ABSTRACT

In recent years, the analysis of exhaled breath condensate samples has been given great weight as a noninvasive methodology of studying physiology and lung diseases. The present study describes a device for measuring exhaled breath condensate that is affordable, easily constructed, portable and suitable for use in the field, as well as allowing the collection of simultaneous samples. The results obtained with this device in terms of the concentrations of pH, hydrogen peroxide and nitrite, metabolites related to inflammatory and oxidative damage, in exhaled breath condensate samples are comparable to those obtained with other devices previously described.

Keywords: Exhalation; Lung diseases; Equipment design.

RESUMO

El análisis de muestras de aire espirado condensado ha cobrado gran relevancia en los últimos años como método no invasivo de estudio de la fisiología y las enfermedades de origen pulmonar. En el presente trabajo se describe un equipo para tomar muestras de aire espirado condensado de bajo costo, fácil de fabricar, de transportar al terreno y que permite tomar muestras en forma simultánea. La concentración de metabolitos relativos a procesos inflamatorios y al daño oxidativo (pH, peróxido de hidrógeno y nitrito) de muestras de aire espirado condensado obtenido con este equipo son comparables a los reportados con otros previamente.

Palavras-chave: Espiración; Enfermedades pulmonares; Diseño de equipo.

In recent years, the analysis of exhaled breath condensate (EBC) has been proposed as a noninvasive methodology of studying lung diseases.­(1) This tool has been used to search for mechanisms as well as to perform clinical follow-up of various pulmonary pathologies.(2,3) Exhaled breath is returned to the environment at a temperature of approximately 37ºC, being saturated with water together with metabolism products and lung epithelial surface derivatives. Cooling of exhaled air by condensers captures part of this exhaled air through a system of hoses and tubes that offer little resistance to respiration. The product of this process is a transparent liquid containing volatile and nonvolatile particles, the concentrations of which are expressed using measures as small as micromoles per liter. Commercial devices have been developed for collecting EBC samples. Among such devices, the most widely used are the RTubeTM (Respiratory Research Inc., Charlottesville, VA, USA) and the ECoScreen (Jaeger GmbH, Hoechberg, Germany). However, there are various models constructed by individual investigators for general use,(4) as well as for use in specific situations such as mechanical ventilation(5) or sample collection from suckling infants.(6) The particular interest of the investigators of the present study was to develop an EBC condenser that is affordable and portable, as well as allowing the collection of simultaneous samples, use in several experimental situations and use in patients with pathologies of pulmonary origin, either in hospitals or in the field, for the follow-up evaluation of exercise tolerance, as well as the effects of air contaminants, altitude and work.

Description of the device

Figure 1 presents a model of the device.



1) Connection to the condenser: Patients can be connected to the condenser via a mouthpiece or a mask. Although the mask is better tolerated by subjects, the mouthpiece allows the formation of approximately twice as much EBC in the same amount of time. For both forms of connection, a saliva trap should be used in order to avoid sample contamination.

2) One-way system: The one-way design allows the device to condense only the exhaled air and avoid contamination with substances in the environment. In our device, we adapted two valves, each measuring 22 mm in diameter (catalog numbers 1664 and 1665; Hudson RCI, Durham, NC, USA).

3) Flexible connector: The flexible connector allows subjects to move and adapt to a more comfortable position without interrupting or increasing resistance to exhalation. This can be constructed using a tube measuring 15 cm in length and 22 mm in internal diameter (catalog number 60-50‑150-1; VBM Medizintechnik GmbH, Sulz, Germany).

4) Flexible heater: The device includes a mesh-covered electrical resistor whose ends are connected to a regulator that maintains the temperature at 37°C, preventing condensation in this area and increasing the collection yield.

5) Glass condenser: A Y-shaped glass condenser is used. The condenser has two upper arms, set at 45° angles, each measuring 120 mm in length and 8 mm in internal diameter. The flexible connector is attached to one end, and a hose, which allows the outflow of air, is connected to the other. At its lower end, the condenser has a third arm, measuring 40 mm in length, to which a plastic tube, which collects the sample, is inserted under pressure. In order to increase the EBC flow collected, more than one glass condenser, joined by flexible connectors, can be used.

6) Cooling system: A box containing crushed ice (−5ºC), a mixture of ice and salt (−15ºC) or ice packs (−15ºC) can be used as a cooling system. The sample volume collected depends on the temperature and on the number of glass condensers used. As a reference, in an adult subject, 1.5 mL can be collected in 15 min when using crushed ice and a single condenser. Approximately twice as much is collected in the same amount of time when using two glass condensers and a mixture of ice and salt.
Note: The device was designed to be portable and to allow easy assembly/disassembly, as well as allowing the collection of simultaneous samples. When collecting samples from patients with pathologies of infectious origin, its parts can be discarded after use. However, the glass condenser can be sterilized and reused.

Sample collection protocol

We recommend that subjects be comfortably seated, at rest and wearing a nose clip. Prior to sample collection, subjects should not eat for one hour and should not smoke for six hours. Due to the lower cost and the greater volume of condensate collected, we recommend that the mixture of ice and salt be used as the cooling method. Total time to collection under these conditions is 10 min or until subjects produce a sample of 1.5-2 mL (Table 1).



