Homocysteine, Ischemic Stroke, and Coronary

Heart Disease in Hypertensive Patients

A Population-Based, Prospective Cohort Study
Liyuan Han, PhD*; Qunhong Wu, PhD; Changyi Wang, PhD*; Yanhua Hao, PhD; Jinshun Zhao, PhD;
Lina Zhang, MD; Rui Fan, MD; Yanfen Liu, MD; Runhua Li, MD; Zhongwei Chen, MD; Tao Zhang,
MD; Sihan Chen, MD; Jianping Ma, MD; Shengyuan Liu, PhD; Xiaolin Peng, PhD; Shiwei Duan, PhD

Background and Purpose—Total homocysteine level (tHcy) is a risk factor of ischemic stroke (IS) and coronary heart
disease. However, the results are conflicting and mainly focused on healthy individuals in developed countries.
Methods—A prospective, population-based cohort study was conducted among 5935 participants from 60 communities in
the city of Shenzhen, China. A Cox regression analysis was applied to evaluate the contribution of tHcy to the risk of
IS and coronary heart disease. The effect of folic acid supplementation on tHcy levels was also evaluated among 501
patients with essential hypertension, who received an average of 2.5 years of folic acid supplementation.
Results—After adjustment for confounding factors, the hazard ratios (95% confidence intervals) of IS caused by
hyperhomocysteinemia were 2.18 (1.65–2.89), 2.40 (1.56–3.67), and 2.73 (1.83–4.08) in the total, male, and female
participants, respectively. Compared with normal levels of tHcy (<15 μmol/L), the hazard ratios (95% confidence
intervals) for IS in the highest tHcy category (≥30 μmol/L) were 4.96 (3.03–8.12), 6.11 (3.44–10.85), and 1.84 (0.52–
6.46) in the total, males, and females participants, respectively. However, we did not observe a significant relationship
between tHcy and the risk of coronary heart disease. The 2.5 years of folic acid supplementation reduced tHcy levels
by 6.7 μmol/L (27.92%) in patients with essential hypertension.
Conclusions—Hyperhomocysteinemia in Chinese hypertensive patients is significantly associated with IS risk but not
coronary heart disease susceptibility, and folic acid supplementation can efficiently reduce tHcy levels.    (Stroke.
2015;46:1777-1786. DOI: 10.1161/STROKEAHA.115.009111.)
Downloaded from http://ahajournals.org by on March 24, 2019

Key Words: coronary heart disease homocysteine hypertension ischemia stroke

S troke is the leading cause of permanent disability in China,1

and ischemic stroke (IS) accounts for almost 80% of all strokes, 2
whereas coronary heart disease (CHD) is a
major worldwide health threat. In tandem with the economic
success of China, the number of individuals in China with IS
and CHD has increased significantly in recent years, with
stroke resulting in 301 million disability-adjusted life-years. 3
CHD in Chinese adults aged 35 to 84 years is predicted to
increase by 64% during the period 2020–2029.4
Although numerous epidemiology studies have researched
the association between hyperhomocysteinemia and IS or CHD,
the results have been conflicting. Most studies have
been retrospective and focused on healthy populations in the
developed world, whereas data from developing countries
remains limited.
Vitamins B12 and B6 and folate are involved in the metabo-
lism of methionine, and deficiency of these B vitamins can cause
elevation of total homocysteine (tHcy) levels. 5 Deficiencies in these
nutrients are more prevalent in developing countries. A meta-
analysis of randomized trials demonstrated that homo-cysteine-
lowering interventions for stroke seem not to have a significant
effect in geographic regions with high dietary folate intake, but may
have a substantial effect in regions with low folate intake, such as
Asia.6 Regarding public health guidelines,

Received February 12, 2015; final revision received May 4, 2015; accepted May 5, 2015.
From the Department of Preventive Medicine, Zhejiang Provincial Key Laboratory of Pathophysiology, School of Medicine, Ningbo University,
Ningbo, China (L.H., J.Z., L.Z., R.F., Y.L., R.L., S.D.); Department of Social Medicine, School of Public Health, Harbin Medical University, Harbin,
China (Q.W., Y.H.); Department of Chronic Disease Prevention and Control, Shenzhen Nanshan Center for Chronic Disease Control, Shenzhen, China
(C.W., Z.C., T.Z., S.C., J.M., S.L., X.P.).
*Drs Han and Wang contributed equally.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.115.
009111/-/DC1.
Correspondence to Qunhong Wu, PhD, Department of Social Medicine, School of Public Health, Harbin Medical University, Harbin 150081, China, E-mail
wuqunhong@163.com or Jinshun Zhao, PhD, Department of Preventive Medicine, Zhejiang Provincial Key Laboratory of Pathophysiology, School of Medicine,
Ningbo University, Ningbo 315211, China, E-mail zhaojinshun@nbu.edu.cn or Shiwei Duan, PhD, Department of Preventive Medicine, Zhejiang Provincial Key
Laboratory of Pathophysiology, School of Medicine, Ningbo University, Ningbo 315211, China, E-mail duanshiwei@nbu.edu.cn.
© 2015 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.115.009111

