Urinary Volatile Compounds Research Articles

Urinary volatile compounds in mycoplasma genitalium infection

Urinary volatile compounds in mycoplasma genitalium infection

Species recognition and the divergences in the chemical and ultrasonic signals between two coexisting Rattus species.

The ability to recognize and differentiate between conspecifics and heterospecifics as well as their signals is critical for the coexistence of closely related species. In the genus Rattus, species are morphologically similar and multiple species often coexist. Here, we investigated the interspecific recognition and signal differentiation of two sympatric rat species, the brown rat (Rattus norvegicus, RN) and the Asian house rat (Rattus tanezumi, RT). In a two-way choice test, both RN and RT females showed a preference for conspecific male rats to heterospecific ones. RT females showed a significant preference for accessible urine of males of same species to those of other species, but not for the inaccessible urine. On the other hand, there were significant differences in the structural characteristics of the ultrasonic vocalization emitted by males of these two rat species. Sodium dodecyl sulphate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and isoelectric focusing electrophoresis unveiled that major urinary proteins (MUPs) in voided urine were more highly expressed in RN males versus RT males. The interspecific differences of urinary volatile compounds were also discussed. In conclusion, female rats had the ability to distinguish between males of either species.

An Optimization of Liquid-Liquid Extraction of Urinary Volatile and Semi-Volatile Compounds and Its Application for Gas Chromatography-Mass Spectrometry and Proton Nuclear Magnetic Resonance Spectroscopy.

Urinary volatile compounds (VCs) have been recently assessed for disease diagnoses. They belong to very diverse chemical classes, and they are characterized by different volatilities, polarities and concentrations, complicating their analysis via a single analytical procedure. There remains a need for better, lower-cost methods for VC biomarker discovery. Thus, there is a strong need for alternative methods, enabling the detection of a broader range of VCs. Therefore, the main aim of this study was to optimize a simple and reliable liquid–liquid extraction (LLE) procedure for the analysis of VCs in urine using gas chromatography-mass spectrometry (GC-MS), in order to obtain the maximum number of responses. Extraction parameters such as pH, type of solvent and ionic strength were optimized. Moreover, the same extracts were analyzed using Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR), to evaluate the applicability of a single urine extraction for multiplatform purposes. After the evaluation of experimental conditions, an LLE protocol using 2 mL of urine in the presence of 2 mL of 1 M sulfuric acid and sodium sulphate extracted with dichloromethane was found to be optimal. The optimized method was validated with the external standards and was found to be precise and linear, and allowed for detection of >400 peaks in a single run present in at least 50% of six samples—considerably more than the number of peaks detected by solid-phase microextracton fiber pre-concentration-GC-MS (328 ± 6 vs. 234 ± 4). 1H-NMR spectroscopy of the polar and non-polar extracts extended the range to >40 more (mainly low volatility compounds) metabolites (non-destructively), the majority of which were different from GC-MS. The more peaks detectable, the greater the opportunity of assessing a fingerprint of several compounds to aid biomarker discovery. In summary, we have successfully demonstrated the potential of LLE as a cheap and simple alternative for the analysis of VCs in urine, and for the first time the applicability of a single urine solvent extraction procedure for detecting a wide range of analytes using both GC-MS and 1H-NMR analysis to enhance putative biomarker detection. The proposed method will simplify the transport between laboratories and storage of samples, as compared to intact urine samples.

Assessment of urinary volatile compounds and proteins in the female goat Capra hircus: A pilot study to reveal potential indicators of oestrus.

Urine samples of female goats in pro-oestrus, oestrus and post-oestrus phases were analysed for finding oestrus-specific volatile compounds using gas chromatograph-mass spectrometry (GC-MS), and proteins using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fourteen urinary volatile were identified covering all three phases among which four compounds, 1-Tetradecanol, n-Pentadecanol, 3-Methylene tridecane and 2-Ethyl-1-dodecene, were unique to oestrus. Also, oestrus urine contained a 25kDa protein, which was totally absent in pro-oestrus urine, and less-expressed in post-oestrus urine. This protein revealed to be complement C3 fragment. This pilot study, for the first time, reveals the difference in urinary volatile compounds and proteins in the female goat during the different phases of oestrous cycle. The four unique volatile compounds and a 25kDa protein that appeared as oestrus-specific in this study warrant further investigation to consider them as urinary biomarkers of oestrus in goats.

