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Homocysteine and risk of coronary artery disease
APPLIED NUTRITIONAL INVESTIGATION
Homocysteine and Risk of Coronary Artery Disease: Folate Is the Important Determinant of Plasma Homocysteine Concentration Bor-Jen Lee, MD, Ping-Ting Lin, MS, Yung-Po Liaw, PhD, Sue-Joan Chang, PhD, Chien-Hsiung Cheng, MD, and Yi-Chia Huang, PhD From the Intensive Care Unit, Critical Care and Respiratory Therapy, Taichung Veterans General Hospital, School of Nutrition, the Department of Public Health, Chung Shan Medical University, and the Department of Biology, National Cheng Kung University, Taiwan, Republic of China OBJECTIVES: The purposes of this study were to study the effects of folate and vitamins B6 and B12 on plasma homocysteine concentration and to estimate the risks for coronary artery disease (CAD) according to quartiles of plasma homocysteine concentration. METHODS: The study was designed as a case-reference observational study. Case subjects (CAD group, n ⫽ 60) were identified by cardiac catheterization to have at least 70% stenosis of one major coronary artery; otherwise, patients were considered for a reference group (n ⫽ 60). Risk factors of cardiovascular disease were recorded, including age, sex, blood lipid profile, hypertension, smoking habits, and drinking habits. Plasma homocysteine, folate, pyridoxal 5⬘-phosphate, and vitamin B12 were measured. RESULTS: CAD subjects had significantly higher mean plasma homocysteine concentrations than did the reference subjects (13.9 ⫾ 4.9 versus 9.1 ⫾ 3.3 mol/L). There were no significant differences between groups with regard to the three B vitamins; however, mean serum folate concentrations for subjects in the highest two quartiles of plasma homocysteine concentration (10.8 –13.8 and ⱖ13.9 mol/L) were significantly lower than those for subjects in the lowest two quartiles (ⱕ8.0 and 8.1–10.7 mol/L). Plasma homocysteine was strongly inversely associated with serum folate in the CAD ( ⫽ ⫺0.166, P ⬍ 0.05), reference ( ⫽ ⫺0.178, P ⬍ 0.001), and pooled ( ⫽ ⫺0.190, P ⬍ 0.001) groups. Age, sex, other confounding factors, and B-vitamin–adjusted odds ratios were significantly increased in the highest quartile of homocysteine concentration (odds ration, 5.54; 95% confidence interval, 0.38 – 81.41). The elevation of 1 ng/mL in serum folate concentration was found to decrease plasma homocysteine by 0.166 mol/L. CONCLUSIONS: Serum folate, but not vitamin B6 or B12, was a strong predictor of plasma homocysteine; while all subjects had adequate B-vitamin status. Folate should be considered as a routine supplementation for individuals who have risk factors for CAD, even for individuals with adequate folate status. Nutrition 2003;19:577–583. ©Elsevier Inc. 2003 KEY WORDS: homocysteine, folate, vitamin B6, vitamin B12, risk factor, coronary artery disease
INTRODUCTION Many studies have shown that plasma homocysteine concentration are significantly higher in patients with coronary artery disease (CAD),1–3 peripheral vascular disease,4,5 and cerebral vascular disease.6,7 A large, prospective, 5-y study, the Physicians Health Study, found that physicians with elevated plasma homocysteine concentrations had a three-fold increase in the occurrence of myocardial infarction than did physicians who had normal plasma homocysteine concentrations.2 Boushey et al.8 indicated that the elevation of 5 mol/L in plasma homocysteine concentration increases the incidence of CAD by 60% to 80%. Thus, hyperhomocystinemia likely is an independent risk factor of CAD.2,8,9
This study was supported by the National Science Council (grant 90-2320B-040-019), Taiwan. Correspondence to: Yi-Chia Huang, PhD, School of Nutrition, Chung Shan Medical University, No 110 Sec 1 Chien-Kuo N. Rd, Taichung, Taiwan 402. E-mail: [email protected] Nutrition 19:577–583, 2003 ©Elsevier Inc., 2003. Printed in the United States. All rights reserved.
Many factors (e.g., age, enzyme deficiencies and mutations, vitamin deficiencies, disease, and drugs) are associated with mild and moderate hyperhomocystinemia; of particular interest are nutritional deficiencies in the vitamin cofactors that are required for homocysteine metabolism: folate and vitamins B12 and B6. Homocysteine is metabolized by two pathways. When methionine is in negative balance, homocysteine is remethylated to form methionine by a methionine-conserving remethylation pathway; this process requires methyltetrahydrofolate as a cosubstrate, methionine synthase, and vitamin B12 as a cofactor. Homocysteine, however, is directed to the transsulfuration pathway when methionine is in excess. During the transsulfuration reaction, homocysteine is irreversibly sulfur conjugated to serine by cystathionine -synthase and cystathionase to it convert to cystathionine and then to cysteine by the pyridoxal 5⬘-phosphate (PLP)– dependent enzymes. Therefore, deficiencies of folate, vitamin B12, or vitamin B6 may account for most cases of hyperhomocystinemia. A large cohort of subjects from the Framingham Study indicated that folate, vitamin B6, and vitamin B12 are important determinants of plasma homocysteine concentrations in a healthy population.10 The Nurses Health Study, with more than 80 000 nurses studied 0899-9007/03/$30.00 doi:10.1016/S0899-9007(02)01098-5
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over 14 y, showed that the risks of mortality and morbidity from myocardial infarction is inversely related to dietary intake of folate and vitamin B6.11 Most cross-sectional, retrospective, case-controlled, and prospective cohort studies8,12–16 have shown that folate and vitamin B12 intake and/or biochemical status strongly and negatively correlate with plasma homocysteine concentration. Thus, folate and vitamin B12 supplementation have the beneficial effect of lowering plasma homocysteine concentration and further reducing the risk of CAD. However, there was no consistent evidence showing the effect of vitamin B6 on plasma homocysteine concentration. Chasan-Taber et al.17 showed that plasma PLP is inversely correlated with homocysteine (r ⫽ ⫺0.29, P ⬍ 0.001) by using the participants in the Physicians Health Study. In a recent randomized, double-blind, placebo-controlled trial, vitamin B6 (1.6 mg/d) was given for 12 wk to 11 healthy elderly subjects after repletion with folic acid and riboflavin. Results showed that vitamin B6 supplementation significantly reduces plasma homocysteine concentration by 7.5%.18 In contrast, some studies have shown that vitamin B6 has no effect on plasma homocysteine concentration. Brattstro¨ m et al.19 studied homocysteine metabolism in 72 patients with occlusive arterial disease and found that plasma PLP was decreased in most patients; however, there was no correlation between plasma PLP and homocysteine concentration. Giraud et al.20 also found that plasma homocysteine concentration did not correlate with vitamin B6 status in 77 Indonesian children. Plasma homocysteine concentration was then suggested not to be an indicator of vitamin B6 status.20 Because most of these studies were done in healthy subjects or patients younger than 60 y with cardiovascular diseases, further studies are warranted to study the effect of B vitamins, especially of vitamin B6, on homocysteine concentration in elderly patients with CAD. Therefore, we investigated the effects of folate, vitamin B6, and vitamin B12 on plasma homocysteine concentration after adjusting risk factors for CAD and estimated the risks for CAD according to the quartiles of plasma homocysteine concentration.
