Mechanisms of ascorbyl radical formation in human platelet-rich plasma.
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 of Asc(∙) 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 was a(H) = 1.88 gauss and g-factor = 2.00627, which was determined to be the Asc(∙). 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 the Asc(∙) signal dose dependently. All enzyme inhibitors showed no obvious antioxidant activity during a Fenton reaction assay. In summary, the obtained data suggest that Asc(∙) formation is associated with NOX, COX, LOX, CYP450, eNOS, and mitochondria in human PRP.
Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wenchang Road, Shihlin Taipei 111, Taiwan ; Graduate Institute of Clinical Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.
Kou-Gi
KG
Shyu
Department of Cardiology, Cathay General Hospital, 280 Jen Ai Road, Section 4, Taipei 106, Taiwan ; Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.
Chao-Chien
CC
Chang
Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.
Yu-Chieh
YC
Yeh
Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan ; Department of Pharmacology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.
Joen-Rong
JR
Sheu
Department of Pharmacology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.
Duen-Suey
0000-0002-1653-992X
DS
Chou
D000975:Antioxidants / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003683:Dehydroascorbic Acid / Q000031:analogs & derivatives* / Q000097:blood / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D053657:Platelet-Rich Plasma / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism
D000975:Antioxidants; D016861:Cyclooxygenase Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D016166:Free Radical Scavengers; D005609:Free Radicals; D016859:Lipoxygenase Inhibitors; D013481:Superoxides; D016718:Arachidonic Acid; D009569:Nitric Oxide; C000820:semidehydroascorbic acid; D003577:Cytochrome P-450 Enzyme System; D019001:Nitric Oxide Synthase; D014969:Xanthine Oxidase; D019255:NADPH Oxidases; D003683:Dehydroascorbic Acid