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Effects of Riboflavin and Folic Acid Supplementation on Plasma Homocysteine Levels in Healthy Subjects
Effects of Riboflavin and Folic Acid Supplementation on Plasma Homocysteine Levels in Healthy Subjects CHERNG ZEE CHUANG, PHD; ADRIENNE BOYLES, BA; BARBARA LEGARDEUR, MPH; JOSEPH SU, PHD; SHANKER JAPA, PHD; ALFREDO LOPEZ-S, MD, PHD
ABSTRACT: Background: Observational studies have shown an inverse relationship between vitamin B2 status and total homocysteine levels, a risk factor for cardiovascular disease. We hypothesize that intervention with riboflavin will lower total homocysteine levels. The total homocysteine lowering by the three genotypes (CC, CT, TT) of methylenetetrahydrofolate reductase polymorphism (677C¡T) was also studied. Methods: The decrease in total homocysteine levels after supplementation with riboflavin (10 mg/d) or folic acid (1 mg/d) for 3 weeks was compared in two groups of healthy subjects (17 per group, matched by age and gender) (Phase 1). Then, both groups received supplementation with folic acid and riboflavin for an additional 3 weeks (Phase 2). Results: During Phase 1, total homocysteine levels were lowered by 2% or 4% after supplementation with riboflavin or fatty acid, respectively, although neither decrease was statistically significant (P ⫽ 0.50 and 0.19). Compared to subjects of CC genotype, total homocysteine lowering in subjects of CT
genotype was approaching significance (P ⫽ 0.059) for the folic acid group, but not for the riboflavin group. After Phase 2, total homocysteine levels were not lowered significantly in either the folic acid (1%) or the riboflavin (2%) group. However, in the folic acid–riboflavin combined group, total homocysteine lowering in subjects of TT type was larger when compared to subjects of CC and CT types (P ⫽ 0.007). Conclusions: Riboflavin supplementation did not lower total homocysteine levels in healthy subjects with CC type of C677T polymorphism. However, supplementation with folic acid or with both folic acid and riboflavin may be important for CT and TT subjects in optimizing their homocysteine metabolism. These findings are relevant in characterizing the factors controlling the high total homocysteine levels for subjects of CT and TT genotypes. KEY INDEXING TERMS: Homocysteine; Riboflavin; Folic acid; Methylenetetrahydrofolate reductase; Intervention. [Am J Med Sci 2006;331(2):65–71.]
H
Vitamins B2 (as flavin adenine dinucleotide [FAD]), B6, B12, and folic acid are important cofactors or substrates of homocysteine metabolism. Blood concentrations of folate and vitamins B6 and B12 have been shown to be determinants of total homocysteine levels.4,5 Supplementation of folic acid alone or with vitamins B6 and B12 has been shown to decrease total homocysteine levels in diseased and healthy populations.6,7 Recent observational studies have indicated that vitamin B2 (riboflavin) may be another determinant of total homocysteine level,8 –12 suggesting that riboflavin may be an additional supplement, as is folic acid, to lower total homocysteine levels. However, intervention studies with riboflavin are needed to confirm the causal relationship between vitamin B2 and total homocysteine levels and to determine whether riboflavin supplementation can be an effective way to lower total homocysteine levels. Only two intervention studies have been conducted, and neither found that riboflavin could lower total homocysteine levels significantly.13,14 Observational studies have also shown that the relationship between vitamin B2 and total homocys-
igh plasma total homocysteine levels are associated with an increased risk of cardiovascular disease, pregnancy loss, and cognitive disorders.1–3
From the Department of Medicine, School of Medicine (CZC, AB, and the School of Public Health (JS), Louisiana State University Health Sciences Center, and Tulane-Charity-LSU General Clinical Research Center (SJ), New Orleans, Louisiana. Submitted for publication March 22, 2005; accepted for publication August 17, 2005. Supported by a Research Enhancement Grant from LSUHSC and in part by both a GCRC grant (NIH Grant #5M01 RR05096-10) and a grant from NCI (R25 #CA047877). Presented in part at the Southern Regional Meeting of the American Federation for Medical Research, February, 2004, New Orleans (Chuang CZ, Boyles A, LeGardeur B, Su J, Lopez-S A. Plasma total homocysteine lowering after supplementation with folic acid and riboflavin in healthy subjects [abstract]. J Investig Med 2004;52:S291.) and at Experimental Biology 2002, April, 2002, New Orleans (Chuang CZ, Boyles A, LeGardeur B, Su J, Lopez-S A. Total homocysteine levels after riboflavin or folic acid supplementation in healthy subjects [abstract]. FASEB 2002;16:A266.) Correspondence: Cherng Zee Chuang, PhD, Louisiana State University Health Sciences Center, Department of Medicine, Section of Nutrition, 1542 Tulane Avenue, New Orleans, LA 70112 (E-mail: [email protected]). BL, AL-S)
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teine levels could be modified by folic acid status9 and by 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism.8 –10 The present study initially investigated the effect of total homocysteine levels after supplementation with riboflavin or fatty acid and then investigated whether subsequent supplementation of both riboflavin and fatty acid would lower total homocysteine levels in those subjects who have been presupplemented with either riboflavin or folic acid. The lowering of total homocysteine levels was also studied in subjects of different MTHFR C677T genotypes. Subjects and Methods Subjects Healthy adult volunteers (N ⫽ 37) were recruited by advertisement (between October 2000 and March 2001) and 34 subjects completed the study. Three subjects did not return to complete the four blood sample drawings during the study, and thus they were dropped from the analysis. Subjects reported to the TulaneCharity-Louisiana State University General Clinical Research Center (GCRC) in the morning to have their blood drawn. Subjects completed a self-reported questionnaire regarding their lifestyle (smoking status, quantity of coffee and/or alcoholic beverage consumption, and amount of physical activity) and medical history. Subjects were excluded if they had a history of diabetes or cardiovascular, liver, renal, or thyroid diseases. The subjects (n ⫽ 12) who were taking any one of the four B vitamins being studied were asked to stop taking supplements for at least 3 days prior to their enrollment in the study. The average length of time from stopping supplements to enrollment in the study was 7.6 days, with a range of 3-15 days. The subjects returned to the GCRC after 1 week for their second blood sample drawing. The average of blood values from the first and second samples was used as the baseline level. Informed consent was obtained and the study was approved by the Institutional Review Board of Louisiana State University Health Sciences Center at New Orleans.
fluorometric detection.15 Plasma levels of folic acid and vitamin B12 were analyzed by radioimmunoassay (Diagnostic Products Corp, USA). Although plasma folic acid level is not as precise an indicator as red blood cell folate level for folic acid status, it has been used as a general and convenient indicator. Owing to limited resources, we measured plasma folate only as the folic acid indicator in this study. The status of vitamins B2 and B6 was analyzed by erythrocyte glutathione reductase activation coefficient (EGRAC) and erythrocyte aspartate aminotransferase activation coefficient (EASTAC), respectively,16 in Pennington Biomedical Research Center. Both tests reflect tissue levels of these vitamins and are not affected by recent dietary intakes. A higher value of either EGRAC or EASTAC indicates a lower vitamin status. All four samples from each individual were analyzed in the same batch. Between-run precision for total homocysteine, folic acid, and vitamin B12 was 8% (9 mol/L; n ⫽ 15), 5% (23 nmol/L; n ⫽ 6), and 5% (450 pmol/L; n ⫽ 6), respectively. The polymorphism of MTHFR C677T was determined by the method of Frosst.17 Plasma creatinine levels were measured by a commercial kit (DMA, USA). Statistical Analysis Statistix software (Analytical Software, USA) was used to perform all statistical analyses. The difference in subject characteristics between the riboflavin and folic acid groups was analyzed by sample t-test, by Kruskal-Wallis test (if the distribution was not gaussian) or by 2 test. The total homocysteine levels and blood status of four B vitamins before and after phase 1 or phase 2 supplementation were compared by paired t-test. The lowering of total homocysteine level stratified by genotypes was analyzed by one-way analysis of variance. The significance levels were chosen at P ⬍ 0.05. Several analyses were performed to assure that subjects who were vitamin B users could be included in the analysis. There was no significance for vitamin B2 and folic acid status at baseline between vitamin B users and vitamin B nonusers (by sample t-test). The results of total homocysteine lowering (mean and P value) during phase 1 for both groups were similar when vitamin B users were excluded from the analysis. In addition, vitamin B2 and folic acid status between blood sampling 1 and sampling 2 were statistically nonsignificant in the riboflavin, folic acid, and combined groups and in the vitamin B users. Therefore, we included vitamin B users in the final analyses.
