An inexpensive UV-LED photoacoustic based real-time sensor-system detecting exhaled trace-acetone.

In this research we present a low-cost system for breath acetone analysis based on UV-LED photoacoustic spectroscopy. We considered the end-tidal phase of exhalation, which represents the systemic concentrations of volatile organic compounds (VOCs) - providing clinically relevant information about the human health. This is achieved via the development of a CO2-triggered breath sampling system, which collected alveolar breath over several minutes in sterile and inert containers. A real-time mass spectrometer is coupled to serve as a reference device for calibration measurements and subsequent breath analysis. The new sensor system provided a 3σ detection limit of 8.3 ppbV and an NNEA of 1.4E-9 Wcm-1Hz-0.5. In terms of the performed breath analysis measurements, 12 out of 13 fell within the error margin of the photoacoustic measurement system, demonstrating the reliability of the measurements in the field.

Effects of Contagious Respiratory Pathogens on Breath Biomarkers.

Due to their immediate exhalation after generation at the cellular/microbiome levels, exhaled volatile organic compounds (VOCs) may provide real-time information on pathophysiological mechanisms and the host response to infection. In recent years, the metabolic profiling of the most frequent respiratory infections has gained interest as it holds potential for the early, non-invasive detection of pathogens and the monitoring of disease progression and the response to therapy. Using previously unpublished data, randomly selected individuals from a COVID-19 test center were included in the study. Based on multiplex PCR results (non-SARS-CoV-2 respiratory pathogens), the breath profiles of 479 subjects with the presence or absence of flu-like symptoms were obtained using proton-transfer-reaction time-of-flight mass spectrometry. Among 223 individuals, one respiratory pathogen was detected in 171 cases, and more than one pathogen in 52 cases. A total of 256 subjects had negative PCR test results and had no symptoms. The exhaled VOC profiles were affected by the presence of Haemophilus influenzae, Streptococcus pneumoniae, and Rhinovirus. The endogenous ketone, short-chain fatty acid, organosulfur, aldehyde, and terpene concentrations changed, but only a few compounds exhibited concentration changes above inter-individual physiological variations. Based on the VOC origins, the observed concentration changes may be attributed to oxidative stress and antioxidative defense, energy metabolism, systemic microbial immune homeostasis, and inflammation. In contrast to previous studies with pre-selected patient groups, the results of this study demonstrate the broad inter-individual variations in VOC profiles in real-life screening conditions. As no unique infection markers exist, only concentration changes clearly above the mentioned variations can be regarded as indicative of infection or colonization.

Protocol for visualizing complex volatile metabolomics data in clinical setups using EDaViS software.

Here, we present a protocol for using Early Data Visualization Script, a user-friendly software tool to visualize complex volatile metabolomics data in clinical setups. We describe steps for tabulating data and adjusting visual output to visualize complex time-resolved volatile omics data using simple charts and graphs. We then demonstrate possible modifications by detailing procedures for the adaptation of four basic functions. For complete details on the use and execution of this protocol, please refer to Sukul et al. (2022)1 and Remy et al. (2022).2.

Origin of breath isoprene in humans is revealed via multi-omic investigations.

Plants, animals and humans metabolically produce volatile isoprene (C5H8). Humans continuously exhale isoprene and exhaled concentrations differ under various physio-metabolic and pathophysiological conditions. Yet unknown metabolic origin hinders isoprene to reach clinical practice as a biomarker. Screening 2000 individuals from consecutive mass-spectrometric studies, we herein identify five healthy German adults without exhaled isoprene. Whole exome sequencing in these adults reveals only one shared homozygous (European prevalence: <1%) IDI2 stop-gain mutation, which causes losses of enzyme active site and Mg2+-cofactor binding sites. Consequently, the conversion of isopentenyl diphosphate to dimethylallyl diphosphate (DMAPP) as part of the cholesterol metabolism is prevented in these adults. Targeted sequencing depicts that the IDI2 rs1044261 variant (p.Trp144Stop) is heterozygous in isoprene deficient blood-relatives and absent in unrelated isoprene normal adults. Wild-type IDI1 and cholesterol metabolism related serological parameters are normal in all adults. IDI2 determines isoprene production as only DMAPP sources isoprene and unlike plants, humans lack isoprene synthase and its enzyme homologue. Human IDI2 is expressed only in skeletal-myocellular peroxisomes and instant spikes in isoprene exhalation during muscle activity underpins its origin from muscular lipolytic cholesterol metabolism. Our findings translate isoprene as a clinically interpretable breath biomarker towards potential applications in human medicine.

