Exogenous Volatile Organic Compounds Research Articles

Exogenous Volatile Organic Compound (EVOC®) Breath Testing Maximizes Classification Performance for Subjects with Cirrhosis and Reveals Signs of Portal Hypertension.

Background: Cirrhosis detection in primary care relies on low-performing biomarkers. Consequently, up to 75% of subjects with cirrhosis receive their first diagnosis with decompensation when causal treatments are less effective at preserving liver function. We investigated an unprecedented approach to cirrhosis detection based on dynamic breath testing. Methods: We enrolled 29 subjects with cirrhosis (Child-Pugh A and B), and 29 controls. All subjects fasted overnight. Breath samples were taken using Breath Biopsy® before and at different time points after the administration of 100 mg limonene. Absolute limonene breath levels were measured using gas chromatography-mass spectrometry. Results: All subjects showed a >100-fold limonene spike in breath after administration compared to baseline. Limonene breath kinetics showed first-order decay in >90% of the participants, with higher bioavailability in the cirrhosis group. At the Youden index, baseline limonene levels showed classification performance with an area under the roc curve (AUROC) of 0.83 ± 0.012, sensitivity of 0.66 ± 0.09, and specificity of 0.83 ± 0.07. The best performing timepoint post-administration was 60 min, with an AUROC of 0.91, sensitivity of 0.83 ± 0.07, and specificity of 0.9 ± 0.06. In the cirrhosis group, limonene bioavailability showed a correlation with MELD and fibrosis indicators, and was associated with signs of portal hypertension. Conclusions: Dynamic limonene breath testing enhances diagnostic performance for cirrhosis compared to static testing. The correlation with disease severity suggests potential for monitoring therapeutic interventions. Given the non-invasive nature of breath collection, a dynamic limonene breath test could be implemented in primary care.

Abstract 3319: Breath-based detection of lung cancer using exogenous volatile organic compound targeting β-glucuronidase in the tumor microenvironment

Abstract Introduction: Numerous studies have sought potential breath biomarkers for lung cancer and many have promising results but, to date, no validated biomarkers with clear connections to cancer metabolism have been discovered. We aim to show the feasibility of an Exogenous Volatile Organic Compounds (EVOC®) Probe targeting tumor-associated extracellular β-glucuronidase, a glycosidase enzyme that usually resides intracellularly within lysosomes in healthy tissues. Material and Methods: To further assess the suitability of β-glucuronidase as a target for a non-invasive breath test, the enzyme activity was evaluated by enzymatic assay in lung cancer PDX specimens and healthy tissues. In addition, β-glucuronidase was assessed by immunostaining a large panel of primary lung cancers, lymph node metastasis and normal lung tissue, including from smokers and those with chronic obstructive pulmonary disease (COPD). We developed a hydrophilic non-cell permeable substrate probe, D5-ethyl-βD-glucuronide (D5-EtGlu), that upon hydrolysis by the target enzyme releases D5-ethanol, a unique volatile reporter molecule. This provides a readout of tumour-associated enzyme activity using breath analysis. Administering D5-EtGlu to mice resulted in tumour-specific release of D5-Ethanol enabling discrimination between healthy and tumour-bearing animals. D5-EtGlu has been administered intravenously to healthy and lung cancer patients followed by breath analysis. A phase 1a clinical trial administered D5-EtGlu to 21 healthy individuals in a single ascending dose study to establish safety and background D5-ethanol levels in healthy individuals. The phase 1b trial is a proof of mechanism study in humans, in which D5-ethanol breath levels in lung cancer patients will be compared to cancer-free individuals receiving the same D5-EtGlu dose. Results and Discussions: Using ex-vivo specimens, higher β-glucuronidase activity was detected in washouts of PDX specimens compared with healthy tissue samples from mice. Higher β-glucuronidase intracellular and extracellular expression was observed in lung cancers compared with lung normal tissue, with low to moderate expression intracellularly in bronchial tissue and in emphysema and COPD. These results reinforce the presence of β-glucuronidase in the tumour microenvironment and its suitability as a target for a breath-probe. A phase 1a resulted in no adverse events and low/no D5-ethanol signal verifying the inaccessibility of D5-EtGlu to intracellular β-glucuronidase. In the phase 1b study, using the highest probe dose, we have detected a D5-ethanol signal in the majority of the cancer patients. Conclusion: β-glucuronidase can be targeted by the non-cell permeable substrate probe D5-ethyl-βD-glucuronide (D5-EtGlu) with potential to be used as a non-invasive breath test for lung cancer diagnosis including a role in screening. Citation Format: Christiaan Labuschagne, Rob Smith, Neelam Kumar, Max Allsworth, Billy Boyle, Sam Janes, Philip Crosbie, Robert Rintoul. Breath-based detection of lung cancer using exogenous volatile organic compound targeting β-glucuronidase in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3319.

