Vitamin Β12 and Folic Acid Effects on Polyamine Metabolism in Rat Liver

90 Bjelakovic G. et a/.: Vitamin B12 and Folic Acid Effects on Polyamine Metabolism in Rat Liver

Pteridines Vol. 17, 2006, pp. 90 - 94

Gordana Bjelakovic, Dusica Pavlovic, Tatjana Jevtovic, Ivana Stojanovic, Dusan Sokolovic, Goran B. Bjelakovic, Jelenka Nikolic, Jelena Basic

Institute of Biochemistry, Department of Hepatogastroenterology, Faculty of Medicine, University of Nis, Serbia

Abstract

Polyamines, spennine, spermidine and putrescinc, small aliphatic nitrous bases, are normal constituents of micro- bial, plant and animal cells, where they fulfill an array of physiological roles. Metabolism of polyamines is associated with growth and differentiation of mammalian cells, spermine related to RNA and spermidine to DNA metabolism. Methyl-cobalamin, the coenzyme of methionine synthetase, catalyses the recycling of homocysteine to methionine using 5-methyltctrahydrofolate. By acceleration of methionine biosynthesis these vitamins may influence spermidine and spermine synthesis. Liver tissue is rich in polyamines and it is the place of vitamin B12 and folic acid deposition. Polyamine oxidase (PAO, EC 1.5.3) and diamino oxidase (DAO, EC 1.4.3.6) participate in the process of degradation and intercon- version of spermine, spermidine and putrescinc. The idea of our work was to examine the effccts of cobalamin and folic acid on the polyamine metabolism. We have examined the amount of spermine, spermidine and putrescine in liver tissue. At the same time we have examined the activities of PAO and DAO, the catabolic enzymes of polyamine metabolism. Our result suggest that the supplementation of experimental animals with vitamin B12 alone or together with folic acid augmentais spermidine and spermine levels in rat liver, at the same time the amount of putrescine does not change. The application of vitamin cobalamin to experimental animals alone increases PAO; the supplementation of experimental animals with vitamin Β12 together with folic acid causes opposite effect - the decrease of PAO activity. DAO activity significantly decreases under the influence of cobalamin and also with cobalamin and folic acid in combination. Our experimental results indicate the importance of cobalamin and folic acid in polyamine metabolism.

Key words: vitamin Β12, folic acid, polyamines. PAO, DAO. rat liver

Introduction to DNA metabolism. Direct binding of polyamines to DNA modulates DNA-protcin interactions. These Polyamines, spermine, spermidine and putrescine, events are important in the molecular mechanisms of are normal constituents of all living cells, prokaryotic, polyamine action on cell proliferation (4-6). Folic acid, eukaiyotic, plant and animal cells, where they fulfill an in the active state as a 5,6,7,8-tetrahydrofolate, and array of physiological roles (1-3). At normal, physio- vitamin B12 in its coenzyme forms, methyl-cobalamin logical pH values, polyamines exist as organic cations. and 5'-deoxyadenosyl cobalamin, participate in a vari- Intracellularly, they are bound electrostatically to ety of enzyme reactions that are intimately related to organic anions, nucleic acids, phospholipids and, may the synthesis of DNA, RNA and proteins (7-10). be, mucopolysacharides. Metabolism of polyamines is Methyl-cobalamin. the coenzyme of methionine syn- associated with growth and differentiation of mam- thetase. catalyses the recycling of homocysteine to malian cells - spermine related to RNA and spermidine methionine using 5-mcthyltetrahydrofolate. By accel-

Corrcspondence to: Gordana Bjelakovic, Institute of biochemistry. Medical faculty. University of Nis, Bul.dr Zorana Djindjica 81, 18000 Nis, Serbia, email: bjelakovicirtjunis.ni.ac.yu

