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Full text of "Report of program activities"

ANALYSIS OF PR0GfL4M ACTIVITILS 
NATIONAL INSTITUTES OF HEALTH 



1956 

I 

i MIIONAL INSTITUTE OF 
ARTIIJUTIS AND METABOUC DISEASES 



NAIIO'M: INSTlTOItS' OF' HEALTH 

FUBLiC HEi\J.:rE SEJIVIC£ 

DEPARTEKIMT OF HEALTH, EDUCATION, AND WELFARE 



Analysis of NIH Program Activities 
January-December 1956 
dl,^. National Institute of Arthritis and Metabolic Diseases 

Laboratory Research 

Introductur;/ ComiiLunts - The coraprehonsion of biological plionomcna results 

from the fusion of information derived from many 
ctis.->iplines. The normal operation of the organism 
may be studied ii'i the intact animal, the isolated intact organ, the sliced 
or homogenized organ, tJic-; individual cell, the sub-eellular particle, 
tl:ie soluble multi-enzymo system or the pur-w individual enzyme. The cheinical 
composition of the substrate, the enzyme which catalyzes, the hormone which 
regulates and the fluid which contains the reaction must all be investigated. 
To gain further insist, the biological system may be insulted, and the 
insult may be an- alteration of diet, the administration of a foreign agent, 
whether microbial or chemical, the applicatiori of some form of energy" or 
other physical stress, or the alteration of normal anatouy by surgery. 

Examples of all of, these and ether approaches will be found in 
the ensuing pagds. One may ask: 'Which of those approaches is the best? 
The ansx-jor quite clearly is that no one approach is superior to all 
others. At a particular time in history, the answer to a particular 
problem will appear to a particular scientist to be most accessible by a 
particular approach. This however .is illusory since each scientist 
necessarily has certain techniques at his comiriaha in which he has re- 
liance and skill. -Each succeeding year presents us with new additional 
techniques, which^ ? s. they are mastered, make older experiments seem 
inexact and luss than rigorous. The recurrent experience has been that 
for a biological truth to be securely established, compatible evidence 
must be obtained froJri biological preparations at various levels of orgarJ.z- 
ation examined by various tecl-iniques. This' of course takes tirae and it 
is a rare event in the historj-of science to see such a truth established 
■within one calendar year. .. '■ 

An example of the effectiveness of. the multidiscipl-ined approach 
will be found, below in'.;the report of the origins and fates of tiie form- 
lEteoaiuino acids. . The very satisfactorj- .progress which we are reporting 
in this area of biochemistry was made- possible because the techniques of 
organic synthesis, chdinical isolation and identification, enzyrae pizrifica- 
tion, chemacal and microbiological analysis, spectrophotometry and nutri- 
tion research were applied to intact animals, to tissues and enzymes 
derived from those, and to microorganisms. 

• A review which covers the events of . a single .calendar yoar, as the 
present one docs^ is an i^rtificial slice of history, resembling, in some 
regards, a single, .fbecjne in a.jaction picture ialm. Its signifir-ance oannot 



/9S4> 



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bo Interpreted v.-ithout a perspective of the past and a vision of the 
future, rh- r.naLypia of i;cicntific experiments uvev such a brxcf span 
is feasible, but^ with rare exceptions, the s>-nthesis of scicntxlic 
findinf^s into scientific truths requires tho consideration oi a iar 
lonjjcr segncnt of time. 

Carbohydrate Chemstrj- and M-tabolism - The chemical structures and rc- 

"^ '^ ~~ — aotions of tho sugars continue 

to be the keystone to the under- 
standing of the ir^tabolisni of these compounds. A number of significant 
advances may be recerded at an organic ohcmical luvcl. A novel intra- 
molecular migration from position 1 to position 2 of a substituont 
benzoyl c-noup on ribosc has been observed, and th^^ existence oi cycxic 
ortho acid esters of sugars has largely b^^en discredited. A new class 
of anhydrosugars has boon discovered, represented by 1,5-anhydro-lJ-U- 
ribofuranose which is at once both a furanose and a pyranose. The 
structure of the intensely sweet natural product, sto-n-oside, has been 
elucidated It is ;•. glycoside wherein two glucose residues are ui 
6(1— > 2) linkage It is thus a sophoroside, and except for sophorose_ 
itoelf the only known member of this class. The conditions determanang 
yields of thn i.ugar anhydrides of many hcxoses and heptuloses have been 
studied, • 

Th2 central role of x^'lulosc 5-phosphate in the oxidative metabo- 
lism of glucose has been est".blished. This pentose has been shown to 
be both a substrate for transkotolase and also the initx'O. pentose 
fonrc^d by this enzyme. IVansketolase, which effects transfer from 
one sugar residue to another of a two-carbon (active glycolaldehyde; 
residue, shown an absolute requirement for the trans-configuratxon at 
C-3 C-U of reactive kotoscs. A bacterial fermentation of xylulose 
5-phosphate has bocn found to require inorganic phosphate and to yield 
acetyl phosphate. Active acetyl is thus produced vdthout j^^^^^^Jj°J 
of coenzyme A. In this «phospnoketolase" reaction, as xn the transketo- 
lase reaction, a requirement for thiamiiiu pyrophosphate was shovjn Sxnce 
the discovery' by NIAMD scientists that ribuLosc \>^^\^'^°^^'^''\l^fj}'^,^^. 
unique primary aoceptor of carbon dioxide in photosyntliesxs, the reactxons 
Sethis suga7havc been a matter of continued interest. A phosphatase 
has been isolated from spinach which preferentially removes one o^ the 
two phosphate groups. This is being further studied in the hope of gain- 
ing access to the presently unavailable ribulose ^-P^^^P^^^^;, J^J"^,!^'^" 
tion whereby another pentose, xylulose, enters mammalxan ^xochernxstry 
has been clarified by the discovery of a kinase xn Ixver whxch catalyzes 
the f o^ti^n of x^^lSiose phosphate. This fi^^g has bearing upon the 
ultdwit.c undcx.<.fc.inding oi' the huiaan rongenxi^xL dr,f-P-^t, rnn.x>suriii. 



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The mctabollsip. of a nxmiber of sugar acids has been' s-bndied, 
Galactvironic acid has boen found to disappear ■when incubated. with rat 
liA'cr extract, yiolding products -presumed to be on the pathway of 
ascorbic acid synthesis. In contrast to findings of others, glucTxronic 
acid is found to be abundantly catabolized by the intact animal. New 
analytical methods xirhich discriminate between glucuronic acicj and 
glucosiduronic acids have been devised in relation to this study. The 
initial step in the metabolism of gluconic acid has been identified as 
conversion to 6-phosphogluconic acid. The enzyme which catalyzes 
this reaction has been purified, its specificity r.nd cof actor require- 
ments have bv-'On identified. By this reaction gluconic acid enters into 
vjell known metabolic channels. 

The application of th>- is>jtope technique to certain problems of 
mechanism of enzyme— catalyzed r -actions has been very revrarding. The 
three hexos>:s, glucose 6-phpsphat ■■, fructose 6-phosphatc arid mannose 
6-phosphatc are interconvertible in the presenc. of appropriate 
mar.imalian isomcrascs. Fructose 6-phosphat<. , t-}.. . r-tohexose, occupies 
an obligator^^ jjiteraediate position betvjeen ta- tv •.. ".Idohexoses. Of the 
two hydrogen atoms boijnd to carbon-1 of fructose, it h'lS been shown by 
use of hydrogen isotopes that one is related uniquely to the glucose . 
transformation vihile the other one is peculiarly labiliaed in the mannose 
transformation. Similarly, in two reactions of dihydroxyacetonej^TDsphate studied 
one of the two hydrogen atoms on' carboii-3 is labilized by aldolase,, tlit; 
other by phosphotriose isoiueraso. 

The metabolism of the important sugar galacuoCw, has been. the 
subjent of intensive study, 'Virtually all of the reactions whereby, 
galactose enters metabolic pathways have b -en delineated. These involve 
phosphoiylatdisa by r specific kinase, interaction of this product in 
the presence of a transferring enzyme with uridine diphosphate glucose 
to yield ur^.-r-ine diphosphate galactose, folloviod by cpiraerization of • . 
the latter compound about carbon-U to yield again the glucose derivative. 
ThiS' latter cpimeriaation has boen shown to require diphosphopyridino 
nucleotide and is therefore believed to proceed via tv.'o oxidoreductive 
steps. Of particular interest arc studies of the metabolic d..fect in 
the congenital disease, galactosemia. In this condition, an inborn 
intolerance toward galactose, galactose 1-phosphate accornulates in the 
tissues where it acts deleteriously. The ultruriate. injuries include 
cataract, mental deficiency, hepatomegaly and other manifestations. 
Assay for the several enzymes possibly invclvGa has rovcaled the complete 
absence from erythrocytes and from liver of the transferring enzyme. and 
normal abundances of all other components. Since the manifestations of 
the disease are probably preventable by elimination of dietary galactose, 
early diagnosis is important. ' In view of the now established, 'tmolfecular" 
nature of the disease and its attribution to a single enzyme defect, such 
early diagnosis by enzymologic assay becomes feasible. The discovery 
that this and several othnr congenital defects may result from the lack 



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of a si/igle enzync provides a useful "marker" in human am cniiiial 
genetic studicG which are being initiated. Of particular interest are 
atteripts to demonstrate, in the spermatozoa of affoctv-d malc£", evi- 
dences of the enzyme deficiency which is present and aemonstrable in 
somatic cells. 

Whereas diabetes (to be considered subsequently) is the most 
frequent import-^nt disturbance of carbohydrate r:etabolism, others, like 
galactosenii ., do jcour and their study is certainly rewarding, Another 
sue 1 congenital defec-^ is pentosuria, in which disease the sugar 
L-xj'lulose appears in la-, urine. Recent studic-s employing chromato- 
graphic techniques have Si own that xylulose, contrary to earlier reports, 
is a normal though minor urinary constituent ana i lueed is but one of 
several sugars found in small amount in nor,..al huinan urine. 

The metabolism of polysaccharides jx rdgh molecular weight has 
also been the subject of study. The detailed nature of the turnover 
of glycogen in muscle and in liver of intact aauiials has been clarified, 
Tt has previously been reported that glycogen is metaboiically inhouio- 
genoous at an intramolecular level. More recent studies reveal that 
this iiihOi.ogeneity is also extended to the intermolecular level. In 
liver it is the jmallest glycogen molecules, in muscle it is the largest 
molecules that are metabolicaLly most active. The characteristics of 
structure of glycogen and of enzyme activity in these two tissues have 
been studied in vitro in an attempt to learn the basis for the in vivo 
differences observed. Another polysaccharide, chondroitin sulfuric acid, 
has also been studied. This material conjugated to protein, a major 
coaponent of the extracellular matrix of cartilage, hence of interest 
in arthritis research, can be extensively degraded in vivo by some as 
yet unide^xtiiice constituent of cinide papaya latex. A product resembl- 
ing chondroitiii sulfate histologically can be observed to enter the 
blood str .rm after papain injection v/hile the several cartilages of the 
body are losin^^ rididity and staining capacity. The nature of the enzyme 
in crude papain responsible for this effect and the identification of 
the producto of its reaction are subjects of present investigation, 

• The microbial fermentations of oxaloacetic acid have been 
studied. An enzyme, oxaloacetic hydrolase has been secured from an 
enrichment culture of i^spergillus niger . Oxalic and acetic acid vrere 
identified as reaction products. The further decomposition of oxalic 
acid may be effected by a second new enzynie, oxalic decarboxylase, which 
has been extracted and extensively purified from Collyvia vcltipes . 

Amino Acids, AjTdne s ^ P urines and Nu cl eic Acids - A coordinatec and fre- 

<iucutly collaborative 
advance may oe recorded 
in the chemistry and biochemistry of the nitrogenous constituents of 
living cells. Especial interest attaches to hydroxy-derivatives. of araino 
acids and amines, 5 -hydroxy-DL- tryptophan has for the first time been 
resolved and all the pharmacologic activity has been found to reside in 



» r; _. 

'r.h'?. L-antipodo, In.- c^iae material was a potent inhibitor of insulin? se 
\t 'T5.0 no more effective in this regard than \<is.s the simpler p~h., 'ro:!Qr- 
iiidole. Other novel indole derivatives which h?ve been identifiea and 
synthesized dre a product derived biologically froin lysergic diethyl 
aiidde and ■another mr.tcrial isolated from Glavjceps .purpurea . The appli- 
cation of color reactions of indoles to his;tologic':l preparations has 
yielded some important information.' Of particular interest was the 
observi^tion of an indole type of material' in the a-cells of the islets 
of LangorhanSj which generate glucagon^ a tryptophan-containing protein. 
No sirailariy staining natcrial could be demonstrated in the p-cells 
xjhxch generate insulin, a protein devoid of tryptophan. Definitive con- 
figurational assigm.ionts have been made to a number of hydroxyamino 
acids. These include the naturally occurring homolog of hydroxyproline, 
5-hydrcxjfpipocolic acid and 6-hydroxylysine, Hydro>:yproline and hydroxy- 
lysine, it will be recalled are found in nature chiefly in collagen, the 
characteristic extracellular protein of connective tissue, A large 
nuraber of r.orivativGS cf hydroxypToline h'fve been synthesized and 
certain o? these, x^^hen tested in a hydroxs^'proline-producing carrot prepa- 
ration, wore found to exhibit striking ^.ntimetabolic activity. Various 



> 



modifications c.nd derivatives of th'- tyrosine and thyroxin molecules 
have also been prepared. Nitro-icrivatives of the imnatural meta - 
tyrosinc have been synthesized and characterized and a series of m- 
diphenyl ethers related to thyroxin have also been prepared. These c:'iri- 
pounds should prove useful in future studies of thyroid function. 

The polysmines, spermine and spermidine, occur abiuidantly and are 
widely distributed in nature. Although found in significant arijunts 
in mamKialian tissues, these substances are .surprisingly to:d.c. Spermine, 
injected into a. renal artersr, produces cl cortical tubular degeneration, 
folloxiied by arterial intimal proliferation and renal atrupny. In the 
hope that knowledge of their metabolism would clarify the:lr toxicology, 
isotopicaily labeled sjjermine and spennidine have been prepared and 
their ia3tabolic fates studied. Liver homogenates transfprm spermine 
into spermidine, and putrescinc. Doubly labeled (G , Jr^) putrescine 
was incorporated biologically into spermine and spermidine, the tlriree- 
carbon side chains arising chiefly from methionine. Perhaps due to its 
basic nature, spermine antagonized the anticoagulant aiction of heparin, 
which is of course strongly acidic. 

Clinical distiirbances in amino acid metabolism have been found 
in relation to the aromatic amino acids phenylalanine, tyrosine and 
trj^tophan more frequently than with other amino acids. For example, 
a specific disturbance in tryptophan metabolism in tiss^'ie.-; of the 
diabetic ajiimal xv-ili be discussed subsoquentlj'-. For tlilo reason among 
otiaers, considcrablf; intp^rost attaches to tjna detailed knowledge of the 
rr-; tabolic pathways of these amino acids. The discovery of a new class 
of enzyraes, "oxygenases", -which catalyze the aerobic oxidation of various 
cyclic amino acid derivatives is of importance . Studies with 0^^ revealed 
that the oxygen incorporated in the hydroyl gi-oup, in uach case, arises 



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^S;>ati. hydroxylase and iMdazoloao^.tic ^'^^ oxidas. Thcs.^z^s 
,-^ r-»^^H -rl-vrtivlv bv ir.lcroorgarasms and cr^ of c&p^»ia-i. cortcoiri 
S^L^rut; noUv;;.^, by r.an3 L yot unknown, nolccul^^ oxygon. Rat 
UvcrrdSo^i-ia contain an .nzymc, n.^ soUilizod, which hyaroxy- 
iTlZ SnSoJSn. in the 3-Position. 'a pyridox-^a-rcquirir^ trar^anonas. 
h^s b. CO purified frori N-uro::porn v-iich trancfon.^ kynureninc ...id 
>:i;ydro;;j^k^.urcrano ii^tri^ufcHIr and xanthurenic acids respectively. 

Tho translornation of aop'.rtic acid into homoserine occurring in 
yoost tos beun studied in detail and her been dissected into vhxec 
successive sU-ps: 

Aspartic acid » p-cspartyl phosphrt^ — ^ aspcxtis-B-seird^dohyde 

^ hOTiOcerino 

The biolot'ical ndgrations of r^cti^yl niorcaptaii have been ojqjlured 
und -^ nui.ibcr of new conip-oujids identiiied. Uoacting ^.ith j-phosphogxyceric 
acid it yields a thiomcthyl oster which is enzyraatin-Oly roauccd to a ^ 
glycorrl thionetliyl ether. Reacting with serine, methyl nercaptan J'l^l-'^ 
S-ri.thyl cystciiie. Ai.iong other products forned in yeast is one tentatively 
identified as L,L-i3-methyl lanthionine. 

Histidine, and its dccarboxj^lation product, hista-minc, continue 
to be subjects of biochordcal stud> . It had i-reyiou^ly been shcvm tnat 
fonr.ijninoglutr..T,ir acid -iris-^s directly frcn nistidine. The next lowor 
honolog f orraijTiiiioaspartic acid has now been ia^ntif led ao a product of 
histamine netabolisn vi-. the steps: 

Histar.inc > irddazoleacetic acid ^ formir.dnoa spar tic acid 

V (in x^sGudomonas ) formyl-ispartic acid 

Tho further clsorvation that fomirdnoglycine is a normal bacterial product 
of purine c-tabolisn extends to three tno list of amino acids known to 
occur iii living cells r.s th:.ir foriuimino derivatives. It therefore becomes 
of pressing iiitcrest to learn of the biological fate of the fomiridno 
group. Fortunately scientists working coll^loratively in several labora- 
tories of h'LJ€) have been able to supply i.trjiy of the needed answers. 
These studies have been carried out with both the glycine and the 
glutardc acid derivatives and eomplete p-jall^aiisin has th'is far buen 
encountered. The lomi:iino group Jias been shown to rdgrate intact to 
tetrahi'drofolic acid, '. r.er.V.er of the vit.airdn E group. Here it is 
decomposed F.t^uwi •'=''., yi'-^ding si if evasively aitimonia and formic acid. 



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over the Sollowlng steps; 

1, Forrairiinogiycino + tetrahydrof olic acid j-, N -fonaiiuinotctra- 

hydrof olic acid + glycine 

2, 5-foi'niii!iiriotctrahydrofoiio odd — ►. 5, lO-mcthonyl tetrahydrof olic 

acid + annaonic: . 

3, 5, 1'^'-nitjthonyl tetrahydrof olic acid — > N -formyl tetrahydrof olic acid, 

h. N '"'-formyl tetrahydrof olic acid + AJP + P^ — » '.'■■' + formic acid + 

tetrahydrof olic acid. 

Attention should bo directed to the previously_^unk'nown intramolticulr-r 
transfer of a substituent on folic acid froiri Ni' to 11^'^, Of ' particular 
interest is. the novel !' substrate phosphorylation" occurring in the hth 
step. This is. also revealed when the sum of the preceding four reactions 
is considcrodj ■" 

Formirainoglycinc + AEP + Pjl — 3> ATP + glycine + forirdc acid+ar,u -.'da 

By virtue of this phosphorylation additional energy for useful work .•.:■• 
made available to the cell from the catatolism of histidine, histamine, 
xantnine and possibly other precursors. 

The pathway of histidine biosynthesis appears to be notably differ- 
ent from that of histidine catabolism. Five steps, coi-nmencing with imida- 
zoleglycorol phosphate, have been identified, to wit: 

Imidazoleglycerol phosphate — > imidazoloacetol phospnate — => histi- 
dinol phosphate hi.'^tldinol — f histidinal — > histidine 

Tl'ie individual enzjirios catalLyzing these steps and their cofactor require- 
ments are cui'-rently being studied. 

Probably the most important advance in the understanding of 
nucleic acid i'lotabolism in the past year has been the development of in- 
fomiation about' polynucleotide phosphorylase. This bacterial onzyine 
catalyzes tixc reversible reaction; 

Nucleoside 5 '-diphosphate ^^ polynucleotide + P^ 

In trie development of this information NIAJ'tD scientists have ,' 
collaborated closely irTith scientists at New Yoi'k University. The new 
synthetic polynucleotides made available by this reaction have been 
tested as substrates for known phosphodiesterases and. nucleases. Pan- 
creatic ribonuclease has .been intonsivelj- investigated in regard to its 
structural substrate requirements. V/ith isotojdc tracers and lytic 
cnzimes of known specificity it has been 'possible to. learn a great deal 
about, the mechanism of the "nucleotide phosphorylase reaction. The first 
evidence that this enaynie may act iri a dogradative sense on natixral . 
nucleic acids was secured by NI/iKD scientists. Likewise the first 



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evidoncvj for tho existence of a mnjTpnalir.n nucioytidu phosphorj-lase w£S 
secured in this let oratory. 

Tho avcilr.bility of varicrus lytic cnzyr.3s of known specificitius 
permits the hope that systcifiatic sequence studios for purines and pj^Ti- 
midines in nucleic acid chains may be achieved. Analogous information 
a!, out amino acid sequences in proteins is of course beccning increas- 
ingly available. Already the first step, the characterization of end- 
groups of polynucleotides, has been achieved by NliJffl investif^ators 
wno now have metiiods to aistingtiish between "$' -ended" and "3 '-ended" 
polynucleotides. 

Nutrition r.nd Vitair.ins - Nutritional studies in recent y^ars have been 

concerned largely with the roles of micrcnutri- 
ents. Tl-ie organic micrcnutrients, vitamins, 
have, in most cases, been found to contribute tj tho synthesis of 
specific cofactcrs required in the operation of certain onzyine-cat.alyzed 
reactions. T!ie n.':ture jf this participation, the bicsynthosis of the 
vitanins thc:mselv«js, the reasons for impeded biosynthesis in those 
species where dietary indispensibility is nanifoijt, the mechanisms of 
degradatj :n of vitajnins, have all been areas of active rcst-arch. 

The folic acid molecule occurs in several modifications in 
nature. Thus in icami.ialian liver there have been found tvic distirict pre- 
cursors of folic acid, prefolic A and prefolic L, which, though inactive 
in microbial assays, are enzyma.tically transformed into mr.terials which 
exhibit folic acid activity. Incubation of folic acid or of citrovorum 
factor, its tetrcjiydrcformyl derivative, with liver extracts results 
in loss of grovrth-promoting activity. In each case an aerobic rupture 
of the bond between the pterin and p-ai.iinobenzoic acid appears to occur. 
From bacteria enzymes hrve been isolated which cleave N-aryl (or N-acyl)- 
L-glutai:iic acid derivatives, folic acid among others. j>.ncther bacterial 
enzyi'ic has been found which hydrolytically removes tb:: 2-amino group 
from the pterin of folic acid. The role of folic acid derivatives in 
metabolism of formimino derivatives of amino acids has been discussed 
elsewhere in this report. An important lead v;as supplied by the 
identification of f ormijriinoglutamic acid, a histidine metabolite, in 
tho urine of folic acid deficient animals. It hrs now been shown that 
histidine, as v/ell as tryptophan, threonine and certain nucleic acid 
derivatives partially counteract the effects of folic acid deficiency. 

Several steps in the biosynthesis of nicotinic acid from 
tryptophan have been further investigated. The oxidations of tryptophan 
and Of 3-hydroxyanthranilic acid are catalyzed by enzymes of the newly 
defined "oxygenase" group. The enzyme picolinic carboxylase and the 
muchanisra of its reaction have been studied. The fact that its activity 
in liver is greatly incruased in diabetes was discovered and explored 
(cf. Diabetes, below). Niacin deficiency in dogs leads to black-tongue. 



- 9 ~ '* 

a disease studied by Goldberger, .The diets he employed were rich in 
corn. Eased upon human nutrition studies, experiments have been under- 
taken to ascertain whether treatment of corn with alkali renders it a 
better source of niacin. Preliminary results indicate this to be the 
case. 

When maintained on vitamin E-free torvLla yeast diets, rats 
develop a fatal hepatic necrosis which may be prevented by an as yet 
unidentified nutritional accessory factor, termed Factor 3. Purifica- 
tion from kidney powder of Factor 3 has been under way for a few years 
at NL'J'ID, and progress is recorded. Two materials of differing 
properties, both exhibiting Factor 3 activity, have been separated and 
considerable concentration of one of these, a-Factor 3, has been 
effected by chromatographic and partition procedures. This product is 
strongly anionic, water-soluble, not extractable with phenol. Factor 3 
deficiency has now been shown to lead to necrotic lesions not only in 
liver, but also in kidneys and skeletal and cardiac muscle, together 
with pancreatic atrophy. The role of vitamin E in the prevention of 
the hepatic necrosis that responds to Factor 3 has also been studied. 
Fourteen synthetic antioxidants have been compared with tocopherol 
both in vivo and in vitro and certain of these, notably N,N'-diphenyl- 
phenylene diamine vjere highly effective as prophylactic agents. Because 
of the finding that the synthetic antioxidants were active in the in 
vitro test, where vitamin E was ineffective, it has been suggested tnr.t 
it is some metabolic product of vitamin E which is the effective agent 
in vivo . This suspicion has been confirmed by the finding that two 
recently isolated metabolic derivatives of vitamin E were effective 
in vitro. 

Whereas most laboratory nutrition studies have of recent years 
been conducted upon rats, it is obviously of interest to secure compara- 
tive data in a variety of other species. Such studies have been under- 
taken in the chick, mouse and guinea pig. Among the recorded findings 
froiri this study is the detection of an xmidentified thermolabile growth 
factor, the absence of a thioctic acid requirement, snd the probable 
absence of a requirement for vitamin E if no dietary fat is allowed, all 
in the chick. An exudative diathesis could be produced in the chick 
when vitamin E and Factor 3 were both withheld. In this species a 
severe vitamin B12 deficiency has been produced in four weeks on a 
purified diet. ' Various analogs of this vitamin have been assayed in the 
deficient chick. The ^-hydroxy analog was about one tenth as potent as 
vitamin B]_2 itself x\ihile the nitrato-, sulfate-, chloro- and acetate* 
cobalamins were fully active. In the chick a relative choline deficioxicy 
could be produced by administration of a competitive inhibitor, 2-amino- 
2-methylpropanol-l. The absence of diet fat was necessary to produce 
fatty liver in choline deficiency in this species. Folic acid deficiency 
can be produced in young mice by dietary means alone. Such mice have 
been found to fail to support the reproduction of lymphocytic chorio- 
meningitis- virua. The virus will reproduce satisfactorily if folic acid 



- 10 - 

is added to the diet. Growth data in the guinea pig suggest that the 
conversion ol tryptophan into niacin occurs in this species, as in nany 
otr:crs. k vitamin B12 rcquirent-nt for optimal growth is present in this 
species, l.hcror.s inositol is not needed on a 30^ casein diet, on purified 
amino acids a definite I'cquiremcnt for inositol is shown by the guinea pig. 

Thf; rapid destruction of vitamin A injected intravenously into 
tJic rat caii be now attributed to an activity jn the blood, especially in 
the stroma of the erythrocytes. This therraolabile, presumably enzymatic 
activity is not inhibited by azide or by cyanide. Chick blood, in contrast 
to rat blood, was almost ineffective in destroying vitamin 1^ in vitro . 

The study of the production of obesity in rats by dietary moans 
has been continued. Such obesity is secured on diets containing hO% of 
fat supplemented with methionine and aureomycin. Pathological studies 
have revealed an osteoart:j?itio type of joint lesion. Organ weights were 
normal except for the adrenals which weigh approximately S^% more in obese 
than in lean animals. Histological changes were also reported both in 
the adrenal and thyroid glands. Blood pressures in obese animals wore 
normal, height r:.>duction studies on obese animals reversed lowest mortali- 
ties when tlic initi.al high-fat diet was offered in lirnitcd amount. 

Many nutritional studies depend upon ultramicroanalytical procedures 
for nutrients or their derivatives and these often are conducted by iracro- 
bial methods. i\ laboratory is operated to carry out such services for 
other investigators, and a number of useful collaborati jns have been under- 
takv-n in this arer.. i'or exaruple, in studies of the effects of ethylene 
oxide upon the diet (see below), in studies of the effect of diabetes upon 
the tolerance to various B vitamins, microbiolot^ical assays were essential. 
ijnong tlio improved methods introduced is the analysis of 5- and 10-fornryl 
derivatives of tetrahydrof olic acid in millimicrogram amounts when in the 
presence of each other, 

Endocrincs and Endocrine Diseases - The general problems of endocrinology 

include isolation identification, puri- 
fication, proof of structiire and ultimate 
synthesis of the products of endocrine glands; development of analytical 
methods for separation and quantitation of such products; the analysis of 
the nature and moJe of action of the hormone upon the target organ; the 
metabolism of the hormone; and the unravelling of the complex interactions 
of the several endocrine glands and associated nerve structures upon each 
other. It is upon the ansv;ers to these problems that understandin?,, 
diagnosis, treatment and prophylaxis of the endocrine diseases should rest. 
In all these areas HLIID scientists have great interest. 

Diabetes is the most frequent as well as the most important 
recognized endocrine disease. Because little is known of the ultimate 
motabolisiii of insulin, because the hypoinsulinism in this state may be 



- 11 - 

rolativG or absolute, beca-uss ins-ulLa antagonists (see below) are Icnown 
to occtir in tho bloodsircm, the development of a method for insulin 
asscT sensitive m the range of physiological concentrations is a problem 
of immodiauc iiri]..ortr.nce. The development of such a method, capable- cf 
detecting as little as 2x10"-^^ moles of insulin is under study. The 
inethod depends upon hypoglycemic effect of insulin upon th.e very sensitive 
diabetic hypophyscctomizod mouse. Even vrith this remarkable sensitivity, 
virtually no insulin was detectable in the blood of mice, Pancreatcctoir/ 
leads tD diabetes of varying severity in various aniraal species. Total 
YTcreatectcmy i^^s not been possible in the past in common laboratory 
r-.. :^nts becaxise of the diffusely scattered arrangement of the gland, A 
vor.- ;:evore diabetes has now been produced in the rat by virtually total 
cxtxrpation of the pancreas. Subsequent hypophysectomy in such diabetic 
rats results in a marked decrease in insulin requirement and an improved 
survival on fasting. The report of others that insulin had specific 
growth-hormone-like functions has been scrutinized. It x-jas found, under 
carefully controlled c;:nditions, that the grovrth-stimulant action cf insTolin 
in the hypophjrsoctciaized rat was entirely attributable to the increase 
in food intrica in response to insulin. 

The earliest antagonist of insulin to attract attention arises in • 
the hypophysis (Houssay) and is now believed to be identical with or 
related to growth hormone. Other agents which have been shovm' to antagonize,- 
directly or indirectly ccrtaii; manifestatioiS of insulin include the 
glucocorticoids, glucagon, insulinase and .■■pccific antibodies to heterolo- 
gous insulin, i.n antagonist identical vjith none of those has been found ■ 
in high frequency in the serui^ of patients in diabetic kctosis. This 
agent, in the a-globuliii fraction cf plasma proteins, disappears in a few ' ■ 
hours after therapy against ketosis is established. Its chemical and 
physical properties, as well as its mode of action are being determined, ' 
In order to evaluate its rclo in the causation cf diabetes, it is soon to 
be tested agaii-ist human insvilin which is being prepared by NL-xMD staff. 
The -Bell-known Increase in tolerance toward insulin in the diabetic wiien 
in keto-acidosis is in large measure explained by the present findings. 

The failure of dietary tryptophan to provide niacin in the 
diabetic rat has previously been reported, iittemjjts to reproduce this 
defect in diabetic dogs or humans have not been successful thus far. 
The defect in the liver of tho diabetic rat has been studied in detail. 
The most striking related enaymological change is an increase, in diabetic 
liver, in the activity of picolinic carboxylas-^, Tnis change in diabetes 
develops gradually after removal of the pancreas, and requires, for its 
development, the presence of adrenal glands. 

Insulin is known to act primariJLy iia i"acilitation of glucose 
utilization by' certain tissues, especially skeletal muscle. Two new 
factors are under study which appear to influence this phenomenon. The 
first of those vias encountered in the course of study of Factor 3 
defj.ciont rats \jhQn it was noted that these animals had an irapaii^ed 



- 12 - 

glucoso toler^rio^. -hv. Glucrsc Tolorsnce Va<itoT (OTF), i.s it has boon 
nrjiod, is not identical with Fp.ctor 3. It is present in kidney and in 
brewer's yecst. It is njt identical vdth any known nutritnt. V^xious 
diets have bucn constructed v/hich arc not necrcgenic yet vxhich lack GIF, 
Its isolation is being pursued. A second factor was encountered when it 
was noted th.-^t peripheral removal of glucose was lowered in the absence 
of the liver. Suitable liver extracts, when adninistcrcd^ Were found 
partially to restore this rate toward normal, Tnis mrterial appears to 
'.'C related to GTF since the extracts of livers of animals whose diets 
were aeficicut in GTF had .hly one quarter of normal activity in restor- 
ing the glucose extraction rate of the hepatectomizcd rat. Gross chcir.ical 
and physicrl properties of this liver su'. stance have '. een detcri.mied end. 
its further st^ dy is being pursued, 

A numVcr of studies relate to the horrioues f ^hr pituitary 
gland. A transpiantallo mouse pituitary tumor which (••oncr-'tes large 
amounts of adronoccrticotrophic hormone (^.CTh) has bceii ir.ui. taincd and 
studied. The AGTH derived from it btsboafi found to contain ,.!l .ophoro 
stimulating hormone (!1SH) activity -ilso. Furthermore, tr.j-iv. ;nt of this 
ACTH v.'ith alkali increased its MSH activity. This findjn,;;, c .jnsidered 
together vjith others, suggests that MSH may l-o a product of 'pcrtlaL 
degradation of ACTH. By application of new chroifvatogrr.pnic methods, 
'thyroid stimulating hormone (TSH) has loon purified throe hundred fold 
from '.eef pituitary glands. By the application of this novel method of 
isolation to the i.iouse pituitary tumors, TSH has r. een identified in this 
material. Physical and chemical studies designed to yield structural 
inform-ation have been undertaken on TSH preparations. The newly developed 
assay method for TSH lino I een applied to human Mood samples, both normal 
and diseased. V.'hercas the human blood levels were either very low or uii- 
detectal.le, high concentrations of TSH vicre found in the Hood of certain 
mice bearing pituitary- tuiaors. 

There has long been a suspicion that ■-he pituitary gland, several 
of the functions of which are to activate other endocrine organs, is itself 
under the directed iiifluence of the hypothalfjriic nuclei. The relations of 
neural and humoral influences, particularly in regard to the question of 
the control of pituitary functiens by nervous impulses, has been the subject 
of a sizable and continuing project. The general procedure has been to 
divide the brain-stem of dogs at predetermined levels and to study, directly 
or indirectly, residual pituitary function, -.s an exar;ple of the t^-pes of 
findings being recorded, it has been noted that dogs, months after 
midbrain transection, fail to respond to surgical and other stresses, as 
normal dogs do, with an outpouring of 'adrenocortical steroids. That the 
animals' adrenal cortices are still responsive is readily demonstrallc 
ly the injection of exogenous i^CTH, which is followed by the expected 
rise in steroid excretion. V/hereas the midbrain-transccted dog appears 
unable to respond to stress by endogenous i.GTH release, in some cases ho • 
apparently generates normal amounts of thyrotrcpliic hormone, aa determined 
by laeasureiacnts of depletion of thyroidal I-^-^, Various ciianges in 



- 13 - 

circulatory and renal functions have 'b^^cn noted to fjllow iriid'urain 
transection, evidenced by reduction of glomerular filtration, renal 
plasraa flow and cardiac output, A marked increase in hemodynamic re- 
sistance in the kidney and in the periphery suggest that a generalized 
arteriolar spasm follows midbrain transection. The mechanism of an 
anomalous creatinuria which follows such surgery is being explored. 

Steroids, I'iiich include hormones of the adrenal cortex, testis 
and ovary,, as well as many other biologically important compounds, have 
lecn the subject of several studies. The application of modern methods 
of separation has permitted analyses of the coiaplex mixtures of steroids 
in urine in health and disease. Among thj abnormal states studied are 
pregnancy. Gushing' s syndrome, Addison's disease, congestive heart fail- 
ure, hepatic cirrhosis j also patients under treatment with ACTH, adrenc- 
corticoids and sj/aithetic analogs. The interconvertibility of M^tacortin 
and Metacortnlone in man was thus demonstrated. Siiailar urinary analyses 
have been cenducted upon dogs subjected to midbrain transection. The 
fecal excretion of steroids has been explored and a 17-ket ;st.er jid nas 
for the first time been demonstrated in this material, Strvctural 
chemical studies on various steroids have revealed: A possible novel 
method of oxidation at carbon-11 of dehydroergosterolj the jccurrenco 
in aniraal bile of sapogenin-like compounds, of a type formerly known 
only in plants j the previously unknown isomerism about carbon-22 in the 
sapogenin side-chainj and' final clarification of the structures of 
tomatidine and solasodine. Earlier mention was made of the natural 
sweetenin; agent, steviosido. In addition to elucidation of its car- 
bohydrate' moiety (q.v.), its aglycone, a steroid relative has -Iso been 
^■■tudied and modifications of its structure have been proposed. Synthesis 
01 ainide^ of colchicine, compounds of therapeutic interest, has been 
studied. 

/jialgcsic Drugs - The quest for analgesic drugs vrl'.ich might be less prone 

to cause luidesirable side effects than drugs presently 
available has been pursued :;t an organic chemical and 
at a pharmacological level. Screening and assay meth^ods for new agents 
have in scrae cases included clinical studies using a double-blind 
technique on patients supplied largely by NCI, L new class Of com- 
pounds, in which the N-methyl of morphine was replaced by a 2-phenylethyl 
group has been described. . VTien certain morphine analogs were thus 
altered, an increase of 50- to 100-fold in activity was found -on 
animal test. High activity and relatively low toxicity' i;erQ also found 
for 2'-h5'-droxy-2,5,9-trimethyl-6:7-benzmcrph3n, a compound newly syn- 
.thcsized by NIj-'.MD scientists. 5-(iii-hydroxyphenyl)-2-methylmorphan, 
■while as active as morphine, has less addiction potential, at least in 
monkeys, 

A large number of derivatives of morphine, analogs have been 
prepared and new synthetic routes have been devised. These have included 
enlargement of the N-containing ring of a codeine derivative, preparation 



lU - 



p 



^a^Utu^cd ;'°phS:nlono3 .^d phenanthrones and of v.-u-xous pxperxdmo 
derivatives, tLse representing portions of Uie prototype '"orphine 
riloc^c New derivatives of thebaine, a .lorphinc congener have been 
rrop'Sd* oihor co.:-.pounds t.hich have been prepared and stuaied mclud. 
ccrtal;; ^fiuoranSs and some hitherto unV:nown ison.crs ol cocaine. 

R.. psr.tcrv. Circulatory and Hen.a_to, ,oieU^PhZ£i£^ " f,:./,::^^^'°c'spS-c- 

tion and rcl;:tcd 

functions have been studied in response to the stress or ^^^^"^^^f ^^^J^" 
rlnp Devices have been designed to perr.dt variations m external rcspira- 
to^'rcsirtanco and in dead space. Studi.c witn th... and other instruments 
using trained underv.ater swix.onors as subjects, have revealed low work 
efficiencies The nvaxUriuin attainable pulmonary minute volur.o and oxygen 
consumption were lower uider these circu:.istanccs than ^ or work in air 
Training of swimmers resulted in either conscious disregard of the CO2 
stir.ulus or a lowered sensitivity of the reopir.-.torj center ^-o CO2. 
Ilso a conscious post-inspiratory ^ause, in th. trained swirmer resulted 
in a lon^r CCo buildup time-. The maximur,: breathing capacity Cl;uJC) w... 
more sensitive toward increased inspiratory than expiratory resistance. 
The respiratory rate for MBC was lowered bv inoreasme this resiot..nce 
but the tidal volume was invariant at the highest ICO for .;aca voluo ol 
resistaiice. 

The effects of low pressures :.t ol::.-^^ted high altitudes hav- b.v a 
further studied. A high-fat diet has beer, shown to increase the mortali. ■ 
of rats followiny exposure to 30,oOO feet sir.ulated high altitude, regard- 
less of whether or not the animals are ob.sc. Cardiovalvular thickening 
CJid the appearance of sterile valvular vegetations are noro strikin- m 
ob.se animrls. Contrary to expectations, surgically induced aortic in- 
sufficiency did not m:'.rkedly interfere with tolerance of the dog toexpos- 
ure to 30,CO0 feet sL-aulated high altitude. At higher altitudes, thu 
stress did increase mortality. 

Insect respiration differs from that of mammals in a number of 
iir.portant regards. Thus it res been shown that no demonstrable oxygen 
debt ensues in bee-moth pupae upon short periods of anoxia. COj release 
by diapausing saturniid moth pupae is discontinuous rather than continu- 
ous and the consequent impo'onding of CO2 docs not interfere with oxygen 
uptake. 

The use of large volumes of buffered saline given orally in lieu of 
parenteral plasma or blood substitutes in the prophylaxis against burn shock 
Sas received complete clinical testing at Lima, Peru. 100 cases vath xOO 
control patients have now been treated and have completely confirmed the 
validity of earlier impressions that this is a very effective mode oi 
therapy Projects related to this study include Kieasurements of hcmodynamxc 



- 15 - 

quantities J t-loctrolytu concentrations m tody fluids and lY-ketosteroid 
excretions in the treated and control groups. Other studies relating 
to shock at 'an animal level showed that promazine and chlorpromazine 
protect "against tourniquet shock, jn. nuraber of other interesting pharma- 
cological effectis of chlorpromazine .have Leen observed, including reduc- 
tion^in body toiiiperature and decrease in si-relling in the injured area. 

Of the patients who survived acute shock in the Lima project, a 
considerable number died in the succeeding three weeks. In virtually 
every case death was attributed to Pseudomonas aeruginosa sepsis. Attempt£= 
to transmit this infection to mice were unsuccessful until and unless 
the animals were either burned or pretroatod with corticosteroids. Now, 
with a ready source of infected laboratory animals, evaluation of modes 
of treatment against this infectious agent is possible and has been under- 
t/^ken. 

Among the plasma substitutes employed today are polyvirjylpyrroli- 
dono (PVF) and dextran. Each of these, injected into a sensitive prepara- 
tion, has bieen shown to cause a drop in blood pressure. The medianisms 
of those actions have been clarified under certain circumstances. When 
dextran is injected into the conscious or etherized mouse, there is^no 
fall in blood pressure but edema occurs. In the barbiturate -treated 
mouse, however, a fall in blood pressure results. FJF injected into the 
dog causes edema and a fall in blood pressure. Either dialysis cf PVP or 
etherization of the dog abolished the hypotension, but edciiia was still 
seen. 

One of the diseases of the blood and blood-forming organs that 
is best linderstood is sickle cell anemia, Tiiis was demonstrated, some 
years ago, to be a "molecular" disease wherein normal hemoglobin is 
replaced by an abnormal hemoglobin "S", Nliu'ID is fortunate in having 
i Bocurf^d t!ie services of one of the scientists responsible for this dis- 
covery, ii number of additional though rarely encountered abnormal 
hemoglobins have been and continue to be detected, with the aid of 
electrophoretic techniques. Of particular interest: in this regard has 
been the resolution of intermediate forms of ferrihemoglobins, oxidation 
products wherein 1, 2 or 3 of the total of four iron atoms are oxidized 
to the trivalcnt state. From the mobility differences between these 
products it has been possible to estimate differences in charge on 
normal and abnormal hemoglobin molecules. 

The plasma of the anemic animal has been shovjn to contain an 
agent which stimulates erythropoiesis. This material, termed "erythropoie- 
tine" remains in the- supernatant solution after alcohol precipitation of 
plasma at pH h to $, i^ctivity is lost after' dialysis at pH h and the 
material has been shown to be thcrmolabilc. Activity is assayed by the 
measurement of incorporation of radioactive iron into erythrocytes of 
irradiated rats. Various tags in addition to iron have been studied and 



•■ . J „ 



-16- 

roinpared. Amonr these ar raCio-chromiujn and di-isopropylfluoro- 
phosph-te-p32. Comparativr studies of the life span of erythrocytes 
have shown that in birds this expectaney is far sh°^ter,xn reptiles, 
far l.n.er, than in ma^.als, this despite the fact that both birds and 
reptiles, in contrast to iru-nBnals, have nucleated red cells. The fact 
that nature red cells have a finite life expectancy and no capacity t. 
replicate makes ter.pting the possibility of segregating the erythrocyte 
population according to age and studying cells of differing ages for the 
chemical or physical bases of aging in this simple system. Two methods 
of segregation, differential centrifugation and differential hemolysis, 
are being explored in this regard, and both anpear to be promising. 
Studies of leucocyte metabolism, especially ir. relation to their reported 
high affinity for insulin, have bprn initiated. 

^'arious Disease P rocesses - ^ variety of disease processes in experiment.! 

anima]:. have, for one reason or another, come 

vnder scrutiny by FIAMD scientists. The 
desirability of securinr counterparts, in small animals, of the various 
forms of arthritis which occur 3ponLaneous> in humans, is obvious. 
The comparative study of different mouse strains has revealed the presence 
of a genetically transmitted lesion veiy cl'sely resembling osteoarthritis. 
This lesion was especially prominent in a congenitally obese strain. 
Py means of diets' mentioned in another section, obesity has been produced 
in rats which then developed osteoarthritis-like joint lesions together 
with adrenal changes. A strain of Streptopacillus has been encountered 
which is virulent in rats. After intravenous inj-jction into yoiuig rats 
an infectious polyarthritis developed. At this stage organisms could be 
recovered from the affectc-d joints, but not from the blood. 

AmonK the viral diseases being studied ie Rous sarcoma. The nature 
of the extension process has been indicated to ue an invasion of the 
virus vrf-iich transforms such normal intramuscular connective tissue as it 
encounters into Rous tumor cells. Newcastle Disease virus has been suc- 
cessfully purified by the antibody-adsorption technique described elsewhere 
in this report. Associcited with the extensive use of monkeys in the 
poliomyelitis (Salk vaccine) project has been th?? necessity of distin- 
guishing poliomytlitis frcri other similar diseases of the monkey central 
nervous system. Several novel diseases of thir e:r->ap have been encoun- 
terpd and tent?tively classified. Another disease Wnich has been studied 
is leprosy. Murine leprosy has been employed as a ec-eening device for 
active antilopral agent and several drugs of promise hnve been encountered. 
Marked susceptibility to bocterial endocarditis after 'nt ravenous in- 
jection of Staphylococcus aureus and Strepto coccus mitis may be 
produced in the do?: by surgical perforation of an aortic 1, aflet. Diffuse 
proliferative rlorrLerulonephritis often develops in the dors receiving 
the latter organism. 

Physical T-'.-surements, Wacromol^cul£^ - There has long bern an interest 

— '^ and ?n activity at NI^.MD in the 

effects of radiant energy upon 
bioloric?l systems of all sorts. The growth of such r.n Activity is heavily 
dependent upon the development of novel measuring instruments and the 
improvement of existing equipment and this Institute hai. a record of 
contributions in this area. Notable improvements achieved in the past 



-17- 
year have included linearization of the wave-length coordinate of the 
Perkin-Elmer spectroscope-'Hodel 13 and conversion of the transmission 
coordinate into the more useful optical density scale. By the applica- 
tior of servo methods to square-wave platinum tlectrode polarograpl'iy, 
an instrttmert has been perfected for the high speed recording of the rate 
of change in oxygen concentration in solutions. Defects in the conven- 
tional sphere techniques for integrating scattered light have led to the 
exploration of other means of accomplishing the same end. 

These improved methods have been applied to various problems of 
biological interest. Differences which hod previously defied detection, 
between the infra-red spectr-^! of lumisterol and erogsterol, have now 
been detected. The infra-red spectra of ATP, ADP and AMP have been com- 
pared, and whereas the first two resemble each other, the last compound 
is distinctly different. This difference is attributable to the presence 
of the pyrophosphjate "energy-rich" bond in ATP and ADP, which is lacking 
in AMP. This attribution is based upon a detailed study of many pyro- 
phosphates in the infra-red spectrometer. In view of the striking 
biochemical importance of these compounds, this new correlation of energy 
content and optical properties should prove of great usefulness. As 
model compounds for theoretical study of infra-red absorption the 12- 
hetoropoly acid salts have served v/ell. Tentative assigraiients of absorption 
bands to specific atom groups in these molecules have been made. 

New ideas, better instruments and improved techniques have all 
assisted in advancing the study of photosynthesis. These studies have 
employed unicellular chlorophyll-containing organisms, chloroplasts de- 
rived from these and lam.ellate particles of chloroplasts, discovered by 
NIAMD scientists, which may be the ultimate photosynthesizing particles. 
Simultaneous measurements have been made of quantity of light abserbed 
at different wave-lengths, changes in oxygen concentration, and fluores- 
cent emission, under a wide variety of circumstances. The oxygen 
generation in response to light has been dissected into two components, 
an initial "burst" which appears to be independent of the presence of 
C02j and a steady state production which requires CO2. An immediate 
"gulp" of oxygen, when illumination is rem.oved, may represent a reversal 
of the burst just noted. Confirmiation of these suggestions awaits the 
completion of equipment to permit continuous recording of CO2 concentra- 
tion, currently under development. Since photosynthetic fixation of 
CO2 is today recognized as merely a specific example of the more general 
fixation of CO2 in which many animal cells also participate, and since 
energy relations, in photosynthesis, are more accessible to direct 
m.easurement, this, study provides an excellent means of attacking a prob- 
lem of very general biological importance. 

A quite different application of spectroscopy to problems of 
biological interest is in the m?tter of hydrogen bonding. It is gen- 
erally conceded today that many macromolecules such as proteins, 
ribonucleic acids, etc., are held together and maintained in their 
typical configurations by hydrogen bonds, wherein a proton in covalent 
linkage to one atom simultaneously occupies an unshared pair of electrons 
of another atom. The helical configuration of many proteins depends up«n 
the presence of such hydrogen bridges and the denaturation of proteins 
is a consequence of rupture of such hydrogen bonds. In order to gain 
more insight into hydrogen bonds, and iheij:* 'thermodynamic properties, 



-18- 

advantage has been taken of the discovery by NI/MD scientists of the 
facts 3) that simple molecules, such as ethanol, exhibit hydrogen 
bonding, and b) that bonded hydrogen atoms makp characteristic con- 
tributions to the infra-red spectrum. The frequencies and intensities 
of absorption in thjs spectrum have been correlated with equilibrium 
constants and heats of formation of hydropen bonds. The application of 
nuclear magnetic resonance spectroscopy to this problem has been ex- 
plored and found to be a useful tool. Hope- fully it will be added to 
the pn^sent complement of available measurements. 

Yet another novel application of spectroscopy hos been in the 
deciphering of certain st(?reochemical problaiis in enzyme chemistry. 
Succinic acid-2,3-ciidouterium has been pnipared by two chemical methods, 
involving the platinum-catalyzed deuteriumaticn of fumaric acid on the 
one hand and maleic acid on the other. The two products, identical by 
chemical test, exhibit quite different infra-red absorption spectra and 
have been assigned the pi^fixes racemic ar>d meso- accordingly. Interest- 
ingly, the product formed by soluble succinic dehydrogenase from fumaric 
acid in DjO resembles the meso- variety, indicating thr.t the attack by 
this enzyme is trans with respect to the double bond to be reduced. 

The selective migration of ions across membranes has been studied 
both in sjmthetic and in biological systems. The use of permselective 
synthetic membranes hr.s been ext.ended. The anomalous behavior of cer- 
tain anion-selective m'-mbranes has suggested a possible mechanism by 
which rapid exch;mee between intra- and extracellular ions mipht occur 
despite high electrical resistance between these two compartments, 
Now membranes of special properties have been prepared using different 
polyelectrolj-les, A theory has been developed to account for ion 
migration against concentration gradients. Some predictions based upon 
this thoorj'' have already been bom out bj'' experiments and vjill be further 
pursued. Theory and experiments have also been developed to account for 
the differential rates of exchange of two different species of ions 
across a nivr'n permselective membrane. 

At n biological level the distribution of sodium and potassium 
across the nei^e cell wall has been a natter of continued study. It 
has been possible, by use of drugs and metabolic inhibitors, to segregate 
a component of potassium flux vjhich is dependent upon metabolic activity, 
active transport, from a second component of passive transport. Since 
cocaine affects the latter, rather than the former, its influence upon 
potassium influx is referred to change in membrane permeability. 
Elevation of the potassium concentration in the external fluid enhanced 
both potassium influx and outflux, the former moi^e than the latter. 

The fine structure of matter may be attacked by physical and 
chemical means. The use of the electron micit>scope is an important 
physical* means to this end. The inci-^asing resolution of such instru- 
ments has permitted the detection of molecular order in proteins of 
molecular w;eight of about 5^^,000, Direct photography of organic compounds 
have revealed spacings as low as 11 A in crj'stals of a molecular weight 
of about 500. This achievement ir-pr^sents a great increase in the power 
of this method. Viruses of- herpes simplex and of vaccinia have been 



-w- 

studied in tissues and essential steps in their growth have been :" ' 
deciphered, ' The X-ray microscope, a relatively unexploited device, 
has developed in usefulness. The rigorous requirement of electron ■ 
microscopy for ijltrathin sections does not obtain here. By appropriate 
selection of X-ray targets to yield soft J.-rays, conventional (5^ micra'^ 
unstained histological sections can be satisfactorily photographed. 

■ Chemical studies of fine structure have relied in part on such 
metjriods as ultra contrifugation, turbidimetry, osmosietTy, electrophoresis, 
diffusion, etc. In addition, methods of chemi.cal analysis and enzyme 
chemj^tiy have been invoked as needed. Comparative analyses of the 
muscle proteins from different species and from different musclfes have 
shown, for exaniple , that tropomyosins from skeletal muscle, uterus and 
■bladder art^ all different. Two elect rophoretically distinct tropomyosins 
coexist in the clam, adductor. That the shortening of muscle models is 
nof due to hj'-drolysis bf ATP has been supported by further evidence, 
and the dependence' of mi^osin adenosinetriphosphatase upon cations has 
been systematically studied. Incidentally this study required. the de- 
velopmeht of : analytical methods for' ATP and ADP when present in the 
same solution. " The structural requirements for sodium and potassium 
binding by'rayosfji have been defined by a theory which is being tested, 
A study of the prdtam.ine salmine has been continued. This protein of 
fish spenn nucleoprotein has been shown to be Inhomogeneous in composi- 
tlbh'. The m.echanism of the fibrinogen-fibrin transf orm.a t ion has been 
further investigated, with attention directed toward the two peptides 
cleaved off of fibrinogen during clotting. The proteolytic nature of' 
thrombin has been confirmed' by the demonstration of C-terminal groups 
in these peptides and by the- di-isopropylfluorophosphatfe inhibition of 
thrombin. The use of a commercial carboxj'peptidase in this reaction led 
to the discovery of a new enzyme in this class, specific for basic- amino 
acicis, ' The decrease in titratable sulfhydryl groups in serum. album.in 
on aging has been studied with respect to anion effects upon its kinetics. 
A, m.echanism for this chemical aging process in pure proteins* has' -been 
suggested. It has been found possible to nitroguanylate intact proteins, 
converting primary,;- amino groups into nitro-guariidino groups. Chromato- 
graphic and colorimetric behaviors, of prsoline, hydroxj^proline,, ^-hj-'droxy- 
pipecolic and pipecolic acids have been .studied. Two new 'amine .acids, 
apparently related to these, have been discox'ered in certain fruits. 
Y-Guanidino butyric acid has been .identified in a variety of fruits. 
This unusual material had previously been reported only in m.arine 
invertebra'tes, ■■■ ■ ■' ' ' • • ' ' ', . 

A recurrent probl^iii for the protein chemist is purification. For 
this reason considc?-rable interest attaches to the development of a 
novel method which' com.bines te'chniqxies of immunochemdstry with those 
of chromatography. Appropriate adso3*hents 'have been rendered highly • 
specific by coating them with specific .antibodies. The method, permits 
"the selective removal from' a protein mix.ture of antigenic contaminants. 
It has already demonstrated its practxc^l usefulness and m.ay develop 
into a procedure of general applicability, 

A form of radiant energy which will doubtless command increasing 
attention of biologists is the cosmic radiation found at very high 



-20« 

altitudts. The idcntificr.tion and measureiP'^nts of energy and charge 
jf h«?a\'y pririary nuclf^i has been preatly aided bv application cf 
photo-enulsicr toclmiquee developed at ?T/>'D. Rockets bearing such 
emulsions to oltituJos of 70,000 to 122,000 fott have returned with 
fog tracks of particlf^s. Analj'sis of these tracks is yielding data of 
both theoretical interest and practical use in aviation medicine. 

Nisccllar'-.?us Items - Histochemistry is the basis for the sciences of 

" histoanatomy and histopathology. Staining; methods, 
originally empirical, have over the years been 
progrcsr.ivcly interprctid in tenns of chenical reactions and identifica- 
tion of tissue roactantSi New methods often evolve rationally, today, 
based upon knowledge of criemical reactions and of tissue constituents. 
To this d-eveiopment' ri.fJlD scientists have made major contributions. On 
evidence supplied bj"- histological studies, three previously confused 
pigments, melnnin, trichoxjinthin and neurcmelanin have been distinguished 
from esQh other. Frcm differences in their eolucilities and reactivities, 
irJ'erences as tc their structures have bf.'.n dravm. Methods cf preparing 
and stainiiig tissues dc-t^mine in large part vjhrt may be seen under the 
microscope. Thus, -the application of frecze-drj'ing technique to kidney 
has revealed pi-otcplasmic extensions of tubule cells into the lumina of 
the proximal oonvclutv-d tubules. Since this phencmencn has not been re- 
ported vxith other fixing methods, it becomes important to ascertain 
whether it represents the true anatomical state or is merely an arti- 
fact of this method cf fixation. A detailed study cf the reaction of 
hematoxylin with keratohyaline has been undertaken, VThereas the oxi- 
dized form of the dye is required, oxygen is not. Oxidi'ied keratohyaline 
granules fail to combinf with reduced dj'-e. The Coons fluorescent anti- 
body technique, which p(;rmits histological localization cf antigens, has 
bcxjn applied to streptococcal hyalurcnidase and to chorionic g^^nadotropin. 
The localization of injected gonadotropin in certain cells of the rat" 
ovarj*- is an important histological demonstration of an endocrinological 
principle. 

Because potassium iodate is preferred in the tropics to potassium 
iodide as en antigoiter agent, it is of interest to determine its long 
term toxicity. In dogs, individual susceptibility tc iodate was found 
to vary greatly. In affected animals, after three months, emesis, 
anbrexia and weight loss were noted. 

>'. hitherto uni*ecognized type of toxicity results from the treatment 
of foodstuffs with ethylene oxide. This vrlatile and reactive substance 
has been used in the pressrvation of foodstuffs. Studies at KIAiMD have 
revealed that diets tr;2atad in this fashion are unsatisfactory for 
support of lift? and grovrth. This is not: due to direct toxicity of ethylene 
oxide, since none of this reagent persists in the diet. Rather it is 
attributable to chemical alterations brought about by reaction between 
ethylene oxir:ie and certain dietary essentials. It could be shown that 
the thiamine activity cf an ethylene oxidr.-"Lr':v';ted ration was markedly 
decreased. 



-21- 

Intramural Research Services - In addition to direct programs designed 

to lead to publishable research, there 
are a ntmber of supporting activities which 
are essential to the research of others, but which do not lead directly 
to publication. The operation of large equipment to permit pilot plant 
scale isolations is such a project. By means of this equipment, several 
tons of horse liver have been processed for concentration of prefolic 
acid. Large quantities of bulbs, leaves, etc., have been processed to 
concentrate materials of interest to workers in WII. 

The Laboratory of Chemistry maintains a micro-analysis laboratoiy 
which this year performed about 10,000 elemental, functional group and 
instrumental analyses for scientists, both within and outside NI/J'iD. 
Included herein are about 1,1^0 infra-red spectra. Improvements in 
technique and the cataloging of the results of these infra-red measure- 
ments are also a part of this operation, A large number of ultraviolet 
spectra he.ve also b-'en run centrally for scientists situated in various 
laboratories of the institute. The mass spectrometer maintained in the 
Laboratory of Biochemisti-y and Metabolism has rendered analytical services 
to scientists situated in NIAMD and in NIAID, Approximately 675 indivi- 
dual samples have been analyzed for abundance of stable isotopes, 
including k15 , C-'-^ and D^ , not counting a large number of reference 
and normal abundance samples. The application cf scanning technique to 
the study of disequilibrium gases has been undertaken. A new accessory 
for the handling of deuteriiam samples has been constructed and placed 
in operation. 

The Lab':ratcry of Pathology maintains a centralized tissue 
preparation service which this year processed in excess of ^3^000 
slides. M.>re than $0 different staining techniques have been employed 
in these preparations. Sections have been secured fr'-m about 3^700 
m'--nkeys for examination for live poliomyelitis virus as a part of the 
Salk vaccine program. From the Division of Indian Health and from the 
Bureau of Prisons materials from about 100 autopsies and 2^00 surgical 
specimens have been prepared and studied. 

During the course cf the year, the Editorial Copimittee has 
processed and approved for publication about 360 manuscripts. The 
function of this committee is two-fold. Firstly, it scrutinizes papers 
for grounds for rejection. Secondly, it offers criticism to the author 
designed to improve the paper. In both of these capacities it has 
functioned admirably. 



-22- 

C vncludi pr ronipents - oince most of the projects described herein are 

continuing proprams, it is not possible to come 
to firm conclusions in a scientific sense about 
them, f^ertain conclusions can, however, be dravTi about the category 
of this Ir.ntitute and the way it is beinp fulfilled by the exertions 
of its scientists. The distribution of efforts amonr the several dis- 
ciplines of basic research in medical sciences is wide and appears in 
general to be a rood and productive distribi tion. Collaborative ven- 
tures in research betv;een two or more basic disciplines, betv^een labor;.'- 
torj' and clinic, are probably on the increase, and this, on the basis 
cf production record, is a pood thing. 

One area v;hich is regarded as of importance, and which should 
be strenf;thened , is the study of the transmission of biological in- 
formation from a cell to its progeny, from a parent to its offspring. 
A growing interest is noted in several laboratories of MIAI'D in various 
aspects of genetics snd the inheritance both of normal and of abnormal 
characteristics. Since much of metabolic disease hfs a familial pre- 
disposition, the imp'»rtance of more knov;lodf%> and understanJing of the 
clier.istry, histology and population distribution of hereditary charac- 
ttristics is manifest. It is expected that future reports nay contain 
the record of mor'- v.-ork in this area. 

A continuing; problem v;ith the scientific direction of an institute 
such as FTAMD is the degree to which it is profitable to try to influence 
the choice of problfsms by our scientists. Certainly the direction ef 
the prof,'rari can be influenced in the selection of n:w staff members to 
fill vacancies crentod eiiher by new positions or by the departure of 
present staff, klco it is possible, bv the Histributior of si^pport 
among the several laboratories, to enhance the pro-iuction in an area 
where this seems dc-sirable. It appears highlv probable, however, that 
per research dollar spent, the greatest return vtIII be secured if tlie 
mature scientist is allowed and encouraped to select the problems on 
which he will vork. It is cu^ belief that the meritorious and exper- 
ienced invistigator will in general be the wisest, judge of his field 
of endeavor. The' mo5^t ijnportant function of the Scientific Directors 
ther<jXore, is in the selection of senior scientists, in their encourage- 
ment, and in the attempt to procure for them thos^. facilities which 
they may require for the fulfillment of their mission. 



AHWUAL REPORT 
January 1 to uecember dl , 1956 

NATioiML Institute or arthritis and MtTAbOLic uiseascS 

clinical Investigatioiis 
Rheumatoid Arthritis 



The research pro 'j ram in rheumatoid arthritis has 
been directed tovvarcs an exploration oi the pathotjenesis, 
patholovjy, iininunoloyica 1 reaction, anci the nature of 
response to a yroup oi new, synthetic 11,17 oxy-jenateu 
steroids. In conjunction with this pro ij rain, studies have 
been done on the physiological disposition oi naturally 
occurrinij adrenocortical steroias in i:ian and the in vitro 
enzymatic metabolism oi steroids. 

The synovial Xluid of rheumatoid arthritis is char- 
acterized by a oeiiciency in the polymerization of 
hyaluronic acic. It is believed that this may indicate 
a metabolic lesion in the synthesis of mucopolysaccaride 
by the synovial tissue. Studies on the biosynthesis of 
hyaluronic acid v^ore, therefore, undertaken employing 
uniformly labelleu tl"* ulucose, incubated with human 
synovial slices ?nd, in subsequent experiments, with 
cell-free extrat-t from human umbilical cord anu 
placenta. Such a system has never been utilized before 
and has been iount. to be superior to and much more 
fruitful than the tissue culture technique. It has been 
demonstrated that synovial slices or placental cell-free 
extract synthesized _in_ vitro hyaluronic acid and that 
certain of the adrenocortical anu synthetic steroids 
inhibit the incorporation of glucose C^''. The effect on 
tnis synthesis oi serum from patients with rheumatoid 
arthritis is now bein<j studied. 

A new concept of the development of morpholoy ical 
lesions in rheumatoid arthritis including arteritis, 
(^ranuloinatous synovitis and subcutaneous nodules has 
been a^ivanced on the basis of intensive hi stopathologi- 
cal studies. The important role of inflammatory 
vascular chan<jes in the development of these lesions has 
been delinea. These original observations have since 
been widely confirmed by others. Hi stopathological 
studies have been extendec in our laboratories during 
the past year to include vascular and other tissue 
changes in synovial membrane, striated muscle, integumen 
anu viscera of other rheumatic and colla-jen diseases. 
The (iiorpholo jical relationship between rheumatoid arthri- 
tis, polyarteritis nodosa, systemic lupus erythematosus, 
systemic scleroderma ana dermatomyositis was defined. 



Thp P'j'jlut inat ion activating factor in serum oi 
patients with rheumatoid arthritis seems to be a protein 
u'ith electrophoretic mobility in the beta globulin range. 
In the serum of almost all nonrheumatoicl arthritics is 
lounu sn inhibitory substance associated ivith plasma 
protein Fraction II of Lohn, which is probably a gamma 
glob'tlin. The isolation and characterization of these 
seruia protein components is desirable, first to enable 
their wore effective use in diagnostic tests for rheuma- 
toid arthritis ana, moreover, to determine the role they 
play in the pathogenesis of the diseas.e. 



Si 
hamper 
me n t o 
a g (J 1 u t 
object 
ox r h e 
also s 
by New 
met hod 
q 'J a n t i 
' u r t h e 
reset i 
optima 
a jglut 
also b 
Isolat 
eva lua 



nee ISO 
e G by 1 
£ met ho 
i tui t i n g 
ive. i 
u lii a t o i d 
I r n j 1 y 
f; a s 1 1 e ' 
appea r 
t a t i V e 
r.more , 
on and 
1 t i me 
inat ion 
e appli 
ion pro 
t i n . 



lalion procedures have been seriously 
ackof precise assay methods, the develop-' 
usior the quantitativeassay ox both 

and inhibitory factors have become a prime 
t has been found that a eu>;lobulin iractiofi 

serum containing aggl ut innt ing . ac t iv ity 

inhibits hemolysis 01 sheep erythrocytes 
s uisease Virus and by Saponins. The 
s to have good potentialities for the 
assay of the aggl ut inat ion act ivat ing factor, 
it has been possible to follow the rate of 
make some observations suggesting an 
of reaction for the sensitizeuisheep cell 

ior best diagnostic use . ■ The met hoa may 
cable to the assay of inhibitory substances, 
cedures can now be subjected to quantitative 



The ant i- inf 1 amiiiatory , metabolic and pi tui tary- 
suppressiny effects of new synthetic 11,17 oxygenated, 
9,J.l halogenated, 1,2 dehydrogenated steroids on patients 
with active rheumatoid arthritis have been studied.' It 
has been observed that the potent sodium- retaining 
eifects of 9-alpha halogenated hydrocortisone can be 
su.DStantially reduced by substituting '^1-desoxy for 
21-hydroxyl yroiip and can be completely overcome by 
substituting a fluorine atom for the hydroxyl subslit- . 
uent at carbon il even vifhen the latter steroid was not 
uehydrogenated at'carbons 1 and J, i-oth new steroids 
were biologically active since they readily suppressed 
secretion of corticotropin by the anterior pituitary. 
However, these compounds lacked the anti-inflaramatory 
potency of preonisone or 9-alpha f 1 uorohydrocort i sone, 
• e are currently engaged in evaluation of delta-one, 
9-alpha fluoro, 16-diacet8te hydrocortisone. Our pre- 
liminary results indicate that this steroid possesses 
about thi; same ant i- inf 1 ammatory potency as prednisone 
anu like the latter does not cause . sodi urn retention or 
potassium loss when given in conventional therapeutic 



-:u 



ooses. Ill the next levj months wo will hi- the lirst to 
stucy a new storoid (synthrsizoQ by Josi'i I'rieo Oi The 
• Squiub institute lOr ..ledical Kesearch), \>hi'h has oxhi- 
bitftl in th» aiireiinl ec loini zed rat impressively enhanced 
ai'. t i- i n . 1 am.na tory and ijlucocorticoid propertits anu hus 
not caust'd ntention oi" sodiun. 



I'K' t ;• h o 1 i s 1'^ ' A d r ^' n p 1 S t "^^ r o 1 d s in 



■lii n 



cort 
tort 
r a p i 
t i me 
i II n 
Prt^i 
a 1 a 
to h 
istr 
equa 
the 
prac 
to i 
i n i u 
Alte 
not 
r t 
istr 
< .\ I*, p 
more 



The 
i son 
i sou 
u ra 
s as 
or 1.1 a 
iinin 
rje 
y dro 
at io 
1 to 
i n r u 
t icn 
n die 
seu 
r th 
poss 
he <: 
at io 
etis 
;ol" 



phy 

e w 
(' e 
te. 
ra 
1 s 
n ry 
1 r a 
cor 
n. 

CO 

Si 
lly 
a t r 
CO r 
e i 
iol 
urt 
n 
the 
d; ■ 



siol 
as s 
X c e p 
Co 
pit. 1 
ubje 

res 
c t io 
t i so 

Hya 
rt i s 
a ' oi 

no 

t hr, 
t i so 
n tra 
c to 
i son 

i CO 

pi a 



J 1 1; a 
t u rj i c 
t t h n 
rt is.o 
y as 
els , 
ults 
n 1 
no in 
rocor 
one c 

.loo 

cort i 
t si i 
n e is 
-a.rt i, 

a C l:'.0 

e to 
r t iso 

sm.) c 



1 (li 
a an 
t it 
ne d 
hy«ir 

0.11 d 

have 
the 

man 
t i so 

11 c (^ 

ril;j. 

sone 
jhtl 
m . • t 
c u 1 a 
n s t r 
hydr 
ne a 
ort i 



spos i t i 

d found 

was mo 
i s .1 p p f a 
ocor t i s 
pa t ii. n t 

be(?n 
cort iso 

after 
n e cone 
nt ra t i 
of cort 

remain 
y more 
abo 1 i lie 
r i M j t c 
ate n n y 
o r t i s 
cetate , 
sone CO 



on a 

to 
t .Tbo 
red 
one 
s wi 
btai 
ne i 
oral 
entr 
n iv i 
i son 
s. 

than 
u to 
t ioii 

si.; 
one, 

the 
nc en 



nu KM' 

be si 

1 i z e d 

from 

idi t 

th ii 

ned t 

s rap 

or i 

a t i on 

t li i ti 

o and 

u v ll 

n ( • - 

hydr 

t c 

n i I ic 

A t 

pi as 

t r r, t i 



ta b 

mi 1 

at 

the 

.; 

ver 
hat 

idl 

h t r 

i n 

one 

at 

a t a 

h V. 1 

OC 

ort 
a n t 
c r 

l:lO 

on 



olio 

a r to 

n mu 

plas 

5 III in 

di se 

y met 

a V I' n 

plas 

hour 

two 

wo 111 

f of 
r t i so 
i sone 
c onv 
oral 
hydro 
by tw 



fate of 
hydro- 

ch more 

ma four 

u tes ) , 

ase. 

est that 

a ii 1 i z e d 

us odmin- 

ma i s 
a ■? t e r ' 

hours 

d seem 

the 

ne. 

, it v.as 

er s i on 

a o 111 i n - 

cort i sone 

en ty or 



Pa 

after 
than e 
1 a bora 
anu ex 
are si 
s i i ij h t 
P 1 .n s in a 
cort ic 
about 
n y tl r c 



r a 1 1 f • 
a (rtor 
xi ste 
tu r i c 
c ret i 
in i 1 a r 
1 y mo 
cone 
s t e r 
.:; to 
ort i s 



1 stu 
e a c c 
o her 
s einp 
o n •:" 
to h 
n^ ra 
en t rs 
one w 
;i mij . 
one i 



le 
i ra 
eto 
loy 

th 
ydr 
pi d 
t io 
as 

pe 

S' 1 



s on corticostorone have been done 

te and reliable analytical method 

lore had been develop <^d in our 

inij isotope dilution. The metabolism 

isnaturallyoccurrin.j adrenal steroid 

©cortisone; however, it is metabolized 

ly -- mean half-time, HO minutes, 

n in normal subjects averares 1.1 i-ig/d, 

found to be synthesize<.i at a rate oi 

r Cfly, whereas the turnover rate for 

7 to .i9 mg,, per day. 



L n z v' ma t i .: Studies on Steroid Substrates 



Studies have been continJeo on elucidating new path- 
ways by whicli steroids are metabolizied and enzymatic 
meciianisms by i.'hich these metabolic changes occur, 
.enzymes or, pa bit o/ reducing various steroids have 



previ 
the p 
Oi th 
a pro 
the r 
carbo 
proce 
trans 
the s 
mecha 
di rec 
prote 



ously 
ast ye 
e -i-'S 
ton is 
euuced 
n 5 as 
ed by 
i e r i 
ubstra 
n i s ni o 
tly by 
in. 



been 

ar it 

(ioubl 

a tta 

pyri 

woul 

a pro 

the 
te GO 
X its 
a py 



iden 
has 
e bo 
cheu 
dine 
d be 
c e s s 
hydr 
able 
kin 
ridi 



t jfi 

bee 

nd q 

to;' 

nuc, 

pre 

of 
oyen 

bon 
d wh 
ne n 



-4- 

ed in 
n f o u n 
f ,,ring 
carbon 
ledt id 
dictea 
1 , 4 gd 

from 
d^ Th 
ere . a 
ucleot 



this laboratory, ijuring 
d that in the reduction 
A ox the steroid nucleus, 
4 and a hydride ion irom 
e is transferred to 

if the reduction did not 
dition and there was direct 
the pyridine nucleotice to 
is is the first biochemical 
double bond is reduced 
ide, rather than a ilavo- 



St 
have r 
conver 
compou 
enzyme 
studie 
becaus 
to a (J 
and we 
R coinp 
Of r ^ a 
types 
uiiijht 



uait s 
e veal 
sion 
no F. 
s are 
s are 
e the 
eneti 
know 
1 icat 
ct ion 
f c 
be re 



on the 
ec that 
01 DOC t 
Turthe 
require 
of iir.po 
aOreno J 
c lack 
, on the 
ed iiietab 
has its 
nipouiids , 
veal in j 



mech 
sepa 

CO 

r trior 
d 10 
rtan 
eni t 
f on 
bas 

01 ic 
coil 
ah 

for 



anism 
rate 
rt ico 
e , it 
r eac 
ce on 
al sy 
e of 
is of 
lesi 
nterp 
i n s i <j 
other 



Lastly; a series of rela 
been studied, and their kine 
that a metabolite of TPNH, n 

hydrpxylqtions. 



01 s 

sy.ste 
stero 

has 
h hyd 

seve 
ndroin 
the h 

our 
on. 
arts 
ht in 
s. 

ted r 
tic a 
ot TP 



teroid hydroxy lation 
ms are involved in the 
ne and compound S to 
been ;found that two 
roxylation. These 
ral counts . First , 
e is, thought to be due' 
ydroxylat inij enzymes 
studies, that this is 
Also, since this type 
in a number of different 
to the mechanism of one 



eactions in liver has 
nalysis has suggested 
W., is involved in these 



Gout 



The research ■ prbc/ram on yout has been concerned 
primarily with the metabolic oritjin of uric acid as 
revealed by the pattern of incorporation of isotopically 
labeled precursors into urinary uric acid anu urinary 
purines in patients with gout :and hyperuricemia of 
various types. clinical studies have also been made of 
the value of in travenou s ( in .contrast to oral) colchicine, 
as a therapeutic aotnt, ,i,n the -inan-ageraent oi acute gout, 

• Althouyh the majority of >jout patients have shown a 
pattern oi incorporation of «jlycine-Wl3 into urinary 
uric acid similar to that found in. the normal, 3 patients 
have been iiound who incorporate glycine— wl5 more promptly 
and to a tjreater extent than do normal individuals in 
ai^reemeat with ^he previous findings. 



-5- 



ylyc 
y <> 'i t 
this 
acid 
t ii n t 
that 

W8S 

suyy 

are 

furt 



t oinp 
i n e - 

y pa 

pa t 

i II 

of 

of 
c X c r 
es t s 
met n 
hr r 
over 



arison oi the pattern o^ gl yc ine-1 -C^ "^ and 
W^-J incorporation into uric acid in the same 
tient has shown an apparent anomaly. While 
i.iit's pattern oi N^^ incorporation into uric 
two different study periods was identical to 
the normal, his pattern ol glycine-1-C^'' was 
an "uver-in corporator," despite the fact that he 
ctiny normal quantities ox uric acid. This 

th;it different portions of the ylycine molecule 
bolizcd differently in this subject and deserves 
stuuy. it suggests that all youty patients may 
-incorporators" when studied iv i t h glyc ine-l-C^ 'i , 



U 
ca r bo 
with 
glyci 
compl 
than 
subj e 
s i inu 1 



siny 
xaiai 
glyc 
nc " 
ete , 
did 
cts 
t a n e 



anoth 

de--i-L 
ine-Nl 
V c r - i 
i n r r 
the no 
and in 
ou s 1 V 



press 
this 
only 
These 
6 e I e r 
b i s y 



ion 
"ove 
part 
res 
t in 
nt he 



1 gly 
r-inc 
i al su 
u 1 1 s s 
a reg 
sis. 



er uric acid precursor, 4-amino-5-imidazole- 
1" (aIC-L^^), administered simultaneously 
->, it vjp.s found that one of tiiese gouty 
ncorporator s" on ivhoin studies arc- now 
porated also more AIC-C^-^ into uric; acid 
rmal. furthermore, although in normal 

gouty patients previously studied, 
administered AIC produced a marked sup- 
cine-N^^ incorporation into uric acid, lo 
rporator" AIC administration resulted in 
ppression of glycine- Nl^ incorporation, 
uygest thatthis gouty patient hasa 
ulatory mechanism for endogenous purine 



The pattern oi giycine- 
uriiiary purines and that of 
uric acid both provided evi 
pathways contributing to ur 
subjects studied. There is 
arisiny from newly formed p 
pathway pri sumably arising 
newly formed purines into t 
subsequent release with nuc 
extent of rapid labeling of 
gouty indi V i dua 1 s af t'er Alv^ 
pnthwny to uric acid may be 
ant in the gouty subjects s 
converse may be true in leu 
the indirect pathway, is s 
isotope content in urinary 
period 5 to 15 days after a 



1-C^'^ incorporation into 

AlC-Cl^ incorporation into 
dence for existence of two 
ic acid formation in all 

a rapid direct pathway . 
urines, and a slower indirect 
from incorporation of the 
issue nucleic acids and their 
leic acid turnover. The 

the body urate pool in the 
-Cl^ suggests that the direct 

quantitatively more import- 
far studied. That the 
kemia, with a predominance of 
uggested by the increased 
adenine and guanine in the 
dinin istrat ion of ylycine-1- 



Indirect evidence suyyestiny thata pathway for 
urate synthesis other than that involving xanthine 
oxioase may be present in the humanhas been deduced 



-6- 



from the time course of appearance of isotope in urinary 
Oxypurines ano uric acid after feeding gl yc ine-l-v;i'i. 
uaxirnal label in^j of uric acid is achieved on the second 
or third day vjith isotope concentrations substantially 
above that of the oxypurines at that time. The curves 
do not indicate e direct precursor-product relationship 
and are consistent Vv>ith the interpretation that most of 
the uric acid formed arises from precursor pools dif- 
ferent from those sampled by the urinary purines. 

Further evidence for a pathway of urate synthesis 
that does not involve xanthine oxidase is proviaed by 
the discovery of a new enzyme in beef liver that pro- 
duces uric acid from uric acid riboside. This' enzyme 
is bein.j purified and characterized, and the detailed 
steps of this metabolic pathway are under investigation. 

Treatment of acute gout with intravenous colchicine 
is at t imes super idr to the oral route of aomi ni strat ion, 
in treatiny '>iO acute attacks, no serious toxic effects 
were found, andin most cases relief was more prompt and 
fewer gastrointestinal symptoms occurred. 

The Physiology of i^iseases of the Thyroid 

Research on the thyroid has been directed towards an 
elucidation of the pe thogienesis of the changes that 
occur in congenital athyreosis (cretinism), and towards 
the biochemical defects in the formation of thyroid 
hormone which result in the development of goiters. In 
addition, the mechanism of action ot thyroid hormone on 
an enzymatic level and on a physio-chemical level has 
been the subject ox considerable work. 



■ A 
has b 
dur in 
Psych 
indiv 
vidua 
level 
elect 
waves 
been 
on th 
respo 
after 
patte 
inf lu 



jrou 
e e n s 
g i\,'hi 
ologi 
i d u a 1 
1 app 

in a 
roenc 

a n u 
d i f f i 
e nor 
nse i 

the 
rn of 
e n c e 



P i. 

tuaie 
ch th 
cal t 

cons 
ears 
11 fa 
ephal 
•J e n e r 
cult 
mal c 
n the 
aamia 

inatu 
of th 



1 n u 1 
d ex 
e re 
esti 
iste 
to h 
cets 
oyra 
al d 
beca 
hild 
EEG 
istr 
rati 
yroi 



V idu 

tens 

spon 

ng h 

ncy 

ave 

of 
m st 
y srh 
use 
ren. 

has 
at io 
on 
d ho 



ai s w 
i vely 
ses t 
as sh 
of de 
been 
Intel 
u d i e s 
yt hmi 
of th 
It 
be e n 
n of 
f bon 
rmone 



ith c 
over 

tre 
own a 
velop 
retar 

1 i g e n 
have 

a. I 
e pau 
is in 

seen 
tri io 
e has 

has 



o n g e n 

a 12 
at men 

rath 
ment . 
ueu t 
ce an 

show 
nterp 
city 
teres 

as e 
dothy 

been 
been 



i ta 1 a 

mont h 

t were 

er unu 

Any 
a b u 
d deve 
n a b s e 
reta t i 
of c on 
t i n g t 
arly a 
ronine 
s t u d i 
noted. 



thy re 
peri 
foil 
sual 
one i 
t the 
1 opme 
nt al 
ons ,h 
trol 
hat p 
s 12 
. Th 
ed an 
but 



osis 
od, 
oweu* 
in t ra- 
noi- 

same ■ 
n t , 
pha 
ave 
data 
artial 
hour s 
e 
d the 



-7- 



malnutrition without thyroid abnormality has been shown 
to result in similarly delayru bone maturation. It is 
hoped to eluciciate the separate roles of growth hormone, 
thyroxine, anurojon, and nutrition on bone maturation. 
The efioct of tri.'atrnent with very larye doses of tl)yrox- 
ine, triiodothyronine and triiodothyroacetic acid has 
been stucjiro. .io particular advantage accrues to any one of 
these aijents. The tjxicology of triiodothyronine in 
children has been delineated. Hypertension has been 
noteci (J Ox V or f tins, of 18 nori.ials) and spider 
an.jior.iata have been seen without evidence of liver 
disease (0 of 9 cretins, '1 oi 10 normals). 

biochemical AbMonflal i t ies in the Formation of 

T h V r o i cJ Hormone as a L a u s e of Goiters 



On 
occur r 
of mo s 
of pat 
cret i n 
1 the 
iodina 
t ra te 
are a 
they a 
they p 
a n i s ni s 
effect 
s t uuie 
c ll a n 'J o 
1.0 mi 
less t 
t r i i od 
c n s i d 
of the 
the t h 
ment io 



the 
iny q 
t ca s 
i e n t s 

has 

t :n z y 
t ion 
iodin 
s t ;) t i 
re of 
e r m i t 

comp 

of c 
d. I 

in i 
lli-u 
han 
1 h y r 
e r ri b 1 

t iiio 
y r i u 
neu a 



ass 

i t 
es 

i.'i 
b'^e 
me 
of 
e w 
s t i 

( 

til 
let 
han 
t w 
od i 
nit 

• *' 

on i 
e d 
ura 

• 

bov 



umpt 
r J e 
of y 
th (J 
n s t 
resp 
tyro 
asp 
coll 
n s i d 
e s t 
el y 
j( s 
as s 
de c 
/ml 
1 a 
n e a 
i scu 
cil 
This 
e an 



ion t 
ns pi 
i t e r 
i t e r 
uciiea 
1: s i b 
sine, 
reser 
y ins 
r o b 1 
u d y 
s e p a r 
in t ii 
liown 
n c e n 
and r 

mill 
Iso w 
s s ion 
types 

has 
u no 



hat i 
ay a 
s in 
s has 

who 
le fo 

alth 
ved, 
i q n i f 
e phy 
f the 
atea 
e lev 
that 
tra t i 
educ t 
i-uni 
n s w i 

as t 

incr 
been 
e 1 f e c 



od ine 
minor 
this 

l> e e n 
showe 
r the 
ouijh 

Hlth 
ican t 
s io 1 

ioui 
from 
el of 
exoye 
n u' h 
ion 
t/ml 
t hou t 
w h e 
ease 
St udi 
t has 



de 

ro 

are 

in 

a a 

i i 
the 
ou.j 

ca 
yic 
de 
org 

aS 
nou 
en 
f t 
by 

ef 
the 
iod 
eu 

be 



f ic i 
K i 
a , a 
i t ia 

con 
r st 

abi 
h ca 
use 
al i 
cone 
anil 
il in 
s TS 
the 
he 1 
a d m i 
feet 
r an 
ide 
in t 
en n 



enc y 
n the 
sy St 
t e d . 
gen i t 
step 

I ity 
ses s 
oi c r 
n t e r e 
e n t r a 
ica t i 

this 

II cau 
level 
evel 
ni str 
. Th 
tithy 
c n c e 
he pa 
oted. 



a n <j n a t 

pa t hoy 
ema tic 

One go 
al a bse 
in the 
to cone 
uch as 
e t i n i s m 
s t , bee 
ting me 
on. Th 

system 
sed no 

01 TSH 
of TSH 
at ion o 
ere has 
r i <i d r 
n t r a t i 
tient 



u r a 1 1 y 
e n e s i s 
study 
i trou s 
nee 

en- 
th i s 

au sc 
ch- 
e 
was 

was 
to 
f 

been 
ugs 
n by 



A further study of euthyroiu adults with goiters has 
been pursued by an j^ vitro approach. After surgical 
removal of the goiters, tliey have been incubated with 
llil and the method has been developed so that this 
tissue will synthesize t hy roglobul in i rom jl-^l (or, as 
in one case, tyrosine Gjl'^), The iodinateu compounds 
after iiydrolysis oj' thyrOjlobulin sho.v no significant 
difference between normal and goitrous ij lands. However, 
.3 previously unknown iouinateu compounds have been found 



-0- 



and their structure is currently under investigation. 
The physical properties of thyroglobul in studied by 
electrophoresis and salting out, have shov>rn no signif- 
icant difference between normal and goitrous glands. 
In one individual with carcinoma of the thyroid, hovjever, 
an iodinated protein was found which was unusually 
soluble in phosphate buffer and which had s different 
elec trophoret ic mobility from normal t hyroglobul in. No 
reason for the development of goiters in these patients 
has yet been found. 

' i'^ebhanism of .Action of Thyroid Hormone 



Siri 

a study 
t i n of 
bio log i 
Electro 
trat ion 
serum w 
have be 
in s e r u 
was fou 
n e p h r s 
there w 
marked 
shown t 
elevate 
From th 
p h y s i 1 
to the 
rate of 
to the 



ce th 
ho s 
free 
cal f 
phore 
of s 
hich 
en de 
m may 
nd to 
is, b 
as ma 
e 1 e V a 
hat t 
d in 
fc se a 
g i c a 
level 
degr 
c 11 c e 



yrox 
been 

or 
luid 
sis" 
ites 
bind 
velo 

be 

be 
ut e 
rked 
t ion 
he a 
preg 
nd 
1 ac 

of 
ad at 
ntre 



ine 

con 
unbo 
s, 

ha s 

on 
s th 
ped 
calc 
6 X 
s sen 

dep 

of 
moun 
nanc 
t iu r 
t i V i 
free 
ion 
tioii 



IS I 

duct 
und 
A ine 

bee 
the, 
yrox 
SO t 
ulat 
10-1 
ti.al 
res s 
free 
t of 
y or 

dat 
ty o 

thy 
of t 

of 



irml 
ed t 
thyr 
thod 
n de 
part 
ine 
hat 
ed. 
iM.' 
ly n 
ion 
thy 
thy 
dur 
a it 
f th 
roxi 
hyro 
free 



y bo 
de 
oxin 

ter 
velo 
i u 1 
can 
the 

The 

Thi 
or ma 
and 
roxi 
roxi 
inQ 

ha s 
yro i 
ne i 
xine 

thy 



und t 
t e r m i 
<;- i n 
med " 
ped s 
ar pr 
be d c 
1 evel 

leve 
s lev 
1. I 
in h y 
ne i n 
n e b i 
treat 

been 
d hor 
n ser 

is. 1 
roxin 



plasma 
ne the c o 
blood and 
K 5:; vers e F 
. t h a t t h 
otein (TB 
t c r 1(1 i n e d . 

of free 

1 of free 
el was V a 
n h y p 1 h y 
pert hy roi 

serum, 
nding. pro 
ment with 

suggeste 
mone i's p 
urn, and t' 
ikewise p 
e iji seru 



protei 
ncentr 

1 h e r 
low 

e cone 
P) in 

Equa 
t hy rox 

thyro' 
riable 
roidi s 
di sm 
•It was 
t e i n w 
, e s t r 
d that 
ro p r t 
hat th 
roport 
m. 



n s , 
a- 



cn- 

t i n s 
i ne 

ji in 

in 
m, 

al so 
a s 
gens. 

the 
ional 
e 
ional 



Studies have also been conducted on the effect of a 
variety of thyroactiva compounds on i sola ted ^ enzyme 
systems. It hES been , shov*n t hat rhammcjl ian , mp.l ic and 
glutamic dehydrogenase are -i nhi bited by IQ-^M thyroxine 
end triiodothyronine, Dinit rophenol has jio effect on 
these enzymes, althou.gh highly halogenatcd phenols and " 
xanthine -dyes, are, also active. In inhibiting these 
enzymes, thie activity of these compounds was roughly '' 
proportional to the degree of halogens plus a decreasing 
order of activity of the halegens in the series of' 
I>Lir?Gl. A number of other pyridine nucleotide linked 
dehydrogenases were shown to be inhibited by thyroxine 
but to r>' lesser extent. Several proteinases Wiere not 
effected as measured by pseudo esterase activ.ity. 



_Q_ 



Whi.ttiLT th. .ri\cts of thyroid hormones on dohydro- 
fjiii.isis is of innjor import nnce in the ultimate action 
of thyroxine- is not known, but these ore the first 
studies to nvenl _i_n vitro .-f foots of thyroxint' in 
isolnt<d in/ymi systt'ms ;jnd, as such, repri:s'.nt <? 
s i 'I n i f i c :• n t y d v o n c e , 

L) i n h e t ly s 

r.i'St.prch in dirbetts hns been directed townrds ornl 
on t id i n bi t i,c drmjs, vitamin metabolism in dinbetus, the 
orfcct of insulin on pentose metabolism, ond galactost 
cfiribotes, bn rbu t r;mi de (BZ-55) hns bc^n found to be 
nctivfy in certsin cesos of diabetes and not effective 
in others, in terras of lowering the Mood sutjarnnd the 
urinnry loss of sugpr. Mowover, the i'irst case of 
death from L;Z-3:3 was noted and reported. On the basis 
of this and subsequent similar reports by others, this 
drug was w i t h d r n w n from use. 

The rnttnbolisin of thiamine, riboflavin, pantothenic 
ccid, nicotinic acid, r.n6 vitamin B^2 have been studied 
in normal individuals, in diabetics ivith and without 
complications. It has been shown that both types 
excrete in the urine n greater than normal amount of a 
test dose of thiamine. Changes in riliollavin and panto- 
thenic acid are inconclusive. There w.re no changes in 
diabetes in the metabolism of vitarain b^o» These data 
are difficult to interpret but suggest ill at there may 
be a defect in dicbetesin the ability to utilize 
tliiarainu. 



T 

invts 
as a 
acts 
gen e r 
V a t i o 
r e s p 
and tl 
erabl 
as mu 
label 
and b 
r ibos 
me lab 
nietab 
gra du 
blood 
in su 1 
cells 
of c e 



he 

t-iG 
me a 

by 

al, 
n s : 
n s i 

-ly 

e i 
ch 

ed 
y c 

b C 

oli 

oli 
all 

in 

an 
rt a 



mt trbol 
a t e d bo 
n s of V 
i 11 c r e a s 
the r e 
for ex 
V to t 
X s e we 
a t e r e s t 
as 60%. 
U - r i L o s 
ompa r i s 
o n 1 1 n t , 
1 s was 
t a 9 is 
y to in 
These s 
on p ( n c 
d furth 
in pent 



i s r.i of a 
t ii as a 
e r i f y i n g 
i n g the 
suits CO 
ample , u 
he effec 
re ins <;; n 
since i 
in the b 
e s h IV e d 
on o f t h 
the p r c- 
demon st- 
being p u 
crease t 
t u d i e s c 
trat ion 
ermore s 
OSes in 



var 
matt 

in 
diff 
n f ir 
-xy 1 
t of 
s it i 
t se 
lood 

sub 
Cl 
sonc 
rate 
r sue 
he r 
on f i 
of s 

carb 



let 

er 
man 
us i 

n a 

ose 

in 

vc, 

er.is 

gi 

s t a 
4 a 
e o 
d, 
d. 
p t e 
rm 
ucja 

St 

ohy 



y of pen 
of i n t r i 

the con 
on space 
nd ex ten 

and L-a 

sulin wh 

u-ribo 

t (^ CPUS 

u c S e , 
n t i a 1 d e 
c t i v i t y 
f s e v e r a 
I u e n t i f 
Insul in 
of loss 
i n ma n t 
r s of a 
the poss 
dratc mo 



>os.e 
n s i c 
cept 

for 
d pr 
rabi 
ore a 
so i 
e a 
Stud 
grnd 
in u 
1 no 
i ca t 

wa s 

of 
he c 
cert 
ibie 
tabo 



s has 
inter 
that 
g 1 u c 
e V i u s 
nose »v 
s i)-a.r 
s f c 
dec re a 
ies wi 
a t i o n 
r i n e w 
n r i bos 
ion of 
shown 
D-ribo 
f feet 
a in ty 
imp or 
lism. 



been 
est and 
insulin 
s e , In 
b s e r - 
ere 

a bin ose 
on s id- 
se of 
th C.14 
to CO., 
i t h 

these 

se fro.-n 

of 

pe into 

t a n c e 



-10- 



Enzyinntic Defect in Galactosemi a 

A specific onzymatic defect was found in the blood 
and liver cells of patients with this disorder. The 
apparently hereditary absence of the enzyme P-gal- 
trans f erase result s in a block in the conversion of 
galactose 1-phosphate to glucose 1-phosphate and 
accounts for the inability ofchildrea with disease to 
utilize milk sugar. A specific spect rophotometr ic 
method for blood has been developed by which the disease 
can be accurately diagnosed. With this diagnosis made 
and the intake of milk avoided, babies with this dis- 
order may now be expected to grow normally and avoid 
the mental and vi sua 1 de feet s which, up to now, have 
been a serious consequence of galactosemia. 

Determination of Basis for "Direct" 
and " I n d i r e ct" Bilirubin Reaction 



have 

for 

diff 

bili 

It h 

that 

rubi 

rub i 

by w 

into 

This 

geni 

whic 

a pr 

met a 



Study 

solve 

some 4 

erence 

rubin 

as now 

d i r e c 

n glue 

n. Th 

hich b 

a for 

wbirk 

tal hy 

h i t ; a 

of ound 

bol ite 



of 
d a 
y 
be 

pig 

be 
t-r 

urp 

e p 
iie 
ra, s 
has 
per 
s b 
■ dc 
s w 



the 
pr 
ear 
twe 
me n 
en . 
eac 
n i d 
roc 

pi 
(lit 

sp 
bil 

egi 
fee 
ith 



met a bo 
b 1 e m w 
s, .The 
e n " d i r 
t s have 
shown b 
ting bi 
e and t 
ess has 
gment s 
able fo 
e c i a 1 s 
i r u b i n e 
n n i n g t 
t in he 

glucur 



1 i s m of 
hich ha 

factor 
e c t " a n 

not be 
y origi 
1 irubin 
li e i n d i 

now , be 
are con 
r excre 
ignif ic 
mia ( t r 
appea 
pat ic a 
on ic ac 



bile 
s bee 
s res 
d, "in 
en cl 
nal w 

is w 
rect 
en CO 
V e r t e 
t ion 
a n c c 
ue Gi 
r the 
bilit 
id. 



pig 

n un 

pons 

dire 

earl 

ork 

ater 

form 

nsid 

d by 

into 

for 

Iber 

t su 

y to 



ment 

der 

ible 

Ct" 

y un 
in 
-sol 

is 
erab 

liv 

the 
the 
t^s 
ch p 

con 



s app 
d i s c u 

for 
react 
derst 
ur la 
uble , 
free 
ly el 
er ce 

bile 
di sea 
disea 
at ien 
jugat 



ears to 

ss ion 

the 

ing 

d , 

boratory 

bili-. 

bili- 

a r i f i e d 

Us 

duct s, 
se cen- 
se) in 
ts have 
e 



D iscovery of New Type of Congenital Hemolytic Anemia 



A jsreviously unknown and unusual form of congenital 
hemolytic anemia was discovered ' by HIAisiD hematologi st s 
in a father and son afflicted by life-long severe 
anemia with jaundice and splenomegaly. in the peripheral 
blood, of the father and son, reticulocytes and mature 
erythrocytes were found to contain one and occasionally 
two round or oval-shaped inclusion bodies. These unusual 
particles were demonstrated by phase microscopy and by 



-11- 

supravital stiiininy with aqucous mothyl violtt, were 
u s !i .-) 1 i y s c I' n in the center of the cell n n d w t • r c found 
to rifiiain in the cell stroma after hemolysis. 

ill both pati»nts with the- rart htmoiytic r.nomia, 
the urihf was found to contain a rare dark brown pigment 
which, on chemical characterization, appeared to belong 
to the iiirsobi 1 i fuchs in group, a type of pigment never 
before isolated from urine, Aftor transfusion of the 
abnormal c<.'lls to the normal recipient, the Irtter 
excreted the samr pigment. This indicated that the 
aneiTiic cells in this disorder hav;- a defective mode of 
breakdown leadin.j to me sobi 1 i f uch s in lormation rather 
than a normal bn.nkr'own to bilirubin. 

New .ilethod for Localizing Obscure Site of, c?n.d 
iiiea sur i ii'i Bl'od L oss from Gastrointestina l Tra c t 

Use of the Cantor tube with periodic aspiration of 
intestinnl contents ts the tube is passed down the 
intestinal tract, in conjunction with chro mate-tagging 
of the patient^s red cells for aid in the detection of 
blood in the intestinal contents, provides a valuable 
tool to clinicians in the search for sites of intes- 
tinal blood loss, Xhe value of this procedure was 
proven in five instances (including one malignancy) in 
which sourciis of blood loss were previously missed by 
standard i.Vc tliods, including radiographic examination 
and lifi- saving surgery was performed, 

LiTLCtivi. Clielnting Agent for Copper in V.'il son's Disease 



Preliminary studies with a preparation of penicilla- 
mine have di.nion s t rated that this is a very effective 
compound for removing copper in \»'ilson's disease. This 
compound, given orally in doses of 4 grams daily for 3 
days will produce a 10- to 20-fold increasL-d excretion 
of coppi.r in the urine, and has an advantage in that it 
can be givun orally. It causes no increased excretion 
of iron in hemochromatosis. 



NATIONAL INSTITUTE OF ARTHRITIS AND IffiTABOLIC DISEASES 
Annual Report - Extramural Programs 

January 1 - December 31; 195^ 



The orderly expansion of the research and training 
grant programs of the Institute noted in previous years 
continued in 1956. Budget increases in both areas per- 
mitted the support of additional worthwhile activities 
as we.ll as some augmentation of current projects where 
indicated. It is particularly gratifying to note that 
the quality of activities financed by these programs re- 
mains at the high level established in earlier years. 



Research Grants Activities 



1956 
Requests: ^ Approval::. 
No . Amount No . Amoun t 



1/ 1/ 



February I956 Council 208 $2,136,868 I6I ^l,^k^,236 

June 1956 Council 395 U, 755, 168 332 3,81+1,067 

November I956 Council 267 2,823,12ij- 225 2,08 



sUl 



870 $9,715,160 7.18-/ $7^1169,01+14- 

In the research grants area, emphasis continues in the 
fields of the arthritides and diabetes mellitus with support 
awarded for projects in all aspects of each, from incidence 
and etiology to modoc; of treatment and rehabilitation, but 
with particular attention focused on studies concerned with 
elucidation of the nature of the underlying causes. For 
example, the chemicaJ. and physical nature of connective 
tissue is being more intensively investigated in an effort 
to broaden our understanding of the presently ill-defined 
and poorly understood group of ailments classified binder the 
title of "arthritis". Similarly, basic studios in diabetes 

— ' Excluding approvals transferred to other Institutes for 
paj'Tnentj excluding all multiples (other-prefix first); ex- 
cluding subsequent withdrawals; and unpaid approvals recon- 
sidered at Juiie meeting for payment . 

2/ 

--' Applicants in I55 institutions in 1+0 states, the District 
of Columbia, and 3 foreign countries. 



mellltus involve the mechanism of action of insulin and of 
glucagon J the relationships of various endocrine glandr- to 
the "biochemical lesion" involved; and the metabolism of 
carbohydrates and fats. 

The Institute is also supporting research in nutrition^ 
li.ver and kidney diseases^ other metabolic defects such as 
galactosemia and phenylpyruvic oligophrenia^ and a wide 
variety of fundamental studies in the fields of endocrin- 
olofZf aiiti metabolism. As of December .1, the program was 
supporting 707 active projects v:ith awards totaling 
$7j 397^155 • I* is anticipated that in the ensuing year 
continued strengthening of the present prograra will occur, 
together with incr^^ased attention to projects in gastro- 
enterology and the aging process. Of interest in this 
siimmary is the observation that of the approxj.niately two 
thousand papers presented at the Federation of American 
Societies for Experimental Biology meeting in Atlantic 
City about one out of every six involved an active grantee 
of this Institute. 



Training Grants Activities 
1956 

1) Graduate Training Grants: 

1/ 1/ 

Requests-^ Approvals-/ 

No. Aiiiount No. Amoujit 

116 $1,639,60)4 111 $1,069,01^1- 



This year reoulted in a marked expansion of the 
graduate training grants prograra supported by NIAMD. 
With the budget for F/Y 1957 almost doubled over 1956, 
support has been recommended for additional training 
centers, and, in most instances, those centers supported 
in the first year of the program have been strengthened. 
The development is demonstrated by a gradual but continu- 
ing increase in the number of trainees actua.lly partici- 
pating, whereas previously the support was expended largely 
in organizing the centers. During this year a total of 
fifty-seven institutions in twenty-nine states and the 



- 3 



District of Columbia received training grant awards, which 
included stipends for fifty-one "indirect" trainees. Pro- 
grams presently in being or recommended for support cover 
interests in arthritis, diabetes and other metabolic 
diseases, hematology, and gastroenterology, and provide 
valuable post-graduate training for yo^ung physicians in 
these areas. 



2) Direct Traineeships: 

■ A total of 106" applications for direct traineeships 
in the amount of $l+30,098 were received. The Traineeship 
Board recommended 86 awards for $353,1^+1 to applicants in 
4^ institutions distributed among 23 states and the District 
of Columbia. 



Fellowship Awards 
1956 


Requests 




1/ 

Approvals-' 


No . AB'ount 




No . Amount 


176 $698,000 




71 $264,5^ 



-' Distributed among 40 institutions in I9 states, the 
District of Columbia and 2 foreign countries. 



Calendar Year 19^6 



PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HE/'.LTH 
INDIVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1 . NIAMD- 1 



SERIAL WUI4BER 
2, NIAOT 3. Nutrition and Endocrinology 



INSTITltt'E OR DIVISION L\BORATORY, BRANCH, OR DEPARTMNT 

k. Nutrition ^, 

SE'CTION OR SERVICE LOCATION (IF OTHER THAN BETHESDAJ 

6. Required nutrients and tbeir met abolic fu ro tion - T he rat 

PROJECT TITLE 



7. Dr. F. S. Daf t 

PRINCI PAL IN^rESTTG\TOR 



8. E. G. Mc Daniel and M, L, Kunde 



OTHER INVESTTGA TORS 

9. IF THIS PROJECT RESEMBLES, COMPLEMi.;NTS, OR PARALLELS RESEARCH DONE 
ELSE(\rHERE IN Tf-^E PUBLIC HEALTH SER\/ICE (WITHOUT INTERCH-.NGE OF PER- 
SONNEL, FACILITIES OR FU^^)S) IDENTIFY SUCH RESEARCH: 

None ^~^ 

10. PROJECT DESCRIPTION: 

Objectives: (a) To discover substances which decrease vitamin 

requirements and to elucidate their mechanism of action. 

(b) To define the functions of amino acids and nucleic acid 
in folic acid metabolism. 