glucose, serum creatinine, total serum cholesterol, high-density lipoprotein [HDL] cholesterol, low-density lipoprotein [LDL] cholesterol, triacylglycerol, alanine aminotransferase, and serum alkaline phosphatase), homocysteine, vitamin B6, vitamin B12, and folate status. Serum and plasma were prepared and then stored frozen (⫺20°C) until analysis. Risk factors of cardiovascular disease were recorded, including age, sex, blood lipid profile (total cholesterol ⱖ 200 mg/dL, HDL cholesterol ⬍ 35 mg/dL, LDL cholesterol ⱖ 130 mg/dL, triacylglycerol ⱖ 200 mg/dL), hypertension (blood pressure ⱖ 140/90 mmHg or currently taking antihypertensive drugs), smoking habits (smoker versus non-smoker, defined as no smoking for at least 3 wk before the study), and drinking habits (drinking, defined as ⬎100 mL/d, versus non-drinking). Biochemical Measurements Plasma homocysteine was measured by using high-performance liquid chromatography according to the method of Araki and Sako.21 The intra-assay and inter-assay of plasma homocysteine variabilities were 3.5% (n ⫽ 5) and 4.8% (n ⫽ 8), respectively. Plasma PLP was determined by high-performance liquid chromatography as previously described.22 The intra-assay and inter-assay of plasma PLP variabilities were 3.2% (n ⫽ 4) and 4.4% (n ⫽ 6), respectively. Serum folate and vitamin B12 were analyzed by using standard competitive immuno-chemiluminometric methods on a Chiron Diagnostics ACS:180 Automated Chemiluminescence System (Chiron Diagnostics Corporation, East Walpole, MA, USA). All analyses were performed in duplicate. Hyperhomocysteinemia was defined as a plasma homocysteine concentration at least 15 mol/L. Vitamin B12 and folate deficiencies were defined as serum concentrations below 169.4 pg/mL, and 2.8 ng/mL, respectively.23 Vitamin B6 deficiency was defined as plasma PLP concentration below 20 nmol/L.23 Statistical Analyses
A case-reference study was conducted from October 2000 to January 2002. Subjects were recruited from the cardiology clinic of the Taichung Veteran General Hospital, which is a 1359-bed teaching hospital in the central part of Taiwan. Patients suspected of having CAD underwent coronary angiography. Case subjects (CAD group, n ⫽ 60) were identified by cardiac catheterization to have at least 70% stenosis of one major coronary artery; otherwise, patients were considered for a reference group. The reference group (n ⫽ 60) consisted of subjects with no history of myocardial infarction and less than 70% occlusion in any coronary artery. All subjects with diabetes (defined by a history of antidiabetic drug use or a fasting plasma glucose concentration ⬎ 140 mg/dL) or liver or renal diseases (identified by serum creatinine and aspartate aminotransferase analyses) were excluded to minimize the influence of other cardiovascular risk factors. The use of medications was recorded. Informed consent was obtained from each subject. The study was approved by the Committee for Ethics of Chung Shan Medical University.
Data were analyzed with SAS 6.12 (SAS Institute, Cary, NC, USA). Differences in subjects’ demographic and health characteristics and the data of biochemical measurements between CAD and reference groups were analyzed by Student’s t test. For categorical response variables, differences between groups were assessed by Fisher’s exact test. Simple regression analyses were used to assess the association of plasma homocysteine concentration with B vitamins (folate, vitamin B6, and vitamin B12) and other risk factors for CAD. Multiple regression analysis with plasma homocysteine concentration as the dependent variable was used to examine the effect of folate, vitamin B6, and vitamin B12 on plasma homocysteine after adjustment for potential confounders and the other two B vitamins. One-way analysis of variance was used to compare differences in serum folate, plasma PLP, and serum vitamin B12 among the quartiles of plasma homocysteine. Adjusted odds ratios (ORs) with 95% confidence intervals (CI) for CAD were calculated from the logistic regression model according to the quartiles of plasma homocysteine concentration across subjects. ORs were computed with adjustment for CAD risk factors and for CAD risk factors plus the other two B vitamins. Statistical results were considered statistically significant at P ⬍ 0.05. Values presented in the text are means ⫾ standard deviation.
Experimental Protocol
RESULTS
MATERIALS AND METHODS Subjects
2
Body mass index (kg/m ) was calculated from height and weight measurements. Blood pressure was measured after a resting period of at least 5 min. Fasting venous blood samples (15 mL) were collected in Vacutainer tubes (Becton Dickinson, Rutherford, NJ, USA) containing an appropriate anticoagulant or no anticoagulant as required to determine some hematologic entities (i.e., plasma
Characteristics of Subjects Characteristics of CAD and reference subjects are shown in Table I. Subjects’ ages ranged from 36 to 92 y, with median ages of 73 y and 67.5 y for the CAD and reference groups, respectively. Unfortunately, we could not recruit age- and sex-matched control
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TABLE I.
TABLE III.
DEMOGRAPHIC AND HEALTH CHARACTERISTICS OF PATIENTS WITH CAD AND REFERENCE SUBJECTS*
SIMPLE LINEAR REGRESSION  OF PLASMA HOMOCYSTEINE WITH RISK FACTORS OF CAD AND B VITAMINS IN INDIVIDUAL AND POOLED CAD AND REFERENCE GROUPS*
CAD (n ⫽ 60)
References (n ⫽ 60)
54/6 71.6 ⫾ 9.7a 70.3 ⫾ 9.1a 165.1 ⫾ 6.2a 25.7 ⫾ 2.7 50.0 201.8 ⫾ 40.2a 47.3 ⫾ 11.5 127.5 ⫾ 35.7a 21.4 138.2 ⫾ 76.9a 38.3 131.1 ⫾ 13.3 74.4 ⫾ 7.4 1.3 ⫾ 0.3a 16.7 26.7
39/21 65.5 ⫾ 9.0b 64.1 ⫾ 9.9b 161.0 ⫾ 8.9b 24.8 ⫾ 3.7 23.3 173.4 ⫾ 36.6b 49.1 ⫾ 15.1 112.0 ⫾ 30.1b 3.3 87.2 ⫾ 43.9b 35.0 133.9 ⫾ 18.5 74.1 ⫾ 12.1 1.0 ⫾ 0.3b 26.7 31.7
Characteristics Male/female Age (y) Weight (kg) Height (cm) Body mass index (kg/m2) Hypercholesterolemia (%) Serum total cholesterol (mg/dL) Serum HDL cholesterol (mg/dL) Serum LDL cholesterol (mg/dL) Hypertriglyceridemia (%) Serum triacylglycerol (mg/dL) Hypertension (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Serum creatinine (mg/dL) Current smoker (%) Drinking (⬎100 mL/d) (%)
* Values are means ⫾ standard deviation. Hypercholesterolemia was defined as serum total cholesterol of at least 200 mg/dL or currently taking cholesterol-lowering drugs. Hypertriglyceridemia was defined as serum triacylglycerol of at least 200 mg/dL. Hypertension was defined as systolic blood pressure higher than 140 mmHg and/or diastolic blood pressure higher than 90 mmHg. Values within a row with different superscript letters are significantly different; P ⬍ 0.05. CAD, coronary artery disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
CAD (n ⫽ 60) Age (y) Sex BMI (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Smoking Drinking Serum total cholesterol (mg/dL) Serum LDL cholesterol (mg/dL) Serum HDL cholesterol (mg/dL) Serum triglyceride (mg/dL) Serum creatinine (mg/dL) Serum folate (mg/mL) Plasma vitamin B6 (nmol/L) Serum vitamin B12 (pg/mL)
0.176† ⫺1.425 ⫺0.150 0.062 ⫺0.035 1.565 ⫺0.167 ⫺0.030 ⫺0.039* ⫺0.036 0.010 9.853‡ ⫺0.166* ⫺0.018 ⫺0.013*
Reference (n ⫽ 60)
Pooled (n ⫽ 120)
0.055 2.094* 0.110 0.031 0.042 1.038 1.038 ⫺0.012 ⫺0.022 0.036 ⫺0.009 4.455† ⫺0.178‡ ⫺0.019 ⫺0.000
0.187‡ 0.374 0.167 0.013 ⫺0.017 0.576 ⫺0.365 0.001 ⫺0.014 ⫺0.000 0.018† 7.660‡ ⫺0.190‡ 0.029 ⫺0.002
* P ⬍ 0.05. † P ⬍ 0.01. ‡ P ⬍ 0.001. CAD, coronary artery disease; BMI, body mass index; HDL, highdensity lipoprotein; LDL, low-density lipoprotein; PLP, pyridoxal 5⬘phosphate.
and over one-fourth of CAD and reference subjects drank alcohol (⬎100 mL/d). Homocysteine and Vitamin Status
subjects. Patients with CAD were older than the reference subjects. Because we adjusted for age and sex when discussing the data, the difference was not expected to affect the results of this study. Both groups were of comparable body size. Patients with CAD had significantly higher serum total cholesterol, LDL cholesterol, and triacylglycerol. Although mean serum creatinine of CAD subjects was significantly higher than that of reference subjects, it was within the normal range, indicating a normal renal function for CAD subjects. With regard to smoking and drinking, 16.7% of CAD subjects and 26.7% of reference subjects smoked regularly
TABLE II. PLASMA TOTAL HOMOCYSTEINE AND B VITAMINS IN PATIENTS WITH CAD AND REFERENCE SUBJECTS*
Fasting plasma total homocysteine (mol/L) Serum folate (ng/mL) Plasma PLP (nmol/L) Serum vitamin B12 (pg/mL)
CAD† (n ⫽ 56)
References (n ⫽ 60)
P
13.9 ⫾ 4.9
9.1 ⫾ 3.3
⬍ 0.001
11.9 ⫾ 9.4 52.2 ⫾ 42.3 380.7 ⫾ 116.7
13.2 ⫾ 8.6 44.6 ⫾ 40.0 510.7 ⫾ 548.0
0.436 0.32 0.085
* Values are means ⫾ standard deviation. † Blood samples could not be obtained from four patients in the CAD group. CAD, coronary artery disease; PLP, pyridoxal 5⬘-phosphate.
