Biol. Chem., Vol. 385, pp. 259–264, March/April 2004 • Copyright  by Walter de Gruyter • Berlin • New York 2004/310030 Measurement of exhaled hydrogen peroxide from rabbit lungs Norbert Weissmann*, Henrik Vogels, Ralph T. Schermuly, Hossein A. Ghofrani, Jo ¨ rg Ha ¨ nze, Ludger Fink, Frank Rose, Werner Seeger and Friedrich Grimminger Department of Internal Medicine, Justus-Liebig- University Giessen, Klinikstrasse 36, D-35392 Giessen, Germany *Corresponding author e-mail: Norbert.Weissmann@innere.med.uni-giessen.de Abstract Exhaled H 2 O 2 is considered an indicator of lung inflam- matory and oxidative stress. Moreover, H 2 O 2 may be involved in signal transduction processes. It is not fully elucidated to what extent (i) H 2 O 2 escapes from the intra- vascular compartment, and (ii) pulmonary H 2 O 2 genera- tion and nasopharyngeal H 2 O 2 generation contribute to exhaled H 2 O 2 . We investigated H 2 O 2 concentrations in breath condensate from isolated buffer-perfused and ventilated rabbit lungs, and from both intubated and spontaneously breathing rabbits with a horseradish per- oxidase/29,79dichlorofluorescin assay. For the perfused lungs, a H 2 O 2 concentration of 58 "19 nM was found. Addition of H 2 O 2 to the buffer fluid resulted in only minute appearance in the exhaled air ( -0.001%). Levels of exhaled H 2 O 2 in intubated rabbits and perfused lungs were virtually identical. Nearly ten-fold higher levels were detected in spontaneously breathing rabbits. Decreasing the inspired oxygen concentration from 21% to 1% resulted in a tendency toward decreased H 2 O 2 exhalation in perfused lungs. In contrast, phorbol-12-myristate-13- acetate (PMA) prompted a ;4-fold increase in H 2 O 2 exhalation. We conclude that the horseradish peroxi- dase/29,79dichlorofluorescin assay is a feasible technique to measure H 2 O 2 in exhaled breath condensate in rabbits. When collecting exhaled air via the tracheal tube, the sig- nal represents pulmonary H 2 O 2 generation with the con- tribution of the remaining body being negligible. Keywords: breath condensate; exhaled; H 2 O 2 ; hypoxic pulmonary vasoconstriction; isolated lung; NADPH oxidase. Introduction H 2 O 2 in exhaled breath condensate has been used as a marker of oxidative stress. Levels of this reactive oxygen species have been shown to be increased in chronic obstructive pulmonary disease (COPD; Dekhuijzen et al., 1996; Nowak et al., 1999), asthma (Antczak et al., 1997; Jobsis et al., 1997; Horvath et al., 1998), cigarette smok- ers (Nowak et al., 1996), pneumonia (Kietzmann et al., 1993; Horvath et al., 1998), bronchiectasis (Loukides et al., 1998), and the adult respiratory distress syndrome (Baldwin et al., 1986; Sznajder et al., 1989; Kietzmann et al., 1993). However, the source from which the expired H 2 O 2 is derived has not been fully elucidated. It remains unclear to what extent the alveolar, the bronchial and the nasopharyngeal spaces, as well as the systemic metab- olism, contribute to the exhalation of this marker. Some studies focused on this aspect (Schleiss et al., 2000). To minimize the portion of nasopharyngeal contribution (Horvath et al., 1998; Jobsis et al., 2000) a mouth rinse, measurement of sputum contamination, and wearing nose clips during exhalation have been used. Being an indicator for inflammatory processes, H 2 O 2 and its precursor superoxide have additionally been sug- gested to play a major role in the signal transduction of hypoxic responses in the lung, especially in hypoxic pul- monary vasoconstriction (HPV). This essential vasore- gulatory mechanism matches lung blood flow with ventilation and thus optimizes pulmonary gas exchange (Fishman, 1976; Voelkel, 1986; Weissmann et al., 2001). Despite the increasing evidence that superoxide and hydrogen peroxide (H 2 O 2 ) are involved in the regulation of HPV, there is still controversy over whether decreased or increased superoxide and H 2 O 2 levels trigger HPV, and from what cellular and intracellular sources such reactive oxygen species are derived (Burke-Wolin and Wolin, 1989; Thomas III et al., 1991; Mohazzab and Wolin, 1994; Grimminger et al., 1995; Mohazzab et al., 1995; Marshall et al., 1996; Weissmann et al., 1998, 2000; Archer et al., 1999; Waypa et al., 2001). To assess pulmonary H 2 O 2 generation in more detail, we developed a sensitive technique for the measurement of exhaled H 2 O 2 levels in isolated, blood-free perfused rabbit lungs using a horseradish peroxidase/29,79dichlo- rofluorescin assay. We investigated the source of exhaled H 2 O 2 by comparing the H 2 O 2 levels in isolated lungs, intu- bated intact animals and spontaneously breathing ani- mals wearing a face mask. Responses of the lung H 2 O 2 generation to hypoxic versus normoxic ventilation and to stimulation with a phorbol ester were characterized. Results Using standard H 2 O 2 solutions, characterization of the applied H 2 O 2 assay revealed a linear correlation between the applied H 2 O 2 concentration and the fluorescence in the range of 0–1400 nM. Specificity for H 2 O 2 was secured by the addition of catalase in parallel measurements. To determine a possible quenching effect of catalase, dif- ferent amounts of this enzyme were added to the sam- ples after 20 min of incubation of standard H 2 O 2 solutions (280 nM) with horseradish peroxidase. Quenching turned out to occur at 28 "7% (150 U/ml catalase), 36 "4% Brought to you by | Georgetown University Authenticated Download Date | 5/24/15 9:55 PM