THE SPARING ACTION OF FAT ON VITAMIN B

THE SPARING ACTION OF FAT ON VITAMIN B VI. THE INFLUENCE OF THE LEVELS OF PROTEIN AND VITAMIN G BY HERBERT M. EVANS, SAMUEL LEPKOVSKY, AND ELIZABETH A. MURPHY (From the Institute of Experimental Biology, University of California, Berkeley) (Received for publication, July 6, 1934) Workers (1) in some other laboratories have encountered difficulties in confirming our preceding work (2) on the sparing relationship between fat and vitamin B. An unsuccessful, though unpublished, attempt in Steenbock s laboratory has resulted in definite progress because it has revealed the fact that fat will not spare vitamin B under all conditions, and we wish here to thank Dr. Steenbock for access to his unpublished data. The diets used in Steenbock s laboratory differ from those used here, as can be seen from the tabulation below. Our diet is No. 720, and Diet 722 is the diet used in Steenbock s laboratory. I Diet 722 Casein... 20 gm. Lard.... 50 Sugar......... 24 Salt Mixture 185 (3). 6 Autoclaved yeast... 300 mg. every other day Cod liver oil*...... 4 drops every other day * Kindly presented by E. R. Squibb and Sons. Diet 720 36 gm. 50 I 0 I 4 10 (approximately 800 mg. daily as determined by food consumption records) 4 drops every other day On Diet 722, no sparing action of fat for vitamin B was noted in Steenbock s laboratory. We confirmed this fact early in 1932. * Aided by grants from the Research Board and the College of Agriculture of the University of California, and the Rockefeller Foundation. 429

430 Fat Sparing Action and Vitamin B. VI The rats died with little or no growth, and usually with symptoms of beriberi at an average age of 64 days. Yet on our Diet 720, run simultaneously, the rats reached an average weight of over 140 gm., with survivors still remaining at 180 days of age. After the rats reached 100 days of age, they began as a group to decline in weight slowly, some of the rats occasionally dying of beriberi after 150 days of age. This is clearly shown in Chart I. The difference in the response of the rats on the two diets was far greater than could logically be expected from the differences in the composition of the two diets. The chief differences in the two diets were the amounts of vitamin G fed and levels of the IGO- y) 140 i 3 320. Q Q s/00-80- GO- 40 553520 OAYS. CHART I. Composite growth curves of two groups of six rats each showing that though both diets contain 50 per cent lard, only the diet with high protein and vitamin G (Diet 720) is capable of sparing vitamin B. protein intake. We, therefore, planned an experiment to determine whether the protein intake or intake of vitamin G or both were responsible for the differences obtained with the two diets. The following variables were studied: (1) high protein, high vitamin G, high fat (Diets 720 and 729); (2) high protein, low vitamin G, high fat (Diets 730 and 728, 300 mg. of autoclaved yeast every other day); (3) low protein, high vitamin G, high fat (Diet 722, 800 mg. of autoclaved yeast); (4) low protein, low vitamin G, high fat (Diet 722, 300 mg. of autoclaved yeast every other day); (5) high protein, high vitamin G, no fat (Diet 731, 800 mg. of autoclaved yeast).

Evans, Lepkovsky, and Murphy 431 Since such large amounts of lard were used in the diet, it was necessary to determine whether any impurities there present could exert the beneficial effect. We therefore prepared a synthetic lard (2); the results of feeding this are reported here. EXPERIMENTAL Composition of Diets-The composition of the diets used in these experiments is listed in Table I. Preparation of Materials-Casein L-XII was prepared by washing 14 kilos of casein twice daily for 5 days with about 80 liters of distilled water containing 80 cc. of glacial acetic acid, and twice daily for 2 days with distilled water. It was then thor- Casein L-XII... Sucrose.... Lard.... Salt Mixture 185 (3).... Autoclaved yeast... Synthetic lard.... All diets were supplemented TABLE Composition - Diet 720 36.0 50.0 4.0 10.0 I of Diets - Diet 722 20.0 24.0 50.0 6.0 36.0 50.0 4.0 % 36.0 4.0 10.0 50.0 Diet 730 36.0 4.0 50.0 36.0 60.0 4.0 with 4 drops of cod liver oil every other day. oughly drained by suspending in a cloth, and later dehydrated with alcohol and ether. The autoclaved yeast was prepared by autoclaving Fleischmann s yeast2 for 6 hours at a pressure of 18 to 20 pounds in layers 2 to 3 inches thick, in pans 18 inches long, 12 inches wide, and 2$ inches deep. Repeated feeding tests of yeast prepared in this way showed that it was free of vitamin B. The synthetic lard was prepared by saponification of lard with 40 per cent KOH in water. Lard was added to the alkali in small portions and agitated with steam until the total amount was 1 The Golden State Company, Ltd., generously provided the casein used in this experiment. 2 The whole dried yeast was generously supplied by The Fleischmann Laboratories of Standard Brands, Inc.

