THE ROLE OF VITAMIN E IN REGULATING THE TURNOVER
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THE ROLE OF VITAMIN E IN REGULATING THE TURNOVER RATE OF NUCLEIC ACIDS* BY JAMES S. DINNING (Fmm the Depurtment of Biochemistry, Xchool oj Medicine, University of Arkansas, Little Rock, Arkansas) (Received for publication, August 12, 1954) A relationship of vitamin E to the metabolism of nucleic acids was sug- gested in an earlier report from this laboratory (1). It was shown that vi- Downloaded from www.jbc.org by guest, on December 30, 2009 tamin E deficiency in the rabbit resulted in the excretion of extra amounts of allantoin. This observation is susceptible to two interpretations. Vi- tamin E deficiency could result in a reduced incorporation of nucleotides into tissue nucleic acids, and a compensatory increase in the rate of purine synthesis could then lead to an increased excretion of allantoin. Alter- natively, vitamin E deficiency could result in an increased rate of turnover of tissue nucleic acids and thus lead to an increased excretion of allantoin Data to be presented in the present report show that vitamin E deficiency in the rat results in an increased rate of incorporation of formate into liver and muscle nucleic acids and suggest that a metabolic r61e of vitamin E is to regulate the turnover rate of nucleic acids. EXPBHIMENTAL Weanling Sprague-Dawley rats of both sexes were given a purified diet consisting of casein 18.6 gm., sucrose 67.4 gm., lard 8 gm., cod liver oil 2 gm., salt mix (2) 4 gm., inositolO.1 gm., choline chloride 0.1 gm., thiamine chloride 0.5 my., riboflavin 0.8 mg., pyridoxine hydrochloride 1 mg., cal- cium pantothenate 2 mg., nicotinic acid 2 mg., 2-methyl-1,4-naphthoqui- none 0.44 mg., and biotin 2.4 y. One group of rats received this diet with- out supplement, and a second group received the basal diet plus oral supplements of oc-tocopherol acetate. The init.ial dose of tocopherol was 4 mg. per rat per week. The dose was increased as the experiment pro- gressed to a final level of 16 mg. per rat per week. The a-tocopherol acetate was administered by dropper from a corn oil solution. After approximately 5 months of feeding, rats were taken from both groups and placed in metabolism cages for the collection of individual 24 hour urine samples. These samples were analyzed for creatine and crea- tinine (3) and allantoin (4). Animals were then injected with 0.2 ml. per * Research paper No. 1004, Journal Series, University of Arkansas. This invest- gation was supported by research grant No. G647(C6), National Institutes of Health, United States Public Health Service. 735 736 VITAMIN E AND NUCLEIC ACIDS 100 gm. of body weight of a solution of sodium C14-formate which contained 1 me. per 25 ml. The specific activity of the formate was 1 mc. per mmole. 4 hours after the injections the rats were killed and samples of liver, small intestines, and skeletal muscle were taken for fractionation by the method of Schneider (5). The nucleic acid extract was freed of trichloroacetic acid (TCA) by heating. An aliquot was evaporated on a planchet for counting, and another aliquot was wet ashed for phosphorus determina- tion. The tissue residue remaining after extraction of acid-soluble ma- terial, lipides, and nucleic acids was considered to be protein. This ma- terial was dried and counted, and corrections were made for self-absorption. All samples were counted with an end window Geiger tube with a window Downloaded from www.jbc.org by guest, on December 30, 2009 thickness of 2 mg. per sq. cm. TABLE I Injhence of Vita.min E Deficiency on Body Weights and on Creatinine, Creatine, and Allantoin Excretion of Rats Mg. excreted per!:: body weight per Animals (10 per group) Average body weight Creatinine Creatine Allantoin cm. Control. . 378 29.2 4.7 110 Vitamin E-deficient.. 336 28.8 9.5 174 In other experiments, livers from five control and five deficient rats were pooled, and the nucleic acids were extracted with hot 10 per cent NaCl after preliminary removal of acid-soluble and lipide material. The nu- cleic acids were precipitated with alcohol, redissolved in alkali, and then fractionated by the alkaline digestion method (6) into ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) fractions. Both fractions were analyzed for pentose, deoxypentose, and phosphorus. The pentose de- terminations indicated that the DNA fractions were uncontaminated with RNA, but that the RNA fraction did contain small amounts of DNA. The fractions were assayed for radioactivity, and appropriate corrections were made for the contamination of the RNA with DNA. Finally, the RNA fractions were hydrolyzed with 1 N HzS04, and the liberated purines were isolated as the copper salts (7). Free purines were regenerated with H2S and dissolved in weak HCl. An aliquot was evaporated for counting, and a separate aliquot was taken for optical density determinations at 260 rnp. The results are reported as counts per minute per micromole of pu- rine. The concentration of purine was calculated from the optical density determinations. J. 6. DINNING 737 Results The data presented in Table I show that vitamin E deprivation had no marked effect on the growth of the rats. The rats deficient in vitamin E did excrete more creatine and allantoin than the control rats. It is to be emphasized that the rats receiving the vitamin E-deficient diet exhibited TABLE II Incorporation of C14-Formate into Tissue Proteins by Control and Vitamin E- Dejicient Rats The results are in countsper minute per mg. Downloaded from www.jbc.org by guest, on December 30, 2009 Animals (5 per group) Small intestine Liver Skeletal muscle Control......................... 