The Effects of Folic Acid Supplementation on Plasma Total Homocysteine Are Modulated by Multivitamin Use and Methylenetetrahydrofolate Reductase Genotypes
Abstract Elevated concentration of plasma total homocysteine (tHcy) is a common risk factor for arterial occlusive diseases. Folic acid (FA) supplementation usually lowers tHcy levels, but initial tHcy and vitamin levels, multivitamin use, and polymorphisms in the gene for 5,10-methylenetetrahydrofolate reductase (MTHFR) may contribute to variability in reduction. We tested the effects of a 3-week daily intake of 1 or 2 mg of FA supplements on tHcy levels in patients with and without coronary heart disease (CHD) who were analyzed for the C677T MTHFR mutation. Prior multivitamin intake and baseline vitamin and tHcy levels were also compared with responsiveness to folate supplementation. Both dosages of FA lowered tHcy levels similarly, regardless of sex, age, CHD status, body mass index, smoking, or plasma creatinine concentration. In non–multivitamin users, FA supplements reduced tHcy by 7% in C/C homozygotes and by 13% or 21% in subjects with one or two copies of the T677 allele, respectively; the corresponding reductions were smaller in users of multivitamins. Moreover, T/T homozygotes had elevated tHcy and increased susceptibility to high levels of tHcy at marginally low plasma folate levels, as well as enhanced response to the tHcy-lowering effects of FA. Although other factors are probably involved in the responsiveness of tHcy levels to FA supplementation, about one third of heterogeneity in responsiveness was attributable to baseline tHcy and folate levels and to multivitamin use.
- Received April 23, 1996.
- Accepted October 18, 1996.
Numerous investigations have demonstrated that the concentration of plasma tHcy is elevated in patients with coronary, cerebral, or peripheral arterial occlusive diseases.1 2 3 4 Plasma/serum homocyst(e)ine [H(e)], or tHcy, is the sum of the concentration of the amino acid homocysteine and the homocysteinyl moieties of the disulfides homocystine and homocysteine-cysteine, whether free or bound to plasma proteins. Homocyst(e)inemia refers to plasma levels of tHcy; hyperhomocyst(e)inemia indicates elevated concentration of plasma/serum tHcy. A comprehensive meta-analysis of 27 studies relating elevated plasma tHcy levels and arterial occlusive diseases was consistent with a causal role of plasma tHcy in the pathogenesis of vascular disease.2 Thus, elevated levels of plasma tHcy are considered to be a common risk factor for arterial occlusive diseases,3 and this risk is graded across the concentration distribution of tHcy.2 FA supplementation (between 0.5 and 10 mg/d),5 6 7 as well as multivitamins containing, inter alia, 0.2 to 0.4 mg FA,8 9 reduces plasma tHcy concentrations. Further studies are required to elucidate the factors responsible for interindividual heterogeneity in responsiveness to folate supplementation. We hypothesized that allelic variations in the gene for MTHFR, an FA-related enzyme, as well as baseline tHcy and vitamin levels, may contribute to that heterogeneity. To test this hypothesis, the effects of a daily supplement of either 1 or 2 mg of FA for 3 weeks on plasma tHcy levels were examined in subjects with or without CHD who were homozygous, heterozygous, or null for the C677T mutation in the MTHFR gene. The modulating effects of multivitamin use and baseline tHcy and vitamin levels on responsiveness to FA supplementation in these subjects also were tested.
Subjects were recruited from internal medicine or family practice physicians associated with Providence St Vincent Hospital (Portland, Ore), from a cohort of patients discharged with the diagnosis of ischemic heart disease (ICD 9 code 410-414), or were self-referred in response to advertisements. The study population included 317 unrelated men and women 45 to 85 years of age at the time of the initial interview. Subjects were excluded from the study if they had received medication(s) within 7 days that may have an effect on tHcy levels (ie, methotrexate, tamoxifen, anticonvulsants, bile acid sequestrants, or nitrous oxide anesthesia). Other exclusion criteria were missed laboratory appointments, ingesting ≥0.8 mg FA daily, or plasma creatinine levels ≥1.7 mg/dL. The study population was limited to 242 subjects in whom MTHFR genotyping was performed, thus fitting in with the main hypothesis of the study. All subjects were advised to continue with their usual medications, including multivitamins, throughout the observation. The study was approved by the Institutional Review Boards of Providence St Vincent Hospital and the Oregon Regional Primate Research Center.