Supplementation After the second blood sample drawing, subjects were randomly divided into two groups (riboflavin and folic acid) matched by age and gender. There were two phases of supplementation in this study (phases 1 and 2). During phase 1, subjects in the riboflavin group received 10 mg riboflavin per day and subjects in the folic acid group received 1 mg folic acid per day for 3 weeks. After the completion of phase 1, subjects in both groups received both folic acid (1 mg/day) and riboflavin (10 mg/day) supplements for an additional 3 weeks (phase 2). Third and fourth blood samples were collected at the end of phases 1 and 2, respectively. All blood samples were drawn with the subjects fasting, and EDTA tubes were used. Plasma samples were separated within 2 hours and stored at ⫺70° C until analysis. The riboflavin supplements (10 mg/capsule) were prepared by a local licensed pharmacist and the folic acid supplements (1 mg/tab) were of commercial brand (Danbury Pharmacal). Three supplements of both riboflavin and folic acid were randomly selected for analysis by high-performance liquid chromatography and their contents were found to be accurate (95-105% and 107-117%, respectively). All participating subjects were advised not to take any vitamin supplements other than the supplements provided by the research staff and to maintain their usual dietary intake during the 6-week period. Biochemical Measurements Plasma total homocysteine level and blood status of B vitamins (folic acid, B2, B6, and B12) were analyzed for the four blood samples from each subject. Plasma total homocysteine levels were analyzed by high-performance liquid chromatography with
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Results The subject characteristics of the riboflavin and folic acid supplemented groups are shown in Table 1. There is no difference between the two groups for any of these characteristics. Phase 1 At the baseline, there are two subjects (12%) in the riboflavin group who show a deficiency of vitamin B2 (EGRAC ⬎1.40; the lower the EGRAC, the better the vitamin B2 status). Their EGRAC decreased below 1.40 after phase 1 supplementation, indicating that supplementation of 10 mg riboflavin for 3 weeks is sufficient to improve vitamin B2 status to normal in this population. In the riboflavin-supplemented group (between A and B in Table 2), EGRAC was significantly lowered from 1.22 to 1.06 (P ⬍ 0.0001) after supplementation. However, total homocysteine levels dropped by only 2% (from 7.81 to 7.67 mol/L), which was not statistically significant (P ⫽ 0.503). For the folic acid supplemented group (between A and B in Table 3), plasma folic acid levels were nearly doubled (from 37.5 to 69.7 2 February 2006 Volume 331 Number 2
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Table 1. Subject Characteristics of the Riboflavin and Folic Acid Supplemented Groups Riboflavin Supplemented Group (n ⫽ 17)
Folic Acid Supplemented Group (n ⫽ 17)
P-valuea
39.3 ⫾ 15.2 23.5 ⫾ 2.8 92.8 ⫾ 23.0 40.8 ⫾ 34.4 1.4 ⫾ 1.4 24.2 ⫾ 23.9 5:12 13:2:2 6:11 10:7 9:5:2 5:7
38.0 ⫾ 13.1 23.4 ⫾ 3.0 98.1 ⫾ 21.2 19.0 ⫾ 26.2 1.3 ⫾ 1.1 21.0 ⫾ 24.0 5:12 11:3:3 6:11 11:6 8:6:2 6:6
0.792 0.902 0.496 0.091 0.932 0.631 1.000 0.753 1.000 0.724 0.928 0.682
Age, year Body mass index Creatinine, mol/L Alcohol,b g/wk Coffee,b cups/d Physical activity,b min/d Men:women White:black:others Vitamin B supplements (Y:N) Nonsmoker:ex-smoker MTHFR (CC:CT:TT)c Contraceptive User (Y:N) a b c
Continual variables were analyzed by sample t-test or by Kruskal-Wallis test and noncontinual variables were analyzed by 2. Variables were analyzed by Kruskal-Wallis test. Two subjects missed MTHFR analysis.
nmol/L) after folic acid supplementation (P ⫽ 0.006). The total homocysteine levels were decreased by 4% (from 7.68 to 7.40 mol/L), although not significantly (P ⫽ 0.189). Surprisingly, EGRAC was significantly increased from 1.20 to1.25 (P ⫽ 0.002), indicating the worsening of vitamin B2 status after folic acid supplementation. Gender stratification in both groups revealed that total homocysteine levels were significantly decreased by 7% (from 7.41 ⫾ 1.24 to 6.87 ⫾ 1.15 mol/L, P ⫽ 0.041) for females in the folic acid group, but were not significant for the other three gender-based supplementation groups. EGRAC was also increased from 1.22 ⫾ 0.16 to 1.27 ⫾ 0.16 (P ⫽ 0.004) for females in the folic acid group. When compared to subjects of CC genotype, subjects of CT genotype in folic acid group (Table 4, P ⫽ 0.059) demonstrated a greater total homocysteine lowering; however, the same did not hold for riboflavin group. Phase 2 For the riboflavin group (between B and C in Table 2), the EGRAC was improved slightly again (from 1.06 to 1.04) at the end of phase 2. Plasma folic acid levels were doubled (from 35.2 to 82.3 nmol/L) after the additional supplementation of folic acid during phase 2. However, total homocysteine levels decreased only by 1% (from 7.67 to 7.57 mol), which
is not statistically significant. In the folic acid group (between B and C in Table 3), plasma folic acid levels reached a plateau (69.7 and 68.3 nmol/L) after continual supplementation of folic acid from phase 1. The EGRAC was significantly improved from 1.25 to 1.08 due to the additional supplementation of riboflavin during phase 2. However, total homocysteine levels dropped only by 2% (from 7.40 to 7.24 mol), which is not statistically significant. In Table 1, the total homocysteine levels did not change much after supplementation with both folic acid and riboflavin in subjects of CC and CT genotypes. However, total homocysteine levels were significantly lowered for subjects of TT genotype in the folic acid–riboflavin combined group when compared to subjects of CC and CT types (P ⫽ 0.007). A scattering plot for total homocysteine changes by MTHFR genotypes in the combined group is shown in Figure 1. Total homocysteine levels were lowered in each of the four subjects with TT genotype after supplementation with both folic acid and riboflavin. Discussion Moderately high total homocysteine levels are associated with an increased risk of cardiovascular disease, pregnancy complications, neural tube defects, and cognitive disorders.1–3 Both lifestyle and
Table 2. Total Homocysteine and Vitamin Status in Riboflavin Group
Plasma total homocysteine, mol/L Plasma folic acid, nmol/L EGRAC EASTAC Plasma cyanocobalamin, pmol/L a
Baseline (A)
Supplementation with Riboflavin (B)
Supplementation with Riboflavin and Folic Acid (C)
P-Valuea between A and B
P-Valuea between B and C
P-Valuea between A and C
7.81 ⫾ 1.53 36.8 ⫾ 12.9 1.22 ⫾ 0.14 2.04 ⫾ 0.23 289 ⫾ 153
7.67 ⫾ 1.26 35.2 ⫾ 12.0 1.06 ⫾ 0.06 2.03 ⫾ 0.18 278 ⫾ 129
7.57 ⫾ 1.79 82.3 ⫾ 51.8 1.04 ⫾ 0.05 2.07 ⫾ 0.19 304 ⫾ 170
0.503 0.383 ⬍0.0001 0.731 0.474
0.592 0.003 0.047 0.182 0.256
0.268 0.003 ⬍0.0001 0.433 0.658
The comparison was analyzed by paired t-test.
