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Summarymethylenetetrahydrofolate reductase polymorphism (MTHFR C677T) is an established determinant of homocysteine plasma level (t-Hcys) while its association with coronary artery disease (CAD) seems to be more limited. In contrast, the association of the substitutions A2756G of methionine synthase (MTR), A66G of methionine synthase reductase (MTRR) and C776G of transcobalamin (TCN) to both t-Hcys and CAD needs to be evaluated further. The objective was to evaluate the association of these polymorphisms with t-Hcys and CAD in a French population. We investigated the individual and combined effects of these polymorphisms and of vitamin B12 and folates with t-Hcys in 530 CAD patients and 248 matched healthy controls. t-Hcys was higher in the CAD group than in controls (11.8 vs 10.4 μM, P median and MTRR AA genotype were two significant independent predictors of CAD with respective odds ratios of 3.1 (95 % CI: 1.8-5.1, P

Transcobalamin (TC) is the plasma transporter that delivers vitamin B12 to cells. We have already reported that HT-29 and Caco-2 cells secrete different TC variants. HT-29 secretes 2 TC isoproteins (codon 259-Pro/Arg [259-P/R]), exhibiting unequal concentrations (TC 259-P > TC 259-R), and Caco-2 cells only secrete the phenotype 259-R. We investigated the relation between phenotypic and genetic TC polymorphism in HT-29 cells transfected with Caco-2 TC complementary DNA and in 159 healthy Caucasians. We found that codon 259-R is buried and, thus, the genetic polymorphism provides no explanation why the TCs from HT-29 and Caco-2 cells have different isoelectric points in nondenaturing isoelectric focusing (IEF). The newly translated TC in HT-29 cells from the Caco-2 complementary DNA recombinant plasmid had the same isoelectric point as the TC constitutively expressed in HT-29 cells, suggesting that TC phenotypic variability involves a specific cell folding of the protein. The codon 259 polymorphism was found to have a biallelic distribution: homozygotes P = 34.6%, heterozygotes R/P = 47.8%, and homozygotes R = 17.6%. In heterozygous samples, the IEF showed that the TC 259-P/TC 259-R ratio = 1.6. The blood apo-TC concentration of 259-P homozygous Caucasians was significantly higher than that of homozygous 259-R (P

The vitamin B 12 status of 20 subjects who were on home parenteral nutrition after surgical or functional small bowelresection and were given 1000 μ g cyanocobalamin every 3 mo was studied by comparing their plasma vitamin B 12 , homocysteine (HS), and methylmalonic acid (MMA) concentrations. The plasma vitamin B 12 concentration (median 145 pmol/L, 95% confidence interval: 123–217) was subnormal in four cases and borderline in four others. In the “low B 12 ” group, the concentrations of the markers of vitamin B 12 deficiency were in the normal range: HS 10.7 μ mol/L (8.0–12.3); and MMA, 0.15 μ mol/L (0.09–0.19). References values were HS, 10.0 μ mol/L (9.4–12.6); and MMA, 0.16 μ mol/L (0.10–0.19). Thus, there were no metabolic signs of vitamin B 12 deficiency in these subjects on parenteral nutrition, despite the fact that their plasma vitamin B 12 levels were low. Analysis of individual data showed that the four patients with low circulating B 12 had markers of intracellular vitamin B 12 deficiency in the normal range.

International audience; Vitamin B12 (cobalamin, cbl) is a cofactor of methionine synthase (MTR) in the synthesis of methionine, the precursor of the universal methyl donor S-Adenosylmethionine (SAM), which is involved in epigenomic regulatory mechanisms. We have established a neuronal cell model with stable expression of a transcobalamin–oleosin chimer and subsequent decreased cellular availability of vitamin B12, which produces reduced proliferation, increased apoptosis and accelerated differentiation through PP2A, NGF and TACE pathways. Anti-transcobalamin antibody or impaired transcobalamin receptor expression produce also impaired proliferation in other cells. Consistently, the transcription, protein expression and activity of MTR are increased in proliferating cells of skin and intestinal epitheliums, in rat intestine crypts and in proliferating CaCo2 cells, while MTR activity correlates with DNA methylation in rat intestine villi. Exposure to nitrous oxide in animal models identified impairment of MTR reaction as the most important metabolic cause of neurological manifestations of B12 deficiency. Early vitamin B12 and folate deprivation during gestation and lactation of a ‘dam-progeny’ rat model developed in our laboratory is associated with long-lasting disabilities of behavior and memory capacities, with persisting hallmarks related to increased apoptosis, impaired neurogenesis and altered plasticity. We found also an epigenomic deregulation of energy metabolism and fatty acids beta-oxidation in myocardium and liver, through imbalanced methylation/acetylation of PGC-1alpha and decreased expression of SIRT1. These nutrigenomic effects display similarities with the molecular mechanisms of fetal programming. Beside deficiency, B12 loading increases the expression of MTR through internal ribosome entry sites (IRES) and down-regulates MDR-1 gene expression. In conclusion, vitamin B12 influences cell proliferation, differentiation and apoptosis in brain. Vitamin B12 and folate combined deficiency impairs fatty acid oxidation and energy metabolism in liver and heart through epigenomic mechanisms related to imbalanced acetylation/methylation. Some but not all of these effects reflect the upstream role of vitamin B12 in SAM synthesis.

