(Circulation. 1999;99:2383-2388.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Hyperhomocysteinemia but Not the C677T Mutation of Methylenetetrahydrofolate Reductase Is an Independent Risk Determinant of Carotid Wall Thickening
The Perth Carotid Ultrasound Disease Assessment Study (CUDAS)
Presented in part at the 70th Scientific Sessions of the American Heart Association, Orlando, Fla, November 9–12, 1997, and published in abstract form (Circulation. 1997;96[suppl I]:I-655).
From the Sir Charles Gairdner Hospital Campus of the Heart Research Institute of Western Australia (B.McQ., M.N., P.L.T., J.H.); the Department of Clinical Biochemistry, PathCentre, QEII Medical Centre (J.P.B.); and the Department of Medicine, University of Western Australia (J.H.), Nedlands.
Correspondence to A/Prof Joseph Hung, University Department of Medicine, QEII Medical Centre, Nedlands, Western Australia, Australia 6009. E-mail jhung{at}cyllene.uwa.edu.au
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Abstract |
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Background—Hyperhomocysteinemia has been identified as a potential risk factor for atherosclerosis. This study examined whether a modest elevation of plasma total homocysteine (tHcy) was an independent risk factor for increased carotid artery intimal-medial wall thickness (IMT) and focal plaque formation in a large, randomly selected community population. We also examined whether vitamin cofactors and the C677T genetic mutation of the methylenetetrahydrofolate reductase (MTHFR) enzyme were major contributors to elevated plasma tHcy and carotid vascular disease.
Methods and Results—In 1111 subjects (558 men, 553 women) 52±13 years old (mean±SD; range, 27 to 77 years) recruited from a random electoral roll survey, we measured fasting tHcy and performed bilateral carotid B-mode ultrasound. For the total population, mean tHcy was 12.1±4.0 µmol/L. Plasma tHcy levels were correlated with IMT (Spearman rank rs=0.31, P=0.0001). After adjustment for age, sex, and other conventional risk factors, subjects in the highest versus the lowest quartile of tHcy had an odds ratio of 2.60 (95% CI, 1.51 to 4.45) for increased IMT and 1.76 (95% CI, 1.10 to 2.82) for plaque. Serum and dietary folate levels and the C677T mutation in MTHFR were independent determinants of tHcy (all P=0.0001). The mutant homozygotes (10% of the population) had higher mean tHcy than heterozygotes or those without the mutation (14.2 versus 12.3 versus 11.6 µmol/L, respectively, P=0.0001). The inverse association of folate levels with tHcy was steeper in the mutant homozygotes. Despite this, the C677T MTHFR mutation was not independently predictive of increased carotid IMT or plaque formation.
Conclusions—Mild hyperhomocysteinemia is an independent risk factor for increased carotid artery wall thickness and plaque formation in a general population. Lower levels of dietary folate intake and the C677T mutation in MTHFR are important causes of mild hyperhomocysteinemia and may therefore contribute to vascular disease in the community.
Key Words: homocysteine • genes • folate • atherosclerosis • ultrasonics
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Introduction |
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Anumber of studies, recently reviewed by Boushey et al,1 have linked elevated plasma total homocysteine (tHcy) levels to atherosclerotic vascular disease affecting coronary, carotid, and peripheral arteries. Several mechanisms for homocysteine-associated vascular disease have been proposed, including potentiation of atherosclerosis through endothelial dysfunction, smooth muscle cell hyperplasia, and increased formation of oxidized lipids.2 3 Previous carotid B-mode ultrasound studies have suggested that hyperhomocysteinemia is a risk factor for carotid atherosclerosis, but these data are limited to a single case-control study4 and cross-sectional studies in older populations.5 6
Plasma tHcy levels are affected by dietary intake of vitamin cofactors in homocysteine metabolism, especially folate, vitamins B6 and B12,7 8 genetic mutations of key metabolic enzymes,2 3 creatine-creatinine synthesis, and renal elimination.8 A genetic mutation (nucleotide C677T, alanine [a] to valine [v] substitution) in the enzyme methylenetetrahydrofolate reductase (MTHFR), which renders the enzyme thermolabile and functionally impaired, has recently been described.9 10 Because this enzyme is involved in the folate-dependent remethylation of homocysteine to methionine, a reduced efficiency may be an important determinant of elevated tHcy in the general population.