Physio-metabolic and clinical consequences of wearing face masks-Systematic review with meta-analysis and comprehensive evaluation.

Background: As face masks became mandatory in most countries during the COVID-19 pandemic, adverse effects require substantiated investigation. Methods: A systematic review of 2,168 studies on adverse medical mask effects yielded 54 publications for synthesis and 37 studies for meta-analysis (on n = 8,641, m = 2,482, f = 6,159, age = 34.8 ± 12.5). The median trial duration was only 18 min (IQR = 50) for our comprehensive evaluation of mask induced physio-metabolic and clinical outcomes. Results: We found significant effects in both medical surgical and N95 masks, with a greater impact of the second. These effects included decreased SpO2 (overall Standard Mean Difference, SMD = -0.24, 95% CI = -0.38 to -0.11, p < 0.001) and minute ventilation (SMD = -0.72, 95% CI = -0.99 to -0.46, p < 0.001), simultaneous increased in blood-CO2 (SMD = +0.64, 95% CI = 0.31-0.96, p < 0.001), heart rate (N95: SMD = +0.22, 95% CI = 0.03-0.41, p = 0.02), systolic blood pressure (surgical: SMD = +0.21, 95% CI = 0.03-0.39, p = 0.02), skin temperature (overall SMD = +0.80 95% CI = 0.23-1.38, p = 0.006) and humidity (SMD +2.24, 95% CI = 1.32-3.17, p < 0.001). Effects on exertion (overall SMD = +0.9, surgical = +0.63, N95 = +1.19), discomfort (SMD = +1.16), dyspnoea (SMD = +1.46), heat (SMD = +0.70), and humidity (SMD = +0.9) were significant in n = 373 with a robust relationship to mask wearing (p < 0.006 to p < 0.001). Pooled symptom prevalence (n = 8,128) was significant for: headache (62%, p < 0.001), acne (38%, p < 0.001), skin irritation (36%, p < 0.001), dyspnoea (33%, p < 0.001), heat (26%, p < 0.001), itching (26%, p < 0.001), voice disorder (23%, p < 0.03), and dizziness (5%, p = 0.01). Discussion: Masks interfered with O2-uptake and CO2-release and compromised respiratory compensation. Though evaluated wearing durations are shorter than daily/prolonged use, outcomes independently validate mask-induced exhaustion-syndrome (MIES) and down-stream physio-metabolic disfunctions. MIES can have long-term clinical consequences, especially for vulnerable groups. So far, several mask related symptoms may have been misinterpreted as long COVID-19 symptoms. In any case, the possible MIES contrasts with the WHO definition of health. Conclusion: Face mask side-effects must be assessed (risk-benefit) against the available evidence of their effectiveness against viral transmissions. In the absence of strong empirical evidence of effectiveness, mask wearing should not be mandated let alone enforced by law. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021256694, identifier: PROSPERO 2021 CRD42021256694.

Advanced setup for safe breath sampling and patient monitoring under highly infectious conditions in the clinical environment.

