A fast chemiluminescent method for H2O2 measurement in exhaled breath condensate
Introduction
Cells lining broncho-alveolar spaces are continuously subjected to many noxious agents which are present in polluted areas [1]. A long-lasting exposure to such noxious agents induces a chronic inflammation, which can develop into different clinical conditions such as chronic obstructive pulmonary disease, or cancer [2], [3]. A common biochemical mechanism that mediates these different pathological processes is the in situ generation of large amount of oxidants, in particular, reactive nitrogen [4] and oxygen species [5], mainly produced by phagocytic cells [6]. Although both oxygen and nitrogen reactive species in physiological conditions exert a regulatory role [7], [8], their increase leads to a vast array of biochemical, cellular and organ dysfunction [9], [10], [11], [12].
One of the most stable forms of the oxygen reactive species is hydrogen peroxide, which is obtained by univalent reduction of molecular oxygen and dismutation of its product, the superoxide anion. The latter reacts with hydrogen peroxide in the Haber–Weiss reaction, producing highly toxic agents [13].
Many papers reported spectrophotometric or fluorimetric methods for H2O2 determination in exhaled breath condensate [14], [15], [16], [17], [18], [19], [20], [21], while only one [22], at our best knowledge, uses a chemiluminescent method. In this paper, we describe a very simple, fast and reliable chemiluminescent method for the determination of H2O2 in condensed expired breath. Moreover, the H2O2 production of smokers and nonsmokers is reported.
Section snippets
Chemicals
Luminol (5-amino-2,3dihydro-1,4 phthalazinedione), was purchased from Sigma (St. Louis, MO, USA). From a 50 mmol/l stock solution in DMSO, a working solution 10 mmol/l in water was prepared.
Type X peroxidase (from horseradish) in 3.2 mol/l ammonium sulphate was obtained from Sigma. The original suspension (2000 U/ml) was diluted 1:500 in water to obtain a working solution of 4 U/ml.
H2O2 (30%) was from Merck (Darmstadt, Germany). The actual H2O2 concentration was calculated from its absorbance
Results
Fig. 1 shows a typical calibration curve of H2O2 determination in the 0–100 μmol/l range (in the insert the 0–1 μmol/l range is shown). As is clearly shown in the figure, the chemiluminescence response is linear for more than three orders of magnitude of H2O2 concentration.
The analytical variability of the luminometer at zero H2O2 concentration was obtained by measuring 20 replicates; the values of means and SD were respectively 3200±442 (CV 13.8 %), with an analytical sensitivity of about 0.01
Discussion
Exhaled breath can give useful information on the normal and/or pathological processes occurring at bronchoalveolar level [16], [23], [24].
A very high amount of reactive oxygen species is produced at the air/lung interface due to the oxidative burden sustained by cells lining alveolar spaces at every respiratory act. Cantin et al. [25] have calculated a rate of H2O2 formation in alveolar spaces of smokers of about 80 nmol ml−1 h−1 (equivalent to a concentration of 80 μmol/l in exhaled breath
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