Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Worldwide Research and Development, Pfizer, Inc., Groton, Connecticut (C.F., L.D., X.H., C.S., M.D.T., R.S.O., H.Z.); and Scienion US, Inc., Monmouth Junction, New Jersey (M.S.).
Drug Metab Dispos. 2013 Oct;41(10):1797-804. doi: 10.1124/dmd.113.053082. Epub 2013 Jul 15.
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 µl/min per milligram compared with a median of 455 µl/min per milligram), but preserved similar AOX1 protein expression levels (approximately 15% less than the median value).
醛氧化酶 1（AOX1）是一种高度表达于肝脏的细胞溶质酶，在代谢含有芳香氮杂杂环取代基的药物方面发挥着关键作用。AOX1 催化的快速代谢可导致药物表现出高清除率、低暴露，从而降低疗效，甚至增加毒性（如果 AOX1 生成的代谢物有毒）。因此，需要开发 AOX1 表达水平与 AOX1 底物清除率之间的相关性。本研究首次建立了一种快速、灵敏、稳健的液相色谱-串联质谱（LC-MS/MS）方法来定量人肝胞质中的 AOX1。该 LC-MS/MS 方法包括一个直接的超滤分级步骤，具有很好的选择性和宽动态范围（5.2 pM 至 20.7 nM）。利用该方法定量了 20 名供体的人肝胞质中的 AOX1 水平，以研究 AOX1 表达的个体差异。未观察到 AOX1 水平在个体或性别之间存在显著差异，尽管男性供体的分布范围比女性供体更宽（分别为 0.74-2.30 pmol/mg 和 0.74-1.69 pmol/mg）。LC-MS/MS 测定的 AOX1 蛋白水平与酶联免疫吸附测定（ELISA）测定的结果一致。一些供体的 AOX1 蛋白水平正常但酶活性低，这可能是由于辅因子缺乏、单核苷酸多态性或同二聚体解离所致。来自慢性饮酒供体的胞质中 AOX1 催化的卡巴唑氧化活性较低（<51 µl/min/mg，而中位数为 455 µl/min/mg），但保留了相似的 AOX1 蛋白表达水平（比中位数低约 15%）。
