Distinguish Esophageal Cancer Cells through VOCs Induced by Methionine Regulation.

Detection of exhaled volatile organic compounds (VOCs) is promising for noninvasive screening of esophageal cancer (EC). Cellular VOC analysis can be used to investigate potential biomarkers. Considering the crucial role of methionine (Met) during cancer development, exploring associated abnormal metabolic phenotypes becomes imperative. In this work, we employed headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) to investigate the volatile metabolic profiles of EC cells (KYSE150) and normal esophageal epithelial cells (HEECs) under a Met regulation strategy. Using untargeted approaches, we analyzed the metabolic VOCs of the two cell types and explored the differential VOCs between them. Subsequently, we utilized targeted approaches to analyze the differential VOCs in both cell types under gradient Met culture conditions. The results revealed that there were five/six differential VOCs between cells under Met-containing/Met-free culture conditions. And the difference in levels of two characteristic VOCs (1-butanol and ethyl 2-methylbutyrate) between the two cell types intensified with the increase of the Met concentration. Notably, this is the first report on VOC analysis of EC cells and the first to consider the effect of Met on volatile metabolic profiles. The present work indicates that EC cells can be distinguished through VOCs induced by Met regulation, which holds promise for providing novel insights into diagnostic strategies.

Qualitative and quantitative rapid detection of VOCs differentially released by VAP-associated bacteria using PTR-MS and FGC-PTR-MS.

Ventilator-associated pneumonia (VAP) is a prevalent disease caused by microbial infection, resulting in significant morbidity and mortality within the intensive care unit (ICU). The rapid and accurate identification of pathogenic bacteria causing VAP can assist clinicians in formulating timely treatment plans. In this study, we attempted to differentiate bacterial species in VAP by utilizing the volatile organic compounds (VOCs) released by pathogens. We cultured 6 common bacteria in VAP in vitro, including Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Staphylococcus aureus, which covered most cases of VAP infection in clinic. After the VOCs released by bacteria were collected in sampling bags, they were quantitatively detected by a proton transfer reaction-mass spectrometry (PTR-MS), and the characteristic ions were qualitatively analyzed through a fast gas chromatography-proton transfer reaction-mass spectrometry (FGC-PTR-MS). After conducting principal component analysis (PCA) and analysis of similarities (ANOSIM), we discovered that the VOCs released by 6 bacteria exhibited differentiation following 3 h of quantitative cultivation in vitro. Additionally, we further investigated the variations in the types and concentrations of bacterial VOCs. The results showed that by utilizing the differences in types of VOCs, 6 bacteria could be classified into 5 sets, except for A. baumannii and E. cloacae which were indistinguishable. Furthermore, we observed significant variations in the concentration ratio of acetaldehyde and methyl mercaptan released by A. baumannii and E. cloacae. In conclusion, the VOCs released by bacteria could effectively differentiate the 6 pathogens commonly associated with VAP, which was expected to assist doctors in formulating treatment plans in time and improve the survival rate of patients.

HS-SPME-GC-MS Untargeted Analysis of Normal Rat Organs Ex Vivo: Differential VOC Discrimination and Fingerprint VOC Identification.

The investigation of volatile organic compounds (VOCs) in human metabolites has been a topic of interest as it holds the potential for the development of non-invasive technologies to screen for organ lesions in vivo. However, it remains unclear whether VOCs differ among healthy organs. Consequently, a study was conducted to analyze VOCs in ex vivo organ tissues obtained from 16 Wistar rats, comprising 12 different organs. The VOCs released from each organ tissue were detected by the headspace-solid phase microextraction-gas chromatography-mass spectrometry technique. In the untargeted analysis of 147 chromatographic peaks, the differential volatiles of rat organs were explored based on the Mann-Whitney U test and fold change (FC > 2.0) compared with other organs. It was found that there were differential VOCs in seven organs. A discussion on the possible metabolic pathways and related biomarkers of organ differential VOCs was conducted. Based on the orthogonal partial least squares discriminant analysis and receiver operating characteristic curve, we found that differential VOCs in the liver, cecum, spleen, and kidney can be used as the unique identification of the corresponding organ. In this study, differential VOCs of organs in rats were systematically reported for the first time. Profiles of VOCs produced by healthy organs can serve as a reference or baseline that may indicate the presence of disease or abnormalities in the organ's function. Differential VOCs can be used as the fingerprint of organs, and future integration with metabolic research may contribute to the development of healthcare.