Case subjects were diagnosed more than 3 months previously with a history of acute myocardial infarction, angina pectoris documented by a cardiologist, percutaneous transarterial coronary angioplasty, or coronary bypass graft surgery (n=140). Control subjects had no history of CHD (n=102). Case and control subjects reported having no history of stroke, intermittent claudication, or peripheral arterial revascularization.
All subjects completed a medical history form, signed an informed consent form, and were then randomized to receive either 1 or 2 mg FA per day for 3 weeks. The need for a placebo group was obviated by previous data that demonstrated stability of tHcy and folate plasma levels during a 6-week interval.7 Subjects were requested to arrive at laboratory appointments in the fasting state (ie, no food after midnight) and were instructed not to take any vitamins on the morning of phlebotomy. During the first appointment, 1-mg FA tablets were given to the subjects, with appropriate instructions. During the second visit, subjects returned their remaining FA tablets for assessment of compliance.
Within 30 minutes of venous blood drawing, plasma was separated in a refrigerated centrifuge at 4°C for clinical chemistry and then frozen for analysis of tHcy by high-pressure liquid chromatography and electrochemical detection as described,10 11 with minor modifications (interassay CV=9.1%) (performed at Oregon Regional Primate Research Center by Dr Malinow). Plasma aliquots were protected from light and frozen at −20°C for radioimmunoassay of FA (CV=7.8%) and vitamin B12 (CV=5.4%) (Bio-Rad Quantaphase II, Bio-Rad Diagnostics) and for radioenzymatic assay of P5′P (CV=14.4%; American Laboratory Product Company, Buhlman Laboratories AG) (performed at Oregon Regional Primate Research Center by Dr Hess). The blood buffy-coat layer was separated, mixed with 3 drops of DMSO, and frozen at −20°C for analysis of the C677T MTHFR polymorphism (performed at Fox Chase Cancer Institute, Philadelphia, Pa, by Dr Kruger). After thawing, DNA was isolated by using Instagene Matrix (Bio-Rad Diagnostics). Isolated DNA was used as the template in a polymerase chain reaction, using 100 ng of the forward and reverse primers as previously described.12 The amplification reaction was performed in a 50-μL volume in 60 mmol/L Tris-HCl, 15 mmol/L (NH4)SO4, 200 μmol/L dNTP, and 5 units of Taq polymerase. The mixture was subjected to 30 cycles of amplification at 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 30 seconds. The polymerase chain reaction products were precipitated with ethanol and digested overnight with HinfI (New England Biolabs). The products were analyzed by 3% agarose gel electrophoresis.
The distribution of study variables was examined using standard exploratory data analysis techniques for independent subjects. Logarithmic transformations were performed to improve normality in some of the study variables (BMI and plasma levels of tHcy, folate, vitamin B12, and P5′P). The distributions of study variables by case-control status was compared using χ2 tests for categorical variables and t test for continuous variables. Statistical significance of changes in tHcy and folate levels was assessed using paired t test. Plasma tHcy and folate levels were compared across categories of folate dose, multivitamin status, and MTHFR genotype using standard ANOVA techniques. Adjustment for potential covariates was carried out using multiple linear and stepwise regression. Mean tHcy decreases after folate supplementation were correlated with the number of T677 alleles in the MTHFR genotypes. All reported probability values are two-sided. Statistical analyses were conducted using SAS, version 6.10 (SAS Institute) and SigmaStat (Jandel Scientific).
The majority of participants (97.4%) were white. Ninety-nine subjects were habitual multivitamin users; intake of FA in current multivitamin users ranged between 0.1 and 0.53 mg/d (mean±SD, 0.384±0.063; n=72). Multivitamins usually contained, inter alia, 2 mg of vitamin B6 and 6 μg of vitamin B12. Multivitamin users reported their duration of intake as “many months” to “several years.”
Most study participants (67%) returned unused FA tablets on their second laboratory visit. The number of remaining tablets was used for compliance assessment; data suggested that subjects consumed 99.7±10.6% of folate supplements.