432 Fat Sparing Action and Vitamin B. VI added; in this way practically complete saponification was obtained. The tota. time required was about 8 hours. The soaps were decomposed with sulfuric acid, the liberated fatty acids were thoroughly washed with hot water, dried, and distilled in vacua at a temperature of 170-190. The distilled fatty acids were esterified with redistilled glycerol with constant stirring, in an atmosphere of carbon dioxide at a temperature of 200-230. Care of Animals-The rats were started on the diets at 21 days of age, and were kept on large wire screens, 2 meshes to the inch, to insure inaccessibility of the rats to their feces. In the groups in which the vitamin G was not mixed with the diet, it was fed out in small boxes provided also with wire screens. The rats were weighed every 5 days, and vaginal smears taken daily except Sunday. Two basic diets were used, one with 20 per cent casein (low protein) and the other with 36 per cent casein (high protein). Two levels of autoclaved yeast were used, 300 mg. every other day (low vitamin G) and 800 mg. daily (high vitamin G). To test the freedom from vitamin B of our casein and autoclaved yeast, we included a control experiment, in which rats were fed the high levels of autoclaved yeast and casein but no lard (Diet 731). Results An examination in Chart II of the growth obtained with Diets 720 (50 per cent fat) and 731 (fat-free) shows unequivocally that fat spares vitamin B; that is, a high fat diet enables a good deal of growth and lengthy survival to take place when no vitamin B is added to the diet. In both of these diets a high level of protein, 36 per cent casein, was fed; 10 per cent autoclaved yeast was fed in Diet 720 as the source of vitamin G and 800 mg. of autoclaved yeast daily were fed with Diet 731. That lard contributes no vitamin B impurity to the high fat diet is shown by the fact that equal or superior growth is obtained with synthetic as contrasted with commercial lard (Diets 729,720, Chart II). Chart II also shows that the sparing action of fat on vitamin B a In this particular case the results obtained with natural lard were for some unknown reason poorer than usual, whereas the results with the synthetic lard (Diet 729, Chart II) are of about the same order as those obtained on several occasions during the last 3 years with natural lard.

Evans, Lepkovsky, and Murphy 433 18.5 NO VITAMIN 8 160 729 SYNTHETIC LARD LOW PROTEIN CHART II. Composite growth curves of groups of six rats each showing the rble of protein, vitamin G, and fat in the growth of rats on vitamin B-free diets. For the low level of vitamin G, 300 mg. of autoclaved Fleischmann s yeast were fed every other day, and 800 mg. daily were fed for the high level of vitamin G. /80. 722 40 -.----.ZO DAYS. - 800 MbM. AOTOCLAVEO FLOSCHMANNS YEAST OAILY -300 MOM. AUTOCLAVED FLEL(CHPL4NhS YEAST EVERY OTHER DAY -----300 MOM. AUTOCLAVED FLE!SCHMANNb YEIST EVERY OTHER DAY CHART III. Composite growth curves of groups of six rats each on high fat diets with vitamin B requirements fully satisfied as 0.2 cc. of an extract, Concentrate 9-G, from rice polish (80 per cent alcoholic extract) (4), showing the r6le played by the level of protein in the diet when vitamin G is reduced from an adequate to an inadequate level. The curves with the dotted lines represent similar experiments which were not included in this series.

434 Fat Sparing Action and Vitamin B. VI is not so simple a phenomenon as we had formerly supposed. From it we can see that the levels of protein and vitamin G play important rales. We are not dealing, therefore, with a simple fat-vitamin B relationship such as we studied in our previous work (2). There the protein and vitamin G did not enter as factors because both were fortuitously given at high levels (36 per cent casein and 10 per cent autoclaved yeast). In Chart III, one can observe that on complete diets, rats grow equally well on 20 per cent casein (Diet 722) and on 36 per cent casein (Diet 720). Yet in the absence of vitamin B (Chart II) with 800 mg. of autoclaved yeast supplying the vitamin G, the lowering of the casein from 36 per cent to 20 per cent is followed by significant biological consequences. Diet 722 containing 20 per cent casein, gives much poorer results than Diet 720 or 729 with 36 per cent casein. Moreover, when an inadequate amount of vitamin G is also fed (150 mg. of autoclaved yeast daily), the difference in protein levels yields further interesting results. Diet 728 with 36 per cent casein (Chart II) still shows evidence of considerable sparing action, but there is no sparing action whatsoever at the low level of casein (Diet 722) when vitamin G is also low. It seems, therefore, that to enable a rat to survive without vitamin B requires the cooperative action of optimal proportions of protein and vitamin G as well as high fat. Attention should be called to the fact that the low protein diet (Diet 722) contains 24 per cent sucrose, whereas the high protein Diets 720, 728, 729, and 730 do not contain any added carbohydrate. The possible significance of sucrose in the phenomenon under investigation is unknown. It is of interest to note the r81e of vitamin G when both the protein and fat are high. In Chart IV, the synthetic lard Diet 730 was fed to rats without any vitamin B or vitamin G additions. At the point noted, vitamin G was given to three of the six rats as an autoclaved aqueous extract of hog liver equivalent to 3 gm. of fresh liver. The liver extract was free from vitamin B as determined by previous test. The rats responded to the vitamin G addition, growing and surviving until the end of the experiment, whereas their controls died of beriberi. We have here the paradox of vitamin G protecting rats from death with symptoms of beriberi supposedly specific for vitamin B deficiency.

