Determination of hydroxyl free radical formation in human platelets using high-performance liquid chromatography with electrochemical detection

F. Blandini, E. Martignoni, R. Ricotti, F. Di Jeso, G. Nappi

Research output: Contribution to journalArticle

Abstract

The formation of the hydroxyl free radical (HFR) can be quantified indirectly, by measuring two products of the hydroxylation of salicylic acid, 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (2,5-DHB). In this study, we used reversed-phase high-performance liquid chromatography with electrochemical (coulometric) detection to measure 2,3- and 2,5-DHB levels in human platelets. The limits of detection of the method were 10 and 5 fmol on column for 2,3-DHB and 2,5-DHB, respectively. We tested the technique by measuring increases in dihydroxybenzoate levels after exposure of platelets to experimentally induced oxidative stress. Then, we measured platelet levels of 2,3- and 2,5-DHB in patients with Parkinson's disease, under therapy with L-DOPA, and in normal subjects. We also measured platelet concentrations of L-DOPA and its major metabolite, 3-O-methyldopa (3-OMD). Parkinsonian patients showed increased levels of both 2,3- and 2,5-DHB. Platelet levels of 2,3-DHB were positively correlated with platelet levels of L-DOPA and 3-OMD. The technique we describe proved simple and extremely sensitive and may represent a useful tool for the study of oxidative stress in humans. Copyright (C) 1999.

Original languageEnglish
Pages (from-to)213-220
Number of pages8
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume732
Issue number1
DOIs
Publication statusPublished - Sep 10 1999

Keywords

  • Dihydroxybenzoate
  • Hydroxyl free radical formation
  • L-DOPA

ASJC Scopus subject areas

  • Chemistry(all)

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