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Papers In Press, published online ahead of print July 1, 2007
J. Lipid Res., doi:10.1194/jlr.D600045-JLR200

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Journal of Lipid Research, Vol. 48, 1637-1646, July 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

The development of human sera tests for HDL-bound serum PON1 and its lipolactonase activity

Leonid Gaidukov and Dan S. Tawfik¹

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of one table and five figures.

Published, JLR Papers in Press, April 13, 2007.

¹ To whom correspondence should be addressed. e-mail: tawfik{at}weizmann.ac.il

Serum paraoxonase (PON1) is a lipolactonase that associates with HDL-apolipoprotein A-I (HDL-apoA-I) and thereby plays a role in the prevention of atherosclerosis. Current sera tests make use of promiscuous substrates and provide no indications regarding HDL-PON1 complex formation. We developed new enzymatic tests that detect total PON1 levels, irrespective of HDL status and R/Q polymorphism, as well as the degree of catalytic stimulation and increased stability that follow PON1's tight binding to HDL-apoA-I. The tests are based on measuring total PON1 levels with a fluorogenic phosphotriester, measuring the lipolactonase activity with a chromogenic lactone, and assaying the enzyme's chelator-mediated inactivation rate. The latter two are affected by tight HDL binding and thereby derive the levels of the serum PON1-HDL complex. We demonstrate these new tests with a group of healthy individuals (n = 54) and show that the levels of PON1-HDL vary by a factor of 12. Whereas the traditionally applied paraoxonase and arylesterase tests weakly reflect PON1-HDL levels (R = 0.64), the lipolactonase test provides better correlation (R = 0.80). These new tests indicate the levels and activity of PON1 in a physiologically relevant context as well as the levels and quality of the HDL particles with which the enzyme is associated.

Supplementary key words blood tests • lactones • lipoproteins • high density lipoprotein-bound enzymes • enzyme stability • atherosclerosis • paraoxonase

Abbreviations: apoA-I, apolipoprotein A-I; DEPCyMC, 7-O-diethyl phosphoryl 3-cyano 4-methyl 7-hydroxycoumarin; DTNB, 5,5'-dithio-bis-2-nitrobenzoic acid; PON1, serum paraoxonase; TBBL, 5-thiobutyl butyrolactone

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