I. INFLUENCE OF AVITAMINOSIS ON ASCORBIC ACID CONTENT OF VARIOUS TISSUES AND ENDOCRINES*

I. INFLUENCE OF AVITAMINOSIS ON ASCORBIC ACID CONTENT OF VARIOUS TISSUES AND ENDOCRINES* BY BARNETT SURE, R. M. THEIS, AND R. T. HARRELSONt (From the Department of Agricultural Chemistry, University of Arkansas, Fayetteville) (Received for publication, April 15, 1939) As a continuation of our investigations on the influence of avitaminoses on the oxidationreduction mechanism of the animal organism, we have studied during the past 3 years the ascorbic acid content of various tissues and endocrines in several vitamin deficiency diseases. Although the rat on satisfactory diets synthesizes ascorbic acid, we have found considerable reductions of this vitamin in certain tissues and endocrines in vitamin A, vitamin B1, and riboflavin deficiencies, thus establishing a definite vitamin interrelationship. The results of these studies are summarized in Tables I to IV inclusive. The preparation of tissue extracts and methods of ascorbic acid determination have been recently described elsewhere (I). It should be mentioned here, however, that the micromethod of Farmer and Abt (2) developed for blood was adopted for endocrines. The composition of the vitamin A and vitamin B1deficient diets has been given in a recent issue of this Journal (3). However, in the repeated vitamin A depletion experiments the ration was modified so that 15 per cent Crisco replaced an equivalent amount of dextrin, in order to insure a sufficiency of vitamin E. Riboflavin deficiency was produced according to the technique of Day and coworkers (). 50 per cent of the pathological animals developed cataract. Vitamin Bg deficiency was produced on a * Research paper No. 621, Journal series, University of Arkansas. i Deceased. 25

26 Vitamin Interrelationships. I diet deficient in all the components of the vitamin B complex, supplemented by daily doses of 20 micrograms of thiamine and 20 micrograms of riboflavin. On this ration the pathological animals either showed prolonged maintenance or lost 1 to 37 per cent of the body weight, and seven out of sixteen avitaminotic rats developed acrodynia. When the vitamin Bg experiments were completed, the W factor of Elvehjem and associates (5) was not yet recognized and we were not certain at that time whether the chick antidermatitis or filtrate factor of Lepkovsky and Jukes (6) should be incorporated in the rat s diet. In all the vitamin A, B1, and Bs studies the paired feeding technique was carried out (7). The control animals either received the same amount of food or were restricted to the same plane of nutrition as the pathological litter mates of the same sex. The pathological animals on the riboflavindeficient diets ingested their food intake ad libitum. Hitherto biochemical changes in avitaminoses were studied in animals that were allowed to exhaust their vitamin reserves during varying stages of a single depletion period. Since Dr. Opper of this department found a vascular disease in the rat by multiple depletions of vitamin A, not encountered in single vitamin depletions (8), it was thought that possibly additional losses of vitamin C might occur in vitamin A and B, deficiencies after repeated depletions. By this technique we have kept several dozen groups of vitamin B1deficient animals alive and in a chronic state of avitaminosis (9) for 6 to months, some animals having had as many as eight depletion periods. During the terminal stage of marked paralysis and convulsions micrograms of thiamine werefound suffirient for recovery of all with the exception of one case. The technique used for producing multiple vitamin A depletions was the one recently described by Opper (8). DISCUSSION To conserve space, all detailed data have been summarized and the final results are expressed as per cent change in the pathological animals compared with their litter mate controls. Because of the fluctuations found in the various groups, the per cent of the animal groups showing decrease, increase, or no change are also indicated, in order that the data could be more critically evaluated.

