Detection of organic hydroperoxides in rabbit lung lavage fluid, but not in lung tissue homogenate, using GSH peroxidase and GSH reductase

A specific method for the detection of organic hydroperoxides in lung lavage fluid (lung surfactant system) and lung tissue homogenate is described. After the inactivation of endogenous GSH peroxidase and GSH reductase and preincubation with catalase, organic hydroperoxides are consumed by addition of GSH and GSH peroxidase. The increase of GSSG, compared to a blank without addition of GSH peroxidase, is measured in a second enzymatic step with GSH reductase. The recoveries of t-butyl hydroperoxide and of peroxidized, free fatty acids added to lavage fluid or to lung homogenate are higher than 85% in each case. The detection limits of this assay for organic hydroperoxides are 0.9 nmol/mg surfactant phospholipid (molar ratio of 0.00066) and 40 nmol/g wet lung weight. The assay detects organic hydroperoxides in the surfactant system of normal rabbit lungs, but not in lung tissue homogenate.