Concentrations of plasma homocysteine and related B vitamins are shown in Table II. CAD subjects had significantly higher mean plasma homocysteine concentration than did reference subjects. Although the mean plasma homocysteine concentration was below 15 mol/L in both groups, the CAD group had a mean plasma homocysteine concentration (13.9 ⫾ 4.9 mol/L) very close to the cutoff value for hyperhomocystinemia. In addition, 35.7% (n ⫽ 20) of CAD patients and 5% (n ⫽ 3) of reference subjects had moderate hyperhomocystinemia (ⱖ15 mol/L). The CAD and reference groups had mean serum folate, plasma PLP, and serum vitamin B12 concentrations higher than the cutoff value. There were no significant differences between groups with regard to the three B vitamins. Associations With Homocysteine Simple regression analyses were performed to understand the relation between homocysteine plus B vitamins and risk factors for CAD; results are shown in Table III. Plasma homocysteine concentration was strongly and inversely associated with serum folate concentration in the CAD, reference, and pooled groups. There was no significant association between plasma homocysteine and plasma PLP in any group. The CAD group showed a negative association between plasma homocysteine and vitamin B12. A positive association was found in the CAD and pooled groups between plasma homocysteine and age or serum creatinine concentration. Plasma homocysteine correlated significantly with sex in the reference group, with LDL cholesterol in the CAD group, and with triacylglycerol in the pooled group. Other risk factors of CAD, including body mass index, blood pressure, smoking, drink-
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Nutrition Volume 19, Numbers 7/8, 2003 TABLE IV.
MULTIPLE LINEAR REGRESSION ANALYSIS WITH PLASMA HOMOCYSTEINE CONCENTRATION AS THE DEPENDENT VARIABLE AFTER ADJUSTING THE POTENTIAL CONFOUNDERS OF CAD AND THE OTHER TWO B VITAMINS* CAD (n ⫽ 56) Vitamins Serum folate (ng/mL) Plasma PLP (nmol/L) Serum vitamin B12 (pg/mL)
References (n ⫽ 60)
Pooled (n ⫽ 116)

P

P

⫺0.0419 ⫺0.00584 ⫺0.0104
0.472 0.606 0.028
⫺0.168 ⫺0.0203 ⫺0.0005
⬍0.001 0.040 0.488
⫺0.166 ⫺0.0065 0.0000
P ⬍0.001 0.452 1.0
* Linear regression coefficients express how the mean of plasma homocysteine concentration varies per unit increase in vitamins after adjusting for age, sex, serum total cholesterol, low-density lipoprotein cholesterol, triacylglycerol, creatinine, and the other two B vitamins. , regression coefficient; CAD, coronary artery disease; PLP, pyridoxal 5⬘-phosphate.
ing, total cholesterol, and HDL cholesterol, did not associate with homocysteine in any group. Relation of Plasma Homocysteine to Vitamin Status To study the effect of B vitamins on plasma homocysteine concentration, multiple regression analysis was performed on the individual and pooled groups (Table IV). Plasma homocysteine concentration was significantly and inversely affected by serum folate concentration after adjusting risk factors for CAD and the other two B vitamins in the reference and pooled groups. An elevation of 1 ng/mL in serum folate concentration decreased plasma homocysteine by 0.166 mol/L. Serum vitamin B12 was significantly correlated with plasma homocysteine concentration only in the CAD group. Plasma PLP, however, had no effect on plasma homocysteine concentration in the individual and pooled groups. Quartiles of Plasma Homocysteine Table V shows the distribution of B vitamins according to the quartiles of plasma homocysteine concentration across subjects. Mean serum folate concentrations for subjects in the first two quartiles of plasma homocysteine concentration (ⱕ8.0 and 8.1– 10.7 mol/L) were significantly higher than those for subjects in the third and fourth quartiles of plasma homocysteine concentration (10.8 –13.8 and ⱖ13.9 M/L). There were no significant differences in the distribution of plasma PLP and serum vitamin B12 concentrations among quartiles of plasma homocysteine concentration.
Odds Ratio for Coronary Artery Disease We calculated risks of CAD by quartiles of plasma homocysteine according to the distribution of all subjects (Table VI and Fig. 1). The age- and sex-adjusted ORs for all CAD and reference subjects increased significantly in the fourth quartile of homocysteine concentration (OR, 12.67; 95% CI, 2.71–59.32). In addition, after adjusting for other risk factors for CAD, homocysteine had a much greater effect on the risk of CAD (OR, 22.95; 95% CI, 2.8 – 188.28). Likewise, when serum folate, plasma PLP, and serum vitamin B12 were included in the regression model, the relation between CAD and homocysteine increased significantly in the third or fourth quartile of plasma homocysteine concentration after adjusting for age and sex. After additional adjustments for all risk factors for CAD plus individual or all B vitamins, the third or fourth quartile of plasma homocysteine showed a lower but still significant effect on the risk for CAD. The important observation was the inclusion of serum folate in the regression model: the OR was about 50 (95% CI, 7.37–334.38) in the fourth quartile of homocysteine concentration after adjusting for age and sex.
DISCUSSION The significant result of this study was that serum folate is the most important determinant of plasma homocysteine concentration if subjects have adequate B-vitamin status. Another important finding was that the cutoff value of hyperhomocystinemia might be considered to decrease to 14 mol/L or even less instead of 15 mol/L or higher.
TABLE V. LEVELS OF B VITAMINS IN ALL SUBJECTS ACCORDING TO QUARTILES OF PLASMA HOMOCYSTEINE CONCENTRATIONS ACROSS SUBJECTS* Plasma homocysteine category (mol/L)
Serum folate (ng/mL) Plasma PLP (nmol/L) Serum vitamin B12 (pg/mL)
ⱕ8.0 (n ⫽ 29)
8.1–10.7 (n ⫽ 30)
10.8–13.8 (n ⫽ 28)
ⱖ13.9 (n ⫽ 29)
17.6 ⫾ 10.5a 53.3 ⫾ 51.0 522.4 ⫾ 464.9
14.4 ⫾ 10.4a 47.3 ⫾ 30.1 527.3 ⫾ 595.6
9.5 ⫾ 6.0b 47.1 ⫾ 49.0 394.2 ⫾ 206.5
8.6 ⫾ 5.4b 45.4 ⫾ 32.5 344.6 ⫾ 148.4
* Values are means ⫾ standard deviation. Values within a row with different superscript letters are significantly different. PLP, pyridoxal 5⬘-phosphate.
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TABLE VI. MULTIVARIATE ADJUSTED ODDS RATIOS OF CORONARY ARTERY DISEASE ACCORDING TO THE QUARTILES OF PLASMA CONCENTRATION ANALYZED BY MULTIPLE LOGISTIC REGRESSION
Adjusted vitamin None
Folate
PLP
Vitamin B12
All
Age and sex adjusted
Quartiles of plasma homocysteine (mol/L)
OR
ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9 ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9 ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9 ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9 ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9
1 1.51 2.69 12.67 1 3.70 9.39 49.65 1 2.25 3.37 17.99 1 2.54 5.16 27.70 1 2.34 5.16 27.60
Age, sex, and other risk factors*
CI
P
0.47–4.82 0.85–8.51 2.71–59.32
0.4859 0.0929 0.0013
0.87–15.77 1.98–44.57 7.37–334.38
0.0767 0.0048 ⬍0.001
0.52–9.83 0.78–14.62 2.95–109.62
0.2806 0.1048 0.0017
0.62–10.42 1.24–21.40 4.51–169.96
0.1951 0.0238 0.0003
0.48–11.40 0.94–28.35 3.60–211.78
0.2921 0.0589 0.0014
OR 1 1.94 5.40 22.95 1 2.05 6.22 27.01 1 1.78 3.68 8.64 1 1.35 3.30 13.86 1 1.24 2.63 5.54
CI
P
0.40–9.52 0.97–30.06 2.8–188.28
0.4138 0.0544 0.0035
0.41–10.37 0.99–38.91 2.93–248.71
0.3849 0.0508 0.0036
0.27–11.72 0.49–27.72 0.76–98.25
0.5481 0.2056 0.0823
0.26–7.08 0.56–19.49 1.62–118.79
0.7232 0.1880 0.0165
0.17–8.94 0.29–24.19 0.38–81.41
0.8313 0.3935 0.2119
* Adjusted for age, sex, serum triacylglycerol, total cholesterol, low-density lipoprotein cholesterol, and creatinine. CI, 95% confidence interval; OR, odds ratio; PLP, pyridoxal 5⬘-phosphate.
