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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-
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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
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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.
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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
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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
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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).
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