Rapid and non-invasive diagnosis of type 2 diabetes through sniffing urinary acetone by a proton transfer reaction mass spectrometry.

Urinary acetone in urine is produced from fat metabolism in human body, which can be accelerated in diabetic patients because of insufficient utilization and storage of glucose. In this study, we tried to develop a novel diagnosis method of type 2 diabetes (T2D) through sniffing urinary acetone by a proton transfer reaction mass spectrometry (PTR-MS). A total of 180 T2D patients and 180 healthy volunteers were recruited from three hospitals for multicenter study. Urine samples were collected in the morning when donators were fasting and stored in glass bottles. Acetone in the headspace of these bottles was qualitatively and quantitatively detected by the PTR-MS in 8 h. Using a threshold of 690.1 ppbv, a diagnostic model was established using urinary acetone with an accuracy of 81.3% (sensitivity: 73.3%, specificity: 89.3%) in hospital Ⅰ. In the verification studies, the accuracies were 92.5% (sensitivity: 88.7%, specificity: 96.2%) in hospital Ⅱ and 83.7% (sensitivity: 76.9%, specificity: 90.4%) in hospital Ⅲ, respectively. The accuracy is comparable to that of clinically used diagnosis methods, fasting plasma glucose (FPG), oral glucose tolerance test (OGTT), and glycosylated hemoglobin A1c (HbA1c) test. The sensitivity for 35 newly diagnosed patients was 85.7%. The newly developed technology is completely non-invasive and much more rapid than clinical FPG, OGTT, and HbA1c tests. It has a promising prospect in clinical use. But the applicability in different human races still need more validations.

Analysis of volatile organic compounds from deep airway in the lung through intubation sampling.

Exhaled volatile organic compounds (VOCs) have been widely applied for the study of disease biomarkers. Oral exhalation and nasal exhalation are two of the most common sampling methods. However, VOCs released from food residues and bacteria in the mouth or upper respiratory tract were also sampled and usually mistaken as that produced from body metabolism. In this study, exhalation from deep airway was first directly collected through intubation sampling and analyzed. The exhalation samples of 35 subjects were collected through a catheter, which was inserted into the trachea or bronchus through the mouth and upper respiratory tract. Then, the VOCs in these samples were detected by proton transfer reaction mass spectrometry (PTR-MS). In addition, fast gas chromatography proton transfer reaction mass spectrometry (FGC-PTR-MS) was used to further determine the VOCs with the same mass-to-charge ratios. The results showed that there was methanol, acetonitrile, ethanol, methyl mercaptan, acetone, isoprene, and phenol in the deep airway. Compared with that in oral exhalation, ethanol, methyl mercaptan, and phenol had lower concentrations. In detail, the median concentrations of ethanol, methyl mercaptan, and phenol were 7.3, 0.6, and 23.9 ppbv, while those in the oral exhalation were 80.0, 5.1, and 71.3 ppbv, respectively, which meant the three VOCs mainly originated from the food residues and bacteria in the mouth or upper respiratory tract, rather than body metabolism. The research results in our study can provide references for expiratory VOC research based on oral and nasal exhalation samplings, which are more feasible in clinical practice.

Analysis of volatile organic compounds in exhaled breath after radiotherapy.

Radiotherapy uses high-energy X-rays or other particles to destroy cancer cells and medical practitioners have used this approach extensively for cancer treatment (Hachadorian et al., 2020). However, it is accompanied by risks because it seriously harms normal cells while killing cancer cells. The side effects can lower cancer patients' quality of life and are very unpredictable due to individual differences (Bentzen, 2006). Therefore, it is essential to assess a patient's body damage after radiotherapy to formulate an individualized recovery treatment plan. Exhaled volatile organic compounds (VOCs) can be changed by radiotherapy and thus used for medical diagnosis (Vaks et al., 2012). During treatment, high-energy X-rays can induce apoptosis; meanwhile, cell membranes are damaged due to lipid peroxidation, converting unsaturated fatty acids into volatile metabolites (Losada-Barreiro and Bravo-Díaz, 2017). At the same time, radiotherapy oxidizes water, resulting in reactive oxygen species (ROS) that can increase the epithelial permeability of pulmonary alveoli, enabling the respiratory system to exhale volatile metabolites (Davidovich et al., 2013; Popa et al., 2020). These exhaled VOCs can be used to monitor body damage caused by radiotherapy.

