5-methyltetrahydrofolate and Vitamin-B-12-Deficiency

Topics: Animals; DNA; Hematopoiesis; Humans; Intestinal Absorption; Intrinsic Factor; Receptors, Cell Surface; Tetrahydrofolates; Vitamin B 12; Vitamin B 12 Deficiency

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

Topics: Alcoholism; Anemia, Hemolytic; Anticonvulsants; Bone Marrow Cells; Bone Marrow Diseases; Cells; Deoxyuridine; Erythrocytes, Abnormal; Female; Fluorouracil; Folic Acid; Folic Acid Deficiency; Formyltetrahydrofolates; Homocysteine; Humans; Hypothyroidism; Lymphocyte Activation; Methionine; Pregnancy; Pregnancy Complications; Statistics as Topic; Tetrahydrofolates; Vitamin B 12; Vitamin B 12 Deficiency

Increased plasma total homocysteine (tHcy) is a risk factor for vascular disease and adverse pregnancy outcomes. Health authorities recommend periconceptional supplementation with 400 micro g folic acid to prevent neural tube defects. Several countries have implemented food fortification with folic acid. However, excessive intake of folic acid could mask an undiagnosed vitamin B-12 deficiency. The biologically active [6S]-5-methyltetrahydrofolate ([6S]-5-MTHF) may be an alternative to folic acid because it is unlikely to mask vitamin B-12 deficiency symptoms.. We compared the tHcy-lowering potential of 2 dosages of [6S]-5-MTHF with that of 400 micro g folic acid during 24 wk of supplementation.. In this double-blind, randomized, controlled intervention trial, 144 female participants were supplemented daily with 400 micro g folic acid, 416 micro g [6S]-5-MTHF, 208 micro g [6S]-5-MTHF, or placebo. Concentrations of tHcy and plasma folate were measured at baseline and at 4-wk intervals.. After supplementation, there was a significant interaction between time and treatment with respect to changes in tHcy and plasma folate (both P < 0.001 by two-factor repeated-measures analysis of variance). The decrease in tHcy did not differ significantly between the 3 supplemented groups (P > 0.05; Tukey's post hoc test). The increase in plasma folate in the group receiving 208 micro g [6S]-5-MTHF was significantly lower than that in the groups receiving 400 micro g folic acid (P < 0.001) or 416 micro g [6S]-5-MTHF (P < 0.05).. [6S]-5-MTHF was shown to be an adequate alternative to folic acid in reducing tHcy concentrations. Supplementation with 416 micro g [6S]-5-MTHF was no more effective than that with 208 micro g [6S]-5-MTHF.

Topics: Adult; Analysis of Variance; Dietary Supplements; Dose-Response Relationship, Drug; Double-Blind Method; Female; Folic Acid; Homocysteine; Humans; Neural Tube Defects; Placebos; Tetrahydrofolates; Treatment Outcome; Vitamin B 12 Deficiency

Food fortification with folic acid has been introduced in several countries for the prevention of neural tube defects. Fortification has lowered total homocysteine (tHcy) concentrations in the US population, a consequence that may have health benefits. However, folic acid fortification could mask vitamin B-12 deficiency. Synthetic L-5-methyltetrahydrofolate (L-MTHF) may be more appropriate than folic acid as a fortificant because it is unlikely to mask the hematologic indicators of vitamin B-12 deficiency.. The objective of the study was to compare the effectiveness of 100 micro g folic acid/d with that of equimolar L-MTHF in lowering tHcy in healthy volunteers.. The study was designed as a 24-wk, randomized, placebo-controlled intervention. Free-living healthy volunteers (n = 167) were randomly assigned to receive a daily supplement containing folic acid (100 microg), L-MTHF (113 microg), or placebo. Blood collected at baseline and at 8, 16, and 24 wk was analyzed for tHcy, plasma folate, and red blood cell folate (RCF) concentrations.. At 24 wk, after adjustment for baseline values, mean (95% CI) tHcy was 14.6% (9.3, 19.5%) and 9.3% (3.7, 14.6%) lower, mean plasma folate was 34% (14, 56%) and 52% (30, 78%) higher, and mean RCF was 23% (12, 35%) and 31% (19, 44%) higher in the L-MTHF and folic acid groups, respectively, than in the placebo group. L-MTHF was more effective than was folic acid in lowering tHcy (P < 0.05). At 24 wk, the increases in plasma folate and RCF concentrations did not differ significantly between the 2 supplemented groups.. Low-dose L-MTHF is at least as effective as is folic acid in reducing tHcy concentrations in healthy persons.

