To investigate the effects of short-term folic acid and/or riboflavin supplementation on serum folate and plasma plasma total homocysteine (tHcy) concentrations in young Japanese male subjects.
In a double blind, randomized controlled trial.
Subjects were randomly assigned to one of four groups and received a placebo (control group), 800 μg/day folic acid (FA group), 8.4 mg/day riboflavin (R group), or both (FAR group) for 2 weeks.
In total, 32 healthy male volunteers aged 20–29 years.
At the end of the 2 week supplementation period, the tHcy concentration decreased significantly in the FA group. Serum folate concentrations had increased between 2.7 and 2.0-fold in the FA and FAR groups, respectively, but the mean within-group changes in serum folate and plasma tHcy concentrations did not differ between these two groups. At the end of the study, alanine amino transferase was decreased in the R and FAR groups, while alanine amino transferase was increased in the FA group.
Supplementation with folic acid, 800 μg/day, for 2 weeks, increased the serum and red blood cell folate concentrations and decreased the plasma tHcy concentrations in healthy young male subjects. Riboflavin supplementation may have blunted the effect of folic acid, which resulted in a diminished reduction of tHcy in our subjects.
An elevated plasma total homocysteine (tHcy) concentration has been recognized as an independent risk factor for cardiovascular disease (Selhub et al., 1995; Graham et al., 1997; Perna et al., 2003). Methylenetetrahydrofolate reductase (MTHFR) is a flavin adenine dinucleotide-dependent enzyme (Guinotte et al., 2003) that catalyzes the primary methyl donor for homocysteine remethylation: 5-methyltetrahydrofolate (5-MTHF). Individuals homozygous for the MTHFR C677T mutation have approximately 50% of the wild-type activity, and this is regarded as a cause of hyperhomocysteinemia (Lathrop et al., 2003).
It has recently been suggested that riboflavin, the precursor for flavin adenine dinucleotide, might act by increasing MTHFR activity, and thereby lower plasma tHcy concentrations (Bates and Fuller, 1986; Ross and Hansen, 1992; McNulty et al., 2002). Examining the effects of folic acid, riboflavin, and the MTHFR genotype on human lymphocytes grown in culture media, Kimura et al. (2004) observed that the tHcy concentration decreased as the riboflavin concentration increased. An inverse association between plasma riboflavin and tHcy concentrations has also been suggested in epidemiological studies (Shimakawa et al., 1997; Hustad et al., 2000; Jacques et al., 2001; Skoupy et al., 2002), and riboflavin is recognized as a determinant of plasma tHcy concentrations. Subjects who frequently consume dairy products, which are typical sources of riboflavin, reportedly have low plasma tHcy concentrations (Gao et al., 2003). High riboflavin intake is regarded as having no harmful effects, and improves hepatic function, including effects on alanine aminotransferase and blood lipids (Rao et al., 1984; Hultquist et al., 1993). Thus, if riboflavin has the effect of conserving folate in homocysteine metabolism, riboflavin alone or in combination with low dose folic acid might be a safer and more comprehensive means of preventing arteriosclerosis.
However, intervention studies have shown no significant plasma tHcy concentration change after supplementation with riboflavin alone (Lakshmi and Ramalakshmi, 1998; McKinley et al., 2002). Reduction of the plasma tHcy concentration by multivitamin administration has been reported (den Heijer et al., 1998; McKay et al., 2000; Sprecher and Pearce, 2002). There is no consensus as to whether riboflavin has homocysteine lowering activity, and the effects of combined riboflavin and folic acid are unclear.
Prevention of hyperhomocysteinemia is necessary early in life, especially in male subjects because of their higher risk of CHD, as compared to female subjects (Noma et al., 2003). Only a few studies have focused on the folate and tHcy status in young male subjects (Fenech et al., 1998; Tamura and Turnlund, 2004), and male subjects aged 20–29 years were reported to have the lowest folate and highest tHcy concentrations among all age groups (Moriyama et al., 2002). Therefore, we examined changes in serum folate and plasma tHcy concentrations in response to short-term folic acid and/or riboflavin supplementation in young male subjects.
Subjects and methods
Subjects and protocol
The major inclusion criteria for this study were as follows: (1) no metabolic or endocrine disease, and no abnormal blood lipid, glucose and hepatic function data on biochemical examination at the beginning of the study; (2) not taking prescription medications; (3) no use of vitamin supplements; (4) smoking of 10 cigarettes/day; and (5) absence of habitual exercise.