Pteridines/Vol. 17/No. 3 Bjelakovic G. et al.: Vitamin Bl2 and Folic Acid Effects on Polyamine Metabolism in Rat Liver 91

eration of methionine biosynthesis, these vitamins may tissue was cut in small pieces and homogenized in ice- influence spermidine and spermine synthesis (11,12). cold water. The homogenates (10% w/v) were cen- Liver tissue is very rich in polyamines and, at the same trifuged at 1500 χ g for 10 min. at 4°C. The resulting time, liver serves as storage place for vitamin B12 and supernatants were used for investigation of enzyme folic acid (6, 7, 13). Polyamine oxidase (PAO, EC activities. 1.5.3) is a key enzyme in polyamine degradation. PAO PAO (spennine-tetrahydiOchloride was used as sub- catalyzes oxidative eleavage of spennine or spermi- strate) and DAO activities (putrescine-dihydrochloride dine giving corresponding amino aldehyde, NH, and was used as substrate) were measured by the spec- H202, potentially toxic agents, inducing apoptosis of trophotometric method of Bachrach and Reches (19), the mammalian cells (14). The normal substrate for modified by Quash et al. (20). One unit of enzyme PAO is Ν1-acetylspermine and Ν1 -acetylspermidinc activity was defined as an increase in optical density of (15). The enzyme is widely distributed in tissues and 0.100 at 660 nm. Total proteins in liver homogenates cells. PAO is present in rat liver peroxisomes (16) and were measured according to the method of Lowry et cytosol of hcpatocytcs ( 17). The idea of our work was al. (21). The obtained results were statistically ana- to examine the effects of cobalamin and folic acid on lyzed using Student's t-test. the polyamine metabolism in rat liver. We have examined the amounts of spermine, spermidine and putrescine in liver tissue. At the same time we have Results examined the activities of polyamine oxidase (PAO) and diamine oxidase (DAO, FX' 1.4.3.6), the catabolic The supplementation of experimental animals dur- enzymes of polyamines metabolism. ing seven days with B12, alone or together with folic Our result suggest that the supplementation of acid, significantly increases spermine and spermidine experimental animals with vitamin Β12, alone or levels in rat liver tissue. Under the influence of vitamin together with folic acid, causes augmentation of sper- Β12 and folic acid putrescine amount does not change midine and spermine levels in rat liver; at the same (Fig. 1). Our results show higher PAO activity in liver time the amount of putrescinc does not change. The tissue of animals receiving vitamin B12 in comparison application of vitamin cobalamin to experimental ani- with the control group. PAO activity in liver of the sec- mals increases PAO activity, while together with folic ond experimental animal group, which received vita- acid it causes opposite effect - decrease of PAO activ- min B12 and folic acid together, was significantly ity. DAO activity significantly decreases under the lower in comparison with the control group and with influence of cobalamin and both cobalamine and folic the first experimental group (Fig. 2). DAO activity sig- acid. Our experimental results indicate the importance nificantly decreases under the influence of cobalamin; of cobalamin and folic acid in polyamine metabolism.

Spermine Spermidine Materials and Methods Putrescine

- In experiments male albino Wister rats, weighing .„ ™ 180-230g, were used. The animals were divided into 5 eoo - : ·: M three groups: one control and two experimental

intramuscularly during seven days, in a daily dose of - • : : 0.5 mg/animal. The second experimental group Χ1 received vitamin B12 (in the same dose) plus folic acid ; :'; ; I (2.5 mg/animal) during seven days. The last doses of : : : —i v'fi;' I vitamins were applied on the seventh day, one hour Control B12 B12+Folic acid before sacrificing. Control group received 0.9 % NaCl instead of vitamins. The animals were killed by decap- Figure 1. Spermine, spermidine and putrescine levels itation. The liver was removed quickly, the excess in liver tissue of rats treated with B12 and folic acid. blood removed by blotting and rinsing with ice cold Vitamin B12 in a single daily dose of 0.5 mg / animal saline The amount of polyamines was investigated i.m. during seven days. The second experimental with butanol extraction followed by electrophoresis. group received vitamin B12 (in the same dose) plus Separated polyamines were identified by ninhydrine folic acid (2.5 mg/animal) during seven days. Control and quantified by spectrophotometric method (18). group received 0.9 % NaCl instead of vitamins; ***p For estimation of PAO and DAO activities, the liver <0.001 compared to controls)

Pteridines/Vol. 17/No. 3 92 Bjclakovic G. et ed.: Vitamin Bl2 and Folie Acid Effects on Polyaminc Metabolism in Rat Liver

