Establishment of a headspace-thermal desorption and gas chromatography-mass spectrometry method (HS-TD-GC-MS) for simultaneous detection of 51 volatile organic compounds in human urine: Application in occupational exposure assessment.

Volatile organic compounds (VOCs) are a group of ubiquitous environment pollutants especially released into the workplace. Assessment of VOCs exposure in occupational populations is therefore a crucial issue for occupational health. However, simultaneous biomonitoring of a variety of VOCs is less studied. In this study, a simple and sensitive method was developed for the simultaneous determination of 51 prototype VOCs in urine by headspace-thermal desorption coupled to gas chromatography-mass spectrometry (HS-TD-GC-MS). The urinary sample was pretreated with only adding 0.50 g of sodium chloride to 2 mL of urine and 51 VOCs should be determined with limits of detection (LODs) between 13.6 ng/L and 24.5 ng/L. The method linearity ranged from 0.005 to 10 µg/L with correlation coefficients (r) of 0.991 to 0.999. The precision for intraday and inter-day, measured by the variation coefficient (CV) at three levels of concentration, was below 15 %, except for 4-isopropyl toluene, dichloromethane, and trichloromethane at low concentration. For medium and high levels, recoveries of all target VOCs were within the standard range, but 1,1-dichloropropene and styrene, which were slightly under 80 % at low levels. In addition, the proposed method has been used to determine urine samples collected in three times (before, during and after working) from 152 workers at four different factories. 41 types of prototype VOCs were detected in workers urine. Significant differences (Kruskal-Wallis chi-squared = 117.18, df = 1, P < 0.05) in the concentration levels of VOCs between the exposed and unexposed groups were observed, but not between the three sampling times (Kruskal-Wallis chi-squared = 3.39, df = 2, P = 0.183). The present study provides an alternative method for biomonitoring and assessing mixed exposures to VOCs in occupational populations.

VOC-based detection of prostate cancer using an electronic nose and ion mobility spectrometry: A novel urine-based approach.

BACKGROUND: Many diseases leave behind specific metabolites which can be detected from breath and urine as volatile organic compounds (VOC). Our group previously described VOC-based methods for the detection of bladder cancer and urinary tract infections. This study investigated whether prostate cancer can be diagnosed from VOCs in urine headspace. METHODS: For this pilot study, mid-stream urine samples were collected from 56 patients with histologically confirmed prostate cancer. A control group was formed with 53 healthy male volunteers matched for age who had recently undergone a negative screening by prostate-specific antigen (PSA) and digital rectal exam. Headspace measurements were performed with the electronic nose Cyranose 320TM. Statistical comparison was performed using principal component analysis, calculating Mahalanobis distance, and linear discriminant analysis. Further measurements were carried out with ion mobility spectrometry (IMS) to compare detection accuracy and to identify potential individual analytes. Bonferroni correction was applied for multiple testing. RESULTS: The electronic nose yielded a sensitivity of 77% and specificity of 62%. Mahalanobis distance was 0.964, which is indicative of limited group separation. IMS identified a total of 38 individual analytical peaks, two of which showed significant differences between groups (p < 0.05). To discriminate between tumor and controls, a decision tree with nine steps was generated. This model led to a sensitivity of 98% and specificity of 100%. CONCLUSIONS: VOC-based detection of prostate cancer seems feasible in principle. While the first results with an electronic nose show some limitations, the approach can compete with other urine-based marker systems. However, it seems less reliable than PSA testing. IMS is more accurate than the electronic nose with promising sensitivity and specificity, which warrants further research. The individual relevant metabolites identified by IMS should further be characterized using gas chromatography/mass spectrometry to facilitate potential targeted rapid testing.

Daily Exposure to Environmental Volatile Organic Compounds Triggers Oxidative Damage: Evidence from a Large-Scale Survey in China.

