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Liver acid-soluble nucleotides in orotic acid-fed rats
826
SHORT COMMUNICATIONS SC 7052
Liver ocid-soluble nucleotides in orotic acid-fed rats
The occurrence of fatty liver on feeding high doses (I % of diet) of orotic acid to weanling rats was shown first by STANDERFER AND I-{ANDLER1. This effect is not counteracted by usual lipotropic factors such as folic acid, vitamin Bz2, methionine and choline. According to HANDSCHUMACHERet al. 2 only adenine supplementation restored the liver lipid concentration to normal. These authors suggest that orotic acid fatty liver is caused by alterations in nucleotide metabolism. In fact, this type of fatty infiltration is accompanied by some changes in pyrimidine metabolism, notably reductions in the phosphorylation of uridine. Additional studies 3 indicate that particle-free preparations from the livers of orotic acid-fed animals have one-third the capacity to convert uridylic acid to cytosine derivatives. RAJALAKSHMI et al. 4 also reported a decrease of the RNA content per unit weight of defatted liver. Since our previous studies have shown that orotic acid at low dietary level (o.oi %) is able to increase the nucleic acids and free nucleotide content 5, notably the cytidine coenzymes6, while a slight reduction in total lipids is present in the liver of vitamin Bz2-deficient chicks, it seemed opportune to study the relationships between nucleotide metabolism and lipid infiltration in the liver of rats weaned on a purified diet containing different doses of orotic acid. In our preliminary trials, orotic acid at low dietary level (from o.ooi--o.I % of diet) was found to be ineffective in producing fatty liver in rats or a significant reduction in liver phospholipids s. In the present study we have examined the effects of dietary orotic acid on liver acid-soluble nucleotides. Male albino rats of the Wistar strain weighing 45-50 g were divided into 4 groups of IO animals each. Rats in Group I received the basal diet composed of vitamin free casein, corn oil, sucrose and mineral and vitamin supplements 1. The other 3 groups were kept on the same diet supplemented with o.ooi, o.I and z % orotic acid, respectively. The animals were killed by decapitation after 4 weeks and their livers were used for the determination of individual nucleotides by chromatography on ion-exchange resin 9. Results are presented in Table I. It is apparent from Table I that the presence of different doses of orotic acid in the diet results in significant changes in the content of nucleotides. In rats fed with o.ooi % orotic acid, the cytidilic and guanylic nucleotides showed a remarkable increase. In those rats given o.I % orotic acid supplement, a diminution of purine nucleotides was observed, which was accentuated upon 1 % supplementation. In this last group, however, an increase in pyrimidine nucleotides was noted, but this increase is totally due to the urydilic nucleotides while the cytidilic nucleotides decrease. The data concerning the fall in the cytidilic nucleotide level in the group with 1 % orotic acid are pratically in accord with those of HANDSCHUMACHERet al. 3, who noted a decreasing conversion of uridine 5-phosphate in cytidine derivatives in vitro under the same experimental conditions. The diminution of the phospholipid content and in part the contemporary increase of neutral fats observeds could be due to these metabolic alterations. In fact, in the groups with o.ooi and o.I % orotic Biochim, Biophys. Acta, 61 (z962) 826-827
827
SHORT COMMUNICATIONS TABLE I E F F E C T OF OROTIC ACID IN VARIOUS DOSES ON R A T - L I V E R A C I D - S O L U B L E N U C L E O T I D E S i~moles o] liver-/ree nucleotides[xoog Compounds
Ur vdilic nucleotides Cytidilic nucleotides Total pyrimidine nucleotides Adenylic nucleotides Guanylic nucleotides Inosinic nucleotides Total purine nucleotides
wet wt.
Basal diet
Basal diet + o.oor % orotic acid
Basal diet + o.z % orotic acid
I96" 60 256 311 80 62 453
185 lO 5 209 323 lO 4 66 493
169 55 224 278 67 58 403
Basal diet + r % orotic acid
358 41 399 165 89 35 289
" The d a t a represent the average of 2 experiments involving 4 animals each.
acid where the content of cytidine nucleotides were not diminished the content of phospholipids was normal and f a t t y livers were not noted. Concerning the adenylic nucleotides, the observed fall in animals fed 1 % orotic acid could be connected with a diminished conversion of adenine to adenine nucleotide as observed by HANDSCHUMACHERet al. 2 in the lO6 o0o × g supernatant solutions of livers from rats fed 1 % orotic acid or with a drain on the available adenine caused by an increase in the pyrimidine pooP. This relationship which exists between the fall in adenine nucleotides and f a t t y liver is not completely clear. However, a connection exists: this is demonstrated by the fact that in groups on 0.o0i-0.I % orotic acid in which the adenylic content is normal, alteration of the hepatic lipid level was not observed. On the other hand, it was noted that these nucleotides are themselves involved in lipid metabolism. In conclusion, from the analysis of the above data, it is clear that the administration of orotic acid in various amounts can determine different effects on the free nucleotide content and therefore on the processes that they regulate.
