© 1995 American Heart Association, Inc.
Articles |
Higher Plasma Homocyst(e)ine and Increased Susceptibility to Adverse Effects of Low Folate in Early Familial Coronary Artery Disease
From Cardiovascular Genetics, Department of Internal Medicine, Cardiology Division (P.N.H., L.L.W., S.C.H., R.R.W.), the Department of Pathology, Associated Regional and University Pathologists (L.L.W., J.W.), Intermountain Health Care (B.C.J.), and the Cardiology Division, LDS Hospital (G.M.V.), University of Utah School of Medicine, Salt Lake City.
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Abstract |
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Abstract To examine the graded risks for coronary artery disease (CAD) associated with plasma homocyst(e)ine [H(e)] and to evaluate the extent to which this risk is mediated by altered vitamin status, we measured plasma concentrations of H(e), vitamins B6 and B12, and folate as well as other coronary risk factors in subjects with early familial CAD and in control subjects. We studied 120 male and 42 female patients with early CAD who were unrelated to each other but were from families in which at least one other sibling had early CAD. Control subjects were 85 men and 70 women with the same age range (38 to 68) as the subjects with CAD at screening. Increasing H(e) was associated with graded increased risks of CAD that appeared consistent with a multiplicative model. Relative odds for CAD were approximately 12.8 in women when those with H(e) levels of 19 µmol/L and above were compared with those with H(e) levels of 9 µmol/L or less (P=.007). For men, the same comparison yielded relative odds of 13.8 (P=.0002). Plasma H(e) remained a strong, independent risk factor after adjustment for standard risk factors and plasma vitamin levels in multiple logistic regression (relative odds, 8.1 for a 10-µmol/L increase in H(e); 95% confidence interval, 3.2 to 20.4; P<.0001). In multivariate ANCOVA the slope of H(e) versus folate was much steeper in subjects with CAD than in control subjects (P=.0035). These data suggest that high plasma H(e) is an important, independent contributor to risk for early familial CAD. Furthermore, subjects with early familial CAD who had low plasma folate levels had exaggerated elevations in plasma H(e), suggesting a possible genetic sensitivity to the detrimental effects of lower folate intake.
Key Words: plasma homocysteine • coronary heart disease • risk factors • genetics • folic acid
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Introduction |
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Total homocysteine in plasma comprises homocysteine bound to protein (70% to 90%), homocystine (homocysteine disulfide), homocysteine-cysteine mixed disulfide, and trace amounts of reduced homocysteine.1 Case-control studies have consistently demonstrated an increased prevalence of elevated plasma H(e) among patients with peripheral vascular disease,2 3 4 5 6 7 cerebrovascular disease,2 4 6 8 9 and CAD.4 6 10 11 12 13 14 15 16 17 18 In addition, persons with elevated plasma H(e) have been found to have thickened carotid artery walls or increased prevalence of carotid artery stenosis as assessed by high-resolution B-mode ultrasound imaging.19 20 Importantly, incidence of newly diagnosed CAD was strongly associated with prospectively measured H(e) in the Physician's Health Study21 and in the Tromsø Study,22 although a Finnish prospective study failed to find any relationship between H(e) and atherosclerotic disease risk, possibly owing to the very low prevalence of elevated H(e) levels in that population.23 However, few if any of these studies have provided data regarding the graded nature of risk associated with increasing levels of plasma H(e) in both men and women, although in prior reports the histograms of the frequency distribution of H(e) levels suggested a graded risk for occlusive arterial disease associated with increasing plasma H(e).12 19 22
Nutritional factors (especially intake of vitamin B6, vitamin B12, and folate),7 11 15 20 21 24 25 26 plasma creatinine (presumably reflecting renal removal),17 and genetic makeup appear to be primary determinants of plasma H(e) concentrations. We have recently reported that plasma H(e) exceeded the 90th percentile for control subjects in 28% of unrelated subjects with early familial CAD. Concordantly high levels of plasma H(e), without vitamin deficiency, were found in 12% of affected sibling pairs. Furthermore, among subjects with early familial CAD plasma H(e) followed a bimodal distribution, even after correction for plasma levels of vitamin B6, vitamin B12, and folate, suggesting that one or more relatively common major genes elevate plasma H(e).17 Remarkably similar results were reported by Genest et al27 among male probands with early CAD and their families, although vitamin levels were not available.
