J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D700022-JLR200 on September 28, 2007

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Journal of Lipid Research, Vol. 49, 245-250, January 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Development of an immunoblot assay with infrared fluorescence to quantify paraoxonase 1 in serum and plasma

Philip W. Connelly1,*,{dagger},§, Graham F. Maguire*, Clive M. Picardo*,{dagger}, John F. Teiber** and Dragomir Draganov{dagger}{dagger}

* Keenan Research Centre in the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Canada
{dagger} Department of Medicine, University of Toronto, Toronto, Canada
§ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
** Department of Internal Medicine, Division of Epidemiology, University of Texas Southwestern Medical Center, Dallas, TX
{dagger}{dagger} Department of Metabolism, WIL Research Laboratories, LLC, Ashland, OH

Published, JLR Papers in Press, September 28, 2007.

1 To whom correspondence should be addressed. e-mail: connellyp{at}smh.toronto.on.ca

Paraoxonase 1 (PON1) requires calcium for activity and is inactivated in the presence of EDTA. Because of this, studies to date have used serum or heparinized plasma for both activity and mass assays of PON1. Whole serum and EDTA plasma were analyzed by SDS-electrophoresis and Western blot using anti-PON1 monoclonal antibody 4C10. Because PON1 has one disulfide and one free cysteine residue, the samples were reduced with dithiothreitol before electrophoresis. Western blot identified a major PON1 band with a molecular mass of ~45 kDa and two minor bands of ~40 and 35 kDa in both serum and EDTA plasma. This established that PON1 is inactive, but structurally intact, in EDTA plasma and suggested that a mass assay could be developed based on SDS-electrophoresis and Western blot. Linearity was established for plasma and for a PON1 standard. Quantification was based on the major PON1 band at 45 kDa. The correlation between serum and plasma PON1 mass was 0.9553. The between-run variation was determined with a serum pool to be 7.8%. The mass of PON1 in serum was significantly correlated with arylesterase activity (r = 0.85). Thus, we have demonstrated the feasibility of measuring PON1 mass in either serum or EDTA plasma.

Supplementary key words Western blot • quantitation • lipoproteins • high density lipoproteins


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