Volatile organic compound emissions from the human body: Decoupling and comparison between whole-body skin and breath emissions

The human body can affect indoor air by emitting volatile organic compounds (VOCs) through breath and skin. However, studies on the quantitative characteristics of skin VOC emissions are scarce compared to those on the characteristics of breath emissions. Moreover, the species and emission rates of VOCs emitted from the breath and whole-body skin of a single person have rarely been reported. In this study, VOCs emitted from the whole-body skin and breath of 14 young adults under controlled conditions were decoupled and measured. The number of VOC species emitted from each study participant's whole-body skin and breath ranged 38–69 and 28–55, respectively, with total emission rates of all detected VOCs of 164–518 and 286–1030 μg/h, respectively. The VOCs in the high-detection rate intervals also had higher emission rates, increasing their importance in human body emissions. There were 9–26 common VOCs and 25–47 different VOCs between the skin and breath emissions. Acetone was detected in both the skin and breath emissions of all study participants, with the highest emission rate in each participant's whole-body emissions. Ketones, siloxanes, aldehydes, and alcohols contributed the most to the whole-body skin emission rates of each study participant, whereas ketones and alkenes alone accounted for most of the breath emission rates. Analysis of the generation mechanisms found that endogenous and exogenous VOCs were dominant in breath and skin emissions, respectively. These results can enrich the qualitative and quantitative characteristics of breath and skin emissions and improve understanding of emission mechanisms.

Identifying robust and reliable volatile organic compounds in human sebum for biomarker discovery

Sebum from sebaceous glands is a rich source of volatile organic compounds (VOCs) that can readily be sampled non-invasively from the surface of skin. The VOC profiles of sebum can then be used to obtain information regarding different medical conditions including diabetes and Parkinson's Disease. However, the effects of sampling approaches and environmental factors on sebum VOC profiles are not established and the confident attribution of VOCs to disease states needs to be free of extraneous influences such as sampling materials and preparatory conditions. Here, we investigated a more standardised skin swab sampling approach for profiling sebum VOCs from healthy human subjects using thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Using a standard GC-MS method for the chemical analysis of sebum swabs, a surprisingly high number of VOCs originate from ‘blank’ medical swab material alone (up to 74 VOCs) and from the ambient environment (up to 29 VOCs) based on control experiments. We found that heat-treatment of medical swabs prior to GC-MS reduced the number of VOCs detected from ‘blank’ swabs and improved the reproducibility of VOC profiling, however significant VOC absorption can still occur from environmental exposure to ambient air. VOCs identified in ‘blank’ swabs consisted predominantly of hydrocarbons, esters, and silicon-based compounds and depended strongly on the material used (cotton and polyester-rayon). Environmental VOCs found to absorb to swabs from the ambient air during sampling included 1-butylheptyl-benzene and hexadecanoic acid methyl ester as well as exogenous VOCs such as isopropyl palmitate and isopropyl myristate. In contrast, sebum VOCs consisted primarily of esters, alcohols, ketones, and aldehydes. 23 and 18 VOCs were identified in sebum collected using polyester-rayon and cotton-based medical swabs, respectively, with 14 VOCs common to both swabs. The effect of subject bathing prior to sebum sampling had minimal impact on the VOC profiles. However, individual differences owing to external factors such as skin type, diet, and exercise will likely influence sebum production. This study highlights the importance of using rigorous controls in sebum sampling, and recommendations are provided for future research involving sebum VOC analysis. For example, the use of sebum sample replicates across multiple days, and the use of control swabs during sample collection is required to confirm the origin and reliability of sebum VOCs. It is anticipated that these recommendations in conjunction with a library of well-established VOCs from medical swabs will further strengthen biomarker identification resulting from sebum VOC analysis.

