"I lungs on the
`
`£22578-2583.
`u ent of aortic
`hypertensive
`
`'3] media
`‘
`n uence of or
`
`a triterpene
`
`1 antiminer—
`. J Steroid
`
`, Chronic
`effects of
`us. 1987;
`
`n the vas-
`‘ vlensian .
`
`.= diuretics
`»~ -rtensi ve
`
`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, l-’.C. Block, C.R. Holzgang,
`P.H. Anderson, D. Seltzer, B. Upson, Q.R. Lin
`
`,Elevated concentration of plasma total homocys-
`Abstract
`teine (tHcy) is a common risk factor for arterial occlusive dis-
`eases. Folic acid (FA) supplementation usually lowers tl-Icy lev-
`els, but initial tHcy and vitamin levels, multivitamin use, and
`polymorphisms in the gene for 5,l0-methylenetetrahydrofolate
`reduetase (MTHFR) may contribute to variability in reduction.
`We tested the effects ofa 3-week daily intake of l or 2 mg of FA
`supplements on tHcy levels in patients with and without coro-
`nary heart disease (CHD) who were analyzed for the C677T
`MTHFR mutation. Prior multivitamin intake and baseline vita-
`min 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—multivita-
`
`min users, FA supplements reduced tHcy by 7% in C/C homozy-
`gotes 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 tllcy and increased susceptibility to high levels of
`tl-Icy 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 lev-
`els to PA supplementation, about one third of heterogeneity in
`responsiveness was attributab e to baseline tHcy and folate lev-
`els and to multivitamin use. (Arterioscler Thrumb Vusc Biol.
`1997;17:1157-1162.)
`
`Key Words 0 homocyst(e)ine 0 gene mutations 0 vitamin
`therapy 0 arterial occlusive diseases
`
`umerous investigations have demonstrated that
`the concentration of plasma tHcy is elevated in
`patients with coronary, cerebral, or peripheral
`arterial occlusive diseases.“ Plasma/serum homocyst-
`(e)ine [H(e)], or tHcy, is the sum of the concentration of
`the amino acid homocysteine and the homoeysteinyl
`moieties of the disulfides homocystine and homocys-
`teine-cysteine, whether free or bound to plasma proteins.
`Homocyst(e)inemia refers to plasma levels of tHcy;
`hyperhomocyst(e)inemia indicates elevated concentra-
`tion of plasma/serum tHcy. A comprehensive meta-anal-
`ysis 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 vascu-
`lar disease? Thus, elevated levels of plasma tHcy are
`considered to be a common risk factor for arterial occlu-
`sive diseases} and this risk is graded across the concen-
`tration distribution of tHcy.2 FA supplementation
`(between 0.5 and 10 mg/d),5'7 as well as multivitamins
`containing,
`inter alia, 0.2 to 0.4 mg FA,“-9 reduces
`plasma tHcy concentrations. Further studies are required
`
`ReeeivcdApril 23, 1996; revision accepted October 18, 1996. ..’~
`From thd_Qregon Regional Primate Research Center, Be-avertonl
`(M.R.M., D.L.H., B.U.), Providence St Vincent Hospital (M.R.1’\If’ K}
`R.A.(_l., P.C.B., C.R.H., P.H.A.) and the Oregon Health Sciences
`University, Portland (M.R.M., P.B.D., D.L.H., R.A.G., P.C.B.,
`C.R.H.), Ore; The Johns Hopkins University, Baltimore, Md
`(F.J.N.); Fox Chase Cancer Institute, Philadelphia, Pa (W.D.K.,
`Q.R.L.); and Carle Foundation Hospital, Urbana, Ill (D.S.).
`Correspondence to M.R. Malinow, MD, Scientist, Oregon
`Regional Pfiimate Research Center, 50’5>l{\1W 185th Ave, Beaverton,
`OR 97U()6.n,— ai
`rnalinowr@ohsu.edu :
`ll
`C
`97'A rieein ]-1eartAssoeiation,h’ic.
`
`to elucidate the factors responsible for interindividual
`heterogeneity in responsiveness to folate supplementa-
`tion. We hypothesized that allelic variations in the gene
`for MTHFR, an FA—rclated enzyme, as well as baseline
`tHcy and vitamin levels, may contribute to that hetero-
`geneity. 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 Cl-ID 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 supple-
`mentation in these subjects also were tested.
