYLGLUTAMIC ACID
suxidine. Repeated production of anemia by bleeding failed to show any
difference between the control dogs and those receiving sulfasuxidine. Re-
generation of hemoglobin after hemorrhage was rapid even in the presence
of 4% sulfasuxidine. This is in marked contrast to the rat, which develops
an impaired capacity to regenerate hemoglobin on a sulfonamide-contain-
ing diet. Deficiencies of PGA were obtained by Krehl ef al. by imposing
the stress of another vitamin deficiency. Dogs kept on a niacin-deficient
diet were found to respond to a few successive single doses of niacin but
then finally failed to respond and died even though niacin was adminis-
tered. In each case the animal was allowed to become deficient again before
the next dose of niacin was administered. If PGA was included in the diet,
the response to niacin was improved and the dog could be alternately
treated with niacin and allowed to become deficient again a large number
of times. This production of deficiency of PGA by a concomitant nicotinic
acid deficiency is reminiscent of the relationship observed between PGA
and riboflavin in the rat.!®
9. Foxrs
Schaefer et al.8° found that foxes kept on a purified type of diet develop
critical symptoms of PGA deficiency in 7 to 14 weeks. These include ano-
rexia, rapid loss of body weight, and reduction of hemoglobin. Total white
cell and erythrocyte counts were reduced to approximately 50% of the
normal value. Administration of 0.25 to 1.0 mg. of PGA per kilogram of
diet resulted in an increase in hemoglobin, white cells, and growth rate.
A yeast preparation containing pteroylglutamic acid conjugate, when fed
at a level calculated to give 0.60 mg. of PGA in the form of the conjugate,
failed to produce any hematological or growth response. A similar yeast
preparation hydrolyzed with kidney enzyme produced a typical increase
in hemoglobin level. This shows that the fox, in contrast to all other animals
thus far observed, is unable to utilize the PGA conjugates present in yeast.
It is also interesting that the fox, although taxonomically related to the
dog, is so susceptible to PGA deficiency, whereas the dog is quite resistant.
Adult foxes as well as growing pups developed PGA deficiency symptoms
on the purified diet. There is evidence that an unidentified factor present
in liver is also required by foxes in addition to PGA.%°
10. INSECTS
Several insects have been observed which require PGA at some stage of
their development. Larvae of the mosquito Aédes aegyptii were found by
88 W. A. Krehl, N. Torbet, J. de la Huerga, and C. A. Elvehjem, Arch. Biochem. 11,
363 (1946).
89 A. EK. Schaefer, C. K. Whitehair, and C. A. Elvehjem, Arch. Biochem. 12, 349
(1947).
IX. EFFECTS OF DEFICIENCY 191
Goldberg et al.°® to require PGA for pupation, xanthopterin or thymine
being ineffective. PGA also increases growth and survival rates, body pig-
mentation, and size of the larvae. PGA has the most marked effect during
the third stage of larval life. Fraenkel ed al.®' showed the need for PGA in
the nutrition of the meal worm Tenebrio molitor. Growth of the larvae of the
carpet beetle was increased by concentrates of PGA (Moore). Grob et al.%
reported that the larvae of rice flour beetle T'ribolium confusum requires two
unknown factors, one of which could be replaced by PGA concentrates.
11. RELATION BETWEEN PGA anp AscorBic AcID
a. PGA and Tyrosine Metabolism
An interesting relationship has been observed between PGA and ascor-
bie acid in the metabolism of tyrosine. Ingestion of large amounts of tyrosine
by scorbutic guinea pigs produces a large increase in the excretion of p-
hydroxyphenylpyruvic acid, p-hydroxyphenyllactic acid, and small amounts
of tyrosine. This apparent defect in tyrosine metabolism disappears on the
administration of small amounts of ascorbic acid (Sealock and Silberstein)
The scorbutic guinea pig is thus unable to oxidatively rupture the benzene
ring. The failure of D-isoascorbic acid to prevent the tyrosyluria except in
doses twenty times that of ascorbic acid indicates that the activity of the
latter is specifically related to its vitamin activity and not simply to its
reducing properties. Painter and Zilva®> found that the excretion of abnor-
mal metabolites began as early as 24 to 48 hours after the ascorbic acid was
withdrawn from the diet and before the tissues became depleted of this
vitamin. It is especially interesting that 3 hours after the administration
of 0.5 g. of tyrosine to guinea pigs on a scorbutic diet an amount of hydroxy-
phenyl compounds equivalent to half the tyrosine appeared, mainly in the
large intestine. Twenty-four hours later none remained in the tissues or the
intestine but instead had appeared in the urine.
Reports that the high excretion of phenolic compounds by patients having
pernicious anemia in relapse was reduced by liver therapy®® prompted the
study of the effect of PGA on the metabolism of tyrosine in the scorbutic
guinea pig. Woodruff et al.8° observed that guinea pigs on a scorbutic diet
and receiving 5% tyrosine excreted 30 to 60% of the dietary tyrosine in
the form of hydroxypheny] derivatives. Administration of either 5 mg. of
90 T,. Golberg, B. De Meillon, and L. Lavoipierre, J. Exptl. Biol. 21, 90 (1945).
1G. Fraenkel, M. Blewett, and M. Coles, Physiol. Zodél. 23, 92 (1950).
2 W. Moore, Ann. Entomol. Soc. Amer. 39, 513 (1946).
9% C. A. Grob, T. Reichstein, and H. Rosenthal, Experientia 1, 275 (1945).
“R.R. Sealock and H. E. Silberstein, J. Biol. Chem. 135, 251 (1940).
% H. A. Painter and S.S. Zilva, Biochem. J. 41, 511 (1948).
96M. E. Swendseid, B. Wandruff, and F. H. Bethell, J. Lab. Clin. Med. 32, 1242
(1947).
192 PTEROYLGLUTAMIC ACID
PGA subcutaneously per day or 25 mg. of ascorbic acid orally or a combina-
tion of the two was equally effective in reducing the excretion of hydroxy-
phenyl derivatives. Only ascorbic acid prevented weight loss and increased
serum ascorbic acid. Pteroyltriglutamic acid was effective, but the cor-
responding diglutamate was ineffective in reducing the excretion of hy-
droxyphenyl compounds in two animals on each compound tested. Ad-
ministration of 5 U.S.P. units of anti-pernicious anemia liver extracts
(approximately 5 y of vitamin By2) was without effect.
The response of scorbutic monkeys to a test load of tyrosine is similar to
that of the guinea pig. Salmon and May” found that PGA was ineffective
in reducing the excretion of hydroxyphenyl compounds even in doses as
high as 95 mg. (40 mg. per kilogram of body weight), half being given orally
and the other half subcutaneously. Subsequently, these same investiga-
tors® found that 1.2 mg. of folinic acid (citrovorum factor) daily given intra-
muscularly was ineffective. Ten milligrams of cortisone, however, injected
intramuscularly for 10 days prevented the excretion of large amounts of
hydroxyphenyl compounds.
An effect of PGA in clinical hydroxyphenyluria of infantile scurvy after
tyrosine ingestion has been observed in approximately half the cases studied
by Govan and Gordon.®? In some cases 5 mg. per day produced an effect.
In another case 10 mg. per day orally was ineffective but 30 mg. intramus-
cularly produced a striking reduction of hydroxyphenyluria. Other cases,
in which 10 to 30 mg. intramuscularly was ineffective, promptly responded
to the administration of ascorbic acid.
Morris et al.!°° also observed hydroxyphenyluria in infants with scurvy
after tyrosine ingestion. Injection of 45 mg. of PGA to one patient was inef-
fective in abolishing the abnormal excretion, but 45 mg. twice daily for
several days (corresponding to 13.7 mg. per kilogram per day) did decrease
the hydroxyphenyluria. A single dose of 30 y of vitamin By, was ineffective,
a finding which is parallel to the ineffectiveness of parenteral liver extract
in the scorbutic guinea pig.
Woodruff!” reported on four cases of tyrosine-induced hydroxyphenyluria
in scorbutic infants which were given PGA intramuscularly in doses of 2
mg. per kilogram of body weight without any effect on tyrosine metabolism.
Apparently the amounts of PGA necessary to reduce hydroxyphenyluria
are very large compared to those for growth of the guinea pig and hemato-
logical response in scorbutic infants. In the guinea pig 20 mg. per kilogram
97 R. J. Salmon and C. D. May, J. Lab. Clin. Med. 36, 591 (1950).
9 R.J.Salmon and C. D. May, Arch. Biochem. 32, 220 (1951).
99 C. D. Govan, Jr., and H. H. Gordon, Science 109, 332 (1949).
100 J. H. Morris, KE. R. Harpur, and A. Goldbloom, J. Clin. Invest. 29, 325 (1950).
101 ©, W. Woodruff, J. Lab. Clin. Med. 36, 640 (1950).
IX. EFFECTS OF DEFICIENCY 193
of body weight per day is effective in controlling hydroxyphenyluria, and
a definite but suboptimal response is procured with 4 mg.!° In Woodruftf’s
study the three patients studied in whom no effect was produced received
between 1.3 and 2.2 mg. per kilogram per day. In cases reported by other
workers the effective doses varied between 3 and 13.7 mg. per kilogram
of body weight per day. Woodruff!" has also observed that in megaloblastic
anemia of infancy as little as 0.2 mg. of PGA will effect a maximal hemato-
logical response. These experiments dramatically illustrate the fact that
vitamin requirements for different functions may vary and that require-
ments can be enormously altered by imposing a metabolic load on the orga-
nism.
b. Relation of PGA and Ascorbic Acid in Megaloblastic Anemia
The role of PGA in megaloblastic anemia of infancy provides additional
information on the relations between ascorbic acid, PGA, and vitamin By.
The effectiveness of PGA in curing the megaloblastic condition of the bone
marrow was demonstrated by Zuelzer and Ogden.!° May et al.! has re-
viewed this work and focused attention on the role of ascorbic acid defi-
ciency in the etiology of this disease. In megaloblastic anemia of infancy
the most frequent symptom is a megaloblastic bone marrow. Examination
of the peripheral blood alone will not always suffice for the recognition of
this syndrome because the circulating red cells are not always macrocytic
and not all macrocytic anemias are associated with megaloblastic marrow.
The majority of the cases occur in patients between the ages of 5 and 12
months, which corresponds roughly to the period of most frequent develop-
ment of clinical signs of ascorbic acid deficiency. Megaloblastic anemia has
been frequently reported as a complication of scurvy.
May and co-workers! experimentally reproduced the condition of
megaloblastic anemia of infancy in monkeys on milk diets used in infant
feeding. Their salient experimental findings are:
1. When ascorbic acid is provided, the diets do not lead to megaloblastic
anemia.
2. If vitamin C is inadequate, all the diets result in megaloblastic anemia.
3. Megaloblastosis of the marrow is quickly eliminated by PGA with-
out addition of vitamin C.
4. Ascorbic acid alone might permit gradual return of the marrow to
normal.
5. Vitamin By: administered intramuscularly does not significantly alter
the megaloblastic bone marrow within 72 hours.
102 W. W. Zuelzer and F. N. Ogden, Am. J. Diseases Children 71, 211 (1946).
103 C.D. May, E. N. Nelson, C. U. Lowe, and R. J. Salmon, Am. J. Diseases Children
80, 191 (1950).
194 PTEROYLGLUTAMIC ACID
6. Vitamin By. plus ascorbic acid administered simultaneously intra-
muscularly effects a prompt correction of the megaloblastic marrow.
7. Vitamin By given intramuscularly prophylactically does not prevent
characteristic development of the megaloblastic marrow.
8. PGA given prophylactically prevents the development of the megalo-
blastic marrow on the vitamin-deficient diets even though scurvy and
anemia develop.
9. Marked reduction in food intake alone leading to severe malnutrition
does not cause anemia or megaloblastic marrow.
The role of vitamin By: in the experimental anemia of monkeys just de-
scribed and in megaloblastic anemia of infancy seems secondary to that of
PGA. Pure vitamin By and liver extracts containing vitamin By». but only
small amounts of PGA are sometimes able to completely cure megaloblastic
anemia in infants.'% Pteroylglutamic acid is almost always effective. In
the monkey vitamin By». is ineffective in preventing the megaloblastosis,
but it is effective when given together with ascorbic acid. It should be borne
in mind that the diets contained milk and hence some PGA. Thus, the
vitamin By. and ascorbic acid together may have increased the effective-
ness of the PGA in the basal diet used.
The basal diets used by the Arkansas workers*®” to produce nutritional
cytopenia in the monkey contained orange juice. Liver extract was unable
to cure the macrocytic anemia in these experiments, which shows that
vitamin By in the presence of vitamin C is ineffective when no PGA is pres-
ent in the basal diet. The failure of May to observe megaloblastic anemia
on his milk diet supplemented with ascorbic acid, whereas Day observed
anemia on his cereal diet with orange, is undoubtedly due to the presence
of some PGA in the milk rations. Thus it appears that the PGA require-
ments of the monkey are increased by a deficiency of ascorbic acid.
The most unique aspect of the scorbutically induced anemia in the mon-
key is its capacity to respond to small quantities of ‘“folinic acid’’ (citro-
vorum factor). Fifteen micrograms of the factor per day in the form of 50
y of concentrate produced the maximum hematological response, whereas
750 y of PGA produced a suboptimal effect.” The folinic acid produced a
prompt reticulocytosis followed by regeneration of the bone marrow to a
normal cellular pattern. Nichol and Welch!” have found that ascorbie acid
functions in the conversion of PGA to citrovorum factor. These data, to-
gether with observations on the high activity of citrovorum factor in the
monkey, suggest that in the absence of adequate levels of ascorbic acid the
104 ©. W. Woodruff, H. W. Ripy, J. C. Peterson, and W. J. Darby, Pediatrics 4, 723
(1949), cited by C. D. May, E. N. Nelson, C. U. Lowe, and R. J. Salmon, Am. J:
Diseases Children 80, 191 (1950).
105 ©, A. Nichol and A. D. Welch, Proc. Soc. Exptl. Biol. Med. 74, 52 (1950).
IX. EFFECTS OF DEFICIENCY 195
“activation” of pteroylglutamic acid is performed inefficiently. This can
be corrected by (a) administering excess precursor which in this case is
PGA, (b) giving small amounts of the end product of the reaction, or (c)
correcting the scurvy. If this theory is valid, one must assume that the con-
version of PGA to citrovorum factor is inefficient even in the presence of
adequate ascorbic acid, since 100 y of PGA produces a submaximal hemato-
logical response on a diet which contains orange juice." It would indeed be
interesting to compare the activities of PGA and CF on nutritional cyto-
penia in monkeys which are receiving adequate ascorbic acid.
12. ENDOCRINE RELATIONSHIPS OF PGA
A fundamental observation regarding the role of PGA in endocrine func-
tion was the finding by Hertz and coworkers!*: 1” that a deficiency of PGA
interfered with the response of chicks to stilbestrol. The normal oviduct
weight of a 100- to 150-g. chick is approximately 25 mg. Injection of 0.5
mg. of stilbestrol per day for 6 days to chicks on a normal stock diet in-
creases the size of the oviduct to approximately 1000 mg. The oviduct
weights of PGA-deficient chicks treated with stilbestrol were 66 to 100 mg.,
whereas in those supplemented with 20 or 50 y of PGA and treated with
stilbestrol they were 637 to 777 mg. It is worthy of note that the oviduct
weights produced by stilbestrol on the stock diet were larger (1090 to 1295
mg.) than those (777 mg.) which could be achieved by large amounts of
PGA on a purified type of diet. This indicates that there are elements in
the stock diet in addition to PGA which mediate the function of stilbestrol.
The function of PGA in the metabolism of stilbestrol is a specific one, as
deficiencies of pantothenic acid, riboflavin, and pyridoxine do not interfere
with the oviduct-stimulating action of stilbestrol on birds of comparable
body weight.
Production of a PGA deficiency by the use of an antagonist ‘x methyl
PGA” also interferes with the effect of stilbestrol in stimulating growth of
the female reproductive tract.1%: 1° The effect of the antagonist could be
completely reversed by PGA. A reduction in the amount of alkaline phos-
phatase in the oviduct of chicks after treatment with estradiol or stilbestrol
has been observed in PGA deficiencies produced by ‘“‘x methyl PGA.’’!!°
Similar results have been observed in monkeys on PGA-deficient diets.
Hertz kept sexually immature monkeys on a purified type of diet for
106 R, Hertz and W. H. Sebrell, Science 100, 293 (1944).
107 R. Hertz, Endocrinology $7, 1 (1945).
108 R. Hertz, Science 107, 300 (1948).
109 AL. Franklin, D. Lewis, E. L. R. Stokstad, and T. H. Jukes, Poultry Science 27,
662 (1948).
410 M. Andrus and M. X. Zarrow, Proc. Soc. Exptl. Biol. Med. 72, 714 (1949).
11 R. Hertz, Proc. Soc. Exptl. Biol. Med. 67, 113 (1948).
196 PTEROYLGLUTAMIC ACID
periods of 43 to 66 days until typical signs of PGA deficiency developed.
They were then given 66 y of estradiol benzoate. Six of the eight deficient
monkeys failed to show the characteristic normal estrogenic response in
the external genitalia. Simultaneous administration of a liver extract con-
taining PGA permitted the normal response to estrogen although pure
PGA was not employed in this experiment. A similar relationship exists in
the estradiol-treated ovariectomized rat.!? The weight of the uterus of the
untreated ovariectomized rat was found to be 25 mg., which was increased
to 84 mg. by injection with estradiol. In the presence of 12.5 y of the an-
tagonist 4-amino PGA, the uterine weight was 32 mg. after estradiol injec-
tion. The effect of 12.5 y of antagonist in decreasing the response to es-
tradiol was reversed by PGA. Corresponding results have been observed
in frogs. Goldsmith et al."° showed that the oviducts of newly metamor-
phased frogs exhibited a marked response to weekly injections of 0.1 mg.
of estradiol benzoate. Administration of PGA increased the response,
whereas 4-amino PGA decreased it. The effect of 4-amino PGA was not
negated by simultaneous administration of PGA.
Evidence regarding the role of PGA in endocrine responses have not
been as clearly defined in the male as they have in the female. Goldsmith
et al.* measured the increase in weight of the seminal vesicles and coagu-
lating glands of testosterone-treated mice. Ten per cent of crude ‘‘x methyl
PGA” in the diet prevented the effect of testosterone, but 6% did not.
Thus a higher level of antagonist is necessary to prevent the testosterone
effect in mice than is required to inhibit the action of stilbestrol in
chicks.!: 1°9 In the rat 4-amino PGA did not interfere with stimulation of
the prostate by testosterone in either the intact immature rat or in the
castrate animal (Brendler™>). It was also observed that 4-amino PGA did
prevent the depressing effect of a-estradiol on the prostate in the intact
adult animal. Thus this PGA antagonist interferes with the tissue response
to estradiol, whether it be the growth stimulation of the oviduct or uterus
in the female, or the depressing effect of estradiol on the prostate in the
male.
No effect of PGA has thus far been observed on the increased comb
growth produced by androgen in the chick. Haque et al."® found that defi-
ciency of PGA, pantothenic acid, nicotinic acid, choline, or vitamin A had
no effect on the increase in comb growth produced by testosterone pro-
12 R. Hertz and W. W. Tullner, Endocrinology 44, 278 (1949).
113}, D. Goldsmith, 8. 8. Schreiber, and R. F. Nigrelli, Proc. Soc. Exptl. Biol. Med.
69, 299 (1948).
114}, D. Goldsmith, H. M. Black, and R. F. Nigrelli, Natwre 164, 62 (1949).
116 H. Brendler, Science 110, 119 (1949).
116 M. KH. Haque, R. J. Lillie, C. S. Shaffner, and G. M. Briggs, Poultry Science 28, 914
(1949).
IX. EFFECTS OF DEFICIENCY 197
pionate. It was also found in confirmation of the work of Hertz! that
PGA deficiency was the only one that interfered with the oviduct weight
produced by estradiol. Zarrow et al." observed that a PGA deficiency
produced either by feeding a purified diet or a commercial chick mash
containing 2 % of crude “‘x methyl PGA” had no effect on the comb stimu-
lation produced by testosterone. Four per cent of this antagonist actually
enhanced the effect of androgen on comb growth.
Dehydroisoandrosterone acetate in relatively large amounts has been
found by Gaines and Totter! to stimulate the growth of S. faecalis and
L. casei in the absence of PGA. Cortisone acetate has been found to support
the growth of Leuconostoc citrovorum in the absence of the citrovorum
factor.1!9
13. Rote or CrrrovorumM Factor IN ANIMAL NUTRITION
Just as leucovorin (synthetic citrovorum factor) can meet the require-
ment of certain microorganisms for PGA, it has also been found that leuco-
vorin is active in promoting growth and hemopoieses in PGA-deficient
chicks and turkeys.’°-!? Experiments with crystalline leucovorin have
shown that, when it is mixed with the ration of chicks or turkeys, it is
considerably less active than PGA; but the two substances have similar
activities when injected. Experiments with leucovorin in chicks may be
compared with numerous studies that have been conducted with leuco-
vorin in anemia in which it has been shown that leucovorin, like PGA, is a
potent hemopoietic agent in pernicious anemia, sprue, and nutritional
macrocytic anemia.!”*
The discovery that leucovorin is more active than PGA in reversing the
toxicity of 4-amino PGA for S. faecalis is paralleled by experiments with
mice in which the same relationship was found.”° There are now many
instances of the ability of leucovorin to counteract the action of 4-amino
PGA or its close chemical derivatives. Thus Burchenal and coworkers!
found that leucovorin when administered prior to 4-amino-10-methyl PGA
completely counteracted the antileukemic effect of this antagonist in mice
17 M. X. Zarrow, I. B. Koretsky, and I. G. Zarrow, Endocrinology 48, 125 (1951).
18 J). 8. Gaines and J. R. Totter, Proc. Soc. Exptl. Biol. Med. 74, 558 (1950).
19 T). 8S. Gaines, H. P. Broquist, and W. L. Williams, Proc. Soc. Exptl. Biol. Med. 17,
247 (1951).
20 AH. P. Broquist, KE. L. R. Stokstad, and T. H. Jukes, J. Biol. Chem. 185, 399 (1950).
21 H. P. Broquist, J. A. Brockman, Jr., M. J. Fahrenbach, E. L. R. Stokstad, and
T.H. Jukes, J. Nutrition 47, 93 (1952).
22 1). G. Hill and G. M. Briggs, Proc. Soc. Exptl. Biol. Med. 76, 417 (1951).
123 'T. D. Spies, G. G. Lopez, F. Milanes, R. L. Toca, A. Reboredo, and R. E. Stone,
Southern Med. J. 48, 1076 (1950).
4 J. H. Burchenal, 8. F. Johnston, H. P. Broquist, and T. H. Jukes, Cancer Research
10, 208 (1950).
198 PTEROYLGLUTAMIC ACID
with Ak, leukemia. The toxic effect of 4-amino PGA for rats,!° the chick
embryo,!”® Drosophila,” and human beings’ has also been demonstrated
to be effectively blocked by CF. It is apparent, then, that 4-amino PGA
may more correctly be termed a CF antagonist than an antagonist of PGA.
a. Enzymatic Conversion of PGA to Citrovorum Factor
From evidence so far published it appears that, before PGA can carry
out its catalytic functions, the organisms must first convert PGA to CF,
and that CF either represents the catalytically active form of PGA or is
more closely related to this form than PGA itself. Nichol and Welch!®
have presented evidence for the existence in liver of an enzyme(s) that is
involved in the conversion of PGA to CF; of particular interest was their
finding that a greater amount of CF was formed when ascorbic acid was
present. Apparently ascorbic acid brings about favorable reducing condi-
tions essential for the conversion. These workers also found that 4-amino
PGA was markedly effective in blocking the conversion of PGA to CF by
liver slices;!° thus it is apparent that 4-amino PGA has two sites of action
in the cell: (1) to block the formation of CF and (2) to compete with CF
for essential reactions in the cell.
The knowledge that CF contains a formyl group is of considerable in-
terest from a biochemical standpoint in that it provides a structural basis
for the concept that CF is concerned with the transfer of the 1-carbon unit
in metabolic reactions. If CF functions by shuttling a formyl group from
one substrate to another, it might be predicted that tetrahydro PGA
might also have activity comparable to CF. Indeed such has been found
to be the case,” for it has been reported that tetrahydro PGA has about
2.5% of the activity of leucovorin for Le. citrovorum and about one-third
the activity of leucovorin in counteracting 4-amino PGA toxicity in the
mouse. However, Elwyn et al.¥° found that the 6-hydrogen atoms of serine
accompanied the 6-carbon atom during the conversion to the methyl
groups of choline and thymine. Such a finding precludes the oxidation of
the 8-carbon to the formate oxidation level unless the 6-hydrogen atoms
are held in escrow by the citrovorum factor and returned to the 6-carbon
atom when the formyl group of CF is reduced back to the methyl group
to be incorporated into choline or thymine.
25 C, A. Nichol and A. D. Welch, Proc. Soc. Exptl. Biol. Med. 74, 403 (1950).
126 W.W. Cravens and E. EH. Snell, Proc. Soc. Exptl. Biol. Med. 75, 43 (1950).
27H. D. Goldsmith and M. H. Harnly, Cancer Research 11, 251 (1951).
28 1. B. Schoenbach, E. M. Greenspan, and J. Colsky, J. Am. Med. Assoc. 144, 1558
(1950).
129 FH Pp, Broquist, M. J. Fahrenbach, J. A. Brockman, Jr., E. L. R. Stokstad, and
T. H. Jukes, J. Am. Chem. Soc. 78, 3535 (1951).
180 T). Elwyn, A. Weissbach, and D. B. Sprinson, J. Am. Chem. Soc. 78, 5509 (1951).
IX. EFFECTS OF DEFICIENCY 199
B. IN MICROORGANISMS
EK. L. R. STOKSTAD
The distribution of organisms requiring PGA is not as widespread as that
of those requiring other growth factors such as biotin, nicotinic acid, or
pantothenic acid. The development of PGA-less mutants is much less fre-
quent than that of other vitamins requiring mutants. Certain classes of
microorganisms, particularly the lactic acid bacteria, have a number of
members which require an external source of PGA. Peterson and Peterson,!*!
in a comprehensive review of the vitamin requirements of bacteria, list 10
bacteria out of a total of 136 as requiring ‘folie acid,” ‘‘eluate factor,”
“vitamin B, ,”’ or “‘L. casez factor.”
Although recognition of the need of animals for PGA antedates that of
microorganisms, it was the discovery of the latter that provided a rapid
assay and hastened the isolation of this vitamin. The nutritive requirements
of lactic acid bacteria for PGA were first pointed out by Snell and Peter-
son.’ They found that an unidentified factor was required which later
proved to be PGA. Later work from this same laboratory showed that
other lactic and propionic acids formed bacteria such as Streptococcus
faecalis R, Lactobacillus delbruckii, and Propionibacterium pentosaceum
also required this factor.!%
In an investigation of the growth factor requirements of a large number
of Enterococci, Niven and Sherman" found 9 organisms requiring PGA out
of a total of 43 studied. Out of 21 strains of Streptococcus lactis, none re-
quired PGA.18°
Tetrahymena gelew is unique among the microorganisms in that it has the
ability to utilize large PGA conjugates which are unavailable to bacteria.
The nutrition of the protozoan has been thoroughly investigated by Kidder
and associates,*: 7 who showed that this organism requires 0.00065 y
PGA per milliliter of culture media for half-maximum growth which is
about three times that required by S. faecalis R.'°8 Rhizopterin, also known
as the “‘S. L. R. factor,’ (N!° formylpteroie acid) which is active for S.
faecalis R but relatively inactive for L. caset was found to be about 0.2 %
as active as PGA for 7. gelew.'*" Pteroyltriglutamic acid and petroylhepta-
131 W. H. Peterson and M. 8. Peterson, Bacteriol. Revs. 9, 49 (1945).
132, E. Snell and W. H. Peterson, J. Bacteriol. 39, 273 (1940).
188 B. L. Hutchings, N. Bohonos, and W. H. Peterson, J. Biol. Chem. 141, 521 (1941).
134 C, F. Niven, Jr., and J. M. Sherman, J. Bacteriol. 47, 335 (1944).
135 C.F. Niven, Jr., J. Bacteriol. 47, 343 (1944).
136 G. W. Kidder, Arch. Biochem. 9, 51 (1946).
187 G. W. Kidder and R. C. Fuller, ITI, Science 104, 160 (1946).
188 HY. L. R. Stokstad, J. Biol. Chem. 149, 573 (1943).
200 PTEROYLGLUTAMIC ACID
glutamic acid were reported by Kidder’ to be approximately as active as
PGA when compared on a molar basis. Thus the ability of this ciliated
protozoan to utilize PGA conjugates and its inability to use pteroic acid
and rhizopterin parallels that of the rat, the chick, and the human.
Clostridium tetani was early reported by Mueller and Miller!®: 4! to re-
quire an unknown growth factor which could be replaced by concentrates
of PGA prepared from liver or by folic acid preparations obtained from
spinach.
1. METABOLISM OF RHIZOPTERIN
A survey of the rhizopterin and PGA requirements of a number of lactic
acid bacteria was made by Stokes et al.!” This showed that all Streptococci
which were stimulated by rhizopterin (N!°-formylpteroic acid) also re-
sponded to PGA. A certain number of organisms, including both Lacto-
bacilli and Streptococci, responded only to PGA, and a third group responded
to neither. They also found that different strains of the same organism
vary widely in their requirements. Thus one strain of S. faecalis can use
either rhizopterin or PGA, another responds to PGA only, and a third
requires neither. The strains that do not require PGA were found to syn-
thesize this factor when grown on a medium devoid of the vitamin.
An interesting observation made by these same investigators was the
finding that certain Enterococci are able to convert rhizopterin into a form
active for L. casei. Presumably this conversion involves the addition of
glutamic acid to rhizopterin to give either PGA or N?!°-formylpteroylglu-
tamic acid. Resting cell suspensions are also able to effect this conversion.!*
A 10-ml. resting cell suspension of S. lactis R or S. zymogenes converts 5 y
of rhizopterin to about 1 y of PGA™* in 3 hours. Two other strains formed
only 0.18 y under similar conditions which shows the wide variation in the
ability of various organisms to effect this conversion. The conversion rate
is increased by the addition of carbohydrates which were able to act as
hydrogen acceptors as evidenced by reduction of methylene blue.
Some interesting facts regarding the assay of PGA in microbial cells were
revealed in this same study.' In the assay of PGA formed from conversion
of rhizopterin by resting cells, the entire cell suspensions were added di-
rectly to the assay medium without any preliminary treatment. The bulk
of the activity was found to reside in the cell. When the cells were auto-
139 G. W. Kidder, Paper presented at the New York Academy of Science, Dec. 7,
1946.
140 J. H. Mueller, P. A. Miller, J. Biol. Chem. 140, 933 (1941).
141 J. H. Mueller, P. A. Miller, Proc. Soc. Exptl. Biol. Med. 49, 211 (1942).
142 JL. Stokes, J. C. Keresztesy, and J. W. Foster, Science 100, 522 (1944).
143 J. 1. Stokes and A. Larsen, J. Bacteriol. 50, 219 (1945).
44 The data have been recalculated on the basis of PGA’s having the same activity
as folic acid of potency 137,000.
IX. EFFECTS OF DEFICIENCY 201
claved with water before being added to the assay medium, only 12% of
the activity was recovered. However, if the cells were autoclaved with
reducing agents such as 0.5 % sodium glycollate, 5% ascorbic acid, or in a
small amount of assay medium, complete recovery was obtained. In each
case the residual PGA remaining in the liquid phase after autoclaving was
resistant to further autoclaving which showed that a labile form existed in
the bacterial cells. This suggests that reducing substances exert a protective
influence during extraction from the cell and emphasizes the difficulties
that may be encountered in the assay of PGA in microbial cells. This
lability of PGA under certain conditions is reminiscent of the acid-labile
PGA found in horse liver by Pfiffner et al.’ This behavior might also be
explained partially by the known properties of the citrovorum factor which
TABLE X
CoMPARATIVE GROWTH RESPONSE OF Le. citrovorum, S. faecalis, AND L. casei TO
LEUCOVORIN AND RELATED COMPOUNDS
Amount of compound required per ml. culture medium for
half-maximum growth in 24 hr., my
Acid-treated Pteroylglutamic
Organism Leucovorin leucovorin® acid
Le. citrovorum 0.15 7.5 30, 000
S. faecalis 0.37 0.38 0.18
L. casei 0.17 0.09
* Prepared by allowing a leucovorin solution (4 mg./ml.) to stand at pH 2 for 24 hours at 25°.
during its conversion to PGA in slightly acid conditions and in the presence
of oxygen goes through certain intermediates, which in turn are converted
to PGA in varying yields, depending on the reducing potential and pH of
the solution.
2. MicrosBioLocicaL AcTIVITY OF CITROVORUM FACTOR
A summary of the microbiological activity of leucovorin, 5-formyl-
5,6,7,8-tetrahydro PGA (synthetic citrovorum factor), and related com-
pounds for several lactic acid bacteria is shown in Table X. It has been
found from a number of microbiological assays that 0.15 my of the an-
hydrous free acid of leucovorin is required for half-maximum growth of
Le. citrovorum;' however, when the vitamin is exposed to mildly acidic con-
ditions as is indicated in the table, only 2 % of the activity remains for Le.
citrovorum. About 200,000 times as much PGA as leucovorin is required
by Le. citrovorum for a comparable growth response; apparently this organ-
ism has lost the ability to convert PGA to CF. It is apparent from the
table that leucovorin has PGA activity for S. faecalis and L. casei, although
46 J.J. Pfiffner, S. B. Binkley, E. S. Bloom, and B. L. O’Dell, J. Am. Chem. Soc. 69,
1476 (1947).
202 PTEROYLGLUTAMIC ACID
twice as much leucovorin as PGA is required to produce half-maximum
growth.
It is evident, then, that in the past when S. faecalis or L. caset has been
employed as the assay organism to determine the pteroylglutamic acid
content of natural materials, these assays have given a figure representing
both PGA and CF. It should be possible to determine with Le. citrovorum
that proportion of the ‘free PGA”’ which exists as CF, since this organism
for all practical purposes does not respond to PGA. Before such differential
assays are performed, consideration should be given to the existence of
bound forms of PGA. There is now considerable evidence that the CF in
fresh liver, and presumably in most other tissues, exists as a conjugate
which behaves in all respects as a polyglutamate analogous to that of
pteroylglutamic acid. Thus Hill and Scott™® reported that a sample of dried
brewer’s yeast which contained initially 1200 CF units per gram was found
to contain 15,300 CF units per gram after incubation overnight at 37° with
a hog kidney enzyme preparation. Moreover this hog kidney preparation
was found to have similar properties to the PGA conjugase known to be
present in hog kidney. Other sources of ‘““CF conjugase” include chicken
pancreas and the enzymes normally present in certain tissues that are
active during autolysis.” “8 There is no evidence at present to indicate
whether ‘‘CF conjugase”’ is identical with PGA conjugase, although it seems
logical that it would be.
Another interesting microbiological property of leucovorin is that, al-
though it appears to be only half as active as PGA for growth of S. faecalis
and L. casei (cf. Table X), when a folic acid antagonist such as 4-amino
PGA is incorporated in the medium, leucovorin is markedly more effective
than PGA in reversing growth inhibition.” In the presence of an equi-
molar amount of leucovorin or PGA about eight times as much 4-amino
PGA is required to reduce growth of S. faecalis to half-maximum in the
presence of leucovorin as is needed in the presence of PGA. Such data im-
ply that CF may be more closely related to the biologically active coen-
zyme than PGA itself.
C. IN MAN
FRANK H. BETHELL
The most conspicuous effect of PGA deficiency in man is macrocytic
anemia associated with megaloblastic erythropoiesis. Normal growth and
1446 © H. Hilland M. L. Scott, Federation Proc. 10, 197 (1951).
147 M. EK. Swendseid, F. H. Bethell, and W. W. Ackermann, J. Biol. Chem. 190, 791
(1951).
148 T,, S. Dietrich, W. J. Monson, H. Gwoh, and C. A. Elvehjem, J. Biol. Chem. 194,
549 (1952).
IX. EFFECTS OF DEFICIENCY 203
differentiation of other hemic elements, the leucocytes and thrombocytes,
are also dependent upon adequate supply of PGA. The integrity of mucosal
surfaces may be impaired in PGA deficiency states with secondary infection
and ulceration. Glossitis, disturbances in gastrointestinal function, and
peripheral neuropathies are sometimes associated with lack of this vitamin.
The increase in appetite, strength, and sense of well-being which attends
the administration of PGA to a severely deficient patient precedes any
appreciable change in peripheral blood values and suggests that the vitamin
participates in many metabolic processes. The predominance of hematologic
manifestations is presumably attributable to the continuous production
and rapid turnover of the cells of the blood.
A wide variety of more or less well-defined clinical entities are associated
with apparent need for PGA. Although lack of this vitamin may constitute
but one facet of a complex dietary deficiency state, many of the disorders
in question are wholly correctible by the administration of PGA alone,
whereas in others, notably addisonian pernicious anemia, PGA has proved
to be an imperfect or secondary form of replacement therapy.
From what is known and has been discussed earlier regarding the natu-
rally occurring forms of PGA, the mode of PGA absorption by the human
being, its metabolic function, and its relationship to other nutritional
factors, a number of theoretical mechanisms may be postulated by which
PGA deficiency states might be presumed to occur. These potential modus
operandi include: (1) primary dietary lack of PGA and CF and their con-
jugates; (2) incomplete breakdown of conjugates to the free and absorbable
forms within the alimentary tract, due, perhaps, to lack of intestinal con-
jugases; (3) impaired absorption due to intestinal pathology, such as
chronic inflammatory disease, hypermotility, and diminished absorptive
surface; (4) non-utilization of PGA after absorption, which may depend
upon lack of other nutritional factors such as vitamin By2, ascorbic acid,
or nitrogen and carbon donors, or which may result from the presence of
competitive or inhibitor substances such as PGA antimetabolites and an-
tagonists. Playing accessory roles which may serve to convert latent or
relative PGA deficiency to overt clinical manifestations are situations in
which there is an increased demand for the vitamin such as pregnancy and
the rapid growth of infancy.
1. NutTRITIoNAL Macrocytic ANEMIA
By this designation is meant the presence of macrocytic anemia with
megaloblastic bone marrow in persons who have a history of dietary inade-
quacy without gastric, intestinal, or hepatic disease. The specific entities
listed below are excluded from this classification. Although the pathogenesis
of such anemia would appear obvious, its interpretation is rendered some-
204 PTEROYLGLUTAMIC ACID
what unsatisfactory by a number of considerations. In the first place, most
of the available food analyses for PGA were performed before the existence
of CF was recognized so that the values reported represent summations
of the growth-stimulating properties of both PGA and CF.° Moreover, in
the case of S. faecalis R assays, there may have been incomplete liberation
of PGA and CF from their conjugates.!°® A disturbing feature of PGA
dietary analyses is the apparent great loss of the vitamin after cooking,
amounting to approximately 50 to 95% of the original activity.®! PGA
determinations on canned foods, except for spinach, were especially low.!*2
These differences between PGA content of fresh raw foods and that of
their cooked or canned counterparts have been attributed to a possible
binding of the vitamin by the tissues.!°° It may be stated, however, that
diets deficient in animal protein and green vegetables are low in PGA and
CF. It is true, of course, that such diets are also low in vitamin By and
in amino acids which are concerned in PGA metabolism, so that a number
of factors may contribute to the manifest deficiency state.
Therapeutic responses to the administration of PGA in cases of nutri-
tional macrocytic anemia were first reported in 19451? and 1946.14) 1° Of
interest is the report of a patient who responded to PGA, relapsed after
discontinuing treatment, and subsequently showed a favorable response to
pteroylheptaglutamic acid (hexaglutamyl conjugate of PGA). The signifi-
cance of this event lies in the reported observations, to be discussed later,
indicating that pernicious anemia patients may be unable to utilize effec-
tively the naturally occurring hexaglutamyl conjugate and that the meta-
bolic defect may be corrected by vitamin B,. administration. It seems
probable, although not clearly proved, that the ‘‘Wills factor” is identical
with PGA including its conjugates. This hemopoietic factor, as supplied by
a preparation of autolyzed yeast (Marmite) was shown by Wills and her
associates to produce responses in cases of tropical macrocytic anemia which
failed to benefit from the prior administration of a purified liver extract
(Anahaemin).!°* !*7 Comparable results were obtained in monkeys.!*-16°
149 V, Cheldelin, A. M. Woods, and R. J. Williams, J. Nutrition 26, 477 (1943).
150 V. Mims, J. R. Totter, and P. L. Day, J. Biol. Chem. 155, 401 (1944).
151 B. 8. Schweigert, A. E. Pollard, and C. A. Elvehjem, Arch. Biochem. 10, 107 (1946).
152 Mf. Ives, A. E. Pollard, C. A. Elvehjem, and F. M. Strong, J. Nutrition 31, 347
(1946).
163 ©. F. Vilter, T. D. Spies and M. B. Koch, Southern Med. J. 38, 781 (1945).
154 T. J). Spies, J. Am. Med. Assoc. 180, 474 (1946).
155 G. A. Goldsmith, J. Lab. Clin. Med. 31, 227 (1946); Federation Proc. 5, 232 (1946).
156 J,, Wills, Brit. Med. J. I, 1059 (1981).
187 T,, Wills and P. D. F. Evans, Lancet II, 416 (1938).
158 T,, Wills and A. Stewart, Brit. J. Exptl. Pathol. 16, 444 (1935).
159 T,, Wills, P. W. Clutterbuck, and B. D. F. Evans, Biochem. J. 31, 2136 (1937).
160 J,, Wills and P. D. F. Evans, Lancet 232, 311 (1937).
IX. EFFECTS OF DEFICIENCY 205
Although many of the patients treated by Wills were pregnant and so do
not fall within the classification of uncomplicated nutritional macrocytic
anemia, it seems proper to regard her cases as primarily attributable to
dietary lack, and to assign to her priority in demonstrating a clear dis-
tinction between the anti-pernicious anemia factor of liver and a hemo-
poietic factor which we now recognize as probably PGA. It must, however,
be borne in mind that nutritional macrocytic anemia varies greatly through-
out the world and that the isolated instances of the condition seen in tem-
perate zones are quite different in associated clinical manifestations and
probably in pathogenesis from endemic deficiency states. Moreover, in most
of the instances reported, no attempt has been made to establish the pri-
mary or major deficiency, whether PGA or vitamin By. .
2. IDIOPATHIC REFRACTORY MEGALOBLASTIC ANEMIA OR
ACHRESTIC ANEMIA
These terms, coined, respectively, by Davis and Davidson! and Israéls
and Wilkinson,'® have been applied to a form of macrocytic anemia with
megaloblastic bone marrow for which no dietary or other etiologic explana-
tion was discoverable and which failed to respond to purified extracts of
liver. The condition has not been generally recognized in the United States
or on the European continent, and its occurrence in Great Britain may
possibly be attributed to unknown factors affecting nutrition if not to
direct dietary deficiency. The pathogenesis of this type of anemia is prob-
ably closely related to the macrocytic anemia of pregnancy on the one
hand, and to that associated with idiopathic steatorrhea or the sprue syn-
drome on the other. As a rule, such patients respond favorably to the ad-
ministration of PGA although they may later relapse while continuing
with the medication. In some instances, liver extract injections, although
initially ineffective, were later successful in maintaining remission.!®
3. MEGALOBLASTIC ANEMIA OF PREGNANCY AND THE PUERPERIUM
Mention has already been made of the cases of tropical macrocytic anemia
complicated by pregnancy observed by Wills and her associates. In temper-
ate climates such anemia, manifested in severe degree, is uncommon, al-
though a comparatively high incidence of mild macrocytic anemia has been
observed in pregnant women whose diets were habitually low in protein of
good quality and presumably also in PGA and vitamin By: .1%: 1% Yet,
opinion is divided on the question of the importance of dietary inadequacy
161 |). J. Davis and L. S. P. Davidson, Quart J. Med. 13, 53 (1944).
162M. C. Israéls and J. F. Wilkinson, Quart. J. Med. 5, 69 (1936).
166 R.H. Girdwood, Edinburgh Med. J. 68, 309 (1951).
164 FW, H. Bethell, J. Am. Med. Assoc. 107, 364 (1936).
165 FH. Bethell, E. Blecha, and J. H. Van Sant, J. Am. Dietet. Assoc. 19, 165 (1943).
206 PTEROYLGLUTAMIC ACID
in the pathogenesis of megaloblastic anemia of pregnancy. Ungley states
that ‘Several of our patients had excellent diets . . .’'°° His further argu-
ment that ‘In others, the diet, although not good, was at least no worse
than that of other women who did not develop anaemia” cannot be given
much weight, since such an observation is common to all clinical nutritional
deficiency states. Certainly, in most of the reported instances of this condi-
tion pre-existing dietary inadequacy has been noted and malnutrition has
characterized every case within the writer’s experience. Such nutritional
abnormalities may well include deficiencies other than PGA itself, such as
amino acids and ascorbic acid which play a role in PGA and CF metabolism.
On the other hand, contributing factors, such as the increased demands of
gestation, possible digestive disturbances, and alterations in the output of
sex hormones, as suggested by Ungley,!®® may play a part in the actual
development of severe macrocytic anemia.
Diminished secretion of intrinsic factor during gestation was proposed
as a cause of macrocytic anemia of pregnancy, on the basis of clinical ex-
periments by Strauss and Castle.!® In the ight of present knowledge, such
an explanation involves a defect in the absorption and utilization of vitamin
By. The same result might, as suggested by these authors, follow a low
dietary intake of extrinsic factor (Biz). However, before the isolation of
PGA and By , numerous observers reported the ineffectiveness of purified
liver extract injections in the treatment of pregnancy macrocytic anemia,
followed by good results obtained from yeast, whole liver, or crude liver
extracts. The earlier therapeutic observations were summarized and ana-
lyzed critically by Watson and Castle.'® Since PGA and Bi. became avail-
able, it has been universal experience that the former constitutes effective
and complete therapy for this type of anemia,!**: 1° whereas vitamin By: is
essentially valueless and may even be detrimental.17°-'™
Patients with macrocytic anemia of pregnancy may exhibit striking
clinical and hematological manifestations in addition to anemia. In a case
observed by the author,!”° spiking fever, glossitis, vulvovaginitis, and
diarrhea were conspicuous. During a period of vitamin By, administration,
166 ©, C. Ungley, Brit. J. Nutrition 6, 299 (1952).
167 M. B. Strauss and W. B. Castle, Am. J. Med. Scz. 185, 539 (1933).
168 J. Watson and W. B. Castle, Proc. Soc. Exptl. Biol. Med. 58, 84 (1945).
169 T,,§. P. Davidson, R. H. Girdwood, and J. R. Clark, Brit. Med. J. I, 819 (1948).
170 F, H. Bethell, M. C. Meyers, and R. B. Neligh, J. Lab. Clin. Med. 38, 1477 (1948).
171 J,, A. Day, B. E. Hall, and G. L. Pease, Proc. Staff Meetings Mayo Clinic 24, 149
(1949).
172, V. Ginsberg, J. Watson, and H. Lichtman, J. Lab. Clin. Med. 36, 238 (1950).
173 R. H. Furman, W. B. Daniels, L. L. Hefner, E. Jones, and W. J. Darby, Am.
Practitioner and Dig. Treatment 1, 146 (1950).
174 ©, C. Ungley and R. B. Thompson, Brit. Med. J. I, 919 (1950).
IX. EFFECTS OF DEFICIENCY 207
these changes were aggravated and pre-existing leucopenia progressed to a
leucocyte count of less than 500 per cubic millimeter. Dramatic sympto-
matic improvement and return of temperature to normal occurred within
48 hours after institution of PGA therapy, followed by rise in the leucocyte
count and subsequent return of all hematologic values to normal. The evi-
dences of severe illness seen in this patient are similar to those observed in
eases of advanced sprue or idiopathic steatorrhea and bear a close resem-
blance to the toxic effects of PGA antagonists, such as aminopterin.
4. MEGALOBLASTIC ANEMIA OF INFANCY
For some reason, perhaps related to the wider use of processed baby
foods, this condition has been seen more commonly in the United States
than in Great Britain or the European continent. It is to be distinguished
from the normoblastic anemia occurring in marasmic infants and, in fact,
children afflicted with megaloblastic anemia are usually well nourished. The
age of development of the blood changes, which include leucopenia and
thrombocytopenia as well as anemia, is usually between 6 months and 2
years. Apparently the condition may result from direct or ‘‘conditioned”’
deficiency of either PGA or vitamin By . Sporadic case reports of this type
of anemia and its response to liver extract injection appear in the literature
prior to the advent of PGA and By: . In the writer’s experience with several
cases seen before PGA or By became available, all responded to liver in-
jection, whether of crude or purified type.
The first clear demonstration of the efficacy of PGA in the treatment of
megaloblastic anemia of infancy was made by Zuelzer'® and Zuelzer and
Ogden."’® These authors stated that the effect of PGA was indistinguishable
from that produced by liver extract in certain cases, but that in others
PGA was effective after failure of response to liver injection. Similar obser-
vations have been made with reference to vitamin By. by Luhby and
associates!””-!79 who obtained responses to PGA or to institution of a diet
of whole milk and vegetables after failure to respond to vitamin By , and
by Woodruff and associates,!*° who reported three instances of megaloblastic
anemia of infancy, one of which responded to PGA after no effect from By ,
whereas the other two were treated successfully with By. by injection.
75 W.W. Zuelzer, J. Am.Med. Assoc. 181, 7 (1946).
176 W.W. Zuelzer and F. N. Ogden, Am. J. Diseases Children 71, 211 (1946).
m7 A.W. Luhby, Health Center J. (Ohio State University) 2, 35 (1948).
m8 A. LL. Luhby and C. A. Doan, Proc. Conf. Preservation Formed Elements and Pro-
teins Blood, Harvard Medical School, Boston (January, 1949).
79 A. L. Luhby and W. E. Wheeler, Health Center J. (Ohio State University) 8, 1
(1949).
180 C. W. Woodruff, H. W. Ripy, J. C. Peterson, and W. J. Darby, Pediatrics 5, 723
(1949).
208 PTEROYLGLUTAMIC ACID
McPherson and associates! and Sturgeon and Carpenter! have reported
good remissions after administration of Biz, although the latter authors
obtained only ‘‘equivocal responses” in two of. their five cases. There have
been no instances reported of failure of response to PGA after adequate
trial.
The existence of a relationship between lack of ascorbic acid and megalo-
blastic anemia of infancy’? receives support from the observation of the
role of this vitamin in the conversion of PGA to CF,!* from the occurrence
of megaloblastic anemia in scorbutic and PGA-deficient monkeys,!**-!8° and
from the apparent decrease in incidence of megaloblastic anemia in infancy
after the addition of ascorbic acid to a popular brand of processed milk
food. However, unqualified application of the findings of zn vitro studies
and animal experiments to the problem of infantile megaloblastic anemia
is unjustifiable, and moreover it has not been shown that CF is more
effective than PGA in the treatment of this form of anemia in humans.
In the present state of our knowledge, it must be concluded that dietary
lack of PGA, vitamin By, or ascorbic acid may play a role in the develop-
ment of megaloblastic anemia of infancy and that it is probable that most
cases are due to a combination of these deficiencies. In any event, PGA
appears to be completely efficacious in the treatment of this form of megalo-
blastic anemia.
5. SPRUE AND IDIOPATHIC STEATORRHEA
These conditions are considered together, since the biochemical defects
are indistinguishable although etiologic factors are undoubtedly different.
In the so-called tropical sprue which was formerly endemic in the southern
part of the United States and parts of Latin America, malnutrition was
almost invariably the rule. Tropical sprue as described by British authors
commonly affects European residents in the tropics and is attributed to a
peculiar environmental effect. Non-tropical sprue or idiopathic steatorrhea
is a sporadic disease of unknown etiology in which there is generally no
history of dietary abnormality or unusual environment. Megaloblastic
anemia is of common occurrence in each of these related conditions, but
the factors responsible for its development may be presumed to vary and
the effects of therapy are known to differ, at least in degree.
Soon after synthetic PGA became available, its effectiveness was demon-
181 A. Z,. McPherson, U. Jonsson, and R. W. Rundles, J. Pediat. 34, 529 (1949).
182 P| Sturgeon and G. Carpenter, Blood 5, 458 (1950).
183 ©, A. Nichol and A. D. Welch, Proc. Soc. Exptl. Biol. Med. 74, 52 (1950).
184 C.D. May, E. N. Nelson, C. U. Lowe, and R. J. Salmon, Am. J. Diseases Children
80, 191 (1950).
185 ©. D. May, HE. N. Nelson, R. J. Salmon, C. U. Lowe, R. I. Lienke, and R: DY
Sundberg, Bull. Univ. Minn. Hosp & Minn. Med. Found. 21, 208 (1950).
186 ©, D. May, R. D. Sundberg, and F. Schaar, J. Lab. Clin. Med. 36, 963 (1950).
IX. EFFECTS OF DEFICIENCY 209
strated in the treatment of endemic American sprue.'7-8* PGA therapy in
three patients with this form of sprue resulted in complete reversal of the
clinical manifestations, including glossitis, atrophy of the lingual papillae,
anorexia, weight loss, and diarrhea with high fecal fat.!°° Maximum reticulo-
cyte responses were noted in 6 to 8 days, erythrocyte, leucocyte, and plate-
let values returned to normal, and megaloblasts and abnormal granulo-
cytes disappeared from the bone marrow. A therapeutic response was
obtained in one case of sprue after the administration of the pure PGA
conjugate, pteroyltriglutamic acid, in a daily dose of 4.9 mg., whereas
another patient responded to the administration of a concentrate of pteroyl-
heptaglutamic acid containing in each daily dose 8.4 mg. of pteroylhepta-
glutamic acid and about 0.3 mg. of PGA.!*! The therapeutic efficacy of
thymine in daily doses of 15 g. has been reported in four cases of sprue,
although the results were less striking than those obtained with PGA.!%
PGA may be regarded as almost universally effective in the treatment
of the megaloblastic anemia of sprue and idiopathic steatorrhea, although
exceptions to this general rule have been reported. Jones et al.!® include
in a series of cases responding to vitamin By one patient with sprue and
megaloblastic anemia who had been receiving PGA, and Girdwood'® men-
tions cases of idiopathic megaloblastic anemia which became refractory to
PGA and developed manifestations of sprue in spite of its continued ad-
ministration. It has been common experience that the dramatic curative
effects of PGA in the syndrome of endemic sprue are rarely observed in the
sporadic form of the disease, idiopathic steatorrhea. In the latter group
megaloblastic anemia is less common and, when present, generally less
severe than in endemic sprue. Furthermore, since there is evidence of asso-
ciated vitamin By, deficiency in many cases of both endemic and sporadic
sprue, it is to be expected that relapse may occur after long-continued
administration of PGA, just as is true of addisonian pernicious anemia.
6. MrGaALoBLAstic ANEMIAS BELIEVED TO BE Dur PRIMARILY
TO Lack OF VITAMIN Bie
This group of conditions includes certain nutritional anemias as well as
pernicious anemia, and megaloblastic anemias associated with fish tape-
'8? W.J. Darby and E. Jones, Proc. Soc. Exptl. Biol. Med. 60, 259 (1945).
88 C. V. Moore, O. 8S. Bierbaum, A. D. Welch, and L. D. Wright, J. Lab. Clin. Med.
30, 1056 (1945).
89 TD. Spies, G. G. Lopez, J. A. Menendez, V. Minnich, and M. B. Koch, Southern
Med. J. 39, 30 (1946).
190 W. J. Darby, E. Jones, and H. C. Johnson, Science 103, 108 (1946).
191 R. M. Suarez, A. D. Welch, R. W. Heinle, R. M. Suarez, Jr., and E. M. Nelson,
J. Lab. Clin. Med. 81, 1294 (1946).
12 T. D. Spies, W. B. Frommeyer, G. G. Lopez, R. L. Toca, and G. Gwinner, Lancet
I, 883 (1946).
193. Jones, W. J. Darby, and J. R. Totter, Blood 4, 827 (1949).
210 PTEROYLGLUTAMIC ACID
worm infestation, and those following gastrectomy, intestinal strictures,
blind loops, and short-circuiting operations or fistulae. Megaloblastic ane-
mia sometimes occurring in cases of chronic liver disease is also probably
due to a “conditioned” deficiency of vitamin Bi. These anemias are dis-
cussed in connection with the pathology of vitamin By , but it is pertinent
at this time to consider certain disturbances of PGA metabolism which
may be encountered in them. Soon after PGA and certain of its conjugates
became available for clinical trial their effect was observed in pernicious
anemia. For a more detailed account of the early therapeutic experiences
with PGA and related compounds, with complete bibliography, reference
should be made to the review of Jukes and Stokstad.!% A reticulocyte
response, a rise in erythrocyte values, a conversion of marrow megaloblas-
tosis to normoblastosis, and symptomatic improvement occurred in essen-
tially every case of pernicious anemia in relapse treated with PGA. The
conjugates of PGA, pteroyldiglutamic and pteroyltriglutamic acids, were
also found to be effective, although the number of patients receiving these
compounds was much smaller than that treated with PGA itself. On the
other hand, the ‘‘hexaglutamyl conjugate,’ also known as pteroylhepta-
glutamic acid, failed to produce responses in several patients treated by
two independent groups of investigators.'”: !°° This is not invariably true,
since a few patients have subsequently been observed who responded in
some degree to the administration of this conjugate.!% The impaired utiliza-
tion of pteroylheptaglutamic acid by patients with pernicious anemia,
which appears to be a quantitative defect, gains special significance from
the fact that a large part of the dietary PGA is in the form of this conjugate.
Demonstration of the effectiveness of PGA in the treatment of pernicious
anemia raised the question of the relationship of the compound to the
active principle in liver known as the erythrocyte maturing factor (EMF).
It was at once apparent that the two substances could not be identical for
the following reasons. First, although liver is a rich source of PGA or re-
lated compounds, the processes of fractionation and purification yielding
the most active anti-pernicious anemia extracts eliminate almost all the
PGA originally present. Second, the weight of the total nitrogenous solids
present in a volume of concentrated parenteral liver extract sufficient to
treat a pernicious anemia patient for 15 days (15 U.S.P. units per milli-
liter) is far less than the quantity of PGA required for the same purpose
(50 to 75 mg.). Third, the relative efficacy of liver extract given by intra-
194 'T. H. Jukes and E. L. R. Stokstad, Physiol. Revs. 28, 51 (1948).
195 WH. Bethel], M. C. Meyers, G. A. Andrews, M. E. Swendseid, O. D. Bird, and
R. A. Brown, J. Lab. Clin. Med. 82, 3 (1947).
196 R. W. Heinle and A. D. Welch, Ann. N. Y. Acad. Sci. 48, 343 (1946).
197 A.D. Welch and R. W. Heinle, Pharmacol. Revs. 8, 345 (1951).
IX. EFFECTS OF DEFICIENCY A ia
muscular injection is about fifty times as great as when administered by
mouth, whereas PGA is almost as effective by the oral as by the parenteral
route of administration. It was not long before even more convincing evi-
dence was at hand clearly distinguishing between PGA and EMF and
demonstrating that the former constituted incomplete therapy for per-
nicious anemia. The occurrence and progression of subacute combined
degeneration of the spinal cord in pernicious anemia patients receiving
PGA was first reported by Vilter ef al.’ Additional reports of similar
observations soon appeared in the literature.'’??°* The accumulated ex-
perience of these authors as well as that of many other clinical observers
enable the following conclusions to be drawn with respect to the effects of
PGA therapy in pernicious anemia. Most, if not all, patients receiving this
medication as the only form of specific treatment ultimately develop some
degree of relapse. The time required for hematologic abnormalities to occur
varies from a few weeks to one year or longer. Glossitis may appear during
PGA administration, even when the blood values are normal. Paresthesias
and evidence of combined system disease may develop insidiously months
after PGA therapy is instituted, or rapidly progressive nervous system
damage may take place during the initial treatment period while the eryth-
rocyte count is rising. Some patients experience glossitis or a fall in blood
values after long-continued PGA administration without ever showing evi-
dence of peripheral or central neuropathy. The mode of action of PGA
with reference to nervous system injury is not well understood. It is the
opinion of some observers that the complication is more likely to develop
in patients whose diets have been poor. In the writer’s experience, no such
correlation has been apparent, and the most rapidly progressive cases of
nervous system involvement occurred in patients who were not severely ill
with anemia and whose dietary histories were good. It is a widely held
impression, which the writer shares, that relatively large daily doses of
PGA, 10 to 20 mg. or more, are especially likely to be associated with the
development of neuropathy. Institution of liver extract or vitamin By»
therapy arrests the progress of the condition and, if begun early, reverses
much of the functional impairment associated with peripheral and central
nervous system disease. There is no evidence that PGA is toxic to nerve
198 C.F. Vilter, R. W. Vilter, and T. D. Spies, Proc. Central Soc. Clin. Research 19,
26 (1946).
199 B. Hh. Hall and C. H. Watkins, J. Lab. Clin. Med. 32, 622 (1947).
200 7,, M. Meyer, Blood 2, 50 (1947).
201 R. W. Heinle and A. D. Welch, J. Am. Med. Assoc. 133, 749 (1947).
202 F. H. Bethell and C. C. Sturgis, Blood 8, 57 (1948).
205 J. F. Ross, H. Belding, and B. L. Paegel, Blood 3, 68 (1948).
208 S. O. Schwartz, 8. R. Kaplan, and B. E. Armstrong, J. Lab. Clin. Med. 35, 894
(1950).
212 PTEROYLGLUTAMIC ACID
tissue or that it is capable of producing nervous system damage in persons
other than patients with pernicious anemia or with related vitamin By»
deficiency states. The explanation of the effect of PGA in these situations
may lie in the aggravation of the By. deficiency through exhaustion of the
small amount of available material. This theory is based in part on the
supposition that vitamin By functions in a number of metabolic pathways
which involve nervous as well as hemopoietic tissue, and it gains credence
from observations on the relationship of vitamin By. to PGA metabolism.
These are discussed in the chapter on vitamin By: .
There remains to be considered the manner in which PGA deficiency
may develop in pernicious anemia and other conditions associated with a
lack of vitamin By. In the first place, it should be emphasized that the
deficiency of PGA may be more apparent than real, since much of the
difficulty may lie in the inability to utilize effectively PGA and its deriva-
tives which are present in the tissues in “bound” or conjugated forms. It
is known that PGA or related compounds, such as conjugates of PGA or
CF are present in the tissues of patients with pernicious anemia in relapse.'®
The impaired utilization of PGA in pernicious anemia may be due to
the presence of inhibiting substances, and one of the functions of vitamin
By. may be to eliminate such inhibitors or overcome their influence.?” The
presence of a substance which inhibits the breakdown of pteroylheptaglu-
tamic acid to free PGA has been demonstrated in certain yeast concentrates,
liver, and spinach.!*° 2° In accordance with this theory, the therapeutic
efficacy of PGA may be due to a mass action effect overcoming the influence
of inhibitors.
On the other hand, some degree of actual PGA deficiency is almost cer-
tainly present in patients with untreated pernicious anemia. The amount
of PGA contained in average diets is small, probably less than 1 mg. daily,
and most of it is in conjugated form. Reference has already been made to
the impaired utilization of pteroylheptaglutamic acid by pernicious anemia
patients. It has been shown that such persons on standard diets excrete
less PGA in the urine than is the case with normal subjects, and that, in
contrast to normal subjects, the administration of yeast concentrates con-
taining pteroylheptaglutamic acid is followed by little or no increase in
excretion of PGA.!%: 2° Following a short period of therapy with concen-
trated parenteral liver extract, pernicious anemia patients excrete free PGA
after oral administration of its hexaglutamyl conjugate in amounts com-
205 R. W. Heinle, KE. M. Nelson, and A. D. Welch, Proc. Central Soc. Clin. Research 19,
27 (1946).
206 M. KH. Swendseid, O. D. Bird, R. A. Brown, and F. H. Bethell, J. Lab. Clin. Med.
Spe OBS (GIG).
207 A. D. Welch, R. W. Heinle, E. M. Nelson, and H. V. Nelson, J. Biol. Chem. 164,
787 (1946).
IX. EFFECTS OF DEFICIENCY 213
parable to the normal rate of excretion.'’®: 2° These observations may be
interpreted as evidence of a defect in PGA metabolism, characteristic of
pernicious anemia, which is corrected by supplying a constituent of liver
extract, presumably vitamin Bi.
Is there reason to suppose that any degree of PGA deficiency may be
present in pernicious anemia patients who have been receiving, by injection,
adequate amounts of liver extract or vitamin By, for a considerable period
of time and whose diets are good? In other words, do these therapeutic
agents completely counteract the factors responsible for impaired utiliza-
tion of dietary PGA in untreated pernicious anemia? Lack of information
prevents categorical replies to these questions. It is true that liver extracts
or vitamin By, constitute apparently complete therapy for pernicious anemia
as measured by usual clinical and hematologic criteria. On the other hand,
the fact that the dietary supply of PGA is almost never large, and the
persistent changes in the alimentary tract characteristic of pernicious ane-
mia, including achlorhydria, altered upper intestinal flora, and sometimes
motility disturbances, provide grounds for believing that absorption of
food PGA may not be optimal in every case. This conclusion is supported
by the impression of some clinicians, including the writer, that supplement-
ing adequate liver extract or vitamin By. therapy with PGA in daily oral
doses of 5 mg. may lead to an increase in sense of well-being and to slight
rises in erythrocyte values.2°> However, evidence to the contrary has also
been presented.”
In cases of macrocytic anemia associated with chronic partial intestinal
obstruction, or anastomoses of the intestine with diminished absorptive
surface and possible blind loops, there may be clear evidence of further
improvement following administration of PGA after initial response to By»
therapy.?!°
7. INDUcED PGA DEFIcIENcY IN MAN
The administration of PGA antagonists for therapeutic purposes would
appear to provide a means of observing the manifestations of pure PGA
deficiency under controlled conditions. However, although some clinical
entities which respond to PGA therapy bear certain resemblances to the
clinical and hematologic picture associated with antagonist administration,
there is no close similarity between the two types of deficiency. The ex-
planation of these differences undoubtedly lies, in part, in the rapidity with
which the effects of such antagonists as 4-aminopteroylglutamic acid
208 F. H. Bethell, Wisconsin Med. J. 51, 1082 (1952).
209 R. B. Chodos and J. F. Ross, Blood 6, 1213 (1951).
10 FH. Bethell, M. E. Swendseid, 8. Miller, and A. A. Cintron-Rivera, Ann. Internal
Med. 35, 518 (1951).
214 PTEROYLGLUTAMIC ACID
(Aminopterin) are induced. Thus the much more rapid turnover of leuco-
cytes and thrombocytes than of circulating erythrocytes is reflected by the
early development of profound leucopenia and thrombocytopenia during
antagonist administration. These changes, if not arrested, lead to the fatal
complications of infection and hemorrhage before the effects of erythro-
poietic depression have time to become manifest in great degree. That
alterations in red cell production, similar to those seen in megaloblastic
anemias, do occur after antagonist administration was first pointed out by
Thiersch and Philips.?!! However, the extensive changes in tissues other
than those concerned with hemopoiesis, especially mucosal ulceration and
hemorrhage, may be attributed to the predominant mode of action of the
4-amino analogs of PGA. The virtual impossibility of reversing the action
of aminopterin and similar antagonists by any amount of PGA which is
feasible to administer had impressed many observers as an argument
against the concept of such compounds being true PGA antimetabolites.
The problem was solved by Nichol and Welch,?!2 who showed that the
conversion of PGA to CF in vitro and in vivo is blocked by the presence of
aminopterin. The major metabolic effect of this compound is, therefore,
inactivation of an enzyme catalyst concerned with the biologically essential
PGA — CF reaction. These authors also showed that the effects of such
antagonists could readily be reversed by the administration of CF, an
observation soon confirmed by others.?!3-?!5
Since the prevention or reversal of the action of aminopterin by CF is
accomplished when the compounds are given in fairly well-defined ratios,
it may be concluded that aminopterin competes with CF in biological sys-
tems which require this factor.
In the words of Welch and Heinle:'” ‘It is evident, therefore, that
aminopterin exerts two quite different types of antagonism. That which
is related to the citrovorum factor is competitive and readily reversible,
while that concerned with folic acid is essentially non-competitive and only
reversible under conditions where very high concentrations of folic acid
are attainable. Obviously, therefore, the practical means of attempting
to reverse the toxic actions of the 4-amino analogues of folic acid involve
the use of citrovorum factor, but the principal reason for the development
of the toxic effects of the analogues when folic acid is administered is the
blockade of the biological utilization of the vitamin.”
211 J. B. Thiersch and F.8. Philips, Proc. Soc. Exptl. Biol. Med. 71, 484 (1949).
212. C, A. Nichol and A. D. Welch, Proc. Soc. Exptl. Biol. Med. 74, 403 (1950).
213 H. P. Broquist, E. L. R. Stokstad, and T. H. Jukes, J. Biol. Chem. 185, 399 (1950).
214 J: H. Burchenal, G. M. Babcock, H. P. Broquist, and T. H. Jukes, Proc. Soc. Expil.
Biol. Med. 74, 735 (1950).
215 H}. B. Schoenbach, E. M. Greenspan, and J. Colsky, J. Am. Med. Assoc. 144, 1558
(1950).
IX. EFFECTS OF DEFICIENCY 215
It is of interest to mention in connection with the discussion of PGA
antagonists that pernicious anemia patients are unusually susceptible to
the action of the 4-amino analogs, which is presumably attributable to
their already defective metabolism or limited supply of PGA. Furthermore,
administration of aminopterin prevents therapeutic response in pernicious
anemia to vitamin By, .”°: ?!6§ This observation suggests that in the growth
of blood cells PGA functions secondarily to vitamin By, a topic which is
discussed further in the section dealing with vitamin Bie.
8. SUMMARY
The pathology of PGA deficiency in man is expressed in a number of
clinical manifestations but especially in altered hemopoiesis, resulting in
anemia, granulocytopenia, and thrombocytopenia. The characteristic
changes in the bone marrow are megaloblastic erythropoiesis, qualitative
abnormalities in granulocytic and megakaryocytic development, and quan-
titative changes in the absolute number and relative proportions of erythro-
cytic and granulocytic elements.
The factors which may contribute to the development of PGA deficiency
states are multiple and complex. Decreased dietary intake of PGA is prob-
ably rarely the sole cause of clinically apparent deficiency, although the
etiologic importance of a poor diet is evident in a group of clinical cate-
gories including nutritional megaloblastic anemia, megaloblastic anemias of
pregnancy and of infancy, and certain cases of the sprue syndrome. Lack
of other dietary constituents may play important roles in contributing to
disturbances of PGA metabolism. Among such substances are vitamin By ,
ascorbic acid, and amino acids.
Deficiency of PGA may result from non-absorption or non-utilization of
the naturally occurring conjugated forms of the vitamin, owing to ability
to liberate the free material. The mechanism involved in this process is
an enzymatic breakdown of a peptide linkage. The enzymes, or conjugases,
responsible for the liberation of free PGA may be quantitatively deficient,
or they may be rendered inert by the presence of inhibitory substances,
which have been demonstrated in animal and plant materials. One of the
metabolic defects present in pernicious anemia and some other related con-
ditions results in relatively poor absorption of dietary PGA, and probably
also in impaired utilization of such amounts of the vitamin as are absorbed
and stored in the tissues.
The metabolic function of PGA involves its enzymatic conversion to a
formyl derivative, the citrovorum factor. The most extreme degrees of
PGA deficiency may be rapidly produced by the administration of the
216 T,, M. Meyer, N. D. Ritz, A. Caccese, J. Rutzky, and A. Sawitsky, Am. J. Med.
Sci. 218, 197 (1949).
216 PTEROYLGLUTAMIC ACID
4-amino analogs of PGA which have the double effect of blocking the
conversion of PGA to CF and of functioning competitively as antagonists
of CF itself. Such therapeutically induced PGA deficiency is employed in
the treatment of selected cases of acute leukemia.
X. Pharmacology
FRANK H. BETHELL
A. ACTIONS AND USES
Pteroylglutamic acid and its analog, the citrovorum factor, play a role
in the synthesis of nucleoproteins. PGA has been termed a hemopoietic
vitamin because its presence in a metabolically active form is required for
the normal development of blood cells. Its therapeutic use is largely con-
fined to certain megaloblastic anemias and nutritional disorders which may
be associated with megaloblastic anemia.' It is the most efficacious form
of therapy available for the megaloblastic anemias of pregnancy and of
infancy, as well as most cases of nutritional megaloblastic anemia and
tropical sprue. It is also employed, with variable degrees of success, in the
management of idiopathic steatorrhea, coeliac disease, and other chronic
intestinal disorders. It should not be used in the treatment of pernicious
anemia, except possibly as an adjunct to adequate liver extract or vitamin
By. therapy. The value of PGA as a dietary supplement in the maintenance
of optimal nutrition has not been established.
B. DOSE
Five to twenty milligrams daily is the usual range of dosage. Ten milli-
grams daily probably produces a maximal effect in most cases, although in
certain refractory cases of megaloblastic anemia much larger doses have
reportedly been required. Parenteral administration of PGA has no ad-
vantage over the oral route except in the presence of persistent diarrhea
or greatly diminished intestinal absorptive surface.
OS AKODOUGIMESY
Pteroylglutamic acid has an extremely low acute and chronic toxicity by
animal tests and produces almost no side reactions even when the dose is
far greater than the therapeutic range.? In man, no unfavorable effects
have been observed except in pernicious anemia when it was employed as
1. H. Bethell, Current Therapy, p. 204. W. B. Saunders Co., Philadelphia, 1952.
2B. K. Harned, R. W. Cunningham, H. D. Smith, and M. C. Clark, Ann. N. Y.
Acad. Sci. 48, 255 (1946).
XI. REQUIREMENTS AND FACTORS INFLUENCING THEM PAYS
the sole form of anti-anemia therapy. In such cases, the neurologic damage
following its use is probably attributable to extreme depletion of vita-
min By 3
XI. Requirements and Factors Influencing Them
A. OF ANIMALS
(See Section [X.A.)
B. OF MAN
FRANK H. BETHELL
The daily requirement of PGA for maintenance of normal nutrition is
unknown, and data applicable to this problem are difficult to obtain. It
would appear, on the basis of food analyses,!* that the daily dietary supply
of PGA is probably less than 1 mg. and, owing to losses in cooking, the
figure may be only a fraction of a milligram. The accuracy and reliability
of food assays for PGA may be impaired by a number of circumstances:
(1) Free PGA may not be released from naturally occurring conjugated
forms which do not support the growth of the test organisms. (2) Interfering
or inhibiting agents may be present. (3) Substances other than PGA which
support the growth of the organisms, such as thymine, may be responsible
for erroneously high values. (4) Of special importance is the fact that most
of the reported food values for PGA were obtained before the existence of
the citrovorum factor (CF) was recognized and its microbial growth-stimu-
lating properties described. Finally, intestinal bacterial synthesis of PGA
or CF may constitute an important source of the vitamin.
On the basis of therapeutic experience in PGA deficiency states, it ap-
pears probable that the total daily requirement of PGA and its analogs
may be supplied by the equivalent of 1 to 2 mg. of the free vitamin. The
demand is presumably greater in pregnancy, in conditions associated with
hypermetabolism, and in disorders characterized by excessive tissue break-
down and regeneration.
7R. W. Vilter, D. Horrigan, J. F. Mueller, T. Jarrold, C. F. Vilter, V. Hawkins,
and A. Seaman, Blood 5, 695 (1950).
1V. H. Cheldelin. A. M. Woods, and R. J. Williams, J. Nutrition 26, 477 (1948).
2M. Ives, A. E. Pollard, C. A. Elvehjem, and F. M. Strong, J. Nutrition 31, 347
(1946).
3 B.S. Schweigert, A. E. Pollard, and C. A. Elvehjem, Arch. Biochem. 10, 107 (1946)
4*R. H. Girdwood, Edinburgh Med. J. 58, 309 (1951).
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CHAPTER 14
PYRIDOXINE AND RELATED COMPOUNDS
(Vitamin B, Group)
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219
220 PYRIDOXINE AND RELATED COMPOUNDS
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I. Nomenclature
ROBERT 8S. HARRIS
Accepted group name: Vitamin Bg
Accepted names: Pyridoxine,! pyridoxal,? pyridoxamine?
1P,. Gyorgy and R. E. Eckhardt, Nature 144, 512 (1939).
2K. E. Snell, B. M. Guirard, and R. J. Williams J. Biol. Chem. 148, 519 (1942).
I. NOMENCLATURE 221
Obsolete names: Adermin®
Antiacrodynia rat factor
Antidermatitis rat factor
Yeast eluate factor!
Factor I°
Factor Y®
Vitamin H’?
Complementary factor’
Empirical formulas: Pyridoxine: CsH),O;N
Pyridoxal: CsH,O3;N
Pyridoxamine: CsH,2.02N>2
Chemical names: Pyrodoxine: 3-hydroxy-4 ,5-dihydroxymethyl-2-methyl-
pyridine
Pyridoxal : 3-hydroxy-4-formy]-5-hydroxymethy1]-2-meth-
ylpyridine
Pyridoxamine: 3-hydroxy-4-aminomethyl-5-hydroxy-
methyl-2-methylpyridine
Structural formulas:
CH.OH CHO CH.NH2
| |
‘a 2 ia HO— pee waa
Nae ete HC i H
N N N
Pyridoxine Pyridoxal Pyridoxamine
3R. Kuhn and G. Wendt, Ber. 61, 1534 (1988).
4C. E. Edgar and T. F. Macrae, Biochem. J. 31, 886 (1937); M. M. El-Sadr, T. F.
Macrae, and C. E. Work, Biochem. J. 33, 611 (1939).
5S. Lepkovsky, T. H. Jukes, and M. E. Krause, J. Biol. Chem. 115, 557 (1936)...
6H. Chick, A. M. Copping, and M. H. Roscoe, Biochem. J. 24, 1748 (1930).
7L. R. Richardson and A. G. Hogan, Missouri Agr. Expl. Sta. Research Bull. 241
(1936): L. E. Booher, J. Biol. Chem. 119, 223 (1937).
8 P. Gyorgy, R. Kuhn, and T. Wagner-Jauregg, Naturwiss. 21, 561 (1933).
222 PYRIDOXINE AND RELATED COMPOUNDS
II. Chemistry
JOHN C. KERESZTESY
A. ISOLATION
1. INTRODUCTION
The apparent and specific chemical properties of vitamin Bs (pyridoxine)
as present in crude concentrates were definitely established by Birch and
Gyorgy.' Within a few years of their publication the nitrogen base postu-
lated as the active vitamin by these authors was isolated in several labora-
tories almost simultaneously. There was no long lapse between the recogni-
tion of the existence of this vitamin and its separation in pure form as had
been the case with the first of the B vitamins. To be sure, pyridoxine is a
much more stable substance than thiamine. Within approximately two
years of the original observations of Birch and Gyorgy, five independent
reports appeared announcing its isolation. Lepkovsky,? Keresztesy and
Stevens,®? Gyérgy,t Kuhn and Wendt,’ and Ichiba and Michi® had suc-
ceeded in isolating pyridoxine from various natural materials. The compound
was now available for the study and the elucidation of its chemistry and
structure which lead directly to its synthesis. It must be noted here that
as early as 1932, Ohdake’ in his systematic study of the constituents of rice
polishings extract had isolated the hydrochloride of an unknown nitrogen
base to which he had assigned the improbable empirical formula
CsHip)NO3-HCl. Its identity with pyridoxine was pointed out later by
Wiardi.’ At this stage in the development of the vitamin, animal assays
were required to follow the biological activities of fractions obtained in the
respective isolation procedures. Such assays are costly in both time and
material as compared with the microbiological assay methods now employed
to follow the fractionation procedures for growth factors. Since pyridoxal
and pyridoxamine are the microbiologically active forms, the question arises
whether pyridoxine itself would have been isolated if the latter assay meth-
ods had been used.
1T. W. Birch and P. Gyérgy, Biochem. J. 30, 304 (1936).
2. Lepkovsky, Science 87, 169 (1988).
3 J. C. Keresztesy and J. R. Stevens, Proc. Soc. Exptl. Biol. Med. 38, 64 (1988).
4P. Gyorgy, J. Am. Chem. Soc. 60, 983 (1938).
®R. Kuhn and G. Wendt, Ber. 71, 780 (1938).
6 A. Ichiba and K. Michi, Sci. Papers Inst. Phys. Chem. Research (Tokyo) 34, 628
(1938).
7S. Ohdake, Bull. Agr. Chem. Soc. Japan 8, 11 (1982).
®P.W. Wiardi, Nature 142, 1158 (1938).
Il. CHEMISTRY 223
2. Meruops or [ISOLATION
a. General
The methods of isolation reported by the various investigators’-* de-
pended upon the use of some of the following properties of pyridoxine: (1)
adsorption on fuller’s earth, or charcoal; (2) solubility in neutral ethanol
or acetone; (3) stability to acid and alkaline hydrolysis; (4) failure to pre-
cipitate with heavy metal salts; (5) formation of an acetyl derivative; and
(6) precipitation with phosphotungstic acid. The sources used were rice
polishings or bran and yeast.
b. Kuhn and Wendt
The method used by Kuhn and Wendt? involved partial purification of a
heat- and alkali-sensitive protein complex in freshly prepared Lebedew
juice from that unique source, Miincher Lowenbrau yeast. Low molecular
weight impurities could be dialyzed away at temperatures below 3°, leaving
behind the vitamin-protein complex. After further purification of the com-
plex as a protein, the prosthetic group of the protein was split off by heat-
ing. Treatment with acetic anhydride yielded a chloroform-soluble acyl
derivative which then was crystallized. After hydrolysis the hydrochloride
was obtained in crystalline form.
c. Lepkovsky
Using a barium hydroxide extract of a fuller’s earth adsorbate prepared
from rice bran extract, Lepkovsky® effected further purification of his fac-
tor I concentrate by removal first of alcohol-insoluble mercury salts, and
then of the water-insoluble lead salts followed by precipitation of the vita-
min with phosphotungstic acid. The phosphotungstate was then crystal-
lized. The vitamin was recovered by decomposing this product with barium
hydroxide and final crystallization of pyridoxine (or factor I, as then named
by this investigator) as presumably the sulfate.
d. Gyérgy
Peter’s eluate,!° a yeast concentrate of thiamine widely used before com-
mercial thiamine chloride was available, served as the starting material for
Gyorgy* in his isolation of pyridoxine. From this he prepared a barium hy-
droxide eluate of fuller’s earth adsorbate. Then this was freed of neutral
alcohol-ethy] acetate-insoluble impurities. Further purification was effected
9S. Lepkovsky, J. Biol. Chem. 124, 125 (1938).
10 H.W. Kinnersley, J. R. O’Brien, R. A. Peters, and V. Reader, Biochem. J. 27.
225 (1933).
224 PYRIDOXINE AND RELATED COMPOUNDS
with aqueous platinic chloride to precipitate impurities. The concentrate
was further treated with phosphotungstic acid, and the vitamin was pre-
cipitated. From the decomposed phosphotungstate pyridoxine was precipi-
tated from alcoholic solution with ether. The vitamin was isolated in erystal-
line form, presumably as the free base.
e. Keresztesy and Stevens
An eluate of a fuller’s earth adsorbate prepared from rice bran extract
was hydrolyzed successively with strong hydrochloric acid and sodium hy-
droxide in the method used by Keresztesy and Stevens.'! After removal of
acetone-insoluble impurities, the concentrate was acetylated and the hy-
drolyzed acetyl derivatives were fractionated with phosphotungstic acid.
The hydrochloride of pyridoxine was recovered from the decomposed phos-
photungstates with acetone from alcoholic solution.
f. Ichiba and Miche
Isolation was accomplished by Ichiba and Michi® by removing impurities
with lead acetate followed by adsorption on acid clay and charcoal. Frac-
tional precipitation of the alcoholic mercuric chloride-soluble fraction re-
sulted in crystals which could be fractionated with alcohol.
B. CHEMICAL AND PHYSICAL PROPERTIES
1. CHEMICAL PROPERTIES
Pyridoxine exhibits the properties of a stable hydroxylated weak nitrogen
base. Birch and Gyo6rgy! found that it was not precipitated from solution
by the heavy metal salts such as those of lead, mercury, silver, or platinum
and not by picric acid but was precipitated by phosphotungstic acid. It was
not inactivated by nitrous acid. Hydrolytic agents such as mineral acids
or aqueous alkali, hot or cold, do not affect the vitamin.” It is stable to
the action of agents such as ethyl nitrite and Fehling’s solution. With fer-
ric chloride, pyridoxine reacts as a phenolic substance giving a reddish-
brown coloration. In alkaline solution, pyridoxine on treatment with 2 ,6-
dichloroquinone chlorimide gives an immediate blue color fading to
reddish-brown. This last reaction was used as the basis of a color test for
the vitamin. 1°
11 J. C. Keresztesy and J. R. Stevens, U. 8S. Pat. 2,287,042 (1943).
2 J.C. Keresztesy and J. R. Stevens, J. Am. Chem. Soc. 60, 1267 (1938).
13). T. Stiller, J. C. Keresztesy, and J. R. Stevens, J. Am. Chem. Soc. 61, 1237 (1939).
14 J. V. Scudi, H. F. Koones, and J. C. Keresztesy, Proc. Soc. Exptl. Biol. Med. 48,
118 (1940).
16 J. V. Scudi, J. Biol. Chem. 139, 707 (1941).
Il. CHEMISTRY 225
2. PHysicAL PROPERTIES
Pyridoxine hydrochloride, CsHj,NO3:HCl, occurs as white platelets,
melting point 204 to 206° with decomposition. The free base, CsHiuNO; ,
melts at 160°. The compound is optically inactive.” Both base and hydro-
chloride readily sublime without decomposition.
The hydrochloride is freely soluble in water but sparingly in alcohol
and acetone.’ The base is soluble in methanol and is not precipitated from
methanol solution by ethyl! ether.!
6
4
9
Ss
x
w
2
0)
2600 3000 3400 3700
nN
Fig. 1. Absorption spectra of vitamin Bs at: O, pH 4; @, pH 5.1; V, pH 6.75
(Stiller et al.13).
Rapid destruction of pyridoxine by light occurs in neutral and alkaline
solutions.!® In 0.1 N HCl there is very little destruction.
The tautomeric properties of pyridoxine are well illustrated by the
changes in its ultraviolet absorption produced by varying the hydrogen
ion concentration.’ The single maximum at 2925 A. at pH 2 diminishes in
intensity at pH 4.5, and concomitantly a new maximum appears at 3275
A. This latter band increases in intensity when the pH is changed to 6.75,
and the 2925 A. maximum disappears but a new band appears at 2560 A.
When the pH is further raised to 10.2 both bands increase in intensity and
16M. Hochberg, D. Melnick, L. Segel, and B. L. Oser, J. Biol. Chem. 148, 253 (1943).
226 PYRIDOXINE AND RELATED COMPOUNDS
shift to shorter wave lengths. Figure 1 illustrates the absorption spectra
of pyridoxine at various pH values.”
The pK (base) value of pyridoxine was found to be 6.2 & 10710!
C. CONSTITUTION
1. INTRODUCTION
Only a short time elapsed between the isolation of the vitamin in crystal-
line form and the recognition of its chemical constitution by two independ-
ent groups of chemists, Stiller, Keresztesy, and Stevens in the United
States, and Kuhn, Wendt, and Westphal in Germany.!”-!°
2. PROOF OF STRUCTURE
a. Stiller, Keresztesy, and Stevens
The establishment of the structure of pyridoxine by Stiller, Keresztesy,
and Stevens!® was based upon the study of the properties of the vitamin as
isolated and its methoxy derivative. The elementary analysis of pyridoxine
(as the free base) gave the empirical formula of CsHi,NO; . Theelectrometric
titration curve of the hydrochloride showed only one break, indicating
that the salt is a monohydrochloride and that its formula must be
CsHyNO3-HCl.” Analysis showed it contained one C-methyl group. O-
methyl and N-methyl groups were absent. Its failure to react with ni-
trous acid, its phenolic reaction with ferric chloride, and its ultraviolet
adsorption characteristics pointed to the probability that the vitamin was
a derivative of 6-hydroxypyridine. Comparison of the ultraviolet absorp-
tion of pyridoxine with that of 2-methyl-3-hydroxy-5-ethylpyridine con-
firmed this point.¥
Methylation of the base with diazomethane resulted in the formation of
the methyl ether, CysHi;0;N, melting point 101 to 102°. The absorption
spectrum showed a single maximum at 2800 A. which was not altered by pH
changes. Oxidation with permanganate yielded two products. One was a
dibasic acid which crystallized with one molecule of water, CgH,O;N, melt-
ing point 208 to 209° (dec.). The other was a lactone, C,H ,O;N, melting
point 209 to 210° (dec.).
Since the dibasic acid gave a negative test with ferrous sulfate, it ap-
peared that neither of the carboxyl groups was in the a-position of the
pyridine ring. Furthermore, the test with resorcinol was positive, and there-
fore the two carboxyl groups must be attached to adjacent carbon atoms.
Thus the dibasic acid could be represented by either of two structures.
17 R. Kuhn and G. Wendt, Ber. 72B, 305 (1939).
18 R. Kuhn, H. Andersag, K. Westphal, and G. Wendt, Ber. 72B, 309 (1939).
19 R. Kuhn, G. Wendt, and K. Westphal, Ber., 72B, 310 (1939).
Il. CHEMISTRY 227
coon COOH
See CH,O, COOH
aie) hig al
(1) (2)
Since the Gibbs test with 2,6-dichloroquinone chlorimide was positive
for pyridoxine, the compound could not be para substituted as regards
the hydroxyl, and therefore position 6 in the ring was unsubstituted. The
structure of the methoxydicarboxylic acid should then be structure 1,
which was confirmed by synthesis by Harris ef al.?°
Pel
‘ , : C:H,OH
CH;—C—CH.—C—CH,0C.H;, + CNCH.—-C—NH, ————>
pyridine as
catalyst
CH,0C:H; eo eos
HCl or fuming \
S—CN H,S0, “S-C=0 —4#NO; O.N¢ S—C=0 proc,
| One | ent | aaa ES
H;C O at 120 H,C O inH:80. H,;C O PCls
N N N 108°
H H H
ee an ae
one -o=0 H.+Pt i @ran H.+Pd ung spe=o NaNO,
HsCh 7 Cl HiC Ny Cl Beco, NG ee
CH.O CHO
\ \ COOH
HO; ‘SY—C=0 CH,N; CH:0¢ ‘S;-C=O Bacmno,,. CH:O¢ SCOOH
| ee | _Bachtndo CHO;
H,;C N7 HH; N” H;C NZ
Synthesis of Dicarboxy Acid derived from Pyridoxine Methyl Ether?°
The vitamin was therefore 2-methyl-3-hydroxy-4 ,5-di(hydroxymethy])
pyridine.
CH,OH
eal aes
CH y /
Pyridoxine
b. Kuhn, Wendt, and Westphal
In a series of three papers, Kuhn, Wendt, and Westphal!7-!* independ-
ently established the structure of pyridoxine. The line of attack was also
20S. A. Harris, E. T. Stiller, and K. Folkers, J. Am. Chem. Soc. 61, 1242 (1939).
228 PYRIDOXINE AND RELATED COMPOUNDS
through the oxidative degradation of the methyl ether of pyridoxine or
adermin, the name used by the German workers. On oxidation of the methyl
ether, the lactone, the same as that of Stiller, Keresztesy, and Stevens,
was obtained. On more vigorous oxidation with hot permanganate, a tri-
carboxylic acid resulted which could be degraded to the dicarboxylic acid.
Since the tricarboxylic acid gave a red color with ferrous sulfate, a test for
pyridine-a-carboxylic acids, and since the dicarboxylic acid did not give a
color with the reagent, it was concluded that the COz had been split off
from the a position on the pyridine ring. Pyridoxine gave a deep blue color
with the Folin-Dennis phenol reagent. On the other hand, this test with
the methyl ether was negative. Thus the compound was a 6-hydroxypyri-
dine derivative. Furthermore, the formation of a lactone of the partially
oxidized methyl ether of pyridoxine indicated that in the vitamin two of
the original hydroxymethyl! groups were located on adjacent carbon atoms
in the pyridine nucleus. The final proof that the dicarboxylic acid oxidation
product of the methyl ether of pyridoxine was identical with synthetic 2-
methyl-3-methoxypyridine-4 ,5-dicarboxylic acid was accomplished by
Kuhn et al.2! by its preparation from 3-methyl-4-methoxyisoquinoline.
c. Ichiba and Michi
Ichiba and Michi?‘ in studying the chemistry of pyridoxine were led
to believe at first that the vitamin was either an a- or y-hydroxypyridine
derivative by the enolic nature of the hydroxyl group. On methylation of
the vitamin with diazomethane they obtained the N-methyl compound
which still gave a red coloration with ferric chloride. The formation of the
N-methyl derivative substantiated the claim of Stiller, Keresztesy, and
Stevens that the vitamin is amphoteric in nature as opposed to the con-
cept of Kuhn and Wendt,” who considered changes in the ultraviolet ab-
sorption spectra as merely reversible shifting of the maxima.
Ichiba and Michi” also synthesized the key compound, i.e., 2-methyl-3-
methoxypyridine-4 ,5-dicarboxylic acid. After preparing 2-methyl-3-meth-
oxy-1-chloroisoquinoline, they reduced this compound to 2-methyl-3-meth-
oxyisoquinoline, which on oxidation yielded the desired dicarboxylic acid
which they found to be identical with that obtained by the oxidation of the
methyl ether of pyridoxine.
21 R. Kuhn, K. Westphal, G. Wendt, and O. Westphal, Naturwissenschaften 27, 469
22 ae and K. Michi, Sci. Papers Inst. Phys. Chem. Research (Tokyo) 35, 73
23 ee and K. Michi, Sci. Papers Inst. Phys. Chem. Research (Tokyo) 36, 1
24 ken and K. Michi, Sci. Papers Inst. Phys. Chem. Research (Tokyo) 36, 173
(1939).
Il. CHEMISTRY 229
O
COOC,H |
ce : es oN O00GHs CH,ONa
Br—GH + K— —_> N—CH
No C% ‘cH
CH; I i
O
7 =
C Cy om
SC—CH; POCI, “SC—CH; CHI C—CH;
NH LN CH,ONa N
¢ ‘a a
O Cl Gl
yee COOH
Cy
Sn+HCl “C—CH; O HOOC?’ OCH,
= SEC! i ewe l
ow’ Sw CH;
Synthesis of Dicarboxy Acid Derived from Pyridoxine Methyl Ether?*
D. SYNTHESIS
a. Kuhn, Westphal, Wendt, and Westphal
With the structure well established in 1939 by the synthesis of the meth-
oxydicarboxylic acid derivative of pyridoxine, the synthesis of the vitamin
was accomplished without delay in the same year. The simplest approach
to the synthesis of pyridoxine was by reconverting this degradation product
to pyridoxine. Kuhn, Westphal, Wendt, and Westphal?! reported their
method for accomplishing this by first converting the dicarboxylic acid to
2-methyl-3-methoxy-4 ,5-dicyanopyridine. On catalytic hydrogenation in
which eight hydrogens were taken up, 2-methyl-3-methoxy-4 , 5-di(amino-
methyl)pyridine was obtained. Nitrous acid converted the aminomethyl
groups to hydroxymethy! groups, the resulting compound being identical
COOH CN CH.NH,
cH,0% \cooH CHOY YCN CH,OY \CH:NH,
per PE Pe
CHAy CHAy / CHa /
CH.OH CH.Br CH.OH
Fy
cH,OY \cH.OH © HOY \CH.Br = HO \CHOH
es ea
CHa CHa / CHayn )
Conversion of Pyridoxine Methyl Ether Dicarboxylie Acid to Pyridoxine*!
230 PYRIDOXINE AND RELATED COMPOUNDS
with the methoxy vitamin. Kuhn and Wendt?> had previously shown that
treatment of the methyl ether of pyridoxine with HBr not only hydrolyzes
the ether but also replaces the hydroxyl groups in the hydroxymethyl
groups with Br which could be replaced by hydroxy] groups by use of silver
acetate. Pyridoxine as its hydrochloride was crystallized from dilute hydro-
chloric acid by the addition of acetone.
b. Harris and Folkers
Following their announcement in 1939?° of their synthesis of pyridoxine,
Harris and Folkers published their original method of synthesis.” They
later described an improved and more practical synthesis.”
The original synthesis started with the condensation of ethoxyacetylace-
tone with cyanoacetamide to yield 3-cyano-4-ethoxymethy]l-6-methyl-2-
pyridone. The following is the graphic representation of the various steps
of the synthesis.
i 7 CH,0C;H,;
ll pyridine SCN HNO;
H
CH.OC.H; CH,OC:H; CH,OC.H,
SCN PCls I~ SCN H.+Pt H.N¢ SCN _#.+Pa
es cl 2Cl “ol 2Cl
toaN ee oy NI
H
CHOC lH: CH.OC.H, CH.Br
OT HONO Boaters HBr ne ee
TELS NZ : H;C SNi 1s} NZ
CH,OH
H,0 HO |CH.0H
AgCl H;C ‘N
HCl
Synthesis of Pyridoxine?’ 28
c. Other Syntheses
Mori and Makino”? reported their synthesis of pyridoxine by practically
the same method used by Harris and Folkers. Later Mowat et al.®°: *! also
25 R. Kuhn and G. Wendt, Ber. 72B, 311 (1939).
26S. A. Harris and K. Folkers, Science 89, 347 (1939).
278. A. Harris and K. Folkers, J. Am. Chem. Soc. 61, 1245 (1989).
*88. A. Harris and K. Folkers, J. Am. Chem. Soc. 61, 3307 (1939).
22S. Mori and K. Makino, Hnzymologia 7, 385 (1939).
30 J. H. Mowat, F. J. Pilgrim, and G. H. Carlson, J. Am. Chem. Soc. 65, 954 (1948).
31G. H. Carlson, U.S. Pat. 2,310,167 (1943.
Il. CHEMISTRY 231
published their synthesis in which 2-methyl-4-carboxy-5-cyano-6-pyridone
amide served as the starting compound. The synthesis of Szabo* consists
in oxidizing 3-methyl-4-methoxyquinoline to the dicarboxylic derivative
of the methoxypyridoxine. This was then converted to the dicyano com-
pound through intermediates. Reduction to the diamino compound followed
by hydrolysis of the ether and treatment with nitrous acid yielded the vita-
min.
More recently applying the method developed for reducing the esters of
heterocyclic carboxylic acids to the corresponding hydroxymethyl com-
pounds, Jones and Kornfeld** directly reduced the 4,5-dicarboxylic ester
of pyridoxine to pyridoxine with lithium aluminum hydride. Excellent yields
were obtained. By this step the complicated conversion of the carboxylic
groups used by others was neatly overcome.
hk. SPECIFICITY
1. GENERAL
Since the recognition that only a part of the vitamin Bg activity of natural
products can be attributed to pyridoxine, vitamin Beg is now used as a class
name to include all compounds having vitamin Bg activity. On the adop-
tion of the report of the Committee on Biochemical Nomenclature of the
American Society of Biological Chemists, pyridoxine is correctly applied
only to the single substance, 2-methyl-3-hydroxy-4 ,5-di(hydroxymethy])-
pyridine. The two other important naturally occurring substances with
vitamin Bg activity are pyridoxal, 2-methyl-3-hydroxy-4-formy]-5-hydroxy-
methylpyridine, and pyridoxamine, 2-methy]-3-hydroxy-4-aminomethyl-5-
hydroxymethylpyridine.
2. PYRIDOXAL AND PYRIDOXAMINE
The existence of other forms of pyridoxine was recognized by Snell et al.*4
as the result of the comparison of microbiological assays on extracts of
natural materials with the values based on chemical and animal assay. In
some instances the discrepancies were as much as several thousandfold.
This highly active form of pyridoxine was given the trivial name, pseudo-
pyridoxine. When pyridoxine was treated with mild oxidizing agents, Car-
penter and Strong*® observed a marked increase in activity for the micro-
organism Lactobacillus casei. Snell**-*8 extended his observation that
#J.L. Szabo, U.S. Pat. 2,359,260 (1944).
%R.G. Jones and E. C. Kornfeld, J. Am. Chem. Soc. 78, 107 (1951).
“EE. E. Snell, B. M. Guirard, and R. J. Williams, J. Biol. Chem. 148, 519 (1942).
% 1,. E. Carpenter and F. M. Strong, Arch. Biochem. 3, 375 (1944).
* i. E. Snell, Proc. Soc. Expl. Biol. Med. 61, 356 (1942).
7 FE. E. Snell, J. Am. Chem. Soc. 66, 2082 (1944).
%® EK. E. Snell, J. Biol. Chem. 164, 313 (1944).
232 PYRIDOXINE AND RELATED COMPOUNDS
autoclaving of pyridoxine with the assay medium or amino acids greatly
increased the activity of pyridoxine for the test organism Streptococcus
faecalis R. and concluded that the products formed by treating pyridoxine
with aminating agents and mild oxidizing agents were, respectively, the
amino and aldehyde derivatives of pyridoxine. By deduction it appeared
that one of the hydroxymethylene groups in either the 4 or 5 position had
been modified. Tests of the compounds synthesized by Harris et al.?% 4°
showed that the compounds were 2-methyl-3-hydroxy-4-aminomethyl-
5-hydroxymethylpyridine and 2-methyl-3-hydroxy-4-formyl-5-hydroxy-
methylpyridine.
CH.NH:; CHO
spas ie
ae a4
Pyridoxamine Pyridoxal
Pyridoxamine was prepared by aminating either the acylated pyridoxine
or, in better yield, by aminating 2-methyl-3-hydroxy-4-methoxymethy]l-5-
hydroxymethylpyridine.
CH.OCH; CH.NH,
HO’ \CH.OH ine HO’ \CH.OH
3
| 120-140° |
CHA y / CHi\y
Owing to the greater reactivity of the methylene group in the 4 position,
this reaction was possible. The isomeric 5-aminomethylpyridine was also
synthesized.
Careful oxidation of pyridoxine with potassium permanganate yielded
an aldehyde which could be separated from the reaction mixture as its
oxime. This was decomposed with nitrous acid and then treated with
ethanol and hydrochloric acid to yield a cyclic acetal which easily hydro-
lyzed to the desired aldehyde. The proof that the formyl group was in the
4 position was accomplished by converting the oxime to the 4-amino com- |
pound by catalytic reduction.
39. A. Harris, D. Heyl, and K. Folkers, J. Biol. Chem. 154, 315 (1944).
40§. A. Harris, D. Heyl, and K. Folkers, J. Am. Chem. Soc. 66, 2088 (1944).
> haa
Ill. INDUSTRIAL PREPARATION 233
III. Industrial Preparation!
JOHN C. KERESZTESY
A. SYNTHESIS
At this writing at least one commercial method for the synthesis of
pyridoxine hydrochloride is based on the method used by Harris and
Folkers.?: * In this method an alkoxyacetylacetone is condensed with cyano-
acetamide to give 3-cyano-4-alkoxymethyl-6 methyl-2-pyridone. This com-
pound on nitration, chlorination, and subsequent reduction yields 2-
methyl-3-amino-4-alkoxymethyl-5-aminomethylpyridine dihydrochloride.
On hydrolysis and diazotization, pyridoxine hydrochloride (2-methy1-3-hy-
droxy-4 ,5-di(hydroxymethyl])pyridine hydrochloride) is obtained.
The major producers of pyridoxine hydrochloride, the only form in which
pyridoxine is commercially available, are the American Cyanamid Co.,
Hoffman-LaRoche, and Merck & Co., Inc.
Bo PURITY STANDARDS, U.S:22
Commercial pyridoxine hydrochloride is produced to meet the following
purity standards.
CsHi,NO;:HCl: a white crystalline powder, stable in air and slowly
affected by sunlight. Melts with some decomposition between 204 and 208°;
1 g. dissolves in 5 ml. of H:O and in about 90 ml. of ethanol; pH about 3.0.
Loss of weight on drying over concentrated sulfuric acid in vacuum desic-
eator should not exceed 0.5 %. Residue on ignition should not exceed 0.1 %.
No ammonia odor when heated at 100° with dilute aqueous caustic and
less than 40 p.p.m. of heavy metals, as Pb.
C. US. PRODUCTION
Year Production, lb. Average value per pound
1940 = $3450 ($5450 initially)
1941 — 1418
1942 = 1022
1943 — 622
1944 4,200 455
1945 2,700 457
1946 10,300 381
1947 12,900 304
1948 10,900 246
1949 10,000 239
1950 17,500 234
1 The author wishes to express his appreciation to Dr. Max Tishler for his assistance
in the assembling of the information of this section.
2U.S. Pat. 2,422,617 (1947).
7U.S. Pat. 2,399,347 (1946).
*U.S. Pharmacopeia, 14th revision, p. 900, 1950.
234 PYRIDOXINE AND RELATED COMPOUNDS
IV. Biochemical Systems
W. W. UMBREIT
A. GENERAL
Pyridoxine, like other vitamins, functions in the form of a coenzyme.
This coenzyme, which has been variously termed codecarboxylase, cotrans-
aminase, etc., is the 5-phosphate of pyridoxal. The various members of the
vitamin Bs group—pyridoxine, pyridoxal, pyridoxamine, and their respec-
tive phosphates—owe their vitamin activity to the ability of the organism
to convert them into the enzymatically active form, pyridoxal-5-phosphate.
Recently several papers have appeared which further extend our informa-
tion. In addition to chemical studies on structure,! the crystalline pyridoxal
and pyridoxamine phosphates! have been prepared.
Pyridoxine is concerned with the activity of a wide variety of enzyme
systems. These catalyze reactions which have apparently very little in
common, except that all are characterized by action of one sort or another
upon amino acids. The known enzyme systems are listed in the following
paragraphs.
B. AMINO ACID DECARBOXYLASES
The properties of the amino acid decarboxylases have been so well de-
scribed in reviews? that no great detail is necessary here. Their distribu-
tion and their relation to pyridoxal phosphate is given in Table I, from
which it is evident that they occur predominantly in bacteria and to a lesser
extent in other organisms. However, even among the bacteria, their dis-
tribution varies greatly from one strain to another. Their physiological
significance is at present unknown. The enzymes decarboxylating asparti¢
acid to B-alanine are probably of nutritional value,**: “4 and those decarboxy-
1M. Viscontini, C. Ebnother, and P. Karrer, Helv. Chim. Acta 34, 1834, 2198, 2199,
2438 (1951); M. Viscontini and E. G. Bonetti, Helv. Chim. Acta 34, 2485 (1951);
J. Baddiley and A. P. Mathias, J. Chem. Soc. 1952, 2583.
1a H}. A. Peterson, H. A. Sober, and A. Meister, J. Am. Chem. Soc. 74, 570 (1952).
2 H. Blaschko, Advances in Enzymol. 5, 67 (1945).
2a KH. F. Gale, Advances in Enzymol. 6, 1 (1946).
31. C. Gunsalus, Federation Proc. 9, 556 (1950).
4Q. Schales, Advances in Enzymol. 7, 513 (1947). |
’O. Schales, in The Enzymes, Vol. 2, Part 1, p. 216. Academie Press, New York, |
1951.
6K. Werle, Z. Vitamin-Hormon-u. Fermentforsch. 1, 504 (1947).
7H. F. Gale, Biochem. J. 34, 392 (1940).
8 E.S. Taylor and E. F. Gale, Biochem. J. 39, 52 (1945).
9H. M. R. Epps, Biochem. J. 89, 42 (1945).
10§. R. Mardasheu and R. N. Etingof, Biokhimia 18, 469 (1948) [C. A. 48, 3065 |
(1949) ]. |
IV. BIOCHEMICAL SYSTEMS 235
TABLE I
SUMMARY OF THE DISTRIBUTION OF THE AMINO Actpb DECARBOXYLASES
Decarboxylase® Bacteria Plants Animals Pyridoxal phosphate?
Arginine + (7, 8, 37) — — +B
‘Histidine + (7, 9-11) ? (12) + (6, 13-20) —
Tyrosine + (21-31) _- + (17, 32-34) +B
Ornithine + (7, 8, 22, 35, — -- +B
36)
Glutamic acid + (22, 35, 37) + (88, 39) + (40, 41) +BPA
Aspartic to 6-alanine + (42-44) — — —
Aspartic to a-alanine + (45) — —- +B
Lysine + (7, 46) — — +B
Dihydroxiphenylalanine — ?(12) + (2,17,47- +A
56)
@ The following decarboxylases are alleged to occur in animal tissues: tryptophan,” cysteie acid,?» >"-59
hydroxyphenylserine® (but not phenylserine), and phenylalanine.® However, in the case of the last two
enzymes, at least, it is quite possible that these are further reactions of tyrosine decarboxylase (which
has been shown to decarboxylate phenylalanine®!) or dihydroxyphenylalanine decarboxylase.
5 Indicates whether pyridoxal phosphate hus been established as a coenzyme: B = bacteria; P =
plant; A = animal.
¢ Extensive efforts 9*"»17 have so far not been ‘able to demonstrate the involvement of pyridoxal
phosphate.
1 A.W. Rodwell, Biochem. J. 48, 39 (1948).
12 E. Werle and A. Raub, Biochem. Z. 318, 538 (1948).
13 2, Werle, Biochem. Z. 282, 292 (1936).
4). Werle and H. Krautzun, Biochem. Z. 296, 315 (1938).
15 EH. Werle and H. Hermann, Biochem. Z. 291, 105 (1937).
16P. Holtz and R. Heise, Arch. Exptl. Pathol. Pharmakol. 187, 589 (1937).
7 P. Holtz, K. Credner, and H. Walter, Z. physiol. Chem. 262, 111 (1939).
18 |). Werle and Kk. Heitzer, Biochem. Z. 299, 420 (1938).
19P. Holtz, R. Heise, and W. Spreyer, Arch. Exptl. Pathol. Pharmakol. 188, 580
(1938).
20 E. Werle, Biochem. Z. 311, 270 (1942).
KE. F. Gale, Biochem. J. 34, 846 (1940).
22. F. Gale, Biochem. J. 35, 66 (1941).
*2H. M. R. Epps, Biochem. J. 38, 242 (1944).
**W.D. Bellamy and I. C. Gunsalus, J. Bacteriol. 46, 575 (1943).
2° T. C. Gunsalus and W. D. Bellamy, J. Bacteriol. 47, 413 (1944).
26W.D. Bellamy and I. C. Gunsalus, J. Bacteriol. 48, 191 (1944).
*7 1. C. Gunsalus and W. D. Bellamy, J. Biol. Chem. 155, 557 (1944).
28 T. C. Gunsalus and W. D. Bellamy, J. Biol. Chem. 155, 357 (1944).
*27W.D. Bellamy and I. C. Gunsalus, J. Bacteriol. 50, 95 (1945).
80 FE. F. Gale and H. M. R. Epps, Biochem. J. 38, 250 (1944).
1 1.C. Gunsalus, W. D. Bellamy, and W. W. Umbreit, J. Biol. Chem. 155, 685 (1945).
® P. Holtz and H. Janisch, Arch. Exptl. Pathol. Pharmakol. 186, 684 (1937).
8% P. Holtz, Z. physiol. Chem. 251, 226 (1938).
EE). Werle and G. Menniken, Biochem. Z. 291, 325 (1937).
% EF. F. Gale, Biochem. J. 39, 46 (1945).
8° J. Baddiley and E. F. Gale, Nature 155, 727 (1945).
236 PYRIDOXINE AND RELATED COMPOUNDS
lating the hydroxyphenylalanines or hydroxyphenylserines may be of sig-
nificance in the formation of arterenol and epinephrin and may bear a rela-
tion to hypertension.*: °°
C. TRANSAMINASES
These enzymes: © catalyze the exchange of amino groups between a-
ketoglutarate and a variety of amino acids yielding glutamate and the cor-
responding keto acid. The reactions between glutamate and pyruvate to
yield alanine and that from glutamate to oxaloacetate to yield aspartate
are reversible. These reactions have recently been extended to a variety of
other amino acids (listed below),*: ®° but these have not yet been shown to
be strictly reversible. It would appear, however, that transamination may
serve as a general mechanism for the synthesis of amino acids from their
keto analogs. Tissues from pyridoxine-deficient animals are low in trans-
37 W. W. Umbreit and IJ. C. Gunsalus, J. Biol. Chem. 159, 333 (1945).
33 Q. Schales, V. Mims, and 8. 8. Schales, Arch. Biochem. 10, 455 (1946).
39 Q. Schales and 8. 8. Schales, Arch. Biochem. 11, 155 (1946).
40 H}. Roberts and 8. Frankel, J. Biol. Chem. 188, 789 (1951).
41H}. Roberts and S. Frankel, J. Biol. Chem. 190, 505 (1951).
428. R. Mardasheu and V. N. Gladkoua, Biokhimiya 18, 315 (1948); [C. A. 42, 8859
(1948) ; 48, 1073 (1949)].
43 T). Billen and H. C. Lichstein, J. Bacteriol. 58, 215 (1949).
44W. E. David and H. C. Lichstein, Proc. Soc. Exptl. Biol. Med. 73, 216 (1950).
45 A. Meister, H. A. Sober, and S. V. Tice, J. Biol. Chem. 189, 577 (1951).
46 EK. F. Gale and H. M. R. Epps, Biochem. J. 38, 232 (1944). |
47 P, Holtz, R. Heise, and K. Ludtke, Arch. Exptl. Pathol. Pharmakol. 191, 87 (1938).
48 P. Holtz, A. Reinhold, and K. Credner, Z. physiol. Chem. 261, 278 (1939).
49 P. Holtz, K. Credner, and A. Reinhold, Arch. Exptl. Pathol. Pharmakol. 193, 688
(1939).
60 P. Holtz and K. Credner, Arch. Exptl. Pathol. Pharmakol. 199, 145 (1942).
51 P, Holtz, Naturwissenschaften 27, 725 (1939).
52 H. Blaschko, J. Physiol. (London) 101, 337 (1942).
53 OQ. Schales and 8. S. Schales, Arch. Biochem. 24, 83 (1949).
54H. W. Page, Arch. Biochem. 8, 145 (1945).
55D. E. Green, L. F. Leloir, and V. Nocito, J. Biol. Chem. 161, 559 (1945).
5° Hf. Blaschko, Biochim. et Biophys. Acta 4, 130 (1950).
57 H. Blaschko, Biochem. J. 36, 571 (1942).
53 F. Bernheim and M. L. C. Bernheim, J. Biol. Chem. 127, 695 (1939).
59 G. H. Sloane Stanley, Biochem. J. 45, 556 (1949).
60 G. Schapita, Compt. rend. soc. biol. 140, 174 (1946).
61 R. W. McGilvery and P. P. Cohen, J. Biol. Chem. 174, 813 (1948).
62 P. P. Cohen, Transaminases: A Symposium on Respiratory Enzymes, p. 210.
University of Wisconsin Press, Madison, Wisconsin, 1942.
68 P, P. Cohen, in The Enzymes, Vol. 1, Part 2, p. 1040. Academie Press, New York,
1951.
64 P,.S. Cammarata and P. P. Cohen, J. Biol. Chem. 187, 439 (1951).
6° J. 1. Feldman and I. C. Gunsalus, /. Biol. Chem. 187, 821 (1950).
IV. BIOCHEMICAL SYSTEMS 237
aminase,®: ® and pyridoxal phosphate proved to be the coenzyme.°*: &9-7
1. Glutamic to pyruvate
2. Glutamic to oxalacetic
3. a-Ketoglutarate to: 6
Glycine Citrulline
Serine Ornithine®®
Threonine Arginine
Cystine Lysine
Cysteine Leucine
Methionine Isoleucine
Valine Phenylalanine
Proline Tyrosine
Histidine Tryptophan
Chemical transaminations between pyridoxal and most amino acids”
have been projected to explain the mechanism of enzymatic transamina-
tion.’® It has now been shown* that animal transaminase is activated by
erystalline pyridoxamine phosphate. This compound requires a long incu-
bation period with the apoenzyme but does not appear to be converted
into pyridoxal phosphate either during this period or during active trans-
amination itself, which seems to be a necessary prerequisite for the “‘shuttle”’
theories proposed’® and which fact, therefore, renders them quite unlikely.
There seems to be little question but that pyridoxal phosphate is concerned
in most reactions of amino acids, but its mechanism of action in these proc-
esses is not yet clear. Pyridoxal phosphate has been shown to be the coen-
zyme of some further transaminases,*! the enzyme synthesizing cystathio-
nine,” that cleaving it to cysteine and a-ketobutyrate,” the serine and
66 J. H. Quastel and R. Witty, Nature 167, 556 (1951).
* F. Schlenk and E. E. Snell, J. Biol. Chem. 157, 425 (1945).
6 S.R. Ames, P. 8S. Sarma, and C. A. Elvehjem, J. Biol. Chem. 167, 135 (1947).
69H. C. Lichstein, I. C. Gunsalus, and W. W. Umbreit, J. Biol. Chem. 161, 311
(1945).
70 A. E. Braunstein and M. G. Kritzmann, Nature 158, 102 (1946).
1K. F. Gale and H. M. R. Tomlinson, Nature 158, 105 (1946).
”®M. Kritzmann and O. Samarina, Nature 158, 109 (1946).
7% F. Schlenk and A. Fisher, Arch. Biochem. 8, 337 (1945).
™F. Schlenk and A. Fisher, Arch. Biochem. 12, 69 (1947).
7D. E. O’Kane and I. C. Gunsalus, J. Biol. Chem. 170, 425 (1947).
7 —). E. O’Kane and I. C. Gunsalus, J. Biol. Chem. 170, 433 (1947).
™W.W. Umbreit, D. J. O’Kane, and I. C. Gunsalus, J. Biol. Chem. 176, 629 (1948).
7% 7—). E. Metzler and E. E. Snell, J. Am. Chem. Soc. 74, 979 (1952).
mE. E. Snell, 2nd Intern. Congr. Biochem., (Paris), pp. 47-63 (1952).
80 A. Meister, H. A. Sober, and E. A. Peterson, J. Am. Chem. Soc. 74, 2385 (1952).
81K. V. Roswell, Nature 168, 104 (1951).
8 F. Binkley, G. M. Christensen, and W. N. Sensen, J. Biol. Chem. 194, 109 (1952).
238 PYRIDOXINE AND RELATED COMPOUNDS
threonine deaminases of Newrospora,® and a D-serine deaminase from bac-
teria.*4
There apparently also exists a transaminase from glutamine to a variety
of keto acids® in which the a-amino group of the glutamine is removed
before that of the amide. This reaction is also catalyzed by pyridoxal phos- —
phate.
D. RACEMASES
Pyridoxal phosphate is the coenzyme of an enzyme catalyzing the forma-
tion of an equilibrium mixture of DL-alanine from either p- or L-alanine.*
There are indications that a variety of such racemases may exist.
BRE TOR ELAN
In addition to decarboxylation and transamination, tryptophan is me- |
tabolized by pyridoxal phosphate enzymes as follows:
1. Tryptophanase:*’ this enzyme catalyses the breakdown of tryptophan
to indole, pyruvate, and ammonia.
Indole ———» Tryptophan ———~» Indole + pyruvate + ammonia (bacteria)
+
serine
Kynurenine ———> Kynurenic acid
3-Hydroxykynurenine —————> Xanthurenic acid
Anthranilic
acid + alanine
3-Hydroxyanthraniliec acid
‘
Quinolinic acid Nicotinic acid
Tryptophan Metabolism
2. In the animal, tryptophan is metabolized through kynurenine accord-
ing to the accompanying scheme. Pyridoxal phosphate is the coenzyme of
the reaction converting kynurenine to anthranilic acid and alanine®: 8° and
83 J. L. Reissig, Arch. Biochem. and Biophys. 36, 234 (1952).
84D. KE. Metzler and EK. E. Snell, J. Beol. Chem. 198, 363 (1952).
85 A. Meister and S. V. Tice, J. Biol. Chem. 187, 173 (1951).
86 W. A. Wood and I. C. Gunsalus, J. Biol. Chem. 190, 403 (1951).
87 W. A. Wood, I. C. Gunsalus, and W. W. Umbreit, J. Biol. Chem. 170, 313 (1947). |
88 A. EK. Braushtein, E. V. Goryachenkova, and T. 8. Paskhina, Biokhimiya 14, 163
(1949) [C. A. 48, 6264 (1949)]. !
89 C. E. Dalgliesh, W. EK. Knox, and A. Neuberger, Nature 168, 20 (1951).
V. SPECIFICITY OF ACTION 239
the step of converting 3-hydroxykynurenine to 3-hydroxyanthranilic acid*®
3. Pyridoxal phosphate is concerned with the formation of tryptophan’’
by the condensation of indole plus serine.*!
F. SULFUR-CONTAINING AMINO ACIDS AND PEPTIDES
Pyridoxal phosphate is the coenzyme of the cystathionine cleavage to
homocysteine and serine.” It is involved, as well, in the cysteine deaminase
by a process which may be identical with the cystathionine cleavage.”
V. Specificity of Action
W. W. UMBREIT
A. COENZYME FORM
Gunsalus and Bellamy! discovered that the rate of tyrosine decarboxy-
lation of Streptococcus faecalis cells was dependent upon the supply of ‘‘pseu-
dopyridoxine’”* in the growth medium. Cells grown in media deficient in
members of the vitamin Bs, group did not decarboxylate tyrosine, but the
resting cells could be activated with respect to this reaction by the addition
of pyridoxal.° When enzyme preparations from such cells were employed,
adenosinetriphosphate (ATP) was necessary in addition to pyridoxal for
activation.® It was possible to obtain active preparations of the coenzyme
in the absence of ATP by treatments designed to phosphorylate the pyri-
doxal.® By purification of yeast fractions showing codecarboxylase activity,
a material was obtained which appeared to contain pyridoxal phosphate.’
The biological properties of the compound were characterized by its acti-
vation of some enzyme systems listed previously, and in those cases studied
the natural material from yeast and the synthetic preparations were inter-
changeable.’ §
90 W. W. Umbreit, W. A. Wood, and I. C. Gunsalus, J. Biol. Chem. 165, 731 (1946).
1). L. Tatum and D. Bonner, Proc. Natl. Acad. Sci. 30, 30 (1944).
# F. Binkley and G. M. Christensen, J. Am. Chem. Soc. 73, 3535 (1951).
% A. E. Braushtein and R. M. Azarkh, Doklady Akad. Nauk. S.S.S. R.71, 93 (1950)
[C. A. 44, 7900 (1950)].
94 R. E. Kallio, J. Biol. Chem. 192, 371 (1951).
1W.D. Bellamy and I. C. Gunsalus, J. Bacteriol. 46, 573 (1943).
21. C. Gunsalus and W. D. Bellamy, J. Bacteriol. 47, 413 (1944).
3 W.D. Bellamy and I. C. Gunsalus, J. Bacteriol. 48, 191 (1944).
*1. C. Gunsalus and W. D. Bellamy, J. Biol. Chem. 155, 557 (1944).
§ 1. C. Gunsalus and W. D. Bellamy, J. Biol. Chem. 155, 357 (1944).
®1.C. Gunsalus, W. D. Bellamy, and W. W. Umbreit, J. Biol. Chem. 155, 685 (1945).
7™W.W. Umbreit, W. D. Bellamy, and I. C. Gunsalus, Arch. Biochem. 7, 185 (1945).
240 PYRIDOXINE AND RELATED COMPOUNDS
The purified coenzyme from synthetic sources contained one phosphorus
per mole of pyridoxal.’ In contrast to pyridoxal, there was no maximum ab-
sorption at 300 my in alkaline solution, which would eliminate the possi-
bility that the phosphate was at position 3. Similar conclusions were reached
in a direct chemical study.!®: 1! Nevertheless, strong claims!?-!® were made
for the activity of the 3-phosphate which were resolved only by a direct
comparison of the various preparations under identical conditions.” '8
Chemical proof of structure®*!!: 1°? including the preparation of a variety
of derivatives has now been given. From the data now available, pyridoxal
phosphate is unquestionably the monophosphoric acid ester of the 5-hy-
droxymethyl group of pyridoxal. A 4-phosphate?? has been postulated to
account for some aspects of spectrum, but this possibility has been elimi-
nated.19-22
B. FORMATION OF THE COENZYME FORM
As mentioned, the only form of vitamin Bg which is active enzymatically
is pyridoxal-5-phosphate. The other members of the vitamin Bs groups owe
their activity to systems capable of converting them into pyridoxal-5-phos-
phate. The interrelationships are illustrated below. The conversion of pyri-
doxine, pyridoxal, and pyridoxamine into pyridoxal phosphate during
erowth of organisms using these materials as sources of vitamin Bs, has
been demonstrated biologically. The conversion of pyridoxal to its phos-
phate by an enzymatic reaction involving ATP®: ?° has been studied. The
conversion of pyridoxamine phosphate to pyridoxal phosphate by way of
8 J. Baddiley and E. F. Gale, Nature 155, 727 (1945).
9T. C. Gunsalus, W. W. Umbreit, W. D. Bellamy, and C. E. Foust, J. Biol. Chem.
161, 743 (1945).
10 J. C. Gunsalus and W. W. Umbreit, Abstr. 110th Meeting Am. Chem. Soc. p. 34B
(1946).
11D). Heyl, 8. A. Harris, and K. Folkers, Abstr. 110th Meeting Am. Chem. Soc. (Chi-
cago), p. 35B (1946).
12 P, Karrer, Schweiz. Z. Pathol. wu. Bacteriol. 10, 351 (1947).
13 P. Karrer, and M. Viscontini, Helv. Chim. Acta 30, 52 (1947).
144 P. Karrer and M. Viscontini, Helv. Chim. Acta 30, 524 (1947).
16 P, Karrer and M. Viscontini, Helv. Chim. Acta 30, 528 (1947).
16 P. Karrer, M. Viscontini, and D. Forster, Helv. Chim. Acta 31, 1004 (1948).
17T. C. Gunsalus and W. W. Umbreit, J. Biol. Chem. 170, 415 (1947).
18 W.W. Umbreit and I. C. Gunsalus, J. Biol. Chem. 179, 279 (1949).
19 TP). Heyl, E. Luz, 8. A. Harris, and K. Folkers, J. Am. Chem. Soc. 78, 3430 (1951).
20 TD). Heyl and S. A. Harris, J. Am. Chem. Soc. 78, 3484 (1951).
21 TD). Heyl, E. Luz, 8. A. Harris, and K. Folkers, J. Am. Chem. Soc. 78, 3486 (1951).
22 T). Heyl, E. Luz, and S. A. Harris, J. Am. Chem. Soc. 78, 3437 (1951).
23 J. Baddiley, E. M. Thain, and A. W. Rodwell, Nature 167, 556 (1951).
24 T.C. Gunsalus, W. D. Bellamy, and W. W. Umbreit, J. Biol. Chem. 160, 461 (1945).
25 W. W. Umbreit and J. G. Waddell, Proc. Soc. Exptl. Biol. Med. 70, 293 (1949).
V. SPECIFICITY OF ACTION 241
an apparently specific type of transamination has also been observed.?°: *7
Not much is known about the enzymes carrying out the series of reactions
d “ f=
illustrated, but there is adequate evidence that they exist.
C. SPECIFICITY AND MECHANISM OF ACTION
In all cases so far known, the enzymatically active form of vitamin Bs
is pyridoxal-5-phosphate and the activity of pyridoxamine phosphate ob-
served occasionally*®*° is due to the presence of auxiliary enzyme systems
converting it to pyridoxal phosphate. It appears that the principal storage
CH,—NH, CHO CH,OH
HOG S\CH.OH le HO (ee
H;C H H;C H He Hi
N N N
Pyridoxamine Pyridoxal Pyridoxine
CHO
7 Ol sian
H;C H
N
Pyridoxal-5-phosphate
(coenzyme form)
CH.—NH, CH,OH
pe oe HO? )CH,OPO.H;
H;C H H;C H
N N
Pyridoxamine Pyridoxine
phosphate phosphate
Interconversion of Members of the Vitamin Bs Group
product of the phosphorylated vitamin is pyridoxamine phosphate but that
it is itself inactive. The fact that only the pyridoxal-5-phosphate is active
in a variety of enzyme systems listed previously makes any general postu-
late of a mechanism of action somewhat premature. Such mechanisms as
have been proposed for either decarboxylation or transamination have
postulated the formation of a Schiff’s base between the 4-aldehyde group
of pyridoxal phosphate and the a-amino group of the amino acid.!: *°-*
26 W.W. Umbreit, D. J. O’Kane, and I. C. Gunsalus, J. Biol. Chem. 176, 629 (1948).
27 A. Meister and 8. V. Tice, J. Biol. Chem. 187, 173 (1951).
28 A. Meister, H. A. Sober, and 8. V. Tice, J. Biol. Chem. 189, 577-591 (1951).
29$. R. Ames, P. S. Sarma, and C. A. Elvehjem, J. Biol. Chem. 167, 135 (1947).
30 H. Blaschko, Advances in Enzymol. 5, 67 (1945).
242 PYRIDOXINE AND RELATED COMPOUNDS
Although such a postulate is a possibility, there seems to be no positive evi-
dence for it and in the case of the transaminases there is some evidence
against 1t.?°
D. ANTAGONISTS
Only one of the vitamin Bs antagonists, desoxypyridoxine, has been
studied by enzymatic techniques.”> This material acts by being itself phos-
phorylated and competing, in the form of desoxypyridoxine phosphate,
with pyridoxal phosphate for the surface of the apoenzyme.
VI. Biogenesis
W. W. UMBREIT
The origin of the vitamin Bs molecule is not known. The transformations
among the various members of the vitamin Bg group are outlined on p. 241.
The original hypothesis of Snell! that, since D-alanine replaced the growth
requirement of certain bacteria for vitamin Bs, condensation between D-
alanine and a 4-carbon dicarboxy acid might serve to form pyridoxal has
not been borne out by subsequent data, since organisms grown on D-alanine
do not contain vitamin B, . Thereappears to be a requirement for D-alanine?
which in the presence of vitamin Bg is met from L-alanine and the pyridoxal
phosphate containing racemase.’
VII. Estimation
HENRY SHERMAN
Since many of the compounds which possess vitamin Bg activity occur in
nature as conjugates which are, in large part, firmly bound to tissue, pre-
31H}. F. Gale, Advances in Enzymol. 6, 1 (1946).
32 T. C. Gunsalus, Federation Proc. 9, 556 (1950).
33 QO. Schales, Advances in Enzymol. 7, 513 (1947).
34 Q. Schales, in The Enzymes, Vol. 2, Part 1, p. 216. Academic Press, New York,
1951.
36H}. Werle, Z. Vitamin- Hormon u. Fermentforsch. 1, 504 (1947).
36 P. P. Cohen, in The Enzymes, Vol. 1, Part 2, p. 1040. Academic Press, New York,
1951.
37 F. Schlenk and E. KE. Snell, J. Biol. Chem. 157, 425 (1945).
38 F. Schlenk and A. Fisher, Arch. Biochem. 8, 337 (1945). |
39 1). Werle and W. Koch, Biochem. Z. 319, 305 (1949).
1). K. Snell and B. M. Girard, Proc. Natl. Acad. Sci. 29, 66 (1948).
2H. EK. Snell, J. Biol. Chem. 158, 497 (1945).
3 W. A. Wood and I. C. Gunsalus, J. Biol. Chem. 190, 403 (1951).
VII. ESTIMATION 243
liminary tissue digestion is usually required for the quantitative extraction
of the active material prior to physical, chemical, and microbiological as-
say.' Melnick and coworkers? have tested various methods for liberating
bound vitamin Bs, and have found the following hydrolytic procedures to
be satisfactory: (1) autoclaving the materials at 15 lb. pressure in 0.055
N H.SO, for 90 minutes; (2) autoclaving at 15 lb. pressure in 2 N H.SO, for
30 minutes; (3) suspending the materials in 4 V HCl at 100° for 60 minutes.
There are modifications of this type of hydrolytic procedure which have
been successfully used; they all employ an acid hydrolysis, since it has been
demonstrated that the three members of the vitamin Bs complex are stable
in acid solution® and unstable in neutral or alkaline solution. Pepsin and
papain digestions in acid media have also been utilized to liberate bound
vitamin Beg.
A. PHYSICAL METHODS
HENRY SHERMAN
1. SPECTROPHOTOMETRIC
Pyridoxine exhibits a typical ultraviolet absorption spectrum which
changes markedly with variations in hydrogen ion concentration.’ °
Melnick eft al.? have shown that pyridoxal and pyridoxamine have similar
absorption curves and behave similarly with changes in pH. They have
used these facts as the basis of their spectrophotometric method for the
assay of vitamin Bg. ‘‘The ultraviolet absorption curves show no maxima
common to all three compounds. However, if readings are taken at 325
mu of solutions at pH 6.75, close estimates of the total amount of the com-
pounds present may be obtained, despite variations in their relative con-
centrations. At that wave length there is an absorption maximum common
to pyridoxine and pyridoxamine, and while pyridoxal absorbs approxi-
mately 20 per cent more light at its 315 mu maximum, at 325 mu all three
compounds absorb to the same extent. The /}%,, values at 325 mw in
pure solutions at pH 6.75 all approximate 440 + 1.5 percent expressed in
terms of the free bases. Spectrophotometric analyses, of course, cannot be
applied unless the test solutions are free from irrelevant light-absorbing
materials, or unless blank solutions containing none of these vitamin Bs
factors are available for evaluating the interference.” This spectrophoto-
1L. Atkin, A. 8. Schultz, W. L. Williams, and C. N. Frey, Ind. Eng. Chem. Anal.
Ed. 15, 141 (1943).
2D. Melnick, M. Hochberg, H. W. Himes, and B. L. Oser, J. Biol. Chem. 160, 1
(1945).
§E. Cunningham and E. E. Snell, J. Biol. Chem. 158, 491 (1945).
‘J.C. Keresztesy and J. R. Stevens, J. Am. Chem. Soc. 60, 1267 (1938).
'E.T. Stiller, J.C. Keresztesy, and J. R. Stevens, J. Am. Chem. Soc. 61, 1237 (1939).
244 PYRIDOXINE AND RELATED COMPOUNDS
metric method has been adapted to pure solutions, but as yet has not been
used successfully with biological materials.
Vacher and Faucquembergue® utilized the change in the shape of the
ultraviolet absorption curve of pyridoxine with pH as the basis for their
assay. They analyzed for pyridoxine in the presence of other substances by
obtaining ultraviolet absorption curves at two or more levels of pH.
2. POLAROGRAPHIC
Pyridoxine produces a double wave when it is reduced at the dropping
mercury electrode, indicating a reduction in two 2-electron steps.” Although
reduction was difficult, satisfactory results were obtained when tetramethyl-
ammonium bromide was the supporting electrolyte. This method, how-
ever, has not yet been used for the estimation of pyridoxine in foods.
B. CHEMICAL METHODS
HENRY SHERMAN
Most of the colorimetric tests that have been used for the assay of the
vitamin Bs group were originally devised when pyridoxine was believed to
be the only member of this biologically active complex. Since pyridoxal
and pyridoxamine are now considered important members of the vitamin
Bs complex and since they are present in natural materials along with other
biologically inactive phenolic compounds which often react similarly, many
of the original concepts concerning the various colorimetric tests have had
to be revised. It seems unlikely that any simple colorimetric test can be ap-
plied toward the assay of all forms of the vitamin Bs complex. At present,
three separate and distinct compounds possess vitamin Beg activity, and
there is some evidence that even these three compounds do not account for
all the biological activity.? The colorimetric procedures which are now avail-
able are most accurate when applied to analytical samples, such as pharma-
ceutical preparations, which contain only pyridoxine. However, if these tests
are employed to assay natural products in which pyridoxine does not con-
stitute the major portion of the vitamin Bs content, considerable caution
must be exercised in interpreting the results.
One class of color reactions includes the formation of cyanine dyestuffs
and phthaleins.
1. CYANINE Dyr Terst®
The cyanine dye test is specific for a-picoline compounds of which the
vitamin Bs group is the only naturally occurring member. It has been ap-
6M. Vacher and D. Faucquembergue, Bull. soc. chim. biol. 81, 1419 (1949).
7 J.J. Lingane and O. L. Davis, J. Biol. Chem. 187, 567 (1941).
8 R. Kuhn and I, Low, Ber. 72, 1453 (1939).
VII. ESTIMATION 245
plied to the determination of pyridoxine. The test depends upon the reac-
tivity of the a-methyl groups. The vitamin must first be converted into the
methyl ether by diazomethane and then into a quaternary pyridine com-
pound by methyl! iodide or dimethylsulfate. The resulting salt, upon heat
treatment with a solution of sodium in ethy] alcohol and then with chloro-
form, produces a violet color, which has absorption maxima at 599 and 555
mu. By this color test, 0.1 mg. of pyridoxine can be detected. It has not
been used extensively in food analysis because of the difficulty encountered
in the preparation of the phenolic ether in quantitative yields.
2. PHTHALEINS®
Stiller and his associates oxidized pyridoxine to the 4,5-dicarboxylie acid
and then fused the resulting acid with resorcinol to obtain the correspond-
ing phthalein, which had a greenish-yellow fluorescence. However, this
method is not readily adaptable to assay procedure because of its lack of
specificity. It can be used, however, to measure pyridoxine in pure solutions.
The most widely used color reactions depend upon the phenolic nature
of the vitamin Bes group. One series of tests involves the active participa-
tion of the phenolic hydroxyl group. Another series of tests depends upon
the coupling reaction which is common to most phenolic compounds. Each
of the three active forms of the vitamin Bs group has a phenolic group in
the 3 position and an unsubstituted position para to this phenolic group.
These two factors favor the condensation of the vitamin with chromogenic
reagents to form dyes which can be measured colorimetrically; this funda-
mental reaction is the basis of the color tests that have been applied most
successfully to the chemical assay of the vitamin Bz, complex.
3. Ferric CHLorRIDE MrEtrHop
When pyridoxine is reacted with a 10% aqueous solution of FeCl, a
red-brown color is formed, indicating the presence of the phenolic hydroxy]
group.* This reaction has been used to estimate pyridoxine in more highly
refined concentrates, by comparing the color developed in the unknown
sample with that of standards.’ Pharmaceutical preparations containing
only pyridoxine of the vitamin B, group are especially amenable to such a
test; other members of the B vitamin group do not interfere.’? No literature
is available on the reactions between pyridoxal or pyridoxamine with FeCl;.
4. PHenou Test with Foutin-DENIS REAGENT!
Pyridoxine, like most phenols, reacts with the Folin-Denis reagent (a
mixture of phosphotungstic and phosphomolybdic acids) to form a blue
*R. D. Greene, J. Biol. Chem. 130, 513 (1939).
0G. G. Villela, Anais assoc. quim. Brasil T, 168 (1948).
1 Q. Folin and W. Denis, J. Biol. Chem. 22, 305 (1915).
246 PYRIDOXINE AND RELATED COMPOUNDS
color which can be measured. This method did not gain wide acceptance due
to its lack of specificity.
5. DtazoTizED SULFANILIC Acip TEstT
Swaminathan” used the color reaction between pyridoxine and diazo-
tized sulfanilic acid as the basis for its estimation. This reaction is not spe-
cific for pyridoxine; complete removal of interfering substances prior to
the coupling reaction is essential. The procedure employed to free extracts
of interfering substances is long and difficult to perform. Protein and
protein derivatives resulting from a pepsin digestion are removed by phos-
photungstic acid, and purines, pyrimidines, and imadizole bases are re-
moved by a silver nitrate and barium hydroxide treatment. The pyridoxine
is then adsorbed on Clarite from a filtrate adjusted to pH 1 to 2, eluted
with hot barium hydroxide, adjusted to pH 6, and then coupled with dia-
zotized sulfanilic acid. This method has been applied to many foods and
to urine samples with excellent results.’ “4
Bina et al.!® improved the chemical procedure with diazotized sulfanilie
acid. They adsorbed pyridoxine on Superfiltrol at pH 3 and then used alka-
line ethyl! alcohol as the selective eluant; this not only aided and simplified
the purification process but also served to stabilize the color which, in the
Swaminathan procedure, started to fade after 5 minutes.
Ormsby et al.'® observed that the three members of the vitamin Bs com-
plex gave different colors when treated with diazotized sulfanilic acid. Pyri-
doxine gives an orange color, pyridoxamine an orange to pink color, and
pyridoxal a bright yellow color. Although the stability of the colors was
limited, absorption spectra could be obtained. It is evident, therefore, that
foodstuffs containing more than one member of the Bs group can produce
a color with diazotized sulfanilic acid which may be difficult to match with
standards prepared from pyridoxine. This suggests why individual colori-
metric tests are not very accurate when applied to natural materials.
6. DiazotizED p-AMINOACETOPHENONE TEST
Brown et al.” introduced this new diazo reaction for pyridoxine; it had
first been used for the determination of thiamine.'® Although the color pro-
duced is not as stable as that developed with diazotized sulfanilic acid, its
sensitivity and specificity are much better. This increased sensitivity is
2M. Swaminathan, Nature 145, 780 (1940).
13M. Swaminathan, Indian J. Med. Research 28, 427 (1940).
14M. Swaminathan, Indian J. Med. Research 29, 561 (1941).
15 A. F. Bina, J. M. Thomas, and E. B. Brown, J. Biol. Chem. 148, 111 (1948).
16 A.A. Ormsby, A. Fisher, and F. Schlenk, Arch. Biochem. 12, 79 (1947).
17K}. B. Brown, A. F. Bina, and J. M. Thomas, J. Biol. Chem. 158, 455 (1945).
18 Hf. J. Prebluda and E. V. McCollum, J. Biol. Chem. 127, 495 (1939).
VII. ESTIMATION 247
probably due to the fact that the color reaction is carried out at a pH of
7.0 to 7.3 instead of at the higher pH of 10 to 11, which is required for the
coupling reaction with diazotized sulfanilic acid. Solutions containing 0.5
to 2.0 y of pyridoxine per milliliter can be measured by this procedure.
The use of diazotized p-aminoacetophenone instead of sulfanilic acid in
the color development permits a simpler and more efficient purification
process. A synthetic resin, Amberlite No. IR-4, is used instead of phospho-
tungstic acid in the purification process prior to the color development.
This resin adsorbs soluble interfering substances, thus preventing their
simultaneous adsorption on Superfiltrol with pyridoxine; the vitamin is
eluted from the Superfiltrol with alkaline ethyl alcohol.
7. DiazotizeD p-NITROANILINE TEST
Swaminathan" used this phenol reagent to assay for pyridoxine in food-
stuffs, but it has not been used by many other investigators. Nassi!® modi-
fied the method somewhat by introducing a blank determination. He ob-
served that pyridoxine, irradiated with ultraviolet light, lost the capacity
to produce an orange color with diazotized p-nitroaniline. He suggested,
therefore, that colorimetric tests be conducted on non-irradiated and ir-
radiated aliquots of the sample; the difference between the two values
would more closely approximate the true pyridoxine content of the sample.
However, considerable caution in interpretation must be exercised in view
of the presence of pyridoxal and pyridoxamine in natural materials.
8. INDOPHENOL TEST
Gibbs” observed many years ago that phenols which had unsubstituted
para positions formed, under the proper conditions, blue indophenol salts.
Pyridoxine gives a positive Gibbs test when reacted with 2 ,6-dichloroqui-
nonechlorimide.® Scudi and his coworkers”! developed an analytical pro-
cedure for the estimation of pyridoxine based upon this indophenol reaction,
which has become the most widely used and accepted of all colorimetric
tests. As little as 0.5 y pyridoxine in | ml. of solution can be analyzed by
this method. However, even this chlorimide reagent, which is more specific
than other reagents, reacts with compounds not possessing Bg activity.
Most of the chemical procedures that have utilized this indophenol reac-
19L. Nassi, Boll. soc. ital. biol. sper. 16, 690 (1941).
20 H. D. Gibbs, J. Biol. Chem. 72, 649 (1927).
21 J. V. Scudi, H. F. Koones, and J. C. Keresztesty, Proc. Soc. Exptl. Biol. Med. 48,
118 (1940).
2 J. V.Scudi, K. Unna, and W. Antopol, J. Biol. Chem. 135, 371 (1940).
23 J. V. Secudi, W. A. Bastedo, and T. J. Webb, J. Biol. Chem. 136, 399 (1940).
4 J. V.Scudi, J. Biol. Chem. 139, 707 (1941).
248 PYRIDOXINE AND RELATED COMPOUNDS
tion depend for their accuracy upon the complete removal of interfering
substances prior to the formation of the color complex.
In this procedure, Scudi* used a two-phase water-butanol solution to aid
in the removal of interfering substances at pH 6.8 to 7.2. However, the
neutralized solution was then weakly buffered with veronal prior to color
development. No provision was made to control interfering compounds
like salts and bases. A borate buffer blank was introduced to correct for
compounds which would react with the chlorimide reagent, but the results
were not very quantitative. Pyridoxine forms a complex with boric acid,
which does not give the indophenol salt. It was originally thought that the
difference in values obtained in veronal and borate buffers would represent
vitamin Be-active materials, but with the discovery of pyridoxal and pyri-
doxamine the borate buffer blank lost its original purpose. Pyridoxal and
pyridoxamine react with the chlorimide reagent to form a blue pigment
even in the presence of borate. Therefore, when all three compounds are
present in natural materials, the total blue color formed with the chlori-
mide reagent is equal to the sum of the members of the vitamin Beg group.
The difference, in the presence and absence of borate, represents the color
contribution of pyridoxine alone, and not the vitamin Beg group.
Bird et al.2°> modified the Scudi colorimetric method so that it was simpler
and more rapid. Pyridoxine was adsorbed on Superfiltrol at pH 3.0, washed,
and then eluted by adding a butanol solution of 2 ,6-dichloroquinonechlori-
mide to the Superfiltrol. Veronal buffer was then added, so that the pH
was raised to 7.8 to 8.0 and the characteristic blue color then developed.
Elution and color development were thus carried out simultaneously.
Hochberg et al.2* developed a method which was based on the coupling
of pyridoxine and the chlorimide reagent in isopropanol, a one-phase sys-
tem. The use of a strong NH,OH-NH,Cl buffer of high basicity and salinity
eliminated the interference caused by different kinds and amounts of bases
and salts in the test solution. The color development reached a maximum
in 60 seconds and was three times as sensitive as other tests; similar tests
required 20 to 40 minutes for complete color development. The use of an
internal standard eliminated the influence of other compounds upon the
rate, extent, and stability of color formation. A borate blank made the re-
action fairly specific for pyridoxine. When this method was applied to bio-
logical materials, the pyridoxine was first adsorbed on Lloyd’s reagent,
eluted, and then submitted to the coupling reaction.” Bottomley has sug-
gested that products which are high in fat content be subjected to an ether
extraction prior to isolation and coupling. Melnick and coworkers,? using
25. D. Bird, J. M. Vandenbelt, and A. D. Emmett, J. Biol. Chem. 142, 317 (1942).
26 M. Hochberg, D. Melnick, and B. L. Oser, J. Biol. Chem. 155, 109 (1944).
27 M. Hochberg, D. Melnick, and B. L. Oser, J. Biol. Chem. 155, 119 (1944).
28 A.C. Bottomley, Biochem. J. 38, V (1944).
VII. ESTIMATION 249
this colorimetric procedure, observed that pyridoxamine and pyridoxal were
about 31 % and 15 % as reactive, respectively, as pyridoxine on an equimolar
basis. They state, therefore, that this method can be used with precision
only when pyridoxine constitutes a major portion of the vitamin Bes content
of the material tested. When applied to metabolically active tissues, like
yeast, which have large amounts of pyridoxal and pyridoxamine, this
method can give only approximate values. Undoubtedly, similar conclusions
may be drawn with respect to other chemical assay procedures.
Canbiick and Lindholm?* have substituted 2 ,6-dibromoquinonechlori-
mide for the chloro compound and have reported good results.
9. CYANOGEN BROMIDE TEST
Sweeney and Hall?** have recently developed a chemical assay procedure
which is based on the K6nig reaction, often used in the chemical assay for
niacin. The pyridine ring is opened by cyanogen bromide and the resulting
product coupled with an aromatic amine to give a colored complex. Al-
though pyridoxine is a pyridine derivative, it reacts very little, if at all,
with the Konig reagent, for it has-an a-methyl group on the pyridine nu-
cleus. However, this methyl group can be removed by first converting it
to the carboxyl group and then heating the resulting a-carboxyliec acid at
120 to 150°. The loss of CO: results in the formation of a compound which
then reacts with the Konig reagent.
In this method, pyridoxine is removed from niacin and other interfering
substances by adsorption on zeolite at pH 7.6 and subsequent elution with
hot normal ammonium hydroxide. The eluate is evaporated to dryness
and oxidized, while being heated, with sulfuric acid and selenium. The
oxidized mixture is neutralized, treated with cyanogen bromide, and then
coupled with sulfanilic acid. The resulting color is read at 480 my. Excel-
lent recovery experiments (90 to 103 %) have been reported.
Pyridoxal gives the same test as pyridoxine, but pyridoxamine gives only
a faint color with this procedure. However, pyridoxamine can be converted
to pyridoxine with nitrous acid in an additional step. In this way, the
procedure affords a means not only for determining the total vitamin Be
complex but also for differentiating pyridoxamine from the other two mem-
bers of the group.
C. BIOLOGICAL METHODS
HENRY SHERMAN
The vitamin B, activity of natural materials is measured most reliably by
animal assay. No preliminary hydrolytic, extractive, and concentrating
procedures are required to liberate the many bound forms of the vitamin
29'T. Canbiick and M. Lindholm, Finiska Kemistsamfundets Medd. 54, 134 (1945).
29a J. P. Sweeney and W. L. Hall, J. Assoc. Offic. Agr. Chemists 36, 479 (1952).
250 PYRIDOXINE AND RELATED COMPOUNDS
so that the resulting total activity may be in a measurable state; the ma-
terial is fed directly to the assay animals. Errors which are inherent in these
chemical manipulations are thus avoided. Moreover, animal assay measures
available, not ‘total,’ vitamin Be.
There are two general methods which have been employed for the bioas-
say of vitamin Bg; one is based upon the cure or prevention of the acrodynia
that is produced on a vitamin Be-deficient diet, the other upon the increase
in weight when graded doses of vitamin Bg are added to the deficient diet.*°
Often, a combination of these two procedures is used. The rat is the experi-
mental animal most widely used.
1. AcRopYNIA Rat TrEst
The first biological assays of vitamin Bs were based entirely, or for the
most part, upon the cure of rat acrodynia produced by the vitamin Be-de-
ficient diet.*!““7 However, the curative procedure was not generally adopted
because of its many inherent weaknesses: (1) factors other than vitamin
Bg are involved in the production and cure of rat dermatitis;**: °° (2) sever-
ity of dermatitis varies with season;*° (8) interpretations of resulting cures
are dependent largely upon subjective influences; and (4) the acrodynia
-annot be produced consistently in the hands of some investigators.” Un-
doubtedly, much of the earlier work was unsatisfactory because the diets
were not completely free from traces of vitamin Beg; a complete synthetic
diet was not available, thus necessitating the inclusion of natural products,
such as ‘‘eluate’”’ and “‘filtrate’’ factors, in the diet. Supplee e¢ al.,*° on the
other hand, have used the onset of acrodynia as the basis of an assay
method. They devised a basal diet which permitted the development of
acrodynia in practically 100% of their animals in six to eight weeks.
30 C. I. Bliss and P. Gyérgy, zn Vitamin Methods, Vol. II, p. 214. Academic Press,
New York, 1951.
31 P. Gyorgy, Biochem. J. 29, 760 (1935).
32. N. Halliday and H. M. Evans, J. Biol. Chem. 118, 255 (1937).
33 G. Lunde and H. Kringstad, Biochem. J. 32, 708 (1938).
34 H. E. C. Wilson and G. K. Roy, Indian J. Med. Research 25, 879 (1938).
35 H. A. Schneider, J. K. Ascham, B. R. Platz, and H. Steenbock, J. Nutrition 18,
99 (1939).
36 M. K. Dimick and C. B. Schrefler, J. Nutrition 17, 23 (1939).
37. J. Reedman, W. L. Sampson, and K. Unna, Proc. Soc. Exptl. Biol. Med. 48, 112
(1940).
383 T, W. Birch, J. Biol. Chem. 124, 775 (1938).
39 H. Sherman, Vitamins and Hormones 8, 55 (1951).
40 P, Gyorgy, J. Nutrition 16, 69 (1938).
'! Hf. Schneider, H. Steenbock, and B. R. Platz, J. Biol. Chem. 182, 539 (1940).
2W.L. Dann, J. Biol. Chem. 128, XVIII (1989).
‘8G. C. Supplee, R. C. Bender, and O. J. Kahlenberg, J. Nutrition 20, 109 (1940).
VII. ESTIMATION 251
2. Rar Growrn Test
Assay procedures which have utilized the growth response have been
more successful. In spite of its greater lack of specificity, it is preferred
today for the biological assay of vitamin Bg.
In the first biological assay procedures by the rat growth method, the
water-soluble vitamins, other than those that were available in crystalline
form, were supplied in the diet by liver extracts, rice polishing concen-
trates, or other natural products.“**® Clarke and Lechycka” replaced the
natural products in the diets with pure vitamins, except for biotin, and
developed a basal ration which could be used successfully to assay for
vitamin Bs, . The vitamins were fed in solution as separate supplements and
given three times each week during both the depletion and assay periods.
Sarma ef al.*8: #9 have modified and improved the method of Conger and
Elvehjem; theirs is now the biological procedure that is being used most
extensively for the bioassay of vitamin Bs. They sought to develop a basal
ration which would permit the minimum growth in the absence of vitamin
Bg, and the maximum growth with optimal amounts of vitamin Bg . Several
carbohydrates (sucrose, dextrin, glucose) and proteins (casein, blood fibrin,
egg albumin) were tried in various combinations to meet this criterion.
The diet finally selected for the bioassay of vitamin Bs had the following
composition: sucrose 75 g., blood fibrin 18 g., salts IV 4 g., corn oil 3 g.,
thiamine 0.2 mg., riboflavin 0.3 mg., nicotinic acid 2.5 mg., calcium pan-
tothenate 2 mg., 2-methyl-1,4-naphthoquinone 1 mg., inositol 10 mg.;
choline chloride 100 mg., and biotin 0.01 mg., per 100 g. of diet. Halibut
liver oil, diluted 1:2 with corn oil, was fed at a level of 2 drops per week,
with a-tocopherol included at 0.5 mg. per drop.
Male rats were placed on this depletion diet for two weeks after which
they were divided evenly with respect to weight into groups of three and
placed on diets containing different amounts of pyridoxine, ranging from
0 to 150 y per 100 g. of ration in increments of 25 7. They were continued
on their respective diets for four weeks. The weight gain per week was
approximately linear up to 75 y of pyridoxine per 100 g. of diet. Twice
this amount was required for maximum weight gains of over 30 g. per week.
The biological activity of pyridoxine, pyridoxal, and pyridoxamine was
determined for three methods of administration. The components of the
vitamin Bs complex were equally active when fed by medicine dropper as
44 C. E. Edgar, M. M. El Sadr, and T. F. Macrae, Biochem. J. 32, 2200 (1938).
45'T. W. Conger and C. A. Elvehjem, J. Biol. Chem. 138, 555 (1941).
46 T,. M. Henderson, H. A. Waisman, and C. A. Elvehjem, J. Nutrition 21, 589 (1941).
47M. F. Clarke and M. Lechycka, J. Nutrition 26, 571 (1943).
48 P.S. Sarma, E. E. Snell, and C. A. Elvehjem, J. Biol. Chem. 165, 55 (1946).
49P.S. Sarma, E. E. Snell, and C. A. Elvehjem, J. Nutrition 38, 121 (1947).
Dae PYRIDOXINE AND RELATED COMPOUNDS
daily supplements to the ration or when injected interperitoneally. How-
ever, pyridoxal and pyridoxamine were approximately 25 % less active than
pyridoxine when the three compounds were mixed with the ration; this
may be due to the increased destruction or utilization of pyridoxal and
pyridoxamine by intestinal bacteria.
Biological materials to be assayed are mixed in the diet at two levels so
that they supply between 25 and 75 y of vitamin Bg per 100 g. of ration.
Weight gain is recorded weekly; after four weeks, the average weight gain
per week is calculated. This value is then compared to a standard curve.
In most cases, higher results were obtained at the lower level of assay.
The values obtained by this method are somewhat lower than values ob-
tained by the yeast growth method, presumably because of the decreased
activity of pyridoxal and pyridoxamine when mixed in the diet.
‘
3. CuIck GRowtH TEST
Prophylactic*® and curative’? techniques have been employed. In the
prophylactic test, day-old white Leghorn chicks were fed the following
vitamin Be-deficient diet: Cerelose 61 g., blood fibrin 18 g., gelatin 10 g.,
salts IV 5 g., CaHPO,,2H.O 1 g., L-cystine 0.3 g., thiamine hydrochloride
0.3 mg., riboflavin 0.6 mg., calcium pantothenate 2.0 mg., choline chloride
150 mg., nicotinic acid 5.0 mg., biotin 0.02 mg., inositol 100.0 mg., folie
acid 0.125 mg., vitamin D; 0.004 mg., a-tocopherol 0.03 mg., 2-methyl-
1 ,4-naphthoquinone 0.05 mg., and vitamin A 1700 I.U. Chicks on this diet
died in two weeks without gaining any weight. Those chicks receiving
vitamin Bs, either as a standard dose or a test food, were allowed to grow
on their respective diets for three weeks. The response was recorded in
terms of body weight. There is no depletion period in this procedure; the
test dose of vitamin Bg is given at the beginning of the assay. Pyridoxal
and pyridoxamine exhibited lower activity than pyridoxine when fed in the
ration, just as in the rat growth test. Growth response was equal for all
three forms of the Bs complex when the vitamins were fed by dropper or
injected interperitoneally.
In the curative test*® chicks are placed on the basal vitamin Be,-deficient
ration for a six-day depletion period. They are then divided into groups of
seven, of equal average weight, and continued on the depletion diet for an
additional five days. The test substance or standard is given in a single
oral dose to each chick and repeated on the following three alternate days.
Basal diet and water are given ad libitum. The chicks are weighed before
ach dose and on the second day after the last dose is given. The basal
vitamin B,-deficient diet used in this test has the following composition:
Cerelose 46.5 g., casein (vitamin free) 25.0 ¢., gelatin 10.0 g., salts IV 5.0 g.,
°oW.H. Ott, Proc. Soc. Exptl. Biol. Med. 61, 125 (1946).
VII. ESTIMATION 253
calcium gluconate 5.0 g., KH2PO, 1.0 ¢., liver extract ‘“L’’ (Wilson and
Co.) 2.0 g., wheat germ oil 4.5 g., 400 D fish liver oil 0.5 g., 1-cystine 0.2
g., choline 0.2 g., inositol 0.1 g., PABA 30.0 mg., thiamine 2.0 mg., ribo-
flavin 2.0 mg., calcium pantothenate 4.0 mg., nicotinic acid 10.0 mg., biotin
0.04 mg., and 2-methyl-1 ,4-naphthoquinone 0.4 mg.
4. Rick Mors Larva Trsr
Sarma®!: ©° observed that the rice moth larva (Corcyra cephalonica St.)
required vitamin Bs for growth, and that growth was proportional to the
amount of vitamin Bs in the diet. An assay procedure based on these ob-
servations was developed. The results obtained with this procedure agreed
well with chemical tests. Pseudopyridoxine (pyridoxal and pyridoxamine)
was equivalent to pyridoxine in activity.
D. MICROBIOLOGICAL METHODS
ESMOND E. SNELL
Vitamin Bs occurs naturally in three unconjugated and several con-
jugated or ‘‘bound” forms. The former are pyridoxal, pyridoxamine, and
pyridoxine. The latter include pyridoxal phosphate and pyridoxamine
phosphate and their combinations with protein, an unidentified conjugate
of pyridoxine present in rice bran concentrate, and possibly additional
compounds. The bound forms show activity in animal assay, but not for
most microorganisms. Hydrolysis to the unconjugated forms must therefore
be carried out preparatory to microbiological assay.**
Pyridoxal and pyridoxamine phosphates are hydrolyzed more rapidly
between pH 1.5 and 2.0 than at lower or higher pH values. Where these
compounds are among the most important bound forms of the vitamin,
low concentrations of acid are superior to higher concentrations for the
liberation of vitamin Bg .*!: °° This situation exists with many, but not all,
natural materials. The procedure of choice for most materials is to heat the
finely divided sample with an excess of 0.055 N H»SO, or HCI at 20 Ib.
pressure in the autoclave for 5 hours. For some materials, higher acid
concentrations are more effective. Preliminary to extensive study of a
single material, therefore, the most effective hydrolytic procedure should
be determined and used.*
Pyridoxal, pyridoxamine, and pyridoxine are equal in activity for animals
51 P.S. Sarma, Indian J. Med. Research 31, 165 (1943).
52 P_S. Sarma, Indian J. Med. Research 32, 117 (1944).
53 1), E. Snell, in Vitamin Methods, Vol. 1, p. 8327. Academic Press, New York, 1950.
54 J. C. Rabinowitz and EB. E. Snell, Ind. Eng. Chem. Anal. Ed.19, 277 (1947).
55S. H. Rubin and J. Scheiner, J. Biol. Chem. 162, 389 (1946).
254 PYRIDOXINE AND RELATED COMPOUNDS
under many conditions®® *7; under others, pyridoxal and pyridoxamine
may show slightly less activity than pyridoxine. The three forms show very
different activities, however, for many microorganisms.°® All lactic acid
bacteria, for example, are unable to utilize pyridoxine for growth; most use
pyridoxal and pyridoxamine, some only pyridoxal.®°® These organisms,
therefore, are not useful for determining the total vitamin Bez content of
natural materials. For many yeasts and molds, however, all three forms
show equal activity;°® consequently, these organisms are most useful for
assay purposes.
Among these latter organisms, assay methods utilizing Neurospora sito-
phila pyridoxineless®*: *8: °° and Saccharomyces carlsbergensis 4228°3, 60-&
have been studied most thoroughly. Both yield excellent results; the latter
procedure, however, is simpler, faster, and more convenient. Growth of the
test organism in the vitamin Be-free medium increases with the vitamin Bg
concentration in the range from 0 to about 0.04 7 per 10 ml. of medium.
Pure vitamin Bg (any of the forms may he used as standard) and samples
to supply the vitamin at several levels within this range are added to indi-
vidual 1 in. by 8 in. tubes containing 4 ml. of medium. Each tube is then
diluted to 9 ml., plugged or otherwise covered, and sterilized by heating in
flowing steam at 100° for 10 minutes. After cooling, | ml. of inoculum
suspension (0.3 mg. of moist yeast) is edded, and the tubes are shaken
mechanically for 16 to 18 hours. Yeast growth is then determined turbidi-
metrically. The responses to various levels of the pure vitamin are plotted
to yield a standard curve, from which the vitamin Bs content of the sample
aliquots can be determined by interpolation. }etails of the procedure have
been treated elsewhere.*: °°
If it is desired to determine pyridoxal plus pyridoxamine, but not pyri-
doxine, Streptococcus faecalis may be used as the assay organism.°*: ®>» 6
Similarly, use of Lactobacillus casei as the assay organism permits the spe-
cific estimation of pyridoxal.®*: © ® Differential assays of this type reveal
that most of the vitamin Bg present in yeast, meats, glandular organs, etc.,
is present as pyridoxal and pyridoxamine, with only traces (if any) of
56 H}. H. Snell and A. N. Rannefeld, J. Biol. Chem. 157, 475 (1945).
57 P.S. Sarma, E. E. Snell, and C. A. Elvehjem, /. Biol. Chem. 165, 55 (1946).
58 J. L. Stokes, A. Larsen, C. R. Woodward, Jr., and J. W. Foster, /. Biol. Chem.
150, 17 (1943).