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Table 3. Total Homocysteine and Vitamin Status in Folic Acid Group
Plasma total homocysteine, mol/L Plasma folic acid, nmol/L EGRAC EASTAC Plasma cyanocobalamin, pmol/L a
Baseline (A)
Supplementation with Folic Acid (B)
Supplementation with Riboflavin and Folic Acid (C)
7.68 ⫾ 1.26 37.5 ⫾ 14.5 1.20 ⫾ 0.14 2.04 ⫾ 0.26 268 ⫾ 110
7.40 ⫾ 1.44 69.7 ⫾ 47.2 1.25 ⫾ 0.15 1.96 ⫾ 0.16 283 ⫾ 116
7.24 ⫾ 1.21 68.3 ⫾ 45.1 1.08 ⫾ 0.12 2.04 ⫾ 0.18 297 ⫾ 149
P-Valuea between A and B
P-Valuea between B and C
P-Valuea between A and C
0.189 0.006 0.002 0.052 0.354
0.431 0.866 ⬍0.0001 0.0005 0.462
0.014 0.003 ⬍0.0001 0.933 0.227
The comparison was analyzed by paired t-test.
genetics are important factors for total homocysteine levels. One of the major determinants of total homocysteine levels is nutritional status in terms of folic acid, vitamin B6, and vitamin B12.4,5 Since vitamin B2 (as flavin mononucleotide) is involved in the formation of active forms of folic acid, vitamin B6 and vitamin B12 in the homocysteine metabolism,18 –20 the significance of vitamin B2 in the control of total homocysteine levels still needs to be determined. Recently, several observational studies have shown an inverse relationship between vitamin B2 dietary intakes and total homocysteine level21,22 or between vitamin B2 status and total homocysteine level in healthy subjects and in patients with end-stage renal disease,8 –12 indicating that vitamin B2 may be another determinant of total homocysteine level. However, results of these association studies cannot support the causal relationship between total homocysteine and vitamin B2 and only intervention studies can confirm the effect of vitamin B2 on the total homocysteine levels. This study did not find a significant decrease (2%) of total homocysteine levels after riboflavin supplementation in these healthy volunteers. Neither do we find a decrease of total homocysteine levels after supplementation with riboflavin in subjects who received pre-supplementation with folic acid or after supplementation with folic acid in subjects who received pre-supplementation with RF. The 2% decrease after riboflavin supplementation is less than the decrease (4%) seen in the age- and gender-
matched folic acid–supplemented group. Although the 4% decrease is statistically nonsignificant in the folic acid group (n ⫽ 17), the decrease in total homocysteine levels (7%) is statistically significant when reanalyzed in only female subjects (n ⫽ 12). In addition, total homocysteine levels were also significantly decreased after 6 weeks of supplementation with folic acid (Table 3, between A and C). This extent of decrease of total homocysteine after folic acid supplementation is smaller than the decrease observed in previous studies,23 probably due to the fortification of cereal grains with folic acid in the United States since January 1998 and/or to the shorter period (3 weeks) of supplementation in this study. The baseline plasma level of folic acid (37 nmol/L or 16 ng/mL) is comparable to studies with blood samples collected after the starting date of folic acid fortification.24 It seems that folic acid is more effective than riboflavin in the lowering of total homocysteine levels. This is understandable, since folic acid functions as a co-substrate while riboflavin (as FAD) functions as a cofactor of enzymes in the homocysteine metabolism. The findings are similar to those of the only two other studies available.10,11 Lakshmi found that the total homocysteine level was lowered from 12.1 to 11.8 mol/L (2.5%, nonsignificant) after riboflavin (10 mg/d, 15 days) supplementation in 10 Asian Indian women with glossitis and angular stomatitis.10 For McKinley’s study in 23 community-living healthy elderly subjects, the total homocysteine lev-
Table 4. Lowering of Total Homocysteine Levels Grouped by MTHFR C677T Genotypes
Folic acid group Riboflavin group Folic acid and riboflavin combined group
Supplementation
CC
CT
TTa
Folic acid, mol/L Folic acid and riboflavin, mol/L Riboflavin, mol/L Folic acid and riboflavin, mol/L Folic acid and riboflavin, mol/L
⫺0.186 ⫾ 0.903 ⫺0.045 ⫾ 0.849 ⫺0.067 ⫾ 0.714 0.119 ⫾ 0.764 0.042 ⫾ 0.784
⫺0.698 ⫾ 0.552 0.077 ⫾ 0.549 0.01 ⫾ 1.00 ⫺0.072 ⫾ 0.892 0.009 ⫾ 0.689
0.918 ⫺1.46 ⫺0.69 ⫺1.12 ⫺1.29 ⫾ 0.41
P-valueb 0.059c 0.051d 0.618c 0.176d 0.007d
a
There are only two subjects of TT genotype for the folic acid or riboflavin group and only their means are shown. Variables were analyzed by one-way analysis of variance. c The comparison was between CC and CT genotypes. d The comparison was between CC, CT, and TT genotypes. b
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Figure 1. Scatter plot for total homocysteine changes versus MTHFR genotypes in the combined group
els changed from 12.00 to 12.57 mol/L (P ⫽ 0.719) after the subjects took 1.6 mg of riboflavin supplements daily for 12 weeks.11 Although riboflavin as FAD is the cofactor of MTHFR, one of the important enzymes in the homocysteine metabolism, the present study and the other two intervention studies do not support the idea that vitamin B2 is an effective nutrient in lowering total homocysteine levels, at least in healthy subjects. Vitamin B2 and several other B vitamins have been fortified in cereal grains in the United States since the 1940s,25 resulting in adequate vitamin B2 status of the general population. This may affect our ability to see the effect of vitamin B2 supplementation on total homocysteine levels in these healthy subjects. Since all three of these intervention studies involved a small number of subjects, there is a possibility that the power is not sufficient to observe a decrease of tHcy after riboflavin supplementation. Future intervention studies in a large population of subjects with suboptimal vitamin B2 status will be necessary to confirm this. The data from this intervention study also show that total homocysteine decrease after riboflavin and/or folic acid supplementation is dependent on the MTHFR C677T genotypes. This is reflected in both the lowering of total homocysteine level in subjects of CT genotypes compared to subjects of CC genotypes in the folic acid–supplemented group during phase 1 and in the lowering of total homocysteine level in subjects of TT type compared to subjects of CC and CT types during phase 2 (supplemented with both folic acid and riboflavin). These findings are in agreement with recent observational and in vitro studies on the relationship between total homocysteine, riboflavin, folic acid, and genotypes of MTHFR C677T. Previous studies have found that higher total homocysteine levels are associated with MTHFR THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
C677T polymorphism when the blood folic acid status is on the lower end of normal values,26 –28 indicating that MTHFR genotypes might modify the relationship between folic acid and total homocysteine levels. Four recent observational studies8 –10,28 indicated that MTHFR C677T could modify the relationship between vitamin B2 and total homocysteine, but one study did not.12 Although the four supporting studies differ in which genotypes can interact with this relationship, all four studies indicate that TT genotype is a modifying factor. Recent in vitro studies with lymphocytes from subjects of CC and TT genotypes indicate that MTHFR is a modifying factor when the supply of folic acid and/or riboflavin is low in the culture medium.29,30 Stern et al found that total homocysteine levels are low in the culture medium and the difference of total homocysteine in the culture medium is not significant between cells of CC and TT types when adequate folic acid and riboflavin are present in the culture medium.29 In contrast, when both folic acid and riboflavin are low in the culture medium, the culture medium total homocysteine levels are high and there is a significant difference of total homocysteine levels in the culture medium between cells of CC and TT types. Kimura’s data30 have shown that the difference of medium total homocysteine levels between cells of CC and TT types is small when cultured in media with high folic acid and high riboflavin concentrations. However, the difference is increased to 2.5- to threefold when lymphocytes are cultured in media with high folic acid/low riboflavin, low folic acid/high riboflavin, or low folic acid/low riboflavin nutrients. This study’s findings are in agreement with these two in vitro studies, indicating that the requirements for folic acid in subjects with CT type and the requirements of folic acid and riboflavin in subjects with TT type are probably higher than those required for CC subjects in the optimization of homocysteine metabolism. Since the baseline total homocysteine levels of healthy subjects in this study are already within the normal values (⬍12 mol/L), the benefits from total homocysteine lowering again with folic acid/riboflavin supplementation in healthy subjects of CT and TT types with normal status of B vitamins probably will be limited. However, the supplementation of folic acid and riboflavin will be of importance for CT and TT subjects who are hyperhomocysteinemic and are at risk for the aforementioned total homocysteine–related diseases.1–3 For example, many studies have shown that total homocysteine levels cannot be normalized in patients with chronic renal disease when supplemented with high doses of folic acid alone or with vitamins B6 and B12.31 An additional supplement of vitamin B2 may help lower total homocysteine levels even more for some of these patients. A limitation of the present findings is the small number (N ⫽ 4) of subjects with TT genotype. 69
