Human liver s9 fraction

Manufactured by Xenotech

Human liver S9 fraction is a subcellular preparation derived from human liver tissue. It contains a mixture of metabolic enzymes and cofactors, primarily cytochrome P450 enzymes, which are involved in the biotransformation of xenobiotics and endogenous compounds. This fraction is commonly used in in vitro studies to assess the metabolic stability and potential for drug-drug interactions of pharmaceutical compounds.

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5 protocols using human liver s9 fraction

Synthesis and Analysis of DNA Adducts

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Enantiopure (S)- and (R)-NNN,^{24 , 25} analytical standards of (R)- and (S)-py-py-dI,^{22 (link)} and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK acetate)^{26 (link)} were synthesized as described previously. Hepatocytes, human liver S9 fraction, and corresponding incubation media were obtained from XenoTech LLC (Kansas City, KS). DNA isolation solutions were purchased from Qiagen (Valencia, CA). 2′-Deoxy[¹⁵N₅]guanosine was procured from Cambridge Isotope Laboratories (Tewksbury, MA). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO).
dGuo was incubated with NNK acetate essentially as described previously.^{27 (link)} Half of the reaction mixture was reduced with NaBH₃CN, and both halves were analyzed for the presence of DNA adducts.

Zarth A.T., Upadhyaya P., Yang J, & Hecht S.S. (2016). DNA Adduct Formation from Metabolic 5′-Hydroxylation of the Tobacco-Specific Carcinogen N′-Nitrosonornicotine in Human Enzyme Systems and in Rats. Chemical research in toxicology, 29(3), 380-389.

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Metabolic Activation of NNN in Human Liver S9

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The human liver S9 fraction (Xenotech) was a pooled mixture isolated from 200 donors (100 male, 100 female, 11–83 years of age, and predominantly Caucasian). The S9 preparation included a recommended protocol for optimal P450 activity, which was adopted in these experiments. Briefly, a buffer was prepared to final concentrations of 50 mM potassium phosphate (pH 7.4), 3 mM MgCl₂, 1 mM EDTA, 1 mM NADPH, 5 mM glucose-6-phosphate, 1 mg/mL calf thymus DNA, 1 U/mL glucose-6-phosphate dehydrogenase, 1 mg/mL human liver S9 fraction, and the appropriate concentration of NNN (2–500 μM). The DNA and all salts were added first and vortexed to ensure full dissolution before enzymes or NNN were added. The final solution (5 mL) was incubated for 24 h at 37 °C. Ice cold IPA (7.5 mL) was added to precipitate the DNA. The DNA was then purified by extraction and analyzed as detailed below.

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Analytical Standards for UHPLC Analysis

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Ultrapure water with a resistivity of 18.2 MΩ·cm at 25 °C was generated with a Millipore™-purification system. Ultrahigh-Performance Liquid Chromatography (UHPLC)-grade MeOH, acetonitrile and formic acid were purchased from Biosolve (Dieuze, France), dichloromethane by Merck (Darmstadt, Germany). The following analytical standards were provided by Sigma–Aldrich (St. Louis, MO, USA): apigenin, benzoic acid, caffeic acid, +/− catechin, chlorogenic acid, cinnamic acid, coumarin, emodin, epicatechin, ferulic acid, isorhamnetin, naringenin, p-coumaric acid, protocatechuic acid, quercetin, quercitrin, rutin, salicylic acid, sinapic acid, β-sitosterol, stigmasterol, syringic acid, tannic acid, taxifolin, theophylline and vanillic acid. Luteolin and procyanidin B2 were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Gallic acid and p-hydroxybenzoic acid were provided by Carl Roth (Karlsruhe, Germany). Human liver S9 fraction and NADPH RS (Regenerating System) were purchased from XenoTech. Medicagenic acid was provided by Phytolab (Vestenbergsgreuth, Germany). All other chemicals and biochemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Peeters L., Van der Auwera A., Beirnaert C., Bijttebier S., Laukens K., Pieters L., Hermans N, & Foubert K. (2020). Compound Characterization and Metabolic Profile Elucidation after In Vitro Gastrointestinal and Hepatic Biotransformation of an Herniaria hirsuta Extract Using Unbiased Dynamic Metabolomic Data Analysis. Metabolites, 10(3), 111.

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Hepatic Microsomal Metabolism Assay

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The following were synthesized at Daiichi Sankyo Co., Ltd. (Tokyo, Japan): Pooled HLM, liver microsomes from mice, rats, dogs, and monkeys, and human liver S9 fraction were purchased from XenoTech (Kansas City, KS). Potassium phosphate buffer (KPB; pH 7.4), cDNA-expressing human P450 microsomes (control and CYP2C8), and NADPH-regenerating system solutions A and B were purchased from Corning (Woburn, MA). CYP1A2-, CYP2C8-, CYP2C9-, CYP2C19-, CYP2D6-, CYP3A4-SILENSOMES, and human plasma were purchased from Biopredic International (Saint Gr egoire, France). Paclitaxel was purchased from Sigma-Aldrich (St. Louis, MO). Dimethylacetamide (DMA) was obtained from Nacalai Tesque (Kyoto, Japan). Saline was obtained from Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan). Methylcellulose 400 (MC) was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Asano D., Hamaue S., Zahir H., Shiozawa H., Nishiya Y., Kimura T., Kazui M., Yamamura N., Ikeguchi M., Shibayama T., Inoue S.I., Shinozuka T., Watanabe T., Yahara C., Watanabe N, & Yoshinari K. (2022). CYP2C8-Mediated Formation of a Human Disproportionate Metabolite of the Selective Na_V1.7 Inhibitor DS-1971a, a Mixed Cytochrome P450 and Aldehyde Oxidase Substrate. Drug metabolism and disposition: the biological fate of chemicals, 50(3).

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Analytical Characterization of Hymenocardia Compounds

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Acetonitrile and methanol (HPLC-quality) and ammonia (25%) were supplied by Fisher Chemical. Pepsin (P-7000, from porcine stomach mucosa, 800-2500 U/mg protein), bile salts (B-8631, porcine), pancreatin (76 190, from porcine pancreas, 149 USP U/mg amylase), sodium dihydrogen phosphate anhydrous (NaH2PO4), disodium phosphate dihydrate (Na2HPO4 • 2 H2O) and tris(hydroxymethyl)aminomethane (TRIS) were purchased from Sigma-Aldrich.
Hydrochloric acid (HCl, 32%), sodium bicarbonate (NaHCO3) and sodium hydroxide (NaOH) were obtained from Merck. Formic acid was purchased from Acros Organics. Human liver S9 fraction and NADPH RS (Regenerating System) were purchased from XenoTech. All chemicals and reagents were of analytical grade and in all experiments deionized water (milliQ, Merck Millipore) was used. Hymenocardine (94%) and hymenocardinol (82%) were isolated from root bark of Hymenocardia acida. The purity of these two compounds was determined by HPLC analysis.The analysis was performed using a XBridge C18 column (4.6  250 mm, 5μm, Waters) and water + 0.1% Formic acid (A) and acetonitrile (B) as mobile phase. The flow rate was 1.0 mL/min and the following gradient was applied: 0 to 5 min 80% A and 20% B, 40 to 45 min 100% B. UV detection was done at 201 nm.

Tuenter E., Bijttebier S., Foubert K., Breynaert A., Apers S., Hermans N, & Pieters L. (2017). In Vitro and In Vivo Study of the Gastrointestinal Absorption and Metabolisation of Hymenocardine, a Cyclopeptide Alkaloid. Planta medica, 83(9).

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