New tool for Gram-negative and Gram-positive bacterial strains differentiation through GCMS direct identification of exogenous VOC metabolites in headspace vials

In this paper, a chromatographic method for the determination of volatile organic compounds (VOCs) as a tool for Gram-positive and Gram-negative bacterial strains identification was described. Direct Headspace gas chromatography coupled with mass spectrometry (Headspace-GC-MS) has been used for the qualitative determination of VOCs emitted by certain bacterial strains (Salmonella Typhimurium, Escherichia coli, Serratia rubidae, Staphyloccocus aureus, Enterococcus casseliflavus, and Enterococcus faecalis). In the first stage of the study, pathogenic bacteria were cultured in broth-specific liquid culture medium directly (lauryl sulfate broth) into headspace vials. Subsequently, the VOC emissions were analyzed by airborne screening in the headspace vials. Thus, the objective was to analyze all the resulting volatile organic compounds in order to select only those compounds, which can be exclusively associated with specific pathogenic bacteria. Qualitative analysis by Headspace-GC-MS has proven to be a non-invasive, accurate, and fast method for identifying certain bacterial strains, based on VOCs emission. A considerable number of volatile organic compounds have been determined in the headspace air, a significant difference being observed between the VOCs emitted by bacterial cultures compared to the culture medium but also between the types of bacterial cultures themselves. The study presents preliminary results, which prove that the identification of the studied pathogenic bacteria is possible, based on the determination of certain types of VOCs in the headspace air of these cultures. This method can be used successfully for the rapid identification of bacterial culture types compared to classical methods.

Breath biopsy early detection of lung cancer using an EVOC probe targeting tumor-specific extracellular β-glucuronidase.

2569 Background: Lung cancer is a leading cause of mortality with 5-year survival less than 20%, largely a result of many cases being diagnosed late. Early detection can increase cancer survival up to 13-fold underscoring the need for effective screening. Targeted Low dose computed tomography (LDCT) has been shown to be effective but its impact to date has been limited due to slow adoption and variable uptake in high-risk populations. Breath analysis represents a non-invasive screening approach either alone or alongside LDCT. Numerous studies have investigated potential endogenous breath biomarkers of lung cancer. Many have produced promising results but to date, no validated biomarkers with clear connections to cancer metabolism have been revealed. We have explored an alternative, probe-based approach based around Exogenous Volatile Organic Compound Probes (EVOC Probes). The probes target tumour associated extracellular b-glucuronidase, a glycosidase enzyme that normally resides within lysosomes. Methods: We use a hydrophilic non cell permeable substrate probe D5-ethyl-βD-glucuronide (D5-EtGlu) that upon hydrolysis by the target enzyme releases D5-ethanol, a unique volatile reporter molecule detectable on breath. This provides a readout of tumour associated enzyme activity using breath analysis. Results: Administering D5-EtGlu to mice resulted in tumour specific release of D5-ethanol, enabling discrimination between healthy and tumour bearing animals. Increased expression of b-glucuronidase in lung cancer tissue and the tumour microenvironment was confirmed with immunohistochemistry (IHC) in clinical samples. A phase 1a clinical trial administered D5-EtGlu to healthy individuals in a single ascending dose study to establish safety and background D5-ethanol levels in healthy individuals. This resulted in no adverse events and low/no D5-ethanol signal verifying the inaccessibility of D5-EtGlu to intracellular b-glucuronidase. The next stage, currently ongoing, is a proof of mechanism in humans. D5-EtGlu is administered intravenously to confirmed lung cancer patients followed by breath analysis. D5-ethanol breath levels will be compared to cancer free individuals receiving the same dose of D5-EtGlu. Conclusions: Non-invasive breath testing has great potential to contribute to diagnosis for lung cancer including a potential role in screening. Our current work is evaluating the use of an administered probe to stimulate tumour-specific enzyme activity and produce a marker detectable on breath. Continued success could result in a sensitive and highly specific method for lung cancer early detection.

Breath-Taking Perspectives and Preliminary Data toward Early Detection of Chronic Liver Diseases.