59 J. L. Stokes, Biol. Symposia 12, 253 (1947).
60 Association of Vitamin Chemists, Methods of Vitamin Assay, 2nd ed., p. 245.
Interscience Publishers, New York, 1951.
61 J,, Atkin, A. 8S. Schultz, W. L. Williams, and C. N. Frey, Ind. Eng. Chem. Anal.
Ed. 15, 141 (1943).
62 J. C. Rabinowitz and E. E. Snell, J. Biol. Chem. 176, 1157 (1948).
63 J. C. Rabinowitz and E. E. Snell, J. Biol. Chem. 169, 631 (1947).
64 J. C. Rabinowitz, N. I. Mondy, and E. E. Snell, J. Biol. Chem. 175, 147 (1948).
IX. OCCURRENCE IN FOODS 255
_ pyridoxine.” In plant tissues, however, all three forms of the vitamin occur
in similar amounts. The significance of this diiference in distribution is not
yet known.
VIII. Standardization of Activity
HENRY SHERMAN
When vitamin Bg was first discovered it was defined as ‘that part of the
vitamin B complex that is responsible for the cure of the specific dermatitis
developed by young rats on a vitamin B-free diet supplemented with puri-
fied thiamine and lactoflavin.’’!: ? The standardization of materials possess-
ing vitamin Bg activity was based upon the curative effect they possessed
against this specific dermatitis or rat acrodynia. Gy6érgy* defined the pro-
visional unit for the estimation of vitamin Bes or the ‘‘rat-day dose” as
“the minimum quantity of the substance that would cause healing of the
specific dermatitis.”” The earlier concentrates possessing vitamin Bg activity
were adsorbates or eluates prepared from yeast or bran extracts. One milli-
liter of a so-called Peters’ eluate! possessed one ‘‘rat-day dose’’ of vita-
min Bs A
When pyridoxine was isolated as the active principle and was synthe-
sized, it became the reference standard; the pyridoxine content of natural
materials, as measured by biological assay, was expressed in equivalent
amounts of the compound. One ‘“‘rat-day dose” of vitamin Bs became
equivalent to approximately 10 y of pyridoxine. Pyridoxine is available
commercially as the hydrochloride; it is a white crystalline powder which
is remarkably stable. A U.S.P. reference standard of pyridoxine hydro-
chloride is available for all assay procedures, chemical, physical, micro-
biological, and biological.
IX. Occurrence in Foods
ESMOND E. SNELL and CHARLES 8S. KEEVIL, JR.
Most analytical figures for the vitamin Bs content of foods are not satis-
factory, primarily because (1) several forms of the vitamin occur naturally
1P. Gyorgy, Nature 133, 498 (1934).
2T. W. Birch and P. Gyérgy, Biochem. J. 30, 304 (1936).
3 P. Gyérgy, Proc. Soc. Expil. Biol. Med. 35, 204 (1936).
4H. W. Kinnersley, J. R. O’Brien, R. A. Peters, and V. Reader, Biochem. J. 27,
225 (1933).
256 PYRIDOXINE AND RELATED COMPOUNDS
that have different activities for different test organisms, and occur in
different ratios in different foodstuffs; (2) the bound forms of these vita-
mins are inactive for many microorganisms and show unusual hydrolytic
behavior, in that they are more rapidly hydrolyzed by low acid concentra-
tions than by high; (3) these facts were not known in earlier studies dealing
with chemical and biological estimation of vitamin B,, and have been
insufficiently considered in many later studies.
Saccharomyces carlsbergensis and Neurospora sitophila respond equally to
each of the three forms of vitamin Bs and, when used with a satisfactory
hydrolytic procedure, should give reliable estimates for the vitamin Bg
content of a sample. Values obtained with these organisms and by rat assay
are listed as “preferred values” in the tables below. The hydrolytic proce-
dure used for liberating the vitamin is indicated by a letter (a—m) in paren-
theses; of these procedures, a and h appear most satisfactory at present.
Figures obtained by rat assay, although listed with the ‘‘preferred
values,”’ are nonetheless subject to considerable error, since on some but
not all rations pyridoxal and pyridoxamine are somewhat less active than
pyridoxine in supporting growth.
The various hydrolytic procedures used for liberating vitamin Bg are
listed in summary form below:
Extractant Procedure
(a) 0.055 N H2SO, , 180 ml./g. finely di- Autoclave at 15 lb. (120°) for 1 hr.
vided sample
(b) 1 N HCI, 40 ml./1-5 g. Autoclave at 15 lb. for 1 hr.
(c) 2 N H28O, , 1:200 dilution Autoclave at 15 lb. for 30 min.
(d) 1 N H.SO, , 50 ml./g. Autoclave at 15 lb. for 30 min.
(e) 20 mg. of papain and of takadiastase Incubate at 37° for 24 hr.
per gram of sample
(f) 2N HCl, 10 ml./g. Autoclave at 20 lb. for 5 hr.
(g) NaOH pretreatment of sample followed by HCI hydrolysis as in (b)
(h) 0.055 N HCl, 180 ml./g. Autoclave at 20 Ib. for 5 hr.
(i) 0.1 N H.SO, at 80° for 30 min.; pepsin digestion at 38° for 24 hr.
(j) No hydrolysis; unknown finely divided, shaken with adsorbent prior to chemical
assay
(k) 0.04 N H.SO,, Autoclave at 15 lb. for 1 hr., then 70 ml. papain-takadiastase
as in (e)
(1) 4.N HCl 100° for 1 hr.
(m) 2% CH;COOH 100° for 2 min.
Table II gives values for the vitamin Bg content of several foodstuffs that
have been assayed repeatedly by different procedures; an idea of the mag-
nitude of the variation observed (due to variations both in procedure and
in the samples themselves) may be gained from it.
In Table III, the vitamin Bs content of a variety of foods, together with
the assay organism and extraction procedure, is summarized. In all cases,
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258 PYRIDOXINE AND RELATED COMPOUNDS
such figures represent the sum of the pyridoxine, pyridoxal, and pyri-
doxamine contents.
In Table IV, results of the only study so far made to determine the dis-
tribution of the individual forms of the vitamin are recorded. The values
for pyridoxine, which usually occurs in the smallest amount, are subject
to by far the largest error. The fallacy in referring to the “pyridoxine”
content of foodstuffs, rather than to their vitamin Bs content, 1s evident
from this table.
These data show that vitamin Bg is widely distributed, and much more
uniformly so than most of the other vitamins. Muscle meats, liver, vege-
tables, whole grain cereals, and especially the bran from cereal grains are
among the best sources; few materials can be classed as really poor sources.
This wide distribution undoubtedly is a reflection of the important and
multiple roles played by the vitamin in anabolic and catabolic reactions
of the amino acids and proteins and probably explains the fact that natu-
rally occurring deficiency diseases due to lack of this vitamin have not so
far been found.
1R. H. Hopkins and R. J. Pennington, Biochem. J. 41, 110 (1947).
2 J. L. Stokes, A. Larsen, C. R. Woodward, Jr., and J. W. Foster, J. Biol. Chem.
150, 17 (1943).
3K. B. Wright, Biochem. J. 39, x (1945).
4P.F. Sharp, J. B. Shields, and A. P. Stewart, Jr., Inst. Food Technol. 6, 54 (1945).
5 J. M. McIntire, B. 8. Schweigert, and C. A. Elvehjem, J. Nutrition 28, 219 (1944).
6 V. H. Cheldelin and R. J. Williams, Univ. Texas Publ. 4237, 105 (1942).
7L. M. Henderson, H. A. Waisman, and C. A. Elvehjem, J. Nutrition 21, 589 (1941).
8 L. Atkin, A. S. Schultz, W. L. Williams, and C. N. Frey, Ind. Eng. Chem. Anal.
Ed. 15, 141 (1943).
9P.S. Sarma, E. E. Snell, and C. A. Elvehjem, J. Nutrition 33, 121 (1947).
10 A. F. Bina, J. M. Thomas, and E. B. Brown, J. Biol. Chem. 148, 111 (1943).
11). B. Brown, A. F. Bina, and J. M. Thomas, J. Biol. Chem. 158, 455, (1945).
12 T,, Siegel, D. Melnick, and B. L. Oser, J. Biol. Chem. 149, 361 (1943).
13 M. Swaminathan, Indian J. Med. Research 28, 427 (1940).
14 J. M. Van Lanen, F. W. Tanner, Jr., and 8. E. Pfeiffer, Cereal Chem. 28, 428 (1946).
15 J. C. Rabinowitz and E. E. Snell, Ind. Eng. Chem. Anal. Ed. 19, 277 (1947).
146A. Z. Hodson, J. Nutrition 27, 415 (1944).
17R. J. Williams, V. H. Cheldelin, and H. K. Mitchell, Univ. Texas Publ. 4237, 97
(1942).
18 §. Morris, G. Herwig, and A. Jones, Analyst 74, 37 (1949).
19P,. R. Burkholder, Science 98, 188 (1943).
20 P. R. Burkholder and I. MeVeigh, Plant Physiol. 20, 301 (1945).
21 M. Hochberg, D. Melnick, and B. L. Oser, J. Biol. Chem. 155, 109 (1944).
22, A.M. Copping, Biochem. J. 37, 12, (1943).
237,. J. Teply, F. M. Strong, and C. A. Elvehjem, J. Nutrition 24, 167 (1942).
24 J.C. Rabinowitz and E. E. Snell, J. Biol. Chem. 169, 631 (1947).
25 P, Burkholder, Science 97, 562 (1943).
26 H. K. Mitchell and E. R. Isbell, Univ. Texas Publ. 4237, 37 (1942).
27 R. J. Williams, V. H. Cheldelin, and H. K. Mitchell, Univ. Texas Publ. 4237, 97
(1942).
2 are? ae — —
IX. OCCURRENCE IN FOODS 259
TABLE III
Tre ViramMiIn Bs CONTENT OF Various FoopstTurrs
Product ‘Vitamin Be content, y/g.* sr of i aac values, va
Dairy Products
Cheese 0.98 (S. cer.) (e)§ a
Egg, hens, dried 2.3 (S. cer.) (c),!2 0.85 (S. cer.) (e)§ —
Egg, hens, fresh 0.48 (S. cer.) (c),!2 0.22 (S. cer.) (e)& —
Milk, cow’s, evaporated 0.62 (S. carls.) (a)8 0.62
Milk, cow’s, dry skim 5.5 (S. carls.) (a)§ 5.5
Milk, cow’s, dry whole See Table II 3.3-8.2
Milk, cow’s, whole See Table IT 0.54-1.1
Milk, goat’s 0.13 y/ml. (S. cer.) (e)27 =
Milk, mare’s 0.01 y/ml. (S. cer.) (e)27 -=
Milk, human 0.08 y/ml. (S. cer.) (e)?7 —
Fish Products
Atlantic mackerel, canned, 2.1 (S. carls.) (a)?8 Pel
: wet solids
‘ Atlantic sardines, canned, 1.6 (S. carls.) (a)?8 1.6
wet solids
; _ Cod, fresh 3.4 (rat)? 3.4
Flounder, fresh 1.0 (S. cer.) -(c)” —
Halibut, fresh 1.1 (S. cer.) (e)® —
Pacific mackerel, canned, 2.7 (S. carls.) (a)*8 DBF)
wet solids
Pacific sardines, canned, 2.8 (S. carls.) (a)*8 2.8
wet solids
Salmon, canned, wet solids 4.5 (S. carls.) (a)?8 4.5
Salmon, fresh 5.9 (rat),”7 0.33 (S. cer.) (e)§ 5.9
Tuna, canned, wet solids 4.4 (S. carls.) (a)*8 4.4
Fruit and Fruit Products
_ Apple, fresh 0.26 (S. cer.) (e)§ _-
_ Banana, fresh 3.2 (S. cer.) (e)® —
Cantaloupe, fresh 0.36 (S. cer.) (e)® —
_ Grapefruit juice, canned 0.08-0.18 y/ml. (S. carls.) (a)?9 0.08-0.18 y/ml.
0.14 (S. carls.) (a)?°
- Grapefruit juice, fresh 0.08-0.18 y/ml. (S. carls.) (a)? 0.08-0.18 y/ml.
Grapefruit sections 0.17-0.24 (S. carls.) (a), 29 0.09 0.17-0.24
; (S. cer.) (e)§
Lemon juice, fresh 0.35 y/ml. (S. carls.) (a)8 0.35 y/ml.
Orange juice, canned 0.16-0.31 y/ml. (S. carls.) (a)?9 0.16-0.31 y/ml.
_ Orange juice, fresh 0.18-0.32 y/ml. (S. carls.) (a)?9 0.18-0.56 y/ml.
. 0.56 y/ml. (S. carls.) (a)§
Orange sections, Valencia 0.31 (S. carls.) (a)?® 0.31
Peaches, canned, wet solids 0.16 (S. carls.) (a)3° 0.16
Peaches, fresh 0.16 (S. cer.) (e)® --
Raisins 0.94 (S. cer.) (e)® -
4
TABLE IlI—Continued
Product
Vitamin Be¢ content, y/g.*
Range of preferred values, y/g.
Strawberries, fresh
Tangerine juice, canned
Tangerine juice, fresh
Watermelon, fresh
Corn
Corn, canned, wet solids
Corn, golden bantam, dry
Corn grits
Corn meal, white
Corn meal, yellow!
Corn steep liquor
Corn, whole yellow
RIcE
Rice, finished
Rice paddy
Rice, raw, husked
Rice, raw, milled
Rice, whole
WHEAT
Wheat bran
Wheat flour, patent
Wheat germ
Wheat germ extract
Wheat, ground seed
Wheat meal
White bread
White flour
Whole wheat bread
MIscELLANEOUS
Barley, malt
Barley, ground seed
Buckwheat, ground seed
Cottonseed meal
Millet
Oats, hulled
Oats, rolled
Rye, ground seed
Sorghum
~ Beef brain, fresh
Beef brain, dry
Beef heart, fresh
Beef heart, dry
0.44 (S. cer.) (e)®
0.33 y/ml. (S. carls.) (a)?9
0.23 y/ml. (S. carls.) (a)?9
0.33 GS. cer.) (e)®
Grain Products
0.68 (S. carls.) (a)3°
7.6 (GS. cer.) (e)3!
2.0 (rat)?
0.67 (S. cer.) (e)§
0
0
.30 y/ml.
.23 y/ml.
3.8 (S. cer.) (c) 5.6 (N. sito.) (g)8 —
9.8 (N. sito.) (b)
See Table IT
3.4
8.1 (N. sito.) (b)*
6.9 (chem.) (i)}8
3.3 (chem.) (i)#
10.3 (chem.) (m)*
See Table II
0.9 (S. cer.) (c)?
See Table II
49.0 (chem.) (j)*4
Dol (Se cen.) (eC). Sad UNesotos) (bo) 2
See Table II
1.0 (S. carls.) (a),8 0.06 (S. cer.)
(e)®
See Table II
4.2 (S. carls.) (a),8 0.58 (S. cer.)
(e)®
11.9 (chem.) (k),! 12.8 (chem.)
(ky
See Table II
30) Ni s2to.)) b)2
TSlNGSe cen.) a(@) 22
3.3 (N. sito.) (b)?
0.92 (S. carls.) (a),3° 0.3 (S. ovt-
formis) (c)4
0.93 (S. carls.) (a),3° 1.5 (S. carls.)
(h),9 2.5 (rat)?
3.0 0S. canls.)x(h)523..7 (Ne seto:)
(b)?
2.1-8.6 (S. carls.) (a)3®
Meat Products
1.6 (S. carls.) (a)®
4.3 (rat)?
See Table II
5.1 (S.. cer.) (e),® 9:2) Gat)?
(N. sito.) (b),32 4.5 (chem.) (m)%
9
3
3
8
8
'G-o8n
4
“al
13
oo
.8-15.7
260
TABLE III—Continued
Product Vitamin Be content, y/g.* Range of preferred values, y/g.
Beef kidney, fresh 3.9 (S. carls.) (a),58.4 (S. carls.) 3.5-9.9
(a),° 9.9 (S. carls.) (h),!5 3.5
(rat)?
Beef kidney, dry 16.5 (rat)? 16.5
Beef liver, fresh See Table II 6.0-7.1
Beef liver, dry See Table II 20.5
Beef muscle, fresh See Table II 2.3-3.2
Beef muscle, dry See Table II 12.5
Beef tongue, fresh 1.3 (S. carls.) (a),5 1.0 (rat)? 1.0-1.3
Beef tongue, dry 3.5 (rat)? 3.5
Chicken, dark meat, fresh <2.0 (rat)? <0
Chicken, breast, fresh 1.3 (S. cer.) (e)® =
Horse muscle, dry 9.8-10.4 (N. sito.) (b)3? 9.8-10.4
Frankfurters 1.3 (S. carls.) (a) 1.3
Lamb liver, fresh 3.0 (rat)? 3.0
Lamb liver, dry 10.3 (rat)? 10.3
Lamb muscle, fresh See Table II 2.5-3.7
Lamb muscle, dry See Table II 9.3-9.7
Lamb stew meat, fresh 2.3 (S. carls.) (a)5 203
Lamb stew meat, dry 6.4 (S. carls.) (a) 6.4
Pork, bacon 2.0 (S. cer.) (e)® —
Pork, heart, fresh 2.9 (rat)? 2.9
Pork, heart, dry 12.8 (rat)? 12.8
Pork, ham, fresh See Table II 3.3-4.8
Pork, ham, dry 19.0 (rat),7 5.1 (S. cer.) (e)§ 19.0
Pork, kidney, fresh 3.3 (rat)? 3.3
Pork, kidney, dry 14.8 (rat)? 14.8
Pork, liver, fresh See Table II 2.9-5.9
Pork, liver, dry 8.2 (rat)? 8.2
Pork, muscle, fresh See Table IT 3.3-6.8
Pork, muscle, dry 13.0 (rat),”? 5.0 (S. cer.) (e)§ 13.0
Sheep heart, fresh 1.6 (rice moth larva) (i)%8 “=
Sheep kidney, fresh 1.7 (rice moth larva) (i)%8 —
Sheep liver, fresh 6.1 (rice moth larva) (i),*8 13.8 —
(chem.) (i)#8
Sheep muscle, fresh 4.6 (chem.) (i),!3 2.4 (rice moth -~
larva) (i),?8 0.43 (S. cer.) (e)®
Veal leg, fresh 3.7 (S. carls.) (a)5 Beil
Veal leg, dry 14.0 (S. carls.) (a)® 14.0
Veal liver, fresh 3.0 (rat)? 3.0
Veal liver, dry 8.4 (rat)? 8.4
Veal muscle, fresh 2.8 (N. sito.) (b),? 2.9 (rat)? 2.8, 2.9
Veal muscle, dry 14.5 (rat)? 14.5
Veal shoulder, fresh 3.0 (S. carls.) (a)5 3.0
Veal shoulder, dry 11.0 (S. carls.) (a)5 11.0
Veal shoulder chop, fresh 3.5 (S. carls.) (a),5 1.3 (S. cer.) (e)§3.5
Veal shoulder chop, dry 12.0 (S. carls.) (a) 12.0
Veal sirloin chop, fresh 4.1 (S. carls.) (a)5 4.1
Veal sirloin chop, dry 14.0 (S. carls.) (a)5 14.0
Veal stew meat, fresh 3.3 (S. carls.) (a)® 3.0
Veal stew meat, dry 12.0 (S. carls.) (a)§ 12.0
261
TABLE I1I—Concluded
Product
Vitamin Be content, y/g.* Range of preferred values, y/g.
Asparagus, green, canned,
wet solids
Beans, French, fresh
Beans, green, canned, wet
solids
Beans, lima, dry
Beets, fresh
Beets greens, fresh
Beet root, fresh
Cabbage, fresh
Cauliflower, fresh
Carrots, canned, wet solids
Carrots, raw
Lettuce, fresh
Mushrooms fresh
Okra, fresh
Onions, fresh
Peas, blackeyed, fresh
Peas, canned, wet solids
Peas, split, dry
Peanuts
Potato, fresh
Spinach, canned, wet solids
Soybeans
Tomato, canned, wet solids
Turnips, fresh
Yams, fresh
Yeast, baker’s, fresh
Yeast, brewer’s, dry
Ale
Beer
Chocolate
Grass, dried (Cerophyl)
Honey
Malt extract
Molasses, blackstrap
Molasses, brown
Sugar, brown
Stout (Mneglish)
Tapioca
Wine, burger
Wine, Tokay
Vegetables
0.30 GS. carls.) (a)39
0.96 (rice moth larva) (i)
0.382 (S. carls.) (a)3°
6.0 GS. cer.) (e)§
0.18 (S. cer.) (e)®
0.37 (S. cer.) (e)®
1.1 (chem.) (i)}8
2.9 (chem.) (i),!3 1.2 (S. cer.) (e),®
2.5 (rice moth larva) (i)38
0.20 (S. cer.) (e)§
0.22 (S. carls.) (a)%°
1.9 (chem.) (i), 2.2 (rice moth
larva) (1),32 1.2 GS. cer.) (e)§
0.71 (S. cer.) (e)®
0.45 (S. cer.) (e)®
0.75 (S. cer.) (e)§
0.63 (S. cer.) (e)§
.9 (S. cer.) (e)®
.46 (S. carls.) (a)3°
.6.(0S. carts.) (a)®, 3.3 (at)?
.0 (S. cer.) (e)®
.6 (chem.) (i),!8 2.5 (rice moth)
(a),82 2.2) GScer.) (e)!
0.6 (S. carls.) (a)3°
See Table II
0.71 (S. carls. )\(a)3°
1.1 (WS. cer.) (e)§
3.2 (S. cer.) (e)®
Yeast Products
1
0
1
3
1
7.0 (S. carls.) (a),86.2 (S. carls) (a)!
See Table II
Miscellanenous
0.5 y/ml. (S. carls.) (c)!
0.5 y/ml. (N. sito.) (b),39 0.60 y/ml.
(S. carls.) (¢)}
0.23 (S. cer.) (e)®
9.2 (S. carls.) (a),° 5.3 (rat), 12.3
GS. carls.) (f)?4
0.04-0.27 (S. carls.) (a) ,49 2.44.8
(chem.)*!
5.4 (N. sito.) (b)?
20.0 (chem.) (1) ,2! 24.9 (S. cer.) (e)??
2.7 (S. cer.) (e)®
0.7 (GS. cer.) (c)*
0.87 (S. carls.) (c)}
3.2 (rice moth larva) (i)38
0.66-2.0 (rat)?
0.72-2.14 (rat)?
40 .0-57.0
0.04-0: 27
5.4
0.87
0.66-2.0
0.70-2.1
* Abbreviations to the assay procedures are as follow: S. cer., Saccharomyces cerevisiae; S. carls., Sac”
charomyces carlsbergensis; N. sito., Neurospora sitophila pyridoxineless; chem., chemical. Hydrolytie pro-
cedures are keyed to the list in the text by the letters in parentheses; references are indicated by super’
seript numbers.
i
IX. OCCURRENCE IN FOODS 263
TABLE IV
PYRIDOXAL, PYRIDOXAMINE, AND PYRIDOXINE CONTENT
oF SeEvERAL Natura MATERIALS*
y/g. of Dry weight
Product Pyridoxal Pyridoxamine Pyridoxinet Total Be
Beef liver, fresh 5.8 + 0.58 | 21.6 + 2.8 —2.5 + 3.3 | 25.0
Celery, fresh 6.4 + 0.7 1.7 + 0.7 (ee les colada
Carrot, fresh Let + 0.2 0.2 + 0.2 5.8 + 0.8 oll
Chicken heart, fresh 6.4/2: 0.7 8.4 + 1.4 —1 + 2 13.8
Chicken liver, fresh 31.8 + 3.3 | 32 + 5.6 0 + 8 63.3
Corn meal, yellow 1.2 + 0.8 0.7 +:0;2 0 + 0.3 1.9
Egg white 0.16 + 0.02 | 0.09 + 0.02 0.8 + 0.07 | 0.55
Egg, whole 4.6 + 0.5 0.84 + 0.5 0 + 0.9 5.4
Egg yolk 9.0 +0.8 2.8 + 1.4 0 + 2 11.8
Fish, frozen 8.2 +£0.8 | 15.3 + 2.1 —1 + 3 22.5
Lemon, whole 2.5 + 0.25); 0.5 +0.3 8.3 +1.6 | 11.3
Lettuce, leaf 10.0 +0.8 |-1 + 2 7.4 41.6 | 16.4
Liver powder, Wilson 1:20} 5.0 + 0.5 | 21.6 + 2.8 12.0 + 4.1 | 38.6
Milk, whole, fresh, y/ml. | 0.26 + 0.02} 0.06 + 0.06 | —0.02 + 0.05] 0.30
Peas, split 0.40 + 0.04} 0.3 + 0.01 1.1 + 0.2 1.8
Pepper, green 6.8 = 0.70 | 37.0 + 4.2 16 + 6.6 | 60.0
Rat kidney 27.2 + 2.5 9.1 + 3.5 8.3 + 4.9 | 44.6
Rat liver 24.0 + 2.5 6.3 + 2.8 7.4 + 4.1 | 37.7
Wheat germ 0.72 + 0.08; 0.5 + 0.01 2.5 + 0.6 3.7
Wheat, whole 1.6 +0.16|] 26 +0.3 TA OS NG 8
Yeast, baker’s, fresh 6.2 + 0.7 insets lee 0 + 2 13.9
Yeast, brewer’s, dry 4.0 + 0.4 | 25 + 2.8 —l + 4 28.0
* From Rabinowitz and Snell.4* Hydrolytie procedure h was employed in all cases.
Tt Caleulated [total Bs-(pyridoxal + pyridoxamine)]. These values are very inexact, since they bear
the cumulative errors of the three values from which they are calculated.
23 J. B. Neilands, F. M. Strong, and C. A. Elvehjem, J. Nutrition 34, 633 (1947).
29 W. A. Krehl and G. R. Cowgill, Food Research 15, 179 (1950).
30 M. Ives, A. E. Pollard, C. A. Elvehjem, and F. M. Strong, J. Nutrition 31, 347
(1946).
31. V. H. Cheldelin and R. L. Lane, Proc. Soc. Exptl. Biol. Med. 64, 53 (1943).
32S. A. Price, Cereal Chem. 23, 318 (1946).
33 V. R. Williams, W. E. Knox, and E. A. Fieger, Cereal Chem. 20, 560 (1943).
34Q. D. Bird, J. M. Vandenbelt, and A. D. Emmett, J. Biol. Chem. 142, 317 (1942).
35 J. M. Cooperman and C. A. Elvehjem, J. Nutrition 27, 329 (1944).
36 F. W. Tanner, Jr., S. E. Pfeiffer, and J. J. Curtis, Cereal Chem. 24, 268 (1947).
37 F. D. Carroll, J. Animal Sci. 9, 139 (1950).
33 P. Sarma, Indian J. Med. Research 32, 117 (1944).
39 B. S. Schweigert, H. E. Sauberlich, C. A. Elvehjem, and C. A. Baumann, J. Biol.
Chem. 165, 187 (1946).
40 G. Kitzes, H. A. Schuette, and C. A. Elvehjem, J. Nutrition 26, 241 (1943).
41M. H. Haydak, L. S. Palmer, M. C. Tanquary, and A. E. Vivino, J. Nutrition 23,
581 (1942).
2 L,.. Perlman and A. F. Morgan, Mood Research 10, 334 (1945).
43 J. C. Rabinowitz and E. E. Snell, J. Biol. Chem. 176, 1157 (1948).
264 PYRIDOXINE AND RELATED COMPOUNDS
X. Effects of Deficiency
AM INSP ANTS
HENRY SHERMAN
The intact green plant is generally able to synthesize adequate amounts
of the B vitamins so that it 1s independent of any external source for these
vitamins.! Most species of the higher plants can grow in the light in media
containing only inorganic salts. At times, however, an external source of
the B vitamins, especially thiamine, niacin, and pyridoxine, may accelerate
growth.
Individual parts of the plant, however, differ widely in their ability to
synthesize these vitamins; those plant tissues which do not synthesize the
vitamins at an adequate rate usually obtain their supply from those parts
that do. Thiamine, niacin, and pyridoxine function as hormones in plants;
they are synthesized in one part of the plant—the leaf—and are trans-
ported, usually in the phloem, to those sections where they are needed.?: ’
Isolated roots of higher plants require thiamine, niacin, and pyridoxine
for growth.! Tomato root tips have been maintained on a solution of min-
eral salts, cane sugar, thiamine, and pyridoxine through twenty-five suc-
cessive passages.t They have also been transferred from liquid media to
agar cultures but only in the presence of pyridoxine.® Isolated roots from
carrot, sunflower, acacia, and Jimson weed also require pyridoxine for
erowth.!
In the intact plant these essential vitamins are synthesized in the leaves
and translocated to the roots. When isolated roots are cultured in media
which are deficient in the above B vitamins, there is evidence of reduced
growth in length. Pyridoxine and thiamine deficiency also result in lowered
cell division activity in the root meristem.!
There is also evidence that thiamine and pyridoxine are also involved in
the process of root growth in cuttings.!
Fries® investigated the role of pyridoxine, thiamine, and biotin in pro-
moting the growth of certain ascomycetes. Pyridoxine was the only vitamin
that was required by all the species investigated. In fact, one species,
Ophiostoma multiannulatum, was used with some success in determining
the pyridoxine content of various malt extracts.
1 J. Bonner and H. Bonner, Vitamins and Hormones 6, 225 (1948).
2 J. Bonner, Am. J. Botany 29, 1386 (1942).
3h, W. Went, Am. Scientist 31, 189 (1943).
4W.J.-Robbins and M. B. Schmidt, Proc. Natl. Acad. Sci. (U. S.) 25, 1 (1939).
5D. Day, Science 94, 468 (1941).
6 N. Fries, Symbolae Botan. Upsaliensis 7, 73 (1943).
X. EFFECTS OF DEFICIENCY 265
B. IN ANIMALS
HENRY SHERMAN
It is comparatively simple to induce vitamin Bg deficiency symptoms in
animals; deficient synthetic diets and/or pyridoxine antagonists, such as
desoxypyridoxine, have been employed for this purpose. A description of
the deficiency varies with the species of animal, but there are a few general
symptoms which appear to be common to most animals. The differences
among the animals appear to be in degree rather than in kind. In general,
vitamin Bg deficiency in animals induces retarded growth, several types of
anemia, epileptiform fits, and characteristic lesions of the skin. The vitamin
complex appears, therefore, to play an important role in blood, nerve, and
skin metabolism.
1. Rats
Poor weight gain is one of the earliest signs of a vitamin Bg deficiency;
this is often apparent within five days.7:§ Accompanying this failure in
growth are usually a decrease in appetite and a marked interference in
efficiency of food utilization.”: ° With this poor growth there is a reduction
in the size of the accessory organs of reproduction and decreased sexual
behavior.!° Simple omission of the vitamin has only a slightly adverse
effect on reproduction." However, the administration of desoxypyridoxine
in the deficiency diet induces a high incidence of resorptions. The growth
of suckling young born from vitamin B,-deficient mothers is retarded
early in the lactation period, and survival to weaning is very rare.
Vitamin B, avitaminosis induces a symmetrical dermatitis or acrodynia
in the peripheral areas of the body such as the tail, paws, nose, mouth, and
ears.U3, 12,18 Very often, this acrodynia is accompanied by an edema of the
corium and a sealiness of the extremities.'* This pathological condition was,
in fact, used as the basis of the first biological assay procedure. There have
been reports, however, that the acrodynia cannot be produced consistently
and that the vitamin only modifies some of the dermal lesions. !® The
7H. Sherman, Proc. Nutrition Symposium Columbia Univ. School of Public Health’
p. 15 (1947).
8H. Sherman, L. M. Campling, and R. 8. Harris, Federation Proc. 9, 371 (1950).
9B. Sure and L. Easterling, J. Nutrition 39, 393 (1946).
10 G. A. Emerson and H. M. Evans, Am. J. Physiol. 129, 352 (1940).
11M. M. Nelson and H. M. Evans, J. Nutrition 43, 281 (1951).
lla F. E. Snell and A. N. Rannefeld, J. Biol. Chem. 157, 475 (1945).
2T. W. Birch, P. Gyérgy, and L. J. Harris, Biochem. J. 29, 2830 (1935).
13 P. Gyorgy and R. E. Eckhardt, Nature 144, 512 (1939).
4 W. Antopol and K. Unna, Proc. Soc. Exptl. Biol. Med. 42, 126 (1939).
16 W.L. Dann, J. Biol. Chem. 128, XVIII (1939).
266 PYRIDOXINE AND RELATED COMPOUNDS
essential fatty acids, linoleic and arachidonic acids, are closely interrelated
with vitamin Bes in the etiology of these skin lesions.
The kidneys and adrenal glands are also adversely affected by a vitamin
Bg deficiency. Very early in the deficiency, the rat develops slight lesions
in the glomerulus, which eventually result in scarring and pitting.®* The
adrenal glands enlarge during the deficiency, especially the zona fascicu-
lata; this leads to an impairment of function which is generally charac-
terized by a decreased rate of water excretion.!>: 1%
Although anemia is not a regular symptom of vitamin Be deficiency
in the rat, there are other indications that vitamin Bg is involved in main-
taining the integrity of the blood system. The regeneration of red blood
cells after hemorrhage is delayed and inadequate in vitamin B,-deficient
rats.16 16a, 16b Deficient rats show a microcytosis, an increase in red blood
cell count, reduction in mean corpuscular diameter, granulocytosis, lym-
phopenia, and many normoblasts.!7 There is a marked reduction in the
ability of the deficient rat to form circulating antibodies to foreign erythro-
cytes:!7!9 this may be due to atrophy of the thymus in these deficient
rats.2° 2! Total body iron and copper are significantly increased in vita-
min Bg deficiency, suggesting an increase in their absorption.”
Vitamin Bg deficiency invariably leads to nervous disorders. Chick et al.
described the appearance of spontaneous convulsive seizures in rats main-
tained for long periods of time on diets deficient in vitamin Bs. These
seizures were characterized by hyperexcitability, circular running, tonic-
clonic convulsions, and a comatose recovery period. These observations
were confirmed by Lepkovsky ef al.24 Daniel et al.?° described similar
seizures in young rats being nursed by mothers maintained on a vitamin
Be-deficient diet. Patton et al.®> observed that spontaneous convulsive
seizures developed in young rats being suckled by mothers on a deficiency
15a T, R. C. Agnew, J. Pathol. Bacteriol. 68, 699 (1951).
1b R. B. Stebbins, Am. J. Physiol. 166, 538 (1951).
ise R. B. Stebbins, Endocrinology 49, 25 (1951).
16 A. Kornberg, H. Tabor, and W. H. Sebrell, Am. J. Physiol. 148, 434 (1945).
16. W. W. Hawkins, B. Lechow, and M. K. Evans, Am. J. Physiol. 170, 155 (1952).
16b W. W. Hawkins and B. Lechow, Rev. can. biol. 11, 65 (1952).
7K. J. Carpenter and E. Kodicek, Brit. J. Nutrition 2, IX (1948).
18 H. C. Stoerk and H. N. Eisen, Proc. Soc. Exptl. Biol. Med. 62, 88 (1946).
19 P. P. Ludovici, A. EK. Axelrod, and B. B. Carter, Proc. Soc. Exptl. Biol. Med. 76,
665 (1951).
20K. H. Busing, Intern. Z. Vitaminforsch., 22, 313 (1950).
21H. C. Stoerk, Proc. Soc. Exptl. Biol. Med. 62, 90 (1946).
22. C, J. Gubler, G. E. Cartwright, and M. M. Wintrobe, J. Biol. Chem. 178, 989 (1949).
23 H. Chick, M. M. El Sadr, and A. N. Worden, Biochem. J. 34, 595 (1940).
24S. Lepkovsky, M. E. Krause, and M. K. Dimick, Science 95, 331 (1942).
25 KH. P. Daniel, O. L. Kline, and C. D. Tolle, J. Nutrition 28, 205 (1942).
26 R. A. Patton, H. W. Karn, and H. E. Longenecker, J. Biol. Chem. 152, 181 (1944).
X. EFFECTS OF DEFICIENCY 267
diet toward the end of the lactation period. These symptoms could be
alleviated by the administration of 10 y of pyridoxine per day, but even
50 y of pyridoxine per day could not protect healthy rats against similar
convulsive seizures induced by sound. The inclusion of 25 to 150 y of
pyridoxine per day in the diets of rats protected them against spontaneous
seizures but not against sound-induced seizures; with higher levels of
pyridoxine the seizures were both delayed and less severe. Davenport and
Davenport”? demonstrated that the pyridoxine-deficient rat showed in-
creased brain excitability measured by the decrease in electroshock thresh-
old. Administration of pyridoxine and/or glutamic acid increased the
threshold, whereas, tryptophan, which intensifies the Bg deficiency, reduced
it. These observations suggest that maintenance of the transaminase sys-
tem is essential for normal brain function.
Vitamin B,-deficient rats often exhibit muscular dystrophy, especially
in the cardiac region.”®: 2°
Since vitamin B, is intimately involved in the decarboxylase*’ and trans-
aminase*! enzyme systems, it is not uncommon to observe disturbances in
protein metabolism in vitamin B, deficiency. Acrodynia is more severe in
pyridoxine-deficient rats fed a high protein diet than in rats fed a low
protein diet.*? Peretti*®: *4 observed that B, deficiency had no effect on
nitrogen balance or nitrogen metabolism, but Hawkins ef al.*® could in-
crease the fasting blood levels of urea and non-protein nitrogen by feeding
pyridoxine-deficient rats on a high protein diet. Beaton et al.*** also demon-
strated a significant increase in the fasting level of blood urea, which,
they claimed, was the result of a true metabolic disturbance and not renal
failure. The avitaminotic rats also exhibited an increased urinary nitrogen
excretion, suggesting an impairment in ability to utilize dietary nitrogen.
Terroine*® depleted young male rats of vitamin Bs on a high protein diet;
when they were then placed on a protein-free diet, the normal rats lost
weight more rapidly than did the deficient animals. Total urinary urea,
ammonia, and amino nitrogen were higher in deficient than in normal rats.
27, V. D. Davenport and H. W. Davenport, J. Nutrition 36, 263 (1948).
28 W. Antopol and C. E. Schotland, J. Am. Med. Assoc. 114, 1058 (1940).
29 R. M. Thomas, E. Mylon, and M. C. Winternitz, Yale J. Biol. Med. 12, 345 (1940).
30 E. F. Gale, Advances in Enzymol. 6, 1 (1946).
31 F. Schlenk and E. E. Snell, J. Biol. Chem. 157, 425 (1945).
32 T,. R. Cerecedo and J. R. Foy, Arch. Biochem. 5, 207 (1944).
33 G. Peretti, Boll. soc. ital. biol. sper. 16, 396 (1941).
34G. Peretti, Boll. soc. ital. biol. sper. 17, 324 (1942).
35 W. W. Hawkins, M. L. MacFarland, and E. W. McHenry, J. Biol. Chem. 166, 223
(1946).
- 35a J. R. Beaton, J. L. Beare, J. M. White, and E. W. McHenry, J. Biol. Chem. 200,
715 (1953).
86 T. Terroine, Arch. sci. physiol. 4, 91 (1950).
268 PYRIDOXINE AND RELATED COMPOUNDS
The excretion of creatinine was higher in deficient rats than in control rats.
Martin®” showed that the toxicity of L-tyrosine was less in pyridoxine-
deficient animals than in control animals.
A derangement in tryptophan metabolism is very common in vitamin
Bg deficiency. Vitamin Bg-deficient rats, or rats receiving desoxypyridoxine,
excrete more xanthurenic acid in the urine than do normal animals, when
they are fed tryptophan.** Such a derangement in tryptophan metabolism
is one of the earliest symptoms of vitamin B, deficiency in higher animals
and man.*? This increased excretion of urinary xanthurenic acid by the
deficient rat is diminished by niacin supplementation.*°
Sheppard and McHenry“ found that in vitamin Beg-deficient rats the
pyridoxine content of liver, kidney, and leg muscle was independent of
protein intake. With a constant protein intake, the pyridoxine concentra-
tion of liver is proportional to dietary pyridoxine concentration up to 25
y per rat per day; the content of pyrodixine in the liver can also be in-
creased by keeping the dietary pyridoxine content constant and increasing
the protein level. Irradiation does not effect the concentration of vitamin
B, in the liver of normal or deficient rats.*?
Many enzyme systems which are intimately involved in the catabolism
of proteins are affected by a vitamin Bg deficiency. The livers of vitamin
Bg-deficient rats have only one-third the kynureninase activity of livers of
normal rats. “* The heart and kidney-cortex tissues of avitaminotic rats
had only 40 % transaminase activity of similar tissues from control animals;
however, the succinic acid oxidase activity of these deficient tissues was
80 to 90 % the activity of normal tissues.“ The glutamic acid decarboxylase
activity of the brain of the vitamin Be-deficient rat is 50 % less active than
that of a normal brain." The zn vitro transfer of sulfur from homocysteine
to serine is retarded with liver extracts of vitamin B,-deficient rats; the
addition of phosphopyridoxal restores this transsulfurization enzyme sys-
37 G. J. Martin, J. Biol. Chem. 166, 389 (1946).
38§. Lepkovsky, E. Roboz, and A. J. Haagen-Smit, J. Biol. Chem. 149, 195 (1943).
89 T,. D. Greenberg, D. F. Bohr, H. McGrath, and J. F. Rinehart, Arch. Biochem. 21,
237 (1949).
M. Heimberg, F. Rosen, I. G. Leder, and W. A. Perlzweig, Arch. Biochem. 28,
225 (1950).
Kk. C. Sheppard and E. W. McHenry, J. Biol. Chem. 165, 649 (1946).
M. L. MacFarland, M. V. Peters, R. M. Ballantyne, and E. W. McHenry, Am. J.
Physiol. 168, 394 (1950).
48 A. EK. Braushtein, 8. V. Goryachenkova, and T. §. Paskhina, Biokhimiya 14, 163
(1948).
a M. Mason and C. P. Berg, J. Biol. Chem. 195, 515 (1952).
443, R. Ames, P. 8S. Sarma, and C. A. Elvehjem, J. Biol. Chem. 167, 135 (1947).
Ma KH. Roberts, F. Younger, and S. Frankel, J. Biol. Chem. 191, 277 (1951).
t
4
ay
X. EFFECTS OF DEFICIENCY 269
tem to normal.*® These livers show a lower rate of cystathionine cleavage
than normal specimens.***: ®> The cysteine desulfhydrase content of liver
extracts of pyridoxine-deficient rats fed a high protein diet is lower than
that obtained from a normal rat.!® This decrease in desulfhydrase concen-
tration takes place a few days before many of the other signs of Bg avita-
minosis occur. Homogenized kidney from vitamin Be-deficient rats had
only one-third the p-amino acid oxidase activity of normal kidney.” A
supplement of p-amino acids decreased nitrogen utilization by vitamin
B,-deficient animals; L-amino acids had no effect. Pyridoxine-deficient rats
gained less water and protein than did rats receiving a complete diet.
A vitamin Bg deficiency causes abnormal fat metabolism.** The carcasses
of pyridoxine-deficient rats contain less fat, and of a higher degree of unsatura-
tion, than the carcasses of normal rats; they also have a higher percentage
of arachidonic acid.*’ The low linoleic acid content of carcass fat of pyri-
doxine-deficient rats is not a specific characteristic of the deficiency, for
caloric and thiamine deficiencies produce similar changes.®*” It has been
demonstrated that vitamin Bg is essential for the conversion of protein
to fat.°! The rate of absorption of lipids from the intestine does not appear
to be affected by the deficiency.*”: ** The livers of pyridoxine-deficient rats,
however, have more fat than those of normal rats.*!: 4° There is a de-
crease in the rate of oxidation of short-chained fatty acids by liver slices
from pyridoxine-deficient rats.*?
Severe vitamin Bg deficiency depressed the basal metabolic rate and
increased the respiratory quotient of rats.°? Riboflavin deficiency had no
effect on the basal metabolic rate. Vitamin Be, deficiency increases the
45 A. E. Braushtein and E. V. Goryachenkova, Doklady Akad. Nauk S.S.S.R. 74,
529 (1950).
45a}. V. Goryachenkova, Doklady Akad. Nauk S.S.S.R. 85, 603 (1952).
45b F. Binkley, G. M. Christensen and W. N. Jensen, J. Biol. Chem. 194, 109 (1952).
46 A. E. Braushtein and R. M. Azarkh, Doklady Akad. Nauk S.S.S.R. 71, 93 (1950).
47K. L. Armstrong, C. Feldott, and H. A. Lardy, Proc. Soc. Exptl. Biol. Med. 73,
159 (1950).
48 L. Voris and H. P. Moore, J. Nutrition 25, 7 (1943).
49 H. Sherman, Vitamins and Hormones 8, 55 (1951).
50 G. Medes, M. V. Mann, and J. B. Hunter, Arch. Biochem. and Biophys. 32, 70
(1951).
51. W. McHenry and G. Gavin, J. Biol. Chem. 138, 471 (1941).
52 G,. Peretti, Boll. soc. ital. biol. sper. 17, 326 (1942).
53 C, W. Carter and P. J. R. Phizackerley, Biochem. J. 49, 227 (1951).
54N. Halliday, J. Nutrition 16, 285 (1938).
55 R.W. Engel, J. Nutrition 24, 175 (1941).
56 P. G. Tulpule and V. N. Patwardhan, Indian J. Med. Research 38, 3 (1950).
57 —D. Orsini, H. A. Waisman, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 51,
99 (1942).
270 PYRIDOXINE AND RELATED COMPOUNDS
specific dynamic action of amino acids such as glutamic acid.®: °°? It is
believed that vitamin Bs deficiency delays the deamination of amino acids.®°
The adult avitaminotic rat cannot adjust to cold very efficiently.°
Vitamin Beg deficiency decreased the concentration of pantothenic acid
in the liver by 78 %, in the kidney by 28 %, and in muscle by 18 %.* Liver
and kidney niacin concentration decreased 15 % and liver riboflavin content
decreased 22 % in vitamin Beg-deficient rats.
2. Micr
In young mice, a vitamin Beg deficiency induces poor growth and paralysis
of the hind legs but no characteristic dermatitis. In adult mice, the defi-
ciency results in failure to maintain body weight and death within two
months. Acute stages of the deficiency in adult mice are often accompanied
by pathological skin lesions and necrotic tails. De Renzo and Cerecedo®
produced an acrodynia in the mouse by feeding a diet deficient in vitamin
B, and desoxypyridoxine. A diet high in casein or an equivalent amount
of methionine aggravated the skin lesions and shortened survival time.
Boutwell et al.** have also been able to produce acrodynia in mice with low
vitamin Bs containing diets.
There is the same close relationship between vitamin Bs and protein
metabolism in mice as there is in rats. Deficient mice excrete xanthurenic
acid, especially when the diet is high in tryptophan. Pyridoxine-deficient
mice fed diets containing 60 % casein lived only one-third as long as those
fed 10% casein. The tissue reserves of pyridoxine in vitamin Be-deficient
mice diminished more rapidly when the diet contained 50% casein than
when it contained only 10 %.®° The mice on the high protein diet lost more
weight and had a higher mortality rate than those on the low protein diet.
This effect was not due to variation in caloric intake, or to unequal urinary
excretion of pyridoxine, or to the tryptophan content of the diets, according
to these authors. At low levels of vitamin B, intake, less vitamin was stored
on the high protein diet, but with high levels of vitamin Bg intake, a high
87). P. Sadhu, Missouri Agr. Expt. Sta. Research Bull. 408, 3 (1947).
59 TD). P. Sadhu and S. Brody, Am. J. Physiol. 151, 342 (1947).
60 J. R. Beaton, R. M. Ballantyne, R. E. Lau, A. Steckley, and E. W. McHenry,
J. Biol. Chem. 186, 93 (1950)
60a H}. H. Ershoff, Proc. Soc. Exptl. Biol. Med. 78, 385 (1951).
61'T, Terroin and J. Adrian, Arch. sci. physiol. 4, 435 (1950)
62 R. J. Williams, R. E. Eakin, E. Beerstecher, Jr. and W. Shive, 7n The Biochem-
istry of B Vitamins. Reinhold Publishing Corp., New York, 1950.
63 H. C. DeRenzo and L. R. Cerecedo, Proc. Soc. Exptl. Biol. Med. 73, 356 (1950).
63a R. K.-Boutwell, H. P. Rusch, and R. Chiang, Proc. Soc. Exptl. Biol. Med. 77,
860 (1951).
64}. C. Miller and C. A. Baumann, J. Biol. Chem. 157, 551 (1945).
65 B.S. Schweigert, H. EK. Sauberlich, C. A. Elvehjem, and C. A. Baumann, J. Biol.
Chem. 165, 187 (1946).
X. EFFECTS OF DEFICIENCY 271
protein diet favored the storage of the vitamin. Mice on deficient diets often
develop fatty livers.®* This condition appeared more rapidly on a high
casein diet than on a low one. The addition of tryptophan to the deficient
diet hastened fatty liver formation.
Silberberg and Levy®® demonstrated that, in young mice deprived of
pyridoxine, cartilage growth and bone formation were inhibited; a high
protein diet accentuated this effect. Interdental bone growth ceased after
2 weeks, and recessive changes progressed in all periodontal structures.®
The mandibular condyle showed slight narrowing of the cartilage cap
after 1 week.
Young Swiss mice which had been fed a special vitamin Be-deficient diet
and given an injection of desoxypyridoxine developed granulocytosis and
lymphopenia.* In leukemic mice of the Ak strain, the deficiency augmented
the leukemic granulocytosis and shortened the survival time.
There was a marked regression of lymphosarcoma implants in deficient
mice.*? Tumor implants failed to develop in mice deprived of vitamin Bg
prior to the inoculation.
3. HAMSTERS
Routh and Houchin” reported that on a vitamin Be-deficient diet the
Syrian hamster developed an “acrodynia-like’’ dermatitis around the
mouth, lost weight, and died within 24 days.
Shwartzman and Strauss”! have produced a vitamin Bg deficiency in the
male Syrian hamster. It was characterized by arrest of growth, diminished
food and water intake, progressive malnutrition, muscular weakness, and
changes of the fur. Increased amounts of xanthurenic acid were excreted
in the urine. Deficient animals died in 12 to 13 weeks. There were no cu-
taneous changes comparable to the classical ‘‘rat acrodynia.” Epileptiform
seizures did not occur in the pyridoxine-deficient hamster, but there were
other symptoms of nervous disorder, such as an ataxic gait, or paresis of
one extremity, and priapism. Autopsy revealed a loss of fat tissue and
atrophy of lymphoid tissues, especially the thymus. The transamination
rate of heart muscle of pyridoxine-deficient hamsters was 30 to 40 % lower
than that of control animals fed ad libitum or restricted amounts of food;
succinoxidase activity was comparable to that of control animals.”
66 R. Silberberg and B. M. Levy, Proc. Soc. Exptl. Biol. Med. 67, 259 (1948).
87 B. M. Levy, J. Dental Research 29, 349 (1950).
6D. R. Weir, R. W. Heinle, and A. D. Welch, Proc. Soc. Exptl. Biol. Med. 72, 457
(1949).
69 H. C. Stoerk, J. Biol. Chem. 171, 437 (1947).
70 J. I. Routh and O. B. Houchin, Federation Proc. 1, 191 (1942).
7G. Shwartzman and L. Strauss, J. Nutrition 38, 131 (1949).
72 G. Shwartzman and H. Hift, J. Nutrition 44, 575 (1951).
Dil Pe PYRIDOXINE AND RELATED COMPOUNDS
4. Docs
Fouts ed al.” first observed a nutritional microcytic hypochromic anemia
in young dogs receiving a diet that was low in vitamin B,. Adult dogs also
developed this condition when placed on a similar diet.”4 The blood plasma
iron concentration progressively increased as the anemia became more
severe.”° It dropped to a low-normal value with pyridoxine therapy. Total
blood copper values were at a low-normal level during the anemia and
increased to normal during pyridoxine administration. The pyridoxine-
deficient dog also exhibited an increased urinary output of urea, ammonia,
uric acid, and creatinine;** xanthurenic acid was excreted in the urine of
these deficient dogs.7® When desoxypyridoxine was fed to dogs, they ex-
hibited atrophy of spleen, thymus, and lymph nodes; the ratio of spleen to
body weight decreased.” They developed epileptiform convulsions. The
lipoid content of the zona fasciculata and zona reticularis of the adrenal
glands decreased, and the glands became larger than normal. The “rat
acrodynia”’ symptoms did not appear in the adult dog. McKibbin e¢ al.78
have shown that young puppies developed a different deficiency picture.
No deficiency symptoms other than anemia, loss of weight, anorexia, and
death were observed in puppies. Adult dogs kept for 300 days or more on
a diet free of vitamin Bs developed symptoms of cardiac failure including
dyspnea, tachycardia, dilatation and hypertrophy of the right ventricle
and the right auricle, accumulation of serous fluid in the thorax and chronic
passive congestion of the liver; degenerative changes were found in the
myelin sheaths of the peripheral nerves and of the spinal cord.79
Vitamin Be-deficient dogs show a decreased ability to convert protein
into carbohydrate, as indicated by the decrease in the urinary dextrose :ni-
trogen ratio in phlorizinized animals on a high protein diet; no such im-
pairment occurs in thiamine and riboflavin deficiencies.®° Dogs receiving a
vitamin Be-deficient diet and desoxypyridoxine show a sharp fall in the
volume of gastric juice.”!
7 P. J. Fouts, O. M. Helmer, S. Lepkovsky, and T. H. Jukes, J. Nutrition 16, 197
(1938).
™P.J. Fouts, O. M. Helmer, and 8. Lepkovsky, J. Am. Med. Assoc. 199, 163 (1940).
*® J.M. McKibbin, A. E. Schaefer, D. V. Frost, and C. A. Elvehjem, J. Biol. Chem.
142, 77 (1942).
761. W. Axelrod, A. F. Morgan, and 8. Lepkovsky, J. Biol. Chem. 160, 155 (1945).
7 C. W. Mushett, R. B. Stebbins, and M. N. Barton, Trans. N. Y. Acad. Sci. 9, 291
(1947).
*® J. M. McKibbin, R. J. Madden, S. Black, and C. A. Elvehjem, Am. J. Physiol.
128, 102 (1939),
9 H.R. Street, G. R. Cowgill, and H. M. Zimmerman, J. Nutrition 21, 275 (1941).
80 P. D. Bartlett and O. H. Gaebler, J. Nutrition 87, 93 (1949).
51 R. A. Forse and D. R. Webster, Surg. Forum, Proc. 36th Congr. Am. Coll. Surgeons,
p- 95, (1950).
X. EFFECTS OF DEFICIENCY 273
5. Pies
Hughes and Squibb™ showed that pigs failed to grow on a vitamin Be-
deficient diet, had rough coats, and had poor appetites. They later devel-
oped epileptiform fits which became more and more frequent. A microcytic
hypochromic anemia was produced.*”: ** Blood hemoglobin and red blood
cell counts were low; the size of the red blood cell was also reduced. The
omission of pyridoxine and pantothenic acid from the diet of pigs led to
the development of abnormal gait and degenerative changes in the periph-
eral nerves, the posterior root ganglia, the posterior roots, and the posterior
funiculi of the spinal cord.**
The pyridoxine-deficient animal continues to absorb iron; tissue iron
is abundant, but its utilization is diminished.** The rate of hemolysis does
not increase. The hemosiderosis of the liver, the spleen, and the bone
marrow of vitamin Be-deficient pigs can be prevented by restricting the
dietary intake of iron. The urinary excretion of iron in pyridoxine defi-
ciency is insignificant and is not altered from the normal. Serum iron is
increased; it is in the ferric state.
Follis and Wintrobe*® investigated the nature of the changes in the
nervous system produced by the deficiency. Ataxia was first manifested as
a slightly high lift of the hind legs, together with a swaying of the hind
quarters in walking. The hind legs often twisted in one direction or an-
other. After 9 or 10 weeks of the deficiency, histologic changes were visible.
Demyelinization of the peripheral nerves occurred (brachial and sciatic);
the nerve fibers with the larger diameters were affected most of all. The
degeneration was characterized by the appearance of small droplets of
neutral fat and by the loss of the fine reticular structure of the myelin
sheath. Later, axis cylinder changes became evident. Myelin degeneration
was found only in the peripheral nerves in the early stages, but in the later
stages necrotic cells were found in the dorsal root ganglia.
Lehrer ef al.’ produced a vitamin Bg deficiency in 2-day-old baby pigs.
Poor appetite was the earliest sign of the deficiency. Within 23 days the
piglets showed muscular incoordination, spastic gait, epileptiform fits,
rough hair coats, a brown exudate around the eyes and impairment of
82 EB. H. Hughes and R. L. Squibb, J. Animal Sci. 1, 320, (1942).
88M. M. Wintrobe, R. H. Follis, Jr., M. H. Miller, H. J. Stein, R. Alcayaga, S.
Humphreys, A. Suksta, and G. E. Cartwright, Bull. Johns Hopkins Hosp. 72, 1
(1943).
84M. M. Wintrobe, M. H. Miller, R. H. Follis, Jr., H. J. Stein, C. Mushatt, and
S. Humphreys, J. Nutrition 24, 345 (1942).
86 G. E. Cartwright, M. M. Wintrobe, and 8S. Humphreys, J. Biol. Chem. 153, 171
(1944).
86 R. H. Follis, Jr. and M. M. Wintrobe, J. Exptl. Med. 81, 539 (1945).
87 W. P. Lehrer, Jr., A. C. Wiese, P. R. Moore, and M. E. Ensminger, J. Animal Sci.
10, 65 (1951).
274 PYRIDOXINE AND RELATED COMPOUNDS
vision. The administration of pyridoxine cured all but the impairment of
eyesight.
Tryptophan metabolism is abnormal; xanthurenic acid is excreted in the
urine.** Fatty livers are often observed in these deficient animals.™
6. MONKEYS
Young rhesus monkeys (Macaca mulatta), weighing 1.5 to 2.0 kg., failed
to grow normally when placed on a vitamin B,-free diet.*° They maintained
their initial weight for 9 months; then a marked weight loss occurred.
During the depletion period, a hypochromic microcytic anemia developed;
nucleated red cells appeared in the blood.** Hemoglobin values dropped.
There was an alteration in ratio of neutrophiles to lymphocytes and a
marked polychromatophia developed. The animals were inactive, weak,
and very irritable. They lost their appetite; they showed thinning of the
fur and some graying of the fur on legs, arms, and back. With pyridoxine
administration, growth was resumed and the blood picture improved. In
most respects, the deficiency symptoms resembled those observed in swine
and dogs. Mushett e¢ al.” observed a similar picture when monkeys were
given desoxypyridoxine.
Greenberg and Rinehart also investigated the symptoms of vitamin Bg
deficiency in the monkey. They showed that the deficient monkey excreted
xanthurenic acid in the urine.’? The blood vitamin Bg level of fourteen
rhesus monkeys fell during the first 2 weeks of the deficiency and remained
for one year at a low concentration of 2 to 3 y per 100 ml. of whole blood.
Control animals receiving 1 mg. of pyridoxine per day had corresponding
blood levels of 5 to 20 y. Rinehart and Greenberg’? maintained five im-
mature monkeys on a synthetic pyridoxine-deficient diet for 544 to 16
months. In the early part of the experiment, there was decreased food
consumption, gradual weight loss, and diminished vigor. After 5 or 6
months, the animals became unkempt, sluggish, and hyperirritable when
disturbed. No convulsions occurred. The hair became thinner and lighter;
hair growth later ceased. Some animals showed edema of the eyelids, and
most developed ‘‘some fissuring of the epithelium of the palms of the hands
and feet.’’ All developed a moderate leukopenia and anemia. Sclerotic
lesions developed in the arteries of all deficient animals; the condition was
8G. E. Cartwright, M. M. Wintrobe, P. Jones, M. Lauritsen, and 8S. Humphreys,
Bull. Johns Hopkins Hosp. 75, 35 (1944).
89 K. B. McCall, H. A. Waisman, C. A. Elvehjem, and E. S. Jones, J. Nutrition 31,
685 (1946).
8% KX. J. Poppen, L. D. Greenberg, and J. F. Rinehart, Blood 7, 436 (1952).
°0 L. D. Greenberg and J. F. Rinehart, Federation Proc. 7, 157 (1948).
%L. D. Greenberg and J. F. Rinehart, Proc. Soc. Exptl. Biol. Med. 70, 20 (1949).
#2 J. F. Rinehart and L. D. Greenberg, Am. J. Pathol. 25, 481 (1949).
X. EFFECTS OF DEFICIENCY 275
more severe in those monkeys which had been longer on the deficient diet.
The lesions were prominent in the coronary arteries, as well as in those of
the kidneys and the pancreas. Advanced changes were found in the arteries
of the testicle of one animal. The authors stated that “it was somewhat
surprising to see an advanced arteriosclerosis in the vessels of an immature
testis.”’ The sclerotic lesions produced in the vitamin B,-deficient monkeys
closely resembled those of man.
The arteriosclerotic lesions appear to be a specific characteristic of a
pyridoxine deficiency, for no such lesions were observed in rhesus monkeys
that were thiamine deficient.%
The subcutaneous injection of desoxypyridoxine induced a microcytic
anemia, leucopenia, and lymphopenia.” The blood-forming constituents
and the fat of the femoral marrow were reduced; the adrenal glands were
also affected, as in dogs.
7. Brros
Jukes“ reported that deficient chicks showed nervous symptoms, charac-
terized by convulsive movements. Lepkovsky and Kratzer®® have described
three stages of the pyridoxine deficiency syndrome. In stage 1, there was an
abnormal excitability; in stage 2, the chick exhibited jerky convulsive
movements; in stage 3, convulsions occurred. These convulsions appeared
after about 12 days on the deficient diet; rats require 4 to 6 months for
similar symptoms to appear. Growth is retarded and is accompanied by
anorexia and extreme weakness. It is significant that the apyridoxie chick
does not develop any dermatitis. Other symptoms of the deficiency include
hyperthrombinemia, decreased clotting time,*® hypoplasia of the spleen,
lymphoid atrophy,” and lowered egg production and hatchability.%7
Turkeys develop nervous disorders on a vitamin Be-deficient diet, charac-
terized by convulsive seizures, but they do not develop any anemia.°**
Severe pyridoxine deficiency in young white Pekin ducklings is character-
ized by a very rapid cessation of growth and severe anemia.*? A chronic
vitamin Bg deficiency in older ducklings caused lack of growth, poor feather-
ing, paralysis, convulsions, and severe microcytic anemia (low hemoglobin,
93 J. F. Rinehart, L. D. Greenberg, and L. D. Ginzton, Blood 3, 1453 (1948).
% T. H. Jukes, Proc. Soc. Exptl. Biol. Med. 42, 180 (1939).
99S. Lepkovsky and F. H. Kratzer, J. Nutrition 24, 515 (1942).
96 T. D. Luckey, G. M. Briggs, C. A. Elvehjem, and E. B. Hart, Proc. Soc. Expil.
Biol. Med. 58, 340 (1945).
7 W.W. Cravens, E. E. Sebesta, J. G. Halpin, and E. B. Hart, Poultry Sci. 25, 80
(1946).
*% F. H. Bird, F. H. Kratzer, V.S. Asmundson, and 8. Lepkovsky, Proc. Soc. Exptl.
Biol. Med. 52, 44 (1943).
99 TD). M. Hegsted and M. N. Rao, J. Nutrition 30, 367 (1945).
276 PYRIDOXINE AND RELATED COMPOUNDS
reduced red blood cell count, low hematocrit, and many young red blood
cells).
8. OTHER ANIMALS
The vitamin B, deficiency symptoms in fox pups are similar to those
in dogs.!°° There is a cessation of growth, anorexia, and reduced hemo-
globin values.
Young rainbow trout (Salmo gairdnerii crideus) develop nervous dis-
orders when vitamin Bg, is omitted from the diet; they also lose their ability
to judge distances.!"!
With the development of synthetic milk diets for young calves, it has
been possible to limit the synthesis of vitamins by microorganisms in
young ruminants. Young calves do not grow on a vitamin Be,-deficient diet.
The excretion of vitamin Bs and its metabolites is lowered.! The deficiency
is further characterized by lack of appetite, anorexia, sluggishness, listless-
ness, poor hair coat (dull and falling out) and, in some cases, epileptiform
fits and death; there was no blood pathology.
C. IN MICROORGANISMS
ESMOND E. SNELL
Pyridoxine, the first member of the vitamin B, group to be recognized,
was isolated by virtue of its vitamin activity for animals in 1938, and was
synthesized in 1939. Its growth-promoting activity for certain yeasts! 1%
and lactic acid bacteria!®®: °° was noted very shortly following its isolation.
It is now known to be required for growth of a wide variety of bacteria,
yeasts, and molds;!7: 1° those not requiring the vitamin appear to synthe-
size it.
Microbiological assay of natural materials with lactic acid bacteria
yielded extremely high values for their apparent “pyridoxine”’ content, as
compared with values obtained by other methods; the existence of addi-
tional compounds with high vitamin Bg activity was therefore postulated.'°°
These were eventually identified as pyridoxal and pyridoxamine.!” It
100 A. K. Schaeffer, C. K. Whitehair, and C. A. Elvehjem, J. Nutrition 34, 131 (1947).
101 B. A. McLaren, E. Keller, D. J. O’Donnell, and C. A. Elvehjem, Arch. Biochem. 15,
169 (1947).
1022 B.C. Johnson, J. A. Pinkos, and K. A. Burke, J. Nutrition 40, 309 (1950).
108 AS. Schultz, L. Atkin, and C. N. Frey, J. Am. Chem. Soc. 61, 1931 (1939).
104 R. E. Eakin and R. J. Williams, J. Am. Chem. Soc. 61, 1932 (1939).
105 H}. F. Moeller, Z. physiol. Chem. 264, 285 (1988).
'06 H. H. Snell and W. H. Peterson, J. Bacteriol. 39, 273 (1940).
07 B.C. J. G. Knight, Vitamins and Hormones 8, 105 (1945).
108 Hy. E. Snell zx Vitamin Methods, Vol. I, p. 8327. Academic Press, New York 1950.
109 H}. KH. Snell, B. M. Guirard, and R. J. Williams, J. Biol. Chem. 143, 519 (1942).
110 KY, E. Snell, J. Am. Chem. Soc. 66, 2082 (1944); J. Biol. Chem. 164, 313 (1944).
X. EFFECTS OF DEFICIENCY ald
became apparent after extensive assays that the lactic acid bacteria were
essentially unable to utilize pyridoxine to satisfy their vitamin Bg require-
ments, the growth-promoting activities previously observed for this com-
pound being due to the chemical transformation of it, in minute yield, to
pyridoxal or pyridoxamine (‘‘pseudopyridoxine’’) by interaction with other
ingredients of the medium during autoclaving."': |? Because of this, many
of these organisms grow suboptimally for lack of vitamin Bes (pyridoxal or
pyridoxamine) in media that contain an excess of pyridoxine.!"*: !!? When
such organisms are grown with small amounts of pyridoxine, therefore, they
may show metabolic incapacities due to vitamin Bg deficiency which are
not apparent when they are grown with equally small amounts of pyridoxal
or pyridoxamine. Examples of this behavior will appear later in the dis-
cussion. Different lactic acid bacteria also vary in the ease with which they
use pyridoxamine and pyridoxal; the former is almost inactive for Lacto-
bacillus caset, the latter is highly active.! Several lactic acid bacteria are
also known for which only the phosphorylated compounds, pyridoxamine
phosphate and pyridoxal phosphate, have high activity.!: 1
Various clostridia so far examined,!!® as well as the protozoan organism
Tetrahymena gelezi,!'® resemble lactic acid bacteria in utilizing pyridoxal or
pyridoxamine far more eifectively than pyridoxine. For most yeasts and
molds, on the other hand, all three compounds have very similar activities,!”
as they do in animals.
A deficiency of vitamin Bs may produce a variety of effects in micro-
organisms in addition to those upon growth. These effects often reflect
themselves in an altered quantitative requirement for the vitamin for
growth under different environmental conditions. Several examples follow.
It was early shown that Streptococcus faecalis, which requires vitamin Be
for growth in a medium based on an acid-hydrolyzed casein supplemented
with tryptophan, could grow without the vitamin if p-alanine were added.!!7
When grown under such conditions, little or no vitamin Bs was synthe-
sized;!!85 however, when cells were grown with vitamin Bs and without
p-alanine, the latter amino acid was synthesized and laid down in the
cells.'9 It was suggested that vitamin Bs (pyridoxal phosphate) served as
11 FE. E. Snell, Proc. Soc. Exptl. Biol. Med. 61, 356 (1942).
12 FE. E. Snell and A. N. Rannefeld, J. Biol. Chem. 157, 475 (1945).
113 B. C. Johnson, Proc. Soc. Exptl. Biol. Med. 55, 199 (1944).
14 W.S. McNutt and E. E. Snell, J. Biol. Chem. 182, 557 (1950).
18 T), Hendlin, M. C. Caswell, V. J. Peters, and T. R. Wood, J. Biol. Chem. 186, 647
(1950).
‘sa M. J. Boyd, M. A. Logan, and A. A. Tytell, J. Biol. Chem. 174, 1013 (1948).
16 G. W. Kidder and V. C. Dewey, Arch. Biochem. 20, 433 (1949): 21, 58 (1949).
7 K. E. Snell, J. Biol. Chem. 158, 497 (1945).
us J.T. Holden, C. Furman, and E. E. Snell, J. Biol. Chem. 178, 789 (1949).
1s J. T. Holden and E. E. Snell, J. Biol. Chem. 178, 799 (1949).
278 PYRIDOXINE AND RELATED COMPOUNDS
a coenzyme for the synthesis of p- from L-alanine;"* this suggestion was
confirmed by the demonstration”? that cells of this organism (and many
other microorganisms) contain an enzyme capable of racemizing L-alanine,
and that pyridoxal phosphate is its coenzyme. Thus in the presence of
vitamin B, such cells synthesize p-alanine; in the absence of vitamin Beg the
deficient cells will grow only if this essential p-amino acid is supplied to
them. Deficient cells grown under the latter conditions contain the apo-
racemase but cannot racemize L-alanine for lack of pyridoxal phosphate.'”°
Cells of Streptococcus faecalis grown either with pu-alanine under the
above conditions or with low levels of vitamin Bs have no detectable tyro-
sine decarboxylase activity, whereas when high levels of vitamin Be are
supplied the activity of this enzyme is high.”! It was this findmg with
vitamin B,-deficient bacteria which led to the identification of pyridoxal
phosphate as (1) the coenzymatic form of vitamin Be!? and (2) the coen-
zyme for the amino acid decarboxylases of both bacterial and mammalian
eells.}23: 124
When cells of S. faecalis are grown with pi-alanine replacing vitamin Beg ,
they are unable to carry out transamination between aspartic and keto-
glutaric acids, but they do so rapidly on addition of pyridoxal or pyridoxal
phosphate.’ This unequivocal demonstration of the role of pyridoxal
phosphate as a coenzyme for the glutamic-aspartic transaminase not only
confirmed a role previously indicated for the vitamin!”®: ”” but showed that
greatly differing amounts of vitamin Bs were required to activate different
vitamin Bes-dependent reactions in the same cells. It had been observed
previously that cells of this organism grown with high levels of vitamin Bg
carried out transamination and decarboxylation of tyrosine, whereas cells
grown with small amounts of the vitamin carried out transamination, but
not decarboxylation.!”8
Several reports’! have pointed out that lactic acid bacteria grown
with pyridoxine in the medium require certain amino acids for growth
that are no longer essential if the pyridoxine is replaced by pyridoxamine
1220 W. A. Wood and I. C. Gunsalus, J. Biol. Chem. 190, 403 (1951).
121 W.D. Bellamy and I. C. Gunsalus, J. Bacteriol. 48, 191 (1944).
122 T, C. Gunsalus and W. D. Bellamy, J. Biol. Chem. 155, 357 (1944).
123. F. Gale, Advances in Enzymol. 6, 1 (1946).
124 H. Werle, Angew. Chem. 68, 550 (1951).
25 H.C. Lichstein, I. C. Gunsalus, and W. W. Umbreit, J. Biol. Chem. 161, 311 (1945).
126 WH}. HW. Snell, J. Am. Chem. Soc. 67, 194 (1945).
127. Schlenk and E. E. Snell, J. Biol. Chem. 157, 425 (1945).
128 P,P, Cohen and H. C. Lichstein, J. Biol. Chem. 159, 367 (1945).
129 M. L. Speck and D. A. Pitt, Science 106, 420 (1947).
130 J. L. Stokes and M. Gunness, Sctence 101, 48 (1945).
131 C, M. Lyman, O. Moseley, S. Wood, B. Butler, and F. Hale, J. Biol. Chem. 167,
177 (1947).
X. EFFECTS OF DEFICIENCY 279
or pyridoxal. It will be apparent from the previous discussion that the
pyridoxine-grown cells were somewhat deficient in vitamin Beg ; the require-
ment for otherwise non-essential amino acids under these conditions is
excellent evidence that vitamin Bs is required for their synthesis. Such
amino acids include cystine for L. casei, L. arabinosus, and S. faecalis;!9
lysine, alanine, and threonine for L. delbrueckii, L. arabinosus, and L.
casez;° and threonine, lysine, alanine, serine, histidine, aspartic acid,
phenylalanine, and tyrosine for L. arabinosus."' In the presence but not in
the absence of ample vitamin Bg , L. arabinosus also synthesizes tryptophan
from indole,’ and S. faecalis synthesizes histidine from imidazole pyruvic
acid.'*’ Indeed, each of the amino acids of casein must be present together
with p-alanine to permit growth of S. faecalis in the absence of vitamin
B,.'* In the presence of ample vitamin Bs, D-alanine and many of the
L-amino acids are synthesized by this organism. It thus appears that vita-
min Bg is intimately involved in synthesis of each of the ‘non-essential’
amino acids in lactic acid bacteria; in their presence, the magnitude of the
vitamin Bs requirement falls either to zero or to such low levels that these
organisms can synthesize the small remaining amount required.
In the presence of adequate amounts of vitamin Bs , many of the essential
amino acids for lactic acid bacteria can be replaced by the corresponding
keto or hydroxy acids; in the absence of the vitamin the keto and hydroxy
acids are without growth-promoting activity. It was shown that the keto
acids were transformed to amino acids by transamination; each of the trans-
aminases involved was vitamin B,s-dependent and hence could not function
in the absence of supplies of this vitamin.!°
Neurospora sitophila pyridoxineless requires approximately ten times as
much vitamin B, for growth in the absence of thiamine as is required when
thiamine is added.’** Vitamin Be,-deficient cultures of this organism thus
require thiamine, which is synthesized when the supplies of vitamin Bs
are more liberal. Although not so interpreted by the investigators, the
observation may indicate an important role for vitamin Bg in the synthesis
of thiamine. A different type of relationship between these two vitamins oc-
curs in Saccharomyces carlsbergensis 4228 and in several similar yeasts!**: 17
in which thiamine suppresses growth. This toxic effect is effectively coun-
132 B.S. Schweigert, J. Biol. Chem. 168, 283 (1947).
138 H. P. Broquist and E. E. Snell, J. Biol. Chem. 180, 59 (1949).
1344 H. P. Broquist and E. E. Snell, unpublished observations.
1386 J.T. Holden, R. B. Wildman, and E. E. Snell, J. Biol. Chem. 191, 559 (1951).
135a J. T,. Stokes, A. Larsen, C. K. Woodward, and J. W. Foster, J. Biol. Chem. 150,
17 (1943).
136 AS. Schultz and L. Atkin, Arch. Biochem. 14, 369 (1947).
137 J. C. Rabinowitz and E. E. Snell, Arch. Biochem. and Biophys. 38, 472 (1951).
280 PYRIDOXINE AND RELATED COMPOUNDS
teracted by vitamin Beg .!’ The mechanism by which excess thiamine causes
a condition of vitamin Beg deficiency in such organisms is not known.
The requirement for vitamin Bg in the synthesis of nicotinic acid, noted
in animals,'* becomes apparent in a widely used assay method for vitamin
Bs which employs Saccharomyces carlsbergensis 4228. This organism grows
very well in the absence of nicotinic acid when vitamin Beg is present in
excess, and consequently nicotinic acid was not included in the initially
proposed assay medium .'*? With suboptimal amounts of vitamin Bez, how-
ever, nicotinic acid gives an “‘extra”’ growth response™®: ! and must there-
fore be added to the medium when this is used for the determination of
vitamin Beg in natural materials.
D. IN MAN
P. GYORGY
Vitamin Beg is required in the nutrition of all species of animals studied.
Its role in metabolic processes is multiple and involves a large number of
biochemical reactions. Deficiency of vitamin Bg in animals manifests itself
first in retardation of growth, which in itself is no indication for lack of a
specific essential nutrient. This is followed by a variety of more character-
istic manifestations of vitamin Bg deficiency, such as cutaneous changes,
microcytic anemia, convulsions, ete.
In general, it is expected that vitamins required by a large number of
vertebrates are also necessary dietary constituents for man. Thus, it ap-
peared to be reasonable to predict that there must be a human dietary
requirement for vitamin B,. However, in contrast to the great majority of
vitamins no pathological or clinical conditions in human beings were known
or identified up to the most recent past which could have been related to
deficiency of vitamin B,. As a matter of fact, the very widespread occur-
rence of vitamin Bg in food products made it a priort improbable that a
major deficiency of vitamin Bs, could develop in man. In the absence of
such clear-cut deficiency conditions for vitamin B, there were three possi-
bilities open to investigate the need for vitamin Bs by man: (1) through
the experimental production of vitamin Bs deficiency; (2) through bio-
chemical procedures which should detect even the initial stage of vitamin
Bes deficiency or a metabolic disarrangement indicating an increased re-
quirement for vitamin B., and (3) through therapeutic tests in specific
clinical conditions.
1388 C. T. Ling, D. M. Hegsted, and F. J. Stare, J. Biol. Chem. 174, 803 (1948).
139 ||, Atkin, A. S. Schultz, W. L. Williams, and C. N. Frey, Ind. Eng. Chem. Anal.
Ed. 15, 141 (1948).
140 R. H. Hopkins and R. J. Pennington, Biochem. J. 41, 110 (1947).
141 J.C. Rabinowitz and E. E. Snell, J. Biol. Chem. 176, 1147 (1948).
X. EFFECTS OF DEFICIENCY 281
Hawkins and Barsky'” attempted to produce deficiency of vitamin Beg in
an adult who subsisted for 55 days on a purified diet and received all neces-
sary known vitamins with the exception of vitamin B,. The basal diet
consisted of a mixture of sucrose, corn oil, vitamin-free casein, salt mixture,
cod liver oil concentrate, and all known water-soluble vitamins, except
pyridoxine. The unpalatability of the mixture made difficult the ingestion
of a sufficient amount to supply the caloric needs of a moderately active
man, with the net result of a definite weight loss. At the level taken the
diet furnished 82.5 g. of protein and 50 mg. of iron daily. It is no wonder
that on such an experimental diet the subject toward the end of the experi-
mental period was complaining of ‘fan unusual degree of depression and
mental confusion.” It may be assumed that the noted beneficial effect of a
supplement of vitamin Bg at this stage of the experiment was due more to a
psychological than nutritional-metabolic effect. The authors themselves
concluded that the experiment revealed no changes which could unequivo-
cally be considered as resulting from a lack of vitamin Bs. Furthermore,
even on the animal experiments it may be definitely stated that the experi-
mental period of 55 days is too short to bring about deficiency of vitamin Beg
in adult animals or man.
More conclusive are the observations reported by Snyderman et al.'%
These authors administered a vitamin Beg-deficient diet for therapeutic
reasons to two mentally defective infants for 76 and 130 days, respectively.
The first changes noted were of biochemical nature. Pyridoxice acid dis-
appeared from the urine, and the total urinary pyridoxine was reduced to
extremely low values ranging from 0.2 to 2 y per day. ‘“‘Subsequently, both
infants lost the ability to convert tryptophan to nicotinic acid, an effect
which was desired in order to block a metabolic path for tryptophan
that might be competing with normal tissue synthesis. A plateauing of the
weight curve occurred 33 and 73 days after the institution of the regime.
On the 76th day one subject developed a series of convulsions which were
promptly relieved by the administration of pyridoxine. The other subject
developed a hypochromic anemia at approximately the 130th day. This
responded dramatically to pyridoxine; a rise of reticulocytes was noted
after 72 hours reaching a peak in 4 days after which red cell count and
hemoglobin rose to normal. Both subjects gained weight normally after
supplementation. In marked contrast to the excellent and prompt clinical
response to the administration of pyridoxine was a delay in the reappear-
ance in the ability to convert tryptophan to nicotinic acid.”
142 W.W. Hawkins and J. Barsky, Science 108, 284 (1948).
1443S. EK. Snyderman, R. Carretero, and L. E. Holt, Jr., Federation Proc. 9, 371
(1950).
282 PYRIDOXINE AND RELATED COMPOUNDS
The development of pyridoxine deficiency may be accelerated in adults
with the use of a pyridoxine analog. Mueller and Vilter described symptoms
of pyridoxine deficiency after prolonged administration of desoxypyri-
doxine." 49 The pyridoxine analog was given to eight patients suffering
from various chronic illnesses in doses of 60 to 150 mg. intramuscularly
daily for periods of 18 to 55 days, while the subjects were maintained on a
diet low in the vitamins of the B complex.
Seborrhea-like skin lesions developed about the eyes, nose, and mouth
within 2 to 3 weeks in seven of the eight patients. In three of the seven the
lesions were quite severe. Half of the patients developed erosions in and
around the mouth resembling cheilosis of riboflavin deficiency and glossitis
resembling morphologically that seen in niacin deficiency. One patient’
developed severe systemic symptoms: nausea, vomiting, weakness, and
dizziness. The subjects with the most severe manifestations of deficiency
were two patients with rheumatoid arthritis.
There was no anemia due to desoxypyridoxine. The total white count
remained unchanged, but in seven of the eight patients there was a mild
absolute lymphocytopenia.
The skin, mucous membrane, and systemic manifestations remained
unchanged when a mixture containing thiamine, riboflavin, and nicotina-
mide was given, but they disappeared 48 to 72 hours after pyridoxine was
administered.
During the period of the administration of desoxypyridoxine there was
no increased excretion of xanthurenic acid in the urine. No tryptophan load
test was performed.
The inhibitory ratio of antimetabolite to metabolite in human beings was
not accurately measured but is at least 1:1.
The pathologic changes and clinical symptomatology observed under
the influence of desoxypyridoxine as well as those seen in infants fed for a
prolonged period of time a vitamin Beg-deficient diet indicate not only the
existing need of man for vitamin Bs but also give an experimental founda-
tion for the clinical and pathologic vitamin Be, deficiency in man.
Even before the above-mentioned experimental clinical studies, claims
were put forward regarding the usefulness of pyridoxine in various patho-
logic conditions in man, with the more or less silent assumption that
primary or secondary lack of vitamin Bs might play a part in the patho-
genesis of these diseases. In other instances pyridoxine was tried thera-
peutically in the clinie on the basis of a possible analogy of the clinical
conditions in question to similar manifestations in experimental vitamin Bs,
144 J. F. Mueller and R. W. Vilter, J. Clin. Invest. 29, 193 (1950).
44a RW. Vilter, J. F. Mueller, H. S. Glazer, T. Jarrold, J. Abraham, C. Thompson,
and V. R. Hawkins, J. Lab. Clin. Med. 42, 335 (1953).
X. EFFECTS OF DEFICIENCY 283
deficiency in animals. The logic of such and similar attempts is certainly
exceedingly thin, and it is no wonder that the observations reported were
very contradictory and apparently often marred by subjective factors,
emanating either from the authors or from the patients.
One group of such reports dealt: with conditions involving the neuro-
muscular system, such as myasthenia, parkinsonism, chorea, epilepsy,
muscular dystrophy, and related diseases. Beneficial results, claimed after
administration of pyridoxine in patients with muscular weakness, “° in
some cases of parkinsonism,'”: 8 even in poliomyelitis, in lightning pains
accompanying tabes dorsalis and in multiple sclerosis, when given intra-
spinally,® or in chorea,'*: '°° and in hypertrophic muscular dystrophy!*!: 1°?
may be contrasted with negative and, in general, better-controlled obser-
rations in parkinsonism,!**-!*° in muscular dystrophy,!°®! in amyotrophic
lateral sclerosis,!®*: '*” and in epilepsy.'®!
The similarity of the cutaneous lesions in experimental pyridoxine de-
ficiency to desquamative and seborrheic or seborrheid lesions in man!
stimulated the use of pyridoxine in the therapy of this widely represented
clinical group of pathologic skin conditions. Negative results!®: !* are
again to some extent matched with beneficial results in seborrheic or sebor-
rheid conditions after treatment with pyridoxine.!*: 1°
145 J’. D. Spies, W. B. Bean, and W. F. Ashe, J. Am. Med. Assoc. 112, 2414 (1939).
46 WY. E. Rosenbaum, 8. Portis, and S. Soskin, J. Lab. Clin. Med. 27, 763 (1941-1942).
147'T. D. Spies, D. P. Hightower, and L. H. Hubbard, J. Am. Med. Assoc. 116, 292,
(1940).
M48 N. Jolliffe, Minnesota Med. 28, 542 (1940).
49S. Stone, Diseases of Nervous System 11, 131 (1950).
160 J. Schwartzman, D. Dragutsky, and G. Rook, J. Pediat. 19, 201 (1941).
1651 W. Antopol and C. E. Schotland, J. Am. Med. Assoc. 114, 1058 (1940).
162 G. E. Donovan, Lancet II, 162 (1940).
153 A.B. Baker, J. Am. Med. Assoc. 116, 2484 (1941).
154 M. A. Zeligs, J. Am. Med. Assoc. 116, 2148 (1941).
1655 W.H. Barker, H. J. Stein, M. H. Miller, and M. M. Wintrobe, Bull. Johns Hopkins
Hosp. 69, 266 (1941).
156 J. W. Ferrebee, W. O. Klingman, and A. M. Frantz, J. Am. Med. Assoc. 116, 1895
(1941).
67 |. M. Eaton, H. W. Woltman, and H. R. Butt, Proc. Staff Meetings Mayo Clinic
16, 523 (1941).
158 A. McBryde and L. D. Baker, J. Pediat. 18, 727 (1941).
159 A. M. Doyle and H. H. Merritt, Arch. Neutrol. Physiat. 45, 672 (1941).
160 A. M. Keith, J. Pediat. 20, 200 (1942).
161 J.T. Fox and G. M. Tullidge, Lancet II, 345 (1945).
162 P. Gyorgy, Arch. Dermatol. and Syphilol. 43, 230 (1941).
168 W. Pehl, Z. Kinderheilk. 61, 613 (1940).
‘6! P. Gyorgy, unpublished observations.
165 N. Jolliffe, L. A. Rosenblum, and J. Sawhill, J. Invest. Dermatol. 5, 143 (1942).
*66C.S. Wright, M. H. Samitz, and H. Brown, Arch. Dermatol. and Syphilol. 47, 651
(1943).
284 PYRIDOXINE AND RELATED COMPOUNDS
The designation ‘‘acrodynia” for the syndrome of experimental pyri-
doxine deficiency in rats has been originally proposed without prejudice
whether the condition in rats is analogous in its etiology with the identically
named condition in children.'®? The name was chosen only on the basis of
the outward similarity of cutaneous manifestations and their distribution
in rats with severe vitamin Bg deficiency and in human acrodynia. As a
matter of fact, the author found pyridoxine without benefit in human ac-
rodynia.'®! However, in contrast to these negative results, beneficial effects
from medication with pyridoxine were claimed in human acrodynia by
Frontali!® and Bose.'®’ The unpredictable and variable course of human
acrodynia makes the so-called positive results less convincing than the
lack of response in other cases on the same medication. Against Frontali’s
further claim of lowered Beg values in blood in acrodynia and their rise after
specific medication, it deserves to be pointed out that the assay methods
for Be, especially for blood, are notoriously far from being satisfactory.
In cases of pellagra, pernicious anemia,!”° and Mediterranean anemia,'!
a slight increase in the granulocytic series of the white blood cells was ob-
served. On the basis of this analogy Cantor and Scott!”: 1 have introduced
the treatment of agranulocytosis with pyridoxine and claimed striking suc-
cess in three cases. These observations were confirmed in agranulocytosis
after treatment with thiouracil’™ and nitrogen-mustard.!”> Inconsistent re-
sponse was noticed by Taylor!® in a case of agranulocytosis after treatment
with thiouracil. During more recent years, after the successful introduction
of intensive antibiotic treatment of agranulocytosis, it became difficult to
assess the value of any other possible therapeutic supplements. One gains,
however, the impression that the original claims regarding the value of
vitamin Bs in the treatment of agranulocytosis are shared at the present
time by few if any hematologists.
Experimental vitamin Be, deficiency may depress the lymphatic tissue
and may also lead to relative lymphocytopenia, especially marked when
lack of vitamin Bg in the diet is combined with the administration of desoxy-
pyridoxine as metabolic antagonist of pyridoxine.’ Gellhorn and Jones!’®
167 'T. W. Birch, P. Gyérgy, and L. J. Harris, Biochem. J. 29, 2830 (1935).
168 Quoted by Bose in ref. 169.
169 R. Bosc, Arch. franc. Pediat. 6, 203 (1949).
170 R. W. Vilter, H. 8S. Schiro, and T. D. Spies, Nature 145, 388 (1940).
171 7. M. Goldman and A. Malvados, J. Clin. Endocrinol. 1, 945 (1941).
72 M.M. Cantor and J. W. Scott, Science 100, 545 (1944).
173 M.M. Cantor and J. W. Scott, Can. Med. Assoc. J. 52, 368 (1945).
4H. H. Fishberg and J. Vorzimer, Proc. Soc. Exptl. Biol. Med. 60, 181 (1945).
75 H. Fleischhacker, Le Sang 21, 368 (1950).
176 H. Taylor, Proc. Roy. Soc. Med. 89, 297 (1945-1946).
177 H. C. Stoerk, J. Biol. Chem. 171, 4387 (1947).
8 A. Gellhorn and L. O. Jones, Blood 4, 60 (1949).
X. EFFECTS OF DEFICIENCY 285
placed three patients with disseminated lymphosarcoma and three cases of
leukemia on a vitamin Be-deficient semi-synthetic, chiefly casein diet, to-
gether with desoxypyridoxine for short periods of up to 14 days. Although
there was evidence of malnutrition in the form of weight loss and weakness,
no specific signs of vitamin B, deficiency developed. Two patients had acute
toxic manifestations after the administration .of large doses of desoxypyri-
doxine. These were characterized by transient epileptiform convulsions.
There were no sequelae and no recurrence of the symptoms when the dose
of the drug was reduced. There was no unequivocal evidence of depression
of hemopoiesis, no significant atrophy of lymphoid tissue, and no signs of
demyelinization of nerves. Tested without a supplementary load of trypto-
phan, the excretion of xanthurenic acid was not increased. These observa-
tions seem to indicate that the restriction of vitamin Bg in the diet together
with desoxypyridoxine for periods up to 2 weeks had no therapeutic effect
in lymphosarcoma and leukemia in human patients.
A large number of investigators studied the effect of pyridoxine medica-
tion in nausea and vomiting of pregnancy. In thirty-seven cases Willis and
his associates'”® have observed very satisfactory results from administra-
tion of pyridoxine (50 mg.). Similar beneficial results were observed by other
authors.'8°83 On the other hand Hesseltine,'*: on the basis of a controlled
study of sixteen cases, concluded that the use of pyridoxine for hyperemesis
gravidarum is valueless and without justification.
It is difficult to assess the results for any medical treatment in a condi-
tion such as nausea and vomiting of pregnancy, greatly influenced by purely
emotional factors. Such scepticism applies equally well to positive as well
as to negative findings.
The same reserved attitude may be observed for the similar syndrome of
radiation sickness. Bergman!** claimed beneficial effect with pyridoxine (in
doses of 100 mg. intramuscularly before and after the surgical procedure)
in the prevention of nausea and especially that of vomiting following gen-
eral anesthesia. However, these results were not confirmed by Kernis and
Stodsky.'8* Greater unanimity is found regarding the treatment of radia-
179 R.S. Willis, W. W. Winn, A. T. Morris, A. A. Newsome, and W.S. Massey, Am. J.
Obstet. Gynecol. 44, 265 (1942).
180 B. B. Weinstein, G. J. Mitchell, and G. F. Sustendal, Am. J. Obstet. Gynecol. 46,
283 (1943).
181 B. B. Weinstein, Z. Wohl, G. J. Mitchell, and G. F. Sustendal, Am. J. Obstet.
Gynecol. 47, 389 (1944).
182 W. M. Silbernagel and O. P. Burt, Ohio State Med. J. 39, 1113 (1943).
183 ©. W. Dorsey, Am. J. Obstet. Gynecol. 68, 1073 (1949).
184 H.C. Hesseltine, Am. J. Obstet. Gynecol. 61, 82 (1946).
185 W. Bergman, Can. Med. Assoc. J. 56, 554 (1941).
186 J,, Kernis and B. Stodsky, Anesthesiology 11, 212 (1950).
286 PYRIDOXINE AND RELATED COMPOUNDS
tion sickness with pyridoxine.!*’-!%8 Various doses were used, from 25 to 200
mg. of pyridoxine. All reports recorded beneficial effects, in some instances
well controlled with placebos.!*!
The greatest difficulty in accepting the beneficial effect of pyridoxine in
conditions such as nausea and vomiting of early pregnancy or following
general anesthesia as well as in radiation sickness is the lack of any direct
indication for a deficiency of vitamin Bg in these conditions. Further, there
is no available proof for a direct pharmacological action of pyridoxine to
explain any clinical beneficial effect after administration of pyridoxine.
As in any deficiency of a vitamin, it wou'd be of great diagnostic value if
biochemical reactions could be found for the assessment of a spe ifiec met-
abohe disturbance connected with the vitamin in question, in this case
with vitamin B,. In this connection the urea level in the blood may furnish
some indication for a specific metabolic abe-ration. 't is known tht fasting
blood urea is significantly less in normal pregnant than in non-pregnant
subjects. The urea level is definitely lower in cases of hyperemesis gravi-
darum than in normal pregnancy, but it may be restored to typical normal
values after the administration of pyridoxine.’* Although the lov level of
fasting blood urea is opposite to the condition found in pyridoxine-defi-
cient rats,!®® the response to a test load of alanine is entirely similar. Blood
urea is increased to a maximum 6 hours after oral administration of 30
mg. of Di-alanine, returning to original values within 12 hours in the non-
pregnant and normal pregnant woman. In women with hyperemesis gravi-
darum, blood urea leve!s rise but fail to decrease significantly bet veen the
sixth and the twelfth hours, thus exhibiting a flat curve. Supportive therapy
alone for 72 hours does not alter the response to pL-alanine, but supportive
therapy supplemented with 120 mg. of pyridoxine produces normal preg-
nancy response to the test.
More specific, at least for a relative vitamin Bg deficiency, is the increased
urinary excretion of xinthurenic acid after a load test of tryptophan.!%*
Without this extra lo d of tryptophan, the excretion of xanthurenie acid,
187 J. R. Maxfield, A. J. Mellwain, and J. KE. Robertson, Radiology 41, 388 (1943).
188 T,, M. Shorvon, Brit. J. Radiol. 19, 369 (1946).
189 H. L. Van Haltern, Radiology 47, 377 (1946).
190 A, Oppenheim and B. Lih, Radiology 47, 381 (1946).
17, D. Scott and G. J. Tarleton, Radiology 47, 386 (1946).
192 W. P. Murphy and M. C. Sossman, Trans. Assoc. Am. Physicians 69, 255 (1946).
193 J. J. Wells and W. C. Popp, Proc. Staff Meetings Mayo Clinic 22, 482 (1947).
194 W. J. McGanity, E. W. McHenry, H. B. Van Wyck, and G. L. Watt, J. Biol.
Chem. 178, 511 (1949).
195 W. W. Hawkins, M. L. McFarland, and E. W. McHenry, J. Biol. Chem. 166, 223
(1946).
196 S$. Lepkovsky, EK. Roboz, and A. J. Haagen-Smit, J. Biol. Chem. 149, 195 (1943).
X. EFFECTS OF DEFICIENCY 287
even in fully developed vitamin Bg deficiency, may stay within normal
limits.!) 78 Greenberg and co-workers!” studied the same phenomenon in
three human subjects on a vitamin Bg-deficient diet for | to 3 weeks (chiefly
casein-sucrose-oil and vitamins), followed by an additional week during
which they received daily supplements of pyridoxine (10 or 15 mg.). The
results summarized in the following tible indicat? that human subjects
may develop early on a vitamin By-deficient diet a derangement of trypto-
TABLE V
XANTHURENIC Actp Excretion!?®
Excretion, mg./24 hr.
Before After
Subject Dietary status Pyridoxine supplement tryptophan tryptophan
D. B. Synthetic diet for 21 days None for 21 days 9.2 271.0
Synthetic diet for 28 days 10mg. per day for last 7 6.2 28.4
days
Natural diet (control 5 mg. every other day 10.1 30.2
period) for 14 days
Synthetic diet for 7 days None for 7 days 4.3 46.8
Synthetic diet for 14 days None for 14 days 7.0 429.0
H.M. Synthetic diet for 21 days None for 21 days 7.4 514.7
Synthetic diet for 28 days 15 mg. per day for 7 5.0 aie ll
days
Natural diet for 38 days 15 mg. per day for 45 9.9 38.9
days
N. K. Natural diet (control None 11.6 47.0
period)
Synthetic diet for 6 days None for 6 days 6.0 31.2
phan metabolism, manifested by the excretion of xanthurenic acid after an
extra load of tryptophan (Table V).
The xanthurenic acid index may be used as a biochemical sign of a latent
vitamin B, deficiency or at least of an increased requirement for vitamin Bg .
The xanthurenic acid index is expressed as the percentage of the dose of
tryptophan, the latter given in an amount of 0.1 g. per kilogram of body
weight.!*%8 In five non-pregnant women Vandelli found'*’’ that the index
ranged from 0.25 to 1.50. In six pregnant women it ranged from 2.13 to
12.4, the higher values tending to occur later in pregnancy. In three preg-
197 J,, D. Greenberg, D. F. Bohr, H. McGrath, and J. F. Rinehart, Arch. Biochem. 21,
237 (1949).
19 FM. Chiancone, Acta Vitaminol. 4, 193 (1950).
199 J. Vandelli, Acta Vitaminol. 5, 55 (1951).
288 PYRIDOXINE AND RELATED COMPOUNDS
nant women who had been treated for nausea and vomiting with pyridoxine,
the index was from 0 to 0.58.
This tryptophan load test should be repeated on a larger number of cases
of pregnancy and extended to other conditions, such as radiation sickness,
in which clinical observations seem to support the therapeutic efficacy of
pyridoxine. Until such more exact biochemical metabolic data become
available, no definite conclusions may be drawn as to the therapeutic value
of pyridoxine in these and similar clinical conditions.
All the above described more or less circumstantial or at least artificially
produced evidence in favor of clinical vitamin Bg, deficiency and, there-
fore, in support of vitamin Bs, requirement for man, has received most re-
cently striking and unequivocal support in direct clinical observations.
Hunt et al2°° observed a young infant in a very serious convulsive state
lasting for several weeks after birth. This infant responded dramatically to
injection of pyridoxine or to larger amounts of pyridoxine-HCl given daily
by mouth (1 to 2 mg.). The basic, puzzling, and unexplained metabolic
defect in this infant has been Bs dependency on a higher level of dietary
requirement.
Even more convincing for the essential nature of vitamin B, in human
dietary was the widespread occurrence of nervous irritability and convul-
sive seizures in young infants in the age period of 6 weeks to 6 months
receiving a proprietary liquid, canned (autoclaved) milk formula. This
formula represents an approximately one-third diluted cow’s milk with
added lactose and fat. Thus, the original vitamin B, content of this formula
is also about one-third of that of undiluted cow’s milk. During autoclaving,
the content of the thermolabile, natural vitamin Bg in this preparation was
further reduced by two-thirds of the original amount and reached a level
of about 60 y per liter, far below the level of vitamin Bs in human milk
(100 to 120 y). In contrast, the same, but spray-dried, powdered, and not
autoclaved, formula showed no appreciable destruction of vitamin Bs, and
the vitamin Be, level remained around 180 y per liter. No cases of nervous
irritability or convulsions were seen in infants receiving the spray-dried
formula with its higher vitamin Be content.
The relation of vitamin Bg to this nutritionally induced convulsive dis-
order is best illustrated by the observation of Coursin?” in a 2144-month-
old infant. This infant received the proprietary liquid formula from birth
without supplements.
““At 4 weeks of age, the baby was noted to be irritable and to have supposed colic
pains with tossing of its head and stiffening of the entire body. Progressive changes
with staring episodes and generalized seizures were noted.® At the age of 244 months,
200 A. D. Hunt, Jr., J. Stokes, Jr.,. W. W. McCrory, and H. Stroud, in press.
201 T). B. Coursin, J. Am. Med. Assoc. in press.
X. EFFECTS OF DEFICIENCY 289
the infant was hospitalized. ...On the 5th day of hospitalization, the infant went
into status epilepticus. The eyes remained fixed and staring, pupils contracted with
no reaction to light. The body was held rigidly with the head thrown back. General-
ized convulsive movements recurred repeatedly with occasional outbursts of an
abnormally high-pitched monotonous cry. There was some evidence of cyanosis about
4 ) A
a . MA Ady . wf | 7 . A y=“ he oF ;
oe Nie’ i , . ’ ’ sa 7 \ wy = a v ¥
- LF + “ 4 wy rad ; /
+ \J u "
:
in j Pine ‘ . a '»
‘ . ~ ” b /
iy on. “4 Whe “, ‘ ' Viui¥ rm | ” ¥ ar, ve mw
Ww, » ¥ yi ' i
* ‘ i ' / a
RF r j vs
rs i ; 4
. A
J ‘ ~ ™ at
A 5 f *\ a “ :
ee 4 . a) N ap 4 4
LP ‘
se ~ a sik :
“A ~ ~ :
va j - } x Dae "
ys a aor ho iV * ws 1A 1 mat Me PT | wer 4
J « ai LIN rs ? ft Ww \ hy fan wy &
vy ‘ \ . Wr OV A
‘ # : = . sf
RP a ‘ .
4, nA
raw, F my, id of adn 7 Aas .
a wy at & Pan Ap ‘ rae icon ee is a2
at AV SL TUT of Ay OW " Ae TAL sy wl WY
Pee NET ABN Ec NAR a a We
LO ; ‘ sec.
- m
\ : 5 r : hes P 450, <i
Ay’ A
\ ee aa 8 | 4 7; eae eo) NW
A ‘al . wet A ¥ yA ¥ “W M 4 f Al AR, Aw a .
r “ : < i 7 5
RO sf ; 7 ve ¥ ‘
Fic. 2. Electroencephalogram during convulsion in an infant on a vitamin B,-de-
ficient milk formula.
ae
af ae aoe a OOO ee nn ee tN Ae ae een
RF pecans te
rJS™ “~ i . fey
wr SAI pI CE ON = a Dn Daa RO
a ae pe
LZ —f bi, a an TS dell VW 5 tS ee
RP ~
pie, | - {~D. = >.
pes Ni neal \y i a ey, ae, —, wg
\ nih Senet : 7
Lan If NOt ee Se ot 4 I UA a _/ MLR Os
Lo { 7mm.
50uv.
ee ee a AAS. i? a, NN pO Ne a : LI nS para NA LON
Fig. 3. Electroencephalogram taken from the same infant (Fig. 2) 3 minutes after
intramuscular injection of 100 mg. of pyridoxine-HCl.
the lips. There was no response to painful stimuli. Pulse was rapid and regular at 180.
Respirations were gasping at 40. The patient was placed in oxygen with some im-
provement of color but no effect on the seizures. He was removed to the EEG labora-
tory and connected by means of needle electrodes to the EEG apparatus. Continuous
recording was undertaken with great technical difficulty due to the child’s muscular
activity. However, successful tracing was obtained with evidence of markedly in-
creased voltage of 200 wv and slowing to 2-3 waves per sec. These appeared in bursts
with recurrently accompanying spikes and lasted for 4-6 sec.... This pattern of
290 PYRIDOXINE AND RELATED COMPOUNDS
activity (Fig. 2) with accompanying clinical seizures was well established when 100
mg. of pyridoxine-HCl was given intramuscularly. For about 1 minute following
injection, there was little change in the EEG. However, by 3 minutes, activity had
definitely diminished and by 4-5 minutes after injection, the EEG became a normal
sleep record (Fig. 3). Clinically, the infant was of good color without added oxygen,
sleeping peacefully and showing no evidence of tremor or rigidity. In 48 hours, al-
though kept on the original formula, there was no evidence of previous difficulties.”’
It is of interest that, as in animals, young infants appear to be more sus-
ceptible to convulsive seizures as manifestations of vitamin B, deficiency
than older children or adults. The critical level of vitamin Bs intake lies
between 50 and 80 y per day.
The biochemical defect characterizing the convulsive disorder in vita-
min Bg deficiency has not yet been elucidated.
Isoniazid may also produce grand mal convulsions in man which are
counteracted by pyridoxine, although perhaps not as completely as the
convulsive seizures occurring in simple, direct vitamin Bg deficiency.2”
XI. Pharmacology
KLAUS R. UNNA
Pyridoxine has an extremely low toxicity. Doses up to 1 g. per kilogram
are tolerated without ill effects by rats, rabbits, and dogs.! The metabolism,
the circulatory and respiratory systems, and isolated smooth muscle organs
of normal animals are not influenced by the vitamin.! Pyridoxine has been
reported to increase the contraction height and work output of perfused
frog muscles.2 This effect was obtained only with concentrations between
0.00005 and 0.005 millimole per liter; a similar effect was observed by the
same authors with thiamine and pantothenic acid in comparable concen-
trations.
Little is known about the toxic effects of pyridoxal and pyridoxamine.
On injection of graded amounts of the different forms of the vitamin into
eggs, 5 mg. of pyridoxal reduced the hatchability more markedly than 5
mg. of pyridoxine, whereas pyridoxamine was without effect im doses of 10
mg.’ Pyridoxamine in doses of 320 mg. per kilogram injected intraperi-
toneally in mice failed to cause toxic effects.*
2022 R. H. Reilly, K. F. Killam, E. H. Jenney, W. H. Marshall, T. Tausig, N. S.
Apter, and C. C. Pfeiffer, J. Am. Med. Assoc. 152, 1317 (1953).
1K. Unna, J. Pharmacol. Exptl. Therap. 70, 1 (1940).
2.N. W. Shock and W. H. Sebrell, Jr., Am. J. Physiol. 146, 399 (1946).
3 W. W. Cravens and E. E. Snell, Proc. Soc. Exptl. Biol. Med. 71, 73 (1949).
4R.E. Parks, Jr.,G. W. Kidder, and V. C. Dewey, Proc. Soc. Exptl. Biol. Med. 79,
287 (1952).
-
XI. PHARMACOLOGY 291
Pyridoxamine has been found to prevent fatal convulsions caused by
thiosemicarbazide in mice; intraperitoneal injections of 320 mg. per kilo-
gram preceding a threefold lethal dose of thiosemicarbazide prevented all
toxic manifestations.’ Pyridoxine and pyridoxal were said to be less effective
antidotes. Toxic effects of semicarbazide were less readily antagonized by
pyridoxamine. In mice and rats, pyridoxine in doses of 6 mg. to 800 mg.
per kilogram raised the convulsive doses of semicarbazide significantly;
intravenous injections of 100 to 400 mg. prevented semicarbazide seizures
in man.® On the other hand, pyridoxine failed to exert an anticonvulsant
effect against seizures induced by metrazol, strychnine, ammonium acetate,
or by electroshock in mice® and by electroshock in rats.®
Pyridoxine, in conjunction with other B vitamins, may also counteract
the anorexia and anemia caused by promin in rats.7
Pyridoxine has been stated to protect mice from death by x-ray radia-
tion, although the leucopenia was not influenced; a similar protection was
obtained with folic acid. On the other hand, a protective effect of pyri-
doxine against injurious effects of P*® could be demonstrated only in pyri-
doxine-deficient mice but not in normal mice.’
Toxic effects of desoxypyridoxine in chicks,!° in rats," and in man’
are readily explained by its conversion into desoxypyridine phosphate and
subsequent competition with pyridoxal phosphate for the apoenzyme.™
The toxic effects of desoxypyridoxine resembling the manifestations of
pyridoxine deficiency were readily obtained when the diet was restricted in
pyridoxine. On an adequate intake of pyridoxine, toxic effects occurred in
rats only after the diet was supplemented with 50 mg. % of desoxypyri-
doxine;” intramuscular injection of 50 mg. caused no ill effects in normal
man.!
Pyridoxine is readily absorbed from the gastrointestinal tract and rapidly
metabolized. Only small fractions of ingested or injected pyridoxine are
excreted as such in the urine. In normal persons on adequate diets a pyri-
doxine activity corresponding to less than 1 yg. per milliliter was found in
the urine.! After intravenous administration of 50 to 100 mg., only 5 to 10%
5B. H. Jenney, R. P. Smith, and C. C. Pfeiffer, Federation Proc. 12, 333 (1953).
6V. D. Davenport and H. W. Davenport, J. Nutrition 36, 139 (1948).
7G.M. Higgins, Am. J. Med. Sci. 207, 239 (1944).
8 A. Goldfeder, L. Cohen, C. Miller, and M. Singer, Proc. Soc. Exptl. Biol. Med. 67,
272 (1948).
9C. Artom, W. E. Cornatzer, and G. T. Harrell, Proc. Soc. Exptl. Biol. Med. 79, 494
(1952).
10 W.H. Ott, Proc. Soc. Exptl. Biol. Med. 61, 125 (1946).
11C¢, C. Porter, I. Clark, and R. H. Silber, J. Biol. Chem. 167, 573 (1947).
122 G. A. Emerson, Federation Proc. 6, 406 (1947).
13 J. F. Mueller and R. W. Vilter, J. Clin. Invest. 29, 193 (1950).
4 W. W. Umbreit and J. G. Waddell, Proc. Soc. Exptl. Biol. Med. 70, 293 (1949).
292 PYRIDOXINE AND RELATED COMPOUNDS
is detected within 1 hour by colorimetric tests as pyridoxine in the urine;!°-!7
recovery of pyridoxine in the urine after ingestion of 8 to 100 mg. ranged
from 4 to 8%.1°: 18:19 Negligible amounts of pyridoxine appear in the
sweat.!® Microbiological assays which differentiate between pyridoxine, pyri-
doxal, and pyridoxamine have shown that normally only minute amounts
of any of the three forms of vitamin are found in urine.”° 4-Pyridoxic
acid,2! the main metabolic product of pyridoxine and also of pyridoxal and
pyridoxamine, accounts for 70 to 90 % of the excretion products measured
after ingestion of either form of vitamin Beg in man.!: 2°: » It has also been
found in the urine of rats, but not of dogs.?* Ingestion of large amounts of
pyridoxine leads to increased excretion of pyridoxal and also of pyridoxa-
mine. On the other hand, no evidence could be obtained showing the con-
version of pyridoxal or pyridoxamine to pyridoxine.”°
Median lethal doses of pyridoxine hydrochloride have been determined
in animals as follows: on intravenous injection in mice (545 mg. per kilo-
gram) and in rats (657 mg. per kilogram); on subcutaneous injection in rats
(3.7 g. per kilogram); and on oral administration in rats (5.5 g. per kilo-
gram).! Tonic convulsions precede death in mice and rats. Doses larger
than 1 g. per kilogram administered to rats, rabbits, and dogs caused in all
three species characteristic manifestations which began with marked im-
pairment of coordination and of righting reflexes within two or three days,
progressing to severe tonic convulsions and death in the stage of paralysis.
Autopsies showed enlargment of the adrenals with occasional hemorrhages
into the cortex. Neurohistologic examination of dogs and rats treated with
2 to 6 g. of pyridoxine per kilogram revealed degeneration of the posterior
columns of the spinal cord and in some cases also a well-marked degenera-
tion of the posterior roots, posterior ganglia, and peripheral nerves.”°
Prolonged daily administration of pyridoxine to rats (25 mg. per kilo-
gram), dogs (20 mg. per kilogram), and monkeys (10 mg. per kilogram)
failed to cause any toxic manifestations or pathologic changes in the tissues.1
Rats receiving 2.5 mg. per kilogram daily were raised through three genera-
tions. Mice tolerated repeated intravenous injections of 100 mg. per kilo-
16 J. V. Scudi, K. Unna, and W. Antopol, J. Biol. Chem. 135, 371 (1940).
16T. D. Spies, R. K. Ladisch, and W. B. Bean, J. Am. Med. Assoc. 115, 839 (1940).
17 J, Flexner and M. R. Chassin, J. Clin. Invest. 20, 313 (1941).
18 Me. Swaminathan, Indian J. Med. Research 29, 561 (1941).
19 B. C. Johnson, T. 8. Hamilton, and H. H. Mitchell, J. Biol. Chem. 158, 619 (1945).
20 J. C. Rabinowitz and E. E. Snell, Proc. Soc. Exptl. Biol. Med. 70, 235 (1949).
21 J. W. Huff and W. A. Perlzweig, J. Biol. Chem. 155, 345 (1944).
22H. Linkswiler and M.S. Reynolds, J. Nutrition 41, 523 (1950).
23 J. W. Huff and W. A. Perlzweig, Science 100, 15 (1944).
24 ©. G. Weigand, C. R. Eckler, and K. K. Chen, Proc. Soc. Exptl. Biol. Med. 44, 147
(1940).
25 W. Antopol and I. M. Tarlov, J. Neuropathol. Exptl. Neurol. 1, 330 (1942).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 293
gram without ill effects.** Prolonged administration of 1 mg. of pyridoxine
to rats subsisting on diets restricted in either thiamine, riboflavin, or panto-
thenic acid failed to aggravate the manifestations of the deficiency state.?®
Since pyridoxine deficiency in experimental animals causes such specific
and striking effects on the skin, on hematopoiesis, and on the central nervous
system, pyridoxine has been given in very large amounts to man for thera-
peutic trials in a large number of conditions. No toxic effects have been
encountered with daily administration over periods of months of 50 to 200
mg. of the vitamin by either the oral, intramuscular, intravenous, or intra-
thecal route.?’-*°
No pharmacodynamic effects of large doses of pyridoxine are established
in man. Administration of large doses in attempts to prevent nausea and
vomiting in radiation sickness and during pregnancy rests, in the absence
of both an experimental basis and carefully controlled studies, on empiri-
cism. It may be remembered that equally large doses of thiamine and of
niacinamide are also recommended for this purpose. Recent observations on
tryptophan metabolism in pregnant women have shown that pyridoxine
prevents the excretion of abnormally large amounts of xanthurenic acid.*!
These findings may be analogous to the observations on tryptophan me-
tabolism in pyridoxine-deficient animals! *? and, hence, imply an altera-
tion during pregnancy of metabolic functions in which pyridoxine is known
to play an essential role. Also, the low fasting blood level of urea of pregnant
women can be restored to normal values by pyridoxine.
XII. Requirements and Factors Influencing Them
A. OF ANIMALS
HENRY SHERMAN
It is difficult to record the precise absolute requirement of animals for
vitamin B,, for there are many factors, external and internal, which in-
fluence this requirement. No single standard set of assay conditions or cri-
teria has received universal acceptance. Since vitamin Bes has at least a
26K. Unna and J. D. Clark, Am. J. Med. Sci. 204, 364 (1942).
27'T. D. Spies, D. P. Hightower, and L. H. Hubbard, J. Am. Med. Assoc. 115, 292
(1940).
28.N. Jolliffe, J. Am. Med. Assoc. 117, 1496 (1941).
29 N. Jolliffe, L. A. Rosenblum, and J. Sawhill, J. Invest. Dermatol. 5, 143 (1942).
30S. Stone, J. Nervous Mental Disease 100, 185 (1944).
31M. Wachstein and A. Gudaitis, J. Lab. Clin. Med. 40, 550 (1952).
82S. Lepkovsky, E. Roboz, and A. J. Haagen-Smit, J. Biol. Chem. 149, 195 (1943).
33W. J. McGanity, E. W. McHenry, H. B. Van Wyck, and G. L. Watt, J. Biol.
Chem. 178, 511 (1949).
294 PYRIDOXINE AND RELATED COMPOUNDS
threefold function in the body, it is conceivable that the requirement for
one function may not be the same as for the other functions. Many times,
growth is used as the criterion of response, but, since this is not specific, it
may not be the appropriate criterion to employ; in a deficiency, biochemical
systems which require vitamin Bs may cease to function long before growth
is affected. The synthesis of vitamin Bs by intestinal bacteria also compli-
cates this entire requirement picture.
1. Rats
The majority of investigators have advocated a pyridoxine requirement
of 10 y per day or 100 y per 100 g. of diet for normal growth.! This value
also applies to the requirement of the cotton rat (Szgmodon hispidus hispi-
dus).2 During lactation, however, this amount is increased to 120 to 200
y per 100 g. of ration.* The vitamin Bs requirement can also be increased
by inducing an experimental hyperthyroidism‘ and by overloading the diet
with thiamine.’ The vitamin Bs requirement does not appear to be increased
in aging rats; thiamine requirement, however, is increased.°®
The three active forms of the vitamin Bs complex are equally effective in
promoting growth and in curing acrodynia when fed by medicine dropper
or injected interperitoneally;7 however, when added to the diet, pyridoxine
seems to be the most active. It has been suggested that intestinal micro-
organisms utilize or destroy pyridoxal and pyridoxamine preferentially.
Linkswiler et al.8 have shown that aureomycin increased the growth of rats
fed limiting levels of pyridoxine, pyridoxal, or pyridoxamine. These three
forms, however, were equally active in promoting growth when fed in the
diet with aureomycin. The authors postulate that the antibiotic may pre-
vent utilization or destruction of the vitamin Bes group by intestinal micro-
organisms, thus increasing the amount available to the rat.
The vitamin Bs requirement also appears to be influenced by the quali-
tative and quantitative amino acid composition of the diet. The feeding of
large amounts of pi-serine® or glycine!” ™ to rats increases their vitamin Bg
requirement. The addition of cystine or methionine to rats fed a vitamin
1R. A. Brown and M. Sturtevant, Vitamins and Hormones 7, 171 (1949).
2 B.S. Schweigert, Vitamins and Hormones 6, 55 (1948).
3C. A. Slanetz, Am. J. Vet. Research 4, 182 (1943).
4V.A. Drill and R. R. Overman, Am. J. Physiol. 185, 474 (1942).
5M. B. Richards, Brit. Med. J. 1, 433 (1945).
6C. A. Mills, Am. J. Physiol. 158, 31 (1948).
7P.S. Sarma, E. E. Snell, and C. A. Elvehjem, J. Biol. Chem. 165, 55 (1946).
8 H. Linkswiler, C. A. Baumann, and EK. E. Snell, J. Nutrition 43, 565 (1951).
9W.H. Fishman and C. Artom, Proc. Soc. Exptl. Biol. Med. 57, 241 (1944).
10H. Pagé and R. Gingras, Trans. Roy Soc. Can. 40, 119 (1946).
1, Pagé and R. Gingras, Rev. can. btol..6, 372 (1947).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 295
Be-deficient diet contaning 15% casein accelerated the deficiency and de-
creased survival time.'? When levels of methionine slightly greater than
that required for normal growth were fed to rats on a limiting amount of
vitamin B,, their growth rate was depressed; other amino acids had no
effect." Cerecedo and Foy" have shown that weanling rats on a 60%
easein diet that was deficient in vitamin Bs, developed more severe skin
lesions earlier than did animals on a lower protein diet. These findings
suggest an inverse relationship between vitamin Bs and protein intake.
The presence in the diet of unsaturated fat appears to spare pyridoxine."
Suboptimal amounts of pyridoxine become optimal when supplemented
with linoleic acid-containing fats.
Beaton et al.'** have demonstrated the importance of other factors in
determining the severity of acrodynia in pyridoxine-deficient rats. Male
rats appeared to develop the deficiency with more rapidity and severity
than female rats, indicating a higher requirement for the former. When
the deficient diet contained an incomplete protein as the only source of
nitrogen, no acrodynia developed, suggesting that the skin lesions develop
under those dietary conditions which allow an increase in body weight
when the deficient diet is supplemented with vitamin B,. On the other
hand, the injection of growth hormone into pyridoxine-deficient animals
aggravated the deficiency symptoms. Adrenalectomy prevented the ap-
pearance of acrodynia in vitamin Bg deficiency.
When rats are maintained at a temperature of 91°F. they require twice
the amount of pyridoxine necessary at 68°F .!
2. Micr
Young weanling mice on a pyridoxine-deficient diet containing 18 to 20%
casein showed a good growth response when the diet was supplemented by
1 y of pyridoxine per gram of diet.!®: 17 When the deficient diet was sup-
plemented by only 0.5 y of pyridoxine per gram of food, the animals grew
at one-half the normal rate. An increase in the protein level of the diet
elevated the requirement for vitamin Bs. Deficient mice on a 60% casein
level lived one-third as long as mice on a 10% casein diet; the mouse re-
quires three times as much pyridoxine on a diet of 60% casein than when
20 % was fed. u-Tryptophan administration decreased the survival time of
2 L. R. Cerecedo, J. R. Foy, and E. C. De Renzo, Arch. Biochem. 17, 397 (1948).
Ma H. J. DeBey, E. E. Snell, and C. A. Baumann, J. Nutrition 46, 203 (1952).
8 7,. R. Cerecedo and J. R. Foy, Arch. Biochem. 5, 207 (1944).
4H. Sherman, Vitamins and Hormones 8, 55 (1951).
Ma J. R. Beaton, J. L. Beare, and E. W. McHenry, J. Nutrition 48, 325 (1952).
1% C.A. Mills, Arch. Biochem. 1, 73 (1942).
i. C. Miller and C. A. Baumann, J. Biol. Chem. 157, 551 (1945).
“WH. P. Morris, Vitamins and Hormones 5, 175 (1947).
296 PYRIDOXINE AND RELATED COMPOUNDS
mice deficient in vitamin Bs, but not as much as casein of equivalent trypto-
phan content; amino acids other than tryptophan also appear to be in-
volved.
The growth rate and survival time of weanling mice receiving 2 y or
more of pyridoxine per gram of food are unaffected by variations of 10 to
60 % in dietary casein. Pyridoxamine and pyridoxal are less active for mice
than pyridoxine, especially on high casein diets.!8
When adult white mice are given a medium dose of x-rays (350 r. in a
single exposure), they succumb within a short time. The survival time can
be prolonged by the daily injection of 50 y of pyridoxine for 7 days preced-
ing and 13 days after irradiation.!9
3. HAMSTERS
The vitamin Bes requirement of the hamster has not been adequately es-
tablished. Schwartzman and Strauss”? have shown that the daily subcutan-
eous injection of 50 y of pyridoxine was sufficient for good growth. As in
rats, the inclusion of corn oil in the diet of vitamin Be-deficient hamsters
delayed the onset of the deficiency symptoms; fat appeared to have a spar-
ing action.
Unpublished data by Fisk”! suggest that the male hamster requires 800
y of pyridoxine per 100 g. of diet, whereas the female hamster requires 400
y per 100 g. of diet.
The hamster’s requirement for vitamin Bs appears to be the highest of
all rodents studied thus far.
4. Docs
No reliable data are available for the vitamin Bs requirement of the dog.
5. Pies
Although the precise requirement is not known, 200 y of pyridoxine per
kilogram of body weight permits the pig to grow normally and prevents the
development of anemia.?? Corn oil does not spare pyridoxine in curing ane-
mia.
6. MoNKEYS
No quantitative data are available for the monkey’s vitamin Bg require-
ment. However, it has been observed that monkeys receiving | mg. of pyri-
18 1}, C. Miller and C. A. Baumann, J. Biol. Chem. 159, 173 (1945).
19 A, Goldfeder, L. Cohen, C. Miller, and M. Singer, Proc. Soc. Exptl. Biol. Med. 67,
272 (1948).
20 G. Schwartzman and L. Strauss, J. Nutrition 38, 131 (1949).
21W. W. Fisk, B. S. Thesis, Massachusetts Institute of Technology, 1951.
22 Gq. EK. Cartwright, M. M. Wintrobe, and 8. Humphreys, J. Biol. Chem. 158, 171
(1944).
XII, REQUIREMENTS AND FACTORS INFLUENCING THEM 297
doxine daily grow normally and do not exhibit any deficiency symp-
toms.”*: ** In all probability, the daily requirement is less than 1 mg.
7. CHICKS
It has been fairly well established that chicks require 200 to 300 y of vi-
tamin Bg per 100 g. of ration for normal growth, egg production, and hatch-
ability.?°-? When linseed oil meal is fed to chicks at a 30 % level, the vitamin
Bg requirement is increased.*> Treatment of the linseed oil meal with water
removes the factor which is responsible for the increased requirement.
The Red Rock breed has a higher requirement for vitamin Bs.2’ When
Red Rock cross chicks were fed diets containing 500 y of vitamin Bs per
10.0
1.0
s
ac
i @ Chick
=
ie’)
£
0.1
on 1 10 100
Body weight, kg.
Fig. 4. Relationship of size of a species to its pyridoxine requirement.
100 g. of ration, vitamin Bg deficiency symptoms appeared. These symptoms
disappeared when the pyridoxine content of the diet was increased. This
variation in requirement may be due to genetic factors.
There is no difference in the chick’s dietary requirement for vitamin Bg
in cool temperatures and in tropical heat.*°
23K. B. McCall, H. A. Waisman, C. A. Elvehjem, and E. 8. Jones, J. Nutrition 31,
685 (1946).
4 J. F. Rinehart and L. D. Greenberg, Am. J. Pathol. 25, 481 (1949).
25 G. M. Briggs, Jr., R. C. Mills, D. M. Hegsted, C. A. Elvehjem, and E. B. Hart,
Poultry Sci. 21, 379 (1942).
26 W. W. Cravens, E. E. Sebesta, J. G. Halpin, and E. B. Hart, Poultry Sci. 25, 80
(1946).
27. H. Kratzer, F. H. Bird, V.S. Asmundson, and 8. Lepkovsky, Poultry Sci. 26,
453 (1947).
28. H. Kratzer and D. E. Williams, J. Nutrition 36, 297 (1948).
29H. L. Lucas, G. F. Heuser, and L. C. Norris, Poultry Sci. 25, 87 (1946).
#C. A. Mills, E. Cottingham, and E. Taylor, Am. J. Physiol. 149, 376 (1947).
298 PYRIDOXINE AND RELATED COMPOUNDS
The pyridoxine requirement of young ducklings is similar to that of
chicks, approximately 250 y per 100 g. of ration.*!
8. OTHER ANIMALS
Fox pups require less than 200 y per 100 g. of ration.*? Rainbow trout
need 100 to 1000 y of vitamin Bg per 100 g. of diet.
Ruminants do not require an external source of vitamin Be, for the vita-
min is synthesized in adequate amounts in the rumen and reticulum of the
sheep and in the rumen of the cow.** Milk from a cow fed a vitamin B-com-
plex deficient diet has as much pyridoxine as milk from cows fed a normal
ration.
It has been fairly well established that with the B vitamins the smaller
species have a higher relative B-vitamin requirement than the larger ones.*4
Figure 4 illustrates this fact with respect to pyridoxine.*®
B. OF MAN
P. GYORGY
As stated in the section dealing with the effects of vitamin Bg deficiency,
there cannot be any doubt as to man’s need for vitamin B,. The human
dietary requirement for vitamin Bs has not yet been definitely established.
It has been estimated to be in the neighborhood of 1.5 mg. per day for
adults,*® or about 0.03 mg. per kilogram per day.*? According to recent
observations on convulsive seizures in young infants receiving a milk
formula low in vitamin Beg, the critical level of minimal requirement seems
to lie between 0.06 and 0.1 mg. per day for infants up to 4 to 6 months of
age—thus about 0.01 to 0.02 mg. per kilogram per day.
Factors influencing the requirement for pyridoxine in man have not yet
been studied. No natural antagonists of pyridoxine are known. It appears
from clinical observation that pregnancy and x-ray irradiation might in-
crease the need for pyridoxine. More direct or indirect evidence is needed
before such a conclusion could be considered as firmly established.
31D. M. Hegsted and M. N. Rao, J. Nutrition 30, 367 (1945).
32 A. K. Schaefer, C. K. Whitehair, and C. A. Elvehjem, J. Nutrition 34, 131 (1947).
33 LL. W. McElroy and H. Goss, J. Nutrition 20, 541 (1940).
34H}. Beerstecher, Jr., Science 111, 300 (1950).
35 R. J. Williams, R. E. Eakin, E. Beerstecher, Jr., and W. Shive, 7n The Biochem-
istry of B Vitamins. Reinhold Publishing Corp., New York, 1950.
36 R. J. Williams, J. Am. Med. Assoc. 119, 1 (1942).
7 R. J. Williams, R. E. Eakin, E. Beerstecher, Jr., and W. Shive, in The Biochem-
istry of B Vitamins. Reinhold Publishing Corp., New York, 1950.
CHAPTER 15
RIBOFLAVIN
I. Nomenclature.
II. Chemistry .
A. Isolation
B. Chemical and Phy cea ropertice :
C. Constitution .
D. Synthesis .
Fe
Chemical Methads!
2. Biochemical Formation .
3. Microorganisms as Producers .
E. Specificity .
ike
2.
Growth Sauulators
Antagonists of Riboflavin . ;
3. Nitroflavins and Basically Snbstituted Teoailosuernee 5
III. Industrial Preparation
IV. Biochemical Systems .
A. Coenzymes : rs
1. General Peneecne: of F en eee mes.
2.
3. Riboflavin Adenine enaleatide
Riboflavin Phosphate.
B. Enzymes
—S —
te GW bo
15:
_—
me Oooeomant our WH
. Old Yellow Rnzy me =
. Synthetic, or Crossed, Wellow Beye
Haas Enzyme
. Diaphorase :
. Straub Flavoprotein, Soluble iterate ee
. Cytochrome C Reductase of Yeast .
. Cytochrome C Reductase of Liver .
. Xanthine Oxidase, Aldehyde Oxidase, Kant hater Oxidase
. Liver Aldehyde Oxidase.
. b-Amino Acid Oxidase
Amino Acid Oxidase, L-Hy tae
: G lycine Oxidase.
. Fumaric Hydrogenase
. Glucose Oxidase of Molds .
Histaminase, Diamino Oxidase
C. Mechanism of Action.
V. Specificity of Action
VI. Biogenesis .
VII. Estimation.
A. on sical and C herent Methods.
. Fluorometric Methods
299
eG Oxidase .
Page
300
301
303
305
309
314
314
322
324
325
325
327
331
332
334
334
336
337
337
339
339
342
343
343
344
346
347
350
351
353
354
355
355
356
356
358
359
361
361
361
361
300 RIBOFLAVIN
2. Colorimetric and Polarigraphic Methods.
3. Enzymatic Methods .
B. Biological Methods
C. Microbiological Methods
VIII. Standardization .
IX. Occurrence in Food
X. Effects of Deficiency
A. In Microorganisms.
B. In Plants .
C. In Insects .
D. In Animals
le Ratsr. ;
2. Dogs and Foxes.
Gly JeseRS} :
4. Young Rarinantae
5. Other Mammals.
6. Birds.
EK. In Man. ;
1. Oral and Facial Tenors of Achoflavinens :
2. Lesions of Scrotum and Vulva
3. Ocular Manifestations
XI. Pharmacology. ;
XII. Requirements and factors nAinenene Gihem
A. Of Animals ;
1. Effect of Dietary Const ruents
2. Effect of Environment
3. Reproduction :
4. Inherent Individual Variations ;
5. Other Factors
Of Man.
I. Nomenclature
ROBERT 8S. HARRIS
Accepted names: Riboflavin (U.S. Pharmacopeia)
Riboflavine (British Pharmacopoeia)
Obsolete names: Vitamin By» Ovoflavin
Vitamin G Lyochrome
Lactoflavin Uroflavin
Hepatoflavin
Empirical formula: Cy7H2»NsO¢
Chemical name: 6,7-Dimethyl-9-(b-1/-ribity])isoalloxazine
Page
368
369
369
372
373
374
377
377
379
379
380
380
384
385
385
386
386
387
388
389
390
391
394
394
396
397
397
397
398
398
ee EaaEEaEEoEEEeEEeEeEeEeoreeeeeeee
Il. CHEMISTRY 301
Structural formula:
ry 7 3 4 5
CH,—CHOH—CHOH—CHOH—CH,0H
H,C is IAF 0
H.C wA 3NH
+
O
II. Chemistry'
THEODOR WAGNER-JAUREGG
Lactoflavin is the original name of riboflavin, still used in Europe. Ovo-
flavin, hepatoflavin, uroflavin, etc., are historical names, indicating the
origin of the preparations, which are chemically identical with lactoflavin.
Riboflavin, the American designation, indicates that the naturally occur-
ring flavin is a derivative of (p-)ribose. This name was adopted in 1952 by
the International Commission for the Reform of Biochemical Nomencla-
ture.
Riboflavin is identical with vitamin B,. In former times, in the United
States, the term vitamin G was also used for this nutritional factor.
After vitamin B; had been obtained in pure form, the isolation of crys-
tallized riboflavin was one of the most fascinating chapters in the chemistry
of the vitamins. For the sake of its historical interest, the story of the dis-
covery of riboflavin will be told here briefly.1*
In 1927, Paul Gyorgy, at that time at the pediatric clinic of the University of
Heidelberg, began investigations on the curative factor for egg white injury, which
he called vitamin H.» Beginning in 1931, he and Edgar Lederer, who worked with
Richard Kuhn at the Kaiser Wilhelm Institute for Medical Research, Heidelberg,
attempted to isolate this vitamin. Vitamin H deficiency in rats is characterized by
a dermatitis. Since pellagra is another avitaminosis connected with skin symptoms,
it seemed useful and interesting to make a comparative study of the nutrition factor
‘ Literature references are given mainly for the last ten years. For earlier and more
references, consult textbooks and reviews, for instance: H. R. Rosenberg, Chem-
istry and Physiology of the Vitamins, pp. 153-194. Interscience Publishers, New
York, 1945; R. J. Williams, R. E. Eakin, E. Beerstecher, Jr., and W. Shive, The
Biochemistry of B-Vitamins, pp. 669-683. Reinhold Publishing Corp., New York,
1950; F. A. Robinson, The Vitamin B Complex, pp. 132-210. John Wiley and Sons,
New York, 1951; G. A. Emerson and K. Folkers, Ann. Rev. Biochem. 20, 584 (1951);
H. Vogel and H. Knobloch, Chemie und Technik der Vitamine, 3rd ed., Vol. 2,
Issues 1 and 2, pp. 129ff. Ferdinand Enke, Stuttgart, 1953.
18 See also P. Gyorgy, Nutrition Revs. 12, 97 (1954).
1 In 1940 P. Gyorgy, V. du Vigneaud, and D. Melville identified this vitamin with
the yeast growth-promoting factor biotin, which had been isolated some years
before by F. Kégl and B. Toénnis.
302 RIBOFLAVIN
connected with this disease. A lack of vitamin Bz, , the heat-stable companion of the
heat-labile vitamin B,; , at that time was considered to be the cause of pellagra. In
the beginning of 1932, the author of this article started the isolation of so-called vita-
min B, at Kuhn’s institute. Gyorgy performed the biological tests on rats, according
to the method of Sherman and Bourquin; later on, when our preparations became
purer, the diet of the animals had to be modified somewhat, since it was lacking not
only in vitamin B, but also in another member of the B-vitamin complex.
The literature contained very little and vague data on the concentration of vi-
tamin B» from yeast and liver, which turned out to be of little value for the pro-
cedure of isolation. For the adsorption of the vitamin, fuller’s earth in acid solution
had been recommended. Another valuable adsorbent soon was found, which adsorbed
vitamin By» already from neutral solution; this was ‘‘Frankonit KL”’ (a bleaching
earth produced by the Pfirschinger Mineralwerke, Kitzingen/Main, Franconia),
which since that time has been used frequently in biochemical work. Before the vita-
min B; investigation, the existence of our sample of this adsorbent had been forgotten
in one of the laboratory closets, after I had tried it with little success for the poly-
merization of isoprene.
None of the known methods was fit for elution of the vitamin from the adsorbent
in yields worth mentioning. But a wrong hypothesis about the chemical nature of
vitamin B» helped me to find the right trail. In one paper of the literature it had been
assumed that vitamin By might contain iron. With regard to the biological properties
of hemin derivatives, one would have been inclined to guess that iron porphyrin
complexes were involved. Since pyridine is a solvent for compounds of this type, this
substance, diluted with water and alcohol, was tried out for the elution of vitamin Bz
adsorbates. This attempt was a full success. The later progress of our investigation
made it clear that iron has nothing to do with vitamin B., but the wrong hypothesis
had proved useful.
The successful elution drove the isolation procedure one essential step forward. It
soon became evident that all eluates, which were active in the animal experiments,
had a greenish-yellow color and showed a yellowish-green fluorescence in the light of
a quartz lamp. Therefore, the idea came up that vitamin Bz» itself might be colored,
and the investigation was continued with attention to this assumption.
It still was difficult to obtain purified preparations of the vitamin from extracts of
yeast, liver, heart, or kidney, because of the presence of large amounts of accom-
panying substances. An 80% methanol extract of egg white turned out to be a much
better starting material. The concentrated, greenish-yellow eluates on precipitation
with AgNO; gave a brownish-red silver salt of the vitamin. In later experiments, a
precipitation with Tl.SO, was inserted for further purification. This salt had been
chosen with regard to the chemical relationship of certain thallous and silver salts.
The first few milligrams of crystallized ‘‘ovoflavin’’ became available for analysis
shortly before Christmas, 1932. The animal tests proved without any doubt the
growth-promoting nature of the substance.
At this stage of the investigation, we received knowledge that the pharmacologist
Ph. Ellinger in Diisseldorf, working on the fluorescence of animal organs, had pre-
pared a colored concentrate from skimmed milk, which obviously was similar to
ovoflavin. Our methods of purification proved to be particularly applicable to whey.
We therefore changed over to this starting material. It was, however, not possible to
handle the large amounts of liquid in the laboratory. Therefore, the first step of the
concentration, the adsorption, was carried through in a large cheese dairy in Bavaria,
With this procedure we soon were able to obtain 1 g. of erystallized “lactoflavin”’
from 5400 1. of whey, thus opening a way for the elucidation of the chemical structure
of vitamin B..
mradd
Il. CHEMISTRY 303
Other investigators had obtained impure preparations of flavin. As early as 1879,
A.W. Blyth isolated from whey a resinous preparation of a red-orange color which he
called ‘‘lactochrom.’’ In 1925, B. Bleyer and O. Kallmann attempted the purification
of the yellow pigment of whey. In 1932, I. Banga and A. Szent-Gyoérgyi obtained a
golden-yellow pigment from heart muscle, whose colored component they called
“eytoflav.”’ In 1933, Ellinger and Koschara described impure, crystalline prepara-
tions of flavin (‘‘lyochrome’’), at the same time that the isolation of pure, crystallized
lactoflavin was published under the authorship of Gyérgy, Kuhn, and Wagner-
Jauregg. The vitamin nature of the pigment was unknown before the investigations of
the latter authors. Shortly after, in 1933, L. E. Booher in the United States described
a concentrate from whey powder with the chemical and biological properties of
riboflavin. Soon other investigators followed with the isolation of riboflavin from
various natural sources.
For the understanding of the biochemical function of riboflavin, the discovery of
the ‘“‘yellow enzyme”? by Warburg and Christian in 1932 was of extraordinary im-
portance. The same authors deseribed lumiflavin, a photochemical degradation
product of riboflavin, which proved of great value for the elucidation of the chemical
structure of riboflavin (Kuhn, Rudy, Wagner. Jauregg, and coworkers, 1933-34).
The synthesis of riboflavin by Kuhn and Weygand in Heidelberg and by Karrer and
his coworkers in Ziirich in 1934 finally confirmed the structural formula.
A. ISOLATION
Three factors mainly govern the possibility of isolating riboflavin from
natural sources in a pure state and in good yield: (1) the concentration in
which the pigment occurs; (2) the amount and kind of accompanying sub-
stances; (3) the form (free or bound) in which the vitamin is present.
1. The concentrations of riboflavin in different natural materials are
listed in a later section. In cases of low concentration, one will usually have
to dispense with the isolation and to confine oneself to the quantitative
determination according to either the vitamin test or one of the fluoro-
metric methods.?
2. Considerable difficulties in the isolation of riboflavin can be caused
by the presence of accompanying substances. As an example the liver might
be mentioned. Although this organ has a very high content of riboflavin,
the isolation of the crystallized substance was rather difficult and was per-
formed only one year after the preparation of riboflavin from other sources
had been described.**: *»
3. Riboflavin occurs in its free, dialyzable form only, in the retina of the
eye, in whey, and in urine. In organs, tissues, and other living cells, ribo-
flavin is present, as riboflavin monophosphoric acid, and as riboflavin
adenine dinucleotide. The two phosphates account for practically all the
riboflavin present in rat kidneys, and 70 to 90% of the total riboflavin in
* For recent developments of the fluorometric methods to determine vitamin Bz» in
plants, see H. Roth, Biochem. Z. 320, 355 (1950).
8@ R. Kuhn and T. Wagner-Jauregg, Ber. 67, 1770 (1934).
8» P. Karrer, H. Salomon, and K. Schépp, Helv. Chim. Acta 17, 419 (1934).
304 RIBOFLAVIN
all tissues is present in the form of the dinucleotide.t Spleen contains an
enzyme that rapidly degrades the phosphate-bound forms of riboflavin to
the free vitamin.°
It has been shown that riboflavin phosphoric acid is able to form loose,
non-dialyzable complexes, for instance with a solution of pseudoglobulin
or albumin from horse serum. The separation of the flavin component and
the protein in this case can be achieved by precipitation of the latter with
ammonium sulfate.®
In order to liberate riboflavin from its natural protein-bound forms, it
is necessary to treat the mashed tissues with suitable solvents at room tem-
perature or at the boiling point of the solvent. Methanol, ethanol, acetone,
undiluted or diluted with water, and aqueous acid solutions have been used
for extraction of the vitamin. For instance, riboflavin from fresh or dried
plants has been extracted in good yields by boiling the material with 70 %
methanol for 45 minutes?
For the isolation of riboflavin from the extracts, it sometimes is useful
first to remove lipids by extraction with ether, in which the vitamin is
insoluble. Salts and glycogen in some cases can be eliminated from ribo-
flavin concentrates by fractionate precipitation with alcohol or acetone.
Impurities from fermentation liquors may be precipitated by means of
acetone, and crude riboflavin can be recovered from the concentrated filtrate
by the addition of more acetone.’ The vitamin can be extracted with butanol
and then precipitated from the extract by addition of petroleum ether.®
In the isolation of riboflavin from whey, the accompanying creatinine has
been removed by picric acid precipitation.
Precipitation of riboflavin occurs with lead acetate and with silver nitrate
in neutral solution, or with phosphotungstic acid in N H.SO,; from the
latter precipitate the phosphotungstic acid can be extracted with amyl alco-
hol. Silver nitrate or mercuric sulfate in acid solution leaves the vitamin in
solution but precipitates some accompanying substances.
Good adsorbents for riboflavin are fuller’s earth in acid solution, Florisil,
Floridin XXF, and Frankonit in neutral solution. One of the best eluents
is pyridine, diluted with aqueous methanol or ethanol;’* ammonia, tri-
ethanolamine, 0.1 N NaOH in 60% ethanol, boiling 60% ethanol, 80%
4O. A. Bessey, O. H. Lowry, and R. H. Love, J. Biol. Chem. 180, 755 (1949).
> J. L. Crammer, Nature 161, 349 (1948); R. S. Comline and F. R. Whatley, zbzd.
161, 350 (1948).
°'T. Wagner-Jauregg and H. Arnold, Biochem. Z. 299, 280 (1938).
7 Merck and Co., British Pat. 621,401 (Aug. 15, 1946) [C.A. 43, 7189 (1949)].
8 Merck and Co., U.S. Pat. 2,355,220 (Aug. 8, 1944) [C.A. 38, 6488 (1944)].
8a P. Gyorgy, R. Kuhn, and T. Wagner-Jauregg (to I. G. Farbenindustrie A. G.),
German Pat. 607,512 (Nov. 22, 1932).
Il. CHEMISTRY 305
acetone, and polyhydrie alcohols’ have been used for elution. Vitamin B»
is adsorbed by charcoal very strongly; however, elution is difficult from
this adsorbate. Adsorption occurs furthermore with lead sulfide, when this
is precipitated in a riboflavin solution; the vitamin can be extracted with
hot water from the precipitate. Riboflavin is not adsorbed by kieselguhr,
kaolin, tale, aluminum oxide, or calcium carbonate.
A combination of precipitation and adsorption methods mostly will be
necessary to isolate pure riboflavin. As examples might be mentioned the
isolation of riboflavin from egg white,!° egg yolk," whey,!: » and urine.”
A general method for the preparation of pure p-riboflavin from natural
sources has been described, which is based on adsorption on fuller’s earth,
fractionation with immiscible solvents and acetone, and crystallization
from an aqueous acetone-petroleum ether mixture; aqueous alcohol solu-
tions have been used for elution of the adsorbates.™
B. CHEMICAL AND PHYSICAL PROPERTIES
Ci7H2N4O¢: molecular weight 376.4; C 54.25%, H 5.36%, N 14.89 %.
Riboflavin crystallizes from 2 N acetic acid, alcohol, water, or pyridine
in fine orange-yellow needles. The decomposition point is 278 to 282°
(darkening at about 240°). Values for the decomposition point between 271
and 293° can be found in the literature. The vitamin is odorless and has a
bitter taste.
Riboflavin is soluble in water only to an extent of 10 to 13 mg. in 100
ml. at 25 to 27.5°, 19 mg. in 100 ml. at 40°, and 230 mg. in 100 ml. at 100°.1®
The vitamin dissolves in ethanol to 4.5 mg. % and is slightly soluble in amyl
alcohol, cyclohexanol, benzyl alcohol, and phenol or amyl acetate. The
impure material has a much higher solubility than the pure substance.
Alkali dissolves the vitamin well, but these solutions are unstable. There
is no solubility in ether, acetone, chloroform, or benzene. Formic acid dis-
solves more than | % of riboflavin.!®
For intravenous administration, sterile, supersaturated solutions of ribo-
flavin in normal saline might be employed. By heating to the boiling
°S. H. Rubin and E. De Ritter, J. Biol. Chem. 158, 639 (1945); Commercial Solvents
Corp., U.S. Pat. 2,343,254 (March 7, 1944) [C.A. 38, 3093 (1944)].
10 R. Kuhn, P. Gyorgy, and T. Wagner-Jauregg, Ber. 66, 576 (1933).
11 P. Karrer, H. Salomon, and K. Schépp, Helv. Chim. Acta 17, 735 (1934).
® R. Kuhn, P. Gyorgy, and T. Wagner-Jauregg, Ber. 66, 1034, (1933); (b) R. Kuhn,
H. Rudy, and T. Wagner-Jauregg, zbid. 66, 1950 (1933).
18 W. Koschara, Ber. 67, 761 (1934).
“RR. D. Greene and A. Black, J. Am. Chem. Soc. 59, 1820 (1937).
°C. C. Tzong, J. Fermentation Technol. (Japan) 24, 56, 187 (1946) [C.A. 44, 5975
(1950) ].
306 RIBOFLAVIN
point, a temporary concentration of 1 mg. per milliliter is said to be at-
tained. The supersaturated solution of riboflavin is fairly stable; it requires
days to crystallize.
In order to obtain more concentrated solutions, riboflavin has been dis-
solved together with other compounds which are capable of increasing its
solubility. N-Methylacetamide, L-tyrosine amide,!® tryptophan,” sodium
acetyltryptophan, urea, nicotinamide, aliphatic amidines, sodium desoxy-
cholate, veratryl alcohol, salts of boric,!®: * mono- and dihydroxybenzoic,
gallic, aminobenzoic, adenylic, and other acids,'® kynurenic and 2-phenyl-
quinoline-4-carbonic acid,'** and propylene glycol with or without the addi-
tion of a monohydroxymonoalkoxybenzaldehyde'!? have been used. Solu-
tions containing a concentration of riboflavin up to about 0.6% can
be prepared in this way. The solubility of riboflavin in nicotinamide solu-
tions at pH 5 increases from about 0.1 % to about 2.5%, when the nicotin-
amide concentration is increased from 5 to 50 %.2° When riboflavin is fused
with an amide such as urea, urethan, or nicotinamide, products are ob-
tained which yield aqueous solutions containing up to 6% of the flavin.?”
Recently a method of solubilizing riboflavin with sodium-3-hydroxy-2-
naphthoate has been developed; the solubility of riboflavin is as high as 7.9 %
in a 10% solution of the solubilizer.24 Borax and alkali are said to give a
complex with riboflavin of the formula C,;HigOsNyNa- Na2BsO7: 10H20.”
Water-soluble derivatives of riboflavin'® include esters with phosphoric,
sulfuric,?"* gallic, aminoacetic, phthalic, succinic,?’ citric, malic, tartaric,
and levulinic acids, and methylol and acetal derivatives.” In the synthesis
of methylol derivative, preparations with as high as 55 % microbiological
activity can be obtained in short reaction time, when only 1 mole of formal-
dehyde is combined with 1 mole of riboflavin. Upon addition of 2 or more
16 Wyeth Inc., U.S. Pat. 2,445,208 (Sept. 13, 1948) [C.A. 42, 6496 (1948)].
17R. A. Harte and J. L. Chen, J. Am. Pharm. Assoc. Sct. Ed. 38, 568 (1949).
7a T), V. Frost, J. Biol. Chem. 145, 693 (1942).
18 Kor literature references, see K. Schoen and 8. M. Gordon, Archives Biochem. 22,
149 (1949).
18a C_§. Runti, Farm. sci. e tec. (Pavia) 7, 344 (1952).
19 American Cyanamid Co., U.S. Pat. 2,449,041 (Sept. 7, 1948) [C.A. 42, 9094 (1948)];
Wyeth Inc., U.S. Pat. 2,449,640 (Sept. 21, 1948) [C.A. 42, 9094 (1948)].
20 T). V. Frost, J. Am. Chem. Soc. 69, 1064 (1947); Abbott Laboratories U. 8S. Pat.
2,407,412 (Sept. 10, 1946) [C.A. 41, 254. (1947)].
20a Merck and Co., U.S. Pat. 2,480,517 (Aug. 30, 1949). [C.A. 48, 9088 (1949)].
21 A. Arnold, M. E. Auerbach, J. R. Shepherd, and 8. D. Sobell of Sterling-Winthrop
Research Institute; see Chem. Eng. News 80, 1414 (1952); Poultry Sci. 31, 350 (1952).
22 Winthrop Chemical Co., British Pat. 560,631, (April 13, 1944) [C.A. 40, 2593 (1946) ];
U.S. Pat. 2,332,548 (Oct. 26, 1943) [C.A. 38, 1849 (1944)].
2a G. B. Stone, Science 111, 283 (1950).
23M. F. Furter, G. J. Haas, and S. H. Rubin, J. Biol. Chem. 160, 293 (1945); Merck
and Co., U. S. Pat. 2,358,356 (Sept. 19, 1944) [C.A. 89, 1514 (1945)).
Il. CHEMISTRY 307
moles of formaldehyde, the activity falls off rapidly. As in the case of tri-
and tetrasuccinates, the sulfate is microbiologically active only after pre-
vious hydrolysis. Riboflavin mono- and disuccinates have vitamin By»
activities for the rat which are 100 % and 65 %, respectively, of riboflavin’s
activity. Both the mono- and diacetone derivatives of riboflavin are active
in the nutrition of rats. Riboflavin-5’-phosphate is fully as active in the rat
as riboflavin (oral and parenteral administration), as well as in the micro-
biological test. The same is true for flavin adenine dinucleotide.
Neutral solutions of riboflavin have a greenish-yellow color. The absorp-
tion spectrum shows characteristic absorption maxima at 475, 445, 359-
372, 268, and 223 mu.” The absorption in the visible part of the spec-
trum has been used for quantitative determination of riboflavin.
Neutral aqueous solutions of riboflavin display intense yellowish-green
fluorescence, with a maximum at 565 my which can be used for quantitative
determination of the vitamin. The fluorescence vanishes on the addition
of acids or alkalis; optimal fluorescence occurs at pH 4 to 8.4 A recent study
of the fluorescence of riboflavin and flavin adenine dinucleotide (FAD)
includes the effect of different quenchers. The relatively weak fluorescence
of FAD may be caused by internal quenching by interaction of the alloxa-
zine and adenine portions of the molecule.”®
Riboflavin has an amphoteric character. Its dissociation constants are
Ka = 6.3 X 10-” and K, = 0.5 X 1075; the isoelectric point corresponds
to a pH of 6.0. The pH of the saturated aqueous solution is approximately
6.74
The optical activity of riboflavin in neutral and acid solutions is exceed-
ingly small. In an alkaline medium, the optical rotation is strongly de-
pendent upon the concentration: {a};'° = —70° (¢ = 0.06 %; 0.1 N NaOH);
fa 21° = —117° (c = 0.5%; 0.1 N NaOH).”* Borate-containing solutions are
strongly dextrorotatory: [a]??? = +340° (pH 12); in this case the rotation
depends only slightly upon the riboflavin concentration.”
Neutral aqueous solutions of riboflavin are relatively heat stable if pro-
tected from light and can be sterilized by autoclaving for a short time;
only slight destruction occurs by heating to 120° for 6 hours. At room tem-
perature (27°) decomposition of buffered solutions (pH 5 and 6) takes place
at rates of 3 and 1.2% per month. No appreciable destruction of the vita-
min can be observed during the cooking of food,”® but when milk in bottles
24 R. Kuhn and G. Moruzzi, Ber. 67, 888 (1934); F. Kavanagh, Arch. Biochem. 20,
315 (1949).
25 G. Weber, Biochem. J. 47, 114 (1950).
25 R. Kuhn, H. Rudy, and F. Weygand, Ber. 68, 625 (1935). For the rule which governs
the rotation of different 9-polyhydroxyalky] flavins, see F. Weygand, Ber. 73, 1278
(1940).
27 R. Kuhn and H. Rudy, Ber. 68, 169 (1935).
*2R. R. Williams and V. H. Cheldelin, Science 96, 22 (1942).
308 RIBOFLAVIN
is exposed to sunlight, more than half of the riboflavin is destroyed within
2 hours.?*: °° The rate of destruction by light becomes higher with increas-
ing temperature and pH.?> Alkali decomposes riboflavin rapidly.
Riboflavin is stable against acids, air, and the common oxidizing agents
(except chromic acid, KMnQO,, and potassium persulfate), bromine and
nitrous acid. The stability of riboflavin has been used for the purification
of the crude synthetic product; in acid solution impurities are oxidized at
a temperature below 100° with use of Cle, H2O2, HNO;, or HCIO3;.22 But
the vitamin is destroyed by hydrogen peroxide in the presence of ferrous
ions.
Reducing agents such as sodium dithionite (Na2S2O,4), zine in acid solu-
tion, catalytically activated hydrogen, and titanous chloride transform
riboflavin in alkaline, neutral, or acetic acid solutions directly into a color-
less dihydroflavin, which is reoxidized on shaking with air. The potential
of an equimolecular mixture of riboflavin and its leuco compound at pH
7.0 is —0.185 volt (—0.146 volt at pH 5.9), pretty much on the negative
side. Combination with the enzyme protein has been shown to raise -the
redox potential from —0.19 volt for p-riboflavin-5’-phosphate to —0.06
volt for the ‘fold yellow enzyme.’
By the action of zinc, tin, or sodium amalgam in strong HCl (pH < 1),
a red reduction intermediate forms which is a semiquinone radical.*> *4
This behavior of riboflavin might be useful for its detection.
Riboflavin gives a red-violet color with concentrated H»SO,, which
changes to yellow on dilution. When heated with 50% NaOH solution,
riboflavin produces a green color, changing to red on dilution.*®
Riboflavin shows a heightened affinity for Fet*.*** It has been found
associated with iron in a protein (conalbumin) which occurs in the avian
blood stream and in egg white.?°>
Bacteriostatic effects of riboflavins have been observed only in the light.
These may be explained possibly by the formation of toxic products and
in part by destruction of needed nutrients. It has been demonstrated that
29 W. J. Peterson, F. M. Haig, and A. O. Shaw, J. Am. Chem. Soc. 66, 662 (1944).
30 J. A. Ziegler, J. Am. Chem. Soc. 66, 1039 (1944).
31 R. Posternack and E. V. Brown (to Charles Pfizer and Co.), U.S. Pat. 2,824,800
(July 20, 1944) [C.A. 38, 221 (1944)].
32 R. Kuhn and P. Boulanger, Ber. 69, 1557 (1936).
33 R. Kuhn and T. Wagner-Jauregg, Ber. 67, 361 (1934). See also K. G. Stern, Bio-
Chem J, 28, 949 (1934).
34 T,, Michaelis, M. P. Schubert, and C. V. Smythe, J. Biol. Chem. 116, 587 (1936).
35 M. Z. Barakat and N. Badran, J. Pharm. and Pharmacol. 3, 501 (1951).
36a A Albert, Biochem. J. 54, 646 (1953); other metal chelates of riboflavin were de-
scribed recently by W. O. Faye and W. E. Lange, J. Am. Chem. Soc. 76, 2199 (1954).
35b J. Bain and H. Deutsch, J. Biol. Chem. 172, 547 (1948).
Il. CHEMISTRY 309
in the presence of riboflavin irradiation causes destruction of tryptophan
and pyridoxine.*®
Riboflavin is practically non-toxic. The toxicity to mice by intraperito-
neal injection amounts to 340 mg. per kilogram.*® The LDs5o9 value for rats,
using the same form of application, is 560 mg. per kilogram.*’ The adminis-
tration of 10 g. per kilogram orally to rats or 2 g. per kilogram orally to
dogs showed no toxic effects.°
C. CONSTITUTION
OH OH OH
V Le lS ars ee
CH,—C—C—C—-CH.0H
onieltae i
H H H
H;C N N
We 7 Sey
7 |
|
6 9
VAN ae a
H;C NN <
|
O
6,7-Dimethy]-9-(p-]’-ribityl)isoalloxazine
The side chain of riboflavin is characterized by the following reactions:
Acetylation with acetic anhydride in pyridine gives a chloroform-soluble
tetraacetate, melting at 242 to 243°. It is easily saponified by diluted alkali
at room temperature; its vitamin By, activity for rats is almost the same as
that of riboflavin. The formation of a tetraacetate indicates the presence
of four hydroxyl groups.
Formation of a diacetone compound indicates that two hydroxyl groups
in pairs are adjacent. Oxidation of riboflavin with lead tetraacetate yields
0.8 mole of formaldehyde. That proves the presence of a primary hydroxy]
group in the a position to a secondary hydroxyl group.
The oxygen-containing part of the side chain of riboflavin can be removed
by irradiation in alkaline solution. The resulting lumiflavin (m.p. 330°),
in contrast to riboflavin, is chloroform-soluble.!”*; >» *8 Irradiation of ribo-
35¢e M. N. Meisel and E. M. Dikanskaya, Doklady Akad. Nauk S.S.S.R. 85, 1377
(1952) [C.A. 47, 2258 (1953)].
36 R. Kuhn and P. Boulanger, Z. physiol. Chem. 241, 233 (1936).
37 K. Unna and J. G. Greslin, J. Pharmacol. 76, 75 (1942).
37a V. Demole, Z. Vitaminforsch. 7, 138 (1938).
88 Q. Warburg and W. Christian, Naturwissenschaflen 20, 980 (1932); Biochem. Z. 266,
377 (1933).
310 RIBOFLAVIN
flavin in neutral or acid solution removes the entire side chain, yielding
lumichrome.*? Lumichrome is formed also by stoichiometrical oxidation of
riboflavin by Pseudomonas riboflavina*® or by mycobacteria.* Deutero-
flavin is a third irradiation product of riboflavin.” > The photochemical
behavior of vitamin B» is demonstrated in the following scheme?!:
——O
Riboflavin
light
acid or neutal alkaline neutral solution,
solution solution absence of Oz
CH;
H;3C N NH H3C N N
NS iN |
ie SW CO SH CO Deutero-
alkali Deutero- 0,
| | | = aes leuco-
NH NH ; flavin
Va NON NEN
H3C N CO H3C N CO
Lumichrome Lumiflavin
6,7-Dimethylalloxazine 6,7, 9-Trimethylisoalloxazine
Lumichrome is the 6,7-dimethyl derivative of alloxazine (I), whereas
riboflavin and lumiflavin are substitution products of the hypothetical
isoalloxazine (II).
H
|
N NH N N
fa ye CO ae
| |
NH NH
NANG 7 Nee
N CO N (x0)
I Il
Alloxazine Tsoalloxazine
In the photolysis of 9-(2’-hydroxyethy])isoalloxazine, alloxazine is formed
39 P, Karrer, H. Salomon, K. Schépp, E. Schlittler, and H. Fritzsche, Helv. Chim.
Acta 17, 1010 (1934).
40 J. W. Foster, J. Bacteriol. 47, 27 (1944); 48, 97 (1944) [C.A. 38, 1761, 5526 (1944)].
10a H. C. Hou, Proc. Soc. Exptl. Biol. Med. 70, 581 (1949).
‘1 The reaction mechanism of the decomposition of riboflavin by light has been dis-
cussed recently by R. Brdicka, Collection Czechoslov. Chem. Communs. 14, 130
(1949) [C.A. 44, 4337 (1950)]; see also ref. 42.
Il. CHEMISTRY OLt
and the side chain produces acetaldehyde, formaldehyde, and an acid,
probably formic acid.”
The alkaline hydrolysis of riboflavine gives urea and 1 ,2-dihydro-6 ,7-
dimethyl-2-keto-1-p-ribityl-3-quinoxaline-carboxylie acid (III). (This acid
has recently been shown to have a depressant action on cardiac and visceral
muscles when injected intravenously in the dog.**) In the case of lumiflavin,
1 ,2-dihydro-2-keto-1 ,6 ,7-trimethyl-3-quinoxalinecarboxylic acid (IV) is
obtained along with urea.» 43» 44
R R
| |
H3C N N H3C N
Ye re WW ‘co SS va eG
| fF Seite | + NH,CONH,
3C N CO H3C N
. III, R = p-ribityl; IV, R = CH;
The oxocarbonic acid (IV) can be decarboxylated by sublimation with
formation of the lactam (V). This, when heated with NaOH, gives 1 ,2-di-
methyl-4-amino-5-methylaminobenzene (VI).
CH; CH;
| |
H;C N H;C N
~~ SS
esol nian) YAU Ss
—CO2 | NaO
C-COOH CH
NY 7 ASS Oy’
H;C N H;3C N
IV V
H;C NHCH;
~
Aas
H;C NH:
VI
On reduction, riboflavin readily takes up two hydrogen atoms with for-
4M. Halwer, J. Am. Chem. Soc. 73, 4870 (1951).
43 A. R. Surrey and F. C. Nachod, J. Am. Chem. Soc. 78, 2336 (1951).
““R. Kuhn and T. Wagner-Jauregg, Ber. 66, 1577 (1933); R. Kuhn and H. Rudy,
Ber. 67, 892, (1934); R. Kuhn, K. Reinemund, and F. Weygand, Ber. 67, 1460
(1934).
4 RIBOFLAVIN
mation of a leuco compound.” » The colorless dihydroriboflavin is reoxi-
dized easily to riboflavin by air. Stronger catalytic hydrogenation of flavins
yields octahydroflavins, which are easily oxidized in alkaline solution by
air to the corresponding hexahydroflavins.”