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A surprising finding of this study is that the vitamin B2 status was significantly worsened (EGRAC increased from 1.20 to 1.25, P ⫽ 0.002) after folic acid supplementation (1 mg/d for three weeks). A similar finding was reported in a recent study of 126 healthy individuals (20-63 years) who were supplemented with 400 g folic acid daily for 4 months.12 The authors suggested that this worsening of vitamin B2 status might be due to the increased turnover of homocysteine metabolism, leading to the increased requirement of riboflavin in cells and/or a shifting of FAD from erythrocyte glutathione reductase to MTHFR. However, EGRAC was not changed (1.24 ⫾ 0.06) after subjects received 400 g folic acid per day for 6 weeks in McKinley’s study.14 The relevance of this topic necessitates further studies in this area. Since folic acid supplement or fortification has been used as an inexpensive means to prevent neural tube defects or to decrease total homocysteine levels, this finding will raise an interesting question: whether vitamin B2 supplements should be administered along with folic acid in long-term folic acid supplementation or fortification. In conclusion, the present study found that riboflavin supplementation did not lower total homocysteine levels effectively in healthy subjects of CC type of C677T polymorphism. However, supplementation with folic acid or with both folic acid and riboflavin may be of specific importance for subjects of CT and TT types in the optimization of their homocysteine metabolism. These findings are relevant in the lowering of high total homocysteine levels for subjects of CT and TT types. Future studies in a large population and with sufficient subjects of TT genotypes are necessary to confirm these results. References 1. Mangoni AA, Jackson SHD. Homocysteine and cardiovascular disease: current evidence and future prospects. Am J Med 2002;112:556–65. 2. Hankey GJ, Eikelboom JW. Homocysteine and vascular disease. Lancet 1999;354:407–19. 3. Ray JG, Laskin CA. Folic acid and homocyst(e)ine metabolic defects and the risks of placental abruption, pre-eclampsia and spontaneous pregnancy loss: a systematic review. Placenta 1999;20:519–29. 4. Malinow MR, Bostom AG, Krauss RM. Homocyst(e)ine, diet, and cardiovascular disease. A statement for healthcare professionals from the Nutrition Committee, American Heart Association. Circulation 1999;99:178–82. 5. Selhub J, Jacques PF, Wilson PW, et al. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 1993;270:2693–98. 6. Ubbink JB, Vermaak WJ, van der Merwe A, et al. Vitamin requirements for the treatment of hyperhomocysteinemia in humans. J Nutr 1994;124:1927-33. 7. den Heijer M, Brouwer IA, Bos GMJ, et al. Vitamin supplementation reduces blood homocysteine levels: a controlled trial in patients with venous thrombosis and healthy volunteers. Arterioscler Thromb Vasc Biol 1998;18:356–61.
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8. Hustad S, Ueland PM, Vollset SE, et al. Riboflavin as a determinant of plasma total homocysteine: effect modification by the methylenetetrahydrofolate reductase C677T polymorphism. Clin Chem 2000;46:1065–71. 9. Jacques PF, Kalmbach R, Bagley PJ, et al. The relationship between riboflavin and plasma total homocysteine in the Framingham offspring cohort is influenced by folate status and the C677T transition in the methylenetetrahydrofolate reductase gene. J Nutr 2002;132:283–8. 10. McNulty H, McKinley MC, Wilson B, et al. Impaired functioning of thermolabile methylenetetrahydrofolate reductase is dependent on riboflavin status: implications for riboflavin requirements. Am J Clin Nutr 2002;76:436–41. 11. Skoupy S, Fodinger M, Veitl M, et al. Riboflavin is a determinant of total homocysteine plasma concentration in end–stage renal disease patients. J Am Soc Nephrol 2002;13: 1331–7. 12. Moat SJ, Ashfield-Watt PAL, Powers HJ, et al. Effect of riboflavin status on the homocysteine-lowering effect of folate in relation to the MTHFR (C677T) genotype. Clin Chem 2003;49:295–302. 13. Lakshmi AV, Ramalakshmi BA. Effect of pyridoxine or riboflavin supplementation on plasma homocysteine levels in women with oral lesions. Natl Med J India 1998;1:171–2. 14. McKinley MC, McNulty H, McPartlin J, et al. Effect of riboflavin supplementation on plasma homocysteine in elderly people with low riboflavin status. Euro J Clin Nutr 2002;56:850–6. 15. Fortin LJ, Genest J Jr. Measurement of homocysteine in the prediction of arteriosclerosis. Clin Biochem 1995;28:155– 62. 16. Bayoumi RA, Rosalki SB. Evaluation of methods of coenzyme activation of erythrocyte enzymes for deficiency of vitamin B1, B2, and B6. Clin Chem 1976;22:327–35. 17. Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111–3. 18. Bates CJ, Fuller NJ. The effect of riboflavin deficiency on methylenetetrahydrofolate reductase (NADPH) (EC1.5.1.20) and folate metabolism in the rat. Br J Nutr 1986;55:455–64. 19. McCormick DB. Two interconnected B vitamins: riboflavin and pyridoxine. Physiol Rev 1989;69:1170–98. 20. Olteanu H, Banerjee R. Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for cloning and mapping of NADPHdependent methionine synthase activation. J Biol Chem 2001;276:35558–63. 21. Jacques PF, Bostom AG, Wilson PWF, et al. Determinants of plasma total homocysteine concentration in the Framingham offspring cohort. Am J Clin Nutr 2001;73:613– 21. 22. Ganji V, Kafai M. Frequent consumption of milk, yogurt, cold breakfast cereals, peppers, and cruciferous vegetables and intakes of dietary folate and riboflavin but not vitamin B-12 and B-6 are inversely associated with serum total homocysteine concentrations in the US population. Am J Clin Nutr 2004;80:1500–7. 23. Homocysteine Lowering Trials’ Collaboration. Lowering blood homocysteine with folic acid based supplements: metaanalysis of randomized trials. BMJ 1998;316:894–8. 24. Rader JI. Folic acid fortification, folate status and plasma homocysteine. J Nutr 2002;132:S2466–70. 25. Backstrand JR. The history and future of food fortification in the United States: a public health perspective. Nutr Rev 2002;60:15–26.
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26. Jacques PF, Bostom AG, Williams RR, et al. Relationship between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 1996;93:7–9. 27. Guttormsen AB, Ueland PM, Nesthus I, et al. Determinants and vitamin responsiveness of intermediate hyperhomocysteinemia (ⱖ40 mol/L): the Hordaland Homocysteine Study. J Clin Invest 1996;98:2174–83. 28. Kim KN, Kim KJ, Chang N. Effects of the interaction between the C677T 5,10-methelenetetrahydrofolate reductase polymorphism and serum B vitamins on homocysteine levels in pregnant women. Eur J Clin Nutr 2004;58:10–6.
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29. Stern LL, Shane B, Bagley PJ, et al. Combined marginal folate and riboflavin status affect homocysteine methylation in cultured immortalized lymphocytes from persons homozygous for the MTHFR C677T mutation. J Nutr 2003;133:2716–20. 30. Kimura M, Umegaki K, Higuchi M, et al. Methylenetetrahydrofolate reductase C677T polymorphism, folic acid and riboflavin are important determinants of genomic stability in cultured human lymphocyte. J Nutr 2004;134:48–56. 31. De Vriese AS, Verbeke F, Schrijvers BF, et al. Is folate a promising agent in the prevention and treatment of cardiovascular disease in patients with renal failure? Kidney Int 2002;61:1199–209.