Case subjects (58% of all study participants) were more likely to be male, older, and former smokers compared with the control group. Cases showed a higher prevalence of the T/T MTHFR genotype. Plasma tHcy was significantly higher in cases than in control subjects. The concentration of plasma vitamins was similar in both groups of subjects (Table 1⇓). Table 1⇓ also shows data in subjects stratified by multivitamin intake. Compared with nonusers, users of multivitamins were leaner and had lower levels of tHcy and higher plasma levels of vitamins.
FA supplementation was about equal in cases and control subjects; dosages of either 1 or 2 mg/d had similar effects on tHcy levels (Table 2⇓). In multivitamin users, folate supplementation reduced initial plasma tHcy levels by <5% (P=not significant). In nonusers of multivitamins, FA supplementation reduced plasma tHcy levels by 10% to 14% (P<.0001). However, reduced levels were somewhat higher than the lower concentrations attained by folate supplementation in users of multivitamins.
The ingestion of either 1 or 2 mg of FA increased plasma folate to approximately similar levels in users and nonusers of multivitamins. Consequently, the relative increases in plasma folate were smaller in users than in nonusers of multivitamins. The percent decrease in plasma tHcy was significantly correlated with percent change in plasma folate (r=.215, P<.01; not shown in tables). The magnitude and statistical significance of results were essentially similar after adjusting for age, log BMI, smoking, sex, CHD status, and plasma creatinine concentration (Table 2⇑).
Current multivitamin users, compared with nonusers, had higher basal levels of folate, P5′P, and vitamin B12 (Table 1⇑). The regression equations and index of correlations for log tHcy versus log plasma vitamin levels in all subjects demonstrated significant negative correlations between log basal tHcy and log plasma vitamin levels (Table 3⇓). The correlation coefficients were −.134 (P<.18), .099 (P=.33), and −.179 (P<.08) in users of multivitamins, and −.432 (P<.001), −.148 (P=.08), and −.216 (P<.01) in nonusers, for log transformed levels of folate, P5′P, and vitamin B12, respectively (not shown in the tables).
Data on effects of FA supplements stratified by multivitamin use in subjects with different MTHFR C677T genotypes are shown in Table 4⇓. In subjects with the C/C genotype, the supplements reduced tHcy levels 3% to 7%. However, tHcy levels decreased 3% to 13% in subjects heterozygous (C/T) and about 10% to 21% in subjects homozygous for the T/T mutation. The effects of FA supplements were more marked in nonusers than in multivitamin users after stratification for the C677T MTHFR polymorphisms. Results observed when subjects were stratified according to their baseline folate concentration, ie, above or below the sample geometric mean (16.1 μmol/L), are shown in Fig 1⇓. Such results resemble those shown in Table 4⇓, since baseline plasma folate concentrations were strongly related to multivitamin intake (see Table 2⇑).
On the basis of the findings shown in Table 4⇑, we hypothesized that subjects with the T/T mutation may be more susceptible to elevated levels of tHcy at marginally low folate levels. To test this hypothesis, baseline levels of tHcy were plotted as a function of baseline folate levels (Fig 2⇓). Levels of tHcy were similar in all subjects at high plasma levels of folate but increased at an accelerated rate (ie, with a steeper slope) at lower folate levels in MTHFR T677 homozygotes compared with C/T heterozygotes or C/C subjects.
A stepwise regression analysis in all subjects indicated that about one third of the heterogeneity of the tHcy response to folate supplementation was attributed to initial plasma folate and tHcy levels, especially in nonusers of multivitamins (Table 5⇓). However, these differences may be due in part to the unequal distribution of MTHFR genotypes in both groups, as shown in Table 2⇑.