Sure, Theis, and Harrelson 27 Influence of Fasting on Ascorbic Acid Content of Tissues and EndocrinesSince inanition plays a significant role in progressive stages of vitamin B1 deficiency, it was of interest to determine the influence of fasting on the ascorbic acid content of tissues and endocrines. One animal was fasted for days, one for 7 days, two for days, and one for 11 days. The results on each animal that was fasted were compared with those of a litter mate control which received our stock Diet 1 () ad Z&turn. The findings were definite and consistent. Fasting for as long as to 11 days showed no effect on the concentration of ascorbic acid in the heart, liver, kidney, lung, spleen, adrenals, thymus, thyroids, and pituitary. InJluence of Supplementing Stock Diet 1 with Ascorbic Acid on Concentration of This Vitamin in Various Tissues and Endocrines In order to determine whether the addition of large amounts of ascorbic acid to the daily diet of rats on our stock diet () (on which we have reared over 95 per cent of our young for the past 19 years) could influence the vitamin C content of tissues and endocrines, the following study was carried out. Three groups of animals in pairs were given daily mg., 8 mg., and 16 mg. of pure crystalline ascorbic a.cid (Merck), respectively, for 66 days. After this period the rats were sacrificed and the ascorbic acid determined in the lung, liver, kidney, and heart. At the same time litter mates were sacrificed that received the same diet but without ascorbic acid supplementation, and the same tissues were analyzed for vitamin C. The amounts of ascorbic acid furnished were 8 to 32 t,imes the prophylactic daily antiscorbutic dose for the guinea pig, and still in the normal rat on a satisfactory diet such large additions exerted no effect on concentration of ascorbic acid in the tissues. It is quite evident, then, that neither alimentation nor amount of food consumed has any influence on the ascorbic acid content of the rat tissue or endocrines. With such results available, marked changes produced by avitaminotic states become very significant. Vitamin B1 and Vitamin A Deficiencies (Tables I and II) Whereas in vitamin B, deficiency the most pronounced reduction of ascorbic acid takes place in the lung, the changes in that tissue in vitamin A deficiency are insignificant. A reduction of 19 and 23 per cent in the liver and kidney respectively in the majority

Vitamin Interrelationships. I of the groups is also of importance. Repeated vitamin BI depletions showed greater losses of vitamin C in the liver and most notably in the kidney, in the latter organ 50 per cent greater reduction having taken place in ascorbic acid because of multiple depletions. Repeated depletions of vitamin BI also showed a marked reduction (27.8 per cent) in the thymus gland. Un TABLE I Injluence of Vitamin B1 Deficiency on Ascorbic Acid Content of Tissues and Endocrines tissue. for all group in patho Tissues or endocrines $ii 1 zzz 1 ;igi: D~~~~~~~~~~~~ Heart.... Liver.... Kidney.... Lung.... Adrenals.... Pituitary.... Thyroids. Heart.. 1 Liver... 1 Kidney.. 1 Lung.. 1 Thymus.., 1 Adrenals. 1 Pituitary.. 9 Thyroids.., 11 1. Single depletion w. mg. per cent 18 0.1 0.16 1.3 23 0.21 0.25 19.0 19 0.13 0.16 23.0 0.20 0.27 35.0 3 3.19 3.61 13.1 6 1.85 1.55 +.9 9 0.28 0.31.7 Repeated 7 0.18 8 0.17 0.18 3.17 1.7 0.27 depletions oer cent per cent per cent. 5 5.6 65.2 30.. 8.2.5 5.3 9.0 6.7 29. 5.9 16.7 83.3. 22.3 33.3 8 1.3 57.0 21.5 21.5 0.26. 92.8 7.2 0.1 75.0 0.o 0.22 29. 85.7 1.3 0.23 27.8 71. 28.6 3.17 ZkO 2.8 35.7 21.5 1.0 +.8 33.3 55.6 11.1 0.29 7. 5.5 27.3 18.2 fortunately, no thymuses were taken for single vitamin B1 depletions, hence no comparison can be made in the case of this endocrine. Since many of the thyroids and pituitaries were used for histopathological studies, we had only a few of these endocrines for vitamin C determinations. That multiple vitamin A depletions produced increased loss of ascorbic acid in the liver is also apparent. The reduction of vitamin C in all the endocrines is