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ABSTRACT: Background: Observational studies have shown an inverse relationship between vitamin B2 status and total homocysteine levels, a risk factor for cardiovascular disease. We hypothesize that intervention with riboflavin will lower total homocysteine levels. The total homocysteine lowering by the three genotypes (CC, CT, TT) of methylenetetrahydrofolate reductase polymorphism (677C¡T) was also studied. Methods: The decrease in total homocysteine levels after supplementation with riboflavin (10 mg/d) or folic acid (1 mg/d) for 3 weeks was compared in two groups of healthy subjects (17 per group, matched by age and gender) (Phase 1). Then, both groups received supplementation with folic acid and riboflavin for an additional 3 weeks (Phase 2). Results: During Phase 1, total homocysteine levels were lowered by 2% or 4% after supplementation with riboflavin or fatty acid, respectively, although neither decrease was statistically significant (P ⫽ 0.50 and 0.19). Compared to subjects of CC genotype, total homocysteine lowering in subjects of CT
genotype was approaching significance (P ⫽ 0.059) for the folic acid group, but not for the riboflavin group. After Phase 2, total homocysteine levels were not lowered significantly in either the folic acid (1%) or the riboflavin (2%) group. However, in the folic acid–riboflavin combined group, total homocysteine lowering in subjects of TT type was larger when compared to subjects of CC and CT types (P ⫽ 0.007). Conclusions: Riboflavin supplementation did not lower total homocysteine levels in healthy subjects with CC type of C677T polymorphism. However, supplementation with folic acid or with both folic acid and riboflavin may be important for CT and TT subjects in optimizing their homocysteine metabolism. These findings are relevant in characterizing the factors controlling the high total homocysteine levels for subjects of CT and TT genotypes. KEY INDEXING TERMS: Homocysteine; Riboflavin; Folic acid; Methylenetetrahydrofolate reductase; Intervention. [Am J Med Sci 2006;331(2):65–71.]
H
Vitamins B2 (as flavin adenine dinucleotide [FAD]), B6, B12, and folic acid are important cofactors or substrates of homocysteine metabolism. Blood concentrations of folate and vitamins B6 and B12 have been shown to be determinants of total homocysteine levels.4,5 Supplementation of folic acid alone or with vitamins B6 and B12 has been shown to decrease total homocysteine levels in diseased and healthy populations.6,7 Recent observational studies have indicated that vitamin B2 (riboflavin) may be another determinant of total homocysteine level,8 –12 suggesting that riboflavin may be an additional supplement, as is folic acid, to lower total homocysteine levels. However, intervention studies with riboflavin are needed to confirm the causal relationship between vitamin B2 and total homocysteine levels and to determine whether riboflavin supplementation can be an effective way to lower total homocysteine levels. Only two intervention studies have been conducted, and neither found that riboflavin could lower total homocysteine levels significantly.13,14 Observational studies have also shown that the relationship between vitamin B2 and total homocys-
igh plasma total homocysteine levels are associated with an increased risk of cardiovascular disease, pregnancy loss, and cognitive disorders.1–3
From the Department of Medicine, School of Medicine (CZC, AB, and the School of Public Health (JS), Louisiana State University Health Sciences Center, and Tulane-Charity-LSU General Clinical Research Center (SJ), New Orleans, Louisiana. Submitted for publication March 22, 2005; accepted for publication August 17, 2005. Supported by a Research Enhancement Grant from LSUHSC and in part by both a GCRC grant (NIH Grant #5M01 RR05096-10) and a grant from NCI (R25 #CA047877). Presented in part at the Southern Regional Meeting of the American Federation for Medical Research, February, 2004, New Orleans (Chuang CZ, Boyles A, LeGardeur B, Su J, Lopez-S A. Plasma total homocysteine lowering after supplementation with folic acid and riboflavin in healthy subjects [abstract]. J Investig Med 2004;52:S291.) and at Experimental Biology 2002, April, 2002, New Orleans (Chuang CZ, Boyles A, LeGardeur B, Su J, Lopez-S A. Total homocysteine levels after riboflavin or folic acid supplementation in healthy subjects [abstract]. FASEB 2002;16:A266.) Correspondence: Cherng Zee Chuang, PhD, Louisiana State University Health Sciences Center, Department of Medicine, Section of Nutrition, 1542 Tulane Avenue, New Orleans, LA 70112 (E-mail: [email protected]). BL, AL-S)
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RF/FA Supplementation and Homocysteine
teine levels could be modified by folic acid status9 and by 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism.8 –10 The present study initially investigated the effect of total homocysteine levels after supplementation with riboflavin or fatty acid and then investigated whether subsequent supplementation of both riboflavin and fatty acid would lower total homocysteine levels in those subjects who have been presupplemented with either riboflavin or folic acid. The lowering of total homocysteine levels was also studied in subjects of different MTHFR C677T genotypes. Subjects and Methods Subjects Healthy adult volunteers (N ⫽ 37) were recruited by advertisement (between October 2000 and March 2001) and 34 subjects completed the study. Three subjects did not return to complete the four blood sample drawings during the study, and thus they were dropped from the analysis. Subjects reported to the TulaneCharity-Louisiana State University General Clinical Research Center (GCRC) in the morning to have their blood drawn. Subjects completed a self-reported questionnaire regarding their lifestyle (smoking status, quantity of coffee and/or alcoholic beverage consumption, and amount of physical activity) and medical history. Subjects were excluded if they had a history of diabetes or cardiovascular, liver, renal, or thyroid diseases. The subjects (n ⫽ 12) who were taking any one of the four B vitamins being studied were asked to stop taking supplements for at least 3 days prior to their enrollment in the study. The average length of time from stopping supplements to enrollment in the study was 7.6 days, with a range of 3-15 days. The subjects returned to the GCRC after 1 week for their second blood sample drawing. The average of blood values from the first and second samples was used as the baseline level. Informed consent was obtained and the study was approved by the Institutional Review Board of Louisiana State University Health Sciences Center at New Orleans.
fluorometric detection.15 Plasma levels of folic acid and vitamin B12 were analyzed by radioimmunoassay (Diagnostic Products Corp, USA). Although plasma folic acid level is not as precise an indicator as red blood cell folate level for folic acid status, it has been used as a general and convenient indicator. Owing to limited resources, we measured plasma folate only as the folic acid indicator in this study. The status of vitamins B2 and B6 was analyzed by erythrocyte glutathione reductase activation coefficient (EGRAC) and erythrocyte aspartate aminotransferase activation coefficient (EASTAC), respectively,16 in Pennington Biomedical Research Center. Both tests reflect tissue levels of these vitamins and are not affected by recent dietary intakes. A higher value of either EGRAC or EASTAC indicates a lower vitamin status. All four samples from each individual were analyzed in the same batch. Between-run precision for total homocysteine, folic acid, and vitamin B12 was 8% (9 mol/L; n ⫽ 15), 5% (23 nmol/L; n ⫽ 6), and 5% (450 pmol/L; n ⫽ 6), respectively. The polymorphism of MTHFR C677T was determined by the method of Frosst.17 Plasma creatinine levels were measured by a commercial kit (DMA, USA). Statistical Analysis Statistix software (Analytical Software, USA) was used to perform all statistical analyses. The difference in subject characteristics between the riboflavin and folic acid groups was analyzed by sample t-test, by Kruskal-Wallis test (if the distribution was not gaussian) or by 2 test. The total homocysteine levels and blood status of four B vitamins before and after phase 1 or phase 2 supplementation were compared by paired t-test. The lowering of total homocysteine level stratified by genotypes was analyzed by one-way analysis of variance. The significance levels were chosen at P ⬍ 0.05. Several analyses were performed to assure that subjects who were vitamin B users could be included in the analysis. There was no significance for vitamin B2 and folic acid status at baseline between vitamin B users and vitamin B nonusers (by sample t-test). The results of total homocysteine lowering (mean and P value) during phase 1 for both groups were similar when vitamin B users were excluded from the analysis. In addition, vitamin B2 and folic acid status between blood sampling 1 and sampling 2 were statistically nonsignificant in the riboflavin, folic acid, and combined groups and in the vitamin B users. Therefore, we included vitamin B users in the final analyses.