`
`Methods
`
`Subjects were recruited from internal medicine or family
`practice physicians associated with Providence St Vincent Hos-
`pital (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 seques-
`trants, or nitrous oxide anesthesia). Other exclusion criteria
`were missed laboratory appointments,
`ingesting 20.8 mg FA
`daily, or plasma creatinine levels 2] .7 mg/dL. The study popu-
`lation 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 observa-
`tion. The study was approved by the Institutional Review
`Boards of Providence St Vincent Hospital and the Oregon
`Regional Primate Research Center.
`
`ACCORD EX 1007
`
`

`
`1158
`
`Arteriosclerosis, Thrombosis, and Vascular Biology Vol 17, No 6 June 1997
`
`Selected Abbreviations and Acronyms
`BMI = body mass index
`CHD = coronary heart disease
`CV = coefficient of variation
`FA = folic acid
`MTHFR = 5, I0-methylenetetrahydrofolate reductase
`P5’P = pyridoxal 5'-phosphate
`tHcy = total plasma homocysteine
`
`Case subjects were diagnosed more than 3 months previously
`with a history of acute myocardial infarction, angina pectoris
`documented by a cardiologist, percutaneous transarterial coro-
`nary angioplasty, or coronary bypass graft surgery (n=l40).
`Control subjects had no history of CHD (n=102). Case and con-
`trol 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 demon-
`strated 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. Dur-
`ing 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 chem-
`istry and then frozen for analysis of tHcy by high—pressure liq-
`uid chromatography and electrochemical detection as
`described,”-“ with minor modifications (interassay CV=9.l%)
`(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=l4.4%;
`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 Can-
`cer Institute, Philadelphia, Pa, by Dr Kruger). After thawing,
`
`DNA was isolated by using Instagene Matrix (Bio-Rad Diag- A
`nostics). Isolated DNA was used as the template in a poly-
`merase chain reaction, using 100 ng of the forward and reverse
`primers as previously described.” The amplification reaction
`was performed in a 50-p.L volume in 60 mmol/L Tris-HCl, 15
`mmol/L (NH4)SO4, 200 umol/L dNTP, and 5 units of Taq poly-
`merase. The mixture was subjected to 30 cycles of amplifica-
`tion at 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for
`30 seconds. The polymerase chain reaction products were pre-
`cipitated with ethanol and digested overnight with Hinfl (New
`England Biolabs). The products were analyzed by 3% agarose
`gel electrophoresis.
`
`Statistics
`
`The distribution of study variables was examined using stan-
`dard exploratory data analysis techniques for independent sub-
`jects. Logarithmic transformations were performed to improve
`normality in some of the study variables (BMI and plasma lev-
`els of tHcy, folate, vitamin B12, and P5’P). The distributions of
`study variables by case-control status was compared using X2
`tests for categorical variables and t test for continuous vari-
`ables. Statistical significance of changes in tHcy and folate lev-
`els was assessed using paired t test. Plasma tHcy and folate lev-
`els 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 (meaniSD, 0.384i0.063;
`n=72). Multivitamins usually contained, inter alia, 2 mg
`of vitamin B6 and 6 ug 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;
`
`TABLE 1. Characteristics of Subjects Stratified by CHD Status and Multivitamin" Use
`CHD, %
`Multivitamin Use, %
`Users
`Nonusers
`(n=99)
`(n=143)
`57.6
`67.8
`
`Control Subjects
`(n=1 02)
`47.1
`
`P
`<.O01
`
`P
`.135
`
`4.3
`61.9
`33.8
`37.1
`
`28.6
`59.3
`12.1
`64.4
`27.4
`9.9
`15.7
`370.6
`50.5
`
`6.9
`38.2
`54.9
`46.1
`
`48.0
`44.1
`7.8
`60.8
`26.8
`8.7
`16.6
`332.8
`53.8
`
`.001
`
`.206
`
`.008
`
`.003
`.31
`.0006
`.53
`.48
`.51
`
`7.1
`46.9
`45.9
`
`52.5
`42.4
`47.5
`9.1
`63.1
`26.1
`8.3
`24.8
`384.7
`77.1
`
`4.2
`55.2
`40.6
`
`61.5
`32.9
`56.6
`10.5
`62.8
`28.0
`10.2
`11.9
`292.6
`39.4
`
`.350
`
`.206
`
`.287
`
`.804
`.001
`<.001
`<.001
`<.O01
`<.001
`
`Cases
`(n=140)
`75.7
`
`Male
`Smoking
`Current
`Former
`Never
`Multivitamin use
`CHD (cases) MTHFR genotype
`C/C
`C/T
`T/T
`Age, y’
`BMI, kg/m2‘
`Plasma tHcy, umol/L‘
`Plasma folate, nmol/L‘
`Plasma vitamin B12, pmol/L*
`Plasma P5’P, nmol/L‘
`*Geometrlc means.
