Oxidative Processes Induced by tert-Butyl Hydroperoxide in Human Red Blood Cells: Chemiluminescence Studies

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A. V. Domanski, E. A. Lapshina, and I. B. Zavodnik^*

Institute of Biochemistry, National Academy of Sciences of Belarus, Bulvar Leninskogo Komsomola 50, 230017 Grodno, Belarus; fax: +375-152-334121; E-mail: hepato@biochem.unibel.by

^* To whom correspondence should be addressed.

Received May 18, 2004; Revision received August 19, 2004

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The erythrocyte is a good model for investigation of the mechanisms of cell damage induced by oxidizing agents. Oxidative damage to cell components and cellular metabolism results in impaired rheological properties of circulating red blood cells and is involved in the development of some pathologies. The aim of the present study was to elucidate further the oxidative processes induced by tert-butyl hydroperoxide (tBOOH) in erythrocytes, identify cellular targets damaged by the oxidant, as well as estimate the energy and stoichiometry of the reactions that occur. The generation of free radicals in the cell was registered using the chemiluminescence technique. The products of oxyhemoglobin (oxyHb) oxidation, changes in intracellular glutathione (GSH) pool, and accumulation of the stable products of membrane lipid peroxidation were concurrently measured. The oxidative processes induced by tBOOH in red blood cells can be described as follows: 1) rapid GSH oxidation (30-60 sec) by glutathione peroxidase; 2) formation of radicals in the reaction between tBOOH and cellular Hb, which are then immediately consumed in lipid peroxidation reactions; 3) generation of chemiluminescence by the radicals formed. Several stages of the oxidative processes can be revealed. The order of the chemiluminescence reaction (n) with respect to oxidant was estimated to be equal to 2.5 at oxidant concentrations less than 0.5 mM and equal to 1.0 at higher oxidant concentrations. The order of the reaction of membrane lipid peroxidation was found to be n = 2.2 at 0.25-0.6 mM tBOOH and n = 0.5 at higher oxidant concentrations. The apparent activation energy of membrane lipid peroxidation was 55.8 ± 6.4 kJ/mol, and that of oxyHb oxidation was 108 ± 16 kJ/mol. It is shown that the interaction of tBOOH and HOCl in erythrocytes is accompanied by changes in both the total number of radicals generated in the cell and the time corresponding to the maximal rate of radical generation.

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KEY WORDS: human red blood cells, oxidative stress, organic hydroperoxide, hypochlorous acid, hemoglobin, chemiluminescence

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