Janet Perry

Cobalamin-Folate Interrelations

Cobalamin deficiency leads to impaired folate function as demonstrated by markedly impaired single-carbon unit transfer into purine, thymidine and methionine. This occurs in the total absence of methylH4folate trapping. In cobalamin deficiency there is impaired synthesis of formylH4folate and raised levels of endogenous formate in blood and liver. FormylH4folate and methionine reverse the effects of cobalamin deficiency. Methionine provides formate via its metabolism to methylthioribose. Recently it has been suggested that the neuropathy of cobalamin deficiency is due to impaired methylation but this was not confirmed. It is likely that defects demonstrated in marrow and liver are also the explanation for the effects of cobalamin deficiency in the CNS.

Methylmalonic Acid Excretion Studies

Summary. Oral valine and isoleucine were equally effective in producing an increased urinary excretion of methylmalonic acid (MMA) in vitamin B12 deficiency but oral MMA itself, methionine and leucine were unsatisfactory.

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.

Abnormal Folate Polyglutamate Ratios in Untreated Pernicious Anaemia Corrected by Therapy

The relative amounts of folate compounds having 1–3‐, 4–, 5– and 6–glutamic acid residues in the molecule were measured in red blood cells. The results were expressed in relation to methyltetrahydrofolate pentaglutamate (5‐glutamic acid residues) which is the analogue present in highest concentration. When the concentration of pentaglutamate is given a value of 100 the relative concentration of folates with 1–3‐, 4‐, 5‐ and 6‐glutamic acid residues in control subjects are 48, 48, 100 and 58 respectively.

Methylthioadenosine serves as a single carbon source to the folate co-enzyme pool in rat bone marrow cells

[ribose-U-14C]Methylthioadenosine (MTA) was prepared by incubating methionine with [14C-U]ATP in the presence of methionine adenosyltransferase and the resulting S-adenosylmethionine was heated to release MTA. Labelled [14C]MTA, when incubated with rat bone marrow cells, yielded [14C]formate which was used in the synthesis of adenine and guanine. Unlike 14C from sodium, formate, serine and glycine, there was no decline in 14C utilization from MTA with bone marrow cells from rats in which cobalamin had been inactivated by exposure to nitrous oxide. It was concluded that methionine via MTA is a significant contributor of single-carbon units at the formate level of oxidation and that this pathway is maintained in cobalamin deficiency.

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‐H4,PteGlu), accumulation of endogenous formate and impaired utilization of [14C]formate. There are contradictory reports on the effect of cobalamin inactivation on CHO‐H4PteGlu synthetase. This study confirms a significant increase in synthetase activity following cobalamin inactivation.