Cobalamin Inactivation Research Articles

The radicalversusionic mechanisms of reduced cobalamin inactivation bytert-butyl hydroperoxide and hydrogen peroxide in aqueous solution

It was demonstrated that antioxidants cannot protect reduced cobalamin against its modification by hydrogen peroxide.

Effect of nitrous oxide on nickel deprivation in rats.

Because nickel may have a biological function in a pathway in which vitamin B12 is important, an experiment was performed to determine the effects of nitrous oxide exposure in rats deprived of nickel. Exposure to nitrous oxide (N2O) causes inactivation of cobalamin and a subsequent decrease in the vitamin B12-dependent enzymes methionine synthase and methylmalonyl CoA mutase. Rats were assigned to dietary groups of 12 in a factorially arranged experiment with dietary variables of nickel (0 or 1 microgram/g) and vitamin B12 (0 or 50 ng/g). After 6 wk, one-half of the rats from each dietary group were exposed to 50% N2O/50% O2 for 90 min/d for the last 28 d of the experiment. Vitamin B12, N2O, or their interaction had numerous effects; classical findings included N2O-induced reduction in plasma vitamin B12 and decreases in the vitamin B12-dependent enzymes. Inactivation of vitamin B12 by N2O, however, did not exacerbate signs of nickel deprivation, possibly because the rats were able to metabolically compensate to N2O exposure.

Monitoring Cobalamin Inactivation During Nitrous Oxide Anesthesia by Determination of Homocysteine and Folate in Plasma and Urine

ERMENS, A. A. M.; REFSUM, H.; RUPREHT, J.; SPIJKERS, L. J. M.; GUTTORMSEN, A. B.; LINDEMANS, J.; UELAND, P. M.; ABELS, J. Author Information

Monitoring Cobalamin Inactivation During Nitrous Oxide Anesthesia

Monitoring Cobalamin Inactivation During Nitrous Oxide Anesthesia

Monitoring cobalamin inactivation during nitrous oxide anesthesia by determination of homocysteine and folate in plasma and urine

The effects of nitrous oxide-induced cobalamin inactivation on homocysteine and folate metabolism have been investigated. Plasma levels of cobalamin, folate, homocysteine, and methionine were determined in 40 patients before and after operation under nitrous oxide anesthesia (range of exposure time, 70 to 720 minutes). Twelve patients anesthetized with total intravenous anesthesia served as control subjects (range of exposure time, 115 to 600 minutes). Postoperative plasma levels of folate and homocysteine increased (p less than 0.001) up to 220% and 310%, respectively, in nitrous oxide-exposed patients, whereas plasma levels of methionine decreased (p less than 0.025). Response occurred after 75 minutes of nitrous oxide exposure. The percentage increase of plasma folate and homocysteine correlated significantly with exposure time (p less than 0.025 and p less than 0.0001, respectively). In eight patients receiving nitrous oxide anesthesia plasma homocysteine levels had not returned to preoperative levels within 1 week (p less than 0.01). Urinary excretion of folate and homocysteine increased during and after nitrous oxide exposure (p less than 0.01 and p less than 0.002, respectively) and correlated with exposure time (p less than 0.01 and p less than 0.005, respectively). It can be concluded that disturbance of homocysteine and folate metabolism by nitrous oxide develops with little delay and return to normal levels requires several days. Elevation of plasma homocysteine levels may therefore be used for monitoring nitrous oxide-induced cobalamin inactivation.

Cobalamin inactivation induces formyltetrahydrofolate synthetase

Loss of cobalamin function produces profound changes in the metabolism of formate. There is impaired synthesis of formyltetrahydropteroylglutamate synthetase (CHO-H 4,PteGlu), accumulation of endogenous formate and impaired utilization of [ 14C]formate. There are contradictory reports on the effect of cobalamin inactivation on CHO-H 4PteGlu synthetase. This study confirms a significant increase in synthetase activity following cobalamin inactivation.