Topics: Adult; Dietary Supplements; Double-Blind Method; Erythrocytes; Female; Folic Acid; Food, Fortified; Homocysteine; Humans; Male; Middle Aged; Neural Tube Defects; Placebos; Tetrahydrofolates; Treatment Outcome; Vitamin B 12 Deficiency

Vitamin B12 is an essential micronutrient for neurological function, as it leads to the regeneration of methionine from homocysteine, which is precursor of biologically active molecule S-Adenosyl Methionine (SAM). Pregnancy is a state of increased demand and delayed postpartum repletion of nutrients may predispose women to depression.. We included women who visited the hospital at 6-weeks postpartum for a regular checkup. Inclusion criteria were age (18-50 years), and willingness to donate venous sample for analysis. Exclusion criteria included previous history of mood disorders or antidepressant medication use, and any systemic illness like hypothyroidism, epilepsy, diabetes, and hypertension. Based on EPDS score of 10 as a cutoff, 217 women with probable postpartum depression (PPD) and equal number of age and BMI matched controls were included. Plasma total vitamin B12, holotranscobalamin (holotc), homocysteine (hcy), methyl malonic acid (MMA), 5-methyl tetrahydrofolate (THF), SAM and serotonin levels were estimated using commercially available ELISA kits. Combined B12 (cB12) score was calculated from study parameters. Multivariate analysis was performed to assess the risk of probable postpartum depression.. Total vitamin B12 and combined B12 score were found to be significantly lower (p = 0.001) and MMA (p = 0.002) and 5-methyl THF (p < 0.001) levels were higher in women with probable depression than women without probable PPD. Women in the lowest vitamin B12 quartile had 4.53 times higher likelihood of probable postpartum depression (p < 0.001). Multivariate analysis demonstrated that decreasing vitamin B12 (OR = 0.394; 95% CI: 0.189-0.822) and cB12 (OR = 0.293; 95% CI: 0182-0.470) and increasing MMA (OR = 2.14; 95% CI: 1.63-2.83) and 5-methyl THF levels (OR = 3.29; 95% CI: 1.59-6.83) were significantly associated with the risk of probable PPD.. Low vitamin B12 may contribute to depressive symptoms in vulnerable postpartum period.

Topics: Adolescent; Adult; Cesarean Section; Cross-Sectional Studies; Depression, Postpartum; Diet; Female; Homocysteine; Humans; India; Methylmalonic Acid; Pregnancy; Pregnancy, Unplanned; Risk Factors; S-Adenosylmethionine; Serotonin; Social Class; Tetrahydrofolates; Transcobalamins; Vitamin B 12; Vitamin B 12 Deficiency; Young Adult

Clinical vitamin B

Topics: DNA Damage; Female; Fibroblasts; Genomic Instability; Glycine Hydroxymethyltransferase; HeLa Cells; Humans; Infant; Tetrahydrofolates; Thymidine Monophosphate; Vitamin B 12 Deficiency