1777
 1778    Stroke    July 2015
China does not emphasize or promote the importance of tak-ing
folic acid routinely; therefore, people in China potentially have
low levels of folic acid and relatively high tHcy levels.
Moreover, different populations have diverse genetic back-
grounds and encounter different environmental risk factors. We
therefore examined the impact of hyperhomocysteinemia on the
risk of IS and CHD and evaluated the effect of folic acid supple-
mentation on tHcy levels in hypertensive patients in China. Of
note, no studies to date have suggested that folic acid supple-
mentation reduces tHcy levels in hypertensive patients.
We hypothesized that hypertension combined with hyper-
homocysteinemia would greatly increase the risk of IS and
CHD. The objectives of this prospective cohort study were to
confirm the association between tHcy levels and the future
risk of IS and CHD in hypertensive patients and to explore
the effect of folic acid supplementation on tHcy levels in
hyper-tensive patients.
Methods
Study Design
This prospective longitudinal study included 5488 hypertensive pa-tients
with a 2.7-year follow-up. The study relied on the hypertension
management information system in community health service centers
(CHSCs). Patients were enrolled from the 60 CHSCs in the Nanshan
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District of the city of Shenzhen, Guangdong Province in southern China.
Six to eight communities were selected from each of the 8 subdistricts in
Nanshan District using a simple random procedure ac-cording to a
sequence of computer-generated random numbers. The baseline survey
was conducted from April 2010 to September 2011. 7 The specific details
of the study participants, recruitment, and base-line data collection have
been described previously.7 Written informed consent was obtained from
all participants. The protocol of this study was approved by the ethics
committees of the collaborating hospitals.
Inclusion and Exclusion Criteria
Inclusion criteria were as follows: (1) patients with essential hy-
pertension ≥20 years of age; (2) local residents who had lived in
Shenzhen for ≥6 months; (3) native Chinese who could be followed-
up without difficulty for at least 3 years; and (4) patients whose
health records had been established in the CHSC.
Exclusion criteria were as follows: (1) patients with secondary
hypertension, cancer, severe liver and kidney disease, or pregnancy;
(2) patients taking folic acid or vitamin B6 or B12 before the follow-
up study; and (3) hypertensive patients with IS or CHD.
During the 2.7-year follow-up, we defined hypertensive patients as
having IS if they had subarachnoid hemorrhage, intracerebral hem-
orrhage, nontraumatic intracranial hemorrhage, or cerebral infarction and
as having CHD if they had myocardial infarction, angina pec-toris, silent
myocardial ischemia, or ischemic heart disease. IS pa-tients were verified
according to symptoms and examination results, including computed
tomography (CT), magnetic resonance imaging, cerebral angiography,
and transcranial doppler ultrasound. CHD in patients was ascertained by
electrocardiography, coronary angiogra-phy, echocardiography, cardiac
CT, and cardiac enzymes.
Data Collection
Patients who fulfilled the inclusion criteria were enrolled consecu-
tively. Trained staff collected the data through physical examination,
questionnaires, biochemical measurement, medical records, and rel-
evant tests.
Physical measurements comprised height, weight, waist circum-
ference, hip circumference, and blood pressure. Body mass index
(BMI) was calculated as weight (kg)/height (m 2) and was ranked into
4 categories: <18=1 (thin), 18 to 24=2 (normal), 24 to 28=3
(overweight), >28=4 (obese). Systolic blood pressure (SBP) and dia-
stolic blood pressure (DBP) were measured using a standard mercury
sphygmomanometer on the right arm of seated participants after a 5-
minute rest. Essential hypertension was defined as a rise in blood
pressure of unknown cause that increased the risk of cerebral, car-
diac, and renal events8 and the absence of secondary causes, such as
renovascular disease, renal failure, pheochromocytoma, aldosteron-
ism, or other causes of secondary hypertension or mendelian forms
(monogenic).9 Patients with essential hypertension were diagnosed
according to SBP ≥140 mm Hg or DBP ≥90 mm Hg or self-reported
use of antihypertensive medication.10
Abnormal total cholesterol (TC) was defined as TC >5.18
mmol/L, abnormal triglyceride (TG) as >1.70 mmol/L, abnormal
low-density lipoprotein cholesterol (LDL-C) as >3.37 mmol/L, and
abnormal high-density lipoprotein cholesterol as <1.04 mmol/L.
Dyslipidemia was defined as any of the followings being abnormal:
TC, TG, LDL-C, or high-density lipoprotein cholesterol according to
Chinese Guidelines on Prevention and Treatment of Dyslipidemia in
Adults.11 Diabetes mellitus was defined according to the current
American Diabetes Association guidelines (fasting plasma glucose
≥7.0 mmol/L and 2-h plasma glucose ≥11.1 mmol/L).12
Questionnaires
Questionnaire responses provided data on the following patient char-
acteristics. (1) Demographic characteristics: name, sex, date of birth,
ethnicity, occupation, education, marital status, community name, ID
number, and so on. (2) Health-related behaviors: smoking, passive
smoking, alcohol consumption, and physical activity. Levels of both
leisure and occupational physical activity were assessed as described in a
previous study.13 Categories of alcohol intake were determined according
to definitions of the National Institute on Alcohol Abuse and
Alcoholism.14 Current smokers were defined as those who smoke >1
cigarette per day; nonsmokers were defined as those who had quit
smoking within the previous 12 months, had never smoked, or smoked
<1 cigarette per day.15 (3) Diet: we adopted the international commonly
used food frequency questionnaire to document the diet of the patients
for the past year. This questionnaire mainly inquired about the intake
frequency of grains, vegetables, fruits, and other foods rich in folic acid
and vitamins. (4) Emotional state: Zung self-rating depression scale was
applied16; if the score was >50, we defined this as depression according to
China’s standards. (5) Disease status and medication use: duration of
hypertension and family history of IS and CHD; use of antihypertensive
medication, including diuretics, re-ceptor blockers, calcium antagonists,
angiotensin-converting enzyme inhibitors, angiotensin II receptor
antagonists, α-blockers, compound preparations, and traditional Chinese
medicine.
Biochemical Measurements
Blood samples were collected by venipuncture from each participant
after overnight fasting. The collection tubes were immediately placed in
an ice container and transferred to the Clinical Laboratory of the Chronic
Diseases Hospital in the Nanshan District within 1 hour. tHcy levels were
measured within 4 h. Additional plasma samples were immediately
frozen and stored at −80°C for future analysis.
Blood tests included fasting glucose, TC, LDL-C, TG, uric acid
(UA), plasma tHcy, and serum creatinine. TC, LDL-C, glucose, and
TG were measured using enzymatic methods, UA was detected by