Methods Employed ; Purified diets with or without specific vitamins, 

and with or without substances which may "spare" 
vitamin requirements are fed to animals. Growth and development, 
physical signs and symptoms, blood changes, tissue conaeritration 
and urinary excretion of vitamins and metabolites are studied. 

Major Findings ; 1, Previous work has shown that large amounts of 

dietary vitamin C or small ajnounts of certain anti- 
biotics decrease markedly the rat's requirement for a variety of B 
vitamins. This ability is shared by some compounds known to inhibit 
"« certain metabolic processes in mammals, such as the compound SKF 525-A, 

2, Parenteral penicillin gets into the gastrointestinal tract 
via the bile. Techniques have been developed to divert the biliary 
secretions from the intestinal tract as an approach to determining 



NIAMD"! -- ^ 

■whether antibiotics exert their vitamin sparing effect in the G-I 
tract or in the tissues, 

3t Large amounts of dietary vitamin C result in an increased 
supply of certain B vitamins in tissues. Previous work showed that 
folic acid deficient rats excrete large amounts of urinary formimino- 
glutaraic acid and that this compound arises from histidine. Further 
work has shown that histidine, tryptophane or threonine, or certain 
nucleic acid derivatives counteract paiPtially the effects of folic 
acid deficiency. 

Significance to NIAMD Research : This basic research may have con- 
siderable significance in elucidating 
the various factors which influence vitamin requirements, threw 
considerable light on vitamin metabolism and offers some hope of 
providing diagnostic tests for specific vitamin deficiencies in man 
and in animals. 

Proposed Course of Project t Current work is directed toward elucida- 
ting the mechanisms of these effects 
using various routes of administration of active substances, vitamin 
balance techniques and dietary manipulations. 



Form No. ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - M/ITIOML INSTITUTES OF HEALTH 
UTOIVIDUAL PROJECT REPCBT 



Part B{ Budget Data 



llo NI/iMD-1 



SERIAL NUMBER 



12, BUDGET DATA; 



ESTIMATED OBLIGATIONS 



MAN YEARS 



FYt^T" 



DIRECT 



REIMBURSEMENT 



TOTAL 



PROF 



CTIiER TOTAL 



$2i|,000 



#9,100 



^33,100 



1,00 



3,66 ii.66 



BUDGETED POSITIONS 



PATIENT DAYS 



PROF 



OTHER 



FY«57" 

1.00 3*66 

13» BUDGET ACTIVITY; 
RESEARCH 

REVIEl-l & APPROVAL 
BIOLOGIC STANDARDS 



TOTAL 



U.66 



[71 ADMINISTRATION 

□ PROl'ESoIOML & 

__ TECHNICAL ASSIST* 

r I ANCE 



□ 



□ 



Ih, IDENTIFY ANY COOPERATING UNITS OF THE PUHJC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONl>ffiL 
FOR THIS PROJECT IN FY 1957. IF COOPER/iTING UNIT IS WITHIN WIH 
INDICATE SERIAL NO(S); 



(Use reverse and addj-tional pages, if necessary) 



\ .1 



Calendar Year 19^6 

RJBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors^ Awards & Publications 1^, NIAMD-l 



SERIAL NUMBER 

16, PUBLICATIONS OTHER TH'i.N ABSTR/\.CTS FROM THIS PROJECT DURING CALENDAR 
YEAR 19^6: 

None 

17, HONORS AND AWARDS: 

None 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - - NATION.AL INSTITUTJiS OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part A, Preject Description Sheet 1. NI/iMD-2 



SERIAL NUMBER 

2, NIAjyro 3. N utrition and Endocrinology 

INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTI4ENT 

it, Nutritian ^, 



SECTION OR SERVICE LOCATION 

Influence -sf accelerated growth on physiological functions - Growth 

6, rate and longevity 

PROJECT TITLE 

(work being completed under supervision of 

7, Dr. J. M. Hundley (Mr. E. G. McDaniel) 

PRINCIPAL INVESTIGATOR 



8. Npne 



OTHER INVESTIGATORS 

9, IF THIS PROJECT RESMBLES, COMPLEMENTS, OR PARilLLELS RESEARCH DONE 
ELSEVJHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PER- 
SONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH; 

None - 

10. PROJECT DESCRIPTION: 

Objectives: To relate various rates of growth to body composition, 
to the development of disease, and to longevity. 

Methods Employed ; Various diets are fed to rats to produce planes 

of growth varying from severe retardation to 
above normal. After active growth has ceased, a loniform diet is 
fed to certain groups so that possible dietary influences will be 
uniform except during the active growth period. Two strains of rats 
are used, one naturally slow growing and the ether exhibiting more 
rapid growth. 

Major Findings ; This study is a long-term project which will 
ccfitinue for another year. No major findings 
have developed thus far. The diets fed have yielded the various 
planes of growth desired. Body composition studies have been 
continued, confirming previous findings that various diets 
influence the percent of body fat as well as producing desired 
differences in lean body mass. 

Significance to Nli'llD Research ; Numerous studies have revealed 

the fact that /imerican children 
are growing at constantly faster rates. It is not known if this 



SERIAL ND. 

is a desirable trend with respect to eventual adult health, longevity, 
and chronic disease prevention. This animal study offers an approach 
to this problem since the experiment is designed to make growth rates 
during childhood the only variable. 

Proposed Course of Pr oj ect ; Studies to be continued as presently 

conducted until all or substantially 
all of the animals have died as the result of natural causes. 
Causes of death are being determined and studied. 



Form No, ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - mTIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11. NIAm-2 



SERIAL NUMBER 



12, BUDGET D/^.TA! 



ESTIMATED OBLIGATIONS 



FI'57" 



DiREc:: 



REUfBriRSEKENT 



?ot;il 



|28,liOO 



!ipLl,300 



139,700 



MAN YEARS 



PROF OTHER TOTAL 



1*83 



2,33 ii.l6 



BUDGETED POSITIONS 



PROF 



OTHER 



T 



OTAL 



PATIENT DAYS 



FY«57 

1.83 2.33 

13. BUDGET ACTIVITYi 
RESEARCH 

REVIEVJ & APPROVAL 
BIOLOGIC STANDARDS 






lul6 



ADMINISTRATION 

PROFESSIONAL & 
TECHNICAL ASSIST- 
ANCE 



□ 

a 



lU. IDENTIFY Ai\]Y COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONi^IEL 
FOR TECS PROJECT IN FY 19^7. IF COOPERATING UNIT IS WITIilN NIH 
INDICATE SERIAL MO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 19^6 



PUBLIC HEALTH SERIVCE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C, Honors, Awards & Publications 1^» NIAtffl~2 

SERIAL NUMBER" 



160 PUBLICATIONS OTHB.R THAN ARSTR/.CTS FROM THIS PRDJECT DURING CALENDAR 
YKAR 1956; 

Hundley, J, Mo Diabetes — overweight: U. So problems, J. Am. Diet, 
Assoc c 32: hll, 19^6. 

Hundley, J, M., Ing, R. B», and Krauss, R. W, Algae as sources of 
lysine and threonine in supplementing wheat and bread diets* 
Science 21: 536, 19^6 » 

Hundley, J. M., Sandstead, H« R., Sampson, A. G., Wbedon, G. D., 
Bakerman, H., and Ing, R« Lysine, threonine and other amino acids 
as supplements to rice diets in manj Amino acid imbalance. Am, 
J, Clin. Nutr. (in press). 

17. HONORS AND AWARDS: 
None 



Calendar Year 19^6 

PUBLIC HEi\LTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. mtamd^"^ 

SERIAL NUMBER 



2n NIAMD 3* Nutrition and Endocrinology ^ 

INSTITUTE OR DIVISION LABORS: TORY, BRANCH, OR DEPARTMENT 

it4> Nutrition ' $» ^ ^ 

SECTION OR SE ' RV ' ICE LOCATION (IF OIHER THAN BIiIthESIJA) 

6, Diabetes - Vitamin-: and amino acid metabolism 



PROJECT TITLE 
7. E. G. McDaniel, J, M. Hundley, F. S. Daft 



PftlNCiPAL INVESTIGATOR^S; 
8, Dr, R. 0. Scow, Dr, S, S. Chernick 



OTHER INVESTIGATORS 

9. IF THIS PROJECT- RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSE^IHERE IN THE PUBLIC HEALTH SER^/ICE (WITHOUT INTERCHANGE OR PER- 
SONNEL, FACILITIES OR FUNDS) IDENTIFY SUCH RESEARCH: 

None 

10. PROJECT DESCRIPTION! 

Objectivesj (a) To study the metabolism of amino acids and vitamins 

in nonnal and diabetic animals and to determine what 
corrective measures may be taken v;ith respect to metabolic defects 
observedo 

(b) To stuc^ niacin deficiency in animals fed large amount.-3 of 
corn. 

Met hods Employed ; (a) In the alloxan diabetic rat there is a 
"" reduced conversion of tryptophan to niacin. The 

studies include the sites of this metabolic defect and the effects of 
various hormones upon the conversion. The effects of diabetes upon 
various metabolites in urine, blood, and tissues are determined by 
use of chemical, chromatographic, and microbiological procedures. 

Experiments are in progress to determine possible similarities 
or differences in the symptoms and effects of experimBntal diabetes 
produced by pancreatectomy or by administration of alloxan or corti- 
sone. 

The effect of dj.abetes upon the ability of the rat to utilize 
trjrptophan for growth and for production of blood and liver pyridine 
nucleotides is being invest igatedj, 



I 



(b) Experiments are being done to determine whether or not 
alkaline treatment of corn (similar to that used in t he prepara- 
tion of corn for human consumption in some areaLS, of the world) has 
any beneficial effect in the prevention of niacin deficiency in 
dogs and rats. 

Major Findings ; (a) The reduced conversion of tryptophan to niacin 

and the elevated liver levels of picolinic acid 
enzyme in alloxan diabetic rats develops over a period of six days 
following alloxan administration. The changes also occur in depan- 
creatized rats but is not apparent daring the first 2l| hours following 
the operation. Dogs made diabetic with alloxan or by removal of the 
pancreas failed to show elevated liver enzymes o Measurement of 
tryptophan to niacin conversion in diabetic dogs has given inconclu'— 
give results due to release of large amounts of niacin from the tissues 
during periods of insulin deprivation. Excretion of urinary 
quinolinic acid (another metabolite of tiyptophan) was decreased in 
diabetic rats, 

(b)Dogs fed high corn diets were able to obtain more niacin from 
alkali treated corn than from untreated corn, as judged by N*- 
methylnicotinamide urinary excretion.. The time necessary to develop 
niacin deficiency (blacktongue) in dogs fed corn diets is decreased 
by substituting an equal amount of gelatin for the cow peas in the 
Goldberger diet. 

The apparent amino acid imbalance resulting from the 
gelatin can be overcome by a mixture of gelatin and zeinc 

Significanc e to NIAMD Resear ch; Numerous complications other than 

abnormal glucose utilization are 
known to be associated with diabetes in humans e It is possible that 
some of these complications may result directly or indirectly from 
abnormal metabslism of proteins, amino acids^, or vitamins, or from 
an inability to utilize in a normal manner some of these factors o 

(b) There &re still large groups of people for whom a major 
part of the diet is corn« Since diets consisting chiefly of corn 
are inadequate in niacin, it is important to determine whether or 
not the availability of niacin may be changed by food preparation 
methods. 

Proposed Course of Project ; (a) Studies are now in progress to 
"~~~~~~~~~" ' determine the sites of other defects 

in the metabolism of tryptophan. The manner in which insulin and 
adrenalectomy restores the tryptophan conversion in the diabetic rat 
will be studied; also, whether tryptophan or its metabolites are 
necessary for a response to insulin^ 

Experiments will be done to determine the source of blood 
glucose which was elevated following the administration of tryptophan 
to diabetic rats, and to determine the rel'itionship between blood 
glucose levels and the metabolism of tryptophane The possible role 
of pyridoxine m these phenomena will be investigated^ 



MIAMD~3 ■ 'i> 
S'EmL MO. 

studies will be done to determine alterations in metabolic 
pathways of other amino acids and vitamins « The effect of abnormal 
tryptophan metabolites upon development of diabetic Qataracts and 
upon the development or progress of the diabetes itself is under 
investigation. 

(b) The effect of alkali treatment of corn will be tested 
for its ability to prevent devlopment of blacktongue, to alter the 
urinary excretion of niacin metabolites in dogs, and to effect growth 
cf rats» Experiments will be done to determine the manner by which 
availability of niacin is increased by such treatment. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEAE? H 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-3 



SERIAL NUMBER 



12 e BUDGET DATA: 



ESTIMATED OBLIGATIONS 



MA.N YEARS 



PROF OTHER TOTAL 



Fit 57" 



DIRECT 



REIMEURSEMCNT 



TOTAL 



|iUl,800 



PROF 



$16,100 



$57,900 



BUDGETED POSITIONS 



OTHER 



?OrAL 



1.67 2.99 

13. BUDGET ACTIVITY; 

RESEARCH []x] 

REVIEW & APPROVAL Ql 

BIOLOGIC STANDARDS []]j 



1.67 



2,99 h.66 



PATIENT DAYS 



1;.66 



ADMINISTRATION 

PROFESSIONAL & 
TECHiaCAL ASSIST- 
ANCE 



□ 



LJ 



lit, IDENTIFY ANY COOPERATING UI\fITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOMEL 
FOR THIS PROJECT IN FY 1957. IF COOPER^lTING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 



PUBLIC HE^ILTH SERVICE - - NATIONAL INSTITUTES OF HEj\LTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 15 c N IAMD-3 



SERIAL NUMBER 

16. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALEKiDAR 
Ya\R 1956: 

McDaniel, Ee G., Hundley, J, M., and Sebrell, W» E, Tryptophan- 
niacin metabolism in alloxan diabetic ratSa J 5 Nutrition 59: 
ii07, 1956. 

17. HONORS AND AWARDS: 

None 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NIAMD-ih 



SERIAL NUMBER 



2. MMB 3" Nutrition a nd ^Endocrinology 

INSTITUTE m DIVISION LABORATORY, BRANCH, OR DEPARTMENT 

4. Nutrition , 5. __™_„__^_______ 



SECTION OR SERVICE LOCATION 

Influence of accelerated growth on physiological functions - Obesity 

6. in animals ^ .,_ ^ ^_ _________™___™__„_™_. 

PROJECT TITLE 

7. Dr. 0. Mickelse n , „__«„___ . 

PRINCIPAL INVESTIGATOR 

8. Mr. A. Anderson and Dr. R. S. Yamampto 
OTHER INVESTIGATORS 

9. IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PER- 
SONNEL, FACILITIES BR FUNDS), IDENTIFY SUCH RESEARCH: 

None 

10. PROJECT DESCRIPTION: 

Objectives ; To determine the bischemical and physiological altera- 
tions that sccur in animals during the development cf 
obesity and the subsequent loss of weight by obese animals. 

Methods Employed : Rats ai'e fed diets of varying fat content (20 to 

63/5) . The growth rates and maximum weights at- 
tained on these diets are studied. Autopsies have been performed on 
most of the rats that have died. Rats reaching a weight ef approxi- 
mately one kg. have been put ©n a variety of reducing regimens. 

Maj®r Findings ; The growth rates of the rats on different levels of 
fat were m.aximal when the diet contained 40^ of fat. 

The ©bese rats showed a mortality rate which was approximately 
two times as great as that of the lean controls fed a stock diet. 
The longevity study will be continued to see if the difference between 
the obese and lean rats is valid. 

Pathological studies showed that the obese rats have an increased 
incidence of an osteoarthritic-like lesion. These lesions were visible 
at the ankle joints on macroscopic examination and at the knee joints 
on microscepic examination. The weights of the heart, kidney, liver, 
spleen, testes, and pituitary gland were the same in both the lean 
cind the ©bese rats. This evidence, together with the observations 



ATTAMn^li - ^ 

SERIAL NUMBER 

reported last year, indicates that the loan and obese rats are similar 
except for the amount of fat in the bodies and the above mentioned 
pathological differences. 

The adrenals in the obese rats weighed approximately one and one- 
half times as much as those in the lean animals. The histological al- 
terations in the adrenals and thyroids were usually such that the obese 
rats could be distinguished readily. These changes in the adrenals 
and thyroid glands again raise the question of whether any metabolic 
abnormalities are associated with the development of obesity. 

The blood pressures of the obese rats are practically the same as 
those of the lean controls. 

The weight reduction studies on the obese rat suggest that a com- 
mercial stock diet containing % whole liver powder is not as good as 
a high fat diet in prsmoting good health. There was a much higher mor- 
tality, as well as a poorer appearance, among the rats being reduced 
on the low fat (commercial stock) diet than among those fed limited 
amounts ©f the high fat diet. 

Significance to NIAMD Research ; Obesity is listed as one of the majsr 

health problems today. The develop- 
ment ®f a means of producing obese animals entirely by dietary procedures 
will permit the study of obesity in animals uncomplicated by other 
physiological changes. The primary emphasis of this work will be on the 
effects of a reduction in body weight in obese animals. 

Proposed Course of the Pro.iect ; Obese rats will be reduced in weight 

to see if their physiological and 
metabolic functions are the same as those of animals that were never 
sbese. These studies will include blood pressure measurements, body 
cempositisn, reproductive capacity, incidence of disease, longevity, etc. 
Attempts will be made to see whether the obesity is the cause or the 
result of the changes in the thyroid and/or adrenal glands. 



Form Ho, CEP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B; Budget Data 



12, BUDGET DATA: 



11, NIAl^D-i; 



SmiAL NUMBER 





ESTIMATED OBLIGATIONS 




mN lEARS 


DIRECT 


REIMBURSEMNT TOTAL 


PROF 


OTIER TcJTAL 


FY«57 ' 








$26,200 


;i]13;300 ^39,500 


1.00 


1.83 2.83 




BUDGETED POSITIONS 




PATIH^T DAYS 


PROF 


OTHER TOTAL 




Fr»57 

1.00 


1,83 2,83 






13 c BUDGET ACTIVITY: 







RESEARCH 

REVIEVJ & APPROVAL 

BIOLOGIC STAwTDARDS 






ADMIMISTMTION 

PROFESSIONAL & 
TECFMCAL ASSIST- 
AI^CE 



a 



m. IDENTIFY ANY COOPER/iTING UNITS OF THE PUETJC HEALTH SERVICE, OR 
OTHER CRGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 195? j IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S); 



(Use reverse and additional pages^ if necessary) 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HE/lLTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publicatisns 15. NIAMD-U 



SERIAL NUMBER 

16. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
CALENDAR YEAR 1956: 

Keys, A., Anderson, J. T. , Mickelsen, 0., Adelson, S. F., and 
Flaminio, F. Diet and seimm cholesterol in man: Lack ©f 
effect of dietary cholesterol, J. Nutrition 59, 39, 1956. 

Mickelsen, 9. Age changes in body comp.?oiti©n. Proc. Univ. 
Mich* Ninth Annual Conference on Aging (in press). 

Hundley, J, M. , and Mickelsen, 0. Malnutrition, including obesity, 
as a cause of chronic disease, Proc. Conference on Preventive 
Aspects sf Chronic Disease (in press). 

Mickelsen, 0., Schaefer, A. E., and Darby, W. J. Harold Russell 
Sandstead (1904-1955). .^. J. Clin. Nutr. 4, 291, 1956. 

17. HONORS Aira AWARDS: 

None 



Calendar Year 1956 

PUBLIC HEALTH SERVrcE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part A a Project Description Sheet 1, miaMD-E 



SERIAL WMBER 
2» NIAMD 3« Nutrition and Endocrinology 



INSllTUTE OR DIVISION LA JURATORY, BRANCH, OR DEPARTMENT 

h» Nutrition 5» 

SECTION OR SERVICE " LOCATION (IF OTHER THAN BETHESDAj 

6. Required nutrients and their metabolic role - The mouse 
PROJECT TITLE 

7. Dr. G. M. Br iggs 

PRINCIPAL INVESTIGATOR 

8 . None 

OTHER INVESTIGATORS 

9. IF THIS PROJECT RESEMBLES, C0?4PLEMENTS , OR ^'IRALLELS RESEARCH DONE 
ELSEWHERE "^N THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PER- 
SONNEI., FACILITIES OR FUNDS) IDENTIFY SUCH RESEARCH: 

None 

10. PROJECT DESCRIPTION: 

Objectives ; To obtain fundamental knovjledge on the nutrition of the 
mouse and to compare nutritional findings obtained from 
other laboratory species with those found in the mouse. 

Methods Employed ; Mice from several strains are started on nutrition 

experiments at as young an age as possibls (12-16 
days, weighing 7-8 grams). TF:ey are fed various experimental diets 
including those designed for other species. Various deficiencies are 
produced by emitting the nutrient in question from the experimental 
diet. In this way deficiencies of folic acid, vitamin P^, pantothenic 
acid, fat, amino acids, etc., are studied » 

Major Findings ; (l) In studJ.es on obesity it was found that the 
combination of the high fat diet (Mickelsen) and 
gold thioglucose treatment produced fatter animals than either treat- 
ment alone. 

(2) Further studies on folic acid deficiency have led to the 
following conclusions; 



^ a. Folic acid deficiency can be produced in the young mouse 
by dietary means alone. This is contrary to most reports in the 
literature, 

b. A more severe folic acid deficiency, as evidenced by poor 
growth and mortalitj'-j, is produced by adding to the diet additional 
gelatin {!%) , and/or methionine {0^%), An amino acid imbalance 
may affect the deficiency. 

c. Studies with Drs. Haas and Stewart have demonstrated the 
necessity of dietary folic acid for the reproduction of the virus 
of lymphocytic choriomeningitis in the mouse, 

d. Preliminary experiments show that aureomycin has a marked 
sparing effect on folic acid requirements in the mouse, which is 
in contrast to findings with the chick in this laboratory. 

Significance to FIAMD Research: Mice have several important 

' advantages for use in nutrition 

experiments inasmuch as they require little space and consume 
relatively small amounts of food„ At the same time, mice have a 
short life span, a fast growth rate, and rather exacting nutri- 
tional requirements. Thus, the project provides an important basic 
tool for the study of nutritional factors associated with infection 
and various metabolic diseases such as arthritis, obesity, blood 
dyscrasias, and various nervous and nutritional disorders. 

Pr oposed Co urse of P roject ; We antici-pate placing emphasis on 

several phases of the folic acid 
problem, including a study of factors involved in producing a 
deficiency and a study of the antibiotic sparing effect. Additional 
studies will be made on the effect of folic acid deficiency in 
various strains of mice on reproduction of viruses and on leukemis^e 
.\dditional studies are planned on the essential fatty acid require- 
ments (with Dr, Bieri) and on the production of a vitamin B-jo 
deficiency (with Dr. Fox), 



Form Noe ORP-1 
October 19^6 
(Attachment I) 



PUELIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B! Budget Data 



11, NIAMD-^ 



SERIAL ITOMBER 



12, BUDGET 


DATA 


• 

ESTIMTED OBLIGATIONS 




MAN YEARS 


DIRECT 




REIMBURSEMENT TOTAL 


PROF 


OTHER TOTAL 


FY«57'" 










$8,300 




$3,700 $12,000 
BUDGETED POSITIONS 


0»33 


0^66 0,99 

PATIEi\iT DAYS 


PROF 




OTHER TOTAL 




FY857 










0.33 




0.66 0.99 






13 • BUDGET ACTIVITY: 







RESEARCH 

REVIEVJ & APPROVAL 

BIOLOGIC STAiMDARDS 



r^ ADMINISTRATION 

□ PROFESS lOML & 

TECraiCAL ASSIST- 

□ ANCE 



D 



□ 



lli-, IDE^lTIFY ANY COOPERATING UNITS OF THE FJBLIC fMLTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUI©S, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S)! 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBLIC HMLTH SERTTICE - - NATIONAL INSTIIUTFS OF HPIILTH 
INDIVIDUAL PROJECT REPORT 



Part C; Honors, Awards & Publications l^t 



16, PJBLICATIONS OTHER THAN ABSTRACTS FROM THIS PFDJECT DURII-iQ CALHiNDAR 
YEAR 19^6 { 

Haas, V. H», Stewart, S, E, and Briggs, G. Mo Folic acid deficiency 
and the sparing of mice infected with the virus of lymphocytic 
choriomeningitis, Jj Virology (in press), 

17. HONORS AND AWARDS: 

None 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIViroAL PROJECT REPORT 



Part A. Project Description Sheet 



2. NI/iMD 



INSTITUTE OR DIVISION 

4. Nutrition 

SECTION OR SERVICE 



9. 



1. NIAMD...I' 

SERIAL NUMBER 

3. Nutrition and Endocrinology 

LABORATORY, BRANCH, OR DEPARTMENT 



LOCATION 



6. Required nutrients and their metabolic role - The Chick 
PROJECT TITLE 

7. Dr. G. M. 



PRINCIPAL INVESTIGATOR 

8, Dr. M. R. Spivey Fox, L. 0. Ortiz, Dr. J. G. Bieri, Dr. M. 
and C. J. Pollard 



Coates, 



OTHER INVESTIGATORS 



IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PAR.ILLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SFJIVICE (WITHOUT INTERCHMGE OF PER- 
SONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESE/iRCH: 

None 



10. PROJECT DESCRIPTION: 

Objectives : To gain more infoimation of a fundsmental nature on 

the biochemistry, physiology, and nutrition of uniden- 
tified factors, essential fatty acids, folic acid, minerals, amino 
acids, and other nutrients in the chick so that this information 
may be compared with the nutrition of other laboratory animals and 
eventually applied to man. 

Methods Employed; By the use of specially prepared highly purified 

diets composed of all known nutrients (except 
the nutrient being studied) groups of young chicks are reared in 
wire-floored cages under standard centrolled conditions. The effect 
of dietary variation is studied by raeasuremont of weight changes, 
faod consumption, and determination of clinical and biochemical 
changes in the animal and its tissues. 

Ma.ior Findings: (l) Additional studies have been made to improve 

the assay for the unidentified chick growth factor 
found in various crude materials such as soybean meal, egg, liver, 
and yeast. The factor appears to be distinct from the "fish", "whey"- 
and "liver" factors of other investigators. It was not present in 
the ash of cn'.de materials and was partially destroyed by heat. The 
factor may be increasing the growth of the chick by some unlcnown 
indirect mechanism since similar growth increases were obtained in 
several trials with a potent antioxidant. Activity cannot be ac- 
counted for by the presence of known amino acids, vitamins, or 
minerals in any of the crude materials. 



NlAMD-6 -^ 
SERIAL NlIMBlJlR 

(2) Thioctic j'^cid . Thioctic acid (lipoic acid), a growth factor 
for certain micri^organisms and repsrted to be a vitoriin for rats and 
chicks, was f®und to be inactive as a vitamin f-or the chick. Thiectic 
acid gave no increases in growth when added to an amino acid diet, a 
casein-gelatin diet, and diets deficient in glycine, thiamine, vitamin 
B5, chsiline, vitamin B]^2j pantothenic acid, or vitamin K. 

(3) Vitamin E . On a diet low ©r devoid of rancid fat, no vitamin 
E deficiency symptoms were obtained in chicks up to 24 weeks of age. 
This is a new finding and raises the question as to the function of vita- 
min E in chick nutrition. (Dr. Bieri is now studying this point.) 

(4) Minerals . Investigators in other laboratories have reported that 
chicks require an "unidentified" mineral for normal growth. We have fsund 
no evidence for such a need but have found that the National Research 
Council's standards for the chick's requirement of known minerals are 
actually inadequate. Studies are in progress on the identity of the 
mineral, or minerals, low by National Research Council standards. 

(5) Antibiotics . In preliminary studies made by Dr. Marie Coates 
it was found that growth could be suppressed in chicks by giving them 
inoculations of gut contents from older birds. The depression could be 
overcome by feeding penicillin. Germ-free tanks (Reynier's) were used 
to carry <?ut these studies under semi-sterile conditions. Penicillin 
feeding under similar circumstances caused significant growth increases 
in 5 out of 8 experiments in our regular chick room. 

(6) Vitamin B^ . We have been producing various degrees of vitamin 
B6 deficiency in the chick. These chicks have low serotonin values in 
the intestinal wall (Drs. Udenfriend and Weissbach, NHI). 

Significance to NIAMD Research ; The project provides basic information 

essential to the complete understanding 
of nutritional and biochemical relatisnships in metabolism and disease. 

Proposed Course of Pro.lect ; Emphasis will be placed on the unidenti- 
fied growth factor for the chick, its 
identity, and relationship to other nutrients. Studies will be con- 
tinued on factors which replace or spare folic acid and en the mineral 
requirements of the chick. 



Form Wo, ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIA,MD«6 



SERIAL NUMBER 



12, BUDGET 


DATA! 








ESTIMTED OBLIGATIONS 




MAN lEARS 


DIRECT 

Fr«5r ■" " 

$20,800 


REimURSEIffiNT TOTAL 


PROF 


OTHER TOTAL 


#9,900 ll;30,7CO 


•67 


1.82 2.ii5-- 




BDTDGETED POSITIONS 




PATIENT DAYS 


PROF 


Oi'HEK TOTAL 




Fr»57 








.67 


1.82 2,h9 






13 • BUDGET ACTIVITY: 







RESEARCH 

REVIH^r & APPROVAL 

BIOLOGIC STANDARDS 



o 
p 



ADfflNISTRATION 

PROFESSIOmi & 
TECHNICAL ASSIST- 
ANCE 



cu 



lit. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTI-ER ORGANIZATIONS^ PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S)! 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 15. NIAMD-4 

SEPJAL NUMBER 

16. PUBLICATIONS OTHER TIl'iN ilBSTRACTS FROM THIS PROJECT DURING 
CALENDAR YE./IR 1956 s 

Briggs, G. M. Inadequacy ,x.f certain salt mixtures used in studies 
of unidentified growth factors for chicks, Peultry Sci. 35, 
740, 1956. 

Briggs, G. M., and Gay, W. I. Public health significance of drugs 
in animal feeds. In "Symposium of Medicated Feeds" edited by 
Welch, H., and Marti-Ibanez , F., Medical Encyclopedia, Inc., 
New York, p 156 (1956). 

17. HONORS AND AWARDS; 

None 



Calendar Year 1956 
PUBLIS HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVimAL PROJECT REI'^ORT 



Part A, Prsject Description Sheet 1, NIAMD-7 

SERIAL mmm 

2. NIAMD ______„______ 3. Nutrition and Endocrinology 

INSTITUTE OR DIVISION LABORATORY, BRi'iNCH, OR DEPiiRTIvffiNT 

4. Nutrition __>_______ 5. _______„ 

SECTION OR SERVICE LOCATION 

Diabetes - Effect of hormones on metabolism of carbohydrates, fat, 

6 . and pr otei n 

PROJECT TITLE 



?• Dr. R. 0. Scow. Dr. S. S. Ckornick 
PRINCIPAL INVESTIGATOR(S) 

8. 



OTHER INVESTIGATORS 

9. IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PAR/.LLELS RESE/lRCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCIL\NGE OF PER- 
SONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 

None 

10. PROJECT DESCRIPTION: 

Objectives ; To detennine the influence of hormones on diabetes and 

©n the metabolism of carbohydrate, fat, and protein; 
also, to determine the nature and mechanism of the protective action 
of remeving the anterior pituitary gland in experimental diabetes. 

Methods Employed : Various endoc,:ine glands (pancreas, hypophysis, 

thyroid, adrenals, and gonad) are removed from 
young experimental animals (rats, guinea pigs, mice, and toads). The 
animals, either treated with various hormones or untreated, are 
studied by varisas physiological and biochemical techniques: meta- 
bolic balance studies (nitrogen, glucose, fat, and ketone bodies), 
glucose tolerance tests, chemical analyses of carcass and tissue for 
various constituents, and effect of different hormones (insulin, 
pituitary hormones) on blood glucose level and utilization. 

Ma.ior Findings ; An operation for removing 99.5^ of the pancreas in 

the rat and the postoperative care necessary for the 
long survival of these operated rats has been developed. (100$S re- 
moval of the pancreas is practically impossible because of the many 
little lobules (8-15) of pancreatic tissue located along the full 
length of the bile duct and because each lobuJ-u has its own duct 
entering the bile duct.) The diabetes that develops in these animals 



NIAMD-7 " ^ 
SERIAL NUMBER 

is very severe and is comparable to that seen in the dcg and cat, the 
most cettmonly used animals in the study pf diabetes-. Within 19 hours 
after pancreatectomy the rat has hyperglycemia (as high as 750 mg/lOO ml.), 
glycosuria, ketonuria, fatty liver, large amounts of fat in the renal 
tubules (intracellular), and gastric retention. If not treated, the 
rat dies within 48 hours in severe diabetic coma. 

In addition to diabetes, the pancreatectomized rat has mfirked im- 
pairment 0f food abssrpticn. Less than 39^ of the dietary nitrogen is 
absorbed without pancreatin and 84% with pancreatin. If only 95^ of 
the pancreas is removed, food absorption is not impaired. 

The insulin requirement by the 99.5% pancreatectomized rat is very 
high. For each gram of diet 2.5 units of regular insulin are needed to 
keep the daily excretion of glucose at less than 0.1 gm. Within 24 
hours after hypophysectomy the insulin requirement was reduced from 2.5 
to 0,4 units/gram of diet. 

The pancreatectomized rat, when fasted, maintains a high blood 
glucose level (300 gm/lOO ml.); excretes glucose and ketone bodies in 
the urine, and survives very few days. At autopsy, large amounts ©f fat 
are found in the liver and the renal tubules. Hypophysectomy not only 
d.oubled the survival time of fasted pancreatectomized rats but also pre- 
vented the appearance of the above signs of diabetes. 

When treated with insulin ajid growth hormone, the pancreatectomized 
rat retained large amounts of nitrogen and gained body weight , Those 
treated with insulin al©ne and tube-fed the same aiaount of focd did net 
graw. When the daily dosage of insulin was reduced from 12 to 1 unit, 
the retention of nitrogen by the growth hsrmone treated rats was re- 
duced t© zere. When the insulin dosage was increased again to the 
initial levels, there was a time lag of several weeks before the growth 
hormsno treated animals retained nitrogen again.^ When the insulin was 
stepped, most ©f the animals died in diabetic coma^ Growth hormone 
als» stimulated nitrogen retention in the hyp--;physectomized-pancreatec- 
tomized rats when insulin was injected. When insulin was withheld and 
the animals were fed, they died within two days. 

It has been reported (Salter and Best) that insulin acted as a 
growth hormone in hypophysectomized rats. Since these workers had ob- 
served a marked increase in food intake in the growing animals, we 
investigated the effect of force-feeding hypophysectomized rats and 
found that such animals, if given 9 to 11 grams of food daily, gained 
36 grams in body weight in 22 days. Similarly treated rats when given 
3 units of insulin daily grew at the same rate. Chemical analyses re- 
vealed that the nature ©f the body weight gain in the two groups was 
similar: 8% of the gain was protein and 60%, fat. (In normal body weight 
gain 20% is protein and 4 to 6% is fat.) It is concluded that insulin 
acted as a growth stimulant only by increasing the food intake of the 
hypophysectomized rats. 



NIAMD-7 ~ '^ 
SERIAL NUMBER 

Significance to NIAMD Research ; The rat has been used very little in 

studies of diabetes except when made 
diabetic by alloxan injections; it has been thought that the rat could 
not become severely diabetic after extirpation of the pancreas. These 
studies clearly demonstrate that after removal of 99.5% of the pancreas 
the rat becomes diabetic within a few hours and, if desired, it can be 
maintained in good health with insulin and tube-feeding as long as re- 
quired. This provides a new and excellent preparation for studying 
diabetes, especially the acute effects of insulin deficiency, in the 
laboratory without the concomitta,nt extra-pancreatic damage that occurs 
when alloxan is used. 

Proposed Course of Pro.iect ; Using the "totally" pancreatectomized rat, 

in vivo and in vitro studies with conven- 
tional and isotopic techniques will be made to determine the role of 
the anterior pituitary and adrenal cortical hormones in various phenomena 
which occur in the diabetic rat: insulin requirement, protein formation, 
relationship of fat in the liver and kidneys to lipemia and to ketone 
body formation, and metabolism of glucose. 



Form Noe 0RP.1 
October 19^6 
(Attachmen-o l) 



PUBLIC PMLTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



Ho MIAMD~7 



SERIAL NUMBER 



RESEARCH 

REVIEW (i APPROVAL 

BIOLOGIC STAiTOARDS 






12, BUDGET DATA 


• 
• 

ESTIMATED OBLIGATIONS 






MAN YEARS 


DIRECT 


REIMBURSEMEi^IT 


TOTAL 


PROF 


OTHER TOTAL 


FY»57 










'M,900 


$19,600 


G6l,500 


1.50 


3*00 U*5o 




BUDGETED POSITIONS 






PATIEIOT DAYS 


PROF 


OTHER 


TOTAL 




FY! 57 










le^O 


3.00 


U.^0 






13 9 BUDGET ACTIVITY; 









ADMINISTRATION 

PROFESSIONAL & 
TECIWICAL ASSIST- 
ANCE 



Cl 



lite IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 19^7-. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S)! 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 15. NIAMD-7 



SERIAL mmm 

16. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
CALENDAR YEAR 1956: 

Hagan, 3. N., and Scow, R. 0. The effect of fasting on muscle 
proteins and fat in young rats of different ages. iim. J. 
Physiol, (in press). 

Scow, R. 0., and Hagan, S. N. Effect sf testosterone propionate 
on myosin, collagen, and other protein fractions in striated 
muscle of gonadectomized rats. Endocrinology (in press). 

Scow, R. 0. "Total" pancreatectomy in the rat: Operation, ef- 
fects, and postoperative care. Endocrinology (in press). 

17. HONORS AND AWARDS; 

None 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 

Individual Project Report 

Part A. Project Description Sheet 1. NIAMD-8 

SERIAL NUMBER 

2- NIAMD 3. Nutrition and Endocrinology 

INSTITUTE OR DIVISION L.^OR.\TORY, BRANCH, OR DEPARTMENT 

A' Nutrition __„ 5. 

SECTION OR SERVICE LOCATION ~ _™ 

6. Required nutrients and their metabolic function - Fat and fat- 
soluble vitamins 
PROJECT TITLE 

7- Dr. J. G. Bieri ^ 

PRINCIPAL INVESTIGATOR 



8. 



C. J. Pollard, Dr. G. M. Br-ip;pHj_ Dr. M. R. Sp ivev Fox. Dr. M. E. Reid 
OTHER INVESTIGATORS " ~~™~" 



9. IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HE/iLTH SERVICE (WITHOUT INTERCHANGE OF PER- 
SONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEiiRCH; 

None 
10. PROJECT DESCRIPTION: 

Objectives; (l) To study the interrelationships among the essential 

fatty acids in normal and fat-deficient animals in order 
to evaluate the metabolic role of these substances. Also, to study 
their effects on other lipid fractions such as cholesterol, 

(2) To determine the requirement for vitamin E in the chick and 
the rat and the dietary factors which influence the requirement. Also, 
to determine the effect of vitamin E on various metabolic processes 

in order to establish whether or not a metabolic function for the 
vitamin exists. 

(3) To elucidate the mechanism whereby vitamin A metabslism is 
regulated in both normal and diseased individuals by determining the 
factors that affect vitamin A and carotene metabolism in the tissues 
Rf animals. 

Methods Employed; (l) Rats, chicks, and guinea pigs are fed puri- 
fied diets containing varying levels of fats or 
pure fatty acids. The changes in tissue polyunsaturated fatty acids 
and other lipid components are then determined. 

(2) The production of vitamin E deficiency in the chick is ex- 
tremely capricious. The effects of various dietary ingredients on 



NIAMD-S '-^ 
SERIAL NUMBER 

the deficiency of vitejnin E is being studied. The deficiency symptoms 
are being correlated with tissue concentrations of the vitamin. A pre- 
vious study which shewed that chicks on a fat-free diet did not require 
dietary vitamin E is being extended to rats. Enzyme activities in nor- 
mal and vitamin E-deficient tissues are being measured in order to 
ebtain information about the possible metabolic rele ^t the vitamin. 

(3) Studies during this year have been concentrated on the fate of 
vitamin A in the blood. Blood from normal oi vitamin A-deficient rats 
and chicks has been incubated under varying conditions with vitamin A 
ana the rate of destruction of the vitamin determined. 

Ma.ior Findings ; (l) In fat-deficient chicks, dienoic and tetraenoic 

a,cids in the tissues decreased and trienoic acid wss 
formed. Feeding linoleic acid partially reversed these changes. Total 
servim lipids were not changed in fat-deficient hens, but serum choles- 
terol may have been elevated. In normal guinea pigs there was very 
little tetraenoic acid in the serum but a considerable amount in the red 
cells. In fat-deficient guinea pigs, although the trienoic acid in- 
creased in the tissues, there was no change in the tetraenoic acid vrith 
the exception of the red cells, in which a decrease occurred. 

(2) Vitamin E was not required in the diet of chicks on a fat-free 
regimen. When chicks were fed a vitamin E-low diet containing a highly 
unsatiirated oil or a high percentage of Torula yeast, a pathological 
condition (exudative diathesis) sometimes developed, which could be pre- 
vented by vitamin E or a concentrate of Factor 3. Analyses of tissues 
from normal and afflicted chicks have not shown any direct relationship 
between the am^^jaut of vitamin E present and the occurrence of the disease. 
The data suggest that th^ action of vitamin E in the tissues is an in- 
direct, or secondary, action. 

(3) Studies have verified a previous report that when vitamin A is 
injected intravenous].y into rats about one-half of the dose cannot be 
accounted for by analysis of the whole animal within a few hoiirs. None 
is excreted. Further investigation revealed that when vitamin A was in- 
cubated with blood, destruction of the vitomin occvirred. The apparent 
enzymatic activity was destroyed by heating. Most of the activity of 
whole blood resides in the red cells and is associated with the stroma. 
Neither cyanide nor azide inhibited the reaction. Blood from young rats 
was considerably more active than that from old rats. Chick blood had 
little activity. 

Signifi cance to NIAI'^D Rese.arch; (l) Additional information about the 

metabolism of the essential fatty acids 
in different animals should contribute considera.bly to our knowledge of 
the role of unsaturated fatty acids in man. A particularly important 
field at present is the relationship between unsaturated fatty acids, 
serum cholesterol, and atherosclerosis. 

(2) Vitamin 7, is of importance in the nutrition of many animals 
but no definite requirement for it has been established in man. Although 
the vitamin is used clinically, there is little evidence that its use 
is beneficial in such cases as chronic abortion, muscular dystrophy, 
and heart disorders. Information on the function of vitamin S in ani- 



NIAMD-8 " .^ 
SERIAL NUMBER 

mals may lead to an understanding of its significance in hioman nutri- 
tion. 

(3) If an enzyme exists in blood which destroys vitamin A, this 
may be an important factor in the regTilation of the blood level of 
this vitamin. It is known that in various disease conditions the 
blood vitamin A falls to subnormal levels. Also, in normal persons 
with an adequate intake of vitajnin A the blood levels of some indi- 
viduals are characteristically low. 

Proposed Course ©f Projects (1) The changes in lipid components of tis- 
sues when pure, individual unsaturated 
fatty acids are fed to normal and fat-deficient animals will be investi- 
gated. The adequacy of linoleic acid and arachidonic acid in fulfilling 
the fat requirement of the guinea pig and rat will be determined. 

(2) Further nutritional studies of vitamin E deficiency in the 
chick will be performed to determine the dietary interrelationships 
involved. Studies at the enzyme level will also be done. Reproduction 
in the rat fed a vitamin E free diet with the minimum requirement of 
pure essential fatty acid will be investigated. 

(3) Fiirther study of the enzyme system in red cells which destroys 
vitamin A will be carried out in order to ascertain its significance 

in the regulation of the blood level of vitamin A. 



Form Mob 0RP»1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - mTIOMAL INSTITUTES OF HEALTH 
II\IDIVIDUAL PROjn^:.CT REPORT 



Part B: Budget Data 



11 ^ NIAMD-8 



SERIAL NUMBER 



12, BUDGET DATA: 



ESTIMA.TED OBLIGATIONS 




FAN YEARS 


DIRECT 


REIMBURSEMENT TOTAL 


PROF 


OTHER TOTAL 


FY«57"'" 








.■pUjiiOO 


^^11;100 $3^,500 


1,00 


2^00 3«00 




BUDGETED POSITIONS 




PATIEOT DAYS 


PROF 


OTHER TOTAL 




Fy?57 








loOO 


2eOO 3*00 






13. BUDGET ACT 


IVITYs 







RESEARCH 

REVIM & APPROVAL 

BIOLOGIC STANDARDS 



I— I -m I 



ADMINISTRATION 

PROFESSIONAL & 
TECHi^IIGAL ASSIST 
ANCE 



□ 



n 



1U» IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
CTHER CR:}ANIZATI0NS^ PROVIDING FUNDS, FACILITIES, OR PERSOiWJEL 
K)R THIt PaOJET'T IN FY 1951 i IF COOPERATING UNIT IS VJITHIN NIH 
INDICATE SffilAL NO(S)! 



(Use reverse and additional pages, if necessaiy) 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HE/iLTH 
INDIViroAL PROJECT REPORT 
Part C: Honors, Awards & Publications 15* NIAMD-8 



SERIAL NUMBER 



16. PUBLICATIONS OTHER THAN ABSTR/tCTS FROM THIS PROJECT DURING 
CALENDAR YEAR 1956; 

Bieri, J. G., Briggs, G. M. , Fox, M. R. Splvey, Pollard, C. J., 
and Ortiz, L. 0. Essential fatty acids in the chick. 
I. Development of a fat deficiency. Proc. Soc. Exp. Biol. 
Med. (in press), 

Bieri, J. G., Pollard, C, J,, and Briggs, G. M. Essential fatty 
acids in the chick. II. Polyunsatiirated fatty acid composi- 
tion of blood, heart, and liver. Arch. Biochem. Biophys. 
(in press) . 

Bieri, J.^G., Pollard, C. J., and Cardenas, R. R., Jr. Utilization 
of vitamin A and carotene by selenium poisoned rats. Proc. 
Soc. Exp. Biol. Med. (in press). 

17. HONORS MD AWARDS; 

None 



Calendar Year 19^6 

PUBLIC HEALW SERVICE - - NATIONAL INSTITUTES OF HEALTH 
IWDIViroiL PROJECT REPORT 
Part A. Project Description Sheet 1. MIAFiD-9 

Serial mqmbrr 



2» NIAMD 3, Nutritio n and E ndo crinol ogy 

INSTITUTE OR DIVISION^ LABO.RATORY, BRANCH, OR DEPARTMEff 

it. Nutrition 5. 

SECTION OR SERVIUE ISMTinirTTFlDfMrTHAr^ElHESDAy 

Required nutrients and their metabolic role - Vitamin B^p* methionine, 
6, choline, and related compou nds 

PROJECT TITLE 



7. Dr, M« R. Spivey Fox 
PRINCIPAL INVESTIGATOR 



8o Dr. Go M« Briggs, Ligia 0» Ortiz, and Dr, 0« Mic kelsen 

OTHER INVESTIGATORg ' "^ — _- ___ 

9. IF TWIS PROJECT RESFMLES, C0MPLEI4ENTS, OR PARALLELS RESFJiRCW DONE 
ELSEWHERE IN THE PUBLIC HE^'iLTT^ SERVICE (WITHOUT INTERCHANGE OF PER- 
SOMEL, FACILITIES OR FONDS) IDENTIFY SUCl RESEARCH: 

None 

10. PROJECT DESCRIPTION: 

Objectives ; To elucidate further the specific functions of each of 

these nutrients and the relationships among them and 
other nutrients. To assess the deteriorative effect of salts in 
experimental diets and to determine ways by which these may be 
minimized. 

Methods Employed ; Deficiencies of vitamin B-.^, methionine, and 

choline have been produced in the young chick 
by dietary omission and, in some cases, by use of a specific 
antagonist. The effect of various nutrients in increasing or 
decreasing the severity of these deficiencies has been studied. 

Mixes of a wide variety of diet constituents and salts have 
been prepared and the development of rancidity and browning has been 
follswed. 

Major Findings ; 1, Vitamin B -,^ 

a, A purified diet has been devised for the first 
time which produces a severe vitamin B-,„ deficiency 

in nondepleted chicks. Vitamin B,p added to this diet results in 

good growth, -^^ 



.1 v.:!- 



•5.!!»H .0: 



>■ . • ■• I 



NIAiyiD"9 •' ^ 

ba Studies with the chick show that the balance of amino acids 
in the diet is extremely critical in vitamin B^p and methionine 
deficiencies. The precise effects of individual amino acids in this 
area are being studied further. 

c. Studies in the chick with various vitamin B analogs show 
that the 5-hydroxy analog of -vitamin B]_2 has about one- tenth the 
activity of vitamin Bnp arid that this analog does not antagonize 
the vitamin. The nitro-, sulfate-, chloro-, and acetato-cobalamins 
have activities similar to vitamin B^^ 'itself, 

d. Since vitamin B, „ is known to function in hemoglobin 
synthesis, an attempt was made to aggravate the vitamin B-in deficiency 
by adding 1% 3-amino-l,2,U-triazole to the diet (there is suggestive 
evidence that porphyrin synthesis might be depressed by this com- 
pound). Preliminary experiments indicate ihat the most severely defi^.^ 
cient chicks were those on the vitamin B12 free diet.> 

2 . Choline and met hi onin e 

a, 2-Amino-2-methylpropanol-lj, a choline antagonist in the 
rat, when fed to chicks caused depression of growth, fatty infil- 
tration of the liver, but no perosis. The adverse effects of the 
antagonist were completely overcome with additional choline, 

bo With a purified diet containing casein, the choline 
antagonist additions were counteracted much more efficiently with 
choline than with betaine. Additional methionine had a negligible 
beneficial effect, 

c. In chicks fed purified diets free of both fat and choline, 
fatty infiltration of the liver was seen at four weeks of age« This 
fatty infiltration did not occur in either of the single deficiencies, 

3 . Rancidity and browning in diet c 

a. Rancidity in experimental die£s is greatly accelerated by 
certain salts. The rancidity-promoting activity of a salt mixture is- 
not necessarily related to the content of salts which are active 
individually. Anhydrous MgSOL markedly protected against the 
acceleration of rancidity caused by MnSOj. in diet mixes,* hydrated 
MgSOh had a negligible effect, 

b. The acceleration of the "browning" reaction in experi- 
mental diets, Tjhich has been associated with the Na2HP0L content 
of certain salt mixes, has been shown to be due to the hygroscopic 
nature of this salt combined with its basicity. 

Significance to NIAMD Research ; A deficiency of vitamin B-ip ^^ 

well as that of other vitamins 
can be produced more readily in the chick than in any other 
experimental animal. The chick, therefore, is ideally suited to 
the study of vitamin B-]_2 and its nutritional and metabolic relation- 
ships to other vitamins and amino acids. Knowledge of vitamin B2_2 
function gained in this manner should ultimately contribute to 
increased understanding of nutritional problems of man. The choline 



smii 



:al no. 

antagonist, 2-amino-2-metbylpropanol-l, affords another approach 
to the study of choline itself and the interrelationships between 
choline, methionine, and vitamin B-, 2» This knowledge may later 
find application in chemotherapy, as has already been the case with 
other antL-metabolites, 

The rancidity and browning which have been associated with 
dietary salts are important factors in experimental diets due to 
the adverse effects of the initial deterioration plus the fact that 
many nutrients may be affected secondarily. In addition, this 
information may have public health significance since similar 
changes may occur in foods consumed by mans 

Propose d Cour s e of Project i To continue along the same lines as 

previously, with emphasis on defining 
functions of the nutrients being studiede 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
IITOIVIDUAL PRO.JECT REPORT 



Part B'. Budget Data 



11, NIAl'ID-? 



SERIAL NUMBER 



12, 


BUDGET 


DATA: 

ESTimTED OBLIGATIONS 






MAN lEARS 




DIRECT 


REIMBURSEMENT 


TOI'AL 


PROF 


OTHER TOTAL 


FI' 


57 












-?^17,000 5^7,600 


;i^ii,6oo 


1,00 


I5I6 2,16 






BUDGETED POSITIONS 






PATIENT DAYS 




PROF 


OTHER 


TOTAL 




Fit 


57 












1,00 


1.16 


2.16 






13. 


BUDGET 


ACTIVITY! 









RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STAITOARDS 



13 ADMINISTRATION Q 

r^ PROFESSIONAL & 

TECI-MICAL ASSIST- 
1;^ ANCE □ 



lU« IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOm\IEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL WO(S)! 



(Use jreverse and additional pages, if necessary) 



Calendar Year 19^6 



PUBLIC HEALTH SER^TICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors, Awards & Publications l5. NlAMD-9 

SERIAL NUMBER 



16, PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALEimAR 
YK4R 19^6 s 

Fox, Mt, R. Spivey, Ortiz, L, 0., and Briggs, G. M, Effect of 

dietary fat on the requirement of vitamin B]_2 by the chick, Proc. 
Soc. Exp. Biol, Med. (in press). 

Rodnan, R, P., Ebaugh, F. G., and Fox, M. R. Spivey, Survival of 
the avian erythrocyte in vivo. Blood, (in press), 

17. HONORS AND AWARDS: 

None 



Calendar Year 19^6 



PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1^ MIAMD-IO 



SERIAL NUMBER 

2. NIAMD 3. Nutrition and Endocrinology 

INSTITUTE OR DIVISION LABORATORY, BR./INCH, OR DEPARTMENT 

k. Nutrition 5« 

SECTION OR SERVECE LOCATION (IF OIHER THAN BETHESDAJ' 

6s Effect of Ethylene Oxide on Foods 

PROJECT TITLE 

7» Dr« Rn S« Yamamot o 

PRINCIPAL INVESTIGATOR 

8, Dr, 0. Mlckelsen 



OTHER INVESTIGATORS 



9. IF THIS PROJECT RESEJIBLES, COMPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PER- 
SOMEL, FACILITIES OR FWNDS), IDENTIFY SUCH RESEARCH: 

None 

10, PROJECT DESCRIPTION: 

Objectives: To determine the nutritional and biochemical changes 

resulting from exposure of diets to ethylene oxide and 
related gases. 

Methods Employed ; Stock diets (Hunt. Club Dog Food), purified diets_, 

and various constituents of the diets are exposed 
to known concentrations of ethylene oxide for definite periods » Th® 
treated diets, or diets prepared from treated constituents, are then 
fed to rats. The growth of these animals is compared to that of 
animals fed the untreated diet. Chemical and microbioloj.;ical analyses 
are made of the diets to see which nutrients are destroyed.. The 
effect of various conditions, such as pH and moisture content of the 
diet during ethylene oxide treatment, concentration of ethylene oxide, 
and duration of treatment, are examinedc 

Major Findings ; Exposure of the purified and stock diets to ethylene 
oxide causes a major nutritional alteration as shown 
by growth failure and, frequently, death of animals fed the treated 
diets. Analyses of the treated diets and ingredients show that the 
primary damage is to the vitamins, especially thiamine c 

Death occurred when rats were fed treated diets containing 
choline chloride or choline bicarbonate, whereas a reduction of 



\ 



MIAMD-10 - l^ 
SERIAL NO. 

growth but no deaths occurred with treated diets containing choline 
citrate or choline bitprtrate. Diets prepared from ethylene oxide 
treated vitamin supplements containing choline chloride reduced the 
growth of rats and produced death in 5 weeks, while diets containing 
choline citrate or choline bitartrate caused normal growth. The 
thiamine content of the diets paralleled rat growth. 

Perfectly dry stock diets showed very little growth inhibition 
following ethylene oxide treatment. Water added to the diets prior 
to ethylene oxide exposure increased the nutritional derangement to 
an extent that was proportional to the concentration of water in the 
diet. 

If the concentration of ethylene oxide used in treating the 
stock diet is increased, death of the rats occurs when fed the stock 
diet so treated, A 3-hour exposure of the puri.fied diet to ethylene 
oxide caused death within 6 weeks, whereas shorter exposure resulted 
in slow growth but no death. The relationship of the amount of 
ethylene oxide used to the amount of diet treated was found to be 
more critical than the amount of ethylene oxide to the size of the 
treatment chamber. 

Significance to NIAMB Research: Ethylene oxide is being used for 

the sterilization of some food 
products. It is being used to a greater extent in the sterilization 
of a variety of pharmaceutical products, especially those which can- 
not be subjected to heat sterilization. 

The nutritional changes produced in diets by ethylene oxide 
treatment, especially the destruction of i/itamins, is of importance 
in nutrition and toxicology. The results continue to emphasize the 
fact that the testing of treated foods should include nutritional 
as well as toxicological studies even though residues of the partic- 
ular agent do not remain in the foodo Testing foods for the presence 
or absence of the original toxicant is not sufficient » 

Proposed Course of Proj ect; We will continue to study the mechanisms 

involved in the destruction of vitamins 
in purified and stock diets treated with ethylene oxide « Also, we 
will attempt to determine the conditions necessary for this destruc- 
tion, as well as to study the compounds formed as a result of 
ethylene oxide treatment. 



Form No* 0RP-.1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11, NIA1«ID-10 



SERIAL NUMBER 



12, BUDGET DATA: 



ESTIMATED OBLIGATIONS 



DIRECT 



REIMBURSEMENT 



TOTAL 



FY-! ^7 



i;i;20,800 



¥9,900 



:a;30,700 



I 



BUDGETED POSITIONS 



1 PROF OTIER 




TOTAL 








FY '57 












1 1,00 1.83 




2.83 








13. BUDGET ACTIVITY: 












RESEARCH 


bJ 




ADMINISTRATION 


n 


REVIEW & APPROVAL 
BIOLOGIC STANDARDS 


i 

n 




P 


ROFESSIONAL St 
TECHNICAL ASSIST- 
ANCE 


-1 



mN YEARS 



PROF OTHER TOTAL 



IcOO 1,83 2,83 



PATIEIC DAYS 



Iho IDENTIFY ANY COOPERATING UNITS OP THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS^ PROVIDING FUNDS^ FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957c IF COOPERATING UNIT IS VJITHIN NIH 
INDICATE SERIAL NO(S)} 



(Use reverse and additional pages, if necessary) 



•vo. 



Calendar Year 19^6 



PUBLIC HEA.LTH Sp-^RVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors, Awards & Publications 1^, NIAMD-IO 

SERIAL NUMHSR" 



16. PUBLICATIONS OTHER THAN ABSTPACTS FROM THIS PROJECT DURING CALENDAR 
YEAR 19561 

Bakerman, H*, Romine, M.j Schricker, J, Aa^ Takahashi, S. M,, and 
Mickelsen, 0. The stability of certain B vitamins when exposed 
to ethylene oxide in the presence of choline chloride. Jo Agricc 
Food Chem, 1;: 956, 1956, 

Mickelsen, 0^ Is toxicology enough for a food protection program? 
J. Am. Diet. Assoc-, (in press) 

Mickelsen, 0« Is toxicology enough? Nutr, Reve (in press), 

17. HONORS AND AWARDS; 

None 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part A, Project Description Sheet 1, NIAMD-11 



SERIAL NUMBER 

2j NIAMD 3-! Nu triti on and E nd ocrinology 

INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTMENT 

kr Nutrition $<> ■ 

SECTION OR SERVICE " LOCATION (IF OTHER THAN BET!-TESDA) 

6 4 N utritional requirements and factors influencing them - The guinea pig 
PROJECT TITLE 

7. Dr^ Mary Et Raid 

PRINCIP/iL INWSTIGATOR ~~" 

8. Dr, G. M. Briggs, Mary G, Martin, Dr, J, Go Bieri 

OTHER INWSTIGATORS 

9. IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR P-'VRALLELS RESE'.RCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PER* 
SONNEL, FACILITIES OR FU^^DS) IDENTIFY SUQI RESEARCH: 

None 

10. PROJECT DESCRIPTION: 

Objectives ; To determine (a) effects of deficiency of different 
nutrients (proteins, carbohydrates, fats, minerals, 
vitamins) in the guinea pig, (b) the quantitative requirement for 
such nutrients, and (c) to study their physiological action, the 
pathological effects of deficiency, and the interrelation with other 
nutrients i 

Methods Employed ; Guinea pigs at 2 to 5 days of age, weighing 

approximately 100 grams, were placed on specially 
prepared experimental diets complete in all nutritional factors known 
to be required by the guinea pig except the one (or ones) under 
investigation. The effect of dietary variation was studied by 
measurement of weekly weight changes and the biochemical and path- 
ological changes in the animal and its t is sues ^ including study of 
the blood picture^ 

Major Findings ; Folic Acid/citrovorum Factor . Results suggest 

that the citrovorum factor is either twice as 
effective as folic acid or that the guinea pig may be able to use 
the D- form of +he compound, A test with pure citrovorum factor 
(L-L- form) wil^ be conducted in an attempt to answer the question. 



MEAMD-ll - L 

Wi acln/Tryptophan Interrelations , With respect to gain in 
weight, 2«5 g. of D^L- tryptophan are equivalent to 20 mgc of niacin 
per kgr, of diet containing 20% purified soybean protein. However, 
the general condition of the animals receiving niacin appeared to be 
somewhat better, possibly because of a lessened tendency to diarrhea. 

Vitamin '^■yi' '^'^'^ ^^^ first time a vitamin B^2 deficiency has 
been produced xn the guinea pig; it results in poor growth and sur- 
vival with no alteration in the blood picture e 

Amino acid diet. Replacing the 20^ soybean protein with an 
equivalent amount~of amino acids that would be contained therein 
resulted in a slower body weight gain^ 

Inositol. K deficiency of inositol has been produced for the 
first time by using the above amino acid diet, 

Celluloseo Studies in progress confirm the need of an unidenti- 
fied Tactor in alfalfa which spares the guinea pig's requirement for 
dietary cellulose^ Attempts to determine the chemical properties are 
in progressa It is known that the ash of alfalfa is inactivee 
Concentrates of the "anti-ulcer" factor from Merck and Coo are inactivec 

S ignificance to NIAi VID Research ; Since the guinea pig, because of 

certain peculiarities in its 
metabolism, may prove to be of special value for the study of meta- 
bolic diseases such as arthritis, symptomatic epilepsy, muscular 
dystrophy, sclerosis, anemias of different types, degenerative 
defects in heart and blood vessels, dental defects, etcc, it is 
highly important to increase our present knowledge of its nutritional 
requirements, 

proposed Course of Project; Studies on the above-mentioned projects 

will be continued with special emphasis 
on the production of deficiencies with amino acid diets (no protein). 



Form No» 0RP«-1 
October 19^6 
(Attachment l) 



FUELIC HEALTH SERVICE - MA.TIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part Bs Budget Data 



11, NIAMD-11 



SERIAL IWIBER 



12 . BUDGET 


DA 


TA: 
ESTIMATED OBLIGATIONS 




MAN YEARS 


DIRECT 




REIMBURSEMENT 


TOTAL 


PROF OTHER TOTAL 


FY '57 










^9,200 




;i^3,900 

BUDGETED POSITIONS 


il3,100 


1,33 1.33 

PATIENT D/iY3 


PROF 




OTHER 


TOTAL 




FY'57 










- 




1633 


1.33 





13 e BUDGET AC TIVITY; 
RESE/iRCH 

REVIEW & APPROVAL 
BIOLOGIC STANDARDS 






ADMINISTRATION j [ 

PROFESSIONAL & 
TECHIMICAL ASSIST- 
ANCE □ 



ll|» IDENTIFY ANY COOPER/lTING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FU1\DS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 195? a IF COOPERATING UNIT IS IJITIDIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBLIC HEi\LTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

PartrCf Honors, Awards & Publications 15. MlAMD-11 



SERIAL NUMBER 

16. RTBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR 
YEAR 19^S: 

Reid, M, E. The nutritional requirement of the guinea pigs The 
National Research Council (in press) ^ 

Reid,. M. Er, The Guinea Pig in Research, Washington, D^ Ce, The 
Human Factors Research Bureau, Ine, (in press). 



J? M^ npp 1 Calendar Year 19^6 

Form No . ORr-i 

December h> 19^6 

(Attachment I) 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet ■ 1 . NIAMD~X2 

^^^ A ^ J ^ SERIAL NUMBER 

National Institute of Arthritis Laboratory of Nutrition 

2. and Metabolic Diseases 3- and Endo crinology 

TOITUTTOR" DIVISION LABORATORY, BRANCH, OR DEPARTMENT 

Section on Experimental 
^* SECTION^OrS ERV/lCE ' LOCATION (IF OTH'iR TH/UV BETHESnAT 

Isolation and General Significance of Factor 3 Against Dietary 

6 . Necrotic_Deg eneration 

PROJECT TITLE 



7 . K Schwarz 



PRINCIPAL INVESTIGATOR 
B.C. Foltz 



OTHER INVESTIGATORS 
9. NO PARALLEL RESEARCH 

10. PROJECT DESCRIPTION 

Objectives: To isolate and chemically identify Factor 3, a vitamin- 
like substance which prevents dietary liver necrosis in 
the rat, and to clarify its biochemical and medical 
significance in general . 

Methods Employed: Fractionation and isolation techniques, worked 

out step by step for Factor 3:. are used for the concentration 

of the active substance from natural sources. Chemical group 
reactions, various physical and chemical analytical methods, 
paper chromatography and spot tests are employed for clarification 
of its characteristics. Dietary liver necrosis in the rat, and 
heart necrosis in the mouse, are produced by vitarain E free 



NIAMD-12 " ^ 2 

SERIAL NO, 

semi synthetic Torula yeast diets. The Factor 3 fractions 
are tested in a prophylactic rat assay against liver necrosis 
geared to a capacity of 30 groups weekly (300 inbred rats). 

Major Fi ndings ; Two different forms of Factor 3 have been recognized^ 
(a-F3 and P-F3) . P~F3 is extractable with phenol from water, 
a-F3 does not enter into the phenol phase. In the preparation 
of Factor 3 concentrates from natural sources the two are found 
in different fractions. A method, the Ba ethanol process, was 
developed for purification of 0-F3. It has been adapted to 
pilot plant scale. For such a-F3 preparations, six subsequent 
steps have been worked out for further purification in the 
laboratory. Workable quantities of concentrated a-F3 are thus 
obtained routinely. 

Concentrated a-F3 is strongly anionic, soluble in water, 
insoluble in organic solvents and stable against oxidation. 
There is no loss of activity on repeated concentration of 
aqueous solutions at reduced pressure. The properties of a-F3 
make possible the application of selective fractionation 
techniques, such as chromatography, for the isolation of the 
active material in crystalline form. 

A study, in collaboration with Dr. W -, DeWitt, NIAID, of 
the effect of the necrosis producing, deficient Torula diet on 
mice was brought to conclusion. A new disease entity, multiple 
necrotic degeneration (MND) was described. Necrosis of the 
heart muscle predominates in KW<, Necrosis of the liver, the 
kidneys and of peripheral muscles, and severe pancreatic atrophy 
accompany the heart damage. The changes are prevented by 
Factor 3 concentrates . 

The pilot plant scale preparation at Merck, Rahway, of 
casein VI, a F3 free casein, was supported by testing a series 
of casein preparations in the prophylactic rat assay. With the 
casein VI, Dr^ J, F. Brock, University of Cape Town, South 
Africa, studied the effect of a F3-free formula on kwashiorkor. 
The latter is a deficiency disease frequent in infants in 
tropical and subtropical areas, but also present in U.S.A. 
The material was capable of initiating cure of the acute 
disease during a 21 day trial similar to untreated casein. 
Whether Factor 3 accelerates cure and x\Thether it is required 
for its consolidation remains to be seen. 

Significance to NIAMD Research; Factor 3 has the characteristics of 
a new B vitamin, is active in preventing marked and fatal 
anatomical lesions in several species and has a demonstrated 
role in maintaining normal tissue respiration. It can be 
anticipated to have an important role in biochemical processes 
and be of significance in human diseases. 



NIAMD-12 -3 

SERIAL NO, 3 

Proposed Course of the Project; Continuation of the current fraction- 
ation and isolation program in order to obtain Factor 3 in pure 
form, to identify its physical and chemical charact-ristics, to 
elucidate its role in metabolisra, and to make it available for 
clinical research. 

Attempts are being made to produce bacterial mutants 
(E. colij Str. faecalis) requiring Factor 3 for growth, to be 
used for the development of a microassay for Factor 3, Long 
term experiments to investigate uncomplicated Factor 3 
deficiencies in rats and mice are under way. A study, with 
Dr. L. Groenberg, U. of Calif., San Francisco, to clarify 
the effect of Factor 3 concentrates on deficient Rhesus 
monkeys is scheduled. The metabolic functions of F3 are 
part of a separate project (see Dr^ Green). Clinical studies 
are planned as soon as a-F3 has been obtained in pure form. 



Form Noo ORP-1 
October 1906 
(Attachment I) 



PUBLIC HEAI,TH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11 9 NIAMD-12 



SERIAL NUMBER 



lii» bUULJiiT Ul 


i. TA ! 








ESTIMATED OBLIGATIONS 


M/lN YEARS 


DIRECT 


REIMBURSEI4ENT 


TOTAL 


PROF OTHER TOTAL 


FY«^7 








119,600 


ii9,700 


^p29,300 


1,33 I9OO 2,33 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHER 


TOTAL 




FY«57 








1.33 


1«00 


2.33 





13* BUDGET ACTIVITY; 
RESEARCH 

REVIEV7 & APPROVAL 
BIOLOGIC STANDARDS 



LaJ ADMINISTRATION 

i 1 PROFESSIONAL & 

TECHNICAL ASSIST- 

iZ2 ^'^^ 



u- 



□ 



lit* IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HE/.LTH SERVICE, OR 
OTIiER ORGANIZATIONS, PROVIDING FUl^iDS, FACILITIES, OR PERSONAL 
FOR THIS PROJECT IN FY 195?^ IF COOPER.\TING UNIT IS WITfflN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



';;;;j 






\/;:^t^^_i- 



Form No. OR^-l Calendar Year 19^6 

(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEA-LTH 
INDIVIDUAL PROJECT REPORT 

Part Ci Honors^ Awards & Publications ■ 1^. NIAMD-12 



S'^L-;.IAL NUMBER 



16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT 
DURING CALENDAR YEAR 19^6: 

1. Schwarzj K.j Chernick^ S. S»j Rodnan, G. P.-j Moej Jc G.^ 
and Mertzj W«3 Dietary Necrotic Liver Degeneration: 
Occurrence of a Specific Metabolic Defect Reversible 

by Intraportal Vitamin Eo Report on the 3rd Inter- 
national Vitamin E Congress j Venice j Italy, Vitamina 
E Atti Del Terzo Congresso Internazionale Venezia, 1955' 

2. Schwarzj K.j The Effect of Antioxidants on Dietary 
Necrotic Liver Degeneration. Proc . Soc . Bxp= Biol, and 
Med. in press » 

3. DeVifitt, W, B>., and Schwarz , Ko, Multiple Dietary Necrotic 
Degeneration in Mice. Heart, Liver j Muscle and Kidney 
Lesions on a Factor 3 Vitamin E-Free Tcrula Yeast Ration •- 
Experientiaj in press. 



17'. LIST HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 1956: 

None . 



Form No. ORP-1 Calendar Year 1956 

(Attachment I) 

PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES 0.F HEALTH 

INDIVIDUAL PROGRESS REPORT 

Part A. Project Description Sheet 1. NIAMD-13 



SERIAL NUMBER 

National Institute of Arthritis Laboratory of Nutrition 
2. and Metabolic Dis e ases 3' and Endocrinology 

INSTITUTE OR DIVISION ' LABORATORY, BRANCH, OR DEPARTMENT 

Section on Experimental 

U • Liver Diseases 5 . - - - 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 

General Characterization of the Dietary Glucose Tolerance Factor (GTF), 

6. and Clarification of its Mode o f Action in Glucose Utilization. 

PROJECT TITLE 

?• Wu Mertz and K» Schwara 



PRINCIPAL INVESTIGATOR 



8. 



OTHER INVESTIGATORS 
9. No Parallel Research 

10, PROJECT DESCRIPTION 

Otejeotives: To clarify, in general, the biochemical and medical 
significance of the glucose tolerance factor (GTF), 
a newly discovered dietary principle necessary for 
maintenance of normal intravenous glucose tolerance 
in rats; to isolate and chemically identify GTF. 

To study the mechanism by which the liver regulates 
the uptake of glucose by tissues . To extract and 
identify the humoral factors from liver which 
accelerate peripheral blood glucose removal, and to 
investigate the interaction of the dietary GTF with 
this mechanism. 



NIAMD-13 ' ^ 

SERIAL NO. 2 

Methods Employed; Glucose removal rates are determined following 
intravenous injection of glucose in fasted rats reared on 
various diets^ especially those deficient in the glucose 
tolerance factor (GTF) . Tests for GTF are performed in pre- 
tested, deficient rats, 18 hours after the supplementation 
of active preparations by stomach tube or by injection. 
Natural sources are assayed with pretested, deficient rats 
after 6 days of dietary supplementation. Conventional ex- 
traction and fractionation techniques are used for elaboration 
of a step-wise concentration process for the isolation of GTF. 

Total abdominal evisceration of rats is performed and 
glucose remioval is measured using continuous infusion with 
and without injection of liver extracts specifically obtained 
from various animals for this purpose. 

Major Findings: Impaired glucose removal rates were discovered in 
rats on a variety of dificient diets, and extracts from natural 
materials were shown to contain a substance which restores the 
deficient glucose tolerance to normal. The effect was originally 
thought to be caused by Factor 3 against liver necrosis. However, 
feeding certain diets containing Factor 3 produced the impaired 
glucose tolerance, and subsequent fractionation of active pre- 
parations led to a clear chemical separation of GTF from Factor 3. 
Active GTF preparations are obtained as byproducts of the Factor 3 
concentration process. They reverse deficient removal rates to 
normal after stomach tubing of 1 mg per 100 g of body weight. 
GTF is not identical with any known dietary factor such as amino 
acids, conventional vitamins or minerals. It is water soluble, 
extractable with isebutanol, apparently of low molecular weight 
and stable against acid hydrolysis. Thus far, GTF is known to 
occur in kidney, and also in Brewer's yeast. Semi -synthetic, 
purified casein diets were found to be deficient in this factor, 
as compared to McCollums lactating diet which maintained optimal 
removal rates . 

A method was developed to obtain extracts from livers cf 
normal animals which are effective in restoring the impaired 
glucose tolerance to normal and to above-normal rates immediately 
after injection of very small amounts. Extracts from livers of 
GTF deficient animals were only l/U as effective. In eviscerated 
rats, peripheral glucose tolerance is approximately l/3 of normal. 
In these animals the dietary GTF is ineffective, whereas the 
active liver extracts produce an increase of glucose utilization 
toward normal. The liver principle, which seems to depend on 
dietary GTF, is water soluble and stable against short heating. 
It is slowly inactivated at room temperature, but not during 
storage at -l^o , 



NIAMD-13 ~ : 
SERIAL NO. 



Si gnificance to NIMD Research; GTF has the diaracteristics of a 
new vitamin t It can> therefore} be expected to be of funda- 
mental biochemical and physiological importance • It may be 
of clinical interest in several human diseases , especially in 
diabeteS) where dietary therapy is essential. 

These studies show that the liver produces a principle 
which enhances peripheral glucose uptake . This opens a new 
approach to the mechanism of blood sugar regulation) which is 
of fundamental importance in physiology and also in disease 
processes, such as diabetes , liver disease 5 and the terminal 
phase of cancer. 

P roposed Course of the Project; Continuation and further develop- 
ment of the fractionation with the aim of obtaining the GTF 
in pure formj to clarify its chemical and physical character- 
istics} and to make it available for clinical research. The 
natural distribution of GTF in dietary ingredients is under 
preliminary investigation o The interference of varying levels 
of fat and other dietary constituents with GTF utilization and 
supply will be studied. Its effects on experimental metabolic 
diseases > such as diabetes > will be analyzed l 

The relation between the dietary GTF and the active liver 
principle will be investigated by assaying of liver extracts 
from rats maintained with; and without; the dietary GTF. 
Attempts will be made to concentrate the active principle from 
liver} using intact rats with impaired glucose removal rates 
for testing. Vfith more purified preparations the mechanism of 
action will be studied on isolated organs. The chemical and 
metabolic relation between the dietary GTF and the active 
principle from liver will ba investigated. 



Form No* ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11 » NIAMD-13 



SERI/tL NUMBER 



12 e BUDGET DATA! 








ESTIMATED OBLIGATIONS 






MAN lEARS 


DIRECT 


REIMBURSEMENT 


TOTAL 


PROF 


OTHER TOTAL 


FYi 57 










116,700 


47,500 

BUDGETED POSITIONS 


$214,200 


0.33 


2,00 2.33 

PATIENT DAYS 


PROF 


OTHER 


TOTAL 




FY'57 










0.33 


2.00 


2.33 







13. BUDGET ACTIVITY; 
RESEARCH 

REVIEW & APPROVAL 
BIOLOGIC STANDARDS 



|n ADMINISTMTION 

071 PROFESSIONAL & 

_J TECHNIC/.L ASSIST - 

r I ANCE 



□ 



□ 



li;« IDENTIFY ANY COOPER/.TING UNITS OF THE PUBLIC HE/xLTH SERVICE^ OR 
OTHER ORGANIZATIONS, PROVIDING FUI\1DS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 195? » IF COOPERi.TING UNIT IS VJITHIN NIH 
INDICATE SERIAL N0(S)8 



(Use reverse and additional pages, if necessary) 



• 'I .• ■ • J 



Form Noe ORP-1 Calendar Year 1956 

(Attachment I) 

PUBLIC HEALTH S^^RVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications i^ . NIAMD~13 



SERIAL NUI'IBER 



16. LIST PUBLICATIONS OTHER THAN A3STRACTS FROM THIS PROJECT 
DURING CALENDAR YEilR 19^6: 

1. Mertz, ¥.3 and Schwarz, K., The Reversal of Respiratory- 
Decline in Necrotic Liver Degeneration by Intraportal 
Antioxidants, Proc. Soc. Exp^ Biol, and Med., in press. 



17. LIST HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT 
DURING CALEI^IDAR YEAR 1956: 

None . 



Form No. ORP-1 Calendar Year 19^6 

(Attachment I) 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HE^XTH 
INDIVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1. miaiv[D~1U 



SERIAL NUMBER 

National Institute of Arthritis Laboratory of Nutrition 

2. a nd Metabolic Dise ases 3» and Endocrinology 

INSTITUTE OR'DIHSION OrB0R.'[TaSY7~BRANCH , OR DEPARfFlENT 

Section on Experimental 
h' Liver Diseases 5. _ _ _ 



SECTION OR SERVICE ' LOCATION (TF"OTHER THAN BETHESDA) 

The Mode of Action of Vitamin Ej and the Metabolic Events in the 

6o D evelopment of Dietary Liver Necrosis 

PROJECT TITLE ^ " 



7 • M . R . Green 



PRINCIPAL INVESTIGATOR 



8, K. Schwarz, W. Mert z and M . G. Piccardo (Guest ) 
OTHER INVESTIGATORS 



9. NO PARALLEL RESEARCH 



10. PROJECT DESCRIPTION 

Objectives: To identify the active form of vitamin Ej and to clarify 
its role in intermediary metabolism. To determine the 
chain of events which lead from nutritional deficiency, 
through metabolic alterations, to the final, acute 
necrotic liver degeneration . 

Methods Employed; Tissue metabolism studies (Warburg technique) were 
performed on slices from livers at various stages of the de- 
ficiency. Effects of antioxidants and of vitamin E derivatives 
on respiratory decline were analyzed in vitro, and also follswing 
intraportal injection. Extracts from liver homogenates were 
prepared and fractionated chemically and by paper chromatography j 
to identify the active forms of vitamin E. Isolated rat livers 



NIAMD-lU ' ^ 2 

SERIAL NO. 

were perfused with isotopxcally labeled tocopherol to study 
uptake and conversion. Ultra thin sections from various 
disease states were prepared and investigated by electron 
microscopy. 

Majer Findings: Using a 2-step Warburg technique > an exact 

comparison was made of the effect of different levels of 
vitamin E injected by different routes . Intraportal in- 
jection produced a dose response curve identical to that 
obtained following peripheral injection of vitamin E in the 
reversal of respiratory decline of liver slices « 

The comparison of vitamin E with lli different synthetic 
antioxidants in three different assay systems was brought to 
a conclusion,, The substances were compared (l) by addition 
to the liver necrosis producing diet (2) by injection into the 
portal vein in a 2-step Warburg technique to analyze their 
potency to reverse respiratory decline j and (3) by addition 
to the Warburg medium to estimate their in vitro effect on 
respiratory decline. Several antioxidants! notably DPPD 
(NjN'-diphenyl-phenylenediamine) were highly effective in 
preventing dietary necrotic liver degeneration. In the 
intraportal injection^ they were equally active in the 
reversal of respiratory decline -, and in the in vitro Warburg 
assay, they were highly effective in preventing the respiratory 
lesion of deficient liver slices. For all three systems, the 
same general scale of comparative activity was found, except 
for vitamin E. The latter is inactive when added to the 
Warburg medium. It was postulated that tocopherol is converted 
into an "active form" in intermediary metabolism o 

Tocopherol derivatives, such as tocopheryl hydro- 
quinone, tocopheryl quinone, and tocopheryl epoxide, were 
assayed and found to be inactive. However, two new, 
recently isolated metabolites of vitamin E, 2-(3-hydroxy- 
3-methyl-5-carboxypentyl) -3j5-6-triraethylben2oquinone and 
its If -lactone, were found highly effective in the i n vitro 
system. 

A rat liver perfusion system was developed permitting 
recycling of small volumes of plasma through isolated livers 
for periods of l/2 to 1 hour. Fractionation and paper 
chromatography methods for liver homogenates were devised 
for the identification of the active vitamin E metabolite » 

A hitherto undescribed histological lesion was discovered 
upon coordinating the metabolic study with electron microscopy. 
Serious changes can be detected in liver cells after 7 days 
on the deficient, liver necrosis producing diet. The ergasto- 
plasmic lamellae degenerate. The ergastoplasm is converted 



SERIAL NO. 

into a cystic structure o The mitochondria become greatly 
swollen, with diminished density of their matrix. The 
external nuclear membrane becomes irregular and partially 
detached from the nucleus . A characteristic degeneration 
of the Golgi apparatus develops after 10 to 13 days of 
dietary treatment. At this tiraej the majority of liver 
cells has undergone the changes of the ergastoplasm and 
also the mitochondria} and respiratory decline is observed. 

Signi ficance to NIAM D Research: It is of basic importance to 
clarify the modi" of action of vitamin E in intermediary 
metabolism about which no conclusive information is avail- 
able at presents From the metabolic studies, information 
may also be obtained on the metabolic role of Factor 3" 
This would be of value in developing an in vitro test system 
for the latter (see project Schwarz), and in defining the proper 
indications for its clinical application v 

It is of general significance to clarify the sequence 
of events which lead from functional enzymatic changes 
through submicrosGopic lesions to final gross necrosis and 
to death- Necrosis is a frequent pathological alteration 
seen in many diseases, and little is known about the 
mechanism through which it develops » This study may be 
expected to contribute to a better understanding of its 
evolution, thus leading to effective ways of prevention 
and treatment 4 

Proposed Course of the P roject; Using labeled tocopherol, and 

chemical fractionation techniques, attempts are being made 
to identify the tocopherol metabolite which is active in 
the metabolism of the liver ;. Cell fractionation and enzyme 
studies are aimed at localizing its site of action which 
from previous experiments is presumed to be related to the 
oxidative systems of intermediary metabolism. Further 
studies will be aimed at the question of interrelation of 
the function of Factor 3 with that of vitamin E. 



Form Noo ORP-1 
October 1956 
(Attachment I) 



PUBLIC HE/.LTH SERVICE - M/.TIONAL Il^TITUTES OF HEALTH 
' INDIVIDUAL PROJECT REPORT 



part Bs Budget Data 



lie MI/iMD-lU 



SERIAL IWMBER 



BUDGET DATA: 

ESTIMATED OBLIG/iTIONS 


' 




M\M YEilRS 


DIRECT REIMBURSEMEIC 


TOTAL 


PROF 


OTHER TOTAL 



$31,800 


$12,100 


$43,900 


1,33 


3.00 h.33 






BUDGETED POSITIONS 






PATIENT DAYS 


PROF 




OTIffiR 


TOTAL 




FY'57 












1.33 




3.00 


U.33 






13* BUDGET 


ACTIVITY: 









RESE/.RCH 

REVIEVr & APPROVAL 

BIOLOGIC STAND/.RDS 



bd 



! I 



AffllNISTRATION fH 

PROFESSIOKJ. & 
TECHI\[I&;L ASSIST- 
ANCE I I 



111. IDENTIFY ANY COOPER.:TIWG UNITS OF THE PUBLIC I-EALTH SERVICE, OR 
OTHER ORGi".NIZ.'.TIONS, PROVIDING FTOffiS, F.:CILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957 » IF COOPERi'.TING UNIT IS WITHIN NIH 
INDICATE SERL'.L NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 195 6 
PUBLIC HEALTH SERVICE -- NATIONAL INSTITUTES OF liEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NIAMD-lg 



SERIAL NUMBER 

2 , NIAM) 3 • ^'^^'ti?^—^''^ EndoGrinology 

INSTITUTE LABORATORY"' 

h . Fractionation and Isola tion 
SECTION"' 

6. Large-scale processing of "biologic al m aterial 
MOJECT~TITLE ~ 

7 . J. C . _Kere_sz te S3'- „ 

PRINCIPAL" INVESTIGATOR ' 

o 
O. 



OTHER INVESTIGATOR 

9. No parallel research 

10. PROJECT DESCR IPTION: 

Objective s; Many problems of importance in the biochem- 
istry of disease require the isolation and identification of 
substances which are present in only tra.ce amounts in the 
particular biological source. It becomes necessary, in 
order to obtain sufficient quantities of the desired compo-ond 
for study; to process large amounts of biological materials , 
such as liver^ brains, excretory products, plant materials, etc, 

Methods emp lo ^rcd : The Itiboi-atory is equipped with 
large-scale apparatus, such as stills, filters, reaction 
and extraction kettles, etc. In most isolation problems the 
original small-scale process has to be modified and developed, 
so that it can be carried out efficiently on the larger scale . 
This adaptation or process development is an important func- 
tion of the laboratory. The degree of participation of the 
laboratory varies according to the needs of the specific 
"oroblems . 



NlAMD-^lg ^^ 
SERIAL NO. 

Maj o r finding s: Several tons of horse liver have been 
processed for prefolic acid concentrate. Large quantities 
of bvilbs, leaves, other plant and animal materials have been 
extracted for the Natural Products Laboratory (Mil). Various 
large scale services were performed for other investigators 
at NIH. 

Signif i cance to arthritis arid M etabolic research: 
Ordinary laboratories lack facilities to effectively carry 
on studies which require the extraction and processing of 
large quantities of biological materials. However;, with the 
equipment and trained personnel of the large scale laboratory, 
such investigations as the isolation of the prefolic acids 
can be undertaken and successfvilly completed. 

Propo sed co u rse of proj ect : The equipment for the safe 
handling of volatile and toxic solvents is being installed. 
With these new facilities, large scale operations can be 
carried on which have been delayed because of the hazards 
with the present equipment. 



Form No, ORP-1 
October 1956 
(Attachment l) 



PUBLIC HE/vLTH SERVICE - NATIONAL INSTITUTES OF HEi'iLTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11. MAMD-l^ 



SERIAI. IWMBER 



12 e BUDGET DA 


TA; 
ESTD-J/.TED OBLICklTIONS 






MkN YE/^S 


DIRECT 


REIMBURSEMENT 


TOTAL 


PROF 


OTHER TOTAL 


FY<57 

$23,900 


i;9,ioo 

BUDGETED POSITIONS 


133,000 


iu5o 


2.00 3^50 

R\TIENT BI.YS 


PROF 


OTHER 


TOTAL 




FY'57 










3.50 


2^00 

rPTITTTlV . 


5.50 







RESafvRGH 

REVIEW & APPROVAL 

BIOLOGIC ST/aNDARDS 



r? administr.;tion Q 

r~\ PROFESS lONilL & 

TECHNICi'.L ASSIST- 
JZ] ^'-NCE □ 



IH, IDENTIFY ANY COOPER/.TING UNITS OF THE. PUBLIC HF'lilLTH SERVICE, OR 
OTHER OR&;NIZi\TIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 195? ^ IF COOPER/.TING UNIT IS WITHIN NIH 
INDIG^TE SERI/.L NO(S): 



(Use reverse and addJ.tional pages, if necessary) 



Calendar Year 195 6 

PUBLIC HEALTH SERVICE -- MTIOML INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 15 . JiiAiiD-jA. 

SERIAl NUS^R" 



16. LIST OF PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS 
PROJECT DURING CALENDAR YEAR 195 6: 

None 



17. LIST HONORS AND AWARDS TO PERSONNEL RELATING TO TILES 
PROJECT DURING CALENDAR YEAR I956: 

None 



Calendar Year 195 ^ 

PUBLIO HEALTH SERVICE - MTIONAL INSTIIUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NIAMD-16 

SERIAL NUMBER 

2 . WIAMD 3 • Nutrition and Endocrinology 



INSTITUTE LABORATORY 

h. Fraction ation & Isolation 
SECTION 

6 . Studies on Fol i c Acid 
PROJECT TITLE 

7. M. Silverman and Jj_ C_. Ifereszte_sy 

PRINCIPAL INVESTIGATORS" 

8 . S_^ Futterman and R. C. Gardine r 

OTHER INVESTIGATORS" 

9. No parallel research 
10. PROJECT DESCRIPTION: 

Studies on folic acid: 

(a) Naturally- occurring foiTns of -,q 

(b) The enzymatic synthesis of N'' and IT tetrahydrofolic 
acid. 

(a) The urinary compounds associated with folic acid 
metaholism in the rat. 

Objectives : To deterraine the role of folic acid in the 
enzymatic systeirs which are related to growth. To identify 
the naturally occurring forms of folic acid and to develop 
new anti-folic compoujads that might be used in the therapy 
of leuiiemia. To identify the compounds associated with 
folic acid metabolism. 



NIAMD-16 " ^ 
SERIAL NO, 



Method s egipj-oygj.: 

(a) The microbiologlcally- inactive form of folic acid in liver, 
hereafter referred to as pre folic acid, can be converted to 

the active citrovorum factor form by incubation with whole 
liver or an enzyme preparation obtained from liver. The 
problem consists of several interrelated parts which include 
purification of the enzme which converts prcfolic to citro- 
voi-um factor by usiial protein fractionation methods and devel- 
opment of a fractionation process for the isolation of pre- 
folic acid suitable for large scale operation. 

(b) Purification of the enzymes from liver which participate 
in the synthesis of citrovorum factor. These include a 
reducing system involving TPW and a fomiyl transferase 
system. 

(c) The isolation of the substance believed to be related 

to isoglutamine in the urine of rats on a folic acid deficient 
diet is continuing, using solvent and clircmatogrEUTi techniques. 

Major findings: A critical method for the extraction 
of pre folic acid from horse liver has been applied to large 
scale operation. Further purification of this crude extract 
was accomplished by adsorption and elution from charcoal, 
fractional solvent precipitations, column chromatography, 
fractionation of ammonium and barium salts. 

Prefolic acid has been found to exist in at least two 
foiTOS. The enzyme systems required to convert two of these 
forms, prefolic A and prefolic B to the microbiologically 
active form are not the same . 

A metabolic derivative of folic acid has been isolated 
from the urine of man. The product, which participates in 
the biosynthesis of purines has been characterized as an 
imidazoline derivative of tetrahydrofolic acid, anhydro- 
6'i'irovorum factor. 

The inactivation of folic acid by liver extracts is due 
to (l) its reduction to tetrahydrofolic acid and (2) spon- 
taneous cleavage of the latter in air at the 9-10 linltage . 
The products are p-aminobenzyylglutamic acid and a pteridine(s) . 
The inactivation of citrovorum factor in the presence of 
1-glutamic acid and a purified liver fraction is due to the 



NIAMD-16 

SERIAL NO. 

following reaction sequence: 

(1) Citrovorum factor - l-glutamic acid enzwe tetrahydro- 

- ' ^ 

folic acid - K formyl-L- glutamic acid. 

/ \ air . 

(2) Tetrahydrofolic acid non-enzymaxic ' p-aminoTDcnzoic acid 

pteridine(s) 

Significa nce to artliritis a nd me taboli c research : The 
isolation and identification of the various metabolites of 
folic acid will result in a better imderstanding of bio- 
chemistry of such specific diseases as some of the anemias 
and leukemias. The mechanisms by which folic acid derivatives 
influence the formation of fundamental components of all living 
cellS; the purines, are being widely studied. This project 
is directed at discovering how the various forms of folic 
acid exist in the body and how they are changed from 
inactive to active f onas . 

Propose d co vi rse of project : The following are the 
goals for the coming year: 

(1) Purification of the DPN dependent enzyme system which 
inactivates folic acid and identification of the pteridine 
end-product. Extension of these studies to formylated and. 
reduced derivatives of folic acid. 

(2) Continuation of attempts to isolate isoglutamine-like 
substance in urine from folic acid deficient rats. 

(3) Purification of the enzyme which converts prefolic acid 
to citrovorum factor. 

(k) Isolation of the prefolic acids. 



Form No. ORP^l 
October 1956 
(Attachment l) 



PUBLIC HEALTH SERVICE - MATIOE-L raSTITUTES OF HE/*L?H 
IWDIVIDU/vL PROJECT REPORT 



Part B: Budget Data 



11» NI/.MD-16 

SERIAL MMBER 



12, BUDGET BL 


t;.j 








ESTIMATED OBLia.TIONS 




M/.N ye;.rs 


DIRECT 


REIMBURSEME'NT TOTuL 


PROF 


OTHER TOTAL 


FYt57" 








1^2^,900 


$12,^00 ip38,UOO 


2eC0 


1^00 3^00 




BUDGETED POSITIONS 


■ 


PATIENT D/.YS 


PROF 


OTHER TOTAL 




FY'57 








2,00 


leOO 3.00 







13* BUDGET ACTIVITY? 
RESE/iP.CH 

REVIEW & APPROVAL 
BIOLOGIC STAND.'.RDS 



U4 
□ 
i I 



ADMIMISTR/^TION 

PROI'^SSIOML & 
TECH1«Q".L ASSIST- 
ANCE 



lli. IDENTIFY ANY COOPER.'.TING UNITS OF THE PUBLIC HE/iLTH SERVICE, OR 
OTHER ORaiNIZ/.TIONS, PROVIDING FUNDS, F/.CILITIES, OR PERSOM>!EL 
FOR T'lIS PROJECT IM FY 19$1» IF COOPER,^.TING UNIT IS WITHIN NIH 
INDIQ'.TE SERL'.L NO(S): 



(Use reverse and additional pages, if necessary) 



PUBLIC I-EALTH SERVICE - NATIONAL INSTITUTES OF HEAI.TH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors, Awards 8s Publications Ig • NIAMD - 16 

SERIAL NU14BER 

16. PUBLICATIONS ; 

Silverman, M. and Gardiner, R. C. 

Labile citrovor-ura factor in urine. J. Bacteriol. 7I; ^33; 1956. 

Silverman, M. and Wright, B. E. 

Microbiological aspects of the de glutamyl derivatives of 

citrovorisn factor and N-^*^ formylfolic acid. 

J. Bacteriol. 72, 373, 1956. 

Silverman, M., Ebaugh, F. E., Jr. and Gardiner, R. C. 
The natiire of labile citrovorum factor in human urine . 
J. Biol. Chem. In press (Nov. 1956)- 

Futterman, S. and Silvemian, M. 

The "inactivation" of folic acid by liver. J. Biol. Chera. 

(In press) . 

Silverman, M., Keresztesy, J. C, Koval, G. J., and 
Gardiner, R. C. 

CitrovorxM factor and the synthesis of formylglutamic acid. 
J. Biol. Chem. (in press). 



17. No honors and awards. 



Calendar Year I956 

PUBLIC HEALTH SERVICE -- MTIOML INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. N iAMD- l? 



SERIAL NUMBER 

2. NIAMD 3. Nutrition and Endoc rinology 

INSTITUTE" LABORATORY 

k. Fractionation and Isolation 
SECTION 

6 . Microbiological as say s for vitamins and aiuino acid s 
PROJECT TITLE 

7. Milton Silveman 



PRINCIPAL INVESTIGATOR 

8 . H. Bake man, M. Roiai nc 
OTHER INVESTIGATORS 

9. No parallel research. 

10. PROJECT DESCRIPTION: 

Objective : Determination of various vitamins and 
amino acids in foodstuffs, body tissues and excretory 
products can be carried out by the use of microbiological 
assays. Samples submitted hy investigators on nutritional 
and allied projects are analyzed for their content of the 
specific vitai;iin or amino acid \uider study. 

Metho ds employed ; The use of microbiological assays 
continues to be of major importance in the studies on 
folic acid. For exai:aple, with svii table test conditions 
one can distinguish between folic acid and its reduced 
derivatives. Further, the concentrations of 5- and 
lO-formyl derivatives of tetrahydrofolic acid in milll- 
microgram amounts can be determined in the presence of 
each other. 

S ignificance to NIAM D re search : These microbiological 
assay procedures are a very' important tool for the meas- 
urement of the concentrations of various cell constituents 
i . e . , amino acids and vitamins . 



NlAMD-l? - ^ 
SERIAL NO. 

The following collaborative studies have been pvuTGuecL 
in the past year: 

( 1 ) With Dr . ._ jVIi ckel sen_^ 

Continuation of the investigation of the destructive 
effects of ethylene oxide on niacin, riboflavin, folic acid, 
pantothenic acid and biotin. The study was extended to include 
obsei'vations on the influence of ethylene oxide vapors in 
dietary proteins. 

Stability of riboflavin under various conditions of storage. 

( 2 ) With Dr. J. Field . 

The excretion patterns of riboflavin and pantothenate 
in diabetic and nomaal hurnans. The influence of insulin on 
these patterns. The determination of these vitamins in the 
diets employed. 

( 3 ) With JDr^_ A . _ E . _Schp.efeT_{lOmp) , 

The influence of high concentrations of sea salt on the 
stability of riboflavin in Korean diets. Riboflavin concen- 
trations in Koi-ean anny rations. 

Proposed course of project: With the extension of lab- 
oratory findings to clinical investigations, there will be 
an increasing use of these assays from clinical materials. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUELIC HEL;LTH service - NATIONAL INSTITUTES OF HE/.LTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11« MIiJffi-17 



SERL.L NUI'IBER 



12, BUDGET m 


T/.: 












EST 


IKl'-TED OBLIGATIONS 






M;;n YE/xRS 


DIRECT 




REIMBURSE^ffiir 


TOTAL 


PROF 


OTHER TOTAL 


FY«57 












lip2U,300 




4ill,200 


^35,^00 


1,50 


2.00 3«50 




BUDGETED POSITIONS 






PATIEIC DAYS 


PROF 




OTHER 


TOTAL 




FY' 57 












1.^0 




2.00 


3.50 







13. BUDGET ACTIVITY*. 



RESEARCH 



REVIEW & APPROVAL 



BIOLOGIC STANDARDS 






ADMINISTRATION 

PROFESS lON/iL & 
TECHl^ICAL ASSIST- 
ANCE 



P 



P 



lU, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING HJNDS, FACILITIES, OR PERSONMEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S)t 



(Use reverse and additional pages, if necessary) 



Calendar Year 195^ 

PUBLIC HEALTH SERVICE -- MTIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C. Honors J Awards & Publications -'-5 'MIMD-II 

SERIAL NO. 

16. PUBLICATIONS: 

Bakerman; H., Roraine^ M.^ Schricker^ J., Takahashi, S. 
and Mickelsen^ 0. The Stability of Certain B Vitamins 
Exposed to Ethylene Oxide in the Presence of Choline 
Chloride. J. of Agricultural and Food Chem. (Accepted 
for Publication) 

17. HONORS AND AWARDS: None 



Calendar Year I956 

PUBLIC HEALTH SERVICE -- NATIONAL INSTITUTES OF HEALTIi 
INDIVIDUAL PROJECT REPORTS 

Part A. Project Description Sheet 1. NIAI^-lQ, 



SERIAL NUMBER 

2. NIAME 3. Nutrition & Endocrino logy 

INSTITUTE ■ LABORATORY 

^' Endocrinology 
SECTION 

6. Insulin Stu dies 
PROJECT TITLE 

7 . Dr. Evelyn Anderson and Dr. Rob ert W. Bate s 

PRINCIPAL INVESTIGATORS 

^' Mrs. France s W. Franc o and Mr. Jerome Cornfield 

other~i!jvestigators 
10. project description: 

Objectives ; To investigate insulin activity in blood. 

Methods a nd Major Findings ; A method has been 
developed for the bioassay of insulin^ using the alloxan 
diabetic hypophysectomized mouse, which is capable of 
detecting as little as 250 uU insiilin. Using the mouse 
as an experimental animal as well as the test animal, 
blood from normal fasted and non-fasted mice, from 
alloxan diabetic mice and from alloxan diabetic hypophy- 
sectomized mice has been transfused . into the assay test 
animal. The conclusion from these findings is that the 
amount of insulin in the blood of any of these experi- 
mental animals is less than 250 uU per ^ ml. blood. 
Another experiment has been to test for insulin in the 
blood of the mouse after a glucose meal. In these 
animals there is very slight evidence of a measurable 
amount of insulin released by the glucose meal. 

Significance to NIAMD research : The findings in this 
research project vovld challenge these findings of others 
who have used an in vitro method for the assay of insulin. 
In the latter work amounts of insulin as high as 13,000 
uU per ml. plasma have been found in normal human beings 
after a glucose meal. 

Proposed course of project; A collaborative work 
with Drs. Sober and Peterson of the NCI is being planned 
in which various fractions of plasma will be assayed 
for insulin. 



Form No a ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B; Budget Data 



11» NIAMD-18 



SERIAL NUI'^ER 



12* BUDGET DATA: 










ESTIMITED OBLIGATIONS 






MAN YEARS 


DIRECT 


REIMBURSEMELIT 


TOTAL 


PROF 


OTHER TOTAL 


FY«57 










||16,000 


16,300 

BUDGETED POSITIONS 


$22,300 


0»66 


1«00 la66 

PATIENT DAYS 


PROF 


OTI-ER 


TOTAL 




FY«57 










0^66 


1,00 


1.66 







13. BUDGET ACTIVITY! 
RESEARCH 

REVIEW & APPROVAL 
BIOLOGIC STANDARDS 



jTl ADMINISTRATION 

[^ PROFESSIONAL & 

TECHNIC/IL ASSIS1 
□ ANCE 



□ 



D 



lli9 IDENTIFY ANY COOPERATBIG UNITS OF T HE PUBLIC HEALTH SERVICE, OR 
OTIER ORGANIZATIONS, PROVIDING FUIMDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 19??. IF COOPERA.TING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year I956 

PUBLIC HEALTH SERVICE — MITOKAL INSTITUTES OF HEALTH 
IKDIVIDUAL PROJECT REPORT 



Part C: Honors, Awards & Publications 15. NIAMD-I 8 

SERIAL NO. 



16. PUBLICATIONS: None. 

17. HONORS AND AWARDS: None. 



Calendar Year 1956 
PUBLIC HEALTH SERVICE -- MTIOWAL INSTITUTES OF HEALTH 
ira)IVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1. NIAMD-19 



SERIAL KmfflER 

2 . NIAMD 3 . Nutr ition and Endocrino legy 

INSTITUTE' LABORATORY 

k , Endocr inology 
SECTION 

6. Study of Anterior Pituitary Hormones - 

Mouse Pituitary !l?aEior: 'Elaborating ACTH an d MSH 
PROJECT TITLE 

7. Dr. Evelyn Anderson^ Dr. Robert Bahn and Dr. Peter 

G. Condliffe _^ 

PRINCIPAL INVESTIGATORS""" 

8. Dr. Jacob Firrth (Har vard Univer s ity) 
OTiffiR INVESTIGATOR 

9. No parallel research. 

10. PROJECT DESCRIPTION: 

Object ives: Dr. Furth found in mice a trans- 
plantable tumor which stimulates the adrenals of the 
host. The purpose of these studies is to establish 
the chemical nature of the MSH and the adrenal stim- 
u3.ating hormone present in the tumor. 

Meth ods employe d: Extracts are made of beef 
pituitary glands and mouse pituitary tuiaors. These 
are fractionated and studied by countorcurrent dis- 
tribution, electrophoresis and chromatography. The 
distribution of hormone in the various fractions is estim- 
ated hy bioassay, i. e., ascorbic acid depletion of the 
adrenals for ACTH and changes in the mclanophorc content 
of isolated frog skin for MSH. 

Major find ings: About one hundred graras of t\;uaor 
powder have been accuraiiLated. In addition to being able 
to stimulate the adrenals, the tumor powder contains MSH. 
One question to be answered is whether the tuiaor contains 
both ACTH and MSH as separate hormone, or whether the 
MSH activity is to be attribvited to the ACTH content, 
sinceit is known that some preparations of ACTH possess 
inherent MSH activity which can be enhanced by treatment 
with alkali. 



SERIAL NO. NIAMD-19 '"^ 



Treatment of tumor extracts with alkali enhances their 
MSH potency, suggesting that the hormone present is more 
like ACTH in this respect. To make further comparisons 
MSH has been prepared from beef posterior pituitary- 
gland and the same procedure is being employed with 
tumor powder. 

Proposed course of proje ct : 

Project completed. 



Form No. ORP-1 
October 1?56 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11, NIAIffl-19 



SERIAL i\fUMEER 



12. BUDGET DATA; 

ESTIMATED OBLIGATIONS 



DIRECT 



REIMBURSEMENT 



TOTAL 



n^^f 



$9,^00 



$li,800 



$lli,300 



13, BUDGET ACTIVITY; 
RESEARCH 

REVIEW & APPROVAL 
BIOLOGIC STAID/JIDS 



MAN YEARS 



PROF OTIiER TOTAL 



0.83 



0.83 





BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHER 


TOTAL 




FI'57 

0.83 


- 


0,83 





ilx3 ADMNISTRATION !_J 

I 1 PROFESSIONAL & 

TECHiCCAL ASSIST- 



111. IDEI^IFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTIER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONi^L 
FOR THIS PROJECT IN FY 19^7. IF COOPERATING UNIT IS VJITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 195 6 

PUBLIC HEALTH SERVICE -- NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors^ Avardp & Publications Ig . MIAMD-19 

SERIAL N0» 



16. PUBLICATIONS: 

Autoncjnous Marimotropic Pituitary Tunors in Micej 
Their Somatotropic Features and Responsiveness 
to Estrogens. Furth^ J., Gadsden, E. L., 
Clifton, K. H., and Anderson, E. Cancer Research 
16: 600-607, 1956. 