Being the proximal matrix, breath offers immediate metabolic outlook of respiratory infections. However, high viral load in exhalations imposes higher transmission risk that needs improved methods for safe and repeatable analysis. Here, we have advanced the state-of-the-art methods for real-time and offline mass-spectrometry based analysis of exhaled volatile organic compounds (VOCs) under SARS-CoV-2 and/or similar respiratory conditions. To reduce infection risk, the general experimental setups for direct and offline breath sampling are modified. Certain mainstream and side-stream viral filters are examined for direct and lab-based applications. Confounders/contributions from filters and optimum operational conditions are assessed. We observed immediate effects of infection safety mandates on breath biomarker profiles. Main-stream filters induced physiological and analytical effects. Side-stream filters caused only systematic analytical effects. Observed substance specific effects partly depended on compound's origin and properties, sampling flow and respiratory rate. For offline samples, storage time, -conditions and -temperature were crucial. Our methods provided repeatable conditions for point-of-care and lab-based breath analysis with low risk of disease transmission. Besides breath VOCs profiling in spontaneously breathing subjects at the screening scenario of COVID-19/similar test centres, our methods and protocols are applicable for moderately/severely ill (even mechanically-ventilated) and highly contagious patients at the intensive care.

Profiling of exhaled volatile organics in the screening scenario of a COVID-19 test center.

Breath volatile organics (VOCs) may provide immediate information on infection mechanisms and host response. We conducted real-time mass spectrometry-based breath profiling in 708 non-preselected consecutive subjects in the screening scenario of a COVID-19 test center. Recruited subjects were grouped based on PCR-confirmed infection status and presence or absence of flu-like symptoms. Exhaled VOC profiles of SARS-CoV-2-positive cases (n = 36) differed from healthy (n = 256) and those with other respiratory infections (n = 416). Concentrations of most VOCs were suppressed in COVID-19. VOC concentrations also differed between symptomatic and asymptomatic cases. Breath markers mirror effects of infections onto host's cellular metabolism and microbiome. Downregulation of specific VOCs was attributed to suppressive effects of SARS-CoV-2 onto gut or pulmonary microbial metabolism. Breath analysis holds potential for monitoring SARS-CoV-2 infections rather than for primary diagnosis. Breath profiling offers unconventional insight into host-virus cross-talk and infection microbiology and enables non-invasive assessment of disease manifestation.

Real-time metabolic monitoring under exhaustive exercise and evaluation of ventilatory threshold by breathomics: Independent validation of evidence and advances.

Breath analysis was coupled with ergo-spirometry for non-invasive profiling of physio-metabolic status under exhaustive exercise. Real-time mass-spectrometry based continuous analysis of exhaled metabolites along with breath-resolved spirometry and heart rate monitoring were executed while 14 healthy adults performed ergometric ramp exercise protocol until exhaustion. Arterial blood lactate level was analyzed at defined time points. Respiratory-cardiac parameters and exhalation of several blood-borne volatiles changed continuously with the course of exercise and increasing workloads. Exhaled volatiles mirrored ventilatory and/or hemodynamic effects and depended on the origin and/or physicochemical properties of the substances. At the maximum workload, endogenous isoprene, methanethiol, dimethylsulfide, acetaldehyde, butanal, butyric acid and acetone concentrations decreased significantly by 74, 25, 35, 46, 21, 2 and 2%, respectively. Observed trends in exogenous cyclohexadiene and acetonitrile mimicked isoprene profile due to their similar solubility and volatility. Assignment of anaerobic threshold was possible via breath acetone. Breathomics enabled instant profiling of physio-metabolic effects and anaerobic thresholds during exercise. Profiles of exhaled volatiles indicated effects from muscular vasoconstriction, compartmental distribution of perfusion, extra-alveolar gas-exchange and energy homeostasis. Sulfur containing compounds and butyric acid turned out to be interesting for investigations of combined diet and exercise programs. Reproducible metabolic breath patterns have enhanced scopes of breathomics in sports science/medicine.

Physiological and metabolic effects of healthy female aging on exhaled breath biomarkers.

