Mechanisms of Ascorbyl Radical Formation in Human Platelet-Rich Plasma

Abstract

Recently, many clinical reports have suggested that the ascorbyl free radical (Asc∙) can be treated as a noninvasive, reliable, real-time marker of oxidative stress, but its generation mechanisms in human blood have rarely been discussed. In this study, we used upstream substances, enzyme inhibitors, and free radical scavengers to delineate the mechanisms ofAsc∙formation in human platelet-rich plasma (PRP). Our results show that the doublet signal was detected in PRP samples by using electron spin resonance, and the hyperfine splitting of the doublet signal wasaH=1.88gauss andg-factor = 2.00627, which was determined to be theAsc∙. We observed that the inhibitors of NADPH oxidase (NOX), cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 (CYP450), mitochondria complex III, and nitric oxide synthase (NOS), but not xanthine oxidase, diminished the intensity of theAsc∙signal dose dependently. All enzyme inhibitors showed no obvious antioxidant activity during a Fenton reaction assay. In summary, the obtained data suggest thatAsc∙formation is associated with NOX, COX, LOX, CYP450, eNOS, and mitochondria in human PRP.