17. HONORS AND AWARDS: None, 



Calendar Year I956 

PUBLIC HEALTH SERVICE -- MTIOML INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NRMD-.20 



SERIAL NUMBER 



2. NIAMD 3. Nut ritio n & Endocri n. 

IWSTIO?UTE LABORATORY 

h . Endocr ino logy 
SECTION 

6. study of Anterior Pituitary Hormones: 
Isolation of Hormones. 

PROJECT TITLE 

7. Dr. Robert W. Bat es and^pr_. Peter G. Condi if fe 
PRINCIPAL IIWESTIGATORS ' "" 

8. Mr. Tulane B. Howard^ Miss Alice M. Laskey 
and Dr. Jacob (H arvard University) 

OTHER INVESTIGATORS '"' ~ 

9. No parallel research. 

10. PROJECT DESCRIPTION: 

Objecti ves: To isolate anterior pituitary hormones 
(thyrotrophin TtsH) adrenotrophin, gonadotrophins, 
growth hormone and prolactin), but especially TSH, from 
pituitary glands, functional transplantable mouse pit- 
uitary tumors and from blood; to determine the chemical 
composition of purified hormone preparations from 
these various sotirces and compare their structure and 
biological properties. 

Methods employed : Extracts are made of pit- 
uitary glands, mouse pituitary tumors and blood. 
These extracts are fractionated and studied by salt 
fractionation, solvent fractionation, isoelectric 
precipitation, preparative electrophoresis, chroma- 
tography and countercurrent distribution. Identifi- 
cation and quantitative estimation of the amino acid 
constitution of purified fractions are made by paper 
and ion exchange chromatography after hydrolysis with or 
without formation of suitable derivatives. The potency 
of all fractions is determined by bioasaay using iodine 
depletion ir. the chick thyroid for thyrotrophin,* 
ascorbic acid depletion of the adrenal of the rat for 
adrenocorticotrophin; cropsac weight increase in 
pigeons for prolactin; seminal vesicle weight increase 



NI AM P-20 ' 
SERIAL KO. 



in rats for follicle stimrilating hormone and ■body- 
weight increase in rats for growth hormone. 

Ma j or findings : TSH has been concentrated from bovine 
pituitary glands^ to the extent of 300 times, with a final 
potency of 6-8 units/mg. Ultracentrifugation of this 
material at pH 7 indicates a single component with a 

sedimentation constant close to 2.7 S. Chromatography 
on carboxymethylcelltilose and electrophoresis on paper 
strips likewise indicate a single component with slight 
impurities. The isolation methods used successfully with 
bovine pituitary glands could not be used for the 
quantitative isolation of mouse TSH from either the 
pituitary tvrnior powder or blood. Preliminary results 
show that the TSH in the tumor bearing mice is either 
chemically different from bovine TSH or is bound to 
different cytoplasmic or plasma proteins. 

Tests for TSH activity have been made on about 25 
samples of human blood. About half of the tests were 
negative, >33 mu/lOO ml. Most of the others were 
borderline, about 50 mu/lOO ml. Blood from an untreated 
cretin contained 100 mvi/lOO ml. and a value of 70 mu/ 
100 ml. was obtained from a case of nodular goiter. 
Blood from thyroide eternized mice with TSH tumors does 
not show high TSH activity until the pituitary tumor 
transplant has started to grow. The blood from mice of 
h different tumor strains contains about 1 unit of TSH 
per ml., which increases to 100,000 mu/ml . when the 
transplantable tumors weigh 2-5 grams. VJhon samples of 
this mouse blood were examined by zone electrophoresis, 
the activity was recovered quantitatively from a zone 
with a mobility between that of the ,^ - and V- globulins. 
When similar samples were examined by cloi'omatography on 
die thylaminocthyl- cellulose, at least \ peaks of TSH 
activity were fotmd. 

A seasonal change was found in the TSH assay. The 
2^4- hour 1I3I uptake of chick thyroids was 65^0 in December 
and Janxiary, and as little as 15^0 in May and June . Tlie 
I-^ ' content of the glands varied in a reciprocal manner. 
The dose levels of TSH for bioassay had to be halved 
during December and January, i. e., the sensitivity or 
the minimum detectable amount was 1 mu instead of the 
usual 2 mu. 

The chromatographic methods employed to study TSH 
have been adapted to prolactin. A purified preparation 
assaying 25 ■'mits/rag. has two components when examined 
on DEAE-cell'olose. 



SERIAL NO. NIAMD-.20 



A variety of transplantable pituitary tumors, 
mostly supplied by Dr. Jacob Furth, have been assayed 
for TSH and prolactin content. No TSH could be 
detected in tumors other than those dependent on a 
radiothyroidectomized host for growth. No prolactin 
was detected in tumors other than those produced by and 
grown in stilbestrol treated mice. 

Significance to NIAIffi research: The mechanisDi of 
action of pituitary hormone's is not, as yet, understood. 
Progress will depend upon availability of pure honnones 
and the accuracy and simplicity of the bioassay pro- 
ced\ires. Progress in this area has been achieved by 
our improved methods for preparation and bioassay 
of the hormones. It is hoped that these methods will 
permit determination of blood levels of TSH in patients. 

Proposed co urse o f project: Efforts to prepare pu.- 
TSH from various raw materials will continue and, when 
successful, full chemical and physical characterization 
will follow. Studies on the effect of TSH on thj-roid 
physiology in the chick will continue. Time course studies 
will be made on the blood levels of TSH in radiothy- 
roidectomizcd mice implanted with TSH producing tumors 
to determine when the blood levels of TSH rise. 
Fractionation studies on mouse blood may load to 
methods for isolating TSH from blood of man where 
the concentration is low. 



Form Noc 0RP«1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - MATIOmL INSTITUTES OF HE/lLTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



lie NIAI€)-20 









SERIAL 


MMBER 


12 » BUDGET DATA: 










ESTIMTED OELIGA^ 
REIMBURSE1-5ENT 


^lONS 




mN YEARS 


DIRECT 


TOTAI. 


PROF OTHER TOBAB 


FY '57' 










^19,000 


$9,700 

BUDGETED POSITIONS 


128,700 




lol6 1,00 2,16 

PATIENT DAYS 


PROF 


OTHER 


TOTAL 




FY' 5? 










1,16 


1,00 


2,16 







13, BUDGET ACTIVITY; 
RESEARCH 

REVIM-I & APPROVAL 
BIOLOGIC STAIiDARDS 



E3 
□ 

P 



ADIlINISTIUTION 

PROIiESSIONAL & 
TECHNICAL ASSISE 
ANCE 



n 



lh» IDENTIFY AI^Y COOPERATING UNITS OF Th^ PUBLIC HEALTH SERVICE^ OR 
OTHER ORGANIZATIONS, PROVIDING FUi-IDS, FACILITIES, OR PERSONAL 
FOR THIS PROJECT IN FY 1957- IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S){ 



(Use reverse and additional pages, if necessary 



Calendar Year 195^ 

PUBLIC HEALTH SERVICE -- NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors^ Awards & Publications 15 . NlAMD-20 

SERIAL NO 



16. PUBLICATIONS: 

Bahn^ Robert C. and Bates, R. W. 
Histologic Criteria for Detection of Prolactin: 
Lack of Prolactin in Blood and Urine of Human 
Subjects. J. Clinical Endocrinology and 
Metabolism 16, 1956, pp. 1337-13^6. 

Condliffe, Peter G. and Bates, Robert W. 
Chromatography of Thyroid Stimulating Hormone 
on Carboxyraethylcellulose . J. Biol. Chem. 
Dec. 1956. (in press). 

BATES, Robert W. and Cornfield, Jerome 
An Improved Assay Method for Thyrotrophin Using 
Depletion of ll3l from the Thyroid of Day-Old 
Chicks. Endocrinology, 1956. (in press). 

17. No honors and awards. 



Calendar Year 1956 

PUBLIC HEALTH SERVICE -- MTIONAI., INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NIAM D-21 

SERIAL NO. 

2 . NIAMD 3 . Nutrit ion & Endocrino logy 

INSTITUTE " LABORATORY 

k. Endocrinology 
SECTlbF 

6. The Influence of the Central Nervous System on 
Metabolic Fmictions - c reatine an d cre atine metabolism 
PR03ilCT~TITLE 

7 . Dr. Kathryn K n ovlton 
PRINCIPAL INVESTIGATOR 

Li . Mr. Leonard H. Kedda 
OTHER' INVESTIGATOR 

9. No parallel research. 

10 . PROJ ECT DESCRIPTIO N; 

Objec tives : To distinguish between extrusion of 
intracellular creatine and failiore of creatine incor- 
poration by tissues as causes of creatinuria, as exam- 
ples, those following transection of the spinal cord 
and accompanying cortisone treatment. 

To learn the course of the effects of hormones on 
such creatinurias, or on normal creatine relationships. 

Creatine labeled with N -^ in the glycinerimoiety i/as 
prepared by conventional techniques. Pi'ocedui'es have been 
worked out for the separation and purification of -urinary 
creatine and creatine . 

Major_ fin dings ; The preparation of creatine labeled 
with N-C5 in the glycine moiety is now nearing completion. 
Glycine containing N-'-^ was prepared by the conventional 
techniques. For preparation of intermediates in the 
synthesis of creatine from glycine, study and mo6.ifi- 
cation of conditions described, in the literature was 
required to produce satisfactory ijroducts in oiir hands. 
Also, instead of the final condensation ef N-^^ labeled 



SERIAL mJI'lBER 

sarcosine with cyanainide to form creatine, we chose the 
reaction in which sarcosine hydrochloride combines with 
methj'-lisothiocarbamide hydriodide to eliminate methyl 
raercaptan and. form creatine . 

The technitjues for separation and purification of 
urinary creatine and creatinine have also been tried out 
for dog urine and modified as necessary. Reduction in the 
acid concentration used for the transformation of creatine 
to creatinine has been found, to give a much more satis- 
factory product. 

Significance to MAIC) rese arc h: Since creatine phos- 
phate"~occupie s a place of some iraportance in intracellular 
energy interchanges of the mammalian body, and since 
several hormones (e. g., androgens, estrogens, adreno- 
cortical steroids with their pituitary colleagues) are 
known to affect creatine-creatinine economy, further 
knowledge of factors and mechanisms of the systems 
involved will contribute basically to oxir understanding 
of metabolic disorders. 

Proposed cours e of pro ject; Creatine isotopically labeled 
withnf^"will~be administered to the normal dog main- 
tained on a constant diet, thus labeling the tissue 
creatine . The creatine and creatinine excreted in the 
urine after the administration of the labeled creatine 
will be separated, piirified, and prepared for the deter- 
mination of their K-^5 abundjance . Urinary excretion of 
those substances will also be followed. The creatinuria- 
provohing xoroccdure then will be instituted and the W-^5 
abundance in both creatine and creatinine followed. 

Later, the source of the creatinuria caused by adreno- 
cortical activity shouJLd be stud.ied as well as the 
effects of androgens and estrogens on creatine- creatinine 
metabolism. These studies will also be accompanied by 
determinations of W-^ abundance in creatine and 
creatinine of muscle. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUHJG HEALTH SERVICE - MTIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROjrCT REPCET 



part B; Budget Data 



11, NIATffi-2 1 ^ 

Serial nuiiber 



12, BUDGET DA 


rAj 








ESTIMATED OBLIGATIONS 




MAN YEARS 


DIRECT 


REIMBURS']t'E!\lT 


TOTAL 


PROF OTHF,R TOTAL 


FY'57 ■■ 








&7,700 


|;9,600 


$27,300 


1,00 1,00 2,00 




BUDGETED POSITIONS 




PATIE>]T DAYS 


PROF 


OTIER 


TOTAL 




Fy'57 








1,00 


1.00 


2,00 





13. BUDGET ACTirETYt 
RESEARCH 

REVIH'I & APPROVAL 
BIOLOGIC STANDARDS 



[xj ADillNISTimTION 

["~J PROFESSIOML & 

TECIffilGAL ASSIST- 
r~| ANCE 



r"i 



□ 



Ik* IDEi\ITIFY AIIY COOPERATING UNITS OF THE PUBLIC lEALTH SERVIC:.^, OR 
OTHER ORGANIZATIONS 5 PROVIDING FU1\!DS, FACILITIES, OR PERS Oi iML 
FOR THIS PROJECT IN FY 1957. IF COOPER/s.TING UNIT IS WITHIN WIH 
INDICATE SERIAL NO(S): 



fnaa T»pvi3-,^RP. anH Adrlit.-i nn^il napes- if necessarv^ 



Calendar Year 195^ 
PUBLIC tffiALIH SERVICE - milOML IWSTITUl'ES OF H35ALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NIAMD-2 2 



SERIAL WO. 
2. IHAMD 3- N & E 



INSTITU^~ lABORATORY 

k . Endoc rinol ogy 
SECTION 

6. The influence of the central nei-vous system on 
Metabolism - CardiovascvLlar-renal jTunction, water 
and electrolyte balance an d al dostero ne e xcr etio n. 
mOJECT"TITLE 

7. Dr. Edward ¥. Hawthorne (Howard University), 

Dr. Hildegard Wilson (Georgetovm) and Dr. Evelyn 
Anderson ^ 



o. 



PRINCIPAL INVESTIGATORS 

Dr. William T. Spence (GeorgetoTO Univer^ityQ 



OTHER im^STIQATOR 
9. No parallel research . 
10. PROJECT DESCRIPTION: 

Objectives: To invostigate; the neural mechanism which 
influenceTelectrolyte and water balance and aldosterone 
secretion. 

Methods cmployBd: The midbrain or spinal cord of 
dogs has~bee"n" transected. Observations wore made on the 
arterial pressure and creatinine excretion. These mea- 
surements were made during the control, operative and 
postoperative period. The dogs are maintained on a 
rigidly controlled diet and daily " intake -output" records 
are kept. Throughout each period determinations are 
made of ai-terial pressure, renal clearances, cardiac 
output, measurements of various fl\iid "spaces" and 
balance studies with respect to nitrogen, sodium and 
water, o.nd creatinine excretion; a method for the analy- ^ 
sis of urinary steroids in dogs which was developed in this 
laboratory (H. W.) is followed. A method for the bioasssy 
of aldosterone using the adrenaloctomized dog with an 



N IAMD-2 g ^ -^ 
SERIAL MJl'ffiER 

explanted bladder also developed in this laboratory (E.W.H.) 

is used. 

Major findings ; The normal dog exoretes very minute 
amounts of aldosterone; when ascites is produced by a 
partial ligature on the vena cava, the aldosterone 
excretion is increased so that sufficient aiaounts are 
present in the urine for a quantitative measurement. 
Midbrain transection did not alter the amount of aldo- 
sterone excreted. 

Other findings were: iiomediately following midbrain 
transeation there occurs a marked reduction (from SO-i'Ofci 
of normal) in effective glomerular filtration, effective 
renal plasma flow, and cardiac output. Associated there- 
with was a marked elevation in calculated total peri- 
pheral and renal resistance. The implication is that 
immediately following midbrain transection in the dog 
there occurs a marked arteriolar spasm. The latter may, 
by increasing the left ventricular work load, lead to 
the shanges in cardiac output, effective renal plasma 
flow, and glomerular filtration rate . 

Sign ifis ance to MAMD rese arch: The prolonged sur- 
vival of dogs with midbrain and cervical cord tran- 
seetiuns affords one an unusual opportunity to study 
the role of the central nervous system in the regu- 
lation of renal and cardiac function. Exactly what 
part the nervous system plays in the regulation of 
normal fluid balance, cardiac output, arterial pressure 
and general renal function is not laaown at the present 
time. These studies bear directly on the mechanism of 
pathologic alterations in the above parameters occurring 
in patients with specific nervous and endocrine dis- 
turbance s . 

Proposed course of pro ject; Studies will be made 
of the acute and chronic alterations in renal and 
cardiac function and the influence of fluid and elec- 
trolyte balance on aldosterone excretion in additional 
dogs with midbrain and spinal cord transection. ¥e 
plan to continue studies of the changes in glanerular 
filtration, renal plasma flow, cardiac output, arterial 
pressure, extracellular fluid space and blood volume, 
along with balance studies Including nitrogen, fluid 
and electrolyte . 



Form No. ORP-1 
October 195^6 
(Attachment I) 



PUBLIC HE/ILTH SERVICE - NA-TIOML INSTITUTES OP HEALTH 
Il'BIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAra-22 



SliRIAL NW5BER 



12, BUDGET DA 


TA! 










ESTIMATED QP.LIGA: 


'IONS 




KM YEARS 


DIRECT 


REIHBURSEIffiNT 


TOTAL 


PROF 


OTHER TOTAL 


FY«57 










$12,300 


^5,100 

BllDGETED POSITIONS 


|il7,l;00 


0.66 


1,00 1,66 

PATIEx^JT n/i.YS 


PROF 


OTHIR 


TOTAL 




FY' 57 










0,66 


1,00 


1,66 






13. BUDGET ACTIVITY! 









RESEARCH 

REVI&J & APPROVAL 

BIOLOGIC STAi\fDARDS 



□ 



ADMINISTRATION 

PROFESSIONAL & 
TECHNICAL ASSIST- 
ANCE 



a 



IMBMMM] 



m, IDH\ITIFY ANY COOPERATING UNITS OF THE PUBLIC HE/iLTH SERVICE, OR 
OTHER ORGANIZATIONS, PiDVIDING RJICOS, FACILITIES. OR TERSONiJEL 
FOR THIS PROJECT IN FY1957. IF COOPERATING UNIT ' IS i/ITHIN NIH 
INDICATE SERIAL NO(S)j 



(Use reverse and additional pages, if necessary) 



Calendar Year 195 ^5 

PUBLIC HEALTH SERVICE — NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part G: Honors, Awards & Publications 1^ <\r TAI^D-?2 

SERIAL NO. 



l6. PUBLICATIONS: (Same publ. cited imder another pro j . ) 

The Effects of Midbrain and Spinal Cord Transection 
on Endo&rine and Metabolic Functions, with Post\ilation 
of a Midbrain Hypothalamico-Pituitary Activating 
System. Anderson, E., Bates, R. ¥., Hawthorne, E. W., 
Haymaker, W., Kiaowlton, K., Rioch, David McK., 
Spence, W. T., Wilson, H. 
Recent Progress in Hormone Research, 1957 (in press). 



17. No honors and awards. 



Calendar Year 195 6 

PUBLIC HEALTH SERVICE -- NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT ■ 

Part A. Project Description Sheet 1. NlAMD-23 _ 

SERIAL NUMBER" 

2. NIM ID 3. Nutrition & Endocrin. 

INSTITUTE 

h . End ocrinology 5 • 

SECTION'""" 

6. The Influence of the Central Nervous System on 
Metabo lic F ii nction s j-^ndocr ine Activity 
PROJECT TITLE "" ~ 

'''• Dr. Hildegard IJ ilson and Dr. Robert W. Bates 
PRINCIPAL IWESTIGATORS ""' " 

u. Dr. Evelyn Anderson^ Dr. David McK. Rioch (Walter 
Reed) J Dr. Webb Hajaaaker (APIP), Dr. William T. 

B Spence (Georgetomi)^ Mr. Jolin Borris (Walter 
Reed ) and yhyj_ ^^"7 M. Garrison (Georgetown) 
OTIiER niVTiJSTIGATORS "" ~' '" "~"~ 

9. No parallel research. 

10. PROJECT DESCRIPTION: 

Objectives: To study the influence of the central 
nervoiis system on ACTII-adi-enal cortical and thjo-oid 
activity. 

Methods emjployed; Hypothalamic -ACTFI-adrenal cor- 
tical activity vas measui'ed in dogs whose midbrain or 
spinal cord was transacted. The iDrocedu-res involved the 
estimation of the steroids in the blood and urine of 
these dogs by a method developed in this laboratory, 
Hypothalamic-TGH- thyroid activity was determined in 
the same dogs by measiu-'ing the rate of I-'-31 depletion 
of the thyL-oid. 

Maj or find ings ; tffidfeiMrt tEansdacbiiiibn cesdi^s tilth 
pefilnHrndntvl3iDckc6fotl;ie'vhypoihaiamipd)Hpitu4±d.rjC'r-adren41on 
cortex system. Five months after the transection there 
was still no rise in steroid production after surgical 
laparotomy without anesthetic. Moreover^ a series of 
other stressful stimuli^ most of which evolied responses 



■NIAME-23 •" ^ 

SERIAL MlJIffiER 

in normal animals, were without effect on the midbrain dog. 
The animal's adrenal cortex was fvLlly able to respond to 
exogenotis ACTII, since a marked rise in steroid excretion 
resulted. Hence, the lack of response must have been due 
to failure of ACTH release. 

The effect of midbrain transection upon the rate of 
disappearance of l-~>'^ from the thyroid (a measure of thy- 
rotrophin release by the pituitary) was determined on tiro 
dogs having a rapid rate of depletion before operation. 
In both dogs depiction of thyroidal I •^ stopped imme- 
diately and for a period of over a xfeok after the tran- 
section. However, in one dog that survived over tliree 
weeks, the rate of depletion of I-^J^ had returned to its 
preoperative level. 

These findings point to an ai-ea in the midbrain 
which acts as an alerting system for the hypothalami co- 
pituitary me chani sin . 

Significance to KIAIC) research: It has been sho\-m 
by Magovm and his associates that the midbrain contains 
part of the reticular activating system which exerts an 
excitatory effect on the thalamus and in turn the cerebral 
cortex. It seems reasonable to expect that some brain stem 
neui'al systems shouJ-d simultaneously activate the h\npo- 
thalamus and through it the pituitary gland so as to allov/ 
adjustments to be made in endoci-ine and metabolic activity. 
The anatomical-physiological findings in this study support 
the concept of a mesencephalic hj'potlialamico- pituitary 
activating system. 

Proposed covg-se of Pi'ojcct: Work is already uiideriray 
on the location of the mesencephalic-hj'pothalamico-pituitary 
activating system. Dogs with electrolytic destruction of 
the dorsal and ventral nuclei of Gudden are being studied. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HtiALTH SERVICE - NATIOML INSTITUTES OF FEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-23 





( 
1. 


3ERmL NUMBER 


12, BUDGET DATA: 






ES 


TIMATED OBLIGATIONS 


FiAN lEARS 


DIRECT 


REHl'BURSEI-iEir TOTAL 


PROF OTHER TOTAL 


FY»57 






;iia9,90O 


;i^9,100 ;;^9,000 


0,66 2,00 2,66 




BUDGETED POSITIONS 


PATIENT DAYS 


PROF 


OTHER TOTAL 





FI«57" 



0,66 



2.00 



2.66 



13 • BUDGET ACTIVITY; 
RESEARCH 

REVIEVI & APPROVAL 
BIOLOGIC STAi^IDARDS 



□ 



ADMNISTRi-lTION 

PROFESSIONAL & 
TECi^EJICAL ASSISE 
ANCE 



r~i 



CZi 



111. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC I-F/XTH SERVICE, OR 
OTHER ORGANISATIONS, PROVIDING EUNDS, FACILITIES, OR PERSOMIEL 
FOR TEES PROJECT IM FY 1957» IF COOPERATING UNIT IS IJITHIN NIH 
IITOICATE SLxRIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 195^ 

PUBLIC HEALTIi SERVICE - MA.TIOKA.L INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors, Awards & Publications 1^, NL''i.MD-.23 

SERI/VL NO. 

16. PUBLICATIONS: 

The effects of midbrain and spinal cord tran- 
section on endocrine and metabolic fvmctions with 
postulation of a midbrain hypothalamico-pituitarji' 
activating system. 

E. Anderson, R. VJ. Bates, E. Hawthorne, ¥. Hayraa-kor, 
K. Knowlton, D. McK. Rioch, W. T. Spence, and 
H. Wilson. 
Recent Progress in Hormone Research, 1957 • (in Press), 



17. HONORS AND AWARDS: None. 



PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1. NL^MD-gL 



SERIAL NUI'IBER 

2. NIAMD 3. Biochemistry t» Metabolism 

INSTITUTE LABORATORY 



4. E nzymes 6. Cell. Biochemistry 5. 

SECTION LOCATION (IF OTHER THAN 

BETHESDA ) 

6. The enzymatic transformation of carbohydrates and their 

PROJECT TITLE intermediary metabolism. 

7. B. L. Horecker . 

PRINCIPAL IWESTIGATOR 

8. P. Z. Smyrniotis to Sept. 1. 195b; A. Ginsburg from Nov. 9,1956 
OTHER INVESTIGATORS 

9. COIvlPLElJENTS OR PARALLELS RESEARCH IN: 

1. NIAfcD-LBM, Section on Intermediary Metabolism 

2. NIAliO-LBM, Section on Metabolic Enzymes 

3. NIAID-LID, Section on Experimental therapeutics 

10. PROJECT DESCRIPTION 

Objectives ; To study the pathways of carbohydrate metabolism 

and their relation to the synthesis of cellular 
components and to cell growth and function. 

Methods Employed ; With enzymes purified from suitable 

sources the individual reactions are 
studied in detail and the reaction products isolated and 
identified. These products are then employed as substrates 
for succeeding steps. VJhere possible, these compounds are 
synthesized with C-*-^ or p32 and the reactions studied in 
intact cells or unfractionated tissue preparations in order 
to evaluate the role of these intermediates in cellular 
metabolism. 

Major Findings ; During the previous year it was shown that 

xylulose 5-phosphate is the substrate for 
mammalian transketolase, as well as for transketolase from 
spinach, indicating the importance of this compound in 



2 



10. FROJECT DESCRIPTION - continued MIkMD-2Ij 



SERIAL NUMBER 



carbohydrate metabolism. This work has now been completed 
with the demonstration that xylulose 5-phosphate is the 
first pentose ester to be formed from hexose phosphate by 
the transketolase reaction. Structural specificity of 
transketolase was defined with the demonstration that 
the trans-cenfiguration on C~3 and C-4 is essential; thus 
D-tagatose C-phosphate was shown to be completely inactive, 
while fructose C-phosphate is rapidly cleaved. 

The mechanism of pentose fermentation in Lactobacillus 
sp. was elucidated with the discovery of an enzymatic 
cleavage involving inorganic phosphate: 

Xylulose 5-phosphate + Pi ^ acetyl phosphate + 

glyceraldehyde 3-phosphate. 

This enzyme has been obtained in nearly pure form from 
extracts of Lactobacillus p entosus and named phospho- 
ketolase. It requires inorganic phosphate, thiamine 
pyrophosphate and sulfhydryl reagents and represents a 
new type of reaction in which vitamin B^ is the coenzyme 
and in which a high energy phosphate compound is found. 
It has now been established that the fermentation of 
pentoses includes the following steps. 

1. Isomerization to the ketose form 

2. Esterif ication to the ketopentose phosphate ester 

3. Conversion to xylulose 5-phosphate 

4. Cleavage by phosphoketolase 

5. Transfer of phosphate from acetyl phosphate to ADP 

6. Conversion of triose phosphate to lactate, yielding 
two more equivalents of ATP. 

The net fermentation equation: pentose > acetate + 
lactate + 2ATP, shows that the energy yield from pentose 
is equal to that derived from the fermentation of hexose. 

In connection with these studies L-arabinose isomerase, 
which catalyzes step 1 for this substrate, was purified 
and the equilibrium mixture shown to contain 10 per cent 
of the pentose as L-ribulose^ It was also shown that 
acetokinase, which-catalyzes step 5, is present only in 
cells grown on pentose and is formed adaptively in response 
to acetyl phosphate, rather than to acetate. Steps 2 and 
3 remain to be worked out; purification of L-ribulose 
kinase is in progress. - 



- a 



IG. PROJECT DESCRIPTION - continued NI;J>ID.^2l4 



SERIAL NUMBER 

Studies on the relation of carbohydrate metabolism to 
nucleic acid synthesis were continued. The ribosidase 
of Lactobaci l lus de lbruckii was purified and shown to be 
specific for (3^D-ribosides, providing a useful analytical 
reagent for this biologically important class of compounds. 
A study was made, in collaboration with Dr. Heppel, of the 
enzymatic splitting of adenine from 5' -adenylic acid in 
Azotobacte r vinelandii . This reaction requires ATP in 
catalytic amounts; however, ATP itself is not split. Ribose 
5-pyrophosphate and ribose 5-triphosphate were synthesized 
and shown not to be intermediates. A very active adenine 
deaminase was also purified from A. vinelandii extracts. 
This enzyme provides a much needed specific and sensitive 
assay method for adenine. 

Studies on the metabolism of erythrose in Alcaligenes faecalis 
were resumed. Evidence in support of an indirect method of 
erythrose 4-phosphate synthesis involving catalytic amount 
of fructose was obtained. The organism was shown to possess 
a highly active fructokinase which has been purified. This 
enzyme has a high affinity for fructose and yields fructose 
6-phosphate. 

Studies on the details of the path of carbon in photo- 
synthesis were continued. The enzymatic hydrolysis of 
ribulose diphosphate in spinach extracts was studied as a 
possible method for the preparation of ribulose 5-phosphate, 
which is still urgently needed. The ribulose diphosphatase 
of spinach has been purified and its properties studied. 
It will also react with fructose diphosphate and sedoheptu- 
lose diphosphate although at reduced rates. Under suitable 
conditions only one of the phosphate residues of ribulose 
diphosphate is removed. 

Significa nce to NIAMD research ; The importance of the 

pentose phosphate pathway 
of carbohydrate metabolism to the basic economy of the cell 
has been confirmed and extended, and evidence has been 
obtained for the existence of additional pathways of carbo- 
hydrate metabolism. These may be of significance in animal 
tissues and a survey will be made for their occurrence. 

Proposed Course of Project : A considerable amount of effort 

will be devoted to a study of 
the mechanism of action of phosphoketolase, since this new 
reaction provides an example of a thiamine pyrophosphate- 
catalyzed reaction which is uncomplicated by other coenzymes. 
Many theories have been advanced for the role of this 



- 4 - 

10. PROJECT DESCRIPTION - continued NLuMD-2U__2_ 



SERIAL IJUlvlBER 

coenzyme in cleavage and condensation reactions but 
little data bearing on this matter is available. 
Further work will also be carried out on the new 
pathways of carbohydrate metabolism and their function 
in various types of cells. 

In addition, toward the latter part of the year a study 
will be initiated on the mechanism of transport of 
carbohydrate across the cell membrane, since knowledge 
of this mechanism may promote an understanding of the 
mechanism of action of insulin. Dr. Horecker is making 
arrangements to spend a year at the Pasteur Institute 
collaborating with Dr. Jacques Monod on a study of 
bacterial carbohydrate transport enzymes. On his return 
the experience gained in this field will be applied to 
an investigation of permability mechanisms in mammalian 
cells. 



Form Mo, ORP-1 
October 19i;6 
(Attachraent I) 



PUr.LIC WAVni SERVICE - W^.TIOML IIJSTITLTJES OF HE/'.LTH 
liffilVIDUAL PIOJGO: REPORT 



Part B: Budget Data 



11, Nni,D-2U 



12, RIJIGET DATA: 



ESTIMATED OBLI ATIOMS 



MAN TEARS 



DIRECT 



RErriBU^SEIffiWT 



TOTAL 



FY 1 57" 



PROF OTFER TarAL 



439,00 



,;l8,600 



■1^57,600 



BUDGETED POSITIOMS 



PROF 



OTHER 



?CTAL 



FY«57" 



2,00 2,00 U,00 



PATIENT DAYS 



2,00 



2,00 



il.OO 



13, BUDG5T ACTIVITY! 
RESrARCH 

REVI0i & APPROVAL 
HCOHCGilC STAi\IDA^yDS 



L.2i 



a 



ADMINISTRATION Q 

PROFESSIONAL & 
TECIiilGAL ASSIST- 
ANCE Q^ 



Ih, IDENTIFY ANY COOPi^RATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER 0RGANI2ATI01.IS, PROVIDING FUNDS, FACILITIES, OR PERSOi«!EL 
FOR THIS PROJECT IN FY 1957. IF COOPJ .RATING UNIT IS WITffiN NIH 
iroiCATE SERIAL NO(S): 

J, Hun^dtz, Postdoctoral Fellov^l, American Cancer Society, 
\intil October 1, 19!?6j E. C, Heath, Postdoctoral Fellow, American 
Heart /xssociation, until November 1, 1956j Y. Takagi, Postdoctoral 
Fellow, Jane Coffin Childs ilemorial Fund, uiitil August 1, 1956; 
G. Donagk, Postdoctoral Fellow, Damon Runyon Memorial Fund, since 
October 1, 1956j D, Burma, Postdoctoral Fellow, Jane Coffin Childs 
Memorial Fund, since September 1, 1956, 



(Use reverse and additional pages, if necessary) 



PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part C: Honors, Awards & Publications 15. NIiu'-lD-2li 



SERIAL NUiABER 

Ic. PUBLICATIONS DURING 195u ; 

"Methods in Enzymology", Vol. 3: Academic Press, New York, 
195o 

The Preparation of Triphosphonucleotide, by 
B. L. Horecker and A. Kornberg, in press. 

The Enzymatic Synthesis of Ribulose Diphosphate and 
Xylulose b-Phosphate, by B. L. Horecker, P. K. Stumpf 
and J. Hurwitz, in press. 

Xylulose 5-Phosphate and the Formation of Sedoheptulose 

7-Phosphate with Liver Transketolase, by B. L. Horecker, 
J. Hurwitz, and P. Z. Smyrniotis, J.A.C.S., 78, o92 
(1956). 

The Biosynthesis of Histidine: Imidazoleacetol Phosphate 
Transaminase, by B. Ames, B. L. Horecker, J. Biol. 
Chem., 220, il3 (1956). 

The Role of Xylulose 5-Phosphate in the Transketolase 

Reaction, by B. L. Horecker, P. Z. Smyrniotis and 
J. Hurwitz, J. Biol. Chem., (.December 1956). 

The Purification of Phosphoketopentoepimerase from 

Lactobacillus pentosus and the Preparation of 
Xylulose 5-Fhosphate, by J. Hurwitz and B. L. 
Horecker, J. Biol. Chem., in press. 

Purification and Properties of a Bacterial Riboside 
Hydrolase, by Y. Takagi and B. L. Horecker, 
J. Biol. Chem., in press. 

Acetyl Phosphate Formation in the Phosphorolytic Cleavage 
of Pentose Phosphate, by E. C. Heath, J. Hurwitz, 
and 3. L. Horecker, J.A.C.S., 78, 5449 (1956). 

The Synthesis of Ribose 5-Pyrophosphate and Ribose 5- 

Triphosphate, by B. L. Horecker, J. Hurwitz, and 
L. A. Heppel, J.A.C.S., in press. 

Adenine Deaminase of Azotobacter vire landii , by L. A. 

Heppel, J. Hurwitz, and B. L. Horecker, J.A.C.3., 
in piess. 



16. PUBLICATIONS - continued NlKi1lJ-2h 

SERIAL NUf/iBER 



An Enzymatic Cleavage of Adenylic Acid Requiring 

Adenosine Triphosphate, by J. Hurwitz, L. A. 

Heppel and B. L. Horecker, submitted to J. Biol. Chem. 

The Enzymatic Cleavage of Adenylic Acid to Adenine and 
Ribose 5-Phosphate, by J. Hurwitz, L. A. Heppel 
and B. L. Horecker, J. Biol. Chem., in press. 

On the Mechanism of CO2 Fixation Leading to Phosphoglyceric 

Acid, by J. Hurwitz, W. B. Jakoby, and B. L. Horecker, 
Biochim. Bioph. Acta, 22, 194 (1956). 



17. HONORS AND Av/ARDS , 1956 ; 

Editor, "Biochemical Preparations" 

Membership Committee, American Society of Biological 
Chemists. 



PUBLIC HE/vLTll SStVICE - - hATIOilAL IlM&TIl'UTES OF HE/lLTH 
Ii DIVIDUAL PKOJIiCT REPORT 
Part A, Project Description Sheet 1„ NI..MD~25 



SEilLlL hUi'lDEll 



2, KIAI1D 3. Biochemistry ex netabolism 

li'.iSTITlJTE ~ ' LABOnAlDRY 

I4, Enzymes '.'::: Ce ll. Bioche mi stry 5. 

SECTION ~" ~ - LOCATIOijTlF^O^HEK THJU-i BEIHESDa) 

6, Studies on the Biosynth esis and Degradation of Pur ines 

moja^T" tit'lt; 

7, Jes se C. jxabinmn.tz 

PRDj'fclPAL MVEoTICiii'rOK 

8, V/. E, Pricer, Jr. 



OTHER liWESTlCATORS 

?, CO.iIPLILiEWTS OR PAiJ-lLLELS Id&iSEARCH Tut 

1. iilfliiD Laboi'atory of Pharmacology'- a. Toxicology, Section on 
Biochanical Pharmacolo^^ 

10, PEDJECT DESCRIPTION 

Objec tives; The oojective of this project is to determine 

of purines. 



biolof^ical mechanisms involved in the metabolism 



Metho ds Employed ; Zinzyraes are purified from suitabJs sources. 
The products of the enzymatic reaction are 
isolated usjjig ion exchnng,e chronatograpiiy or paper chromatog- 
raphy and ars identii'ied from tiisir spectral characteristics, 
by specific chemical and enayiiatlc assays, and by comparison to 
compounds prepared by synthetic means. These products, prepared 
enzyi.Htically or synthetically are employed in studying further 
enzyi;atic reactions. 

Major Findings: The major findings during the past year have 
' been concerned with the metabolism of iorm- 

imino glycine and tetrahydrofolic acid, .ui enzyme has been pur- 
ified from the oacterial cells which cataly2:.es a reaction in 
which formimino,_,lycine, a product formed fx'om x.-inbhine, reacts 
with tetrahydrofolic acid to yield 5-fori.iimino-tetrahydrofolic 
acid. This previously unloriovm folic acid derivative iias novj 
beai isolated. 5-Formii:iiJio-tetrahydroiolic acid is acted on by 
a second enzy^ie, which has also been partially purified, to 
yield 5, 10-m.ec.heuyl- tetrahydrofolic acid, a coi.ipound irhich had 
been known through chemicrO. sjTithesis, but which had not been 
demonstrated iu a biolO;;^icpl systeia. i^ third enzyme brin^.s 
about the h;>drolysis of 5,10-iisthenyl-toti'ahydrofolic acid to 
lO-formyl-tebrah^'-drofolic acid. This product, when 



PKOJKGT DESCI^iPTlOiJ - Continued 



SERiAL liUil 



I-iUiiBER 



incubated i-jith extracts ox ths orf-anism, adenosine diphosphate and 
inorganic phosphate, foiT.is adeaooine triphosohat-, a key conpound 
in all living or,[,anisms involved in the biological utilization of 
the ener^ resultin^^ from Metabolic activity. The overall process 
reprosaits a new catalytic role of the folic acid coenzyiie in 
wiiich the "C]_" :^i'Oup of formiiuiiio -glycine can be utilized for anino 
acid and purine synthesis or for the production of energy. Ihese 
reactions are suimiarized oy the following equations j 

(1) fomiinpjio:^lycine + tetrahydroiolic acid >> ^ -f onnii.iino- 

tetrahydrofolic acid + glycine 

(2) 5-foriaiiiinctetrahydrofolic acid v $,10-riethenyl- 

tetrahydrofolic acid + NHo 

(3) 5^10 men thenyl- tetrahydroiolic acid p 10-forr.iyl- 

tetraliydroiolic acid 

(U) 10' -fori.iyltetraaydrofolic acid + iJ)P + P_j_ >vATP + tetra- 

hydrofolic acid + fornic acid 



Suin of equations I-I4: 

(5) fomimino glycine + ADP + P^ > ATP + Foriaic acid + 

glycine + NHn 

Significance to IllA-iD Research ; Folic acid is recognised as a 

vitar.iin required for the growth 
of riBny animal species and for blood fonaation. It has been impli- 
cated by previous work in the biosynthesis of purines, thyiaine, 
serine and histidine, liost of the specific enz7i'.Tatic reactions in 
which it is involved, hoirever, are not laioi'/n. 

The metabolic defect involved in a folic acid deficiency in ani- 
mals xjas first sur;gested o'j the vDrk of Silverman and co-v;orkers 
of this Institute -Jx) reside in tlie transfer of the foniixiino group 
but the specific aizynatic reactions afiected resisted elucidation. 
On the basis of the reactions discovered in the bacterial systei:i, 
using the methods developed in this study, another group of KIH 
scientists has shotrn that these enzyi.ies also occiir in raai.u.ialian species. 
The excretion of fori.iii.iino .glutamic acid in folic deficient rats has 
now been fully explained. 

Further vrork on tiio i.iechanism o^. actj.on of folic acid derivatives 
is L.iportant for a more rationcl ai^proach to the problems of nutri- 
tional deficiencies and the mode of action of chemotherapcutic agents. 
Folic acid antagonists have shovm proi.iise in the treati.ient of leu- 
kemia and it is ii^iperative that further f-uidsi.iental ini'ormation on 
the function o." ■'■,1 .; ': vitamin be obtain ^d. 



10, PKOdLCT DrSCklPTlOl: _ Continued MIri.MD-2$ -3 



:ZI:IkL i:Ui-k;3l 



Proposed Course of Projec t; Problei.is uhich shall be investigated 

during tiic next calendar year include: 

(1) the identification of the compounds and enzymatic reactions 
involved in the conversion oi' , 10 -I'orayltetrahydro folic acid, 
ADP and inorganic phosphate to iv'iF, foriiic acid and tetrahydro- 
folic acidj 

(2) the utilization of th^j microbial enzyiae systens for the 
complete degradation and isolation of each carbon and nitro^^en 
atoi:i of the purine molecule; and 

(3) the general occiuToiice and significance of p-foriiiii-.iino- 
tetrahydrofolic acid in other bioloi=,ical syst3ms and its possi- 
ble role ill the biosynchesis of guanine and histidine. 



Form No, ORP-1 
October 1956 
(Attachment I) 



PUBLIC I-EALTH SERVICE - NA?IOi«I/'.L INSTI^OTES OF HEALTH 
INDIVIDUAL PROJEC? REPORT 



part B: Budget Data 



11, NIAMD-25 



SERIAL lIUliLER 



12. BIlDCr-^;: DA 


TA: 








ESTTl'I/VTED OFLIGATIONS 




IIAN YEARS 


DIRi^CT 


'.1EI^03URSi•:■?'iEl^lT 


TOTivL 


P'lOF OTHER TOTAL 


FY'57 








$28,100 


•■^12,500 


:.,li0,600 


2,00 1,00 3.00 




BUDGETED POSITIONS 




PATIEiC DAIS 


PROF 


othl;r 


TOTAL 





FY«57 

2,00 1,00 

13, BUDGir ACTIVITY; 
RESEARCH 

REVIE''! &. APPilOVAL 
BIOLOGIC STANDAl^.DS 



3.00 



nn ADMIMISTR/VTION 

f~"1 PROFESSIONid, & 

TECffillCAL ASSIST - 
I I ANCE 






m, ID:r:>iTIFY ANY COOPERf.TIMG UNITS 01^ THE PUBLIC m-MJUil SERVICE, OR 
OTHER ORGANIZATIOIIS, PROVIDTNQ PTJITOS, FACILITIES, OR PERSOFireL 
FOR T:-n:s PROJECT IM FY 19^7. IF C''jOPE'"JTING UNIT IS WITHIN NIH 
liOICATE SERLiL NO(S): 



(Use reverse and additional pages, if necessary) 



PUBLIC HEALIH SEPt\/ICE - - WATIOHaL USTITUTES Oi^ HEALTH 

ILjDIvIDUaL P...OJEv.T itEPORT 

Part C: Honors, Awards Ik Publications l5. MLiiO-2$ 

oEuIAL iTUiiiSER 

16. PUSLICaTIOHS DUnU'lG 19^6: 

ATP Foriiiation Acconpanyintj Forninino glycine utilization, by J. C, 
Eabinointz and W. E, Pricsr, Jr., J. Aia. Chen, Soc. , 78, l5l3 
(1956) ~ 

Enzymatic Synthesis of N ^Fornyltetrahydrofolic Acid and its ruole 
in ATP Fornation Durinji, Fori.iii.iino glycine Dey?adation, by J. C. 
Rablnowits and W. E, Pricer, Jr, , J. Aia, Chen, Soc., 78, I1I67 
(1956) "~ 

Fomininotetrahydroiolic Acid and Anhyurofornyltetrahydrofolic Acid 
as InternediatGS in the r'ori.iation of 1! -Fon.iyltetrahydrofolic 
Acid, bv J. C. uabinowitz and W, E, Pricer, Jr, , J. Aia, Chen. 
Soc, 78, 5702 (1956) 

Internediate Steps in the Forroylation of Tetrahydrofolic Acid by 
Fori.iiminoglutai.iic Acid in Rabbit Liver, by H, Tabor and J. C, 
habino'.fitz, J. An. Cherii. Soc,, 78^ 5705 (195") 

Purine Fernentation by Clo striding cylindrosporunp 7, Foniiiiino- 
glycine, oy J, C. h.abinowitz and W, E, Pricer, Jr,, J. Biol. 
Cha,i., 222, 537 (1956) 

17. HOlvORS AiiD Ai;AitDS 1956: 
li one 



PUBLIC HEALTH SERVICE - - NATIONAL DiSTITUTES OF HEALTH 
B'IDIVIDUAL PROJECT REPORT 
Part, A. Project Description Sheet 1. N LaMD-26 



SKliIAL K UMBER 

2, NIAi© 3. Biochemistry & Metabolism 

11 STITUTE LAPORATOHY 

k. I'hzymes °x Cel l, biochsm. 5 . 

SECT ion "■ ' LOCATIOM (Ii=" OIHER THAi-J BErriESDn) 

6, Studies on the structu r e, biosynthesis and intermediary m e tabolism 

PROJECT Tfi'LE oT nucleic acids and nucleotides 

7, Dr. Leon A. Heppel 



PRINCIPAL HjVESTIukTOR 
8, Dr. Russell J, Hilmoe 



O'iiU^ Bi VESTIGATORS 

9, ho parallel research in F, H. S, 

10. PRO JEC T DSbCR IP TIQM 

Objectives ; The objective of this project is to discover pathv;ays for 

the biosynthesis and breakdovm of nucleic acids aid smaller 
polynucleotides. 

Methods Employeda with enzymes purified from suitable sources various 
biosynthetic and degradative reactions are carried 
out. The products are studied by means of paper chromatography, paper 
electrophoresis, chemical analysis, enzymatic analysis, ion-exchange 
coliimn chromatOj-Taphy and isotope tracer methods. 

i'lajor Finding s; The i:iost important contribution of nucleic acid 

enzymology of the last several years was the dis - 
covery of polynucleotide phosphorylase in A zotobacter vinelandii 
(Oclioa et al., Liti^ausr and Kornberg, Dcei'sl* This bacterial enzjTue 
catalyzes the reversible synthesis of ribonucleic acid-like polymers. 
In collaboration with Professor S. Ochoa -che stnictures of these 
pol'^Tiiers have been determined and the reaction mechanism studied. 
These polymers may have but a single base or any combination of bases. 
This has made available new substrates for nucleases and phospho- 
diesterases and as a result, it has boon possible to study the speci- 
ficity requirements of such enzyiaes more completely than ever before. 
In particular, the action of pancreatic ribonuclease on pyrimidine 
polymers has been studied in great detail and new ijitermediates in 
the hydrolysis have been isolated. 

Studies on nucleotide sequence have been undertaken. Starting with 
p-^ -labeled ADP and unlabeled UDP a polymer containing adenylic and 
uridylic acid units was synthesized and degraded enzymatically and 



10. PROJECT DESCRIPTION - Continued Ni;ii''ID-26 



SIRlaL I^iUi-iBEli 

32 
chemically. It was possible, bGcauE.i of the presence ox' p-^ , to obtain 

evidence bcarj.ng on tho average nuclGotldj sequence;, Pri;lii:iin;^ry evi- 
dence indicates that the units are polyiaoriaed in random I'ashion, 

Tho first evidence that natm-ally occurring nucleic acids (virus and 
yeast) undergo net phosphorolysis vjjth bacteriiil polynucleotide 
phosphorylase was obtained in this laboratoiy. This is reaction (l). 

polynucleotide + inorganic phosphate ^ nucloosido $' -diphosphate ^ > 

A nov; class of small ribopol^Tiucleotidcs was discovered last year, 
contaiiiit^g 5' -phosphomonoester end groups. These are formed from the 
adenylic acid polj'ner by thj action of a nuclease purified from liver 
nuclei. Bacterial onzjTnes do not form such compounds. These small 
polynucleotides undergo phosphorolysis by poljuucleotide phosphorylase, 
both from Azotobacter vin^ landii and S, coli . nn example is shovm 
(reaction T), 

(3) 

trinucleotide + inorganic phosphate ~r=::^ dinucljotido + ADP 

The tetranucl;.otide is active in this reaction also, but the "5 '-ended" 
dinucleotide is inactive, heraovrl of th j ^'-phosphomonoester group 
destroys activity, aiid small polynucleotides with 3'-phosphomonoe3ter 
groups also are not phosphorolyzud. Those observations arc Important 
for they establish the structures needed for the action of poljTiuclootide 
phosphorylase -and may provide a clue to the nature of the primer. The 
advanta{-js of be:aig able to study reversible poljmucleotide synthesis 
with small molecules are obviousj the structure of all reaction components 
are laiovjn. 

iifter the discovery of polynucletodio phosphorylase in bacteria a 
determined effort was made by Ochoa as well as by others to lind 
such an en:c,ymatic system for polynucleotide sjmtheeis in animal 
tissues. These trials all ended in failure, probably because 
nucleic acid sjTithesis in aniraal tissue extracts is a sloi-r reaction 
aiid degradative enzym'3& are so povjerful. In this laboi'atory not 
phosphorolysis of polyTiucl.:otides has been obtained with a fraction 
fi'om guinea pii^ liver nucl.i. This fraction also catalyzes tho 
incorporation of inorganic p-' into ADP, just as does bacterial 
polynucleotide phosphorylase. Purification of this enzyi.ie is under vfay, 

btudies of the mechanism of action of nucleases vjith 0-^" arc nearly 
complete. Throe otnor projects completed this j^ear (with Dr. Hun-zitz 
tmd Dr, Horecker) are: 

(1) Purification and e &udy of a bacterial enzyme which specifi- 
cally cleavjs ;idenosine-5' -phosphate to adjnin: ?uid ribose- 
5 ' -phosphate, 

(2) Enzymatic studi.s on ribosj pyrophosphate end riboc .i 
triphosphate. 

(3) Purification and study of a highly specific bacterial adenase. 



10. PROJECT DEdtRIPTION - Continu'-;d NKMD-26 



Oii'.LJ. i.Ui-iBER 

Signil'icf.n c:; t o HL^dP Ro5-:;:.rch ; It is cxpcctod th:;t the^o studios 

will pro'/xdj inforri2tion on tho 
mechanisms of nucloov.ido :.n6 nucleic r.cid synthasis and breakdoim, 
Tho importance of thiiso substances in coll oconomy is emphasized by 
tho groat attention noi^ bein^ paid to them by investigators in all 
fields of biology end .rudicino. 

Pr oposed Cours'^ ^' oi Pi'ojoct ; rurther studios on th. mechanism of the 

polynucleotide phosphorylaso reaction 
arc contemplated, especially usin^ small ribopol^-nucleotides as 
substrates, ■ Further purification and study oi the system vjhich 
s;/nthesizes polyiiucleo tides in liver will be carried out. The 
interostijiy and unusual phosphodiesterase from liver wiiich forms 
small polynucleotides with 5' -phosplioraonoest.jr groups will bo 
studied iLn [greater detail. 



i 



Form Mo, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC lE^VLTH SEIIVICE - NATIONAL INSTITUTES OF HiL^MTH 
Ii®IVn)UAL PROJECT REPORT 



Part B: Budget Data 



11. NIAMD-26 



SEillAL IWIIBER 



12, BUjjGE^^ da 


TA: 








ESTITIATED 0!: LIGATIONS 




Ki^uM YEAiiS 


DIRECT 


REIlIBURSEIfEir 


TOTAL 


PROF a: HER TOTAL 


F/«57 








132,500 


^?1U,900 


,rU7,l|.00 


2,00 1.00 3.00 




BUDGIJTED POSITIONS 




PATIEiC DAYS 


PROF 


an HER 


TOTAL 




FY'57 ■ 








2,00 


1.00 


3.00 




13. BUDGET ACTI'/ITY: 







RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STAilDAl^S 



SADIilNISTRATIOH 

IZ! 



! ^i PROFESS lOM/lL Ik 

TECItMICAL ASSIST- 
ANCE 



a 



m. IDEICIFY ANY COOPEPJ.^ING WIITS OF THE PUBLIC lEALTH SERVICE, OR 
OTIER ORGANIZATIONS, PROVmiNG FUNDS, FACILITIES, OR PERSOMffiL 
FOR TiaS PROJECT IN FY 19^7. IF CO OPERi^.TI NG UOT? IS 'JITHIN NIH 
INDICATE SERIAL NO(S)i 

Dr, D. E, Xoshland, Jr., Brookhaven National Laboratory 

Dr, S« Ochoa, Department of Biochemistry, Nevr York University 

Dr, f'taxine Singer, Postdoctoral P.H.S. Fellov/, NIAIiD, Since Oct, 1, 19$6 

Dr, B, L, Ffcireoker, NIA>ID 

Dr, J, Ilin-fitz, American Cancer Society - until Oct, 1, 1956 



(Use reverse and additional pages, if necessary) 



PUBLIC HKilH 6EkVICE - - NATIOlJuL INSTITUTES OF HE.iTH 
IlJDIVIDU.i PJiOJECT REPORT 

■ NLJ1P-?6 



Part C. Honoi'Sj Awr.rds u Publications SE^.LX iruriBER 

16. PUBLIC.iTIONS DUiXriG 1956: 

Parii'ication and Proportijs of oplson RibonucloaoO, by 
II. L. Kaplan and L. a. Hoppclj J. Biol. Chon. 222, 90? (1956). 

Enzynatic oyuthosis of iiibonucl iic ^.cid - - Like Polyjiucleotid js, 
by S. Ochoa and L. iw Hoppol, Johns Hopkins Syiaposiiun on Ghci.iical 
Basis of iiGr.;dityj in pr.:)ss. 

udonino DGaninaso of ..zotob.'.ckcr Vinclondiij by L. jw Hqppolj 
J. Hun-fitz and B. L. Horockor, J.u.C.S., in prjss. 

The Synthesis of Riboso 5-Pyrophosphatj and Itiboso 5- Triphosphate, 
by B. L. ilorjckor, J. Hurvjitz and L. i^. ujppcl, J.*..C.S., in press. 

The Enzynatic Cloavaj'C of ^.djnylic ..cid to ..donino and Ribose 
5-Phosphatc,- 

by L, A» Hoppol, , J, iiurwitz, end B. L. Horeckcr, J. Biol, Chom., in 

press, 

"Small Polyribonucleotides vjith ^'-Phosphononoester End-Groups", by 
L. ii. Keppcl, P. J. Ortiz and S. Ochoa, Science 123, Ul5 (1956), 

17, i'io honors or a\-j?.rds in 1956, 



PUBLIC HE.J.TH SERVICE - - NnllOlJ.iL INSTITUTES OF HE.XTH 
BIDIVIDU.i PROJECT REPORT 

Part A, Project Description Shoot 1. NDiMD-g? 



suit LI UUI'iBER 

2, NLUjffl 3. Liochoiaistry &: nctabolisii 

ni\iSTITUTE ■ " " L.BOR.VrO;J 

h, Enc ynos St Co ll. Biochcm. 5. 



SECTION ■ " LOC.VnOlj (Ii'' OTHER THiJ-i BETHESDA) 

6, Carbohydrate untabolism in HaTnrnalian Tissuoa 

PROJECT TITLE 

7, Dr. Gilbort ,.shwell 



FRBICIP..L Ii'Ai;STI(i.TOR 
Joan Iliclonan 



01'HER KVESna/iORS 

9, No parallel 

10. PROJECl DESCRIPT ION 

Objectives: The previously described finding of Dr- xylulose phosphate 

as an intermediate in mammalian pentose metabolism has 
prompted further investigation into the biochemical reactions of this 
compound and a study of the metabolic pathways mediated by ±t, v;ith 
particular reference to pentosuria and uronic acid metabolisra. 

i'icthods employe d! In £,encral, the methods employed involve an enzymatic 

or colorimetric d ^termination of substrate utilization 
or product formation. This is usually followed by purification of the 
enzj.'me system involved. Isolation of the reaction products is attempted 
by use of paper and columnar ci'iromato;,raphy and final identification 
made by preparation of the appropriate crystalline derivatives. 

Major Fin dings: Work on the purification and properties of the spleen 

enzyme rosponsiolo j. or the interconvor^ion of xrylulose- 
and ribulose-phoophate has been completed. The 200 
fold purixi;d enzyr.ie vjas shovm to '/ield m. equilibrium value of l,k for 
the reaction, Ribulose-^-P <■■ ■ ~ Xylulose-5-P. £-x;Aulokinasc, 
the enzyme xhich converts D- xylulose to its phosphoric acid ester, has 
been loiuid in a livor c;ctract. This finding, correlated viith work in 
other laboratories, establishes a new pathway for tii ,: conversion of 
L-xj'luloS'' to glucose and provides a rational basis for the better 
understanding of the lon^; knov/n clinical picture of esscaitial pentosuria. 

Very recently, an extract of rat liver iias been shoi-m to metabolise 
^-g^alacturonic acid in the presence of reduced triphosphopyridino 
nucleotid":. The product has not bjcn idjntified as yet but is pre- 
sumed to b.j L-galactonolactono, a known intermediate in the bio- 
synthesis of~ascorbic acid. 



10. PROJECl DESCRlPliOk Continuod MIiiMD-27 ' "-^ 

siti.L KUi-fiER 

SlfcnJficancG to KLJiD Re search; It is n;-cpGCt d th.it i study of thcso 

coi.ipounrtc; .v.nJ i,h.i pathways by v;hich 
thoy are utilizad vjill contribute to oui' laiowlodgo of the overall 
metabolic process and liius lead to a more rational approach to the 
midorstaitiing and treatment of the patliolOt,ical alterations in 
m :tabolism. 

Propos ed Com\"o of Project ; !.urthcr vjoric on thj purification and 

properties of D-:rylulokinase is indi- 
cated and is in pro-ress. In addition, the reaction products of 
tiie g-galactui'onic acid metabolisiiig enzyme are under investigation 
in the hope that new information on the biosj-n thesis of ascorbic 
acid in mar.unalLi will be forthcoming. Those studies may also pro- 
vide information on the etiology of pentosuria and the normal 
function of this metaoolic pathway. 



Font! No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SKRVIGE - mTIONAL lUSTITUTES OF HC/^.LTH 
INDIVIDUAL PROJECT REPORT 



part B) Budget Data 



11, NI/'.I-iD-27 



SERLiL imSTR 



12, BUDGET DATA: 






ESTIMITED OBLIGATIONS 




MAN YEARS 


DIRECT REIMEintS ¥EIY: 


TOTAL 


PROF irffiR TOTAL 


FY«57 






^,,30,200 IJiim^OOO 


m,^oo 


1,00 2,50 3,$0 


BUDGETED POSITIONS 




PATIENT DAYS 


PROF OTIER 


TOTAL 




FY»57 






IcOO 2t50 


3c^0 




13. HJTiGET ACTIVITY! 







RESEARCH 

REVIEl'J & APPROVAL 

BIOLOGIC STANDARDS 






ADMINISTRATION 

PROFESSIONAL & 
TECHNICAL ASSISE 
A'\ICE 



a 
a 



Iht IDEi\!TIFY A'JY CO0PLR';TING UNITS OF TrfF, PUBLIC HEALTH SSR.VICE, OR 
OTHER OriGANIZ.'',.TIONS , PROVIDING FUNDS, FACILrTIE;J. OR PERSONi^EL 
FOR THIS PROJECT IN' FY 193"7, IF COOPER/iTHm UNIT IS '■ffTHIN NIH 
INDICATE SuRIiil NO<S)s 



(Use reverse and additional pages, if necessary) 



Part C. Honorsj ..wirdG &: Publications NIia1D-27 



16. PuBLIC;.TiaMS 

1. Enzymatic Pliosphorylation ox D-xylulo;; :• in Livor, by J, Hickman 
and G. ..jhwjll^ J.x..C.S„, 78, "^Deccaiibcr Jp, 195o 

2. aizynatic Fori.iiitioa of Xyluloso-^-F'nosphato from RibosG-5- 
PhosphatG in Sploonj by G. ..siiuoll and J. Hiclcinan, J. Biol, 
Chcm. , In pros s 

3. Colorimotric DjteiTaination of Sugars ;, by C, .^sln.'oll in 
"hcthods in En^jTiiology", edited by Goldwick and KapJiin, 
Vol., Ill, in prosG. 

17. Mo honors or a;/ards iii 19$6o 



PUBLIC IF.ALTTI SERVICE - - MTIOML IT'PiTITUTES OF HF-MJH 

IFDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NIkI4D-28 

SERI/X 'IIMRER 

2. rational Institute of Arthritis and Ket?bolic Diseases 
INSTITUTE 

3. Laborator^.^ of Piochemistry and Metabolistn 
labor; TORY 

h. Intemediary Metabolism 5. 

SECTION 

6. The Stereochemistry" of Enzj-Tnatic T?--duction 
PROJECT TITLE 

7. Miss J. D. Benedict, Dr. D. Stetten, Jr., Dr. U. Liddell 
and Dr. E. D. Becker 

PRINCIPAL INVESTIGiVTO^S 

8. None 

OTNER irn?;sTIG;.TORS 

9. None 

R'TtAIiEL RESR,?.RCH 

10. PROJECT DESCRIPTION 

Objectives - The objective of this project is to study 
the mechanism of enzymatic reduction of double bonds in compounds 
which are of metabolic importance. 

Methods Emplo^z-od - Unsaturated compounds are treated with 
deuterium in the presence of the appropriate enzi,Tne in the re- 
duced state. The deuterium labeled product of the reaction is 
isolated and a suitable derivative is prepared for infra-red 
analysis. This spectrum is then compared with that of a refer- 
ence compound of knovm configuration. 

P'ajor bindings - Preliminary'' results in a study of the 
fumarate-succinate reduction indicate that the deuterated suc- 
cinate is a meso form rather than a racemic mixture. 

Si nificance to NI^MD Research - The enzjTnatic reactions 
being studied are involved in the s^mthesis and breakdown of both 
carbohydrates and fats. It is felt that an extension of the 
knowledge of the meclianism of these reactions will be of value. 

Proposed Course of Project - The fumarate-succinate reaction 
will be further investigated in an attempt to validate the pre- 
liminary findings. The crotonyl coenzjnne A - butyryl coenzyme A 
reaction will also be studied. 



i 



Form No, ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOMAL INSTITITES OF HEALTH 
INDIVIDU/:L PROJECT REPORT 



Part B: Budget Data 



12, BUDGET D/iTA: 



11, HiAiro-28 



SEfilAL NUilBER 





ESTIIiATED OBLIG/ITIONS 




MAH YEARS 


DKECT 


REii' burse: rjiT 


TOTAL 


PROF OTHER TOTAL 


FI'57 








;^7,600 


,,.8,800 


^6,iiOO 


1.50 0.50 2,00 




BUDGETED POSITIONS 




PATIEiC DAYS 


PROF 


OTHER 


TOTAL 




FY«57' ■ 








1.50 


0,50 


2,00 




13<i BUDGET ACT'IVITYj 







RESEARCH 

REVIEl'J & APPROVAL 

BIOLOGIC STAi^mA'IDS 



nr i ADmmSTRATION Qj 

r~~! PROFESSIONAL & 

TECHIIIGAL ASSIST- 
□ A-NCE □ 



lli. IDENTIFY AiiY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROiriDING FUNDS, FACILITIES, OR PERSOffiEL 
FOR THIS PROJECT IN FY 1957. IF COOPEPJ.TING UNIT IS VITHIN i\IIH 
BIDICATE SERIAL NO(S) : 



(Use reverse and additional pages, if necessary) 



PTIFLIC HFALTP SEWTCE - - F/TION/.L irSTITUTFS OF HEALTH 

IMDIVIDUAL PROJECT RFPORT 

Part C. Honors, Awards and Publications l5. MKMD-28 

SERI/.L iRHDF.R 

16. PUBLICATIONS F10M THIS PROJECT - None. 

17. KONO.RS AND AWi'.RDS - None. 



PUELIC HE/.LTH SERVTC^' - - i'cTTOK/L irSTTTHTES OF KE/.LTH 

IMDIVIDU/.L PROJECT RrpoRT 

Part ... Project Description Sheet 1, MIkiiD-29 

SF.Ri;.L I'UTIBER 

2 . National Institute of Arthritis and Netrbolic Diseases 
B'STITUTE 

3« Laborat ory of I'iochemistry and Metabolis m 
ll. Inttnnediaiy Metabolism 5. 



SECTION 

6 . E nzymatic Conversions of Sugars 
'-■ROJF.CT TTTLT? 

7. j)r. Y. J. Topper, Dr. n. t^Ioom and Dr. S. Segal 
Pi'IFCIPAL BnreSTIGATORS 



•one 



OT!ni,R IlvWSTIGATORS 

9. None 

FARi'XLEL RESEARO-' 

10. PROJECT DESCRIPTION 



Objectives - The objective of this project is to studj"- 
the enzymatic mechanisms by which sugars are metabolized and 
synthesized. 

Methods Employed - (l) Kon-isotopic substrates are exposed 
to the biological catalysts in the presence of D2O or T2O and 
the distribution of isotope in the products is determined; (2) 
deuterated or tritiated substrates are incubated with the enzymes 
and the redistribution of the isotope is then studied; (3) bacteria 
are grown in the presence of cl'j-labeled carbon sources and the 
labeling pattern in the bacterial carbohydrates is studied. 

Major Findings - The interconversion of glucose-6-phosphate 
and fructose-6-phosphate , catalj'-zed by phosrihon''lase isomerase, 
proceeds via an enediol intermediate, and a detailed mechanism 
to account for this over-all isomerization has been proposed. 
It has also been shoxm that of the two hydrogen atoms on C-1 of 
fructose-6-phosphate, one is activated by phosphoplucose isomerase 
and the other is activated by phosphomannose isomerase. On the 
basis of such stereospecificity and other stereochemical con- 
siderations it is concluded that. fructose-6-phosph?te can form 
either a cis cr trans enediol whereas glucose-6-phosiiihate and 
mannose-6-phosphate can form only one or the other of the two 
geometrical isomers. 



NL.?'D-29 " -" 

SE1L\L NO. 

It has been demonstrated tlist muscle- aldolase car activate 
the alpha hydrogen of dihydroxj'-acetone phosphate in the absence 
of any acceptor. Furthermore, the hydrotren which is labilized 
by aldolase is different from the h.ydrogen which is labilized 
by phospnotriosc isomerase, A detailed mechanism for the tv;o 
reactions has been proposed. 

Significance to ?'i;j^[D Research - The onzjTiKS studied 
mediate important steps in carbohydrate metabolism. It is felt 
that the elucidation of these and other enzj^Tiiatic reactions v;ill 
be of aid in the understanding and treatment of various metabolic 
diseases. 

Proposed Courso of Pro .ject - The mechanism of biosjoithesis 
of L-fucose by Acrobncter neropenes will be investif^ated. 



Form No. ORP-1 
October 19^6 
(Attachment I) 



PUBLIC lEALTH SERVICE - WATIOMAL INSTITUTED OF HiilALTH 
INDIVIDUAL PROJECT REPORT 



Part B: aidget Data 



11. NI/>.MD-29 



SERIAL NUl'IEER 



12, BU13GET DA 


TA: 






ESTIMATED OBLiaiTIOWS 


MAN YEARS 


DIRECT 


REB'iBURSEMLIWT TOTAL 


PROF CTHiLR TOTAL 


FY '57 






1*19,300 


$8,900 .|,>28,200 


1.00 1.50 2.50 




BUDGETED POSITIONS 


PATIEiMT DAYS 


PROF 


OTHER TOTAL 





FY«57" 



1.00 



1.50 



13. BUDGj:? ACTIVITY; 
RESEARCH 

REVIEl-J & APPROVAL 
BIOLOGIC STANDARDS 



2,50 



[^1 ADim«STRATI0N 

I I PROFESSIONAL & 

TECHNICAL A3SIST- 



□ 



lii. IDErlTIFY ANY COOPER/.TING UNTTS OF THE, PUFLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUi^DS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 195?. IF COOPERATING UlMiT I^ WITHIN NIH 
BIDICATE SF;RLA.L NO(S): 



(Use reverse and additional pages, if necessary) 



PUBLIC HE/.LTK SERVICE - - NATIONAL INSTITUTES OF KF„''.T,TH 

IFDB'IDUAL PROJECT REPORT 

Part C. Honors, Avre.rds &; Publications 15, NKMD-29 

oERKL I'TJMBER 

16. PUBLICATIONS FROM THIS PROJECT 

Stetten, D., Jr. and Bloom, B., The Metabolism of the 
Amidine Group of Arginine in the Intact Rat, Jc Biol. Chem. 
220 , 723 (1956) 

Rlooin, B., Filter Paper Support for Mounting and Assay 
of Radioactive Rrecipitates, Analy. Chem. 28, I638 (1956). 

Bloom, B. and Topper, Y. J., On the Mechanism of / ction 
of Aldolase and Phosphotriose Isomcrase, Science 12Li , 982 (1956) 

Topper, Y. J., On the Mechanism of Action of Phosphotriose 
Isomerase and Phosphomannose Isomerase, J. Biol. Chem. (in 
press) . 

17. HONORS AND AWARDS 

Lectureships at Michigan State, Purdue and Vanderbilt 
Universities. 



PUBLIC HEALTK SF"VICE - - NATIOWJ. IfSTITUTES OF KE.'LTH 

INDIVIDUAL PPDJECT RITORT 

Part A. Project Description Sheet 1. Ni;u'ID-30 

SERI/.L NUMBER 

2. N.?tional Institute of ..rthritis and Ketabolic Diseases 
INSTITUTE ., 

3., Laboratory of T^dochemisti^'- and Metabolism 

L\?OR..TORY 

h. Intermediary Metabolism ^, 

SECTION 

6 . Human Diabetes and Studies of Insulin an d Insulin :" ntaponists 
PROJT^ICT TITLE 

7. Dr. J. B. Field, Dr. F. Tietze, Dr. G. W. Brovm and 
Dr. D. Stetten, Jr. 

PRINCIPAL IT'nni,STIG...TOPLS 

8. None 



OTHER IN^rPSTIGi-.TORS 



9. Section on Clinical Endocrinology, NIA-MD 
C00PET7A.TING UNIT 

10. PROJECT DESCPJPTION 

Objectives - Purification and characterization of human 
insulin and insulin antagonist found in human serum during dia- 
betic acidosis. Estimation of glucose in biological fluids 
through the use of glucose oxidase. 

Methods Employed - The absence of physiological activity 
of insulin in the presence of antagonist sera obtained from patients 
in diabetic acidosis is determined by observing the lack of aug- 
mentation of glycogenesis produced in the isolated rat diaphragm 
as compared to controls with no added insulin. Chemical, physical 
and enzymatic methods are used in an attempt to concentrate and 
characterize this insulin antagonist. The effect of the antagonist 
on human insulin will be investigated. The isolrtion of insulin 
from human pancreas obtained at autopsy is being carried out ac- 
cording to well docujTiented procedures employed in the large scale 
commercial extraction of this protein from non-human sources. 
In addition, 'alternative methods of isolation (e.g., paper and 
column chromatography) possibly more applicable to small quantities 
of starting material are being explor<:d, 

T'ydrogen peroxide produced by the action of the mold enzyme, 
glucose oxidase , on glucose is determined by use of the reagent, 
potassium titanium oxalate. The method is being developed for 



-2- 

NlAl-iD-30 • '" ^ 

"serial f'o. 

rapid sp^ctrophotornetric determination of rlucose through the 
formation of a highily stable colored complex. 

Pntient Material 



Male Adults 


h 


li5 days 


Fomale / dults 


1 


30 days 


Outpatients 


3 


5 OPD visits 



Mn.ior I indings - It has beon established th:;t those 
patients in diabetic acidosis who require exalted amounts of 
insulin for treatment have a humoral insulin anta[:onist. This 
antagonist is not related to the lowered sorum pi' or the in- 
creased circulating adrenal cortical steroids ^ It v/as not 
found in two acromegalic diabetic pati':'nts who v;ere insulin re- 
sistant. It migrates electrophoreticallj' with the alpha globulins, 
is not a lipoprotein, and is devoid of insulinase activity. 
It does not interft.re with the binding of insulin by the rat 
diaphragm and is still active even though the diaphragm was pre- 
viously exposed to insulin^ It was stable ■fcrtien kept frozen, but 
was inactivated by heating to 100° C for h minutes. It was not 
inactivated by tr^fpsin digestion and had no glucagon activity. 
Within 6 to 10 hours after insulin treatment was started, it was 
no longer possible to depionstrate this material in serum. 

In agreement with a procedure published recently for the 
isolation of insulin from small quantities of calf pancreas, it 
has been found that paper chromatography of crude extracts of 
human pancreas may lead to the isolation of purified material 
possessing hormonal activity. However, the yield of actiTc- 
material so isolated has proven insufficient for adequate char- 
acterization of the protein hormone by physico-chemical methods. 
An investigation of the possible use of sjTithetic ion exchange 
resins for the isolation of insulin has revealed that the protein 
is strongly adsorbed by phosphor;/lated cellulose, a cation ex- 
changer, in the pH region 1-3 o Grj''stalline insulin has b.-en 
successfully chroma tographed on the adsorbent employing 0.5 N HCl 
as the eluting fluid. The application of this method to the iso- 
lation of insulin from crude sources is presently being investigated. 

Hydrogen p.roxide reacts with potassium titanium oxalate 
(K2TiO (oxalate) 2) to form an oxalate compoimd with a character- 
istic absorption at 385 millimicrons. Maximum color intensity 
is reached when the molar ratio of the fairly soluble titanium 
salt to hydrogen peroxide equals one. The rate of formation of 
hydrogen peroxide produced by the enzymatic oxidation of glucose 
may be followed spectrophotometrically. Glucose yielded 
stoichiometric amounts of hydrogen peroxide upon the conversion 
of glucose to gluccnolactone. 

Significance to NIA^Il ^ese-'rch - The discoverj'' of an insulin 
antagonist in sera of patients with diabetic acidosis h-'S ?'iven 
some insight into the increased insulin requirement exhibited by 



-3- 

NL^dMD-30 • - 
SERIAL NO. 

such patients. FurthcT characterization of this m?tcrial could 
give much needed inf oritr tion regarding the mode of action of 
insulin ■ind the factors which determine insulir. dosage in diabetes. 

Studies of the composition and arrangement of the amino 
acids of pork, sheep, and beef insiilin have revealed only minor 
structural differences among th:-sc three species. The effective- 
ness of these insulins in the control of human diabotes implies 
that these similarities extend also to human insulin. It is the 
purpose of this phase of the investigation to examine this question. 

The highly specific ensyTiictic reaction for determination 
of glucose should be useful for an accurate estimation of blood 
and urine glucose levels-. It lias become apparent that older 
glucose methods often yield false high values, necessitpting 
a reappraisal of such methods and development of a method of 
greater specificity. 

Proposed Course- of T?csearch - Further attempts are beinr 
made to define this antaronist and the mechanism of its production 
and action. Its effectiveness apainst human insulin will also 
be tested. ^Refinements in the <Tlucose oxidase-titanium method 
are under way to shorten the time course of the reaction. 



Form No. ORP.l 
October 1956 
(Attachment I) 



PUBLIC HEi\LTH SERVICE - NATIOW/.L INSTITUTES OF HEALTH 
INDIVinUAI, PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-30 



SmihL iNlUl-iBFil 



12, BlITGET 


DA 


TAj_ 
ESTHIATED OBLIG/^.TIOWS 






MAN YEARS 


DIRECT 




REIIjBURSa-.ENT 


TOTAL 


PROF 


OTI-KR TOTAL 


FY«57 

$36,900 




^J17,300 


if5l4,200 


2.50 


1.50 li.OO 






BUDGETED POSITIONS 






PATIENT DAYS 


PROF 




OTHER 


TOTAL 1 




FI'57 












2.50 




1.50 


li.OO 






n. HinrrK? 


AT 


I'TVTTYt 









RESEARCH 

REVIEl^iJ & APPROVAL 

BIOLOGIC STANDARDS 



m ADIUNISTR/vTION □ 

I I PROFESSIONAL & 

TEC'iNICAL ASSIST- 
□ ANCE □ 



lU. IDENTIFY AI\rY COOPERATING UNITS OF THE PUBLIC HEALTH S ERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUiTOS, FACILITIES, OR PERSOifWEL 
FOR THIS PROJ^JCT IN FY 1957. IF COOPERATING UNIT IS VJITHIN NIH 
IiroiCATE SERL-iL NO(S): 

Section on Clinical Endocrinology, MIAMD. 



(Use reverse and additional pages, if necessary) 



PUBLIC HFALTH SFPVICE - - R TI0^7L INSTITUTES OF ?FALTH 

INDIVIDUAL PPOJECT iTFPORT 

Part C. Honors, Awards ■'/ Publications l5. MI''^i'ID-30 

SF^I/X nm^'FR 

16. PUPLICATTONS FROM THIS PROJECT 

Stetten, D., Jr., and Field, J. E., Humoral Antaponism 
to Insulin. Diabetes 5, 321 (1956). 

Field, J. B., and Stetten, D., Jr,, Hurroral Insulin 
Antagonism /.ssociated vTith Diabetic Acidosis, /m. J. V'ed, 
21, 339 (1956), 

Field, J. B. , and Stetten, D., Jr., Studies on Humoral 
Insulin Antagonists in Diabetic Acidosis. Diabetes 5, 391 (1956), 

17. HOT'ORS AI'TD AWARDS - None. 



PUBLIC HEALTH SERVICE - - NATIONAL I!"STITtITFS OF HF.'.LTH 

iroiVIDUAL PROJECT PT^PO^T 

Part A. Project Description Sheet 1. NIitMD-31 

SE^t;x >7F';BER 

2 , National Institute of /.rthritis and Metabolic Diseases 
INSTITUTE 

3. Laboratory of Bioch.'mistry and Metabolism 
LABOnj'-TORY 

Ij. Intermediary'- Metabolism ^. 

SECTION 

6. The Metabolism of Glucuronic y.cid 
PROJECT TITLE 

7. Dr. F. Eisenberg, Jr» 
PRIl'CIP/i imrESTIGATOR 

8 . None 



OTHE,R INVESTIGATOR 

9. None 

R-.RilLLEL RESEARCH 

10. PROJECT DESCRIPTION 



Objectives - The objective of this project is to elucidate 
the metabolic pathways of utilization of glucuronic acid in the 
mammal. 

Methods Employed - C-^^-labeled glucuronate was injected 
into rats. T>espiratory carbon dioxide was collected and assayed 
for radioactivity. Urine was collected for the study of labeled 
end products of metabolism. 

Major Findings - In contrast to our ovm in vitro findings 
and to other in vivo studies showing that glucuronate is meta- 
bolically relatively inert, glucuronate hr.s been found to be 
rapidly catabolized to carbon dioxide in vivo , .-s much as 70% 
of the injected Cl^ appears as CCp in six hours. This effect, 
however, is extremely variable with often as little as 3.5^ of 
the injected dose appearing as 002* 

In connection with the glucuronate study, a method has 
been developed for the assay of gluci.u:'onic acid in the presence 
of glucosiduronic acids. The method consists of converting glu- 
curonic acid to glucuronolactone by means of hot glacial acetic 
acid. Glucuronolactone is then measured by the hydroxylamine- 
FeClo method of Lipmann and Tuttle. Since glucosiduronic acids 
fail to lactonize under these conditions the method serves to 



-2- 

NLa4D-31 ' "^ 
SERL-\L KO. 

measure glucuronic acid directlj''. Heretofore, there has been 
no direct method available for the assay of f rt e glucuronic acid 
in the presence of conjugated glucuronic acid. The method is 
not suitable for the determination of glucuronic acid in the 
presence of 1-0-acyl glucuronic acids since they are hydrolyzed 
under the conditions of lactonization. 

Significance to ^'I'.MD Resenrch - In view of the wide 
distribution of glucuronic acid in connective tissue, a defect 
in the metabolism of this compound may be associated with arthritis 
and other diseases of that tissue. In order to recognize any de- 
rangement at the biochemical level in the diseased state it is 
essential to study the normal course of metabolism. 

Proposed Course of Project - Further work is under way 
either to eliminate or to explain the variability in the com- 
bustion of glucuronate. The urine will be analyzed for the 
presence of possible intermediates in the oxidation. 



Form No, ORF-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF fEALTH 
INDIVIDUAL PROJj.',CT REPORT 



part B: Budget Data 



11. NIAfffi- 31 

SERLIL mi-£ER 



RESE^^CH 

REVIEl-J & APPROVAL 

BIOLOGIC STArTDARDS 



12, BUDGET 


DATA: 






ESTIMATED OrLIGATIOI-IS 


MAN YFJ.RS 


DIRECT 


REI'IBURSEIIENT TOTAL 


PROF OTIER TOTAL 


FY«57 






■^9,300 ^8,700 $28,000 


1,00 1,50 2.50 




HIDGETED POSITIONS 


PATIENT DAYS 


PROF 


OTI-ER I'OTAL 




FY»57 






1,00 


1.5;o 2.^0 




13. BUDGET 


ACTIVITY: 





m ADMINISTR/iTION 

\ I PROFESSIOML & 

TECHi\!ICAL ASSISI 
\ } ANCi. 



□ 



Zh* IDEl'ITIFY ANY COOPERATING UNITS OF TIE PUBLIC HEALTH SERVICE, OR 
OTIER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOItlEL 
FOR THIS PROJECT IN FY 1957, IF COOPER/iTING Ui^IIT IS iJITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



PUBLIC HEALTH SERVICE - - RTIOMy.L INSTITUTES OF HEALTH 

INDIVIDUAL i^T^aiFCT REPORT 

Part C. Honors, Awards <f- Publications 15. NLiMD-31 

SEP.IAL I-UMEER 

16. PUBLICATIONS FRCM THIS PROJECT 

Eisenberg, F., Jr., and Field, J. P.., The Enz^inatic 
Hydrolysis of Glucuronolactone, J. Biol. Chem. 222, 293 (1956) 

Bloom, B., Eisenberg, F., Jr., and Stetten, D., Jr., 
Occurronce of Kon-Fnbden-iieyerhof Pathways in the Intact 
Rat, J. Biol. Chem. 222, 301 (1956). 

Eisenberg, F., Jr., and de la Haba, D. S., A Direct 
Method for the Determination of Glucuronic Acid in the 
Presence of Conjugated Glucuronides, Arch. Biochem. & 
Biophys . ( in press) . 

17. HONORS AND AWARDS - None. 



PUBLIC HF.ALTH SERVICE - - Nf TTOTOI, INSTITUTES OF HE/>LTH 

INDTVIDU/L PROJECT ^POTP 

Part f. . Project Description Sheet 1. NIm'-ID-32 

?. National Institute of Arthritis and Ketabolic Diseases 
INSTITUTE. ■ ~~ 

3 . Laboratory of Riochemistr;','- and Me tabolism 
LAEORlTOra: 

U. Intermediary'- Metabolism 5. . 

SECTION 

6. Study of Gluconic Acid Metabolism 
PHOJECT TITLE 

7. Dr. I. G. Leder 



PRINCIPAL n^'ESTIGATOR 

8. None 

OTHER BiVESTIGATOP.S 

9. None 

PAP'U,EL RESE/Rai 

10. PROJECT DESCRIPTION 



Objectives - The object of this project is to elucidate 
the mechanism by which gluconic acid is utilized in manTmalian 
tissues. 

Methods Employed - The en:^-Ttie which catalyzes the initial 
metabolic step in the utilization of gluconic acid is obtained 
in cell free extracts and purified. The kinetics, stoichiometry 
and general properties of the reaction are studied spectrophoto- 
metrically and the reaction products identified chromatographically. 

Major Findings - An enzyme which catalyzes the conversion 
of gluconic acid to gluconic acid-6-phosphate according to equa- 
tion (1) has been extracted from hog kidney and purified 
approximately 80-fold, 

ATP + D-gluconic acid — — v D-gluconic acid-6-phosphate + ADP (1) 

4- f + t 

UTP can replace ATP and Mn , Zn , Co"^ or Ca can substitute for 

Mg* with decreasing effectiveness. The enzjone appears to be 
completely specific for D-gluconic acid. An interesting relation- 
ship between the concentrations of ATP and Mg has been revealed. 



-2- 



NI/iMD-32 ^ 
SF.'^I/J,, MO. 

+ 
At each level of ATP the optimal nolar concentration of Mp is 
one-half that of ATP, higher concentrations of Mg* being inhibi- 
tory. This suerests that a Mg -(/TP)2 complex may be the active 
phosphorylating accent. Kinotic studies have indicated that be- 
sides functioning as a phosphon''l"tinf' agent, n? servns to 
protect the enzjone from inhibition by Cu* ions , presurriably by 
chelation. The enz^/me has been used for the quantitative deter- 
mination of D-p;luconic acid. 

Significance to ?'L.?'iD Research - Gluconic acid may be 
present in the human as a result of the action of liver glucose 
oxidase or of the clinical administration of calcium gluconate. 
Findings show how this component is gear-->d to the established 
glucose-6-phosphat8 oxidative pathway of carbohydrate metabolism 
and thus add to our understanding of the patterns of carbohydrate 
metabolism in normal and diseased states. 

Proposed Course of Research - It is felt that this study 
has been brought to a successfxil conclusion. 



Form No. ORP-1 
October 19% 
(Attachment l) 



PUBLIC HEALTH SERVICE - WA?I ':)HAL INoTII'UTEvS OF HEALTH 
liOIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NTAl-lD-32 



SERUIL flUiffiER 



12, BimGi:? 


DA 


TA: 












ESTIIATED O'LIG/.TIOMS 


mN YEARS 


DIRECT 




REIMBURSEI>'Ta-lT 


TOTAI 


PROF 


OTHER TOTAL 


FY '57 












$10,500 




$14,000 


.tflil,500 


0.50 


1.00 i.5o 






BUDGEThlD POSITIOJB 






PATIEwT DAYS 


PROF 




OTflER 


TOTAL 




FI»57 












0.50 




1.00 


1.50 






13. BUDGET 


ACTIVITY: 









RESEARCH 

REVIEl-J & APPROVAL 

BIOLOGIC STANDARDS 



[3 ADMIWISTRATION 

j~~i PROFESsIOML & 

TECroilCAL ASSIST 
I \ ANCE 



a 
□ 



lU. IDEiriFY ANY COOPER/iTING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIOilS, PROVIDTOG FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPER^'TING UNIT IS VJITHM MIH 
BIDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



o:; 



PUBLIC fffi/iLTK SERVICE - - NtTIOWX B'STITUTKS OF HE/.LIK 
INDIVIDUAL PROJECT ^FPORT 

Part C. Honors, Awnrds & Publications 15. NLi.MD-32 

SEPTAL I'UT'IBFP 

16. PUBLIC/ TIONS FROM TPTS PROJECT 

Leder, I. G., Fop Kidney Gluconokinase. J. ^iol. 
Cher., (in press) . 

17. HOFORS AFD AWARDS - Fone, 



PUBLIC HEALTI' SERVICE - - >L;TI0NAL irSTITUTFS CF FlE.\LTfT 

iroiVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. jjTA'ff)-^'^ 

SE"^L"L NU^'TPER 

2. Nat ional Institute of Arthritis and Metr.bolic Diseases 
T?^TITUTE 

3, Laboratory of Biochemistry and Metp.bolisiti 

LA per; TCP Y 

I). Intermedia r'l'- Metnbolism 5» 

S^;CTION 

6, A Study of the Effect of Papaya Extracts on Mainmalian Cartilage 
PROJECT TITLE 

7, Dr. I. G. Leder and Dr. S. S. Spicer 
PRINCIPAL IFVT^.STIGATORS 

8, Dr. D. Stctten, Jr. and Dr. J. H. Bryant 
OTKP.R INVESTIGATORS 

9 • None 



PARALLEL RESE/.RCH 
10. PROJECT DESCRIPTION 



Objectives - To elucidate the mechanism whereby the 
injection of crude papaya extract induces structural alterations 
in the cnrtilagenous tissue of the mammal. To purify the papaya 
component responsible for this action. To explore the applica- 
bility of this reaction to the study of mucopolysaccharides in 
extracts and in situ . 

Methods Employed - The gross manifestation of the effect 
of crude extracts of papaya latex, namely/ the collapse of the 
ears of young rabbits, is further explored bjr histochemical 
and pathological studies y In vitro experiments are performed 
with cartilage slices and with various extracts of cartilage. 
The nature of the reaction and its products are investigated 
by paper chrcmatcgraphy and electri^phorc'sis and by chemical 
analysis fcr split products of mucopolysaccharides. The active 
component of papaya extract is purified by standard techniques. 
of protein fracti.-nation. 

Major Findings - It has been reported that the injection 
of crude extracts of papaya latex, but net of crystalline papain, 
results in the collapse i.f the ears of young rabbits. These ob- 
servations have been ccnfirmed. Histochemical studies of biopsy 
material have indicated that the metachromatic material of the 
ear cartilage, presumably chondroitin sulfate, increases in dye 



-2- 

NIMD-33 ' '^ ^ 
SERUX NO. 

binding capacity iindo.T acid cnditi'ns shortly after the 
administrati'n -f crude papain. During the subsequent fcur 
hours the metachramatic material disappears frcm the cartilage. 
Experiments perfcrmed with chcndrritin sulfate and vdth ear carti- 
lage slices and extracts in vitro have indicated that incubation 
with crude papain dees noT'lead te any extensive degradation of 
the mucopolysaccharides, nor tc the release of inorganic sulfate. 

vSignificance to NI\HD Research - It is hoped that this 
approach to the study of the metabolism of mucopolysaccharides 
will bring us closer t an understanding of the changes that 
take place in these ccmp^unds during disease states in man, 
particularly the diseases of connective tissue. 

Proposed C'.urse :'f Project - Further studies will be 
carried out in vivo and in vitro . It is hoped to purify the 
active agent in papaya and to elucidate the nature of its effect 
en connective tissue, enzymologically and histochemically. 



Form No. ORP-1 
October 1956 
(Attachment l) 



PUBLIC HEALTH SERVICE - MTIONAI, B'STITUTES OF HEALTH 
INDP/IDUAL PROJECT REPORT 



Part B: Budget Data 



11. NBMD-33 



S:tiRIAL miMBER 



12, BUDGE? 


D/i 


TA: 
ESTBtlTED OBLIGATIONS 






mi] YEARS 


DIRECT 




RED-iBUIlSFiiErj 


TOTAL 


PROF 


OTPER TOTAL 


FY«57 












$7,000 




vii2,700 
BUDGETED POSITIONS 


i9,700 


0.50 


0.50 1.00 

PATIENT DAYS 


PROF 




OTB^R 


TOTAL 




Fr«57 












0.50 




0.50 


1.00 







13. EUDGI^T ACTIVITY ; 
RESEARCH 

REVIEW & APPROVAL 
BIOLOGIC STANDARDS 



be i AttllNIS'rRATION 

i I PROFESSIONAL & 

TECHNICAL ASSIST- 
r~| ANCE 






lU, IDMTIFY AM COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZ/iTIONS, PROVIDING FUNDS, FACILITIES, OR PERSOTI^ffiL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS VJITHIN NIH 
liTOICATE SERIAL NO(S): 



(Use reverse and additional pap.es, if necessary) 



PUBLIC HEALTH SERVICE - - F/ TIOK'.L IFSTITUTES OF F^-.LTK 

iroiVIDUAL PROJECT TPO^T 

Part C. Honors, Awards & Publications 1$. NIkI-ID-33 

SEPTAL flJi-IBEH 

16. PUBLICATIONS FRCf; THIS PROJECT - None, 

17. HONORS Am AWARDS - Ncne. 



PUBLIC HE/'.LTH SERVICE - - NATIOWL IfiSTITUTES OF HEALTH 

INDI'fJDW'L PPOJECT RF^OTT 

Part A. Project Doecription Sheet 1. nIj;mD-^Ii 

SFRi;.L MUI'^PER 

2 , National Institute of Arthritis and Metabolic Diseases 
INSTITUTE 

3. Labors to r;^/- of ^biochemistry and Metabolism 
LABORi'.TOW 

h. Intermediary Metabolism 5. ^__ 

SECTION 

6. Metabolism of Glycogen 
PROJECT TITLE 

7. Dr. M. R. Stetten 
PRINCIPAL II^rs^STIG; TOR 

8. Dr. P. Stetten, Jr. and Mr, H. M. Katzen 
OTHER INVESTIGATORS 

9 r None 



PAR/lLLEL resfj-.rch 

10. PROJECT DESCRIPTION 



Objectives - To gain insight into the normal structure 
and metabolic turnover of the glycogen of liver and muscle . 

Methods Employed - Glycogen is isolated from the livers 
and muscles of rats and rabbits by methods involving extraction 
with acid or alkali. Samples are separated into fractions of 
varying size by differential centrifugation or precipitation 
and molecular weight determinations carried out by light scat- 
tering. Branching patterns are determined by means of periodate 
oxidation and enzymatic degradation. Affinities of larse and 
small molecules for muscle phosphorylase are determined by kinetic 
studies using various fractions of glycogen. 

Radioactive glucose is administered to rats or rabbits and 
the radioactivity of the isolated glycogf^n studied as a function 
of solubility and of anatomical distribution. 

Glucose-1-POi -C"'-'^ has been prepared from radioactive starch 
and is being used in in vitro studies of the mechanism of glyco- 
gen synthesis as catalyzed by muscle and liver phosphorj''lase in 
the presence or absence of branching and debranching enzymes. 



-2- 

NlKMD-3li •' '^ 
SE'^LIL NO. 

Major Findings - Glycof^en is metabolically inhomogeneous, 
not only as previously shown, within individual molecules, but 
also within a given population of molecules. It has been shown 
that liver and muscle glycogen differ qualitatively in their 
metabolic behavior in vivo . In the peripheral replacement of 
glucose residues it is the larger molecules of muscle and the 
smaller molecules of liver that are most actively involved. 
In vitro studies Viave shorn thrt this in vivo difference cannot 
'clearly be attributed to differences in branching pattern or the 
kinetics of action with muscle or liver phosphorylase in vitro . 
Fractions of both liver and muscle glycogen which are least 
readily extractable with TCA are found to be the most metabolically 
active sugf^esting that anatomical distribution in relation to 
other cellular constituents may be a determining factor in the 
rate of turnover. The methods conventionallj'- used for the iso- 
lation of glycogen result in a product which is considerably 
degraded. Extraction with cold trichloroacetic acid produces 
less degradation than hot KOH. In the absence of air the split- 
ting by alkali is minimized. 

Significance to Nlf^JlD Research - Alterations and defects 
in the vjay the body metabolizes various carbohydrates have been 
found to be characteristic of certain nutritional states, drug 
actions and metabolic diseases. Any additional knowledge as to 
how carbohydrates are normally handled may be expected to con- 
tribute to a better understanding of the nature of these conditions 
and diseases. 

Proposed Course of P roj ect - Further studies will be 
carried out in an attempt to understand the in vivo differences 
between the metabolic behavior of liver and muscle glycogen. 
A method for isolation using conditions which will give a pro- 
duct more nearly like "native glycogen" will be sought. The 
metabolism of glycogen will be studied as a function of anatomical 
distribution within organs and cells. 



Foot No, ORP-1 
(Attachment I) 



PUBLIC HEALTH SERVICE - MTIOMAL II-JSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, MIAMD~3l; 



SEltlAL NU>iBER 



IZ, bUiJuJiT Ui 


■i : A. : 








ESTIMATED OBLIGATIONS 


IIAM YEARS 


DIRECT 


REIMBURSEt'IEl^r 


TOTAL 


PROF OTHER TOTAL 


FY«57 








$22,800 


i:fl0,100 


$32,900 


loOO 1,66 2,66 




BUDGETED POSITIONS 




PATIEOT DAYS 


PROF 


OTHER 


TOTAL 




FY '57' 








1.00 


1.66 


2.66 




13, BUDGET ACTIVITY: 







RESEARCH 

REVIEIfJ & APPROVAL 

BIOLOGIC STANDARDS 



jT] ADMINISTRATION ( | 

I I PROFESSIONAL k 

TECm«GAL ASSI3T- 

O AHCE □ 



m. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HE^iLTH Sja^VICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUI^IDS, FACILITIES, OR PERSONiffiL 
FOR THIS PROJECT IN FY 19^7. IF COOPEPATING UOTT IS '//ITHIN NIH 
INDICATE SiiRIAL NO(S)j 



(Use reverse and additional pages, if necessary) 



PUBLIC HEALTH SERVICE - - W.TIOFAL B'STITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part C. Honors, Awards & Publications 1$. UIAMD-3U , 

SERL^L ITOIBER 

16. PUBLICATIOKS FROM THIS PROJECT 

Stetten, D., Jr., and Stetten, M. R., Glycogen Turnover, 
in "Essays in Biochemistry'-", Wiley, New York (1956). 

Stetten, M. R., Katzen, H. M. and Stetten, D., Jr., 
Metabolic Tnhomogeneity of Glycogen as a Function of f'ole- 
cunar Weight, J. Biol. Chem. 222, 587 (1956). 

17. HONORS AID AWARDS - Hone. 



PUBLIC Hi:..LTP SFRVICE - - NATIONAL INSTITUTES OF H'^ALTH 

INDIVIDUAL PROJECT m.;pO'''T 

Part A. Project Description Sheet 1. NInMD-3g 

SERL.L NUI'IBER 

2 . National Institute of Art h ritis and Metabolic Diseases 
INSTITUTE 

3. Laboratory of Biochemistry and Metabolism 

Ufcvratory 

h. Intermediary Metabolism 5« 

SECTION 

6 . Mass Spectrometric Analyses of S table Isotopes 
PROJECT TITLE 

7. Mr« A. L. Kenney 

PRIFCIPAL II^r'.rESTIGATOR. 

8. Mrs. D. L. Lowory 
OTh-ER im-'ESTIGATGR 

9. Similar items in National Cancer Institute and National 
Heart Institute 

PARALLEL RESE.' RCH 

10. PROJF.CT DESCRIPTION 

Objectives - The purpose of this operation is to supply 
investigators with measurements of the abundances of stable 
isotopes in gaseous mixtures, 

Methods Employed - Sampli.s are prepared by the following 
methods: Nitrogen is liberated from ammonium salts by the 
action of sodiiom hypobromite in an evacuated sj'^stem. Carbon 
is converted to carbon dioxide by wet or drj'- combustion and is 
liberated from bariinn carbonate by the action of perchloric acid 
in an evacuated system. Hydrogen is recovered from the dry 
combustion of organic materials as water. This water is in turn 
treated \<n.th hot zinc to liberate hydrogen gas. The measurements 
of isotope ratios on these gases are conducted upon a mass 
spectrometer, Consolidated Engineering Company Model ^liOl, 

Ma.jor Findings - ; nalyses on unknown samples have been 
conducted at the rate of approximately 875 per year. This 
figure does not include the very large number of known samples 
and normal abundance determinations which art continuously 
being performed, A special method has been perfected and 
apparatus devised to permit the routine analysis of hydrogen 
samples for deuterium. 



-2- 



SERIiiL NO. 

Significance to T"L'.?T) Research - The samples for 
analysis have arisen in various Laboratories of the Institute. 
In addition to the laboratories of the Section on Intermediciy 
Metabolism, analytical service has been rendered to Dr. Herbert 
Tabor, Laboratory of Pharmacology and Toxicology, Dr. Jarvis 
Seegmiller and Dr. Leonard Laster, Clinical Investigations 
Branch, and Dr. Harry Eagle of NI/iID. This analytical service 
permits sci."ntists to use the stable isotopes interchangeably 
with radioactive isotopes in tracer studies o Two advantages 
of stable isotopes may be mentioned. Of certain elements such 
as nitrogen and oxygen, no radioactive isotopes of useful hrlf- 
Ijfe are available. In experiments designed for human sub.iects 
the use of stable isotopes is free of the hazards of toxic 
radiation. 

Proposed Course of Project - Regular anrlytical service 
xfill be continued indefinitely. Projects underway and in view 
will probably involve the addition of analyses for 0^° to the 
present schedule. The analysis of isotopic mixtures in which 
the components are present in other than equilibrium proportions 
is being explored and will shortly be put to research use. 



Form No, ORP-l 
October 19^6 
(Attaclment I) 



PUBLIC HEALTH SERVICE - NATIOMaL INSTITUTES OF HE/iLTH 
IITOIVIDUAL PROJECT REPORT 



part Bt Budget Data 



11, NTAMD-3£____ 
Sr,RIAL NU]-1I£R 



12, FUlXiET DATAi 



ESTH-IATED OBLIGATIONS 


mN YEARS 


DIRECT 




REI!.BURSEi'IEMT 


TCTAL 


PROF CCHra TOTAL 


FY'57 










$114,200 




^,100 

BU/GETED POSITIONS 


^20,300 


2.00 2.00 

PATISOT D/.YS 


PROF 




OTIER 


TOTAL 




FY«57 










- 




2,00 


2.00 





13, BUDGET^ ACTIVITY; 

RESEARCH [Yl 

REVIH'J & AP~-ROVAL [^ 

BIOLOGIC STANDARDS ( \ 



ADMINISTR/^TION 

PROFESSIOmi & 
TECHIHCAL ASSIST- 

ANCE 



a 
n 



lU. IDENTIFY Ai^IY COOPERATING UNITS OF THE PUBLIC HE/>.LTH SERVICE, OR 
OTHER ORGANIZATIONS, PROViODING FUiTOS, FACILITIES, OR PERSONIIEL 
FOR THIS PROJECT IN FY 19^7. IF COOPERjlTING Ui>IIT IS V/ITIICN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



RIPLIC HEALTH SFWICE - - N/TIOF/IL II'STITUTFS OF F^/ LTH 

INHVIDUAL PROJFCT pf.PORT 

Part G. Honors, Awards Ik Publications 15. NLiiMD-35 

SERI/.L rOJMBER 

16. PUBLICATIONS FROM THIS PROJECT - None.. 

17. HOr.'ORS AND A.WinDS - I 'one. 



PUBLIC HEALTH SE'^VICE - - NATIONAL INSTITUTES OF HEALTH 

INDIT'IDUAL Pf'OJECT REPORT 

Part A. Project Description Sheet 1. MIn:€)-36 

PROJECT NUMBER 

2. Hatioral Institute of Arthritis and Ketabolic Diseases 
INSTITUTE 

3, Laboratory of Biochemistry and Metabolism 
LABORATORY 

h. Intermedia ly Metobolism 5. 

SECTION 

6 . Physiology of Erythrocytes 

PROJECT TITLE 

7. Dr. Y. J. Topper, Dr. 3. Bloom and Dr. E. R. Simon 
PRINCIP/iL INVESTIGi' TORS 

8 . None 



OTHTR INVESTIGATORS 

9. None 

PARf.LI.EL I'ESEARai 

10. PROJECT DESCRIPTION 



Objectives - The objectives of this project are to studjr 
the aging process in erythrocytes, their permerbility character- 
istics and general enzjinatic constitution. 

Methods Employ ed - Red c^lls are tapped in vivo X'jith Fe^ 
and are then fractionated on the bnsis of differential osmotic 
fragility and differential centrifugation. The specific radio- 
activity of the fractions provides a measure of the relative 
ages of the cells contained therein. Standr.rd enzyite methods 
are utilized in comparing the enzymatic constitution of the dif- 
ferent age groups. 

Major Findings - A new method has been developed for sepa- 
rating leucocytes from erj't.hrocytes . It has been found that two 
groups of young red cells are prssent in a normal cell population, 
one representing the most and the other representing the least 
osmotically fragile erythrocytes. 

Significance to NIAM) ''esearch - It is believed that an 
investigation of the structural and biochemical alterations which 
red cells experience during their normal life span will assist in 
the understanding of degenerative changes observed in other tissues. 

Proposed Course of Project - The two types of young 
erythrocytes will be further studied from a chemical and enzymatic 
standpoint. In addition, attempts will be made to characterize 



-2- 



NKMD-36 """^ 
SERIi\L NO. 

and determine the mechanism of action of erythrocytin. Also, 
red coll fragility as it relates to primaquine sensitivity 
will be investigated. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NA.TIONj\L INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAIT>-36 



SERIAL NUMBER 



12, BUDGET DATA; 






EST 


BiA"ED OBLIGATIONS 




MAN YEARS 


btaECT 


REEIBURSEICNT 


TOTAL 


PROF OTHER 'TOTAL 


FY'bY 








^j28,000 


$1.12,500 


^0,500 


2.00 1.00 3.00 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHER 


TOTAL 




FI«57 








2,00 


1,00 


3,00 




13, BUDGET ACTIVITY: 







RESEARCH 

REVIEW &. APPROVAL 

BIOLOGIC STAi^IDARDS 



t x\ 



ADIiliaSTRATION 



PROFESSIONAL & 
TECffi'IIGAL ASSIST. 
Q ANCE 






m. IDEOTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTIER ORGArCEZATIONS, PROVIDING TONDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WrJHIH NIH 
IITOICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



PUBLIC FT^/.LTF SERVICE - - NATIONAL INSTITUTES HF IFfLTH 

INDTVIDUfL P'^CJECT PEPORT 

Part C. Honors, Awards & Publications l5. Nlil'l D-36 

SERI/iL NUi-IRER 

16. PUBLICATIONS FROM THIS PROJECT - None. 

17. HONORS L¥D AWARDS - Hone. 



Calendar Year 1956 

PUBLIC HE/JLTH SERVICE - MTIOW/X IISTITUTES OF HE/iLTH 
INDWIDWJ. PROJECT REPORT 



Part A, Project Description Shk..et 1, MIiJ-lD-37 

SERIAL NUMBER 



2. KI/JiD 3i Biochemistry and Metabolism 

INSTITUTE OR DIVISION iZBORAlDRY, BR/iMCH OR DEPT. 



km Metabolic Enz;','TTE s 5o 

SEC HON OR SERVICE K)GATION (IF OTiER Tlu'.M RtirriESDA 



6, Metabolic enzymes with specia l refere nce to hereditary changes . 
FROJECT TITLE 



7 . Herman M. Kalcka r 

PRINCIPAL INVESTIG/.TOR 



8, Elizabeth xMamell 
OTHER INVESTIGATORS 



9. IF TrilS mOJECT RESEMBLES, COI^IPLEffiNTS, OR FARi".LLELS RESE/iRCH 
DONE ELSEiflffiFtE IN IHE FQBLIC HEALTH SERVICE (WTHOUT IN'IER- 
CH/JIGE OF PERSOi'INEL, FACILITIES OR FUmS) IDENTIFY SUCH 
RESE/JICH: (BY SERIAL NO.(S) IF l-JITHIN NIH). 