To further evaluate the contribution of elevated tHcy to atherosclerotic vascular disease, we examined the independent association between fasting tHcy and carotid artery intima-media thickening and focal plaque formation in a large, randomly selected community population, with a broad age range (27 to 77 years) and an equal male-to-female ratio. We also examined whether the C677T MTHFR mutation and vitamin cofactors were important determinants of plasma tHcy and carotid vascular disease in this population.
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Methods |
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Subjects
Subjects were original participants in the 1989 Australian National Heart Foundation (NHF) Perth Risk Factor Prevalence Survey.11 This was a random electoral roll survey of 2000 people from the Perth, Western Australia, metropolitan area, with equal numbers of men and women and equal numbers of subjects in each decile of age between 20 and 70 years. Repeat electoral roll and death record matching in May 1995 established a current address for 1807 living subjects. All of these were invited to attend our study clinic between June 1995 and December 1996, and 1111 subjects (61% of those eligible) agreed to participate. Subjects who had previous carotid artery surgery were excluded. The age-adjusted prevalence of risk factors in the present study population was similar to that reported for the entire 1989 cohort.11 Written informed consent was obtained from all study participants. The study protocol was approved by the Institutional Ethics Committee of the University of Western Australia.
Laboratory Measurements
In all subjects, a fasting venous blood sample was obtained.
Special care was taken with the samples for tHcy analysis, with
the plasma separated by centrifugation shortly after
venipuncture and transport in ice-cooled containers.
Fasting tHcy was determined by reverse-phase high-performance
liquid chromatography after treatment with
tributylphosphine, deproteinization, and fluorogenic derivatization by
the method of Araki and Sako.12 The interassay coefficient
of variation was 6% in our laboratory.13 Total
cholesterol, HDL cholesterol, and
triglyceride levels were determined enzymatically with a
Hitachi 747 autoanalyzer. LDL cholesterol was
calculated with Friedewald's method.14 Plasma
creatinine levels were also measured. DNA was extracted by
the salt phenol chloroform method from the cells of the buffy coat.
MTHFR genotype was determined by
HinfI digestion of the PCR products.10
Serum folate was determined by immunoassay with ACS:180 (Chiron
Diagnostics).
Risk Factor and Dietary Assessment
A self-administered questionnaire similar to that used by the
1989 Australian NHF Risk Factor Prevalence Survey was used to
record a history of hypertension, hyperlipidemia,
diabetes, angina pectoris, myocardial infarction, stroke, or a family
history of premature-onset coronary or cerebrovascular disease
(by age 55 years) in first-degree relatives.11 Smoking
lifetime exposure by pack-years was calculated. Anthropomorphic
measurements and the lower of 2 resting sitting blood pressures,
measured with a mercury column manometer, were recorded by a
trained research nurse. All subjects completed a self-administered
semiquantitative food-frequency questionnaire prepared by the CSIRO
Division of Human Nutrition, Australia,15 with responses
reviewed by a research nurse on the day of their visit. The average
daily intake of folate and vitamins B6 and
B12, adjusted for supplemental vitamin use, was
calculated by the CSIRO Dietary Assessment by Computer
Program.15
Carotid Ultrasound Examination
Bilateral carotid B-mode ultrasound was performed by 2 trained
sonographers using a 7.5-MHz annular phased-array transducer on an
Interspec (Apogee) CX 200 ultrasound machine. Scans were performed
according to a standardized protocol similar to that used by Salonen et
al.16 The characteristic echo interfaces on the far wall
of the distal common carotid artery were optimized and recorded on
super VHS videotape, along with an ECG lead, for subsequent offline
analysis. A thorough search of the distal common carotid
artery, carotid bulb, and internal and external carotid arteries was
also made to determine the presence of focal plaque and/or calcific
deposits. Plaque was defined as a clearly identified area of focal
increased thickness (
1 mm) of the intima-media layer.
