Hilary J. Powers

Effect of riboflavin status on the homocysteine-lowering effect of folate in relation to the MTHFR (C677T) genotype.

BACKGROUNDnRiboflavin (vitamin B(2)) is the precursor for FAD, the cofactor for methylenetetrahydrofolate reductase (MTHFR). MTHFR catalyzes the formation of 5-methyltetrahydrofolate, which acts as a methyl donor for homocysteine remethylation. Individuals with the MTHFR 677C-->T mutation have increased plasma total homocysteine (tHcy) concentrations, particularly in association with low folate status. It has been proposed that riboflavin may act together with folate to lower plasma tHcy, particularly in individuals with the thermolabile MTHFR T variant.nnnMETHODSnWe measured B-vitamin status and plasma tHcy in 126 healthy individuals 20-63 years of age (42 CC, 42 CT, and 42 TT MTHFR genotypes) at baseline and after three interventions (4 months): placebo plus natural diet; daily 400 microg folic acid supplement plus natural diet; and increased dietary folate to 400 microg/day.nnnRESULTSnAt baseline and after nutritional intervention, lower riboflavin status was associated with increased plasma tHcy concentrations. Plasma tHcy was 2.6 micromol/L higher in the lowest plasma riboflavin quartile compared with the highest (P <0.02) and was 4.2 micromol/L higher in the highest erythrocyte glutathione reductase activation coefficient (EGRAC) quartile compared with the lowest (P <0.001). This effect was not restricted to those with the T allele. Folic acid given as a 400 microg/day supplement appeared to exacerbate a tendency toward riboflavin deficiency, as suggested by an increase in the proportion of individuals with EGRAC > or =1.4 from 52% to 65% after supplementation (P <0.05).nnnCONCLUSIONSnFolate and riboflavin interact to lower plasma tHcy, possibly by maximizing the catalytic activity of MTHFR. The effect may be unrelated to MTHFR genotype.

Folate protects against oxidative modification of human LDL.

Elevated plasma total homocysteine is considered to be a graded risk factor for cardiovascular disease. Folate, through its homocysteine-lowering potential, may therefore be protective. Folate, however, may have protective effects independent of homocysteine-lowering. We have measured the effects of folate on Cu-catalysed oxidative damage to the unsaturated lipids in human LDL. Experiments were carried out in the presence of citrate, and followed increases in absorption at 234 nm, which measures the amount of conjugated diene produced. There is a lag time during which endogenous antioxidants are oxidised, followed by rapid oxidation of lipid. Addition of 0-6 microm - 5-methyltetrahydrofolate produced a dose-dependent increase in the lag time, suggesting that folate may have a direct anti-oxidant role in vivo, which is independent of any indirect effects through lowering of homocysteine levels.

Sensitivity of markers of DNA stability and DNA repair activity to folate supplementation in healthy volunteers.

We have previously reported that supplementation with folic acid (1.2u2009mgu2009day−1 for 12 week) elicited a significant improvement in the folate status of 61 healthy volunteers. We have examined effects of this supplement on markers of genomic stability. Little is known about the effect of folate supplementation on DNA stability in a cohort, which is not folate deficient. Preintervention, there was a significant inverse association between uracil misincorporation in lymphocyte DNA and red cell folate (P<0.05). In contrast, there were no associations between folate status and DNA strand breakage, global DNA methylation or DNA base excision repair (measured as the capacity of the lymphocyte extract to repair 8-oxoGua ex vivo). Folate supplementation elicited a significant reduction in uracil misincorporation (P<0.05), while DNA strand breakage and global DNA methylation remained unchanged. Increasing folate status significantly decreased the base excision repair capacity in those volunteers with the lowest preintervention folate status (P<0.05). Uracil misincorporation was more sensitive to changes in folate status than other measures of DNA stability and therefore could be considered a specific and functional marker of folate status, which may also be relevant to cancer risk in healthy people.

Folate Status and Aberrant DNA Methylation Are Associated With HPV Infection and Cervical Pathogenesis