None 



10, PROJECT DESCRIPTEON 

Objectives ; A further expansion of the studies on galactose 
metabolism and its aberrations in human heritable di.scases and in 
mutations in microorganisms. Experiments on bacterial mutants 
may give some insight into the mechanism of synthesis of specific 
enzj^nic proteins. Likewise, since it has been found that the genes 
which pxe closely linked control the Sj'-nthesis of the same enzyme, 
the relation betvrecn gene action and enzyme synthesis can be 
studied. 



Calendar Year 1956 

PUBLIC HE/iTH SERVICE - MnONAL INSTITUTES OF HEALTH 
IMDIVIDU/X PROJECT REPORT 



Part A, Project Description Shuet !• inJiMD-37 

SERIAL NUMBER 



2, NIAMJD 3. Biochomistry fnd Metabolism 

INSTITUTE OR DIVISION ' L/lBORAIDRY,' BR/iNCH OR DEPT, 



it. Metabolic EnzynBS $, 



SECTION OR SERVICE LOC/IfTON (IF OTHER THAN BETHESDA 



6, Metab oli c enzymes with special reference to hereditary changes . 
PROJECT TITLE 



7. Herman M. Kalckar 

PRINCIPAL IWESTIG/.TOR 

8. Elizabeth Maxwell 
OTHER I CTESTl GATORS 



9. IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR FAR/J,LELS RESEARCH 
DONE ELSEVJHErtE IN 'IHE PUBLIC HEALTH SERVICE (WITHOUT INTER- 
CH/JIGE OF PERSOI^ELj FACILIHES OR FUmS) IDENTIFY SUCH 
RESE/uHCH: (BY SERIAL NO,(S) IF WTHIW NIH), 

None 



10. PROJECT DESCRIPnON 

Obje c tives ; A further expai'ision of the studies on galactose 
metabolism and its aberrations in human heritable diseases and in 
mutations in microorganisms. Experiments on bacterial mutants 
may give some .insight Into the mechanism of synthesis of specific 
enzymic proteins. Likewise, since it has been found that the genes 
which are closely linked control tlic synthesis of the same enzyme, 
the relation betvjeen gene action and enzyme synthesis can be 
stud3.ed. 



- 2 - 

NIaMD-37 ' "'^ 
SERIAL NMEER 

10, PROJECT DESCRIPTION - contimicd 

Work on heritable metabolic disturbances in mammals also 
prompted us to extend our studies to heterozygous individuals 
and to study the metabolism of the gametes. The latter with 
the special purpose of studying the possible existence of 
phenotypic segregation in gametes. 

The biological role of the enzymic conversion of glucose (as 
UDPGlucose) into galactose ( UDPGalactosc) for growth and develop- 
ment deserves major attention. A continuation of the study on the 
biochemical mechanism of this conversion is also planned with 
special reference to the properties of the enzyme protein. 

Metabolic Enzymes of Galactose Metabolism 

A, The Mature of Enzyme Defect in Congenital Galactosemia 

The author has, in collaboration with Drs. Elizabeth P, 
Anderson and Kurt J, Isselbacher, continued the studies on human 
congenital galactosemia, a disease of infancy and childhood. 
About twenty cases of this disease have been under study. It was 
found that the disease originates from a defect in an enzyme 
which catalyzes the incorporation of a-galactose-1-phosphate 
(Gal-l-P) into uridine diphosphoglucose (UDP- glucose), giving 
IIDP- galactose. The enzyme is called Gal-l-P uridyl transferase, 
(See last year's report from OTir group), Tliis enzjTne was first 
described by one of us (H,M,K,) three years ago while studying 
the enzymes of galactose metabolism in yeast adapted to this 
sugar. We found the enzyme abvmdant in hemolysates in normal 
indnviduals and in liver homogenatcs (obtained from humans post 
mortem). In galactosemia it has been possible to obtain liver 
biopsies from two cases. The defect in Gal-l-P uridyl transferase 
in congenital galactosemia is as highly conspicuous in liver 
samples as it is in hemolysates (including hemolysates from blood 
of the umbilical cord of newborn galacto.'^eid.c infants). The other 
enzymes of galactose metabolism were found to be present, 

B, Clinical Metabolic Studies 

In order to see whether a galactoseinic individual is able to 
metabolize tracer amounts of galactose, a metabolic balance study 
on such an individual was initiated. This study was performed in 
collaboration with Drs, Frank Eisenberg Jr. and Kurt J, Isselbacher, 
The subiect was a 2ii-year old male vjith congenital galactosemia. 
Pure C-^^- labelled galactose (from Dr, H, Isbcll, National B\ireau 
of Standards) was used as a marker and menthol as a trap for the 
glucosiduronic acid which might be formed. The occurrence of C-'-'^ 



- 3 - 



MIii ro-17 



SERIAL NUMBER 

10. PROJECT DESCRIPTION - continued 

in the urinojry urea was ^^sed as a measure of conversion of C 
galactose to C02« Mo radioactivity could be detected in the urea. 
However, it was found that a distinct amount of the radioactive 
galactose did appear as C-'-^ menthol glucosiduronic acid. The red 
cells (but not the blood plasma) retained radioactivity, presumably 
due to accumulation of galactose- 1-phosphate, The capacity to 
metabolize galactose was, however, ha.ghly restricted, amounting 
to about 2 per cent of a normal adult. It is possible that the 
small residual galactose metabolism may follow a related pathway 
which may or may not be idential to the normal main pathway for 
galactose metabolism. The results of the studies on the whole 
individual, as well as those on the liver biopsies, using C^'^- 
labelled galactose are in good agreement. lioreover, they demon- 
strate that the methods using radioactive galactose are more 
sensitive than is the enzymatic assay on blood. The latter can 
detect activity down to 3 per cent of normal values, but not 
lower, 

C e D iagnostic Assay for Congenital Galactosemia 

Tlie method used for the determination of Gal-l-P uridyl 
transferase in hemolysates constitutes a basis for a diagnostic 
assay of congenital galactosemia. A routine method has been 
described; this method should be possible to perform in a well- 
equipped hospital laboratory, (A laboratory kit will apparently 
soon be commercially available). The test can also be pc^rformed 
on red cells hemolysate from umbilical cord blood. An internal 
standard by which the corresponding pyrophosphorylase of UDPGlucose 
is also assayed should guarantee against spurious interpretations 
of results or lack of activity due to improper handling of the 
blood specimen. The enzyme Gal-l-P uridyl transferase has been 
found to be present within normal range in hemolysates from a 
large nimiber of physiological and pathological cases such as 
pregnancy, mill-c allergies, chorioepithelioma, hepatitis and liver 
cirrhosis and transient gruLactose intolerance. Our method which 
employes only a few ml. of blood may make the old galactose load- 
ing test partially or completely superfluous. The latter method 
is hazardous, especially in young infants, and is less specific 
than the new test, 

D, Metabolic Pattern in F\jrc Mce Strains 

The study of a h^;ri table metabolic 'error' like galactoseinia 
made us wonder about the possibility of finding signs of phcnotypic 



1-1 



NIAMD--37 
SERIAL NUIIBfiR 



10, PROJECT DESCRIPnON - continued 

segregation in spermatozoa of individuals which ccrry a trait, i.e., 
are heterozygous with respect to a gene involved in production of 
a metabolic enzyme. It is knoim that many animal strains can show 
heritable metabolic patterns. Law and his group at National Cancer 
Institute showed some yeajrs ago that certain strains of mice 
possess a high concentration of P-glucuroaidase in the liver; 
other strrins have concentration levels which are much lower (one 
tenth of the high level). The inheritajice of high P- glucuronidase 
titer in liver corresponds, as shown by Law, Morrow & Greenspan, 
to that of one single dominant gene. We have continued these 
studies, focusing our interest toward tlie enzymology of the 
spermatozoa, Drs. L. Law and M, Potter, National Cancer Institute, 
have kindly furnished us mice of pure strains, A P-glucuronoside 
of methylujTibelliforone indiich exhiibit fluorescence was used as sub- 
strate. This enabled us to carry out the studies on washed 
spermatozoa from a few individuals of a pure strain. It was found 
that P-glucuronidasc is present in the spermatozoa, Methylation 
of the carboxyl group of the umbellif crone glucosidoronic acid 
made the substrate much loss efficient. The relation between 
activity ajid strains, with speciaJ. emphasis on the hybrid strains, 
is under study. 

E . Micro biological Studie s on Bacterial Mutants of Galactose 
Metabolis m 

In order to learn more about the rG].rtionship between genes 
rjid enzymes of galactose metabolism, studies on microorganisms were 
initiated. Dr. J, Lederberg, University of Wisconsin has furnished 
us with a nujdber of mutant strains of E, coli (K22) which have 
been rendered incapable of using galactose as a carbon source for 
growth. This was done mainly by transduction witli a virus. 
Dr, Kiyoshi Kurahashi, in our group, has identified the various 
genotypes furnished as belonging to two phenotypical classes, 
(i) Mutants blocked prior to phosphorylation, (ii) Mutants 
blocked after the phosphorylation step of galactose. The latter 
which comprised h mutants were all identified as defect in 
Gal-l-P uridjrl transferase, the same enzyme ^hich is defect in 
human congenital galactosemia. It was found that mixing of 
extracts from mutants blocked in different enzymes restorc-d com- 
plete capacity to metabolize free galactose. Conversely, mixtures 
of extracts from mutants blocked in tlio production of the same 
enzyme (but genetically different) gave no trace of reactivation. 
These findings furnished an independent and highly sensitive 
phenotypic identification of the genotypes. Moreover, the outcome 



- 5- 



10, PROJECT DESCRIPTION - continued 



NLaMD-37 



also showed that there is no recombination of gene products (in 
this case incomplete enzyme proteins) in cell lysatcs. 

Drs, Albert Wahba, K. Kurahashi and the author have studied 
the effect of galactose on growth of E. coli mutants blocked 
either before or after phosphorylation of galactose, Tliis study 
was also initiated as a resiilt of our studies on congenital 
galactosemia, in which case the mutation is the result of a 
mutation of a gene necessary for the synthesis of the enzyme 
Gal-l-P ui^idyltransferase. It was found th^t growth of mutants 
of the latter category which are blocked in Gal~l-P uridyl trans- 
ferase are markedly siippresscd if a small amount of galactose is 
added to a growth medium containing glucose and ammonia or amino 
acids. This suppression of growth by addition of galactose to 
glucose does not occur at all in the mutants blocked prior to 
galactose phosphorylation or in the wild type in which all the 
enzymes of galactose metabolism are active. These observations 
might lead to a better understandi.ng of the pathogenesis of the 
cellular lesions in human congenital galactosemia. The method 
may also furnish a convenient method for classification of pheno- 
typcs of bacterial mutants of galactose metabolism. The physio- 
logical implication of these studies may have bearings on many 
problems of growth, 

F, The Mechanism of Galactose to Glucose Con ve rsion 

The conversion of the glucose to galactose is a process of 
primary importance for growth, Tliis enables the organism to use 
galactose as fuel and to use glucose as a source of sugar for 
galactosidcs such as lactose or brain galactolip^ds, 

Leloir and his group in Buenos Aires have found tliat the con- 
version of glucose to galactose taJ-res place according to the 
equation: uridine diphospho galactose — dJ iun.dine diphospho glucose, 
Dr, E, Maxwell in our group succeeded xn purifjdng the enzyme 
which catalyzes this step. The source was calf liver, Tlie puri- 
fication made it possible to study the properties of the enzyme. 
Dr. Maxwell discovered that diphosphopyridine nucleotide (DPN) is 
a cof actor for the enzyme. Tliis finding indicates that an 
oxidation reduction is involved rather than a walden inversion. 
The conventional name galacto-waldenase was therefore replaced by 
a new end, in our opinion, more correct name, UDPGalactose-Ii- 
epimerase which leaves a margin for many mechanisms, aiiong them 
oxn.data.on reduction. Studies using liydrogen-labelled water 
(deuterium water, tritium water) show a complete lack of exchange 



6 - 



NIAMD-37 " ''■■' 
SERIAL NUMBER 



10. PROJECT DESCRIPTION - continued 

of carbon- bound hydrogen vdth tiiat of water, Tritimn labelled 
pyridine nucleotides have also been made and arc under study, A 
recent study in the scanm.ng spectrophotofluorometcr indicates 
the presence of protein- bound reduced DPN in UDPGal-U-epimerasc, 

Human erythrocyte lysates contain an inhibitor which com- 
pletely suppresses the activity of the li-epimeraso present in 
these cells. By addition of a great excess of DPN it is possible 
to reactivate the enzyme. It should be added that the concen- 
tration of DPN in filtrates of hcmolysates which have not received 
extra DPN is sufficiently large to render purified U-epimerase 
preparations fully active. This can be shown by fractionation of 
the filtrate by norite. The nature of tlie erythrocyte inhibitor 
is under study. 

G, Metabolism of Uridine Diphospho N-Acetyl Glucosamine in v.i » 
antigen produced in E. coli . 

V,i, antigen is a characteristic constituent of a number of 
highly virulent bacteria of the coli-tjrphoid group. Dr. M. Landy 
and liis coworkers at Walter Reed Hospital have shoxm that v,i, 
antigen contains N-acctyl glucosamine as the main constituent. It 
would be of great interest to understand ti:c biosynthesis of this 
compound, 

Dr« Robert Micat has continued his research on the metabolism 
of uridine diphospho-N-acetyl g].ucosaminc (one of the known complex 
amino sugar compounds) in strains of E. coli viiich synthesize v,i, 
antigen. Ho has demonstrated tlic formation of a blue fluorescent 
compound. By merjis of carbon lii-labelling either of the acetyl 
group or of the hoxose part^ he was able to sliow that both moities 
axe part of the fluorescent compound. The studies w:.ll be con- 
tinued at Duke University School of Medicine by Dr. '^eat who 
received a position there September 1st and hence terminated his 
fellowship in our section, 

Sigm.f icance to NI/iMD research ; A study of the mechajiJsm of carbo- 
hydrate metabolism in health end disease is of interest for a 
closer understanding of normal and pathological growth and develop- 
ment in higher and lower organisms. 



PUBLIC HF^/iTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part B: Budget Data 11 « NIi^MD-37 



SERIAL mJiIBER 
12: BUDGET DATA 



13, BUDGET ACTIVITY! 



lli, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORG/J^IIZATIONS, PROVIDING FUNDS, F/.CILITIES, OR PERS0N1>]EL 
FOR THIS PROJECT IN FY 195?. IF COOPERATING UNIT IS WITHIN MH 
II^HDICATE SERIAL NO(S)s 

Elizabeth P. Anderson - Postdoctoral Fellow, American Canocr 
Society until July 1, 1956* 

Robert W. Wheat - Postdoctoral Fellow, United States Public 
Health Service until September 1, 1956, 

Kiyoshi Kurahashi - Postdoctoral Fellow, Jane Coffin Childs 
Memorial Fund, since September 1, 19^5* 

Albert Wahba - Postdoctoral Fallow, Drjaon Runyon Memorial Fund, 
since September 1, 1956* 

Hugette Szulmajster - Postdoctoral Fellow CL.ntre National de 
la Recherche Scientifique, since October 1, 1956» 

Bodll Waage-Jenson - trainee under the /onerican-Scandinavian 
Foundation tlirough a grant-in-aid from the Eli Li].ly 
Laboratories until July 1, 1956. 

Kurt J. Isselbacher, Arthritis &■ Rheumatism Branch, M/JID \intil 
July 1, 1956. 

Frank Eisenburg Jr., Section on Intermediary Metabolism, NIAMD, 



Form No, ORP-1 
October 19^6 
(Attaciiment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
iroiVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAMD-37 









SERIAL 


iWilHER 


12. BUDGET DA 


TAs 
ESTIMATED OHLIG/VTIONS 






mN YEAIiS 


DIRECT 


REII'iBORSEMENT 


TOTAL 


PROF OTHER TOTAL 


FI'57 










^70,900 


$32,300 
BUDGETED POSITIONS 


&03,200 




2.00 2,00 luOO 
PATIENT DAYS 


PROF 


CTHFfl 


TOTAL 




FY»57 

2,00 


2,00 


ll.OO 






13. BUDGET AC 


nviTY: 









RESEARCH 

REVIH'.f & APPROVAL 

BIOLOGIC STAiTOA-^DS 



tlTi ADfflNISTRATION 

I i PROFESSIONAL & 

TECHNICAL ASSIST- 
i 1 ANCE 



□ 



□ 



lU, IDEl^TIFY ANY COOPElPuATIMG UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER 0RGANILATI0M3, PROVIDING FUNDS, FACILITIES, OH PERSOiflffiL 
FOR T'lIS PROJECT IN FY1957. IF COOPERi.TIHG UNIT IS UITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



PUBLIC I-IE;J,TH SSRVICE - NATIOiV.L INSHTUTES OF HEALTH 
IiroiVIDUAL PROJECT REPORT 

Part C: Honors, /.wprds & Publicrtions 1^. NIkMD-37 



SERIAL NUMBER 

16, LIST PaBLIC.'TIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
CALENDAR YE/Jl 19^6: 

SoniG Consider." tions Concerning the Nrture of the Enzymic Galacto- 
VJaldenasc Conversion, by Herman M, Kalckar and Elizabeth S, 
Ifejcwcll, Biochc-m, ct BiophySg Acta, in press. 

Diphosphopyridinc Nucleotide, A Cofactor for Galacto-Waldcnasc, 
by Elizabeth S. Maxwt.ll, J. Im. Chem, Soc., 78, lO?!; (19^6). 

Congenital Galactoseriiia, A Single Enzymatic Block in Galactose 
Metabolism, by K, J. Isselbachcr, E. P. /nderson, K, Kiirahashi 
and H. H. Kalckar, Science, 123, 3198 (1956), 

Enzymatic Formation of Uridj.ne Diphosphoglucuronic .'cid, by 
J, L, Strominger, E, S. Maxvjell, J, /xelrod and H, M, Kalckar, 
J, Biol, Chem,, in press. 

Some Properties of Uridine Diphosphoglucose Dehydrogenase, by 
E, S, Maxwell, H, M, Kalckar and J. L, Strominger, /irohives of 
Biochemistry & Biophysics, Linderstr/m-Lang Anniversary Volume, 
in press. 

Biological Problems Posed by Studies on Mutants of Galactose 
Metabolism in Man and Microorganism, by Hiirraan M, Kalckar, 
Science, in press. 

Defect in the incorporation of Galactoso-1-phosphatc into 
nucleotides in Liver Tissue from Patients iri.th Congenital 
Galactosemia, by Elizabeth P. Anderson, H. i'l, Kalckar and 
Kurt J, Issolbacher, Science, in press. 

Enzyme Formation in Galactose Negative Mutants of E. coli 
by K, Kurahashi, Science, in press. 

Studies on the M(;tabolism of C^-Labelled Galactose in a 
Galactosemic Indiviaual, by Frank Eisenberg Jr., Kurt J, 
Isselbacher and Herman M, Kalckar, Science, in press. 



17, HONORS AND miRBS: 

None 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - NATIONi\L INSTITUTES OF HEALTH 
INDIVIDU/JL PROJECT REPORT 

Part A. Project Description Sheet 1. NIiJ^lD--38 



SERI/J. NUIIBER 



2. mWD 3, Biochemistry and Metabolism 

INSTITUTE OR DIVISION LABORATORY, BR/NCH OR DEFT, 



Ue Metabolic Enzymes ^, 

SECTION OR SERVICE L0CATI0N"T1F OTHER TRi'.N BETHESDA 

a^ Metabolism of inositol and its derivates. 

b) The biosj-nthesis of desoxyribose in bacterio-phage 

6» infected E. coli. 

PROJECT TITLE 



7» Arthur Weissbach 

PRINCIPAL INVESTIGATOR 



8, None 

OTHER j;nvestigators 



9. IF THIS PROJICCT REvSEMBLES, CCMPLE,MENTS, OR P/JU.LLELS RESEARCH 
DONE ELSEl-JIiERE IN TR-E PUBLIC HEALTH SERVICE (WTHOUT INTER- 
CHiUIGE OF PERSONNEL, FACILITIES OR FUI\roS) IDENTIFY SUCH 
RESEARCH! (BY SERI/J, NO.(S) IF VilTHIN NIH), 

None 

10, PROJECT DESCRIPTION 

a) To study the metabolism of myo inositol and its 
derivatives in plants and animals. 

The metabolic fate and biosynthetic origin of inositol are, 
at present, unknown though this compound is ubiquitous in all 
species. Though attempts to demonstrate the phosphorylation of 
inositol by ATP in cell-free systems have not been successful in 
our work, evidence has been obtained for an enzyme in yeast which 
catalyzes the formation of ATP from /DP in the presence of inositol 



- 2 - 

MKMD-38 " ^" 
SERIAL NUMBER ~ 

10, PROJECT DESCRIPTION - continued 

hexapho sphate (phytic acid). The details of this reaction are 
as yet unclear. It is tentatively visualized as a transphorylase 
reaction in which a phosphate from phytic acid is transferred to 
ADP in the presence of a "pulling system" such as the hexokinase 
reaction, UDP will not replace ADP as a phosphate acceptor in 
tliis system which seems also to require Mg"*"*", 

inositol + ADP — )• inositol + ATP Glucose GlucoEe-6-P 

hexaphosphate pentaphosp^hate hexoKmase' 

(?) 



The enzyme catalyzing this reaction (l) has been partially 
purified by standard procedures and the mechanism of the reaction 
is being studied with the aid of isotopes, chromatography, 
inhibitors, etc. 

In conjunction i-rith this work the inositol dehydrogenase 
system of Goldstone and Magasanik has been adapted for the 

enzymr.tic assay of inositol at low levels (,01 ^ ,0^ |J.M), 

As suggested by the work of Franzl et al_. this enzyme has been 
found to be iniiibited by low levels of colchicine (lQ~h M), a 
mitotic poison, though the significance of this is unclear. 

b) In normal E. coli cell, only about 20;l of the pentose 
synthesized is of the desoxy type. After infection W).th the 
even T phages, the E, coli cell produces only dasoxypentose. 
Using 12"*" phage and E, coli cells, the synthesis of desoxj'- 
pentose is being studied in cell-free systems and, shortly, in 
protoplasts. Preliminary experiments have indicated a low level 
of synthesis in cell-free extracts of phage-infected E, coli 
cells. It is so low, however, that protoplasts prepared from 
E, coli cells by the penicillin method of Lederberg are being 
investigated in this regard instead. Since this work has Just 
started, no findings can be presented, ITie study of the 
mechanism of the biosynthesis of desoxyribose portion of DN/i. 
in such systems is the object of this investigation which in- 
volved the preparation, handling and assay of both the T2 pliages 
and their host cells. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEA.L?H SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PR0J13GT REPORT 



Part D: Budget Data 



11. NL\MD-38 



SERIAL MUHEER 



12, HJDGET DA 


rA: 








ESTI1L\TED OPLIO/.TTONG 


FAN YEARS 


DIRECT 


REIMEURSEM^IWT 


TOTAL 


PROF OTHER TOTAL 


FY«57 








^28,800 


m,^oo 


i:ii;2,300 


1.00 1,00 2.00 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHER 


TOTAL 




FI'57 








1,00 


1.00 


2,00 





13. BUDGET ACTIVITY ; 
RESEARCH 

REVIEW & APPROVAL 
BIOLOGIC STANDARDS 



nTi ADMINISTRATION 

i i PROFESSIONAL & 

TECMIGAL ASSIST- 
{ i ANCE 






111, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZ/iTIONS, PROVIDING FUi\IDS, FACILITECS, 0". PERSOIJI-IEL 
FOR Tins PROJECT IN FY 1957. IF COOPER/iTING UNIT IS TITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and addj.tional pajes, if necessary) 



PUBLIC HEALTH SERVICE - NATIONAL li'STITQTES OF HE/iLTH 
irroiVIDU/i PROJECT REPORT 

P/JIT 0: Honors, Awards &t. Publications l5. MKMD-33 



SERIAL MUIffliLR 



16, LIST PUPSLIC; nONS OTHER TII/IN ABSTRACTS FROM ffllS PROJECT DURING 
CALENDAR YEIR 1956: 

None 



17, LIST HONORS /KD AW/JIDS TO PERSONlffiL RELAHNG TO THIS PROJECT DURING 
CALEND/Jl YEm 1956: 



None 



Calendar Yecr 19^6 
PQBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF IffiALTH 
INDIVIDUAL PROJECT REPORT 
Pr.rt A. Project Description Sheet 1. NIAMII-39 



SERIAL NUI'IBER 



2, NIAMD 3. Biochemistry and Metabolism 

INSTITUTE OR DIVISION LABORATORY, BRANCH OR DEFT. 



k. Metabolic Enzynss ^^ 5. 



SECTIOFOR" SERVICE " LOCAnON (IF OTIiER TMN BETHESDA 



6, Th e possible role of sugar nucleotides as glycosyl donor s. 

mo JECT ■ TITLE 



7, Victor Gin s burg 



PRINCIPAL liWESTIGATOR 
8, None 



OTHER IWESnCATORS 



9. IF 'EilS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLELS RESEARCH 
DOfJE ELSEWHERE IN THE PUBLIC HEALlIi SERVICE (WITHOUT INTER- 
CH/NGE OF PERSONi\lEL, FACILITIES OR FUNDS) IDENTIFY SUCH 
RESEARCH (BY SERIAL NO.(S) IF WTHIN NIH)o 

None 

10. PROJECT DESCRIPTION 

Objectives - To study the possible implication of sugar 
nucleotides as intermediates in the metabolism of polysaccharides 
and other glycosyl compounds. 

Methods Employed - The usual methods of biochemical research 
will be employed. These will include specific methods for the 
assay of sugar nucleotides that wore developed previously in this 
section. 

Major Findings - This project is just starting. 

Significance to tlie program of the in stitute - The metabolism 
of complex carbohydrates is virtually unlcnown, Iny contribution 
to the understanding of the biochemistry of these carbohydrates 
would enhance the imderstanding of cellular metabolism 



Form No, ORP-1 
October 19^6 
(Attachment l). 



PUBLIC HEALTH SERVICE - NATIONAL TOSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPCRT 



Part B: Budget Data 



11. NIAMD~39 

SERIAL NUMBER 



12. BUDGET DATA: 








ESTIMTED OBLIGATIONS 




mn YEARS 


DIRECT 


REB-EUIISEM^NT 


TOTAL 


PROF OTHER TOTAL 


FY»57 








$20,iiOO 


4'i9,800 


;;^30,200 


1,00 1,00 2,00 




BUDGETED POSITIONS 




PATIEir DAYS 


PROF 


OTHER 


TOTAL 




FY»57 








1,00 


1.00 


2,00 




13, BUDGET AC 


TIVTTY: 







RESEARCH 

REVIEVJ & APPROVAL 

BIOLOGIC STAi\!riAPIlS 



^ ADMINISTRATION 

j j PROFESSIONAL & 

TECHiJICAL ASSISI 
I I ANCE 



LJ 



□ 



lU, IDEI>riFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONi>IEL 
FOR THIS PROJECT IN FY 1957. IF COOPER/lTING UNIT IS VIITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



> 



Ih 



PUBLIC HE/JLTH SERVICE - NATIONAL IHSHTUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications l5, NIiVMD-39 



SERIAL NUMBER 



16. LIST PUBLIC/ TTONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
CALEKiD/Jl YS;JI 19^6: 

None 



17, LIST HONORS AND AW;.KDSTO PERSONl'JEL REK.TING TO THIS PI^JECT DURING 
CAXENDAR YEAH 1956: 



None 



Calendar Year 1956 
PUBLIC tIEALTH SERVICE - NATIONAL INSTITUTES OF liEALTH 
INDIVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1. NLiMD-UO 



SERIAL MJMBEI^ 
2. NIAMD 3. Laboratory of Chemistry 

DISTITUTE OR DIVISION LABORATORY 

h. S ection on Carboh^'drates 5. 

SECTION OR SERVICE LOCATION (IF OTHER THAI\I BETHESDA) 

6. Carbo hydrates for Biochemical Research 

PROJECT TITLE 

7 . Hewitt G. Fletcher, Jr. 

PRINCIPAL II^IVESTIGATOR 

8. Robert K. Ness, H. B. Wood, Jr., E. Vis (Fellow), H. W. Diehl 

OTHER INVESTIGATORS 

9. Parallel Research: None 
10. PROJECT DESCRIPTION 

Objectives : 

A. To investigate new synthetic pathways for the preparation of 
carbohydrates and carbohydrate derivatives of biochemical 
importance . 

B. To improve kno^ra. procedures for the preparation of carbohydrate 
substances needed for biochemical research. 

C. To provide (insofar as facilities and time peraiit) NIH biochemi- 
cal and clinical investigators with such carbohydrates as are 
needed. 

Recent Progress : The main effort of this project has been devoted to 
topics involving D-ribose and its distant relative 2-deoxy-D-ribose. 
The areas of effort may be grouped under three headings. 

A. Avcyl Migration in Ribose Derivatives. 

Hydrolysis of 2,3. 5-trl-O- benzoyl -D-ribofuranosyl bromide 
gives a mixture of 2 ,3!5-"tri-0-benzoyl-i3-D-ribose and 1,3,5- 
tri-O-benzoyl-a-D-ribose. This newly discovered migration, of 
an acyl group from C2 to C]_; is the first of its tj'pe to be 
conclusively demonstrated and not only maizes 2-substituted ri- 
bose derivatives relatively accessible but may also have impor- 
tant implications in other areas of carbohydrate chemistry. 
Through this research the cyclic ortho-acid structure, fre- 



PROJECT DESCRIPTION (Cont'd) 



NL.MD-UO 



SERIAL NUl^IBER 



quently postulated in the past for various carbohydrate deriva- 
tives is rendered exceedingly unlikely. The most widely 
acQepted example of a oyclia ortho acid (a monobenzoyl-D-talose) 
was first reported from aii«ther laboratory, nearly twenty years 
ago. Re -examination of the substance by the original author has 
now revealed that the proposed structure is impossible. Repeti- 
tion of the preparation by the NIH group has given none of the 
expected substance but an isomer which has been shown to be 1-0- 
benzoyl-a-D-talopj'raiiose . 

B. Intra- and Intermolecular Condensations with Ribose. 

The condensation of D-ribose with benzaldehyde under vari- 
ous conditions has been found to afford no less than six well- 
defined crystalline substaraces. One of these has been shovm to 
be 2,3-0-benz3'lidene-p-D-ribofuranose, a substance of consider- 
able potential value in the synthesis of ribofuranosides of 
biochemical interest. Other products were found to be benzyli- 
dene derivatives of a monomeric and of a dimeric anhydride of 
ribose. Through these the structures of the puzzling "mono- 
acetoneanliydroriboses" reported some twenty years ago by P. A. 
Levene and his associates have been elucidated. One of these, 
1.5-anhydro-|3-D-ribofuranose, is the first adequately charac- 
terized member of a new class of carbohydrate derivative and; 
incidentally, is both a furanose and a pyranose. 

In the course of these studies the condensation of D-glucose 
with benzaldehyde was re-studied and the yield of i|, 6-0 -benzyl i- 
dene-D-glucose, a substance which has proved of great value in 
the synthesis of glucose derivatives, raised from 12^ to it2^. 
Tvra di-0-benzylidene-D~glucoses were found; subsequent investi- 
gation has sho-vm them to be the 1,2:^1-, 6- and 1,2: 3, 5- isomers. 
The latter opens an attractive pathway to the synthesis of 6- 
substituted glucose derivatives. 

C. The Preparation of 2-Deoxy-D-rlbose. 

The synthesis of 2-deoxy-D~rlbose from D-glucose via the 
metasaccharinic acids which was developed by J. C. Sowden is 
being studied. Preliminary work has afforded a substantial 
quantity of pure 2-deoxy-D-ribose for the Laboratory of Bio- 
chemistry and Metabolism and has allowed a redetermination of 
the physical constants of this important substance. 

Propo sed Course of Project : 

1. The general topic of pentose anhydrides opened by the "discovery" 
of l;5-anhydro- P-D-rlbofuranose will be investigated further. 

2. New methods for the selective substitution of sugar molecules ^ 
particularly that of D-ribose will be explored. 



PROJECT DESCRIRCION (Cont'd) ''' " 



SERIAL IIUMBER 



3. An attempt will be made to improve the synthesis of 2-deoxy-D- 
i-j.bose and thus make this unusually important sugar more 
readily available for chemical and biochemical research. 



Form No. ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - MTIONAL IHSTITITES OF ML^AL?H 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAI«.D-IiO 



SERIAL NUMBER 



12, BUDGET DA 


TA: 






ESTIFATRD OBLIGATIONS 


MAN YEARS 


DIRECT 


REIMBURSElIEtJT TOTAL 


PROF aniER TOTAL 


FI'57 






^50,600 


;a;19,200 5^69,800 


3,^0 2.00 SSO 




BUDGETED POSITIONS 


PATENT EA.YS 


PROF 


OTfER TOTAL 




T!",'tq7 







3.50 



2.00 



13. BUDGET ACTIAriTYs 
RESEARCH 

REVIEJ & APPROVAL 
BIOLOGIC STAf.IDARDS 



5.50 



[]73 ADMINISTRiiTION 

I i PROFESSIOmL & 

TECffillC/LL ASSIST. 
{ i ANCE 



□ 



□ 



lli. IDEiCIFY ANY COOPERATIIIG UNITS OF TfE PUiDLIC HEALTH S ,RVICE, OR 
OTHER ORGAHI'^ATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR T'HS PROJECT IN FY 19^7, IF COOPERi.TING UNIT IS 17ITHIN NIH 
INDICATE SERI/vL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 19 5 6 

PUBLIC HEALTH SERVICE - NATION.'\L INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part C: Honors, Awards & Publications 15.NIAMD-hO 



SERIAL HU^IBER 

16. LIST OF PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURraG 
CALEiroAR YEAR I956: 

R. K. Ness and H. G. Fletcher, Jr. 

The Conversion of l,i|-,6-Tri-0-benzoyl--2 3-0"(l-benzyloxybenzylidene)- 

p-D-fructofur.anose to 1 k 6-Tri-0-benzoyl-2,3-0-(l-ethoxybenzyli- 

dene)-p-D-fructofuranose by Acidic Ethanol. ~ 

J. Am. Chem. Soc. , 78, 1001 (1956) 

R. K. Ness and H. G. Fletcher, Jr. 

E\'idence that the Supposed 3,5 ■Di-0-benzoyl-l,2-0-(l-hydroxybenzyli- 
dene)-Q;-■D-ribose is Actually 1,3,5-Tri-O-benzoyl-a-D-ribofuranose. 
J. Am. Chem. Soc, 78, i;710 (1956) 

H. B. Wood. Jr., H. W. Diehl and H. G. Fletcher, Jr. 

Some Products Arising from the Condensation of D-Ribose with Benz- 

aldehyde. 2,.3-0-Ben2ylidene-P--D-ribofuraiiose a^id Di-(2;3-0-Benzyli- 

dene-D-ribofuranose) 1;5' :1 ' ,5-dianhydride. 

J. Am. Chem. Soc, 78, it-715 (I956) 

E. Vis and H. G. Fletcher, Jr. 

l,5-Aiili5'dro-P-D-riboxuri.inose and the 'Monoacetone Anhydroriboscs " 

of Levene and Stiller 

J. Am. Chem. Soc. , In Press 

H. B. Wood, Jr. , H. W. Dieh]. and H. G. Fletcher. Jr. 

1.2:4; 6-Di-0-benzylidenc-a"D-glucopyranose and Improvements in the 

Preparation of 4, 6-0-Benzylidene-D-glucopyranose. 

J. Am. Chem. Soc. , In Press 

17. HONORS AND AV/ARDS: None 



Calendar Year I956 
PUBLIC HEALTH SERVICE •■ NATIONAL INSTITUTES OF HEALTH 
INDIVIIXJAL PROJECT REPORT 
Part A. Project Description Sheet 1. NIaMD-UI 



SERIAL lO-IBER 



2. NIAMD 3. Laboratory of Chemistry 

INSTITUTE OR DIVISION LABORATORY 

h. Section on Carbohydrates 5- 



SECTION OR SERVICE LOCATION 

6. Cheiaistry ox" Steviosidc and Rela.ted Substances 



PROJECT TITLE 



Hewitt G. F l .ctcher , Jr. 



PRINCIP.U IN^-ESTIG/iTOR 
8. Erik Vis (FellovQ 



OTHER INVESTIG.YL'ORS 

9. PARALLEL RESEARCH: None 

10. PROJECT DESCRIPTION 

Objectives : The objective of this project is to elucidate the struc- 
ture and configuration of the carbohydrate moieties of stevioside, 
the sweet, non-toxic principle of the Paraguayan plant Stevia 
Rebaudiana (Bertoni) and to investigate the ixnique chemistry of some 
simpler analogs of stevioside. 

Recent Progress : The work of the previous years (1954 and 1955) had 
revealed the structure and mode of attachment of the sugar moieties 
of steviosidc. The isolated, ester-linked glucopyranosyl residue 
was shown to have a p-liiikage. However, since stevioside has been 
found to resist the action of all stereospecific glucosidases (eraul- 
sin diastase etc.) the two anomeric configurations of the disac- 
charide (2-O-D-glucopyrcuiosyl-D-glucopyranosyl) moiety remained in 
doubt. Treatment of steviolbioside heptaacetate with hjnirogen 
bromide in glacial acetic acid has now led to a derivative of 2-0- 
P-D-glucopyranosyl-D-glucose and thus one (and probably both) of~the 
anomeric linkages in the disaccharide portion of stevioside are p. 
The disaccharide 2-0-p-D-glucopyranosyl D-glucose is known also as 
sophorose and stevioside is thus a sophoroside. Only one other 
sophoroside has as yet been discovered in nature although the di- 
saccharide itself has been detected among the products fonaed from 
concentrated glucose solutions through the action of eraulsin. 

Proposed Course of Project : With this work the project is considered 

to be temiinated. 



Fonn No. ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - WA?IOilAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11, NIALro-hl 

SERHL I^IUi:;BLR 



12, BUDGET DATA: 








ESTIMATED OBI.Ii:iATIONS 




IAN YEARS 


DIRECT 


REII'iBURSEMENT TOTAL 


PROF 


CTHLR TOTAL 


FY'57 








$29,300 


$11,100 filio,Uoo 


1,00 


3.00 li.OO 




BUDGETED POSITIONS 




PATIEro DAYS 


PROF 


OTHER TOTAL 




FY«57 








2.00 


3.00 5.00 







13, BUDGET ACTIVITY; 
RESEARCH 

REVIEIaJ & APPROVAL 
BIOLOGIC STAlvEARDS 



Hcl ADMINISTRATION 

! I PROFESSIONAL & 

TECHNICAL ASSIST- 
I I • ANCE 



□ 

ED 



lU, IDEi\lTIFY ANY COOPERATING UNITS OF THE PUPLIC HEALTH SERVICE, OR 
OTHER ORGAOTZATIONS, PROVIDING FUlvIDS, FACttlTIES, OR P^LRSOm^lEL 
FOR THIS PROJECT IN FY 19?7. IF COOPERATING Ul\fIT IS VJITHEN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pa^es, if necessary) 



Calendar Year 1956 



PUBLIC flEALTH SERVICE - NATIOIIAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 15- N LiMD-ul 



SERIAL I-IUMBER 

16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURnJG 
CALEDID.AR YE/J^ 1956: 

H- B. Wood;, Jr., and H. G. Fletcher, Jr. 

The AnoLieric 2,3 > 4, 6-Tetra-O-acctyl-l-O-mesitoyl-L-glucopyranoses 

and Their Behavior with Alkali. 

J. Am. Chem. Soc. , 78, 207 (1956) 

H. B. Wood, Jr. and H. G. Fletcher, Jr. 

Migration of a Mesitoyl Group from C]_ to C2 in a-D-Glucopyranose. 

Derivatives of 2-0-Mesitoyl-D-glucose. 

J. Am. Chem. Soc. , TS,, 28^+9 (1956) 

E. Vis and H. G. Fletcher, Jr. 

Stevioside IV. Evidence that Stevioside is a Sophoroside. 

J. Am. Chem. Soc. , 78, ^^709 (1956) 

17. HONORS AND AW/JIDS: None. 



Calender Year 1956 

PUBLIC lEE/iTH SERVICE - U7J!I0LL'\L INSTITUTES OF flEAI.TH 

IHDIVirUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NL^M )-h? 

SEhlPL IIUMBER 

2. MIAMD 3- Laboratory of Chemistry 

DESTITUTE " L/LBOR/a:ORY 

h. Section on Carbohj-drr.tes S- 



SECTION LOCATION 

6. Higher- carbon sugars, anhydro sugars^ sugar alcohols a ) :d their 
PROJECT TITLE 

derivatives 



7. nelson K. Richtrayer 



PRDJCIPAL liWESTIGATOR 

8 . James 17. Pratt, Laura C. Stewart, Enimaiiuol Zissis 

OTIiER INVESTIGATORS 

9. PARALLEL RESEARCH: Hone 

10. PROJECT DESCRIPTION 

Objectives : To e\'olve generalizations relating the physical and 
chemical properties of the groups of substances named in the project 
title to their configurations aj:id conforr.iations. 

Re cent Progres s: In continuation of our studies on the formation of 
monomeric nonreducing aaihj'dro sugars by the action of acids on hex- 
oses , heptoses, ajid heptuloscs , research in 19?6 included a study of 
the effect of temperature on the extent of anhydride formation; in 
general, it appears that an increase in temperature results in aji 
approach toward equimola.rity at equilibrium. Of especial signifi- 
cance was the preparation of 1 , 6-anhydro-3-deoxy-p-D- arabln o-hexo- 
pyranose (sirup) and l,6-aniiydro-3"deoxy-p-D-ribO"hexopj'a'ano3e (sirup) 
and their crystalline diacetates; of crystalline 3"deoxy-D- ribo - 
hexose_, and of 2,7-aiiliydro-P-D--mnno-heptulopja"anose by the action of 
acid on D- manno -heptulose and of rlkali on the nev? phenyl g-D- manno - 
heptulopyranoside. The structure of the tri-0-methylenevolemitol has 
been reasonably well established as l,3:2,5:'^-j6-tri-0-methylene-D- 
glycero -D- ng.nno -heptitol , in agreement with the prediction on the 
basis of conformational axialysis by J. A. Hills [Chen. S: Ind. , 633 
(I95if)]. 

Current and Projected Investigations : Continuation of these and 
closely related topics. 



Fom No. ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATION/iL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAMD-h2 



SERI^VL iWMBER 



12, BUDGET DA 


TAS 










ESTIMATED OBLIGATIONS 






MN YEARS 


DIRECT 


REIWBURoEMEl'TT 


TOTAL 


PROF 


{TlMi TOTAL 


FY»57 










$51i,600 


,^2 0,2 00 
BUDGETED POSITIONS 


ii;7U,8oo 


li.OO 


1.00 5.00 

PATIENT DAYS 


PROF 


OTHER 


TOTAL 




FY'57 

U.OO 


1.00 


5.00 







13. BUDGET ACTIVITY: 
RESEARCH 

REVIEVJ & APPROVAL 
BIOLOGIC STANDARDS 



HI 

□ 
p 



ADMINISTRATION 

PROFESSIONAL & 
TECHNIC/iL ASSIST- 
ANCE 



□ 



a 



lli, IDENTIFY ANY COOPER/iTING UNITS OF TIE PUBLIC lE/'.LTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDE-IG FUiTOS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS IJITHIN MIH 
liTOICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



PUBLIC HE.^LTH SERVICE - IIATIONAL INSTITUTES OF HEiYLTH 
INDIVIDUAL PROJECT REPORT 
Part C: Honors, Awards & Publications 15- I'IlAi'ID-U2 



SERI/X lOffiER 



16. PUBLIC/a?IONS OTHER THAJM ABSTRACTS FROM THIS PROJECT DURING 
CALEITOAR YEAR 195 6: 

Laura C. Stewart,, Emmanuel Zissis and Nelson K. Riclitmyer 

Die Bildung von 2.7-Anhydro-p-D- gluco -heptulop>T:aiose durch Ein- 

wirkung von Saure auf D-gluco-Heptulose und von iUkali auf Phenyl - 

g-D-g luco -heptulopyranosld. 

Chem. Ber., 89; 535-5^'^ (1956) 

Nelson K. Richtmyer and James W. Pratt 

Sedoheptulose — Its Rotation, Reducing Power, Ecj.uilil>rium with Sedo- 

heptulosan in Acid Solution, and Crystalline Hexaacetatej Also 

Crystalline 2,7-Anliydro-P-D-cltro-heptulofuranose . 

J. Am. Chem. Soc. ,, 78> ^^17-^^721 (1956) 

17. HONORS AND AWARDS: None 



Calendar Year 195*5 
PUBLIC HEALTH SERVICE - NATIOILU INSTITUTES OF HEALTH 
IIIDIVIDUAL PROJECT REPORT 
Part A. Pi'ojcct Djscription Sheet 1. NL.>iD-U3 



SERI.AL ilUMBER 

2. HIAMD 3- Latjorctory of Chemistry 

INSTITUTE OR DIVISION LABORATORY 



k. Section on Carbohydrates 5. 

SECTION LOCATION 

6. Micror-nalytical La"boratory 



PROJECT TITLE 
7. William C. /dford 



PRINCIP/i INVESTIGATOR 

PaiJ_a M. P arisiuS; E-/elyn G. Peako, B^-ron Bacr, Lillian J. Wyngarde n 
OTHER INVESTIGATORS 
(to Aug. 26), Rosemary L. O'Brien (from Aug. 20), VJillio jn R. Jones__ 

Charlies G. Rems"burg 



9. P/iRMUHL RESE/J^CH: None 
10. PROJECT DESCRIPTION 

The microaiialytical laboratory is a searvice organization which 
provides necessary analj-tical services for research personnel of the 
NationrJ. Institutes of Health. The scope of this work is summarized 
as follows: 

1. About 10,000 elemental, functional group cjxd instrumental 
analyses were performed during the past year. These, with the ap- 
proximate number of each, include: carbon and hydrogen (2,500), 
nitrogen by Dumas, KjeldalxL and Nessler methods (2,300), reducing 
sugar (800), halogens (15O), phosphorus (ij-50), lactic acid (2,200 
or 1,100 in duplicate), functional groups such as methoxyl acetyl, 
carboxyl.. active hydrogen (150), weight loss-moisture-ash etc. (h^O) , 
metaJ-S such as sodium, potassium, iron, barium and antimony (50) , 
miscellaneous (350) _ infra-red spectra (l,150), and optical rota- 
tions (150). Recipients of this service include approximately 100 
research workers of the NIH staff. In addition ^ analyses are per- 
formed for governmental agencies outside the NIH, insofar as they 
can be handled without interfering with the progress of NIH research. 
Agencies which have received such service at various times include 
the Naval Medical Research Institute, Bethesda, Md. the Tuberculosis 
Research Laboratory, U.S.P.H.S., New York, the National Bureau of 
Standai-^s, the Naval Research Laboratory., .'\nacostia, Marylcuid State 
Board of Health, Bcatimore, and the Food & Drug Administration, 
Washington D. C. 



PROJECT DESCRIPTIOH (Cont'd) NlKlnD-U3 



SERIAL IWMBER 

2. A laboratory operated by Mr. Jones provides infrared spectra 
for the Carbohydrate Section of the Laboratory of Chemistry and also 
for personnel of other Institutes insofar as his vrark load and sched- 
ule permit. 

3. A large number of special and inorganic analyses were per- 
formed by Mr. Remsburg who also maintained a variety of standard 
solutions for the use of researchers. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - MTIOWAL INSTFJUTES OF HEALTH 
IflDIVIDUAL PROJECT REPORT 



Part Bs Budget Data 



11, Niwm-U3 



12, BUDGET DATA! 










ESTH'IATED OBLIGATIONS 






MAN YEARS 


DIRECT 


REEIBUl^SEI'IENT 


TOTAL 


PROF 


OTHER TOTAL 


FY«57 










161,900 


.ii23,200 
BUDGETED POSITIONS 


$85,100 


2,00 


5.00 7.00 

PATIEiC DAYS 


PROF 


OTHER 


TOTAL 




FY«5? 










2, 00 


5.00 


7.00 







13. BUDGET A C TIVIT Y! 
RESEARCH 

REVIEl^J & APPROVAL 
BIOLOGIC STANDARDS 



[3 ADMNISTRATION 

I I PROFESSIONAL & 

TECHNICAL ASSIST- 
I I ANCE 



□ 



□ 



lit, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING RIi\roS, FACILITIES, OR PERSONi^lEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



1 



Cclendar Yeur 195 6 
PUBLIC HEALTH SERVICE - N/iTIONAL IWSTITUTE,S OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part C: Honors, Awards & Publications 15. NL1MD-I43 



SERIAL IWMBER 
16. PUBLIC7\TI0WS : None 
17- HONORS AI\ID AW/iRDS: None 



Calendar Year 1956 
RJBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
iroiVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1, M iijviD-LLi 



mmhhh ^ 

(Serial Ilumber) 



2. NIAJ'D 3. Laboratory of Chtmistry 

U. Section on Analgesics 5» 

6. Chemical Strixctnre and An alge sic Action 

Project Title "" '" ■"■ 

7. Principal Investigator: Nathan B. Eddy 8. None 

9. 

10, Project Description: 

Obj ectives; To ascertain the relation of chemical structure to 
analgesic J addictive and toxic properties and the possible inter- 
relationships of these properties j with the ultimate goal of obtain- 
ing more adequate drug relief of pain with greater safety. 

M?.*^^.^...E'^^::_?l?.2iii^* ^^ screening program (hot-plate method) 
has been continued not only ''dth new synthetic compounds prepared oy 
chemists in this Section but with compounds supplied by industrial 
laboratories and by another Institute (Heart). Recent emphasis has 
been on compounds in which the laethyl on the nitrogen has been 
replaced by phenylethj^ or closely allied radicals. 

Ve have also cooperated inth the Laboratories of Mental Health 
in the study of enzymatic N-demethylation of narcotics and the 
possible mutual relationship of this phenomenon and of analgesic 
action to tolerance developinent. 

A program has been worked out recently for a cooperative study 
with an industrial laboratory on possible tranquilizing effect of 
drugs prepared in our laboratories. 

In the clinic the first of our acute evaluation studies of the 
clinical effectiveness of a new analgesic agent have been completed. 
This study was run parallel w1.th tvro similar studies with the same 
agent at other institutions. 

There have also been cooperative studies on addiction liability 
with the Lexington group and vdth Dr. M, H. Seevers at the University 
of Michigan. 



SEHL.L NO. 



Significant findings include: 

1) Activity 50-100 times that of morphine in some of the afore- 
mentioned N-(2-phenylethyl) compounds (mouce test). Conclusions can 
be dravrn as to the optimal influence of these substituents, 

2) High activity (comparable to morphine) and relatively Iot-; 
toxicity in 2' -hydroxy-2,5'jS'-trimethyl-6:7-benzmorphan, a new com- 
pound synthesized in our laboratories. 

3) Activity approaching that of morphine was found in one 
compound of fifteen alkaloids derived from amaryllidaceae. It 
showed high toxicity. 

U) Clinical results checked favorably with those obtained else- 
where in the cooperative study mentioned in this connection. 

5) 5-(Tn-Hydroxyphenyl)-2-riiethylmorphan, a compound synthesized 
in our laboratory and of activity comparable to morphine in mice, 
has been found to possess a good deal less addiction potential than 
morphine in monkeys (University of Michigan studies). 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOi-IAL INSTITUTES OF HFALTH 
ira)IVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAl-m-liU 



SERIAL MJMBER 



12, BUDGET DATA; 








ESTIMATED OBLIG/'.TIONS 




MAN YEARS 


DIRECT 


REIHBURSEMFJr TOTAL 


PROF 


OTKi'.R TOTAL 


FY '57 








^5,800 


$6,300 $22,100 


0.50 


2.00 2,50 




BUDGETED POSITIONS 




PATIEiW DAYS 


PROF 


OTHER TOTAL 





BY«57' 



0.50 



2,00 



2.50 



13, BUDGET ACTIVITY! 
RESE/lRCH 

REVIEW & APPROVAL 
BIOLOGIC STAND/iRDS 






ADMNISTRATION 

PROFESSIOmL & 
TECHNICAL ASSIST- 
ANCE 



□ 



Q 



lU. IDENTIFY AI^ COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOffilEL 
FOR T-ilS PROJECT IN FY 1957, IF COOPEliATING UNIT IS VJITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse or additional pages, if necessary) 



Part C: Honors, Awards & Rxbllcations. ]_^ NIAMD-UU 

(Serial Number) 

16. Eddy, N. B. "The search for new analgesics", J. Chronic 
Pis., h, ^9, (19^6). 

Eddy, N. B. "Addiction liability of analgesics: tests and 
results", J, Am. G eriatrics Soc. , h, 177 (19^>6). 

Eddy, N. B., Halbach, H. and Braenden, Olav J. "Synthetic 
substances mth morphine-like effect. Relationship between 
analgesic action and addiction liability, with a discussion 
of the chemical structure of addiction producing substances". 
Bull. i;id . Hlth. Org. lU, 353 (1956). 

Eddy, fJ, B. "The history of the development of narcotics". 
Law and Contemp orary Pr oblems, (^;uarterly publication by Duke 
University School of I^smJ, In press. 

Ferrine, T. D. and Eddy, K. B. "The preparation and analgesic 
activity of U-carbethoxy-Ij-phenyl-l-( 2-phenylethyl)-piperidine 
and related compounds", J. Org . Chem ., 21, 125 (1956). 

Eddy, N. B. "Synthetic narcotic drugs". Union Signal, 82, 63 
(1956). — 

17. Reappointed, for an aciditional 5-year term, to the expert 
panel of tlie ^.orld Health Organization on matters pertaining 
to drugs liable to produce addiction. 

Secretary of the Committee on drug addiction of the National 
Research Council. 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 

I^roIv^)UAL project report 

Part A. Project Description Sheet 1, NLJ4D-U5 

i[Serial NumberT 

2« NIAI'-ID 3» Laboratory of Chemistry 

U. Section on Analgesics 5» 

6. Study of Analgesic Action in Humans i-rith Pathological Pain. 

7. Nathan B. Eddy 8. Joseph Cochin 

9. 

10. Project description: 
Objectives: 

a) Evaluation of new analgesic agents in patients with persistent 
pain. 

b) To determine whether or not development of tolerance and 
pliysLcal dependence as the result of prolonged analgesic medication 
can be held in abeyance by the simultaneous administration of a 
narcotic antagonist. 

Methods and results: 

a) By the dou.ble blind technique and the administration to the 
same patient of one or more roiinds of a quintet of agents, which is 
made up of two doses of a standard analgesic, a placebo, and two 
doses of the new agent, we have been collaborating in the evaluation 
of dl-li,U-diphenyl-6-piperidine-3-heptadone (piperidyl methadone). 
It appears to have approximately one half of the anali^esic effective- 
ness of morphinej that is, its effective dose is twice that of the 
optimal dose of morphine, but in our experience its use has been 
attended by the appearance of no side effects. ! e have begun 
evaluating a depot preparation of morphine, comparing it to ordinary 
parenteral preparations of morphine. This has just recentl?/ been 
initiated and our results are still too fragmentary to permit any 
conclusions to be drawn. 

Twenty patients have been made available to us by clinical 
secti ons of the National Cancer Institute and two patients by clini- 
cal sections of NIAID. 



IJe have been able so far, at their request, to use members of the 
regular nursing staff as observers for the collection of data. This has 
not always been satisfactory and any increase in the number of patients 
available for study would mal-ce the services of a fu].l-time nurse observer 
essential. 

b) This part of the project has not yet begun because no patients 
have been available. It is becoming increasingly important (see below) 
to get tills project under way and a request has been made for the assign- 
ment of h beds in the Clinical Center for this purpose, patients to be 
admitted specifically for this study and cared for jointly by ourselves 
and the Metabolic Division of the Cancer Institute. 

One of us (N. B. Eddy) has elaborated a completely detailed project 
outline for the study under (b), which has been very highly commended by 
the Statistical Division, OD. As the result of conferences here and 
abroad this protocol has been accepted and will be follox^ed in joint 
investigation of the problem at Montefiore Hospital in New York, Eppen- 
dorfer I'Srankenhaus in Hamburg, Germany, and at the Psychiatric Clinic in 
Berlin, Germany, as well as our Clinical Center. The data from all of 
these institutions are to be pooled and analyzed together. Hence the 
emphasis on the desirability of getting the project going here parallel 
to its pursuit at the other centers. 

The clinical staff are to an increasing extent consulting us on 
problems of pain relief and management of patients who seem to be already 
tolerant to narcotic drugs. Ve have also aided in withdrawing patients 
whose pain is no longer a major problem but whose physical dependence on 
narcotics precluded abrupt termination of these agents. 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - NATIONAL BISTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part Aj Project Description Sheet 1^ NIiiMD-'li6 ,— — 

'^' (Serial Number) 

2. NIAMD 3. Laboratory of Chemistry 

k» Section on Analgesics $. 

6. The Synthesis of the Unknown Isomers of 1-Cocaine. 

7. Stephen P. Findlay 8. None 

9. 

10. Project Description; 

The synthesis of racemic alio cocalilne and of racemic allo- 
pseudococafne has been completed (see last year's report), and 
the resolution of these racemates is in progress. These sub- 
stances, which are closely related to atropine as well as 
l-cocaine, are of theoretical importance to both organic and 
pharmacological chemistry. 

The raechanism whereby amines and amine salts catalyze the 
condensation reactions of aldehydes has not heretofore been 
elucidated. A mechanism for such reactions based on theoretical 
considerations and kinetic data for similar reactions has been 
developedj and its applicability to such diverse phenomena as the 
Robinsonian biological synthesis, the Mannich and Knoevenagel 
reactions, the Strecker amino acid synthesis, the conversion of 
nitriles to ortho esters, and the ammonium clxLoride catalyzed 
synthesis of acetals is being investigated. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUELIC lEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-U6 







SERIAL 


l\IUMffiR 


12, BUDGET DATAs 








ESTIMATED OBLIGATIONS 






MAN YEARS 


DIRECT REIMBTIRSEMErIT 


TOTAL 


PROF OTHER TOTAL 


FY«5t 








i^9,100 $3,000 


:i;,12,100 




1.00 - 1.00 


BUDGETED POSITIONS 






PATIEOT DAYS 


PROF OTHER 


TOTAL 




FY«57 








1,00 


1.00 







13 t BUDGET ACTIVITYi 
RESEARCH 

REVIH'J &■ APPROVAL 
BIOLOGIC STANDARDS 



fx] ADMINISTRATION 

i 1 PROFESS lOmL & 

TECHNICAL ASSIST- 
□ ANCE 



a 



a 



lU, XDEiriFY ANY COOPERATING UICETS OF THE PUBLIC HEALTH SERVICE, CR 
OTHER ORGANIZATIONS, PROVTOING FUNDS, FACILITIES, OR PERSONNEL 
FOR Tins PROJECT IN FY 19^7. IF COOPERATING Ul^IT IS VJITHIN NIH 
INDICATE SERIAL NO(S)! 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

Part C: Honors, Awards and Publications 1$, NI/iMD-[t6 

(Seri al Number ) 

16 . Publi cati ons : 

Findlay, Stephen P. "The Conversion of Certain I^roles to ol, 
5-Alkane-Dioxijnes", ^, Org, Chem ., ^, 6hh (l??6), 

Findlay, Stephen P. "The Synthesis of Racemic Allococa5!ne and 
Raceriiic Allopseudococaxne", J, Org . Chem. , 21, 711 (1956). 



Calendar Year 19^6 

Part A; Project Description Sheet 1. NIjLMD''li7 ^__ 

(Serial Number ) 

2, NIAMD 3» Laboratory of Chemistry 

U. Section on Analgesics 5» 

6. Alternative Syntheses of 6 : 7-benzmorphan derivatives 

7. Edward M. Fry 8. None 

9' 

10. Project Description: 

Objective: To develop new and versatile ^ntheses for the 
title compounds. 

The strong analgesic activity of the Compound I (R=R'=CH^) 
makes it desirable to synthesize other similar compounds in which 
the R radicals are varied as well as configurational isomers. The 
Gretje synthesis (cf . E. L. May progress report, 1956) is not of 
general utility and attempts to develop other synthetic approaches 
are being made. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL WSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



il, NIAMD-U7 

Sm^L NUI4E1ER 



12, BUDGET 


DATA; 

ESTIMATED OBLIGATIOMS 






MAN TEARS 


DIRECT 


REIMEURSEMi:i\iT 


TOTAL 


PROF 


OTHER TOTAL 


Fyt57 










^^9,^00 


*3,000 

BUDGETED POSITIONS 


:ipl2,500 


1,00 


1,00 
PATIEilT DAYS 


PROF 


OTHER 


TOTAL 




FY»57 










1,00 


- 


1.00 






13. BUDGET ACTIVITY ; 









RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STAhlDARDS 



nn ADMNISTRATION Q 

I I PROFESSIONAL & 

TECHNICAL ASSIST- 



lli, IDEI^JTIFY ANY COOPEHATING UNITS OF THE PUTLIC I-EALTH SERVICE, OR 
OTHER ORGANIZATIONS, PRO^'IDING FUITOS, FACILITIES, OR PERSOmffiL 
FOR THIS PROJECT IN FY 19^7* IF COOPERATING U1\IIT IS WITHH^ NIH 
Ii\IDICATE SERI/IL NO(S); 



(Use reverse and additional pages, if necessary) 



SErSl^NO. Calendar Year 1956 

Part C; Honors, Awards and Publications: 
16 . Public ati ons : 

Cohen and Fry: "Some reactions of 3-substituted hydantoins", 
J. Am. Cherg . Soc . (In press). 



Calendar Year 1906 

PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 

INDr/IDUAL PROJECT REPCRT 

Part A. Project Description Sheet 1. NL.MD-U8 

(Serial Number) 
2. NIAM) :5, fjaboratory of Chemistry 

U, Section on Analgesics 5» 

6. Sjmthetic Analgesic Agents 

7. Everette L, May 8. None 
9. 

10. Project description: 

Objective: Synthesis of new drugs for relief of pain and 
possible utility as tranquilizing agents. 

Methods: Synthesis of a hydroxylated 3-ring benzmorphan system> 
containing a major portion of the morphine skeleton, was accomplished 
by a modification of the well-knovin Grewe synthesis. The substance 
showed considerable activity, which was brought equal to that of 
morphine by generation of a quaternary carbon atom at the critical 
position analogous to that in morohine. Extension of the work to 
analogs of the very active piperidine derivatives alphaprodine and 
betaprodine showed that the position of methyl and propionoxy groups 
in these di^ugs is optimal, and transposition results in practical 
loss of activity. Indeed it x-ras found that changes in what were 
formerly regarded as unimportant minor groups may cause profound 
change in activity. The replacement of N-raethyl in the morphine 
series by N-phenylethyl may give a 10-fold increase. 

Major findings: Significant analgesic activity in relatively 
simple benzmorphan derivatives; extraordinary activity in N-phenyl- 
ethyl morphine types, in desomorphine about 100 times that of morphinej 
a vastly improved preparative method for these phenylethyl types. 

Projected work: will involve optical resolution of some of the 
racemic substances described. 



Form Mo, OPR-1 
October 1956 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTmrES OF HEALTH 
IITOIVIDUAL PROJECT REPORT 



part B; Budget Data 



11. NIMn>r.U8 

SERIAL NUMBER 



12, BUDGET 


DATA: 






ESTIFATED OBLIGATIONS 


MAM YEARS 


DIRECT 


REII-ffiURSEI'iENT TOTAL 


PROP OTHER TOTAL 


FY«57 ' 






^15,600 


$5,000 i,2 0,600 


1.00 1,00 2.00 




BUDGETED POSITIONS 


PATIEOT DAYS 


PROF 


OTHER TOI'AL 




FY«57 






1,00 


1.00 2.00 




i-^. RimaKT 


ACTIVITY! 





RESEARCH 

REVIEliT & APPROVAL 

BIOLOGIC STANDARDS 






ADMINISTRATION 

PROFESSIONAL &; 
TECffllGAL ASSIS1 
ANCE 



a 



LJ 



lii, IDEIMTIFY AM COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONInIEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 19^6 

Part C: H onors. Awards and Publications. l5. jij, ,n-)|fl 

CSeri al Nvmber ) 

16 . Publi oati ons : 

May, E. L. , "Structiires Related to Morphine. V. Azabicyclodecanes 
Derived from 2-(m-Iiethoxyphenyl')-cyclohexanone."i,J.6ree Chem aj gl» 
223 (1956). ~ ~ '■ 

May, E. L., "Structures Related to Morphine. VI. N-(2-Phenylethyl) 
Derivatives of some Phenyl and Benzmorphans." J. Org. Chem. , 21, 
889 (1956). ~ 

May, E. L. "Structures Related to Morphine. VII, Piperidine Deriva- 
tives and Examples of Failure in the Knoevenagel Reaction." J. Org . 
Chem . (In press). 

Popovici, Geschickter, May and Mosettig. "Gonadal Effects of 
trans-Decahydroouinolino Alcohols." J. Org, Chem . (In press). 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
BIDIVIDUAL PROJECT REPORT 

Part A: Project Description Sheet J, NLiMD-L9 

(Serial Number) 

2. NIAMD 3. Laboratory of Chemistry 

U* Section on Analgesics 5» 

6. Aminofluorenes as Synthetic Analgesics 

7. James G. Miirphy 6. None 

9. 

10. Project Description: 

Objectives and Methods: To determine the influence of the tri- 
cyclic fluorene nucleus on potential synthetic analgesics. For this 
purpose, fluorine itself was aminoalkylated by standard methods. 
The resulting dimethylaminoethylfluorene was found to have some 
analgesic action. Since the presence of a phenolic hydroxyl usually 
increases such action, the model experiment was extended to methoxy- 
fluorene successfully, but the cleavage of methoxyl to the desired 
hydroxyl is still in progress. 



Form No. ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOMAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11, NIAIID - h9 



SERIAL NUilBER 



FI«57' 



BUDGET DATA: 

ESTIMATED OBLIGATIONS 




MAN YEARS 


DIRECT 


REIIffiURSEMEOT 


TOTAL 


PROF OTHER TOTAL 


§8,200 


i'.3,000 


iS)ll,200 


1,00 - 1,00 





BUDGETED POSITIONS 


PATIENT DAYS 


PRO'F 


OTHER TOTAL 




•57 

1,00 


1,00 





13, BUDGET ACTIVITY; 
RESEARCH 

REVIEV/ & APPROVAL 
BIOLOGIC STAiTOARDS 



fxl ADMINISTRATION 

I I PROFESS lOmL & 

TECH1«CAL ASSIST- 



tj 



lU. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOM'JEL 
FOR TfflS PROJECT IN FY 19^7, IF COOPERi.TING UNIT IS VJITHIN NIH 
INDICATE SERIAL NO(S)j 



(Use reverse and additional pages, if necessary) 



N L-IMD-U9 ^„^, 

SERI^.L NO. Calendar Year 1956 



Part C: Honors, Awards and Publications: None 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



1. NIiJ4D>$0 

(Serial IvHimber) 

3. Laboratory of Chemistry 

5. 

8 . None 



Part A. Project Description Sheet 

2. NIAMD 

U. Section on Analgesics 

6. Analgesic Drugs 

7. Theodore D. Perrine 

9. 

10. Project Description: 

Objectives: Preparation of new analgesicsj and basic research 
on morphine dhgraistry. 

Methods: 

1. Enlargement of the nitrogen-containing ring in the 
codeine series. The ring in lO-hydroxydihydrocodeine has success- 
fully been opened, additional methylene group added, and closed 
again. Final steps incomplete. 

2. The presence of a quaternary carbon atom in definite 
relation to basic nitrogen seems essential for analgesic action. 
Fundamental studies are being conducted on methods of generating 
such quaternary carbons in the presence of other labile but 
necessary functional groups. 

3. Phenoxyacetates of the morphine series have been found 
to have markedly prolonged analgesic action. The series of such 
compounds is being extended. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOML INSTITUTES OF HEALTH 
IIMDIVIjjUAL project REI'ORT 



part B: Budget Data 



11, NiArm-^o 
Serial iwmber 



12. BUDGET DA 


TA: 










EST 


IIIATED OBLIGATIONS 




MAN YEARS 


DITtECT 




REIMBUIiSFJ^'!i;NT TOTAL 


PROF 


OTIER TOTAL 


FY»57 










$10,200 




5iU,ooo iin)i.,2oo 


1,00 


1,00 




BUDGETED POSITIONS 




PATIEOT DAICS 


PROF 




OTHER TOTAL 




FY' 57 










1,00 




1,00 







13. BUDGET ACTIVITY; 
RESEARCH 

REVIEW & APPROVAL 
BIOLOGIC STAimARDS 



nn ADMINISTRATION ^^ 

I I PROFESSIOmL & 

TECmWCAL ASSIST- 
□ ANCE □ 



lU, IDENTIFY Ai'JY COOPERATING UNITS OF THE PUBLIC HFALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUWS, FACILITIES, OR PERSONl^L 
FOR TfflS PROJECT IN FY 19^7, IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL WO(S): 



(Use reverse and additional pages, if necessary) 



SERIi.L NO. Calendar Year 1956 

Part C: Honors, Awards and Publications 
16. Pablications; 

Perrine, T. D. and Eddy, N. B. "The preparation and analgesic 
activity of U-carbethoxy-U-phenyl-l-(2-phenylethyl)-piperidine 
and related compounds", J. Org . Chem., 21, 12^ (19^6). 



Calendar Year 19^6 



public health service - national institutes of health 
iivDH'idual project report 



Part A: Project Description Sheet 1. NL^i-ID-^l ^__ 

(Serial Number) 

2. WIAMD 3. Laboratory of Chemistry 

km Section on Analgesics 5» 

6. Basic Research on Thebaine Chemistry. 

7. Lyndon F. Small 

8 . None 

9- 

10» Project Description: 

Thebaine is a very reactive morphine alkaloid, and the source of 
several important narcotics. It reacts vjith methyl hypobromite to 
add the elements of methanol, and the product of hitherto unknown 
structure has been converted into a series of hydroxy ketones and 
dihydroxycodeine derivatives of theoretical and perhaps practical 
interest. The reaction has been extended to dihydrothebaine and 
similar types. Project incomplete. 



Form No, CEP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11. NIAMD-gl 

SERIAL NUifflER 



12 , BUDGET 


DATA: 

ESTD4ATED OBLIGATIONS 






MAN YEARS 


DIRECT 


REIMHJRSfeMENT 


TOTAL 


PROF 


OTHER TOTAL 


FI«57 










|j6,^00 


$2,100 
BUDGETED POSmONS 


$8,600 


0,50 


0,50 

PATIENT DAYS 


PROF 


OTHER 


TOTAL 




FY '57 










0.50 


•• 


0.50 







^3» HJDGET ACTIVITY; 
RESEARCH 

REVIEI-J & APPROVAL 
BIOLOGIC STA^IDARDS 



T^l ADMINISTRATION 

I I PROFESS lOML & 

. TECHNICAL ASSIST- 
ED ANCE 






Hi. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PR-OVIOKG FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 195?. IF COOPER/iTING UNIT IS VJITHIN NIH 
INDICATE SERIAL NO(S): 

Merck & Con^jany and Mallinclcrodt Chemical Works, 



(Use reverse and additional pages, if necessary) 



SERIAL ^0. Calendar Year 1956 



Part C: Honors, Awards, and Publications: None 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part A: Project Description Sheet 

2. NIAMD 

U. Section on Analgesics 

6. Hydroxylated codeine derivatives 

7. Lyndon F. Sraall 

8. Lewis J. Sargent 



1, NL-.MD-52 

(Serial Number) 

3. Laboratory of Chemistry 

5. 



10. Project Description: 

The action of osmium tetroxide on unsaturated derivatives of the 
codeine series results in the introduction of two nevf hydroxyl groups. 
The reaction has been applied to all available drugs of this type. 
In those tested so far, the result has been a diminution of analgesic 
activity. Project completed. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOML INSTITUTES OF HEALTH 
I1€)IVIDUAL PROJECT REPORT 



Part B: Badget Data 



11, NIAMD..^g 

SERIAL NUMBER 



12, BUDGET DATA: 








ESTB'iATED OBLIGATIONS 






MAN YEARS 


DIRECT REU'EUl^SEIffil^ 


TOTAL 


PROF 


OTHER TOTAL 


FY»57 








5^11,900 $it,000 


V15,900 


1,00 


1.00 


BUDGETED POSITIONS 






PATIEi^ DAYS 


PROF OTHER 


TOTAL 




FI«57 








1.00 


1>00 






13, BUDGET ACTIVITY: 









RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STANDARDS 



Lx] ADMINISTRATION Q 

I ! PROFESSIOML & 

TECffNICAL ASSIST- 
□ ANCE □ 



lli, IDENTIFY ANY COOPER/'-TING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOMviEL 
FOR THIS PROJECT IN FY 1957, IF COOPER.e.TINa UNIT IS WITHIN NIH 
IITOICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



NIaMD-52 
SERIAL NO. 

Calendar Year 1956 
Part C: Honors, j^.wards, and Publications: None 



MIAMD-g2 
SERIi;L NO. 

' Calendar Year 1956 

Part C: Honors, /.wards, and Publications: None 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A: Project Description Sheet 1. NKMD-g3 — ^ 

(Serxal Number; 

2. NIAMD 3. Laboratory of Chemistry 

U, Section on Analgesics 5« 

6. Relationship of structure to analgesic action in the morphine 

series. 

7. Lewis J. Sargent 8. None 

9. 

10. Project description: 

Objectives_and_methods: Proof of structure of the drug 
Metopon, as'well as" the influence of position of ketone and 
alcoholic oxygen in new drugs of a similar type. This involves 
a difficult synthesis of nitrogen-free degradation products 
which have been obtained directly from Metopon, and vhlch will 
determine the relative position of methyl and carbonyl groups 
in this drug. The introduction of a new hydroxyl group in 
Dicodide, which results in Eucodal, has a strong intensifying 
action, but nothing is known of the effect of position of this 
hydroxyl. New dihydroxy derivatives have been prepared by the 
action of osmium tetroxide on unsaturated centers, and keto 
hydroxy tjjpes from transformations of thebaine or dicodide. 
Polyhydrox?/ compounds so far tested seem less active than the 
simpler types. 



Form No. ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEA.LTH SERVICE - NA.TIOI>IAL INSTITUTES OF HEALTH 
IrlDIVIDUAL PROJECT REPORT 



Part B; Budget Data 



11, NIAMD-.$3 

SERIAL NUMffiR 



12, BUDGET 


DATA: 










— 


ESTIMATED OBLIGATIONS 






MAN YEARS 


DIRECT 


REIHBURSEIffiOT 


TOTAL 


PROF 


OTHER TOTAL 


FY'-^T' '" 










$5,200 




$2,100 
HJDGETED POSITIONS 


17,300 


0.50 


0,50 

PATIEWT DAYS 


PROF 




OTHER 


TOTAL 





FY '57" 



0.50 



13. BUDGET ACTIVITY: 



o.5o 



RESEARCH 

REVIEl'J g£ APPROVAL 

BIOLOGIC STANDARDS 



CD 



ADMINISTRATION 



PROFESSIONAL & 
TECHIMICAL ASSIST- 
ANCE 



a 



□ 



llu IDEI'inFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORG/iNIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS VJITHIN NIH 
BIDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



December 195^ 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NIAMD-g$ 



SERIAL NUMBER 
2. NIAMD 3. Chemistry 



INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTMENT 

k. Steroid 5« ^__ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 

6. The Structure of the Aglycone of Stevioside. 

PROJECT TITLE 

7. Erich Mosettig, Fred Dolder and Heinz F. Lichti 

PRINCIPAL INVESTIGATORS 

8. No other investigators. 

9. No parallel research done elsewhere. 

10. PROJECT DESCRIPTION ; This is a continuation of the project given under 
the same heading in the report of December 195^ and December 1955 • 

Objective ; To determine the fine structure of the aglycone. 

Methods Employed ; In regard to the structure of "steviol" and 
"isosteviol" there still remain to be definitely established the 
positional and steric location of the carboxyl, the hydroxyl and 
methylenic group of steviol. Certain experiments which could not be 
explained by the structure of "steviol" as proposed by Erich Mosettig 
and W. R. Nes (Stevioside II) show that the fourth hydroaromatic ring 
must be otherwise connected than it was accepted by the former in- 
vestigators. In accordance to I.R. spectra, steviol seems to possess 
a gem-dimethyl group in the i)-po6ition. Appropriate chemical reactions 
as well as physical measurements are being employed for establishing 
these finer structural points. 

Significance : The steviol-isosteviol structure has become of very 
acute interest since it could be tied up with the garrya alkaloids and, 
more recently, also with cafestol. 



Form No. ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOML INSTITUTES OF HEALTH 
lilDIVIDUAL PROJECT REPCTIT 



Part B: Budget 



11, NIAMD-55 



SERIAL NUMBER 



FIf$T 



1.33 



1,00 



2.33 



13.. B UTOET ACTIVITY; 
RESEARCH 

REVI0/J Ir APPROVAL 
BIOLOGIC STAilDARDS 



12, BUDGET DATA: 






ESTimTED OBLIGATIONS 




MAN YEARS 


T^TRRC^ HEIl'IBURRRW.WT 


TOTA^ 


PROF ormR TOTAI' 


FY«57 






Cil8,800 $7,100 


$25,900 


1.33 1.00 2.33 


BUDGETED POSITIONS 




PATIEITT DAYS 


PROF OTHER 


TOTAL 





nri ADiilNISTRATION 

!3~1 PROFESS 1 0^!AL & 

TECm-IICAL ASSIST- 
I i ANCE 






lU, IDENTIFY ANY COOPERATING UNITS OF TIE PUBLIC HEALTH SERVICE, OR 
OTILER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 19^7. IF COOPERATING UNIT IS VJITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



December 1956 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 15 . HLiMD-3'g 



SERIAL NUMBER 

16. No publications from this project during calendar year 1956. 

17. No honors and awards relating to this project during calendar yeeir 1956. 



I >,:., 1^'= ■ 



December 195^ 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

fart A. Project Description Sheet 1. NLJ'tD-56 



SERIAL NUMBER 



2. NIAMD 3. Chemistry 



INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARUvJENT 



k. Steroid 5. 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 



6. The Structure of Cholegenin and iBocholegenin. 



PROJECT TITLE 



7. E. Mosettig and M. J. Thompson 



PRINCIPAL INVESTIGATORS 
8. I. Scheer 



OTHER INVESTIGATORS 

9. No parallel research done elsewhere. 

10. PROJECT DE SCRIPTION; 

Objective ; The verification of the structure of cholegenin and iso- 
cholegenin as proposed by Spring et al. (Glasgow, 195^ )• 

Methods Employed ; Failure to isolate the material from American 
ox-bile restricts experimentation to extremely limited material 
provided by Professor Spring. Oxidative and reductive degradation 
of the cholegenins. Attempts to transform them to known plant 
sapogenins or derivatives. 

Significance ; a) Tine occurrence of close relatives of steroidal 
sapogenins in animal organism is unique. 

b) If the cholegenin structure is correct, a direct 
proof of the existence or non-existence of the C-22 isomerism in 
the (unopened) steroidal sapogenin could be elaborated. 



'V'V'i' ■.r:':'^- 



V. ■;:;;/•!.,' 



■)•;■., ■•; • ■■•a; v., ( 



.f ( V ■: ■ » 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
II€)IVIDUAL PROJECT REPORT 



part B: Budget Data 



11, NIA MD~^6 

SERIAL ITOI^lBER 



12. BUDGET DATA! 



ESTIMATED OBLIGATIONS 



FYl$7" 



DIRECT 



REIMBURSEMENT 



TOTAL 



^?19,900 



$7,100 



(57,000 



FY! 57" 



PROF 



BUDGETED POSITIONS 



OTHER 



TOTAL 



.83 



1.50 



2.33 



13* BUDGET ACTIVITY; 
RESEARCH 

REVIEVJ i, APPROVAL 
BIOLOGIC STAiroARDS 



MAN YEARS 



PROF OTHER TOTAL 



.83 1.50 2.33 



PATIEiW DAYS 



{X~\ ADMIlrtSTRATIOM 

□ PROFESSIONAL & 

TECHNICAL ASSISE 
I i ANCE 



□ 



□ 



Hi, IDi- CITIFY ANY COOPER/.TING WJITS OF THE PUBLIC HEALTH SERVICE. OR 
OTHER ORGANIZATIONS, PROVIDING FUI©S, FACILITIES. OR PERSONNEL 
FOR T MS PttOJICCT IN FY 1957. IF COOPERjiTING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 

Departmenb of Chemistry, "the Royal Technical College, Glasgow, 
Scotland. 



(Use reverse and additional paf^es, if necessary) 



December 19^6 



PUBLIC HEALTH SERVICE - - NATIOML INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors, Awards & Publications l5, NIrJ4D-56 

SERIAL NUMBER 



16, PUBLICATIONS OTHER TH/IN ABSTRACTS FRCM THIS PROJECT DURING CALENDAR 
YEAR 1956: None 

Paper pertaining to report of last year: 

I, Scheer and M, J, Thompson and E, Mosettig: "5-Cholestene-3P,26- 
diol," J, Am. Chem. Soc,, 78, li733 (1956). 

17. No honors or awards relating to this project during calendar year 1956* 



December 1956 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NLiMD-57 



SERIAL NUMBER 



2. NIAMD 3. Chemistry 



INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTMENT 

k. Steroid 5. 



SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 

6. The C-22 isomerism of a new class of dihydrosapogenins. 

PROJECT TITLE 

?. E. Mosettig, I. Scheer and M. J. Thompson 

PRINCIPAL INVESTIGATORS 

8. No other investigators, 

9. No parallel research done elsewhere. 

10. PROJECT DESCRIPTION : 

Objectiv e; Study of the stereochemistry of the sapogenin side chain. 

Methods Employed ; In the reduction of kryptogenin in acidic media four 
compounds have been obtained; dihydrotigogenin, tigogenin, and two 
new sapogenin derivatives. It is shown that the new compounds are 
isomeric at C-22 and are 22,26-epoxycholestane-3P, l6-diol and 22,26- 
epoxy-22-isocholestane-33, l6-diol. The preparation of cholestane-3P, 
22,26-triol and cholestane-3P,26-diol-22-one is described. The 
latter was converted to 22,26-epoxycholestan-33-ol and 22,26-epoxy- 
22-isocholestan-3P-ol. 

Significance ; Demonstration of the possibility of C-22-i6omerism 
in steroidal sapogenins. 






;"::i^ji':;;;i 



,\i--:fiiAiii\ 






!." ■■ -.'liy- < T Jfi'^:'. ' 'r'i ,\'^y•^''^'■'^'■ 



>;tt^iKI.:>:g);5-i>,'^' 



Form No, ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEA.lt H SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B! Budget Data 



11. NIAI-ffl-57 



SERIAL iWMIER 



12, BUDGET DA 


TA: 








ESTIFATED OBLIGATIONS 




MAN YEARS 


DIRECT 


REIMHJRSEMENT TOTAL 


PROF 


OTHER TOTAL 


FI«57 








as, 300 


ili7,100 :i^5,U00 


.83 


1.50 2,33 




BUDGETED POSITIONS 




PATIEI>!T DAYS 


PROF 


OTHER TOTAL 




Fr»$7 








.83 


1.50 2.33 







13. BUDGET ACTIVITY; 
RESE/iRCH 

REVIEV; I'. APPROVAL 
BIOLOGIC STANDARDS 



f3f| ADMINISTRATION Q 

I i PROFESS lOmL & 

TECm^IGAL ASSIST- 
□ ANCE L_| 



ll;. IDEI^ITIFY ANY COOPER/s.TIl\fG UNITS OF THE PUHJC HEALTH SERVICE, (E 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOIMNEL 
FOR THIS PROJECT IN FY 1957. IF COOPER/.TING UNIT IS WITIilN NIH 
IITOICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



December 1956 

PUBLIC HEALTH SERVICE --- NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 15'N KMD-57 



SERIAL NUMBER 
16. No publications pertaining to this project during Calendar Year 1956. 



17. HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR 
YEAR 1956: 

Part of lecture presented (by Erich Mosettig) before Joint Meeting of 
Chemical and Pharmaceutical Societies of Basel (June 1956). 



ij-M' 



■- iriOIvHIi^.; ii; 



J-.- 



December 19^6 



PUBLIC HEA.LTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part A, Project Description Sheet 1, UU.MD-^Q 

SERIAL NUMBER 



2t NIAMD 3. Chemistry 

fflSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTMENT 

k» Steroid 5» 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA^ 

6, Study of Fecal Steroids 

PROJECT TITLE ' 

7« Erich Heftmann 

PRINCIPAL INVESTIGATOR 

8. Erich Mosettig 

OTHER INVESTIGATORS 

9» No parallel research done elsewhere, 

10, PROJECT DESCRIPTION ; 

Objective ; To identify the 17-ketosteroids in feces. 

Methods Employed ; Fractions from stool extract which give the 
Zimmermann test for ketosteroids have been further purified, using 
partition and adsorption chromatography and molecular distillation. 
While none of the fecal steroids have so far been isolated in 
crystalline form, a crystalline semicarbazone of one compound has 
been obtained. Infrared analyses and paper chromatograms indicate 
that the substances which are being purified are indeed 17-keto- 
steroids, One of them resembles 3a-hydroxyetiocholan-17-one, 

Significance ; For an understanding of steroid metabolism in man it 
is of fundamental importance to know which steroids are excreted by 
way of the feces. The present investigation is the first indication 
that 17-ketosteroids may be so excreted. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
IITOIVIDUAL PROJECT REPCRT 



part B: Budget Data 



Jl. NIAMD-58 



SERIAL NUMBER 



12, BUDGET DATA: 




ESTIMATED OBLIGATIONS 


MAN lEARS 


DIRECT REIMBURSEMEIIT TOTAL 


PROF OTHER TOTAL 


FI'57 




$9,1|00 13,000 .1jil2,ii00 


0.50 0.67 1.17 


BUDGETED POSITIONS 


PATIENT DAYS 



FI«?7" 



PROF 



OTIER 



TOTAL 



0.^0 



0.67 



13. BUDGET ACTIVITYi 
RESEARCH 

REVIS'J & APPROVAL 
BIOLOGIC STAlViDARDS 



1.17 



[^ ADMINISTRATION 

□ PROFESSIONAL & 

TECHNICAL ASSIST- 
I I ANCE 



n 



lU. IDENTIFY ANY COOPER/iTING UNITS OP THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING PUIOS, FACILITIES, OR PERSONi\IEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN MH 
BDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



December 19^6 



PUBLIC HEALTH SERVICE - - NATIOML DISTITUTES CF HEALTH 
ETOIVmUAL PROJECT REPORT 



Part C; Honors, Awards & Publications I5, NlAMDtr58 

§ERI!iL NUIPER 



169 No publications during calendar year 1956. 



17. No honors or awards to personnel relating to this project during 
calendar year 19^6, 



December 19^6 



PUBLIC HEALTH SERVICE - - NATIONAL BISTITUTES OF HEALTH 
INDIVmUAL PROJECT REPORT 



Part A, Project Description Sheet Jf NI>.MD-$9 

§ERIA,L' NUMBER 



2. NIA^© 3. Chemistry 

INSflTUTE"OR DIVISION LABORATORY, BRANCH, OR DEPARTMENT 

kt Steroid 5» _____^ - 

SECTION OR SEkVIClE ^ LOCATION {IF OTHER THAN BETHESDA; 



6, Analysis of Individual Corticosteroids in Urine 
PROJECT TITLE 



7» David F« Johnson 



PRINCIPAL INVESTIGATOR 

8e Erich Heftmann 

OTHER INVESTIGATORS ' """ 

9» No parallel research done elsewhere, 

10. PROJECT DESCRIPTIOH ; 

Objective t Application of the Heftmann- Johns on method to the 
determination of individual adrenal corticosteroids in urine. 

Methods Employ ed; Heftmann, E,, and Johnson, D. F, : Anal. 
Chem, 26, 519 JX9Sh) with modifications indicated in last annual 
reportc 

Significance ; A study of the corticosteroid excretion patterns in 
normal men and women, in pregnancy. Gushing' s syndrome, Addison's^ 
disease, congestive heart failure and cirrhosis as well as in patients 
treated with adrenocorticotrophic honnone, adrenocortical hormones, 
and synthetic hormone analogs has demonstrated interesting quantita- 
tive clianges in the composition of the urinary corticosteroid fraction. 
From this study it seems evident that much remains to be learned about 
the significance of changes in the excretion of individiial hormones 
in health and disease. The interoonversion of meticorten and 
meticortolone in vivo has been demonstrated. The substance with an 
absorption maximum at 273 njfi, described in the last annual report has 
been identified as caffeine. 



Form No. ORP-1 
October 19^6 
(Attachment I) 



PUBLIC !EALTH SERVICE - ^lATIOML BISTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B; Budget Data 



11, NIAM)-59 



SERIAL NUl-IBER 



12, BUDGET DATA: 






ESTIMATED OBLIGATIONS 




MAN YEARS 


DIRECT REIMBURSEMENl' 


TOTAL 


PROF OTHER TOTAL 


FY»57 






.'Ja3,700 $5,000 


^118,700 


1.50 - 1.50 


BUDGETED POSITIONS 




PATIENT DAYS 


PROF OTHER 


TOTAL 




FY«57 






1,50 


1.50 




13, HJDGET ACTIVITY; 







RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STAlffiARDS 



□ 



ADMNISTRATION 

PROFESSIONAL & 
TECIMICAL ASSIS1 
ANCE 



n 



□ 



Hi, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OT'HER ORGANIZATIONS, PROVIDBIG FUTOS, FACILITIES, OR PERSONrJEL 
FOR THIS PR0JE;CT IN FY 1957, IF COOPERATING UNIT T- VIETHIN NIH 
INDICATE SFHIAL NO(S): 



Dr. Mosettig and Dr. Bunim - for samples. 

Dr. Mosetti, for work on identification of isolated material. 



(Use reverse and additional pages, if necessary) 



December 19^6 



PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors, Awards & Publications 3,5, NIiiMD-59 

SERIAL NUI'BER 



16, PUBLICATIONS OTHER THAN ABSTRACTS FROM THE PROJECT DURING CALENDAR 
YEAR 1956: 

Johnson, D, F,, Heftmann, E,, and Hayden, A, L, : "Determination 
of Individual Adi'enocortical Steroids in Urine," Acta Endocrinol,, 
1956, in press, 

17, HONORS AND AWARDS RELATING TO THIS PROJECT DURDMG CALENDAR YEAR 1956 i 

This work has been presented at the Symposium of the Division of 
Biological Chemistry of the American Chemical Society, 129th 
Meeting, Dallas, Texas, on April 10, 1956 and at the Second 
International Congress of Clinical Chemistry, New York, New York 
on September lli, 1956, 



■ «•. i!! ;.-.•«' '.- 



'■v. 



v^^X.:: 



December 195^ 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1. NL.MD-60 



SERIAL NUMBER 
2. NIAMD 3. Chemistry 



INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTMENT 

k. Steroid 5. 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 

6. Approaches to the Synthesis of Cortisone 

PROJECT title' 

7. William R. Nes and C. W. Shoppee 

PRINCIPAL INVESTIGATORS 

8. No other investigators. 

9. No parallel research done elsewhere. 

10. PROJECT DESCRIPTION ! 

O bjective ; To elucidate the hydrolysis of dehydroergosteryl 
p-toluenesulfonate and ascertain whether it constitutes a method 
for the introduction of oxygen at C-11 in the steroid nucleus. 

Methods Employed ; Dehydroergosteryl p-toluenesulfonate was pre- 
pared and its hydrolysis studied by conventional chemical and 
spectroscopic methods. It was found the reaction is kinetically 
controlled and that thermodynamic equilibration results in a 
reversal rearrangement which takes place in the initial reaction. 
The product was characterized and evidence was obtained that oxygen 
may have been introduced in the 11-position. 

Significance ; If the product could be shown by further work to be 
definitely a C-11 oxygenated steroid, cortisone and other adreno- 
corticoids could be prepared by this new route and obviate more 
cumbersome chemical methods already known. 



Fom No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIOML INSTITUTES OF HEALTH 
Ii«3IVIDUAL PROJECT REPORT 



part B: Budget Data 



11, NIAI;iD-60 

SERIAL NUI4BER 



12, BUDGET DATA: 








ESTIMATED OBLIGATIONS 




MN YEARS 


DIRECT 


REIMBURSEfffiiMT TOTAL 


PROF 


OTHER TOTAL 


FYt57 








^10,200 


•,pi+,000 'i,ilU,200 


.50 


1.00 1,50 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHER TOTAL 




FY«$7 

1,50 


1.00 2.50 






1 ^. RTinrnr.f An'"- 


rVT'TiY. 







RESEARCH 

REVI&J & AP'^ROVAL 

BIOLOGIC STANDARDS 



m 



ADMINISTRATION 



□ 



I i PROFESSIONAL & 

TECffNIGAL ASSIST- 
□ ANCE □ 



111, IDEiCIFY km COOPERATING UNZTS OF TIffi PUBLIC HEALTH SERVICE^ OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SPJRIAL NO(S)! 

University of iJ'ales, American Cancer Society and the 
Anna Fuller iAind. 



(Use reverse and additional pages, if necessary) 



December 195^ 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 15 . ML^iO-60 



SERIAL NUt-IBER 



16. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURINO CALENDAR 
YEAR 1956: 

Pertaining to last years report, I wish to include the following two 
papers : 

William R. Nes; "The Anthrasteroid Rearrangement. III. The Pathway 
in the Conversion of Dehydroergosterol to Anthraergostapentaene-'-", 
J. m. Chem. Soc, 76, 193 (1956). 

William R. Nes, Robert Kostic and Erich Mosettig: "The Anthrasteroid 
Rearrangement. IV. The Preparation of Several New Anthrasteroids 
and Some Observations on the Dehydrobromination of 7-Bromo-A^-steroids ," 
J. Am. Chem. Soc, 78, 14-36 (1956). 

Publications pertaining to this report: 

William R. Nes and C. W. Shoppee, "The 3,5-Cyclosterold Rearrangement," 
Journal of the Chemical Society (London), in press. 

17. NO HONORS OR AWARDS RELATING TO THIS PROJECT DURING CALENDAR YEAR 1956. 



December 1956 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet I, NIAMD-61 



SERIAL NUMBER 
2. NIAMD 3. Chemistry 



INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTMENT 



k. Steroid 5« 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 



6. Stereochemistry of the Side Chain of Steroidal Alkaloids 
■ 



PROJECT TITLE 



7. Yoshlo Sato 



I 



PRINCIPAL INVESTIGATOR 



H. George Latham, J r. 
OTHER INVESTIGATORS 



9« No parallel research done elsewhere. 

10. PROJECT DE SCR IPTION ; 

Objective ; The strucural elucidation of steroidal alkaloids. 

P Methods Employed ; The reaction of a solution of zinc chloride-acetic 
anhydride-acetic acid upon the steroidal alkaloids tomatidine and sola- 
sodine leads to the production of a diacetylated anhydro derivative in 
each case. The structure of these compounds has been ascertained 
through various reactions to be 2-(20-allopregnanyl)5-niethyl-Al-tetra- 
hydropyridine and 2-(20yl-preanen(5) )5-iDethYl-A^-tetrahydropyridlne. 
They are probably in equilibrium with the A ^^2) -isomer. 

The steroidal sapogenins (neotigogenin and tlgogenin) have pre- 
viously been converted into tomatidine and solasodine. We have now 
conversely converted these steroidal alkaloids into steroidal sapo- 
genln derivatives, namely dihydroneotigogenin and dlhydrotlgogenin, 
through the rearrangement of the respective N-nltroso-N-acetyl dlhydro 
and tetrahydro derivatives. 






:: ■:, .VH/;^UV 



December 1956 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 

NIi.iffl-61 ' "^ INDIVIDUAL PROJECT REPORT 

SERInL NO. 

Part A. Project Description Sheet (Continued) Page 2 

10, PROJECT DESCRIPTION (Cont'd) : 

Signific ance; The structural elucidation of these steroidal alkaloids and 
their interrelationship with the steroidal sapogenins is of classical 
fundamental importance in the field of steroid chemistry. 

Through degradative studies of the steroidal alkaloids it may 
become feasible to utilize these substances as a potential source for the 
production of biologically active hormones including cortisone and its 
derivatives. They may also be potential hypotensives, fungicides and 
bactericides. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOML INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-.61 

SERIAL NUMBER 



FY '57" 



FY«57' 



BUDGET DA 


TA: 








ESTH^ATED OaiGATIONS 




MAN YEARS 


DIRECT 


REIHBURSE^ENT TOTAL 


PROF 


OTI-IFS TOTAL 


;|^0,200 


^^,100 ^28,300 


2,00 


2.00 




BUDGETTD POSITIONS 




PATIENT DAYS 


PROF' ■ 


OTHER TOTAL 




1 
2.00 


2,00 






inrrF," A(7T 


IVITY: 







RESEARCH 

REVIEl'J & APPROVAL 

BIOLOGIC STArlDARDS 



f1?l ADMINISTRATION Q 

r~\ PROFESoIOtlAL & 

TECffNIGAL ASSIST- 
□ ANCE □ 



Ih* IDEi\^IFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH S.'JIVICE, CR 
OTHEI^ ORGKiJIZATIONS, PROVIDING FU^HDS, FACILITIES, OR PERSOit'EL 
FOR THIS PROJECT IN FY l?^?. IF COOPER/.TIHG UNIT IS WITIHN NIH 
INDICATE S:ilIAL NO(S): 



(Use reverse and additional pages, if necessary) 



December 1956 

PUBLIC HEALTH SERVICE - - NATIOi^lAL MSTITUTES OF HEALTH 
Ii©IVIDUAL PROJECT REPORT 

Part C: Honors, Awardg & Publications 15. NIi>MD-6l 



SERB.L NUMBER 



16. PUBLICATIONS OTHER THAN ABSTRACTS FRCM THIS PROJECT DURING CALENDAR 
YEAR 1956 t 

Y, Sato and H, G, Latham, Jr, : "New Dihydro Derivatives of Tomatidine 
and Solas odine," J. Am, Chem, See., 78, 3l50 (1956), 

Y, Sato, H, G, Latliam, Jr., and I, Scheer: "Conversion of Tigogenin 
and Neotigogenin into l6,22-Epoxycholest-25-en-3p-ol and 16,22- 
Epoxycholestan-3p-ol," J. Org, Chem. 21, 689 (1956), 

17, HONORS AND AWARDS REUTBJG TO THIS PROJECT DURII\IG CALENDAR YEAR 1956: 

In April, 1956, Dr. Yoshio Sato was invited by the National 
Congress of the Japanese Pharmaceutical Society as guest 
speaker at Kyushu, Japan, 



I 



December 195^ 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NKMD-62 



SERIAL NUMBER 



2. NIAMD 3. Chemistry 



INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTMENT 

h. Steroid 5, 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 

6. The Reaction of Colchicine with Amines . 

PROJECT TITLE 

7. William R. Nes and Hans Lettre. 

PRINCIPAL INVESTIGATORS 

8. No other investigators. 

9. No parallel research done elsewhere. 

10. PROJECT DESCRIPTION ; 

Objective ; To obtain quantitative data on the preparation of 
colchicamides. 

Methods Employed ; The rate of reaction of colchicine with ammonia 
and various amines was studied spectrophotometrically. The rate was 
dependent on the structure of the amine and was correlated with 
theoretical studies on analogous systems. The reactions were found 
to proceed to completion and the products were readily isolated. 

Significance ; The investigation resulted in a simple preparation 
of pure derivatives of colchicine which are more potent antimitotic 
agents than the parent substance. These derivatives are of 
interest in the investigation of the growth of cancer cells. 



?i::;<iMUi-: .iAi 






ivJV 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
IITOIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAM)-62 



SERIAL l\IUr4BER 



12, BUDGIoT 


DA 


TA: 
ESTIMATED OBLIGATIONS 




MAN YEARS 


DIRECT 




REIMBURSEMENT TOTAL 


PROF 


OTHI:.R TOTAL 


FY»57 








1^9,200 




$3,000 iii>12,200 
BUDQETED POSITIONS 


.^0 


,67 1.17 

PATISl'lT DAYS 


PROF 




OTIIE ; TOTAL 




TT'57 










0,50 




0,67 1,17 







13, BUDGET ACTIVITYi 
RESEARCH 

REVIEl^r & ApmOVAL 
BIOLOGIC STANDARDS 



a 



ADMINISTRATION 

PROFESSIONAL k 
TECffivIICAL ASSIST- 
ANCE 



□ 



II4. IDENTIFY ANY COOPEMTMG UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTI-ER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PrRSOWlEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS VJITHIN NIH 
IJ\1DICATE SJIRIAL NO(S): 



University of Heidelberg, American Cancer Society and 
the Anna Puller SXind, 



(Use reverse and additional pages, if necessary) 



December 1956 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 15. NL'.MD-62 



SERIAL NUMBER 
16. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR 

William R. Nes and Hans Lettre; "Concerning Polyvalent Ammonium 
Compounds," Liebigs Annalen der Chemie, 598, 65 (1956). 

IT. No honors and awards relating to this project during Calendar Year 1956. 



December 1956 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Shee-| 1. NLiMD-63 



SEI^JAL NUMBER 
2. NIAMD 3. Chemistry 



INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARMENT 

h. Stero id 5. 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 

6. Infrared spectroscopic studies. 

PROJECT TITLE 

7. H. K. Mill er 

PRINCIPAL INVESTIGATOR 

8. No other investigators. 

9. No parallel research done elsewhere. 



PROJECT DESCRIPTION ; Approximately eight hundred infrared spectra 
were made to support Investigations in progress in the Steroid Section. 
An attempt to correlate infrared spectra of morphine compounds is 
progressing slowly as time permits. Morphine compounds of sufficient 
purity are being collected and their spectra are being taken, both in 
the conventional sodium chloride prism range and with the lithium 
fluoride prism. Potassium bromide pelleting technique has been 
employed and efforts are under way to improve the technique, both 
with regard to speed and to our specific requirements. An effort is in 
progress to have an infrared microscope fabricated so that much smaller 
samples may be examined in the infrared range of the spectrum. Most 
of the spectra collected by this laboratory have been catalogued and 
further efforts at systematization of spectra for easy reference and 
convenient use are proceeding. 



December 1956 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NL.MD-63a 



SERIAL NUMBER 
2. NIAMD 3. Chemistry 



INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTMENT 



k. Steroid 5» 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 



6. Ultraviolet spectroscopic services. 



PROJECT TITLE 



T* Mrs» Anne H. Wright 
PRINCIPAL INVESTIGATOR 



8. No other investigators. 

9. No parallel research done elsewhere. 

10. PROJECT DESCRIPTION ; Mrs. Wright is in charge of the Gary Machine and 
has during the past year carried out determinations for members of this 
section and for members of the other sections of the Laboratory of 
Chemistry, in particular, of the Analgesics and Metabolite Section. 
Occasionally there are also determinations to be done for scientists 
outside of this laboratory. The number of determinations is approxi- 
mately 2-10 per day, depending largely on the amount of information 
available in regard to physical and structural properties of the 
compounds to be determined. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - mTI JilaL INSTITUTES OF HD\LTH 
I1€)IVIDUAL PROJECT REPORT 



part B: Budget Data 



II, NIAM&.63 

SERIAL NUMBER 



12, BUDGET DATA: 



ESTIMATED OBLIGATIONS 



FY>57" 



DIRECT 



RED'tBUR.SErTEMT 



[•OTAL 



$13,200 ;,,!^,000 



;,1;18,2C0 



BUDGETED POSITIONS 



FY«57" 



PROF 



OTHER 



:OTAL 



MAN YEARS 



PROF OTIiER TOTAL 



1,00 ,67 1,67 



PATIEI^ DAYS 



1,00 



.67 



1.67 



13. BUDGET ACTIVITY; 
RESEARCH 

REVIHJ & APPROVAL 
BIOLOGIC STAhlDARDS 



nn ADMINISTRATION 

1 1 PROFESSION/iL & 

TECHi\IICAL AS3IS1 
□ ANCE 



a 



Ih, IDENTIFY A.IY COOPERATING UNITS OF THE PUBI.IC HFALTH SERVICE, OR 
OTPER ORGANIZATIONS, PROA'IDING FUNDS, FACILITIES, OR PERSONi>IEL 
FOR THIS PROJECT IN FY 19^7. IF COOPER/TING U^NIT IS WITHIN NIH 
INDICATE SEltHL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - - MTIOI^IAL INSTITUTES OF HEALTH 

INDIVIDUAL PRO>JECT REPORT 

Part A. Project Description Sheet 1 . ML\MD-5L i 

SERIAL NUlffiER 

2 . MIAI€) 3 . laboratory of Chemistry 

INSTITUTE OR DIVISION LABOPATORY, BRANCH, OR DEPARTMEIJT 



Section on Metaho 1 i tes 5 • 



SECTION OR SERVICE LOCATION (IF OTPIER THAN BETHESDA) 

6 . (Lahile) Metabolites and Antimetabolites of Amino Acids 

PROJECT TITLE 

7 . Bernhard Witkop 

PRINCIPAL INVESTIGATOR 

8 . L. A. Cohen. A. A. Pat c hett, G. F. Holland, K. Freter (V.S.) 

