BMC Public Health 2008, 8:72 http://www.biomedcentral.com/1471-2458/8/72 Cadmium shows androgen and estrogen-like activities in vitro and in vivo, and it disrupts the male endocrine sys- tem in animal models. There is conflicting evidence regarding whether cadmium increases or decreases testo- sterone production in experimental models [4-6], with decreases more commonly seen after a single, large dose injection of cadmium and increases more commonly seen after chronic oral cadmium exposure [7]. Studies investi- gating the association of cadmium and sex steroid hor- mones in men have been inconsistent, finding either no association [8-10], or a positive association between cad- mium and testosterone levels [11-13]. Inconsistencies in these studies may be due to small sample sizes, to differ- ences in study design, or to inadequate control of con- founders such as tobacco use, a major source of cadmium [14] that is also associated with higher testosterone levels in men [15,16]. The purpose of the current analysis was to evaluate the association of urinary cadmium levels, a biomarker of long term and ongoing cadmium exposure, with serum concentrations of sex steroid hormones (total testoster- one, total estradiol, androstanediol glucuronide [AAG], estimated free testosterone, and estimated free estradiol) and sex hormone-binding globulin (SHBG) in the Third National Health and Nutrition Examination Survey (NHANES III). Methods Study Population NHANES III was a stratified, multistage probability survey designed to select a representative sample of the civilian non-institutionalized US population [17]. 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 hor- mones. Serum for hormone assays was available for 1,470 participants (75%). After excluding 25 participants miss- ing 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 par- ticipants missing data for other covariates, the final sam- ple 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 Institu- tional 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. Data Collection Demographic, household income, physical activity, ciga- rette smoking, and alcohol consumption data were col- lected using standardized questionnaires during an in- home interview [17]. 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 [18]. Serum cotinine was measured using liquid chromatography/atmospheric pressure ionization tandem mass spectrometry [19]. Atomic absorption spec- trometry was used to measure blood lead as described by Sassa and colleagues [20] and serum selenium as described by Lewis and colleagues [21]. All materials used for collecting and processing urinary cadmium specimens were screened for possible cadmium contamination [22]. 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. Uri- nary cadmium was measured by graphite furnace atomic absorption with Zeeman background correction, using the CDC modification of the method of Pruszkowska and colleagues [23]. Since NHANES III only collected spot urine samples, all analyses were performed using creati- nine 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 ana- lyzer [24]. Sex Steroid Hormones Participants in the morning examination session fasted overnight before having blood drawn. After centrifuga- tion, serum was aliquotted and stored at -70°C. Serum concentrations of testosterone, estradiol, AAG (a metabo- lite 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 immu- noassays on the 2010 Elecsys autoanalyzer (Roche Diag- nostics, Indianapolis, IN). AAG was measured by an enzyme immunoassay (Diagnostics Systems Laboratories, Webster, TX). The detection limits of the assays were 0.02 Page 2 of 7 (page number not for citation purposes)