Sure, Theis, and Harrelson 29 also evident in the animals depleted of vitamin A repeatedly. However, additional data are necessary for the findings on the pituitary and thyroids to be conclusive (Table II). TABLE Influence of Vitamin A DeJiciency on Ascorbic Acid Content of Tissues and Endocrines II Tissues or endocrines No. of B~O PS Average ascorbic acid per gm. fresh tissue for all groupa Change in pathological groups Animal groups showing Decrease Increase,bF;g, Heart... Kidney.... Liver.... Lung.... Cerebrum.... Lens.... Thymus... Adrenals.... Pituitary... Thyroids.... 9 13 18 5 9 Single depletion T7. m7. per cent per een ti per cent 0. 0.12 2 77.7 0.11 0.1 27.3 75.0 0.21 0.22.5 5.6 0.19 0.20 5.3 72.6 0.20 0.22 5.0 8 0.25 0.25 +0 25.0 0.29 0.36 2.1 75.0 3.52 3.68.5 75.0 1.20 1.30 8.2 75.0 0.25 0.23 +.0 5 oer cent 11.1 11.2 25.0 38.1 7.3 22.2 5.2 2 5 25.0 25.0 25.0 25.0 25.0 25.0 Repeated depletions Heart. Kidney. Liver... Lung. Spleen. Eyes... Skeletal muscle. Thymus. Adrenals. Pituitary.. Thyroids. 6 16 16 15 7 5 12 16 7 5 7 7 0.15 0.19 0.20 0.29 0.2 0.25 0.3 0.3 0. 9 6 6 0.26 0.31 3.15 3.6 1.05 1.32 0.26 0.30 f0 5 16.7 33.3 26.6 68.7 18.8 12.5 5.0 81.3 12. 6.3.2 6.6 13. f0 57.1 2.9 +.0 2 5 3 f0 2 19.2 75.0 16.6 8. 12. 93.7 6.3 25.8 57.1 2.9 15.3 8 2 Ribo$avin Deficiency (Table III)The greatest losses of ascorbic acid in tissues occurred in riboflavin deficiency, which was to the extent of 1 to 5 per cent in the lung, kidney, and liver. In the endocrines, the thymus suffered the greatest change, a reduction of 85.7 per cent. The thyroids showed a 20.7 per cent reduction..

250 Vitamin Interrelationships. I The results on the endocrines need some comment. Frequently the thymus in the riboflavindeficient animal was very small, in some cases not over 20 mg., hence greater difficulties were often encountered in obtaining as accurate titrations as with the larger thymuses of the heavier litter mate controls. On the other hand, similar situations arose in the case of the thymus in the animals that were depleted of vitamin B1 both once and repeatedly. We conclude that the tremendous reduction in ascorbic acid content of the thymus gland in riboflavin deficiency compared with that found in vitamin B1 deficiency is significant. A similar criticism may be applied to the titrations of the pituitary and Heart,.. Liver... Kidney. Lung. Thymus... Adrenals. Pituitary. Thyroids... TABLE Injluence of Ribojlavin Dejiciency on Ascorbic Acid Content of Tissues and Endocrines verage ascorbic cid per gm. fresh No. of z Tissues or endocrines grd ps ssue for all group! I Change 31 i n pathostudied. logical Pathc groups logical Control I 9 mg. 7 0.18 0.11 0.17 0.21 3.63 1.19 0.29 III WJ. per cent per cent 7 f0 33.3 0.26. 90.o 0.16 5. 9 0.2 1.2 8 0.39 85.7 9 3.95 8.6 5 1.1 +.1 5 0.35 20.7 80.O I Animal groups showing NO change per cent 22.3.0.0 thyroids (in the various avita nnoses) which frequ ntly weighed only 2 to mg. in the avitaminotic animals. The lack, however, of uniformly great losses of vitamin C in these endocrines in the pathological animals of the various avitaminoses would point against errors inherent in the micromethod of titration. Vitamin Bs DeJiciency (I able IV)Since the results of these studies are expressed as mg. of ascorbic acid per gm. of fresh tissue, it might be argued that the greater weights of the control animals than the pathological, which influenced substantially the weights of the tissues and endocrines, had a determining influence on their ascorbic acid content. That this is not the case is clear

