Mol/ml recombinant CYP2J2 was mixed with 2 pmol/ml rat cytochrome P450 reductase (CPR), 1 pmol/ml cytochrome b5, buffer containing 100 mM potassium phosphate (pH 7.four), and 50 mM DLPC on iceCYP2J2 Activity, Induction, and Inhibition in Cardiomyocytessystem. Ten microliters from the sample was injected on a Phenomenex (Torrance, CA) Aeris PEPTIDE XB-C18 column (1.7 mm, 150 2.ten mm). The mobile phases consisted of aqueous phase A: 0.1 formic acid in H2O, and organic phase B: 0.1 formic acid in acetonitrile. The samples were analyzed applying the following gradient: mobile phase B: 0 minutes, 3 ; three minutes, 30 ; 5 minutes, 1050 ; 8.four minutes, 50 ; 8.four.five minutes, 500 ; 8.five.five minutes, 90 ; 9.510 minutes, 90 ; one hundred.5 minutes, 3 . The column was re-equilibrated to initial circumstances for 1 minute along with the flow rate was 0.three ml/min. The source temperature was 350 , the capillary charge was 3500 V, and gas flow was five l/min.Leptomycin B The CYP2J2-specific peptide sequence monitored was VIGQGQQPSTAAR. Standards for mass spectrometry were custom ordered from and synthesized by Thermo Fisher (Rockford, IL). Similarly, the heavy-labeled peptide made use of as an internal typical was synthesized using a heavy (13C6, 15N4) arginine residue at the C-terminal end on the fragment (+10 Da), also by Thermo Fisher. The transitions monitored were 656.85 . 602.33 (CYP2J2 fragment) and 661.9 . 612.1 (synthesized peptide internal standard). The protein content material was determined using a normal curve containing the following concentrations of synthesized unlabeled peptide (nM): 0, 0.5, 1, two.5, 5, 10, 25, 50, one hundred, 500. The internal standard concentration was the identical as above (50 nM). Kinetic Parameters of CYP2J2-Mediated Metabolism in Human Cardiomyocytes. Experiments to decide Km and Vmax of terfenadine and astemizole hydroxylation by the cells were carried out in triplicates. Kinetic parameters were measured under established linearity for cell density and time. Cells have been plated in 96-well plates at an approximate density of 100,000 cells per well and allowed to adhere for the plate for 24 hours in 100 ml of total media. The cells were then washed with phosphate-buffered saline (100 ml) and dosed with terfenadine or astemizole in serum-free media [100 ml, containing 0.1 dimethylsulfoxide (DMSO)] at varying concentrations (0, 0.02, 0.05, 0.1, 0.two, 0.five, 1, two, 5, 10, 25, 50, and one hundred mM). Immediately after 2 hours of incubation at 37 , the reaction was quenched by the addition of acetonitrile (one hundred ml) containing 0.1 mM midazolam as internal standard. Vigorous pipetting was then used to facilitate cellular detachment in the plate and lysis.VV116 The samples have been centrifuged (3500g, ten minutes), and 150 ml was transferred to a new 96-well plate for spectrometric analysis.PMID:24293312 To rule out prospective involvement by CYP3A4 or CYP2C8, we also conducted activity experiments with probe substrates for CYP3A4 and CYP2C8. The incubations were carried out as outlined for Km and Vmax determination of CYP2J2 above but employing midazolam (three mM) or amodiaquine (2 mM) as probe substrates for CYP3A4 and CYP2C8, respectively, rather of terfenadine. Metabolite Detection and Quantification. Metabolites and parent were quantified on a Sciex API4000 liquid chromatography andem mass spectrometry (LC-MS/MS; Applied Biosystems) connected to a Shimadzu HPLC System (LC-10AD, SCL-10A) equipped having a CTC PAL Autosampler (LEAP Technologies, Carrboro, NC). Ten microliters of supernatant was injected on an Agilent Zorbax XDB C8-column (two.
