Breath research in times of a global pandemic and beyond: the game changer.

In contrast to blood and urine samples, breath is invisible and ubiquitous in the environment. Different precautions are now necessary beyond the usual 'Universal Precautions'. In the era of COVID-19, breath (especially the aerosol fraction) can no longer be considered as harmless in the clinic or laboratory. As Journal of Breath Research is a primary resource for breath-related research, we (the editors) are presently developing safety guidance applicable to all breath research , not just for those projects that involve known COVID-19 infected subjects. We are starting this process by implementing requirements on reporting safety precautions in research papers and notes. This editorial announces that authors of all new submissions to JBR henceforth must state clearly the procedures undertaken for assuring laboratory and clinical safety, much like the existing requirements for disclosing Ethics Committee or Institutional Review Board protocols for studies on human subjects. In the following, we additionally make some recommendations based on best practices drawn from our experience and input from the JBR Editorial Board.

Lipid peroxidation in cardiac surgery: towards consensus on biomonitoring, diagnostic tools and therapeutic implementation.

This review focuses on oxidative stress and more specifically lipid peroxidation in cardiac surgery, one of the fundamental theories of perioperative complications. We present the molecular pathways leading to lipid peroxidation and integrate analytical methods that allow detection of lipid peroxidation markers in the fluid phase with those focusing on volatile compounds in exhaled breath. In order to explore the accumulated data in the literature, we present a systematic review of quantitative analysis of malondialdehyde, a widely used lipid peroxidation product at various stages of cardiac surgery. This exploration reveals major limitations of existing studies in terms of variability of reported values and significant gaps due to discrete and variable sampling times during surgery. We also appraise methodologies that allow real-time and continuous monitoring of oxidative stress. Complimentary techniques highlight that beyond the widely acclaimed contribution of the cardiopulmonary bypass technology and myocardial reperfusion injury, the use of diathermy contributes significantly to intraoperative lipid peroxidation. We conclude that there is an urgent need to implement the theory of oxidative stress towards a paradigm change in the clinical practice. Firstly, we need to acquire definite and irrefutable information on the link between lipid peroxidation and post-operative complications by building international consensus on best analytical approaches towards generating qualitatively and quantitatively comparable datasets in coordinated multicentre studies. Secondly, we should move away from routine low-risk surgeries towards higher risk interventions where there is major unmet clinical need for improving patient journey and outcomes. There is also need for consensus on best therapeutic interventions which could be tested in convincing large scale clinical trials. As future directions, we propose combination of fluid phase platforms and 'metabography', an extended form of capnography-including real-time analysis of lipid peroxidation and volatile footprints of metabolism-for better patient phenotyping prior to and during high risk surgery towards molecular prediction, stratification and monitoring of the patient's journey.

Standardization of the collection of exhaled breath condensate and exhaled breath aerosol using a feedback regulated sampling device.

Exhaled breath condensate (EBC) and associated exhaled breath aerosols (EBA) are valuable non-invasive biological media used for the quantification of biomarkers. EBC contains exhaled water vapor, soluble gas-phase (polar) organic compounds, ionic species, plus other species including semi- and non-volatile organic compounds, proteins, cell fragments, DNA, dissolved inorganic compounds, ions, and microbiota (bacteria and viruses) dissolved in the co-collected EBA. EBC is collected from subjects who breathe 'normally' through a chilled tube assembly for approximately 10 min and is then harvested into small vials for analysis. Aerosol filters without the chilled tube assembly are also used to separately collect EBA. Unlike typical gas-phase breath samples used for environmental and clinical applications, the constituents of EBC and EBA are not easily characterized by total volume or carbon dioxide (CO2) concentration, because the gas-phase is vented. Furthermore, EBC and associated EBA are greatly affected by breathing protocol, more specifically, depth of inhalation and expelled breath velocity. We have tested a new instrument developed by Loccioni Gruppa Humancare (Ancona, Italy) for implementation of EBC collection from human subjects to assess EBC collection parameters. The instrument is the first EBC collection device that provides instantaneous visual feedback to the subjects to control breathing patterns. In this report we describe the operation of the instrument, and present an overview of performance and analytical applications.

