NHANES III was a stratified, multistage probability survey designed to select a representative sample of the civilian non-institutionalized US population . NHANES III included two phases (phase I: October 1988 – October 1991 and phase II: September 1991 – October 1994), each capable of independently producing unbiased national estimates. Within each phase, participants were randomly assigned to either a morning or afternoon/evening examination. The present study was conducted among men aged ≥ 20 years participating in the morning examination session of phase I (N = 1,967). The study was restricted to participants in the morning session to reduce extraneous variation due to diurnal production of sex hormones. Serum for hormone assays was available for 1,470 participants (75%). After excluding 25 participants missing data for urinary cadmium, 11 participants missing data for urinary creatinine, 7 participants missing data for serum testosterone, 5 participants missing data for serum SHBG, 9 participants missing data for serum albumin, 11 participants missing data for serum cotinine, and 140 participants missing data for other covariates, the final sample included 1,262 men. The protocol for NHANES III was approved by the National Center for Health Statistics of the Center for Disease Control and Prevention Institutional Review Board. All participants gave written informed consent. The assay of stored serum specimens for the Hormone Demonstration Program was approved by the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health and the National Center for Health Statistics, Centers for Disease Control and Prevention.
Demographic, household income, physical activity, cigarette smoking, and alcohol consumption data were collected using standardized questionnaires during an in-home interview . Whole body electrical resistance was measured using a Valhalla Scientific Body Composition Analyzer (model 1990B; Valhalla Scientific, Inc., San Diego, CA) and prediction equations were used to predict percent body fat . Serum cotinine was measured using liquid chromatography/atmospheric pressure ionization tandem mass spectrometry . Atomic absorption spectrometry was used to measure blood lead as described by Sassa and colleagues  and serum selenium as described by Lewis and colleagues .
All materials used for collecting and processing urinary cadmium specimens were screened for possible cadmium contamination . A spot urine specimen was collected and shipped at -20°C to the NHANES laboratory at the National Centers for Environmental Health at the Centers for Disease Control and Prevention in Atlanta, GA. Urinary cadmium was measured by graphite furnace atomic absorption with Zeeman background correction, using the CDC modification of the method of Pruszkowska and colleagues . Since NHANES III only collected spot urine samples, all analyses were performed using creatinine corrected urinary cadmium values (urinary cadmium divided by urinary creatinine concentrations, expressed as μg/g) to account for between participant differences in urine dilution. Urinary creatinine was measured using the method of Jaffe with a Beckman ASTRA automated analyzer .
Sex Steroid Hormones
Participants in the morning examination session fasted overnight before having blood drawn. After centrifugation, serum was aliquotted and stored at -70°C. Serum concentrations of testosterone, estradiol, AAG (a metabolite of dihydrotestosterone), and SHBG were measured in 2005 at the laboratory of Dr. Nader Rifai at Children's Hospital in Boston, MA. Sex steroid hormone levels and SHBG are stable after multiple freeze-thaw cycles [25, 26]. Testosterone, estradiol, and SHBG levels were quantified using competitive electrochemiluminescence immunoassays on the 2010 Elecsys autoanalyzer (Roche Diagnostics, Indianapolis, IN). AAG was measured by an enzyme immunoassay (Diagnostics Systems Laboratories, Webster, TX). The detection limits of the assays were 0.02 ng/mL for testosterone, 5 pg/mL for estradiol, 0.33 ng/mL for AAG, and 3 nmol/L for SHBG. The coefficients of variation (CV%) for quality control specimens included during the analyses of the NHANES III samples ranged from 5.8 to 5.9% for testosterone, 2.5 to 6.7% for estradiol, 5.0 to 9.5% for AAG, and 5.3 to 5.9% for SHBG. Free testosterone concentrations were estimated from measured testosterone, SHBG, and albumin, while free estradiol concentrations were estimated from measured estradiol, SHBG, and albumin [27, 28].
Participants were categorized into quartiles of urinary cadmium based on the weighted population distribution. Age and race-ethnicity adjusted means and percentages were calculated by linear regression for continuous covariates and logistic regression for dichotomous covariates.
Due to skewed distributions of sex steroid hormones, we calculated adjusted geometric means by quartile of cadmium using multiple linear regression on log-transformed hormone levels. We also present analyses for molar ratios of estradiol to testosterone, testosterone to SHBG, and estradiol to SHBG. Initial models adjusted for age and race-ethnicity. Subsequent models further adjusted for smoking status and serum cotinine. Additional models further adjusted for household income, physical activity, alcohol consumption, percent body fat, blood lead, and serum selenium. Tests for linear trend across quartiles of cadmium were computed by including an ordinal variable with the median of each quartile of cadmium in the linear regression models. Due to the importance of smoking in determining cadmium levels and the possibility of overadjustment, we additionally present models separately for never, former, and current smokers.
Data were analyzed using SUDAAN (version 9.0; Research Triangle Institute, Research Triangle Park, NC) to account for the complex NHANES sampling design, including unequal probabilities of selection, over-sampling, and non-response.