The intima-media thickness (IMT) was defined as the distance between
the characteristic echoes from the lumen-intima and media-adventitia
interfaces,17 as shown in Figure 1
. End-diastolic images were
digitized, and a semiautomated edge-detection software program was used
to identify leading-edge echo-interface points from the far wall of the
distal 1 cm of the common carotid artery.18 Three
end-diastolic images were analyzed from both the
right and left distal common carotid arteries at a site free of any
discrete plaque, and measurements were averaged to give the mean IMT.
Repeat measurement of randomly selected scans revealed no significant
variation in the IMT measurement obtained during any specific time
period of the study. Quality control measures included repeat scans on
a subset of 30 subjects on 2 separate occasions 7 to 10 days apart. The
intraobserver coefficient of variability was 2.9% for sonographer 1
and 4.8% for sonographer 2. The interobserver coefficient of
variability was 5.9%.
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Statistical Analysis
Mean IMT was treated as a continuous as well as a categorical
variable, with those above the 80th percentile of IMT for the total
cohort (>0.8 mm) classified as increased IMT. Spearman rank
correlations were used to describe the association of continuous risk
factors, including tHcy, with mean IMT. Determinants of tHcy were
assessed by stepwise linear regression. Logarithmic transformation of
some biochemical variables, including tHcy, was performed to
normalize the distribution. Stepwise logistic regression was used to
test the independent relation between sex-specific tHcy quartiles
(independent variable) and increased IMT or focal plaque (dependent
variables). The tHcy quartiles were entered into the regression
analysis, and odds ratios were estimated after adjustment for
other confounding risk variables. Analysis was performed
with SAS statistical software.19 ANOVA was used to compare
mean values between groups, and if overall significance was
demonstrated, intergroup differences were assessed by multiple range
testing. Statistical significance was taken as a 2-sided
P<0.05. Results are expressed as mean±SD or proportions
with 95% CIs unless otherwise stated.
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Results |
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Subjects
The clinical characteristics of the study population divided into men (n=558) and women (n=553) are shown in Table 1
. A similar prevalence of
conventional risk factors was observed between men and women, except
that men had higher blood pressure and waist-to-hip ratio, lower HDL
cholesterol, and higher triglyceride levels
than women (Table 1). A previous history of myocardial
infarction or stroke was recorded in 7% of the total
population.
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tHcy and Carotid Atherosclerosis
Figure 2 shows the frequency
distribution of plasma tHcy by sex. The mean tHcy concentrations were
12.9±3.1 µmol/L in men (range, 7.6 to 40.5) versus
11.2±3.8 µmol/L in women (range, 6.0 to 35.9)
(P=0.001). In men, the cutoff thresholds for tHcy quartiles
were 
10.5, 10.6 to 12.4, 12.5 to 14.4, and >14.4 µmol/L, and
in women, they were 8.4, 8.5 to 10.5, 10.6 to 12.8, and >12.8
µmol/L.
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In the overall population, the average carotid artery mean IMT was
0.71±0.14 mm, and focal plaque was identified in 26% of
subjects. Although men had a higher mean IMT than women (0.73±0.14
versus 0.69±0.13 mm, P=0.001), women still composed
41% of subjects with increased IMT and 43% with focal plaque. Plasma
tHcy correlated to IMT (Spearman rank
rs=0.31, P=0.0001), with a
strength of association similar to that of most other conventional risk
factors (Table 2).
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Figure 3 shows that the prevalence of
thickened IMT and focal plaque increased progressively across
sex-specific tHcy quartiles (P=0.001, test for trend in both
men and women). Before tHcy was entered into the model, logistic
regression had selected (in order) age, systolic blood
pressure, smoking pack-years, LDL cholesterol, waist-to-hip
ratio, and hypertension history as independent determinants of
increased IMT (all P<0.05). Sex and plasma
creatinine were no longer significant predictors once other
risk variables were included, although we continued to include sex
in the logistic model. For plaque, similar risk variables were
selected, with the addition of a history of vascular disease and
diabetes mellitus (all P<0.05). After adjustment for all
these risk variables, tHcy remained a significant predictor of
increased IMT (P= 0.0005) and plaque (P= 0.018).
Table 3 shows the adjusted odds ratios
for increased IMT and plaque between sex-specific tHcy quartiles. When
the highest and lowest quartiles of tHcy were compared, an increased
odds ratio of 2.60 (95% CI, 1.51 to 4.45) for thickened IMT and 1.76
(95% CI 1.10 to 2.82) for plaque remained.
