Bianca Fuhrman1, Nina Volkova, Michael Aviram. 1. Rambam Medical Center, Rappaport Family Institute for Research in Medical Sciences and Technion Faculty of Medicine, Haifa, Israel. Fuhrman@tx.technion.ac.il
Abstract
OBJECTIVE: The high-density lipoprotein (HDL)-associated paraoxonase-1 (PON1)/free PON1 ratio is lower in diabetic patients in comparison with healthy controls. Because diabetes is associated with increased oxidative stress, we hypothesized that a labeled recombinant PON1 (rePON1) would detect differences in HDL capacity to bind PON1 under specific experimental conditions, such as oxidation, addition of polyphenols, or in vivo dosing of diabetic patients with polyphenols. METHODS: In the present study we determined labeled rePON1 binding to HDL under various oxidative conditions by using polyacrylamide gel electrophoresis for the separation of free labeled rePON1 from HDL-bound labeled rePON1. RESULTS: The HDL-rePON1/free rePON1 ratio gradually decreased as the extent of HDL oxidation increased, and the antioxidants vitamin E or pomegranate juice (PJ) inhibited the redistribution of rePON1. PJ or its purified polyphenols, punicalagin, gallic acid, or ellagic acid, increased rePON1 binding also to non-oxidized HDL. Further, rePON1 associated more efficiently with HDLs isolated from diabetic patients after PJ consumption versus HDLs isolated before PJ consumption. CONCLUSIONS: We conclude that 1) oxidative stress impairs binding of fluorescein isothiocyanate-labeled rePON1 to HDL and 2) PJ polyphenols directly increase the HDL-rePON1 association beyond their antioxidative effect. Copyright 2010 Elsevier Inc. All rights reserved.
OBJECTIVE: The high-density lipoprotein (HDL)-associated paraoxonase-1 (PON1)/free PON1 ratio is lower in diabeticpatients in comparison with healthy controls. Because diabetes is associated with increased oxidative stress, we hypothesized that a labeled recombinant PON1 (rePON1) would detect differences in HDL capacity to bind PON1 under specific experimental conditions, such as oxidation, addition of polyphenols, or in vivo dosing of diabeticpatients with polyphenols. METHODS: In the present study we determined labeled rePON1 binding to HDL under various oxidative conditions by using polyacrylamide gel electrophoresis for the separation of free labeled rePON1 from HDL-bound labeled rePON1. RESULTS: The HDL-rePON1/free rePON1 ratio gradually decreased as the extent of HDL oxidation increased, and the antioxidants vitamin E or pomegranate juice (PJ) inhibited the redistribution of rePON1. PJ or its purified polyphenols, punicalagin, gallic acid, or ellagic acid, increased rePON1 binding also to non-oxidized HDL. Further, rePON1 associated more efficiently with HDLs isolated from diabeticpatients after PJ consumption versus HDLs isolated before PJ consumption. CONCLUSIONS: We conclude that 1) oxidative stress impairs binding of fluorescein isothiocyanate-labeled rePON1 to HDL and 2) PJ polyphenols directly increase the HDL-rePON1 association beyond their antioxidative effect. Copyright 2010 Elsevier Inc. All rights reserved.