M. Lumb

SELECTIVE INACTIVATION OF VITAMIN B12 IN RATS BY NITROUS OXIDE

Exposure of rats to nitrous oxide rapidly inactivated the cytosol enzyme, methionine synthetase, but the mitochondrial enzyme, methylmalonyl CoA mutase, seemed to be unaffected, although both enzymes require vitamin B12.

Studies on the haemopoietic toxicity of nitrous oxide in man

Summary. Nitrous oxide inactivates vitamin Bi2 and in man can produce a megaloblastic anaemia. Haematological and biochemical changes were studied in nine surgical patients ventilated with 70% N20 for up to 24 h and in three control patients. There was a rise in the numbers of hypersegmented neutrophils in peripheral blood following N2O. Serial bone marrow aspirates showed gross megaloblastic change after 24 h of N20 which had reverted to normoblastic but dyserythropoietic haemopoiesis by 1 week. Giant forms of early myeloid precursors were also seen after 24 h ventilation with N20 but by 1 week abnormalities were evident in more mature cells, metamyelocytes and segmented neutrophils. Megalo‐blastosis was associated with abnormal dU suppression which showed a correction pattern similar to that seen in vitamin Bi2 deficiency. Administration of N20 was also associated with a progressive rise in serum folate and fall in serum methionine levels. No similar patterns were seen in the three control patients.

The substrate for folate polyglutamate biosynthesis in the vitamin B12- inactivated rat

Abstract Rats were maintained for 24 hours in a chamber containing 50% N2O in order to inactivate cobIalamin. Control animals breathed air. Folate analogues [0.01μmol] labelled with either 3H or 14C, were given IP and their hepatic uptake and conversion into folate polyglutamate measured. There was impaired hepatic uptake of folate by N2O-treated animals varying from 21% of that in controls given H4PteGlu to 69% with 10-CHO-H4PteGlu. There was no detectable formation of folate polyglutamate in the N2O-treated animal with H4PteGlu and 5-CH3-H4PteGlu but ‘normal’ polyglutamate synthesis with 5-CHO-H4PteGlu, 10-CHO-H4PteGlu and 5,10-CH-H4PteGlu.

Impaired deoxyuridine utilization in the B12-inactivated rat and its correction by folate analogues

Abstract Rats were kept in an atmosphere of 50% N2O in order to inactivate cob(I)alamin. There was an impaired utilization of deoxyuridine for DNA synthesis by marrow cells from these animals. The defect was not improved by the addition of hydroxocobalamin. Formylated tetrahydrofolates corrected the defect but tetrahydrofolate and 5-methyltetrahydrofolate produced either little or no improvement. Thus formyltetrahydrofolates overcome both the impairment of folate polyglutamate synthesis [11] and the impaired deoxyuridine utilization which follows N2O-induced oxidation of the B12 coenzyme.

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.

Urinary folate loss following inactivation of vitamin B12 by nitrous oxide in rats

Summary. Rats were injected with [2‐14C]H4PteGlu daily for 3 d and thereafter one group left in air and a second group in an atmosphere of nitrous oxide/oxygen (1/1). Nitrous oxide inactivates cobalamin. The N2O‐treated rats excreted large amounts of L. casei‐active folate into the urine. The urinary folate co‐chromatographed with authentic 3H‐labelled 5‐methyltetrahydrofolate. Both groups of animals excreted 14C‐labelled breakdown products in the urine but there was no evidence of increased folate catabolism in the N2O‐treated rats. It was concluded that the folate deficiency that develops in the N2O‐treated rat is due to massive urinary loss of folate. This appears to be secondary to impaired cellular uptake of folate which leads to a raised plasma folate level.