The concentrations of vitamin B12, its analogs, and the haptocorrin and transcobalamin carriers in 21 patients suffering from Crohn's disease and a group of controls (20 adults) were measured. There were no significant differences in the mean values for vitamin B12, total corrinoids (vitamin B12 + analogs), or vitamin B12 or total corrinoids bound to haptocorrin or transcobalamin of the Crohn's and control patients. There was a significant increase in the binding capacity of transcobalamin in the Crohn's patients compared to the controls (P

Due to the fact that the existence of vitamin B12 analogs in biological materials has been much debated, we realized on plasma, bile, and feces series of experiences to demostrate that analogs assayed by radioisotopic method in these biological media using intrinsic factor and haptocorrin as binders could not be extraction products or dosage artifacts. To this purpose, exposure to light was used to transform all corrinoids (true B12 + analogs) to their hydroxo forms. After that all corrinoids were transformed with KCN by cyanilation to their cyano forms. This permitted us to separate corrinoids on high performance liquid chromatography into only two peaks. These peaks will be subsequently identified by radioisotopic dilution assay as cyanocobalamin α and/or β cyanocobinamides. Because the binding ability of an intrinsic factor to corrinoids is limited to its cobalamin forms, our method enabled us to emphasize the existence in biological materials of corrinoids that only recognize haptocorrin (analogs of vitamin B12) and not intrinsic factors.

Resume La vitamine B12, egalement appelee cobalamine, est un micronutriment synthetise par la flore bacterienne. Elle est active sous forme de deux coenzymes, la methylcobalamine et la deoxyadenosylcobalamine. Son assimilation met en jeu deux proteines de transport intraluminal, l'haptocorrine qui est degradee par les proteases pancreatiques et le facteur intrinseque gastrique qui permet l'endocytose recepteur-dependante de la vitamine au niveau distal de l'ileon. Le dosage de la vitamine B12 serique n'est pas toujours suffisant pour etablir une carence et il faut recommander le dosage de l'acide methylmalonique et de l'homocysteine seriques. Les carences sont le plus souvent dues a un syndrome de malabsorption, mis en evidence par le test de Schilling. Elles sont particulierement frequentes dans la population âgee. Les causes gastriques regroupent la maladie de Biermer mais aussi la gasdtrite chronique avec defaiut de liberration de la B12 alimentaire. Parmi les autres causes de alabsorption, les plus frequentes sont la pullulation microbienne etles causes iatrogenes medicamenteuses, radiotherapies ou chirurgicales. Enfin, il existe un certain nombre de causes genetiques de carences cellulaires qui affectent soit l'endocytose du facteur intrinseque ou de la transcobalamine, soit le transport intracellulaire et la conversion en coeynzyme actif de la vitamine. Selon les etiologies, le traitement substitutif peut etre administre par voie orale, intramusculaire ou par perfusion. Certaines causes genetiques de carence cellulaire requierent specifiquement l'administration de l'un des deux coenzymes actifs.

High-performance capillary electrophoresis (HPCE) was compared for the identification and determination of corrinoids (hydroxy-, cyano-, 5′-desoxyadenosyl- and methyl-cobalamin and cyano-cobinamide) with high-performance liquid chromatography (HPLC). The within-run reproducibility of the retention times in HPCE and HPLC were similar (2.4 and 2.2%, respectively). The detection limit in HPCE was 20 μg/ml. HPLC can be used, in combination with radioisotope dilution assay, when very low concentrations (100 pg/ml) have to be determined in biological material. HPCE is more efficient than HPLC for the identification of corrinoids after conversion into the CN-cobalamin and CN-cobinamide forms.

