Elevated plasma homocysteine level is associated with ischemic stroke in Chinese hypertensive patients

EJINME-02714; No of Pages 7 European Journal of Internal Medicine xxx (2014) xxx–xxx

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European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim

Original Article

Elevated plasma homocysteine level is associated with ischemic stroke in Chinese hypertensive patients☆ Chang-yi Wang a,b, Zhong-wei Chen b, Tao Zhang b, Jun Liu a, Si-han Chen b, Sheng-yuan Liu b, Li-yuan Han c, Zhao-hui Hui d, Yu-ming Chen a,⁎ a

Guangdong Provincial Key Laboratory of Food, Nutrition and Health, School of Public Health, Sun Yat-sen University, Guangzhou, 510080, People's Republic of China Shenzhen Nanshan Center for Chronic Disease Control, Shenzhen, 518054, People's Republic of China Department of Preventive Medicine, Medical School of Ningbo University, Ningbo, 315211, People's Republic of China d Shenzhen Xili People's Hospital, Shenzhen, 518054, People's Republic of China b c

a r t i c l e

i n f o

Article history: Received 6 November 2013 Received in revised form 19 April 2014 Accepted 19 April 2014 Available online xxxx Keywords: Homocysteine Hypertension Ischemic stroke Coronary heart disease

a b s t r a c t Background: Accumulating data suggest that hyperhomocysteinemia is associated with the risk of ischemic stroke (IS) and coronary heart disease (CHD) in the general population, but the relationship remains unclear in hypertensive patients. We examined the association of total homocysteine (tHcy) with IS and CHD in hypertensive patients. Methods: A total of 5935 Chinese hypertensive patients were recruited in a community-based cross-sectional study from 60 communities in Shenzhen, China. Plasma tHcy was quantitatively measured using the enzyme cycle method. Conventional risk factors for IS and CHD were obtained through questionnaire interviews and physical examinations. We included cerebral infarction, embolism and small-vessel disease as IS; and myocardial infarction, angina pectoris, coronary revascularization, and cardiac arrest as CHD. IS and CHD were retrospectively adjudicated by specialists via interviews, hospital records or relevant tests. Results: Significantly higher values of tHcy were observed in IS patients than in non-IS controls among both men and women. Greater tHcy level was dose dependently associated with an increased risk of IS presence in women, men and them combined (p-trend: 0.002, 3.8 × 10−4 and 0.001). The odds ratios (95% CI) of IS for tHcy ≥ 30 (vs. b15) μmol/L were 2.84 (1.73–4.34) in men, 4.41 (1.62–9.15) in women, and 2.86 (1.72–4.75) in their combination after adjusting for other main risk factors of IS. We did not find any significant association between tHcy and presence of CHD after the adjustment for covariates. Conclusions: Plasma homocysteine level is positively associated with the presence of IS, but not CHD, in Chinese hypertensive patients. © 2014 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

1. Introduction Cardiovascular and cerebrovascular diseases are the leading causes of death in China, accounting for over 40% of all deaths [1]. The current data state that there is a positive association between total homocysteine (tHcy) and risk of hypertension [2,3], ischemic stroke (IS) [4–11] and coronary heart disease (CHD) [4,11–14], but interventional trials with folate supplementation have so far failed to verify the association [15]. A meta-analysis of prospective studies estimated that a reduction of 3 μmol/L tHcy would decrease the risk of CHD by 18% and stroke by

☆ This work was supported by Shenzhen Nanshan Bureau of Science and Technology (2010058). ⁎ Corresponding author at: Department of Medical Statistics & Epidemiology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, People's Republic of China. Tel.: +86 20 87330605; fax: +86 20 87330446. E-mail address: [email protected] (Y. Chen).

24% [16]. Eikelboom et al. [10] reported a strong association between increasing plasma tHcy and IS caused by artherosclerosis (particularly of the large arteries), but not cardioembolic or other etiologic subtypes. Li et al. [17] found that C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene was associated with increased risk of cerebral thrombotic stroke in Chinese. These studies suggested different etiologic reasons and polymorphisms of MTHFR gene might change the susceptibility of IS and the tHcy-IS association However, due to the limited sample size in their study, the effect of tHcy on IS and CHD remains inconclusive in Chinese. Elevated tHcy is known to increase oxidative stress, diminish vasodilation, stimulate the proliferation of vascular smooth muscle cells, alter the elastic properties of the vascular wall and thus increase the risk for atherosclerosis [18]. Atherosclerosis caused by hypertension is the most important risk factor for IS and CHD. Graham et al. [19] found that the odds ratios of vascular disease in male, were 2.2 for the highest quartiles (versus remainder) of tHcy, 3.9 for hypertension, and over 10.0 for both. Bogdanski et al. [20] found that tHcy might increase carotid

http://dx.doi.org/10.1016/j.ejim.2014.04.011 0953-6205/© 2014 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Wang C, et al, Elevated plasma homocysteine level is associated with ischemic stroke in Chinese hypertensive patients, Eur J Intern Med (2014), http://dx.doi.org/10.1016/j.ejim.2014.04.011