OTHER INVESTIGATORS 

9. IF THIS PROJECT RESEMBLES, COMPIEMETJTS, OR PARALLEI^ RESEARCH DONE 
ELSEWHERE IN THE PUBLIC HEALTH SERVICE (VrtTHOUT INTERCHANGE OF PER- 
SONNEL, FACILITIES OR FUNDS) IDENTIFY SUCH RESEARCH: (BY SERIAL 
NO. (S) IF raTHIN NIH). 

Serotonin Problem: S. Udenfriend, NHI 

LSD Metabolism: E. E. Evarts, J. Axelrod, NIMH 

Collagen Research: K. A. Piez, NIDR 

Analysis of Amino Acids: P. Irreverre, KIAl-D 

Oxidation Mechanisms of Enzymes: 0. Haj^ishi, NIAMD 

Preciorsor of Formiminoglycine : J. Rabinovitz, NIAMD 

10. PROJECT DESCRIPTION 

Project : Development of synthetic and analytical procediires to 
prove the presence of novel intermediates in the catabolism of amino 
acids which are too labile to be detected by custoraar;'.'- routine methods. 

Ob.lectives : To show that a detailed knowledge of the oxidation 
mechanism ef heterocycJ.ic, aromatic and aliphatic model compounds 
makes possible the synthesis of missing or novel intermediates in 
the break-down of physiologically important factors such as amino 
acids, ergot alkaloids, purines, vitarai.ns etc. The availability of 
such intermediates obtained by synthesis makes it possible for the 
biochemist, pharmacologist and clinician to gain new insight into 
metabolic processes. 

Methods Employed : New routes are being developed for the 
synthesis of compounds which so far have not been synthesized because 



NL.riD-61i 
SEEIAL NO. 

10. PROJECT DESCRIPTION (Cont'd) 

the difficiilties encountered have heen too great or because the 
knovrledge for the postuJ.ation of such compounds has been lacking. 
Special nev techniques are employed in an attempt to simulate 
in vitro normal and abnormal oxidation processes occurririg in vivo . 
Physico-chemical methods are being used and adapted to the study of 
the more subtle aspects of reactions and interactions of functional 
groups in model compounds as vel]. as in higher molecuJ.ar complexes. 
Additional cooperative projects with biochemical, pharmacological 
and clinical groups on the campus are being projected or have been 
started . 

Major Findings : In the field of indole chemistry the resolution 
of the new vital h^.-droxyamine acid, 5 -hj/droxjz-trj'P'tophan via the 
brucine salts of the N-carbobenzyloxy-5-benzyloxy-tryptophan was 
achieved by K. Freter. The enzjonatic and pharm-acological tests of 
the antipodes were conducted Independently by Drs. Udenfriend and 
Weissbach and established the complete inactivity of the D-amino 
acid and the fact that the entire physiological activity rests in 
the L-compound. Of a great number of hydroxyi.ndoles submitted to 
Dr. Martha Vaughan (WHi) for testir^ as inhibitors of insulinase 
5-hydroxyindole was as active as 5 "hydroxy- L-trj'p)tophan whereas 
5 -hydroxy-D- tryptophan was without an^r effect. The reputedly psycho- 
tomimetic adrenolutin inhibited insulinase. The first m.etabolite 
of LSD obtained by the action of liver microsomes by Dr. J. Axelrod, 
NIMH, was synthesized via hydrolytic cleavage of the disulfide of 
LSD by Dr. K. Freter and proved to be the oxindole analog of LSD. 
Another indole metabolite isolated in the Lilly laboratories from the 
culture medium of the ergot-producing fungus Claviceps purpurea was 
shown to be 2,2-bis[3' -indyl]-indoxyl. Complete X-ray analysis of 
the t\ro pure diastereoisomeric a,5-di-p-iodobenzoyl-o-hydroxy- threo 
and erythro-L-ornithine lactones, prepared by Dr. T. Beiler, Stetson 
University, has been started by Dr. Dorothy Crowfoot Hodgkin, 
Cambridge, England. The corresponding derivatives of the homologous 
3-hydroxj'-lysine lactones are being prepared. Tlie complete absolute 
configuration of natural 5-hydroxy-L-pipecolic acid from, dates has 
been elucidated by C. M. Foltz. HMson's Lactone Rule has been 
applied to / and o-hydroxyami.no acids and led to the posta^.ation 
of the erythro-configToration for natural 6-h;i'droxylysine from 
collagen. Dr. A. A. Patchett finished the preparation of a large 
n\OTber of new derivatives of hydroxyproline which have all been 
tested in the tissue cultures of "collagen "-producing carrot tissue 
by Prof. F. C. Steward, Cornell University. Antimetabolic activities 
were shown bj^ some compounds in dilution of 1:10"°. The structural 
elucidation (C. M. Foltz) of the two diastereoisomeric D-threo- and 
D-er;rthro-l-phenyl-l,2-epoxj'propanes from ephedrine and Y-ephedrine, 
respectively, opened the way for the preparation of new l,l*-tetra- 
hydrooxazines as potential anorexigenic agents. Synthetic approaches 



SERIAL NO. 

10, PROJECT DESCRIPTION (Cont'd) 

to the smal3. cyclic disulfide -peptide contained in insiilin have 
been started by L. A. Cohen and G. F. Holland. Quebrachamine, 
an indole alkaloid, chemically related to the mctabollcally 
stimulating indole alkaloid ibogainc, has been investigated and a 
preliminary structure has been proposed. The mechanism of the 
oxidation of catechol, a bacterial metabolite of tryptophan, to 
cis , cis -muconic acid by pyrocatechase (O. Hayalshi) has been 
studied in model oxidations by A. A. Patchett. 

Significance to Metabolic Diseases Research : Ver-/ recent 
biochemicaJ. and clinical investigations have led to surprising 
findings concerning the etiology of certain long-lmown diseases. 
Hereditary, nutritional or environmental factors produce deviations 
from standard metabolic patterns of ani.no acid degradation. In 
particular the follomng diseases have been associated with nevr or 
known amino acid metabolites: schizophrenia and other mental dis- 
orders, diabetes, and phenylpyruvic oligophrenia. 

The new 5-hydroxytryptam;i.ne syndrome, the growing importance of 
serotonin in the central ner'/ous system and in psychic disorders 
and the metabolic fate of LSD malie imperative the preparation of 
many new hjTiroxyindole derivatives. Tlie process of aging of 
fibrous proteins, viewed from a chemist's standpoint, may be due 
to loss of enzymatic specificity in the biogenesis of diastereoiso- 
meric amino acids or to abnormal oxidation reactions leading to 
reactive keto amino acids capable of forming cross-linkages and 
poljTneric proteins. Tlie exact analysis of many collagen samples will 
be needed to prove or disprove such a hypothesis. 

Proposed Course of Project : Tlie chemistry of the bviilding 
stones of collagen will be further investigated, first on the leve]. 
of the single ami.no acids, then in peptides and finally in protein, 
such as collagen from mammals, "collagen" from plant tissue, 
gelatin, elastin, e.lastoidin. Four types of reactions deserve 
special interest: 1. Selective oxidation of hydroxyl groups to 
reactive keto amino acids. 2. Fonnation of cross linkages by 
a.ldolization and crotonization taking place between peptide strands. 
3. Selective reduction of oxidized gelatin or col.lagen to protein 
containing unnatural allohydroxyamino acids, h, /aialysis of 
collagen samples (with K. A. Piez) of various age groups and of 
normal and pathological origin (chondrosarcoma, lupus and other 
collagen diseases) for the presence of abnomnl amounts and t;rpes 
of hydroxyamino acids. The sjmthesis of three new related metabolites 
is being attempted by K. Freter: 1. The cyclic precursor of 
formiminoglycine (FIG) isolated by J. Rabinowitz, viz. , ^(5H)- 
imidazolone. 2. The cyclic precursor of formiminoaspartic acid, 
a metabolite of histamine (0. Ha;>'aishi and H. Tabor). 3. Tlie 
cyclic precursor of formiminoglutamic acid, a metabolite of histidine 



I 



SERIAL NO. 

2.0. PROJECT DESCRIPTION (Cont'd) 

(H. Tabor et al.). 

In extension of stMies with Dr. S. L. Frless, Naval I-fedical 
Center, on the Acetylcholinesterase Surface, polyfunctional sub- 
strates such as tetraacetates of quatcrnized amj.no sugars will be 
Investigated as substrates for the enzyme. 



Form No. ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - m?IOWAL IHSTITjTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



12. BUDGET DATA J 



11, NIAm-61i 



SERIAL iJUIiBER 





ESTD'iATED OBLIGATIONS 






I-IAW YEARS 


DIRECT 


REIMBURSEMENT 


TUTAL 


PROF 


OTHER TOTAL 


FY '57 










|6ii,700 


^i;2[i,200 i 
BUDGETED POSITIONS 


,88,900 


3.50 




3.00 6,50 
PATIENT DAYS 


PROF 


OTHER 


TOTAL 





3.50 3.00 

13. BUDGET ACTIVITY! 
RESEARCH 

REVIEl'J & APPROVAL 
BIOLOGIC STANDARDS 



6,50 



nn AffilNISTRATION 

T~\ PROFESSIONAL St. 

TECHI\IICAL ASSIST- 
O ANCE 






lit. IDEl^lTIFY ANY COOPLRATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, 0;l PERSON lEL 
FOR THIS PROJECT IN FY 1957» IF COOPE.RkTING UNIT IS VHTHIN NIH 
INDICATE SERIAL NO(S)! 



S. Udenfriend, NHI 
J. Axelrod, NIMH 
K. A. Piez, NIDR 
P, Irreverre, NIA^D 
0. Hayaishi, NIAMD 
J. Rabinowitz, NIAIffi 



Prof, F. C. SteiTard, Cornell University 
Dr. T, Beilei', Stetson University 
S. L» Eciess, Naval uedical Center 



(Use reverse and additional pages, if necessary) 



Calendar Yoar 1956 

PUBLIC HEALTH SERVICE - - NATIONAL- INSTITUTES OF HE/vLTH 

INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & PuMicationo 1$. NLu'-iD-6U 

SERIAL NUMBER 

16. LIST PUBLICATIONS OTHER TttlW j\BSTRACTS FROM THIS PROJECT DURING 
CALENDAR YEAR I956: 

Sjmthesis of 5-Hydroxypipecolic Acid and Scpai^tion of Its 
Diastereoisomers. Science, 123, 81l-2-8!+3 (1956). L. A. Cohen, 
F. Irreverre, C. A. Piez, B. Hitkop and H. L. Wolff. 

Imine Enamine Systems and the Mechanism of their Oxidation. 
Infrared Diagnosis of the Hydrochlorides of Oi-ganic Bases. III. 
Oxidation Mechanism XVII. J. /uii. Chem. Soc, 78, 2873-2882 
(1956). B. Witkop. 

Quinol Intermediates in the Oxidation of Phenols and Their Re- 
arrangements. Oxidation Mechanisms XVIII. J. Am. Chera. Soc, 
78, (1956) (in press). S. Goodwin and B. Witkop. 

Tlae Conversion of L-Histidine into (Allo)H^droxyprolinc via threo - 
and erythro-7-hydroxyorni thine . J. Am. Chem. Soc, 78, 2882-2893 
(1956). B. Witkop and T. W. Beiler. 

Studies on the Stereochemistry of Ephedrine and Y-Ephedrine. 
J. Am. Chem. Soc, 78, (1956), in press. B. Witkop and C. M. 
Poltz . 

The Stereochemistry of the l-Phenyl-l,2-propanediols and of a- 
Isoephedrine . J. Am. Chem. Soc, 78, (1956), in press. C. M. 
Foltz and B. Witkop. 

Studies on Hydroxj'proline . J. An. Chen, Soc, 78, (1956), in 
press. A. A. Patchett and B. Witkop. 

Configuration of 5-Hydroxypipecolic Acid from Dates. J. jto. Chem. 
Soc, 78, (1956), in press. B. Witkop and C. M. Fo^tz. 

Metaholism of Lysergic Acid Diethylamide. Nature I78, I'j-S-l^'j-, 
(1956). J. Axelrod, R. 0. Brady, B. Witkop and E. V. Evarts. 

The Application of Hudson's Lactone Rule to 7- and 5-H^'drox>Timino 
Acids and the Question of the Configuration of S-Hydroxjr-L- lysine. 
Experientia, 12, 372 (1956). B. WitL.op. 

The Acetylcholinesterase Surface. VII. Interference vrith Surface 
Binding as Reflected hy Enzymatic Response to Turicine, Betonicine 
and Related Heterocyc.Tes. J. /jn. Chem. Soc, (in press). S. L. 
Friess, A. Ji. Ritchett and B. VJithop. 



NlAlg)-6U 

SEPaiAL NO. 

16. LIST OF PUBLICATIONS (Cont'd) 

Stiodies on the Cliemical and EnzjTmtic Oxidation of Lysergic 
Acid Diethylaniidc. J. Am. Chem. Soc. (in press). K. Froter, 
J. .\xelrod and B. Witkop. 

Qucbrachaniinc I. J. jto. Chom. Soc. Bernliai-xl Witkop. (in press), 

Biochemical and Iliarraacological Studies with D and L-5-Hydroxy- 
tryptophan. Proc. of the Soc. for Eqierimental Biol, and 
Medicine. Kvirt Frcter, Herbert Weissbach, Sidney Udenfriend and 
Bernhard Witkop. (in press). 

Some Reactions of 3-Substituted Hydantoins. J. Am. Chem. Soc, 
78, 5863 (19^6). L. A. Cohen and E. M. Fry. 

17. HONORS AND AWARDS: None 



Calendar Year 1956 

;^IP.LIC ffALTH SER''ICE -;:- -;t liATIOHAL INSTITUTES OV Hi./lLTH 

INDIVIDUAL prtOJECT RKPO IT 

Part A. Project Description Sheet 1- HL.14D-6 5 

SERIAL UmWR 

2. National Institute of 3 -Pathology f-i 
Arthritis and Iletabolic Diseases Histochemistry 
INSTITUTE Oil DI'ISIOW LABORATORY. BimilCH 

OR DEPARTiCNT 

3. Histochemistry 5. 

SECTION OR SERVICE LOCATIOil (IF OTHER 

THAiJ BETHESDA 

6. Researches in H istochemistry 

PROJECT TITLE" 

?. R. D. Lillie 



PRINCIR'VL INVXSTiGATOR 

8. S« S . Spicer» J. Bo Lo n^jley^ and G^ Gt Glenner 

OTHE.^ IN./ESTIGATORS 

9. IF THIS PROJECT RESEMBLESs COHPLEIIENTSj OR PARALLELS 
RESEARCH DONE ^.L3E^^«hRE IN THE PUBLIC HEALTH SERVICE 
('JITHOUT INTERCHAI^IGE of PERSONNEL* FACILITIES OR 
FUNDS)* IDENTIFY SUCH RESEARCH: (BY SERIAL NO. (S) 
IF WITHIN NIH). 

10. PROJECT DESCRIPTION: 

Researches in histochemistry* carried out by four 
workers of doctor's grades are independent to a consid- 
erable extent* but also very considerably interrelated; 
with much interchange of information* and common use of 
technical personnel. 

LILLIE - Major attention to detailed histochera- 
ical study of the pigments and enterochromaffin and 
chromaffin substances with greatest emphasis this year 
on the raelanins.-. Three distinct pigments in this group 
can now be characterized: Melanin; the ocular and epi- 
dermal pigment* appears to be an acid substance of prob- 
able indolic structure and with a quinhydrone groupings 
a rather readily alkali soluble and acid methanol sol- 
uble yellow hair pit^ment desi^-nated as trichoxanthin* 
and human neuromelanin. Neither of the two latter pig- 
ments appear to possess o-quinhydrone groupinj^S" Hew 
methods for demonstration of indoles in tissues have 
been demised* methods for other aromatic amino acid 
localization are under stuJyc Improved raetnods for 



NI..MD-6$ - ^j 

SERIAL NUIIBER 

10, (Cont'd) 

lipofuscin and melanin identification have been devised. - 

GLENNER - Participation in the indole method re- 
searclij studies on bilirubin localization methods* on the 
demonstration of glucagon in islet cells* evolution of a 
method designed to localize monoaiaine oxidase* and an 
improved Mann technic for pituitary cells: Drn Glenner 
is also making cooperative studies on the histochemical 
nature of the so-called "microsome fraction" of the al- 
buminogenic cel]s of the foirl oviduct with Drs. R- M> 
Hendler I^JHI* and A= J-. Dalton; NCI« 

LONGLEY - 1) Studies of the reduction product 
of ditetrazolium salts used as hydrogen accepters in 
histochemical methods for various reductases have shovjn 
that these compounds* as presently a-<failable* are grossly 
impure. The principal contaminant is a monotetrazoliura 
which is also reduced under the same conditions r The 
significance of quantitative results based on the use of 
these has been established, (Burtneri- Bahn* and Longley). 
2) Preparation of a review on functional and morpholog- 
ical sites in the vertebrate kidney has been continued 
(Longley). 3) The histochemistry and the site of origin 
of certain renal adenomatous hyperplastic lesions occxirring 
in mice inoculated with extracts from tissue cu?.tures of 
spontaneously occurring parotid gland tumors is being 
investigated (Longley and Stewart* IICI-LB). h) It has 
been observed that in histological sections of kidney 
tissue prepared by the method of freezint-, and drying 
that the convoluted portions of the proximal tubule 
generally show many protoplacmic-- extensions and nuclei 
within the lumen. In view of the rapidity iidth which 
tissue elements are immobilized by this technique* it 
is of interest to determine whether the basis of this 
phenomenon lies in artifactual displacement of these 
nuclei or whether it represents an unreported feature 
of renal tubular anatomy^ Experiments are in hand to 
clarify this point (Longley and Burstone > MIDR-H;P)- 

SPICER - Investigations concerning the mechanism 
of the reaction of keratohyaline with hematoxylin point 
to the importance of metal complex formation^ Thus a 
chelating r.gent such as versene strongly interferes with 
the reactions salts of Cu and Fe enhance it. Since the 
oxidized form of the dye reacts with reduced keratohyaline 



NL'iMD-65 - 3 
SMIivL NUMBER 

10 (Cont'd) 

under anaerobic conditions j it appears unlikely that the 
reaction depends on enzymatic polyphenol oxidase activity 
in the keratohyaline granules = Iloreover oxidized j^ranules 
fail to combine with the reduced form of the dye> indi- 
cating T.hat only the oxidized hematoxylin reacts \jith the 
tissue components. 

Attempts to develop a histocheraical method for 
detecting reducing sugars have thus far met vdth failures 
due primarily to difficulty in achieving anhydrous con- 
ditions for the reaction. 



Form Moo ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SEIVICE - NATI 'ML INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAl''iD>65 



SERIAL HUMBER 



12, BUDGET DATA: 



FY«57" 



DIRECT 



^^95,200 



FY '57' 



PROF 



6.00 



ESTIMATED OBLIGATIONS 



REIMBURSEI'f; :NT 



?OTAL 



i|.i;3,500 



•'iil38,700 



BUDGETED POSITIONS 



OTI-ER 



TOTAL 



5oOO 



11,00 



13. BUDGET ACTIVITY; 
RESEARCH 

REVIEW & APPROVAL 
BIOLOGIC STANDARDS 



s 



MAN YEARS 



PROF OTiILH TOTAL 



6.0 5,0 



PATIENT DAYS 



ADMINISTRATION 



Q PROFESSIONAL & 

TECHNICAL ASSISI 
ANCE 



1 ' 



11,0 



lU, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSON EL 
FOR THIS PROJECT IN FY 1957 < IF COOPEWiTING UNIT IS WITHIN NIH 
liTOICATE SERIAL NO(S): 

Various minor cooperations with professional personnel have 
been notedj Credit is generally shared in publications, but no 
exchange of funds, facilities or personnel is entailed. 



(Use reverse and additional pages, if necessary) 



Calendar Year 19^6 

PUBLIC HtiALTH SER^aCE - - IlATIOM/iL INSTITUTES OF HEALTH 
INDIVIDUAL PRO.TECT RI PORT 

Part C: Honors* Awards i Publications. l5,NI/iMD-65 



jEiilAL UUIfflER 



16. LIST PUBLICATIONS OTHER THA1\I ABSTiiACTS FRQi THIS 
PROJECT DURING CALENDAR YEAR 1956: 

Longley^ J. B. : Alkaline phosphatase in kidneys of 
agloraerular fish. Science 123;l[i2-lli3> 1956. 

Longley. J. B. : Histochemical or histolo!.;ical tech- 
niques? Bull. N.Y. Acad. Med. 32:83-85$ 1956. 

Longley- J. B. and Fisher? E. R.: A histochemical basis 
for changes in renal tubular function in yoxing raiceo 
Quartc J. Plicro Sci- 97:187-195> 1956. 

Fullmer > H. M.. and -L-illie > R. D. ; Some aspects of the 
mechanism of orcein staining^ J<. Histochera- Cyto- 
chemo U:6it-68> 1956v 

Fullmer* H. H. and Lilliej R. D.: A selective stain 
for elastic tissue (orcinol-new fuchsin). Stain 
Techn. 3l!27-29> 1956c 

Lillie* .'(. Dj5 The p-dimethylaminobenzaldehyde reaction 
for pyrroles in histochemistry: Melanins* entero- 
chromaffin? zymogen granules* lensa J. Histochem. 
Cytochem, U}120-150) 1956. 

Lillie* R> D.s The periodic acid Schiff reaction in 
pathology, Aiao J. Clin, Path.- 26:305, 1956. 

Lilliej R. D. ; Phenolic oxidative activities of the 
skin: Some reactions of keratchyalin, and tricho- 
hyalin. J. Histochem, Cytochem^ ii:3l8-330j 1956 

Lillie* R. Do: A Nile blue staining technic for the 
differentiation of Melanin and lipofuscins . Stain 
Techn, 31:l5l-l53* 1956. 

Lillie? R. D. : The mechanism of Nile blue staining of 
lipofuscins. J. Histochem. Cytochemc [ii377-38l> 1956. 

Lillie* R- D<. i Reversal of cyanide blockade of aldehyde 
reactions by reoxiaation v;ith periodic acido J« Histo- 
chemo Cytochemo hth79-hQO} 1956 j 



OJ'tij}.-- 



NniMD-65 ->?- 
SERI'\L NmtT3ER 

16. (Cont'd) 

Accepted for 19b7 publication up to November 13 » 15^56: 

Burtner* Helen J.> Bahn. R. C.' and Lon;-ley> J. B. : 
Observations on the reduction and quantitation of 
neotetrazolium. J. Histochemo Cytochenis 

Bahns R. C. and Lonjleyj J- B. : Quantitative effects 
of a mercurial diuretic on the distribution of renal 
succinic dehydroj^enase in the rat. J. Pharmacol. 
Exp. Ther. 

Lilliej R. D. : The xanthydrol reaction for pyrroles and 
indoles in histochemistry: Zymogen ..jranules) lens> 
enter ochromaf fin and melanins.- J. Histochem. 
Cytochera. 

GlenneD G. G. ? The simultaneous deirion strati on of 
bilirubins hemosiderin and lipofuscin pigment in 
tissue sections.' Am. J. Clin. Fath» 

Glennerj G. G^ and Lilliej R- D. : The histochemical 
demonstration of indole derivatives by the post- 
coupled p-benzylidene reaction^ J. Histochem^ 
Cytochemt 

Lilliej R. D. j Henson? J. G- j and Burtner? H. J.; 
Metal reduction reactions of the me Ian ins : Silver 
and ferric ferricyanide reduction by various 
reagents in vitro. 

Lillie ) R. D. ' Metal reduction reactions of the 
melaninss His tocheraical studies. 

Lilliej Rr D.J Ferrous ion uptakes A specific 
reaction of the melanins 

Lilliej R. D. s Trichoxanth^nj the yellow granular 
pigment of guinea pig hair follicles and hairs- 

17. LIST HOIIORS AllD AWARDS TO PERSONNEL RELATING TO 
THIS PROJECT DURING CALENDAR YEAR 1956: 

Dr. R. Dc Lillie was elected Vice-President of the 
Histi-.chemical SoA^ty-< Dr. R- D. Li?i.lie v;as 
fcleo ot'd Koncrary member of the Argentine pathol- 
ogical Societyc 



Calendar Year 1??6 

PUBLIC HEALTH SERVICE - - MTlOML I^!STITUTES OF HEAIIFH 

INDIVIDUAL PROJECT REPORT 

Part A, Project Description Sheet 1. MtiMD~66 

SiRIAL I.IUM3ER 
National Institute of 
2, Arttoitis and Hetabolic Diseases 3. Pa thology a n d Histochemistry 

Institute or division UBOKATORYrmiiCH, cr departi>£mt 

U. Pathologic Anatomy 5. 

SECTION OR SERVICE LOCATIOL. (.IF OTH£..i TMiJ BaiTHliSDA; 

6, As Pathologic lesions induced by spermine and related substances - 

fechanism of induction. 

B. Human pathologic studies; di agnostic - research. 

PROJECT TITLES 

7. L. L« Ashburn 



PRINCIPAL li-rVESTIGAT OR 
8. Celia Tabor and Sanford Rosenthal 



OTHEPl INVESTIGATORS 

9. IF THIS PROJECT RESiKBLES, COI'IPLEIEiJTS, OR PARALLELS RESEARCH DOi® 
ELSE/JHExffi IN THE PUBLIC HEALTH SERVICE (.WITHOUT IWTERCEiNOE OF ?jJi- 
SOIMEL, FACILITIES OR FUNDS). 

Coroplements spermine studies in Laboratory of Pharmacology and Toxi- 
cology, ICAIID. 

10. PROJECT DESCRIPTION 

A, Spermine studies 

Objective s; Study of pathologic lesions produced by speriidne and 
related substances; mechanism of their induction. 

Methods Employed: Administration of test substance and inactive 
TcontroIJ material by various routes (subcutaneous, intra- 
aortic and intra-arterial (renal) and serial study of organs 
by gross and histologic methods. 



SERIAL ilO. 

10. (Cont'd.) 

Major Findings ; Injection of spermine into one renal artery is 
followed by cortical tubular degeneration with or \d.thout 
areas of infarction and followed by progressive arterial 
intimal proliferation, narrovjing of arterial lumina and 
renal atrophy. In areas of atrophy there is generally 
marked increase in number and degree of granularity of the 
granular cells of the juxtaglomerular apparatus; the oppo- 
site kidney is not involved. Intra-aortic spermine injection 
(with right renal artery clamped) produces diffuse lesion in 
left renal cortex X\rithout areas of infarction. Occasionally 
right kidney shows changes even though "protected" by claiip 
during injection. Lvidence suggests that tubular degeneration 
(the early changes) is due either to direct "toxic" effect of 
spermine or to cortical ischeirda brought about by arterial 
constriction at a level distal to arcuate vessels; medullary 
tubules generally uninvolved. 

Significance to the Program of the Institute s The study attempts 
to gain basic information on the physiologic and pharmacologic 
effects of spermine and its related and breakdoim products » 

Proposed Course of Project : Continue experiment along same general 
lines in attempt to determine whether renal lesions are 
brought about, at least in part, by temporary (3-U hrs) induced 
constriction of cortical arteries. Also study of cardiac 
lesion will be continued. Function of the granular cells in 
juxtaglomerular apparatus will be studied. 

B. Hioman Pathology 

Diagnostic: Service to various Federal Hospitals, mainly those of 
the Division of Indian Health and Bureau of Prisons - approx- 
imately 2,500 surgicals and 100 autopsies were studied and 
reported. 

Resear ch : Continued indexing of tumors of the Aiaerican Indian. 

Analysis of data xcLll begin in early 1957. Preliminary exa- 
mination of data suggests preponderance of certain tumor types 
in this ethnic group 



Form No» ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL Ii'ISTITuTES 01'' HEALTH 
Ii\!DIVinUAL PROJiTCT RiiPO.lT 



part B: Budget Data 



11, NIAi 03-66 



3E:lIiiL wUIiBER 



12, BUDGET DATA: 



DIR^.CT 



FY'57" 



ESTn'ATED OBLIGATIONS 



R'3IMBURS3i '^WT 



TOTAL 



MAN YEARS 



PROF ar»i;R T(rAL 



:'ii2l,700 



,100 



$29,800 



1..00 1.00 



2,00 



FY' 57' 



PROF 



1»00 



BUDGET "D POSITIONS 



OTHER 



1,00 



?OTAL 



2,-00 



PATIENT DAYS 



13. BUDGET' ACTIVITY; 
RESEARCH 

REVIEl-J & APPROVAL 
BIOLOGIC STANDAPiDS 






ADrilNISTRATION 

PROFESS lOi'IAL & 
TECHiilCilL ASSIST- 
ANCE 



c 



lUs IDENTIFY AilY COOPERATLrn UNITS OF THE PUll IC HiMLTH SERVICE, OR 
CTHCR ORGANI'^ATIONS, PROVIDING FU>IDS, FACILITIES, OR P: RSONi'EL 
FOR T'nS PROJECT IN FY 1957: IF COOPERATING UNIT IS VHTHIN IttH 
IiroiCATE SrRIAL NO(S)i 



(Use reverse and additional pa es, if necessary) 



■->S' 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 

IiroiVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications !$■, H]>-''^-66 

SERIAL ilUI-IHER 

16. PUBLICATIONS: 
None . 

17. HONCRS AND AwARDS: 
Noneo 



Calendar Year 1956 

PUBLIC .^ilALTH Si^RVICE - - liATIOi^AL liJSi'ITUT^o OF HEALTH 

INDIVIDUAL ?.:OJLCT iiETuYi! 

Part A, Project Description Sheet 1. HLJiD~67 

SiaAL iHJiiBi: 
National Institute of 
2, Arthritis and Metabolic Diseases 3. Patholoa^ and Histocher.iistry 

INSTITUTE 'v'.i DIVISION yiBQlATO:!, "il./CH, ' . D-,PAiiTi .-.ifl' 

1;. Pathologic Anatomy 5« 

SECTION OR SEiiVICE LOC.'.ilui' (IF aiV^fi THAx.' :ETPi,Sjj/0 

6. Preparation of stained tissue section for various research personnel 

of IHH (other than NCll. 

PROJECT TITLE 

7. yjTa Roy Reed - Head of Tissue Preparation Laboratory 

PRINC IPAL I iWESTI GAT OR 

8. See Item 6. ^____ 

OTHSR IWESTIGATO.IS 

9. IF THIS PROJECT RoSJ^LcS, Cuff liik.i ITS, OR PA.IALLLLS ..iESEAilCH DONE 
^LSEnHEilE IN TfE PUBLIC liALTH Sj. ...'TCI ( .jITHOUT li'ir^RCIANGE OF PER--- 
SOWNEL, FACILITIES OR K'lIDS). 

10. PROJECT DESCRIPTION: 

This histopathologic preparation laboratory receives, sections aiid 
stains animal tissue in support of various research projects con- 
ducted in many of the Institutes ;, other than iICI. In some instances 
(notably jolio vaccine control program), ;jhole specimens are received 
and technicians select appropiate blocks for processing. 

Tills laboratory also makes siivdlar preparations of human tissue 
received from a large number of Federal hospitals in connection ^-rlth 
a diagnostic service. 



SLRI AL ;!0. 



10. (Cont'd.) 









Stained 












Slides 


Special 






Ac 


cessioned 


Routine 


Stains 


Snares 


Aniinals 




7,323 


9,252 






Surgicals 




2,298 


it, 819 


15,561 


21,721 


Autcjpsied 




Ihh 


1,956 







Total 



The statistical report above represents a 12 month period and includes 
latest available figures. 

Hematoxylin and eosin is the routine stain in this laboratory. Special 
stains include the follovjing: 

Alkaline phosphatase} allochi^ome stain; Altiiian's mitochondria stain; 
azure-eosin; Bauer stain; Bodian' s protargol silver teclinique; 
Bowie's stain; Bro\-m-Brenn stain for bacteria; Ciaccio sudan black 
for lipoids; De Galantha silver method for urates; Diazo-safrardn; 
Dominici; Dunn-Thonpson hemoglobin stain; Ferrocyanide-HCl reaction 
for hemosiderin; Feulgen reaction; kite's stain for lepra bacilli; 
Foot's modification of Hortega's silver carbonate stain; Giemsa stain; 
Gallocyanin method for nerve tissue; Gomori' s chrome alum hematoxylin; 
Gomori's aldehyde luchsin; Gomori' s riiethenaridne silv^vr; combined 
Gomori methods for demonstration of alpha and beta cells of pancreatic 
islets; Gram acetone; Gram meigert; Lerner's rapid Gram stain for 
tissue; Gridley's stain for f'jngi; Goodpasture's stain for encephali- 
tozoon; Hale's method for acid mucopolysaccharides; lieidenliain' s iron 
hematoxylin; Heidenhain' s "azan" variant; Higl-iman-nallory amyloid stain; 
Hucker Conn Crystal violet stain; Luxol blue-cresylecht violet tech- 
nique; Hasson's trichrome; lielanin stain (Bahn's diamine silver); 
Mayer's mucihematein; Mayer's mucicarmine; fiyelin sheath; Orcein stain 
for elastic tissue; Phosphotungstic acid-hematoxylin; Parson's stain 
for Negri bodies; Periodic acid Schifi reaction; r^egaud's iron hemato- 
xylin for mitochondria; Ixinehart-Aodul-Kaj Method for acid mucopoly- 
saccharides; Toluidin blue; Thionin; Van Gieson's technique; Von 
Kossa's technique for calcium salts; warthin-Starry for soirochaetes; 



siJRiAL r;o. 



10. (Cont'd.) 



inayson's stain; ..eigert's difierential stain for fibrin; wilder stain 
for reticulum; jright and Craighead for encephalitozoon and toxo- 
plasrua; Ziehl-iiielson carbol fuchsin for acid fast bacilli; Schmorl's 
ferric ferricyanide reduction teclinique. 

All of the staining procedures given above were performed during the 

past year. 



Form No. ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIO.iaL INSTITUTES OF HKA.LTH 

iN]3iviDUAL itojt;ct REPCRT 



Part B: Budget Data 



11. illiiiu)— 6? 



SERI/.L iM'iEER 



12, BUDGET DATA: 



ESTIMATED OBLIG/'-TIONS 



FY' 57' 



DIRECT 



REinnURSEl'^^F 



TOTAL 



$33,900 



^3,200 



.^7,100 



BUDGETED POSITIONS 



FY'57~ 



PROF 



OTI-ER 



TOTAL 



7.00 



7.00 



13. BUDGET ACTIVITY; 
RESEARCH 

REVIEW & APPROVAL 
BIOLOGIC STAiJDARDS 



liAN YEARS 



PROF OTHER TOTAL 



7.00 7.00 



PATIE^^ DAYS 



ADFilNIbTRATION 

PROFESSIOML & 
TECHiilGi ASSIST- 
A;.ICE 



lU, IDEiCIFY ANY ^OPERATING UMTS OF THE rUET.IC HEALTH SERVICE, OR 
OTHER ORGANIZATIOKS, PROVIDI G MJiMDS, F.f.CILITI S, OR PER30 ."^^lEL 
FOR THIS PROJECT IM FY 19^7. IF COOPER. TING UJv'IT IS 'JITHIH WIE 
liroiCATE SERI/'.L MO(S)p 



Division of Biologies Standai'dSo 



(Use reverse and additional pa^es, if necessar;.-) 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - - iJATIUML li^BTITUTES OF .lEALlTi 
INDIVIDUAL PROJECT R^ORT 
Part C: Honors, Awards t Publications 15. NI/lMD~67 

b£.iiAL miim.Li 

16. PUBLICATIONS: 
lione. 

17, HONCRS & A'.j'ARDS; 
None . 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - - MTIOWAL IKSTITUTi.S OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A, Project Description Sheet 1^ NLJ4D-68 

SERIAL ffUl'IEER 

National Institute of 
2. Arthritis and Metabolic Diseases 3. Pathology and Histochemistry 

INSTITUTE OR DIVISION LABCRATORY, BRAf'CH, UR DEPARTiiLin' 

U, Pathologic Anatomy 5» , 

SECTION CR SERVICE LGCATI 0NTIF~"0THER THAN BETKESDA; 

63 Study of Mechanisms Involved in Infectious Diseases. 

FROJECT TITLE ' ~~ 



7» Eo M. Lerner II 



PRINCIPAL liWESTIGATOR 



8e Elizabeth Verder, Alexis Shelokov, V» Haas, Roger Cole, Leon Sokoloff . 
OTHER INVESTIGATORS 

9, IF THIS PROJECT RES-IJ-IBLES, CO^PLE^'EWTS, OR PARALLELS RESEARCH DOiffi 
ELSEvmHERE IN THE PUBLIC HEALTH SLRVICE (WITHOUT liHi^xiCIiAU^E OF PER- 
SONI^ffiL, FACILITIES OR FUNDS). 

This project coitplements, and is part of, research done by units 
described in lU, below, with interchange of personnel and facilities. 

10. PROJECT DEoCRIPTIOM : 

Objectives: To study the course of natural infections in man, and 
natural and artificially induced infections in animals, em- 
ploying bacteriological, oathological, immunological, and 
chemical methods, to elucidate factors influencing host-parasite 
relationships ,. 

A. Streptobacillus moniliformis Infections. 

B. Tissue Antibodies for Streptococcus in Rlieumatic Fever. 

C. Modification of Lyrti^hocytic Choriomeningitis Infections in lice. 



10, (Cont'd.) NL .MD-68 " I 

Serial ko. 



Methods Employed ; Ac - Specialized cultural tecliniques to isolate 
strains of Streptobacillus - Injection of amimals, and bacte- 
riological, "TiiiimirioTogicaTJ and histolor;ical study for identi- 
fication of the microorganism, screening of virulent strains, 
etc. 

B. - Extraction of tissue from cardiac biopsies and quanti- 
tation of tissue fluids. Immunological testing of tissue 
fluids and blood. 

C. - Pathological study and virus titration of rnice infected 
with LCM virus and treated. 

Major Findings } A, - Several strains of Streptobacillu s isolated 
from lung and middle ear infections in rats, and adapted to 
grow on simplified culture media r Antigens prepared from ba- 
cillary phase employed to measure plasma opsonins in guinea 
pigs and rats. Normal animal have a small amount of opsonin 
for this microorganism. 

One recently isolated strain has appeared virulent for rats. 
Intravenous inoculation produced a polyarthritis in 5-7 days 
in young rats, in a very high percentage of animals, vjhereas 
subcutaneous inoculation was ineffective. This was reprodu- 
cible consistently. The iTiicroorganism xias recovered from 
affected joints, but not from unaffected joints or blood. The 
joints showed arthritis, and no lesions were found elsewhere. 
Plasma opsonins of infected rats have been somewhat elevateds 

B^ - C-reactive protein, al^tistreptolysin-C titre, and C- 
polysaccharide precipitin have shown to date very slight or 
no increase in heart tissue fluids over serum levels. 

C. - In a small series, mice receiving amethopterin after 
intracerebral LCH virus have shown either negative or extre- 
mely slight lesions, although virus was recovered in high 
titre from the brain. Preliminary experiments suggest that 
PGA-deficient diet or in utero immunization cause some decrease 
in incidence or severi'Ey of lesions o 



10. (Cont-dO niim-68 - . 

SERIAL WO. 



Significance to the P rog ram of the Institu te: A, - A reproducible , 
high incidence, infectious arlhritis in rats has been demons- 
tratedo This apparently affects joints exclusively, unlike 
the arthritis concomitant vjith generalized lesions in other 
bacterial infections. Some immunological response to the in- 
fecting agent has been demonstrated, although of relatively 
low degree » This would appear to provide an experimental model 
for study of the infectious etiology of arthiritis, and a pos- 
sible basis for definition of the role of hypersensitivity in 
this disease. 

B. - Presence or absence of specific reactions to the Strepto - 
coccus in involved tissues, compared to serum, vd.ll aid in 
evaluating the hypothesis tliat this microorganism and hyper- 
sensitivity are involved in the pathogenesis of rheumatic fever. 

Cf ~ Modification or prevention of active disease after LCM 
infection, despite persistence of live virus, vd.ll afford nev; 
information regarding this and other viral infections, as vjell 
as possible new approaches to therapy. 

Proposed Course of P roject; Av- - Continue to study experimentally 
produced infected" animals, to determine vjhether hypersensiti- 
vity can produce arthritic lesions.- Pathologic study of de- 
velopment and regression of the lesions , Develop a more 
sensitive immunological technique than is presently available 
for these microorganisms. Explore infectivity of the filte- 
rable "L" forms, and immunological roeans of their identifi- 
cation. 

B= - Continue study of streptococcal immunological reactions 
in rheumatic fever tissues. 

C. - Continue study of modified lynphocytic choriomeningitis 
in micec 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIOW.L INSTITUTES OF HEALTH 
IITOIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAIiD-68 



SERIAL IWMBER 



13. BUDGET ACTIVITY 
RESEARCH 

REVIEW !k APPROVAL 
BIOLOGIC STANDARDS 



12, BUDGET DATA 


» 

ESTIMATED OBLIGATIONS 


MAN YEARS 


DIRECT 


REIMBURSEffilviT TOTAL 


PROF or PER TOTAL 


FI157 

$16,100 


#6,100 |ii22,200 
BUDGETED POSITIONS 


1,00 1.00 2.00 
PATXEMT DAYS 


PROF 


OTHER TOTAL 




FYf57 

1.00 


1,00 2,00 





Q] ADHINISTR/TION 

'\~"J PROFESSIONAL & 

TECIMICAL ASSIST - 
( I AWCE 



□ 



□ 



111, IDENTIFY Ai>lY COOPERATING UNITS OF THE PUH.IC lEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FU^DS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS V/ITHIN NIH 
INDICATE SERIAL NO(S): 

NIAID - Laboratory of Infectious Diseases - Provides facilities 
and personnel as needed. 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - - MTIOML INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards k Publications 1$. NIAMD-68 

SERIAL NUMffiR 

16. PUBLICATIONS: 
None. 

17. HONORS AND AWARDS: 
Nones 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - - IteTIOWAL Il&TITUTES OF HEALTH 

IiroiVIDUAL PROJECT R^PO.tT 

Part A. Project Description Sheet 1. NIm'-LD-69 

SERIAL IJUl-IBiLR 

National Institute of 

2. Arthritis and Metabolic Diseases 3c Pathology and Histoch emistry 

INSTITUTE CR DIVISION ' LADORilTORY, BRANCH, uR DEPA^TIiEiZr 

kf Patholog ic Anatoiny 5» , 

SECTIOFCR SERVtdE LOCATION [IF OTHER THAN BI^THESUA; 

6. A. study of experimental endocarditis and glomerulonephritis in dogs. 

Bo High altitude studies. 

C. Toxicity of iodides and iodates. 

D. Pathology of trypanosomiasis in rabbits. 

PROJECT TITLES 

B« Highman 



PRINCIPAL INVESTIGATOR 



8. P. D, Altland (Projects A and B); J., Roshe (Project A); S, E. .Jebster 
(Project C); E. J. Tobie (Project B) c 

CTkEft ir^ESTTMTc^ 

9. IF THIS PROJECT RESEI-I3LES, COiIPLEliEl'?rS, OR PARALLELS RESEARCH DOi>JE 
ELSEijHERE in THE PUBLIC HEALTH SERVICE (u'lTHOUT IWTKiCHAKGE OF PEi^- 
SON^ELj FACILITIES OR FUNDS). 

No known parallel research in tte Public Health Service. 

10. PROJECT DESCRIPTION ; 

Project A, Study of experimental endocarditis and glomerulonephritis 
in dogSt 

Objectives : To study endocarditis and glomerulonephritis 
in dogs viith aortic insufficiency, the development 
and regression of the lesions, and their response 
to therapyc 



10. (Cont'd.) NIaMD-69 



SERIAL no. 



Methods Emplo yed; Aortic insufficiency and an associated 
marked susceptibility to endocarditis is induced in 
dogs by perforating an aortic leaflet- An infection 
resembling acute and subacute bacterial endocarditis 
is induced consistently in such dogs by intravenous 
injection of cultures of Staphyl oc oc c us aureus and 
Strepto coccus mitis respectivelyo Dogs given S, 
mitis of^en develop a diffuse proliferative glomeru- 
lonephritis. The effects of a Hufnagel valve (in- 
serted before the bacterial injection), cortisone, 
and antibiotic therapy have been under study .i 

Hajor Findi ngs; A single intravenous injection of 83,333 
units of sodium peaicillin G and 10 mg. of strepto- 
mjrcin sulfate per kg. prevented staphylococcal endo- 
carditis in dogs with aortic insufficiency when given 
within 8 hours after a single bacterial inoculation. 
A longer delay gave variable results » Four similar 
antibiotic injections daily for $ days arrested the 
disease if treatment was begun within kQ hours after 
injecting staphylococci. Treatment delayed 72 hours 
or longer gave variable results. The disease often 
recurred shortly after cessation of treatment, or, 
if the disease was arrested, subsequent postmortem 
studies often revealed slight to marked valvular 
deformities r, A I-Iufnagel valve offered no striking 
protection against either endocarditis or glomerulo- 
nephritis . 

Significance to the Pr ogram of the Inst itute; Our findings 
reemphasize the importance of early treatment of endo- 
carditis and may shed light on some causes of treatment 
failures in human endocarditis and on the pathogenesis 
of glomerulonephritis. 

Proposed Course of Proje ct; Studies are continuing on the 
" effects of cortisone therapy, on the course of the 
untreated infection in dogs given S, mitis , and on 
the effects of antibiotic therapy in dogs given 
Staph y lococcus aureus. 

Project B. High altitude studies. 

Objective s; To study the effects of discontinuous expo- 
sure to simulated high altitudes (hypoxia) on various 
animals . 



10. (Cont'd.) HIAi4D-69 ' ^ 

SiRIAL WO. 

Methods Enployed ; Anirnals are exposed several hours 
daily in a low pressure altitude chamber to a 
simulated altitude of or above 25,000 feet. Studies 
have been made recently on dogs vn.th and vrLthout 
abnormal cardiac valves and on noririal and obese rats, 

fejor Finding s; The incidence of severe lesions involving 
the cardiac valves was less in normal rats e;:posed 
repeatedly to 25,000 feet than in similarly exposed 
obese rats and higher than in similarly exposed normal 
dogs. Dogs with aortic or mitral insufficiency or 
endocarditis tolerated single exposures to 3^,000 and 
36,000 feet less favorably than control dogs without 
valvular inpairment. 

Si gnificance to the Program of the Institute : The findings 
emphasize the inportance of species "diTferences and 
possibly nutritional factors in evaluating the effects 
of high altitude. They may be significant to those 
concerned vjith aviation medicine, health of liigh al- 
titude inhabitants, and laboratory investigation of 
cardiorenal disorders = 

Proposed Course of Projec t; The effects on do^s of re- 

peated daily exposures to 30,000 feet is under study., 

Projects C and D: Projects are continuing. No major findings to 
report. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIOi^IAL INSTITUTES OF HEAL'TH 
DDIVIDUAL PAOJECr REPORT 



part B: Budget Data 



11. niai:d-69 



SERIAL NUMBER 



RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STANDARDS 



12. BUDGET DATA: 








ESTIMTED OBLIG/iTIOWS 






HAN YEARS 


DIRECT REIi-BUESEICEiC 


TOTAL 


PROF 


OTHER TOI'AL 


FY157 








$m,6oo ij.5,000 


;;i;19,600 


1,00 


1,00 


BUDGETED POSITIONS 






PATIENT DAYS 


PROF OTIER 


TOTAL 




Fr'57 








1.00 


1,00 






13. BUDGET ACTIVITY! 









Q ADiilNISTRATION 

r~"| PROEESSIOML & 

TEGHi\TICAL A,SSIST- 
f] AiJCE 



a 



□ 



1U» IDENTIFY ANY COOPERATING TOUTS OF T^E PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZA^'IONS, PROVIDING FUIMDS, FACILITIES, OR PERSOiWEL 
FOR THIS PROJECT Iw'i'Y 1957- IF COOPERATING U"\IIT IS iJITHIN NIH 
INDICATE SIRIAL NO(S)'. 

Laboratory of Physical Biology, Section on Physiology, WIAI'iD. 

Clinic of Surgery, Naiaonal Heart Institute. 

Laboratory of Ir.armac-jlogy and Toxicology, Section on Pharmacology, NIAIJD. 

Laboratory of Tropical Diseases, National Institute of Allergy and 

Infectious Diseases. 



(Use r'^verse and additional pages, if necessary) 



Calendar Year 19 $6 

PUBLIC HMLTH SERVICE - - MTIONAL INSTITUTES OF ViZALTU 

INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards &i Publications 15. MLJ''iD-69 

SERIAL NUl-IBER 



16. PUBU CATIONS: 

Tobie, Ea J,, and Highinan, Bo ; Influence of the amino nucleoside 
of puromycin on the course and pathology of trypanosome infections 
in rabbits and raice. Am, Jo Trop» Medo and Hygc ^:50ii-5l5j 1956, 

Highman, B., Roshe, J., and Altland, PcDo: Production of endo- 
carditis with Staphylococcus aureus and Streptococcus mitis in 
dogs with aortic insufficiencyc Girculalion Research U:250-256^ 
1956. 

Altland, P* D., and Highman, B. j Effects of high altitude expo- 
sures on dogs and on their susceptibility to endocarditis. Sub- 
mitted for publication to Aviation i'ledicine. 



17. HONORS AND AWARDS: 
None . 



Calendar Year 1?56 

PUBLIC HEALTH SERVICE - - NATIOIIAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT H^PORT 

Part A. Project Description Sheet i, MLJ'ffi)-70 

SERIAL rni'MR 

National Institute of 

2. Arthritis and Metabolic Diseases 3» Pathology and Histochemistry 

INSTITUTE on DIVISION LABORATORY, IRAiJCH, OR DEPART!' iEi'IT 

k* Pathologic Anatomy ^. _______^ 

SECTION OR SERVICE LOCATION (IF OTHER THAU BlTHESDA) 

6. Conparative pathology 

PROJECT TITLE 

7« Ladd N. Loomis 

ffilMflli'AL IWESTTOATOft 

8. A. Vj, Pratt, R. Rn k\filliams, Jt Davidson, F. Abinanti. 
(METTMISTrSArnS 

9. IF THIS PROJECT RESEMBLES, COl'PLEfiEWTS, OR PAkALLELS Ri;,SEARCH DONE 
ELSEvv-HERE IN THE PUBLIC HEALTH SEiiVICE (vi/ITHOUT INTi:. .CHANGE OF PER- 
SOi\INEL, FACILITIES OR FUNDS). 

No parallel research in Public Health Service. 

10. PROJECT EESCRIPTION: 

Comparative Pathology, both as individual and cooperative research. 

(a) Rous sarcoma of chickens (in cooperation with Dr. A. vj. Pratt 
NCI). 

. Objectives ; The nature and progression of the early ske- 
letal muscle lesions produced by the Rous sarcoma in 
the chicken. 

Methods Employed ; Microscopic examination of the early 
muscle lesions are being made. Suitable photographs 
for illustrative plates have been taken. 



10, (Cont'd.) NL;MD-70 . ■ 

SERIAL iIO. 

Findings : The muscle lesions in the Hous tumor-bearing 
chicks have a characteristic pattern of invasion 
into normal muscle elements o The virus apparently 
converts the normal intramuscular connective tissue 
elements into .lous tumor cells ; thus invading by- 
conversion and extension. 

Significance to the Program of the In stitute; This study 
will add to our information on the Rous tumor with 
unknown significance for the cancer field. 

Proposed Course of Project ; This project is comjjleted; 
manuscript in preparation, 

(B) Production of Newcastle disease virus (i'J,D,V,) for purification 
and assay, (in cooperation with Dr. H, R. Williams IHAilD). 

Objectives ; Purification of N,D«Vc by immunological 

methods, including the use of cellulose materials as 
an antigen-antibody bearer. 

Methods Employed ; Newcastle Disease virus (N.D,V.) v;as 

harvested from chick embryos and was ultracentrifuged. 
The ultracentrifuged virus material is exposed to 
powdered cellulose coated with anti -allantoic fluid 
rabbit sera. A "before and after" calculation can be 
made, using HI^q ratios to nitrogen values in con- 
junction with hemagglutination titres, that iden- 
tifies virus purification. 

Findings: Some evidence of significant i'.D.V. virus 
purification has been determined. Secondly, the 
i-I.D.V. preparations are used for an assay method 
(agglutination-activating factor) on sera of rheu- 
matoid arthritic patients. 

(C) Serotonin toxicity in rats (in cooperation with Dr. John 
Davidson NHI). 

Objective s ; To see if abnormally elevated serotonin- 
blood levels produce cardiac lesions in rats. 



10. (Cont'd.) ... , 

S^xTCAL WO. 

Methods Employ ed; Ten Spragne-Dawley rats were given 

intraperi^neal injections of 5-HydroKy-trytophan 

(a serotonin precursor) t\jice daily. Tvio rats were 
sacrificed on the 30th, 60th, 90th and 120th day. 
Their organs, principally their hearts, vjere exa- 
mined microscopically using differential stains as 
well as standard stains. A total of approximately 
2,000 sections were examined including sections from 
15 control rats. 

Findings ; No significant findings. 

(d) Monkey Safety Tests of the Salk Polio Vaccine continues. 

Objectives ; Examination of cervical and lumbar cord 
sections, from Salk vaccine inoculated Rhesus 
monkeys, for evidence of live polio virus. 

A total of 3,700 monkeys were examined during the 
period November 1, 1955 x.o October 31^ 1956. 

Differential diagnostic problems were encountered 
during the past year due to C.N.S, lesions other 
than poliomyelitis. These have tentatively been 
classified as "Dengue-like"; "solitary tract in- 
fections"; or "radiculo-encephalorayelitis". 

These problem monkeys are examined for the distri- 
bution of lesions in the cerebrum, cerebellum, 
midbrain, and medullary areas, as vrell as within 
the spinal cord. The rete sting of these monkeys _ 
was made in cooperation with Drc Francis Abinanti, 
D.B.S. 

(E) Neuropathology of dogs. 

Objec tive s; Classification of the various CMS lesions 
of~aogs is being made. Many of these lesions are 
rare and there is little information about them. 

Meth ods Employed ; A careful post-mortem examination is 
made 4 



10. (Cont'd.) NLiMD-.7 i 

SERIAL TiS." 

A systematic microscopic examination of the brain 
and spinal cord is made using standard histo- 
pathologic techniques and some special stains. 
Routine sanples of the other organs are examined 
microscopically. 

Findings: The conditions encountered vary from virus 
diseases to traumatic conditions and tumors r T'./o 
rare brain tumors were found,. Also a case of acute 
hemorrhagic pancreatitis and one congenital aortic 
valvular heart condition were found at post-mortem. 

Proposed Course of Project ; The survey is continuing and 
the interesting material will be published. 



Form No. ORP-1 
October 1956 
(Attacliment I) 



PUBLIC HEALTH SERVICE - IIATIOML Fi^ISTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAIID-70 



SmiAL i^IUMBER 



12, BUDGET DATA 










ESTIMATED OBLIGATIONS 




MAN YEAltS 


DIRECT 


RI^H'IBURSEMEiir TOTAL 


PROF 


OTIER TOTAL 


FYS57 








|15,600 


!!J6,1C0 is2 1,700 


loOO 


2.00 3.00 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHER TOTAL 




FY«57 








1.00 


2.00 3.00 






13. BUDGET ACTIVITY? 







RESEARCH 

REVID/f & APPROVAL 

BIOLOGIC STANDARDS 



r^n ADMINISTRiVTION 

( i PROFESSIOML & 

TECHJIGAL ASSIS: 
I i AI^ICE 






lU. IDENTIFY AlvIY COOPER/ITING U1«TS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORG/\NIZATIONS, PROVIDING FUWJG, 1?ACILITIES, OR PER.SOIn&IEL 
FOR Tins PROJECT IN FY 1957. IF COOPER/.TING UNIT IS WITHIN NIH 
INDICATE SERIAI, NO(S): 

Laboratory of Physiology, Radiation Physiology Section, National 

Cancer Institute, Dr, As \h Pratt, 
Laboratory of Physical Biology, NIAiC Dr.; R, H, VJilliams . 
General Medicine and Experimental Therapeutics Branch, National Heart 

Institute. Dr. John D. Davidson, 
Laboratory of Viral products, Division of Biologies Standards, Dr. 

Francis Abinanti. 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PU3LIC HE/.LTH SERVICr. - - liATIOLAL li'STITUTiS OF HEALTH 

Ii€lIVIDUAL PilOJECT PffipCRT 

Part C: Honors, Awards ?i Publications 1$> NLu-ID-70 

SEIIIAL IJUiiBER 



16. PUBLICATIONS: 

Histogenesis of Rous Sarcoma I, Induced by partially Purified 
viruSf J, of the National Cancer Institute 17:101-121, 1?56» 



17. HONORS km AV/AUDS: 
None. 



Calendar Year 19^6 
PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part A« Project DescriptiGn Sheet 1, NL.?ID-71 



SERIAL NUlilBER 
National Institute of 
2« Arthritis a nd Metabolic Diseases 3a Pathology and Histochemistry 
INSTITUTE" LA BORA TORY 

k» Hematology 5: ^__^ 

SECTION LOCATION (IF OTHER THAN BETHt^SDA 

6, Regulation of hemopoiesis 

PROJECT TITLE 

7, Frederick Stohlman, Jro, and George Breche r 

PRINCIPAL INVESTIGATOR(S} 

8, None 

OTHER INVESTIGATORS 

9, IF THIS PROJECT RESEI.IBLES, COIvIPLEliENTS , OR PARALLELS RESEARCH DONE 
ELSK'TIERE IN THE PUBLIC HEALTH SERVICE (WTHOUT INTERCHANGE OF PER- 
SONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH. 

Dr, S. Je Sarnoff ( ), Laboratory of Cardiovat-cular Hemo- 

dynamics and Dr, F. Stohlman, Jr. ( ) are collaborating in 

the study of anemia produced by the mechanical damage to red 
cells in experimental animals and men carrying plastic heart 
prosthesis, 

19. PROJECT DESCRIPTION 

Objectives : Study of normal iron and red cell tumoverj 
witn~special reference to the identification of 
substances capable of stimulating red cell pro- 
due tion ( ery thr cpoie tine ) , 

Methods 5j;nplc3''i=:d I Substanoos capable of stimulating red 
c e lT~ pr oci'^c ti o;:i are assayed by injecting them into 
irr.-.dxated rats which respond better than normal 
anrujials to sucr* stimulation- The response is 
mef'.r'ia'ed hy tho incorporati<;in of radioactive iron 
int.-. red cells , It his been shown that under ir.he 



NL.MD-71 



SERIAL NUMBER 



10. (Cont'd.) 



cell production. Other methods employed for the 
measurement of red cell production include the 
clearance of radio-iron^ and survival studies of 
red blood cells using radio-chromium and DFP^^ 
as tracers. Iron absorption will be studied by 
a surviving preparation of rat intestine, using 
radio-irono 

Major findings ; 

le "Erythropoietine" has been shown to remain 
in the supernate after acidification of the plasma to 
pH U or 5 and alcohol precipitation at these pHs , The 
activity is lost on dialysis at pH h and is heat labilee 
This is in contrast to the findings of Borsook, who has 
described a heat-stable factor in anemic rabbit plasma 
capable of stimulating erythropoiesL^^ in rats and mice, 

2» To date, we have been unable to obtain positive 
results with Borsook's method in our assay procedure. 

3* The effect of radiation on mature red cells has 
been shown to be an indirect one. It appears due to a 
change in the plasma induced by whole bouy irradiation, 

k» DFP-^ (di-isopropylfluorophosphate) used as red cell 
tracer in dogs has given survival curves that are similar 
in shape but not identical with those obtained by differ- 
ential agglutination. 

These initial studies indicate a shortened life 
span of the DFP tagged cells but absence of the type of 
"elution" seen with radio-chromium. 

Significance te NIAMD Research ; Anemia is a common compli- 
cation of arthritis and certain metabolic diseases and 
may be refractory to treatment. A better understanding 
of regulation ©f erythropoiesis should result eventually 
in improved therapy. 

Proposed course of project ; Although the best available 

assay methods for erythropoietine are painfully slow and 
systematic study of plasma fractions with this assay 
both laborious and time consuming, it is proposed to 



Form No. ORP-1 
October 1956 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIOimL IIISTITUTES OF HEALTH 
liTOIVIDUAL mOJECT REPCRT 



Part B: Budget Data 



11. NIAfiD-71 







SERIAL NiJl-iBER 


12. BUDGET DATA: 






ESTIMATED OBLIGATIONS 


MAN YEARS 


DIRECT 


REIMBURSErENT TOTAL 


P::0? OTHER TOTAL 


FI«57 






•V31,900 


;J12,100 5ii[tli,000 


1.00 li.OO 5.00 




BUDGETED POSITIONS 


PATIEiC DAYS 


PROF 


OTHER TOTAL 




FY'57 






1,00 


ll.OO 5.00 




13. BUDGET ACTIVITY: 





RESEARCH 

REVIEl'J & APPROVAL 

BIOLOGIC STANDARDS 



□ 



ADMIWISTR/lTIOW 

PROFESSIOML Zt 
TECHNIC/iL ASSIST- 
ANCE 



□ 



LJ 



lU. IDENTIFY Ai^TY COOPER/iTING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNiJS, FACILITIES, OR PERSOiHIEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WTHIW NIH 
INDICATE SERIAL NO(S): 

Dr. S, J, Sarnoff, Laboratory of Cardiovascular Hemodynaiaics and 
Dr. F. Stohlman, Jr. are collaborating in the study of anemia produced 
by the mechanical damage to red cells in ej;p3rimental aniwals and men 
carrying plastic heart prosthesis. 



(Use reverse and additional pages, if necessary) 



ar;,yA?i; 



NIAMD-71 '3 
SERIAL NUMBER 



lOo (Cont'd.) 



concentrate our major effort again in this direction 
because of the potential importance of this plasma 
factor. We also wish to continue o\ir attempts to repro- 
duce findings on erythropoietine from other laboratories 
in order to determine whether the different assay pro- 
cedures measure different factors. 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part C, Honors, Awards & Publications 1^, N LiMD-71 

SERIAL NUMBER 

16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROJ' THIS PROJECT DURING 
CALENDAR YEAR 19^6: 

Stohlman, Fo, Jr.: Red Cell Survival in the Dog Determined by 
a Method of Diffeiential Agglutination Employing Canine Anti-A 
Serum. J, of Lab. & Clin. Med,, 1|7:83, 19^6. 

Stohlman, F., Jr., and Schneidemian, M, : Application of the 
Cr-* Technic to the Study of Experimental Hemolysis in the Dog, 
J, of Labo & Clin, Med,, 1^7:72, 19%. 

Stohlman, F,, Jr., and Brecher, Get The Stimulation of Ery- 
thropoiesis in Sublethally Irradiated Rats by a Plasma Factor, 
Proc, Soc, Exp, Biol,, & Medc, 91:1, 19% „ 

Stohlman, F,, Jr^, Sarnoff, S, Jo, Case, R, B., and Ness, A. T,: 
Hemolytic Syndrome Following the Insertion of a Lucite Ball- 
Valve Prosthesis into the Cardiovascular System. Circulation 
13:586, 1956, 

Stohlman^ Ft, Jr., and Brecher, G, : The Erythropoietic 
Response to Hypfflxia, Proc, Int, Soc, of Hem^ In press, 

Brecher, G. , Schneiderman, M^. , and Vifilliams, G, Z.: 
Evaluation of an Electronic Red Cell Counter^ Am. Je 
Path, Dec, 19^6 o 

Stohlman, F,, Jr,, and Brecher, G, : Humoral Regulation III» 
Effect of Exposure to Simulated Altitude;, Submitted to 
Proc, of Soc, of Exp, Biolc & Med, 

17, LIST HONORS AND AlVARLS TO PERSONNEL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 19^6: 

None, 



i?6,..i;,;: 



. -.-n*"',*- »eV iff 



» V 



Calendar Year 1956 

PUBLIC HEALTH SERVICE NAnONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A, Project Description Sheet I4 NIAKD-72 

SERIAL NUJ,fflER 

National Institute of 
2o Arthritis and Metabolic Diseases 3e Pathology and Histochemistry 

INSTITUTE OR DIVISION LABORATORY 

kt Hematology 5^ 

SECTION ■ LOCATION (IF OTHER THAN BETHESDA 

6 . Physiology of white cells in diabetes 

PROJECT TITLE 

7. Jane Shohl and George Brecher 

PRINCIPAL INVESTIGATOR(s1 

8. None 

OTHER INVESTIGATORS 

9. IF THIS PROJECT RESEMBIilS, COJIPLEMENTS , OR PARALLEI^ RESEARCH DONE 
ELSEV^fHERE IN THE PUBLIC HEALTH SERVICE (XHTHOUT INTERCHANGE OF PER- 
SONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 

This project complements investigations by Dr, Gorsach ( ), 
Laboratory of Clinical Investigations , National Institute of Micro- 
biology; whose studies will be concerned primarily with the clearing 
mechanism of bacteria in diabetics rather than with the specific 
function of white blood cells 

10. EROJECT DESCRIPTION 

Objectives ; Characterization of the metabolic abnormality 
of white cells in diabetes and the possible relation- 
ship to the susceptibility to infections. As employed^ 
diabetes will be produced experimentally in rabbits by 
alloxan. The metabolism of the white cells will be 
studied using chemical and tracer meth;-ids. The role of 
demonstrable abnormalities of white cells in the 
increased susceptibility to infection of diabetic 
animals and men will be studied. 

Major Findings : Only preliminary findings are available 
of this project started a year ago. 



NLJ"ID-7? 



, SERIAL NO. 

10. (Cont'd.) 

Significance to MIAhU Researc h: The susceptibility of 
diabetics to infection is one of the unsolved 
puzzles of this important disease. 

Future Course o f the Project ; In view of the lack of 
space and facilities for glycogen turnover studies 
in our section, this project will be discontinued 
in its present form. 



Form No, ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOML BISTIT'JTES OF HEALTH 
BIDIVIDUAL PROJECT REPORT 



part Bs Budget Data 



12, BUDGET DATA: 



11. NIAMD»72 



SERIAL I>JUI"1BER 





ESTIMATED OPLIGA.TIOi\IS 




HAM YEARS 


DIRECT 


REDIBURSEMENT TOTAL 


PROF 


otw:r total 


FI'57 








;iJ10,800 


f:,!4,000 :;^U,800 


loOO 


1,00 




BUDGETED POSITIONS 




PATiEi^rr days 


PROF 


OTHER TOTAL 




FY«57 








1,00 


1,00 







13. BUDGET ACTIVITY; 
RESEARCH 

REVIEl-J & AP.'^ROVAL 
BIOLOGIC STANDARDS 






ADMINISTR/iTION 

PROFESSIONAL & 
TEGHi«CAL ASSIS1 
AWCE 






111, IDENTIFY AflY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUJJDS, FACILITIES, OR PERSOMiEL 
FOR THIS PROJECT IN FY 195?. IF COOPER/iTIMG UNIT IS WTTHIN NIH 
liTOICATE SERIAL NO(S): 

This project complements investigations by Dr» Gorsuch, Laboratory of 
Clinical Investigations, National Institute of Microbiology, vriiose 
studies will be concerned primarily with the cleai^in^ mechanism of 
bacteria in diabetics rather tlian \iri.th tlie specific function of white 
blood cells. 



(Use reverse and additional pages, if necessary) 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 1$. NL;;iD-72 

SERIAL NUI'iBLR 

160 LIST PUBLICATIONS OTHER THAN ABSTFACTS FROM THIS PROJECT 
DURING CALENDAR YEAR 19^6: 

None, 

17, LIST HONORS AND AWARDS TO PERSONIffiL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 19^6: 

None. 



PUBLIC HEALTH SliHVICL - NATIONAL IiMSTITyXES OF HEALTH 
IIJUIVIDUAL PROJhCT RtiPORT 



Part A. Project Description Sheet 1. |iJfflrI2___-_ 

Hlp^UiU ;i. Pgtholo.-:y ana Hi st oclic i.ii st ry 

INSTITUTE LAJiOKATOKY 



^, Hematology 5. 

SLOTIOW LOCATION 

6. PatiiOyenesis oi txper imenta 1 Arthritis and 

Pathol o-iv 1 Rhe 'iniFl, i_sj!i 

Pr.uJiicT TITLE.- 



7 . 


Leon Sokoloi.;. i'.. i- . 




PklNtlPAL INVESTI-SATOa 


a. 


iJone 



OTHER IiJVESTIiATOKS 
9. uo parallel research in the Public Health Servise. 
1 . PHO JEV.T uESGllIPTIOH 

The iinaings reportec in last year's project 
analysis have been expancetl: 

1) A second instance ol i^ran Jlorr.atoiis 
synovitis in a patient with b o e c k ' s sarooic! has 
been atuciiea. 

i) Synovial biopsy correctly establ ishei'. the 
cia<jnosis oi rneu.natoid arthritis rather than .C) o ;i t 
in one patient in who:ii tm aiainosis ivas ancertain. 
it reaCiiriiied a diagnosis oi C;0ut in a patient 
incluaeu in last year's report, who had certain 
clinical .'eatures su'jvjsstive oi rheu..iatoic 
arthritis. 

J) m a patient originally consiceret to have 
scleroderiiia with rhe j.viato i dl i!ce arthritis, biopsies 
revealec. normal synovial tissue anc non sc lerot ic ^ 
suin. The case is now regaroec. as one o. Jerner' s 
svncroine . Hi stolojicel xindin.is in the joints have 
not been reporter. Tho observation, thouij'i negative, 
is valuable in evaluating the pa tlio jene s i s oi the 
joint c;e:.oriuiits in this condition , 



NIAMD-73 ■• - 

SERIivL NO. ^) ^^ amputated linger with scleroderma, 

obtained irom another institution, has been worked 
up histoloijically. The joints were normal. Thus, 
although synovitis occurs in some patients" 
peripheral joints, it is not necessarily present in 
digital joints covered by involved skin. 

5) in a toe previously aifecteu twice by acute 
gout, no residual urate deposits were present at 
necropsy. The histological linuimjs in the joints 
in the intercrit ical phases ox acute g out have not 
been reportea bexore. 

Pathological and hi stopatho 1 oy ical studies 
performed during 1956 and, in aduition, those done 
during the preceding three years, but not incluued 
in the corresponding annual reports, /ollow: 

g Joints dissected 

Inst itut ion Skin uuscle , at autopsy or 
vial 1 ^ . 

alter amputatio n 

Clinical v^enter 60 27 :56 23 

Johns Hopkins 4 4 4 1 

Naval i-ieaical 2 

i.it. AltoVeteransl6 11 65 1 

Walter Reed Army __1 _1 ^ _0 

Total 81 4\j 107 2'o 

In aduition, 11 auricular appendages obtained 
at mitral commissurotomy Jor a collaborative 
project with Ldward Lerner and Fioger Cole (iJlAlu) 
have been processed and studied. 



Form No, ORP-1 
October 19$6 
(Attachment I) 



PUBLIC HEALTH SilRVICE - NATIOi^IAL IMSTITUTEG OF HEALTH 
BIDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-73 



SERIAL i>IUrlDER 



RESEARCH 

REVIE14 &i APPROVAL 

BIOLOGIC STANDARDS 



12, BUDGET DATA 










ESTIITATED OBLIGATIONS 




FiAN YEARS 


DIRECT 


PEIMEURSE^ENT TOTAL 


PROF 


OTIK/i TOTAL 


FZ«57 








|;10,700 


.i(ii.,000 ii^lU.TOO 


0.50 


1.00 1,50 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHER TOTAL 




FYt^? 








0,50 
i'3_ ■nrrnnT?rn Apm-n 


1,00 1.50 







□ 



ADMINISTR/.TION 

PROFES.IOmL 5t 
TECirNIGAL ASSIST- 
ANCE 



□ 



lZI 



Ik* IDEi^IFY Ai>Ty COOPE.mTING UNITS OF THE PUBLIC HEALTH S.OTICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSON.'EL 
FOR THIS PROJECT IN FY 1957. IF COOPFJl/iTING UNIT IS ^iIT'lN NIH 
INDIQ.TE SERIAL NO(S): 



(Use reverse and additional pa^es, if necessary) 



Part »^. ilonors. Awards o Pu bl ioations 



1 5. NIhI'II-73 



ShUlAL NUi.iBLli 

1 6 , Publications During calenaar Yrar 1956 

L, Soi;oloj.i.'„ Some Aspects O- thp Patholoyy oi 
tollayen Liiseases. bull. i^l. Y„ Acao, l-ieo, 32 ; 
760-7t)Y, 19 JO. 

L. Soicoloi... biopsy as a Diagnostic Procedure in 
Rheumatic liiseaseSo Lull. Rheuw, biso J,: 
Suppl. i , 1936, 

L. Solcolo.'j ano J. Jo bunira. Vascular Lesions 
in Rheuiiiatoid Arthritis, J. chronic bis. (in 
preparation). 

L, Soi;olo;ii\ Patholoi^y Oi Gout, Wetabolisin 
(In preparation). 

17, Honors and Awards 

Pemberton uecturer, Philadelphia, October -il , 1956. 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

NI>ii4D-7li 



Part A. Project Description Sheet 1^. 

SERIAL NUIvIBER 
National Institute of 

2, Arthritis and Metabolic Disease s 3. Pathology and Histochemistry 
INSTITUTE OR DIVISION LABORATORY ~ 

h» Hematology $a 

SECTION LOCATION (IF OTHZR THAN BETHESUA 

6 . Pathogenesis of experimental arthritis and patholo g y of rheumatism 
PROJECT TITLE 

7. Leon Sokoloff 

PRINCIPAL INVESTIGATOR 

8. Emmanuel Silverstein 

OTHER im^ESTI GATORS 

9. IF THIS PROJECT RESEfBLES, COMPLEI lENTS , OR PARALLELS RESEARCH DONE 
ELSEl'ifHKRE IN THE PUBLIC HEALTH SERVICE ('"ITHOUT INTERCHANCE OF PER- 
SONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 

Studies on tissue antibodies in rheumatic fever with special 
reference to the streptococcus (in collaboration with Dr. E, M, 
Lerner of LPH, NIAIID ( ), and R. M. Cole, NMI ( ). 
His topathology of the synovial membrane in arthritis will be 
carried out in collaboration with the Laboratory of Clinical 
Investigations, 

10. PROJECT DESCRIPTION: 

O bjectives : Investigation of factors influencing develop- 
ment of degenerative joint disease and non-infectious 
inflammatory arthritis in small laboratory animals. 
Study of his topathology of the synovial membrane in 
human arthritis. 

Methods: Histologic and papain-maceration examination 
of small laboratory animals have been used to es- 
tablish the presence of osteoarthritis , 



SERIAL MUIBER 



10. (Cont'd.) 



The roles of spontaneous (genetic) and dietarily 
induced obesities as modified by amount of physical 
activity, and of genetic constitution in the 
development of spontaneous osteoarthritides are 
being studied. Attempts to produce noninfectious 
arthritides by foreign protein and hormonal 
factors are under way. 

Major Findings ; An osteoarthritis closely resembling 
the human disease has been shoivn to correlate 
strikingly with the presence of obesity^ whether 
spontaneous or dietary. 

Signific an ce to NIAuiD R esearch; The economic im- 

portance of disability due to cnronic arthritic 
degeneration can hardly be exaggerated- 

The correlation of the incidence of arthritides 
in mice vath specific etiologic factors indicates 
that these lesions are not inevitable concomitants 
of ageing and that hence preventive measures may 
become available. 

Rheumatoid arthritis and certain of the rheumatic 
diseases are characterized by apparently non- 
infectious chronically progressive inflammation 
of joints. The present search for a counterpart 
of this disease in animals, if successful, vfill 
provide the means of studying not only the patho- 
genesis but also suitable treatment of this chronic 
and disabling disorder. 

Future Course of the Project ; Because the experiments 
noted above are long term type, it is estimated 
that the dietary studies on mice will require an 
additional two years for completion. These studies 
are being conducted in collaboration virith Dr. 
i'ickelsen. Approximately 600 mice are presently 
being maintained on the several dietary regimens o 
Approximately 200 Fl hybrid mice are presently 
being maintained for a comparable period. Thyroid 
histology and function will be studied ivith Dr. J. 
Rail to evaluate the role of the thyroid of mice 



NL J-iD-7U -X 
SERIAL NlTrlBEir 



10, (Cont'd.) 



in epiphyseal ageing and osteoarthritis o The 
procedures enumerated under methods vdll be used 
in an attempt to produce the counterpart of 
rheumatoid arthritis in laboratory animals : The 
possible role of hypersensitivity and infection 
with Streptobaoillus moniliformis in the lesions 
will be studied in collaboration with Dr. Lerner, 



Form No, ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL roSTITUTES OF HEALTH 
INDIVIIJUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAI1D-7U 









SERIAL mViBL 


R 


12. BUDGET m 


TA: 
ESTIMATED OBLIGATIONS 






mN YEARS 


DIRECT 


REIffiURSEMEHT 


TOTAL 


PROF 


OTHER TOTAL 


FY' 57 








.'^25,500 


1-9, 100 
BUDGETED POSITIONS 


$314,600 


lo50 


2.00 3.50 
PATIEir DAYS 


PROF 


OTIER 


TOTAL 




FY'57 










1.50 


2,00 


3.50 






13. BUDGET ACTIVITY; 









RESEARCH 

REVIElif & APPROVAL 

BIOLOGIC STANDARDS 



□ 
p 



ADMNISTR/iTION 

PR0FES3I0ML & 
TECHNICAL ASSIST- 
ANCE 



□ 



□ 



lU, IDEiriFY ANY COOPERATING UNITS OF THE PUETJC HE/iLTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONiiEL 
FOR TEES PROJECT IN FY 1957, IF COOPt;RATING UNIT IS l/ITHIN NIH 
UraiCATE SERIAL NO(S): 

Studies on tissue antibodies in rheumatic fever with special 
reference to the streptococcus (in collaboration mth Dr. E. M, 
Lerner of NIAMD, and Dr, R. ^'^ Cole, ma.) HLstopathology of the 
synovial membrane in arthritis will be carried out in collaboration 
with the Laboratory of Clinical Investigations, 

(Use reverse and additional pages, if necessary) 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part Cs Honors, Awards & Publications l^c NLJ-iP-TU 

SERIAL NUi.iBER 

16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
CALENDAR YEAR 19^6: 

1, Sokoloff, Lj : Natural History of Degenerative Joint Disease 
in Small Laboratory Animals. 1, Pathologic Anatomy of 
Degenerative Joint Disease in Mice, A-M.A. Arch, Path* 62, 
118-128, 1956, 

2, Sokoloff, L,, and Jay, G, E, Jr,: 2r Epiphyseal Maturation 
and Osteoarthritis d£ the Knee of Mice of Inbred Strains, 
A.M.Ac Arch, Pathc 62, 129-135, 19^6, 

3, Sokoloff, L,, and Jay, G. E. Jr^,: 3, Variations in 
Epiphyseal Maturation in the Head of the Femur Among Inbred 
Strains of Mice, A.MoA. Arch, Path. 62, I36-I39, 19^6, 

li. Sokoloff, L,, and Jay, G, E, Jr,: Degenerative Joint 
Disease in the Laboratory Rat; A.M.Ao Arch. Path, 62, 
Iii0-m2, 1956. "~ 

5o Sokoloff, L„ I Some Aspects of the Pathology of Collagen 
Diseases. Bull, N, Y, Acad. Med. 32, 76O-767, 1956, 

6, Sokoloff, Lc : Biopsy as a Diagnostic Procedure in 

Rheumatic Diseases, Bull, Rheum., Dis, 7, Suppl, 2, 1956, 

17, LIST HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 1956c 

Pemberton lecturer, Philadelphia, October 31j 1956, 



;;.,.r«»i.v»"-- 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1, NL';MD-75 



SERIAL NUIBER 
National Institute of 
2« Arthritis and Metabolic Diseases 3. Pathology and Histcchemist ry 
INSTITUTE OR DIVISION LABORATORY 

Ii, Hematology 5. 

SECTION LOCAnON (IF OTHER THAN BETHESUA j 

6, Studies of dietary factors and embryonic growths as affecting rat 
PROJECT TITLE 

leprosy and experimental amyloidosis, 

7. George L. Fite 



PRINCIPAL INVESTIGATOR 
8» None 



OTHER IWfESTI GATORS 

9. IF THIS PROJECT RESEl/IBLES, COllPLEIiJENTS , OR PAPJ^LLELS RESEARCPI DONE 
ELSEi^HERE IN THE PUBLIC HEALTH SERVICE (^nTHOUT INTERCHANGE OF 
PERSONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH. 



10. PROJECT DESCRIPTION 

A. Project for 19^7. 

1. To investigate the effect of vitamin E deficiency 
on rat leprosy, following the lead of Bergel and 
Mason in their work on human leprosy, in which 
they reported enhancement of the infection in 
rats fed E-deficient diets rich in cod liver oil. 
This work, if true, may be of great significance, 
and appears of readily susceptible analysis using 
rat leprosy. 



Ni^.ID..75 '^ 
SERIAL NUlvBER 



10. (Cont'd.) 



2. To explore further the unreported vrork of H, S. 
N. Greene in transplantation of lepromas to 
brain tissue. It is planned to explore this 
field widely: by implantation of infected 
embryonic tissues into non-susceptible animals, 
not only into brain, but testis and other 
organs, 

3. To examine critically the infectivity of M, 
Leprae Iiurium when stored in various menstruums 
in the frozen state. 

U, To analyse experimental amyloidosis in animals 
using several dietary procedures and rat leprosy 
as the immediate causative agent. 

Significance to NIAtD Research ; A direct attack on 
basic problems in nutrition as related to 
chronic infectionc 

B, 19^6 Activities : 

1. Cooperative investigation of morphologic aspects of 
experimental liver necrosis with Drt, K. Schwarz 
(LNE) in relation to various dietary factors. This 
entails a considerable amount of histologic study 
of livers and other viscera of rats ■ 

2» Leprosy biopsies from Caroline Islands examined as 
a consultant to Leonard Wood Memorial Foundation 
and inters ervice committee on leprosy. 

This service has also demanded a good deal of time 
in conferential planning of leprosy research and 
teaching with Dr„ Binford et al. 



Form No« QRP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SLRVICE - NATIONAL INSTITUTES OF HE/.LTH 
INDIVIDUAL PROJECT RITQRT 



part B: Budget Data 



11. MIAIffi-7^ 



SmiAL WiiBm 



12, HJDGET DATA 


1 








ESTimTED OBLIGATIONS 




MAN YEARS 


DIRECT 


REIMBURSEMEJr TOTAL 


PROF 


OTH}<:n TOTAL 


FY'57 








■4'i-7j900 


■47,100 ;ij,25,000 


1.00 


1.00 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHER TOTAL 




FY'57 











1,00 


1. 


.00 








13. 


BUDGET ACTIVITY: 














RESEARCH 


,y 




ADHffllSTR/vTION 


L_ 




REVI&J & APPROVAL 
BIOLOGIC SrAi\IDARDS 


D 




PROFE 
TEC 
ANC 


SSIOkIAL & 
fflJIGA.L AS3IST- 
E 


1 



Ih. IDEiriFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, CR 
OTIER ORGAmZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONaEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN MIH 
INDICATE SERIAL NO(S): 

This project is undertaken vjith the knovjledge and approval of the 
PHS Interbureau Advisory Committee on Leprosy, and supplements, but 
does not parallel, that of Dr. C. H, Binford at CDC. 



(U-re reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBUC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications 1^. NLJ-lD-75 

SERIAL NUlfdER 

16. PUBLICATIONS: 

Fite, Gt, and Wade, H,: The Contribution of Neisser to the 
Establishment of the Hansen Bacillus as the Etiologic Agent of 
Leprosy and the So-Called Hansen-Neisser Controversy. Internat, 
J. Leprosy. 

Habermann, R., ''Williams, F. Jr^, and Fite, G. : Characteristic 
Inclusion Bodies in Viral Diseases of P.:an, Animals, and Birds ^ 
An exhibit. 

Fite, G. : Leprosy, Society, and Hansen's Diseaset Int. J. 
Leprosy, In press. 

No author listed, but originally authored by Gc L^ Fite: 
Conference Report. Progress and Potentials in Leprosy Research* 
Public Health Reports, 71, 993, 19%. 

16, HONORS AND AWARDS: 

None, 



Calendar Year 19^6 
PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1, NI>'iMD-76 



SERIAL KUivBER 
National Institute of 
2, Arthritis a nd Metabolic Diseases 3. Pathology and Histochemistry 

INSTITUTE LA BORA TORY 

U, Hematology $» 

SECTION LOCATION (IF OTHER THAN BETHESDA 

6, Studi es en normal and abnormal human hemoglobins 

PROJECT TITLE 

7. H. A. Itano 

PRINCIPAL INVESTIGATOR 

8, Elizabeth Robinson 

OTHER INVESTIGATORS 

9. IF THIS PROJECT RESEI'JBLES, COflPLEMENTS , OR PARALLELS RESEARCH DONE 
ELSEi-'T-ERE IN THE PUBUC HEALTH SERVICE (iTITHOUT INTERCHANGE OF PER- 
SOroiEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH: 

No parallel research in P, H. S, 

10. PROJECT DESCRIPTION: 

Objectives : To study the physical chemistry, biochemical 
genetics, and clinical significance of the normal 
and abnorm-al human hemoglobins e 

Methods employed : Moving boundary electrophoresis; 

spectrophotometry; kinetic and equilibrium measvire- 
ments with pH meter and spectrophotometer. 

Major find ings: (a) Hyposthenuria in sickle cell disease 
is reversible if sickling cells are replaced by 
normal cells before permanent renal damage has taken 
place. Keitel, Thompson, and Itano 

(b) Intennediate forms of carbonmonoxy- and ferri- 
hemoglobin, namely, molecules with 1, 2, and 3 
hemes oxidized have been demonstrated by moving 



NIjJ-ID-76 'i- 
SERIAL NOT.I3ER 



(Cont'd.) 



boimdary electrophoresis and spectrophotometry, 
Itano and Robinson, 

(c) Mobility differences obtained from (b) have 
been used to estimate the differences in charge 
among the normal and abnormal hemoglobins, Itano 
and Robinson, 

(d) Samples from other laboratories have been 
checked with results obtained here on previous 
samples. Samples containing the rare hemoglobins 
D, G, and J have been received and confirmed, 
Itano and Robinson, 



Form No. ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOMAL INSTITUTES OF HEALTH 
IITOIVIDUAL PROJECT REPORT 



Part B ! Budget Data 



11. i\rDUiD-7 6 _ 
SERIAL WMWB. 



RESEARCH 

REVIEV/ & APPROVAL 

BIOLOGIC STAITOARDS 



12, BUDGET DATA 


• 






ESTIMATED OBLiavTIONS 


MAN YEA IS 


DIRECT 


RED'IBURSEMEUT TOTAL 


PROF OTHER TOTAL 


FY557 

$20,^00 


^i)7,100 ^1,27, 600 


1,00 1,00 2,00 




BUDGETED POSITIONS 


PATIENT DAYS 


PROF 


OTHER TOTAL 




FY557 

1,00 


1.00 2,00 




13. BUDGET A err 


iTITY: 





Dli 



Lj 



ADiilNISTRATION [^| 

PROFESSIOl-IAL & 
TECm«CAL ASSIST- 
ANCE [|3| 



lii. IDEi^lTIFY MY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUIMDS, FACILrTIEo, OR PERSONi\!EL 
FOR THIS PROJECT IN FY 19^7. IF COOPEit'iTING UNIT IS VOTHIN NIH 
INDICATE SERIAL NO(S): 

NIH: Laboratory of Kidney and Electrolyte Metabolism. - H« 
Keitel Children's Hospital, Los Angeles, California - P, 
Sturgeon and Vl, Bergren, 



(Use reverse and additional pages, if necessary) 



Calendar Year 19^6 

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part C: Honors, Awards & Publications l^c NL VMD-76 

SERIAL NUlviBER 

16» LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT 
DURING CALENDAR YEAR 1956: 

Thorup, 0, Ae, Itano, H, A., iJlTieby, M., and Leavell, B. S,; 
Hemoglobin J. Science, 123:889, 19^6, 

Itano, H. A., Bergren, Vf, R,, and Sturge--.n, P, : The abnormal 
human hemoglobins. Medicine, 3^:121-159, 1956. 

Itano, H, A,: The hemoglobins. Annual Review of Biochemistry, 
25:331-318, 1956. 

Itano, H, As, and Robinson, E, Demonstration cf Intermediate 
forms of carbonmonoxy- and ferrihemoglobin by moving boundary 
electrophoresis. J. Am. Chem. Soc, (Submitted for publication 
Novc 2, 1956) 

17. LIST HONORS AND AMRDS TO PERSONNEL RELATING TO THIS PROJECT 
DURING CALENDAR YEAR 1956: 

None. 



Forn Mo. ORP-1 Calendar Year 19^6 

October 1956 Pape 1 

PUBLIC HE/iLTH SERVICE — riATIObl/i INSTITUTES OF HEALTH 

INDIVIDUAL PkO,ECT REPORT 

Part A, Project Description Sheet 1, 



2. N ational ^ Institute of Arthritis an d Metabol ic Di sc;ascg 

"IwstTTute 

3 . Laboratory o f Pharmacology ax\d Toxicolo;^-/ 

Li\BOr(J\TOuY 

h, Pharmac olog:y ^. 

SEGTIOM '" LOCATIOH'Ci'f OTHEii TRTiM 



3ETHESn'0 

6. The physiological function, intermediary metabolism, 
and pathological effects of spermine, sp'crmidine, and 
related amines . 

PROJECT TTtlS 

7. Drs. S. M. Rosenthal, Celia W. Tabor, R. C. Greene 

PRINCIPAL IlWESTIGAfORS ' ' 

3 • Drs. H. Tabor, E. L. Jackson, and L. L. Ashburn (f rom LPH) 
"OTPIER I WESTI GATORS 

9. IF THIS mOJECT RESEMBLES, COMPLEWEiOTS, OR P/JIALLELS 
RESEARCH DOME ELSEWHERE IN THE PUBLIC HEALTH SERVICE, 
WITHOUT IMTERCII/vMGE OF PEI^SOMMEL, FACILITIES OR FUI'IDS, 
IDEOTIFY SUCH RESFARCH: (BY SERIAL NO(S) IF WITHIN i'lM) 

Does not apply 

10. PROJECT DESCRIPTION 

Objectives; Spermine and spermidine have been found 
to be potent nephrotoxic af:ents. Analyses of animal 
tissues, and of plant and bacterial cells, have demonstrated 
a -wide distribution, frequently in high concentration. 
Studies ar3 conducted to elucidate its metabolism, 
physioloi;;ical function, its relation to renal disease, 
to spcrmatozoal physiology, and to bacterial defense 
mechanisms . 

Methods Erripl oycd; (1) The physiological effects 
of the degradation products (with an enzyme purified 
from beef plasma) have been further stiidiod. (2) 
Simplified chromatographic methods have made possible 
the rapid estimation of these amines in tissue extracts, 
body fluids, and in plants and bacteria. (3) Adminis- 
tration to animals by various routes (including in- 
jection into the renal arter-/)j to produce renal changes 



Form No. ORP-1 Calendar Year 1956 

October 191^6 Pa^c 2 

PUBLIC HMLTH SJaiVICE — W/iTIOWAL INSTITUTPJS OF HEALTH 

INDIVIDUAL ITiOJECT REPOIiT 

10. PROJECT Dl'SCKIPTIOM (co nt'd) NL .MD-77 - -^ 

Sat'l/.L ilUllBER" 

and hypertension. (I4) With isotopic labeled (C"*"^ and N^^) 
putrescine, spermidine aiid spermine, the intermediary 
metabolism of these compoiinds can be followed. (5) 
Kutant organisms requiring; tliosc amines for growth arc 
being sought. An A. spiirgill ns mutant (Sneatih) is being 
studied. 

Major Findings: (1) C"'"'^' labulc^d spermine and 
spermidine were proparcdj labeled both in t)ie terminal 
carbon, and in the central (putr^jscinc) carbon (mth 
Dr. H. Tabor and Dr. E. L. Jac'rson). (2) With these 
preparations degradation by tissue homogenates, 
particularly in liver, was shown. Spermidine and 
putrcscino ^^^cre the cldef products from spermine. This 
demonstrates putrescinc as a normal constituent of 
tissues. (3) Degradation of these C-'-'* compounds by the 
purified enzyme (amine oxidase) indicate random split- 
ting, with long chained amino aldehydes, putrescinc, 
ammonia, and H2O2 being formed, (h) With C^'i - N^^ 
label, putroEcine has been shown to be incorporated as 
a unit in the biosynthesis of spermine and spermidine. 
Studies id.th G^^ labeled methionine indicate that this 
is the chief donor of the 3 carbon terminal moiety of 
the molecule (Dr. Greene). (5) An extended study of 
the polyamine content of bacterial and plant cells re- 
vealed high concentrations in most of them. The re- 
lative amounts of putrcscine, spermine or spermidine 
are influenced by the pH during growth. (6) Spermidine 
has been shown to exist in the cell as an acid" labile 
conjugate, the nature of which has not been established. 
(7) Further pharmacological studies of spermine re- 
vealed a diuretic action in amounts that produce no 
demonstrable toxic effects. Also spermine antagonizes 
the anticoagulant action of heparin upon heparinizcd 
blood. 

Signi.ficanc> j to MIAMlJ Resea rch; Spermine and 
spermidine are widely distributed in mammalian and 
plant tissues and no previous work has demonstrated 
any physiological function or relation to diseases. 
It is believed that tliis work will throw light on 
their biochemical significance, and that a causative 
I'elationship may be shoi-ni to some types of human 
renal disease 



Form No. OllP-l Calendar Year 193>'6 

October 1956 Paj-u 3 

PUBLIC HMLTH SErL\n:CE — W.TIOMAL INSTITUTES 01'' HET^LTH 

lilDIVIDUAL PROJECT REPORT 

10. PP.OJroT DESCRIPTIO rT (cont'd) N KMD-77 ' ^ 

- — SFTtlAL immEil ~ 

Proposed Course of P roject; (1) Study the- 
characteristics of chronic lesions produced by spermine 
and spcriiiidinG, and their relation to human d;lseases. 
(2) To establish the relationship of the pathological 
effects to the acute renal lesions which result froi.i 
shock and eclarfipsia. (3) To establish the signifi- 
cance of the spermatocidal and bacteriocidal actions 
in terms of spermatozoal physiology and of bacterial 
defense mechanisms, (I4) Further to elucidate thi^ 
intcriaediary metabolism of these amines by the use of 
mutant bacteria and isotcpic labeling. (5) Phariaa- 
cologica]. effects^ including renal function studies, 
folloidnp spermine administration, (6) To determine 
the state in which these amines exist in bound form 
in thu cells, and the factors concerned in their 
liberation. 



Form No, ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - MATinmL IIJSTITUTES OF HE/-.LTH 
IivrDIVinUAL PUUJRCT REPORT 



Part B: Budget Data 



11. NIAfID-77 



SERIAL iWIiBF.R 



RESEARCH 

REVIEl'l & APPROVAL 

BIOLOGIC STANDARDS 



12, BUDGET DATA: 










ESTIMATED LIG/VT: 


[ONS 




MAN YEARS 


DIRECT 


REIliBURSBIEKiT 


TOTAL 


PROF 


OTHER TOTAL 


FY'57 










$1;0,300 


$1^,200 
BUDGETED POSITIONS 


tf55|500 


2.Q3 


1.66 h,h9 
PATIEir DAYS 


PROF 


OTHER 


TOTAL 




FYt57 










2.83 


2.66 


^,h9 






13. BUDGET ACTIVITY: 









a 



AmiNISTRATION 

PROFESSIONAL & 
TECHinCAL Assis-: 
ANCE 



iZI 



□ 



lli. IDENTIFY ANY C00P::RATING UNITS OF THE PUBLIC lEALTH S:;RVICE, OR 
OTHER ORGAWIZaTIOITS, PROVIDING FUiTOG, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPER/.TING UIOT IS VJITHIN NIH 
IiroiCATE SERRL NO(S): 



(Use reverse and additional pages, if necessary) 



Form OUP-1 Calendar Yuar 19^56 

October 1956 Page 5 

PUBLIC HE/iLTH SERVICE — NATIOI'AL INSTITUTES OF HFjILTH 

IWniVIDU/iL HtOJECT REPORT 

Part C. Honors, Awards and Riblications 15. NIAIffl- 7Z 



SFlJAL i.rUI'IBHt 



16. LIST PUBIIC'TIOIIS OTHETi THAN ABSTR/iGTS FToDH THIS PROJECT 
DUPiING CALENDAR YF^'di 1956: 

Rosenthal, S. M. £ind Tabor, Cclia W, "The pharmacology 
of spermine and spermidine. DistirLbution and excretion." 
J. Pharm. & Exp. Thcrap., 1956:16. 

Tabor, Cclia W. and Rosenthal, S. M. "The pharmacology 
of spermine and spermidine. Some effects on animals 
and bacteria." J. Pharm, & Exp. Therap., 1956:16. 

17. LIST HO-^IORS AND AWARDS TO PERSONNEL REUTIIIG TO THIS 
PROJECT DURING CALEIIDAR YEAR 1956: 

None 



Form No. ORP-1 Calendar Year 1956 

October 1956 Page 1 

PUBLIC HEALTH SERVICE — MTIOIWX INSTITUTES OF HEALTH 

INDIVIDUAL I'ROJECT REPORT 



Part A. Project Description Sheet 1. TJiMMD-Tfl 

silHIAL i'iulIBER 

2. TIational Institute of Arthritis and Metabolic Diseases 
inSTITUTE 

3. Laborator:v'- of Pharnacoloa^ and Toxicology 

L/xBOMTORY 
k. Pharmacology 5. Bethesda, lid, and Lima, Peru 

SECTIO:i ■^~ LOCATIOIJ (IF OTHER THAN 

BETHESDA) 

6. M echanisms aiid therapy of trauraatic shock 
PROJECT TITLE 

7 . Drs. R. Carl liillican, Kehl Markley, and S. M. Rosenthal 
PRIIICIPAL IIIVESTIGATORS 

8. Mr. John Rust 



OTHEF: iriVESTIGATOR 



9. IF THIS PROJECT RESEMBLES, C0MPLEI^1EI>!TS, OR PARALLELS 
RESEilRCH DONE ELSH'JHERE IN THE ETTBLIC HEALTH SERVICE, 
WITHOUT IIITERCHAMGE OF PSRSOHlIEl, FACILITIES OR FUNDS, 
IDEITIFY SUCH RESK'JICH: (BY SEKLXL NO(S) IF WITHIil NIH) 

Does not apply 

10. PROJECT DESCRIPTION 

Objectives ; (1) Study of effectiveness and mechanisms 
of action of certain drugs against shock. (2) Cause and 
therapy of late deaths in bums. (3) Evaluation of 
effective chemotherapy of fatal Pseudomonas infection in 
cortisone-treated mice, (k) Clinical evaluation in 
bum shock: (a) effectiveness of large volumes of 
saline solutions | (b) study of Pseudomonas iiif action 
following bumsj (c) studj'' of the effects on blood 
volume, plasma proteins and electrolj-tes, and on 
renal function and excretion of large volumes of 
sodium solutions administered to normal subj ects; 
(d) fliiid and electrolyte changes in natives at high 
altitude, follomng major surgery. 



Form No. ORP-1 Calendar Year 19^6 

October 1?56 Paf^e 



o 



PUBLIC HE/iLTH SERVICE — MTIOIIAL IlISTITUTES OF HaUTH 

lilDIVIDUAL PROJECT REPORT 

10, PROJECT DliSCHIPTIOII (cont'd) MI /J^lD-78 

" — " SeRL'J. I>IUri3ER 

Methods Ernployed; (1) Standardized traumatic shock 
produced in mice by application of tourniquets to hind 
legs or dipping animals in hot water. Mortality studies, 
analyses of fluid distribution and measurements of 
bleeding volume, hematocrit and plasma protein levels 
employed to study mechanisms for effectiveness of 
drug therapy in treatment of shock, (2) Cortisone 
pretreatment used to increase the susceptibility of 
mice to Pseudomonas aei-uginosa infection permitting 
the use of small infective doses of organisms and 
producing infections fatal 2 to 7 days after inocu- 
lation. Mortality employed to study effectiveness 
of aiitibiotics and other antibacterial agents in 
treatment of Pseudomonas infection. (3) The clinical 
study is divided into two parts. On an alternate case 
basis J (a) large volumes of saline solutions chiefly 
by mouth are compared with plasma therapy plus glucose 
solutions (b) large volumes of saline are compared 
mth similar therapy supplemented by adequate plasma. 
The clinical studj' includes mortality, fluid and 
electroljdie balances, hematological and hemodynamic 
measurements, and rate of gastrointestinal absorption 
in shoe!:. 

Major Findings; Ex perimental shock. (1) Promazine 
(a phenothiazine derivative) given prophylactically has 
a therapeutic effect in tourriiquet shock. Similar 
findings observed for chlorpromazine and chlorpromazine 
sulfoxide. Other phenothiazine derivatives with 
predominaiitly antihistaminic properties (plienergan 
and pyrathiazine ) have no therapeutic effect. (2) With 
chlorpromazine treatment there occurs; (a) marked re- 
duction in body temperature (b) decrease in the 
eweUing in the area of injury and (c) an increase in 
the bleeding volume and lowering of plasma proteins 
ai:id hematocrit (indicating a movement of extravascular 
fluid into the circulation). Ml these positive find- 
ings may be related to therapeutic effect. (3) In 
Pseudomonas aeruginosa in.fection in cortisone-treated 
mice various antibiotics were compared as to their 
effects on survival. Certain of them, polymyxin B, 
clxLoramphenicol, and oxytetracycline, are effective 
but at maximally tolerated doses. Combined therapy 
was additive but not synergistic. 



Form No. ORP-1 Calendar Year 1956 

October 19?6 Pa?^e 3 

PUBLIC HE/VLTH SERVICE — mTIOII/J. INSTITUTES OF HE.\LTH 

IITOIVIDU/J. PROJECT REPORT 

10. PROJECT DESCRIFTIOH (cont'd) NIjJfiD-TB :■ 

SERIAL mJI-3ER 

Clinical Study (Lima Project). The effective- 
ness of large volumes of saline given orally and 
parenterally was demonstrated in a series of 100 cases, 
using 100 alternate cases treated -with plasma (Ev-'ans 
Formula) for comparison. The mortality curves were 
similar. 

As a part of this study several related problems 
have been completed and are in press or being submit- 
ted: 

1. A detailed studj' of hemodynairiic changes, of 
blood and tu'-ine, fluid and electrolytes, of 17-keto- 
steroid excretion and eosinophile changes following 
burns, comparing results in the saline and in the 
colloid groups. 

2. The effects of large voliimes of saline given 
orally to normal volunteers, were studied. In contrast 
to reports in the literature the majority of adminis- 
tered fluid and sodium was excreted within 2I4 hours. 
Glomerular filtration rate increased 32 per cent while 
renal blood flow did not change. 

3. A high percentage of burned patients died 
■within 3 weeks and the cause was demonstrated to be 

a Pseudomonas septicemia, characterised by an \iniisual 
sld.n lesion and by multiple abscesses. 

h. Natives at high altitude have a compensatory 
polycj.-bhemia. Their ability to undergo stress, and 
the fluid and electrolyte changes folloT-ang stress 
(major operations) were studied. The hemodynamic 
and electrolyte changes were similar to those observed 
at sea level. 

Proposed Course of Project; (1) Continued study of 
the meclianism of action and effectiveness of drugs in 
traumatic shock. (2) Further experimental and clinical 
work on causes and treatment of delayed death follovjing 
extensive burns. (3) Studies on the possible role of 
the bacterial factor in traumatic shock. (I4) Peru 
Project . The evaluation of plasma therapy in burn 
shock is being made by comparing the effectiveness of 
large volunies of saline supplemented by adequate 
plasma therapy in an alternate group. 



Form No. ORP-1 Calendar Tear 1956 

October 1?56 Page h 

PUBLIC HSILTH SERVICE — N^lTIOrtlL IHSTITUTES OF IffiALTH 

IIJDIVIDUAL PROJECT RETORT 



10. PR OJECT DESCRIFTIOII (cont'd) NIAMD-78 '/ 
■~~~ "SERL'vL lyUl-IBER 

A studj'- of the causes and treatment of 
Pseudomonas infections in these burn cases is being 
set up. This will include an application of the 
laboratory findings of the various results of 
therapy in animals viith this infection (cortisone- 
treated mice). A conplete bacteriological and 
immunological study -vri-ll supplement these trials, 
which iri.ll be made on aji alternate case basis. The 
relation of excessive steroid secretion to increased 
susceptibility to infection will bo studied in 
these human cases of burns. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SEiWICE - NATIOML IKBTITUTES OF HEALTH 
liTOIVIDUAL PROJEC? R^J^ORT 



part B: Budget Data 



11. NIAllD-78 



RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STANDAPODS 









SiiRIAL mmBER 


12, BirOGET nA' 


:a; 








ESTIf'ATED OBLIdlTIONS 




MAN YEARS 


DIRECT 


R'^Br'URSEI'EMT 


TOTAL 


PROF OTHER TOTAL 


FY»57 








$Il9,0C0 


$19,200 


$68,200 


3.50 2.67 6.17 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTIER 


TOTAL 




FY'57 






h.^o 


2.67 


7.17 




13. HJDGET ACTIVITY: 







GO 

a 



ADi'IINISTRATION 

PROFESSIONAL & 
TECHNICAL ASSIST- 
ANCE 



□ 



U 



m, IDEiriFY ANY COOPr.RATING UNITS OF THE PUBLIC HE^MTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUiTOS, FACILITIES, OR PERSONNEL 
FOR T^^IS .PROJECT IN FY 1957. IF COOPEii/.TING UNIT IS WITHIN HIH 
liroiCATE SERIAL NO(S): 

Research Grant - NIH (Lima, Peru Project) "G-3123. 
,;'C5 currently active. 