Volatile organic compounds (VOCs) are ubiquitous environmental pollutants and have been implicated in adverse health outcomes. In this study, concentrations of 11 VOC metabolites (mVOCs) and three oxidative stress biomarkers (8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-guanosine, and dityrosine) were determined in 205 urine samples collected from 12 cities across mainland China. Urinary ∑11mVOC concentrations ranged from 498 to 1660 ng/mL, with a geometric mean (GM) value of 1070 ng/mL. The factorial analysis revealed that cooking, solvents, and vehicle emissions were the three primary sources of VOC exposure. A significant regional variation was clearly found in ∑11mVOC concentrations across four regions in China, with high urine VOC concentrations found in North and South China (GM: 1450 and 1340 ng/mL). The multiple linear regression model revealed that most mVOCs were significantly positively correlated with three oxidative stress markers (ß range: 0.06-0.22). Mixture effect regression showed that isoprene, crotonaldehyde, acrolein, and benzene were the strongest contributors to oxidative stress. Approximately 80% of the participants have HQ values greater than 1.0 for 1,3-butadiene and benzene, suggesting that their exposure doses were close to potential adverse health effects. Our findings provide comprehensive information on human exposure and potential health risks of VOCs in China.

Detection of urological cancers by the signature of organic volatile compounds in urine, from dogs to electronic noses.

PURPOSE OF REVIEW: Urine volatile organic compound (VOC) testing for early detection of urological cancers is a minimally invasive and promising method. The objective of this review was to present the results of recently published work on this subject. RECENT FINDINGS: Organic volatile compounds are produced through oxidative stress and peroxidation of cell membranes, and they are eliminated through feces, urine, and sweat. Studies looking for VOCs in urine for the diagnosis of urological cancers have mostly focused on bladder and prostate cancers. However, the number of patients included in the studies was small. The electronic nose was the most widely used means of detecting VOCs in urine for the detection of urological cancers. MOS sensors and pattern recognition machine learning were more used for the composition of electronic noses. Early detection of urological cancers by detection of VOCs in urine is a method with encouraging results with sensitivities ranging from 27 to 100% and specificities ranging from 72 to 94%. SUMMARY: The olfactory signature of urine from patients with urological cancers is a promising biomarker for the early diagnosis of urological cancers. The electronic nose with its ability to recognize complex odors is an excellent alterative to canine diagnosis and analytical techniques. Nevertheless, additional research improving the technology of Enoses and the methodology of the studies is necessary for its implementation in daily clinical practice.

A Novel Urine Test Biosensor Platform for Early Lung Cancer Detection.

Lung cancer is the leading cause of cancer-related mortality worldwide. Early detection is essential to achieving a better outcome and prognosis. Volatile organic compounds (VOCs) reflect alterations in the pathophysiology and body metabolism processes, as shown in various types of cancers. The biosensor platform (BSP) urine test uses animals' unique, proficient, and accurate ability to scent lung cancer VOCs. The BSP is a testing platform for the binary (negative/positive) recognition of the signature VOCs of lung cancer by trained and qualified Long-Evans rats as biosensors (BSs). The results of the current double-blind study show high accuracy in lung cancer VOC recognition, with 93% sensitivity and 91% specificity. The BSP test is safe, rapid, objective and can be performed repetitively, enabling periodic cancer monitoring as well as an aid to existing diagnostic methods. The future implementation of such urine tests as routine screening and monitoring tools has the potential to significantly increase detection rate as well as curability rates with lower healthcare expenditure. This paper offers a first instructive clinical platform utilizing VOC's in urine for detection of lung cancer using the innovative BSP to deal with the pressing need for an early lung cancer detection test tool.

Urinary metabolites of multiple volatile organic compounds among pregnant women across pregnancy: Variability, exposure characteristics, and associations with selected oxidative stress biomarkers.

Volatile organic compounds (VOCs) are a group of pollutants pervasive in daily life with identified adverse health effects. However, no study has investigated the variability in VOC metabolites during pregnancy and their relationships with oxidative stress biomarkers in pregnant women. In the present study, the variability of 21 selected VOC metabolites was examined and their relationships with three selected oxidative stress biomarkers measured in spot urine samples at three trimesters of 1094 pregnant women were analyzed. Nineteen VOC metabolites were ubiquitous in the urine samples with detection rates ranging from 75.9% to 100%. Monohydroxybutenyl mercapturic acid (MHBMA) and s-phenyl mercapturic acid (PMA) had detection rates lower than 1.00%. Intraclass correlation coefficients (ICCs) of the detected analytes at three trimesters ranged 0.07-0.24, and the concentrations were highest in the first trimester. Higher concentrations of some VOC metabolites were related with participant characteristics including higher pre-pregnancy body mass index (BMI), lower education level, unemployment during pregnancy, multiparity, and sampling season of summer or winter. In repeated cross-sectional analyses, interquartile range (IQR) increases in the 19 detected VOC metabolites were positively related with 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-hydroxyguanosine (8-OHG), and 4-hydroxy nonenal mercapturic acid (HNEMA) with the estimates ranging from 9.00% to 204%. The mixture effect of the VOC metabolites on the oxidative stress biomarkers was further assessed using weighted quantile sum regression (WQS) models and the results showed that the WQS index of VOC metabolite mixture was significantly associated with 8-OHdG (ß: 0.37, 0,32, and 0.39 at the 1st, 2nd, and 3rd trimester, respectively), 8-OHG (0.38, 0.32, and 0.39) and HNEMA (1.21, 1.08, and 1.10). Glycidamide mercapturic acid (GAMA), and trans,trans-muconic acid (MU) were the strongest contributors of the mixture effect on 8-OHdG, 8-OHG, and HNEMA, respectively. Overall, urinary concentrations of the VOC metabolites during pregnancy were strongly associated with the oxidative stress biomarkers.

