Effect of temperature control on the metabolite content in exhaled breath condensate
Graphical abstract
Introduction
The non-invasive, quick, and safe collection of exhaled breath condensate (EBC) makes it a candidate as a diagnostic matrix for use in personalized health monitoring devices. Single-use, sterile EBC collection device components can be mass produced cheaply and used safely in non-medical settings. The recent advances in microfluidic lab-on-a-chip analysis and cloud-based data analysis algorithms may make prescreening of a number of diseases possible in a short time period at a small fraction of the current cost.
EBC is a complex matrix which has been shown to have a chemical composition resembling that of the extracellular lung fluid [[1], [2], [3], [4], [5]]. This biological sample is rich with a wide variety of compounds including: non-volatile biomolecules aerosolized from the airway lining fluid and water-soluble volatile compounds, proteins, lipids, antibodies, and carbohydrates. In total, in humans, EBC contains up to 2000 different compounds [[6], [7], [8]]. EBC analysis may not precisely measure solute concentrations in native airway fluid; however, if concentrations of certain compounds differ enough between a healthy and diseased state, EBC analysis can be a potential diagnostic tool [7]. Some individual compounds or a set of compounds can be reflective of diseased state and are called biomarkers. Currently, nitric oxide (NO) [[9], [10], [11], [12]], hydrogen peroxide (H2O2) [[13], [14], [15], [16]], and acetone, measured from breath condensate, are the three most studied and used compounds for diagnostic biomarkers of inflammatory responses in the respiratory system. Concentrations of lipids can also be measured from EBC, including fatty acids, steroids, eicosanoids, and their subclasses, such as prostaglandins or isoprostanes [17,18]. For instance, 8-isoprostane detected in EBC, is considered a biomarker of oxidative stress and antioxidant deficiency, showed differences between healthy smokers and patients with COPD [19]. Plasma lysozyme was found to be significantly higher in patients with adult respiratory distress syndrome (ARDS) as compared to healthy patients [20]. Other low-volatility compounds such as enzymes have been considered as effective biomarkers of illness diagnosis. The pH of EBC can also serve as a simple but robust biomarker of various lung diseases [[21], [22], [23]]. The compounds, including volatile organic compounds (VOCs) present in EBC are not limited to the respiratory system but may originate from blood borne biogenic compounds, and can be representative metabolites of a wide range of systemic processes [24,25]. Patients with and without lung cancer, regardless of the cancer stage, were discriminated using 22 VOCs including alkanes, alkane derivatives, and benzene derivatives [26]. A combination of eight VOCs was sufficient to discriminate between asthmatic and healthy children [27].
EBC analysis has some current limitations; the persistent problem is the lack of standardization in the collection methods, the collection devices, and the sample analysis [[28], [29], [30]]. The collection procedure and hardware design are known to significantly affect the metabolomic content of the EBC sample [31,32]. A number of parameters were examined: effect of sampling duration, breathing pattern, collected fraction of the exhaled breath (alveolar end tidal versus total expired volume), collection device material, condensation temperature, contamination from saliva, sample transfer, and storage [[33], [34], [35]]. The design and performance of commercially available EBC samplers such as the Rtube™ (Respiratory Research, Inc., Austin, TX, USA), EcoScreen® (Erich Jaeger GmbH, Hoechberg, Germany), and TurboDECCS (MEDIVAC, Parma, Italy) were compared to answer some of the questions about sample collection procedure and device choice [[36], [37], [38]].
In our previous work [38], we compared the performance of an engineered EBC collection device with that of RTube™ and TurboDECCS®. Though the three devices equilibrated in the volume of collected EBC sample, the EBC samples differed in the metabolomic content. The engineered device collected EBC samples that contained less saliva but higher number of compounds. There were some design differences responsible for that; different collection temperatures, different surface materials, and different saliva filtering mechanism.
The engineered device had a PTFE housing (duct) and a glass condenser surface cooled by dry ice pellets; it warmed up from −56 °C to - 30 °C in a 10 min sampling period. RTube™ and TurboDECCS® had polypropylene condenser surfaces and significantly differed in collection temperature and its stability. RTube™ warmed from −56 °C to 0 °C during a 10 min sampling period and TurboDECCS® warmed from −7 °C to 6 °C during the first minute of breath sampling [38]. The surface properties of the collector surface are also known to have an effect on the recovery of the metabolomic content of EBC.