quantitative determination with uricase, creatinine was detected by
the Jaffe method, and a circulating enzymatic method was adopted
for the measurement of Hcy. All of these indicators were tested with
an automatic biochemical analyzer (Hitachi 7080).
Folic Acid Intervention
Intervention patients had tHcy levels ≥15 μmol/L during the baseline
survey and agreed to participate in the intervention program. Patients
took a daily dose of 0.8 mg folic acid, 2 tablets each time (0.4 mg per
tablet; Beijing SL Pharmaceutical, H10970079), or took 1 enalapril
maleate/folic acid tablet daily (10 mg enalapril maleate, 0.8 mg folic
acid; Shenzhen Osa Pharmaceutical, H20103723). In the follow-up
 Han et al  Hcy, IS, and CHD in Hypertensive Patients   1779
intervention, CHSC clinicians evaluated the intake of folic acid and
enalapril maleate/folic acid tablets by documenting the medication time,
dosage, adverse reactions, compliance, and withdrawal time and reasons.
Patients who took folic acid or enalapril maleate/folic acid tablets for >1
month were defined as the exposure group; patients with adverse effects
were not included in the exposure group.
Statistical Analysis
Qualitative data are expressed as proportion (%) and were ana-lyzed
using the chi-squared test. Quantitative data are presented as
mean±standard deviation; variables with normal distributions were
analyzed by the Student’s t test, whereas variables with skewed distri-
butions were analyzed using the Mann–Whitney U test. We used the
Markov Chain Monte Carlo method of multiple imputation to ensure
that the imputed data set had a monotone missing pattern through 20
imputations. The multiple imputation method is commonly used and is
flexible for variables with missing values. 17 The tHcy levels were
divided into 3 categories (normal, <15 μmol/L; mildly elevated, 15–30
μmol/L; moderately elevated, 30–100 μmol/L),18 tHcy levels ≥15
μmol/L were defined as hyperhomocysteinemia.18
The hazard ratios (HRs) and 95% confidence intervals (CIs) of IS
and CHD were calculated according to the tHcy levels using the Cox
regression model. The model comprised the following: M0, not
adjusted; M1, adjusted for 13 factors (including age, sex, education,
smoking, alcohol consumption, BMI, physical activity, diabetes mel-
litus, depression, family history of stroke, years of hypertension, an-
tihypertensive medication, and use of folic acid); M2, adjusted for 18
factors (including the above 13 factors, SBP, TC, glucose, TG, and
LDL-C). Based on model M2, we calculated the area under the curve
value under the receiver-operating characteristic (ROC) curve to
evaluate the effect of the model. Power analysis was simulated with
Power and Sample Size Calculation software.19
Results
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General Characteristics
As shown in Figure I in the online-only Data Supplement,
among the total of 5935 participants as of April 2011, 202
pre-sented with IS and 270 with CHD (25 had both IS and
CHD) and were thus excluded. Of the remaining 5488
participants with an average follow-up of 2.7 years, 327 were
lost to fol-low-up (5.96%): 138 persons refused to continue,
81 returned to their home town, 65 provided the wrong
telephone number or wrong address, 29 moved, and 14 died
(not of IS or CHD). Ultimately, 5161 participants were
successfully followed and included in this prospective study.
Baseline Characteristics
Table 1 presents the baseline characteristics of the
participants according to sex. Of the 5488 participants, 2712
were males (49.42%) and 2776 were females (50.58%).
There were no differences between males and females
concerning physical activity, depression, antihypertensive
medication, and baseline SBP. Education, smoking, alcohol
consumption, salt intake, oil intake, vegetable intake, fruit
intake, years of hypertension, age, BMI, DBP, and levels of
TC, LDL-C, UA, glucose, and creatinine showed statistically
significant differences between males and females (P<0.05).
Comparison of Baseline Characteristics Between
Participants Lost and Not Lost to Follow-Up
To assess the information bias of the participants lost to follow-
up, we compared the baseline characteristics between those
lost and not lost. Table I in the online-only Data Supplement
demonstrates that only fruit intake, physical activity, and
SBP differed significantly between the 2 groups (P<0.05).
Comparison of Indicators Between Baseline and
Follow-Up
Table II in the online-only Data Supplement shows that age,
BMI, waist circumference, SBP, and levels of TC, LDL-C,
TG, glucose, creatinine, and tHcy were significantly higher
after 2.7 years of follow-up than at baseline, although DBP
and UA levels were significantly lower.
After following-up the 5488 participants for 2.7 years, we
identified 197 with IS and 7 who died of IS. We also observed
157 with CHD and 5 who died of CHD. Another 14 died for
reasons other than IS or CHD. The main type of IS was cere-bral
infarction (mainly hemorrhagic IS, 75%), intracerebral
hemorrhage (17%), subarachnoid hemorrhage (5%), non-
traumatic intracranial hemorrhage (1%), and other (2%). The
proportions of CHD types were angina pectoris (76%), myo-
cardial infarction (17%), silent myocardial ischemia (4%),
ischemic heart disease (2%), and sudden death (1%).
Association of tHcy With IS and CHD
We analyzed the tHcy levels based on sex and age (Table 2).
The mean tHcy levels were 13.60 μmol/L in all participants,
15.96 μmol/L in males, and 11.70 μmol/L in females. Males
had higher tHcy levels and a higher prevalence of hyperho-
mocysteinemia than females in each age group (P<0.0001).
Of note, tHcy levels and prevalence of
hyperhomocysteinemia increased with age.
The participants were further divided into 2 groups based
on tHcy levels (<15 and ≥15 μmol/L). Table 3 shows that
after 2.7 years of follow-up, the incidence of IS was 3.82%
in hypertensive patients, 6.18% in the exposure group (tHcy
≥15 μmol/L), and 2.84% in the control group (tHcy <15
μmol/L). The HRs (95% CIs) of IS caused by hyper-
homocysteinemia were 2.18 (1.65–2.89), 2.40 (1.56–3.67),
and 2.73 (1.83–4.08) for all participants, males, and females,
respectively.
After 2.7 years of follow-up, the incidence of CHD was
3.04% in hypertensive patients, 3.16% in the exposure group
(tHcy ≥15 μmol/L), and 2.92% in the control group (tHcy
<15 μmol/L). The HRs (95% CIs) of CHD caused by
hyperhomo-cysteinemia (tHcy ≥15 μmol/L) were 1.46 (1.06–
2.02) in all participants, 1.19 (0.76–1.88) in males, and 2.26
(1.40–3.64) in females (Table 3). According to power
calculations, our sample size provided >80% power to detect
the relative asso-ciations of tHcy with IS and CHD risk.
Different tHcy Levels and the Prediction of IS
and CHD
Table 4 demonstrates that under the M2 model (adjusted by 18
factors), the HRs (95% CIs) for IS in the highest tHcy category
(≥30 μmol/L) compared with normal levels of tHcy (<15
μmol/L) were 4.96 (3.03–8.12) in all participants, 6.11 (3.44–
10.85) in males, and 1.84 (0.52–6.46) in females. Compared
with the reference group (tHcy <15 μmol/L), the HRs (95% CIs)
of hyperhomocysteinemia (tHcy ≥15 μmol/L)
 1780    Stroke    July 2015

