Administrative Supplement: Glutathionylated Products of Radical-Induced Lipid Oxidation in Inflammatory Disease

Case Western Reserve University, Cleveland OH

Investigators

Abstract

Abstract An administrative supplement is requested for the repair of a key tool for the project, a triple quad LC-MS/MS. This funding opportunity came just in time as this important equipment has been broken for over a month. This instrument is needed for quantitative detection of products gener- ated by free radical-induced lipid oxidation in the retina, e.g., pseudo leukotrienes (øLTs), and structurally similar leukotrienes (LTs). Our primary tool for assessing øLT and LT generation is LC-MS/MS with isotope dilution quantitation. Understanding the generation and cellular responses to free radical-induced lipid oxidation products is complex owing to their diversity and ability to mimic enzymatically generated receptor agonists, e.g., LTs. We discovered glutathione (GSH) derivatives produced from oxidatively truncated arachidonyl phospholipids that are structural and functional analogues of cysteinyl (Cys) LTs. We refer to them as øLTs. The project is testing the hypotheses that øLTs contribute to retinal pathology and physiology in a rat model of light-induced retinal degeneration. øLTs are present in human retina, are produced in vivo consequent to oxidative insult, and exhibit LT-like biological activities. Therefore, øLTs can elicit cellular responses erroneously presumed to be induced exclusively by CysLTs. Consequently, they are a confounding factor for interpreting previous studies on the involvements of LTs in retinal pathology and physiology. The research is testing the hypotheses that øLTs and related GSH derivatives of oxidatively truncated docosahexaenoyl phospholipids contribute to receptor-depen- dent inflammatory cytokine signaling, retinal edema, pathological neovascularization, or physio- logically important initiation of retinal pigment epithelium (RPE) autophagy. The potential patho- physiological significance of these glutathionylated products of lipid oxidation lies in the possibility of their ubiquitous generation in high levels, e.g., compared to those of CysLTs. We are testing the conclusion of preliminary studies that øLTs are LT receptor agonists and will determine if their ability to induce the expression of IL-13 and TGF-b1 promotes LT biosynthesis. Because cross reactivity may confound the interpretation of immunoassays for LTs, we must quantitate both øLTs and LTs by LC-MS/MS. We will determine signaling pathways, e.g., with RNA-Seq studies, activated by glutathionylated products of free radical-induced lipid oxidation addressing the questions: do they promote inflammation in ocular pathology or initiate autophagy in RPE cells and how do they do it? Molecular level insights into the role of oxidative stress in the pathogenesis of inflammatory eye diseases such as AMD can facilitate development of therapeutic interventions that ameliorate the progression of these common but disabling diseases.