Determination of hydroxyl free radical formation in human platelets using high-performance liquid chromatography with electrochemical detection.
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.
Blandini
F
F
Laboratory of Functional Neurochemistry, Neurological Institute C. Mondino, Pavia, Italy. blandini@tin.it
Martignoni
E
E
Ricotti
R
R
di Jeso
F
F
Nappi
G
G
D000328:Adult; D000978:Antiparkinson Agents / Q000032:analysis / Q000627:therapeutic use; D001792:Blood Platelets / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry / Q000379:methods; D005260:Female; D006801:Humans; D062385:Hydroxybenzoates / Q000378:metabolism; D017665:Hydroxyl Radical / Q000032:analysis*; D007980:Levodopa / Q000378:metabolism* / Q000627:therapeutic use; D008297:Male; D008875:Middle Aged; D010300:Parkinson Disease / Q000188:drug therapy / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000978:Antiparkinson Agents; D062385:Hydroxybenzoates; D017665:Hydroxyl Radical; D014443:Tyrosine; D007980:Levodopa; C009135:2,3-dihydroxybenzoic acid; C008404:3-methoxytyrosine