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C. Wang et al. / European Journal of Internal Medicine xxx (2014) xxx–xxx

2.2. Questionnaire interview and examination

6203 patients screened 212 were excluded

65 secondary hypertension 33 hemorrhagic stroke 31 had IS/CHD over 2 years 22 liver or kidney failure 8 cancer 53 user of vitamin B6 or B12

5991 eligible patients invited

56 refused

5935 completed the interviews and tests

A structured interview was used to collect demographic and socioeconomic information, and information about smoking, alcohol drinking, leisure and occupational physical activities, a detailed history of hypertension and medications used, family history of IS and CHD, and other co-morbidities. Face-to-face interviews were conducted by trained physicians in the CHSC. Both leisure and occupational physical activity levels were assessed, as did in previous studies [22]. Drinking categories were determined according to the content of National Institute on Alcohol Abuse and Alcoholism (NIAAA) [23]. In addition, the Zung self-rating depression scale (ZSDS) was used to assess their depression status [24]. Depression was defined as the standard score of ZSDS ≥50 in Chinese. Duration of hypertension was calculated from the date of hypertension diagnosis to IS/CHD diagnosis for the IS or CHD cases, or to the date of visit for non-cases. All participants had confirmed hypertension of 5.1 (mean) (range: 0–21) years before the IS/CHD. Systolic and diastolic blood pressures were measured using a standard mercury sphygmomanometer on the right arm of seated participants after a 5-minute rest. Height (no shoes, nearest 0.1 cm), weight (light indoor clothes, nearest 0.1 kg), and waist and hip circumferences were also determined. Body mass index (BMI) was calculated as weight (kg)/height (m2).

Fig. 1. Flowchart of participant selection. IS: ischemic stroke; CHD: coronary heart disease.

2.3. Biochemical measurement

artery intima–media thickness by reducing the number of endothelial progenitor cells in patients with newly diagnosed hypertension. These results suggested that the effect of tHcy on IS and CHD may be different in hypertensive patients from those in healthy individuals. However, limited studies examined the association in hypertensive patients. Therefore, we investigated the association of plasma tHcy with the risk of IS and CHD with a cross-sectional study in 5935 Chinese hypertensive patients.

2. Material and methods

Fasting blood samples were drawn from the antecubital vein into siliconized tubes. Special care was taken with samples for homocysteine analysis. The tubes were immediately placed in an ice container and transferred to the Clinical Laboratory of Chronic Diseases Hospital in the Nanshan District. Blood samples for all of the analyses were centrifuged within 15 minutes at room temperature at 3000 rpm. All of the tHcy analyses were carried out within 4 h. Additional plasma samples were stored at −70 °C for further analysis. 2.3.1. Lipid profile The plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL), glucose, and triglycerides (TG) were quantitatively measured using enzymatic methods by the automatic biochemical analyzer HITACH 7080.

2.1. Subjects This cross-sectional study recruited 5935 Chinese hypertensive patients aged over 20 years from 60 community health service centers (CHSC) selected using a two-stage sampling method in Nanshan district, Shenzhen, China from April 2010 to September 2011 (Fig. 1). Eight sub-districts were selected in Nanshan district, and six to eight communities were then selected from each sub-district using a simple random procedure according to the sequence of computer-generated random numbers. All the participants with hypertension diagnosed in one of five collaborating hospitals according to at least three blood pressure tests at different time and registered in the electric information system of CHSC. They were invited to participate in the study by physicians through their phone numbers or family addresses in the electric information if they were eligible. We collected the data through questionnaire interview, examination, biochemical measurement, clinical records and relevant tests. Participants were required to be native Chinese who had lived in Shenzhen for over 6 months. The hypertensive patients had been diagnosed according to definition of values ≥ 140 mm Hg SBP and/or ≥ 90 mm Hg DBP [21] and were registered in a CHSC by their family address. The exclusion criteria included secondary hypertension, hemorrhagic or cardio embolic stroke, liver and kidney failure, cancer, pregnancy, a history of IS or CHD over 2 years, and being a user of folic acid, vitamin B6 or vitamin B12. Written informed consent was obtained from all patients. The ethical committees of the collaborating hospitals approved the study.

