He study design was entered into the SetupX database [17]. Plasma samples

He study design was entered into the SetupX database [17]. Plasma samples were aliquotted and maintained at 280uC until use, at which point 30 ml of plasma samples were thawed, extracted and derivatized [18]. Briefly, 15 ml aliquots were extracted with 1 ml of degassed acetonitrile:isopropanol:water (3:3:2) at 220uC, centrifuged and decanted with subsequent evaporation of the solvent to complete dryness. A clean-up step with acetonitrile/water (1:1) removed membrane lipids and triglycerides and the supernatant was again dried down. Internal standards C8 30 FAMEs were added and the sample was derivatized with methoxyamine hydrochloride in pyridine and subsequently by MSTFA (Sigma-Aldrich) for trimethylsilylation of acidic protons. A Gerstel MPS2 automatic liner exchange system was used to inject 1 ml of sample at 50uC (ramped to 250uC) in splitless mode with a 25 sec splitless time. An Agilent 6890 gas chromatograph (Santa Clara, CA) was used with a 30 m long, 0.25 mm i.d. Rtx5Sil-MS Tubastatin A site column with 0.25 mm 5 diphenyl film; an additional 10 m integrated guard column was used (Restek, Bellefonte PA). Chromatography was performed at a constant flow of 1 ml/min, ramping the oven temperature from 50uC for to 330uC over 22 min. Mass spectrometry used a Leco Pegasus IV time of flight mass (TOF) spectrometer with 280uC transfer line temperature, electron 86168-78-7 site ionization at 270 V and an ion source temperature of 250uC. Mass spectra were acquired from m/z 85?00 at 20 spectra/sec and 1750 V detector voltage. Result files were exported to our servers and further processed by our metabolomics BinBase database. All database entries in BinBase were matched against the Fiehn mass spectral library of 1,200 authentic metabolite spectra using retention index and mass spectrum information or the NIST05 commercial library. Identified metabolites were reported if present with at least 50 of the samples per study design group (as defined in the SetupX database). Quantitative data were normalized to the sum intensities of all known metabolites and used for statistical investigation.Materials and Methods SubjectsPlasma samples and associated clinical data were collected as part of the Pharmacogenomic Evaluation of Antihypertensive Response (PEAR) study, which is a prospective, randomized, parallel group titration study undertaken in primary care patients with mild to moderate essential hypertension. The objectives and design of the PEAR study have been described previously [16]. Subjects of any self-defined race or ethnicity, aged 17?5 years, were enrolled at the University of Florida (Gainesville, FL), Emory University (Atlanta, GA), and the Mayo Clinic (Rochester, MN). The study was approved by the institutional review board at each institution, and all participants provided informed, written consent prior to being screened for study participation. Enrolled subjects had newly diagnosed, untreated or treated hypertension; if treated, the antihypertensive(s) was discontinued with a minimum washout of 18 days. Briefly, exclusion criteria included diastolic blood pressure (DBP) .110 mm Hg or systolic blood pressure (SBP) .180 mm Hg, secondary hypertension, cardiovascular disease, diabetes, renal insufficiency (serum creatinine .1.5 in male patients and .1.4 in female patients), liver enzymes .2.5 times the upper limit of normal, and treatment with BP-raising drugs, among others.Genotyping of Lipase Candidate GenesGenotypes of 16 lipase genes were obtained from the Illum.He study design was entered into the SetupX database [17]. Plasma samples were aliquotted and maintained at 280uC until use, at which point 30 ml of plasma samples were thawed, extracted and derivatized [18]. Briefly, 15 ml aliquots were extracted with 1 ml of degassed acetonitrile:isopropanol:water (3:3:2) at 220uC, centrifuged and decanted with subsequent evaporation of the solvent to complete dryness. A clean-up step with acetonitrile/water (1:1) removed membrane lipids and triglycerides and the supernatant was again dried down. Internal standards C8 30 FAMEs were added and the sample was derivatized with methoxyamine hydrochloride in pyridine and subsequently by MSTFA (Sigma-Aldrich) for trimethylsilylation of acidic protons. A Gerstel MPS2 automatic liner exchange system was used to inject 1 ml of sample at 50uC (ramped to 250uC) in splitless mode with a 25 sec splitless time. An Agilent 6890 gas chromatograph (Santa Clara, CA) was used with a 30 m long, 0.25 mm i.d. Rtx5Sil-MS column with 0.25 mm 5 diphenyl film; an additional 10 m integrated guard column was used (Restek, Bellefonte PA). Chromatography was performed at a constant flow of 1 ml/min, ramping the oven temperature from 50uC for to 330uC over 22 min. Mass spectrometry used a Leco Pegasus IV time of flight mass (TOF) spectrometer with 280uC transfer line temperature, electron ionization at 270 V and an ion source temperature of 250uC. Mass spectra were acquired from m/z 85?00 at 20 spectra/sec and 1750 V detector voltage. Result files were exported to our servers and further processed by our metabolomics BinBase database. All database entries in BinBase were matched against the Fiehn mass spectral library of 1,200 authentic metabolite spectra using retention index and mass spectrum information or the NIST05 commercial library. Identified metabolites were reported if present with at least 50 of the samples per study design group (as defined in the SetupX database). Quantitative data were normalized to the sum intensities of all known metabolites and used for statistical investigation.Materials and Methods SubjectsPlasma samples and associated clinical data were collected as part of the Pharmacogenomic Evaluation of Antihypertensive Response (PEAR) study, which is a prospective, randomized, parallel group titration study undertaken in primary care patients with mild to moderate essential hypertension. The objectives and design of the PEAR study have been described previously [16]. Subjects of any self-defined race or ethnicity, aged 17?5 years, were enrolled at the University of Florida (Gainesville, FL), Emory University (Atlanta, GA), and the Mayo Clinic (Rochester, MN). The study was approved by the institutional review board at each institution, and all participants provided informed, written consent prior to being screened for study participation. Enrolled subjects had newly diagnosed, untreated or treated hypertension; if treated, the antihypertensive(s) was discontinued with a minimum washout of 18 days. Briefly, exclusion criteria included diastolic blood pressure (DBP) .110 mm Hg or systolic blood pressure (SBP) .180 mm Hg, secondary hypertension, cardiovascular disease, diabetes, renal insufficiency (serum creatinine .1.5 in male patients and .1.4 in female patients), liver enzymes .2.5 times the upper limit of normal, and treatment with BP-raising drugs, among others.Genotyping of Lipase Candidate GenesGenotypes of 16 lipase genes were obtained from the Illum.

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