Supplementation After the second blood sample drawing, subjects were randomly divided into two groups (riboflavin and folic acid) matched by age and gender. There were two phases of supplementation in this study (phases 1 and 2). During phase 1, subjects in the riboflavin group received 10 mg riboflavin per day and subjects in the folic acid group received 1 mg folic acid per day for 3 weeks. After the completion of phase 1, subjects in both groups received both folic acid (1 mg/day) and riboflavin (10 mg/day) supplements for an additional 3 weeks (phase 2). Third and fourth blood samples were collected at the end of phases 1 and 2, respectively. All blood samples were drawn with the subjects fasting, and EDTA tubes were used. Plasma samples were separated within 2 hours and stored at ⫺70° C until analysis. The riboflavin supplements (10 mg/capsule) were prepared by a local licensed pharmacist and the folic acid supplements (1 mg/tab) were of commercial brand (Danbury Pharmacal). Three supplements of both riboflavin and folic acid were randomly selected for analysis by high-performance liquid chromatography and their contents were found to be accurate (95-105% and 107-117%, respectively). All participating subjects were advised not to take any vitamin supplements other than the supplements provided by the research staff and to maintain their usual dietary intake during the 6-week period. Biochemical Measurements Plasma total homocysteine level and blood status of B vitamins (folic acid, B2, B6, and B12) were analyzed for the four blood samples from each subject. Plasma total homocysteine levels were analyzed by high-performance liquid chromatography with
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Results The subject characteristics of the riboflavin and folic acid supplemented groups are shown in Table 1. There is no difference between the two groups for any of these characteristics. Phase 1 At the baseline, there are two subjects (12%) in the riboflavin group who show a deficiency of vitamin B2 (EGRAC ⬎1.40; the lower the EGRAC, the better the vitamin B2 status). Their EGRAC decreased below 1.40 after phase 1 supplementation, indicating that supplementation of 10 mg riboflavin for 3 weeks is sufficient to improve vitamin B2 status to normal in this population. In the riboflavin-supplemented group (between A and B in Table 2), EGRAC was significantly lowered from 1.22 to 1.06 (P ⬍ 0.0001) after supplementation. However, total homocysteine levels dropped by only 2% (from 7.81 to 7.67 mol/L), which was not statistically significant (P ⫽ 0.503). For the folic acid supplemented group (between A and B in Table 3), plasma folic acid levels were nearly doubled (from 37.5 to 69.7 2 February 2006 Volume 331 Number 2
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Table 1. Subject Characteristics of the Riboflavin and Folic Acid Supplemented Groups Riboflavin Supplemented Group (n ⫽ 17)
Folic Acid Supplemented Group (n ⫽ 17)
P-valuea
39.3 ⫾ 15.2 23.5 ⫾ 2.8 92.8 ⫾ 23.0 40.8 ⫾ 34.4 1.4 ⫾ 1.4 24.2 ⫾ 23.9 5:12 13:2:2 6:11 10:7 9:5:2 5:7
38.0 ⫾ 13.1 23.4 ⫾ 3.0 98.1 ⫾ 21.2 19.0 ⫾ 26.2 1.3 ⫾ 1.1 21.0 ⫾ 24.0 5:12 11:3:3 6:11 11:6 8:6:2 6:6
0.792 0.902 0.496 0.091 0.932 0.631 1.000 0.753 1.000 0.724 0.928 0.682
Age, year Body mass index Creatinine, mol/L Alcohol,b g/wk Coffee,b cups/d Physical activity,b min/d Men:women White:black:others Vitamin B supplements (Y:N) Nonsmoker:ex-smoker MTHFR (CC:CT:TT)c Contraceptive User (Y:N) a b c
Continual variables were analyzed by sample t-test or by Kruskal-Wallis test and noncontinual variables were analyzed by 2. Variables were analyzed by Kruskal-Wallis test. Two subjects missed MTHFR analysis.
nmol/L) after folic acid supplementation (P ⫽ 0.006). The total homocysteine levels were decreased by 4% (from 7.68 to 7.40 mol/L), although not significantly (P ⫽ 0.189). Surprisingly, EGRAC was significantly increased from 1.20 to1.25 (P ⫽ 0.002), indicating the worsening of vitamin B2 status after folic acid supplementation. Gender stratification in both groups revealed that total homocysteine levels were significantly decreased by 7% (from 7.41 ⫾ 1.24 to 6.87 ⫾ 1.15 mol/L, P ⫽ 0.041) for females in the folic acid group, but were not significant for the other three gender-based supplementation groups. EGRAC was also increased from 1.22 ⫾ 0.16 to 1.27 ⫾ 0.16 (P ⫽ 0.004) for females in the folic acid group. When compared to subjects of CC genotype, subjects of CT genotype in folic acid group (Table 4, P ⫽ 0.059) demonstrated a greater total homocysteine lowering; however, the same did not hold for riboflavin group. Phase 2 For the riboflavin group (between B and C in Table 2), the EGRAC was improved slightly again (from 1.06 to 1.04) at the end of phase 2. Plasma folic acid levels were doubled (from 35.2 to 82.3 nmol/L) after the additional supplementation of folic acid during phase 2. However, total homocysteine levels decreased only by 1% (from 7.67 to 7.57 mol), which
is not statistically significant. In the folic acid group (between B and C in Table 3), plasma folic acid levels reached a plateau (69.7 and 68.3 nmol/L) after continual supplementation of folic acid from phase 1. The EGRAC was significantly improved from 1.25 to 1.08 due to the additional supplementation of riboflavin during phase 2. However, total homocysteine levels dropped only by 2% (from 7.40 to 7.24 mol), which is not statistically significant. In Table 1, the total homocysteine levels did not change much after supplementation with both folic acid and riboflavin in subjects of CC and CT genotypes. However, total homocysteine levels were significantly lowered for subjects of TT genotype in the folic acid–riboflavin combined group when compared to subjects of CC and CT types (P ⫽ 0.007). A scattering plot for total homocysteine changes by MTHFR genotypes in the combined group is shown in Figure 1. Total homocysteine levels were lowered in each of the four subjects with TT genotype after supplementation with both folic acid and riboflavin. Discussion Moderately high total homocysteine levels are associated with an increased risk of cardiovascular disease, pregnancy complications, neural tube defects, and cognitive disorders.1–3 Both lifestyle and
Table 2. Total Homocysteine and Vitamin Status in Riboflavin Group
Plasma total homocysteine, mol/L Plasma folic acid, nmol/L EGRAC EASTAC Plasma cyanocobalamin, pmol/L a
Baseline (A)
Supplementation with Riboflavin (B)
Supplementation with Riboflavin and Folic Acid (C)
P-Valuea between A and B
P-Valuea between B and C
P-Valuea between A and C
7.81 ⫾ 1.53 36.8 ⫾ 12.9 1.22 ⫾ 0.14 2.04 ⫾ 0.23 289 ⫾ 153
7.67 ⫾ 1.26 35.2 ⫾ 12.0 1.06 ⫾ 0.06 2.03 ⫾ 0.18 278 ⫾ 129
7.57 ⫾ 1.79 82.3 ⫾ 51.8 1.04 ⫾ 0.05 2.07 ⫾ 0.19 304 ⫾ 170
0.503 0.383 ⬍0.0001 0.731 0.474
0.592 0.003 0.047 0.182 0.256
0.268 0.003 ⬍0.0001 0.433 0.658
The comparison was analyzed by paired t-test.
