Determination of picomole quantities of hydroperoxides by a coupled glutathione peroxidase and glutathione disulfide specific glutathione reductase assay.
A procedure to quantitate picomole amounts of hydroperoxides based on GSSG formation is described. Hydroperoxides are incubated with GSH and glutathione peroxidase, and the GSSG formed is measured by a GSSG-specific glutathione reductase recycling assay. Prior to analysis the remaining GSH is removed with N-ethylmaleimide. N-Ethylmaleimide inhibition of the recycling assay is prevented by alkaline hydrolysis of the N-ethylmaleimide, without GSSG hydrolysis, at pH 11. The method is rapid, 30 min, with a limit of detection of 142 pmol calculated by linear regression analysis. 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.
Allen
K G
KG
Department of Food Science and Human Nutrition, Colorado State University, Fort Collins 80523.
Huang
C J
CJ
Morin
C L
CL
D016718:Arachidonic Acid; D001095:Arachidonic Acids; D055598:Chemical Phenomena; D002621:Chemistry; D005978:Glutathione / Q000031:analogs & derivatives*; D019803:Glutathione Disulfide; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; D008054:Lipid Peroxides / Q000032:analysis*; D009994:Osmolar Concentration
D001095:Arachidonic Acids; D008054:Lipid Peroxides; D016718:Arachidonic Acid; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; D005978:Glutathione; D019803:Glutathione Disulfide