N05033: Evaluation of riboflavin status as modulator of homocysteine response to folate in relation to MTHFR genotype and endothelial function

Study Duration: May 2001 to April 2002

Contractor: University of Wales, College of Medicine

Low dietary intake of the vitamin folate is associated with an increased risk of neural tube defects, cardiovascular disease and cancer. Elevated plasma homocysteine concentrations are a marker of low folate status and have been shown to be a risk factor for cardiovascular disease. A genetic variant in the way that the body processes folate is common in the UK population (methylenetetrahydrofolate reductase (MTHFR) TT genotype ~12%). Individuals with this variant require a higher intake of folate to maintain a low homocysteine concentration. The vitamin riboflavin (B2) is required to provide the cofactor for the folate metabolising enzyme MTHFR. Recent studies have reported that low riboflavin status is associated with raised plasma homocysteine with some evidence that this effect may be confined to those subjects with the genetic variant in MTHFR combined with a low intake of folate from the diet.

Rationale and Objectives

It was hypothesised that subjects with the genetic MTHFR T variant may require higher intakes of folate and riboflavin derived cofactors to overcome the partial metabolic block and maintain low concentrations of plasma homocysteine. The effect of riboflavin status on homocysteine concentrations by folate and folic acid supplements has not been evaluated previously in an intervention study. This may have important health implications for advising on folate and riboflavin intakes in relation to reducing risk of folate related diseases (neural tube defects, cardiovascular disease and cancer).

The study assessed the influence of riboflavin in interacting with folate to lower plasma homocysteine in subjects with different MTHFR genotype status. Blood samples and dietary questionnaires were utilised from previous studies funded by the FSA (Projects N05002 and N05006). The original studies explored the effect of dietary intervention to increase folate intake on plasma homocysteine concentration and vascular endothelial function in individuals with different MTHFR genotypes [Pullin et al (2001) & Ashfield-Watt et al (2002)].

Study subjects

Healthy subjects aged 18-65 were recruited in South Wales. Exclusion criteria were: smoking, history of cardiovascular disease or epilepsy, vitamin supplements, drugs known to interfere with folate metabolism or pregnancy. All subjects provided written, informed consent. Ethical approval was obtained from the Local Research Ethics Committee.

Study design

126 subjects (42TT, 42CT, and 42CC) were recruited to a double-blind Latin square cross-over intervention study. Each subject underwent 3 x 4 month interventions in random order.

Interventions

1) Control diet: Subjects were asked consume their normal diet but to avoid folate-fortified foods and to take a placebo tablet daily.

2) Natural folate enriched diet: Subjects were encouraged to consume their usual diet plus additional folic acid fortified foods and naturally folate rich foods to achieve a total of at least 400 micrograms per day.

3) Supplement: Subjects were asked to consume the same diet as in intervention 1, but to take a 400 micrograms folic acid supplement daily.

Each intervention was carried out double blind such that neither the nutritionist nor the subject knew which treatment (control or supplement) they were receiving. Subjects attended for assessment on 4 occasions. Baseline measurements were made at visit 1 and post intervention measurements at visits 2, 3, and 4. Each visit consisted of blood sampling, dietary interview and assessment of vascular endothelial function. Blood samples stored from this previous FSA funded study enabled us to investigate the potential effect of riboflavin on folate and homocysteine metabolism. Samples were analysed by three different collaborators, who have the relevant expertise to carry out these analyses.

a) Plasma riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) by an HPLC assay successfully developed for this project in Cardiff.

b) Erythrocyte Glutathione Reductase Activation Coefficienct (EGRAC). A well established measure of riboflavin status. High activation coefficients indicate low riboflavin status. Spectrophotometric assay carried out in Sheffield.

c) Red Cell Folate assay. Microbiological assay was attempted as this is reported to be unaffected by genotype in contrast to protein binding assays which can give misleading results on red cells. Assays were carried out in Norwich. However the results were all very low indicating that the samples for this analyte were not suitable probably due to inadequate concentrations of antioxidant ascorbate for prolonged storage. The plasma folate assays carried previously out on samples in Cardiff using a protein binding method (IMX) are valid as these samples were stored for a short period and the protein binding method is suitable for plasma.

d) Homocysteine measured by enzymatic immunoassay (IMX) method in Cardiff.

Riboflavin nutritional status of subjects at baseline
The mean dietary intake of riboflavin in the study population was 1.45(0.51) milligrams per day. Overall, thirty percent of the study population at baseline did not achieve the UK reference nutrient intake (RNI) for riboflavin intake.

Biochemical measures of riboflavin status
Riboflavin concentrations increased with age and EGRAC level was also associated with age but not to gender or genotype.

Riboflavin, Homocysteine, Folate status and MTHFR genotype at baseline
Plasma homocysteine was significantly inversely correlated with plasma riboflavin and significantly positively associated with EGRAC. Plasma homocysteine was 2.6 micromols per litre higher in the lowest plasma riboflavin quartile compared to the highest and was 4.2 micromols per litre higher in the highest EGRAC quartile compared to the lowest.

Subjects with the TT genotype had significantly higher homocysteine concentrations compared with either the CT or CC genotypes. Plasma folate was the lowest in TT group compared with either the CT or CC genotypes. CT individuals had intermediate values. When split by by genotype there was an association between Hcy and plasma riboflavin or EGRAC in the CC and TT subjects but not in the CT group.

