Direct measurement of hydrogen peroxide release from rat alveolar macrophages: artifactual effect of horseradish peroxidase.
Van Scott-MR; Miles-PR; Castranova-V
The validity of the method used to measure the release of hydrogen- peroxide (7722841) (H2O2) from alveolar macrophages obtained from male Sprague-Dawley-rats, involving the horseradish-peroxidase (HRP) catalyzed oxidation of scopoletin by H2O2, was examined to discover the reason for inconsistent results reported in the literature. It was found that Type II HRP stimulated resting macrophages to release H2O2. The amount of H2O2 released by rat alveolar macrophages following stimulation with Type II HRP was found to depend on the length of preincubation of cells at a temperature of 37.5 degrees-C and the timing of Type II HRP addition. The addition of zymosan particles did not result in additional release of H2O2 following stimulation with Type II HRP. Myeloperoxidase did not stimulate the release of H2O2 from rat alveolar macrophages, and H2O2 release was evident following stimulation with zymosan. Some other pure HRP preparations also failed to stimulate the release of H2O2 from rat alveolar macrophages and were recommended to monitor zymosan stimulated release of H2O2. Type II HRP was also shown to stimulate the release of H202 from guinea-pig alveolar macrophages. Since the results indicated that Type II HRP stimulated the release of H2O2 from alveolar macrophages from both rats and guinea-pigs, the authors conclude that the enzyme is not the best catalyst for this assay.
NIOSH-Author; Peroxides; Analytical-methods; Chemical-analysis; Oxidative-enzymes; Laboratory-animals; In-vitro-studies; Alveolar-cells; Phagocytes; Peroxidases
Appalachian Laboratory for Occupational Safety and Health, NIOSH, Department of Physiology, West Virginia University, Morgantown, West Virginia, Document No. 0291Y, 26 pages, 15 references