Formate metabolism in the cobalamin‐inactivated rat

Endogenous formate levels in blood and liver were assayed in rats both after inactivation of cobalamin (Cbl) by exposure to N2O as well as in air-breathing controls. The uptake of [14C]formate by tetrahydrofolate (H4folate) in bone marrow cells and liver homogenate and the incorporation of [14C]formate into purine, pyrimidine, methionine, serine and choline, was measured. There was a significant accumulation of endogenous formate following Cbl inactivation. There was impaired utilization of [14C]formate for single unit carbon (C1 unit) transfers mediated by folate in Cbl-inactivated tissues, other than for synthesis of adenine. The impairment was not accompanied by any accumulation of labelled methylH4folate indicating that methylfolate trapping played no part in impaired single carbon unit transfer. The effect of Cbl lack was a failure to form formylH4folate so that formate accumulated. The reason for this is not known.

Effect of cobalamin inactivation on folate metabolism of leukemic cells

Exposure to nitrous oxide inactivates the cobalamin coenzyme of methionine synthetase, an essential enzyme in folate metabolism. Hemopoietic cells are especially dependent on the function of cobalamin for the folate-dependent synthesis of thymidylate (dTMP). Inhibition of methionine synthetase may therefore be of potential value in the treatment of hematological malignancies. In the present study we investigated the effect of nitrous oxide induced cobalamin inactivation on folate metabolism of fresh leukemic cells and the human myelomonocytic cell line U937. Cells were exposed to nitrous oxide for 20 h. Subsequently they were subjected to the deoxyuridine suppression test (dU test), which measures the disturbance of folate-dependent dTMP synthesis. In all bone marrow samples, cobalamin inactivation resulted in a 200% increase of the dU test value, implicating a decreased de-novo synthesis of dTMP. Incubation of leukemic cells with methotrexate, 5-fluorouracil or cycloleucine induced similar increases of the dU test values which could be further raised to 400% with the addition of N 20 exposure. Prolonged experiments with U937 cells revealed that the disturbance of folate metabolism aggravated up to 48 h of nitrous oxide exposure. It can be concluded that cobalamin inactivation in human leukemic cells results in disturbed folate-dependent dTMP synthesis. Moreover, effects of several drugs interfering with folate metabolism can be enhanced.

Impaired formylation and uptake of tetrahydrofolate by rat small gut following cobalamin inactivation.

The effect of inactivation of cobalamin by N2O on the intestinal absorption of folate was studied using rat everted gut sacs. Further, in view of uncertainties about the presence of methionine synthetase in gut [1], this enzyme was measured. Everted gut sacs were incubated with [2-14C]tetrahydrofolate, and the subsequent appearance of labelled formyl- and methyl [14C] tetrahydrofolate in everted segments of small intestine of rats was studied. Considerable methionine synthetase activity was present in washed everted gut sacs but not in gut segments in the absence of such treatment. Methionine synthetase activity declined after exposure to N2O, which oxidizes and inactivates cob(I)alamin. Folate uptake by gut sacs was not affected by 24 h exposure of the animals to N2O but fell significantly after 7 days exposure. There was a significant fall in the amount of formyltetrahydrofolate formed after cobalamin inactivation and this was reversed by supplying either methionine, methylthioadenosine or sodium formate. Serine had no effect. The data support the hypothesis that methionine and methylthioadenosine act by supplying single carbon units at the formate level of oxidation.

Enhanced therapeutic effect of methotrexate in experimental rat leukemia after inactivation of cobalamin (vitamin B12) by nitrous oxide.

