| > u l l i lI WI I 146 231 COCARBOXYLASE AND THRAMENE iN TISfiiUES 0F RATS RECEWENG QIFFEREHT CONCENTQETWMS CIF 'E’HSAMWE EN THE WET I Timsia {m the Emma M M. 3. l‘fiLECE-‘HGJTIJ‘E STATE CC‘LLEGE ‘ L. ..-. ,. mun mmm Ffiakazm ”35:21 0-169 This is to certify that the thesis entitled Cocarboxylese and Thiamine in Tissues of Rats Receiving Different Concentrations of Thiamine in the Diet presented by John H. Flokstra has been accepted towards fulfillment of the requirements for _£._S._ degree inmis ’6 ry MZjor professor [hm May 22, 1950 _.“-_._._._._.., .___ _, TV COCARBOXYLASE.AND THIAMINE IN TISSUES OF RATS RECEIVING DIFFERENT CONCENTRATIONS OF THIAMINE IN THE DIET By John Hilbert Flaketre A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture anthpplied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1950 CHEMISTRY om. TC: \2 . 3 'I avg-1.1 ( ACKNOWLEDGMENT The author wishes to express his thanks to Doctor Richard U. Byerrum for his helpful and encouraging suggestions given during the work on this problem and the preparation of this manu- script; also to Doctor Carl A. Hoppert and others who have aided in this work. ********** *tttttty *****1 sets an as CONTENTS Page INTRODUCTION......................................... HISTORICAL........................................... EXPERIMENTAL......................................... Animals and Diets............................... Manome'bric Techniques.........n..........u...- Analytical Method for Thiamine and Cocarboxylase RmUL'I'Seeeeeeeeeeeeeeeeeeeeeeeeeee‘eeeeeeeeeeeeeeeooeo Enzyme Experiments.......o.....................o Thiamine Intake and Growth of Rats Receiving Experimental Diets...eeeeeeeeeeeeeeeeoeeeeeeee ADRIYSIB Of Tissuueeeeeeeeeeeeeeeeeeeeeooeeeeee CONCI'USIONSCCOOO0.00.00.00.0000000000000IOOO0.00.0... BIBLIOGRAPMOOOOCOOOOOOOO0.00000000000000000000...... l 2 4‘10! 10 10 11 12 17 19 INTRODUCTION In recent years considerable work has been done to indicate the participation of the water soluble vitamins, as coenzymes, in enzyme systems. Lohmann and Schuster (1) observed in 1937 that the pyro- phosphoric acid ester of thiamine, which they called cocarboxylase, was a coenzyme in the system which catalyzes the decarboxylation of pyruvic acid. Ochoa and Peters (2) showed that the cocarboxylase con- tent of tissues decreased when thiamine-deficient diets of polished rice were fed to rats and pigeons. They also indicated that the activ- ity of the pynrvate carboxylase enzyme system in yeast depended on the quantity of the cocarhexylase and also thiamine present. Most of the investigation for ascertaining the animal requirement for thiamine has been based on the rate of growth of animls receiving different quantities of the vitamin in the diet. Since it is now recognized that thiamine functions as a coenzyme and activator for pyruvate carboxylase, a more accurate index of the animal thiamine re- quirement might be a measurement of the change in concentration of co- carboxylase and thiamine in the tissues of animals fed different quantities of vitamin Bl. The present investigation was begun to observe the effect of known concentrations of thiamine in purified diets on the quantity of cocarboxylase or thiamine in the tissues of rate, since no such systematic study has been node. In addition, a comparison of vitamin B1 requirement, based on observed growth, with the tissue cocarboxylase and thiamine content was made. HISTORICAL There appears to be no doubt that thiamine is intimately related to carbohydrate metabolism in animals. In experimental animals having a thiamine deficiency, abnormlly large amounts of pyruvic and lactic acids accumlate in tissues and body fluids. A more precise analysis of the relationship betwaen thiamine and carbohydrate metabolism was made when Lehmann and Schuster (l) discovered that