5-methyltetrahydrofolate and Anemia--Megaloblastic

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.

Topics: Anemia, Megaloblastic; Animals; Central Nervous System Diseases; Coenzymes; Folic Acid; Folic Acid Deficiency; Formates; Humans; Methionine; Models, Biological; Tetrahydrofolates; Thymidine; Vitamin B 12; Vitamin B 12 Deficiency

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Anemia, Megaloblastic; Animals; Central Nervous System; Deoxyuridine; Disease Models, Animal; Drug Interactions; Enzyme Activation; Folic Acid; Folic Acid Deficiency; Histidine; Humans; Liver; Methionine; Methylmalonyl-CoA Mutase; Nitrous Oxide; Oxidation-Reduction; Pteroylpolyglutamic Acids; Purines; Serine; Tetrahydrofolates; Thymidylate Synthase; Vitamin B 12; Vitamin B 12 Deficiency

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Anemia, Megaloblastic; Animals; Humans; Liver; Macaca fascicularis; Methionine; Methylmalonyl-CoA Mutase; Nervous System Diseases; Nitrous Oxide; Rats; S-Adenosylhomocysteine; S-Adenosylmethionine; Tetrahydrofolates; Vitamin B 12 Deficiency

The critical disturbance of folate metabolism caused by vitamin B12 deficiency which results in megaloblastic anaemia remains controversial. Vitamin B12 is required in the methionine synthase reaction in which homocysteine is converted to methionine and methyl tetrahydrofolate (methyl THF) to THF. The 'methyl-folate trap' hypothesis suggested that failure of demethylation of methyl THF with consequent deficiency of folate co-enzymes derived from THF is the crucial lesion caused by vitamin B12 deficiency. A more recent theory suggested that reduced supply of methionine leads to reduced availability of 'activated formate' and hence of formyl THF and it is this defect that results in failure of folate co-enzyme synthesis. The present results, based on deoxyuridine suppression tests on 103 cases of megaloblastic anaemia, show that THF itself is equally capable of correcting the failure of thymidylate synthesis in vitamin B12 deficiency as in folate deficiency. Although not as effective as formyl THF in correcting the dU blocking test in vitamin B12 deficiency, this is equally so for the correction of the test by THF compared with formyl THF in folate deficiency. The results therefore favour the theory that it is in the supply of THF and not of 'active formate' or formyl THF that vitamin B12 plays a critical role in folate metabolism.

Topics: Anemia, Megaloblastic; Bone Marrow; Cells, Cultured; Deoxyuridine; DNA; Female; Humans; Leucovorin; Male; Megaloblasts; Tetrahydrofolates; Thymidine Monophosphate; Vitamin B 12; Vitamin B 12 Deficiency

Folate deficiency and megaloblastic anemia occur in chronic renal failure. However, the possible role of intestinal malabsorption as a cause of the reported deficiency has not been investigated. Therefore, we examined the intestinal absorption of 5-methyltetrahydrofolate in rats made uremic by subtotal nephrectomy using in vivo perfusion technique and in vitro everted sac technique. The results were compared with those obtained in a group of sham-operated rats with normal renal function. The amount of 5-methyltetrahydrofolate absorbed in vivo was significantly lower in the uremic animals as compared to the control group. In contrast, no significant difference was found in the absorption of 5-methyltetrahydrofolate in vitro in the two groups. To mimic the uremic environment, the in vitro studies were repeated using jejunal sacs from normal animals filled with either buffer solution, or sera from uremic patients before and after dialysis. Their results showed a marked suppression of 5-methyltetrahydrofolate absorption with predialysis sera and a significant improvement with post dialysis sera. We conclude that intestinal absorption of 5-methyltetrahydrofolate is impaired in uremia. The results of the in vitro experiments suggest that the observed transport defect is due to some influence of uremic environment rather than to an acquired intrinsic defect of enterocytes in uremia.