Folate deficiency has serious consequences for the fetus. Folic acid fortification of food addresses this problem. However, clinical consequences of vitamin B-12 deficiency may be worsened by high folic acid intakes, perhaps as a direct result of unmetabolized folic acid, which does not occur naturally in body tissues.. We attempted to attribute associations that we previously found between higher folate status and anemia and cognitive test performance to circulating unmetabolized folic acid or 5-methyltetrahydrofolate (5MeTHF).. The subjects (n = 1858) were senior participants in the US National Health and Nutrition Examination Survey (1999-2002) who had normal renal function and reported no history of stroke, recent anemia therapy, or diseases of the liver, thyroid, or coronary arteries. Subjects had undergone a phlebotomy, a complete blood count, and cognitive and dietary assessments.. Circulating unmetabolized folic acid was detected in approximately 33% of the subjects and was related to an increased odds of anemia in alcohol users. In seniors with a serum vitamin B-12 concentration <148 pmol/L or a plasma methylmalonic acid concentration > or =210 nmol/L, the presence compared with the absence of detectable circulating unmetabolized folic acid was related to lower cognitive test scores and lower mean cell volume. In the same subgroup, higher serum 5MeTHF was related to an increased odds of anemia and a marginally significantly decreased odds of macrocytosis. In seniors with a normal vitamin B-12 status, a higher serum 5MeTHF concentration was related to higher cognitive test scores.. Results of this epidemiologic study were somewhat consistent with reports on the folic acid treatment of patients with pernicious anemia, but some findings were unexpected.

Topics: Aged; Aging; Anemia; Anemia, Macrocytic; Blood Cell Count; Cognition; Cognition Disorders; Cystatin C; Female; Folic Acid; Hemoglobins; Homocysteine; Humans; Linear Models; Male; Methylmalonic Acid; Multivariate Analysis; Nutrition Surveys; Tetrahydrofolates; United States; Vitamin B 12 Deficiency

Haematological sequellae of vitamin B12 deficiency are attributed to disturbed DNA synthesis, but vitamin B12 itself plays no role in DNA biosynthesis. A proposed explanation for this is the methylfolate trap hypothesis. This hypothesis states that B12 deficiency impairs overall folate metabolism because 5-methyltetrahydrofolate (5MTHF) becomes metabolically trapped. This trap results from the fact that 5MTHF can neither be metabolised via the methionine synthase pathway, nor can it be reconverted to its precursor, methylenetetrahydrofolate. Other manifestations of the methylfolate trap include cellular folate loss because of shorter 5MTHF polyglutamate chains and global hypomethylation. The methylfolate trap has never been demonstrated in humans. We describe a patient with B12 deficiency who was homozygous for the common methylenetetrahydrofolate reductase (MTHFR) C677T mutation. We analysed red blood cell (RBC) folate vitamers and global DNA methylation by liquid chromatography (LC) in combination with tandem mass spectrometry, and 5MTHF polyglutamate length by LC-electrochemical detection. Compared to post-B12 supplementation values, homocysteine was higher (52.9 micromol/l vs. 16.8 micromol/l), RBC folate was lower (268.92 nmol/l vs. 501.2 nmol/l), the 5MTHF fraction of RBC folate was much higher (94.5% vs. 67.4%), polyglutamate chain length was shorter (more tetra- and pentaglutamates), and global DNA methylation was 22% lower. This is the first time that virtually all features of the methylfolate trap hypothesis have been demonstrated in a human with vitamin B12 deficiency.

Topics: Chromatography, Liquid; DNA Methylation; Erythrocytes; Folic Acid; Homocysteine; Homozygote; Humans; Hydroxocobalamin; Male; Mass Spectrometry; Methylenetetrahydrofolate Reductase (NADPH2); Middle Aged; Tetrahydrofolates; Vitamin B 12; Vitamin B 12 Deficiency