Chemical determinations

Various parameters, such as markers of inflammation, remodeling and tissue oxidative damage, have been determined in EBC ­samples­.­(2,3) Using the EBC condenser described in the present study, the concentrations of hydrogen peroxide, nitrite and pH were determined by different methods (Table 1), as were the concentrations of malondialdehyde,(7) 8-­isoprostane and protein (data not shown). Although our condenser was not directly compared with others, the values revealed by the chemical analysis of the samples collected using the EBC condenser described here are at a level similar to previously reported values obtained using other condensers in healthy subjects (Table 1). In the future, this comparison should be carried out in order to allow a statistical analysis, which was not performed in the present report.
Regarding the influence of the type of EBC condenser on the results, there have been reports in which other condensers were used indicating differences for parameters such as pH,(8-9) whereas others show no differences for aldehydes (malondialdehyde, hexanal, heptanal or nonanal).(10) Similarly, one group of authors,(11) using four different types of condensers, found no differences in condensate volume, nor in the concentration of hydrogen peroxide, 8-isoprostane or cytokine. This suggests that, in the search for standardization of EBC sample collection, it is necessary to standardize and describe the conditions under which samples are collected and handled, this probably being more important than is the type of condenser used. To date, there is no definite evidence that one condenser is more appropriate than others for collecting reproducible samples as suggested in the American Thoracic Society and the European Respiratory Society consensus on EBC collection methodology.(2)

In summary, the device described in the present study corresponds to a low cost system that has disposable parts and allows the collection of simultaneous samples from subjects under various environmental and experimental situations, providing results similar to those obtained with other EBC condensers previously described.

Acknowledgments

We wish to thank Mr. Luis Pizarro Zýñiga for his assistance in sample collection and chemical analysis. We would also like to thank Dr. Claus Behn for his comments and for facilitating the performance of the present study.

References

1. Hunt J. Exhaled breath condensate: an overview. Immunol Allergy Clin North Am. 2007;27(4):587-96; v.

2. Horváth I, Hunt J, Barnes PJ, Alving K, Antczak A, Baraldi E, et al. Exhaled breath condensate: methodological recommendations and unresolved questions. Eur Respir J. 2005;26(3):523-48.

3. Kharitonov SA, Barnes PJ. Exhaled biomarkers. Chest. 2006;130(5):1541-6.

4. van Beurden WJ, Harff GA, Dekhuijzen PN, van den Bosch MJ, Creemers JP, Smeenk FW. An efficient and reproducible method for measuring hydrogen peroxide in exhaled breath condensate. Respir Med. 2002;96(3):197-203.

5. Walsh BK, Mackey DJ, Pajewski T, Yu Y, Gaston BM, Hunt JF. Exhaled-breath condensate pH can be safely and continuously monitored in mechanically ventilated patients. Respir Care. 2006;51(10):1125-31.

6. Moeller A, Franklin P, Hall GL, Horak F Jr, Wildhaber JH, Stick SM. Measuring exhaled breath condensates in infants. Pediatr Pulmonol. 2006;41(2):184-7.

7. Araneda OF, García C, Lagos N, Quiroga G, Cajigal J, Salazar MP, et al. Lung oxidative stress as related to exercise and altitude. Lipid peroxidation evidence in exhaled breath condensate: a possible predictor of acute mountain sickness. Eur J Appl Physiol. 2005;95(5‑6):383-90.

8. Leung TF, Li CY, Yung E, Liu EK, Lam CW, Wong GW. Clinical and technical factors affecting pH and other biomarkers in exhaled breath condensate. Pediatr Pulmonol. 2006;41(1):87-94.

9. Prieto L, Ferrer A, Palop J, Domenech J, Llusar R, Rojas R. Differences in exhaled breath condensate pH measurements between samples obtained with two commercial devices. Respir Med. 2007;101(8):1715-20.

10. Corradi M, Rubinstein I, Andreoli R, Manini P, Caglieri A, Poli D, et al. Aldehydes in exhaled breath condensate of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2003;167(10):1380-6.

11. Rosias PP, Robroeks CM, Kester A, den Hartog GJ, Wodzig WK, Rijkers GT, et al. Biomarker reproducibility in exhaled breath condensate collected with different condensers. Eur Respir J. 2008;31(5):934-42.

12. Nowak D, Kalucka S, Białasiewicz P, Król M. Exhalation of H2O2 and thiobarbituric acid reactive substances (TBARs) by healthy subjects. Free Radic Biol Med. 2001;30(2):178-86.

13. Hunt JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TA, et al. Endogenous airway acidification. Implications for asthma pathophysiology. Am J Respir Crit Care Med. 2000;161(3 Pt 1):694-9.

14. Nightingale JA, Rogers DF, Barnes PJ. Effect of inhaled ozone on exhaled nitric oxide, pulmonary function, and induced sputum in normal and asthmatic subjects. Thorax. 1999;54(12):1061-9.

15. Paget-Brown AO, Ngamtrakulpanit L, Smith A, Bunyan D, Hom S, Nguyen A, et al. Normative data for pH of exhaled breath condensate. Chest. 2006;129(2):426-30.

16. Gay CA, Gebicki JM. Perchloric acid enhances sensitivity and reproducibility of the ferric-xylenol orange peroxide assay. Anal Biochem. 2002;304(1):42-6.

17. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982;126(1):131-8.



About the authors

Oscar Florencio Araneda Valenzuela
Professor of Human Physiology. Universidad Mayor School of Medicine, Santiago, Chile.
Maria Paulina Salazar Encina
Pediatrician at the Luis Calvo Mackenna Hospital, Santiago, Chile.


Study carried out at the Universidad Mayor School of Medicine, Santiago, Chile.
Correspondence to: Oscar Araneda Valenzuela. Camino la Pirámide, 5750, Huechuraba, Santiago, Chile.
Tel 56 02 3281295. E-mail: oscar.aranedav@mayor.cl
Financial support: none.
Submitted: 17 February 2008. Accepted, after review: 26 May 2008.