Clinical utility of breath ammonia for evaluation of ammonia physiology in healthy and cirrhotic adults.

Blood ammonia is routinely used in clinical settings to assess systemic ammonia in hepatic encephalopathy and urea cycle disorders. Despite its drawbacks, blood measurement is often used as a comparator in breath studies because it is a standard clinical test. We sought to evaluate sources of measurement error and potential clinical utility of breath ammonia compared to blood ammonia. We measured breath ammonia in real time by quartz enhanced photoacoustic spectrometry and blood ammonia in 10 healthy and 10 cirrhotic participants. Each participant contributed 5 breath samples and blood for ammonia measurement within 1 h. We calculated the coefficient of variation (CV) for 5 breath ammonia values, reported medians of healthy and cirrhotic participants, and used scatterplots to display breath and blood ammonia. For healthy participants, mean age was 22 years (±4), 70% were men, and body mass index (BMI) was 27 (±5). For cirrhotic participants, mean age was 61 years (±8), 60% were men, and BMI was 31 (±7). Median blood ammonia for healthy participants was within normal range, 10 µmol L(-1) (interquartile range (IQR), 3-18) versus 46 µmol L(-1) (IQR, 23-66) for cirrhotic participants. Median breath ammonia was 379 pmol mL(-1) CO2 (IQR, 265-765) for healthy versus 350 pmol mL(-1) CO2 (IQR, 180-1013) for cirrhotic participants. CV was 17 ± 6%. There remains an important unmet need in the evaluation of systemic ammonia, and breath measurement continues to demonstrate promise to fulfill this need. Given the many differences between breath and blood ammonia measurement, we examined biological explanations for our findings in healthy and cirrhotic participants. We conclude that based upon these preliminary data breath may offer clinically important information this is not provided by blood ammonia.

Monitoring breath markers under controlled conditions.

Breath analysis has the potential to detect and monitor diseases as well as to reduce the corresponding medical costs while improving the quality of a patient's life. Herein, a portable prototype, consisting of a commercial breath sampler modified to work as a platform for solid-state gas sensors was developed. The sensor is placed close to the mouth (<10 cm) and minimizes the mouth-to-sensor path to avoid contamination and dilution of the target breath marker. Additionally with an appropriate cooling concept, even high sensor operating temperatures (e.g. 350 °C) could be used. Controlled sampling is crucial for accurate repeatable analysis of the human breath and these concerns have been addressed by this novel prototype. The device helps a subject control their exhaled flow rate which increases reproducibility of intra-subject breath samples. The operation of this flame-made selective chemo-resistive gas sensor is demonstrated by the detection of breath acetone.

Breath ammonia and ethanol increase in response to a high protein challenge.

Quantifying changes in ammonia and ethanol in blood and body fluid assays in response to food is cumbersome. We used breath analysis of ammonia, ethanol, hydrogen (an accepted standard of gut transit) and acetone to investigate gastrointestinal physiology. In 30 healthy participants, we measured each metabolite serially over 6 h in control and high protein trials. Two-way repeated measures ANOVA compared treatment (control versus intervention), change from baseline to maximum and interaction of treatment and time change. Interaction was significant for ammonia (p < 0.0001) and hydrogen (p < 0.0001). We describe the dynamic measurement of multiple metabolites in response to an oral challenge.

Changes in the concentration of breath ammonia in response to exercise: a preliminary investigation.

Breath ammonia has proven to be a difficult compound to measure accurately. The goal of this study was to evaluate the effects that the physiological intervention, exercise, had on the levels of breath ammonia. The effects of vigorous exercise (4000 m indoor row) in 13 participants were studied and increases in breath ammonia were observed in all participants. Mean pre-exercise concentrations of ammonia were 670 pmol ml(-1) CO2 (SD, 446) and these concentrations increased to post-exercise maxima of 1499 pmol ml(-1) CO2 (SD, 730), p < 0.0001. The mean increase in ammonia concentrations from pre-exercise to maximum achieved in conditioned (1362 pmol ml(-1) CO2) versus non-conditioned rowers (591 pmol ml(-1) CO2) were found to be statistically different, p = 0.029. Taken together, these results demonstrate our ability to repeatedly measure the influence of exercise on the concentration of breath ammonia.

