Exhaled markers of oxidative stress in the airways

Sammanfattning: Breath contains a number of different molecules, some of which may have potential use as markers for oxidative stress in the airways during chronic airway inflammation. The major objectives were to develop and validate methods for quantifying five potential exhaled markers of oxidative stress: malondialdehyde (MDA) and free 3-nitrotyrosine (3-NT) in exhaled breath condensate (EBC) and ethane, pentane and isoprene in exhaled air, and to test the applicability of the methods. A further goal was to evaluate the effect of different collection variables on ethane, pentane and isoprene levels and to explore whether these compounds are formed in the airways. The developed MDA method was based on derivatization with thiobarbituric acid, high-performance liquid chromatography (HPLC) and fluorescence detection. The limit of detection (LOD) was low (4.1 nM) and the response was linear in the concentration range examined (0.020-1.0 µM). Within-assay precision was 4% and between-assay precision 9%. Samples could be stored for one month at 20C and three months at 80C without losses. The method for determining exhaled ethane, pentane and isoprene involved pre-concentration on a multibed sorbent tube, thermal desorption and endcut gas chromatography (GC) with flame ionization detection (FID). The LODs were 5, 2 and 6 pmol/sample for ethane, pentane and isoprene, respectively. The analyte responses were linear and the repeatability was 7%, 10% and 12%, for the respective analytes. Certified levels of ethane and pentane agreed well with the measured levels. Ethane and pentane were stable for up to six days of storage in sample tubes. Isoprene levels were not stable during storage in the sample tubes used here, but using a modified tube sorbent, levels were stable for up to two days. Moreover, a method employing GC/negative chemical ionization (NCI)/tandem mass spectrometry (MS) was established for quantifying 3-NT in EBC. The detection limit was 0.56 pM (corresponding to 3 amol/µL injected) and a good linear relationship between added (0.025-5.0 nM) and measured 3-NT levels was observed. Within-day and between-day precision was 11% and 12%, respectively. No artifactual nitration was observed during sample processing. 3-NT levels were much lower compared to reported levels, based on immunochemical measurements.The possible production of ethane, pentane and isoprene in the airways was evaluated by studying the flow-dependency of analyte concentrations, as well as concentrations in dead space and alveolar air after subjects held their breath. The concentration of these substances in breath obtained after inhaling purified air was also examined. To this end, novel collection equipment was constructed. The major fractions of exhaled ethane, pentane and isoprene seem to be of systemic origin, but there was a tendency for ethane to be slightly flow-dependent in subjects with asthma, i.e. the concentration decreased at a higher flow-rate in subjects with asthma. When measuring ethane and pentane, it is important to take ambient air concentrations into account. In conclusion, valid methods to quantify MDA and 3-NT in EBC, as well as for ethane, pentane and isoprene in exhaled air, were developed. These methods may be feasible tools for monitoring airway oxidative stress.