Exposure to volatile organic compounds and polycyclic aromatic hydrocarbons is associated with the risk of non-alcoholic fatty liver disease in Korean adolescents: Korea National Environmental Health Survey (KoNEHS) 2015-2017.

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent liver diseases among adolescents. Several animal studies have suggested that volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) increase NAFLD risk. However, few epidemiological studies have confirmed the association between VOCs, PAHs and NAFLD in the general adolescent population. Therefore, we analyzed 798 adolescents from the Korean National Environmental Health Survey (KoNEHS), 2015-2017, to examine the associations of urinary metabolites of VOCs and PAHs with serum alanine aminotransferase (ALT) activity and NAFLD prevalence. We performed linear regression, logistic regression, and Bayesian kernel machine regression (BKMR) to evaluate the association of urinary VOCs and PAHs metabolites with ALT levels and NAFLD prevalence. After adjusting for all covariates, urinary benzylmercapturic acid and 2-hydroxyfluorene levels were found to increase ALT activity and NAFLD prevalence. Additionally, the BKMR analyses showed a significantly positive overall effect on ALT activity and NAFLD prevalence with urinary concentrations of VOCs and PAHs metabolites, with 2-hydroxyfluorene as the biggest contributor. Our study suggests that exposure to low-level VOCs and PAHs may have a detrimental effect on NAFLD risk in adolescents. Given the increasing prevalence of NAFLD in adolescents, future cohort studies are confirmed to comprehend the effect of these chemicals on NAFLD risk.

Development of a headspace-solid phase microextraction gas chromatography-high resolution mass spectrometry method for analyzing volatile organic compounds in urine: Application in breast cancer biomarker discovery.

BACKGROUND AND AIM: Breast cancer (BC) is the leading cause of cancer-related death in females. The development of non-invasive methods for the early diagnosis of BC still remains challenge. Here, we aimed to discover the urinary volatile organic compounds (VOCs) pattern of BC patients and identify potential VOC biomarkers for BC diagnosis. METHODS: Urine samples were analyzed by headspace-solid phase microextraction (HS-SPME) combined with gas chromatography-high resolution mass spectrometry (GC-HRMS). To assure reliable analysis, the factors influencing HS-SPME extraction efficiency were comprehensively investigated and optimized by combing the Plackett-Burman design (PBD) with the central composite design (CCD). The established HS-SPME/GC-HRMS method was validated and applied to analyze urine samples from BC patients (n = 80) and healthy controls (n = 88). RESULTS: A total number of 134 VOCs belonging to distinct chemical classes were identified by GC-HRMS. BC patients demonstrated unique urinary VOCs pattern. Orthogonal partial least squares-discriminant analysis (OPLS-DA) showed a clear separation between BC patients and healthy controls. Eight potential VOC biomarkers were identified using multivariate and univariate statistical analysis. The predictive ability of candidate VOC biomarkers was further investigated by the random forest (RF) algorithm. The candidate VOC biomarkers yielded 76.3% sensitivity and 85.4% specificity on the training set, and achieved 76.0% sensitivity and 92.3% specificity on the validation set. CONCLUSIONS: Overall, this work not only established a standardized HS-SPME/GC-HRMS approach for urinary VOCs analysis, but also highlighted the value of urinary VOCs for BC diagnosis. The knowledge gained from this study paves the way for early diagnosis of BC using urine in a non-invasive manner.

Patterns of volatile organic compounds in excrements of preterm neonates.