Real-time monitoring of endogenous lipid peroxidation by exhaled ethylene in patients undergoing cardiac surgery.

Pulmonary and systemic organ injury produced by oxidative stress including lipid peroxidation is a fundamental tenet of ischemia-reperfusion injury, inflammatory response to cardiac surgery, and cardiopulmonary bypass (CPB) but is not routinely measured in a surgically relevant time frame. To initiate a paradigm shift toward noninvasive and real-time monitoring of endogenous lipid peroxidation, we have explored pulmonary excretion and dynamism of exhaled breath ethylene during cardiac surgery to test the hypothesis that surgical technique and ischemia-reperfusion triggers lipid peroxidation. We have employed laser photoacoustic spectroscopy to measure real-time trace concentrations of ethylene from the patient breath and from the CPB machine. Patients undergoing aortic or mitral valve surgery-requiring CPB (n = 15) or off-pump coronary artery bypass surgery (OPCAB) (n = 7) were studied. Skin and tissue incision by diathermy caused striking (> 30-fold) increases in exhaled ethylene resulting in elevated levels until CPB. Gaseous ethylene in the CPB circuit was raised upon the establishment of CPB (> 10-fold) and decreased over time. Reperfusion of myocardium and lungs did not appear to enhance ethylene levels significantly. During OPCAB surgery, we have observed increased ethylene in 16 of 30 documented reperfusion events associated with coronary and aortic anastomoses. Therefore, novel real-time monitoring of endogenous lipid peroxidation in the intraoperative setting provides unparalleled detail of endogenous and surgery-triggered production of ethylene. Diathermy and unprotected regional myocardial ischemia and reperfusion are the most significant contributors to increased ethylene.

Fast and accurate exhaled breath ammonia measurement.

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Factors influencing breath ammonia determination.

Amongst volatile compounds (VCs) present in exhaled breath, ammonia has held great promise and yet it has confounded researchers due to its inherent reactivity. Herein we have evaluated various factors in both breath instrumentation and the breath collection process in an effort to reduce variability. We found that the temperature of breath sampler and breath sensor, mouth rinse pH, and mode of breathing to be important factors. The influence of the rinses is heavily dependent upon the pH of the rinse. The basic rinse (pH 8.0) caused a mean increase of the ammonia concentration by 410 ± 221 ppb. The neutral rinse (pH 7.0), slightly acidic rinse (pH 5.8), and acidic rinse (pH 2.5) caused a mean decrease of the ammonia concentration by 498 ± 355 ppb, 527 ± 198 ppb, and 596 ± 385 ppb, respectively. Mode of breathing (mouth-open versus mouth-closed) demonstrated itself to have a large impact on the rate of recovery of breath ammonia after a water rinse. Within 30 min, breath ammonia returned to 98 ± 16% that of the baseline with mouth open breathing, while mouth closed breathing allowed breath ammonia to return to 53 ± 14% of baseline. These results contribute to a growing body of literature that will improve reproducibly in ammonia and other VCs.

Clinical breath analysis: discriminating between human endogenous compounds and exogenous (environmental) chemical confounders.

