Quantitative analysis of 8‐isoprostane and hydrogen peroxide in exhaled breath condensate

The airways of smokers are exposed to high levels of oxidative stress, caused by oxidants and free radicals inhaled with cigarette smoke and reactive oxygen species produced by activated inflammatory cells 1, 2. The oxidative burden of the smoker's airways can be investigated using traditional methods of sampling alveolar fluids or bronchial secretions, such as sputum induction and bronchoscopy with bronchoalveolar lavage. Sampling of these lung secretions is, however, invasive, and this may influence oxidative status markers. Recently, collection of exhaled breath condensate (EBC) has been suggested as a simple and easily repeatable procedure for monitoring airway inflammation and oxidative stress 3. The method entails cooling exhaled air, which results in condensation. This EBC contains aerosolised airway epithelial lining fluid particles and volatile compounds. There is increasing evidence that exhaled markers may reflect biochemical changes in airway lining fluid 3. The collection of EBC might, therefore, be useful in identifying smokers at a greater risk of developing smoking-related pulmonary diseases and investigating the potential effects of extra antioxidants. A number of studies have focused on markers of oxidative processes in the EBC of smokers, including hydrogen peroxide 4–6, 8‐isoprostane 7, nitrogen oxides 8–10 and nitrosothiols 11. However, it should be emphasised that the analysis and interpretation of EBC results are not straightforward. Several methodological issues regarding sampling and the analytical techniques used for EBC need to be taken into account, such as issues related to the collection procedure and the analysis of oxidative markers in EBC.

The aim of the present study was to determine whether 8‐isoprostane and H₂O₂ are detectable in nonconcentrated EBC from healthy male smokers, when measured using immunochemical and colorimetric assays, respectively. In addition, the reproducibility of these measurements was investigated.

Subjects and methods

Results

8‐isoprostane and H₂O₂ concentrations were below the LOD in many samples (table 1⇓). 8‐isoprostane was detectable in EBC samples from only three smokers on all three occasions within 1 week (mean 5.3, 4.0 and 4.5 pg·mL⁻¹). These three smokers were not different from the others with respect to age, body mass index and cumulative cigarette consumption. Eight of the 12 smokers exhibited 8‐isoprostane concentrations above the LOD of 3.9 pg·mL⁻¹ for at least one measurement (36% of all EBC samples contained detectable 8‐isoprostane). The concentration of 8‐isoprostane in these samples ranged 3.9–10.6 pg·mL⁻¹.

None of the smokers showed detectable concentrations of H₂O₂ for all three measurements within 1 week, but 11 of the 12 subjects exhibited H₂O₂ concentrations above the LOD of 0.31 µM for at least one measurement (47% of all EBC samples contained detectable H₂O₂). The concentration of H₂O₂ in these EBC samples ranged 0.31–0.49 µM.

The reproducibility of the measurements was calculated: the intraclass correlation coefficient was 0.33 for 8‐isoprostane and −0.34 for H₂O₂. However, these values were calculated from only a few detectable concentrations.

Separate spiking experiments were performed by adding 3.1–50 pg·mL⁻¹ 8‐isoprostane or 0.5–10 µM H₂O₂ to fresh and thawed (after 5 h at −80°C) EBC collected from a healthy subject. These experiments, which were performed in duplicate, showed a recovery of exogenous 8‐isoprostane of 83.2–109.5% for fresh samples and 114.5–145.6% for thawed samples. Whereas, recovery of exogenously added H₂O₂ ranged 69.9–129.0% for fresh samples and even reached >200% in thawed samples.

Discussion

The present report shows that levels of 8‐isoprostane and H₂O₂ cannot be reproducibly assessed in EBC from healthy smokers because of their low concentration and/or the lack of sensitivity of the available assay methods.