BACKGROUND: As neonates are susceptible for many diseases, establishing noninvasive diagnostic methods is desirable. We hypothesized that volatile organic compounds (VOCs) could be successfully measured in diaper samples. METHODS: We performed a feasibility study to investigate whether ambient air-independent headspace measurements of the VOC profiles of diapers from premature infants can be conducted using ion mobility spectrometer coupled with multi-capillary columns (B & S Analytik GmbH). RESULTS: We analysed 39 diapers filled with stool (n = 10) or urine (n = 20) respectively, using empty diapers as a control (n = 9). A total of 158 different VOCs were identified, and we classified the content of the diapers (urine or stool) according to their VOC profiles with a significance level of p < 0.05. CONCLUSIONS: We have developed a novel method to study headspace VOC profiles of biosamples using ion mobility spectrometry coupled with multi-capillary columns. Using this method, we have characterized the VOC profiles of stool and urine of preterm neonates. Future studies are warranted to characterize specific VOC profiles in infections and other diseases of the preterm neonate, thus establishing quick and noninvasive diagnostics in the routine care of the highly vulnerable preterm and term neonates.

Changes in the urine volatile metabolome throughout growth of transplanted hepatocarcinoma.

Trained detection dogs distinguish between urine samples from healthy organisms and organisms with malignant tumors, suggesting that the volatile urine metabolome contains information about tumor progression. The aim of this study was to determine whether the stage of tumor growth affects the chemical differences in the urine of mice and to what extent the "olfactory image of disease" perceived by dogs coincides with the "image of disease" recorded by the mass spectrometer. We used a novel laser ionization mass spectrometry method and propose a mass spectrometric analysis without detailed interpretation of the spectrum of volatile metabolomes in urine. The mass spectrometer we use works without sample preparation and registers volatile organic compounds in air at room temperature without changing the pH of the sample, i.e. under conditions similar to those in which dogs solve the same problem. The experimental cancer models were male BDF-f1 hybrid mice transplanted with hepatocarcinoma tissue, and similar mice transplanted with healthy liver tissue were used as controls. Our data show that both dogs and our proposed laser mass spectrometry method are able to detect both the entire spectrum of volatile organic compounds associated with the disease and minor changes in this spectrum during its course.

Investigation of urinary volatile organic compounds as novel diagnostic and surveillance biomarkers of bladder cancer.

BACKGROUND: The diagnosis and surveillance of urothelial bladder cancer (UBC) require cystoscopy. There is a need for biomarkers to reduce the frequency of cystoscopy in surveillance; urinary volatile organic compound (VOC) analysis could fulfil this role. This cross-sectional study compared the VOC profiles of patients with and without UBC, to investigate metabolomic signatures as biomarkers. METHODS: Urine samples were collected from haematuria clinic patients undergoing diagnostic cystoscopy and UBC patients undergoing surveillance. Urinary headspace sampling utilised solid-phase microextraction and VOC analysis applied gas chromatography-mass spectrometry; the output underwent metabolomic analysis. RESULTS: The median participant age was 70 years, 66.2% were male. Of the haematuria patients, 21 had a new UBC diagnosis, 125 had no cancer. In the surveillance group, 75 had recurrent UBC, 84 were recurrence-free. A distinctive VOC profile was observed in UBC patients compared with controls. Ten VOCs had statistically significant abundances useful to classify patients (false discovery rate range 1.9 × 10-7-2.8 × 10-2). Two prediction models were evaluated using internal validation. An eight-VOC diagnostic biomarker panel achieved AUROC 0.77 (sensitivity 0.71, specificity 0.72). A six-VOC surveillance biomarker panel obtained AUROC 0.80 (sensitivity 0.71 and specificity 0.80). CONCLUSIONS: Urinary VOC analysis could aid the diagnosis and surveillance of UBC.

A prediction model using 2-propanol and 2-butanone in urine distinguishes breast cancer.