2.3.2. Uric acid, creatinine, and homocysteine The serum level of uric acid (UA) was determined quantitatively with the uricase method, the creatinine was determined quantitatively with the Jaffe reaction method, and the automatic biochemical analyzer (HITACH 7080) was used to measure the total homocysteine level in serum by enzymatic cycling assay. 2.4. Case definition and adjudication 2.4.1. Ischemic stroke All participants answered a stroke symptoms questionnaire, which included questions about changes in vital status, neurological and cardiac symptoms, and all intervals of hospitalizations. We included cerebral infarction, embolism and small-vessel disease as IS [25]. The stroke subtypes (ischaemic, intracerebral haemorrhagic, and subarachnoid haemorrhagic) were determined independently adjudicated retrospectively by two neurologists (Z.W.C. and Z.H.H.) using a standardized manner based on the clinical assessment and neuroimaging (CT or MRI), without knowing the level of homocysteine. We excluded patients with transient ischemic attacks, intracerebral haemorrhagic or subarachnoid haemorrhagic stroke. 2.4.2. Coronary heart disease We included a history of myocardial infarction, angina pectoris, coronary revascularization, and cardiac arrest as CHD [4]. CHD was

Please cite this article as: Wang C, et al, Elevated plasma homocysteine level is associated with ischemic stroke in Chinese hypertensive patients, Eur J Intern Med (2014), http://dx.doi.org/10.1016/j.ejim.2014.04.011

C. Wang et al. / European Journal of Internal Medicine xxx (2014) xxx–xxx

ascertained by means hospital records, interviews, exercise tests, angiography, and any other relevant evidence that helped the diagnosis and retrospectively confirmed by two cardiologists (TZ and SHC). 2.5. Statistical analysis Study participants were classified into normal (b 15 μmol/L), moderate (15–30 μmol/L) and intermediate-to-severe (≥30 μmol/L) hyperhomocysteinemia according to clinical cutoffs of plasma tHcy [26]. For group comparisons, t-tests/ANOVA was used for the continuous variables with a normal distribution; Mann–Whitney U test was used for the others with skewed distributions; and chi-square tests/logistic regression analysis was used for the categorical variables. The odds ratios (OR) and 95% confidence intervals (95% CI) of IS and CHD were estimated according to the homocysteine levels with logistic regression models. We estimated the p values for trend by using the median tHcy values of tHcy categories. The interactions between homocysteine levels and subgroups stratified by age, sex, depression, family history of stroke/CHD, years of hypertension, antihypertensive medication or SBP were examined in the logistic regression model. OR for each 5-μmol/L increment was also estimated using homocysteine as a continuous variable. The logistic model performance was assessed using the area under curve (AUC) by receiver operating characteristic (ROC) analysis. In the multivariate models, we adjusted for the major risk factors of stroke or CHD as the covariates. Two logistic regression models were used to assess the association between homocysteine and IS/CHD. In addition, we

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calculated the net reclassification improvement (NRI) from multiple logistic models with and without tHcy levels according to Pencina et al. [27]. Reclassification tables were constructed using b5%, 5%-10%, 10%– 20%, and ≥ 20% risk categories based on the study by Veeranna et al. [28]. The multiple imputation (MI) method was used to deal with the missing data in 197 patient records with different missing values. MI is commonly used and flexible when multiple variables have missing values [29]. We used the Markov Chain Monte Carlo (MCMC) method of MI procedure to make the imputed data set have a monotone missing pattern through 20 imputations. The regression method was used for all continuous variables and the logistic method was used for all class variables. All of the probability values for the statistical significance tests were two tailed. All of the analyses were performed with the use of SAS 9.1 software (SAS Institute Inc., Cary, NC, USA).

3. Results Among 5935 participants, 2928 were male (49.33%) and 3007 were female (50.67%). The subjects had a mean (SD) age of 59.0 (12.1) years. In both men and women, the tHcy levels were higher in patients who ate less fruit, used antihypertensive drugs or drunk alcohol. In both men and women, higher tHcy concentrations were associated with older age, longer time of hypertension, and higher levels of SBP, UA and creatinine (Table 1).

Table 1 Characteristics of patients by categories of plasma homocysteinea. Characteristics

Total homocysteine, μmol/L Median Range Education, % bMiddle School Middle/High School ≥College Smoking status, % Never Past Current Alcohol consumptionb, % Never Light Moderate Physical activityc, % Low Moderate High Diabetes, % Fruit intake, ≥200 g/d, % Depression, % Antihypertensive drugs, 10−6 Age, y, mean (SD) Years of hypertension, y, mean (SD) SBP, mm Hg, mean (SD) DBP, mm Hg, mean (SD) Body mass index, kg/m2, mean (SD) Cholesterol, mmol/L, mean (SD) LDL cholesterol, mmol/L, mean (SD) Uric acid, μmol/L, mean (SD) Triglycerides, mmol/Ld Glucose, mmol/L, mean (SD) Creatinine, μmol/L, mean (SD)

Total homocysteine (men)

Total homocysteine (women)

b15 (n = 1568)

15–30 (n = 1122)

≥30 μmol/L (n = 238)

