Scientific Core

ANALYTICAL AND BIOCHEMICAL CORE (CORE B) ABSTRACT The overall goal of the biochemical synthesis and analytical core (Core B) will be to provide state-of-the-art measurements of isolevuglandins (isoLGs) and related lipid metabolites, provide IsoLGs for participants of the PPG and use state of the art high energy mass spectroscopy to define specific peptides that are IsoLG adducted. Core B will analyze the amount of IsoLG-lysyl protein adducts (IsoLG-Lys) in cells and tissues of both experimental animals (projects 1, 2 and 4) and in cells and plasma of human subjects (all projects). Because IsoLGs so rapidly react with lysine residues of proteins, unreacted IsoLG cannot be measured in biological systems. Thus, for overall quantification of IsoLGs in cells and tissues we quantify IsoLG-lys. Core B is also perfecting high energy mass spectroscopy to define specific peptides and proteins adducted by IsoLG and will use this to detect specific targets of IsoLG adduction in humans and experimental models. A second approach for detection and quantification of IsoLG adducts will involve the use of the single chain antibody D- 11, which detects IsoLG adducted to proteins and peptides, independent of the peptide backbone. When needed, core B will provide PPG investigators with fluorescently tagged D-11 for use in flow cytometry and immunostaining. Core B will also synthesize IsoLG active analogs including alkynyl-IsoLG, and the inactive synthetic precursor of IsoLG, IsoLG-dimethoxyacetal (IsoLG-DMA) and anhydro-IsoLG for use in cultured cells and experimental animals. Core B will measure levels of 2-hydroxybenzylaime in plasma and tissues from both experimental animals and plasma of humans in all projects. The methods and reagents provided by Core B are highly unique, and have been developed by the Core Director, Dr. Davies and other Project investigators during the past decade. It is essential that the Project Directors have access to these resources for successful completion of the PPG.