A mathematical model is developed for the folate cycle based on standard biochemical kinetics. We use the model to provide new insights into several different mechanisms of folate homeostasis. The model reproduces the known pool sizes of folate substrates and the fluxes through each of the loops of the folate cycle and has the qualitative behavior observed in a variety of experimental studies. Vitamin B(12) deficiency, modeled as a reduction in the V(max) of the methionine synthase reaction, results in a secondary folate deficiency via the accumulation of folate as 5-methyltetrahydrofolate (the "methyl trap"). One form of homeostasis is revealed by the fact that a 100-fold up-regulation of thymidylate synthase and dihydrofolate reductase (known to occur at the G(1)/S transition) dramatically increases pyrimidine production without affecting the other reactions of the folate cycle. The model also predicts that an almost total inhibition of dihydrofolate reductase is required to significantly inhibit the thymidylate synthase reaction, consistent with experimental and clinical studies on the effects of methotrexate. Sensitivity to variation in enzymatic parameters tends to be local in the cycle and inversely proportional to the number of reactions that interconvert two folate substrates. Another form of homeostasis is a consequence of the nonenzymatic binding of folate substrates to folate enzymes. Without folate binding, the velocities of the reactions decrease approximately linearly as total folate is decreased. In the presence of folate binding and allosteric inhibition, the velocities show a remarkable constancy as total folate is decreased.

Topics: Allosteric Site; Animals; Dose-Response Relationship, Drug; Folic Acid; Homeostasis; Humans; Kinetics; Liver; Methionine; Methotrexate; Models, Biological; Models, Chemical; Models, Theoretical; Tetrahydrofolate Dehydrogenase; Tetrahydrofolates; Thymidylate Synthase; Time Factors; Up-Regulation; Vitamin B 12 Deficiency

We report on a child in whom severe nutritional vitamin B12 deficiency was exacerbated by a genetic impairment of the folate cycle, causing reduced CSF concentrations of the methyl group donor 5-methyltetrahydrofolate. Some patients with vitamin B12 deficiency may benefit from high dose folic acid supplementation, even if plasma concentrations are high.

Topics: Breast Feeding; Coma; Dehydration; Diet, Vegetarian; Folic Acid; Humans; Infant; Male; Methylenetetrahydrofolate Reductase (NADPH2); Muscle Hypertonia; Mutation; Oxidoreductases Acting on CH-NH Group Donors; Polymorphism, Genetic; Tetrahydrofolates; Vitamin B 12 Deficiency

Total fasting plasma homocysteine (tHcy), homozygosity for the C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene and for the A2756G mutation of the methionine synthase (MS) gene, vitamin B12 and folate plasma levels were evaluated in 170 consecutive patients (89 M, 81 F; mean age 41 +/- 12 yrs) with documented early-onset thrombosis (89 venous, 69 arterial, 12 both; mean age at first episode 36 +/- 11 yrs), and in 182 age- and sex-matched healthy control subjects. Moderate hyperhomocysteinemia (HHcy, tHcy >19.5 microM in men and >15 microM in women) was detected in 45 patients (26.5%) and in 18 controls (9.9%, Mantel-Haenszel OR and 95% C.I. after stratification for arterial or venous thrombosis: 3.25, 1.78-5.91). The 677TT MTHFR genotype was not significantly more prevalent in patients (27.6%) than in controls (21.4%, RR = 1.42: 0.84-2.41), and markedly contributed to HHcy (Mantel-Haenszel RR after stratification for case/control status: 8.29, 4.61-14.9). The 2756GG MS genotype, observed in 4 patients (2.4%) and 8 controls (4.4%), was not associated to HHcy. tHcy was negatively correlated to folate and vitamin B12 levels, with better correlation found in subjects with the 677TT mutation (r = -0.42 and -0.25) than with the 677CC or CT MTHFR genotype (r = 0).37 and -0.11). However, folate was similar in patients and controls and vitamin B12 was higher in patients (460 +/- 206 vs. 408 +/-185 pg/ml, p = 0.011). In a generalized linear model, 44% of the variation in tHcy levels was explained by folate and vitamin B12 levels, the MTHFR genotype, gender, and by the interaction of the MTHFR genotype with folate (p < or =0.028); the interactions of vitamin B12 with the MTHFR genotype, gender and patient/control status also significantly contributed to the variation in tHcy levels (p < or =0.028). A 4-week administration of 5-methyltetrahydrofolate (15 mg/day) markedly lowered plasma tHcy in 24 patients with MTHFR 677TT genotype, but the response to treatment correlated with vitamin B,2 levels (p = 0.023). Subjects carrying the MTHFR 677TT genotype have higher folate and vitamin B12 requirements irrespective of the A2756G polymorphism of the MS gene. Yet unidentified abnormalities of MS or of any of the enzymes participating in the synthesis of methylated vitamin B12 may play an important role in the phenotypic expression of moderate hyperhomocysteinemia.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Adolescent; Adult; Age of Onset; Aged; Amino Acid Substitution; Case-Control Studies; Fasting; Female; Folic Acid; Gene Frequency; Genetic Heterogeneity; Homocysteine; Humans; Hyperhomocysteinemia; Italy; Male; Methylenetetrahydrofolate Reductase (NADPH2); Middle Aged; Nutritional Requirements; Oxidoreductases Acting on CH-NH Group Donors; Point Mutation; Risk Factors; Sex Factors; Tetrahydrofolates; Thrombophilia; Thrombosis; Vitamin B 12; 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