N N
Ss ~
yi Ww; CO VE Wwe CO
, - Be ,
NH NH
SEAN NN Yi
N CO NH CO
Hexahydroflavin Octahydroflavin
Three intermediate compounds have been obtained in the crystalline
state by stepwise reduction of riboflavin to leucoriboflavin. They consist
of molecular compounds of reduced and unreduced and radical-like inter-
mediate molecules.*: 4® In verdoflavin 1 mole of riboflavin and | mole of
monohydroriboflavin (with a free valence) are associated; chloroflavin is
probably partly free monohydroriboflavin and partly a quinhydrone,
formed of riboflavin and leucoriboflavin; rhodoflavin contains the hydro-
chlorides of leucoriboflavin and monohydroriboflavin:
R Consumption
| onyeen WEDS
N N Onda ian
SS
aN ve V7, CO
| 0
NH
YIN Sf
N CO
Flavin (yellow)
tl
4° P. Karrer and R. Ostwald, Rec. trav. chim. 57, 500 (1988).
46 R. Kuhn and R. Strébele, Ber. 70, 747, 753 (1987).
II. CHEMISTRY
Verdoflavin (bronze-green)
iN
R
N NH
y, Y “eo
ee a
N CO
Chloroflavin (grass-green)
i
R
i NH N NH
TC GEN UB
llewiee?- clean
Pern /\/s an
NH CO NE Co
tl
R
SNH
vy \/ co
|
ae IN a
NH CO
Leucoriboflavin (colorless)
313
Consumption
of moles
oxygen upon
oxidation
0.25
0.50
314 RIBOFLAVIN
By analysis of the titration curves, Michaelis and Schwarzenbach”
showed that in solution, at low concentration including the physiological
concentration range, there is an intermediate form of reduction which is
entirely represented by a free radical. The maximum ratio of this to the
total dye is 0.10 at pH 4.62, and 0.14 at pH 6.92, at 30°. In higher concen-
trations, a partial dimerization of the radical to a bimolecular compound
takes place. No other molecular species on an oxidation level between
flavin and dihydroflavin could be detected in solution.
On reduction of the yellow enzyme (riboflavin phosphate attached to a
specific protein) at 0° by the reduced triphosphopyridine nucleotide (co-
enzyme), under anaerobic conditions, a red intermediate is obtained which
possesses the same absorption spectrum as rhodoflavin. Thus the red reduc-
tion form of riboflavin can be produced under nearly neutral conditions.
D. SYNTHESIS
1. CoemicaL Metruops
In 1891, O. Kiihling synthesized alloxazines by condensation of o-phenyl-
enediamine hydrochloride with alloxan.
NH, NE N NH
+ aed
OC NH & yy, NH
~ OY SAA
NH, CO
Using the same principle, R. Kuhn and P. Karrer worked out methods
for the synthesis of flavins, based on o-xylene, D-ribose, and alloxan as
CH.OH CH.OH
|
HOCH HOCH
HOCH HOCH
I III
HOCH HOCH
II
CH, CH,
| HO N |
H;C NH SOUZA H;C N
C CO \4A\/ ae
|
so C NH” | ie
Yo we VAN NGA
H;C NH, O (ere) H;C N (ere)
47 T,, Michaelis and G. Schwarzenbach, J. Biol. Chem. 128, 527 (1938).
Il. CHEMISTRY 315
starting materials. Riboflavin (III) could be obtained by condensation of
1 ,2-dimethyl-4-amino-5-(p-l’-ribitylamine)benzene (I) with alloxan, which
reacts in its lactim form (II).
The reaction is carried out in acid solution. Boric acid as a catalyst in-
creases the yield considerably.*: *° Other catalysts are H2S, SnCl., or allox-
antin in the presence of 1 mole of HCI.°°
Four representative examples of riboflavin synthesis are given in the
following, which differ in the preparation of the intermediate I.
(1) This intermediate can be prepared by condensation of o-nitroxylidine
(IV) with p-ribose®! and catalytic reduction of the formed riboside (V)
to the diamine (I). The yield was 16% riboflavin, calculated on the
amount of ribose used.
Sena ; see
HC NH, H.C NHCHCH (OH) CH;
Ne lA
IV aed cl | Vv at
VIN ON
HC NO: HC NO,
Recently, 6,7-diethyl-9-(p-l’-ribity])isoalloxazine has been prepared by
this method.*
(2) o-Nitrochlorobenzenes have been reacted with amino sugars or amino
alcohols, and the condensation product was hydrogenated to the diamine.*!
Poor yields are obtained with sugars containing four and five hydroxyl
groups, but sugars with shorter chains (n < 4) give satisfactory yields.
The required glycamines can be prepared by hydrogenation of the cor-
48 R. Kuhn, Angew. Chem. 49, 6 (1936).
49 R. Kuhn and F. Weygand, Ber. 68, 1282 (1935).
50 Hoffmann-La Roche and Co., British Pat. 628,410 (Aug. 29, 1949) [C.A. 44, 4935
(1950) ].
51 By condensation of aromatic amines with p-ribose, two isomers are obtained which
have been considered to be the corresponding N-arylribofuranosylamines (A) and
N-arylribopyranosylamines (B); A is converted to B in the presence of water,
[L. Berger and J. Lee, J. Org. Chem. 11, 75, 84, 91 (1946); G. P. Ellis and J. Honey-
man, Nature 167, 239 (1951); J. Chem. Soc. 1952, 1490 2053]. The NasSO, complexes
of arylamine-N-p-ribopyranosides can be hydrogenated to the corresponding
ribitylamines in excellent yield [Hoffmann-La Roche, Inc., U.S. Pats. 2,384,102,
2,384,105 (Sept. 4, 1945) [C.A. 40, 600, 2854 (1946)].
52 R. Kuhn and R. Strébele, Ber. 70, 773 (1937).
53 J. P. Lambooy, J. Am. Chem. Soc. 72, 5225 (1950).
54 P. Karrer, H. Salomon, K. Schépp, and E. Schlittler, Helv. Chim. Acta 17, 1165
(1934); P. Karrer, E. Schlittler, K. Pfaehler, and F. Benz, zbid. 17, 1516 (1934).
316 RIBOFLAVIN
(ll INEL@H. (CHOE) Cit OF
YEE VA
4+ H2NCH:(CHOH)nCH20H 4H
| —HCl 7a | a:
VN VN
NO, NO,
NHCH.2(CHOH), CH.0OH
\
NH:
responding sugars in liquid ammonia containing 3 % of water, over a Raney
nickel catalyst at 85° and 200 p.s.i.*°
9-(6-Hydroxyethy])isoalloxazine,** —6-nitro-9-(6-hydroxyethy])isoaloxa-
zine, 9-(8-diethylaminoethyl])isoalloxazine, 6-nitro-9-(8-diethylaminoethy])
isoalloxazine, and other basically substituted isoalloxazines®® have been
prepared by this method. 9-(Dialkylaminoalky])isoalloxazines, the free
bases, differ chemically from riboflavin by their solubility in organic sol-
vents, for instance CHC] .
(3) Another method for the synthesis of substituted 2-nitroanilines which
are needed for the synthesis of riboflavin is the condensation of substituted
o-dinitrobenzene with sugar amines. For instance, o-dinitroxylene and riba-
mine are condensed in aqueous alcoholic solution and catalytically reduced
to the corresponding diamine. The over-all yield of riboflavin amounted to
4.5% of the ribose used.*”
H3C NO2 H3C NHR H3C NH-R
NIN Ne WA
| +H:NR , | +H
Ye NYS. VON AS VAN
H;C NO» H;3C NO, H;C NH,
3-Methylriboflavin®® and, recently, 6 ,7-dichloro-9-(1’-p-sorbityl)isoalox-
azine and its analogs have been synthesized by this method; a variant uses
substituted o-iodonitro-benzenes as starting materials.°?
(4) A fourth method of riboflavin synthesis starts with the condensation
of 3 ,4-xylidine with p-ribose by boiling the amine and the sugar in alcoholic
55 R. B. Flint and P. L. Salzberg, U.S. Pat. 2,016,962 (1932); F. W. Holly, E. W. Peel,
K. Folkers et al. J. Am. Chem. Soc. 72, 5416 (1950); 78, 332 (1951); 74, 4047 (1952).
56 F. Kipnis, N. Weiner, and P. E. Spoerri, J. Am. Chem. Soc. 69, 799 (1947).
*7 RR. Kuhn and F. Weygand, Ber. 68, 1001 (1935).
68 R. Kuhn, K. Reinemund, F. Weygand, and R. Strébele, Ber. 68, 1765 (1935).
59 FW. Holly, E. W. Peel, R. Mozingo, J. J. Cahill, F. R. Koniuszy, C. H. Shunk,
and K. Folkers, J. Am. Chem. Soc. 72, 5416 (1950); 74, 4047, 4251 (1952).
Il. CHEMISTRY 317
solution.®: © The 3 ,4-xylidine-N-p-riboside formed is catalytically reduced
without isolation of the reaction product prior to hydrogenation.” Karrer
and Meerwein® have shown that coupling with phenyldiazonium salt gives
the corresponding azo dye, with a yield of 92 % of the theoretical amount.
The reduction to (2-amino-4,5-dimethylphenyl)-p-1’-ribamine can be per-
formed with 85 % of the theoretical yield.*
0
——-
| OH OH OH
Ee
HC NH; HC NH—C—C—C—C—CH:
ON OE, Li: ae bc
+ p-ribose | af sl Jel isl +H
pa aN a=
H;C H;C
H;C NHCH>2(CHOH);CH:0H
SG
| +CsHsN2Cl ,
NN
H;C
H3C NHCH:(CHOH);CH20H
ES
+H,
i
H;3C N=N—C Hs
H,C NHCH:2(CHOH);CH;0H
( |
FOS a
H;C NH.
This method can be used for industrial preparation of riboflavin. The
yield obtained is very high, 38 % calculated for ribose. The method is limited
to the synthesis of 6,7-substituted flavins, because only m,p-disubstituted
aniline derivatives couple with diazonium salts in the ortho position.
60 P. Karrer et al., Helv. Chim. Acta 18, 1435 (1935); W. A. Wisansky and 8. Ansbacher,
J. Am. Chem. Soc. 63, 2532 (1941).
61 R. Kuhn and L. Birkhofer, Ber. 71, 621 (1938).
62 Hoffman-La Roche, Inc., U. 8. Pat. 2,477,560 (Aug. 2, 1949) [C.A. 44, 169 (1951)].
68 P| Karrer and H. Meerwein, Helv. Chim. Acta 18, 1130 (1935); 19, 264 (1936).
64 P. Karrer, Helv. Chim. Acta 30, 2101 (1947).
318 RIBOFLAVIN
p-Ribose, needed for the riboflavin synthesis described, can be obtained
either from natural sources or by synthetic methods. It has been prepared
by hydrolysis of yeast nucleic acid.*® From 2 kg. of yeast, only 1 to 2 g.
of pure D-ribose have been obtained via yeast nucleic acid and guanosine.
The synthetic method starts with glucose, which, via calcium gluconate,
is converted to p-ribose through the following steps: D-arabinose, dia-
cetyl-arabinal, p-arabinal. The latter, by oxidation with perbenzoic acid,
gives a mixture of D-arabinose and p-ribose, with a yield of 10 to 17 %.*8> %
The sirupy ribose prepared by this method can be obtained crystallized
by conversion to aniline-N-p-ribofuranoside and subsequent hydrolysis
(Berger and Lee**).
Recently processes have been developed whereby ribose can be prepared
directly by electrolytic reduction of ribonolactone. The corresponding acid
can be obtained by rearrangement of arabonic acid, which usually is pro-
duced by the oxidation of corn sugar in alkaline solution with oxygen or
air. By a newer method of the Northern Regional Research Laboratory,
calcium arabonate is obtained with 85 % yield by electrolytic oxidation of
2-ketogluconate.”
Since the preparation of D-ribose forms a bottleneck in the synthesis of
riboflavin, methods have been developed which avoid the use of ribose.
F. Weygand® in 1940 showed that it is possible to use D-arabinose for
the synthesis of riboflavin. N-p-Arabinoside of xylidine (I) is transformed
by a so-called Amadori isomerization into the isoarabinose derivative II,
which under alkaline conditions (possibly favoring the keto form) can be
hydrogenated to the intermediate III of the riboflavin synthesis. The
yield is about 13 % of the pentose used.
Later, processes of technical importance were developed which avoid the
primary use of pentoses altogether and operate with p-ribonic acid or its
lactone. This sugar acid can be obtained by pyridine epimerization of D-
arabonic acid, which in its turn is prepared from D-glucose.
In the procedure of Pfizer and Co.,®° p-ribonamide is acetylated and the
65 H. Bredereck, Ber. 71, 408 (1938); H. Bredereck, M. Kothnig, and E. Berger, Ber.
73, 956 (1940).
66 M. Gehrke and F. X. Aichner, Ber. 60, 918 (1927); W. C. Austin and F. L. Humoller,
J. Am. Chem. Soc. 56, 1152 (1934); T. Reichstein and M. Steiger, Helv. Chim. Acta
19, 189, 193 (1936).
67 ©, L. Mehltretter, in ‘“Crops in Peace and War,”’ Yearbook Agr. U. S. Dept. Agr.
1950-51, 782.
68 F. Weygand, Ber. 78, 1259, 1264 (1940). Based on a similar principle is the modified
synthesis of riboflavin described by V. M. Berezovskil, V. A. Kurdyukova and N.,
A. Preobrazhanskil, J. Appl. Chem. (U.S.S.R.) 22, 527, 533 (1949) [C.A. 44, 2530
(1950)]. A variant of Weygand’s method forms the object of a patent of the Miles
Laboratories, Inc., British Pat. 594,949 (Nov. 24, 1947) [C.A. 42, 2630 (1948)].
69 Pfizer and Co., Inc., British Pats. 545,360 (May 21, 1942), 551,401 (Feb. 25, 1943)
[C.A. 38, 5845, 2344 (1944)], 585,212 (Feb. 3, 1947) [C.A. 41, 3815 (1947)].
II. CHEMISTRY
319
ian CH.OH
|
ai ae
aA 75° heh cox Pt + Ha
O | Amadori | alkaline
HCOH isomerization CO solution
| | |
i rie
H;C NH H;C NH
V4 “SAN
| x Ul
FN
H;C H;C
CH,.0H
|
HOCH
HOCH
HOCH
CH,
H3C NH
er
III
yi
H;C
reaction product is converted into tetraacetylribonic acid by treatment
with nitrous acid, which then is reacted with PCI; to form the acid chloride.
This is reduced to give tetraacetyl-p-ribose, palladium supported on BaSO,
being used as a catalyst. Hydrogenation of tetraacetyl-p-ribose in the pres-
ence of o-4-xylidine, with Raney nickel or platinum as catalyst, yields
tetraacetyl-1-p-ribityl-o-4-xylidine, which finally is coupled with a phenyl-
diazonium salt. A similar method uses tetrabutyril-p-ribonamide as a start-
ing material.7°
A somewhat different method starts with p-ribonolactone, prepared from
D-arabonic acid via p-ribonic acid.” The lactone is reacted with xylidine,
70 Merck and Co., U.S. Pat. 2,424,341 (Sept. 22, 1947) [C.A. 42, 211 (1948)].
1M. Tishler, N. L. Wendler, K. Ladenburg, and J. W. Wellman, J. Am. Chem. Soc.
66, 1328 (1944); Merck and Co., U.S. Pat. 2,420,210 (May 6, 1947) [C.A. 41, 5548
(1947)]; Hoffman-La Roche Inc., U.S. Pats. 2,488,881, 2,488,883 (March 3, 1948)
[C.A. 42, 5048 (1948)].
320 RIBOFLAVIN
and the ribonic xylidide, after acetylation, is chlorinated to the imidochlo-
ride, which can be reduced smoothly to the amine and then be deacetylated.
HC NH:
ve
Jpzibonplactone SR INEIC O (CHO) CHE Ort =>
Vi RNHCO(CHOCOCH,),CH,OCOCH; —“—>
HC RN=C(CHOCOCH,),CH,0OCOCH; —2>
RNH=> Cl
RNHCH,.(CHOCOCH;) ;CH,OCOCH; 22 —>
RNHCH.(CHOH);CH,OH
In another procedure 3 ,4-dimethylaniline and tetraacetyl-p-ribonitrile”
are subjected to catalytic reductive coupling and the resulting acetylated
amine is deacetylated.
Alloxan, which was needed for the earlier synthesis of riboflavin, can be
obtained by oxidation of uric acid or barbituric acid.
The newer methods use barbituric acid or its derivative directly. The
condensation of an appropriate o-aminoazo compound with barbiturie acid
can be carried through in the presence of a weak organic acid, such as acetic
acid.”
R
|
HC NHR UPR H;C NON
DN Ui yo OC CO % Ufa re ‘eo
| “oe ti | = | + ArNH
H2C NH NH
ESVES Bs IR Ry
H;C N==NaAr CO H;C N CO
R = v-Ribityl; Barbiturie acid Riboflavin
R = tetraacetyl-p-
ribityl
L-Lyxoflavin recently was synthesized according to this method.”
Previously, Bergel et al.7> used a method to convert N-p-ribityl-o-4-
? K. Ladenburg, M. Tishler, J. W. Wellman, and R. D. Babson, J. Am. Chem. Soc.
66, 1217 (1944).
78M. Tishler and G. H. Carlson, U.S. Pat. 2,350,376 (1944) [C.A. 38, 4963 (1944)];
M. Tishler, K. Pfister, 3rd, R. D. Babson, K. Ladenburg, and A. J. Fleming,
J. Am. Chem. Soc. 69, 1487 (1947).
™D. Heyl, E. C. Chase, F. R. Koniuszy, and K. Folkers, J. Am. Chem. Soc. 78,
3826 (1951).
7°. Bergel, A. Cohen, and J. W. Haworth, J. Chem. Soc. 1945, 165; Hoffman-La
Roche and Co., British Pats. 550,169 (Dec. 28, 1942) [C.A. 38, 1247 (1944)], 550,836
(Jan. 27, 19438) [C.A. 38, 1752 (1944)].
Il. CHEMISTRY ool
xylidine into riboflavin by coupling with diazotized aniline and shaking the
resulting azo compound with excess alloxantin or dialuric acid in an atmos-
phere of nitrogen, finally oxidizing any leucoriboflavin by shaking with air.
A method involving neither ribose nor alloxan is the following:7® 3 ,4-
dimethylaniline (I) is reductively condensed in the presence of a palladium
catalyst with tetraacetyl-p-ribononitrile (II) with loss of NH3. Ribonitrile
can be prepared from ribonic acid via the amide. The formed N-tetraacetyl-
p-ribitylamino-3 , 4-dimethylaniline (III) is coupled with p-nitrophenyldia-
zonium chloride, and the product is reduced in the presence of a platinum
catalyst to 1-N-tetraacetylribitylamino-2-amino-4 ,5-dimethylbenzene (IV).
This compound is then condensed with 5,5-dichlorobarbituric acid (V)
to form tetraacetylriboflavin (VI) which is then hydrolyzed to riboflavin.
CH,OAc CH,OAc
|
II (HCOAe); UI (HCOAe);
|
CN CH.
I + |
H;C NH, H;3C NH
Sa
+He
=NHi as
rf He
H.C H;C
CH,OAc CH,OAc
|
(HCOAc); (HCOAc);
IV VI
CH. V CH:
| |
H3C NH axe H;C N
Va ore co NANO SAaneo
| ss Cl1.C NH = | ue
yy Ae we VO BN aE
H;3C NH. CO 3C N CO
6 ,7-Dimethyl-9-benzylisoalloxazine can be formed by heating 5,5-di-
chlorobarbituric acid in pyridine with 1-benzylamino-2-amino-4,, 5-dimeth-
ylbenzene. Similarly, 5-amino-N-ribityl-o-4-xylidine and 5 ,5-dichlorobarbi-
turic acid gives riboflavin in excellent yield.
Synthesis of Riboflavin-5'’~phosphate (flavin mononucleotide, FMN). The
phosphorylation of riboflavin with phosphoryl chloride in pyridine provides
76M. Tishler and J. W. Wellman (to Merck and Co.), U.S. Pat. 2,261,608 (Nov. 4,
1941) [C.A. 36, 1050 (1942)]; M. Tishler, J. W. Wellman, and K, Ladenburg, J. Am.
Chem. Soc. 67, 2165 (1945).
a2e RIBOFLAVIN
a method for small-scale preparation of riboflavin-5’-phosphate. The origi-
nal method of Kuhn and Rudy” yields mainly a cyclic phosphate, ribo-
flavin-4’ ,5’-phosphate, as shown by Forrest and Todd.” Acid hydrolysis
of the cyclic ester gives riboflavin-5’-phosphate, which is identical with the
natural riboflavin phosphate.
OH
OH OH OH
CH.—C—C—C—CH,O0P=O
| H H
OH
H;C N N
| | D-Riboflavin-5’-phosphate
H3C N CO
A more complicated way of synthesis was carried out previously by con-
version of riboflavin into the 5-trityl ether, acetylation, removal of the
ether group, and phosphorylation of the exposed 5’-hydroxyl group with
phosphorous oxychloride; hydrolysis then gave p-riboflavin-5’-phosphate.”
Dichlorophosphoric acid seems to be a more useful reagent for the
phosphorylation of riboflavin than phosphorous oxychloride; FMN has
been prepared by this method in quantities greater than milligrams.*°
The most recent method for the synthesis of riboflavin-5’-phosphoric
acid uses anhydrous metaphosphorie acid as a phosphorylating agent.5%
FMN gives a crystallized monodiethanolamine salt with a water solubil-
ity of more than 200 times that of riboflavin.
2. BIocHEMICAL FORMATION
1 ,2-Dimethyl-4 ,5-diaminobenzene (I) seems to be the first biogenetic
precursor of vitamin Bey and vitamin By, both substances containing I
as a structural element.*!
7 R. Kuhn and H. Rudy, Ber. 68, 383 (1935).
73 H.S. Forrest and A. R. Todd, J. Chem. Soc. 1950, 3295. The cyclic riboflavin-4’ ,5’-
phosphate seems to be contained in a new flavin nucleotide (FAD-X), isomeric
with flavin adenine dinucleotide (FAD). It cannot be stated at present whether
FAD-X occurs naturally or is produced artificially during the isolation of FAD;
F. M. Huennekens, D. R. Sanadi, E. Dimant, and A. I. Schepartz, J. Am. Chem.
Soc. 75, 3611 (1953).
79 R. Kuhn, H. Rudy, and F. Weygand, Ber. 69, 1548, 1974, 2557 (1936).
80 ],. A. Flexser and W. G. Farkas, XIJth Intern. Congr. Pure Appl. Chem. New
York, Abstr. Papers p. 71, 1951; Chem. and Eng. Chem. 1951, 3947.
80a M. Viscontini, C. Ebnéther, and P. Karrer, Helv. Chim. Acta 35, 457 (1952).
81 TZ). W. Woolley, J. Exptl. Med. 93, 13 (1951); Proc. Soc. Expil. Biol. Med. 76, 745
(1950).
Il. CHEMISTRY 323
2-Amino-4,5-dimethyl-1l-ribitylaminobenzene (II) might be the next
intermediate of the biosynthesis of riboflavin since in the presence of al-
loxan it is a potent stimulator of Lactobacillus casei. This substance is used
for the synthesis of riboflavin also by Mycobacterium tuberculosis.®*
As a higher natural intermediate, 6,7-dimethyl-9-p-ribosidoflavin (IIT)
also has been considered, yielding the vitamin by reduction of the N-
glycoside linkage.*® According to Weygand,® not only N-p-riboside, but
also an N-p-arabinoside could be involved, being transformed by an Am-
adori rearrangement to the p-ribose derivative.
H;C NH, H,C NHCH.(CHOH);,CH.OH
va
[om on |
HC—C—@—_C_ CH .or
| H EP on
H.C N N
\
VA Vl rere
| III
LPF OFE
HC N O
5'-p-Riboflavin-p-glucopyranoside (riboflavinyl glucoside) has been pre-
pared by incubation of riboflavin with an enzyme obtained from rat liver®
No function has so far been ascribed to this new derivative of riboflavin.
Recently light has been thrown on the mechanism of riboflavin biosyn-
thesis in Ashbya gossypii by using isotopic compounds.* C'-formate intro-
duced into the culture medium gave rise to riboflavin tagged in the carbon-2
position. The tracer atom from C'-bicarbonate ended up in the carbon-4
position. C4H;COOH, CH;C“OOH, and totally labeled glucose produced
riboflavin containing C™ in both the side chain and o-xylene portions of the
molecule.
The formation of ribose-5-phosphoric acid and the corresponding ketose,
82H. P. Sarett, Federation Proc. 4, 101 (1945); J. Biol. Chem. 162, 87 (1946).
8a M.I. Smith and E. W. Emmart, J. Immunol. 61, 259 (1949).
88 L. G. Whitby, Nature 166, 79 (1950); Biochem. J. 50, 433 (1952).
8G. W. E. Plaut, Chem. Eng. News 1632 (April 20, 1953).
324 RIBOFLAVIN
ribulose-5-phosphoric acid, by enzymatic oxidation of 6-phosphogluconic
acid could be demonstrated.5**
3. MICROORGANISMS AS PROoDUCERS®
Different natural sources have been used for the production of vitamin
B, by fermenting microorganisms. Whey and other milk by-products have
been treated with lactose-fermenting yeasts, especially Saccharomyces
fragilis, or with Clostridium butylicum, several species of Lactobacilli, or
molds. Molasses or other carbohydrate mashes were fermented with vari-
ous strains of butanol-producing Clostridia, especially Cl. acetobutylicum;
among the bacteria, this microorganism is one of the best producers of ribo-
flavin. Riboflavin is formed by numerous strains of Mycobacterium tubercu-
losis. In Mycobacterium smegmatis up to 3.6 mg. of riboflavin is formed per
100 mg. of the dried cells.°**
A majority of the varieties of the yeast species Candida produce substan-
tial amounts of riboflavin when glucose is used as the carbon source.
Candida guilliermondia and Candida flarert were found to produce high
yields of riboflavin on a simple synthetic medium of low cost.*® The use of
the Candida germs for commercial exploitation is very difficult because of
their extremely low tolerance for iron. 2,2’-Dipyridyl has been recom-
mended to control the iron content in fermentation media’
In 1935 Guilliermond, a French mycologist, observed that Hremothecium
ashbyzi in laboratory cultivation produced a yellow pigment which formed
crystals in the thread-like cells. The microorganism originally was isolated
as a pathogen for cotton plants in the Belgian Congo. The pigment has
since been identified as riboflavin. High yields of this vitamin have been
observed in fermentations with H. ashbyiz, using sugar and nitrogenous
compounds in a deep fermentation method, with formation of up to nearly
500 mg. per liter of medium.*°
Ashbya gossypii also was found to be of value in the microbiological
production of riboflavin.’ In shake cultures on a medium containing 4%
glucose, 0.5% peptone, and 2.5% corn steep liquor solids, titers of 1000
mg. per liter were obtained. A large variety of factors influencing biosyn-
thesis of riboflavin in submerged aerobic cultivation by A. gossypii has
been studied.®
84a B. L. Horecker, P. Z. Smyrniotis, and J. E. Seegmiller, J. Biol. Chem. 193, 383
(1951).
85 For more details and references, see F. A. Robinson, footnote 1, pp. 148-154, and
J. M. Van Lanen and F. W. Tanner, Jr., Vitamins and Hormones 6, 163 (1948).
85a R. L. Mayer and M. Rodbart, Arch. Biochem. 11, 49 (1946).
86 H. Levine et al., Ind. Eng. Chem. 42, 1176 (1950).
86a R. Hickey, U.S. Pat. 2,425,280 (Aug. 5, 1947) [C.A. 41, 6668 (1947)].
87 J. Wickerham, M. H. Flickinger, and R. M. Johnston, Arch. Biochem. 9, 95 (1946).
88 Ff, W. Tanner, Jr., C. Vojnovich, and J. M. Van Lanen, J. Bacteriol. 58, 737 (1949).
Il. CHEMISTRY 325
Among the molds, the following have been recorded as flavin-producing:
Aspergillus niger, Aspergillus flavus, Penicillium chrysogenum, and species
of Fusarium.
For recovery of the vitamin from the fermented liquors adsorption-elu-
tion methods have been used. Another possibility is the precipitation of
reduced forms of riboflavin from nutrient media by the metabolic reducing
action of certain bacteria, particularly a group of generally avirulent strep-
tococci.’’ For instance, up to 90 % of the dissolved riboflavin can be ob-
tained as a red-orange, amorphous precipitate with Streptococcus faecalis,
under anaerobic conditions” (cf. p. 314). Instead of bacterial reduction,
reducing chemicals have been used, for instance, sodium dithionite
(NasS20,-2H.O), stannous, titanous, chromous, and vanadous salts.%! These
methods make use of the fact that the various reduced forms of riboflavin
are much less soluble in water than the oxidized form. The solubility in 1
ml. of water is for riboflavin 100 to 130 y, for verdoflavin 50 y, for chloro-
flavin 20 y, and for leucoriboflavin approximately 8 y.°!*
Ek. SPECIFICITY
Some of the more important relations between chemical constitution
and biological activity will be discussed here. Details about the specificity
of flavin enzymes can be found in a later section.
1. GrowTH STIMULATORS
Riboflavin tetraacetate and diacetone riboflavin are active for rats,
probably as a result of hydrolysis in the organism; the tetraacetate is, how-
ever, inactive on lactic acid bacteria. Riboflavin-5’-phosphate and flavin
adenine dinucleotide not only are growth-promoting in rats but both were
also as effective as riboflavin for growth and acid production of L. helveticum.
The flavinyl glucosides are inactive.
It appears that only flavins which serve as a sole source of growth-pro-
moting flavin (in the absence of suboptimal amounts of riboflavin) for
L. casei and B. lactis acidi are capable of supporting growth in animals.®
To possess this activity, the flavin must be a ribityl derivative and be sub-
stituted in either the 6 or 7 position or both by a methyl group or by an ethyl
group in the 6 position, together with a methyl group in the 7 position.
6 ,7-Diethyl-9-(p-1’-ribityl)isoalloxazine recently has been found capable
89 G. E. Hines, Jr. (to Commercial Solvents Corp.), U. S. Pat. 2,387,023 (Oct. 16,
1945) [C.A. 40, 428 (1946)]; G. W. McMillan (to Commercial Solvents Corp.), U.S.
Pat. 2,367,646 (Jan. 16, 1945) [C.A. 39, 3400 (1945)].
°R. J. Hickey, Arch. Biochem. 11, 259 (1946).
"G. E. Hines, Jr. (to Commercial Solvents Corp.), U. S. Pat. 2,367,644 (Jan. 16,
1945) [C.A. 39, 3399 (1945)].
#8 S$. J. Shimizu, J. Fermentation Technol. (Japan) 28, 139 (1950) [C.A. 47, 1755
(1953).
326 RIBOFLAVIN
of serving as the sole source of flavin in the growth of Lactobacillus casei ;%*
however, this flavin has an antiriboflavin activity in the growing rat.
7-Ethyl-9-(p-1’-ribityl)isoalloxazine is effective only in the presence of
suboptimal amounts of riboflavin.%
Riboflavin
antagonist:
NA NZ Be a
\F (
“hey UNA \ \ yawN CNG
H;C.2 H;Ce H3C
H;C H;C
CH;
Riboflavin 6-Methyl- 7-Methyl- 6-Ethyl-7- 6,7-Diethyl- Isoribo-
flavin flavin methylflavin flavin flavin
Biologically Active Riboflavin Homologs
Absence of substituents in both the 6 and 7 positions of riboflavin is con-
nected with high toxicity.*® For biological activity, the imino group in the
3 position has to be unsubstituted.
Substituents in any other than the 6 and 7 positions on the benzene
ring destroy and in certain cases reverse the vitamin activity. For instance,
isoriboflavin, an isomer of riboflavin, which has its two methyl groups in
the 5 and 6 positions, is an antagonist of vitamin B..
From the examples given, it is obvious that only slight alterations of
the ring substituent in the riboflavin structure can be made without the
loss of vitamin activity.
Ojsllst™ Jal
|
The following t-araboflavins, R—CH,—C—-C—-C—CH;0H, possess
Ht OnOn
some little stimulating activity for rats and lactic acid bacteria in the
presence of suboptimal amounts of riboflavin: 6,7-dimethyl-9-(1-1’-
arabityl)isoalloxazine (u-araboflavin), 6,7-trimethylene-9-(L-1’-arabity])-
isoalloxazine, 6,7-tetramethylene-9-(L-1’-arabityl)isoalloxazine,** 6-meth-
yl-7-amino-9-(L-1’-arabityl)isoalloxazine; the p-arabityl derivative corre-
sponding to the latter substance is said to be about half as effective as
riboflavin in promoting the growth of B2-avitaminotic rats.
92 J. P. Lambooy, J. Biol. Chem. 188, 459 (1951). |
%a H. V. Aposhian and J. P. Lambooy, Proc. Soc. Exptl. Biol. Med. 78, 197 (1951); J.
Nutrition 47, 539 (1952). |
9% P. Karrer and T. H. Quibell, Helv. Chim. Acta 19, 1034 (1936).
9% R. Kuhn, H. Vetter, and W. Rzeppa, Ber. 70, 1307 (1937).
95 Siimi Nishida, C.A., 45, 7127 (1951); 46, 6715 (1952).
Il. CHEMISTRY 327
It has been suggested®* that the small vitamin By, activity of L-arabo-
flavin might be due to contamination with riboflavin, formed during the
synthesis by an Amadori rearrangement (see p. 318). In rats receiving 10
y of riboflavin per day t-araboflavin in higher doses (200 y per day) reduces
the growth very slightly.’
Of the other stereoisomers of riboflavin which have been synthesized,
‘ag OH
only p-xyloflavin, R—CH.—C—-C—-C—CH,0H,, is said to produce a slow
Cee
HOHE
gain of weight in vitamin B.-deficient rats.°®
OH OHH
ly
L-Lyxoflavin, R—CH.—C—-C—C—-CH,—OH, was isolated from hu-
edly:
ishastehe 6a
man myocardium in 1949 and has been made synthetically.* It is devoid
of riboflavin activity in rats when tested by the standard assay, but in a
rat assay for unidentified vitamins in liver and other source materials, as
well as for Lactobacillus lactis, u-lyxoflavin has shown growth-promoting
or vitamin activity.!°° Lyxoflavin seems to be the first exception from the
generally accepted rule that flavins isolated from different natural sources
are chemically identical with p-riboflavin; but its natural existence has
not been definitely confirmed.)
Besides the mentioned riboflavin isomers, L-1’-ribityl-, p-1/-arabityl-,
and p-l’-lyxitylflavin have been prepared synthetically; none of these
substances has vitamin By activity.
2. ANTAGONISTS OF RIBOFLAVIN
Diethylflavin, 6 ,7-diethyl-9-(p-1’-ribityl)isoalloxazine, has already been
mentioned as an antagonist of riboflavin in the growing rat.°?*
Isoriboflavin, 5,6-dimethyl-9-(p-1’-ribityl)isoalloxazine, in a daily dose
of 2 mg. in rats almost completely inhibits the growth-promoting effect of
10 y of riboflavin per day. The inhibitory effect can be prevented entirely
96 F, Weygand, Ber. 73, 1259, 1264 (1940).
97H. von Euler and P. Karrer, Helv. Chim. Acta 29, 353 (1946).
9}. Sodi Pallares and H. Martinez Garza, Arch. Biochem. 22, 63 (1949); Arch. Inst.
cardiol. Méx. 19, 753 (1949) [C.A. 44, 5368 (1950)].
99 TP). Heyl, E. C. Chase, F. Koniuszy, and K. Folkers, J. Am. Chem. Soc. 73, 3826
(1951).
100 G. A. Emerson and K. Folkers, J. Am. Chem. Soc. 73, 2398, 5383 (1951). Mary 8.
Shorb, Proc. Soc. Exptl. Biol. Med. 79, 611 (1952). Compare, however, E. E. Snell,
O. A. Klatt, H. W. Bruins, and W. W. Cravens, Proc. Soc. Exptl. Biol. Med. 82, 583
(1953).
101T. S. Gardner, E. Wenis, and J. Lee, Arch. Biochem. and Biophys. 34, 98 (1951).
328 RIBOFLAVIN
by the daily administration of 40 y of riboflavin. Two milligrams of isori-
boflavin daily restricts the growth of riboflavin-deficient rats much more
than does the deficiency of the vitamin alone.!” In ZL. casei isoriboflavin
showed no signs of competitive inhibition of riboflavin.?°
p-Araboflavin evidently is an antagonist of riboflavin. Two hundred
micrograms per day decreases the rate of growth of rats receiving 10 y
of riboflavin per day to such an extent that no growth takes place by the
third week.”
Another strong antagonist of riboflavin is p-galactoflavin, 6,7-dimethyl-
ORGHE Hs OE
ect peticecd
9-(p-1’-dulcityl)isoalloxazine, R—CH.—C—-C—C—C—CH.OH. Im a dose
ee aloe
H OHOHH
of 2.16 mg. per day, it inhibits completely the response of the animals to
10 y of riboflavin daily and markedly inhibits the response to 40 y of vita-
min By» daily. The inhibitory effect is almost, but not completely, prevented
by 200 y of riboflavin daily.1%
Dichloroflavin, 6 ,7-dichloro-9-(p-1’-ribityl)isoalloxazine (I), inhibits the
growth of Staphylococcus aureus, Streptobacterium plantarum, and Bacillus
lactis acidi, but not of yeast. The inhibition is competitively prevented
by riboflavin. Dichloroflavin has an oxidation reduction potential of Hyp =
—0.095 volt (pH 7); for riboflavin it is Hy) = —0.185 volt (pH 7). This
difference has been regarded as an explanation for the inhibitory behavior
of dichloroflavin; perhaps the vitamin analog cannot function like ribo-
flavin in the oxidation—reduction reactions which are catalyzed by the ribo-
flavin coenzymes.'"* Twelve other halogen-substituted flavins with various
sugar chains in the 9 position have been found to be less effective as ribo-
flavin antagonists than dichloroflavin, tested with Streptobactertum plan-
tarum P 32.14
The growth of Hremothecitwm ashbyii, which produces riboflavin, is in-
hibited by dichloroflavin, without reduction of its riboflavin production.!°
Therefore, the inhibition of this microorganism probably is not to be as-
sociated directly with displacement of the vitamin.
It has been shown that certain 9-substituted 6,7-dichloroisoalloxazines
are inhibitors of D-amino acid oxidase.!% However, neither dichloroflavin
102 G. A. Emerson and M. Tishler, Proc. Soc. Exptl. Biol. Med. 55, (1944).
103 G. A. Emerson, E. Wurtz, and O. H. Johnson, J. Biol. Chem. 160, 165 (1945).
1088 R. Kuhn, F. Weygand, and E. F. Moller, Ber. 76, 1044 (1943).
104, Weygand, R. Lowenfeld, and KE. F. Moller, Ber. 84, 101 (1951).
105 W. H. Schopfer, Intern. Z. Vitaminforsch. 20, 116 (1948).
106 R. B. Barlow, J. Chem. Soc. 1951, 2225.
Il, CHEMISTRY 329
nor its 5’-phosphate, even in a 1LOO0-fold excess over riboflavin, has an
influence on the activity of p-amino acid oxidase or xanthine oxidase.!?°*
OH OH OH
Cis—C-_u—_C-—_CH,0H
leh Jab) dst
Cl N N
WS
4 co
| I
NH
Ne eee
Cl N CO
Dichloroflavin
OHH OH OH
CH;—6—_C_C__ €—CE.0n
H OHH
Cl N bah
| | ait
FAN
Cl N CO
Dichloro-p-sorboflavin
For one substance of this type, 6,7-dichloro-9-(1’-p-sorbity])isoallox-
azine (II), a special biological effect has been observed.*? It exhibits no
significant inhibition of riboflavin microbiologically and zn vivo in rats.
However, it is effective in producing regression of established lympho-
sarcoma implants in mice. The p-ribityl, L-arabityl, and p-dulcityl anaiogs
of compound IT show, respectively, slight, questionable, and no carcinolytic
activity. 6-Chloro-9-(1/-p-sorbity])isoalloxazine appeared to have some ac-
tivity in several tests. Other riboflavin analogs in which the substituents
in the 6 and 9 positions were varied gave questionable or negative results.
Lettré!°® first demonstrated that certain flavins, which are antagonists
of riboflavin, have an antimitotic effect. Later, regression of lymphosarcoma
and decreased growth rate of spontaneous mammary carcinomas in mice
106a P. Karrer and H. Ruckstuhl, Bull. schweiz. Akad. med. Wiss. 1, 236 (1945).
106b H. Lettré, Angew. Chem. 53, 363 (1940); H. Lettré and M. E. Fernholz, Ber. 73,
436 (1940).
330 RIBOFLAVIN
and of Walker carcinomas in rats deficient in riboflavin were reported.!%
In some cases, the animals were rendered strongly deficient in riboflavin
by feeding one of the riboflavin antagonists, isoriboflavin, galactoflavin, or
diethylflavin, along with a diet deficient in riboflavin. The activity of di-
chloro-p-sorboflavin (II) shows that the mechanism of regression of the
lymphosarcoma need not necessarily occur through riboflavin inhibition.
Lumiflavin, the photolysis product of riboflavin, is either an inhibitor or
a stimulator of the utilization of riboflavin or flavin adenine dinucleotide
by L. casei, depending upon the relative amounts of lumiflavin present.1%
The phenazine analog of riboflavin, 2,4-diamino-7 ,8-dimethyl-10-(v-
ribityl)-5,10-dihydrophenazine (I),!°° antagonizes the action of vitamin
By, in riboflavin-requiring bacteria. The dinitrophenazine derivative from
which I is prepared produces mild riboflavin deficiency in mice. Adequate
amounts of riboflavin overcome the effects of the compound.
CH;
CH.(CHOH);,CH,0H |
| NHCH(CH:);N(C2H;)2HCl
Hi@ N NH, H;CO |
NG SIR, ON 4
| I II
TaN VA VSN NY,
H;C NH Cll
NH,
Atabrin (Mepacrine)
Many substances with antimalarial activity were found to inhibit the
growth-promoting effect of riboflavin on microorganisms, for instance mepa-
crine (atabrin) (II),!!°'™ substituted pyrimidines,!” and quinine. How-
ever there is no strong parallelism between the antimalarial and the anti-
vitamin function; for instance, proguanil (paludrine) does not antagonize
riboflavin. The structural similarity of certain antimalarials to the flavin
nucleus is probably not directly correlated to their antimalarial action.
Mode of Action of Riboflavin Antagonists.“4 At least three ways are pos-
107 H,. P. Morris and W. van B. Robertson, J. Natl. Cancer Inst. 3, 479 (1943); H. C.
Stoerk and G. A. Emerson, Proc. Soc. Exptl. Biol. Med. 70, 703 (1943).
108 H. P. Sarett, Federation Proc. 4, 101 (1945); J. Biol. Chem. 162, 87 (1946).
109 J). W. Woolley, J. Biol. Chem. 164, 31 (1944); H. P. Sarett, J. Biol. Chem. 162,
87 (1946).
110 J. Madinaveitia, Biochem. J. 40, 373 (1946).
111 M. Silverman and E. A. Evans, Jr., J. Biol. Chem. 150, 265 (1943).
12 F. H.S. Curd and F. L. Rose, J. Chem. Soc. 1946, 343, 362, 366, 370; A. R. Todd et
al., ibid, 1946, 357.
113 J,, Hellermann, A. Lindsay, and M. Bovarnick, J. Biol. Chem. 163, 553 (1946).
14H). B. Kearney, J. Biol. Chem. 194, 747 (1952).
II. CHEMISTRY 331
sible in which a riboflavin antagonist might bring about growth inhibition:
(a) by competition with the prosthetic group of flavoenzymes; (b) by com-
petitively inhibiting the phosphorylation of riboflavin; and (c) by being
enzymatically converted to an analog of riboflavin-5-phosphate or flavin
adenine dinucleotide, in which form they might competitively inhibit
flavoenzymes.
It has been shown that yeast flavokinase, the enzyme which catalyzes
the phosphorylation of riboflavin, also phosphorylates p-araboflavin and
dichloroflavin to the corresponding nucleotides. The effects of these phos-
phorylated riboflavin analogs on flavoenzymes have not yet been studied.
Isoriboflavin, galactoflavin, and sorboflavin neither are phosphorylated
by flavokinase nor do they inhibit the enzymatic phosphorylation of ribo-
flavin by flavokinase. This enzymatic process is inhibited by lumiflavin,
when present in excess over riboflavin.
3. NITROFLAVINS AND BASICALLY SUBSTITUTED I[SOALLOXAZINES
Two 6-nitroflavins have been synthesized :"°
CH.CH,OH CH:.CH:2N(C2Hs)2
N N N N
LAR UN ee
I | | es ae
NH NH
oN i Ge
O.N N CO O.N N CO
6-Nitro-9-(6-hydroxyethyl)- 6-Nitro-9-(8-diethylaminoethy])-
isoalloxazine isoalloxazine
Chemotherapeutic tests with substance I did not show noticeable success.
The structural similarity between riboflavin and atabrin suggested the
synthesis of basically substituted isoalloxazine derivatives as possible anti-
malarials. The following types have been prepared.°*®: !1°-118
R’”CH(CH:2),N(C2Hs)2 CH,CHOHCH.NR,»
i N H;C ‘ N R= o
; Ve ae Se ae Wa ‘co or C2H;
Ill a | | | 1G res 9g
\ NE \ ae NH
N co H;C N rere)
45 H. Hippchen, Ber. 80, 263 (1947).
16 FE. King and R. M. Acheson, J. Chem. Soc. 1946, 681.
17 R. R. Adams, C. A. Weisel, and H. 8. Mosher, J. Am. Chem. Soc. 68, 883 (1946).
u8 H. Burkett, J. Am. Chem. Soc. 69, 2555 (1947).
Bae RIBOFLAVIN
They were found to be devoid of antimalarial activity and did not show
the tendency to inhibit the metabolic effects of riboflavin. 9-(6-Diethyl-
aminoethyl)isoalloxazine hydrochloride was ineffective in mice infected
with Trypanosoma nagana."
Summary. The preceding discussions show that only slight changes in the
chemical constitution of riboflavin can be made without loss of vitamin By
activity. This fact is in agreement with the high chemical specificity of vi-
tamins in general. In replacing the ribityl side chain by other sugar alcohols,
or by alteration of substituents in the benzene ring of the isoalloxazine
nucleus, derivatives can be obtained which are antagonists of riboflavin.
No derivatives of flavins effective against pathogenic organisms have yet
been developed but carcinolytic activity was found among chloro-p-sorb-
oflavins.
III. Industrial Preparation
THEODOR WAGNER-JAUREGG
Pure crystallized riboflavin for therapeutic purposes is made by chemical
synthesis. It is difficult to indicate actual manufacturing processes, since
they are held as confidential. The best index of determining the methods
used in the industry is the patent literature.!
Concerning the pentose component, methods of riboflavin synthesis with-
out the use of p-ribose are of interest, since none of the known syntheses
of p-ribose are simple and economical. Different syntheses of N-(p-ribityl)-
3,4-dimethylaniline, the key intermediate in the riboflavin synthesis, have
been developed which do not involve p-ribose (p. 318). The introduction
of the second amino group can be performed with good yield by the method
of Karrer and Meerwein (p. 317).
With regard to the isoalloxazine formation, different patented methods
have been described: (1) requisite diamine and alloxan; (2) requisite amino-
azo compound and barbituric acid;? (3) requisite aminoazo compound and
dialluric acid in the presence of a hydrogen transfer catalyst (in this method,
reduction to the diamine occurs); (4) requisite diamine and dichlorobarbitu-
ric acid. Examples have been given in the preceding section (pp. 315ff.).
Vitamin Bs concentrates suited to enrich poultry and livestock feeds can
be prepared more cheaply by fermentation processes. During World War
! A list of patents on vitamin B» can be found in H. Vogel and H. Knobloch, Chemie
und Technik der Vitamine, 3rd ed., Vol. 2, Issues 1 and 2, pp. 197-214. Ferdinand
Enke, Stuttgart, 1953.
2M. Tishler, K. Pfister, 3rd, R. D. Babson, K. Ladenburg, and A. J. Fleming,
J. Am. Chem. Soc. 69, 1487 (1947).
II. INDUSTRIAL PREPARATION 333
II, preparation of riboflavin was started from the residues of butanol-ace-
tone fermentation with Clostridium acetobutylicum.
Currently, most of the commercial riboflavin production by aerobic
fermentation is probably obtained by biosynthesis with HMremothecium
ashbyzi, in submerged culture with continuous aeration and agitation. Pat-
ents covering this process were filed by several firms,*: 4 and conditions for
the production of riboflavin by Ashbyiz have been reported.® By the use of
E. ashbyii grown on solid media (germ rice and germ wheat), the vitamin
Be production on an industrial scale is said to have reached a maximum at
20,000 y per gram (2 %).®
A medium containing grain stillage from the ethanol fermentation,
cerelose and 1 % peptone is excellent for the cultivation of A. gossypii to
produce yields of riboflavin as high as 15,000 y per gram.’ Studies of ribo-
flavin production for commercial preparation by fermentation using the
yeast Ashbya gossypit upon a pilot-plant scale were made recently.’ Corn
steep liquor, peptone, and tankage are suitable nitrogen sources with the
fermentation of nutrient mashes.®*: !°
In 1943, more than 75,000 lb. of crystalline riboflavin were produced
from all sources. It is priced at about $100 to 1380 per kilogram today,
whereas in 1938 the price was $7,945 a pound."!
Besides the pharmaceutical use, small amounts of vitamin By» are incor-
porated in most bread flours and breakfast foods and in nearly all poultry
and hog feeds.
3 Commercial Solvents Corp., U. S. Pat. 2,202,161 [C.A. 34, 6676 (1940)]; British
Pat. 527,478 (April 138, 1939). Chem. Trade J. 113, 26 (1943).
4Commercial Solvents Corp., U. S. Pats. 2,483,855 (Oct. 4, 1949) [C.A. 44, 2698
(1950) ], 2,498,549 (Feb. 21, 1950) [C.A. 44, 4630 (1950)]; Hoffmann-La Roche, Inc.,
U.S. Pat. 2,493,274 (Jan. 3, 1950) [C.A. 44, 2698 (1950)]; Don H. Larson (to Com-
mercial Solvents Corp.) U.S. Pat. 2,615,829 (Oct. 28, 1952) [C.A. 47, 834 (1953)].
°C. Chin, Hakké Kégaku Zasski, 25, 140 (1947) [C.A. 44, 7384 (1950)].
6R. Takata, J. Japan. Biochem. Soc. 20, 130 (1948) [C.A. 44, 8063 (1950).
7K. L. Smiley, M. Sobolow, F. L. Austin, R. A. Rasmussen, M. B. Smith, J. M.
Van Lanen, L. Stone, and C. 8. Boruff, Ind. Eng. Chem. 48, 1380 (1951).
8V. F. Pfeifer, F. W. Tanner, Jr., C. Vojnovich, and D. H. Traufler, Ind. Eng.
Chem. 42, 1776 (1950).
9 Merck and Co., British Pat. 640,452 (July 19, 1950) [C.A. 44, 9622 (1950)].
10 For more information on the microbiologic production of riboflavin, see the re-
port of D. Perlman, W. E. Brown, andS. B. Lee, Ind. Eng. Chem. 44, 1996 (1952)
and Production of Riboflavin by Fermentation in “Industrial Fermentations,”’
(L. A. Underklofer, R. J. Hickey, eds.), Vol. 2. Chemical Publishing Co., New York,
1954.
uF. W. Tanner, Jr., and V. F. Pfeifer, in ‘‘Crops in Peace and War,”’ Yearbook Agr.
U.S. Dept. Agr. 1950-51, 763.
334 RIBOFLAVIN
IV. Biochemical Systems
M. K. HORWITT
Knowledge of the close relationship between vitamins and biological oxi-
dations may be said to date from 1932, the year in which Warburg and
Christian! discovered the first flavoprotein. This compound, often referred
to as the ‘“‘old yellow enzyme,” which they obtained from the aqueous ex-
tract of bottom yeast was soon separated? into a protein and a yellow
prosthetic group. Stern and Holiday,’ using spectroscopic methods, found
that the prosthetic group of Warburg’s yellow enzyme was a derivative of
alloxazine. This fact, when combined with the observations of Ellinger
and Koschara,‘ Booher,® and Kuhn e¢ al.® on the correlations between vita-
min B, and a water-soluble yellow-green fluorescent pigment, was soon
corroborated by the synthesis of riboflavin by the Kuhn’: ® and Karrer®
schools. Theorell’s!® demonstration that Warburg’s enzyme contained one
molecule of phosphate and Kuhn, Rudy, and Weygand’s" proof of consti-
tution of riboflavin-5-phosphoric acid were the concluding steps in a fasci-
nating story of the first separation, identification, and synthesis of the
prosthetic group of an enzyme.
A. COENZYMES
All flavoproteins can be characterized as specific proteins which contain
either flavin mononucleotide or flavin dinucleotide as prosthetic groups, or
coenzymes. The flavin mononucleotide, riboflavin phosphate, is not in the
strict sense a nucleotide, since the compound is derived from p-ribitol
rather than from p-ribose."' The location of the phosphoric acid at the 5
position has been definitely established.” To date at least three flayvopro-
teins with enzymatic activity have been shown to contain the mononucleo-
tide. These are Warburg’s yellow enzyme, cytochrome ¢ reductase, and
L-amino acid oxidase.
Flavin adenine dinucleotide (FAD) is isoalloxazine adenine dinucleotide.
1Q. Warburg and W. Christian, Biochem. Z. 254, 488 (1982).
20. Warburg and W. Christian, Biochem. Z. 266, 377 (1933).
3K. G. Stern and E. R. Holiday, Ber. 67, 1104, 1442 (1934).
4P. Ellinger and W. Koschara, Ber. 66, 315, 808 (1933).
5 L.. E. Booher, J. Biol. Chem. 102, 39 (1933).
6 R. Kuhn, P. Gyorgy, and T. Wagner-Jauregg, Ber. 66, 317, 576, 1034 (1933).
7R. Kuhn, K. Reinemund, H. Kaltschmitt, R. Strébele, and H. Trischmann, Natur-
wissenschaften 23, 260 (1935).
8 R. Kuhn, K. Reinemund, F. Weygand, and R. Strébele, Ber. 68, 1765 (1935).
9P. Karrer, K. Schépp, and F. Benz, Helv. Chim. Acta 18, 426 (1935).
10H. Theorell, Biochem. Z. 272, 155 (1934).
11 R. Kuhn, H. Rudy, and F. Weygand, Ber. 69, 2034 (1936).
22 P. Karrer, P. Frei, and H. Meerwein, Helv. Chim. Acta 20, 79 (1987).
IV. BIOCHEMICAL SYSTEMS 335
ee O
CH:—C—C—C—CH.,0— P—OH
ORE oa a |
mE O
H
ies aN
Riboflavin-5-phosphate
Its structure has not been proved by synthesis. According to Warburg and
Christian," it is composed of one adenine, one flavin, two pentose, and two
phosphoric acid molecules and may be visualized as a combination of ribo-
flavin phosphate and adenylic acid with the elimination of one molecule
of water.
isl H
Oar
nee Oo -— | —O— Clie
| 0 0 |
HOo—C—iH 7 CH
| |
isl€—\O—— al HOCH
| Oo. |
HO—C—E HOCH
| |
CH. CH N
| eae
N N NC Cr
ipa a ve
oie eeeat
oe \
NO NOt a
eee eee
] C
O |
N
H.
Flavin adenine dinucleotide
The enzymatic phosphorylation of riboflavin by an enzyme in yeast
named flavokinase has recently been reported by Kearney and Englard."4
'8Q. Warburg and W. Christian, Biochem. Z. 298, 150 (1938).
4}. B. Kearney and S. Englard, J. Biol. Chem. 198, 821 (1951).
336 RIBOFLAVIN
The reaction catalyzed by this enzyme is:
Riboflavin + ATP — Riboflavin-5-phosphate + ADP
The mechanism of transformation of riboflavin phosphate to flavin ade-
nine dinucleotide is not known, but it has been shown to occur in human
blood cells.!®
1. GENERAL PROPERTIES OF FLAVIN COENZYMES
Riboflavin phosphate and flavin adenine dinucleotide resemble the
parent vitamin, riboflavin, in many respects. They exhibit the same char-
acteristic yellow color and yellow-green fluorescence. Reduction with hypo-
sulfite, platinum, or H2 will reduce riboflavin and its coenzymes to colorless
compounds which will reoxidize to their original state when shaken with
air. If reduced in strongly acidic solution, a red intermediate is formed
H
O Basie
nate Gite SS group
OH O
Bee
HCOOH Protein
eae
N_,N~
al nig C=O Acid
gy = Trou
|
O
Riboflavin-5-phosphate Apoenzyme
which has the properties of a semiquinoid radical.!® In solution they are
essentially unstable. This decomposition is influenced by light, heat, and
pH, riboflavin being rapidly decomposed in strongly alkaline solutions.
Riboflavin phosphate is considerably more soluble in water than free
riboflavin and can be precipitated by various salts. It is hydrolyzed quite
slowly in weakly alkaline solutions, quite rapidly in acid solutions, and by
phosphatases such as a-glycerophosphatase. Riboflavin phosphate com-
bines with specific proteins,” the apoenzymes, by attachment at the phos-
phorie acid group and at the slightly acidic imino group in the 3 position.
The typical fluorescence of riboflavin is dependent upon the presence of
a free 3-imino group, and neither 3-substituted riboflavin nor the enzyme
systems will fluoresce.
15 J. R. Klein and H. I. Kohn, J. Biol. Chem. 186, 177 (1940).
16. Haas, Biochem. Z. 290, 291 (1937).
17 R. Kuhn and H. Rudy, Ber. 69, 2557 (1936).
IV. BIOCHEMICAL SYSTEMS 337
2. RIBOFLAVIN PHOSPHATE
Banga ef al.’ obtained a yellow substance from heart muscle in 1932
which may have been the first preparation of riboflavin phosphate. How-
ever, the yellow enzyme which Warburg and Christian! obtained from
yeast in the same year was more thoroughly investigated. They purified an
aqueous extract of autolyzed bottom yeast by treatment with lead sub-
acetate, removed excess lead with phosphate, and precipitated the yellow
enzyme in the form of a viscous oil at low temperature with carbon dioxide
and acetone. After reprecipitation from acetone and precipitation with
methanol at 0°, a dry product was obtained which could be readily disso-
ciated into a colorless protein and a yellow prosthetic group. The latter
was later proved to be riboflavin phosphate.
Theorell!® has shown that the combination of riboflavin phosphate with
the apoenzyme could be reversibly dissociated as follows: When a solution
of the yellow enzyme was dialyzed against 0.02 N HCl at 0°, the dialyzate
was slowly decolorized. The colored group (riboflavin phosphate) passed
through the membrane, and the colorless protein remained behind. The
protein was changed to a metaprotein by its contact with the dilute acid
(i.e., precipitated when brought to pH 7), but when the protein was di-
alyzed against water to remove all traces of hydrochloric acid, 50 to 70%
of the metaprotein was renatured. This renatured protein was now capable
of recombination with the coenzyme riboflavin phosphate to produce a
complex with all the properties of the original yellow enzyme.
The combination between the coenzyme and the apoenzyme*® takes
place in a stoichiometric manner as shown in Fig. 1.
The reversible dissociation procedure of Theorell, which requires several
days for its completion, has been replaced by a simpler method by Warburg
and Christian”! in which a good yield was obtained in about 1 hour. To a
solution of yellow enzyme, purified by electrophoresis, an equal volume of
saturated ammonium sulfate was added. Sufficient 0.1 N hydrochloric acid
was added to this solution at 0° to shift the pH to about 2.8. The colorless
precipitate formed contained 78 % of the apoenzyme in native form while
the coenzyme remained in the supernatant fluid. Resynthesis was accom-
plished by merely remixing the components.
3. RIBOFLAVIN ADENINE DINUCLEOTIDE
The dinucleotide is widely distributed in animal tissues and in micro-
organisms. It has been isolated from liver, kidney, muscles, tumor tissue,
yeast,!2 13. 22-24 and Neurospora.”®
18 T. Banja, A. Szent-Gyoérgyi, and L. Vargha, Z. physiol. Chem. 210, 288 (1932).
19H. Theorell, Biochem. Z. 275, 344 (1934).
20 H. Theorell, Biochem. Z. 278, 263 (1935).
21Q. Warburg and W. Christian, Biochem. Z. 298, 368 (1938).
338 RIBOFLAVIN
Since riboflavin is necessary for the growth of many bacteria and is a
common component of plant products, it is logical to assume that the
dinucleotide is a common constituent of the cells of most living things.
Yeast offers the most convenient source for preparation. Warburg and
Christian" extracted yeast at 75°; the filtrate was two-thirds saturated
with ammonium sulfate and extracted with phenol. The phenol extracts
were mixed with ether, and the dinucleotide extracted with water. The
ether was removed by evacuation, the aqueous solution acidified with nitric
acid to about pH 2, and the dinucleotide precipitated as the silver salt. The
precipitate was resuspended in water and decomposed with hydrogen sul-
hl [ea a
70
mm.3 Oz used up in 10 minutes
0 1.4 2.8 4.2 5.6 7.0 8.4 9.8 12 1216 240) 154
7 of the active group
Fria. 1. Synthesis of the Warburg-Christian flavoprotein. The activity of the en-
zyme increases as more of the prosthetic group, riboflavin phosphate, is added to
form the original catalytically active flavoprotein (Theorell?°).
fide. The dinucleotide, which was almost completely absorbed on the silver
sulfide precipitate, was eluted with dilute barium acetate. The eluates were
mixed with ammonium acetate solution and concentrated to dryness 7m
vacuo. By taking advantage of the fact that the barium salt of the dinucleo-
tide is twice as soluble at 60° as at room temperature, it was possible to
separate the barium salts of the dinucleotide from the barium salts of the
contaminating adenine nucleotides.
The similarities of the dinucleotide to riboflavin phosphate in color,
fluorescence, and reversible reduction and oxidation have been discussed
(p. 336). It is less stable than riboflavin because of its tendency to hydro-
22 P. Karrer, P. Frei, B. H. Ringier, and H. Bendas, Helv. Chim. Acta 21, 826 (1938).
23 QO. Warburg, W. Christian, and A. Griese, Biochem. Z. 295, 261 (1938).
24 QO. Warburg, W. Christian, and A. Griese, Biochem. Z. 297, 417 (1938).
25 N. H. Horowitz, J. Biol. Chem. 154, 141 (1944).
IV. BIOCHEMICAL SYSTEMS 339
lyze to riboflavin phosphate and adenylic acid. The absorption spectra
of riboflavin and flavin adenine dinucleotide are given in Fig. 2."
B. ENZYMES
The classification of flavoproteins based upon their behavior during in
vitro experiments may lead to erroneous conclusions about their intracellu-
lar function. Relatively slight changes in the method of isolation may pro-
duce flavoproteins with different properties and with sluggish activities not
normally associated with cellular action. Until more definitive information
becomes available, one should not discount the suspicion that an artifact
10
8
n~
S
x
_ 3 6
Ele
5
2
Sai es
8
s
e
So
eae
<=
0 ! :
230 270 310 350 390 430 470 510 550
Wavelength in mz
Fic. 2. Absorption spectra of riboflavin (—————) and flavin adenine dinu-
cleotide (---------- ). (Warburg and Christian". )
may have been produced in some of the isolation procedures. During the
development of our present knowledge of flavoproteins a system of nomen-
clature has evolved which binds specific proteins to specific substrates.
Whether or not such activities are as represented, it is necessary to charac-
terize the individual enzymes in this manner in order to appreciate not
only the work which has been done but also that which will be reported.
Table I summarizes the known flavoproteins and some of their charac-
teristics.
1. OLD YELLOW ENzYME
a. Preparation and Properties
The discovery, isolation, and some of the properties of the yellow oxida-
tion enzyme have been discussed in the sections describing the coenzyme
riboflavin phosphate (see p. 337).
340
RIBOFLAVIN
Since homogeneous preparations contain 0.66% riboflavin phosphate,
and since ultracentrifuge measurements of the molecular weight give a
figure?® of about 70,000, there can be only one molecule of prosthetic ribo-
flavin phosphate per molecule of flavoprotein. Elementary analysis of the
enzyme by Theorell”’ yields typical figures for a protein: C
51.5 %;
H = 7.37%; N = 15.9%; P = 0.048%; S = 1.0%. The specific rotation;
aliesis 30°.
TABLE I
FLAVOPROTEINS
Ap-
ee es prox.
Enzyme Source thetic Reducing system eee: pees
group Wane
ber
Warburg yellow enzyme Bottom yeast RP H2-DPN or H2-TPN Os, M.B.® 50
Crossed yellow enzyme Synthetic FAD | H2-DPN or H2-TPN Oz 35
Haas enzyme Bottom yeast FAD | He-TPN M.B. >50
Diaphorase, coenzyme fac- | Various FAD | H2DPN M.B. 8500
tor, straub flavoprotein
Cytochrome ¢ reductase of | Brewer’s yeast RP H2-TPN Cytochrome ec | 1300
yeast
Cytochrome ec reductase of | Pig liver FAD | H2-TPN Cytochrome ¢ | 1150
liver
Xanthine, aldehyde, xan- | Milk FAD | Hypoxanthine, xan- M.B., O2 300-550
thopterin oxidase thine; aldehyde; xan-
| thopterin; He-DPN
Aldehyde oxidase Liver FAD | Aldehydes Oz 550
p-Amino acid oxidase Kidney, liver FAD | p-Amino acids Oz 2000
t-Amino acid, t-hydroxy acid| Kidney, bacte- | RP t-Amino acids, lactie | O2 6
oxidase ria acid, ete.
Glycine oxidase Kidney, liver FAD | Glycine Ov
Fumaric hydrogenase Yeast FAD | Reduced dyes (leuco- | Fumarie acid | 2700
methylene violet)
Glucose oxidase | Penicillium FAD | Glucose | Oz
notatum |
Histaminase, diamino oxi- | Kidney FAD | Di- and polyamines Oz
dase
* RP = riboflavin phosphate; FAD = flavin adenine dinucleotide.
6 M.B. = Methylene blue.
Kuhn and Desnuelle*s“° obtained results which were approximately the
same except that they found 0.48 % sulfur instead of 1.0 %. Hydrolysis of
the apoenzyme by boiling with dilute sulfuric acid and analyses of the
for amino acids gave the following values: arginine
resulting hydrolyzate
26R. A. Kekwick and K
27 H. Theorell, Biochem
.O. Pedersen, Biochem. J. 30, 2201 (1936).
. Z. 290, 293 (1937).
28 R. Kuhn and P. Desnuelle, Ber. 70, 1907 (1937).
29 R. Kuhn and P. Desnuelle, Z. physiol. Chem. 251, 14 (1938).
80 R. Kuhn and P. Desnuelle, Z. physiol. Chem. 251, 19 (1938).
IV. BIOCHEMICAL SYSTEMS 341
8.25%, histidine 2.75%, lysine 13.7%, tyrosine 7.75%, phenylalanine
5.75 %, tryptophan 4.86%, cystine 0.48 %, glutamic acid 7.17%, and as-
partic acid 2%. Since only 20 % of the total sulfur could be accounted for
as cystine, it is probable that other sulfur-containing amino acids are pres-
ent in the protein. According to these analyses, which account for about
66 % of the total nitrogen, the yellow enzyme contains approximately*! 33
molecules of arginine, 13 molecules of histidine, 66 molecules of lysine, 40
molecules of proline, 30 molecules of tyrosine, 24 molecules of phenylalanine,
17 molecules of tryptophan, 34 molecules of glutamic acid, 12 molecules of
aspartic acid, and only 1 to 2 molecules of cystine.
b. Mechanism of Action of Yellow Enzyme
After Barron and Harrop*®: * found that methylene blue could catalyti-
eally increase the respiration of erythrocytes, Warburg and Christian*
repeated the experiménts, using hexose monophosphate (Robison ester) as
a substrate in an extract of horse erythrocytes. From this extract they were
later able to separate a thermolabile substance, an enzyme designated as
zwischenferment (Robison ester dehydrogenase), and a thermostable ‘‘zwi-
schenferment-coferment”’ (triphosphopyridine nucleotide) to form a com-
plete, iron-free, respiratory chain capable of reacting with molecular oxy-
gen. The isolation of the yellow enzyme from bottom yeast! led to the
chemical characterization of the thermolabile substance in blood cells
which became known as the yellow oxidation enzyme.
Warburg*® has stated that in respiring cells the yellow enzyme cannot
successfully compete with hemin enzymes which react with oxygen at a
much greater rate. In hemin-free cells, e.g., in facultative anaerobic lactic
acid bacteria, the respiration is catalyzed by the yellow enzyme which es-
tablishes direct contact with molecular oxygen.** However, the “turnover
number” of the old yellow enzyme (the number of times that a molecule
of enzyme is both oxidized and reduced in a minute) under optimum con-
ditions at 38° in pure oxygen is only 55.?° At the low oxygen tensions which
exist in animal tissues, flavoprotein would hardly be autoxidizable. This
would mean that the old yellow enzyme, at best, fulfills a highly special-
ized, not yet understood role in normal cell respiration, that it may react
with other unknown acceptors, or that it is an artifact?! of preparation.
The classical method of describing the action of the old yellow enzyme
31 C. Oppenheimer and K. G. Stern, Biological Oxidation, p. 192. Nordemann, New
York, 1939.
#2 E.S. G. Barron and G. A. Harrop, J. Exptl. Med. 48, 207 (1928).
8% E.S.G. Barron and G. A. Harrop, J. Biol. Chem. 79, 65 (1928).
34 OQ. Warburg and W. Christian, Biochem. Z. 242, 206 (1931).
8° OQ. Warburg and W. Christian, Biochem. Z. 238, 131 (1931).
3% OQ. Warburg, Naturwissenschaften 22, 441 (1934).
342 RIBOFLAVIN
is based upon the fact that a mixture of hexose monophosphate, Robison
hexose monophosphoric enzyme (zwischenferment), and coenzyme II
(TPN) does not react with molecular oxygen. When the old yellow enzyme
is added to this mixture, the following reactions take place:
H OH
a4
C
|
HC—OH
|
HOC—H 0 = TEN ee. a ener
| Tri
phospho-
likanel pyridine
bots
HC OH nucleotide
HC—O—P=O
H
OH
COOH
HC—0
HOCH
HC-—Om
|
HCO OH
H—C—O—P=
H
OH
H.-TPN + flavoprotein — TPN + H_-flavoprotein Phosphogluconie acid
H,»-Flavoprotein + O2 — flavoprotein + H2O>»
As previously stated, this cycle of oxidation and reduction is relatively
slow even when pure oxygen is used.
2. SYNTHETIC, OR CROSSED, YELLOW ENZYME
The property of the flavin coenzymes which makes it possible for them
to form complexes with foreign apoenzymes was utilized by Warburg and
Christian?! to make the first synthetic flavoprotein. They combined the
protein part of the old yellow enzyme with flavin adenine dinucleotide and
showed that the resulting holoenzyme could, like the old yellow enzyme,
catalyze the oxidation of both di- and triphosphopyridine nucleotides. How-
ever, the catalytic activity was even less than the slow rates shown by the
old yellow enzyme.
IV. BIOCHEMICAL SYSTEMS 343
3. Haas ENZYME
In 1938, shortly after Warburg and Christian! ?3» 7 had identified flavin
adenine dinucleotide as the prosthetic group of p-amino acid oxidase, Haas**
isolated a flavoprotein from bottom yeast which contained isoalloxazine
adenine dinucleotide as the prosthetic group. The catalytic properties
were qualitatively the same as those of the old yellow enzyme. Haas’ en-
zyme was somewhat more rapidly reduced by reduced triphosphopyridine
nucleotide, but the rate achieved was still too slow to be of biological im-
portance. At physiological tensions of oxygen this flavoprotein was virtually
non-oxidizable, and in reconstructed systems it required an intermediate
carrier, like methylene blue, for its reaction with molecular oxygen.
The Haas enzyme contained about 0.7 % flavin adenine dinucleotide.
The molecular weight was estimated as 70,000. The specific protein could
not form a catalytically active complex with riboflavin phosphate.
4, DIAPHORASE
The term diaphorase (from the Greek édcadepav = transfer) is applied
to a group of flavoproteins capable of catalyzing the oxidation of reduced
pyridine nucleotides. Diaphorase, also called coenzyme factor, was dis-
covered independently by von Euler and Hellstrom*® and by Dewan and
Green.*’: 4! It has since been found in bacteria, yeast,” plants,* blood,“
milk, animal muscle,*’: #: 4° brain, kidney, intestine, thyroid, and pla-
centa.
Preparations of diaphorase are essentially alkaline phosphate extracts of
ground tissues. These crude preparations are more active* per milligram
of dried weight than Warburg’s pure old yellow enzyme. Most diaphorases
are associated with insoluble particles which complicate the task of puri-
fication.
The prosthetic group in diaphorase is isoalloxazine adenine dinucleotide.
Heating to 60° does not destroy the enzymatic activity. Solutions of di-
aphorase are yellow and show a green fluorescence. In this respect they
differ from other flavoproteins which do not fluoresce. The addition of
sodium dithionate or reduced diphosphopyridine nucleotide decolorizes the
enzyme solution.
87 Q, Warburg and W. Christian, Biochem. Z. 296, 294 (1938).
8% EK. Haas, Biochem. Z. 298, 378 (1938).
89H. von Euler and H. Hellstrém, Z. physiol. Chem. 252, 31 (1938).
*0 J. G. Dewan and D. E. Green, Nature 140, 1097 (1937).
“1 J.G. Dewan and D. E. Green, Biochem. J. 32, 626 (1938).
“2D. E. Green and J. G. Dewan, Biochem. J. 32, 1200 (1938).
“8 H.S. Corran and D. E. Green, Biochem. J. 32, 2331 (1938).
“ H. von Euler and G. Giinther, Z. physiol. Chem. 256, 229 (1938).
“6 H. von Euler and K. Haase, Naturwissenschaften 26, 187 (1938).
344 RIBOFLAVIN
The general diaphorase reaction for the oxidation of either the di- or
triphosphopyridine nucleotide is as follows:
H.-DPN + diaphorase — DPN + reduced diaphorase
Reduced diaphorase + methylene blue
— Diaphorase + leucomethylene blue
Leucomethylene blue + O2 — Methylene blue + H.O2
Abraham and Adler*® have indicated that there are two separate di-
aphorases, one for reduced coenzyme I and one for reduced coenzyme II.
5. STRAUB FLAVOPROTEIN, SOLUBLE DIAPHORASE
The chemical nature of the diaphorases was clarified when Straub‘: 4
succeeded in separating a soluble flavoprotein from pig heart. This prepa-
ration had all the catalytic properties of diaphorase, and it was concluded
that they were the same compound in different physical states. The essen-
tial step of the preparation was the heating of the crude diaphorase prepa-
ration at pH 4.6 in 2% ammonium sulfate and 3 % ethyl alcohol at 48° for
10 to 15 minutes. Some 14 % of the enzyme activity went into solution and
could be centrifuged from the insoluble particles.
Straub’s flavoprotein is much more stable to heat than diaphorases which
are attached to insoluble particles. The soluble flavoprotein from the pig
heart can be heated to 80° without destroying its catalytic activity. Boiling
the solution liberates the prosthetic group, flavin adenine dinucleotide.
According to Corran et al.,*° the oxidation of both reduced coenzyme I
and reduced coenzyme II can be catalyzed by this flavoprotein. Abraham
and Adler‘® showed that the heart flavoprotein had very little activity
against reduced coenzyme II, and it is now customary to catalog the oxi-
dation of reduced diphosphopyridine nucleotides as a specific function of
soluble diaphorase.
The catalytic effect of heart flavoprotein on oxygen uptake in lactic
systems and in malic, tissue phosphoric, and a-glycerophosphoric enzyme
systems has been stressed by Corran e¢ al. The turnover number of this
enzyme was found to be 8500.
6. CyTocHROME C REDUCTASE OF YEAST
In the living cell cytochrome ¢ is reversibly reduced and oxidized. Cyto-
chrome oxidase provides a mechanism for the participation of oxygen in
46), P. Abraham and EH. Adler, Biochem. J. 34, 119 (1940).
47 FB. Straub, Nature 148, 76 (1939).
48. B. Straub, Biochem. J. 33, 787 (1939).
49 H.S. Corran, D. E. Green, and F. B. Straub, Biochem. J. 33, 793 (1939).
IV. BIOCHEMICAL SYSTEMS 345
this preparation, but the re-reduction of cytochrome e¢ will not take place
in vitro unless an enzyme capable of reducing cytochrome ¢ is added. Several
flavoproteins capable of reducing cytochrome ec have been isolated.°? One
of these is cytochrome ec reductase, which, like Warburg’s yellow enzyme,
has the ‘‘mononucleotide”’ riboflavin phosphate for a prosthetic group.
This enzyme, which was isolated by Haas ef al.°! in 1940 and which
catalyzes the transfer of hydrogen between reduced triphosphopyridine
nucleotide and cytochrome c, is not to be confused with either the di-
aphorases, which can oxidize diphosphopyridine nucleotide, or with Haas’
flavoprotein from yeast, which, like the diaphorases, contains riboflavin
dinucleotide as the prosthetic group.
a. Preparation and Properties of Cytochrome C Reductase
A specially prepared dried brewer’s yeast®! was extracted in 20° water
for 33 hours. After centrifuging, the resulting supernatant solution was
51% saturated with ammonium sulfate at pH 4.5. The resulting precipitate
was suspended in 31 % ammonium sulfate, in which the enzyme is soluble;
again precipitated with 51% ammonium sulfate; freed of salt by dialysis;
precipitated with ethanol; adsorbed on aluminum hydroxide gel and eluted
with alkaline ammonium sulfate; adsorbed on tricalcium phosphate gel and
eluted with pH 6.1 phosphate buffer; and finally adsorbed on aluminum
hydroxide gel and eluted with alkaline ammonium sulfate, from which solu-
tion it was precipitated with 70 % saturated ammonium sulfate. The cyto-
chrome ec reductase so obtained in 1940 was considered to be 87 % pure. It
was estimated that the dried yeast contained 0.6 g. of this flavoprotein per
kilogram.
The first preparations of cytochrome c reductase were very unstable in
environments not considered harmful to other flavoproteins, which may
explain why it was not isolated at an earlier date. The enzyme lost 30 % of
its activity in 2 days at 0° and would have been completely destroyed
after 24 hours in 33% acetone, a condition under which Warburg and
Christian prepared their old yellow enzyme. Its molecular weight is about
75,000. Its prosthetic group, riboflavin-5-phosphate, can be interchanged*!
with that of the old yellow enzyme. This fact placed this coenzyme in a
more significant position as a biologically important compound, since prior
to the isolation of cytochrome ¢c reductase it was suspected that riboflavin-
5-phosphate might be an artifact.
In 1942, Haas ef al.®? improved the procedure of isolation and obtained
50 J. B. Sumner and G. F. Somers, Chemistry and Methods of Enzymes, p. 271.
Academic Press, New York, 1947.
5! E. Haas, B. L. Horecker, and T. R. Hogness, J. Biol. Chem. 136, 747 (1940).
82 EB. Haas, C. J. Harrer, and T. R. Hogness, J. Biol. Chem. 148, 341 (1942).
346 RIBOFLAVIN
a stable product by lyophilization which was 98 % pure, and had eight-fold
better yield. This enzyme reacted 10° times faster with cytochrome than
with oxygen, so it was concluded that the direct action of the reductase
with oxygen was of no physiological importance.
b. Mechanism of Action of Cytochrome C Reductase
Reduced cytochrome c¢ reductase reacts with oxidized cytochrome ¢
(CyFet**) according to the equation.
Reduced Cy c¢ reductase + 2CyFett* >
Cy c reductase + 2CyFe** + 2Ht
When hexose monophosphate (Robison ester), Robison ester dehydrogen-
ase (zwischenferment), and coenzyme II (TPN) are added to a solution
containing cytochrome c and cytochrome ¢ reductase,
Glucose-6-phosphate + TPN + H,O _ 2wischenferment_.
H.-TPN + phosphogluconic acid
H.-TPN + cytochrome c reductase —
TPN + reduced cytochrome ec reductase
which in turn will reduce cytochrome ec.
7. CyTocHROME C REDUCTASE OF LIVER
Proof of the reduction of cytochrome c in animal tissue by reduced coen-
zyme II (H2-TPN) was not reported until 1949, when Horecker® isolated
TPN cytochrome c reductase from pig liver. This flavoprotein has flavin
adenine dinucleotide as a prosthetic group but otherwise is quite similar
to the cytochrome c reductase obtained from yeast by Haas, Horecker, and
Hogness.
a. Preparation
The details of the preparation of acetone liver powder and its subsequent
extraction, trypsin digestion, salt precipitations, gel adsorptions, and fur-
ther purifications are described by Horecker.**
b. Properties
The enzyme can easily be split into the protein and flavin adenine di-
nucleotide prosthetic groups. The protein fraction can be reactivated by
either flavin adenine dinucleotide or riboflavin phosphate. Of interest is the
fact the apoenzymes of both yeast and liver cytochrome ¢ reductases form
53 B. L. Horecker, J. Biol. Chem. 183, 593 (1950).
IV. BIOCHEMICAL SYSTEMS 347
more active compounds with riboflavin phosphate than with flavin adenine
dinucleotide.
The reaction of liver cytochrome ¢ reductase with oxygen is less than 2%
as fast as the reaction with cytochrome c.
The turnover number of the liver enzyme is 1140 (molecular weight
68,000), as compared to a similar turnover number of 1300 (molecular
weight 78,000) for the yeast enzyme.
8. XANTHINE OXIDASE, ALDEHYDE OXIDASE, XANTHOPTERIN OXIDASE
The enzymatic oxidation of hypoxanthine and xanthine to uric acid in
the presence of tissue brei and oxygen was recognized by Spitzer®‘ in 1899.
Burian®® named this enzyme xanthine oxidase. In 1902, Schardinger®® ob-
served that if formaldehyde and methylene blue were added to fresh milk
in the absence of oxygen the methylene blue was rapidly decolorized. These
two enzymatic processes remained unrelated until 1922, when Morgan et
al.” showed that milk was a rich source of xanthine oxidase. The work of
Dixon and Thurlow,°** Booth,*? and Ball®: ® supported the suggestion that
the same enzyme is involved in both reactions. Corran and Green® isolated
a flavoprotein from cow’s milk which showed no activity as a xanthine-
aldehyde oxidase but could catalyze the oxidation of reduced coenzyme I.
Then Corran et al.® prepared a milk flavoprotein which catalyzed the oxi-
dation of hypoxanthine, aldehydes, and reduced coenzyme I, and showed
that although all three activities were associated with the same flavoprotein
they could be differentially inactivated. Although the Michaelis constant
(K,,) and the kinetics of the three substrates are not identical, the addition
of aldehyde specifically inhibits the oxidation of purine and vice versa.
Either the same active group is concerned in the activation of both purines
and the aldehydes, or else there are two active groups in such close juxta-
position in the protein molecule that activity at one interferes with activity
at the other.®
a. Preparation
Ball®™ has prepared xanthine oxidase from unpasteurized cream, since
the enzyme was adsorbed on the fat globules. After the cream was shaken
with one volume of 0.2 M NasHPO, at 38° for 2 hours, the fat was re-
moved by centrifugation at 0°. The enzyme solution was digested with
commercial lipase for 314 hours at 38°, clarified with 0.5 M calcium chlo-
ride, and centrifuged. The solution was 60% saturated with ammonium
sulfate, and after standing overnight at 0° the enzyme was centrifuged
down, dissolved in one-tenth the original volume of water, and further
purified by precipitating with 33 % ammonium sulfate.
54W. Spitzer, Pfliigers Arch. ges. Physiol. 76, 192 (1899).
348 RIBOFLAVIN
The preparation by Corran et al. started with whole milk and included
precipitation in 13 % aleohol and adsorption on alumina as well as precipi-
tation with ammonium sulfate. Although their process was a bit more com-
plicated and gave a lower yield, the product obtained had a catalytic activ-
ity which was about 1000 times greater than that of milk.
Xanthine oxidase can also be prepared from pig’s liver.®
b. Properties of Xanthine Oxidase
The xanthine oxidase prepared by Ball® had an isoelectric point at about
pH 6.2. The addition of cyanide alone to xanthine oxidase caused an irre-
versible inhibition. If cyanide and substrate were added simultaneously,
no inhibition occurred. The mode of action of cyanide, first noted by
Szent-Gyorgyi® and later studied by Dixon and Keilin,®* remains unex-
plained. This inhibition was utilized by Corran et al.® to eliminate the
xanthine-aldehyde without affecting the diaphorase activity. This diapho-
rase activity to reduced coenzyme I has been confirmed by Ball and Rams-
dell. ® :
The molecular weight of Ball’s preparation was calculated as 74,000. It
was golden brown in color and did not fluoresce in ultraviolet light. The
flavin adenine dinucleotide prosthetic group could be split off from the
apoenzyme by dialyzing against running water for 2 weeks.
c. Action of Xanthine-Aldehyde Flavoproteins
Hypoxanthine and xanthine are oxidized to uric acid in the presence of
the oxidase:
HN—C=O HN—C=O
|| ou [| a
(HO)HC C—N O=C C—N
rae + 2k
x XS
CH CH
VA Vl
EN CaN HN CaN
Hydrated hypoxanthine Xanthine
HN—C=O HN—C=O
H |
O= INI O=—=C C—N
—> a + 2H
CH(OH) C=O
ie
HIN C——N ENS CaN
H
Hydrated xanthine Uric acid
IV. BIOCHEMICAL SYSTEMS 349
in the presence of air, H2O2 is formed. Dixon® has reported on the relative
velocity with which nine different purines can be oxidized. Xanthine oxi-
dase can catalyze the anaerobic dismutation of xanthine to hypoxanthine
and uric acid:*°
2 Xanthine @ Hypoxanthine + uric acid
There is no specificity toward aldehydes.” Any non-toxic aldehyde can
be oxidized by this flavoprotein.
R OH O
es VA
PENS ~
H OH
RCHO + H:0 —
d. Xanthopterin Oxidase
Wieland and Liebig™ described the presence in liver and milk of an en-
zyme which catalyzes the oxidation of xanthopterin (2-amino-4,6-dihy-
droxypteridine). Kalekar and Klenow” found that xanthine and xanthop-
terin oxidases of cream are inhibited by certain preparations of folic acid.
Later, Kalckar et al.7? showed that this inhibition was due to 2-amino-4-
hydroxypteridine-6-aldehyde, a fission product of folic acid formed by
irradiation. Studies of the inhibitory effects of this and other compounds
by Hofstee,4 Lowry et al.,7> and Williams and Elvehjem’® showed that
55 R. Burian, Z. physiol. Chem. 43, 497 (1905).
56 F. Schardinger, Z. Untersuch. Nahr. u. Genussm. 5, 1113 (1902).
57 E. J. Morgan, C. P. Stewart, and F. G. Hopkins, Proc. Roy. Soc. (London) B94,
109 (1922).
88 M. Dixon and S. Thurlow, Biochem. J. 18, 971, 976, 989 (1924).
59 V. H. Booth, Biochem. J. 32, 494 (1938).
60 FE. G. Ball, Sczence 88, 131 (1938).
61. G. Ball, J. Biol. Chem. 128, 51 (1939).
62 H.S. Corran and D. E. Green, Biochem. J. 32, 2231 (1938).
68H. S. Corran, J. G. Dewan, A. H. Gordon, and D. E. Green, Biochem. J. 33, 1694
(1939).
64D. E. Green, Mechanisms of Biological Oxidation, p. 97. University Press, Cam-
bridge, 1940.
65 A Szent-Gyorgyi, Biochem. Z. 173, 275 (1926).
66 M. Dixon and D. Keilin, Proc. Roy. Soc. (London) B119, 159 (1936).
87 F. G. Ball and P. A. Ramsdell, J. Biol. Chem. 181, 767 (1939).
68M. Dixon, Enzymologia 5, 198 (1938).
69T). E. Green, Biochem. J. 28, 1550 (1934).
70V. H. Booth, Biochem. J. 29, 1732 (1935).
71H. Wieland and R. Liebig, Ann. 555, 146 (1944).
72H. M. Kalckar and H. Klenow, J. Biol. Chem. 172, 349 (1948).
73H.M. Kalekar, N. O. Kjeldgaard, and H. Klenow, J. Biol. Chem. 174, 771 (1948).
™4B.H. J. Hofstee, J. Biol. Chem. 179, 633 (1949).
350 RIBOFLAVIN
xanthine oxidase and the xanthopterin oxidase of cream are identical.
2-Amino-4-hydropteridine is also oxidized by xanthine oxidase.7®
9. Liver ALDEHYDE OXIDASE
While attempting to prepare the xanthine oxidase of pig liver, Gordon
et al.” isolated a flavoprotein which specifically catalyzed the oxidation of
aldehydes to their corresponding acids. The prosthetic group was flavin
adenine dinucleotide. Unlike its counterpart in milk, this aldehydase showed
no activity toward either hypoxanthine or reduced coenzyme I.
a. Preparation
The removal of four main colored impurities—hemoglobin, xanthine oxi-
dase flavoprotein, a Fe(OH);-protein complex, and catalase—served as the
working hypothesis upon which Gordon ef al.” based their isolation. Hemo-
globin was separated by precipitating the aldehydase with half-saturated
ammonium sulfate. Xanthine oxidase was destroyed by heating at 48° for
5 minutes in 25 % alcohol. The orange-red Fe(OH)s3-protein complex (prob-
ably ferritin) is slightly less soluble than the aldehyde oxidase in ammonium
sulfate solutions. Repeated fractionations between 20 and 35 % saturations
of ammoniacal ammonium sulfate solutions removed all the iron complex.
The removal of catalase proved most difficult, and, since some catalase
accompanies the aldehyde enzyme in the course of many salt fractionations,
adsorptions, solvent precipitations, cataphoresis, ete., it was believed that
catalase formed a compound with the aldehyde enzyme which could not
be resolved by known methods.
b. Properties of Liver Aldehyde Oxidase
Purified preparations of the enzyme are yellowish brown in color. This
color is partly bleached on reduction with hydrosulfite and restored after
shaking in air. It contains 0.17% flavin phosphate and has a turnover
number of about 550 with acetaldehyde. The flavin adenine dinucleotide
prosthetic group can be split off by boiling, acidification to less than pH
4, prolonged dialysis against water, or exposure to fat solvents, ete.
Gordon et al.” reasoned that their liver flavoprotein was different from
the milk xanthine-aldehyde-dihydrocoenzyme I oxidase of Corran et al.*®
because the liver enzyme was somewhat less soluble in ammonium sulfate
solutions and was rapidly and irreversibly inactivated by dialysis against
water at O°, whereas the milk enzyme was stable over a period of days.
Furthermore, the cyclical reduction and oxidation of the flavin group in
7° OQ. H. Lowry, O. A. Bessey, and BE. J. Crawford, J. Biol. Chem. 180, 389, 399 (1949).
76 J. N. Williams and C, A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 71, 303 (1949).
7 A. H. Gordon, D. E. Green, and V. Subrahmanyan, Biochem. J. 34, 764 (1940).
IV. BIOCHEMICAL SYSTEMS 351
the liver enzyme could be easily demonstrated, whereas there was no clear
evidence of such a mechanism in the case of the milk enzyme.
Another difference between liver and milk xanthine oxidase was studied
by Richert e¢ al.,> who showed that antabuse (tetraethylthiuram disulfide)
inhibited the rat liver oxidase but did not affect milk xanthine oxidase.
Richert and Westerfeld’®: 8° and Remy and Westerfeld®! have made an
extensive study of the xanthine oxidase activities of a variety of animal
tissues.
c. Mechanism of Action of Liver Flavoprotein
The liver aldehydase oxidizes a variety of aldehydes such as acetaldehyde,
propaldehyde, butaldehyde, crotonaldehyde, benzaldehyde, salicylalde-
hyde, and glycollic aldehydes. Formaldehyde is not oxidized, but this may
be due to the denaturing effect of formaldehyde on the enzyme. The action
between aldehyde and oxygen is not direct but proceeds as follows:
Crotonaldehyde + flavoprotein —
Crotonie acid + reduced flavoprotein
Reduced enzyme + O, — Enzyme + H.O»
The presence of catalase in the enzyme preparation causes all the perox-
ide to be decomposed to water and oxygen.
Oxidation-reduction indicators such as methylene blue, nitrate, and cyto-
chrome ec can be reduced by the enzyme, but the catalytic reductions of
nitrate and cytochrome c¢ are too slow to be of physiological significance.
10. p-Amino Actp OXIDASE
The existence of this enzyme was in dispute for years before Krebs: 5°
extracted it from the kidney and liver of rats. He clearly distinguished it
from L-amino acid oxidase in 1935.54 Warburg and Christian’ showed that
the prosthetic group of this enzyme is flavin adenine dinucleotide. Negelein
and Brémel* isolated the apoenzyme portion in a high degree of purifica-
tion and estimated molecular weight at 70,000. There is no absolute agree-
7D. A. Richert, R. Vanderlinde, and W. W. Westerfeld, J. Biol. Chem. 186, 261
(1950).
7D. A. Richert and W. W. Westerfeld, Proc. Soc. Bxptl. Biol. Med. 76, 252 (1951).
80 W. W. Westerfeld and D. A. Richert, 7. Biol. Chem. 184, 168 (1950); 192, 85 (1951).
81 C, Remy and W. W. Westerfeld, J. Biol. Chem. 198, 659 (1951).
8 H. A. Krebs, Z. physiol. Chem. 217, 191 (1933).
83H. A. Krebs, Z. physiol. Chem. 218, 157 (1933).
84H. A. Krebs, Biochem. J. 29, 1620 (1935).
85 ©. Negelein and H. Brémel, Biochem. Z. 300, 225 (1939).
aoe RIBOFLAVIN
ment on the rates at which different amino acids are attacked,** since tech-
niques and preparations differ, but it is generally acknowledged that methi-
onine shows the highest rate of oxidation and that glycine, lysine, and
glutamic acid are not oxidized at all. Amino acids with the amino group in
the 6 position, e.g., 8-alanine and B-aminobutyric acid, and dipeptides such
as alanyl glycine and leucyl glycine are inactive as substrates.
p-Amino acid oxidase is present in most animal organs, kidney and liver
being the best sources. There is an active D-amino oxidase in Neurospora.?*
Kisch®” has claimed that there are at least three different p-amino acid
oxidases, depending on the species of animal from which the kidney extracts
are prepared.
The physiological role of this enzyme is not understood, since the amino
acids which occur in cells are all of levo configuration. It has been postulated
that it is present to destroy p-amino acids formed by the racemization of
the natural amino acids.
a. Preparation of D-Amino Acid Oxidase
Since it is not inhibited by drying or by treatment with many organic
solvents, it is simple to prepare D-amino oxidase by extracting ground tissue
(usually kidney) with acetone and drying zn vacuo." The tissue powder
can be extracted with water.
b. Mechanism of Action
The oxidation of p-amino acids based upon the determination of oxygen
uptake, ammonia, and keto acids was represented as*
NH; he ;
R—c— coon rede P=—6— COOH see R—C— COOH NEE
H
However, hydrogen peroxide is formed during the oxidation; so the reaction
in highly purified preparations devoid of catalase may be written as fol-
lows 2288
NH, O
(1) CH;—C—COOH + flavin + H.O — CH;C—COOH + H.-flavin
lal
(2) H.-flavin + O2.— H2O2 + flavin
I
(3) CH;CCOOH + H:02.— CH;COOH + CO2+ H:20
The turnover number of b-amino acid oxidase is about 2000.
86 J. R. Klein and P. Handler, J. Biol. Chem. 139, 103 (1941).
87 B. Kisch, Enzymologia 12, 97 (1936).
IV. BIOCHEMICAL SYSTEMS 353
11. u-AmIno Acip OxipAsn, L-Hyproxy Acip OxIDAsE
Although its activity in tissue had been investigated by many workers
before and after Krebs’*?-** classical studies, it was not until 1944 that
Stumpf and Green*® obtained a cell-free preparation of L-amino acid oxidase
from Proteus vulgaris. The following year, the first electrophoretically
homogeneous L-amino acid oxidase was prepared by Blanchard ef al.®*: °°
from rat kidneys. A distinguishing feature of this preparation was that
riboflavin monophosphate was the prosthetic group. Later, these workers®!
showed that this enzyme was able to catalyze the oxidation of L-a-hydroxy
acids. The apparent specificity of the enzyme for the L-antipodes was at-
tested by the fact that L-lactic acid is completely oxidized, whereas DL-
lactic acid is only 50% oxidized.
Another water-soluble L-amino oxidase is found in snake venom. This
has been studied by Zeller and Maritz® and Singer and Kearney® and
may be a flavoprotein.
a. Preparation
Blanchard et al.®° started with a cold homogenate of 1.5 kg. of rat kidney
which was dewatered and dried with the aid of cold acetone. They used an
extensive series of extractions and precipitations from salt solutions, in-
cluding stepwise fractionations between 30 and 60% ammonium sulfate
saturations.
b. Properties
Lt-Amino acid oxidase is more difficult to extract from ground tissue than
p-amino acid oxidase. It is more rapidly denatured by organic solvents, is
inhibited by cyanide, and has only a small fraction as much activity as
p-amino acid oxidase in liver or kidney slices.*
The enzyme is electrophoretically homogeneous,*® but in the ultracen-
trifuge two components appeared. The lighter component (molecular
weight 138,000) contained 2 molecules of the riboflavin phosphate. The
heavier component (molecular weight 555,000), which seems to be an ag-
gregate of 4 molecules of the lighter component, had 8 molecules of flavin.
The flavin content was 0.66 %. The enzyme from rat kidney and liver cata-
88 P. K. Stumpf and D. E. Green, J. Biol. Chem. 158, 387 (1944).
89M. Blanchard, D. E. Green, V. Nociti-Carroll, and 8. Ratner, J. Biol. Chem.
155, 421 (1944).
9M. Blanchard, D. E. Green, V. Nociti-Carroll, and S. Ratner, J. Biol. Chem.
161, 583 (1945).
* M. Blanchard, D. E. Green, V. Nociti-Carroll, and 8. Ratner, J. Biol. Chem. 163,
137 (1946).
# E. A. Zeller and A. Maritz, Helv. Chim. Acta 27, 1888 (1944); Helv. Physiol. Acta 8,
C48 (1945).
% T. P. Singer and E. B. Kearney, Federation Proc. 8, 251 (1949).
354 RIBOFLAVIN
lyzed the oxidation of the following thirteen natural amino acids to their
keto derivatives: leucine, methionine, proline, norleucine, norvaline, phenyl-
alanine, tryptophan, isoleucine, tyrosine, valine, histidine, cystine, and
alanine.
The specificities of the t-hydroxy acid oxidase are similar to those of the
L-amino acid oxidase;*! there is a pronounced relationship between the
length of the carbon chain and the rate of oxidation. The turnover number
of L-amino oxidase is very low (about 6).
c. Mechanism of Action
In the presence of oxygen as hydrogen acceptor, one molecule of oxygen
is taken up for each molecule of amino acid oxidized, and one molecule
each of keto acid, NH;, and H2O2 are formed. Methylene blue can replace
oxygen as the hydrogen acceptor.
12. GLYCINE OXIDASE
Prior to the isolation of glycine oxidase by Ratner et al.,** three enzymes
were known which could deaminate amino acids oxidatively: D-amino acid
oxidase, L-amino acid oxidase, and t-glutamic dehydrogenase. None of
these has any activity toward glycine.
a. Preparation
Glycine oxidase, which was found in the liver or kidney of all animals
tested, was extracted from an acetone powder of pig kidneys and precipi-
tated with 30 % saturated ammonium sulfate. The precipitate was dissolved
in water and precipitated twice in 24% monopotassium acid phosphate.
The enzyme preparation retained activity for 7 to 10 days at 0.5°. It was
rapidly destroyed (80 % in 12 hours) by dialysis against water.
b. Properties
Solutions are pale greenish yellow but water-clear. These can tolerate
3 minutes’ exposure to 55° without appreciable loss of activity. Flavin
adenine dinucleotide is the prosthetic group, and it can be reversibly sepa-
rated from the apoenzyme. Glycine oxidase catalyzes the oxidation of
glycine and N-monomethylglycine (sarcosine), but not other substituted
products or peptides of glycine.
c. Mechanism of Oxidation
The oxidation of glycine (where R is H) or sarcosine (where R is CHs)
%S. Ratner, V. Nocito, and D. E. Green, J. Biol. Chem. 152, 119 (1944).
IV. BIOCHEMICAL SYSTEMS 355
is as follows:
CH:NHRCOOH + oxidized enzyme — CH=NRCOOH + reduced enzyme
CH=NRCOOH + H.0 — CHOCOOH + NHR
Reduced enzyme + O2 — oxidized enzyme + H20.2
Methylene blue can be reduced when the reaction is carried out anaerobi-
cally.
13. Fumaric HypDROGENASE
The only example to date of a flavoprotein enzyme system in which an
organic substrate is a hydrogen acceptor was discovered in yeast by Fischer
and Eysenbach® and Fischer et al.%° Fumaric hydrogenase from yeast
catalyzes the reduction of fumaric acid to succinic acid by the reduced
forms of various oxidation-reduction dyes. Fumaric acid seems to be a
specific hydrogen acceptor, but to date no naturally occurring hydrogen
donor has been found for this reaction.
The enzyme is present as an impurity in the preparation of Warburg
and Christian’s old yellow enzyme, from which it can be separated by
cataphoresis in alkaline solutions.** The prosthetic group is flavin adenine
dinucleotide.
The reduction of fumarate was demonstrated as follows:
COOH
Leuco- Be Coon CH, . :
methylene + | = | 2 bass ie
violet HOOC—C—H CH,
“id
COOH
Fumarie acid Succinie
acid
Fumarate can be replaced in this system®> by maleate, crotyl alcohol,
phenylerotyl alcohol, and geraniol. Hyposulfite can replace the reduced
dyes as reductants of the enzyme.
The turnover number of this reductase is about 2700.
14. Guucose OxIDASE OF MOoLps
Penicillium notatum produces a flavoprotein which oxidizes glucose to
gluconic acid in the presence of oxygen. Because hydrogen peroxide accu-
mulates as a result of its action, it was prematurely classified as an anti-
%° F. G. Fischer and H. Eysenbach, Ann. 530, 99 (1937).
98 F. G. Fischer, A. Roedig, and K. Rauch, Naturwissenschaften 27, 197 (1939).
356 RIBOFLAVIN
biotic, notatin.*” The enzyme apparently occurs in many fungi. This en-
zyme shows a pronounced specificity for glucose®’ and has very little or no
activity for about fifty other sugars tested.
H OH COOH
a
C HCOH
HCOH HOC—H
HOCH (07 —-0:-- EO > ) Hic On Heo:
HCOH He— OH
|
HC HC—_0OH
H
HC—OH
H
p-Glucose p-Gluconic acid
Methylene blue cannot be used instead of oxygen. If hydrogen sulfide*®
is added to poison the catalase in crude extracts, there is a quantitative
formation of hydrogen peroxide.
15. HISTAMINASE, DIAMINO OXIDASE
The flavoprotein nature of histaminase was first noted by Swedin!0%: 1%
in 1943, but a subsequent report by Leloir and Green!” did not confirm
his findings. Kapeller-Adler'® has presented evidence to show that this
enzyme prepared from hog kidney contains flavin adenine dinucleotide.
This flavoprotein will catalyze the oxidation of di- and polyamines to
amino aldehydes. Although it has a much higher affinity for histamine,
it will also act on cadaverine, putrescine, and agmatine.
According to Zeller,!°*: °° the action of histaminase is represented as
follows:
R—CH.NH2 + O2 + H:0 — RCHO + NH; + H:02
7 C, EK. Coulthard, R. Michaelis, W. F. Short, G. Sykes, G. E. H. Skrimshire, A. F.
B. Standfast, J. H. Birkinshaw, and H. Raistrick, Biochem. J. 39, 24 (1945).
9% K. Keilin and E. F. Hartree, Biochem. J. 42, 221 (1948).
99 W. Franke and F. Lorenz, Ann. 532, 1 (1937).
100 B, Swedin, Acta Med. Scand. 114, 210 (1943).
101 B, Swedin, Arkiv Kemz. Minerol. Geol. 17A, 27 (1944).
102 T,. F. Leloir and D. E. Green, Federation Proc. 5, 144 (1946).
103 R. Kapeller-Adler, Biochem. J. 44, 70 (1949).
104}. A. Zeller, Helv. Chim. Acta 21, 880 (1988).
105 HY. A. Zeller, Advances in Enzymol. 2, 93 (1942).
IV. BIOCHEMICAL SYSTEMS 357
C. MECHANISM OF ACTION
When the various enzyme systems in which riboflavin is a coenzyme are
alternately reduced and oxidized, it is believed that they function by ac-
cepting and donating hydrogen atoms one at a time. Several intermediates
of the reduced and unreduced molecules!’ have been postulated, but the
existence of such intermediates has not been proved by titration curves.!”
Since the quinhydrone'® is the only form that exists in dilute aqueous solu-
tion, the transfer of hydrogen atoms may be indicated as:
|
N N
HONG Bat SS ail. Stabilized
a EAC ie +H. semi- —TH,
‘si | —H quinoid ‘=
et \ GON pa radical
N C
O
A recent study by Singer and Kearney’? demonstrated that isoalloxazine
derivatives, in the absence of added proteins, are capable of catalyzing the
reaction between reduced pyridine nucleotides and cytochrome c. They did
not imply that this process, which is less efficient than the enzymatic reac-
tions, has a significant biological role, but they hoped that the reactions
provided a chemical basis for the function of the isoalloxazine nucleus as
a mediator of electron transfer between dihydropyridine nucleotides and
cytochrome c or molecular Os. The most effective compound in this non-
enzymatic reduction is isoriboflavin, followed by riboflavin, riboflavin phos-
phate, and flavin adenine dinucleotide.
106 R. Kuhn and R. Strébele, Ber. 70, 753 (1937).
107 T,. Michaelis and G. Schwartzenbach, J. Biol. Chem. 123, 527 (1988).
108 R. Kuhn and P. Boulanger, Ber. 69, 1557 (1936).
109 TP. Singer and E. B. Kearney, J. Biol. Chem. 183, 409 (1950).
358 RIBOFLAVIN
V. Specificity of Action
M. K. HORWITT
Riboflavin, riboflavin-5-phosphate,! and flavin adenine dinucleotide? are
the only naturally occurring flavins which have been found to have vitamin
Bz activity. They are equally effective in promoting the growth of rats and
Lactobacillus caset.
At this writing, the new vitamin activity of L-lyxoflavin, the 9-1-1’-
lyxityl sterioisomer of riboflavin, is being investigated. This compound has
been reported as a constituent of heart muscle,’ but its significance awaits
confirmation.*
Synthetic derivatives of isoalloxazine which have been found to have
riboflavin activities include:
(a) 7-Methy1-9-(p-1'-ribity])isoalloxazine®7
(b) 6-Methy1-9-(p-1’-ribityl)isoalloxazine®7®
(c) 6-Ethyl-7-methyl-9-(p-1’-ribityl)isoalloxazine®
These three compounds were approximately one-half as active as ribo-
flavin in growth tests on rats. Compound c was almost as active as riboflavin
in stimulating the growth of lactic acid bacteria,? but compounds a and b
stimulated the bacterial growth only moderately.
Among the synthetic arabityl derivatives which have been studied are:
(d) 6 ,7-Dimethyl1-9-(p-1’-arabityl)isoalloxazine’
(e) 6 ,7-Dimethy1-9-(L-1’-arabityl)isoalloxazine!
(f) 6-Methyl-9-(1-1’-arabityl)isoalloxazine®
(q) 6 ,7-Trimethylene-9-(L-1’-arabity])isoalloxazine’
(h) 6 , 7-Tetramethylene-9-(L-1’-arabity])isoalloxazine®
1P. Gyorgy, Proc. Soc. Exptl. Biol. Med. 35, 207 (1936).
2H. P. Sarett, J. Biol. Chem. 162, 87 (1946).
3. Sodi Pallares and H. Martinez Garza, Arch. Biochem. 22, 63 (1949).
4'T.S. Gardner, E. Wenis, and J. Lee, Arch. Biochem. 34, 98 (1951).
5 P. Karrer and T. H. Quibell, Helv. Chim. Acta 19, 1034 (1936).
6 P. Karrer, H. von Euler, M. Malmberg, and K. Schépp, Svensk. Kem. Tidskr. 47,
153 (1935).
7P. Karrer and F. M. Strong, Helv. Chim. Acta 18, 1343 (1935).
8 R. Kuhn, H. Vetter, and H. W. Rzeppa, Ber. 70, 1302 (1937).
91). E. Snell and F. M. Strong, Enzymologia 6, 186 (1939).
10 H. von Euler, P. Karrer, and M. Malmberg, Helv. Chim. Acta 18, 1336 (1935).
11P,. Karrer, H. Salomon, K. Schépp, F. Benz, and B. Becker, Helv. Chim. Acta 18,
908 (19385).
VI. BIOGENESIS 359
p-Araboflavin (compound d) inhibits growth and increases the mortality
rate” more than the absence of riboflavin alone. The other arabityl deriva-
tives can sustain life in rats at a diminished growth rate if given in rela-
tively large amounts.
Isoriboflavin [5 ,6-dimethyl-9-(p-1’-ribityl)isoalloxazine] is an isomer of
riboflavin which, if given to rats at levels of 2 mg. per day, will counteract
the growth-promoting effects of 40 y of riboflavin. It is interesting that
isoriboflavin has no inhibitory effect on Lactobacillus caset.
Riboflavin tetraacetate’ and both the mono- and diacetone derivatives™
are fully active in supporting rat growth, probably owing to hydrolysis in
the mammalian organism, but they are inactive for lactic acid bacteria.
Replacement of the p-ribityl group by a glucosidic group" results in a total
loss of biological activity. The monomethylol derivative!’ prepared by
reacting riboflavin with formaldehyde retains about half the original activ-
ity. Riboflavin mono-, di-, tri-, and tetrasuccinates!® vary in rat growth
activity as 100, 65, 21, and 0%, respectively. Substitution of a methyl
group in the 3 position” results in complete loss of vitamin activity.
Generally speaking, the activity of esterified derivatives of riboflavin
may vary with the ability of the test organism to effect hydrolysis of the
ester; the 3 position must remain unsubstituted, and substitution in the
6 or 7 position is necessary.
When the 6 and 7 positions are not substituted, the compounds are
toxic.'8 A recent review by Woolley!’ has highlighted the increased interest
in the inhibitory analogs of the vitamins.
VI. Biogenesis
M. K. HORWITT
Riboflavin is synthesized by most higher plants, yeasts, and lower fungi,
and by some bacteria. The tissues of higher animals are unable to synthe-
size this vitamin, but the gastrointestinal tract of many of these animals
harbor bacteria which may be capable of providing riboflavin for their host.
% H. von Euler and P. Karrer, Helv. Chim. Acta 29, 353 (1946).
R. Kuhn, H. Rudy, and F. Weygand, Ber. 68, 625 (1935).
™R. Kuhn and K. Strébele, Ber. 70, 747 (1937).
1K. Schoen and 8. M. Gordon, Arch. Biochem. 22, 149 (1949).
16M. F. Furter, G. J. Haas, and 8S. H. Rubin, J. Biol. Chem. 160, 293 (1945).
R. Kuhn, K. Reinemund, F. Weygand, and R. Strébele, Ber. 68, 1765 (1935).
*®R. Kuhn and P. Boulanger, Z. physiol. Chem. 241, 233 (1936).
DPD. W. Woolley, A Study of Antimetabolites. John Wiley and Sons, New York,
1952.
360 RIBOFLAVIN
The riboflavin content of the milk of cows and goats! is many times the
amount in the feed, as the result of synthesis by organisms which inhabit
the rumen of these animals.
Observations that the fecal contents of rats,? fowls,*? and man*: > may
have more riboflavin than the food ingested have stimulated research on
the nutritional usefulness of intestinal synthesis. It has been common labo-
ratory knowledge that coprophagia by rats must be avoided if nutritional
deficiencies are to be produced. Whether the riboflavin formed by micro-
organisms is absorbed from the lower intestinal tract in significant quan-
tities is not certain. Najjar et al.4 have reported that there was a rise in
urinary riboflavin after normal subjects were given enemas containing 20
mg. of riboflavin, but Everson et al.* found no increase in urinary excretion
of riboflavin after administering 2 mg. by retention enema. The concept of
low utilization of the riboflavin of intestinal bacteria is supported by
studies’ indicating the relative non-availability of the vitamins in ingested
yeast.
Although riboflavin is required as a growth factor for a large number of
microorganisms,’ most of them are able to synthesize more than their
requirement. Microbiological production of riboflavin by Clostridium
acetobutylicum has been promoted as a commercial source.’ There have
been reports on riboflavin production by the yeast Hremothecitum ashbyit'®
and by Candida guilliermondia.": * Certain molds (A shbya gossypii) pro-
duce and excrete so much riboflavin that yellow crystals are formed about
the mycelium.”
An investigation of riboflavin synthesis by the bacterial flora of the
human intestine has been made by Burkholder and McVeigh." The organ-
isms studied were Escherichia coli, Proteus vulgaris, Bacterium aerogenes,
Alcaligenes faecalis, Bacillus mesentericus, and Bacillus vulgatus. EH. colt,
1L. W. McElroy and H. Goss, J. Nutrition 20, 527 (1940).
2W.H. Griffith, J. Nutrition 10, 667 (1935).
3 W. F. Lamoureux and R. 8. Schumacher, Poultry Sci. 19, 418 (1940).
4V.A. Najjar, G. A. Johns, G. C. Medairy, G. Fleischmann, and L. E. Holt, Jr., J.
Am. Med. Assoc. 126, 357 (1944).
5 C, W.. Denko, W. E. Grundy, N. C. Wheeler, C. R. Henderson, G. H. Berryman,
T. E. Friedemann, and J. B. Youmans, Arch. Biochem. 11, 109 (1946).
6G. Everson, E. Wheeler, H. Walker, and W. J. Coulfield, J. Nutrition 35, 209
(1948).
7H. T. Parsons, A. Williamson, and M. L. Johnson, J. Nutrition 29, 373 (1945).
8 W. H. Peterson and M.S. Peterson, Bacteriol. Revs. 9, 49 (1945).
9C.F. Arzberger, U.S. Pat. 2,326,425 (Aug. 10, 1943).
10 A. Raffy and M. Fontaine, Compt. rend. 201, 1005 (1937).
11P, R. Burkholder, Proc. Natl. Acad. Sci. U. S. 29, 166 (1943).
122, W. Tanner, C. Vojnovich, and J. M. Van Lanen, Science 101, 180 (1945).
13 A. Raffy, Compt. rend. soc. biol. 126, 875 (1937).
144P.R. Burkholder and I. MeVeigh, Proc. Natl. Acad. Sci. U. S. 28, 285 (1942).
VII. ESTIMATION 361
normally a dominant organism in human intestinal flora, produced the
most riboflavin. Reviews by Knight!® and Van Lanen and Tanner!® on
growth factors in microbiology have covered other aspects of riboflavin
synthesis by microorganisms.
The synthesis of riboflavin in green plants is of major importance in
supplying human riboflavin requirements. The locus of this synthesis is
not known, but there is apparently a higher concentration in the leaves
than in the remainder of the plant.
It is apparent that the biosynthesis of riboflavin phosphate and flavin
adenine dinucleotide from riboflavin is a property possessed by all living
organisms which have need of this vitamin.!7: '§
The work of Plaut!® has thrown some light on the mechanism of ribo-
flavin biosynthesis by Ashbya gossypiz. The addition of C'-formate to flask
cultures of yeast gave rise to riboflavin tagged in the carbon-2 position.
The tracer atom from C'-bicarbonate ended up in the carbon-4 position.
C“H;COOH, CH;C“OOH, and totally labeled glucose produced riboflavin
containing Cin both the side chain and o-xylene portions of the molecule.
VII. Estimation
D. M. HEGSTED
The most commonly used methods for the estimation of riboflavin are
undoubtedly the various modifications of the fluorometric and microbio-
logical methods. These methods have largely replaced the more laborious,
expensive, and generally less exact biological assays using chicks and rats.
In fact, the early development of the rapid and fairly satisfactory methods
discouraged the development of careful animal assays for riboflavin. Colori-
metric and polarigraphic methods have been proposed, but these do not
appear to be generally applicable to biological materials.
A. PHYSICAL AND CHEMICAL METHODS
D. M. HEGSTED
1. FLuoRoMEetTrRic Mretruops
The properties of riboflavin have been discussed in an earlier section of
this chapter. Those which have been utilized for the separation and estima-
B.C. J.G. Knight, Vitamins and Hormones 3, 108 (1945).
16 J. M. Van Lanen and F. W. Tanner, Jr., Vitamins and Hormones 6, 163 (1948).
7 E. B. Kearney and 8. Englard, J. Biol. Chem. 193, 821 (1951).
1% J. R. Klein and H. I. Kohn, J. Biol. Chem. 136, 177 (1940).
19G.W. E. Plaut, Federation Proc. 12, 254 (1953)
362 RIBOFLAVIN
tion by fluorometry are: stability in acid solution, adsorption on fuller’s
earth from dilute acid, elution from fuller’s earth by dilute pyridine, sta-
bility to weak oxidizing agents, reversible reduction to the leuco form by
hydrosulfite, destruction by visible light or ultraviolet light, transforma-
tion to lumiflavin by irradiation in alkaline solution, insolubility in CHCl;
whereas lumiflavin is soluble in this solvent, solubility in benzyl alcohol
and butanol-pyridine mixtures, and a characteristic fluorescence which is
optimum between pH 6 and 7 and completely depressed in strong acid or
alkali. The object of the various methods is to effect a sufficient separation
from other material so that the fluorescence measured is that of riboflavin
alone.
a. Methods Utilizing the Fluorescence of Riboflavin
Practically all the modifications of the fluorometric method in common
use have been described in detail by Stiller! and Jones.” The completeness
of these reports makes any extensive description of the technical details
unnecessary at this time and allows less reference to the original literature
than would otherwise be necessary. Readers are referred to these presenta-
tions for a description of the methods. It appears more useful to direct this
discussion toward an evaluation of the methods available in so far as this
is possible.
The principle steps of the fluorometric analysis include extraction of the
sample, removal of interfering materials, and measurement of the ribo-
flavin in the resultant extract. Reference to standard amounts of riboflavin
and correction for the fluorescence of suitable blanks must be included in
the latter step.
(1) Extraction. The usual procedure is a hot dilute acid extraction, or
enzymatic hydrolysis, or both. The acid is ordinarily between 0.04 and
0.25 N. Several acids have been used, such as H,SO,4, HCl, and H3PO,, at
either boiling temperature or in the autoclave. The enzymatic treatment
may be principally the action of phosphatases or nucleotidases (takadia-
stase, clarase, mylase, etc.) to free riboflavin from the nucleotides, or it
may also include a proteolytic enzyme, usually papain, to liberate ribo-
flavin bound to proteins. Since the fluorescence of flavin adenine dinucleo-
tide is relatively much less than that of free riboflavin®: 4 and much of the
riboflavin may appear in this form®” the desirability of sufficient hydrolysis
1K. T. Stiller, zn Vitamin Methods, Vol. I. Academic Press, New York, 1950.
2 J. H. Jones, in Vitamin Methods, Vol. II. Academic Press, New York, 1951.
3H. B. Burch, O. A. Bessey, and O. H. Lowry, J. Biol. Chem. 175, 457 (1948).
4G. Weber, Biochem. J. 47, 114 (1950).
5 J. L. Crammer, Nature 161, 349 (1948).
6O. A. Bessey, O. H. Lowry, and R. H. Love, J. Biol. Chem. 180, 755 (1949).
7K. Yagi, J. Biochem. (Japan) 38, 161 (1951).
VII. ESTIMATION 363
appears obvious. If only free riboflavin is present (pharmaceuticals), no
special extraction should be necessary. The method of Scott et al.’ utilizes
acid-acetone as the extraction medium. Acid methanol has also been used.®
Many workers find an acid extraction alone sufficient,!: 1° yet Watts
et al." believe that some of the apparent losses in meats on storage may
represent simply insufficient extraction, and the use of acid extraction fol-
lowed by enzymatic hydrolysis is common practice.
(2) Purification. The procedure in almost universal use is a brief per-
manganate oxidation introduced originally by Koschara!” to remove inter-
fering pigments. Excess permanganate is removed by H»Os, usually within
2 minutes. However, Klatzien et al.!® recently reported the production of
a non-specific blue fluorescence by this oxidation of certain materials,
Leviton" found that in the presence of appreciable amounts of ferrousiron,
riboflavin was readily destroyed by H»O» oxidation. Hence, even the use
of this common procedure is subject to certain limitations.
Several methods employ the adsorption of riboflavin on fuller’s earth,"
such as Florisil used by Conner and Straub,'® followed by elution with pyri-
dine-acetic acid solution. Hoffer e¢ al.” indicated some possible limitations
to this procedure. Often the values for the same material decrease as the
size of the sample is increased. It was thought that this represented losses
(incomplete adsorption or elution) on the column or possibly adsorption
of riboflavin on residues. These authors also reported that the native ribo-
flavin in wheat flour extracts is less readily adsorbed than pure riboflavin.
In general, the methods which utilize the Florisil adsorption also utilize
permanganate oxidation either before or after the adsorption and elution.
Whereas permanganate oxidation after the elution is much more effective
in decreasing the pigmentation, the non-specific pigment said to be pro-
duced by permanganate oxidation in some products" may be separated
from riboflavin by adsorption on Florisil.
The original procedure of Hodson and Norris!’ and the more recent modi-
fication by Rubin et al.!° includes a reduction of riboflavin and other pig-
§M. L. Scott, F. W. Hill, L. C. Norris, and G. F. Heuser, J. Biol. Chem. 165, 65
(1946).
9A. P. Jansen, Rec. trav. chim. 69, 1275 (1950).
1D. B. Morell, Australian. J. Exptl. Biol. Med. Sc. 25, 251 (1947).
"1 B.M. Watts, D. H. Peng, and N. C. Esselbaugh, J. Biol. Chem. 172, 707 (1948).
2 W. Koschara, Z. physiol. Chem. 282, 101 (1935).
1% C. Klatzien, F. W. Norris, and F. Wokes, J. Pharm. and Pharmacol. 1, 915 (1949).
4A. Leviton, J. Am. Chem. Soc. 68, 835 (1946).
1 J. W. Ferrebee, J. Clin. Invest. 19, 251 (1940).
16R.T. Conner and G. J. Straub, Ind. Eng. Chem. Anal. Ed. 18, 385 (1941).
™ A. Hoffer, A. W. Alcock, and W. F. Geddes, Cereal Chem. 21, 515 (1944).
% A. Z. Hodson and L. C. Norris, J. Biol. Chem. 181, 621 (1939).
S. H. Rubin, E. De Ritter, R. H. Schuman, and J. C. Bauernfeind, Ind. Eng. Chem.
Anal. Ed. 17, 136, (1945).
364 RIBOFLAVIN
ments by hydrosulfite and stannous chloride. The riboflavin is then reoxi-
dized to the fluorescent form by shaking with air. Many other pigments
are not reoxidized by this procedure. Slater and Morell?° have modified
the method of Najjar! and applied it to a variety of products. In this pro-
cedure (described in detail by Jones”), the riboflavin is extracted into
butanol-pyridine mixture after the permanganate oxidation. Anhydrous
Na.SO, is used to effect the transfer and separation. This method, which
has not had general application, appears to have advantages and to deserve
broader trial.
Pigments and fluorescent materials which are not separated from ribo-
flavin by purification treatment must be taken into account by appropriate
blanks. The two parts of the method are therefore inseparable and cannot
be easily discussed alone. The criticisms of the hydrosulfite reduction,
which is usually used later in the analysis to obtain the blank reading, are
discussed below.
(3) Fluorometric Measurement. Although visual comparison of samples
with appropriate standards under ultraviolet light has been used,! it is
obviously much less satisfactory than the use of a photofluorometer and
may be impossible in the presence of interfering materials. A number of
fluorometers are available, equipped with appropriate light source, filters,
etc. Optical parts must be of low fluorescent glass. (See Loofbourow” for
a short discussion of riboflavin fluorometry.) Since riboflavin is sensitive
to light, a secondary standard of fluoroscein is used to adjust the sensitivity
of the instrument, and readings are performed rapidly. A ‘‘standard curve,”
such as is commonly used in colorimetry, is usually not applicable; there-
fore the instrument must yield an essentially straight-line relationship be-
tween riboflavin concentration and reading over the range used. This range
is limited, since self-quenching occurs at high concentrations, probably
above 0.13 y per milliliter.2? Perhaps the instrument developed by Lowry”
deserves special mention because of its stability and adaptability over wide
ranges of riboflavin concentration.
(4) Standards.: Probably the preferred method of relating fluorometer
reading to the amount of fluorescence in the unknown is the inclusion of a
“recovery sample” with each unknown. The increment in reading caused
by the added riboflavin is used to calculate the riboflavin in the sample.
This method automatically allows for losses in preparation (assuming that
the riboflavin in the sample behaves as that added; see Hoffer et al."),
adsorption of either incident or fluorescent light by colored samples, and
20H. C. Slater and D. B. Morell, Biochem. J. 40, 644 (1946).
21V. A. Najjar, J. Biol. Chem. 141, 355 (1941).
22 J. R. Loofbourow, Vitamins and Hormones 1, 143 (1943).
23 O. H. Lowry, J. Biol. Chem. 178, 677 (1948).
VII. ESTIMATION 365
“quenching” of the fluorescence of riboflavin by various materials in the
extract.'!: 4 The disadvantage of the method is that the time and work are
doubled and errors in the recovery sample are recorded in the unknown
also. As indicated above, the recovery from the Florisil column may be
critically related to the amount of riboflavin in the sample. Many workers
compromise (see Stiller!) and effect considerably saving in time by reading
the sample, adding a known amount of riboflavin, and taking another read-
ing. After dilution is accounted for, the increment in fluorometer reading
is used as a measure of the fluorescence of riboflavin in that extract and the
content in the sample is calculated from it.
Essentially all workers have come to use one or the other of the internal
standards mentioned above, since the fluorometer reading is influenced not
only by the amount of riboflavin but by the other materials in the extract.
The increment in reading caused by a constant amount of riboflavin will
generally fall in about the same range, however, and samples which show
widely divergent values should be viewed with suspicion.
(5) Blank Readings. Since most biological materials do not yield extracts
which contain only riboflavin as the fluorescent material, blank determina-
tions for each sample are ordinarily required and simple reagent blanks do
not suffice. Reagents must be sufficiently purified to yield low fluorescent
extracts, of course. The procedure which is common to most methods!
is the reduction of riboflavin to the leuco form with sodium hydrosulfite,
assuming that only riboflavin is reduced and that no fluorescent materials
are produced in the reduction. Others recommend strong alkali*!:*> or
acid*® to suppress the riboflavin fluorescence, although these have not been
widely used as yet. Another, and probably more specific, method is the
destruction of riboflavin by irradiation with artificial light or sunlight.
After an extensive comparative study of seven methods for the determina-
tion of riboflavin in urine, Morell and Slatter?’ find serious fault with the
hydrosulfite blank. They conclude: ‘‘Methods using the hydrosulfite blanks,
even those involving adsorption on Florisil, give high answers because they
include ‘apparent riboflavin,’ i.e., compounds other than riboflavin which
fluoresce in the same spectral region and the fluorescence of which is de-
stroyed by hydrosulfite.
“Urine contains not only ‘apparent riboflavin’ but also precursors of
‘apparent riboflavin’ which are changed to ‘apparent riboflavin’ by NasS.Ox-
SnCl, reduction, KMnO, oxidation, or during adsorption on Florisil.’”’ The
authors feel that their method utilizing a blank obtained by sunlight irradia-
24H. Kahler and E. P. Davis, Proc. Soc. Exptl. Biol. Med. 44, 604 (1940).
25 B. A. McLaren, 8S. Cover, and P. B. Pearson, Arch. Biochem. 4, 1 (1944).
26 A. Gourevitch, Bull. soc. chim. biol. 30, 711 (1948).
*7T). B. Morell and E. C. Slater, Biochem. J. 40, 652 (1946).
366 RIBOFLAVIN
tion is much superior. De Ritter et al.8 also compared several methods for
the determination of riboflavin in urine and concluded that the method
of Slater and Morell gave results most comparable to the microbiological
assay. Pearson and Schweigert?? found the fluorometric method to give
very large errors when applied to the urine of sheep or goats given large
doses of riboflavin when compared to the microbiological assay, but rat
and human urine gave comparable results by either method.
(6) Comment. It may probably be taken as a truism that, when many
methods are in use for the determination of the same substance, none is
really satisfactory. This may be a little severe in the present case, since
very few authors have attempted careful examination of several methods.
The work of Morell and Slater?’ *7 and that of De Ritter et al. are com-
mendable exceptions. Most studies compare ‘their’? method with another,
usually the microbiological. It is certainly safe to conclude that no ‘‘best
method” for all materials can be selected at this time. Several of the meth-
ods undoubtedly give reasonably accurate and comparative values when
applied to materials of a generally similar origin, meats for example, and
which contain reasonable amounts of riboflavin. On the other hand, many
materials of plant origin are very low in riboflavin and yield highly colored
extracts containing numerous and varied pigments. These present numer-
ous problems, and, indeed, each extract to some degree may present a special
problem. Not only is the problem difficult but it has received relatively less
study, since such materials are not usually important riboflavin sources in
the diet and the knowledge that they are low may often be a sufficient
answer. On the other hand, as Morell and Slater point out, the principal
interest in urinalysis is usually in the samples which are very low and where
methods fail or become undependable. Their work appears promising here.
b. Methods Utilizing the Fluorescence of Lumiflavin
The transformation of riboflavin into the chloroform-soluble derivative,
lumiflavin, by irradiation in alkaline solution would appear to offer promise
as a specific method of determination, especially if combined with some of
the procedures now in common use. It is said! *° that the transformation
is not quantitative and difficult to standardize, but very few workers in
the United States appear to have investigated its possibilities. Foreign
workers continue to improve and use the method. Roth,*! Jansen,® and
28H. De Ritter, M. E. Moore, E. Hirschberg, and S. H. Rubin, J. Biol. Chem. 175,
883 (1948).
27 P, B. Pearson and B. 8. Schweigert, J. Nutrition 34, 443 (1947).
30 P. Karrer, 7n Ergebnisse der Vitamin und Hormonforschung, Vol. II, p. 381.
Akademische Verlagsgesellschoff, Leipzig, 1939.
31 H. Roth, Biochem. Z. 320, 355 (1950).
VII. ESTIMATION 367
others: ** have described methods in recent years. In general the procedure
includes an acid extraction, preliminary CHCl; extraction, photolysis in
alkaline solution (pH 13 to 14 or 0.56 N NaOH), reacidification to pH 4.5,
and extraction of the lumiflavin into CHCl; . Illumination with a 200-watt
bulb appears as satisfactory as ultraviolet light, and destruction does not
occur with over-illumination.? Permanganate oxidation after photolysis**
may be desirable. There is disagreement as to whether one simple extraction
is sufficient®: ** or whether continuous extraction must be used.°®
Although some of the technical operations appear cumbersome for rou-
tine use, this is probably only a reflection of the lack of effort to improve
and simplify the procedures. More intensive study and comparison with
other methods is needed.
c. Microfluorometric Methods
Burch et al.’ developed methods for serum, red blood cells, and whole
blood which require only 25 cubic mm. of material. Riboflavin in the white
cells may be obtained with only 0.1 ml. of blood. The amounts of free ribo-
flavin, riboflavin phosphate, and flavin adenine dinucleotide (FAD) may
also be determined. Under the conditions used, the relative fluorescence
of FAD is only 14% of riboflavin. By determinations before and after hy-
drolysis (5 % trichloroacetic acid at 38° for 20 hours) the amount of FAD is
obtained. Riboflavin mononucleotide is practically absent from serum and
may be ignored. Hydrosulfite reduction is used to obtain blank readings.
This method has proved extremely useful, and as yet no serious criticisms
or improvements have been recorded. The small volumes required make
it especially useful for field surveys in nutrition. Serum riboflavin values
have been shown to be related to riboflavin intake in experimental animals,*4
especially the FAD content, which fluctuates less and falls later than the
free riboflavin level. Whole blood levels are less useful,**: *° since the ribo-
flavin content is greatly influenced by the number of red cells. The diagnos-
tic value in human beings remains to be proved but appears promising.
d. Estimation of Riboflavin Nucleotides
Bessey et al.*° developed a method for the fluorometric determination of
riboflavin, riboflavin mononucleotide, and flavin adenine dinucleotide in
tissues. The differential fluorescence of FAD as compared to riboflavin and
32 A. Fujita and K. Matsuura, J. Biochem. (Japan) 37, 445 (1950).
33K. Yagi, Igaku to Seibutsugaku 18, 264 (1951) [C.A. 45, 9591 (1951)].
81K. Suvarnakich, The Measurement of Riboflavin Deficiency, Thesis. Harvard
School of Public Health, 1951.
35 A. E. Axelrod, T. D. Spies, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 46,
146 (1941).
36 OQ. A. Bessey, O. H. Lowry, and R. H. Love, J. Biol. Chem. 180, 755 (1949).
368 RIBOFLAVIN
the mononucleotide, which fluoresce equally, is utilized for the estimation
of FAD. Benzyl alcohol extraction*”: * is used to separate riboflavin from
the nucleotides. From 70 to 90% of the riboflavin in tissues was found to
be present as FAD, and essentially all the remainder was the mononucleo-
tide. Fujita and Matsuura® used benzyl alcohol separation to distinguish
between riboflavin and esterified riboflavin and examined a large number
of plant and animal tissues.
Crammer’? described the separation of the three forms by paper chroma-
tography using ‘‘collidine” or butanol-acetic acid as the developing solvent.
He detected only FAD in heart, brain, kidney, and liver, whereas only free
riboflavin was found in the spleen. These results are not consistent with
those of Bessey et al. The latter authors report the distribution of riboflavin
compounds in one sample of spleen to be similar to that found in other tis-
sues mentioned above. Yagi’ has extended the qualitatively chromato-
graphic method of Crammer to a quantitative estimation of the three con-
stituents. After separation, the position on the paper was determined under
ultraviolet light, and the spots extracted and analyzed by the lumiflavin
procedure. It would appear that his results are comparable to those re-
ported by Bessey et al.
Since the only known functions of riboflavin involve the nucleotides
rather than free riboflavin, these methods should be of great use and inter-
est. Critical evaluation of the various procedures must, of course, await
further comparative work.
2. COLORIMETRIC AND POLARIGRAPHIC METHODS
Since riboflavin is a colored compound, it can obviously be determined
by colorimetry.!: 4° A few authors apparently use this procedure," but it
is about 100 times” less sensitive than the fluorometric method and there-
fore not applicable to most biological materials. Its usefulness in the assay
of pharmaceuticals should apparently depend upon the equipment of the
laboratory and the specificity of the separation from other yellow materials
as compared to separation from fluorescent materials. Recently” a colori-
metric method based on the production of a red color in the presence of
silver nitrate or Deniges reagent has been described. Sensitivity is much
below that of fluorometric methods.
37 A. HWmmerie, Nature 141, 416 (1938).
38 A. Kmmerie, Rec. trav. chim. 58, 290 (1939).
39 J. C. Crammer, Nature 161, 349 (1948).
40 Hf. R. Rosenberg, Chemistry and Physiology of the Vitamins, Interscience Pub-
lishers, New York, 1945.
4.) Weygand and K. Wacker, Alin. Wochschr. 24/25, 488 (1947).
2M. Z. Barakat and N. Bodran, J. Pharm. and Pharmacol. 3, 501 (1951).
VII. ESTIMATION 369
A polarigraphie method was described by Lingane and David. This
method also appears less sensitive than those in common use and has had
very little application.
3. EnzyMAtTic MererHops
In their paper on the isolation of the prosthetic group of D-amino acid
oxidase, flavin adenine dinucleotide, Warburg and Christian* describe an
assay method for this riboflavin derivative. This consists in the measure-
ment of oxygen uptake when the material to be assayed is added to an
appropriate buffered solution containing the protein moiety of the enzyme
and pu-alanine as the substrate. The method has been used by various
groups: 4® and was modified to measure the apoenzyme instead of the
prosthetic group by Negelein and Bromel.*” Recently Comline and What-
ley*® have used the method to study the destruction of FAD by an en-
zyme in animal tissues, particularly in spleen.
The method used by Kuhn and Rudy*? for the estimation of the prosthe-
tic group of the yellow enzyme, riboflavin mononucleotide, appears to have
had little use. This also utilizes oxygen uptake but with hexose phosphate
as the substrate, the specific protein of the yellow enzyme, and appropriate
coenzymes to complete the oxidizing system. Oxygen uptake is a function
of the amount of riboflavin phosphate added.
B. BIOLOGICAL METHODS
D. M. HEGSTED
The Bourquin and Sherman®® rat assay was the first method for the esti-
mation of what was then called vitamin Bz or G to receive rather wide ac-
ceptance. When pure riboflavin became available, it was shown that this
method gave quantitative responses to graded doses of riboflavin within
certain limits of growth. Although biological assays have since been im-
proved, the early development of the microbiological assay apparently satis-
fied most of the requirements for a biological method, and the riboflavin
assay has never been subjected to the study and refinement which some of
the other vitamin assays received. The perfect assay diet would be one in
which all the nutrients required by the assay organism, with the exception
43 J. J. Lingane and O. L. Davis, J. Biol. Chem. 137, 567 1941).
44Q. Warburg and W. Christian, Biochem. Z. 298, 150 (1938).
45S. Ochoa and R. J. Rossiter, Biochem. J. 33, 2008 (1939).
46 J. R. Klein and H. I. Kohn, J. Biol. Chem. 136, 177 (1940).
47 EF. Negelein and H. Brémel, Biochem. Z. 300, 225 (1939).
48 R.S. Comline and F. R. Whatley, Nature 161, 350 (1948).
47 R. Kuhn and H. Rudy, Ber. 69, 2557 (1936).
50 A. Bourquin and H. C. Sherman, J. Am. Chem. Soc. 68, 3501 (1931).
370 RIBOFLAVIN
of riboflavin, are present in adequate amounts. The numerous diets which
have been used since the original Bourquin-Sherman diet have been tabu-
lated by Day and Darby,*! and the details of the biological assay are dis-
cussed by these authors. The diets have been made more complete with
the passage of time, the availability of more crystalline vitamins, and more
knowledge of the rat’s nutritional requirements. Undoubtedly further im-
provements could now be made.
The usual assay is made with weanling rats which are fed the riboflavin-
low diet for 2 or 3 weeks until growth has practically ceased in the majority
of the animals. They are then carefully divided according to weight, sex,
etc., and groups are fed various levels of the test material. Other groups
receive standard amounts of riboflavin. At the end of the test period the
growth of the standard groups is used to construct a dose-response curve
from which the riboflavin content of the unknown is determined. Bliss and
Gyorgy” and Day and Darby* have discussed these assays and the calcu-
lation of results in detail. Assays utilizing young chicks have also been
used®!: 53 in which the general procedure is similar, although the diet must
be devised with the nutritional requirements of chicks in mind.
It is commonly assumed that the bioassay which utilizes larger animals
has peculiar advantages over other methods, since it measures the response
of the vitamin in an organism similar to man or at least more similar than
is L. caset. It may be well to point out that this is not necessarily so. There
is ample evidence that the response of the rat to known amounts of ribo-
flavin is markedly influenced by the composition of the diet with which it
is fed. Specifically, the amount and kind of carbohydrate and fat are known
to be important,®*4 and other unsuspected relationships may exist. The level
of these may not be controllable when low-potency materials are assayed.
Thus one reaches the same position found in all assays to date, namely that
accurate figures for low-potency materials may probably not be obtained.
Furthermore, it would appear logical that these and other dietary inter-
relationships will be true of the human species as well. If so, the riboflavin
potency of a given material will vary with the diet with which it is fed as well
as with the composition of the material itself. This rather unsatisfactory
state of affairs must be accepted as the nature of the thing, and the bioassay,
even if accurately done, appears to offer no advantages except for research
studies on the physiology of riboflavin. The more rapid and accurate
methods discussed elsewhere should, and undoubtedly will, be favored.
51 P. L. Day and W. J. Darby, in Biological Symposia, Vol. XII, Estimation of the
Vitamins. Jaques Cattell Press, Lancaster, Pa., 1947.
82 C, I. Bliss and P. Gyérgy, in Vitamin Methods, Vol. II. Academic Press, New
York, 1951.
53TH. Jukes, J. Nutrition 14, 223 (1937).
64G. J. Mannering, D. Orsini, and C. A. Elvehjem, J. Nutrition 28, 141 (1944).
VII. ESTIMATION 371
Summary
The author has attempted to point to some of the faults of existing meth-
ods for riboflavin assay as a stimulus for further development in the field
rather than a condemnation of the present procedures. Many of the present
methods are adequate to obtain the kind of knowledge most generally
sought, which is a more or less quantitative idea of the riboflavin content
and with special interest in high-potency materials. Methods designed for
a special tissue, serum for example, may be quite satisfactory. It is not to
be assumed, however, that success with one material, or even several,
guarantees the method for general use. The following report may well be-
come a classic example of what may befall the unsuspecting worker who
makes a simple analysis. Baude et al.** reported that the riboflavin content
of sow’s milk as determined by a fluorometric method designed for cow’s
milk®® was quite low, approximately 0.5 y per milliliter, and more stable
to light than the riboflavin in cow’s milk.” Microbiological studies in an-
other laboratory failed to confirm this figure, and this discrepancy led to a
collaborative effort®* to uncover the difficulty. The same sample of milk
was analyzed by several methods with the following results: original fluoro-
metric assay, 0.4 y per milliliter; microbiological assay,®*® 1.2 to 1.5 y per
milliliter; chick assay, 1.2 to 1.8 y per milliliter; and rat assay, 1.6 to 2.0
y per milliliter. It was provisionally decided that the fluorometric method
did not yield complete extraction, and the sample was then hydrolyzed with
H.SO, at pH 1 for an hour, incubated with takadiastase, and the assay com-
pleted by the method of Slater and Morell. This also yielded low results,
but it was found that if the final extraction into butanol-pyridine was omit-
ted higher values were obtained. Thus it appeared that the riboflavin did
not behave normally and did not completely enter this solvent. Finally, an
assay by the method of Bessey, Lowry, and Love was said to yield results
comparable to those of the microbiological assay. Unfortunately the story
is not complete at the present time, but it appears that at least part of the
riboflavin of sow’s milk is present in a form which does not behave as does
previously known compounds. It should be borne in mind that this example
is with a material which does not yield highly colored extracts and that
cow’s milk has been analyzed successfully for riboflavin many times by
fluorometric methods.
55 R. Baude, S. K. Kon, and 8. Y. Thompson, J. Dairy Research 14, 414 (1945-46).
56K. M. Henry, J. Houston, and 8. K. Kon, Biochem. J. 34, 607 (1940).
57 R. Baude, M. E. Coates, K. M. Henry, 8. K. Kon, S. J. Rowland, 8. Y. Thomp-
son, and D. M. Walker, Brit. J. Nutrition 1, 64 (1947).
58 V. E. Davis, R. MacVicar, C. B. Ross, C. K. Whitehair, A. A. Heidebrecht, R.
Baude, M. E. Coates, K. M. Henry, S. K. Kon, 8S. Y. Tompson, and F. Wilby,
Nature 165, 522 (1950).
59H. A. Kornberg, R. 8. Langdon, and V. H. Cheldelin, Anal. Chem. 20, 81 (1948).
ote RIBOFLAVIN
C. MICROBIOLOGICAL METHODS
BK. E. SNELL
Riboflavin was found to be a growth factor for certain lactic acid bac-
teria® ®! shortly after its isolation as a vitamin for rats.” Relatively few
microorganisms that require this vitamin are known; most of these are
lactic acid bacteria or closely related organisms.
The use of a strain of Lactobacillus casei for the quantitative determina-
tion of riboflavin was suggested by Snell and Strong® in 1939. The proce-
dure recommended has been widely and successfully used since that time,
both in its original form and in several slightly modified forms. Indeed,
the subsequent widespread use of microorganisms for quantitative assay
work was greatly stimulated by the success of this method. The original
assay medium is identical with that later recommended by the Association
of Official Agricultural Chemists.*t The medium adopted by the U. 8.
Pharmacopeia® differs only in the concentration of glucose, which has been
increased from 1.0 to 3.0%. Procedural details, precision, reliability, and
specificity of the method are given in numerous treatises. -®
A medium based on peptone, glucose, yeast extract, acetate, and inor-
ganic salts and freed from riboflavin by treatment of the crude ingredients
with light, alkali, or lead acetate followed by hydrogen sulfide is used.
Growth of L. casei in this medium increases with the riboflavin concentra-
tion in the range from 0 to about 0.20 y per 10 ml. of medium. Pure ribo-
flavin and samples to supply riboflavin at several levels within this range
are added to individual tubes containing 5 ml. of the double-strength me-
dium. Hach tube is then diluted to 10 ml., capped, autoclaved, cooled, and
inoculated. Response of the test organism is customarily determined by
acid titration after 72 hours incubation at 37°. Turbidimetric estimations
of growth can be made as early as 24 hours. Interpolation of the response
obtained to several different known amounts of sample onto the curve
obtained by plotting the responses to increasing amounts of pure riboflavin
60S. Orla-Jensen, N. C. Otte, and A. Snog-Kjaer, Zentr. Bakteriol. Parasitenk., Abt.
IT, 94, 434 (1936).
61H), H. Snell, F. M. Strong, and W. H. Peterson, Biochem. J. 31, 1789 (1937).
62 R. Kuhn, P. Gyorgy, and T. Wagner-Jauregg, Ber. 66B, 317, 576, 1034, 1577, 1950
(1933).
63), EH. Snell and F. M. Strong, Ind. Eng. Chem. Anal. Ed. 11, 346 (1939).
64 A. R. Kemmerer, J. Assoc. Offic. Agr. Chemists, 27, 541 (1944).
66 U. S. Pharmacopeia 14th Revision, p. 752, 1950.
66 Ff}, Ey. Snell, 7n Vitamin Methods, Vol. 1, p. 8327. Academie Press, New York, 1950.
67 Association of Vitamin Chemists, Methods of Vitamin Assay, 2nd ed., p. 245.
Interscience publishers, New York, 1951.
68 F. M. Strong, in Biological Symposia, Vol. XII, Estimation of the Vitamins, p.
143. Jaques Cattell Press, Lancaster, Pa., 1947.
VIIl. STANDARDIZATION 373
gives figures from which the average riboflavin content of the sample is
readily calculated.
The extraction of riboflavin offers little difficulty because of the looseness
with which it is bound, its stability to heat and acid, and the fact that the
known bound forms, riboflavin phosphate and flavin adenine dinucleotide,
have the same activity as riboflavin for the test organism. Autoclaving the
finely divided sample with a tenfold excess of 0.1 N HCl at 15 lb. pressure
for 15 to 20 minutes has proved very effective. The cooled mixture is di-
luted and filtered through paper to remove traces of fatty acids, which
interfere with the response to riboflavin. Properly diluted aliquots of the
filtrate, adjusted to pH 6.8 to 7.0, are used for assay. All operations, during
both extraction and assay, should be carried out in darkness or subdued
light, since riboflavin is readily destroyed by light.
Accuracy of the procedure is attested by agreement of the results obtained
for a great variety of materials with those obtained by chemical and rat
growth procedures.®: °° A micro modification of the procedure that permits
determination of riboflavin in amounts from 0 to 0.004 y has been de-
scribed.**: 69
Most other microbiological procedures for riboflavin also utilize L. casei
as the test organism in a slightly modified medium.®* An exception is the
procedure of Kornberg et al.,°°: 7° which employs a strain of Leuconostoc
mesenteroides said to be some fifty times more sensitive to riboflavin than
is L. caset. These methods may prove useful for special applications. They
have not, however, been tested nearly as widely as the Snell-Strong proce-
dure recommended above.
VIII. Standardization
M. K. HORWITT
With the elucidation of the chemical nature of riboflavin, the need for
a standard of activity for vitamin B» or G became less urgent. The present
U.S.P. Reference Standard is a recrystallized sample of riboflavin obtainable
from the U.S.P. Reference Standards Committee. Comparisons of purified
riboflavin with the older units of activity showed that one Bourquin-Sher-
man rat growth unit,! the daily addition of which will produce an average
gain of 3 g. per rat per week, was equal to about 2.5 y of riboflavin.’ *
69 O. H. Lowry and O. A. Bessey, J. Biol. Chem. 155, 71 (1944).
70H. A. Kornberg, R.S. Langdon, and V. H. Cheldelin, Anal. Chem. 20, 81 (1948).
1A. Bourquin and H. C. Sherman, J. Am. Chem. Soc. 58, 3501 (1931).
20. A. Bessey, J. Nutrition 15, 11 (1988).
374 RIBOFLAVIN
Von Euler? had proposed a unit which consisted of 5 y of riboflavin, the
amount which produced an increase in weight of 0.8 to 1.0 g. per day in
young rats. A Cornell unit® was defined as the growth effect on chicks
equivalent to that produced by 1 y of riboflavin.
The need for standards of biological activity continues to exist, especially
in the study of derivatives of riboflavin. As an example, consider the assay
of a very water-soluble riboflavin derivative prepared by Stone.® Fluoro-
metric assay of the material yielded a value of 57.2% riboflavin; micro-
biological assay by the U.S.P. XIII revision method yielded a value of
33 % riboflavin. The biological assay by the standard rat growth method
indicated the riboflavin potency was almost nil.
IX. Occurrence in Food
M. K. HORWITT
The best sources of riboflavin are milk, egg white, liver, heart, kidney,
and growing leafy vegetables. Beef muscle, veal, apricot, tomato, and
poultry muscle are good sources. Fish muscle, unenriched grains, and leg-
umes, although relatively poor sources, supply important minimal amounts
to the average regimen. Yeast, the richest natural source of riboflavin, is
not normally a major component of non-therapeutic diets.
The primary factors to be evaluated in a consideration of the stability
of riboflavin in food products are the effects of heat, light, elution, and the
intracellular reactions which take place during storage.
The relative heat stability of riboflavin is a fortunate property which
favors its preservation by ordinary cooking procedures.! Even the addition
of bicarbonate to a pH of 8.8-does not appreciably increase the loss of ribo-
flavin during short cooking procedures.” The major losses which occur dur-
ing home cooking or commercial canning operations are probably attribu-
table to the extraction of the vitamin by the water used in the cooking or
3H. von Euler, P. Karrer, E. Adler, and M. Malmberg, Helv. Chim. Acta 17, 1157
(1934).
4H. von Euler, Institut international di Chimie Solvay, Sixiéme Conseil de Chemie,
rapport et discussions sur Les Vilamines et les Hormones, p. 198. Paris, 1938.
5L. C. Norris, H. S. Wilgus, A. T. Ringrose, V. Heiman, and G. F. Heuser, Cornell
Univ. Agr. Exptl. Sta. Bull. 660, 3 (1936).
6G. B. Stone, Science 111, 283 (1950).
1 H.-Levine and R. E. Remington, J. Nutrition 18, 525 (1937).
2C. H. Johnston, L. Schauer, 8. Rapaport, and H. J. Deuel, Jr., J. Nutrition 26,
227 (1943).
IX. OCCURRENCE IN FOOD
Food
Cereal products:
Refined
Whole grain and enriched
Dairy products:
Cheese, Cheddar
Cheese, cottage
Cream, light
Custard
Egg
Ice cream
Milk
Buttermilk, skim
Whole
Desserts:
Cake, plain, chocolate
Cookies, plain
Pie crust
Puddings
Fish:
Cod, haddock, cooked
Halibut, herring, whitefish
Salmon, canned
Fruits:
Banana
Cantaloupe
Citrus
Yellow
Meat:
Beef, lamb, veal, cooked
Fowl, cooked
Liver, cooked
Luncheon meats, cooked
Pork, ham
TABLE II
RIBOFLAVIN CONTENT OF REPRESENTATIVE Foops!®
Approximate measure
1 slice bread (30 g.)
1g cup cereal (20 g.)
3 soda crackers (20 g.)
1 slice bread (30 g.), 14 cup cereal
(20 g.), 2 graham crackers (20
z)
1 cu. in.
4g cup
4g cup
+g cup
1 medium
¥g cup
1 cup
1 cup
1 piece (75 g.)
2 medium
46 shell, single crust
+2 cup
1 medium serving
1 medium serving (75 g.)
1 medium serving
1 small
44 melon, 444-in. diameter
4 grapefruit, 1 medium orange
Fresh (100 g.)
Dried (30 g.)
1 medium serving
1 medium serving
1 small serving
2 slices sausage, minced ham,
dried beef, 14 frankfurter (30
g.)
1 medium serving, cooked
Riboflavin, mg.
376 RIBOFLAVIN
TABLE IIl—Concluded
Food Approximate measure Riboflavin, mg.
Sweets:
Candy bar 1-2 oz. chocolate-coated bar 0.17
Molasses; sorghum 1 tablespoon 0.03
Vegetables:
Cabbage, cooked 24 cup 0.05
Cauliflower, cooked 28 cup (70 g.) 0.04
Corn, parsnips, cooked 14 cup corn; 1 large parsnip 0.09
Green and yellow:
Asparagus, cooked 26 cup 0.174
Broccoli, cooked 28 cup Onl5¢
Carrots, cooked 22 cup 0.05
Green beans, cooked 1g cup 0.102
Leafy greens, cooked 2 cup spinach, turnip, kale, 0.21¢
other greens
Peas, fresh, cooked, canned 16 cup 0.142
Sweet potato, cooked 1g large 0.062
Potato, cooked 1 small (100 g.) 0.03
Tomato, fresh, canned or juice 1g cup; 1 small tomato 0.08
Other, commonly served raw 2 pieces celery; 8 slices cucumber; 0.03
l¢ head lettuce
@ For canned, reduce by one-half.
blanching operations.*-* These losses are usually less than 20%, and they
can be further minimized if the cooking fluids are consumed.
Losses of riboflavin due to exposure to light during cooking may prove
to be an important economic loss. Cheldelin et al.’ have shown large losses,
up to 48 % , incurred in the cooking of eggs, milk, and pork chops in uncoy-
ered dishes under conditions where there was no loss of riboflavin when
cooking dishes were covered. The loss in milk*-!° which is allowed to stand
in glass containers on the consumer’s doorstep may be as high as 85 % after
2 hours exposure to bright sunlight.!! This may be compared with practic-
3H. L. Mayfield and M. T. Hedrick, J. Am. Dietet. Assoc. 25, 1024 (1949).
4J.M. McIntire, B.S. Schweigert, L. M. Henderson, and C. A. Elvehjem, J. Nutri-
tion 25, 148 (1948).
5 J. R. Wagner, F. M. Strong, and C. A. Elvehjem, Ind. Eng. Chem. 39, 985 (1947).
6 W. A. Krehl and R. W. Winters, J. Am. Dietet. Assoc. 26, 966 (1950).
7V. A. Cheldelin, A. M. Woods and R. J. Williams, J. Nutrition 26, 477 (1948).
8 R. R. Williams and V. H. Cheldelin, Science 96, 22 (1942).
9J. A. Ziegler, J. Am. Chem. Soc. 66, 1039 (1944).
10W. J. Peterson, F. M. Haig, and A. O. Shaw, J. Am. Chem. Soc. 66, 662 (1944).
11 A.D. Holmes and C. P. Jones, J. Nutrition 29, 201 (1945).
X. EFFECTS OF DEFICIENCY Sit
ally no loss of riboflavin during pasteurization procedures,” where light
effects are minimal.
The losses of riboflavin during storage either by quick freezing’: 4 or in
sterilized containers'®-" are relatively small.
In the absence of liver and yeast, which contain from 2 to 4 mg. of ribo-
flavin per 100 g., the riboflavin content of the average diet is usually re-
lated to the amount of animal protein consumed. Unenriched cereal prod-
ucts are poor sources. Green and yellow vegetables, although relatively
high in riboflavin, are not usually eaten in sufficient amounts to supply a
major portion of the daily requirement. The preparation of experimental
diets low in riboflavin has been discussed by Horwitt et al.!§
Table Il contains representative amounts of riboflavin in foodstuffs.
This table is adapted from the Leichsenring and Wilson'® short method of
dietary analysis. It is based upon their compilation of data published by
Watt and Merrill?® and Bowes and Church.?! The figures presented are
averages, often of a wide range of analytical results, and should be re-
garded as working estimates which will vary with the geography, season,
and method of preparation.
X. Effects of Deficiency
A. IN MICROORGANISMS
E. E. SNELL
Shortly after the isolation of riboflavin as a vitamin for animals, Orla-
Jensen and coworkers! showed that the compound also was an essential
growth factor for many lactic acid bacteria. This finding was rapidly con-
2 A.D. Holmes, J. Am. Dietet. Assoc. 20, 226 (1944).
13 B.S. Schweigert, J. M. McIntyre, and C. A. Elvehjem, J. Nutrition 26, 73 (1943).
144M.S. Rose, J. Am. Med. Assoc. 114, 1356 (1940).
1°G. Adams and 8. L. Smith, U. S. Dept. Agr. Misc. Publ. 536, (1944).
16 J. F. Feaster, J. M. Jackson, D. A. Greenwood, and H. R. Kraybell, Ind. Eng.
Chem. 38, 87 (1946).
17TD).8. Moschette, W. F. Hinman, and E. G. Halliday, Ind. Eng. Chem. 39, 994 (1947).
8M. K. Horwitt, G. Sampson, O. W. Hills, and D. L. Steinberg, J. Am. Dietet. As-
soc. 25, 591 (1949).
9 J. M. Leichsenring and E. D. Wilson, J. Am. Dietel. Assoc. 27, 386 (1951).
20 B. K. Watt and A. L. Merrill, U. S. Dept. Agr. Handb. 8 (1950).
21 A. de P. Bowes and C. F. Church, Food Values of Portions Commonly Used, 6th
ed. College Offset Press, Philadelphia, 1946.
1S. Orla-Jensen, N. C. Otte, and A. Snog-Kjaer, Zentr. Bakteriol. Parasitenk.,
Abt. IT 94, 434 (1936).
378 RIBOFLAVIN
firmed,? and the effect of several variations in the riboflavin structure on
the bacterial response was determined.* The response proved very specific,
and on these grounds a microbiological assay for the vitamin was developed
and proposed.‘ Both growth and acid production of lactic acid bacteria are
proportional to riboflavin in the suboptimal range of concentrations.‘ ®
Riboflavin is required as a growth factor by fewer microorganisms than
are most of the other vitamins;°: ° besides the lactic acid bacteria, however,
many of the hemolytic streptococci, some propionic acid bacteria, some
clostridia, and some luminescent bacteria require it. Few if any naturally
occurring yeasts or other fungi have been found that require riboflavin;®
mutants of Neurospora crassa that require it have, however, been obtained,’
and all organisms so far examined that do not require preformed supplies
of this vitamin synthesize it.*» © Indeed, the synthesis of riboflavin by cer-
tain fungi, e.g., by Ashbya gossypit and related organisms, constitutes a
commercial method for the production of this vitamin.’
Aside from decreased growth in the absence of sufficient supplies, few
other effects of riboflavin deficiency in microorganisms have been described.
From the role of this vitamin as a hydrogen carrier, it might be expected
that, as in higher animals, the level of certain oxidative enzyme systems
would be depressed during growth on suboptimal supplies. That this is
true is shown by investigations of Doudoroff® with the luminescent orga-
nism, Photobacterium phosphorescens. Cultures grown on a yeast autolyzate
agar frequently produced, as variants, dull or dark colonies; these lumi-
nesced more brightly when riboflavin was added. The same “‘dark”’ colonies
were stimulated in growth on a riboflavin-deficient medium by additions
of this vitamin. Apparently the dull variants had lost the ability to synthe-
size sufficient riboflavin for their needs; this was not true of the original
bright colonies. In this case, amounts of riboflavin sufficient for growth of
the ‘‘dull” variants were insufficient to permit maximum luminescence.
That riboflavin-enzymes are among those concerned in light production by
such luminescent organisms has been proved by Eyring and Johnson.!°
At low concentrations, many fatty materials show a “sparing effect”’
on the requirement of lactic acid bacteria for riboflavin;® this effect, which
is sometimes troublesome in microbiological assays, may possibly indicate
2. E. Snell, F. M. Strong, and W. H. Peterson, Biochem. J. 31, 1789 (1937).
3. E. Snell and F. M. Strong, Enzymologia 6, 186 (1939).
4. E. Snell and F. M. Strong, Ind. Eng. Chem. Anal. Ed. 11, 346 (1939).
5. EB. Snell, in Vitamin Methods, Vol. I, p. 327. Academic Press, New York, 1950.
6B. C. J. G. Knight, Vilamins and Hormones 3, 105 (1945).
7G. W. Beadle and E. L. Tatum, Am. J. Botany 32, 678 (1945).
8 F. W. Tanner, Jr., C. Vojnovich, and J. M. Van Lanen, J. Bacteriol. 58, 737 (1949).
9M. Doudoroff, Enzymologia 5, 239 (1938).
10 fF. H. Johnson and H. Eyring, J. Am. Chem. Soc. 66, 848 (1944).
X. EFFECTS OF DEFICIENCY 379
that riboflavin participates in fat synthesis by these organisms. If the
fatty materials required were supplied preformed, the requirement for ribo-
flavin might well be decreased thereby.
B. IN PLANTS
Ma HORWILE,
Riboflavin is apparently synthesized by higher plant life,!!: as evidenced
by its distribution in our vegetable foods. There is no recorded evidence of
riboflavin deficiency in plants.
According to Galston,' riboflavin determines the photo6dxidation of in-
doleacetic acid and may be regarded as a photoreceptor in light-growth re-
actions. Ferri has emphasized the fact that the induction of the photoinac-
tivation of indoleacetic acid is a property common to many fluorescent
substances. It has been known that the photodxidation of indoleacetic acid
could be determined by eosin! and that eosin-treated roots yielded less
auxin than untreated ones.!® It will be interesting to observe the develop-
ment of this subject.
C. IN INSECTS
M. K. HORWITT
It has been apparent for some time" that riboflavin is essential for many
insects. Among those whose requirements have been studied are the larvae
of the flesh fly, Sarcophagia sp.,!8 the cockroach, Blattella germanica, the
larvae of the confused flour beetle, Tribolium confusum,?®:?* Drosophila
larvae,” and the yellow fever mosquito, Aédes aegypti.”
Fraenkel and Blewett,?4 who have made extensive studies of the nutri-
tional requirements of beetles, have shown that riboflavin is required by
Tribolium and Ptinus, but that Lasiodermia, Sitodrepa, and Silvanus do
not need riboflavin in their diet because of the presence of intracellular
symbiotic microorganisms which synthesize the vitamin.
1 P. R. Burkholder, Science 97, 562 (1943).
2 J. Bonner, Botan. Gaz. 103, 581 (1942).
13 A.W. Galston, Science 111, 619 (1950).
4M. G. Ferri, Arch. Biochem. 31, 127 (1951).
15 F. Skoog, J. Cellular Comp. Physiol. 7, 227 (1935).
16 P. Boysen-Jensen, Planta 22, 404 (1934).
17 R. Craig and W. M. Hoskins, Ann. Rev. Biochem. 9, 617 (1940).
18 G. Di Maria, Arch. zool. ital. 25, 469 (1938).
19C. M. McCay, Physiol. Zoél. 11, 89 (1938).
20 G. Frobrich, Z. vergleich. Physiol. 27, 336 (1939).
21K. Offhaus, Z. vergleich. Physiol. 27, 384 (1939).
2K. L. Tatum, Proc. Natl. Acad. Sci. U. S. 25, 490 (1939).
23.W. Trager and Y. SubbaRow, Biol. Bull. 75, 75 (1938).
*4G. Fraenkel and M. Blewett, J. Exptl. Biol. 20, 28 (1943).