cocarboxylase, the coenzyme of the pyruvate carboxylase system, was the pyrophosphoric ester of thiamine. In accord with this finding Lipschitz, Potter and Elvehjem (3) indicated that a thiamine deficiency produced first a general disturbance of carbohydrate metabolism which later caused a gross pathological effect in which the animl developed lesions in the nervous system. All the work done to determine the requirement of thiamine for animals has been based on the results of growth studies. Light and Cracas (4), studying the amount of thiamine required for norml growth of different strains of rats, reported a variation in requirements from one strain to another. Arnold and Elvehjem (5) produced thiamine deficiency symptom in rats on a variety of low-vitamin B1 diets. Initially the growth rate increased as the vitamin B1 concentration of the diet was increased if the basal ration provided an adequate supply of the other essential factors. Rats receiving a synthetic low-vitamin Bl ration plus an additional 80-100 micrograms of vitamin Bl per 100 grams of ration grew at what was considered to be a normal rate. These .- same workers concluded (6) that for most species of animals the re- quirement for thiamine, as based on growth, was approximately one micro- gram of vitamin Bl per gram of dietary solids. Ordinarily, in what may be considered to be normal synthetic diets this concentration is doubled to assure a sufficient supply of thiamine. It was demonstrated as early as 1932 that there was a relationship between dietary vitamin Bl and the tissue concentration of both cocar- boxylase and thiamine. Simola (7) showed that a vitamin Bl-free diet had little, if any, effect on the rat liver and brain cozymase activity (the enzyme systems involved in the conversion of glucose to pyruvate), but that the cocarboxylase content of these organs appeared to be de- creased under these conditions. Westenbrink (8) reported a reduction in the vitamin content of rat tissues after feeding for five weeks on a vitamin Bl-free diet. Leong (9) and Schultz et al (10), studying the influence of the amount of vitamin 31 ingested on the amount that could be stored in rat tissues, found that an intake of from Sis-90 micrograms per day (gave minim storage. Since apparently the main function of thiamine was that of a pre- cursor of cocarboxylase, a more significant method of tissue analysis was to compare the cocarboxylase as well as the thiamine content of tissues of aninnls fed different diets. Westenbrink and Goudsmit (11), using the thiochrome method, were among the first to do this. They found that the free thiamine‘content of most tissues in a norml rat was small compared to the cocarboxylase content. Ochoa and Peters (12,2) -3- came to conclusions similar to those of Westenbrink and Goudsmit. Using the yeast enzyme (mnometric) method of analysis, they showed that there is much less thiamine than cocarboxylase in the liver and brain of the norml rat. The concentration of cocarboxylase was re- ported to be reduced in all tissues in the avitaminous rat, especially in the brain. In contrast to this work Salcedo et a1 (13) pointed out that, unlike other tissues, the brain maintains its cocarboxylase con- centration for a considerable period. In view of the differences in results concerned with tissue con- centrations of cocarboxylase and thiamine, it is apparent that a sys- temtic study of the relations between the'intake of vitamin Bl in a purified diet and tissue concentration of thiamine and cocarbowlase would be desirable. In addition, it appears that a comparison should be made between the growth rate and tissue concentrations of cocarboxylase and thiamine as an index of ascertaining vitamin Bl requirements. A solution of these two problems was the aim of the present study. EXPERIMENTAL Animals