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Detection of salicylate and its hydroxylated adducts 2,3- and 2,5-dihydroxybenzoic acids as possible indices for in vivo hydroxyl radical formation in combination with catechol- and indoleamines and their metabolites in cerebrospinal fluid and brain tissue

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Author: Sloot, W.N. · Gramsbergen, J.B.P.
Institution: Instituut CIVO-Toxicologie en Voeding TNO
Source:Journal of Neuroscience Methods, 1-2, 60, 141-149
Identifier: 82591
doi: DOI:10.1016/0165-0270(95)00005-F
Keywords: Biology · Animal · Biogenic Amines · Catechols · Chromatography, High Pressure Liquid · Electrochemistry · Hydroxybenzoic Acids · Hydroxyl Radical · Indoles · Magnesium · Male · Manganese Poisoning · Neostriatum · Rats · Rats, Wistar · Reference Standards · Salicylic Acid · Salicylic Acids · Spectrophotometry, Ultraviolet · Support, Non-U.S. Gov't


It has been suggested that salicylate (SA) hydroxylation can be used to detect hydroxyl radical formation in vivo. Here we describe a rapid and sensitive HPLC method using ultraviolet absorbance (UV) and electrochemical detection (EC) to detect SA (UV), its hydroxylated adducts 2,3- and 2,5- dihydroxybenzoic acids (DHBA) and catechol in combination with catechol- and indoleamines and related metabolites (EC) in one isocratic run. These compounds were measured in acidified cerebrospinal fluid (CSF) and perchlorate extracts of striatal tissues of untreated and SA-loaded rats (300 mg/kg SA, i.p.). Peaks were identified by comparing retention times of samples and standards, by adding standards to biological samples, by voltamograms, and by comparing chromatograms of manganese (Mn2+)-injected striata of SA-loaded rats with several control conditions. Six hours after unilateral injection of 0.4 μmol Mn2+ into striatum, 2,3-DHBA and 2,5- DHBA levels in striatum were respectively 4- and 7-fold increased as compared to non-injected (contralateral) striata, suggesting in vivo hydroxyl radical formation. In addition, dopamine and serotonin levels were depleted in Mn2+-injected striata by 46% and 64%, respectively. In CSF of Mn2+- injected rats, DHBA/SA ratios were not significantly changed as compared to those of control rats. In conclusion, the described technique can be applied to study in vivo hydroxyl radical formation in direct relation with dopaminergic and serotonergic neurotransmitter changes during neurotoxic processes.