Exposure to nitrous oxide interferes selectively with the coenzyme function of vitamin B12 and causes inactivation of methionine synthetase, with subsequent impairment of folate metabolism and reduction of cellular proliferation. In a rat leukemia model (BNML) we investigated the combined administration of nitrous oxide, inactivating vitamin B12, and methotrexate (MTX), a folate antagonist inhibiting the enzyme dihydrofolate reductase. Through different mechanisms, both agents decrease the availability of tetrahydrofolate, and subsequently of other reduced folates, with increased impairment of folate-dependent synthesis of thymidylate. Effects on leukemic growth and on hematological values in rats demonstrated enhancement of the therapeutic effect of MTX by exposure to nitrous oxide. With several treatment schedules, the results of combined treatment were seen to be better than additive when compared with the effects of single agents. In particular, pretreatment of leukemic rats with nitrous oxide for 3 days before administration of MTX appeared effective. With higher doses of MTX, concomitant exposure to nitrous oxide even resulted in toxic effects. These findings were in accordance with the results of some metabolic studies performed in leukemic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Cobalamin inactivation decreases purine and methionine synthesis in cultured human lymphoblasts.

Cobalamin (vitamin B12) is required by two enzymes in mammalian cells, specifically methionine synthetase (N5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) and methylmalonyl CoA mutase (EC 5.4.99.2). Cobalamin deficiency in humans leads to megaloblastic anemia which is probably secondary to decreased methionine synthetase activity and to subacute combined degeneration of the spinal cord which appears to be secondary to decreased methylmalonyl CoA mutase activity. A decrease in methionine synthetase activity should cause 5-methyltetra-hydrofolate to accumulate because this folate is a major storage form of intracellular folate and its production from 5, 10-methylene tetrahydrofo-late is essentially irreversible. in vivo. These were the data that originally formulated the methylfolate trap hypothesis to describe cobalamin deficiency. Trapping intracellular folates as 5-methyltetrahydro-folate should decrease intracellular purine synthesis and thus lead to decreased DNA synthesis and ultimately to megaloblastosis. However, not all of the data concerning B12 deficiency are totally explainable by the folate trap and animal studies only partially support this mechanism.

Role of folate dependent transformylases in synthesis of purine in bone marrow of man and in bone marrow and liver of rats.

The activity of the two folate dependent enzymes supplying carbon 2 and carbon 8 of the purine nucleus was assayed in the bone marrow of rats and man, as well as in rat livers. The activity of both enzymes was several fold greater in marrow than in liver. Inactivation of cobalamin by exposure to nitrous oxide did not affect the enzymes in rat marrow cells, although an appreciable effect on hepatic enzymes was found. The depression of hepatic glycinamide ribotide (GAR) transformylase in rats exposed to nitrous oxide was prevented by supplying a formate precursor, methylthioadenosine. There was a considerable rise in the activity of GAR transformylase in human marrow cells from patients with megaloblastic anaemia due to cobalamin deficiency but no change in activity in marrow from patients deficient in folate.

Turnover of the methyl moiety of 5-methyltetrahydropteroylglutamic acid in the cobalamin-inactivated rat

AbstractThe metabolism of the methyl group of 5-methyltetrahydrofolate was studied in rats in which cobalamin had been inactivated by exposure to nitrous oxide and in air-breathing control animals. Methylfolate labeled with [14C] in the methyl group and with [3H] in the pteridine- PABA portion was injected and the disappearance of [14C]H3- relative to [3H]folate was measured in liver. The half-time of the methyl group in the livers of control rats was two hours. There was no turnover of the methyl group for the first 72 hours after cobalamin inactivation. After 72 hours, there was a slow turnover of the methyl group, with a half- time of 43 hours. In control rats, it is assumed that the methyl group was metabolized by transfer to homocysteine to form methionine. In cobalamin-inactivated rats, it was shown that methylfolate was used as the substrate for forming folate polyglutamate, and analogues with 3, 4, and 5 glutamic acid residues were present. It is likely that oxidation of the methyl group by methylene tetrahydrofolate reductase occurs from folate polyglutamate containing six and seven glutamic acid residues, (Brody et al, Biochemistry 21: 276, 1982), since we were unable to demonstrate labeled methyl in longer chain analogues.