Menthol, a unique urinary volatile compound, is associated with chronic inflammation in interstitial cystitis

Chronic inflammation is a potential systemic risk factor for many bladder dysfunctions, including interstitial cystitis (IC). However, the underlying mechanism through which a healthy bladder protects itself from inflammatory triggers remains unknown. In this study, we identified odor compounds in urine obtained from IC patients and healthy controls. Using comprehensive solid-phase microextraction-gas chromatography-time-of-flight-mass spectrometry (SPME-GC-TOF-MS) profiling and bioinformatics, we found that levels of urinary volatile metabolites, such as menthol, were significantly reduced in IC patients, compared to healthy controls. In an attempt to understand the mechanistic meaning of our volatile metabolites data and the role of menthol in the immune system, we performed two independent experiments: (a) cytokine profiling, and (b) DNA microarray. Our findings suggest that lipopolysaccharide (LPS)-stimulated inflammatory events, such as the production and secretion of inflammatory cytokines (e.g., TNF-α, IL-6, and IL-1β) and the activation of NF-κB and associated proteins within a large signaling network (e.g., Akt, TLR1, TNFAIP3, and NF-κB), are suppressed by the presence of menthol. These findings broaden our knowledge on the role of urinary menthol in suppressing inflammatory events and provide potential new strategies for alleviating both the odor and inflammation associated with IC.

Urinary volatile compounds differ across reproductive phenotypes and following aggression in male Siberian hamsters

Chemical communication plays an integral role in social behavior by facilitating social encounters, allowing for the evaluation of social partners, defining territories and advertising information such as species and sex. Odors provide information about the social environment for rodents and other mammals; however, studies identifying chemical compounds and their functions have thus far focused primarily on a few species. In addition, considerably less attention has been focused on how environmental factors and behavioral context alter these compounds during periods of reproductive quiescence. We examined the effects of photoperiod and social context on chemical communication in the seasonally breeding Siberian hamster which displays modest territorial aggression during long “summer-like” days, but increased aggression in short “winter-like” days. We collected urine samples from long- and short-day male hamsters to investigate how photoperiod and subsequent changes in reproductive phenotype alter urinary volatile compound profiles. Next, we identified changes in urinary compounds before and after an aggressive encounter. Male hamsters exhibited a diverse urinary profile across photoperiods; however, long-day reproductive males showed higher levels of individual compounds when compared to short-day non-reproductive males. In addition, individual compounds were altered following an aggressive encounter; some changed only in long days whereas others changed regardless of photoperiod. Further, aggression and circulating levels of testosterone were positively correlated with urinary compounds in long-, but not short-day males. These findings suggest both photoperiod- and aggression-specific physiological regulation of urinary compounds in this species and contribute to a greater understanding of chemical communication more broadly.

Detection of estrus in Indian blackbuck: Behavioural, hormonal and urinary volatiles evaluation

