Reductase Assay

A procedure to quantitate picomole amounts of hy-droperoxides based on GSSG formation is described. Hydroperoxides are incubated with GSH and glutathi-one peroxidase, and the GSSG formed is measured by a GSSG-specific glutathione reductase recycling assay. Prior to analysis the remaining GSH is removed with iV-ethylmaleimide. IV-Ethylmaleimide inhibition of the recycling assay is prevented by alkaline hydrolysis of the iV-ethylmaleimide, without GSSG hydrolysis, at pH 11. The method is rapid, 30 min, with a limit of detec-tion of 142 pmol calculated by linear regression analy-sis. Stoichiometric amounts of GSSG are produced in response to hydroperoxides. An application of the method is shown with air oxidation of arachidonic acid solutions over 4 h at room temperature. The method is sufficiently sensitive to quantitate the low amounts of hydroperoxides present in fresh arachidonic acid samples. 0 1990 Academic Press, Inc. Lipid peroxidation has been implicated in a variety of pathological processes (1,2) and free fatty acid hydroper-oxide concentrations control both the activation and the irreversible inhibition of prostaglandin (PG)3 H syn-thase, the common and rate-controlling step of tissue PG production (3,4). Numerous methods have been employed to detect the process of lipid peroxidation and to quantitate lipid hy-droperoxide concentrations (1). However, most of these 1 To whom reprint requests should be addressed