Aberrant DNA methylation is a recognized feature of human cancers, and folate is directly involved in DNA methylation via one-carbon metabolism. Previous reports also suggest that folate status is associated with the natural history of human papillomavirus (HPV) infection. A cross-sectional study was conducted to test the hypothesis that folate status and aberrant DNA methylation show a progressive change across stages of cervical pathology from normal cells to cervical cancer. Additionally, we postulated that a gene-specific hypermethylation profile might be used as a predictive biomarker of cervical cancer risk. DNA hypermethylation of seven tumor suppressor genes, global DNA hypomethylation, systemic folate status, and HPV status were measured in 308 women with a diagnosis of normal cervix (n = 58), low-grade cervical intraepithelial neoplasia (CIN1; n = 68), high-grade cervical intraepithelial neoplasia (CIN2, n = 56; and CIN3, n = 76), or invasive cervical cancer (ICC; n = 50). Lower folate status was associated with high-risk HPV infection (P = 0.031) and with a diagnosis of cervical intraepithelial neoplasia or invasive cervical cancer (P < 0.05). Global DNA hypomethylation was greater in women with invasive cervical cancer than all other groups (P < 0.05). A cluster of three tumor suppressor genes, CDH1, DAPK, and HIC1, displayed a significantly increased frequency of promoter methylation with progressively more severe cervical neoplasia (P < 0.05). These findings are compatible with a role for folate in modulating the risk of cervical cancer, possibly through an influence over high-risk HPV infection. DAPK, CDH1, and HIC1 genes are potential biomarkers of cervical cancer risk. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2782–9)

Antioxidant supplementation with or without B-group vitamins after acute ischemic stroke: a randomized controlled trial.

BACKGROUNDnEvidence shows that there is a rapid increase in the production of markers of oxidative damage immediately after acute ischemic stroke and that endogenous antioxidant defenses are rapidly depleted, thus permitting further tissue damage. Several studies point to an antioxidant effect of B-group vitamins and a pro-oxidant effect of elevated total plasma homocysteine (tHcy).nnnMETHODSnTo test whether supplementary antioxidants with or without B-group vitamins during this critical period enhance antioxidant capacity or mitigate oxidative damage, ninety-six acute ischemic stroke patients within 12 hours of symptom onset were randomly assigned to receive either daily oral 800 IU (727 mg) vitamin E and 500 mg vitamin C (n = 24), or B-group vitamins (5 mg folic acid, 5 mg vitamin B(2), 50 mg vitamin B(6), and 0.4 mg of vitamin B(12); n = 24), both vitamins together (n = 24), or no supplementation (n = 24) for 14 days. Treatment groups and controls were matched for stroke subtype and age. Blood was obtained before treatment, at day 7, and day 14 for measurements of plasma or blood vitamin status, plasma total antioxidant capacity (TAOC), malondialdehyde (MDA), tHcy and C-reactive protein (CRP).nnnRESULTSnSupplementation with antioxidant vitamins and B-group vitamins separately or together significantly increased the plasma concentration of vitamin C, E, pyridoxal phosphate (B(6) status), red blood cell folate, and improved a measure of B(2) status (red cell glutathione reductase activation coefficient [EGRAC]), compared with the control group. Plasma TAOC increased significantly in the antioxidant treatment groups compared with the nonsignificant decline seen in the control group. tHcy concentrations decreased in subjects who received B-group vitamins and the control group compared with the rise seen in those who received antioxidants alone. There was a significant reduction in plasma MDA concentration in the 3 treatment groups, in contrast to the increase seen in the control group; however, the changes were most evident in antioxidant groups. CRP concentrations (a marker of tissue inflammation) were significantly lower in the 3 treatment groups compared with the control group. There were no additive or synergistic effects of antioxidants and B-group vitamins together on any outcome measure.nnnCONCLUSIONSnAntioxidants supplementation with or without B-group vitamins enhances antioxidant capacity, mitigates oxidative damage, and may have an anti-inflammatory effect immediately postinfarct in stroke disease.

Elevated plasma homocysteine elicits an increase in antioxidant enzyme activity.

Elevated plasma homocysteine is considered to be a risk factor for cardiovascular disease. The mechanisms for this effect are not fully understood but there is some evidence for a role for reactive oxygen species (ROS). This study was conducted to explore the effects of elevated plasma total homocysteine (tHcy) concentration on activity of antioxidant enzymes in the circulation. The study group consisted of 10 patients with inherited defects of homocysteine metabolism, from whom 41 blood samples were collected over a period of six months. Blood samples were also collected from 13 of their obligate heterozygous parents. For data analysis samples were classified as those with plasma tHcy < 20 μM or ≥ 20 μM. The activity of erythrocyte superoxide dismutase (SOD) and plasma glutathione peroxidase (GSHPx) was elevated in samples with plasma tHcy > 20 μM. Moreover, a significant correlation was demonstrated between plasma GSHPx activity, plasma glutathione peroxidase protein and plasma tHcy. In vitro studies confirmed that this observation was not due to a simple chemical enhancement of enzyme activity. Homocysteine protected GSHPx from loss of activity following incubation at 37°C. A similar effect was seen with another thiol-containing amino acid, cysteine. Results suggest that elevated plasma tHcy represents an oxidative stress, resulting in an adaptive increase in activity of antioxidant enzymes in the circulation.

Riboflavin deficiency in the rat: effects on iron utilization and loss.