12.2 2.2–14.9

18.1 15.0–29.9

43.5 30.0–184.8

7.0 68.8 24.3

7.5 67.1 25.4

12.6 66.8 20.6

73.0 3.5 24.5

73.1 2.7 24.2

67.7 2.2 30.2

57.2 29.0 13.8

61.8 21.8 16.5

55.9 28.2 16.0

8.0 75.3 16.8 8.3 75.2 28.1 78.6 55.8 (12.4) 4.6 (3.2) 133 (14) 85 (10) 24.8 (2.9) 4.93 (0.98) 2.95 (0.76) 370 (88) 1.62 (1.13–2.35) 5.71 (1.34) 85.2 (11.8)

6.7 75.2 18.1 7.6 67.7 26.5 85.0 60.6 (12.7) 5.7 (3.3) 134 (15) 84 (11) 24.6 (2.9) 4.89 (0.95) 2.9 (0.77) 386 (96) 1.54 (1.10–2.26) 5.69 (1.51) 93.9 (21.2)

8.0 77.3 14.7 6.3 63.8 30.3 81.1 56.7 (16.0) 5.1 (3.4) 136 (17) 85 (10) 24.7 (2.9) 4.83 (0.99) 2.91 (0.78) 393 (103) 1.62 (1.19–2.20) 5.47 (1.24) 96 (21.0)

P

b15 (n = 2504)

15–30 (n = 463)

≥30 μmol/L (n = 40)

10.7 2.6–14.9

17.4 15–29.8

38.7 31.1–83.0

5.1 66.7 28.2

6.1 68.5 25.5

12.5 55.0 32.5

98.6 0.2 1.2

98.1 0.3 1.6

95.0 0.8 4.2

88.7 7.2 4.1

90.9 3.9 5.2

95.0 5.0 0.0

6.2 74.1 19.8 8.4 81.4 28.1 78.4 59.2 (10.7) 4.7 (3.0) 133 (15) 83 (10) 24 (3.1) 5.29 (1.06) 3.07 (0.83) 304 (80) 1.51 (1.11–2.17) 5.62 (1.22) 66 (10.1)

6.1 75.2 18.8 5.8 76.7 33.7 84.9 65.6 (11.0) 7.9 (5.1) 135 (17) 81 (11) 23.9 (3.2) 5.25 (1.08) 3.02 (0.82) 339 (100) 1.57 (1.14–2.26) 5.53 (1.04) 78.7 (22.9)

10.0 60.0 30.0 10.0 70.0 25.0 85.0 65.5 (10.8) 7.6 (5.2) 137 (19) 83 (11) 24.1 (3.2) 4.87 (1.03) 2.75 (0.77) 319 (94) 1.54 (1.20–2.19) 5.41 (0.87) 79.4 (28.8)

0.028

0.134

0.201

0.134

0.001

0.043

0.525

0.516 2.1 × 10−6 0.414 2.0 × 10−4 2.6 × 10−21 4.6 × 10−4 2.1 × 10−4 0.229 0.118 0.256 0.218 1.6 × 10−6 0.373 0.045 4.3 × 10−45

p

0.345

0.152 0.014 0.045 0.004 1.5 × 10−32 4.8 × 10−18 4.1 × 10−4 0.037 0.879 0.038 0.028 1.5 × 10-15 0.700 0.225 8.3 × 10-79

SBP = systolic blood pressure; DBP = diastolic blood pressure. a Categorical variables are described by % and evaluated by chi-square tests; continuous variables were described by means (SD) and examined by analysis of variance. b Never, light and moderate drinkers: men: 0, b7 and ≥7 drinks/week; women: 0, b4 and ≥4 drinks/week. c Low, moderate and high physical activity: b1 time, 1–3 times and N3 times leisure physical activities per week. d Medians (quartile range), analyzed using nonparametric tests.

Please cite this article as: Wang C, et al, Elevated plasma homocysteine level is associated with ischemic stroke in Chinese hypertensive patients, Eur J Intern Med (2014), http://dx.doi.org/10.1016/j.ejim.2014.04.011

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C. Wang et al. / European Journal of Internal Medicine xxx (2014) xxx–xxx

Table 2 Plasma total homocysteine levels and prevalence of hyperhomocysteinemia (≥15 μmol/L) by sex. Men N

Women Homocysteine, μmol/L Median (quartile range)

Ischemic stroke Non-cases Cases Coronary heart disease Non-cases Cases

2928

14.6 (12.0–18.7)

2817 111

14.5 (12.0–18.5) 17.3 (13.1–25.7)

2813 115

14.6 (12.0–18.6) 14.7 (12.2–20.0)

Hyperhomocysteinemia p

n (%)

p

1360 (46.5) 1.3 × 10−4

Homocysteine, μmol/L Median (quartile range)

3007

11.2 (9.5–13.7)

2916 91

11.2 (9.5–13.6) 12.7 (10.8–15.0)

2852 155

11.2 (9.5–13.6) 11.9 (10.2–14.9)