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) and women (
) across sex-specific plasma tHcy
quartiles. Bars indicate 95% CIs. Cutoff thresholds for tHcy quartiles
(1 to 4) were
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tHcy, Vitamin Cofactors, and MTHFR
Genotype
There was a strong graded inverse association between tHcy and
quartiles of serum folate and dietary folate (ANOVA
P=0.0001), as shown in Table 4. A similar inverse association
was found between tHcy and dietary intake of vitamin
B6 (ANOVA P=0.0001) and vitamin
B12 (ANOVA P=0.026), although the
difference was primarily between the lowest and highest quartiles of
vitamin intake.
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The C677T mutation of MTHFR was present in heterozygous (av) form in 47% (n=519) and homozygous (vv) form in 10% (n=111) of subjects. Genotype frequencies conformed to the Hardy-Weinberg equilibrium. There was no difference in the prevalence of the C677T mutation in men compared with women or between quartiles of vitamin intake. Mutant homozygotes had a higher mean tHcy level than heterozygotes or those without the mutation (14.2 versus 12.3 versus 11.6 µmol/L, respectively, P=0.0001). The mean serum folate level was also lower in vv than in aa homozygotes (6.3 versus 7.8 µg/L, respectively, P=0.001).
Figure 4 shows that for each
genotype (aa, av, vv) there
was an inverse relationship between tHcy level and folate quartile (all
P<0.025 for trend), but this was steeper for the
vv homozygotes. The mean difference in tHcy level from the
highest to the lowest quartile of serum folate in vv
homozygotes was 5.1 µmol/L (95% CI, 1.6 to 8.6 µmol/L)
compared with 2.1 µmol/L (95% CI, 1.2 to 3.0 µmol/L) in
aa subjects. A similar interaction between
MTHFR genotype and dietary folate intake was
observed. However, subjects with serum folate levels in the highest
quartile displayed no significant differences in tHcy levels between
MTHFR genotypes.
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Independent determinants of tHcy concentration selected by stepwise
linear regression were (in order) serum folate, age, plasma
creatinine, dietary folate intake, MTHFR
genotype, and male sex, which together explained 
39% of the
total variance in tHcy levels
(R2=0.39, P=0.0001). Serum
folate level alone accounted for 12% and MTHFR
genotype 3% of the total variance in tHcy levels. Dietary
vitamin B6 and B12 intakes
were no longer independent determinants of tHcy after folate was
included in the model. Despite their association with tHcy
concentration, neither the serum or dietary folate nor the
MTHFR genotype was selected by logistic
regression as an independent predictor of increased carotid IMT or
plaque, even when analysis was confined to those in the lowest
quartile of dietary or serum folate.
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Discussion |
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In this study, plasma tHcy concentration was found to be a graded and independent risk factor for carotid wall thickening and focal plaque formation in a large, randomly selected community population. Folate status and the MTHFR genotype were significant determinants of tHcy levels in the overall population but were not independent predictors of carotid wall thickening or plaque.
tHcy and Carotid IMT
There is good biological evidence to suggest that elevations of
tHcy may promote the development of
atherosclerosis.2 3 We used B-mode carotid
ultrasound as a well-validated technique for the detection of
subclinical atherosclerosis.20 21 22
In particular, carotid wall thickening measured by B-mode
ultrasound has been shown to correlate strongly with standard risk
factors and focal plaque formation.20 21 22 Previously, the
Atherosclerosis Risk in Communities
study,4 from a case-control sampling of study participants
46 to 64 years old, reported an increased odds ratio for thickened IMT
for subjects in the top compared with the bottom quintile of tHcy.
However, this association was no longer significant after adjustment
for conventional risk factors. Selhub et al,5 in a
cross-sectional study of 1041 elderly subjects 67 to 96 years old,
demonstrated a 2-fold increase in the risk of carotid stenosis
(25%) for subjects in the highest versus lowest quartile of tHcy
after adjustment for conventional risk factors. Most recently, the
Rotterdam Study,6 in a cross-sectional study of 630
subjects 55 years old, demonstrated an increased prevalence of
thickened IMT, but not plaque, in subjects whose tHcy was in the upper
15th percentile.
