Philippe Gerard

Nutritional and genetic determinants of vitamin B and homocysteine metabolisms in neural tube defects: A multicenter case–control study†

Neural tube defects (NTDs) are severe congenital malformations due to failure of neural tube formation in early pregnancy. The proof that folic acid prevents NTDs raises the question of whether other parts of homocysteine (Hcy) metabolism may affect rates of NTDs. This French case‐control study covered: 77 women aged 17–42 years sampled prior to elective abortion for a severe NTDs (cases) and 61 women aged 20–43 years with a normal pregnancy. Plasma and erythrocyte folate, plasma B6, B12 and Hcy were tested as five polymorphisms MTHFR 677 C → T, MTHFR 1298 A → C, MTR 2756 A → G, MTTR 66 A → G and TCN2 776 C → G. Cases had significantly lower erythrocyte folate, plasma folate, B12 and B6 concentrations than the controls, and higher Hcy concentration. The odds ratio was 2.15 (95% CI: 1.00–4.59) for women with the MTRR 66 A → G allele and it was decreased for mothers carrying the MTHFR 1298 A → C allele. In multivariate analysis, only the erythrocyte folate concentration (P = 0.005) and plasma B6 concentration (P = 0.020) were predictors. Red cell folate is the main determinant of NTDs in France. Folic acid supplement or flour fortification would prevent most cases. Increased consumption of vitamins B12 and B6 could contribute to the prevention of NTDs. Genetic polymorphisms played only a small role. Until folic acid fortification becomes mandatory, all women of reproductive age should consume folic acid in a multivitamin that also contains B12 and B6.

Alcoholic Cirrhosis and Cobalamin Metabolism

The cobalamin status of 27 patients suffering from alcoholic cirrhosis and 20 control subjects was analyzed. Plasma cobalamin (p < 0.005), total corrinoids (p < 0.005) and their analogs (p < 0.05) were all significantly elevated in the cirrhosis patients. These differences were due to increased haptocorrin (HC)-bound corrinoid (p < 0.02), which could be explained by a deficient hepatic clearance of cobalamin bound to HC. The increase in the concentration of true cobalamin was greater than that of its analogs. There were positive correlations between cholestasis (serum alkaline phosphatase) and plasma analog concentrations (p < 0.05), HC-bound cobalamin (p < 0.005) and total corrinoids bound to HC (p < 0.005). The plasma concentrations of the indicators of cobalamin deficiency, homocysteine (p < 0.05) and methylmalonic acid (p < 0.001), were increased, which could indicate poor cellular penetration of vitamin B12 or a defect in the activation of the two vitamin-B12-dependent enzymes.

Effects of vitamin B12 and folate deficiencies on DNA methylation and carcinogenesis in rat liver.

Abstract Deficiencies of the major dietary sources of methyl groups, methionine and choline, lead to the formation of liver cancer in rodents. The most widely investigated hypothesis has been that dietary methyl insufficiency results in abnormal DNA methylation. Vitamin B12 and folate also play important roles in DNA methylation since these two coenzymes are required for the synthesis of methionine and S-adenosyl methionine, the common methyl donor required for the maintenance of methylation patterns in DNA. The aim of this study was to review the effects of methyl-deficient diets on DNA methylation and liver carcinogenesis in rats, and to evaluate the role of vitamin B12 status in defining carcinogenicity of a methyl-deficient diet. Several studies have shown that a methyl-deficient diet influences global DNA methylation. Evidence from in vivo studies has not clearly established a link between vitamin B12 and DNA methylation. We reported that vitamin B12 and low methionine synthase activity were the two determinants of DNA hypomethylation. Choline- or choline/methionine-deficient diets have been shown to cause hepatocellular carcinoma in 20–50% of animals after 12–24 months. In contrast, the effect of vitamin B12 withdrawal, in addition to choline, methionine and folate, induced hepatocellular carcinoma in less than 5% of

Cobalamin malabsorption due to nondegradation of R proteins in the human intestine. Inhibited cobalamin absorption in exocrine pancreatic dysfunction.