Evidence has accumulated that molecular defects in two enzymes involved in H(e) metabolism explain some of the excess high plasma H(e) seen in subjects with CAD. Clarke et al4 found that 18 of 60 subjects with CAD had free H(e) serum levels after methionine loading within the range of levels in obligate heterozygotes for cystathionine ß-synthase deficiency. Israelsson et al11 reported similar results in a smaller group of patients with CAD. A thermolabile genetic variant of the second enzyme, MTHFR, has been reported to be significantly more common among subjects with early CAD than in control subjects and is associated with elevated H(e) levels.28 29 30 Recently a mutation that apparently causes the thermolabile MTHFR defect was identified. It was found to be extraordinarily common, with a gene frequency of 38% (with 12% homozygotes) in unselected French-Canadian blood bank samples.31
We were interested in determining whether the association between plasma H(e) and CAD risk was graded and to what extent this risk might be explained by differences in vitamin status in subjects with CAD versus control subjects. Measurement of plasma concentrations of vitamin B12, vitamin B6, and folate in control subjects as well as in subjects with CAD allowed us to examine these questions, thus extending our previous observations. We have confirmed that plasma H(e) is an independent CAD risk factor and now report that strong graded risks associated with increasing plasma H(e) are present in both men and women. In addition, we find evidence for an interaction that leads to a steep increase of plasma H(e) among subjects with CAD as plasma folate levels decrease below the highest quintile.
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Methods |
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Study Participants
Subjects with early familial CAD included 120 men and 42 women who had survived a myocardial infarction, percutaneous transluminal angioplasty, or coronary artery bypass grafting before age 55 for men or age 65 for women. Each of these subjects had a sibling confirmed to have early CAD by the same definition. Only unrelated subjects with CAD were included in the present study. These were selected as the sibling with the earliest onset of CAD in families with multiple screened siblings. Control subjects were 85 men and 70 women who were either selected from a random population sampling or were spouses of hypertensive siblings who had participated in previous studies in our clinic. Control subjects from the two sources were confirmed to have comparable risk factor distributions before they were combined into one group. Further details of the selection methods have been reported.17
For purposes of this study, ages at screening of both subjects with CAD and control subjects were constrained to be from 38 to 68 years so as to be entirely overlapping. This study was approved by the Institutional Review Board of the University of Utah Medical Center. All subjects gave their written informed consent before participating in the study.
We used the following clinical definitions to categorize subjects. (1) A participant was considered to have hypertension if he or she was taking antihypertensive medication with a prior physician diagnosis of hypertension or if the mean of two supine diastolic blood pressures taken with a Critikon Dynamap automated blood pressure machine was greater than 95 mm Hg. (2) Diabetes was considered present if a prior physician diagnosis had been made or if the fasting serum glucose upon screening was greater than 7.77 mmol/L (140 mg/dL). (3) Cigarette smoking was dichotomized as "ever" or "never," with "ever smoking" defined as the subject's having smoked daily for 1 year or more. Many patients had quit smoking after onset of their CAD: hence the designation as "ever" smoking rather than as "current" and "former." (4) Patients being treated with lipid-lowering drugs included those taking bile acid sequestrants, niacin (at least 1000 mg daily), 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitors, or fibrates.
Laboratory Methods
H(e) was measured as described by Malinow et al3
with minor modifications.17 In brief, H(e) was measured in
EDTA plasma that had been separated from chilled whole blood within 2
hours of collection after subjects had fasted overnight. Plasma was
stored at -70°C until analysis. Samples were treated with 6
mol/L urea and sodium borohydride (NaBH4) to liberate free
H(e), which was quantified by electrochemical detection after
high-pressure liquid chromatography. All H(e)
determinations were performed in the Associated Regional and University
Pathologists, Inc, laboratories. Details regarding the accuracy and
precision of our assay have been previously
reported.17
Blood samples were collected after the subjects had fasted for 12 to 16 hours, according to guidelines of the Lipid Research Clinics Program's manual of laboratory operations.32 Lipids were measured by use of a microscale procedure developed in our laboratory.33 Our lipid laboratory participates in the standardization program of the Center for Disease Control in Atlanta, Ga.
All vitamin assays were performed on plasma collected as described above from fasting subjects. Vitamin B6 (as pyridoxal-5'-phosphate) was determined by a radioenzymatic method with a reported normal range of 20 to 93 nmol/L.34 The CIBA-Corning automated chemiluminescence system was used for the quantitative determination of vitamin B12 and folate in plasma.35 Vitamin B12 levels of 148 to 664 pmol/L are considered normal, while values of less than 66 to 74 pmol/L are evidence of frank deficiency. The normal range for folate is 6.8 to 36 nmol/L.
