Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington.
Department of Medicine (Division of Medical Genetics), University of Washington, Seattle, Washington.
Curr Protoc. 2021 Jan;1(1):e25. doi: 10.1002/cpz1.25.
Human paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme with antioxidant, anti-inflammatory, and antiapoptotic roles. The ability of PON1 to hydrolyze specific organophosphate (OP) compounds and prevent accumulation of oxidized lipids in lipoproteins has prompted a large number of studies investigating PON1's role in modulating toxicity and disease. Most of these studies, however, have only focused on PON1 single nucleotide polymorphism analyses and have ignored PON1 activity levels, arguably the most important parameter in determining protection against exposure and disease. We developed a two-substrate activity assay termed "PON1 status" that reveals both the functional PON1 genotype and plasma PON1 activity levels. While our previous studies with PON1 status demonstrated that both PON1 functional genotype and enzymatic activity levels obtained exclusively by determining PON1 status are required for a proper evaluation of PON1's role in modulating OP exposures and risk of disease, the original PON1 status assay requires the use of highly toxic OP metabolites. As many laboratories are not prepared to handle such toxic compounds and the associated waste generated, determination of PON1 status has been limited to rather few studies. Here, we describe a PON1 status protocol that uses non-OP substrates with a resolution equivalent to that of the original PON1 status approach. We have also included useful suggestions to ensure the assays can easily be carried out in any laboratory. The protocols described here will enable a proper examination of the risk of exposure or susceptibility to disease in PON1 epidemiological studies without the need to handle highly toxic substrates. © 2021 Wiley Periodicals LLC. Basic Protocol: Determining PON1 status using non-organophosphate substrates Support Protocol 1: Experimental pathlength determination Support Protocol 2: PON1 DNA genotyping for the Q192R (rs662) polymorphism.
人对氧磷酶 1（PON1）是一种高密度脂蛋白相关酶，具有抗氧化、抗炎和抗凋亡作用。PON1 水解特定有机磷（OP）化合物的能力以及防止脂蛋白中氧化脂质积累的能力促使大量研究调查 PON1 在调节毒性和疾病中的作用。然而，这些研究大多仅集中在 PON1 单核苷酸多态性分析上，而忽略了 PON1 活性水平，这可能是决定对暴露和疾病的保护最重要的参数。我们开发了一种双底物活性测定法，称为“PON1 状态”，它揭示了功能性 PON1 基因型和血浆 PON1 活性水平。虽然我们之前使用 PON1 状态的研究表明，PON1 功能基因型和仅通过确定 PON1 状态获得的酶活性水平对于正确评估 PON1 在调节 OP 暴露和疾病风险中的作用都是必需的，但原始的 PON1 状态测定法需要使用剧毒的 OP 代谢物。由于许多实验室都没有准备好处理这些有毒化合物及其产生的相关废物，因此 PON1 状态的测定仅限于少数几个研究。在这里，我们描述了一种 PON1 状态方案，该方案使用非 OP 底物，其分辨率与原始 PON1 状态方法相当。我们还包括了一些有用的建议，以确保这些测定可以在任何实验室中轻松进行。这里描述的方案将使 PON1 流行病学研究能够正确检查暴露或对疾病的易感性的风险，而无需处理剧毒底物。 © 2021 威利父子公司。基本方案：使用非有机磷底物确定 PON1 状态 支持方案 1：实验路径长度的确定 支持方案 2：PON1 Q192R（rs662）多态性的 DNA 基因分型