380 RIBOFLAVIN
The American cockroach, Periplaneta americana,?? and Tineola bissel-
liella?® accumulate much more riboflavin in the malpighian tubes than can
be accounted for by the diet.
D. IN ANIMALS
MOK HORWITT
Riboflavin is essential for growth and normal health for all animals. Its
restriction has been studied in nearly all species which are related to hu-
man economics. The primary effect of riboflavin restriction is the cessation
of growth. Because it is a fundamental constituent of animal tissue, new
tissue cannot be formed unless a minimum amount of riboflavin is available.
It is therefore necessary not only for growth but also for tissue repair.”
The amounts of riboflavin needed for normal growth have been discussed
in the section on animal requirements. When less than these requirements
is provided, a variety of pathological trends become evident. Wolbach and
Bessey”® have published an excellent review of the tissue changes in vita-
min deficiencies, in which they summarized most of the studies reported
prior to 1942. The large majority of the older papers on the Be vitamin dealt
with mixed deficiencies and often described syndromes which are not seen
in modern work with a ‘‘pure” deficiency of riboflavin. At present, most
of our interpretations of ariboflavinosis in animals are based upon experi-
ments which deal with the removal of all, or nearly all, of the riboflavin
from the diet. The application to man of such work is limited by the fact
that one rarely, if ever, observes a human deficiency syndrome due to the
complete absence of dietary riboflavin. More work on the effect of long-
standing suboptimal intakes of riboflavin is indicated.
1. Rats
Goldberger and Lilly?’ were apparently the first to describe symptoms of
riboflavin deficiency in the rat, as a result of a study in which they at-
tempted to produce rat pellagra. They reported a severe ophthalmia and a
bilateral, symmetrical alopecia, which almost completely denuded the head,
neck, and trunk. The dermatitis which these authors also observed has
since been shown to have been complicated by a pyridoxine deficiency.*°: *!
Whereas the lesions of pyridoxine deficiency are characterized by a florid
25 R. L. Metealf and R. L. Patton, J. Cellular Comp. Physiol. 19, 373 (1942).
26 R. G. Busnel and A. Drilhon, Compt. rend. 216, 213 (1943).
27M. K. Horwitt, O. W. Hills, C. C. Harvey, E. Liebert, and D. L. Steinberg, J.
Nutrition 39, 357 (1949).
28.$..B. Wolbach and O. A. Bessey, Physiol. Revs. 22, 233 (1942).
29 J. Goldberger and R. D. Lilly, Public Health Repls. (U.S.) 41, 1025 (1926).
30 P. Gyorgy, Nature 138, 498 (1933).
31 P. Gyorgy, Biochem. J. 29, 741 (1935).
X. EFFECTS OF DEFICIENCY 381
dermatitis of the extremities and a swelling of the ears, the lesions of
ariboflavinosis are less specific and slower to develop. An eczematous con-
dition of the skin especially affects the nostrils and eyes. The eyelids be-
come denuded of hair and may be stuck together with a serous exudate.
Conjunctivitis, blepharitis, corneal opacities, and vascularization of the
cornea are common manifestations of rat ariboflavinosis.*’-*> The ques-
tion of specificity of cataract formation, first reported in 1931 by Day et al.**
in rats deficient in what was then known as vitamin G, has not yet been
resolved. There is an apparent inverse relationship between cataract for-
mation*’-*° and the amount of riboflavin in the diet. There also seems to be
a relationship between corneal opacities and amino acid deficiencies.*°- In
a study of the growth of the eye during riboflavin and tryptophan defi-
ciencies, Pirie*® noted that the eye continued to grow at a normal rate, so
that the deficient animals had relatively large eyes in undersized bodies.
Before the pathology of choline deficiency was recognized, there were
frequent reports of hepatic injury as a consequence of riboflavin defici-
ency.**: # It is now assumed that the fatty livers associated with deficien-
cies of B complex vitamins are not directly related to riboflavin deple-
tion.”*» 46
A variety of neuropathological changes have been reported, and it is
likely that the proportions of fat, carbohydrate, and protein in the diet,
as well as the severity of the riboflavin depletion, may play an important
part in determining the exact nature of the pathology. Partial paralysis of
the legs of the rat are produced more easily on a high fat ration.‘”: 48 In its
severe form this paralysis is characterized by degeneration of the myelin
8% H. Chick, T. F. Macrae, and A. N. Worden, Biochem. J. 34, 580 (1940).
33. R. Richardson and A. G. Hogan, Missouri Agr. Expt. Sta. Research Bull. 241
(1936).
34 B. Sure, J. Nutrition 22, 295 (1941).
35H. R. Street, G. R. Cowgill, and H. M. Zimmerman, J. Nutrition 22, 7 (1941).
36 P. L. Day, W. C. Langston, and C. 8. O’Brien, Am. J. Ophthalmol. 14, 1005 (1931).
37 P. L. Day, W. J. Darby, and W. C. Langston, J. Nutrition 18, 389 (1937).
33 Q. A. Bessey and S. B. Wolbach, J. Exptl. Med. 69, 1 (1939).
39M. M. El-Dadr, Chemistry & Industry 58, 1020 (1939).
40 P. B. Curtis, S. M. Hauge, and H. R. Kraybill, J. Nutrition 5, 503 (1932).
H.S. Mitchell and G. M. Cook, Proc. Soc. Exptl. Biol. Med. 36, 806 (1937).
2 W.K. Hall, L. L. Bowles, V. P. Sydenstricker, and H. L. Schmidt, Jr., J. Nutri-
tion 36, 277 (1948).
43 A. Pirie, Brit. J. Nutrition 2, 14 (1948).
4 P. Gyorgy and H. Goldblatt, J. Hrptl. Med. 70, 185 (1989).
48 G. Gavin and E. W. McHenry, J. Biol. Chem. 182, 41 (1940).
46 P. Gyorgy and H. Goldblatt, J. Haptl. Med. 72, 1 (1940).
47 J. H. Shaw and P. H. Phillips, J. Nutrition 22, 345 (1941).
** R.W. Engel and P. H. Phillips, Proc. Soc. Exptl. Biol. Med. 40, 597 (1939).
382 RIBOFLAVIN
sheaths of the sciatic nerves, axis cylinder swelling, and fragmentation.
Myelin degeneration and gliosis in the spinal cord have also been observed.
Histological changes in the skin have been described by Wolbach and
Bessey”: *°: °° as follows: ‘“‘We find that the initial responses are in the epi-
dermis and its appendages. The vascular engorgement, so characteristic
of pyridoxine deficiency, does not occur. The epidermis as a whole shows
little change other than a moderate hyperkeratosis. In some locations there
is sight hyperplasia of the epidermis, particularly of the snout and sides
of the head, possibly related to scratching. Sebaceous glands, including the
Meibomian glands of the eyelids, become somewhat atrophic. There is an
increased rate of shedding of hair which we believe to be the result of separa-
tion of the cornified anchoring cells from the epithelial sheaths. The out-
standing and thus far, to us, distinctive feature of the deficiency is the
effect upon regeneration of hair follicles and hair formation. In the late
stage of the deficiency, regeneration of the hair follicles does not occur or
is incomplete. Follicles engaged in hair formation during the establishment
of the deficiency undergo atrophy and for a time continue to form imper-
fect hair. The atrophy is apparent in all parts of the hair follicle but is
more evident in the matrix. The cuticular cells continue longest but undergo
atypical cornification. Thus various degrees of retardation of hair produc-
tion are found in a given area of skin; complete suppression, hair roots
represented by loosely packed columns of cornified fusiform cells and hair
roots consisting of medulla with imperfectly formed cortical substance.
Sharply flexed or buckled hair follicles are common, presumably occasioned
by the lack of support normally afforded by the forming hair shaft or root.
In cross section, the hair roots are often oval or flat in outline. The micro-
scopic appearances account satisfactorily for the gross appearances of the
sparsely distributed hair. The gross impression of thickening of the skin
may be accounted for by the persistence of many atrophic regenerated
follicles because these may and often do extend to the depth of normal ac-
tive follicles (1.e., to the muscle panniculus), and owing to their number,
should affect the texture of the skin. In 48 hours after riboflavin therapy,
there is marked restoration of normal appearances of the follicles and in
72 hours the epithelium of the follicle has assumed normal appearances.
The matrix cells respond first.”
According to Kornberg et al.! rats fed a diet deficient in riboflavin de-
veloped granulocytopenia in about 5.0% of the cases and, less frequently,
anemia. The granuloeytopenia responded to folic acid more frequently than
to riboflavin. However, the anemia observed was alleviated in more rats
49S$. B. Wolbach, J. Am. Med. Assoc. 108, 7 (1937).
50S. B. Wolbach and O. A. Bessey, Science 91, 559 (1940).
51 A. Kornberg, F. S. Daft, and W. H. Sebrell, Arch. Biochem. 8, 481 (1945).
X. EFFECTS OF DEFICIENCY 383
by riboflavin than by folic acid. There was a hyperplasia of bone marrow
in the riboflavin-deficient rats which was indistinguishable from that seen
in rats with folic acid deficiency. There was also an atrophy of the lymph-
oid tissue and no evidence of blood formation in the spleen. Further study
of these observations has led to the assumption that riboflavin deficiency’
can cause anemia in the rat.
The importance of riboflavin in the reproductive cycle has been quite
apparent to animal breeders. Its absence from the diet of rats may result
in anestrus, and if riboflavin is not restored within 10 weeks, the damage
becomes irreparable.*® Female rats bred on a deficient diet by Warkany®®: 7
gave birth to litters one-third of which had congenital skeletal malfor-
mations including shortening of the mandible, tibia, fibula, radius, and
ulna, fusion of the ribs, sternal centers of ossification, fingers, and toes,
and cleft palate. There were no abnormal young when riboflavin was added
to the diet.®® Nelson et al.*® did not observe skeletal abnormalities at birth
in the litters of their riboflavin-deficient rats. The principal changes which
appeared in their deficient animals were retarded development of the
epiphyses, progressive decrease in the width of the epiphyseal cartilage,
increased hyalinization of its matrix, and calcification and separation of the
epiphyseal cartilage from the marrow cavity by a thin layer of bone. Hema-
topoietic tissue was replaced by fat in all the rats after they had been on
the deficient diet for 144 days.
Riboflavin has been found to be protective to the rat against the rickett-
siae of murine typhus.®° Chronic riboflavin deficiency is often accompanied
by a type of pediculosis against which riboflavin seems to have a specific
effect.
It has been shown that there is no appreciable loss of appetite during
riboflavin deficiency. On the contrary, there is a relatively increased food
intake during the final stages of riboflavin deficiency.**: ® Pair-fed control
rats grew much faster than the riboflavin-depleted rats. Consequently,
riboflavin has been associated with an increased economy of food utiliza-
tion.
52 W. H. Sebrell, Federation Proc. 8, 568 (1949).
53 C. F. Shukers and P. L. Day, J. Nutrition 25, 511 (1943).
54 A. Kornberg, H. Tabor, and W. H. Sebrell, Am. J. Physiol. 145, 54 (1945).
88K, H. Coward, B. G. E. Morgan, and L. Waller, J. Physiol. 100, 423 (1942).
56 J. Warkany and R. C. Nelson, Science 92, 383 (1940).
57 J. Warkany, Vitamins and Hormones 3, 73 (1945).
88 J. Warkany and E. Schraffenberger, Proc. Soc. Exptl. Biol. Med. 54, 92 (1943).
°? M. M. Nelson, E. Sulon, H. Becks, and H. M. Evans, Proc. Soc. Exptl. Biol. Med.
66, 631 (1947).
60 H. Pinkerton and O. A. Bessey, Science 89, 368 (1939).
$1 P. Gyorgy, Proc. Soc. Exptl. Biol. Med. 38, 383 (1938).
62 B. Sure and M. Dichek, J. Nutrition 21, 453 (1941).
384 RIBOFLAVIN
2. Does AND FoxEs
Sebrell®: ® was probably the first to critically evaluate the pathological
state in dogs now known to be due to a deficiency of riboflavin. Among the
signs noted was the characteristic ‘“‘yellow liver” due to fatty infiltration.
Later, during studies of canine blacktongue, it was shown that riboflavin
often prevents death in animals on blacktongue-producing diets.®-* With
inadequate riboflavin, collapse, coma, and death occurred in less than 102
days. The onset was sudden and characterized by ataxia, weakness, and
loss of deep reflexes, so that the dog was unable to stand. The animal ap-
peared fully conscious and without pain prior to final collapse, as though
death were due to a cellular asphyxia brought on by a failing chemical
mechanism.”* This collapse syndrome was also noted by Street and Cowgill®
in dogs on diets which contained not more than traces of riboflavin.
Street et al.,°°> while investigating neurological manifestations of ribo-
flavin deficiency, were not able to confirm the finding of fatty degeneration
of the liver in their dogs. They noted myelin degeneration in peripheral
nerves and in the posterior columns of the spinal cord, becoming more ex-
tensive with the length of the period on the deficient diet. Opacities of the
cornea were also noted. The suggestion of these authors that inanition was
the cause of previously published reports of fatty liver cannot be reconciled
with the observations of Potter e¢ al.,®°° who noted no fatty liver in their
inanition-control dogs, whereas their riboflavin-deficient, choline-supple-
mented diets produced typical friable, fatty, yellow livers. Also noted was a
dry, flaky dermatitis, usually accompanied by a marked erythema on the
hind legs, chest, and abdomen, and a purulent discharge from the eye,
which was associated with a conjunctivitis. This was followed in a few
days by vascularization of the cornea, which in several dogs went on to
corneal opacification.
Anemias have been noted in riboflavin-deficient dogs,°*: °° 7° but it
remains a question whether this is a specific part of the deficiency syndrome.
The deficiency symptoms which develop in the fox closely resemble those
63 W. H. Sebrell, Public Health Repts. (U.S.) 44, 2697 (1929).
64 W. H. Sebrell, Natl. Insts. Health Bull. 162, Part 3, 23 (1933).
65 W. H. Sebrell, D. J. Hunt, and R. H. Onstott, Public Health Repts. (U.S.) 52,
235 (1937).
66 W. H. Sebrell, R. H. Onstott, and D. J. Hunt, Public Health Repts. (U.S.) 52, 427
(1937).
67 W. H. Sebrell and R. H. Onstott, Public Health Repts. (U.S.) 53, 83 (1988).
68 WH. R. Street and G. R. Cowgill, Am. J. Physiol. 125, 323 (1939).
69 R. L. Potter, A. E. Axelrod, and C. A. Elvehjem, J. Nutrition 24, 449 (1942).
70 H. Spector, A. R. Maass, L. Michaud, C. A. Elvehjem, and E. B. Hart, J. Biol.
Chem. 150, 75 (1948).
X. EFFECTS OF DEFICIENCY 385
observed in the dog. Loss of weight, muscular weakness, coma, opacity of
the lens, and fatty infiltration of the liver have been reported.”
a. EIGs
The similarity of By deficiency in the pig to human pellagra was noted
by Hughes” in 1938, and subsequent reports have highlighted the economic
importance of adequate riboflavin in the diet of swine.”: 7 Patek et al.7
characterized riboflavin deficiency in the pig as a syndrome including re-
tarded growth, corneal opacities, dermatitis, changes in the hair and hoofs,
and terminal collapse associated with hypoglycemia. These pigs showed
changes in the corneal epithelium, lenticular cataracts, hemorrhages of the
adrenals, and lipoid degeneration of the proximal convoluted tubules of the
kidneys. Mitchell e¢ al.7° did not find any cataracts or corneal changes but
did note anorexia and vomiting in their riboflavin-deficient pigs. They
considered the absolute and relative neutrophilic granulocyte concentra-
tions in the blood as the most sensitive indices of riboflavin deficiency.
4. YounG RuMINANTS
It is generally agreed that ruminants can meet most of their requirement
of B-complex vitamins by intestinal synthesis. However, during the first
days after birth the rumen of the young animal has not yet reached func-
tional capacity, and unless riboflavin is supplied to the feed of young dairy
calves, definite signs of riboflavin deficiency develop. Synthetic milk diets
have been devised which do not favor normal rumen function.” On such
diets the dairy calf develops hyperemia of the buccal mucosa, lesions in the
corners of the mouth, along the edges of the lips, and around the navel, loss
of appetite, scours, excessive salivation and lacrimation, and loss of hair.77-79
Pounden and Hibbs*® have observed that the type of ration fed to calves
was a controlling factor in the development of riboflavin-producing flora
and fauna*! in the rumen.
1A. E. Schaefer, C. K. Whitehair, and C. A. Elvehjem, J. Nutrition 84, 131 (1947).
72 E. H. Hughes, Hilgardia 11, 595 (1938).
73 E. H. Hughes, J. Nutrition 20, 233 (1940).
™M. M. Wintrobe, Am. J. Physiol. 126, 375 (1939).
15 A.J. Patek, Jr., J. Post, and J. Victor, Am. J. Physiol. 188, 47 (1941).
76H. H. Mitchell, B. C. Johnson, T. S. Hamilton, and W. T. Haines, J. Nutrition 41,
317 (1950).
7 A.C. Wiese, B. C. Johnson, H. H. Mitchell, and W. B. Nevens, J. Nutrition 33,
263 (1947).
7% R.G. Warner and T.S. Sutton, J. Dairy Sci. 31, 976 (1948).
7G. J. Brisson and T.S. Sutton, J. Dairy Sci. 34, 28 (1951).
80 W. D. Pounden and J. W. Hibbs, J. Dairy Sci. 30, 582 (1947).
8! W. D. Pounden and J. W. Hibbs, J. Dairy Sci. 31, 1041 (1948).
386 RIBOFLAVIN
Riboflavin deficiency has also been observed in young lambs reared on
artificial diets.
5. OTHER MAMMALS
Mice show effects**: 8 quite similar to those described for rats. A histo-
logical basis for the inhibition of lengthwise growth in riboflavin-deficient
animals has been suggested in studies of endochondral ossification in mice.*®
Rhesus monkeys** develop a freckled type of dermatitis on face, hands,
legs, and groin, and a hypochromic, normocytic anemia, both of which are
improved by riboflavin administration. Fatty livers which cannot be re-
lated to inanition have also been demonstrated in these monkeys.
The similarity between periodic ophthalmia in horses during the course
of which corneal vascularization and cataracts frequently occur, and ribo-
flavin deficiency in experimental animals, has suggested a possible common
etiology.’ It has been reported® that riboflavin is effective in preventing
the appearance of equine periodic ophthalmia but that it does not influence
the course of the disease in established cases. Studies of horses on diets
low in riboflavin®? have shown a correlation between their urinary excretion
and their dietary intake.
6. Brrps
The economic importance of poultry raising has stimulated much excel-
lent research on the vitamin requirements of birds. The needs of chicks
and fowls for growth and egg laying have been assayed by many investi-
gators.°°-°8 During these studies peculiar pathological syndromes have been
82 R. W. Luecke, R. Culik, F. Thorp, Jr., L. H. Blakeslee, and R. H. Nelson, J. Ant-
mal Sci. 9, 420 (1950).
83§. W. Lippincott and H. P. Morris, J. Natl. Cancer Inst. 2, 601 (1942).
84 P. F. Fenton and G. R. Cowgill, J. Nutrition 34, 273 (1947).
85 B. M. Levy and R. Silberberg, Proc. Soc. Exptl. Biol. Med. 63, 355 (1946).
86 J. M. Cooperman, H. A. Waisman, K. B. McCall, and C. A. Elvehjem, J. Nutrz-
tion 80, 45 (1945).
87 T. C. Jones, F. D. Mauer, and T. O. Roby, Am. J. Vet. Research 6, 67 (1945).
88 TC. Jones, T. O. Roby, and F. D. Maurer, Am. J. Vet. Research 7, 403 (1946).
89 P. B. Pearson, M. K. Sheybani, and H. Schmidt, Arch. Biochem. 3, 467 (1944).
90 F. H. Bird, V. S. Asmundson, F. H. Kratzer, and 8. Lepkovsky, Poultry Sct. 25,
A7 (1946).
91 R. M. Bethke and P. R. Record, Poultry Sct. 21, 147 (1942).
2 W. Bolton, J. Agr. Sct. 34, 198 (1944). |
% T,. F. Leloir and D. E. Green, Federation Proc. 5, 144 (1946).
4 W. W. Cravens, H. J. Almquist, R. M. Bethke, L. C. Norris, and H. W. Titus,
Recommended Nutritional Allowances for Poultry, National Research Council,
1946.
95 HW. L. Lucas, G. F. Heuser, and L. C. Norris, Poultry Scz. 25, 137 (1946).
96 T. H. Jukes, E. L. R. Stokstad, and M. Belt, J. Nutrition 38, 1 (1947).
97 J. C. Fritz, W. Archer, and D. Barker, Poultry Sci. 18, 449 (1939).
9% TD. M. Hegsted and R. L. Perry, J. Nutrition 35, 411 (1948).
“a
X. EFFECTS OF DEFICIENCY 387
observed which not only have advanced our understanding of riboflavin
deficiency but also have been of major importance in the discovery of the
more recently described vitamins of the B complex.
Phillips and Engel*® have observed in chicks specific pathology in the
main peripheral nerve trunks, characterized by degenerative changes in
the myelin sheaths of the nerve fibers, which was quite similar to that
seen during riboflavin deficiency in rats” on high-fat diets. A prolonged,
mild deficiency produced a characteristic ‘“‘curled-toe paralysis” in chick-
ens.°°
Riboflavin deficiency in turkeys**: !°°: !°! produced a severe dermatitis.
Hegsted and Perry® did not observe any characteristic gross signs of ribo-
flavin deficiency in the duckling; the animals failed to grow and died within
a week.
Ek. IN MAN
M. K. HORWITT
A syndrome resembling pellagra (pellagra sine pellagra) has been known
for centuries, but its relationship to the diet was first recognized by Stannus
in 1911.1: 1° His findings were generally confirmed and augmented by
Bahr! (1912 to 1914) in Ceylon, Scott! (1918) in Jamaica, Moore!®: 17
(1930) in West Africa, Landor and Pallister!®’ (1935) in Singapore, and
Ackroyd and Krishnan?’ (1936) in South India. Yeast products were first
used therapeutically by Goldberger and Tanner! (1925) in their classical
studies on induced pellagra, and by Fitzgerald!!! (1932), who reported an
outbreak in an Assam prison of ulcerations at the angles of the mouth
which were benefited by 1 oz. of yeast daily.
The first suggestion that two separate dietary factors might be concerned
in the production of clinical pellagra came from Goldberger e¢ al." in 1918,
and what appears to have been riboflavin deficiency was produced on a
99 P. H. Phillips and R. W. Engel, J. Nutrition 16, 451 (1938).
100 §, Lepkovsky and T. H. Jukes, J. Nutrition 12, 515 (1936).
101 T. H. Jukes, Poultry Sct. 17, 227 (1938).
1022 H.S. Stannus, Trans. Roy. Soc. Trop. Med. Hyg. 5, 112 (1912).
103 H. S. Stannus, Trans. Roy. Soc. Trop. Med. Hyg. 7, 32 (1913).
106 P. H. Bahr, A Report on Researches on Sprue in Ceylon, 1912-1914. Cambridge
University Press, London, 1915.
105 H. H. Scott, Ann. Trop. Med. 12, 109 (1918).
106 TF). G. F. Moore, West African Med. J. 4, 46 (1930).
107 TJ). G. F. Moore, J. Trop. Med. 42, 109 (1939).
108 'V. J. Landor and R. A. Pallister, Trans. Roy. Soc. Trop. Med. Hyg. 19, 121 (1935).
109 W. R. Ackroyd and B. G. Krishnan, Indian J. Med. Research 24, 411 (1936).
110 J. Goldberger and W. F. Tanner, Public Health Rept. (U.S.) 40, 54 (1925).
1G. H. Fitzgerald, Indian Med. Gaz. 67, 556 (1932).
2 J. Goldberger, G. A. Wheeler, and E. Sydenstricker, J. Am. Med. Assoc. 71, 944
(1918).
388 RIBOFLAVIN
casein diet by Goldberger and Tanner.!!® A clear-cut separation between
these two deficiency states was not made until 1938, at which time both
nicotinic acid and riboflavin were available. Sebrell and Butler™: '™* studied
a group of patients on a diet low in riboflavin and nicotinic acid and showed
that the manifestations of pellagra sine pellagra were due to riboflavin de-
ficiency.
1. ORAL AND FactaL LESIONS OF ARIBOFLAVINOSIS
The changes observed by Sebrell and Butler!!*-"!> “consisted of lesions
on the lips, which began with a pallor of the mucosa in the angles of the
mouth. This pallor was soon followed by maceration; and within a few
days superficial linear fissures, usually bilateral, appeared exactly in the
corner of the mouth. These fissures showed very little inflammatory reac-
tion, remained moist, and became covered with a superficial yellow crust,
which could be scraped off without bleeding. In some instances these linear
fissures showed a tendency to extend onto the skin of the face but did not
extend into the mouth.”’
In addition, there was a ‘‘scaly, slightly greasy, desquamative lesion on
a mildly erythematous base in the nasolabial folds, on the alae nasi, in the
vestibule of the nose and occasionally on the ears and around the eyelids,
especially at the inner and outer canthi.”’
In the years subsequent to the above presentation there have been many
“confirmatory” reports stemming from clinical observations of ‘‘aribo-
flavinosis.’"®1?5 On the other hand, the failure of several groups to dupli-
cate Sebrell and Butler’s results and the frequency of angular stomatitis
refractory to riboflavin therapy led to some skepticism regarding the syn-
3 W.H. Sebrell and R. E. Butler, Public Health Repts. (U.S.) 58, 2282 (1988).
114 W. H. Sebrell and R. E. Butler, Public Health Repts. (U.S.) 54, 2121 (1939).
115 W. H. Sebrell, Human Riboflavin Deficiency, 7n Biological Action of the Vitamins,
p. 73. University of Chicago Press, Chicago, 1942.
116 V, P, Sydenstricker, L. E. Geeslin, C. M. Templeton, and J. W. Weaver, J. Am.
Med. Assoc. 118, 1698 (1939).
117 P, Manson-Bahr, Lancet II, 317, 356 (1940).
118 R, W. Vilter, S. P. Vilter, and T. D. Spies, J. Am. Med. Assoc. 112, 420 (1939).
119 J. W. Oden, L. H. Oden, Jr., and W. H. Sebrell, Public Health Repts. (U.S.) 54,
790 (1939).
120 T, D. Spies, W. B. Bean, and W. F. Ashe, Ann. Internal Med. 12, 1830 (1939).
121'T, D. Spies, R. W. Vilter, and W. F. Ashe, J. Am. Med. Assoc. 113, 931 (1939).
122 N. Jolliffe, H. D. Fein, and L. A. Rosenblum, New Engl. J. Med. 221, 921 (1939).
123 H. D. Kruse, V. P. Sydenstricker, W. H. Sebrell, and H. M. Cleckley, Public
Health Repts. (U.S.) 55, 157 (1940).
124 V. P. Sydenstricker, W. H. Sebrell, H. M. Cleckley, and H. D. Kruse, J. Am. Med.
Assoc. 114, 2487 (1940).
125 TD. Spies, W. B. Bean, R. W. Vilter, and N. E. Huff, Am. J. Med. Sci. 200, 687
(1940).
X. EFFECTS OF DEFICIENCY 389
drome.'?® It is now apparent that the cause of failure in those studies in
which the typical picture was not produced was either the brevity of the
experimental period” or too high a level of riboflavin in the diet.'!?5-1%°
Williams ef al.!*8 fed a diet containing between 0.8 and 0.9 mg. per day for
over 9 months, and Keys et al.!° gave 1.0 mg. for 5 months without pro-
ducing any clinical changes. Horwitt e¢ al.° fed a diet containing between
0.8 and 0.9 mg. of riboflavin daily for over 2 years, and only one of twenty-
two subjects showed any signs (angular stomatitis) which might be attrib-
uted to a lack of riboflavin. However, when these workers!*!-!8 reduced the
riboflavin intake to 0.55 mg. per day, a level only slightly higher than that
used by Sebrell and Butler, incontrovertible signs of ariboflavinosis ap-
peared in less than 6 months. Subsequent studies have confirmed these
observations and have indicated that the course of the development and
healing of the lesions was not altered by low dietary levels of nicotinic acid
(6 mg.) and tryptophan (250 mg.).
The oral lesions which are generally accepted to be part of the clinical
picture of riboflavinosis may be summarized as follows: angular stomatitis,
fissures in the angles of the mouth which resemble perleche, cheilosis, in-
volvement of the vermilion border of the lips including vertical fissuring,
and crusting and desquamation of the mucous membrane. Glossitis, includ-
ing the magenta tongue, may be seen, but “‘pure”’ riboflavin deficiencies
have been produced! without such defects.
The characteristic facial lesions include seborrheic accumulations in the
folds of the skin, especially in the nasolabial folds. Mild infections of the
upper respiratory tract may initiate an inflammation of the nostrils and
spread as a weeping, crusty lesion over the skin of the septum. Fissures may
appear in the nasolabial folds.
2. LESIONS OF SCROTUM AND VULVA
Stannus'™: 1% was the first to record that scrotal involvement may be
the initial sign of deficiency (nineteen of one hundred cases of ‘“‘pellagra”’).
26 M. Ellenberg and H. Pollack, J. Am. Med. Assoc. 119, 790 (1942).
27 J. J. Boehrer, C. E. Stanford, and E. Ryan, Am. J. Med. Sci. 205, 544 (1943).
88 R. D. Williams, H. L. Mason, P. L. Cusick, and R. M. Wilder, J. Nutrition 25,
361 (1943).
29 A. Keys, A. F. Henschel, O. Mickelsen, J. H. Brozek, and J. H. Crawford, J. Nu-
trition 27, 165 (1944).
180M. K. Horwitt, E. Liebert, O. Kreisler, and P. Wittman, Bull. Nat. Research
Council (U.S.) 116 (1948).
131 B.A. Zeller, Advances in Enzymol. 2, 93 (1942).
12 OQ. W. Hills, E. Liebert, D. L. Steinberg, and M. K. Horwitt, Arch. Internal Med.
87, 682 (1951).
188 M. K. Horwitt, C. C. Harvey, O. W. Hills, and E. Liebert, J. Nutrition 41, 247
(1950).
4M. K. Horwitt, Personal communication (1952).
390 RIBOFLAVIN
Sydenstricker™®: !*° noted an itching dermatitis of the scrotum or vulva in
patients with pellagra. Purcell'®” described a scrotal dermatitis that im-
proved with riboflavin treatment. Mitra'** reported a urogenital lesion
among Indians which responded to riboflavin. Goldberger and Wheeler™®
showed that six of their eleven patients exhibited scrotal dermatitis before
any other lesions of pellagra appeared. In a recent study! scrotal dermatitis
was the most frequently observed symptom of riboflavin deficiency; twelve
of fifteen subjects had it, either mildly or severely. Typically, this began
as a patchy redness associated with scaling and desquamation of the super-
ficial epithelium of the anterior surface of the scrotum. The median com-
missure was uninvolved in most of the patients. The more prolonged and
severe cases showed a lichenification of the involved areas. The far-advanced
lesion became quite raw and extended up the shaft of the penis or to the
inner aspects of the thigh. The response to treatment with 6 mg. of ribo-
flavin per day was prompt, and in two cases with severe inflammation it
was dramatic. This recent study emphasizes the question of the role of
ariboflavinosis in the development of those scrotal and vulval lesions which
have been considered characteristic of pellagra.
3. OcuLAR MANIFESTATIONS
In experimental animals vascularization of the cornea and involvement
of the lids are early and constant findings.**: *8: 4° In man, ocular pathology
is not constant, but it may occur in a high percentage of cases. Conjuncti-
vitis, lacrimation, and burning of the eyes have been observed as manifesta-
tions which have been cured by riboflavin by sufficient investigators to be
non-controversial; corneal vascularization in human riboflavin deficiencies
has not been noted so often under controlled conditions.
Spies and his associates’®: '?! were among the first to note that the ocular
lesions were cured by riboflavin administration. Sydenstricker and co-
workers!!® reported that photophobia was associated with conjunctivitis.
Reduced visual acuity, itching, a sensation of roughness of the eyelids,
keratitis, and mydriasis have also been reported.'* 124) 141-148
Rubeosis iridis has been suggested as a manifestation of deficiency which
135 V. P. Sydenstricker, Am. J. Public Health 31, 344 (1941).
136 V. P. Sydenstricker, Ann. Internal Med. 14, 1499 (1941).
137 F. M. Purcell, Trans. Roy. Soc. Trop. Med. Hyg. 35, 323 (1942).
138 K. Mitra, Indian Med. Gaz. 78, 330 (1948).
139 J. Goldberger and G. A. Wheeler, Public Health Service, Hygienic Lab. Bull. 120,
116 (1920).
140 R. KE. Eckardt and L. V. Johnson, Arch. Ophthalmol. 21, 315 (1939).
141 H.C. Hou, Chinese Med. J. 59, 314 (1941).
42 PH. Pock-Steen, Geneesk. Tijdschr. Ned.-Indié 79, 1986 (1939).
143 P, B. Wilkinson, Lancet II, 655 (1944).
XI. PHARMACOLOGY 391
can be cured by riboflavin."4 Vascular networks of the iris were markedly
improved after only 2 days of riboflavin supplementation.
Kruse and colleagues"*: 4 reported corneal vascularization in forty-five
of forty-seven patients with riboflavin deficiency. Proliferation and en-
gorgement of the bulbar conjunctival capillaries of the limbar plexus were
considered by them to be the earliest and most common sign of aribo-
flavinosis. As a consquence of many controversial reports,!*-!*! the signifi-
cance of these observations is not clear. No evidence of corneal vasculariza-
tion was noted by the Elgin group!” despite frequent slit-lamp examinations
of subjects before, during, and after experimental riboflavin deficiency.
XI. Pharmacology
M. K. HORWITT
The low solubility of riboflavin may be responsible for its relative innocu-
ousness. Unna and Greslin! found that oral administration of 10 g. per
kilogram to rats and 2 g. per kilogram to dogs produced no toxic effects.
Giving 340 mg. per kilogram to mice intraperitoneally, which is 5000 times
the therapeutic dose, or the equivalent of 20 g. per day for a man, had no
apparent effect.2-* The rat LDso for riboflavin following intraperitoneal ad-
ministration was 560 mg. kilogram.’ Death, which was due to kidney con-
cretions, occurred in 2 to 5 days. Similar results were obtained by Antopol®
after intraperitoneal administration of 125 to 500 mg. per kilogram of the
sodium salt. In addition, cytological changes were noted in the heart,
pancreas, and pituitary gland, and the adrenals were markedly congested.
Since crystalline concretions of riboflavin were readily detectable in the
ureter and bladder within a few hours after a saturated solution of riboflavin
“4 A. S. Stannus, Trans. Ophthalmol. Soc. United Kingdom 62, 65 (1942).
45 J. B. Youmans, E. W. Patton, W. D. Robinson, and R. Kern, Trans. Assoc. Am.
Physicians 57, 6 pp. (1942).
46 H.R. Sandstead, Public Health Repts. (U.S.) 57, 1821 (1942).
147 JT). Vail and K. W. Ascher, Am. J. Ophthalmol. 26, 1025 (1943).
43 FF. Tisdall, J. F. McCreary, and H. Pearce, Can. Med. Assoc. J. 49, 5 (19438).
149 J. F. McCreary, J. V. V. Nicholls, and F. F. Tisdall, Can. Med. Assoc. J. 61, 106
(1944).
160. §. Stannus, Brit. Med. J. II, 103 (1944).
151 J. G. Scott, J. Roy. Army Med. Corps 82, 133 (1944).
1K. Unna and J. G. Greslin, J. Pharmacol. Exptl. Therap. 76, 75 (1942).
2R. Kuhn and P. Boulanger, Z. physiol. Chem. 241, 233 (1936).
3R. Kuhn, Klin. Wochschr. 17, 222 (1938).
4V. Demole, Z. Vitaminforsch. 7, 138 (1938).
5 W. Antopol, J. Med. Soc. New Jersey 39, 285 (1942).
392 RIBOFLAVIN
was given intravenously, Selye® studied bilaterally nephrectomized rats to
learn more about the role of the gastrointestinal tract in the absorption and
excretion of riboflavin. He noted that excess riboflavin was rapidly excreted
into the small intestine, especially the duodenum. Destruction of ribo-
flavin proceeded slowly, if at all, in an isolated loop of duodenum, but
quickly in an isolated large intestine. The bile does not function in the elimi-
nation of this vitamin. If the intestinal canal and kidneys are removed,
the tissues of the rat cannot destroy or eliminate any significant percentage
of large doses of intravenously administered riboflavin.
The riboflavin lost in sweat under tropical conditions’ has been considered
of nutritional importance. However, the amounts which can be proved pres-
ent in sweat are too small to be significant.®: °
Riboflavin is excreted predominantly in the feces, which contain not only
the part contributed by the intestinal walls but also that which is synthe-
sized by intestinal bacteria.
Urine contains riboflavin," riboflavin phosphate,” and a compound called
uroflavin," a derivative which has been reported to be more soluble and to
contain more oxygen than riboflavin. The methods of analysis ordinarily
used for the estimation of riboflavin in urine do not distinguish between
these compounds, since they have similar fluorimetric and microbiological
activities.
The amount of riboflavin in the urine will vary with the recent dietary
intake and with tissue storage. The correlation between urinary excretions
and dietary intake is discussed on p. 401. Urinary excretion of riboflavin
will also be affected by marked alterations in nitrogen balance.'*-!® Less
is excreted in the urine, on a given intake, when tissue growth is rapid, as
during convalescence after severe trauma,” during lactation,'® or after
administration of testosterone propionate;!® more is excreted after severe
6 H. Selye, J. Nutrition 25, 137 (19438).
7D.M. Tennent and R. H. Silber, J. Biol. Chem. 148, 359 (1948).
8 OQ. Mickelsen and A. Keys, J. Biol. Chem. 149, 479 (1943).
9F. Sargent, P. F. Robinson, and R. E. Johnson, J. Biol. Chem. 158, 285 (1944).
10C, W. Denko, W. E. Grundy, N. C. Wheeler, C. R. Henderson, G. H. Berryman,
T. E. Friedemann, and J. B. Youmans, Arch. Biochem. 11, 109 (1946).
11 A. EK. Axelrod, T. D. Spies, C. A. Elvehjem, and V. Axelrod, J. Clin. Invest. 20,
229 (1941).
122A. Kmmerie, Acta Brev. Neerl. Physiol. Pharmacol. Microbiol. 8, 116 (1938).
13.W. Koschara, Z. physiol. Chem. 232, 101 (1935).
14H. P. Sarett and W. A. Perlzweig, J. Nutrition 25, 173 (1948).
15H. P. Sarett, J. R. Klein, and W. A. Perlzweig, J. Nutrition 24, 295 (1942).
16 HT. Pollack and J. J. Bookman, J. Lab. Clin. Med. 38, 561 (1951).
17W. A. Andrea, V. Schenker, and J. 8. L. Browne, Federation Proc. 5, 3 (1946).
18 C, Roderuck, M. N. Coryell, H. H. Williams, and I. G. Macy, J. Nutrition 32, 267
(1946).
19W. T. Beher and O. H. Gaebler, J. Nutrition 41, 447 (1950).
XI. PHARMACOLOGY 393
burns or surgical procedures where protein losses indicate cellular decom-
position.”°
The riboflavin content of the blood is relatively constant?!’ (approxi-
mately 40 y per 100 ml.) when measured by microbiological techniques.
However, since the ingestion of riboflavin can cause a 30% increase in the
flavin adenine dinucleotide content of the red blood cell,” it is likely that
the use of improved methods” will show a correlation between dietary and
erythrocyte content.
Although there is no appreciable storage capacity of riboflavin in animal
tissues, it is apparent that the amount can vary, since the organs of animals
will lose as much as two-thirds of their original content when the animals
are fed riboflavin-deficient diets.2°?§ A combined protein and riboflavin
deficiency is especially effective in decreasing the riboflavin content of the
tissues of the growing rat.?? Diets on which signs of clinical ariboflavinosis
are observed are usually low in protein.
The concept of a rational pharmacology based upon antivitamins, which
was pioneered by Woolley,*® has greatly stimulated the search for ribo-
flavin antimetabolites. Wright and Sabine*! have shown that flavin adenine
dinucleotide lowered the atabrin inhibition of tissue respiration and of p-
amino acid oxidase. This, and a similar observation by Haas® with respect
to cytochrome reductase, led Hellerman, et al.** to quantitize the metabolic
antagonisms of antimalarials like atabrin and quinine.*!: *° The phenazine
analog of riboflavin,** as well as isoriboflavin,*” when fed to mice and rats
produced ariboflavinosis.
20 H. Pollack and 8. L. Halpern, Therapeutic Nutrition, National Research Coun-
cil, 1951.
21 A. EK. Axelrod, T. D. Spies, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med. 46,
146 (1941).
22 F. M. Strong, R.S. Feeney, B. Moore, and H. T. Parsons, J. Biol. Chem. 137, 363
(1941).
*3M. K. Horwitt, E. Liebert, O. Kreisler, and P. Wittman, Bull. Natl. Research
Council (U.S.) 116, 1948.
24 J. R. Klein and H. I. Kon, J. Biol. Chem. 136, 177 (1940).
25 H. B. Burch, O. A. Bessey, and O. H. Lowry, J. Biol. Chem. 175, 457 (1948).
26 R. Kuhn, H. Kaltschmitt, and T. Wagner-Jauregg, Z. physiol. Chem. 282, 36 (1935).
27 F. Vivanco, Naturissenschaften 28, 306 (1935).
8 A. E. Axelrod, H. A. Sober, and C. A. Elvehjem, J. Biol. Chem. 134, 749 (1940).
29 J. W. Czaczkes and K. Guggenheim, J. Biol. Chem. 162, 267 (1946).
30D. W. Woolley, Science 100, 579 (1945).
31C. I. Wright and J. C. Sabine, J. Biol. Chem. 155, 315 (1944).
32. Haas, J. Biol. Chem. 155, 321 (1944).
33 |. Hellerman, A. Lindsay, and M. R. Bovarnick, J. Biol. Chem. 168, 553 (1946).
34 J. Madinaveitia, Biochem. J. 40, 373 (1946).
8° F. H. Johnson and I. Lewin, Science 101, 281 (1945).
36 PD). W. Woolley, J. Biol. Chem. 164, 31 (1944).
37 G. A. Emerson and M. Tishler, Proc. Soc. Exptl. Biol. Med. 55, 184 (1944).
394 RIBOFLAVIN
The effects of riboflavin derivatives on the growth of neoplasms have
been receiving increasing attention. Antopol and Unna** and Miller and
Miller*® have shown that large amounts of riboflavin retarded the oceur-
rence of pathological changes in liver produced by p-dimethylaminoazoben-
zene. Pollack et al.“° noted that the concentration of riboflavin was lower
in tumor than in normal tissue. Severe riboflavin deficiency decreased the
growth rate of carcinomas": *” in mice. This could be accomplished either
by riboflavin deprivation or by administering” isoriboflavin or galactoflavin.
Diethyl-riboflavin® and 6,7-dichloro-9-(1’-p-sorbityl)isoalloxazine“ were
effective in reducing the growth of tumors in rats.
XII. Requirements and Factors Influencing Them
M. K. HORWITT
Any statement regarding the amount of riboflavin required must be a
compromise depending upon variations in the heredity, growth, environ-
ment, age, activity, and health of the organism. The synthesis of riboflavin
by the host and the differences in the availability of the vitamin from di-
verse sources must also be considered. With all these variables it is unlikely
that there will ever be complete agreement among the workers in this field.
Why, for example, only three out of fourteen men on identical diets low in
riboflavin,! living in the same environment for over a year, should show
relatively severe signs of ariboflavinosis, and why three others in the same
group should show no signs of deficiency at all, are questions which will
plague the investigator for years to come. The concept of “individual sus-
ceptibility” is an excuse which covers our ignorance and confounds those
responsible for statements of recommended allowances.
A. OF ANIMALS
Table III presents representative published requirements of some mam-
mals, birds, and fish. It is apparent from the data listed that there is not
38 W. Antopol and K. Unna, Cancer Research 2, 694 (1942).
39 H. C. Miller and J. A. Miller, Cancer Research 7, 468 (1947).
40M. A. Pollack, A. Taylor, J. Taylor, and R. J. Williams, Cancer Research 2, 739
(1942).
41H. P. Morris and W. van B. Robertson, J. Natl. Cancer Inst. 3, 479 (1943).
4 H.C. Stoerk and G. A. Emerson, Proc. Soc. Exptl. Biol. Med. 70, 703 (1943).
48H. V. Aposhian and J. P. Lambooy, Proc. Soc. Exptl. Biol. Med. 78, 197 (1951).
44. W. Holly, E. W. Peel, R. Mozingo, and K. Folkers, J. Am. Chem. Soc. 72, 5416
(1950).
1M. K. Horwitt, O. W. Hills, C. C. Harvey, E. Liebert, and D. L. Steinberg, J.
Nutrition 39, 357 (1949).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 395
TABLE III
Some Reported RIBOFLAVIN REQUIREMENTS
Amount Reference
Mice 1.5 y/g. food 2,30
4 y/day 4
0.4-0.6 mg./100 g. body wt. 5
Rat 2-3 y/g. food 6,°7
7.5 y/day 8
10 y/day 9
18 7/day 10
Dog 60-100 7/kg. body wt./day 114
100-200 y/kg. body wt./day 12
Swine 20-66 7/kg. body wt./day 13
1.7 mg./kg. feed 14
Fox 1.24.0 y/g. diet 15
Horse 44 y/kg. body wt./day 16
Holstein calf 1.0 mg./kg. feed 17
Monkey 25-30 y/kg. body wt./day 18
Chick 2.75-3.25 y/g. food 19
2.9-3.6 y/g. food 20, 21
Poult 3.25-3.75 y/g. food 19
3-4 y/g. food 22525
Duck 3 y/g. food 24, 25
Trout 5-15 y/g. food 26
6-9 y/g. food 27
2H. P. Morris and W. B. Robertson, J. Natl. Cancer Inst. 3, 479 (1948).
3W.C. Langston, P. L. Day, and K. W. Cosgrove, Arch. Ophthalmol. 10, 508 (1933).
4S. W. Lippincott and H. P. Morris, J. Natl. Cancer Inst. 2, 601 (1942).
5 P. F. Fenton and G. R. Cowgill, J. Nutrition 34, 273 (1947).
6 H. C. Sherman and L. N. Ellis, J. Biol. Chem. 104, 91 (1934).
7C. A. Mills, Arch. Biochem. 2, 159 (1943).
8 J. W. Czaczkes and K. Guggenheim, J. Biol. Chem. 162, 267 (1946).
*G. C. Supplee, R. C. Bender, and O. J. Kahlenberg, J. Nutrition 20, 109 (1940).
10 G. F. Mannerling, M. A. Lipton, and C. A. Elvehjem, Proc. Soc. Exptl. Biol. Med.
46, 100 (1941).
1R.L. Potter, A. E. Axelrod, and C. A. Elvehjem, J. Nutrition 24, 449 (1942).
2H. R. Street and G. R. Cowgill, Am. J. Physiol. 125, 323 (1989).
13. H. Hughes, J. Nutrition 20, 233 (1940).
4 FE. H. Hughes, E. W. Crampton, N. R. Ellis, and W. J. Loeffel, Recommended
Nutrient Allowances for Swine, Report of Committee on Animal Nutrition, Na-
tional Research Council, 1944.
15 A. E. Schaefer, C. K. Whitehair, and C. A. Elvehjem, J. Nutrition 34, 131 (1947).
16 P. B. Pearson, M. K. Sheybani, and H. Schmidt, Arch. Biochem. 3, 467 (1944).
7H. H. Draper and B. C. Johnson, J. Nutrition 46, 37 (1952).
18 J. M. Cooperman, H. A. Waisman, K. B. MeCall, and C. A. Elvehjem, J. Nutri-
tion, 30, 45 (1945).
19 F. H. Bird, U.S. Asmundson, F. H. Kratzer, and 8. Lepkovsky, Poultry Sci. 25,
47_(1946).
396 RIBOFLAVIN
any unanimity of opinion. However, if one considers the ratio of riboflavin
to food intake, it can be noted that 2 to 3 mg. of riboflavin per kilogram of
diet seems to satisfy most of the suggested requirements. Such a figure is
only of approximate value, since the protein, fat, and carbohydrate pro-
portions of the diet will vary widely.
1. Errect oF DIeTaRy CONSTITUENTS
It has been shown! **: °° that the fat content of the diet has a marked
effect on the riboflavin requirement of the rat. Thus, the replacement of
the dextrin in a rat diet with isocaloric amounts of fat increased the amount
of riboflavin needed for growth and enabled the production of more severe
deficiency symptoms. Riboflavin appeared to play no part in the synthesis
of fat, carbohydrate, and protein.**: *! Extra fat gained by rats on high-
riboflavin diets may result from the sparing of dietary fat through more
efficient utilization of dietary energy.® Reiser and Pearson* found that
cottonseed oil in the diet of chicks increased the requirement of riboflavin.
Rats fed a diet containing only 2 % of calories as fat required half as much
riboflavin as rats fed the standard 20 % fat diet, and rats on the latter diet,
in turn, required half as much riboflavin as those fed 40 % of their calories
as fat.$
It is not possible to vary the amount of fat in a diet without changing
the relative concentration of the other constituents. The effects of high-fat,
low-fat, high-protein, and low-protein diets on riboflavin requirements of
the rat were studied simultaneously by Czaczkes and Guggenheim.* They
noted that rats on low-protein diets needed at least twice as much ribo-
flavin as animals kept on a ‘‘normal” diet. These authors believe that the
20 LL. C. Norris, H. 8. Wilgus, A. T. Ringrose, V. Heiman, and C. F. Heuser, Cornell
Univ. Agr. Expt. Sta. Bull. 660, 3 (1936).
*1R.M. Bethke and P. R. Record, Poultry Sci. 21, 147 (1942).
22,W. W. Cravens, H. J. Almquist, R. M. Bethke, L. C. Norris, and H. W. Titus,
Recommended Nutritional Allowances for Poultry, National Research Council,
1946.
23'T, H. Jukes, E. L. R. Stokstad, and M. Belt, J. Nutrition 33, 1 (1947).
24 J. C. Fritz, W. Archer, and D. Barker, Poultry Sci. 18, 449 (1989).
25 T). M. Hegsted and R. L. Perry, J. Nutrition 35, 411 (1948).
26 B. A. McLaren, E. Keller, D. J. O’Donnell, and C. A. Elvehjem, Arch. Biochem.
15, 169 (1947).
27 A.V. Tunison, D. R. Brockway, J. M. Maxwell, A. L. Dorr, and C. M. McCay,
N. Y. State Conservation Dept. Cortland Hatchery Rept. No. 11 (1942).
28 J. H. Shaw and P. H. Phillips, J. Nutrition 22, 345 (1941).
29 G. J. Mannerling, D. Orsini, and C. A. Elvehjem, J. Nutrition 28, 141 (1944).
30 HK. W. McHenry and G. Gavin, J. Biol. Chem. 125, 653 (1938).
31H. W. McHenry and G. Gavin, J. Biol. Chem. 188, 471 (1941).
32 T,. Voris and H. P. Moore, J. Nutrition 25, 7 (1948).
33 R. Reiser and P. B. Pearson, J. Nutrition 38, 247 (1949).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 397
different requirements for riboflavin are due to differences in the amounts
of riboflavin which are synthesized in an available form.
Recent studies by Everson eé al.*! have stressed the importance of com-
plete digestion in evaluating the availability of riboflavin in various foods.
Working with young women, they observed that a larger proportion of
riboflavin was available from ice cream than from legumes or almonds.
Large doses of ascorbic acid or aureomycin can prevent or delay signs of
riboflavin deficiency in rats. Daft and Schwarz** have reported that ribo-
flavin-deficient rats died as expected, but that on identical diets plus 2 %
ascorbic acid or 20 mg. % aureomycin litter mates survived.
2. Errect oF ENVIRONMENT
Workgng with rats at environmental temperatures of 90 and 68° F.,
Mills’ concluded that the dietary concentration of riboflavin needed for
maximum growth was not altered by temperature. Mitchell et al.,*° using
pigs as their experimental animals, have claimed that the riboflavin require-
ment is greater (2.3 p.p.m. at 42°F.) at low temperatures than at high
temperatures (1.2 p.p.m. at 85° F.).
3. REPRODUCTION
Barrett and Everson® indicated that the need for B vitamins increased
rapidly as pregnancy*® progressed in the rat. Hogan and Anderson*®’ showed
that a synthetic diet slightly inadequate for growth was seriously inade-
quate for lactation. It is reasonable to expect lactation to increase the re-
quirement, since logically the need for mother and offspring is greater than
that of the mother alone.
4. INHERENT INDIVIDUAL VARIATIONS
Those who have worked with animals in nutritional studies are acutely
aware of the individual variations which will occur, even in closely inbred
litter mates. Fenton and Cowgill’ have highlighted this problem by study-
ing the riboflavin requirements of two inbred strains of mice. Mice of the
Cs; strain showed maximum growth when the diet contained 0.4 mg. of
riboflavin per 100 g., whereas those of the A strain required a dietary level
of 0.6 mg. At a 0.2-mg. level the C;; mice had lowered red cell counts and
34 G. Everson, E. Pearson and R. Matteson, J. Nutrition 46, 45 (1952).
35 F. §. Daft and K. Schwarz, Federation Proc. 11, 200 (1952).
36 H. H. Mitchell, B. C. Johnson, T.S. Hamilton, and W. T. Haines, J. Nutrition 41,
317 (1950).
37 M. Barrett and G. Everson, J. Nutrition 45, 493 (1951).
38 G. Everson, E. Williams, E. Wheeler, P. Swanson, M. Spivey, and M. Eppright,
J. Nutrition 36, 463 (1948).
39 A. G. Hogan and G. C. Anderson, J. Nutrition 36, 437 (1948).
398 RIBOFLAVIN
less riboflavin in their muscle and liver than those of the A strain on the
same diet.
5. OrgeR Factors
There have been suggestions that the growth requirements of male and
female rats are different,” the over-all effects of riboflavin deficiency being
more prominent in the male. Unlike thiamine deficiency, the lack of ribo-
flavin is not associated with severe anorexia; thus, appetite is not so im-
portant a factor in riboflavin depletion studies. The interrelationships be-
tween riboflavin and other vitamins of the B group have been studied.*°
B. OF MAN
In the absence of experimental data on human subjects, the estimation
of riboflavin requirements is based upon average dietary consumptions or
upon extrapolations of data from animal experiments. Calculation of aver-
age consumption is not a satisfying procedure, since different locales will
show great variations depending upon dietary habits and the availability
of riboflavin-rich foods. Attempts to calculate man’s needs from data on
rat growth tend to give figures which are too high to be practical. It is
therefore necessary to test vitamin requirements on man, himself.
It is logical to expect that the minimum requirements of human beings
would be much more variable than for the inbred laboratory animal. That
this is the case was illustrated by Horwitt et al.,!: 4‘ who studied fifteen men
on a diet providing 0.55 mg. of riboflavin per day. Three of the men de-
veloped relatively severe dermatological lesions, nine men showed mild
symptoms of ariboflavinosis, and three others had no symptoms at all.
Excretion studies which compare the amount of riboflavin intake with
the amount excreted in the urine have been used for many years as a means
of estimating human requirements. Much confusion has resulted from this
approach because there is, as yet, no agreement upon how much riboflavin
should be excreted before the intake is considered adequate.
In some of the older studies on riboflavin excretion” the diet was not
considered adequate if it contained less than 2 mg. of riboflavin per day.
Consequently, urinary excretions of less than 500 meg. per day were desig-
nated as deficient. More recent comparative studies on the amounts of
riboflavin excreted in the urine on different levels of intake have shown that
40 K, Bhagvat and P. Devi, Biochem. J. 45, 32 (1949).
41Q. W. Hills, E. Liebert, D. L. Steinberg, and M. K. Horwitt, Arch. Intern. Med.
87, 682 (1951).
4“ A. Emmerie, Nature 138, 164 (1936).
43 FM. Strong, R.S. Feeney, B. Moore, and H. T. Parsons, J. Biol. Chem. 187, 363
(1941).
44°V. H. Feder, G. T. Lewis, and H. 8. Alden, J. Nwtrition 27, 347 (1944).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 399
a reserve of riboflavin cannot be maintained by men at levels of intake be-
low 1.1 mg. per day* on a diet containing approximately 2200 cal. Brewer
et al.4® calculated the requirement of women to be 1.3 to 1.5 mg. per day
on a diet providing 2100 to 2300 cal. per day.
The recommended daily allowances of the Food and Nutrition Board
of the National Research Council state that 1.8 mg. of riboflavin is ade-
quate for a 70-kg. adult man, and 1.5 mg. for a 56-kg. adult woman. The
assumption was made that increased work and greater than average caloric
consumption do not increase the need for riboflavin. The allowances during
the latter half of pregnancy and during lactation were increased to 2.5 and
3.0 mg., respectively. There is as yet no proof that more than the normal
daily allowance is required during pregnancy. This problem was reviewed
by Oldham et al.” If one estimates the total amount of riboflavin stored
during the gestation period, it seems likely that an additional 0.2 mg. per
day should satisfy the needs for growth during pregnancy. The increased
allowance for lactation makes ample provision for the amount in human
milk, which contains about 0.5 mg. riboflavin per liter.
The recommended allowances for children are graduated in accordance
with the growth rate at different ages. It has been recommended that chil-
dren from 1 to 3 years be allowed 0.6 mg. per day, and that children 10 to
12 years be allowed 1.8 mg. per day. During the rapid period of growth
from 13 to 15 years, it has been recommended that both girls and boys
receive 2.0 mg. of riboflavin per day. It is apparent that, since these al-
lowances are adjusted for growth requirements, the actual need of an in-
dividual will vary with his or her own pattern of growth.
* * * * *
One of the major goals of all the research described in this chapter is to
determine how much riboflavin is required by man for optimum nutritional
health. The techniques used may be classified under four headings: (1) ob-
servations of the repair of pathology by riboflavin administration; (2)
survey studies of the nutritional status of population groups; (3) experi-
mental production of riboflavin deficiency; and (4) evaluation of urinary
excretion of riboflavin in health and disease.
Riboflavin deficiency states ordinarily noted by the clinician, whose pri-
mary obligation is to facilitate the repair of apparent pathology, do not
often present adequate opportunities to assay the individual’s need for
riboflavin. The important contributions of these observations are in the
classifications of conditions which can be healed by riboflavin, usually given
46M. K. Horwitt, C. C. Harvey, O. W. Hills, and E. Liebert, J. Nutrition 41, 247
(1950).
46 W. Brewer, T. Porter, B. Ingalls, and M. A. Ohlson, J. Nutrition 32, 583 (1946).
47H. Oldham, B. B. Sheft, and T. Porter, J. Nutrition 41, 231 (1950).
400 RIBOFLAVIN
in amounts which are far in excess of the daily requirement. Without these
classifications the researcher in nutrition would not know what to look for.
Excellent examples of the population survey type of study have been
described by Goldsmith* and by Wilkins and Sebrell.*? Wilder®® has sum-
marized the extensive surveys of malnutrition in Newfoundland®!-* to
show how the enrichment of flour benefited the population. But, although
these surveys were of great value to our understanding of the nutrition of
population groups, they are difficult to interpret in terms of the require-
ment of the individual.
The experimental production of riboflavin deficiency has been successful
only in those studies in which levels of 0.55 mg. of riboflavin per day or
less have been fed. Those investigators who provided 0.8 mg. per day to
their subjects could not produce signs of ariboflavinosis. Nevertheless, no
nutritional authority has yet suggested that 0.8 mg. is adequate for optimal
health. Rather, one recommends amounts which provide somewhat more
than the minimal daily need as fortification against unknown contingen-
cies. The concept that riboflavin cannot be stored may not be entirely
correct, since even at dietary levels of 0.5 mg. per day about 6 months
must elapse before signs of ariboflavinosis appear.
One of the more important advantages of depletion studies is the oppor-
tunity provided for simultaneous investigation of urinary excretion. Since
the urinary excretion is a reflection of the dietary intake and the dietary
intake is, in the last analysis, the cause of riboflavin deficiency, it is under-
standable why so much effort has been devoted to the study of riboflavin
in urine.
Load tests, in which a known amount of riboflavin is administered and
the percentage excreted is determined, are useful means of estimating the
degree of saturation of the tissues. Although the usual procedure is to ad-
minister riboflavin in the post-absorptive state and to analyze the riboflavin
excreted during the following 4 hours, a 24-hour collection may be consid-
ered a load test if the dietary intake during that period is known. Gold-
smith has reviewed the literature on the use of urinary excretion tests in
48G. A. Goldsmith, Federation Proc. 4, 263 (1945).
49 W. Wilkins and W. H. Sebrell, Mederation Proc. 4, 258 (1945).
50 R. M. Wilder, Federation Proc. 9, 562 (1950).
51G. A. Goldsmith, W. J. Darby, R. C. Steinkemp, A. S. Beam, and E. McDevitt,
J. Nutrition 40, 41 (1950).
52 J. D. Adamson, N. Jolliffe, H. D. Kruse, O. H. Lowry, P. E. Moore, B.S. Platt,
W.H. Sebrell, J. W. Tice, F. F. Tisdall, R. M. Wilder, and P. C. Zameenik, Can.
Med. Assoc. J. 52, 227 (1945).
583. W.R. Aykroyd, N. Jolliffe, O. H. Lowry, P. E. Moore, W. 1. Sebrell, R. E. Shank,
F. F. Tisdall, R. M. Wilder, and P. C. Zamecnik, Can. Med. Assoc. J. 60, 329
(1949).
54 G. A. Goldsmith, Federation Proc. 8, 553 (1949).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 401
the evaluation of riboflavin nutrition. Oldham ef al.°> and Pollack and
Bookman** have shown that increased amounts of riboflavin are excreted
by subjects in marked negative nitrogen balance. Therefore, in those spec-
ial cases where tests for riboflavin excretion are made on patients who are
eatabolizing abnormal amounts of their own tissues, the excretion datamay
TABLE IV
ComMPaARISON oF UrtNARY EXCRETION witH DartLy INTAKE OF RIBOFLAVIN. ExceprT
FOR THOSE ON THE 0.55-Ma.-Dtet, tHe Data Listed REPRESENT PLATEAU
LEVELS WuHicH WERE REACHED IN 10 WEEKS OR LESS ON THE
INTAKE DESIGNATED?
Riboflavin excretion, 24 hr. Excretion during 4 hr.
Number of Duration of following subcutaneous
subjects on each Riboflavin diet at time of Ingested injection of 1 mg. of
intake intake, mg. analyses, weeks? Amount, 7 riboflavin, % riboflavin, y
15 0.55 15 il eS aul 9.3 23 +: 15°
ala 0.75 12 73 + 5 9.7 56 + 34
12 0.85 15 76 + 38 8.9 58 + 22
28 eat 13 97 + 62 8.8 70 + 35
39 1.6 100 434 + 185 26.5 227 + 146
12 2.15 10-78 714 + 293 33.2 297 + 124
13 2.59 2-44 849 + 258 33.3 298 + 172
13 3.55 1.5 1714 + 3004 48 .34 373 + 90?
* At an 0.85-mg. intake or higher the figures obtained at 15 weeks remain at the same level for at least 2
years more. At a 0.75-mg. intake most of the subjects remained at the level designated for 6 months, at which
time they were supplemented. A few individuals who remained longer on this diet showed no further change,
but whether or not their excretions would have decreased further with additional time is not known.
® The time of analyses for diets providing 0.75, 0.85, and 1.1 mg. of riboflavin represents the first time
plateau levels were obtained which were repeated during subsequent months. The results recorded for the
diets providing 1.6, 2.15, and 2.55 mg. are averages of voluminous data obtained during the period of time
indicated.
© After 7 weeks.
4 These results were obtained from the same individuals who received the 2.55-mg. intake. After 62 weeks
on this intake, their supplementation was further increased to provide a 3.55-mg. intake. The values for the
24- and 4-hr. excretion periods were obtained 114 and 3 weeks, respectively, after the change in supplementa-
tion.
be higher than normally expected. There is a high degree of agreement in
the results of excretion studies by different laboratories of subjects on low
levels of riboflavin intake,**: *7- but the variations are greater when the
55 H. Oldham, E. Lounds, and T. Porter, J. Nutrition 34, 69 (1947).
°6 H. Pollack and J. J. Bookman, J. Lab. Clin. Med. 38, 561 (1951).
57 C. W. Denko, W. E. Grundy, N. C. Wheeler, C. R. Henderson, G. H. Berryman,
T. E. Friedemann, and J. B. Youmans, Arch. Biochem. 11, 109 (1946).
88 R.D. Williams, H. L. Mason, P. L. Cusick, and R. M. Wilder, J. Nutrition 25, 361
(1943).
59 A. Keys, A. F. Henschel, O. Mickelsen, J. H. Brozek, and J. H. Crawford, J. Nutri-
ton 27, 165 (1944).
60 V.A. Najjar and L. E. Holt, Bull. Johns Hopkins Hosp. 69, 476 (1941).
402 RIBOFLAVIN
test dose or daily intake is more than | mg.‘*: %-6 Table IV presents a
summary of the urinary excretions obtained by the Elgin group*® at differ-
ent levels of dietary intake.
51H. G. Oldham, F. A. Johnston, 8. C. Kleiger, and H. Hedderich-Arismandi, J.
Nutrition 27, 435 (1944).
6 M. V. Davis, H. G. Oldham, and L. J. Roberts, J. Nutrition 32, 143 (1946).
68 A. K. Axelrod, T. D. Spies, C. A. Elvehjem, and V. Axelrod, J. Clin. Invest. 20,
229 (1941).
64 F. T. Lossy, G. A. Goldsmith, and H. P. Sarett, J. Nutrition 45, 213 (1951).
65 C. A. Conners, R. E. Eckardt, and L. V. Johnson, Arch. Ophthalmol. 29, 956 (1943).
6 R. EK. Johnson, C. Henderson, P. F. Robinson, and F. C. Consolazio, J. Nutrition
30, 89 (1945).
CHAPTER 16
THIAMINE
I. Nomenclature.
IIT.
IV.
Watt:
IX.
. Chemistry .
A. Isolation :
B. Chemical and Phy Seal Beanentice ,
C. Constitution .
D. Synthesis .
E. Specificity
Industrial Preparation
A. From Natural Sources .
1. Low Concentrates from Rice Beank or Polen :
2. Low Concentrates from Yeast
3. High Concentrates from Rice Germs . ;
4. Isolation of Crystalline Thiamine from vatural Sorirres ;
. Biosynthesis of Thiamine in Yeast .
. Synthesis .
. Patent Situation :
Commercial Forms and Baniey
. Production and Prices
ekerical Systems .
A. Enzymes ;
1. The Unitarian Wey
Seo daw
2. Coupling of Pyruvie Acid Ondation aa Ehaconarilaiine :
3. Inhibitors and Activators .
B. Coenzymes é :
1. Chemical roca ;
2. Enzymic Synthesis
. Specificity of Action
A. The Essential Metals
B. Thiamine Pyrophosphate
C. Substrate Specificity .
. Biogenesis .
Pit.
Estimation.
A. Physical
B. Chemical .
C. Biological .
D. Fermentation
E. Microbiological .
Standardization of Activity
Occurrence in Food iF é
A. Occurrence of Thiamine and iis Phosphate Cc ppennde ;
B. Occurrence of Natural Antithiamines and Thiaminases in Roads E
403
Page
404
404
404
406
406
406
408
409
409
409
410
410
410
411
411
424
425
425
426
426
433
434
436
437
438
438
442
442
442
443
445
448
448
448
450
450
451
453
454
454
458
404 THIAMINE
Page
Xx -Effects:of Deficiency 5.2. « § 9. 5 cts wo. Se ce
A> Im-Microorganisms: . §: < 3 « «'« « = 4 4 a 2 ) Seen
B= In*Animals. 0.0 a0 we hae, Be Se Oe
Cine Mian 3-5, 9.3 es nays Sch Me, Oe ieee et Seen ree
XS Pharmacology: 2.4 yee eo 4 ee
XII. Requirements and Factors Influencing Them : . . . | +.) eae
A. Of Animalse .0. 2 214 fee Coe Saas Se bo Re
IB. OF Mane V6.0. 9 ho a en be” 8 Sas Se
I. Nomenclature
ROBERT 8S. HARRIS
Accepted name: Thiamine (U.S. A.)
Aneurin (Brit. Pharm.)
Obsolete names: Vitamin B,
Oryzamin
Torulin
Polyneuramin
Vitamin F
Antineuritic vitamin
Antiberiberi vitamin
Empirical formula: Ci2Hi;NsOSCl- HCl
Chemical name: 3-(4-Amino-2-methylpyrimidyl-5-methyl)-4-methyl-5-6-
hydroethylthiazolium chloride hydrochloride
Structure:
HCl CH;
N==C—NH, Cz—3C—CH;
B él Yee |
H;C—C C—CH.—N3
Ih ol Ne
N—CH (0) a
CH,0H
II. Chemistry
B. C. P. JANSEN
A. ISOLATION
After Eijkman’s discovery that polished rice was the cause of polyneu-
ritis in birds and of beriberi in men, Grijns,! his successor in Batavia, was
1G. Grijns, Geneesk. Tijdschr. Ned. Indié 41, 3 (1901). (Dutch; afterwards trans-
Il. CHEMISTRY 405
the first to state that these diseases were the result of a “partial hunger,”
a deficiency of some unknown substance that is present in very small quan-
tities in the outer layers (the silverskin) of the rice. Grijns called this sub-
stance “the protective substance”; Grijns also made the first attempts to
isolate this substance from an extract from the silverskin of the rice. He
succeeded in getting highly active fractions, but did not obtain a pure sub-
stance. About ten years later Casimir Funk, working in the Lister Insti-
tute in London, obtained a crystalline substance from rice polishings.? He
coined the word “‘vitamine’’—an amine essential for life—for it. It was
proved afterwards however, that this product had no antineuritic activity,
and consequently it was not the desired substance. As it was shown sub-
sequently that several ‘“‘vitamines’’ were not amines at all, Drummond?
proposed that the final ‘‘e” of the name ‘‘vitamine’’ be dropped.
In 1926 Jansen and aa working in the same laboratory where
Eijkman and Grijns had made their researches, obtained the crystalline
substance having a great antineuritic activity. They sent 40 mg. of it to
Eijkman, who was at that time in the Netherlands. Eijkman was able to
confirm the prophylactic and curative activities of this substance against
avian polyneuritis.° Eijkman confessed that, before he received these crys-
tals, he had doubted if the vitamin would be a normal chemical single sub-
stance. Jansen and Donath succeeded in the isolation because they used
the finding of Seidell® that the antineuritic substance is adsorbed by fuller’s
earth, and furthermore they found a fairly quick method (i.e., 10 days)
for testing the antineuritic activity of the different fractions by ine use of
small rice birds (Munia maja).
About ten years later several workers in the United States, Germany,
and England improved the method of isolation and thereby obtained suffi-
cient quantities to establish its structural formula and to find methods for
its synthesis (Williams and Cline,’ Grewe,’ Andersag and Westphal,’ and
Todd and Bergel.!”)
lated into English: Prof. Dr. G. Grijns: Researches on Vitamins 1900-1911. J.
Noorduijn en Zoon, Gorinchem, 1935.)
2C. Funk, J. Physiol. (eadon) 43, 395 (1911); Die Vitamine. J. F. Bergmann,
Miinchen and Wiesbaden, 1922.
8 J.C. Drummond, Biochem. J. 14, 660 (1920).
*B.C.P. Jansen and W. F. Donath, Proc. Roy. Acad. Sci. Amsterdam 29, 1390-1400
(1926).
§C. Eijkman, Proc. Roy. Acad. Sci. Amsterdam 80, 376 (1927).
8A. Seidell, Public Health Repts. (U. S.) 31, 364 (1916).
7R. R. Williams and J. K. Cline, J. Am. Chem. Soc. 58, 1504 (1936).
5 R. Grewe, Z. physiol. Chem. 242, 89 (1936).
*H. Andersag and K. Westphal, Ber. 70, 2035 (1937).
1” A. R. Todd and F. Bergel, J. Chem. Soc. 1937, 364.
406 THIAMINE
B. CHEMICAL AND PHYSICAL PROPERTIES
Thiamine hydrochloride crystallizes into colorless, monoclinic needles,
which have a melting point of about 250°, a characteristic smell, anda slightly
bitter taste. These crystals are stable to the atmospheric oxygen. They are
very soluble in water, much less so in alcohol, and insoluble in ether, and
other fat solvents. Thiamine chloride hydrochloride crystallizes from alco-
holic aqueous solutions as the hemihydrate, Ci2Hi7ON.SCI-HCl-44H.0.
Thiamine hydrochloride in water forms a strongly acid solution pH of a
5 % solution about 3.5; in solution with a pH less than 5 it is fairly stable
to heat and oxidation; this solution shows two absorption bands in the ultra-
violet at 235 and 267 mu.
At a pH of 5 or higher, it is destroyed by autoclaving, and at a pH of 7
or more by boiling or merely storing at room temperature.
By treatment with sulfite it is readily split into the pyrimidine and thia-
zole parts.
In a highly alkaline solution thiamine is oxidized by ferricyanide to thio-
chrome (cf. Section VI).
C. CONSTITUTION
The work of the above-mentioned investigators has shown that the thia-
mine molecule consists of a pyrimidine compound and a thiazole compound,
connected by a CH, bridge. The structural formula of the thiamine hydro-
chloride is:
H CH; CH,-CH,OH
| |
C Ci 5C
Yi 4
NT Cee Oo ——N3
> all IN
CH;—C CNH.—HCl Cl C2— 8
Ni |
N H
The elucidation of the constitution was greatly relieved by the discovery
of Williams et al." that thiamine is quantitatively split by sulfite in faintly
acid solutions into the pyrimidine and the thiazole halves:
Cy2Hy30N4SCl. “f NassO; => C.sH oN 3803 o CsHsNSO + 2NaCl
D. SYNTHESIS
The synthesis of thiamine has been performed in different ways. It is
possible to synthesize the pyrimidine nucleus and the thiazole nucleus sepa-
11 R. R. Williams, E. R. Buchman, and A. E. Ruehle, J. Am. Chem. Soc. 57, 536
(1935).
II. CHEMISTRY 407
rately and afterwards to connect both parts. It is also possible to synthesize
one of the nuclei with an extra side branch and afterwards to build up the
other ring from this side branch.
For synthesizing the pyrimidine part, ethyl formate and 6-ethoxyethyl-
propionate may be condensed with Na.
H-COOC:H; + CH:0C:H;-CH.-COOC.H; + Na — NaOCH
C—CH,0C.H;
|
COOC.H;
This product is condensed with acetamidine.
HC—ONa NH: N=C
| pew
C—CH.OC.H; cL C—CH; ar CH;:-C C—CH.OC.H;
COOC.H; NH N=-CH
The hydroxy] group is converted into an NH: group by treating first with
POCI; and then with NH3;. The ethoxy group is converted into a bromide.
Thus the final product is:
NH, : HBr
The thiazole moiety may be synthesized in several ways (cf. Buchman”
and Clarke and Gurin'’). The method of Buchman consists in condensing
thioformamide with bromoacetopropanol.
NH, N——C.-CH,
| ! !
C—H + CH;-CO-CHBr.CH,-CH,0H —> HC C-CH,-CH,0H
S S
By heating the hydrobromide of the pyrimidine compound with the
thiazole compound, the thiamine hydrobromide is formed.
The bromide hydrobromide may be converted into the chloride hydro-
2 E.R. Buchman, J. Am. Chem. Soc. 58, 1803 (1936).
18H. T. Clarke and S. Gurin, J. Am. Chem. Soc. 57, 1876 (1935).
408 THIAMINE
NH,-HBr
NC 1 (Clo(CBla
|
CHC C-CHoBr + H—C C-CH.-CH,OH —
ee
N—CH S
NH,-HBr
iN——C Br
ee
wea C—CH.—N———_C -CH;
eel | |
N—CH lal C-CH,:-CH,0H
SY
Ss
chloride by treating with AgCl or by precipitating the practically insol-
uble thiamine picrate and disolving it in hydrochloric acid.
EK. SPECIFICITY
The activity of thiamine seems to be very specific. Even small alterations
in the molecule give inactive substances or diminish the activity 100 or
1000 times or actually produce antagonistic effects. Most instructive in this
respect is the work of Emerson and Southwick. They replaced the methyl
group in position 2 in the pyrimidine ring of thiamine by other alkyl groups.
Replacement by ethyl does not change the activity, as measured by rat
experiments; replacements by propyl gives a definite reduction of the ac-
tivity in pigeon tests. When the methyl is changed into an n-butyl group,
the activity 1s reversed. However, Schopfer!® and Schultz,!® some years
before the work of Emerson and Southwick, had established that a thiamine
having in the second position of the pyrimidine nucleus an ethyl in place
of a methyl group has a greater activity than normal thiamine on Phyco-
myces and on animals. Their relative activity is expressed by the ratio of
ethylthiamine to methylthiamine having the same physiological activity.
This ratio for Phycomyces was found to be 0.83:1.0 (Schopfer). The ratio
for the pigeon is 0.85:1.0 (Schultz). The discrepancies with the results of
Emerson and Southwick may be due to the inaccuracy of the animal experi-
ments.
Barton and Rogers, in the book of R. J. Williams et al.,” give a huge
14G, A. Emerson and P. L. Southwick, J. Biol. Chem. 160, 169 (1945).
15 W. H. Schopfer, Compt. rend. soc. phys. et hist. nat. Genéve pp. 58, 64 (1941).
16 Ff. Schultz, Z. physiol. Chem. 265, 113 (1940).
17 A.D. Barton and L. L. Rogers in R. J. Williams, R. E. Eakin, E. Beerstecher, Jr.,
and William Shive, The Biochemistry of B Vitamins, pp. 684-702. Reinhold Pub-
lishing Corp., New York, 1950.
III. INDUSTRIAL PREPARATION 409
number of examples of the influence of modifications in the pyrimidine or in
the thiazole moiety of thiamine on the biological activity of these thiamine
analogs. They arrive at the following conclusion: ‘‘as a result of these tests
it is evident that the thiamine molecule can undergo very little modifica-
tion without extensive loss of vitamin B, activity.”
Williams and Cline'® were able to establish that the synthetic thiamine
hydrochloride was identical with the natural in composition, ultraviolet ab-
sorption and antineuritic potency. Eckler and Chen!’ in elaborate pharma-
ecological studies compared the curative doses and the minimum lethal
doses of natural and synthetic thiamine. This work confirmed the identity
of both substances.
In several microorganisms the thiamine may be replaced by one or both
of its pyrimidine and thiazole moieties.?? Abderhalden?! has shown that
higher animals too could sustain on the thiazole + pyrimidine moieties
instead of thiamine itself. One would think that this might be brought about
by the phenomenon of “‘refection,”’ i.e., the synthesis of thiamine by the
microorganisms in the gut, as was found by Fridericia et al.22 But E.
Abderhalden and R. Abderhalden* stated that tissue extracts are capable
of synthesizing thiamine from the pyrimidine and thiazole parts—only to a
very small extent, however, up to about | % of the theoretical amount.
III. Industrial Preparation
H. M. WUEST
A. FROM NATURAL SOURCES
1. Low CoNCENTRATES FROM RICE BRAN OR POLISHINGS
Effective thiamine preparations go back as far as 1910, when Robert R.
Williams under the direction of E. B. Vedder prepared the first crude ex-
tracts from rice bran in Manila which were used in the treatment of beriberi
in the Philippine Islands.! The preparation, called tikitiki extract (or just
tikitiki, the Tagalog word for bran) is still used in the Philippines, and
similar preparations are sold in Japan.
In this country preparations of this kind were first made about 1930 by
18 R. R. Williams and J. K. Cline, J. Am. Chem. Soc. 59, 216 (1937).
19C. R. Eckler and K. K. Chen, Proc. Soc. Exptl. Biol. Med. 35, 458 (1937).
20 W. H. Schopfer, Ergeb. Biol. 16, 1 (1939).
21 R. Abderhalden, Pfliigers Arch. ges. Physiol. 243, 762 (1940).
2 |, S. Fridericia, P. Freudenthal, 8. Gudjonsson, G. Johansen, and N. Schoubye,
J. Hyg. 27, 70 (1928).
23. Abderhalden and R. Abderhalden, Pfliigers Arch. ges. Physiol. 248, 85 (1939).
1E. B. Vedder, Beriberi, p. 405. William Wood & Co., New York, 1913.
410 THIAMINE
the Vitab Corporation in Emeryville, California, with an activity up to 50
units per milliliter. Colman? and Arnold and Schreffler* improved the ex-
traction and the taste of the preparation. It is still used today: U. 8.
Pharmacopeia XIV (1950, p. 515) describes the extract with 0.06 mg. of
B, per milliliter.
2. Low CONCENTRATES FROM YEAST
By plasmolysis of yeast, coagulation of the protein material and con-
centration of the aqueous solution Light and Frey! developed a method of
preparing yeast concentrates with a thiamine content up to 300 units per
gram.
3. HicgH CONCENTRATES FROM RicE GERMS
A highly improved method for isolating crystalline thiamine from rice
polish was publishedin May 1934 by Williams e¢ al.° By using a richer source
than rice bran or polish, namely rice germs, F. Hoffmann-La Roche Ltd.
in Basle, Switzerland (F. Elger, A. J. Frey, and H. M. Wuest) developed a
method yielding concentrates with 25,000 to 30,000 units per gram. This
product was used in 1935-1937 for the multivitamin preparation Nestrovit
(vitamins A, B;, C, and D in a basis of dried skimmed milk, sugar, and
cocoa butter); 1200 to 1500 kg. of rice germs were worked up daily.
4. ISOLATION OF CRYSTALLINE THIAMINE FROM NATURAL SOURCES
The isolation of crystalline thiamine by Jansen and Donath®, by Windaus
et al.,’ and by Williams et al.° are today only of historical interest. Williams®
gives a good characterization of the efforts in his book: “It is doubtful
whether the isolation and identification of any other substance in the
history of biochemistry have cost as much labor as have these operations
as applied to thiamin. The first gram of the pure vitamin must have costan
ageregate of several hundred thousand dollars’ (written in 1988).
In spite of the improvements of the methods *" pure natural thiamine
2H. B. Coleman, U.S. Pat. 2,369,775 (February 20, 1945).
3A. Arnold and C. B. Schreffler, U.S. Pat. 2,390,679 (December 11, 1945).
*R. F. Light and C. N. Frey, U.S. Pat. 2,184,748 (December 26, 1939); British Pat.
428,044 (April 30, 1935).
5R.R. Williams, R. E. Waterman, and J. C. Keresztesy, J. Am. Chem. Soc. 66, 1187,
1189 (1934); U.S. Pat. 2,049,988 (August 4, 1936).
6° B.C. P. Jansen and W. F. Donath, Proc. Koninkl. Akad. Wetenschap. Amsterdam
29, 1390 (1926); Mededeel. Dienst Volksgezondheid, Ned.-Indié 16, 186 (1926).
7A. Windaus, R. Tschesche, H. Ruhkopf, F. Laquer, and F. Schultz, Nachr. Ges.
Wiss. Gottingen IIT p. 207 (1982).
§R. R. Williams, and T. D. Spies, Vitamin B; (Thiamin) and Its Use in Medicine
p. 138. The Maemillan Co., New York, 1988.
9A. Seidell, J. Biol. Chem. 82, 633 (1929).
Ill. INDUSTRIAL PREPARATION 411
could not compete with the synthetic product; its isolation on an industrial
scale never started.
B. BIOSYNTHESIS OF THIAMINE IN YEAST
Using selected trains of Saccharomyces cerevisiae Schultz et al. could show
that thiamine has a stimulating effect on the growth of these yeasts. The
two moieties of the thiamine, thiazole and pyrimidine, had a similar stimu-
lating effect, especially when used together.!® The authors developed their
observation to a technical process for the production of yeast with high
vitamin content.!® Such yeasts (with 200 units per gram, wet basis) were
sold by Standard Brands under the trade name Hi-Yeast for making en-
riched bread (1938-1943). In dry form the preparation is still used for
pharmaceutical purposes.
C. SYNTHESIS
If the isolation of thiamine from natural sources is interesting, the
history of the industrial synthesis is dramatic, as from 1935 three groups
were in the final race: in this country Williams et al.; in Germany Andersag
and Westphal in the laboratories of the I. G. Farbenindustrie in Elberfeld;
and Todd and Bergel in England. The scientific priority without any doubt
falls to Williams with his first publication of the complete synthesis in
August 1936.7 The claims of priority for Andersag and Westphal made by
H. Horlein'® were based on their earlier patent applications, which were
published much later. Todd and Bergel!’ published their synthesis about
eight months after Williams, only to find out that Andersag and Westphal’?
had already filed a German patent application for the same process more
than a year previously.
10H. W. Kinnersley, J. R. O’Brien, and R. A. Peters, Biochem. J. 27, 232 (1933); 29,
716 (1935).
17. R. Cerecedo and D. J. Hennessy, J. Am. Chem. Soc. 59, 1617 (1937); L. R. Cere-
cedo and F. J. Kaszuba, ibid. 59, 1619 (1937); L. R. Cerecedo and J. J. Thornton
ibid. 69, 1621 (1937); L. R. Cerecedo, U.S. Pat. 2,114,775 (April 19, 1938).
2 R.D. Greene and A. Black, J. Am. Chem. Soc. 59, 1395 (1937).
18 H. R. Rosenberg, Chemistry and Physiology of the Vitamins, p. 118. Interscience
Publishers, New York, 1942.
4A. S. Schultz, L. Atkin, and C. N. Frey, J. Am. Chem. Soc. 59, 948, 2457 (1937);
U.S. Pat. 2,249,789 (July 22, 1941).
16 AS. Schultz, L. Atkin, and C. N. Frey, J. Am. Chem. Soc. 60, 490 (1938).
16 Standard Brands, British Pats. 532,013 (January 15, 1941); 529,825 (September 25,
1941); A. S. Schultz, L. Atkins, and C. N. Frey, U.S. Pats. 2,262,735 (November
11, 1941); 2,233,956 (November 9, 1943) ; 2,354,281 (July 25, 1944).
7R.R. Williams and J. K. Cline, J. Am. Chem. Soc. 58, 1504 (1936)
18H. Horlein, Z. physiol. Chem. 253, 82 (1938).
19 AR. Todd and F. Bergel, J. Chem. Soc. 1937, 364.
20H. Andersag and K. Westphal, German Pat. 685,032 ex. 3 (filed January 29, 1936,
issued Nov. 16, 1939).
412 THIAMINE
Williams’ process grew in the hands of the chemists of Merck and Co.,
Rahway, New Jersey; the ring closure of Todd and Bergel was adopted
industrially by Hoffmann-La Roche in their three plants in Basle, Nutley,
New Jersey, and Welwyn, Herts. Andersag and Westphal’s method has
not yet reached the production in tons.
The established structure of thiamine, opens three possibilities for the
synthesis:
1. Building up the pyrimidine ring and the thiazole ring separately, then
connecting both by quaternization to the thiazolium ring.
2. Building up the pyrimidine ring with the group —CH»—NH, in the
5 position, elongating this side chain, and forming the second ring by ring
closure to the thiazolium compound.
3. Building up the thiazole ring, quaternization to thiazolium by elonga-
tion with a suitable side chain, and ring closure of the pyrimidine ring.
All three ways are described in literature. The first method is used by
Williams et al. and by Andersag and Westphal: as the two ring systems are
built up separately, the best conditions for the ring closure can be chosen,
namely, alkaline condensation for the pyrimidine and acid medium for
the thiazole nucleus. The quaternization goes easily and gives a nearly
theoretical yield, an important factor for the success of the method.
Todd and Bergel follow the second possibility: the elongation of the side
chain is done with the simple and elegant step of thioformylation of the
amine, the ring closure to the thiazolium being performed in an acid
medium, both reactions giving excellent yields.
The third possibility seems to have only slight chances, as pyrimidine
rings are best built up in an alkaline medium, a condition highly unfavor-
able for the thiazolium ring. This way is described for a thiamine homolog
in a French patent,” but no experimental data are given. There is no evi-
dence that the pyrimidine ring closure was performed.
The following discussion of the industrial synthesis is based on the
technical point of view, with special emphasis on the reported yields.
For practical reasons the molecule is divided into four groups.
A B C
1 ' '
leas ee ee
CH — CO) (6) Ce Ole Ny
i (1)! (6) (7) \ (2) QQ)
Cl =
217. G. Farbenindustrie, A. G., French Pat. 816,482, p. 5 (May 3, 1937).
Ill. INDUSTRIAL PREPARATION 413
A and B serve to build up the pyrimidine ring, C and D for the ring
closure to the thiazole or thiazolium nucleus. All three industrial syntheses
use these four groups as stepping stones to the end product; in Todd and
Bergel’s synthesis the nitrogen for the thiazolium ring switches to group
B which is then elongated to group D.
1. Group A, Two CARBON AND Two NITROGEN ATOMS
The natural starting material for the left half of the pyrimidine ring is
NH
Vi
acetamidine CH;—C , Which is easily available from acetonitrile in
~
NH,
two steps: the nitrile is converted to acetimino ether by ethanolic hydrogen
chloride in the absence of water, the ether being converted to acetamidine
hydrochloride with ethanolic ammonia. Yield is 80 to 91% in small
batches.” Under optimal conditions, the yield may be raised to 95%.
Instead of acetamidine the acetimino ethyl ether can be used if amino
methylene malonitrile® serves as group B. The use of thioacetamide and
amino methylene malonitrile’* does not offer special advantages. Acetoni-
trile, the starting material, can be obtained from dimethylsulfate and
sodium cyanide. In this country it is available as a commercial item.
2. Group B, Four Carson ATOMS AND ONE oR Two Amino Groups
(4)C
The carbon skeleton with four carbon atoms permits quite a number of
variations. The chain of the carbon atoms 4, 5, and 7 can be provided by
the esters of ethoxypropionic acid, succinic acid, malonic acid, and malo-
nitrile; carbon atoms 6 is added in most of the cases by a formyl group,
either as such or as the methylene ether. The substitution in 7 may lead
to a halogen or to an amino group (synthesis of Todd and Bergel’®).
a. Indirect Introduction of the Amino Group in the 4 Position
Cline ef al.2> start from ethoxypropionic ester (obtained from ethyl
acrylate by addition of ethanol with sodium ethylate) and convert it with
2 Organic Syntheses, Vol. 22, p. 5. John Wiley and Sons, New York, 1941.
230. Hromatka, Merck & Co., U.S. Pat. 2,235,638 (March 18, 1941); Merck & Co.,
German Pat. 667,990 (November 24, 1938); F. Hoffmann-La Roche and Co., A. G.,
British Pat. 486,414 (June 2, 1938).
*4F. Hoffmann-La Roche and Co., A. G., British Pat. 546,624 (July 22, 1942).
25 J. K. Cline, R. R. Williams, and J. Finkelstein, J. Am. Chem. Soc. 59, 1052 (1937).
414 THIAMINE
sodium and ethyl formate to sodioformylethoxypropionate, using the crude
CH.—CH—COOEt — EtO—CH.—CH.—COOEt
+ HCOOEt + Na — EtO—CH.—C: (CHONa)COOEt
sodium salt for the ring closure. In the form given by these authors the
method is not satisfactory, as formylation and ring closure (see under c)
give very poor yields.
Todd and Bergel!® formylate cyanoacetic ester in acetic anhydride with
ethyl orthoformate to ethoxymethylene cyanoacetic ester.2° No yield is
CN—C: (CHOEt)COOEt
given, but from the abundant work of Claisen it can be assumed that the
reaction goes well.
An alternative route of the same authors is slightly longer but offers
certain advantages at the ring closure. Here ethyl malonate is condensed
with ethyl orthoformate in the presence of zine chloride with a yield of
about 60 %, as shown by Claisen.””
EtOOC—C: (CHOEt)COOEt
In the method of Andersag and Westphal’? one more carbon atom is
added by starting from ethyl succinate to formyl ethyl succinate. The
yield of this formylation (60 to 70%) is satisfactory (Wislicenus ef al.?°).
EtOOC—CH.—CH (CHO)COOEt
b. Direct Introduction of the Amino Group in the 4 Position
The more reactive the methylene group in this series, the better the
yield at the formylation. Grewe*®® formylated malonitrile with orthoformie
ester in acetic anhydride to ethoxymethylene malonitrile
HC(OEt); + CN—CH.,—CN = CN—C:(CHOEt)CN + 2EtOH
As Diels et al.*! have shown, a yield of 75 % of ethoxymethylene malonitrile
can easily be obtained.
Starting material for malonitrile is chloroacetic acid which is converted
to ethyl cyanoacetate with 77 to 80 %.*° The next step, cyanoacetamide,
gives a yield of 86 to 88 %,** and the conversion of the amide to the nitrile
26 G. de Bollemont, Compt. rend. 128, 1340 (1899); Bull soc. chim. de Paris 25, 20
(1901).
27 T,, Claisen, Ann. 297, 76 (1897).
28 HT. Andersag and K. Westphal, Ber. 70, 2035, 2086 (1937).
29 W. Wislicenus, IH. B6klen, and F. Reuthe, Ann. 868, 347 (1908).
0 R. Grewe, Z. physiol. Chem. 242, 89 (1986).
310. Diels, H. Gartner, and R. Kaack, Ber. 35, 3141 (1922).
2 Organic Syntheses Vol. 1, p. 254. John Wiley and Sons, New York, 1941.
83 Organic Syntheses, Vol. 1, p. 179, John Wiley and Sons, New York, 1941.
Cr
Ill. INDUSTRIAL PREPARATION 41!
is performed with phosphorus pentachloride at 90°; the evolution of large
amounts of hydrogen chloride starts, and the malonitrile is distilled 7m vacuo
as soon as it is formed; yield 70% (Hesse™). Ethyl cyanoacetate is com-
mercially available in this country.
3. Rina CLOSURE TO THE PYRIMIDINE, A B
bp]
The ease with which the pyrimidine nucleus is formed varies consider-
ably and therefore also the yields. In the most favorable cases no condens-
ing agent is necessary, as the alkalinity of the free acetamidine is sufficient
to close the ring. In other cases sodium ethylate or sodium hydroxide is
used for the ring closure, and in one special case it even happens in neutral
or acid solution. Normally the formyl and cyano (or carbethoxy) groups
react at the same time, the ring closure taking place in one step. Todd and
Bergel!® have isolated as an intermediate an acrylo compound which could
be used for the ring closure in two different ways.
a. Indirect Introduction of the Amino Group in the 4 Position
Williams and Cline” reacted ethyl sodioformyl-6-ethoxypropionate with
acetamidine hydrochloride and sodium ethylate in ethanol, forming
2-methyl-4-hydroxy-5-ethoxymethylpyrimidine. The yield, as quoted by
these authors, is extremely poor, only 3.5%. If the process has been used
for technical purposes, it must have been radically improved.
By treatment with phosphorus oxychloride the hydroxyl group in the
4 position is replaced by chlorine (yield 70%), which is exchanged with
ethanolic ammonia under pressure by the amino groups (yield 70%)
finally the ethoxy group in the 7 position is replaced by bromine (hydrogen
bromide in glacial acetic acid, yield 90%). Starting from acetonitrile and
ethyl acrylate, this synthesis of the pyrimidine moiety requires eight steps.
With the exception of formylation and ring closure the yields are good and
the reactions show no special difficulty.
When Todd and Bergel!® reacted free acetamidine with ethyl ethoxy-
methylene cyanoacetate, they obtained colorless needles of ethyl a-cyano-
B-acetamidinoacrylate (yield 46 %), and all efforts to close the ring directly
at various temperatures and various amounts of sodium ethylate gave no
satisfactory results. Treatment of the intermediate by boiling with aqueous
2.5% sodium hydroxide closed the ring (yield 37%), giving a total yield
for the ring closure of only 17 %.
By replacement of the hydroxyl group with chlorine (60 to 70% yield)
and the amino group, 2-methyl-4-amino-5-cyanopyrimidine was obtained
with a yield of 40%. Finally the cyano compound was catalytically re-
4B. C. Hesse, Am. J. Chem. 18, 723 (1896).
416 THIAMINE
duced*®® to 2-methyl-4-amino-5-aminomethylpyrimidine, the last step in
this group for their synthesis.
Not satisfied with the results, Todd and Bergel found a much better
ring closure with ethyl ethoxymethylene malonate and acetamidine in
presence of 1 mole of sodium ethylate; the ring is closed witha yield of 60 %.
NH: COOEt N=C—OH
CH,;-C + C—COOEt — CH;—C o Coe
|
NH CHOEt NCH
After the replacement of the hydroxyl group by chlorine (yield 65%),
the 4-chloro compound is treated with concentrated aqueous ammonia at
room temperature.
The yield of amination is 65%, and the following dehydration of the
amide to the nitrile yields 50 %. The hydrogenation to the diamine is done
as above.
Starting from acetonitrile, ethyl cyanoacetate, or malonate and ethyl
orthoformate, both methods of Todd and Bergel have eight steps. The
yields of the second method were promising, but both methods were aban-
doned when still better ways became available.
Andersag and Westphal’s procedure® is interesting in that they could
perform a Hofmann degradation in the pyrimidine series, a reaction which
the American and British workers had tried without success. The condensa-
tion of acetamidine with ethyl formyl succinate in hot alcoholic solution
gives ethyl 2-methyl-4-hydroxypyrimidyl[5]acetic ester, which is converted
via the 4-chloro compound to 2-methyl-4-aminopyrimidyl|[5]acetamide.
This amide is treated with an ice-cold aqueous solution of bromine in 2.5
N potassium hydroxide and heated, yielding 2-methyl-4-amino-5-amino-
methylpyrimidine in a nearly quantitative yield. The diamide dihydro-
chloride is then treated with a hot solution of sodium nitrite to remove the
amino group in the 7 position, and the resulting 2-methyl-4-amino-5-hy-
hydroxymethylpyrimidine is converted with hydrogen bromide in acetic
acid to 2-methyl-4-amino-5-bromomethylpyrimidine dihydrobromide. As
with the exception of the Hofmann degradation no yields are given for the
nine different steps, it is not possible to evaluate the method. It seems that
the I. G. Farbenindustrie has not used it technically.
b. Direct Introduction of the Amino Group in the 4 Position
If ethyl a-cyano-$-acetamidinoacrylate (Todd and Bergel!®) is not treated
with alkali but heated in water (with or without a slight amount of acid),
the ring closes in a different way, the carbon atom of the cyano group enter-
ing the ring and leaving the carbothoxy group untou ched.
III. INDUSTRIAL PREPARATION 417
2-Methyl-4-aminopyrimidyl[5|carbonic acid is formed*®> with a yield of
65 to 75%. By transformation of the carbethoxy group to the amide and
eyano group the way to the diamine is opened. It is remarkable that the
neutral or acid ring closure goes so well. The drawback of the method is the
low yield in the formation of the acrylic acid (46 %, see above).
The best manner of the ring closure with direct introduction of the amino
group was first described by Grewe.*® When the alcoholic solutions of free
acetamidine and ethoxymethylene malonitrile are united, the mixture
solidifies at once under formation ef the crystalline cyano compound.
NH CHOEt N—CH
Grewe himself gives no yield; in the laboratories of Elberfeld*® a yield of
69 % was obtained.
Ethoxymethylene malonitrile can be quantitatively converted to amino-
methylene malonitrile;*! the condensation with acetimino ethyl! ether gives
the best yield for the pyrimidine ring closure reported in literature,”
NH CN N=C—NH,
! | ee
CH—C 8+ C—CN — CH;—C C—CN + EOH
| | |
OEt H.N—CH N—CH
namely 88%. The resulting 2-methyl-4-amino-5-cyanopyrimidine was
catalytically hydrogenated by Grewe*® with palladium charcoal as a catalyst
and glacial acetic acid with dry hydrogen chloride as a solvent. No yield is
given by Grewe, but it can be assumed from similar hydrogenations of
nitriles that it is very good.
Glacial acetic acid in the presence of hydrogen chloride is not a very
pleasant medium for high-pressure hydrogenation as it corrodes even
normally acid-resistant metals considerably. The difficulty can be overcome
by specially constructed autoclaves.
4. Group C, Five CarBon ATOMS FOR THE THIAZOLE RING
The possibilities for the synthesis of the thiazole ring are nearly as
numerous as those of the pyrimidine ring, and the different groups in the
three countries together with industrial laboratories have developed more
than half a dozen ways for the five carbon atoms.
35 Chinoin, Budapest, British Pat. 538,743 (August 14, 1941).
36 T. G. Farben, Werk Elberfeld, technical directions for Bi, as of July 1, 1943. Micro-
film FD 20/46, Docket No. 63,986. H. M. Stationary Office, Photogr. Section.
418 THIAMINE
The normal way of builing up a thiazole ring starts with a-halogenocar-
bonyl compounds, and all practical syntheses for the 4-methyl-5-6-hy-
droxyethyl thiazole use the pentanone chain, where X is a halogen, and R
CH;—CO—CHX—CH.—CH>2R
is a hydroxyl, ethoxyl, O-acyl, or halogen. As the final thiazole derivative
requires a free 8-hydroxyethyl group and the exchange of an ethoxyl by
hydroxyl is not too easy, it 1s obvious that compounds with the free hy-
droxyl or O-acyl are superior; the use, of y-aceto-y-chloropropylethy] ether
as described by Clarke and Gurin*’ as early as 1935 is therefore out of the
question for technical purposes.
If y-acetopropylacetate is brominated in ether, bromine enters mainly
in the y-position and y-aceto-y-bromopropylacetate is obtained (Andersag
and Westphal*®*). The bromination can be done under very mild conditions,
CH;—CO—CHBr—CH:—CH,0COCH;
i.e., in glacial acetic acid with a mixture of bromine and pyridine hydro-
chloride.*® Whatever the conditions of direct halogenation with chlorine or
bromine, the possibility of substitution on other carbon atoms of the chain
is given and any purification of these mixtures of halogenated ketones is
difficult.
If a second carbonyl is present at carbon atom 3, however, the chlorina-
tion can be performed with sulfuryl chloride and only substitution in 3
position takes place. Buchman*® has shown that a-acetobutyrolactone can
be chlorinated with sulfuryl chloride to a-chloro-a-acetobutyrolactone with
a very good yield (83 %). When this lactone was decarboxylated by boiling
with concentrated hydrochloric acid, Buchman expected y-chloro-y-aceto-
propyl alcohol and reported a yield of 73%. As Stevens and Stein*? have
CH,;—CO—CHCI—CH:—CH:0H or CH;—C(OH)—CHCI—CH:—CH:—O
|
shown, only a small part of the reaction product is the free aleohol; by far
the larger part forms an ether from 2 moles of the expected alcohol by the
elimination of 1 mole of water. The yield was about 13 % alcohol and 62%
CHCl— ©CO—.CH;
CH:
OCH:
|
|
CH;—C—CHCI—CH,—CH:—0
| |
37 H. T. Clarke and 8. Gurin, J. Am. Chem. Soc. 57, 1876 (1935).
38 Roche Products, F. Bergel, and A. Cohen, British Pat. 554,428 (July 2, 1948);
Xoche Products and F. Bergel, British Pat. 550,197 (December 29, 1942).
39. R. Buchman, J. Am. Chem. Soc. 58, 1803 (1936).
40 J. R. Stevens and G. A. Stein, J. Am. Chem. Soc. 62, 1045 (1940).
Ill. INDUSTRIAL PREPARATION 419
ether, totaling 75%. Both compounds can be used for the synthesis of the
thiazole, but the chlorine in the free alcohol is much more reactive than
the chlorine in the ether. With the bromo compound, the alcohol is even
less stable in its free form and converts spontaneously to its ether. The
same authors found that a boiling solution of 1% HCl is more satisfactory
than the concentrated acid.
If the saponification and decarboxylation are done with hydrochloric
acid in glacial acetic acid with just the calculated amount of water and
acetic anhydride is added after the decarboxylation, excellent yields of
y-chloro-y-acetopropylacetate are obtained (93 to 95%, Low and Smith*').
The use of the acetate was already proposed by Todd et al.,‘? but the
low yield in the first step of their process (sodium ethyl acetoacetate and
8-bromoethyl acetate, 25%) was not encouraging.
The saponification of a-chloro-a-acetobutyrolactone with sulfuric acid
in alcohol at 40 to 50° leads to the formation of a ring ketal, namely 2-
methyl-2-ethoxy-3-chlorotetrahydrofurane in a yield of 70 % (Klingenfuss**)
CH,:——CHCl
| |
CH, C—CH;
SS OR oa
O OEt
These ketals do not react with ketone reagents (semicarbazide), but their
chlorine is reactive and they can be used for the synthesis of the thiazole
ring.
The starting material for Buchman’s method, a-aceto-y-butyrolactone,
is easily obtainable* from sodium ethyl acetoacetate in alcohol and ethylene
oxide, with a yield of 60%. Both raw materials, ethyl acetoacetate and
ethylene oxide, are commercial items. Starting from them, only three steps
are required to the necessary 3-halogenopentanolone|4]; the yields of these
steps are good to excellent.
5. Group D, NITROGEN, CARBON ATOM 2, AND SULFUR
a. For Closing the Thiazole Ring
Thioformamide, NH,—CH=S, is the easiest source for the remaining
three atoms of the thiazole ring. Willstitter and Wirth*® reacted formamide
“| Roche Products, J. A. Low, and R. J. Smith, British Pat. 606,026 (August 5, 1948).
#2 A. Todd, F. Bergel, and A. Jacob, J. Chem. Soc. 1936, 1555.
48 M. Klingenfuss, U.S. Pat. 2,123,653 (July 12, 1938); British Pat. 496,801 (December
6, 1938).
447. L. Knunyantz, G. W. Chelintzew, and E. D. Ossetrava, Compt. rend. Acad. Sci.
U.R.S.S.1, 312 (1934) [C. A. 28, 4382 (1934)].
46 R. Willstatter and T. Wirth, Ber. 42, 1911 (1909).
420 THIAMINE
in ethereal solution under cooling with phosphorus pentasulfide and got the
crude product with a yield of 40 to 45 %. As thioformamide is very unstable
in crystalline state, only a concentrate is prepared for technical purposes.
Gabriel improved the method a little by using larger amounts of phosphorus
pentasulfide,*® but even then the crude product contained only 36% of
sulfur (theory 52.5%). Most of the workers in the thiamine field have use
the Willstétter-Gabriel method.
Todd et al.47 claim a superior way by reacting free dithioformic acid in
ether with aqueous ammonia. A yield as high as 50 % is claimed in a United
States patent.”
CHSSH + 2NH; = CHSNH, + NH,SH
Dithioformic acid is technically available,‘® but it is not known if the
method is used for the production of thioformamide.
b. For Closing the Thiazolium Ring
If, instead of thioformamide, compounds with a substituted amino group
are used, thiazolium salts are obtained instead of thiazole. The compound
necessary for this kind of thiamine synthesis, 2-methyl-4-amino-5-thio-
formylaminomethylpyrimidine, can be obtained via formylation of 2-
methyl-4-amino-5-aminomethylpyrimidine; as has been known for a long
time,°° the 4-amino group remains untouched and only the 5-formylamino-
methyl compound is formed. This in turn can be transformed to the thio-
formylaminomethyl by means of phosphorus pentasulfide,** but the yields
of this step are extremely poor.
It was one of the greatest improvements in the synthesis of thiamine
when Todd et al. found a simple method for the thioformylation of amines.‘
Dithioformic acid is obtained as potassium salt when potassium sulfide
(from potassium hydroxide and hydrogen sulfide) in alcoholic solution is
HCCl; + 2K,8 = HCSSK + 38KCl
reacted with chloroform.’® The yield in the hands of the British workers
was about 45 % in the form of recrystallized salt.
46 S$. Gabriel, Ber. 49, 1115 (1916).
47 AR. Todd, F. Bergel, and Karimullah, Ber. 69, 217 (1936); A. R. Todd, F. Bergel,
Karimullah, and R. Keller, J. Chem. Soc. 1937, 361.
48 M. Hoffer, U.S. Pat. 2,220,243, ex. 4 (November 5, 1940).
49 Levi, Atti reale accad. naz. Lincet 32, 569 (1923).
50 §, Gabriel and J. Colman, Ber. 34, 1246 (1901); C.O. Johns, Am. Chem. J. 41, 58 (1908).
III. INDUSTRIAL PREPARATION 421
Amines of any kind react with aqueous or alcoholic solutions of dithio-
RNH, + HCSSK = RNH—CHS + KSH
formates under very mild conditions. In the case of aniline or aminoquino-
line the yield is quantitative; no yields are given for 2-methyl-4-amino-5-
aminomethylpyrimidine, but by analogy it must be assumed that the
yield is good. As sodium dithioformate is technically easily available with
good yields, this process offers a minimum of inconvenience for part 3 of
the synthesis of thiamine. The bad odor of unwanted sulfur compounds in
the reaction can be overcome by suitable engineering (closed reaction
vessels, centrifuges, and good ventilation of the working rooms).
6. C + D, Rina CLosvureE To THE THIAZOLE RING
The ring closure between this formamide and the halogenated ketones
takes place easily (e.g., mixing the two components in the presence of a
small amount of alcohol and keeping the mixture for three days at room
temperature), but the yields were not too good at the beginning. Buchman
got 50% 4-methyl-5-6-hydroxyethyl thiazole,*® about the same yield as
Clarke and Gurin® obtained for the 5-8-ethoxy ethyl compounds. Using the
pure “ether” instead of the alcohol, Stevens and Stein®! could increase the
yield to 70%. It can be assumed that the condensation of y-chloro-y-
acetopropylacetate with thioformamide gives still better results.
There was no lack of experiments to replace the thioformamide by raw
materials of easier accessibility. Ammonium dithiocarbaminate (from am-
monia and carbon dilsufide) condenses with the halogenated ketone to
2-mercapto-4-methyl-5-6-acetoxyethyl thiazole (excllent yields are
claimed, but no figures are given); Spiegelberg®? has shown that the mer-
captol group can be replaced by hydrogen when it is oxidized with 30%
hydrogen peroxide in acid solution to the sulfinic acid. Sulfur dioxide is
split off and oxidized by more hydrogen peroxide to sulfuric acid. The yield
is more than 75%; in the case of 2-mercapto-4-methyl-5-6-chloroethy]
thiazole it is 87 %. Even with the relatively high price of the 30 % hydro-
gen peroxide, the process seems promising for technical use.
7. Linkinc THE Two RING SystTEMs, QUATERNIZATION OF THE
THIAZOLE TO THIAZOLIUM
Linking the two ring systems together (the final step of the thiamine
synthesis) offers little difficulty, especially when the pyrimidine compound
with bromine in the 7 position is used.
J. R. Stevens and G. A. Stein, J. Am. Chem. Soc. 62, 1046 (1940).
*® H. Spiegelberg, British Pat. 492,637 (September 23, 1938); U. S. Pat. 2,179,984
(November 14, 1939).
422 THIAMINE
N= CONE HBr CHs oy.CH.0H
I rae
N—=C—NH-HBr CEs CH.—CH.OH
|
CH:—C C—CH:—N.
| | lraee
INS CE Br
Willams et al.”® did the first reported experiment with 150 mg. of 2-
methyl-5-bromomethyl-6-aminopyrimidine hydrobromide and got a yield
of 45% when they heated the bromo compound with 4-methyl-5-6-hy-
droxyethyl thiazole in the presence of 0.2 ml. of butanol for 15 minutes at
120°. Stein et al.®® got 67% for the 2-ethyl homolog of thiamine. If light
petrolatum is used as the medium and the heating is done under optimal
conditions, a nearly quantitative yield may be obtained. The petrolatum
is removed after the reaction with ether, and the crystalline product is
ready for the conversion to the chloride hydrochloride (thiamine). Andersag
and Westphal*> follow the same procedure (no solvent, 30 minutes at 120
to 180°), but no yield is given in their paper.
To replace the bromine ions by chlorine ions, Williams et al. use an
aqueous suspension of silver chloride and shake the bromide solution with
it for a half-hour. The ion exchange is quantitative, and no losses of thi-
amine occur at this step. After filtration of the silver bromide, the aqueous
solution of thiamine is concentrated to a small volume and alcohol is added;
thiamine crystallizes in the form of needles. After one or two crystalliza-
tions from 90 % ethanol the crystals are dried and ready for delivery. From
the silver bromide the silver may be recovered by reduction with zine dust
in aqueous suspension and the excess zine removed by diluted acetic acid.
The carefully washed silver is then ready for conversion into silver nitrate
and in turn to silver chloride. The bromide hydrobromide can also be pre-
cipitated with an aqueous solution of picric acid and the crystalline thi-
amine picrate decomposed with 10% hydrochloric acid.*t The picric acid is
removed by filtration and the small amount in solution by extraction with
ether. The process does not offer any advantages which would justify the
hazard of picric acid and an ether extraction.
The conversion can be avoided if 2-methyl-5-chloromethylpyrimidine
hydrochloride is reacted with the thiazole. The laboratories of the I. G.
53 G. A. Stein, W. L. Sampson, J. K. Cline and J. R. Stevens, J. Am. Chem. Soc. 63, 2061
(1941).
‘41. Hoffmann-La Roche and Co., Ltd., Basle, Swiss Pat. 197,717 (August 1, 1938).
Ill. INDUSTRIAL PREPARATION 423
Farbenindustrie** used this process in 1943 (methyl acetamide as a solvent
heating 1 hour at 115°), but the yield of pure thiamine in batches of 3 kg.
was only 45%. The use of the bromo compound for the quaternization
followed by the ion exchange to the chloride hydrochloride seems to be
much superior.
8. THrazotium RinG CuLosurE TO THIAMINE
The model experiments of Todd et al.” for the thiazolium ring closure
e.g., N-phenylthioacetamide and chloroacetone (without a solvent at 80°),
gave quantitative yields. But when they heated y-chloro-y-acetopropanol
with 2-methyl-5-thioformylamine pyrimidine in dioxane (b.p. 101°), n
thiamine could be obtained; heating at 140° without a solvent gave con-
siderable resinification, darkening, and only a small yield. The results were
much better when y-chloro-y-acetopropylacetate was used; after heating
at 115 to 120° for a few minutes, the ring closed in a smooth reaction.
N=C—NH; CH, CH:—CH.OCOCH;
wer!
SF ‘ CH:—NHCHS + CO—CHCI =
N—CH
thiamine monochloride + CH;COOH
At this temperature the acetyl group was saponified and thiamine (as
chloride) was obtained directly. No indication regarding the yield is given.
By using y-bromo-y-acetopropylbenzoate, Andersag and Westphal” could
lower the temperature for the ring closure to 100°, but here again figures
for the yield are lacking. Roche Products (Welwyn Garden City, Herts)
uses y-bromo-y-acetopropylacetate for their actual manufacturing process.*®
The reaction between 2-methyl-2-ethoxy-3-chlorotetrahydrofurane and
the thio compound, as described by Klingenfuss,°* used even milder condi-
tions: 95 % formic acid or 80% acetic acid as a solvent and reaction tem-
peratures of 40 to 50° (10 to 20 hours). If calcium bromide was added to the
reaction mixture, a yield of 54% of thiamine was obtained.
A similar yield (57%) is obtained when the thio compound is reacted
with a mixture of 2-methyl-2,3-dichlorotetrahydrofurane in formic acid
with pyridine; the pyridine splits off hydrogen chloride, and the inter-
mediate reacts under ring closure. It is obvious that this process®? was de-
veloped to circumvent existing patents, but it does not offer any advan-
tages.
§§ A Factory for Vitamin B,; Production, Ind. Chemist, June 1947, 359-68.
86M. Klingenfuss, U.S. Pat. 2,127,446 (nigast 16, 1938).
57 Chinoin, Budapest, British Pat. 609,803 (October 7, 1948).
424 THIAMINE
A Japanese circumvention process is even more clumsy: it starts from
2-methyl-4-amino-5-formylaminomethylpyrimidine and introduces a mer-
capto group into the pentanolone chain (7 ,y-diaceto-y-mercaptopropanol).
(CH;—CO). C(SH)CH2»—CH.OH
For the ring closure to thiamine (at 100 to 110° in formic acid containing
hydrogen chloride) a yield of 40 % is claimed.*8
Dy PATENT Sera TOM
As the patent laws vary from country to country, the patent situation
for every new important compound shows variations in different countries.
In the case of thiamine these variations are especially pronounced, as the
three groups mentioned above filed their basic patent applications within
a short time. Additional applications originated from the laboratories of
Roche in Basle and Welwyn, the Merck laboratories at Rahway and Darm-
stadt, and some outsiders.
In this country the Williams-Buchman group (assignors to Research
Corporation, New York) predominates and has a strong position on the
basis of composition of matter claims, protecting not only the important
intermediates of the pyrimidine and thiazole groups but also the halo-
genated pentanolone chain for the thiazole ring closure. It is interesting
that the product claims for 2-methyl-4-amino-5-aminomethylpyrimidine
and 4-methyl-5-6-ethoxythiazole of Williams are based not on their syn-
thesis but on the degradation of thiamine, a procedure which has no
value for building up the end product but which was essential for the
knowledge of the structure and formed the basis of the subsequent synthesis
of these intermediates. In Germany the I. G. Farbenindustrie has the
strongest position on the basis of the patents of Andersag and Westphal
which try to claim all possible ways, including such remote possibilities as
the fluoro compounds as halogenated intermediates. These inventors were
also successful in getting patent protection for the Todd and Bergel process
in Germany, Great Britain, and the United States, as their patent applica-
tion for this process was filed in Germany as early as January 29, 1936. The |
strong patent position of Hoffmann-La Roche is based on their claims for
the thioformylation of amines and the ring closure to the thiazolium ring.
In the United States the whole field from the first patented intermediates _
to the end product is covered by about 35 patents. The first application —
for the end product of Williams and Cline was filed June 15, 1936, whereas —
Andersag and Westphal had filed their first German application for the |
same step on January 29 of the same year (but with the correct position _
of the alkyl groups only as of March 24, 1936 as shown by the Brit. Pat. |
58 T, Matukawa and M. Ohta, U.S. Pat. 2,184,720 (December 26, 1939).
= aq
IV. BIOCHEMICAL SYSTEMS 425
Spec. 471416). The priority of the German inventors under the patent
convention is not contested.
The first United States patent granted for the synthesis of thiamine fell
to Klingenfuss (August 16, 1938), whereas the basic patent of Williams and
Cline was issued only five years later (September 7, 1943).
The circumvention patents of the outsiders did not lead to the industrial
use; no thiamine produced on the basis of such processes has appeared on the
market (1953).
EK. COMMERCIAL FORMS AND PURITY
Thiamine is sold commercially in the form of two salts, the hydrochloride
and the mononitrate. The hydrochloride is official in most of the countries;
U.S. Pharmacopoeia XIV (1950, p. 621) and British Pharmacopoeia (1953,
p. 42) describe the product in details and the requirements of purity. An
especially pure product is sold for the preparation of ampuled solutions.
The mononitrate is not hygroscopic and therefore is preferred in the food
industry. Its preparation is described by a Canadian patent;°? for the salt
itself see U.S.P. XIV, third supplement, p. 11. Its pharmaceutical behavior
was investigated by Macek et al.®°
-F. PRODUCTION AND PRICES
The United States, England, and Switzerland are the big producers of
thiamine. The following figures show the importance of this vitamin in the
ow. 8.%
production sales value price per kg.
lbs lbs $ }
1951 268 , 000 183,000 12,040,000 145.00
1952 224,000 145,000 9,574,000 146.00
The price for hydrochloride and mononitrate is the same; there is no
higher price for the special product for ampules. Since December 1952, the
price has remained stable at $135.00 per kg.
IV. Biochemical Systems
B. C. P. JANSEN
A. ENZYMES
Everyone who experiments on animals on thiamine-deficient diets is
struck by the fact that these animals lose weight every day, while their
59 EB. W. Schoeffel (to Merck and Co. Inc.), Canad. Pat. 469,559 (Nov. 21, 1950).
6° Thomas J. Macek, B. A. Feller, and E. J. Hanus, J. Am. Pharm. Assoc. Sci. Ed.
39, 365-69 (1950).
61 U.S. Tariff Commission, reports 175 (1951) and 190 (1952) (Synth. Org. Chemicals).
426 THIAMINE
eo
food intake is reduced to a low level. After they receive a small fraction of a —
milligram of thiamine, however, their appetite is restored at once and their —
weight increases enormously. This led to the supposition that the vitamin —
has something to do with cell metabolism. Several workers investigated the
influence of thiamine on the metabolic rate of tissues.
Westenbrink! has reviewed all the investigations published between 1920
and 1932 and has analyzed the data from fifteen publications on this sub-
ject. About half the workers give a positive, and the other half a negative,
answer. However, after statistically evaluating the work, Westenbrink
found that in only two of the fifteen investigations the metabolism of vita-
min-rich tissues was significantly higher than in vitamin-poor ones. He
himself could find no difference.? The definite answer to this question came
from the brilliant work of Peters and his school in Oxford. Peters realized
that the development of opisthotonus, which is a sign of the last stages of
polyneuritis in pigeons and fowls, proved that the disorder in thiamine-
deficient animals affects both the peripheral and the central nervous system.
In 1929 Peters and Kinnersley investigated the metabolism of polyneuritie
pigeons; the only abnormality they could find was a slight increase in the
amount of lactic acid in the brain of pigeons in opisthotonus. Peters also
studied the oxygen uptake by minced brain from pigeons in opisthotonus
in comparison with that of normal birds. The great difference from many
of his predecessors was that he added lactic acid (Kinnersley and Peters’)
or in later experiments sugar or pyruvic acid to the brain (Peters and Sin-
clair, Peters and Thompson,® Thompson,® and Peters’). In a long series of |
experiments Peters demonstrated that thiamine plays a decisive part in
carbohydrate, and especially in pyruvic acid, metabolism. Peters definitely
established the fact that the metabolism of brain from polyneuritic pigeons,
as measured by oxygen uptake in Barcroft-Warburg tubes, was less than
that of the brain of normal pigeons. Furthermore, Peters demonstrated that
the addition of a solution of thiamine to minced brain from polyneuriti¢
pigeons suspended in pyruvic acid solution increased the oxygen uptake
of this mixture (catatorulin effect of thiamine). This was the first instance _
of a chemical substance catalyzing an organ tissue preparation (Passmore
et al8). The work of the Oxford school was confirmed by Sherman and
Elvehjem.’ Further research led to the concept that the catalysis affecting
1H. G. K. Westenbrink, Arch. néerl. physiol. 17, 239 (1982).
2H. G. K. Westenbrink, Arch. néerl. physiol. 17, 549 (1932).
3H. W. Kinnersley and R. A. Peters, Biochem. J. 24, 711 (1930).
4R. A. Peters and H. M. Sinclair, Biochem. J. 27, 1910 (1983).
5R. A. Peters and R. H.S. Thompson, Biochem. J. 28, 916 (1934).
6R.H.S. Thompson, Biochem. J. 28, 909 (1934).
7R. A. Peters, Biochem. J. 30, 2206 (1936).
8 R. Passmore, R. A. Peters, and H. M. Sinclair, Biochem. J. 27, 842 (1933).
9 W.C. Sherman and C. A. Elvehjem, Am. J. Physiol. 117, 142 (1936).
IV. BIOCHEMICAL SYSTEMS 427
the oxygen uptake was due not to thiamine itself but to a compound that
was synthesized from the free vitamin (Peters et al.!° and Westenbrink and
Polak"). The nature of this compound became clear from another series of
investigations. Neuberg and his collaborators (Neuberg and Karezag! and
Neuberg and Rosenthal’*) found an enzyme in yeast which decarboxylates
pyruvic acid to acetaldehyde. They called it carboxylase. Simola' showed
that the tissues of rats on a thiamine-deficient diet had a greatly reduced
content of carboxylase.
All the known thiamine-containing enzymes (holoenzymes) consist of a
protein part that is usually called the apoenzyme and a coenzyme of lower
molecular weight, the thiamine pyrophosphate (or sometimes thiamine tri-
or polyphosphates). All these enzymes catalyze either a pure decarboxyla-
tion process or an oxidative decarboxylation reaction.
Both take part in the metabolism of pyruvic acid or, more generally
speaking, in the metabolism of ‘a-oxycarboxylic acids.
We distinguish between pure carboxylases and oxidative carboxylases,
which are usually called pyruvic dehydrogenases. The carboxylases are
present in yeast and other microorganism, whereas animal tissues contain
pyruvodehydrogenases.
Whether the carboxylase and the pyruvodehydrogenase are identical or
are different enzymes will be discussed below.
Auhagen!®: '® demonstrated that carboxylase could be rendered inactive
by washing with a phosphate buffer solution at pH 7 to 8. The activity was
restored by adding an extract of boiled yeast. So he assumed that yeast con-
tains a coenzyme, which he called cocarboxylase. Lohmann and Schuster!”
succeeded in isolating this cocarboxylase in a pure, crystalline state, and
they proved that it is the pyrophosphoric ester of thiamine. This coenzyme
+ alkali-washed yeast + magnesium ions decomposes pyruvic acid ac-
cording to the reaction:
carboxylase
CH;COCOOH — CH;CHO + CO,
Peters and his pupils demonstrated that the cocarboxylase is the active
form of thiamine in tissue oxidation, also (Banga et al.!8: !9).
The bond between the apo- and the cocarboxylase at neutral or slightly
R.A. Peters, H. Rydin, and R. H.S. Thompson, Biochem. J. 29, 53 (1935).
"1H. G. K. Westenbrink and J. J. Polak, Rec. trav. chim. 56, 315 (1937).
® C. Neuberg and L. Karezag, Biochem. Z. 36, 68 (1911).
%C. Neuberg and P. Rosenthal, Biochem. Z. 51, 128 (1913).
MP.E. Simola, Biochem. Z. 254, 229 (1932).
#1. Auhagen, Z. physiol. Chem. 204, 149 (1931).
#F. Auhagen, Biochem. Z. 258, 330 (1933).
™ K. Lohmann and P. Schuster, Biochem. Z. 294, 188 (1937).
% TI. Banga, S. Ochoa, and R. A. Peters, Nature 144, 74 (1939).
97. Banga, S. Ochoa, and R. A. Peters, Biochem. J. 33, 1109 (1939).
ae
428 THIAMINE
acid pH is very firm. Washing with water and dialysis does not remove a
trace of thiamine pyrophosphate. At pH about 8, the dissociation becomes
total, so that by washing with an alkaline phosphate buffer the thiamine
pyrophosphate can readily be removed. Also at pH 4.6 the thiamine pyro-
phosphate may be split off (Stumpf et al2°). One exception, however, has
been found in the enzyme that brings about the condensation of acetalde-
hyde (Green et al.'), for the coenzyme is completely dissociated even in
neutral solutions (Stumpf”).
Stumpf assumes that the thiamine pyrophosphate enzymes can be classi-
fied into two general groups, depending on the relative degrees of dissocia-
tion. The first group, to which the majority of the known carboxylases
belong, is not dissociated between pH 4.6 and 7.8. In the second group the
enzyme is dissociated even in neutral solutions.
Ochoa and Peters (Ochoa”*) found that the addition of thiamine to apo-
carboxylase in the form of alkali-washed yeast greatly enhanced the capac-
ity of this apoenzyme for the production of carbon dioxide after addition
of the coenzyme thiamine pyrophosphate and magnesium. The activation
was maximal with doses of thiamine twenty times larger than the thiamine
pyrophosphate. Instead of thiamine itself also the pyrimidine part of
the molecule 2-methyl-4-aminopyrimidyl-5-methylaminodihydrochloride
could be used (Ochoa and Peters™). This proved that the activation is not
caused by synthesis of the added thiamine to thiamine pyrophosphate.
This stimulation by thiamine of the resynthesis of thiamine pyrophosphate
was much greater in baker’s than in brewer’s yeast (Lipton and Elvehjem**).
Westenbrink and his collaborators succeeded in giving an explanation for
this activation (Westenbrink and van Dorp?’ and Westenbrink et al.)
They found a powerful phosphatase in yeast; this phosphatase is capable of
hydrolyzing the phosphoric esters of thiamine in a very short time. The
thiamine does not actually stimulate the carboxylase system but only in-
hibits the phosphatase that destroys the carboxylase.
When the coenzyme once is bound to the protein, thiamine pyrophos-
phate is resistant to this phosphatase (Westenbrink et al.?8). This is in ac-
20 P. K. Stumpf, K. Zarudnaya, and D. E. Green, J. Biol. Chem. 167, 817 (1947).
21D, E. Green, W. W. Westerfeld, B. Vennesland, and W. E. Knox, J. Biol. Chem. 145,
69 (1942).
2 P.K. Stumpf, J. Biol. Chem. 159, 529 (1945).
23 §. Ochoa, Natwre 141, 831 (1988).
248. Ochoa and R. A. Peters, Biochem. J. 32, 1501 (19388).
25 M.A. Lipton and C. A. Elvehjem, J. Biol. Chem. 186, 637 (1940).
26 AH. G. K. Westenbrink and D. A. van Dorp, Nature 145, 465 (1940).
27 H. G. K. Westenbrink, D. A. van Dorp, M. Gruber, and H. Veldman, Enzymologia ©
9, 73 (1940).
28 AH. G. K. Westenbrink, A. F. Willebrands, and C. E. Kamminga, Enzymologia 9,
228 (1940).
=
IV. BIOCHEMICAL SYSTEMS 429
cordance with the observation of Lipmann,”’ confirmed by Lipton and
Elvehjem® and by Westenbrink, that thiamine is without any effect when
it is added to the yeast suspension some minutes after the addition of co-
carboxylase, for then the cocarboxylase is destroyed by the phosphatase.
Westenbrink eé al.*! demonstrated the high enzymic activity of this phos-
phatase: in 10 seconds it dephosphorylated a large part of the cocarboxy-
lase. The objection of Lipton and Elvehjem*® that phosphate, the other
product of the splitting of thiamine pyrophosphate, did not inhibit the
phosphatase action on cocarboxylase could be answered by the assumption
that in this case there is no shift of the equilibrium according to the law
of mass action, but a competitive inhibition. It appeared later that ani-
mal phosphatases are not inhibited by thiamine but by phosphate ions
(Westenbrink e¢ al.*?).
The synthesis of carboxylase by bringing together alkali-washed yeast,
magnesium, and thiamine pyrophosphate was thoroughly studied by
Parvé.® Even when a large amount of thiamine is added, a great deal of
the thiamine pyrophosphate is hydrolyzed before it is bound to the protein
and becomes resistant to the phosphatase. Therefore, only part of the thia-
mine pyrophosphate is resynthesized to carboxylase; at most about 25 %.
In all respects, the effect of washing at pH 6.2 or 5.6, the resynthesis at
pH 6.8, the dissociation at pH 7.8, and the activity per microgram of thia-
mine pyrophosphate left the resynthesized carboxylase equally as stable
as the original holoferment. Only one difference was found. The maximum
activity, measured by the production of carbon dioxide from pyruvate, is
about pH 5.7 for the native carboxylase and pH 6.2 for a mixture of alkali-
washed yeast + magnesium ions + thiamine pyrophosphate. This differ-
ence is easily explained, for the optimum pH for the resynthesis is 6.8. In
the mixture of alkali-washed yeast + coferment, two reactions are going
on simultaneously; the synthesis of the holoenzyme and the action of this
enzyme on pyruvate. So pH 6.2 is a compromise between pH 5.7 and 6.8.
Lipton and Elvehjem gave another explanation for the stimulation of the
formation of carboxylase by thiamine; they postulated that a substance in
yeast, probably a protein other than the enzyme protein, adsorbs the co-
carboxylase and in this way inhibits the formation of the holoenzyme car-
boxylase. The addition of excess thiamine saturates this material and thus
permits the adsorption of the thiamine pyrophosphate on the apoenzyme.
Most of their experiments could be interpreted just as well by the supposi-
*9F. Lipmann, Enzymologia 7, 142 (1939).
M.A. Lipton and C. A. Elvehjem, J. Biol. Chem. 136, 637 (1940).
1H. G. K. Westenbrink, D. A. van Dorp, and M. Gruber, Ree. trav. chim. 60, 185
(1941).
® H.G.K. Westenbrink, E. P.S. Parvé, and J. Goudsmit, Enzymologia 11, 26 (1943).
*® bE. P.S. Parvé, Thesis, Scheltema en Holkema, Amsterdam, 1945.
430
TABLE I
THIAMINE
REACTIONS CATALYZED BY THIAMINE PYROPHOSPHATE ENZYMES
Reactions
References
Pyruvic acid — Acetaldehyde + CO,
Pyruvic acid + oxygen — CO: + H2O
Pyruvic acid + oxygen — Acetic acid + COs
Pyruvic acid + phosphate + oxygen S$
Acetylphosphate + CO, + HO
Pyruvic acid + phosphate + H,0 — Acetyl-
phosphate + H», + COs:
Pyruvic acid + phosphate — Acetylphos-
phate + HCOOH
Pyruvie acid + CO. — Oxalacetic acid
Pyruvic acid + oxygen — Citric acid
Pyruvic acid + oxygen —> Acetolactic acid
Pyruvic acid + O, > a-Ketoglutaric acid
Pyruvic acid + O: —> Succinie acid
Pyruvie acid + (Oz) — Carbohydrate
Pyruvic acid > Formation of acetylcholine
2 Pyruvic acid — Acetoin + 2CO,
Acetaldehyde — Acetoin
Pyruvic acid + acetaldehyde — Acetoin
+ COz
2 Pyruvic acid S$ a-Acetolactic acid + CO2
Acetic acid + O2 — CO: + H20
a-Ketobutyric acid + O2 — Propionic acid
+ COz
a-Ketovaleric acid + O2 — Butyric acid +
CO,
a-Ketoisovaleric acid — Isobutyric alde-
hyde + COs
a-Ketobutyric acid — Propioin + CO,
a-Ketoglutaric acid — Succinice semialde-
hyde + CO,
a-Ketoglutarie acid + O2 — Suceinie acid
2 Diacetyl + 2H20 — Acetoin + 2 acetic acid
Lohmann and Schuster,” Kensler e¢
al.,?> Cajori*®
Peters and his school, see Peters,%?
Stumpf et al.?°
Lipmann,*® Still,3® Stumpf,?? Stumpf
et al.2°
Lipmann‘’?
Werkman”
Utter and Werkman,*! Kalnitzky and
Werkman®
Krebs and Eggleston,*? Smyth
Sober et al.,4° Barron et al.46
Barron et al.‘
Barron et al.47 >
Barron et al.47
Barron eé al.*7
Mann and Quastel,*8 Minz*9
Silverman and Werkman,*® Green et
al. ,2%+ >t Cajori?6
Green et al.?!
Gross and Werkman®
Utter and Werkman,*! Kalnitzky and
J
al
:
,
,
Juni’
Quastel and Webley*!: 5
Long and Peters,°® Stumpf”?
Long and Peters (56)
Green et al.®!
Green et al.2!
Green et al.2!» 51
Stumpf et al.,2° Barron et al.”
Green et al.®8
———
34H. R. Buchman, E. Heegard, and W. G. Kerckhoff, Proc. Natl. Acad. Sci. U. 8. |
26, 561 (1940).
39 C. J. Kensler, N. F. Young, and C. P. Rhoads, J. Biol. Chem. 148, 465 (1942).
36 F. A. Cajori, J. Biol. Chem. 148, 357 (1942).
37-R. A. Peters, Bull. soc. chim. biol. 28, 700 (1946).
38. Lipmann, Hnzymologia 4, 65 (1937).
39 J. L. Still, Biochem. J. 35, 380 (1941).
IV. BIOCHEMICAL SYSTEMS 431
tion of Westenbrink and van Dorp as by that of Lipton and Elvehjem.
There was one experiment that was explained more easily by the Wisconsin
than by the Amsterdam investigators: Lipton and Elvehjem demonstrated
that by incubating the apoenzyme with cocarboxylase at 30° the recovery
of carboxylase gradually increased as the incubation period increased. How-
ever, Westenbrink and his collaborators could not confirm these results
with the kind of yeast they had at their disposal.
Buchman et al.** found a competitive inhibition of thiamine pyrophos-
phate by the thiazole pyrophosphate portion of the cocarboxylase molecule.
Therefore they assumed that the thiamine pyrophosphate is bound to the
apoenzyme through the pyrophosphate group which is common to both the
cocarboxylase and the inhibitor.
We know a great many reactions that are catalyzed by thiamine-con-
taining enzymes; they are listed in Table I. Little is known as yet of the
question whether all these reactions are catalyzed by different enzymes
(containing different apoenzymes).
These enzymes have not yet been obtained in a pure, crystalline state.
All are protein-thiamine pyrophosphate-magnesium compounds. Fairly
pure preparations have been made from yeast.*': ®** The composition of both
preparations did not differ very much; per mole of thiamine pyrophosphate
they contained 1 g. of atom of magnesium and about 75,000 g. (1 mole?)
of protein.
40 F. Lipmann, J. Biol. Chem. 155, 55 (1944).
41M. F. Utter and C. H. Werkman, Arch. Biochem. 2, 491 (1943).
42 G. Kalnitzky and C. H. Werkman, Arch. Biochem. 2, 113 (1948).
48 H. A. Krebs and L. V. Eggleston, Biochem. J. 34, 1383 (1940).
44D. H. Smyth, Biochem. J. 34, 1598 (1940).
45H. A. Sober, M. A. Lipton, and C. A. Elvehjem, J. Biol. Chem. 134, 605 (1940).
46 E.S.G. Barron and C. M. Lyman, J. Biol. Chem. 141, 951 (1941).
47.8. G. Barron, C. M. Lyman, M. A. Lipton, and J. M. Goldinger, J. Biol. Chem.
141. 957 (1941).
4 P. J.G. Mann and J. H. Quastel, Nature 145, 856 (1940).
49 B. Minz, Compt. rend. soc. biol. 140, 412 (1946).
50M. Silverman and C. H. Werkman, J. Biol. Chem. 138, 35 (1941).
51D. E. Green, D. Herbert, and V. Subrahmanyan, J. Biol. Chem. 138, 327 (1941).
52. N. H. Gross and C. H. Werkman, Antonie van Leewwenhoek J. Microbiol. Serol. 12,
17 (1947).
53. Juni, Federation Proc. 9, 396 (1950).
J. H. Quastel and D. M. Webley, Biochem. J. 35, 192 (1941).
55 J. H. Quastel and D. M. Webley, Biochem. J. 36, 8 (1942).
55 C. Long and R. A. Peters, Biochem. J. 33, 759 (1939).
57 B.S. G. Barron, J. M. Goldinger, M. A. Lipton, and C. M. Lyman, J. Biol. Chem.
141, 975 (1941).
53D. E. Green, P. K. Stumpf, and K. Zarudnaya, J. Biol. Chem. 167, 811 (1947).
59 F. Kubowitz and W. Liittgens, Biochem. Z. 307, 170-2 (1941).
432 THIAMINE
As we have not at our disposition pure, crystalline enzyme preparations,
the activity has to be related to one mole of the coenzyme, the thiamine
pyrophosphate. Peters and his collaborators stated that the activity of the
carboxylase in yeast and that of the pyruvodehydrogenase in brain disper-
sions were of the same order. The pyruvodehydrogenase at 30° catalyzes
the uptake of 1500 moles of O2 per minute, computed per mole of thia-
mine pyrophosphate.
Some years later, Green et al.,°! and almost simultaneously Kubowitz
and Liittgens,®°® using a fairly purified enzyme preparation from yeast,
found that this carboxylase preparation catalyzes the decarboxylation of
900 and 700 moles of pyruvic acid at 30° and 20°, respectively, per minute
per mole of thiamine pyrophosphate.
On the whole, the purity of the pyruvodehydrogenases from animal tis-
sues is less. It is difficult to obtain a solution free from cell particles. There-
fore we do not know whether the oxidative carboxylations are brought about
by enzymes that contain only thiamine pyrophosphate + magnesium as
the coenzyme part, or whether these enzymes contain also another oxidative
coenzyme.
Green ef al. and Stumpf et al.” obtained pyruvodehydrogenase prepara-
tions that were free from flavin adenine dinucleotide and from cytochrome e.
Stumpf et al. could demonstrate that pyruvic and a-ketoglutaric oxidases
from pigeon breast muscle were different enzymes. The most cogent evi-
dence came from summation experiments: by adding a-ketoglutaric acid
to a pyruvodehydrogenase preparation that was saturated with pyruvic
acid, an extra carbon dioxide production was obtained.
Table I indicates that many different reactions are catalyzed by thiamine
pyrophosphate enzymes and that most of them attack pyruvic acid or
other a-keto fatty acids.
It may be remarked that pyruvic acid stands at the crossroads of the
carbohydrate metabolism; it is the end point of the anaerobic processes,
and the starting point of the decarboxylation and oxidation reactions and
of the reversal of the anaerobic chain of reactions. This attack on pyruvie
acid may be a simple decarboxylation, an oxidative decarboxylation, a
carbon dioxide fixation, a dismutation, or a condensation. We may assume
that the exact type of reaction depends upon the protein, the apoenzyme,
with which the thiamine pyrophosphate is combined.
In the experiments with tissues or crude extracts, the enzyme system con-
tained several other enzymes and catalytic substances, such as the Cy
dicarboxylic acids, besides the carboxylase. In their initial work Long and
Peters®® concluded that there is a distinct difference between the systems
in yeast and in the animal tissue. The yeast system decarboxylates only
the pyruvic acid, whereas in the tissue oxidation accompanies decarboxyla-
IV. BIOCHEMICAL SYSTEMS 433
tion. Animal tissues contain a pyruvic acid dehydrogenase. From these re-
sults they inferred that the apoenzymes are different in yeast and in animal
tissues.
Thus, thiamine pyrophosphate can act in yeast as the coferment of pure
carboxylase, whereas in animal tissues and in several bacteria it acts as the
coferment of a pyruvic dehydrogenase. Watt and Krampitz,°’* using C%O»
with isotopic C™ as a tracer, were able to demonstrate that the transforma-
tion of pyruvic acid into a-acetolactic acid + COs is a reversible reaction.
1. THe UNITARIAN VIEW
Coenzymes commonly catalyze different reactions, depending upon the
nature of the apoenzyme. However, the difficulty with thiamine pyrophos-
phate is that the two kinds of reactions are so widely different from a chemi-
eal point of view—one is oxidative and the other is non-oxidative. Also,
none of the mechanisms proposed for the oxidative action of thiamine
pyrophosphate has proved to be satisfactory (see below). Krebs drew at-
tention to this difficulty, and he was the first who advanced an attractive
unitarian theory, bringing all the different activities of thiamine under one
single denominator. Evans,°® working in Krebs’ laboratory, had shown
that minced pigeon liver was able to oxidize pyruvate to a-ketoglutarate.
From experiments to elucidate the mechanism of this reaction, Krebs and
Eggleston* were led to the assumption that the primary step is an assimila-
tion of carbon dioxide to pyruvic acid, a Wood and Werkman reaction.™» ©
CO, + CH;COCOOH — COOHCH.COCOOH
The oxalacetate formed in this way is converted into citrate and a-keto-
glutarate according to Krebs’ citric acid cycle. Krebs and Eggleston as-
sumed that the first of this cycle of reactions, the formation of oxalacetate,
(the reverse of “‘decarboxylation’’) is catalyzed by thiamine pyrophosphate.
In this way, the vitamin is not directly concerned with the oxidation of
pyruvate but with a reaction preliminary to the oxidation; the oxalacetate
acts as a hydrogen carrier. However, the experimental evidence for this
theory was not very convincing. Their chief argument was based on the
demonstration with suspensions of pigeon liver that pyruvate utilization
was greatly increased by suspending it in bicarbonate solution instead of
in phosphate buffer and by adding carbon dioxide to the gas phase, whereas
carbon dioxide and bicarbonate have no general effect on processes in liver
suspensions.
59a JT). Watt and L. O. Krampitz, Federation Proc. 6, 301 (1947).
60K. A. Evans, Jr., Biochem. J. 34, 829 (1940).
AH. G. Wood and C. H. Werkman, Biochem. J. 30, 48 (1936).
8 H. G. Wood and C. H. Werkman, Biochem. J. 32, 1262 (1938).
434 THIAMINE
Krebs also demonstrated that acetoacetate formation is somewhat re-
duced in livers of thiamine-deficient pigeons and that addition of the vita-
min restored this formation.
Furthermore Smyth, as a collaborator of Krebs, prepared cultures of
Staphylococcus aureus or S. albus that were deficient in thiamine. The pyru-
vate metabolism of these microorganisms was much lower than that of
vitamin-saturated cells. Added thiamine greatly enhanced this pyruvate
metabolism. In this respect thiamine could be replaced by oxalacetate. This
effect could not be obtained in Staphylococci grown in vitamin-sufficient
media. So these experiments are in accordance with the assumptions that
thiamine catalyzes the formation of oxalacetate and that this substance
acts as a hydrogen carrier in the dismutation of pyruvate. At the end of
his article Krebs drew attention to the work of Ruben and Kamen,® who
were able to demonstrate, with the aid of radioactive carbon, that animal
tissues were able to assimilate carbon dioxide. In the following years several
investigators firmly established the fact of the assimilation of carbon diox-
ide by animal tissues (e.g., Evans and Slotin,* Solomon et al.,®° and Utter
and Wood"). The role of thiamine pyrophosphate in this process is not
definitely proved, however, and there are several experimental data which
cannot be brought into line with this unitarian view of Krebs. Barron et
al.,°” in contradiction to the results of Smyth with bacteria, stated that the
condensation reactions of pyruvate in tissues of avitaminotic animals were
accelerated by thiamine but not by oxalacetate. The results of Green with
the purified carboxylase preparations are not easily reconciled with the
Krebs’ theory.
2. CouPLING OF Pyruvic ACID OXIDATION WITH PHOSPHORYLATION
The coupling of oxidations with phosphorylations with high-energy and
low-energy phosphate bonds cuts the flow of energy of the oxidation into
fractions (Lipmann®). In this way it might be thought that the difference
between oxidizing and non-oxidizing enzymes becomes less important.
Lipmann?®: * studied the oxidation of pyruvate by an extract from Bacillus
delbriickit (Bact. acidificans longissimum). The pyruvic acid is oxidized to
acetic acid and carbon dioxide.
CH;COCOOH + 402. — CH;COOH + CO,
The reaction requires inorganic phosphate and adenylic acid. The inorganic
63 S$. Ruben and M. D. Kamen, Proc. Natl. Acad. Sci. U. S. 26, 418 (1940).
64. A. Evans, Jr. and L. Slotin, J. Biol. Chem. 141, 439 (1941).
65 A. K. Solomon, B. Vennesland, F.W. Klemperer, J. M. Buchanan, and A. B. Hastings,
J. Biol. Chem. 140, 171 (1941).
66 M.F. Utter and H. G. Wood, J. Biol. Chem. 164, 455 (1946).
67 F. Lipmann, Advances in Enzymol. 1, 99 (1941).
od
IV. BIOCHEMICAL SYSTEMS 435
phosphate disappears during the process, and an equivalent amount of
adenosine polyphosphate is formed. Thus the oxidation generates an energy-
rich phosphate bond. The primary oxidation product of the pyruvic acid
behaves like acetyl phosphate. Synthetic acetyl phosphate is able to trans-
fer its high-energy phosphate bond to adenylie acid. From these facts
Lipmann formulated the oxidation process.
CH;COCOOH + H;PO, + O2. — CH;COOPO;H2 + COs + H2O2
Afterwards Lipmann* calculated the bond energy of acetyl phosphate. As
it is some 3 keal. higher than the average energy of 12 keal. of the energy-
rich bond, it will amount to around 15 keal. The need of inorganic phos-
phate for pyruvate oxidation in brain also was demonstrated (Banga
et al.8: 1%), However, Ochoa et al.® could find no indication that acetyl
phosphate is an intermediate in the oxidation of pyruvic acid by brain or
by muscle. Thus there are many carboxylases that require inorganic phos-
phate for their action. However, there are exceptions to this rule: Stumpf”
studied the pyruvic acid oxidation by Proteus vulgaris, which contains an
enzyme that, in the presence of thiamine pyrophosphate and magnesium,
specifically catalyzes the oxidation decarboxylation of pyruvic acid to
acetic acid and carbon dioxide with liberation of considerable free energy:
CH;COCOOH + 40, — CH;COOH + CO,
No disappearance of pyruvic acid could be observed under anaerobic
conditions and no carbon dioxide was produced. This enzyme from Proteus
vulgaris is equally active in the absence, as in the presence, of inorganic
phosphate. In several ways Stumpf could demonstrate that the assumption
that trace amounts of phosphate should react with pyruvic acid to form
acetyl phosphate, which could then act as a catalyst, was not in accordance
with the facts. Similarly the pyruvic oxidase of Escherichia coli (Still**) does
not require the presence of inorganic phosphate. Ochoa® thoroughly studied
the coupling of phosphorylation with oxidation of pyruvic acid in the brain
and the oxidation of the a-ketoglutaric acid’® by a preparation from the
heart of the cat. He confirms the observation that inorganic phosphate
and adenylic acid or adenine triphosphate are required for the oxidation.
The rate of oxygen uptake by the dehydrogenase from cat heart depended
upon the concentration of inorganic phosphate. However, it is not certain
that this preparation contained a thiamine pyrophosphate enzyme.
Kalnitzky and Werkman,®” working with extracts of Escherichia coli,
found that inorganic phosphate was essential for the activity of its pyruvic
8S. Ochoa, R. A. Peters, and L. A. Stocken, Nature 144, 750 (1939).
69S. Ochoa, J. Biol. Chem. 188, 751 (1941).
70S. Ochoa, J. Biol. Chem. 155, 87 (1944).
Py 4)
436 THIAMINE
acid hydrogenase. Utter and Werkman* showed that the apparently “hy-
droclastic”’ action of this extract
CH;COCOOH + H.O — CH;COOH + HCOOH
was really a phosphoroclastic” split. Utter et al.” with the aid of C¥ as a
tracer demonstrated that this reaction
is reversible. This reversibility shows that a thiamine pyrophosphate en-
zyme can catalyze a synthesis, as well as a breakdown, of pyruvate.
Acetic acid is formed by the dephosphorylation of acetyl phosphate.
The acetyl phosphate that is formed by the oxidative decarboxylation of
pyruvic acid by the action of thiamine pyrophosphate is a higher reactive
compound. Lipmann was able to prepare acetyl phosphate synthetically.
However this product was inactive. So the enzymatic intermediate must
have a somewhat different structure.
Most of the thiamine-enzyme preparations from animal sources are easily
sedimented, e.g., by centrifuging 30 minutes at 15,000 or 20,000 r.p.m.
(Ochoa” and Green et al.*8). Some of the enzyme preparations from bacteria
seem to be more resistant to centrifugal force. Thus Silverman and Werk-
man? state that the carboxylase from Aerobacter aerogenes remained in the
supernatant solution after centrifuging for 1 hour at 250,000 to 300,000
r.p.m. With a preparation from Proteus vulgaris, Stumpf” found that cen-
trifugation for 1 hour in a gravitational field of 9700g sedimented only a
small part of enzyme. However, centrifugation for 1 hour in a gravitational
field of 100,000g sedimented all active material. There seems to be a differ-
ence between the different enzymes.
3. INHIBITORS AND ACTIVATORS
Most of the preparations show no inhibition by 3.3 to 10 X 10% M
iodoacetate, fluoroacetate, fluoride, or malonate (Green et al.*! Green et
al." Stumpf,” and Stumpf et al.2°). Some preparations are more or less
inhibited by iodoacetate and fluoride. Thus the enzyme from Aerobacter
aerogenes is inhibited 85% by 7 X 10-* M iodoacetate, and 36% by 5 X
10-* VW fluoride (Silverman and Werkman®’), and the diacetyl mutase from
pigeon breast muscle was inhibited 39% by 3.38 X 10-° MW iodoacetate
(Green et al.°8).
Salts of heavy metals were found to be strong inhibitors for the cocar-
boxylase from yeast (Green e¢ al.°'). A 7/10,000 concentration of AgNO,
71M. F. Utter, C. H. Werkman, and F. Lipmann, J. Biol. Chem. 154, 723 (1944).
IV. BIOCHEMICAL SYSTEMS 437
CuSO, , or Hg(NO;) gave complete inhibition, but 1//10,000 ZnSO, showed
no inhibition at all.
Several other inhibitors are described for carboxylases. Cajori®® found
inhibition for carboxylase from yeast by hemin; the enzyme was activated
by cysteine. Kensler et al.*° showed inhibition of yeast carboxylase by the
split products of N ,N-dimethylaminobenzene; they confirmed the stimu-
lation by cysteine.
Stumpf,” working with a pyruvic oxidase from Proteus vulgaris, found
that inorganic pyrophosphate in 3.4 < 10-° VW concentration inhibits the
enzyme activity; this inhibition is reversed by 0.7 X 10-* M thiamine pyro-
phosphate. He ascribed this inhibition by inorganic pyrophosphate to its
reversible union with the magnesium-protein complex, thus blocking a simi-
lar union with thiamine pyrophosphate. Carboxylase activators were dem-
onstrated by Greenberg and Rinehard.” They found that cysteine, NaHSOs,
and phenylhydrazine were able to activate the thiamine pyrophosphate
enzymes. Tauber’ found that sodium cyanide is a good activator. All these
compounds combine with aldehyde and ketone groups.
B. COENZYMES
Until recently only one thiamine-containing coenzyme was known, 1.e.,
thiamine pyrophosphate. Thiamine itself and thiamine monophosphate are
without any coenzyme activity.
Several years ago, some French authors’** prepared thiamine triphosphate
and thiamine polyphosphates.
In thiamine pyrophosphate the pyrophosphoric acid is attached to the
thiazole group of the thiamine molecule. Thus the formula of pyrophosphate
is
ate Gr Cn,
| ee
CH;—C C—CH,—N—C 0 )
pe | \ | !
N=C C—CH,—CH.0—P—O0—P—OH
Y
H.N HC—S OH OH
Roux ef al. assumed that in thiamine triphosphate the third phosphate
group is attached to the NHz of the pyrimidine. Thus it has the following
formula.
72 TD). M. Greenberg and J. F. Rinehart, Proc. Soc. Exptl. Biol. Med. 43, 495 (1940).
7H. Tauber, The Chemistry of Enzymes, p. 250. John Wiley and Sons, New York,
1950.
738 H. Roux, Y. Feysseire, and G. Duchesne, Bull. soc. chim. biol. 30, 592 (1948).
438 THIAMINE
eee Cl CH,
| rd
CH;—-C—C-——-CH, N—¢ O O
iy N
N=C 0 C—CH,—CH.0O—P—O—P—OH
Peel id |
HN—P—OH H—C—S OH OH
|
OH
In the polyphosphates still more phosphate groups are attached to the
pyrimidine nucleus. The name cocarboxylase hs been reserved for the thia-
mine pyrophosphate.
Cocarboxylase can be synthesized from thiamine chemically or enzymi-
cally. However, because carboxylase is not dissociated (see below), the en-
zymic synthesis stops when the apoenzyme is saturated. No preparative use
can therefore be made of this reaction.
1. CHEMICAL PROCEDURE
Lohmann and Schuster” described a method for preparing the cocarboxy-
lase. Weyland and Tauber” gave an extensive description for the prepara-
tion of thiamine pyrophosphate which substance Weil-Malherbe” synthe-
sized by treating the thiamine bromide with silver pyrophosphate. Karrer
and Viscontini’® improved the method of Weyland and Tauber. The strong
hydrochloric acid that is present during the procedure of Weyland and
Tauber” tended to hydrolyze a large part of the thiamine pyrophosphate
to monophosphothiamine. Therefore Karrer and Viscontini’® improved the
method by preparing the phosphate of the cocarboxylase. In this way they
reduced the acidity of the solution and so obtained a yield that was 55%
of the calculated one.
2. ENZYMIC SYNTHESIS
Yeast, bacteria, and animal tissues contain systems that are able to
phosphorylate thiamine. The first reports (von Euler and Vestin”; and
Lohmann and Schuster”) needed confirmation because thiamine strongly
stimulates cocarboxylase activity (see Section IV A, p. 425). Ochoa and Pe-
ters** and Ochoa et al.® studied the phosphorylation process in animal
tissues. They found that liver shows a particularly good phosphorylating
capacity. Slices, brei, and ‘‘dispersions” were equally active. Brain and
muscle were much less active; preparations from duodenal mucosa (pig)
74 J. Weyland and H. Tauber, J. Am. Chem. Soc. 60, 2263 (1938).
75 FH. Weil-Malherbe, Biochem. J. 34, 980 (1940).
76 P. Karrer and M. Viscontini, Helv. Chim. Acta 29, 1901 (1946).
77 AH. von Kuler and R. Vestin, Naturwissenschaften 25, 416 (1937).
IV. BIOCHEMICAL SYSTEMS 439
showed no activity at all. Ochoa thinks that the reaction
Thiamine + 2 phosphate — Thiamine pyrophosphate
is reversible. Oxygen is essential for a reaction to the right. The optimum
pH is about 8.5. Goodhart and Sinclair,’® working with blood from avitamin-
ous pigeons, demonstrated that the nucleated blood cells can phosphorylate
thiamine.
D. Siliprandi and N. Siliprandi* demonstrated that in rats and also in
human beings’*» insulin is essential in the conversion of thiamine to cocar-
boxylase.
A cell-free protein preparation containing the apocarboxylase has been
made from yeast (Weil-Malherbe’’). He was able to demonstrate that
phosphorylation of thiamine occurs only in the presence of adenylpyrophos-
phorie acid or such reactions as entail its intermediate formation.
Leuthardt and Nielsen’** succeeded in preparing a purified apoenzyme
for the transformation of thiamine into cocarboxylase. This apoenzyme
needs the addition of adenosinetriphosphate and magnesium ions. The
optimal pH of the purified enzyme was found to be between pH 6.8 and
pH 6.9.
Phosphopyruvie acid can act as a phosphate donor for the synthesis of
cocarboxylase in the presence of catalytic amounts of adenylic acid or
adenylpyrophosphate. Thus a direct transfer of phosphate from adenyl-
polyphosphate is the mechanism for phosphorylating thiamine. These
results were confirmed by Lipton and Elvehjem*® and by Quastel and
Webley.®® The synthesis does not take place via the monophosphate
(Weil-Malherbe’”’), for the production of thiamine pyrophosphate goes faster
from thiamine itself than from its monophosphate. The synthesis of co-
carboxylase is almost completely inhibited by 0.005 M iodoacetic acid, and
only slightly inhibited by 0.04 7 sodium fluoride (Lipton and Elvehjem*').
Westenbrink and his collaborators studied the synthesis of cocarboxylase
by living yeast cells (Westenbrink et al.**). By adding a large excess of thia-
mine to the yeast, much more thiamine pyrophosphate is synthesized than
can be bound to the apocarboxylase present in the yeast; it is probably
73 R.S. Goodhart and H. M. Sinclair, Biochem. J. 33, 1099 (1939).
78a J). Siliprandi and N. Siliprandi, Nature 168, 422 (1951).
7b DZD. Siliprandi and N. Siliprandi, Nutrition Revs. 10, 214 (1952).
79H. Weil-Malherbe, Biochem. J. 33, 1997 (1939).
79a F. Leuthardt and H. Nielsen, Helv. Chim. Acta 35, 1196 (1952).
80 M. A. Lipton and C. A. Elvehjem, Nature 145, 226 (1940).
81M. A. Lipton and C. A. Elvehjem, Cold Spring Harbor Symposia Quant. Biol. 7,
184 (1939).
82 H.G. K. Westenbrink, E. P. 8. Parvé, and H. Veldman, Biochim. et Biophys. Acta
1, 154 (1947).
440 THIAMINE
bound to some other yeast protein. Contrary to the supposition of Sperber,**
Westenbrink e¢ al.*? proved that the synthesis of carboxylase by living yeast
cells is not a simple reversion of the action of the phosphatase, the enzyme
that decomposes the cocarboxylase.
As we take into account the tremendous activity of the newly discovered
cobalamin (vitamin Biz), we must not exclude the possibility that the car-
boxylase preparations of animal cells contain a small quantity of a power-
ful oxidizing coenzyme other than thiamine pyrophosphate.
At the International Biochemical Congress in Paris in July, 1952, there
was held a symposium on the tricarboxylic acid cycle (the citric acid cycle) .*4
Most of the participants in this meeting considered it established that the
oxidation of pyruvic acid is performed by a thiamine pyrophosphate-con-
taining protein with other coenzyme(s) as the oxidizing agent(s). The oxi-
dizing agent might be the protogen® or a-lipoic acid that was isolated by
Reed et al.°® in crystalline form. Green® states that the pyruvic oxidase
and other a-keto fatty acid oxidases require at least four prosthetic groups
to catalyze the oxidative decarboxylation of their substrates: (1) thiamine
pyrophosphate, (2) magnesium ions, (3) pyridine nucleotide, and (4) coen-
zyme A.
So the decarboxylation and the oxidation of pyruvic acid are separate
steps in the oxidation of pyruvate. Only the first step, the decarboxylation,
is catalyzed by the thiamine pyrophosphate as a coenzyme. The product
of decarboxylation seems to be not free acetaldehyde but an enzyme- or
coenzyme-bound acetaldehyde derivative which, upon transfer of the acet-
aldehyde-group to coenzyme A (see pantothenic acid page) undergoes
oxidation as an acetaldehyde-CoA compound. (cf. Ochoa, p. 81 in ref. 84).
Langenbeck*® made model experiments on enzyme reactions. Referring to
these experiments, Weil-Malherbe®’ thinks that a Schiff base is first formed
and that this base undergoes an intramolecular oxidation-reduction process.
No experimental evidence was given for this view. On the contrary Stern
and Melnick®® presented experimental data against the Langenbeck cycle.
Lipmann”! reduced thiamine with platinum black and Hy» or by NaS2Ox.
He assumed that a dehydro derivative might be formed which forms a redox
83H}. Sperber, Naturwissenschaften 29, 765 (1941).
84 Symposium on the tricarboxylic acid cycle held at the Second International Con-
gress of Biochemistry, Paris, 1952.
8 J). J. O’Kane and I. C. Gunzalus, J. Bacteriol. 56, 499 (1948).
86 T,. J. Reed, Sczence 114, 93 (1951).
87 J). EK. Green, Science 115, 661 (1952).
88 W. Langenbeck, Hrgeb. Enzymforsch. 2, 314 (1983).
89 H. Weil-Malherbe, Nature 145, 106 (1940).
99 K. G. Stern and J. L. Melnick, J. Biol. Chem. 181, 597 (1939).
1. Lipmann, Nature 188, 1097 (1936).
IV. BIOCHEMICAL SYSTEMS 441
system with the vitamin. In 1938, however, Lipmann and Perlman” showed
that the activity of thiamine was lost after dehydrogenation. Stern and
Melnick®® pointed out that the sulfite, formed in the reaction of thiamine
with sodium dithionite, must split the vitamin molecule (Williams ef al.**).
This was proved by Karrer et al.“* and Karrer and Viscontini.”® Further-
more, Stern and Melnick’? showed that reduction of thiamine with activated
hydrogen gave a product that was not autoxidizable and was biologically
inactive when tested on polyneuritic pigeons. Cautious reduction of thia-
mine pyrophosphate with activated hydrogen, however, gave a dihydro-
cocarboxylase that also was not autoxidizable but that was active in curing
polyneuritic pigeons,
On the other hand Zima and Williams’® and Zima et al.*® oxidized the
sodium salt of thiamine with iodine and obtained a product which they
assumed was formed from two molecules of thiamine, in which the S—H
of the thiazole half of the molecule is transformed to S—S. Thus, it appeared
that the thiamine/thiamine disulfide should form a reduction-oxidation
system just as does cysteine/cystine or glutathione and its disulfide.
Karrer and Viscontini,’* *? however, chemically synthesized the thia-
mine disulfide pyrophosphate. This was combined with the apoenzyme, the
alkali-washed yeast. The product was inactive with pyruvic acid. There-
fore the disulfide form of cocarboxylase is not the active coenzyme. Never-
theless, the disulfide may be active in the animal organism, but only by
preliminary reduction to thiamine itself. Similarly, Peters** found that the
disulfide is active only after reduction. But the fact that 7m vitro tissue prep-
arations are able to reduce the disulfide appears, in Peters’ opinion, to
leave room for the suggestion of Williams and Zima that the disulfide may
play a part in the dehydrogenation.