Table 1.  The Baseline Characteristics of the Followed 5488 Subjects According to Sex

Variables Males (n=2712) Females (n=2776) P Value Total (n=5488)

Education, %
<Middle school 203 (7.49%) 150 (5.40%) 0.0008 353 (6.43%)
Middle/high school 1846 (68.07%) 1863 (67.11%) 3709 (67.58%)
  ≥College 663 (24.45%) 763 (27.49%) 1426 (25.98%)
Smoking, % 745 (27.47%) 41 (1.48%) <0.0001 786 (14.32%)
Alcohol consumption, % 1139 (42.00%) 307 (11.06%) <0.0001 1446 (26.35%)
Salt intake, %
  Low (<6 g/d) 457 (16.85%) 531 (19.13%) 0.0066 988 (18.00%)
  Moderate (6–13 g/d) 1959 (72.23%) 2000 (72.05%) 3959 (72.14%)
  High (>13 g/d) 296 (10.91%) 245 (8.83%) 541 (9.86%)
Oil intake, %
  Low (<25 g/d) 361 (13.31%) 456 (16.43%) <0.0001 817 (14.89%)
  Moderate (25–40 g/d) 2029 (74.82%) 2081 (74.96%) 4110 (74.89%)
  High (>40 g/d) 322 (11.87%) 239 (8.61%) 561 (10.22%)
Vegetables intake, %
  Low (<300 g/d) 210 (7.70%) 161 (5.75%) 0.0014 371 (6.72%)
  Moderate (300–500 g/d) 1845 (67.68%) 1862 (66.55%) 3707 (67.11%)
  High (>500 g/d) 671 (24.61%) 775 (27.70%) 1446 (26.18%)
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Fruit intake, ≥200 g/d, % 1934 (71.31%) 2237 (80.58%) <0.0001 4171 (76.00%)
Physical activity, %
<1 time/week 400 (14.75%) 349 (12.57%) 0.054 749 (13.65%)
  1–3 times/ week 1380 (50.88%) 1429 (51.48%) 2809 (51.18%)
  >3 times/ week 932 (34.37%) 998 (35.95%) 1930 (35.17%)
Depression, % 729 (26.88%) 779 (28.06%) 0.327 1508 (27.48%)
Years of hypertension, %
<2 522 (19.25%) 602 (21.69%) 0.0026 1124 (20.48%)
2–5 834 (30.75%) 733 (26.40%) 1567 (28.55%)
5–10 640 (23.60%) 668 (24.06%) 1308 (23.83%)
  ≥10 716 (26.40%) 773 (27.85%) 1489 (27.13%)
Antihypertensive drugs 2179 (80.35%) 2176 (78.39%) 0.0728 4355 (79.35%)
Diabetic 277 (10.21%) 260 (9.37%) 0.2905 537 (9.78%)
High TC 170 (6.27%) 355 (12.79%) <0.0001 525 (9.57%)
High TG 736 (27.14%) 638 (22.98%) 0.0004 1374 (25.04%)
High LDL 501 (18.47%) 639 (23.02%) <0.0001 1140 (20.77%)
Age, y 57.07±13.04 59.68±11.01 <0.0001 58.39±12.13
BMI, kg/m2 24.74±2.92 23.94±3.08 <0.0001 24.33±3.03
SBP, mm Hg 133.54±14.65 132.96±15.52 0.1525 133.25±15.1
DBP, mm Hg 84.9±10.52 82.49±10.23 <0.0001 83.68±10.44
TC, mmol/L 4.92±0.97 5.28±1.06 <0.0001 5.10±1.03
LDL, mmol/L 2.94±0.76 3.06±0.82 <0.0001 3.00±0.8
UA, μmol/L 378.11±92.21 308.47±84.62 <0.0001 342.89±95.06
TG, mmol/L* 0.52±0.58 0.46±0.52 <0.0001 0.49±0.56
Glucose, mmol/L 5.69±1.42 5.59±1.19 0.0058 5.64±1.31
Creatinine, μmol/L 89.11±17.38 67.76±13.49 <0.0001 78.31±18.85
The qualitative data were presented as (%), analyzed with χ2 tests; The quantitative data were presented as mean±SD, analyzed with
Student’s t test. BMI indicates body mass index; DBP, diastolic blood pressure; LDL, low density lipoprotein; SBP, systolic
blood pressure; TC, triglyceride; TG, total cholesterol; and UA, uric acid.
*The distribution of TG was skewed; therefore, we analyzed it with nonparametric test.
 Han et al  Hcy, IS, and CHD in Hypertensive Patients   1781