`
`
`
`
`
`'b~!'-i!'?<lr%Il!-:l‘eI—-E:-=1!-Iif4-yr
`
`
`
`2.L‘-'P'_h"'-»
`
`

`
`
`
`Malinow et al Folic Acid Supplements and Plasma Total Homocysteine
`
`
`
`1159
`
`
`TABLE 2. Effects of 1 or 2 mg FA Supplements on tHcy and Folate Levels Stratified by
`Multivitamin Use (Unadjusted Data)*
`Multivitamin Users
`
`tHcy, nmol/L
`Baselinef
`Follow-up1'
`Relative delta, %:c
`P§
`Folate, nmol/L
`Baselinej
`Follow-upf
`Relative delta, %1:
`P§
`
`1 mg FA
`(n=54)
`
`2 mg FA
`(n=45)
`
`8.4
`7.9
`-4.5
`.08
`
`25.4
`42.4
`91.4
`.0001
`
`3.2
`7.8
`-3.3
`.15
`
`24.2
`36.4
`72.2
`.0001
`
`P
`
`.57
`.35
`.74
`
`.60
`.10
`.34
`
`Multivitamin Nonusers
`1 mg FA
`2 mgFT
`(n=69)
`(n=74)
`
`10.4
`as
`-13.7
`.0001
`
`10.9
`32.7
`251.3
`.0001
`
`10.0
`3.3
`-10.4
`.0001
`
`12.9
`34.9
`215.0
`.0001
`
`P
`
`.43
`.92
`.30
`
`.09
`.46
`.25
`
`P, Users vs Nonusers
`7 _
`1 mg FA
`2 mg FA
`
`.0003
`.056
`.009
`
`.0001
`.005
`.0001
`
`.0001
`.018
`.022
`
`.0001
`.64
`.0001
`
`
`
`
`
`
`
`
`
`‘Significance of findings did not change appreciably after adjustment for age, log BMI, smoking, sex, CHD status,
`and plasma creatinine concentration.
`TGeometric mean.
`¢Arithmetic mean.
`§Paired ttest between baseline and follow-up values.
`
`
`
`
`
`vitamin 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 sub-
`jects heterozygous (C/T) and about 10% to 21% in sub-
`jects 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 sub-
`jects were stratified according to their baseline folate
`concentration, ie, above or below the sample geometric
`mean (16.1 ttmol/L), are shown in Fig 1. Such results
`resemble those shown in Table 4, since baseline plasma
`folate concentrations were strongly related to multivita-
`min 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 lev-
`els (Fig 2). Levels of tHcy were similar in all subjects at
`high plasma levels of folate but increased at an acceler-
`ated rate (ie, with a steeper slope) at lower folate levels
`in MTHFR T677 homozygotes compared with C/T het-
`erozygotes or C/C subjects.
`A stepwise regression analysis in all subjects indi-
`cated 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 dif-
`ferences may be due in part to the unequal distribution of
`MTHFR genotypes in both groups, as shown in Table 2.
`
`TABLE 3. Regression Equations and Index of
`Correlations for Log Baseline tHcy (Dependent Variable,
`(nmol/L) (n=242)
`
`Independent Variable (Baseline)
`log Folate,
`log P5’P,
`log vitamin B12,
`nmoVL
`nmol/L
`pmol/L
`1.241
`1.092
`1.585
`Intercept
`-2231
`-0701’
`-244:
`Slope‘
`—.475i
`—.166T
`-3131:
`Correlation
`‘Slopes were essentially similar after adjustment for age,
`smoking, sex, case status, and plasma creatinine.
`1P<.01; iP<.O01.
`
`log BMI,
`
`.data suggested that subjects consumed 99.7i10.6% of
`jlfolate supplements.