We performed a 2-week randomized, double-blind intervention trial. Initially, 40 healthy male Japanese volunteers, aged 20–29 years and living in a metropolitan area, were randomly assigned to the control group or one of three supplementation groups; the folic acid (FA), riboflavin (R) and folic acid plus riboflavin (FAR) groups. The subjects took their supplements three times a day just after each meal. Since, it has been recognized that oral administration of the vitamin just after a meal is more effective for its absorption than in fasting state, and that higher doses of riboflavin increases urinary excretion rate of riboflavin (Jusco and Levy, 1967). Furthermore, we considered that administration of supplements just after each meal would make subjects remember to take it properly.
The dose of folic acid was 800 μg/day (Nature Made Folic Acid, Otsuka Pharmaceuticals), and that of riboflavin was 8.4 mg/day (Nature Made Vitamin B2, Otsuka). Lactose tablets served as the placebo. To account for the risk of excess folic acid administration, the dosage of folic acid was set at the upper limit of the recommended dietary allowance for Japanese adults, assuming a daily dietary intake of 200 μg/day of folate from food. The dosage of riboflavin was based on previous riboflavin supplementation studies (Inada et al., 1989), in which the riboflavin status remained unchanged even after riboflavin supplementation of 7 mg/day for 2 weeks. Furthermore, riboflavin deficiency has been treated with 10 mg/day of riboflavin without ill effects (Boisvert et al., 1993).
During the study, all subjects were asked to live as they usually would, but without travel or intense exercise. They were also required to monitor their daily intakes using a checklist. The subjects recorded whether or not they ate meals and took the supplements as well as their physical condition. Compliance was monitored by counting the remaining tablets and by inspection of the checklist. Eight participants had low compliance, because they skipped breakfast nearly everyday. Therefore, we excluded them from the subjects, leaving 32 participants who were ultimately found to be eligible for the study. The Japan Women's University Human Ethics Committee approved this study, and written informed consent was obtained from all participants.
Sample collection and processing
Fasting blood samples were collected from all subjects at baseline and at the end of the study. Each subject fasted for 12 h after the last supplement intake following supper on each day prior to blood collection.
Plasma for tHcy and glucose measurements was collected in EDTA·Na2-coated tubes to which sodium fluoride had been added. The plasma was separated within 60 min of collection by centrifugation at 3000 g for 15 min at 4°C, and stored at −80°C until analysis. Plasma tHcy concentrations were measured by HPLC using a Hitachi F-1080 fluorescence detector (Hitachi High Technologies Ltd, Japan) and a Shimadzu LC-VP system (Shimadzu Ltd, Japan), by adapting the method of Frick et al. (2003).
Serum samples were allowed to clot at room temperature and then centrifuged at 1000 g for 10 min at room temperature. Hepatic function and the serum concentrations of total cholesterol (TC), HDL-cholesterol (HDL-C) and triacylglycerol (TG) were determined using biochemical analysis. LDL-cholesterol (LDL-C) was calculated according to the Friedwald formula, as TC-(HDL-C)–TG/5. Serum folate and vitamin B12 concentrations were measured using an electric chemiluminescence method (Elecsys 2010; Roche Diagnostics, GmbH). Samples for red blood cell (RBC) folate measurement were collected in heparin-coated tubes and analyzed microbiologically (Tamura, 1990). Samples for total vitamin B2 concentrations in whole blood were collected in EDTA·Na2-coated tubes, and the vitamin B2 concentrations were measured using the fluorescent lumiflavin method (Yoshida et al., 1961). DNA for genotyping was extracted from whole blood to which EDTA·Na2 had been added, using a kit (GenTLE, TaKaRa Biochemicals, Japan), and the presence of the MTHFR C677T genotype was determined with a polymerase chain reaction (PCR) and Hinf I restriction enzyme digestion, as described by Frosst et al. (1995). The presence of the C677T mutation within the MTHFR gene was detected by the appearance of a 175-base pair fragment on a 2% agarose gel, which was stained with ethidium bromide and viewed under ultraviolet light. Blood pressure was measured on the right arm using a mercury sphygmomanometer, with the participants in a sitting position and after 5 min of rest.
Dietary records were kept for 3 days before blood collection at baseline and at the end of the study. Dietary intakes were confirmed in an interview by a trained dietitian. The mean daily nutritional intakes were analyzed (Excel-Eiyokun software; Kenpakusha Co., Ltd, Tokyo) based on the table of nutrient contents published by the Japanese Science and Technology Agency (5th edition). Thiamine and riboflavin in-takes, which affect energy metabolism, were calculated per 1000 kcal of energy consumed. Other results are shown as per standard body weight, based on a body mass index (BMI)=22 kg/m2.
