The development of human sera tests for HDL-bound serum PON1 and its lipolactonase activity.
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.
Gaidukov
Leonid
L
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Tawfik
Dan S
DS
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D043303:Aryldialkylphosphatase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000378:metabolism; D004796:Clinical Enzyme Tests / Q000379:methods*; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood*; D010755:Organophosphates / Q000378:metabolism; D014468:Umbelliferones / Q000378:metabolism
C523443:7-O-diethylphosphoryl-3-cyano-4-methyl-7-hydroxycoumarin; D008075:Lipoproteins, HDL; D010755:Organophosphates; D014468:Umbelliferones; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D015107:4-Butyrolactone