Safe and noninvasive methods for breast cancer screening with improved accuracy are urgently needed. Volatile organic compounds (VOCs) in biological samples such as breath and blood have been investigated as noninvasive novel markers of cancer. We investigated volatile organic compounds in urine to assess their potential for the detection of breast cancer. One hundred and ten women with biopsy-proven breast cancer and 177 healthy volunteers were enrolled. The subjects were divided into two groups: a training set and an external validation set. Urine samples were collected and analyzed by gas chromatography and mass spectrometry. A predictive model was constructed by multivariate analysis, and the sensitivity and specificity of the model were confirmed using both a training set and an external set with reproducibility tests. The training set included 60 breast cancer patients (age 34-88 years, mean 60.3) and 60 healthy controls (age 34-81 years, mean 58.7). The external validation set included 50 breast cancer patients (age 35-85 years, mean 58.8) and 117 healthy controls (age 18-84 years, mean 51.2). One hundred and ninety-one compounds detected in at least 80% of the samples from the training set were used for further analysis. The predictive model that best-detected breast cancer at various clinical stages was constructed using a combination of two of the compounds, 2-propanol and 2-butanone. The sensitivity and specificity in the training set were 93.3% and 83.3%, respectively. Triplicated reproducibility tests were performed by randomly choosing ten samples from each group, and the results showed a matching rate of 100% for the breast cancer patient group and 90% for the healthy control group. Our prediction model using two VOCs is a useful complement to the current diagnostic tools. Further studies inclusive of benign tumors and non-breast malignancies are warranted.

Optimization of training and measurement protocol for eNose analysis of urine headspace aimed at prostate cancer diagnosis.

More than one million new cases of prostate cancer (PCa) were reported worldwide in 2020, and a significant increase of PCa incidence up to 2040 is estimated. Despite potential treatability in early stages, PCa diagnosis is challenging because of late symptoms' onset and limits of current screening procedures. It has been now accepted that cell transformation leads to release of volatile organic compounds in biologic fluids, including urine. Thus, several studies proposed the possibility to develop new diagnostic tools based on urine analysis. Among these, electronic noses (eNoses) represent one of the most promising devices, because of their potential to provide a non-invasive diagnosis. Here we describe the approach aimed at defining the experimental protocol for eNose application for PCa diagnosis. Our research investigates effects of sample preparation and analysis on eNose responses and repeatability. The dependence of eNose diagnostic performance on urine portion analysed, techniques involved for extracting urine volatiles and conditioning temperature were analysed. 192 subjects (132 PCa patients and 60 controls) were involved. The developed experimental protocol has resulted in accuracy, sensitivity and specificity of 83% (CI95% 77-89), 82% (CI95% 73-88) and 87% (CI95% 75-94), respectively. Our findings define eNoses as valuable diagnostic tool allowing rapid and non-invasive PCa diagnosis.

Hope for Ostomates: A Carbon and Zeolite Impregnated Polyester Fabric Inhibits Urine Odor in Cancer Patients: A Randomized Experimental Study.

OBJECTIVE: Many individuals with bladder cancer have undergone a surgical urostomy and often complain of being self-conscious of the unpleasant smell of their own urine. The focus of this study was to test the efficacy of a pouch cover made of a carbon and zeolite containing polyester material to inhibit the smell of urine by comparing two trained dogs' response time in detecting volatile organic compounds (VOCs) in urine, with and without the fabric covering the samples. METHODS: This study used a randomized, blinded experimental design to evaluate the efficacy of a fabric to interfere with two highly trained dogs' ability to detect specific VOCs present in the urine of prostate cancer patient. Ninety urine samples were analyzed in this study. RESULTS: Prior to the experiment, both dogs accurately detected VOCs in the uncovered test urine samples of men with prostate cancer with a sensitivity and specificity of nearly 100%. Both dogs recognized the "uncovered" urine samples of men with prostate cancer within two seconds. When the test sample was covered with the study fabric, the test urine samples were detected within 30-40 seconds and in some instances the dogs were not able to identify the covered samples, whatsoever. CONCLUSION: The findings of this study demonstrate that the carbon and zeolite containing polyester fabric did significantly interfere with the ability of the dogs to detect VOCs in urine of men with prostate cancer. The fabric may show promise as a pouch cover in controlling offensive urine odor which many ostomates experience.

Sniffer dogs can identify lung cancer patients from breath and urine samples.