Table 2.  The tHcy Levels of Different Sex and Age

Hyperhomocysteinem
tHcy, μmol/L (≥15 μmol/L)
n means±SD P Value n,%
Total 5935 13.60±1.52 1863 (31.39)
Males 2928 15.96±1.55 <0.0001 1360 (46.45)
Females 3007 11.70±1.38 503 (16.73)
Males
<50 y 859 15.49±1.65 <0.0001 329 (38.30)
50–60 y 677 15.03±1.54 279 (41.21)
60–70 y 772 15.64±1.48 367 (47.54)
  ≥70 y 620 17.81±1.52 385 (62.10)
Females
<50 y 558 10.28±1.38 <0.0001 51 (9.14)
50–60 y 772 10.70±1.34 68 (8.81)
60–70 y 1057 11.94±1.35 172 (16.27)
  ≥70 y 620 13.74±1.40 212 (34.19)
Total
<50 y 1417 13.20±1.62 <0.0001 380 (26.82)
50–60 y 1449 12.55±1.49 347 (23.95)
60–70 y 1829 13.46±1.43 539 (29.47)
  ≥70 y 1240 15.64±1.49 597 (48.15)
The distribution of tHcy was skewed. tHcy indicates total homocysteine level.

were 1.82 (1.17–2.84) in all participants, 2.19 (1.40–3.43) in Effect of Folic Acid Supplementation on tHcy Levels
males, and 1.60 (1.09–3.19) in females. Among the 5488 hypertensive patients in the follow-up
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Furthermore, under the M2 model, there was no significant
difference in the risk of CHD between participants with the
highest tHcy category (tHcy ≥30 μmol/L) and those with nor-
mal levels of tHcy (<15 μmol/L). In addition, compared with the
group with tHcy <15 μmol/L, there was no significant asso-
ciation between hyperhomocysteinemia and the risk of CHD.
ROC Analysis
Based on the M2 model, the area under the curve of the ROC
curve using the logistic regression model (prediction of dif-
ferent levels of tHcy leading to IS) was 0.71 to 0.74. After
eliminating tHcy from the model, the logistic regression
(area under the curve of ROC) was 0.70 to 0.73. Using the
same model but with CHD instead of IS, the area under the
curve of the ROC did not differ from that of IS to any great
extent (data not shown).
study, 501 took folic acid for an average duration of 2.5
years. Table 5 reveals that compared with the
nonintervention group, age, UA, and creatinine were higher
in the intervention group, whereas there was no significant
difference between the 2 groups regarding BMI, waist
circumference, SBP, DBP, TC, glucose, TG, and LDL-C.
Figure demonstrates that after taking folic acid for 2.5 years,
compared with baseline data, the plasma tHcy level in the
intervention group declined 6.7 μmol/L on average. Folic acid
supplementation exerted the most significant effect in the first 3
months, after which tHcy levels declined at a slower pace. In the
nonintervention group, tHcy level did not vary extensively.
Discussion
Vascular risk is stronger in individuals with hyperten-sion
combined with hyperhomocysteinemia. 20 However,

Table 3.  The Associations of Hyperhomocysteinemia With the Risk of IS and CHD

Control group, tHcy Exposure group

<15 μmol/L (Reference) (tHcy ≥15 μmol/L)
N n=3656 n=1505 HR (95%CI) P Value
IS-Males 2565 33 (2.26%) 57 (5.17%) 2.40 (1.56–3.67) <0.0001 9
IS-Females 2596 71 (3.24%) 36 (8.96%) 2.73 (1.83–4.08) <0.0001 10
IS-Total 5161 104 (2.84%) 93 (6.18%) 2.18 (1.65–2.89) <0.0001 19
CHD-Males 2565 41 (2.80) 34 (3.08) 1.19 (0.76–1.88) 0.6789 7
CHD-Females 2596 58 (2.64) 24 (5.97) 2.26 (1.40–3.64) 0.0005 8
CHD-Total 5161 99 (2.71) 58 (3.85) 1.46 (1.06–2.02) 0.0294 15
CHD indicates coronary heart disease; HR, hazard ratio; IS, ischemic stroke; and tHcy, total homocysteine level.
 1782  Stroke  July 2015

