Arteriosclerosis, Thrombosis, and Vascular Biology
`atvb.ahajournals.org
`Arteriosclerosis, Thrombosis, and Vascular Biology. 1997; 17: 1157-1162
`doi: 10.1161/ 01.ATV.17.6.1157
`Articles
`The Effects of Folic Acid Supplementation on Plasma Total
`Homocysteine Are Modulated by Multivitamin Use and
`Methylenetetrahydrofolate Reductase Genotypes
`
`M.R. Malinow, F.J. Nieto, W.D. Kruger, P.B. Duell, D.L. Hess, R.A. Gluckman,
`P.C. Block, C.R. Holzgang, P.H. Anderson, D. Seltzer, B. Upson, Q.R. Lin
`
`+
`
`Author Affiliations
`
`Correspondence to M.R. Malinow, MD, Scientist, Oregon Regional Primate Research
`Center, 505 NW 185th Ave, Beaverton, OR 97006. E-mail malinowr@ohsu.edu
`
`Abstract
`
`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.
`
`Key Words:
`homocyst(e)ine
`gene mutations
`vitamin therapy
`arterial occlusive diseases
`
`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
`1 2 3 4
`diseases. 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
`2
`pathogenesis of vascular disease. Thus, elevated levels of plasma tHcy are
`3
`considered to be a common risk factor for arterial occlusive diseases, and this risk
`2
`is graded across the concentration distribution of tHcy. FA supplementation
`5 6 7
`(between 0.5 and 10 mg/d), as well as multivitamins containing, inter alia, 0.2
`8 9
`to 0.4 mg FA, 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,
`
`ACCORD EX 1007
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`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.
`
`Methods
`
`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
`7
`of tHcy and folate plasma levels during a 6-week interval. 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-
`10 11
`pressure liquid chromatography and electrochemical detection as described,
`
`
`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
`12
`reverse primers as previously described.
` 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.
`
`Statistics
`
`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
`2
`case-control status was compared using χ 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).
`
`Results
`
`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.
`
`Table 1.
`
`Characteristics of Subjects
`Stratified by CHD Status and
`Multivitamin Use
`
`View this table:
`In this window In a new window
`
`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.
`
`Table 2.
`
`Effects of 1 or 2 mg FA
`Supplements on tHcy and Folate
`Levels Stratified by Multivitamin
`Use (Unadjusted Data)*
`
`View this table:
`In this window In a new window
`
`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).
`
`Table 3.
`
`View this table:
`
`

`
`Regression Equations and Index
`of Correlations for Log Baseline
`tHcy (Dependent Variable, (μmol/L) (n=242)
`
`In this window In a new window
`
`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⇑).
`
`Figure 1.
`
`Effect of MTHFR genotype on
`efficacy of FA supplements.
`Subjects were stratified by basal
`plasma folate levels above the
`sample geometric mean (A) and
`equal to or below the sample
`geometric mean (B). Arrows
`indicate change in tHcy after FA
`treatment. A, C/C n=35, C/T
`n=65, T/T n=12. B, C/C n=54,
`C/T n=62, T/T n=13. % indicates change relative to baseline
`concentration after FA treatment. p, paired t test: tHcy, basal vs follow-
`up. ▪, t test: T/T vs C/C.
`
`View larger version:
`In this page In a new window
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`
`Table 4.
`
`Changes in tHcy Induced by FA
`Supplements Stratified by
`Multivitamin Use
`
`View this table:
`In this window In a new window
`
`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.
`
`Figure 2.
`
`Log baseline tHcy plotted vs log
`baseline plasma folate levels.
`Regression equations for MTHFR
`genotypes are as follows: C/C,
`y=1.210−.199x (r=.482,
`P<.001; n=128); C/T,
`y=1.222−.212x (r=.440,
`P<.0001; n=89); and T/T,
`y=1.429−.359x (r=−.618,
`P<.001; n=25).
`
`View larger version:
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`
`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⇑.
`
`Table 5.
`
`Stepwise Regression Analysis of
`tHcy Response to Folate
`Supplementation*
`
`Discussion
`
`View this table:
`In this window In a new window
`
`This study demonstrated that multivitamin users had lower baseline levels of tHcy
`8
`than nonusers, in agreement with results previously reported by Brattstrom et al
`9
`and Pietrzik et al. 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
`13
`lacking FA.
`
`
`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
`5
`or 10 mg/d of FA in CHD patients, 0.5 or 5.0 mg/d in subjects with tHcy<16
`6
`7
`μmol/L, and 0.65 or 5 mg/d in hyperhomocyst(e)inemic individuals. Our results
`2
`and the data analyzed by Boushey et al 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.
`14 15 16
`Kang et al
`
`
` 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
`12 17
`of the MTHFR protein.
`
` The frequency of the homozygous form (T/T) of this
`17
`polymorphism was 12% in French Canadians and 12% to 15% in populations of
`18
`European, Middle Eastern, and Japanese origin.
` The frequency of homozygotes for
`the T677 allele in 60 Dutch patients with arterial occlusive diseases was 15%,
`19
`compared with 5.2% in 111 control subjects.
` However, such differences between
`CHD cases and control subjects were not confirmed by Deeb and Motulsky
`20
`21
`(unpublished observations), by Schwartz et al,
` or by Wilcken et al.
` 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
`22
`associations. Hopkins et al
` 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
`23
`tHcy has been established in the detailed study of Jacques et al.
` 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
`7 24 25
`betaine,
`
` 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
`22
`Fig 2⇑), as reported earlier,
` and they may have higher folate requirements to
`23
`regulate tHcy, as proposed by Jacques et al.
` 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
`
`=body mass index
`BMI
`CHD =coronary heart disease
`CV
`=coefficient of variation
`FA
`=folic acid
`MTHFR=5,10-methylenetetrahydrofolate
`reductase
`P5′P =pyridoxal 5′-phosphate
`tHcy =total plasma homocysteine
`
`Acknowledgments
`
`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.
`
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