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THE CONCENTRATIOH OF RIBOFLAVIR AND ITS PHOSPHORYLATED DHIVATIV'ES IN THE TISSUES OF RATS FLD VARYING WANTITIES 0F VITAMIN B, By Lucile Ellen Decker A THESIS Shh-itted to the School of Graduate Studies of’Hichigtn State College of Agriculture and.Applied Science 1n.pertiel fulfillnent of the requirenentl for the degree of MASTER OF SCIENCE Department of Chemistry 1952 [1-L'£5V ACKNOWLEDGMEHT The euthor viehee to expreu her uincere appreciation to Doctor Richard D. Byerrun for hie guidance during the course of this etudy, end to the other numbers of the Chemistry department for their edvice and c00peretion. “W" MW W W H 'I’ fuwygagf‘” TABLE OF CONTENTS PAGE mmwcrmn..... Aniaala and D10ta...... Analytical flatbed for Riboflavin, llavin Honomeleotide and Fh‘in Din‘mlaotidoee000000000000000000000000000000000. cmuon or mmt.eeeeeaaeeeeeaeaaeaeeOOOOOOI00000000000 WLTSOCOOOOOOOOOOCOOOOOOOO...0......000.00.00.00OOOOOOOOOOOCCOO Gm'th R‘t. or R‘t...0000000000OOOOOOOOCOOOOOOOOO0.0.0.0.... Flavin Dimmleotida, Plavin Honouucleotide and Free Ribo- flavin Conant-renal: in name... ammo” or mrsOOOOOOOOeoooooeeeeeeeeeeeaooeaeaoeoooeeeoaea 'tmin 3. Int“. “d M ““0...OOOOOOOOOOOOOOOOOOOOOOO Flavin Dirmcleotida and Flavin Mononucleotide plus Free Ribo- fllmln Concentration in Tinne- ‘ sugge'ud m‘m MmmentOOOOOIO0.00000COICIOOODOOOOO. MHOOOOOOOIOOOOCOOOOOOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 'nuOWODOOO000.00.000.00...0....O0.00.00...0.00.00.00.00... 1 3 10 10 E15 16 16 18 26 27 28 1.13 01‘ TABLES PAGE momm CONTENT OF EXPERIMENTAL DIETS..................... AVERAGE 13mm INTAKEAHD AVERAGE URIEHT GIIHS.............. AVERAGE 111300le (FTISwEs.....................nun..... AWGE m +FREE RIBOFLLVII CONTEST O? rxswse............. AVERAGE TOTAL BIBOFLAVIII CONTENT OF TISSIES.................. 13 19 2O 21 LIST OF FIGJRES um PAGE 1. The Relation Between Growth Response and the Riboflavin Inuk....0......OCOOOOOOOO0.0...O...OOOOOOOOOOOOOOOOOCOOOO. 17 2. ll'he Relation Betveen the FAD Content of Tissues and the Diet.” Vim“ B'Oeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 22 3. The Relation Between the Total Riboflavin Content of Tissues md the D1.m Vim“ 33.0.0.0.OOOOOOOOOOOOOOOO99.0.00... 23 INTRODUCTION IN'I'ROZIICTION Flavins as a class of biological piments have been known since about 1879 (l) . However, their importance was not fully recognised until the work in 1932-33 by Slant-Gyorgyi (2) and Harburg and Christian (3) on “cytoflav' or the “yellow enzyme“. Following this, the active components of the flavins were found to be phosphorylated derivatives of riboflavin, ' either flavin adenine dinucleotide (FAD) or flavin nononucleotide (m) . I In 1933 (h) riboflavin was isolated, crystallized and shown to have vita-in activity for the rat. The importance of riboflavin in regard to its redo: properties was then recognised. In addition to these properties, it was found to stinu- late growth and to prevent loss of hair (alOpecia) , inflammation of the conjunctiva and certain other pathological conditions (5) . It has been shown that the concentration of certain of the enzyme listens requiring the participation of riboflavin or a derivative are dilinished in the riboflavin deficient mind. In addition, several authors have measured the MD concentration in tissues of nomal and A deficient rats. However, nest of the work on the nutritional requirement at vitsnin B. has been concerned with growth measurements. It would seen of interest to see whether the tissue levels of the coenzymes MD and new light not be used as a nore critical requirement index and also to con- pare s reQuirenent fer vitanin B, based on these levels with the growth criterion. The purpose of the present study, therefore, was (a) to measure the eencentrations of riboflavin and its pimsphoryleted derivatives in tissues of rats fed synthetic diets with varying riboflavin concentrations and (b) te coupare the dietary vitamin B, requirement based on tissue levels with that based on growth. 3 HISTORICAL HISTORIC ” The biochemical studies on.riboflevin first began in the enzyme field, rather than the nutrition field. A role for riboflavin-containing ensyees as hydrogen carriers was first shown when Warburg and Christian (3) isolated their 'Old fellow Enzyme“ froa yeast. Isolation of the pure seensyne froa this enzyme systen showed it to be flavin aononucleotide (6). (This system is not specific since‘FAD may be substituted for'EHN as the eesasyne with, however, lass activity.) Lmanino acid oxidase is another easy-e which requires specifically FM] and it has been isolated from rat kidney and liver (7,8). This systee catalyses the oxidation of about thirteen enino acids and also catalyses dehydrogenation of Loelphe hydroxy acids having structures related to the amino acids (9). An enzyae which specifically catalyses the reaction between reduced fPN and cytochreee c has been isolated by Has, Borecker and Hogans (10) and called 1TH.