Table 4.  HRs (95% CI) of IS and CHD for Different Levels of tHcy

Hcy, μmol/L*

Hcy ≥15 μmol/L

<15 μmol/L (reference) 15–30 μmol/L ≥30 μmol/L P trend† vs <15 μmol/L‡

IS-Male
Case 33 33 24
Person-year 3947 2438 540
M0§ 1.00 1.48 (1.01–2.45) 5.91 (3.41–10.22) <0.0001 2.40(1.56–3.67)
M1§ 1.00 1.31 (0.78–2.19) 6.04 (3.43–10.66) <0.0001 2.13(1.49–2.87)
M2§ 1.00 1.50 (0.91–2.48) 6.11 (3.44–10.85) <0.0001 2.19(1.40–3.43)
IS-Female
Case 71 33 3
Person-year 6294 996 89
M0§ 1.00 2.93 (1.91–4.51) 2.99 (0.89–10.26) <0.0001 2.73 (1.83–4.08)
M1§ 1.00 1.85 (1.17–2.91) 1.94 (0.56–6.89) <0.0001 1.96 (1.41–2.51)
M2§ 1.00 1.82 (1.15–2.87) 1.84 (0.52–6.46) 0.0100 1.60 (1.09–3.19)
IS-Total
Case 104 66 27
Person-year 10 241 3434 629
M0§ 1.00 1.70 (1.22–2.36) 4.48 (2.68–7.00) <0.0001 2.18 (1.65–2.89)
M1§ 1.00 1.66 (1.18–2.32) 5.00 (3.07–8.15) <0.0001 2.03 (1.48–2.77)
Downloaded from http://ahajournals.org by on March 24, 2019

M2§ 1.00 1.66 (1.18–2.33) 4.96 (3.03–8.12) <0.0001 1.82 (1.17–2.84)

CHD-Male
Case 41 24 10
Person-year 3947 2438 540
M0§ 1.00 0.94 (0.579–1.57) 1.82 (0.90–3.70) 0.2783 1.19 (0.76–1.88)
M1§ 1.00 0.77 (0.45–1.29) 1.68 (0.81–2.91) 0.6410 0.91 (0.57–1.47)
M2§ 1.00 0.73 (0.43–1.24) 1.53 (0.73–2.78) 0.8183 0.87 (0.54–1.40)
CHD-Female
Case 58 19 5
Person-year 6104 996 89
M0§ 1.00 2.00 (1.18–3.40) 6.58 (2.45–17.64) <0.0001 2.26 (1.40–3.64)
M1§ 1.00 1.13 (0.61–2.07) 4.62 (1.43–14.87) 0.0533 1.34 (0.77–2.34)
M2§ 1.00 1.09 (0.59–2.01) 4.25 (1.32–13.73) 0.1250 1.3 (0.74–2.27)
CHD-Total
Case 99 43 15
Person-year 10 051 3434 629
M0§ 1.00 1.26 (0.87–1.81) 2.47 (1.41–4.33) 0.0038 1.46 (1.06–2.02)
M1§ 1.00 0.98 (0.43–1.45) 2.25 (0.95–3.92) 0.0850 1.53 (0.67–2.87)
M2§ 1.00 0.95 (0.41–1.41) 2.10 (0.89–3.74) 0.1424 1.49 (0.89–2.58)
CHD indicates coronary heart disease; HR, hazard ratio; IS, ischemic stroke; and tHcy, total homocysteine level.
*The categories of 15–30 μmol/L and ≥30 μmol/L were combined because of limited number of participants.
†Estimated using the category numbers (1, 2, and 3) as a continuous variable.
‡The categories of 30–100 μmol/L and ≥100 μmol/L were combined because of limited number of participants.
§Please refer to the article for the factors adjusted for model M0, M1, and M2.

few studies have focused on the effects of tHcy on IS and
CHD in hypertensive patients. In this population-based,
prospective cohort study, we hypothesized that hyperho-
mocysteinemia might increase the risk of IS and CHD in
Chinese hypertensive patients. Our findings indicate that
hyperhomocysteinemia is associated with IS but not CHD in
hypertensive patients and that folic acid supplementa-
tion reduces tHcy levels. To our knowledge, this
prospec-tive cohort study is the first to show a
potential association
of tHcy level with IS susceptibility in hypertensive patients in
China.
Both our baseline survey and cohort study indicated that elevated
tHcy was associated with IS, and the effect of higher tHcy levels on IS
risk tended to be stronger in females. Consistent with our findings, Hung
et al showed that resistant hypertension was associated with a higher
risk of IS, especially in women and elderly patients. 21 Moreover, Yan et
al reported that women with mild hypertension had a higher risk of
stroke
 Han et al  Hcy, IS, and CHD in Hypertensive Patients   1783