Healthy aging driven physio-metabolic events in females hold the key to complex in vivo mechanistic links and systemic cross talks. Effects from basic changes at genome, proteome, metabolome, and lipidome levels are often reflected at the upstream phenome (e.g., breath volatome) cascades. Here, we have analyzed exhaled volatile metabolites (measured via real time mass spectrometry based breathomics) data from 204 healthy females, aged between 07 and 80 years. Age related substance-specific differences were observed in breath biomarkers. Exhalation of blood-borne endogenous organosulfur, short-chain fatty acids, alcohols, aldehydes, alkene, ketones and exogenous nitriles, terpenes, and aromatics have denominated interplay between endocrine differences, energy homeostasis, systemic microbial diversity, oxidative stress, and lifestyle. Overall marker expressions were suppressed under daily oral contraception. Young homosexual/lesbian adults turned out as breathomic outliers. Previously proposed disease-specific breath biomarkers should be reevaluated upon aging effects. Breathomics offers a noninvasive window toward system-wide understanding and personalized monitoring of aging i.e., translatable to gerontology.

Effects of COVID-19 protective face masks and wearing durations on respiratory haemodynamic physiology and exhaled breath constituents.

BACKGROUND: While assumed to protect against coronavirus transmission, face masks may have effects on respiratory-haemodynamic parameters. Within this pilot study, we investigated immediate and progressive effects of FFP2 and surgical masks on exhaled breath constituents and physiological attributes in 30 adults at rest. METHODS: We continuously monitored exhaled breath profiles within mask space in older (age 60-80 years) and young to middle-aged (age 20-59 years) adults over the period of 15 and 30 min by high-resolution real-time mass-spectrometry. Peripheral oxygen saturation (S pO2 ) and respiratory and haemodynamic parameters were measured (noninvasively) simultaneously. RESULTS: Profound, consistent and significant (p≤0.001) changes in S pO2 (≥60_FFP2-15 min: 5.8±1.3%↓, ≥60_surgical-15 min: 3.6±0.9%↓, <60_FFP2-30 min: 1.9±1.0%↓, <60_surgical-30 min: 0.9±0.6%↓) and end-tidal carbon dioxide tension (P ETCO2 ) (≥60_FFP2-15 min: 19.1±8.0%↑, ≥60_surgical-15 min: 11.6±7.6%↑, <60_FFP2- 30 min: 12.1±4.5%↑, <60_surgical- 30 min: 9.3±4.1%↑) indicate ascending deoxygenation and hypercarbia. Secondary changes (p≤0.005) to haemodynamic parameters (e.g. mean arterial pressure (MAP) ≥60_FFP2-15 min: 9.8±10.4%↑) were found. Exhalation of bloodborne volatile metabolites, e.g. aldehydes, hemiterpene, organosulfur, short-chain fatty acids, alcohols, ketone, aromatics, nitrile and monoterpene mirrored behaviour of cardiac output, MAP, S pO2 , respiratory rate and P ETCO2 . Exhaled humidity (e.g. ≥60_FFP2-15 min: 7.1±5.8%↑) and exhaled oxygen (e.g. ≥60_FFP2-15 min: 6.1±10.0%↓) changed significantly (p≤0.005) over time. CONCLUSIONS: Breathomics allows unique physiometabolic insights into immediate and transient effects of face mask wearing. Physiological parameters and breath profiles of endogenous and/or exogenous volatile metabolites indicated putative cross-talk between transient hypoxaemia, oxidative stress, hypercarbia, vasoconstriction, altered systemic microbial activity, energy homeostasis, compartmental storage and washout. FFP2 masks had a more pronounced effect than surgical masks. Older adults were more vulnerable to FFP2 mask-induced hypercarbia, arterial oxygen decline, blood pressure fluctuations and concomitant physiological and metabolic effects.

Deficiency and absence of endogenous isoprene in adults, disqualified its putative origin.