In vivo studies demonstrate that the pancreatic enzymes and the ionic environment in the upper gastrointestinal tract are essential determining factors for transport and absorption of cobalamin in man. Jejunal fluid was aspirated from healthy human volunteers after administration of cyano[57Co]cobalamin preparations. Immunochemical analysis of the aspirates demonstrated that all isotopic vitamin was transferred to a protein that is identical to the gastric intrinsic factor in terms of molecular mass (57,500), ionic nature (mean pI, 5.09), and reactivity with anti-intrinsic factor sera. However, in the aspirates from patients with exocrine pancreatic dysfunction the vitamin was found to be coupled > 60% to a protein identical to R proteins in terms of molecular mass (125,000), ionic nature (mean pI, 3.51), and reactivity with anti-R protein and anti-intrinsic factor sera. The preferential transfer of cobalamin to R proteins in the patients and to intrinsic factor in healthy subjects was associated, respectively, with low and normal levels of pancreatic enzymes in the intestine and these in turn were paralleled respectively by impaired and normal ileal absorption of cobalamin. These findings confirm the suggestion that the formation of unabsorbable cobalamin complexes may be the reason of impaired vitamin absorption in exocrine pancreatic insufficiency. Observations made with other selected patients demonstrate: (a) that decreased enzyme activity and nondegradation of R proteins may also be due to nonactivation of pancreatic zymogens in an acidic pH of the intestinal juice the vitamin transported to the jejunum couples to intrinsic factor when pancreatic function is normal, and to intrinsic factor and R protein in exocrine pancreatic insufficiency. The observations made with these selected patients may explain why not all patients with exocrine pancreatic insufficiency develop imparied cobalamin absorption, and also why the malabsorption is corrected by the administration of bicarbonate in certain patients.

Homocysteine and related genetic polymorphisms in Down’s syndrome IQ

Objective: Down’s syndrome (DS) is the most frequent genetic cause of Alzheimer-type dementia. Its metabolic phenotype involves an increased trans-sulphuration of homocysteine. The aim of the present study was to investigate the influence of homocysteinaemia (t-Hcys), folate, vitamin B12, and related polymorphisms on intelligence quotient (IQ) in DS. Methods: The IQ of 131 patients with trisomy 21 from a specialist centre in Sicily was determined and classified according to DMS-IV. The effects of age, folate, vitamin B12, t-Hcys, and genetic polymorphisms on IQ were evaluated separately and in combination using regression analyses. Results: IQ was significantly lower in DS patients with t-Hcys >7.5 μmol/l (median) and in those who were carriers of methylenetetrahydrofolate reductase (MTHFR) 677 T allele and of methylenetetrahydrofolate reductase 677 T and transcobalamin 776 G combined alleles (p = 0.0013, p = 0.0165, and p = 0.0074, respectively). The IQ correlated significantly with t-Hcys and folate in single and multiple regression analyses, independently of age. In addition, t-Hcys >9.6 μmol/l (upper quartile) was found to be associated with low IQ (<40, median of study group) with an odds ratio of 2.61 (p = 0.0203). The odds ratio was increased by threefold in carriers of MTHFR 677T allele. The MTHFR 677T allele/transcobalamin 776 G allele combination was associated with the risk of DS patients to have an IQ less that the median with an odds ratio of 2.68 (95% CI 1.26 to 5.70, p = 0.0104). Conclusion: This study found evidence of an association between t-Hcys and MTHFR 677 T and transcobalamin 776 G alleles with IQ in patients with DS. The association may be related to a defective remethylation of homocysteine, affecting IQ.

Constitutive nitric oxide synthase inhibition combined with histamine and serotonin receptor blockade improves the initial ovalbumin-induced arterial hypotension but decreases the survival time in brown norway rats anaphylactic shock.