and Diets Male and female albino rats of a strain developed in the Michigan State College Chemistry Department approximately 35 days old were used as the experimental animals. The rats, at the beginning of the feeding experiments, weighed between 50 and 75 grams. Initial and final weights were measured. ‘ Purified diets which differed only in their thiamine content were fed these animals for a period of about four weeks. All of the experi- mental diets contained the following censtituents: casein (vitamin free) 18%, dextrose 74%, corn oil (Ihzola) 4%, and salt mixture (14) 4%. Vitamins dissolved in 70% ethyl alcohol were added by dripping the alcoholic solution from a pipet on the casein and allowing the casein to dry. The following vitamins were added per hundred grams of all the diets: riboflavin 0.5 mg., pyridoxine 0.2 11%., choline chloride 50.0 mg., calcium pantothenate 2.8 mg. F'our experimental diets were then devised each containing the above constituents and in addition a given amount of thiamine Mdro- chloride as shown on Table I. Vitamins A, D, E and K were supplied by feeding each animal two drops (approxinntely 0.1 ml.) of haliver oil per week. These diets were fed in special cups to prevent spilling and the approximate weigit 'of diet consumed by the experimental animals was measured. Distilled water was supplied for drinking. -5- TABLE I EXPERIMENTAL DIETS CONTAINING DIFFERENT CONCENTRATIONS OF THIAMINE HYDROCHLORIDE ‘ Diet Number Thiamine drochloride mg. 00 go diet 1 0.0 2 0.1 3 _ 0.2 4 0.4 -5- Manometric Techniques The influence of cocarboxylase and thiamine concentrations upon yeast pyruvate carboxylase activity was measured, using the Warburg- Barcroft apparatus, by observing the volume of carbon dioxide liberated in the decarboxylation of a substrate of sodium pyruvate. The enzyme preparation was prepared by washing brewers yeast once with water, three times with a 0.13 M phosphate buffer of pH 7.9, and once again with redistilled water. A suspension (10 mg./ml.) of the washed yeast was made in 0.05 M phosphate buffer of pH 6.2 and one ml. was added to the flask. 0.1 m1. of MnClz-MgClz solution containing 20/44 g. of Mn‘H and 100 /q g. of Mg" was then added. 0.05? mM._ of the substrate, sodium pyruvate, was added in 0.2 ml. of water to the flask sidearm. Cocarboxylase and thiamine were added in the desired concentrations and buffer of pH 6.2 to give a total volume in the flask of 3.0 ml. After a ten minute thermal equilibration period at 30° C. , zero time readings were nude on the nanometers and the reaction was initiated by tipping the flask to mix enzyme and substrate. Volume measurements were then mde at convenient intervals during the time required for the reaction. Analytical Method for Thiamine and Cocarboxylase The thiamine and cocarboxylase concentrations in rat liver, mscle and brain were measured. Rats which had eaten the experimental diets for a specified period were decapitated and the liver, brain and muscle from a hind leg were removed immediately and frozen by placing on solid carbon dioxide. ’ These tissues were kept frozen until they were ready to be analyzed. -7- Analyses were made, using a modification of the method of Hennessey and Cerecedo (15). This method involves measuring the fluorescence of the thiochrome produced from thiamine by oxidation with alkaline potassium ferricyanide. The thiochrome from free thia- mine is soluble in isobutanol, phosphorylated thiochrome from cocar- boxylase is not; thus a separation of the two can be made. Free thiamine - The weighed tissue sample was homogenized using .the Potter-Elvehjem technique (16). It was then extracted with about 19 ml. of 0.1 N sulfuric acid in a boiling water bath for 30 minutes. The suspension was cooled, diluted to 25 ml. with water, mixed thoroughly, and centrifuged. Total