Direct measurement of hydrogen peroxide release from rat alveolar macrophages: artifactual effect of horseradish peroxidase.
Van Scott-MR; Miles-PR; Castranova-V
Exp Lung Res 1984 Jan; 6(2):103-114
A study was made of the validity of a method for measuring hydrogen- peroxide (H2O2) release from alveolar macrophages. The method usually used for such studies involved the horseradish-peroxidase (HRP) catalyzed oxidation of scopoletin by H2O2. It was found that release of H2O2 and luminol catalyzed chemiluminescence were stimulated in rat alveolar macrophages by type-II HRP at concentrations usually used in the HRP/scopoletin method. The length of time the cells were preincubated at 37.5 degrees-C and the time at which type-II HRP was added determined the amount of H2O2 released. The cells did not release additional H2O2 upon exposure to zymosan particles following stimulation with type-II HRP. An alternative catalyst, myeloperoxidase, did not stimulate H2O2 release and could be used to measure H2O2 release from rat alveolar macrophages. Resting H2O2 release was negligible using myeloperoxidase. Zymosan induced H2O2 release could be monitored by use of more pure HRP preparations which do not stimulate alveolar macrophages to release H2O2. The data indicated that type-II HRP stimulated H2O2 release from rat and guinea-pig alveolar macrophages but not from New-Zealand-white-rabbit alveolar macrophages or human blood leukocytes. The authors conclude that type-II HRP is not the catalyst of choice for this assay. These findings explain conflicting results obtained in earlier studies and support the view that rat alveolar macrophages release minimal amounts of H2O2 at rest and that stimulation can be brought about by exposure to zymosan.
NIOSH-Author; Alveolar-cells; Cell-biology; Lung-cells; Laboratory-techniques; Particulates; Peroxides; In-vitro-studies; Laboratory-animals; Peroxidases
Experimental Lung Research