We have extended these observations by studying a large, randomly selected general population, equally distributed between men and women and across a broad age range. We found that tHcy levels correlated with mean IMT with a strength of association similar to that of most traditional risk factors. More importantly, we found that there was an independent association of tHcy with increased IMT and plaque, even after adjustment for age, sex, and other conventional risk factors. The cutoff values for the upper tHcy quartiles in our population are in line with those indicated to be associated with an increased risk of vascular disease.1 5 Although we studied a younger population, the odds ratios for increased IMT (2.60) and focal plaque (1.76) for subjects in the highest versus lowest tHcy quartile were similar to those found for carotid stenosis in the elderly Framingham Heart Study cohort.5
tHcy, Vitamin Cofactors, and MTHFR Mutation
We found a strong graded inverse association between tHcy
concentration and the level of serum folate and dietary folate intake,
similar to that noted by others.7 From the lowest to
highest quartile of serum and dietary folate level, there was an
average of 2 to 4 µmol/L change in mean tHcy concentration, in
accordance with the difference suggested by Boushey et
al.1 There was also an inverse but weaker association of
dietary vitamin B6 and B12
intakes with plasma tHcy level. The 10% prevalence of homozygotes for
the MTHFR C677T mutation was in accord with the 10% to
14% reported in other populations.10 23 24 Mutant
vv homozygotes had significantly higher tHcy levels than
av heterozygotes or aa homozygotes. Because
MTHFR is involved in the folate-dependent remethylation
of homocysteine, a low serum or dietary folate level was found to
exacerbate hyperhomocysteinemia associated with this mutation.
Conversely, increased folate intake could counter the effect of this
mutation, because there was no difference in tHcy levels between
genotypes in those who had a high serum folate concentration. A
degree of folate "wastage" was also suggested by a lower mean serum
folate level in the vv subjects relative to the other
genotypes despite equivalent dietary folate intakes, a finding
also reported by Harmon et al.23 Thus, individuals
with the homozygous mutant MTHFR allele may require
a higher than usual folate intake to correct the associated
hyperhomocysteinemia.
Some case-control studies have reported that homozygosity for the C677T
mutation was associated with premature coronary artery
disease,25 26 whereas other studies have not found an
association.24 27 28 Although the MTHFR
genotype and serum folate were significant determinants of
tHcy, together accounting for 15% of the total variance in tHcy
levels, they were still not independent predictors of increased carotid
IMT or plaque in our study population. This is not surprising, given
that the link between vascular disease and the C677T mutation or folate
is likely to be mediated in large part through their effects on
homocysteine metabolism.5 This is also the
case for creatinine, which, despite being a significant
determinant of tHcy level, was not an independent predictor of
increased IMT. Furthermore, the MTHFR mutation will most
likely have greater impact on vascular disease in populations that are
more folate-deficient.
In conclusion, our study adds further weight to the evidence that tHcy is an independent graded risk factor for atherosclerosis in a general population. Thus, measurement of plasma tHcy may contribute to vascular risk assessment in individuals as well as in population studies. Lower dietary intake of folate and the C677T MTHFR mutation are important causes of mild hyperhomocysteinemia in the community. Although the impact of proposed folate supplementation programs remains to be studied, it is likely that adoption of measures to increase dietary folate intake sufficiently to lower plasma tHcy levels in the general community will have favorable public health effects. The C677T MTHFR mutant genotype may need to be taken into account in decisions on optimal doses of folic acid required to lower the tHcy concentration.
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Acknowledgments |
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This study was supported by grants-in-aid from the National Heart Foundation of Australia (G 94P 4232) and Healthway, the Western Australian Health Promotion Foundation. We appreciate the technical assistance provided by Elsie Yu, Katherine Loh, and Marcus Sommerville, Heart Research Institute, Sir Charles Gairdner Hospital and Clive Hunt, Christine Chin, and Jody Burley, PathCentre, Queen Elizabeth II Medical Center.
Received November 18, 1998; revision received February 3, 1999; accepted February 16, 1999.