The gold standard method for chronic liver diseases diagnosis and staging remains liver biopsy, despite the spread of less invasive surrogate modalities based on imaging and blood biomarkers. Still, more than 50% of chronic liver disease cases are detected at later stages when patients exhibit episodes of liver decompensation. Breath analysis represents an attractive means for the development of non-invasive tests for several pathologies, including chronic liver diseases. In this perspective review, we summarize the main findings of studies that compared the breath of patients with chronic liver diseases against that of control subjects and found candidate biomarkers for a potential breath test. Interestingly, identified compounds with best classification performance are of exogenous origin and used as flavoring agents in food. Therefore, random dietary exposure of the general population to these compounds prevents the establishment of threshold levels for the identification of disease subjects. To overcome this limitation, we propose the exogenous volatile organic compounds (EVOCs) probe approach, where one or multiple of these flavoring agent(s) are administered at a standard dose and liver dysfunction associated with chronic liver diseases is evaluated as a washout of ingested compound(s). We report preliminary results in healthy subjects in support of the potential of the EVOC Probe approach.

Blood, urine and semen Volatile Organic Compound (VOC) pattern analysis for assessing health environmental impact in highly polluted areas in Italy

Volatile Organic Compound (VOC) analysis is usually applied in pollution assessment by checking for toxic or harmful volatile compounds in air, water and soil samples. In this study, exogenous VOCs and their derivatives, metabolized by cells, were valued into specific body fluids. In particular, the VOC profiles of blood, urine and human semen samples collected from young men living in two high pollution areas in Italy, i.e. Land of Fires and Valley of Sacco River, were fingerprinted. The analysis is based on Headspace Solid Phase MicroExtraction (HS-SPME) followed by Gas Chromatography-Mass Spectrometric detection (GC-MS). The volatile composition of the three body fluids showed that some VOCs are in common between blood, urine and human semen samples, whereas others are present only in a body fluid. Some compounds, as well as also some chemical classes show a higher affinity for a specific body fluid. Statistical analysis allowed to discriminate the two contaminated areas and identify those compounds which significantly contribute to the two areas classification. Some of these compounds are toxic and found prevalently in Valley of Sacco River samples, correspondingly to sperm analysis results for young men living in this zona worse than those living in Land of Fires.

Volatile organic compounds in a headspace sampling system and asthmatics sputum samples

The headspace of a biological sample contains exogenous volatile organic compounds (VOCs) present within the sampling environment which represent the background signal. This study aimed to characterise the background signal generated from a headspace sampling system in a clinical site, to evaluate intra- and inter-day variation of background VOC and to understand the impact of a sample itself upon commonly reported background VOC using sputum headspace samples from severe asthmatics. The headspace, in absence of a biological sample, was collected hourly from 11am to 3pm within a day (time of clinical samples acquisition), and from Monday to Friday in a week, and analysed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Chemometric analysis identified 1120 features, 37 of which were present in at least the 80% of all the samples. The analyses of intra- and inter-day background variations were performed on 13 of the most abundant features, ubiquitously present in headspace samples. The concentration ratios relative to background were reported for the selected abundant VOC in 36 asthmatic sputum samples, acquired from 36 stable severe asthma patients recruited at Glenfield Hospital, Leicester, UK. The results identified no significant intra- or inter-day variations in compounds levels and no systematic bias of z-scores, with the exclusion of benzothiazole, whose abundance increased linearly between 11am and 3pm with a maximal intra-day fold change of 2.13. Many of the identified background features are reported in literature as components of headspace of biological samples and are considered potential biomarkers for several diseases. The selected background features were identified in headspace of all severe asthma sputum samples, albeit with varying levels of enrichment relative to background. Our observations support the need to consider the background signal derived from the headspace sampling system when developing and validating headspace biomarker signatures using clinical samples.

Detection of Microbial Nitroreductase Activity by Monitoring Exogenous Volatile Organic Compound Production Using HS-SPME-GC-MS

Development of a rapid approach for universal microbial detection is required in the healthcare, food and environmental sectors to aid with medical intervention, food safety and environmental protection. This research investigates the use of enzymatic hydrolysis of a substrate by a microorganism to generate a volatile organic compound (VOC). One such enzyme activity that can be used in this context is nitroreductase as such activity is prevalent across a range of microorganisms. A study was developed to evaluate a panel of 51 microorganisms of clinical interest for their nitroreductase activity. Two enzyme substrates, nitrobenzene and 1-fluoro-2-nitrobenzene, were evaluated for this purpose with evolution, after incubation, of the VOCs aniline and 2-fluoroaniline, respectively. Detection of the VOCs was done using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) with obtained limits of quantitation (LOQ) of 0.17 and 0.03 µg/mL for aniline and 2-fluoroaniline, respectively. The results indicated that both enzyme substrates were reduced by the same 84.3% of microorganisms producing the corresponding volatile anilines which were detected using HS-SPME-GC-MS. It was found that nitroreductase activity could be detected after 6–8 h of incubation for the selected pathogenic bacteria investigated. This approach shows promise as a rapid universal microbial detection system.

