Characterization of free radicals formed from COX-catalyzed DGLA peroxidation

Abstract

Like arachidonic acid (AA), dihomo-γ-linolenic acid (DGLA) is a 20-carbon ω-6 polyunsaturated fatty acid and a substrate of cyclooxygenase (COX). Through free radical reactions, COX metabolizes DGLA and AA to form well-known bioactive metabolites, namely, the 1 and 2 series of prostaglandins (PGs1 and PGs2), respectively. Unlike PGs2, which are viewed as proinflammatory, PGs1 possess anti-inflammatory and anticancer activities. However, the mechanisms linking the PGs to their bioactivities are still unclear, and radicals generated in COX–DGLA have not been detected. To better understand PG biology and determine whether different reactions occur in COX–DGLA and COX–AA, we have used LC/ESR/MS with a spin trap, α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN), to characterize the carbon-centered radicals formed from COX–DGLA in vitro, including cellular peroxidation. A total of five types of DGLA-derived radicals were characterized as POBN adducts: m/z 266, m/z 296, and m/z 550 (same as or similar to COX–AA) and m/z 324 and m/z 354 (exclusively from COX–DGLA). Our results suggest that C-15 oxygenation to form PGGs occurs in both COX–DGLA and COX–AA; however, C-8 oxygenation occurs exclusively in COX–DGLA. This new finding will be further investigated for its association with various bioactivities of PGs, with potential implications for inflammatory diseases.