Barron and Lyman* showed that the thiamine pyrophosphate was more
resistant to oxidation and reduction than thiamine itself. They concluded
that the action of thiamine pyrophosphate as a component of enzyme sys-
tems does not involve reversible oxidation and reduction. They were led
to postulate that the phosphorylated thiamine acts as the integral part of
the activating protein. After summarizing all the work that has been per-
formed until now, we must conclude that much more experimental work
2 F. Lipmann and G. Perlman, J. Am. Chem. Soc. 68, 2574 (1938).
%R.R. Williams, R. E. Waterman, J. C. Kuesztesy, and E. R. Buchman, J. Am.
Chem. Soc. 57, 536 (1935).
%P. Karrer, W. Graf, and J. Schrucki, Helv. Chim. Acta 28, 1523 (1945).
9° QO. Zima, and R. R. Williams, Ber. 78, 941 (1940).
6 O. Zima, K. Ritsert, and Th. Moll, Z. physiol. Chem. 267, 210 (1941).
7 P. Karrer and M. Viscontini, Helv. Chim. Acta 29, 711 (1946).
9% R.A. Peters, Nature 158, 707 (1946).
442 THIAMINE
is necessary, preferably with pure pyruvodehydrogenase preparations, to
establish the mechanism for the oxidative activity of thiamine pyrophos-
phate.
V. Specificity of Action
B. C. P. JANSEN
A. THE ESSENTIAL METALS
Most of the carboxylases are thiamine pyrophosphate-magnesium-pro-
tein compounds (Ochoa!). Magnesium may be replaced by manganese.
Green et al.” using a preparation from top brewer’s yeast, made an elaborate
study of the replacement of magnesium by other metals. All the divalent
TABLE II
Metal, 1 mg./3.3 ml. CO: , mm.3 Metal, 1 mg./3.3 ml. COz, mm.
No addition 23 Zn*+ 282
Megtt 875 Catt 558
Mntt 850 Fett 550
Cot 694 Altt+ 50
Cdt* 422 Fettt 39
metals tested could replace magnesium. The trivalent form of iron and the
trivalent aluminum were practically inactive (Table IT).
Stumpf? in his pyruvic oxidase from Proteus vulgaris replaced the Mg by
Mn-, Fe-, Ni-, Zn= or Co-, but Ca=, Ba=, Cd= and the trivalent Fe= and
Al were ineffective. Green et al.4 stated that their preparation of diac-
etyl mutase from pigeon breast muscle did not require magnesium or any
other divalent metal.
B. THIAMINE PYROPHOSPHATE
In pure preparations not containing a phosphorylating system, thiamine
pyrophosphate cannot be replaced by thiamine, monophosphothiamine,
pyrithiamine (Stumpf*), diphosphopyridine nucleotide, triphosphoropyri-
dine nucleotide, flavin dinucleotide, adenosinetriphosphate, or pyridoxal
phosphate (Green e¢ al.‘).
1§..Ochoa, Biochem. J. 38, 1262 (1939).
2D. E. Green, D. Herbert, and V. Subrahmanyan, J. Biol. Chem. 188, 327 (1941).
3P. K. Stumpf, J. Biol. Chem. 159, 529 (1945).
4D. E. Green, P. K. Stumpf, and K. Zarudnaya, J. Biol. Chem. 167, 811 (1947).
V. SPECIFICITY OF ACTION 443
Thiamine triphosphate and thiamine polyphosphates have an activity
somewhat similar to that of thiamine pyrophosphate, but they quanti-
tatively are much less active; their cocarboxylase activity amounts to only
30 % of that of thiamine pyrophosphate.*: °
Velluz et al.’ tried to restore the carboxylatic activity of apocarboxylase,
obtained by washing yeast with an alkaline phosphate solution. From
four to five times more thiamine triphosphoric acid than thiamine pyro-
phosphate was required to saturate the washed yeast. The resynthesized
enzymatic system developed 80% of the activity of the one rebuilt with
thiamine pyrophosphate.
Plotka et al.8 studied the action of thiamine triphosphoric acid on the
heart. On the excised frog’s heart thiamine triphosphoric acid exerts a
slight positive inotropic action on the normal organ and restores the regu-
larity of the fatigued heart. On the rabbit’s heart in situ, thiamine triphos-
phoric acid protects the organ against fibrillation induced by faradization.
Thiamine pyrophosphate also exhibits some antifibrillatory properties but
much less than thiamine triphosphoric acid. Plotka et al. in the same article
discussed the problem of the existence of thiamine triphosphoric acid in organ-
isms, and they think it is justifiable to consider that thiamine triphosphoric
acid plays a role in the special metabolism related to nerve impulse trans-
mission.
C. SUBSTRATE SPECIFICITY
Green et al.2 studied the activity of a purified carboxylase from yeast on
different substrates (Table III).
Thus the a-ketonic acids, in addition to pyruvic acid, are decarboxylated
also, but the higher homologs are attacked at a lower rate.
The specificity of carboxylases from animal tissues was somewhat differ-
ent (Green et al.’). These preparations had no action on oxalacetic acid,
mesoxalic acid, a-ketocaproic acid, or phenylpyruvie acid. They decarboxy-
lated a-ketobutyric acid under formation of propioin, according to the equa-
tion
2CH;-CH2-CO-COOH — CH;3-CH2-CHOH-CO-CH2CH; + 2CO2
and a-ketoglutarice acid to succinic semialdehyde and COz:
COOH-CH.2-CH2-CO-COOH — COOH-CH2:CH2-CHO + CO,
5M. Herbain, Bull. soc. chim. biol. 32, 784 (1950).
6 H. Roux and A. Callandre, Bull. soc. chim. biol. 32, 793 (1950).
7L. Velluz, G. Amiard, and J. Bartos, J. Biol. Chem. 180, 1137 (1949).
8C. Plotka, M. Peterfalvi, R. Jequier, and L. Velluz, Am. J. Physiol. 158, 279
(1949).
®D. E. Green, W. Westerfeld, B. Vennesland, and W. E. Knox, J. Biol. Chem. 145,
69 (1942).
444 THIAMINE
The decarboxylation of a-ketoglutaric acid took place with the same
velocity as that of pyruvie acid. Green et al. made it probable that the
pyruvo(apo)carboxylase and the a-ketoglutaro(apo)carboxylase were differ-
ent enzymes. For if thiamine pyrophosphate was split from the carboxylase
by washing at pH 4.6, the maximum rate for carbon dioxide development
with pyruvate as a substrate was found by the addition of 2 y of thiamine
pyrophosphate per milliliter of Green’s preparation. With a-ketoglutarate
as a substrate, the maximum reaction velocity was attained only when 300
y of thiamine pyrophosphate per milliliter was added. A third enzyme
seemed to be present in Green’s preparation, an enzyme for the condensa-
tion of acetaldehyde to acetoin. For maximum activity it required 30 y
of thiamine pyrophosphate per milliliter. Each enzyme perhaps has a very
TABLE III
DECARBOXYLATION OF a-KeEetTonic AcipS BY YEAST CARBOXYLASE
Substrate, 0.04 COz , mm.3
Pyruvie acid 596
a-Ketoisovaleric acid 525
Oxalacetie acid 191
a-Ketoisocaproic acid 24
a-Ketoglutaric acid
Phenylpyruvie acid
a-Hydroxyphenolpyruvie acid
Acetoacetie acid
No addition
Ss Sis) 2)
high specificity for different substrates and may be catalyzed by different
apoenzymes plus the same coenzyme.
The pyruvic oxidase, prepared by Stumpf* from Proteus vulgaris proved
to be very specific. It oxidizes only pyruvic acid; a-ketobutyric, a-ketocar-
poic, a-ketoglutaric, acetoacetic, glyoxylic, and lactic acids are not at-
tacked. The enzyme preparation oxidized formic acid and phenylpyruvic
acids, even in the absence of thiamine pyrophosphate. Therefore, the oxi-
dation of these two acids cannot be attributed to the thiamine pyrophos-
phate-pyruvie acid oxidase. The diacetyl mutase preparation of Green et
al.4 from animal tissues was found to be highly specific for diacetyl. Glyoxal,
methylglyoxal, dipropionyl, benzil, cyclohexamedione-1,2, alloxan, and
cyclohexanehexone were not attacked by the enzyme. However, the same
preparation catalyzes the oxidative decarboxylation of pyruvic acid and
of a-ketoglutaric acid (Stumpf et al.!°). Here again, the a-ketoglutaric oxi-
dase, after splitting off the thiamine pyrophosphate, required a larger
10 P| K. Stumpf, K. Zarudnaya, and D. E. Green, J. Biol. Chem. 167, 817 (1947).
VI. BIOGENESIS 445
amount of thiamine pyrophosphate than the pyruvic oxidase. Here they
found still more cogent evidence bearing on the non-identity of the pyruvic
acid and the a-ketoglutaric oxidase from summation experiments. For ex-
ample, with concentrations of substrates that were adequate to saturate
the respective enzymes, a-ketoglutaric acid was oxidized at the rate of
59 mm.* of OQ» per 30 minutes, whereas the rate for pyruvic acid was 37
mm.* in the same period. The rate with both substrates together was 92
mm.*, which represents a virtual summation of the two rates. Were a single
enzyme involved, the rate in the presence of both substrates would be ex-
pected to be in between the two rates, but never greater than the rate of
either.
VI. Biogenesis
B. C. P. JANSEN
The biogenesis of the active part of the thiamine enzymes comprises
three stages:
1. The synthesis of thiamine itself.
2. The synthesis of the pyrophosphate of thiamine, the cocarboxylase.
3. The synthesis of the whole enzyme, the carboxylase.
For the second and third stages see Section IV A. In this section we shall
discuss only the first stage.
Thiamine is synthesized by plants and also by a number of microorgan-
isms. Usually it is assumed that no thiamine is synthesized by animal tis-
sues. However, E. Abderhalden and R. Abderhalden! demonstrated that
animal tissues were capable of synthesizing thiamine from a solution con-
taining both the pyrimidine and the thiazole moieties. But this was true
only to a slight extent for about 1 % of the theoretical amount was formed
(see page 409). R. Abderhalden? protected rats and pigeons on a thiamine-
free diet from deficiency symptoms by giving the animals large quantities
of the pyrimidine and the thiazole moieties. Therefore it seems that animal
tissues have a trifling capacity to couple the pyrimidine and thiazole parts
into thiamine.
In plants the thiamine generally is abundant in seeds and often in green
leaves and in roots.
As there is a close connection between microorganisms, particularly
mycorhizal fungi, and many plants (and often microorganisms are essential
for plants), it is difficult to state which organisms are the most important
1. Abderhalden and R. Abderhalden, Pfliigers Arch. ges. Physiol. 248, 85 (1939).
*R. Abderhalden, Pfliigers Arch. ges. Physiol. 248, 762 (1940).
446 THIAMINE
for thiamine synthesis. In any event the growth of some plant roots, e.g.,
tomato roots, or pea roots, is greatly stimulated by extremely small amounts
of thiamine (10~!° g. or less per milliliter). Organic manure and fertile soils
contain appreciable amounts of thiamine. Experiments under sterile con-
ditions have demonstrated that plants are able to synthesize thiamine.
Most of it is produced in the young green parts of the plant. In experiments
with plants, however, we must take into account the thiamine that is stored
in the plants. Bonner? was able to grow freshly isolated pea roots in a pure
synthetic medium containing inorganic salts and sucrose. If such roots were
subcultured by the removal of 10-mM. tips into fresh medium and particu-
larly if this procedure were repeated several times, thiamine was found to
be essential for growth; 0.2 y of thiamine per milliliter of the culture medium
gave optimum growth, and even 0.002 y per milliliter had a marked stimu-
lating effect upon the growth of these roots.
The microorganisms vary considerably in their ability to synthesize thia-
mine. Schopfer* established the following groups:
1. Organisms autotrophic for thiamine: Abszdia repens.
2. Organisms requiring only pyrimidine: Rhodotorula rubra type.
3. Organisms requiring only thiazole: Mucor Ramannianus type.
4. Organisms requiring both pyrimidine and thiazole: Phycomyces type.
5. Organisms requiring the entire molecule of thiamine: Glawcoma-Phyto-
phthora type.
A separate group of organisms which are partially inhibited by excess
thiamine: Rhizopus nigricans and other Rhizopus species.
Williams et al.> give a list of those macro- and microorganisms that re-
quire either the pyrimidine or the thiazole moiety, those that need both
components, and those that do not require any component at all.
By an (artificial) symbiosis of two microorganisms it is possible to per-
form a synthesis of the thiamine molecule, if each organism is able to syn-
thesize a different nucleus.
Schopfer* gives the examples of Mucor ramannianus and Rhodotorula
rubra. When each of these is inoculated separately on a thiamine-free syn-
thetic medium, neither is able to develop. But when they are inoculated
together, they grow luxuriantly. This proves that Rhodotorula synthesizes
thiazole which it supplies to Mucor rammannianus and, conversely that
Mucor ramannianus synthesizes pyrimidine which it furnishes to Rhodo-
torula.
(ep)
3 J. Bonner, Science 85, 163 (1937).
4W. H. Schopfer, Plants and Vitamins, pp. 110, 114. Chronica Botanica Co., Wal-
than, Mass., 1949.
5 R. J. Williams, R. HE. Eakin, E. Beerstecher, and W. Shive, The Biochemistry of
Vitamins, p. 686. Reinhold Publishing Corp., New York, 1950.
VI. BIOGENESIS 447
From our knowledge of the chemical steps that are used in the thiamine
synthesis, we may assume that an organism completely autotrophic for
thiamine must be able to effect the synthesis of the pyrimidine and thiazole
moieties and to condense pyrimidine and thiazole into thiamine.
About the pyrimidine synthesis zn vivo we are not well informed. There
are indications that the thiazole part may be synthesized by the condensa-
tion of methionine, acetaldehyde and ammonia.*: ‘4 In this way a-amino-f-
(4-methylthiazole-5)-propionic acid is formed.
It is known that certain microorganisms can perform transformations
analogous to the conversion of this acid to the thizaole part of thiamine.‘
Bonner and Buchman’ stated that pea roots synthesize thiazole from the
precursors thioformamide and chloroacetopropyl alcohol that are used for
the zn vitro synthesis. When these two precursors of thiazole are given along
with the pyrimidine moiety of thiamine to pea roots, growth is as good as
when thiamine is supplied. In the in vitro synthesis chloroacetopropy] alco-
hol is essential; the pea roots are able to use also acetopropy! alcohol.
in vitro = a
HCSNH, + CH;COCHCICH,CH,0H ———} | ieee
in vivo He ©—CH,
eee
N
Thioformamide chloroacetopropylalecohol Thiazole part of thiamine
in vitro
HCSNH,; + CH;COCH.CH.CH,OH ~~~? Thiazole part of thiamine
im vivo
Acetopropylalcohol
Wild Neurospora strains are able to synthesize thiamine. Tatum in his
extensive experiments on mutants of Neurospora obtained four strains that
had lost the ability to synthesize thiamine. Accumulation of a pyrimidine
component by mutant 18558 and of both pyrimidine and thiazole compo-
nents by 9185 was shown in bioassays. So Tatum and Bell’ showed that
the evidence obtained with strains 9185 and 18558 is consistent with the
view that the synthesis in Neurospora takes place through the production
of the thiazole and pyrimidine moieties, with their subsequent coupling.
Woolley and White? studied the inhibition of the growth of a number of
microorganisms by the antithiamine, pyrithiamine. They found that the
inhibition of the growth of microorganisms by pyrithiamine is related to
their requirements for thiamine or its components. They found furthermore,
6C. R. Harrington and R. C. C. Moggridge, Biochem. J. 34, 685 (1940).
7 J. Bonner and E. R. Buchman, Proc. Natl. Acad. Sci. U. S. 24, 431 (1938).
8. L. Tatum and T. T. Bell, Am. J. Botany 33, 15 (1946).
9D. W. Woolley and A. G. C. White, J. Exptl. Med. 78, 489 (1943).
448 THIAMINE
among the bacteria, yeast, and fungi investigated, that the growth of those
requiring intact thiamine is inhibited by a much lower concentration of
pyrithiamine than those which respond to one or both components, whereas
those with no thiamine requirements were not appreciably inhibited. In
Neurospora there seemed to be no definite correlation between capacity for ~
synthesis of thiamine and its components and the degree of inhibition of
pyrithiamine.
Several authors have demonstrated a favorable influence of light on the
biosynthesis of thiamine. Bonner and Greene!” found, for example, that the
thiamine content of pea plants kept in the dark did not increase, whereas
in the light this content rose rapidly.
VII. Estimation
B. C. P. JANSEN
A. PHYSICAL
Thiamine may be estimated by its absorption in the ultraviolet at 265
mu. This method has been used in the laboratory of von Muralt! in Bern
by Lithy? for measuring the thiamine content of nerve fibers.
To distinguish the thiamine from other substances (nuclein components)
absorbing at 265 my, the absorption may be measured before and after
destroying the thiamine by ferricyanide.
B. CHEMICAL
Barger et al.’ showed that thiamine on oxidation with ferricyanide in
alkaline solution is transformed into thiochrome, see facing page.
This thiochrome is characterized by a strong blue fluorescence. Jansen
found that in a very strong alkaline solution and with a suitable quantity
of ferricyanide the transformation is practically quantitative. The thio-
chrome is extracted from the aqueous solution by isobutanol. Thus the
fluorescence of the thiochrome, which is accurately measured in a fluoro-
meter, may be used as a yardstick for the thiamine.’
It could be expected that this method is not only sensitive but also spe-
cific for use in biological fluids and extracts. Bouman® found, however, that
10 J. Bonner and J. Greene, Botan. Gaz. 100, 226 (1938).
1A. von Muralt, Die Signaliibermittlung in Nerven. Birkhiuser, Basel, 1946.
2H. Lithy, Helv. Physiol. Acta 4, C20 (1946).
3G. Barger, F. Bergel, and A. R. Todd, Nature 136, 259 (1935).
4B.C.P. Jansen, Rec. trav. chim. 55, 1046 (1936).
5 J. Bouman, Z. Vitaminforsch. 19, 386 (1948).
VII. ESTIMATION 449
H
va
C Cl
> aes
N ie 2 en ee
Ee. C—NH:-HCl H-C C—C.H,0OH
Se ee
N S
Thiamine
H He
fog eee
C C
UN aes
|» "lhl a
CH;-C C C C—C,.H,0H
Se Be ys
Thiochrome
blighted potatoes (infected with Phytophtera) contain a substance with a
blue fluorescence which is soluble in isobutanol. Also urine and other bio-
logical fluids contain interfering substances. In such cases a preceeding
adsorption of thiamine to Decalso, fuller’s earth, or other suitable adsorb-
ents, is necessary.
More elaborate descriptions of this method are available.*:7 By the use
of a suitable fluorometer 0.01 7 of thiamine or less may be determined by
the thiochrome method. In biological fluids and extracts a large part of the
thiamine is present in the form of pyrophosphate. By oxidizing this it is
transformed to the pyrophosphate of thiochrome. This is insoluble in iso-
butanol. Therefore the pyrophosphate must first be hydrolyzed. This hy-
drolysis can be performed by takadiastase or by a phosphatase, e.g., from
yeast.
Burch et al.7* elaborated a micromethod to determine thiamine and thia-
mine phosphates in very small quantities of blood and blood cells.
Prebluda and McCollum: *) devised a method using the color production
of a diazotized aromatic amine (e.g., p-aminoacetophenone) with thiamine.
The method has been further developed by Melnick and Field.!° This
§ Association of Vitamin Chemists, Methods of Vitamin Assay, p. 69. Interscience
Publishers, New York, 1947.
7P. Gyorgy, Vitamin Methods, Vol. 1. Academic Press, New York, 1950.
7@ H. B. Burch, O. A. Bessey, R. H. Love, and O. H. Lowry, J. Biol. Chem. 198, 477
(1952).
8H. J. Prebluda and E. V. McCollum, Science 84, 488 (1936).
°H. J. Prebluda and E. V. McCollum, J. Biol. Chem. 127, 495 (1939).
107). Melnick and H. Field, J. Biol. Chem. 127, 505, 515, 531 (1939).
450 THIAMINE
azocoloric method is less sensitive and less specific than the thiochrome
method, but it is somewhat simpler and quicker and may be useful in
special instances, e.g., for clinical determination in urine."
C. BIOLOGICAL
Obviously the earliest methods that were used for thiamine estimation
were biological ones. In the beginning when polished rice, an easily obtain-
able thiamine-free food, was taken as a basal diet, hens and cocks were used
as experimental animals. Soon smaller birds, especially pigeons, were taken,
and Jansen worked with very small birds, nonnetjes (unia maja). When
mammals (rats) were the experimental animals, polished rice could no
longer be used as a basal diet because of its deficiencies in other nutrients.
Therefore a complex basal diet was essential. The experiments may be pro-
phylactic or curative. Both are apt to give not very accurate results, and
both require large amounts of thiamine-rich extracts. These are serious
handicaps, and therefore when the chemical and microbiological methods
were brought about, the biological methods were not applied very much
any longer. The advantages of the biological methods are that they do not
require cumbersome extractions and that they give specific results. For de-
tails see, e.g., Coward” and Gyorgy."
D. FERMENTATION
Schultz et al. found that yeast fermentation is enhanced by the pres-
ence of free thiamine. They utilized this fact for a quantitative method for
the estimation of thiamine. Used with a Warburg apparatus, the method
is suitable for a microestimation.®
Nearly all the thiamine in blood and in animal tissues is present in the
form of thiamine pyrophosphate (cocarboxylase). This cocarboxylase can
be measured by its activity as a coenzyme. Ochoa and Peters!® were the
first to use this method for the quantitative determination of thiamine and
cocarboxylase in boiled tissue extracts. Goodhart and Sinclair” applied
this method to the determination of cocarboxylase in blood. All the blood
cocarboxylase was found within the blood cells, particularly in the nucleated
cells.
11M. Hochberg and D. Melnick, J. Biol. Chem. 156, 53 (1944).
2 Kk. H. Coward, The Biological Standardisation of Vitamins. William Wood and
Co., Baltimore, 1947.
13 P, Gyérgy, Vitamins Methods, Vol. II, pp. 45, 179, 448. Academic Press, New York,
1951.
144 A,S. Schultz, L. Atkin, and C. N. Frey, Ind. Eng. Chem. Anal. Ed. 14, 35 (1942).
16 7,, Atkin, A. S. Schultz, and C. N. Frey, J. Biol. Chem. 129, 471 (1939).
16S. Ochoa and R. A. Peters, Biochem. J. 32, 1501 (1988).
17 R.S. Goodhart and H. M. Sinclair, Biochem. J. 33, 1099 (1939).
VII. ESTIMATION 451
Westenbrink'’ worked out a micromethod which permits the determina-
tion of about 0.00005 y of cocarboxylase and about 0.0005 y of thiamine
separately in a mixture of both compounds.
Westenbrink ef al.'* used alkali-washed yeast as an adsorbant and at the
same time as the apoenzyme for the cocarboxylase preparation from blood.
In this way, using the ‘‘cartesian diver”’ technique, it was possible to deter-
mine the cocarboxylase content of 0.01 ml. of blood (Parvé?).
EK. MICROBIOLOGICAL
In 1935 the Swiss investigator Schopfer found that the growthof the mould
Phycomyces blakesleeanus required the presence of thiamine.?! He used this
fact to develop‘a method for the estimation of thiamine, by measuring its
influence on the growth of Phycomyces blakesleeanus.”
This method is very sensitive and especially suitable for series analysis of
foodstuffs, ete. A drawback is the fact that it takes 8 to 10 days before
growth is complete. The split products of thiamine, the pyrimidine plus the
thiazole part, are active also. (For further details, see ref. 7, p. 485.)
In his search for the components of Wildiers’ “bios,” Williams and
Roehm” found that thiamine greatly stimulates the growth of yeast (Sac-
charomyces cerevisiae). So thiamine may be estimated by its influence on
the growth of yeast. Here again the pyrimidine and the thiazole parts are
both active on yeast growth.”
In 1944 Sarett and Cheldelin introduced the use of Lactobacillus fermenti.
This microorganism is extremely sensitive to traces of thiamine.?®: 2°
In this way it is possible to determine quantities of 5 to 50 my of thiamine.
Cocarboxylase is about 30% more active than an equimolecular quantity
of thiamine. The pyrimidine and thiazole moieties are inactive if the incuba-
tion is not prolonged beyond the usual time of 16 to 18 hours at 37°. (For
details and for the use of other lactic acid bacteria, see ref. 7, p. 372.)
Often chemical or microbiological methods are used for thiamine estima-
tion in the urine of men or animals to establish the state of thiamine nutri-
tion. Mickelsen et al.,” in their extensive research on the thiamine excre-
18H. G. K. Westenbrink, Enzymologia 8, 97 (1940).
19H. G. K. Westenbrink, E. P. 8. Parvé, A. C. van der Linden, and W. A. van den
Broek, Z. Vitaminforsch. 18, 218 (1948).
20 E. P.S. Parvé, Thesis, Scheltema en Holkema, Amsterdam, 1945.
21 W. H. Schopfer, Z. Vitaminforsch. 4, 67, 187 (1935).
22, W.H. Schopfer, Ergeb. Biol. 16, 1 (1939).
22R. J. Williams and R. R. Roehm, J. Biol. Chem. 87, 581 (1950).
24R.J. Williams, J. R. McMahan, and R. EE. Eakin, Univ. Texas Publ. 4157, 31 (1941).
25H. P. Sarett and V. H. Cheldelin, J. Biol. Chem. 155, 153 (1944).
26H. P. Sarett and V. H. Cheldelin, J. Nutrition 30, 25 (1945).
27 Q. Mickelsen, W. O. Caster, and A. Keys, J. Biol. Chem. 168, 415 (1947).
452 THIAMINE
tions of normal young men on controlled intakes of thiamine from 0 up to
16 mg. per day, found that at intake levels between 0.7 and 1 mg. of thia-
mine per day the thiamine excretion may be used as an indicator of the
TABLE IV
COLLABORATIVE RESULTS BY THE RECOMMENDED THIOCHROME METHOD
Thiamine found, y/g.
Wheat Dried yeast
Malt Wheat flour, Yeast
Laboratory extract germ 85% extract Sample 1 Sample 2 Sample 3
A Metl 22 55 29 17 45
ID) 4.0 21 3.8 54 20 47
EK 24 ‘14 54
F 15
G 3.9 21 52 27
I 32
J Ba Zl 21 3.8 61 28
K 16
L 3.9 20 47 yA 19 57
M ono 23 3.8 39
N 3.6 37 47
Mean 3.62 Pall 3} SAO M683) 29.1 16.8 50
TABLE V
ReEsutts By BroLtocicaL AND MicrosioLoGcicaL MErTHuops
Thiamine found, y/g.
Dried yeast
Wheat anne
Malt Wheat flour Yeast Sample Sample Sample
Laboratory Method used extract germ (85%) extract 1 2 3
K Rat growth 45
N Bradyeardia 20 27
Ie Rat growth 17 54
F Yeast fermentation Bag) Al 03) 38
J Lactobacillus fermentum 4.1 23 58
Mean value of the collaborative fluori- 3.62 21.3 3.75 51.3 29.1 16.8 50
metric results (from Table IV)
intake. From 1 mg. up to about 5 mg., however, it is better to use the pyra-
mine excretion. Pyramine is the pyrimidine-like component of the thia-
mine molecule which is excreted in the urine. The pyramine content of the
urine was determined by a modification of the yeast fermentation method
of Schultz et al.4 The standard used in these determinations was 2-methyl-
4-amino-5-ethoxymethylpyrimidine hydrochloride.
VIII. STANDARDIZATION OF ACTIVITY 453
The different methods of estimation vary widely in principle and even
the same method (chemical, biological, etc.) may be carried out in different
ways. Thus, for obtaining a reliable extract, different proteinases may be
used, combined with extraction with diluted hydrochloric acid, sulfuric
acid, or sodium acetate solution. The extracts may be purified for removal
of interfering substances in different ways. Several different methods are
used for the oxidation of thiamine to thiochrome. Therefore it is possible
that the results of the thiamine estimation of one laboratory do not agree
very well with those of another institute.
Therefore in many countries the results of different laboratories have
been compared. The most recent investigation in this respect probably is
that of the Aneurine Panel of the Subcomittee on Vitamin Estimations of
the British Analytical Methods Committee of the Society of Public Analysts
and Other Analytical Chemists.*® The panel prepared large homogeneous
amounts of malt extract, wheat germ, wheat flour (85 % extraction), yeast
extract, and three different samples of dried yeast. Representative samples
of these thiamine-containing foodstuffs were sent to the collaborating lab-
oratories in England and in the Netherlands. A detailed description of the
recommended thiochrome method was given.”® For the results, see Tables
IV and V.
From these results we see that the various results in the different labora-
tories are not in perfect agreement. Particularly the biological methods
gave divergent results. On the other hand the agreement of the results of
the thiochrome and the microbiological methods was not too bad.
VIII. Standardization of Activity
B. C. P. JANSEN
Before the large-scale production of pure, crystalline thiamine hydro-
chloride, it was decided at the Vitamin Standards Conference of the Com-
mission on Biological Standardization of the Health Organization of the
League of Nations held at London in 1931 that a certain quantity (10 mg.)
of an acid clay activated by an extract from rice polishings should be used
as an international unit of antineuritic vitamin B, (at that time the name
of thiamine).! This activated acid clay was prepared in the Medical Labora-
tory at Batavia (now Djacarta) and had already been shown to be a very
stable material. In experiments with pigeons and rats it appeared that 10
mg. of this activated acid contains about 3 y of thiamine hydrochloride.
*8 Analytical Methods Committee, Analyst 76, 127 (1951).
‘Medical Research Council, Vitamins: A Survey of Present Knowledge, p. 313.
His Majesty’s Stationery Office, London, 1932.
454 THIAMINE
Thus 1 international unit of thiamine is equivalent to 3 y of thiamine
hydrochloride.
Afterwards Keresztesy and Sampson? found that this standard prepara-
tion was not ideal; they demonstrated that the preparation contained twice
as much thiamine as was indicated in the animal experiments, the animal
organism being able to elute only one-half of the adsorbed thiamine.
Now that the crystalline synthetic thiamine hydrochloride is abundantly
available, the pure synthetic preparation itself is taken as a standard, and
the quantity of thiamine usually is expressed not in international units but
in milligrams of thiamine.
Before the isolation of the thiamine some authors had recommended
units based on rice polish or yeast. Of course these are now obsolete, and
a comparison with the pure vitamin is only approximately possible. With
this restriction we can say that 1 y of crystalline thiamine hydrochloride is
equivalent to 0.166 Smith curative unit or to 0.666 Chase-Sherman unit or
to 0.333 Roscoe unit.
IX. Occurrence in Food
B. C. P. JANSEN
A. OCCURRENCE OF THIAMINE AND ITS PHOSPHATE
COMPOUNDS
In contrast to other vitamins, e.g., the fat-soluble vitamins and ascorbic
acid, thiamine has a widespread distribution in food. It is entirely absent
only from oils and fats, cassava, and refined sugar. On the other hand we
do not know of any foods that are as rich in thiamine as certain fish liver
oils are in vitamins A and D. In animal experiments brewer’s yeast is used
as a rich source of thiamine (and other B vitamins).
In connection with this lack of abundantly thiamine-containing foods it
is important, as stated by Friedemann et al.,! that the intestinal absorption
of thiamine hydrochloride by normal human subjects is extremely limited.
The maximum which could be orally taken without an increase of fecal
thiamine is about 5 mg. per day.
A large part of thiamine in vegetable products is in the form of thiamine
itself; in animal tissues it is present largely in the form of its phosphate or
pyrophosphate derivative (as cocarboxylase). With regard to the nutri-
tive value of the food, it does not matter in which form thiamine is present,
2 J. C. Keresztesy and W. L. Sampson, Proc. Soc. Exptl. Biol. Med. 36, 686 (1937).
1T, EK. Friedemann, T. C. Kmieciak, P. K. Keegan, and B. B. Sheft, Gastroenter-
ology 11, 100 (1948).
|
i
IX. OCCURRENCE IN FOOD 455
because in the animal organism the thiamine is easily phosphorylated (by
an adenosinetriphosphate-containing enzyme) and dephosphorylated by a
powerful phosphatase.
Thiamine is present as such in blood plasma, but as pyrophosphate in
the corpuscles. In human blood only about 10 % of the thiamine is present
as such in the plasma; the rest is in the corpuscles. Normal blood of a well-
fed person contains about 6 to 12 y of thiamine per 100 milliliters. The mean
values of the thiamine pyrophosphate content of well-nourished men were:
1.5 y per 10" red cells; 290 y per 10" leucocytes; for women: 1.3 y per 10"
red cells; 270 y per 10" leucocytes.” *
As thiamine is fairly resistant to high temperature and Oz in a weakly
acid milieu, it is clear that most processed foods contain thiamine. However,
in alkaline and even in the neutral state the resistance to heat is not great.
Therefore, if processing of neutral foodstuffs includes treatment at or
above 100°, a substantial loss of thiamine takes place, dependant upon the
duration of the heating. Furthermore, in the cereal grains thiamine is found
especially in the outer grain layers, and therefore machine-milled rice is
very poor in thiamine. Thus it is obvious that beriberi is a disease of rice
and cassava (manioc)-eating countries,’ > as here a great deal of the popula-
tion depends on rice or cassava as their main source of calories.
For the thiamine content of the different foods we may refer to tables
as published elsewhere.* Special consideration will here be given to those
foods that are either particularly rich in thiamine or that forming the main
food item for many people.
a. Cereals
(1) Wheat. The thiamine content of wheat depends especially on the
variety and to a lesser extent on the conditions under which it is grown,
i.e., the quality of the soil, the use of fertilizers, etc. Usually the content is
between 500 and 800 y of thiamine per 100 g. of whole wheat.
Thiamine is very unevenly distributed in the kernel. The content of
the inner layer, the endosperm, is the lowest, about 30 y per 100 g. The
aleuron layer and the germ are much richer in thiamine; by far the richest
part is the scutellum, the small layer between the germ and the rest of
the kernel. This small layer alone, consisting approximately of 1.5% of
the whole kernel, contains 60 % of its thiamine (Hinton’). The scutellum of
rye, corn, and barley has a similar high content of thiamine (Hinton).
2G. Smits and E. Florijn, Biochim. et Biophys. Acta 3, 44 (1949).
8H. G. K. Westenbrink and J. Goudsmit, Arch. néerl. physiol. 23, 79 (1938).
4A. Dubois and G. Corin, Bull. soc. pathol. exotique 7, 402 (1914).
5 B.C. P. Jansen, Repts. Dutch-Indian Med. Service I, 1 (1923).
6B. K. Watt and A. L. Merrill, U. S. Dept. Agr., Handbook 8.
7J. J. C. Hinton, J. Soc. Chem. Ind. 61, 143 (1942).
8 J. J. C. Hinton, Biochem. J. 38, 214 (1944).
456 THIAMINE
In the modern milling machines (high milling) the process can be regu-
lated in such a way that several fractions are obtained corresponding to
the different layers of the kernel. For the preparation of white bread usually
a fraction is taken that contains all the endosperm plus so much of the
other parts that about 70 % of the kernel is present in this fraction. We call
this a 70 % extraction flour. The thiamine content of this flour is about 60
y per 100 g. In wartime in England, Holland, and in several other countries
a flour of about 85 % extraction was used for bread making. The thiamine
content of this fraction was about 240 y per 100 g.
In the process of bread-making yeast is added, so that the thiamine con-
tent increases somewhat, but because of the high baking temperature part
of the thiamine, especially in the crust, is destroyed. White bread contains
approximately 40 y of thiamine per 100 g., whole meal bread about 180
y, and the 85 % extraction war bread about 120 y. To the ‘‘enriched”’ bread
so much synthetic thiamine has been added that the total thiamine amounts
to 110 to 180 vy per 100 g.
(2) Rye. Rye bread usually is made of the whole kernel. Sometimes flour
is used. The thiamine content of rye bread is somewhat lower than that of
the corresponding wheat bread.
(3) Rice. The thiamine content of rice is lower than that of wheat, namely
about 300 y per 100 g. In home-pounded rice, most of the thiamine remains.
In machine-milled rice, only about 30 y per 100 g. is left. Rice is always
thoroughly washed before cooking. This reduces the thiamine content still
further.
In the native process of parboiling, the rice is first soaked in water, then
boiled and dried. This product is milled. The resulting parboiled rice con-
tains nearly all the thiamine of the whole rice kernel. About the same proc-
ess 1s used in industry for the preparation of ‘‘converted”’ rice. This also
contains nearly all the thiamine of the original rice. ‘‘Enriched”’ rice con-
tains about 500 y of thiamine per 100 g.
(4) Corn. The thiamine content of corn is not very high. Its chief de-
ficiency is that of niacin and tryptophan. Therefore, pellagra is the disease
of the corn-eating countries. However, in some cases of pellagra clinical
signs of a thiamine deficiency may be found. For the prevention and ther-
apy of pellagra an extra addition of thiamine, besides niacin, is necessary.
b. Legumes
Peas and beans are among the foods richest in thiamine. They contain
about 500 to 600 y of thiamine per 100 g. After the discovery that beriberi
was caused by the use of polished rice, “katjang idjo,” a kind of green peas,
was employed in the prevention of beriberi.®
Soybeans are just as good a source of thiamine as the common pulses.
IX. OCCURRENCE IN FOOD 457
In Southeast Asia soybeans are often fermented by the mould Aspergillus
oryzae. The product is digestible and palatable. However, it has lost a con-
siderable part of its thiamine. So the fermentation process does not mean
an improvement in regard to thiamine (Jansen®),.
c. Nuts
Nuts are pretty rich in thiamine (300 to 500 y per 100 g.). Coconuts con-
tain only traces of thiamine.
d. Potatoes
In countries where potatoes are much in use, as in Western Europe,
potatoes may fulfill a large part of the thiamine need of the population be-
cause of their high thiamine content. This content does not appear to be
very high, 1.e., 80 to 100 y of thiamine per 100 g., but potatoes contain
about 75 to 80% of water. Thus, in terms of dry matter, the content is
four to five times as large.
Boiling potatoes reduces the thiamine content very little. Boiling peeled
potatoes in a large quantity of water produces a slight reduction, but al-
most all the thiamine will remain in the potatoes and only traces are found
in the water, provided that the boiling ceases when the potatoes are done.
If however, the boiling has been prolonged until the potatoes fall apart,
then the thiamine is extracted by the water.
These factors are of consequence for such European countries as the
Netherlands where potatoes form a large part of the diet and thus con-
stitute’ an important source of thiamine.
On account of the fact that the potato was one of the chief food items
in wartime in the Netherlands, an investigation was made into the thiamine
content of different varieties of potatoes, raised under different conditions
of soil, manure, etc. More than 100 varieties were analyzed.’ It appeared
that the thiamine content of different samples varied from 40 y to 160 y
per 100 g. of fresh potato. However, in the varieties that were in common
use the content amounted to 85 to 100 y per 100 g.
e. Meat and Fish
The vitamin content of the organs, particularly liver and kidney, is
higher than that of the muscles. This is also the case with thiamine. Beef
contains about 120 y of thiamine per 100 g. Since thiamine plays such an
important part in muscle activity, however, it is obvious that the thiamine
content is highest in those muscles which perform most of the work, as,
e.g., the breast muscles in flying birds.
9A. J. Reestman, B. C. P. Jansen, and J. Bouman, Landbouwkund. Tijdschr. 55,
711 (1943).
458 THIAMINE
It is a remarkable fact that the meat of pigs is very rich in thiamine.
Pork is one of the richest sources of thiamine. Lean pork contains nearly
1 mg. of thiamine per 100 g. Fish on the whole contains somewhat less
thiamine than beef, but the difference is not very large. In eggs, most of
the thiamine is in the yolk; the thiamine content of fresh yolk is about 300
y per 100 g.
f. Milk
Taking into account the fact that the amount of dry matter in fresh
cow’s milk is only 18%, the thiamine content, about 45 y per 100 g., i
fairly good. Human milk contains only one-third as much thiamine as
cow’s milk. Part of the thiamine in human milk is in the form of pyro-
phosphate and of orthophosphate.'°
Kon (in Reading, England) estimated the influence of pasteurization and
sterilization on cow’s milk that is used for feeding infants. Pasteurization
of the milk reduces the thiamine content about 10%, and the more drastic
sterilization procedures may destroy 30% or more.!! If we compare the
content of this sterilized milk with the recommendation of the Food and
Nutrition Board of the American National Research Council for infants,
we see that the margin of safety for thiamine in the sterilized milk used
as an infant food is small. On the other hand, in breast milk the thiamine
content is still lower. Holt et al.,! who themselves estimated the thiamine
requirement of infants, draw attention to this fact. They suppose that
perhaps in infants fed with breast milk part of the required thiamine is
synthesized by the microflora in the gut.
Fortunately most infant foods are acidified (by churning or by the addi-
tion of an acid), and in these preparations the thiamine is much more stable.
B. OCCURRENCE OF NATURAL ANTITHIAMINES
AND THIAMINASES IN FOODS
Not so much is known about natural antithiamines in food. There is
quite a literature, however, on thiaminases. The first indications of a harm-
ful influence from the consumption of fresh-water fish were found on a
silver fox farm of a Mr. Chastek. These foxes developed a paralysis that
was afterward called the Chastek paralysis. Green et al.'*» 4 demonstrated
that this disease arises in foxes on a diet containing 10 % or more of fresh
108. de Jong, Enzymologia 10, 253 (1942).
1 A K. Kon, Brit. Med. Bull. 5, 170 (1947).
12 [,, meets Holt, Jr., R. L. Nemir, S. E. Snyderman, A. A. Albanese, K. C. Ketron,
L. P. Guy, and R. Carretero, J. Nutrition 37, 53 (1949).
13 R. G. Green, W. E. Carlson, and C. A. Evans, J. Nutrition 21, 243 (1941).
144 R. G. Green, W. E. Carlson, and C. A. Evans, J. Nutrition 23, 165 (1942).
IX. OCCURRENCE IN FOOD 459
uncooked fish and that it could be prevented or cured by giving thiamine.
Woolley’ and Spitzer et al.'® found that a heat-labile principle, probably
an enzyme from an extract of carp viscera, was able to inactivate a thi-
amine solution zn vitro. Krampitz and Woolley” showed the enzyme activ-
ity of the fish principle by isolating the split products from thiamine:
4-methyl-5-hydroxyethylthiazole and 2-methyl-4-amino-5-hydroxymethyl-
pyrimidine.
Sealock and Livermore* realized that this reaction gave rise to hydrogen
ions.
a
N—CH Cl C=C—C:,H;0OH
| ll ve
eH,—C C—CH;—N + Hs) —
eer N
N—C—NH;-HCl H-C—S§
CH;
|
N—C-H-HCl Cl C—=C—C.H;0OH
| | |Z
CH;—C C—CH.0OH + N + Ht
a
N=CH Can
fe
H
By using a bicarbonate buffer in a Warburg apparatus Sealock and Liver-
more’® were able to study the kinetics of the reaction by measuring the
carbon dioxide that was set free by the hydrogen ions. Probably this same
reaction might be used to estimate the thiaminase content of foods. This
enzyme, the thiaminase, is present in several species of fishes, mostly in
fresh-water fishes (Deutsch et al.!*), but also in the Atlantic herring and
in aquatic animals like the clam (Melnick et al.?°). Jacobsohn and Azevedo?!
found that the tissues of shrimps and some mussels are rich in thiaminase,
but crabs do not contain it. Redi and Giri” could demonstrate the presence
of two different thiaminases in fresh-water mussel. Thus the substances
15D. W. Woolley, J. Biol. Chem. 141, 997 (1941).
16 E. H. Spitzer, A. J. Combes, C. A. Elvehjem, and W. Wisnicky, Proc. Soc. Expil.
Biol. Med. 48, 376 (1941).
77. O. Krampitz and D. W. Woolley, J. Biol. Chem. 152, 9 (1944).
1 R.R. Sealock and A. H. Livermore, J. Biol. Chem. 166, 379 (1944).
19 H. F. Deutsch and A. D. Hasler, Proc. Soc. Exptl. Biol. Med. 58, 63 (1943).
20 1). Melnick, M. Hochberg, and B. L. Oser, J. Nutrition 30, 81 (1945).
*1 K.P. Jacobsohn and M. D. Azevedo, Arch. of Biochem. 14, 83 (1947).
2K. K. Redi and K. V. Giri, Enzymologia 18, 281 (1949).
460 THIAMINE
exercising an antithiamine activity in fish and other sea food are thiamin-
ases. Some reports have been published on plants that show an antithiamine
activity, but it has not yet been established whether these plants contain
antithiamines or thiaminases.
Weswig et al.?> found that ferns (Pteris aquilina) have an antagonistic
activity against thiamine. Horses fed a fodder containing large amounts of
ferns show signs of polyneuritis and can be cured by thiamine.
Bhagvat and Devi™ found antithiamine activity in extracts from rice
polishings, beans (Phaseolus radiatus), mustard seed, etc.
Somogyi,’ in a comprehensive investigation of antithiamine factors, not
only confirmed their existence in carp viscera but also demonstrated that
extracts of organs of warm-blooded animals (rabbits or chicks) have a
considerable antithiamine activity. The greatest activity was observed with
extracts of heart and spleen. Liver extract showed a moderate activity,
whereas the extract of intestines of warm-blooded animals had only a slight
action in contrast to that of carp extracts. The active principles obtained
from warm-blooded animals are thermolabile and do not dialyze, so prob-
ably they contain thiaminases.
X. Effects of Deficiency
B. C. P. JANSEN
A. IN MICROORGANISMS
Of the effects of thiamine deficiency on microorganisms, not much is
known. The microorganisms that cannot synthesize thiamine can be used
for the microbiological estimation of thiamine.
B. IN ANIMALS
As far back as 1892 Eykman published his results about the signs of
nerve degeneration in fowls that were fed with thiamine-poor polished rice.
From this work started the whole vitamin research. Thus the degeneration
of the peripheral nerves was the first pathological symptom noted. Eykman
stained the nerves with Marchi solution, and he thought that in the poly-
neuritic animals the axis of the nerves was degenerated. Half a century
afterward in an extensive examination of the peripheral nerve fibers in
23 P. H. Weswig, A. M. Freed, and J. R. Haag, J. Biol. Chem. 165, 737 (1946).
24K. Bhagvat and P. Devi, Indian J. Med. Research 32, 131 (1944).
25 J. G. Somogyi, Die Antianeurin-factoren. Hans Huber, Bern, 1952.
X. EFFECTS OF DEFICIENCY 461
thiamine deficiency Swank and Prados! made the observation that the
first neuronal histologic change in thiamine-deficient pigeons is degeneration
of the distal part of the axon, and changes in myelin are secondary to this,
and further that opisthotonus (the characteristic manifestation of acute
thiamine deficiency in pigeons) may not be attended by any definite neuro-
logic lesions.
About thirty years after the work of Eykman, Peters in Oxford, England,
was able to demonstrate that not only the peripheral nerves, but also the
central nervous system was affected by a thiamine deficiency. From that
work of Peters resulted a large part of our knowledge of the role thiamine
plays in carbohydrate, especially in pyruvate, metabolism. It is probable
that most of the pathology of thiamine deficiency is due to a disturbance
in the carbohydrate metabolism. The comprehensive work of Peters and
his school on the details of the biochemical action of substances causing
pathological effects and in particular in trying to understand the initial
changes from thiamine deficiency has led him to call these initial changes
“biochemical lesions.’ Peters studied the epistothonus signs induced in the
rice-fed pigeon by thiamine deficiency. The epistothonus signs clear up very
quickly when thiamine is given, and there is no detectable histological
damage at this stage. It is in this sense an example of a pure ‘“‘biochemical
lesion.”’
The clinical symptoms of thiamine deficiency are connected with the
metabolic disturbances. How close the connection between both is, is not
precisely known. These symptoms are nearly the same in different animals.
Usually there are signs of lameness, of convulsions, accompanied in pigeons
with head retraction and in rats with walking in a circle, and of “biochemical
lesions.”’ Other signs are anorexia, reduction of growth or decline in weight,
and emaciation. As Drummond has emphasized, many of these signs are
not independent from each other. Thus the anorexia may be the cause of
the decline in weight. In rats the heart rate is reduced:’ in normal rats the
rate is 500 beats per second; in severe deficiency it is not more than 250
to 300. This fact was used by Birch and Harris* as an indication of the
severity of thiamine deficiency in rats. (In human beings just the opposite
takes place; thiamine deficiency leads to tachycardia For an extensive
investigation on the pathology of thiamine deficiency in monkeys, see, e.g.,
Rinehart ef al.
1R. L. Swank and M. Prados, Arch. Neurol. Psychiat. 47, 97 (1942); M. Prados and
R. L. Swank, Arch. Neurol. Psychiat. 47, 626 (1942).
2?R. A. Peters, Proc. Roy. Soc. Med. 41, 781 (1948).
3T. W. Birch and L. J. Harris, Biochem. J. 28, 602 (1934).
‘J. F. Rinehart, L. D. Greenberg, and M. Friedman, Am. J. Pathol. 23, 879 (1947).
462 THIAMINE
As thiamine in the form of thiamine pyrophosphate is necessary for the
metabolism of pyruvate, one would expect that the amount of pyruvate
in blood, and perhaps also in urine, may be increased in thiamine deficiency.
Platt® and Platt and Lu® indeed found a large increase of bisulfite-binding
substances, consisting mainly of pyruvic acid, in the blood of beriberi pa-
tients. Some hours after the administration of thiamine to the patients the
amount of the bisulfite binding substances dropped to normal. In animal
experiments Thompson and Johnson’ found that pigeons and rats with
symptoms of acute thiamine deficiency had a high blood pyruvate level.
One would be inclined to think that these symptoms of acute thiamine
deficiency might be caused by a pyruvate poisoning of the animal. The
work of de Jong,® however, makes this supposition highly improbable.
De Jong devised a micromethod for determining the pyruvate level of a
small drop of blood; he thus was able to execute several determinations in
the course of the development of polyneuritis in the animals. In this way
he proved that the symptoms of acute polyneuritis in pigeons developed
before the rise of pyruvate, and the disappearance of the symptoms after
the thiamine administration preceded the return of the pyruvate level to
normal.
The results of this work of de Jong seem to demonstrate that the poly-
neuritis signs are independent of the pyruvate metabolism and, for that
reason, of the catalytic action of thiamine pyrophosphate.
From the work of Loewi and of Dale we know that a chemical substance,
the acetylcholine, plays a role in the transmission of nerve stimuli.
Binet and Minz’ showed that the acetylcholine not only plays a role in
the transmission of the stimulus from the end of the nerve to the effective
organ but that it works also in the nerves themselves. The acetylcholine
content of the nerves was increased after electric stimulation. However the
addition of an extract of the non-stimulated nerve to that of the stimulated
nerve intensified the action of this extract, or of a solution of pure acetyl-
choline. This points to another substance present in the extract of the
stimulated nerve. The authors, considering that thiamine deficiency leads
to polyneuritis, assumed that the active substance of the stimulated nerve
might be this vitamin. So they compared the activity of the substance of
the stimulated nerve with that of thiamine. They found that their activities
in this respect were the same. They further showed that thiamine also
stimulates the activity of acetylcholine in the isolated intestine of the rat
5B. 8. Platt, Trans. Roy. Soc. Trop. Med. Hyg. 31, 493 (1938).
6 B.S. Platt and G. D. Lu, Biochem. J. 38, 1525 (1939).
7R. H.S. Thompson and J. R. Johnson, Biochem. J. 29, 694 (1935).
8S. de Jong, Arch. néerl. physiol. 21, 465 (1986).
9L. Binet and B. Minz, Compt. rend. soc. biol. 117, 1027 (1934).
X. EFFECTS OF DEFICIENCY 463
and in the circulatory organ of the cat, in the absence of the eserine that
inhibits the enzyme cholinesterase.
The liberation of thiamine (or a thiamine derivative) by nerve action was
for the first time demonstrated by Minz.!° Lwoff'! had devised a highly
sensitive microbiological method for the estimation of very small quantities
of thiamine, using Flagellatae. With this method Minz compared the libera-
tion of thiamine from excised resting ox nerves with that of electrically
stimulated nerves. He was able to prove that the stimulated nerves deliv-
ered much more thiamine than the resting nerves (four to eight times as
much). Shortly afterward these results were confirmed and extended by
von Muralt. This Swiss investigator has given a review of all the work,
mostly from his own laboratory, on thiamine and peripheral neurophysi-
ology.” Von Muralt and his collaborators quickly froze exited nerves or
resting nerves in liquid air. The frozen nerves were ground in a mortar and
extracted for only 10 minutes with Ringer’s solution. The thiamine content
of the extracts was estimated by several different methods. The extracts of
the stimulated nerves were richer in thiamine than the extracts of the
resting nerves. This means that thiamine in the exited nerves is in such a
state that more can be extracted by Ringer’s solution in 10 minutes than
can be obtained from a corresponding sample of unexcited nerves. The
different methods for estimating the liberated thiamine yielded on the whole
practically the same results; however, the yeast ferment method of Atkin,
Schultz, and Frey showed much lower values for the thiamine content of
both excited and resting nerves; and also the difference between excited
and unexcited nerves had disappeared. It was not yet possible to give an
explanation for this discrepancy. Furthermore it was difficult to draw con-
clusions from these experiments because A. Wyss and F. Wyss" in the
laboratory of von Muralt found that by poisoning the nerves by mono-
iodoacetic acid more thiamine is obtained in the extract of resting nerves
than in the extract of excited nerves. In most experiments thiamine exerts
an inhibiting effect on the vagus or acethycholine action on the heart. By
replacing certain groups in the thiamine molecule by other groups, the
effect is mostly reduced but not abolished. It is amazing, however, that
the thiamine pyrophosphate has no action at all!
From all these facts it may be assumed that thiamine or a thiamine
compound plays a role in neurophysiological activity.
However, there is as yet no convincing evidence that the neurological
active substance is thiamine itself or one or more derivative(s) of thiamine.
1B. Minz, Compt. rend. soc. biol. 127, 1251 (1938).
1M. Lwoff, Compt. rend. soc. biol. 128, 241 (1938).
2A. yon Muralt, Vitamins and Hormones 5, 93 (1947).
13 A. Wyss and F. Wyss, Experientia 1, 160 (1945).
464 THIAMINE
Von Muralt proposes that as long as this uncertainty exists this substance
be called ‘‘the second Vagusstoff,” because Loewi, before he understood the
exact nature of the chemical mediator in the heart, called it ““Vagusstoff.”’
Thus we have two well-established facts concerning the pathology of
thiamine in animals. The first is the activity of thiamine pyrophosphate
as a coenzyme in the carbohydrate metabolism; the second is the role of
thiamine or a derivative of thiamine (the ‘‘second Vagusstoff”’) on the
neurophysiological activity. We do not yet know whether these facts are
closely connected, or whether they are quite independent from each other.
From the fact that the thiamine pyrophosphate lacks the neurophysiological
action of thiamine it is probable that the neurophysiological activity is dif-
ferent from the activity on the carbohydrate metabolism.
On the other hand we know that thiamine pyrophosphate is active in
the production of acetic acid which is essential to restore the active acetyl-
choline from the inactive choline that is formed from acetylcholine by the
action of the cholinesterase.
C. IN MAN
In the beginning of vitamin research it was easy to compose a diet for
the study of thiamine deficiency. With fowls or pigeons as experimental
animals, polished rice, after being washed in running water to remove the
last traces of thiamine, was a suitable diet. Polished rice, however, not only
lacks thiamine, but it also shows a shortage of many other nutrients. There-
fore when other experimental animals, e.g., rats, were used, it appeared
necessary to add these other nutrients (proteins, mineral salts, nearly all
other vitamins) or to compose a complete, synthetic diet.
Williams et al.,!4 in their experiments to investigate the signs of a pure
thiamine deficiency in volunteers on a thiamine-poor diet, found no signs
of edema in their experimental persons, whereas the natives in rice-consum-
ing countries recognize beriberi because of the signs of edema in these
patients.
Therefore, to study the effects of a pure thiamine deficiency it is neces-
sary to provide a diet that contains all nutrients in physiological amounts,
except thiamine. Since we do not yet know all the essential nutrients, it is
very difficult to compose a suitable synthetic diet, not considering the cost
of some nutrients! So it is better to choose a good natural diet in which only
the thiamine is destroyed. In these diets the thiamine is often destroyed
by autoclaving for several hours at pH 5. But by this procedure other nu-
trients are damaged too. A more specific way to destroy the thiamine is to
MR. D. Williams, H. L. Mason, B. F. Smith, and R. M. Wilder, Arch. Internal Med.
69, 721 (1942).
X. EFFECTS OF DEFICIENCY 465
treat the diet, or the thiamine-containing parts of the diet, with sulfite.!®» '
Presumably the most specific method of destroying the thiamine would be
treatment with thiaminase. Smith and Proutt!” stated that cats fed a diet
consisting exclusively of thiaminase-rich raw carp developed all the signs
of the thiamine deficiency characteristic for this animal. To counteract
specifically the activity of thiamine, the antithiamines, e.g., pyrithiamine
or oxythiamine, can be used. Woolley'’ was able to demonstrate that at
least one of the activities of pyrithiamine consists in the antagonizing of
the synthesis of cocarboxylase—the active form of thiamine in carbohy-
drate metabolism. Woolley and White! state that, whereas mice fed a
ration free of thiamine develop no characteristic symptoms of thiamine
deficiency, the same animals, on administration of pyrithiamine, do show
many of these symptoms. Therefore the best way to study the effects of
uncomplicated thiamine deficiency presumably is the use of antithiamines
and thiaminases, added to an otherwise optimal diet. A great difficulty is
the detection of the first signs of a deficiency. This is important because
beriberi is not found in Western countries, but it is possible that even here
many persons suffer from a mild thiamine deficiency. Usually the excretion
of thiamine into the urine per 24 hours is determined, or during 3 hours
after giving a measured dose of thiamine. Also the blood thiamine level is
used as a yardstick. However, this gives only a vague indication.?® Swank
and Jasper?! compared encephalograms of normal pigeons with those of
thiamine-deficient birds. An increase in the brain potentials occurred slowly
in the thiamine-deficient pigeons and preceded the development of clinical
signs. Shortly before the appearance of preopisthotonus the amplitude of
the brain potentials became three times as high as during the control
period. The administration of thiamine to pigeons with preopisthotonus
caused return of the brain waves to normal.
Horwitt and Kreisler,” from their work on patients on diets with differ-
ent thiamine levels, tried to devise an index of carbohydrate metabolism,
correlating the levels of glucose, lactic acid and pyruvic acid in the blood
of the patient after a measured exercise and the carbohydrate metabolism
which is influenced by the thiamine intake. This carbohydrate index was
15 R. R. Williams, R. E. Waterman, J. C. Keresztesy, and E. R. Buchman, J. Am.
Chem. Soc. 57, 536 (1935).
16 A. §. Schultz, L. Atkin, C. N. Frey, and R. R. Williams, J. Am. Chem. Soc. 63,
632 (1941).
177). C. Smith and L. M. Proutt, Proc. Soc. Exptl. Biol. Med. 56, 1 (1944).
1 DT). W. Woolley, J. Biol. Chem. 191, 43 (1951).
19T). W. Woolley and A. G. C. White, J. Biol. Chem. 149, 285 (1948).
20 J. k. Kirk and M. Chieffi, J. Gerontol. 5, 236 (1950).
21 R. L. Swank and H. H. Jasper, Arch. Neurol. Psychiat. 47, 821 (1942).
22M. K. Horwitt and O. Kreisler, J. Nutrition 37, 411 (1949).
466 THIAMINE
thought to be an indication of the state of thiamine nutrition before any
clinical signs of thiamine deficiency occurred.
Mouriquand and Coisnard”’ observed that pigeons on a thiamine-poor
diet demonstrated a fall in the chronaxy of the nerves and that this fall
begins before the clinical signs appear.
XI. Pharmacology
KLAUS R. UNNA
Thiamine has been shown to produce a variety of pharmacologic effects.
It should be borne in mind that these effects have been obtained in experi-
mental animals maintained on adequate diets only on parenteral adminis-
tration of thiamine in doses several thousand times larger than those
required for optimum nutrition. These pharmacologic effects in animals
have no counterpart in the therapeutic use of the vitamin in man.
Death after intravenous injection of thiamine in animals is due to de-
pression of the respiratory center.‘* The heart is still beating at the time
of cessation of the respiration. Artificial respiration enables the animals to
survive otherwise lethal doses;? doses of thiamine resulting in concentra-
tions of 7 to 10 mg. % in the blood were fatal to dogs (under ether anes-
thesia), whereas blood levels of 36 mg.% were tolerated when artificial
respiration was provided.
Rapid intravenous injections of 5 to 50 mg. per kilogram cause a transient
fall in blood pressure in cats and dogs which increases with increasing
dosage of thiamine. The fall in blood pressure is not influenced by atropine
or antihistamines; it may be accentuated after adrenergic blockade with
dibenamine.®: © There is evidence that the fall in blood pressure is due to
thiamine acting at several sites: on the vascular smooth muscle itself, on
the vasomotor center, and on the heart. Perfusion experiments on the
rabbit’s ear and on various arterial areas in dogs have shown that part of
the vasodilator effect obtained was due to the acidity of the highly con-
centrated thiamine solution. Experiments on decapitated cats in which the
23 G. Mouriquand and J. Coisnard, Presse méd. 1944, 277.
1H. Molitor and W. L. Sampson, #. Merck’s Jahresber. 51, 3 (1936).
2G. Hecht and H. Weese, Klin. Wochschr. 16, 414 (1937).
3 J. A. Smith, P. P. Foa, H. R. Weinstein, A. S. Ludwig, and J. M. Wertheim, J.
Pharmacol. Exptl. Therap. 98, 294 (1948).
4T. J. Haley, Proc. Soc. Exptl. Biol. Med. 68, 153 (1948).
5H. Mazella, Arch. intern. pharmacodynamie 86, 484 (1951).
6S. H. Jaros, A. L. Wnuck, and E. J. de Beer, Ann. Allergy 10, 291 (1952).
i
XI. PHARMACOLOGY 467
hypotensive effect of thiamine was markedly diminished indicate that the
vasodilatation may be of central origin. A moderate decrease in heart rate
following the injection of large doses of thiamine may contribute to a minor
extent to the fall in blood pressure.
Thiamine has little, if any, effect on the isolated heart of the frog!) ? 7-9
or of the turtle.s Whether the bradycardia observed in dogs’: *: !° is caused
by an action of thiamine on the cardiac vagus or on the medullary centers
remains undecided. Studies on the dog heart lung preparation® have failed
to show any change in heart action with concentrations of thiamine far
exceeding those which produced hypotension in the intact dog.
Thiamine is without effect on the isolated intestine of rats, rabbits, and
guinea pigs and on the guinea pig uterus.!: §: " The claim that acetylthi-
amine has an acetylcholine-like effect on the gut” has not been confirmed.!!
In large concentrations thiamine inhibits the action of nicotine on the iso-
lated intestine of rabbits and guinea pigs without interfering with responses
to acetylcholine or epinephrine; the thiazole moiety of the vitamin, 4-
methyl-5-hydroxyethylthiazole, has similar effects.!®? Thiamine also pre-
vents the rise in blood pressure induced by nicotine." It blocks transmission
of nerve impulses through the superior cervical ganglion.'®
Besides its ganglionic depressant action at large doses, thiamine, in still
larger doses, depresses the transmission of impulses to the skeletal muscle
at the neuromuscular junction.": '® In accord with this curare-like action,
it has been found to depress the response of the skeletal muscle to acetyl-
choline.'®: !° Curarizing effects could be demonstrated in intact mammals
only under artificial respiration following excessive, otherwise lethal doses
of thiamine. Curare-like paralysis of the respiratory muscle is not the cause
of death by intravenous injection of thiamine, since the diaphragm responds
to direct and indirect electrical stimulation at the time respiration has
ceased.? 4-Methyl-5-hydroxyethylthiazole was found to have curare-like
action similar to thiamine.?°
7P. Kaiser, Pfliiger’s Arch. ges Physiol. 242, 504 (1939).
8 V. Erspamer, Arch. intern. pharmacodynamie 63, 261 (1939).
9. M. Boyd and R. W. Dingwall, Quart. J. Pharm. Pharmacol. 14, 209 (1941).
10 R. Tislowitz and I. Pines, Klin. Wochschr. 16, 923 (1937).
1 R. Dufait, Arch. intern. pharmacodynamie 66, 274 (1941).
! R. Kuhn, T. Wieland, and H. Huebschmann, Z. physiol. Chemie 259, 48 (1939).
18K. Unna and E. P. Pick, J. Pharmacol. Exptl. Therap. 81, 294 (1944).
4. P. Pick and K. Unna, J. Pharmacol. Exptl. Therap. 87, 138 (1946).
1° H. Mazella and N. Ferrero, Arch. intern. pharmacodynamie 82, 220 (1950).
16 V. Demole, Kongressber. XVI Intern. Physiol. Kongr. II, 19 (1988).
7 J. A. Smith, P. P. Foa, and H. R. Weinstein, Science 108, 412 (1948).
18 C. Torda and H. G. Wolff, Proc. Soc. Exptl. Biol. Med. 56, 89 (1944).
197). P. Sadhu, Am. J. Physiol. 147, 233 (1946).
20 J. A. Smith, P. P. Foa, and H. R. Weinstein, Am. J. Physiol. 155, 469 (1948).
468 THIAMINE
Excessive doses of thiamine may produce bronchoconstriction in dogs.?!
Application of a 2 to 10% solution of thiamine directly to the motor cortex
of dogs caused generalized convulsions; this effect was not obtained with
either of the two moieties of the thiamine molecule.”
Since thiamine or a thiamine-like substance has been reported to be re-
leased together with acetylcholine on electrical stimulation of cholinergic
nerves," 24 numerous studies have been carried out with the object of
studying a possible interdependence of the effects of thiamine and acetyl-
choline. Thiamine was found to potentiate the effects of acetylcholine on
the leech muscle?®: 2° and on other preparations (for references, see Minz?’).
The required concentrations of thiamine were large and far beyond those
found in normal tissues. Other studies on isolated organs (intestine, uterus,
leech muscle, frog heart, frog rectus muscle), however, have failed to dem-
onstrate any sensitizing effect of thiamine on the action of acetylcholine;
at concentrations of 1 to 10 mg. % in the nutrient solution, thiamine de-
pressed the effects of acetylcholine.”: *:" Thus, the influence which thi-
amine may exert on the reactivity of the tissues to acetylcholine appears
not to be sufficiently substantiated to allow general conclusions. Thiamine
in large concentrations inhibits cholinesterase.**: °° To what extent this
action may be involved in some of the pharmacodynamic effects of thi-
amine is difficult to assess. Lacking data on the actual acetylcholine levels
in the tissues of thiamine-treated animals, there is little reason to compare
thiamine to such a potent cholinesterase inhibitor as eserine. Elucidation
of the interdependence between acetylcholine and thiamine has already
been initiated at the biochemical level; an interdependence with regard to
the pharmacological effects of the vitamin has yet to receive unequivocal
substantiation.
The lethal doses of thiamine by various routes of administration have
been determined in a number of species.! On intravenous injection the
lethal doses in mice were 125 mg. per kilogram; in rats, 250 mg. per kilo-
gram; in rabbits, 300 mg. per kilogram; and in dogs, 350 mg. per kilogram.
The ratios of the lethal doses on intravenous injection to those on subcu-
21M. Post and J. A. Smith, Am. J. Physiol. 163, 742 (1950).
22M. V. Dias, Science 105, 211 (1947).
23 B. Minz, Compt. rend. soc. biol. 127, 1251 (1938).
24 A. von Muralt, Nature 152, 188 (1943).
25 B. Minz and R. Agid, Compt. rend. 205, 576 (1937).
26. yon Bruecke and H. Sarkander, Arch. exptl. Pathol. w. Pharmakol. 195,
218 (1940).
27 B. Minz, La transmission chimique de l’influx nerveux, p. 155. Flammarion,
Paris, 1947.
28D. Glick and W. Antopol, J. Pharmacol. Exptl. Therap. 65, 389 (1939).
29 W. Sullmann and H. Birkhauser, Schweiz. med. Wochschr. 69, 648 (1939).
XI. PHARMACOLOGY 469
taneous and oral administration were found to be 1:6:40. These data on
lethal doses in mice and rabbits have been confirmed.2 In monkeys, intra-
venous administration of 200 mg. per kilogram failed to elicit any symp-
toms,' and only 600 mg. per kilogram caused the first toxic symptoms.?
It is interesting to note that dogs and particularly monkeys are less sensi-
tive than rodents. Lethal doses of thiamine mononitrate as determined in
mice and rabbits were not significantly different from those of thiamine
hydrochloride.*
On intravenous injection of 50 mg. per kilogram daily for a period of 4
weeks, rabbits failed to show loss in weight, or other toxic manifestation.
No pathologic tissue changes were found on autopsy.2 Rats have been
maintained for three generations on a daily intake of 0.08 to 1.0 mg. of
thiamine, i.e., doses up to one hundred-fold of the daily requirement for
the vitamin, without any untoward effects.*° Other observations that daily
subcutaneous injections of 0.1 mg. of thiamine, though not affecting growth,
caused impairment of lactation and cannibalism and decreased fertility in
the second generation*! can hardly be taken as evidence of thiamine toxicity,
since these experiments were inadequately controlled, and the same mani-
festations were obtained in rats without thiamine injections on adding
manganese chloride to that particular diet.*? In the light of subsequent
discovery of other nutritional factors essential for the rat, these effects were
more likely due to inadequacy of the diet than to the injections of thiamine.
Prolonged daily administration of 1 mg. of thiamine to weanling rats main-
tained on a diet deficient in another B vitamin (riboflavin, pyridoxine, or
pantothenic acid) failed to cause significant effects on the weight of these
animals or on the manifestations of their deficiency state.**
The data on acute toxicity and the absence of evidence of cumulative
toxicity give evidence for the very large therapeutic margin of thiamine.
The ratio between the daily requirement for thiamine and its lethal dose
has been variously estimated at from 600 to 70,000 (depending on species
and route of administration).
No toxic effects of thiamine administered by mouth have been reported
in man. Parenterally, doses of 100 to 500 mg., in single and repeated injec-
tions, have been given to patients.**-*’ Toxic or other effects have not been
8° R. R. Williams and T. D. Spies, Vitamin B,; (Thiamine) and Its Use in Medicine,
p. 286. The Macmillan Co., New York, 1938.
1 DP. Perla, Proc. Soc. Exptl. Biol. Med. 37, 169 (1937).
#2 —D. Perla and M. Sandberg, Proc. Soc. Exptl. Biol. Med. 41, 522 (1939).
38K. Unna and J. D. Clark, Am. J. Med. Sci. 204, 364 (1942).
C.D. Aring and T. D. Spies, J. Neurol. Psychiat. 2, 335 (1939).
35. L. Stern, Am. J. Surg. 39, 495 (1938).
36N. Jolliffe, J. Am. Med. Assoc. 117, 1496 (1941).
7 A. Ruskin and G. M. Decherd, Jr., Am. J. Med. Sci. 213, 337 (1947).
470 THIAMINE
noted on many thousands of injections by either the subcutaneous, intra-
muscular, intraspinal, or intravenous route in doses which in many cases
were from one hundred to two hundred times larger than the daily main-
tenance dose. These excessive amounts have been well tolerated apparently
without any noticeable or measurable effects on circulation, respiration, or
other organ systems. A nut-like taste has been reported on injection of
large amounts of thiamine; this taste sensation has been used as criterion
for the measurement of circulation time by intravenous injection of 300 mg.
of thiamine.*”
In relatively rare instances, thiamine has caused reactions resembling
anaphylactic shock in man. Such reactions have been recorded in over two
hundred cases in the world literature (for extensive case references, see
Jaros et al.®). All reactions have occurred exclusively on parenteral adminis-
tration. They consist in their milder form of a feeling of burning and
warmth, urticaria, weakness, restlessness, sweating, nausea, tightness of
the throat and chest, dyspnea, hypotension, and tachycardia. In more
severe cases the symptoms may rapidly progress to angioneurotic edema,
cyanosis, pulmonary edema, hemorrhage into the gastrointestinal tract,
and collapse. Five cases of sudden death following intravenous or intra-
muscular injection of thiamine have been reported.**“! The signs and
symptoms of these reactions” are those of anaphylactic shock. Their onset
follows the injection within minutes. The patient may, in milder cases,
recover quickly. Treatment directed against the symptoms generally con-
sists in injection of epinephrine, artificial respiration, administration of
oxygen, and analeptics such as caffeine. The occurrence of these reactions
and their severity is not related to the dose of thiamine injected, which has
varied between 5 and 100 mg. The great majority of patients in which
these reactions have been observed had previously tolerated parenteral
injection of equal amounts of thiamine without any untoward effects. Thus,
they apparently developed a hypersensitivity to thiamine. Only in rare
instances*: 44 have such reactions been observed on the first known injection
of thiamine. Most of the reactions have been reported after four to ten or
more preceding injections.
These reactions are caused by thiamine and not by other solutes, sol-
vents, or preservatives in the solutions which were injected, since they
38 C, A. Mills, J. Am. Med. Assoc. 117, 1501 (1941).
39 T. M. Reingold and F. R. Webb, J. Am. Med. Assoc. 130, 491 (1946).
40 Fornara, cited by F. Dotti, Minerva med. 1, 720 (1949).
41 J. Arias, Rev. Med. Peruana 22, 160 (1951).
4 C. G. Weigand, Geriatrics 5, 274 (1950).
43M. M. Mitrani, J. Allergy 15, 150 (1944).
44 J. Seusing, Klin. Wochschr. 29, 394 (1951).
XI. PHARMACOLOGY 471
have occurred alike with preparations from different manufacturers and
also with aqueous solutions of crystalline thiamine hydrochloride.
Since the symptomatology of these reactions is consonant with most, if
not all, aspects of anaphylactic shock, the most likely explanation for the
mechanism of the reactions seems to be an anaphylactic one. In many of
the cases the observers have obtained immediate whealing on intradermal
injection of thiamine, and, in some, positive transference of the sensitivity
has been accomplished. Since the manifestations of the thiamine reactions
are those known to occur with certain immunologic alterations and since
these patients have been shown to have such immunologic alterations, it
would appear reasonable to associate the two. It is conceivable that a
combination of thiamine with protein develops which is antigenic to the
host. The evidence is, at present, not conclusive, and the anaphylactogenic
properties of thiamine require further investigation. If thiamine is an ob-
ligatory whealing agent,*® positive intradermal tests may not be valid proof
of individual sensitivity. Attempts to sensitize rabbits by massive and
repeated injections of thiamine have failed.*®
Positive patch tests obtained in individuals with these reactions do not
present immunologic evidence for the immediate anaphylactic type of
reaction. They rather indicate the existence of the delayed eczematous
type of hypersensitivity which would be the immunologic substrate for the
contact dermatitis type of reaction. In persons handling pharmaceutical
preparations of thiamine, occurrence of contact dermatitis on the hands
and forearms has been observed.”
Recently, on the basis of the similarity of hypotensive effects of thiamine,
acetylcholine, and histamine on intravenous injection in dogs, the sugges-
tion has been made®: * that overdoses of thiamine may cause an accumu-
lation of acetylcholine in excessive quantities in tissues which in turn may
be responsible for the occurrence of the untoward reactions observed in
man. Such an explanation lacks experimental evidence: the sudden onset
of these reactions, lack of correlation to dose administered, limitation to
parenteral injection, the manifestations comprising the entire spectrum of
anaphylactic signs and symptoms, and other facts militate against such an
assumption.
45 F. Kalz, J. Invest. Dermatol. 5, 135 (1942).
‘eT. J. Haley and A. M. Flesher, Science 104, 567 (1946).
“7 F.C. Combes and J. Groopman, Arch. Dermatol. and Syphilol. 61, 858 (1950).
4S. H. Jaros, Ann. Allergy 9, 133 (1951).
472 THIAMINE
XII. Requirements and Factors Influencing Them
A. OF ANIMALS
B. C. P. JANSEN
The animal body is unable to store thiamine to any large extent. An
adult human body does not contain more than about 30 mg. of thiamine.
As the body continually loses thiamine in the urine, feces, and perspira-
tion, it needs a constant supply.
It is difficult to find an exact criterion for measuring the requirement of
an animal. The growth curve of young animals is most frequently used as
a criterion. However, a drawback is that the curve indicating the influence
of the thiamine content of the diet on the growth of the animal is an asymp-
totic one. Thus it is difficult to fix the maximum (or “‘normal’’) growth.
Other methods used are the influence of the diet on the thiamine content
of the blood or on normal or abnormal metabolism, i.e., on the pyruvic acid
content of the blood.
As a thiamine deficiency produces anorexia, Cowgill! used the ‘‘normal
appetite” as a criterion.
Furthermore it is assumed that a certain (minimum) amount of thiamine
is essential to keep an animal alive, to promote normal growth, and to
protect it from polyneuritis. On the other hand, the work of Rasmussen
et al2 and of Foster et al.* clearly demonstrated that mice are more resistant
—or, as Schneider* puts it, less susceptible—to a certain strain of polio-
myelitis virus if the thiamine content of the diet is reduced to an amount
below the content that is required in other respects. Therefore it seems that
a diet which may be considered thiamine deficient gives these animals
better protection against poliomyelitis.
It is obvious that a great many factors exert influence on the requirement
of thiamine in animals. The factors studied in the greatest detail are:
1. Size of the animal.
2. Composition of the diet.
3. Physical state of the animal (hyperthyroidism, pregnancy, lactation,
fever, age, etc.).
4. Climate (temperature).
Intestinal microflora.
Individual genetic factors.
Performance of muscular work.
MD
1G. R. Cowgill, The Vitamin B Requirement of Man. Yale University Press. New
Haven, 1934.
2 A. F. Rasmussen, H. A. Waisman, C. A. Elvehjem, and P. F. Clark, J. Infectious
Diseases 74, 41 (1944).
’C. Foster, J. H. Jones, W. Henle, and F. Dorfman, J. exptl. Med. 80, 257 (1944).
471. A. Schneider, Vitamins and Hormones 4, 35 (1946).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 473
(1) Size of the Animal. We know that the rate of the metabolism of an
animal depends upon its body surface. Thus we expect that the require-
ment of thiamine, an agent in carbohydrate metabolism, will also depend
on the surface area of the body. Cowgill, in studying the thiamine require-
ments of mice, rats, pigeons, dogs, and human beings, found that their
requirement is proportional to their weight.°-%
As the metabolism is connected with the amount of calories an animal
consumes per day, it is probably better not to indicate the amount of thi-
amine an animal needs per day but to record the thiamine content of the
food (or, still better, the relation between thiamine and carbohydrate
intake: see below).
For the thiamine requirements of different kinds of animals, see: for the
growing rat, Brown and Sturtevant;> for the guinea pig, Mannering;® for
the mouse, Morris;’ for chicks, Bird;$ and for pigeons, Bird.$
(2) Composition of the Diet. Thiamine plays a role in carbohydrate me-
tabolism. Thus, in the first place, the thiamine requirement depends on the
carbohydrate content of the diet. More than two decades ago Evans and
Lepkovsky’® found the ‘‘thiamine-sparing” action of fats. Several other
authors confirmed this action.
Proteins" and alcohol: * also have a thiamine-sparing action. These
components of the diet may depress the thiamine requirement practically
to zero. The most probable deduction from this fact is that thiamine is
probably not involved in the enzyme system necessary for the metabolism
of fats, ete. This was confirmed by the work of de Caro and Rindi.* These
authors produced a state of athiaminosis in rats by feeding them a thiamine-
deficient diet, demonstrated by a rise in the pyruvic acid level of their
blood. Addition of fat to the diet reduced the pyruvic acid level to normal.
Part of the thiamine-sparing action probably is caused not only by the
reduction in carbohydrates in the diet, but also by microbial syntheses of
thiamine in the gut (see Section XII A (5).
We may mention here also the presence of antithiamines or of thiamin-
ase, each of which increases the requirement for thiamine.
®R. A. Brown and M. Sturtevant, Vitamins and Hormones 7, 176 (1949).
6G. J. Mannering, Vitamins and Hormones 7, 207 (1949).
7H. P. Morris, Vitamins and Hormones 5, 176 (1947).
8H. R. Bird, Vitamins and Hormones 5, 166 (1947).
9H. R. Bird, Vitamins and Hormones 5, 169 (1947).
10H. M. Evans and 8S. Lepkovsky, Science 68, 298 (1928); J. Biol. Chem. 83, 269
(1929).
uW. J. Dann, Federation Proc. 4, 153 (1945).
2 J. V. Lowry, W. H. Sebrell, F. S. Daft, and L. L. Ashburn, J. Nutrition 24, 73
(1942).
13 W. W. Westerfeld and E. A. Doisy, Jr., J. Nutrition 80, 127 (1945).
4 7,. de Caro and G. Rindi, Nature 167, 114 (1951).
474. THIAMINE
(3) Physical State of the Animal. As thyroxine regulates the (basal)
metabolism, it is to be expected that hyperthyroidism or the feeding of
extra doses of thyroxine will increase the requirements of thiamine. The
work of several investigators has confirmed this supposition (e.g., Him-
wich et al.,!° Cowgill and Palmieri,!® Drill and Sherwood,” and Peters and
Rossiter.!® A review of this work is given by Drill.'*
It is obvious also that pregnancy, in particular during the latter half, and
lactation increase the requirements of thiamine. There are reports that the
thiamine requirement of a rat successfully nursing a litter is five times as
large as normal (Evans and Burr,?° Sure,?! and Sure and Walker?’).
Mills et al.** have demonstrated that the thiamine requirement of rats per
gram of diet increases greatly with old age. The most probable explanation
for this fact is the supposition that the efficiency of thiamine utilization is
diminished.
Gerrits” observed that 38 infants, 0 to 244 months of age, never excrete
thiamine in the urine, independent of their nutrition. Hamil et al.,2° work-
ing with an improved method for the determination of thiamine, also found
low values for the thiamine in the urine during the first days of life. In this
respect it is interesting that the thiamine pyrophosphate content of the
blood of newborn infants is much higher than the content of the blood of
adults.”°?
(4) The Climate (Temperature). Kline et al.?® stated that by raising the
environmental temperature from 78°F. to 90°F. the thiamine requirement
of the rat is decreased.
Hegsted and McPhee” later found that on lowering of the environmental
temperature the thiamine requirement of rats increased considerably. At
78°F. the requirement of adult rats amounted to 164 to 168 y of thiamine
per 1000 non-fat calories; at 55°F. the figures were 191 to 203 y.
15H. KE. Himwich, W. Goldfarb, and G. R. Cowgill, Am. J. Physiol. 99, 689 (1932).
16 G. R. Cowgill and M. L. Palmieri, Am. J. Physiol. 105, 146 (1933).
17V. A. Drill and C. R. Sherwood, Am. J. Physiol. 124, 683 (19388).
18 R. A. Peters and R. J. Rossiter, Biochem. J. 33, 1140 (1989).
19V. A. Drill, Physiol. Revs. 28, 355 (1943).
20 H. M. Evans and G. O. Burr, J. Biol. Chem. 76, 263 (1928).
21 B. Sure, J. Biol. Chem. 76, 685 (1928).
22. B. Sure and D. J. Walker, J. Biol. Chem. 91, 69 (1930).
23 C. A. Mills, E. Cottingham, and E. Taylor, Arch. Biochem. 9, 221 (1946).
24W.B. J. Gerrits, Thesis, Amsterdam Noord-Hollandsche Uitgever Maatschappij,
1940.
25 B. M. Hamil, M. N. Coryell, C. Roderuck, M. Kaucher, E. Z. Moyer, M. E. Harris,
and H. H. Williams, Am. J. Diseases Children 74, 434 (1947).
%« F). Floryn, and H. Strengers, Acta Physiol. Pharmacol. Neerl. 2, 100 (1951).
26. L. Kline, L. Friedman, and E. M. Nelson, J. Nutrition 29, 35 (1945).
27D. M. Hegsted and G. S. McPhee, J. Nutrition 41, 127 (1950).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 475
This is in agreement with the results of the work of Ershoff,?> who found
that rats could survive on a thiamine-deficient diet for an average of 64.7
days at 23° (about 74°F.), whereas on the same diet the average surviving
time was only 27.6 days at 2° (approximately 36°F.).
Furthermore Sarett and Perlzweig?" demonstrated that with a thiamine-
rich diet the tissues laid down by rats at 91°F. were twice as rich in thi-
amine as the tissues from rats given the same diet at 75°F.
On the other hand Mills*®: *! found that rats require twice as much thi-
amine at 91°F. as at 65°F. He explains this by the heavy perspiration at
the higher temperature. In experiments on chicks, Mills ef al.*? were able
to establish the fact that the thiamine content of the diet required for pro-
tecting the animals from polyneuritis was three times as high at 90°F. as
at 70°F. Mills** points out that 2 to 3 weeks are required for metabolic
adaptation to heat, and he believes that the neglect of this fact may explain
the different results of Kline et al.
Edison et al.** from their experiments came to the conclusion that the
thiamine requirements for the growth of rats in a tropical environment
(90°F. and 70 % relative humidity) were not greater and may be less than
in temperate conditions (72°F. and 50% relative humidity).
Considering these conflicting results, it is obvious that other factors also
change at different temperatures, so that it is not a simple problem to find
the sole influence of the temperature. Kline et al.?* tried to eliminate the
influence of different levels of food intake at different temperatures by
giving the thiamine-free diet and the additional thiamine separately.
Not so much from the sum total of all these results but more from a
priort reasoning an optimal temperature for a minimum thiamine require-
ment probably will be found; above and below this temperature the re-
quirement will be higher. However, it is to be expected that this optimum
temperature will not be a fixed one but will also depend on other factors,
e.g., on humidity. At all events there seems to be a great difference in
thiamine requirement at varying temperatures.
(5) Intestinal Microflora. Several of the B vitamins are synthesized by
the microorganisms in the gut, some of them to such an extent that this
synthesis may replace the intake by food.
In some cases this is also true for thiamine. Thus as far back as 1915
8B. H. Ershoff, Arch. Biochem. 28, 299 (1950).
29H. P. Sarett and W. A. Perlzweig, J. Nutrition 26, 611 (19438).
30 C. A. Mills, Am. J. Physiol. 183, 515 (1941).
31 C, A. Mills, Proc. Soc. Exptl. Biol. Med. 64, 265 (1943).
32 C. A. Mills, E. Cottingham, and E. Taylor, Am. J. Physiol. 149, 376 (1947).
33 C. A. Mills, Nutrition Revs. 4, 95 (1946).
34 A. QO. Edison, R. H. Silber, and D. M. Tennent, Am. J. Physiol. 144, 648 (1945).
476 THIAMINE
Theiler et al.8° demonstrated that ruminants may be sustained on a thi-
amine-deficient food: the thiamine is produced by the flora of the rumen.
Fridericia eé al.°° found that rats that normally need the thiamine from
their food can produce sufficient thiamine in their intestines, if the diet
contains a large amount of fresh potato starch. They called this phenome-
non “‘refection.”’
Under normal circumstances, however, all non-ruminant higher animals
depend on their diets for their supply of thiamine. Apparently no one has
yet undertaken the experiments to feed animals on a carbohydrate-free diet
to see whether those animals and also the next generation can normally live
a whole lifetime without thiamine. However, Dann*’ was able to maintain
rats for more than a year (about half the lifetime of a rat) on a thiamine-
free, carbohydrate-free synthetic diet. It is possible that the refection—the
production of thiamine by the microorganisms in the gut—in this case is
sufficient to produce enough thiamine for protein and fat metabolism.
(6) Individual Genetic Factors. Practically all initial research on nutrient
requirements has been performed with large groups of animals or human
beings.
Already Ancel Keys, in his carefully conducted experiments with healthy
volunteers who were maintained for several months under strictly con-
trolled conditions, observed that one ‘‘normal”’ person may excrete two or
even three times as much thiamine as another “normal” person on exactly
the same diet (Mickelson et al.*). These volunteers were all ‘‘normal”’
young men with no history, signs, or symptoms of nutritional, digestive,
or metabolic peculiarities. Just recently, Williams*® has stressed the fact
that individual requirements may differ widely. Thus the need for thiamine
in man may vary from 0.5 to 1.5 mg. daily. Therefore it is possible that
the quantity contained in a certain nutrient, which is sufficient for the
average person or animal, may be too low for some individuals, depending
on their genetic makeup. Williams coined the term ‘‘genetotropic diseases”
for diseases that are caused by a genetically larger requirement of a nutri-
ent in a certain individual.*® Everyone experimenting with animals knows
that even in largely inbred rats individual requirements are widely different.
Therefore it is important to work with groups of at least eight to ten, but
preferably with even larger groups of animals, to obtain reliable average
35 A. Theiler, H. H. Green, and P. R. Viljoen, S. African Direct. Vet. Research Resp.
pp. 3-4 (1951).
86 L. S. Fridericia, P. Freudenthal, 8. Gudjonsson, G. Johansen, and N. Schoubye,
J. Hyg. 2%, 70) (1928).
37 W.. J. Dann, Federation Proc. 4, 153 (1945).
38 QO. Mickelsen, W. O. Caster, and A. Keys, J. Biol. Chem. 168, 415 (1947).
89 R. J. Williams, L. J. Berryand, and E. Beerstecher, Jr., Arch. Biochem. 28, 275
(1949).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 477
results for the requirements of animals. Light and Cracas*® determined the
thiamine requirements of different strains of white rats; one strain needed
twice the amount of thiamine as another strain to obtain the same growth
rate.
(7) Performance of Muscular Work. We know that thiamine is essential
for carbohydrate metabolism. Therefore animals doing heavy muscular
work should require more thiamine than those at rest. Of course this holds
true only if carbohydrates are metabolized. Otherwise thiamine require-
ments are not increased during heavy work. This has been demonstrated
recently by a series of experiments by Gruber and Ruys.*! They compared
the thiamine pyrophosphate contents in breast muscle, heart, and liver of
carrier pigeons which had performed an uninterrupted flight of about 140
miles with the corresponding contents of resting pigeons. Considering the
work expenditure and the available carbohydrates in the bodies of the
pigeons, Gruber and Ruys calculated that at least 80% of the calories for
the flight must have come from fat metabolism, and only a very small
percentage was supplied by carbohydrates. In accordance with these facts,
the thiamine content of the organs proved not to have been decreased by
the heavy work expenditure during the flight.
Taking into account all these factors that influence the thiamine require-
ment (and there are several others on which research has been scanty or
nil), it is clear that it is impossible to state precisely the daily required
amount for a certain animal. Even so it is possible to indicate a certain
quantity for the requirement per 100 g. of diet containing at least 60% of
carbohydrates. This content is about 100 to 150 y. There is a surprising
agreement between different investigators for different kinds of animals:
for pigeons,” for rats,* for chicks, ** and even for man.*®
As with other nutrients, life is possible at different levels of thiamine
intake. To find out the optimal intake, Byerrum and Flokstra*” determined
the thiamine and the thiamine pyrophosphate content of liver, muscle, and
brain of rats fed on different levels of thiamine. As the level of thiamine
was increased up to 200 y per 100 g. of food, the thiamine pyrophosphate
increased; beyond that level no further increase was found. Normal growth
took place even on 100 y of thiamine per 100 g. of food. Therefore for
4 R. F. Light and L. J. Cracas, Science 87, 90 (1938).
41M. Gruber and C. A. J. Ruys, Acta Physiol. et Pharmacol. Neerl. 2, 106 (1951).
42 R. L. Swank and O. A. Bessey, J. Nutrition 22, 77 (1941).
43 G. C. Supplee, R. C. Bender, and O. J. Kahlenberg, J. Nutrition 20, 109 (1940).
44. A. Arnold and C. A. Elvehjem, J. Nutrition 15, 403 (1938).
45'T. H. Jukes and H. Heitman, Jr., J. Nutrition 19, 21 (1940).
46 Recommended Daily Dietary Allowances, Nutrition Revs. 6, 319 (1948).
47 R. U. Byerrum and J. H. Flokstra, J. Nutrition 48, 17 (1951).
478 THIAMINE
maximal cocarboxylase content of these tissues twice the amount is re-
quired as is needed for normal growth. We cannot tell whether this maximal
intake of thiamine has any advantage for the animal or not.
B. OF MAN
W. H. SEBRELL, JR.
The principal factors influencing thiamine requirements are the carbo-
hydrate and the calorie intake. The requirement for thiamine is reduced
when fat forms a large part of the diet, but for practical purposes the
thiamine need may be based on the total calorie intake.
An early appraisal of the thiamine requirements of man was made by
Cowgill! in 1934. This appraisal was based on an analysis of dietary data
in the literature in relation to the occurrence of beriberi. On the basis of
this data the minimum intake of thiamine necessary to prevent beriberi is
not less than 0.28 mg. per 1000 cal. (60-kg. man on 2500 cals.) or a total
per day of 0.7 mg.
Elsom et al. in a study on women volunteers concluded that 0.65 mg.
per day was the minimum intake necessary to maintain health.
Melnick,*’ using saturation tests, reported that adults required 0.35 mg.
per 1000 cal. or 0.875 mg. per day on a 2500-cal. diet.
Williams ef al.°° found that an intake of 0.22 mg. of thiamine per 1000
cal. caused a slow depletion of tissue reserves and with an intake of 0.45
mg. per 1000 cal. there was a slight depletion of cocarboxylase.
Keys et al.®! studied the performance of normal young men on controlled
thiamine intakes and found that for a period of 10 to 12 weeks no benefit
of any kind was observed with intakes of more than 0.23 mg. of thiamine
per 1000 cal. (intake 3050 + 200 cal. per day).
In 1944 Holt” critically reviewed the studies of experimental thiamine
deficiency by Williams, Elsom, Keys, and Najjar and their collaborators
and reinterpreted the data to conclude that the minimum thiamine require-
ment of an adult man on a diet of natural foods lies between 0.17 and 0.23
mg. per 1000 cal. He concludes that a range of intake between 0.24 mg.
and 0.44 mg. per 1000 cal. appears to protect against thiamine deficiency.
Oldham et al.** found no change in blood thiamine levels with intakes
above 0.2 mg. per 1000 cal.
48K. O’S. Elsom, J. G. Rheinhold, J. T. L. Nicholson, and C. Chornoch, Am. J.
Med. Sci. 203, 569 (1942).
49 T). Melnick, J. Nutrition 24, 139 (1942).
50 R: D. Williams, H. L. Mason, and R. M. Wilder, J. Nutrition 26, 71 (1943).
51 A. Keys, A. F. Henschel, O. Mickelsen, and J. M. Brozek, J. Nutrition 26, 399
(1948).
52 7. E. Holt, Jr., Federation Proc. 3, 171 (1944).
53 H. G. Oldham, M. V. Davis, and L. J. Roberts, J. Nutrition 32, 163 (1944).
XII. REQUIREMENTS AND FACTORS INFLUENCING THEM 479
Foltz et al.,°* using four medical students under observation in a hospital,
found that a daily intake of 0.2 mg. per 1000 cal. resulted in deficiency
symptoms within 8 weeks. It is their opinion that the minimum daily
requirement of thiamine for young adult men is from 0.33 to 0.45 mg. per
1000 cal.
Keys et al.®° in further studies on men with restricted intake of the B
vitamins found that an intake of 0.185 mg. of thiamine per 1000 cal. (daily
intake 3300 cal.) was slightly less than entirely adequate.
Glickman et al.°® found a daily intake of 0.4 mg. per 1000 cal. entirely
adequate.
In an attempt to resolve some of the differences of opinion concerning
the minimum human requirement for thiamine, a study was conducted by
Horwitt et al.’ under the auspices of the National Research Council. These
investigations showed that 0.4 mg. of thiamine (0.18 mg. per 1000 cal.) was
. below the minimal requirement of relatively inactive men on 2200 cal.
daily.
On the basis of all the evidence available, therefore, the opinion of the
National Research Council® that the minimal thiamine requirement for
adults is 0.23 mg. or more per 1000 cal. is well founded. On this basis and
in order to allow a suitable factor of safety for individual variation, differ-
ences in type of diet, and variations in body stores, which are never large
and easily depleted by various stresses, an intake of 0.5 mg. of thiamine
per 1000 cal. is reeommended by the National Research Council as a safe
allowance for adults at ordinary low levels of calorie intake.
The thiamine requirement of the infant on a calorie basis is similar to
that of the adult. Knott et al.*® concluded that young infants have a mini-
mum thiamine requirement of approximately 0.2 mg. daily, which can just
be met if its mother’s milk contains 20 y or more of thiamine per 100 ml.
They suggest that 0.4 mg. of thiamine per kilogram may be a practical
standard for the ordinary needs of the young infant. The average thiamine
content of human milk was found to be about 0.15 mg. per liter. A more
critical analysis showed 0.2 mg. per liter in a group of women whose infants
were receiving no other milk in contrast to an average of 0.09 mg. per liter
in the milk of women whose infants required supplementary feeding.
54. E. Foltz, C. J. Barborka, and A. C. Ivy, Gastroenterology 2, 323 (1944).
55 A. Keys, A. Henschel, H. L. Taylor, O. Mickelsen, and J. Brozek, Am. J. Physiol.
144, 5 (1945).
66 N. Glickman, R. W. Keeton, H. H. Mitchell, and M. K. Fahnestock, Am. J.
Physiol. 146, 538 (1946).
57M. K. Horwitt, E. Liebert, O. Kreisler, and P. Wittman, Bull. Natl. Research
Council (U. S.), 116 (1948).
58 Recommended Dietary Allowances, Natl. Research Council (U. S.) Reprint and
Circ. Ser. 129 (1948).
59 K. M. Knott, 8S. C. Kleiger, F. W. Scheutz, and G. Collins, J. Pediat. 22, 43 (1943).
480 THIAMINE
Holt et al.©° found the thiamine requirement of seven infants to vary
between 0.14 mg. and 0.20 mg. per day on the basis of a urinary excretion
test. With an average thiamine content of cow’s milk of 0.35 to 0.4 mg.
per liter, an infant weighing 7 kg. is calculated to receive at least 0.3 mg.
of thiamine a day, but this makes no allowance for destruction by heat in
pasteurization or sterilization. The margin of safety is, therefore, regarded
as small by Holt and coworkers in the case of either sterilized milk or breast
milk, since the latter contains roughly only half as much thiamine as cow’s
milk.
The meager data available on the thiamine requirement in pregnancy
and lactation®-® indicate that in relation to calories the requirement may
be considered to be in the same proportion as for infants and adult men.
60. E. Holt, Jr., R. L. Nemir, S. E. Snyderman, A. A. Albanese, K. C. Ketron,
L. P. Guy, and R. Carretero, J. Nutrition 37, 53 (1949).
61 H. Oldham, B. B. Sheft, and T. Porter, Federation Proc. 6, 416 (1947).
62 M. Kaucher, E.’Z. Moyer, A. J. Richards, H. H. Williams, A. L. Wertz, and 1.G
Macy, Am. J. Diseases Children 70, 142 (1945).
68 C, Roderick, H. H. Williams, and I. G. Macy, J. Nutrition 32, 249 (1946).
Hr
LY;
We
Wal.
WIFE.
CHAPTER 17
THE TOCOPHEROLS
. Nomenclature .
. Chemistry
A. Chemistry of Vitamin E : ,
B. Derivatives and Their Phy eplécic at Action :
C. Physical Properties.
D. Products of Oxidation .
Industrial Preparation .
A. Source Materials.
1. Natural Tocopherols
2. Synthetic Tocopherols.
B. Procedures .
1. Natural Mecanberale
2. Synthetic Tocopherols. i
C. Properties and Potency of Available Pree Ee
1. Natural Tocopherols
2. Synthetic Tocopherols.
D. Production Control Tests
Biochemical Systems.
A. Absorption and iste vobeen:
B. Storage . :
C. Antioxygenic enon
tin Vivo .
2. In Viiro . ‘
D. Sparing Action on V 7 amin A :
Estimation . s
A. Quantitative Deena Silom
1. Chemical assay
2. Bioassay.
Occurrence in Food .
Effects of Deficiency
A. In Animals.
1. Introduction ;
. Male Reproductive Syster nih
Female Reproductive System
. Muscular System.
Nervous System .
. Vascular System.
. Other Manifestations .
B. In Man . :
1. General Considerar
2. Vitamin E in Early Life .
3. Vitamin E in Later Life .
SS oaripe Bec
481
. 512
. 514
. 514
. 514
5 GHITe
. 519
. 522
. 532
. 584
. 538
. 1
. O41
. 545
. dol
482 THE TOCOPHEROLS
Page
WILL: (Pharmacology. s,. t..cadewen «ley Sa eae egeelh aie etl vices tee oie eae
A. cEtypervaitaminosis H. . . 4. were teh «+, af Rel a ne rr
B.-Mode-of Administration s..:- + 2) 7%. «= ese ye
©; Metabolic Stressin Animals “725° < :.° = 5, See ete
DeTherapeutic’ Usex 8st Se Owe 2084 2 Gh eee
1. Heart Disease. . . Aes aie Ue sel 02
2. Peripheral Vascular Disenee! bay lat ok! is eye ees 0
3. Menopausal Syndrome. . . ook +) ee o aie Onnon
4. Primary Fibrositis and Related Tneoncers ee ih Oe
5. Diabetes: Mellitus: . 2. 3 2 0. af i 4 te ere
6. General Comments. .. . i a, ot a, Bae
IX. Requirements and Factors nina Mhem: sve. # ep) ot
Aj OF Amimals fie. so Ga Go ie eh SS! oS San cubes os bir
Be Of Man’. stesso e eS ms. ss oe ners ee, = pa ely 0
I. Nomenclature
ROBERT 8S. HARRIS
Accepted names: Vitamin E
Tocopherol
Obsolete names: Factor “X”’
Antisterility vitamin
Empirical formulas: a-Tocopherol: CogH5002
8-Tocopherol: CogHasO2
y-Tocopherol: CogHasOo
6-Tocopherol: Co7H4602
Chemical names: a-Tocopherol: 2,5,7 ,8-tetramethyl-2-(4’ ,8’ , 12’-trimeth-
yldecyl)-6-chromanol, or 5,7 ,8-trimethyltocol
8-Tocopherol: 2 ,5 ,8-trimethyl-2-(4’ ,8’ , 12’-trimethylde-
cyl)-6-chromanol, or 5,8-dimethyltocol
y-Tocopherol: 2,7 ,8-trimethyl-2-(4’ ,8’ , 12’-trimethylde-
CUAL MOEN, or 7 ,8-dimethyltocol
6-Tocopherol: 2 ,8-dimethyl-2-(4’ ,8’ , 12’-trimethylde-
cyl)-6-chromanol, or 8-methyltocol
Structures:,
CH;
H;C | CH;
, |
\A\~ g \__(oH,).CH(CH,),CH(CH,);CHCH;
CH; CH; CH;
6 3
VON EN A
Or. 4
CH;
a-Tocopherol
Il. CHEMISTRY 483
ta | | |
( / ON _(CH,),CH(CH.),CH(CH,);CHCH,
LOSS
HO >|
CH;
B-Tocopherol
CH;
H.C He CH; i a ae
a /ON|_(cH.);CH(CH;);CH(CH,),CHCH,
PO NAL
HO
y-Tocopherol
CH;
| CH; | |
/ON\_(CH,),CH(CH,),CH(CH,),CHCH,
CH; CH; CH;
By
HO
6-Tocovherol
II. Chemistry
HENRY A.*MATTILL
Vitamin E was first recognized by at least three groups of investigators!~*
as a fat-soluble substance necessary for reproduction in the rat. In place of
the original ‘‘X factor,’’! the designation of “‘E”’ was suggested by Sure,”
since this was the next letter in sequence following the identification of the
antiricketic vitamin as a separate entity. After the isolation of vitamin E
in purity,’ the name tocopherol was proposed (from the Greek, tokos (off-
spring), pherein (to bear), and ol, to signify an alcohol). Because of the
multiple nature of vitamin E, the new name is used in the plural in the
generic sense, and it is supplied with Greek letter prefixes, a, 8, etc., for
1H. M. Evans and K. 8S. Bishop, J. Metabolic Research 3, 233 (1923); J. Am. Med.
Assoc. 81, 889 (1923).
2B. Sure, J. Biol. Chem. 58, 693 (1924); 62, 371 (1924); 63, 211 (1925).
3H. A. Mattill, J. S. Carman, and M. M. Clayton, J. Biol. Chem. 61, 729 (1924).
4H. M. Evans, O. H. Emerson, and G. A. Emerson, J. Biol. Chem. 118, 319 (1936).
484 THE TOCOPHEROLS
designating the individual specific types. Without these prefixes the term
tocopherol is considered synonymous with tocopherols and vitamin E.
Unique in the history of vitamins was the organization of two interna-
tional symposia dealing with vitamin E. The first, in London in 1939, was
sponsored by the Nutrition Panel of the Society of Chemical Industry; the
second, in New York ten years later (lacking one week), under the auspices
of the New York Academy of Sciences and marking about the twenty-fifth
birthday of vitamin E. The volume of the proceedings of the first® (edited
by Bacharach and Drummond) contains less than one hundred pages (fif-
teen papers and discussions). That of the second® (edited by Mason) is
almost five times this size. These publications not only illustrate the ad-
vantages of such conferences for crystallizing opinions and guiding the
course of further investigation, but they are also enduring records of prog-
ress. They contain valuable bibliographies, as do the many reviews to
which reference will be made. Especially useful are the annotated bibliog-
raphies prepared by Merck and Company, Inc.’ (1925-1941) and Distilla-
tion Products Industries® (1940-1950), which also contain references to
suggested clinical applications and to many of the patents which have been
granted for the synthesis of tocopherols, their starting materials and de-
rivatives, and their use, alone and with synergists, in the stabilization of
oils, fats, and other autoxidizable substances.
Had the physiological role of vitamin E not been extended far beyond
the confines of reproduction, the manner of its action would still be an
intriguing problem, unsolved after a quarter of a century. So many connec-
tions have been established with other biological processes that even a
brief survey of them is little short of bewildering. Attempts at coordinating
the activities of vitamin E under some fundamental and unifying concept
have not yet been rewarded. An understanding of its chemical nature and
properties, although this has progressed much farther, is not complete.
A. CHEMISTRY OF VITAMIN E
The first work on its chemical nature® indicated that, like vitamins A
and D, vitamin E was to be found in the unsaponifiable portion of certain
fats, that it was somewhat vulnerable to saponification, and that it was
destroyed by bromination and acetylation but not by hydrogenation. Vac-
uum distillation caused considerable decomposition, but solvent partition
as between pentane and 92% methanol effected some concentration.
®> Vitamin EK, a Symposium. Heffer and Sons, Cambridge, 1940.
6 Vitamin E. Ann. N. Y. Acad. Sci. 62, Art. 3, 63-428 (1949).
7 Alpha Tocopherol, Merck and Co., Inc., May and December, 1941.
§ Vitamin EK. Distributed by the National Vitamin Foundation, 150 Broadway,
New York 7, New York (1950, 1952).
9H. M. Evans and G. O. Burr, Mem. Univ. Calif. 8, 176 pp., (1927).
II. CHEMISTRY 485
Another approach to the understanding of its chemistry was opened by
the observations that diets ordinarily adequate for reproduction gave
sterile animals if the diets contained certain fats which readily become
rancid or if the usual mixed diets were treated with ethereal ferric chlo-
ride.!°- From the unsaponifiable portion of several vegetable oils and plant
extracts concentrates were prepared!® which contained antioxidants (called
inhibitols'!® because of the presence of o- or p-hydroxyl groups) and also
vitamin E,!": '8 as judged by bioassays.
From these observations, either of two conclusions could be drawn: (1)
that vitamin E was readily oxidized and was protected by the accompany-
ing antioxidants, or (2) that the vitamin and the antioxidant were the
same substance. The latter view was favored by the fact that the two could
apparently not be separated, but it was not acceptable because the biologi-
cal and antioxygenic activities were not parallel from one concentrate to
another. The uncertainty and confusion were resolved by the demonstra-
tion’? that there were several substances with unequal vitamin E activity
and whose antioxygenic action also varied but in the opposite direction.
The several kinds of vitamin E have been shown to be the principal
antioxygenic components of natural fats, and their possible function as
“biological antioxidants” has been the subject of many discussions and
publications.?°
The parallel observations on the vitamin E and the antioxidant content
of concentrates! confirmed the earlier findings of Evans and Burr? except
for the biological inactivation of vitamin E by acetylation; the presence of
a hydroxyl group was demonstrated, and the benzoic acid ester as well as
the acetylated compound was shown to be biologically active. A previously
observed band in the ultraviolet spectrum with maximum at 2940 A. was
believed not to be related to vitamin H,” since concentrates from other
sources, such as palm oil, showed the same band but were biologically
inactive.
10L. T. Anderegg and V. E. Nelson, Ind. Eng. Chem. 18, 620 (1926); V. E. Nelson,
R. L. Jones, G. Adams, and L. T. Anderegg. ibid. 19, 840 (1927).
1H. M. Evans and G. O. Burr, J. Am. Med. Assoc. 88, 1462 (1927).
12H. A. Mattill, J. Am. Med. Assoc. 89, 1505 (1927).
18 J. Waddell and H. Steenbock, J. Biol. Chem. 80, 431 (1928).
144M. W. Taylor and V. E. Nelson, Proc. Soc. Exptl. Biol. Med. 27, 764 (1930).
15 H.S. Olcott and H. A. Mattill, J. Biol. Chem. 93, 59, 65 (1931); J. Am. Chem. Soc.
58, 1627 (1936).
16 AW. A. Mattill, J. Biol. Chem. 90, 141 (1931).
17H. 8. Olcott and H. A. Mattill, J. Biol. Chem. 104, 423 (1934).
1 H.S. Olcott, J. Biol. Chem. 107, 471 (1934).
19H. §. Olcott and O. H. Emerson, J. Am. Chem. Soc. 59, 1008 (1937).
20 Trans. Ist-5th Confs. on Biol. Antioxidants, New York (1946-1950).
21H. S. Oleott, J. Biol. Chem. 110, 695 (1935).
22 A. J.P. Martin, T. Moore, M. Schmidt, and F. P. Bowden, Nature 134, 214 (1934).
486 THE TOCOPHEROLS
Drummond and his colleagues”? prepared very potent concentrates from
wheat germ oil, being the first to use selective adsorption on aluminum
oxide. They confirmed many of the previous observations including this
maximum adsorption at 2940 A. with a minimum at 2670 A. Whether it
was characteristic of the vitamin or of an associated substance, the band
‘“nersisted” in proportion to the vitamin activity when this was destroyed
by ultraviolet radiation.
Pure vitamin E was first isolated by Evans and the Emersons? from the
unsaponifiable matter of wheat germ oil. By the use of cyanic acid on the
unsaponifiable fraction, three solid amorphous allophanates were obtained.
The alcohol regenerated from one of these, when given in a single dose of
3 mg., always enabled vitamin E-deficient rats to bear young. It was named
a-tocopherol. Of the alcohols obtained from the other two allophanates,
one had less biological potency (later called 8), the other none at all. a-To-
copherol was a light-yellow viscous oil which could not be crystallized, but
its conversion to another erystalline derivative (with p-nitrophenyl iso-
cyanate), followed by reconversion to the allophanate and regeneration of
the alcohol, left its biological activity unimpaired. The peak of its absorp-
tion band was at 2980 A., and microanalysis indicated a provisional formula,
C29H5902. Soon thereafter the same a-tocopherol was isolated from cotton
seed oil,” and later?® still another active substance, y-tocopherol, was ob-
tained from the same source. Further investigation revealed that only
a-tocopherol is present in lettuce and that palm oil is qualitatively similar
to cottonseed oil and contains no 6-tocopherol.
In several other laboratories, tocopherols were isolated from wheat germ
oil, the 8 by Todd and his coworkers”* in London, neotocopherol by Karrer
and his colleagues?’ 28 in Zurich, and cumotocopherol by John?’ in Gét-
tingen, the name indicating that pseudocumohydroquinone (trimethyl-
hydroquinone) was produced from it by thermal decomposition. Both
products were probably identical with 8-tocopherol.*° The preponderance
of B-tocopherol in European wheat germ oil and of the a form in that of
California has not been explained.
23 J. C. Drummond, E. Singer, and R. J. MacWalter, Biochem. J. 29, 456, 2510 (1935).
24Q. H. Emerson, G. A. Emerson, and H. M. Evans, Science 88, 421 (1936).
25 QO. H. Emerson, G. A. Emerson, A. Mohammad, and H. M. Evans, J. Biol. Chem.
122, 99 (1937).
26 AR. Todd, F. Bergel, and T. 8S. Work, Biochem. J. 31, 2257 (1937).
27 P. Karrer, H. Salomon, and H. Fritzsche, Helv. Chim. Acta 20, 1422 (1937).
28 P. Karrer and H. Salomon, Helv. Chim. Acta 21, 514 (1988).
29 W. John, Z. physiol. Chem. 250, 11 (1937).
30 F. Bergel, A. R. Todd, and T. 8. Work, J. Chem. Soc. 1938, 253; F. Bergel, A.
Jacob, A. R. Todd, and T. 8. Work, bid. 1938, 1375; F. Bergel, A. M. Copping, A.
Jacob, A. R. Todd, and T. 8. Work, zbid. 1938, 1382.
II. CHEMISTRY 487
The successful chemical identification of a-tocopherol by Fernholz*! be-
gan with the isolation of duroquinone (tetramethylhydroquinone) from the
products of its thermal decomposition. Dehydrogenation by selenium* also
produced it, and the British workers* obtained traces of it from concentrates
along with pseudocumoquinone from pure 8-tocopherol.
Fernholz’s suggestion*®! that a-tocopherol might be a mono ether of
duroquinone was shown to be untenable by them*®® and by John,**: *4
Karrer,® Drummond,** and their coworkers, most of whom proposed a
coumaran or chroman nucleus with a side chain (Karrer) of isoprene resi-
dues as in phytol (the alcoholic portion of chlorophyll). Fernholz*’ also
concluded from the dissimilarities of the absorption spectra of ethers of
duroquinone and of tocopherol that this did not have a simple ether struc-
ture. Thermal decomposition of tocopherol produced not only durohydro-
quinone but also an unsaturated aliphatic hydrocarbon. Mild oxidation of
this substance by chromic acid produced a lactone, C2:;H4902, whose free
hydroxy acid was so readily relactonized that a tertiary hydroxyl had to
be postulated. More vigorous oxidation yielded dimethylmaleic anhydride,
an acid CysH32.02, along with a ketone CisH3.0, diacetyl, and acetone.
Particularly the formation of a 16-carbon acid limited the number of pos-
sible structures for the lactone. After close and skillful organic chemical
reasoning, the structure he proposed for a-tocopherol was that of a substi-
tuted 6-hydroxychroman, with a long aliphatic side chain attached to the
pyran ring.
Intensive work in other laboratories confirmed the presence of a chroman
nucleus; degradation studies by John and his coworkers*® demonstrated this
nucleus with two substituents on carbon 2 (adjacent to the chroman oxy-
gen), thus providing a tertiary ether ring and optical activity, and Smith
and his coworkers*® came to the same conclusion by still different organic
chemical procedures and reasoning.
Meantime, still favoring the coumaran structure, but undecided, Karrer
and his coworkers*® accomplished an almost quantitative condensation of
31. Fernholz, J. Am. Chem. Soc. 59, 1154 (1937).
2 C.S. McArthur and E. M. Watson, Science 86, 35 (1937).
% W. John, E. Dietzel, and P. Giinther, Z. physiol. Chem. 252, 208 (1938).
4 W. John, Z. physiol. Chem. 262, 222 (1938).
35 P. Karrer, H. Salomon, and H. Fritzsche, Helv. Chim. Acta 21, 309 (1938).
86 A. R. Moss, W. F. J. Cuthbertson, J. F. Danielli, and J. C. Drummond, J. Soc.
Chem. Ind. (London) Transactions 57, 133 (1938).
37}. Fernholz, J. Am. Chem. Soc. 60, 700 (1938).
% W. John, E. Dietzel, P. Giinther, and W. Emte, Naturwissenschaften 26, 366 (1938).
89. I. Smith, H. E. Ungnade, and W. W. Prichard, Science 88, 37 (1938).
49 P. Karrer, H. Fritzsche, B. H. Ringier, and H. Salomon, Helv. Chim. Acta 21, 520
(1938).
488 THE TOCOPHEROLS
trimethylhydroquinone with phytyl bromide, zinc chloride being the cata-
lyst. Except for lacking optical activity the product had physical and chem-
ical properties identical with those of naturally occurring vitamin E.
Later*!: ” this synthetic product was shown also to have practically the
same physiological properties as vitamin E, and thus a unique event hap-
pened in organic-biochemical history—the synthesis of a complex sub-
stance before its actual constitution was known!
A span of approximately fifteen years had elapsed between the first recog-
nition of the existence of vitamin E and the synthesis of physiologically
active tocopherol. The last two years of this period witnessed as rapid a
succession of significant events as has ever been seen in any field of bio-
chemical endeavor.
The Karrer synthesis with phytyl bromide was duplicated by Smith and
his colleagues without a catalyst* and by the use of dienes (phytadiene)“
and of allyl and crotyl compounds and butadiene.*> For the construction
of the phytol chain, John and Pini“** used (tetra)farnesol, Smith and Sprung“
used citral.
Further proof for the existence of a chroman nucleus was provided by
permanganate oxidation,*® which produced the same decisive lactone, and
later by another method of synthesis, via carbinols,*® and by the quantita-
tive conversion of tocopherol (from purified tocopherylquinone via a ter-
tiary halide) into the diacetate.°°
For 6-tocopherol, John? and also Karrer et al.*° had proposed the same
general structure as that of the a-tocopherol, less one of the methyl groups,
and John** had concluded from the products of hydriodic acid treatment
of 8-tocopherol that the methyl groups were in the 5,8 positions. Emerson*!
confirmed the general structure by obtaining the same lactone from both
B- and y-tocopherol. Both these dimethyl compounds had been synthesized
from dimethylhydroquinones and phytyl bromide,*: * the 8 from the para
41 P. Karrer and V. Demole, Schweiz. med. Wochschr. 68, 954 (1938).
42 P. Karrer, H. Fritzsche, B. H. Ringier, and H. Salomon, Helv. Chim. Acta 21, 820 ©
(1938).
43. I. Smith and H. E. Ungnade, J. Org. Chem. 4, 298 (1939).
447,. I. Smith, H. E. Ungnade, H. H. Hoehn, and 8. Wawzonek, J. Org. Chem. 4,
311 (1988).
451, I. Smith, H. E. Ungnade, J. R. Stevens, and C. C. Christman. J. Am. Chem.
Soc. 61, 2615 (1939; L. I. Smith and J. A. King, zbid. 68, 1887 (1941). |
46 W. John and H. Pini, Z. physiol. Chem. 278, 225 (1942).
477,. I. Smith and J. A. Sprung, J. Am. Chem. Soc. 65, 1276 (1943).
48 QO. H. Emerson, Science 88, 40 (1988).
49 J,. I. Smith and H. C. Miller, J. Am. Chem. Soc. 64, 440 (1942).
50M. Tishler and N. L. Wendler, J. Am. Chem. Soc. 68, 1532 (1941).
51 QO. H. Emerson, J. Am. Chem. Soc. 60, 1741 (1938).
62 F', Bergel, A. M. Copping, A. Jacob, A. R. Todd, and T. 8. Work, J. Chem. Soc.
1938, 1382.
Il. CHEMISTRY 489
and the y from the ortho, and the products had biological activity com-
parable with that of the respective tocopherols. By degradation studies
the structure of y-tocopherol was confirmed. The suggestion®® that the un-
substituted tocopherol be called tocol was generally adopted; a-, B-, and
y-tocopherol are 5,7 ,8-trimethyl-, 5,8-dimethyl-, and 7 ,8-dimethyltocol,
respectively. A fourth tocopherol, 8-methyl-(6-)tocol, is the principal to-
copherol in soybean oil°® and occurs, normally, in small amounts, in ren-
dered hog fat.*”
Increased yields from natural sources were obtained by preliminary
chromatographic adsorption of unsaponified wheat germ oil and also of
the subsequent unsaponifiable portion,®® as well as by fractional crystalliza-
tion.®? Natural a- and y-tocopherol were both obtained in crystalline form
from 2.5% methyl! alcohol solution at —35°,® and their extinction coeffi-
cients were not changed significantly by crystallization. 8-Tocopherol could
not be crystallized, but crystals of its azobenzene-4-carboxylate were ob-
tained as orange plates. The practicability of molecular distillation was
demonstrated ;®!-® the process will be discussed elsewhere.
B. DERIVATIVES AND THEIR PHYSIOLOGICAL ACTION
The always engaging question as to the relation of organic structure to
physiological action prompted the preparation of many closely related
derivatives of the tocopherols. Various esters were made, some of them
beautifully crystalline, including those with fatty acids and with benzoic,
succinic, and phosphoric acids.*-® Lower homologs®® and higher homologs
53 A. Jacob, M. Steiger, A. R. Todd, and T. S. Work, J. Chem. Soc. 1939, 542.
54 OQ. H. Emerson and L. I. Smith, J. Am. Chem. Soc. 62, 1869 (1942).
55 P. Karrer and H. Fritzsche, Helv. Chim. Acta 21, 1234 (1938).
56 M. H. Stern, C. D. Robeson, L. Weisler, and J. G. Baxter, J. Am. Chem. Soc. 69,
869 (1947).
57 J. R. Chipault and W. O. Lundberg, Arch. Biochem. 12, 317 (1947).
58 A. R. Moss and J. C. Drummond, Biochem. J. 32, 1953 (1938).
59 W.S. Singleton and A. E. Bailey, O7l & Soap 21, 224 (1944).
60 C. D. Robeson, J. Am. Chem. Soc. 65, 1660 (1943).
61 F. W. Quackenbush, H. Gottlieb, and H. Steenbock, Ind. Eng. Chem. 38, 1276
(1941).
6K. C. D. Hickman, Chem. Eng. News 20, 1561 (1942).
68M. E. Wall, Ind. Eng. Chem. 41, 1465 (1949).
64V. Demole, O. Isler, B. H. Ringier, H. Salomon, and P. Karrer, Helv. Chim. Acta
22, 65 (1939).
65 P. Karrer, Helv. Chim. Acta 22, 334 (1939).
66 P. Karrer and G. Bussmann, Helv. Chim. Acta 23, 1137 (1940).
* 1,. I. Smith, W. B. Renfrow, Jr.,and J. W. Opie, J. Am. Chem. Soc. 64, 1084 (1942).
68 J. G. Baxter, C. D. Robeson, J. D. Taylor, and R. W. Lehman, J. Am. Chem.
Soc. 65, 918 (1943).
69 A. Jacob, F. K. Sutcliffe, and A. R. Todd, J. Chem. Soc. 1940, 327.
490 THE TOCOPHEROLS
containing one or two ethyl groups in place of the methyl’? (or of H in f
5,7-tocol) and allyl and crotyl derivatives” were prepared, as well as com- —
pounds containing synthetic phytol or fewer isoprene units in the side
chain.”? With pseudocumol as starting material and by the use of Grignard —
reactions, a wide variety of derivatives can be made, containing a side
chain other than the phytyl group; with cetyl the product was called iso- —
a-tocopherol.”
Most of these compounds were tested for their physiological action by
the method of rat assay (see below). Even with the earlier and less precise
procedures, certain general conclusions were readily apparent and are still
valid. If the activity of natural a-tocopherol is set at 100, that of natural
B-tocopherol is 40, and that of y-tocopherol is 8 (or less). 7° Figures for
the synthetic products were reported’® as 100:25:19. 6-Tocopherol has only
1 % of the biological activity of the a.°® By contrast, nuclear dealkylation
increases antioxygenic action! ” and resistance to atmospheric oxidation,®®
but if the distinction between antioxygenic activity, as usually measured
in accelerated tests at 70° to 100°, and antioxidant potency measured at
body temperature is valid, the latter and the biological activity run paral-
lel.78 Optical activity (residing in carbon 2) is very slight, but the potencies
of the synthetic dl compounds (@ and #) were found to be two-thirds and
one-half, respectively, of those of the natural.’® For the cure of nutritional
muscular dystrophy in rabbits, the ratios of potency for the a, 6, and y
varieties were reported as 100:30:20, and the synthetic a and y about 90
and 30% of the natural.®°
The biopotency of the esters is equal to that of the free alcohols, or even
slightly greater, because the esters are not autoxidizable. The use of the
crystalline succinate was suggested as a vitamin E standard” in place of
70 P. Karrer and O. Hoffmann, Helv. Chim. Acta 22, 654 (1939); 28, 1126 (1940); P.
Karrer, H. Fritzsche, and R. Escher, ibid. 22, 661 (1939); P. Karrer and R.
Schlapfer, cbid. 24, 298 (1941).
71 P, Karrer, R. Escher, H. Fritzsche, H. Keller, E. H. Ringier, and H. Salomon,
Helv. Chim. Acta 21, 939 (1938).
72 P. Karrer and K. A. Jensen, Helv. Chim. Acta 21, 1622 (1938); P. Karrer and H.
Fritzsche, ibid. 22, 260 (1939); P. Karrer and B. H. Ringier, ibid. 22, 610 (1939);
P. Karrer, H. Koenig, B. H. Ringier and H. Salomon, zbid. 22, 1139 (1939); P.
Kerrer and K. 8. Yap, bid. 23, 581 (1940).
73, W. John, P. Giinther, and F. H. Rathmann, Z. physiol. Chem. 268, 104 (1941).
74M. Joffe and P. L. Harris, J. Am. Chem. Soc. 65, 925 (1943).
75 L,. Weisler, J. G. Baxter, and M. I. Ludwig, J. Am. Chem. Soc. 67, 1230 (1945).
76 H. Gottlieb, F. W. Quackenbush, and H. Steenbock, J. Nutrition 25, 433 (1943).
77 C. Golumbic, J. Am. Chem. Soc. 68, 1142 (1941).
73K. L. Hove and Z. Hove, J. Biol. Chem. 156, 623 (1944).
79 P_L. Harris, J. L. Jensen, M. Joffe, and K. HE. Mason, J. Biol. Chem. 156, 491 (1944).
80 1}, L. Hove and P. L. Harris, J. Nutrition 38, 95 (1947).
Il. CHEMISTRY 491
the oily liquid dl-acetate originally proposed,*! 1 mg. of which was one
international unit (I.U.). In terms of this unit, the biological equivalents
of dl-a-tocopherol, d-a-tocopherol, and d-a-tocopheryl acetate are 0.68,
0.92, and 1.36 I.U. per milligram, respectively.”
Substitution of ethyl for one or two methyl groups lessened the activity
slightly, but compounds obtained by replacement of the two o-methy]
groups (7, 8) in a-tocopherol by tri- or tetramethylene rings were inactive
in 50-mg. doses.’ When the 2-methyl group was replaced by an ethyl or a
propy! group, biological activity was slightly reduced.** Of several methoxy-
tocols, only the 5,7-dimethyl-8-methoxytocols had any biological activity
(60 mg. was completely protective), and this was less than that of 5,7-
dimethyltocol.*°
The character of the side chain is highly specific; reduction of the number
of isoprene units, even by one, is inactivating, but the four units may be of
natural or synthetic origin; the asymmetry of the phytol residue is unim-
portant.*®
An aminotocopherol (NH, in place of OH) was biologically equivalent
to the hydroxyl-containing compound,* whether as such or after biological
replacement is not known. Methyl and ethyl ethers and the allophanates
are inactive.”!
A naphthotocopherol obtained as a by-product in the synthesis of vita-
min K has the biological properties of both the vitamins, the adequate rat
dose exceeding that of 8-tocopherol.**: °°
Many of the compounds described in the comprehensive review of vita-
min E (up to 1940) were demonstrated to have biological activity in
massive doses, 50 to 100 mg.*! Not only are such amounts unphysiological,
but the results may be suspect, because the control of the bioassay was
inadequate according to present practice. Recently some of these com-
pounds were shown to be inactive biologically.®
81 FE. M. Hume, Nature 148, 472 (1941); Quart. Bull. Health Organisation League Na-
tions 9, 436 (1940-1941).
82 P. L. Harris and M. I. Ludwig, J. Biol. Chem. 179, 1111 (1949); 180, 611 (1949).
8 P. Karrer and A. Kugler, Helv. Chim. Acta 28, 436 (1945).
84 P. Karrer and H. Stihelin, Helv. Chim. Acta 28, 438 (1945).
85 P. Karrer and K. Diirr, Helv. Chim. Acta 32, 1361 (1949).
86 P. Karrer and H. Rentschler, Helv. Chim. Acta 26, 1750 (1943).
87 L. I. Smith, W. B. Renfrow, and J. W. Opie, J. Am. Chem. Soc. 64, 1082 (1942).
88M. Tishler, L. F. Fieser, and N. L. Wendler, J. Am. Chem. Soc. 62, 1982 (1940).
89M. Tishler and H. M. Evans, J. Biol. Chem. 139, 241 (1941).
9 L. I. Smith, Chem. Revs. 27, 287 (1940).
" H. M. Evans, O. H. Emerson, G. A. Emerson, L. I. Smith, R. E. Ungnade, W. W.
Prichard, F. L. Austin, H. H. Hoehn, J. W. Opie, and 8. Wawzonek, J. Org. Chem.
4, 376 (1939).
® P. D. Boyer, M. Rabinovitz, and E. Liebe, J. Biol. Chem. 192, 95 (1951).
492 THE TOCOPHEROLS
C. PHYSICAL PROPERTIES
The absorption maxima, in the ultraviolet, of a-, B-, and y-tocopherol
are given as 292, 297, and 298 mp, respectively ;%* those of the synthetic
compounds are the same except for the y, which was at 300 mu; the ex-
tinction coefficients”®: *8. % are 70 to 73.7, 86 to 87, and 90 to 93.
Infrared spectra of some natural and synthetic tocopherols, their esters,
tocopherylquinone, and hydroquinone were obtained by Rosenkrantz;%* %
bands near 8.6 » and 10.9 uw are characteristic of tocopherols. Much may
be expected from the application of spectrophotometric methods in the
elucidation of structure of the tocopherols, their oxidation products, and
derivatives.
The specific rotations of the a, 6, and y compounds in ethyl! alcohol are
+0.32, +2.9, and +2.2, respectively; in benzene those of the a and y are
—3.0 and —2.4.%
D. PRODUCTS OF OXIDATION
Tocopherylquinone, the first stable oxidation product of tocopherol, is a
yellow compound and is obtained by the action of ferric chloride or silver
a-Tocopherylquinone
nitrate. It was identified by John* and is quite without activity for the
rat.59 8-99 The same quinone was produced by Karrer ef al. with various
agents;!0° with gold chloride a potentiometric titration was made the basis
of a quantitative determination.!™ This quinone can be reduced to tocoph-
erylhydroquinone by the use of palladium in alcohol or of zine in glacial
acetic acid and is unstable unless acetylated. In strong acid solution with
93 J. G. Baxter, C. D. Robeson, J. D. Taylor, and R. W. Lehman, J. Am. Chem. Soc.
65, 918 (1943).
94H. Rosenkrantz, J. Biol. Chem. 178, 489 (1948).
°° H. Rosenkrantz and A. T. Milhorat, J. Biol. Chem. 187, 83 (1950); J. Am. Chem.
Soc. 72, 3304 (1950).
96 P. Karrer and A. Geiger, Helv. Chim. Acta 28, 455 (1940).
97 C. Golumbic and H. A. Mattill, J. Biol. Chem. 134, 535 (1940).
*® M.D. Wright and J. C. Drummond, Biochem. J. 34, 32 (1940).
99 R. R. Ridgeway, J. C. Drummond, and M. D. Wright, Biochem. J. 34, 1569 (1940).
100 P. Kerrer, R. Escher, H. Fritzsche, H. Keller, B. H. Ringier, and H. Salomon,
Helv. Chim. Acta 21, 939 (1938).
101 P, Karrer and H. Keller, Helv. Chim. Acta 21, 1161 (1938) ; 22, 253, 617 (1939).
)
Il. CHEMISTRY 493
OH
H,;,C HO
CH
H;C ii VA 3
CicHss
HO a
CH; 7 \
a«-Toc opherylhydroquinone
a reducing agent, the original tocopherol can be regenerated by reduction
and cyclization.”
The biological inactivity of tocopherylquinone is an indication that this
reaction does not occur in vivo, at least not as regards its effect on repro-
duction.
Tocopherylhydroquinone is also inactive in the rat,!% but when it was
given intravenously every 5 days in a water emulsion! to dystrophic rab-
bits it was equivalent to a-tocopherol itself, the quinone being less effec-
tive. The conclusion was drawn that the hydroquinone was the anti-
dystrophic agent, and, being unstable, it was oxidized before it could be
converted to tocopherol for storage purposes.
With more vigorous oxidation of tocopherol, as with nitric acid, a red
color develops. Since its depth is proportional to the amount of tocopherol,
it was proposed as the basis of a quantitative method.!°* This “red” qui-
none, which is biologically inactive!” and has resisted all attempts at
crystallization, was first assigned a p-quinone structure.!%: !°° It remained
for Smith and his colleagues!® to show that by the action of nitric acid
any substituent on carbon 5 is replaced by oxygen and that the substance
was a chroman-5 ,6-quinone.
CH;
Hea WOONG a
| Ci¢H33
UNOS
Omnis
O
Chroman-5 ,6-quinone
12 W. John, E. Dietzel, and W. Emte, Z. physiol. Chem. 257, 173 (1939).
103 A Issidorides and H. A. Mattill, J. Biol. Chem. 188, 313 (1951).
#4 H. Rosenkrantz and A. T. Milhorat, Proc. Soc. Exptl. Biol. Med. 74, 468 (1950).
10° J.B. Mackenzie, H. Rosenkrantz, 8. Ulick, and A. T. Milhorat, J. Biol. Chem. 183,
655 (1950).
106M. Furter and R. E. Meyer, Helv. Chim. Acta 22, 240 (1939).
107 C. Golumbic, J. Am. Chem. Soc. 64, 2337 (1942).
08 W. John and W. Emte, Z. physiol. Chem. 261, 24 (1939).
09 P. Karrer, H. Fritzsche, and R. Escher, Helv. Chim. Acta 22, 661 (1939).
no y,. I. Smith, W. B. Irwin, and H. E. Ungnade, J. Am. Chem. Soc. 61, 2424 (1939).
494 THE TOCOPHEROLS
The course of oxidation of the tocopherols in autoxidizing fats depends
on the nature of the fat. In animal fats (containing added a-tocopherol) —
}
:
f
7
q
and in vegetable fats, where tocopherols occur naturally, tocoquinones —
were the immediate products, but the chroman-5 ,6-quinone appeared only
in vegetable fats.!!! The red quinone appears to have its origin in 7 ,8-di-—
methyltocol (y)," in which position 5 is occupied by H. This is more
a
fj
a
-
4
J
readily oxidized than a methyl group, but when pure a-tocoquinone is
gently heated in methanol containing traces of synergistic acids, such as—
phosphoric, sulfuric, tartaric, and others, chroman-5 ,6-quinone is formed
along with other unidentified oxidation products of the nature of quinones.'4 —
At the same time some a-tocopherylquinone is converted to a-tocopherol, —
as demonstrated by antioxygenic and biological activity and by absorption
spectrum. This is a kind of dismutation, as if the acids catalyzed the dis-
placement of the 5-methyl group. This behavior is consistent with the
more rapid production of the red quinone from y-tocopherol by the action
of silver nitrate and may ultimately contribute to an understanding of
the unsolved kinetic problem of synergism of ascorbic, phosphoric, and
other polyhydroxy acids with tocopherols.!4"° Further oxidation products
have been!” and doubtless remain to be discovered and identified, along —
?
with dimers, but it is doubtful that any of them beyond tocopherylquinone
will prove to play a role in biological processes.
By analogy with simpler quinols, the transitory existence of a semi-
quinone or free radical could be assumed. Proof of this was provided by
the ingenious‘ technique used by Michaelis and Wollman.!!8 A solution of
tocopherol in suitable organic solvents and brought to the temperature of
liquid air assumed the consistency of glass. When this was irradiated with
ultraviolet light, it developed an orange-red color and absorption bands not
characteristic of the quinone. Both the color and the absorption bands dis-
appeared when the temperature was slightly elevated. Recently, fresh evi-
dence for the existence of the free radical of tocopherol has been obtained
in the author’s laboratory.!° Univalent oxidation does not involve the
opening of the ring, and if the formation of this semiquinone is not con-
cerned in the biological action of tocopherol, it certainly finds its place in
any explanation of the antioxygenic action of tocopherols in vitro.
111 ©, Golumbiec, O7l & Soap 20, 105 (1943).
12 ©, EK. Swift, G. E. Mann, and G.S. Fisher, Orl & Soap 21, 317 (1944).
113 A. Issidorides, J. Am. Chem. Soc. 73, 5146 (1951).
14 C, Golumbic and H. A. Mattill, J. Am. Chem. Soc. 68, 1279 (1941).
115 ©, Golumbic, O7l & Soap 19, 181 (1942); 23, 184 (1946).
16.V, P, Calkins, J. Am. Chem. Soc. 69, 384 (1947).
17 W. John and W. Emte, Z. physiol. Chem. 268, 85 (1941).
118 T,, Michaelis and 8. H. Wollman, Science 109, 313 (1949); Biochim. et Biophys.
Acta 4, 156 (1950).
119 G, E. Inglett, Unpublished data.
III. INDUSTRIAL PREPARATION 495
Still another partial oxidation product, a-tocopheroxide, was obtained
by Boyer and his associates’: ! by oxidizing a-tocopherol with two equi-
valents of ferric iron in the presence of a ,a-dipyridyl. If treated immediately
by ascorbic acid, the product could be reduced to a-tocopherol, as shown
by the absorption spectrum, but after some days it was irreversibly con-
verted to tocopherylquinone; in the ultraviolet region its peak is at 2370
A.; in the infrared it lacks the characteristic absorption of the OH groups
at 30 A. Its chemical properties are those of an epoxy compound, the epoxy
CH;
2,5,7,8-Tetrameth yl-2(4,8, 12-trimethyltridecyl)-9, 10-epoxy-6(5H)-chromanone
group probably being in the 8,9 position. Given intraperitoneally in the
rat test, it had about one-ninth the activity of a-tocopherol, by mouth only
one-thirtieth, perhaps related to its rapid oxidation to the inactive toco-
pherylquinone. Tocopheroxide may be the second step in the oxidation of
tocopherol to its quinone.
III. Industrial Preparation
PHILIP L. HARRIS
A. SOURCE MATERIALS
1. NaturAL TOCOPHEROLS
Corn, cottonseed, soybean, and wheat germ oils with tocopherol con-
tents ranging from about 0.1% to 0.3% constitute the starting material
for practically all natural tocopherol preparations. These oils represent a
tremendous potential raw material source but one which challenges the
manufacturer in his efforts to increase the tocopherol concentration a hun-
dredfold, to at least 30 % and sometimes to 100 %, for use in pharmaceutical
products.
The tocopherols in corn oil are largely y-tocopherol, those in cottonseed
oil are a- and y-tocopherols in about equal amounts, those in soybean oil
20 P. D. Boyer, M. Rabinovitz, and E. Liebe, Ann. N. Y. Acad. Sct. 52, 188 (1949).
121 P. D. Boyer, J. Am. Chem. Soc. 73, 733 (1951).
496 THE TOCOPHEROLS
are a, y, and 6 forms, and those in wheat germ oil are a- and B-tocopherols.!4
Consequently, concentrates made from any of these oils represent a prepa-
ration of the particular predominant tocopherol or tocopherols of the origi-
nal oil. The tocopherols in the commercial concentrates of mixed tocopherols
are standardized to contain 50% a-tocopherol and the remainder y- plus
6-, with only negligible amounts of 6-tocopherol.
The preparation of concentrates of the a form of tocopherol is of prime
interest, since this form possesses the highest physiological activity. How-
ever, concentrates of the individual B-, y-, and 6-tocopherols can be pre-
pared.5-?
2. SYNTHETIC TOCOPHEROLS
Trimethylhydroquinone and phytol or phytyl chloride are most often
mentioned as source chemicals for the preparation of synthetic, racemic
a-tocopherol. However, the synthesis can be carried out with a variety of
other starting materials,’ ° but apparently with considerably more diffi-
culty and less efficiency.
Synthetic 6- and y-tocopherols, with two methyl groups, and synthetic
6-tocopherol, with only one methyl group, on the chroman ring, can be
prepared from the properly constituted dimethyl- and monomethylhydro-
quinones, respectively, and phytol.
B. PROCEDURES
Details of commercial operations in the vitamin E field have not been
published. However, the following references cover procedures which should
be usable.
1. NatrurRAL TocoPHEROLS
Saponification, with subsequent extraction and concentration of tocoph-
erols in the non-saponifiable fraction, is a feasible method for preparing
low potency preparations. It is used, for example, to make about an eight-
fold concentration of the tocopherols of wheat germ oil (Table I). Similarly,
direct, hot ethanol extraction of wheat germ, followed by low-temperature
1k). L. Hove and Z. Hove, J. Biol. Chem. 156, 601 (1944).
2M. L. Quaife, J. Biol. Chem. 175, 605 (1948).
3F. Brown, Biochem. J. 51, 237 (1952).
4F. Brown, Biochem. J. 52, 523 (1952).
5 J. G. Baxter, C. D. Robeson, J. D. Taylor, and R. W. Lehman, J. Am. Chem.
Soc. 65, 918 (1948).
6 C. D. Robeson, J. Am. Chem. Soc. 65, 1660 (1943).
7M. H. Stern, C. D. Robeson, L. Weisler, and J. G. Baxter, J. Am. Chem. Soc. 69,
869 (1947).
8 L. I. Smith, Chem. Revs. 27, 287 (1940).
9W. John and H. Pini, Z. physiol. Chem. 278, 225 (1942).
Ill. INDUSTRIAL PREPARATION 497
treatment of the extract, which freezes out much of the extracted lipid ma-
terial, and by removal of solvent, yields a tocopherol-enriched oil.!”
The details of procedure and equipment used for the solvent extraction
of vegetable oils with liquid propane or other hydrocarbons have been de-
scribed but without much information concerning the potency of the con-
centrates obtained.!!- Also, fractional crystallization of cottonseed oil from
solvents yields a tocopherol-containing fraction.“
Adsorption chromatography is used to prepare relatively pure individual
TABLE I
DESCRIPTION OF SOME TyPicaL CoMMERCIALLY AVAILABLE
TocoPHEROL PREPARATIONS
Tocopherol
Content, mg./g. ~
io-
Ree Specific | Absorption |potency,
Product Total a-To- Color and form index, gravity, Spee L.U./g.
tocoph- copherol 20° q29 E! % (rat
erol or ny 4 1cm. {sterility
tocoph- hk el test)
A eral fe acetate
Natural
Wheat germ oil’eoncentrate (1| 16 10 | Dark-brown vis- — — = 10
to 8) | cous oil
Natural mixed tocopherol con-| 500 250 | Red viscous oil 1.4972 |0.9320- | 39 at 294 230
centrates (Type 4-50) 0.9550) my
d-a-Tocopheryl acetate con- | Approx.) 350 | Brownish-yellow 1.4864 |0.9320- | 22.4 at 475
centrates (Type 6-35) 500 viscous oil 0.9550; 284 my
d-«-Tocopheryl acetate (Type) 1000 1000 | Light-yellow vis- |1.4940- |0.9500—- | 40-44 at 1360
6-100) cous oil 1.4985! 0.9640 284 mp
Synthetic
dl-a-Tocopherol (racemic) 1000 1000 | Clear viscous oil /|1.5030—- |0.9470- | 71-76 at 680
| which darkens 1.5070} 0.9580) 292 my
readily
dl-a-Tocopheryl acetate 1000 1000 | Clear yellow vis- |1.4958- |0.9545- | 42.5 + 1 1000
(racemic) | | cous oil 1.4972) 0.9665) at 285.5
my
tocopherols, usually on a small scale, as in analytical procedures.*: !® How-
ever, chromatography can be adapted to large-scale production.'!*"5
Molecular distillation of tocopherol-containing oils has proved to be an
efficient and practical procedure for preparing potent concentrates of natu-
10W. D. McFarlane, U.S. Pat. 2,497,317 (Feb. 14, 1950).
11N. D. Embree and N. H. Kuhrt, U.S. Pat. 2,454,692 (Nov. 23, 1948).
12 A. W. Hixson and R. Miller, U.S. Pat. 2,508,387 (May 23, 1950).
13 W. M. Leaders and F. A. Norris, U.S. Pat. 2,521,234 (Sept. 5, 1950).
4 W.S. Singleton and A. E. Bailey, Oil & Soap 21, 224 (1944).
16 A. Emmerie, Ann. N. Y. Acad. Sci. 52, 309 (1949).
16 Q. Isler, Chimia (Switz.) 5, 249 (1951).
7 J. C. Drummond, British Pat. 517,401 (Jan. 29, 1940).
18 J. G. Farbenindustrie, German Pat. 717,483 (Jan. 29, 1942).
498 THE TOCOPHEROLS
ral tocopherols. In this process, heat-labile tocopherols are distilled without
destruction at relatively low temperature and pressure, and under condi-
tions of very short thermal exposure. Concentrates of 14 to 18 % tocopherols
are readily obtained from soybean oil!® and also from leaf meal extracts."
Further processing permits even greater concentration of tocopherols.2!™4
Trimethylhydroquinone
+
a
CaaS
HO ie CH CH CH;
Ce CH.—CH,—CH,—C*H—CH.,.— CH,—CH,— C*H— CH,—CH,—CH,— CH— CH;
CH;
Phytol
P2Os | 140°
HO-—C* ~C CH, CH: CH, CH;
Ax 9 ~ CH= CH: — CH, — C*H— CH: —CH,— CH: C*H —CH— CH, CH, — CH— CH
| CH;
CH;
Racemic a-Tocopherol
Fie. 1. Synthesis of a-tocopherol. The asterisks indicate points of asymmetry
giving rise to a mixture of isomers. The natural occurring form is d-a-tocopherol.
2. SYNTHETIC ‘TOCOPHEROLS
Condensation of trimethyl hydroquinone and phytol under the condi-
tions shown in Fig. 1 is conveniently carried out with almost quantitative
yields of racemic a-tocopherol.?? This and similar reactions used commer-
cially are modifications of Karrer’s and of Smith’s classic original synthesis
in 1938.26-28
19 F. W. Quackenbush, H. Gottlieb, and H. Steenbock, Ind. Eng. Chem. 33, 1276
(1941).
20 M. E. Wall, Ind. Eng. Chem. 41, 1465 (1949).
21K. C. D. Hickman, Chem. Eng. News 20, 1561 (1942).
22'T. R. Olive, Chem. & Met. Eng. 61, 100 (1944).
23. W.S. Singleton and A. E. Bailey, Oil & Soap 21, 157 (1944).
24 J. Green and P. R. Watt, J. Sci. Food Agr. 1, 157 (1950).
25 F. von. Werder, U.S. Pat. 2,230,970 (Feb. 4, 1941).
26 P. Karrer, H. Fritszche, B. H. Ringier, and H. Salomon, Helv. Chim. Acta 21,
520, 820 (1938).
27 P. Karrer, H. Fritsche, B. H. Ringier, and H. Salomon, Nature, 141, 1057 (19388).
8 ||. I. Smith, H. E. Ungnade, and W. W. Prichard, Science 88, 37 (1938).
Ill. INDUSTRIAL PREPARATION 499
C. PROPERTIES AND POTENCY OF AVAILABLE PREPARATIONS
1. NarurRAL TocoPHEROLS
Wheat germ oil concentrates and unsaponifiable fractions are primarily
of historical interest, since, in the early days of tocopherol use, these prep-
arations alone were available. However, there is even yet some manufac-
ture of wheat germ oil concentrates, and, as shown in Table I, they are
brown oil preparations relatively susceptible to oxidative destruction and
possessing only about 10 1.U. of vitamin E activity per gram.
The second listed tocopherol preparation in Table I is typical of a type
of mixed tocopherol concentrate prepared from vegetable oils in which
the total tocopherol content may be 300 mg. per gram to 600 mg. per gram
and the a form of tocopherol constitutes at least 50% of the total. Conse-
quently, in the example listed, Type 4-50 concentrate contains 250 mg.
of a-tocopherol per gram. Since this is d-a-tocopherol in the free alcohol
form, 250 mg. furnishes a biopotency of (250 * 0.92) 230 I.U. per gram.?°
Furthermore, the antioxidant potency of these concentrates is very high
because of the free tocopherol content.
d-a-Tocopheryl acetate concentrate, Type 6-35, is representative of a
series of preparations in which the tocopherol is present in the acetic acid
ester form, has practically no antioxidant activity, but does have excep-
tional stability and high biopotency. The example in Table I supplies 475
I.U. of vitamin E per gram concentrate as the result of its content of 350
mg. of d-a-tocopheryl acetate per gram (850 X 1.36).°: *!
The last natural-type product in Table I is pure d-a-tocopheryl ace-
tate, a light-yellow viscous oil with the highest biopotency of any vitamin
E product, 1360 I.U. per gram.**
2. SYNTHETIC TOCOPHEROLS
The two synthetic preparations available are clear viscous oils with a
biological potency of 680 I.U. per gram for the a-tocopherol*! and 1000
I.U. per gram (by definition) for the a-tocopheryl acetate. Synthetic a-
tocopherol and a-tocopheryl acetate are racemic mixtures of from two to
eight isomers, depending upon whether natural or synthetic phytol was
used in the synthesis (Fig. 1). The separation and isolation of the natural
d isomer of a-tocopherol can be effected by resolution of the bromocamphor-
sulfonates.®
Esters of natural or synthetic a-tocopherol, other than the acetates, and
tocopheramine* are occasionally prepared. For example, succinates, acid
29 P. L. Harris and M. I. Ludwig, J. Biol. Chem. 180, 611 (1949).
30 P. L. Harris and M. I. Ludwig, J. Biol. Chem. 179, 1111 (1949).
31 P.L. Harris, Ann. N. Y. Acad. Sci. 62, 342 (1949).
8 P. Karrer, U.S. Pat. 2,215,398 (Sept. 17, 1940).
33 L. I. Smith, W. B. Renfrow, Jr., and J. W. Opie, J. Am. Chem. Soc. 64, 1082 (1942).
500 THE TOCOPHEROLS
succinates, fatty acid esters, nitrobenzoates, and phosphates of a-tocoph-
erol are described**~*° but have remained laboratory products.
D. PRODUCTION CONTROL TESTS
Considerable experience with various physical and chemical techniques
permits the generalization that the Emmerie-Engel reaction*! is the method
of choice for production control in which total tocopherol values suffice.2-
However, procedures employing ceric sulfate titration of tocopherols, modi-
fications of Kofler’s method,*® serve a useful purpose in control analyses on
pure tocopherols. The quantity of a-tocopherol in the preparation, how-
ever, is the important question and is obtained by determining the non-a-
tocopherol moiety by the nitroso method and subtracting this from the
amount of total tocopherols determined separately.‘* Direct determination
of a-tocopherol, after removal of non-a-tocopherols by careful, arbitrary
washing with sulfuric acid, is used by a regulatory agency in inspection
control assays.”
Products containing tocopherol esters must be carefully saponified be-
fore analyzing for total and individual tocopherols. Quantitative hydrolysis
is possible if proper precautions to prevent tocopherol loss and destruction
are observed. For example, exclusion of air during reaction and neutraliza-
tion and use of an antioxidant are necessary.‘?*
Bioassays of final products and praticularly of new types of preparations
are desirable, since the chemical methods do not distinguish the various
unnatural isomers of a-tocopherol from the natural d isomer. The physio-
34 F, Hoffmann-La Roche & Co., Swiss Pat. 208,851 (May 16, 1940).
35 F. Hoffmann-La Roche & Co., German Pat. 711,248 (Aug. 28, 1941).
36 F. Hoffmann-La Roche & Co., German Pat. 712,748 (Oct. 2, 1941).
37 F. Hoffmann-La Roche & Co., Swiss Pat. 216,825 (Sept. 15, 1941).
Pe KMarrer, Ues. bata 2,colel25 (Heb. lle 194):
39 L. I. Smith, W. B. Renfrow, Jr., and J. W. Opie, J. Am. Chem. Soc. 64, 1084 (1942).
40 U. V. Solmssen and J. Lee, U.S. Pat. 2,457,932 (Jan. 4, 1949).
41 A. Emmerie and C. Engel, Rec. trav. chim. 58, 283 (1939).
# J. G. Baxter, Biol. Symposia 12, 484 (1947).
48. W. Rawlings, Oil & Soap 21, 257 (1944).
44H. W. Rawlings, N. H. Kuhrt, and J. G. Baxter, J. Am. Oil Chemists’ Soc. 25,
24 (1948).
45M. Kofler, Helv. Chim. Acta 30, 1053 (1947).
46M. L. Quaife, J. Biol. Chem. 175, 605 (1948).
471). G. Chapman, P. Lichon, and J. A. Campbell, J. Am. Pharm. Assoc. Sct. Ed.
40, 379 (1951).
48K. Ritsert, H. Merck’s Jahresber. 55, 13 (1943).
49'P. Canbick and B. Wallenberg, Svensk Farm. Tidskr. 50, 477 (1946).
50 'T. Moore and J. Tosic, Biochem. J. 87, xiv (1948).
5tR. A. Dunford, Can. Chem. Process Inds. 35, 47 (1951).
52 HY, Eden and V. H. Booth, Food Manuf. 25, 279 (1950).
IV. BIOCHEMICAL SYSTEMS 501
logical response of carefully controlled test animals to administration of
tocopherol preparations according to standard procedures® is the ultimate
measure of potency and a desirable quality control test to be conducted on
composite production samples.
IV. Biochemical Systems
HENRY A. MATTILL
A. ABSORPTION AND METABOLISM
The absorption of tocopherols, like that of other fat-soluble substances
is greater from oil solution than from vegetable tissues.! There is no evi-
dence that esterification is a feature of the absorption of vitamin E as it is
of vitamin A; indeed, when the ester is fed to rats, the serum contains the
free alcohol, suggesting rather rapid hydrolysis;? but only about half as
much tocopherol is found in the serum as when free tocopherol is given. The
rise in human serum was reported to be about the same after the ingestion
of either form.’ The presence of bile salts is essential; in rats* and in dogs?®
with bile fistula the amount of tocopherol absorbed from a good diet was
sufficiently reduced to cause demonstrable deficiency.
On a normal intake, absorption is fairly complete. After a daily dose of
3.5 mg. ina rat, 3 to 15% of it appeared in the feces; after a large dose, as
much as 25 %.® Its presence in the urine could be demonstrated only spec-
trophotometrically under those conditions but not chemically; also, no
tocoquinone was demonstrable.’
Methylation of any of the lower homologs to the trimethyl a variety is
unlikely in view of the appearance of y-tocopherol in the eggs following the
feeding of it to laying hens.’: ° The deposition in eggs after feeding the three
tocopherols was 10:2.5:1 for a:8:y. The feeding of a-tocopherol to milch
53 K, E. Mason and P. L. Harris, Biol. Symposia 12, 459 (1947).
1C. Engel and J. T. Heins, Acta Brevia Neerl. Physiol. Pharmacol. Microbiol. 18,
37 (1943).
*C. Engel, Acta Brevia Neerl. Physiol. Pharmacol. Microbiol. 11, 18 (1941).
3K. L. Hove, Trans. 1st Conf. on Biol. Antioxidants, New York p. 51 (1946).
4 J. D. Greaves and C. L. A. Schmidt, Proc. Soc. Exptl. Biol. Med. 37, 40 (1937).
5K. M. Brinkhous and E. D. Warner, Am. J. Pathology 17, 81 (1941).
6’W. F. J. Cuthbertson, R. R. Ridgeway, and J. C. Drummond, Biochem. J. 34,
34 (1940).
7L. R. Hines and H. A. Mattill, J. Biol. Chem. 149, 549 (1943).
§M.L. Quaife, W. J. Swanson, M. Y. Dju, and P. L. Harris, Ann. N. Y. Acad. Sci.
52, 300 (1948).
9M. Y. Dju, M. L. Quaife, and P. L. Harris, Am. J. Physiol. 160, 259 (1950).
502 THE TOCOPHEROLS
cows (10 g. daily) raised the tocopherol content of the milk to 0.068 mg.
per gram of fat (from 0.025), whereas a mixture of y- and 6-tocopherols
changed it to 0.0834 mg. per gram of fat (from 0.022). Studies on normal
adults confirmed this idea; a-tocopherol increased the level of serum to-
copherols more quickly and for a longer time than vy. There appears to be
a selective deposition of a-tocopherol in animal tissues and fluids.
The catabolic pathway is not known, but presumably it is via tocoph-
erylquinone, the first stable oxidation product; more of this than of
tocopherol was reported in dog plasma.!° It has recently been found in the
stools,!! but it was not demonstrable in rat liver, muscle, or urine.’
The presence of some tissue tocopherol in a bound form has been in-
ferred from the additional amounts variably obtained by acid alcohol ex-
traction, after the usual treatment with alcohol-petroleum ether (Skel-
lysolve B) mixture.”?’ ” Furthermore, the normal plasma content (0.9 to
1.2 mg.%) is only slowly extracted by ether or hydrocarbon solvents,
whereas prior or simultaneous use of ethyl alcohol rapidly extracts it;!
measurements of intrinsic viscosity and other evidence favor the view that
conjugates are formed with native proteins, most successfully with lipid-
free bovin plasma albumin, such that a concentration of 2 to 3 mg. of to-
copherol per milliliter of a 2% protein solution can be readily achieved as
a stable slightly opalescent solution. The advantages of this procedure
over the use of the unphysiological phosphate or succinate are obvious.
B. STORAGE
The capacity of various rat tissues to accumulate tocopherol was first
demonstrated by Mason" in extensive trials with the bioassay method.
When the diet contained minimal amounts of vitamin E the liver stored
one-half to one-fourth as much as the skeletal muscles, body fat, and visceral
organs. With a high intake, the liver stored 14 times as much as at the lower
intake, the other tissues only 3 to 4.5 times. The liver is thus the chief re-
pository of vitamin E when its intake is high, and the amount found there
may be the best index of previous intake.
C. ANTIOXYGENIC ACTION
1. In Vivo
Because of the increased stability of fats containing vitamin E, the pos-
sibility of enriching the fat stores by adding tocopherols to the diet has
10 J. V. Scudi and R. P. Buhs, J. Biol. Chem. 146, 1 (1942).
11. Rosenkrantz, A. T. Milhorat, and M. Farber, J. Biol. Chem. 192, 9 (1951).
2H. Kaunitz and J. J. Beaver, J. Biol. Chem. 166, 205 (1946).
13$. R. Ames and H. A. Risley, Ann. N. Y. Acad. Sci. 52, 149 (1949).
144K, E. Mason, J. Nutrition 28, 71 (1942).
—
IV. BIOCHEMICAL SYSTEMS 503
been explored. It was first demonstrated experimentally in rats!" that
the induction period of rendered abdominal fats could be prolonged in this
manner, the maximum deposition of the tocopherols being achieved 7 to
10 days after single doses. a-Tocopherol was slightly more effective than
y-tocopherol (the opposite is true zn vitro), 8-tocopherol being intermediate.
The quantities of the three forms in the fat, as found by chemical determina-
tion, varied in the same direction, suggesting that the rate of absorption
might be the dominant factor.
Similar protection was imparted to the fat of rabbits on a purified diet
(but not on a natural ration) by feeding or injecting tocopherols at high
levels;8 pork fat was also protected in this manner when tocopherols were
fed to baby pigs in sufficient amounts over a 12-week period.” In stored
turkeys the peroxide value of the fats and the development of unpleasant
flavors were inversely related to the tocopherol content of the tissues,
which was increased by extra feedings.”°
2. In Vitro
The prevention of oxidized flavors in milk has been correlated with in-
creased tocopherol content of the milk produced by feeding.”! The factors
responsible for the production of these unacceptable qualities are many and
complex, and much study has been given to the usefulness of various sta-
bilizers, including tocopherol by itself and in various combinations,” to
prevent deterioration in the quality of stored milk, butter, and other fat-
containing foods and food products. Tocopherol with ascorbic, citric, and
phosphoric acids (or their acid salts) are the principal naturally occurring
synergists”-*° in milk, but they may not be effective in aqueous systems.
Vitamin A and carotene in small concentrations,”® cephalin,?” methionine,”
15 W. O. Lundberg, R. H. Barnes, M. Clausen, and G. O. Burr, J. Biol. Chem. 153,
265 (1944).
16 W. QO. Lundberg, R. H. Barnes, M. Clausen, N. Larson, and G. O. Burr, J. Biol.
Chem. 168, 379 (1947).
7G. O. Burr, W. O. Lundberg, and J. R. Chipault, Ozl & Soap 28, 382 (1946).
18 R. Major and B. M. Watts, J. Nutrition 35, 103 (1948).
19 T,. E. Carpenter and W. O. Lundberg, Ann. N. Y. Acad. Sci. 52, 269 (1949).
20 J. E. Criddle and A. F. Morgan, Proc. Soc. Exptl. Biol. Med. 78, 41 (1951).
21.V.N. Krukovsky, J. K. Loosli, and F. Whiting, J. Dairy Sct. 32, 196 (1949).
22H. A. Mattill, Oil & Soap 22, 1 (1945).
23H. S. Oleott and H. A. Mattill, J. Am. Chem. Soc. 68, 2204 (1936).
24C. Golumbie and H. A. Mattill, J. Am. Chem. Soc. 68, 1279 (1941).
25V. P. Calkins and H. A. Mattill, J. Am. Chem. Soc. 66, 239 (1944).
26 B. R. Monaghan and F. O. Schmitt, J. Biol. Chem. 96, 387 (1932).
27H. 8. Olcott and H. A. Mattill, Oil & Soap 18, 98 (1936).
28D. F. Clausen, W. O. Lundberg, and G. O. Burr, J. Am. Oil Chemists’ Soc. 24,
403 (1947).
504 THE TOCOPHEROLS
sulfhydryl groups,?’ and crude sources of vitamin B complex*® are known
to be effective in other systems. Complications are increased by the factor
of actual and relative concentrations; in more than minimal amounts, caro-
tene becomes a pro-oxidant,*!~** and there is an optimum concentration of
tocopherol for most effective stabilization.**: *°
The deterioration of fats and oils in natural and prepared foods and its
prevention by tocopherols and synergists are, chemically, still in the em-
pirical stage.** In general, the most effective fat antioxidants have a normal
oxidation potential between 848 and 484 millivolts.*’ A difference in po-
tential must exist between antioxidant and synergist, and the oxidation of
the synergist by the oxidized form of the antioxidant must be faster than
the oxidation of the antioxidant by the fat peroxides, which, in turn, must
be faster than the oxidation of the synergist by the fat peroxides.
D. SPARING ACTION ON VITAMIN A
Closely allied to the antioxygenic action of the tocopherols is their long-
discussed sparing action on vitamin A. The first indications of this physio-
logical property were the observations of Moore®* and of Bacharach*® to the
effect that the vitamin A reserves in the livers of rats kept for a long time
on E-deficient diets were much lower than those of rats receiving supple-
ments of vitamin E. Tocopherol concentrates prevented the autoxidation
of carotene zn vitro and increased the biological response of vitamin A-de-
ficient rats to minimal quantities of carotene.*? The vitamin A requirement
is thus partly dependent on the adequacy of dietary vitamin E.
