Paraoxonase-1 (PON1) Status Analysis Using Non-Organophosphate Substrates

Abstract

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 PON1₁₉₂ genotype and plasma PON1 activity levels. While our previous studies with PON1 status demonstrated that both PON1₁₉₂ 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.

Keywords: PON1 activity assay; PON1 status; non-organophosphate substrates; paraoxonase-1.

Figure 1.

PON1-mediated hydrolysis of the substrates used for the non-OP PON1 status assay, phenyl acetate (high-salt arylesterase activity) and 4-(chloromethyl)phenyl acetate (CMPAase activity).

Figure 2.. Comparison of the (A) toxic OP and (B) non-OP PON1 status assays using the same population (n = 183).

Samples were run in triplicate, and each data point corresponds to the average value of the pathlength-corrected triplicate. Activity results are displayed in enzymatic units (U/L for panel 2A and U/mL for panel 2B). The PON1 status carried out in panel A corresponds to the original PON1 status that uses toxic OP substrates (paraoxon and diazoxon) (Furlong et al., 2006; Richter & Furlong, 1999; Richter et al., 2004, 2008, 2009). Panel B displays enzymatic activity results using the non-OP PON1 status described here (Richter et al. 2008 and 2009). In both panels, the open circle data points correspond to individuals homozygous for PON1_192Q (QQ), the solid square data points correspond to individuals heterozygous for PON1_192QR (QR), and the open triangle data points correspond to individuals homozygous for PON1_192R (RR). Reproduced with permission from Richter et al., 2008.

Figure 3.. High-salt arylesterase activity rates (mOD/min) over the total duration of the assay (4 min or 240 s).

The activity rates of the top and middle plasma samples are not linear for the entire 4 min. Therefore, a shorter range of time (80 s) that ensures activity rates for all plasma samples are linear should be selected (vertical line). OD: optical density.