It is believed that cigarette smoking causes an increase in levels of free radicals and reactive oxygen species, and that oxidative status may be monitored by analysis of EBC markers 3. 8‐isoprostane, a prostaglandin F_2α isomer that is formed by free radical lipid peroxidation of arachidonic acid, may be a quantitative marker of in vivo oxidative stress in the airways of smokers 14. Montuschi etal. 7 previously investigated the effect of smoking on 8‐isoprostane levels using a similar condensing device (the commercially available EcoScreen®) and immunochemical assay (with the same LOD) to those used in the present study. They observed higher mean concentrations of 8‐isoprostane in 12 healthy smokers (24.0±2.6 pg·mL⁻¹), as well as in 10 healthy nonsmokers (10.8±0.8 pg·mL⁻¹), than observed in the present study (5.4±2.0 pg·mL⁻¹ for all subjects combined). The difference in concentration in the smokers might be due to differences in baseline characteristics. The present smokers were younger and had a lower cumulative cigarette consumption than the smokers of the study of Montuschi et al. 7 (44 versus 60 yrs; 29 versus 34 pack-yrs).

It might be argued that the broad range of age (24–61 yrs) and cumulative cigarette consumption (3–57 pack-yrs) of thepresent smokers implicates variation in susceptibility to oxidative stress and might subsequently affect the level of 8‐isoprostane measured in EBC. However, whether age andcumulative cigarette consumption indeed increase EBC 8‐isoprostane concentrations has not been addressed in previous studies.

In the present study, smokers refrained from smoking for a period of only 2 h prior to producing EBC, whereas, in other studies on 8‐isoprostane in EBC, smokers abstained from smoking for ≥12 h. However, Montuschi et al. 7 have shown that a short period of abstinence from smoking enhances exhaled 8‐isoprostane concentrations. This cannot, therefore, explain the low 8‐isoprostane concentrations detected in the present experiments.

The concentrations of 8‐isoprostane found in the present healthy smokers resembled more closely those found in healthy nonsmoking subjects (4.5±0.5 pg·mL⁻¹; n=15; eight males; mean age 42 yrs) in the study of Carpagnano et al. 15, also analysed using the same EIA kit. The investigators reported that 8‐isoprostane was detectable in the EBC of all subjects. However, figure 1 of their report shows that four of their 15 healthy subjects exhibited 8‐isoprostane concentrations below the LOD (3.9 pg·mL⁻¹). These results and the present findings indicate that more sensitive assays than the sole commercially available EIA kit are required to detect 8‐isoprostane concentrations in EBC from healthy (smoking) subjects in a more accurate manner. In addition, data obtained by immunoassay need to be compared with data obtained by more sensitive methods such as mass spectrometry. Moreover, the specificity of the available EIA kit should be further investigated.

Isoprostanes are poorly volatile substances, and, consequently, it may be expected that their concentrations in EBC will be very low in healthy smokers. The condensate samples were not enriched by extraction or lyophilisation, because the possibility that the method used for enrichment of the samples might influence 8‐isoprostane measurement could not be excluded. In addition, previous studies did not enrich EBC samples for measurement of 8‐isoprostane concentrations 7. However, in a recent abstract of van der Meer etal. 17, all EBC samples were concentrated three-fold using vacuum centrifugation, and a mean concentration of 5.2 pg·mL⁻¹ was observed in healthy subjects.