- cytochrona c-rsductsse. Its prosthetic group is also flavin.aone- nucleotide. IAD sets as the eoensyae or presthetie group of several easy-es of biological interest. (A) D—uinc acid oxidase which catalyzes the oxide-- tion.of all the cc.non.alpha-D-aaine acids except lysine, via.inino acids to alphaeketo acids can use only no as its coenzyle (11). The biolOgicel funstion.ef this ensyes is unknown since although it occurs in.aost tissues , its substrates are the unnatural isomers. (B) Glycine is dominated in the presence of glycine oxidase which occurs in the liver and kidneys of most mammalian species, however, rat kidney appears to contain none (12). (C) In 1932, Schardinger (13) discovered an enzyme in milk which catalyzed the oxidation of formaldehyde to formic acid. Morgan, Stewart and Hopkins, twenty years later (111) , {band that certain animal tissues and milk contained a catalytic system which led to the oxidation of either xan'thine or hypoxanthine under aerobic or anaerobic conditions which may be the same enzyme as that dis- covered by Schardinger. Ball in 1939 (15) isolated a flavoprotein fron I111: with the property of s coensyne for nnthine oxidase. One of the two prosthetic groups obtained in this preparation seems to be FAD. Corrsn and Green (16) using a different method of preparation reported the isolation of a flavcprotein from milk which catalysed the oxidation ef aldehydes and which was not identical with zanthine oxidaee. Hot-gen (17) investigated the distribution of santhine 0:1nd in different anieal tissues and found that mthine oxidase and aldehyde oxidase activity were ilvsriably present together. Because of these observations, the Question of the possible identity of nnthine oxidase with aldehyde cxidase has been brought up by nay. Dimn and Thurlow (18) and Booth (19) have con- cluded that xanthinc and aldehyde oxidase are the same enzyme. Booth (19) has suggested that the ensyne be called nnthine oxidase, even when speaking of its participation in aldehyde reactions. However, the ques- tion ef possible identity has not been definitely settled. the presence in liver of an aldehyde oxidase which nay be separated fro. liver asnthine oxidase has been reported by Gordon, Green and Subrabawan (20). The prosthetic group for the aldehyde oxidase is flavin adenine dinucleotide. The fact that the milk flavoprotein apparently catalyses the oxidation of three different substrates, purines, aldehydes and dihydro coensyne I while liver aldehyde oxidaso catalyzes only one of these reactions has led these authors to suggest that the silk ensyee nay be a close association of three enzymes which have not before been resolved. L ' (D) The idea that a special ensyne was required for the transport of electrons between Coensyne III-reduced (JPN-H.) and the cytochrome was arrived at independently by Adler (21) and Bowen and Green (22). An ensyne which could nediate in the oxidation of mm '8. has been described variously as 'coensyee factor“ by Devan and Green and 'diaphorase" by we:- and Hellstrcl (23). In the reduction of cytochrone c by reduced on in mind tissues, it is not known whether the reaction is catalysed by a single any-e or the Joint action of two systens. The ensyne which does catalyse it say be specifically designated as IPl-cytoohrone s-reductase (2h) even though the relation between it and the diaphorases resales to be settled. (E) Straub (25) has isolated a soluble flavOprotein from heart nuscle which Corren, Green and Straub (26) describe as a soluble 'diaphorase'. (P) less (27) has isolated a yellow ensyne fro. yeast in which the protein portion differs free that of Harbor; and Christian since PAD is the specific coensyne. (G) in ensyse having FAD as the coensync which catalyses the hydrogenation of fuaric acid to succinic acid has been discovered by Fischer (28) , but whether thie reaction ie the physiological function of the enayne hae not been eetabliehed. Since riboflavin ie part of the coenzyne in the catalyet for the reactione deecrihed above it would be logical to presume that a de- ficiency of it might lead to a decreased enzyme concentration in animal tieelee. That thie ie true he been ehawn for the nnthine oxidaee con- tent of rat liver (29) and the D-enino acid oxidaee content of liver and kidney (30) . When rate were maintained on a vitamin B. deficient diet, Ochoa and Roeeiter (31) found that the un content wee eignificantly 1... in the liver and heart of thoee animale than in the cane organe of normal rate. Beeeey, Lewry and Love (32) have found that a deficient diet decreaeed coneiderably the tieeue concentration of riboflavin in all forne, namely: free, no and Pill eepeoially in liver, kidney and heart. the poet obvim effect of a «rare riboflavin deficiency ie the failure of chi-ale to grow. The Bourguin-Sherean procedure involving leaeurenent of rat growth ueing a etandard diet containing different quanti- tiee of riboflavin hae been need encceeefnlly ae a bioaeeay method for the autumn content of varioue materiale (33) . Within cot-tun unite of growth thie diet elicite a quantitative reeponee to graded done of l' the vitamin. In addition, the growth nethod hae aleo been need u a criterion for eetabliehing the vit-in dietary requirement. 10 ,u g riboflavin/day hae been euggeeted ae a ninim requirement by m1» (310 for railing young weanling rate to naturity. Being purified dicta, £111. (35) found that a diet containing 3 Mg/gran diet wee adeQuate to support normal activity in adult rats and enabled then to live a nonal life epan. However, 10 flg/gran diet appeared to confer additional benefite on the offepring,‘and to give increaeed reproduction. Hannering (36) has euggeeted a aininun requirement of 18 ,ug per day on a low fat diet. Hayfield and Hedrich (37) place the daily riboflavin require-ant for unanim- effioiency of protein utilisation at about 5 fl 3. Uaing the criterion of naintenance ef tieeue level concentration for a period of h to 12 weeka, Ceachea and Guggenheim (38) found that on a eynthetic diet 1.5 {mg/day waa sufficient for growth of fifty gran rate, although optinal growth wae not obtained with thia mount of 3.. It has aleo been of interest to ace how the conpoeition of the diet influencee the require-out. Caachee and Guggenhein (38) ehowed that the level of fat, protein or carbohydrate in diete ieocalorio with the normal influenced the requirement. On a low protein diet, tieaue levela were It naintained