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 S. R. Thompson, P. A. McCaskie, J. P. Beilby, J. Hung, M. Jennens, C. Chapman, P. Thompson, and S. E. Humphries IL18 Haplotypes Are Associated with Serum IL-18 Concentrations in a Population-Based Study and a Cohort of Individuals with Premature Coronary Heart Disease Clin. Chem., December 1, 2007; 53(12): 2078 - 2085. [Abstract] [Full Text] [PDF]
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 Wei Hsi Chen, Hung Sheng Lin, Yi Fen Kao, Min Yu Lan, and Jia Shou Liu Hyperhomocysteinemia Relates to the Subtype of Antiphospholipid Antibodies in Non-SLE Patients Clinical and Applied Thrombosis/Hemostasis, October 1, 2007; 13(4): 398 - 403. [Abstract] [PDF]
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 A. Faggiano, D. Melis, R. Alfieri, M. De Martino, M. Filippella, F. Milone, G. Lombardi, A. Colao, and R. Pivonello Sulfur Amino Acids in Cushing's Disease: Insight in Homocysteine and Taurine Levels in Patients with Active and Cured Disease J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6616 - 6622. [Abstract] [Full Text] [PDF]
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 J. Hung, B. M. McQuillan, C. M. L. Chapman, P. L. Thompson, and J. P. Beilby Elevated Interleukin-18 Levels Are Associated With the Metabolic Syndrome Independent of Obesity and Insulin Resistance Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1268 - 1273. [Abstract] [Full Text] [PDF]
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 S. Hickling, J. Hung, M. Knuiman, K. Jamrozik, B. McQuillan, J. Beilby, and P. Thompson Impact of voluntary folate fortification on plasma homocysteine and serum folate in Australia from 1995 to 2001: a population based cohort study J. Epidemiol. Community Health, May 1, 2005; 59(5): 371 - 376. [Abstract] [Full Text] [PDF]
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 F. Aucella, M. Margaglione, E. Grandone, M. Vigilante, G. Gatta, M. Forcella, M. Ktena, A. De Min, G. Salatino, D. A. Procaccini, et al. The C677T methylenetetrahydrofolate reductase gene mutation does not influence cardiovascular risk in the dialysis population: results of a multicentre prospective study Nephrol. Dial. Transplant., February 1, 2005; 20(2): 382 - 386. [Abstract] [Full Text] [PDF]
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T. A. Manolio, E. Boerwinkle, C. J. O'Donnell, and A. F. Wilson Genetics of Ultrasonographic Carotid Atherosclerosis Arterioscler. Thromb. Vasc. Biol., September 1, 2004; 24(9): 1567 - 1577. [Abstract] [Full Text] [PDF]
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 C. M. L. Chapman, J. P. Beilby, B. M. McQuillan, P. L. Thompson, and J. Hung Monocyte Count, But Not C-Reactive Protein or Interleukin-6, Is an Independent Risk Marker for Subclinical Carotid Atherosclerosis Stroke, July 1, 2004; 35(7): 1619 - 1624. [Abstract] [Full Text] [PDF]
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 A. Hassan, B. J. Hunt, M. O'Sullivan, R. Bell, R. D'Souza, S. Jeffery, J. M. Bamford, and H. S. Markus Homocysteine is a risk factor for cerebral small vessel disease, acting via endothelial dysfunction Brain, January 1, 2004; 127(1): 212 - 219. [Abstract] [Full Text] [PDF]
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 Y. Aso, K.-i. Okumura, K. Takebayashi, S. Wakabayashi, and T. Inukai Relationships of Plasma Interleukin-18 Concentrations to Hyperhomocysteinemia and Carotid Intimal-Media Wall Thickness in Patients With Type 2 Diabetes Diabetes Care, September 1, 2003; 26(9): 2622 - 2627. [Abstract] [Full Text] [PDF]
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 D. S Wald, M. Law, J. Morris, N. J Wald, J. Hung, J. P Beilby, M. W Knuiman, and M. Divitini Folate and risk of cardiovascular disease BMJ, May 10, 2003; 326(7397): 1035 - 1035. [Full Text]