Our CAD group showed significantly higher plasma homocysteine concentration than the reference group. Approximately 20% (23 of 116) of subjects had a homocysteine concentration of at least 15 mol/L. The prevalence was lower than in the elderly population from the Framingham Study,10 indicating that approximately 29% of the elderly population had homocysteine concentrations higher than 14 mol/L. However, among our 23 hyperhomocystinemia subjects, 20 (87%) were CAD patients. Hyperhomocystinemia seems to be prevalent in patients with CAD. To understand the distribution of plasma homocysteine in our population, plasma homocysteine was divided into quartiles based on results from all subjects. We noticed that previous studies used percentile values from control samples to identify elevated homocysteine levels in patients with cardiovascular disease.10,15,24 Our reference subjects, however, were not a generally healthy population; they had a clinical profile severe enough to undergo cardiac catheterization. Therefore, we did not think it is reasonable to look at risks for CAD by quartiles of plasma homocysteine according to the distribution of the reference group. In the present study, we found that the risk of CAD was significantly increased in subjects with plasma homocysteine levels of at least 13.9 mol/L, in agreement with Joosten et al.25 who identified 13.9 mol/L (from the mean ⫾ 2 standard deviations among health subjects) as the cutoff value for an elevated homocysteine concentration. In the Framingham Heart Study, Selhub et al.10 used 14.0 mol/L as the cutoff value for hyperhomocystinemia. Selhub et al.24 also suggested that the risk of stenosis is increased in subjects with homocysteine concentration between 11.4 and 14.3 mol/L. Robinson et al.16 indicated the cutoff value for hyperhomocystinemia is associated with an increased likelihood of CAD. By this definition, they found that a homocysteine concentration of 14 mol/L produces a CAD OR of 4.8 (P ⬍ 0.001). Dalery et al.26 used a 90th percentile level (12.16 mol/L for women and 15.55 mol/L for
men) for homocysteine in control subjects to define an elevated homocysteine level in CAD patients. We might reconsider reducing the cutoff value to 14 mol/L or even less instead of 15 mol/L or higher for hyperhomocystinemia based on the findings from previous10,16,24 –26 and the present studies. Many potential confounding factors for CAD (i.e., age, sex, total cholesterol, and LDL cholesterol) have been associated with increased plasma homocysteine concentration.1,26,27 Decreased renal function is also associated with mild and moderate hyperhomocystinemia.28,29 Although our subjects were free of diabetes and liver and renal diseases, age, sex, total cholesterol, LDL cholesterol, triacylglycerols, and creatinine were adjusted to rule out any possible influences of those confounding factors on plasma homocysteine. In the past decade, much attention has been paid to the relations between plasma homocysteine and deficiencies of B vitamins. Ubbink et al.30 found that subjects with hyperhomocystinemia (⬎16.3 mol/L) have significantly lower plasma PLP, vitamin B12, and folate than do subjects with normal homocysteine concentration. Somewhat in disagreement with those of previous studies,10,16,26,30,31 we found no correlation between homocysteine and vitamin B6 or vitamin B12. Serum folate, however, was significantly, inversely correlated with plasma homocysteine concentration in the CAD and reference groups; subjects with elevated homocysteine concentration (ⱖ13.9 mol/L) had significantly lower serum folate concentrations than did subjects in the lowest two quartiles of plasma homocysteine concentration. In addition, our results showed that folate, not vitamin B6 or B12, predicts plasma homocysteine after adjusting those potential confounding factors for CAD. Previous studies32,33 have postulated that S-adenosylmethionine (an activator for the enzyme cystathionine -synthase) favors folate and vitamin B12– dependent homocysteine remethylation to methionine in the fasting state; vitamin B6 deficiency does not affect S-adenosylmethionine, and homocys-
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FIG. 1. Multivariate adjusted odds ratio for CAD according to the quartiles of plasma homocysteine concentration analyzed by multiple logistic regression. (A) No adjustment of vitamins. (B) Folate is adjusted. (C) Pyridoxal 5⬘-phosphate is adjusted. (D) Vitamin B12 is adjusted. (E) The B vitamins are adjusted. *P ⬍ 0.05; 95% confidence intervals are presented within parentheses. CAD, coronary artery disease.
teine is then removed by the remethylation pathway. Vitamin B6, therefore, mainly affects homocysteine after methionine loading. Thus, folate is considered a more important determinant of plasma homocysteine. Another possible explanation for our not observing an effect of vitamin B6 and B12 on homocysteine might be that our CAD and reference subjects had adequate vitamin B6 and B12 status, whereas their folate status was significantly lower in the highest two quartiles of plasma homocysteine. Plasma homocysteine thus was not affected by vitamin B6 and B12 but might accumulate due to the lower folate status. This idea is consistent with previous studies32–34 indicating that vitamin B6 supplementation does not affect homocysteine concentration in healthy subjects if they have normal vitamin B6 status. Our study supported findings from previous studies15,35 indicating that serum folate status is a much stronger determinant of plasma homocysteine than vitamins B6 and B12. In addition, folate intake also might be an important indicator of plasma homocysteine. Selhub et al.10 indicated that individuals with folate intakes up to 280 g/d nevertheless have elevated plasma homocysteine. Rimm et al.11 found that the lowest risk of CAD was among subjects with a folate intake above 400 g/d. We did not assess vitamin B6, vitamin B12, and folate intakes in the present
study, because there was a lack of information on the folate content for Chinese foods; therefore, the intake of folate could not be determined. Further study is warranted to study the amount of dietary folate that should be recommended to individuals with risk factors for CAD to reduce their plasma homocysteine concentrations and decrease their risks of CAD. In conclusion, there was a strong and inverse association between plasma homocysteine and serum folate concentration for individuals with risk factors for CAD. However, vitamin B6 and B12 had no effects on plasma homocysteine concentration in individuals with an adequate vitamin status.
REFERENCES 1. Kang SS, Wong PW, Cook HY, Norusis M, Messer JV. Protein-bound homocyst(e)ine. A possible risk factor for coronary artery disease. J Clin Invest 1986;77:1482 2. Stampfer MJ, Malinow MR, Willett WC, et al. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA 1992;268:877
Nutrition Volume 19, Numbers 7/8, 2003 3. Montalescot G, Ankri A, Chadefaux-Vekemans B, et al. Plasma homocysteine and the extent of atherosclerosis in patients with coronary artery disease. Int J Cardiol 1997;60:295 4. Boers GH, Smals AG, Trijbels FJ, et al. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med 1985;313:709 5. Taylor LM Jr, DeFrang RD, Harris EJ Jr, Porter JM. The association of elevated plasma homocyst(e)ine with progression of symptomatic peripheral arterial disease. J Vasc Surg 1991;13:128 6. Brattstro¨ m LE, Hardebp E, Hultberg BL. Moderate homocysteinemia—a possible risk factor for arteriosclerotic cerebrovascular disease. Stroke 1984;15:1012 7. Brattstro¨ m L, Lindgren A, Israelsson B, et al. Hyperhomocysteinaemia in stroke: prevalence, cause and relationships to type of stroke and stroke risk factors. Eur J Clin Invest 1992;22:214 8. Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocyst(e)ine as a risk factor for vascular disease: probable benefits of increasing folic acid intakes. JAMA 1995;274:1049 9. Wilcken DEL, Wilcken B. The pathogenesis of coronary artery disease: a possible role for methionine metabolism. J Clin Invest 1976;57:1079 10. Selhub J, Jacques PF, Wilson PWF, Rush D, Rosenberg IH. Vitamin status as primary determinants of homocysteinemia in an elderly population. JAMA 1993; 270:2693 11. Rimm EB, Willett WC, Hu FB, et al. Folate and vitamin B-6 from diet and supplements in relation to risk of coronary heart disease among women. JAMA 1998;279:359 12. Chu RC, Hall CA. The total serum homocysteine as an indicator of vitamin B-12 and folate status. Am J Clin Pathol 1988;90:446 13. Lindenbaum J, Savage DG, Stabler SP, Allen RH. Diagnosis of cobalamin deficiency: II. Relative sensitivities of serum cobalamin, methylmalonic acid, and total homocysteine concentrations. Am J Hematol 1990;34:99 14. Morrison HI, Schaubel D, Desmeules M, Wigle DT. Serum folate and risk of fatal coronary heart disease. JAMA 1996;275:1893 15. Verhoef P, Stampfer MJ, Buring JE, et al. Homocysteine metabolism and risk of myocardial infarction: relation with vitamin B6, B12, and folate. Am J Epidemiol 1996;143:845 16. Robinson K, Mayer EL, Miller DP, et al. Hyperhomocysteinemia and low pyridoxal phosphate. Common and independent reversible risk factors for coronary artery disease. Circulation 1995;92:2825 17. Chasan-Taber L, Selhub J, Rosenberg IH, et al. A prospective study of folate and vitamin B6 and risk of myocardial infarction in US physicians. J Am Coll Nutr 1996;15:136 18. McKinley MC, McNulty H, McPartlin J, et al. Low-dose vitamin B-6 effectively lowers fasting plasma homocysteine in healthy elderly persons who are folate and riboflavin replete. Am J Clin Nutr 2001;73:759 19. Brattstro¨ m L, Israelsson B, Norrving B, Bergqvist D, Tho¨ rne J. Impaired homocysteine metabolism in early-onset cerebral and peripheral occlusive arterial disease. Atherosclerosis 1990;81:51
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20. Giraud DW, Driskell JA, Setiawan B. Plasma homocysteine concentrations of Indonesian children with inadequate and adequate vitamin B-6 status. Nutr Res 2001;21:961 21. Araki A, Sako Y. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr 1987;422:43 22. Bates CJ, Pentieva KD, Matthews N, Macdonald A. A simple, sensitive and reproducible assay for pyridoxal 5⬘-phosphate and 4-pyridoxic acid in human plasma. Chin Chim Acta 1999;280:101 23. Food and Nutrition Board, Institute of Medicine. Dietary reference intakes. Thiamin, riboflavin, niacin, vitamin B-6, folate, vitamin B-12, pantothenic acid, biotin, and choline. Washington, DC: National Academy Press, 1998 24. Selhub J, Jacques PF, Bostom AG, et al. Association between plasma homocysteine concentrations and extracranial carotid-artery stenosis. N Eng J Med 1995; 332:286 25. Joosten E, van den Berg A, Riezler R, et al. Metabolic evidence that deficiencies of vitamin B-12 (cobalamin), folate, and vitamin B-6 occur commonly in elderly people. Am J Clin Nutr 1993;58:468 26. Dalery K, Lussier-Cacan S, Selhub J, et al. Homocysteine and coronary artery disease in French Canadian subjects: relation with vitamins B12, B6, pyridoxal phosphate, and folate. Am J Cardiol 1995;75:1107 27. Nygård O, Vollset SE, Refsum H, et al. Total plasma homocysteine and cardiovascular risk profile. The Hordaland Homocysteine Study. JAMA 1995; 274:1526 28. Arnadottir M, Brattstrom L, Simonsen O, et al. The effect of high-dose pyridoxine and folic acid supplementation on serum lipid and plasma homocysteine concentrations in dialysis patients. Clin Nephrol 1993;40:236 29. Hultberg B, Andersson A, Sterner G. Plasma homocysteine in renal failure. Clin Nephrol 1993;40:230 30. Ubbink JB, Vermaak WJH, van der Merwe A, Becker PJ. Vitamin B-12, vitamin B-6, and folate nutritional status in men with hyperhomocysteinemia. Am J Clin Nutr 1993;57:47 31. Robinson K, Arheart K, Refsum H, et al. Low circulating folate and vitamin B6 concentrations: risk factors for stroke, peripheral vascular disease, and coronary artery disease. Circulation 1998;97:437 32. Ubbink JB, van der Merwe A, Delport R, et al. The effect of a subnormal vitamin B-6 status on homocysteine metabolism. J Clin Invest 1996;98:177 33. Selhub J, Miller JW. The pathogenesis of homocystinemia: interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Am J Clin Nutr 1992;55:131 34. Bosy-Westphal A, Holzapfel A, Czech N, Mu¨ ller MJ. Plasma folate but not vitamin B12 or homocysteine concentrations are reduced after short-term vitamin B6 supplementation. Ann Nutr Metab 2001;45:255 35. Ubbink JB, Vermaak WJH, van der Merwe A, et al. Vitamin requirements for the treatment of hyperhomocysteinemia in humans. J Nutr 1994;124:1927
Homocysteine and Risk of Coronary Artery Disease: Folate Is the Important Determinant of Plasma Homocysteine Concentration Bor-Jen Lee, MD, Ping-Ting Lin, MS, Yung-Po Liaw, PhD, Sue-Joan Chang, PhD, Chien-Hsiung Cheng, MD, and Yi-Chia Huang, PhD From the Intensive Care Unit, Critical Care and Respiratory Therapy, Taichung Veterans General Hospital, School of Nutrition, the Department of Public Health, Chung Shan Medical University, and the Department of Biology, National Cheng Kung University, Taiwan, Republic of China OBJECTIVES: The purposes of this study were to study the effects of folate and vitamins B6 and B12 on plasma homocysteine concentration and to estimate the risks for coronary artery disease (CAD) according to quartiles of plasma homocysteine concentration. METHODS: The study was designed as a case-reference observational study. Case subjects (CAD group, n ⫽ 60) were identified by cardiac catheterization to have at least 70% stenosis of one major coronary artery; otherwise, patients were considered for a reference group (n ⫽ 60). Risk factors of cardiovascular disease were recorded, including age, sex, blood lipid profile, hypertension, smoking habits, and drinking habits. Plasma homocysteine, folate, pyridoxal 5⬘-phosphate, and vitamin B12 were measured. RESULTS: CAD subjects had significantly higher mean plasma homocysteine concentrations than did the reference subjects (13.9 ⫾ 4.9 versus 9.1 ⫾ 3.3 mol/L). There were no significant differences between groups with regard to the three B vitamins; however, mean serum folate concentrations for subjects in the highest two quartiles of plasma homocysteine concentration (10.8 –13.8 and ⱖ13.9 mol/L) were significantly lower than those for subjects in the lowest two quartiles (ⱕ8.0 and 8.1–10.7 mol/L). Plasma homocysteine was strongly inversely associated with serum folate in the CAD ( ⫽ ⫺0.166, P ⬍ 0.05), reference ( ⫽ ⫺0.178, P ⬍ 0.001), and pooled ( ⫽ ⫺0.190, P ⬍ 0.001) groups. Age, sex, other confounding factors, and B-vitamin–adjusted odds ratios were significantly increased in the highest quartile of homocysteine concentration (odds ration, 5.54; 95% confidence interval, 0.38 – 81.41). The elevation of 1 ng/mL in serum folate concentration was found to decrease plasma homocysteine by 0.166 mol/L. CONCLUSIONS: Serum folate, but not vitamin B6 or B12, was a strong predictor of plasma homocysteine; while all subjects had adequate B-vitamin status. Folate should be considered as a routine supplementation for individuals who have risk factors for CAD, even for individuals with adequate folate status. Nutrition 2003;19:577–583. ©Elsevier Inc. 2003 KEY WORDS: homocysteine, folate, vitamin B6, vitamin B12, risk factor, coronary artery disease
INTRODUCTION Many studies have shown that plasma homocysteine concentration are significantly higher in patients with coronary artery disease (CAD),1–3 peripheral vascular disease,4,5 and cerebral vascular disease.6,7 A large, prospective, 5-y study, the Physicians Health Study, found that physicians with elevated plasma homocysteine concentrations had a three-fold increase in the occurrence of myocardial infarction than did physicians who had normal plasma homocysteine concentrations.2 Boushey et al.8 indicated that the elevation of 5 mol/L in plasma homocysteine concentration increases the incidence of CAD by 60% to 80%. Thus, hyperhomocystinemia likely is an independent risk factor of CAD.2,8,9
This study was supported by the National Science Council (grant 90-2320B-040-019), Taiwan. Correspondence to: Yi-Chia Huang, PhD, School of Nutrition, Chung Shan Medical University, No 110 Sec 1 Chien-Kuo N. Rd, Taichung, Taiwan 402. E-mail: [email protected] Nutrition 19:577–583, 2003 ©Elsevier Inc., 2003. Printed in the United States. All rights reserved.