with 20 ,ug/day utd even then eecondery deficiency effeote were preeont. On a high protein diet, the vitenin requirement was 15 [A g/day, the high fat diet "Quired 20 /1g/day while the low fat diet maintained tieaue levele on only 3.5 lug/day. According to theee authore, the variance in require-ant nay be due to either netabolic factore or differencee in the amount of inteetinal ayntheeie of riboflavin, or both. the mall mount required by the low fat diet could be attributed to inoreaeed inteetinal eyntheeie and the amount required by the high fat III! the high protein diete due to the poeeibility of B; entrance in beth protein and fat notabolian. Schweigert, 33 3., (39) red various diets consisting of either high carbohydrate, high fat or high protein, each with high (so fag/day) and low (8 #g/day) intakes of vitamin 83. They etate that the fat, carbo- hydrate, or protein content did not affect the tissue concentrations when the one level of B, was fed. This does not agree with results given by Coachea and Guggenheil for the diets containing 8 [Hg/day, however, the high B. concentration was nuch higher than any which Czachee and Guggenheim fed and night, therefore, give Optimal mounts in the tissues. It should be noted that the carbohydrate used by Czachee and Guggenheim was rice flour, as compared with the use of sucrose by Schweigert which might account for the differences on the low B, diets. In addition to the effecta shown by various protein, carbohydrate and fat levels, the type of each used is important. Mannering (ho) has shown that dextrin, narkedly, and cornstarch, moderately, stmlate ' bacterial synthesis of B. in the rat intestine, probably by providing a more favorable mediun for bacteria and , in this way, their use in diets reduces the dietary requirement. Sucrose which is absorbed more rapidly than the other two does not promote thie synthesis. Rieeen, Schweigert and Elvehjen (bl) showed that on ingestion of 30 [ug/day the total riboflavin level of the liver was a function not only of protein intake, but also methionine and cystine content of the diet. About one third of the increased riboflavin content of the liver caused by greater protein intake could be accounted for by ingestion of increased anounte of methionine, but none of the increase could be accounted for by ingeetion of increased cystine . The intake of vitaain 0 ha been ahown to control total riboflavin atorage in liver, kidney and adrenala of the guinea pig (142). Hovever, aince rate are able to ayntheaiae their on vitamin C and guinea pigs ean not, thia new be of no eignificance in the rat. Sure and Ford (10) found an interrelationship between thianine and riboflavin indicating that the efficiency of riboflavin utilisation vaa decreaeed in the abaence of thiuine, but that the reveree vae not true. It may be noted then that the requirement of the rat for vita-in B. varies depending on the diet coupoeition, age of the anilala and other factora. In view of the importance of the riboflavin containing enzyme ayateu it me of inter-eat, firet, to measure the tiaaue concentration of the riboflavin coenlylee and, eeoond, to compare the dietary vitanin require- nent for their optilal concentrationa with the requirenent for youth . EXPERIHENTAL 10 EXPERIMENTAL Angels end Diets Isle end tenele elbino rets or e etrein developed in the Hiohigen Stete College Chemistry Deperhent were 28 to 35 deys old when selected for these emerinents. The eninele which were divided into groups of eight with eech group heving en equel nusber of seles end reuslee weighed between 16 end 50 gas. The ever-ego initiel end tinel group weights ere mum in m1. :1. I hrified diets men differed only in the vitamin a. content were fed to the and. 33 m tor epprexieetely 31 den. The bees]. diet, e nodiIieetion er the Bomuin-Shenen diet (33) conteined the following eonstiteents by weight: eesein (vitamin tree” 18%, sucrose 68%, corn oil (Inele) 71, selt Iixture (NJ) 15% end Boughex 3i. A Vito-in supple- sent eensisted of the following mounts per 100 grens or diet: thinin hydrochloride 500 m, eieotinio eoid 2,000 ,ug, pyridoxine hydrochloride 250 Ml: no.1. (folio eeid) 100 ,ug, end eholine chloride 100 :3. These mediseolndinSOIethenolendeddedto theeeseinbyseensere pipette. After the oeeein bed dried the other oonetituents were edded. The vita-in B. eontent o: the diets wee veried by sddition or the proper eliquot of e solution of orystelline riboflsvin“ in 1101 ethanol-Hutu to the eesein es in the use of the other vita-in eupplements. The W 4...... .._._ * Vitenin-tree test eesein (114.9% I, 0.1]; #3 riboflavin per gram) sennfeotered by Geaerel Bioeheeioels Inc... Chsgrin tells, Ohio. " ommnm rimmm obteined rm Eestanen lode]: Coupeqy, c.r. grade. riboflevin content of the diets is shown in table I. The diets were then stored in derk bottles in e refrigeretor. Vitsnins A end D were fed es three drape of cod liver oil per at every other dey. Distilled weter was supplied for drinking. A cerotul record of food consumption was nede. Speciel food cups were weighed every two deys end food spilled wee collected on peper towels end eleo weighed. The eninele were kept in individuel, reieed eegee end the roon tupereture ninteined between 75 end 78° r. Amman Method for Bibotlsvin, m end an After seoritioin; the eninele using ether, the tissues were removed es quickly es possible end inedietely froeen between sheets of solid cerbon dioxide. Until they were used for the enelysee, the tissues were kept trosen st -1o° C . The method used for enelysie wee essentielly thet of Beseey, Lowry and Love (32). It depends on the feet thet when PAD is split to flewin phosphete or tree'riboflevin there is en increese in fluorescence. Also, an sen be distinguished free free ribotlsvin on the beeis or its distri- bution coefficient between weter end beneyl eloohol. Since riboflevin solutions ere sensitive to light, ember glasswere wee used end en Best-lea eefety leap with Urstten Filter 0A used for illusi- netion es such es possible. All resgents were nede up with glue re- distilled weter end redietilled triehloroeoetic eeid wee used throughout. In the cold room, the tissue eenple wee extrected with e large volue of ice wster, using e Werinz Blender. After the sddition or en equal TABLE]: mom VIN CONTENT O! EIPERDIEN‘I‘AL DIEI'S 50 100 200 .4 ON mire-atop boo TABLE 11 AVERAGE mm INTAKE 13D AVERAGE WEIGiT W8 W Diet Aversge Daily Averege Deily Aversge Averege Averege tuber Riboflevin Food Intake Initiel Totel Deily Inteke‘ Height Weight Height Geined Geined As no 3- a. 3......