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J. P. Beilby, C. C.J. Hunt, L. J. Palmer, C. M.L. Chapman, J. P. Burley, B. M. McQuillan, P. L. Thompson, and J. Hung Apolipoprotein E Gene Polymorphisms Are Associated With Carotid Plaque Formation but Not With Intima-Media Wall Thickening: Results From the Perth Carotid Ultrasound Disease Assessment Study (CUDAS) Stroke, April 1, 2003; 34(4): 869 - 874. [Abstract] [Full Text] [PDF]
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 A. de Bree, W. M. Verschuren, A.-L. Bjorke-Monsen, N. M. van der Put, S. G Heil, F. J. Trijbels, and H. J Blom Effect of the methylenetetrahydrofolate reductase 677C->T mutation on the relations among folate intake and plasma folate and homocysteine concentrations in a general population sample Am. J. Clinical Nutrition, March 1, 2003; 77(3): 687 - 693. [Abstract] [Full Text] [PDF]
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F. Orio Jr., S. Palomba, S. Di Biase, A. Colao, L. Tauchmanova, S. Savastano, D. Labella, T. Russo, F. Zullo, and G. Lombardi Homocysteine Levels and C677T Polymorphism of Methylenetetrahydrofolate Reductase in Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 673 - 679. [Abstract] [Full Text] [PDF]
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T. H Rissanen, S. Voutilainen, K. Nyyssonen, R. Salonen, G. A Kaplan, and J. T Salonen Serum lycopene concentrations and carotid atherosclerosis: the Kuopio Ischaemic Heart Disease Risk Factor Study Am. J. Clinical Nutrition, January 1, 2003; 77(1): 133 - 138. [Abstract] [Full Text] [PDF]
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 A. De Bree, W. M. M. Verschuren, D. Kromhout, L. A. J. Kluijtmans, and H. J. Blom Homocysteine Determinants and the Evidence to What Extent Homocysteine Determines the Risk of Coronary Heart Disease Pharmacol. Rev., December 1, 2002; 54(4): 599 - 618. [Abstract] [Full Text] [PDF]
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 T. Rissanen, S. Voutilainen, K. Nyyssonen, and J. T. Salonen Lycopene, Atherosclerosis, and Coronary Heart Disease Experimental Biology and Medicine, November 1, 2002; 227(10): 900 - 907. [Abstract] [Full Text] [PDF]
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 M. Klerk, P. Verhoef, R. Clarke, H. J. Blom, F. J. Kok, E. G. Schouten, and and the MTHFR Studies Collaboration Group MTHFR 677C->T Polymorphism and Risk of Coronary Heart Disease: A Meta-analysis JAMA, October 23, 2002; 288(16): 2023 - 2031. [Abstract] [Full Text] [PDF]
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 S. Cuomo, P. Guarini, G. Gaeta, M. de Michele, F. Boeri, J. Dorn, M.G. Bond, and M. Trevisan Increased carotid intima-media thickness in children-adolescents, and young adults with a parental history of premature myocardial infarction Eur. Heart J., September 1, 2002; 23(17): 1345 - 1350. [Abstract] [Full Text] [PDF]
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H. Adachi, Y. Hirai, Y. Fujiura, H. Matsuoka, A. Satoh, and T. Imaizumi Plasma Homocysteine Levels and Atherosclerosis in Japan: Epidemiological Study by Use of Carotid Ultrasonography Stroke, September 1, 2002; 33(9): 2177 - 2181. [Abstract] [Full Text] [PDF]
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 K. K. Koh Effects of estrogen on the vascular wall: vasomotor function and inflammation Cardiovasc Res, September 1, 2002; 55(4): 714 - 726. [Full Text] [PDF]
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P. A. Ashfield-Watt, C. H Pullin, J. M Whiting, Z. E Clark, S. J Moat, R. G Newcombe, M. L Burr, M. J Lewis, H. J Powers, and I. F. McDowell Methylenetetrahydrofolate reductase 677C->T genotype modulates homocysteine responses to a folate-rich diet or a low-dose folic acid supplement: a randomized controlled trial Am. J. Clinical Nutrition, July 1, 2002; 76(1): 180 - 186. [Abstract] [Full Text] [PDF]
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 B Shannon, S Gnanasampanthan, J Beilby, and B Iacopetta A polymorphism in the methylenetetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability Gut, April 1, 2002; 50(4): 520 - 524. [Abstract] [Full Text] [PDF]
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