Breath Biopsy Assessment of Liver Disease Using an Exogenous Volatile Organic Compound-Toward Improved Detection of Liver Impairment.

INTRODUCTION:Liver cirrhosis and its complication — hepatocellular carcinoma (HCC) — have been associated with increased exhaled limonene. It is currently unclear whether this increase is more strongly associated with the presence of HCC or with the severity of liver dysfunction.METHODS:We compared the exhaled breath of 40 controls, 32 cirrhotic patients, and 12 cirrhotic patients with HCC using the Breath Biopsy platform. Breath samples were analyzed by thermal desorption–gas chromatography–mass spectrometry. Limonene levels were compared between the groups and correlated to bilirubin, albumin, prothrombin time international normalized ratio, and alanine aminotransferase.RESULTS:Breath limonene concentration was significantly elevated in subjects with cirrhosis-induced HCC (M: 82.1 ng/L, interquartile range [IQR]: 16.33–199.32 ng/L) and cirrhosis (M: 32.6 ng/L, IQR: 6.55–123.07 ng/L) compared with controls (M: 6.2 ng/L, IQR: 2.62–9.57 ng/L) (P value = 0.0005 and 0.0001, respectively) with no significant difference between 2 diseased groups (P value = 0.37). Levels of exhaled limonene correlated with serum bilirubin (R2 = 0.25, P value = 0.0016, r = 0.51), albumin (R2 = 0.58, P value = 5.3e-8, r = −0.76), and international normalized ratio (R2 = 0.29, P value = 0.0003, r = 0.51), but not with alanine aminotransferase (R2 = 0.01, P value = 0.36, r = 0.19).DISCUSSION:Exhaled limonene levels are primarily affected by the presence of cirrhosis through reduced liver functional capacity, as indicated by limonene correlation with blood metrics of impaired hepatic clearance and protein synthesis capacity, without further alterations observed in subjects with HCC. This suggests that exhaled limonene is a potential non-invasive marker of liver metabolic capacity (see Visual abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A388).

Breath-based monitoring of liver metabolism using exogenous volatile organic compounds - towards improved detection

Breath-based monitoring of liver metabolism using exogenous volatile organic compounds - towards improved detection

Sequestration of Exogenous Volatiles by Plant Cuticular Waxes as a Mechanism of Passive Associational Resistance: A Proof of Concept.

Numerous plant-derived volatile organic compounds (VOCs) induce the expression of resistance-related genes and thereby cause an “associational resistance” in neighbouring plants. However, VOCs can also be sequestered by plant cuticular waxes. In case that they maintain their biological activity, such sequestered VOCs could generate a “passive” associational resistance that is independent of any gene expression in the receiver. As a proof of concept, we used major components of the cuticular wax layers of the tree, Parkinsonia praecox, and conidia of Colletotrichum lindemuthianum, a fungal pathogen that has not been reported to infect P. praecox. Wax layers were re-constituted on glass slides and exposed to each of 20 pure VOCs for 1 d and then to ambient air for 1 d or 15 d. Gas chromatography-mass spectrometry (GC-MS) analyses showed that all 20 VOCs were sequestered by the re-constituted wax layers. Exposure to 18 of the VOCs significantly inhibited the germination of C. lindemuthianum conidia on these wax layers after 1 day of exposure to ambient air. Four of the VOCs: 4Z-heptenol, farnesene, limonene, and 2E-decenal, inhibited germination rates to less than 25% of the controls. After 15 d, all VOCs were still detectable, although at strongly reduced concentrations, and no significant inhibition of conidial germination could be detected anymore. Exogenous VOCs can be sequestered by the components of plant cuticular waxes and maintain their biological activity, at least over a certain time span: an effect that could generate a transient “passive associational resistance” to pathogens.