— l 0.0 Mb 116.6 2.8 0.1 2 1 .0 3 .9 M .S h .6 O .1 3 2.9 5.8 1.6.6 19.8 0.6 h 6.6 6.6 1.5.6 29.5 1.0 5 18.6 9.3 h9.9 71.6 2.5 6 30.0 10.0 h7.h 101.5 3.2 'l h2.8 10.1 50.1; 121.7 3.9 ' Calculsted Iron eotuel food consumption. 1h volume of 20% trichloroecetic ecid, the solution wee centrifuged end en eliqnst ef the supernetent neutrelised with one fourth its voluee of h 1! 1.11130“ Another eliquot wee incubated overnight et 38° 0. end then neutrelised. The fluorescence of the suspensions were neesured es soon D possible eftor neutrelisetion, using the Coleman Electronic Photo- fluorcneter (Models 12 end 12 B) . The concentretion of riboflavin in the eliquot was read free e . stenderd curve propered by using verious concentretions of riboflevin dissolved in e weter solution containing trichloroecetio ecid end [.90. is the sens proportions es in the final tissue extrect. The pH of this listure which is ebout 6.6 is important since the fluorescence of ribo- fleVie chengee with pH, being et e nexinun between p8 5.9 end 7.? (1.5) . the velidity of using the stenderd curve wee checked by teking reed- ins on e tissue extrect eliquot before me efter e known count of riboflevin bed been eddsd. These enelyses using the etenderd curve speed Within the emerisentel error with the mount of riboflevin edded. Celculetion of Result; In e highly concentrated salt solution FAD (celculeted es riboflevin) hes e fluorescence equel to 15% of that of riboflavin. we (calculated ee riboflsvin) end free riboflevin have equal fluorescence. If the Ippll'b ent riboflevin of the initiel senple - R1 end the epperent riboflevin in the hydrolysed emple - 3‘, then PAD - at " Bi . The totel riboflevin z, e no + non no (as e free riboflevin): in attempt was node to determine the free riboflavin by the honey]. elcohol extraction nethod (32). This wee unsuccessful since the amount of free riboflevin was too small to be reed securately with the photo- fluoroneter used. Therefore, the PM + free riboflevin wee celculeted es the difference between the total riboflavin and the PAL". In order to test the velidity of using frozen tissue emples, ribo- flevin enalyses were nede on tissues before freezing end efter freezing end storing for e few weeks. There was no appreciable effect on the tissue concentretion either due to freezing or to storing while frozen. RESULTS 16 RESJLTS Growth Rete of Reta At the end of the experimental period, the rete on diete l, 2 end 3 Med typicel eyuptoee of e vitamin B. deficiency. The fur coet wee oily looking end uneven end there wee e coneidereble reddieh eocumletion of porplvrin on the peel, noee end whiekore. Two enieele on diet 1 died before the experinentel period an completed, the remaining enieele on thie diet end diet 2 were well end either had geimd very little weight er hed loet weight ee ehown in teble 11, page 13. lei-ele on diet 3 geined eore weight but reeeehled the ebove eninele in eppeerence. leverel eninele on diet 14 eleo ehoved theee eyeptoee but to e leeeer 6081'”. the enieele on diete 5, 6 end 7 were ell lerge heelthy looking enieele with good for eoete et the end of the feeding period. the gein in weight of theee enhele inereeeed ee the ingeeted B. inane-ed; the lugeet gein, 3.9 (./dey, wee on diet 1 (I43 lug riboflevin conned per dey). the growth rete fer nelee ie generally greeter then thet for fuelee. Thie new be eqnelieod either by eelecting ell of one eex for the experieent or, no in the preeent etudy, by heving en «and amber of both cone. On the lower diete there wee no noticeehle growth difference between the urn, hcwuer, on diete S, 6 end 7 the growthof the eele rete wee ebove thet 9‘ “3' fuel". The everege weight geine ere ehown in figure 1. 17 L06 OF THE DOSE 0.4 0.8 /.2 /.6 ' I ‘ l ' I ‘ F ‘ Q4r- 5 § - \' . K53- ‘~. :2: e \ 352- L‘. - I b/_ \ 0 :E: e GD 0 1 l 5 1 l l l l L /0 2'0 30 4'0 VITAMIN 32 INTAKE Qua/DAY) Figure l The relation between growth response and the riboflavin intake. Weight gain is expressed as the average group gain in grams/day; riboflavin intake is expressed as ((g vitamin Bg/day and logarithm of the dose in micrograns. I :T-= vitamin B2 intake ;(g/day ml: logarithm of the dose 18 The vituin B. inteke which no celenleted fro- the food consumption ie given in teble II. It will be noted thet the enount of vitenin B. con-_ need on diet 2 wee very uell end theee rete end thoee receiving no a. were elnoet the one ee fer ee growth end eppeerenoe were concerned. PAD, Ill end Free Riboflevin Concentretion in Tieeuee The PAD end totel riboflevin were detereined ee deecribed before. Since the free riboflevin wee too enell to be eccuretely neeenred the venue of m «- free ere equel to the difference between the totel ribo- nun end the PAD. However, it wee epperent thet the lergeet pert of the difference between totel riboflevin end MD ie m. The reeulte of the time enelyeee