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Table 3. Total Homocysteine and Vitamin Status in Folic Acid Group
Plasma total homocysteine, mol/L Plasma folic acid, nmol/L EGRAC EASTAC Plasma cyanocobalamin, pmol/L a
Baseline (A)
Supplementation with Folic Acid (B)
Supplementation with Riboflavin and Folic Acid (C)
7.68 ⫾ 1.26 37.5 ⫾ 14.5 1.20 ⫾ 0.14 2.04 ⫾ 0.26 268 ⫾ 110
7.40 ⫾ 1.44 69.7 ⫾ 47.2 1.25 ⫾ 0.15 1.96 ⫾ 0.16 283 ⫾ 116
7.24 ⫾ 1.21 68.3 ⫾ 45.1 1.08 ⫾ 0.12 2.04 ⫾ 0.18 297 ⫾ 149
P-Valuea between A and B
P-Valuea between B and C
P-Valuea between A and C
0.189 0.006 0.002 0.052 0.354
0.431 0.866 ⬍0.0001 0.0005 0.462
0.014 0.003 ⬍0.0001 0.933 0.227
The comparison was analyzed by paired t-test.
genetics are important factors for total homocysteine levels. One of the major determinants of total homocysteine levels is nutritional status in terms of folic acid, vitamin B6, and vitamin B12.4,5 Since vitamin B2 (as flavin mononucleotide) is involved in the formation of active forms of folic acid, vitamin B6 and vitamin B12 in the homocysteine metabolism,18 –20 the significance of vitamin B2 in the control of total homocysteine levels still needs to be determined. Recently, several observational studies have shown an inverse relationship between vitamin B2 dietary intakes and total homocysteine level21,22 or between vitamin B2 status and total homocysteine level in healthy subjects and in patients with end-stage renal disease,8 –12 indicating that vitamin B2 may be another determinant of total homocysteine level. However, results of these association studies cannot support the causal relationship between total homocysteine and vitamin B2 and only intervention studies can confirm the effect of vitamin B2 on the total homocysteine levels. This study did not find a significant decrease (2%) of total homocysteine levels after riboflavin supplementation in these healthy volunteers. Neither do we find a decrease of total homocysteine levels after supplementation with riboflavin in subjects who received pre-supplementation with folic acid or after supplementation with folic acid in subjects who received pre-supplementation with RF. The 2% decrease after riboflavin supplementation is less than the decrease (4%) seen in the age- and gender-
matched folic acid–supplemented group. Although the 4% decrease is statistically nonsignificant in the folic acid group (n ⫽ 17), the decrease in total homocysteine levels (7%) is statistically significant when reanalyzed in only female subjects (n ⫽ 12). In addition, total homocysteine levels were also significantly decreased after 6 weeks of supplementation with folic acid (Table 3, between A and C). This extent of decrease of total homocysteine after folic acid supplementation is smaller than the decrease observed in previous studies,23 probably due to the fortification of cereal grains with folic acid in the United States since January 1998 and/or to the shorter period (3 weeks) of supplementation in this study. The baseline plasma level of folic acid (37 nmol/L or 16 ng/mL) is comparable to studies with blood samples collected after the starting date of folic acid fortification.24 It seems that folic acid is more effective than riboflavin in the lowering of total homocysteine levels. This is understandable, since folic acid functions as a co-substrate while riboflavin (as FAD) functions as a cofactor of enzymes in the homocysteine metabolism. The findings are similar to those of the only two other studies available.10,11 Lakshmi found that the total homocysteine level was lowered from 12.1 to 11.8 mol/L (2.5%, nonsignificant) after riboflavin (10 mg/d, 15 days) supplementation in 10 Asian Indian women with glossitis and angular stomatitis.10 For McKinley’s study in 23 community-living healthy elderly subjects, the total homocysteine lev-
Table 4. Lowering of Total Homocysteine Levels Grouped by MTHFR C677T Genotypes
Folic acid group Riboflavin group Folic acid and riboflavin combined group
Supplementation
CC
CT
TTa
Folic acid, mol/L Folic acid and riboflavin, mol/L Riboflavin, mol/L Folic acid and riboflavin, mol/L Folic acid and riboflavin, mol/L
⫺0.186 ⫾ 0.903 ⫺0.045 ⫾ 0.849 ⫺0.067 ⫾ 0.714 0.119 ⫾ 0.764 0.042 ⫾ 0.784
⫺0.698 ⫾ 0.552 0.077 ⫾ 0.549 0.01 ⫾ 1.00 ⫺0.072 ⫾ 0.892 0.009 ⫾ 0.689
0.918 ⫺1.46 ⫺0.69 ⫺1.12 ⫺1.29 ⫾ 0.41
P-valueb 0.059c 0.051d 0.618c 0.176d 0.007d
a
There are only two subjects of TT genotype for the folic acid or riboflavin group and only their means are shown. Variables were analyzed by one-way analysis of variance. c The comparison was between CC and CT genotypes. d The comparison was between CC, CT, and TT genotypes. b
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Figure 1. Scatter plot for total homocysteine changes versus MTHFR genotypes in the combined group
els changed from 12.00 to 12.57 mol/L (P ⫽ 0.719) after the subjects took 1.6 mg of riboflavin supplements daily for 12 weeks.11 Although riboflavin as FAD is the cofactor of MTHFR, one of the important enzymes in the homocysteine metabolism, the present study and the other two intervention studies do not support the idea that vitamin B2 is an effective nutrient in lowering total homocysteine levels, at least in healthy subjects. Vitamin B2 and several other B vitamins have been fortified in cereal grains in the United States since the 1940s,25 resulting in adequate vitamin B2 status of the general population. This may affect our ability to see the effect of vitamin B2 supplementation on total homocysteine levels in these healthy subjects. Since all three of these intervention studies involved a small number of subjects, there is a possibility that the power is not sufficient to observe a decrease of tHcy after riboflavin supplementation. Future intervention studies in a large population of subjects with suboptimal vitamin B2 status will be necessary to confirm this. The data from this intervention study also show that total homocysteine decrease after riboflavin and/or folic acid supplementation is dependent on the MTHFR C677T genotypes. This is reflected in both the lowering of total homocysteine level in subjects of CT genotypes compared to subjects of CC genotypes in the folic acid–supplemented group during phase 1 and in the lowering of total homocysteine level in subjects of TT type compared to subjects of CC and CT types during phase 2 (supplemented with both folic acid and riboflavin). These findings are in agreement with recent observational and in vitro studies on the relationship between total homocysteine, riboflavin, folic acid, and genotypes of MTHFR C677T. Previous studies have found that higher total homocysteine levels are associated with MTHFR THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
C677T polymorphism when the blood folic acid status is on the lower end of normal values,26 –28 indicating that MTHFR genotypes might modify the relationship between folic acid and total homocysteine levels. Four recent observational studies8 –10,28 indicated that MTHFR C677T could modify the relationship between vitamin B2 and total homocysteine, but one study did not.12 Although the four supporting studies differ in which genotypes can interact with this relationship, all four studies indicate that TT genotype is a modifying factor. Recent in vitro studies with lymphocytes from subjects of CC and TT genotypes indicate that MTHFR is a modifying factor when the supply of folic acid and/or riboflavin is low in the culture medium.29,30 Stern et al found that total homocysteine levels are low in the culture medium and the difference of total homocysteine in the culture medium is not significant between cells of CC and TT types when adequate folic acid and riboflavin are present in the culture medium.29 In contrast, when both folic acid and riboflavin are low in the culture medium, the culture medium total homocysteine levels are high and there is a significant difference of total homocysteine levels in the culture medium between cells of CC and TT types. Kimura’s data30 have shown that the difference of medium total homocysteine levels between cells of CC and TT types is small when cultured in media with high folic acid and high riboflavin concentrations. However, the difference is increased to 2.5- to threefold when lymphocytes are cultured in media with high folic acid/low riboflavin, low folic acid/high riboflavin, or low folic acid/low riboflavin nutrients. This study’s findings are in agreement with these two in vitro studies, indicating that the requirements for folic acid in subjects with CT type and the requirements of folic acid and riboflavin in subjects with TT type are probably higher than those required for CC subjects in the optimization of homocysteine metabolism. Since the baseline total homocysteine levels of healthy subjects in this study are already within the normal values (⬍12 mol/L), the benefits from total homocysteine lowering again with folic acid/riboflavin supplementation in healthy subjects of CT and TT types with normal status of B vitamins probably will be limited. However, the supplementation of folic acid and riboflavin will be of importance for CT and TT subjects who are hyperhomocysteinemic and are at risk for the aforementioned total homocysteine–related diseases.1–3 For example, many studies have shown that total homocysteine levels cannot be normalized in patients with chronic renal disease when supplemented with high doses of folic acid alone or with vitamins B6 and B12.31 An additional supplement of vitamin B2 may help lower total homocysteine levels even more for some of these patients. A limitation of the present findings is the small number (N ⫽ 4) of subjects with TT genotype. 69
