Recently, carotid B-mode ultrasound imaging has been

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1 Plasma Homocysteine Levels and Atherosclerosis in Japan Epidemiological Study by Use of Carotid Ultrasonography Hisashi Adachi, MD, PhD; Yuji Hirai, MD; Yoshihisa Fujiura, MD; Hidehiro Matsuoka, MD, PhD; Akira Satoh, MD; Tsutomu Imaizumi, MD, PhD Background and Purpose We examined whether hyperhomocysteinemia is an independent risk factor for increased carotid artery intimal-medial wall thickness (IMT) in a large, randomly selected community in Japan where the dietary habit is different and the incidence of coronary artery disease is lower compared with those of western countries. Methods In 1111 cases (452 men, 659 women) aged years old (range, 40 to 94 years) recruited from a population-based survey performed in 1999, we measured fasting plasma total homocysteine levels and performed bilateral carotid B-mode ultrasound. The participants underwent measurements of other blood chemistries (total cholesterol, HDL cholesterol, glycosylated hemoglobin A 1c, and creatinine). Results For the total population, the mean total homocysteine level was 10.9 mol/l. Total homocysteine levels were higher in men than in women and increased with aging. With multiple linear regression analysis after adjustments for age and sex, the most powerful determinant of total homocysteine levels was serum creatinine (P 0.001). With multiple stepwise regression analysis after adjustments for age, sex, and other confounding factors, total homocysteine was significantly (P 0.05) related to IMT. Furthermore, when mean values of IMT adjusted for age, sex, and other related factors were analyzed across total homocysteine quartiles, IMT (P 0.05) showed a significant trend as total homocysteine level increased. Conclusions Plasma total homocysteine levels in Japan are similar to those reported in western countries. Mild hyperhomocysteinemia is an independent risk factor for increased carotid artery wall thickness in Japan as well. (Stroke. 2002;33: ) Key Words: atherosclerosis epidemiology homocysteine intima-media thickness Recently, carotid B-mode ultrasound imaging has been used for the quantitative evaluation of carotid atherosclerosis. 1 3 With the use of this method, studies showed that elevated plasma total homocysteine is a risk factor for atherosclerosis. 4 8 However, their data are limited to a single case-control study with a small number of cases 4 and crosssectional studies in elderly subjects. 5,6 Most of the studies were performed in the western countries. 4,5,7,8 To elucidate the relationship between hyperhomocysteinemia and atherosclerosis, it may be necessary to examine this relationship in a different population with a large number of cases in which the dietary habit and other environmental or cultural backgrounds are different from those of western countries. Another issue that needs to be clarified is the relationship between plasma total homocysteine and blood pressure (BP) because some previous studies 9 11 reported a positive relationship between them; however, data were also limited to a small number of cases. We therefore investigated the interrelationships among total homocysteine, hypertension, and carotid atherosclerosis in a general population with a large number of cases in Japan in which the dietary habit is different, the incidence of coronary artery disease is lower, and the incidence of cerebrovascular disease is higher compared with western countries. Subjects and Methods Subjects We performed an epidemiological study in 1999 in a farming community in southwestern Japan (Tanushimaru town). The total population aged 40 years in this district was 3463 persons (1647 men and 1816 women). A total of 1920 persons (794 men and 1126 women) received a health examination. Because plasma total homocysteine levels are strongly affected by diet, we performed blood tests in only 1111 subjects (452 men and 659 women) on an overnight fast (of 1920 subjects). Data Collection The subjects medical history, use of alcohol, and smoking were ascertained by questionnaire. Alcohol intake and smoking were classified as current habitual use or not. Height and weight were measured, and body mass index (kilograms per meter squared) was calculated as an index of obesity. Blood pressure was measured twice with the subjects in the supine position. The second BP after 5 deep breaths with the fifth phase diastolic pressure was used for analysis. Hypertensive subjects Received August 31, 2001; final revision received December 18, 2001; accepted February 27, From the Third Department of Internal Medicine and Cardiovascular Research Institute, Kurume University School of Medicine, Kurume, Japan. Correspondence to Hisashi Adachi, MD, PhD, Third Department of Internal Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume, , Japan. hadac@med.kurume-u.ac.jp 2002 American Heart Association, Inc. Stroke is available at DOI: /01.STR