ere given in teblee III, IV end V end ehown grephicelly in figuree 2 end 3. The eoeneyne concentretione were celcnleted ee ribeflevin end ell were expreeeed ee lug of riboflevin per gre- of wet tieeue weight einoe previous experinente of thie type heve eholn thet eelculetione beeed on either #g/gren of dry weight or ,ug/gren of nitrogen give precticeny the one picture ee theee beeed on wet tieeue weight. Tiuuee rm diet 2 enilele were not enelyted ee the uount of vitenin B. ingeeted wee no 101! end theee eninele were much the one u diet 1 eninele. If one enlinee the effect of dietery I. concentretion on the MD end m + free riboflevin concentretione in eech tieene it ie noted thet the level of theee conetituente in brein ie neerly independent of dietery B . ‘ eencentretion. There ere nell differencee noted but beceuee of the la! hub of riboflevin end ite phoephorylated derivetivee the experilentel TABLE III AVERAGE FAD CONTW’II OF TISSJES Diet Brein ,ug/g freeh' tieeue 1.93 I 0.06 2.16 2 0.06 2.39 3 0.05 1 3 h 2.25 i 0.06 S 6 2.08 z 0.16 1 2.27 .t 0.06 Heart ,ug/g mu? # tieeue 9.1.7 I 0.65 10.10 3 0.1.6 12.06 3 0.35 13.96 t 0.39 13.19 t 0.57 13.75 : 0.1.1 Kidney lug/g fresh. tieeue 12.68 t 0.52 16.22 .t 0.71. 1h.h6 : 0.71 15.89 i 2.21 15.81: I 1.03 16.9h .t 0.99 1.9 Liver ,ug/g freeh' tieene 10.61 t 0.39 12.38 I 0.57 11.71 3 0 .hz 17 .11. t 1.32 22 .07 f. 0 .82 19.37 1’ 0.35 in g m) (celculeted ee riboflevin) “ The 3 ie one etenderd devietion fron e neen of 6, 7 er 8 veluee (would be expected to include 67$ of ell values) . 20 TABLE IV AVERAGE m + FREE RIBOFLAVIN CONTENT OF TISSUES W Diet Brain Heart . Kidney ' Liver Huber ,Ug/g freeh“ In g/g freeh‘ 1/ 3/3 fruh' rug/g fresh. tieene tieeue ’ tiuue time 1 1.01 ' 2.51. 5.3.3 2.23 3 0 .915 2 .52 7 .19 2 .99 h 1 .08 3 .03 11 .21 3 .77 s 1.21. has 1M1 1.1.9 ' 6 1 .12 3 .93 , 15 .20 6 .05 7 1 .09 3 .13 111 .711 5 .57 * m (celculeted u riboflevin) . free riboflevin. AVERAGE TOTAL RIBOFLAVII CONTENT 0? 713311138 TABLE? 21 Diet Brein Heart Kidney Liver """" “$.22“ “(5.21“ ”“632“ “”5133“ 1 2.91. 3 0.05 12.01 2 0.36 18.11 2 0.1.3 12.81. 2 0.1.6 3 3.10 t 0.011 12.95 t. 0.33 23.141 2 0.73 15.37 1: 0.61; h 3.33 1 0.08 15 ...09 0 .50 25.67 3 0.99 15.1.8 1 0.52 5 3.63 1 0.06 18.3. 1 0.1.1 30.66 2: 0.62 211.53 2: 1.99 6 3.20 t 0.01. 17 .12 1 0.16 31.05 f. 1.01 28.12 t 0.91 1 3.36 z 0.02 16.93 1’. 0.116 31.68: 1.51 2h.9h : 0.36 “The - ie one standard deviation from e new of 6, * Totel riboflevin (calculeted ee riboflevin). 7 or 8 veluee (would be expected to include 671 of e11 veluee) . M Q \ U: \ FAD we. /6. TISSUE) Q 01 22 1 J 1 1 L 1 /O 20 30 40 V/TAM/N 82 INTAKE GHQ/DAY} Figure 2 The relation between the FAD content of tissues and the dietary vitamin Bg. FAD (calculated as riboflavin) is expressed as 14g FAD/g. fresh tissue weight (average) and riboflavin intake as (kg vitamin Bz/day (average). ‘5 = brain, 3 = heart, = kidney, u = liver (i1 01 G. TISSUE) an O N U. 6/ 611' Q \ \ Q 01 T0 TAL R/BOFLA VIN 0| 23 (If/”‘0 M“ O _- ,,////’ /,/ (D u— . ‘ (3 I p.- y~r15"""—rfi ##15"7 C, D 1 L 1 i 4, .L . . /0 20 30 4o wm MIN 32 INTAKE Qua/DAY) Figure 3 The relation between t}e total riboflavin content of tissues and the dietary vitamin B2. Total riboflavin is expressed as 11g riboflavin/g. fresh tissue weight (average) and riboflavin intake as $43 vitamin Bg/day (average). 1- = brain, D = heart, = kic‘jney, {29 = liver error of deterlinetion ie probebly lerge enough to account for these. In liver, hewwver, the firm level of FAD increeeed with increeeing mounts ef dietery B. up to en inteke of ebout 30 /_./g/d&}*. Lerger intakes of vitamin B. did not increeee the FAD further, in feet, 0. wall drop in its tieeue level wee noted. Approxinetely the me findinge were observed for m: + free riboflevin concentration in liver. In hurt the pettern wee einilar to thet of liver where both the FAD end the m + free inereeeed Up to en inteke of 19 /L(g/dey (diet 5), but on diete 6 end 7 the FAD remained the one while the my free concentra- tion dropped elightly. Il'he FAD concentretion in kidney increased considerably on a. daily mm of epproxinetely 3 ,ug (diet 3) drapped slightly on diet I; end then peduelly leveled off. The not + free riboflevin inoreeeed nore peduelly with the B. intake up to e deily intake of 19 ,ug (diet 5) end then lerger mount- did not inoreeee the no: in the kidneye. .. It in intereeting thet e deficiency greetly effected the M e free riboflevin concentretion in kidney end liver, but not to euch en extent in heert. It my doc be noted thet thet perticuler coeneyne concentreo tion in kidney end liver tieeuee of deficient enimele wee about half of A thet in the tieenee of eninele fed diet 7 (113 'u‘g/dny). fheee reenlte obteined for tieeue concentretione of riboflevin end ite pheepheryleted derivetivee efter feeding rete e diet edequete in vit-in B. egree euentielly with theee reported by Beeeey, Lowry end Love (32). but ere 1mi- then theee given by Cohen end Roeeiter (31) . The nethod of enelyeie used in this study is the sme es that used by the fewer euthore. Ochoe end Roesiter used en enzynetic method of analysis which any help to eccount for the greet differences. The growth rates obtained were somewhat lower than those which have been reported for the sane dietary B, intake by Burch, :3... _e_l. (1.6) , but in egreenent with those given by Edgar end Hecrae (117). In the present study intestinal synthesis was kept et e nininum by the use of sucrose es the dietary carbohydrate. The use of e different diet by Bessey, .23. _e_l_. which would ellow increeeed synthesis of riboflavin in the intestine may eccount for the higher growth rete which they obteined with the «we dietery inteke of vitenin Ba. mmssxou or we 26 