Determination of paraoxonase 1 status and genotypes at specific polymorphic sites.

The procedures for determining paraoxonase (PON1) status and for determining PON1 genotypes for polymorphisms in coding and important regulatory regions are described. PON1 status is determined by a functional two-substrate analysis of plasma PON1 activities. Differences in catalytic efficiency of the PON1192Q and PON1192R alloforms result in the clear separation of all three phenotypes at position 192 (Q/Q, Q/R, R/R) and at the same time, the two-substrate analysis indicates activity levels of PON1. Because the enzyme activity levels are as important as the polymorphic genotypes, this two-substrate analysis of PON1 status provides the most relevant information for investigating the association of PON1 genetics with susceptibilities to disease, organophosphorus insecticide sensitivity, and pharmacokinetic status of drug metabolism. Genotyping of polymorphic sites alone fails to provide this important information but can be useful for gene frequency determination and forensic analysis. Analytical procedures for determining PON1 status and genotypes are described.

Expression of human paraoxonase (PON1) during development.

BACKGROUND: Paraoxonase (PON1), a HDL-associated enzyme, protects against toxicity from specific organophosphorus compounds and oxidized lipids. Common polymorphisms in the PON1 gene have been identified and characterized in the coding region, 5' regulatory region and 3' UTR. The Q192R coding region polymorphism determines substrate-dependent differences in catalytic efficiency of hydrolysis. The -108CT polymorphism in the 5' regulatory region has a significant effect on PON1 expression, with the -108C allele expressing on average twice the level of plasma PON1 as the -108T allele. In addition to the effects of regulatory and coding region polymorphisms on PON1 levels and activity, plasma PON1 levels are also developmentally regulated. Since PON1 levels are important in determining resistance to specific organophosphorus compounds, the time course of appearance of PON1 in newborns is of great interest. RESULTS: We report here that PON1 levels plateau between 6 to 15 months of age, and that variability in the age at which PON1 levels plateau is quite variable among individuals. In mice and rats, plasma PON1 activity reaches a plateau at 3 weeks of age. In mice that lack endogenous PON1, human transgenes encoding either PON1(Q192) or PON1(R192) under the control of the human PON1 regulatory sequences exhibited a similar time course of expression as that seen in wild-type mice, indicating conservation of the developmental regulatory elements between mouse and human PON1.