The effect of gas standardisation on exhaled breath condensate pH

To the Editors:

We have read with interest the American Thoracic Society/European Respiratory Society Task Force document on exhaled breath condensate (EBC) 1. EBC pH is emerging as a potential biomarker in respiratory disease. Gas standardisation (or de-aeration) of EBC with argon is commonly performed to remove carbon dioxide prior to pH measurement 2–4. It has been argued that CO₂ is unwelcome “noise” in the sample and, although the completeness of CO₂ removal has not been confirmed, gas-standardised EBC pH is stable and provides reproducible measurements 3, 4. However, some authors regard CO₂ as a relevant component of EBC and have measured pH without gas standardisation 5, 6. These variations in methodology make comparison between studies difficult.

We recently reported a mean change in pH after gas standardisation of 0.94 4, and this pH was stable at room temperature. Despite this rise in EBC pH, samples from patients with respiratory disease may remain acidic. Furthermore, gas standardisation may have little effect on the pH of very acidic samples 3, 7. These observations indicate that the effect of gas standardisation is variable, and that there may still be stable acids present in the sample after presumed removal of CO₂. Therefore, we measured EBC pH pre- and post-gas standardisation to investigate the relative contributions of CO₂ and other acids to EBC pH. We also investigated the stability of EBC pH samples left at room temperature without gas standardisation.

EBC was collected from a total of 30 chronic obstructive pulmonary disease (COPD; 19 males, mean age 63 yrs, 15 current smokers, mean forced expiratory volume in one second (FEV₁) 62% predicted) and 20 asthma patients (nine males, mean age 50 yrs, zero current smokers, mean FEV₁ 96% pred). pH was measured prior to and following gas standardisation with argon as previously described 5. In samples collected from six COPD patients, pH was measured immediately and after 30 min and 3 h standing at room temperature without gas standardisation. Informed consent was obtained and the local ethics committee approved the study.

The mean (95% confidence interval (CI)) increase in pH post-argon was 0.91 (0.81–1.01; p<0.00001) and 0.92 (0.77–1.06; p<0.00001; fig. 1⇓) in asthma and COPD, respectively. In asthma, there was a significant correlation between pre-argon pH and subsequent change in pH (r = -0.77; p<0.0001), which was described by the equation y = -0.58x+4.8. Six samples from COPD patients had a pre-argon pH of <6, with the change in pH post-argon ranging -0.05–1.75. Exclusion of these six samples resulted in a similar correlation (r = -0.67; p = 0.0004) and equation (y = -0.6x+4.9). The pH of samples measured after standing at room temperature was significantly higher than those measured immediately; the mean (95% CI) increase was 0.20 (0.11–0.28) after 30 min and 0.67 (0.47–0.86) after 3 h.

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Fig. 1—

Exhaled breath condensate pH pre- and post-argon gas standardisation in asthma and chronic obstructive pulmonary disease (COPD) patients.

EBC pH is unstable at room temperature, presumably because CO₂ diffuses out of solution. This raises practical difficulties, as pH measurements may vary if not performed immediately. In contrast, the pH of argon de-aerated EBC samples is stable at room temperature 4.

Gas standardisation caused a significant increase in EBC pH, with a mean change of ∼1, which is similar to a previous study 8. However, we have shown that the effects of gas standardisation are more complex and can be related to the pre-argon value. At pre-argon pH >6, the effect is predictable and described by similar equations in asthma and COPD patients. Gas standardisation removes more CO₂ in samples with lower pre-argon pH values.

The effect of gas standardisation in samples with pre-argon pH <6 was unpredictable. Gessner et al. 7 also noted that argon de-aeration may have very little effect on the pH of samples with an initial pH of <6. This suggests that either dissolved CO₂ is not being removed, or that the pH is mainly determined by other acids. If we assume that the change in pH after gas standardisation is a surrogate for CO₂ concentration, it has been shown that this varies between individuals. Acidic exhaled breath condensate pH could be caused by airway acidification at any level from the mouth to the alveoli. The source and nature of exhaled breath condensate acidification remain controversial 9, 10. The use of gas standardisation allows us to differentiate the probable contribution of CO₂ and that of other, as yet unknown, acids to exhaled breath condensate pH.