This study demonstrated that multivitamin users had lower baseline levels of tHcy than nonusers, in agreement with results previously reported by Brattstrom et al8 and Pietrzik et al.9 Moreover, multivitamin users had higher baseline concentration of plasma folate, P5′P, and vitamin B12. FA supplements lowered tHcy in nonusers of multivitamins, whereas the decreases of tHcy were minimal in multivitamin users, since, as indicated above, the relative tHcy changes were inversely proportional to the baseline folate and directly proportional to the baseline tHcy levels. However, the tHcy levels attained after folate supplementation in users of multivitamins were somewhat lower than those reached in nonusers of multivitamins, perhaps due to interactions with other vitamins, the longer duration of multivitamin intake, or factors not identified in this study. Nonetheless, it seems likely that FA is mainly responsible for the effects of multivitamins on tHcy, since decreases in tHcy levels were not observed in subjects ingesting a multivitamin lacking FA.13
Our data show that supplementary doses of 1 or 2 mg of FA decreased tHcy levels equally, regardless of sex, age, presence of CHD, BMI, smoking, or plasma creatinine concentration. Whether dosages higher than 2 mg/d or a longer-term observation would have greater effects needs to be established. Previous studies, including some that were also short term, showed similar tHcy reductions with 2.5 or 10 mg/d of FA in CHD patients,5 0.5 or 5.0 mg/d in subjects with tHcy<16 μmol/L,6 and 0.65 or 5 mg/d in hyperhomocyst(e)inemic individuals.7 Our results and the data analyzed by Boushey et al2 suggest that significant reduction of tHcy levels may be achieved by ingesting a supplement of about 400 μg of FA daily. Consequently, it may be considered to exclude from certain clinical trials participants who report current use of multivitamins on a regular basis.
The enzyme MTHFR catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, thus transferring a methyl group to cobalamin, which in turn, donates a methyl group for the conversion of homocysteine to methionine. Kang et al14 15 16 reported the presence of a common homozygous thermolabile form of MTHFR in 5% of white controls and in 17% of CHD patients. The DNA mutation responsible for the heat-labile variant has been identified as a C-to-T mutation at nucleotide 677, which substitutes a valine for alanine at position 114 of the MTHFR protein.12 17 The frequency of the homozygous form (T/T) of this polymorphism was 12% in French Canadians17 and 12% to 15% in populations of European, Middle Eastern, and Japanese origin.18 The frequency of homozygotes for the T677 allele in 60 Dutch patients with arterial occlusive diseases was 15%, compared with 5.2% in 111 control subjects.19 However, such differences between CHD cases and control subjects were not confirmed by Deeb and Motulsky (unpublished observations), by Schwartz et al,20 or by Wilcken et al.21 In our series, tHcy was higher in subjects homozygous for the T677 allele, and T677 homozygotes were more prevalent in CHD patients than in non-CHD subjects (12.1% versus 7.8%, respectively). Whether the disparity with other reported series is due to differences in genetic pools or other undetermined factors needs further study.
Our findings demonstrated a significant negative correlation between tHcy and basal levels of folate, P5′P, and B12; the simultaneous intake of these vitamins in multivitamins may be involved in interactions that could partially account for these associations. Hopkins et al22 measured plasma tHcy, folate, and vitamins B6 and B12 in subjects with early familial CHD and in control subjects. Their data suggested a “possible genetic sensitivity” to the detrimental effects of low folate intake. The relationship between folate status, MTHFR polymorphism, and plasma tHcy has been established in the detailed study of Jacques et al.23 Our data suggest that basal levels of plasma vitamins and tHcy, as well as the effects of folate supplements on tHcy levels, are significantly influenced by multivitamin use. Moreover, subjects homozygous for the T677 MTHFR allele had larger decreases in plasma tHcy levels after FA supplementation, whereas the FA supplements induced smaller tHcy decreases in homozygotes for the C677 allele, especially in subjects with higher baseline folate levels.
It could be broadly surmised that individuals in whom tHcy levels are not lowered by FA supplementation may be more likely to lack the T677 MTHFR allele. In those subjects, additional treatment with other agents, such as pyridoxine, cobalamin, or betaine,7 24 25 may be advisable. Subjects homozygous for the T677 allele were more likely to have elevated levels of tHcy in the presence of low folate status (see Fig 2⇑), as reported earlier,22 and they may have higher folate requirements to regulate tHcy, as proposed by Jacques et al.23 Our data suggest that decreases in tHcy associated with folate supplementation are related to prior intake of multivitamins, baseline tHcy, and folate plasma concentration. These factors may account for about one third of the heterogeneity of the response of tHcy to FA supplementation. Additionally, the response to folate supplementation is affected by the number of T677 alleles in the gene for MTHFR, and thus, subjects with the T/T genotype showed the most robust response to the tHcy-lowering effects of FA. It is likely that other factors not considered in our study may also be involved in that response. Further research is necessary to delineate those interactions to formulate a rational approach to the clinical management of patients at risk for arterial diseases.