Studies by Hickman and his colleagues*! showed that the growth-pro-
moting action of vitamin A and of carotene in rats was equally enhanced
by the simultaneous oral administration of any of the tocopherols; in hu-
man subjects fecal excretion of added dietary carotene and of other re-
ducing materials was increased when tocopherols or tocoquinones were also
29 P. Gyorgy, E. T. Stiller, and M. B. Williamson, Science 98, 518 (1943).
30 R. Tomarelli and P. Gyorgy, J. Biol. Chem. 161, 367 (1945).
31 C. R. Thompson and H. Steenbock, Arch. Biochem. 4, 15 (1944).
32H. O. Kunkel and W. L. Nelson, J. Biol. Chem. 183, 149 (1950).
33H. Heftmann, J. Am. Oil Chemists’ Soc. 24, 404 (1947).
34C. E. Swift, W. G. Rose, and G. 8. Jamieson, Ozl & Soap 19, 176 (1942).
35 C. Golumbic, O7l & Soap 20, 105 (1948).
86 Deterioration of Fats and Oils, Quartermaster Corps Manual No. 17-7. Commit-
tee on Food Research, Chicago, 1945.
37 C, Golumbic, O7l & Soap 28, 184 (1946).
38'T. Moore, Biochem. J. 34, 1321 (1940).
39 A. L. Bacharach, Quart. J. Pharm. Pharmacol. 18, 138 (1940).
40 FB. W. Quackenbush, R. P. Cox, and H. Steenbock, J. Biol. Chem. 145, 169 (1942).
41 K.C.D.Hickman,M. W. Kaley, and P. L. Harris, J. Biol. Chem. 152, 308, 313, 321
(1944).
IV. BIOCHEMICAL SYSTEMS 505
fed. As mentioned earlier,” vitamin E increased the utilization of vitamin
A in chicks, and the administration of 1 g. of tocopherol per day to New
Zealand cows raised the levels of carotene and vitamin A in their milk
fat.* This sparing effect must be taken into account in any assessment of
the vitamin A value of a diet.**: *
These facts and the ineffectiveness of the acetate ester, previously ob-
served": 4° and later confirmed,” suggested an antioxygenic action of tocoph-
erols in and near the alimentary tract rather than in the liver.*® Such limi-
tation on the site of action of tocopherols does not explain the effectiveness
of otherwise suboptimal quantities of essential fatty acids in preventing
the fatty acid deficiency syndrome in rats when they receive tocopherol,*
which has not been confirmed,®*° or the increased stability of tissue fats.
This sparing action of tocopherols is definite, and the conflicting results
are doubtless due to uncontrolled variables, including the form and amounts
of vitamins A and E administered,*!: * the manner in which they are
given,**: ** and the presence of inositol®° or of stabilizers such as cephalin®®
or xanthophyll.*”
The biological implications of this point of view were recently emphasized
by Dam and his colleagues,°*-®° who found that methylene blue, thiodi-
phenylamine, and Antabuse (tetraethylthiuram disulfide), when added to
vitamin E-deficient diets containing cod liver oil, caused an increased deip-
osition of vitamin A in the livers of chicks. The first two substances d d
the same in rats. When cod liver oil was replaced by lard, or when the diet
contained no fat, methylene blue had no effect on the vitamin A storage.
#2 H. Patrick and C. L. Morgan, Poultry Sci. 22, 397 (1943) ; 28, 525 (1944).
48W. A. McGillivray, J. Dairy Research 19, 119 (1952).
44K. C. D. Hickman, P. L. Harris, and M. R. Woodside, Nature 150, 91 (1942).
4° K. Guggenheim, Biochem. J. 38, 260 (1944).
465A. LL. Bacharach, Quart. J. Pharm. Pharmacol. 18, 138 (1940).
47M. C. Miles, E. M. Erickson, and H. A. Mattill, Proc. Soc. Exptl. Biol. Med. 70,
162 (1949).
“8H. Popper, F. Steigmann, and H. A. Dyniewicz, Gastroenterology 10, 987 (1948).
49 FE. L. Hove and P. L. Harris, J. Nutrition 31, 699 (1946).
50 L,. Anisfeld, S. M. Greenberg, and H. J. Deuel, Jr., J. Nutrition 45, 599 (1951).
1G. R. Halpern and J. Biely, J. Biol. Chem. 174, 817 (1948).
52 J. M. Lemley, E. A. Brown, O. D. Bird, and A. D. Emmet, J. Nutrition 34, 205
(1947).
53 R. M. Johnson and C. A. Baumann, J. Biol. Chem. 175, 811 (1948).
54H. J. Deuel, Jr., S. M. Greenberg, and E. E. Savage, J. Nutrition 48, 371 (1951).
55 P. Handler, J. Biol. Chem. 162, 77 (1946).
56 G. C. Esh and T.S. Sutton, J. Nutrition 36, 391 (1948).
57 P. Goldhaber, L. Zacharias, and V. E. Kinsey, J. Nutrition 42, 453 (1950).
88H. Dam, I. Prange, and E. Sondergaard, Acta Pharmacol. Toxicol. 8, 1 (1952).
59H. Dam, I. Prange, and E. Sondergaard, Acta Pharmacol. Toxicol. 8, 23 (1952).
0 H. Dam and H. Granados, Acta Pharmacol. Toxicol. 8, 47 (1952).
506 THE TOCOPHEROLS
The protective effect is believed to be in the tissues, since the vitamin A |
of the unsupplemented cod liver oil diet did not diminish during storage
of the food or under conditions resembling those in the alimentary tract.
Of particular interest was the observation that methylene blue could par-
tially replace tocopherol in preventing the sterility of female rats on vita-
min E-deficient diets. Vitamin E is thus a biological antioxidant, or it can
maintain the integrity of certain enzymatic redox systems in which methy-
lene blue can replace the vitamin.
V. Estimation
HENRY A. MATTILL
A. QUANTIATIVE DETERMINATION
Quantitative methods for the determination of tocopherols include chemi-
cal, physical, and biological procedures. All are described and critically
evaluated in two recent volumes.!
1. CHEMICAL ASSAY
The chemical methods are based largely on the assay of oxidation prod-
ucts. The most complete oxidation is that obtained in the Furter-Meyer
method with nitric acid, the color of the chroman-5 ,6-quinone being de-
termined spectrophotometrically or colorimetrically. Volumetric titration
methods have been developed with ceric sulfate? and lead tetraacetate.’
By means of the latter, almost pure tocopherylquinone can be prepared.
A procedure was also devised for the simultaneous determination of to-
copherol, tocopherylquinone, and vitamin K,*:° involving oxidation to
quinone, reduction to the respective hydroquinones, and the use of 2,6-
dichloroindophenol. Immediate diminution of the blue color is a measure
of vitamin K; subsequent slower reduction is due to tocopherylhydroqui-
none.
The most versatile and commonly used method is based on the oxidation
of tocopherol to tocopherylquinone by ferric chloride in the presence of
a,a-dipyridyl, the resultant ferrous chloride being measured by the red
color produced.® The blue color produced on the addition of potassium fer-
1P, Gyorgy, Vitamin Methods. Academic Press, New York, 1950, 1951.
2H. Schulek and P. Rozsa, Z. anal. Chem. 126, 253 (1948).
3 A. Issidorides, J. Am. Chem. Soc. 78, 5146 (1951).
4 J. V. Scudi and R. P. Buhs, J. Biol. Chem. 146, 1 (1942).
5.N.R. Trenner and F. A. Bacher, J. Biol. Chem. 187, 745 (1941).
6 A. Emmerie and C. Engel, Rec. trav. chim. 57, 1351 (1938).
V. ESTIMATION 507
ricyanide can also be used.’ Interfering substances such as vitamin A and
other materials readily oxidized under the conditions must be removed by
various means,’ such as selective adsorption on Floridin X$° or clay,!° treat-
ment with concentrated sulfuric acid," hydrogenation,” or high-vacuum™
or molecular distillation" of the tocopherols from extracts. Or the deter-
mination can be made before and after acetylation, the difference being a
measure of tocopherol.!®
With minor modifications, this method has had wide use; typical ex-
amples are cited of its application to oils,!* foodstuffs,!’ plant!® and ani-
mal*°* tissues and serum.”°~*!
In the presence of fats, the amount of color produced is greatly depressed,
for reasons as yet unknown; the effect varies with the nature and concen-
tration of the fat, and the interference is, of course, avoidable by prelimi-
nary separation of the unsaponifiable portion, with due care to prevent
loss due to the high temperature of saponification.
The less sensitive potentiometric method by which tocopherol is oxidized
to tocopherylquinone with gold chloride* is subject to the same interfer-
7P. Meunier and A. Vinet, Compt. rend. 211, 611 (1940); Ann. chim. anal. et chim.
appl. 28, 145 (1941).
8 J. G. Baxter, Biol. Symposia 12, 484-507 (1947).
9 A. Emmerie and C. Engel, Rec. trav. chim. 58, 283 (1939).
10 P. Meunier and A. Vinet, Bull. soc. chim. biol. 24, 365 (1942).
u W. E. Parker and W. D. McFarlane, Can. J. Research, B18, 405 (1940).
2M. L. Quaife and R. Beihler, J. Biol. Chem. 159, 663 (1945).
13 J. Glavind, H. Heslet, and I. Prange, Z. Vitaminforsch. 18, 266 (1948).
144M. L. Quaife and P. L. Harris, Ind. Eng. Chem. Anal. Ed. 18, 707 (1946).
15 A. Emmerie and C. Engel, Z. Vitaminforsch. 18, 259 (1943).
16 A. Emmerie, Rec. trav: chim. 59, 246 (1940); 60, 104 (1941).
17 J. Tosic and T. Moore, Biochem. J. 39, 498 (1945).
1 M. L. Quaife and P. L. Harris, Anal. Chem. 20, 1221 (1948).
19M. E. Wall and E. G. Kelley, Ind. Eng. Chem. Anal. Ed. 18, 198 (1946).
20M. L. Quaife and M. Y. Dju, J. Biol. Chem. 180, 263 (1949).
21H. B. Devlin and H. A. Mattill, J. Biol. Chem. 146, 123 (1942).
22 L. R. Hines and H. A. Mattill, J. Biol. Chem. 149, 549 (1948).
23 H. Kaunitz and J. J. Beaver, J. Biol. Chem. 166, 205 (1946).
24M. L. Quaife, W. J. Swanson, M. Y. Dju, and P. L. Harris, Ann. N. Y. Acad. Sct.
52, 300 (1949).
25 A. Emmerie and C. Engel, Rec. trav. chim. 58, 895 (1939).
26 A. Emmerie, Rec. trav. chim. 61, 305 (1942).
27 G. G. Mayer and H. Sobotka, J. Biol. Chem. 148, 695 (1942).
78 A.S. Minot, J. Lab. Clin. Med. 29, 772 (1944).
29M. L. Quaife and P. L. Harris, J. Biol. Chem. 156, 499 (1944).
30M. L. Quaife, N.S. Scrimshaw, and O. H. Lowry, J. Biol. Chem. 180, 1229 (1949).
31M. Farber, A. T. Milhorat, and H. Rosenkrantz, Proc. Soc. Exptl. Biol. Med.
79, 225 (1952).
82H. Kaunitz and J. J. Beaver, J. Biol. Chem. 156, 653, 661 (1944).
508 THE TOCOPHEROLS
ence by other reducing substances. At the dropping mercury electrode,*
the 8- and y-tocopherols are oxidized at more positive potentials than is
the a, and the latter can be determined, polarigraphically, within 5% in
the presence of oils or of traces of cholesterol.*®> The apparent oxidation-
reduction potential of a-tocopherol seems to be between the normal oxi-
dation potentials of mono- and dimethylhydroquinones,** approximately
+200 millivolts.° The potentials of the four tocopherols (@ to 6) are re-
ported as +273, +3848, +348, and +405 millivolts, respectively.”
None of these methods distinguishes between all the different tocopherols,
and, since their biological activity is unequal, various chemical methods
were sought by which to confirm or replace the tedious bioassay. Chromat-
ographic separation of the pure tocopherols from mixtures of them is in-
complete.** In one of the differentiating chemical methods,*® advantage was
taken of the fact that at 15° B- and y-tocopherols produce only about half
the color (Emmerie and Engel) which they would produce at 35°, whereas
temperature has no effect on color production by a-tocopherol.
A differential method for 6-tocopherol was developed, *° 41 depending
on the fact that with the ferric chloride-dipyridyl reagent 6-tocopherol
gives a slow steady increase in color intensity (in 10 min.) after the initial
rapid oxidation is completed (244 min.). This probably indicates that 6-
tocopherol is oxidized beyond the p-quinone stage. The absorption spec-
trum of AgNO;-oxidized 6-tocopherol has a broad maximum, suggesting
a mixture of reaction products. With nitric acid oxidation the product has
an absorption maximum at 373 my which might be made the basis for
spectrophotometric differentiation from the other tocopherols whose maxi-
mum is at 460 to 480 mu.
An actual separation is partially secured by utilizing the fact that -
tocopherol does not couple with diazotized o-dianisidine.” A differential
method for the determination of mixtures of a-, y-, and 6-tocopherols*
depends on the relative difference in color intensity of the coupled y- and
6-tocopherols in alkaline solutions, measured at two wavelengths.
All the tocopherols except the a react with nitrous acid to form nitroso
33 P. Karrer, W. Jaeger, and H. Keller, Helv. Chim. Acta 28, 464 (1940).
34 T,. I. Smith, L. J. Spillane, and I. M. Kolthoff, J. Am. Chem. Soc. 64, 447, 646 (1942).
35 J. J. Beaver and H. Kaunitz, J. Biol. Chem. 152, 363 (1944).
36 C, Golumbiec and H. A. Mattill, J. Biol. Chem. 134, 535 (1940).
37 W. Wacks, Biochem. Z. 319, 561 (1949).
38 H. Emmerie, Ann. N. Y. Acad. Sci. 52, 309 (1949).
39H}. L. Hove and Z. Hove, J. Biol. Chem. 156, 601 (1944).
40G. S. Fisher, Ind. Eng. Chem. Anal. Ed. 17, 224 (1945).
41M. H. Stern and J. G. Baxter, Anal. Chem. 19, 902 (1947).
4M. L. Quaife, J. Am. Chem. Soc. 66, 308 (1944).
43 J,, Weisler, C. D. Robeson, and J. G. Baxter, Ind. Eng. Chem. Anal. Ed. 19, 906
(1947).
V. ESTIMATION 509
derivatives." The 6-, y-, and 6-nitrosotocopherols can be separated by
simple chromatography, estimated separately, and the difference between
their sum and the total as determined by the Emmerie and Engel method
is a-tocopherol.*? Recently*® countercurrent distribution has been employed
for the separation of tocopherol from tocopherylquinone in stools. The
a, 6, and 6 compounds have been cleanly separated by reversed-phase
paper chromatography, but the 8 and y isomers could not be.”
Fluorometry has also been applied, preferably to the unsaponifiable mat-
ter of fats after chromatographic separation; the nitric acid oxidation prod-
uct is treated with o-phenylenediamine to form the fluorescent phenazine.*®
If the purely physical methods for estimation of the tocopherols are limi-
ted to their absorption spectra in the ultraviolet region, the first and ap-
parently only attempt to apply the technique quantitatively to natural
products*® showed that unidentified substances seriously interfere both
before and after saponification.
2. BIOASSAY
Tocopherol, the ‘‘fertility”’ vitamin, owes its name to the first demonstra-
tion of vitamin E deficiency, the failure of laboratory rats to procreate.
This was a chance observation, in Pasteur’s sense, and the existence and
indispensability of vitamin E might have been discovered in connection
with several other unnatural and diseased conditions now known to be
produced when it is lacking. Indeed, deprivation of vitamin E is followed
by a more baffling array of physiological abnormalities in different species
than has ever been encountered with a single vitamin. None of these dis-
orders is as suitable for purposes of bioassay as the restoration of fertility
in a vitamin E-deficient female rat. In one or another of its modifications,
this circumstantial, tedious, and demanding procedure has been the guide
and milestone in the exploratory work on the distribution and chemical
nature of vitamin E.
To be valid, such a bioassay must meet several criteria.®°
1. Fertilization by a normal male must be established by the finding of
sperm in the vaginal contents or of a vaginal plug.
44 J. V.Scudi and R. P. Buhs, J. Biol. Chem. 146, 1 (1942).
45M. L. Quaife, J. Biol. Chem. 175, 605 (1948).
46H. Rosenkrantz, A. T. Millhorat, and M. Farber, J. Biol. Chem. 192, 9 (1951).
47 F. Brown and K. L. Blaxter, Chemistry & Industry 1951, 633.
48 M. Kofler, Helv. Chim. Acta 25, 1469 (1942) ; 26, 2166 (1943) ; 28, 26 (1945): 30, 1053
(1947).
H. Lieck and H. Willstaedt, Svensk Kem. Tidskr. 57, 134 (1945) [C.A. 40, 4759
(1946) ].
49T. Moore and K. R. Rajagopal, Biochem. J. 34, 335 (1940).
50 A. L. Bacharach, E. Allchorne, and H. E. Glynn, Biochem. J. 31, 2287 (1937).
510 THE TOCOPHEROLS
2. The 4- to 5-day estrous cycles, having been regular before fertiliza-
tion, as determined by vaginal smears, should cease.
3. Implantation should be demonstrated by the placental sign or vaginal
blood leak about the twelfth day of pregnancy.
4, On the twentieth or twenty-first day, the animal casts a litter if vita-
min E supplies have been adequate, or her weight declines steeply but not
abruptly, indicating resorption of the young. If there is any question of the
fertility of the animal as such, an adequate dose of vitamin E should be
given after another positive mating, and it is also essential to recognize
a pseudopregnancy.
Because of the possibility of initial or first litter fertility due to vitamin
E stores, a resorption gestation was once considered a necessary step be-
fore any animal was used for test. It was demonstrated, however, that the
animal was a less sensitive indicator after a resorption gestation, and the
practice first recommended by Mason and Bryan*! is now generally fol-
lowed: the rats to be used for assay purposes are reared on a vitamin E-
deficient diet beginning in their nursling period. Animals whose vitamin E
storage is limited to that obtained through placental and mammary trans-
fer, on such a regime, never show first litter fertility. In any case, placental
transfer is very limited, whereas mammary transfer, especially after ad-
ministration of large doses of tocopherol, has been demonstrated in lactat-
ing rats and in other species”: ** to be considerable, particularly in colos-
trum.*!: °° Tocopherols in early human milk are reported as 0.13 to 3.6
mg. per 100 ml., with an average of 0.14 mg. in later milk.®® A later study”
confirmed the mammary transfer of tocopherol; premature infants on for-
mulas low in vitamin EK demonstrated a rapid decline in serum tocopherol
levels. During the first six days after birth, the figures for breast-fed infants
increased much more rapidly than those for bottle-fed.
A further improvement in the direction of standardization®® was the ex-
amination of the uterus at the sixteenth day by laparotomy, a positve re-
sponse being the presence of two or more viable fetuses with at least four
implantation sites. The presence of placental scars or of resorbing fetuses
(or of both) testifies to the positive character of the mating and the in-
51K. E. Mason and W. L. Bryan, Biochem. J. 32, 1785 (1938).
52 K. E. Mason and W. L. Bryan, J. Nutrition 20, 501 (1940).
53 A. M. Pappenheimer, H. Kaunitz, and C. Schogoleff, Proc. Soc. Exptl. Biol. Med.
55, 229 (1944).
54, Whiting and J. K. Loosli, J. Nutrition 36, 721 (1948).
55 T). B. Parrish, G. H. Wise, and J.S. Hughes, J. Dairy Sci. 30, 849 (1947); Ann. N. Y.
Acad. Sci. 52, 251 (1949).
56 M. L. Quaife, J. Biol. Chem. 169, 513 (1947).
57 $. W. Wright, L. J. Filer, Jr., and K. E. Mason, Pediatrics 7, 386 (1951).
58K. EH. Mason, J. Nutrition 28, 59 (1942).
a
V. ESTIMATION DL
adequacy of the test dose. The assay period is thereby shortened, and there
is no temptation to use the animal for further and possibly unreliable as-
Says.
There is some question as to the time and manner of administering the
test dose, whether on the fourth, fifth, and sixth days,® or on each of the
first ten days,®° after conception; the latter should be advantageous if more
efficient use can be made of a smaller dose.*! The test substance is ordinarily
administered by mixing it with a small portion of the diet under conditions
that assure its consumption or by giving it, dissolved in a suitable oil
(olive), with a medicine dropper.
Enough animals must be used (perhaps ten) on each of several levels of
ingestion (three or four) to permit statistical treatment of the results which
relate dosage to litter efficiency either on a curve™ or more simply by the
method of probits.® Litter efficiency is suitably expressed in terms of mean
fertility dose, which is the least amount of tested substance on which one-
half of the animals give a positive response as defined above. A series of
animals on pure tocopherol or an ester of it should accompany the several
series of the unknown substance under test.
The results of bioassays made by this standardized procedure are more
reliable than earlier results, many of which were disappointing in their
wide divergence; thus, the mean fertility dose for synthetic racemic dl-a-
tocopherol acetate was 0.56 to 1.71 mg.** The MFD of natural a-tocopherol
(and its succinic acid ester) given by mouth is now 0.75 mg.® The “unit”
for vitamin E as originally proposed®™ was 1 mg. of tocopherol acetate in
olive oil, an amount greater than the presently accepted MFD for this sub-
stance.
Other criteria for the basis of a bioassay have included increase in ma-
ternal body weight during gestation,® the number of offspring delivered
at term, whether living or dead,®® or the percentage of placental implants
resulting in birth of living offspring.* The disappearance of symptoms of
exudative diathesis in chicks,® the reduction of creatinuria in rabbits suf-
fering from muscular dystrophy, the prevention of hemolysis due to an
59 M. Joffe and P. L. Harris, J. Am. Chem. Soc. 65, 925 (1943).
60 P. L. Harris, J. L. Jensen, M. Joffe, and K. E. Mason, J. Biol. Chem. 156, 491 (1944).
61C. A. Cabell and N. R. Ellis, J. Nutrition 23, 633 (1944).
6 A. L. Bacharach, Biochem. J. 32, 2017 (1938).
68 K. EK. Mason and P. L. Harris, Biol. Symposia 12, 459 (1947).
6 KE. M. Hume, Nature 148, 472 (1941); Quart. Bull. Health Organisation League
Nations 9, 436 (1940-1941).
6° H. Gottlieb, F. W. Quackenbush, and H. Steenbock, J. Nutrition 25, 433 (1943).
665 A. L. Bacharach and E. Ailchorne, Biochem. J. 32, 1298 (1938).
67. S. Palmer, Ind. Eng. Chem. Anal. Ed. 9, 427 (1937).
8 H. Dam, J. Glavind, I. Prange, and J. Ottesen, Kgl. Danske Videnskab. Selskab.
Biol. Medd. 16, No. 7 (1941).
512 THE TOCOPHEROLS
alloxan-like substance (considered later), and the prevention of testicular
atrophy in the male rat®® have also been suggested as possible methods of
bioassay of vitamin E. The interesting suggestion to use the transparent
crustacean Daphnia magna for rapid detection and evaluation of vitamin
FE” seems never to have been further developed.
VI. Occurrence in Food
HENRY A. MATTILL
The distribution of the tocopherols is probably wider than that of any
other vitamin, certainly more extensive than that of the other fat-soluble
types. They occur as free alcohols, not as esters, and except for their con-
sequent rapid oxidation in experimental rations containing certain unsatu-
rated fats their discovery might have been long delayed. Before the develop-
ment of trustworthy chemical methods for the separate estimation of each
of the tocopherols, the application of the arduous bioassay had provided
much information on the occurrence of vitamin E in various plants and in
animal tissues. Subsequent chemical determinations have added many
items,!’ 2? most of which can be found in the usual food tables. The richest
dietary sources are the vegetable oils and cereal products containing them,
butter and margarine, eggs and liver, legumes and greens.
In plant tissue the tocopherols usually accompany carotene or highly
unsaturated fatty acids (in seed oils),? substances readily oxidized in air.
There appears to be a significant correlation between the amounts of linoleic
acid and tocopherol found in fats and oils.t As was pointed out,°® this as-
sociation of tocopherol and unsaturated fatty acids in vegetable tissues
and the presence of relatively impermeable cellulose walls afford consider-
able protection against autoxidation, whereas in animal tissue there are
no barriers to the diffusion of oxygen. If there is likelihood of peroxidation
of the fats in animal tissue and if the tocopherols act as stabilizers, one
would expect to find a higher concentration of them in tissues of high fat
content. In the rat,! except for the pituitary and suprarenals, this appears
to be true, but none of the many rat tissues examined is completely devoid
69 M. L. Herraiz and J. C. Radice, Ann. N. Y. Acad. Sci. 52, 88 (1949).
70 A. Viehoever and I. Cohen, Am. J. Pharm. 110, 297 (1938).
1M. L. Quaife, Ann. N. Y. Acad. Sci. 52, 300 (1949).
2P. L. Harris, M. L. Quaife, and W. J. Swanson, J. Nutrition 40, 367 (1950).
3 K.-M. Bradway and H. A. Mattill, J. Am. Chem. Soc. 56, 2405 (1934).
4). L. Hove and P. L. Harris, J. Am. Oil Chemists’ Soc. 28, 405 (1951).
5K. C. D. Hickman and P. L. Harris, Advances in Enzymol. 6, 469 (1946).
VI. OCCURRENCE IN FOOD 513
of tocopherols when the intake is abundant.®:* Lard® and fish oils’ may
contain small amounts. Bacterial synthesis of tocopherol in the intestine!®
is unlikely;" indeed, the feeding of sulfaguanidine in a diet high in cod liver
oil protected rats against peroxide-produced yellow-brown coloration of
adipose tissue,” perhaps due to an in vivo antioxygenic effect of the sulfa
drug.
The amount found in cereal products depends primarily upon the ex-
tent of milling, since the tocopherols are mostly in and near the embryo;
in durum wheat the non-germ tocopherol is fairly uniformly distributed
throughout the endosperm. In animal products the content increases with
the rising level of vitamin E in the diet, especially in the liver and body
fats, as recently found in swine; more tocopherol was found in the blood
cells than in the plasma. The severity of processing and length of storage
to which the foodstuffs have been subjected are major factors, whatever
the source. In dairy cows the level of plasma tocopherols seems to be directly
related to the intake, whether this varies as the result of natural seasonal
supply or of supplementary feedings.!*: !® The tocopherol content of hu-
man serum seems to vary similarly.”
As mentioned in connection with mammary transmission,'*~*! the amount
of vitamin E in milk is closely dependent on the amount in the diet and is
greater in colostrum than in later milk. Recently, in a series of human milk
samples representing various donors and stages of lactation, the figures
were 0.10 to 0.48 mg. per 100 ml.;” the mean was 0.24 mg., or about 80 y
per gram of fat, and throughout the period of lactation (8rd through 37th
6 WV. F. J. Cuthbertson, R. R. Ridgeway, and J. C. Drummond Biochem. J. 34, 34
(1940).
7J.R. Chipault, W. O. Lundberg, and G. O. Burr, Arch. Biochem. 8, 321 (1945).
8 N. Simmonds, J. E. Becker, and E. V. McCollum, J. Nutrition 1, 39 (1928).
°C. D. Robeson and J. G. Baxter, J. Am. Chem. Soc. 65, 940 (1948).
10 J. Pindborg, Nature 164, 493 (1949).
11P, L. Harris, Nature 165, 572 (1950).
12H. Granados, E. Aaes-Jorgensen, and H. Dam, Experientia 6, 150 (1950).
13 FD. S. Binnington and J. S. Andrews, Cereal Chem. 18, 678 (1941).
14 J. W. Bratzler, J. K. Loosli, V. N. Krukovsky, and L. A. Maynard, J. Nutrition
42, 59 (1950).
15 F.C. van der Kaay, G. H. B. Teunissen, A. Emmerie, and M. van Eekelen, Ann.
N.Y. Acad. Sci. 52, 276 (1949).
16 C, E. Latschar, G. H. Wise, D. B. Parrish, and J. S. Hughes, J. Nutrition 38,
503 (1949).
17 J. Couperus, Nederl. Tijdschr. Geneesk. 87, 541-547 (1943) [C.A. 38, 4296 (1944)].
18 K. E. Mason and W. L. Bryan, J. Nutrition 20, 501 (1940).
19, Whiting and J. K. Loosli, J. Nutrition 36, 721 (1948).
20 —D. B. Parrish, G. H. Wise, and J. 8S. Hughes, J. Dairy Sci. 30, 849 (1947).
21M. L. Quaife, J. Biol. Chem. 169, 513 (1947).
22 PL. Harris, M. L. Quaife, and P. O’Grady, J. Nutrition 46, 459 (1952).
514 THE TOCOPHEROLS
week), the values were correlated with the fat content. Pasteurization
caused no loss. Evaporated cow’s milk and whole milk powder contained
23 to 40 y of tocopherol per gram of fat; proprietary infant foods, 29 to
ly:
VIL. Effects of Deficiency
KARL E. MASON
A. IN ANIMALS
1. INTRODUCTION
a. General Considerations
More than a quarter century has passed since the existence of vitamin E
(a-tocopherol) was definitely established. Its first recognized function as
an antisterility factor for the laboratory rat has been overshadowed by its
demonstrated need for maintenance of structural and functional integrity
of skeletal muscle, cardiac muscle, smooth muscle, and, in some animals,
the peripheral vascular system. Tocopherols play an important role as
intracellular antioxidants, related especially to the stabilization of ingested
fats and possibly of products arising in the metabolic synthesis and deg-
radation of lipids, and they may also function in a detoxifying capacity.
Morphologie alterations arising in the course of vitamin E deficiency may
well represent localized reactions of particularly susceptible tissues to loss
of these vital antioxidants or, secondarily, to dysfunction of enzyme sys-
tems in which tocopherols actively participate.
The histopathologic lesions of vitamin E deficiency are remarkably
varied, they represent morphologic alterations in a number of unrelated
tissues, they seem not to be related to dysfunction of any specific type of
cell or tissue, and they are of such a nature that restoration of normal
morphology rarely occurs after tocopherol therapy, even though the physi-
ological or biochemical disturbances are corrected. Most lesions are de-
pendent upon fat in the diet, and their onset and intensity are accentuated
in proportion to the amount and degree of unsaturation of the fat used.
There are the possibilities that unsaturated fats destroy dietary traces of
the vitamin in the diet or the gut, that they or their oxidation products
produce a direct cell injury which is superimposed upon that due to lack
of vitamin E, and that excess utilization of tocopherols to stabilized un-
saturated fats being incorporated into cell lipids hastens depletion of tissue
tocopherols and the precipitation of deficiency manifestations (which may
or may not be related to lipid metabolism of the cells involved). Conceivably,
VII. BFFECTS OF DEFICIENCY 515
the true picture may represent a combination of these postulated interac-
tions. With these possible interactions in mind, the symptomatology and
histopathology of experimental vitamin E deficiency may be more com-
prehensible.
TABLE II
LISTING OF REFERENCES DESCRIBING HISTOPATHOLOGIC CHANGES AFTER VITAMIN E
DEFICIENCY IN VARIOUS SPECIES OF ANIMALS
(The numbers refer to references cited throughout the chapter.)
Reproductive system Musculature
Animal species | Rae ee
eae ie va ere Ae Skeletal Cardiac Smooth
Monkey 21 75 75 75 75
Dog 24 24, 6la 21 115, 116
Rat 3, 13-20 14, 32, 33 | 1, 4, 50, 51, | 4, 90-92 1, 2, 4, 5, | 52, 52a, 71, | 33, 114
53-57, 70- 92-97 74, 77-80,
74, 80, 90, 100b, 101,
92, 10la 10la
Mouse 29 25, 26, 37 | 27, 28; 29 28 29
Hamster 21 21 57a, 76 21 21 21
Cotton rat 21 21
Mink 62
Rabbit 23 57, 63a, 65, | 68a, 84, 85
67-69a
Guinea pig 22 38, 39 63a, 66, 82a | 63a, 86 100a
Sheep 58, 59, 59a, 89 | 58, 89
Goat 63a 63a
Cattle 60, 64a, b, c | 64a, b, ec, 64a
87, 88
Horse 61
Pig 24a 24a
Chick 30, 30a 57, 64 100 57, 100, 81, | 102-105,
106-109 110-113
Duck 63
Turkey 57 81, 99
Pheasant 57, 100
Guppy fish 31 [rol
b. Histopathologic Lesions in Different Species
Those structural and functional derangements which appear to be most
characteristic of avitaminosis E, whether experimentally induced or oc-
curring naturally, are listed in Table IJ, and under each are cited references
to pertinent literature dealing with the types of tissue dysfunction observed
in various animal species. It will be noted that lesions of skeletal muscles
constitute the most universal finding, and that recorded alterations of
other organs and tissues are rather spotty. The absence of recorded lesions
in tissues or organs of certain species does not necessarily mean that a
need for vitamin E on the part of these structures does not exist; for the
most part, it indicates either that the muscular lesions are so pronounced
516 THE TOCOPHEROLS
that the duration and degree of depletion necessary to bring about other
tissue dysfunctions have not been attained (as in the rabbit and in cattle),
or that only a deficiency state during early life of the species has been
studied (as in the mink, sheep, pig, horse, duck, turkey) and that little or
nothing is known of the effects of prolonged, chronic deficiency during adult
phases of life.
c. Pigment
Since frequent reference will be made to an acid-fast pigment often as-
sociated with lesions of vitamin E deficiency, it seems appropriate at this
point to discuss briefly its nature and possible origin. Martin and Moore!
first called attention to the occurrence of this pigment in uterine smooth
muscle, skeletal muscle, sex glands, and other organs and tissues of rats
maintained for prolonged periods on low E diets, and commented on its
insolubility, inert and iron-free nature, and the brownish discoloration of
the affected tissues. Its brownish-yellow fluorescence was also recognized.?
Although its major site of formation appears to be in the musculature, it
eventually comes to be acquired by macrophages of the adjacent connective
tissues and distributed rather widely throughout the reticulo-endothelial
system.*®: + Chemical analysis of the pigment in rat uteri suggests the pres-
ence of oxidation products of protein.? Histochemical studies® suggest a
lipofuchsin type of pigment derived through peroxidation and polymeriza-
tion of unsaturated fats; except for certain differences in its oxidation po-
tential, it is undistinguishable from a yellowish-brown, waxy pigment com-
monly observed in association with nutritional cirrhosis of rats on low
protein diets and first characterized by Lillie et al.,° who proposed the term
“ceroid” for it. In paraffin sections of tissues exposed to various fixatives
and fat solvents, both pigments have similar acid-fast, sudanophilie and
other staining reactions. Recent histochemical studies’: * further support
the theory that ceroid arises through the autoxidation of unsaturated lipids
pathologically accumulated in cells having an insufficiency of biological
antioxidants. Victor and Pappenheimer® have pointed out that failure to
1A.J.P. Martin and T. Moore, J. Hyg. 39, 648 (1939).
2 'T. Moore and Y. L. Wang, Biochem. J. 87 Proc. i (1948); Brit. J. Nutrition 1, 53
(1947).
3K. KH. Mason and A. F. Emmel, Yale J. Biol. Med. 17, 189 (1944).
4K. E. Mason and A. F. Emmel, Anat. Record 92, 33 (1945).
5H. Elftman, H. Kaunitz, and C. A. Slanetz, Ann. N. Y. Acad. Sci. 52, 72 (1949).
6R. D. Lilhe, L. L. Ashburn, W. H. Sebrell, F. S. Daft, and J. V. Lowry, Public
Health Repts (U.S.) 57, 502 (1942).
7W.G. B. Casselman, J. Exptl. Med. 94, 549 (1951).
8R. D. Lillie, Stain Technol. 27, 37 (1952).
9 J. Victor and A. M. Pappenheimer, J. Haptl. Med. 82, 375 (1945).
VII. EFFECTS OF DEFICIENCY Bly
provide sources of vitamin E, combined with the presence of cod liver oil,
in cirrhosis-producing diets is the primary cause of ceroid production, and
that tocopherol supplements suppress or prevent its formation. Pigment
accumulation in adipose tissue in low-E rats! (see p. 538), which provides
the closest morphologic counterpart to ceroid formation in the fatty in-
filtrated liver, is dependent upon the presence in the diet of fatty acids
having chain lengths of at least 18 carbon atoms and at least two unsatu-
rated bonds and is accentuated as chain length and unsaturation are in-
creased.!! The presence of peroxides in the adipose tissue! undoubtedly
plays a role in pigment formation. The evidence thus indicates that the
pigment of vitamin E deficiency and ceroid of nutritional cirrhosis are very
similar, if not identical. It should also be emphasized that this pigment
accumulates at the same sites and has much the same characteristics as
the so-called ‘‘wear and tear” pigment normally found to a limited extent
in the adrenal cortex, sex glands, and elsewhere in the body*: * For these
reasons care must be exercised in relating the occurrence of acid-fast pig-
ment to a state of avitaminosis E.
2. Mate REPRODUCTIVE SYSTEM
a. Rats
In male rats depleted of vitamin E from early life the seminiferous epi-
thelium shows no injury until active spermatogenesis begins, during the
third month of life, when a progressive and relatively rapid degeneration of
the epithelium occurs. Although there is close agreement among investiga-
tors regarding the distinctive character and irreparable nature of the histo-
pathologic changes,'*” there has been no satisfactory elucidation of the
underlying metabolic disturbances. The latter are so profound that vitamin
E therapy must be given 10 to 15 days prior to first appearance of histo-
logic injury in order to give full protection; therapy begun at intermediate
periods results in protection of certain seminiferous tubules but progressive
degeneration in others, or degeneration in all tubules, depending upon the
10K. E. Mason, H. Dam, and H. Granados, Anat. Record 94, 265 (1946).
uy. J. Filer, Jr., R. E. Rumery, andK. E. Mason, Trans. 1st Conf. on Biol. Antioxi-
dants, Josiah Macy, Jr. Foundation, New York, p. 67 (1946).
” H. Dam and H. Granados, Acta. Physiol. Scand. 10, 162 (1945).
18K. E. Mason, J. Exptl. Zool. 45, 159 (1926); Am. J. Anat. 52, 153 (1933).
14H. M. Evans and G. O. Burr, Mem. Univ. Calif. 8, 1 (1927).
16 A. Juhdsz-Shaffer, Virchow’s Arch. path. Anat. u. Physiol. 281, 3 (1931); 286, 834
(1932).
16 A. Ringsted, Dissertation, Underségelser over testis’ histopathologi ved E-avi-
taminose; en eksperimentel-morfologisk studie. Nyt Nordisk Forlag, Copen-
hagen, 1936.
17 C, Engel and L. H. Britschneider, Intern. Z. Vitaminforsch. 18, 58 (1943).
518 THE TOCOPHEROLS
interval. Degeneration is delayed 30 to 40 days by a single dose of 0.5 to
1.0 mg. of tocopherol fed on the 15th day of life;!® yet daily doses of between
0.5 and 0.75 mg. are necessary to protect the testis in rats reared 16 to 17
months on E-low diets.”° Prolonged deficiency has no effect on the weight
of accessory sex glands,”° indicating that hormonal functions of the testis
are not impaired.
The degenerative process, which reaches completion in 2 to 3 weeks,
affects seminiferous tubules unequally as it progresses. It is characterized
by the following sequence of events: (1) inhibition of spermatogenesis, as-
sociated with abnormal swelling and fusion of mature sperm; (2) marked
diminution in sperm, and nuclear chromolysis in spermatids and second-
ary spermatocytes; (3) extensive sloughing of germ cells, and fusion of many
into large multinucleate cells; (4) nuclear fragmentation and hydropic de-
generation of remaining germ cells; (5) eventually the shrunken tubules
are lined by a vacuolated, fibrous Sertoli syncytium. During the degenera-
tive process many germ cells are sloughed and transported to the epidi-
dymis; others undergo dissolution in situ. Moderate amounts of acid-fast
pigment are usually demonstrable in the Sertoli syncytium and in macro-
phages of the interstitial connective tissue.’ Leydig cells are normal. The
testes are grossly atrophied, brownish, flabby, and watery when cut.
b. Other Species
In the hamster there occurs a much more gradual degeneration of the
germinal epithelium, accompanied by accumulation of much acid-fast pig-
ment in the germ cells and macrophages of the interstitial tissue, but, unless
injury reaches an advanced stage, vitamin E therapy results in relatively
successful restoration of the germinal epithelium.”! Testicular degeneration,
associated with some pigment, occurs also in the guinea pig if a chronic
deficiency is maintained such that symptoms of muscular dystrophy are
kept minimal until after sexual maturity,” but therapeutic response has
not been studied. Testis damage resembling early phases of injury in the
rat has been observed in the rabbit,” the dog,” and the monkey.*! There
is also a suggestion that the germinal epithelium of young pigs is altered
18K. EK. Mason, Am. J. Physiol. 181, 268 (1940).
19 H. Kaunitz, A. M. Pappenheimer, and C. Schogoleff, Am. J. Pathol. 20, 247 (1944).
20 H. M. Evans and G. A. Emerson, J. Nutrition 26, 555 (1943).
21K. EK. Mason, Unpublished studies.
22 A. M. Pappenheimer and C. Schogoleff, Am. J. Pathol. 20, 239 (1944).
23 M. L. Chevrel and M. Cormier, Compt. rend. 226, 1854 (1948); Ann. Endocrinol.
10, 19 (1949).
24K. M. Brinkhous and E. D. Warner, Am. J. Pathol. 17, 81 (1941).
VII. EFFECTS OF DEFICIENCY 519
by vitamin E deficiency.*** The testis of the mouse, on the other hand, is
remarkably resistant; most investigators have observed no injury after
deficiency periods up to 14 months;?°* however, Menschik et al.2° report
marked atrophy of the germinal epithelium after about 18 months. There
is no testis injury in Florida cotton rats depleted to the point of showing
marked muscular dystrophy.”! In lower forms, testis degeneration has been
reported only in cockerels*’: *°* and in the guppy fish.*! The varied response
of the testis of different animals to vitamin E deficiency represents an inter-
esting but little-understood phenomenon.
3. FEMALE REPRODUCTIVE SYSTEM
a. Fetal Resorption
Intrauterine death and resorption of the fetus in well-nourished rats
represents the phenomenon primarily responsible for the discovery of vita-
min E and the basis for its subsequent bioassay and identification. The
histopathology is presented in the classic monograph of Evans and Burr™
and the later studies of Urner.** All reproductive events are normal up to
the time of implantation, which occurs at about the 7th day after insemi-
nation in the rat. Several days later there is retardation of fetal develop-
ment, diminished hemopoietic activity in yolk sae and liver, and rarefac-
tion of the allantois and mesenchymal tissues of the embryo proper. Either
the allantoic placenta fails to properly differentiate and invade the maternal
decidua, or else the latter offers unusual resistance to this invasion. In
either case, impaired vascular relationships between fetal and maternal com-
ponents of the placenta appear to be responsible for asphyxia, starvation
and death of the fetus. This is followed by rapid necrosis and resorption of
the fetus, more or less persistent but not severe uterine bleeding, and grad-
ual regression of the placenta until little more than a blood clot remains
24a F. B. Adamstone, J. L. Krider, and M. F. James, Ann. N. Y. Acad. Sci. 52, 260
(1949).
25 W.L. Bryan and K. E. Mason, Am. J. Physiol. 181, 263 (1940).
26 M. Goettsch, J. Nutrition 28, 513 (1942).
27 A. M. Pappenheimer, Am. J. Pathol. 18, 169 (1942).
28 C. E. Tobin, Arch. Pathol. 50, 385 (1950).
29 Z. Menschik, M. K. Munk, T. Rogalski, O. Rymaszewski, and T. J. Szezesniak,
Ann. N.Y. Acad. Sct. 52, 94 (1949).
30 F. B. Adamstone and L. E. Card, J. Morphol. 56, 325; 339 (1934); Anat. Record
84, 499 (1942).
30a HY. H. Herrick, I. M. Eide, and M. R. Snow, Proc. Soc. Exptl. Biol. Med. 79, 441
(1952).
31H. W. Cumings, Jr., Dissertation, University of Illinois (1940); Anat. Record 84
Proc. 499 (1942).
32 J. A. Urner, Anat. Record 60, 175 (1981).
520 THE TOCOPHEROLS
at term. There is also the interesting observation that offspring of rats
dosed at critical periods after the 8th day of gestation sometimes show
rather extensive umbilical and skeletal defects.**4
Fetal resorption can be prevented by administration of adequate vitamin
E at any time during the first week of pregnancy. If the dosage is critical
there may be delivery of dead as well as viable fetuses, the latter rarely
surviving more than a few days. Less adequate dosage delays fetal death
and resorption for varying periods. Under the latter conditions, fetuses at
about the 16th day of pregnancy frequently show pronounced changes in
the vascular system (stasis, distention, thrombosis, local hemorrhages)
and generalized ischemia prior to death, but no obvious lack of hemo-
poiesis.**
With prolonged depletion of vitamin E beyond the first few months of
reproductive life, there is a progressive increase in vitamin EF) requirements
for the completion of established pregnancies, and also a progressive inter-
ference with implantation of the ovum (i.e., decreased fecundity rate) as
age progresses.*4: > Evidence points to a uterine and not an ovarian dys-
function.*®
Fetal death and resorption quite comparable to that in the rat, although
not studied in great detail, occur in the mouse”: 2° and in the hamster.”!
That in the mouse has been attributed to impaired production of histio-
trophe.*” In the guinea pig there may occur necrosis of the placenta and
fetal death,** or abortion due to premature separation of the placenta.*®
Defective development of the chick embryo deprived of vitamin E will be
discussed later (p. 534).
b. Ovary
In rats reared for a year or more on E-low diets, there are no alterations
in behavioral estrus, ovulation, fertilization of the ovum, or its early de-
velopment and tubal transport.*® Histologically, macrophages of the ovarian
stroma progressively accumulate pigment arising at sites of follicular atresia
and luteal regression,? a phenomenon which is much more marked in the
hamster.?! In resorbing rats there is premature regression of corpora lutea,
33 K. E. Mason, Yale J. Biol. Med. 14, 605 (1942); and 7n Essays in Biology (in honor
of H. M. Evans), p. 401, University of California Press (1948).
338 B. H. Thomas and D. W. Cheng, Proc. Iowa Acad. Sci. 59, 218 (1952).
34 G, A. Emerson and H. M. Evans, J. Nutrition 18, 501 (1989).
35 H. Kaunitz and C. A. Slanetz, Proc. Soc. Exptl. Biol. Med. 66, 334 (1947); J.
Nutrition 36, 331 (1948).
36 R. J. Blandau, H. Kaunitz, and C. A. Slanetz, J. Nutrition 38, 97 (1949).
37 P. Soumalainen, Nature 165, 364 (1950).
38 A. M. Pappenheimer and M. Goettsch, Proc. Soc. Exptl. Biol. Med. 47, 268 (1941).
39 A. Ingelman-Sundberg, Acta Endocrinol. 2, 335 (1949).
VII. EFFECTS OF DEFICIENCY 521
but this is secondary to early termination of pregnancy.*® Otherwise, most
investigators have failed to find any morphologic changes in the rat ovary,
except in animals rather advanced in age where senility changes tend to
confuse the picture. On the other hand, well-controlled observations on
mice"! indicate that lack of vitamin E results in fewer primordial ova, less
interfollicular tissue, smaller but more numerous corpora lutea, and ab-
sence of neutral fat but marked increase in insoluble lipid complexes. Effects
on the ovary of other species have not been reported. Histologic changes
occurring in the uterus in vitamin E deficiency are discussed in a later sec-
tion (p. 531).
c. Other Endocrines
There is no convincing evidence that vitamin E deficiency exerts any
direct effect upon the function or structure of the anterior pituitary. There
exists a rather extensive and controversial literature, much of which has
been reviewed elsewhere.*°*: *: 4% Alterations observed in the basophiles of
the anterior pituitary of male rats, resembling those following castration,
are regarded as secondary to the testicular degeneration;***® they are ab-
sent in deficient female rats.” The pituitary of male chicks, showing testi-
cular injury of vitamin E deficiency, shows changes similar to those of the
male rat.*°* Thyroid hypoplasia reported in low-E rats, and attributed to
disturbed anterior pituitary functions, seem explicable on the basis of
a relative inadequacy of dietary iodine.*®
The adrenal cortex undergoes no significant change as a result of vitamin
E deficiency other than an accumulation of acid-fast pigment which closely
resembles the “wear and tear’’ pigment normally present to a limited extent
in the zona reticularis and may reflect diminished ability of the low-E ani-
mal to effectively stabilize cortical lipids of certain types. The accumulation
of acid-fast pigment is especially marked in the mouse?’: 4° and occurs to a
limited extent in the rat and hamster.”
40 B. H. Ershoff, Anat. Record 87, 297 (1943).
41 Z. Menschik, Quart. J. Exptl. Physiol. 34, 97 (1948).
42K. E. Mason, in Sex and Internal Secretions, Chapter 22. Williams & Wilkins
Co., Baltimore, 1939.
48K. E. Mason, Vitamins and Hormones 2, 107 (1944).
44, A.A. Koneff, Anat. Record 74, 383 (1939).
4° C. Biddulph and R. K. Meyer, Am. J. Physiol. 132, 259 (1941); Endocrinology 30,
551 (1942).
46 P. A. T. Tibirica, J. Dutra de Olivera, and A. Aguiar, Hospital O. (Rio de Janeiro)
26, 585 (1944).
47S. I. Stein, J. Nutrition 9, 611 (1935).
48M. M. O. Barrie, Lancet 233, 251 (1937).
49C, E. Tobin and J. P. Birnbaum, Arch. Pathol. 44, 269 (1947).
522 THE TOCOPHEROLS
4. MuscuLar SYSTEM
a. Skeletal Muscle
Nutritional muscular dystrophy constitutes the most universal manifes-
tation of vitamin E deficiency (Table II) and signifies a vitally important,
but as yet unknown, function of tocopherol in the metabolism of skeletal
muscle, Although the histopathologic changes vary considerably from spe-
cies to species, and even at different age periods in the same species, there
is still a fundamental pattern of change which is expressed as an acute
type of reaction in young animals and as a chronic type in adult animals.
The former type, of which the ‘“‘late lactation”? paralysis of rats is typical,
has received most attention because of ease of production experimentally
and occasional spontaneous occurrence in domestic animals. Since com-
bined placental and mammary transfer of tocopherol is often barely suffi-
cient to meet the daily needs of the young offspring of mammals, inade-
quacy of dietary tocopherol during the lactation and early post-lactation
periods, when there is unusually rapid growth and maturation of muscle
fibers, can be expected to have a particularly devastating effect. The dys-
trophic changes occuring in adult animals bear a close resemblance to the
lesions of human muscular dystrophy and to the hyaline necrosis (Zenk-
er’s degeneration) of muscle which follows prolonged febrile states, such
as typhoid fever in man. It should also be kept in mind that at any given
stage of vitamin E depletion there are certain differences in the extent to
which the dystrophic process affects different muscles in the same animal,
or even different regions of any one muscle.
(1) Late-Lactation Paralysis. When vitamin E reserves of lactating rats
are critically low, the suckling young frequently exhibit a generalized paral-
ysis, usually between the 18th and 25th days of life.°? This often appears
rather suddenly. There is clenching of the forepaws, weakness and drag-
ging of the extremities, inability to recover posture when placed on their
backs, diminution of respiration and body temperature, listlessness and |
death. Spontaneous recovery may occur when symptoms are mild. Vitamin
E therapy prevents the symptoms if given as late as the 15th day of lacta-
tion, but it has little or no beneficial effect once symptoms have appeared.
Spontaneous recovery with retention of residual paralysis has been ob-
served,” *! but it is of rare occurrence. Lesions of the brain and spinal
cord have been described®”: *?* but not confirmed. Most investigators favor a
purely myogenic origin of the dystrophic process. Immobilization of a
muscle by section of its nerve or its tendon prior to the 18th day markedly
50 H. M. Evans and G. O. Burr, J. Biol. Chem. 76, 273 (1928).
51H. M. Evans, J. Mt. Sinat Hosp. N. Y. 6, 233 (1940).
52M. D. Lipshutz, Rev. Neurol. 65, 221 (1936).
52a W. de Gutierrez-Mahoney, Southern Med. J. 34, 389 (1941).
VII. EFFECTS OF DEFICIENCY 523
Ser)
by inte ue
of “ay
Mt
Fia. 2. Skeletal muscle from Vitamin E-deficient rat 22 days old, showing “‘late-
lactation’’ paralysis. Note the strands of fragmented muscle fibers separated by areas
of edema and leucocytie infiltration. A few normal fibers can be seen at the periphery
of the muscle (left). * 100. (From Mason.°***)
Fic. 3. Skeletal muscle from same rat, at higher magnification, showing an area
where a group of fibers have undergone necrosis. Portions of two fibers, still possessing
cross striations, can be seen at left (S), and several basophilic, spindle-shaped, multi-
nucleate strands representing early stages in regeneration of new fibers at (R). Else-
where there is an intermingling of macrophages (M), fibroblasts (F), muscle nuclei
with investments of myoplasm (X), endothelial cells and faint outlines of capillaries.
A fibroblast in mitosis is seen at lower right. * 440. (From Mason.°*)
524 THE TOCOPHEROLS
protects against dystrophy.®? Death is usually ascribed to serious impair-
ment of the respiratory musculature, but other metabolic dysfunctions
may be involved. The following description is based upon the reports of
Olcott,** Telford e¢ al.,°° Pappenheimer,**: °* *” and Mason*”.
Grossly, the skeletal muscles are pale, ischemic, moist, and sometimes
grayish and gritty owing to calcium deposition. Microscopically, there is
widespread interstitial edema, leucocytie infiltration and segmental frag-
mentation of muscle fibers (Fig. 2). The affected segments show loss of cross
striations, amitotic proliferation of sarcolemma nuclei, hyaline necrosis of fi-
brillar substance, nuclear fragmentation, rupture of sarcolemma sheaths, and
removal of debris by invading macrophages. In the densely cellular areas
marking the point of breakdown of fiber segments, there appear many baso-
philic, fusiform, or band-like strands with centrally located nuclei; these
represent young, regenerating fibers presumably arising from plasmodial
masses and, perhaps, from intact muscle nuclei with investing sarcoplasm
released in the degenerative breakdown of the fibers (Fig. 3). Edema and leu-
cocytic infiltration diminish in a few days, and regenerating fibers become
increasingly numerous. The frequent presence of a layer of normal fibers
at the periphery of the muscle suggests that proximity to good vascular
supply may retard the dystrophic process.°*: °°
Weanling rats low in E but exhibiting no symptoms of paralysis, and
rats showing spontaneous recovery from paralysis, usually show consider-
able muscle damage histologically.**: °> Similar muscle changes, unassoci-
ated with external symptoms, have been observed in newborn rabbits,*
in prepubertal mice,” and in young pigs.*4* The muscular dystrophy ob-
served in ‘‘stiff-lamb” disease,®* °° °°" in ‘“‘white muscle disease” of young
calves,®’ and in a similar syndrome in the foal,®! all of which occur under
53 A.M. Pappenheimer, J. Mt. Sinai Hosp. N. Y. 7, 65 (1940); Physiol. Revs. 23, 37
(1948).
54H. S. Olcott, J. Nutrition 16, 221 (1938).
557. R. Telford, G. A. Emerson, and H. M. Evans, Proc. Soc. Exptl. Biol. Med. 41,
291 (1939); 45, 135 (1940).
56 A.M. Pappenheimer, Am. J. Pathol.15, 179 (1939).
57 A. M. Pappenheimer, On Certain Aspects of Vitamin E Deficiency, American
Lecture Series No. 17, Charles C Thomas, Springfield, Ill., 1948.
57a K. EK. Mason, Proc. 1st & 2nd Med Conf. (1951-52) Muscular Dystr. Ass’ns Amer.,
New York p. 94.
58 J. P. Willman, S. A. Asdell, and P. Olafson, Cornell Agric. Exp. Sta. Bull. 608, 3
(1934).
59 J. P. Willman, J. K. Loosli, S. A. Asdell, F. B. Morrison, and P. Olafson, J. Animal
Sci. 4, 128 (1945): Cornell Vet. 86, 200 (1946).
599. H. Draper, M. F. James, and B. C. Johnson, J. Nutrition 47, 583 (1952).
60 T,. R. Vawter and E. Records, J. Am. Vet. Med. Assoc. 110, 152 (1947).
61T. C. Jones and W. O. Reed, J. Am. Vet. Med. Assoc. 118, 170 (1948).
VIl. EFFECTS OF DEFICIENCY 525
farm conditions and seem undoubtedly due to inadequacy of vitamin E
in early life, closely resembles the early paralysis of laboratory mammals;
the same is true of muscle changes observed in puppies,®!* mink,” ducks,
goats,®* and chicks® (where definate lesions have been observed only
in the pectoral muscles), and guppy fish.*! Of particular interest are the
classic studies of Blaxter and coworkers,®**-* who describe in detail the
symptomatology, gross pathology, biochemical alterations, and_histo-
pathology of a muscular dystrophy produced in Ayrshire calves reared on
a low-E diet. The syndrome is indistinguishable from the long recognized
‘white muscle’ disease of calves. Dietary cod liver oil accentuated the
muscle lesions and also nullified otherwise protective doses of a-tocopherol,
as is also true of other herbivorous animals (guinea pig, rabbit, goat).
They describe swelling of muscle fibers, fragmentation, evidence of increased
breakdown or impaired synthesis of muscle globulins, and massive sarcolem-
mic proliferation which they think reflects an attempt to maintain con-
stant the volume of the dystrophic muscle; it is their opinion that in differ-
ent species the latter is accomplished by varying degrees of sarcolemmic
proliferation and fatty infiltration, thus accounting for much of the species
variation in general character of the muscle lesions observed.
For a more detailed review and discussion of vitamin E as it applies
to the nutrition of farm and laboratory animals, the reader is referred to
the recent review by Blaxter and Brown.‘
(2) Dystrophy in Rabbit and Guinea Pig. Herbivorous animals as a whole
appear to be particularly susceptible to withdrawal of vitamin E, and also
to the presence of unsaturated fats in the diet.®*: ® It was in the guinea
pig and rabbit that nutritional muscular dystrophy was first experimentally
produced by Goettsch and Pappenheimer,®® although its relationship to lack
6la H. D. Anderson, C. A. Elvehjem, and J. E. Gonce, Jr., Proc. Soc. Exptl. Biol.
Med. 42, 750 (1939).
62 K. E. Mason and G. R. Hartsough, J. Am. Vet. Med. Assoc. 119, 72 (1951).
53 A. M. Pappenheimer and M. Goettsch, J. Exptl. Med. 59, 35 (1934).
63a J,. LL. Madsen, C. M. McCay, and L. A. Maynard, Proc. Soc. Exptl. Biol. Med. 30,
1434 (1933); Cornell Univ. Agr. Expt. Sta. Mem. 178, 3 (1935).
6§ H. Dam, I. Prange, and E. Sondergaard, Acta Pathol. Microbiol. Scand. 31, 172
(1952).
64a kK L. Blaxter, P. S. Watts and W. A. Wood, Brit. J. Nutrition 6, 125 (1952).
6b KX. L. Blaxter and W. A. Wood, Brit. J. Nutrition 6, 144 (1952).
6ic A. M. Macdonald, K. L. Blaxter, P.S. Watts, and W. A. Wood, Brit. J. Nutrition
6, 164 (1952).
64d KL. Blaxter, W. A. Wood, and A. M. MacDonald, Brit. J. Nutrition 7, 34 (1953).
sie Kk. L. Blaxter, F. Brown, and A. M. MacDonald, Brit. J. Nutrition 7, 105 (1953).
64f Kk. L. Blaxter and F. Brown, Nutrition Abstr. & Rev. 22, 1 (1952).
65 L. L. Madsen, J. Nutrition 11, 471 (1936).
66 M. Goettsch and A. M. Pappenheimer, J. Exptl. Med. 54, 145 (1931).
526 THE TOCOPHEROLS
of vitamin E was not clearly established until a later date.*> ® Experimental
work with this group of animals has centered largely around the rabbit,
because of the ease with which symptoms can be produced and made to
disappear by vitamin E therapy and the sequence repeated as often as
desired.®? In respect to this striking responsiveness to tocopherol therapy,
which involves rapid biochemical and morphologic repair, the dystrophic
process in the rabbit differs from that in the rat. On the whole, the muscle
lesions described in rabbits and guinea pigs more closely resemble the acute
or explosive type exemplified by late-lactation paralysis than the more
chronic type described below. However, it seems probable that the basic
alterations occurring within the muscle fibers are fundamentally similar in
all instances. Recent studies®®* indicate that the initial disturbance, which
precedes loss of cross striations or other microscopic change, involves loss
of actomyosin or alterations of the submicroscopic pattern in which acto-
myosin is organized.
(3) Adult, or Chronic, Dystrophy. In young rats which spontaneously
recover from late-lactation paralysis there is, within a week or so, a dra-
matic diminution in the intensity and extent of the muscle lesions. At one
month of life only occasional fibers are dystrophic; the remaining muscu-
lature is normal and shows little or no connective tissue replacement of
degenerated fibers. With continued deficiency there is progressive involve-
ment of more and more fibers, usually in groups such that a patchy distri-
bution of lesions results, which may be extensive enough to cause locomotor
disabilities by the fifth or sixth month of life. In rats whose vitamin E re-
serves prevent the occurrence of early lesions, those of the later type may
not be evident microscopically until the fourth or fifth month, and gross
evidence of dystrophy not apparent until the eight to tenth month of life.
Gross and microscopic details of adult dystrophy in rats have been given
by Ringsted,’° Einarson and Ringsted,’ Evans et al.,” Knowlton et al.,”
67 C. G. Mackenzie and E. V. McCollum, Science 89, 370 (1939): J. Nutrition 19,
345 (1940). |
68 C. G. Mackenzie, J. B. Mackenzie, and E. V. McCollum, Science 94, 216 (1941);
J. Nutrition 21, 225 (1941).
69 C. G. Mackenzie, Proc. Soc. Exptl. Biol. Med. 49, 313 (1942).
69a M. Aloisi, A. Ascenzi, and E. Bonetti, J. Pathol. Bacteriol. 64, 321 (1952).
70 A. Ringsted, Biochem. J. 29, 788 (1935).
711. Hinarson and A. Ringsted, Effect of Chronic Vitamin EF Deficiency on the Nery-
ous System and the Skeletal Musculature in Adult Rats. Levin and Munksgaard,
Copenhagen, 1938.
2H, M. Evans, G. A. Emerson, and I. R. Telford, Proc. Soc. Exptl. Biol. Med 38,
625 (1938).
7G. C. Knowlton, H. M. Hines, and K. M. Brinkhous, Proc. Soc. Exptl. Biol. Med.
41, 453 (1939); 42, 804 (1939).
VII. EFFECTS OF DEFICIENCY O24
Mackenzie ef al.,°* Pappenheimer,®® Martin and Moore,! Monnier,” and
Mason and Emmel.' The first evidence of paresis is a waddling and slightly
incoordinated gait; later there is pronounced straddling of the hind legs,
hyperflexion of digits, flabbiness and weakness of the musculature involving
the adductor thigh muscles particularly, dragging of the hind legs, ina-
bility to walk or stand, distinct ataxia, pronounced muscle atrophy, de-
formity of feet, and general and localized sensory disturbances. The ani-
mals are hump-backed, unkempt in appearance; ulcerations of the skin
are common. The skeletal muscles are atrophic, somewhat dry or gritty,
and somewhat brownish in color. Once the adult paresis is well-established,
prolonged vitamin E therapy does no more than arrest the process and
improve the growth and well-being of the rat; that is, there results a per-
manent paralysis of about the degree present at the beginning of
therapy.*: 7 * This is quite different from the rabbit, where paresis can
be made to disappear and reappear repeatedly by careful regulation of the
vitamin E intake,°®® and the late-lactation paralysis in rats where E therapy
is of little or no avail once symptoms appear.
The histopathologic picture (Fig. 4) differs from that of late-lactation
paralysis chiefly in the relatively smaller number and widely scattered lo-
cation of affected muscle fibers. Edema, fragmentation, and leucocytie infil-
tration are less conspicuous features. Necrosis tends to involve longer fiber
segments, but the degenerative and regenerative processes, although proceed-
ing perhaps at a somewhat slower rate, are quite similar to those characteriz-
ing late-lactation paralysis. Sarcolemma nuclei appear to undergo amitotic
proliferation and become irregularly distributed in the fiber, followed by
the appearance of coarse interfibrillar granules and vacuoles, loss of cross
striations, breakdown of fibrillar substance, and removal of debris by in-
vading macrophages. The latter are often marked by accumulations of
acid-fast pigment which commonly appears in the degenerating fibers.
Usually the point of segmental degeneration is marked by an area of high
cellular concentration which, as in the case of early dystrophy, contains
considerable numbers of free muscle nuclei with their investment of sarco-
plasm and strongly basophilic multinucleate strands which represent early
phases of a regenerative reaction. The basophilic strands give rise to slender
muscle fibers possessing rows of centrally placed nuclei, arising presumably
through rapid amitotic divisions; such fibers are quite common, and de-
generating fibers absent, in muscles from animals given vitamin E therapy
73a C. G. Mackenzie, J. B. Mackenzie, and E. V. McCollum, Proc. Soc. Exptl. Biol.
Med. 44, 95 (1940).
74M. Monnier, Compt. rend. soc. phys. et hist. nat. (Geneve) 57, 252 (1940); Intern. Z.
Vitaminforsch. 11, 235 (1941).
528 THE TOCOPHEROLS
after periods of dystrophy. Changes of fundamentally the same type occur in
the mouse,” ** the hamster*”* and cotton rat,?! the dog,” and the monkey.7°
Certain differences observed in the histopathologic picture in various labo-
ratory animals may be attributable to species differences in the extent and
rate of the degenerative and regenerative processes in dystrophic muscle.
(4) Interrelationships with the Nervous System. Except for conflicting opin-
5 : es i 4 Me
Fia. 4. Skeletal muscle of hamster after chronic Vitamin E deficiency (200 days),
showing areas of necrosis of muscle fibers (N) and other fibers in various stages of
regeneration. The latter vary from elongated, basophilic, multinucleate strands (R)
to fibers which are normal, except for an irregular distribution of nuclei in chain-like
rows located more or less centrally in the fiber (X.). In some areas, degenerated fibers
have been replaced by fatty tissue (F). X 125. (From Mason.°*")
ions concerning the rat, the dystrophic muscle lesions of vitamin E-de-
ficient animals are considered to be purely myogenic. The early onset of
the lesions, their biochemical nature and response to E-therapy,” frequent
occurrence of similar alterations in cardiac muscle are in accord with this
assumption. It should be mentioned, however, that in most instances
the nervous system has not been carefully studied. On the other hand,
much attention has been given to the nervous system of the rat. In their
pioneer studies on the paralysis of adult rats reared for many months
on low-E diets, Einarson and Ringsted’! described lesions in the dorsal
75K. EK. Mason and I. R. Telford, Arch. Pathol. 48, 363 (1947).
76 O. B. Houchin and H. A. Mattill, J. Biol. Chem. 146, 301, 309, 313 (1942).
i lll
VII. EFFECTS OF DEFICIENCY 029
columns, dorsal nerve roots, and ventral horn cells of the spinal cord and
stated (p. 134 in ref. 71) that ‘*... the muscular changes in many of the
animals, especially in the early stage of the affection, show several points
of decided resemblance to the picture of muscular dystrophy, ie., a myog-
enous muscular atrophy. Gradually, as the disease in these animals is
progressing, however, the muscular changes increasingly assume the typi-
cal neurogenous appearance so that finally the muscular features alone
would be evidence enough of the presence of an amyotrophic lateral sclero-
sis. ... On the other hand, we have not been able to exclude the existence
of a mixed picture, 1.e., the possibility that the muscular changes may in
part be dystrophic and partly be due to the spinal changes.”’ Similar con-
clusions were reached by Monnier. Although, in a study of comparable
material, some investigators” have found no lesions of the nervous system,
others®: *° have observed demyelinization and gliosis in the posterior col-
umns and dorsal nerve roots, but not the alterations in the ventral horn
cells and pyramidal tracts described by Einarson and Ringsted. In a recent
and detailed ‘‘criticizing review” of the literature on this subject, Einarson®®
admits that in their earlier report’ there was overemphasis of ‘‘the myo-
pathogenetic significance of the spinal cell changes,” and he expresses
the opinion that “vitamin E on the whole acts simultaneously on the mus-
culature and the nervous system, due to some physico-chemical effects it
exerts in metabolism.” Until there is better agreement regarding lesions
of ventral horn cells (see p. 533), it is impossible to say with certainty that
an atrophy of spinal origin is superimposed upon a true myopathy.
Peripheral nerves and motor end plates are reported as normal in nutri-
tional myodegeneration of ducklings,*! guinea pigs, * °* and young®® and
old?® rats; but Telford* finds that loss of end plates, secondary to degenera-
tion of muscle fibers, occurs in young rats. Einarson and Ringsted™ ob-
served some atrophic muscle spindles in dystrophic muscle of adult rats
which they imply may be related to alterations noted by them in ventral
root fibers.
77 A. Wolf and A. M. Pappenheimer, Arch. Newrol. Psychiat. 48, 538 (1942).
#% C, N. Luttrell and K. E. Mason, Ann. N. Y. Acad. Sci. 52, 113 (1949).
79. N. Malamud, M. M. Nelson, and H. M. Evans, Ann. N. Y. Acad. Sci. 52, 135 (1949).
80 |,. Einarson, Acta Psychiat. et Neurol. Scand. Suppl. 78,9 (1952).
81 A. M. Pappenheimer, M. Goettsch, and E. Jungherr, Storrs Agr. Expt. Sta. Bull.
229 (1939).
82 W. M. Rogers, A. M. Pappenheimer, and M. Goettsch, J. Exptl. Med. 54, 167
(1931).
82a H. Chor and R. E. Dolkart, Arch. Pathol. 27, 497 (1939).
83 T. R. Telford, Anat. Record 81, 171 (1941).
530 THE TOCOPHEROLS
An important recent development is the demonstration that a-tocoph-
erylhydroquinone, which is a potent antidystrophy compound, has no
antisterility activity®*: > and undergoes little or no conversion to a-tocoph-
erol in vivo. Another oxidation product of tocopherol, a-tocopheryl-
quinone, is also antidystrophic but appears to have no antisterility activity.
Thus a-tocopherol, the antistenility vitamin, may represent a provitamin
for other compounds functioning as antidystrophy vitamins. An excellent
discussion of these and other aspects of experimental muscular dystrophy
has been presented by Mackenzie.**¢
b. Cardiac Muscle
Hyaline necrosis and replacement fibrosis of cardiac muscle, closely re-
sembling the changes occurring in skeletal muscle, have been observed in
the vitamin E-deficient rabbit,®*: > 8° guinea pig, 4: 8° young calf, >> ©
cow,®*”: & sheep,*® 8° goat,®* rat,* 9°? mouse,” hamster,”! and cotton rates
The lesions are rapid in onset in herbivorous animals (rabbit, guinea pig,
sheep, and cattle), not associated with acid-fast pigment, and frequently
the cause of sudden death through myocardial failure. This is in strik-
ing contrast to other laboratory animals (rat, hamster, and cotton rat)
in which extensive focal necrosis and scarring of the myocardium, with
accumulation of pigment in muscle fibers and macrophages, may exist
for many months without serious effects. Electrocardiographic changes of
varying degrees, indicative of myocardial damage without involvement of
the conducting system (Purkinje fibers), have been found in the rat, guinea
pig, rabbit, cattle, and monkey. The most dramatic picture is seen in sudden
collapse of cattle in cardiac failure, usually with little or no symptomatology
prior to exitus. The phenomenon in the rabbit has received the
most thorough study, largely through the careful studies of Gatz and
Houchin,™ who provide an excellent description of the electrocardiographic
and histopathologic changes. They believe that a phase of increased muscle
metabolism, as reflected in increased O. consumption similar to that in
83a A. Issidorides and H. A. Mattill, J. Biol. Chem. 188, 313 (1951).
8b J. B. Mackenzie and C. G. Mackenzie, Proc. Soc. Exptl. Biol. Med. 84, 388 (1953).
se ©. G. Mackenzie, In Symposium on Nutrition, Johns Hopkins Press, Baltimore
(1953).
84 A. J. Gatz and O. B. Houchin, Anat. Record 110, 249 (1951).
85 J, H. Bragdon and H. D. Levine, Am. J. Pathol. 25, 265 (1949).
86S. Americano Freire and B. Figueiredo Magalhaes, Rev. brasil. biol. 3, 91 (1948).
87'T. W. Gullickson and C. E. Calverley, Science 104, 312 (1946).
88 T’. W. Gullickson, Ann. N. Y. Acad. Sci. 52, 256 (1949).
89 R. Culik, F. A. Bacigalupo, F. Thorp, Jr., R. W. Luecke, and R. H. Nelson, J.
Animal Sci. 10, 1006 (1951).
90S. Americano Freire, Brasil-méd. 55, 308 (1941).
1G. J. Martin and F. B. Faust, Haptl. Med. and Surg. 5, 405 (1947).
2 W. Ruppel, Arch. exptl. Pathol. Pharmakol. 206, 584 (1949).
VII. EFFECTS OF DEFICIENCY Sea!
skeletal muscle, precedes the onset of morphologic changes. The latter are
characterized by interstitial edema, infiltration of neutrophiles and mononu-
clear cells, hyaline necrosis of muscle fibers in association with coagulative
necrosis and vacuolation of the sarcoplasm, gradual loss of myofibrillae, and
appearance of wide constriction bands. Cardiac capillaries and larger vessels
are prominently distended with blood; and sometimes small hemorrhages
occur. Usually the necrosis is patchy in its distribution. Neither Purkinje
fibers, neurons, nor fibers of the autonomic plexi show any changes. When
severe, the lesions are grossly visible as circumscribed grayish areas. Lesions
are most extensive in the peripheral myocardium of the ventricles but occur
also in the papillary muscles, the interventricular septum, and the atrial
musculature.
c. Smooth Muscle
Rats deprived of vitamin E for several months exhibit a yellowish dis-
coloration of the uteri which gradually increases to a chestnut brown color
as deficiency progresses, due to the accumulation of brownish, fluorescent,
acid-fast pigment granules in the smooth muscle cells and macrophages of
the myometrium.!:?) 4: > > A similar but somewhat less pronounced
change occurs in the smooth musculature of the fallopian tube, cervix,
vagina, seminal vesicle, prostate, vas deferens, ureter, trabeculae and cap-
sule of the spleen, small intestine, bronchi, and uterine and plumonary
peiis:+*)*» 92-94
The uterine changes constitute a prototype for those observed in smooth
muscle elsewhere. Pigment granules appearing first at each pole of the nu-
cleus gradually push the myofibrillae peripherally, eventually distending
and even distorting the muscle cells such that they are difficult to distin-
guish from intervening pigment-laden macrophages. It is more presumed
than established that much of the pigment in macrophages is derived from
muscle cells undergoing necrosis; some of it may be released to macrophages
without breakdown of the cells. Macrophages containing large globules of
pigment, produced perhaps by alteration and concentration of smaller pig-
ment granules, become numerous and conspicuous in the intermuscular
connective tissue and in the outer zone of the endometrium. Endometrial
fibrosis seems not to be accentuated.°®
Uterine pigmentation does not occur in rats ovariectomized before pu-
berty, but does appear if such rats are given estrogen treatment;*? once
93 W. Hessler, Intern. Z. Vitaminforsch. 11, 9 (1941).
%V. Demole, Intern. Z. Vitaminforsch. 8, 338 (1939); Schweiz. med. Wochschr. 71,
1251 (1941).
95 J. C. Radice and M. L. Herraiz, Ann. N. Y. Acad. Sct. 52, 126 (1949).
96 J. Lopes de Faria, Ann. N. Y. Acad, Sct. 52, 121 (1949) ; Hospital O (Rio de Janeiro)
29, 583 (1946).
97 W. B. Atkinson, H. Kaunitz, andC. A. Slanetz, Ann. N. Y. Acad. Sci, 52, 68 (1949).
ae THE TOCOPHEROLS
established, however, it is but little influenced by prolonged tocopherol
therapy, pregnancy, castration, or hormone treatment. Although uterine
pigmentation does not appreciable modify the response of the uterus to
drugs which act either upon the autonomic nerve supply or as direct muscle
stimulants,” or interfere with the course of pregnancy if adequate vitamin
E is provided after mating, it may be to some degree responsible for the
lowered incidence of fertile matings, due to impaired implantation, and for
the increased requirements of successful gestation in long-term E-deficient
rats.29: 34-36, 98
There is a remarkable species variation with respect to the response of
smooth muscle to vitamin E depletion. In the monkey the pigment changes
are especially marked in vascular smooth muscle but occur also in the small
intestine, gall bladder, urinary bladder, and bronchi.’? Although not ex-
tensively studied in other species, the lesions appear to be limited largely
to the urinary bladder, blood vessels, and small intestine in hamsters, and
to the small intestine in the dog, but are absent in the cotton rat.?! In the
mouse only brownish discoloration of the uterus has been reported.?’ Pig-
mentation of smooth muscle has not been observed in association with other
lesions of vitamin E deficiency in herbivorous animals or in birds. These
findings suggest that from a metabolic standpoint, at least, smooth muscle
may differ widely from species to species, and even in different organs of the
same species. In avian species, turkey poults show a patch hyaline necrosis
of the smooth musculature of the gizzard, with inflammatory reactions and
replacement fibrosis,°? °° as the only recognized manifestation of vitamin
E deficiency; similar changes of a milder type have been reported in the
ehick.!°°
5. NERVouUS SYSTEM
Studies on the effects of vitamin E deficiency upon the nervous system,
based chiefly upon the rat, have given rather controversial results (see re-
view by Einarson*®). In rats showing late-lactation paralysis, Lipshutz*?
reported extensive cerebrospinal lesions involving especially the vestibular
and tectospinal pathways and dorsal sensory columns, and de Guiterrez-Ma-
honey®”* has described rather widespread cellular hyperchromasia and loss
of Nissl pattern; other investigators,** °® however, report no neuropatho-
logic changes in such animals.
Demyelinization and gliosis in dorsal nerve roots and dorsal sensory col-
umns (cuneatus and gracilis) of the spinal cord of adult rats subjected to
% A. Fuhr, R. E. Johnson, H. Kaunitz, and C. A. Slanetz, Ann. N. Y. Acad. Sci. 52,
83 (1949).
% ff. Jungherr and A. M. Pappenheimer, Proc. Soc. Exptl. Biol. Med. 87, 520 (1937).
100 KH. Jungherr, Ann. N. Y. Acad. Sci. 52, 104 (1949).
VII. EFFECTS OF DEFICIENCY 533
prolonged vitamin E deficiency, as first reported by Einarson and Ringsted”!
and Monnier™ and considered related to tabes-like symptoms in such ani-
mals, although questioned by some investigators,” have been well substan-
tiated by others.”: 7°
Einarson and Ringsted’ also described extensive degeneration of ventral
horn cells and, in a few cases, demyelinization of the pyramidal tracts,
leading to a picture suggestive of a combination of tabes dorsalis combined
with spinal muscular atrophy or amyotrophic lateral sclerosis. Eimarson*?
now considers the pyramidal tract lesions a misinterpretation, due to faulty
comparison with control material. The ventral horn changes described also
by Monnier™ but not observed by other investigators’7-7° who have searched
for them, are characterized by alterations in Nissl substance leading to
lipodystrophy and irreparable atrophy of motor horn cells, those located
more centrally and at the venous end of the capillary bed where hypoxia
is greatest being more affected than the more peripheral cells. Similar changes
in dystrophic guinea pigs have been reported.!0°* Einarson®?: !°°> thinks that
when vitamin E is inadequate adenylic acid, necessary for formation of
high-energy phosphate bonds, is not properly liberated from cytoplasmic
nucleotides and that accumulation and decomposition of the latter lead to
the degenerative cell changes observed. Failure to use, or to properly apply,
the gallocyanin-chromalum stain, having a selective affinity for nucleic
acids, may explain the failures of others to confirm these findings.*® Pos-
sibly the widespread cellular hyperchromasia described by de Gutierrez-
Mahoney** in ventral horn cells and elsewhere in the nervous system, and
the accumulation of acid-fast pigment observed by Pappenheimer and
Victor and Einarson!’™ in motor cells of the cord and medulla of old
vitamin E-deficient rats, represent changes of a similar type but revealed
by other staining methods.
Mackenzie et al.”** report marked tremors in adult paralyzed rats and,
following a shrill note, an outburst of activity terminating in collapse with-
out convulsions or loss of consciousness. Hamsters on low-E diets have been
reported!”!» to show a sudden onset of incoordination, hyperexcitability, and
violent reactions to external stimuli such as touch or noise, culminating in
collapse, unconsciousness, stupor, and death; tocopherol orally effected
spectacular recovery in some instances. Jn the writer’s experience, and that
of others who have studied E-deficiency in the hamster, this phenomenon
has never been observed.
100a MT. Ekblad and G. Wohlfart, Z. ges. Neurol. Psychiat. 168, 144 (1940).
100b [,. Einarson, Acta. Orthopaed. Scand. 19, 55 (1949).
101 A. M. Pappenheimer and J. Victor, Am. J. Pathol. 22, 395 (1946).
wis J, Kinarson, J. Neurol. Neurosurg. Psychiat. 16, 98 (1953).
101b J. W. Hamilton and A. G. Hogan, J. Nutrition 27, 213 (1944).
534 THE TOCOPHEROLS
Lesions of the cerebellum and cerebrum resulting from vitamin E defi-
ciency in birds appear to be secondary to vascular changes and, for that
reason, are discussed in the following section.
6. VASCULAR SYSTEM
a. Chicks
Most of the lesions observed in vitamin E-deficient chicks appear to be
the result of alterations in the peripheral vascular system, especially the
rapillary bed. Like the skeletal muscle lesions of the duck, the chick, and
the pheasant’: ®. 6,100 and gizzard necrosis of the turkey and the
chick,*'> °°) °° referred to previously, these manifestations are rarely seen
in chicks after the second month of life. Since they occur spontaneously
in the field with variable frequency,*!: 1°: ! they are of economic as well
as academic importance.
(1) Embryonic Mortality. According to Adamstone,!” inadequate vitamin
E in the chick egg results in embryonic death at about the fourth day of
incubation, due to disintegration of blood vessels of the blastoderm, hemor-
rhage into the coelom and exocoelom, and cellular proliferations in the blas-
toderm which interrupt the vitelline circulation. If this critical period is
passed, there may be spontaneous rupture of vascular channels at various
sites within the embryo, usually associated with clusters of pycnotic his-
tiocyte-like cells at the points of extravasation. Whether these cells are
responsible for the vascular rupture or represent a protective reaction at
the site of injury has not been satisfactorily established.
(2) Haudative Diathesis. Chicks reared from hatching on low-E diets
usually exhibit a state of exudative diathesis or of nutritional encephalo-
malacia, or sometimes both, during the first two months of life. The two
manifestations appear to be secondary to dysfunctions of the capillary
bed, sometimes regress spontaneously, and are influenced considerably by
variations in fats and other dietary components.
Exudative diathesis, as described by Dam and Glavind,!” is characterized
grossly by the appearance of large patches of subcutaneous edema on the
breast and abdomen, and less frequently on the neck, legs, or wings. These
represent local subcutaneous and interfascial accumulations of a plasm-like
fluid frequently tinged greenish by decomposed hemoglobin. The affected
tissues show edema, hyperemia, and increased permeability of the capil-
laries as indicated by increased absorption of intravenously injected Trypan
blue. The subcutaneous tissue at the site of old lesions retains a buff color
102 F. B. Adamstone, J. Morphol. 52, 47 (1981); Arch. Pathol. 31, 622 (1941).
108 TT, Dam and J. Glavind, Nature 148, 810 (1939); Skand. Arch. Physiol. 82, 299
(1939).
a
VII. EFFECTS OF DEFICIENCY 535
for some time.!™ Bird and Culton!®® describe more severe manifestations
of a similar disorder in which edema of brain and lungs, marked distention
of heart and pericardium, generalized ascites, and coronary and intestinal
hyperemia usually terminate in stupor and death of the chicks. No histo-
pathologic study of the localized lesions of exudative diathesis has been
reported.
(3) Nutritional Encephalomalacia. This disorder of the nervous system,
as described by Pappenheimer et al.®°7: 5!) 1°. 1% and by Adamstone,! is
characterized by motor incoordination, ataxia, head retraction, coarse
tremors, opisthotonos, prostration with legs spastic and claws strongly
flexed, somnolence, stupor, and death. At necrospy there is gross swelling,
flattening, irregular distortion, and greenish-brown discoloration of the
cerebral convolutions. Similar lesions frequently occur in the cerebrum,
midbrain, and medulla. They vary from small focal areas to large confluent
patches of ischemic necrosis. The cerebellar lesions, microscopically, show
edema and disruption of cellular and fibrillar elements of the gray matter,
degenerative necrosis of Purkinje cells and the small cells of the granular
layer, capillary thrombi which are especially abundant where the blood
vessels make a pronounced right-angle turn at the Purkinje cell level, and
small hemorrhages in the cortical white matter. Although capillary throm-
bosis seems to be the primary cause of the ischemic necrosis, it is possible
that some of the symptomatology may be secondary to prolonged vasocon-
striction or vasomotor paralysis of larger blood vessels.! During spontaneous
or induced recovery there is active ingrowth of new blood vessels, gliosis
and reparative reorganization in the softened tissues, and appearance of
phagocytes with brownish pigment. The symptoms and lesions described
are identical to those of ‘‘crazy chick disease,” long recognized by poultry-
men in brooder-stage chicks*!: 1°
Jungherr,!°° who has given particular attention to the seasonal occurrence
and histopathology of this spontaneous disease, which is often a more
chronic disorder than that produced experimentally, reports that in associa-
tion with, and sometimes independent of, this ischemic necrosis there occurs
extensive fibrosis of the cerebellum. Furthermore, the ischemic and fibrotic
lesions are often associated with large areas of increased vascularity in the
medulla, the midbrain, and the thalamus, accentuated by adventitial cell
proliferation and intervascular gliosis, which he regards as a ‘‘new morpho-
logic expression of subacute E-avitaminosis in chickens.”
10 H. Dam, J. Nutrition 27, 193 (1944).
105 H.R. Bird, and T. G. Culton, Proc. Soc. Exptl. Biol. Med. 44, 543 (1940).
106 A. M. Pappenheimer and M. Goettsch, J. Exptl. Med. 58, 11 (1931).
107 A. Wolf and A. M. Pappenheimer, J. Exptl. Med. 54, 399 (19381).
108 F. B. Adamstone, Arch. Pathol. 31, 603 (1941); 43, 301 (1947).
536 THE TOCOPHEROLS
(4) Influence of Variations in Diet. According to Dam,!% exudative dia-
thesis is rare and encephalomalacia never occurs in chicks fed purified
E-deficient diets containing no added fats; furthermore, dietary unsatur-
rated fats accentuate both manifestations, and by varying the type and
proportion of fats and other components of the diet it is possible to produce
these two manifestations separately or concomitantly. Essentially the same
diet may give a very different incidence of the two symptoms in the hands
of different investigators.!°° The presence of substances such as ascorbic
acid,!°? xanthophyll," or redox substances of various types, by virtue
of their capacity to replace tocopherol as an antioxidant in the gastrointesti-
nal tract or elsewhere, may confer considerable protection against the symp-
toms. Thus it appears that in addition to lack of vitamin E there is a delicate
balance of dietary factors which operate to determine the onset and severity
of the vascular dysfunctions and the anatomic site at which they occur.
In this connection reference may be made to the lymphoblastoma-like
growths observed by Adamstone!!? in the liver, the intestine, and other
visceral organs of the chick, sometimes leading to hemorrhage by invasion
of blood vessels, when cod liver oil or sardine oil, but not halibut liver oil,
was added to diets previously treated with ferric chloride to destroy traces
of vitamin E. On the other hand, additions of halibut liver oil led to an
anemia-like condition presumably resulting from extensive erythrophag-
ocytosis in the liver."% These findings have not been confirmed or satis-
factorily explained.
b. Mammals
(1) Hemorrhage. The vascular stasis and hemorrhage occurring in the
rat fetus, as described in an earlier section (p. 520), bears a certain resem-
blance to the vascular dysfunctions in chicks. So also do the interfascial,
subcutaneous, and thymic hemorrhages observed during the second month
of life in rats reared from birth on low-E diets high in total unsaturated
fatty acids of cod liver oil,!* and also the hemorrhages into cranial and
visceral cavities, lungs, and intestine observed by Elvehjem et al. in
puppies born of mothers fed mineralized milk diets low in vitamin E. As
in the chick studies just discussed, there are again the three factors to be
109 T,. Zacharias, P. Goldhaber, and V. E. Kinsey, J. Nutrition 42, 359 (1950).
110 P. Goldhaber, L. Zacharias, and V. E. Kinsey, J. Nutrition 42, 453 (1950).
11 Af. Dam, I. Kruse, I. Prange, and E. Sondergaard, Acta Physiol. Scand. 22, 299
(1951).
1122, F. B. Adamstone, Am. J. Cancer 28, 540 (1986); Arch. Pathol. 31, 717 (1941).
13 F. B. Adamstone, Arch. Pathol. 31, 613 (1941).
14 R. E. Rumery, Thesis, Histological and biochemical reactions of the immature
rat to diets low in tocopherols (vitamin E) and high in unsaturated fatty acids.
University of Rochester, 1952.
15 C, A. Elvehjem, J. E. Gonce, Jr., and G. W. Newell, J. Pediat. 24, 486 (1944).
—————
VII. EFFECTS OF DEFICIENCY ban
considered—inadequate vitamin E, early age of the animal, and some type
of metabolic stress or local insult of tissues. Whether the latter relates to
fat peroxides or to other metabolites as well is not known. There is also
the question of whether in the young organism the vascular bed is peculiarly
susceptible to injury by such metabolic factors, or whether the metabolic
factors differ qualitatively or quantitatively as biochemical processes
mature.
Of particular interest in this connection are the studies of Holman!!®
demonstrating that a necrotizing arteritis in dogs, produced by induction
of renal insufficiency after prior feeding of a high fat diet, can be retarded
or prevented by tocopherol. Here vitamin E seems to protect against chemi-
cal injury resulting from the presence of abnormal lipids deposited in the
vascular wall. Difference of opinion exists as to whether a-tocopherol does!
or does not!® protect dogs against stilbestrol-induced thrombocytopenic
purpura. In rats tocopherols prevent increased capillary fragility due to
a-irradiation.!!°
(2) Hemolysis. Gyérgy and Rose have shown that hemoglobinuria, intra-
vascular hemolysis, and death occurring in alloxan-treated rats are readily
prevented by dietary tocopherols, although the diabetic phenomena are
not affected;”° and that dialuric acid, a decomposition product of alloxan,
and several related compounds produce hemolysis of erythrocytes.!!: !
a-Tocopherol, through its function as an antioxidant, protects the red cell
against this chemical injury, perhaps by counteracting a free radical or
peroxide formed as an intermediate of the oxidation-reduction system of
dialuric acid and alloxan. On the basis of these reactions they have de-
veloped an hemolysis test which can be applied either in vivo or in vitro
as a measure of the biologic activity of tocopherols and of vitamin E de-
pletion in the rat.!? ’** Modifications of this test have demonstrated in-
creased tendency to hemolysis in red cells of vitamin E-deficient monkeys
(unpublished observations in the writer’s laboratory) and in those of pre-
mature infants,!* full-term infants, and newborn rats.'! Although hemolysis
16 R. L. Holman, Proc. Soc. Exptl. Biol. Med. 66, 307 (1947); Southern Med. J. 42,
108 (1949).
17 F. Skelton, E. Shute, H. G. Skinner, and R. A. Waud, Science 108, 762 (1946).
18s A. J. Richtsmeier, M. Spooner, and O. O. Meyer, Proc. Soc. Exptl. Biol. Med. 65,
298 (1947).
119 §. R. Ames, J. G. Baxter, and J. Q. Griffith, Jr., Intern. Z. Vitaminforsch. 22, 401
(1951).
120 P, Gyérgy and C. 8. Rose, Science 108, 716 (1948).
121 ©, §. Rose and P. Gyoérgy, J. Nutrition 39, 529 (1949); Blood 5, 1062 (1950).
122 P. Gyérgy and C. S. Rose, Ann. N. Y. Acad. Sci. 52, 231 (1949).
1228 C. S. Rose and P. Gyérgy, Am. J. Physiol. 168, 414 (1952).
123 AH. H. Gordon, and J. P. de Metry Proc. Soc. Exptl. Biol. Med. 79, 446 (1952).
124 P. Gyorgy, G. M. Cogan, and C.S. Rose, Am. J. Diseases Children 82, 237 (1951).
538 THE TOCOPHEROLS
can be produced by many other compounds and can be prevented by other
antioxidants, a-tocopherol is unquestionably a most effective natural
intracellular antioxidant.
It is generally accepted that vitamin E deficiency does not influence the
peripheral blood picture or hematopoietic functions. However, Dinning”®
has recently reported a pronounced leucocytosis in low-E rabbits, respond-
ing to tocopherol therapy, which he concedes may reflect leucocytic infiltra-
tion of dystrophic muscle.
7. OTHER MANIFESTATIONS
a. Adipose Tissue
Prolonged vitamin E deficiency in rats results in considerable diminution
in body fat, accounting in part for the plateau in body weight occurring
during early adulthood and for the rather emaciated appearance of rats
showing advanced stages of paresis. Menschik et al.2°: 1° have studied this
phenomenon in mice, reporting that adipose tissue develops normally during
early life but that after about 9 months of deficiency little or no body fat
is evident—except for the brown glandular fat of the interscapular region
which is often increased in amount and of deeper brown color than in con-
trol mice. They were led to conclude that vitamin E deficiency interferes
with the ability to deposit dietary fats as neutral fats, related perhaps to
the fact that both ingested and stored fat undergo abnormal or abortive
metabolic changes.
A strikingly different reaction occurs in the adipose tissues of the rat
when low-E diets contain high levels (about 20%) of cod liver oil!® or
highly unsaturated fractions of this oil,!?’7 or methyl esters of linseed, corn,
or soybean oils.!! Under such circumstances there is a brownish discolora-
tion of the subcutaneous and intraperitoneal adipose tissue. Peroxides are
usually demonstrable either by chemical! or histochemical! methods.
Microscopically,!*: ” the adipose tissue first appears studded with yellow-
ish-brown islets. The latter represent clusters of fat cells in various stages
of development in which small fat globules and the peripheral portions of
larger fat vacuoles are composed of acid-fast pigment; pigment-laden mac-
rophages are also present in increased numbers. At later stages these cells,
and possibly other connective elements, participate in complex foreign-
125 J. §. Dinning, Proc. Soc. Exptl. Biol. Med. 79, 231 (1952).
26 Z. Menschik, Edinburgh Med. J. 51, 486 (1944).
27 H. Granados, K. E. Mason, and H. Dam, Acta. Pathol. Microbiol. Scand. 24, 86
(1947).
8 H. Granados and H. Dam, Acta Pathol. Microbiol. Scand. 27, 591 (1950).
29 J. Glavind, H. Granados, 8S. Hartmann, and H. Dam, Experientia 5, 84 (1949).
130 H. Dam, Ann. N. Y. Acad. Sci. 52, 194 (1949).
VII. EFFECTS OF DEFICIENCY 539
body type of reactions leading to the formation of large, pigment-laden
giant cells which eventually dominate the picture. Tocopherol therapy
arrests the process but in most locations brings about no more than a lim-
ited reduction in pigment and very little change in other cellular reactions.
Similar alterations of adipose tissue have been observed in the mouse,?8
hamster,”! pig,!!: ? and mink;®: '* it is of interest that the naturally oc-
curring ‘‘yellow fat disease” or ‘‘steatitis’’ of mink, frequently causing seri-
ous losses of kits prior to the pelting season, fits into this picture. In all
instances a dietary intake of highly unsaturated fats and inadequacy of
vitamin E have been involved. The most satisfactory explanation for the
histopathologic reactions described above is that unsaturated dietary fats
incorporated into adipose tissue cells which lack sufficient tocopherol as
an antioxidant to stabilize them, or to counteract peroxides which accumu-
late in the tissues, undergo polymerization or combine with cell proteins,
or both, to form acid-fast pigment which provokes giant cell reactions and
perhaps a certain amount of cell necrosis.
b. Liver
Certain histopathologic changes in the liver have been observed in a
few species, but, in most instances, lack of vitamin E is merely one of several
factors involved. In chicks there has been reported a phenomenon of ‘‘ery-
thropagocytosis,’’ in which the liver shows brownish discoloration and,
microscopically, enlargement of hepatic cells, widening of sinusoids, and
much hemosiderin in hepatic and Kupffer cells; there is also hyperplasia of
myeloid tissue." Its occurrence only when ferric chloride-treated diets are
supplemented with halibut liver oil raises questions as to its specificity.
After prolonged vitamin E depletion in mice (14 months or more)
Menschik e¢ al.**: * have noted progressive accumulation of coarse “‘lipo-
proteic”’ globules, swelling, and nuclear pycnosis in hepatic cells; they also
describe sinusoidal dilation, extravasation of erythrocytes or obvious hemor-
rhage, hemosiderin in Kupffer cells, and disorganization of the parenchyma.
Histochemically, the lipoproteic globules are composed of a mixture of un-
saturated fatty acids, phospholipids, and cholesterol, probably combined
with protein, resembling the ‘‘ceroid’’ of nutritional cirrhosis referred to
before and suggesting abnormal metabolic changes in liver fat.
In nutritional cirrhosis (diffuse hepatic fibrosis) of rats fed diets low in
lipotropic factors (methionene, choline) there is an extensive fatty infiltra-
tion of the liver followed by a progressive deposition of fibrous tissue in the
131 K. L. Robinson and W. E. Coey, Nature 168, 997 (1951).
122 J. R. Gorham, N. Boe, and G. A. Baker, Cornell Vet. 41, 332 (1951).
183 J. R. Gorham, G. A. Baker, and N. Boe, Vet. Med. 46, 100 (1951).
184 Z. Menschik and T. J. Szezesniak, Anat. Record 108, 349 (1949).
540 THE TOCOPHEROLS
form of irregular trabeculae with extensive disorganization of the paren-
chyma. If such diets are low in vitamin E, as was usually the case in the
early studies, the livers become grossly brownish-yellow in color and on
histologic examination show accumulations of ceroid pigment in cells of
the parenchyma and fibrotic areas, especially in the latter.® > 6 As
mentioned previously (p. 516) this ceroid is generally indistinguishable
from the acid-fast pigment of vitamin E deficiency. Tocopherols have no
influence on the fatty or fibrotic changes but retard or prevent ceroid for-
mation.’ The cellular rections which occur in areas of fatty infiltration of
hepatic cells are essentially the same as those described above for adipose
tissue and indicate the need for tocopherols to prevent undesired oxidative
changes in the infiltrating lipids. Dietary unsaturated fats such as cod liver
oil accentuate ceroid formation.!*” Except for dissemination of much of this
pigment to the lungs, the histopathologic changes in the musculature and
other tissues are typical of those seen in rats fed low-E diets containing
adequate protein.
A different type of liver injury, known as acute or massive hemorrhagic
necrosis, occurs in rats reared on diets low in vitamin E and deficient in
sulfur-containing amino acids (alkali-treated casein or low casein diets)!*8-™!
or containing as their protein component certain yeasts (high yeast _
diets)?" which lack an unidentified protective substance (factor 3 of
Schwarz)!* present in most American yeasts. There is a vast and confusing
literature on this subject, which now warrants the statement of Schwarz!
that ‘‘a simultaneous lack of 3 factors—cystine, vit. EK and factor 3—1s a
prerequisite for the development of dietary necrotic liver degeneration,
and each one of them alone can protect.” It is not yet clear how each of
these substances acts in protecting the rat against the rather sudden onset
of massive necrosis and hemorrhage in the liver!®®: '!-!* or massive lung
hemorrhages,'° which cause death. In addition to widespread centrolob-
ular necrosis of the liver, there have been noted dystrophic changes in
skeletal muscles,'’’: ' ulcers of the forestomach,'*: '* kidney lesions, and
hemorrhage in lymph nodes and intestine.
135 P, Gydérgy, Am. J. Clin. Pathol. 14, 67 (1944).
136 HH. Popper, P. Gyérgy, and H. Goldblatt, Arch. Pathol. 37, 161 (1944).
137 M. Wachstein, Proc. Soc. Exptl. Biol. Med. 59, 73 (1945).
1388 P, Gyérgy and H. Goldblatt, J. Hxptl. Med. 89, 245 (1949).
139 K. Schwarz, Z. physiol. Chem. 281, 106 (1944); 283, 106 (1948); Ann. N. Y. Acad.
Sci. 52, 225 (1949).
140}. L. Hove, D. H. Copeland, and W. D. Salmon, J. Nutrition 39, 397 (1949).
141 M. Goettsch, J. Nutrition 44, 443 (1951).
42 QO, Lindan and H. P. Himsworth, Brit. J. Exptl. Pathol. 31, 651 (1950).
143 P, Gyorgy, C. S. Rose, R. M. Tomarelli, and H. Goldblatt, J. Nutrition 41, 265
(1950).
144K. Schwarz, Proc. Soc. Exptl. Biol. Med. 77, 818; 78, 852 (1951).
VII. EFFECTS OF DEFICIENCY 541
c. Kidney
A progressive nephrosis of the kidney occurs in low-E rats, involving
isolated convoluted tubules of the outer cortex after 3 to 4 months of de-
ficiency and becoming quite generalized by the tenth month.! The glomeruli
show little change, but the tubular epithelium becomes coarsely granular,
irregular in contour, and separated from the basement membrane to form
an amorphous pale-staining layer. In advanced stages, the loops of Henle
and even the collecting tubules are involved. It is surprising that the rats
can survive with such widespread nephron damage. Acid-fast pigment has
been observed in the tubular epithelium of the rat,‘ !° monkey,”® and mink™
in the absence of the nephrotic changes just described.
d. Tooth Depigmentation
Depigmentation of the maxillary incisors of the rat is recognized as a
common manifestation of vitamin E deficiency; mandibular incisors are
also involved if dietary protein is low.° This iron-containing, non-fluores-
cent pigment is continuously formed and deposited by the enamel organ
as the incisor is worn away by attrition. The depigmentation is secondary
to atrophic changes in the enamel organ. 7 According to Pindborg,™®
there is edema and disorganization of the papillary layer, probably caused
by capillary damage in this region, followed by epithelial derangement and
cyst formation in the ameloblast layer. There is also a progressive deposi-
tion of acid-fast pigment in macrophages of the highly vascular periodontal
connective tissue.” After vitamin E therapy, function of the enamel organ
is restored and newly deposited enamel acquires its normal color. Depig-
mentation of maxillary incisors occurs also in the hamster,” but no histo-
pathologic studies have been reported.
B. IN MAN
1. GENERAL CONSIDERATIONS
A state of avitaminosis E has not been shown to occur in man. There
are some similarities between certain manifestations of vitamin E defi-
ciency in experimental animals (such as fetal resportion, dystrophic changes
in skeletal muscle) and certain clinical disorders in man (habitual abortion,
progressive muscular dystrophy); yet, it has not been established that the
latter are either due to lack of vitamin E or benefited by therapeutic use of
the vitamin. On the other hand, there has arisen an extensive but decidedly
145 'T. Moore, Biochem. J. 37, 112 (1943); Ann. N. Y. Acad. Sci. 52, 206 (1949).
146 J. T. Irving, Natwre 160, 122 (1942).
47 H. Granados, K. E. Mason, and H. Dam, J. Dental Research 24, 197 (1945); 25,
179 (1946).
48 J.J. Pindborg, J. Dental Research 29, 212 (1950); 31, 805 (1952).
542 THE TOCOPHEROLS
controversial literature regarding the therapeutic efficacy of vitamin E
(a-tocopherol) in a variety of clinical disorders which often have little in
common with the deficiency syndrome in lower animals and which are not
associated with any known inadequacy of vitamin E in the diet or in body
tissues. If tocopherol actually does exert a beneficial effect in such disorders,
the high dosage levels reported as necessary suggest a true pharmacologic
action on particular tissues or local regions of the body, perhaps through
some influence upon deranged metabolic processes at the affected site.
The distribution of vitamin E in various tissues and organs of man, at
a concentration quite similar to that in lower mammals, implies that it
serves some useful purpose. Depletion of body stores of vitamin E in lower
animals produces a variety of symptoms and histopathologic changes which
suggest that vitamin E functions as an important intracellular antioxidant;
whether it has other metabolic functions remains to be established. Regard-
less of how it participates in the metabolic economy of the animal organism,
it is a logical assumption that it functions in like manner in metabolic
processes of man, and that certain of the symptoms and lesions character-
izing the deficiency state in animals would also occur in man, provided
that comparable dietary conditions prevailed for a sufficiently long period
of time. On the basis of animal experimentation, where we have some notion
of the relations between previous tissue storage and rate of metabolic utili-
zation (or depletion) of tocopherol as a function of time, the possibility
that adult man ever reaches a state of uncomplicated avitaminosis E seems
rather remote. However, during early infancy, before tissue storage becomes
an important factor, there is real possibility of tocopherol inadequacy, as
will be discussed later.
Deficiency states are of two general types: natural deficiency, due to
inadequate intake of a nutrient over prolonged periods of time which, in
the case of fat-soluble vitamins, are measured in terms of years, if previous
intake and body storage have been reasonably normal; and ‘“‘conditioned”’
deficiency, arising through factors which chronically diminish the absorp-
tion or storage or increase the rate of metabolic utilization of the vitamin.
Deficiency of a fat-soluble vitamin rarely appears as a true clinical syn-
drome in adult man; however, states of suboptimal nutrition with respect
to such vitamins are recognized, although they are usually complicated
by other dietary inadequacies. To establish whether suboptimal or marked
deficiency of vitamin E occurs in man there must be acquisition of many
data on the vitamin E status of normal individuals and of others whose
status might be considered suboptimal on the basis of dietary habits or
organic disease. Such data must relate primarily to four major aspects of
vitamin FE nutriture: (1) the dietary intake over a period of years, (2) the
extent of intestinal absorption and excretion, (3) capacity for tissue storage
VII. EFFECTS OF DEFICIENCY 543
and (+) rate of metabolic utilization. In such considerations, it is customary
to express vitamin E in terms of d-a-tocopherol, which constitutes about
90% of the total tocopherol in human tissues and seems to be absorbed
more efficiently than, or preferentially to, the non-a@ forms which represent
about one-half the usual dietary supply."
a. Dietary Intake
Chemical and biological analyses indicate that tocopherols are present
in essentially every article of diet; yet, the richest sources such as vegetable
oils, unmilled cereals, and eggs constitute a much smaller proportion of the
usual dietary than animal products, vegetables, and fruits, which represent
relatively poor sources of a-tocopherol. It has been estimated that the aver-
age American dietary provides approximately 14 to 19 mg. of d-a-toco-
pherol, daily,!®° and that the better diets probably do not provide more
than 25 mg.™° Furthermore, the recommended diet of the National Re-
search Council (1945) and certain therapeutic diets (low sodium, diabetic)
would convey only about one-half this amount.? The same is true of the
average diet in Holland.!*! The estimates do not take into consideration
the losses of tocopherol which may occur during storage, commercial hand-
ling, and cooking, concerning which very little information is yet available.
b. Intestinal Absorption and Excretion
The question of how efficiently the gastrointestinal tract absorbs ingested
tocopherol has received scant attention. It has been recognized that little
or none is excreted in the urine! 1 and that appreciable amounts are
lost in the feces.!*?-!54 The tocopherol content of bile is of about the same
magnitude as that of the blood, and undoubtedly some absorbed tocopherol
is re-excreted into the intestinal tract via the biliary tract.) 1? On the
basis of bioassay tests, Hickman ef al.!** concluded that in normal human
subjects the ratio between ingested and excreted tocopherol is of the order
of 4 to 1.2. On the other hand, recent studies of Klatskin and Molander,!®
based on chemical analyses for fecal tocopherol and a larger number of
subjects, indicate that normal individuals excrete approximately two-thirds
of ingested tocopherol in the feces; they are also of the opinion that there
is little or no destruction in the gastrointestinal tract, and concur with
others that intestinal synthesis of tocopherol is quite unlikely. On the as-
149 M. L. Quaife, W. J. Swanson, M. Y. Dju, and P. L. Harris, Ann. N. Y. Acad. Sci.
52, 300 (1949).
1460 PL. Harris, M. L. Quaife, and W. J. Swanson, J. Nutrition 40, 367 (1950).
151 C, Engel, Ann. N. Y. Acad. Sci. 62, 292 (1949).
152 G. Klatskin and D. W. Molander, J. Clin. Invest. 31, 159 (1952).
183 G. Klatskin and D. W. Molander, J. Lab. Clin. Med. 39, 802 (1952).
154 K. C. D. Hickman, M. W. Kaley, and P. L. Harris, J. Biol. Chem. 152, 321 (1944).
544 THE TOCOPHEROLS
sumption that approximately 50% of ingested tocopherol is absorbed, it
appears that a normal adult on an average American diet would have a net
absorption of about 7 to 10 mg. of d-a-tocopherol, daily.
c. Tissue Storage
Most tissues and organs of man contain tocopherol, chiefly in the form
of a-tocopherol, as first reported by Abderhalden who carried out a series
of analyses on organs from human fetuses, newborn infants, and
adults. These findings have been generally verified and extended by
other studies!®*»? which begin to give a fairly satisfactory picture of to-
IRABIAR Ul
EstTiIMATED CONTENT OF ToTaL TocOHPEROLS IN HUMAN SUBJECTS
Tissue Woman, mg. Man, mg.
Fat 6180 1885
Muscle 269 285
Blood 45 64
Liver 33 45
Pancreas 10 i
Spleen 7 4
Heart 4 3
Kidney 10 2
Uterus 2
Lung 12
Testis 2
Total 6560 2309
Total on basis of 50 ke. $120
Total on basis of 70 kg. 3440
@ From Quaife and Dju.!56
copherol distribution and concentration in man. Unlike vitamin A, which
is stored largely in the liver, tocopherol is stored chiefly in the adipose
tissue. On the basis of tocopherol content of a wide variety of tissues from
two healthy adults, both cases of accidental death, total body storage has
been estimated to be 3.4 g. for a 20-year-old male and 8 g. for a 40-year-old
female (Table III). Accepting 5 g. as the average body storage in well-
nourished adults, an individual of 70 kg. body weight would possess ap-
185 R, Abderhalden, Intern. Z. Vitaminforsch. 16, 309, 319 (1945).
156M. L. Quaife and M. Y. Dju, J. Biol. Chem. 180, 263 (1949).
157 K: EH. Mason, M. Y. Dju, and L. J. Filer, Jr., Federation Proc. 11, 449 (1952).
1688 M. Y. Dju, K. E. Mason, and L. J. Filer, Jr., Htwdes Néo-natales 1, 49 (1952).
159 KE}. Mason and M. Y. Dju, Nutrition Symposium Series, National Vitamin Foun-
dation, New York, 7, 1 (1953).
VII. EFFECTS OF DEFICIENCY 545
proximately 70 mg. of tocopherol per kilogram of body mass. For compari-
son there are reported levels of 5.6 mg. per kilogram in a newborn infant
analyzed in toto, and of 3.1 mg. per kilogram in fetuses of 2 to 6 months
gestation age (average of 20 fetuses).!*” 1°85 Analyses of separate tissues and
organs of man, as discussed later (p. 555) suggest that the tocopherol con-
tent of adipose tissue provides a much more reliable index of vitamin E
nutriture than that of other tissues.
d. Metabolic Utilization
The metabolic utilization of a-tocopherol, which can be regarded as the
difference between net absorption and net storage over any given period
of time, has at present a rather intangible value. It is possible to control
and measure net absorption with a certain degree of accuracy, but total
storage or changes in storage over periods of time cannot be measured
or estimated in a living individual. Of greater importance is an under-
standing of those metabolic and other factors which influence the rate
of utilization of tocopherol in man, both as it applies to the body as a
whole and to isolated organs and tissue. At the present time we have es-
sentially no information on this question and are only able to draw certain
inferences from experimental studies in which factors such as increasing
age, diets high in unsaturated fats, and other types of metabolic stress
exert a noteworthy effect on the total body economy of vitamin E.
2. VITAMIN E IN EaARuty LIFE
During recent years considerable attention has been given to the vitamin
E status of man during prenatal and early postnatal life. Tocopherol con-
centration is low in tissues of the fetus and newborn infant!®*: 17-9 but
somewhat higher in the placenta!” 1°8: 16°. 16 which, as in lower mammals,
appears to have but a limited capacity to transfer tocopherol to the fetus.
Tocopherol levels are also low in cord blood!®*-!%° and in blood of the new-
born,'®: 1 generally ranging from one-third to one-fifth those of the
mother; yet, maternal blood levels show a natural increment (see Table IV,
160 R, Abderhalden, Schweiz. med. Wochschr. 75, 281 (1945).
161 G. Athanassiu, Klin. Wochschr. 24, 170 (1946); 25, 362 (1947).
162 J. Varangot, H. Chailley, and N. Rieux, Compt. rend. soc. biol. 187, 210 (1943);
138, 24 (1944).
163 W. Neuweiler, Intern. Z. Vitaminforsch. 21, 83 (1949).
164 J. V. Straumfjord and M. L. Quaife, Proc. Soc. Exptl. Biol. Med. 61, 369 (1946).
165 P| Cattaneo and A. Mariani, Rend. ist. swper. sanita 18, 424 (1950).
166 F. Gerloezy, B. Beneze, J. Szenasy, and D. Kunez, Experientia 7, 427 (1951).
167 W. T. Moyer, Pediatrics 6, 893 (1950).
168 S. W. Wright, L. J. Filer, Jr., and K. E. Mason, Pediatrics 7, 386 (1951).
546 THE TOCOPHEROLS
p. 552) during the latter part of gestation.'®: 16: 169-171 The reason for this
is not clear; it may help to overcome the restricted placental transfer of
tocopherol, or it may reflect a physiologic preparation for more effective
transfer of tocopherol to the milk, better to prepare the infant for the exi-
gencies of early extrauterine existence. Human breast milk has a consider-
ably higher content of tocopherol than does cow’s milk;!®: 17-4 further-
more, in both species colostrum is much richer in tocopherol than is later
milk.
There is thus unquestionable evidence that the newborn infant has a
rather small endowment of tocopherol at birth. Analysis of one full-term
infant in toto!” 18 has indicated a total content of about 25 mg., or ap-
proximately the daily intake of an adult on a high-quality diet. It also ap-
pears that tocopherol concentration in tissues shows no significant increase
during the first three years of postnatal life.!°* It is pertinent at this point
to call attention to the fact that certain manifestations of vitamin E de-
ficiency have been produced only during relatively early phases of life of
the species, and that in producing states of experimental vitamin E de-
ficiency it has always been a practice to initiate the deficient diet early
in life, before any appreciable tissue storage has occurred, because of the
recognized difficulty in depleting tissue reserves of the vitamin. The ques-
tion then arises as to whether the seemingly precarious tocopherol status
of the newborn infant provides any basis for a natural, or a conditioned,
deficiency state during infancy or early childhood.
a. Susceptibility of Erythrocytes to Hemolysis
Gyorgy and his associates!”>: 17° have demonstrated that erythrocytes of
low-E adult rats, and of newborn rats from mothers on stock diets, are
readily hemolyzed zn vivo or in vitro by small amounts of dialuric acid,
alloxantin (both reduction products of alloxan), or hydrogen peroxide, and
that small amounts of a-tocopherol protect the cells against these effects.
In the writer’s laboratory, it has also been shown that erythrocytes of
vitamin H-deficient monkeys hemolyze when exposed to small amounts of
169 N.S. Scrimshaw, R. B. Greer, and R. I. Goodland, Ann. N. Y. Acad. Sci. 52, 312
(1949).
170 W. J. Darby, M. E. Ferguson, R. H. Furman, J. M. Lemley, C. T. Ball, and G. R.
Meneely, Ann. N. Y. Acad. Sct. 52, 328 (1949).
71 T,, Rauramo, Acta Obstet. Gynecol. Scand. 24, 193 (1944); 27, Suppl. 2 (1947).
172 W. Neuweiler, Intern. Z. Vitaminforsch. 20, 108 (1948).
173 M. L. Quaife, J. Biol. Chem. 169, 513 (1947).
74 P. L. Harris, M. L. Quaife, and P. O’Grady, J. Nutrition 46, 459 (1952).
175 C.S. Rose and P. Gyorgy, J. Nutrition 39, 529 (1949); Blood 5, 1062 (1950); Am. J.
Physiol. 168, 414 (1952).
176 P, Gyorgy, G. M. Cogan, and C.S. Rose, Am. J. Diseases Children 82, 237 (1951);
Proc. Soc. Exptl. Biol. Med. 81, 536 (1952).
VII. EFFECTS OF DEFICIENCY 547
alloxantin having no effect upon the erythrocytes of controls fed the same
diet supplemented with tocopherol.
Furthermore, Gyorgy et al.'”® report that the red blood cells of full-term
infants at birth show mild hemolysis when exposed to small amounts of
hydrogen peroxide, that incubation of washed erythrocytes with a-to-
copherol makes them resistant to this effect, and that tocopherol fed to the
infant (but not when given to the pregnant mother) accelerates the dis-
appearance of this fragility which normally occurs during the first week or
so of postnatal life. They speculate that ‘the ‘physiologic’ vitamin E de-
ficiency of the fetus and the newborn may have practical clinical implica-
100 M. C!
30
% 80
HEMOLYSIS 70
60
10 20 30 40 S50 10 20 30 40 20 3 40 50 60
AGE IN DAYS
Fic. 5. Effect of a-tocopherol administration, beginning at time indicated by
arrow, upon the susceptibility of erythrocytes to hemolysis (H2O2) in four premature
infants (from Gordon and de Metry!’).
tions in Rh-incompatibility as well as in the development of erythroblas-
tosis in newborn infants of diabetic and prediabetic mothers.” Of additional
significance is the recent report of Gordon and deMetry!” describing he-
molysis of red cells, by the hydrogen peroxide test, in premature infants
bottle-fed for periods up to 30 days after birth, and disappearance of this
fragility within 2 to 5 days after tocopherol administration to the infant
(Fig. 5). They emphasize the low tocopherol content of most artificial
formulas for infants, compared to that of breast milk, as have Wright et
al.,1®° who showed that in bottle-fed full-term infants the postnatal increase
in serum tocopherol is much more gradual than in breast-fed infants (Fig.
6), and that in bottle-fed prematures during the first month or so of post-
177 Hf. H. Gordon and J. P. deMetry, Proc. Soc. Exptl. Biol. Med. 79, 446 (1952).
548 THE TOCOPHEROLS
natal life there is a gradual decline in serum tocopherols until they reach
levels comparable to those which in the experimental animal are associated
with manifestations of vitamin E deficiency (Fig. 7). It seems, therefore,
that the bottle-fed premature infant, denied the benefit of placental trans-
fer of vitamin E during the latter phases of gestation, physiologically
handicapped from the standpoint of suckling and other postnatal adapta-
tions, and usually reared on a low-fat formula (because of poor tolerance
for fats) rarely providing more than one-fourth to one-fifth the tocopherol
present in breast milk, represents the nearest approach to a natural avita-
minosis EH in man. It still remains to be determined whether the low-E
status of the premature or full-term newborn is etiologically related to any
HAS
©
NON NURSING MOTHERS
WI2O 7 5Snie ge eee elele en
=
ie}
5@O1.5] NURSING "MOTHERS
. fe) to)
O8 geeeeeeee
2t-|0| BREASTFED INFANTS
= ° eee
O05 Reena ae os
BOTTLE-FED INFANTS
2 4 6 +4 oO8
DAYS MONTHS
Fic. 6. Mean serum tocopherol levels for bottle-fed and breast-fed full-term
infants, and their mothers, during the first postpartum week and at periods of 1 to 4
and 5 to 8 months postpartum (from Wright ef al.'*).
specific disorders or dysadaptations of the young infant, and also whether
the maintenance of abnormally low levels of serum tocopherol in prema-
tures for a considerable period of time before the institution of a mixed
diet has any harmful influence upon the subsequent health of the infant.
A few clinical states which have been explored from this general standpoint
justify comment, even though the results are equivocal.
b. Retrolental Fibroplasia
In view of what has been said regarding the tocopherol status of the
fetus and the newborn infant, it is natural that considerable attention has
been given to the possible implication of an inadequacy of vitamin E in
the etiology of retrolental fibroplasia.!”*-!8! This disorder is characterized by
178 Retrolental Fibroplasia, Ret. 2nd M & R Pediat. Research Conf., Columbus (1951).
179 W. C. Owens and E. U. Owens, Am. J. Ophthalmol. 32, 1631 (1949).
VII. EFFECTS OF DEFICIENCY 549
unusual proliferative activity in retinal capillaries, followed by edema and
small hemorrhages which rupture into the vitreous, separation of the retina,
and, with further proliferation, the formation of a disorganized membrane-
like mass in the vitreous. Its etiology is unknown, but it is generally re-
garded as related to some metabolic disorder of prenatal or early postnatal
life.
There are many observed facts which provide quite logical reasons for
suspecting that inadequacy of vitamin E might be involved, as outlined
by Owens and Owens.!” Although the latter investigators found no signifi-
cant difference in serum tocopherol levels between infants with normal eyes
0.5
04
oD
3
Se
o
Tes
fo)
0.2
=
3
® O01
0
1-10 11-20 21-30 31-40
Hospital days
Fra.7. Mean level of serum tocopherols in artificially fed premature infants during
the first 40 days postpartum (from Wright ef al.18).
and those who developed retrolental fibroplasia, prophylactic tocopherol
therapy given to alternate premature infants with birth weights of 3 pounds
or less gave results which were at least encouraging; so also did the use of
a-tocopherol in infants showing early stages of the disease. Unfortunately,
however, their subsequent experience,* and that of other investiga-
tors,'8°: 8! has raised doubt as to whether tocopherol has therapeutic
value in preventing or ameliorating the lesions. Capillary changes due
to physiologic immaturity and electrolyte imbalance’ and failure to main-
tain proper oxygenation'’’ during early postnatal life are currently under
consideration.
180 W. E. Kinsey and J. F. Chisholm, Jr., Am. J. Ophthalmol. 34, 1259 (1951).
181 W. O. La Motte, Jr.. G. S. Tyner, and H. G. Scheie, Arch. Ophthalmol. 47, 556
(1952).
182 A.C. Krause, Am. J. Ophthalmol. 34, 1003 (1951).
183 TS. Szewezyk, Am. J. Ophthalmol. 34, 1649 (1951); 35, 301 (1952).
550 THE TOCOPHEROLS
c. Other Implications
There are suggested alterations in the peripheral vascular system during
early phases of life in connection with certain manifestations of experi-
mental vitamin E deficiency; for instance, exudative diathesis and nutri-
tional encephalomalacia in young chicks, vascular stasis and hemorrhage
in the fetus of low-E rats, the reported effect of tocopherol on vascular
resistance in guinea pigs, and stilbestrol-induced purpura in dogs. To these
may be added the reported, but as yet unestablished, effects of tocopherol
in thrombocytopenic purpura and in thromboembolic phenomena in adult
man as discussed in a later section. On the basis of these reported findings,
clinicians have been led to test the efficacy of a-tocopherol in certain dis-
orders of infancy with results which are somewhat questionable.
Impressed by the frequency with which fetal death in prematures, espe-
cially in those with birth weights under 1.5 kg., can be related to cerebro-
meningeal hemorrhages due to multiple rupture of capillary vessels, Min-
kowski!*4: 18 has attempted to measure the vascular resistance of such
infants (on the basis of petechiae produced by vacuum cup applied to skin
of the back) and its response to vitamin P-like substances and to a-to-
copherol. When mothers were given large doses (600 to 900 mg.) of a-to-
copherol several hours prior to premature delivery, there was a definite
increase in vascular resistance of the premature infant as compared to that
observed in infants of comparable weight from untreated mothers: tocoph-
erol also lessened the visibility of the capillary network of the skin, as
visualized by the capillaroscope.!** There were four instances of intracranial
hemorrhages in the treated group, and twelve in the untreated group, each
represented by 105 infants; Minkowski considers these results suggestive
of beneficial effects but recognizes the need for additional data before more
conclusive statements can be made.
It has been reported! 87 that a-tocopherol given to pregnant, Rh-nega-
tive mothers protects the infant against erythroblastosis, possibly through
reduced permeability of the placenta to the Rh antigen, as indicated by
marked reduction of antibody titer in the maternal plasma.
Gerloczy, who was for a period convinced that a-tocopherol, largely
through a diuretic effect, greatly benefited premature infants with sclere-
dema, has since reported!** that subsequent experience has led him to doubt
the efficacy of tocopherol therapy. Tocopherol is said to be effective in the
treatment of erythredema (Pink disease)!8® and in effecting increased ap-
181 A. Minkowski, Arch. franc. pediat. 6, 276 (1949); Ann. Paediat. 174, 80 (1950).
185 A. Minkowski, Le Sang 22, 701 (1951).
186 B.S. Ten Berge, Med. T'ydschr. Geneesk. 94, 3417 (1950).
187 B. S. Ten Berge and F. J. J. Van Assen, Med. Tydschr. Verlosk. 50, 38 (1950).
188 F’. Gerloczy, Paediat. danub. 6, 83 (1949).
189 G. Forsyth, Med. J. Australia I, 78 (1941).
, i i ET EE
VII. EFFECTS OF DEFICIENCY 551
petite and improved body growth in infants failing to respond to other
measures.!*" 1° These observations, based on a small number of subjects,
require confirmation.
3. VITAMIN E IN Later LIFE
As pointed out earlier, it seems rather unlikely that a natural deficiency
of vitamin E ever occurs in man during adolescence or adulthood. On the
other hand, it is conceivable that a ‘‘conditioned”’ deficiency might arise
as a result of (1) prolonged and severe impairment of fat absorption, (2) an
inherited defect of metabolism affecting the capacity of certain tissues or
organs properly to utilize the vitamin, or (3) certain types of metabolic
stress which greatly increase requirements for the vitamin or its rate of loss
from tissue stores.
There is no known symptomatology of avitaminosis E in man. Therefore,
any evaluation of the tocopherol status of man depends largely upon in-
formation pertaining to (1) dietary history, (2) plasma tocopherol levels,
(3) tocopherol concentration in tissues, and (4) the demonstration of histo-
pathologic changes which are comparable to those characterizing the ex-
perimental deficiency state in other primates and lower mammals. Dietary
histories are not particularly informative in the case of vitamins which are
stored rather tenaciously by tissues. Chemical and histopathologic analyses
of tissues and organs are limited to postmortem material or to small samples
obtained through biopsy. Blood levels represent the interplay of many
factors and are not necessarily indicative of the tocopherol status of the
individual as a whole; however, they constitute the most widely used cri-
terion for evaluating vitamin E nutriture in man.
a. Plasma (or Serum) Tocopherols
(1) Normal Levels. Most of our information on plasma tocopherols is
based upon blood samples analyzed by the method of Quaife and Har-
ris,!* 1% utilizing the Emmerie and Engel color reaction. A micromethod
later developed by Quaife et al. and requiring minute samples of blood
has been of particular value in studies with infants!®: !°° and will undoubt-
edly come into much wider use. The values given by these methods are
generally higher and more consistent in the hands of different investigators
than are those reported by European workers employing other chemical
procedures.
190 J. Frey, Klin. Wochschr. 27, 348 (1949).
191 G. W. Schmidt, Arch. Kinderheilk. 1388, 178 (1950).
192 M. L. Quaife and P. L. Harris, J. Biol. Chem. 156, 499 (1944).
193 M. L. Quaife and R. Biehler, J. Biol. Chem. 159, 663 (1945).
194 M. L. Quaife, N. S. Scrimshaw, and O. H. Lowry, J. Biol. Chem. 180, 1229 (1949).
195 J,. J. Filer, Jr..S. W. Wright, M. P. Manning, and K. E. Mason, Pediatrics 8, 328
(1951).
502 THE TOCOPHEROLS
Plasma tocopherol levels in newborn infants are approximately one-
third to one-fourth those found in healthy adults. Average values of 0.23
mg. % (S.D. + 0.13), 0.37 mg. % (S.D. + 0.15),% and 0.43 meng
(S.D. + 0.12)'* have been reported. For young and adolescent children,
values of about 1 mg. % are considered normal.!** Plasma tocopherol levels
for healthy adults usually fall within the range of 1.0 to 1.2 mg. % (see
Table IV). During pregnancy there is a rather pronounced increment in
plasma tocopherols,!*: 1%) 16-171 which declines only gradually during lac-
TABLE IV
PiasMA TocopHEROL LEVELS IN MAN
Individuals studied Plasma tocopherols,®
mean and S.D.
No. Type Age range mg. % Reference
13 Normal 17-48 1.20 + 0.22 192
70 Normal 1.04 + 0.32 198
350 Clinic patients 0.78 + 0.26 198
21 Normal 109-0217 199
42 Patients, unselected 0.92 + 0.29 199
62 Patients, cardiacs 0.94 + 0.35 199
23 Normal 20-40 1.23 + 0.31 200
57 Patients, convalescent 17-86 1.02 + 0.37 200
43 Patients, liver disorder 27-75 0.95 + 0.32 200
26 Normal 16-39 0.90 + 0.19 197
162 Patients, infirmary 40-101 0.93 197
20 Normal 22-55 1.26 202
11 Pregnancy, early Lt, S=* O19 164
12 Pregnancy, late 1.62 + 0.31 164
54 Pregnancy, delivery 1.70 + 0.30 164
17 Pregnancy, 0-8 wk. 1.05 =. 0.27 169
36 Pregnancy, 9-16 wk. 1.02 + 0.25 169
36 Pregnancy, 17-24 wk. 1.29 + 0.32 169
33 Pregnancy, 25-32 wk. 1.38 + 0.30 169
75 Pregnancy, 33-40 wk. 1.51 + 0.40 169
@ Method of Quaife and coworkers.192, 193
tation.1®: 171 A tendency for values to increase with age has been
reported.!7° 197
(2) Effect of Disease. Compared to normal healthy adults, clinic pa-
tients! and convalescent hospital patients randomly selected: 199, 200
196 AS. Minot, J. Lab. Clin. Med. 29, 772 (1944).
197 M. Chiefi and J. E. Kirk, J. Gerontol. 6, 17 (1951).
198 P- L.. Harris, K. C. D. Hickman, J. L. Jensen, and T. D. Spies, Am. J. Public
Health 36, 155 (1946).
199 J. M. Lemley, R. G. Gale, R. H. Furman, M. E. Cherrington, W. J. Darby, and
G. R. Meneely, Am. Heart J. 37, 1029 (1949).
200 G, Klatskin and W. A. Krehl, J. Clin. Invest. 39, 1528 (1950).
VII. EFFECTS OF DEFICIENCY 553
have somewhat lower levels of plasma tocopherol (Table [V). Patients with
liver disease also tend to show low values,2°’: 2°! but they are not signifi-
cantly different from those of convalescent patients with no evidence of
liver disease;!®2: 2°° the same is true of cardiac patients.'*? Lower-than-usual
values are also commonly observed in diseases where intestinal absorption
is defective, as in sprue,?°: 2° celiac disease, fibrocystic disease of the pan-
creas, biliary obstruction, and diarrhea associated with achlorhydria.!”°: 1%
This is in accord with experimental evidence that surgical production of a
DISEASES ASSOCIATED WITH
HYPERCHOLESTEROLEMIA
a
PREGNANCY
MISCELLANEOUS METABOLIC
AND INFECTIOUS DISEASES
HEALTHY, ADULTS;
0.5] ABSORPTIVE DEFECTS
ie)
Mg-"/ooce.
Fic. 8. Range of plasma tocopherols in man in relation to good health, pregnancy,
and certain broadly classified diseases (from Darby ef al.'7°).
bihary fistula leads to a state of vitamin E deficiency in the rat and dog.
On the other hand, higher-than-usual values are frequently observed in
diseases associated with hypercholesteremia and in cardiovascular disease
and pregnancy, both of which are often associated with increased blood
lipids.!° (Fig. 8). These deviations from normal might be explained, accord-
201 H. Popper, A. Dubin, F. Steigmann, and F. P. Hesser, J. Lab. Clin. Med. 34, 648
(1949).
202 W. J. Darby, M. E. Cherrington, and J. M. Ruffin, Proc. Soc. Exptl. Biol. Med. 68,
310 (1946).
203 W. J. Darby, E. Jones, H. F. Warden, and M. M. Kaser, J. Nutrition 34, 645
(1947).
554 THE TOCOPHEROLS
ing to Darby et al.,!”° on the basis of differences in intestinal absorption or
differences in lipid-carrying power of the blood.
The effectiveness of intestinal absorption of vitamin EK has been meas-
ured by the tocopherol ‘‘absorption curve” (also termed “‘tolerance curve,”
“tolerance test,’ ‘blood persistence curve’’). Subjects are given a single,
large, oral dose of tocopherol (usually 5 to 20 mg. of a-tocopherol per kilo-
gram of body weight) and blood samples are taken at 0, 3, 6, 9, 12, and
24 hours. The curve obtained by plotting the tocopherol values, or the
planimeter measurement of the area under the curve, provides a basis for
comparing individual responses. Low response curves have been reported
in some, but not all, cases of liver disease,?°°: 2° in primary fibrositis,?* and
in sprue;!*: 20, 203 low responses have been observed in cases of fibrocystic
disease of the pancreas, diarrhea and cirrhosis in infants, and in children
with celiac syndrome and lupus erythematosus, and rather high responses
in metabolic disorders with associated hypercholesteremia.!%°
Low response curves may not necessarily reflect impaired intestinal ab-
sorption. The fate of tocopherol, once it has passed the intestinal barrier,
may be influenced by inadequacy of a lipid or protein ‘‘carrier’’ to which
it may be bound either for purposes of transport or for functional activity?”
or by other biochemical alterations of the blood or tissues. Klatskin and
Molander,'*? who have studied the combined picture of absorption and
fecal excretion in normal, convalescent, and cirrhotic individuals, feel that
the low plasma tocopherol level in cirrhotics is ‘‘more closely related to
tissue depletion than to an impairment of absorption or utilization.” Their
observation that cirrhotics excrete a considerably smaller fraction of ad-
ministered tocopherol than do healthy adults suggests also the possibility
that in patients with impaired hepatic functions much less tocopherol is
re-excreted via the biliary tract.’ Although bile from patients with opera-
tive biliary fistulas has about the same tocopherol content as blood,!?: 2%
it is not known whether this represents a true excretion product or a phase
in an enterohepatic circulation of tocopherol.
From what has been said, it appears that tendencies for plasma tocoph-
erol to reach different levels in a variety of diseases may reflect previous
dietary intake, effectiveness of absorption and excretion, state of tissue de-
pletion, or biochemical changes in the blood which influence its carrying
capacity for tocopherol. Low levels observed in diseases where a significant
and prolonged impairment of fat absorption is indicated may sometimes
reflect a low vitamin E status of the body as a whole, although they may
rarely represent degrees of depletion commensurate with typical biochemi-
‘al or histopathologic manifestations of avitaminosis E.
204 C. L. Steinberg, Med. Clin. N. Amer. 30, 221 (1946).
206 K. C. D. Hickman and P. L. Harris, Advances in Enzymol. 6, 469 (1946).
—— -—. 4
VII. EFFECTS OF DEFICIENCY 555
b. Tissue Tocopherols
Tissue levels of tocopherols, especially those of the major storage depots,
are undoubtedly the most reliable index of vitamin E nutriture. The usual
method for analysis of tocopherols in foods and tissues?°* is laborious and
time-consuming. Tissues obtainable are limited to postmortem material,
except where generous biopsy samples can be secured. Although d-a-tocoph-
erol is the predominant type found in tissues, variable but usually not
very large amounts of y- and 6-tocopherols are also present ;!°*°? however,
results are generally expressed in terms of total tocopherols.
Information concerning tocopherols in human tissues is limited to anal-
yses carried out by Abderhalden!®® on a variety of tissues from fetuses,
newborn, and adults, those of Quaife and Dju!** on two cases of accidental
death, and a more extensive series of analyses covering the period from
early fetal life to old age,!*-!* carried out in the writer’s laboratory. The
results of these studies indicate that tocopherols are widely distributed in
human tissues from early fetal life to advanced old age. Tocopherol levels,
expressed as milligrams per 100 g. of fresh tissue, are low in fetuses of 2 to
6 months’ gestation age, and only slightly higher in premature and full
term infants at birth. The data obtained are in accord with other evidence
that during the early postnatal period of life states of suboptimal vitamin
E nutriture may occur. During early postnatal life, tissue levels tend to
increase slowly unless suppressed by disease of various types. During child-
hood and adolescence they reach levels comparable to those of adults
which, for muscle, heart, liver, and certain other visceral organs, are ap-
proximately twice those at birth, and for adipose tissue are considerably
higher than in other tissues, with the exception of the adrenal. Both pitui-
tary and testis contain about four times as much tocopherol per unit of
fresh tissue as do other visceral organs, but only about one-third as much
as the adrenal. During the latter few decades of life there appears to be a
tendency for tocopherols to diminish somewhat in liver and in adipose
tissue. It is also of interest that total tocopherols (expressed as milligrams
per 100 g. of fresh tissue) in skeletal muscle, heart, liver, and other visceral
organs, except the endocrine glands mentioned, are of about the same order
as observed in the circulating blood.
On the other hand,when tocopherols are expressed as milligrams per
gram of extractible fat the values for adipose tissue are considerably lower
than for most of the tissues just mentioned, whereas the values for pituitary,
testis, and adrenal appear to be considerably higher. Although tocopherols
in adipose tissue appear to increase during early life on the basis of tocoph-
erolsper unit of fresh tissue, and todiminish in terms of tocopherols per unit of
extractible fat, from a quantitative standpoint the adipose tissue of the
203 M. L. Quaife and P. L. Harris, Anal. Chem. 20, 1221 (1948).
556 THE TOCOPHEROLS
body appears to represent the major site of tocopherol deposition and may
therefore best reflect the tocopherol status of the body as a whole.
c. Histopathology
The histopathologic lesions of experimentally induced deficiencies often
have a close counterpart in the corresponding deficiency state in man. This
is true of vitamin C and of vitamins A, D, and K, insofar as the period of in-
faney and early childhood is concerned; however, differences in responsive-
ness of the adult organism and the tenacity with which its tissues retain
traces of the fat-soluble vitamins seldom permit outspoken symptomatology
or histopathologic changes. It is notably difficult to induce manifestations
of vitamin E deficiency in mature animals that have built up considerable
stores of tocopherol. Furthermore, the fact that tocopherol storage is chiefly
in the adipose tissue, and thereby much less subject to interference by dis-
ease processes such as influence storage of vitamin A in the liver, may ex-
plain why there is so little evidence of vitamin E inadequacy in man other
than during early infancy, as discussed previously.
Histopathologic approaches to the question of vitamin E deficiency in
man have been generally twofold. One is based upon the occurrence of an
acid-fast pigment commonly associated with the lesions of experimental
vitamin E deficiency; the other relates to striking similarities between the
muscle lesions of progressive muscular dystrophy in man and those common
to the various species of animal in which vitamin E deficiency has been
produced.
(1) Acid-Fast Pigment (Ceroid). An acid-fast pigment, sometimes re-
ferred to as ceroid, occurs in the smooth and striated musculature and
becomes disseminated throughout the reticulo-endothelial system, after
prolonged vitamin E deficiency in certain animals; in organs such as the
sex glands and the adrenal, where small amounts of this pigment occur
normally, there may be a conspicuous increase. Excess of intracellular,
unsaturated lipids or fat peroxides and inadequacy of tocopherols as anti-
oxidants are considered primary factors in the genesis of this pigment. A
comparable pigment has been described by Wolf and Pappenheimer?” in
rarious parts of the central nervous system, and by Pappenheimer and
Victor?’ in a variety of other tissues and organs from routine autopsies.
They report that, in general, the occurrence of acid-fast pigment in human
tissues tends to be associated with hepatic cirrhosis and hemochromatosis,
celiac disease, pancreatic fibrosis, and non-tropical sprue, except for its
occasional location about focal degenerative lesions such as atheromatous
placques and areas of follicular atresia. It is of interest that acid-fast pig-
207 A. Wolf and A. M. Pappenheimer, J. Neuropathol. Exptl. Neurol. 4, 402 (1945).
208 A. M. Pappenheimer and J. Victor, Am. J. Pathol. 22, 395 (1946).
VII. EFFECTS OF DEFICIENCY 557
ment, some of which may be derived from red blood cells,”’? is commonly
associated with atheromatous lesions of the aorta and other vessels,2°": 2!°
and that atheromatous aortas are said to contain peroxides*!! such as are
associated with sites of ceroid formation in vitamin [-deficient animals.
A ceroid-like pigment in the human ovary has also been described by
Brenner?” and by Reagan,?!’ usually in association with follicular atresia,
and regarded as an oxidation product of vitamin A (or carotene) and un-
saturated fats, catalyzed perhaps by lipoxidase of the ovarian tissues.
It should be made clear that in the studies describing ceroid in human
tissues no claim is made that an avitaminosis E is involved. Ceroid may
arise in tissues where, in association with unsaturated fats, there are local
oxidative disturbances similar to those occurring in vitamin E deficiency
but due to other causes; there is also the possibility of a localized destruc-
tion of intracellular tocopherol due to metabolic stress or chemical insult
even though the tocopherol status of the body in general is normal.
There are several reports which present more definite suggestions that
human beings may approach a state of conditioned avitaminosis EK. Pappen-
heimer and Victor?’ have presented in considerable detail the postmortem
findings on four individuals exhibiting chronic nutritional disorders (idio-
pathic hypoproteinemia, gastrocolic fistula subsequent to gastroenteros-
tomy, non-tropical sprue, and chronic jejunitis with cirrhosis) in which
there was found an abundance of acid-fast pigment having much the same
localization as that seen in vitamin E-deficient animals. In all cases, there
was pronounced pigmentation of the muscular coats of the esophagus,
stomach, and small intestine, such as seen in the E-deficient monkey and
in dogs with biliary fistulae. Acid-fast pigment was also noted in liver cells,
Kupffer cells, uterine muscle, phagocytes of ovarian stroma, cardiac and
skeletal muscle, media of small arteries, and the Sertoli syncytium and
interstitial cells of the degenerate testes of the one male of the series.
Tverdy et al.2* present a detailed clinical history and postmortem findings
in a case of non-tropical sprue, in which they noted acid-fast pigment with
essentially the same distribution in intestinal smooth muscle, liver, degen-
erate testes, and macrophages of various organs and tissues. Histopatho-
logic changes were also noted?** in the central nervous system, including
209 W. S. Hartroft, Am. J. Pathol. 28, 526 (1952).
210 R. C. Burt, Am. J. Clin. Pathol. 22, 185 (1952).
21 J. Glavind, 8. Hartmann, J. Clemmesen, K. E. Jessen, and H. Dam, Acta Pathol.
30, 1 (1952).
212 §. Brenner, S. African J. Med. Sci. 11, 173 (1946).
213 J. W. Reagan, Am. J. Obstet. Gynecol. 59, 433 (1950).
214 G. Tverdy, A. L. Froehlich, and B. Fierens, Acta Gastro-enterol. Belg. 12, 221
(1949).
24a T,, van Bogaert and G. Tverdy, Monatsschr. Psychiat. Neurol. 120, 301 (1950).
558 THE TOCOPHEROLS
the presence of much acid-fast pigment, especially in relation to blood
vessels and the choroid plexus epithelium. They consider these changes to
be strongly indicative of an avitaminosis E associated with sprue.
Reference has been made (p. 544) to the low serum tocopherol levels and
flat type of absorption curve observed in sprue, celiac disease, and similar dis-
orders.!** Also of interest is the comment of Frazer?!’ that ‘‘Dietary inade-
quacy is certainly not a common cause of vitamin deficiency in the sprue
syndrome. It may account for the occasional case of vitamin E deficiency,
especially since oxidative rancidity of fats may be one of the precipitating
causes in tropical sprue.”
It is unfortunate that in the five cases referred to above no data could
be obtained on serum tocopherol levels prior to death or on tissue levels
at postmortem. With lack of evidence of this type, the findings reported
can be considered only highly indicative that a conditioned state of avita-
minosis KE may sometimes occur in man as a result of chronic diseases
which seriously interfere with the absorption of fats and fat-soluble vita-
mins.
(2) Progressive Muscular Dystrophy. Many investigators have been im-
pressed by the striking similarity between the skeletal muscle lesions of
progressive muscular dystrophy and dermatomyositis in man, and those of
nutritional myodegeneration which represent the most characteristic
manifestation of vitamin E deficiency in experimental animals (p. 552).
This applies also to myocardial lesions, which constitute a rather charac-
teristic finding in progressive muscular dystrophy”!®: 7” as they do in many
animal species deficient in vitamin E.
There is also evidence of a common biochemical defect, in the form of
urinary excretion of ribose-phosphorus-containing complexes which appear
to be rather specific for human muscular dystrophy”: ?8* and which occur
also in dystrophic vitamin E-deficient rabbits.?!° The suggestion that these
complexes may reflect disturbances in nucleotide metabolism is in keeping
with other evidence of disturbed nucleic acid metabolism in the vitamin
K-deficient rabbit?° and monkey.” Of particular interest is the observa-
tion of Minot et al.?'8 that ‘“Spentose-containing complexes were detected in
the urine of an apparently normal 3 year old brother of one of our patients
216 A, C. Frazer, Brit. Med. J. II, 731 (1949).
216 W. G. Nothacker and M. G. Netsky, Arch. Pathol. 50, 578 (1950).
27 J. Zatuchni, E. KE. Aegerter, L. Molthan, and C. R. Shuman, Circulation 3, 846
(1951).
218 A.S. Minot, H. Frank, and D. Dziewiatkowski, Arch. Biochem. 20, 394 (1949).
218a W.-F,. Orr and A. S. Minot, Arch. Neurol. Psychiat. 67, 483 (1952).
219 A. §. Minot and M. Grimes, J. Nutrition 39, 159 (1949).
220 J. M. Young and J. S. Dinning, J. Biol. Chem. 193, 743 (1951).
221 J. S$. Dinning, L. D. Seager, and P. L. Day, Federation Proc. 10, 380 (1951).
a
VII. EFFECTS OF DEFICIENCY 559
with muscular dystrophy. There was a familial history of the disease, as
one older brother of our patient and a maternal uncle had already died after
running a typical course. Within the next 2 years this younger brother de-
veloped the typical clinical picture of rapidly progressing muscular dys-
trophy. It is possible that disintegration of muscle cells was already in
progress before the appearance of detectable dysfunction. It is also con-
ceivable that some inborn anomaly of vitally important nucleotide me-
tabolism is responsible for the deterioration seen in clinical dystrophy.”
A generalized aminoaciduria observed in muscular dystrophy’” may reflect
muscle breakdown and not a primary metabolic disturbance.
It is beyond the scope of this chapter to review the numerous reports
affirming or denying the therapeutic usefulness of wheat germ products or
tocopherol in human muscular dystrophy. Over a period of more than 15
years the results have been predominantly negative. This is not surprising
in view of the normal levels of tocopherol found in the blood!®*: 7° and
in various tissues and organs. #3 Jt is possible, however, that in indi-
viduals with muscular dystrophy tocopherols cannot exert their normal
functions in skeletal muscles, either because of a metabolic block such as
referred to by Minot or because of an inability to effectively convert to-
copherols to their hydroquinones or to other compounds which exert the
antidystrophy effects of vitamin E. It is this general approach which has
been followed by Milhorat e¢ al.,22*: 24*) 24 who report that dl-a-tocopheryl-
hydroquinone and related compounds are effective in reducing creatinuria
in dystrophic patients and also in curing dystrophy of vitamin E deficiency
in rabbits. Unquestionably, this constitutes an important area for continued
investigation.
Other investigators”*2’ are of the opinion that wheat germ oil and to-
copherol are effective in treating those diseases of muscle which they clas-
sify as collagen diseases; namely, dermatomyositis and menopausal dys-
trophy. The latter term has been proposed by Shy and McEachern”” for
222 S. R. Ames and H. A. Risley, Proc. Soc. Exptl. Biol. Med. 68, 131 (1948).
223 A. S. Minot and H. E. Frank, Am. J. Diseases Children 67, 371 (1944).
223 KE. Mason, M. Y. Dju, and S. J. Chapin, Federation Proc. 12, 422 (1953); Proc.
Ist & 2nd Med. Conf. (1951-52) Muscular Dystr. Ass’ns. Amer., New York p. 94.
224 AT. Milhorat, J. B. Mackenzie, S. Ulick, H. Rosenkrantz, and W. E. Bartels,
Ann. N.Y. Acad. Sci. 52, 334 (1949).
224a J. B. Mackenzie, H. Rosenkrantz, S. Ulick, and A. T. Milhorat, J. Biol. Chem.
183, 655 (1950).
24b A. T. Milhorat, Proc. 1st & 2nd Med. Conf. (1951-52) Muscular Dystr. Ass’ns.
Amer., New York p. 78.
225 T). McEachern, Bull. N. Y. Acad. Med. 27, 3 (1951).
226 R. Rabinovitch, W. C. Gibson, and D. McEachern, J. Neurol. Neurosurg. Psychiat.
14, 95 (1951).
227 G. M. Shy and D. McEachern, J. Neurol. Neurosurg. Psychiat. 14, 101 (1951).
560 THE TOCOPHEROLS
a type of myopathy occurring predominantly in women, but occasionally
in men, at the age of about 40 years or thereafter, and thought to be a
close counterpart of the dystrophy in vitamin E-deficient animals; others
classify this condition as myositis.?"* Both diseases are said to respond also
to cortisone therapy.”” The observations” regarding dermatomyositis are
in accord with the favorable response to wheat germ oil reported previously
by Milhorat et al.?’ There is still a question whether wheat germ products
possess effective substances apart from tocopherols. It has been suggested?”
that one such substance, inositol, can interact with tocopherol in the gastro-
intestinal tract to form a condensation product which diminishes the
creatinuria of progressive muscular dystrophy; conceivably, a metabolic
mechanism of this type could be defective or absent in patients with dys-
trophy.
On the basis of different patterns of inheritance, Tyler and Stevens”: 2%
are of the opinion that most cases of progressive muscular dystrophy can
be grouped under two major headings: the childhood type, occurring only
in males and inherited as a sex-linked recessive, and the fascioscapulo-
humeral type, occurring in both sexes and inherited as a Mendelian domi-
nant. It is as yet impossible to say whether the same or different metabolic
defects of metabolism are involved, or the extent to which the metabolic
defect in each case may eventually be related to oxidation or other conver-
sion products of tocopherol.
d. Disorders of Reproduction
The discovery of vitamin E and the long-established method for its bio-
assay are based upon the phenomenon of fetal resorption in the rat. It is
not surprising that over the past twenty-five years there have appeared
numerous clinical reports on the therapeutic use of vitamin E in habitual
abortion, threatened abortion, threatened miscarriage, premature labor,
and eclamptic states. The results are conflicting and confused by differ-
ences in definition, vitamin E dosage, and extent to which other therapeutic
measures are employed and often not recorded. As is so often the case with
unestablished therapeutic agents, favorable reports considerably exceed
those which relay negative findings.
(1) Habitual Abortion. An habitual aborter is usually defined as one who
has spontaneously aborted before the 16th week during three successive
pregnancies. It is estimated that 4% of all spontaneous abortions are ha-
228 1). Denny-Brown, Can. Med. Assoc. J. 67, 1 (1952).
29 A. 'T. Milhorat, F. C. Weber, and V. Toseani, Proc. Soc. Exptl. Biol. Med. 43, 470
(1940).
230 A, T. Milhorat and W. E. Bartels, Science 101, 93 (1945).
231, H. Tyler and F. E. Stevens, Ann. Internal Med. 32, 640 (1950); 35, 169 (1951).
232, EH. Stevens and F. H. Tyler, Am. J. Human Genetics 8, 111 (1951).
VII. EFFECTS OF DEFICIENCY 561
bitual, and that about 10% of all pregnancies terminate in abortion,
amounting to 240,000 yearly in the United States.2** Bacharach,2** in a
statistical analysis of reported cases of habitual abortion treated with
vitamin E up to 1940, felt that the chance of a successful pregnancy was
definitely increased by this therapeutic measure. Hertig and Livingstone?*®
later state: “Vitamin E, judging from the literature, has an important effect
on the favorable outcome of pregnancy in cases of habitual abortion—this
in spite of the fact that the average human dietary cannot be shown to be
deficient in vitamin E.”’ Only about 16 % of habitual aborters show plasma
tocopherol levels below the average normal range, and these are effectively
raised to normal by as little as 25 mg. of a-tocopherol, daily.“* An 80%
salvage in 211 patients with from three to eleven previous abortions, by
correction of contributory conditions of varied type, has been reported by
Javert et al.*” The present status of the problem is well summarized in
their statement: ‘‘There is such a maze of literature that proper cognizance
cannot be taken of all the pertinent articles. As the reader reviews them
in order to develop his own philosophy, let him be reminded of three im-
portant matters: the high percentage of success irrespective of which vita-
min, hormone or method is employed; the lack of specific information as to
the pathogenesis of human spontaneous abortion . . .”’
(2) Threatened Abortion and Miscarriage. Evan Shute, although dubious
about the merits of vitamin E in habitual abortion, has reported its thera-
peutic usefulness in threatened abortion and threatened miscarriage,”**: 2%9
premature labor, abruptio placentae,“ and non-eclamptic late-tox-
emias of pregnancy,”” and is of the opinion that tocopherol may in some
way counteract the effects of high blood estrogen rather than compensate
for a true avitaminosis E. Other clinicians have reported similar success
with tocopherol, frequently combined with progesterone therapy; the rea-
son that so much doubt still exists concerning these claims is due not so
much to other reports in the negative as it is to failure to, or inability to,
satisfactorily validate these clinical experiences by control data or by basic
information regarding the tocopherol status of the patients. The most
238 C, Mazer and 8. L. Israel, Diagnosis and Treatment of Menstrual Disorders and
Sterility, 3rd edition. Hoeber, New York, 1951.
234 A. L. Bacharach, Brit. Med. J. I, 890 (1940); I, 567 (1948).
*35 A. Hertig and R. A. Livingstone, New Engl. J. Med. 280, 797 (1944).
236 K. Delfs and G. E. S. Jones, Obstet. Gynecol. Survey 3, 680 (1948).
237 C. T. Javert, W. F. Finn, and H. J. Stander, Am. J. Obstet. Gynecol. 57, 878 (1949).
238 FE}. Shute, Urol. and Cutaneous Rev. 47, 239 (1943).
239 K, Shute and W. E. Shute, J. Obstet. Gynaecol. Brit. Empire 49, 534 (1942).
240 HB, Shute, Am. J. Obstet. Gynecol. 44, 271 (1942); J. Obstet. Gynaecol. Brit. Em-
pire 62, 571 (1945).
241. Shute, Surg. Gynecol. Obstet. 75, 515 (1942).
242 EH}. Shute, Am. J. Surg. 71, 470 (1946).
562 THE TOCOPHEROLS
thorough study in the latter sphere'®’ reports no significant changes in
plasma tocopherol in cases of threatened abortion, prematurity, pre-
eclampsia, or essential hypertension complicating pregnancy, except for
patients with abortion during the 17th to 24th weeks of pregnancy; the
interpretation which may be placed upon this latter finding is questionable
until more data are available. Whether data on the tocopherol content of
aborted fetuses and placentas would contribute to a better understanding
of this perplexing problem remains to be determined.
(3) Sterility in the Male. The other classic manifestation of experimental
vitamin E deficiency, namely, testicular degeneration in the rat, has no
known counterpart in man. Although Shute’ holds the opinion that vita-
min i therapy causes an increase in sperm count and enhances the possi-
bilities that infertility in males can frequently be overcome by vitamin E
therapy, largely through improvement in the number and quality of the
sperm, Williams** and Farris*° report that vitamin E concentrates have
no significant effect on sperm concentration, motility, or cytologic aberra-
tions in infertile men. Three other studies, yielding somewhat contradictory
results, are summarized by Swyer.?!
VIII. Pharmacology
KARL E. MASON
As so aptly stated by Mattill,! “None of the vitamins has been associated
with as wide a variety of biological processes as vitamin E, or has been
functionally related to so many basically different physiologic and chemical
reactions. ... The search for some unifying principle or correlating idea
as to the manner of action has not been rewarding.” Largely through the
pioneer researches of Mattill and his associates, the tocopherols have come
to be recognized as widely distributed and important biological antioxi-
dants, both in vive and in vitro. Compared to other tocopherols (6, y, 4),
a-tocopherol possesses the greatest biological activity im vivo and is re-
garded as the prototype of vitamin E. It represents 90% or more of the
tocopherols in animal tissues. Whether ingested as the natural d or the
synthetic dl form, or esters of the same, it is absorbed as d-a-tocopherol;
the latter is probably bound to lipids or proteins during transport in the
blood stream and deposited as such within the cell.
243 1}, Shute, Urol. and Cutaneous Rev. 48, 423 (1944).
244 W. W. Williams, Vrans. Am. Soc. Study Sterility 3, 67 (1947).
245 KY. J. Farris, Ann. N. Y. Acad. Sct. 52, 409 (1949).
26 G.I. M. Swyer, Brit. J. Nutrition 3, 100 (1949).
1H. A. Mattill, Nutrition Revs. 10, 225 (1952).
——————
VIII. PHARMACOLOGY 563
a-Tocopherol, like ascorbic acid, is one of the few antioxidants capable
of passing the intestinal barrier, reaching intracellular sites, and exerting
regulatory control over cell oxidations. Despite hopes and a certain amount
of evidence that tocopherol might prove to function in some oxidation-re-
duction system, or participate in some specific manner in certain enzyme
systems, no clear-cut claims can yet be made; nor has there been produced
indisputable evidence that a-tocopherol exerts biological effects unrelated
to its well-recognized function as an intracellular antioxidant.
A. HYPERVITAMINOSIS E
No state or syndrome of hypervitaminosis E has been described, nor is
there evidence that tocopherols per se exert any deleterious effect in ani-
mals or man. Demole? has shown that mice will tolerate oral doses of 50
g. per kilogram, and rats doses of 4 g. per kilogram daily for 2 months.
Adult humans have tolerated oral doses of 1 g. per day for months, or larger
doses for shorter periods, with no undesirable effects. Clinical literature
contains references to complaints of gastric distress and other symptoms
in patients on much smaller dosage levels; these are probably related to
fatty substances present in tocopherol concentrates or, in some instances,
to psychic factors.
B. MODE OF ADMINISTRATION
The natural and synthetic forms of a-tocopherol and their acetate esters
are viscous oils. Intramuscular injections, frequently used in clinical prac-
tice, have sometimes led to painful reactions locally and to oleogranulomas
at a later date;’ ‘‘solubilized’’ preparations may be less reactive in these
respects. Tocopherol ointments have been used to only a limited extent.
In animals, implanted pellets of the crystalline esters (palmitate, succinate,
and phosphate) produce marked local tissue reactions;* so also does injec-
tion of the slightly water-soluble phosphate ester. Neither in animals nor
in man are there reliable data concerning the relative effectiveness of ab-
sorption and utilization of tocopherols administered in these various ways,
as compared to oral dosage which appears to be the most effective mode
of administration.
C. METABOLIC STRESS IN ANIMALS
There is a considerable body of evidence that a-tocopherol has a re-
markable capacity to protect experimental animals against a variety of
metabolic stresses, including those induced by anoxia, high intake of un-
saturated fats, low protein intake, restricted intake of other vitamins (A,
2V. Demole, Intern. Z. Vitaminforsch. 8, 338 (1939).
3C. L. Steinberg, Ann. N. Y. Acad. Sci. 52, 380 (1949).
4C. E. Tobin, Proc. Soc. Exptl. Biol. Med. 73, 475 (1950).
564 THE TOCOPHEROLS
Be, and essential fatty acids), and the toxic or otherwise detrimental effects
of such substances as alloxan, silver nitrate, o-cresyl phosphate and carbon
tetrachloride. In many instances, additional tocopherol means the difference
between death of the animal or continued survival in good health. Pre-
sumably these beneficial effects relate to the antioxidant functions of to-
copherol, operating sometimes in the intestinal tract and at other times at
sites where local tissue injury would otherwise occur. Furthermore, tocoph-
erol can enhance the curative action of critical amounts of vitamin A
and essentially fatty acids, possibly by protecting them up to the point
of intestinal absorption.
D. THERAPEUTIC USE
There exists an extensive but rather controversial literature dealing with
the therapeutic efficacy of tocopherol in a wide variety of disease states,
many of which have little or nothing in common with experimentally in-
duced manifestations of vitamin E deficiency and are not associated with
any evidence of a significant inadequacy of tocopherol in the patient. Fur-
thermore, the effects reported are usually obtained only with relatively
high doses over a period of many weeks or months. As expressed by Hick-
man? in an interesting review of this subject, ‘“The discrepancy between
the few milligrams a day that suffice to maintain the majority of people
in health and the hundreds of milligrams being used clinically gives cause
for serious thought. ... Only time and continued study can resolve the
dosage paradox with this vitamin .. ..”’
Those who report benefit in certain clinical disorders usually stress the
importance of sustained, high daily dosage, generally amounting to between
200 and 600 mg. of a-tocopherol; this represents about twelve to thirty-six
times the average daily intake from diet. There is also the common obser-
vation that only in a certain proportion of patients suffering from a spe-
cified disorder is there a significant remission of symptoms, the remainder
showing no benefit other than perhaps an improved sense of well-being and
physical vigor. Observations such as these suggest that in certain instances
high tocopherol dosage may, through its capacity to regulate and enhance
intracellular oxidations, greatly improve states of lowered or otherwise
altered metabolism at various localized sites in tissues and organs of the
body. The location of the latter, and their relation to the etiology and se-
quelae of the disease entity under consideration would, of course, vary
widely from patient to patient. In other words, as in its protective effect
in enabling the experimental animal to resist and overcome the effects of a
variety of metabolic stresses, the clinical value of tocopherol may he in
large part in its ability to counteract localized metabolic and toxic stresses
> K.C. D. Hickman, Record Chem. Progr. 9, 104 (1948).
VIII. PHARMACOLOGY 565
which may be either primary or secondary to, or quite independent of, a
primary disease process.
lL. Heart DISEASE
Vogelsang, Shute, and Shute®!? report that intensive tocopherol therapy
usually ameliorates or abolishes the clinical signs and symptoms (anginal
pain, exertional dyspnoea, fatiguability, lowered exercise tolerance) of coro-
nary, rheumatic, and hypertensive heart disease, and also hastens the reso-
lution of coronary thrombi and the recovery of damaged cardiac tissue.
These effects are attributed to a pharmacologic action of a-tocopherol in
decreasing the oxygen requirement of cardiac muscle, in helping to resolve
and also in preventing intravascular thrombi, and in improving the func-
tional state of the capillary bed. Although, as they point out, there is con-
siderable experimental evidence to support such postulations, there are
also certain etiologic and other differences between cardiac disease in man
and the experimentally induced lesions of vitamin E deficiency. Clinical
reports!!® which might be considered as supporting these claims record a
total of 109 cases of heart disease, of which 40 were regarded as being bene-
fited to a variable degree by tocopherol therapy. On the other hand, another
series of clinical reports,!”-’ representing observations on a total of 252
6° A.B: Vogelsang, E. V. Shute, and W. E. Shute, Med. Record 160, 21, 163, 279 (1947);
161, 83, 155 (1948).
7W. E. Shute, E. V. Shute, and A. B. Vogelsang, Med. Record 160, 91, 230 (1947);
Ann. Internal Med. 30, 1004 (1949).
8 W. E. Shute, Swmmary 1, 13 (1949); 3, 19 (1951).
9W. E. Shute and E. V. Shute, Swmmary 2, 3 (1950).
10 A. L. Pascoe and W. E. Shute, Summary 1, 50 (1949).
1M. B. Molotchick, Med. Record 160, 11 (1947).
2 J,. Pin, Contribution to the Study of the Physiological and Therapeutic Properties
of Vitamin E. Maurice Lavergne, Paris, 1947.
18K. P. Ball, Lancet, I, 116 (1948).
