Rosemary Deacon

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.

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.

Alpha-Thalassaemia Trait in Various Racial Groups in the United Kingdom: Characterization of a Variant of Alpha-Thalassaemia in Indians*

SUMMARY. Patients whose red‐cell indices are suggestive of thalassaemia trait, but who have a normal haemoglobin electrophoretic pattern, may be carriers of αthalas‐saemia. A diagnosis of αthalassaemia trait was made in 44 such patients, using the incorporation of [3H]leucine by reticulocytes to measure the relative rates of synthesis of the α and β‐chains of adult haemoglobin. Patients with αthalassaemia trait had a reduced rate of synthesis of the αchains, with a mean α/β specific activity ratio of 0.79 ± SD 0.07. The mean α/β specific activity ratio of 20 control subjects was 1.06 ± SD 0.08. The diagnostic value of the haemoglobin H (Hb H) preparation was assessed in proven αthalassaemia heterozygotes of various races. A high proportion of ‘false negative’results in Indian and Negro heterozygotes indicated that the Hb H preparation is a highly unreliable screening test for use in a multi‐racial population. There was no significant difference in the mean level of Hb A2 in αthalassaemia heterozygotes (2.0 ± SD 0.6) compared with that of the control group (2.1 ± SD 0.5). Comparison of data from patients with α and β‐thalassaemia traits showed that αthalassaemia trait is the milder disorder, in terms of its effects on red‐cell morphology, red‐cell indices and degree of globin chain imbalance. Amongst individual patients with αthalassaemia trait, there was no correlation between the α/β specific activity ratio and the red‐blood‐cell (RBC) count, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH). This suggests that the α/β ratio cannot be used to distinguish between carriers of a mild gene (‘silent carriers’) and carriers of a more severe disease.

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.

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.

The effect of folate analogues on thymidine utilization by human and rat marrow cells and the effect on the deoxyuridine suppression test

The deoxyuridine (dU) suppression test assesses the capacity of marrow cells or activated lymphocytes to convert exogenous dU into thymidine. In addition, the effect of the added cobalamin or folate on the result is used to determine the nature of the deficiency in megaloblastic marrows. This study shows that folates have a considerable effect on the uptake of labelled thymidine by marrows from patients with megaloblastic anaemia in the absence of added dU. Less thymidine was taken up by megaloblastic marrow cells if they were first incubated with 5-formyltetrahydrofolate, 10-formyltetrahydrofolate or tetrahydrofolate. 5-Methyltetrahydrofolate increased thymidine uptake in cobalamin-deficient marrows and reduced thymidine uptake in folate-deficient marrows. These results could be explained if added folates facilitated utilization of endogenous dU. However, the addition of folates or cobalamin did not affect the validity of a dU suppression test with human marrow. More marked changes were present on the addition of folates to rat marrow cells both from control and nitrous oxidetreated animals.