thiamine - A weighed tissue sample was homogenized and ex- tracted with 19 ml. of 0.1 N sulfuric acid for 30 minutes. The sus- pension was cooled and 0.1 g. of Clarase, a phosphatase preparation which hydrolyzes cocarboxylase to free thiamine, in 1.25 ml. of 2.5 M sodium acetate solution was added. This was diluted to 25 ml. with water and mixed, the final pH being about 4.7. The sample was then kept at 45-5o° c. for at least two hours or at 40° c. overnight and centrifuged just before the analysis was to be made. Determination of thiochrome - A 5 m1. portion of the solution obtained after following either procedure described above was put into each of three glass-stoppered reaction vessels: A, B and C. To A, the blank,was added 3 ml. of 15% sodium hydroxide; to B, the sample, was added 3 ml. of alkaline ferricyanide solution (1.5 ml. 1% potas- sium ferricyanide diluted to 50 ml. with 15% sodium hydroxide); to C, the internal standard, was added 0.5 ml. of a solution of thiamine hydrochloride containing 0.5}! g. thiamine hydrochloride and 3 m1. of alkaline ferricyanide solution. Fifteen ml. of purified isobutanol were added to each vessel, they were stoppered and shaken vigorously for one and one-half minutes. The vessels were then cen- trifuged for about four minutes. The upper isobutanol layer containp ing the thiochrome from thiamine was then drawn off with a pipette and blown into a test tube containing 2 g. of anhydrous sodium sulfate. The fluorescence of the three tubes was then measured on a Coleman Electronic Photofluorometer. The difference in fluorescence between tubes B and A.was duerto the thiamine in the sample. The difference in fluorescence between.tubes C and B was due to the 0.51%4g. of thia- mine added. Knowing these two values and the sample weight, the con- centration .of thiamine in the sample was calculated. The difference between the free thiamine and total thiamine is due to phosphorylated thiamine, or cocarboxylase. The concentration of thiamine in each case was calculated and the difference was multi- plied by the proper factor to obtain cocarboxylase concentration. -9- RESULTS Enzyme Experiments Cocarbozglase and Enzyme Activity - Some information about the effect of added cocarboxylase upon the activity of yeast pyruvate carboxylase was obtained by the Warburg technique. Figure 1 shows the effect of added cocarboxylase upon the activity of the carboxy- lase system found in washed brewers yeast. The amount of added cocarboxylase in microgranm is plotted against the reciprocal of the time in seconds required to obtain an evolution of 200/41. of carbon dioxide in excess of the blank. A total evolution of 1272 [(1. of carbon dioxide was theoretically possible. It is seen that, within the limits of concentration of cocarboxylase used, the activity of the enzyme system as measured by carbon dioxide evolution is propor- tional to the amount of cocarboxylase added. Thiamine and W Activijy - In Figure 2 the activating effect of added thiamine upon a yeast pyruvate carboxylase system containing a constant amount of cocarboxylase is shown. Micrograms of thiamine added are plotted against the reciprocal of the time in seconds re- quired to obtain an evolution of 100 f4 1. of carbon dioxide. Thiamine increased the activity of the system only when cocarboxylase was added to the flask, otherwise no increase in carbon dioxide production above that of the blank was noted. A marked increase in activity was noted as thiamine was increased up to a concentration of about 10/14 g. of added thiamine; more than this amount seemed to have little further activating effect. -10- I 1 Y 1: 44° .06» . a 8 9- -‘ um 21 , m .04, _ E. d y. ‘\ - .02 ‘ b .