Evans, Lepkovsky, and Murphy 435 In Chart III, the role of the protein level is illustrated when vitamin B is fully adequate, but vitamin G inadequate. The high protein diet containing 36 per cent casein (Diet 728) is little better than the diet containing 20 per cent casein (Diet 722) when 800 mg. daily of autoclaved yeast are fed, but when only 150 mg. of autoclaved yeast are fed daily, Diet 728 is definitely better, and the higher protein level is the only factor to which this superiority can be ascribed. This is in agreement with the observations of Sherman and Derbigny (5). 20-20 DAY.5 CHART IV. Role of vitamin G upon the high fat Diet 730 (synthetic lard) in the absence of vitamin B. The rats were started without vitamins B and G. At the point T vitamin B-free autoclaved liver extract, fed as the source of vitamin G, was given to three rats. The other three rats declined and died with typical symptoms of beriberi. DISCUSSION In their paper on the studies of vitamin G with special reference to the protein intake, Sherman and Derbigny (5) write, And in so far as the pellagra problem is to be regarded as nutritional, one should think not in terms of a choice between protein theory and vitamin theory, but rather of a theory broad enough to take account of the possible participation of more than one nutritional factor. The problem which has engaged our attention for several years, the sparing of vitamin B, is one in which several factors are now known to participate. They are fat, protein, and vitamin G. The fullest effect is possible only when all these factors are,

436 Fat Sparing Action and Vitamin B. VI as high as is possible in the diet. This is accomplished by the total exclusion of carbohydrates. The question may well be raised, what effect is produced by the total exclusion of carbohydrates. As early as 1914 Funk (6) correlated the severity of vitamin B deficiency symptoms with the amount of carbohydrates in the diet. Just what is actually the effect of totally excluding carbohydrates cannot be very well determined from the data at hand. We do know from Chart II, however, that the exclusion of the bulk of carbohydrate from the diet (Diet 722 + 150 mg. of autoclaved yeast daily) does not necessarily in any way improve the vitamin B deficiency. It must be accompanied by other important changes in the quantitative make-up of the diet with respect to the protein, fat, and vitamin G. Data are accumulating which indicate an importance also in the qualitative make-up of the fats. Some have been published (7) and other data bearing this out will be treated elsewhere. The cooperative action of protein, fat, and vitamin G to enable the rat to make considerable growth and to survive for a long time upon vitamin B-free diets is an example of the possible physiological interrelationship of these factors. Closer attention will have to be given the possible relationships of similar physiological factors in nutritional investigations. Another question raised by the data presented is what constitutes the proper level of intake of any given factor. What, for example, is the correct protein level for optimal nutrition? Obviously, the data presented teach us there is no absolute value for protein intake. Thus, with all factors satisfied, as good growth is obtained upon 20 per cent casein as with 36 per cent (Chart III), but in the same chart, there are curves to show that when vitamin G is inadequate, superior growth is obtained with the higher level of protein. A given level of vitamin B, to give another example, may be adequate on high fat diet but no longer adequate on a fat-free diet (2). These instances would seem to justify the statement that the balance of the dietary constituents determines whether a given level of any factor will give optimal performance. SUMMARY 1. For fat to exert its optimal sparing action upon vitamin B, both the protein and vitamin G must be high.

Evans, Lepkovsky, and Murphy 437 2. The participation of these three factors and their interrelationship in sparing vitamin B are discussed. BIBLIOGRAPHY 1. Gregory, E., and Drummond, J. C., 2. Vitaminforsch., 1, 257 (1932). 2. Evans, H. M., and Lepkovsky, S., J. Biol. Chem., 83, 269 (1929). 3. McCollum, E. V., and Simmonds, N., J. Biol. Chem., 33,63 (1918). 4. Evans, H. M., and Lepkovsky, S., J. Nutrition, 3, 353 (1931). 5. Sherman, H. C., and Derbigny, I. A., J. Biol. Chem., 99, 165 (193233). 6. Funk, C., Z. physiol. Chem., 89,378 (1914). 7. Evans, H. M., and Lepkovsky, S., J. Biol. Chem., 96, 179 (1932).

THE SPARING ACTION OF FAT ON VITAMIN B: VI. THE INFLUENCE OF THE LEVELS OF PROTEIN AND VITAMIN G Herbert M. Evans, Samuel Lepkovsky and Elizabeth A. Murphy J. Biol. Chem. 1934, 107:429-437. Access the most updated version of this article at http://www.jbc.org/content/107/2/429.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/107/2/429.citation.full.h tml#ref-list-1