Aldehyde Oxidase 1 (AOX1) in Human Liver Cytosols: Quantitative Characterization of AOX1 Expression Level and Activity Relationship

Abstract

Aldehyde oxidase 1 (AOX1) is a cytosolic enzyme highly expressed in liver and plays a key role in metabolizing drugs containing aromatic azaheterocyclic substituents. Rapid metabolism catalyzed by AOX1 can cause a drug to exhibit high clearance, low exposure, and hence decreased efficacy or even increased toxicity (if AOX1 generated metabolites are toxic). There is a need to develop the correlation between AOX1 expression levels and AOX1-substrate clearance. A fast, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify AOX1 in human liver cytosol for the first time. This LC-MS/MS method includes a straightforward ultrafiltration fractionation step and gives great selectivity and wide dynamic range (5.2 pM to 20.7 nM). The AOX1 levels in human liver cytosols of 20 donors were quantified using this method to investigate individual differences in AOX1 expression. No significant individual or gender differences in AOX1 levels were observed, although male donors exhibited a broader distribution than female donors (0.74-2.30 pmol/mg versus 0.74-1.69 pmol/mg, respectively). The AOX1 protein levels measured by LC-MS/MS were consistent with those measured by an enzyme-linked immunosorbent assay. Several donors have a normal AOX1 protein level but low enzyme activity, which might be due to cofactor deficiency, single nucleotide polymorphism, or homodimer dissociation. Cytosols from donors with chronic alcohol consumption had low AOX1-catalyzed carbazeran oxidation activities (<51 ml/min per milligram compared with a median of 455 ml/min per milligram), but preserved similar AOX1 protein expression levels (approximately 15% less than the median value)

Aldehyde Oxidase Activity in Donor-Matched Fresh and Cryopreserved Human Hepatocytes and Assessment of Variability in 75 Donors

“…Interestingly, evaluation of the medical history data of the donors revealed that three of the five low-activity donors had histories of extensive alcohol abuse up until time of death. This acute effect of alcohol abuse was also recently reported by scientists at Pfizer (Fu et al, 2013). The exact mechanism of this alcohol effect on AO activity was (Fu et al, 2013).…”

“…This acute effect of alcohol abuse was also recently reported by scientists at Pfizer (Fu et al, 2013). The exact mechanism of this alcohol effect on AO activity was (Fu et al, 2013). Thus, the presence of AO protein apparently does not correlate with activity, which complicates the assessment of variability factors.…”

Comparative transcriptomics of hepatic differentiation of human pluripotent stem cells and adult human liver tissue

“…UGT2B4 is a member of the UDP glucuronosyltransferase family, which consists of phase II drug metabolism enzymes (10,33). AOX1 is an oxidase that plays a key role in the metabolism of xenobiotics and drugs containing aromatic azaheterocyclic substituents (12). Finally, CYP2E1, a member of the cytochrome P450, is a phase I drug-metabolizing enzyme (33), and differences in epigenetic regulation of CYP2E1 between hPSC-HEP and primary hepatocytes have been determined (36), which may account for the impaired activity of this enzyme.…”

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

“…Unfortunately, the levels between the two methods vary greatly from 0.74-2.3 pmol/mg cytosolic protein (Fu et al, 2013) to 21-40 pmol/mg cytosolic protein . The discrepancy, whether due to enzyme source, preparation, or differences in the quantification method, warrants a number of further assessments to reach a consensus and provide a best practice for quantifying AO.…”