- 1 I_ l A l 0 50 l00 ISO 200 THIAMINE ( AG. ADDED) Figure 2. The Influence of Added Thiamine Upon'Washed Yeast Pyruvate Carboxylase Activity. Enzyme = 10 mg. washed yeast in 1 ml. 0.05 M phosphate buffer' H 6.2; substrate 2 6.25 mg. sodium pyruvate added in 0.2 ml. H20 0.057 mM. sodium pyruvatgl; activators = 20/xg. Mnfik as Mh012~4H20 in 0.1 m1. H20, lOO,ug.Mg as M3C1206H 0 in 0.1 ml. H20; cocarboxylase = 2/ug. added in 0.1 ml. €20; thiamine made up so that 0.2 ml. gave desired amount, 0.05 M phOSphate buffer pH 6.2 to give a final volume of 3.0 ml. l/time a reciprocal of time in seconds required for evolution of loo/Ml. 002. An attempt was made to use the manometric technique as an ana- lytical method for thiamine and cocarboxylase. Due to difficulty in obtaining a source of yeast which gave consistent, duplicable results, this method was not found adaptable to such a quantitative measurement. However, the data presented in Figures 1 and 2 do indicate that the yeast enzyme activity is proportional to the amount of cocarboxylase present and also that thiamine in the presence of cocarboxylase exerts an activating influence upon the enzyme system. Thiamine Intake and Growth of Rats Receiving Experimental Diets Figure 3 shows the food intake of a typical rat receiving Diet No. 1 (thiamine free). The intake increased for about nine days, then fell sharply and gradually decreased until the end of the experimental period. Rats on this diet gained weight for just under two weeks, then lost weight, and at the end of the experiment the average weight was slightly less than the original weight. Thiamine deficiency symptoms appeared after about three weeks on a vitamin Bl-free diet. The food intake of a typical rat fed Diet No. 2 (1144 g. thiamine hydrochloride/g. of diet) is given on Figure 4. A steady increase in food intake was observed. Rats on this dietary level gained weight steadily and no thiamine deficiency symptoms were apparent. 0n Figure 5 is shown the daily food intake of a typical rat fed Diet No. 3 (containing 2 /"( g. thiamine hydrochloride/g. of ration). The food intake increased steadily and a gradual gain in weight was observed. Both average food intake and gain in weight were slightly -11- I T 1 I r 1 l6' ‘ __ m _ z r “‘ '2“ VLH ‘ p. <[ a - DU 0 8’ - C) —j_‘ C) t _ h. as 4' — y w - - a 4 _1 () r 1 r' ' v"‘ I * ‘ ’1‘ O 5 I0 I5 20 25 30 DAYS Figure 3. Daily Food Intake for a Typical Rat Receiving a Thiamine Free Diet ( Diet No. 1 ) _ Rat No. 2 Initial weight = 64 g. Final weight = 66 g. Average food intake / day = 6.2 g. I 1 1 fi I —1 l6* "J _ —J v— - :2: " F“ :2th a ‘ I- «1 an _l 3 8’ 77'" " <3 ~ r— z ML .5 4* ‘ '- -1 .o I i T j 0 5 l0 1'5 2'0 25 so DAYS Figure 4. Daily Food Intake for a Typical Rat Receiving Diet No. 2 Rat No. 22 Initial weight : 76 g. Final weight : 178 g. Average food intake / day = 10.8 g. body weight a .95 Average mg. vitamin B1 hydrochloride / kg. G.FOOD EATEN '6__ I I l U I 1 L T _ l2" ‘ 8*rtfl ‘ 4r ~ 0 ‘ l I I I O 5 K) I5 20 25 DAYS Figure 5. Daily Food Intake for a Typical Rat Receiving Diet No.3 Rat N0. 12 Initial weight : 54 g. Final Weight a 179 g. Average food intake / day : 11.9 g. Average mg. vitamin B1 hydrochloride / kg; body weight : 2.05 higher than in the case of rats receiving Diet No. 2. These rats appeared to be in excellent condition at the end of the experiment. Figure 6 gives the daily food intake of a rat receiving 4,44g. thiamine hydrochloride/g. diet, Diet No. 4. The average daily intake is slightly lower than that of rats receiving Diet No. 3 and about the same as that of rats fed Diet No. 2. The gain in weight is steady and the average gain is slightly higher than that of rats on Diet No. 2, but slightly less than that of the rats receiving Diet No. 3. Table II shows the average gain in weight and average daily food intake, comparing the four dietary levels. The average initial weight is given for purposes of comparison. The average