Many factors (e.g., age, enzyme deficiencies and mutations, vitamin deficiencies, disease, and drugs) are associated with mild and moderate hyperhomocystinemia; of particular interest are nutritional deficiencies in the vitamin cofactors that are required for homocysteine metabolism: folate and vitamins B12 and B6. Homocysteine is metabolized by two pathways. When methionine is in negative balance, homocysteine is remethylated to form methionine by a methionine-conserving remethylation pathway; this process requires methyltetrahydrofolate as a cosubstrate, methionine synthase, and vitamin B12 as a cofactor. Homocysteine, however, is directed to the transsulfuration pathway when methionine is in excess. During the transsulfuration reaction, homocysteine is irreversibly sulfur conjugated to serine by cystathionine -synthase and cystathionase to it convert to cystathionine and then to cysteine by the pyridoxal 5⬘-phosphate (PLP)– dependent enzymes. Therefore, deficiencies of folate, vitamin B12, or vitamin B6 may account for most cases of hyperhomocystinemia. A large cohort of subjects from the Framingham Study indicated that folate, vitamin B6, and vitamin B12 are important determinants of plasma homocysteine concentrations in a healthy population.10 The Nurses Health Study, with more than 80 000 nurses studied 0899-9007/03/$30.00 doi:10.1016/S0899-9007(02)01098-5
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over 14 y, showed that the risks of mortality and morbidity from myocardial infarction is inversely related to dietary intake of folate and vitamin B6.11 Most cross-sectional, retrospective, case-controlled, and prospective cohort studies8,12–16 have shown that folate and vitamin B12 intake and/or biochemical status strongly and negatively correlate with plasma homocysteine concentration. Thus, folate and vitamin B12 supplementation have the beneficial effect of lowering plasma homocysteine concentration and further reducing the risk of CAD. However, there was no consistent evidence showing the effect of vitamin B6 on plasma homocysteine concentration. Chasan-Taber et al.17 showed that plasma PLP is inversely correlated with homocysteine (r ⫽ ⫺0.29, P ⬍ 0.001) by using the participants in the Physicians Health Study. In a recent randomized, double-blind, placebo-controlled trial, vitamin B6 (1.6 mg/d) was given for 12 wk to 11 healthy elderly subjects after repletion with folic acid and riboflavin. Results showed that vitamin B6 supplementation significantly reduces plasma homocysteine concentration by 7.5%.18 In contrast, some studies have shown that vitamin B6 has no effect on plasma homocysteine concentration. Brattstro¨ m et al.19 studied homocysteine metabolism in 72 patients with occlusive arterial disease and found that plasma PLP was decreased in most patients; however, there was no correlation between plasma PLP and homocysteine concentration. Giraud et al.20 also found that plasma homocysteine concentration did not correlate with vitamin B6 status in 77 Indonesian children. Plasma homocysteine concentration was then suggested not to be an indicator of vitamin B6 status.20 Because most of these studies were done in healthy subjects or patients younger than 60 y with cardiovascular diseases, further studies are warranted to study the effect of B vitamins, especially of vitamin B6, on homocysteine concentration in elderly patients with CAD. Therefore, we investigated the effects of folate, vitamin B6, and vitamin B12 on plasma homocysteine concentration after adjusting risk factors for CAD and estimated the risks for CAD according to the quartiles of plasma homocysteine concentration.
glucose, serum creatinine, total serum cholesterol, high-density lipoprotein [HDL] cholesterol, low-density lipoprotein [LDL] cholesterol, triacylglycerol, alanine aminotransferase, and serum alkaline phosphatase), homocysteine, vitamin B6, vitamin B12, and folate status. Serum and plasma were prepared and then stored frozen (⫺20°C) until analysis. Risk factors of cardiovascular disease were recorded, including age, sex, blood lipid profile (total cholesterol ⱖ 200 mg/dL, HDL cholesterol ⬍ 35 mg/dL, LDL cholesterol ⱖ 130 mg/dL, triacylglycerol ⱖ 200 mg/dL), hypertension (blood pressure ⱖ 140/90 mmHg or currently taking antihypertensive drugs), smoking habits (smoker versus non-smoker, defined as no smoking for at least 3 wk before the study), and drinking habits (drinking, defined as ⬎100 mL/d, versus non-drinking). Biochemical Measurements Plasma homocysteine was measured by using high-performance liquid chromatography according to the method of Araki and Sako.21 The intra-assay and inter-assay of plasma homocysteine variabilities were 3.5% (n ⫽ 5) and 4.8% (n ⫽ 8), respectively. Plasma PLP was determined by high-performance liquid chromatography as previously described.22 The intra-assay and inter-assay of plasma PLP variabilities were 3.2% (n ⫽ 4) and 4.4% (n ⫽ 6), respectively. Serum folate and vitamin B12 were analyzed by using standard competitive immuno-chemiluminometric methods on a Chiron Diagnostics ACS:180 Automated Chemiluminescence System (Chiron Diagnostics Corporation, East Walpole, MA, USA). All analyses were performed in duplicate. Hyperhomocysteinemia was defined as a plasma homocysteine concentration at least 15 mol/L. Vitamin B12 and folate deficiencies were defined as serum concentrations below 169.4 pg/mL, and 2.8 ng/mL, respectively.23 Vitamin B6 deficiency was defined as plasma PLP concentration below 20 nmol/L.23 Statistical Analyses
A case-reference study was conducted from October 2000 to January 2002. Subjects were recruited from the cardiology clinic of the Taichung Veteran General Hospital, which is a 1359-bed teaching hospital in the central part of Taiwan. Patients suspected of having CAD underwent coronary angiography. Case subjects (CAD group, n ⫽ 60) were identified by cardiac catheterization to have at least 70% stenosis of one major coronary artery; otherwise, patients were considered for a reference group. The reference group (n ⫽ 60) consisted of subjects with no history of myocardial infarction and less than 70% occlusion in any coronary artery. All subjects with diabetes (defined by a history of antidiabetic drug use or a fasting plasma glucose concentration ⬎ 140 mg/dL) or liver or renal diseases (identified by serum creatinine and aspartate aminotransferase analyses) were excluded to minimize the influence of other cardiovascular risk factors. The use of medications was recorded. Informed consent was obtained from each subject. The study was approved by the Committee for Ethics of Chung Shan Medical University.
Data were analyzed with SAS 6.12 (SAS Institute, Cary, NC, USA). Differences in subjects’ demographic and health characteristics and the data of biochemical measurements between CAD and reference groups were analyzed by Student’s t test. For categorical response variables, differences between groups were assessed by Fisher’s exact test. Simple regression analyses were used to assess the association of plasma homocysteine concentration with B vitamins (folate, vitamin B6, and vitamin B12) and other risk factors for CAD. Multiple regression analysis with plasma homocysteine concentration as the dependent variable was used to examine the effect of folate, vitamin B6, and vitamin B12 on plasma homocysteine after adjustment for potential confounders and the other two B vitamins. One-way analysis of variance was used to compare differences in serum folate, plasma PLP, and serum vitamin B12 among the quartiles of plasma homocysteine. Adjusted odds ratios (ORs) with 95% confidence intervals (CI) for CAD were calculated from the logistic regression model according to the quartiles of plasma homocysteine concentration across subjects. ORs were computed with adjustment for CAD risk factors and for CAD risk factors plus the other two B vitamins. Statistical results were considered statistically significant at P ⬍ 0.05. Values presented in the text are means ⫾ standard deviation.
Experimental Protocol
RESULTS
MATERIALS AND METHODS Subjects
2
Body mass index (kg/m ) was calculated from height and weight measurements. Blood pressure was measured after a resting period of at least 5 min. Fasting venous blood samples (15 mL) were collected in Vacutainer tubes (Becton Dickinson, Rutherford, NJ, USA) containing an appropriate anticoagulant or no anticoagulant as required to determine some hematologic entities (i.e., plasma
Characteristics of Subjects Characteristics of CAD and reference subjects are shown in Table I. Subjects’ ages ranged from 36 to 92 y, with median ages of 73 y and 67.5 y for the CAD and reference groups, respectively. Unfortunately, we could not recruit age- and sex-matched control
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TABLE I.
TABLE III.
DEMOGRAPHIC AND HEALTH CHARACTERISTICS OF PATIENTS WITH CAD AND REFERENCE SUBJECTS*
SIMPLE LINEAR REGRESSION  OF PLASMA HOMOCYSTEINE WITH RISK FACTORS OF CAD AND B VITAMINS IN INDIVIDUAL AND POOLED CAD AND REFERENCE GROUPS*
CAD (n ⫽ 60)
References (n ⫽ 60)
54/6 71.6 ⫾ 9.7a 70.3 ⫾ 9.1a 165.1 ⫾ 6.2a 25.7 ⫾ 2.7 50.0 201.8 ⫾ 40.2a 47.3 ⫾ 11.5 127.5 ⫾ 35.7a 21.4 138.2 ⫾ 76.9a 38.3 131.1 ⫾ 13.3 74.4 ⫾ 7.4 1.3 ⫾ 0.3a 16.7 26.7
39/21 65.5 ⫾ 9.0b 64.1 ⫾ 9.9b 161.0 ⫾ 8.9b 24.8 ⫾ 3.7 23.3 173.4 ⫾ 36.6b 49.1 ⫾ 15.1 112.0 ⫾ 30.1b 3.3 87.2 ⫾ 43.9b 35.0 133.9 ⫾ 18.5 74.1 ⫾ 12.1 1.0 ⫾ 0.3b 26.7 31.7
Characteristics Male/female Age (y) Weight (kg) Height (cm) Body mass index (kg/m2) Hypercholesterolemia (%) Serum total cholesterol (mg/dL) Serum HDL cholesterol (mg/dL) Serum LDL cholesterol (mg/dL) Hypertriglyceridemia (%) Serum triacylglycerol (mg/dL) Hypertension (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Serum creatinine (mg/dL) Current smoker (%) Drinking (⬎100 mL/d) (%)
* Values are means ⫾ standard deviation. Hypercholesterolemia was defined as serum total cholesterol of at least 200 mg/dL or currently taking cholesterol-lowering drugs. Hypertriglyceridemia was defined as serum triacylglycerol of at least 200 mg/dL. Hypertension was defined as systolic blood pressure higher than 140 mmHg and/or diastolic blood pressure higher than 90 mmHg. Values within a row with different superscript letters are significantly different; P ⬍ 0.05. CAD, coronary artery disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
CAD (n ⫽ 60) Age (y) Sex BMI (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Smoking Drinking Serum total cholesterol (mg/dL) Serum LDL cholesterol (mg/dL) Serum HDL cholesterol (mg/dL) Serum triglyceride (mg/dL) Serum creatinine (mg/dL) Serum folate (mg/mL) Plasma vitamin B6 (nmol/L) Serum vitamin B12 (pg/mL)
0.176† ⫺1.425 ⫺0.150 0.062 ⫺0.035 1.565 ⫺0.167 ⫺0.030 ⫺0.039* ⫺0.036 0.010 9.853‡ ⫺0.166* ⫺0.018 ⫺0.013*
Reference (n ⫽ 60)
Pooled (n ⫽ 120)
0.055 2.094* 0.110 0.031 0.042 1.038 1.038 ⫺0.012 ⫺0.022 0.036 ⫺0.009 4.455† ⫺0.178‡ ⫺0.019 ⫺0.000
0.187‡ 0.374 0.167 0.013 ⫺0.017 0.576 ⫺0.365 0.001 ⫺0.014 ⫺0.000 0.018† 7.660‡ ⫺0.190‡ 0.029 ⫺0.002
* P ⬍ 0.05. † P ⬍ 0.01. ‡ P ⬍ 0.001. CAD, coronary artery disease; BMI, body mass index; HDL, highdensity lipoprotein; LDL, low-density lipoprotein; PLP, pyridoxal 5⬘phosphate.