The effect of azidothymidine (Zidovudine, AZT) on pyrimidine (thymidine, deoxyuridine, and thymidine triphosphate) incorporation into DNA in folate- and/or vitamin B12-deficient and normal human bone marrow cells was studied to investigate whether such vitamin deficiency affects susceptibility to AZT-induced hematologic toxicity. Bone marrow cells from 12 patients were studied: 5 had folate and/or vitamin B12 deficiency; 7 controls included 5 with anemia related to chronic disease and 2 with iron deficiency. At 0.2 microM AZT (3 hr, 37 degrees C), the approximate pharmacologic serum trough level, pyrimidine incorporation into DNA was suppressed by 12 to 19% in folate- and/or vitamin B12-deficient cells and by 16 to 23% in normal cells. At 2.0 microM AZT (3 hr, 37 degrees C), the approximate pharmacologic serum peak level, this was suppressed by 15 to 40% in folate- and/or vitamin B12-deficient cells and by 32 to 47% in controls. Deoxyuridine incorporation into DNA was inhibited significantly greater than thymidine at 2.0 microM AZT (3 hr, 37 degrees C) in both groups. Inhibition of deoxyuridine incorporation was not reversed with methyltetrahydrofolate or vitamin B12. There tended to be less striking suppression by AZT of deoxyuridine incorporation into DNA in bone marrow cells from vitamin B12-deficient patients, which was made more striking by adding vitamin B12. This suggests that some of what passes for "AZT damage" to bone marrow cells may in fact be coincident deficiency of vitamin B12. AZT inhibition of DNA synthesis in 3 hr bone marrow cultures is relatively consistent in a variety of hematologic disorders. As approximately two-thirds of AIDS patients appear to be in negative balance with respect to folate and/or vitamin B12, the fact that AZT-induced inhibition of pyrimidine incorporation into DNA is occurring in cells which may be megaloblastic, i.e., in a state of impaired DNA synthesis, suggests that these cells may be more susceptible to AZT toxicity. The data also support the notion that AZT inhibition results predominantly from termination of DNA chain elongation. Whether folate or vitamin B12 supplementation may partially overcome apparent "AZT inhibition" of DNA synthesis (hematologic toxicity) and whether the benefit of such therapy exceeds the risk will require further study.

