title: Why do most human liver cytosol preparations lack xanthine oxidase activity?
abstract: When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.
authors.affiliation: Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., K.V.C., S.N., C.A.J.-J., J.P.J.); Department of Pharmaceutics, University of Washington, Seattle, Washington (K.E.T., T.W.); Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.C., E.G.S.); Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden (S.C.S.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer Inc., La Jolla, California (M.Z.).
authors.forename: John T
authors.initials: JT
authors.lastname: Barr
authors.forename: Kanika V
authors.initials: KV
authors.lastname: Choughule
authors.forename: Sahadev
authors.initials: S
authors.lastname: Nepal
authors.forename: Timothy
authors.initials: T
authors.lastname: Wong
authors.forename: Amarjit S
authors.initials: AS
authors.lastname: Chaudhry
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Joswig-Jones
authors.forename: Michael
authors.initials: M
authors.lastname: Zientek
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Strom
authors.forename: Erin G
authors.initials: EG
authors.lastname: Schuetz
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Thummel
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Jones
mesh_terms: D042931:Aldehyde Oxidase / Q000378:metabolism; D000493:Allopurinol / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010117:Oxypurinol / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D010477:Perfusion; D053719:Tandem Mass Spectrometry; D046509:Tissue Culture Techniques / Q000379:methods; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D000493:Allopurinol; D014969:Xanthine Oxidase; D042931:Aldehyde Oxidase; D010117:Oxypurinol