EBC consists mainly of water and volatile and water-soluble organic compounds, such as H₂O₂. These substances are able to reach the exhaled breath and can easily be detected in EBC. The detectable concentration of H₂O₂ in the present male smokers was within the concentration range of all previous observations in healthy smokers (0.10–0.75 µM) 5. This wide range of exhaled H₂O₂ concentration may be explained, in part, by the variety of condensing devices of different design that were used for the collection of EBC samples or by differences in the analytical methods used for measurement of H₂O₂. For example, Guatura et al. 18 explained their higher levels of H₂O₂ (0.75 µM) in healthy subjects by the use of a face mask and nasal breathing. The most frequently used devices for EBC collection are Teflon-lined tubes in buckets of ice, a specially designed double-walled glass vessel that is placed in ice or liquid nitrogen, or double-jacketed cooling tubes. The material of the cold tubes (glass or Teflon), as well as the length of the tube, may be important for the capture of the compounds under examination. The present study employed a new commercially available device (Ecoscreen®), which is composed of a lamellar condenser (aluminium, length ∼10 cm) and an electric refrigeration system. Before recommending its use, the performance of this device should be tested further and compared with other condensers. Furthermore, a standardised method for analysing EBC H₂O₂ is still lacking. The choice of reaction substrate (tetramethylbenzidine, p-hydroxyphenylacetic acid or homovanillic acid), as well as use of fresh versus thawed EBC samples, might explain another part of the variation in H₂O₂ concentration. In a study by van Beurden et al. 4, a different assay was used, with a reported LOD of 0.02 µM, which was lower than that reported in the present study (0.31 µM). This assay was based on the H₂O₂-dependent oxidation of p-hydroxyphenylacetic acid to a highly fluorescent dimer. The p-hydroxyphenylacetic acid reagent was added within 30 min after collection, the samples frozen at −70°C and the highly fluorescent dimer measured after 2 weeks. However, another study assessing EBC H₂O₂ reported an LOD of 0.1 µM using the same analytical method as van Beurden et al. 4 used 19. Conversely, Horvath etal. 20 and Ganas et al. 8 reported a LOD of H₂O₂ of ∼0.1 µM using the same analytical method as in the present study. Moreover, two previous studies using an assay based on H₂O₂-dependent oxidation of homovanillic acid reported an LOD of 0.1 µM 5. The different measurement techniques and reported LODs of H₂O₂ in the available studies may hamper adequate comparison between these study results and indicate the need for standardisation of analysis of EBC samples.

Although the present study contradicts published studies regarding the levels of 8‐isoprostane and H₂O₂ in EBC, the present authors are confident that every effort was made to avoid error during all phases of the study. After 15 min of breathing through the condenser, the samples were transported in <5 min to a −80°C freezer and stored there until analysis. All analyses were performed within 2 months after collection.

In the present study, several analytical problems that might interfere with the assessment of 8‐isoprostane and H₂O₂ in EBC were addressed. All of the guidelines of the manufacturer of the EIA kit were followed, including processing a positive control (maximum binding absorbance), which was in the expected range. The assay plates for 8‐isoprostane and H₂O₂ were also read at different times after addition of the reagent and no differences were observed. In a separate experiment, different concentrations of the reaction substrate (tetramethylbenzidine) were added to the H₂O₂ assay. This influenced neither the results nor the LOD. Finally, recovery experiments (using fresh and thawed (after 5 h at −80°C) samples) were also performed to assess whether components of EBC might mask the detection of 8‐isoprostane and H₂O₂. Recoveries of 83.2–109.5% (fresh samples) and 114.5–145.6% (thawed samples) were obtained for 8‐isoprostane. Whereas, recovery of exogenously added H₂O₂ ranged 69.9–129.0% for fresh samples and even reached >200% in thawed samples.

Owing to the lack of sensitivity of the methodology used in the present study, reproducibility could not be assessed in a reliable manner. Recently, two studies, by van Rensen et al. 22 and van der Meer et al. 17, reported that the levels of 8‐isoprostane or H₂O₂ measured in EBC on two consecutive days were not reproducible in patients with asthma or healthy volunteers. They used the intraclass correlation coefficient to assess the reproducibility of their measurements, which resulted in values of −0.35 (asthma) and 0.08 (healthy) for 8‐isoprostane and −0.14 (asthma) and −0.08 (healthy) for H₂O₂. The poor reproducibility of the present study confirms the results of van Rensen et al. 22 and van der Meer et al. 17.

In conclusion, levels of 8‐isoprostane and hydrogen peroxide cannot be reproducibly assessed in exhaled breath condensate from healthy smokers because of their low concentrations in most exhaled breath condensate samples and/or the lack of sensitivity of the assays used. In order to use these exhaled breath condensate markers for research purposes or as possible diagnostic test for the identification ofsmokers at higher risk of developing smoking-related pulmonary diseases, more sensitive assays for the detection of low concentrations are essential. In addition, the performance of different exhaled breath condensate collection devices needs to be compared.