Detection of β-alanyl aminopeptidase as a biomarker for Pseudomonas aeruginosa in the sputum of patients with cystic fibrosis using exogenous volatile organic compound evolution.

A novel, rapid and sensitive analytical method has been developed and applied to 105 sputum samples from patients with cystic fibrosis, including 5 samples from post-lung transplant patients. This new method is specifically targeted to measure β-alanyl aminopeptidase activity which is characteristic of some important Gram-negative pathogens. Of relevance to this study are Pseudomonas aeruginosa and pathogens of the Burkholderia cepacia complex both of which are commonly associated with respiratory infections as well as increased morbidity and mortality in adult cystic fibrosis patients. The analytical method involves the addition of a novel enzyme substrate (i.e. 3-amino-N-(3-fluorophenyl)propanamide) that interacts with β-alanyl aminopeptidase to generate an exogenous volatile organic compound 3-fluoroaniline (LOD 0.02 μg mL−1; LOQ 0.06 μg mL−1). 3-Fluoroaniline was determined at 20 times above its calculated limit of quantification in the sputum samples by HS-SPME-GC-MS and then the results compared with standard culture methods and bacterial identification using MALDI-TOF-MS. Detection of 3-fluoroaniline was possible after only 8 h incubation of the sputum samples with a 95% success rate; this increased to 100% at 24 h which was well within the typical routine timeframe of 48 h. To our knowledge, this is the first demonstration of detection of P. aeruginosa by use of a custom-designed substrate to liberate a detectable and unique VOC. The very high negative predictive value (100% in this study) means such an assay could be appropriate as a screening technique for patients who are not yet colonized by this pathogen.

Volatile organic compounds and lung cancer: a tight link useful for diagnosis

The human body generates thousands of volatile organic compounds (VOCs) that can be excreted thought breath, skin, urine or feces, related to endogenous process and influenced by exogenous VOCs (1). Endogenous VOCs (e.g., isopropanol and benzene) are low molecular weight compounds produced by cell metabolism and represent the endpoint of cellular process (i.e., gene expression, mRNA transcription and protein activity).

Use of exogenous volatile organic compounds to detect Salmonella in milk

Rapid, sensitive, and selective detection and identification of pathogenic bacteria is required in terms of food security. In this study, exogenous VOCs liberated by Salmonella strains have been identified and quantified via head space-solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) in milk samples. The specific enzymes targeted for detection and/or differentiation of Salmonella were C8 esterase, α-galactosidase and pyrrolidonyl peptidase using the following enzyme substrates: 2-chlorophenyl octanoate, phenyl α-d-galactopyranoside and L-pyrrollidonyl fluoroanilide, respectively. Detection of the exogenous VOCs, 2-chlorophenol, phenol and 3-fluoraniline was possible with typical limits of detection of 0.014, 0.045 and 0.005 μg/mL, respectively and correlation coefficients >0.99. The developed methodology was able to detect and identify Salmonella species within a 5 h incubation at 37 °C by the detection of the liberated VOCs. It was found that the milk samples tested were Salmonella free.

Human breath as a source of VOCs in the built environment, Part II: Concentration levels, emission rates and factor analysis

This study aimed to understand the volatile organic compound (VOC) emission levels and possible influencing factors in human exhaled breath via quantitative analysis. Using the experimental method developed previously, breath samples of 117 healthy human subjects were collected and analyzed. Concentration levels of 60 VOCs with the highest detection rates (DRs) were analyzed, and statistical results of emission rates of these VOCs are provided in a table for easy reference. In general, endogenous VOCs were found to have higher concentrations than exogenous VOCs. Furthermore, concentrations and emission rates of these VOCs were statistically compared for gender, different age groups, and smoking exposure types. The results of the univariate analysis showed that these three factors may yield a significant difference in concentration and emission of some, but not all, VOCs. The regression model showed that, for most VOCs, active/passive smoking and being male or of older age were most likely associated with higher emission levels. Age was emphasized to be a significant factor influencing VOC emissions through exhaled air in this study.