4 C.N. J. Gram and V. Schmidt, Nord. Med. 37, 82 (1948).
19 N. Agadjanian, J. Méd. Paris 68, 29 (1948); Swmmary 3, 50 (1951).
16 J. Dedichen, Nord. Med. 41, 324 (1949).
17 S$. Baer, W. I. Heine, and D. B. Gelfond, Am. J. Med. Sci. 215, 542 (1948); Ann.
N.Y. Acad. Sci. 52, 412 (1949).
18 H. Levy and E. P. Boas, Ann. Internal Med. 28, 1117 (1948).
‘9D. H. Makinson, 8. Oleesky, and R. V. Stone, Lancet I, 102 (1948).
20 G. L. Baum and W. Stein, Wisconsin Med. J. 48, 315 (1949).
21 C. K. Donegan, A. L. Messer, E. 8. Orgain, and J. M. Ruffin, Am. J. Med. Sci.
217, 294 (1949).
22 H. Eichert, Southern Med. J. 42, 717 (1949).
*3 J. Travell, S. H. Rinzler, H. Bakst, Z. H. Benjamin, and A. L. Bobb, Ann. N.Y.
Acad Sci. 52, 345 (1949).
4H. P. Rush, Calif. Med. 71, 381 (1949).
257.8. Ravin and K. H. Katz, New Engl. J. Med. 240, 331 (1949).
566 THE TOCOPHEROLS
patients, record no recognizable benefit resulting from this therapeutic
procedure. Many variables, some controllable and others not, enter into
such clinical studies and markedly influence the final conclusions, which
often fall far short of a critical evaluation of patient response in terms of
what the result might have been in the absence of tocopherol therapy. Until
many of these variables are resolved and more reliable criteria of responses
established, the question of the merits of tocopherol therapy in heart dis-
eases must be considered an unsettled one.
2. PERIPHERAL VASCULAR DISEASE
Rather remarkable effects of high dosage of a-tocopherol in the manage-
ment of vascular disorders such as indolent ulcers, early grangrene of the
extremities, thromboangiitis obliterans, thrombophlebitis, phleobothrom-
bosis and cerebral thrombosis, and also burns, have been reported by Shute
et al.;°: 5-*4 these effects are attributed to antithrombic, thrombocytolytic,
and capillary-vasodilative functions of tocopherol. Others report beneficial
effects in cases of thromboangiitis obliterans,*°: °° leg ulcers,*’*? and phlebi-
tis,4° but contrary views are also expressed.‘!“** Increased peroxide content
of subcutaneous tissues in cases of vascular disease of the extremities, sug-
gestive of a local deficiency of vitamin E,‘° and the reported effects of to-
copherol on experimentally induced femoral thrombi‘* and in prevention
of arterial lesions” in dogs, are of interest in this connection.
26M. E. Eisen and H. Gross, N. Y. State J. Med. 49, 2422 (1949).
27 H. Berger, N. Y. State J. Med. 50, 441 (1950).
8 . V. Shute, A. B. Vogelsang, F. R. Skelton, and W. E. Shute, Surg. Gynecol.
Obstet. 86, 1 (1948); Seminar 1, 3 (1949).
29 A. B. Vogelsang, E. V. Shute, and W. E. Shute, Med. Record 161, 83 (1948).
30}. V. Shute, Ann. N. Y. Acad. Sci. 52, 358 (1949).
31 W. KE. Shute and E. V. Shute, Seminar 1, 47 (1949).
3 KH. V. Shute, J. Obstet. Gynaecol. Brit. Empire 58, 843 (1951).
33 H. V. Shute and W. E. Shute, Am. J. Surg. 84, 187 (1952).
34 W. R. Cameron, Seminar 8, 9 (1951).
35 A. H. Ratcliffe, Lancet 257, 1128 (1949).
36 A.M. Boyd, A. H. Ratcliffe, R. P. Jepson, and G. W. H. James, J. Bone and Joint
Surg. 31B, 325 (1949).
37 C. Stritzler, Ann. N. Y. Acad. Sci. 52, 368 (1949).
88 H. Siedenstopf and A. Kruger, Med. Klin. 44, 1060 (1949).
89 A. Bijdendijk and F. J. Noordhoek, Nederl. Tijdschr. Geneesk. 95, 1039 (1951).
40 R. Castagna and G. Impallomeni, Bol. Soc. Piemont Chir. 18, 155 (1948).
410. L. Pennock, Ann. N. Y. Acad. Sci. 52, 413 (1949).
“TL. L. Pennock and A. M. Minno, Angiology 1, 337 (1950).
43, A. Wegner, Dermatol. Wochschr. 128, 385 (1951).
‘4 H. [. Lippmann, in discussion of paper by E. V. Shute, ref. 30.
4 K. E. Jessen, J. Glavind, 8. Hartmann, and H. Dam, Acta. Pathol. Microbiol.
Scand. 23, 73 (1951).
46G. Enria and R. Ferrero, Arch. Sci. Med. 91, 23 (1951).
47 R. L. Holman, Southern Med. J. 42, 108 (1949).
OE
VIII. PHARMACOLOGY 567
A practical application of the observation that a-tocopheryl phosphate
has antithrombic activity has not been established. Kay,*® in reviewing
an extensive experience, states: ‘It is conceivable, though not yet proven
statistically, that the administration of alpha tocopherol and calcium will
reduce the incidence of postoperative phlebothrombosis and pulmonary
embolism.”’ This question has been carefully reviewed elsewhere,*°-®? with
essentially the same conclusions. The reported effect of tocopherol in coun-
teracting stilbestrol-induced purpura in dogs** has not been confirmed ;*4
its reputed antipurpuric action in thrombocytopenic purpura in man®®:*6
warrants further study.
3. MENOPAUSAL SYNDROME
a-Tocopherol, at dosage levels considerably lower than those employed
in other clinical disorders, is reported to relieve symptoms of the menopause
and to be especially useful where estrogens are contraindicated.*’-*” Perloff ®
reports that results with tocopherol compare very favorably with those
obtained with hormone therapy, which seems contradictory to the postula-
tion® that vitamin E is antiestrogenic. On the other hand, the more recent
report of Blatt et al.,®°° based on a careful comparison of the effects of estro-
gens, phenobarbital, tocopherol, and placebos on 748 climacteric patients,
concludes that a-tocopherol is no more effective than placebo therapy in
controlling menopausal symptoms.
48K. L. Zierler, D. Grob, and J. L. Lilienthal, Am. J. Physiol. 153, 127 (1948).
49 J. H. Kay, Yale J. Biol. Med. 23, 515 (1951).
50 Nutrition Revs. 10, 46 (1952).
51 W. H. Seegers, in Trans. 4th Conf. on Blood Clotting and Allied Problems, New York,
p. 157 (1951).
827.8. Wright, Circulation 5, 161 (1952).
53 F. R. Skelton, E. V. Shute, H. G. Skinner, and R. A. Waud, Science 103, 762 (1946).
5¢ A. J. Richtsmeier, M. Spooner, and O. O. Meyer, Proc. Soc. Exptl. Biol. Med. 65,
298 (1947).
55 EB. V. Shute, Urol. and Cutaneous Rev. 50, 732 (1946).
56 A. B. Vogelsang, Med. World (London) 64, 448 (1946).
67 A. M. Hain and J. C. B. Sym, Brit. Med. J. II, 8 (1943).
6 EK. V. Shute,. Brit. Med. J. II, 526 (1943).
59C. J. Christy, Am. J. Obstet. Gynecol. 50, 84 (1945).
69H. E. Ferguson, Virginia Med. Monthly 75, 447 (1948).
*! B. B. Rubenstein, Federation Proc. 7, 106 (1948).
62 H. C. McLaren, Brit. Med. J. II, 1378 (1949).
§§R.S. Finkler, J. Clin. Endocrinol. 9, 89 (1949).
64 W. H. Perloff, Am. J. Obstet. Gynecol. 58, 684 (1949).
65 N. R. Kavinoky, Ann. Western Med. Surg. 4, 27 (1950).
66S. H. Sikkema, Rocky Mount. Med. J. 48, 550 (1951).
67H. A. Gazan, N. Y. State J. Med. 52, 1289 (1952).
8. V. Shute, Am. J. Obstet. Gynecol. 35, 970 (1938); J. Endocrinol. 2, 173 (1940).
6° M.G. H. Blatt, H. Wiesbader, and H. 8. Kupperman, Arch. Inlernal Med. 91, 792
(1953).
568 THE TOCOPHEROLS
4. Primary FIBROSITIS AND RELATED DISORDERS
Under this heading are included conditions generally regarded as meta-
bolic disorders of the connective tissues and represented by Dupuytren’s
contracture, Peyronie’s disease, and generalized involvement of muscles
such as seen in myositis, fibromyositis, muscular rheumatism, lumbago,
and bursitis. The reported beneficial effects of tocopherol therapy have been
ascribed to effects upon the vascular bed or upon altered connective tissues,
perhaps through correction of some localized metabolic disturbances in the
involved tissues.
Steinberg’s observations’? that Dupuytren’s contracture frequently re-
sponds favorably to high tocopherol dosage have been extended by him?*: 7!
and confirmed by certain investigators”: ”* but not by others.“ 7 It is
noteworthy that in the two most recent studies, where tocopherol was the
sole form of treatment and plaster casts were used to measure changes in
flexion deformity, definite though moderate improvement was observed
in 23 of 26 affected hands in one study” and no improvement noted in 58
hands in the other.”°
Peyronie’s disease (fibrous infiltration of the intercavernous septum of
the penis), which is not uncommonly associated with Dupuytren’s con-
tracture,’®-79 is said to respond favorably to tocopherol therapy,” 7%? al-
though negative or negligible responses are not infrequent. Similar results
have been reported in the treatment of urethral stricture®® and interstital
cystitis.8°° No contrary findings have as yet been reported.
There is also the interesting observation of Edgerton et al. *! that tocoph-
erol frequently relieves the pain associated with keloids when other meas-
ures have failed but does not prevent the development of these areas of
dense scar tissue. This is attributed to softening of the scar tissue, perhaps
through correction of underlying metabolic or vascular disturbances. This
70 C. L. Steinberg, Med. Clin. N. Amer. 30, 221 (1946).
1 C. L. Steinberg, Arch. Surg. 68, 824 (1951).
72 G. R. Thomson, Brit. Med. J. II, 1382 (1949); Glasgow Med. J. 30, 329 (1949).
73 J. EK. Kirk and M. Chieffi, Proc. Soc. Exptl. Biol. Med. 80, 565 (1952).
74 R. A. King, J. Bone and Joint Surg. 318, 443 (1949).
75H. J. Richards, Brit. Med. J. I, 1828 (1952).
76'T. Katz-Galatzi, Acta Med. Orient. 8, 193 (1949); cited by W. W. Scott, Yearb.
Urol., p. 320 (1950).
7 J. I. Waller and W. C. Dreese, J. Urol. 68, 623 (1952).
78 W.W. Scott and P. L. Seardino, Southern Med. J. 41, 173 (1948).
79 P_ L. Seardino and W. W. Scott, Ann. N. Y. Acad. Sci. 52, 390 (1949).
80 P. L. Seardino and P. B. Hudson, Ann. N. Y. Acad. Sci. 52, 425 (1949).
8020 R. H. Van Duzen and R. Mustain, J. Urol. 65, 1033 (1951).
81 M. T. Edgerton, Jr., E. M. Hanrahan, and W. B. Davis, Plastic Reconstr. Surg. 8,
224 (1951).
ail,
VIII. PHARMACOLOGY 569
general hypothesis has also been offered as an explanation of the favorable
response observed in other types of primary fibrositis.
Myositis and other disorders usually included in the category of gen-
eralized primary fibrositis have been reported to be benefited by tocopherols
whether administered orally: *’ or in the form of an ointment.**: *° If
other clinicians have had a different experience, they appear not to have
reported it.
Burgess and Pritchard*® are of the opinion that tocopherol, through some
unexplained action upon the connective tissues, exerts beneficial effects in
a variety of collagenoses such as chronic discoid lupus erythematosus, scle-
roderma, and granulomatous ulcers. Although others have reported favor-
able results in granuloma inguinale’ and in lupus erythematosus (when
combined with pantothenic acid therapy*’), still others have recorded nega-
tive or questionable results.’°- Only further research can clarify this con-
troversial question.
5. DiaBrETES MELLITUS
a-Tocopherol administration is said to considerably reduce the insulin
requirements of diabetics,**: “* the effect being attributed to improved glyco-
gen storage in muscle cells rather than to potentiation of insulin action.
On the other hand, other investigators,°°-*’ who have studied this question
with particular care, have reported only negative results.
82 C. L. Steinberg, Am. J. Med. Sci. 201, 347 (1941); N. Y. State Med J. 42, 773 (1942) ;
J. Bone and Joint Surg. 24, 411 (1942); Ann. Internal Med. 19, 136 (1943).
83 C, J. Sutro and L. J. Cohen, Arch. Surg. 42, 1065 (1941).
84 M. Ant, NV. Y. State J. Med. 45, 1861 (1945) ; Industrial Med. 15, 399 (1946) ; Rhewma-
tism 6, 114 (1950).
85 M. M. Kessler, J. Med. Soc. New Jersey 48, 164 (1951).
86 J. F. Burgess and J. E. Pritchard, Lancet II, 215 (1948); Arch. Dermatol. and
Syphilol. 57, 605, 953 (1948); Can. Med. Assoc. J. 59, 242 (1948).
87 T. Cochrane, Brit. J. Dermatol. Syphilol. 62, 316 (1950).
88 A. Welsh, Arch. Dermatol. and Syphilol. 65, 137 (1952).
89 R. D. Sweet, Lancet II, 310 (1948).
90 J. Morgan, Brit. J. Dermatol. Syphilol. 63, 224 (1951).
% H. H. Sawicky, Arch. Dermatol. and Syphilol. 61, 906 (1950).
2 F. Pascher, H. H. Sawicky, M. G. Silverberg, M. Braitman, and N. B. Kanof,
J. Invest. Dermatol. 17, 261 (1951).
% U. Butturini, Giorn. clin. med. (Parma) 26, 90 (1945); Ann. N. Y. Acad. Sci. 52,
397 (1949).
% A. Vogelsang, Med. Record 161, 363 (1948); J. Clin. Endocrinol. 8, 883 (1948); Ann.
N.Y. Acad. Sci. 52, 406 (1949).
9. H. Bensely, A. F. Fowler, M. V. Creaghan, F. Zahalan, F. Lax, H. Berish, and
E. K. MeDonald, Can. Med. Assoc. J. 61, 260 (1949).
96° G. M. Guest, Ann. N. Y. Acad. Sci. 62, 411 (1949).
7 H. Pollack, K. E. Osserman, J. J. Bookman, M. Ellenberg, and J. Herzstein, Am.
J. Med. Sci. 219, 657 (1950).
nr
~I
(=)
THE TOCOPHEROLS
6. GENERAL COMMENTS
Without reference to numerous other clinical reports, many of them un-
substantiated or unchallenged, dealing with beneficial effects of tocopherol
in a variety of other human ills, it will be apparent that the status of a-to-
copherol as a chemotherapeutic agent rests on an extensive but decidedly
controversial literature. Out of the array of conflicting evidence, there
seem to emerge indications that in certain disease processes there may occur
more or less local metabolic disturbances which may lead to increased
needs for, or even to local destruction of, tissue tocopherol, especially in-
volving the connective tissues and the related capillary bed; conceivably,
such local disturbances which may not be compensated for by normal body
stores of tocopherol can be overcome by high and sustained tocopherol
therapy. Whether this represents a glimmer of a pharmacologic action of
tocopherol, and whether functions of a-tocopherol other than its generally
accepted role as an intracellular antioxidant would be necessary to explain
such effects, remain for future research to decide.
IX. Requirements and Factors Influencing Them
HENRY A. MATTILL
A. OF ANIMALS
From what has been said, it is obvious that no categorical statements
can be made as to the requirements of different species or for the prevention
of a particular nutritional impairment in any of them. Even with a highly
purified basal diet, the results are subject to considerable biological varia-
tion, and with natural foodstuffs the minute variations implied in the ex-
perimental work described in the preceding pages can produce profound
effects, depending on the nature and history of each component. A diet
high in fat, particularly unsaturated fat, appears to increase the need for
vitamin E.!* Any statement regarding the MFD (mean fertility dose)
(0.75 mg. of a-tocopherol) for the rat holds only for the specific conditions
of the test. The daily needs of the two sexes are about equal, despite the
fact that the male requirement begins early (40th to 50th day of life),
whereas that of the female arises only after conception.® The minimal daily
1H. Gottlieb, F. W. Quackenbush, and H. Steenbock, J. Nutrition 25, 433 (1943).
2G. A. Emerson and H. M. Evans, J. Nutrition 27, 469 (1944).
3H. Dam, H. Granados, and I. Prange, Acta Physiol. Scand. 18, 161 (1949).
4H. M. Bruce, J. Hyg. 48, 171 (1950).
5K. E. Mason, Am. J. Physiol. 131, 268 (1940).
IX. REQUIREMENTS AND FACTORS INFLUENCING THEM 571
requirement is 45 to 49 of the MFD for adult female rats. The minimum
amounts for prevention of muscle dystrophy in nursling rats from E-low
mothers depends on the time when supplementation begins; the longer this
is delayed, as between the 10th and 17th day, the more is required.® There
are no essential differences between the antisterility and dystrophy-pre-
venting potencies.’ As the daily dosage is reduced below 0.75 mg., the
length of the reproductive period is reduced.’ The gradual decline in pro-
ductivity of rat colonies may often be traced to the inadequacy of the vita-
min E supplies in commercial feeds. Such premature sterility appears to
have no effect on total span of life.’ For the rabbit the daily amount of a-
tocopherol to prevent and cure dystrophy has been given as 0.2 to 0.4 mg.
per kilogram;!° the minimum need of the laying hen is 1.2 mg. per day"!
and of the guinea pig 3 mg.”
Like many other functions, that of vitamin E might be quantitatively
related to the 0.7 power of the body weight. On this basis the daily require-
ment of an adult person of 70 kg. would be perhaps 30 mg. According to
surveys in Holland" and in the United States the average individual in-
take of a-tocopherol is 6 mg. per 1000 cal. or less,!® but the tocopherols of
vegetables are much less available than those in oils,” and they may not
all be the a type.
Few, if any, sufficiently controlled experiments have been made to es-
tablish the requirements of domestic animals that are known to need vita-
min E. Their natural food ordinarily supplies it in abundance, and the
preparation of a ration devoid of it, as by treatment with ferric chloride,
introduces other variables and may also fail of its purpose.
The moot question has been whether domestic animals on their natural
feeds require supplements of vegetable oils or of tocopherol in order to yield
the best returns in rate of growth and in reproduction. Sterility in cows
was reported to have been successfully treated with wheat germ oil,!® the
6H. M. Evans and G. A. Emerson, Proc. Soc. Exptl. Biol. Med. 44, 636 (1940).
7M. Goettsch and A. M. Pappenheimer, J. Nutrition 22, 463 (1941).
8 H. M. Evans and G. A. Emerson, J. Nutrition 26, 555 (1943).
°C. M. McCay, G. Sperling, and L. L. Barnes, Arch. Biochem. 2, 469 (1943).
10S. H. Eppstein and S. Morgulis, J. Nutrition 22, 415 (1941).
11M. Y. Dju, M. L. Quaife, and P. L. Harris, Am. J. Physiol. 160, 259 (1950).
122 F. A, Farmer, B. C. Mutch, J. M. Bell, L. D. Woolsey, and E. W. Crampton, J/.
Nutrition 42, 309 (1950).
13 P, L. Harris, Ann. N. Y. Acad. Sci. 52, 240 (1949).
14C, Engel, Ann. N. Y. Acad. Sct. 52, 292 (1949).
15 7,. E. Booher and I. T. Behan, J. Nutrition 39, 495 (1949).
16P. L. Harris, M. L. Quaife, and W. J. Swanson, J. Nutrition 40, 367 (1950).
17 ©, Engel and J. T. Heins, Acta Brevia Neerl. Physiol. Pharmacol. Microbiol. 18,
37 (1943).
18 P. Vogt-Mgller and F. Bay, Vet. J. 87, 165 (1931).
572 THE TOCOPHEROLS
assumption being that for some unknown reason a deficiency may have
existed, notwithstanding dietary abundance. There are numerous and more
recent statements both in support and in denial of this contention as ap-
plied to all farm animals. The problem is complicated by the fact that their
need of vitamin I) for reproduction has not been clearly demonstrated ;!9
the young, through lack of enough E in their milk, suffer degeneration of
voluntary or cardiac muscle, from which they may or may not recover if
untreated. Tocopherol given to the young effects a cure. The dietary in-
adequacy in the dams can be due to the lack of green feed, the use of re-
stricted rations, degerminated cereals, or sophisticated concentrates. There
is increasing evidence that the responses of animals to tocopherol differ,
on the farm as well as in the laboratory.?°: 7!
Many more experiments, carefully controlled and with guarded conclu-
sions, like those reported on swine,” ?* must be performed before the re-
quirements of domestic animals are known.
B. OF MAN
KARL E. MASON
Requirements of tocopherol for man have not been satisfactorily estab-
lished and can be inferred in only an approximate manner on the basis of
what is known concerning dietary intake, effectiveness of absorption, rate
of utilization, extent of excretion, and tissue storage. An interesting dis-
cussion of the various factors operating between nature’s deposition of to-
copherol in plant foods and the subsequent transfer of tocopherol across the
intestinal barrier in man has been presented by Hickman and Harris.”
It is reported that part of the dietary tocopherol is oxidized to quinones in
the intestine.?° Others,?® who feel that there is very little destruction in the
intestine, indicate that perhaps more than half of ingested tocopherol may
be excreted in the feces. The measurement of fecal tocopherols is a difficult
procedure because of the presence of many interfering substances. Progress
has been made,” 7-6 and it is hoped that improved methods will make pos-
sible more informative tocopherol-balance studies.
19'T. W. Gullickson, Ann. N. Y. Acad. Sci. 52, 256 (1949).
20 J. K. Loosli, Ann. N. Y. Acad. Sci. 52, 2438 (1949).
211). B. Parrish, Ann. N. Y. Acad. Sci. 52, 251 (1949).
22 T,. EH. Carpenter and W. O. Lundberg, Ann. N. Y. Acad. Sci. 52, 269 (1949).
23 F. B. Adamstone, J. L. Krider, and M. F. James, Ann. N. Y. Acad. Sct. 52, 260
(1949).
24K. C. D. Hickman and P. L. Harris, Advances in Enzymol. 6, 469 (1946).
25 H.-Rosenkrantz, A. T. Milhorat, and M. Farber, J. Biol. Chem. 192, 9 (1951).
26 G. Klatskin and D. W. Molander, J. Clin. Invest. 31, 159 (1952); J. Lab. Clin. Med.
39, 802 (1952).
IX. REQUIREMENTS AND FACTORS INFLUENCING THEM 573
As previously stated (p. 543), the average daily intake for adult man
appears to be about 14 to 19 mg. of d-a-tocopherol. On the basis of tocoph-
erol analyses of foods it has been estimated”? that on diets considered
low, average, and high in vitamin E there would be provided, respectively,
1, 4, and 10 mg. of a-tocopherol for infants; 4, 10, and 20 mg. for children;
5, 15, and 35 mg. for adults; and 3, 10, and 15 mg. for aged persons. When
consideration is given to losses due to storage, cooking, inactivation in the
gut, and fecal excretion, the latter representing by far the major loss, the
total available for metabolic utilization and storage might be reduced by
one-third more; this would give a net absorption of approximately 5 to 7
mg. of d-a-tocopherol daily for an average adult whose daily intake amounts
to about 20 mg. per day. The extent to which requirements vary with age
is not known. One might expect that, in terms of milligrams of tocoph-
erol per kilogram of body weight, the requirements during infancy and
childhood would be greater than during adult life. This would be in
keeping with the evidence of slow acquisition of body storage stores during
childhood and adolescence.” ?° It is not clear whether lowered tissue stor-
age observed in old age’: 2° represents diminished dietary intake or in-
creased needs to compensate for the generally lowered oxidative state of
body tissues. On the experimental side, there is evidence of increased re-
quirements for reproductive function as age progresses.
The requirements for vitamins are influenced by much the same condi-
tions which necessitate increased intake of vitamin A; namely, conditions
which chronically impair the absorption of fats from the gastrointestinal
tract. It seems reasonable to assume also that those factors which have
been shown to accentuate deficiency manifestations in animals, most of
which can be considered to represent types of metabolic stress (excess in-
take of unsaturated fats, low protein intake, ingestion of chemical sub-
stances such as o-cresyl phosphate or silver nitrate), can be regarded as
potentially capable of increasing human requirements as well.
27K. C. D. Hickman, Record Chem. Prog. 9, 104 (1948).
28 K. E. Mason, M. Y. Dju, and L. J. Filer, Jr., Federation Proc. 11, 449 (1952).
, 79 K. E. Mason and M. Y. Dju, Nutrition Symp. Ser., National Vitamin Foundation,
New York 7, 1 (1953).
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CHAPTER 18
NEW AND UNIDENTIFIED GROWTH FACTORS.
VERNON H. CHELDELIN
Page
I. Lipoie Acid (Thioetiec Acid, Pyruvate Oxidation Factor, Protogen, Ace- :
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The body of information that has formed the basis for the preceding
chapters has enabled us to obtain a clear outline of the chemistry and func-
tion of several of the vitamins that have been discussed. Although the
pioneer work on vitamins was done only half a century ago, the intensity
of research, particularly during the past two decades, has so overcome the
time disadvantage that several vitamin systems are now as well or better
understood than many of the more classical compounds of biochemical im-
portance.
The purpose of the present discussion is mainly to provide the reader with
an up-to-date account of the newly suspected and recognized members of
the biocatalyst family—those which have been reported but have not yet
been sufficiently developed to merit separate consideration. However, in
view of the speed with which problems in this field are being carried forward,
the members of the ‘‘new factors” group must obviously be transient ones.
Compounds listed in the present article may within a few years be expected
either to emerge as fully characterized vitamins or to be dropped altogether
as duplications are discovered and the occasional artifact or combined effect
of known factors is recognized and eliminated. New members are mean-
while being added at a rapid rate.
I. Lipoic Acid (Thioctic Acid, Pyruvate Oxidation Factor,
Protogen, Acetate Replacement Factor)
This compound is selected for initial consideration because its chemistry
and some of its biological functions have become well outlined and because
its development typifies both the intensity of study and the diversity of
systems that are being employed in the search for new growth factors. Two
-—
219
=
576 NEW AND UNIDENTIFIED GROWTH FACTORS
bacterial and one protozoal growth systems and one bacterial oxidative
system were under study in the four laboratories that independently re-
ported the existence of this compound.
Priority for the discovery of lipoic acid is assigned to Guirard, Snell,
and Williams! and to Kidder and Dewey,?: * who at an earlier date noted a
combined growth effect that was later shown‘ to be due to lipoie acid, py-
ridoxal, and copper ions. Guirard et al. observed! that the stimulatory effect
of acetate upon the growth of Lactobacillus casei could be replaced by small
quantities of natural extracts such as liver, yeast, or dried grass juice. They
concentrated the active material 44-fold from yeast. The following year,
O’Kane and Gunsalus® found that an unidentified factor from yeast (later
called pyruvate oxidation factor®) was necessary in resting cells of Strep-
tococcus faecalis, for oxidation of pyruvate, or its anaerobic dismutation to
lactate, acetate, and carbon dioxide. Preparations were obtained that were
approximately 200 times as potent as the starting material (yeast extract).
Independently of these investigations, Stokstad et al.4 fractionated the natu-
ral supplements described by Kidder and Dewey?’ * for the nutrition of
Tetrahymena geleii and found that the necessary unknowns could be reduced
to two. Both of these unknowns produced the same biological response, and
it was concluded that they were two forms of the same compound. They
named the substance ‘‘protogen”’ because of its effect upon the protozéon.
Later,’ these workers found that a strain of Corynebacterium also required
protogen for growth. Finally, Kline and Barker® described what appears
to have been lipoic acid as a growth factor for Butyribacterium rettgert.®
They concentrated the factor approximately 200-fold from yeast and dem-
onstrated the existence of at least three forms.
The probable identity of protogen, the acetate replacing factor, and the
pyruvate oxidation factor was reported by Snell and Broquist,!° who showed
a high degree of correlation to exist among the relative potencies of different
concentrates for acetate replacement, pyruvate oxidation, and Tetrahymena
growth. The compound had been found to be fat-soluble, and because of
this and its adsorption characteristics it was also thought to be similar to
1B.M. Guirard, E. E. Snell, and R. J. Williams, Arch. Biochem. 9, 381 (1946).
2V. C. Dewey, Biol. Bull. 87, 107 (1944).
3G. W. Kidder and V. C. Dewey, Arch. Biochem. 8, 293 (1945).
4.1L. R. Stokstad, C. E. Hoffmann, M. A. Regan, D. Fordham, and T. H. Jukes,
Arch. Biochem. 20, 75 (1949).
5D. J. O’Kane and I. C. Gunsalus, J. Bacteriol. 54, 20 (1947).
6D. J. O’Kane and I. C. Gunsalus, J. Bacteriol. 56, 499 (1948).
7E. L. R. Stokstad, C. E. Hoffmann, and M. Belt, Proc. Soc. Exptl. Biol. Med. 74,
571 (1950).
8]. Kline and H. A. Barker, J. Bacteriol. 60, 349 (1950).
9 J. C. Gunsalus, L. Struglia, and D. J. O’Kane, J. Biol. Chem. 194, 859 (1952).
10. KE. Snell and H. P. Broquist, Arch. Biochem. 28, 326 (1949).
I. LIPOIC ACID 577
vitamin By; ;§: 1!» ° however, the latter identity has not been confirmed ex-
perimentally.!2"
The vitamin was first isolated in pure form by Reed, Gunsalus, ef al.;"°
the term ‘“‘lipoic acid’’ was also introduced at this point to describe the
(fat-soluble) compound as it was obtained from liver after hydrolysis, pos-
sibly from ester or amide combination. The isolation procedure from yeast™
disclosed the existence of five chromatographically distinct forms of the
factor. These have been characterized by Gunsalus ef al.° as: (1) a bound
form, (2) a water-soluble, fat- solvent-insoluble form, referred to as ‘“‘water-
soluble complex,’ and three fat-soluble varieties referred to as (8) a weak
acid, (4) a strong acid, and (5) a neutral substance. The first form was
found very widely distributed; the second was observed principally after
enzyme digestion. The three fat-soluble compounds remained after hydroly-
sis with acid or alkali. However, Reed ef al.'*: 1° reported only two forms
after hydrolysis. This may possibly have been due to production of some
forms during operational treatments, or to conversion of natural conjugates
(e.g., esters or amides) to the two principal compounds remaining. The
two forms, when bioautographed on paper with water-saturated butanol,
exhibited Ry values of 0.90 and 0.60. The Ry 0.60 form was largely con-
verted to the Ry 0.90 variety (a more polar substance). A similar intercon-
version was reported by the Lederle group in their isolation of protogen;'®
they observed a marked increase in water solubility and designated the
process protogen A — protogen B.
The potency of pure lipoic acid (the Ry 0.60 form, called a-lipoic acid)"
was found to be 250,000 times that of yeast, based on the stimulation pro-
vided in the S. faecalis test for pyruvate oxidation. As a replacing agent for
acetate in L. casez, lipoic acid was 15,000,000 times as potent; half-maxi-
mum growth was provided by 1.7 X 10~® y per milliliter of culture medium.
This compound thus ranks with biotin, vitamin B,., and the folic acid
group as an extremely active biocatalyst.
Studies on the structure of the lipoic acids have been both intensive and
numerous. The picture of the interrelationships among the various forms
has rapidly emerged, although the important details are not yet all worked
1. E. Snell and L. D. Wright, Ann. Rev. Biochem. 19, 307 (1950).
122, V. H. Cheldelin, Nutrition Revs. 9, 289 (1951).
128 Since this manuscript was prepared, a report has been issued indicating a relation-
ship between vitamin B,; and orotic acid [L. Manna and 8. M. Hauge, J/. Biol.
Chem. 202, 91 (1953)].
13 T,. J. Reed, B. G. De Busk, I. C. Gunsalus, and C. 8. Hornberger, Jr., Science 114,
93 (1951).
4 7,. J. Reed, B. G. De Busk, P. M. Johnston, and M. E. Getzendaner, J. Biol. Chem.
192, 851 (1951).
15 T,. J. Reed, M. E. Getzendaner, B. G. De Busk, and P. M. Johnston, J. Biol.
Chem. 192, 859 (1951).
578 NEW AND UNIDENTIFIED GROWTH FACTORS
out. Reports during the past year have been issued from fhe Lederle Labor-
atories'®!9 and jointly from the universities of Texas and Illinois and the
Eli Lilly Laboratories.?°*> These all point tothe lipoic acids as a family of di-
thiodctanoie acids and their derivatives. The first synthesis of a pure com-
pound with lipoic acid activity was carried out by the Lederle group, who
prepared the cyclic 6,8-dithiooctanoic acid, which they called 6-thioctic
acid.!® This was accomplished by condensing ethyl adipyl chloride with
ethylene to yield A’ ,6-ketooctenoate, and converting the latter in consecu-
tive steps to 8-thiol-6-hydroxyoctanoic acid (with thioacetic acid), 6,8-
dithioloctanoie acid (thiourea in HI), and finally the cyclic disulfide (I. in
KI). The product was confirmed as being identical with protogen A, after
earlier work had suggested the 5,8 ring structure for the factor.!8 Syn-
thetic pu-6-thioctic acid appeared from the tabular data presented!® to be
roughly half as active in pyruvate oxidation as the isolated lipoic acid of
Reed et al. Protogen B(G-lipoic acid) may be presumed to be the corre-
sponding (mono) sulfoxide.'®: !8» 2° This structure appears consistent with
the observed transformations (protogen A — protogen B) during isolation,
and with the properties of the two compounds.
The need for lipoic acid for the oxidation or dismutation of pyruvate
places this catalyst in the same particular area of metabolism as diphos-
phothiamine (DPT) and coenzyme A (CoA). The precise enzymatic role of
lipoic acid has not yet been determined, although important indications
were provided by the findings?* 9 that ‘“lipothiamide” (LT) and ‘‘lipothia-
16H}. L. Patterson, J. A. Brockman, Jr., F. P. Day, J. V. Pierce, M. E. Macchi, C. E.
Hoffmann, C. T. O. Fong, E. L. R. Stokstad, and T. H. Jukes, J. Am. Chem. Soc.
73, 5919 (1951).
17 J, A. Brockman, Jr., E. L. R. Stokstad, E. L. Patterson, J. V. Pierce, M. Macchi,
and F. P. Day, J. Am. Chem. Soc. 74, 1868 (1952).
1 M.W. Bullock, J. A. Brockman, Jr., E. L. Patterson, J. V. Pierce, and E. L. R.
Stokstad, J. Am. Chem. Soc. 74, 1868 (1952).
19M. W. Bullock, J. A. Brockman, Jr., E. L. Patterson, J. V. Pierce and E. L. R.
Stokstad, J. Am. Chem. Soc. 74, 3455 (1952).
20 L. J. Reed, B. G. De Busk, I. C. Gunsalus, and G. H. F. Schnakenberg, J. Am.
Chem. Soc. 73, 5920 (1951).
21C. S. Hornberger, Jr., R. F. Heitmiller, I. C. Gunsalus, G. H. F. Schnakenberg
and L. J. Reed, J. Am. Chem. Soc. 74, 2382 (1952).
21. J. Reed, Q. F. Soper, G. H. F. Schnakenberg, S. F. Kern, H. Boaz, and I. C.
Gunsalus, J. Am. Chem. Soc. 74, 2383 (1952).
23 T,. J. Reed, I. C. Gunsalus, G. H. F. Schnakenberg, Q. F. Soper, H. E. Boaz, 8. F.
Kern, and T. V. Parke, J. Am. Chem. Soc. 75, 1267 (1953).
24 7,. J. Reed, B. G. De Busk, C. S. Hornberger, Jr. and I. C. Gunsalus, J. Am.
Chem. Soc. 75, 1271 (1953).
25 C. 8. Hornberger, Jr., R. F. Heitmiller, I. C. Gunsalus, G. H. F. Schnakenberg,
and L. J. Reed, J. Am. Chem. Soc. 75, 1273 (1953).
°6 L. J. Reed and B. G. De Busk, J. Am. Chem. Soc. 74, 3457 (1952).
I. LIPOIC ACID 579
mide pyrophosphate” (LTPP) were active in the oxidative decarboxylation
of pyruvate. These materials were obtained in a reaction mixture of a-
lipoic acid or its acid chloride with thiamine and thiamine pyrophosphate,
respectively. The biologically active products possessed Ry values identical
with those of two of the forms present in natural materials. Both gave nega-
tive thiochrome tests and positive azo tests, and the authors suggested that
the carboxyl group of lipoic acid was conjugated with the pyrimidine amino
group of thiamine. The test system used was a mutant strain of Hscher-
ichia coli that would not respond to either vitamin, alone or mixed together,
but which grew luxuriantly on LT or LTPP. Soluble enzyme preparations
from the mutant catalyzed the anaerobic dismutation of pyruvate or the
oxidation of a-ketoglutarate only when LTPP was supplied (LT was inac-
tive in the latter system). These reactions thus reveal a definite enzymatic
role for lipoic acid in oxidation of a-keto acids, as a molecular conjugate
with thiamine, and point to LTPP as the actual coenzyme. The authors”
proposed, partly on the basis of previous knowledge of the mechanism of
pyruvate oxidation in #. coli,*°: *! that LTPP participates according to re-
action 1:
Pyruvate (a-Ketoglutarate) + LTPP + DPNt+ —
\ Acetyl LTPP (succinyl LTPP) + CO. + DPNH + H+ (1)
In the presence of coenzyme A, the acyl group could then be transferred
as follows:
Acetyl LTPP (succinyl LTPP) + CoA —
Acetyl CoA (suecinyl CoA) + LTPP (2)
This scheme thus provided a plausible order of participation for LTPP
and CoA, although it did not per se clarify the question of whether LTPP
is needed for decarboxylation or dehydrogenation. Evidence bearing on
this is provided in other studies on FZ. coli and S. faecalis,?~** as follows:
1. The complete system for the pyruvate — acetate + CO» conversion
in £. coli requires DPT, diphosphopyridinenucleotide (DPN), CoA, Mgt,
and lipoie acid (presumably as LTPP).
27 L. J. Reed and B. G. De Busk, J. Am. Chem. Soc. 74, 3964 (1952).
23 |. J. Reed and B. G. De Busk, J. Biol. Chem. 199, 873 (1952).
29 L. J. Reed and B. G. De Busk, J. Biol. Chem. 199, 881 (1952).
80S. Korkes, J. R. Stern, I. C. Gunsalus, and 8. Ochoa, Nature 166, 439 (1950).
31 §. Korkes, A. del Campillo, I. C. Gunsalus, and 8. Ochoa, J. Biol. Chem. 193, 721
(1951).
2 D—. J. O’Kane, J. Bacteriol. 60, 449 (1950).
83 M. 1.Dolin and I. C. Gunsalus, J. Bacteriol. 62, 199 (1951).
34M. I. Dolin and I. C. Gunsalus, Federation Proc. 11, 203 (1952).
35 T, C. Gunsalus, J. Cellular Comp. Physiol. Suppl. 1, 41, 113 (1953).
36 B. G. De Busk and L. J. Reed, Federation Proc. 12, 193 (1953).
580 NEW AND UNIDENTIFIED GROWTH FACTORS
2. When ferricyanide replaces oxygen as the final electron acceptor, CoA
and DPN may be by-passed, although the requirement for DPT and lipoic
acid remains.
3. When no oxidation occurs, as in acyloin formation, CoA, DPN, and
lipoic acid may all be omitted; however, DPT is still required.
From these observations it appears that in these organisms LTPP may
be involved in dehydrogenation at a very early stage in the breakdown of
pyruvate. This is in line with the report by Sanadi et al.*’ that pig heart a-
ketoglutaric oxidase contains up to 6 moles of lipoic acid and about 1 mole
of cocarboxylase per mole of enzyme, but no DPN or CoA. Schemes sug-
gesting possible sequences of reactions have been proposed,*®: **-4° which
may be summarized approximately as follows:
O —
_ DPT+, Mg (1)
CH;COCOO- ———> |_CH;—C:_}| DPTt + CO,
(acetaldehyde — DPT complex)
This complex may then be oxidized by lipoic acid, presumably in the
disulfide form. Whether this step involves the lipoic moiety for the first
time, or whether LTPP is the actual acceptor in reaction 1, is speculative.
In either case the cyclic disulfide may react to produce an acyl lipoate:
=
s-
O ace: 0 Hy
| S-—C—CHR | -~oHR
[CH;—C:]- ————> CH;—C~S—CH, | (2)
S=
Hi 0 a
|
ZL pin
CH;—C ~S—CH, CH;—C~S—CoA + H.C CHR (8)
oxalacetate OH- Jos:
H,
C
H.G7 >CHR :
Citrate Ac~PO, CH;,COOH l + DPNH
(4) (5) (6) (7)
According to this scheme, the acetyl CoA produced in reaction 3 is removed
37 —D. R. Sanadi, J. W. Littlefield, and R. M. Bock, J. Biol. Chem. 197, 851 (1952).
388 M. Calvin and P. Massini, Experientia 8, 445 (1952).
39 M. Calvin, Chem. Eng. News 31, 1735 (1953).
40 T,. J. Reed, Symposium on Metabolic Significance of Vitamins, American Insti-
tute of Nutrition, Federation Proc. 12, 558 (1953).
"aa ae
I. LIPOIC ACID 581
as “‘acetate” via such reactions as 4, 5, or 6. CoA-SH is simultaneously re-
generated for use in reaction 3, whereas the oxidized functional form of
lipoate reappears in reaction 7 for use in reaction 2. Through the use of
flavoproteins and oxygen, or lactic dehydrogenase, DPN* is made available
for re-use in reaction 7. When ferricyanide is the electron acceptor, an alde-
hyde-LTPP complex may be supposed to react according to equation 8:
Os OT
| + 2%Fe(CNo
CHs—Cr eh LRP = (CH —C + LTPPt + 2Fe (CN) .«™ (8)
OHS
CH;COOH
Alternately, an aldehyde-DPT complex may combine with a second mole-
cule of aldehyde to produce acetoin.
The foregoing reactions explain fairly well most of the observed require-
ments for lipoic acid, DPT, CoA, and DPN in the oxidation of pyruvate
(but not free acetaldehyde or ethanol*') by Z. coli. and S. faecalis. However,
the need for LTPP when oxidation proceeds, in contrast to DPT when acy-
loins are formed, is not clearly explained. It is possible that the same apoen-
zyme might bind either co-factor. This has indeed been suggested,*® and an
experimental verification of this concept would permit a simpler presenta-
tion of reactions 1 and 2 above.
These studies thus suggest that lipoic acid possesses at least three im-
portant functions in £. coli: first, as a primary (co)dehydrogenase in the
oxidation of a-keto acids; second, as an acyl transferase; and third, in the
transfer of energy through the formation of a “high-energy” S-acyl bond,
which can be transferred to CoA and utilized in various biosynthetic reac-
tions. This full complement of activities may be restricted to certain orga-
nisms, however, since it has been reported*: # that in Tetrahymena pyruvic
dehydrogenase activity was unaffected by the removal of lipoic acid from
the system; only the acylation process was impaired by this treatment. It
would appear that lipoic acid is not the primary electron acceptor from
pyruvate in this organism. A role, as yet undescribed, for lipoic acid in
animal systems is suggested by its presence in a-ketoglutaric®” and pyruvic
oxidases,** although dietary requirements for the factor have not been es-
tablished.” An active function in animal pyruvic dehydrogenase systems
is further hinted through the work of Peters et al.,*® Stocken and Thomp-
41H. D. Barner and D. J. O’Kane, J. Bacteriol. 64, 381 (1952).
# G.R. Seaman, Proc. Soc. Exptl. Biol. Med. 80, 308 (1952).
43 G. R. Seaman, Proc. Soc. Exptl. Biol. Med. 82, 184 (1953).
44D. E. Green, Science 115, 661 (1952).
4° E. L. R. Stokstad, H. P. Broquist, and E. L. Patterson, Federation Proc. 12, 430
(1953).
46R. A. Peters, H. M. Sinclair, and R. H. 8S. Thompson, Biochem. J. 40, 516 (1946).
582 NEW AND UNIDENTIFIED GROWTH FACTORS
son,” and Gunsalus.*> The two former groups observed that the vesicant
arsenical Lewisite (CHCI=CH—AsCl.) was particularly effective against
the pyruvic oxidase system in pigeon brain, and that the attack was pri-
marily upon —SH groups in this system. Reversal of Lewisite oxide poison-
ing by BAL (2,3-dimercaptopropanol) was shared by other dithiols, but
not by monothiols. It was then shown*® that lipoic acid, ike BAL (but un-
like glutathione), was highly active in overcoming arsenate and arsenite
inhibition of apopyruvate dehydrogenase activity in S. faecalis. Whether
the lipoic acid functioned in the latter study as catalyst or substrate is not
clear; however, the high reversing power of lipoic acid is noteworthy (0.04
y was somewhat less active than 100 y of BAL). It is quite possible that
lipoic acid is one of the first compounds in the pyruvic oxidase system to be
inactivated by Lewisite.*”*
On the basis of the belief that lipoic acid possesses codehydrogenase func-
tions, Calvin and his associates**: *9: 48 have suggested the intriguing possi-
bility that this coenzyme may participate in the primary quantum con-
version act of photosynthesis. It was observed*? that the incorporation of
CO, into members of the citric acid cycle in green algae was strongly in-
hibited by light. This was at first interpreted as being due to the mainte-
nance (in light) of too low a concentration of a key intermediate required
for entry into the cycle, and, more recently, as a possible depletion of the
supply of oxidized lipoic acid available to oxidize pyruvate. The key reac-
tion is envisaged’ as
ne a
Chlorophyll (activated) -+- S——S — Chlorophyll (ground state) +S 8
whereby lipoie acid is maintained as thiyl-free radicals due to light-induced
47 7,. A. Stocken and R. H. 8. Thompson, Biochem. J. 40, 5385 (1946).
47a The postulated activity of lipoic acid in the dithiol form as an anti-arsenical
raises some questions as to the correctness of the proposed structures of LT and
LTPP,?° if the latter are assumed to be the active forms of the compound. Thus,
with a lipoie acid sulfur-protein link ruled out because of the low reversing power
of monothiols, only the pyrophosphate group remains for ready binding to the
apoenzyme. Such a linkage, however, would be expected to be weaker than that
known to exist between lipoic acid and protein.’: !4 Similar reservations apply to
the manner of attachment of LTPP to the apoenzyme during transport of the C2
unit to CoA. Other possibilities are: (a) rupture of the thiazole ring and attachment
to protein or thiamine through the S atom; (b) function of lipoie acid without thi-
amine in the arsenate reversing system; (c) an alternate structure of LT which
would permit attachment through the carboxyl or amino groups. Further ex-
periments are needed to determine the exact relationships of the various moieties
in lipoic acid systems.
48M. Calvin and J. A. Barltrop, J. Am. Chem. Soc. 74, 6153 (1952).
49 A. A. Benson and M. Calvin, J. Exptl. Bot. 1, 63 (1950).
II. CARNITINE 583
electronic excitation. In the absence of light, reoxidation of the free radicals
would occur, through DPN, flavoproteins, and other oxidants, including
finally CO,.
Such a proposal implies that the ‘“‘reduction”’ of lipoie acid (disulfide) by
electronic excitation proceeds more rapidly than its reduction by pyruvate.
Since the /y’ of the operating lipoic acid coenzyme has not yet been re-
ported, a comparison cannot be made; however spectrophotometric evi-
dence is presented**: * to show that the energy needed to rupture the S—S
bond in 6,8-thioctie acid (30,000 to 40,000 cal. per mole) is approximately
equal to the energy available for photosynthesis when one quantum of light
is absorbed. By contrast, 5,8-thioctic acid and other disulfides required
55,000 to 70,000 cal. per mole for similar cleavage. The peculiar instability
of 6,8-thioctic acid was thought to be due to strain within the ring. This is
further suggested by the yellow color of the latter compound; the other
disulfides examined were colorless. Further indirect evidence supporting
this concept was provided by model systems, whereby zine porphyrin or
tetralin could be used to reduce the disulfide photochemically. Finally, when
algae were treated with 6,8-thioctic acid, oxygen production was acceler-
ated, using quinone as the oxidizing agent. 5 ,8-Thioctic acid was ineffective.
These suggestions, if verifiable by direct experiments, will consititue a
major step toward a full understanding of the photosynthetic process.
The interest that the discovery of lipoic acid has engendered will doubt-
less continue for some time; in addition to further studies on the chemistry
of the vitamin, the enzymic aspects of the problem deserve major atten-
tion. The finding in lipoic acid of a catalytically active thiol has emphasized
again the importance of this class of compounds to biochemical systems.
The realization that CoA and other thiols possess acetylating power in
in vitro systems and that the thiol bond is probably of the “high-energy”
variety®’- focuses interest upon the lipoic acid system as a possible parti-
cipant in energy conservation and transport from pyruvate to ATP and
other eventual acceptors.
II. Carnitine (Vitamin B,)
In 1947 Fraenkel and Blewett*! observed that yeast extracts contained
two apparently unfamiliar growth factors which were required by the
mealworm Tenebrio molitor. One of the factors, adsorbable on Norit, was
found to be folic acid; the other, a ‘‘filtrate factor,’’ was regarded as a new
50 F, Lynen and E. Reichert, Angew. Chem. 68, 47 (1951).
51 F. Lynen and E. Reichert, Ann. 574, 1 (1951).
52 J. Baddiley and E. M. Thain, J. Chem. Soc. 3425 (1951).
83 T. Wieland and E. Bakelmann, Angew. Chem. 64, 59 (1952).
54G. Fraenkel and M. Blewett, Biochem. J. 41, 469 (1947).
584 NEW AND UNIDENTIFIED GROWTH FACTORS
growth principle. It was designated vitamin By because of its ready water
solubility and its importance to the insect.
Assays of vitamin By were carried out by placing 4-week-old 7’. molitor
larvae on a purified cassein-salts-glucose-cholesterol-vitamins diet, and
noting the growth response to sources of the vitamin over a 4- to 6-week
period. Using this assay, the vitamin was found in yeast, whey, and many
animal tissues. Vegetables, with the exception of wheat germ, were poor
sources. Isolation of the vitamin was effected from liver and whey®*: °°
by adsorption on and elution from fullers’ earth, extraction into phenol,
chromatography on alumina columns, and countercurrent extraction with
phenol-dilute HCl. The purified material was found upon characterization,
degradation, and synthesis studies to be identical with carnitine [(CHs)3-
N+—CH.CH(OH)CH.COO-], the trimethyl betaine of $-hydroxy-y-
aminobutyrie acid.
The purified growth factor was active at levels of 0.37 to 0.75 y per gram
of diet. This places the requirement at the catalyst level of activity, de-
spite the fact that in several animal tissues the carnitine content approaches
0.1% (it represents up to 3% of the total water-soluble ‘‘extractives”’ of
skeletal muscle). The high activity for growth, in spite of the high content
in tissues, is reminiscent of choline. Moreover, the similarity in structure
to choline and the thetins suggests the possibility that carnitine may serve
as a methylating agent 7m vivo. Carter et al.,°® acting on this supposition,
have tested crotonobetaine (a dehydration product of carnitine) and £-
hydroxy-y-aminobutyric acid as possible replacements for carnitine in
Tenebrio. Only the latter compound was active, at levels of 12 to 24 y per
gram of diet. The authors therefore suggested that carnitine may partici-
pate in transmethylation reactions in animal tissues. A similar postulate
was made earlier for carnitine in the human being®”: *®8 on the basis of an
increased excretion of methylated pyridinium compounds (described as
trigonelline) after administration of carnitine. Thus, even though only a
few insect species closely related to Tenebrio (e.g., Palorus ratzeburgi?®)
appear to require an exogenous supply of carnitine, the interesting pos-
sibility that this compound may take a place among the group of import-
ant transmethylating agents in higher animals has been raised by this
work with lower forms. It is not unreasonable to hope that future researches
into the growth requirements of other phyla may bring to light additional
55 G. Fraenkel, Arch. Biochem. and Biophys. 34, 468 (1951).
56 H. Ki. Carter, P. K. Bhattacharyya, K. R. Weidman, and G. Fraenkel, Arch. Bio-
chem and Biophys. 38, 405 (1952).
57 W. Ciusa and G. Nebbia, Bull. inst. polytech. Jassay 3, 181 (1948).
583 W. Ciusa and G. Nebbia, Acta Vitaminol. 2, 49 (1948).
59 G. Fraenkel, Arch. Biochem. and Biophys. 34, 457 (1951).
III. PEPTIDES 585
essential metabolites and meanwhile expand our knowledge in a relatively
undeveloped area of comparative biochemistry.
III. Peptides
The question of nutritional equivalence of amino acids and intact pro-
teins has been raised often since the classical review of the subject by Rose.®
He and his colleagues had shown that, although rats could grow on a puri-
fied diet containing known amino acids, much better performance could
be elicited with complete proteins. During the decade that followed, im-
provements in supplemental rations and in the availability of many of the
common amino acids made possible continuing improvements in purified
diets. The greater growth that resulted materially reduced the margin of
superiority of intact proteins over amino acid mixtures.
Strepogenin.®!-§§ Against this background, considerable interest was cre-
ated by the discovery by Woolley and Sprince®™: ® of a peptide-like fraction
of natural materials that was necessary for growth of a strain of hemolytic
streptococci. The material, which was called strepogenin, was also shown
to be needed for early growth of L. casei and to promote growth in mice.*
However, its structure has remained unknown, and to the writer’s know-
ledge no pure samples of strepogenin have been prepared. It is probably a
mixture, perhaps of structurally closely related peptides. The best infor-
mation has been derived indirectly from degradation studies of insulin,’?: 7
which was thought to have a strepogenin-like pattern in a portion of its
structure, together with inhibition studies on lycomarasmin, the tomato-
wilting agent of Fusariwm lycopersicti SACC.
Lycomarasmin activity in tomatoes could be duplicated by tripeptides
containing serine, glycine, and aspartic acid.”? These peptides were also
antagonistic to strepogenin for L. casez. Serylglycylglutamic acid was then
synthesized and found to possess strepogenin activity, about one-fortieth
that of strepogenin concentrates. It was concluded that the latter peptide
may be a fragment or a relative of strepogenin.
60 W. C. Rose, Physiol. Revs. 18, 109 (1938).
61 Nutrition Revs. 4, 273 (1946).
62 Nutrition Revs. 5, 218 (1947).
63 Nutrition Revs. 6, 223, 277 (1948).
641). W. Woolley, Ann. Rev. Biochem. 16, 376 (1947).
65. L. R. Stokstad and T. H. Jukes, Ann. Rev. Biochem. 17, 474 (1949).
66). EH. Snell and L. D. Wright, Ann. Rev. Biochem. 19, 305 (1950).
57D. W. Woolley, J. Exptl. Med. 73, 487 (1941).
8 H. Sprince and D. W. Woolley, J. Exptl. Med. 80, 213 (1944).
9D. W. Woolley, J. Biol. Chem. 162, 383 (1946).
10T). W. Woolley, J. Biol. Chem. 171, 443 (1947).
71D. W. Woolley, J. Biol. Chem. 179, 593 (1949).
72D. W. Woolley, J. Biol. Chem. 166, 783 (1946).
586 NEW AND UNIDENTIFIED GROWTH FACTORS
Support for the concept of an intrinsic growth-promoting property as-
sociated with the serine-glycine-glutamic acid structure is provided by the
experiments of Chattaway and coworkers.”*: 4 They found that extracts of
liver and yeast contained growth-promoting agents for C. diphtheriae
gravis, S. faecalis R., and L. casei, which upon concentration proved to be
of peptide nature. Two peptides, labeled P; and Pz, contained the bulk of
the activity; P, upon hydrolysis was found to yield serine, glycine, and
glutamic acid. Finally, the experiments of Kreh] and Fruton’> have con-
firmed the activity of L-serylglycyl-L-glutamic acid for L. casez and have
shown that the closely related .L-seryl-L-alanyl-L-glutamic acid was in-
active.” Also inactive were several related peptides of glutamic acid,
glycine, and tyrosine.
Peptides of other amino acids have also been shown to be more active
than their constituent moieties in supporting microbial growth. Malin et
al. reported that certain peptides of glycine were utilized more readily
by several lactobacilli than was glycine itself. Simmonds and Fruton ob-
served’®: 8° that a prolineless mutant of #. coli was more responsive to any
of several proline peptides tested than it was to proline, and that an isolated
species of Alcaligenes, termed ‘‘SF”’’,*' required leucyl peptides for growth;
with these in the medium, no other nitrogen or carbon source was needed.
Other peptide requirements have been demonstrated by Snell e¢ al.;°-*4 in
a medium in which p-alanine satisfied the vitamin Bg requirement, L. casei
could be shown also to depend upon a peptide factor for its nutrition. Frae-
tionation of the factor from partly hydrolyzed casein produced a mixture
73 F. W. Chattaway, F. C. Happold, and M. Sandford, Biochem. J. 38, 111 (1944).
74 F, W. Chattaway, D. E. Dolby, D. A. Hall, and F. C. Happold, Biochem. J. 45,
592 (1949).
75 W. A. Krehl and J. 8. Fruton, J. Biol. Chem. 178, 479 (1948).
75a Although recent reports on the structure of insulin’®: ” fail to reveal a serine-
glycine-glutamic acid sequence, the closest relatives are a cysteine-glycine-glu-
tamic acid, and a cysteine-glycine-serine series. Both of these are found in the
‘Dhenylalanine”’ fraction of insulin rather than in the “‘glycine”’ fraction where
strepogenin activity was first reported.7° However, the similarity of the first se-
quence listed here to the strepogenin-active compound (serine replaced by cys-
teine) may warrant the testing of additional peptides.
76 F, Sanger and H. Tuppy, Biochem. J. 49, 481 (1951).
77 F, Sanger and EL. O. P. Thompson, Biochem. J. 58, 366 (1953).
73 R. B. Malin, M. N. Camien, and M.S. Dunn, Arch. Biochem. and Biophys. 32, 106
(1951).
79 S$. Simmonds and J. 8. Fruton, J. Biol. Chem. 174, 705 (1948).
80 §. Simmonds and J. 8S. Fruton, J. Biol. Chem. 180, 635 (1949).
81.§,. Simmonds and J. S. Fruton, Sczence 109, 561 (1949).
82 AH. Kihara, W. G. McCullough, and FE. E. Snell, J. Biol. Chem. 197, 783 (1952).
83 H. Kihara and E. E. Snell, J. Biol. Chem. 197, 791 (1952).
84 H. Kihara, O. A. Klatt, and E. E. Snell, J. Biol. Chem. 197, 801 (1952).
Ill. PEPTIDES 587
of dipeptides, thought to be the alanyl and tyrosyl peptides of valine, leu-
cine, and isoleucine. Also, Sloane and McKee*® have shown that the Staphy-
lococcus albus factor of Hughes*® is replaceable by an intact plasma protein
fraction, the activity of which may be due to special peptide structures,
especially those of cysteine.
Several influences appear to be operative in determining the response of
organisms to peptides; the most obvious is the need for a particular unit
per se either because of a more rapid transfer into the cell or because of a
paucity of appropriate conjugating enzymes to bring about its biosynthesis
from the amino acids. Apart from this, it has been shown that in L. casez,®*
when b-alanine was present in the medium it inhibited the utilization of the
L isomer; however, D-alanine had no effect on L-alanine peptides. A similar
effect was then suggested for other systems, i.e., an antagonism among cer-
tain related amino acids that may not be experienced when peptides are
employed instead. The destructive action of tyrosine decarboxylase™ upon
free tyrosine, but not on its peptides, was also noted and offered as an ex-
planation of the greater response of S. faecalis to tyrosine peptides. Finally,
it should be pointed out that many peptides have been shown to be less
active than their constituents.’ 8° These may simply become digested, as-
similated, and resynthesized into protein patterns in which the peptide
sequences in question may not appear at all. Some peptides (in addition
to the antibiotic polypeptides) actually delay or inhibit bacterial growth,°°
perhaps by interference with the synthesis of peptides and proteins within
the cells.
The role of strepogenin and other peptides in animal nutrition is doubt-
ful. Although Womack and Rose®*® were able to produce more rapid weight
gains inratsfed intact protein (casein) than in those maintained on nineteen
amino acids, these differences have been eliminated by employing acid-
hydrolyzed casein supplemented with tryptophan and cystine (Ramasarma
et al.*!). The casein hydrolyzate was devoid of strepogenin activity. Other
workers*?: * have also shown that properly balanced amino acid mixtures
supported good growth of mice and that these mixtures were not improved
85 N. H. Sloane and R. W. McKee, J. Am. Chem. Soc. 74. 987 (1952).
86 T. P. Hughes, J. Bacteriol. 23, 437 (1932).
87 J. S. Fruton and 8. Simmonds, Cold Spring Harbor Symposia Quant. Biol. 14, 55
(1949).
88 V. Nurmikko and A. I. Virtanen, Acta Chem. Scand. 5, 97 (1951).
89S. Simmonds, J. I. Harris, and J. 8. Fruton, J. Biol. Chem. 188, 251 (1951).
99 M. Womack and W. C. Rose, J. Biol. Chem. 162, 735 (1946).
1G. B. Ramasarma, L. M. Henderson, and C. A. Elvehjem, J. Nutrition 38, 177
(1949).
9% —. Brand and D. K. Bosshardt, Abstr. 114th Meeting, Am. Chem. Soc. p. 38C (1948).
93K. H. Maddy and C. A. Elvehjem, J. Biol. Chem. 177, 577 (1949).
588 NEW AND UNIDENTIFIED GROWTH FACTORS
by the addition of strepogenin-rich proteins. It is possible that lower taste
acceptability may have been chiefly responsible for the poorer performance
on amino acid mixtures.*!: °* The need for special peptides such as strepo-
genin thus seems to be best established for microbial species. Even with
these, the proportion of peptides that is utilized per se is probably very
small; in L. casez, strepogenin activity has been claimed for glutamine
(although this is not in agreement with the findings of others%**8), In
|
spite of the technical difficulties involved, a major demand in this area of —
investigation continues for purified fractions, so that the nutritional and
biochemical roles of these compounds may be further realized.
IV. Lyxoflavin
In 1947, Pellares et al.®° isolated a pentose from human heart, which they
identified as lyxose. Later,!°° these workers reported the isolation of lyxo-
flavin from the same source, although this was challenged by subsequent
work.!0
This close relative of riboflavin (and of the corresponding moiety of
vitamin Bi.) was viewed by Emerson and Folkers!”: 1% as a possible new
member of the B complex, although they recognized that the experimental —
evidence for the reported existence of lyxoflavin was not as rigorous as might
be desired. They therefore devised a ration based on soybean meal as the
major constituent, to which had been added 0.5 % desiccated thyroid, and
observed the rate of growth of rats on this diet. The growth-depressing
effect of large doses of thyroid was overcome by extracts of liver and by
fish meal, or alternatively by synthetic lyxoflavin. The lyxoflavin effect
was shown not to be due to conversion to riboflavin. The ability of lyxoflavin
to completely replace the effect of liver or fish meal gave support for its
classification as a vitamin for the rat, and its general importance was fur-
ther suggested by the findings that it also stimulated swine and chick
growth.! 1% Tt seemed possible from this work that lyxoflavin might be
94 7,. P. Snipper, Ph.D. Thesis, Oregon State College, 1951.
95 H. T. Peeler, L. J. Daniel, L. C. Norris, and G. F. Heuser, J. Biol. Chem. 177, 905
(1949).
% L. D. Wright and H. R. Skeggs, J. Bacteriol. 48, 117 (1944).
97 #}, Kodicek and 8. P. Mistry, Biochem. J. 51, 108 (1951).
9 EH}. Kodicek and 8S. P. Mistry, Biochim. et Biophys. Acta (In Press).
99). S. Pellares, F. V. Orozco, and J. R. Carvallo, Arch. inst. cardiol. Mex. 17,
575 (1947).
100 #}, S. Pellares and H. M. Garza, Arch. Biochem. 22, 63 (1949).
101'T. §. Gardner, HE. Wenis, and J. Lee, Arch. Biochem. and Biophys. 34, 98 (1951).
102 G. A. Emerson and K. Folkers;.J. Am. Chem. Soc. 73, 2398 (1951).
103 G, A. Emerson and K. Folkers, J. Am. Chem. Soc. 78, 53888 (1951).
104 R.C. Wahlstrom and B. C. Johnson, J. Animal Sci. 10, 1065 (1951).
106 H, W. Bruins, M. L. Sunde, W. W. Cravens, and E. E. Snell, Proc. Soc. Ezpil.
Biol. Med. 78, 535 (1951).
Pe
VI. FACTORS REQUIRED IN UNHEATED GROWTH MEDIA 589
related to or identical with the ‘stress factor’ in liver observed by
Ershoff,!°® which was capable of counteracting the growth-depressing effect
of thyroid in rats fed a casein diet.
However, Ershoff has since shown!” that lyxoflavin was ineffective as
an antithyrotoxic factor when this diet was used. Since the diets employed
in the two laboratories differed considerably in composition (soybean-dex-
trose versus casein-sucrose), the possibility has been raised (Cooperman
et al.'s) that lyxoflavin may act as a stimulant in liver factor synthesis by
the intestinal flora when the soybean-dextrose diet is employed. Finally,
the latter authors were able to demonstrate a slight replacement of ribo-
flavin in rat diets and L. casei growth media, and they concluded that the
existing evidence did not warrant the classification of lyxoflavin as a new
vitamin. Microbiological evidence is inconclusive, for lyxoflavin is stimula-
tory both in its own right! and as an adjuvant for riboflavin,!: 1° or is
inhibitory.!°° Thus, the question whether lyxoflavin may be a member of
the B complex remains unsettled. Of greater importance, however, is the
question whether lyxoflavin can function in metabolism in any unique fash-
ion. Further experiments are in order to definitely establish the natural oc-
currence of lyxoflavin, its possible presence in flavoproteins, and its effect
upon growth and metabolism.
V. Coenzyme III
Proteus vulgaris was shown by Singer and Kearney"®: “ to require a
previously undescribed cofactor for oxidation of cysteine-sulfinic acid to
cysteic acid. Isolation of the factor from bakers’ yeast produced a nucleo-
tide which was thought by the authors to be identical with nictotinamide-
ribose-(5)-pyrophosphate. Because of the similarity to the other nico-
tinamide coenzymes, the name coenzyme III was tentatively assigned.
Coenzyme III was found in high concentration in yeast and in liver and
kidney mitochondria.” In each of these systems, sulfinic dehydrogenase
activity was demonstrated, and a fairly general requirement for this co-
factor in cysteine metabolism appears possible.
VI. Factors Required in Unheated Growth Media
In 1933 Orla-Jensen! observed that many lactic acid bacteria would not
grow properly upon carbohydrates that had been sterilized in distilled
106 B. H. Ershoff, Proc. Soc. Exptl. Biol. Med. 78, 459 (1950).
107 B. H. Ershoff, Proc. Soc. Exptl. Biol. Med. 79, 469 (1952).
108 J. M. Cooperman, W. L. Marusich, J. Scheiner, L. Drekter, E. De Ritter, and S. H.
Rubin, Proc. Soc. Exptl. Biol. Med. 81, 57 (1952).
109 M.S. Shorb, Proc. Soc. Exptl. Biol. Med. 79, 611 (1952).
110}. B. Kearney and T. P. Singer, Biochim. et Biophys. Acta 8, 698 (1952).
11 T. P. Singer and E. B. Kearney, Biochim. et Biophys. Acta 8, 700 (1952).
12 T, P. Singer and E. B. Kearney, Federation Proc. 12, 269 (1953).
us A.D. Orla-Jensen, J. Soc. Chem. Ind. 52, 374 (1933).
590 NEW AND UNIDENTIFIED GROWTH FACTORS
water and added aseptically to a sterile yeast-casein medium. Heating of
glucose with the medium resulted in normal growth, as did heating of small
amounts of methylglyoxal, furfural, or pentoses with the yeast-casein mix-
ture. These observations have been repeated and extended in later
years,!4-"7 so that it appears, that at least two types of transformation occur
during heating: one is the expulsion of oxygen and/or the formation of re-
ducing substances,!: '-?! whereas the other involves interaction of car-
bohydrate with phosphate and/or the nitrogenous components of the me-
dium. The compounds so produced were presumed to act as the stimulatory
agents.
Studies in this laboratory!” have revealed that products formed by heat-
ing glucose with inorganic phosphate and amino acids will greatly stimu-
late the growth of Lactobacillus gayoni 8289 (strain 45; cf. ref. 123 for de-
scription of organism) during a 12-hour incubation period. In a series of
experiments, glycine was found to be consistently superior to other amino
acids as a precursor of active material, whereas alanine produced substances
that were strongly inhibitory. N-Glucosylglycine was then synthesized as
the ethyl ester! and found to be as active as equal weights of yeast extract
in a filter-sterilized medium, at levels up to | mg. per 10 ml. of culture. —
Higher levels of yeast produced greater stimulation, whereas amounts of
glucosylglycine above 5 mg. became inhibitory. However, when glucosyl-
glycine was heated separately and added aseptically to the basal medium,
additional growth stimulation was provided which approached, although
it did not equal, the growth on yeast extract. Of approximately twenty
species of lactic acid bacteria tested, three others were stimulated by un-
heated glucosylglycine: Streptococcus zymogenes 10100, Lactobacillus acido-
philus (O.S.C. strain), and Leuconostoc mesenteroides P63. L. gayont F20
responded only after the compound had been heated. Other organisms,
such as L. gayont 8289, strain 49,'° L. casei, and Saccharomyces cerevisiae
14K. L. Smiley, C. F. Niven, Jr. and J. M. Sherman, J. Bacteriol. 45, 50 (1943).
115 J. C. Rabinowitz and E. E. Snell, J. Biol. Chem. 169, 631 (1947).
16 H). H. Snell, KE. Kitay, and E. Hoff-Jgrgenson, Arch. Biochem. 18, 495 (1948).
17 C, E. Hoffman, E. L. R. Stokstad, B. L. Hutchings, A. C. Dornbush, and T. H.
Jukes, J. Biol. Chem. 181, 635 (1949).
118 W. EK. Shive, J. M. Ravel, and R. E. Eakin, J. Am. Chem. Soc. 70, 2614 (1948).
19 V, Kocher, Intern. Z. Vitaminforsch. 20, 369 (1949).
1220 R. D. Greene, A. J. Brook, and R. B. McCormack, J. Biol. Chem. 178, 999 (1949).
121 T,. K. Koditschek, D. Hendlin, and H. B. Woodruff, J. Biol. Chem. 179, 1093 (1949).
122 T), Rogers, T. E. King, and V. H. Cheldelin, Proc. Soc. Exptl. Biol. Med. 82,.
140 (1953).
123 V. H. Cheldelin and A. P. Nygaard, J. Bacteriol. 61, 489 (1951).
124M. L. Wolfrom, R. D. Schuetz, and L. F. Cavalieri, J. Am. Chem. Soc. 71, 3518
(1949).
VII. GUINEA PIG ANTISTIFFNESS FACTOR 591
LM did not respond at all to glucosylglycine under these conditions, al-
though they were stimulated by yeast extract.!2°
The relative superiority of glycine over other amino acids in producing a
biologically active material with glucose, and the strong inhibition produced
with alanine, appear to confer a measure of specificity upon the heat activa-
tion reaction and to raise the possibility that one or at most a few factors
may be involved. Presumably these represent conversion products from
glucosylglycine, since the latter compound possesses relatively low activity.
The role of phosphate is not yet clear. Further research should disclose the
nature of these factors, as well as their relation to the products of the Mail-
lard “browning reaction.’’?° The latter appears to be a more general reac-
tion between carbohydrates and amino acids, resulting in a net loss of
nutritional value of the amino acids toward higher animals.!”7
VII. Guinea Pig Antistiffness Factor
A syndrome in guinea pigs was described several years ago, whereby these
animals developed characteristic joint stiffness on diets high in milk
(Wulzen and Bahrs"*: 2°), When adequate greens were given, the animals
maintained normal health. Intermediate degrees of stiffness at the wrist
joints were detected in the animals on the milk diets, and an assay method
was developed which attempted to place the stiffening (produced at least
in part by the deposition of calctum phosphate in the joints) on a quantita-
tive basis. Constituents of the diet were sought which might protect the
animals against the onset of stiffness, and it was believed (van Wagtendonk
and Wulzen"®) that such a protective factor could be obtained in pure form,
either from raw cream or from sugar cane juice. On the basis of this ap-
proach, the existence of a nutritional principle, the ‘‘antistiffness factor,”
was claimed, and methods were given for its isolation from cream!° and
from cane juice.™!
The existence of the syndrome has been confirmed by other experi-
menters'*: '** and has been described in detail in a review of the subject by
25 T). Rogers, T. E. King, and V. H. Cheldelin, unpublished.
26 7, C. Maillard, Compt. rend. 154, 66 (1912).
7 AR. Patton, Nutrition Revs. 8, 193 (1950).
128 A.M. Bahrs and R. Wulzen, Proc. Soc. Exptl. Biol. Med. 33, 528 (1936).
29 R, Wulzen and A. M. Bahrs, Am. J. Physiol. 133, 500P (1941).
180 W. J. Van Wagtendonk and R. Wulzen, Arch. Biochem. 1, 373 (1943).
31 W. J. van Wagtendonk and R. Wulzen, J. Biol. Chem. 164, 597 (1946).
182 J. J. Oleson, E. C. Van Donk, 8S. Bernstein, L. Dorfman, and Y. SubbaRow, J/.
Biol. Chem. 171, 1 (1947).
133 H. G. Petering, L. Stubberfield, and R. A. Delor, Arch. Biochem. 18, 487 (1948).
592 NEW AND UNIDENTIFIED GROWTH FACTORS
the principal authors." The condition is accompanied by extensive calcifica-
tion in the joints, body wall and cavity, and upper skeleton; profound
changes have been observed in the skull and teeth. Hearing is impaired
in the affected animals. Numerous changes have also been recorded for —
calcium, phosphorus and protein levels in the blood.!4
Beyond this, it is difficult, if not impossible, to make further positive
statements regarding the “antistiffness factor,’ owing in the main to the fact
that the assay method was developed and used without adequate controls.
Later, a critical examination of the assay by Christensen et al.!®° and an ex-
amination™® of the data of Oleson et al.! revealed that the assay method
could not distinguish between concentrations of active materials that dif-
fered by five- or even tenfold. These discoveries necessarily vitiated the claims —
based on the wrist stiffness assay, whether for isolation of an active principle
or for correlation of stiffness with peculiarities in metabolism. The fact that
pure compounds have been isolated from cane juice!!: *7) 8 thus simply
reflects the success of chemical separations of materials (chiefly steroid)
in the ether-extractable fractions of sugar cane, with no connection between
the chemical separations and any physiological index.
In spite of the unsatisfactory character of many of the studies, it ap-
pears possible that a variety of steroids may possess some antistiffness
potency. Thus, positive results have been claimed for stigmasterol and
various esters of ergostanol and ergostenol (although this is denied by the
work of Smith e¢ al.!°°). All these tests suffer from the inadequacies of the
assay described above. It seems, however, that the assay is capable of de-
tecting advanced stages of the condition; addition of these sterols to the
“deficient”’ guinea pig diet over a period of several months might serve
qualitatively to establish whether or not the compounds in question can
serve as antistiffness agents. Finally, attention should be given to the ques-
tion whether this is a nutritional or (perhaps more likely) a pharmacologi-
eal principle, i.e., a condition brought about by high levels and imbalances
of caletum and phosphorus in the diet. When cotton rats were maintained
on diets high in calcium and phosphorus but low in several other minerals,
especially magnesium, a similar ‘“‘calcinosis’? was observed, which led to
extremely high (23 to 36 %) ash contents in the heart tissue.° The condi-
134 W. J. van Wagtendonk and R. Wulzen, Vitamins and Hormones, 8, 70 (1950).
135 B, E. Christensen, M. B. Naff, V. H. Cheldelin, and R. Wulzen, J. Biol. Chem. 175,
275 (1948).
136 In F. J. Stare, Nutrition Revs. 6, 107 (1948).
137 T). H. Simonsen and W. J. van Wagtendonk, J. Biol. Chem. 170, 239 (1947).
138 H, Rosenkrantz, A. T. Milhorat, M. Farber, and A. E. Milman, Proc. Soc. Expil.
Biol. Med. 76, 408 (1951).
139 §. E, Smith, M. A. Williams, A. C. Bauer, and L. A. Maynard, J. Nutrition 38,
87 (1949).
140 M.A. Constant and P. H. Phillips, J. Nutritzon 47, 317 (1952).
al
VIII. MISCELLANEOUS FACTORS 593
tion, as in guinea pigs, was alleviated by feeding oatmeal but was aggra-
vated by increasing the phosphate content of the diet. Vitamin E was
ineffective. Microscopic appearance of the tissue lesions'! was reported to
be similar to those in guinea pigs.'”
The gross similarities in guinea pig stiffness and human arthritis confer
a continuing interest upon this unusual disease. It is hoped that studies will
be resumed which will aim at the development of sound analytical proce-
dures and the establishment of the nutritional and metabolic relationships
that may exist.
VIII. Miscellaneous Factors
The recent literature contains many depositions describing factors for
growth, reproduction, and intermediary metabolism. The evidence is fa-
vorable that one or more of these will prove to be of major importance. This
is particularly true in the field of poultry nutrition,» “* where at least
ten reports of new factors have been issued. However, caution is urged in
accepting the entire group as bona fide factors, for most of them have not
yet been purified or sufficiently cross-checked against other factors to deter-
mine what aliases may exist. In addition to probable duplications, the newer
members of the Biz group (see Chapter 3) as well as antibiotics may be
responsible for some of these effects. Artifacts may be obtained owing to
interplay of known factors, e.g., amino acid and amino acid-vitamin im-
balances; such derived foods as processed soybean meals, which are used
in many poultry diets, may vary considerably in their composition. Finally,
the possibility exists that uncontrolled pathogenic organisms in the intesti-
nal tracts of animals may influence performance in a non-uniform manner,
particularly where mobility is restricted, as in poultry batteries.
A list of several of these and other newly described factors is given in
Table I. Where possible, pertinent information is given regarding the sus-
pected identities which reflects the opinions of either the listed authors or
the present writer. Finally, for convenience in summarizing some of the
older literature, several earlier ‘‘vitamins’”’ and related materials are listed
in Table II. These have either been discarded altogether or have been
shown to be replacable by one or more known compounds. It is unlikely
that these terms will ever be used again, for the growth factors of the future
will probably be given names in keeping with their chemical structure or
biological function.
Certain trends in future research on new growth factors may be adum-
brated by an analysis of the current list. Thus, factors for rat nutrition are
_ 41M. A. Constant, P. H. Phillips, and D. M. Angevine, J. Nutrition 47, 327 (1952).
142 P.N. Harris and R. Wulzen, Am. J. Pathol. 26, 595 (1950).
M3 H. Menge, G. F. Combs, P.-T. Hsu, and M.S. Shorb, Poultry Sci. 31, 237 (1952).
_ 44 A. Menge and G. F. Combs, Poultry Sci. 31, 994 (1952).
594 NEW AND UNIDENTIFIED GROWTH FACTORS
SomE AppITIONAL UNIDENTIFIED FAcTOoRS
TABLE I
Factor
References
Remarks
Growth factors for chicks
From bran
From yeast
From fish meal, yeast
From liver
From whey
From grass juice
From fermentation products
From peanut meal
Factors for egg production and
hatchability
From liver, whey
Growth and antiperosis agents
for turkeys
From yeast, whey
From liver, fish meal, whey
Antipancreatie fibrosis factor
for ducks
From cornstarch
Antiliver, kidney necrosis fac-
tors for rats
From cornstarch
From yeast
Growth factor for rats
From fish solubles
“Stress factors’”’ for rats
From liver
“Stress factors’’ for mice
From liver, cottonseed
Growth factors for mink
From liver, whey
Monkey antianemia factor
Growth factor for foxes
From liver, whey
Growth factor for corn borer
Potato eelworm hatching factor
Growth factor for Treponemata
From enzymatic protein di-
gests
Cravens et al.145
Cravens et al.145
Savage et al.146
Combs et al.,'47 Menge et al.
143, 148
Menge et al.143, 148
Kohler and Graham150, 151
Stokstad et al.153
Young et al.155
Couch et al.156
Scott1s7
Atkinson and Couch, Me-
Ginnis et al.,459 Briggs,160
Menge and Combs"4
Miller161
Baxter!62
Schwarz!63 (‘‘factor 3’’)
King and Haugel®
Ershoff106
Bosshardt and Huff!67
Schaefer e¢ al.,188 Tove et al.169
Cooperman et al.171
Schaefer et al.173
Beck et al.174, 175
Calam et al.176
Eagle and Steinman!®9, 181
Probably numerous duplicatio
(see text)
Related to orotic acid?
Thought to be identical with grass
juice factor for guinea pigs and
ratsl52
Effect may be produced by anti-
bioties!54
Two factors claimed157
Effects most pronounced when sup
plemented with antibiotics!59
May be related to factor for ducks,
abovelél
May be related to Biz 4
Required during feeding of thyroid
extract; Biz partly active,16. 16
also lyxoflavin (see text) 1% 4
Similar to above; synthesis of atl
acids may be controlled by fac-
tors!§
Four claimed; one is probably Bit
Deficiency induced through defi-
ciency of several individual vita-_
mins; curable by folie acid bu'
believed non-identical!”*
v
Basal diet probably deficient in
Big; similar to mink diet!
Leaf extracts including grass jui
concentrates!®? are active
Low molecular weight acid-la
tone;!77 may be similar to ger-
mination factors for Striga an
Orobanche'?9
Replaceable by serum albumin
which functions as a carrier for
oleate or other lipids!$
Factor
TABLE I—Concluded
References
Growth factors for Trypano-
soma cruzt
From blood
Growth factor for Trichomonas
vaginalis
From pancreas
Growth factors for Pilobolus
Growth factors for Clostridia
Growth factor for Microbacte-
rium flavum
Growth factor for pleuropneu-
monia-like organisms
GePPEO)
Staphylococcus albus factor
(SSF)
Growth factors for lactobacilli
Alkali-stable factors for lacto-
bacilli
Lactobacillus leichmanii factor
Lactobacillus bifidus factors
Growth factor for Fusobacteria
Growth factor for Pythiogeton
Coenzyme of alcoholic fermen-
tation
Citric acid oxidation factor
Formic hydrogenlyase cofactor
Bacterial deaminase cofactor
Coconut milk factor
McRay et al.184
Sprince et al.185
Hesseltine et al.,!8° Pages?
Knight and Fildes,'89 Jones
and Clifton!99
Bishop et al.192
Tang et al.193
Hughes®é
Kitay and Snell!%5
Robinson e¢ al.,19° Ostling and
Nyberg!7
Peeler and Norris!%8
Gyllenberg ef al,%® Shorb
and Veltre,!8%b Gyoérgy et
al,8*
Omata!93
Perlman?0
Ohlmeyer,”! Tria and Barna-
bei202
Foulkes?%
Lichstein and Boyd?
Williams and Christman?°5
Van Overbeek et al.,2°° Du-
hamet and Gautheret,?0
Steward and Caplin?
VIII. MISCELLANEOUS FACTORS 595
Remarks +
Reportedly a complex derivative
of hemoglobin"
Iron porphyrins slightly active ;!7
“coprogen” isolated,'8° probably
related to ‘‘ferrichrome”’ an or-
gano-iron pigment!8§
May be peptide mixture,!*! possibly
strepogenin-like!
Low molecular weight basie pro-
tein!
Glycoprotein with special S-con-
taining peptide structure® (see
text)
Mixtures, probably peptides
May be related to Biz, although not
identical
May be related to above factors
from various sources; may be
oligosaccharides
Probably a nucleotide!
Probably a nucleotide?
May be derived from fatty acids?
Produced by H2SOu degradation of
carbohydrates; functionally re-
lated to biotin, adenylie acid ;2°
may be similar to heat-produced
growth factors"
Stimulates plant root growth; in-
doleacetic,2°? naphthaleneacetic
acids?!9 partly active, but not
identical with factor?!
45 W. W. Cravens, H. W. Bruins, M. L. Sunde, and E. FE. Snell, Federation Proc. 10,
379 (1951).
46 J. EK. Savage, B. L. O’Dell, H. L. Kempster, and A. G. Hogan, Poultry Sci. 29, 779
(1950).
147 G. F. Combs, C. W. Carlson, R. F. Miller, H. T. Peeler, L. C. Norris, and G. F.
Heuser, J. Biol. Chem. 182, 727 (1950).
48 H. Menge, G. F. Combs, and M. 8S. Shorb, Poultry Sci. 28, 775 (1949).
49 T. D. Wright, J. W. Huff, H. R. Skeggs, K. A. Valentik, and D. K. Bosshardt,
J. Am. Chem. Soc. 72, 2312 (1950).
150 G. O. Kohler and W. R. Graham, Poultry Sci. 30, 484 (1951).
151 G, O. Kohler and W. R. Graham, Poultry Sci. 31, 284 (1952).
152 G. O. Kohler, C. A. Elvehjem, and E. B. Hart, J. Nutrition 15, 445 (1938).
163 F. L. R. Stokstad, T. H. Jukes, J. Pierce, A. C. Page, Jr., and A. L. Franklin,
J. Biol. Chem. 180, 647 (1949).
64 EL. R. Stokstad and T. H. Jukes, Proc. Soc. Exptl. Biol. Med. 73, 523 (1950).
596
TABLE II
NEW AND UNIDENTIFIED GROWTH FACTORS
NAMES FOR VITAMINS AND GROWTH Factors, Now DiscARDED OR BELIEVED
IDENTICAL WITH OTHER KNOwN COMPOUNDS
Factor
References
Remarks
Bs
Bio and Bu
Bus
Bw; factor W
Li, Le
Factors R, S (for chick growth)
Vitamin T (torutiline)
T factor, for Lactobacillus fer-
menti
Factor U
Factor V, for Hemophilus
Factor X
Factor Y
Growth factors for guinea pigs:
GPF-1
GPF-2
GPF-3
Gizzard erosion factor
Factors for L. helveticus and S.
lactis: HL 1, 2, 3, and 4
L. gayont factor
Xanthine oxidase factor
Williams and Waterman?!2
Reader?!4
Carter et al.216
Centanni?!8
von Euler et al,?19
Briggs et al.220
Norris and Majnarich22!
Hogan et al.223
Lunde and Kringstad,226 Elve-
hjem et al.227
Lunde and Kringstad226
Nakahara et al.229
Schumacher et al.?82
Goetsch235
Metcalf et al,237
Stokstad and Manning*89
Davis”!
Boas248
Chick e¢ al.244
Woolley and Sprince?45
Almquist and Stokstad,?46
Bird et al.247
Barton-Wright et al.,249 Colio
and Babb250
Cheldelin and Riggs®®!
Westerfeld and Richert255
Probably pantothenic acid?!3
Replaceable by mixtures of ar-
ginine, glycine, and cystine®!5
Presumed identical with Be?! or
nicotinic acid2!7
Prevented digestive disturbances
in pigeons; probably a mixture28
Adenylic acid?!9
Probably a mixture of Bie and the
folie acid group
Announced as metabolite of xan-
thopterin, but later work failed
to confirm earlier results?22
Antiperosis factor in chicks, re-
placeable by manganese?4 and
choline225
Regarded as identical with bio-
tin,?8 although properties also
resemble pantothenic acid phos-
phates, coenzyme A
Anti-gray hair factor for rats; prop-
erties resemble bound forms of
pantothenie acid
Factors from yeast, liver reportedly
related to anthranilie acid, ad-
enosine, necessary for normal lac-
tation230, 231
R = probably related to folie
acid,233 § = probably identical
with strepogenin?%4
From insects; mixture of folie acid
group, Bis, desoxyribosides*%é
From tomato juice; may be iden-
tical with thiamine’
Probably a mixture containing
Bo?49 and folie acid activity?
Diphosphopyridine-nucleotide™*”
Equivalent to biotin?’
Equivalent to pyridoxine?
Identical with folie acid?5
Replaceable by cellulose plus am-
ino acids*45
Similar to strepogenin™5
Replaceable by B12*48
Chloroform-soluble fractions from
liver; may be related to folie acid
group, also lipoie acid
Partly replaced by nucleotides,?5?
plus high levels of folie acid,?%
plus glutamine or asparagine;
glucosylglycine active (see text
122)
Replaceable by inorganic molyb-
date256
oe
.
+
~
Ls
VIII. MISCELLANEOUS FACTORS 597
very few; with the discovery of vitamin By» and its inclusion in rat diets,
dramatic deficiencies could no longer be produced with purified rations fed
165 R. J. Young, M. B. Gillis, and L. C. Norris, J. Nutrition 60, 291 (1953).
186 J. R. Couch, O. Olcese, B. G. Sanders, and J. V. Halick, J. Nutrition 42, 473 (1950).
167 M. L. Scott, Poultry Sci. 81, 175 (1952).
8 R. L. Atkinson and J. R. Couch, J. Nutrition 44, 249 (1951).
159 J. McGinnis, L. R. Berg, J. R. Stern, M. E. Starr, R. A. Wilcox, and J. S. Carver,
Poultry Sci. 31, 100 (1952).
160 G. M. Briggs, Trans. Am. Assoc. Cereal Chemists 10, 31 (1952).
161 OQ. N. Miller, J. Nutrition 50, 13 (1953).
162 J. H. Baxter, J. Nutrition 34, 333 (1947).
163 K. Schwarz, Proc. Soc. Expil. Biol. Med. 78, 852 (1951).
16 R. H. King and 8. M. Hauge, Arch. Biochem. 24, 350 (1949).
165 J. J. Betheil and H. A. Lardy, J. Nutrition 37, 495 (1949).
166 G. A. Emerson, Proc. Soc. Exptl. Biol. Med. 70, 392 (1949).
167 TJ). K. Bosshardt and J. W. Huff, J. Nutrition 50, 117 (1953).
168 A. E. Shaefer, S. B. Tove, C. K. Whitehair, and C. A. Elvehjem, J. Nutrition 35,
157 (1948).
169 S$. B. Tove, R. J. Lalor, and C. A. Elvehjem, J. Nutrition 42, 433 (1950).
70 W. L. Leoschke, R. J. Lalor, and C. A. Elvehjem, J. Nutrition 49, 541 (1953).
171 J. M. Cooperman, H. A. Waisman, K. B. McCall, and C. A. Elvehjem, J. Nutrition
30, 45 (1945).
172.8. C. Smith and C. A. Elvehjem, J. Nutrition 45, 583 (1951).
73 A. EK. Schaefer, C. K. Whitehair, and C. A. Elvehjem, J. Nutrition 35, 147 (1948).
4 §$.D. Beck, J. H. Lilly, and J. F. Stauffer, Ann. Entomol. Soc. Amer. 42, 483 (1949).
178 §. D. Beck, J. Gen. Physiol. 36, 317 (1953).
176 C. T. Calam, H. Raistrick, and A. R. Todd, Biochem. J. 45, 513 (1949).
7 C. T. Calam, A. R. Todd, and W. S. Waring, Biochem. J. 45, 520 (1949).
478 R. Brown, A. W. Johnson, E. Robinson, and A. R. Todd, Proc. Roy. Soc. (London)
B136, 1 (1949).
79 R. Brown, A. D. Greenwood, A. W. Johnson, A. R. Lansdown, A. G. Long, and
N. Sunderland, Biochem. J. 52, 571 (1952).
180 H. Eagle and H. G. Steinman, J. Bacteriol. 56, 163 (1948).
181 H. G. Steinman and H. Eagle, J. Bacteriol. 60, 57 (1950).
182 H. G. Steinman, H. Eagle, and V. I. Oyama, J. Bacteriol. 64, 265 (1952).
183 V. IT. Oyama, H. G. Steinman, and H. Eagle, J. Bacteriol. 65, 609 (1953).
164 W. L. McRay, E. R. Noble, and E. L. Tondenold, Science 115, 288 (1952).
85 H. Sprince, E. L. Gilmore, and R. 8. Lowry, Arch. Biochem. 22, 483 (1949).
186 C, W. Hesseltine, C. Pidacks, A. R. Whitehill, N. Bohonos, B. L. Hutchings, and
J. H. Williams, J. Am. Chem. Soc. 74, 1362 (1952).
187 R. M. Page, Am. J. Botany 39, 731 (1952).
88 J. B. Neilands, J. Am. Chem. Soc. 74, 4846 (1952).
69 B.C. J. G. Knight and P. Fildes, Brit. J. Exptl. Pathol. 14, 112 (1933).
199 ., W. Jones and C. E. Clifton, J. Bacteriol. 65, 560 (1953).
191 G. M. Shull and W. H. Peterson, Arch. Biochem. 18, 69 (1948).
19 R. W. Bishop, M.S. Shorb, and M. J. Pelezar, Jr., Proc. Soc. Exptl. Biol. Med. 81,
407 (1952).
13 F. F. Tang, H. Wei, D. L. McWhirter, and J. Edgar, J. Pathol. Bacteriol. 40, 391
(1935).
194 P.F. Smith and H. E. Morton, Arch. Biochem. and Biophys. 38, 23 (1952).
598 NEW AND UNIDENTIFIED GROWTH FACTORS
in the usual manner. Even without Bi» , the deficiency state often became —
pronounced only when an abnormal stress was placed on the animal, e.g.,
195 KH}. Kitay and E. E. Snell, J. Bacteriol. 60, 49 (1951).
196 FA. Robinson, B. W. Williams, and L. H. Brown, J. Pharm. and Pharmacol. 4,
27 (1952).
197 G, Ostling and W. Nyberg, J. Pharm. and Pharmacol. 5, 46 (1953).
1988 H. T. Peeler and L. C. Norris, J. Biol. Chem. 188, 75 (1951).
198a Ht. Gyllenberg, M. Rossander, and P. Roine, Acta Chem. Scand. 7, 694 (1953). |
198b M.S. Shorb, and F. A. Veltre, Poultry Sci., 32, 924 (1953).
1%8¢ G, F. Springer and P. Gyorgy, Federation Proc. 12, 272 (1953).
1984 C, §. Rose, R. Kuhn, and P. Gyorgy, Federation Proc. 12, 428 (1953).
199 R. R. Omata, J. Bacteriol. 65, 326 (1953).
200 T). Perlman, Am. J. Botany 38, 652 (1951).
201 P. Ohlmeyer, J. Biol. Chem. 190, 21 (1951).
202 HY. Tria and O. Barnabei, Boll. soc. ital. biol. sper. 27, 133 (1951) [C. A. 47, 1196
(1953) ].
203 HY}. C. Foulkes, Biochem. J. 49, LX (1951).
204 H.C. Lichstein and R. B. Boyd, Proc. Soc. Exptl. Biol. Med. 79, 308 (1952).
205 V. R. Williams and J. F. Christman, J. Bacteriol. 65, 238 (1953).
206 J. van Overbeek, M. E. Conklin, and A. F. Blakeslee, Sczence 94, 350 (1941).
207 |, Duhamet and R. J. Gautheret, Compt. rend. soc. biol. 144, 177 (1950).
208 F.C. Steward and 8S. M. Caplin, Science 118, 518 (1951).
209 R. 8S. deRopp, J. C. Vitueci, B. L. Hutchings, and J. H. Williams, Proc. Soc. Exptl.
Biol. Med. 81, 704 (1952).
210 G. Segretain, Compt. rend. soc. biol. 235, 1342 (1952).
211 J. R. Mauney, W.S. Hillman, C. O. Miller, F. Skoog, R. A. Clayton, and F. M.
Strong, Physiol. Plantarum 5, 485 (1952).
2122, R.R. Williams and R. E. Waterman, J. Biol. Chem. 78, 311 (1928).
213 J. G. Lee and A. G. Hogan, Missouri Agr. Expt. Sta. Bull. 342 (1942).
214 V. Reader, Biochem. J. 23, 689 (1929).
215 G. M. Briggs, T. D. Luckey, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 150,
11 (1948).
216 C, W. Carter, H. W. Kinnersley, and R. A. Peters, Biochem. J. 24, 1844 (1930).
217 'T. F. Macrae and C. K. Edgar, Biochem. J. 31, 2225 (1937).
218 KH}. Centanni, Biochim. e terap. sper. 22, 137 (1935); cited in H. R. Rosenberg,
Chemistry and Physiology of the Vitamins, p. 522. Interscience Publishers, New
York, 1945.
219 H. yon Euler, F. Schlenk, L. Melzer, and B. Hégberg, Z. physiol. Chem. 258, 212
(1939).
220 G. M. Briggs, T. D. Luckey, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 148,
163 (1943).
221 HY, R. Norris and J. J. Majnarich, Sctence 109, 32, 33 (1949).
222 HY. R. Norris, personal communication.
223 A. G. Hogan, N. B. Guerrant, and H. L. Kempster, J. Biol. Chem. 64, 113 (1925).
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225 'T. H. Jukes, J. Biol. Chem. 184, 789 (1940).
226 G. Lunde and H. Kringstad, J. Nutrition 19, 321 (1940).
227 C, A. Elvehjem, C. J. Koehn, and J. F. Oleson, J. Biol. Chem. 115, 707 (1936).
228 HT. R. Rosenberg, Chemistry and Physiology of the Vitamins, pp. 197, 469. Inter-
science Publishers, New York, 1945.
iJ
=
as
op
VIII. MISCELLANEOUS FACTORS 599
by feeding thyroid extract. In the future, metabolites essential for the rat
and certain other species may be best revealed through the application of
external stresses to induce deficiencies. Similar stress devices have been
employed successfully upon microbial populations, through the use of anti-
metabolites, and the creation of mutants that are incapable of carrying
out normal metabolic reactions. Pteroyl glutamic acid (Chapter 13) and p-
hydroxybenzoic acid (Chapter 12) were discovered by these means. Since
the limit to the number of derived systems that may be produced through
these approaches is remote, they offer many possibilities for discovering
pathways of metabolism that are at present obscured because of the ab-
sence of suitable test systems. Turning again to ‘‘normal”’ strains of or-
ganisms, the fruitful researches on Tenebrio and other insects may signal a
shift of interest among investigators toward the lesser known phyla of the
229 W. Nakahara, F. Inukai, and 8. Ugami, Science 87, 372 (1938).
230 W. Nakahara, F. Inukai, and 8S. Ugami, Sci. Papers Inst. Phys. Chem. Research
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436 (1948).
231 S. Ugami, Bull. Inst. Phys. Chem. Research (Tokyo) 21, 1006 (1942); cited in B. L.
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232 A. E. Schumacher, G. F. Heuser, and L. C. Norris, J. Biol. Chem. 135, 313 (1940).
233 T. H. Jukes and E. L. R. Stokstad, Physiol. Revs. 28, 51 (1948).
234 M. L. Scott, L. C. Norris, and G. F. Heuser, J. Biol. Chem. 167, 261 (1947).
235 W. Goetsch, Experientia 3, 326 (1947).
236 A. Wacker, H. Dellweg, and E. Rowold, Klin. Wochschr. 29, 780 (1951) [C. A. 46,
5155 (1952)].
237 TD). Metcalf, G. J. Hucker, and J. C. Carpenter, J. Bacteriol. 51, 381 (1946).
238 H. P. Sarett and V. H. Cheldelin, J. Biol. Chem. 165, 153 (1944).
239 FE. L. R. Stokstad and P. D. V. Manning, J. Biol. Chem. 125, 687 (1938).
40 B. L. R. Stokstad, P. D. V. Manning, and R. E. Rogers, J. Biol. Chem. 132, 463
(1940).
241 T). J. Davis, J. Infectious Diseases 21, 392 (1917).
242 A. Lwoff and M. Lwoff, Proc. Roy. Soc. (London) B122, 352 (1937).
243 M.A. Boas, Biochem. J. 21, 712 (1927).
244 HT. Chick, A. M. Copping, and M. H. Roscoe, Biochem. J. 24, 1748 (1930).
245 TI). W. Woolley and H. Sprince, J. Biol. Chem. 153, 687 (1944).
46 H. J. Almquist and E. L. R. Stokstad, Nature 187, 581 (1936).
247 H.R. Bird, C. A. Elvehjem, and E. B. Hart, J. Biol. Chem. 114, x (1936).
248 C. W. Mushett and W. H. Ott, Poultry Sci. 28, 850 (1949).
249 FE. C. Barton-Wright, W. B. Emery, and F. A. Robinson, Biochem. J. 39, 334
(1945).
250 T,. G. Colio and V. Babb, J. Biol. Chem. 174, 405 (1948).
251 V. H. Cheldelin and T. R. Riggs, Arch. Biochem. 10, 19 (1946).
252 B,. L. Hutchings, N. H. Sloane, and E. Boggiano, J. Biol. Chem. 162, 737 (1947).
253 A. P. Nygaard and V. H. Cheldelin, J. Bacteriol. 61, 497 (1951).
254 D. Rogers, T. E. King, and V. H. Cheldelin, Federation Proc. 11, 454 (1952).
255 W. W. Westerfeld and D. A. Reichert, J. Biol. Chem. 184, 163 (1950).
256 E. C. DeRenzo, E. Kaleita, P. Heytler, J. J. Oleson, B. L. Hutchings, and J. H.
Williams, J. Am. Chem. Soc. 75, 753 (1953).
600 NEW AND UNIDENTIFIED GROWTH FACTORS
animal kingdom, with the probability that many important new meta-
bolites may be exposed to view, and the assurance that our knowledge of
comparative biochemistry will be greatly enriched. Though many of these
metabolites may come to fall outside the biocatalyst class, it is neverthe-
less the continued unveiling of metabolic machinery to which the investi-
gators in this field are dedicated, and their progress will be followed keenly
by all who would gain an insight into the economy of the cell.
Author Index
Numbers in parentheses are footnote numbers. They are included to indicate the reference when
an author’s work is cited but his name does not appear on the page.
A
Aaes-Jorgensen, E., 513
Aberhalden, E., 409, 445
Aberhalden, R., 409, 445, 544, 545 (155),
555
Abraham, E. P., 344
Abraham, J., 282
Acheson, R. M., 331
Ackermann, W. W., 20, 70, 129, 202
Ackroyd, W. R., 387
Adams, G., 485
Adams, R.., 3, 9
Adams, R. R., 331
Adamson, J. D., 400
Adamstone, F. B., 515 (24a, 30, 102, 108,
112, 113), 519, 524 (24a), 534, 535, 536,
539 (113), 572
Adler, E., 344, 373 (3)
Adrian, J., 270
Aegerter, E. E., 558
Agadjanian, N., 565
Agarwala, S. C., 24, 26 (48), 35 (45), 36
(62)
Agid, R., 468
Agnew, L. R. C., 266
Aguiar, A., 521
Ahmann, F. F., 7
Aichner, F. X., 318
Albanese, A. A., 458, 480
Albert, A., 4, 308
Albertson, N. F., 9
Alcayaga, R., 273, 274 (83)
Alcayaga, S., 273
Alcock, A. W., 363, 364 (17)
Alden, H. S., 398, 402 (44)
Aliminosa, L., 52, 53 (9)
Allchorne, E., 509, 511
Allen, W., 111
Allfrey, V., 162
Allfrey, V. G., 95
Allgeier, A. M., 147, 175
601
Almquist, H. J., 386, 395 (22), 596
Aloisi, M., 515 (69a), 526
Americano Freire, 8., 515 (86, 90), 530
Ames, 8. R., 237, 241, 268, 537, 502, 559
Amiard, G., 443
Anderegg, L. T., 485
Andersag, H., 226, 227 (18), 405, 411, 412,
414, 416, 420 (28), 422, 423, 424
Anderson, C. R., 60, 79
Anderson, G. C., 397
Anderson, H. D., 515 (61a), 525
Anderson, R. C., 28 (17), 53, 103, 109 (30),
143 (20)
Andrea, W. A., 392
Andrews, C. H., 79
Andrews, D. H., 3
Andrews, G. A., 47, 61, 85, 210, 212 (195),
213 (195)
Andrews, J. 8., 513
Andrus, M., 195
Andus, L. J., 77
Anfinsen, C. B., 35 (48)
Angevine, D. M., 593
Angier, R. B., 28 (12, 13, 14, 15), 39, 71,
95, 96, 98 (21, 24), 99 (24), 101, 103
(24), 104 (27), 105, 106 (27, 34), 108
(33, 35), 109 (27), 118, 114 (23), 115
(23), 121, 122 (1), 128 (2), 143 (@, 3, 5,
6, 15, 16), 149 (57), 154 (57), 161
Anigstein, L., 61, 79 (11, 12, 13, 14)
Anisfeld, L., 505
Anker, W., 77
Annoni, G., 55
Ansbacher, S8., 37, 38, 41, 42, 52, 56, 58,
59, 83, 317
Ant, M., 569
Antopol, W., 247, 265, 267, 283, 292, 391,
394, 468
Antoshkiw, T., 4, 5
Aposhian, H. V., 326, 327 (92a), 394
Apter, N. S., 290
602
Aramburu, T., 148 (17)
Archdeacon, J. W., 50, 54
Archer, W., 386, 395 (24)
Arias, J., 470
Aring, C. D., 469
Armstrong, B. E., 211
Armstrong, K. L., 269
Arnall, F., 4
Arnold, A., 306, 410, 477
Arnold, H., 304
Arnold SW... 0le 7927)
Arth, G. E., 28 (17), 103, 109 (30), 143 (20)
Artom, C., 291, 294
Arzberger, C. F., 360
Ascenzi, A., 515 (69a), 526
Ascham, J. K., 250
Ascher, K. W., 391
Asdell, S. A., 515 (58, 59), 524, 530 (59)
Asenjo, C. F., 173
Ashe, W. F., 283, 388, 390 (120, 121)
Ashburn, LL. ., 71, 171, 174; 473, 516,
540 (6)
Ashenbrucker, H., 187
Asmundson, V.S., 275, 297, 386, 395
Astwood, E. B., 40, 57
Athanassiu, G., 545
Atkin, L., 248, 254, 257 (8), 259 (8), 260
(8), 262 (8), 276, 279, 280, 411, 450,
452 (14), 462, 465
Atkinson, R. L., 594
Atkinson, W. B., 515 (97), 531
Auerbach, M. E., 306
Auhagen, E., 9, 73, 427
Austin, F. L., 491
Austin, W. C., 318
Avakian, 8., 7, 9 (59), 153
Axelrod, A. E., 147, 266, 367, 384, 392,
393, 395, 402
Axelrod, H. E., 272
Axelrod, V., 392, 402
Aykroyd, W. R., 400
Azarkh, R. M., 239, 269
Azevedo, M. D., 459
B
Babb, V., 596
Babcock, G. M., 214
Babson, R. D., 320, 332
Bacharach, 484
Bacharach, A. L., 504, 505, 509, 511, 561
Bacher, F. A., 506, 508 (5)
AUTHOR INDEX
Bacigalupo, F. A., 515 (89), 530
Baddiley, J., 234, 239 (8), 240, 583
Bader, M. N., 61, 79 (11, 12, 13)
Badran, N., 308
Baer, 8., 565
Bahr Py He sod
Bahrs, A. M., 591
Bailey, A. E., 489, 497, 498
Baily, J., 43, 48 (2), 53
Bain, J. A., 308
Bakelmann, E., 588
Baker, A. B., 283
Baker, G. A., 539
Baker, L. A., 72 (53)
Baker, L. D., 283
Bakst, H., 565
Baldini, M., 55
Ball, C. T., 546, 552 (170), 553 (170), 554
(170)
Ball, E. G., 35 (48), 348
Ball, K. P., 565
Ballantyne, R. M., 268, 270
Ballard> Me Pied
Ballard, S. A., 120
Bambas, L., 106
Bang, “J., 75
Banga, I., 337, 427, 428, 485
Banga, J., 303
Bantz, A. C., 22, 35
Barakat, M. Z., 308, 368
Barborka, C. J., 479
Bardos, T. J., 110, 111, 114 (73), 118
Barger, F. L., 118
Barger, G., 448
Barker, D., 386, 395 (24)
Barker, H. A., 576, 577 (8), 582 (8)
Barker, W. H., 283
Barlow, O. W., 58, 63
Barlow, R. B., 328
Barltrop, J. A., 582, 583 (48)
Barnabei, O., 595
Barner, H. D., 581
Barnes, L. L., 571
Barnes, R. H., 503
Barrett, M., 397
Barrie, M. M. O., 521
Barritt, A. S., 58, 62 (61), 83
Barron, HE. 8. G., 341, 480, 484, 441
Barsky, J., 281
Bartels, W. E., 559, 560
Bartlett, P. D., 272
AUTHOR INDEX
Barton, A. D., 408
Barton, M. N., 272, 274 (77), 275 (77)
Barton-Wright, E. C., 596
Bartos, J., 448
Bastedo, W. A., 247
Baude, R., 371
Bauer, A. C., 592
Bauernfeind, J. C., 363
Baum, G. L., 565
Baumann, C. A., 45, 110, 185, 262 (89),
270, 271 (65), 294, 295, 296, 505
Baur, E., 15
Baxter, J. G., 489, 490 (56), 492, 493 (93),
496, 497 (5), 500, 502 (7), 507, 508, 513,
537
Baxter, J. H., 594
Bay, F., 571
Beadle, G. W., 23, 26 (41, 42), 32, 33, 34,
35 (2, 3, 46), 72 (61), 378
Beam, A. 8., 400
Bean, W. B., 283, 292, 388, 390 (120)
Beard, 8. H., 10
Beare, J. L., 267, 295
Beaton, J. R., 267, 270, 295
Beaver, J. J., 502, 507, 508
Beck, S. D., 594
Becker, B., 358
Becker, J. E., 511
Becks, H., 383, 384 (59)
Beerstecher, B., Jr., 298
Beerstecher, E., 298, 446
Beerstecher, E., Jr., 69, 79, 143 (25),
149 (25), 270, 298, 301, 408, 476
Behan, I. T., 571
Beher, W. T., 392
Beihler, R., 507
Beilstein, F., 6
Beinert, H., 72 (60)
Belding, H., 211
Bell, J. M., 571
pew, CL. 447
Bellamy, W. D., 235, 239, 240 (6), 278
Belt, M., 126, 130, 155, 156, 178, 187, 386,
387 (96), 395 (23), 576
Bencze, B., 545
Bendas, H., 337 (22)
Bender, R. C., 250, 395, 477
Bendlich, A., 114, 115 (86)
Ben-Ishai, Ruth, 131
Benjamin, Z. H., 565
Bennett, L. L., Jr., 133
603
Bennett, M. A., 140
Bennett, M. J., 22
Benseley, EK. H., 569
Benson, A. A., 582
Benz, F., 315, 316 (54), 334, 358
Berezovskil, V. M., 318, 323 (68)
Berg, C. P., 268
Berg, L. R., 594 (159)
Bergeim, O., 11, 21
Bergel, F., 320, 405, 411, 412, 413, 414,
415, 416, 418, 419, 420, 423 (47), 424,
448, 486, 487 (30), 488 (53), 492 (26)
Berger, E., 318
Berger, H., 565 (27)
Berger, L., 315, 318
Bergman, W., 285
Bergmann, E. D., 131
Berish, H., 569
Berman, L., 57, 84
Bernheim, F., 235 (58)
Bernheim, M. L. C., 235 (58)
Bernstein, §., 591, 592 (132)
Berryman, G. H., 46, 47 (24), 53, 360, 392,
401
Berryrand, L. J., 476
Bessey, O. A., 304, 349 (75), 362, 367 (3),
368, 371, 373, 380, 381 (28), 382 (28),
383, 384 (28), 390 (88), 398, 449, 477
Best, M. M., 79 (15)
Betheil, J. J., 187, 594 (165)
Bethell, F. H., 47, 61, 85, 143 (12), 159,
191, 202, 205, 206, 210, 211, 212 (195),
213 (195), 215, 216
Bethke, R. M., 386, 395 (21, 22)
Beyer, K. H., 14, 39, 40, 49, 72 (54)
Bhagvat, K., 398, 460
Bhattacharyya, P. K., 584
Bichel, J., 85
Biddulph, C., 521
Biehler, R., 551, 552 (193)
Bielschowsky, F., 68
Biely, J., 505
Bierbaum, O. §., 209
Biesele, J. J., 160
Bijdendijk, A., 566
Billen, D., 234 (43), 235 (43)
Bina, A. F., 246, 257 (10, 11), 260 (11)
Binet, L., 462
Binkley, F., 237, 239, 269
Binkley, S. B., 92, 93 (10), 94, 99 (10),
100 (10), 164, 165, 176, 178, 201
604
Binnington, D. S., 513
Biocca, E., 85
Birch, T. W., 4, 222, 224, 250, 255, 265,
284, 461
Bird, F. H., 275, 297, 386, 395
Bird, H. R., 138, 473, 515 (105), 535, 596
Bird, O. D., 36 (60), 40, 92, 94, 143 (12,
13, 14), 152 (68), 159, 164, 165, 166,
176, 177, 178, 210, 212 (195), 213 (195),
248, 260 (84), 505
Birkhauser, H., 468
Birkhofer, L., 317
Birkinshaw, J. H., 356
Birnbaum, J. P., 521
Bishop, K. 8., 483
Bishop, R. W., 595 (192)
Bissell, A., 57
Black, A., 305, 410 (12)
Black, S., 38, 71, 75,2171, 172, 189, 196,
272
Blakeslee, A. F., 595 (206)
Blakeslee, L. H., 386
Blanchard, K. C., 27, 28, 30 (4), 67
Blanchard, M., 16, 28, 3538, 354 (91)
Blandau, R. J., 520, 532 (36)
Blaschko, H., 234, 235 (2, 52, 56, 57), 241
Blatt, M. G. H., 567
Blaxter, K. L., 509, 515 (64a, 64b, 64c),
525, 530 (64a, b, c)
Blecha, E., 205
Blewett, M., 147, 191, 379, 583, 584 (54)
Bleyer, A., 303
Bliss, C. I., 250, 370
Bliss, E. A., 68
Block, F., 48
Bloom, E. S., 28 (16), 92, 93 (10), 94, 99
(10), 100 (10), 101 (28), 102, 103 (28),
143 (14), 164, 165, 176, 177, 178, 201
Bloomberg, B. M., 47
Blyth, A. W., 303
Boas, E. P., 565
Boas, M. A., 596
Boaz, H., 578
Bobb, A. L., 565
Bock, R. M., 580, 581 (37)
Bodran, N., 368
Boe, N., 539
Boehrer, J. J., 389
Bohonos, N., 91, 93, 94, 108 (15), 175, 177,
199, 595 (186)
Bohr, D. F., 268, 287
AUTHOR INDEX
Boéklen, E., 414
Bollman, J. L., 62, 82
Bolton, W., 386
Bomskoy, C., 145, 146 (31)
Bond, T. J., 110, 111,194 (3)
Bone, J. F., 187
Bonetti, E., 515 (69a), 526
Bonetti, G., 234
Bonner, D., 239
Bonner, H., 264
Bonner, J., 78, 264, 379, 446, 447 (3), 448
Booher, L. E., 334, 571
Booker, L. E., 303
Bookman, J. J., 392, 401, 569
Booth, V. H., 347, 349, 500
Boothe, J. H., 28 (12, 13, 14, 15), 39, 71,
95, 96, 98 (21, 24), 99 (24), 101, 103
(24), 104 (27), 105, 106 (27, 34), 107,
108 (88, 35), 109 (27), 118, 114 (28),
115 (23), 121, 122 (1), 12312) eone
3, 5, 6, 15, 16), 149 (57), 154 (57), 161
Borasi, M., 66
Borek, E., 44
Boruff, C. 8., 333
Bosc, R., 284
Bosse, M. D., 147
Bosshardt, D. K., 587, 594 (149, 167)
Bottomley, A. C., 248
Boulanger, P., 308, 309, 326 (86), 357,
359, 391
Bouman, J., 448, 457
Bourke, A. R., 79 (10)
Bourne, G. C., 79 (82)
Bourquin, A., 302, 369, 370, 373
Boutwell, R. K., 270
Bovarnick, M., 330
Bowden, F. P., 485
Bowles, L. L., 381
Boyd, A. M., 566
Boyd, E. M., 467
Boyd, M. J., 277
Boyd, R. B., 595
Boyer, P. D., 491, 495
Boysen-Jensen, P., 379
Bradway, E. M., 512
Bragdon, J. H., 515 (85), 530
Braitman, M., 569
Brand, E., 587
Brandaleone, H., 60, 83
Brandenberg, R. O., 148 (17)
AUTHOR INDEX
Bratton, A. C., 11, 19, 20, 28, 31, 96, 98
(22), 162
Bratzler, J. W., 513
Braushtein, A. E., 237, 238, 239, 268, 269
Bray, H. G., 46, 47
Brdicka, R., 310
Bredereck, H., 318
Brendler, H., 196
Brenner, 8., 557
Bressler, B., 166
Brewer, W., 399
Briggs, G. M., 177, 196, 197, 275, 297, 594,
596 (215, 220)
Briggs, G. M., Jr., 33, 39, 42, 90, 176
Brinkhous, K. M., 501, 515 (24, 73), 518,
526, 528 (24)
Brisson, G. J., 385
Britschneider, L. H., 515 (17), 517
Brockman, J. A., Jr., 111, 113 (76), 197,
198, 577 (16), 578
Brockway, D. R., 395 (27)
Brody, 8., 270
Brémel, H., 351, 352 (85), 369
Brook, A. J., 590
Broquist, H. P., 89, 110, 111, 113 (76), 114
(73), 121, 136, 137, 159, 197, 198, 214,
279, 576, 581
Brown, E. A., 505
Brown, E. B., 113, 246, 257 (10, 11), 260
(11)
Brown, E. V., 308
Brown, F., 496, 509, 525
Brown, G. B., 114
Brown, G. M., 40
Brown, H., 283
Brown, L. H., 595 (196)
Brown, O. W., 3
Brown, R., 594 (178, 179)
Brown, R. A., 59, 92, 94, 143 (12, 13, 14),
152, 153 (68), 166, 168, 176, 177, 178,
210, 212 (195), 213 (195), 294, 473
Brown, W. E., 333
Browne, J.S. L., 392
Brozek, J., 478, 479
Brozek, J. H., 389, 401
Bruce, H. M., 570
Bruce, W. F., 177, 178 (42)
Bruckner, A., 10
Bruins, 327
Bruins, H. W., 327, 588, 594 (145)
Bryan, W. L., 510, 513, 515 (25), 519
605
Buchanan, J. M., 132, 434
Buchman, E. R., 406, 407, 418, 421, 424,
430, 431, 441, 447, 465
Buchwald, K. W., 4, 5
Buck, J. S., 7, 9 (55)
Buckholder, P. R., 379
Buhs, R. P., 502, 506, 509
Bullock, M. W., 578
Burch, H. B., 362, 367, 393, 449
Burchenal, J., 161
Burchenal, J. H., 143 (7), 151, 152, 159,
160, 161, 197, 214
Burchenal, J. R., 151, 152 (66), 160 (66),
161 (66), 347
Burgess, J. F., 369
Burk, D., 42, 61
Burke, K. A., 276
Burkett, H., 331
Burkholder, P. R., 163, 257 (19, 20, 25),
360
Burnett, W. B., 9
Burr, G. O., 474, 484, 485, 503, 513, 515
(14, 50), 517, 519, 522
Burstein, C. L., 55
Burt, O. P., 285
Burt, R. C., 557
Biising, K. H., 266
Busnel, R. G., 380
Bussmann, G., 489
Bustad, L. K., 42, 187
Butler, B., 278, 279 (131)
Butler, R. E., 388
Butt, H. R., 61, 81, 82 (51), 283
Butturini, U., 569
Byerrum, R. U., 477
Bywater, W. G., 57
C
Cabell, C. A., 511
Caccese, A., 215
Cahill, J. J., 316, 329 (59)
Cain, C. K., 152
Cajori, F. A., 480, 437
Calam, C. T., 594 (177)
Calkins, D. G., 28 (16), 94, 148 (14), 177,
178
Calkins, V. P., 494, 503
Callandre, A., 443
Calverley, C. E., 515 (87), 530
Calvin, M., 580, 582 (38, 39), 583 (39, 48)
Cameron, W. R., 566
606
Camien, M. N., 586
Cammarata, P. S., 236
Campbell, B., 62, 85
Campbell, C. J., 94, 143 (14), 166, 168,
176, 177,178
Campbell, J. A., 500
Campling, L. M., 265
Canbiack, T., 249, 500
Cantarow, A., 48
Cantor, M. M., 284
Caplin, S. M., 595
Card, L. E., 515 (30), 519
Carlson, C. W., 594 (147)
Carlson, G. H., 230, 320
Carlson, W. E., 458
Carman, J. S., 483
Carpenter, G., 208
Carpenter, J. C., 596 (237)
Carpenter, K. J., 266
Carpenter, L. E., 231, 503, 572
Carretero, R., 281, 458, 480
Carrol, F. D.; 261 «37)
Carter, B. B., 266
Carter, C. W., 269, 296
Carter, H. E., 584
Cartwright, G. E., 147, 186, 187, 188 (76),
266, 273, 274 (83), 296
Carvallo, J. R., 588
Carver, J. 8., 594 (159)
Cash, R. L., 79 (16)
Casselman, W. G. B., 516
Castagna, R., 566
Caster, W. O., 451, 476
Castle, W. B., 143 (7), 206
Caswell, M. C., 277
Cattaneo, P., 545, 546 (165), 552 (165)
Cavalieri, L. F., 114, 115 (86), 590
Cavill, G. W. K., 85
Cei, C., 55
Centanni, E., 596
Cerecedo, L. R., 44, 52, 147, 175, 189, 267,
270, 295, 410 (11)
Chaiet, L., 109, 143 (21)
Chaikoff, I. L., 41, 57
Chailley, H., 545
Chandler, J. P., 47, 53
Chapin, 8. J., 559
Chapman, D. G., 500
Chapman, O. D., 182
Charalampous, F. C., 44, 45, 52
Charipper, H. A., 41, 50, 54, 57 (27)
AUTHOR INDEX
Chase, E. C., 320, 327
Chassin, M. R., 292
Chattaway, F. W., 35 (87), 586
Cheetham, H. C., 3
Cheldelin, V., 217
Cheldelin, V. H., 22, 163, 204, 257 (6, 17),
259 (6, 27), 260 (6, 31), 261 (6), 262
(6), 307, 308 (28), 373, 376, 451, 577,
590, 591, 592, 596 (122, 238, 253, 254)
Chelintzew, G. W., 419
Chen, J. L., 306
Chen, K. K., 53, 292, 293 (24), 409
Cheng, D. W., 520
Cherbuliez, E., 10
Cherrington, M. E., 188, 191 (80), 552
(202), 553 (199), 554 (202), 558 (202)
Chevrel, M. L., 515 (23), 518
Chiancone, F. M., 287
Chiang, R., 270
Chick, H., 266, 381, 596 (244)
Chiefh, M., 465, 552, 568
Chin, C., 333
Chipault, J. R., 489, 503, 513
Chisholm, J. F., Jr., 548 (180)
Chodat, F., 78
Chodos, R. B., 213
Chor, H., 515 (82a), 529
Chornoch, C., 478
Christensen, B. E., 592
Christensen, C. M., 85
Christensen, E., 6
Christensen, G. M., 237, 239, 269
Christian, W., 303, 309, 334, 335, 337 (1,
13, 23, 24) 338, 339, 341 (21), 342, 348
(23), 348, 351, 352, 369
Christman, C. C., 487
Christman, J. F., 595
Christy, €. J., 567
Chung. FLW, 797)
Cintron-Rivera, A. A., 218
Ciusa, W., 584
Claisen, L., 414
Clark, F. M., 35 (35, 36)
Clark, I., 291, 292 (11)
Clark, J. D., 298, 469
Clark, J. R., 206
Clark, M. C., 216
Clark, P. F., 472
Clarke, H. T., 407, 418, 421
Clarke, M. F., 251
Clausen, D. F., 503
I ———— SS
AUTHOR INDEX
Clausen, M., 503
Clayton, M. M., 488
Clayton, R. A., 595 (211)
Cleckley, H. M., 388, 390 (124), 391 (123,
124)
Clement, D. H., 61,79 (41)
Clemmesen, J., 557
Clifton, C. E., 595
Climenko, D. R., 38
Cline, J. K., 405, 409, 411, 412, 413, 422,
424, 425
Clinton, R. O., 7, 9 (55)
Clowes, G. H. A., 42, 72 (53)
Clutterbuck, P. W., 180, 204
Coates, M. E., 371
Coburn, A. D., 16, 28
Cochrane, T., 569
Coey, W. E., 539
Cogan, G. M., 537, 546, 547 (176)
Cohen, A., 320, 418
Cohen, F. L., 3
Cohen, I., 512
Cohen, L., 291, 296
Cohen, L. J., 569
Cohen, P. P., 13, 14 (3, 4, 5), 49, 53, 235
(61), 236, 241 (36), 278
Coisnard, J., 466
Colby, R. W., 187
Coleman, H. B., 410
Coles, M., 191
Colio, L. G., 596
Collins, G., 479
Colman, J., 420
Colsky, J., 198, 214
Combes, A. J., 459
Combes, F. C., 471
Combs, G. F., 593, 594 (143, 148)
Comline, R. S., 304, 369
Conger, TL: W.,; 251
Conklin, M. E., 595 (206)
Conn, J. B., 109
Conner, G. H., 42
Conner, R. T., 363
Conners, C. A., 402
Consolazio, F. C., 402
Constant, M. A., 592, 593
Contab, M. A., 89 (1), 90
Cook, G. M., 381
Cooperman, J. M., 42, 184, 185, 260 (35),
386, 395, 589, 594
Cope, A. C., 7, 9 (52, 53, 54)
607
Copeland, D. H., 540
Copping, A. M., 257 (22), 486, 487 (30),
488, 596 (244)
Cordy, D. R., 42
Corin, G., 455
Cormier, M., 515 (23), 518
Cornatzer, W. E., 291
Corran, H. §8., 343, 344, 347, 348, 350 (49)
Coryell, M. N., 392, 474
Cosgrove, K. W., 295
Costello, M., 60
Cosulich, D. B., 28 (12), 39, 71, 96, 98 (21,
24), 99 (24), 101, 103 (24), 104 (27),
105, 106 (27, 34), 108 (33, 35), 109 (27),
111, 113 (76), 114 (23), 115 (28, 29, 90),
116 (90), 117 (90), 118 (29, 90), 119
(90, 95), 120 (90, 95), 121, 122 (1),
123 (2), 143 (15, 16), 149, 155 (68),
156 (77), 157 (58, 74)
Cottingham, E., 297, 474, 475
Couch, J. R., 594
Coulfield, W. J., 360
Coulthard, C. E., 356
Couperus, J., 513
Coursin, D. B., 288
Coward, K. H., 388, 450
Cowdry, E. V., 30 (20), 31
Cowgill, G. R., 41, 259 (29), 260 (29), 272,
381, 384 (35), 386, 395, 397, 472, 474,
478
Cox, sR. Pe, 004
Cracas, L. J., 172, 477
Craig, J. A., 40
Craig, R., 379
Crammer, J. L., 304, 362, 368
Crampton, E. W., 395, 571
Cravens, W. W., 158, 159, 198, 275, 290,
297, 327, 386, 395 (22), 588, 594
Crawford, B., 52, 53 (9)
Crawford, E. J., 349 (75)
Crawford, J. H., 389, 401
Creaghan, M. V., 569
Credner, K., 235 (48, 49, 50)
Criddle, J. E., 503
Cross, M. J., 1385, 136 (37)
Cruickshank, A. H., 51, 55
Cuellar, F. T., 56
Culik, R., 386, 515 (89), 530
Culton, T. G., 515 (105), 535
Cunha, T. J., 42, 187
Cunningham, E., 243
608
Cunningham, R. W., 216
Cunnings, H. W., 515 (81), 519, 525 (31)
Curtis, A. C., 47, 85
Curtis, D., 7, 9 (56, 57)
Cushing, J. E., 34, 35 (15), 76
Curd gH HS. (330
Curtis, J. J., 260 (86)
Curtisul bs 3c
Cusick, P. L., 389, 401
Cuthbertson, W. F. J., 487, 501, 513
Cutting, W. C., 11, 20
Cutts, N.S., 35 (63, 55)
Czaczkes, J. W., 393, 395, 396 (8)
D
Dabrowska, W., 144, 164
Dack, G. M., 183
Daft, F. S., 39, 71, 189, 148 Q), 147. @Q),
W7AlS iGP4, lliges, Wek Gls, US) ISO) (IED),
382, 397, 473, 516 540 (6)
Dale, 462
Dalgliesh, C. E., 238
Dam, H., 505, 511, 513, 515 (64, 103, 104,
111), 517, 525, 534 (64), 535, 536 (104),
538 (10, 12), 541 (10), 557, 566, 570
Dana, A. 58., 188
Danciger, J. A., 79 (20)
Daniel, E. P., 266
Daniel, L. J., 149, 152 (55), 1538, 154 (70),
177, 588
Danielli, J. F., 487
Danielopolu, D., 56
Daniels, T. C., 6
Daniels, W. B., 206
Danne Ee ee os. lO
Dann, W. J., 250, 265, 473, 476
Dansi, A., 10, 72 (63)
Darby, W. J., 90, 91 (2), 182, 188, 191 (80),
194, 206, 207, 209, 370, 381, 400, 546,
552 (170, 202), 553 (199), 554 (202,
203), 558 (202)
Davenport, H. W., 267, 291
Davenport, V. D., 267, 291
David, W. E., 234 (44), 235 (44)
Davidson, C. 8., 148 (7)
Davidson, L. 8. P., 205, 206
Davis, B. D., 16, 17, 18 (3), 35 (88, 39), 81
Davis, D. J., 596
Davis, E. P., 365
Davis, H. C., 61, 79 (28)
Davis, H. J., 79 (87, 38)
AUTHOR INDEX
Danis; Wed. 205
Davis, M. V., 401 (62), 478
Davis, O. L., 244, 369
Davis, V. E37
Davis, W. B., 568
Day, D., 264
Day, °P., 577 (6) ans
Dey, lel, Ca, 78
Day, L. A., 206
Day, P: L., 90, 91, 93,137; 138) 142aeane
143 (8), 146, 147 (88), 148, 163, 164,
180, 181, 182, 183, 184 (54), 194, 204,
370, 381, 383, 390 (86), 395, 558
de Beer, E. J., 466, 470 (6), 471 (6)
DeBey, H. J., 295
de Bollemont, G., 414
De Busk, B. G., 577, 578°:03, 27.28eeo
579, 582 (14, 26)
Decherd, G. M., Jr., 469, 470
de Caro, L., 473
Dedichen, J., 565
de Elio, Javier, F., 56
deGrunigen, A., 103
de Gutierrez-Mahoney, W., 515 (52a),
522, 532
deHaahn, P. G., 68, 69 (29), 70 (29), 127,
128 (10), 129 (10), 185 (10)
Deiss, W. P., 53
de Jong, 8., 458, 462
de la Huerga, J., 190
del Campillo, A., 579
Delfs, E., 561
Dellweg, H., 596 (236)
Delor, R. A., 591
De Meillon, B., 191
de Metry, J. P., 537, 547
Demole, V., 309, 391, 467, 488, 489, 515
(94), 531, 563
Denis, W., 245
Denko, C. W., 46, 47, 53, 360, 392, 401
Denny-Brown, D., 560
Denton, C. A., 138
DeRenzo, E. C., 270, 295, 596 (256)
De Ritter, E., 805, 363, 366, 589
Dermer, O. C., 10
de Ropp, R. 8., 159, 595 (409)
Desnuelle, P., 340
Deuel, H. J., Jr., 374, 505
Deutsch, H. F.,, 308, 459
Devi, P., 398, 460
DeVilbiss, L. A., 59, 84
AUTHOR INDEX
Devlin, H. B., 507
Dewan, J. G., 348, 347 (63), 348 (63)
Dewey, V. C., 143 (11, 18), 159, 277, 290,
291 (4), 576
Dhellemmes, G., 56
Dias, M. V., 468
Diaz, L. A., 59, 84
Diaz-Rivera, R. 8., 79 (18)
Dichek, M., 383
Dicken, D. M., 22, 26 (28), 30 (19), 31,
34, 35 (17), 67, 77
Diczfalusy, E., 79
Diels, O., 414, 417 (31)
Dietrich, L. 8., 202
Dietzel, E., 487, 493
Di Ferrante, N., 48
Dikanskaya, E. M., 309
Dimant, E., 322
Di Maria, G., 379
Dimick, M. K., 250, 266
Dingwall, R. W., 467
Dinning, J. S8., 137, 138, 142, 538, 558
Dixon, M., 348, 349
Dju, M. Y., 501, 507, 548, 544, 545 (157,
158, 159), 546 (157, 158, 159), 555
(157, 158, 159), 559 (159), 571, 573
Doan, C. A., 181, 182, 184, 195 (64), 207
Doisy, E. A., Jr., 44, 473
Dolby, D. E., 586
Dolin, M. I., 579
Dolkart, R. E., 515 (82a), 529
Donath, W. F., 405, 410
Donegan, C. K., 565
Donovan, G. E., 283
Dorfman, F., 472
Dorfman, L., 591, 592 (132)
Dornbush, A. C., 590
Dorr, A. L., 395 (27)
Dorsey, C. W., 285
Dotti, F., 470
Doub, L., 4, 5 (28), 107
Doudoroff, M., 378
Doyle, A. M., 283
Dragutsky, D., 283
Draper, H. H., 395, 515 (59a), 524, 530
(59a)
Dreese, W. C., 568
Dreger, E. E., 9
Dreisbach, P. F., 106, 107, 124
Drilhon, A., 380
Drekter, L., 589
609
Drill, V. A., 294, 474
Druce, J. Go F.,.7
Drummond, J. C., 405, 461, 484, 486, 487,
489, 492 (58), 497, 501, 513
Dry, T. J., 61, 62, 81, 82 (51)
Drysdale, G. R., 138
Duane, R. B., 61, 79 (85)
Dubin, A., 553, 554 (201)
Dubois, A., 455
Duchesne, G., 437
Dudley, H. W., 115
Dufait, R., 467, 468
Duhamet, L., 595
Dumm, M. E., 45
Dunford, R. A., 500
Dunn, M. S., 586
Durel, P., 63
Diirr, K., 491
Dutra de Olivera, J., 521
du Vigneaud, V., 61, 301
Dyer, H. A., 63
Dyniewicz, H. A., 505
Dziewiatkowski, D., 558
E
Eakin, R. E., 20, 69, 70, 79, 103, 110 (31),
129, 130, 148 (24, 25), 149 (25), 156,
270, 276, 298, 301, 408, 446, 451, 590
Eagle, H., 594 (182, 183)
Eaton, L. M., 283
Easterling, L., 265, 288 (9)
Ebnother, C., 234, 322
Eckardt, R. E., 390, 402
Eckert, H. W., 20
Keke, R. S., 51, 60, 79
Eckhardt, R. E., 265
Keckler, C. R., 292, 293 (24), 409
Eden, E., 500
Edgar, C. E., 251, 596 (217)
Edgar, J., 595 (193)
Edgerton, M. T., Jr., 568
Edison, A. O., 475
Edwards, P. C., 149, 153 (60), 154 (60)
Edwards, R. R., 138
Efimov, N. I., 78
Eggleston, L. V., 430, 433
Eichert, H., 565
Eide, I. M., 115 (30a), 519, 521 (30a)
Eigen, E., 121
Hijkman, C., 405
Hinarson, L., 515 (71, 80, 100b, 101a), 526,
610
527 (71), 528; 529) (71), 532, 533 (715
80)
Einhorn, A., 7
Eisen, H. N., 266
Eisen, M. E., 565 (26)
Ekblad, M., 533
Ekstrand, T., 27, 36 (59)
El-Dadr, M. M.., 381
Elftman, H., 515 (5), 516, 531 (5)
Elion, G. B., 149, 150, 151
Ellenberg, M., 389, 569
Eller, J. J., 59, 84
Ellinger, A., 44, 52, 54
Ellinger, P., 334
Ellinger, Ph., 302, 303
Ellis, G. P., 315
Ellis, L. N., 395
Ellis, N. R., 395, 511
ElSadr, M. M., 251, 266
Elsom, K. O’S., 478
Elvehjem, C. A., 33, 38, 39, 42 (85), 43,
59, 71, 75 (89), 90, 156, 165, 171, 172,
175, 176, 184, 185, 189, 190, 202, 204,
217, 237, 241, 251, 252 (48), 254, 257
(5, 7, 9, 23), 259 (7, 28, 30), 260 (5, 7,
9, 30, 35), 261 (5, 7, 9), 262 (, 9, 30,
39, 40), 268, 269, 270, 271 (65), 272,
274, 275, 276, 294, 297, 298, 349, 367,
370, 376, 377, 384, 385, 386, 392, 393,
395 (26), 396 (10), 402, 426, 428, 429,
430 (45), 481, 439, 459, 472, 477, 515
(61a, 115), 525, 536, 587, 588 (91),
594 (152, 168, 169, 170, 171, 172, 173),
596 (215, 220, 227, 247)
Elwyn, D., 137, 138, 198
Embree, N. D., 497
Emerson, G. A., 38, 52, 59, 174, 185, 265,
291, 301, 327, 328, 330, 393, 394, 408,
483, 486, 491, 492 (89), 515 (20, 55, 72),
518, 520, 524, 526, 532 (20, 34), 570,
571, 588, 589 (103), 594 (103, 166)
Emerson, K., Jr., 11, 20
Emerson, O. H., 483, 485, 486, 488, 489,
490 (19), 491, 492 (89)
Emerson, §., 76
Emmart, E. W., 323
Emmel, A. F., 515 (8, 4), 516, 517 (8, 4),
520 (3), 527, 580 (4), 531 (4), 541 (4)
Emmerie, A., 368, 392, 398, 497, 500, 506,
507, 508, 509, 513
Emery, W. B., 596 (249)
AUTHOR INDEX
Emmett, A. D., 92, 94, 164, 166, 168, 176,
177, 178, 248, 260 (34), 505
Emte, W., 487, 493, 494
Engel, C., 500, 501, 506, 507, 509, 515 (17),
517, 5438, 571
Engel, R. W., 269, 381, 387
Englard, 8., 335, 361
English, A., 140
Enria, G., 566
Ensminger, M. E., 273
Eppright, M., 397
Eppright, M. A., 18, 34, 163
Epps, H. M. R., 235 (9, 46)
Eppstein, S. H., 571
Epstein, M., 156
Ercoli, G., 55
Erickson, EK. M., 505
Erlenmeyer, H., 75
Ershoff, B. H., 38, 51, 54, 59, 270; 475,
) §21, 589, 594
Erspamer, V., 467, 468
Escher, R., 490, 492, 493
Esh, G. C., 505
Esselbaugh, N. C., 363
Etingof, R. N., 235 (10)
Evans, B. D. F., 180, 204
Evans, E. A., Jr., 330, 433, 434, 458
Evans, H. M., 38, 175, 250, 265, 383, 384
(59), 478, 474, 483, 484, 485, 486, 491,
492 (89), 515 (14, 20, 50, 51, 55, 72,
79), 517, 518, 519, 520, 522, 524, 526,
529, 532 (20, 34), 533 (79), 570, 571
Evans, M. K., 266
Everson, G., 360, 397
Eykman, 460, 461
Eyring, H., 378
Eysenbach, H., 355
Kyster, H. C., 7
Fabiano, A., 59
Fachini, G., 56
Fahnestock, M. K., 479
Fahrenbach, M. J., 28 (12), 39, 71, 89,
101, 104 (27), 105, 106 (27, 34), 108
(83, 35), 109 (27), 111, 113° @G) eaters
122 (1), 123 (2), 148 (15, 16), 155; 157
(74), 197, 198
Failey, R. B., 53
Fairbridge, R. A., 24, 26 (44)
Falco, E. A., 126, 149, 150, 151 (65)
AUTHOR INDEX
Farber, M., 502, 507, 509, 572, 592
Farber, 8., 159
Farkas, W. G., 322
Farmer, F. A., 177, 571
Farris, E. J., 562
Faucquembergue, D., 244
Faust, E. C., 61
Faust, F. B., 515 (91), 530
Fay, J., 147
Feder, V. H., 398, 402 (44)
Feeney, R.8., 393, 398
Feldman, L. I., 236
Feldott, C., 269
Feller, B. A., 425
Fellows, E. J., 9
Fenton, P. F., 41, 386, 395, 397
Fenwick, F., 4
Ferguson, F. C., 160
Ferguson, H. E., 567
Ferguson, M. E., 546, 552 (170), 553 (170),
554 (170)
Fernholz, M. E., 329, 487
Ferrebee, J. W., 283, 363
Ferrero, N., 467
Ferrero, R., 566
Ferri, M. G., 379
Feysseire, Y., 437
Fieger, E. A., 260 (33)
Field, H., 449
Fierens, B., 557
Fieser, L. F., 491
Figueiredo Magalhaes, B., 515 (86), 530
Fildes, P., 37, 67, 595
Filer, L. J., Jr., 510, 517, 538 (11), 544,
545 (157, 158), 546 (157, 158), 547 (168),
548 (168), 549 (168), 551 (168), 552
(168), 553 (195), 554 (195), 555 (157,
158), 558 (195), 573
Finkelstein, J., 413, 422 (25)
Finkelstein, N., 52, 53 (9)
Finkler, R. 8., 567
Finland, M., 11, 51, 60
Finn, W. F., 561
Fischel, E. E., 61, 79 (35)
Fischer, F. G., 355
Fischer, G., 2
Fischer, G. 8., 494
Fischer, H. O. L., 16
Fischer, O., 120
Fishberg, E. H., 284
Fisher, A., 237, 241 (88), 246
611
Fisher, C. V., 68
Fisher, G. 8., 508
Fishman, W. H., 294
Fisk, W. W., 296
Fitzgerald, G. H., 387
Fleischhacker, H., 284
Fleischmann, G., 360
Fleming, A., 21
Fleming, A. J., 320, 332
Flesher, A. M., 471
Flexner, J., 292
Flexser, L. A., 322
Flickinger, M. H., 324
Flinn, L. B., 79 (19)
Flint, R. B., 316
Flokstra, J. H., 477
Flora, J. O., 79 (15)
Florijn, E., 455, 474
Flynn, E. H., 28 (18), 111, 113, 114 (84),
115 (84), 120 (84)
Foa, P. P., 466, 467
Folin, O., 245
Folkers, K., 28 (17), 103, 109 (80), 143
(20), 227, 229 (26, 27, 28), 230, 232,
233, 240, 301, 316, 320, 327, 329 (59),
394, 588, 589 (103), 594 (103)
Follis, R. H., Jr., 273, 274 (83)
Foltz, E. E., 479
Fong, C. T. O., 577 (16), 578
Fontaine, M., 360
Fordham, D., 103, 576
Foreman, W. W., 3
Fornara, 470
Forrest, H. S., 116, 123, 322
Forse, R. A., 272
Forster, D., 240
Forsyth, G., 550
Fosdick, L. 8., 9
Foster, A. O., 66
Foster, C., 472
Foster, J. W., 200, 254, 257 (2), 260 (2),
261 (2), 262 (2), 279, 310, 328 (40)
Foulkes, E. C., 595
Foust, C. E., 240
Fouts, P. J., 272
Fowler, A. F., 569
Fox, C. L., Jr., 20, 70, 129, 130
Fox, H. J., 143 (7)
Fox, J. T., 283
Foy, J. R., 267, 295
Foye, W. O., 308
612
Fraenkel, G., 147, 191, 379, 583, 584 (54)
Francis, A. W., 6
Frank, H., 558
Frank, H. E., 559
Franke, W., 356
Frankel, 8., 235 (40, 41), 268
Franklin, A. L., 41, 57, 126, 127, 130, 139
(7), 155, 156, 157, 158 (85), 166, 187,
195, 196 (109), 594 (153)
Frantz, A. M., 283
Fraser, L. E., 62, 79 (20), 83
Frazer, A. C., 558
Freed, A. M., 460
Freeman, M. V., 43, 48 (2), 53
Frei, P., 334, 337 (12, 22)
Freudenthal, P., 409, 476
Frey, C. N., 172, 248, 254, 257 (8), 259
(8), 260 (8), 262 (8), 276, 280, 410, 411,
450, 452 (14), 462, 465
Frey, J., 551
Fridericia, L. 8., 409
Friedemann, T. E., 46, 47 (24), 53, 360,
392, 401, 454
Frieden, E., 92
Friedericia, L. §., 476
Friedgood, H. B., 59, 84
Friedman, L., 474, 475 (26)
Friedman, M., 461
Fries, N., 35 (47), 130, 264
Fritz, J. C., 386, 395 (24)
Fritzsche, H., 310, 486, 487, 488 (35), 489,
490, 492, 493, 498
Frobrich, G., 379
Froehlich, A. L., 557
Frommeyer, W. B., 140, 209
Frost, D. V., 38, 176, 272, 306
Fruton, J. S., 586, 587
Fuhr, A., 532
Fujita, A., 367, 368
BullenssAy ies 20
Fuller, H., 79 (21)
Fuller, R. C., III, 148 (22), 199
Funk, C., 405
Furman, C., 277
Furman, R. H., 206, 546, 552 (170), 553
(170, 199), 554 (170)
Furter, M., 493
Furter, M. F., 306, 359
AUTHOR INDEX
G
Gabriel, S., 420
Gaebler, O. H., 272, 392
Gaines, D. 8., 197
Gale, E. F., 234, 235 (7, 8, 46), 237, 239
(8), 241 (31), 267, 278
Gale, R. G., 552, 553 (199)
Galston, A. W., 379
Gardner, T. S., 327, 358, 588
Gartner, H., 414, 417 (31)
Garza, H. M., 588
Gass, 8. R., 40
Gates, M., 145
Gatz, A. J., 515 (84), 530
Gautheret, R. J., 597 (207)
Gavin, G., 269, 381, 396
Gazan, H. A., 567
Gazzola, A. L., 28 (15), 108, 143 (6)
Geddes, W. F., 85, 363, 364 (17)
Geeslin, L. E., 388, 390 (116)
Geffen, C., 162
Gehrke, M., 318
Geiger, A., 492
Geiman, Q. M., 35 (48)
Geitner, P., 6
Gelfond, D. B., 565
Gellhorn, A., 284, 287 (178)
George, W. L., 156
Geraci, J., 107
Gerloczy, F., 545, 550
German, H. L., 179
Gerrits, W. B. J., 474
Gershberg, H., 47, 48
Gerstl, G., 3, 43, 53
Gerstung, R. B., 68
Getzendaner, M. E., 20, 70, 129, 577, 582
(14)
Gibbs, H. D., 247
Gibbs, W., 53, 55, 227
Gibson, W. C., 559
Giese, A. C., 6
Gillespie, J. M., 24 (45), 26 (44, 51), 32,
35, 67
Gillis, M. B., 594 (155)
Gilman, E., 4
Gilman, H., 7
Gilmore, E. L., 595 (185)
Gingras, R., 294
Ginsberg, V., 206
AUTHOR INDEX
Ginzton, L. D., 274
Girdwood, R. H., 205, 206, 209, 212 (163),
217
Giri, K, V., 459
Gladkoua, V. N., 235 (42)
Glavind, J., 507, 511, 515 (103), 534, 538,
557, 566
Glazer, H. S., 282
Glazko, A. J., 162
Glick, D., 468
Glickman, N., 479
Glynn, H. E., 509
Goetchius, G. R., 72 (65)
Goetsch, W., 596
Goettsch, M., 515 (26, 63, 66, 81, 106), 519,
520 (26), 524, 525, 529, 534 (63, 81),
535 (81), 540, 571
Golberg, L., 191
Goldacre, R., 4
Goldberg, B., 161
Goldberg, 8. D., 7, 9 (49, 50)
Goldberger, J., 387, 390
Goldblatt, H., 381, 540
Goldbloom, A., 192
Goldfarb, A. R., 3, 43, 53
Goldfarb, W., 474
Goldfeder, A., 291, 296
Goldhaber, P., 505, 515 (109, 110), 536
Goldin, A., 161
Golding, J. S. R., 57
Goldinger, J. M., 430 (47, 57), 434 (57)
Goldman, A. A., 113
Goldman, L. M., 283
Goldsmith, E. D., 41, 50, 54, 57 (27), 158,
196, 198
Goldsmith, G. A., 204, 400, 402
Golumbic, C., 490, 492, 493, 494, 503, 504,
508
Gonce, J. E., Jr., 515 (61a, 115), 525, 536
Good, R. A., 62, 85
Good, T. A., 62, 85
Goodhart, R. 8., 439, 450
Goodland, R. I., 546, 552 (169), 562 (169)
Goodwin, J. F., 84
Gordon, A. 8., 41, 50, 54, 57 (27)
Gordon, A. H., 347 (63), 348 (63), 350
Gordon, H. H., 192, 537, 547
Gordon, M., 16, 17, 20, 70, 103, 110 (31),
129, 143 (24)
613
Gordon, 8. M., 306, 359
Goryachenkova, I. V., 238, 268, 269
Gorham, J. R., 539
Goss, H., 298, 360
Gottlieb, H., 489, 490, 498, 511, 570
Gots, J. 8., 1381, 132
Goudjonsson, 8., 409
Goudsmit, J., 429, 455
Gourevitch, A., 365
Govan, C. D., Jr., 192
Graber, M., 52, 53 (9)
Graf, W., 441
Graham, W. R., 594 (150, 151)
Gram, ©: N. J, 565
Granados, H., 505, 513, 517, 588 (10, 12),
541 (10), 570
Greaves, J. D., 501
Green, D. E., 16, 28, 72 (56), 235 (55),
237 (55), 348, 344, 347 (64), 349, 350
(49), 353, 354 (91), 356, 386, 428, 430
(20), 431 (51), 4382, 434, 486 (20), 440,
442, 443, 444, 581
Green, H. H., 476
Green, H. N., 21, 26 (28), 68
Green, J., 498
Green, M. N., 20, 69
Green, R. G., 458
Greenberg, D. M., 437
Greenberg, G. R., 131, 182
Greenberg, L. D., 268, 274, 275, 287, 297,
461
Greenberg, 8. M., 505
Greene, J., 448
Greene, R. D., 245, 305, 410 (12), 590
Greenspan, E. M., 198, 214
Greenwood, A. D., 594 (179)
Greer, R. B., 546, 552 (169), 562 (169)
Gregersen, M. I., 20
Greiff, D., 60, 79 (22)
Grekin, R. H., 47, 85
Greslin, J. G., 309, 391
Grewe, R., 405, 414, 415, 416 (30), 417
Griese, A., 337 (23, 24), 343 (23)
Griffith, J. Q., Jr., 537
Griffith, W. H., 360