Moderate hyperhomocysteinemia is a risk for neural tube defect and neurodegenerative and vascular diseases and has nutritional, metabolic, and genetic determinants. Its prevalence in sub-Saharan Africa remains unknown.Our goal was to evaluate the prevalence of hyperhomocysteinemia and the influence of nutritional, metabolic, and genetic determinants in savanna and coastal regions of Togo and Benin.Volunteers were recruited from coastal (C groups; n = 208) and savanna (S group; n = 68) regions. Vitamin B-12, folate, total homocysteine (tHcy), cystatin C (a marker of glomerular filtration), and inflammatory and nutritional protein markers were measured in plasma, and the methylenetetrahydrofolate reductase (MTHFR) 677C--T and 1298A--C polymorphisms and the methionine synthase 2756A--G polymorphism were examined in genomic DNA.Moderate hyperhomocysteinemia (tHcy15 micromol/L) was recorded in 62.3% and 29.4% of the subjects from the coast and savanna, respectively (P0.0001). A histogram distribution of tHcy in the coastal groups showed a distinct group, C2 (15% of the total group), with tHcy28 micro mol/L. Folate6.75 nmol/L (lower quartile) and MTHFRCT/TT genotype were the 2 main risk factors for moderate hyperhomocysteinemia in the whole population [odds ratios: 5.3 (95% CI: 2.5, 11.2; P0.0001) and 4.9 (1.6, 14.8; P = 0.0048), respectively] and in the C2 group [odds ratios: 15.9 (4.5, 56.8; P0.0001) and 9.0 (2.3, -35.2; P = 0.0017), respectively]. Cystatin C was another potent risk factor in the C2 group.A high prevalence of hyperhomocysteinemia in coastal West Africa, related to folate concentrations and the MTHFR 677 T allele, suggests the need to evaluate the influence of hyperhomocysteinemia on disease in this area.

5,10-Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) are two of the key enzymes in the folate/vitamin B12-dependent remethylation of homocysteine to methionine. The frequencies of MTHFR single nucleotide polymorphisms (SNPs), 677C-->T, 1298A-->C, 1317T-->C and of MTR, 2756A-->G, have been widely studied in Caucasians, but they have never been reported simultaneously in a large population from Sub-Saharan Africa. Presently, we report the prevalence of these SNPs and their relationship to homocysteine in 240 subjects recruited in West Africa. The frequencies of the mutant genotypes 677TT (0.8%) and 1298CC (2%) were lower than that usually observed in Caucasians, while the frequency of the mutant 1317CC was higher (16%). We formed a systematic association of the mutated MTHFR 677C-->T SNP with a 1298A/1317T common haplotype. The MTHFR mutant genotype 677TT was associated with an intermediate hyperhomocysteinemia (92.4 +/- 6.0 micromol/l) higher than that described in Caucasians. The 2756A-->G SNP in the MTR was similarly distributed in Africans compared to Caucasians. In conclusion, the MTHFR 677TTor 1298CC genotypes are much rarer in Africans than in Caucasians. The 677TT low frequency may be related to the high effect of this mutation on homocysteine metabolism in the environmental conditions of this African region.

The human colon adenocarcinoma cell line HT29 can be adapted to 10(-7)- 10(-4) M concentrations of methotrexate (MTX). Cells adapted to 10(-4) M MTX have an enterocyte-like phenotype with DHFR gene amplification. Presently, we hypothetized that an increased expression of folate binding protein (FBP) may participate to the MTX resistance of 10(-4) MTX HT29 cells. The cDNA FBPalpha/beta-actin ratio of amplified transcripts was 4.8- and 1.5- fold higher in 10(-4) and in 10(-7) M MTX HT29 respectively, than in standard type HT29 cells. An increase of transcript level was observed when decreasing folic acid concentration. PI-PLC cleaved 7.7 times more membrane FBP in 10(-4) M than in 10(-7) M MTX and wild type HT29 cells. In contrast to 10(-7) M MTX cells, growth of 10(-4) M MTX cells was dependent on folic acid concentration and abolished at a concentration lower than 0.9 microM. In conclusion, the adaptive mechanism of HT29 cells resistant to 10(-4) M MTX is the result of the synergistic overexpression of both DHFR and FBPalpha. Overexpression of FBPalpha may be related to the enterocyte-like phenotype of the cells.

Glomerular filtration is one of the major determinants of plasma total homocysteine (tHcy). To evaluate the respective roles of residual glomerular filtration (by measuring a specific protein marker, cystatin C), genetic polymorphisms and nutritional status in tHcy blood levels in end-stage renal disease patients (ESRD) under hemodialysis and supplemented with folate, we measured tHcy, folate, vitamin B 12 (B 12 ), creatinine, cystatin C, albumin and C-reactive protein and determined the polymorphism of methylenetetrahydrofolate reductase (MTHFR) (C677T and A1289C) and of methionine synthase (MS) (A2756G) in 114 ESRD patients before hemodialysis and 76 control subjects. All patients received a folate supplementation of 700 μg/day. Hyperhomocysteinemia was observed in all patients and exceeded the upper normal limit by 2-fold in 52.4% of the patients. Serum folate was significantly increased and the B 12 level was not different from controls. Folate, Cystatin C and creatinine were significantly correlated to tHcy, while no correlation was found between tHcy, albumin and C-reactive protein. No difference in genotype frequency between ESRD patients and controls was found for MTHFR A1289C and MS A2756G. The MTHFR 677TT genotype was less frequent and was associated with a significantly higher tHcy level in patients. Folate and residual glomerular filtration estimated by cystatin C and creatinine levels were two independent determinants of tHcy in ESRD patients. These data suggest that hyperhomocysteinemia is a consequence as well as a complicating factor of renal failure.