Analysis of Listeria using exogenous volatile organic compound metabolites and their detection by static headspace\u2013multi-capillary column\u2013gas chromatography\u2013ion mobility spectrometry (SHS\u2013MCC\u2013GC\u2013IMS)

Listeria monocytogenes is a Gram-positive bacterium and an opportunistic food-borne pathogen which poses significant risk to the immune-compromised and pregnant due to the increased likelihood of acquiring infection and potential transmission of infection to the unborn child. Conventional methods of analysis suffer from either long turn-around times or lack the ability to discriminate between Listeria spp. reliably. This paper investigates an alternative method of detecting Listeria spp. using two novel enzyme substrates that liberate exogenous volatile organic compounds in the presence of α-mannosidase and d-alanyl aminopeptidase. The discriminating capabilities of this approach for identifying L. monocytogenes from other species of Listeria are investigated. The liberated volatile organic compounds (VOCs) are detected using an automated analytical technique based on static headspace–multi-capillary column–gas chromatography–ion mobility spectrometry (SHS–MCC–GC–IMS). The results obtained by SHS–MCC–GC–IMS are compared with those obtained by the more conventional analytical technique of headspace–solid phase microextraction–gas chromatography–mass spectrometry (HS–SPME–GC–MS). The results found that it was possible to differentiate between L. monocytogenes and L. ivanovii, based on their VOC response from α-mannosidase activity.

A chromatographic approach to distinguish Gram-positive from Gram-negative bacteria using exogenous volatile organic compound metabolites

This paper utilized L-alanine aminopeptidase activity as a useful approach to distinguish between Gram-negative and Gram-positive bacteria. This was done using two enzyme substrates, specifically 2-amino-N-phenylpropanamide and 2-amino-N-(4-methylphenyl)propanamide which liberated the volatile compounds aniline and p-toluidine, respectively. Two complementary analytical techniques have been used to identify and quantify the VOCs, specifically static headspace multicapillary column gas chromatography ion mobility spectrometry (SHS-MCC-GC-IMS) and headspace solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC–MS). Superior limits of detection were obtained using HS-SPME-GC–MS, typically by a factor of x6 such that the LOD for aniline was 0.02μg/mL and 0.01μg/mL for p-toluidine. In addition, it was also possible to determine indole interference-free by HS-SPME-GC–MS at an LOD of 0.01μg/mL. The approach was applied to a range of selected bacteria: 15 Gram-negative and 7 Gram-positive bacteria. Use of pattern recognition, in the form of Principal Component Analysis, confirmed that it is possible to differentiate between Gram-positive and Gram-negative bacteria using the enzyme generated VOCs, aniline and p-toluidine. The exception was Stenotrophomonas maltophilia which showed negligible VOC concentrations for both aniline and p-toluidine, irrespective of the analytical techniques used and hence was not characteristic of the other Gram-negative bacteria investigated. The developed methodology has the potential to be applied for clinical and food applications.

Factors that influence the volatile organic compound content in human breath

Background. Thousands of endogenous and exogenous volatile organic compounds (VOCs) are excreted in each breath. Inflammatory and deviant metabolic processes affect the level of endogeneous VOCs, which can serve as specific biomarkers for clinical diagnosis and disease monitoring. Important issues that still need to be tackled are related to potential confounding factors like gender and age and endogenous and exogenous factors, like f.i. smoking. Methods. The aim of this study was to systematically access the effect of endogenous and exogenous factors on VOC composition of exhaled breath. In the current study breath samples from 1417 adult participants from the LifeLines cohort, a general population cohort in the Netherlands, were collected and the total content of VOCs was measured using gas chromatography-time-of-flight-mass spectrometry. Breath samples were collected in Groningen and transferred to carbon tubes immediately. These samples were then shipped to Maastricht and measured in batches. VOCs profiles were correlated to 14 relevant characteristics of all participants including age, BMI, smoking and blood cell counts and metabolic parameters as well as to 16 classes of medications. Results. VOCs profiles were shown to be significantly influenced by smoking behavior and to a lesser extent by age, BMI and gender. These factors need to be controlled for in breath analysis studies. We found no evidence whatsoever in this 1417 subjects’ cohort that white blood cell counts, cholesterol or triglycerides levels have an influence on the VOC profile. Thus they may not have to be controlled for in exhaled breath studies. Conclusion. The large cohort of volunteers used here represents a unique opportunity to gauge the factors influencing VOCs profiles in a general population i.e. the most clinically relevant population. Classical clinical parameters and smoking habits clearly influence breath content and should therefore be accounted for in future clinical studies involving breath analysis.