Rosias et al. [35,37] studied the effect of the condenser surface coatings on measurement of biomarkers in EBC. Five condenser coatings (silicone, glass, aluminum, polypropylene, and Teflon) were compared using the EcoScreen® device. Adhesive properties of different condenser coatings influenced the eicosanoids and proteins measurements in EBC. Silicone and glass coatings were shown to be more efficient for measurement of 8-isoprostane or albumin in EBC. The relative importance of hardware parameters, e.g. temperature level versus surface material, and their effects on the content of EBC samples needs to be quantified with a rigorous set-up where both tested parameters are highly controlled.
The contamination with saliva needs to be minimized because oral microbiome contributes a wide variety of metabolites that may obscure biomarkers originating in the lungs [39]. The level of saliva contamination in collected EBC samples was different; the engineered device had the least level of saliva contamination.
The previous studies that used different devices are informative and give some common points for estimation but lack to define the relative importance of design parameters because the compared devices differ in surface material temperature, and saliva filtering [32,36,38].
While all design parameters (saliva trap, material choice, breath flow, chamber design, and heat transfer) have their effect on the metabolomic content of EBC, here we investigate the significance of the condensation temperature while keeping other parameters constant. A custom EBC sampling device was used [38]. A refrigeration-based cooling system with an accurate temperature control was constructed for this experimental investigation. The EBC samples were collected at incremental temperatures, between 5 °C and −56 °C, from one group of volunteers, with the one device, and with the same procedure. The volatile fraction of the EBC samples was analyzed with a gas chromatography coupled to mass spectroscopy (GC-MS) method and the non-volatile fraction with high performance liquid chromatography-mass spectrometry (HPLC-MS) methods. Knowing some guidelines for the choice of an optimal temperature for EBC collection will be very useful for engineering future portable platforms for EBC analysis. From an engineering point of view, the temperature level may directly determine the power requirements. From a diagnostic point of view, it may affect the metabolomic content of EBC samples.
Section snippets
EBC sampler and experimental hardware
A custom built EBC sampling device [38] was used in this study with a modification in the cooling method. Fig. 1 shows the main components of the device. Its design employs a number of engineering solutions to make it optimal for collection of EBC samples [38]. Chemically inert materials (PTFE and glass) were used for parts that are in contact with the biological sample. An engineered mass-momentum-based flow filter was used to reduce sample contamination with saliva droplets carried with the
Condenser surface temperature optimization
The device was calibrated for temperature accuracy. Fig. 4a shows measured temperature values with no forced air flow through the condenser tube; both ends of the tube were open to the ambient air (20 °C, 40% RH). Eight thermocouples were distributed, in a helix arrangement, on the inner side of the glass tube along its length. The significantly higher reading from TC8 is due to its position at the end of the tube. We suspect that it had a poor thermal contact with the cold condenser surface
Conclusions
This work contributes to the prior knowledge in the field of EBC analysis and helps to standardize the collection protocol that will determine the design of an optimal EBC sampling hardware in terms of operating temperature. Here, we considered the sole effect of collection temperature that might be a viable primary parameter for the purpose of standardization among different devices [5,32,42,45,46]. The experimental results corroborate the findings of previous studies about effects of
Acknowledgments
Partial support was provided by: NIH award U01 EB0220003-01 [CED, NJK]; NIH National Center for Advancing Translational Sciences (NCATS) through grant#UL1 TR000002 [CED, NJK]; NIH award UG3-OD023365 [CED, NJK]; NIH award 1P30ES023513-01A1 [CED, NJK]; NIH award 1K23HL127185-01A1 [MS]; Office of Naval Research grant #N-00014-13-1-0580 [CED, BCW, MS]; and The Hartwell Foundation [CED, NJK]. Student support was provided by NIH award T32 HL07013 [KOZ] and NIH award #P42ES004699 [KOZ]. The contents
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