Table 5.  The Differences of Intervention Group (Folic (CVD) risk.25 Here, we observed that elderly women tended
Acid Supplementation) and Nonintervention Group to have higher tHcy levels, perhaps because of the lower
Intervention Group Nonintervention
estro-gen levels in older women.
Variables (n=501) Group (n=4660) P Value At present, the beneficial effect of folic acid supplementa-
tion remains controversial. It is reported that supplementation
Females
with vitamins B6 and B12 plus folic acid could reduce blood
Age, y 58.08±13.14 61.15±12.43 <0.0001 pressure in patients with essential hypertension. 26 Indeed, a
BMI, kg/m2 25.00±2.96 24.72±2.74 0.0945 substantial body of evidence supports a role of plasma homo-
Waistline, cm 89.47±9.33 88.77±9.06 0.1939 cysteine in the pathogenesis of hypertension. 27 However, an
SBP, mm Hg 134.55±14.59 134.98±14.71 0.6138 updated Cochrane review demonstrated that clinical trials using
DBP, mm Hg 84.39±10.59 83.97±10.6 0.4907 low and high doses of folic acid, vitamin B12, and vita-min B6
had not shown any clinical benefit 28 and claimed that the effects
TC, mmol/L 4.97±0.98 4.91±0.89 0.2515
of folate might be influenced by the duration of treatment. 29 The
LDL, mmol/L 3.00±0.77 2.96±0.75 0.3035
average duration of folic acid supplementa-tion in our study was
UA, μmol/L 374.61±92.11 382.4±94.91 0.1448 2.5 years, which reduced the tHcy level by an average of 6.7
TG, mmol/L* 0.54±0.58 0.49±0.54 0.1814 μmol/L (27.92%). Our results therefore suggest that folic acid
Glucose, mmol/L 5.84±1.46 5.77±1.28 0.3332 supplementation can be beneficial for hypertension by reducing
Creatinine, μmol/L 90.91±15.99 96.59±22.94 <0.0001 the level of tHcy.
Males Before adjusting for the traditional risk factors of CVD,
Age, y 61.05±11.03 64.05±11.51 0.0011 we noted a significant association between tHcy and the risk
of CHD in females, but this association did not remain after
BMI, kg/m2 24.21±3.10 24.19±3.10 0.9341
adjusting for the risk factors. A meta-analysis revealed that
Waistline, cm 84.8±9.36 85.29±9.79 0.5280
lifelong moderate homocysteine elevation had little or no
SBP, mm Hg 134.07±15.55 134.71±14.67 0.6203 effect on CHD.30 However, in contrast to our study, another
DBP, mm Hg 81.80±10.15 81.23±10.04 0.4988 study declared that serum tHcy was an independent predictor
TC, mmol/L 5.34±1.04 5.35±1.05 0.8750 of CHD in the elderly population.31
LDL, mmol/L 3.12±0.83 3.15±0.82 0.5952 The major constituent of CVD in western populations is
UA, μmol/L 305.7±84.08 327.89±94.02 0.0016
CHD; however, it is stroke in the Chinese population. 32 A ran-
domized trial demonstrated that multivitamin supplementation
TG, mmol/L* 0.47±0.52 0.45±0.46 0.6323
Downloaded from http://ahajournals.org by on March 24, 2019

decreased the mortality of stroke, 32 but not CHD in Chinese

Glucose, mmol/L 5.74±1.21 5.69±0.86 0.4537
individuals.33 Genetic variants of the methylenetetrahydrofo-late
Creatinine, μmol/L 70.19±14.1 77.44±17.51 <0.0001 reductase gene can modulate tHcy levels; the TT genotype of the
Total methylenetetrahydrofolate reductase C677T gene is related to
Age, y 59.63±12.17 62.04±12.22 <0.0001 greater risk of IS6, but not CHD,34 and the T allele is more
BMI, kg/m2 24.58±3.06 24.55±2.86 0.8307 frequent in Asians than Europeans.6 The above may partially
explain the significant association between tHcy and IS, but not
Waistline, cm 87.02±9.63 87.70±9.42 0.1321
CHD in our study.
SBP, mm Hg 134.30±15.10 134.9±14.68 0.4010
Elevated tHcy level is a clotting factor, which is capable
DBP, mm Hg 83.03±10.44 83.13±10.50 0.8494
of increasing the risk of stroke >4-fold in patients with atrial
TC, mmol/L 5.16±1.03 5.05±0.96 0.013 fibrillation.35 Stroke and myocardial infarction have differ-
LDL, mmol/L 3.06±0.80 3.02±0.77 0.2295 ent pathogenesis. Most IS are embolic and because of cere-
UA, μmol/L 338.5±94.47 365.64±97.84 <0.0001 bral venous thrombosis with infarction from stasis. Elevated
TG, mmol/L* 0.50±0.55 0.48±0.52 0.3619 tHcy levels increase thrombosis and the risk of
  Glucose, mmol/ L 5.79±1.33 5.74±1.16 0.4498 cardioembolic strokes, whereas myocardial infarctions are
mainly caused by plaque rupture and occlusion of a coronary
Creatinine, μmol/L 80.05±18.24 90.7±23.16 <0.0001
artery, with the thrombosis being secondary to the
BMI indicates body mass index; DBP, diastolic blood pressure;
occlusion.36 There is a strong positive association between
LDL, low density lipoprotein; SBP, systolic blood pressure; TC,
triglyceride; TG, total cholesterol; and UA, uric acid.
stroke from atrial fibrilla-tion and age, and tHcy levels above
*The distribution of TG was skewed; therefore, we analyzed it 14 μmol/L are present in 40% of patients >80 years.37
with nonparametric test. Previous studies have indicated that vitamin B therapy might
reduce the risk of stroke but not coronary events 38 and that the
than men and showed that a 10 mm Hg increase in SBP was nonsignificant effect of vitamin B therapy might be related to
associated with a 38% increased risk of stroke in women. 22 The metabolic deficiency of vitamin B12 and impaired renal
Framingham Heart Study also suggested an overall higher function.39 A randomized controlled trial demonstrated that renal
lifetime stroke susceptibility in women. 23 Estrogen and estro- failure may alter the response to vitamin B ther-apy. 40 Vitamin B
gen–progestogen therapy can reduce homocysteine levels in therapy is helpful in patients with good renal function, but
harmful in patients with markedly impaired renal function. 39
menopausal women.24 Another study suggested that higher
Metabolic B12 deficiency is present in 30% of patients with
levels of estrogen–homocysteine conjugates could lead to a
vascular disease who are >70 years of age,
lowering of homocysteine-induced cardiovascular disease
1784    Stroke    July 2015
 patients. Unlike Europe and the United States, China
does not

