Marilyn H. Hill

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 (1.2 mg day−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.

Correcting a marginal riboflavin deficiency improves hematologic status in young women in the United Kingdom (RIBOFEM)

BACKGROUND Moderate riboflavin deficiency is prevalent in certain population groups in affluent countries, but the functional significance of this deficiency is not clear. Studies have indicated a role for riboflavin in the absorption and use of iron. OBJECTIVE We investigated the effect of riboflavin supplementation on hematologic status in a group of moderately riboflavin-deficient women aged 19-25 y in the United Kingdom. DESIGN One hundred twenty-three women with biochemical evidence of riboflavin deficiency [erythrocyte glutathione reductase activation coefficient (EGRAC) >1.40] were randomly assigned to receive 2 or 4 mg riboflavin or a placebo for 8 wk. Measurements of hematologic status were made pre- and postsupplementation, and dietary intakes were also assessed; iron absorption was measured in a subgroup of women. RESULTS One hundred nineteen women completed the intervention. The use of a riboflavin supplement for 8 wk elicited a significant improvement in riboflavin status with a dose response (P < 0.0001). For women who received supplemental riboflavin, an increase in hemoglobin status correlated with improved riboflavin status (P < 0.02). Women in the lowest tertile of riboflavin status at baseline (EGRAC >1.65) showed a significantly greater increase in hemoglobin status in response to the supplement than did women in the first and second tertiles (P < 0.01). Dietary iron intake and iron absorption did not change during the study. CONCLUSIONS Moderately poor riboflavin status can affect iron status: the lower the riboflavin status, the greater the hematologic benefits of improving status. The results also suggest that consideration should be given to raising the currently accepted EGRAC threshold for deficiency. This trial was registered at controlled-trials.com as ISRCTN35811298.

Responses of biomarkers of folate and riboflavin status to folate and riboflavin supplementation in healthy and colorectal polyp patients (the FAB2 Study).

Epidemiologic data suggest that increasing folate intake may protect against colorectal cancer. Riboflavin may interact with folate to modulate the effect. A double-blind randomized placebo-controlled intervention study (the FAB2 Study) was carried out in healthy controls and patients with colorectal polyps (adenomatous and hyperplastic) to examine effects of folic acid and riboflavin supplements on biomarkers of nutrient status and on putative biomarkers of colorectal cancer risk (DNA methylation and DNA damage; to be reported elsewhere). Ninety-eight healthy controls and 106 patients with colorectal polyps were stratified for the thermolabile variant of methylene tetrahydrofolate reductase, MTHFR C677T, and were randomized to receive 400 μg of folic acid, 1,200 μg of folic acid, or 400 μg of folic acid plus 5 mg of riboflavin or placebo for 6 to 8 weeks. Blood samples and colon biopsy samples were collected for the measurement of biomarkers of folate and riboflavin status. Supplementation with folic acid elicited a significant increase in mucosal 5-methyl tetrahydrofolate, and a marked increase in RBC and plasma, with a dose-response. Measures of riboflavin status improved in response to riboflavin supplementation. Riboflavin supplement enhanced the response to low-dose folate in people carrying at least one T allele and having polyps. The magnitude of the response in mucosal folate was positively related to the increase in plasma 5-methyl tetrahydrofolate but was not different between the healthy group and polyp patients. Colorectal mucosal folate concentration responds to folic acid supplementation to an extent comparable to that seen in plasma, but with a suggestion of an upper limit. (Cancer Epidemiol Biomarkers Prev 2007;16(10):2128–35)

A vitamin B-12 supplement of 500 μg/d for eight weeks does not normalize urinary methylmalonic acid or other biomarkers of vitamin B-12 status in elderly people with moderately poor vitamin B-12 status.

