Changes in the levels of glutathione in phagocytosing human neutrophils

Abstract

The tripeptide γ-glutamyl-cysteinyl-glycine, or glutathione, protects cells against oxidative damage by its ability to react with free radicals and peroxides. From studies with human neutrophilic granulocytes deficient in glutathione metabolism, it is known that the protection by this compound is essential for the proper functioning of these cells. Previous studies have yielded conflicting estimates of reduced glutathione (GSH) and oxidized glutathione (GSSG) in human neutrophils. We have optimized three independent assays to measure glutathione in about 106 human neutrophils. With these methods we have measured the level of GSH and GSSG at rest, during phagocytosis, and during incubation of the cells with a hydrogen-peroxide-generating system. GSH was measured as a rate-limiting cofactor in the formaldehyde dehydrogenase reaction, in the nonspecific colorimetric reaction with 5,5'-dithiobis-(2-nitribenzoic acid) after cell extraction with sulfuric acid, and in the latter reaction coupled to the glutathione reductase reaction. The first two methods gave falsely high values for GSH; other sulfhydryl compounds probably contributed to the result. With the third method, which measures the sum of GSH plus GSSG, 14.2 ± 2.2 nmole GSH + GSSG/107 neutr ophils (mean±SD, n=21) were found. GSSG was measured in the glutathione-reductase-coupled reaction after binding GSH to N-ethylmaleimide, followed by separation of GSSG from free N-ethylmaleimide over Sephadex G-10. A level of 0.6±0.8 nmole/107 neutrophils (mean±SD, n=12) was found. Subtraction of this value from the value for GSH+GSSG leads to an estimation of about 13 nmole GSH/107 neutrophils. During phagocytosis, the level of GSH decreased by about 30% in 15 min. This decrease was dependent on the generation of reactive oxygen compounds, because it was not observed in the neutrophils of a patient with chronic granulomatous disease. The amount of GSSG did not increase proportionally: only about 40% of the GSH that disappeared was recovered as GSSG. From the stability of the GSH levels during phagocytosis (when large amounts of oxidative products are generated) and during incubation of neutrophils with a H2O2-generating system, we conclude that these cells have a large capacity to keep glutathione in the reduced form. Thus, the glutathione redox system comprises a very potent system for protection of phagocytic leukocytes against oxidative stress.