The determination of the reproductive status is one of the most important factors for effective wild life conservation and management, and effective use of assisted reproductive techniques like artificial insemination, in vitro fertilization/embryo transfer depends on the knowledge of the basic reproductive physiology. In this context the reproductive status of female blackbucks (Antelope cervicapra L.) was assessed by behaviour and determination, sex steroid hormones in faeces and urinary volatile compounds. The male and female blackbucks exhibited as many as 31 different reproductive/courtship behaviour patterns. Particularly, the males showed a more extensive repertoire: i.e. 23 behavioural patterns by territorial males, 11 by bachelor males and 4 by females. The behaviours such as, mounting, Flehmen, clockwise and anticlockwise movements were significantly higher in male blackbuck when exposed to estrus. By contrast, such courtship behaviours were completely absent in male when exposed to diestrus. It clearly indicates that, the estrus female produces specific chemical cues (pheromone) through urine, which would involve in attracting the conspecifics. In addition, the average faecal oestrogen concentration was significantly higher (p<0.05) during the estrus faecal than the proestrus and diestrus periods. In contrast, the faecal progesterone concentration was significantly higher (p<0.05) during the diestrus faecal sample than that of proestrus and estrus faecal sample. Twenty-eight volatiles are identified, across the three reproductive phases (i.e. proestrus, estrus and diestrus) of sexually mature and prepubertal females. Amongst, the compounds 2-methyl-3-butyn-2-ol, 3,7-dimethylnonane, 3-phenyl-2-propen-1-ol and 2-hydroxybenzoic acid occurred only during estrus which may be considered as marker for detection of estrus which would ultimately help for artificial insemination in captive condition. The findings of the present study suggest that the non-invasive approaches like reproductive behaviours, faecal steroids and estrus-specific urinary volatiles could serve as good indicators for detection of estrus for blackbuck.

Urinary Volatile Compounds as Biomarkers for Lung Cancer

Lung cancer is a leading cause of deaths in cancer. Hence, developing early-stage diagnostic tests that are non-invasive, highly sensitive, and specific is crucial. In this study, we investigated to determine whether biomarkers derived from urinary volatile organic compounds (VOCs) can be used to discriminate between lung cancer patients and normal control patients. The VOCs were extracted from the headspace by solid-phase microextraction and were analyzed by gas chromatography time-of-flight mass spectrometry. Nine putative volatile biomarkers were identified as elevated in the lung cancer group. Receiver operating characteristic curve analysis was also performed, and the markers were found to be highly sensitive and specific. Next we used principal component analysis (PCA) modeling to make comparisons compare within the lung cancer group, and found that 2-pentanone may have utility in differentiating between adenocarcinoma and squamous cell carcinomas.

Identification of urinary sex pheromones in female buffaloes and their influence on bull reproductive behaviour

The present study was carried out to identify the urinary sex pheromones of buffaloes and their role in relation to bull reproductive behaviour. Urinary samples were collected from 12 buffalo heifers at four different phases of estrous cycles. Fourteen compounds were identified throughout the cycle, which included phenol, ketone, alkane, alcohol, amide, acid and aldehyde. Among the 14 chemical profiles, three compounds were only found on the day of estrus, viz. 1-chlorooctane, 4-methylphenol and 9-octadecenoic acid. Behavioural investigation clearly showed that bulls were attracted and exhibited repeated flehmen behaviour towards the 4-methyl phenol. The bulls displayed penile erection and mounting behaviour while exposed to 9-octadecenoic acid. However, the other compound, 1-chlorooctane, did not influence such sexual behaviours. The present results provide evidence that the estrus-specific urinary volatile compounds appear to be sex pheromones which initiate the bull’s reproductive behaviour.

Urinary Volatile Compounds as Biomarkers for Lung Cancer: A Proof of Principle Study Using Odor Signatures in Mouse Models of Lung Cancer

A potential strategy for diagnosing lung cancer, the leading cause of cancer-related death, is to identify metabolic signatures (biomarkers) of the disease. Although data supports the hypothesis that volatile compounds can be detected in the breath of lung cancer patients by the sense of smell or through bioanalytical techniques, analysis of breath samples is cumbersome and technically challenging, thus limiting its applicability. The hypothesis explored here is that variations in small molecular weight volatile organic compounds (“odorants”) in urine could be used as biomarkers for lung cancer. To demonstrate the presence and chemical structures of volatile biomarkers, we studied mouse olfactory-guided behavior and metabolomics of volatile constituents of urine. Sensor mice could be trained to discriminate between odors of mice with and without experimental tumors demonstrating that volatile odorants are sufficient to identify tumor-bearing mice. Consistent with this result, chemical analyses of urinary volatiles demonstrated that the amounts of several compounds were dramatically different between tumor and control mice. Using principal component analysis and supervised machine-learning, we accurately discriminated between tumor and control groups, a result that was cross validated with novel test groups. Although there were shared differences between experimental and control animals in the two tumor models, we also found chemical differences between these models, demonstrating tumor-based specificity. The success of these studies provides a novel proof-of-principle demonstration of lung tumor diagnosis through urinary volatile odorants. This work should provide an impetus for similar searches for volatile diagnostic biomarkers in the urine of human lung cancer patients.