RF/FA Supplementation and Homocysteine
A surprising finding of this study is that the vitamin B2 status was significantly worsened (EGRAC increased from 1.20 to 1.25, P ⫽ 0.002) after folic acid supplementation (1 mg/d for three weeks). A similar finding was reported in a recent study of 126 healthy individuals (20-63 years) who were supplemented with 400 g folic acid daily for 4 months.12 The authors suggested that this worsening of vitamin B2 status might be due to the increased turnover of homocysteine metabolism, leading to the increased requirement of riboflavin in cells and/or a shifting of FAD from erythrocyte glutathione reductase to MTHFR. However, EGRAC was not changed (1.24 ⫾ 0.06) after subjects received 400 g folic acid per day for 6 weeks in McKinley’s study.14 The relevance of this topic necessitates further studies in this area. Since folic acid supplement or fortification has been used as an inexpensive means to prevent neural tube defects or to decrease total homocysteine levels, this finding will raise an interesting question: whether vitamin B2 supplements should be administered along with folic acid in long-term folic acid supplementation or fortification. In conclusion, the present study found that riboflavin supplementation did not lower total homocysteine levels effectively in healthy subjects of CC type of C677T polymorphism. However, supplementation with folic acid or with both folic acid and riboflavin may be of specific importance for subjects of CT and TT types in the optimization of their homocysteine metabolism. These findings are relevant in the lowering of high total homocysteine levels for subjects of CT and TT types. Future studies in a large population and with sufficient subjects of TT genotypes are necessary to confirm these results. References 1. Mangoni AA, Jackson SHD. Homocysteine and cardiovascular disease: current evidence and future prospects. Am J Med 2002;112:556–65. 2. Hankey GJ, Eikelboom JW. Homocysteine and vascular disease. Lancet 1999;354:407–19. 3. Ray JG, Laskin CA. Folic acid and homocyst(e)ine metabolic defects and the risks of placental abruption, pre-eclampsia and spontaneous pregnancy loss: a systematic review. Placenta 1999;20:519–29. 4. Malinow MR, Bostom AG, Krauss RM. Homocyst(e)ine, diet, and cardiovascular disease. A statement for healthcare professionals from the Nutrition Committee, American Heart Association. Circulation 1999;99:178–82. 5. Selhub J, Jacques PF, Wilson PW, et al. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 1993;270:2693–98. 6. Ubbink JB, Vermaak WJ, van der Merwe A, et al. Vitamin requirements for the treatment of hyperhomocysteinemia in humans. J Nutr 1994;124:1927-33. 7. den Heijer M, Brouwer IA, Bos GMJ, et al. Vitamin supplementation reduces blood homocysteine levels: a controlled trial in patients with venous thrombosis and healthy volunteers. Arterioscler Thromb Vasc Biol 1998;18:356–61.
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8. Hustad S, Ueland PM, Vollset SE, et al. Riboflavin as a determinant of plasma total homocysteine: effect modification by the methylenetetrahydrofolate reductase C677T polymorphism. Clin Chem 2000;46:1065–71. 9. Jacques PF, Kalmbach R, Bagley PJ, et al. The relationship between riboflavin and plasma total homocysteine in the Framingham offspring cohort is influenced by folate status and the C677T transition in the methylenetetrahydrofolate reductase gene. J Nutr 2002;132:283–8. 10. McNulty H, McKinley MC, Wilson B, et al. Impaired functioning of thermolabile methylenetetrahydrofolate reductase is dependent on riboflavin status: implications for riboflavin requirements. Am J Clin Nutr 2002;76:436–41. 11. Skoupy S, Fodinger M, Veitl M, et al. Riboflavin is a determinant of total homocysteine plasma concentration in end–stage renal disease patients. J Am Soc Nephrol 2002;13: 1331–7. 12. Moat SJ, Ashfield-Watt PAL, Powers HJ, et al. Effect of riboflavin status on the homocysteine-lowering effect of folate in relation to the MTHFR (C677T) genotype. Clin Chem 2003;49:295–302. 13. Lakshmi AV, Ramalakshmi BA. Effect of pyridoxine or riboflavin supplementation on plasma homocysteine levels in women with oral lesions. Natl Med J India 1998;1:171–2. 14. McKinley MC, McNulty H, McPartlin J, et al. Effect of riboflavin supplementation on plasma homocysteine in elderly people with low riboflavin status. Euro J Clin Nutr 2002;56:850–6. 15. Fortin LJ, Genest J Jr. Measurement of homocysteine in the prediction of arteriosclerosis. Clin Biochem 1995;28:155– 62. 16. Bayoumi RA, Rosalki SB. Evaluation of methods of coenzyme activation of erythrocyte enzymes for deficiency of vitamin B1, B2, and B6. Clin Chem 1976;22:327–35. 17. Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111–3. 18. Bates CJ, Fuller NJ. The effect of riboflavin deficiency on methylenetetrahydrofolate reductase (NADPH) (EC1.5.1.20) and folate metabolism in the rat. Br J Nutr 1986;55:455–64. 19. McCormick DB. Two interconnected B vitamins: riboflavin and pyridoxine. Physiol Rev 1989;69:1170–98. 20. Olteanu H, Banerjee R. Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for cloning and mapping of NADPHdependent methionine synthase activation. J Biol Chem 2001;276:35558–63. 21. Jacques PF, Bostom AG, Wilson PWF, et al. Determinants of plasma total homocysteine concentration in the Framingham offspring cohort. Am J Clin Nutr 2001;73:613– 21. 22. Ganji V, Kafai M. Frequent consumption of milk, yogurt, cold breakfast cereals, peppers, and cruciferous vegetables and intakes of dietary folate and riboflavin but not vitamin B-12 and B-6 are inversely associated with serum total homocysteine concentrations in the US population. Am J Clin Nutr 2004;80:1500–7. 23. Homocysteine Lowering Trials’ Collaboration. Lowering blood homocysteine with folic acid based supplements: metaanalysis of randomized trials. BMJ 1998;316:894–8. 24. Rader JI. Folic acid fortification, folate status and plasma homocysteine. J Nutr 2002;132:S2466–70. 25. Backstrand JR. The history and future of food fortification in the United States: a public health perspective. Nutr Rev 2002;60:15–26.
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26. Jacques PF, Bostom AG, Williams RR, et al. Relationship between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 1996;93:7–9. 27. Guttormsen AB, Ueland PM, Nesthus I, et al. Determinants and vitamin responsiveness of intermediate hyperhomocysteinemia (ⱖ40 mol/L): the Hordaland Homocysteine Study. J Clin Invest 1996;98:2174–83. 28. Kim KN, Kim KJ, Chang N. Effects of the interaction between the C677T 5,10-methelenetetrahydrofolate reductase polymorphism and serum B vitamins on homocysteine levels in pregnant women. Eur J Clin Nutr 2004;58:10–6.
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29. Stern LL, Shane B, Bagley PJ, et al. Combined marginal folate and riboflavin status affect homocysteine methylation in cultured immortalized lymphocytes from persons homozygous for the MTHFR C677T mutation. J Nutr 2003;133:2716–20. 30. Kimura M, Umegaki K, Higuchi M, et al. Methylenetetrahydrofolate reductase C677T polymorphism, folic acid and riboflavin are important determinants of genomic stability in cultured human lymphocyte. J Nutr 2004;134:48–56. 31. De Vriese AS, Verbeke F, Schrijvers BF, et al. Is folate a promising agent in the prevention and treatment of cardiovascular disease in patients with renal failure? Kidney Int 2002;61:1199–209.
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