3.5 × 10−4 1290 (45.8) 70 (63.1)

0.528

p 3.2 × 10−6

0.937 1307 (46.5) 53 (46.1)

The median tHcy and the prevalence of hyperhomocysteinemia were significantly higher in IS subjects than in non-IS subjects, but no significant differences were noted between CHD and non-CHD subjects (Table 2). Both the univariate and multivariate analyses showed dosedependent relations between serum tHcy and IS risk. After adjusted for age, sex (for total subjects), education, depression, smoking, alcohol consumption, body mass index (BMI), physical activity, diabetes, family history of stroke (or CHD), years of hypertension, and antihypertensive medication, the odds ratios (95% CIs) for IS in the highest tHcy category (≥30 μmol/L), comparing the normal levels of tHcy, were 2.84 (1.73– 4.34) in men, 4.41 (1.62–9.15) in women, and 2.86 (1.72–4.75) in total. The corresponding odds ratios (95% CI) for IS per 5 μmol/L increase in tHcy were 1.10 (1.05–1.16), 1.11 (1.02–1.23), and 1.10 (1.05–1.15) in men, women, and both combined, respectively. Similar

N

0.003

results were obtained when present systolic blood pressure, serum cholesterol, LDL, triglycerides and glucose were further adjusted for. The crude risk of CHD tended to increase but the multivariate-adjusted risk tended to decrease with an increase in tHcy. However, no significant OR was observed (all p N 0.05) (Table 3). Adding tHcy in the model, the logistic model performance (AUC of ROC) substantially improved in the model 1 (from 0.704 to 0.739) and model 2 (from 0.702 to 0.744) in the identification of IS in women, but ameliorated little (AUC increases b 0.01) in men for IS. Upon examining the reclassification properties of tHcy for IS, we observed an improvement in the net risk stratification (NRI = 0.072, p b 0.05). Almost no improvement was observed for the CHD identification (data not shown).

Table 3 Odds ratios (95% CI) of IS and CHD for each levels of plasma homocysteine a. Total homocysteine (tHcy)

Ischemic stroke Male Non-case/case, n Crude Model 1 Model 2 Female Non-case/case, n Crude Model 1 Model 2 Total Non-case/case, n Crude Model 1 Model 2 Coronary heart disease Male Non-case/case, n Crude Model 1 Model 2 Female Non-case/case, n Crude Model 1 Model 2 Total Non-case/case, n Crude Model 1 Model 2

OR per 5 μmol/L tHcy

p-Trend b

b15 μmol/L (referent)

15–30 μmol/L

≥30 μmol/L

1527/41 1.00 1.00 1.00

1073/49 1.70 (1.12–2.59) 1.32 (0.85–2.04) 1.30 (0.84–2.02)

220/18 3.05 (1.72–5.40) 2.84 (1.73–4.34) 2.61 (1.70–4.10)

1.09 (1.04–1.14) 1.10 (1.05–1.16) 1.10 (1.05–1.16)

6.4 × 10−6 2.8 × 10−4 3.8 × 10−4

2436/68 1.00 1.00 1.00

442/21 1.70 (1.03–2.81) 1.13 (0.67–1.92) 1.15 (0.68–1.96)

35/5 5.12 (1.95–13.47) 4.41 (1.62–9.15) 4.50 (1.64–9.59)

1.20 (1.06–1.35) 1.11 (1.02–1.23) 1.12 (1.02–1.24)

3.1 × 10−4 0.003 0.002

3963/109 1.00 1.00 1.00

1515/70 1.68 (1.24–2.28) 1.20 (0.87–1.68) 1.19 (0.86–1.66)

255/23 3.28 (2.06–5.23) 2.86 (1.72–4.75) 2.87 (1.73–4.79)

1.10 (1.06–1.15) 1.10 (1.05–1.15) 1.10 (1.05–1.15)

9.9 × 10−8 9.3 × 10−4 0.001

1506/62 1.00 1.00 1.00

1078/44 0.99 (0.67–1.47) 0.73 (0.49–1.10) 0.72 (0.48–1.09)

229/9 0.96 (0.47–1.95) 0.78 (0.38–1.63) 0.75 (0.36–1.56)

1.00 (0.93–1.07) 0.98 (0.90–1.07) 0.98 (0.89–1.07)

0.910 0.194 0.154

2386/118 1.00 1.00 1.00

430/33 1.55 (1.04–2.31) 0.89 (0.58–1.35) 0.88 (0.57–1.34)

36/4 2.25 (0.79–6.42) 1.37 (0.46–4.10) 1.34 (0.45–4.03)

1.17 (1.05–1.3) 1.02 (0.89–1.17) 1.01 (0.88–1.16)

0.012 0.868 0.812

3892/180 1.00 1.00 1.00

1508/77 1.10 (0.84–1.45) 0.81 (0.61–1.09) 0.80 (0.59–1.074)

