The Third National Health and Nutrition Examination Survey (NHANES III) was conducted in 1988–1994 on a nationwide multi-stage probability sample of approximately 40,000 persons from the civilian, non-institutionalized population aged 2 months and over of the United States excluding reservation lands of American Indians. Of these, 31,311 were examined. Serum tHcy was measured only in the second half of the survey (Phase II), itself a representative sample of the U.S. population. The analyses of serum tHcy concentration in this report are restricted to 560 Mexican American men aged 40–74 years examined with valid serum tHcy and data on history of doctor diagnosed diabetes measured in the survey. The analyses of serum tHcy and fasting serum insulin are restricted to 186 men examined in the morning after fasting 9 to 24 hours with valid serum tHcy and insulin data, no history of diabetes, and not taking insulin or oral hypoglycemic agents. Numbers of persons in various correlation and regression analyses that follow may vary slightly due to differing numbers with missing values on selected other variables. Details of the plan, sampling, operation and response have been published as have procedures used to obtain informed consent and to maintain confidentiality of information obtained [20, 21].
Demographic data, medical history including doctor-diagnosed diabetes mellitus, and behavioral information was collected prior to the examination by household interview. US Office of Management and Budget race categories and Mexican American ethnicity were determined by self report . Examinations were carried out in a mobile examination center. Blood samples were obtained at the examination center. Blood in a red-top Vacutainer tube was allowed to stand for 45 min at room temperature to allow complete clotting and clot retraction. Samples were centrifuged at 1500 × g for 30 min at 4 degrees Celsius. Samples were frozen at -20 degrees Celsius. Vitamin B12 and folate in serum and folate in red blood cells were measured by the Division of Environmental Health Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, using the Quantaphase II Folate/B12 radioassay kit (Bio-Rad Laboratories, Hercules, CA) [22, 23]. Surplus serum samples were stored at -70 degrees for 8–36 months before analysis for total homocysteine concentration at the the US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University using the reverse-phase high-performance liquid chromatography (HPLC) and fluorescence detection method of Araki and Sato [22, 24, 25]. This assay measures total homocysteine, including both reduced and oxidized forms. Quality control methods are described elsewhere . Previous analyses have shown that length of time fasting did not affect tHcy concentration in this sample . Values for tHcy were missing if examined persons had no blood sample obtained or if insufficient surplus serum was available.
Frozen serum was sent to The Missouri Diabetes Diagnostic Laboratory and stored at -70 C until analysis for serum insulin concentration. Insulin radioimmunoassay (RIA) was performed using the Pharmacia Insulin RIA kit (Pharmacia Diagnostics AB, Uppsala, Sweden) for the majority of samples. (Prior to November, 1990, RIA kits purchased from Cambridge Laboratories, Cambridge, Massachusetts and its successor Ventrex, Inc., Cambridge, Massachusetts were used. Based on simultaneous analyses using all three assays, results from these kits were converted to Pharmacia equivalence.) Quality control procedures included the reanalysis of 5% of specimens randomly selected either within-assay or between assay and the analysis of batch specimens consisting of four levels of control pools before and after all survey specimens. The internal reference range for fasting serum insulin in non-obese, nondiabetic adults (mean age 28.1 years) was 3.08–11.92 uIU/mL . The cross-reactivity of Pharmacia insulin antibody with proinsulin is approximately 40%. Concentration of C-peptide in serum was determined using RIA in a 3-day, batch, sequential-saturation method with two incubations . The internal reference range for fasting serum C-peptide was 0.266–1.079 pmol/mL. Frozen plasma was sent to the Missouri Diabetes Diagnostic Laboratory for determination of plasma glucose using a modified hexokinase enzymatic method on the Cobas Mira Chemistry System (Roche Diagnostic Systems, Inc, Montclair, New Jersey). Within and between-assay quality control procedures were used. During the 6 years of the survey the coefficient of variation of the method was 1.6–3.7% . Glycated hemoglobin (HbA1c) in whole blood was determined using a high-performance liquid chromatographic assay on the Diamat automated HPLC system, model 723 (Bio-Rad Laboratories, Hercules, CA). The upper limit of normal for HbA1c in this system has been defined as 6.1% .
Technicians measured height to the nearest 0.1 centimeter, weight to the nearest 0.01 kg, triceps, subscapular, suprailiac and mid-thigh skinfold thickness to the nearest 0.1 millimeter and waist and buttocks circumference to the nearest 0.1 centimeter as described in detail elsewhere [20, 27, 28]. With the sample person standing at minimal respiration, waist circumference was measured in a horizontal plane at the level of the high point of the iliac crest to the nearest 0.1 cm. Hip circumference was measured in a horizontal plane at the maximum extension of the buttocks. The following were computed: waist-to-hip circumference ratio (WHR), and body mass index (BMI = weight /height2, kg/m2). Extensive descriptive data on diabetes, glucose tolerance, height, weight, BMI and obesity prevalence as well as serum tHcy, folate, and vitamin B12 in the NHANES III population have been published elsewhere and will not be duplicated here [9, 18, 19, 21, 23, 25, 28].
The plan of the present analyses was as follows. Detailed descriptive statistics and measures of association were computed initially using the Statistical Analysis System (SAS) . Pearson partial correlation was used to assess the association of the natural logarithm (LN) of serum tHcy concentration with other variables controlling for age or age and BMI . Correlation analysis results are presented because of their familiarity, ease of interpretation by a wide audience, and use in previous reports that may be compared to the present one. Multivariate logistic regression analysis was used to estimate models for controlling for confounding of the association of LN serum tHcy with history of doctor-diagnosed diabetes mellitus . Linear multivariate regression analysis was used to estimate models for controlling for confounding of the association of LN serum tHcy with fasting serum insulin . Only variables with pre-specified hypotheses were entered into the regression models. Following these preliminary analyses, analyses were performed using techniques that incorporated sampling weights and design features of the survey [29, 30]. Population estimates for means and percentiles of variables were produced using weighted SAS or SUDAAN procedures . Pearson correlations of LN serum tHcy with other variables were performed using SAS weighted analysis and all statistical testing and variance estimation were performed using the PROC LOGISTIC and PROC REGRESS procedure for regression models in the SUDAAN system [23, 24].