daily food intake calculated on a per kilogram body weight basis shows very little dif- ference between the four groups. In addition the average food intake on the last day is listed. That of the thiamine-free group was below its average daily intake, but those of the other groups, as might be expected, are higher than their average daily intakes, due to the increased weight of the rats. Analysis of Tissues Free Thiamine Content - In Table III are presented the data from analyses of three rat tissues for free thiamine. These data were cal- culated on a per gram fresh weight, per gram dry weight, and a per gram nitrogen basis. The free thiamine in the liver increased gradually as the amount of thiamine in the diet increased up to the level of Diet No. 3. A comparatively large amount of free thiamine was found in the -12- 6. F000 EATEN '6.- I I I f I F - flfi l2 *- —‘ *7— F" fi—'— 8 - r—4 “a 0 I T , I I I O 5 l0 I5 20 25 30 DAYS Figure 6. Daily Food Intake for a Typical Rat Receiving Diet No. 4. Rat No. 33 Initial weight = 69 g. Final weight = 170 g. Average food intake / day - 11.5 g. Average mg. vitamin B1 hydrochloride / kg. body weight = 3.68 TABLE II AVERAGE INTAKE AND WEIGHTS OF RATS RECEIVING EXPERIWTAL DIETS Thiamine Average Average Average Average Daily Average Content of Initial Weight Daily Intake per Intake Diet Weight Gained Intake Kilogram Last Day Body Weight Thiamine Free 68 g. -l.9 g. 6.2 g. 93.0 g. 2.4 g. 1 lug./g. 65 96 10.7 97.5 12.3 Z/ug./g. 57 115 11.6 94.7 14.5 4 ,qg./g. 64 102 10.6 92.2 13.0 -13.. AVERAGE TISSUE CONTENT OF FREE THIAMINE AND COCARBOXYLASE TABLE III Free Thi ami ne Cocarboxylase Diet Tissue ,«g./g. As-fso Mao/so Also/6. #6-76- ,us-fe N0. fresh dry N fresh dny N weight weight weight weight 1 liver 0.04* i.06** 0.12 1.2 0.70‘ 1.16 2.20 21.4 1 muscle 0.02 1.04 0.06 0.6 0.45* 2.50 1.74 15.1 1 brain 0.10* 1.16 0.45 5.1 1.50* 1.56 6.77 76.0 2 liver 0.45 1.46 1.41 15.7 2.46* 11.25 7.76 75.7 2 muscle 0.22 1.26 0.64 6.4 0.52* 1.55 1.27 9.5 2 brain 1.27 1.61 5.72 64.5 2.15* 1.62 9.61 106. 5 liver 0.66 $.50 2.76 26.4 6.27 11.57 19.7 191. 5 muscle 0.07 1.25 0.27 2.4 1.45 1.60 5.46 41.6 5 brain 1.09 1.91 4.94 55.5 4.25 11.71 19.1 214. 4 liver 2.94 12.07 9.25 69.7 6.22 $2.25 19.5 169. 4 muscle 0.94 2.90 5.61 27.2 1.51 21.02 5.05 56.1 4 brain 1.19 11.01 5.57 60.4 5.56 11.50 15.2 172. * Indicates a significant difference from.va1uer1isted for Diet No. 3 by observation or "t" test for significance. as The $313 1 standard deviation from a mean of 6 values (would be expected to include 67% of all values). -14- liver of animals receiving Diet N0. 4. In nuscle tissues the free thiamine content was quite low for rats fed Diet Nos. 1, 2 and 3. The experimental error in these cases may be large, since such a swell amount of thiamine was present. A higher concentration, however, was noted in the muscle of animls fed Diet No. 4. In the brain of animals fed a vitamin Bl-free diet practically no free thiamine was found. The brain of rats fed the other dietary levels of thiamine had a higher thiamine concentration and the amount present was about the same for all dietary levels. Calculations were made to determine whether the free thiamine con- tent of tissues of rats fed Diet Nos. 1, 2 and 4 was statistically significantly different than that of rats fed Diet No. 3, the thiamine level which gave minim growth. No significant difference was ob- served between the free thiamine content of liver, 11116016, or brain of rats fed Diet Nos. 2 or 4 and those receiving Diet No. 3. The free thiamine content of both liver and brain of rats on Diet No. 1 was significantly different from that of rats on Diet No. 3. There was no significant difference between these two dietary levels in the free thiamine content of mscle tissue. These data are also presented in graphical form