Plasma vitamin B-12 is the most commonly used biomarker of vitamin B-12 status, but the predictive value for low vitamin B-12 status is poor. The urinary methylmalonic acid (uMMA) concentration has potential as a functional biomarker of vitamin B-12 status, but the response to supplemental vitamin B-12 is uncertain. A study was conducted to investigate the responsiveness of uMMA to supplemental vitamin B-12 in comparison with other biomarkers of vitamin B-12 status [plasma vitamin B-12, serum holotranscobalamin (holoTC), plasma MMA] in elderly people with moderately poor vitamin B-12 status. A double-blind, placebo-controlled, randomized 8-wk intervention study was carried out using vitamin B-12 supplements (500 μg/d, 100 μg/d, and 10 μg/d cyanocobalamin) in 100 elderly people with a combined plasma vitamin B-12 <250 pmol/L and uMMA ratio (μmol MMA/mmol creatinine) >1.5. All biomarkers had a dose response to supplemental vitamin B-12. Improvements in plasma vitamin B-12 and serum holoTC were achieved at cobalamin supplements of 10 μg/d, but even 500 μg/d for 8 wk did not normalize plasma vitamin B-12 in 8% and serum holoTC in 12% of people. The response in uMMA was comparable with plasma MMA; 15-25% of people still showed evidence of metabolic deficiency after 500 μg/d cobalamin for 8 wk. There was a differential response in urinary and plasma MMA according to smoking behavior; the response was enhanced in ex-smokers compared with never-smokers. uMMA offers an alternative marker of metabolic vitamin-B12 status, obviating the need for blood sampling.

Erythrocyte pyridoxamine phosphate oxidase activity: a potential biomarker of riboflavin status?

BACKGROUND Riboflavin status is commonly measured by the in vitro stimulation of erythrocyte glutathione reductase with flavin adenine dinucleotide and expressed as an erythrocyte glutathione reductase activation coefficient (EGRAC). However, this assay is insensitive to poor riboflavin status in subjects with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Because G6PD deficiency is common in parts of the world where ariboflavinosis is endemic, it is important to have a measure of riboflavin status that is unaffected by differences in G6PD status. OBJECTIVE The objective was to further develop and validate a fluorometric assay for pyridoxamine phosphate oxidase (PPO) activity as a measure of riboflavin status. DESIGN A fluorometric assay was optimized for the flavin-dependent enzyme PPO in erythrocytes. Hemolysates from a previous riboflavin intervention study (2- and 4-mg riboflavin supplements) were used to investigate the responsiveness of the method to changes in riboflavin intake. RESULTS PPO activity and the PPO activation coefficient (PPOAC) were used to assess riboflavin status. Both PPO activity and PPOAC responded to riboflavin supplements (P < 0.01), but only PPO showed a dose response (P < 0.001). The change from baseline to after the intervention in PPOAC and PPO enzyme activity was significantly inversely correlated (P < 0.001). Both PPO activity and PPOAC were strongly correlated with EGRAC (P < 0.001). Additionally, both PPOAC and EGRAC showed a significant inverse correlation with dietary riboflavin intake (P < 0.01); PPO activity was positively correlated with riboflavin intake (P < 0.01). CONCLUSION PPO activity could be used as a biomarker for measuring riboflavin status, especially in populations with a high prevalence of G6PD deficiency. This trial is registered at www.isrctn.org as ISRCTN35811298.

Study Protocol: Randomised controlled trial to investigate the functional significance of marginal riboflavin status in young women in the UK (RIBOFEM)

BackgroundThe functional significance of moderate riboflavin deficiency as it is currently assessed is not well understood. Animal and human studies have suggested a role for riboflavin in the absorption and mobilisation of iron and as such may be important in maintaining haematological status. Recent National Diet and Nutrition Surveys in the United Kingdom have shown that young women in particular are at risk of moderate riboflavin deficiency and low iron status.Methods/DesignA randomised placebo controlled intervention trial was conducted to investigate the effect of riboflavin supplementation on various measures of haematological status in a group of moderately riboflavin deficient young women aged 19 to 25 years. Women who were low milk consumers were initially screened for riboflavin status as assessed by the erythrocyte glutathione reductase activation coefficient assay (EGRAC). One hundred and twenty three women with EGRAC values >1.40 were randomised to receive 2 mg, 4 mg riboflavin or placebo for 8 weeks. In addition 36 of these women were randomly allocated to an iron bioavailability study to investigate the effect of the intervention on the absorption or utilisation of iron using an established red cell incorporation technique.DiscussionOne hundred and nineteen women completed the intervention study, of whom 36 completed the bioavailability arm. Compliance was 96 ± 6% (mean ± SD). The most effective recruitment strategy for this gender and age group was e-communication (e-mail and website). The results of this study will clarify the functional significance of the current biochemical deficiency threshold for riboflavin status and will inform a re-evaluation of this biochemical threshold.Trial RegistrationCurrent Controlled Trials Registration No. ISRCTN35811298