Statistical Analysis
The SAS statistical software package was used for
data analysis (SAS Institute, Inc). Statistical
analyses on triglycerides were done after
logarithmic transformation to normalize the distribution.
Analyses involving H(e) were done both with and without log
transformation. Because results were virtually identical, we have
reported the untransformed results. Statistical tests included
Student's t test, 
2 test, Fisher's
exact test, Pearson's correlation, and stepwise multiple logistic
regression. ANCOVA to test for interaction and to adjust for potential
confounding was performed with the SAS GLM program.
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Results |
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Clinical characteristics of unrelated subjects with early familial CAD and control subjects are shown in Table 1
. Despite
completely overlapping age ranges in subjects with CAD and control
subjects, the subjects with CAD were on average 8 years older, and the
proportion of men was higher in this group than in the control group.
In addition, there were marked differences in the prevalence of
hypertension and diabetes. Subjects with CAD had significantly higher
BMI, blood pressure, and levels of serum glucose, plasma total
cholesterol, triglyceride, and VLDL
cholesterol. Plasma HDL cholesterol was lower
among subjects with CAD. Plasma H(e) was significantly higher in
subjects with CAD than in control subjects. Measured plasma LDL
cholesterol levels were not different in subjects with CAD
compared with control subjects, probably because of treatment. More
than half of the subjects with CAD (52%) were being treated for high
cholesterol (34% were taking drugs), whereas only 6% of
the control subjects were on a diet and none were taking lipid-lowering
medication.
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To estimate the effect of increasing H(e) on risk, we categorized
subjects with CAD and control subjects by plasma H(e) concentration,
and relative odds were calculated as shown in Fig 1.
There was a progressive increase in risk among both men and women as
H(e) rose above 9 µmol/L. H(e) levels of 13 µmol/L and above were
associated with significantly increased risk in both men and women. The
Mantel-Haenszel test for trend was strongly positive in men
(P=.00003) and women (P=.00002). Although this
test examines the linear component of trend, risks appeared to be
multiplicative, with relative odds increasing to 12 or more in both men
and women when plasma H(e) was greater than 18 µmol/L.
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9 µmol/L defined as
baseline, 95% confidence intervals (CIs), and P values. For
calculation of the relative odds for women in the highest H(e)
category, 1 was added to each cell. Fisher's exact test was used to
calculate the P value for this category.
Multiple stepwise logistic regression was then performed to evaluate
the effects that other risk factors might have on the strength of the
association between plasma H(e) and CAD risk. Potential risk factors
included in the model were age, sex, BMI, cigarette smoking,
hypertension, diabetes, plasma total cholesterol, measured
LDL cholesterol, triglycerides (natural
log–transformed), HDL cholesterol, plasma vitamin
concentrations, and plasma H(e). Results are shown in Table 2. Plasma H(e) entered third into the model, after age
and cigarette smoking. Other significant risk factors were diabetes,
hypertension, and plasma triglycerides. A 10-µmol/L
increase in H(e) was associated with an 8.1-fold increase in risk after
adjustment for other risk factors (95% confidence interval, 3.2 to
20.4; P<.0001), in good agreement with the categorical
univariate analyses. Although plasma vitamin
B6 concentration was lower in subjects with CAD than in
control subjects, no vitamin concentration approached significance as a
risk factor after plasma H(e) entered into the model in the stepwise
logistic regression. Because results were virtually identical when
log-transformed H(e) was used, only the untransformed results are
presented.
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Correlations between H(e) and plasma vitamin concentrations in subjects
with CAD and control subjects are given in Table 3. Of
note is the strong intercorrelation among all the vitamins themselves
as well as between the vitamins and plasma H(e). The
univariate correlation between plasma H(e) and folate
appeared to be the strongest of the correlations of H(e) with the
vitamins in both subjects with CAD and control subjects.
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To test for potential environmental interaction with disease status, we
performed an ANCOVA with plasma H(e) concentration as the dependent
variable and age, sex, BMI, creatinine, vitamins, and
interaction terms between vitamin levels and case status (subjects with
CAD versus control subjects) as independent variables.