Topics: Anemia, Megaloblastic; Animals; Biological Transport; Folic Acid Deficiency; In Vitro Techniques; Intestinal Absorption; Male; Rats; Rats, Inbred Strains; Tetrahydrofolates; Uremia

We describe an inborn error of vitamin B12 metabolism in an infant who had severe developmental delay, megaloblastic anemia, and homocystinuria. There was no evidence of methylmalonic aciduria or deficiency of folate or vitamin B12. Treatment with hydroxocobalamin, but not with cyanocobalamin and folic acid, resulted in rapid clinical and biochemical improvement. Cultured fibroblasts showed an absolute growth requirement for methionine, defective incorporation of radioactivity from [14C]5-methyltetrahydrofolate into protein, and normal incorporation of radioactivity from [14C]propionate, thus assigning the intracellular defect to methionine synthesis. The proportion of intracellular methylcobalamin in the fibroblasts was decreased, but that of 5'-deoxyadenosylcobalamin was normal. Methionine synthetase activity in cell extracts was normal, as was cobalamin incorporation into cultured cells. This defect differs from those described previously in being limited to methylcobalamin accumulation and defective use of 5-methyltetrahydrofolate by intact cells with normal activity of methylmalonyl CoA mutase.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Amino Acid Metabolism, Inborn Errors; Anemia, Macrocytic; Anemia, Megaloblastic; Cobamides; Fibroblasts; Homocystinuria; Humans; Hydroxocobalamin; Infant, Newborn; Male; Methionine; Propionates; Tetrahydrofolates; Vitamin B 12

The role of vitamin B12 in the folate dependent biosynthesis of thymidine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting 'de novo' thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)-thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6-3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6-3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, N6-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl-FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl-FH4.

Topics: Anemia, Macrocytic; Anemia, Megaloblastic; Bone Marrow; Deoxyuridine; DNA; Folic Acid; Folic Acid Deficiency; Humans; Leucovorin; Tetrahydrofolates; Thymine Nucleotides; Vitamin B 12; Vitamin B 12 Deficiency

The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10-methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10-methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; 5,10-Methylenetetrahydrofolate Reductase (FADH2); Adult; Aged; Alcohol Oxidoreductases; Anemia, Macrocytic; Anemia, Megaloblastic; Bone Marrow; Bone Marrow Cells; Deoxyuridine; DNA Polymerase II; DNA-Directed DNA Polymerase; Female; Folic Acid; Humans; Hydroxocobalamin; Leucovorin; Male; Methylenetetrahydrofolate Reductase (NADPH2); Middle Aged; Tetrahydrofolates; Thymidine; Vitamin B 12; Vitamin B 12 Deficiency

We studied two brothers (J.R. and M.R.) with the cobalamin D variant of congenital methylmalonic aciduria-homocystinuria, whose previously reported lack of megaloblastic anemia conflicted with current concepts of cobalamin's role in DNA synthesis and the "methyltetrahydrofolate (MTHF) trap" hypothesis. Both subjects were indeed hematologically normal, although J.R. had a mean corpuscular volume of 96 fl. However, both demonstrated abnormalities in the deoxyuridine suppression test. J.R. had an abnormal suppression value of 21.0% (normal less than 10%) that was correctable by adding hydroxocobalamin or folic acid in vitro but not MTHF. M.R. had normal suppression (8.9%), but demonstrated worsening (18.6%) when MTHF was added. J.R.'s classical deoxyuridine suppression pattern of cobalamin deficiency thus supports the trap hypothesis. However, his lack of comparable morphological changes suggests that impaired de novo thymidylate synthesis and the trap hypothesis, though valid, may not fully account for the megaloblastic maturation accompanying cobalamin deficiency. Equally noteworthy was the deleterious effect of MTHF on M.R.'s marrow, suggesting its potential usefulness as an in vitro "stress test" for latent cobalamin abnormality.

Topics: Adolescent; Adult; Anemia, Megaloblastic; Bone Marrow; Deoxyuridine; Homocystinuria; Humans; Male; Metabolism, Inborn Errors; Methylmalonic Acid; Tetrahydrofolates; Thymidine; Vitamin B 12

Folate analogues were added to human bone marrow cells to determine their effect on deoxyuridine utilization in the deoxyuridine suppression test. Formyltetrahydrofolates fully corrected the impairment of dU utilization in pernicious anaemia marrows but tetrahydrofolate was relatively ineffective. All these analogues were effective in megaloblastic marrows from folate deficient patients. Formyltetrahydrofolates also enhanced dU utilization by normal human marrows whereas methyltetrahydrofolate reduced its use. In terms of the methylfolate trap hypothesis, the expectation that cobalamin-deficient marrows would be able to use tetrahydrofolate normally was not realized.

Topics: Anemia, Megaloblastic; Anemia, Pernicious; Bone Marrow; Bone Marrow Cells; Deoxyuridine; Folic Acid; Formyltetrahydrofolates; Humans; Hydroxocobalamin; Methylation; Tetrahydrofolates