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2 protocols using acetamidophenol

1

Characterization of Cytochrome P450 Activity

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Human hepatic microsomes were obtained from Xenotech (Lenexa, KS, USA) and expressed CYP1A2, CYP2C9*1 +OR, CYP2C19 +OR, CYP2E1+OR+ cytochrome b5, CYP2D6*1+OR, and CYP3A4 +OR were purchased from Gentest (Woburn, MA, USA). The designation ‘+OR’ signifies that the preparation contained supplemental, cDNA-expressed cytochrome P450 reductase. Ketoconazole, dextromethorphan, dextrorphan, diclofenac, phenacetin, acetamidophenol, testosterone, 6-β-hydroxy-testosterone, midazolam, quinidine, sulfaphenazole, and nicotinamide adenine dinucleotide phosphate (NADPH) were acquired from Sigma Chemical Co (St Louis, MO, USA). S-(+)-mephenytoin, 4′-hydroxy-mephenytoin, 1′-hydroxy-midazolam, and 4′-hydroxy-diclofenac were obtained from Ultrafine Chemicals (Manchester, UK). 3-N-benzylnirvanol was purchased from Gentest. All other chemicals were the highest grades available.
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2

Comprehensive Metabolite Quantification Assay

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Caffeine, allopurinol, paracetamol-glucuronide, paracetamol sulphate, high-purity trifluoroacetic acid, salicyluric acid, N,N-diethyl-paraphenylenediamine (DEPPD), sodium acetate, metaphosphoric acid (MPA) and hydrogen peroxide were supplied by Sigma Chemicals. Paracetamol-mercapturate was supplied by Toronto Research Chemicals. HPLC grade acetonitrile and water was supplied by Burdick & Jackson. Ferrous sulphate was supplied by Labchem. Acetamidophenol, phenylbutyric acid, 2,3 & 2,5 dihydroxy benzoic acid, NADPH, GSH, GSSG and catechol were all obtained from Sigma-Aldrich. Ethyl acetate and diethyl ether were purchased from Merck. N,O Bis(trimethylsilyl)fluoro acetamide (BSTFA) and trimethylchlorosilane (TMCS) were supplied by Sigma Aldrich. 2,4,6-tris-2-(pyridyl)-1,3,5-triazine (TPTZ) and Ferri Chloride (FeCl3.6H2O) were also supplied by Sigma Chemicals. Carnitine and acylcarnitine reference standards and deuterated analogues were obtained from Cambridge Isotope Laboratories.
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