The Oxidation of Catecholamines and 6-Hydroxydopamine by Molecular Oxygen: Effect of Ascorbate

Abstract

Comparative kinetic studies on the oxidation of catecholamines (CA) (dopamine (DA), epinephrine (EP). norepinephrine (NEP)) serving as a neuromediator in the sympathetic nervous system, 3,4-dihydroxyphenylalanine (DOPA) and 6-hydroxydopamine (6-OHDA), a wellknown neurotoxic agent, were performed in the presence of ascorbate (AscH^- ) in 50 mᴍ phosphate buffer, pH 7.40, at 37 °C by using a Clark electrode, EPR and the absorption spectroscopy. The oxidation of CA and DOPA alone was found to be a self-accelerating process, with quinone products (Q) acting as autocatalysts. The rate of oxygen consumption (R_ox) increased with time and reached a steady-state level. A starting value of R_ox increased in the order: EP < DOPA ≈ NEP ≪ DA ≪ 6-OHDA, whereas a steady-state value of R_ox changed in the order: DOPA < DA < NEP ≪ EP ≪ 6-OHDA. The changes in R_ox with time were found to correlate with the resistance of primary Q to the intramolecular cyclization.

The effect of AscH^- on CA oxidation depended dramatically on whether AscH^- was added to non-oxidized or preoxidized CA. Added to non-oxidized CA and DOPA, AscH^- inhibited their oxidation (but not that of 6-OHDA). For the case of DA, a pronounced lag period was observed by both a Clark electrode and spectrophotometrically. The addition of AscH^- to preoxidized CA, DOPA and 6-OHDA induced an increase in R_ox a steadystate concentration of the ascorbyl radical. The kinetic behaviour of the systems was determined by two major factors: 1) AscH^- suppressed the formation of Q, a catalyst for CA oxidation, most likely due to the reaction of AscH^- with the semiquinone formed from CA; 2) Q derived both from CA and 6-OHDA catalyzed AscH^- oxidation. The elevated cytotoxicity of 6-OHDA was found to be in part caused by the condition that 6-OHDA oxidation was not inhibited by AscH^- and by the high efficiency of 6-OHDA as a redox cycling agent in combination with A scH⁻. These observations explain the very pronounced and prolonged cytotoxicity of 6-OHDA even at low concentrations that increases at elevated concentrations of AscH^- .