Turnover of the methyl moiety of 5-methyltetrahydropteroylglutamic acid in the cobalamin-inactivated rat

The metabolism of the methyl group of 5-methyltetrahydrofolate was studied in rats in which cobalamin had been inactivated by exposure to nitrous oxide and in air-breathing control animals. Methylfolate labeled with [14C] in the methyl group and with [3H] in the pteridine- PABA portion was injected and the disappearance of [14C]H3- relative to [3H]folate was measured in liver. The half-time of the methyl group in the livers of control rats was two hours. There was no turnover of the methyl group for the first 72 hours after cobalamin inactivation. After 72 hours, there was a slow turnover of the methyl group, with a half- time of 43 hours. In control rats, it is assumed that the methyl group was metabolized by transfer to homocysteine to form methionine. In cobalamin-inactivated rats, it was shown that methylfolate was used as the substrate for forming folate polyglutamate, and analogues with 3, 4, and 5 glutamic acid residues were present. It is likely that oxidation of the methyl group by methylene tetrahydrofolate reductase occurs from folate polyglutamate containing six and seven glutamic acid residues, (Brody et al, Biochemistry 21: 276, 1982), since we were unable to demonstrate labeled methyl in longer chain analogues.

Cobalamin inactivation decreases purine and methionine synthesis in cultured lymphoblasts.

The megaloblastic anemia of cobalamin deficiency appears secondary to decreased methionine synthetase activity. Decreased activity of this enzyme should cause 5-methyltetrahydrofolate to accumulate intracellularly, and consequently, decrease purine and DNA synthesis; this is the basis of the "methylfolate trap" hypothesis of cobalamin deficiency. However, only some of the clinical and biochemical manifestations of cobalamin deficiency can be explained by the methylfolate trap. We investigated cobalamin deficiency by treating cultured human lymphoblasts with N2O since N2O inhibits methionine synthetase activity by inactivating cobalamin. We found that 4 h of N2O exposure reduced rates of methionine synthesis by 89%. Rates of purine synthesis were not significantly reduced by N2O when folate and methionine were present at 100 microM in the medium; however, at the physiologic methionine concentration of 10 microM, N2O decreased rates of purine synthesis by 33 and 57% in the presence of 100 microM folate and in the absence of folate, respectively. The dependency of rates of purine synthesis on methionine availability would be expected in cells with restricted methionine synthetic capacity because methionine is the immediate precursor of S-adenosylmethionine, a potent inhibitor of 5-methyltetrahydrofolate synthesis; methionine serves as a source of formate for purine synthesis; and rates of purine synthesis are dependent on the intracellular availability of essential amino acids. We conclude that cobalamin inactivation decreases purine synthesis by both methylfolate trapping and reduction of intracellular methionine synthesis.

Effect of cobalamin inactivation on folate-dependent transformylases involved in purine synthesis in rats.

N2O oxidizes and inactivates cob[I]alamin, and animals exposed in this way serve as models for cobalamin 'deficiency'. Such animals show a fall in activity of glycinamide ribotide transformylase and a rise in that of 5-amino-4-imidazolecarboxamide ribotide transformylase. The fall in glycinamide ribotide transformylase activity was prevented by parenteral 5'-methylthioadenosine derived from methionine. Methylthioadenosine in turn is converted into formate. Activity of glycinamide ribotide transformylase recovers after 7 days despite continued N2O inhalation, and this is probably related to restoration of methionine synthesis by induction of betaine:homocysteine transmethylase.

Tissue S-adenosylmethionine levels in fruit bats (Rousettus aegyptiacus) with nitrous oxide-induced neuropathy.