Creatinine (P<.0001), folate
(P<.0001), case status (P<.0001), vitamin
B12 (P=.01), and the term for interaction
between case status and folate (P=.0035) were found to be
significantly associated with plasma H(e). Thus, plasma H(e)
remained significantly different in subjects with CAD versus control
subjects even after vitamin levels were controlled for (13.2±0.30
µmol/L [least-squares mean±SEM] in subjects with CAD versus
10.5±0.29 µmol/L in control subjects). Furthermore, the significant
interaction term between case status and folate level suggests that the
slope between plasma H(e) and folate is different in subjects with CAD
compared with control subjects. These two slopes were estimated by
nesting of folate within case status by use of ANCOVA. The slope
between plasma H(e) and folate in subjects with CAD was -0.167 (SEM,
0.024; P=.0001), and the corresponding slope among controls
was -0.068 (SEM, 0.021; P=.002). The two regression lines
are plotted in Fig 2. Note that the lines intersect at a
plasma folate level of approximately 60 nmol/L, well within the
observed range of plasma folate in both subjects with CAD (4.8 to 73
nmol/L) and control subjects (4.5 to 92 nmol/L).
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The interaction between plasma folate and case status is further
illustrated in Fig 3. Least-squares means and standard
errors shown were obtained by ANCOVA, with folate (and other vitamin)
quintiles substituted as categorical variables for the continuous
variables in the model above. Quintile cutpoints were determined
for the control group with the same cutpoints applied to the cases.
Power is, of course, lost when categories are substituted for a
continuous variable. Nevertheless, the results are
presented as a graphical representation of the more
formal interaction analysis above. Note in Fig 3 that plasma
H(e) levels do not increase consistently at lower plasma
vitamin B6 and vitamin B12 levels, as might be
expected, because the stronger correlation with folate (which in turn
correlates with vitamin B6 and vitamin B12) has
been included in the model.
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Discussion |
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We have found plasma H(e) concentration to be a strong, graded, independent risk factor for early familial CAD in both men and women. Furthermore, plasma concentrations of vitamin B6, vitamin B12, and folate, which correlate strongly with plasma H(e), did not explain the risk associated with plasma H(e). Nevertheless, plasma vitamin concentrations, especially that of folate, were important correlates of plasma H(e) among both subjects with CAD and control subjects. In addition, a significantly steeper slope between plasma folate and H(e) was found among subjects with CAD. Indeed, the difference in plasma H(e) concentration between subjects with CAD and control subjects might have disappeared if all the subjects with CAD had had plasma folate levels in the highest quintile.
Other studies have noted a fairly strong negative correlation between plasma H(e) and vitamin intake or plasma vitamin levels (vitamin B6, vitamin B12, and particularly folate) among free-living persons.7 11 21 24 25 36 Of note was a stronger correlation between folate and H(e) among subjects with CAD compared with control subjects recently reported by Mansoor et al.7 However, the present study is the first to specifically note an interaction, which was manifested by a significantly steeper slope in the regression lines between folate and H(e) in subjects with CAD compared with control subjects. Our findings thus suggest a considerably greater sensitivity to decreasing folate concentration among subjects with CAD.
Marked sensitivity to folate status, moderately elevated plasma H(e) when plasma folate is in the lower range, and significantly increased risk for early CAD has been noted among persons carrying the thermolabile variant of the MTHFR gene.37 38 Indeed, 29% of CAD patients under age 5028 and 17% to 18% of unselected CAD patients (mean age, 59 years) displayed this recessive trait compared with only 5% of control subjects.29 39 Homozygosity for this mutation results in a reduction of approximately 50% in basal MTHFR activity and mild to moderate hyperhomocyst(e)inemia, although plasma H(e) may range from as low as 4.3 µmol/L to as high as 38 µmol/L.30 39 When exposed to mild heat (46°C for 5 minutes), thermolabile MTHFR retains only 7% to 17% of initial activity compared with 20% to 50% for normal MTHFR.39 In a recent small survey in which direct genetic techniques were used, 12% of unselected samples from French-Canadian blood bank controls were found to be homozygous carriers for the MTHFR mutation leading to heat lability.31
An increase among our subjects with early familial CAD in the prevalence of homozygous carriers of the thermolabile MTHFR defect leading to enhanced sensitivity to folate is an attractive hypothesis. Alternatively, increased prevalence of other genetic defects leading to hyperhomocyst(e)inemia may also conceivably lead to enhanced folate sensitivity. Indeed, patients with cystathionine ß-synthase deficiency are routinely treated with folate even though there is no inherent defect in their folate-dependent pathway.40 Such patients also appear to be at increased risk for premature vascular disease.4 Recent progress in identifying causal mutations for both the defective MTHFR and cystathionine ß-synthase genes could facilitate screening among patients with early familial CAD.31 41 42 43 44
