Chapter 3. Subjects and Methods (Figure 2)
3.3 Homocysteine and long-term vascular events (Chin-Shan cohort)
The participants were enrolled in the Chin-Shan Community Cardiovascular Study, a prospective community-based study of risk factors and cardiovascular consequences.3, 96,
97 In brief, the study was started in 1990 with an initial cohort 3602 participants, who were recruited on the basis of official registrations. This study was approved by the institutional review boards of the National Taiwan University. The study collected information regarding medical history, the results of physical examination and
laboratory tests, and an assessment of health status that included evidence of stroke and cardiovascular disease since baseline assessment and the follow-up period. Among the participants, 2117 subjects had homocysteine measurement during 1994 to 1995, in whom 2009 were free from history of stroke, cardiovascular disease and cancer. We collected detailed information about lifestyle factors including alcohol intake, smoking, and regular exercise, as well as data regarding socioeconomic status and family history of stroke and coronary heart disease (CHD). With regard to the follow-up schedule, we gathered information about stroke, cardiovascular events and deaths through monthly collections of official death certificate documents, by annual questionnaires, and by house-to-house visits.
3.3.2 Ascertainment of events
The study outcomes were stroke, CHD and all-cause death. Stroke was defined as a
sudden neurological deficit of vascular origin that lasted longer than 24 h and was proved by evidence from an imaging study 98. Transient ischemic attacks were not included in this definition. Incident CHD cases were defined as non-fatal myocardial infarction, fatal CHD, and hospitalization for percutaneous coronary intervention and coronary artery bypass surgery. Fatal CHD was considered to have occurred if fatal myocardial infarction was confirmed by hospital records, if CHD was listed as the cause of death on the death certificate or was the underlying and most plausible cause of death
99. Deaths from any cause were identified from official certificate documents and further
verified by house-to-house visits.
3.3.3 Measurements of serum homocysteine and other biochemical variables The procedures of blood sampling have been reported elsewhere 98, 100, 101. In brief, all venous blood samples drawn after a 12-h overnight fast were immediately refrigerated and transported within 6 h to the National Taiwan University Hospital. Serum samples were then stored at -70 ºC until analysis. Serum Hcy were collected into tubes
containing ethylenediaminetetraacetic acid and were measured by fluorescence
polarization immunoassay (Abbott IMx System) 93, 94, which correlated well with high performance liquid chromatography. The coefficients of variation were within 5.2% 95. 3.3.4 Statistical methods
Participants were classified by quartiles of Hcy concentration. Continuous variables were presented as mean (SD) or median values, and categorical data were presented in contingency tables. ANOVA and the chi-square test were used to test differences between quartiles. Relationships between baseline Hcy concentrations and blood pressure, fasting glucose, body mass index and lipid profiles were evaluated with age- and sex-adjusted Spearman partial correlation coefficients.
Incidence rates for stroke, CHD and all-cause death were calculated for each Hcy
quartile by dividing the number of cases by the number of person-years of follow-up.
We estimated the relative risk (RR) and 95% confidence interval (CI) by the
multivariate Cox proportional hazards models. We specified 4 models for estimating the RRs of events in higher Hcy quartiles relative to the lowest quartile. In model 1, we estimated the univariate RR of Hcy with the first quartile as the reference. In model 2, we adjusted for age and sex variables. In model 3, we additionally adjusted for body mass index, educational level, occupation, alcohol intake, smoking, and regular exercise.
In model 4, we adjusted for the presence/absence of hypertension and diabetes at baseline, family history of stroke and coronary heart disease, continuous variables of total cholesterol, triglyceride, HDL-C and LDL-C concentrations along with
adjustments for the variables in model 3. We modeled Hcy concentrations as quartiles to avoid the assumption of linearity and to reduce the effects of outliers, and we used median Hcy concentrations for the categories to test for linear trends across quartiles. In secondary analyses, we constructed receiver-operating-characteristic (ROC) curves to generate the optimal cutoff point with highest Youden’s index for the occurrence of stroke, CHD and death. We then calculated hazard ratios for stroke, CHD and death for high and low Hcy level using aforementioned cutpoints. The multivariate-adjusted hazard ratios were presented as the model 4.
To test the feasibility of the cutpoints of Hcy, ROC curves were further plotted again for full adjusted model (age, sex, body mass index, educational level, occupation, alcohol intake, smoking, regular exercise, hypertension, diabetes and family history of stroke and coronary heart disease) with Hcy and for that without Hcy. The
discrimination of models with and without events were assessed by comparing the c statistics for CHD and death respectively, where Hcy level was treated as binary variable with aforementioned cutpoints. In addition, the increased discriminative value
of Hcy was further examined with the method described by Pencina et al.102. Two statistics, including, net reclassification improvement, and, integrated discrimination improvement, were presented with the priori meaningful risk categories set as follows:
the 10-year estimated low, median and high risk rate were defined at 5%, 15% and 25%
for stroke; 10%, 25% 30% for CHD ; and 5%, 10% 20% for death 103.
All statistical test were 2-tailed with type I error of 0.05, and P values <0.05 were considered statistically significant. Analyses were performed with SAS software (version 9.1; SAS Institute).
3.4 Homocysteine lowering clinical trial on Alzheimer’s disease