Percentages of MTHFR genotypes and smokers in the four groups were compared using a χ2 test, and other baseline characteristics were compared with a Mann–Whitney U test. Changes in each parameter from the baseline were compared using Wilcoxon's signed rank test. To minimize the potential influence of differences in baseline values, analysis of covariance (ANCOVA) was used, with the BMI and plasma tHcy concentrations as covariates. The significances of individual differences among the four groups were evaluated using the Fisher LSD post hoc test. Analyses were conducted using SPSS for Windows version 10.0.5J (SPSS Inc., Japan). All statistical tests were two-sided and significance was defined as P<0.05.
The average age of the 32 subjects was 22. 0±1.5 years and BMI averaged 21.4±1.4. The BMI of the FA group was lower than that of the FAR group. There were no significant differences in blood pressure or MTHFR genotype frequency among the four groups. Seven of the subjects had a smoking habit, with an average daily tobacco consumption of seven cigarettes (range: 5–10), but there were no significant differences in the percentage of smokers among the groups (Table 1). Supplement consumption compliance during the study was 95.4±0.6%.
At baseline, the dietary energy, total fat, riboflavin and folate intakes for all subjects were 32.4±6.6 kcal/kg, 25.6±6.6%, 0.566±0.072 mg/1000 kcal, and 4.2±2.2 μg/kg, respectively (Table 2). During the intervention period, no significant changes in dietary intake were observed, except for the higher folate and/or riboflavin intakes resulting from supplementation.
The blood vitamin and tHcy concentrations of the subjects are shown in Table 3. Mean baseline serum and RBC folate concentrations averaged 12.5±5.2 and 453±109 nmol/l, respectively, and the average plasma tHcy concentration was 7.2±3.5 μmol/l. The control subjects had lower tHcy concentrations than the R group, and six subjects (four: CC, two: TT) had mild hyperhomocysteinemia, over 12 μmol/l based on the report of Graham et al. (1997) on the European Concerted Action Project.
After the intervention, the serum and RBC folate concentrations were increased between 2.7 and 1.4-fold in the FA and between 2.0- and 1.2-fold in the FAR group. The mean within-group changes in serum folate concentrations in the FA and FAR groups were significantly different from those in the other two groups, and this trend remained after adjustment for baseline plasma tHcy concentrations and BMI. In the FA group, plasma tHcy concentrations were markedly decreased, while no significant difference was seen in the FAR group. However, changes in plasma tHcy concentrations were not different between the FA and FAR group. In our study, number of subjects with TT genotype in FA and FAR groups were one and two, respectively. The serum folate concentrations of the subjects with TT genotype also increased from 8.8 to 24.2 nmol/l in the FA group, 10.1 to 21.2 and 4.4 to 20.9 nmol/l in the FAR group. In addition, the plasma tHcy concentrations of them were decreased from 13.1 to 12.0 μmol/l in the FA group, 6.8 to 5.1 and 7.3 to 6.5 μmol/l in the FAR group. In the R group, no significant changes in serum folate and plasma tHcy concentrations were observed. Neither serum riboflavin nor vitamin B12 concentrations changed during the study.
Biochemical data for the subjects were within normal ranges, and there were no differences among the groups at baseline (Table 4). Serum glucose and TG concentrations were unchanged in all groups during the study (data not shown). In the R group, TC and LDL-C concentrations were decreased with no reduction in HDL-C concentration, after intervention. A decrease in TC and LDL-C concentrations was observed in the FAR group, but it did not reach statistical significance.
The mean changes in ALT concentrations based on ANCOVA, with BMI and the plasma tHcy concentrations as covariates, revealed significant differences in ALT concentrations in the R and the FAR groups from that of the FA group. ALT was increased in the FA group, but decreased in the R and FAR groups. Difference of MTHFR genotypes and smoking habits had no effect to the result.
Greater improvements in folate and tHcy status were obtained with our present short-term, low dosage (800 μg/day) folic acid supplementation than in earlier studies using 5–10 mg of folic acid for periods of 6 weeks (Landgren et al., 1995; Klerk et al., 2002) and 2 months (Brouwer et al., 1999). Serum folate and plasma tHcy concentrations were relatively low in our young male subjects, though six participants showed mild hyperhomocysteinemia at the beginning of the study, and four of the six had no genetic risk factors. After the supplementation period, mean serum folate concentrations in the FA group were increased 2.7-fold, and in the FAR group, there was a mild increase of 2.0-fold. The FA group showed a significant decrease in plasma tHcy with no change in the serum vitamin B12 concentration, which affects tHcy status. Brouwer et al. (1999) also reported that 2 weeks of folic acid supplementation at 500 or 250 μg/day increased serum folate concentrations 1.9- and 1.6-fold, respectively, in their healthy young Dutch subjects. Thus, our results support the notion that folic acid supplementation is an easy way to rapidly increase the serum folate concentration including subjects with TT genotype.