`Case subjects (58% of all study participants) were
`"more likely to be male, older, and former smokers com-
`pared 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. Com-
`pared 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 con-
`trol 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 multivita-
`mins, FA supplementation reduced plasma tHcy levels by
`' 10% to 14% (P<.O00l). 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 rela-
`tive 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<.Ol; 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 creati-
`nine 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 corre-
`lations for log tHcy versus log plasma vitamin levels in
`all subjects demonstrated significant negative correla-
`tions between log basal tHcy and log plasma vitamin
`levels (Table 3). The correlation coefficients were —.134
`(P<.18),
`.099 (P=.33), and —.l79 (P<.08) in users of
`multivitamins, and —.432 (P<.0O1), —.148 (P=.08), and
`-.2l6 (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 multi-
`
`.ad Diag.
`1 a poly-
`d reverse
`reaction
`:-HCl, 15
`Faq poly-
`mplifica-
`72°C for
`vere pre-
`rlfl (New
`2 agarose
`
`ling stan-
`zlent sub-
`improve
`lsma lev-
`utions of
`
`using X2
`)us vari-
`ilate lev-
`)late lev-
`e dose,
`standard
`ates was
`)I1. Mean
`orrelated
`ypes. All
`analyses
`ute) and
`
`white.
`1 users;
`
`ranged
`i0.063;
`a, 2 mg
`vitamin
`nonths”
`
`sed FA
`nber of
`ssment;
`
`
`
`
`
`
`
`

`
`1160
`
`Arteriosclerosis, Thrombosis, and Vascular Biology Vol 1 7, No 6 June 1997
`
`A.
`
`B.
`
`
`
`
`
`Plasmatotalhomocysteine(timol/L)
`
`
`
`
`
`Plasmatotalhomocysteine(umol/L)
`
`P
`* .002
`**< .001
`I .015 (vs C/C)
`
`crr
`
`T/T
`
`c/c
`
`crr
`
`T/T
`
`MTH FR C677T Genotype
`
`MTH FR C677T Genotype
`
`FIG 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, Cfl' n=65, T/T n=‘l2. B, C/C n=54, C/T n=62, T/T n=13. % indicates change relative to baseline concentration after FA
`treatment. p, paired ttest: tHcy, basal vs follow-up. I, ttest: T/T vs C/C.
`
`Discussion
`
`This study demonstrated that multivitamin users had
`lower baseline levels of tHcy than nonusers, in agreement
`with results previously reported by Brattstrom et all‘ and
`Pietrzik et a1.9 Moreover, multivitamin users had higher
`baseline concentration of plasma folate, P5'P, and Vitamin
`B12. FA supplements lowered tHcy in nonusers of multi-
`vitamins, 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 lev-
`els. However, the tHcy levels attained after folate supple-
`mentation in users of multivitamins were somewhat lower
`
`than those reached in nonusers of multivitamins, perhaps
`due to interactions with other vitamins, the longer dura-
`tion 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.”
`
`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 creati-
`nine 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 reduc-
`tions 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 umol/L,“ 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 ug of FA daily.
`Consequently, it may be considered to exclude from cer-
`tain 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—methyltetrahydro-
`folate, thus transferring a methyl group to cobalamin,
`which in turn, donates a methyl group for the conversion
`
`c/c
`(n=42)
`
`P
`
`P‘
`
`c/c
`(n=47)
`
`P
`
`P’
`
`.16
`.53
`.031
`
`.16
`.75
`.037
`
`TABLE 4. Changes in tHcy Induced by FA Supplements Stratified by Multivitamin Use
`Multivitamin Users
`Multivitamin Nonusers
`C/T
`T/T
`T/'I'
`CIT
`(n=81)
`(n=15)
`(n=10)
`(n=47)
`
`MTHFR Genotype
`tHcy, umol/L
`Baselinet
`Follow-upt
`Relative delta, %1;
`P§
`‘Adjusted for age, log BMI, smoking, sex, CHD status, and plasma creatinine concentration.
`1'Geometric mean.
`1Arithmetic mean.
`§Paired ftest between baseline and follow-up values.
`
`8.3
`7.9
`-3.2
`.26
`
`8.4
`8.0
`-3.3
`.19
`
`8.1
`7.2
`-10.2
`.004
`
`.91
`.53
`.48
`
`.65
`.15
`.33
`
`9.9
`9.0
`-7.1
`.0.22
`
`10.0
`8.5
`-13.1
`.0001
`
`11.8
`9.1
`-20.9
`.0002
`
`

`
`
`
`(pmol/L)
`LogBaselinetHcy
`
`
`Malinow er al
`
`Jove the
`aatment.
`after FA
`
`or 2 mg
`of sex,
`1 creati-
`
`2 mg/d
`' effects
`1g some
`' reduc-
`,5 0.5 or
`0.65 or
`s.7 Our
`suggest
`chieved
`
`A daily.