Grijns, G., 404, 405
Grimes, M., 558
Grob, C. A., 191
Grob, D., 566
614
Groopman, J., 471
Gross, H., 565 (26)
Gross, N. H., 430
Gross, P., 147
Gruber, M., 428, 429, 477
Gruen, D. M., 4
Grundy, W. E., 46, 47 (24), 53, 360, 392,
401
Gualco, 8., 57
Gubler, C. J., 266
Gudaitis, A., 293
Gudjonsson, S., 476
Guerrant, N. B., 596 (223)
Guest, G. M., 569
Guggenheim, K., 393, 395, 396 (8), 505
Guirard, B. M., 39, 163, 231, 276, 277
(109), 576
Gullickson, T. W., 515 (87, 88), 530, 572
Gunderson, C., 89
Gunness, M., 278, 279 (130)
Gunsalus, I. C., 234, 235 (37), 236, 237
238, 239, 240 (6), 241 (32), 242 (26),
278, 576, 577, 578 (13), 579, 580 (85),
582
Giinther, G., 343
Giinther, P., 487, 490
Gunzalus, I. C., 440
Gurin, S., 407, 418, 421
Guy, L. P., 458, 480
Gwinner, G., 209
Gwoh, H., 202
Gyorgy, P., 38, 222, 223, 224, 250, 255, 265,
283, 284 (164), 301, 302, 303, 304, 305,
307 (12a), 309 (12a), 310 (12a), 334,
346 (6), 358, 370, 372, 380, 381, 383,
449, 450, 504, 506, 537, 540, 546, 547
H
Haag, J. R., 460
Haagen-Smit, A. J., 27, 30 (8), 268, 293
Haagen-Smith, A. J., 286
Haas, E., 336, 348, 345, 346, 393
Haas, G. J., 306, 359
Haase, K., 343
Haehner, E , 124
Haig, F. M., 376
Hain, A. M., 567
Haines, W. T., 385, 397
Hale, F., 278, 279 (131)
Haley, T. J., 466, 467, 469, 471
Halick, J. V., 594 (156)
AUTHOR INDEX
Hall, B. E., 206, 211
Hall; D. A., 586
Hall, W. K., 381
Hall, W. L., 249
Halliday, N., 250, 269
Halmagyi, D., 21, 57
Halpern, G. R., 505
Halpern, S. L., 393
Halpin, J. G., 275, 297
Halwer, M., 311
Ham, T. H.; 143:(7)
Ham, W. E., 42
Hamil, B. M., 474
Hamilton, C. R., 58, 63, 64 (9)
Hamilton, H. L., 60, 61, 79 (9, 23)
Hamilton, J. W., 533
Hamilton, T.S8., 31, 49, 53, 292, 385, 397
Hancock, E. M., 7, 9 (52, 53, 54)
Handler, P., 352, 505
Hanrahan, E. M., 568
Hansen, H. L., 9
Hanus, E. J., 425
Hanze, A. R., 107
Happold, F. C., 35 (87), 586
Haque, M. E., 196
Hardeastle, A. B., 66
Harding, W. M., 156
Hardy, E. M., 7, 9 (54)
Hare, H. A., 53, 55
Harned, B. K., 216
Harnly, M. H., 158, 198
Harper, A. E., 164
Harpur, E. R., 192
Harrell, G. T., 291
Harrer, C. J., 345
Harringdon, C. R., 447
Harris, A. E., 182
leleneatsy, dj, il, atsy¥/
Harris, J. S., 68
Harris, L. J., 4, 265, 284, 461
Harris, M. E., 474
Harris, P. L., 490, 491, 499, 501, 504, 505
(41), 507, 511, 512, 518, 543, 546, 551,
552 (192), 554, 555, 571, 572
Harris, P. N., 42, 58, 63, 593
Harris, R. S., 265
Harris, 8. A., 28
(20), 227, 229
233, 240
Harrop, G. A., 341
Harrow, B., 44, 52
(17), 103, 109 (30), 143
(26, 27, 28), 230, 282,
AUTHOR INDEX
Hart, E. B., 39, 42 (85), 90, 176, 185, 275,
297, 384, 594 (152), 596 (215, 220, 247)
Hart, W. M., 48
Hartcroft, W. S., 557
Harte, R. A., 306
Hartmann, §., 538, 557, 566
Hartree, E. F., 356
Hartsough, G. R., 515 (62), 525, 539 (62),
541 (62)
Harvey, C. C., 380, 389, 398 (1), 399, 401
(45), 402 (45)
Haskins, F. A., 16
Hasler, A. D., 459
Hastings, A. B., 434
Hauge, S. M., 381, 577, 594
Hausmann, K., 140
Hawkins, V. R., 140, 217, 282
Hawkins, W. W., 266, 267, 272 (35), 281,
286, 287 (195)
Haworth, J. W., 320
Haydak, M. H., 262 (41)
Hazard, H., 77
Hecht, G., 20, 466, 467, 469
Hedderich-Arismandi, H., 401 (61)
Hedrick, M. T., 376
Heegard, E., 430, 431 (34)
Hefner, L. L., 206
Heftmann, E., 504
Hegsted, D. M., 44, 45, 52, 175, 178, 275,
280, 297, 298, 386, 387 (98), 395 (25),
474
Heidebrecht, A. A., 371
Heiman, V., 374, 395 (20)
Heimberg, M., 268
Heine, W. I., 565
Heinle, R. W., 140, 147, 148 (46), 156,
161, 188, 209, 210, 211, 212, 213 (205),
214, 271
Heinrich, M. R., 159
Heinrich, R. A., 143 (14)
Heins, J. T., 571
Heins, P. L., 501
Heise, F. H., 78
Heise, R., 235 (47)
Heitman, H., Jr., 477
Heitmiller, R. F., 578
Heitzer, K., 235
Hellermann, L., 330, 393
Hellstrém, H., 343
Helmer, O. M., 272
Henderson, C., 402
Henderson, C. R., 360, 392, 401
Henderson, F. G., 53
Henderson, L. M., 38, 59, 251, 257 (7),
259 (7), 260 (7), 261 (7), 587, 588 (91)
Hendlin, D., 277, 590
Hendricks, W. J., 61, 79 (24)
Henle, W., 472
Hennessy, D. J., 410 (11)
Henningsen, A. B., 60
Henry, K. M., 371
Henschel, A. F., 389, 401, 478, 479
Hensel, M., 44, 52, 54
Herbain, M., 443
Herbert, D., 480 (51), 431 (51), 432 (51),
436 (51), 442, 443 (2)
Hermann, H., 235
Herraiz, M. L., 512, 515 (95), 531
Herrick E. H., 515 (80a), 519, 521 (30a)
Hertig, A., 561
Hertz, R., 195, 196 (108), 197
Herwig, G., 257 (18), 260 (18)
Herzstein, J., 569
Heslet, H., 507
Hesse, B. C., 415
Hesser, F. P., 553, 554 (201)
Hesselbach, M. L., 42
Hesseltine, H. C., 285
Hesseltine, C. W., 595
Hessler, W., 515 (93), 531
Heuser, G. F., 90, 149 (55), 152 (55), 153,
154 (70), 177, 178 (42), 297, 363, 374,
386, 395 (20), 588, 594 (147), 596 (224,
232, 234)
Heyl, D., 7, 9 (54), 232, 240, 320, 327
Heytler, P., 596 (256)
Hibbs, J. W., 385
Hickey, R. J., 324, 325, 333
Hickman, K. C. D., 489, 498, 504, 505
(41), 512, 548, 552, 554, 564, 572, 573
Hift, H., 271
Higgins, G. M., 174, 291
Hightower, D. P., 283, 293
Hildebrandt, H., 54
Hill, A. J., 6
Hill, C. H., 202
Hill, E. G., 197
Hill, F. W., 177, 363
Hillman, W. S., 595 (211)
Hills, O. W., 380, 389, 390 (132), 391 (132),
394, 398 (1), 401 (45), 402 (45)
Himdsworth, H. P., 540
616
Himes, H. W., 248, 244 (2), 248 (2)
Himwich, H. E., 474
Hindmarsh, M. M., 77
Hines siG 0H. Jr, 325
Hines, H. M., 515 (73), 526
Hines, L. R., 501, 507
Hinton, J. J. C., 455
Hippchen, H., 331, 332 (115)
Hirata, Ji., 3
Hirschberg, E., 366
Hitchings, G. H., 126, 149, 150, 151 (65)
Hixson, A. W., 497
Hoaglund, R. J., 82
Hochberg, M., 225, 243, 244 (2), 248 (2),
257 (21), 262 (21), 450, 459
Hodson, A. Z., 166, 257 (16), 363
Hoehn, H. H., 488, 491
Hoff-Jgrgenson, E., 590
Hoffer, A., 363, 364
Hoffer, M., 420
Hoffman, C. E., 130, 576, 577 (16), 578,
590
Hoffmann, O., 490
Hofmann, A. W., 3, 10 (8)
Hofstee, B. H. J., 349
Hogan, A. G., 90, 91, 92, 147, 168, 178,
175, 176, 178, 381, 397, 533, 594 (146),
596 (213)
Hoégberg, B., 596 (219)
Hogness, T. R., 345, 346
Holden, J. T., 277, 278 (119), 279
Holiday, E. R., 334
Holland, B. R., 136
Holly, F. W., 316, 329 (59), 394
Holman, R. L., 515 (116), 537, 566
Holmberg, B., 4
Holmes, A. D., 376
Holt, L. E., Jr., 281, 360, 401, 458, 478, 480
Holtz, P., 235 (47, 48, 49, 50, 51)
Honeyman, J., 315
Hopkins, F. G., 145, 347 (57)
Hopkins, R. H., 257 (1), 262 (1), 280
Horecker, B. L., 324, 345, 346
Horlein, H., 411
Hornberger, C. 8., Jr., 577, 578 (13)
Horowitz, N. H., 387 (25), 351 (25)
Horrigan, D., 140, 217
Horwitt, M. K., 380, 389, 390 (132), 391
(132), 398, 394, 398, 399, 401 (45), 402
(45), 465, 479
Hoskins, W. M., 379
AUTHOR INDEX
Hou, H. C., 310, 390
Houchin, O. B., 271, 515 (76, 84), 529, 530
Housewright, R. D., 25, 26 (49), 31, 35,
36 (28), 68
Hove, E. L., 490, 496, 501, 505, 508, 512,
540
Hove, Z., 490, 496, 508
Howard, J. W., 79 (19)
Howard, M. E., 12
Hrubesch, A., 146
Hsu, P.-T., 593, 594 (143)
Hubbard, L. H., 283
Hubbard, R. S., 27, 28 (9), 293
Hucker, G. J., 596 (237)
Hudson, P. B., 568
Huebschmann, H., 467
Huennekens, F. M., 322
Huff, J. W., 292, 594 (149, 167)
Huff, N. E., 388
Hughes, E. H., 273, 385, 395
Hughes, J. S., 510, 513
Hughes, T. P., 587, 597
Hull, R., 116
Hultquist, M. E., 28 (12), 39, 71 (50),
73, 101, 104 (27), 105, 106 (27, 34), 107,
108 (83, 35), 109 (27), 111, 113 (76),
114, 115 (90), 116 (90), 117 (90), 118
(90), 119 (90, 95), 120 (90, 95), 121,
122 (1), 123 (2), 124, 148 (15, 16),
149 (59), 155, 156 (77), 157 (74)
Hume, E. M., 491, 511
Humoller, F. L., 318
Humphreys, 8., 186, 273, 274 (83), 296
Hunt, A. D., 288
Hunt, D. J., 384
Hunter, J. B., 269
Hutchings, B. L., 28 (12, 18, 14, 15), 39,
71, 91, 92, 93, 94, 95, 96, 98 (21, 24),
99 (24), 101, 103 (24), 104 (27), 105,
106 (27, 34), 108 (33, 35), 109 (27),
113, 114 (23), 115 (23), 121, 122 (1),
123 (2), 143 (2, 3, 5, 6, 8, 10, 15, 16),
149, 154 (57), 158, 161, 162, 175, 177,
184, 199, 590, 595 (186, 209), 596 (252,
256)
Hutchison, D. J., 159
Hutner, S. H., 35 (51)
I
Ichiba, A., 222, 223 (6), 224, 228, 229 (23)
Ichniowski, C. T., 56
AUTHOR INDEX
Impallomeni, G., 566
Ingalls, B., 399
Ingelman-Sundberg, A., 515 (39), 520
Inglett, G. E., 494
Inukai, F., 596 (229, 230)
Irving, J. T., 541
Irwin, W. B., 493
Isbell, E. R., 257 (26)
Isbell, H., 23, 24 (39, 40), 26 (40), 27, 30
(1, 2), 31 (1), 35 (41, 48, 44), 46, 47
(23)
Isler, O., 489, 497
Israel, 8S. L., 561
Israéls, M. C., 205
Issidorides, A., 498, 494, 506, 530
Ivanovies, G., 79
Ives, M., 204, 217, 259 (380), 260 (30), 262
(30)
Ivy, A. C., 479
J
Jacob, A., 419, 486, 487 (30), 488 (53), 489
Jacobs, K., 281
Jacobsohn, K. P., 459
Jaeger, W., 507 (83)
Jaffé, M., 6
James, G. W., III, 79 (25)
James, G. W. H. 566
James, M. F., 43, 186, 188, 515 (24a, 59a),
519, 524 (24a), 530 (59a), 572
Jamieson, G. 8., 504
Janisch, H., 235
Janota, M., 183
Jansen, A. P., 363, 366, 367 (9)
Jansen, B. C. P., 405, 410, 448, 455, 456
(5), 457
Jaros, S. H., 466, 470, 471 (6)
Jarrold, T., 217, 282
Jasper, H. H., 465
Javert, C. T., 561
Jeffreys, C. E. P., 27, 30 (8)
Jenkins, R. L., 9
Jensen, J. L., 490, 511, 552
Jensen, K. A., 57, 490
Jensen, W. N., 269
Jenney, E. H., 290, 291
Jeppson, J., 6
Jepson, R. P., 566
Jequier, R., 443
Jessen, K. E., 557, 566
Joffe, M., 490, 511
617
Johansen, G., 409, 476
John, W., 486, 487, 488, 490, 492, 493, 494,
496
Johns, G. A., 360
Johnson, A. W., 594 (178, 179)
Johnson, B. C., 31, 48, 49, 53, 92, 186, 188,
276, 277, 292, 385, 395, 397, 515 (59a),
524, 530 (59a), 588
Johnson, F. H., 378, 393
Johnson, H. C., 209
Johnson, J. R., 462
Johnson, L. V., 390, 402
Johnson, M. L., 360
Johnson, O. H., 72 (56), 328
Johnson, R. E., 392, 402, 532
Johnson, R. M., 505
Johnson, T. B., 113
Johnston, C. H., 374
Johnston, F. A., 401 (61)
Johnston, J., 3, 6, 7 (39)
Johnston, P. M., 577, 582 (14)
Johnston, R. M., 324
Johnston, 8S. F., 151, 152 (66), 160 (66),
161 (66), 197
Jolliffe, N., 283, 293, 400, 469
Jones, A., 257 (18), 260 (18)
Jones, B. F., 174
Jones, C. P., 376
Jones, E., 206, 209, 553, 554 (203)
Jones, E. S., 274, 297
Jones, G. E. S., 561
Jones, J. H., 362, 364, 472
Jones, L. O., 284, 287 (178)
Jones, L. W., 595
Jones, M. J , 33, 35 (8, 12), 68, 71, 72 (58),
74 (27, 28), 79 (58), 134, 135 (33), 186
(38)
Jones, P , 274
Jones, R. G., 28 (18), 118, 114 (84), 115
(84), 120 (84), 231
Jones, R. L., 485
Jones, T. C., 386, 515 (6)
Jonsson, U., 208
Juhasz-Shiffer, A., 515 (15), 517
Jukes, T. H., 110, 111, 113 (76), 114 (73),
126, 127, 130, 139 (7), 143 (19), 155,
156, 157, 158 (85), 166, 178, 187, 195,
196 (109), 197, 198, 210, 214, 272, 275,
370, 386, 387 (96), 395 (23), 477, 576,
577 (16), 578, 585, 590, 594 (153, 154),
596 (225, 233)
618
Jungherr, E., 515 (81, 99, 100), 529, 532,
534 (81, 99, 100), 535 (81)
Juni, E., 431
Justice, D. H., 41
K
Kaack, R., 414, 417 (31)
Kaezka, E. A., 28 (17), 103, 109 (30), 143
(20)
Kahlenberg, O. J., 250, 395, 477
Kahler, H., 365
Kaiser, A., 10
Kaiser, P., 467, 468
Kalckar, H. M., 349
Kaleita, E., 596 (256)
Kaley, M. W., 504, 505 (41), 543
Kallio, R. E., 239
Kallmann, O., 303
Kalnitzky, G., 480, 435
Kaltschnitt, H., 334
Kalz, F., 471
Kamen, M. D., 434
Kamminga, C. E., 428
Kancher, M., 474
Kanof, N. B., 569
Kao, C.H., 10
Kapeller-Adler, R., 356
Kaplan, N. O., 39, 45
Kaplan, S. R., 211
Karezag, L., 427
Karimullah, 420, 423 (25)
Karn, H. W., 266
Karnofsky, D. A., 158, 160
Karrer, P., 72 (64), 107, 123, 234, 240,
241 (12), 308, 305, 310, 312, 314, 315,
316 (54), 317, 322, 326, 327, 328 (97),
329, 334, 337 (12, 22), 358, 359, 366,
373 (3), 438, 441, 486, 487, 488, 489,
490, 491, 492, 493, 498, 499, 500, 507
(33)
Kaser, M. M., 5538, 554 (203)
Kaszuba, F. J., 410 (11)
Katz, K. H., 565
Katz-Galatzi, T., 568
Kaucher, M., 374, 480
Kaunitz, H., 502, 507, 508, 510, 515 (5,
19, 97), 516, 518, 520, 531 (5), 532
(35, 36)
Kavanagh, F., 307
Kavinoky, N. R., 567
Kay, J. H., 567
AUTHOR INDEX
Kazenko, A., 144, 164
Kearny, E. B., 330, 335, 353, 357, 361, 589
Keegan, P. K., 454
Keeton, R, W., 479
Keilin, D., 348
Keilin, K., 356
Keith, A. M., 283
Keith, C. K., 137, 138, 142 (42)
Kekwick, R. A., 340
Kelemen, E., 21, 57
Keller, E., 43, 276, 395 (26)
Keller, H., 490, 492, 507 (83)
Keller, R., 420, 423 (25)
Kelley, B., 188
Kelley, E. G., 507
Keltch, A. K., 72 (53)
Kemmerer, A. R., 372
Kempster, H. L., 178, 594 (146), 596 (223)
Kensler, C. J., 61, 430, 437
Keresztesy, J. C., 41, 90, 108, 109, 114,
120, 121 (89), 143 (21, 23), 200, 222,
223 (3), 224, 225 (8, 12), 226 (12), 228,
243, 245 (4, 5), 247 (5), 410, 412, 454,
465
Kerkhoff, W. G., 480, 431 (84)
Kem Gr, 4515
Kern, R., 391
Kern, 8S. F., 578
Kernis, L., 285
Kerrer, P., 490, 492
Kessler, M. M., 569
Ketron, K. C., 458, 480
Keys, A., 389, 392, 401, 451, 476, 478, 479
Kidder, G. W., 148 (11, 18, 22), 159, 199,
200, 277, 290, 291 (4), 576
Kihara, H., 586, 587 (83, 84)
Killam, K. F., 290
King, C. G., 95, 162
King, F. E., 9, 331
King, H., 79
King, J., 10
King, J. A., 488
King, R. A., 568
King, R. H., 594
King, T. E., 590, 591, 596 (122, 254)
Kinnersley, H. W., 223, 255, 410 (10),
426, 596 (216)
Kinney, J. M., 142
Kinsey, V. E., 505, 515 (109, 110), 536,
548 (180), 549
Kipnis, F., 316, 331 (56)
AUTHOR INDEX
Kipping, F. B., 113
Kirby, F. G., 79 (26)
Kirch, E. R., 11, 20
Kirchner, J. G., 27, 30 (8)
Kirk, J. E., 465, 552, 568
Kirsanova, V. A., 124, 155
Kisch, B., 352
Kitay, E., 590, 595
Kitzes, G., 262 (40)
Kjeldgaard, N. O., 349
Kjerulf-Jensen, K., 57
Klaeui, H., 107
Klatt, 327
Klatskin, G., 543, 552, 553 (152, 200), 554,
572
Klatt, O. A., 586, 587 (84)
Klatzien, C., 363
Kleiger, S. C., 401 (61), 479
Klein, J. R., 336, 352, 361, 369, 392, 393
Klemperer, F. W., 434
Klenow, H., 349
Kilinck, G. H., Jr., 58, 63
Kline, L., 576, 577 (8), 582 (8)
Kline, O. L., 266, 474, 475
Klingenfuss, M., 419, 423, 425
Klingman, W. O., 283
Klotz, I. M., 4
Kmieciak, T. C., 454
Knight, B. C. J. G., 276, 361, 378, 595
Knobloch, H., 301, 332
Knott, E. M., 479
Knowles, J. L., 141
Knowlton, G. C., 515 (73), 526
Knox, W. E., 238, 260 (33), 428, 430 (21),
436 (21), 443
Knunyantz, I. L., 419
Koch, M. B., 204, 209
Koch, W., 241 (39)
Kocher, V., 590
Kodicek, E., 266, 588
Kodischek, L. K., 590
Koehn, C. J., 596 (227)
Koelle, G. B., 56
Koenig, H., 490
Kofler, M., 500, 509
Koft, B. W., 33
Kogl, F., 301
Kohler, A. R., 159
Kohler, G. O., 594 (152)
Kohn, H. I., 68, 336, 361, 369
Kolson, J., 147, 180, 184 (54)
619
Koltay, M., 57
Kolthoff, I. M., 508
Kon, H. I., 393
Kon, 8. K., 371, 458
Koneff, A. A., 521
Koniuszy, F. R., 316, 320, 327, 329 (59)
Koones, H. F., 224, 247
Koretsky, I. B., 197
Korkes, 8., 579
Kornberg, A., 71, 172, 178, 174 (15, 16),
190 (15), 266, 382, 383
Kornberg, H. A., 371, 373
Kornfeld, E. C., 231
Korte, F., 108
Koschara, W., 145, 146, 303, 305, 334, 363,
392
Koser, 8. A., 25, 26 (49), 31, 35, 36 (28),
68
K6thnig, M., 318
Kovaes, K., 57
Krahl, M. E., 42, 72 (53)
Krampitz, L. O., 438, 459
Kratzer, F. H., 275, 297, 386, 395
Krause, A. C., 549
Krause, M. E., 266
Krautzun, H., 235
Kraybill, H. R., 381
Krebs, H. A., 351, 352 (84), 353, 430, 433
Krehl, W. A., 190, 259 (29), 260 (29),
376, 552, 553 (200), 586
Kreisler, O., 389, 393, 465, 479
Krider, J. L., 186, 188, 515 (24a), 519,
524 (24a), 572
Krider, M. M., 76
Kringstad, H., 250, 596
Krishnan, B. G., 387
Kritzmann, M. G., 237
Kruger, A., 566
Krukovsky, V. N., 508, 513
Kruse, H. D., 388, 390 (124), 391 (123,
124), 400
Kruse, I., 515 (111), 536
Kubota, T., 106, 123
Kubowitz, F., 481, 432
Kuesztesy, J. C., 441
Kueter, K. E., 58
Kugler, A., 491
Kuh, E., 28 (12), 39, 71 (50), 73, 101, 104
(27), 105, 106 (27, 34), 108 (33, 35),
109 (27), 121, 122 (1), 128 (2), 143
(15, 16), 155, 156 (77)
620 AUTHOR INDEX
Kuhl, W. J., Jr., 47, 48
Kihling, O., 314
Kuhn, R., 72 (607), 222, 223, 226, 227,
228, 229, 230 (21), 244, 301, 302, 303,
304, 305, 307 (12a, b), 308, 309 (12a,
b), 310 (12a, b) 311 (12b), 312 (12a,
b), 314, 315, 316, 317, 318 (58), 322,
323 (46), 326 (36), 327 (48), 328, 334,
336, 340, 346 (6), 357, 358, 359, 369,
372, 391, 393, 467
Kuhrt, N. H., 497, 500
Kumler, W. D., 4, 5, 6
Kunez, D., 545
Kunkel, H. O., 504
Kupperman, H. 8., 567
Kurdyukova, V. A., 318, 323 (68)
Kushida, M. N., 151, 152 (66), 160 (66),
161 (66)
Kwisda, A., 6
L
Lacaz, C. S., 85
Ladenburg, K., 319, 320, 321, 332
Ladisch, R. K., 292
Lake, H. 8., 46, 47 (20)
Lalor, R. J., 594 (169, 170)
Lambooy, J. P., 315, 325 (53), 326, 327
(92a), 394
Lamoureux, W. F., 360
La Motte, W. O., 548 (181), 549
Lampen, J. O., 10, 18, 22, 24 (46), 25, 26
(53), 27, 30°), 31"(8), 33, 90. (8; 12,
30, 33), 68, 71, 74 (27, 28), 184, 135
(83), 1386
Landor, V. J., 387
Landy, M., 20, 22, 23, 25, 26 (28), 30 (19),
31, 34, 35 (17, 18), 36 (16, 58), 46, 47,
67, 68, 72 Ol), 77
Lane, R. L., 260 (81)
Langdon, R. 8., 373
Lange, W. E., 308
Langenbeck, W., 440
Langham, W., 3
Langston, W. C., 90, 91 (2), 182, 381, 390
(36), 395
Lansdown, A. R., 594 (179)
Lansford,; M., 118
Laquer, F., 410
Lardy, H. A., 137, 138, 269, 594 (165)
Larkum, N. W., 20, 23 (14), 25 (14), 34,
36 (16, 58), 67, 68 (11)
LaRosa, W. V., 21
Larsen, A., 200, 254, 257 (2), 258, 260 (2),
261 (2), 262 (2), 279
Larson, Don H., 333
Lascelles, J., 134, 135, 136 (37)
Laskowski, M., 144, 164
Laskowski, 8S. C., 7, 9 (55), 92
Laszlo, D., 41, 161
Latschar, C. E., 513
Lau, R. E., 270
Laufer, S., 11, 21, 36
Lauritsen, M., 274
Lautemann, E., 16
Lavoipierre, L., 191
Lawrence, C. A., 72 (65)
Lax, F., 569
Lazzell, C. L., 3
Leaders, W. M., 497
Lechow, B., 266
Lechycka, M., 251
Leder, I. G., 268
Lederberg, J., 17
Lederer, E., 301
Lederer, M., 21
Lee, C. T., 62
Lee, J., 315, 318, 327, 358, 500, 588
Lee, J. G., 596 (213)
Lee, S. B., 333
Lehmann, J., 78, 79
Lehman, R. W., 489, 492, 493 (93), 496,
497 (5), 502 (7)
Lehrer, W. P., Jr., 273
Leichsenring, J. M., 375 (19)
Leloir, L. F., 235 (55), 237 (55), 356, 386
Lemley, J. M., 505, 546, 552 (170), 553
(170, 199), 554 (170)
Leonard, C. 8., 63
Leonian, L. H., 32, 35 (1, 50)
Leoschke, W. L., 594
Lepkovsky, S., 222, 223 (2), 266, 268, 272,
275, 286, 293, 297, 386, 387, 395, 473
Lerner, S. R., 41, 57
Lesis, R. W., 3
Lettré, H., 329
Leuchtenberger, C., 41, 108, 161
Leuchtenberger, R., 108, 161
Leuthardt, F., 439
Levaditi, C., 72 (62)
Levi, 420
Levine, H., 324, 374
Levine, H. D., 515 (85), 530
Ee
AUTHOR INDEX
Leviton, A., 363
Levy, B. M., 271, 386
Levy, H., 565
Levy, M. D., 61, 79 (27)
Lewin, I., 393
Lewin, R. A., 35
Lewis, D., 195, 196 (109)
Lewis, G. T., 398, 402 (44)
Lewis, H. B., 44
Lewis, J. C., 23, 26 (35), 27, 30 (6), 31 (6),
34, 35 (40), 46, 47, 68, 71 (13)
Lewis, W. L., 3
Lewisohn, R., 108, 161
Lichon, P., 500
Lichtfield, J. T., Jr., 19
Lichtman, H., 206
Lichstein, H. C., 234 (48, 44), 235 (48, 44),
237, 278, 595
Liddle, L. M., 7
Liebe, E., 491, 495
Liebert, E., 380, 389, 390 (132), 391 (132),
393, 398 (1), 399, 401 (45), 402 (45),
479
Liebig, R., 349
Lieck, H., 509
Lienke, R. I., 208
Light, R. F., 172, 410, 477
Lih, B., 286
Lilienthal, J. L., 567
Lillie, R. D., 516, 540 (6)
Lillie, R. J., 177, 196
Lilly, J. H., 594 (174)
ibalky, Vi2.G.,32;°35 (1, 50)
Lindan, O., 540
Lindholm, M., 249
Lindlar, H., 107
Lindsay, A., 330
Ling, C. T., 280
Lingange, J. J., 244, 369
Link, K. P., 75, 172
Linkswiler, H., 292, 294
Lipmann, F., 39, 45, 429, 430, 434, 435,
436, 440, 441
Lippincott, 8. W., 386, 395
Lippman, H. I., 566
Lipshutz, M. D., 515 (52), 522, 532
Lipton, M. A., 395, 428, 429, 430 (45, 47,
57), 431, 434 (57), 439
Little, P. A., 160
Littlefield, J. W., 580, 581 (37)
Livermore, A. H., 459
621
Livingstone, R. A., 561
Locke, E., 160
Lockwood, J. 5., 21, 66
Loeffel, W. J., 395
Loewi, 462, 464
Logan, M. A., 277
Lohmann, K., 427, 430, 438
Lond, B. S., 89 (1), 90
Long, A. G., 594 (179)
Long, C., 430, 432
Long, P. H., 68
Gong Reo
Longenecker, H. E., 266
Loofbourow, J. R., 364, 368 (22)
Loomis, T. A., 27, 28
Loesli, J. K., 508, 510, 513 (59), 524, 530
(59), 572
Lopes de Faria, J., 515 (96), 531
Lopez, G. G., 1438 (17), 197, 209
Lorenz, F., 356
Lossy, F. T., 402
Lounds, E., 401
Lourie, E. M., 65
Love, R. H., 304, 362, 367, 368 (36), 371,
449
Low, I., 244
Low, J. A., 419
Lowe, C. U., 193, 194, 208
Lowell, F. C., 11, 60
Lowenfeld, R., 328
Lowry, J. V., 472, 516, 540 (6)
Lowry, O. H., 304, 349, 362, 364, 367 (3),
368 (36), 371, 373, 398, 400, 449, 507,
551
Lowry, R. S., 595 (185)
Lu, G. D., 462
Lucas, H. L., 297, 386
Luckey, T. D., 33, 39, 42 (85), 90, 185,
275, 596 (215, 220)
Ludes, H., 124
Ludke, K., 235 (47)
Ludovici, P. P., 266
Ludwig, A. 8., 466, 467
Ludwig, M. I., 490, 491, 499
Luecke, R. W., 386, 515 (89), 530
Luhby, A. L., 207, 208 (179)
Lundberg, W. O., 489, 503, 513, 572
Lunde, G., 250, 596
Lundquist, F., 49, 53
Lustig, B., 3, 48, 53
Liithy, H., 448
622
Liittgens, W., 431, 432
Luttrell, C. N., 515 (78), 529, 533 (78)
Luz, E., 240
Lwoff, A., 596 (242)
Lwoff, M., 463, 596 (242)
Lyman, C. M., 278, 279 (130), 430 (46, 47,
57), 434 (57), 441
Lynch, H. M., 21
Lynen, F., 583
Lynn, G., 3
Lythgoe, B., 35 (87)
Lytell, A. A., 277
M
McArthur, C. S., 487
McBryde, A., 283
McCabe, M. M., 168
McCall, K. B., 184, 185, 274, 297, 386, 395,
594 (171)
McCay, C. M., 379, 395 (27), 515 (63a),
525, 5380 (63a), 571
McChesney, E. W., 38, 58, 63
McCollum, E. V., 40, 71, 75 (40), 246, 449,
513, 515 (67, 68), 526, 527, 533 (73a)
McCormack, R. B., 590
McCreary, J. F., 391
McCrory, W. W., 288
McCulloch, E. C., 42
McCullough, W. G., 586
McDevitt, E., 400
Macdonald, A. M., 515 (64c), 525, 530
(64c)
McDonald, E. K., 569
McEachern, D., 559, 560 (227)
McElroy, L. W., 298, 360
McElroy, W. D., 34, 35 (15)
MacFarland, M. L., 267, 268, 272 (35),
286, 287 (195)
McFarlane, W. D., 497, 507
McGanity, W. J., 286, 293
MecGillvray, W. A., 505
McGilvery, R. W., 18, 14 (8, 4, 5), 49, 236
(61)
McGinnis, J., 594
McGinty, D. A., 57
McGlohon, V. M., 40
McGrath, H., 268, 287
McGregor, M. A., 42
McHenry, E. W., 267, 268, 269, 270, 272
(35), 286, 287 (195), 293, 295, 381, 396
Mcllwain, A. J., 286
AUTHOR INDEX
McIntire, F. C., 176
Mackenzie, C. G., 40, 57, 71, 75 (40), 515
(67, 68), 526, 527 (69), 530, 533 (73a)
McIntire, J. M., 38, 59, 257 (5), 260 (5),
261 (5), 262 (5), 376; 377
McKee, R. W., 35 (48), 587, 595 (85)
Mackenzie, J. B., 40, 57, 71, 75, 4938, 515
(67, 68), 526, 527 (69), 530, 533 (73a),
559
McKibbin, J. M., 71, 75 (89), 171, 272
McKinney, G. R., 14
McLaren, B. A., 48, 276, 365, 395 (24)
McLaren, H. C., 567
McLendon, P. A., 79 (87, 38, 42, 43)
MacLeod, C. M., 21, 22, 47, 66, 68 (3)
McMahan, J. R., 451
McMaster, L., 6, 7
MeMillan, G. W., 325
McNally, P. A., 74
MeNutt, W.S., 277
McPhee, G. 8., 474
McPherson, A. Z., 208
McRay, W. L., 595
MeVeigh, I., 163, 257 (20), 360
MacVicar, R., 371
MacWalter, R. J., 486
MeWhirter, D. L., 595 (193)
Ma, R., 35 (52)
Maass, A. R., 156, 189, 384
Macchi, M. E., 577 (16), 578
Macek, Thomas, J., 425
Machlin, L. J., 188
Macrae, F. F., 251, 381, 596 (217)
Macy, I. G., 392, 480
Madden, R. J., 272
Maddy, K. H., 587
Madinaveitia, J., 330
Madsen, L. L., 515 (68a, 65), 525, 530
(63a)
Magis, C., 20
Maier, J., 60, 77, 79
Maiese, M. R., 4, 5
Maillard, L. C., 590
Main, E., 60, 83
Majnarich, J. J., 596
Major, R., 502
Majoros, M., 57
Makino, K., 15, 230
Makinson, D. H., 565
Malamud, N., 515 (79), 529, 533 (79)
Malin, R. B., 586
AUTHOR INDEX
Mallette, M. F., 152
Mallory, M. E., 93
Malmberg, M., 358, 373 (8)
Malvados, A., 283
Mann, G. E., 494
Mann, M. V., 269
Mann, P. J. G., 480
Manna, L., 576
Mannering, G. J., 370, 473
Mannerling, G. F., 395, 396 (10)
Manning, M. P., 551, 553 (195), 554 (195),
558 (195)
Manning, P. D. V., 90, 91, 175, 596 (240)
Manson-Bahr, P., 388
Marangoni, B. A., 55
Mardasheu, S. R., 235 (10, 42)
Mariani, A., 545, 546 (165), 552 (165)
Maritz, A., 353
Maroney, J. W., 61, 79 (28)
Marshall, E. K., Jr., 11, 19, 20, 28, 31, 96,
98 (22), 162
Marshall, J. H., 35
Marshall, W. H., 290
Martin, (A. J. ob. 485.515)(1)). 516, 527,
Bam); p4) (1)
Martin, A. R., 72 (52, 59)
Martin, G. J., 38, 39, 41, 56, 57, 58, 59,
ha6a, 7h, (0; oo, 1495 153) 155; 178,
268, 515 (91), 530
Martinez Garza, H., 327, 358
Martino, L., 59
Marusich, W. L., 589
Mason, H. L., 389, 401, 464, 478
Mason, K. E., 490, 501, 502, 510, 511, 513,
515 (3, 4, 13, 18, 21, 25, 33, 62, 75, 78),
516, 517 (3, 4), 518, 519 (21), 520
(8; 21, 25), 521 (21),/523 (67a), 524,
525, 527, 528 (21), 529, 530 (4, 21, 75),
531 (4), 532 (21, 75), 533 (78), 538
(10, 11), 539 (21, 62), 541 (4, 10, 62,
75), 544, 545 (157, 158, 159), 546 (157,
158, 159), 547 (168), 548 (168), 549
(168), 551 (168), 552 (168), 553 (195),
554 (195), 555 (157, 158, 159), 558 (195),
559 (159), 570, 573
Mason, M., 268, 484
Massey, W. S., 285
Massini, P., 580, 582 (38)
Mathias, A. P., 234
Matsuura, K., 367, 368
Matteson, R., 397
623
Mattill, H. A., 483, 485, 492, 493, 494, 501,
503, 505, 507, 508, 512, 515 (76), 529,
530, 562
Mattis, P. A., 72 (54), 76
Mattocks, A. M., 149 (60), 153 (60), 154
(60)
Matukawa, T., 424
Mauney, J. R., 595 (211)
Mauer, F. D., 386
Maxfield, J. R., 286
Maxwell, J. M., 395 (27)
Maxwell, M. E., 24, 26 (44)
May, C. D., 185, 192, 193, 194 (72), 208
May, H. B., 85
May, M., 118
Mayer, G. G., 507
Mayer, R. L., 12, 15, 33, 324
Mayfield, H. L., 376
Maynard, L. A., 518, 515 (63a), 525, 530
(63a), 592
Mazella, H., 466, 467
Mazer, C., 561
Mazur, A., 44, 52
Medes, G., 269
Meerwein, H., 317, 334, 337 (12)
Mehltretter, C. L., 318
Meigen, W., 6
Meinke, W. W., 136
Meisel, M. N., 309
Meister, A., 234, 235 (45), 237, 238, 241
Melnick, D., 225, 248, 244 (2), 248, 257
(12, 21), 259 (12), 260 (12), 262 (12,
21), 449, 450, 459, 478
Melnick, J. L., 440, 441
Melzer, L., 596 (219)
Melville, D., 301
Mendairy, G. C., 360
Meneely, G. R., 546, 552 (170), 553 (170,
199), 554 (170)
Menendez, J. A., 209
Menge, H., 593, 594
Menniken, G., 235
Menschik, Z., 515 (29), 519, 521 (29), 532
(29), 538, 539
Mentzer, C., 72 (62)
Merlo, P., 55
Merrill, A. L., 455
Merritt, H. H., 283
Messer, A. L., 565
Metcalf, D., 596
Metcalf, R. L., 380
624. AUTHOR INDEX
Metzler, D. E., 237, 238
Meunier, P., 507
Meyer, H., 145, 146 (81)
Meyer, L. M., 211, 215
Meyer, O. O., 537, 567
Meyer, R. E., 493
Meyer, R. K., 521
Meyers, M. C., 47, 85, 206, 210, 212 (195),
213 (195), 215
Michael, A., 6
Michaelis, L., 4, 308, 312 (84), 314, 357,
494
Michaelis, R., 356
Michaud, L., 156, 189, 384
Michi, K., 222, 223 (6), 224, 228, 229 (23)
Mickelsen, O., 389, 392, 401, 451, 476, 478,
479
Milanes, F., 148 (17), 197
Miles, M. C., 505
Milhorat, A. T., 492, 493, 502, 507, 509,
559, 560, 572, 592
Miler, A. K., 40, 71, 133
Miller, C., 291, 296
Miller, C. O., 595 (211)
Miller, C. 8., 132
Miller, D. R., 18
Miller, E. C., 45, 270, 295, 296, 394
Miller, H., 34, 35 (15), 84
Miller, H. C., 488
Miller, J. A., 394
Miller, J. K., 52
Miller, M. H., 273, 274 (83), 283
Miller, O. N., 594
Miller, P. A., 200
Miller, R., 497
Miller, R. F., 594 (147)
Miller, 8., 213
Mills, C. A., 294, 295, 395, 397, 470, 474,
475
Mills, R. C., 33, 39, 42 (85), 176, 297
Milman, A. E., 592
Milner, M., 85
Mims, V., 91, 92, 93, 143 (8), 147, 148, 163,
164, 165, 166, 180, 181, 184 (54), 204,
236 (38)
Minkowski, A., 550
Minnich, V., 158, 209
Minno, A. M., 566
Minot, A. 8., 507, 552, 558, 559 (196)
Minz, B., 430, 462, 463, 468
Mirick, G. S., 25, 68
Mirone, L., 147, 189
Mistry, S. P., 588
Mitchell, G. J., 285
Mitchell, G. W., Jr., 51, 55
Mitchell, H. H., 31, 49, 58, 292, 385, 397,
479
Mitchell, H. J., Jr., 188
Mitchell, H. K., 16, 23, 24 (89, 40), 26
(40), 27, 30 (1, 2), 31 (1), 35 (48, 44),
46, 47 (23), 68, 90, 91, 92, 124, 257
(17, 26), 259 (27)
Mitchell, H. 8., 381
Mitchell, W. R., 35 (36)
Mitra, K., 390
Mitrani, M. M., 470
Mizukami, S., 106, 123
Mizutani, M., 4
Moeller, E. F., 276
Moersch, G. W., 152, 153 (68)
Moggridge, R. C. C., 447
Mohammad, A., 486
Molander, D. W., 548, 552, 553 (200), 554,
572
Molitor, H., 52, 466, 467, 468, 469
Moll, Th., 441
Moller, E. F., 72 (60), 328
Molotchick, M. B., 565
Molthan, L., 558
Monaghan, B. R., 503
Mondy, N. I., 254
Monnier, M., 515 (74), 527, 529 (74), 533
(74)
Monson, W. J., 202
Moore, A. E., 160
Moore, B., 393, 398
Moore, C. V., 158, 209
Moore, D. G. F., 387
Moore, H. P., 269, 396, 397 (82)
Moore, J. A., 40
Moore, M. E., 366
Moore, P. E., 400
Moore, P. R., 185, 273
Moore, R. C., 38
Moore, T., 485, 500, 504, 507, 509, 515
(1, 2), 516, 527, 531 (1, 2), 541 (1)
Moore, W., 191
Moragues, V., 60, 79
Morell, D. B., 368, 364, 365, 366
Morgan, A. F., 262 (42), 272, 503
Morgan, B. G. E., 383
Morgan, C. L., 505
AUTHOR INDEX
Morgan, E. J., 347
Morgan, J., 569
Morgulis, 8., 571
Mori, M., 10
Mori, 8., 230
Morpurgo, E., 56
Morris, A. T., 285
Morris, C. J., 21
Morris, H. P., 295, 330, 386, 394, 395, 473
Morris, J. E., 192
Morris, 8., 257 (18), 260 (18)
Morrison, F. B., 515 (59), 524, 530 (59)
Morton, H. E., 595 (194)
Moruzzi, G., 307
Moseley, O., 278, 279 (131)
Mosher, H. S., 331
Moss, A. R., 487, 489, 492 (58)
Moss, J., 153, 155
Mouriquand, G., 466
Mousset, H., 69
Moyer, E. Z., 474, 480
Moyer, W. T., 545, 552 (167)
Mowat, J. H., 28 (12, 13; 14,15), 39, 71,
95, 96, 98 (21, 24), 99 (24), 101, 103
(24), 104 (27), 105, 106 (27, 34), 108
(33, 35), 109 (27), 113, 114 (23), 115
(3), 121, 122: (1); 123 (2), 143 @; 3, 5,
6, 15, 16), 149 (57), 154 (47), 161, 230
Mozingo, R., 28 (17), 103, 109 (80), 148
(20), 316, 329 (59), 394
Mueller, J. F., 140, 217, 282, 287 (144), 291
Mueller, J. H., 200
Muenzen, J. B., 44, 52
Mulliken, S. P., 10, 11
Munk, M. K.., 515 (29), 519, 521 (29), 532
(29), 538 (29), 539 (29)
Muntz, H. H., 79 (29)
Muratore, F., 55
Murphy, W. P., 286
Murray, A., III, 3
Murray, E. S., 51, 60, 61, 79 (8, 30, 41)
Mushatt, C., 273
Mushett, C. W., 174, 185 (18), 272, 274,
75 (77), 596 (248)
Mustain, R., 568
Mutch, B. C., 571
Myers, M. C., 61
N
Nachod, F. C., 311
Naff, M. B., 592
625
Nafziger, H., 124
Nagata, Y., 11
Najjar, V. A., 360, 364, 401, 478
Nakahara, W., 11, 596 (230)
Nassi, L., 247
Neale, F. C., 46, 47 (20)
Nebbia, G., 584
Negelein, E., 351, 352 (85), 369
Neilands, J. B., 259 (28), 595 (188)
Neligh, R. B., 206, 215
Nelson, E. M., 148, 209, 212, 213 (205),
474, 475 (26)
Nelson, E. N., 193, 194, 208
Nelson, H. G., 74
Nelson, H. V., 212
Nelson, M. M., 175, 265, 383, 384 (59),
515 (79), 529, 533 (79)
Nelson, R. C., 383
Nelson, R. H., 386, 515 (89), 530
Nelson, V. E., 485
Nelson, W. L., 504
Nemir, R. L., 458, 480
Nesheim, R. O., 188
Neter, E., 27, 28 (9)
Netsky, M. G., 558
Neuberg, C., 427
Neuberger, A., 238
Neumann, A. L., 188
Neumann, F. W., 76
Neuweiler, W., 545, 546
Nevens, W. B., 385
Newell, G. W., 515 (115), 536
Newman, E. V., 174
Newman, J. R., 79 (81)
Newsome, A. A., 285
Nichol, C. A., 194, 198, 208, 214
Nicholls, J. V. V., 391
Nicholson, J. T. L., 478
Nielsen, E., 38, 71, 189
Nielsen, H., 439
Niementowski, St., 120
Nigrelli, R. F., 196
Niles, J. O., 79 (32)
Nimmo-Smith, R. H., 134, 135
Nitti, F., 69
Niven, C. F., Jr., 199, 590
Noble, E. R., 595 (184)
Noble, W. M., 34
Nociti-Carroll, V., 235 (55), 237 (55), 353,
354 (91)
Noll, H., 75
626
Noordhoek, F. J., 566
Norris, E. R., 146, 596 (222)
Norris, F. A., 497
Norris, F. W., 363
Norris, L. C., 90, 149 (55), 152 (55), 153,
154 (70), 177, 178 (42), 297, 363, 374,
386, 395 (20, 22), 588, 594 (147, 155),
595, 596 (224, 232, 234)
Northey, E. H., 28 (12), 39, 71 (50), 73,
101, 104 (27), 105, 106 (27, 34), 108
(33, 35); 109 (7), 121, 122 (1), 128
(2), 143 (15, 16)
Nothacker, W. G., 558
Nottebohm, E., 6
Novelli, G. D., 39
Nurmikko, V., 587
Nyberg, W., 595
Nygaard, A. P., 590, 596 (253)
O
O’Banion, E. E., 76
O’Brien, C. 8., 381, 390 (36)
O’Brien, J. R., 223, 255, 410 (10)
Ochoa, 8., 369, 427, 428, 435 (18, 19), 436,
438, 439, 442, 450, 579
O’Dell, B. L., 28 (16), 92, 93 (10), 94, 99
(10), 100 (10), 101 (28), 102, 103, 143
(14), 147, 165, 168, 176, 177, 201, 594
(146)
Oden, J. W., 388
Oden, L. H., Jr., 388
O’Donnell, D. J., 43, 276, 395 (26)
Oesper, R. E., 7
Offhaus, K., 379
Ogden, F. N., 193, 207
O’Grady, P., 513, 546
Ohdake, S., 222
Ohlmeyer, P., 595
Olafson, P., 515 (58, 59)
Olcott, H. S., 515 (54)
Ohlson, M. A., 399
Ohta, M., 424
O’Kane, D. E., 237
O’Kane, D. J., 237, 241, 242 (26), 440,
576, 577 (9), 579, 581
Olafson, P., 524, 530 (59)
Olcese, O., 594 (156)
Olcott, C. T., 172
Olcott, H. S., 485, 490 (19), 491 (21), 503,
524, 532 (54)
Oldham, H. G., 399, 401 (61, 62), 478, 480
AUTHOR INDEX
Oleesky, S., 565
Oleson, J. F., 596 (227)
Oleson, J. J., 143 (10), 149 (57), 154 (87),
158, 160, 177, 591, 592 (182), 596 (227,
256)
Olive, T. R., 498
Omata, R. R., 595
O’Meara, R. A. Q., 74
Onstott, R. H., 384
Opie, J. W., 489, 491, 499, 500
Oppenheim, A., 286
Oppenheimer, C., 341
Orgain, E. S., 565
Orla-Jensen, S., 372, 377
Orlan-Jensen, A. D., 589, 597 (118)
Ormsbee, R. A., 35 (48)
Ormsby, A. A., 246
Orozco, F. V., 588
Orr, J. H., 68
Oyerey Ih oe) aa 7
Orr, W. F., 558
Orsini, D., 269, 370, 396
Ortega, L. G., 161
Oser, B. L., 225, 243, 244 (2), 248 (2), 257
(12, 21), 259 (12), 260 (12), 262 (12,
21), 459
Osserman, K. E., 569
Ossetrava, E. D., 419
Ostling, G., 595
Ostwald, R., 312
Oswald, A., 54
Oswald, E. J., 20, 23 (14), 25 (14), 34, 36
(16, 58), 67, 68 (11)
Ott, W. H., 252, 291, 596 (248)
Otte, N. C., 372, 377
Ottesen, J., 511
Overman, R., 294
Overman, R. 8., 75, 172
Owens, E. U., 548, 549
Owens, W. C., 548, 549
Oxford, A. E., 35 (33)
Oyama, V. I., 594 (182, 183)
P
Paegel, B. L., 211
Paganelli, V., 160
Page, A. C., Jr., 594 (153)
Pagé, E., 294
Page, E. W., 2385 (54)
Page, R. M., 595
Paget, M., 56
AUTHOR INDEX
Painter, E. E., 20
Painter, H. A., 191
Pallister, R. A., 387
Palmer, L. S., 262 (41), 511
Palmieri, M. L., 474
Pappenheimer, A. M., 510, 515 (19, 22, 27,
38, 53, 56, 57, 63, 66, 77, 81, 99, 101,
106, 107), 516, 518, 519, 520, 524 (27),
525 (57), 527, 528 (27), 529 (56), 532
(56), 533 (77), 534 (57, 68, 81, 99), 535
(57, 81), 540 (9), 556, 557 (208), 571
Park, C. R., 24 (47), 35 (1)
Parke, I. V., 578
Parker, J. G., 48
Parker, R. P., 111, 113 (76), 114, 115 (90),
116 (90), 117 (90), 118 (90), 119 (90,
95), 120 (90, 95)
Parker, W. E., 507
Parks, R. E., Jr., 159, 290, 291 (4)
Parrish, 1D. B:, 510, 513, 572
Parrott, E. M., 90, 91, 175
Parsons, H. T., 360, 393, 398
Parvé, E. P.S., 429, 439, 440 (82), 451
Pascher, F., 569
Paschkis, K. E., 48
Pascoe, A. L., 565
Pashkina, T. 8., 238, 268
Passmore, R., 426
Pasternak, R. L., 111
Patch, E. A., 39, 40, 72 (54)
Pateck, A. J., Jr., 385
Patrick, H., 505
Patterson, E. L., 577 (16), 578, 581
Patterson, P. A., 158
Patton, A. R., 590
Patton, E. W., 390
Patton, R. A., 266
Patton, R. L., 380
Patrono, V., 57
Patwardhan, V. N., 269
Pauli, R., 72 (56)
Pearce, H., 391
Pearson, E., 397
Pearson, P. B., 179, 366, 386, 395, 396
Pease, G. L., 206
Pedersen, K. O., 340
Peel, E. W., 316, 329 (59), 394
Peeler, H. T., 588, 594 (147), 595
Peet, C. H., 9
Pehl, W., 283
Pelezar, M. J., Jr., 595 (192)
627
Pellares, I. S., 588
Peng, D. H., 363
Pennington, D., 23, 27, 30 (5), 31 (5), 35
(42)
Pennington, R. J., 257 (1), 262 (1), 280
Pennock, L. L., 566
Perault, R., 72 (62)
Peretti, G., 267, 269
Perez-Santiago, E., 79 (18)
Perla, D., 469
Perlman, D., 333, 595
Perlman, G., 441
Perlman, L., 262 (42)
Perloff, W. H., 567
Perlzweig, W. A., 268, 292, 475
Perosa, L., 55
Perry, R. L., 386, 387 (98), 395 (25)
Peterfalvi, M., 443
Petering, H. G., 124, 591
Peters, L., 58, 64
Peters, M., 61, 79 (24)
Peters, M. V., 268
Peters, R. A., 223, 255, 410 (10), 426, 427,
428, 430, 432, 435 (18, 19), 488 (68),
441, 450, 461, 474, 581, 596 (216)
Peters, V. J., 40, 277
Petersen, R., 108
Peterson, E. A., 234, 237
Peterson, J. C., 194, 207
Peterson, M. S., 199, 360
Peterson, W. H., 10, 13, 22, 24 (46), 25,
26 (43, 53), 27, 30 (8), 31 (3), 35 (30,
33, 34, 45), 36 (62), 90, 91, 175, 199,
276, 360, 372, 378, 595 (191)
Peterson, W. J., 308, 376
Pfaehler, K., 315, 316 (54)
Pfeifer, V. E., 333
Pfeiffer, C. C., 290, 291
Pfeiffer, S. E., 257 (14), 260 (36)
Pfiffner, J. J., 28 (16), 92, 93, 94, 99, 100,
101 (28), 102, 103 (28), 164, 165, 176,
177, 178, 201
Pfister, K., 3rd., 320, 332
Philips, F. S., 158, 160, 214
Phillips, P. H., 381, 387 (47), 396, 592,
593
Phizackerley, P. J. R., 269
Pick, Bi: P., 487
Pidacks, C., 595 (186)
Pierce, J., 166
Pierce, J. V., 577 (16), 578, 594 (153)
628
Pike, R. M., 68
Pilgrim, F. J., 230
Pin, L., 565
Pindborg, J., 513, 541
Pines, I., 467
Pini, H., 488, 496
Pinkerton, H., 60, 79 (22), 383
Pinkos, J. A., 276
Pirie, A., 381
Pitt, A. A-;A9
Pitt, D. A., 278, 279 (129)
Pitt-Rivers, R., 57
Platt, B. S., 400, 462
Platz, B. R., 250
Plaut, G. W. E., 137, 138, 323, 361
Plentl, A. A., 140
Plotka, C., 448
Plotz, H., 61, 79 (9)
Pock-Steen, P. H., 390
Pohland, A., 28 (18), 113, 114 (84), 115
(84), 120 (84)
Polak, J. J.; 427
Poling, C. E., 38
Pollack, H., 389, 392, 393, 401, 569
Pollack, M. A., 394
Pollard, A. E., 204, 217, 259 (30), 260
(380), 262 (30)
Pope, H., 36 (61)
Popp, A., 84
Popp, W. C., 286
Popper, H., 505, 540, 553, 554 (201)
Popper, K. J., 274
Porter, C. C., 291, 293 (11)
Porter, J. W., 46, 47 (24), 53
Porter, T., 399, 401, 480
Portis, S., 288
Post, J., 385
Post, M., 468
Postage, J. R., 35
Posternack, R., 308
Potter, R. L., 384, 395
Pounden, W. D., 385
Power, F. W., 44
Purcell, F. M., 390
Putignano, T., 55
Prados, M., 461
Prange, I., 505, 507, 511, 515 (64, 111),
525, 534 (64), 536, 570
Prebluda, H. J., 246, 449
Preobrazhanskil, A., 318, 323 (68)
Price, S. A., 260 (82)
AUTHOR INDEX
Prichard, W. W., 487, 491, 498
Pring J) Ne
Pringle, A., 149, 153 (56), 154 (56)
Pringle, R. B., 130
Pritchard, J. E., 569
Proutt, L. M., 465
Purrman, R., 145
Q
Quackenbush, F. W., 489, 490, 498, 504,
511, 570
Quaife, M. L., 496, 500, 501, 507, 508, 509,
510, 512, 513, 543, 544, 546 (164), 551,
552 (164, 192, 193), 555, 571
Quastel, J. H., 77, 237, 430, 439
Quibell, T. H., 326, 358
Quick, A. J.; 16
R
Rabinovitch, R., 559
Rabinowitz, J. C., 253, 254, 255 (62), 257
(15), 261 (15, 24), 263, 279, 280 (137),
292, 590
Rabinowitz, M., 491, 495
Rachele, J. R., 142
Radice, J. C., 512, 515 (95), 531
Raffy, A., 360
Rainbow, C., 35 (53, 54, 55)
Raistrick, H., 356, 594 (176)
Rajagopal, K. R., 509
Rakoff, A. E., 48
Raleigh, G. W., 79
Ralli, E. P., 45
Ramasarma, G. B., 587, 588 (91)
Ramsdell, P. A., 348
Rannefeld, A. N., 254, 265, 277
Ransone, B., 172
Rao, M. N., 275, 298
Rapaport, 8., 374
Rasmussen, A. F., 472
Rasmussen, R. §., 120
Ratcliffe, A. H., 566
Rathmann, F. H., 490
Ratner, S., 16, 28, 353, 354 (91)
Raub, A., 235
Rauch, K., 355
Rauramo, L., 546, 552 (171)
Ravel, J. M., 70, 103, 110 (31), 118, 180,
143 (24), 156, 590
Ravenel, S. F., 61, 79 (83, 34)
AUTHOR INDEX
Ravenscroft, P. H., 3
Ravin, I. §., 565
Rawlings, H. W., 500
Reader, V., 223, 255, 596
Reagen, J. W., 557
Reasenberg, J. R., 7, 9 (51)
Reboredo, A., 197
Record, P. R., 386, 395 (21)
Records, E., 515 (60), 524
Redi, K. K., 459
Reed, G. B., 68
Reed, L. J., 440, 577, 578 (13, 27, 28, 29),
579, 580, 581 (40), 582 (14, 26)
Reed, R. W., 68
Reed, W. O., 515 (61), 524
Reedman, E. J., 250
Rees, O. W., 3
Reestman, A. J., 457
Regan, M., 127, 1389 (7)
Regan, M. A., 576
Register, U. D., 185
Regnier, J. L., 7, 9 (58)
Reichert, D. A., 596 (255)
Reichert, E., 583
Reichstein, T., 191, 318
Reilly, R. H., 290
Reinemund, K., 311, 316, 318 (58), 334,
359
Reingold, I. M., 470
Reinhold, A., 235 (48, 49)
Rheinhold, J. G., 478
Reiser, R., 396
Reissig, J. L., 238
Remington, R. E., 374
Remy, C., 351
Renfrow, W. B., Jr., 489, 491, 499, 500
Rentschler, H., 491
Reuthe, F., 414
Reynolds, M. §., 292
Rhoads, C. P., 61, 430, 487 (85)
Ribeiro, F., 78
Richards, A. J., 480
Richards, G. V., 38
Richards, H. J., 568
Richards, M. B., 294
Richards, R. K., 50, 54, 55, 58, 59
Richardson, L. R., 171, 173, 178, 381
Richert, D. A., 351
Richtsmeier, A. J., 537, 567
Rickes, E. L., 90, 109, 143 (21, 23)
Rideal, E. K., 9
629
Ridgeway, R. R., 492, 501, 513
Ridgway, L. P., 158
Riegel, EK. R., 4, 5
Rieux, N., 545
Rigaud, M., 120
Riggs, T. R., 45, 596
Riley, E., 77
Rindi, G., 473
Rinehart, J. F., 268, 274, 275, 287, 297,
437, 461
Ringier, B. H., 337 (22), 487, 488, 489, 490,
492, 498
Ringk, W. F., 7, 9 (50)
Ringrose, A. T., 374, 395 (20)
Ringsted, A., 515 (16, 70, 71), 517, 526,
527 (71), 528, 529 (71), 533 (71)
Rinzler, 8. H., 565
Ripy, H. W., 194, 207
Risley, H. A., 502, 559
Ritchey, M. G., 30 (20, 21), 31
Ritsert, K., 441, 500
Ritz, N. D., 215
Robbins, E. B., 49, 54
Robbins, M., 164
Robbins, M. L., 79 (10)
Robbins, W. J., 35 (52), 264
Roberts, E., 235 (40, 41), 268
Roberts, E. C., 69, 127, 135 (8), 168
Roberts, L. J., 401 (62), 478
Robertson, E. I., 177
Robertson, J. E., 286
Robertson, W. van B., 330, 394, 395
Robeson, C. D., 489, 490 (56), 492, 493
(93), 496, 497 (5), 502 (7), 508, 513
Robinson, 1., 151, 152 (66), 160 (66), 161
(66), 594 (178)
Robinson, F. A., 301, 324, 595, 596 (249)
Robinson, J. D., 7
Robinson, K. L., 539
Robinson, P. F., 392, 402
Robinson, W. D., 391
Roboz, E., 268, 286, 293
Roby, T. O., 386
Rodbart, M., 324
Rodda, H. J., 116
Roderuck, C., 480
Roderuk, C. E., 392, 474
Rodwell, A. W., 235, 240
Roedig, A., 355
Roehm, R. R., 451
630
Roepke, R. R.; 33, 35 (8, 12), 135, 136
(38)
Rogalski, T., 515 (29), 519, 521 (29), 532
(29), 538 (29), 539 (29)
Rogers, D., 590, 591, 596 (254, 122)
Rogers, L. L., 111, 126, 131 (5), 408
Rogers, R. E., 596 (240)
Rogers, W. M., 529
Roholt, K., 40
Rominger, E., 145, 146 (31)
Rook, G., 283
Roscoe, M. H., 596 (244)
Rose, C. 8., 537, 540, 546, 547 (176)
Rose, F. L., 72 (52, 59), 330
Rose, H. M., 61, 79 (85)
Rose, W. C., 585, 587
Rose, W. G., 504
Rosen, F., 268
Rosenbaum, E. E., 283
Rosenberg, H. R., 301, 368, 410 (13), 596
(218, 228)
Rosenblum, H., 62, 79 (20), 83
Rosenblum, L. A., 283, 293
Rosenkrantz, H., 492, 493, 502, 507, 509,
559, 572, 592
Rosenthal, H., 191
Rosenthal, P., 427
Ross, CoB: 371
Ross, J. B., 148 (7)
Ross, J. F., 211, 218
Ross, S., 79 (86, 37, 38, 42, 43)
Rossiter, R. J., 369, 474
Roswell, E. V., 237
Roth, B., 111, 113 (76), 114, 115 (90), 116
(90), 117 (90), 118 (90), 119 (90, 95),
120 (90, 95), 155
Roth, H., 308, 304 (2), 366
Rothman, 8., 6, 60
Routh dials. 27
Roux, H., 4387, 448
Rovenstine, EH. A., 55
Rowland, S. J., 371
Rowold, E., 596 (236)
Roy, G. K., 250
Rozsa, P., 506
Rubbo, 8. D., 24 (45), 26 (44, 51), 32, 35,
67
Ruben, §., 434
Rubenstein, B. B., 567
Rubin, J., 6, 60
Rubin, M., 26 (53), 72 (55)
AUTHOR INDEX
Rubin, 8. H., 253, 305, 306, 359, 363, 366,
589
Ruckstuhl, H., 329
Rudy, H., 303, 305, 307 (12b), 309 (12b),
310 (12b), 311 (12b), 312 (12b), 322,
334, 336, 359, 369
Ruegamer, W. R., 156, 184, 185, 189
Ruehle, A. E., 406
Rif, H., 15
Ruffin, J. M., 552 (202), 553, 554 (202),
558 (202), 565
Ruhkopf, H., 410
Rumery, R. E., 515 (114), 517, 536, 538
(11)
Rundles, R. W., 208
Runti, C. 8., 306
Rupp, W., 108
Ruppel, W., 515 (92), 530, 531 (92)
Rush, H. P., 270, 565
Ruskin, A., 469, 470
Russell, P. B., 149, 150, 151 (65)
Russo, H. F., 14, 39, 40, 49, 72 (54)
Rutzky, J., 215
Ruyeyea Ande. 47
Ryan, E., 389
Rydin, H., 427
Rymaszewski, O., 515 (29), 519, 521 (29),
532 (29), 538 (29), 539 (29)
Rzeppa, W., 326, 358
S)
Sabine, J. C., 393
Sadhu, D. P., 270, 467
Sab Pee al@
Sala, G., 66
Salamon, I. I., 18
Salassa, R. M., 62, 82
Salkin, I., 79 (29)
Salomon, H., 303, 305, 310, 315, 316 (54),
358, 486, 487, 488 (35), 489, 490, 492,
498
Salmon, R. J., 192, 193, 194, 208
Salmon, W. D., 141, 540
Salvador, U. J., 7, 9 (55)
Salzberg, P. L., 316
Samarina, O., 237
Samitz, M. H., 283
Sampath, A., 160
Sampson, W. L., 38, 59, 250,, 422 454, 466,
467, 468, 469
Sanadi, D. R., 322, 580, 581 (37)
AUTHOR INDEX
Sanchez Ruiz, F., 79 (39)
Sandberg, M., 469
Sanders, B. G., 594 (156)
Sandground, J. H., 58, 62, 63, 64 (9), 66
Sandstead, H. R., 391
Sanford, M., 35 (37), 586
Sanger, F., 586
Santos, J. J.,79 (18)
Sanz Sanchez, Felix, F., 56
Sarett, H. P., 33, 35 (7), 133, 134, 323,
330, 358, 392, 402, 451, 475, 596 (238)
Sargent, F., 392
Sarkander, H., 468
Sarma, P. S., 237, 241, 251, 252 (48), 253,
254, 257 (9), 260 (9), 261 (9, 38), 262
(9), 268, 294
Saslaw, S., 181, 182, 183, 184, 195 (64)
Sauberlich, H. E., 110, 120, 185, 262 (39),
270, 271 (65)
Saunders, B. C., 10
Saunders, K. H., 7
Savage, E. E., 505
Savage, J. E., 594
Sawhill, J., 283, 293
Sawicky, H. H., 569
Sawitsky, A., 215
Seardino, P. L., 568
Schaar, F., 185, 194 (72), 208
Schaefer, A. E., 141, 189, 190, 272, 276,
298, 385, 395, 594
Schales, O., 234, 235 (38, 39, 53), 241 (33,
34)
Schales, S. S., 235 (38, 39, 53), 236 (4)
Schapita, G., 235 (60)
Schardinger, F., 347
Schauer, L., 374
Scheie, H. G., 548 (181), 549
Scheifley, C. H., 61, 81, 82 (51)
Scheiner, J., 253, 589
Schenker, V., 392
Schepartz, A. I., 322
’ Scheutz, F. W., 479
Schiro, H. S., 284
Schlapfer, R., 490
Schlenck, F., 237
Schlenk, F., 13, 237, 241 (37, 38), 246, 267,
278, 596 (219)
Schleyer, W. L., 65
Schlittler, E., 310, 315, 316 (54)
Schmidt, C. L. A., 501
Schmidt, G. W., 551
631
Schmidt, H., 386, 395
Schmidt, H. L., Jr., 381
Schmidt, M., 485
Schmidt, M. B., 264
Schmidt, V., 40, 565
Schmied-Kowarzik, V., 100, 106, 108 (26),
124
Schmitt, F. O., 503
Schmitt, J. A., 124
Schnakenberg, G. H. F., 578
Schneider, H. A., 250, 472
Schnitzer, R. J., 65
Schoeffel, E. W., 425
Schoen, K., 306, 359
Schoenbach, E. B., 161, 198, 214
Schoenheimer, R., 140
Schogoleff, C., 510, 515 (19, 22), 518
Schopfer, W. H., 77, 328, 408, 409, 446,
447 (4), 451
Schopp, H., 334
Schépp, K., 303, 305, 310, 315, 316 (54),
358
Schotland, C. E., 267, 283
Schoubye, N., 409, 476
Schraffenberger, E., 383
Schreffler, C. B., 250, 410
Schreiber, 8S. S., 196
Schrucki, J., 441
Schubert, M. P., 308, 312 (34)
Schuchardt, G. §., 49
Schuette, H. A., 262 (40)
Schuetz, R. D., 590
Schulek, E., 506
Schulman, M. P., 132
Schultz, A. S., 243, 254, 257 (8), 259 (8),
260 (8), 262 (8), 276, 279, 280, 411, 450,
452, 462, 465
Schultz, F., 408, 410
Schultz, F. W., 479
Schumacher, A. E., 90, 596
Schumacher, R. §., 360
Schuman, R. H., 363
Schuster, P., 427, 430, 438
Schwab, J. L., 181, 182, 183 (62)
Schwartz, 8S. O., 211
Schwartzenbach, G., 357
Schwartzman, G., 296
Schwartzman, J., 283
Schwarz, K., 397, 540, 594
Schwarzenbach, G., 314
632
Schweigert, B. S., 165, 178, 179, 204, 217,
257 (5), 260 (5), 261 (5), 262 (5, 39),
270, 271 (65), 279, 294, 366, 376, 377
Schwyzer, R., 107, 123
Scott, C. C., 49, 54
Scott, E. G., 61, 79 (19, 28)
Scott, H. H., 387
Scott, J. W., 284, 391
Scott, L. D., 286
Scott, M. L., 149 (55), 152 (55), 153, 154
(70), 177, 178 (42), 202, 363, 594, 596
(234)
Scott, W. W., 568
Scrimshaw, N.S., 507, 546, 551, 552 (169),
562 (169)
Scudi, J. V., 224, 247, 248, 292, 502, 506,
509
Seager, L. D., 558
Sealock, R. R., 459
Seaman, A., 140, 217
Seaman, G. R., 581
Seaman, W., 111
Sealock, R. R., 191
Sebesta, E. E., 275, 297
Sebrell, W. H., 71, 143 (9), 147 (9), 171,
172, 173, 174 (15, 16), 190) (15), 195,
266, 290, 382, 383, 384, 388, 390 (124),
391 (123, 124), 400, 473, 516, 540 (6)
Seeger, D. R., 28 (12), 39, 71 (50), 73,
101, 104 (27), 105, 106 (27, 34), 107,
108 (88, 35), 109 (27), 121, 122 (1),
123 (2), 148 (15, 16), 149, 155, 156
(77), 157
Seegers, W. H., 567
Segel, L., 225
Segretain, G., 595 (210)
Seidell, A., 410
Selbie, F. R., 54, 67
Semb, J., 28 (12, 13; 14, 15), 39; 71, 95,
96, 97, 98 (21, 24), 99 (24), 101, 103
(24), 104 (27), 105, 106 (27, 34), 108
(33, 35), 109 (27), 118, 114 (23), 115
(23), 1215 122) (1), 12372) 1435(53..5;
6, 15, 16), 149 (57), 154 (57), 161
Serri, F., 85
Sevag, M. G., 20, 33, 69
Shaffner, C. S., 196
Sharp, E. F., 257 (4), 260 (4)
Sharpe, G., 156
Sheft, B. B., 454
Seegmiller, J. E., 324
AUTHOR INDEX
Seidell, A., 405
Sensen, W. N., 237
Seusing, J., 470
Seyle, H., 392
Shank, R. E., 400
Shaw, A. O., 376
Shaw, J. H., 381, 387 (47), 396
Sheft, B. B., 399, 480
Shepherd, J. R., 306
Sheppard, E. C., 268
Sherman, H., 250, 265, 269, 295
Sherman, H. C., 302, 369, 370, 373, 395
Sherman, J. M., 199, 590
Sherman, W. C., 426
Sherwin, C. P., 44, 52
Sherwood, C. R., 474
Sherwood, R. M., 179
Sheybani, M. K., 386, 395
Shields, J. B., 257 (4), 260 (4)
Shikata, K., 35 (32)
Shimizu, 8. J., 325
Shive, W., 17, 20, 28 (18), 40, 69, 70, 79,
103, 110 (31), 111, 113, 114 @4see
115 (84), 118, 120 (84), 126, 128, 129,
130, 131 (5), 135 (8), 148 (24, 25), 149,
156, 270, 298, 301, 408, 446, 590
Shock, N. W., 290
Shorb, M. S., 327, 589, 593, 594 (143, 148),
595 (192)
Shorr, H. L., 147, 188
Short, W. F., 356
Shorvon, L. M., 286
Shriner, R. L., 6
Shukers, C. F., 147, 180, 182, 184 (54), 383
Shull, G. M., 35 (34), 595 (191)
Shuman, C. R., 558
Shunk, C. H., 316, 329 (59)
Shute, E., 537, 561, 562, 565, 566 (9), 567
Shute, W., 561, 565, 566 (9)
Shwartzman, G., 271
Shy, G. M., 559, 560 (227)
Sibley, M., 110, 114 (74)
Sica, Ac J, L475 lio
Sickels, J. P., 28 (12), 39, 71 (50), 73, 101,
104 (27), 105, 106 (27, 34), 108 (33,
35), 109 (27), 121, 122 @), 235ee
143 (15, 16)
Siedenstopf, H., 566
Siegel, L., 257 (12), 259 (12), 260 (12), 262
(12)
Sieve, B. F., 59, 60, 83, 84 (62)
AUTHOR INDEX
Sievers, O., 79
Sikkema, 8. H., 567
Silber, R. H., 291, 293 (11), 392, 475
Silberberg, R., 271, 386
Silbernagel, W. M., 285
Silberstein, H. E., 191
Siliprandi, D., 439
Siliprandi, N., 439
Silver, P. H., 57
Silverberg, M. G., 569
Silverman, M., 114, 120, 121 (89), 330, 430,
436
Simmonds, N., 513
Simmonds, 8., 586, 587
Simmons, R. W., 146
Simmonsen, D. H., 592
Simola, P. E., 427
Simpson, R. E., 165
Sims, E. 8., 166
Sinclair, H. M., 426, 439, 450, 581
Singer, E., 486
Singer, M., 291, 296
Singer, T. P., 353, 357, 589
Singleton, W.S., 489, 497, 498
Sirks, J. L., 72 (57)
Sjogren, B., 27, 36 (59)
Skeggs, H. R., 20, 23 (15), 25 (15), 39,
67, 76, 147, 588, 594 (149)
Skelton, F., 537
Skelton, F. R., 566, 567
Skinner, H. G., 537, 567
Skipper, H. E., 133, 149 (60), 153 (60),
154 (60), 161
Skoog, F., 379, 595 (211)
Skrimshire, G. E. H., 356
Slanetz, C. A., 294, 515 (5, 97), 516, 520,
531 (5), 5382 (85, 36)
Slater, E. C., 364, 365, 366
Sloane, N. H., 91, 94, 143 (8), 177, 184,
587, 595 (85), 596 (252)
Sloane Stanley, G. H., 235 (59)
Slobodkin, N. H., 93, 108 (15), 177
Slotin, L., 434
Smadel, J. E., 61, 79 (9)
Smiley, K. L., 333, 590
Smith, B. F., 464
Smith, C. W., 120
Smith, D. C., 465
Smith, D. T., 36 (61)
Smith, G. B. L., 7, 9 (51)
Smith, H. D., 216
633
Smith, H. W., 52, 53 (9)
Smith, J. A., 466, 467, 468
Smith, J. M., Jr., 28 (12), 39, 71 (50), 73,
101, 103, 104 (27), 105, 106 (27, 34),
108 (33, 35), 109 (27), 111, 113 (76),
114, 115 (29, 90), 116 (90), 117 (90),
118 (29, 90), 119 (90, 95), 120 (90, 95),
121, 122 (1), 123 (2), 143 (15, 16), 149
(59), 155 (58), 156 (77), 157 (58, 74)
Smith, L. I., 487, 488, 489, 491, 493, 496,
498, 499, 500, 508
Smith, M. B., 333
Smith, M. I., 323
Smith, P. F., 595 (194)
Smith, P. K., 48, 48 (2), 53, 61, 79 (10,
36, 40)
Smith, R. J., 419
Smith, R. P., 291
Smith, R. T., 82
Smith, S. C., 184, 185, 594 (172)
Smith, S. E., 592
Smits, G., 455
Smyrniotis, P. Z., 324
Smyth, D. H., 430, 434
Smythe, C. V., 308, 312 (34)
Snell, E. E., 23, 40, 68, 90, 91 (6), 92, 124,
158, 159, 162, 163, 168, 198, 199, 231,
237, 238, 241 (37), 242, 243, 251, 252
(48), 253, 254 (53), 255 (62), 257 (9,
15, 24), 260 (9), 261 (9, 15), 262 (9, 24),
263, 265, 267, 276, 277 (109), 278 (119),
279, 280 (187), 290, 292, 294, 295,
327, 358, 372, 373 (63, 66), 378, 576,
577, 585, 586, 587 (83, 84), 588, 590,
594 (145), 595
Snipper, L. P., 588
Snog-Kjaer, A., 372, 377
Snow, M. R., 115 (30a), 519, 521 (30a)
Snyder, J. C., 51, 60, 61, 79 (7, 8, 30, 41),
81
Snyderman, 8. E., 181, 281, 458, 480
Sobell, S. D., 306
Sober, H. A., 234, 235 (45), 237, 241, 393,
430
Sobolow, M., 333
Sobotka, H., 507
Sodi Pallares, E., 327, 358
Sollman, T., 56 % &
Solmssen, U. V., 500
Solomon, A. K., 434
Soloweitchik, 8., 78
634
Somers, G. F., 345
Somogyi, J. G., 460
Sondergaard, E., 505, 515 (64, 111), 525,
534 (64), 536
Soper, Q. F., 578
Sorkin, H.,75 .
Soskin, 8., 283
Sossman, M. C., 286
Soumalainen, P., 515 (87), 520
Southwick, P. L., 28 (17), 103, 109 (380),
143 (20), 408
Spangler, J. M., 61
Speck, M. L., 278, 279 (129)
Spector, H., 384
Spensley, P. C., 9
Sperber, E., 440
Sperling, G., 571
Spicer, 9. es, cl wih
Spiegelberg, H., 108, 421
Spies, T. D., 140, 143 (4, 17), 197, 204, 206
(154), 209, 211, 283, 284, 292, 293, 367,
388, 390 (120, 121), 392, 393, 402, 410,
412, 469, 552
Spillane, L. J., 508
Spink, W. W., 20, 28, 25, 67
Spitzer, E. H., 459
Spitzer, W., 347
Spivey, M., 397
Spoerri, P. E.,7,9 (50), 316, 331 (56)
Spooner, M., 537, 567
Sporn, E. M., 185
Sprague, K. L., 76
Spreyer, W., 235
Sprince, H., 189, 585, 595, 596
Sprinson, D. B., 187, 188, 198
Sprung, J. A., 488
Squibb, R. L., 273
Sreenivasan, A., 165
Stéhelin, H., 491
Stamp, T. C., 21, 66, 67
Stander, H. J., 561
Standfast, B., 356
Stanford, C. E., 389
Stannus, H. §., 387, 389, 391
Stare, F. J., 178, 280, 592
Starling, D., 149 (60), 153 (60), 154 (60)
Starr, M. E., 594 (159)
Stauffer, J. F., 594 (174)
Stebbins, R. B., 266, 272, 274 (77), 275
(77)
Steckley, A., 270
AUTHOR INDEX
Steele, J. M., 60, 83
Steenbock, H., 250, 485, 489, 490, 498,
504, 511, 570
Steenken, W., Jr., 78
Steers, E., 33
Steiger, M., 318, 488 (53), 489
Steigmann, F., 505, 553, 554 (201)
Stein, G. A., 418, 421, 422
Stein, H. J., 273, 274 (83), 283
Stein, 8S. L., 521
Stein, W., 565
Steinberg, C. L., 554, 563, 568 (3), 569
Steinberg, D. L., 380, 389, 390 (1382),
391 (132), 398 (1)
Steinkemp, R. C., 400
Steinman, H. G., 594 (180, 181, 182, 183)
Stekol, J. A., 137
Stern, E. L., 469
Stern, J. R., 579, 594 (159)
Stern, K. G., 308, 334, 341, 440, 441
Stern, M. H., 489, 490 (56), 496, 508
Stetten, M. R., 20, 70, 129, 130
Stevens, F. E., 560
Stevens, J. R., 222, 223 (8), 224, 225 @,
12), 226 (12), 228, 248, 245 (4, 5), 247
(5), 418, 421, 422, 488
Steward, F. C., 595
Stewart, A., 90, 182, 204
Stewart, A. P., Jr., 257 (4), 260 (4)
Stewart, C. P., 347 (57)
Still, J. L., 480, 435
Stiller, E. T., 224, 225, 226, 227, 228, 243,
245 (5), 247 (5), 362, 363 (1), 364 (1),
365, 366 (1), 504
Stock, C. C., 151, 152 (66), 160 (66), 161
(66)
Stockell, A. K., 188, 191 (80)
Stocken, L. A., 485
Stodsky, B., 285
Stoerk, H. C., 266, 271, 284, 330, 394
Stokes, J., Jr., 288
Stokes, J. L., 90, 109, 125, 140, 143 (23),
200, 254, 257 (2), 260 (2), 261 (2), 262
(2), 278, 279 (130)
Stokstad, E. L. R., 28 (12, 18, 14, 15), 39,
71, 90, 91, 92, 93, 94, 95, 96, 98 (21, 24),
99 (24), 101, 103 (24), 104 (27), 105,
106 (27, 34), 108 (15, 33, 35), 109 (27),
110, 111, 113 (76), 114 (23, 78), 115
(23), 121, 122 (1), 123 (2), 125, 126,
127, 130, 189, 148 (2, 3, 6, 8, 10, 15, 16,
AUTHOR INDEX
19), 149 (57), 154 (57), 155, 156, 157,
158 (85) 161, 162, 166, 175, 177, 178,
184, 187, 195, 196 (109), 197, 198, 199,
210, 214, 386, 387 (96), 395 (23), 576,
577 (16), 578, 581, 585, 590, 594 (154),
596 (233, 240)
Stoll, R., 78
Stone, G. B., 306, 374
Stone, L., 333
Stone, M. A., 41
Stone, R. E., 143 (4, 17), 197
Stone, R. V., 565
Stone, S., 283, 293
Strait, L. A., 5
Strandskov, F. B., 26 (52), 72 (55)
Straub, F. B., 344, 350 (49)
Straub, G. J., 363
Straumfjord, J. V., 545, 546 (164), 552
(164)
Strauss, E., 11, 51, 60
Strauss, L. 271, 296
Strauss, M. B., 206
Street, H. R., 272, 381, 384, 395
Strehler, B. L., 34
Streigthoff, F., 20, 22, 23 (14), 25 (14), 34,
35 (18), 36 (16, 58), 67, 68 (11)
Strengers, H., 474
Strength, D. R., 141
Strickland, A. G. R., 27, 30 (8)
Stritzler, C., 566
Strébele, R., 312, 315, 316, 318 (58), 323
(46) , 334, 357, 359
Strong, F. M., 204, 217, 231, 257 (23),
259 (28, 30), 260 (30), 262 (30), 358,
372, 373 (63), 376, 378, 393, 398, 595
(211)
Stroud, H., 288
Struglia, L., 576, 577 (9)
Stubberfield, L., 591
Stubbs, J. J., 34
Stumpe, W. M., 58, 62 (61), 88
Stumpf, P. K., 353, 428, 430 (58), 432 (58),
435, 436 (58), 437, 442, 444 (4)
Sturgeon, P., 208
Sturgis, C. C., 211
Sturtevant, M., 59, 294, 473
Suarez, R. M., 209
Suarez, R. M., Jr., 209
SubbaRow, Y., 28 (12, 13, 14, 15), 39, 71,
92, 94, 95, 96, 98 (21, 24), 99 (24), 101,
103 (24), 104 (27), 105, 106 (27, 34),
635
108 (33, 35), 109 (27), 114 (23), 115
(23) 7,121,122) (1), 123 (2), 148 (2:3,
5, 6, 15, 16), 149 (57), 154 (57), 158,
160, 161, 379, 591, 592 (132)
Subrahmanyan, V., 350, 430 (51), 431
(51), 432 (51), 4386 (51), 442, 443 (2)
Sudborough, J. 8., 10
Sugiura, K., 61, 160
Suksta, A., 273, 274 (83)
Sullivan, C. D., 50, 54
Siillman, W., 468
Sulon, E., 383, 384 (59)
Simi Nishida, 326
Sumner, J. B., 345
Sundberg, R. D , 185, 194 (72), 208
Sunde, M. L., 588, 594 (145)
Sunderland, N., 594 (179)
Supplee, G. C., 250, 395, 477
Sure, B., 38, 265, 288 (9), 381, 383 (34),
474, 483
Surrey, A. R., 311
Sustendal, G. F., 285
Sutcliffe, F. K., 489
Sutherland, G. L., 118
Sutherland, J. E., 156
Sutro, C. J., 569
Sutton, T. S., 385, 505
Suvarnakich, J., 367
Swain, G., 72 (59)
Swaminathan, M., 246, 247, 257 (13), 260
(13), 261 (13), 262 (13), 292
Swank, R. L., 461, 465, 477
Swanson, P., 397
Swanson, W. J., 501, 507, 512, 543, 571
Swedin, B., 356
Sweeney, J. P., 249
Sweet, R. D., 569
Swendseid, M. E., 143 (12, 13), 152, 153,
159, 166, 191, 202, 210, 212 (195), 213
(195)
Swift, C. E., 494, 504
Swingle, K. F., 147
Swyer, G. I. M., 562
Sydenstricker, E., 387
Sydenstricker, V. P., 381, 388, 390 (116,
124), 391 (123, 124)
Sykes, G., 356
Sym, J. C. B., 567
Szabo, J. L., 231
Szezesniak, T. J., 515 (29), 519, 521 (29),
532 (29), 538 (29), 539 (29)
636
Szenasy, J., 545
Szent-Gyorgyi, A., 303, 337, 348
Szewezyk, T.8., 549
ay
Tabone, J., 20, 69
Tabor, C. W., 48, 48, 53
Tabor, H., 172, 266, 383
Takagi, S., 106, 123
Takata, R., 333
Tamayo, M. L., 56
Tamura, J. T., 67
Tang, F. F., 595
Tanos, B., 21
Tanner, F. W., Jr., 257 (14), 260 (36),
324, 333, 360, 361, 378
Tanner, W. F., 387
Tanquary, M. C., 262 (41)
Tarantini, A., 55
Tarleton, G. J., 286
Tarlov, I. M., 292
Tatting, B., 147, 187
Tatum, A. L., 58, 66
Tatum, E. L., 23, 26 (41, 42), 30 (20, 21),
Bil 32.33, 34, 05) (203) c2(O)h 239)
378, 379, 447
Tauber, H., 11, 21, 36, 437, 438
Tausig, T., 290
Taylor, A., 394
Taylor, E., 297, 474, 475
Taylor, E. C., Jr., 152
Taylor, E. 8., 235 (8)
Taylor, H., 284
Taylor, H. L., 479
Taylor, J., 394
Taylor, J. D., 489, 492, 493 (93), 496, 497
(5), 502 (7)
Taylor, L. W., 179
Taylor, M. W., 485
Telford, I. R., 515 (55, 72, 75), 524, 526,
528, 529, 530 (75), 541 (75)
Templeton, C. M., 388, 390 (116)
Ten Berge, B.8., 550
Tennent, D. M., 392, 475
Teply, L. J., 162, 257 (23)
Terp, P., 49, 53
Terroine, T., 266, 270
Terzian, L. A., 77
Teunissen, G. H. B., 513
Thain, E. M., 240, 583
Theiler, A., 476
AUTHOR INDEX
Theorell, H., 334, 337, 338, 340, 341 (20)
Thiersch, J. B., 158, 160, 188, 214
Thomas, B. H., 520
Thomas, J. M., 246, 257 (10, 11), 260 (11)
Thomas, R. M., 267
Thompson, C., 282
Thompson, C. R., 504
Thompson, E. O. P., 586
Thompson, R. C., 23, 24, 26 (40), 27, 30
(1, 2), 31 (1), 35 (48, 44, 49), 46, 47, 206
Thompson, R. H. §., 426, 427, 462, 581
Thompson, 8. Y., 371
Thomson, G. R., 568
Thornton, J. J., 410 (11)
Thorp, F., Jr., 386, 515 (89), 530
Thorpe, W. V., 46, 47 (20)
Thurlow, 8., 347
Mibinica, BaeAj Lh.) 521
Lice, J. Wr, 400
Tice, S. V., 235 (45), 238, 241
Tichenor, C. J., 79 (42, 43)
Tierney, N. A., 61, 79 (41, 44, 45)
Tillson, E. K., 14, 40, 49
Tinker, J. F., 114
Tisdall, F. F., 391, 400
Tishler, M., 233, 319, 320, 321, 328, 332,
393, 488, 491, 492 (89)
Tislowitz, R., 467
Titus, H. W., 386, 395 (22)
Tobias, E B., 158
Tobie, W. C., 72 (58), 79 (58)
Tobin, C. E., 515 (28), 519, 521, 528 (28),
530 (28), 539 (28), 563
Toca, R. L., 143 (4), 197, 209
Todd, A. R., 35 (87), 116, 322, 330, 405,
411, 412, 413, 414, 415, 416, 419, 420,
423, 424, 448, 486, 487 (80), 488 (53),
489, 492 (26), 594 (176, 177, 178)
Todd, C. W., 79 (19)
Toepfer, E. W., 171
Tolle, C. D., 266
Tolman, L., 155
Tomarelli, R., 504, 540
Tomlinson, H. M. R., 237
Tondenold, E. L., 595 (184)
Tonnis, B., 301
Torbet, N., 190
Torda, C., 56, 467
Toseani, V., 560
Tosic, J., 500, 507
AUTHOR INDEX
Totter, J. R., 91, 93, 138, 143 (8), 146, 147
(88), 148, 168, 166, 180, 181, 184 (54),
197, 204, 209
Tove, 8. B., 594 (168)
Trager, W., 379
Traube, W., 115
Traufler, D. H., 333
Travell, J., 565
Trenner, N. R., 109, 506, 508 (5)
Tria, E., 595
Trischmann, H., 334
Trufanov, A. V., 124, 155
Tschesche, R., 75, 108, 145, 146, 410
Tullidge, G. M., 283
Tullner, W. W., 196
Tulpule, P. G., 269
Tung, W. L., 10
Tunison, A. V., 395 (27)
Tuppy, H., 586
Tuttle, L. C., 39
Tverdy, G., 557
Tyler, F. H., 560
Tyner, G. §., 548 (181), 549
Tzong, C. C., 305, 306 (15)
U
Ugami, S., 11, 596 (229, 230, 231)
Ulick, 8., 493, 559
Ullmann, F., 3
Umbreit, W. W., 235 (37), 237, 238, 239,
240 (6), 241, 242 (25, 26), 278, 291
Underkofler, L. A., 22, 35, 333
Ungley, C. C., 206
Ungnade, H. E., 487, 488, 491, 493, 498
Unna, K., 38, 59, 247, 250, 265, 290, 291
(1), 292 (1), 293, 309, 391, 394, 467, 469
Upton, A. C., 51
Urner, J. A., 515 (32), 519
Utter, M. F., 430, 434, 436
Uyeo, 8., 106, 123
Uzbachian, J. B., 3
¥
Vacher, M., 244
Vail, D., 391
Valentik, K. A., 594 (149)
Vallance-Owen, J., 85
Van Assen, F. J. J., 550
van de Langerijt, J., 128
Vandelli, I., 287
637
Vandenbelt, J. M., 4, 5 (28), 99, 101 (28),
102, 103 (28), 164, 248, 260 (34)
van den Broek, W. A., 451
Van den Ende, G., 79
van der Kaay, F. C., 513
Vanderlaan, W. P., 57
Vanderlinde, R., 351
van der Linden, A. C., 451
Vander Werff, H., 149, 150, 151 (65)
Van Donk, E. C., 591, 592 (132)
van Dorp, D. A., 428, 429, 431
Van Duzen, R. E., 568
van Eekelen, M., 513
van Goor, H., 77
Van Haltern, H. L., 286
Van Lanen, J. M., 257 (14), 324, 333, 360,
361, 378
van Nouhuys, F., 175
van Overbeek, J., 595
Van Sant, J. H., 205
Van Wagtendonk, W. J., 591, 592 (131)
Van Wyck, H. B., 286, 293
Varangot, J., 545
Vargha, L., 337
Vawter, L. R., 515 (60), 524
Vedder, E. B., 409
Veldman, H., 428, 439, 440 (82)
Velluz, L., 443
Venkataraman, A., 44
Venkataraman, P. R., 44
Vennesland, B., 428, 430 (21), 434, 436
(21), 443
Verly, W. G., 142
Verwey, W. F., 40
Vestin, R., 438
Vetter, H., 326, 358
Victor, J., 385, 515 (101), 516, 533, 540
(9), 556, 557 (208)
Viehoever, A., 512
Viljoen, P. R., 476
Villela, G. G., 245
Vilter, C. F., 140, 204, 211, 217
Vilter, R. W., 140, 211, 217, 282, 284, 286
(144), 291, 388, 390 (121)
Vilter, S. P., 388
Vincent, J. M., 85
Vinet, A., 507
Virtanen, A. I., 587
Viscontini, M., 234, 240, 322, 488, 441
Vitucci, J. C., 595 (209)
Vivanco, F., 393
638
Vivino, A. E., 262 (41)
Vivino, J. J., 20, 23 (15), 25 (15), 67
Voegtlin, C., 63
Vogel, H., 301, 332
Vogelsang, A. B., 565, 566, 567, 569
Vogt-Mgller, P., 571
Vojnovich, C., 324, 333, 360, 378
Voleani, B., 131
von Bruecke, F., 468
von Kuler, H., 72 (64), 327, 328 (97), 343,
358, 359, 373 (3), 374, 438, 596
von Muralt, A., 448, 463, 464, 468
von Werder, F., 498
Voris, L., 269, 396, 398 (32)
Vorzimer, J., 284
Voss, E., 58, 66
WwW
Wachstein, M., 293, 540
Wacker, A., 100, 108 (26), 124, 596 (236)
Wacker, K., 368
Wacks, W., 508
Waddell, J., 485
Waddell, J. G., 240, 242 (25), 291
Wagner, J. R., 376
Wagner-Jauregg, T., 303, 304, 305, 307
(12a, b), 308, 309 (12a, b), 310 (12a,
b), 311 (12b), 312 (12a, b), 334, 346
(6), 372, 393
Wahlstrom, R. C., 588
Waisman, H. A., 38, 42, 59, 185, 251, 257
(7), 259 (7), 260 (7), 261 (7), 269, 274,
297, 386, 395, 472, 594 (171)
Wakim, K. G., 76
Walker, 330
Walker, D. J., 474
Walker, D. M., 371
Walker, H., 79 (25), 360
Walker, J., 123
Wall, M. E., 489, 498, 507
Wallenberg, B., 500
Waller, C. W., 28 (12, 13, 14, 15), 39, 71,
95, 96, 97, 98 (21, 24), 99 (24), 101, 103
(24), 104, 105, 106 (34), 108 (33, 35),
109, 118, 114 (23), 115 (23), 121, 122
(1), 123, 143: (2, 3) 5, 6; 15, 16); 149
(57), 154 (57), 155, 157 (74), 161
Waller, J. I., 568
Waller, L., 383
Walter, H., 235
Wandruff, B., 191
AUTHOR INDEX
Wang, Y. L., 515 (2), 516, 531 (2)
Warburg, O., 303, 309, 334, 335, 337 (1, 13,
23, 24), 338, 339, 341 (21), 342, 343
(23), 348, 351, 352, 369
Warden, H. F., 553, 554 (203)
Waring, G. B., 159, 161
Waring, W.S., 594 (177)
Warkany, J., 383
Warner, E. D., 501, 515 (24), 518, 528 (24)
Warner, R. G., 385
Waterman, R. E., 410, 412, 441, 465, 596
Watkins, C. H., 211
Watson, E. M., 487
Watson, J., 206
Watt, B. K., 455
Watt, D., 433
Watt, G. L., 286, 293
Watt, P. R., 498
Watts, B. M., 363, 503, 515 (64a, 64c)
Watts, P.S., 515 (64a, c), 525, 530 (64a, c)
Waud, R. A., 537, 567
Wawzonek, S., 488, 491
Wayne, E. J., 84
Weaver, J. W., 388, 390 (116)
Webb, F. R., 470
Webb, T. J., 247
Weber, F. C., 560
Weber, G., 307, 362, 365 (4)
Webley, D. M., 4380, 439
Webster, D. R., 272
Weese, H., 466, 467, 469
Wegner, A., 566
Wei, H., 595 (193)
Weidman, K. R., 584
Weigand, C. G., 292, 293 (24), 470
Weil-Malherbe, H., 438, 439, 440
Weiner, N., 316, 331 (56)
Weinstein, B. B., 285
Weinstein, H. R., 466, 467
Weir, D. R., 156, 161, 271
Weisblat, D- I., 107
Weisel, C. A., 331
Weisler, L., 489, 490 (56), 496, 508
Weiss, K. W., 137
Weiss, 8., 137
Weissbach, A., 198
Welch, A. D., 39, 71, 75, 76 (45), 93, 182,
140, 147, 148 (46), 156, 161, 172, 188,
194, 198, 208, 209, 210, 211, 212, 218
(205), 214, 271
Wellman, J. W., 319, 320, 321
AUTHOR INDEX
Wells, J. J., 286
Welsh, A., 569
Wendler, N. L., 819, 488, 491
Wendt, G., 72 (60), 222, 223, 226, 227, 228,
229, 230 (21)
Wenis, E., 327, 358, 588
Went, F. W., 264
Werkman, C. H., 430, 483, 435, 486
Werle, E., 234, 235 (6), 241 (35, 39), 278
Werner, A. E. A., 21
Wertheim, J. M., 466, 467
Wertz, A. L., 480
Westenbrink, H. G. K., 426, 427, 428,
429, 431, 439, 440, 451, 455
Westerfeld, W. W., 44, 351, 428, 480 (21),
436 (21), 443, 473, 596 (255)
Westphal, K., 226, 227, 228, 229, 230
(21), 405, 411, 412, 414, 416, 420 (28),
422, 423, 424
Westphal, O., 228, 229, 230 (21)
Weswig, P. H., 460
Weygand, F., 100, 106, 108 (26), 124, 303,
307, 311, 315, 316, 318 (58), 322, 323,
327, 328, 334, 359, 368
Weyland, J., 438
Whatley, F. R., 304, 369
Wheeler, E., 397
Wheeler, G. A., 387, 390
Wheeler, H. L., 7
Wheeler, N. C., 360, 392, 401
Wheeler, W. E., 207, 208 (179)
Whitby, L. G., 323
White, A. G., 48
White, A. G. C., 447, 465
White, J. M., 267
Whitehair, C. K., 189, 190, 276, 298, 371,
385, 395, 594 (168, 173)
Whitehill, A. R., 595 (186)
Whiting, F., 503, 510, 513
Whitmore, W. F., 7, 9 (49)
Whitney, D. M., 79 (14)
Wiardi P. W., 222
Wickerham, J., 324
Wicks, L. F., 30 (20, 21), 31
Widnmann, E., 2, 3, 6
Wiebelhaus, V. D., 14
Wiedling, S., 78
Wieland, H., 349
Wieland, T., 467, 583
Wiesbader, H., 567
Wiese, A. C., 273, 385
639
Wiesel, L. L., 58, 62, 83
Wilbrand, J., 2,3
Wilby, F., 371
Wilcox, R. A., 594 (159)
Wilder, R. M., 389, 400, 401, 464, 478
Wildman, R. B., 279
Wilgus, H. 8., 374, 395 (20), 596 (224)
Wilhoyte, K. M., 14
Wilkins, W., 400
Wilkinson, J. F., 205
Wilkinson, P. B., 390
Willebrands, A. F., 428
Williams, B. W., 595 (196)
Williams, D. E., 297
Williams, E., 397
Williams, J. H., 595 (186, 209), 596 (256)
Williams, J. N., 349
Williams, M. A., 592
Williams, R. D., 389, 401, 464, 478
Williams, R. H., 84
Williams, R. J., 13, 34, 69, 79, 90, 91 (6),
92, 143 (25), 149 (25), 217, 231, 257 (6,
17), 259 (6, 27), 260 (6), 261 (6), 262
(6), 270, 276, 277 (109), 298, 301, 376,
394, 403, 446, 451, 476
Williams, R. R., 38, 307, 308 (28), 376,
405, 406, 409, 410, 411, 412, 413, 415,
423 (25), 424, 425, 441, 465, 469, 596
Williams, V. R., 260 (33), 595
Williams, W. L., 197, 243, 254, 257 (8),
259 (8), 260 (8), 262 (8), 280
Williams, W. W., 562
Williamson, A., 360
Williamson, J., 65
Williamson, M. B., 504
Willis, R.S., 285
Willman, J. P., 515 (58, 59), 524, 530 (59)
Wills, L., 89, 90, 180, 182, 200, 204, 205
Willstaedt, H., 509
Willstatter, R., 419, 420
Wilson, E. D., 375 (19)
Wilson, H. E., 181, 182, 183 (62), 184, 195
(64)
Wilson, H. E. C., 250
Wilson, J. E., 142
Wilson, K., 163
Wilson, M. F., 148
Wilson, W., 115
Windaus, A., 410
Wing, J. F., 6
Winkelblech, K., 4
640
Winkler, K. C., 68, 69 (29), 70 (29), 127,
128 (10), 129 (10), 135 (10)
Winn, W. W., 285
Winsten, W. A., 121
Winternitz, M. C., 267
Winters, R. W., 376
Wintrobe, M. M., 147, 186, 187, 188 (76),
266, 273, 274 (83), 283, 296, 385
Wirth, T., 419
Wisansky, W. A., 56, 58, 83, 317
Wise, G. H., 510, 513
Wislicenus, W., 414
Wisnicky, W., 459
Wittle, E. L., 40, 99, 152, 153 (68)
Wittman, P., 389, 393, 479
Witty, R., 237
Wnuck, A. L., 466, 470 (6), 471 (6)
Wohl, Z., 285
Wohlfahrt, G., 533
Wokes, F., 363
Wolbach, 8S. B., 380, 381 (28), 382 (28),
384 (28), 390 (38)
Wolf, A., 515 (77, 107), 529, 533 (77), 535,
556
Wolf, D. E., 28 (17), 103, 109, 143 (20)
Wolf, H. J., 145, 146
Wolf, L. M., 162
Wolff, H. G., 56, 467
Wolfram, D. J., 79 (15)
Wolfrom, M. L., 590
Wollman, 8. H., 494
Woltman, H. W., 283
Womack, M., 587
Wood, H. G., 4338, 434
Wood, P. B., 46, 47 (20)
Wood, S., 278, 279 (131)
Wood, T. R., 277
Wood, W. A., 238, 239, 242, 278, 515 (64a,
64b, 64c), 525, 5380 (64a, b, c)
Wood, W. B., Jr., 25 (47), 35 (31)
Woodruff, C. W., 188, 191, 192, 193, 194,
207
Woodruff, H. B., 590
Woods, A. M., 204, 217, 376
Woods, D. D., 37, 67, 73 (5), 1380, 134, 135,
136 (37)
Woods, I. F., 42
Woodside, M. R., 505
Woodward, C. R., Jr., 254, 257 (2), 260 (2),
261 (2), 262 (2), 279
AUTHOR INDEX
Woolley, D. W., 15, 180, 149, 153 (56),
154 (56), 189, 322, 330, 359, 393, 447,
459, 465, 585, 586 (70), 596
Woolpert, O. C., 181, 182, 183 (62)
Woolsey, L. D., 571
Worden, A. N., 266, 381
Work, T. 8., 486, 487 (30), 488 (53), 489,
492 (26)
Wright, C. I., 393
Wright, C.S., 283
Wright, E. B., 257 (3)
Wright, I. S., 567 ,
Wright, L. D., 20, 23 (15), 25 (15), 39, 67,
71, 76 (45), 93, 147, 150, 172, 209, 577,
585, 58., 594 (149)
Wright, M. D., 492
Wright, M. E., 7, 9 (54)
Wright, S. W., 510, 545, 547 (168), 548,
549, 551 (168), 552 (168), 553 (195),
554 (195), 558 (195)
Wright, W. B., Jr., 155, 156 (74)
Wulzen, R., 591, 592 (191), 593
Wurtz, H., 328
Wyeno, J., 72 (51)
Wyss, A., 463
Wyss, F., 463
Wyss, O., 26 (52), 32, 35 (1), 72 (55)
Y
Yagi, K., 362, 367, 368
Yamagulin, S8., 35 (82)
Yamaki, T., 35 (82)
Yamamoto, K., 15
Yap, K.S., 490
Yegian, D., 79
Yeomans, A., 51, 60, 61, 79 (41, 46)
Youmans, A. 8., 79
Youmans, G. P., 79
Youmans, J. B., 46, 47 (24), 53, 360, 391,
392, 401
Young, J. M., 558
Young, N. F., 480, 437 (35)
Young, R. J., 594
Younger, F., 268
Z
Zacharias, L., 505, 515 (109, 110), 536
Zahalan, F., 569
Zalokar, M., 33, 76
AUTHOR INDEX
Zamecnik, P. C., 400
Zarafonetis, C. J. D., 47, 51, 53, 60, 61,
79 (7, 8, 30, 41), 85
Zarudnaya, K., 428, 430 (20, 58), 432 (58),
436 (20, 58), 442, 444 (4)
Zarrow, I. G., 197
Zarrow, M. X., 195, 197
Zatchuni, J., 558
Zeligs, M. A., 283
Zeller, E. A., 56, 353, 356, 389
641
Ziegler, J. A., 308, 376
Zierler, K. L., 567
Zilli, K., 48
Zilva, S. 8., 191
Zima, O., 441
Zimmerman, A.; 68
Zimmerman, H. M., 272, 381, 384 (35)
Zinder, N., 17
Zook, E. G., 171
Zuelzer, W. W., 193, 207
Subject Index
A
Acetaldehyde,
condensation of, 444
oxidation, 581
formation of, 427
in thiamine biogenesis, 447
Acetamidine, in thiamine synthesis, 413
Acetarsone, p-aminobenzoic acid and, 63
Acetate, 17, 44, 464
formation from pyruvate,
579-580
in riboflavin synthesis, 323, 361
Acetate replacement factor, see Lipoic
acid
Acetoacetic acid, 444
in thiamine deficiency, 434
Acetobacter suboxydans, in p-amino-
benzoic acid estimation, 22-23
a-Aceto-y-butyrolactone, in thiamine
synthesis, 418-419
Acetoin, 44
formation of, 444, 581
a-Acetolactic acid, formation of, 433
Acetopropyl alcohol, in thiamine bio-
genesis, 447
p-Acetotoluide, oxidation of, 3
Acetylcholine, 56
thiamine and, 462-464, 467, 468, 471
Acetyldiiodotyrosine, 57
Acetylmethy] acetate, 44
Acetyl phosphate,
bond energy of, 485
in pyruvate oxidation, 435
Acetylthiamine, 467
Acetylthyroxine, 57
Achromotrichia,
in vitamin deficiency, 37-40, 41
p-aminobenzoic acid and, 59-60, 83-84
Acriflavin, 65
Acrodynia rat test, in pyridoxine esti-
mation, 250
Actomyosin, in muscular dystrophy, 526
Acute hemorrhagic necrosis, tocopherol
and, 540
434-436,
643
Acyloin, formation, coenzymes required,
580
Adenine, 22, 25, 40, 151
effect in pteroylglutamic
ficiency, 139-140, 187
in growth of microorganisms, 124-125
in pteroylglutamic acid estimation, 162
in sulfonamide reversal, 68-69
synthesis, pteroylglutamic acid and,
139-139
Adenosine,
effect in pteroylglutamic acid de-
ficiency, 139
in sulfonamide reversal, 69
Adenosine diphosphate, 439
Adenosine polyphosphate, in pyruvate
oxidation, 435
Adenosine triphosphate, 239, 240, 442
in p-aminohippuric acid formation, 14
in detoxification, 44-45
in thiamine phosphorylation, 439
Adenylie acid, 44, 439
in pyruvate oxidation, 434-435
in sulfonamide reversal, 77
in tocopherol deficiency, 533
Adermin, see Vitamin Bs
Adipose tissue, in tocopherol deficiency,
538-539
ADP, see Adenosine diphosphate
Adrenal gland, vitamin Bg, and, 266, 272,
292, 295
Age, effect on thiamine requirement, 474
Agmatine, oxidation of, 356
Agranulocytosis, vitamin B, and, 284
Alanine, 34, 236, 286
as inhibitor, 590-591
racemization of, 238
requirement for, 279
B-Alanine, 234
p-Alanine, 586
in utilization of L-alanine, 587
vitamin Bs, and, 277-278
Alcohol, effect on thiamine requirement,
473
Aldehydes, oxidation of, 349
acid de-
644. SUBJECT INDEX
Aldehyde oxidase,
mechanism of action, 351
preparation, 350
properties, 350-351
Alloxan, 44, 444, 564
effect on growth, 323
in riboflavin synthesis, 314-315
preparation of, 320
tocopherol and, 537, 546
Alloxantin, tocopherol and, 546
A-Methopterin, see also 4-Amino-N?°-
methylpteroylglutamic acid
effect on purine synthesis, 133
p-Aminoacetophenone, in thiamine esti-
mation, 449-450
Amino acid decarboxylases, coenzyme
of, 234-236
p-Amino acid oxidase, 393
coenzyme of, 343
in flavin nucleotide estimation, 369
in vitamin Bg deficiency, 269
mechanism of action, 352
preparation, 352
riboflavin analogs and, 328-329
specificity of, 351-352
t-Amino acid oxidase,
coenzyme of, 334
preparation, 353
properties, 353-354
specificity and mechanism of action,
353-354
Amino acids, 33
in old yellow enzyme, 340-341
pteroyl derivatives of, 154-155
synthesis, pteroylglutamic acid and,
136-137
p-Aminobenzoic acid,
absorption of, 52
acetylation in disease, 48
as sun-screening agent, 6, 60
biochemical systems of, 13-16
biogenesis of, 16-19
by microorganisms, 34-36
bound forms of, 27-28
chemistry, 2-12
color reactions of, 11-12
degradation of, 6
derivatives of, 10
detoxification of, 44, 52-53
diazotization of, 7, 11, 19-21
differentiation from isomers, 11-12
effect of ionization on requirement, 32
effects of deficiency,
in bacteria, 32-36
in chicks, 42
in fish, 43
in hamsters, 42
in mice, 41-42,
in rats, 37-41 |
in swine, 42-43
effect on,
aminoimidazole utilization, 132
pteroylglutamic acid synthesis, 133-
134
trypanosomes, 65
tumors, 41-42
estimation of,
chemical methods, 19-21
microbiological methods, 21-27
excretion, 52-53
in human, 46-49
in lower animals, 43-46
halogenation of, 6-7
in amino acid synthesis, 136
industrial preparation, 12-13
inositol and, 38
in pteroylglutamic acid estimation,
161-162
in purine synthesis, 129
in rhizopterin, 109
in sulfanilamide requirement, 76-77
in sulfonamide resistance, 66-68
ionization of, 4
nomenclature, 2
occurrence in foods, 27-31
oxidation of, 25-26
paper chromatography of, 21
pentosides and hexosides of, 10
peptides of, 9, 13-14
pharmacology of, 52-62
properties of, 3-6
salt and esters of, 7-10, 65
sulfonamide reversal and, 66-79, 127-
128, 172
therapeutic activity of, 79-85
therapeutic uses of, 59-62
toxicity, 53-55
in humans, 51-52
in lower animals, 49-51
tyrosinase and, 15
ultraviolet absorption of, 4-6
SUBJECT INDEX
urinary metabolites of, 46, 47-48
vitamin By. and, 18-19
p-Aminobenzoylglutamic acid, 15, 16,
33, 34, 113
in folie acid, 98-99
in folic acid synthesis, 104, 107-108, 122
in sulfonamide reversal, 73-74
p-Aminobenzoyl-p-glutamie acid, 155
p-Aminobenzoylglycine, effect on
growth, 25
4-Amino-9, 10-dimethylpteroylglutamic
acid, as inhibitor, 157, 160
2-Amino-4,5-dimethyl-1-ribitylamino-
benzene, effect on growth, 323
Aminoethanol, in choline synthesis, 141
p-Aminohippuric acid, 21, 46, 53
biosynthesis of, 14
2-Amino-4-hydroxy-5-formy1-10-methyl-
5, 6, 7, 8-tetrahydropteroylglu-
tamic acid, reaction with nitrous
eyentol, ANE
2-Amino-4-hydroxy-6-methylpteridines,
in pteroylglutamic acid synthesis,
105, 107-108, 122-123
2-Amino-4-hydroxy pteridine -6-alde-
hyde, 349
2-Amino-4-hydroxypteridine-6-carbox-
yliec acid,
formation from pteroylglutamic acid,
96
in pteroylglutamic acid estimation, 162
2-Amino-4-hydroxypteridines, as inhibi-
tors, 152-153
2-Amino-4-hydroxy-6-pteridyl-methyl-
(4’-methyl-5’-hydroxyethylthiaz-
olium)bromide, 15
5(4)-Amino-4(5)-imidazolecarboxamide,
20
accumulation of, 69-70
in purine synthesis, 128-132
synthesis, formate and, 139
Aminomethylene malononitrile, in thi-
amine synthesis, 417
4-Amino-9-methylpteroylglutamic acid,
as inhibitor, 160
4-Amino-N!°-methylpteroylglutamic
acid, see also A-methopterin
as inhibitor, 160
a-Amino-§ - (4-methylthiazole-5) - propi-
onic acid, in thiamine biogenesis,
447
645
p-Aminophenylactie acid,
as growth factor, 32
effect on thyroid gland, 41
Aminopterin, see also 4-Aminopteroyl-
glutamic acid,
citrovorum factor and, 110
effect on purine synthesis, 130, 133
methionine synthesis and, 137
resistance to, 159
4-Aminopteroylalanine, 157
4-Aminopteroylaspartic acid, as in-
hibitor, 160
4-Aminopteroylglutamic acid, see alsa
Aminopterin
as inhibitor, 160
citrovorum factor and, 197-198
derivatives, as inhibitors, 157-159
effect in humans, 213-215
estradiol and, 196
reversal of, 157-158
2-Aminopurine, as inhibitor, 151
p-Aminosalicylic acid, 21
p-aminobenzoic acid and, 78-79
Aminotocopherol, activity of, 491
5-Aminouracil, as inhibitor, 150
Ammonium acetate, pyridoxine and, 291
Amyleaine, 8
Anaphylactic shock, thiamine and, 470-
471
Anemia,
achrestic, 205
p-aminobenzoic acid and, 55
in vitamin B,-deficiency, 281
megaloblastic,
ascorbie acid and, 193-195
idiopathic, 205
of infancy, 207-208
of pregnancy, 205-207
pteroylglutamie acid and, 193-195
vitamin By and, 193-195, 209-213
nutritional, macrocytic, 203-205
Anesthesine, 8
Anesthetics, derived from p-aminoben-
zoic acid, 7-10
Aneurin, see Thiamine
Anhydroleucovorins, formation of, 119-
120
N-(2-Anilinoethyl)-formanilide,
blance to leucovorin, 118-119
resem-
646
Antabuse,
effect on oxidases, 351
vitamin A and, 505
Anthranilie acid, 20
formation of, 238
Antiacrodynia rat factor, see Vitamin Be
Antiberiberi vitamin, see Thiamine
Antibiotics, 593
Anticanitic factor, see p-Aminobenzoic
acid
Antidermatitis rat factor, see Vitamin Beg
Anti-gray hair factor, see p-Aminoben-
zoic acid
Antimonials, p-aminobenzoic acid and,
58
Antineuritic vitamin, see Thiamine
Antisterility-vitamin, see Tocopherols
Antithiamines, occurrence of, 460
Arabinose, in riboflavin synthesis, 318
Araboflavins,
effects on growth, 326-327, 328, 358-359
phosphorylation of, 331
Arachidonic acid, vitamin Bs and, 266,
269
Arginine,
in sulfonamide reversal, 69
pteroylglutamic acid and, 137
Arsanilie acid, p-aminobenzoic acid and
63
Arsenicals, p-aminobenzoie acid and, 58,
62-66
Arsenite, as enzyme inhibitor, 14
y-(p-Arsenosopheny])-butyric acid, 65
Arsphenamine, 58
Arterenol, 236
Ascorbie acid, 15
in anemia, 208
in citrovorum factor formation, 198
in detoxification, 63
in monkey anemia, 185
in pteroylglutamic acid assay, 201
in tocopherol deficiency, 536
relation to pteroylglutamic acid, 191-
195
riboflavin and, 397
tocopherol and, 494, 495, 503
Ashbya gossypii, in riboflavin formation,
324, 333
Aspartic acid, 9, 236
in sulfonamide reversal, 69
SUBJECT INDEX
lycomarasmin and, 585
requirement for, 279
Aspartic acid decarboxylase, 234
Atabrin, 129, 331
flavin adenine dinucleotide and, 393
riboflavin and, 330
ATP, see Adenosine triphosphate
Atropine, 466
Aureomycin, 80
riboflavin and, 397
vitamin Bs and, 294
Azide, as enzyme inhibitor, 14
B
Barbituric acid, in riboflavin synthesis,
320
Benemid, 40
effect on p-aminohippuric acid syn-
thesis, 14
Benzenearsonic acid,
acid and, 63
Benzil, 444
Benzimidazole-2-methyl-p -aminobenzo-
ylglutamie acid, activity of, 153
Benzocaine, 8
Benzoic acid, detoxification by, 64
Benzylsuccinate, 64
Beriberi, 404
Betaine, choline and, 141
B, Factor, see p-Aminobenzoic acid
Bile, tocopherol and, 501
Biliary obstruction, tocopherol and, 553
Biotin, 24, 35
formate and, 137
in pteroylglutamic acid deficiency, 186
in sulfonamide reversal, 173
pantothenic acid and, 39
sulfanilamide and, 71, 133
Bismuthyl compounds, detoxification
of, 58, 66
Blacktongue, riboflavin and, 384
Blood,
p-aminobenzoic acid and, 55
in pteroylglutamic acid deficiency,
171-173, 180-182
in riboflavin deficiency, 382-383, 384
in vitamin Bg deficiency, 266, 272, 273,
274, 275
riboflavin in, 393
Blood pressure, thiamine and, 466-467
Borate, in pyridoxine estimation, 248
p-aminobenzoie
SUBJECT INDEX
Borax, detoxification of, 66
British Anti-Lewisite, 582
5-Bromouracil,
as inhibitor, 150
in growth of microorganisms, 126
Butesin, 8
Butylene glycol, 44
Butyn, 8
Butyribacterium retigert, lipoic acid and,
576
C
Cadaverine, oxidation of, 356
Calcium, dietary, in stiffness syndrome,
592
Calf,
tocopherol deficiency in, 524, 525, 530
vitamin B, deficiency in, 276
Candida spp., in riboflavin formation, 324
Carbarsone, p-aminobenzoic acid and,
62, 63
Carbohydrate, effect on
requirement, 473, 478
Carbohydrate index, in thiamine de-
ficiency, 465-466
Carbon dioxide,
assimilation by tissues, 434
formation from pyruvate, 484-436
in riboflavin synthesis, 323, 361
in serine synthesis, 136
Carbonic anhydrase, sulfonamides and,
77
Carbon tetrachloride, tocopherol and, 564
Carboxylase, 434
activators of, 436-437
distinction from pyruvic dehydrogen-
ase, 432-433
inhibitors of, 436-437
in thiamine deficiency, 427
sedimentation of, 436
substrate specificity of, 443-445
Carboxypeptidase, 164
Carboxysulfathiazole, 177
Carnitine,
assay of, 584
discovery of vitamin function, 583-584
isolation of, 584
Caronamide, 40
Carotene,
in antioxygenic action, 503-504
tocopherol and, 504-506, 512
thiamine
647
Catalase, 351, 356
aldehyde oxidase and, 350
Catatorulin effect, 426
Celiac disease,
ceroid and, 556
tocopherol and, 553, 554
Cephalin, in antioxygenic action, 503-504
Cereals, thiamine in, 455-456
Ceric sulfate, in tocopherol assay, 506
Ceroid,
in human tissues, 556-558
nature of, 516
tocopherol and, 539-540
CF, see Citrovorum factor
Chastek paralysis, thiamine and, 458-459
. Chick,
p-aminobenzoic acid deficiency in, 42
pteroylglutamie acid deficiency in,
175-180
pteroylglutamic acid requirement of,
176-178
pyridoxine requirement of, 297
riboflavin deficiency in, 387
tocopherol deficiency in, 519, 532, 534—
536, 539
vitamin B, deficiency in, 275
Chick growth test, in pyridoxine estima-
tion, 252-253
Chloramphenicol, 80
y-Chloro-y-acetopropylacetate, in thi-
amine synthesis, 423
Chloroacetopropy! alcohol, in thiamine
biogenesis, 447
Chloroflavin, 312
Cholesterol, 508, 539
Choline, 45, 198, 584
effect on growth, 141
synthesis from serine and glycine,
137-138
synthesis, vitamins and, 140-142
tocopherol and, 539-540
Cholinesterase, 463, 464, 468
p-aminobenzoic acid and, 56
Choline pteroylglutamic
acid deficiency, 142
Chroman-5,6-quinone,
formation from tocopherol, 493-494
in tocopherol assay, 506
Chromatography, in tocopherol assay,
508, 509
oxidase, in
648
Chromotrichia factor, see p-Aminoben-
zoic acid
Chronaxy, in thiamine deficiency, 466
Circulatory system, see also Vascular
system, p-aminobenzoic acid and,
55
Citric acid, 433
tocopherol and, 503
Citrovorum factor, see also Leucovorin
aminopterin and, 158-159
chemistry of, 110-121
conjugates of, 202
function of, 198
in animal nutrition, 197-198
in leucocytes, 159
in monkey anemia, 185-186
in reversal of growth inhibition, 202
in reversal of pteroylglutamic acid
analogs, 214-215
in scorbutic anemia, 194-195
in serine synthesis, 136
in sulfonamide reversal, 135
location of formyl group, 114-120
location of hydrogen, 113-114
microbiological activity of, 201-202
reduction stage of, 111-114
relation to leucovorin, 120-121
synthesis of, 111
tyrosine and, 192
Clostridium acetobutylicum, in p-amino-
benzoic acid estimation, 24—25
Cobalamin, see Vitamin Bye
Cocaine, 9
Cocarboxylase, 427, see also Thiamine
pyrophosphate
Codecarboxylase, 234
Coenzyme A, 578
achromotrichia and, 39-40
in pyruvic acid oxidation, 440
regeneration from acetyl form, 580-581
Coenzyme I, see Diphosphopyridine
nucleotide
Coenzyme II, see Triphosphopyridine
nucleotide
Coenzyme III, function of, 589
Colorimetry, in riboflavin estimation,
368-369
Complementary factor, see Vitamin Beg
Conalbumin, 308
Cortisone,
p-aminobenzoic acid and, 58, 62, 83
SUBJECT INDEX
myositis and, 560
pteroylglutamic acid and, 197
tyrosine and, 192
Corynebacterium, lipoic acid and, 576
Cotransaminase, 234
“Crazy chick’”’ disease, 535
Creatinine, 268, 272
Creatinuria, 511, 559, 560
o-Cresyl phosphate,
564, 573
Crotonobetaine, 584
Curare, resemblance to thiamine, 467
Cyanide, 437
as enzyme inhibitor, 14, 15
Cyanine dye test, for pyridoxine, 244-245
Cyanogen bromide, in pyridoxine esti-
mation, 249
Cycloform, 8
Cyclohexanedione-1,2, 444
Cyclohexanehexone, 444
Cyclopropane, 55
Cystathionine,
cleavage, pyridoxine and, 239, 269
synthesis of, 237
Cysteie acid, formation of, 589
Cysteine, 237
in enzyme activation, 437
peptides, 587
Cysteine deaminase, 239
Cysteine desulfhydrase, in vitamin Beg
deficiency, 269
Cysteine-sulfinic acid, oxidation of, 589
Cystine,
hemorrhagic necrosis and, 540
requirement for, 279
vitamin Bs and, 294-295
Cytochrome,
riboflavin and, 357
pyruvic dehydrogenase and, 432
Cytochrome ¢ reductase, 393
coenzyme of, 334, 344-345, 346-347
mechanism of action, 346
preparation and properties, 345-346,
347
Cytosine, 125
tocopherol and,
D
Dehydroisoandrosterone acetate, pter-
oylglutamie acid and, 197
5-Dehydroshikimie acid, in aromatic
biosynthesis, 18
SUBJECT INDEX
Dermatomyositis, tocopherol and, 559—
560
Desoxypyridoxine, 265, 270, 275, 284-285
as antagonist, 242, 282
toxicity of, 291
Desoxyribosides, 130
requirement for, 181-132
Deuteroflavin, 310
Diabetes, tocopherol and, 569
Diacetone riboflavin, in growth stimula-
tion, 325
Diacetyl mutase, 442
inhibitors of, 436
specificity of, 444
Dialuric acid, tocopherol and, 537, 546
Diamine oxidase, 356
2,4-Diamino-5-p-chlorophenoxypyrimi-
dine, as inhibitor, 150-151
2,4-Diamino-6-hydroxy-5-formamido-
pyrimidine, resemblance to leuco-
vorin, 115
2,6-Diamino-7-methylpurine, 152
2,4-Diaminopteridine, as inhibitor, 152-
153
2,6-Diaminopurine, as inhibitor, 151-152,
160, 161
2,4-Diaminopyrimidines, as inhibitors,
150-151
o-Dianisidine, in tocopherol assay, 508
Diaphorase, coenzyme of, 343-344
Diarrhea, tocopherol and, 553, 554
Diazotized p-aminoacetophenone, in
pyridoxine estimation, 246-247
Diazotized, p-nitroaniline, in pyridoxine
estimation, 247
Diazotized sulfanilie acid, in pyridoxine
estimation, 246
Dibenamine, 466
a,8-Dibromobutyraldehyde, 155
2,3-Dibromopropionaldehyde, in
acid synthesis, 104, 106, 122
Dichloroflavin,
effects on growth, 328-329
phosphorylation of, 331
2,6-Dichloroindophenol, in
assay, 506
2,6-Dichloroquinonechlorimide,
folic
tocopherol
in pyridoxine estimation, 247-249
reaction with pyridoxine, 224, 227
Dichloro-p-sorboflavin, 330
649
6,7-Diethyl-9-(p-1'-ribityl)isoalloxazine,
330
effects on growth, 325-326
Dihydropteroylglutamic acid, reduction
potential of, 111
Dihydroriboflavin, 312
5, 10-Diformyl-5,6,7 ,8-tetrahydropter-
oylglutamie acid, formation of, 120
Dihydrouracil, 114
2,3-Dihydroxyacrylaldehyde, see
ductone
3,5-Diiodo p-aminobenzoic acid, in thy-
rotoxicosis, 84
Diiodotyrosine, 41, 57
2,3-Dimereaptopropanol, 582
p-Dimethylaminoazobenzene, riboflavin
and, 394
p-Dimethylaminobenzaldehyde, reaction
with p-aminobenzoic acid, 11, 21
N,N-Dimethylaminobenzene, carboxyl-
ase and, 437
p-Dimethylaminobenzoie acid,
violet absorption of, 5
1,2-Dimethyl-4,5-diaminobenzene, in
riboflavin biosynthesis, 322
Dimethylhydroquinones, in tocopherol
synthesis, 488-489, 496
N,N-Dimethyl-1-naphthylamine, as
coupling agent, 20
9,10-Dimethylpteroylglutamic acid, as
inhibitior, 156-157
o-Dinitrobenzene, in
thesis, 316
Diphosphopyridine nucleotide, 342, 442,
583
in p-aminohippuric acid synthesis, 14
in pyruvate oxidation, 579-580
oxidation of, 344
regeneration of oxidized form, 580-581
Diphosphothiamine, see Thiamine pyro-
phosphate
Dipropionyl, 444
a,a-Dipyridyl, in tocopherol assay, 506,
508
Disease,
p-aminobenzoie acid and, 85
treatment with vitamin By , 282-290
Dithiocarbaminate, in thiamine syn-
thesis, 421
Dithioformie acid, in thiamine synthesis,
420-421
Re-
ultra-
riboflavin syn-
650
Dithiodctanoie acid, see Lipoic acid
Dog
pteroylglutamic acid deficiency in,
189-190
riboflavin deficiency in, 384
tocopherol deficiency in, 518, 528, 532
vitamin Bg deficiency in, 272
DPN, see Diphosphopyridine nucleotide
Duck,
pteroylglutamic acid requirement of,
178
pyridoxine requirement of, 298
riboflavin deficiency in, 387
vitamin B, deficiency in, 275-276
Duroquinone, formation from a-toco-
pherol, 487
Dysentery, in pteroylglutamic
deficiency, 183-184
acid
E
Electroshock, pyridoxine and, 291
Encephalograms, in thiamine deficiency,
465
Endocrine glands, in
ficiency, 521
Epinephrine, 55, 56, 236
thiamine and, 467
Eremothecium ashbyii, in riboflavin for-
mation, 324, 333
Ergostenol, as antistiffness factor, 592
Erythredema, tocopherol and, 550
Erythroblastosis, tocopherol and, 547
550
Erythrocyte maturing factor, 210-211
Eserine, 468
Essential fatty acids, tocopherol and,
505, 563-564
Estradiol, pteroylglutamic
195-196
Jstrogen, tocopherol and, 561, 567
Ethanol, oxidation, 581
Ethoxypropionic acid, in thiamine syn-
thesis, 4138-414
Ethyl adipyl chloride, in lipoie acid
synthesis, 578
N-Ethyl-1l-naphthylamine, as coupling
agent, 20
7-Ethyl-9-(p’-1’-ribityl)isoalloxazine ef-
fect on growth, 326
Ethylthiamine, activity of, 408
tocopherol de-
acid and
SUBJECT INDEX
Exudative diathesis, in tocopherol de-
ficiency, 534-535
Eye, in riboflavin deficiency, 381, 384,
390-391
Factor Bx , 596
Factor GPF-1, 596
Factor GPF-2, 596
Factor GPF-3, 596
Factor HL, 1,2,3,4, 596
Factor P, see p-Aminobenzoic acid
Factor R, 596
Factor 8, 596
Factor T, 596
Factor, U, 175, 596, see also Pteroyl-
glutamic acid
Factor V, 596
Factor W, 596
Factor X, 596, see also Tocopherol
Factor Y, 596, see also Vitamin Bs
Factor I, see Vitamin Be
Factor 3, liver necrosis and, 540
FAD, see Flavin adenine dinucleotide
Fat,
effect on riboflavin requirement, 378-
379, 396
effect on thiamine requirement, 473,
478
effect on tocopherol assay, 507
in tocopherol deficiency, 514, 536, 537
metabolism, in vitamin Bg deficiency,
269
tocopherol and, 570, 573
Fatty acids,
in ceroid production, 517
unsaturated, 539
Fermentation L. casei factor, see Pteroyl-
triglutamate
Ferric chloride,
in pyridoxine estimation, 245
in tocopherol assay, 506, 508
Ferricyanide,
as oxygen acceptor, 580
in thiamine estimation, 448
in tocopherol assay, 506-507
Fibrositis, tocopherol and, 568-569
Fish,
p-aminobenzoic acid deficiency in, 43
thiamine in, 457-458
SUBJECT INDEX
Flavin adenine dinucleotide, 393, 442
as coenzyme, 342, 348, 348, 350, 351,
354, 355, 356, 357
analogs of, 331
estimation of, 8367-368
fluorescence of, 307, 362
in growth stimulation, 325, 330, 358
isolation of, 338-339
pyruvie dehydrogenase and, 432
structure of, 334-335
Flavin coenzymes, chemical properties
of, 336
Flavin mononucleotide, 345, 353, 357
analogs of, 331
as coenzyme, 334
estimation of, 367-368
in growth stimulation, 325, 358
isolation of, 337
synthesis of, 321-322
Flavokinase,
action of, 335-336
effect on riboflavin analogs, 331
Flavoproteins, 581, 583
prosthetic groups of, 334
Fluoride, 436, 439
as enzyme inhibitor, 14
Fluoroacetate, 436
Fluorometry, in riboflavin estimation,
361-368
FMN, see Flavin mononucleotide
Folacin, see Pteroylglutamic acid
Folic acid, see Pteroylglutamic acid
Folin-Denis reagent, in pyridine estima-
tion, 245-246
Folinic acid, 28, 40, 75, 599, see also Citro-
vorum factor, Leucovorin
Formaldehyde, 351
Formate,
incorporation in resistant leukemia,
161
in purine synthesis, 138-139
in riboflavin synthesis, 323, 361
in serine synthesis, 136, 137
oxidation of, 444
rhizopterin and, 109
4-Formylamino-5-imidazolecarbox-
amide, in purine synthesis, 131
N!°-Formylpteroic acid, 103, 199
activity of, 144
metabolism of, 200-201
651
N'°-Formylpteroylglutamie acid, 103
effect on inhibitors, 110-111
in serine synthesis, 136
reduction potential of, 111
10-Formy1-5,6,7,8-tetrahydropteroyl-
glutamic acid, see Citrovorum fac-
tor, Leucovorin
Fox,
pteroylglutamie acid deficiency in, 190
pyridoxine requirement of, 298
riboflavin deficiency in, 384-385
vitamin B, deficiency in, 276
Fumarie hydrogenase, coenzyme of, 355
Furfural, as growth factor, 590
G
Galactoflavin, 330, 394
effect on growth, 328
Gastrointestinal tract, p-aminobenzoic
acid and, 55-56
Gizzard erosion factor, 596
Glucose, 15
heated, as growth factor, 589-590
in riboflavin synthesis, 323, 361
in ribose synthesis, 318
phosphorylation of, 14
Glucose oxidase, action of , 355-356
N-Glucosylglycine, as growth factor,
590-591
Glutamic acid, 9, 16, 236
combination with p-aminobenzoic acid,
28
in sulfonamide reversal, 69
in vitamin B, deficiency, 267
pteroylglutamic acid and, 137
Glutamic acid decarboxylase, in vitamin
B, deficiency, 268
Glutamic-aspartic transaminase, coen-
zyme of, 278
Glutamic dehydrogenase, 354
Glutamine, 9
strepogenin and, 588
_ Glutathione, 11, 582
in detoxification, 63
Glycerol diacetate, 44
Glycine, 9, 62, 70
conjugation with p-aminobenzoic acid
14
in choline synthesis, 137-138
in detoxification, 44
652
in growth factor production, 590-591
in purine synthesis, 130
in salicylate detoxification, 82
in serine synthesis, 136
in thymine synthesis, 138
lycomarasmin and, 585
peptides, utilization of, 586
pteroylglutamic acid and, 137
toxicity, 138
vitamin Bes and, 294
Glycine oxidase,
mechanism of action, 354-355
preparation, 354
properties, 354
Glycylglycine, 9
Glycogen, 569
Glycollate, in pteroylglutamic acid as-
say, 201
Glyoxal, 444
Glyoxylic acid, 444
Growth, in vitamin B, deficiency, 265,
270, 273, 274
Growth factors, unidentified, 594-595
Guanidine, formation from folie acid, 95—
96
Guanine, 22, 25, 40
in growth of microorganisms, 124-125
in sulfonamide reversal, 69, 77
synthesis, pteroylglutamic acid and,
138-139
Guanosine, 318
Guinea pig,
pteroylglutamic acid deficiency in, 189
tocopherol deficiency in, 518, 520, 525,
530
Guinea pig antistiffness factor, isolation
of, 591
H
Haas enzyme, coenzyme of, 343
Hamster,
p-aminobenzoic acid deficiency in, 42
tocopherol deficiency in, 518, 520, 530,
532, 533, 539, 541
vitamin B, deficiency in, 271
vitamin By requirement of, 296
Hatchability, tocopherol deficiency and,
534
Heart, in vitamin Bg deficiency, 272, 274—
275
Heart disease, tocopherol and, 565-566
SUBJECT INDEX
Heat, pyridoxine and, 295
Heme, pteroylglutamie acid and, 137
Hemin, 437
Hemolysis, tocopherol and, 537, 546-548
Hemorrhage, in tocopherol deficiency,
586-537, 550
Hepatoflavin, see Riboflavin
Hexahydroflavins, 312
Hexose monophosphate, 341, 342
Hippuric acid,
formation of, 16
pteroylglutamic acid and, 137
Histaminase, specificity of, 356
Histamine, 55-56
thiamine and, 471
Histidine,
in sulfonamide reversal, 69
requirement for, 279
Homocystine, 239, 268
in methionine synthesis, 137, 140-142
Homogentisic acid, 48
Horse, ophthalmia, riboflavin and, 386
Human,
pteroylglutamic acid deficiency in,
202-216
riboflavin deficiency in, 387-391
riboflavin requirement of, 398-402
tocopherol deficiency in, 541-562
vitamin Bg deficiency in, 280-290
vitamin Bs requirement of, 298
Hydroquinone, 15
achromotrichia and, 40, 41, 59
u-Hydroxy acid oxidase, specificity of,
353, 354
6-Hydroxy-y-aminobutyrie acid, in
growth of Tenebrio, 584
3-Hydroxyanthranilice acid, 239
p-Hydroxybenzoie acid, 18, 24, 599
detoxification by, 64
esters, 65
formation of, 16
in p-aminobenzoic acid reversal, 81
6-Hydroxychroman, tocopherol and, 487
3-Hydroxykynurenine, pyridoxal phos-
phate and, 238-239
Hydroxyphenylalanine, 236
p-Hydroxyphenyllactie acid, 191
p-Hydroxyphenylpyruvie acid, 191
Hydroxyphenylserine, 236
5-Hydroxyuracil, as inhibitor, 150
SUBJECT INDEX
Hypercholesteremia, tocopherol and,
553, 554
Hyperthyroidism, vitamin Bs and, 294
Hypoxanthine, 125, 126, 350
in sulfonamide reversal, 68-69
oxidation of , 347, 348
Hypoxanthine desoxyriboside,
pterin and, 158
amino-
I
Idiopathic steatorrhea, pteroylglutamic
acid and, 208-209
Individual variation,
riboflavin requirement and, 397-398
thiamine requirement and, 476-477
Indole, 20, 238, 239
Indoleacetic acid, riboflavin and, 379
Indophenol, in pyridoxine estimation,
247-249
Inhibition analysis,
in folie acid function, 126-127
in sulfonamide reversal, 69-70
Inosine, in sulfonamide reversal, 69
Inosinie acid, synthesis of, 131-132
Inosinic transformylase, citrovorum fac-
tor and, 132
Inositol, 34, 35
p-aminobenzoic acid and, 38, 41-42
in muscular dystrophy, 560
tocopherol and, 505
meso-Inositol, in aromatic biosynthesis,
16
Insects,
pteroylglutamic acid requirements of,
190-191
riboflavin and, 379-380
Insulin, 569
in detoxification, 44
in thiamine phosphorylation, 439
strepogenin and, 585
Todoacetate, 436, 439
as enzyme inhibitor, 14
Isoalloxazines, synthesis of, 315-316
p-Isoascorbie acid, in tyrosine metabo-
lism, 191
Isoglutamine, 9
Isoleucine, in sulfonamide reversal, 69
Isoleucovorin chloride, formation of,
118, 119-120
Isoniazid, pyridoxine and, 290
653
Isoriboflavin, 330, 357, 393, 394
effect on growth, 326, 327-328, 359
K
a-Keto acids,
; decarboxylation of, 443
oxidation of, 579, 581
a-Ketobutyric acid, 237, 444
decarboxylation of, 443
a-Ketocaproic acid, 443, 444
a-Ketoglutaric acid,
decarboxylation of , 443-445
formation from pyruvate, 433
oxidation of, 485, 579
transamination of, 236
a-Ketoglutaric oxidase,
coenzyme content of, 580
pyruvic dehydrogenase and, 432
Kidney,
in tocopherol deficiency, 541
in vitamin Bg, deficiency, 266
Kynureninase, in vitamin Bg deficiency,
268
Kynurenine, 20
metabolism of, 238
L
Lactation,
riboflavin requirement and, 397
thiamine requirement and, 474
vitamin Bg and, 294
Lactic acid, 44, 444, 465
production from, pyruvate, 576
thiamine and, 426
Lactic dehydrogenase, 581
Lactobacillus arabinosus, in p-aminoben-
zoic acid estimation, 23
Lactobacillus casei,
in pteroylglutamic acid estimation,
167-168
in riboflavin assay, 372-373
lipoic acid and, 576
requirement for peptides, 586-587
Lactobacillus fermenti, in thiamine esti-
mation, 451
Lactoflavin, see Riboflavin
Larocaine, 8
Late-lactation paralysis, 522-525
LBF, see Pantetheine
Legumes, thiamine in, 456-457
654
Leucine, 9
peptides, requirement for, 586
Leucoriboflavin, 312
Leucovorin, see also Citrovorum factor
isomers of, 120-121
reaction with nitrous acid, 118
Leukemia,
pyrimidine analogs and, 151
vitamin Bs and, 284-285
Lewisite, pyruvic oxidase and, 581-582
Light, effect on riboflavin in foods, 376-
377
Linoleic acid,
tocopherol and, 512
vitamin Bs and, 266, 269, 295
Lipoic acid,
discovery of, 576
disulfide, bond energy of, 583
forms of, 577
free radical, in photosynthesis, 582-583
function of , 579-580, 581
isolation of, 577
Lewisite and, 582
potency of, 577
regeneration of oxidized form, 580-581
structure of, 577-578
synthesis of, 578
Lipoic acid sulfoxide, 578
Lipothiamide, properties of, 578-579
Lipothiamide pyrophosphate, properties
of, 579
Liver,
in riboflavin deficiency, 384
in tocopherol deficiency, 539-540
Liver disease,
ceroid and, 556
tocopherol and, 553, 554
Lumichrome, formation from riboflavin,
309-310
Lumiflavin, 303, 362
effects on growth, 330
formation from riboflavin, 309
in riboflavin assay, 366-367
Lupus erythematosus, tocopherol and,
554, 569
Lycomarasmin, strepogenin and, 585-586
Lyochrome, see Riboflavin
Lysine,
in sulfonamide reversal, 69
requirement for, 279
SUBJECT INDEX
Lyxoflavin,
effects on growth, 327, 358
isolation of, 588
synthesis of, 320
Lyxose, isolation of, 588
M
Magnesium,
carboxylase and, 442
dietary, in stiffness syndrome, 592
Malic acid, in detoxification, 44
Malonie acid, 436
as enzyme inhibitor, 14
in thiamine synthesis, 413, 414
Malononitrile, in thiamine synthesis,
413, 414-415
Mapharsen, 58
Mapharside, 63, 65
Meat, thiamine in, 457-458
Melanine, 48
Menopause, tocopherol and, 567
Mepacrine, see Atabrin
Metals, in carboxylase activation, 442
Mesoxalic acid, 443
Methanol, utilization for choline syn-
thesis, 142
Methionine, 135-136, 198
p-aminobenzoic acid and, 34
Methionine,
effect in pteroylglutamic acid defi-
ciency, 139
in antioxygenic action, 503-504
in sulfanilamide requirement, 76-77
in sulfonamide reversal, 69, 127-128
in thiamine biogenesis, 447
in vitamin Beg deficiency, 270
synthesis,
pteroylglutamic acid and, 137, 174
vitamins and, 140-142
tocopherol and, 539-540
vitamin Bg and, 294-295
2-Methyl-4-aminopyrimidy!-5-methyl-
aminodihydrochloride, in carbox-
ylase activation, 428-429
(Methylbis-6-chloroethyl)amine, effect
on p-aminohippurie acid synthesis,
14
2-Methy]-5-bromomethyl-6-aminopyrim-
idine, linkage with thiazole, 421-
423
SUBJECT
5-Methyleytosine, in growth of microor-
ganisms, 126
5-Methyl-2,4-diaminopurine, 126
Methylene blue, vitamin A and, 505-506
Methylglyoxal, 444
as growth factor, 590
4-Methyl-5-8-hydroxyethyl thiazole,
linkage with pyrimidine, 421-423
5-Methylisocytosine, in growth of micro-
organisms, 126
N'°-Methylpteroic acid, as inhibitor, 127
X-Methylpteroylglutamic acid, 187
as inhibitor, 110, 126-127, 155-156, 161
stilbestrol and, 195
7-Methylpteroyl-p-glutamic acid, as in-
hibitor, 155
9-Methylpteroylglutamic acid, as in-
hibitor, 156-157
2-Methyl-5-thioformylamine pyrimidine
in thiamine synthesis, 423-424
Methylthiouracil, 57
_ Metrazol, pyridoxine and, 291
Microorganisms,
intestinal,
riboflavin and, 359-361
thiamine and, 475-476
pteroylglutamic acid and, 199-200
riboflavin requirement of, 377-378
thiamine and, 446
Milk, thiamine in, 458
Mink,
pteroylglutamiec acid deficiency in, 189
tocopherol deficiency in, 539, 541
Monkey,
pteroylglutamic acid deficiency in,
180-186
pteroylglutamic acid requirements of,
184
pyridoxine requirement of, 296-297
riboflavin deficiency in, 386
tocopherol deficiency in, 518, 528, 530,
532, 537, 541
vitamin B, deficiency in, 274-275
Monkey anti-anemia factor, possible
identity of, 185-186
Monoeaine, 8
Monomethylhydroquinone, in tocopherol
synthesis, 496
Mouse,
p-aminobenzoic acid deficiency in, 41-
42
INDEX 655
pteroylglutamic acid deficiency in, 189
riboflavin deficiency in, 386
tocopherol deficiency in, 519, 520, 521,
528, 530, 532, 539
vitamin Bs, deficiency in, 270-271
vitamin Bs requirement of, 295-296
Muscle, p-aminobenzoic acid and, 56
Muscular dystrophy, 525-526
chronic, 526-528
human, tocopherol and, 558-559
inheritance of, 560
Muscular system,
in tocopherol deficiency,
cardiac, 530-531
skeletal, 522-529
smooth, 531-532
Myositis, tocopherol and, 559-560, 569
N
Naphthotocopherol, activity of, 491
N-(1-Naphthyl) ethylenediamine, as
coupling agent, 20
Nausea, vitamin B, and, 285-286, 291
Neoarsphenamine, 58, 65
detoxification of, 63
Nervous system,
p-aminobenzoic acid and, 56
in muscular dystrophy, 528-529, 532-
534, 535
in riboflavin deficiency, 381-382, 384,
387
in thiamine deficiency, 460-461
in vitamin Bg deficiency, 266-267, 271,
272, 273, 275, 281
vitamin B, and, 288-290
Neurospora crassa, in p-aminobenzoic
acid estimation, 23-24
Niacin, see Nicotinic acid
Niacinamide, 11, 293
Nicotinamide-ribose-pyrophosphate, as
coenzyme, 589
Nicotine, thiamine and, 467
Nicotinic acid, 11, 35, 65, 160, 456
deficiency, pteroylglutamic acid and,
190
in vitamin B, deficiency, 270
vitamin Bg and, 280, 281
Nitric acid, in tocopherol assay, 506
p-Nitrobenzoic acid,
effect on growth, 25
656
detoxification by, 64
reduction of, 2-8, 12
p-Nitrobenzoylglycine, effect on growth,
25
o-Nitrochlorobenzene, in riboflavin syn-
thesis, 315
6-Nitroflavins, 331
Nitrogen mustard, see Methylbis-(6-
chloroethyl)amine
N?!°_Nitrosopteroylglutamic
tivity of, 103
Nitrosotocopherols, in assay, 508-509
p-Nitrotoluene, 12
5-Nitrouracil, as inhibitor, 150
o-Nitroxylidine in riboflavin synthesis,
315
Norit eluate factor, 175-176, see also
Pteroylglutamic acid
Notatin, see Glucose oxidase
Novocaine, 8
Nucleic acids,
effect on conjugase, 166
in ribose preparation, 318
Nucleotides, in muscular dystrophy, 558-
559
Nutritional encephalomalacia, tocoph-
erol and, 535
O
Octahydroflavins, 312
Oils, as tocopherol sources, 495-496
Old yellow enzyme, see also Yellow en-
zyme
chemical properties, 340
mechanism of action, 341-342
preparation, 334
Oral lesions, in pteroylglutamic acid de-
ficiency, 182-183
Oryzamin, see Thiamine
Ovoflavin, see Riboflavin
Oxalacetic acid, 448
formation from pyruvate, 433
transamination of, 236
Oxythiamine, 465
acid, ac-
P
PABA, see p-Aminobenzoic acid
PAHA, see p-Aminohippuric acid
Paludrine,
as inhibitor, 150-151
riboflavin and, 330
SUBJECT INDEX
Pancreatic fibrosis,
ceroid and, 556
tocopherol and, 553, 554
Panthesine, 8
Pantocaine, 8
Pantothenic acid, 11, 17, 35, 37, 44-45,
59, 160
achromotrichia and, 37-40, 41, 84
in detoxification, 45
in vitamin Bg deficiency, 270
pteroylglutamiec acid and, 39, 173
tocopherol and, 569
pL-N-Pantoyl-n-butylamine, 17
Pellagra, 456
Penicillin, 17, 129
Pentoses, as growth factors, 590
Peptides, as growth factors, 585-588
Pernicious anemia, pteroylglutamic acid
and, 210-213
PGA, see Pteroylglutamie acid
Phenylacetic acid, detoxification by, 64
Phenylalanine, 11, 17, 18
requirement for, 279
o-Phenylenediamine,
in riboflavin synthesis, 314
in tocopherol assay, 509
Phenylglycine, 64
Phenylhydrazine, carboxylase and, 437
Phenylpropionic acid, detoxification by,
64
Phenylpyruvic acid, 448
oxidation, 444
Phloroglucinol, in aromatic biosynthesis,
gl
Phosphatase, 440
p-aminobenzoic acid and, 56-57
carboxylase activity and, 428-429
Phosphate, in pyruvate oxidation, 434—
436
Phosphogluconic acid, in pentose forma-
tion, 323-324
Phospholipids, 539
Phosphopyruvie acid, 439
Phosphoric acid, tocopherol and, 494, 503
Phosphorus, dietary, in stiffness syn-
drome, 592-593
Phosphorylase, p-aminobenzoie acid in,
13
Photosynthesis, lipoie acid and, 582-583
Phthaleins, in pyridoxine estimation, 245
Phycomyces, in thiamine estimation, 451
——
———
SUBJECT INDEX
Phytol, in tocopherol synthesis, 496, 498
Phytyl bromide, in tocopherol synthesis,
488
Phytylehloride, in tocopherol synthesis,
496
Pigment, in tocopherol deficiency, 516-
517, 531-532, 538-539
Plants, vitamin Bg deficiency in, 264
Polarography, in vitamin Bs estimation,
244
Poliomyelitis, thiamine and, 472
Polyneuramin, see Thiamine
Polyneuritis, 404
Polypeptidase, p-aminobenzoic acid in,
13
Pontocaine, 8
Potatoes, thiamine in, 457
Pregnancy,
thiamine requirement and, 474
tocopherol level in, 552, 553
vitamin Bg and, 285-286, 287-288, 293
Procaine, 8, 55
p-aminobenzoic acid and, 58
Progesterone, tocopherol and, 561
Proguanil, see Paludrine
Proline, peptides, utilization of, 586
Promin,
p-aminobenzoic acid and, 78
pteroylglutamic acid and, 174
pyridoxine and, 291
Promizole, pteroylglutamic acid and, 174
Propesin, 8
Propioin, formation of, 443
Prothrombin, 55
Protein,
association with pteroylglutamic acid,
94-95
effect on conjugase, 166
effect on riboflavin requirement, 396-
397
effect on thiamine requirements, 473
metabolism, vitamin B,s and, 267-268,
270-271
pteroylglutamic acid and, 174
vitamin B, and, 295
Protogen, see also Lipoic acid
in pyruvic acid oxidation, 440
Pseudopyridoxine, 231, 277
Pteridines,
substituted, as pteroylglutamic acid
analogs, 152-153
657
Pteroic acid, 16, 33, 34
activity of, 144
in sulfonamide reversal, 74
synthesis of, 15, 104
Pteroylaminoadipie acid, activity of, 155
Pteroylaminopimelic acid, activity of,
155
Pteroylaspartiec acid, as inhibitor, 154-
155, 160
Pteroyldiglutamic acid, activity of, 142-
144
Pteroylglutamic acid, 28, 42, 583
achromotrichia and, 39
p-aminobenzoic acid and, 138, 32-34,
133-136
antagonists, 149-161
resistance to, 160-161
biochemical systems of, 124—142
chemistry of, 91-121
citrovorum factor and, 110
cleavage of, 15-16
conjugates,
hydrolysis of, 163-167
isolation of, 93-95
utilization by fox, 190
utilization by Tetrahymena, 199-200
conversion to citrovorum factor, 198
degradation of, 95-99
derivatives, synthesis of, 108
effects of deficiency,
in animals, 171-198
in human, 202-216
in microorganisms, 199-202
effect on feathers and pigmentation,
176
endocrine relationships, 195-197
estimation of,
chemical methods, 161-162
microbiological methods, 162-168
extraction from tissues, 163
formation from formylpteroic acid,
200-201
glycine toxicity and, 138
hydrogenation of, 101-103
inability to replace p-aminobenzoic
acid, 135-136
in p-aminobenzoic acid estimation, 24
in choline synthesis, 137-138
industrial preparation of, 121-124
in foods, 168-171
effect of cooking, 204
658
in methionine synthesis, 1387, 140-142
in purine synthesis, 138-139
in radiation sickness, 291
in serine synthesis, 136-138
in sulfonamide reversal, 68-69, 70, 71
isolation of, 91-93
mechanism of action, 128-142
nomenclature of, 89-91
pharmacology of, 216-217
polyglutamates, 28
isolation of, 93-94
proof of structure, 96-97
properties of, 99-104
reaction with nitrous acid, 103, 118
reduction potential of, 111
relation to ascorbic acid, 191-195
relation to other vitamins, 173-174
release, xanthopterin and, 147-149
requirements, of human, 217
riboflavin and, 382-383
specificity of, 109-121
specificity of action, 142-161
standardization of activity, 168
synthesis of, 15, 104-108
toxicity of, 216-217
utilization in pernicious anemia, 212
Pteroylheptaglutamic acid, 178, 209, 210
in pernicious anemia, 212-213
Pteroyltriglutamic acid, 33, 34, 160, 177,
178, 181, 209, 210
effect on tumors, 161
Purines, 33, 135-136, 167
pteroylglutamic acid and, 124-127
in p-aminobenzoic acid estimation, 22,
25
substituted, as pteroylglutamic acid
antagonists, 151-152
Putrescine, oxidation of, 356
Pyramine, estimation, 452
Pyridine nucleotide, in pyruvic acid oxi-
dation, 440
Pyridoxal, see also Vitamin Be
activity,
for chick, 252
for microorganisms, 254, 276-277
for rat, 251-252
differentiation from pyridoxine, 248
formation of, 231-232
in tyrosine decarboxylation, 239
toxocity of, 290
SUBJECT INDEX
Pyridoxal phosphate, 442
as coenzyme, 234, 278
formation of, 240-241
in growth of microorganisms, 277
in transamination, 237-238
mechanism of action, 241-242
structure of, 240
Pyridoxamine, see also Vitamin Bg
activity,
for chick, 252
for microorganisms, 254, 276-277
for rat, 251-252
differentiation from pyridoxine, 248-
249
formation of, 231-232
toxicity of, 290
Pyridoxamine phosphate,
as cotransaminase, 237
in growth of microorganisms, 277
Pyridoxic acid, 281
excretion of, 292
pteroylglutamic acid and, 178
Pyridoxine, 34, 35, see also Vitamin Bg
biochemical systems, 234-239
coenzyme form of, 234
excretion of, 291-292
industrial preparation, 233
in growth of microorganisms, 276-277
pharmacology of, 290-293
proof of structure, 226-229
purity standards, 233
synthesis of, 229-231
toxicity of, 292-293
Pyrimidines,
pteroylglutamic acid, and, 124-127
ring closure in thiamine synthesis, 415-
417
substituted,
as pteroylglutamic acid antagonists,
150-151
riboflavin and, 330
Pyrithiamine, 442
action of, 465
as inhibitor, 447-448
Pyruvate oxidation factor, see Lipoic
acid
Pyruvie acid, 44, 238
carboxylation of, 433
decarboxylation of, 443-445
dismutation, 579
SUBJECT INDEX
formation from acetate, 436
in thiamine deficiency, 462, 465
oxidation,
coenzymes required, 440
phosphorylation and, 434-436
thiamine and, 426-427
transamination of, 236
Pyruvie dehydrogenase, 427
distinction from carboxylase, 432-433
Pyruvie oxidase, 435
lipoic acid in, 581
Q
Quinic acid, in aromatic biosynthesis, 16
Quinine, riboflavin and, 330, 393
Quinoxaline-2-carboxylyl-p-aminoben-
zoylglutamic acid, activity of, 153
R
Rabbit, tocopherol deficiency in, 518,
525-526, 530
Racemases, coenzyme of, 238
Radiation sickness, vitamin Bs and, 285-
286, 291, 298, 296
Rat,
p-aminobenzoic acid deficiency in, 37-
41
assay,
for riboflavin, 369-370
for tocopherol, 509-511
pteroylglutamie acid deficiency in,
171-175
pyridoxine requirement of, 294-295
riboflavin deficiency in, 380-383
tocopherol deficiency in, 517-518, 519-
520, 526-527, 530-533, 537, 538-539,
540, 541
vitamin Bs deficiency in, 265-270
Rat growth test, in pyridine estimation,
251-252
Reductone,
in pteroylglutamic acid synthesis, 122
in sulfonamide reversal, 74
Reproductive system,
female, in tocopherol deficiency, 519-
521
male, in tocopherol deficiency, 517-519
tocopherol and, 560-562
Reproduction,
in riboflavin deficiency, 383
659
in vitamin Be deficiency, 265
pteroylglutamie acid and,
179-180
riboflavin requirement and, 397
Retrolental fibroplasia, tocopherol and,
548-549
Rheumatic fever, p-aminobenzoic acid
and, 51-52, 55, 61-62, 81-83
Rhizopterin, 28, see also N}-Formyl-
pteroic acid
chemistry of, 109
Rhodoflavin, 312
formation of, 314
Ribamine, in riboflavin synthesis, 316
Riboflavin, 160
adsorption of , 304-305
analogs,
mode of action, 330-331
phenazine, 330, 393
antagonists of, 327-331
biochemical systems, 334-357
biogenesis of, 359-361
by fungi, 360
blanks, in assay, 365-366
bound forms, 304
hydrolysis of, 362-363
chemistry of, 301-332
constitution, 309-314
degradation products of, 809-311
derivatives, activity of, 306-307, 358,
359
effects of deficiency,
in animals, 380-387
in human, 387-391
in insects, 379-380
in microorganisms, 377-379
in plants, 379
estimation,
biological methods, 369-371
microbiological methods, 372-373
physical and chemical methods, 361-
369
excretion of, 392
extraction of, 304, 373
fluorescence of, 307
history of, 301-803
in detoxification, 45
industrial preparation, 332-333
in sulfonamide reversal, 77
in vitamin Bg deficiency, 270
174-175,
660
isolation of, 303-305
load test, 400-402
lyxoflavin and, 588, 589
mechanism of action, 357
nomenclature and formula, 300-301
occurrence in food, 374-377
pharmacology of, 391-394
precipitation of, 304
properties of , 305-309
pteroylglutamic acid and, 174
purification for assay, 363-364
reduction of, 308
requirements,
of animals, 394-398
of human, 3898-402
solubilizing agents for, 306
specificity of, 325-332
specificity of action, 358-359
stability of, 307-308
standardization of, 373-374
standards in assay, 364-365
synthesis,
biological, 322-324
chemical, 314-322
microbiological, 324-325
toxicity of, 309, 391-392
Riboflavin adenine dinucleotide, 303-
304, see also Flavin adenine di-
nucleotide
Riboflavin monophosphate, 303-304, see
also Flavin mononucleotide
Riboflavin tetraacetate, in growth stimu-
lation, 325
Ribose,
in riboflavin synthesis, 314-316
preparation of, 318
Ribose phosphate,
formation of, 323-324
Ribulose phosphate, formation of, 323-
324
Rice bird, in thiamine assay, 405
Rice bran, concentrates of, 409-410
Rice germ, concentrates of, 410
Rice moth larva test, in pyridoxine es-
timation, 253
Rickettsial diseases, 12
p-aminobenzoie acid and, 51, 60-61,
79-81
Rocky Mountain spotted fever, p-amino-
benzoic acid and, 79
SUBJECT INDEX
Rous sarcoma, vitamin deficiency and,
159-160
Ruminants,
riboflavin deficiency in, 385-386
vitamin Bs, and, 298
Ss
Salicylates, 55
p-aminobenzoic. acid and, 61-62, 82
Salicyluric acid, 62
formation of, 82
Sarcosine, oxidation of, 354
Scleredema, tocopherol and, 550, 569
Semicarbazide, pyridoxine and, 291
Serine, 135-136, 239, 268
in choline synthesis, 137-138
in methyl group formation, 198
in purine synthesis, 130
in sulfonamide reversal, 70, 127-128
in thymine synthesis, 138
lycomarasmin and, 585
requirement for, 279
synthesis, pteroylglutamie acid and,
136-138
vitamin Bs and, 294
Serine deaminase, coenzyme of, 237-238
Serum,
tocopherol levels,
in disease, 552-554
normal, 551-552
Serylalanylglutamie acid, strepogenin
and, 586
Serylglycylglutamic acid, strepogenin
and, 585
Shikimie acid,
in p-aminobenzoic acid reversal, 81
in aromatic biosynthesis, 17-18
Size, influence on thiamine requirement,
473
Skin,
in riboflavin deficiency, 380-381, 382,
384, 388-390
in vitamin B, deficiency, 265-266
Spetrophotometry, in vitamin Bg esti-
mation, 243-244
Sprue, 183
ceroid and, 556, 557-558
pteroylglutamic acid and, 208-209
tocopherol and, 553, 554
Staphylococcus albus, peptide require-
ment of, 587
SUBJECT INDEX
Stibosan, 66
“Stiff-lamb”’ disease, 524
Stigmasterol, as antistiffness factor, 592
Stilbestrol,
p-aminobenzoic acid and, 58
pteroylglutamic acid and, 195-196
tocopherol and, 537, 567
Straub flavoprotein, properties of, 344
Strepogenin,
discovery of, 585
in growth of mammals, 587-588
Streptococcus faecalis, in pteroylglutamic
acid estimation, 167-168
Stress,
growth factors and, 498-599
tocopherol and, 563-564
Strychnine, pyridoxine and, 291
Succinic acid, in thiamine synthesis, 413,
414
Succinic acid oxidase, in vitamin B, de-
ficiency, 268, 271
Succinic semialdehyde, formation of, 443
Succinylsulfathiazole, 39, 40, 177, 186, 187
p-aminobenzoic acid and, 75-76
Sulfadiazine, 129
Sulfaguanidine, 39, 40, 57, 177
reversal by p-aminobenzoie acid, 75
tocopherol and, 513
Sulfamerazine, 176
Sulfanilamide, 11, 15, 19, 21, 24, 45
as growth essential, 76-77
pteroylglutamic acid and, 127-128, 133
Sulfanilic acid, 15, 40
Sulfapyridine, 15, 21
Sulfasuxidine, see Succinylsulfathiazole
Sulfathalidine, 186
Sulfathiazole, 15
p-aminobenzoic acid and, 60
synergism with 2,4-diaminopteridine,
153
uptake by bacteria, 75
Sulfhydryl groups,
in antioxygenic action, 503-504
“Sulfonamide inhibitor,’’ in p-amino-
benzoic acid estimation, 21-22
Sulfonamides, see also specific compounds
p-aminobenzoic acid and, 66-79
effect on plants, 77-78
effect on malarial mosquito, 77
in pteroylglutamic acid deficiency, 168,
171-173
661
reversal, 66-79
by pteroylglutamie acid, 134-135
Swine,
p-aminobenzoic acid deficiency in, 42~
43
pteroylglutamic acid deficiency, 186—
189
pyridoxine requirement of, 296
riboflavin deficiency in, 385
tocopherol deficiency in, 518-519
vitamin Bes deficiency in, 273-274
Synthetase, definition of, 14
ak
Taka-diastase, in pteroylglutamic acid
estimation, 163
Teeth, in tocopherol deficiency, 541
Temperature,
effect on thiamine requirement, 474-
475
riboflavin requirement and, 397
Tenebrio molitor, growth of, 583-584
Testosterone,
pteroylglutamic acid and, 196-197
riboflavin and, 392
Tetracaine, 8
Tetrahydropteroylglutamiec acid,
activity of, 198
in serine synthesis, 136
reduction potential of, 111
Tetrahymena, lipoic acid and, 576
Thetins, 584
Thiaminase,
effect on thiamine, 459
in food, 458-460
pteroic acid and, 15
Thiamine, 11, 34, 35, 298
absorption of, 454
activity of reduced form, 440-441
biochemical systems, 425-442
biogenesis of, 445-448
chemistry of, 404-409
constitution of, 406
diazotized, 21
diets deficient in, 464-465
effects of deficiency,
in animals, 460-464
in human, 464-466
in microorganisms, 460
estimation,
biological, 450
662
chemical, 448-450
fermentative, 450-451
microbiological, 451-453
physical, 448
in carboxylase activation, 428-431
in detoxification, 45
industrial preparation,
biosynthetic, 411
from natural sources, 409-411
patent situation, 424-425
synthesis, 411-424
in peripheral nerves, 463-464
isolation of , 404-405, 410-411
nomenclature, 404
occurrence in food, 454-458
oxalacetate and, 434
pharmacology of, 466-471
properties of, 406
pyridoxine and, 279-280, 294
requirements,
of animals, 472-478
of human, 478-480
specificity of , 408-409
standardization of activity, 453-454
synthesis, 406-408
by animal tissues, 409
toxicity of, 466, 468-470
Thiamine disulfide pyrophosphate, ac-
tivity of, 441
Thiamine monophosphate, 437, 442
in thiamine pyrophosphate synthesis,
439
Thiamine polyphosphate, 437-438
activity of, 443
Thiamine pyrophosphate, 578, 579
as coenzyme, 427
dissociation from apoenzymes, 427-431
essential metals, 442
estimation, 450-451
in determining thiamine requirement,
477-478
in pyruvate oxidation, 435
occurrence of, 454-455
specificity of action, 442-445
synthesis,
chemical, 438
enzymatic, 438-442
unitarian view of action, 433-434
Thiamine pyrophosphate enzymes, reac-
tions catalyzed by, 480
SUBJECT INDEX
Thiamine triphosphate, 437
activity of, 443
Thiocaine, as anesthetic, 9
Thiochrome, in thiamine estimation,
448-449
Thiochrome pyrophosphate, 449
Thioctic acid, see Lipoic acid
5,8-Thioctic acid, 583
Thiodiphenylamine, vitamin A and, 505
Thioformamide,
in thiamine biogenesis, 447
in thiamine synthesis, 419-420
Thiosemicarbazide, pyridoxamine and,
291
Thiouracil, 57, 174, 284
effect on thyroid gland, 40-41
Thiourea, pteroylglutamic acid and, 174
Threonine,
effect in pteroylglutamic acid defi-
ciency, 139, 174
in purine synthesis, 130
in sulfanilamide requirement, 76-77
requirement for, 279
Threonine deaminase, coenzyme of, 237—
238
Thymidine, 130, 156
Aminopterin and, 158
citrovorum factor and, 110
effect in pernicious anemia, 140
in sulfonamide reversal, 127-128
pteroylglutamie acid and, 125
Thymine, 33, 130, 134, 135-186, 150, 156
Analogs, as inhibitors, 150
effect in pernicious anemia, 140
effect in pteroylglutamic acid defi-
ciency, 139-140, 187, 209
in growth of microorganisms, 124-126
in pteroylglutamic acid assay, 167
in sulfonamide reversal, 68, 70
synthesis from serine and glycine, 138
Thymine gly¢ol, 114
Thyroid, lyxoflavin and, 588
Thyroid gland, p-amino benzoic acid
and, 40-41, 57
sulfaguanidine and, 40
Thyrotoxicosis, p-aminobenzoic
and, 84-85
Thyroxine, 41, 57, 174
p-aminobenzoic acid and, 64-65
effect on thiamine requirement, 474
acid
SUBJECT INDEX 663
Tocopherols,
absorption of, 501-502, 543-544
antioxygenic action of, 490
in vitro, 503-504
in vivo, 502-503
biochemical-systems, 501-506
bound forms of, 502
catabolism of, 502
chemistry, 483-495
chromatography of, 497
derivatives, 489-491
dietary intake of human, 543
distillation, 497-498
effects of deficiency,
in animals, 514-541
in human, 541-562
esters, activity of, 490-491
estimation,
biological, 509-512
chemical, 506-509
excretion of, 543-544
extraction of , 496-497
in adults, 551-562
industrial preparation of, 495-501
in infants, 545-551
isolation of, 484-486
isomers,
differential assay of, 508
relative activity of, 490
resolution of, 499
mode of administration, 563
nomenclature and formulas, 482-483
occurrence in foods, 512-514
oxidation of, 492-495
pharmacology of, 562-570
physical properties, 492
potency,
of natural products, 499
of synthetic products, 499-500
production control tests, 500-501
requirements,
of animals, 570-572
of human, 572-573
semiquinone formation, 494
sidechain in activity of, 491
source materials, 495-496
storage of, 502
therapeutic use, 564-570
tissue levels, 555
tissue storage of, 544-545
toxicity of, 563
a-Tocopherol, proof of structure, 487-488
8-Tocopherol, structure of, 488
a-Tocopheroxide, properties of, 495
Tocopherylbydroquinone,
activity of, 493, 530
muscular dystrophy and, 559
Tocopherylquinone,
activity of, 492-493, 530
in plasma, 502
in tocopherol assay, 506
Torulin, see Thiamine
TPN, see Triphosphopyridine nucleotide
Transaminases,
coenzyme of, 236-238
in vitamin Bg activity, 268, 271
Transsulfurization, in vitamin Bez de-
ficiency, 268-269
2,4,5-Triamino-6-hydroxypyrimidine,
155
in folic acid synthesis, 104-106, 122, 124
Trichochromogenic factor, see p-Amino-
benzoic acid
Trimethylhydroquinone, in tocopherol
synthesis, 488, 496, 498
Triphosphopyridine nucleotide, 314, 341,
342, 442
oxidation of, 344, 345, 346
Trout,
pyridoxine requirement of, 298
vitamin B, deficiency in, 276
Tryparsamide, p-aminobenzoic acid and,
63
Tryptophan, 16, 17-18, 20, 270-271, 281,
456
effect in pteroylglutamic acid defi-
ciency, 139
formation of, 259
in sulfonamide reversal, 69
in vitamin B, deficiency, 267, 268, 295-
296
metabolism of, 238-239
requirement for, 279
Tryptophanase, coenzyme of, 238
Tryptophan test, in vitamin Be, defi-
ciency, 286-288
Tsutsugamuchi disease, p-aminobenzoic
acid and, 79
Tumors,
p-aminobenzoic acid and, 61
inositol and, 41-42
664
pteroylglutamie acid analogs and, 159-
161
pteroyltriglutamate and, 108
riboflavin analogs and, 329-330
riboflavin and, 394
vitamin Bs, and, 271, 284-285
Turkey,
pteroylglutamic acid requirement of,
178
riboflavin deficiency in, 387
tocopherol deficiency in, 532
vitamin Beg deficiency in, 275
Tutocaine, 8
Typhus,
p-aminobenzoic acid and, 79
riboflavin and, 383
Tyrosinase, 15, 48
p-aminobenzoic acid and, 56
Tyrosine, 11, 17-18, 24, 48, 268
ascorbic acid and, 191
in pteroylglutamic acid deficiency, 187
peptides, requirement for, 587
pteroylglutamic acid and, 191-192
requirement for, 279
Tyrosine decarboxylase,
coenzyme of, 239
in vitamin Be deficiency, 278
peptide requirement and, 587
Tyrosyl compounds, excretion in vita-
min deficiency, 188
U
Uracil, 125, 150
effect in pernicious anemia, 140
in pteroylglutamic acid deficiency, 187
Urea, 11
pyridoxine and, 286
Uric acid, 320, 348
Urine, riboflavin compounds in, 392-393
Uroflavin, see Riboflavin
riboflavin and, 392
Uropterin, see Xanthopterin
V
Valine, in sulfonamide reversal, 69, 128
Vascular disease, peripheral, tocopherol
and, 566-567
Vascular system, in
ciency, 534-538
Verdoflavin, formation of, 312
tocopherol defi-
SUBJECT INDEX
Vitamin A,
ceroid and, 557
in antioxygenic action, 503-504
in tocopherol assay, 507
tocopherol and, 504-506, 563-564
Vitamin B, , 175, 176, see also Pteroylglu-
tamic acid conjugate, Pteroylhep-
taglutamie acid
Vitamin B, conjugase, in pteroylglu-
tamic acid estimation, 163-166
Vitamin B, , 596
Vitamin B;, see Carnitine
Vitamin By , 596
Vitamin Bx , see p-Aminobenzoiec acid
Vitamin B, , see Thiamine
Vitamin Bz , see Riboflavin
Vitamin B; , 596
Vitamin By, , 596
Vitamin B; , 596
Vitamin Be, see also, Pyridine, Pyridoxal,
Pyridoxamine
antagonists, 242
biogenesis of , 242
chemistry, 222-232
effects of deficiency,
in animals, 265-276
in humans, 280-290
in microorganisms, 276-280
in plants, 264
estimation,
biological methods, 249-253
chemical methods, 244-249
microbiological methods, 253-255
physical methods, 2438-244
experimental deficiency in humans,
281-282
hydrolysis of bound forms, 242-248,
253, 256
isolation, 222-224
nomenclature and formulas, 220-221
occurrence in foods, 255-263
properties of, 224-226
requirements,
of animals, 293-298
of human, 298
specificity of, 231-232
specificity of action, 239-242
standardization of activity, 255
tocopherol and, 563-564
Vitamin B;, 596
Vitamin Bs , 596
SUBJECT INDEX
Vitamin Bio , 596
Vitamin By, , 596
Vitamin j2 , 160, 440, 593, 597
biosynthesis of, 322
deficiency, 188-189
effect on purine requirement, 131
glycine toxicity and, 138
in amino acid synthesis, 136
in anemia, 203, 205, 206, 207, 209, 211-
213
in choline synthesis, 137-138
in methionine synthesis, 140-142
in pteroylglutamic acid deficiency, 186,
187, 188
pteroylglutamic acid and, 194-195
reproduction and, 175
synthesis of, 18-19
tyrosine and, 192
Vitamin B,3 , 576-577
Vitamin By, , 596
“Vitamine’’, 405
Vitamin E, see also Tocopherols
Vitamin F, see Thiamine
Vitamin G, see Riboflavin
Vitamin H, see p-Aminobenzoic acid
Vitamin H, see Vitamin Bg
Vitamin K, 491, 506
sulfonamides and, 172
Vitamin L, 596
Vitamin L, , 596
Vitamin M, 180, see also Pteroylglutamic
acid
Vitamin P, 550
Vitamin T, 596
Vitamins B, in antioxygenic action, 503-
504
WwW
‘“White-muscle’’ disease, 524, 525
Work, effect on thiamine requirement,
477
x
Xanthine, 22, 25, 125
in sulfonamide reversal, 69
oxidation of, 347, 348
Xanthine oxidase, 350, 351
preparation, 347-348
properties, 348
riboflavin analogs and, 328-329
Xanthine oxidase factor, 596
Xanthophyll, tocopherol and, 505, 536
Xanthopterin,
hematopoietic effect of, 145-147
in pteroylglutamic acid estimation, 162
in pteroylglutamic acid deficiency, 187
isolation and sources of, 145
relation to pteroylglutamic acid, 145-
149
reproduction and, 175
Xanthopterin oxidase, inhibition of, 349
Xanthurenic acid, 282, 285
vitamin Bs and, 268, 270, 271, 272, 274,
286-288, 293
o-Xylene, in riboflavin synthesis, 314
3,4-Xylidine, in riboflavin synthesis,
316-317
Xyloflavin, effect on growth, 327
yg
Yeast,
concentrates of, 410, 411
in thiamine estimation, 450-451
Yeast eluate factor, see Vitamin Bg
Yellow enzyme,
in flavin nucleotide estimation, 369
resolution of, 337
synthetic, 342
“‘Yellow-fat’’ disease, 539
Z
Zwischenferment, 341, 342
— p+
=
. Seesee
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