Folate and cobalamin.
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Anemia, Megaloblastic↗
Biomedical subjects
I Chanarin
Publications and source records attributed to I Chanarin.
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.
5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
Significance of lymphocytosis in adults.
11/19 adults with a persistent increase in the total lymphocyte count, but with neither lymphodenopathy, hepatomegaly, nor splenomegaly, proved to have a monoclonal increase in B lymphocytes indicative of chronic lymphatic leukaemia (CLL). All remaining 8 patients had increased numbers of T-lymphocytes, more often of the helper than of the suppressor subsets. All the patients with a lymphocyte count in excess of 10 000/microliter, but only 5/13 patients with a lymphocyte count below 10 000/microliter had CLL. In adults a sustained lymphocytosis is thus not an adequate basis for the diagnosis of early CLL, which requires the demonstration of a monoclonal population. The T-lymphocytosis is likely to be reactive rather than neoplastic in nature.
Adult↗
Conversion of endogenous cobalamins into microbiologically-inactive cobalamin analogues in rats by exposure to nitrous oxide.
Rats were exposed to a mixture of oxygen and nitrous oxide (1/1) for up to 18 d. There was a marked fall in both the serum and liver cobalamin levels. There was a significant rise in the concentration of microbiologically-inactive cobalamins in serum 24 h after exposure to N2O. The proportion of microbiologically-inactive cobalamin in liver increased throughout the period of exposure. We were unable to show a similar effect in sera from patients inhaling N2O for 24 h, nor by exposing rat plasma to N2O for 24 h. It was concluded that microbiologically-inactive cobalamins are produced in vivo from endogenous cobalamins and that one form appears to be a product of cobalamin oxidation.
Animals↗
The mechanism of the anaemia in rheumatoid arthritis: effects of bone marrow adherent cells and of serum on in-vitro erythropoiesis.
Clonal assays for erythroid progenitors (BFU-e and CFU-e) were used to study 20 patients with rheumatoid arthritis, 15 of whom had anaemia of chronic disease and five of whom were haematologically normal. The numbers of bone marrow BFU-e and CFU-e in the anaemic patients did not differ significantly from those in normal controls. Macrophages were removed from the bone marrow by a combination of adherence and buoyant density centrifugation over a sucrose gradient and the resulting fractions were cultured alone or together with autologous adherent cells in BFU-e assays. Co-culture with adherent cells significantly increased colony growth in both the controls and in seven of eight anaemic patients studied. Serum from 14 anaemic patients and from five non-anaemic patients was added to cultures of bone marrow or to control peripheral blood 'null' cells. Anaemic serum uniformly either inhibited or failed to stimulate BFU-e growth under these conditions. Serum from non-anaemic patients and from 10 healthy controls stimulated BFU-e growth from 'null' cells to an equal degree.
Adult↗
Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-adenosylmethionine metabolism in the rat.
Inhalation of nitrous 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 cobalamin-dependent pathway, on tissue levels of methionine, S-adenosylmethionine and on related enzymes. The level of methionine in liver fell initially after exposure to N2O but was restored to pre-N2O levels after 6 days despite continuing N2O exposure. Brain methionine fell within 12 h of N2O exposure but the fall was not significant. The restoration of methionine levels is accompanied by an increase in activity of betaine homocysteine 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 N2O inhalation was continued. There was an initial rise in liver S-adenosylmethionine levels followed by a steady fall to 40% of its initial level after 11 days of N2O 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.
5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
Effect of nitrous oxide-induced inactivation of vitamin B12 on glycinamide ribonucleotide transformylase and 5-amino-4-imidazole carboxamide transformylase.
Exposure to nitrous oxide (N2O) in vivo is accompanied by oxidation of cob[I]-alamin to the inactive cob[III]alamin [1]. There is loss of methionine synthetase activity [2] and evidence of depressed supply of single carbon units at the formate level of oxidation [3,4,5]. We measured the effect of inactivation of B12 on the folate-dependent transformylases concerned in purine synthesis. After 24 h exposure to N2O there was a significant fall in glycinamide ribonucleotide transformylase (EC 2.1.2.2) and a significant increase in 5-amino-4-imidazole carboxamide transformylase (EC 2.1.2.3).
Acyltransferases↗
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.
Animals↗
Effects of chronic exposure to nitrous oxide on methionine synthase activity.
Hepatic methionine synthase activity has been determined in rats continuously exposed (24 h day-1 and 7 day week-1) to concentrations of nitrous oxide ranging from 500 to 50 000 p.p.m. for periods ranging from 24 h to 28 days. The inactivation of this enzyme was dependent upon both time and concentration of nitrous oxide exposure, but there was no statistical evidence that exposure continuing beyond 48 h increased the effect. However, the dose-response curve for 24 h was significantly different from the curve for pooled data from exposures lasting 2-28 days. These latter data indicated no significant effect with 450 p.p.m. and an ED50 of 5400 p.p.m. Significant inhibition was detected at 1000 p.p.m. These results suggest that the limit of exposure of 25 p.p.m. recommended by the American National Institute of Occupational Safety & Health may be unduly restrictive.