We performed this study to determine whether riboflavin administration increases the serum folate concentration and decreases the tHcy concentration. Flavin adenine dinucleotide, a coenzyme of MTHFR, probably facilitates the reaction by which administered folic acid is converted to 5, 10-MTHF and then further to 5-MTHF. Unexpectedly, the folate increasing and tHcy lowering effects of riboflavin suggested by previous reports (Shimakawa et al., 1997; Hustad et al., 2000; Jacques et al., 2001; Skoupy et al., 2002) were not seen in the present study. Previous reports have demonstrated an inverse relation between riboflavin and tHcy in subjects with poor riboflavin status such as the elderly (Jacques et al., 2001) and those with genetic risk factors (Hustad et al., 2000). In our subjects, however, riboflavin and folate concentrations were within normal range including subjects with TT genotype. Furthermore, despite similar folate intakes and serum folate concentrations, our subjects had lower plasma tHcy concentrations than those in the Dutch studies with subjects of similar age (den Heijer et al., 1998; Brouwer et al., 1999). Flavin adenine dinucleotide also acts as a coenzyme for lipid and energy metabolisms; the adverse effects of a high fat-riboflavin deficient diet on lipid metabolism have been documented in rats (Liao and Huang, 1986). A Western diet characterized by higher intakes of red meat and several foods high in fat, might contribute to high plasma tHcy and low serum folate concentrations (Fung et al., 2001). Therefore, lipid intake might influence folate metabolism via the consumption of flavin adenine dinucleotide. These observations support the suggestion that ethnicity and lifestyle influence the folate status response to folate intake (Ubbink et al., 1993; Maruyama et al., 2004; Nurk et al., 2004; Perry et al., 2004). Thus, our subjects' low fat intakes might have affected their overall low plasma tHcy concentrations and could explain the effects of riboflavin being unremarkable. In this intervention trial, we should have tried to increase 5, 10-MTHF with riboflavin and folate containing foods such as green vegetables instead of folic acid supplements.
Apparently blunted changes in serum folate and plasma tHcy resulted from concomitant administration of riboflavin in the present study. The FAR group showed a significant increase in serum folate, but the serum folate concentration change was less remarkable in the FAR group than in the FA group. One possible explanation for this observation is that folic acid supplementation is probably less effective in subjects with high serum folate concentrations. A study of the high dose folic acid supplementation suggested the existence of a lower plateau (van Oort et al., 2003). However, the folate concentration was not particularly high in our subjects. Furthermore, folic acid coexisting with riboflavin is reportedly easily catabolized to para-aminobenzoylglutamate (Biamonte and Schneller, 1951). In the FAR group, administered folic acid might have been partly catabolized before absorption.
In addition, interestingly, we found ALT to be decreased in the R and FAR groups, but increased in the FA group. It is reasonable to speculate that riboflavin suppresses folate action. The tendency for ALT to be increased in the FA group may reflect the promotion of homocysteine remethylation in the liver, associated with the elevated folate concentrations. Our study period was short and ALT concentrations never exceeded the normal range. However, in a study of 800 μg/day folic acid supplementation for 12 weeks, subjects' serum folate concentrations increased 5.8-fold (van Oort et al., 2003). It has been suggested that excessive folic acid administration masks the diagnosis of cobalamin deficiency (Dickinson, 1995), which is also important for homocysteine metabolism. Furthermore, promotion of in-stent restenosis after high dose folic acid has been observed (Lange et al., 2004). It was recently suggested that high dose folic acid might promote DNA hypomethylation and tumor growth (Baggott et al., 1992; Bills et al., 1992). These results highlight the necessity of determining the maximum tolerable dose of folic acid, taking into consideration effects on liver function. It appears to be worthwhile to obtain a moderate increase in the serum folate concentration. More research is needed to determine whether concomitant administration of riboflavin and folate might be a useful way to prevent the harmful effects of excess folic acid supplementation, and whether administering extra riboflavin might diminish the risks of excess folic acid supplementation.
In conclusion, 2-week daily dietary supplementation with 800 μg folic acid apparently improved the folate and tHcy status with the elevation of ALT in young male subjects, although these effects might have been blunted by riboflavin. Therefore, the dosage level, supplementation duration and the effects of combining folic acid with other vitamins require further consideration. More studies are necessary to investigate the interaction between folic acid and riboflavin, and the effects of dietary intakes of these vitamins.
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Araki, R., Maruyama, C., Igarashi, S. et al. Effects of short-term folic acid and/or riboflavin supplementation on serum folate and plasma total homocysteine concentrations in young Japanese male subjects. Eur J Clin Nutr 60, 573–579 (2006) doi:10.1038/sj.ejcn.1602351
- short-term supplementation
- folic acid
- serum folate
- young males
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