and over one-fourth of CAD and reference subjects drank alcohol (⬎100 mL/d). Homocysteine and Vitamin Status
subjects. Patients with CAD were older than the reference subjects. Because we adjusted for age and sex when discussing the data, the difference was not expected to affect the results of this study. Both groups were of comparable body size. Patients with CAD had significantly higher serum total cholesterol, LDL cholesterol, and triacylglycerol. Although mean serum creatinine of CAD subjects was significantly higher than that of reference subjects, it was within the normal range, indicating a normal renal function for CAD subjects. With regard to smoking and drinking, 16.7% of CAD subjects and 26.7% of reference subjects smoked regularly
TABLE II. PLASMA TOTAL HOMOCYSTEINE AND B VITAMINS IN PATIENTS WITH CAD AND REFERENCE SUBJECTS*
Fasting plasma total homocysteine (mol/L) Serum folate (ng/mL) Plasma PLP (nmol/L) Serum vitamin B12 (pg/mL)
CAD† (n ⫽ 56)
References (n ⫽ 60)
P
13.9 ⫾ 4.9
9.1 ⫾ 3.3
⬍ 0.001
11.9 ⫾ 9.4 52.2 ⫾ 42.3 380.7 ⫾ 116.7
13.2 ⫾ 8.6 44.6 ⫾ 40.0 510.7 ⫾ 548.0
0.436 0.32 0.085
* Values are means ⫾ standard deviation. † Blood samples could not be obtained from four patients in the CAD group. CAD, coronary artery disease; PLP, pyridoxal 5⬘-phosphate.
Concentrations of plasma homocysteine and related B vitamins are shown in Table II. CAD subjects had significantly higher mean plasma homocysteine concentration than did reference subjects. Although the mean plasma homocysteine concentration was below 15 mol/L in both groups, the CAD group had a mean plasma homocysteine concentration (13.9 ⫾ 4.9 mol/L) very close to the cutoff value for hyperhomocystinemia. In addition, 35.7% (n ⫽ 20) of CAD patients and 5% (n ⫽ 3) of reference subjects had moderate hyperhomocystinemia (ⱖ15 mol/L). The CAD and reference groups had mean serum folate, plasma PLP, and serum vitamin B12 concentrations higher than the cutoff value. There were no significant differences between groups with regard to the three B vitamins. Associations With Homocysteine Simple regression analyses were performed to understand the relation between homocysteine plus B vitamins and risk factors for CAD; results are shown in Table III. Plasma homocysteine concentration was strongly and inversely associated with serum folate concentration in the CAD, reference, and pooled groups. There was no significant association between plasma homocysteine and plasma PLP in any group. The CAD group showed a negative association between plasma homocysteine and vitamin B12. A positive association was found in the CAD and pooled groups between plasma homocysteine and age or serum creatinine concentration. Plasma homocysteine correlated significantly with sex in the reference group, with LDL cholesterol in the CAD group, and with triacylglycerol in the pooled group. Other risk factors of CAD, including body mass index, blood pressure, smoking, drink-
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Nutrition Volume 19, Numbers 7/8, 2003 TABLE IV.
MULTIPLE LINEAR REGRESSION ANALYSIS WITH PLASMA HOMOCYSTEINE CONCENTRATION AS THE DEPENDENT VARIABLE AFTER ADJUSTING THE POTENTIAL CONFOUNDERS OF CAD AND THE OTHER TWO B VITAMINS* CAD (n ⫽ 56) Vitamins Serum folate (ng/mL) Plasma PLP (nmol/L) Serum vitamin B12 (pg/mL)
References (n ⫽ 60)
Pooled (n ⫽ 116)

P

P

⫺0.0419 ⫺0.00584 ⫺0.0104
0.472 0.606 0.028
⫺0.168 ⫺0.0203 ⫺0.0005
⬍0.001 0.040 0.488
⫺0.166 ⫺0.0065 0.0000
P ⬍0.001 0.452 1.0
* Linear regression coefficients express how the mean of plasma homocysteine concentration varies per unit increase in vitamins after adjusting for age, sex, serum total cholesterol, low-density lipoprotein cholesterol, triacylglycerol, creatinine, and the other two B vitamins. , regression coefficient; CAD, coronary artery disease; PLP, pyridoxal 5⬘-phosphate.
ing, total cholesterol, and HDL cholesterol, did not associate with homocysteine in any group. Relation of Plasma Homocysteine to Vitamin Status To study the effect of B vitamins on plasma homocysteine concentration, multiple regression analysis was performed on the individual and pooled groups (Table IV). Plasma homocysteine concentration was significantly and inversely affected by serum folate concentration after adjusting risk factors for CAD and the other two B vitamins in the reference and pooled groups. An elevation of 1 ng/mL in serum folate concentration decreased plasma homocysteine by 0.166 mol/L. Serum vitamin B12 was significantly correlated with plasma homocysteine concentration only in the CAD group. Plasma PLP, however, had no effect on plasma homocysteine concentration in the individual and pooled groups. Quartiles of Plasma Homocysteine Table V shows the distribution of B vitamins according to the quartiles of plasma homocysteine concentration across subjects. Mean serum folate concentrations for subjects in the first two quartiles of plasma homocysteine concentration (ⱕ8.0 and 8.1– 10.7 mol/L) were significantly higher than those for subjects in the third and fourth quartiles of plasma homocysteine concentration (10.8 –13.8 and ⱖ13.9 M/L). There were no significant differences in the distribution of plasma PLP and serum vitamin B12 concentrations among quartiles of plasma homocysteine concentration.
Odds Ratio for Coronary Artery Disease We calculated risks of CAD by quartiles of plasma homocysteine according to the distribution of all subjects (Table VI and Fig. 1). The age- and sex-adjusted ORs for all CAD and reference subjects increased significantly in the fourth quartile of homocysteine concentration (OR, 12.67; 95% CI, 2.71–59.32). In addition, after adjusting for other risk factors for CAD, homocysteine had a much greater effect on the risk of CAD (OR, 22.95; 95% CI, 2.8 – 188.28). Likewise, when serum folate, plasma PLP, and serum vitamin B12 were included in the regression model, the relation between CAD and homocysteine increased significantly in the third or fourth quartile of plasma homocysteine concentration after adjusting for age and sex. After additional adjustments for all risk factors for CAD plus individual or all B vitamins, the third or fourth quartile of plasma homocysteine showed a lower but still significant effect on the risk for CAD. The important observation was the inclusion of serum folate in the regression model: the OR was about 50 (95% CI, 7.37–334.38) in the fourth quartile of homocysteine concentration after adjusting for age and sex.
DISCUSSION The significant result of this study was that serum folate is the most important determinant of plasma homocysteine concentration if subjects have adequate B-vitamin status. Another important finding was that the cutoff value of hyperhomocystinemia might be considered to decrease to 14 mol/L or even less instead of 15 mol/L or higher.