BACKGROUND: Lung cancer is the most common oncological cause of death in the Western world. Early diagnosis is critical for successful treatment. However, no effective screening methods exist. A promising approach could be the use of volatile organic compounds as diagnostic biomarkers. To date there are several studies, in which dogs were trained to discriminate cancer samples from controls. In this study we evaluated the abilities of specifically trained dogs to distinguish samples derived from lung cancer patients of various tumor stages from matched healthy controls. METHODS: This single center, double-blind clinical trial was approved by the local ethics committee, project no FF20/2016. The dog was conditioned with urine and breath samples of 36 cancer patients and 150 controls; afterwards, further 246 patients were included: 41 lung cancer patients comprising all stages and 205 healthy controls. From each patient two breath and urine samples were collected and shock frozen. Only samples from new subjects were presented to the dog during study phase randomized, double-blinded. This resulted in a specific conditioned reaction pointing to the cancer sample. RESULTS: Using a combination of urine and breath samples, the dog correctly predicted 40 out of 41 cancer samples, corresponding to an overall detection rate of cancer samples of 97.6% (95% CI [87.1, 99.9%]). Using urine samples only the dog achieved a detection rate of 87.8% (95% CI [73.8, 95.9%]). With breath samples, the dog correctly identified cancer in 32 of 41 samples, resulting in a detection rate of 78% (95% CI [62.4, 89.4%]). CONCLUSIONS: It is known from current literature that breath and urine samples carry VOCs pointing to cancer growth. We conclude that olfactory detection of lung cancer by specifically trained dogs is highly suggestive to be a simple and non-invasive tool to detect lung cancer. To translate this approach into practice further target compounds need to be identified.

Searching for Potential Markers of Glomerulopathy in Urine by HS-SPME-GC×GC TOFMS.

Volatile organic compounds (VOCs) exiting in urine are potential biomarkers of chronic kidney diseases. Headspace solid phase microextraction (HS-SPME) was applied for extraction VOCs over the urine samples. Volatile metabolites were separated and identified by means of two-dimensional gas chromatography and time of flight mass spectrometry (GC × GC TOF MS). Patients with glomerular diseases (n = 27) and healthy controls (n = 20) were recruited in the study. Different VOCs profiles were obtained from patients and control. Developed methodology offers the opportunity to examine the metabolic profile associated with glomerulopathy. Four compounds found in elevated amounts in the patients group, i.e., methyl hexadecanoate; 9-hexadecen-1-ol; 6,10-dimethyl-5,9-undecadien-2-one and 2-pentanone were proposed as markers of glomerular diseases.

Exploratory Study Using Urinary Volatile Organic Compounds for the Detection of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) biomarkers are lacking in clinical practice. We therefore explored the pattern and composition of urinary volatile organic compounds (VOCs) in HCC patients. This was done in order to assess the feasibility of a potential non-invasive test for HCC, and to enhance our understanding of the disease. This pilot study recruited 58 participants, of whom 20 were HCC cases and 38 were non-HCC cases. The non-HCC cases included healthy individuals and patients with various stages of non-alcoholic fatty liver disease (NAFLD), including those with and without fibrosis. Urine was analysed using gas chromatography-ion mobility spectrometry (GC-IMS) and gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS). GC-IMS was able to separate HCC from fibrotic cases with an area under the curve (AUC) of 0.97 (0.91-1.00), and from non-fibrotic cases with an AUC of 0.62 (0.48-0.76). For GC-TOF-MS, a subset of samples was analysed in which seven chemicals were identified and tentatively linked with HCC. These include 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene (2TMS derivative), 2-butanone, 2-hexanone, benzene, 1-ethyl-2-methyl-, 3-butene-1,2-diol, 1-(2-furanyl)-, bicyclo(4.1.0)heptane, 3,7,7-trimethyl-, [1S-(1a,3ß,6a)]-, and sulpiride. Urinary VOC analysis using both GC-IMS and GC-TOF-MS proved to be a feasible method of identifying HCC cases, and was also able to enhance our understanding of HCC pathogenesis.

Volatile scent chemicals in the urine of the red fox, Vulpes vulpes.