`‘om cer—
`t use of
`
`of 5,10-
`lhydro-
`ualamin,
`lversion
`
`'o.o
`
`1.5
`1.2
`0.8
`0.4
`Log Baseline Plasma Folate (nmol/L)
`
`.FIG 2. Log baseline tHcy plotted vs log baseline plasma folate
`levels. Regression equations for MTHFR genotypes are as fol-
`lows: C/C, y=1.210—.199x (r=.482, P<.001; n=128); C/T,
`~ y=1.222—.2‘l2X (r=.440, P<.0O01; n=89); and T/T, y=1.429—
`...359x (r=-.618, P<.0O1; n=25).
`
`of homocysteine to methionine. 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 muta-
`tion at nucleotide 677, which substitutes a valine for ala-
`‘ttine at position 114 of the MTHFR protein.”~17 The fre-
`quency of the homozygous form (T/T) of this
`‘polymorphism was 12% in French Canadians” and 12%
`to 15% in populations of European, Middle Eastern, and
`Japanese origin.” 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.“’ However, such differences between CHD
`cases and control subjects were not confirmed by Deeb
`and Motulsky (unpublished observations), by Schwartz
`.et al,2° 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%, respec-
`tively). Whether the disparity with other reported series
`is due to differences in genetic pools or other undeter-
`rmined factors needs further study.
`Our findings demonstrated a significant negative corre-
`lation between tHcy and basal levels of folate, P5’P, and
`B12; the simultaneous intake of these vitamins in multivi-
`ftamins may be involved in interactions that could partially
`account for these 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 detrimen-
`ital 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 211.23
`Eur 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
`Bllpplementation, whereas the FA supplements induced
`
`All subjectsf
`Log basal tHcy
`Log basal folate
`Current nonusers of
`multivitaminst
`Log basal tHcy
`Log basal folate
`Current users of
`multivitamins:
`.81
`.0048
`1.42
`.079
`.282
`Log basal tHcy
`*Results were essentially similar when the MTHFR genotype data were
`stratified for C/C vs C/T or T/T, and C/C or CH’ vs T/T.
`Tvariables not in model: log P5’P, log vitamin B12, and C677T poly-
`morphism.
`:l:Variab|es not in model: log P5’P,
`phism, and log folate.
`
`log vitamin B12, C677T polymor-
`
`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,7-2435 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,” and they may
`have higher folate requirements to regulate tHcy, as pro-
`posed 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.
`
`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 Insti-
`tutes of Health. We are grateful to members of the cardiology
`section and to internists and family practice physicians of Provi-
`dence 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.
`
`References
`1. Kang SS, Wong PWK, Malinow MR. Hyperhomocyst(e)inemia as 21
`risk factor for occlusive vascular disease. Annu Rev Nutr. 1992;12:
`259-278.
`2. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantita-
`tive assessment of plasma homocysteine as a risk factor for vascular
`disease: probable benefits of increasing folic acid intake. JAMA.
`1995;274:1049-1057.
`3. Malinow MR. Hyperhomocyst(e)inemia: a common and easily
`reversible risk factor for occlusive atherosclerosis. Circulation.
`1990;8l :2004-2006.
`
`
`
`Folic Acid Supplements and Plasma Total Homocysteine
`
`1161
`
`TABLE 5. Stepwise Regression Analysis of tHcy
`Response to Folate Supplementation*
`Fl
`R‘
`SE
`
`P
`
`[3
`
`.549
`.588
`
`.589
`.627
`
`.302
`.345
`
`.347
`.394
`
`1.90
`1.84
`
`<.0OO1
`<.O001
`
`2.11
`2.04
`
`<.0O01
`.0014
`
`1.0
`
`1.0
`
`

`
`1162
`
`Arteriosclerosis, Thrombosis, and Vascular Biology Vol I 7, No 6 June 1997
`
`4.
`
`5.
`
`Mayer EL, Jacobsen DW, Robinson K. Homocysteine and coronary
`atherosclerosis. JAm Coll Cardiol. 1996;27:517-527.
`Landgren F, Israelsson B, Lindgren A, Hultberg B, Andersson A,
`Brattstrom L. Plasma homocysteine in acute myocardial infarction:
`homocysteine-lowering effect of folic acid. J Intern Med.
`1995;237:381-388.
