Comprehensive Determination of Isoprostanoid Metabolism

PROJECT SUMMARY Oxidative stress (OxS) is a biochemical process that leads to damage of cellular lipids when endogenous redox homeostasis is disrupted. OxS is mechanistically linked with the physiology of aging and age-related diseases, such as cardiovascular disease, neurodegeneration, cancer, frailty, diabetes, and SARS-CoV-2. The

identification of biomarkers to measure OxS is thus of significant public health revelance in order to better understand disease mechanisms and target potential therapies. F2-Isoprostanes (F2-IsoPs) are formed from the oxidation of the cellular lipid arachidonic acid and considered to be excellent biomarkers of OxS. Currently,

F2- IsoPs are being used as outcome measures in more than 80 active clinical trials worldwide. Further, the NIA- sponsored Interventions Testing Program (ITP) has identified nine agents that significantly increase lifespan, and four of those agents are known to decrease F2-IsoPs. While F2-IsoPs have proven to be useful

biomarkers of OxS, our laboratory has obtained evidence to support the hypothesis that metabolites of F2- IsoPs more accurately reflect endogenous OxS than unmetabolized F2-IsoPs in certain biological settings. Yet, F2-IsoP metabolites are rarely quantified in clinical studies. F2-IsoP-like molecules (F-isoprostanoids) are

made from the oxidation of other polyunsaturated fatty acids (PUFA), including adrenic, eicosapentaenoic (EPA), and docosahexaenoic acids. These compounds are proving to be useful biomarkers in neurodegenerative conditions and age-related macular degeneration, but their metabolism has never been studied. The central hypothesis of this proposal is that understanding the metabolism of F-isoprostanoids is

critical for the accurate and complete quantitation of these urinary biomarkers in aging and OxS-related diseases. In Specific Aim 1, we will use human liver microsomes to identify metabolites of several F- isoprosatnoid isomers generated from the free radical oxidation of PUFA. Metabolites will be identified using

mass spectrometry (MS). In Specific Aim 2, we will establish and validate a robust LC/MS method for the quantification of F-isoprostanoid metabolites, from Specific Aim 1, in human urine. For this purpose, we will utilize urine samples from The Fatty Acid Desaturase Activity, Fish Oil, and Colorectal Cancer Prevention

Study (FnADAFO), a randomized clinical trial that was completed in 2018 at Vanderbilt. Subjects recruited in this study were supplemented with olive oil or marine fish oil for six months, so these urine samples are ideal for validation of this metabolite quantification. We anticipate that the completion of this application will redefine

our understanding F2-IsoP metabolism and, for the first time, define a strategy to comprehensively assess this important biomarker of lipid peroxidation. Overall, these studies will change how the field evaluates endogenous OxS and lipid peroxidation, thus setting the stage for future applications examining the role of

isoprostanoids in human physiology and the pathogenesis of disease.