Distinguish oral-source VOCs and control their potential impact on breath biomarkers.

By means of glass bottle sampling followed by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) technique, the change characteristics of volatile organic compounds (VOCs) in breaths, between before gargling and after gargling, were investigated, respectively, in 41 healthy subjects and 50 esophageal cancer patients. Using an untargeted strategy, 143 VOC chromatographic peaks were enrolled in the statistical analysis. Based on the orthogonal partial least squares discriminant analysis (OPLS-DA), the VOC variations after gargling for each breath test group were obtained according to the combined criteria of variable importance in projection (VIP > 1.5), Wilcoxon signed-rank test (P < 0.05), and fold change (FC > 2.0). When gargled, the levels of indole, phenol, 1-propanol, and p-cresol in the breath of healthy people decreased; meanwhile, for esophageal cancer patients, the declined VOCs in breath were indole, phenol, dimethyl disulfide, and p-cresol. Particularly, these substances were previously reported as breath biomarkers in some diseases such as esophageal, gastric, thyroid, breast, oral, and lung cancers, as well as certain non-cancer disorders. The present work indicates that expiratory VOCs involve the prominent oral cavity source, and in the breath biomarkers study, the potential impact that originates from oral volatiles should be considered. In view of the present results, it is also proposed that gargle pretreatment could eliminate possible interference from the oral cavity VOCs that might benefit breath biomarker investigation. Gargle pretreatment helps to distinguish oral-source VOCs and control their potential impact on breath biomarkers.

Variable VOCs in plastic culture flasks and their potential impact on cell volatile biomarkers.

In order to find out cancer markers in human breath, in vitro cell culture is often used to study the characteristic volatile organic compounds (VOCs). In the cell culture process, disposable vessels are frequently adopted. However, these vessels are normally made of plastic, and they have the possibility to release some VOCs, which may interfere with the cell-specific volatiles and even can result in an incorrect conclusion. In this study, by using glass cell culture flasks as control, the headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) analyses of the VOCs in plastic cell culture flasks were systematically carried out for the first time. A total of 35 VOCs were detected in five brands of flasks. In each flask, there were between 13 and 25 volatile compounds. Furthermore, the components and packaging bag of each flask were also sampled and analyzed by HS-SPME-GC-MS. The results show that the flask cap, septum, flask body, and packaging bag exhibit respectively different volatile behaviors. The former two parts release the most volatiles which have obvious contributions to the headspace gases in the flasks, while the flask body mainly liberates styrene. For different flasks packed within the same bag, the headspace analyses show that their residual VOCs are inconsistent with each other. Moreover, the residual VOCs in the same flask are variable in three consecutive days. These results indicate that the multiple flasks in parallel cell culture experiments, or the same flask with different cell culture durations, will produce an indelible disturbance to the cell-specific VOCs. In addition, among the 35 VOCs detectable in five brands of empty plastic flasks, 15 VOCs were previously reported as characteristic VOCs from lung cancer, melanoma, cervical cancer cells, or normal cells. This is an alert that, when using plastic flasks, it must be careful to treat the possible interference from the background VOCs in the flasks. This study demonstrates that the cell culture tool needs to be standardized, and the clean glass or metal vessels are strongly recommended for usage when studying cell volatile biomarkers. Graphical abstract.

On-line monitoring human breath acetone during exercise and diet by proton transfer reaction mass spectrometry.

AIM: This study aims to develop a method for monitoring fat loss by detection of breath acetone. METHODS: A new method combining direct breath sampling system and proton transfer reaction MS (PTR-MS) was developed for on-line detection of breath acetone. The breath acetone of 272 volunteers was detected respectively when exercise or diet. RESULTS: Exercises perennially can make the breath acetone increase by 40-130%. Dinner fasting for 1 week can make it increase by 140%. CONCLUSION: Exercise and diet are two useful methods to lose fat. Determination of breath acetone concentration using the direct breath sampling system-proton transfer reaction MS can help design scheme of exercise and diet for losing weight.