BACKGROUND: Isoprene (C5H8) is a clinically important breath metabolite. Although, hundreds of studies have reported differential expressions in isoprene exhalation as breath biomarker for diverse diseases, the substance couldn't enter to clinical practice as diagnostic marker. Moreover, many experimental/basic observations upon breath isoprene remained unrelated to the corresponding pathophysiological effects on its putative metabolic origin (i.e. mevalonate pathway). Here, we investigated the fundamental reason that hindered the rational interpretation and translation of this marker from basic to clinical science. METHODS: Via high-resolution mass-spectrometry based breathomics in 1026 human subjects, we discovered adults with significant deficiency (order of magnitude lower than the normal) and complete absence of breath isoprene. We prospectively applied real-time breathomics, quantitative gene expression analysis of the mevalonate pathway enzymes, lipid-profiling and hemodynamic monitoring on those isoprene deficient subjects and controls. Additionally, the subject with absence of isoprene was followed up throughout different phases of her womanhood. RESULTS: In contrast to convention, we witnessed that adults can live healthy without exhaling isoprene or with significant deficiency. This rare phenotype represents a recessive inheritance. Despite physio-metabolic changes during menstrual cycle (that is known to profoundly affect isoprene exhalation) and profoundly increased plasma cholesterol during pregnancy and after childbirth, isoprene remained absent. All genes of mevalonate pathway enzymes were normally expressed in all participants, without any down-regulation or compensatory up-regulation. CONCLUSIONS: Absence/deficiency of isoprene despite normal lipid profiles and no mevalonate pathway malfunction disqualifies the long-believed metabolic origin of isoprene from cholesterol biosynthesis. Thus, clinical translation of breath isoprene expressions should not be generally attributed to corresponding pathophysiological effects onto mevalonate/cholesterol pathway. Our finding has refined and optimized the clinical interpretation of isoprene as biomarker in volatile metabolomics and breathomics. Future studies will address the correct metabolic origin of isoprene to imply this important marker to routine practice.

Exhaled breath compositions under varying respiratory rhythms reflects ventilatory variations: translating breathomics towards respiratory medicine.

Control of breathing is automatic and its regulation is keen to autonomic functions. Therefore, involuntary and voluntary nervous regulation of breathing affects ventilatory variations, which has profound potential to address expanding challenges in contemporary pulmonology. Nonetheless, the fundamental attributes of the aforementioned phenomena are rarely understood and/or investigated. Implementation of unconventional approach like breathomics may leads to a better comprehension of those complexities in respiratory medicine. We applied breath-resolved spirometry and capnometry, non-invasive hemodynamic monitoring along with continuous trace analysis of exhaled VOCs (volatile organic compounds) by means of real-time mass-spectrometry in 25 young and healthy adult humans to investigate any possible mirroring of instant ventilatory variations by exhaled breath composition, under varying respiratory rhythms. Hemodynamics remained unaffected. Immediate changes in measured breath compositions and corresponding variations occurred when respiratory rhythms were switched between spontaneous (involuntary/unsynchronised) and/or paced (voluntary/synchronised) breathing. Such changes in most abundant, endogenous and bloodborne VOCs were closely related to the minute ventilation and end-tidal CO2 exhalation. Unprecedentedly, while preceded by a paced rhythm, spontaneous rhythms in both independent setups became reproducible with significantly (P-value ≤ 0.005) low intra- and inter-individual variation in measured parameters. We modelled breath-resolved ventilatory variations via alveolar isoprene exhalation, which were independently validated with unequivocal precision. Reproducibility i.e. attained via our method would be reliable for human breath sampling, concerning biomarker research. Thus, we may realize the actual metabolic and pathophysiological expressions beyond the everlasting in vivo physiological noise. Consequently, less pronounced changes are often misinterpreted as disease biomarker in cross-sectional studies. We have also provided novel information beyond conventional spirometry and capnometry. Upon clinical translations, our findings will have immense impact on pulmonology and breathomics as they have revealed a reproducible pattern of ventilatory variations and respiratory homeostasis in endogenous VOC exhalations.

Changes of Exhaled Volatile Organic Compounds in Postoperative Patients Undergoing Analgesic Treatment: A Prospective Observational Study.