28.3 15.3 2.6 Vitamin E-deficient. 25.5 17.3 3.0 TABLE III Concentration of Nucleic Acid Phosphorus in Tissues from Control and Vitamin E-Deficient Rats The results are in mg. of P per 100 gm. Animals (5 per group) Small intestine Liver Skeletal muscle Control.................... 67.6 76.0 16.0 Vitamin E-deficient. 54.4 70.4 16.6 TABLE IV Incorporation of C14-Formate into Nucleic Acids by Control and Vitamin E- Dejicient Rats The results are in counts per minute per micromole of P. Animals (5 per group) Small intestine Liver Skeletal muscle Control......................... 150.5 14.6 10.8 Vitamin E-deficient . . 140.5 33.8 16.5 no gross signs of the deficiency. The data in Table II demonstrate that vitamin E deprivation did not affect> the incorporation of formate into protein of the three tissues studied. The concentration of nucleic acid phosphorus of small intestine, liver, and skeletal muscle was not signifi- cantly affected by deprivation of vitamin E, as shown in Table III. The data recorded in Table IV were obtained by the Schneider frac- tionation procedure (5). Vitamin E defi ciency did not affect the incor- poration of formate into the nucleic acids of the small intestines. The 738 VITAMIN E AND NUCLEIC ACIDS incorporation of formate into the nucleic acids of liver and skeletal muscle was considerably increased in the vitamin E-deficient rats, and was most marked in the liver. The data in Table V show that vitamin E deficiency resulted in an increased incorporation of formate into both RNA and DNA of liver tissue. The results of experiments in which copper purines were isolated are presented in Table VI. RNA purines isolated from livers of vitamin E- deficient rats exhibited considerably higher specific activity than those from control rats. TABLE V Downloaded from www.jbc.org by guest, on December 30, 2009 Incorporation of C14-Formate into Liver Ribonucleic Acid and Deoxyribonucleic Acid by Control and Vitamin E-DeJicient Rats The results are in counts per minute per micromole of P. Animals RNA DNA Control................................. 13.5 8.5 Vitamin E-deficient.. 36.2 12.8 TABLE VI Incorporation of C14-Formate into RNA Purines by Control and Vitamin E-Dejkient Rats Animals Control...................... 28.3 Vitamin E-deficient, 82.0 DISCUSSION The rate of incorporation of formate into nucleic acids as observed in these experiments should be directly related to turnover rates. There was no marked difference in concentration of tissue nucleic acids between control and vitamin E-deficient rats. Also the fact that the specific ac- tivities of protein of all three tissues studied and of nucleic acids of the small intestines were not affected by vitamin E deficiency suggests that there was no marked change in the size of the formate pool. Finally, a short time interval between formate injection and sacrifice of the animals was chosen so that the activities of the tissue nucleic acids should reflect rates of incorporation. Two considerations are important in appraising these results. The vi- tamin E-deficient rats exhibited no gross deficiency signs, and the tissue most drastically affected in terms of nucleic acid turnover rates was liver, J. S. DINNING 739 a tissue which is not considered to be structurally affected by vitamin E deficiency. It is believed that the regulation of the turnover rates of nucleic acids is a primary metabolic function of vitamin E. The precise enzymatic reaction involved remains to be elucidated. SUMMARY Vitamin E deficiency in the rat results in an increased incorporation of formate into the nucleic acids of liver and of skeletal muscle. This is considered to reflect an increased turnover rate of nucleic acids in these tissues as a result of vitamin E deficiency. It is suggested that the regu- lation of turnover rates of nucleic acids is a primary metabolic function of Downloaded from www.jbc.org by guest, on December 30, 2009 vitamin E. BIBLIOGRAPHY 1. Young, J. M., and Dinning, J. S., J. Biol. Chem., 193, 743 (1951). 2. Hubbell, R. B., Mendel, L. B., and Wakeman, A. J., J. Nub-., 14, 273 (1937). 3. Folin, O., J. Biol. Chem., 17, 469 (1914). 4. Young, E. G., and Conway, C. F., J. Biol. Chem., 143, 839 (1942). 5. Schneider, W. C., J. Biol. Chem., 161, 293 (1945). 6. Schmidt, G., and Thannhauser, S. J., J. Biol. Chem., 161, 83 (1945). 7. Hitchings, G. H., and Fiske, C. H., J. Biol. Chem., 140, 491 (1941).