In a stepwise linear regression model plasma homocysteine was shown to be significantly determined by four factors: plasma folate, MTHFR TT genotype, EGRAC and creatinine.

Effect of fortified foods on riboflavin status
Riboflavin intake was increased by 0.4 milligrams per day in the period which included fortified foods from 1.45 + or - 0.51 to 1.85 + or - 0.65 milligrams per day (P < 0.001). The intake of riboflavin in the control (1.24 + or - 0.52 milligrams per day) and supplement (1.19 + or - 0.40 milligrams per day) periods were similar and are both significantly lower than the baseline period (P < 0.001) which is consistent with the advice given to avoid fortified cereals during these periods.

Riboflavin status and vascular endothelial function
Riboflavin status at baseline or following increased intake from fortified foods had no significant effect on vascular endothelial function as assessed by flow-mediated dilatation.

Effect of increased folic acid intake on riboflavin status
Folate intake increased following the dietary intervention by 228 micrograms per day from 254 + or - 83 micrograms per day at baseline to 482 + or - 127 micrograms per day. The folic acid supplementation period increased folate intake relative to baseline by 307 micrograms to 561 + or - 98 micrograms per day. Folate intake dropped during the control phase (216 + or - 82 micrograms per day) when fortified cereals were excluded from the diet. Plasma riboflavin was significantly lower in the folic acid supplementation group. EGRAC was significantly lower following the dietary regime when compared to baseline or control and showed a borderline significant increase following the supplement regime.

Riboflavin status assessed by either plasma riboflavin or EGRAC was reduced following supplements with folic acid alone. The folic acid supplement regime increased the proportion of subjects with an EGRAC of below 1.4 from 52% at baseline to 65% following folic acid supplements. This value (1.4) is often regarded as a conservative cut-off for satisfactory riboflavin status.

These studies provide further information on the relative value of different biochemical markers of riboflavin status. The best biochemical indicator of riboflavin status appears to be EGRAC with plasma riboflavin being a less reliable indicator overall. Plasma FMN and FAD do not appear to be useful markers of riboflavin status.

Age and gender differences in riboflavin intake and biochemical markers of status were consistent with National trends suggesting a high prevalence of low riboflavin status in the general population. Although riboflavin intake estimates are commonly reported to correlate strongly with EGRAC values, the latter may tend to overestimate the prevalence of deficiency, a pattern repeated in the study reported here.

The fortified diet intervention led to an increase in both folate and riboflavin intake showing that fortified cereals can have a significant impact on the intake of those vitamins.

The finding that plasma homocysteine levels were inversely related with plasma riboflavin and positively associated with EGRAC confirms that riboflavin is an important dietary influence on this compound in addition to dietary folate. The suggestion from previous studies that this effect was confined to only those with T variant in MTHFR was not confirmed by this study.

The observation that the degree of homocysteine lowering following fortified foods (which increased both folate and riboflavin intake) is comparable to that from a higher dose of folic acid and suggests that both vitamins have an additive or synergistic effect on homocysteine lowering. However, these results could also be partly explained by a plateau effect with higher dose folic acid with respect to homocysteine lowering.

The observation that folic acid supplementation of 400 micrograms per day for 3 months produced a significant decrease in riboflavin status was an interesting and unexpected finding. This would suggest that folic acid supplementation alone may increase the rate of turnover of flavins thereby exacerbating any tendency to riboflavin deficiency.

The lack of any association between riboflavin status at baseline or following increased intake form fortified foods with vascular endothelial function suggests that increasing riboflavin intake may not have additional benefits to the cardiovascular system.

In conclusion our results show

1. A high prevalence of low riboflavin status in the UK population.

2. A synergistic effect of riboflavin and folate on homocysteine lowering which is related to MTHFR genotype for folate but possibly not for riboflavin.

3. Preliminary evidence that folic acid supplements alone may have an effect to reduce riboflavin status.

Further studies are required particularly to investigate further the possible effect of folic acid on riboflavin status.

Pullin CH, Ashfield-Watt PAL, Burr ML, Clark ZE, Lewis MJ, Moat SJ, Newcombe RG, Powers HJ, Whiting JM, McDowell IFW. Optimisation' of dietary folate or low-dose folic acid supplements lower homocysteine but do not enhance endothelial function in healthy adults irrespective of methylenetetrahydrofolate reductase (C677T) genotype. J American College of Cardiology. 38 : 1799-1805 (2001).

Ashfield-Watt PAL, Pullin CH, Whiting JM, Clark ZE, Moat SJ, Newcombe RG, Burr ML, Lewis MJ, Powers HJ, McDowell IFW Methylenetetrahydrofolate reductase (C677T) genotype modulates blood folate and homocysteine responses to a folate rich diet or low dose folic acid supplement: a randomised controlled trial. Am J Clin Nutrition 76:180-186 (2002).

Sanderson P, McNulty H, Mastroiacovo P, McDowell IF, Melse-Boonstra A, Finglas PM, Gregory JF 3rd (2003) Folate bioavailability: UK Food Standards Agency workshop report. Br J Nutr. 90, 473-479.

Stuart J Moat1, Pauline AL Ashfield-Watt1, Hilary J Powers, Robert G Newcombe and Ian FW McDowell The effect of riboflavin status on the homocysteine lowering effect of folate in relation to MTHFR genotype Clin Chem 49: 295-302 (2003)

Contact: Dr Alison Tedstone
Tel: 020 7276 8929
Email: alison.tedstone@foodstandards.gsi.gov.uk