2 2178 Stroke September 2002 were defined as those with BP 140 and/or 90 mm Hg or those receiving antihypertensive medication. Blood was drawn from the antecubital vein for determination of lipids (total cholesterol and HDL cholesterol), glycosylated hemoglobin A 1c (HbA 1c ), creatinine, and total homocysteine levels in the morning after 12-hour fast. Fasting blood samples were centrifuged within 1 hour after collection. Plasma total homocysteine was measured by high-pressure liquid chromatography. 12 Other chemistries, such as serum total cholesterol, HDL cholesterol (enzymatic assay method), HbA 1c (ion-exchange high-performance liquid chromatography), and creatinine (enzymatic assay method) were measured at a commercially available laboratory (Kyodo Igaku Laboratory, Fukuoka, Japan). Carotid intimal-medial wall thickness (IMT) of the common carotid artery was determined with the use of duplex ultrasonography (SSA-380A, Toshiba) with a 10-MHz transducer, with the subject in the sitting position with the head upright. Longitudinal B-mode images at the diastolic phase of the cardiac cycle were recorded by a single trained technician who was blinded as to the subject s background. The images were magnified and printed with a highresolution line recorder (LSR-100A, Toshiba). Measurements of IMT were made by the same technician using fine slide calipers at 3 levels of the lateral and medial walls 1 to 3 cm proximal to the carotid bifurcation. These 6 combined near- and far-wall measurements were averaged. Interobserver and intraobserver variations were 3.8% and 4.2%, respectively. 13 This study was approved by the Ukiha branch of the Japan Medical Association, by the mayor, and by the welfare section of Tanushimaru district. All participants gave informed consent. Statistical Methods Results are presented as mean SD. The medications for hypertension, hyperlipidemia, and diabetes were coded as dummy variables. Multiple linear regression analysis was performed for determinants of total homocysteine levels adjusted for age, sex, and confounding factors. Multiple linear regression analysis was also performed for determinants of carotid IMT measurement adjusted for age, sex, and confounding factors. The IMT levels by quartiles of the increasing total homocysteine values were compared by ANCOVA, adjusted for age, sex, and other related covariates. We confirmed that all of the parameters were not skewed distributions. Statistical significance TABLE 1. Clinical Characteristics of Subjects Characteristics Men (n 452) Women (n 659) Age, y Body mass index, kg/m Systolic BP, mm Hg Diastolic BP, mm Hg Total cholesterol, mmol/l HDL cholesterol, mmol/l HbA 1c,% Creatinine, mol/l Homocysteine, mol/l IMT, mm Alcohol intake, % yes Current smoking, % yes Medication Hypertension, % yes Hyperlipidemia, % yes Diabetes mellitus, % yes Values are mean SD or percentage, unless indicated otherwise. Figure 1. Distributions of plasma homocysteine levels in men and women. thcy indicates total homocysteine. was defined as P All statistical analyses were performed with the use of the SAS system. Results Characteristics of the 1111 subjects are presented in Table 1. Figure 1 shows distributions of plasma total homocysteine levels in men and women. Figure 2 shows mean total homocysteine levels stratified by age groups in men and women. With the exception of men aged 40 to 49 years, plasma total homocysteine levels increased with age in both men and women, and they were significantly higher (P 0.001) in men than in women in each age group except people aged 80 years. These results indicate that age and sex are major determinants of plasma total homocysteine, consistent with previous studies. Table 2 shows determinants of total homocysteine levels by multiple linear regression analysis, adjusted for age and sex. Systolic and diastolic BP, HbA 1c (inversely), creatinine, and smoking were significantly related to total homocysteine levels. Of these, creatinine showed the strongest significance. Then, by the use of multiple stepwise regression analysis, after further adjustments for creatinine, smoking, age, HbA 1c (inversely), and sex were significantly related to plasma total homocysteine (Table 3). Blood pressure no longer showed a significant association. Table 4 shows determinants of IMT with the use Figure 2. Mean plasma homocysteine levels stratified by age group in men and women.

3 Adachi et al Homocysteine and Atherosclerosis 2179 TABLE 2. Multiple Linear Regression Analysis for Determinants of Plasma Homocysteine Levels Adjusted for Age and Sex Variables 95% CI Body mass index, kg/m , Systolic BP, mm Hg , Diastolic BP, mm Hg , Total cholesterol, mmol/l , HDL cholesterol, mmol/l , HbA 1c,% , Creatinine, mol/l , Alcohol intake, % yes , Current smoking, % yes , Antihypertensive medication, % yes , Each row reports a separate multiple regression model for a determinant of plasma total homocysteine levels adjusted for age and sex. of multiple linear regression analysis adjusted for age and sex. Body mass index, systolic and diastolic BP, antihypertensive medication, HDL cholesterol (inversely), HbA 1c, creatinine, and total homocysteine levels were significantly related to IMT. Plasma total homocysteine was a determinant of IMT even after adjustments for age, sex, and other confounding factors (Table 5). Finally, mean values of IMT adjusted for age, sex, HDL cholesterol, systolic BP, and antihypertensive medication are presented across total homocysteine quartiles (Figure 3). IMT (P 0.05) showed a significant trend as total homocysteine level increased. Discussion Our results showed that the mean value and distribution of total homocysteine in Japan are similar to those in western countries 12 and that total homocysteine is an independent determinant of carotid atherosclerosis. 4 8 Determinants of Plasma Total Homocysteine Levels Normal values of fasting plasma total homocysteine levels are between 5 and 15 mol/l, 12 and the mean values of total homocysteine are 13 mol/l for men and 10 mol/l for women in western countries. 