Anaphylactic shock accidents after allergen exposure are frequent. After immunization with ovalbumin (OVA), a common dietary constituent, we evaluated the efficacy of pretreatment with histamine-receptor or serotonin-receptor blockers administered alone or in combination with a nitric oxide synthase inhibitor (L-NAME) on OVA-induced anaphylactic shock in Brown Norway rats. Animals were allocated to the following groups (n = 6 each): control (0.9% saline); diphenydramine (15 mg kg−1); cimetidine (20 mg kg−1); diphenydramine + cimetidine; dihydroergotamine (50 &mgr;g kg−1); diphenydramine + cimetidine + dihydroergotamine; L-NAME (100 mg/kg) alone or associated with diphenydramine, cimetidine, diphenydramine + cimetidine, dihydroergotamine, or diphenydramine + cimetidine + dihydroergotamine. Mean arterial blood pressure (MABP), heart rate (HR), and survival time were monitored for 60 min following treatment. The shock was initiated with i.v. OVA. The MABP drop after i.v. OVA was worsened by diphenydramine and was modestly attenuated by cimetidine, dihydroergotamine, or both together. L-NAME potentiated slightly the effects of cimetidine and dihydroergotamine by lessening the initial MABP decrease, but this transient effect was not sufficient to prevent the final collapse or to improve survival time. Decreased vasodilatory (prostaglandins E2), increased vasoconstrictory (thromboxane B2) prostaglandins, and unchanged leukotriene C4 concentrations were contributory to the overall hemodynamic changes. Thus, the combined blockade of vasodilator mediators (histamine, serotonin, and nitric oxide) slowed the MABP drop in anaphylactic shock, but did not improve survival. More studies are needed to understand these discordant effects.

Crohn's disease and vitamin B12 metabolism

The concentrations of vitamin B12, its analogs, and the haptocorrin and transcobalamin carriers in 21 patients suffering from Crohns 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 Crohns and control patients. There was a significant increase in the binding capacity of transcobalamin in the Crohns patients compared to the controls (P<0.001), but there was no difference in the binding capacities of haptocorrin. The serum concentrations of the markers of vitamin B12 status, homocysteine and methylmalonic acid, showed an increase (P<0.01) in homocysteine in the Crohns disease patients, but no change in methylmalonic acid. As the hyperhomocysteinemia was associated with normal folate concentrations, there may have been a defect in the activation of the enzyme due to altered intracellular vitamin B12 status.

Hyperhomocysteinemia is related to residual glomerular filtration and folate, but not to methylenetetrahydrofolate-reductase and methionine synthase polymorphisms, in supplemented end-stage renal disease patients undergoing hemodialysis.

Abstract 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 B12 (B12), 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 B12 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.

Blood transcobalamin levels in malignant hepatoma.

Alpha-Fetoprotein (AFP) levels measured by RIA show a strong sensitivity for the biological diagnosis of malignant hepatoma (MH). However, this parameter lacks specificity. Previous observations of an alteration in vitamin B12 metabolism in the presence of hepatoma led us to study vitamin-B12-binding proteins. Vitamin B12, also called cobalamin, is transported in the blood by two proteins or transcobalamins: one is haptocorrin (HC), which is linked to most of the cobalamin, and the other is transcobalamin II, which is involved in tissue exchanges. In this work, the levels of AFP and transcobalamins were determined by RIA and radioisotope dilution assay, respectively. They were measured in patients with MH (group A) and in patients with other liver diseases (group B). Compared with group B, group A showed a significant increase in total serum HC (p less than 0.005). In conclusion, it was observed that MH is accompanied by increased levels of HC. The origin of these changes could be due to either an increase in HC synthesis or a catabolic defect.

Increased serum corrinoids correlates with disease severity and IgA levels in alcoholic cirrhosis

Relationship between increased serum cobalamin level and liver disease have been recently reported. In this work, levels of total corrinoids, cobalamin (vitamin B12) and cobalamin analogues and levels of IgA were determined by radioisotope dilution assay and nephelometric laser analyses. They all have been measured in superior vena cava, inferior vena cava and hepatic vein of controls and of alcoholic cirrhotic patients grouped according to the Child-Pugh classification. Compared with normal subjects, venous blood content of total corrinoids, of cobalamin and of IgA in alcoholic cirrhotics increased significantly with the severity of the disease (p less than 0.01). In severe, moderate, and mild alcoholic cirrhosis total corrinoids and cobalamin were, respectively, about 5-, 2-, and 1.5-fold higher than in controls, whereas IgA was 3-, 2.5- and 1.5-fold higher, respectively. The serum IgA level was significantly correlated with the level of seric saturated haptocorrin (r = 0.54; p less than 0.01) and with the seric total corrinoids (r = 0.39; p less than 0.01). In the absence of significant hepatic cytolysis, the enhanced level of seric corrinoids in cirrhosis could be partly explained by a competitive inhibition of the liver uptake of haptocorrin by circulating asialoglycoproteins, including IgA.