Assessment and treatment of postoperative pain can be challenging as objective examination techniques to detect and quantify pain are lacking. We aimed to investigate changes of exhaled volatile organic compounds (VOCs) in patients with postoperative pain before and after treatment with opioid analgesics. In an observational study in 20 postoperative patients, we monitored for postoperative pain, hemodynamic parameters, and catecholamines before and during treatment. VOCs in the patients were determined by direct real-time proton transfer reaction time-of-flight mass spectrometry prior (0 min) and after piritramide application (15 min as well as 30 min). Cardiovascular variables changed and norepinephrine levels decreased during treatment. The VOCs acetonitrile (<0.001), acetaldehyde (p = 0.002), benzopyran (p = 0.004), benzene (p < 0.001), hexenal (p = < 0.001), 1-butanethiol (p = 0.004), methanethiol (p < 0.001), ethanol (p = 0.003), and propanol (p = < 0.001) changed significantly over time. Patients with Numeric Rating Scale (NRS) < 4 showed a significantly lower concentration of hexenal compared to patients with NRS > 4 at the time points 15 min (45.0 vs. 385.3 ncps, p = 0.047) and 30 min (38.3 vs. 334.6 ncps, p = 0.039). Breath analysis can provide additional information for noninvasive monitoring for analgesic treatment in postoperative patients.

Unravelling the Potential of Salivary Volatile Metabolites in Oral Diseases. A Review.

Fostered by the advances in the instrumental and analytical fields, in recent years the analysis of volatile organic compounds (VOCs) has emerged as a new frontier in medical diagnostics. VOCs analysis is a non-invasive, rapid and inexpensive strategy with promising potential in clinical diagnostic procedures. Since cellular metabolism is altered by diseases, the resulting metabolic effects on VOCs may serve as biomarkers for any given pathophysiologic condition. Human VOCs are released from biomatrices such as saliva, urine, skin emanations and exhaled breath and are derived from many metabolic pathways. In this review, the potential of VOCs present in saliva will be explored as a monitoring tool for several oral diseases, including gingivitis and periodontal disease, dental caries, and oral cancer. Moreover, the analytical state-of-the-art for salivary volatomics, e.g., the most common extraction techniques along with the current challenges and future perspectives will be addressed unequivocally.

Non-Invasive Assessment of Metabolic Adaptation in Paediatric Patients Suffering from Type 1 Diabetes Mellitus.

An analysis of exhaled volatile organic compounds (VOC) may deliver systemic information quicker than available invasive techniques. Metabolic aberrations in pediatric type 1 diabetes (T1DM) are of high clinical importance and could be addressed via breathomics. Real-time breath analysis was combined with continuous glucose monitoring (CGM) and blood tests in children suffering from T1DM and age-matched healthy controls in a highly standardized setting. CGM and breath-resolved VOC analysis were performed every 5 minutes for 9 hours and blood was sampled at pre-defined time points. Per participant (n = 44) food intake and physical activity were identical and a total of 22 blood samples and 93 minutes of breath samples were investigated. The inter-individual variability of glucose, insulin, glucagon, leptin, and soluble leptin receptor relative to food intake differed distinctly between patients and controls. In T1DM patients, the exhaled amounts of acetone, 2-propanol, and pentanal correlated to glucose concentrations. Of note, the strength of these correlations strongly depended on the interval between food intake and breath sampling. Our data suggests that metabolic adaptation through postprandial hyperglycemia and related oxidative stress is immediately reflected in exhaled breath VOC concentrations. Clinical translations of our findings may enable point-of-care applicability of online breath analysis towards personalized medicine.

Natural menstrual rhythm and oral contraception diversely affect exhaled breath compositions.