(Use reverse and additional pages, if necessary) 



Form No. ORP-1 Calendar Year 1956 

October 15>56 Page 6 

PUBLIC HBUTH SER/ICE — KATIOWlL IIISTITUTES OF HE.'VLTH 

iroiVIDUilL PROJECT REPORT 

Part C. Honors, Amrds and l^iblications 15. NKMD-78 

SERL^L ilUi'IBHl 

16. LIST PUBLICATIONS OTIffiR THAN ABSTRi\CTS FRDH THIS 
PROJECT DURING Ci'^XEI'MR YET.R 19^6: 

Hillica:!, R. Carl, Rust, John R., Verder, Elizabeth, 
and Rosenthal, Sanf ord M. "Experimental chemotherapy 
of Pseudomonas infection I. Production of fatal in- 
fections in cortisone-treated mice." Antibiotics 
Annual, 193'6-57. In press. 

Narvaes, E. and Mariaey, Kehl. "Post -operative water 
and electrolyte changes in natives of high altitudes." 
J. Applied Physiol. In press. 

Markley, Kehl, Bocanegra, Manuel, Morales, G., and 
Chiappori, M. "Oral sodium loading in non-hydratod 
normal individuals." J. Clin. Investigation. In 
press . 

Harklej'-, Kehl, Gurmendi, G., Chavez, P. M., and Baaan, 
A. "Fatal Pseudomonas septicemias in burned patients." 
Arch. Surg. In press. 

Markley, Kehl, Bocanegra, M., Bazan, A., Tenple, R., 
Chiappori, M., Tlorales, G., and Carrion, A. "Clinical 
evaluation of saline solution therapy in burn shock." 
J. A. M. A. 1956, I6l:lli65. 

17. LIST HOIDRS AND AWiuRDS TO PERSOMEL RELilTING TO THIS 
PROJECT DURING CALMTDilR YSIR 1956: 

None. 



Forra No. ORP-1 Calendar Year 19^6 

October 1956 Pan;o 1 

PUBLIC HBILTH SERVICE — I-MIOlFiL IHSTITUTES OF HE/.LTH 

IlffilVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. HL'u€)-79 

SE[tt\L iIU14BER 

2 . jyT ational Institute of Arthritis and Metabolic Diseases 

Tmstit^jte 

3 . Laborator?/ of Pharmacology and ToyJcology 
L'lBOR-ITORY 

k. Pharmacology 5. . 

SBCTIo'R LOCATION (IE OTHER TJL\H BETtTSDA) 

6. 1. Studies on the chcmistr:^ of m-tyrosine, especially 
as related to its iodo and nitro derivatives. The 
synthesis of some new meta diphenyl ethers having iodo 
and nitro substituents. 

2. The sjmthesis of C^-labeled sperrdne and C ^- 

labeled spermidine. 

PROJECT TITLES 

7. E. L. Jackson 8, None 



PRINCIPAL INVESTiaiTOR OTHER IrlVESTICLlTORS 

9. IF THIS PROJECT RESSyBLES, CDMPLEMEI>ITS, OR Pi'JljULELS 
RESETJICH DONE ELSS;#IERE IN THE PUBLIC HE/.LTH SERVICE, 
WITHOUT INTERCHTiNGE OF PIESONNEL, FACILITIES OR RUmS, 
IDEIOTIIT SUCH RESB\RCH: (BY SERIAL NO(S) IF WITHIN NIH) 

Does not apply 

10. PROJECT DESCRIPTIO N 

O bjectives; Project 1. Prior to the studies of 
this project no nitro derivative of m-tjrrosine was 
knowi:! and only one iodo derivative oT unknoxm structure 
had been discovered. Since iodo and nitro derivatives 
of L-tj'-rosine are biologically important compoundSj 
fundamental studies are being conducted on the synthesis 
and strtacture of such derivatives of m- tyrosine. These 
compounds provide intermediates for tEc synthesis of 
analogs of the L-tyrosine derivatives. The new meta 
diphenyl ethers can be used for the synthesis of nitro 
and iodo meta diphenyl ethers having variable side 
chains. 

Project 2. The work on this project 
in 1956 T-ras limited to finishing some details and the 
preparation of a manuscript on a new method for the 



Form No. ORP-1 Calendar Year 1956 

October 1956 Pape 2 

PUBLIC HfilLTH SEKVICE — W.TIOMAL INSTITUTES OF HE,1LTH 

INDIVIDUAL PROJECT REPORT 

10. mOJECT DESCRIPTION (cont'd ) NI^4D-79 ■- 

SERIAL WUiiBER 

synthesis of spermidine. This manuscript has been 
accepted for publication by the J. Org. Cham. 

Ilajor Findings ; The nitration of ri- tyrosine has 
yielded three isomers of B-(3-hydrQxydanitrophonyl)- 
D,L-alanine from which were prepared the three cor- 
responding p-(3 -nethoxydinitrophenyl ) -D, L-alanines . 
The results have established the orientation of the 
nitro groups in two of the isomers of p-(3-hydroxj'- 
dinitrophenyl)-D,L-alanine formed in the nitration 
of m- tyrosine. From the rules of orientation the 
third dinitro-ru-tyrosine is probably .G-(3-hydroxy- 
2j[i-dinitrophenyl)-DjL-alanine. 

The synthesis of some nex-r meta diphenyl ethers was 
started. 2, 6-Di]iitro>-3-(p-methoxyphenoxy) -benzyl 
alcohol was prepared by reduction of the aldehyde 
group of 2j6-dinitro-3-(p-methoxyphenoxy)-benzaldehyde. 
A new crystalline form oT 3-hydrox3'--2,6-dinitro- 
benzaldehyde has been obtained. 

Significance to I'lIjUm Research; The new nitro 
derivatives of m-tj'-rosine provide intermediates for 
the synthesis of the corresponding iodo derivatives 
of m-tjTOsine and for the synthesis of phenyl ethers 
of These nitro and iodo derivatives of m-tyrosine. 
The new meta diphenyl ethers arc intermediates that 
can be used for the synthesis of nitro and iodo meta 
diphenyl ethers ha^/ing variable side chains. This 
type of diphenyl ether is of interest in thyroid studies, 
especially as related to the problem of thjToxine-like 
actj.vity and chemical structure. Some of the derivatives 
of m-tyrosine arc of interest in certain types of enzyme 
study, such as the substrate specificity of tyrosinase 
and mammalian EO PA -decarboxylase. 

Proposed Course of Pix),-ject; (1) From 2,6-dinitro- 
3 - ( p-methox:,TDhenoxy ) -b en zaldchyde it is planned to 
attempt the synthesis of such compounds as 2,6-diiodo- 
3-(£-hydrox3'--3tj5t_diiodophenox3'-)-benzoic acid and 
p-[3-P-hydrox3^-3 • , 5 • -diiodophenoxy )-2, 6-diiodophenyl] - 
D,L-aIanine. (2) To study the oxidation of M-acetyl-8- 
(3-hydroxy-ii,6-diiodophcnyl)-D,L-alanine, since the 
oxidation of N-acetyl-3,5-diiodotjTrosine is known to 
yield N-acetyl -thyroxine. 



Form No, ORP-1 
October 1956 
(Attachiaent l) 



PUBLIC HEALTH SrJtVICE - NATIOMAL INSTITITES OF lEALTH 
Ii©IVIDUAL PHOJEC? REPORT 



part Bj Budget Data 



11. NIAMD-79 









SOiLlL iWHDiJl 


12, BUDGET DATA: 








ESTIiATED OBLIGATIOi 


IS 


MAN YEARS 


DIRECT 


REIiiBURSEIlLNT 


TOTAL 


PROF OTIER TOTAL 


IT '57 








;(f8,700 


(f3,000 


Ul,700 


0,67 - 0.67 




EUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHliR 


TOTAL 




¥i^$l 








0,67 


- 


0.6? 




13. BUDGET A 


3TIVITY: 







RESmRCH 

REViai & APPROVAL 

BIOLOGIC STAiTOAilDS 



}~n ADIHNISTRATION 

I I PROFESS lOM/.L !k 

TECHNICAL ASSIST- 
Pj ANCE 



□ 



□ 



lU. IDENTIFY ANY OIOPEIW.TIWG UNITS OF THE PUBLIC HEALTH S...RVICi], OR 
OTHER ORGANIZATIONS, P'"?.OVIDING FUiOS, FACILITIE.5, OR PERSOfFi^L 
FOR T IS PROJECT IN FY 19^7. IF COOPERi.TING UNIT IS ^ffTHIN NIH 
INDICATE SERIAL NO(S); 



(Use reverse and additional pages, if necessary) 



Form No. ORP-1 Calendar Year 19^6 

October 19^6 Page U 

PUBLIC HB'JLTII SHIVICE — MTIOIIU INSTITUTES OF HE.\LTH 

IiroiVIDUAL PROJECT REPORT 

Part C. Honors, Avrards and PuW.ications l5. NLiMD-79 

SERLYL ;iUi-IBER 

16. LIST PUBLICilTIONS OTHER THAN /3STRACTS FROM THIS 
PROJECT DURING C.UEI^ia\R YEAR 1956: 

Jackson, E. L. "The sjmthesis of p-(3-Hethox3^-2,6- 
dinitrophcnyl)-D,L-alanino and P-(3-Mcthoj:3'-li,6- 
dinltrophcnia)-D,L-alanine. Proof of the structures 
of two isoiTieric dinitro-m-tyrosines". J. An. Chem. 
Soc, 79 (1957). In press. 

Jackson, E, L. "The preparation of spca'raidine and 
riionoacctylspornidine". J. Ore. Chcin., 21:1956. 

17. LIST HOIIORS AIID AW/lRDS TO PERSONrlEL RELATING TO THIS 
PROJECT DURING CILEI'ID/Jl YEVR 1956: 

None . 



Porn Mo. ORP-1 Calendar Year 1956 

October 195'6 Page 1 

PUBLIC HMLTH SERVICE~WATIOMi\L INSTITUTES OF HE/O^TH 

IMDIVIDUAL PROJECT REPORT 



Part A. Project Description Sheet 1. NL J4D-80 

Serl"il nji'im 

2. National Institute of Arthritis and Metabolic Diseases 
INSTITUTE 

3. Laboratoi-y of Pharmacology and Toxicology 

"lTboratory 

i4. Pharmacolog y 5. 

SECTION LOCATION (IF OTHER THIN BETHESai) 

6. The Chemotherapy of Mouse Leprosy 
PROJECT TITLE 

7. Dr. Y. T. Chang 8. None 



PRINCIPAL INVESTIdlTOR OTHER IIWESTIGilTORS 

9. IF THIS PROJECT RESHIBLES, COMPLEMENTS, OR PilRALLELS 
RESE^JiCH DONE ELSEWHERE IN THE PUBLIC HEilLTH SERVICE, 
WITHOUT INTmCH/iNGE OF PERSONNEL, FACILITIES OR FUI\IDS, 
IDENTIFY SUCH RESB^iRCH: (BY SERIAL IIO(S) IF WITHIN 
NIK) 

Does not apply 

10. PROJECT DESCRIPTION 



Objectives ; The toxicology and evaluation of 
therapeutic effectiveness of drugs in mouse leprosy. 
The tissue culture of intracellular parasites. 

Major Findings ; Paromomycin and two long chain 
keto fatty acids, i.e., 9,12-diketo-lO-octadecenoic acid 
and 12-keto-lO-octadecenoic acid, showed suppressive 
activity in mouse leprosy. The short-term (3 weeks) 
screening technique, employing mesenteric spreads and 
acid fast counts of liver and spleen of the mouse, 
revealed good results with the more active compounds, 
but poor responses to the less active compounds. Some 
groxith of Mycobacterium leprae murium was found in the 
tissue ciolture of monocytes, obtained from peritoneal 
exudate of leprous mice. 



Form No. ORP-1 Calendar Year 19?6 

October 1956 Page 2 

public he/llth service— nj^tioiul institutes of hetilth 
iijdividu;ll project report 

10. PRQJIDT DESCRIFTIOI'T (cont'd) MKMD-80 ' - 

SERUtL MTOffiER ' 

Significance to III/iMD Research ; The leprosy studies 
are earned out in cooperation with The American Leprosy 
Foundation (Dr. Chang is on a FelloxJship from them). 
The results are applied to their clinical evaluation 
studies. 

Proposed Course of Project; Continuation of 
evaluation of drugs in mouse leprosy, using both 
long-term and short-term techniques. Continuation 
in the study of tissue culture of intracellular 
parasites. Bioassay of diaminopimelic acid, a 
metabolite of mycobacteria, in leprous tissues and 
in tissue cultures. 



Form No. ORP-1 
October 19^6 
(Attaclunent I) 



PUBLIC HEALTH SERVICE - mTIOmL INSTITUTES OF HEALTH 
i;nDIVIl-)U/lL PROJECT REPORT 



Part B: Budget Data 



11. NIAMD-80 







SERML 


IWMBER 


12, BUDG.h.T DATA 










ESTIMTED OBLIGATIONS 




I IAN YE/.RS 


DIRECT 


REIffBURSEMErj TOTAL 


PROF OTHER TOTAL 


FY»57 








$3,800 


#1,000 $ii,800 




0,67 0.67 




BUDGETED POSITIONS 




PATIEIC D/lYS 


PROF 


OTHER TOTAL 




FYt57 








"* 


0,67 0.67 







13. BUDGET ACTIVITY 
RESEARCH 

REVIEVJ & APPROVAL 
BIOLOGIC STANDARDS 






ADIEENISTRATION 

PROFESSIONAL & 
TECfttllCAL ASSIST- 
ANCE 



□ 



□ 



II4. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HE/\LTH SERVICE, OR 
OTHF^R ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOrffEL 
FOR THIS PROJECT IN FY 193'7. IF COOPERATING UNIT IS i-.lTinN NIH 
INDICATE ST.RIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Form Mo. ORP-1 Calendar Year 1956 

October 1956 P^c ^ 

PUBLIC HEALTH SERVICE — MTIOr'AL INSTITUTES OF HEALTH 

II\!DIVIDUAL PROJECT REPORT 

Part C. Honors, Awards and Publications 15. NL'J'lD-80 

SERIAL IIUIIBER 

16. LIST PUBLIG.\TIOMS OTHER TEI^J ABSTR.\CTS PTOH THIS PROJECT 
DURING CiiLEl-!DAR YEAR 1956: 

Mchlcr, Alan H. and Chang, Y. T. "Biological reactions 
of fluoroacetylcholine." Arch. Biochem. & Biophys. 
1956, 62:293-298. 

Chang, Y. T. "Chemotherapy of mi^rrine leprosy. V. The 
effects of various combinations of Ujli'-diaminodiphenyl 
sulfone (DDS), streptonycin and isoniazid (isonicotinic 
acid hydrazide) on mouse leprosy." Internat. J. Leprosy. 
In press. 

Chang, Y. T. "Chemotherapy of murine leprosy. VI. 
The effects of isonicotinylhydrazone of 2-carboxy- 
methoxy-3-methoxybenzaldehyde (Compound 373) and 
isonicotinylhydrazone of 2-carboxymethoxybcnzaldehyde 
(Conpound 37?) on mouse leprosy." Internat. J. 
Leprosy. In press. 

Chang, Y. T. "Chemotherapy of murine leprosy. VII. 
The effect of cycloserine (Seromycin) on mouse leprosy." 
Internat. J. Leprosy. In press. 

17. LIST HONORS AflD AWARDS TO PERSOFrlEL RELATING TO THIS 
PROJECT DURING C/iLELmJl YE.R 1956: 

None 



Porn No. ORP-1 Calendar Year 13^6 

October 1956 Page 1 

PUBLIC HEALTH SERVICE — MATIOML IHSTITUTES OF HEALTFI 
II\IDIVIDUAL PliDJECT REPORT 



Part A. Project Description Sheet 1, NLJ4D-8 1 

SERIAL fIU14BER 

2. Mational In stit ute of Arthritis an d Metabolic Diseases 
liJStlTUTE 

3 . Labor atory of Pharmac ology and^ Toxicolory 

LABOIiATORY 

h. Pharmacolop;y 5. _^__^ 

SECTTOM ■" LOCATIOifTiF OTHER THAN BETHESDA) 

6. Metabolic processes drug action and physical chemistry 
related to electrolyte distribution and bioelectrical 
phenomena as exemplified by nerve . 

PROJECT TifLE 

7. Dr. A. M. Shanes 8. Mr. M. D. Herman and 

PRINCIPAL INVESTIGATOR Dr. C. Paul Bianchi 

(NIH F ellow) 

OTHER INVESTIGATORS 
9. IF THIS PROJECT RESEMBLES, COMPLEMENTS, OR PARALLES 
RESEARCH DOrlE ELSEWHERE IN THE PUBLIC HEALTH SERVICE 
(WITHOUT INTERCIiANCE OF PERSONNEL FACILITIES OR FUI^IDS), 
IDEIWIFY SUCH RESEARCH: (BY SERIAL 1\!0(S) IF VffTHIN NIH). 

Does not apply 

10. PROJECT DESCRIPTION 

O bjectives : To determine the concentrations and 
rates of entry and exit of sodium, potassium, and chloride 
in nerve - measured by flaiae photometry and radioisotope 
techniques - as affected by metabolic inhibition, meta- 
bolites, dru s, and ions. Modifications of the bio- 
electrical characteristics under these conditions are 
also being followed to determine their possible relation 
to the physiolo,',ical ions. 

Methods Enp loyed; Desheathed sciatic nerves of the 
toad, which our studies have demonstrated to be remarkably 
stable in vitro for Ion:; periods of time, are exposed to 
experimental conditions for varying intervals of time, 
usually in the presence of radioisotopes or after the 
radioisotopes have been taken up by the tissue. The rate 
of uptalce or rate of loss of the isotopes is compared 
TO.th controls to evaluate experimental effects. The Beckman 
flame spectrophotometer provides data on the sodium and 



Form No. ORP-1 Calendar Year 19136 

October 193'6 Page 2 

PUBLIC HEALTH SERVICE — HA.TIONAL INSTITUTES OF HBULTH 

IflDIVIDUAL PROJECT REPORT 



10. PROJE CT DESCRIPTIOM (cont]_d) NLvMD-8l >- 
SERIAL NUMBER 

potassium content of the tissue. Restin- and action 
potentials are I'olloxired potentiometrically and Vjy cathode 
ray oscillO',;raphy usin •• specially desi'-'ned circulation 
units that minimize changes in potassium concentration 
of the interstitial fluid which would occur under our 
experimental conditions. 

Major Findings: Estimates of the extracellular 
space of our preparations based on the rapid component 
• of k'^^ _ f;3'? and K - Na exchanp:G are larger than those 
obtained by equilibration with trace quantities of C^'^ 
labeled sucrose. 

Metabolic inhibition (usually anoxia combined with 
iodoacetate treatment) causes (a) an increase in 
potassium outflux of about ^0%, (b) a decrease in 
potassium infliu:, (c) no chan,r;e in either sodium or 
chloride outflux, and (d) a ^0% increase in sodium in- 
flux. 

Cocaine reduces potassium influx and outflux. Under 
aerobic conditions the effect is small but significant. 
During anoxia, which reduces potassium inflioc to about 
1/3 - l/).i and raises outflux about S0%, the effectiveness 
of the anaesthetic is proportionately much greater on 
both fluxes. 

Cocaine causes a sli[;ht increase in membrane 
potential which may be transitory in uninhibited prepa- 
rations. The drur^ greatly slows the decline of the 
membrane potential which normally'' occurs in the absence 
of oxyg-en and does not interfere with the post-anoxic 
recovery of the potential which follows upon return of 
the nerves to oxygen. 

Fieduction of the sodium concentration of the 
medium approximately halves potassium influx under aerobic 
conditions. If the nerves are previously inhibited by 
lack of oxyi<cn (usually combined with iodoacetate poison- 
inr;), the only effect of decreasing the sodium content 
of the medium is to slightly increa se the potassium in- 
flux, an action which is a trifli-' more marked vriicn 
sucrose rather than choline chloride replaces sodium 
chloride in the milieu. 



Form Mo. ORP-1 Calendar Year 1956 

October 19^6 Pago 3 

PUBLIC HEALTH SERVICE — MTIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

10. PROJECT DESCRIPTION (cont'd ) NIAMD-81 ' ^ 

" ^ ~ SERIAL NUMBER 

Si:' -: ni.ficance to H I MD Research ; Our studies of the 
unidirectional fluxes demonstrate that metabolic in- 
tei'-feroncG induces potassium leakage from nerve fibers 
by concomitantly reducing the rate of uptake and 
accelerating the rate of outward movement of this ion. 
These data, together mth those on sodium and chloride, 
suggest that nortaally about 3A of potassium entry 
occurs in exchange with intracellular sodium as a 
consequence of active transport (i.e., ionic movement 
against electrochemical gradients brought about by 
metabolic viork) and thereby balances the inward leak 
of sodium and outward leak of potassium. Metabolic 
inhibition suppresses active transport of sodium and 
potassium and increases loakiness to both sodium and 
potassium. Data in the literature for other cells, 
although not as complete as desirable, suggest the same 
processes are operative in other tissues, e.g., muscles 
and red cells. 

Our findings with cocaine suggest it reduces leakiness 
to the ions without marked action on active transport. 
Lowered sodium, on the other hand, appears to act like 
metabolic inhibition in suppressing active transport; 
this is consistent with the respiratory depression 
known to occur in low sodium. 

These studies therefore direct attention to 2 
distinct effects of metabolic derangement, namely, 
depression of active transport of ions and increased 
permeability to ions. The latter is subject to some 
measure of control with cocaine, and past experience 
sug-osts that "stabilizers" in general (e.g., local 
anaesthetics, antihistartiinics) could slow down or delay 
cellulai" malfiijactionin^ resulting from ionic imbalance. 
These studies also suggest the desirability of ex- 
ploring the possibility that physiological substances 
such as hormones and other drugs may act through their 
effects directly on ionic permeability, rather than or 
in addition to action on cellular enzymes. Effects 
might then be secondarily through alterations of 
membrane potential, of metabolic reactions dependent 
on the ionic content of the cells, or of rates of entry 
and exit of metabolites. 



Form Mo, ORP-1 Calendar Year 1956 

October 1956 Pare i^ 

PUBLIC HEilLTH SEEiVICE — MTIONAL li'ISTITUTES OF HEALTH 

iroiVIDUAL PROJECT REPORT 

10 . F ROJIiCT DESCRIFTIO M ( cont'd) NL\MD-8l '^ 

SERI/iL IJUMBER 

Pro posed Course of Project: (1) The movement of 
sodium, chloride, and potassium isotopes will continue to 
be follovjed uiider similar 'Experimental conditions sup- 
plemented by further examination of the effects of 
alterations in the ionic milieu, (2) Further cor- 
relations will be attempted between bioelectrical 
phenomena and the chemical findings. Since in the 
squid giant axon the transitory inward and outward 
currents of the active fiber are correlated with sodium 
and potassium movement, we hope to explore the action 
of cocaine on these phenomena. (3) An investigation 
i^^ill be begun on the possible role of carbohydrate 
metabolism in electrolyte distribution and transport, 
particularly as affected by related hormones. (Ii) Ex- 
ploratory studies will be begun on the possible nature 
of drug interaction with cells which alter permeability 
characteristics. 



Form No, ORP-1 
October 1956 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAI'D-ei 



SERIAL NUMBER 



12, BUDGET DATA: 



ESTIIiATED OBLIGATIONS 



FY«57" 



DIREC: 



REIMBURSEtENT 



:OTAL 



^ii21,100 ;|8,100 



$29j200 



BUDGETED PGSI1 


IONS 






PATIENT DAYS 


PROF OTIER 






TOTAL 




Fy'57 










2,00 






2,00 




13. BUDGET ACTIVITY! 










RESEARCH 




|x 


ADMINISTRATION f 


RllViaJ & APPROVAL 
BIOLOGIC STAHDARDS 




LJ 


PROFESSIONAL St 
TECHNICAL ASSIST- 
ANCE [ j 



MAN YEARS 



PROF CTIffiR TOTAL 



2.00 - 



2.00 



lit, IDENTIFY AM COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, CR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONi\IEL 
FOR THIS PROJECT IN FY 19^7. IF COOPERC.TIMG UNIT IS VJITHIN NIH 
INDICATE S"::,RBL NO(S)s 

NIMH 

Rr search Fellowship 



(Use reverse and additional pages, if necessary) 



Form Ko. ORP-1 Calendar Year 1^56 

October 19^6 Page 6 

PUBLIC HEALTH SERVICE — mTIOMAL INSTITUTES OF HEALTH 

INDIVIDUAL HiOJECT REPORT 

Part C. Honors, A^jards and Pablications 15. NL lN TO -SI 

16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT 
DURING CALEI^IDAR YEAR 19^6 1 

Shanes, A. M, After-potentials in nerve. In _ Electro- 
physiolo^ of the Heart . Annals, N, Y, Acad. 'Sci,' tn 
press, 

Shanes, A. M, The distinction between effects of 
metabolic transport and passive transfer of ions. 
Science 12h:72l4, 1956. 

Shanes, A, M, Ionic transfer in a vertebrate nerve. 
University of Wisconsin Press, In press, 

17, LIST HONORS Mm AWARDS TO PERSOTOIEL RELATING TO THE 
PROJECT DURING CAI^ENU'VR YEAR 19^6 » 

Dr, Shanes has been asked to serve as presiding chairman 
and leader of the panel discussion at one of the sessions 
of the Aj'lAS Synposiura 'i Evolution of nervous control from 
primitive organisms to man", (Dec. 29, 1956). 

Dr, Shanes has been invited to prepare a review on the 
role of ions in nerve functioning' and in drug action 
for Pharmacological Reviews, 1957. 

Dr. Shanes served as an observer at the International 
Physiological Congress in Brussels (July 30-August h, 
1956) at the request of the American Physiological 
Society ajid U, S. National Committee of the Inter- 
national Union of Physiological Sciences, 

Dr, Shanes was an invited guest speaker as follows: 
(1) at two seminars in Neurological Sciences, part 
of the Medical Faculty Training Program, Graduate 
School of Medicine, ■ University of Pennsylvania 
(Oct. 29, 30, 1956) J (2) at a seminar in the Department 
of Zoophysiolo,gy, University of Copenhagen (August 20, 
1956); (3) at a seminar in the Department of Pharmacology 
and Physiology, Temple University, Philadelphia (May 17, 
1956 )| (ti) at the conference, "Physiology and Pharmacology 
of Nervous Tissue", organized by and held at the Chemical 
Walfaro Laboratories, Edgewood, Md, (April 13, 1956); (5) 
at the Biophysics Colloquium at Johns Hopkins Univ., 
Baltimore (Feb. 2k, 1956)j (6) at the conference, "Electro- 
phj-^iology of the Heart", organized and held by the I^ Acad. 
Sci, (Feb. 17, 1956); (7)at the Biology Seminar, Princeton 
Univ., N, J. (Jan. 13, 1956); (8) Dr. Shanes is Secretary of 
the Society of General Physiologists, 



■■'■ ' ■■ ■•.1.^'", 



-•y-'fM.-: 



Form No. ORP-1 

October 1956 Calendar Year 1956 



PUBIIC HE/ilTH SERVICE - - Ni,TION;.L INSTITUTES OP' HLi.LTH 
INDIVID Ui.l PROJECT REPORT 

Part I.. Project Description Sheet 1. MIAMD-82 

SERi;JL NUMBER 

2. National Institute of i.rthritiis & lietabolic Diseases 
INSTITUTE 

3. Pharmacology and Toiicology 
L-.BOR/A'ORT 

k. Biochemical Pharriiacology 
SECTIOl! 



5. 



LOCTION (IF OTHER TIUN BETIffiSa.) 



6, Histidine, Hist amine & Related Imidazoles & ^'.mines 
PiiOJECT TITLE 

7. Herbert Tabor 



PRIflCIP.'.i INVE.jTICL'.TOR 

8. B. i.mes, H. Bauer, and L. Vlyngarden 
OTHER IIIVESTIG/.TORS 

9. IF THIS PROJECT RESEMBLES, CCFiPLEMENTS, OR PARi.LLElS 
RESLLRCH DONE ELSEl.'HERL IN THE PUBIIO HLi.lTH SERVICE 
(l-IEiOLT Ii'TERCH.'.NGE OF PERSONi^L, F/.CIXITIES OR FUNDS) 
lELNTIFY SUCH RESE..RCH: (BY SERL'.L NO.(S) IF V.ITHIN NIH). 

The degradation of formiminoglutamic acid (cf . #10) is 
related to formimino glycine degradation in Clostridia 
extracts by Dr. J. Rabinowitz (KIj.MD). 

10. ProJECT DESCRIPTION 

Objoctivcs ; To study the biosynthesis, intermediary 
mebabolism, and pharmacological activity of these com- 
pounds in order to understand better their physiological 
and pathological role. 

Major l^indings ; /.. Further work has been done on 
the nature of the intermediate steps in the metabolism of 
histidine and histandne. j. new synthesis of formimino- 



Form No, ORP-1 

October 1956 Calendar Year 1956 

fJTAMD-82 - l- 



SERIAL NUMBER 



glutajaic and of f ormiminoaspartic acid has been developed; 
these compounds are now available in large amounts. The 
former compound is an intermediate in the degradation of 
histidine, while the latter is now shown to be an inter- 
mediate in the pathway histamine — > imidazoleacetic — > 
I ormiminoaspartic — * formylaspartic acid (in Psuudomonas 
vjith Dr. 0. Hayaishi). 

Particular emphasis has been accorded to the in vitro 
and in vivo metabolism of formimino glutamic acid. This 
compound, which accumulates in folic-deficient animals, is 
degraded by a series of liver enzymes as foDJLows: 

(1) Formimino-glutamic acid + tetrahydrofolic acid — ^ 
Formiminotetrahydrofolic acid + [glutamic acid] 

(2) Formiminotetrahydrofolic acid — »• 5A0""iethenyl- 
tetrahydrofolic acid + [NH3] 

(3) 5,10-mv,thenyl-tcti-ahydrofolic acid — »■ 10-formyl- 
tetrahydrofolic acid 

[These reactions are similar to those found by Dr. J. 
Rabinowitz for formminoglycine metabolism by Clostridia 
extracts,] 

B. Further studies on histidine biosynthesis have 
been carried out by Dr. Ames. 

(1) Imidazoleglycerol phosphate — >• imidazoleacctol 
phosphate, 

(2) Ir.d.dazoleacetol phosphate — r histidinol phosphate. 

(3) Histidinol phosphate — ♦ Histidinol 
(ii) Histidinol — » Histidinal 

(5) Histidinal — ^ Histidine 

During the past year, special attention has been 
dii'ected to reaction 1, a dehydrase, to reaction 2, which 
is a transaminase type enzyme with a pyridoxal phosphate 
cof actor and to rtaction 3, a histidinol phosphate phos- 
phatase. 

C. (See project report of Dr. S. M. Rosenthal for 
the use of isotopicrlly-labeled putrescine.) 



Form No. ORF-1 

October 1956 Calundar Year 1956 

NL'^ID-82-3 



SERIj.L NUl>iBER 



Significance to NL'.^ID Rescarch t Histidinc is an 
essential amino acid, and enters into many important metabolic 
relationships, The C-2 of the imidazole ring is important 
in "one-carbon metabolism," and is closely related to studies 
on the role of folic acid in metabolism. Changes in his- 
tidine metabolism, have been described upon alterations in 
the hormonal pattern, particularly in rheumatoid arthritis. 
Histamine is a powerful pharmacological agent which is 
normally present in tissues. It appears to be very 
important in gastric secretions, neurohormonal reaction to 
stress, certain typos of neural transmission, and in 
allergy and anapliylaxis. Histamine appears to be very 
important in stimulating the adrenal-anterior pituitary 
system. It is anticipated that further studios on its 
metabolic inter-relationship will aid in clarifying hista- 
mine's role in these phenomena. 

Proposed Course of Project : I. Histamine Metabolism 

j'.. Nature of other urinary metabolites after administration 
of G-'-'^ histamine. 

B. Enzymatic and chemical syntheses of imidazoleacetio 
acid riboside. 

C. Enzymatic degradation of imidazoleacetic acid. 

D. Physiological and pharmacological studies as indicated 
during the development of the project. 

E. Histajiiinc metabolism in a mouse mast-cell tumor. 

II. Histidine Metabolism 

Purification of various enzymes involved in conversion 
of histidinc to glutamic acid. Particular emphasis 
will be directed towards one-carbon transfer studies 
(in v itro rxid i n vi vo ) , and the role of various folic 
acid derivatives. I n vivo and in vitro studios with 
formiminoglutamic acid. 

B. Biological syntheses and degradation of carnosinc, 

anserine, ergothioneine, and other related imidazoles. 
Particular emphasis on studies concerned with the 
biosynthesis of histidinc. 



- 3 



Form No, ORP-1 
October 19^6 
(Attaclmient l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11. WIAMD-82 



SERIAL NU11BER 



RESEAIiCH 

REVIEl'J & APPROVAL 

B'OLOGIC STANDARDS 



12, BUDGET DATA 


» 






ESTBttTED OPLIGATIONS 


MAN YEARS 


DIRECT 


REDIBURSEMENT TOTAL 


PROF OTHER TOTAL 


Fy«57 ■ 






$li5,200 


,';;17,100 $62,300 


2,00 2,33 U.33 




BUDGETED POSITIONS 


PATIENT DAYS 


PROF 


■ ■ OTHER TOTAL ^ 




FI«57 






2.00 


3.33 5.33 




13. HJDGET ACTIVITY: 





nn ADMINISTRATION tZD 

[~J PROFESSIONAL & 

TECffNICAL ASSIST- 
□ ANCE □ 



lU. IDEtTTIFY ANY COOPEiykTING UNITS OF TIE PUBLIC HEALTH S7RVICE, CR 
OTHER ORGANIZATIONS, PROVIDING FU1\!DS, FACILITIES, OR PERSONIEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Form No, ORP-1 

October 1956 Calendar Year 1956 



PUBLIC HEALTH SERVICE - - NATI0I\1A1 INSTITLTES OF HEALTH 
IM)IVIDUA1 PROJECT REPORT 

Part C: Honors, Awards & Publications 15. NI/uMD-82 

SERIAL NUMBER 

16. PLBLI CATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
CALENDAR YEAR 1956: 

(1) Tabor, H. The Fate of Histamine in the Body. Ciba 
Foundation Symposium on Histamine. (1956) 

(2) Ames, B. N. and Horecker, B. L. The Biosynthesis 
of Histidines Imidazoleacetol Phosphate Trans- 
aminase. J. Biol. Cham. 220, 113 (1956) 

(3) Bauer, H. and Tabor, H. Cyanomethylimidazole and 
Imidazoleacetic Acid Hydrochloride, Biochem. Prepara- 
tions. (1956) In press. 

ik) Tabor, H. and Rabinowitz, J. Intermediate Steps in 
the Formylation of Tetrahydrofolic Acid by Formimino- 
gLutamic Acid in Rabbit Liver. J. Am. Cham. See. 
78, 5705 (1956) 

(5) Tabor, H. and Rabinowitz, J. C. Formiminoglycine, 
Formimino-L-Aspartic Acid, Formimino-L-Glutamic 
Acid. Biochem. Preparations. (1956) In press, 

(6) Ames, B. N. The Biosynthesis of Histidine: 
L-Histidinol Phosphate Phosphatase. J. Biol, 
Chem. (1956) In press. 



17. AV/ARDS: 
None. 



- 5 - 



Form No. ORP-1 

October 1956 Calendar Year 1956 



PUaiO HEALTH SERVICE - - NATIONAL li^TITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet It NKMD-83 

SERIi.L NUMBER 

2. National Institute of Arthritis 'jj tetabolic Diseases 

INSTITLT'E 

3. Pharmacology and Toxicology 

L\BOR^\TCRI 

1;. Biochemical Pharmacology 
SECTION 



5. 



LOC.TION (IF OTHER THAN BETHESDA) 



6. Biosynthesis of Nicotinic Acid 
PROJECT TITLE 

7. i.lan H. fehler 



PRINCIPAL investig;.tor 

8. Catherine Rhodes 
OTHER lOTESTIGATOP 

9. PHDJECT DESCRIPTION 



Objectives ; To isolate the individual steps in the 
sequence of reactions resulting in nicotinic acid forma- 
tion, to study the properties of the enzymes involved, 
and to describe the intermediate metabolites. VJith the 
reactions available, to study the relation of these enzymes 
to altex-ed metabolic conditions. 

Methods Employed ; Enzymes are obtained from various 
sources and purified by the variety of methods currently 
used in this field. Chemical and physical, especially 
spectrophotometric, methods are used to measure enzyme 
activity and to identify products. Possible substrates 
and products are synthesized by conventional organic 
chemical techniques. Isotopic coit^Dounds are synthesized 
and radioactivity measured to follovj the course of 



Form No. ORP-1 ^.. 

October 19^6 Calendar Year 1956 



SERIi.L NUMBER 



reactions in vivo and in vitro , i.nimals are treated to 
produce altered metabolic states, and enzymes from such 
animals are assayed. 

Major Findings ; The natures of three enzymatic reactions 
involved in tryptophan metabolism were further elucidated. 
In collaboration with Drs. Rothberg and Kayaishi, it was 
found that both tryptophan peroxidase and 3-hydroxyanthranilic 
oxidase are enzymes of the newly-defined class of oxygenases. 
Further insight into the nature of tryptophan peroxidase was 
gained by the elimination of a-J3-dihydroxytryptophan as a 
possible intermediate. The nature of picolinic carboxylase 
was studies with C-^^-labeled substrate in collaboration 
with Dr. May, and the reaction found to be a simple 
decarboxylation. 

The nature of the increase in level of picolinic 
carboxylase in the livers of diabetic rats was investigated 
in collaboration with >ir. McDaniel. It was found that the 
increase occurs with rats made diabetic by pancreatectomy 
as well as by alloxan administration. The presence of 
adrenal hormones is essential for the increase, but adrenal 
hormones given in excess to non-diabetic animals cause only 
a small paii; of the change found in diabetic animals. 

The reactions leading to nicotinic acid formation 
were further investigated with C^^-labeled substrates. 
3-Hydro;cyanthranilic acid was synthesized in collabora- 
tion with Dr. i'jay, one preparation had the label in the 
carboxyl group and the other had the label in the 2 and 
3 carbons of the ring. The metabolism of these conpounds 
in intact animiols shows more extensive oxidation and 
decarboxylation than occurs with any of the known 
products, quinolinic, nicotinic or picolinic acids. 

Significance to Nliu'g) Research ; Two lines of inquiry 
are related to NI/J'^iD research. One is a study of the 
reactions that influence niacin metabolism in order to 
gain more insight into the biochemistry of this vitamin. 
The other is the analysis of the effect of diabetes on 
liver enzymes, which may give information about the 
nature of the metabolic lesions in this disease. 

Proposed Course of Project ; It is proposed to study 
the nature of the change in enzyme levels in the livers 
of animals in altered hormonal states in three ways: 



- 2 - 



Form No. ORP-1 

October 1956 Calendar Year 1956 

NIAMD-83 -■ 



SERIAL NUIffiER 



(l) by further evaluating the influences of endocrine removal 
and supplementation in the intact animal; (2) by studying 
changes in isolated livers maintained in a perfusion 
apparatus; and (3) by purifying the enzyme picolinic carboxylase 
to a point where it will be possible to learn by amino aoid 
incorporation studies whether the change represents increased 
synthesis of the enzyme or decreased destruction. 

It is proposed to study the biosynthesis of nicotinic 
acid by using C-'-'^-labeled 3-hydroxyanthranilic acid (a 
known precursor) as a substrate in various systems in an 
attempt to find conditions under which it forms nicotinic 
acid. The possibility that quinolinic acid is an inter- 
mediate in this conversion is being investigated in collabo- 
ration with Dr. Jakoby by attempting to synthesize ring- 
labeled quinolinic acid that will be used as a substrate in 
varLous systems. 



- 3 - 



Form No, ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOM/IL INSTITUTES OF HEALTH 
Ii\nDIVIDUAL PROJECT REPORT 



Part B: Budget Data 




11. 


NIAra)-83 

SEIilAt, NUI^BER 


12. BUDGET DATA; 








ESTIIIATED OBLIGATIONS 






l^N YEARS 


DIRECT REIMPURSEMT'INT 


TOTAL 




PROF OTIER TOTAL 


FY'57 

$2l,l;00 !if8,100 


^,.29,500 




1.00 1.33 2.33 


BUDGETED POSITIONS 






PATIENT DAYS 


PROF OTHER 


TOTAL 






Fityi 








1*00 1,33 


2.33 






T?- nnrr;!?^ Ai^TUTn^v. 









RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STANDARDS 



P 
P 



ADfllNISTRATION 

PROFESSIONAL & 
TECHi«CAL ASSIST- 
ANCE 



lD 



□ 



Ik, IDEi-riFY AMY COOPER/iTING UNITS OF TIE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUi\IDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 19^7. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S)! 



(Use reverse aiid additional pages, if necessary) 



Form No. ORP-1 

October 1956 Calendar Year 1956 



PUBLIC HEALTH SERVICE - - NATIOIiAi INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part 0: Honors, Awards &c Publications 15. NLllD-83 

SERIAL NUI-iBER 

16. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
G^JiEI-IIli'.R YEAR 1956: 

(1) nehler, A. H, Ifetabolism of Tryptophan. Amino Acid 
Ifetabolisn. Johns Hopkins Press. (1955) 

(2) I-fehler, /. . H. Formation of Picolinic and Quinolinic 
Acids Following Enzymatic Oxidation of 3-Hydroxy- 
anthranilic j.cid. J. Biol. Chem. 218 , 2ia (1956) 

(3) i'lShler, A. H. and Chang, Y. T. Biological Reactions 
of Fluoroacetylcholine. i'.rch. Biochem, and Biophys. 
62, 293 (1956) 

(k) i'fihler, A. H. and May, E. L. Studies with Carboxyl- 
labeled 3-Hydroxyanthranilic and Picolinic Acids In 
12;V0 and In Vitro . J. Biol. Chem, (1956) In press. 

17. AWARDS: 
None. 



- 5 - 



Form No. ORP-1 

October 1956 Calendar Year 1956 



PUBIIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEAiTH 
INDIVIDUAL PROJECT REPORT 

Part A, Project Description Sheet 1. NI/J-1D-8U 

SERIAL NUMBER 

2. National Institute of Artliritis & Metabolic Diseases 
INSTITUTE 

3. Pharmacology and Toxicology 

LABORATORY 

kt Bioctiemical Pharmacology 

SECTION 



5. 



LOCATION (IF OTHER THAN BETHESDA) 



6. Enzymatic Reactions Involved in the Synthesis of Metabolic 

Derivatives of Asparbic Acid and Ifethyl Mercaptan 

PROJECT TITLE 

7. Simon Black 



PRINCIPAl INVESTIGATOR 



8. Mrs. Phyllis F. Downey (Previously Mrs. N. G. VJright) and 

i:ir. Edith C. Wolf f 

OTHER INVESTIGATORS 

9. IF THIS PROJECT RESET^HLES, COMPiEi^ENTS, OR PAR/ilLElS 
RESEARCH DONE EiSEWlJERE IN THE PUBLIC HEALTH SERVICE 
(TfJllTIOUT INTERCHANGE OF PERSOMEL, FACILITIES OR FWIDS) 
IDENTIK SUCH RESEj\RCH: (BY SERIAL NO.(S) IF WITHIN NIH). 

None, 

10. PROJECT DESCRIPTION: 

Objectives ; ;. long term objective is the discovery 
of enzymatic mechanisms involved in the synthesis of con- 
stituents of living tissue, particularly of proteins. 

Methods ; Cell-free enzyme extracts of yeast, as well 
as of plant and animal tissues, are tested for their 
ability to convert aspartic acid or methyl mercaptan to 



Form No. ORP-1 

October 1956 Calender Year 1956 



NIjiMD-8U 



-%- 



SERIAL NUIffiER 



new compounds. Chemical, chromatographic, radiochemical, 
and radioautographic methods are used. When new substances 
are found the meclianisms of their formation are elucidated 
by classical enzymological methods. 

Major Findi ngs; It has been found that aspartic acid 
is converted to Tiomoserine in yeast through three enzymatic 
steps involving the newly discovered intermediates, p-aspartyl 
phosphate and aspartic-|3-semialdehyde. 

It has also been found that methyl mercaptan is 
enzymatically incorporated into the previously unltnown 
methylthiol ester of 3-phosphoglyceric acid, and that the 
latter is enzymatically converted to glyceryl methylthiol 
ester, also ne;wly discovered, which has been isolated in 
crystalline form. An additional enzyme has been discovered 
in yeast which catalyzes the phosphorylation of glyceric 
acid by ATP to yield 3-phosphoglyceric acid, 

S-methyl cysteine has been found as an enzymatic 
product of methyl mercaptan and L-serinc. At least ten 
derivatives of methyl mercaptan are formed by yeast, most 
of which are not yet identified. In addition to S-mcthyl 
cysteine one of them has been tentatively identified as 
L,L,B-methyl lanthionine which was previously unknown. 

S ignific a nce to NIAMD Research ; Knowledge of the 
intimate chemical transformations in living cells will 
serve as a basis for better understanding of the nature 
of disease and its more intelligent treatment. 

Proposed Course of Project ; The immediate plan is 
to learn the relata. on of the newly found derivatives of 
methyl -mercaptan to established biochemical processes. 



Form No. ORP-l 
October 19^6 
(Attachment l) 



PUBLIC HEALTH 3:.:RVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11. NIAMD-8U 



SERIAL NUi-iBER 



12, BUDGET DATA 


1 








ESTIMTED OBLIGATIONS 




MAN YEARS 


DIRECl' 


REIMBURSEI'ENT 


TOTAL 


PROF OTIiER TOTAL 


FT'57 

(li31,200 


JiilljlOO 


^U2,300 


1.00 2.33 3.33 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


O^HF.R 


TOTAL 




FY«57 

1,00 


2,33 


3.33 





13. BUDGET ACTIVITY; 
RESEARCH 

REVIEl'J & APPROVAL 
BIOLOGIC STANDARDS 



g3 ADMINISTRATION Q 

I I PROFESSIONAL & 

TECHNICAL ASSIST- 
I I ANCE □ 



lU. IDEMTIFY ANY COOPER.V:iNG UOTTS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGAI\IIZATIONS, PROv/IDING FUi\IDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPER..TING UNIT IS l-lTHIN NIH 
INDICATE SIRIAL NO(S): 



(Use reverse and additional pages, if necessary) 



I'orm No. ORP-1 

October 1956 Calendar Year 1956 



PUBLIC HE/JiTH SERVICE - - N^-.TIONi.! INSTITLTES OF HEiATH 
INDIVIDLVA PROJECT HEPOHT 

Part C: Honors , jiWards & Publications 15 . NI/1MD-8I4 



SLRi;.L NUMBER 



16. PUHLICATIONS OTHER THi.N /.BSTRjXTS FROM THIS PROJECT DURING 
CiAENDAR YEj'.R 1956: 

(1) Black, S, and Wright, N. G. Enzymatic Formation of 
Glyceryl and Phosp ho glyceryl Methylthiol Esters, 

J. Biol. Chem. 221_, 171 (1956) 

(2) Wolff, E. C, Black, S. and Downey, P. F. Enzymatic 
Formation of S-Kethyl Cysteine. J. ^.m. Chem. Soc, 
78 (November, 1956) 

(3) Black, S, L Mcroanalytical I'-iethod for .'.cetic and 
Other Violatile /.cids, in "Methods in Enzymology" 
(iicademic Press), Vol. 3. In press. 



17. AWARDS: 
None. 



- h - 



Calendar Year 1956 

PUBLIC HiiALTH SERVICE— MTIOML INSTITUTES OF tiiALTH 

ifdividual project report 

Part A. Project Description Sheet 1. NI/JO-8g 

serihl kg. 

2, National Institute of Arthritis ik tetabollc Diseases 

INSTITUTE 
h. Toxicology 3. Pharinacology &. Toxicology 

"""";: laboratory 

section 

5. :J ^ 

lS^TION (IF 'drHER TMM BETHESDAj 

6. hetabolic studies on folic acid and related cum- 

pounds . _^ 

PROJECT TITLE 

7. Dr. 0. Hayaish i 

PRINCIPAL INVESTIGATOR 

8. ^r. Bruce Levenberg and Dr. w. B. Jakoby 

OTHER INVESTIGATORS 

9. IF THIS PROJECT RESEMBLES, COMPLEffiNTS, OR PARAL- 
LELS RESEiVRCH DON"E ELSEk\fHERE IN THE PUBLIC HEALTH 
SERVICE (w/ITHOUT INTERCHANGE OF PERSON'N'EL, FACILI- 
TIES OR FUNDS) IDENTIFY SUCH RESEARCH: (BY SERIAL 

NO.(S) IF VjITHEN NIH). 

Does not apply 

10. PROJECT DESCRIPTION ; 

(a) Research projects 

'•^joctlves; To elucidate the mechanism of biosyn- 
thesis, intermediate metabolism and degrada- 
tion of folic acid and related conpounds, 

Methods j^^mployed; Through the enrichment culture 
technique, bacterial strains iifhich rapidly 
metabolize folic acid were obtained from soil 
and animal feces. Highly active preparations 
of "adaptive enzymes" were produced by these 
microorganisms when they were grown on speci- 
fix substrates. By the use of such highily 
active enzyme preparations, the detailed met- 
abolic pathways were investigated. 



NIAMD-8£_- 
SERIi^L NO. 

- 2 - 

Major Findings » Extracts of an organism isolated 
by enrichment culture techniques possess a 
deacylase which catalyzes the hydrolysis of 
N-acyl ( or N-aryl)-L-glutamic acid deriva- 
tives, such as f clic acid, N-acetyl-L-glu- 
tamic acid and N-(p-nitrobenzoyl)-L-glutamic 
acid. This deacylase has been separated from 
other acylamino acid-cleaving activities 
present in the original extract, 

■^n enzyme has been partially purified 
from extracts of a second soil micro-organism, 
which hydrolyzes the 2-amino group of several 
naturally occurring pterines, resulting in 
the formation of the corresponding hydroxy- 
derivatives. The enzyme, which is distinct 
from a very potent guanase present in the 
same extracts, catalyzes the deamination of 
folic acid and 2-amino-l4-fvdrQxypteridine-6- 
carbcocylic acid, but does not attack leucovor- 
in or aminopterin. 

In collaboration with Dr, Elizabeth Ander- 
son of the Ni.;I, a successful attempt has been 
jTiade to obtain a soluble enzyme system carry- 
ing out purine synthesis de novo in extracts 
of ascites-form plasma-cell leukemias in mice. 
The anti-carcinogens L-azaserine and 6-diazo- 
5-oxo-L-norleucine have been found to exert 
a powerful inhibitory action upon the enzymatic 
step leading to the formation of 5-amino-imi- 
dazole ribotide,one of the intermediate com- 
pounds in purine biosynthesis. The mechanism 
of inhibition of this reaction in both sensi- 
tive and resistant strains of the tumor is at 
present under investigation. 

Very dilute solutions of guanine and 
other purines maintained at pH's from 5 to 7 
for 20 hours or longer were found to undergo 
rather extensive degradation, acconpanied by 
the complete loss of absorption of light in 
the ultra-violet region and the evolution of 
3 moles of ammonia per mole of guanine 
destroyed. Further studies are now in pro- 
gress to determine the nature of this rather 
surprising degradative process. 



NlaMD-Bg J 
SERL.L NO. 

- 3 - 

Significance to the program of the Institute; Vit- 
amins have been found to play an important 
role for the etiology of certain metabolic 
diseases, Studies on the metabolism of folic 
acid may throw some light on the understand- 
ing of these diseases and may ultimately lead 
to a better treatment, 

Proposed Course of Project; Metabolism of folic 
acid will be studied by enzymes from micro- 
bial and mammalian origin. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SmVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-85 

SERIAL MMBER 



12, BUDGET DATA: 






ESTiraTED OBLIGATIONS 


MAN YE/iRS 


DIRECT 


REII'iHJRSEiENT TCTiiL 


PROF OTIffiR TOTAL 


FY«57 






^13,300 


)i5,000 118,300 


1,16 .66 1,82 




BUDGETED POSITIONS 


PATIEir DAYS 


PROF 


OTHER TOTAL 




fY'57 .. 






2,16 


.66 2.62 




13. BUDGET AC: 


IVITY: 





RESEARCH 

REVI&J & APPROVAL 

BIOLOGIC STAiWARDS 



LZl 



ADMINISTRATION 



[___! PROraSSIONAL & 

TECHNICAI. ASSIST- 
[~" | ANCE 



□ 

a 



liu IDEi^JTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR Pi:RSONi\IEL 
FOR THIS RWJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE S.iiIAL NO(S): 

Dr, E. Anderson (NCI) is collaborating on the azaserine 
problem, Dr, G. Tomlcins, A^il, NIAJ'D, has been collaborating 
on Viadril metabolism. 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBLIC HEALTH SER\?ICE— NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPCRT 

Part C. Honors, Awards & Publications l5. N^'^MD-85 

SERIAL NO. 

16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS 
PROJECT DURING CALENDAR YEAR 1956. 

yi. B. Jakoby and Ci, Tomkins, An iinzymatic Detoxi- 
fication Mechanism for Viadril, Science, 123 , 9I4O 
(1956) 

17. LIST HONORS AND AWARDS TO PERS0N1\'EL RELATING TO 
THIS PROJECT DURING CALENDAR YEAR 1956. 

None 



Calendar Year 19^6 

PUSLIC HEALTH SERVICE — NATIONAL INSTITUTES CF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A, Pr9;)ec| Desoript.i9n Sheet 1. NIuMD-86 

SERIAL NO. 

2 National Institute of Arthritis ^ Metabolic Diseases 

Ij, TcQcicology 3. PharmacoloQr & Toxicology 

SECTION LABORATORY 



5. 



LOCATION (IF OTIER THAN BETtESDA) 



6. Metabolism of aromatic amino acids and related compounds 
PROJECT TITLE 

7. Dr. 0, Hayaishi 

PRINCIPAL INW2STIGAT0R 

8. Dr. M. Katagiri, Dr. Y. Saito, and Dr. W. 5. Jakoby 
OTHER INVESTIGATORS 

9. IF THIS PROJECT I'iESEffiLES, COf^PLEMENTS, OR PARALLELS RE- 
SEARCH DONE ELSEWHERE IN THE PUBLIC HEALTH SERVICE (v^ffTH- 
OUT INTERCHANGE OF PERSONNEL, FACILITIES OR FUNDS) IDENTIFY 
SUCH RESEARCH: (BY SERIAL NO.(S) IF WITHIN KIH). 

Does not apply 

10. PROJECT DwSCRIPTIONi 

(a) Research projects 

Objectives ; To study the metabolism and pharmacological 

activity of aromatic amino acids and related compounds. 

Methods Ji-mployed; Through enrichment culture and adaptive 
enzyme technique, enzymes were isolated and purified 
from various microorganisms which catalyze chemical 
transformation of biologically important aromatic com- 
pounds. Chemical and spectrographic methods were used 
together with 02^° and H^0^° to study intimate mechan- 
isms of individual reactions. 

Major Findings; Through the use of Qxygen-l8 (a heavy 
oxygen isotope) atmospheric oxygen was shown to be 
directly utilized and incorporated into organic 



Form Wo. ORP-1 
October 1956 
(Attachment l) 



PUBLIC lEALTH SEiWICE - MA?IOMAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11, NIAMD-86 

SERIAL IWMBER 



12, BUDGET DATA 


t_ 






ESTIIiATED OBI.IGATIONS 


MAN YEARS 


DIRECT 


REIMBURSEMENT TOTAL 


PROF OTHER TOTAL 


FY»57 






m,^oo 


^5,000 $18,200 


1,16 ,66 1.82 




BUDGETED POSITIONS 


PATIENT DAYS 


PROF 


OI'IffiR TOTAL 




FY 157 






1,16 


1,66 2.82 




1 •^- prinr,F" km 


JTT'Y' 





RESEARCH 

REVIEW & AP PROVAL 

BIOLOGIC STAfJDAEDS 



(^ 1 ADMINISTRATION [^ 

I I PROFESSIONAL & 

TECHNICAL ASSIST — 
I i ANCE Qj 



111, IDENTIFY AI\!Y COOP'',R/.TING UNITS OF THE PUBLIC HEALTH SERVICE, CE 
OTHER OR:iANIZATIONS, PROVIDING FUi\IDS, FACn.ITIES, OR Pi^RSOififfiL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS iflTHIN NIH 
INDICATE S",RIAL NO(S): 

Dr. S, Rothberg, Nl-EE, 10?:^ , has been collaborating in 0-^° 
experiments, 

Dr, A, H. nehler, Dr. H, Tabor, (Biochemical pharmacology 
Section) ur» H, Posner and Dr. S, Udenfriend, from NHI, Dr. B, 
Witkop and Dr. A, Patchett (Laboratory of Chemstry), 

Similar. type of vjork on the metabolism of imidazole acetic 
(Use reverse and additional pages, if necessary) 



»>■ 



-2- 

NIkrg)-86 
SERIAL WU14B131 

lli. IDEilTIi^ AbJY COOP-;RA':iNG UMTS, .TC, (continued): 

acid has been carried out in collaboration with Dr« 
H, Tabor and details vjill be reported from the 
Section on BiocherdcaX Pharmacology, 



Calendar Year 1956 

PU3LIC HEALTH SERVICE — NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part C, Honors, Awards & Publications 15. NLJ'ID-86 

SERIAL NO. 

16. LIST PUBLICATJQNS giiW-B. Tf^N 4?STRACTS FROM THIS PROJECT 
DURING CALENDAR YEAR 1956| 

W. 3, Jakoby and D, M, Bonner, Kynurenine Transaminase 
from Neurospora, J. 5iol. Chem., 222, 68? (1956). 

W. B, Jakoby, The inability of phenylalanine to serve as 
a precursor of indole in Neurospora, Biochim, Biophys. 
Acta, 21, 390 (1956). 

M. Katagiri and 0, Hayaishi, Enzymatic degradation of 
p-ketoadipic acid,, J, Biol, Chem, (in press). 

17. LIST HONORS AND AWARDS TO PERSONNEL RELATING TO THIS 
PROJECT DURING CALENDAR YEAR 1956. 

Dr, 0, fiayaishi was Chairman of a Symposium on the t-nzy- 
matic Activation of Molecular Oxygen at the Fall meeting 
of the American Chemical Society in Atlantic City, N. J., 
on September 20, 1956. 



Calendar Year 1956 

PUBLIC HLALTH SERVICE—MTIOMAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJl.CT itEPQiT 

Part A, Project Description Sheet 1, NLiMD-67 

SERIAL IMO. 
2, National Institute of Arthyltls k Metabolic Diseases 
INSTITUTE"""" ■ ~ ■ "■' "^ 

h, 'J oxic ology 3. Pharmacology & Toxicolog y 

SnXJTION LABORATORY 



5. 



LOCATION (IF OTHER THAN BETHESDA) 



6, Metabolism of dicarbaxyllc acids 
PROJECT TITIE 

7, Pr, 0. Hayaishl 

PRINCIPAL INVESTIGATOR 

8, Dr, H, Shimazono, Dr, M, Katagiri, Dr. W, B, Jakoby 

and Dr. Y, Saito. 

OTHER INVESTIGATORS 

9, IF THIS PROJECT RESEt'iBLES, CCMPLEiyJENTS, OR PAIiALLELS 
RESEARCH DONE ELSEw/JiERE IN TliE PUBLIC HEALTH SERVICE 
(raiHOUT INTERCHANGE OF PERSCNNEL, FACILITIES OR 
FUiMDS) IDENTIFY SUCH RESEARCH: (BY SERIAL NO.(S) IF 
l-jITHIN NIH) 

Does not apply 

10, PROJECT DESC OPTION; 

(a) Research projects 

Objectives: To elucidate the mechanism of biosyn- 
thesis and degradation of biologically inport- 
and dicarboxylic acids and related compounds. 

Methods Employed; Through the enrichment culture 
technique, bacterial strains wliich rapidly 
metabolize dicarboxylic acids x^ere obtained 
from soil, liLghly active preparations of 
"adaptive enzymes" were prcduced by these mi- 
croorganisms when they were grown on specific 
substrates, iiy tlie use of such highly active 
enzyme preparations, the detailed metabolic 
pathways were investigated. 



NKMD~87 
SERL'iL NO. 



2 ~ 



heohanlsms of COp fixation in photosyn- 
thesis T-Jere investigated vdth a purified enzyme 
preparation and deuterium or tritium labeled 
water. 

Major Findings; A new enzyme, tentatively designated 
as oxaloacetic hydrolase, was isolated from 
Aspergillus nlger and v;as partially purified, 
i^in^"*" was required for the hydrolytic cleavage 
of oxaloacetate and oxalic and acetic acids 
were identified as products of the reaction. 

Oxalic decarbcQcylase was isolated from 
Colly-yja veltipes, a wood-destroying fungus, 
and was purified about ijOO fold. Properties 
of this enzyme and the kinetics of the reaction 
were investigated. 

The ene-diol of ribulosediphpspnate was 
shown to be a likely intermediate.', in the 
reaction forining phosphoglyceric acid from 
ribulosediphosphate, 

Significance to the program of the Instltutet In 
spite of the recent progress in the knowledge 
of fatty acid metabolism, little has been 
understood about the metabolism of dicarboxy- 
lic acids. Oxalic acid has been knoim for 
years to be widely distributed in men^ plants 
and microorganisms and together with malonic 
acid, exliibits high toxicity in men. 

The present work has elucidated the bio- 
logical origin and the metabolism of oxalic 
acid and has led to a discovery of three new 
enzymes. One concerning the synthesis and 
the other two concerning the breakdoim of 
oxalic acid. 

Proposed Course of Project; Further studies on 

the enzymes and metabolism of oxalate, malonate 
and adipate will be pursued. Oxaloacetic liy- 
drolase will be purified in order to study the 
mechanism of the reaction. 



Form Mo, ORP-1 
October 19^6 
(Attachment l) 



PUBIIC HEALTH SERVICE - MATIOM/.L INSTITUTES OF HEALTH 
IiroiVIDUAL PROJECT REPCET 



Part B: Budget Data 



11. NIAlvlD-87 



SERIAL NUMBER 



12, BUDGET DA 


:a: 








ESTIi'..TEU OPLIGATIONS 




MN YEARS 


DIlffiCT 


:iKIiIBURSEi;:,NT TOTAL 


PROF 


OTHLR TOTAL 


FY»57 








121,300 


$8,100 $29,^00 


1.66 


1.33 2.99 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTIffiR TOTAL 




FY«57 








1.66 


1.33 2.99 






13. BUDGET ACTI-ITY: 







RESEARCH 

REVIEVJ &: AP^\ROVAL 

BIOLOGIC STANDARDS 



£^1 ADffiNISTRATION Q 

1 I PROFESSIOML & 

TECroilCAL AS3IST- 
! I ANCE □ 



lii. IDEiriFY AiIY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 

OTHER ORGANIZ/lTIONS , PROVIDING FUiTOS, FACILITIES, OR PERSONl>iEL 

FOR THIS PnOJ^,CT IN FY' 19^7. IF COOPERATING UNIT IS V'lTHIN NIH 
INDICATE SERIAL NO(S): 

Dr. J, Hurwitz and ur, B, L, Horecker have collaborated on 

the CO2 fixation problemc They ai^e \Tith the Laboratory of 
Biochemistry and Uetabolism. 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBLIC HEALTH SERVICE—NATIOI^AL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part G: Honors, Awards &; Publications 1$. HLJ4D-87 

SERIAL NOt 

16, LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS 
PROJECT DURING CALENDAR YEAR 1956. 

0, Hayaishi, H, Shlmazono, M, Katagiri, and 
i, Saito, Enzymatic formation of oxalate and ace- 
tate from oxaloacetate,, J, ^m, Chem, Soc,, 78, 
5126 (1956). 

V;, 3, Jakoby, E, Qhmura and 0, Hayaishi, Enzymatic 
decarboxylation of oxalic acid,, J, Biol, Chem., 
222 , h35 (1956). 

J. Hurwitz., W, 3, Jaloby, B. L. Horecker, The 
mechanism of CO. fixation leading to phosphof^lyceric 
acid, Biochim. Biophys. Acta, 22, tStJ W56) 

W, B, Jakoby, D, Brummond, and S, Ochoa., Formation 
of 3-phosphoglyceric acid by carbon dioxide fixa- 
tion with spinach leaf enzymes,, J. Biol, Chem., 
218, 811 (1956). 

17, LIST HONORS AMD A'WARDS TO PERSONNEL REUTING TO 
TfflS PROJECT DURING CALENDAR YEAR 1956: 

None 



t. * 



Calendar rear 1956 

PUBLIC HEALTH SERVICE— NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A, Project Description Sheet 1. NIAMD-63 

SERIAL NO. 

2, National Institute gf Aythrjtj-P &- Metabolig Diseases 

li. Toxicology 3. Pharmacology k Toxicology 

SECTION LABORATORY 



5. 



LOCATION (IF OTHER THAN BETHESDA 



6, Toxicologic studies of iodates, the cellular local- 
ization of hyaluronidase 

PROJECT TITLE 

7, Dr. S, H, Ivebster, Dr, W, F, von Oettingen, and 

Dr. E. W. grnmart 

PRINCIPAL IIWESTIGATORS 

8, Mr. E. F. Stohlman and Mr. C. R. Brubaker 
OTHER INVESTIGATORS 

9, IF THIS PROJECT RESEMBLES, COMPLEMENTS, (B PARAL* 
LELS RESEARCH DONE ELSEWHEi^fi IN THE PUBLIC HEALTH 
SERVICE (WITHOUT INTERCHANGE OF PERSONNEL, FACIL- 
ITIES OR FUNDS) IDENTIFY SUCH RESEARCH} (BY SERIAL 
NO.(S) IF WITHIN NIH). 

Does not apply 

10, PROJECT DESCRIPTION 

(a) Research projects 

Objectives { (A) The toxicology of iodates as a 
basis for use in iodized salt to prevent 
goiter, 

(B) Because of the technical difficulties 
and pathological and biological significance 
of the antigens studied the program has been 
broken down into a series of projects: 



NIaMD-88 '- ^ 
SERIi.L NO. 

- 2 - 

I, "'Studies on Streptococcal hyaluronidase and 
antihyaluronidase, (l) The localization of 
sites of absorption of streptococcal hyaluron- 
idase (Group C) with fluorescent antibody," 
(2) iitudies on site of absorption of Strepto- 
coccal hyaluronidase in tissues of the eye, 

II. "The Localization of Injected Human Chorionic 
Gonadotropin in the Rat Ovary," Drs, A. 
Breslow, E. W, Eiranart Sc H. Aitman, 

Methods Employed; Administration of iodates and 

iodides to experimental animals and subsequent 
examination by hematological, ophthalmological 
and histopathological methods. Use of fluores- 
cein labeled antibody and fluorescence micro- 
scopy. 

Major Findings; The emetic action of KIO^ is pro- 

nounced, increasing vdth the amount administered. 
Sodium bicarbonate, fed simultaneously with the 
iodate, appears to decrease the irritating 
action of the latter. Individual susceptibil- 
ity to dogs to the iodate varies greatly. The 
cliief toxic effects noted in affected animals, 
after 3 months of the experiment, are emesis, 
anorexia, and loss of weight. Methemoglobin 
formation appears negligible (Webster). 

ii survey of the clinical toxicity of var- 
ious drugs was made to evaluate their hazards 
in clinical and industrial use (von Oettingen) . 

Using the Coons fluorescent antibody tech- 
nique the sites of absorption of the enzyme has 
been localized in particular cells and areas 
of the cell in the lung, spleen, kidney and 
liver. In the eye tissue deposition has been 
found in the nuclei of the corneal epithelium 
and in areas of skin about the eye. The de- 
position of the antigen is in highly specific 
cells and tissue areas, blocking action of 
unconjugated antihyaluronidase globulin in 
paired sections identifies these fluorescent 
sites from other fluorescence present. 

Studies by similar techniques on sections 
of rat ovaries following injection with 



nl;md-88 - •' 

serLlL no. - 3 - 

chorionic gonadotropin reveal the presence of 
the hormone in specific cells (^-mmart), 

Significance to the program of the Institute; T he 
use of iodate to lodinize salt is a problem 
of injjortance in tropical countries, where un- 
stable and hygroscopic properties of KI pre- 
sent a difficulty. The findings of this study 
are expected to show whether KIO, is a safe 
substitute or the unstable KI at present used 
to iodize cooking salt and salt-feed mixtures 
for cattle. 

'Studies with hyaluronidase: :may throw some 
light on the etiology and treatment of arthri- 
tis and rheumatic diseases. 

Proposed Course of Project; The chronic toxicity 
feeding study will be continued for another 9 
months. Blood, urine and ophthalmoscopic exam- 
inations will be carried out every three months, 
iilectroretinograms and photographs of fundi 
will be repeated at the end of the experimental 
period in cooperation with Drs, Gunkel and 
Von Sallmann, (Webster), 

ourvey of the toxicologic evaluation of 
drugs will be continued, (von Oettingen) , 

Further studies with this enzyme are being 
carried out in various tissues. With new 
equipment not yet received it will be possible 
to emphasize precisely the details of the lo- 
calization of the antigen to the morphology of 
the cell, and to study in greater detail the 
blocking action of the antihyaluronidase glob- 
ulin, i^ttempts will be made to obtain more 
purified hyaluronidase for these studies 
(with Dr, t/illiams), (iimmart). 



Form No, ORP-1 
October 1956 
(Attachment l) 



PUBLIC HEALTH SmVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11, NIAMD-88 







SERIAL NUli'BER 




12, BUDGT DATA: 








ESTiri/lTED OBLIGATIONS 




MAN YEARS 




DIRECT REIMHJRSEI'tENT 


TOTAL 


'ROF OTHER 


TOTAL 


FY'57 








$66,900 $25,300 


$92,200 


l;,00 2,00 


6,00 


BUDGETED POSITIONS 




PATIEi^ DAYS 




PROF OTHER 


TOTAL 




FY'57 








U.OO 2,00 


6,00 






1^. RTrTr;i;"T^ ir'^'T^rr'^Vt 









RESEARCH 

REVIEl'if & APPROVAL 

BIOLOGIC STANDARDS 



(T"! ADMINISTRATION □ 

i I PROFESSIOML & 

TECHNICAL ASSIST- 



Ih, IDENTIFY MY COOPER/:TING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGiiNIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOmffiL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS VJITHIN NIH 
INDICATE SERIAL NO(S)! 

Dr. Ben ttLghman, NIAOT (Pathology) and Dr. Ralph Gunkel, NIITOB (Retinal 
Examnations) (with JJr. Webster) 

Dr, R. ^K Cole, ma, J, B. Lon:iley, NIAiiD, L, V, Sallman, liTOB, 
A. Breslow, DBS, and H. Altraan, NCI (mth Dr. Qmnart). 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 
PUBLIC lEALTH bERVICii— MATIOML IKiSTITUTLS OF HEaLTH 

iivdividual project report 

Part C: Honors, Awards & Publications 1$, NLJ'ID-eS 

SERIAL NO. 

16. LIST PUBLICaTIOIvS OTHER THi\K ABSTiiACTS FROM Ti-EES 
PROJECT DURING CALENDAR TEAR 1956: 

W. F, von Oettingen, \^ol, I, Kcute Toxicities. 
editor, ifi, b, apector, W, b. baunders Comp,, Phila,, 
1956, 

"f. i', von Oettingen, Review of the book "The Alka- 
loids, Chemistry and Physiology by R, H. F. mnske, 
i^cience, 123, 8o5 (1956). 

W. F, von Oettingen, Review of "Gewerbliche \^er- 
giftungen" by L, Telecky, A. M, A.^ Arch. Ind. 
Health, 13, 199 (1956). 

Iv. F. von Oettingen, Review of the book "kViederbel- 
ebung" by H. Kilian a. a Dohnhardt, A. H. A. Arch. 
Ind, Health, 13, 197 (1956). 

!^. w, Emmart and L, R, Crisp,, Improved Freeze Dry- 
ing and Embedding Apparatus for Frozen Tissues., 
The Review of Scientific Instruments, Vol. 27. No, 5. 
315 (1956). 

i^. w/. Einmart. Transactions of the First Conference 
on Glaucoma, mcy Foundation, December 1955. 

17. LIST HONORS AND AWARDS TO PERSONNEL RELATING TO 
THIS PROJECT DURING CALENDAR YEAR 1956. 

None 



Calendar Xear 1956 



PUBLIC HErtLTH oEi^VICE NaTIONkL IrJoTITUTEb OF HE/J.TH 

INDIVIDUkL PilOJECT liEPORT 
Part H, Project Description Sheet 1, NIaMD-89 



oZRIhL IM-:BER 

2, Nlah'D 3, Laboratory of Physical Biology 

INSTITUTE OH DIVIijIpIT ^ - Ij^BOIUTORY, BRANCH, OR DEPhRTI'iENT 

4. Physiology 5. ______^ 

SECTION OR oERVICE LOCATION (IF OTHER THi.N BETHEoDa) 

6. Pulmonary ventilation 

Pr.OJECT TITLE — — __ 

7. Heinz Specht 



PRIKCIPnL INVESTIGATOR 

8. Howard F. Brubach, Roscoe G. Bartlett. Jr. 

OIKER IIJVEoTIGaTORo 

9. IF THIS PROJECT REuEIlBLEo, COi^iPLEtiENTo, OR PaRaLLELo RE JEkRCH DONE 
ELoE^TiERS IN THE PUBLIC HEaLTH oERVICE C ITKOUT INTERCHANGE OF 
PERSONNEL, F.-.CILITIES OR FUNDS), IDENTIFY SUCH REoEhRCH: 

(BY oERIriL NO. (S) TF ' I'THIW KIH), 

Mo parallel research in PHS. 

10. PROJECT DESCRIPTION 

Studies of piilmonaiy ventilation and its cost in higher than 
normal density atmospheres are in progress utilizing underwater 
facilities of this Laboratory. 

Ob.jectives ; The principal objective of the over-all project is 
to explore ne^f phenomena regarding breathing behavior with 
regard to their physiological significance. In order to 
accomplish this various studies of liiysiology must be under- 
taken in order to accurately control the experimental situa- 
tion. Since various observations on past studies on sub- 
merged subjects indicate a. limiting effect on maximum rate of 
pulmonary ventilation by the dense environment provided by 
water, the degree to v^ich this contributes to the cost of 
ventilation is being studied, Measures of normal ventilatory 
capacity are being explored in order to form a base for assess- 
ment of effects of abnormal environments. 



- 2 - NT;.^m-89 



SERIAL ITOHiER 



10. PROJECT DESCRIPTION - CONTINUED 



Methods Employed ; Subjects are studied for oxygen consumption, 
maximum breathing capacity and other physiological responses 
by means of volumetric devices made for the purpose. A 
device for using various external inspiratory and expiratory 
resistances has been made and used as well as a device for 
employing various degrees of added dead space to the respira- 
tory tract both for use in air and submerged. 

Ka.-jor Findings ; Studies of the AMork efficiency and respiratory 
response of trained underv^ater swimmers v.ere further analyzed. 
Work efficiencies v«ere low (2-8/o). At low swim speeds (.6 and 
,7 knot) average efficiencies from swimner to swimmer varied 
markedly (2-8^) while at higher swim speeds (.7-1.2 knot) the 
average efficiencies varied less (3-5/o). The maximum attain- 
able respiratory responses to an extended exercise period (20 
minutes) in underv/ater swijiimlng v/ere much lo^jer than those re- 
ported in the literature for work in air. Thus maximxam pul- 
monary minute volumes were usually much less than 80 liters 
and the maxim\mi oxygen consiomption was only about 100 liters/ 
square meter body surface/hour. A comparison of the response 
of trained and untrained swimmers to the exercise of underwater 
swimming revealed two factors which could account for the 
elevated end-tidal CO2 levels in the trained sv/imr^ers; (1) The 
trained sv;iminers had a significantly lower oxygen ventilation 
equivalent (liters pulmonary ventilation/liter oxygen consumption) 
due to (a) a conscious disregard of the CO2 stimulus, and/or 
(b) a lov.ered sensitivity of the respiratory centers to CO2. 
(2) Consciously produced post-inspiratory pauses resulting in 
a longer CO2 build up time. 

The maximum breathing capacity (f-BC) has been determined 
^^dth various expiratory and inspiratory resistances (singly 
and cojiibined) at breathing rates of 4-196 per minute. A given 
resistance in the inspiratory duct reduced the MBC more than 
if it v;ere in the expiratory duct. At low resistance levels 
the combined resistances lowered the I-IBC little more than the 
inspiri-tory resistance alone, nt higher resistance levels 
the effect of inspiratory and expiratory resistances were 
more nearly additive. The breathing rate at which the highest 
MBC occurred v;as lov/ered by increasing the resistance. Thus 
it was about 100 breaths per minute at the lowest resistance 
level and about 40 breaths per minute at the highest resistance 
level. The relationship vras such that the tidal volume was 
the same at the highest MBC at each resistance level. The IIBC 
increased rapidlj' over the lower breathing rates and fell off 
slowly at rates of over 120. Because a four fold increase in 
the expiratory airway resistance lowered the 1;BC little it was 
concluded that special low resistance testing equipment was 
not necessary for the normal subject. 



- 3 - NKI4D-89 



SEfiI/-.L NmiBER 

10. PROJl^CT DEbCRIPTION - CONTINUED 

Ma.jor Findings (cont'd): 

The work of breathing in air compared to that of sub- 
merged subjects is in pro ress and has yielded data on 3 sub- 
jects. Differences are observed in the direction expected 
id.th regard to submerged vvork, but equivocal findings have 
so far resulted as regards the effect of the rate of breathing. 

Significance to WlriliD Research ; In order to extend the under- 
standing of both normal and abnormal respiratory gas exchange 
it has become necessary to explore the significance of 
phenomena beyond those used classically for measuring pul- 
monary function. Inasmuch as physical environment affects 
such phenomena the study of atmospheric density effects on 
breathing vdll ultimately form a nev; set of criteria for 
judging the level of normal or abnormal behavior. 

Proposed Course of Project ; The Office of Naval Research has 
requested us to continue these studies and it is planned 
that the main area of investigation will be in the study 
of cost of breathing under water, limitations on submerged 
breathing, and the elimination of such limitations by suit- 
able devices to be developed. 



Form No, ORP-l 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIOML niSTITUTES OF HEALTH 
I1\1DIVIDUAL PROJECT REPORT 



part B: Budget Dat^ 11, NIAMD-89 

SERRL IWMBER 


12, BiroGET DATA 


4 
• 






ESTimTED OBLIilATIOWS 


MAM YEARS 


DLRECT 


REIMBURSEI EI^ TOTAL 


PROF OTHER TOTAL 


JY«57 ' 






ii38,800 


$1U,200 ^53,000 


3.00 1,33 h.33 




BUDGETED POSITIONS 


PATIEivT DAYS 


PROF 


OTIKR TOTAL 




FY«57 






U.OO 


1.33 5.33 




13. BUDGET ACTIVITY: 





RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STAi^IDARDS 



1^: ADMINISTRATION □ 

j i PROFESSIOmL & 

'. TECHNICAL ASSIST- 

1 I ANCE □ 



lU. IDENTIFY ANY COOPERivTING UNITS OF THE PUBLIC HEALTH SERVICE, 01 
OTHER ORGANIZATIONS, PROVIDING FUiTOS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN BY 1957. IF COOPEfWiTING UNIT IS WITHIN NIH 
INDICATE S.Ei^HL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBLIC KEviLTH SERVICr NaTION/I INSTITUTSo OF HT/J^TH 

hmdividuaL project report 

Part C: Honors, ..vjards & Publications I5. NIAI4D-89 

5EKI..L NUI-'iBER 

16. LIoT PUBLIPhTIQNo OTHER THhN ABSTRkCTo F'la: THIb PROJECT DURING 
CALE^DHR IEiiR'l956;" 

Goff, L. G. , H. F. Bi-ubach, H. Specht and N. braith. Effect of 
total immersion at various temperatures on oxygen uptake at 
rest and during exercise, J, Applied Physiol., 9: (1) 59-61, 
July, 1956. 

Goff, L. G. , Roberto Frassetto and H. Specht, Oxygen require- 
ments in underwater s^^ri^lming. In pi^ss - J. Applied Physiol. 

Goff, L. G, and R, G. Bartlett, Jr. Elevated end-tidal CO2 in 
the trained underwater swimmer. In press - J. applied Physiol, 

Specht, K,, L, G. Goff, H. F, Brubach and R. G, Bartlett, Jr. 
Work efficiency and respiratory response of trained vinderwater 
swimmers using a modified SCUB/i. In press - J. Applied Physiol. 

Goff, L. G. , H. F. Brubach and H. Specht. Measurements on 
respiratory responses and work efficiencj'' of underwater swimmers 
utilizing improved instrumentation. Submitted to J. Applied 
Physiol, 

Bartlett, R. G, Evidence for 'diffusion respiration' in rhythmic 
breathing. Submitted to J. -'ipplied Physiol. 

Bartlett, R. G, and F. H. Quimby. Heat balance in restraint 
(emotionally) induced hypothermia. Approved by NlAl>D Editorial 
Board. 

17. LIST HONORS aND A'-'AiiDS TO PZRoOMEL iffil^.TING TO THIS PROJECT 
DURING CALENDAR YEAR 1956: 

None. 



Calendar Year 1956 

PUBLIC HEiiLTH SERVICE - - NhTIONhL INoTITUTEb OF HEhLTH 
INDIVIDUAL PROJECT REFOilT 
Part A. Project Description Sheet 1. NKMD-90 



oERI/vL NU13ER 

2. Nlrtt^ID 3. Laboratory of Physical Biology 

INSTITUTE OR DIVISION LABORATORY, BRhNCH, Oii DEPaRTI^iENT 

k. Physiology 5. 

LOCATION (IF OTHER TlluU BETHEoDa) 

6. Effects of hypoxia on physiologic mechanisms 

PROJi;CT TITLE 

7. Paul D. Alt land 

PRIiVCIP.i IN'\/ESTIGaT0R 

8. Edwin C. Thompson. I.ilton Parker. Edna Devlin 

OTIVZR INVESTIGATORS 

9. IF THIo PROJECT REoEJlBLES, COKPLEIIENTS, OR PaRaLLELS REoEaRCH 
DONE ELSE HEi-(E IN THE PUBLIC HEaLTH SERVICE (VITHOUT INTEiICHaNGE 
OF PERSONNEL, FACILITIES OR FUNDS), IDENTIFY oUCH REoEaRCH: 

(BY oERLiL NO.(S) IF UITHIN NIH). 

No parallel research in PHS, 

10. PxiOJ^^CT DESCRIPTION 

Objectives ; Subproject a. The fundamental mechanisms which 
influence altitude tolerance are unknown. Studies have been 
conducted in order to determine the importance of obesity in 
rats to survival at high altitudes. Particular emphasis has 
been placed on survival to acute exposures to 30,000 feet. 

Subproject b. The altitude tolerance of individuals 
with cardJDvascular disease is xinknown. Experiments have been 
conducted in order to determine the survival of dogs with 
heart disease when exposed to high altitudes. 

Subproject c. The use of radioisotopes has made 
possible the study of life of red blood cells. The longevity 
of the nucleated erjiihrocytes of birds have been studied as 
a part of a larger program to determine the phylogenesis of 
erythrocyte longevity. 

Methods Employed ; Altitude exposures conducted in 2 decompres- 
sion chambers. Physiologic and radiobiologic methods have 
been employed. 



- 2 - MI/iMD-90 



SERIhL IJUI-IBER 
10. PxHOJECT DESCRIPTION - CONTINUED 

M a.jor Findings : Exposure of 2 strains of rats (Sprague-Dav/ley 
and Osborn-J.endei) fed a high fat-diet to 30,000 feet simu- 
lated altitude has shovm that all such rats, irrespective of 
the degree of obesity, die vdthin 86 minutes, ouch treat- 
ment as preoxygenation or slowed rate of ascent, designed to 
minimize aeroembolism, faile4 tq improve the survival, otock- 
fed rats v^ere much less susceptible ^q the delet^erious effects 
of decompression hypoxia. Obese rats exposed repeatedly to 
25,000 feet simulated altitude for short daily intervals 
showed increasol incidence of cardiovalvular thickening and 
presence of mitral valvular vegetations of nonbacterial origin. 

Dogs with surgically induced aortic insufficiency and 
mitral insufficiency shovred high tolerance to exposures to 
30,000 and 32,000 feet. At 34,000 and 36,000 feet, however, 
there is an increase in the mortality of dogs vdth the cardiac 
lesions as compared vdth unoperated controls. 

By use of radioactive carbon it has been found that the 
life of the chicken erythrocyte is 20 days and the duck is 39 
days. This shows that, iinlike the nucleated erythrocyte of 
cold-blooded animals, the nucleated erythrocytes of warm- 
blooded animals are short lived. 

Significance to NL'JtD Research ; Contributes facts concerning the 
importance of obesity and heart disease to svirvival linder the 
stress of hypoxia and reduced atmospheric pressure. Results 
suggest the possibility that hypoxia may hasten development of 
cardiovascular disease in obese animals. Provides data on the 
comparative physiology of blooa formation. 

Proposed Course of Pro.ject ; Studies on mechanism of altitude 
tolerance. Experiments on hypoxic tolerance of dogs with 
cardiac defects will be continued. Study of influence of 
hypoxia on physiology of reproduction in the dog will be 
continued. 

Studies on physiology of blood formation and erythro- 
cyte longevity will be continued v\d.th emphasis on (a) nature 
of erythropoietic stiniulus in poikilotherms, (b) longevity 
of erythrocyte in turtles, toads, and of the fetal erythro- 
cytes of mammals. 



Form No.l 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - MATIOML INSTITUTES OF HEALTH 
INDIVIDUAL PROJi';CT REPORT 



Part B: Budget Data 



11. MIAMI)-90 



SERIAL NUMBER 



L2, BUDGi^T data 


• 






ESTIKATED OBLIGATIONS 


MAN YEARS 


DIRECT 


reimbursei#;nt total 


PROF OTHER TOTAL 


1^1 57 






^.33,200 


$;,12,100 i^^,300 


1.00 U.33 5.33 




BUDGETED POSITIONS 


PATIEir DAYS 


PROF 


OTFER TOTAL 




FY>57 






1.00 


U.33 5.33 




13. BUDGET ACTIVITY: 





RESEARCH 

REVISE & APPROVAL 

BIOLOGIC STANDARDS 



m ADMINISTRJVTION 

| ~~| PROFESSIOmL & 

TECHiIGAL ASSIS: 
□ ANCE 






lU. IDEiriFY ANY COOPERiiTING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHiIR ORGAIUZATIONS, PROVIDING FUiTOS, FACILITIES, OR PERSONNEL 
RJR THIS PROJECT IN FY 195?. IF COOPERATING UNIT IS V^ITHIN iCH 
INDICATE SERIAL NO(S): 

Studies xrith obese animals conducted with cooperation of Dr, 
Olaf Miclcelsen of Section of Biochemistry and Physiology of Nutrition, 

NIAiiD. 

Studies on dogs >dth aortic insufficiency are being conducted 
;d.th Dr. Joseph Roshe (formerly of Surgery Branch, ^^abional Heart 
Institute - noij at Department of Surgery, University of Indiana 
Medical Center, under special services contract for 6 months) and 
(Use reverse and additional pages, if necessary) 



6 -2- 



NIA>m-90 
SERIAL imiWR 



II4. IDENTIFY ANY COOPERATING UNITS, etc, (continued) 

Dr. Benjamin itigliman , Section on Histochemistry and pathology, 

NIAIiD, 

Studies on longevity of erythrocytes are being conducted 
with Dr, Ivirlvland Brace of ijeneral Radiobiology Section, 
i^tional Cancer Institute* 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - - WaTIONkL INoTI'fUT£5_. OF HEaLTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Av^ards k Publications 15. NLi^iD-90 



oERIkL mh'.bER 



16. LIST PUBLIC..TIPN§ QTHEJ^ '^k^ Aap?rij.CTb FRQi' THIS PROJECT DURING 

Calendar year 1956: 

Highman, B, , J. Roshe and P. D, Altland. Production of endo- 
carditis ^^^ith vjtaphylococcus aureus and otreptococcus mitis in 
dogs vd.th aortic insufficiency. Circulation Research, 4: 250, 
1956. 

Brace, K, C, and P. D, iiltland. Life span of the duck and 
chicken erythrocyte as determined with C-*-^. Proc, Soc, Exp, 
Biol, Led., 92: 615, 1956, 

Altland, P. D. and B, Highman, Effects of high altitude ex- 
posures on dogs and on their susceptibility to endocarditis. 
Submitted to J, Aviation Medicine, 

17. LIuT HONORS nlCD A 'ARDS TO PExioONNEL RELi^TING TO THIS PROJECT 

DURING Cia::r,ND/Ji ye.-j:{ 1956: 

None, 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - - i^rtTIONjJ. INoTITUTEo OF HE.-XTH 

INDIVIDU.i PROJECT .iEFO^T 

Part A. Project Description Sheet 1. ITLlI-ID-$i1 



<JJ^ 



E..I..L Wl±,Eli 



2. NIAI-ID 3. Laboratory of Physical Biology 

IFSTirUTE On DI\^ISIuN LaBOrtnTORY, BIW^ICH, OR DEP/.RTi.Li.T 

4. Physiology 5. 

SECTION OR oERVICE LOCi.TIOi^I (IE OIHER TH>J^ BETHE^Da) 

6. Some biophysical and biochemical aspects of insect respiration 
PROJECT TITLE 

7. John B. Buck 

PaiNCIP..L INVESTIGATOR 

Stanley Friedman, 

8. Margaret L. Keister, Helen D. Park, David P« McCarthy 

OTHEil INVESTIGATORS 

9. IF THIS Pi^JECT RESK-BLES, COl^iPLEl'ENTo, OR P.JliiLLSLS REbEaRCH 
DONE ELSE"-HExtE IN THE PUBLIC HEjaLTH SERVICE ('.I'THOUT INTERCKnNGE 
OF PZRoONNEL, FACILITIES OR FUNDo), IDENTIFY oUCH HEoEaRCH: 

(BY SSRI.lL NO.(S) IF ' ITHIN NIH). 

No parallel research in PHS. 

10. PROJECT DEoCRIPTION 

Objectives ; Our unit is pursuing several related aspects of 

insect respiration, among which are (1) carbohydrate metabolism, 
with special reference to the mobilization ijid utilization of 
glycogen, (2) metabolism during the transition from aerobic to 
anaerobic conditions and (3) relation between diffusion and 
mass flow in gas transfer. 

Methods Employed ; Carbohydrate metabolism is being followed by 
chromatographic methods of identification and assay of various 
types of blood sugars, chemical isolation of tissue glycogen, 
and respirometry of intact organisms and of tissues. The 
aerobic-anaerobic transition zone is being studied by measur- 
ing oxygen uptake before, during and after exposure to various 
partial pressures of oxygen. The biophysical aspects of gas 
transfer involve dimensional study of the respiratory system, 
respirometry and computation. 



2 - NI/uMD-91 



SERL-L [MloVR 



10. PROJECT DESCRIPTION - COKTIIWED 



Ma.ior Findinpis ; The work on intennediary metabolism is just 
beginning and has been mainly devoted to methodology. 
Glycogen has been successfully isolated from fly muscle, and 
a device for stimulating flight of flies while v.ithin a 
respirometer flask has been developed. In studies on bee- 
moth pupae given a period of anoxia it has been found that no 
oxygen debt is accumulated but rather that development is 
lengthened by a corresponding amount as if metabolism v/ere in 
total abeyance during anoxia. Gas exchange studies on dia- 
pausing saturniid moth pupae indicates that by restricting 
the respirc-tor;'- valves and storing CO2 in body buffers the 
organism is able to impound CO2 for long periods vdthout inter- 
fering vdth O2 uptake. By a combination of diffusion and mass 
flovr any observed CO2/O2 flux ratio can be accounted for, as 
a rationale for the observed cycles of CO2 retention and sudden 
release it has been shoi.m theoretically that water conservation 
can be enhanced. 

Significance to NlitLS Research : The studies on intermediary 
metabolism are specifically oriented to contribute to the 
Institute' s diabetes program. It is hoped that they may 
shed light on the basic mechanism of glycogen mobilization. 
The anoxia study has already indicated that insects may show 
interesting differences from manmals in regard to oxygen 
debt accumulation and anaerobic metabolism. The biophysical 
study, it is hoped, can contribute to the theoretical know- 
ledge of gas transfer mechanisms in respiratory systems. 

Proposed Course of Project ; The direction of the investigations 
under way was indicated in some detail in last year's report 
and is also suggested in the "Objectives" and "Methods" sec- 
tions above. 



Form No. ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - MTIOWAL INSTITUTES OF HCALTH 

livroiviDUAL mo<rECT report 



Part B: Budget Data 



11, NIAI"D-91 



SERIAL MUMEER 



12. HIDGET DATA: 








ESTIMATED OBLIGATIONS 




mw YtARS 


DIRECT IlEIl'IBmSEIIEir 


TOTAL 


PROF 


OTHER TOTAL 


FY«57 








#UO,600 &5,200 


•1?^5,800 


ii.OO 


1.33 5.33 


HJDGETED POSITIONS 






PATIENT DAYS 


PROF 01" HER 


TOTAL 




FY»57 








U.OO 1.33 


5.33 






13. BUDGET ACTI^'ITYj 









RESEARCH 

REVIEl'J & APPROVAL 

BIOLOGIC STAKTDARDS 



□ 



ADMINISTRATION 

PROFESS lOmL & 
TECHNICAL ASSIST • 
ANCE 



□ 
P 



lU. IDEiCIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVIG^^, OR 
OTHER ORGANIZATIONS, PROVIDING FUI^IDS, FACILITIES, OR PERSOlfi^lEL 
FOR THIS P^^OJECT IN FY 195?. IF COOPERATING UNIT IS iJITHIN NIH 
INDICATE S RIAL N0(3): 



(Use reverse and addj.tional pages, if necessary) 



Calendar Year 1956 

PUBLIC HEALTH SEruVICE NaTIOKaL INoTITUTES OF HEaLTK 

liv'DIVIDUaL PROJECT REPORT 
Part C: Honors, . v;ards & Publications I5. NIhMD-91 



SERIiiL iM'JiEh 



16. LIST PUBLICaTIOFS OTtiTR TH.J-; aBSTR..CTS FROli THK PROJECT DURING 
C/iLEIiD>iR YEaR 1956: 

Buck, J. ti. Triggering of insect spiracular valve. To be 
published in volume on "Physiological Triggers" for the Soc. 
of Gen. Physiologists - in press. 

Hastings, J. V/. and J. B. Buck. The firefly pseudoflash in 
relation to pliotogenic control. Biol, Bullc , 111: (1) 101-113, 
August 1956. 

Park, h. D, i;odif ication of X-ray injury to Hydra littoralis 
by post irradiation treatment v:ith magnesium sulfate and 
glutathione. To appear in October 1956 issue of Biol, Bull. 



17. LIOT HONORS iJvD /;,'aRDS TO PERoOlVTffiL RELATING TO THIS PROJECT 
DURING ChIMDaR YEaR 1956: 

Dr. Buck elected to Executive Coirmiittee, Am. Soc. of Zoologists, 



Calendar Year 1956 

PUBLIC HE;J.'rH SERVICE Ki/^TION^iL INSTI'l-UTEo OF HE-^LTH 

INDIVIDUnL PROJECT REPORT 

Part h. Project Description Sheet 1. MKMD-92 

SERLJL MUI;BER 

2. MIAMD 3. Laboratory of Physical Biology 

INSTITUTE OR DIVISION LHbOR.iTORY, bR.JCCH, On DEPkRTI lEr'T 

4. Physiology 5, 



SECTION OR SERVICE LOCATION (IF OTHER THM BETKEoDh) 

The circulatory response to injection of the synthetic plasma 

6. expanders, dextran and pol;'nAinylpyrrolidone 

PROJECT TITLE 

7. Louise H, I'iarshall 



PRINCIPAL IIVVEoTIGhTOR 
8, Charles H. Hanna 



OTHER INVEoTIGhTORS 



9. IF THIS PROJECT REoEI.BLE:?, COKPLEMEfJTo, Oil PaRhLLELS liESEiiRCH 
D0I>JE else HERE IN THE PUBLIC HEaLTH SERVICE (VJITHOUT INTERCHANGE 
OF P^RSOM'!EL, FaCILITIEo OR FUWDo), IDENTIFY SUCH RZoEm»I: 
(BY SERIAL NO. (S) IF ' ITHIN NIH). 

No parallel research in PHS, 

10. PROJECT DESCRIPTION 

Objectives ; This year's vork has been directed toi-ards investi- 
gation of the circulatory response of sensitive species to 
dextran and polyvinj'lpyrrolidone, both used as synthetic plasma 
expanders. In particular, the viork has differentiated betvreen 
the vasodepressor reaction and that due to capillary permeability 
changes, and has sought to establish conditions under which one 
reaction occurs v;ithout the other. 

Methods Employed : See below. 

Major Findings ; ^Tiile direct measurements of arterial blood 

pressures were being made, dextran was injected into conscious 
or anesthetized rats. The conscious rats were prepared with 
indvrelling carotid cannulae twenty-four hours previously. It 
was found that in conscious animals, or those vjhich had ether, 
there v/as little or no fall in blood pressure after dextran 
v;as injected intravenously, but edema and extravasation of 
dye (T-1824) appeared. Under barbiturate anesthesia, the 
blood pressure decrease occurred, while the gross signs v.'ere 
variable in appearance. Dialysis of dextran solution did 
not alter its ability to produce blood pressure changes or 
gross signs in rats anesthetized with barbiturates. 



NIAMD-92 



SERLiL HULBER 



10. PROJECT DESCRIPTION - CONTINUED 

Ka.jor Findings (cont'd): 

Polyvinylpyrrolidone adiiiinistered intravenoxosly to dogs 
produced a fall in blood pressure and gross symptoms of in- 
creased capillary permeability (edema) whether the animals 
were conscious or anesthetized with pentothal. nfter ether, 
however, the blood pressure decrease was abolished, and the 
appearance of edema was variable, nfter dialysis, poly- 
vinylpyrrolidone lost its vasodepressor activity v/hen injected 
into dogs, but the ability to produce edema was not altered. 

Significance to MJ'liD Research ; Since it has been shown by others 
that these two polymers are histamine-liberators in the rat 
and dog respectively, the present studies are more generally 
significant than is apparent in that they amplify the available 
information regarding the physiological conditions of histamine 
release in the body. 

Proposed Course of Project ; The question of whether or not other 
histamine releasers confer an acute refractoriness to dextran 
in rats and to polyvinylpyrrolidone in dogs vdll be subjected 
to experiment. 



Form No, ORP-1 
October 1?56 
(Attachment I) 



PUBLIC HDILTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL Pi^OJECl' REPORT 



Part R: Budget Data 



11. NIAMD-92 



SERIAL NUIIBER 



12 , BUDGET 


DATA: 












ESTIi'iATED OF-LIGATIOMS 






Mil YEARS 


tlRECT 




R^IMBURSEWNT 


T rjT AL 


PROF 


OTIER TOTAL 


FY»57 












$23,100 


^9,100 


ip32,200 


1,00 


2.50 3.50 






BUDGETED POSITIONS 






PATIENT DAYS 


PROF 




OTffi^ 


TOTAL 




FI»57 












1,00 




2,50 


3.5o 







13. BUDGET activity ;; 
RESEARCH 

REVIEW & APPROVAL 
BI'lLOGIC STANDARDS 



DO 
ilj 



ADMINISTRATION 

PROPESSIOT'!/vL k 
TKCinnCAL ASSISI 
ANCE 



P 

□ 



lU, IDEriTIFY AM COOP^Ri'.TING UNITS OF TIE PUBLIC HEALTH SERVIC:., OR 
OTHER OI^GANIZ/iTIONS, PROVIDING FU'^IDS, FACILITIL3, OR r^CRSONI^IEL 
FOR Tins PROJILCT IN FY 1957. IF COOPERjlTING UNIT IS '-JITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 



PUBLIC miALTti SKWICE - - NKnOWii liMbTITUlEo OF mhLTH 
IWDIVIDUmL P.'^OJLCT HEPO.'^T 

Part C: Honors, awards and Publications 15. BLJ'lI-92 



bSRI-i NUi.BER 



16. LIST PUBLIC^nONd OTHER IHaN ABSTRriCTo FHCJ. IHI^ PROJECT 
DURIi-JG CALZKDnR YEaR 1956: 

I-arshall, L. H. and C, K, Hanna. Direct measurement of 
arterial blood pressure in the guinea pig. I-roc. 3oc. Exp. 
J.ed., 92: 31, 1956. 

17. LIST HONORS ..MD ii'iARDS TO PEH^O:\ZL .IELkTIMCJ TO THL; PROJECT 

du?jj;g CkLend.lR tl^ 1956: 

None, 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - - MhTIONaL li^ioTITU'IES OF HE..LTH 
INDIVIDUiiL PROJECT REPORT 
Part A. Project Description Sheet 1, NTA?^..9'^ 



bERI.iL UUiloER 

2. NiAlD 3. Laborator^^ of Physical Biolo.(?y 

INSTITUTE 0^ DIVISION LnBORATORY, riR.^u.CH, OR DEPaRTIDIK'T 

4. Physiology 5. 

SECTION OR oERVIGE LOCATION (IF OTHi:.R TO,>K DETHESDa) 

6, Ionization studies in the upper atmosphere 

PROJECT TITLE 

7. Herman Ya/jpda 

PRINCIPAL BIVESTIGr.TOR 

8, Mardalee B. Dickinson 

OiHER EJVESTIGaTORS 

9. IF THIS PROJECT RESEIBLES, COI:PLlJ-ENTS, OR PazIaLLELS RESS.JICH 
DONE ELSE HERE IN THE PUBLIC KEhLTH SERVICE (UITHOUT IMTERC?i^iJGE 
OF PERoOKiCL, FACILITIES OR FUNDS), IDEivTIFY oUCH RESE.-diCH: 

(BY oERI/.L NO. (S) IF I THIN NIH). 

No parallel research. 

10. PROJECT DESCRIPTION 

Objectives : Evaluation of the flux, energy and charge spectra 
of the heavy primary nuclei of the cosmic radiation, laying 
particular emphasis on the terminal portion of the trajec- 
tories which are believed to have the most pronounced 
biological action on living tissues. 

Methods Employed ; Exposure of emulsions in rockets and high 
altitude balloons. To secure more direct information, of 
interest to the U. S, Air Force, emulsions have also been 
flo\\fn embedded in piiantoms of the h\aman skull, and as 
external monitors attached to living mice and guinea-pigs. 

Major Findings : The frequency of heavy primary thindoim hits 
emulsion and tissue have been measured over an altitude range 
of 70,000 to 122,000 ft. This data permits interpolation 
to the intensity prevalent in exposures to personnel flying 
present day jet planes and extrapolation to conditions in 
free space of interest in the forseeable future. T\\e data 
has been reported at a meeting of the /.ero-Medical Associa- 
tion and a complete report has been accepted for publica- 
tion in the Journal of aviation i edicine. 



- 2 - NL.MD-93 - •'■ 

SERIAL MUMBLE 

10. PROJECT DESCRIPTION - C0K'TII\IU3D 

Major Findings (cont'd): 

The continuation of our rocket exposures at Vhite Sands 
has brought to light the possible existence of a new type 
of heavy primary radiation v^lth a collision cross section 
considerably greater than geometric. This has been described 
at the N, Y, meeting of the American Physical Society (Bulle- 
tin A, P.O. beries II, 1, 64, 1956) and has aroused interest 
ainjng physicists in view of the similar behavior of the 
anti-proton recently discovered at Berkeley, The possible 
significance of this observation has been discussed by 
several theoreticians at the 6th Annual Rochester Conference 
on High Energy Physics and published in their Proceedings 
(interscience Publishers 1956). 

Significance to KIAI^ID Research ; Basic investigations in 

nuclear phj^sics with significance to the health of military 
personnel flying at altitudes above 60,000 ft. 

Proposed Course of Project ; Development of special monitoring 
techniques permitting the precise tracing of an individual 
heav^'' primary particle into living tissue. This permits 
the histologist to better locate potential sites of bio- 
logical damage. Preliminary studies are under way in 
cooperation \iith Dr. ' ebb Haymaker of the ^rmed Forces 
Institute of Pathology (damage to brain and eye tissue) and 
with Dr. Chase of Broim University on pigmentation changes 
in the hair of black mice exposed to cosmic radiation at 
high altitudes. 

We have also instigated a program of flying eggs and 
egg white, serum albumen, etc., on the theoretical possi- 
bility that sufficient thermal energy is liberated in the 
terminal portion of heavy primaries to cause the coagula- 
tion of the transparent protein. It may be possible to 
discern these filaments of coagulated matter by means of 
high-povfer darkfield microscopy. Their diameter would 
provide a direct estimate of the radius of potential 
biological action of the heavy cosmic ray primaries. 



Form No. ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - NA?IUWAL Ti'ISTITUTES OF HEALTH 
II\roiVIDU/a PROJEG? REPORT 



Part B: Budget Data 



11, NIAI-D-93 



RESEARCH 

REVIEW Jc APPROVAL 

BIOLOGIC STA.i:DAlfflS 





SERML NUi-IBER 

1 




12, BUDGET DATA: 








ESTIMATED OBLIGATIOLTS 




MAN YEARS 


DIRECT 


REIMBUR.S]JIfi;NT TOTAL 


PROF 


OTI-ER TOTAL 


FY'57 








$19,100 


$■7,100 426,200 


1.00 


1,50 2,50 




RIDGETED POSITIO'HS 




PATIENT DAYS 


PROF 


GTHCR TOTAL 




FY'57 








1,00 


1.50 2.50 






13. BUDGET ACT 


IVITY: 







P^l ADinWISTRATIOW 

□ PROFESBIOmi & 

tecmical assis". 
lZI ance 






lli. IDENTIFY ANY COOPERATING UNITS OF TfE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUi\IDS5 FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING Ui'HT IS VdTHIN IMIH 
IITOICATE SimAL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 

PUBLIC EAL'n- SERVICE - - NATION,-.L n;^TITUTEo OF HEaLTH 
INDIVIDUAL P.10JIXT REPORT 

Part C: Honors, y^wards and Publications 15. NLtMD-93 

c:>LiU/vL KUlJil^il 

16. LIST PUBLIChTIOIJS OTHER THkN aBSTR^CTo FROl. THIo PROJ::CT 
DURIilG ChLENDAiI YEhR 1956: 

Yagoda, H. The tracks of nuclear particles. Scientific 
American, 194: (5) 40-47, i ay 1956. 

