Abstract
Lipid hydroperoxides are the primary products of lipid peroxidation. Therefore, many investigators have tried to detect lipid hydroperoxides in biological samples to obtain direct evidence for free radical injury in vivo or to measure the degree of oxidative stress. The chapter introduces a chemiluminescence-based high-performance liquid chromatography (HPLC) assay of lipid hydroperoxides for these purposes. The assay has several advantages: (1) it is very sensitive, with the lower limit of detection being about 0.1 pmol or less, (2) interference by biological antioxidants is avoided because hydroperoxides and antioxidants can be separated by HPLC, and (3) information on which lipid class is oxidized can be obtained. The chemiluminescence-based HPLC assay is based on light emission during the hydroperoxide-induced oxidation of isoluminol. The reaction is catalyzed by metal ions. An HPLC mobile phase, which can dissolve lipid hydroperoxides, is mixed with a 50% aqueous methanol solution containing 50 m M sodium borate (pH 10), 1 m M isoluminoI, and 5 mg/liter microperoxidase after passing through an HPLC column.
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