Volatile organic compounds (VOCs) in exhaled breath originate from current or previous environmental exposures (exogenous compounds) and internal metabolic (anabolic and catabolic) production (endogenous compounds). The origins of certain VOCs in breath presumed to be endogenous have been proposed to be useful as preclinical biomarkers of various undiagnosed diseases including lung cancer, breast cancer, and cardio-pulmonary disease. The usual approach is to develop difference algorithms comparing VOC profiles from nominally healthy controls to cohorts of patients presenting with a documented disease, and then to apply the resulting rules to breath profiles of subjects with unknown disease status. This approach to diagnosis has a progression of sophistication; at the most rudimentary level, all measurable VOCs are included in the model. The next level corrects exhaled VOC concentrations for current inspired air concentrations. At the highest level, VOCs exhibiting discriminatory value also require a plausible biochemical pathway for their production before inclusion. Although these approaches have all shown some level of success, there is concern that pattern recognition is prone to error from environmental contamination and between-subject variance. In this paper, we explore the underlying assumptions for the interpretation and assignment of endogenous compounds with probative value for assessing changes. Specifically, we investigate the influence of previous exposures, elimination mechanisms and partitioning of exogenous compounds as confounders of true endogenous compounds. We provide specific examples based on a simple classical pharmacokinetic approach to identify potential misinterpretations of breath data and propose some remedies.

Forced expiratory capnography and chronic obstructive pulmonary disease (COPD).

This report proposes a potentially sensitive and simple physiological method to detect early changes and to follow disease progression in obstructive pulmonary disease (COPD) based upon the usual pulmonary function test. Pulmonary function testing is a simple, although relatively insensitive, method to detect and follow COPD. As a proof-of-concept, we have examined the slope of the plateau for carbon dioxide during forced expiratory capnography in healthy (n = 10) and COPD subjects (n = 10). We compared the change in the rate of exhalation of carbon dioxide over time as a marker of heterogeneous ventilation of the lung. All subjects underwent pulmonary function testing, body-plethysmography, and forced exhalation capnography. The subjects with COPD also underwent high-resolution computed tomography of the chest. Regression lines were fitted to the slopes of the forced exhalation capnogram curves. There was no difference in the mean levels of exhaled carbon dioxide between the COPD and the healthy groups (p > 0.48). We found a significant difference in the mean slope of the forced exhalation capnogram for the COPD subjects compared to the healthy subjects (p = 0.01). Most important, for the COPD subjects, there was a significant positive correlation between the slope of the forced exhaled capnogram and a defined radiodensity measurement of the lung by high-resolution computed tomography (r(2) = 0.49, p = 0.02). The slope of the forced exhalation capnogram may be a simple way to determine physiological changes in the lungs in patients with COPD that are not obtainable with standard pulmonary function tests. Forced exhalation capnography would be of great clinical benefit if it can identify early disease changes and at-risk individuals.

Off-axis integrated cavity output spectroscopy with a mid-infrared interband cascade laser for real-time breath ethane measurements.

Cavity-enhanced tunable diode laser absorption spectroscopy is an attractive method for measuring small concentrations of gaseous species. Ethane is a breath biomarker of lipid peroxidation initiated by reactive oxygen species. A noninvasive means of quickly quantifying oxidative stress status has the potential for broad clinical application. We present a simple, compact system using off-axis integrated cavity output spectroscopy with an interband cascade laser and demonstrate its use in real-time measurements of breath ethane. We demonstrate a detection sensitivity of 0.48 ppb/Hz(1/2).

Changes in oxidative stress during outpatient surgery.

Reactive oxygen species are associated with tissue inflammation and injury. Our laboratory has demonstrated that ethane, a stable product of lipid peroxidation, in exhaled breath can be used to measure total body oxidative stress. Herein patients were studied who underwent outpatient surgery, laproscopic bilateral tubal ligation (BTL, n = 10) and anterior cruciate ligament (ACL, n = 10) repair of the knee. These surgical procedures were expected to involve mild degrees of ischemia and reperfusion. In each of these cases propofol, an intravenous anesthetic with antioxidant properties, was used. Breath ethane was measured as a biomarker of oxidative stress that occurred at reperfusion of ischemic tissue. Data were analyzed by one-way analysis of variance. Clinically relevant concentrations of propofol were unable to completely block the increase in oxidative stress following reperfusion in either of these minor surgeries. Breath ethane increased significantly after reperfusion in both the BTL (p = 0.03) and the ACL (p = 0.005) patients. Also, the increase in oxidative stress was related to the time of ischemia.