HOW VITAMIN B12 ACTS

The manner in which lack of cobalamin (vitamin BI2) in man leads to megaloblastic anaemia and a neuropathy has led to many hypotheses but little hard fact. At a biochemical level cobalamin is a cofactor in two pathways, i.e. the methylation of homocysteine to yield methionine (methionine synthetase reaction) and the conversion of a product of the metabolism of propionic acid, leucine and valine, namely methylmalonic acid into succinic acid (methylmalonyl CoA-mutase reaction) (Poston & Stadtman, 1975; Babior, 1975). Disturbance of conversion of methylmalonic acid occurs as an inborn error of metabolism with excretion of large amounts of this substance in the urine. These chddren do not show any of the features of cobalamin deficiency and, for this and other reasons, methylmalonic acid is not believed to be significant in producing either megaloblastic anaemia or the characteristic neuropathy (Mahoney & Rosenberg, 1975). The observation that the anaesthetic gas nitrous oxide (N20) produced megaloblastic changes in the marrow by an effect on cobalamin, transformed the situation (Lassen et a l , 1956; Amess et a l , 1978). It had already been shown that transition-metal complexes of which cobalamin was one, activated and cleaved N 2 0 (Banks et a l , 1968). At the same time the cobalamin itself was oxidized. In practice, N 2 0 is a specific inactivator of the enzyme, methionine synthetase, of which both cobalamin and folate are co-factors. Thus ‘biochemical’ pernicious anaemia can be produced in any convenient experimental animal by short-term exposure to N 2 0 . Current research suggests that the sequence of changes that arise from vitamin BIZ deficiency are as follows: (1) The primary defect, which has been recognized for many years, is failure to make methionine from homocysteine. (2) Methionine is needed as a source of formate derived by oxidation of methionine methyl. (3) Formate with tetrahydrofolate forms formyltetrahydrolate and this is converted into folate polyglutamate, the folate coenzyme. Formate, too, is essential for purine synthesis and after reduction, for conversion of deoxyuridine to thymidine. (4) Methionine is also needed to produce S-adenosylmethionine which is a donor of methyl groups. Lack of S-adenosylmethionine may be important in the nervous system. Failure of methiortine synthesis (Fig 1). Impaired activity of the enzyme, methionine synthetase, has been demonstrated in marrow from patients with untreated pernicious anaemia (Taylor et al, 1974; Sauer & Wilmanns, 1977) as well as in the N20-treated experimental animal (Deacon et al, 1978). In the rat the activity of the enzyme falls after 30 min exposure to N20 and is barely detectable after 6 h. There is no recovery as long as exposure to N 2 0

The effect of nitrous oxide-induced inactivation of vitamin B12 on the activity of formyl-methenyl-methylenetetrahydrofolate synthetase, methylene-tetrahydrofolate reductase and form iminotetrahydrofolate transferase

Summary Inhalation of nitrous oxide leads to oxidation of cob(I)alamin and inactivation of methionine synthetase [2] of which cob(I)alamin is a coenzyme. There is cessation of folate polyglutamate synthesis from tetrahydrofolate but this remains intact with formyltetrahydrofolate as substrate [7]. The activity of formyltetrahydrofolate synthetase is increased following exposure to N 2 O while that of methenyltetrahydrofolate cyclohydrolase is depressed The activities of methylenetetrahydrofolate dehydrogenase, methylenetetrahydrofolate reductase and formiminotetrahydrofolate transferase are unchanged. The data suggest a failure of supply of formate. The depression of cyclohydrolase activity further blocks interconversion between methylene groups derived from serine and formate.

The effect of nitrous oxide-induced inactivation of vitamin B12 on serine transhydroxymethylase

Summary The cobalamin cofactor in methionine synthetase undergoes rapid oxidation in rats breathing N 2 O. The activity of methionine synthetase falls to very low levels and there is impaired synthesis of folate polyglutamate and impaired methylation of deoxyuridine. The last two effects are overcome by supplying tetrahydrofolate carrying a formate substituent but not by tetrahydrofolate itself. The serine-glycine reaction is a major source of single carbon units. The enzyme concerned, serine transhydroxymethylase was unimpaired in both marrow and liver of rats exposed to N 2 O and hence single carbon units at the formaldehyde level of oxidation are available to the N 2 O-treated rat. The failure of formaldehyde carbon (methylene) to substitute for formate in the N 2 O-treated rat is probably due to depressed cyclohydrolase activity preventing oxidation of methylene to formate.

The effect of nitrous oxide-induced inactivation of vitamin B12 on thymidylate synthetase activity of rat bone marrow cells

Abstract Exposure to nitrous oxide (N2O) in vivo is accompanied by oxidation of cob[I]alamin to the inactive cob[III]alamin [1] and to loss of methionine synthetase activity [2]. There is a steady increase in thymidylate synthetase activity in marrow collected from rats exposed to N2O and this returns to normal on restoring the animals to an air environment.