TABLE V. LEVELS OF B VITAMINS IN ALL SUBJECTS ACCORDING TO QUARTILES OF PLASMA HOMOCYSTEINE CONCENTRATIONS ACROSS SUBJECTS* Plasma homocysteine category (mol/L)
Serum folate (ng/mL) Plasma PLP (nmol/L) Serum vitamin B12 (pg/mL)
ⱕ8.0 (n ⫽ 29)
8.1–10.7 (n ⫽ 30)
10.8–13.8 (n ⫽ 28)
ⱖ13.9 (n ⫽ 29)
17.6 ⫾ 10.5a 53.3 ⫾ 51.0 522.4 ⫾ 464.9
14.4 ⫾ 10.4a 47.3 ⫾ 30.1 527.3 ⫾ 595.6
9.5 ⫾ 6.0b 47.1 ⫾ 49.0 394.2 ⫾ 206.5
8.6 ⫾ 5.4b 45.4 ⫾ 32.5 344.6 ⫾ 148.4
* Values are means ⫾ standard deviation. Values within a row with different superscript letters are significantly different. PLP, pyridoxal 5⬘-phosphate.
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TABLE VI. MULTIVARIATE ADJUSTED ODDS RATIOS OF CORONARY ARTERY DISEASE ACCORDING TO THE QUARTILES OF PLASMA CONCENTRATION ANALYZED BY MULTIPLE LOGISTIC REGRESSION
Adjusted vitamin None
Folate
PLP
Vitamin B12
All
Age and sex adjusted
Quartiles of plasma homocysteine (mol/L)
OR
ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9 ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9 ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9 ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9 ⱕ8.0 8.1–10.7 10.8–13.8 ⱖ13.9
1 1.51 2.69 12.67 1 3.70 9.39 49.65 1 2.25 3.37 17.99 1 2.54 5.16 27.70 1 2.34 5.16 27.60
Age, sex, and other risk factors*
CI
P
0.47–4.82 0.85–8.51 2.71–59.32
0.4859 0.0929 0.0013
0.87–15.77 1.98–44.57 7.37–334.38
0.0767 0.0048 ⬍0.001
0.52–9.83 0.78–14.62 2.95–109.62
0.2806 0.1048 0.0017
0.62–10.42 1.24–21.40 4.51–169.96
0.1951 0.0238 0.0003
0.48–11.40 0.94–28.35 3.60–211.78
0.2921 0.0589 0.0014
OR 1 1.94 5.40 22.95 1 2.05 6.22 27.01 1 1.78 3.68 8.64 1 1.35 3.30 13.86 1 1.24 2.63 5.54
CI
P
0.40–9.52 0.97–30.06 2.8–188.28
0.4138 0.0544 0.0035
0.41–10.37 0.99–38.91 2.93–248.71
0.3849 0.0508 0.0036
0.27–11.72 0.49–27.72 0.76–98.25
0.5481 0.2056 0.0823
0.26–7.08 0.56–19.49 1.62–118.79
0.7232 0.1880 0.0165
0.17–8.94 0.29–24.19 0.38–81.41
0.8313 0.3935 0.2119
* Adjusted for age, sex, serum triacylglycerol, total cholesterol, low-density lipoprotein cholesterol, and creatinine. CI, 95% confidence interval; OR, odds ratio; PLP, pyridoxal 5⬘-phosphate.
Our CAD group showed significantly higher plasma homocysteine concentration than the reference group. Approximately 20% (23 of 116) of subjects had a homocysteine concentration of at least 15 mol/L. The prevalence was lower than in the elderly population from the Framingham Study,10 indicating that approximately 29% of the elderly population had homocysteine concentrations higher than 14 mol/L. However, among our 23 hyperhomocystinemia subjects, 20 (87%) were CAD patients. Hyperhomocystinemia seems to be prevalent in patients with CAD. To understand the distribution of plasma homocysteine in our population, plasma homocysteine was divided into quartiles based on results from all subjects. We noticed that previous studies used percentile values from control samples to identify elevated homocysteine levels in patients with cardiovascular disease.10,15,24 Our reference subjects, however, were not a generally healthy population; they had a clinical profile severe enough to undergo cardiac catheterization. Therefore, we did not think it is reasonable to look at risks for CAD by quartiles of plasma homocysteine according to the distribution of the reference group. In the present study, we found that the risk of CAD was significantly increased in subjects with plasma homocysteine levels of at least 13.9 mol/L, in agreement with Joosten et al.25 who identified 13.9 mol/L (from the mean ⫾ 2 standard deviations among health subjects) as the cutoff value for an elevated homocysteine concentration. In the Framingham Heart Study, Selhub et al.10 used 14.0 mol/L as the cutoff value for hyperhomocystinemia. Selhub et al.24 also suggested that the risk of stenosis is increased in subjects with homocysteine concentration between 11.4 and 14.3 mol/L. Robinson et al.16 indicated the cutoff value for hyperhomocystinemia is associated with an increased likelihood of CAD. By this definition, they found that a homocysteine concentration of 14 mol/L produces a CAD OR of 4.8 (P ⬍ 0.001). Dalery et al.26 used a 90th percentile level (12.16 mol/L for women and 15.55 mol/L for
men) for homocysteine in control subjects to define an elevated homocysteine level in CAD patients. We might reconsider reducing the cutoff value to 14 mol/L or even less instead of 15 mol/L or higher for hyperhomocystinemia based on the findings from previous10,16,24 –26 and the present studies. Many potential confounding factors for CAD (i.e., age, sex, total cholesterol, and LDL cholesterol) have been associated with increased plasma homocysteine concentration.1,26,27 Decreased renal function is also associated with mild and moderate hyperhomocystinemia.28,29 Although our subjects were free of diabetes and liver and renal diseases, age, sex, total cholesterol, LDL cholesterol, triacylglycerols, and creatinine were adjusted to rule out any possible influences of those confounding factors on plasma homocysteine. In the past decade, much attention has been paid to the relations between plasma homocysteine and deficiencies of B vitamins. Ubbink et al.30 found that subjects with hyperhomocystinemia (⬎16.3 mol/L) have significantly lower plasma PLP, vitamin B12, and folate than do subjects with normal homocysteine concentration. Somewhat in disagreement with those of previous studies,10,16,26,30,31 we found no correlation between homocysteine and vitamin B6 or vitamin B12. Serum folate, however, was significantly, inversely correlated with plasma homocysteine concentration in the CAD and reference groups; subjects with elevated homocysteine concentration (ⱖ13.9 mol/L) had significantly lower serum folate concentrations than did subjects in the lowest two quartiles of plasma homocysteine concentration. In addition, our results showed that folate, not vitamin B6 or B12, predicts plasma homocysteine after adjusting those potential confounding factors for CAD. Previous studies32,33 have postulated that S-adenosylmethionine (an activator for the enzyme cystathionine -synthase) favors folate and vitamin B12– dependent homocysteine remethylation to methionine in the fasting state; vitamin B6 deficiency does not affect S-adenosylmethionine, and homocys-
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Nutrition Volume 19, Numbers 7/8, 2003
FIG. 1. Multivariate adjusted odds ratio for CAD according to the quartiles of plasma homocysteine concentration analyzed by multiple logistic regression. (A) No adjustment of vitamins. (B) Folate is adjusted. (C) Pyridoxal 5⬘-phosphate is adjusted. (D) Vitamin B12 is adjusted. (E) The B vitamins are adjusted. *P ⬍ 0.05; 95% confidence intervals are presented within parentheses. CAD, coronary artery disease.
teine is then removed by the remethylation pathway. Vitamin B6, therefore, mainly affects homocysteine after methionine loading. Thus, folate is considered a more important determinant of plasma homocysteine. Another possible explanation for our not observing an effect of vitamin B6 and B12 on homocysteine might be that our CAD and reference subjects had adequate vitamin B6 and B12 status, whereas their folate status was significantly lower in the highest two quartiles of plasma homocysteine. Plasma homocysteine thus was not affected by vitamin B6 and B12 but might accumulate due to the lower folate status. This idea is consistent with previous studies32–34 indicating that vitamin B6 supplementation does not affect homocysteine concentration in healthy subjects if they have normal vitamin B6 status. Our study supported findings from previous studies15,35 indicating that serum folate status is a much stronger determinant of plasma homocysteine than vitamins B6 and B12. In addition, folate intake also might be an important indicator of plasma homocysteine. Selhub et al.10 indicated that individuals with folate intakes up to 280 g/d nevertheless have elevated plasma homocysteine. Rimm et al.11 found that the lowest risk of CAD was among subjects with a folate intake above 400 g/d. We did not assess vitamin B6, vitamin B12, and folate intakes in the present
study, because there was a lack of information on the folate content for Chinese foods; therefore, the intake of folate could not be determined. Further study is warranted to study the amount of dietary folate that should be recommended to individuals with risk factors for CAD to reduce their plasma homocysteine concentrations and decrease their risks of CAD. In conclusion, there was a strong and inverse association between plasma homocysteine and serum folate concentration for individuals with risk factors for CAD. However, vitamin B6 and B12 had no effects on plasma homocysteine concentration in individuals with an adequate vitamin status.
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