Natural menstrual cycle and/or oral contraception diversely affect women metabolites. Longitudinal metabolic profiling under constant experimental conditions is thereby realistic to understand such effects. Thus, we investigated volatile organic compounds (VOCs) exhalation throughout menstrual cycles in 24 young and healthy women with- and without oral contraception. Exhaled VOCs were identified and quantified in trace concentrations via high-resolution real-time mass-spectrometry, starting from a menstruation and then repeated follow-up with six intervals including the next bleeding. Repeated measurements within biologically comparable groups were employed under optimized measurement setup. We observed pronounced and substance specific changes in exhaled VOC concentrations throughout all cycles with low intra-individual variations. Certain blood-borne volatiles changed significantly during follicular and luteal phases. Most prominent changes in endogenous VOCs were observed at the ovulation phase with respect to initial menstruation. Here, the absolute median abundances of alveolar ammonia, acetone, isoprene and dimethyl sulphide changed significantly (P-value ≤ 0.005) by 18.22↓, 13.41↓, 18.02↑ and 9.40↓%, respectively. These VOCs behaved in contrast under the presence of combined oral contraception; e.g. isoprene decreased significantly by 30.25↓%. All changes returned to initial range once the second bleeding phase was repeated. Changes in exogenous benzene, isopropanol, limonene etc. and smoking related furan, acetonitrile and orally originated hydrogen sulphide were rather nonspecific and mainly exposure dependent. Our observations could apprehend a number of known/pre-investigated metabolic effects induced by monthly endocrine regulations. Potential in vivo origins (e.g. metabolic processes) of VOCs are crucial to realize such effects. Despite ubiquitous confounders, we demonstrated the true strength of volatolomics for metabolic monitoring of menstrual cycle and contraceptives. These outcomes may warrant further studies in this direction to enhance our fundamental and clinical understanding on menstrual metabolomics and endocrinology. Counter-effects of contraception can be deployed for future noninvasive assessment of birth control pills. Our findings could be translated toward metabolomics of pregnancy, menopause and post-menopausal complications via breath analysis.

Smell of cells: Volatile profiling of stem- and non-stem cell proliferation.

Bacterial and cell cultures are known to emit a large number of volatile organic compounds (VOCs). Conventional biochemical methods are often destructive, time-consuming and expensive. In contrast, VOC analysis of headspace over cultures may offer a non-destructive alternative for the monitoring of cell proliferation and metabolism. VOC profiles from cultures of murine pluripotent stem cells and fibroblasts were assessed every 24 h for 3 days. Pure cell media were measured as parallel controls. VOC analysis was highly standardized with respect to time of measurement and phases of cell growth. Cultures were grown in custom-made inert boxes. In order to determine the effects of fresh media supply on VOC emissions, both cell types were cultured with and without daily media exchange. VOCs from headspace were preconcentrated by means of needle trap micro-extraction and analysed by gas chromatography-mass spectrometry (GC-MS). Murine pluripotent stem cells emitted increasing concentrations of thiirane and methyl-methoxy-hydroxy-methyl-amine (MMHA). Substance concentration correlated with cell numbers. Murine fibroblasts did not emit thiirane or MMHA. Concentrations of aldehydes, especially benzaldehyde, were lower in both cell cultures than in pure media samples. Daily media exchange resulted in higher cell numbers, but had no major effects on VOC concentrations emitted from the cells. Investigation and monitoring of volatile substances such as thiirane and MMHA may enhance the understanding of stem cell properties and lead to a destruction-free characterization of pluripotent stem cells.

Applied upper-airway resistance instantly affects breath components: a unique insight into pulmonary medicine.

Respiratory parameters such as flow or rate have complex effects on the exhalation of volatile substances and can hamper clinical interpretation of breath biomarkers. We have investigated the effects of progressively applied upper-airway resistances on the exhalation of volatile organic compounds (VOCs) in healthy humans. We performed real-time mass-spectrometric determination of breath volatiles in 50 subjects with parallel, non-invasive hemodynamic monitoring, breath-resolved spirometry and capnometry during controlled tidal breathing (12 breaths/min). Airway resistance was increased by changing the mouthpiece diameters from 2.5 cm to 1.0 cm and to 0.5 cm. At the smallest diameter, oxygen uptake increased (35%↑). Cardiac output decreased (6%↓) but end-tidal PCO2 (8%↑) and exhalation of blood-borne isoprene (19%↑) increased. Carbon dioxide production remained constant. Furan, hydrogen sulphide mirrored isoprene. Despite lowered minute ventilation (4%↓) acetone concentrations decreased (3%↓). Exogenous acetonitrile, propionic acid, isopropanol, limonene mimicked acetone. VOC concentration changes could be modelled through substance volatility. Airway resistance-induced changes in hemodynamics, and ventilation can affect VOC exhalation and thereby interfere with breath biomarker interpretation. The effects of collateral ventilation, intra-alveolar pressure gradients and respiratory mechanics had to be considered to explain the exhalation kinetics of CO2 and VOCs. Conventional breath sampling via smaller mouthpiece diameters (≤1.0 cm, e.g. via straw in Tedlar bags or canisters, etc) will immediately affect VOC exhalation and thereby mislead the analysis of the obtained results. Endogenous isoprene may probe respiratory muscle workload under obstructive conditions. Breath-gas analysis might enhance our understanding of diagnosis and management of obstructive lung diseases in the future.