12 Although a population-based study with a small number of cases (n 474) has been reported from Japan, 6 our study is the first to report plasma total homocysteine levels of a large number of cases (n 1111) in a general population in Japan. The mean values TABLE 3. Multiple Stepwise Regression Analysis for Determinants of Plasma Homocysteine Levels Variables Coefficient 95% CI P Creatinine , Current smoking , Age , HbA 1c , Sex , Systolic BP , Diastolic BP , r TABLE 4. Multiple Linear Regression Analysis for Determinants of IMT Adjusted for Age and Sex Variables 95% CI Body mass index, kg/m , Systolic BP, mm Hg , Diastolic BP, mm Hg , Total cholesterol, mmol/l , HDL cholesterol, mmol/l , HbA 1c,% , Creatinine, mol/l , Homocysteine, mol/l , Alcohol intake, % yes , Current smoking, % yes , Antihypertensive medication, % yes , Each row reports a separate multiple regression model for a determinant of IMT adjusted for age and sex. of our data were 12.6 mol/l for men and 9.8 mol/l for women. Thus, the plasma total homocysteine levels in our population are similar to those in western countries despite differences in diet and other genetic and environmental factors. Our report is somewhat contradictory to that which demonstrated a positive association between plasma total homocysteine levels and cardiovascular mortality, in which the plasma total homocysteine level was reported as 8 mol/l in 20 Japanese men. 14 Previous studies indicated that age and sex are major determinants of plasma total homocysteine levels. 5,7,12 We also demonstrated similar findings. With the exception of men aged 40 to 49 years, plasma total homocysteine increased with age in both men and women (Figure 2). Several reasons for high total homocysteine levels in those aged 40 to 49 years must be considered. As shown in Table 3, smoking was one of the most powerful determinants of total homocysteine. Accordingly, the most likely reason for high total homocysteine in subjects aged 40 to 49 years is the high prevalence of smokers in this age group compared with the other age groups (53.3% versus 35.5%: P 0.01). Taken together, age and sex are major determinants of plasma total homocysteine levels. TABLE 5. Multiple Stepwise Regression Analysis for Determinants of IMT Variables Coefficient 95% CI P Age , Systolic BP , HDL cholesterol , Sex , Antihypertensive medication , Homocysteine , HbA 1c , Creatinine , Body mass index , r

4 2180 Stroke September 2002 Figure 3. Mean IMT values, adjusted for age, sex, systolic BP, HDL cholesterol, and antihypertensive medication, stratified by quartiles. thcy indicates total homocysteine. Our study showed that creatinine was the strongest related variable for total homocysteine. It is known that renal function affects plasma total homocysteine levels. 15 However, many previous studies did not consider renal function in the analysis of plasma total homocysteine levels. 4,10,11 It may be interesting to note that HbA 1c was inversely related to total homocysteine. This inverse association may seem puzzling, but it has been demonstrated that the mean plasma level is normal or low in insulin-dependent diabetes mellitus and non insulin-dependent diabetes mellitus patients Indeed, Wollesen et al 16 showed that renal hyperfiltration is the cause of the lower than normal mean plasma total homocysteine levels in populations of diabetic patients and concluded that glomerular filtration is rate limiting for renal clearance of homocysteine. Although we did not examine glomerular filtration in our study, the inverse relationship may well be explained by the greater glomerular filtration in subjects with higher HbA 1c. As demonstrated in Table 3, smoking was the second major determinant of total homocysteine level; this finding is consistent with those of western countries. 20,21 It is not well known how smoking is related to hyperhomocysteinemia. However, it is suggested that smoking may reduce vitamin B 6 in the body, which is one of the vitamins involved in the mechanism for homocysteine metabolism. 22 Determinants of IMT By multiple linear regression analysis (Table 4), total homocysteine was significantly related to IMT after adjustments for age and sex. To further examine this relationship, we performed the following 2 analyses. First, we evaluated determinants of IMT using multiple stepwise regression analysis (Table 5). After adjustments for age, sex, and other confounding factors, plasma total homocysteine levels were positively and independently associated with carotid IMT. Second, as shown in Figure 3, IMT showed a significant dose-dependent relationship among quartiles of total homocysteine levels. Taken together, our results indicate that hyperhomocysteinemia is a significant and independent risk factor for IMT. Positive associations were also found between IMT and known risk factors for carotid atherosclerosis such as BP (the strongest for systolic BP) and low HDL cholesterol. Consistent with previous reports, 1,2 hypercholesterolemia and smoking status were not risk factors for carotid atherosclerosis in our study. In conclusion, we found that plasma total homocysteine levels in Japan are similar to those in western countries and that high plasma total homocysteine levels are related to carotid atherosclerosis in Japan Acknowledgments This study was supported in part by the Kimura Memorial Heart Foundation, Fukuoka, Japan. We are grateful to members of the Japan Medical Association of Ukiha, the elected officials and residents of Tanushimaru, and the team of physicians for help in performing the health examinations. We wish to thank Drs K. Fukuda and A. Shibata (Department of Public Health, Kurume University School of Medicine), Drs K. Toyomasu and N. 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