Istituto di Chimica Biologica, University o/ Bologna, Bologna (Italy)
M. MARCHETTI
P. PUDDU C. M. CALDARERA
1 S. ~B. STANDERFER AND P. H A N D L E R , Proc. Soc. Exptl. Biol. Med., 90 (1955) 270. 2 ~R. E. HANDSCHUMACHER, "~V. A. CREASEY, J. J. JAFFE, C. A. PASTERNAK AND L. HANKIN, Proa. Natl. Acad. Sci. U. S., 46 (196o) 178. 3 ]~. E. HANDSCHUMACHER, W. A. CREASEY AND L. HANKIN, Federation Proc., 19 (196o) 31o. 4 S. RAJALAKSHMI, D. S. R. SARMA AND P. S. SARMA, Biochem. J., 80 (1961) 375. 5 M. YiARCHETTI, C. M. CALDARERA AND G. MORUZZI, Giorn. biochim., 9 (x96o) 378. 6 M. •ARCHETTI, C. M. CALDARERA AND G. MORUZZI, Biochim. Biophys. Acta, 55 (1962) 218. 7 M. MARCHETTI, P. PUDDU AND C. M. CALDARERA, Biochim. Biophys. Acta, 59 (1962) 475. 8 M. MARCHETTI, P. PUDDU AND C. M. CALDARERA, Giorn. Biochim., I I (1962) 309 . 9 ]R. B. HURLBERT, H. SCHMITZ, A. F. BRUMM AND V. R. POTTER, J . Biol. Chem., 209 (1954) 23.
Received August 9th, 1062 Biochim. Biophys. Acta, 61 (1962) 826-827
SHORT COMMUNICATIONS SC 7052
Liver ocid-soluble nucleotides in orotic acid-fed rats
The occurrence of fatty liver on feeding high doses (I % of diet) of orotic acid to weanling rats was shown first by STANDERFER AND I-{ANDLER1. This effect is not counteracted by usual lipotropic factors such as folic acid, vitamin Bz2, methionine and choline. According to HANDSCHUMACHERet al. 2 only adenine supplementation restored the liver lipid concentration to normal. These authors suggest that orotic acid fatty liver is caused by alterations in nucleotide metabolism. In fact, this type of fatty infiltration is accompanied by some changes in pyrimidine metabolism, notably reductions in the phosphorylation of uridine. Additional studies 3 indicate that particle-free preparations from the livers of orotic acid-fed animals have one-third the capacity to convert uridylic acid to cytosine derivatives. RAJALAKSHMI et al. 4 also reported a decrease of the RNA content per unit weight of defatted liver. Since our previous studies have shown that orotic acid at low dietary level (o.oi %) is able to increase the nucleic acids and free nucleotide content 5, notably the cytidine coenzymes6, while a slight reduction in total lipids is present in the liver of vitamin Bz2-deficient chicks, it seemed opportune to study the relationships between nucleotide metabolism and lipid infiltration in the liver of rats weaned on a purified diet containing different doses of orotic acid. In our preliminary trials, orotic acid at low dietary level (from o.ooi--o.I % of diet) was found to be ineffective in producing fatty liver in rats or a significant reduction in liver phospholipids s. In the present study we have examined the effects of dietary orotic acid on liver acid-soluble nucleotides. Male albino rats of the Wistar strain weighing 45-50 g were divided into 4 groups of IO animals each. Rats in Group I received the basal diet composed of vitamin free casein, corn oil, sucrose and mineral and vitamin supplements 1. The other 3 groups were kept on the same diet supplemented with o.ooi, o.I and z % orotic acid, respectively. The animals were killed by decapitation after 4 weeks and their livers were used for the determination of individual nucleotides by chromatography on ion-exchange resin 9. Results are presented in Table I. It is apparent from Table I that the presence of different doses of orotic acid in the diet results in significant changes in the content of nucleotides. In rats fed with o.ooi % orotic acid, the cytidilic and guanylic nucleotides showed a remarkable increase. In those rats given o.I % orotic acid supplement, a