DISCUSSION OF RESULTS Vitssin B. Inteke end Growth lists It wes shown thet when other dietary cosponents ere held st constent concentretion increasing the snount of vitssin B. ingested increesee the rste of growth. PM of the differences in growth of rets on diets 1; end 3 es oompsred with those on diets 1 end 2 night be expleined by the in- creased food consumption on the two former diets. This could not be the cese with the minds on diets S, 6 end 7 since eversge food consmption wes slnost the sens for these three groups. The inebility to grow when eninels ere fed the vitssin B. deficient diets lsy be due either to the inebility to preperly utilise the food esten or simply to s lowered food intake resulting fros enerexie; Rats receiving e B. deficient diet heve been shown to exhibit s reel depression of food utilisation es conpered with peir-fed rets supplemented with 20 /u._g 3. per day, sccording to Sure (I48) . The differences in food utilisetion result in put from differences in completeness of food oxidstion es ribo- flsvin is en essentisl component of shout 'e dozen flsvoproteins. This ssy explein the differences in growth found on diets 5, 6 end 7, where, slthough the rats ste sheet the use ssount of food, the growth increesed es dietery vitenin B, increeeed. It is difficult to set s definite figure for annual growth rate of rsts but s growth approeching h.0 grens per dsy is considered very good. Edger end Hecrse obteined growth gains of h.3 to 13.6 grsns per day for 27 nales en a purified ration with 50 )ug vitsnin B. given as an additional supplement (147). However, in a group composed of equal numbers of sales and females, it is thought that 3.9 grams per day, the average group gain on diet 7, is close to the nuisun obtainable with this synthetic diet. FAD and mu + Free Riboflavin Concentration in Tissues Of the four tissues studied, brain tissue was affected least by a Vitamin B, deficiency, heart somewhat lore and kidney and liver were affected nost. If one compares the decrease in coensyne concentration caused by a deficiency of vitsnin B. (i.e. comparing tissue levels of aninals receiving diet 1 with those receiving diet 7) it will be seen that in heart the [AD concentration is lowered relatively more than the m, whereas in liver and kidney the reverse is true. This night suggest um the aninal body attempts to saintsin the concentration of that particu- lar riboflavin containing coeneyne which is here important to the functions carried set by each ergen, er in the case of the heart, that the ability te synthesise FAD tron m, which is an ability of nest cells of noraal aninsls (to), is impaired. The intake of vitenin B. necessary to naintain the tissue levels varies with the organ and with the particular coensyneg the amount needed to maintain kidney no is relatively shall, a larger intake is required for both an and MD in heart, while kidney FMN and liver MD and m all require about 30 fig per day. The tissue eoemyne concentrations, especially in the latter groups exhibit deviations which nay be explained in part by the influence of 28 body stores at the beginning of the experiment, to seasonal variation (the experimental period extended from October to April), or to other unknown variables. Since the liver is concerned to a great extent with netsbolisn and the coensyne concentrations in liver do not reach the Optimal range until the elount of vitamin B. ingested (19-3) /.(.g/day) is enough to cause nor-a1 growth and “well being“ the liver coenzyne concentrations night he used to give an indication of the nutritional status of the animal. Mined Dieteg;ReQuirenent An attuupt was ads to compare the dietary requirement for vitamin B, based on (a) that necessary for growth and (b) that necessary for mainte- nance of coensyee tissue levels. On this synthetic diet, which was selected to elininate as much intestinal bacterial synthesis of ribo- flavin as possible aaximn growth required an intake of somewhat more than h} fig per day. It should be noted that the increased growth was not attributed to a larger food consumption, but to increased utilisation of ingested food. for an optimal tissue level of both FAD and m the necessary intake would use to be around 30 (Hg per day (the amount received by animals on diet 6) which is lower than that necessary for naadmun growth. However, taking into consideration both criteria it is suggested that an adequate dietary intake of vitain B. would be 30 to ho ‘xiig per day, or 3 to 1; lug per gran of diet. SHINE! l. 3. 29 MARY 'e'hen rats are fed a diet containing no added vitamin B. growth ceases. Between an intake of 6.6 and M /Ll_g B; per day the growth response with respect to the log of the dose is linear. The PAD and the ms + free riboflavin concentration in brain is alnost independent of the anount of B . in the diet. However, in kidney and liver coensyee concentrations were alnost doubled as the concentration of the intake was increased free no added B, to 30 [1.1g B. per day. In the heart nuscle of deficient aninals the MD is decreased relatively more than the ma, whereas in kidney and liver tissues the reverse is true . The vita-in intake necessary to maintain tissue levels varies with the organ and the eoensyne. In general, an intake of 30 fig per day is sufficient to naintain optimal concentrations in all tissues. A dietary requirement for growing rats on a synthetic diet of the type employed in this study is suggested. For optimal coensyne tissue levels the intake should be around 30 I1:g B, per day and for maximm growth the intake should be somewhat higher than 143 /L a. g per day. For both cptinal tissue levels and growth it would seem that 30 to 140.11g B, per day or 3 or 14 mg per gran of diet would be adequate. BIBLIOGPAPHI BIBLIO GRAPH! 