Oral or nasal breathing? Real-time effects of switching sampling route onto exhaled VOC concentrations.

There is a need for standardisation in sampling and analysis of breath volatile organic compounds (VOCs) in order to minimise ubiquitous confounding effects. Physiological factors may mask concentration changes induced by pathophysiological effects. In humans, unconscious switching of oral and nasal breathing can occur during breath sampling, which may affect VOC patterns. Here, we investigated exhaled VOC concentrations in real-time while switching breathing routes. Breath from 15 healthy volunteers was analysed continuously by proton transfer reaction time-of-flight mass spectrometry during paced breathing (12 breaths min-1). Every two minutes breathing routes were switched (Setup-1: Oral â†’ Nasal â†’ Oral â†’ Nasal; Setup-2: OralinNasalout â†’ NasalinOralout â†’ OralinNasalout â†’ NasalinOralout). VOCs in inspiratory and alveolar air and respiratory and hemodynamic parameters were monitored quantitatively in parallel. Changing of the breathing routes and patterns immediately affected exhaled VOC concentrations. These changes were reproducible in both setups. In setup-1 cardiac output and acetone concentrations remained constant, while partial pressure of end-tidal CO2 (pET-CO2), isoprene and furan concentrations inversely mirrored tidal-volume and minute-ventilation. H2S (hydrogen-sulphide), C4H8S (allyl-methyl-sulphide), C3H8O (isopropanol) and C3H6O2 increased during oral exhalation. C4H10S increased during nasal exhalations. CH2O2 steadily decreased during the whole measurement. In setup-2 pET-CO2, C2H6S (dimethyl-sulphide), isopropanol, limonene and benzene concentrations decreased whereas, minute-ventilation, H2S and acetonitrile increased. Isoprene and furan remained unchanged. Breathing route and patterns induced VOC concentration changes depended on respiratory parameters, oral and nasal cavity exposure and physico-chemical characters of the compounds. Before using breath VOC concentrations as biomarkers it is essential that the breathing modality is defined and strictly monitored during sampling.

Drug detection in breath: non-invasive assessment of illicit or pharmaceutical drugs.

Breath analysis not only holds great potential for the development of new non-invasive diagnostic methods, but also for the identification and follow up of drug levels in breath. This is of interest for both, forensic and medical science. On the one hand, the detection of drugs of abuse in exhaled breath-similar to the well-known breath alcohol tests-would be highly desirable as an alternative to blood or urine analysis in situations such as police controls for drugged driving. The non-invasive detection of drugs and their metabolites is thus of great interest in forensic science, especially since marijuana is becoming legalized in certain parts of the US and the EU. The detection and monitoring of medical drugs in exhaled breath without the need of drawing blood samples on the other hand, is of high relevance in the clinical environment. This could facilitate a more precise medication and enable therapy control without any burden to the patient. Furthermore, it could be a step towards personalized medicine. This review gives an overview of the current state of drug detection in breath, including both volatile and non-volatile substances. The review is divided into two sections. The first section deals with qualitative detection of drugs (drugs of abuse), while the second is related to quantitative drug detection (medical drugs). Chances and limitations are discussed for both aspects. The detection of the intravenous anesthetic propofol is presented as a detailed example that demonstrates the potential, requirements, pitfalls and limitations of therapeutic drug monitoring by means of breath analysis.