Figure. The differences of total homocysteine level (tHcy)

between the intervention group (folic acid supplementation) and
nonintervention group.

thus higher doses of B12 are necessary for these patients. 41

However, high doses of cyanocobalamin may increase the lev-
els of thiocyanate in patients with renal failure. 42 Elevation of
tHcy and asymmetrical dimethylarginine increases vascular
risk in patients with renal failure.43 Methylcobalamin reduces
the levels of tHcy and asymmetrical dimethylarginine in dialy-
sis patients,44 whereas cyanocobalamin does not lower levels
of asymmetrical dimethylarginine. Therefore, Spence has
suggested that methylcobalamin instead of cyanocobalamin
should be used in patients with renal impairment.39
A previous study tested the sensitivity, specificity, and
accuracy of carotid-color Doppler in detecting dolichoca-
rotids (DCs) and explored the 5-year incidence of cerebro-
CVD (transient ischemic attack, IS, myocardial infarction,
and cardiovascular death) in patients with DCs. The findings
Downloaded

related to cerebro- vascular symptoms. 45 In addition, a positive

suggested that patients with DCs were susceptible to poten-

tially disabling and fatal events and that presence of DCs was
from

association existed between DCs and nonischemic dilated car-

diomyopathy, and women were more likely to develop DCs. 46
.http://ahajournals

Future studies should pay attention to the role of DCs in the

development of stroke.
In addition, elevated heart rate is a physiopathological
state that can affect CVD risk.47 Heart rate reduction is already
regarded as a therapeutic target in several cardiac conditions,
org

such as ischemic heart disease and heart failure. Ivabradine

onby March 24,

reduces the heart rate and is a reliable pharmacological drug

for the reduction of morbidity and mortality related to coronary
artery diseases and chronic heart failure.48 Hyperpolarization
and cyclic nucleotide channels may also be important targets
for the reduction of heart rate.49
2019

The advantages of this study were as follows. (1) We stud-

ied a large community-based sample of hypertensive patients,
and participants were homogeneous regarding their genetic
background and environmental exposure. (2) Data were col-
lected under rigid quality control, and important covariables
were measured and controlled in the analysis. (3) The pro-
spective cohort study enabled us to obtain a less biased asso-
ciation between exposure variables and outcome events. (4)
The findings of our study have potential value in diverse cir-
cumstances. At present, most of the literature concerning the
relationship between tHcy and susceptibility to IS and CHD
has involved healthy individuals rather than hypertensive
emphasize or promote the importance of taking folic acid rou-tinely;
thus, people in China potentially have lower levels of folic acid and
relatively high tHcy levels. It is therefore neces-sary to gather data from
the Chinese population to provide a comprehensive evaluation of the
potential influence of tHcy and folic acid on the risk of developing IS
and CHD. (5) We have controlled for many traditional risk factors
related to IS and CHD and have applied hierarchical analysis to explore
the sensitivity and ensure the reliability of the results.
The limitations of this study also need to be noted. (1) The
representativeness of the population may be a limitation because
only the patients who were registered in the health management
system of CHSCs were included in our cohort study; patients in the
early stages of hypertension were not recruited. (2) Selection bias
may be an issue because we did not include the healthy population.
(3) Although we con-sidered many traditional risk factors, there
may be other confounding factors not included in this study.
Circulating homocysteine is influenced by many factors, such as
nutri-tional deficiencies, lifestyle factors, physiological condi-tions,
and drugs. IS and CHD are complex diseases that are influenced by
both genetic and environmental factors and their interactions. Future
functional research is needed to elaborate the mechanisms of the
interaction between tHcy and folic acid in hypertensive patients. (4)
Many types of IS and CHD exist; owing to the limited number in
each sub-group, we did not perform subgroup analysis regarding the
different forms of IS and CHD. (5) Another limitation is the
possible recall bias during data collection. However, we have tried
to minimize recall bias through rigorous training in sur-vey methods
and application of standard operating proce-dures during data
collection.

Summary
This population-based, prospective cohort study suggests that it is
necessary to replenish folic acid in Chinese patients with
hypertension. Strategies for the prevention and treatment of
hypertension, including folic acid supplementation, are neces-sary to
prevent stroke.22

Acknowledgments
We are grateful for the help of the doctors and nurses in the above-
mentioned health centers in data and sample collection.

Sources of Funding
The research was supported by grants from Shenzhen Nanshan Bureau of
Science and Technology (2010058), K.C.Wong Magna Fund in Ningbo
University, the National Natural Science Foundation of China (81402745),
the Natural Science Foundation of Ningbo City (2014A610268), the Key
Program of Education Commission of Zhejiang Province (Z201017918), the
Natural Science Foundation of Zhejiang Province (LQ13H260002),
Zhejiang Province Scientific Research Projects of Education (Y201326971),
the Ministry of Education, Humanities and Social Sciences project
(14YJC630046), China Medical Board Health 2020 Project (08–929), and
China Medical Board Distinguished Professorships Project (G16916400/
F510000).

Disclosures
None.
 Han et al  Hcy, IS, and CHD in Hypertensive Patients   1785
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