[ I Control I I I Control Γ-Π B12 L'i' Vi B12 B12+Folic acid ! B12+Folic acid 2 0 -

o CL öl 10- iE. 'tz ^ 115 -

0.0 -i μ- - •) ι , ο υ -ι 1 \ 1 1 2 3 4 5 t; Control B12 B12 + Folic acid

Figure 2. Polyaminc oxidase activity in liver tissue of Figure 3. Diamine oxidase activity in liver tissue of rats treated with B12 and folic acid. Vitamin Bi: in a rats treated with B12 and folic acid. Vitamin B12 in a single daily dose of 0.5 nig / animal i.m. during seven single daily dose of 0.5 mg / animal i.m. during seven days. The second experimental group received vitamin days. The second experimental group rcccived vitamin B12 (in the same dose) plus folic acid (2.5 mg/animal) Β12 (in the same dose) plus folic acid (2.5 mg/animal) during seven days. Control group received 0.9 % NaCl during seven days. Control group received 0.9 % NaCl instead of vitamins; *p <0.05 compared to controls) instead of vitamins; **p <0.01, *p <0.05 compared to controls) al so the supplementation of experimental animals with cobalamin and folic acid in combination decreases spermidine and spermine. Rovena Matthews's investi- DAO activity (Fig. 3). The experimental results indi- gation of the precise structure of methionine synthase cate the importance of cobalamin and folic acid in represents that two binding sites exist in the enzyme polyaminc metabolism. molecule - one for folic acid and the other for cobalamine (33. 34). Methionine synthase appears to be equally impaired in folate deficiency, as well as in Discussion cobalamin deficiciency (34). The activity of cobalamin-dependent methionine Aliphatic polyamines, spermine and spermidine, as synthase is also under the influence of the presence of well as their precursor, diamine putrescine. are normal nitric oxide (NO), which is well known inhibitor of cell constituents. At physiological pH values, methionine synthase (35). These literature data explain polyamines exist as organic cations. Polyamines inter- our interest for the investigation of vitamin B12 and act electrostatically and covalently with various folic acid effects on polyamine metabolism. The macromolecules and, as a consequence, play an essen- increased spermine and spermidine biosynthesis in our tial role in the cell metabolism. They are intraeellular- experiment could be discussed as a consequcncc of the ly bound electrostatically to nucleic acids in nucleus presence of high amounts of cobalamin and folic acid. and ribosomes, and to phospholipids present in plasma The obtained results potentiate that intake of high cell membrane (22 -25). Metabolism of polyamines is doses of these vitamins accelerate the synthesis of associated with growth and proliferation of mam- spermidine and spermine probably by increasing malian cells, influencing DNA-protein interactions, methionine synthase activity and raising the amount of which is the basic mechanism of their action on cell methionine, the precursor of spermidine and spermine proliferation (26-30). Vitamin B12, cobalamin, partici- biosynthesis. The metabolism of polyamines is under pates as methyI-cobalamin and 5'-dcoxyadenosyl the influence of numerous cell compounds. The cobalamin in metabolism of mammalian cells (31). biosynthesis of polyamines is highly regulated by the Physiological functions of folic acid are closely linked activities of two key decarboxylases, ornithine decar- to metabolic functions of vitamin Β12. Methyl-cobal- boxylase (ODC) and S-adenosylmethionine decar- amin and tetrahydrofolic acid participate in the synthe- boxylase (AdoMetDC). Both enzymes have key cys- sis of DNA, RNA and proteins (32). As coenzymes of teine residues - Cys360 in ODC and Cys82 in methionine synthase, cobalamin and folic acid partici- AdoMetDC. These residues react readily with NO, pate in the transfer of methyl group to homocysteine, which is therefore a potent inactivator of polyamine forming methionine (32) Methionine, in the form of S- synthesis (36). On the other hand, NO inhibits methio- adenosylniethionine, is required for the biosynthesis of nine synthase by binding to cobalt in cobalamin (37).

Pteridines/Vol. 1 7/No. 3 Bjelakovic G. eral.: Vitamin B12 and Folic Acid Effects on Polyamine Metabolism in Rat Liver 93

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