in Figure '7. Cocarboxylase content - The concentrations of cocarboxylase on a per gram fresh weight, per gram dry weight and a per gram nitrogen basis are given for tissues from rats on all four dietary levels on Table III. With increasing amounts of thiamine in the diet, the tissue concentration of cocarboxylase increased more rapidly than that of -15.. THIAMINE N MG. THIAMINE INTAKE Figure 7. The Relation Between Dietary Thiamine and the Free Thiamine Content of Tissues. Thiamine intake is expressed as mg. vitamin B1 hydrochloride / kg. body weight / day (average). /Yg. thiamine is expressed as jug. vitamin B hydrochloride / g. fresh weight of tissue (average). 0: live . 9: muscle. 0: brain. free thiamine, up to a certain limit for each tissue. In the liver the cocarboxylase content increased with an increase in dietary thia- mine up to about the Diet No. 3 level of thiamine. More dietary thiamine apparently gave no higher cocarboxylase concentration in the tissue. A similar observation was made with the muscle and brain tissues, although not such a high concentration of cocarboxylase was obtained in these tissues. Significant differences were calculated, comparing the tissue cocarboxylase concentrations of rats receiving Diet No. 3 with those of the rats receiving other dietary levels of thiamine. The liver, muscle and brain cocarboxylase contents of rats fed Diet Nos. 1 and 2 were found to be significantly different than the tissue contents of rats fed Diet N0. 3. Liver, 11116016 and brain cocarboxylase contents of the rats on Diet No. 4 were not significantly different than those of rats fed Diet N0. 3. This information is also shown on Figure 8. To check the validity of using frozen tissues in this work com- parisons were undo of the thiamine and cocarboxylase concentrations of fresh tissue and the same tissue after freezing. Results were the same for fresh and frozen tissue, within the limits of experimental erro re m ‘25 - ...| )- x o ' 1 04" c: < 8 Q L.— 03 3 AG. 63 1 J 1 1 1 1 l l 0‘ I 2 . 5 4 THIAMINE INTAKE Figure 8. The Relation Between Dietary Thiamine and the Cocarboxylase Content of Tissues. Thiamine intake is expressed as mg. vitamin B1 hydrochloride / kg. body weight / day (average). .xwg. Cocarboxylase is expressed asxxg. Cocarboxylase / g. fresh weight of tissue (average). 0:: liver. .a muscle. 0:: brain. CONCLUSIONS An attempt was rude to ascertain the effect of cocarboxylase and of thiamine on the activity of the pyruvic acid decarboxylase enzyme system found in yeast. It was noted that the activity of the enzyme system was proportional to the amount of cocarboxylase present when other variables were kept constant. At constant cocarboxylase con- centration the addition of small amounts (up to 10,“ g./10 mg. yeast) of thiamine caused further activation of the enzyme system. These results agree essentially with the findings of Ochoa and Peters (2). In the thiamine feeding experiments it was shown that the aver- age daily food intake was slightly greater for rats receiving two micrograms of thiamine hydrochloride per gram of diet than for those receiving greater or less concentrations of thiamine. A gain in weight, followed by a slightly larger loss was observed in rats receiving vita- min Bl-free diets. The largest average gain in weight was found in the group of animls receiving two micrograms of thiamine hydrochloride per gram of diet, although animals fed other dietary levels of thiamine also showed weight gains which were not significantly different from these. These data corroborate the findings of Arnold and Elvehjem (6), which indicate that one microgram of thiamine per gram of dietary solids provides "normal" growth for rate. The concentration of free thiamine in tissues increased with in- gestion of larger quantities of thiamine. In animals