1. Bly'th, A. 14., 'The Composition of Covs' Milk in Health and Disease ." J. Chen. ace. 2 530-539 (1879). 2. Bangs, I. and Beent-Gyorgyi, 1., “fiber Co-rermente, Wuserstoffe donatoren and Arsenvergiftung der ZellatimingdI Biochem. Z . 336. 203-211; (1932) . 3. (a) Hes-burg, 0. end Christian, 31., "fiber sin neues Oxydations ferment and sein Abscrptionsspektun' Biechen. z. 32 138 (1932). (b) Uarburg, o. and Christian, $1., ‘lJber das gelbe Ferment und Seine Wirkmgen.’ Biochen. 2. age 377 (1933). h. Kuhn, 11., Gyorgy, P., Wagner-Jauregg, Th. 'Uber Lactoflavin, den rarbstoff der Holke.‘ Ber., £63 1031’. (1933). 5. Sure, B., 'Pwther Observations on Riboflavin as a Food Factor in Economy of hoe Utilisation.‘ J. Nutrition. 3; 295-301 (191d) . 6. Kuhn, 1., Rudy, 3., and Heygand, 7., “fiber die Bildung eines lunstlichen Penuents aus 6.7-Dinetrql-9-1-araboflavin—Sh phosphors'a'ure.' Ber. Dent. Chen. use. .632 203).; (1936). . 7. Blanchard, 11., m'een, D. 3., Iocito, V., and Ratner, 8., 'l-Anino Acid Oxidase of Aninal Tissue: J. Biol. Chen. 155 1.21 (191.11). 8. Blanchard, 11., Green, D. 3., Hocito, 7., and Ratner, 8., "Isolation of 14mm Acid OxidaseJ' J. Biol. on... .131 583 (191.5). 9. Blamhard, 11., Green, D. E., locito, 1., and Battier, 8., 'l-Hydroxy Acid one». .' J. Bisl. Chm. L622 137 (191.6) . 10. Bass, 3., Borecker, B. L., Hogness, T. R., "The Enzymatic Reduction of Ci‘tochgs'cse C Cytochrome c Reductnse.‘ J. Biol. Chan. 26 7h? 19 . 11. (a) Val-burg, o. and Christian, w., ”Cooternent der dailanin-Oxydase.‘ niece-e. 2., 2 6 291. (1938). (b) Usrburg, O. and Christian, 111., 'Isolierung der prosthetischen Gruppe der d-Aninosaure amass.“ Biochen. 2. 325 261 (1938) . 12. Ratner, 8., locite, 1., and Green D. E., “Glycine Oxidase.' J. Biol. cm. 12 119 (19h). 31 13. lehsrdisger, 1., 2. Untersuch lahr. u. Genusu. 2 22 (1902). lb. Morgan, B. J., Stewart, C. P., and Hopkins, 1'. 0., 'Anaerobic and aerobic oxidation of nethine and hypexanthine, by tissues and by lilk.‘ Proc. Roy. Soc. (undon) 1325 109-131 (1922). 15. Ball, E. 0., 'xsnthine Oxidasea Purification and Properties.“ J. Biol. Chen. 1;; 51 (1939). 16. Cori-an, H. 8. and Green, D. 3., 'i'he Isolation of a Plavoprotein from Ows' Milk! Biochcn. J. 2 2231(1938). 11. larger, 3. J. , "The Distribution of Ianthine Oxidae." Biochen. J. g 1282 (1926). 18. Diana, 11. and Thu-lee 3., 'Studies on Xanthine Oxidase 11. The Dyna-ice cf the duo System) Biochem. J. 18 976-988 (1921) . 19. Booth, 1'. H., 'The Identity of lanthinc Ozidaee and the Schardingcr any») Bioeheu. J. 39 1132 (1935). * Death 1. H. "The specificity of lanthine Oxidase." Biochem. J. 23 9). (1958) . 20. Gordon, A. H. Green, D. E., Subrahnany ”Liver Aldehyde an 1., Oxidase. Biochem J. 2!; 7611-7711 (19110). 21. idler E., V. Euler, H. and Hellstron, H. 'Ensymio hydro n-carriers in Huecle.‘ mm Kali lineral. oooi. 1323 so. 38 1937). 22. Devan, J. G. and Green, D. E., "Coensyne Factor-«A lee Oxidation Catalyst." Biochea. J. 626 (1938) . 'Coensyne Factor of least) Bioch. J. 2 (1938) . 23. V. Euler, H. and .Hellstr‘o'n, H., 'Zur lenntnis dsr Bneynatischen ' Rae‘sersgffuber' tracer in Huskel.' Z. thwsiol. Chan. .22 31 193 . ‘ 2h. Fetter, V. 8., 'fhe Assn of Animal tissues for Respiratory Easy-es I. 1116)). Halic Dehydrogenase Systan." J. Biol. Chem. 165 311 19 . 25. Straub, F. 3., 'Iselation ad Properties of a l’lavoprctein fro: Heart Huscle ‘l‘issuoedI Bioohu. J. 21 787 (1939). 26. Corran, I. 8., Green, D. B. and atraub, l'. B., 'On the Catalytic Function of Heart Flavoprotein! Biochen. J. .21 793 (1939) . 32 27. Bass, 2., 'Isolierungeines neuen gelben Pements.‘ Biochen. z. 229 378 (1938). 28. Fischer, 1'. 0., Roedig 1., and Ranch, K. "tharic hydrogenase, a yellow emyxne .' Natureissenschai‘ten. 21 197 (1939) . 29. Axelrod, L. E. and Elvehjen, C. A. "The Xanthine Oxidasc Content of Rat Liver in Riboflavin Deficiency." J. Biol. Chem. .139 725-738 (1911) . 30. Axelrod, A. 5., Sober, H. A. and Elvehjen, C. 1., "The d-Anino Acid Oxidase Content of Rat Tissues in Riboflavin Deficiency.“ J. Biol. Chem. _1_3_1. 71.9 (191.0). * 31. Ochoa, S. and Rosaiter, R. J., 'Flavin-adeninedimlcleotide in Rat Tissues." Biochen. J. 33 200' (1937). 32. Bessey, O. 1., Lcsry, 0. 3., and Love, 11. 11., "The Fluoronetric Hsasm'enent of the Nucleotides of Riboflavin and Their Concentra- tion in Iissues.‘ J. Biol. Chen. 1813 755-769 (1919). 33. Bourquin, A. and Sherman, H. c. "Detemination of Vitamin a (8.) ." J. “a Ch”. &cap 22 3301-3501) (1931) e 311. Bupplee, G. 0., Bender, R. 0., Kahlenberg, O. J., “The Effect of Couplinenting Factors on the Quantitative Response and Specificity of Vite-in I... J. lutrition. 29 109-123 (191.0). 35. Ellis, 1.. 3., Inachinsky, Sherman, H. 0., I'Experimemts Upon the - Bitnificanoe of Liberal Levels of Intake of Riboflavin.“ J. lutrition. 22 153-160 (191.3). 36. warring, G. J., Lipton, I. A. and Elvehjsa, C. 1., |'Relation of Dietary Fat to Riboflavin Requirement of moving Rats.’ Proc. Soc. Exptl. Biol. and Bad. 11.5.: 100-th (191.1). 37. Hayfield, H. L. and Hedrick, K. 2., “The Effects of Various Levels of Thiamine and Riboflavin Intake Upon the Utilisation of Casein Supplemented with Hethionino.‘ J. lutrition. 31 1175-86 (1969). 38. Csaches, J. W. and Guggenhein, 1., "The Influence of Diet on the Ribo- flavin leteboli- of the Rat.‘ J. Biol. Chem. _1_6_2_ 267-71. (191.6). 39. Schweigort, B. 8., McIntire, J. 11., and Elvehjen, 0. 1., “Effect of the Composition of the Diet on the Vitamin Content of Rat Tissues.‘ Arch. Bioehen. 3 113 (19113). 33 1.0. Hannering, G. J., Orsini, D., Elvehjen, C. 1., “Effect of the Composition of the Diet on the Riboflavin Requirement of the int.