No abstract

A simple, sensitive, non-stimulated assay was developed to measure the superoxide anion concentration in whole blood, using an ultra-sensitive chemiluminescence (CL) analyzer and lucigenin amplification. The assay system can be performed without leukocyte isolation or stimulant administration. The blood CL levels of healthy males (362.8 +/- 337.7 counts /10 sec) were not different from those of females (335 +/- 308.7 counts/10 sec) (p=0.64), whereas the CL levels in whole blood in patients with acute pancreatitis (2522 +/- 2014 counts/10 sec) were significantly higher than those of healthy controls (p<0.001). This assay system may be valuable in the future for quantitative measurement of reactive oxygen species in various disorders.

Oxygen-derived free radical injury has been associated with several cytopathic conditions. Oxygen radicals produced by chondrocytes is an important mechanism by which chondrocytes induce matrix degradation. In the present study, we extend these observations by studying oxidative processes against osteoblasts. Osteoblasts were mixed in in vitro culture with 200 microM menadione. The cytotoxic effect of menadione-induced oxidative stress was monitored by lucigenin- or luminol-amplified chemiluminescence, tetrazolium assay and immunocytochemical study. Results showed that adding menadione induces an oxidative stress on osteoblasts, via superoxide and hydrogen peroxide production, that can be eradicated by superoxide dismutase (SOD) and catalase in a dose-dependent manner. Catalase and the appropriate concentration of dimethyl sulfoxide have a protective effect on cytotoxicity induced by menadione, whereas SOD does not. Menadione-treated osteoblasts have a strong affinity for annexin V, and the nuclei are strongly stained by TUNEL (TdT-mediated dUTP nick-end labelling). The results suggest that menadione-triggered production of reactive oxygen species leads to apoptosis of osteoblasts.