fed thiamine-free or low thiamine level diets, the bzain retained a larger concentration -1 7.. of free thiamine and cocarboxylase than the other tissues examined. This is in agreement with the work of Salcedo et a1 (13), but differs from the results obtained by Ochoa and Peters (2). An increase in the tissue cocarboxylase concentration was not found in animals fed more than two micrograms of thiamine hydrochloride per gram of diet. However, if an analogy my be drawn from yeast ex- periments to the situation in rat tissue, it is apparent that increas ing thiamine concentrations will increase the pyruvate carboxylase activity even if the cocarboxylase concentration is not increasing. A mximum concentration of cocarboxylase was found in tissues of rats fed two microgranm of thiamine hydrochloride per gram of diet. since the main physiological function of thiamine is believed to be that of a precursor for cocarboxylase, it is significant that the amount of thiamine giving ”normal" growth (l/ug./g. of diet) does not give the largest concentration of cocarboxylase in tissues. If the concentra- tion of cocarboxylase in tissues is to be used as an index of the thiamine requirement, it is apparent that a minimm dietary level of two micrograms per gram of diet is necessary for an optinum ~coenzyme concentration. -13- (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) BIBLIOGRAPHY Lohmann, K. and Schuster, P. "Cocarboxylase? Biochem. Z. 294, 166-214 (1957). Ochoa, S. and Peters, R. A. "Vitamin B 'and Cocarboxylase in Animal Tissues." Biochem. J. 5g, 501-15 (1956). Lipschitz, M. A., Potter, V. R. and Elvehjem, C. A. "The Metabolism of Pyruvic Acid in Vitamin B Deficiency and Inanition." J. Biol. Chem. 123, 267—8l (1938). Light, R. F. and Cracas, L. J. "Vitamin B Requirements of Different Strains of White Rats." Science 81, 90 (1938). Arnold, A. and Elvehjem, C. A. "Studies on the Vitamin 81 Requirements of Growing Rats.” J. Nutrition 15, 429-43 (1956). "' Arnold, A. and Elvehj em, C. A. "Influence of the Composition of the Diet on the Thiamine Requirement of Dogs." Am. J. Physiol. 126, 269-96 (1959). Simola, P. E. "Cozymase and Cocarboxylase Content of the Rat Organisms Suffering from B Avitaminosis." Biochem. Z. 254, 229-44 (1952). Westenbrink, H. G. K. "Vitamin B Contents of the Organs of White Rats Fed 011 Norml and Vitamin Bl-Free Diets." Arch. neerland. physiol. 11, 560-77 (1952). Leong, P. C. "The Maximum Storage of Vitamin B in the Tissues of the Rat." Biochem. J. a, 367-72 (1937). SChUltz, Ae 8., Light, Re Fe, Ora-0&8, Le Jo and Aikin, Le "The Concentration of Vitamin B in the Tissues of the Rat." J. Nutrition 11, 143-49 (1839). Westenbrink, H. G. K. and Goudsmit, J. "Quantitative Measure- ment of Vitamin B1 by the Thicchrome Reaction." Nature 142,150-1 (1956). Ochoa, S. and Peters, R. A. “Quantitative Measurement of Vita- ' min B and its Phosphoric Esters and Their Synthesis in Anima Tissues.“ Nature 142, 556 (1956). (13) (14) (15) (16) Salcedo, J., Najjar, V. A., Holt, L. E. and Hutzler, E. W. "The Relation Between‘Urinary Excretion and Tissue Con- centration of Thiamine in Rats." J. Nutrition 36, 307-13 (1946). ' '— Phillips, P. H. and Hart, E. B. "The Effect of Organic Dietary Constituents Upon Chronic Fluorine Toxicosis in.the Rat." J. Biol. Chem. 109, 657-65 (1955). Hennessey, D. J. and Cerecedo, L. R. "The Determination of Free and Phosphorylated Thiamine by a Modified Thicchrome Assay.” J. Am. Chem. Soc. _6_1_, 179-65 (1959). Potter, V. R. and Elvehjem, C..A. "A.Modified Method for the Study of Tissue Oxidations." J. Biol. Chem. 114, 495-504 (1956). -2 O— .39 284498 T612.39 284498 F629 Flokstra Cocarboxylase and thia- mine in tissues of rats receiving different conc- entrations of thiamine in the diet. 4 13-31; v“ 1 11111111111111 MANN \l _ 3 1293 02446 8500 .