Yagoda, H. Frequency of thindovm hits by heavj^ primary ^ 
nuclei in emulsion and tissue. J. aviation 1 edicine - in 
press. 

Yagoda, H, Book review, "Physical technioues in biological 
research" - Oster and Pollister. science, 123, 741, April 
27, 1956. 

Yagoda, H. Book review, "The Viking Rocket Gtory" by K. \!. 
Rosen. The bcientific lonthly, 82: (5) 267, I^ay 1956. 

Yagoda, H. Book review, "Radiation Dosimetry" by Hine and 
Brovmell. ocience, 124, 896, November 2, 1956. 

Yagoda, H. Book review, "The Men Behind the Space Rockets" 
by H. Gartmann. The bcientific lonthly - in press. 

17. LIbT HONO.Cb AiD a'.'aRDo TO PE1^0i\,NEL RELATING TO THIo PROJECT 
DURING C^LEKD^R YE.Jl 1956: 

Dr. Yagoda invited to present special papers at (a) ;.ero- 
medical association - Chicago, (b) U. S, Naval Research 
Laboratory seminar, (c) U. S. Bureau of standards seminar, 
and (d) Univ. of Laryland Physics Colloquim seminar. 

Dr. Yagoda acted as consultant for the iiand Corporation on 
upper atmosphere research and frequency of micro-meteorite 
hits. 

Dr. Yagoda' s services requested as consultant for U. S. 
Air Force on Project "Lan-High". 

Dr. Yagoda invited by National Research Council to participate 
in the International Geophysical Year Cosmic Ray Program. 



Calendar Year 1956 

PUBLIC HEALTH SiiRVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part A. Project Description Sheet 



l.iOiiiEiSiL 



SERIAL NUiBER 



2. NIA^'D 3* Laboratory of Physical Biolegy 

INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARK-IENT 



4. Secti o n en Photo biology 5. 

SECTION OR oERVICE LOCATION (IF OTHER THAN BETHEoDii) 



6, Mechanism of Photo Action. 
PROJECT TITLE 



7. Dr. F. S. Brack ett 



PRINCIPAL INVESTIGATOR 



Molecular Structure - Dr. Umer Liddel and Dr. Edidn Becker 
Cellular Action - Dr. Rodn^ Olson and Mrs. Estelle Engel 
Molecular Action - Dr. Norman Sharpless and Miss Dolores Gregory 
Dr. Chamey (from 9/17/56) 

8. Mr. Sh owke ir __^ 

OTHER IrJVESTIGATORS 



9. IF THIS PROJECT liZSJflBLES, COMPLEl'iENTS, OR PARALLELS Rii-dEARCH DONS 
SLSffi.'HERE IN THE I^UBLIC HEALTH SIEVICE ('.ITHOUT Ii-JTIECHaMGE OF PER- 
SONNEL, FACILITIES OR FUNDS) IDEt^'TIFY SUCH RESEARCH: 

No parallel research. 



- 2 - 

SERIAL NUMBER 
10. PROJECT DESCRIPTION: 

Objectives: To gain an understanding ^f the mechanism by which 
radiation affects living cells. 

Emphasis is placed upon processes in which radiation 
contributes essentially to the requirements of living cells 
(in contrast to the destructive acticns of photo-oxidation 
and photc-ionization) . 

Important mechanisms of particular concern are: 

Hydrogai transfer and acceptance. 
C arbo:qyla tio n . 
Phosp horj'-latio n . 
Photo-isomeri zatio n , 

The approach to these broad objectives is through a 
series of projects of more specific and limited objectives 
in association with the investigators indicated. 

Methods Employed; Most of the vcrk of this Section depends to a 
considerable extent upon the original development of instru- 
ments and methods. Advances in the methods and techniques of 
data reduction have been an important aspect of cur instru- 
mental W0l4i. 

Not only are more powerful methods valuable as timesavers 
and so in widening the scope viiich can be encompassed but it 
is found that they reveal types of information which vould 
othermse be missed. Our pioneering in this field dates far 
back of the present recognized trend. I'e introduced the first 
infrared recording at high resolution, the first linearizing by 
cams of the frequency scale, the first programming of slit \^ddth 
and length. Methods of time integration of radiant power were 
perfected. 

Now commercially available caas can replace those cut by 
hand. By developing servo methods we can eliminate mrach of 
the programming. Recent progress includes: 

1) Linearizing - Design of means to adapt constant 
frequency cams frsm P&3 Model No. 21 to our recently purchased 
Model 13 have been worked out and proved successful. Because 
of numerous requests this information is being published. 
Transformation of transmission scale to optical density is 
still the subject of study. 



3 - 



I'iThMyi-g) 



SERIAL NUx^IBER 
PROJECT DESCRIPTION (CONTD): 

Methods Employed (Contd): 

2) Differentiat ing - Servo methods have been adapted to 
recording of oxygen concentration by application of the square 
wave HBthod of platiniun electrode polarography and the record- 
ing of the rate of diange of concentration has been accom- 
plished, together vdth a seven-fold improvement in tiine 
resolution. This has opened vp vjhole new fields of study 

in transient oxygen exchange, 

3) Spatial I ntegration - Spatial integration of 
scattered radiation has been explored, oonparing traverse 
methods iN/lth the time honored integrating sphere. Serious 
defects in the conventional sphere technique have been 
discovered. Zonal integration of scattered light is being 
studied, 

Ma.ior Findings ; To be discussed under the separate project 
heads , 

Significance to Research of the Institute ; A basic under- 
standing of the laechanlsra of the action of radiation in 
cells vculd answer some of the most fundamental problems 
of living things, such as how energy is stored and made 
available. Almost as fundaiaental is the role of radiation 
in steroid transformation, particularly production of 
Vitar.jin D, vdiich appears to be a very general requirement. 
Hydrogen bonds are no doubt of key importance in biological 
processes, ijhile our findings appear remote from immediate 
medical practice, advances In biological understanding 
usxmlly bear fruit sooner than one can anticipate. 

Proposed Cou r se of Project ; Integration of optical density in 
spectral bands over the frequenqy range of component contri- 
bution is the logical basis for quantitative evaluation of 
spectra. An analogue breakdown into band conponents may be 
the right answer aid offers the possibility of developing 
models of nolecular significance. It is hoped that this 
field can be explored. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIOi-IAL INSTITUTES 0? HEALTH 
IIOIVIDUAL PROJECT REPORT 



Part B: Budget 


Data 11, NIAMD-9U 






SE 


ilAL NUiiBER 




12: BUDGET DATA 


• 








ESTIMATED OBLIGATIONS 




MAN YE/iRS 


DIRECT 


REIMBimSEMENT TOTAL 


PROF 


OTIffiR TOTAL 


FY»57 








$33,200 


1.12,100 |il5,300 


1,67 


2.00 3.67 




BUDGETED POSITIONS 




PATIF.NT DAYS 


PROF 


OTHER TOTAL 




FY'57 








2.67 


2,00 ii.67 







13. BUDGET ACTIVITY; 

RESEARCH g^ 

REVI5VJ & APPROVAL Q 

BIOLOGIC STAilDARDS \~~\ 



ADMINISTR/>.TION 

PR0FES';'.IJNAL & 
TECH,jICAL ASSIST- 
ANCE 



□ 



□ 



lU, IDEivITIFY ANY COO?ER/'/:iNG UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS PROVIDING FINDS, FACILITIES, OR PERSONiM. 
FOR T^HS PROJECT IN FY ly5'7. IF COOPER/iTING UNIT IS iriTHTN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year I956 



PUBLIC HEALTH oERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors, Awards & Publications 15. NL.i-iD-9U 

oiiRIAL NUiiB£R 



16. LIST PUBLICATIONS OTHSR THAN ABSTRACTS FROM THIS PROJECT DURING 
CiiLLl©AR YEAR 1956: 

Brack ett, F. S., J. H. Daniel and R. G. Crickard, Recording 
Oxygen Concentration and Rate of Exchange. Submitted for 
publication in Review of Scientific Instruments. 

Mattem, C. F. T., Brackett, F. S., and Olrcn, B. J., Determi- 
nation of nuiiiber and size of particles by electrical gating. 
I. Blood cells. J. of Applied Physics (in press). 

Book Review of "Essentials of biological and medical physics", 
F. S. Brackett, Sciaice 123: (3207), 1086-108? (1956). 

Book Review of "Physical Techniques in Biological Research, 
Vol. 1. Optical Techniques", F. S. Brackett, Rev, Sci, Inst. 
27 (5), 319 (1956). 

Book Review of "Recent advances in optics", F, S. Brackett, 
Rev. sci. Inst., 2? (6), 404 (1956). 



17. LIST HONORS AND AVARDS TO i^ERSONNEL RELATIi^G TO THIS PROJECT DURIIC 
CALENDAR YEAR 1956: 

None. 



Calendar Year 1956 

PUBLIC HEALTH SLRVICE - NATIONAL irETITUTES OF HEALTH 
INDIVIDUAL PROJECT ItEi'ORT 

Part A. Project Description Sheet 1. NL'.MD~9'^ 



SERL.L NO. 



2. NIAM D 3. Laboratory of Physical B iology 

INoHTUTE OR DIVISION LADORAIDRY," BRAI^JCH, OR DEPaRTI'IEIW 



4, Photobiology 5. 

SECTION OR SrJRVTCE ' LOCAHO'n (if OTHKR THaN BETHEoDA) 



6, Molecul ar st ructi^re d etermined ty spectroscop ic me thods 

PROJECT nTLE 



7. Umer Liddel 



PRINCIPAL IKTVESTIGATOR 



8, Edvan D. Becker 



OTHER INVESTIGATORS 



9. IF THIS PROJECT RESI^iBLES, GOIiPLEMENTS, OR PARALLELS RESEARCH DONE 
ELSE HERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PER- 
SONI^"EL, FACILITIES OR FUNDS) IDx3^TIFY oUCH RESEARCH: 

None. 



10. PROJECT DESCRIPTION: 

Ob^ iectives ; (1) An understanding of the forces between atoms 
in molecules of biological in^ortance. (2) Development of 
precision analytical methods for certain components cf 
molecules of biological importance. 



- 2 - 

PROJECT DESCRIPTION: (CONTD) bERIAL t«JUiviBER 

Methods Employed ; Absorption of light in the infrared region 
of the spectrum is generally recognized as providing valuable 
information on the composition and structure of molecules. 
Since the infrared spectrum is particularly sensitive to 
hydrogen bond forhiation we have found that the study of 
infrared spectra provides a useful tool for investigating 
such interactions. 

Through the courtesy of Varian Associates we have been 
able to ei(plore the newer technique of nuclear magnetic 
resonance. This method appears to be capable of furnishing 
val liable supplementaiy information on hydrogen bonding forces. 



Ma,ior Findings : Much of our effort has been directed toward the 
determination of equilibrium constants and heats of formation 
for hydrogen bonds in several simple alcohols. We have been 
able to correlate these thermodynamic quantities with frequency 
and intensity features of the spectra in order to provide a 
convenient and rapid method of estimating thermodynamic 
quantities from the spectra of oonplex molecules. Our nuclear 
magnetic resonance studies of ethanol have been successfully 
correlated with infrared spectral resiits. By means of 
infrared and nuclear magnetic resonance studies we have 
found that weak hydrogen bonding exists between chloroform 
molecules. Our preliminary studies of intraiiiolecular 
hydrogen bonds in such conpounds as aldol and dihydroxy- 
anthraquinones have revealed sigiif leant differences from 
the intenuolecular alcohol bonds mentioned above. 



Significanc e to Research of the Institute ; Protein molecules are 
generally thought to be held together b^y- hydrogen bonds. The 
close resemblance in behavior (sensitivity to temperature and 
chemical environment) betvreen proteins and the simple hydrogen 
bonding systems now under study strongly suggests the inportance 
of hydrogen bonds in determiiiing the properties of proteins, 
V/e believe that a basic knowledge of the characteristics of 
hydrogen bonding wLU provide a better understanding of 
biological processes and may suggest structural modifications 
in proteins that can be used to control such processes. 



Proposed Course of Project : lie plan to continue these investi- 
gations along several lines: (1) The studies already begun 
on intramolecular hydrogen bonding will be continued. (2) 
The techniques developed in the study of hydixigai bonding between 



- 3 - 

PROJECT DESCRIPTION: (CONT'D) NL>liD-95 



SERIAL IWi-BER 



Proposed Co urse of Pro.jec t; (Cont ' d) 

alcohol molecules will be applied to investigations of 
hydrogen bonding in related, but more complex systems. The 
correlations made between thermodynamic and spectroscopic 
quantities will be extended. (3) Modifications of spectre- 
scopic instruments are planned to facilitate these studies. 
(4) The spectral analysis of small sajnples by means of an 
infrared microscope will be exj^lored. (5) A request has 
been submitted for the purchase of nuclear magnetic 
resonance equipment. Ve plan to use this equipment to 
extend the present scope of molecular structure investi- 
gations , 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIOI^IaL IKS?I?JTES OF HEALTH 
IiroiVIDUAL PROJEC? REPOR? 



part B: Budget Data 



11. MIA.MD-95 



SLHLiL iJUillER 



12. BUDGiL? Bi-. 


TA: 








ESTIi'ift.?ED OBLIGATIONS 





VAV. YEARS 


DIRECT 


RHKBURSEi^riT TOTAL 


PROF 


OTHER TCTaL 


FY'57 








^21,000 


V8,100 $29,100 


1,00 


1,00 2,00 




BUDGETED POSITIONS 




PATiniT DAIS 


PROF 


OT'HER TOTAL 




FI«57 








1,00 


1,00 2.00 






13. BUDG"T AC 


TIHTY: 







RESEARCH 

REVIEl'f & APPROVAL 

BIOLOGIC STAkmRDS 



W 



ADMINISTRATION 



PROFESSIOML & 
TECH::IGAI AS3IS': 
I i AKCE 






lh» IDEIITIFY AM COOPERJl^'ING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
0TH:R ORGANIZATIONS, PROVIDING FUNDS, FAGIL.ITIE3, OR PERSCMEL 
FOR TIIS PROJECT IN FY 1957. IF COOPER..TING UNIT IT 'JITHIil NIH 
rivEDICATE SERIAL NO(S): 



(Use reverse and additional pages if necessary) 



Calendar Year 1956 

PUBLIC tiE/iLTH SERVICE - NATIOM;\L INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT RSPORT 

Part C: Honors, Awards & Publications 15. m t,'M n-p5 

SERIAL NUMBER 



16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 

CAL:a^]Di.ui year 1956: 

Liddel, Umer and Edwin D. Becker, Letter to the Editor, "Tempera- 
ture Dependent Absoi^ption by CHoOH and CHCL, in the 3 Micr,-:n 
Region", J. Chera. Phys. 2^, 173' (1956). 



17, UST HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING 
CALi©AR YEAR 1956: 

None. 



Calendar Year 1956 

PUBLIC I-EaLTH oEIiVICE - - NaTIONaL INSTI'IUTEo OF HiiliiLTH 
Ir;DIVIDU«L PROJECT iffiPORT 

Part .i. Project Description Sheet 1. NT>iMn-9(^ 



SERIaL im.beh 

2. M.J-D 3. Laboratory of Physical Biolo/^y 

INoTITUTE OR DIVIoION LiiBORnTORY, Biu.iJCH, OR DEPARTf^NT 

4. Photobiology 5. 

cECTIO?J OR oERVICE LOCATION (IF OTH.'-R 'fflAi'.' BETHESDa) 

Effects of radiant energy on the oxidative metabolism and 

6. chemosynthesis in living cells ______ 

PROJECT TITLE 

7. Rodney A. Olson __^ ——— 

PRIirciPAL INVESTIGaTOiI 

8. Robert G. Crickard (until 10-27-56 resignation); Estelle Engel 
OTHER INVEbTIGATORS 

9. IF THIi- PROJECT REoEUBLSo, COMPLEl'lENTi) , OR PmiALLELo tEoEaRCH 
DOIS. jA^ir HERE IW THE PUBLIC HEi.LTH oERVICE C ITHOUT INTER- 
CH:UMGE OF PERoOKNEL, FaCILITISd OR FUNDo), IDEIJTIFY ^UCH 
RESEARCH: 

None. 

10. PROJECT DESCRIPTION: 

Objectives : To study the specific effects of radiant energy on 
cellular oxidative metabolism and chemosynthesis utilizing 
the ultraviolet, visible, and infrared regions ©f the 
spectrum. To determine the role and identity of enzjTnes, 
photosensitizers, and pigments in the photochemical and 
other reactions involved. In addition, to identify the 
intracellular morphological changes associated vdth such 
metabolic effects in order to localize reaction sites and 
participating cell structures or inclusions. 

M ethods Employed ; Cell suspensions of unicellular organisms 
(Chlorella, etc. ) cultured aseptically under various experi- 
mental conditions are irradiated in monochromator systems 
designed to provide homogeneous radiation of all cells. 
Simultaneous measurements of oxygen concentration and the 
rate of oxygen metabolism during prescribed radiation and 
dark intervals under various conditions of intensity, 
wavelength, dark adaptation substrate availability, pigment 



- 2 - NL.MD-96 



t 

10. PriOJI^;CT DESCRIPTION (CONT'D): 

Objectives (Cont'd ) : 

density and specific enzyme inhibitors are provided at 3- 
second intervals by a fast electronic pen recording 
polarographic instrument developed for this purpose (see 
Braclcett). These measui'ements when correlated with optical 
deteniiinations in these cells of the concomitant transient 
changes in energy absorbed, selective absorption, quenching 
of fluorescence, and luminescence due to back reaction pro- 
vide quantum; and kinetic data leading to the identity and 
nature of the photochemical and dark reactions involved. 
High resolution monochromatic photoiricrography combined with 
histochemical technioues provide morphological data concern- 
ing the sites of action vjithin the cell. 

Major Fjndii],(7s : Continued study of the short period (3-9 

seconus) transient responses of oxygen metabolism to light 
and dark under a wide variety of controlling factors in 
cell suspensions of Ch lorella has provided infonriation 
which should lead to further identification of the metabolic 
intermediates and the reactions involved. This has been 
accomplished by a complete and improved reconstruction of 
the appaiatus (during the moving and refitting of the 
laboratory) providing (1) increased light intensity well 
beyond the saturation level for oxygen evolution, (2) avail- 
ability of this intensity level at vjavelengths 4050 8, 4350 
°, 5490 2, 5780 2, (3) enhanced sensitivity and stability 
of oxj'gen metabolic rate measurements, and (4) simultaneous 
measurement of transient changes in the level of chloro- 
phyll fluorescence which accompany the oxygen changes during 
light. 

A prelajTiinary comparison of the response of the initial 
burst of oxygen on illumination with the subsequent steady 
state rate under varying conditions of temperature, light 
intensity and wavelength, specific inliibitors, and sub- 
strate availability establishes the separate identity of the 
two processes involved. The b\irst process appears to be 
photochemical in nature, occurring at all wavelengths but 
saturating at a much lower intensity than the steady state 
process. Similar conclusions are offered by the response 
of simultaneous fluorescence transients to these varying 
conditions. 

Ijhile the oxygen burst effect persists in the absence 
of CO2 and the steady state does not, careful analysis of 
rise time characteristics of transient curves vn.th and 
without CO2 do not show conclusively a distinct segrega- 



- 3 - uinm-96 ■■ ■'■' 

10. P:iOJECT DE&CrilPTION (CONT'D): 
Major Findings (Cont'd) ; 

tion of the burst process from carboxylation. Since 
complete absence of residual intracellular CO2 cannot be 
assured, adequate evidence for this hypothesis awaits 
precise simultaneous rate measurements of CO2 uptake in 
cell suspensions for which no method currently exists. 

Similarly, evd.dence for or against the presumption 
that the immediate gulp of oxygen on the removal of 
illumination is a reversal of the burst reaction awaits 
simultaneous measurement of CO2 exchange rates. In this 
respect an electrolytic in situ method for the measure- 
ment of CO2 in aqueous solutions is being explored. 

Much of the work with specific inhibitors has been 
delayed by complications involving the indirect action 
of these substances on the behavior of the oxygen elec- 
trode, oteps have been taken to eliminate these effects 
by the construction of special electrodes which exclude 
such materials. 

The parallel investigation of cell particulate and 
chloroplast morphology and physiology in C hlorella has 
been advanced by a nevj method of isolating in quantity 
intact C hlorell a chloroplasts, Fxu-ther selective dis- 
integration of these chloroplasts yields a suspension 
of lamellate particles which conform in size and shape 
to the lanimelar distribution of chlorophyll in the 
intact chloroplast (observed in this study) by monochromatic 
photomicrography in the maxima of the absorption bands 
and fluorescence photomicrography. The pigments 
(carotinoids as well as chlorophylls) appear tc be con- 
centro.ted on these laramelar units. Exceedingly nigh 
resolution photomicrographs in the 4050 2 region have 
been attained by suspension in Bovine plasma albumen 
with matching refractive index. Cell particulates of 
this type have been used in the oxygen metabolism 
studies but their routine use awaits evaluation of 
techniques for their complete isolation from the fev; 
but omnipresent intact cells. 

Significance to Resea r ch of the Institute : Unicellular 
organisms with special metabolic adaptation for the 
utilization of radiant energy in chemosynthesis carry 
out to a marked degree but by different pathways the 
same general metabolic reactions which occur in the 
more specialized cells of higher organisms. During 



_ 4 - NIAMD-56 - *y 

^ SERiAlTNiri'.'bEii 

PROJECT DESCRIPTION (CONT'D): 

Sif^nificance to Research of the Institute ; 

the synthesis of proteins, fats, oils, steroids and other cell 
components some mechanism for CO2 assimilation is now con- 
sidered to be operable in the normal metabolism of all cells. 
In cells not adapted to utilize radiant energy the energy 
source for this process is provided chemically via phosphate 
cr other energy transferring systems. Radiant energy thereby be- 
comes a tool by which specific metabolic pathways may be induced 
in photo synthetic organisms without changing in any other way 
the physical or chemical environment of the cell. Use of the 
appropriate wavelength and intensity of radiation may provide 
considerable choice in the degree of participation by knovm pig- 
ments in both synthesis and oxidative reactions while the dura- 
tion and intensity of light allows for a kinetic study of the 
carboxylation-decarboxylation equilibrium. Information in this 
direction leads to a better understanding of the more subtle 
metabolic pathways which characterize anomalies in physiologically 
diseased cells. 

P roposed Course of Pro.ject ; Immediate emphasis on further systematic 
study of the effect on O2 transients of physical factors (intensity, 
wavelength, temperature, etc.) and physiological factors (Op and 
CO2 concentrations, glucose and other substrate concentrations, and 
specific inhibitors). Determination of possible linkage of 
transients with dark oxidative phosphorylation and photophosphory- 
lation via specific effects of phosphorylating decoupling agents. 
Determination of all these factors on the behavior of other 
transients (CO2, quenching of fluorescence, luminescence, etc.) 
and subsequent correlation with O2 transients. Substitution of 
cell particulate components (chloroplasts, etc.) for intact cells 
in above studies in order to isolate specific component reactions 
and to allow addition of various phosphoric esters unable to 
penetrate the intact cell. Continuation of cellular morphological 
studies and histochemical techniques for the determination of 
cellular sites of action. Further development of CO2 and "ion 
exclusion" electrodes. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HI';ALTH service - NATIONAL INSTITUTES OF HE/iLTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAI€)-96 



SERIAL NUMBER 



RESEARCH 

REVIKU & APPROVAL 

BIOLOGIC STANDARDS 



12, BUDGET DATA: 










ESTIMATED OBLIGATIONS 






MAW YEARS 


DIRECT 


REIMBURSEMEl>C 


TOTAL 


PROF 


OTHER TOTAL 


IY<57 










^U,300 


$9,100 
BUDGETED POSITIOI^B 


$33,UOO 


2»67 


2.67 

PATIEir DAYS 


PROF 


OTHER 


TOTAL 




FY«57 










3.67 


M 


3,67 






13. BUDGPr ACTIVITY: 









lH! 



ADIilNISTRATION 

PROFESSIOML & 
TECHi^lICilL ASSIST- 
ANCE 



□ 



111. IDEiCIFY ALIY COOPERATING UIOTS OF THE PUH.IC HE/iLTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING l^NDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF C ..OPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and addititional pages, if necessary) 



Calendar Year 1956 

PUBLIC HEALTH oE.WICE - - NaTIONhL INoTIl-UTEo OF HEALTH 

INDIVIDUAL PROJECT RJ^PORT 

Part C: Honors, Awards & Publications 15. NIaMD-96 

oERIi.L NUl;BEIi 

16. LKT PUBLICATIONS OTHER THaN ABcTrUCTb FROli THIS PROJECT 
DUxRING CaLEIJDaR YEaR I956: 

None, 

17. LIoT HONORS AND aUaRDS TO PERSONNEL RELxTIX TO THIo PROJECT 

DURING Calendar ye^ 1956: 

None, 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1 . NI/.M D-?7 

bERIAL NUi'IBER 



2. NIAI'^JD 3. Laborator y o f Physical Biology 

INSTITUTE OR DIVISION LAPORhTORY, BRANCH, OR DEPARTMENT 



4. Section on Photobiology 5. ____. 

SECTION OR SERVICE LOCATION Ti"F OTHER TtlAN B:lTHLSDA) 

Investigations of the action of radiant energy on biologically 

6 . important compounds. 

PROJECT TITLE 



7. Dr. Norman E. Sharpless 



PRINCIPAL INVESTIGATOR 



Mrs. Janet Munday, until resignation 5-1-56 
8. Miss Dolores Gregory, from 8-27-56 



OTHER IWESTIGATORS 



9. IF THIS PROJECT RESEi'iBLES, COi'IPLEi-IENTS, OR PARALLELS RESEARCH DONE 
ELSEjHERE IN THE PUBLIC HEALTH SERVICE (VilTHOUT INTERCHANGE OR PER«- 
SONNEL, FACILITIES OR PUNDS) IDENTIFY SUCH RESEARCH: 

None. 



10. PROJECT DESCRIPTION: 

Objectives: The objectives of this project are the establishment 
of the structures of various biologically important intermediates 
in photobiology and to study their kinetics and other physical 
chonical properties. 

I'iethods Employed ; Ultraviolet or visible radiation is the tool 
employed to effect any alterations in the materials, and 
spectroscopy in the ultraviolet, visible and infrared 
regions is the major method used to evaluate any changes 
occurring. 



- 2 - 

UB.m-9 7 • 

SERIAL iWiiBr^ 



PROJECT DESCRIPnON (CONTD): 



Fa,1or Findi ngs ; The available literature on the infrared spectra 
cf ergosterol aid lumisterol (as acetates) indicated that 
both compounds liave identical spectra. Using the unacetylated 
compounds we have been unable to confirm this. It would be 
surprising if tvo compounds, even as closely related as 
ergosterol and luinisterol should have the same spectra. 

An investigation of calcium and phosphorous metabolism 
controllirjg vitajnins leads naturally to interest in the 
so-called "high energy phosphate bond". An investigation 
of the infrared spectra of adenosine nxjno-, di- and 
triphosphates showed that while the di- and triphosphates 
had reasonably similar spectra, adenosine monophosphate was 
s;if ficiently different, especially in tlie 1700 cm~l region 
and the 1000 cm"-^ region to warrant further investigation. 
Since the structural differences in these compounds reside 
in the presence or absaice of pyrophosphate linkages, this 
infrared spectra investigation has been started from this 
angle. 

A series of inorganic pyrophosphates was synthesized 
and their infrared spectra run. The spectra showed in 
general a strong band in the region of 1060-1121 cm"^; a 
moderate band in the region of 923-979 cnrl; a doublet 
group at 990-1011 and 1026-1050 crn-l; and a low band at 
722-733 cm-1. 

There were individual differences also. Some ccmpounds 
showed a band at about 1200 cm~l (lig, Co, Zn and Ni), vihlle 
the others did not (Na, Ba, Cd and Mn) . 

Manganese pyrophosphate was an excgition to the general 
rule, showing only a very sin^jle spectmm, with three bands, 
at 962, 1103 and 1142 cm"!. 

The investigation of the infrared spectra of the 
12-heteropoly acid salts has been completed and is ready 
for publication. An equation has been derived making it 
possible to calculate extinction coefficients for potassium 
bromide pellets. Using this equation it has been possible 
to give intensities for the various bands in the spectra, 
pennitting a quantitative con?)arison between related conpounds. 
Utilizing this, it was shoi-m that conpounds in the heteropoly 
acid series having the same central atoms have more nearly 
alike spectra than those with the same surrounding atoms. In 
this series also the spectmm of a manganese compound. 



- 3 - 

NL.MD~97 ' 

SK-tlAL NUiBER 

PROJECT DESCRIPTION (CONTDj: 

Majo r Findings (Contd ) : 

aramoniuni 12-inangano)iJolybdate was found to differ vddely 
from the spectra of the other compounds investigated, 
being simpler and less defined. 

Tentative assignments have been made relating some 
infrared bands to vibrating atom groups vdthin these 
molecules . 

Signific ance to the Researcli of the Institute : Fundaiaental 
investigations into the structures and reactivities of 
biologically importait molecules are basic to the under- 
standing of their actions in the cell or on the organism, 
as well as being necessary for the development of similarly 
acting substances or antagonists. 

Propose d Cou rse of Proje ct; A conprehensive continuation of the 
kinetics and mechanism of the vitamin D series, transfor- 
mation, utilizing infrared and ultraviolet spectroscopy for 
establishing the structures of various intermediates and 
related conpounds. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
BIDIVIDUAL PnOJECT REPORT 



Part B; Budget Data 



11. NIAi€)-97 



SERIAL NUi»mER 



RESEARCH 

REVIHJ & APPROVAL 

BIOLOGIC STANDARDS 



QJ ADMINISTRATION 

1 i PROFESSIONAL & 

TECHrtlC/iL ASSIST- 
HZ! ANCE 



12. BUDGET DATA 


I 








EST MATED OBLIGATI ONS 




ITAN YEARS 


DIRECT 


REIMBURSEMEKiT TOTjM, 


PROF 


OTHER TOTAL 


FY«57 








$iU,ioo 


?J5,000 iil9,100 


1.00 


»67 1.67 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTFIER TOTAL 




FY'57 








1,00 


.67 1.67 






13. BUDGET ACTIVITY; 










Hi. IDEAlTIFY ANY C00P::RATING UNITS OF THE PUBLIC HE/\LTH SERVICF,, OR 
OTFER ORGANIZATIONS, PROVIDING FUiTOS, FACILITIES, OR PERSON'NEL 
FOR THIS PR0JI3CT IN FY 19^7. IF COOPER/ITING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessajry) 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPCET 

Part C: Honors, Awards & Publications 15, N LiIffi -97 



alMAL MUIiBER 



16. UST PUBLICATIONS OTHER THAN ABSTRACTS FROM THId PROJECT DURING 
Ci-iLSNDAR YEAR 1956: 



None, 



17. LIST HONORS AND AVIARDS TO PERSOmiEL RELATING TO THIS PROJECT DURING 
CALENDAR YEAR 1956: 



None. 



Calendar Year 1956 

PUBLIC HEALTH SERVICE — NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. Ki;iMD-96 



SERIAL NUMBER 

National Institute of Arthritis 

2- and Met,-..b olic Diseases 3. If,boratori_of Physical Biology 

INSTITUTE OR DIVISION LABORATORY, bii/J^CH, OR DEPARTIffiNT 

4. Molecular Biophysics 5. 



SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA) 

6. Investigation of the macromolecular organization of livi ng matter, 
PROJECT TITLE " ^ ~" 



^r.Jlalph W. G. Wi;]3koff 



PRINCIPAL INVESTIGATOR 



Dr. L. W. Labaw Dr. C. E. Challice Persis Griffin 
8. Mr. V. M. Mosley (Visiting Scientist) Karen Zehner 



OTHER IMESTIGATORS 



9. IF THIS PROJECT RESEMBLES, COMPLEIvIENTS , OR PARALLELS RESEARCH DONE 
ELSEWHERE IN THE PUBLIC IIE.ILTH SERVICE (WITHOUT INTERCHANGE OF PER- 
SONNEL, FACILITIES OR FUNDS) IDENTIFY SUCH RESEARCH: (BY SERIAL 
NO. (S) IF WITHIN NIH). 



NO PARALLEL RESEARCH IN PH3 



10, PROJECT DESCRIPTION: 



Objectives: To gain information about the macromolecules that are 
essential constituents of living matter, to see how they are 
arranged in the structures they form and to see how this 
arrangement is altered by infectious and degenerative disease. 
To study certain of these macromolecules (such as viruses) in 
purified form after isolation from the living material. 



NIi.MD-98 - ■ 
SERLkL NO.' 



- 2 - 



10. PROJECT DESCRIPTION - CONTINUED 

Methods Employed: The electron ndcroscopy of microorganisms, 
cells, and tissues in suspension or thinly sectioned; the 
physicochemical characterization of macromolecular components 
isolated from such material using electron microscopy. X-ray 
diffraction, and similar established techniques; the develop- 
ment of new physical procedures, including X-ray microscopy, 
to further such characterization. 

Major Findings: The work done in Bethesda during the last year 
has proceeded along the following main lines: 

(1) There has been a continuing steady improvement in the 
resolution of our electron microscopes through the develop- 
ment and use of modifications of the commercial instruments 
at our disposal. This has permitted us successfully to 
record the molecular order in crystals of proteins having 
molecular weights of ca 50,000 and particles of a diameter 
of ca 30 A. It has also permitted the direct photography 
of molecular spacings of ca 11 A in organic crystals of a 
molecular weight of only about 500. It is to be anticipated 
that such success with substances having molecules far 
smaller than those of the proteins previously investigated 
will lead to important extensions of high resolution electron 
microscopy as the requisite techniques are further developed. 

(2) Collaborative studies are continuing with the Pasteur 
Institute on the development of viruses within tissues. 
During the past year the principal investigator spent 
several weeks at the Institute in Paris working on this 
project and using for the purpose the new and much superior 
Siemens electron microscope recently installed there. 
Considerable progress has been made over the past year in 
extending knowledge of how the virus of herpes simplex 
develops within the tissues of several hosts and a 
significant advance has been made in deciphering essential 
steps in the groirth of the virus of vaccinia. 

(3) Very important progress has been made in developing 
methods of X-ray microscopy so that they can be applied to 
unstained tissue sections no thicker than those routinely 
employed for optical microscopy. By arranging existing 
microscopes for operation in vacuo and by employing X-ray 
targets of very light metals satisfactory photographs have 
been obtained of sections of soft tissues, such as kidney. 



NL^MD-98_ 
SERIAL NOc 



- 3 - 



10. PROJECT DESCRIPTION - CONTINUED 

Major Findings: 

no more than 5 micra thick. A series of studies 
employing these techniques and dealing '.d.th the 
development of teeth is now being carried out in 
collaboration vfith the Dental Institute. 

iU) Further collaborative work with the Dental 
Institute has resulted in important improvements 
in the methods required for routinely cutting the 
ultrathin sections required for high resolution 
electron microscopy of tissues. 



Form No. ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HE/U.TH S^JIVICE - NATIONAL INSTITUTES OF HFALTH 
ItJDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAI.iD-98 



SERIAL Nm-IEER 



12. BirDGlvT DAT 


Aj. 








ESTIiATED OBLIGATIONS 




MAW YEAHS 


DIRECT 


REIMBURSEMENT TCTAL 


moF 


OTHI.R TOTAL 


FY '57 








$63,600 


;it2U,200 $87,800 


3o33 


3.50 6.83 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTIER TOTAL 




FY»57 








U.33 


3=50 7.83 






13. BUDGET ACT 


IVITY: 







RESEARCH 

REVIEIJ &. APPROVAL 

BIOLOGIC STANDARDS 






ADI'ilNISTRATIOW 



PROFESSIONAL & 
TECIKICAL ASSIS1 
ANCE 



□ 



lU. IDENTIFY ANY COOPERATING UNITS OF THE PUBIJC HEALTH SJ31VICE, OR 
OTHER ORGANIC- ATI ONS, PROVIDING FURiDS, FACILITIES, OR PERSONNEL 
FOR THIS PROjr.CT IN FY 1957. IF COOPLRj-.TING UNIT IS liTTTHIN NIH 
INDICATE SE^miL N0(S): 



(Use reverse and additional pages, if necessary) 



Calendar Year 1956 
- 5 - 

PUBLIC HEALTH SEiWICE — NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C. Honors, Awards & Publications 15. N L:MP-98 



SERIAL NUMBER 

16. PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
CALENDAR YEAR 1956: 

Labaw, L. W. and Wyckoff , R. W. G., The electron microscopy 
of mnute crystalline detail. Proc. of the Royal Academj^ 
of Sciences (Netherlands). ~ IN PRESS 

Labaw, L. W, and Wyckoff, R. W. G., The structure of 

southern bean mosaic virus protein crystals. Archives 
of Biochemistry and Biophysics. — IN PRESS 

Mosley, V. M., Scott, D. B. and Wyckoff, R. w. G., The X-ray 

microscopy of thin tissue sections. Science, 12^: 683, 1956. 

Wyckoff Ralph W. G. , Viruses and macromolecules studied 
with the electron microscope and ultracentrif uge . 
IRE Transactions on Medical Electronics, PGME-6, 
pp. 4.9-55, October, 1956. (Paper read at the S^posium 
on Biophysics, American Physical Society, Annual Meeting 
hold m the Hotel New Yorker, New York, Wednesday, 
February 1, 1956) . "^ ' 

Wyckoff Ralph W. G La Structure des Cristaux Macromole'culaires 
au Microscope Electronique. The Bulletin of the French 
Society of Optics. (This is a condensation of a lecture 
IMPRESS ^^^ ^^^^^"^ Institut, Paris, France, May 1956). - 

17. LIST HONORS AND AWARDS TO PERSONNEL RELATING TO miS PROJECT 
DURING CALENDAR YEAR 1956: ^"Uj£,tX 

NONE 



PUBLIC HEALTH S:aRVICE - NATIONAL INSTITUTES OF HEALTH 
IiroiVIDUAL PROJECT REi'ORT 

Part A. Project Description Sheet 1, NLJ4D- 99 



SERIAL NUiiBER 



2. NIAMD 3. Laboratory of Physical Biology 

INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DlilP ARTi-iENT 



4. Section on Moleoular Biophysics 5. 



SECTION OR SERVICE LOCATION (IF OTHoR THAi^ BjJTHEoDA 



The Physical Chemistiy of Manbranes and Complex Manbrane Systems 

6 , of Biologic a l Interest 

PROJECT TITLE 



7. Karl SoUner 



PRINCIPAL INVESTIGATOR 



Dr. Joseph vfagner. Research Associate on the Visiting Scientist 
Program until Sept, 7, 1956| Dr. Norman Gershfeld, teiminated as 
of November 1956; Dr. Marc Lewis, Post-Do ctcrate Fellow. 
8, (Dr. Rex N eihof was on leave of absence throughout the year. 

OTHER INVESTIGATORS 



9. IF THIS PROJECT RESEMBLES, OOliFiEI'ENTS, OR PARALLELS RESEARCH DONE 
ELSE'.JHERE IN THE PUBLIC HEALTH SERVICE (ittTHOUT ICTERCHANGE OF PER- 
SONNEL, FACILITISb OR FUNDS) IDIj^ITIFY SUCH RESEARCH: 

None. 



niM D-9 9 



SERIAL mi'EER 
10. PROJECT DESCRIPTION: 

Objectives ; A physicocheraical study of membranes and 

membrane mcdel systems vdth the purpose cf providing a rational 
phy si CO chemical basis for the elucidation of numerous phenomena 
in living organisms, for instance, electrolyte balance and electro- 
lyte distribution, the accumulation of electrclytes in living 
cells, cell and nerve potentials, and electrophysiology in general. 



Methods Employed : The preparation of membranes of highly character- 
istic and specific electrochemical properties (the msthcds having 
been worked out by the principal investigator and his collabk^ra- 
ters), and the investigation cf these membranes, and cf membrane 
systems in which such membranes are the functional part, by 
physicochemical, especially electrochemical methods, such as 
potential and resistance measurements, also ty chemical 
analytical procedures, including radioactive tracer methods. 



Ma.jer Findings ; The rate of self-exchange of ions across perm- 
selective membranes, as rgsorted last year, can be predicted 
quantitatively from a knowledge of the electrical resistance 
cf the membranes under the same conditions, except in the case 
of certain anion-selective membranes prepared vdth synthetic 
cationic polyelectrolytes. VJith such membranes the rates 
of self -exchange of ions are much higher than those calcu- 
lated from the membrane resistance. The cause of this anomaly 
was found, in cooperation with Dr. ijagner, to be due not to 
a peculiarity of the bulk phases of these membranes but to 
some as yet poorly understood interfacial process vdiich is 
rate-determining in the electrical transfer of ions across 
the phase boundaries but not in the spontaneous exchange of 
ions across the same phase boundaries ty thermal motion. 
This result might be of some general significance in cellular 
physiology if it should occur with cell membranes because it 
could provide a mechanism which yields a rapid exchange of 
ions between the interior of the cell and the sur n^ionding milieu, 
viiile keeping the interior of the cell and the milieu separated 
electrically. 

The anomalous behavisr of the anion-selective membranes 
prepared with synthetic cationic polyelectrolytes makes them 
\insuitable for a variety of model studies in spite of their 
extraordinarily high ionic selectivity. It therefore was 
necessary to prepare some other anion selective membranes of 
extreme selectivity which behave normally and at the same 
time give the desired extreme degree of ionic selectivity, 
of the order of 10,000:1 and better in ,01 N solution. Such 
membranes were obtained by the use cf specially purified 
protamine, in cooperation with Dr. LewLs and Dr. Gcttliet, 



c ^ 



c _ 



- 3 - 



NIAJ^D"?? 



-99 ^ 



SERIAL NUl'BER 
PROJECT DESCRIPTION: (CONTtp) 

Ma.jor Findings ; (Cont'd) 

Theoretical wark was continued on a recently (1955) 
published theory of the accumulation of electrolytes — both 
of anions and cations simultaneously — against concentration 
gradients. The theory was generalized to be applicable to 
systems in v^hich the high resistance part of the model of the 
call which accumulates electrolyte resides in the cell membrane. 
It could be demonstrated that membranes of extremely high 
ionic resistance vhich nevertheless yield high rates of 
exchange of ions across their thickness are feasibleby 
virtue of a variety of mechanisms, and that such membranes 
may be combined in model systems which must show the phenome- 
non of ion accumulation. Preliminary e:;q3eriments have 
already borne out the essential correctness of the theoretical 
predictions. 

The rates of exchange of two different species of ions 
across permselective meuibranes were studied in cooperation 
with Dr. Gottlieb and a theory tentatively developed which 
permits calculation of the rates of this "allo-exchange" from 
the rates of self-exchange across the same membrane of the 
two species of ions under consideration. This approach seems 
to be a promising basis for the development of a theory of the 
absolute reaction rates of ionic processes occurring across 
membranes . 



Significance to Research of the Institute ; In order to under- 
stand electrolyte relationships in living cells and tissues, 
it is necessary to have accurate information on meirbrane 
model ^sterns inhich, under carefully cent relied known 
conditions, reproduce at least some of the major in vivo 
phenomena. The work of recent years, particularly 
the study of polyionic potentials and the construction of 
an in vitro model of electrolyte accumulation, together with 
the before-described effects on flux rates in related phenomena 
have brought us significantly nearer to an understanding and 
an in vitro reproduction of the type of effects which 
ultimately must govern the in vivo osmotic behavior of cells 
and tissues. The work already carried out seems to indicate 
that even fairly conplex membrane systems, similar to those 
found in living nature, may prove in the foreseeable future 
amenable to a complete and quantitative physicochemical 
analysis. 



\2"J::; 



4 - 



mkm-9±sjL 



SERIAL NUIBER 
PROJECT DESCRIPTION: (CONT'D) 



Proposed Course ef Pro.ject : Continuation cf the theoretical 
vrork on electrolyte accumulation; construction and e^qsloration 
of experimental model systems to verify this theory. Finishing 
up the work on the preparation of inproved permselective 
protamine collodion membranes. Further vic-rk en membrane 
equilibria, particularly certain peculiarities of membrane 
equilibria in ionizing solvents. Continuation of the work on 
absolute rates of exchange of ions across permselective 
membranes from the experimental and theoretical point cf view. 
Theoretical and ej^jerimental studies on the relative rates of 
flux of several coexisting species of ions cf the same charge 
across permselective membranes in continuation of previous 
WDrk on the electrical potentials, "polyionic potentials", 
arising in such systems. A prelindjiary study concerning the 
forces involved in the formation of regular structures by means 
of the exploration of long range forces of attraction and 
repulsion between microscopic and macroscopic particles. (The 
e^q^erimental techniques have been worked out and tested by the 
senior investigator before coming to NIH.) These latter studies 
are designed to furnish an insight into the physical forces which 
create organized structures of various levels of complexity and 
size in living systems. 



Form No. ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - MTIOi^L INSTITUTES OF HEALTH 
li'iDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NI/i^D-?? 



SERI/iL blUIffi.Jl 



12. BUDGET DATA 


■ 










ESTIMATED OBLIGATIONS 






MAN YEARS 


DIRECT 


REII-BURSEM2WT 


TOTAL 


PROF 


OTIIER TOTAL 


FY'57 










$28,itOO 


;;in 1,100 ^39,500 


2o50 


,50 3.00 




BUDGETED POSITIONS 






PATIEir DAYS 


PROF 


OTHER 


TOTAL 




FY'57 










3.50 


.50 


It. 00 






13. BUDGET ACTIVITY: 









RESEARCH 

REVIEIJ &. APPROVAL 

BIOLOGIC STANDARDS 



\ir\ ADMINISTRATION 

r7'~| PROFESSIONAL & 

tecil:ical assist- 
I I ance 



□ 



n 



lU. IDEI^TIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICr^, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOlt^EL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING ..iNTT IS WITHIN NIH 
INDICATE SERIAL i>JO(S): 

Dr. Melvin Gottlieb, Laboratory of Iddney and .:ilectrol5rte 
Metabolism, i^ational Heart Institute, up to June 30, 1956, at vjhich 
date he resigned from NIH. Dr. Gottlieb cooperated on a voluntary 
basis v;ith the principal investigator on the studies of absolute re- 
action rates in membrane systems p Dr. Gottlieb still continues on a 
voluntary basis in the theoretical work necessary and in the v/riting 

(Use reverse and additional paj^es, if necessary) 



3-2. 



NIA.Mp~99 
SERIAL WWiBER 



lilo IDENTIFY ANY COOPERATBIG UNIT ETC. (continued): 

of the papers which will summarize the work™ 

Dr. Eugene Grim of the Uepaxtment of Physiology, University 
of Minnesota, for a ten-day period, to work on the preparation 
of two manuscripts dealing with work done in earlier yeai-s by 
the senior investigator and Dr. Grim at IIIH. 

Dr« Charles \h Carr, of the Department of Physiological 
Chemistry, University of Minnesota, for two and one-half months ' 
XTOrk on a grant from the Lalor Foundation, to study electro- 
osmotic effects across mosaic membranes composed of exclusively 
anion-selective and exclusively cation-selective parts; also, to 
discuss earlier (19U3 and 19^6) woi^k on thermo-osmosis. 



Calendar Year I956 

PUBLIC IffiALIH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 

Part C: Honors, Avards & Publications 15, NIkMn^<)<^ 



SERIAL NUIffiER 



16. LIST PUBLIC An ONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING 
CALENDAR YEAR 1956: 

Dray, Sheldon and Karl Sollner, An experimental test in tw>-icnic 
systeiDS with permseleotive membranes of the theory of dynamic 
polyionic potentials across membranes rf ideal icnic 
selectivity, Biochim. Biophys , Acta 22, 213-219, I956. 

Dray, Sheldon and Karl Sollner, An experimental test in three- 
and four-ionic ^sterns vdth permselective meidjranes of the 
theory of (fynamic polyionic potentials across membranes of 
ideal icnic selectivity^ Biochim. Bicphys, Acta 22, 220-225, 
1956. 

Sollner, Karl and Rex Neihof ;, A possible mechanism of the selective 
accumulation of ions by living cells. Arch, of Biochem, and 
Biophys., 62, 507-508, 1956. 

Gottlieb, Melvin, Rex Neihof and Karl Sollner, Preparation 
and properties of strong base type collodion matrix mem- 
branes, J. Phys. Chem., in press. 

Neihof, Rex and Karl Sollner, The physical chemistry of the 
differential rates of permeation of ions across porous 
membranes. Discussions of the Farad, Soc. 94-lCl, 1956, 

in press. 

Neihof, Rex and Karl Sollner, The transitory overshooting of 
final equilibrium concentrations in memtrane systems i^tiich 
drift toward the Gibbs-Dcnnan membrane equilibrium, J. Phys, 
Chem., in press. 

Sollner, Karl, Book Review of Ultrasonic Engineering with 
Particular Reference to High Power Applications, J. Colloid 

Sc, n., 291, 1956. 



17 » LIST HONORS AND AWARDS TO PERSONNEL RELATIM} TO THIS PROJECT DURING 
CALENDAR YEAR 1956: 

None. 



Calendar Year 1956 

PUBLIC HEALTH SERVICE - NATIONAL IWSTITUTSS OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part A. Project Description Sheet 



1. NL^MD-100 



SERIAL mu!-:b?;r 



NIAMD 



INSTITUTE 



3 . Laboratory of Physical Biology 
LABORATORY, BixANCH, OR DH-ARTMENT 



U. Physical Biochemistry 5. 

SECTION LOCATION (IF OTHER THAN BETHESDA) 



6. The mechanism of the orderly aggregation of proteins in 

building up biological structures. 

PROJECT TITLE 



7. Dr. Koloman Laki and Dr. H. A. Saroff 
PRINCIPAL INVESTIGATCRS 



Dr. W. J. Bowen 

Dr. D. R. Kominz 

Dr. W. R. Carroll 

Dr. R. B. Simpson 

8. Dr. J. A. Gladner 



Dr. F. Irreverre 
M. J. Callanan 

E. R. Mitchell 
A . R . Hayden 

F. M. Saad 



E. N. Smith 
J. W. Healy 
Dr. L. B. Nanninga, 
Visiting Scientist 



OTHER liWESTIGATORS 



9. IF THIS PROJECT RESEffiLES, COMPLEtENTS, OR PARALLELS PJSE/lRCH 
DONE ELSE\rHERE IN THE PUBLIC HEALTH SERVICE ('/ITHOUT II'TER- 
CHANGE OF PERSONNEL, FACILITIES OR FUNDS) IDENTIFY SUCH 
RESEARCH: (BY SERIAL NO.(S) IF V/ITHIN NIH) . 



None 



NIAMD-^lOO • 
SERIAL NWIBER 



10. PROJECT DESCRIFTION 

Objectives : 

The objective of this project is to study the / 
mechanism of polymerization of proteins .in order to g,c.j.n 
insight as to how cells build up network structures. The 
structures involved in muscular contraction and blood 
coagulation are the results of protein poljTuerization. The 
main part of this project is, therefore, devoted to the 
study of proteins involved in muscular contraction and blood 
coagulation. 

Methods Employed : 

The method of attack is both direct and indirect. In 
the direct attack the proteins of muscular contraction and 
blood coagulation are separated from tlieir native milieu 
and are studied under arbitrary' conditions which are 
selected to reveal properties of interest. (Isolation; 
characterization: size and stiape of the molecules; deter- 
mination of chemical composition; modifications brought 
about by changing specific groupings either chemically or 
enzymatically, ) In the indirect attack studies are made on 
some other already better known proteins to gain information 
before the direct attack is made. In these studies the 
procedures of biochemistry and physicochemistry are employed. 
For example: paper and ion-exchange chromatography, 
enzymology, ultra centrifugal analysis, osmometry, light- 
scattering measurements, electrophoresis, the diffusion 
measurements, etc. 

Major Findings : 

In order to investigate the relationship between myosin 
and tropomyosin, the determination of am.ino acids of the 
muscle proteins isolated from muscles of various vertebrate 
and invertebrate animals was continued. 

In addition to being different from striated muscle 
tropomyosin, uterine tropomyosin was found to be different 
from smooth muscle (bladder) tropomyosin. 



- 3 - 

NIAMD-100- ■'" 
SERIAL NUiffiER 

10. PROJECT DESCRIPTION - CONTINUED 

It was found that in the adductor muscle of the clam 
there were tw3 types of tropomyosins differing mainly in 
their electrical charge. The one carryixig the lower 
charge, called tropomj''osin A, was found in abundance in 
the clam muscle (Kominz, Saad, LakiJ. 

The chemical properties of this tropomyosin A were 
found to show resemblance to the properties of "paramyosin" 
of the clam muscle (Laki, Kominz, Saad, Szent-Gyorgyi) . 

A method for determining the concentration of ATP and 
of ADP in common solution was devised. 

The effect of magnesium on the activity of myosin 
adenosine triphosphatase was found to be regulated by the 
accompanying salt concentration and it is indicated that 
the vastly different effect of magnesium from that of 
calcium is related to the type of binding betv/een the two 
metals involved and the myosin. 

Further evidence was found that the shortening of 
muscle models caused by the addition of ATP is not due 
to hydrolysis of the ATP (Bowen). 

Work on binding of ions Mg, Ca, K, CI to heavy and 
light meromyosin was continued, 'ifftien these experiments 
were extended to myosin it was found that myosin binds 
Mg as the meromyosins do, but to a smaller extent than 
was expected (Nanninga) . 

A theory has bean developed to explain the binding 
of potassium and sodium ions to myosin. In this theory 
matched carboxyl-imidazole and carboxyl-amino groups are 
considered as chelating sites for the binding (Saroff, 
Lewis) . 

Studies on the physical properties and the micro- 
heterogeneity of the sm.all, basic protamine, salmine, was 
continued. Recent results have clearly demonstrated an 
unusual tj'pe of ion binding by salmine. Numerous com- 
mercial and laboratory preparations of salmine have shown 



SEHIriL i\IU!iBAa 

10. PiiOJiiCT UESCuIPTIOK - COlYTIilUiiD 

physical characteristics of a homogeneous protein, but 
have always given amino acid analyses incompatible id-th 
a homogeneous substance, indicating that the nucleo- 
protein of the fish siTerm contains several prote'^Jis, 
similar in size and physical properties, but slightly 
different in amino acid composition (Carroll, Callanan, 
Mitchell) . 

Fractionation experiments have been carried out on 
carrot tissue culture as well as on the extracts of wild 
carrot roots. The object of this study is the isolation 
in pure form of a protein rich in hydroxyproline . The 
chemical and physicochemical properties of this protein 
will be compared to those of collagen (irreverre, Witkop, 
Stewart . 

Studies on the chemical composition of the two 
peptides released during the clotting of fibrinogen were 
continued. Using the enzyme carboxypeptidase revealed 
that these peptides have C-terrninal end-groups, indicating 
a proteolytic cleavage as the mschanism for their release. 

The likelihood of such a mechanism was strengthened 
by the finding that the nerve gas DFP inhibited thrombin 
(Laki, Gladner). 

In the experiments where cOiiimercial preparations of 
carboxypeptidase were used to degrade protein, it was 
found tliat basic amino acids were split off. These 
findings led to the recognition of a new carboxypeptidase 
specific for the release of basic amino acids (Gladner, 
Faulk) . 

The natuxe of the sulfhydryl group in serum albumin 
has been under continued study. It vras found that anions 
such as Cl~ and N0~- affect the rate of decrease of SH 
content. Oxygen also was found to affect the change, but 
not directly (Saroff, Simpson). 

The reagent, S-methyl isothionitro-urea, was found 
to react with both amino acids and proteins in the pH 
region 8 and 9. With this reagent the amino groups of 
a protein are changed to a nonpolar nitro-^anidino 
group (Saroff, Evans). 



- 5 - 

SERIAL MJl^'iBER 

10. PrtOJECT DSlCrllPTION - COOTIMED 

A quantitative colorimetric method far the determi- 
nation of proline, hydro:<ypro].ine, 5-hydroxypipe colic 
acid, pipecolic acid and baikiain (A,5--dehydropipecolic 
acid) and an ion-exchange Ciiromatogranhic procedure for 
their separation vra.s devised (Piez, Irreverre). 

Two new compounds behavirig lilce cyclic imino acids 
were found in some edible fruits. One of these, an acid 
stable compound behaves like one of the isomers of 
piperidine dicarbacylic acids (Irreverre, 3i?Lber) . 

To achieve further identification, several 
piperidine. mono and dicarboxylic acid derivatives have 
been synthesized (Irreverre, Evans). 

Gamma-guanido butyric acid had been identified in 
fruits like dates, bananna, papaya, inango, viatermelon, 
Persian melon, and strawberry, as well as in three 
species of nosquitoes, and in silkvrorju pupae. Since 
gamma-.^uanido butyric acid va.s found by other workers in 
marine invertebrates, it is of interest to trace the ex- 
tent of the occurrence of tliis compound in the plant and 
the animal kingdom (Irreverre). 

Significance to NIAI^iD R esearch : 

When part of the protoplasm or the whole cell (as in, 
e.g., cell division or muscular contraction) performs 
mechanical work, a network structure is built up at least 
temporarily, mainly thiough an orderly polymerization of 
globular proteins. This structure then reacts with the 
surrounding material and by utilizing metabolic energy 
(stored in ATP e.g.) performs work (muscular contraction, 
ameoboid movement). In order to understand this "mechano- 
chemical coupling" (the interaction of structure with the 
surrounding) and its disorders , we must know how such 
structures are built up. Muscular contraction and blood 
coagulation are examples of processes where structures are 
built up through protein polymerization. Such knov/ledge 
eventually will lead us to the understanding of certain 
diseases of muscle. Study of blood clotting, in addition 



- 6 - 

NTAMn-inn 

SERIAL NWIBER 

10. PrtOJECT Dj33CRIPTI0N - COITTINUED 

to supplying clues for protein polymerization, gives us 
better understanding of the disorders of blood clotting. 

When both direct and indirect approa.ch leads to 
some specific disease (e.g. hemophilia, rheumatoid 
arthritis) the advantage offered by studying the disease 
is utilized to the extent profitable. 

Proposed Course of Project : 

In the next calendar year the project vdll be pur- 
sued along the lines presented above. 



Form Mo. ORP-1 
October 19^6 
(Attacliment I ) 



PUBLIC HEALTH SERVICE - NATIOML INSTITUTES OF HEALTH 
INDIVIDUAL HiOJECT REPORT 



Part B: Budget Data 



11, NIAMD-100 





SE 


RIAL WUI'BER 




12: BUDGET DATA 


• 
• 








ESTimTED OBLIGATIONS 




14AN YEARS 


DIRECT 


REIMBURSF.MENT TOTAL 


PROF 


OTHER TOTAL 


FY'57 








$152,700 


$58,1100 f;ii211,100 


10.00 


8.67 18,67 




BUDGETED POSITIONS 




PATIENT' DAYS 


PROF 


OTHER TOTAL 




Fyi$7 








11.00 


8.67 19,67 






13. BUDGET ACTOTITY: 







RESEARCH 

REVIEIJ & APPROVAL 

BIOLOGIC STANDARDS 



i I 



ADICENISTRATION 

PROFESS lOML & 
TECHNICAL ASSIST- 
ANCE 






111, IDEiCIFY kW£ COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERoONi'JEL 
FOR THIS PROJECT IN FY 1957. IF COOPRiV.TING UNIT IS WITHIN NIH 
INDIQITE S~.RIAL i«(S): 

Mr, Robert L, Silber, Chemistry Instructor, Evansville High School, 
Evansville, Indiana (cooperating with Irreverre), 

Dr, Karl Piez, ^^ational Institute of Dental Research (cooperating ;ri.th 
Dr. Irreverre), 

Dr« Bernhard Witkop, Laboratory of Chemistry, NL^MD (cooperating with 
(Use reverse and additional pages, if necessary) 



.2- 



NIA?€I-100 

SERIAL NUMEER 

lUo lUEMTIFY ANY COOPERATING UNIT, E?C. (continued): 

Irreverre ) , 

Dr. F. C, Stexrard, Department of Botany, Cornell University, 
Ithaca, M. Y, (cooperating idth Irreverre), 

Dr, A. ^, Szent-Gyorgyi, Institute for liuscle Research, 
iJoods Hole, Mass. (cooperating with Laki, Kominz, Saroff), 

Dr. J, L. Folk, National Institute of Dental Research (co- 
operating with Gladner, Laid.)? 



Calendar Year 1956 

PUBLIC HEALTH SKiiVICE - NATIONAL IiGHTUTES OF HEALTH 
INDIVIDUAL PKOJECT HEPORT 



Part C: Honors, Awards & Publications 15. HI jj-ID-IOO 

SERIAL i-ilJI£ER 



16. LIST PUBLICATIONS OTH?]R THAN ABSTRACTS FROM THIS PHOJECT 
DURING CALENDAR YEAR 1956: 

Bowen, W. J., and Korwin, T. D.: The kinetics of myo- 
kinase. II. Studies of heat denaturation, the effects 
of salts, and the state of equilibrium. Arch. Biochem. 
and Biophys. 64, 278-284, October 1956- 

Bowen, W. J.: Adenosinetri phosphate and the shortening 
of muscular models. To appear in Symposium on Bio- 
colloids, 9th Annual Research Conference, Gatlinburg, 
Tenn., April 12-14, 1956. 

Bowen, 'V. J., and Kenmi, T. D.; A simple method for 
assaying adenosine triphosphate and adenosine di- 
phosphate in mixtures. J. of Biol. Chem. 220, 9-14, 
May 1956. 

Blum, J., Kericin, T. D., and Bowen, 'i. J.: Dependence 
of length of muscle fibers upon ATP concentration. 
Arch. Biochem. k Biophys., in press. 

Gladner, J. A., and Laki, K. The inhibition of thrombin 
by diisopropyl phosphorofluoridate. Arcli. Biochem. 
and Biophys, 62, 501-503, June 1956. 

Kominz, D, R., Saad, F., Gladner, J. A., and Laki, K.: 
^lammalian Tropomyosins. Jircn. Biochem. and Biophys., 
in press. 

Laki, K., and Kitzinger, C: Heat clianges during the 
clotting of fibrinogen. Nature, in press. 

Laki, K.: Studies on the composition of contractile 
muscle proteins. To appear in Symposium on Bio- 
colloids, 9th Annual Research Conference, Gatlinburg, 
Tenn., April 12-14, 1956. 



MI;MD~100 - :•■ 
SERIAL NUMBEit 

16. PUBLICATIONS - COIJTIiNlUED 



Mihalyi, E., Laki, K., Knoller, M. I,: Nucleic acid and 
nucleotide content of myosin preparations. Arch. 
Biochem. and Biophys., in press. 

Nanninga, L. B.: The binding of magnesium, calcium and 
chlorine ions to heavy and light meromyosin. Arch. 
Biochem. and Biophys., in press. 

Lewis, M. S., and Saroff, H. A,: The binding of ions to 
the muscle proteins. Measurements on the binding of 
potassium and sodium ions to myosin A, myosin B, and 
actin. J. of Am. Chem. Soc, in press. 

Piez, K. A., Irreverre, F., and Wolff , H. L,: The separa- 
tion and determination of cyclic imino acids. J. Biol. 
Chem., in press. 



17. LI3T HONORS AND AJ/uiDS TO FiliSOlaViiL iuilLyiTIiiG Tu THIS 
PROJJ.CT DURIi'jG CiiLENDAR YEAR 1956. 



None 



PUuLio HL.ALTH SbiiVIUi. - UATICiJmL IlvSTiTUTLS • HcALTH 
IitluIVlbUAL P1.0Jl,^.T UliPOUT 



Part A. Project oes'- ription ohcet i . NLiMD-101 



>- • 



H lA i.iL» 3 . Physical liolo-iv 

iNSTlTOTi:, LAotitATOitY 

Physical uiochemistry 5, 



6. isolatioii arni characterization o- the A jCjluti nation 

Activating I' actor in Kheuiiiatoid Arthritis 

PiwcJi^oi TITLi:; 

7. » . i< . 1'.' i 1 1 i a III s , 1.1 . u . 
PuiiJuiP/iL iiJV..STIOAT0R 

8. S, S, otone, J. Jenkins, j, Irancois, t= Israel, 
J. J. Lunim. t^u o. , 



OTHi.lt liJVcjriuATOuS 

9. i\lo parallel research in the Public Health service. 

1 . Pi.OJ.ioT ..i^SuniPTION 

Ob i Lct ive : Tlie ajylutination activating factor 
occurs in th<t serum c patients vvith rheuinatoio 
arthritis and about 20 to 30 per cent oi patients 
Viith various "collayen" diseases. Less than ^ive 
per cent Oi patients with other types o_ arthritis 
or ivith other Diseases show detectable activity in 
the serum. The factor seeiis to be a protein with 
elec trophoretic mobility in the beta ylobulin ranije, 
In the seruin oi almost all nonrheumatoio arthritics 
is found an inhibitory suli stance associated with 
plasma protein •""'raction II Oj. Cohn, which is 
probably a ijamma ylorjulin. The isolation ana char- 
acterization 01 these ;serum protein components is 
Desirable, first to enable their more oifoctive use 
in ciajnostic tests .or r hi; u;iia toi d arthritis anu, 
moreover, to aeter.uine the role they play in the 
pathogenesis of the cisease. 

^jince isolation procedures have been seriously 
hampered by lacK 0173%*^?^^ met hoas , the cevelopment 
Oi methocs for the quantitative assay 0. both 
ayylutinating and inhibitory factors have btcomr a 
prime ODjective 01 this study. 



NIAMD»10i___ . ^ 

SERIAL NO. 

10, (cont inueti) 

Methods : The sensitize ti shoep coll arjglutina- 
tion inothotl oi : aaler ana Rose, and inouiJieii by 
Ziii, is employe a lor clinical detection of the 
activity in rheumatoiu serum. Also, some seini- 
quantitative studies usinj the inhioitory activity 
of certain Iraction 11 (Cohn) preparations have been 
useti. Two new methods are in thi, process o' 
development. In addition, a considerable nuraoer of 
electrophoretic studi(?s oi active ayijlatinatiny 
preparations and inhibitory preparations have been 
perf ormeu. 

Patient material : Patients ivith and without 
rheumatoio arthritis in the clinical wards of iiUALiU 
have been usea lor some detaileu _iji vivo studies oi 
substances which showeo promisinj inhioitory 
activity in vitro . 

Sera Ivom patients irom the blinical Center, iit. 
Alto Veterans Administration Hospital, Walter Piece 
Army Hospital, George .ashinrjton University Hospital, 
Johns Hopkins Hospital and other sour(;es have been 
tested xor sensitized sheep cell ayglutinatory 
activity in continuinij cooperative programs with 
these institutions. 

Kia i or Findings : 

1) It has been louna that a euylobulin Traction 
of serum containing a-jylutinatini^ activity slso 
stronijly inhibits hemolysis oi sheep erythrocytes by 
Newcastle's uisease Virus and by Saponins^ The 
method appears to have yood potentialities for the 
quantitative assay of the agglutination activa^inij 
factor, Farthermore , it has oeen possiole to loliow 
the rate of reaction and make some observations 
su9L,estin(j an optimal time of reaction ior the sen- 
sitized sheep cell ag jl ut ina i.ion ior b^st uiatjnostic 
use. The methoc may also De applicable to the assay 
of inhibitory substances. Isolation procedures can 
now be subjected to quantitative evaluation. 

2) Some early work with Newcastle's Jisease 
Virus "sensitizeo" with specific antiijouy indicates 
that a much simpler assay methoo may soon become 
available. Preliiiinary stjcies indicate that the 
reaction can oe iolloweo by icinetic niethods ano may 
provide further improvements in technique ano in 
unuerstanciny oi mechanisiiis involved in the reaction. 



NL-inP-lOl 



J) Studies Ox complexin.^ between Cuhn Fraction 
H anti euglobulins x'rom rheumatoid and normal sera 
indicate bindiny Ox part Ox Cohn fraction II to 
both types Oi euijlobulia« Thus, the absence of 
precipitation in the normal serum (Epstein and 
Johnson procedure) with Cohn Fraction II iroy be 
partially or wholly aue to the presence oi. inhib- 
itory substances in the normal seru.n. A decrease 
in free or .unctional inhibitory substances in the 
rheumatoid scrum would at least partially account 
xor the precipitin reaction observed by Lpstein 
and Johnson. 

S i M n i 1 i c a n c e ; The projram provides an approach 
to a more usexul diaijnostic method Tor diiierenti- 
atiny rheumatoid arthritis from other arthritides. 
It also may proviue an insight into the pa tho<jenesi s 
of rheumatoid arthritis. 

The secono oi the assay methods may prove to be 
a special case oi a wore general type ox reaction 
which can be applied to the quantitative determin- 
ation ox minute amounts oi proteins, viruses, and 
anti bodies. 

Proposeci course oi the Proiect ; 

1) I'urther developments of quantitative assay 
methods now in progress, especially the kinetic 
methods. 

^) Use Ox the assay methods to follow isola- 
tion procedures for the agglutinating factor and 
its inhibition. 

vJ) Further attempts to characterize chemically 
anu lunctionally the components of the protein 
system involved in the sensitized sheep cell 
phenomenon. 



''}:. srJ y 



■^'•iifi.-- 



I : 



Form Mo, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEAL?H SERVICE - NA.TIOML INSTITUTES OF HEAI.TH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. KI AMD-101 

SerMTiwmber 



12, BUDGET DATA: 








ESTII1A.TED OBLIGATIONS 




MAN YEARS 


DIRECT 


REIMBUliSSM^ir TOTAL 


PROF 


OTHT-:!! TOTAL 


FY«57 








$21,300 


$8,100 s^p29,itOO 


0.83 


2.17 3.00 




BUDGETED POSITIONS 




PATIEOT DAYS 


PROF 


OTHER TOTAL 




FY«57' 








0,83 


2.17 3.00 






T^- RTITYIT^'^ AH" 


^nrT'TV- 







RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STAITOARDS 



a 



ADMINISTRATION 

PROFESS IOi\IAL Ss. 
TECHJIGAL ASSIST- 
ANCE 






lU. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC KiALTH SERFICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACr.ITIES, Oil PERSOM^iEL 
FOR THIS PROJECT IN FY 19^7. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



'■:■'(■■■ 



^»>-' 



Part C: HONORS, AWARDS t^ PUBLICATIONS 

15. NL'uMD-lOl 

SLFJIAL NUi.iBEK 

16. PUbLICATXONS OTHER THAN ABSTRACTS DURING CALliNUAR 
YEAH 1956; 

a. R. Williams - Sheep bell Agglutinins in 

Rheumatoid Arthritis. Clinical 
Proceedings ox the children's 
Hospital (ashing ton, b. c.) 
li:97 (1956). 

17. HONORS ANu A'uARDS: 
None. 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - NATIONM. INSTITUTES OF HE;lLTH 
INDIVIDUAL PliOJECT REPORT 

Part A. Project Description Sheet 1. NL:MD-102 



SERIAL miiBilAi 



2 NIAMD 3. Laboratory of Physical Biology 

INSTITUTE OR DIVISION LABORATORY, BRmUGH, OR DEPARTMEOT 



Physical Biochemistry - 
4. Group on Immuno chemistry 5» 



SECTION LOCATION (IF OTHii-R THaN Br-TIESDA) 



6. Immunochemical approaches to the purification and characteriza- 

tion of proteins. 

PROJECT TITLE 



7. Dr. R. R. Williams 



PRINCIPAL INVESTIGATOR 



S. S. Stone 
Daniel Francois 
Clarence Isreal 
R. L. Evans 



8 . OTHER IWESTIGaTORS 

9. IF THIS PROJECT RESffiBLES, COi'iPLEIffiNTS, OR PAKALLSLS RESivmCH 
DONE ELSElMiRE IN THE PUBLIC HEALTH SiirtVICE (WITHOUT ICTrit- 
CHANGE OF PERSONliEL, FACILITIES OR FUldS) IDEOTIFY SUCH 
RESEARCH: (BY SEKIAL N0.(3) IF WITHIN NIH) . 

Related projects in NIAID and NCI. 

Dr. Geoffrey Edsall and Elmer Becker of Vfelter Reed General 

Hospital collaborating with Dr, H. a. Sober. 

Dr. Sheldon Dray - characterization of allergens. 



2 - 



Ni;iMD-102 



SERIAL NUMBiili 



10. PROJECT DESCRIPTION 

A. Objectives : 

1. Development and application of ijiimunochemical 
methods for the purification of proteins. 

2. Application of these and supplementary methods 
to the determination and characterization of proteins 
of biochemical interest. 

B. Methods Employed : 

1. Methods involving interactions of antigens and 
antibodies on the surface of suitable supporting media. 

2. Conventional immunochemical procedures. 

3. New immunochemical methods being developed by 
this group and elsewhere. 

4. Chromatography of constituents of substances 
investigated. 

C. Ma.jor Findings : 

1. A general procedure for the purification of pro- 
teins has been developed. It makes available the specific 
combining groups of an antibody for combination with their 
specific antigens without requiring the dissociation of 
the antigen -antibody complex. This has been demonstrated 
in a model system with mixtures of bovine serum albumin 
and bovine gamma globulin by adsorbing albumin to a 
suitable adsorbent, reacting the albumin with anti- 
albumin and using the residual reactive sites to remove 
albumin from artificial mixtures of albumin and gairima 
globulin. It has been demonstrated that the antigen- 
antibody complex isolated in this manner contains only 
traces of non-antigen or antibody protein and little or 
no enzymatic activity which would alter proteins which it 
would be desirable to isolate in a highly purified form. 



- 3 - 

Ni;.MD-102 -3 
SERIAL NUlnBER 

10. PROJECT DSSC.-ilFTION - CONTMUED 

2. The method has been successfully applied by 

S. S. Stone to the removal of traces of subtilisin from 
repeatedly recrystallized carboxypeptidase. The sub- 
tilisin contaminant apparently arises from the growth 
of the omni-present B. subtilis in the course of isola- 
tion of carboxypeptidase and has been confusing to in- 
vestigators interested in C-terminal amino acid of 
proteins . 

3. Papers have been submitted on items 1 and 2. 

4. In the coiirse of the above a simple, conveniently 
prepared, cellulose anion exchange medium was developed 
by precipitating chitosan on powdered cellulose. This 
has good protein adsorbing capacity and resembles closely 
some polysaccharide protein complexing systems in living 
organisms . 

5. The above studies make possible the study of 
antigen-antibody reactions in stages and with the elimi- 
nation of secondary aggregation reactions. 

D. Significance : 

1. The above methods can be applied to a number of 
difficult problems in protein purif icatioi . Examples of 
possible applicable situations are (a) adaptive enzymes 
of various organisms, (b) new enzymes and possibly to 
genes arising from spontaneous or induced mutations, and 
(c) isolation of foreign proteins such as allergens or 
viruses from the host. 

E. Proposed Course of Pro.ject ; 

1. Application of the methods described above to the 
purification of other proteins <, 

(a) A cooperative project ^d-th Dr. L. N. Loomis 
of Pathologic Anatomy for the purification of 
Newcastle's disease virus is under way. 



- 4 - 

NL>ID-102 -V 
SERIAL NUMBER 

10. PROJECT DESCRIPTION - CONTINUED 

(b) A second project vdth Dr. E. H. Emimrt for 
the isolation of streptococcal hyaluronidase has been 
initiated. 

(c) Use of the above methods in studying the 
binding of peptide fragments of bovine albumin to its 
antibody are contemplated. It is hoped these may 
provide some data on the fine structure of the antibody 
combining sites of bovine serum albumin in terms of 
amino acid sequences involved. 

(d) Other studies such as cross-reactions of 
related antigens in terms of antigen binding instead 
of the conventional cross-precipitation now employed. 

(e) Application of some present methods being 
developed on the rheumatoid arthritis studies to a 
general method for the estimation of minute concentra- 
tions of proteins in biological materials 



Form" No, ORP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - MTIOWAL INSTITLPTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NTAMD-102 





s: 


RIAL l^IUMBER 




12, BUDGET DATA 


I 








ESTIMATED OELIGATIONS 




mn YEARS 


DIRECT 


REIMBUItSaiENT TOTAL 


PROF 


GTi-ER TOTAL 


FY'57 








$19,300 


$7,100 $26,UOO 


1,1*9 


.83 2.32 




BUDGETED POSITIONS 




PATIENT DAYS 


PROF 


OTHER. TOTAL 




FY»57 








1.U9 


.83 2,32 















13. BUDGET ACTIVITY; 
RESEARCH 

REVIEVJ & APPROVAL 
BIOLOGIC STAI\1DARDS 



fxl ADMINISTR/iTION 

{ I PR0FES3I0N/'.L & 

TECHNICAL ASSIST- 
1 I AMCE 






II4. IDENTIFY AilY C00PF31ATING UNITS OF THE PUBLIC HICAITH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSON EL 
FOR THIS PROJECT IN FY 1957. IF COOPE'iii.TING UNIT IS vflTHEN NIH 
IiroiCATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



. 4. ' ;<! 



Calendar Year 1956 

PUBLIC HEALTH SEIWICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part C: Honors, Awards & Publications i5. NIiaMD-102 

SERIAL NU^SER 



16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS 
PROJECT DURING CALE!®AR YEAR 1956: 

None 



17 , LIST HONORS AND AWARDS TO PERSONffiL RELATING TO THIS 
PROJECT DURIiC CALENDAR YEAR 1956: 



PUBLIC ?IEALTH SERVICE — NA'^OMAL INSTITUTES OF HEALTH 
INDIVIDUAL PRO.IECT REPCRT 



Part A. Project Description Sheet 



2. NIAMD 



INSTITUTE OR DIVISION 

k. Clinical Investigations 
SECTION OR SERVICE 





1. NLJ^D-103c 




SERIAi. NUrBm 


3. 


ART. IRITIS u RHLUmTISM 




Lj\B, BR..KCH, OR DLFARTJElf 


5, 





LOCATION IF OTHER TilAN 
SiLT'tiESDA 



6. Trial of New Anti-Rheumatic Dru^^s 
PROJECT TITLE 

7. Dr. Joseph J. Bunim 

PRINCIPAL INVESTIGATOR 

8. Drs. Roger L, Black, K. Lemone Yielding, Ernest Simon, and 
Ralph E. Peterson 

OTHER INVESTIGATORS 

9. No parallel research elsewhere in Public H^^alth Service 

10. PROJECT DESCRIPTION 

This project is C'-ncerned with the development of newer and 
better methods of management of rheumatic diseases. In the cal- 
endar year 1956, a total of patients have been studied. Further 
observations have been made using prednisone, prednisolone, A-'-,9a- 
fluorohydrocortisonei and the new steroids. A-'-, 21-deso>qy-9a- 
fluorohydrocortisone, 21-desoxy-9a,21-difluorohydrocortisone, and 
Ao, 21-desoxy-21-fluorohydrocortisone have been evaluated. Special 
attention was directed to (1) anti-inflammatory, (2) metabolic, 
(3) hormonal effects, and (k) adverse side effects. 

In studies on prednisone and prednisolone, continued observa- 
tions have been made on the original group of 15 rheumatoid arth- 
ritis patients treated with these compoiands. Side-effects in this 
group followed for up to 2 years and in another group of 21 fol-" 
lowed for varying periods of time include: 

Fractures 7 

Peptic ulcers $ 
Mental changes 5 
Rapid spread of tuber- 
culosis with death 1 
Diabetes 1 



SER:UiL NLJ^-a03c 

Project Description (cont'd) 

The appearance of ecchymosis has "".een noted in 10 of the 15 closely 
followed patients. 

Six patients with scleroderma treated with prednisone or 
prednisolone have been followed for 18 months, with no S'-rious 
side effects. 

Studies have been conducted using /^-'■,21-desoxy-9a-fluoro- 
hydrocortisone in h patients. This steroid V7as found to be un- 
satisfactory as an anti-rheumatic agent in doses as high as 100 
mg, daily, although 6o mg. daily was sufficient to suppress ad- 
renal function and to cause hypopotassemia and sodium retention. 

Metabolic studies on one patient piven 50 mg, daily of 21- 
desoxy-9a,21-difluorohydrocortisone revealed an inadequate anti- 
inflammatory effect, but no significant sodium or potassium 
changes. 

Studies with the steroid ^ ,21-desoxy-21-fluorohydrocortisone 
are in progress. 

It is to be hoped that the NIAMD may contribute to the de- 
velopment of more effective means for management of rheumatic 
diseases. By careful observation of anti-inflammatory, mineralo- 
corticoid, and hormonal effects, as well as aide-effects, of new 
synthetic steroids, it is hoped to obtain further information 
concerning the relationship of chemical structure, biological 
activity, and clinical effects. 



Form Mo, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-103 (C) 
SERIAL iWi'IBLR 



12, BUDGET DATA: 

ESTU'l/iT.ED OBLIGATIONS 




fAN YEARS 


DIRECT REIMBURSEHEifJ TOTAL 


PROF 


OT}Ij::R T.JTy.L 


FY '57 

$60,000 m9,90Q $239,900 


3o33 


1.00 li,33 



BUDGETED POSITIONS 



FYt57" 



PROF 



OTIER 



TOTAL 



U.33 



1,00 



5.33 



13. BUDGET ACTIVITY; 
RESEARCH 

REVIEl'7 & APPROVAL 
BIOLOGIC STANDARDS 



2162 






PATIENT DAYS 



ADMIi^JISTRATIOM 

PROFESS lOML & 
TECHNICAL ASSIST- 
ANCE 



C 



lU, IDENTIFY ANY COOPLRiiTING UNITS OF THE PUBLIC HiJ,ALTH S^RIICE, OR 
OTHER ORGANIZATIONS, PROVIDI'IG FUNDS, TACILITIES, OR PERSOiTiffiL 
FORT ITS PROJECT IN FY 19^7. IF COOPr;R/'.TING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S)s 



(Use reverse and additional pares, if necessary) 



SERIAL ML..MD--103C 

Part C, Honors, Awards, and Publications 

16. Publications ; 

1. Rodnan, G. P., Black, R. L,, Bollct, A. J., and Bunim, J. J. 
Observations on the Use of Prednisone in Patients with Progressive 
Systemic Sclerosis (Diffuse Scleroderma). Ann. Int. lied., Iili:l6- 
29, 1956. 

2. Bunim, J, J. The Use of Prednisone in Rheuiriatic Diseases 
(Editorial). J. Chronic Dis., 3:53'7, 1956. 

3. Bunim, J. J,, Harvey, A. McG., Bollet, A. J., Hilbish, 

T. F., Van Scott, E., Sokoloff, L., and Brecher, G. Systemic Lupus 
Erythematosus. Circulation, lij:125, 1956. 

li, Bunim, J. J., and Black, R. L. Connective Tissue (Collagen) 
Diseases. Ann. Rev, of Med^ , In press. 

5. Bunim, J. J. Clinical Uses and Hazards of the Adrenal 
Hormones and Analogues in the Management of Rheumatic Diseases. 
Bull. N. Y. Acad. Med., In press. 

17. Honors and Awards : 

Dr. J. J, Bunim was elected to membership in the Association 
of American Physicians in May 1956. 

Dr. Bunim was invited to give principal lectures or papers on 
the following occasions: 

University of Virginia Medical College (January, 1956) 

Postgraduate Course at Mt, Sinai, Miarrd (I'lay 1956) 

Staff Conference at Roosevelt Hospital, , New York (Hay 1956) 

American College of Physicians, Boston (June 1956) 

Annual Postgraduate Fortnight of New York Academ^y of Iiedi- 

cine (October 1956) 
Postgraduate Course at Kansas City University College of 

Medicine (November 1956) 
Monsanto Chemical Company, ~St. Louis Medical Society 

Conference on Aspirin, St. Louis (November 1956) 

Dr. Bunim has been reappointed for another term of three years 
to the Research Committee and to the Ifedical and Scientific Com- 
mittee of the Arthritis and Rheum^.tism Foundation, 



PUBLIC HEALTH SERVICE — NATIONAL I\iSTITUTES OF HEALTH 
INDIVIDUAL FROJECT REPORT 

Part A. Project Description Sheet 1. NIAMD-lOUc 



SERLiL NUiai:.R 



2. NLU^iD, CI 3. Arthritis and Rhcuimtism 

INSTITUTE OR DIVISION LaB, BR,v::CH, OR DEPARTFiENT 

h. Steroid 5. 



SECTION OR SERVICE LOCATION IF OTHER TfL^a! 

BETHESDA 

5. Fet.^bolisn of Adrenal Steroids 
PROJECT TITLE 

?. Dr. Ralph E. Peterso n 

PRINCIPAL INVE,STIGATOR 

3, Charles Pierce, Aurora Karrer, Margaret Bollier, Bernard 
Kliman, Georj^e Nokos 
OTHER INVESTIGATORS 

9. No parallel research elsewhere in Public Health Service 

10. PROJECT DESCRIPTION ; 

Objectives: Physiological disposition, metabolic fate, and rate 
of synthesis of various steroids in man. 

Significance to IvfLiIP Research ; The studies on physiological dis- 
position and pharmacology are important in regard to the tr3at- 
mcnt of patients with steroids. The studies on turnover rate 
should make it possible for the first time to determine the 
actual production of hydrocortisone and corticosterone by the 
adrenals in various disease states and in normal subjects. The 
studies in methodology are necessary to make it possible to more 
reliably carry out all of these studies. 

Nothods Employed; Major Findings: Utilizing radioactive labeled 
steroids and impi'oved methods for biochemical analysis of 17- 
hydroxi'-steroids and 17-ketostcroids developed in this labor:'.tory, 
the rate of endogenous synthesis, turnover rate, physiological 
disposition, and metabolic fate of various adrenal steroids have 
been determined both in the exprrimental animal and in man during 
health and disease. Isotopic hydrocortisone, cortisone and corti- 
costerone were administered to normal man in large or trace 



SERIAL Nlhl-iD-lOUc - 



Methods Employed; Major Findings (cont'd) ; amounts, intra v.. no us I.7 
or orally, and were verj^ rapidly excr^ ted, almost entirely in the 
urine. Fifty per cent appeared in the urine within the first 6 
hours, additional 30)t in the next 18 hours, and 10^ more in the 
following 2U hours. About 60,^ of administered steroid was excreted 
as glucuronide. Only $% to 10^ appeared in the feces, via biliary 
excretion. Thus 9S% of the administered radioactivity was re- 
covered. No isotope was demonstrated after 72 aoiju^s and no sig- 
nificant quantity appeared in the expired cirbon dioxide. 

It was discovered that the adrenal cortex in normal man syn- 
thesizes from 17 to 29 nig. of hydrocortisone daily, that the 
miscible pool consists of 1.1 to 2,l\ mg, and that this pool is 
turned over approximately 10 times a day. Following adrenocorti- 
cotropin stimulation, the rate of svnthesis increased to an average 
of 1^0 mg. pnr day. Turnover rate studies have been done in pa- 
tients with thyroid dis'-ase and liver disease. Patients with 
thyrotoxicosis have been shown to have an increased turnover of 
hydrocortisone and patients with myxedema have been shown to have 
a decreased turnover. Those defects are altered by appropriate 
therapy for the treatment of the disease. Patients with cirrhosis 
of the liver have been shown to have a decreased turnover rate of 
hydrocortisone. A diurnal variation in the rate of synthesis of 
hydrocortisone was shown. In a normal subject stressed by the 
injection of Piromen, a 5-fold increase in the turnover of hydro- 
cortisone was found. Prior 4dministration of aspirin abolishes 
this effect. Hyirocorti.sone in either pharmacological or physio- 
logical (trace) amounts was found to have a mean half-life of 
11^ minutes. The rate of disappearance of hydrocortisone from 
the plasma was markedly delayed in patients with liver disease and 
slightly delayed in patients with myxedema. It was much accelerated 
in patients with thyrotoxocGsis. The following three very polar 
steroids have been isolated, crystallized^ and identified as 
urinary metabolites of hydrocortisone: A^-pregnene-6p,ll|?,17a,21- 
tetrol~3r2 0-dicne ; Ay-pregnenc-lip , 17 a, 20j3 , 21-tetrol-5-one ; and 
A^-pregnene-lip,17a,21a,21--tetrol-3-one, The latter steroid had 
not previously been isolated from human urine. 

The physiological disposition and metabolic fate of cortisone 
were studied and found to be similar to hydrocortisone except that 
cortisone was metabolized at a much more rapid rate. Cortisone 
disappeared from the plasma h times as rapidly as hydrocortisone 
(23 to 35 minutes), in normal subjects and in patients with liver 
disease. Preliminary results have been obtained that suggest that 
a large fraction of the cortisone is rapidly metabolized to hydro- 
cortisone in man after oral or intravenous administration. Hydro- 
cortisone concentration in plasma is equal to cortisone concentra- 



SERIAL NL.MD-lOhc 



Methods Employed; Major Findings (cont'd ): tion v;ithin onu hour 
after the infusion of POO mg. of cortisone and at 2 hours practi- 
cally no cortisone remains. Our data would seem to indicate 
that slightly more than one-half of the infused cortisone is 
metabolized to hydrocortisone. After the intra-articular in- 
jection of cortisone, it was not possible to demonstrate any sig- 
nificant conV'Tsion of the cortisone to hydrocortisone. After 
oral administration of cortisone acetate, the plasma hydrocorti- 
sone exceeds the plasma cortisone concentration by 20 or more fold. 

Parallel studies on corticosteronc have been done after a 
more accurate end reliable an.al:/tical method than existed hereto- 
fore had been developed in our laboratories employing isotope 
dilution. The metabolism and excrttioii of this naturally occur- 
ring adrenal steroid are similar to hydrocortisone; however, it 
is metabolized slightly more rapidly — mean half-time, 80 minutes. 
Plasma concentration in normal subjects averages 1.1 agt in con- 
trast to a mean plasma hydrocortisone concentration of l5 U.g%. 
Corticosterone was found to ')e sjmthe sized at a rate of about 2 
to 3 nig, per day, whereas the tiu'nover rate for hydrocortisone 
is 17 to 29 mp. per day. 

Studies have been carried out on the effect of amphenone 
on adrenal cortical synthesis. These studies clearly demonstrate 
that amnhenone causes a decreased synthesis of both h"drocorti- 
sone and corticosterone in normal subjects and in patients with 
adrenal cortical carcinom'^.. There does net appear to be a 
significant decrease in the total urinary 17-ketosteroids. 

Studies have also been carried out on the effect of salicy- 
lates on adronal function, using various parameters for adrenal 
function. It was not possible to show thr:t salicylates altered 
either adrenal cortical function or the metabolism of adrenal 
cortical s+.croidiio 

Preliidnary investic^rtions have begun on the aeveloprrent of 
an isotope derivative metxiod for the measurjment of aldosterone 
in body fluids. If successful, this procedure will be applied to 
the determin- tion of other steroids previously difficult to assay 
because of their very low concentrations in body fluids. 

Proposed Course of Project ; (1) Additional data on the motabolic 
fate, rate of synthesis, and plasma l:.vels of corticosterone in 
man. (2) Additional data on the in vivo and in vitro transforma- 
tion of cortisone to hydrocorticoneT r3) Further studies on the 
isolation and identification of thj metabolites of hydrocortisone 
in the urine and perhaps qualitative and quantitative studies on 
the patterns of these metabolites in urinij in normal and disease 
states, (h) Development of a new method for assay of aldosterone 
in biological fluids. 



Form No, ORP-1 
October 19^6 
(Attaclment l) 



PUBI.IG HEALTH S^^VICE - NATIONAL mS?ITUTES OF IFAJ/rH 
BJDIVIDUAL PROJECT REPCET 



Part B: Budget Data 



11, NBMD-IOU (6) 
SiiEIAL KWiKH" 



REVIEW & APPJ^OVAL 
BIOLOGIC STANDARDS 



Pt: 1 ADfD;wISTR/'TION 



□ 



PROrES^IOLIAL & 
TECHMICAL ASSIST- 
ANCE 



12. BIJDG':? DATA 










ESTB'ATED OBLIGATIONS 




MAN YEARS 


DIRECT 
FI»57 


IffiItfflU.1SEm';NT TOTAL 


PROF 


OTHER TOTAL 


¥^1,000 


;pl03,900 .Vl5li,900 


2,00 


5»00 7.00 




BUDGETED POSITIONS 


l?5l 


PATIENT DAYS 


PROF 


othhe total 




I'T;57 








3.00 


5c00 8,00 






13. BUDGET ACTIVITY: 










lU. IDENTIFY AMY G00Pi;,R.^TING UNITS OF THE PUBLIC lEALTH SERVICE, OR 
0:Kh.R organizations j EIOVIHING funds, facilities, OR PERSOMEL 
FOR TI!IS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
iroiCATK S>^IAL N0(S): 



(Use x'everse and additional pa.^;es, if necessary) 



SERIAL ML.MD-l Oltc 

Part C. Honors, Awirds, and Fxiblications : 

16. PUBLIC/vTIONS ; 

Peterson, R. E. , and Wyngaarden, J. B, The Miscible Pool 
and Turnover Rate of Hydrocortisone in Kan. J. Clin. Inv' st,, 
35:552-561, 1956. 

Peterson, R, E,, Karrer, A., and Guerra, S. L,, Evaluation 
of the Silber-Porter Procedure for the Determination of Plasma 
Hydrocortisone,, Anal. Chcm. , In press. 

Pierce, C. E., and Peterson, R. E. Liquid-Liquid Continuous 
Extractor for Solvents Heavier than Water. Anal. Chem. ,26:2029- 
2030, 1956. 

Peterson, R. E. The Identification of Corticosteronc in 
Human Plasma and its Assay by Isotope Dilution. J. Biol. Ch'-m., 
In press. 

McDonald, R. K., Weise, V. K., and Peterson, R. E. Effect 
of Aspirin and Reserpine on the Adrenocortical Response to 
Piromen in Man. Proc, Soc, Exper. Biol. & Med., In press, 

17. HONORS Al^ro AWARDS: None 



P'JBLIC HEALTH SERVICE — N'aTIONAL INSTITUTES OF HEALTH 
nOr/IDUAL PROJECT REPORT 



Part A., Project Description Sheet 1. NI..j4D-105c 

SERIAL NUMBER 

2. NIAI-D 3. Metabolic Diseases Branch 



INSTITUTE OR DIVISIOI^I LAB, BRAMCH, OR DEPARTbiENT 

h. Hematology 5. 



SECTION OR SERVICE LOCATION IE OTH^R THAN 

BETHESDA 

6. Metabolism of Bile Pigments 
PROJECT TITLE 

7. Dr. Rudi Schmid 



PRINCIPAL INVESTIGATOR 



8. Lydia Hammaker - full time as of June 8, 1956 . 
OTflER INVESTIGATORS 

9. No parallel research in Public Health Service 

10. PROJECT DESCRIPTION 

Objectives ; To study the nature of direct reacting bilirubin 
and the mode of excretion of bilirubin into bile and urine. 

Methods Employed ; Bilirubin from normal bile and from jaundiced 
serum and urine was diazotized; then the diazo compounds obtained 
were separated by paper chromatographic methods. The isolated 
azopigmcnts were then hydrolyzed and the products isolated and 
quantitated. 

Patient Material; 



Major Findings ; Work in this project has revealed that the "direct 
reacting" ijater-solublo bilirubin in jaundiced serum and urine, 
and in normal bile, is bilirubin glucuronide, whereas the "in- 
direct-reacting" bilirubin of jaundiced scrum is free bilirubin. 
These results were obtained by studying the stable dipyrromcthone 
diazonium pigm-ents derived from treatment of biological fluids 
with an excess of diazotized sulfanilic acid purification, and 



SERIAL ^JL'.f'D-lO^'c 



separation by paper chromator^raphy. It was observed that direct 
reacting bilirubin of suruni, bile, and urine resulted in azo- 
pipments, which were conjugated with glucuronic acid, 3y heat- 
iiig the conjugated azopigments in hydrochloric acid or by in- 
cubating them with p-glucuronidase, free glucuronic acid and 
free azopipnent were obtained in a 1:1 molar ratio. The latter 
compound was found to be identical with tlie azopigiaents, result- 
ing from diazotization of "indirect-reacting" bilirubin and 
crystalline bilirubin. In human bile most of the bilirubin is 
excreted as glucuronide, In obstructive and hepatogenous (re- 
gurgitation) jaundice, glucuronic acid conjugated bilirubin gains 
access to the blocd, where it is detectable as "direct-reacting" 
bilirubin, Electrophoretically both bilirubin fractions, the 
free ("indirect") and the conjugated ("direct") migrate v;ith the 
scrum albumin. 

Material from two patients with severe congenital Gilbert's 
disease has been studied and the observation made that these 
patients have a defect in the ability to conjugate compounds with 
glucuronic acid; in addition to bilirubin, the defect appears to 
involve other metabolites which are excreted as glucuronides. 
Hcpatectomized dogs (surgery carried out by NHI) have been found 
to be unable to form and excrete bilirubin glucuronide. 

Significance to MIMD Research ; Although it has long been known 
that in jaundiced serum bilirubin occurs in two different forms 
of reacting pigments, a "direct" and an "indirect", the factors 
responsible for this difference have not been understood. The 
identification of direct-reacting bilirubin as bilirubin glu- 
curonide appears to have solved a problem which has been under 
discussion for some kO years. The validity of the present work 
has been substantiated by a group of British workers who approached 
the problem differently but obtained essentially similar results. 
Althous^h it is now clear that the liver has to conjugated biliru- 
bin with glucuronide in order to get it into a form suitabl'^ for 
excretion, a number of additional problems are unsolved and need 
further study. 

Proposed Course of Project : Studies will be undertaken to deter- 
mine the mechanism of conjugation in the livrr in cooperation with 
Dr. Julius Axelrod from NIMH. A strain of rats has been obtained 
and is presently being bred in our Animal Procurement Section, 
which appears to have a defect very similar to human Gilbert's 
disease, i.e., the rats are unable to conjugate bilirubin with 
glucuronic acid, Extensive study will be made on these rats in 
an attempt to define the extent of the defect, particularly vjith 
respect to other m.etabolites. Studies will be undertaken in 
hcpatectomized dogs in an attempt to isolate the yellow pigment 
which accumulates in the plasma and is excreted in the urine. 
Preliminar}/ studies have shown that this is not bilirubin glu- 
curonide nor free bilirubin. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SEiWICE - NATIOML INSTITUTES OF HE/iLTH 
IITOIVIDUAL PKDJECT REPORT 



Part B: Budget Data 



11. NIAiro-10^ (C) 







SH 


^lAL tlUl^PER 




12 , BUDGJ!:T 


DATA: 










ESTDI/.TED Oi'XIGATIONS 




MAN YEARS 


DIRECT 




REII'iBURSEfiGNT TOTAL 


PROF 


OTICR TOTAL 


FI>57' 










Vll,700 


i;;l3,ooo ;,ii2U,700 


1.00 


1,00 2.00 






BUDGETED POSITIONS 


162 


PATIENT DAYS 


PROF 




OTIffiR TOTAL 




FY'57 










1.00 




1,00 2.00 






13. BUDGET 


ACTIVITY: 







RESFARGH 

REVIEJ & APPROVAL 

BIOLOGIC STANDARDS 



|y [ ADMINISTRATION 

I ^i PROFESSIOML &. 

TECHNICAL ASSISI 
I j AWCE 






lii. IDEICIFY Al'IY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER OHGAi^ZATIOHS, PROVIDING FUWS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 19^7. IF COOP RATING UNIT IS WITHIN WIH 
INDICATE SiJlIAL NO(s): 



(Use reverse and additional pages, if necessary) 



SERIAL NL-MD-lOSc 
Part C. Honors, Awards, and Publications 
16. FU3LJCATIG"!S 



Direct-Reacting Bilirubin Glucuronide, in Serum, Bile, rnd 
Urine, R. Schmid. Science, 121^:76-77, 1956. 

Glukuronsaure-konjugicrtes Bilirubin, das "direkt rcagierende" 
Bilirubin in Serum, Harn, und Galle. R. Schmid. Schweizerischen 
Medizinischen Wochenschrift, 56:775, 1956, 



17. HOiJORS AND AWARDS: None 



PUBLIC EEALTd SERVICE — NA-^IONAL INSTITUTES OF HE-ALTH 

II'IDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. NLu''ID-106c 

SERL\L NUI'BER 

2. NIAMD ?. Metabolic Diseases Branch 



INSTITUTE OR DIVISION LA3, BRANCH, OR DEPARTMENT 

lie Hematology 5. 



SECTION OR SERVICE LOCATION IF OTHER T}-jj.i 

BETHE3DA 

6. Studies in Anemia 



PROJECT TITLE 
7. Dr. Rudi Schmid 



PRBICIPAL INVESTIGATOR 

Dr. Ted Clemens, Jr. 
OTHER INVESTIGATORS 



9, No parallel research elsewhere in Public Health Service 

10. PROJECT DESCRIPTION 

Objectives ; 1. Development of a method for quant itating and more 
accurately localizing the source of blood loss from the gastro- 
intestinal tract. 

2. Characterization of a new type of congenital 
and familial hcmol^'tic anemia. 

Methods Employed ; 1. Quantification of gastrointestinal blood 
loss was studied by means of oral administration of radioactive 
chromate tagpred red blood cells to normal subjects and neasure- 
m.ent of amounts of radioactivity in the stools. In patients with 
known blood loss their own red blood cells were tagged with 
radioactive chromate, put back into their bloodstream and the 
radioactivity of the stools measure. Study was made of the use- 
fulness of a Cantor tube down the intestinal tract, followed by 
periodic aspiration and testing for presence of blood and radio- 
activity r.s the tube was passed down the intestine, 

2, Characterization of the new type of hemolytic anemia was 
attem.pted by a variety of standard hematological tests and, in 
addition, by study of the red cells with supravital staining. 



SERIAL NI«;'ID-106c 



Methods Employed (cont'd) ; phase microscopy, and ultraviolet 
absorDtion microscopy. Fnctors influencing the life of the ab- 
normal cells were studied by tr;:.nsfusion of a small amount of 
those cells tagc^cd with radio-chroi'atfj and obs'.:rvation of the 
disappearance rate of tagged cells, of mature cells with inclu- 
sion bodies, of reticulocytes, and of sidcrocytcs. An unusual 
urinary- i^ipment was chemically isolated and ef Torts made to 
crystallize it. 

Patient Material: 



Major Findings ; 1. The study of gastrointi.stinal blood loss 
quantific ition and source; localization has resulted in the develop- 
ment of a valuable clinical diagnostic tool. One finding of great 
importance from the mr.'-isurement of radioactivity in the stools 
following oral administration of tagged blood is that patients 
with chronic blood loss from the gastrointestinal tr.ct evidently 
lose much greater amounts of blood than have been estimated from 
present commonly used clinical tests. 

In several anemic patients passage of the Cantor tube re- 
vealed sources of significant blood loss; upon surgical exaiij:i.a- 
tion hitherto undetected malignancies were found and removed. In 
those patients previous careful x-ray studies had been negative 
and the amounts of Hood loss esti"iated by the usual clinical 
tests had been regarded as unimportant by present standards. 

2, A previously unknown and unusual form of congenital hemo- 
lytic anemia was discovered by NIAMD hematologists in a father 
and son afflicted by life-long sevre anemia with jaundice and 
splenomegaly. In the peripheral blood first of the father and 
later of the son reticulocytes and mature er'/throcytes were found 
to contain one and occasionally two round or oval-shaped inclu- 
sion bodies. These unusual particles were demonstrated by phase 
microscopy and by supravital staining with aqueous methyl violet, 
were usually seen in the center of the coll, and were found to 
remain in the cell stroma after hemolysis. 

Transfusion of the inclusion body-containing red cells into 
a normal recipient showed rapid r:;moval of inclusion bodies and 
siderocytes from the recipient's circulation so that by 14 days, 
none could be found, ^rom measurement of chroma to-tagged cells, 



SERL'lL NLliD-106c 



Major Findings (cont'd) ; however, appi'oximatel^- 30,'i of the trans- 
fused red cells renriined in the circulation. Because of known 
unreliability of the chromate tagging method with red cells of 
sliort li.fe span, the significance of this difference was not clear. 

In both patients with the rare hemolytic anemia, tho urine 
was found to contain a rare dark brown pigment which, on chemical 
character izf.tion, appeared to belong to the mesobilifuscin group, 
a type of pigment never before isolated from urine. After trans- 
fusion of the Rbnormal cells to the normal recipient, the latter 
excreted the same pigment. This indicated that the anemic cells 
in this disorder have a defective mole of brf-^akdown leading to meso- 
bilifuscin formation rather th^n a normal breakdown to bilirubin. 

Significance to NRFD Research ; 1. Use of the Cantor tube with 
periodic aspiration of intestinal contents as the tube is passed 
down the intestinal tract, in conjunction with chromate -tagging of 
the patient's red cells for aid in the detection of blood in the 
intestinal contents, provides a valuable tool to clinicians in the 
search for sites of intestinal blood loss. The value of this pro- 
cedure was proved in 5 instances (including 1 malignancy) in which 
sources of blood loss were previously missed by standard methods, 
including r idiographic examination, and life-saving surgery v?as 
performed. 

2, The categorical interest of NIAMD in disorders of the 
blood has been furthered by the discovery by NLiffl hematologists 
of a previously unknown form of congenital hemolj-iiic anemia. This 
anemia, found in a father and son, was found to be characterized 
by red, cell inclusion bodies j such bodies have been previously 
noted only in toxic states and in premature or defective infants. 
By coincidence a similar case was found by hematologists at the 
Walter Reed Army Medical Center and paired publication was ar- 
ranged. The study of this rare disorder yielded thr subsidiary 
finding that these cases excrete in the urine a pigment regularly 
found in small quantities in ff.cos but never previously noted in 
urine. An abnormal and highly unusual form of hemoglobin break- 
down has thus been uncovered. 

Proposed Course of Project ; 1. Additional cases of chronic blood 
loss anemia will be studied with Cantor tube technique in an effort 
to refine the technique and to define more fully the' range of its 
usefulness. 

2, Attempts will be made to define the nature of the metabo- 
lic defect in the red cells of 'nclusion body-congenital hemolytic 
anemia; preliminary studies suggest a defect in glutathione content 
or reduction. The rare urinary pigment will be further character- 
ized in collaboration with Dr. Walter Siedel in Frankfurt, Germany, 
an outstanding organic elimist in the field of pyrrol metabolism. 