265/13 1.06 (0.60–1.89) 0.94 (0.51–1.72) 0.91 (0.49–1.67)

1.01 (0.96–1.07) 1.00 (0.93–1.07) 0.99 (0.92–1.06)

0.540 0.293 0.233

a

The categories of 30–100 μmol/L and ≥100 μmol/L were combined due to limited number of participants. Estimated using the category numbers (1, 2 and 3) as a continuous variable, the same for those values in Table 4. Model 1: adjusted for age, sex, education, smoking, alcohol consumption, BMI, physical activity, diabetes, depression, family history of stroke (or CHD), years of hypertension, and antihypertensive medication use. Model 2: further adjusted for present SBP, serum cholesterol, glucose, triglycerides, and LDL. b

Please cite this article as: Wang C, et al, Elevated plasma homocysteine level is associated with ischemic stroke in Chinese hypertensive patients, Eur J Intern Med (2014), http://dx.doi.org/10.1016/j.ejim.2014.04.011

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Total Hyperhomocysteinemia n (%)

Homocysteine, μmol/L

N p

Hyperhomocysteinemia

Median (quartile range)

503 (16.7)

5935

12.7 (10.5–16.1)

5733 202

12.7 (10.4–16.0) 14.5 (11.8–19.8)

5665 270

12.7 (10.5–16.1) 13.1 (10.7–16.8)

p 1.5 × 10–8

0.027 480 (16.5) 23 (25.3)

5.0 × 10−6

0.145

No significant interactions between homocysteine and the above stratified factors on the presence of IS and CHD were found (Table 4). 4. Discussion In this community-based cross-sectional study, we found that moderately elevated serum tHcy was significantly associated with higher risk of IS among Chinese hypertensive patients. The association did not alter substantially after adjustment for cardiovascular risk factors.

p

1770 (30.9) 93 (46.0)

0.014 466 (16.3) 37 (23.9)

n (%) 1863 (31.4)

0.481 1773 (31.3) 90 (33.3)

In this study, the tHcy-IS association was independent of a number of risk factors (age, education, alcohol intake, smoking, physical activity, depression and cardiometabolic factors). Most of these factors are well-established risk factors of CVD in previous studies [30]. In nonhypertensive individuals, elevated plasma tHcy levels seem to be a stronger risk factor for IS [4–6,31], in which the odds ratios ranged between 2.0 and 4.4. A nested case–control study reported that the multivariate-adjusted odds ratio (95% CI) comparing the extreme quartile (≥ 11.0 vs. b7.0 μmol/L) of tHcy for IS was 3.89 (1.60 to 9.46) in 11846 Japanese [31]. Similar result was observed

Table 4 Multivariate-adjusted odds ratios of IS and CHD by plasma homocysteine levels in subgroups. OR (95% CI) by total homocysteine (μmol/L) b15 (referent) Ischemic stroke (IS) Age, y b60 1.00 ≥60 1.00 Depression No 1.00 Yes 1.00 Family history of stroke No 1.00 Yes 1.00 Years of hypertension, y b5 1.00 ≥5 1.00 Antihypertensive medication use No 1.00 Yes 1.00 Systolic blood pressure, mm Hg 140–160 1.00 160–180 1.00 ≥180 1.00 Coronary heart disease (CHD) Age, y b60 1.00 ≥60 1.00 Depression no 1.00 yes 1.00 Family history of CHD No 1.00 Yes 1.00 Years of hypertension, y b5 1.00 ≥5 1.00 Antihypertensive medication use No 1.00 Yes 1.00 Systolic blood pressure, mm Hg 140–160 1.00 160–180 1.00 ≥180 1.00

p-Trend

15–30

≥30

1.56 (0.74–3.26) 1.18 (0.82–1.71)

2.77 (0.87–8.81) 3.40 (1.92–6.01)

0.067 0.001

1.36 (0.89–2.07) 1.16 (0.68–1.98)

4.32 (2.41-7.74) 1.25 (0.40–3.94)

3.6 × 10−5 0.549

1.21 (0.85–1.74) 2.01 (0.86–4.73)

3.11 (1.81–5.33) 14.12 (1.86–107.39)

0.009 0.012

1.29 (0.60–2.78) 1.25 (0.86–1.80)

5.16 (2.08–12.76) 2.73 (1.46–5.11)

0.003 0.007

0.56 (0.14–2.20) 1.32 (0.94–1.85)

1.70 (0.31–9.53) 3.39 (1.99–5.77)

0.997 1.0 × 10−4

1.12 (0.63–2.01) 1.14 (0.67–1.93) 1.35 (0.72–2.55)

3.44 (1.31–9.01) 1.98 (0.83–4.77) 4.56 (1.86–11.20)

0.079 0.202 0.005

1.05 (0.53–2.07 0.85 (0.62–1.18)