Iron absorption and daily loss of Fe were measured in riboflavin-deficient (B2-) Norwegian hooded rats and controls (B2+). Animals were fed on a test meal extrinsically labelled with 59Fe and whole-body radioactivity measured for 15 d. Riboflavin deficiency led to a reduction in the percentage of the 59Fe dose absorbed and an increased rate of 59Fe loss. All post-absorption 59Fe loss could be accounted for by faecal 59Fe, confirming that the loss was gastrointestinal. Fe concentrations and 59Fe as a percentage of retained whole-body 59Fe were higher in the small intestine of riboflavin-deficient animals than their controls, 14 d after the test meal. A separate experiment demonstrated that riboflavin deficiency was associated with a significant proliferative response of the duodenal crypts of the small intestine. These observations may explain the enhanced Fe loss in riboflavin deficiency.

Riboflavin deficiency and iron absorption in adult gambian men

Iron absorption from 3.38 mg 58Fe was measured in riboflavin-deficient Gambian men with haemoglobin (Hb) less than 11.5 g/dl before and after oral riboflavin therapy, and the results compared with a group not receiving riboflavin. Riboflavin status (as determined by erythrocyte glutathione reductase activation coefficient) and Hb increased in teh riboflavin-supplemented but not placebo group. Plasma ferritin levels were low and did not change in either group. There was very wide variation in percentage iron absorption between individuals and also within single individuals on two separate occasions but no measurable change with riboflavin supplementation. The results of the study indicate that the efficiency of iron utilization is impaired in riboflavin deficiency, but that iron absorption is unaffected.

Blood Folate Status and Expression of Proteins Involved in Immune Function, Inflammation, and Coagulation: Biochemical and Proteomic Changes in the Plasma of Humans in Response to Long-Term Synthetic Folic Acid Supplementation

We used plasma proteomics to identify human proteins responsive to folate status. Plasma was collected from subjects treated with placebo or 1.2 mg of folic acid daily for 12 weeks in a randomized controlled trial. Homocysteine and folate were measured by immunoassay and uracil misincorporation by electrophoresis. The plasma proteome was assessed by 2-D gel electrophoresis, and proteins were identified by LC MS/MS. 5-methylTHF increased 5-fold (P = 0.000003) in response to intervention. Red cell folate doubled (P = 0.013), and lymphocyte folate increased 44% (P = 0.0001). Hcy and uracil dropped 22% (P = 0.0005) and 25% (P = 0.05), respectively. ApoE A-1, alpha-1-antichymotrypsin, antithrombin, and serum amyloid P were downregulated, while albumin, IgM C, and complement C3 were upregulated (P < 0.05). More than 60 proteins were significantly associated with folate pre- and postintervention (P < 0.01). These were categorized into metabolic pathways related to complement fixation (e.g., C1, C3, C4, Factor H, Factor 1, Factor B, clusterin), coagulation (e.g., antithrombin, alpha-1-antitrypsin, kininogen) and mineral transport (e.g., transthyretin, haptoglobin, ceruloplasmin). Low folate status pre- and post-treatment were associated with lower levels of proteins involved in activation and regulation of immune function and coagulation. Supplementation with synthetic folic acid increased expression of these proteins but did not substantially disrupt the balance of these pathways.

Hyperhomocystinemia in children with inflammatory bowel disease.

Objectives Thromboembolism is a significant cause of morbidity and mortality in patients with inflammatory bowel disease (IBD). Plasma total homocysteine (tHcy) is a risk factor for vascular disease and has been implicated as a mediator of thromboembolic events in adults with IBD. The authors studied the link between tHcy and IBD in children, in whom associations may be clearer, and investigated associations with plasma von Willebrand factor antigen, a marker of vascular damage. Methods This cross-sectional study included 43 patients with IBD (27 Crohn disease, 9 ulcerative colitis, and 7 indeterminate colitis) and 46 control subjects from a pediatric gastroenterology clinic. Plasma tHcy, plasma 5-methyl tetrahydrofolate, red cell folate, plasma vitamin B12, plasma von Willebrand factor antigen, and methylene tetrahydrofolate reductase (MTHFR) genotype (for the C677T mutation) were measured. Results Plasma tHcy concentrations were higher in children with IBD than in control subjects, when corrected for age (P < 0.05), and plasma tHcy was negatively correlated with plasma 5 methyl tetrahydrofolate (P < 0.0005). Plasma 5 methyl tetrahydrofolate and age were the main predictors of plasma tHcy. Neither MTHFR genotype nor von Willebrand factor showed any association with any other measure, and there were no differences between children with IBD and control subjects. Conclusions Elevated plasma tHcy is a consequence of IBD in children, probably mediated by poor folate status associated with diet or the pathophysiology of the disease.