- J. Nutrition. g_8_ 111-155 (1911.). 111. Riescn, H. V. Schweigert, B. 8., Elvehjem, C. 1., "The Effect of the Level of Casein, Cystine and Methionine Intake on Riboflavin Retention and Protein Utilisation by the Rat ." Arch. Biochem. 22 357-955 (19116). 112. Cinino, 8., 'Intervitanin correlation: relation between ascorbic ‘aci: :nd riboflavin.‘ Boll. soc. ital. biol. sper., 33 291-3 19 6 . 113. Bore, 3., Ford, 2. '11., Jr., “Vitanin Interrelationships II. i'hisnin and Riboflavin interrelationships in metabolism” J. Biol. Chen. 11.6, 21.1 (191.2). 1111. Hank, P. B. and Oscar, B. L., “A Modification of the Osborne-Mendel Salt 111nm.” Science. 11. 369 (1931). 1.5. Ellinger, P. and Holden, ll. 'Quenching Effect of Electrolytes on the Fluorescence Inteneit of Riboflavin and '1‘hiochronc.‘ Biochan. J. 3_8_ 1117 (191,111) . 1.6. Burch, H., Bessey, 0. and Lorry, 0)., 'Fluoronetric Measurements of Riboflavin and its natural Derivatives in kall Quantities of Blood Seru- and Cells.” J. Biol. one... 115 1.57-1.70 (191.8). 117. Edgar, C. 3., Hanna, 1'. l‘. and Vivanco, 1., "the Water-Soluble B-Vituins VII Growth-Promoting Properties of Lacto navinJ' J. Biochem. 2 379-585 (1937) . 118. Sure, D., 'Purther Observations on Riboflavin as a Food Factor in Economy of Food Utilisation.“ J. Hutrition. 23 295 (191.1) . 1.9. Klein, J. R. and Iohn, H. I. ”the Synthesis of Flavin-adenine Dinucleotide from Riboflavin by Hman Blood Cells in Vitro and in Vivo.' J. Biol. Chem. 126 177-89 (1910) . (I'IVII. .. cot: tron/1130" Hmcm. v1}: Lt) 1:: .‘EE’ 5'5': n‘II'iEVRIVifz‘ .; "H ""1 '1" , 5?; .7. own “. ‘.A-"D'I" V'g- Ifi' -v In). 3N "J C3}, hr- m; whit-.3, it-ii..,r? any-(:4: «b C}? ‘VIT'N‘B‘: :32 By Lucile 31132:. the alter A351 £1.13."- I'Tt C? Jubmittod to the 3021001 of Graduate :5tudken or kichigr’n state College of :ogicnlture one .A polled Sloiozaoe in partial fulfillment of the re'guirenerto for the degree at lA’LWfl 3???) 21.2303” Demrtmt of Chemistry 1953 AM m .’U ...c , ”pkg“. . n3... 3‘ 9"“, T" . art’s} a. we“ v. y Mi“. V‘N’r 'r‘? .',._ _. PL. C ,-" , .w .TI.5;. v.51? ) .. ‘ .1": ‘5... ‘5 4. . .) "o: J. a a b} :3 Mr)“ may», 1 ’e :V'vv I h. org“:- 9 y ’5 ~ n.“ . ‘7‘ v" ft"!!- ”Y” 'ul 0! " 1'.‘\. I v... ‘ w s) .3 ' H C} n 1 r 0.. «9.1 trace-5' ‘y M w’n ;u~ '3 at» -... «I . 2 I“? 's n. ‘ . 4 .c. Lucille Ellen flanker c tho tiracmen of the riot the vitamin, ribofl::xvi:-:, in £31223 in tho free form and also to {12171.1 odor-.139:11:-:.1cl.wtida (19.3) and flovin macromolecular (3‘33? . Thane florin rzuc'lootic‘iee not no contangmen in various n..;;}r.9 nyotom. It hm: boo-n 33:07:... timt- t} .9 tissue 00:09. *5 cticnn of cert-2:1... niacifio 95.93.99 system, 1.41.. Deanna acid. oxiwoa mvd now-thine 0115.62.59 end of the aborta‘rzao W70 in {prom}. mare Gi:.$iia'17Md during a vitnmin 33 dietary deficiency. Varieties criteria have been not no to detom‘ne the of" want rec- quiremert for vitamin 333, ouch us the cmmt neoeocory for mint-o. 22¢.- .09 of tote). riboflavin Home levels! or t! at :zecaoaoory for minimize saw-9th and life open. them has 22015 been or. attempt to comlntc the diatom! revguiremerzt with the oytiml common: timme level. It ma: thank-«jut to be of interest to one how the m: and the FAD concentrations in tits-um were affected. by feeding vex-art 5; omur‘to of vitorzin $13, and to escort: in the dietary intoke of vitmziz. 55;; wooing-y for m: ogmiml tiaizue co- crazy» concentration. More. the :mrmee of t?» moor-.1; otuoy ms: (91 to :9.»me tho oo- .oert‘r‘etims of riboflavi- u. :1 its "hm”: .rvixtm doriwtiv» a in tissue» of rats: fed says. t...otic diet-3 .dth voryr-i' g. ribofl: ”vi": omcnvtmtimo and (b) to commie the diet-tar“; Vits'a.z.ain BE remix-meat Food on timtua 19711913 with that banned or; gumtho Tho composition of the diet affects tha dietary reauimxaant for riboflavin. A amniotic diet which was a modification of the Bourquino 311911“ any diet for 11891319 83 ms chosen to minimize as much as mania masons sources of riboflavin, such as that from 1:21;”. 131ml synthuia. Fiat: wore fad the ayvthatio dicta for approximteiy four weaken. After sacrificing the: wink, the brain, kidmy. liver and heart than» we quickly frozen mad later analyzed for no and F353? 4- mo titan-Vin. ‘11:. method 9! analysis depends on the: fact that in. I highly concentrat- ted salt solution PAD has a {horseman afgual to 153 of that of 2159- flavin and F35?! and free riboflavin have 0:13.131 fluorescenco. It was not possible to determine uomzratoly the very small amount of free riboflavin Want, thus tho ms + free were calculated by utterance. 0f the tissum analyzed, brain oomzym concentrations wart about irtdapendont of the dietary vitamin Ba. In the heart of dotiOiont an- imla tha PM) is decreased relatively mom than the: ram. whamus in kidney and limr the rovema is trua. An intake of apgzroximtely 3/‘8 Why is sufficient to maintain kidnoy 27".-3 oozmamtz‘atioaa on the ammo 1m). as those: of tha animals meoiviu; the highest intake (w/Aa; 83/ day}. ms {- in. is not mintaizsod until an intiikfl 01‘ 1.9 lug Bg/dny is attained. In 117.1“ an. intakn of around 30 [15 33/65:)! is nocaaanry for optiml coxzcentmtiom of hath ooenzyzma, 91.11. in heart 19 fl; Bg/dzy in sufficient for both. fixing mximun growth as a criterion tha requirement is different- Tha highest intake gave growth which, it was felt, closely approached the maximum attainable with this synthetic dint. 'ihsmforo, the dietary thniromont for maximum growth would be aomawhat higher than 43/(5 32/ day. There was an increase in growth obtained with each increase in ‘vitamin Ba intake over 19/4g/day which Gould not be attributed to an inorcasad food consumption. MICHIGAN STATE UNIV HHHuI 3 1293 30 RSITY Jim lTlllfilWlTl'Es 6 4 097