Topics: Bone Marrow; Deoxyuridine; DNA; Folic Acid Deficiency; Humans; Pyrimidines; Tetrahydrofolates; Thymidine; Vitamin B 12 Deficiency; Zidovudine

We have studied the uptake of both propionate and leucine in a chorionic villus sample from a fetus at risk for cblC disease. The ratio of propionate to leucine incorporation (x 10(-3] was 3.85 +/- 0.27 (n = 8) in the at risk sample, and 2.8 +/- 0.14, and 3.1 +/- 0.15 (n = 4) in two control samples. The finding of an unaffected fetus was confirmed by the absence of methylmalonic acid in amniotic fluid or maternal urine in the second trimester, and after birth by study of cultured fibroblasts from the baby. Because of the reported variability in propionate incorporation in chorionic villus biopsies, however, we recommend that chorionic villus sampling be confirmed by amniocentesis.

Topics: Adult; Biopsy; Carbon Radioisotopes; Chorionic Villi Sampling; Female; Fetal Monitoring; Fibroblasts; Humans; Infant; Infant, Newborn; Leucine; Male; Pregnancy; Propionates; Risk Factors; Skin; Tetrahydrofolates; Tritium; Vitamin B 12 Deficiency

We performed studies in 25 patients with low serum cobalamin levels who had few if any clinical or hematologic findings of cobalamin deficiency. All but three had morphologically normoblastic hematopoiesis, and 15 were not even anemic. None of those tested excreted methylmalonic acid or homocystine. Nevertheless, the dUST identified metabolic abnormalities in 18 of the 25 cases. In vitro additives were essential in the dUST. Especially noteworthy was MTHF, whose addition unmasked an otherwise undetectable dUST abnormality in four cases. Why MTHF appears to act as a "stress test" in this setting is unknown but deserves further attention. Seven patients had early forms of classical malabsorptive states such as pernicious anemia, defined by abnormal Schilling test results. Among the rest, seven of 13 patients displayed malabsorption of protein-bound cobalamin despite normal absorption of free cobalamin by the Schilling test. In two patients, initially normal Schilling test results became abnormal the following year. These findings demonstrate that seemingly falsely low serum cobalamin levels often indicate subtle biochemical cobalamin deficiency. Early stages of pernicious anemia or other classical malabsorptive states are sometimes responsible for such subtle deficiency. However, malabsorption confined to protein-bound cobalamin is an equally common cause. Current concepts of cobalamin deficiency and the absorptive defects that can cause it should be expanded to include atypical defects requiring newer methods of identification.

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow; Deoxyuridine; Female; Folic Acid Deficiency; Humans; In Vitro Techniques; Intestinal Absorption; Male; Middle Aged; Prospective Studies; Protein Binding; Tetrahydrofolates; Vitamin B 12; Vitamin B 12 Deficiency

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.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Animals; Male; Nitrous Oxide; Rats; Rats, Inbred Strains; Tetrahydrofolates; Time Factors; Vitamin B 12 Deficiency

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

Cobalamin-deficient L1210 mouse leukemia cells were propagated on [3H]folate under conditions designed to completely label the intracellular folate compounds. The latter, present almost entirely as polyglutamate forms, were converted to the corresponding monoglutamates by treatment with gamma-glutamylcarboxypeptidase; the monoglutamates were subsequently separated and identified by chromatography on Sephadex G-25. 5-Methyltetrahydrofolate accounted for approximately 70% of the total folate pool (17.8 nmol/10(9) cells), while the remainder was comprised largely of 5-formyltetrahydrofolate (13.0%), and tetrahydrofolate (9.5%). The identity of 5-methyltetrahydrofolate were further confirmed by chromatography on DEAE-cellulose and by its quantitative conversion to tetrahydrofolate after treatment with a highly purified preparation of methionine synthetase (5-methyltetrahydrofolate:homocysteine methyltransferase, EC 2.1.1.13). In contrast, cobalamin-replete cells contained only 5.3% 5-methyltetrahydrofolate, along with 30% each of the other three reduced folate compounds. These results provide direct experimental proof of the "methyl trap" hypothesis, which predicted that 5-methyltetrahydrofolate should accumulate in cobalamin-deficient cells.