The red fox is a highly adaptable mammal that has established itself world-wide in many different environments. Contributing to its success is a social structure based on chemical signalling between individuals. Urine scent marking behaviour has long been known in foxes, but there has not been a recent study of the chemical composition of fox urine. We have used solid-phase microextraction and gas chromatography-mass spectrometry to analyze the urinary volatiles in 15 free-ranging wild foxes (2 female) living in farmlands and bush in Victoria, Australia. Foxes here are routinely culled as feral pests, and the urine was collected by bladder puncture soon after death. Compounds were identified from their mass spectra and Kovats retention indices. There were 53 possible endogenous scent compounds, 10 plant-derived compounds and 5 anthropogenic xenobiotics. Among the plant chemicals were several aromatic apocarotenoids previously found in greater abundance in the fox tail gland. They reflect the dietary consumption of carotenoids, essential for optimal health. One third of all the endogenous volatiles were sulfur compounds, a highly odiferous group which included thiols, methylsulfides and polysulfides. Five of the sulfur compounds (3-isopentenyl thiol, 1- and 2-phenylethyl methyl sulfide, octanethiol and benzyl methyl sulfide) have only been found in foxes, and four others (isopentyl methyl sulfide, 3-isopentenyl methyl sulfide, and 1- and 2-phenylethane thiol) only in some canid, mink and skunk species. This indicates that they are not normal mammalian metabolites and have evolved to serve a specific role. This role is for defence in musteloids and most likely for chemical communication in canids. The total production of sulfur compounds varied greatly between foxes (median 1.2, range 0.4-32.3 µg 'acetophenone equivalents'/mg creatinine) as did the relative abundance of different chemical types. The urinary scent chemistry may represent a highly evolved system of semiochemicals for communication between foxes.

A Caenorhabditis elegans behavioral assay distinguishes early stage prostate cancer patient urine from controls.

Current methods for non-invasive prostate cancer (PrCa) detection have a high false-positive rate and often result in unnecessary biopsies. Previous work has suggested that urinary volatile organic compound (VOC) biomarkers may be able to distinguish PrCa cases from benign disease. The behavior of the nematode Caenorhabditis elegans has been proposed as a tool to take advantage of these potential VOC profiles. To test the ability of C. elegans Bristol N2 to distinguish PrCa cases from controls, we performed chemotaxis assays using human urine samples collected from men screened for PrCa. Behavioral response of nematodes towards diluted urine from PrCa cases was compared to response to samples from cancer-free controls. Overall, we observed a significant attraction of young adult-stage C. elegans nematodes to 1:100 diluted urine from confirmed PrCa cases and repulsion of C. elegans to urine from controls. When C. elegans chemotaxis index was considered alongside prostate-specific antigen levels for distinguishing cancer from cancer-free controls, the accuracy of patient classification was 81%. We also observed behavioral attraction of C. elegans to two previously reported VOCs to be increased in PrCa patient urine. We conclude nematode behavior distinguishes PrCa case urine from controls in a dilution-dependent manner.

Feasibility of integrating canine olfaction with chemical and microbial profiling of urine to detect lethal prostate cancer.

Prostate cancer is the second leading cause of cancer death in men in the developed world. A more sensitive and specific detection strategy for lethal prostate cancer beyond serum prostate specific antigen (PSA) population screening is urgently needed. Diagnosis by canine olfaction, using dogs trained to detect cancer by smell, has been shown to be both specific and sensitive. While dogs themselves are impractical as scalable diagnostic sensors, machine olfaction for cancer detection is testable. However, studies bridging the divide between clinical diagnostic techniques, artificial intelligence, and molecular analysis remains difficult due to the significant divide between these disciplines. We tested the clinical feasibility of a cross-disciplinary, integrative approach to early prostate cancer biosensing in urine using trained canine olfaction, volatile organic compound (VOC) analysis by gas chromatography-mass spectroscopy (GC-MS) artificial neural network (ANN)-assisted examination, and microbial profiling in a double-blinded pilot study. Two dogs were trained to detect Gleason 9 prostate cancer in urine collected from biopsy-confirmed patients. Biopsy-negative controls were used to assess canine specificity as prostate cancer biodetectors. Urine samples were simultaneously analyzed for their VOC content in headspace via GC-MS and urinary microbiota content via 16S rDNA Illumina sequencing. In addition, the dogs' diagnoses were used to train an ANN to detect significant peaks in the GC-MS data. The canine olfaction system was 71% sensitive and between 70-76% specific at detecting Gleason 9 prostate cancer. We have also confirmed VOC differences by GC-MS and microbiota differences by 16S rDNA sequencing between cancer positive and biopsy-negative controls. Furthermore, the trained ANN identified regions of interest in the GC-MS data, informed by the canine diagnoses. Methodology and feasibility are established to inform larger-scale studies using canine olfaction, urinary VOCs, and urinary microbiota profiling to develop machine olfaction diagnostic tools. Scalable multi-disciplinary tools may then be compared to PSA screening for earlier, non-invasive, more specific and sensitive detection of clinically aggressive prostate cancers in urine samples.