The Nitric Oxide/Heme Protein Complex as a Biologic Marker of Exposure to Nitrogen Dioxide in Humans, Rats, and In Vitro Models

In an effort to develop a biologic marker of exposure to nitrogen dioxide (NO2), we investigated the in vivo formation of a complex between heme proteins and nitric oxide (NO). In aqueous solution, NO2 disproportionates to NO and nitrate. The NO binds to the iron of heme proteins to form an electron spin resonance (ESR)-detectable complex. We have shown that when rat liver, lung, or nasal microsomes are exposed to 20 ppm NO2 in vitro, an ESR signal attributable to an NO/heme protein complex is detected. After inhalation exposure of rats to 20 ppm NO2 for 6 h, this same ESR signal was detected in microsomes prepared from the exposed rats' lungs or liver; microsomes prepared from the nasal tissue failed to yield any detectable signal. When we lavaged the lungs of rats exposed for 6 h to 0, 5, 10, 20, or 30 ppm NO2 and isolated the bronchoalveolar cell pellets, the NO/heme protein complex was detected in the cell pellets. We were able to demonstrate a dose-dependent relationship between the ESR signal intensity of the NO/heme protein complex and the NO2 exposure concentration. Finally, we used ESR to examine bronchoalveolar lavage cell pellets obtained from human volunteers exposed to either 1.5 or 4 ppm NO2, for 20 min every other day, for six exposures. No signal was found in any of the samples taken 3 wk prior to NO2 exposure, but an ESR signal attributable to the NO/heme protein complex was detected in every sample obtained after the 4 ppm NO2 exposure and in five of eight samples obtained after the 1.5 ppm NO2 exposure. We were able to demonstrate a linear relationship between the ESR signal intensity of the human NO/heme protein complex and the NO2 exposure concentration. Our results indicate that the NO/heme protein complex may be a useful biologic marker for NO2 exposure.