**2. Subjects and Methods**

A total of 1323 asymptomatic Chinese adults (mean age 47.1 ± 11.7 years and 47.5% male) in Southern China (Hong Kong, Macau, Pan Yu, n = 395) and Northern China (Yu County in Shanxi and Three Gorges Territories of Yangtze River, n = 928) were studied in 1996–2007, as part of the international collaborative Chinese Atherosclerosis in the Aged and Young Project (CATHAY Study). The study protocol and some related findings have been reported previously [14–18].

All recruited subjects were apparently healthy. They were not known to have hypertension, diabetes mellitus or metabolic syndrome, had no major vascular, hepatic or renal disease, and were not taking any regular medications, including vitamin supplementation. Nearly all subjects (>95%) were local born residents and the other migrated to the county for over 10 years. After fasting for 14 h and signing written informed consent, their cardiovascular risk profiles, including smoking, body mass index (BMI), waist circumference, waist hip ratio (WHR), systolic and diastolic blood pressure (SBP, DBP) were measured. On recruitment, blood was taken once for fasting lipid profile (total, high and low lipoprotein cholesterol, TC, HDL-C, LDL-C and triglycerides TG), creatinine, vitamin B12, folate, and fasting total homocysteine (HC). Fasting glucose was measured by haemstix and HC was evaluated on stored frozen sample by enzymatic immune assay (Abbott IMX analyses, Abbott Peak, IL, USA). Blood was assayed in batches at the The Hospital Central Corde de Januarie, Macau, and The Prince of Wales Hospital, Hong Kong, currently accredited by the USA laboratory centres. MTHFR genotypes were evaluated by PCR technique at the Li Hysan research laboratory of the Chinese University of Hong Kong. Metabolic Syndrome (MS) was diagnosed according to International Diabetes Federation (IDF) criteria [19,20].

Our research study and informed consent form were reviewed and approved by our institutional research ethics committee of The Chinese University of Hong Kong (CREC 2000-108). This study complied with the 1995 and 2003 Helsinki Declaration for human studies.
