Journal of Experimental & Clinical Cancer Research 2009, 28:135 http://www.jeccr.com/content/28/1/135 of 4 ng/ml [15]. Among the study participants whose PSA level was higher than 4 ng/ml, we ultimately included in the control group only those who tested negative at the prostate biopsy. We requested and obtained the pertinent medical records from the urologists. tural information about the analytes by fragmenting the parent ions inside the mass spectrometer and identifying the resulting daughter/fragment ions. We processed the resulting data and quantified the estrogen metabolites using the QuanLynx software (Waters). For each case, four control subjects were randomly chosen after matching for age (within a 3-year-range), race and date of recruitment. The independent variables of interest, namely 2-OHE1, 16α-OHE1 and the 2-OHE1 to16α- OHE1 ratio, were available for 110 controls and 26 cases, thus we conducted the present analysis on 136 subjects. Hormonal Determinations For standardization purposes, we collected morning spot urine between 7:00 a.m. and 9:00 a.m. from all partici- pants. We then transferred the aliquoted urine samples to the Eppley Institute, University of Nebraska Medical Center (UNMC), and stored them at -80°C until analysis. Each sample was thawed only once prior to analysis. We handled urine samples identically and located them in the laboratory runs randomly. All laboratory personnel were blinded in regards to case-control status. All of the study samples were analyzed in duplicate. Two-milliliter aliq- uots of urine were partially purified throughout solid phase extraction (SPE) with a phenyl cartridge (Varian, Palo, Alto, CA) and ultra-performance liquid chromatog- raphy/tandem mass spectrometry (LC/MS-MS). Analytes were identified based on their retention time and tandem mass spectrometry. Standards of the catechol estrogens 2- OHE1(E2) and 16α-OHE1(E2) were purchased from Ster- aloids Inc. (Newport, RI). To avoid the artifacts and errors introduced by maintain- ing the urine samples at 37°C for 8 hours, we carried out all the analyses without glucuronidase/sulfatase treat- ment. We adjusted urine samples to pH 7 with 1 M NaOH or 1 M HCl. We performed the LC/MS analyses through a Waters Acquity ultra-performance liquid chromatography (UPLC) system connected with a high performance Quat- tro Micro triple quadruple mass spectrometer designed for LC/MS-MS operation. We performed the analytical sepa- rations on the UPLC system using an Acquity UPLC BEH C18 1.7 μm column (1 × 100 mm) at a flow rate of 0.15 ml/min. We then moved the elutions from the UPLC col- umn to the Quattro Micro mass spectrometer. The ionization method used for MS analysis was Electro- spray ionization (ESI) in both the positive ion (PI) and negative ion (NI) mode with an ESI-MS capillary voltage of 3.0 kV, an extractor cone voltage of 3 V, and a detector voltage of 650 V. We performed the MS-MS in the multi- ple reaction monitoring (MRM) mode to produce struc- To calculate limits of detection, we injected various con- centrations of the analytes to LC/MS-MS. The detection limit was considered to be the injected amount that resulted in a peak with a height at least two or three times higher than the baseline. The detection limits of 2-OHE1 and 16α-OHE1 were 18 fmol and 349 fmol, respectively. Intra-assay coefficients of variation for 2-OHE1 and 16α- OHE1 were 3.2% and 3.0%, respectively. Inter-assay coef- ficients of variation were 1.9% and 3.5%, respectively. We had previously measured the intra- and inter-individ- ual variability for 2-OHE1, 16α-OHE1 determinations and their ratio over a one year period [13]. The intra-class correlation coefficients (ICCs) and lower limit of 95% CI (in parentheses) were 0.70 (0.46), 0.63 (0.35) and 0.78 (0.62), respectively. We had previously provided a detailed description of the procedures related to the relia- bility assessment [13]. Systematic Review We conducted a systematic search of the literature to iden- tify additional studies published up to August 2009 which examined the association between estrogen metabolites and Pca risk using our standard methods [19-22]. We searched MEDLINE (January 1966 onwards) and EMBASE (January 1980 onwards). An expert librarian designed a search strategy combining terms for estrogens, estrogen metabolites and prostate specific antigen (PSA) with terms for Pca (available upon request). We screened titles and abstracts in duplicate using the following inclusion criteria: observational studies investigating prostate can- cer risk in relation to estrogen metabolism. We included studies providing at least one measure of either urinary or circulating levels of 2-OHE1, 16α-OHE1 and the 2-OHE1 to 16α-OHE1 ratio. Statistical analysis We examined distributions for all variables of interest by determining the frequencies, mean, median and measures of variance. To evaluate the statistical significance of the unadjusted associations between case/control status and participants' characteristics, we used either Fisher's exact tests or Pearson's chi-square tests for categorical variables. The 2-OHE1 and 16-αOHE1 urinary levels were standard- ized by total urinary creatinine. We used unconditional logistic regression to compute crude and adjusted odds ratios (OR) and 95% confident interval (CI) of Pca in rela- tion to 2-OHE1, 16-αOHE1 and the ratio of 2-OHE1 to Page 3 of 8 (page number not for citation purposes)