diminution of purine nucleotides was observed, which was accentuated upon 1 % supplementation. In this last group, however, an increase in pyrimidine nucleotides was noted, but this increase is totally due to the urydilic nucleotides while the cytidilic nucleotides decrease. The data concerning the fall in the cytidilic nucleotide level in the group with 1 % orotic acid are pratically in accord with those of HANDSCHUMACHERet al. 3, who noted a decreasing conversion of uridine 5-phosphate in cytidine derivatives in vitro under the same experimental conditions. The diminution of the phospholipid content and in part the contemporary increase of neutral fats observeds could be due to these metabolic alterations. In fact, in the groups with o.ooi and o.I % orotic Biochim, Biophys. Acta, 61 (z962) 826-827
827
SHORT COMMUNICATIONS TABLE I E F F E C T OF OROTIC ACID IN VARIOUS DOSES ON R A T - L I V E R A C I D - S O L U B L E N U C L E O T I D E S i~moles o] liver-/ree nucleotides[xoog Compounds
Ur vdilic nucleotides Cytidilic nucleotides Total pyrimidine nucleotides Adenylic nucleotides Guanylic nucleotides Inosinic nucleotides Total purine nucleotides
wet wt.
Basal diet
Basal diet + o.oor % orotic acid
Basal diet + o.z % orotic acid
I96" 60 256 311 80 62 453
185 lO 5 209 323 lO 4 66 493
169 55 224 278 67 58 403
Basal diet + r % orotic acid
358 41 399 165 89 35 289
" The d a t a represent the average of 2 experiments involving 4 animals each.
acid where the content of cytidine nucleotides were not diminished the content of phospholipids was normal and f a t t y livers were not noted. Concerning the adenylic nucleotides, the observed fall in animals fed 1 % orotic acid could be connected with a diminished conversion of adenine to adenine nucleotide as observed by HANDSCHUMACHERet al. 2 in the lO6 o0o × g supernatant solutions of livers from rats fed 1 % orotic acid or with a drain on the available adenine caused by an increase in the pyrimidine pooP. This relationship which exists between the fall in adenine nucleotides and f a t t y liver is not completely clear. However, a connection exists: this is demonstrated by the fact that in groups on 0.o0i-0.I % orotic acid in which the adenylic content is normal, alteration of the hepatic lipid level was not observed. On the other hand, it was noted that these nucleotides are themselves involved in lipid metabolism. In conclusion, from the analysis of the above data, it is clear that the administration of orotic acid in various amounts can determine different effects on the free nucleotide content and therefore on the processes that they regulate.
Istituto di Chimica Biologica, University o/ Bologna, Bologna (Italy)
M. MARCHETTI
P. PUDDU C. M. CALDARERA
1 S. ~B. STANDERFER AND P. H A N D L E R , Proc. Soc. Exptl. Biol. Med., 90 (1955) 270. 2 ~R. E. HANDSCHUMACHER, "~V. A. CREASEY, J. J. JAFFE, C. A. PASTERNAK AND L. HANKIN, Proa. Natl. Acad. Sci. U. S., 46 (196o) 178. 3 ]~. E. HANDSCHUMACHER, W. A. CREASEY AND L. HANKIN, Federation Proc., 19 (196o) 31o. 4 S. RAJALAKSHMI, D. S. R. SARMA AND P. S. SARMA, Biochem. J., 80 (1961) 375. 5 M. YiARCHETTI, C. M. CALDARERA AND G. MORUZZI, Giorn. biochim., 9 (x96o) 378. 6 M. •ARCHETTI, C. M. CALDARERA AND G. MORUZZI, Biochim. Biophys. Acta, 55 (1962) 218. 7 M. MARCHETTI, P. PUDDU AND C. M. CALDARERA, Biochim. Biophys. Acta, 59 (1962) 475. 8 M. MARCHETTI, P. PUDDU AND C. M. CALDARERA, Giorn. Biochim., I I (1962) 309 . 9 ]R. B. HURLBERT, H. SCHMITZ, A. F. BRUMM AND V. R. POTTER, J . Biol. Chem., 209 (1954) 23.
Received August 9th, 1062 Biochim. Biophys. Acta, 61 (1962) 826-827