`. den Heijer M, Brouwer IA, Blom HI, Gerrits WBJ, Bos GMJ. Lower-
`ing of homocysteine blood levels by means of vitamin supplementa-
`tion. Ir Med J. l995;164:4. Abstract.
`. Ubbink JB, Vermaak WJH, van der Merwe A, Becker PJ, Delport R,
`Potgieter HC. Vitamin requirements for the treatment of hyperhomo-
`cysteinemia in humans. J Nutr. 1994;124:1927-1933.
`. Brattstrom L, Lindgren A, lsraelsson B, Andersson A, Hultberg B.
`Homocysteine and cysteine: determinants of plasma levels in middle-
`aged and elderly subjects. J Intern Med. 1994;236:633—64l.
`. Pietrzik K, Kierkes J, Bung P, Kroesen M. Effect of low dose vitamin
`supplementation on homocysteine levels. Ir J Med Sci. l995;164:16.
`Abstract.
`
`. Malinow MR, Kang SS, Taylor LM, Wong PWK, Coull B, Inahara
`T, Mukerjee D, Sexton G, Upson B. Prevalence of hyperhomo-
`cyst(e)inemia in patients with peripheral arterial occlusive disease.
`Circulation. 1989;79: 1 180-1188.
`. Malinow MR, Sexton G, Averbuch M, Grossman M, Wilson D,
`Upson B. Homocyst(e)inemia in daily practice: levels in coronary
`artery disease. Caron Artery Dis. l990;l:2l5-220.
`. Goyette P, Sumner JS, Milos R, Doncan AMV, Rosenblatt DS,
`Matthews RG, Rozen R. Human methylenetetrahydrofolate reduc-
`tase:
`isolation of DNA, mapping and mutation identification. Nat
`Genet. 1994;7:195-200.
`. Seltzer D, Anderson PH, Hess DL, Upson BM, Malinow MR. Folic
`acid lowers plasma homocyst(e)ine without changing plasma creati-
`nine or uric acid, or the urinary excretion of homocyst(e)ine. FASEB
`J. l995;9:A320. Abstract.
`. Kang SS, Passen El, Ruggie N, Wong PWK, Sora J. Thermolabile
`defect of methylenetetrahydrofolate reductase in coronary artery dis-
`ease. Circulation. l993;88:l463—1469.
`. Kang SS, Wong PWK, Susmano A, Sora J, Norusis M, Ruggie N.
`Therrnolabile methylenetetrahydrofolate reductase: an inherited risk
`factor for coronary artery disease. Am J Hum Genet. 1991;48:536-545.
`. Kang SS, Wong PWK, Zhou J, Sora J, Lessick M, Ruggie N,
`Greevich G. Thermolabile methylenetetrahydrofolate reductase
`
`in patients with coronary artery disease. Metabolism.
`611-613.
`
`l988;87.
`5'
`
`. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG;
`Boers GHJ, den Heijer M, Kluijtmans LAJ, van den Heuvel LP
`Rozen R. A candidate genetic risk factor for vascular disease: a com 7
`mon mutation in methylenetetrahydrofolate reductase. Nat Genet;
`1995;10:111-113.
`. Motulsky AG. Nutritional ecogenetics: homocysteine-related a1-re.
`riosclerotic vascular disease, neural tube defects, and folic acid. Am 1
`Hum Genet. l996;58:l7-20.
`
`. 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 hyperhomo.
`cysteinemia: a common mutation in the methylenetetrahydrofolate,
`reductase gene is a genetic risk factor for cardiovascular disease. Am,
`JHum Genet. l996;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 myocar.
`dial infarction among young women. Circulation. 1996;93:621_,
`Abstract.
`
`. Wilcken DEL, Wang XL, Sim AS, McCredie M. Distribution in.‘
`healthy and coronary populations of methylenetetrahydrofolatei
`reductase (MTHFR) C677T mutation. Arterioscler Thromb Vase
`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.
`Arteriascler Thramb Vasc Biol. 1995;l5: 1314-1320.
`. Jacques PF, Bostom AG, Williams R, 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. l996;93:7-9.
`. Franken DG, Boers GHJ, Trijbels FJM, Kloppenborg PWC. Treat-
`ment of mild hyperhomocysteinemia in vascular disease patients:
`Arteriascler Thramb. 1994;14:465-470.
`. Wilcken DEL, Wilcken B, Dudman NPB, Tyrrell PA. Homocystin-
`uria: the effects of betaine in the treatment of patients not responsive
`to pyridoxine. NEngl JMed. 1983;309:448-453.