Hyperhomocyst(e)inemia appears to aggravate atherosclerosis by at least three general mechanisms: endothelial cell toxicity,45 46 47 48 increased platelet adhesiveness,45 48 and modification of clotting factors.40 49 50 51 52 Early consequences of H(e) toxicity to endothelial cells may be impairment of their protective capacity to secrete nitric oxide.48 Interestingly, although H(e) caused a dose-dependent decrease in DNA synthesis in cultured human endothelial cells, similar increases in H(e) stimulated growth of cultured smooth muscle cells, potentially promoting the growth of atherosclerotic plaques.53 In one study, plasma H(e) and fibrinogen were correlated and some or all of the increased risk associated with elevated H(e) appeared to be mediated through increased plasma fibrinogen.16 Although elevated levels of H(e) have been hypothesized to lead to increased production of oxidized lipoproteins,54 55 no increase in cholesterol hydroperoxide was found in the HDL of patients with homozygous cystathionine ß-synthase deficiency.56 H(e) increases the affinity of Lp(a) for fibrin, which may inhibit plasmin, thereby further favoring thrombosis.57
Although subjects with CAD and control subjects had the same age range, mean age and sex distributions were considerably different. The greater average age of the subjects with CAD probably contributed importantly to the much higher prevalence of hypertension and diabetes among them compared with control subjects. However, age had minimal effects on plasma H(e) or plasma vitamin concentrations. Furthermore, neither age nor sex influenced the significance of plasma H(e) level as an independent CAD risk factor. Although we were able to screen relatively few women with early familial CAD, this study is, to our knowledge, the first to demonstrate graded, markedly increasing risks associated with plasma H(e) in women. Our results in men corroborate similar observations in previous studies.15
The relationship between plasma vitamin and H(e) levels has been examined in a considerably larger population of primarily healthy elderly subjects from Framingham, Mass.25 In that study, mean levels of H(e) increased strikingly in the lowest two deciles of plasma vitamin concentration. In contrast, we observed what appeared to be a more linear relationship between vitamin concentration and H(e). However, our population appeared to be better nourished than the elderly Framingham subjects—having higher mean plasma vitamin concentrations and different quintile cutpoints—with the result that our observations were more comparable to those made in the upper six or seven deciles of the Framingham population. In these higher ranges, the relationship between plasma vitamin and H(e) concentrations appeared quite linear in the Framingham population as well. Even more striking effects of vitamins on H(e) might therefore be expected in persons with a lower vitamin status than those included in the present study.
Although plasma total and LDL cholesterol concentrations were not significant risk factors in this evaluation, more than half of the patients with CAD were treated for hyperlipidemia, many with potent lipid-lowering drugs. Thus, our results cannot be taken as evidence against the significance of total or LDL cholesterol as a CAD risk factor. In fact, after maximum lifetime total cholesterol was estimated by substitution of the historically reported highest previous total cholesterol (when available from participant questionnaires) for screened total cholesterol, estimated maximum total cholesterol did become a highly significant risk factor. Furthermore, HDL cholesterol entered into this model while triglyceride did not. The association with plasma H(e), was, if anything, slightly strengthened in this model (results not shown). Though this estimate of maximum total cholesterol may be biased (because patients treated for high cholesterol will be much more aware of their previous cholesterol levels than will untreated persons), these results do further confirm the independence of plasma H(e) as a CAD risk factor.
In summary, we have confirmed a strong, graded, independent risk for early familial CAD associated with increasing levels of plasma H(e). In addition, our patients with CAD had increased sensitivity to the effects of plasma folate on plasma H(e) compared with control subjects. These results may have considerable public health importance if persons predisposed to early coronary disease due to elevated plasma H(e) could readily and conveniently normalize their plasma H(e) by folate supplementation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants HL-47651, HL-21088, and HL-47466 from the National Heart, Lung, and Blood Institute, Bethesda, Md.
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Footnotes |
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Reprint requests to Dr Paul N. Hopkins, Cardiovascular Genetics Research Clinic, 410 Chipeta Way, Rm 161, Salt Lake City, UT 84108.
Received April 17, 1995; accepted June 16, 1995.
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