Topics: Animals; Folic Acid; Leukemia L1210; Mice; Structure-Activity Relationship; Tetrahydrofolates; Vitamin B 12 Deficiency

Topics: Animals; Binding, Competitive; Chiroptera; gamma-Aminobutyric Acid; Glutamates; Kainic Acid; Neurons; Receptors, Cell Surface; Receptors, GABA-A; Receptors, Glutamate; Receptors, Kainic Acid; Synaptic Membranes; Tetrahydrofolates; Vitamin B 12 Deficiency

Recent work has shown that several folates interact with excitatory kainic acid receptors in the mammalian brain and appear to have agonist activity at these receptors. Since kainic acid is a potent neurotoxin it is possible that folates share this toxicity and that high levels of folates result in neuronal damage. Levels of methyltetrahydrofolate are markedly elevated in vitamin B12 deficiency, a disease associated with neuronal destruction. We propose that this destruction occurs as a result of a neurotoxic action of methyltetrahydrofolate. Injection of kainic acid into the basal ganglia of experimental animals produces a pattern of damage similar to that found in patients dying of Huntington's chorea. It is possible that the underlying defect in this disease resides in the pathways of folate metabolism such that a neurotoxic excess of folates accumulates in the central nervous system. Such a disease process might be arrested by antifolate drugs.

Topics: Animals; Brain; Chemical Phenomena; Chemistry; Chiroptera; Folic Acid; gamma-Aminobutyric Acid; Humans; Huntington Disease; Kainic Acid; Models, Biological; Rats; Receptors, Cell Surface; Receptors, Kainic Acid; Tetrahydrofolates; Vitamin B 12 Deficiency

Prior exposure of rats to a mixture of nitrous oxide/oxygen (80/20) for 20 to 24 hours and feeding rats a diet deficient in vitamin B-12 and methionine for 23 weeks resulted in a 86 to 90% decrease in the activity of the vitamin B-12 dependent enzyme, 5-methyltetrahydrofolate-homocysteine methyltransferase (EC 4.2.99.10). In addition, deficient animals excreted some 15 times as much methylmalonic acid as controls indicating impaired functioning of the vitamin B-12-dependent enzyme, methylmalonyl CoA mutase (EC 5.4.99.2). Hepatocytes prepared from rats deficient in vitamin B-12 and methionine and from nitrous oxide-treated rats showed no impairment of the membrane transport of 5-methyltetrahydro-[G-3H]folic acid when compared to the respective controls. This observation was true both for the initial rate of entry and for the ability of these cells to accumulate this folate derivative. These findings support the "methyl trap" hypothesis and apparently rule out the "cellular transport" hypothesis for the folate-vitamin B-12 metabolic interrelationship.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Animals; Biological Transport; Cell Membrane; Liver; Male; Methylmalonic Acid; Methylmalonyl-CoA Mutase; Methyltransferases; Nitrous Oxide; Rats; Tetrahydrofolates; Vitamin B 12 Deficiency

[2-14C]Tetrahydropteroylglutamic acid and 5-[G-3H]methyltetrahydropteroylglutamic acid were given intraperitoneally to fruit bats. Folate polyglutamates were formed in the liver from both substrates in different amounts and at different rates. The methylfolate pool appeared to remain separate from the tetrahydrofolate pool. More polyglutamate was formed from tetrahydropteroylglutamic acid than from 5-methyltetrahydropteroylglutamic acid. There was a fall in the folate content of the liver in the vitamin B-12-deficient bat and a more rapid incorporation of folates into polyglutamates but thereafter a more rapid loss of the labelled folate from liver.

Topics: Animals; Chiroptera; Folic Acid; Liver; Pteroylpolyglutamic Acids; Tetrahydrofolates; Vitamin B 12 Deficiency