Form Mo, ORP-1 
October 1956 
(Attachment l) 



PUI3LIC HEALTH SERVICE - NATKliJAL INSTITUTES OF HFALTH 
INDIVIDUAL PROJSai' RilPCRT 



Part B: Budget Data 
12, HJDGET DATA: 



11. NRMD -106 (C) 
SJCRIAL 'MUl-lBER'" 



ESTIMATED OBLIG/iTTONS 



FY '57' 



DIRECT 



REITTBURSFJIENT 



TOTAL 



$16,500 



;A05,2O0 



$121, 700 



BUDGETED POSITIONS 



PROF 



OTHER 



FY557" 

3.00 

13 e BUDGET ACTIVITY! 
RESEARCH 

REV10.'r & APPROVAL 
BIOIOGIC STAI^iDARDS 



tTj 



TOTAL 



3.00 



MN YEARS 



PROF 



2.00 



1269 



ADIUMISTRATION 

PROFESSIOMAL & 
TECHHIC/i ASSISI 
AWCE 



OTHER TOTAL 



2,00 



PATIEir DAYS 



rzi 
□ 



lU. IDEiriTY Am C00?i.R(\TING m«TS OF TIE PUBLIC IEaLTH SERVICE, OR 
OTBCR ORGAillZ/iTIONS, PROVIDIUG FUimS, FACILITIES, OR PERSOl-UffiL 
FOR THIS PROJJT,CT TM FY 195?. IF COOPERaTTHG UiOT IS WITHIN MH 
IITOICATE SERI/vL HO(s): 



(Use reverse and additional pages, if necessary) 



SERIAL NL.1.D-106C 
Part C. Honors, Awards, and Publications 

16. PUBLICATIONS : 

Familial Hemolytic Anemia with Spontaneous Erythrocyte In- 
clusion Bodies. R, Schjnid, G. Brecher, G. Z. Williams, and 
T. Clemens, Jr. (Submitted to the Proceedings of the Fifth 
International Congress of Hematology, Boston, August, 1956). 

17. HONORS AND AWARDS: None 



FU3LIC HjiALTH SERVICE ~ NATIOML INSTITUTES '"F KLXLTH 
I>)DIVIDUAL PROJECT REPORT 
Part A. Project Description Sheet 1. NL.MD-107c 



SERIAL NUI3ER 
2, NIAI©, CI 3. Arthritis and Rheum tism 



INSTITUTE OR DIVISION U3, BRANCH, OR DEPhRTI-ENT 

Jj. Steroid 5. 

SECTION OR S£RVICE LOCATION IF uTHii-R TrUN 

BETHESDA 

6 . Studies in Heavy Metal Storage Diseases 
PROJECT TITLE 

7. Dr. Marvin J. Seven 
PRnCIPAL INVIiSTxGATOR 

8. Dr. Ralph E, Peterson 

OThER INT'ESTIGaTCRS 

9. No parallel research elsewhere in Public Health Service 

10. PROJECT DESCRIPTION 

Objectives : Search for better chelating agents for removal of 
heav\r metal ions associated vrith their excessive disposition in 
various heavy metal storage diseases in man, 

Ile thods Emiployed ; Administration of chelating agents to man, and 
evaluation of their usefulness by assays of the urinary excrc.ticn 
of the hrav?^ metal ions. 

Major Findings : Versene, ,pareiiterally administered, was found to 
be only mildly effective in the rem.oval of iron in hemochroma- 
tosis and copper in Wilson's disease. Versenol was found to be 
only slightly superior to Versene in the- removal of iron in hi^mo- 
chromatosis; however, in Wilson's disease this com.pound was found 
to be superior to Versene for the reincv:::! of copper. These agents 
arc only effective when given intravenously and this of course 
is a major disadvantage. They are without effect when j:,iven 
orally. If f_;iven intramuscularly thoy ire very painful. An 
ethyl ester of Versenol was invi^stigated, but this too resulted 
in some local irritation at the injection site, and was only 
moderately effective in the removal of copper in Wilson's disease. 



SERIAL NL.MD-107C 

Major Findings (cont'd) ; 

This compound given orally will produce n 10 to 20-fold increased 
excretion of copper in the lur-ine, and has ttj adv.ntare in thit it 
can be piven or::lly. It causes no increasi^d excretion of iron in 
hemochromatosis. The average daily dose of p^^nicillamine is Li gm 
for a three day period. 

Preliminary studies have been made with the use of an intra- 
muscular proparntion of iron Vcrsenol for the tre-i-tmcnt of iron 
deficiency anemia . This preparation has been found to be a very 
effective agent for rapidly increasing the body stores of ironj 
however, to date it has been difficult to keep the preparation 
stable t Unless a stable preparation can be found, this compound 
cannot be utilized in man. 

An improved and simplified method for the assay of urine 
copper and iron has been worked out. 

Significance to MBIMD Research; It is the hope that an effective 
orally administt'rud chelating agent can be found, and that it can 
function as a valuable therapeutic agent in the treatment of 
heavy metal storage diseases. 

Proposed Course of Project ; No further studies are planned be- 
cause the senior investigator has tcrmin".ted his fellowship. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBTJC rffiAL?H SiRVICE - mTIOl'lAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAI-ID-IO? (C) 



SERRL NUMBER 



12 » HJDGET 


DA' 


tL 


















EST 


imTED OBLIGATIONS 






mN YEARS 


DIRECT 








REIiiEURSEiiEWT 


TOTAL 


PROF 


OT'IffiR TOTAL 


FYI57 
















072,00 








m,3oo 


i^6,5oo 


1.50 


i.co 2.50 








BUDGETED POSITIONS 




9^1 


PATIEOT DAYS 


H101-' 








CTHJ'K 


TOTAL 




FY '57 
















1.50 








1,00 


2.50 







13. BUDGET ACTIVITY'. 
RESEARCH 

REVIEl'J & APPROVAL 
BIOLOGIC STAHDARDS 



[Z! 



ADJilNISTR/^.TION 



l_ 1 PROI'ESSIONAL & 

J" TECIIillCAL ASSIST. 

I ANCE 



D 



111, IDENTIFY A >IY COOPER/.TING UNITS OF THE PUBLIC IF^LTH SFJlVICii, OR 
OTIiER 0;[GANI2ATI0NS, PROVIDING FUNDS, FACILITIES, OR PERSONl'EL 
FOR THIS PROJECT IN FY 1957. IF COOP ;iATING UNIT IS WITICEN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



SliRIAL nL.l©-107c 



pp.rt C. Honors, Awi.rds, and Publications 

16. PUBLICATIONS: None 

17. WJARDS AND HONORS: None 



PUBLIC IDV'.LTH SEUVICE lUvTIOlIAL Jj'STITUTr? Q? I'^ALTTI 



iLinr/muAL piojt^ct liEPoiiT 



Part A. Project Description Sheet 1. N Liffl-108c 

Serial ilumber 

2, III/ vID 3. CH nical Invcstic ;jtions_ 

Institute or Division ' Laboratory, Branch or Dept, 

^' • Ai-thritis C: Rheumatis m Er , $ , 

Se'ction or Service Location 



6, Internediar^^ Ririne iieta bolism in Gout 

Project Title 

7 , J. F. oee{ ;mi ller, ii. D, 

I'ri.ncipal Investigator 

8 . Leonard Laster, M. P.. L ois Liddle, Lthel Love,IIeil Goodsell 

Other Investigators 

9, No parallel research in Public Health Service 



10, Pi'oject Description: 

The metabolism of uric acid is under study in :outy 
patients using the general approach of determining the 
fate of isotopically labeled precursors of uric acid, or 
labeled uric acid, folloTJing their administration to 
normal and gouty individuals. 'Jith the pattern of incor- 
poration of glycine I:-'--^ into uxinary uiuc acid as a 
"screening study, x/e have found three gouty patients uho 
incorporate ^^Jycine more prom.ptly and to a greater extent 
than do normal subjects or a majority of the gouty patients, 
lie have studied two of thef-e "over incorporators," using 
another Drecvirsor of uric acid, and the inotope data are 
available for one subject at this tii'ie. He incorporated 
i}-amno-5-imidazolecarboxa!iiide-Gl3 into uric acid to a 
greater extent than normal. It was found in previous 
studies of healthy and routy subjects th^t the simultaneous 
administration of AIC-cl3 and glycine W-^ produced an in- 
hibition of ::lycine incorooration into urate. In the 
"over-incorDorator" the glycine incorporacion v:as only 
partially inhdlUted, suggesting a possible defect in the 
regulatory mechanism for overall purine biosynthesis. 

In all our studies we have also determined the 
subject's urate pool size and turnover rate, using uric 



Page 2 of Project Description T-heet N LJiD-10 6c " '- 

"S'erial LluSber 



acid C-'^'. Such data mil aid in tlie interpretation of the 
AIC incorporation data, Follouin;'] the injection of the 
uric acid C-'-'*, a given per cent of radio activity is not 
reco^^ered in the urinary iiric acid. If this loss can be 
attributed to uricolysis, then v:e find an average of 30^ 
uricolj^sis in normal subjects id-th a range of 30-5^/-^ in 
pouty subjects. Ue vrill investigate uricolysis in greater 
detail by administering: utIc acid with extremely higli iA-' 
content and measuring the recovery of N^^ in urinaiy urea, 
aminorLia, la-ic acid and total nitrogen, Ue also plan 
fujrther investigations in vitx-'o and in vivo of the regulatory 
mechanism for purine biosynthesis. In addition, uric acid 
precursors other than AIC id-ll be used to e:cplore the bio- 
chemical defect in gouty patients. 

We have investigated the influence of colchicine on 
uric acid formation from glycine N-'--^, In tuo patients 
studied thus far, colchicine appears to increase the 
iiTimediate incorporation of glycine into uric acid to a 
small extent. This finding needs further study. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SEHVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJlsCT RLPORT 



Part B: Budget Data 



11 NIAliD-108 (C) 



SEHIAL NUiffiER 



12, BUDGET DATA 


I 








ESTPiATED Or.LIGATIGNS 




MA.H YEARS 


DIRECT 


REIIviEURSEI-Ei>JT TOrAL 


PROF 


OTiER TOTAL 


FY«57 








^16,800 


$23,^00 C:ll|0,300 


.33 


2,00 2.33 




HroOETED POSITIONS 


285 


PATIENT DAYS 


PROF 


OTHER TOTAL 




FI«^7 








0,33 


3.00 3.33 






1^- RUrrrF,T AC"T 


\rr'!'Y! 







RESEARCH 

REVIEl'/ & APPROVAL 

BIOLOGIC STANDARDS 



CZj 



ADMINISTR/vTION 

PlOFES.lIOmL & 
TECHNICAL /.SSIST- 
ANCE 






lU. IDE JTIFI Ai^ COOPr,R..TIiIG UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTIER ORGAi-flZilTIONS, PROVIDING FUNDS, "AGILITIES, OR PEREOi\riJEL 
FOR THIS PROJl^CT IN FY 1957. IF COOPERATING UNIT IS WITHIN HIH 
INDICATE SERW-L NO(S): 



(Use reverse and additional pages, if necessary) 



Part C. Honor s, Mrard3 :•■ Fublicatio n3 iS.JJI^'D-lOGc 



serial number 



16, Publicatio ns fro m this project d ui-itiK 1956t 

J. '-. Ceeeniller, DeW. Stetten, J. J. Dunim, L. 
Sokoloff, L. Lacter aiv' J. B. •/:mcaarden, Clinical and 
iJetrbolic Aspects of Gout, /un. J. of ledicine. In press. 



17. Honors and Awards: Itone 



POBT.IC irALT!! STTJVTCE HATIOM/.L INSTITUTES OP H)->.LTII 



IimiVTDU/X PROJECT RFPOr.T 



Part A. Project description Sheet 1 ?.'!/. MP- 109c 

Serial ifunber 



2, MIAM) 3. Clinical Investigations 

Inatltute or Division Laboratory, Dranch or Dept. 

li. Arthritis ' ilheumatis'n '3r .5. 

."icction or Service Location 

6. ttetaboHc ajul Tiierafeutic Studies of Gouty Arthritis 

lYojcct Title 

and Hyperuriceiiia 



7. J. r, oeccwillor. ij, U., liconarrl L-ister, ■.. 
Principal Investicators 



n. 



8, K. I^pyone Yicl-Un . X.J.. Joseph MoGuirc, .:.':),, 

Other Investigators 

liOls I.id'le. Fthel Love 

5ther Investigators 

9, iio parallel research in Public Health Service 



10, Project Description: 

We are establishing normal values for the level of 
scrum uric acid and the ''aily urinary urate excretion in 
hunan subjects under controlle*' conditions, as detennined 
with an accurate enzymatic method for urate assay. V7e are 
tryinc to correlate these values with clinical features 
of pout and further studies will be pursued to obtain 
statistically valid conclusions. 

In an evaluation of the effectiveness of intravenous- 
ly administered colchicine, ve firK? that this route of 
administration may at tines be superior to the oral route 
in the manarenent cf acute fout, ^'e have encountered no 
serious toxic effects in treating 20 acute attaclts, r-elief 
vaa more prompt in the majority of tlie patients, and in 
some cases jiiprovcnent occurred after oral medication had 
failed. In general there were fe\7er gastrointestinal 



Pace 2 of Project Description "heet NLJ-in-lOSc 

Serial "rJurfcer 



— '■- s follor;ing the intravenous administratton of 
Jtic doses of colchicine, A fe:' cases did not 
rca^ojiri to this fona of Wierapy, Further studies of 
this problem are planned. 

In Uro cases of leulcenia ;dth associated hyper- 
uricemia, AIC-C -^ v;as incorporated into uric acid in a 
pattern indlcotinf a rapid and a sloir contribution of 
irotope to urinary uri.c acid. The data for urate pool 
size and turnover rate detenninations in these patients 
are not yrt available. It is hoped that by comparing uric 
aci'" nctalx>lism in persons \;ith such diseases to f^outy 
patients, inaicl^t can be pained into ihe metabolic abnor- 
fiality of cout. 

In one patient >rlth essential hy^jei-uricemia, a 
relative of a gouty patient re have already studied, 
2ly':inc JH-' '.'as incorporated into urinary uric acid in a 
pattern differinc from the normal in tliat peak incoiTKDra- 
tion of isotope vras not observed until the l?th day alter 
administration, './c vrill continue to search for a 
blocticmical difference in nvirine metabolism betv;een 
patients '.fho ehmi only essential hyperuricemia and those 
individuals ^dth hyT>€niric€.nia uho also suffer attacks 
of acute pout. 



' 



fbm No. GRP-1 
October 1956 
(Attaclmcnt I ) 



PUBLIC HEALTH SFilVICE - NATIONAL INSTITUTES (F HEALTH 
riDIVITUAL PROJECT REPCRT 



Part Bi Budget Data 



11. IlIA!tL^109 (C) 
^RIAL NUWHER" 



12, HJDOE? DATA 


J_ 








ESTr •" -^ '^'^' ■:'^'JNS 




HAN YEARS 


DIRECT 


TOTAL 


PROF 


or HE?. TOTAL 


FY«57 








v21,800 


i97,200 ^^119, 000 


3.00 


1.00 1^.00 




BUDGETED POSITIONS 


1168 


I'ATIEUT DAYS 


plioP 


OTHER Tv-TTkL 




FTf«^7 








3.00 


1,00 U.oo 






13. Bum? Arri 


/I?Yt 







RES LARCH 

REVm/ & APPROVAL 

BIOLOGIC STAlfDARDS 



fx"l ADI.INISTlUiTION 

I I PRoiT-s::i >i;;i.L & 

TEOKillGAL ASSIS: 
[ j AHCE 






Hi. IDErriFY AiJY COOPr.R/.TINO UiJITS OF THE i^UFLIC HEALTH SERVICE, OR 
OTHER ORQA-JIZ/.TIOIIS, F'.OVTnr.IO FUNDS, FACIUTIEo, OR PiaSONNEL 
FOR T:fI3 P;0J:CT m FY 19!57. IF COOPERATING UNIT IS WITHIN NIH 
IiiDia-.Tl-: 3 .RDvL N0(3)s 



(Use reverse and additional pages, if necessary) 



Part C, Honors, Av/ards t Publications NL>MD-10yc 

t Seria!r"FKJiiiiber" 



1^1. Publi carbons from this project durir^ 1956! 



DeW, Stetten, J. H. Talbott, J. E, Scecniller, J. B. 
'./yngaarrlen and L. Laster, "Pathogenesis of Gout" (letter) 
iletabolism. In press. 



17. Honors and Awards: lione 



PUBLIC iir/j.TH sT:nvicF. jjATioii/j . insTinms oj ij-alth 

ItDIVmUAL PT^Of CT R "^RT 



Part A. Project description Hheet 1. NIAWVllOo 

Serial Nurfcer 



2 hflAlD 3 Clinical Investigations 

* Tnstltut!e or T^raWdi" laEoretory, Branch or iJepi. 

li. Arthritis '-. rainumatism Dr. 5. 



Section or Service location 

6. BiocheiTi ical_Rcactignn Tn volvinr Uric Aci d 

Project Title 

7, Leona rd Lostcr, K. D, 

T'ri."nc~ipal Tirvcstif'.ator 

/'.Iberta Dlair 



8, J, "f neerminer. ]: P.. /•■ 

Other Invcnt'.rstors 

9, Ho parallel researd* in Public liealth Ser\d.ce 

10, Project Description: 

Studies of vric ac-sj biosynthesis ar/- -..e nh^'siolocy 
of this comom.nd suefcst that it mny not be an ^"ert end 
pro^»uct as it is naxt considered. It was shoA-n in 1922 that 
beef rM hlood cells contain uric acid riboside. A chronat- 
ograohic rKtl^.o^' lian now been devised to isolate this 
corrouiyl in its pure fona and usinf it as a substrate, a 
ne^r enayne has been isolated fx^m anirml tianue tl>at splits 
the riboside into uric ac5d anrl another compound, T.iis 
ensyw is b^ ifiel in ordrr .hat the reversibility 

of the react br studied and the product identified. 

v/hen this is corTpleted, it is proposed to c© -^n to study 
the f onwtion or exisU^nce of uric acid ribotide, a com- 
pound as yet uni:nown, and to det^rrrine uhethrr the ^'ray-fB 
an\ cnth-.rays involved in this study ),ave an>- apnlicabillty 
to nomal rtv^ r.'outy hurians, CornTounrls nuch as the ribo- 
side and ribotide of uric acid nay be involved i'.i the 
transport of Viis compound across tJw tubules of the 
kidney an\ this problem will be investigated. 



Form No. 0'>P-1 
October 19^6 
(Attachment I) 



PUBLIC HE^XTH SLRVICE - NATIONAL IHSTITITES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part Bi Budget Data 



11. NIA}4D-110 (C) 



SERL.L NWiEFR 



12. njlBFr: DAT^ki 



BF£(i? 



ESTry.TED 0?LTi;.TIONS 



TOTi.L 



FY»5f 



ia3,7oo 



e5|Ooo 



^8,700 



Flf'57' 



ITCTT 



E'lLOSTED POSITIONS 

t5nf"i 



TOTT 



1,00 



2,00 



3oOO 



13. Wiry-": //rnviTYi 

FEHE'.;.^:! 

REVIEJ'/ !: APPIiOVAL 

BIOLOGIC STAtHDAlUDS 



PROF 



1.00 



Pxl ATHIMISTR/vTIOH 

rn PR(>FHk3SIORL & 

:'ECHIfIC/.L ASSIST- 
( I AKCR 



MAIJ YE/.PS 



OTftJl TOTAL 



2.00 3.00 



PATIENT DAYS 






Hi. IDE rTTFY AJ'Y C00F-.:1'.:TJG U'hITS OF TIE i^UBLIC HE/.LTH SJ^VICE, CR 
(TUP. 0?.G«\ir[:/.TIONS, PROVTDIirO »TWnS, KTiCILITIK, OR PERSOWiCL 
FOR THIS PROJECT Irl FY 19^7. IF COOPER/.TING UNIT IS WITHIH iHH 
INDICATE SERIAL i)0(S)j 



(Use reverse and additional pages, if necessary) 



Part C. Honors, /.'.rarrta *. PubLi cations 1<. NL.?-ffi-110c 



16, I\ibli cations! Jbne 

17, lienors zni Airards: Ihne 



PUBLIC HEALTH SFRVICE II/lTIONAL INSTITUTES OF I-JEALTH 



liDWIDUAL PROJEiGT REPORT 



Part A. Project Description .Sheet I J^LiMD-ll lc 

Serial Number 



2. NIAi ro 3. Clin ical Inve sti gations 

Institute or Division Laboratory, Branch or Dept, 

b. Arthritis c; Rheumatism Br, $, 



Section or Service Location 

6. Biochemical Studies of Metabolic Diseases 



Project Title 

7. J. E. Se egmiller, M. D, ^ Leonard La ster, M. D^ 

Principal Investig-ator 

8. Ethel Lo ve 

Otlier Investigators 

9. Para-llel research in Public Health Service 

Parallel research being done by Dr. Frank Eisenberg, 
NIAI®, of the Laboratory of Biochemistry and i'fetabolism, 

10. Project Description: 

A study to identify trace quantities of carbohydrates 
present in hujran urine is being pursued in cooperation iilth 
Dr. Frank Eisenberg^ NIAJO. Dr. Eisenberg is reporting 
on this oroject. 

We are investigating the precise nature of the 
metabolic defect associated with alkaptonuria. Indirect 
evidence supports the concept that the snzyrne, homogen- 
tisate oxidape^ is absent in patients afflicted with this 
disorder. Because we have been unable to demonstrate the 
presence of this enzyrae in readily available biological 
materi.als, including saliva, seruiii^ white cells, and whole 
blood from normal individuals, biopsy material from other 
tissues Trill need to be used to demonstrate the absence 
of the enzyme in alkaptonuria patients , We are now con- 
sidering the use of liver biopsy to obtain enzyrae- containing 
tissue. 

Using a rat liver homogenatc preparation of homogen- 
tisate oxidase, We have found no inhibitor of this enzyme 
in the seruiii of an alkaptonuric subject. The urine, 
however, may possess a small degree of inhibitory action 
as compared to the urine of a normal patient. 



Page 2, Project Description Sheet HL-J'iD-lllc 

Serial Nwaber 



The presence of large araountv-s of homo gentis ate in 
the urine of these patients presents an opportunity for 
investigation of aromatic ring biosynthesis in the human, 
using isotopically labeled precursors. 

A standardized method for the separation of the 
ultraviolet absorbing cationic components of urine by ion 
exchange resin has been developed. This method will be 
used for preliminary screeni.ng for abnormal metabolites 
in a variety of diseases. 

In one normal volunteer made mildly thyrotoxic with 
triiodothyronine, the pattern of glycine N^-H incorporation 
into uric acid was very similar to that observed when the 
patient was euth3''roid. 



Fom Mo, ORP-1 
October 19^6 
(Attachment l) 



PUaiG HEALTH SF£IVICE - MATIOML li^ISTITUTEC OF lEALTH 
IriDIVIKJAL PHOJl^CT REPCET 



part B: Bu.dget Data 



11, MI/vMD-ni (C) 



SERI/iL NUMBER 



12: BUliGi;? m.?A 


I 








T^STrfl/lTED OPLICaTIOKS 




im YEP.RS 


DHIECT 


RF,EinjRSEI'5EWT TOTAL 


PROF 


OTHER TOTAL 


FYt57"" 








$10,700 


^19,000 129,700 


1.00 


1,00 2.00 




BUDGETED POSITIONS 


230 


PATTEi^iT DAIS 


PitOF 


OTHER TOTAl 




FYI$7 








1,00 


1,00 2^00 






13. BUDGET ACTH 


JlTJi 







RESEARCH 

REVIE.J &. APPROVAL 

BIOLOGIC STANDARDS 






ADMINISTR/iTION 

PROrES SIGNAL & 
TECffiUCAL ASSIST- 
ANCE 






lit, IDEfiTIFY ANY G00PERiiTIi\FG UNITS OF TfE PUBLIC lEALTH S^mVIGE, OR 
OTHER ORGAtHZATIOHS,, P lOVIDIWG FUi\IDS, FACILITIES, OR PERSONiEL 
FOR THIS PtOJECT IN FY 1957. IF COOPIIRiiTING UNI? IS WITHIN NIH 
INDICATE SFEIAL NO(S)j 



(Use reverse and additional pages, if necessary) 



Part C. Honors. hiRvds !k Piibli cations l5.iPi®liii£ 

Serial Number 



16, Ribli cations: None 



17, Honors and Awaids: None 



Public Health Service National Institutes of Health 



II^r/IDUAL PROJECT REPORT 



Part A. Pro.l ection Description Sheet 1. NIaMD-112c 

Serial Number 

2, i'II/LL€) 3» Clinical Investigations 

Institute or Division Laboratory, Branch or Dept, 



h. Arthritis & Rheumatism Br, 5. 



Section or Service Location 

6 . Studies on the E nzym atic Metabolism of Steroids 

Project Title' 

7. Gordon l i. Tomk ins, 11. D, 

Principal investigator 

8 . Joseph McGuire 3 M.D,j Patricia Hichaelj Jean Curran, D.Mason 

Other Investigators 

9. No parallel project in P. H. S, 



10. Project Description : 

The objectives of these studies are to elucidate nevj 
pathways by which steroids are metabolized, to study the 
enzymatic mechanisms by xrtiich these metabolic changes occur, 
and, ultimately, to get an insight into how steroids exert 
their dramatic biological effects. The methods employed 
involve techniques for handling enzyme proteins, as well 
as the totally different methods used in steroid chemistry 
for the isolation, identification and, in some cases, 
synthesis of substrates or end products. 

Considerable progress has been made in several aspects 
of this work since the preceding report. Previously, we 
had identified enzymes capable of reducing (and thereby 
inactivating) various steroids. Further work on the 
characterization of the substrate specificity of these 
enzymes, as well as the mechanism of their action, have 
yielded interesting preliminary results. It has been 
found that in the reduction of the h-$ double bond of 
ring A of the steroid nucleus, a proton is attached to 
carbon h and a hydride ion from the reduced pyridine 
nucleotide is transferred to carbon $ as xrould be predict- 
ed if the reduction did not proceed by a process of l,i! 
addition and there was direct transfer of the hydrogen 
from the pyridine nucleotide to the substrate double bond. 



HnJiD-112c 



Serial Number 



Page 2 - Proje ct Description She et 

This is the first biochemical mechanism of its kind i-rhere 
a double bond is reduced directly by a pyridine nucleotide, 
rather than a flavo protein. 

Studies on the mechanism of steroid hydroxylatlon have 
revealed that separate systems are involved in the conver- 
sion of DOC to corticostei-one and compound S to compound F. 
Furthermore, it has been found that 2 enzymes are required 
for each hydroxj^lation. These studies are of importance 
on several counts. First, because the adrenogenital syn- 
drome is thought to be due to a genetic lack of one of the 
hydroxylating enzymes and we know on the basis of our 
studies that this is a complicated metabolic lesion. Also, 
since this type of reaction has its counterparts in a 
number of different types of compounds, an insight into 
the mechanism of one might be revealing for others. 

Lastly, a series of related reactions in liver has 
been studied, and their kinetic analysis has suggested 
that a metabolite of TPNH, not TPN, is involved in these 
hydroxylations , 

Pro jected c ourse of project: Many reactions not 
yet comnletely studied have been encountered in liver, 
which will be elucidated in the future. These are reduc- 
tion of C21 hydroxymethyl group to a methyl, ring 
hydroxjrlation, partial aromatisation. 

The metabolic defect in adrenogenital syndrome will 
also be investigated at an enzyine level. 



Form No, QRP-1 
October 1956 
(Attacliment I) 



PUBLIC HEALTH SERVICE » NATIomL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, MIAMD-112 (C) 



SERIAL NUI4IER 



RESEARCH 

REVIEl-J & APPROVAL 

BIOLOGIC STANDARDS 



CiH ADIIJNISTRATION 

□ PROFESSIONAL & 

TECHNICAL ASSIST- 

□ ANCE 



12, BUDGET DATA 


s_ 








ESTIMA.TED ODLIGATIONS 




MAN YEARS 


DIRECT 


REIMBURSEIIEiC' TOTAL 


PROF 


OTHER TOTAL 


FY '57 








$2^,100 


$11,200 $36,300 


1.17 


1.33 2,$0 




BUDGETED POSITIONS 





PATIENT DAYS 


PROF 


OTHER TOTAL 




FY '5 7 








2.17 


2.33 ll»50 






13. BUDGET ACTIVITY! 







□ 



□ 



lit. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING F'JNDS, FACILITIES, OR PERSOIfflEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S)! 



(Use reverse and additional pages, if necessary) 



Part C; Honors , ATjards "; Piibli cations Ig. M LxI"ED- 112a 

Serial Number 



3.6, Publications from this project durj.ng 19^6: 

Tomliins, G., A ifemvnalian 3-0. Hydroxys tero id Dehydrogenase, 
J. B. C., 218: 1)37, 1956, 

Tomkins, G,, Enzymatic Reactions in Steroid Metabolism, 
Progress in Hormone Research, 12: 12^, 1956, 

Jakoby, ^J. , and Tomkins, G. M. , Enzymatic Detoxification 
Mechanism for Viadril. Science, 123: 9hO, 1956, 

Tomlcins, G. M., The Enzymatic Reduction of ■.■:A^'-3-Ketosteroids, 
J. B. C. In press. 

Liddle, G. R., Tomkins, G, M, , and Richard, J, E,, Studies 
of Structure-Function Relationships of Stei-oids : The 
2-iJethyl-Gorticosteroid.'3. Metabolism V, 381!., 1956. 



17. Honors and Awards: None 



Pu blic Health Service Not ional Ins titutes_of_Health 

IMDI VIDUAL PROJTCT_RrTORT 

Part A, Pro.jec t Descrip tion Sheet l. NIi.MD- 113c 

Serial Niuuber 

2. NIAID 3. Clinical Investigations 

Ynstitute or Division Laboratory^Branch or Dept, 

h, .Arthritis C: Rhe-uunatism hv,$ . 



Section or Service Location 

6. Biosynthesis of Hyaluronic Acid 
Project~l^.tle 

^ . K . Lemone Jfielding, M. D. 

Principal Investigator 

8 . Prnrdnn I' ^ Tornlcin s. I-'. P., Joseph J . Bunim, II. D. 

Other Investigators 

9. No parallel project in P. H . S, 



10, Pro.ject Description! 

Incubation of fresh human synovial tissue slices mth 
glucose ClG has yielded a labeled com-^ound having the 
chemical and nhysical characteristics of hyaluronic acid. 
Certain of the adrenal cortical and synthetic steroids were 
found to inhibit this incorporation of glucose C ''. Serum 
from patients idth rhemaatoid arthritis has not produced 
consistent effects. Work is in progress to elucidate the 
mechanism of steroid inhibition. Furthermore, a cell free 
system has been prepared frop. human umbilical cords which 
will incorporate glucose C^'- into hyaluronic acid. Studies 
of joint fluid suggest that certain changes in the struc- 
ture or metabolism of hyaluronic acid are characteristic 
of rheumatoid arthritis. It would appear, then, that a 
thorough understanding of the formation of hyaluronic acid 
should°provide a promising approach to the problem of 
rheumatic diseases. 



Form Wo, ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEA.L::H service - imTIOML INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



part B: Budget Data 



11, 1\IIAI©-113 (G) 



SERIAL NUIfflER 



RESEARCH 

REVIEl'J & APPROVAL 

BIOLOGIC STANDARDS 



12, BUDGET 


DATA 


• 

ESTimTED OBIJGATIONS 




MAN YEARS 


DIRECT 




RElMBURSEIffiNT TOTAL 


PROF 


OTlER TOTAL 


FY«57 

^i;7,liOO 




^^3,800 §11^200 


.66 


.66 






BUDGETED POSITIONS 





PATIENT DAIS 


PROF 




OTHER TOTAL 




FY«57 










,66 




,66 






13. BUDGET 


ACTIVITY! 







13 An^IWISTRATION 

rn PROFESSIONAL & 

TECHtJIG/'-L ASSIST- 
Y^ ANCE 



a 
a 



111, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVmiNG FUi^IDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S); 



(Use reverse and additional pages, if necessary) 



Part C. Honors, Awards aucl Publications 1$, NLiMD-113c 

Serial Number 



16, Publicabions from this project during 1956: None 



17, Honors and Awards: None 



Calendar Year 19^6 

PUBLIC liEALTH SERVICE - - milOIJAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1» MIaMD-IIUc 

SERIAL NUi^B^ 

2, MIAMD 3. Metabolic Diseases Branch 

INSTITUTE OR DIVISIOK LABORATORY, BRANCH, OR DEPARTJIEWT 

h. 5. ^ 

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHESDA} 



6 • Total Ene rgy Metabolisra; Studies in Health and Disease 
PROJECT TITLE 



7, G. Dona?.d VJhedon 

MiNCIPAL INVESTIGATOR 

8, Ernest E. Huber, Jr, and Ronald Ho Thompson 
OTHER I':!VE3TIGAT0RS 



10 6 P ROJEC T DESCRIPTION 
Obj ectives ; 

1) To establish by complete actual measurement a technique 
of total energy balance which can be applied to various clinical 
problems and to fundamental physiological problems of energy 
metabolism not now understoodj 

2) To study the influence on tctal energy consumption and 
balance of various factors, including climate and the endocrine 
hormones 3 

3) To investigate the characteristics of energy balance and 
their influence on the nutritional state of patients in pertinent 
disease conditions, such as obesity and cancer. 

Meth ods Employed ; Indirect human ealorimetry by means of 
expire3~air analysis in the Metabolic Chamber, metabolic balance 
determinations, caloric analysis of dietary intalce and excreta. 



* 2 - 

NLiMD-llUc 
SERIAL IIW'JBER 

Patient Material ; 

6 patient days 



Major Findings ; Work on this project during the past 
calendar year has principally involved installation, prepara- 
tion and modification of various apparatuses and considerable 
pre-experimental testing. Tests of normal subjects have 
demonstrated the rapid response and sensitivity of the general 
analyzer system and the feasibility of use of a plexiglass- 
vinyl hood being constantly exhausted for continuous measure- 
ment of respiratory gas concentrations, a system not previously 
used in human metabolism studies « A second hood, more suitable 
for lengthy studies, was then constructed, fitted mth a small 
microphone for easy intercommunication between subject and in- 
vestigator, and tested for suitability in use during feeding 
(liquid diets) and sleeping at night. Carbon dioxide analyzers 
have been installed and modified electronically by E. E. Huber 
to provide linear response; this was an important, useful modi- 
fication apparently not known to the company manufacturing this 
analyzer. The Metabolic Chamber itself has been fitted with 
equipment necessary for reasonable comfort in studies expected 
to last several days» Apparatus has been assembled and con- 
structed for use in metabolic rate and work efficiency studies 
in a project on the metabolic effects of thyroid hormonal aiia- 
logues. In November, the first alcohol check experiments were 
carried outo 

In preparation for construction of the direct gradient 
calorimeter to be used in later studies, two test panels bear- 
ing multiple coDper-constantan thermocouples have been made by 
the NIH Instrument Shopj these will be compared for speed and 
stability of response. 

Significance to MIAMD Research ; Previous studies of total 
energy metabolism were concerned with the influence of various 
diets on normal metabolism and were carried out with chambers 
in which oxygen consumption could not be measured directly* 
Other total energy data have been baaod on measureraents of food 
consumption or on brief measurements of activity metabolism 
extrapolated to twenty-four hours. The NIAl'ID Metabolic Chamber 
presents an ujiique facility for the exact and complete measure- 
ment of all important expired and excreted products in the 
study of energy metabolism over long periods of tim.e. Study 



^3" 

with this technique has not been made previouslj'' of the effeots 
on total energy of various physical and endocrine factors or of 
disease states. An example of a disease in which energy metabo- 
lisiti may be altered, to be approached with the Metabolic Chamber, 
is obesity or overweight which pi'edisposes many to the develop- 
ment of diabetes, heart diaease, hypertension and atherosclerosis* 

Proposed Course of Project ; Experimental work during the 
next year will include studies of metabolic rate and work ef» 
ficiency first in normal subjects and then in patientsj studies 
in the latter w5.11 be part of an investigation of the metabolic 
effects of thyroid hormonal analogues. VJork will also be under- 
taken in normal subjects to establish the technique of energy 
balance over periods of several days; later this technique mil 
be applied in studies of disease. Additional developmental work 
xd.ll be carried out toward construction of the direct gradient 
calorimeter within the existing chamber. 



Form No« OIIP-1 
October 1956 
(Attachment I) 



PUBLIC HEALTH SERVICE - WATIOMAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part Bs Budget Data 



11, NIAMD-III4 (C) 



SERIA.L NUlfflER 



Fit $7 



FY '57 



BUDGET DATA! 






ESTHmTED OBLIGATIONS 




FiAW YEARS 


DIRECT REIMEURSEICiNT TOTAL 


PROF 


OTHER TOTAL 


$26,200 - ^p26,200 


2.33 


1.00 3.33 


BUDGETED POSITIONS 


6 


PATIEIOT DAYS 


PROF OTiffiR TOTAL 




2.33 1.00 3.33 






BUDGET ACTIVITY: 







RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STANDARDS 



'nr \ AmiNIATRATIOM 

I 1 PROFlilSSIONAL & 

TECFMic/i.L ASSIST- 
' I ANCE 



□ 



lU, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HE/lLTH SERVICE, OR 
OTHER, ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOIWIEL 
FOR THIS PROJECT IN FY 19^7. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL HO(S): 



(Use reverse and additional pages, if necessaiy) 



Part C. Honors, Awards & Publications iS* NTi. ^O-llkc 

SERIAL iTOlBER 

16, Pablications ; None 

17, Honors and Auards; Mone 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - - MTIOliAL INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet 1. WIPJ^ID-ll^c .. 

SERIAL NUMBER 

2, N IM1D 3. Metabolic Diseases Branch 

INSTITUTE OR DIVISION LABORATORI, BRANCH, OR DEPARTMENT 

'SeICtIon OR gfiflVlfifi ' WCAfloM (If otMEr than bethesua; 



6, studies in Bone Metabolism 



PROJECT TITLE 
7, G, Donald I'Jhodon and Robert Pj Heaney 



8. Jean Ciirran 



OTHER DIVEST IGATORS 

9. 

10, PROJECT DESCRIPTION ; 

Obj ectives t 

1» To investigate the factors affecting mineral storage 
and loss in demineralizing bone diseases, with particular at- 
tention to the relative influences of adrenal cortical steroids, 
gonadal steroids and dietary levels of minerals. 

2, To investigate tha rates of mineral deposition and 
amounts of bone undergoing active exchange with body fluids, 
in various bone disorders. 

Methods Employed ; 

1, Metabolic balance studies under rigid dietary control 
in patients with various demineralizing bone diseases, noting 
the effects on nitrogen, calcium and phosphorus balsuices of 
adrenal cortical steroids, of gonadal steroids and of various 
dietary levels of calciijm and phosphorus. 

2. Determination of pool size, turnover rate and deposi- 
tion rate of calcium in patients with various bone disorders, 
using tracer doses of radioactive calcium. 



- 2 - 

SERIAL M'-IBSR 

Patient Material ; 

1J|)|6 patient days 

Major Findings ; 

1, Detailed metabolic balance studies in a group of 

six patients with demincralization of the spine (sstesporosis) 
have sho-wn a consistent trend suggesting import.ance of abundant 
amounts of dietary calciujn in the treatment or prevention of 
this condition. Since the disease Is now regarded as a disorder 
of protein tissue metabolism and not of mineral metabolism, 
present therapeutic attitudes scorn the value of dietaiy minerals 
and favor adjninistration of anabolic gonadal hormones. In the 
present studies these hormones have not consistently favored 
calcium storagej the possibility has been raised that ineffective- 
ness of these agents in our liands might be related t© previoiis 
saturation of deposition processes by the continuing abundance 
of mineral which we have provided. 

Administration of gonadal steroids to t'.TO patients with 
osteoporosis and active rheumatoid artliritis, requiring adrenal 
cortical steroid for control of their joint disease, brought about 
definite nitrogen storage in the face of the known anti-anabolic 
action of adrenal cortical steroids but failed to influence cal- 
cium balance to any noticeable extent, 

2, Tracer radioactive isotope studies have been carried 
out on eight patients with analyses complete on three, so that 
the data is prelirainary in nature. The miscible pool of calcium 
appears to be approid.matelj'' 10 gm., much larger than the laaox-m 
amount of extra-osseous calciuxa in the body yet considerably 
smaller than the total body calcium. In vintreated liypopara- 
thyroidism the pool size and turnover rate of calcium are re- 
duced. The turnover rates in all patients studied thus far are 
much larger than can be accounted for by dietary intalce and 
excretory loss, so that it is expected that mth additional 
data it Td.1.1 be possible to calculate and evaluate actual rates 
of new bone deposition. 

Sign ificance to NIAI- ID Research; Deritineralization of the 
skeleton is a comriion accompaniment of many incapacitating chronic 
diseases ab all ages, including severe rheumatoid arthritis. Al- 
though gonadal steroid hormones administered parenterally are 
reported as favoring mineral storage, universal agreement on this 
point does not exist, nor is there adequate information as to the 



- 3- 



effects of oral gonadal steroids which are riore commonly pre- 
scribed in practice for the treatment of osteoporosisa Since 
a high incidence of the latter bone disorder has been particu?- 
larly noted in patients idth active rheumatoid arthritis under 
management with adrenal cortical steroids, and the latter com- 
pounds are being more and more Td.dely prescribed, it is becom- 
ing increasingly important to obtain information which xri.ll 
aid in understanding more clearly the relative influences of 
adrenal cortical and gonadal steroids and of dietary minerals 
upon mineral metabolism. The radioactive isotope tracer method 
is being developed in the hope that it will prove to be a val- 
uable supplement to the metabolic balance techniqu.e in the 
study of mineral disorders. 



Proposed Course of Project : The studies will be continued 
in a number of additional ps.tients, employing the methods de- 
scribed, in order to obtain information which, it is hoped, will 
ultimately enable us to deal effectively vjith the extensive 
pixiblems outlined. 



Fom No, ORP-l 
October 1?56 
(Attachment l) 



PUBLIC HEALTH SERVICE - i'tATIONAL INSTITUTES OF HEALTH 
II\IDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11. NIAMD-»ill^ (G). 



s^.i/lL number 



12, BUDGET DATA: 












ESTIMTED OBLIG/i.TIONS 








MAN EARS 


DIRECT 


REBIBURSEi-ffii^JT 


TOTAL 




PROF 


OTHER TOTAL 


FI»57 

$35,600 


$120,700 


$156,300 




1.00 


5.00 6.00 


BUDGETED POSITIONS 




1/|) 


6 


PATIENT DAIS 


PROP 


OTHER 


TOTAL 






FI«57 












1,00 


5.00 


6.00 









13. BUDGET ACTIVITY; 
RES.SARCH 

REVIEiyJ & APPROVAL 
BIOLOGIC STANDARDS 



CD 
D 



ADI'HNISTRATION f^ 

PROFSSSIOML & 
TEGHMICAL ASSIST- 
ANCE Q 



lU. IDEWTIFI AMI COOPE^TING UNITS OF THE PUBLIC HEALTH SERVICE, CR 
OTIffiR ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOMjEL 
FOR THIS PROJECT IN FI 1957. IF COOPERATING UNIT IS WITHIN NIH 
DHDICATE SERIAL N0(S): 



(Use reverse and additional pages, if necessary) 



Part C. Honors, Awards & Rablications 1^. g^^^-^^j^^^^ 

16. Publications : None 

17, Honors and Awards: None 



Calendar Year 1956 

PUBLIC HS/vLTH S'.IRVICE - - NATIOiIAL INoTITUTJS OF lEiiLTH 
E3DI7IDUAL PROJECT REPOilT 

Part A. Project Description Sheet 1. NIii.MD-ll6c 

SEfllAL NUMBSR 

2, MI/u)'!D 3» Iletabolic Diseases Branch 

!r:.iSTrruTs or division laboPuItory, brimgii, or departi-'iEsmt 

h, 5. , . 

gfcflOirOR'SSRVICS LOCATICN (IF OTin.i T.K.N BLTtlKSDA) 

6, Iletab olic Effects o f Adrena l -Cortical Steroids 

ERdJZCT TITLE 



7, G, Donald 'Jhedon 

PRINCIPAL 'IW'STIG/VTOR 



8« Robert P. Hegiiey, Roger L. Black and Jean Gurran 

OTHER i:'W'::r;TIG;iTORS 

9, This project cornpleraents (and is cooperative witli) "Trial of New 
Anti-RheuTiiatic Drugs". 

lOo PROJECT DEoCRIPTION ; 

Objectives: To evaluate the metabolic effects of various 
new synthetic adrenal-cortical steroids with reside ct to sodlujra, 
potassium and nitrogen excretion and in selected instances mth 
respect to calciuxa and phosphorus balanofli.. Effective anti- 
Inflainrnatorj^ action does not qualify a new steroid for ijide 
clinical trial in rheumatoid artliritis unless certain metabolic 
side-effects can be shovm to be minor or iabsent. Tlie particular- 
ly undesirable effects most often encountered are sodium and 
water retention, and potassium and nitrogen loss. 

Methods E mployed ; Under rigid dietary control short-term 
metabolic s'tudies (six weeks) are made of the effect of new- 
synthetic adrenal steroids on the urinary excretion of nitro- 
gen, sodium and potassium and on the blood levels of the latter 
two elements. VJhen short-term studies suggest acceptability 
of the compound with resnect to the metabolism of these ele- 
ments, more lengthy studies "are carried out in selected patients 
for the long-term effects of the steroids on the complete meta- 
bolic balance of these elements ajid of calcium and phosphorus* 



2 - 



SiSlAL WWIBER 



Patient Material i 211 patient days 

Ma.i or Findings ; 

1» Studies on the effects of prednisone and prednisolone 
have shoT/in minor changes in sodium and potassium excretion in- 
dicating that these steroids are more acceptable for long-tenn 
use than their earlier analogues, cortisone and hydrocortisone. 
The definite increase in nitrogen excretion can be prevented 
from leading to negative nitrogen balance if protein intalce is 
kept at high levelse Despite the clinical experience of a 
hlgji incidence of vertebral compression fractures in patients 
td-th rheurrtatoid arthritis on prednisone and prednisolone, 
certainly suggesting serious mineral losses from the skeleton, 
metabolic balance studies for the effects of these steroids on 
calcium and phosphorus balance in three patients have been 
singularly inconclusive. 

2. Delta-1, 9-alpha fluoro-hydro cortisone was shown to 
have little influence on nitrogen excretion but marked though 
varying effects on sodium (retention) and potassium (loss). 
Three balance studies consistently shov-red an increase in urinary 
calcium and decrease in fecal calcium without appreciable change 
in the overall balance, suggesting an increase in intestinal 
absorption of this element. 

3. Delta-1, 21-desoxy, 9-alpha fluoro--liydrocortisone in 
two patients showed sharp but poorly maintained sodium re- 
tention, slight irregular increases in potassium excretion 
and a tendency toward nitrogen retention, the latter effect 
probably not significant. 

k» In one patient 21-desoxy, 9-alpha 21-dlfluoro~hydro- 
cortisone (PG-lj.) in doses up to 50 mgra. daily yielded data 
which suggested the possibility of a slight salt-losing effect 
but no other changesj this steroid had no significant anti- 
inflammatory action when given by mouth in doses of 100 rag./day. 

5. Delta-1, 21-desoxy, 21~fluoro-hydrocortisone in one 
patient has brought about no significant changes in sodium, 
potassium and nitrogen excretion. 



-3 - 



NI/.MD-ll6c ■ 'J 
SERIiU. MikBER 



Sifr nificance to NIAI'ID Research ; This study is cooperative 
with "Trial of Mew .tati-Rheumatic Drugs", MIAI'D, and is important 
primarily in indicating whether effective anti-rheumatic steroids 
may be safely given to patients over considerable periods of 
time with respect to metabolic effects. Of additional importance 
is the fact that determination of the metabolic action of steroids 
under study may yield information T^iiich will give useful leads to 
chemists engaged in the sjTithesis of various cortisone-like 
steroids g 

Proposed Co urse of Pt;pj]e£t: This project vail be continued 
during the coming year "Song present lines, with particular stress 
on electrolyte effects of new steroids and attention to raineral 
effects in the more promising compounds shoiim to lack significant 
undesirable action on electrolytes* Additional stu(^ is neces- 
sary to clarify the action of nrednisone and prednisolone on 
calcium metabolism. 



Form No. ORP-1 
October 1956 
(Attachment l) 



PUBLIC HE.A.LTH SERVICE - WATIOmL INSTITUTES OF lEALTH 
INDIVIDUAL PRG.JECT RtPORT 



Part B; Budget Data 



11, NHM3-116 (C) 



SEtilAL WUl^BER 



12; BUDGET 


DATA 


1 

ESTD'ATED OBLIGATIONS 




MAN YEARS 


DIRECT 




REIH'BURSEIIENT TOTAL 


HIOF 


OTHER TOTAL 


FYI57 










$19,600 




t^l7,,800 $37,UOO 


1.16 


1.32 2.U8 






BUDGETED POSITIONS 


211 


PATIENT DAYS 


PROF 


OTHER TOTAL 




Fr»?7 










1,16 




1.32 2.it8 






13. BUDGET . 


ilGTr 


JllYt 







RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STANDARDS 



iTI Ani-ilMISTRATION 

I ) PROFESSIONAL & 

TECHNICAL ASSIS": 
I I AKGE 






lU. IDEN'TIPT ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUi\[DS, FACILITIES, OR PERSOJfl\lEL 
FOR THIS PROJECl' IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
irDICATE SERIAL NO(S)- 



(Use reverse and additional pages, if necessary) 



Part Ge Honors, Awards & Publications 



l$t NIiiMD-ll6c 
SERIAL IPS' 



16« Publications : None 



17. Honors and Awards; None 



Calendar Year 19^6 

PUBLIC lEilLTII SERVICE - - MTIONAL BISTITUT'lS OF HE.'iLTH 

TDIVIDUAL PROJECT REFQRT 

Part A, Project Description Sheet 1. NIaMD-117c 

SERIAL JTOMBER 

2, NIAilD 3» Metabolic Dis eases Branch 

I?feTITU^S OR DIVISIOi'I iABORATORY, BP^MCH, OR DEPAim-iE'WT 

h* ____________ ^« ,,_,. 

aECi'loT^t or'S^rVICS Lb'CATior (if other t:i/iN BETIiSSDA; 

6, PROJECT TITLE ; Effect of Sulfon yl urea perivatives on Inter** 
mediary Metabolism in llan* 



7» Thomas F» liVawley 



8, Stan ton Segals Gr Donald Vfl-iedon and Mary Margaret Camiis 

OTHER I>!VESTIGAT0RS 

9. 
10, PROJECT DESCRIPTION 

Objectives: To corapare the changes in carbohydrate metabo- 
lism accompanying the hypoglycewic-inducing effects of the 
sulfonylurea derivatives xri.th those observed follomng insulin 
as a possible means of deterraining the locus or loci of action 
of the sulfonylurea derivatives. 

Methods Emplo yed ; The shorb-term effects of the sulfo- 
nylui^ea derivative, orinase, have been studied using intra- 
venous administration of sodium orinase. Glucose loads have 
been given both before and after the adrainistration of orinase. 
Blood levels of glucose, phosphate, potassium^ pyruvic acid 
and orinase liave been determined. In the sai'ie subjects the 
studies have been repeated using insulin and, except for blood 
orinase, making the same deterirlnationsi 

Patient Mater ial: 
311 patient days 



- 2 - 

NKIffl-llTc - 
SERIAL MJl-S'ZR 

Major Findings ; Sulfonylurea derivatives (orinase), given 
intravenously, produced a ^-60% lowering of the blood sugar in 
30 minutes, followed by a rise due to secondary factors similar 
to those occurring after insulin. The magnitude of the increase, 
hovrever, was somewhat less* Glucose assimilation coefficients 
were increased as with insulin, vllth oral glucose given follow- 
ing orinase administration, rise in blood sugar was less than 
that following insulin and may suggest sor,Te interference by 
orinase in the intestinal absorption of glucose. 

A somewhat greater and raore prolonged faU. in serum phos- 
phate occurred after orinase than occurred with insulin, ¥ith 
insulin, there was a rise in serum pyruvic acid accompanying 
the utilization and disappearance of glucose. Ho^-rever, trith 
orinase, there tjas a fall in pyruvic acid in spite of the de- 
cline in blood glucose. 

The findings to date indicate that there are differences 
in glucose utilization under the influence of orinase as com- 
pared to insulin. Opposite effects in pyruvic acid metabolism 
suggsst that orinase does not act primarily by increasing 
peripheral utilization of glucose. The effects observed, 
although not exclusively, indicate that orinase enhances 
hepatic disposition of glucoso. It appears that the sulfo- 
nylurea derivatives have more than one site of action rela- 
tive to the production of a lowering of blood sugar. 

Significance to ]^IIAI4D Resear ch; Since differences 
evidaitly exist in the mode of glucose utilization under the 
influence of insulin and orinase, it is possible to conclude 
that these agents are not interchangeable. In attempting to 
manage diabetic patients mth orinase, therefore, it may be 
anticipated that some effects will be observed vjhich are siof- 
flciently different from those related to insulin to make 
management solely with these new compounds an entirely new 
undertaking. Added experience and further studies are needed 
to determine ^Aiether the effect of orinase to lower blood 
sugar may be the only beneficial influence of such compounds 
and whether accompanying effects maj^ be undesirable over a long 
period of administration. The work should contribute additional 
basic information for better understanding of the altered carbo- 
hydrate metabolism in diabetes • 



- 3 - 



Proposed C ourse of Project : 



mm-ii7c_ 
s2;rial mm::R 



1« studies using the same techniques will be continued 
on a larger sample of individuals. Since no studies of this 
type have been done in diabetics or patients with liver 
disease, these are planned, 

2. Because it is laiOTrjn that insulin affects the distri- 
bution of certain pentoses in animals (Goldstein and Levine) 
and in raan (Segal and V,^Tigaarden) it is planned to conduct 
similar studies with the so-called insulin responsive sugars 
using sodium orinase rather than insulin, 

3a To determine more accurately the overall metabolic 
effects of sodium orinase, studies of R,Q» and energy expendi- 
ture are contemplated using the Metabolic Chamber and observ- 
ing the comparative effects of insulin and sulfonylttrea deriva- 
tives in both normals and diabetics* 



Form No, ORP-1 
October 1956 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
Ii\lDIVIDUAL PROJECT REP(aiT 



Part Bt Budget Data 



11. NIAffi)-117 (C) 



SERIAL NUMBER 



12. BUTWET DATA 


• 








ESTIMATED OBLIGATIONS 




mi] YEARS 


DIRECT 


REIMBURSE^EI\IT TOTAL 


PROF 


OTHER TOTAL 


FYS 57 








121,800 


!ii25,800 .1fli7,600 


1.33 


1.00 2,33 




BUDGETED POSITIONS 


311 


PATIENT -DAYS 


PROP 


OTHER Ta:AL 




Fi?57 








1.33 


1.00 2,53 







13. BUICET ACTIVITY; 

RESEARCH [xj 

REVIEW & APPROVAL P] 

BIOLOGIC STANDARDS (~"1 



ADMINISTRATION 

PROFESSIONAL & 
TECHNICAL ASSIST- 
ANCE 



P 

□ 



lU. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PEfiSONJIEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Part C, Honors, Awards & Publications 1^« F(„^:^"^W'; lu 
16, Publications i None 



17, Honors and Awards; None 



Calendar Year 1956 
PUBLIC HEALTH SERVICE - - NATIOML INSTITUTES OF HEALTH 

INDIVIDUAL PROJECT REPORT 

Part A. Project Description Sheet lo Nl/xfiP-llSc 

2, IIIAT^ ___«„ 3* Arthritis and Rheiunatism Branch 

Institute or division ILabopjitori, braiNCH, or departmeiit 

I;. _________ 5. 

sEdi'ioil OR s£;^.vica location (if other" tean bethesda; 

6 . a Study of the 5h;grmatic Defect in Galactosemia 

PROJECT TITLE 

'^» Kurt J, Isselbacher 

P^ICIPAL lOTESTIGATOR 

8. Herraan Kalckar and Elizabeth Anderson ^ 

OTHER INVESTIGATORS 

9, 

10, PROJECT DESCRIFn ON; 

Objectives ; To determine the nattire of the underlj^ing 
defect in the disease galactosemia or galactose diabetes. 

Methods Employed : Methods consisted of in vitro studies 
on biologic materials in blood and liver tissues obtained from 
normal and galactosemic patients. These tissues were then 
subjected to enzymatic assays with specific reference to the 
presence or absence of the enzyme galactose-phosphate-uridyl- 
transf erase (P-Gal-transferase). Radioactive galactose and 
galactose 1-phosphate for in vivo and in vitro studies were 
also employed. In one study a patient xras given tracer amounts 
of C-^'^ galactose to determine the extent of galactose metabolism 
in galactosemia. 

Patient Material ; 

93 patient days 

Major Findings ; During the past year the study has 
attempted to elucidate the defect in the disease galactosemia 
(galactose diabetes) . It has been observed that erytlirocytes 
of galactosemic subjects show an accumulation of galactose-1- 
phosphate, either after galactose ingestion or upon the incu- 
bation of the red cells mth galactose. Normal red cells under 



- 2 



similar conditions do not demonstrate this accumulation. 
This suggested that the defect in this disease was due to a 
block in the conversion of galactose- 1-phosphate to glucose-1- 
phosphate» It has been demonstrated that the cause of this 
block is due to a lack of a specific enzjine called P-Gal 
Transferase. This enzyme is present in normal red cells but 
is completely absent in the red cells, and almost completely 
absent from the liver, of 16 galactosemic subjects tested* 
As a result of these observations, a specific spectrophoto- 
metric enzymatic assay test has been developed by means of 
which the disease galactosemia can accurately be diagnosed 
without resorting to the potentially hazardous galactose 
tolerance test. 

Si gnificance t o NIA.riD Research ; This project has followed 
the special interest of iWAI-t) "in enzyme chemistry and inter- 
mediary carbohydrate metabolism. It represents collaboration 
between a clinician familiar vrith ttie disease galactosemia, yet 
interested in intermediary carbohydrate metabolism, and a 
laboratory group engaged in the study of enzymatic processes. 

Galactosemia or galactose diabetes is an unusual disease 
characterized by an inability to utilize the sugar of milk so 
tliat babies with this disorder develop poorly and, if they sur- 
vive, have severe degrees of mental retardation, impaired 
vision, etc. The discovery of a specific enzymatic defect in 
this disease leading to the development of a specific spectro- 
photometric enzymatic assaj-- test -tdll enable the ready diag- 
nosis of this disorder in young infants and, by providing 
nourishment to these babies from sources other than milk, mil 
enable physicians to save for hsalthjr life many children now 
doomed to serious defects and premature death. 

Proposed Course of Project ; Follovj-up studies are being 
conducted on the efficacy of milk restriction in several galacto- 
semic patients studied to date. 



Form No, ORP-1 
October 19^6 
(Attachment l) 



PUBIJC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
niDIVIDUAL PROJECT REPCRT 



P; B: Budget Data 



11. NIAI€)-118 (G) 



SERIAL NMBER 



12, BUmET 


DATA! 












ESTB'ATED OBLIGATIONS 






MN YEARS 


DIRECT 


REIMBURSEimWT 




TOTAL 


PROF 


OTHER TOTAL 


FY '57^ 












118,500 


$7,800 




$26,300 


1»00 


1,00 2,00 




BUDGETED POSII 


IONS 




93 


PATIENT DAYS 


PROP 


OTIER 




TOTAL 




FY '5 7 












1»00 


1,00 




2,00 






13, BUDGET 


ACTIVITY: 











RESEARCH 

REVIEVJ & APPROVAL 

BIOLOGIC STAflDARDS 



P 



ADMINISTR/lTION 

PROFESSIONAL & 
TECHl\IIGAL ASSIST- 
ANCE 



P 

a 



lU, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC ffiALTH SERVICE, OR 
OTHER ORGANIZATIONS, HIOVIDING FUNDS, FACILITIES, OR PERSONI^L 
FOR THIS PROJECT IN FY 19^7, IF COOPERtlTING UNIT IS WITHIN MIH 
INDICATE SERIAL N0(s): 



(Use reverse and additional pages, if necessary) 



Part C. Honors, Awards & Publications 15. .N T^^llgg^^ 

16, PUBLICATIONS ; 

1, Kalckar, H, M., Anderson, E. P., and Isselbacher, K. J«: 
Galactosemia, A Congenital Defect in Nucleotide Transferase: 
Preliminary Reports Proco Nat. scad. 3ci., k2_:h9, 1956. 

2, Isselbacher, K. J., Anderson, E. P., Kurahashi, K., and 
Kalckar, H. M. r Congenital Galactosemia, A Single Enzymatic 
Block in Galactose Metabolism. Science 123 : 6 35 > 1956. 

3, Kalckar, H. M., Anderson, E. P, and Isselbacher, K, J.s 
Galactosemia, A Congenital Defect in a Nucleotide Transferase. 
Biochem, Biophys. Acta, 20:262, 1956. 

17 e Honors and Awards : 

None 



Calendar Year 1956 
PUBLIC HEALTH SEIJ7ICE - - NA.TTOIIAL INSTITUTES OF HEilLTH 

IIOIVIDUAL PROJECT REPORT 

Part A, Project Description Sheet 1» NIiiMD-119c 

SSRBL MJKBER 

2. wmi) 3. 

■E^STiTtrta 0R'"bT7t's"ibM" 

k, 5. ^ 

SECTION OR SERVICE LOCATION ^IF OTHER TilAN BSTHSSbA) 

6. PROJECT TITLE ; Stud y of the Normal and Abnormal Physiology of 
the ' For med "EXements of th e Bl oodt 

7» Fre derick Stohli^ an, Jr, fLab. Path. & Histochem,,5 er.Y?n Hftmatnln fnr^ 
WTi-ICIPAJL IIWESTIGATOE 



8. 



nTHEiniNiVSSTTGATOlS" 



9. IF THIS PROJECT R£SS^ffiLE3, COIiHLa'ENTS, OR PARALLELS RESEARCH 
DONE ELSEWHERE IN THE PUBLIC H:ALTH SE .:^/ICE (VJITHOUT INTER- 
CHANGE OF PERSON :EL, FACILITIES OR FUl^S) IDENTIFY SUCH 
RESEARCH, 

Dr, S» J. Sarnoff, Laboratory of Cardiovascular Hemo- 
djmainics, NHIj is collaborating in the study of anemia pro- 
duced by the mechanical damage to red cells in experimental 
animals and men carrying plastic heart prosthesis. Basic 
work on this project is also the subject of study by F, 
Stohlraan, Jr», and G, Brecher, Laboratory of Pathology and 
Histochemistry. 

10, PROJECT DESCRIPTION; 

Objectives : Study of factors contributing to the pro- 
duction and destruction of formed elements of the blood in 
normal and disease states. 

Methods Employe d: Aside from routine determination of 
formed elements in the peripheral blood, these consist of 
measurement of red cell survival and differential agglutina- 
- tion with Cr^l, red cell production i-iith Fe^^ .yp-tate, and 
platelet function with a variety of standard coagulation 
tests. 



- 2 - 

MI^.MD -119c ■"- 

In addition^ the ability of the marrow of patients vdth 
refractory type anemas to respond normallj'' to standard 
stimuD-i, phlebotomy, hypertransfusion and steroids is being 
studied. 

Patient Material ; 
35 patient days 

Major Findings ; 

1. A hemolytic process follomng insertion of lucite 
prosthesis for the surgical correction of acute aortic valve 
insufficiency has been established and defined* 

2, In some patients with refractory anemia it has been 
established that the anemia is due to an alteration in the 
regulatory mechanism of er;;'thropoiesis, rather than an in- 
ability of the marrow to produce red cells, as iias been previ- 
ously thought to be the case. 

Sign i ficance to HIAIID Research : Anemia is a common com- 
plication of arthritis and certain metabolic diseases and may 
be refractory to treatment. A better understanding of tlie 
regulation of erythropoiesis should eventually result in im- 
proved therapy. Additionally, the demonstration of hemolysis 
folloi'Ting insertion of valvular prostheses is of importance 
physiologic-illy as well as dictating the postoperative therapy 
in these cases. Modif:'/ing the hemolysis is of the utmost 
importance in the further development of valvular prostheses 
for the definitive treatment of valvular heart disease, a 
sequelae of some rheumatic diseases. 

P roposed Course of Project ; Patients with refractory 
anemia will continue to be studied in an effort to delineate 
the physiologic derangements responsible for the anemia. When 
sufficiently pvtre "erythropoietine" is developed in the course 
of our basic research, its effectiveness will be studied in these 
patients. Attempts at modifying hemolysis in patients folloxii^ 
ing insertion of valvular prosthesis will be continued. 



Form Mo, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
IITOIVIDUAL ffiOJECT REPORT 



Part B: Budget Data 



11, NIAMD 119 (C) 



SERIAL NWIBER 



12. BUDGET 


DATA 


ESTIMTED OELIG/ITIOMS 




MAW ffiARS 


DIRECT 




REIMHJR3EMEMT TOTAL 


PROF 


OTHER TOTAL 


FY'57 










$U,700 




$3,U00 $8,100 


.33 


.33 






BUDGETED POSH IONS 


3^ 


PATIEIIT DAYS 


PROF 




OTHER TOTAL 




FY»57"' 










.33 




,33 







13. BUDGET ACTIVITYt 
RESEARCH 
REVIEW &t. ^P PROVAL 
BIOLOGIC STANDARDS 



[Hi 



ADMIMISTRATIOM 

PROFESSIOML & 
TECmilCAL ASSIST- 
ANCE 



a 
a 



lU, IDEl^IPY ANY COOPERATING UNITS OF TFE PUBLIC HEALTH SER'^/'ICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSOMlffiL 
FCE THIS PROJECT IN FY 19$?, IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL N0(S)8 

Dr« S, J. Sarno-ff, Laboratory of Cardiovascular Heraodynamics, NIH, 
is collaborating in the study of anemia produced by the mechanical 
damage to red cells in experimental animals and men carrying plastic 
heart prosthesis, Basic work on this project is also the subject of 
study by F, Stohljnan, Jr., and G. Brecher, Laboratory of Pathology and 
Histochemistry, 



(Use reverse and additional pages, if necessary) 



Part C, Honors, Awards & Publications 1$, NL.ID-liPc 

SERIAL ITOMBER 

16, PUBLICATIONS ; 

!• Stohlman, F., Jr., Sarnoff, S. J., Case, R, B,, 
and Ness, A. T, : Hemolytic Syndrome Follo'.d.ng the Inser- 
tion of a Lucite Ball-Valve Prosthesis into the Cardio- 
vascular System. Circulation 13:586, 1956. 

2o Stohlman, F., Jr., and Sarnoff, S. J»: Comparison 
of Hemolysis Following the Insertion of Lucite and Silastic 
Ball-Valve into the Circulatory System. Proc, Int, Soc, of 
Hem« In press. 

17 • Honors and Awards ; 
None 



P-JILIG HEALTH SERVICE - - MATIOMAI. lilSTITUTES OF HE.xLTH 
IITOIVIDUiJ. PROjr:CT REPORT 1956 



Part .g. Project Description Sheet 1. N]JiMD-120c 

SERIAL iiaMBER 

2. NI.iI€> 3. Clinical Di vision 

INSTITUTE , LiiBOR/iTORY 

^« Clin ical Endocriiiolopy Br . 5. __^ 

3 :CTiOM LOCATION 

6 . Studies in Cr etinism. 
PEOJ::jT TITLE 

7 . D r. J. S. RgJ.1 

PRnCIRiL" INVES 'mATOR 

8 . Dr. Char les L ewallon, Dr. Daniel FedcrLnn 
OTI-IER I;.fVESTIG^.TORS 

9. No parallel research in Public Health Service 
10. P ROJECT D ESCRIPTION 

Method 

Clinical material consisted of 6 athyreotic cretins 
andt[«jo goitcrous cretins^ The techniques consisted of 
kinetic studies with I -'"^ labeled iodide and I'^-^-'- labeled 
thyroxine. Further tcchnifiuos involved qualitative and 
quantitative analysis of iodide compounds by cltromatography. 

Results 

a) Athyreotic cretins: Clinical data included 
electro-encephalographic recordings of changes frora thyroid 
deficiency ajid the responses of these to v;^ious thyroidal 
hormones . The rapidity of response of this index ajid of 
changes in serum cholesterol and serura alkaline phosphate 
was compared. The dose of thyroxine, triiodothyronine , or 
triiodothyroacetic acid necessary for maximal response was 
studied. Careful quantitative studies of bone maturation 
were done. In two cretins, hypertension was discovered 
which was aggravated by thyroid hormones. 

b) Goiterous cretins: One cretin who had a thyroid 
which concentrated iodide, but which was unable to organify 
iodide, was studied. This is the second case of this 



NIAMD-.120C - '- 



abnormality reported in the world literature. The 
kinetics of iodide metabolism in this patient was 
studied with particular respect to thyroid plasma io- 
dide clearance, renal clearance and iodide spaces. 
These cases present the only situation in which the 
thjToid iodide concentrating mechanism can be studied 
isolated from the mechanisms of organification and hydro- 
lysis. Of considerable interest, therefore, was the 
finding that pituitary thyroid stimulating hormone did 
not affect the concentration of iodide in this patient. 
This seemed to sug;/;est that TSH has no effect on iodide 
concentration. The effects of triiodothyronine and 
propyl thiouracil on this thyroid were also studied. 
Another goitrous cretin was studied whose thyroid had 
no affinity for iodide. In spite of absence of function 
in this thyroid, it re';ressed after treatment with triiod- 
othj''roninc , suggesting responsivity to TSH, 

Future Studie s 

It is proposed to s tudy other subjects with en- 
larged thyroids and hypothyroidism in an attempt to 
define the biochemical altera tions responsible for the 
h^/^Dothyroidism, It is also planned to study various 
selected patients to determine the relative rates of degra- 
dation of thyroxine to see if this parameter may influence 
goiter development and/or h^.^pothyroidism. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPCHT 



Part B: Budget Data 



11, NIAMD-120 (C) 



SERIAL IWMBER 



12, BUDGET DATA: 








ESTIITATED OBLIGATIONS 




MAN YEARS 


DIRECT 


REDIBURSEI'iENT TOTAL 


PROF 


OTHER TOTAL 


FY* 5 7 








$ill,600 


|15U,U00 $196,000 


2,17 


2,00 It,17 




BUDGETED POSITIONS 


18^0 


PATIENT DAYS 


raoF 


OSIER TOTAL 




FI«57 








2,17 

T ■DTmnirm r, prp- 


2,00 U,17 







RESEARCH 

REVIEl'J & APmOVAL 

BIOLOGIC STAi\!DARDS 



[^ ADMINISTRATION 

I I PROFESSIONAL & 

TECHNICAL ASSIS:: 
1 I ANCE 



n 



Ik* IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTIER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONAL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN WIH 
INDICATE SERIAL NO(S) : 



(Use reverse and additional pages, if necessaiy) 



\ .- 



NM4D-120C 
SERIAL mmER 

Part C, Honors, A wards &c Publica t iens 

16, Pulplications 

Rail, J, E, The RoIg ®f Radioactive Iodine in the 
Diagnosis of Thyroid Disease 

17, Honors and Awards 
None 



?(.'.:l.TG i'li.Iff:; 3. i'lCE - - NiiTIONAI, INSTITUTES OF HEAI.TH 
INDIVIDUAL PROJECT JSPORT 19^6 



Part A. Proje ct Descriptio n Sheet 1, NIAi4D»-121c 

SERIAL mm'R 

2, NLU-D 3. Clinical Division 



IIJSTITUTE LABORATORY 

U, C linical End oc rino logy'- Br. 5. 

SECTION LOGA TION 

6 , Studies on th e Thyroxi nc-Bindi ng Protein of Serum 
PROJECT TITLE 

7 « Jacob Robbins, H ,D. 

PRIriCIPAL i'NffiSTIG-.TOR 

8. J. E> Rail. M,D. 

OTirJR Il'J^.'ESTIGATORS 

9, No parallel research in Public Health Services 

10« .PROJECT .DESCRIPTION 

This is a continuation of the project undertaken 
to elucidate the role in thyroid physiology of the 
interactions between thyroid hormone and scrum protein. 
The method employed include chromatographs, a method of 
zone electrophoresis specifically developed for the pur- 
pose, and radioactive labeling techniques. The patients 
studied have included subjects with hyperthyroidismj hy- 
pothyroudism, cretinisms, nephrosis, and pregnancy. The 
latest named patients were hospitalized at the D, C, Gen- 
eral Hospital, A variety of aiimal specimens have also been 
exajiiined. The major findinfjs are: (l) the thyroxLnc- 
binding capacity/' of the th;5rroxinc -binding alpha globulin 
(TBP) is elevated in pregnancy and in hypothyroidism, de- 
pressed in nophrosis and unchanged in hyperthyroidism, 
(2) the clinical '.thyroid status of the patients correlates 
more closely with the unbound thjyroxine concentration in 
scrum than with the total thyroxine (or PBI), (3) the 
normal unbound thyroxine concentration, which has been 
determined by indirect calculation and involves certain 
assumptions, appears to be extremely small (6 x 10 M), 
(U) various animal species differ greatly in the patterns 
of thyroxine -binding in serum. 



SERIAL mmrs. 



The findings, in .addition to providing now inform- 
ation on a littlc-studicd aspect of thryroid physiology, 
have some practical significr.noG in clinical medicine. 
They explain the paradoxical levels of serum iodine which 
occur in certain disorders, and indicate that thyroid 
hormone availcibility to the tissues may be modified by 
extra thjrroidal mechanisms. 

Proposed studies include: (l) investigation of 
some of the assumptions used in the calculation in order 
to put thorn on a firmer bases, (2) development of a 
simpler method for measuring thyroxino-blndlng capacity, 
(3) measurement of th3T?oxLne-blnding in other diseased 
states. 



Form Mo. ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - MA.TIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPCRT 



part B; Budget Data 



11, NmMD-121 (C) 



SERIAL mmBER 



12, BUDGET DATA 


■ 










ESTimTED OBLIGATIONS 




MAN YEARS 


DtaECT 


RED'IBURSEI'IEOT 


TOTAL 


PROF 


OTHER TOTAL 


FI»57 










.f2li,li00 


l3Li,600 


$59,000 


1.33 


1.00 2,33 




BUDGETED POSITIONS 




U19 


PATIENT DAYS 


PROF 


OTHER 


TOTAL 




Fr'57 










2,33 


1,00 


3.33 






13. BUDGET ACTIVITY: 









RESEARCH 

REVIEVJ & APPROVAL 

BIOLOGIC STAIW.RDS 






ADMINISTRATION 

PROFESS I OML & 
TECHNICAL ASSIST 
ANCE 



CD 
□ 



111, IDEOTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDED FULIDS, FACILITIES, OR PERSOinINEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



NLvMD-121c 
Part G. Public a tions. Awards & Honors SERL.L NO. 



16 , Publicati ons 

Reverse Flow Zone Electrophoresis, A Method for Deter- 
mining Thj^roxinG-Bindinf' Capacity of Serum Protein^, 
by Dr. J, Robbins, Arch, Biochom, and Biophys, 63, 
U6l, 1956. "" 

The Inter-action of Thyroid Hormones and Protein in 
Biologicr.l Fluids, by Dr. J,, Robbins a nd J, E, Rail, 
Recent Progress in Hormone Rc6earch, iia press, 

17. Honors 

None 



PUBLIC HEALTH SERVICE - - NATIONAL INSTHTO'ES OF ffi5ALTH 
INDIVIDUilL PROJECT REPORT 19^6 



Part A. Description Sheet 1 . NIiJ4D-122c 

SERi;.L MJI'BER 

2. NI/iMD 3. Clinic .il Division 

INSTITUTE ■" L'lBORATORY 

h. Clinical Endo cr inol og y Br. 5. 

S^;GTI0N ~ ~ LOC/--TIOM 

6 , Tho Effect of t he Th^rroid Hon nonc:s on Dehydrogenases In Vitro 
PROJECT TITLE 

7. Jan Wolff, M.D., Ph.D. 
PRINCIPAL IN '7ESTIGAT0R 

8, Hone 

OTHER Ij\p/ESTlLrATORS 

9. No parallel research in Public Health Service, 

10. PROJECT DESGRIPI'IOK 

It is hoped by tho in vitro approach to gain insight 
into the fundamental iiEchansims of the action of the thyroid 
hormones. This is carried out by comparing initial rates of 
reaction of various purified or crystalline dohydrop,cnases 
upon their respective substrates in the presence and absence 
of added thyroxine, triiodothjrronine and a number of nnal- 
oguGS and related compounds. 

At a concentration of 10"5 M, thyroxine is able to 
inliibit oxidation or reduction reactions by the following 
dehj^drogenases: j^east alcohol dehydro?:cnase , raamiiialian 
malic, glutamic, trioscphosphate, glucose-6-phosphate and 
lactic dehydrogenases. Triiodothyronine, its a.cetic acid 
analogue, and a number of thyromiinetic , congeners have sim- 
ilar effects, although not all at the same concentrations, 
A studjr of the structural requirements forthis effect has 
been partially completed cjid shows a rou';;h correlation to 
the degree of halo<3cn substitution on halophenols and xan- 
thene dyes. In the thyronine series, there is a rough cor- 
relation with biological (in vivo) compounds tested. 

Attempts to study the nature of the inhibition are 
imcomplete. It is reversible by the criteria of Ackerman 
and Potter but falls between perfect competitive and per- 



NIiAT)..122c - — 

SERIAL mmm. 



feet non-competitive inhibition vjhcn ?JicilyzGd by Line- 
■Kreavcr and Burk plots. The Kj values are in the 
neighborhood of 10~5. Attempts to determine the affinity 
constants trj^ equilibrium ancdysis, ultrafiltration or 
ultracentrifugation have been inconclusive so far. 

Studies have also been carried out to dctcmine 
iiThether other tjrpes of enzymes are equally affected. The 
oxidation of hypoxanthinc by xmthine oxidase is not in- 
fluenced by concentration of thyroxine effective for 
dehydrogenases. Results with horseradish peroxidase ai-e 
equivocal - due to difficulty in finding an assay suit- 
able for this purpose, Investi'^ation of a number of other 
readily available cnzATJies in planned. 

It is hoped to conclude the first phase of the stucfy 
with the above material. Late , we hope to identify the 
site of attachment of tl^^rroxine, in view of the fact that 
the dehydrogenases studied probably all contain zincj and to 
compare the affinity constajits with those determined by 
physical methods. 



Form Mo, ORP-1 
October 1956 
(Attachment l) 



PUBLIC HEALTH SERVICE - NATIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B; Budget Data 



11, NIA^D-122 (C) 



SERIAL WUMSIR 



12 , HJDGET 


DA^ 


As. 

ESTimTED OBLIGATIONS 




mW YEARS 


DIRECT 




REfflBURSEMNT TOTAL 


PROF 


OTHER TOTAL 


Fy»57 










12^,000 




111,200 136,200 


1,00 


2.00 3,00 






BUDGETED POSITIONS 





PATIEOT D/^YS 


PROF 




OTHER TOTAL 




F/«57" 










1,00 
13 « BUDGET 


AC^ 


2.00 3.00 

IVITY: 







RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STANDARDS 



[Tj ADMINISTRATION 

Pj PROFESSIONAL & 

' . lEGHMIGAL ASGIST- 

i I AMGE 






lU, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PEUSONIIEL 
FOR THIS PROJECT IN FY 195? . IF COOPERATING UNIT IS VJITfflN NIH 
INDICATE SERIAL NO(S)! 



(Use reverse and additional pages, if necessary) 



.MD-1 2?.e 



Part G. Publications, Awards & Honors 
16, Publicrtions 

Wolff, J, Inliibition of Malic Dchydrogonase by Thyro-dnc 
Fed. Proc., March 19^6 

17 9 Honors 
N<me 



PUBLIC HEALTH SER^^ICE - - "'ATITJ/J; I'-'STTTUTES OF HS..LrH 
imi^rCDiLIL PFOJECT REPORT 1956 



P"Ei ■''^. Project Description Sheet 1. NI iiMD- 123c 

SERI-Jj WU'BER 

2, NX -if) 3. Clinical D ivision 



I-STITUTE L 30R..T0RY 

^» Clinical Endocrinolo;:/ Br, 5. 



S^:CTION LOaTION 

6 , S tudies of Pentose M cta^ 'olisri in Man 
PROJECT TITLE 

7 , Stanton Sog:oJ.. J.-irics B. Vcmr-a rdcn 
FPJ. ^CIRJ., Irnr.;STIG..7rOF;S 

8. Joseph Foley, Alb c;rba Blair 

OTH R I~-nESTIG..TOHS 

9. No rese rch in Public Health Service, 
10, PROJl'XT DESCRIPTION 

The object of tliis study has been to obtain inform- 
ation on the distribution, cxcr.-tion and metabolism of 
pentoses in man and the effects of insulin ther on. Pre- 
viously, it has been demonstrated that xrhen the pentoses 
D-^.ylose, D-.?rabinose, and L-arabinose were infused into 
m?ii, the rate of disappearance from blood was proportional 
to the concentration, IJlicn insulin was given, a marked 
decrease of the blood levels of the pentoses D-xylose rnd 
L-arabinosc was seen. During thu p -st ^--oar, studies of 
the biolo'ic beha-'ior of the sug^-rs D-i^ocose and. D-ribosc 
were perform.ed. D-l^ocose behaves like D-arabinose and is 
unresponsiv.. to insulin administration. These findings agree 
with animal studies and suggest that insulin increases the 
volume of distribution of D-:!^/lose and L-?rabinose. 

D-ribose differs from all oth r pentoses in its 
behavior xjhen infused into the boc^, A plot of decreas- 
ing blood levels revesls a two phase curve with only the 
second phase reveriing ch^mces with tiirK2 proportional to 
concentration. I'Jhile D-x-rlose and L-arabinose cause 
incre.'Ses in blood sugar (no change with D-xylose or D- 
ai'abinose occurs), D-ribose causes a marked decrease of 



imiffi-123c__j~- 
SERIAL FJl-iBFJl 



30% to 60% in blood gli.icosc which appears to be similar 
to insulin administration, /ilso, D-ribosc excretion in 
the urine is much loss than the other pentoses. Ribose 
responds to insulin administration not be a sudden fall 
in blood levels, majdmal in 30 minutes as with xylose^ 
but by an increase in gcneri'J. rate of disappearance over 
many minutes, 

G-'-^ labeled D-jQ'-iose and D-ribose have been admin- 
istered and results indicate significant metabolism of 
both these sugars. Thirteen per cent of Xjrlose G ^ 
activity was found in thr- expired air in six hours and 
16°^ of D-ribosc label w as found here. Over S0% of the 
Xylose appeared in the urine unchanged, D-ribose studies 
reveal that the urin.3ry C^ activity is much greater than 
can be accounted for by ribose present, indicating metabo- 
lites axe present. This is confirmed by the fact that 
while ribose levels of blood fall rapidly, the fall of 
Cl^ appeared in a m.a:draal concentration in cjcpircd GOn 
in hS minutes, which is similar to results found with 
glucose. However, D-ribose gives maximal labeling of 
CO2 in 2 hours, vrhich indicates much slower metabolic 
process for ribose. 

Studies are to be continued to determine the kinetics 
of ribose utilization as well as the pathx^ays of its meta- 
bolism. An attempt will be made to ascertain the cause 
of the marked blood glucose falls associated with ribose 
adiainistrrtion, as well as the nature of the labeled 
material found in the excreted urine. Studies will be 
done to >. lucidate the metabolism of D and L-arabinose 
and D-lyxose, using labeled material. 

These studies have demonstrated that in man, insulin 
has effects on handling of other sugars than D-glucose, 
Since pentoses are believed to be intermediates in the 
oxidativo pathway of metabolism, these studies will con- 
tribute to elucidating such a mcchauiism in man. 



Form Mo, ORP-1 
October 19^6 
(Attachment I) 



PUHJC HEALTH SERVICE - MTIONAL INSTITUTES OF IWM. TH 
INDIVIDUAL PROJECT REPORT 



Part B{ Budget Data 



11. NIAMD~]23 (C) 
SERIAL NUM'ffiR 



12. BUDGET 


DATA 


EST IMATED OBLIQflTI ONS 




MAN YEARS 


DIRECT 




REBIBUI^SEMENT TOTAL 


PROF 


OTHER TOTAL 


FY«57 










$2ii,900 




^^29,100 ^^5U,000 


147 


1.33 2.50 






BUDGETED POSITIONS 


356 


PATIENT DAYS 


PROF 




OTHER TOTAL 




FI«^7 










1.17 

1 o ■DTTnnTT'm 


nmn 


1.33 2,50 

TTTIV- 







RESEARCH 

REVIH-I & APPROVAL 

BIOLOGIC STAICARDS 



X 



D 



ADMINISTRATION 

PROFESSIOmi & 
TECffiCTCAL ASSISI 
AWCE 



□ 



ll;, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC lEALTH SERVICE, OR 
OTHER ORGANIZATIO.NS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 1957. IF COOPERilTING UNIT IS WITHIN NIH 
II\!DICZTE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



N I/JID-123C 

SERIAL iIUtlBER 



Part C, Honors, Awards & Ribllcations 



16, Publicatio ns 

Segal^ Stantonj >^ngairdcn, James 3,; Foley, Joseph, 
The Effect of Insulin on Blood Levels of Infused Pen- 
toses in Man, Fed. Proc, 1$, 1956. 
Abstract paper submitted for publication, 

Segal, Stejiton; Blair, lilberta, and Wyngaarden, James B, 
An Enzymatic Spcctophotomctric Method for the Determina- 
tion of P'mric Acid in Blood. hB, 137, 1956, 

Iffynga-^xden, "James B.j Segal, Stanton, and Folejr, Joseph. 
Physiologic Disposition and Metabolic Fate of Infused 
Pentoses in Man, Submitted. 



17, Honors 
None 



PUBLIC HEALTH SEKVICE MJI0m._ lUSTITlJ^^ 



iroiVIDTIAL PROJECT REPORT 



Part A. Project Description Sheet 1. MTJiMD-12!ic , 

Serial Humber 

2 MIA.1* 3. Clinical In vestigations _ 

* Institute or Division Laboratory, branch or Dept, 



ll. C linical Endocrinology . _ _ 5. ^ 

Section or Service ' Location 

6 . Urinary Purines in G-out 

Project Title 

7. James B. \']7,Tigaarden^ M. D. 

Principal Investigator 

8 . Alberta Blair, Janet Schuck^ John T. Dunn 

• ~ Other Investigators 

9. IIo parallel research in Public Health Service 

10. Project Description: 

O bjectives - To define the major pathways of uric acid 
synthesis in normal subjects, in patients with g out and 
in patients with :ipukemia (or related disorders). To ex- 
plore possible accessory pattoiays of urate formation, not 
involving xanthine as the sole and immediate precursor oi 
\iric acid , 

Methods employed - Patients x/ere given C^^-labeled 
glycine, adenine or hjToxanthine , and the labeling pattern 
of various urinary purine bases were studied and compared 
with that of uric acid. Urinary purines were precipitated 
from urine as copper salts, redissolved in PICl, and sep- , 
arated individually by sequential elution from Dowex-50-H^ 
resin columns. Adenine, guanine, hypoxanthme , xantlime, 
and 7-methyl guanine irere obtained in crystalline form, 
as was uric acid. Their isotope composition xms then de- 
termined and plotted against the day of the sample. Ob- 
servations were made for periods up to 3 weeks from the 
point of administration of the tracer compound. 

In evaluating accessory pathways, reactions involving 
8-hydroxylation of purines were studied. Methods involved 
standard enzymatic incubations and spectrophotometnc 



Page 2 of Project Description Sheet NKMD-12 l;c ' ^ 

Serial Jlumber 

measToreraents, coliimn and paper chromatographic separation 
techniques, and tracer approaches. 

Patient Ifeterial - During calendar year 19$6, two 
control, tvro gouty and one leulcemic subject were st^idied. 
In addition, one leulcemic patient given C-'^'-labeled 
amino-imidazolecarboxam3.de by Drs, Seegmiller and Laster 
(I'TIAIO) was studied in conjunction with them. 

Major Finding s - Evidence obtained from the pattern 
of labeling of urinary piorines suggests the existence in 
all subjects of txio classes of pathways generating viric 
acid. The one probably involves immediate cleavage of 
a portion of ner^rly synthesized nucleotides (reflected in 
maximal first day labeling of certain urinaiy purines); 
the other probably involves cleavage of nucleotides 
generated by catabolism of nucleic acids (reflected in 
later labeling of purine bases, particularly notable in 
the leukemic subjects). The finding that peak urate en- 
richment norm^ally is not reached for sor.ie li8 hours after 
peak urinary hypoxanthine enrichment when glycine-1-C-^ 
is fed, suggests that an accessory pathway for uj?ate syn- 
thesis involving intermediates other than hypoxanthine 
and xanthine may exist. The prompt conversion, of infused 
hypoxanthine-8-C^' to uric acid (peak U. A, G""-'' at 10 
hours) provides additional support for the concept that 
not all uric acid normally formed involves this intermed- 
iate. 

In the leulcemic subjects evidence was found for 
significant enhancement of nucleic acid turnover, mani- 
fested in an abnormal degree of enrichment of precursor 
purines (particularly adenine) during the 5 to 1^ day 
portion of the studies. 

A rather surprising finding was that of extremely 
marked labeling of adenine and guanine in urine on the 
first day of the glycine study in the lei:il<:er(iic individual. 
This pattern xfas reflected in unusually high labeling of 
hypoxanthine and xanthine on day 1, but the values here 
were less strilcingly unusual. However, the entire pattern 
suggests that synthesis of uric acid by direct route (i.e., 
not via nucleic acids) was also augmented in this subject. 
These results further raise the question whether compounds 
containing adenine and/or guanine may not represent the 
transport forms of precursor piu^ines, since results from 
Abrams* laboratory suggests that bone marrow utilizes 
preformed purines in formation of erythropoietic nucleic 
acids considerably more efficiently than it does glycine. 



Page 3 of Project Description Sheet NI/i.MD-12hc " -^ 

Serial ifuraber 

Results in the gouty individuals do not yet perriiit 
a clear statement regarding abnormalities of \arate synthet- 
ic pathirays, Ti-jo findings, however, are of note, (l) In 
one gouty subject, not excreting excessive quantities of 
urate in urine (avg.= l(35j range 38I4-517 mg. jjer day), and 
without renal disease, there T-ras an excessive incorporation 
of G^ from glycine but not of N^? from glycine into uj'in- 
ary uric acid. This is the first indication of a possible 
metabolic defect in this type of gouty subject, and further 
these results raise important questions regarding quali- 
tative differences in the handling of different atoms of 
the glycine molecule in gout, (2) In both types of gouty 
subjects (i.e., those excreting normal and those excreting 
excessive quantities of uric acid), there was prompt label- 
ing of certain precursor purines. In one overexcretor, 
hypoxanthine appeared to be labeled somewhat in excess of 
normal, whereas in one normal excretor, 7-methyl guanine 
Xiras very highly labeled on daj'' 1. These results raise 
several possibilities regarding possible precursors of 
the urate arising by direct routes, and suggest areas in 
which to concentrate future studies. 

In study of reaction models, 2 , 6-diamino-purine was 
oxidized to 2,6-diamino-8-hydroxypurine, and the oxidation 
of adenine to 2,8-dihydroxyadenine by xanthine oxidase was 
shown to involve 8-hydroxyadem.ne as the intermediate, 
Mlk xanthine oxidase was found to contain weak guanase 
and nucleoside phosphorylase activities. 

Significance t o program of IJIAID - These studies have 
contributed to the advances in our knowledge of purine 
metabolism, relating in a general waj'" to gout, a subject 
of considerab3.e interest mthin KIAi'lD, 

Proposed co\jrse - The principle investigator has 
left the tllAKD, but plans to continue similar studies at 
Dulce University, attemptin;'; especially to gain inforraa- 
tion on the pattern of purine labeling in the early hours 
in gouty subjects who incorporate excessive C-'^ from 
gl^'"cine into uric acid, and who synthesize excessive qioan- 
tities of uric acid. Such studies may help define the 
particular nucleotide pathways and free base patlit'iays in- 
volved in the metabolic defect of gout. 



Form No. ORP-1 
October 19^6 
(Attachment l) 



PUBLIC HEALTH SERVICE - MTIONAL INSTTrUTES OF HEALTH 
INDIVIDUAL PROJf;CT REPORT 



part Bs Budget Data 



11, NIAM>12U (q) 



SERIAL NUi'BER 



12. BUDGET DATA 


ft 








ESTIMATED OBLIGATIONS 




MAN YEARS 


DIRECT 


REIMBURSEMENT TOTAL 


PROF 


OTIER TOTAL 


Fl«57 








112,00 a 


gL3,U00 '^25,1100 


.50 


1,00 1,50 




BUDGETED POSITIONS 


172 


PATIENT DAYS 


PROF 


OTIER TOTAL 




FY«57 

1.50 


1.00 2.5o 






13, BUDGET ACTIVITY} 







RESEARCH 

REVIEW & APPROVAL 

BIOLOGIC STArJIlARDS 



iZ! 



ADIffNISTRATION 

PROFESSIONAL & 
TECmiGAL ASSIST- 
ANCE 






lit, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTHER CRGANIZATIONS, PROVIDING FUNDS, FACILITE^.S, OP PERSONNEL 
FOR TT-iS PROJECT IN FY 1957. IF COOPERii.TI NG UNIT IS WITHIN NIH 
INDICATE SERIAL NO(S): 



(Use reverse and additional pages, if necessary) 



Part C. Honors, Awards Si. Publications lg. NIi.I-ffl-12lic 

Serial Number 

16, Publications from this project du.ring 19^ 6, 

Ifyngaarden, J. B., 2,6-Diaminopurine as Substrate and 
Inhibitor of Xanthine Oxidase, J. Biol. Chem. In press. 

IJyngaarden, J. B., and Dunn, J. T,, 8-Ify-droxyadenine 
as the Interriiediate in the Oxidation of Adenine to 2,8- 
Dihydroxyadenine by Xanthine Oxidase, Arch, Biochem. 
Biophys. In press, 

Ifyngaai-Hien, J, B,, Intermediary P\irine Metabolism 
and the Metabolic Defects of Gout, Metabolism, In press, 

Seegmiller, J, E., Stetten, De^/,, Bunim, J. J., 
Sokoloff, L., Laster, L., and I/yngaarden, J, B,, Metabolic 
and Clim.cal Aspects of Gout, Am. J. Medicine, In press, 

Stetten, DeW, , Talbott, J, H., Seegmiller, J, E,, 
¥yngaarden, J, B,, and Laster, L,, Pathogenesis of Gout 
(letter) Metabolism. In press. 



17, Honors and Awards: None 



PUBLIC HE.JLTH SERvriCE - - MATIOj>iAL T'STITU'TES OF KF.^LTH 
INDIVIDUAL PROJECT REPORT 1S>56 



Part A. Projec t Descrip ti on Sheet 1, N Ii.i''ID-12$c 

— - . - SJilRLJ. iJUfBER 

2, Nli-J-B 3. Clinical Investigations 

INSTITUTE L;;B0R..T0RY 

^« Clini cal Endocrino lo^ 3r. 5. 

LOCi.'TION" 

6 . Studies_ in Diabetes 
K.OJSCT TITLE 

7. Dr. James 3. F_i_Gld_ 

PRI ■ GIP: JL I..n^.3Tir':.:T0R 

8 . fir. Dmiiel D. Fede _r:iian 
OIHER IM^TES i/IG.iTORS 

9. No parallel research in Riblic Health St^rvice, 
10. PROJECT JBSGRIFTION 

Obj_c_ctivcs 

1) To study the metabolism of some of the B vitamns 
in diabetes as compared to normal. In a ddition, comparison 
will be made between diabetes with neuropathy, retinopathy, 
and renal lesions and diabetes without these complications. 

2) To s-oudy the effect of an oral sulfonamide deri- 
vative (carbutajiiide) in the treatment of diabetes. 

Methods Smpl oyed 

Patients divided into three .■^ roups (l) norm.als, 2) 
diabetics x\dth complications, 3) diabetes without ccmpli- 
cations) are jlacod on a standa d diet of 35 calories pnr 
Kg. ajTl const;^nt kno^m ajnounts of Thirotiin, riboflavin, 
pantothenic acid, niacin, ajid vitamd-n B-[_2» J^fter a period 
of two Xfeelcs on this diet, they exo ivcn a test dose of 
5 mg. of Tliiaiiiin, 5 ng, of riboflavin, 50 mg. of p^into- 
thenic acid, 50 mg. of Nicotinic acid, and 50 I'rng. of 
vitrjrtin Bq_2. The urinary excretion of these vitajnins is 
measui-cd both before, and during the test dose and these 
are used as the basis of comparison, jidequacy of urine 
collections is checked by urinary creatinine determinations. 



SERIi'iL MUi-IBER 



The diabetic patients a re controlled as well as possible 
■with insulin. The study is then repeated with the diabetics 
off insulin for two to tlTrc> days and the normals are put 
on a 500 caloric diet and restricted fluid to maicc the 
weight loss similar to the diabetics off insulin. 

Diabetic patients whose dia.botcs could not be con- 
trolled hy diet alone were placed on carbutamide after sev- 
eral days control period during which time, 2U hour urinaiy 
''glucose excretion, fasting and post-prandroJ. blood sugars 
were measured as well as renal, thyroid, adrenal, and liver 
functions. These sa me parameters were measured while on 
carbutajnide. 

Clinical M aterial 

1073 patiiant ;days *'- ■ 3 



Maj or Fi ndings 

The diabetics without complications seem to excrete 
more of a test dose of Thiamin than do the diabetics with 
complications end normals. This difference was not seen 
when their pre-test daily excretion patterns are exajnincd. 
There is no diffei-ences between the three groups in regards 
to riboflavin excretion. There is the sijg cstion that 
diabetics xjith no complications excrete more pantothenic 
acid than the other two groups, but tliis finding is not 
definite yet. The diabetics with complications excrete 
less of the test dose of nicotinic acid than the normals 
and the diabetics xd-thout complications. 

In some; middle-aged, mild diabetics who have not had 
di?.betes or insulin therapy for a long time, it was possible 
to reduce the urinarj'- glycosuria, fasting blood sugar levels 
Jgy using carbutamide. There was no evidence of any change in 
thyroid, a drenal, or liver function. However, one patient 
died whjLlc taking the drug and at autopsjr shoxred eosinophilic 
interstitual r^^ocarditis and gr.anulom.atous lesions in the 
liver, spleen and port "i lymph nodes wliich were interpi-eted 
as hjrpei-scnsitivity to the sulfonajnide derivative. This pat- 
ient had cosinophilia, fever and back pain prior to death. 
One acromcg'ilic dia'ictic iijith diabetes responded to the drug 
and one did not. 



SEltlAL i^LII'IBER 



SignficancG 

The lack of a cori-Glatiun botwncn the excretion pattern 
of the B vitanins and the presence of diabetic complications 
corrobora.tes the clinical observation that large doses of 
these vitaanins arc of questionable benefit in the treatment 
of meuropathy. It has been shown that during periods of 
poor control of the diabetes the excretion of B vitariiins is 
greatly increased and thus it is possible tha.t when the 
diabetes is poorly rcgulatedj a relative vitamin deficiency 
can exist. 

The occurrence of sever, fatal reactions to car- 
but.aniide has lead to an evaluation of the toxicity of 
these drugs and the subsequent mthdrawaJ. of some of them 
from clinical" testing and human use. 

Proposed Co urse 

To obtain more information on the excretion of these 
vit?jnins in normal and dia.botics. 



Form No, ORP-1 
October 19^6 
(Attachment I) 



PUBLIC HEALTH SERVICE «- NA.TIONAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-12^ (C) 
SERIAL NUl'IBER 



13, BUDGET ACTIVIT Y; 
RESEARCH 

REVIEW & AP PROVAL 
BIOLOGIC STANDARDS 



12, BUDGET DATA 


I 








ESTIMATED OaiGATIONS 




MAN YEARS 


lilRECT 


REDlBlESEi-'iEIC TOTAL 


PROF 


OTHER TOTAL 


FY«57 








$19,800 


;I589,UOO :i3.09,20O 


1,^0 


1.^0 




BUDG.ETED POSITIONS 


1073 


PATIEJIT DATS 


PROF 


OTHER TOTAL 




FYt^T" 








1.50 


1.^0 

rmv. 




^ 









ADI'IINISTRATION 

PROFESSIONAL & 
TECMICAL ASSIST. 
ANCE 






lU, IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR 
OTI-ER ORGANIZATIONS, PROVIDffiG FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 195)'?. IF COOPERATING UNIT IS WITHIN NIH 
INDICATE SERIAL MO(S): 



/■ti. 



*j « -J jj 4. J *...**n »».-.^M £ j> « »„A»..\ 



SERIAL NUffiER 



Part C. Publications, /Mards & Honors 



16, Publications 

Field, J. B, and Federman, D, Sudden Death in a Diabetic 
During Treatment with Carbutamide (BZ-55) Diabetes in Press 

Field, J, B, and Foderman, D. Effect of Carbutamide in 
the Diabetes associated ^^^ith Acromegaly, Diabetes in Press 

Field, J. B. and Woodson, M. L. Effect of an Oral I^o- 
glycemic Drug (carbutaxiido) in Glycoi^cn Deposition by 
Isolated Rat Hemidiaphragms , Proc, Soc,, E:;qDer. Biol, and 
Mcd« in press. 



PUBLIC HEALTH SFJIVICE - - mTIOW.L INSTITUTES OF HEALTH 
Il^IDIVIDUAL PROJiiCT REPORT 
part A. project Description Sheet 1. ™^||^. 

2. Ma^tAnnal_Tnstitu te of Arthritis and M etabolic Diseases 
INSTITUTE 

3, Administration U. ^* _ ■ 

DEPARTMENT 

6, Office of the Director, National Institute of Arthritis and 
Metabolic Dise ases^ 

PROJECT TITLE" 

7, Dr. Floyd S. Daft, Director; Dr. G. Donald iJhedon, Assistant 
Directors H, G . Ba ylis, Executive Officer 

PRINCIPAL INVl'iSTIGATORCS) 



8. 



9. 



10, PROJECT DESCRIPTION 

T-he activities of the National Institute of Arthritis and 
Metabolic Diseases include two major areas: (l) Intramural Programs 
Oaboratory and Clinical Research; and (2) Extramural Programs (Fellow- 
sSps, Research and Training Grants.) The Office of the Director is 
responsible for planning and directing the overall administration of 
the Institute, in conducting, fostering and coordinating investigation 
of the cause/prevention, diagnosis, and treatment of arthritis, rheum- 
atism, and metabolic diseases; for maintaining effective °Pf f J-^g ^J" 
lationships with other Institutes of the National Institutes of Health, 
with other units of the Public Health Service and the Department of 
Health, Education, and ^lelfare, other governmental ^S^^^^^^J* ^f P^^Jf 
and private agencies carrying on related functions. The Office of the 
Director also participates in determining policies governing the 
National Institutes of Health, 

During 1956, the Director, vjith the cooperation and advice of 
his Staff, completed certain organizational changes in intramural 
research operations, which insulted in a total structure of six 
laboratories and twenty-one sections. Also, the realignment of 
responsibilities in the Office of the Director resulted m the crea- 
tion of the position of Assistant Director. 

The Office of the Director sponsored several cooperative 
Conferences in collaboration with officials of the American Rheuma- 
tism Association and the Arthritis and Rheumatism Foundation, .hese 
included the Third Interim Scientific Session of the American Rheum- 
atism Association and the Second National Conference on Research and 
Education in the Rheumatic Diseases, 



Form No, ORP-1 
October 1956 
(Attachment l) 



PUBIJC ffiALTH SERVICE - MTIOMAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part Bs Badget Data 



11, NIA.MD-126 



SERIAL NUMBER 



12, BUDGET DATA;. 



ESTIMATED OELIGATIONS 



FYS 57" 



DIREGl' 



REIMBURSEMENT 



TOTAL" 



$85,000 



^3.67,000 



$252,000 



BUDGETED POSITIONS 



FI»57" 



PROF 



OTHER 



"tco?al' 



U.oo 



8.00 



13, BUDGET A C TIVITY: 
RESEARCH 

REVIEtl & APPROVAL 
BIOLOGIC STANDARDS 



1 1 

□ 



PROF 



12.00 



ADMINISTRATION 

PROFESSIONAL & 
TECiraiCAL ASSIST- 
ANCE 



TM YEARS 



OTHER TOTAL 



U.OO 8.00 12,00 



PATIENT mYS 






lU. IDENTIFY km COOPER/iTING UNITS OF THE PUBLIC HEi^.LTH SERVICE, OR 
OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR I^RSONbIEL 
FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WTTHIW NIH 
INDICATE S ERIAL NO(S) : 



(Use reverse and additional pages, if necessary) 



Form No. ORP-1 
October 19^6 
(Attachment l) 



FUBTJC HEALTH SERVICE - mTIOKAL INSTITUTES OF HEALTH 
INDIVIDUAL PROJECT REPORT 



Part B: Budget Data 



11, NIAMD-126 



SERIAL NUMBER 



12, BUDGET DATA: 



ESTIMATED OELIGATIOMS 



TOTAL" 



I'M YEARS 



FY! 57' 



DIREC"J 



REIMBURSEMENT 



PROF 



OTHER TOTAL 



FY»57' 



f 8 5, 000 


$167,000 

BUDGETED POSITIONS 


$2^2,000 


U.oo 


8,00 12,00 

PATIENT KlYS 


PRCF 


OTHER 


TOTAL 




il.OO 


8.00 


12,00 






BUDGET ACT 


IVITYf 









RESEARCH 

REVIEl/l & APPROVAL 

BIOLOGIC STANDARDS 



□ ADMINISTRATION 

rn PROFliSSIONAL & 

TECHNICAL ASSIST- 
i 1 AMCE 






lU. IDEICIFY AiW COOPER/iTING UNITS OF TJIE PUBLIC HEA.LTH SERVICE, OR 
OTHER ORGA.NIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL 
FOR THIS PROJECT IN FY 19^7. IF CCOPERATING UNIT IS yiTHIN NIH 
INDICATE S ERIAL NO (S) ! 



(Use reverse and additional pages, if necessary) 



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