The effect of cobalamin inactivation by the anaesthetic gas nitrous oxide on the concentration of S-adenosylmethionine (Ado Met) in brain and liver of fruit bats (Rousettus aegyptiacus) was examined. Test animals exposed to N2O-oxygen (50:50, v/v) developed ataxia and paralysis leading to death after an average of 9.8 weeks (n6). Animals receiving pteroylmonoglutamic acid supplements in the diet became ataxic earlier (mean 8.8 weeks) while those receiving methionine supplements survived for significantly longer periods (12.5 weeks, P less than 0.01). Plasma cobalamin levels indicated severe depletion of cobalamin stores in N2O-exposed animals. The mean concentration of Ado Met in the brain of N2O-treated bats was nearly 50% higher than that of untreated controls. Ado Met levels in treated bats receiving pteroylmonoglutamic acid or methionine supplements were respectively 18 and 25% higher than in controls. In contrast, the concentration of Ado Met in the liver of all the N2O-treated groups was slightly lower than in controls. These results suggest that the N2O-induced neuropathy in the fruit bat is not related to a depletion of Ado Met in the nervous system.

Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-asenosylmethionine metabilism in the rat

Inhalation of nitrous oxide oxidises cobalamin and, in turn, inactivates methionine synthetase which forms methionine from homocysteine and which requires cob[I]alamin as a co-factor. This study was planned to determine the effect of virtual cessation of methionine synthesis via a cobalamn-dependeent pathway, on tissue levels of methionine, S-adenosylmethionine and on related enzymes. The level of methionine in liver fell initially after exposure to N 2O but was restored to pre-N 2O levels after 6 days despite continuing N 2O exposure. Brain methionine fell within 12 h of N 2O exposure but the fall was not significant. The restoration of methionine levels is accompanied by an increase in activity of betaine homoysteine methyltransferase in liver but this enzyme was not detected in brain. The activity of methionine synthetase remained very low in both liver and brain as long as N 2O inhalation was continued. There was an initial rise in liver S-adenosyl-methionine levels followed by a steady fall to 40% of its initial level after 11 days of N 2O exposure. However, there was no change in the level of S-adenosylmethionine in brain during this period. The data indicate that either brain meets its requirement by increased methionine uptake from plasma or that there are alternate pathways in brain for methionine synthesis other than those requiring a cobalamin coenzyme.

Increased urinary excretion of formiminoglutamic acid in nitrous-oxide-treated rats and its reduction by methionine.

Inhalation of nitrous oxide oxidises cob(I)alamin and inactivates methionine synthetase of which cobalamin is a co-enzyme. The biochemical changes in the rat following exposure to nitrous oxide resemble in some detail the changes present in patients with untreated pernicious anemia due to deficiency of cobalamin. There is a marked increase in the excretion of formiminoglutamic acid in the urine following exposure to nitrous oxide. A significant decrease is produced, while on N2O, by giving methionine. The explanation for these findings is discussed in the light of recent data on the effects of cobalamin inactivation.

Cobalamin inactivation by nitrous oxide produces severe neurological impairment in fruit bats : Protection by methionine and aggravation by folates

Nitrous oxide, which inactivates cobalamin when administered to fruit bats, results in severe neurological impairment leading to ataxia, paralysis and death. This occurs after about 6 weeks in animals depleted of cobalamin by dietary restriction, and after about 10 weeks in cobalamin replete bats. Supplementation of the diet with pteroylglutamic acid caused acceleration of the neurological impairment - the first unequivocal demonstration of aggravation of the neurological lesion in cobalamin deficiency by pteroylglutamic acid. The administration of formyltetrahydropteroylglutamic acid produced similar aggravation of the neurological lesion. Supplementation of the diet with methionine protected the bats from neurological impairment, but failed to prevent death. Methionine supplementation protected against the exacerbating effect of folate, preventing the development of neurological changes. These findings lend support to the hypothesis that the neurological lesion in cobalamin deficiency may be related to a deficiency in the methyl donor S-adenosylmethionine which follows diminished synthesis of methionine.