Sure, Theis, and Harrelson 251 from the results obtained in vitamin B, deficiency. In this avitaminosis large differences in body weight, and therefore respective differences in weights of tissue and endocrines, were observed between the pathological and control rats; yet, no noteworthy changes were found in ascorbic acid in any of the tissues or endocrines in this deficiency disease. From the nature of the results reported in this paper it is evident that one avitaminosis, produced on a diet satisfactory in every respect with the exception of one vitamin, leads to exhaustion of the reserves of another vitamin. Since the rat, which is recognized to synthesize vitamin C or ascorbic acid, suffers such tremendous Tissues or endocrines (16 groups in each) Heart... Kidney.... Liver.... Thymus.... Adrenals.... Pituitary.... Thyroids.... TABLE In$uence of Vitamin Be Deficiency on Ascorbic Acid Content of Tissues and Endocrines Average ascorbic acid per gm. fresh tissue for all groups Pathological WI. ml. 5 5 0. 0. 0.18 0.19 0.21 0.19 2.97 2.9 1.29 1.29 0.2 0.23 7 IV Change in pathological gro ps ~ per cent _ f0 f0 5.5 +9.5 +1.0 f0 +.1 / 13eorease Animal groups showing pm cent IlUX%We NO change per cent per cent 31.2 37.6 31.2 37.5 62.5 5 5 3.7 5 6.3 31.2 62.5 6.3 31.2 56.2 12.6 3.7 5 6.3 losses of this vitamin in vitamin B1 and riboflavin deficiencies, and in multiple vitamin A deficiency, the question arises, does the human beriberi victim require greater amounts of ascorbic acid? Is this also true of the numerous cases of mild beriberi in this country among infants, growing children, and adults? Also, are greater needs of vitamin C indicated in human cases of vitamin A deficiency? That human ca,ses of vitamin A deficiency often do not respond to vitamin A therapy, unless supplemented by ascorbic acid or riboflavin, has been recently reported by Kimble and Gordon (11). Findings of this nature should be taken into consideration by practicing physicians and clinicians in their daily experiences with vitamin therapy.

252 Vitamin Interrelationships. I SUMMARY 1. Fasting for as long as to 11 days produces no changes in the concentration of the ascorbic acid content of tissues or endocrines in the albino rat. 2. The ingestion of to 16 mg. of ascorbic acid per day by the rat on a satisfactory diet does not result in an increase of the concentration of this vitamin in tissues or endocrines. 3. In vitamin B, deficiency the most significant changes in ascorbic acid were found in the lung, kidney, and liver, as follows: lung 35.0 per cent, kidney 23.0, liver 19.0. Repeated vitamin B, depletions produced additional heavy losses of vitamin C in the kidney and liver: kidney 75.0 per cent, liver.. Of the endocrines the only significant change was 27.8 per cent reduction in the thymus.. In vitamin A deficiency the only changes in ascorbic acid found were: heart, nine groups, 2 per cent; kidney, four grow, 27.3; and thymus, four groups, 2.1. However, there were too few groups for the kidney and thymus for the results to be considered conclusive. In multiple vitamin depletions, however, sufficient groups (twelve to sixteen) were available for the kidney, liver, thymus, and adrenals to demonstrate positively the following changes: kidney 26.6 per cent,, liver 5.0, thymus 19.2, and adrenals 12.. In seven groups in which the pituitaries and five in which the thyroids were studied, 25.8 and 15.3 per cent were found respectively. 5. In riboflavin deficiency the following losses of vitamin C in endocrines and tissues were found. They represent the greatest reductions of ascorbic acid in single vitamin depletions: liver. per cent, kidney 5., lung 1.2, thymus 85.7, and thyroids 20.7. No changes were observed in the adrenals or pituitary. 6. In vitamin Bg avitaminosis only, no noteworthy changes occurred in ascorbic content of either tissues or endocrines. BIBLIOGRAPHY 1. Sure, B., and Theis, R. M., Endocrinology, 2, 672 (1939). 2. Farmer, C. J., and Abt, A. F., Proc. Sot. Exp. Biol. and Med., 32, 1635 (1935). 3. Sure, B., and DeWitt, J. B., J. Riol. Chem., 126, 287 (1938).

Sure, Theis, and Harrelson 253. Day, P. L., Darbey, W. J., and Livingston, W. C., J. Nutrition, 13, 389 (1938). 5. Elvehjem, C. A., Koehn, C. J., Jr., and Oleson, J. J., J. Biol. Chem., 116, 707 (1936). Frost, D. V., and Elvehjem, C. A., J. Biol. Chem., 121, 255 (1937). 6. Lepkovsky, S., and Jukes, T. H., J. Biol. Chem., 11, 9 (1936). 7. Sure, B., and Smith, M. E., J. Nutrition, 6, 155 (1932). 8. Opper, L., Proc. Sot. Exp. Biol. and Med., 0,9 (1939). 9. Sure, B., Endocrinology, 23, 575 (1938).. Sure, B., J. Biol. Chem., 7, 9 (1927). 11. Kimble, M. S., and Gordon, E. S., Proc. Am. Sot. BioZ. Chem., J. BioZ. Chem., 128, p. lii (1939).