1.18 (0.34–4.11) 0.93 (0.46–1.88)

0.797 0.429

0.85 (0.58–1.25) 0.97 (0.61–1.55)

1.02 (0.48–2.14) 0.90 (0.30–2.73)

0.608 0.841

0.89 (0.64–1.24) 0.76 (0.37–1.57)

0.98 (0.48–2.00) 1.21 (0.32–4.56)

0.609 0.774

0.87 (0.46–1.65) 0.88 (0.63–1.23)

0.63 (0.15–2.74) 1.16 (0.58–2.29)

0.498 0.784

0.76 (0.19–3.06) 0.91 (0.67–1.24)

NA. 1.09 (0.59–2.02)

0.460 0.819

1.37 (0.83–2.23) 0.70 (0.44–1.14) 0.67 (0.38–1.18)

1.88 (0.64–5.56) 0.47 (0.14–1.57) 1.19 (0.45–3.14)

0.126 0.077 0.532

p-Interaction

0.98

0.15

0.38

0.35

0.70

0.66

0.18

0.66

0.50

0.97

0.40

0.13

Covariates adjusted: see Model 2 in Table 3, except for the stratified variables. NA: not available due to limited sample size.

Please cite this article as: Wang C, et al, Elevated plasma homocysteine level is associated with ischemic stroke in Chinese hypertensive patients, Eur J Intern Med (2014), http://dx.doi.org/10.1016/j.ejim.2014.04.011

6

C. Wang et al. / European Journal of Internal Medicine xxx (2014) xxx–xxx

in another nested case–control study of 80 age- and sex- matched pairs from Israel [4]. In our study, we found that serum tHcy is strongly associated with IS in hypertensive patients, who are prone to strokes. The prevalence of hyperhomocysteinemia was 31.4% in our study population, which was higher than that of their healthy counterparts [32]. Previous studies showed that hyperhomocysteinemia was associated with smoking, alcohol consumption, physical inactivity, higher blood pressure and serum creatinine, lower plasma concentrations of folate, vitamin B12, and vitamin B6 [32]. Enalapril may also increase in plasma tHcy among the hypertensive patients [33]. Plasma tHcy levels were higher in Chinese than in American [34], which may be due to more folic acid fortified food in the United States [35]. In consistent with the study by Graham et al. [19], we found that the tHcy-related risk of IS tended to be higher in women than in men. Estrogens could decrease tHcy levels [36] through formation of estrogen–homocysteine conjugates [37]. Table 1 showed women in higher quartiles had older ages, and might have lower level of estrogens. In our study, we did not observed significant association between tHcy and CHD in hypertensive participants. Similar results were found by a case–control study [38] and a prospective cohort study [39] in healthy individuals. However, many other studies found that elevated plasma tHcy was associated with greater risk of CHD in nonhypertensive individuals. A prospective cohort study with 2009 participants found that tHcy was positively associated with risk of CVD events and death [12]. Another 24-year prospective study revealed that tHcy was an independent risk factor for acute myocardial infarction in 1368 women in Gothenburg [13]. These conflicting results may be due to the differences in the major CVD, which is CHD in the Western populations, but is stroke in Chinese [34]. A randomized trial showed that multivitamin supplementation significantly decreased the mortality of stoke [40], but not CHD in Chinese [34]. The association of tHCY-IS and tHCY-CHD might also be affected by the polymorphisms of MTHFR gene. TT genotype of the MTHFR C677T gene was associated with greater risk of IS [16], but not CHD [41], while the T allele is more frequent in Asians than in individuals of European descent [16]. In addition, the definitions of CHD varied in these studies; some included angina pectoris, myocardial infarction (fatal, acute, incident), and coronary artery disease. Currently, the precise mechanisms of hyperhomocysteinemia on the risk of IS and CHD are not clear. Several hypotheses have been proposed. Hyperhomocysteinemia might increase the risk of ischemic stroke via the following mechanisms: (1) increasing arterial blood pressure [5]; (2) by causing endothelial dysfunction by increasing oxidant stress and decreasing the release of nitric oxide, impairing vasodilation [42]; (3) inducing oxidative injury to vascular endothelial cells and impairing the production of nitric oxide, a strong vascular relaxing factor, from the endothelium [43]; or (4) enhancing platelet adhesion to endothelial cells, thus promoting the growth of vascular smooth muscle cells [44]. This study had some limitations. It was a cross-sectional study based on retrospective data, a causal relationship cannot be determined due to the unclear or even inverted time sequence: that is, the homocysteine levels were measured up to 2 years after the occurrence of IS or CHD. Also, the selection bias could not be excluded in our study since some patients with severe IS or CHD may be not registered in the CHSC, which may underestimate the association. In addition, normal subjects (without hypertension) were not included in our study. The ORs of IS/ CHD would be much lower in our study as compared to subjects with low tHcy and hypertension than compared those with low tHcy only. Third, IS/CHD might be influenced by a large amount of genetic and environmental factors. Although a large number of important risk factors of IS/CHD were adjusted for in our analyses, residual confounding from genetic factors and unknown or poorly measured determinants of IS/CHD could not be completely ruled out. The use of a single tHcy measurement to classify patients might underestimate the strength of