Selected Abbreviations and Acronyms
|BMI||=||body mass index|
|CHD||=||coronary heart disease|
|CV||=||coefficient of variation|
|tHcy||=||total plasma homocysteine|
This study was aided by grants from the Nohlgren/Menne Fund (Providence St Vincent Medical Center Foundation), from Merck Inc, and grant P51RR00163-34 from the National Institutes of Health. We are grateful to members of the cardiology section and to internists and family practice physicians of Providence St Vincent Medical Center for allowing us to conduct this study in their patients, to Liqun Wang for performing some of the MTHFR genotyping, and to Andrea Irvin-Jones for data management and editorial assistance.
Kang SS, Wong PWK, Malinow MR. Hyperhomocyst(e)inemia as a risk factor for occlusive vascular disease. Annu Rev Nutr. 1992;12:259-278.
Malinow MR. Hyperhomocyst(e)inemia: a common and easily reversible risk factor for occlusive atherosclerosis. Circulation. 1990;81:2004-2006.
den Heijer M, Brouwer IA, Blom HJ, Gerrits WBJ, Bos GMJ. Lowering of homocysteine blood levels by means of vitamin supplementation. Ir Med J. 1995;164:4. Abstract.
Ubbink JB, Vermaak WJH, van der Merwe A, Becker PJ, Delport R, Potgieter HC. Vitamin requirements for the treatment of hyperhomocysteinemia in humans. J Nutr. 1994;124:1927-1933.
Pietrzik K, Kierkes J, Bung P, Kroesen M. Effect of low dose vitamin supplementation on homocysteine levels. Ir J Med Sci. 1995;164:16. Abstract.
Malinow MR, Kang SS, Taylor LM, Wong PWK, Coull B, Inahara T, Mukerjee D, Sexton G, Upson B. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation. 1989;79:1180-1188.
Malinow MR, Sexton G, Averbuch M, Grossman M, Wilson D, Upson B. Homocyst(e)inemia in daily practice: levels in coronary artery disease. Coron Artery Dis. 1990;1:215-220.
Seltzer D, Anderson PH, Hess DL, Upson BM, Malinow MR. Folic acid lowers plasma homocyst(e)ine without changing plasma creatinine or uric acid, or the urinary excretion of homocyst(e)ine. FASEB J. 1995;9:A320. Abstract.
Kang SS, Passen EI, Ruggie N, Wong PWK, Sora J. Thermolabile defect of methylenetetrahydrofolate reductase in coronary artery disease. Circulation. 1993;88:1463-1469.
Kang SS, Wong PWK, Zhou J, Sora J, Lessick M, Ruggie N, Greevich G. Thermolabile methylenetetrahydrofolate reductase in patients with coronary artery disease. Metabolism. 1988;87:611-613.
Kluijmans LAJ, Lambert PWJ, van den Heuvel WJ, Boers GHJ, Frosst P, Stevens EMB, van Oost BA, den Heifer M, Trijbels FJM, Rozen R, Blom HJ. Molecular genetic analysis in mild hyperhomocysteinemia: a common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease. Am J Hum Genet. 1996;58:35-41.
Schwartz SM, Siscovick DS, Malinow MR, Rosendaal FR, Beverly RK, Psaty BM, Reitsma PH. Hyperhomocyst(e)inemia, a mutation in the methylenetetrahydrofolate reductase gene, and risk of myocardial infarction among young women. Circulation. 1996;93:621. Abstract.
Wilcken DEL, Wang XL, Sim AS, McCredie M. Distribution in healthy and coronary populations of methylenetetrahydrofolate reductase (MTHFR) C677T mutation. Arterioscler Thromb Vasc Biol. 1996;16:878-882.
Hopkins PN, Wu LL, Hunt SC, James BC, Vincent GM, Williams RR. Higher plasma homocyst(e)ine and increased susceptibility to adverse effects of low folate in early familial coronary artery disease. Arterioscler Thromb Vasc Biol. 1995;15:1314-1320.
Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, Selhub J, Rozen R. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation. 1996;93:7-9.
Franken DG, Boers GHJ, Trijbels FJM, Kloppenborg PWC. Treatment of mild hyperhomocysteinemia in vascular disease patients. Arterioscler Thromb. 1994;14:465-470.