5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
Impaired chemiluminescence and bactericidal killing by neutrophils from patients with severe cobalamin deficiency.
Neutrophil function was examined in 24 patients with megaloblastic haemopoiesis due to vitamin B12-or folate deficiency. Defective bactericidal activity was demonstrated in the B12-deficient group only and was associated with normal phagocytic activity but impaired intracellular killing. Luminol amplified luminescence was consistently low in B12-deficient subjects with RBC counts of less than 2 X 10(12)/l. These defects were not seen in the folate-deficient cases although isolated abnormalities were present in those who were also alcoholic.
Adult↗
Management of megaloblastic anaemia in the very young.
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Anemia, Macrocytic↗
Studies on the haemopoietic toxicity of nitrous oxide in man.
Nitrous oxide inactivates vitamin B12 and in man can produce a megaloblastic anaemia. Haematological and biochemical changes were studied in nine surgical patients ventilated with 70% N2O 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 N2O 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 N2O but by 1 week abnormalities were evident in more mature cells, metamyelocytes and segmented neutrophils. Megaloblastosis was associated with abnormal dU suppression which showed a correction pattern similar to that seen in vitamin B12 deficiency. Administration of N2O 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.
Anesthesia, General↗
Solid-phase vitamin B12 assays using polyacrylamide-bound intrinsic factor and polyacrylamide-bound R-binder.
A new solid phase vitamin B12 assay is described using intrinsic factor to measure microbiologically-available B12 and R-binder to measure total B12. The solid phase reagent consists of intrinsic factor coupled to polyacrylamide beads and salivary R-binder coupled to polyacrylamide beads. The assay is simple to perform and separates completely sera from controls and patients with megaloblastic anaemia due to B12 deficiency.
Acrylic Resins↗
Separation of cobalamin analogues in human sera binding to intrinsic factor and to R-type vitamin B12 binders.
Intrinsic factor (IF) and cobalamin-R-binding protein (R-binder) linked to polyacrylamide beads were used to absorb cobalamins from solutions and serum extracts. Both binding agents were equally effective in removing [57Co]B12 from aqueous solution. IF was more effective than R-binder in removing [57Co]B12 added to a serum extract. All endogenous cobalamins detectable in serum by saturation analysis assay were removed by absorption onto R-binder. Absorption with IF removed microbiologically-active cobalamins but left behind analogues assayable with an R-binder. However, when absorption with IF was continued the concentration of R-binding cobalamins steadily declined indicating that IF bound both types of cobalamins through the binding was less avid for the microbiologically-inactive analogues than for microbiologically-active cobalamins. Finally, the R-binding analogues in serum were carried on transcobalamin I and none was detectable on transcobalamin II. The absorption studies establish the presence of two types of cobalamins one binding preferentially to IF and the other preferentially to R-binder. Only the former is detected by microbiological assay.
Absorption↗
Chronic cobalamin inactivation impairs folate polyglutamate synthesis in the rat.
Nitrous oxide, by inactivating cobalamin in vivo, produces a suitable animal model for cobalamin 'deficiency.' The synthesis of folate polyglutamate with tetrahydrofolate as substrate is severely impaired in the N2O-treated rat, but is normal with formyltetrahydrofolate as substrate. Methionine restores the capacity of the N2O-treated rat to utilize tetrahydrofolate the minimum effective dose being 16 mumol. S-Adenosylmethionine was somewhat less effective than methionine but 5'methylthioadenosine, a product of S-adenosylmethionine metabolism, was significantly more effective than methionine in correcting the defect in folate polyglutamate synthesis. 5'Methylthioadenosine is metabolised to yield formate. It is suggested that these compounds have their effect in correcting folate polyglutamate synthesis by supplying formate for the formylation of tetrahydrofolate. Formyltetrahydrofolate, at least in the cobalamin-inactivated animal, is the required substrate for folate polyglutamate synthesis. Cobalamin is concerned with the maintenance of normal levels of methionine and this in turn is a major source of formate through S-adenosylmethionine and 5'methylthioadenosine.
Adenosine↗
Urinary methylmalonic acid excretion.
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Humans↗
Formes frustes in myeloproliferative disorders. Identification by the growth of an endogenous erythroid clone in vitro in patients with arterial vascular disease.
The myeloproliferative disorders, including polycythaemia rubra vera, arise as a result of a single-cell mutation. A characteristic of the abnormal haemopoietic clone is that it can form erythroid colonies in vitro in the absence of added erythropoietin. Such endogenous erythroid clones were consistently found in two of seven patients with peripheral vascular disease. These two patients had mean platelet counts of 600 X 10(9)/l and 630 X 10(9)/l. Culture of blood and bone-marrow cells from patients with raised platelet counts secondary to a variety of other disorders failed to yield such colonies. The presence of endogenous erythroid clones provides early evidence of a myeloproliferative disorder.
Adult↗