the relevant associations due to regression dilution [45]. And a variety of drugs might affect the blood tHcy concentration. Although we collected the information about the study participants' antihypertensive medications, other types of medications were not recorded at the enrollment. Finally, generalizability was limited due to the fact that the study was performed in only one region of China. 5. Conclusion In conclusion, our findings suggest that serum tHcy level is associated with IS but not CHD in a sample of 5935 Chinese hypertensive patients. Prospective studies are needed to confirm these findings in patients with hypertension. Learning points • Homocysteine level was associated with age, drinking, fruit intake, SBP, and levels of uric acid and creatinine in hypertensive patients. • The median values of serum tHcy were significantly higher in IS subjects than in non-IS subjects, but did not differ significantly between CHD and non-CHD subjects. • The odds ratios (95% CI) of IS for subjects with intermediate-to-severe hyperhomocysteinemia compared with the normal level were 2.84 (1.73-4.34) in men and 4.41 (1.62-9.15) in women after adjusting for main risk factors of IS. Conflict of interest The authors state that they have no conflicts of interest. Acknowledgments We are grateful for the help of the doctors and nurses in the abovementioned health centers in data and sample collection. References [1] Ministry of Health of China. Health Statistics Annual of China; 2010. [2] Lu H, Lu ZH, Li PG, Wang YY, Yan ZY. Elevated homocysteine and hypertension in Xinjiang Province, China. Ethn Dis 2010;20:7–10. [3] Sen U, Mishra PK, Tyagi N, Tyagi SC. Homocysteine to hydrogen sulfide or hypertension. Cell Biochem Biophys 2010;57:49–58. [4] Tanne D, Haim M, Goldbourt U, Boyko V, Doolman R, Adler Y, et al. Prospective study of serum homocysteine and risk of ischemic stroke among patients with preexisting coronary heart disease. Stroke 2003;34:632–6. [5] Cui R, Moriyama Y, Koike KA, Date C, Kikuchi S, Tamakoshi A, et al. Serum total homocysteine concentrations and risk of mortality from stroke and coronary heart disease in Japanese: the JACC study. Atherosclerosis 2008;198:412–8. [6] Rueda-Clausen CF, Cordoba-Porras A, Bedoya G, Silva FA, Zarruk JG, Lopez-Jaramillo P, et al. Increased plasma levels of total homocysteine but not asymmetric dimethylarginine in Hispanic subjects with ischemic stroke FREC-VI sub-study. Eur J Neurol 2012;19:417–25. [7] Wu XQ, Ding J, Ge AY, Liu FF, Wang X, Fan W. Acute phase homocysteine related to severity and outcome of atherothrombotic stroke. Eur J Intern Med 2013;24:362–7. [8] Tu WJ, Zhao SJ, Liu TG, Yang DG, Chen H. Combination of high-sensitivity C-reactive protein and homocysteine predicts the short-term outcomes of Chinese patients with acute ischemic stroke. Neurol Res 2013;35:912–21. [9] Ashjazadeh N, Fathi M, Shariat A. Evaluation of homocysteine level as a risk factor among patients with ischemic stroke and its subtypes. Iran J Med Sci 2013;38:233–9. [10] Eikelboom JW, Hankey GJ, Anand SS, Lofthouse E, Staples N, Baker RI. Association between high homocyst (e)ine and ischemic stroke due to large- and small-artery disease but not other etiologic subtypes of ischemic stroke. Stroke 2000;31:1069–75. [11] Sun NL, Xi Y, Yang SN, Ma Z, Tang CS. [Plasma hydrogen sulfide and homocysteine levels in hypertensive patients with different blood pressure levels and complications]. Zhonghua Xin Xue Guan Bing Za Zhi 2007;35:1145–8. [12] Sun Y, Chien KL, Hsu HC, Su TC, Chen MF, Lee YT. Use of serum homocysteine to predict stroke, coronary heart disease and death in ethnic Chinese. 12-year prospective cohort study. Circ J 2009;73:1423–30. [13] Zylberstein DE, Bengtsson C, Bjorkelund C, Landaas S, Sundh V, Thelle D, et al. Serum homocysteine in relation to mortality and morbidity from coronary heart disease: a 24-year follow-up of the population study of women in Gothenburg. Circulation 2004;109:601–6.

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Please cite this article as: Wang C, et al, Elevated plasma homocysteine level is associated with ischemic stroke in Chinese hypertensive patients, Eur J Intern Med (2014), http://dx.doi.org/10.1016/j.ejim.2014.04.011