This is consistent with that observed previously in HLM (24). assay and the levels of EXE, 17-DHE and 17-DHE-Gluc were quantified by UPLC/MS in patients urine and plasma. A 39-fold decrease ((*2/*2) subjects vs. subjects with the (*1/*1) genotype. The plasma levels of 17-DHE-Gluc was decreased 29-fold ((*2/*2) genotype vs. subjects with (*1/*1) genotype. The levels of Caffeic Acid Phenethyl Ester plasma EXE-adjusted 17-DHE was 28% higher ((*2/*2) genotype vs. subjects with the (*1/*1) genotype. These data show that UGT2B17 is the major enzyme responsible for 17-DHE-Gluc formation and that the copy number variant may play a role in inter-individual variability in 17-DHE levels study have strongly implicated UGT2B17 as the major enzyme responsible for the glucuronidation of 17-DHE (24). A polymorphic whole-gene deletion of the gene has been recognized with an allelic prevalence of ~30% in Caucasians (27C30), and this copy number variant (CNV) was associated with decreased formation of Caffeic Acid Phenethyl Ester 17-DHE-Gluc in human liver microsomes (HLMs) (24). The goal of the present study was to examine the effect of the CNV on 17-DHE-Gluc formation and 17-DHE levels TaqMan? Copy Number Assay were purchased from Life Technologies (Carlsbad, CA, USA). Subjects and samples Ninety-six post-menopausal Caucasian breast cancer patients (age range: 35 to 89 y) with ER+ breast tumors taking 25 mg CBL2 EXE daily (orally) and one healthy volunteer not taking EXE (used as a control) were recruited from your breast oncology medical center at the Penn State Hershey Malignancy Institute into this study. All recruited subjects provided Caffeic Acid Phenethyl Ester blood (10 cc) and urine (up to 50 mL). Patients were excluded from the study if they had been given EXE concurrently with adjuvant chemotherapy or if they were taking other adjuvant endocrine therapies, or were on chronic corticosteroid or megestrol acetate therapies. Approval was obtained from the Institutional Review Table at Penn State University with informed consent obtained from all subjects and with all specimens being Caffeic Acid Phenethyl Ester de-identified. Specimens were obtained 4~6 hours after last pill ingestion by a trained nurse coordinator after patients had been taking EXE for at least 28 days. Pretreatment medical histories including a comprehensive list of current medications and results of physical and laboratory examinations were also collected for each subject. Blood was separated by differential centrifugation and buffy coat was used to extract genomic DNA. Aliquoted urine samples and plasma fractions of blood samples were stored at ?80C until analysis. Sample preparation Genomic DNA was purified from blood samples using PureLink ? Genomic DNA Kits. DNA quantity and purity were decided photometrically at 260 nm and 280 nm using the Thermo Scientific Nanodrop 2000 spectrophotometer (Waltham, MA, USA). For EXE metabolite analysis, a 50-L aliquot of each urine sample was first spiked with 10 L of a mixture of deuterium-labeled internal requirements in methanol, including D3-EXE (0.17 M), D3-17-DHE (1.7 M) and D3-17-DHE-Gluc Caffeic Acid Phenethyl Ester (1.1 M). Ninety L of methanol was then added to extract EXE and its metabolites. After vortexing and subsequent centrifugation at 16,100 g for 10 min at 4C, an aliquot of 50 L of supernatant was transferred to a sample vial for analysis by ultra-pressure liquid chromatography (UPLC)/mass spectrometry (MS). For analysis of plasma, 10 L of each plasma sample was first mixed with 10 L of a mixture of deuterium-labeled internal standards as explained above. Eighty L of methanol was then added to precipitate proteins. After vortexing and subsequent centrifugation at 16,100 g for 10 min at 4C, an aliquot of 50 L of supernatant was transferred to a sample vial for analysis by UPLC/MS. UPLC/MS conditions For the simultaneous analysis of EXE, 17-DHE and 17-DHE-Gluc in urine and plasma, samples prepared as explained above were analyzed using a UPLC/MS system (Waters), consisting of an Acquity UPLC pump, an Acquity sample manager-FTN, an ACQUITY UPLC BEH column C18 (2.1100 mm, 1.7 m particle size), and a XEVO G2-S QTOF mass spectrometer. UPLC was performed at a circulation.