Comparison of Urinary Scents of Two Related Mouse Species, Mus spicilegus and Mus domesticus

Whereas the house mouse (Mus domesticus) has been studied extensively in terms of physiology/behavior and pheromonal attributes, the evolutionarily related mound-building mouse (Mus spicilegus) has received attention only recently due to its divergent behavioral traits related to olfaction. To date, no chemical studies on urinary volatile compounds have been performed on M. spicilegus. The rationale for our investigations was to determine if there are differences in urinary volatiles of intact and castrated M. spicilegus males and to explore further whether this species could utilize the same or structurally similar pheromones as the male house mouse, M. domesticus. The use of capillary gas chromatography/mass spectrometry (GC-MS) together with sorptive stir bar extraction sampling enabled quantitative comparisons between the intact and castrated M. spicilegus urinary profiles. Additionally, through GC-MS and atomic emission (sulfur-selective) detection, we identified qualitative molecular differences between intact M. spicilegus and M. domesticus. A series of volatile and odoriferous lactones and the presence of coumarin were the unique features of M. spicilegus, as was the notable absence of 2-sec-butyl-4,5-dihydrothiazole (a prominent M. domesticus male pheromone) and other sulfur-containing compounds. Castration of M. spicilegus males eliminated several substances, including delta-hexalactone and gamma-octalactone, and substantially decreased additional compounds, suggesting their possible role in chemical communication. Some other M. domesticus pheromone components were also found in M. spicilegus urine. These comparative chemical analyses support the notion of metabolic similarities as well as the uniqueness of some volatiles for M. spicilegus, which may have a distinct physiological function in reproduction and behavior.

Urinary Volatile Profiles of the Deermouse (Peromyscus maniculatus) Pertaining to Gender and Age

Biological aspects of chemical communication in the deermouse have been extensively documented in the past. To provide a broad characterization of the urinary volatile compounds (potential chemosignals), the urine samples from adult and young animals of both sexes, as well as from castrated animals, were examined. The volatiles were extracted through a dynamic headspace technique and analyzed by combined capillary gas chromatography–mass spectrometry. The major constituents have been identified as various ketones, aldehydes, pyrazines, pyridines, quinolines, a thiazole, an amide, and a nitrile. While no chromatographic peaks were found to be unique to gender or age, quantitative analyses revealed that numerous compounds exhibited significant variations among the different groups. Their endocrine dependencies suggest a potential chemosignaling function.

Variations of equine urinary volatile compounds during the oestrous cycle.

Equine urine was analysed by capillary gas chromatography. The volatile profiles from oestrous and dioestrous samples were compared to establish any qualitative or quantitative difference that may have potential value in olfactory communication. Forty-five different volatile compounds were detected. Of these, 17 major compounds were common to all chromatograms. The chemical profile of oestrous urine was distinguished by the presence of a unique peak that was not present in dioestrous samples. Numerous constituents exhibited endocrine dependence: while the concentrations of seix peaks increased at oestrus, the concentrations of another five peaks decreased at the same time. Since oestrous urine, but not dioestrous urine, has been shown to elicit sexual behaviour in the stallion, the unique peak, together with the peaks that were present in increased concentration at oestrus, may represent important chemical signals that stallions use to detect urinary 'oestrous odours'. Statistical analyses also indicated that the relative ratios (normalized peak areas) of many peaks changed significantly across the oestrous cycle: the rations increased in nine peaks, decreased in six peaks, and remained constant in two peaks at oestrus.

Speculating about pyrazines

Of the various types of alerting signals found in nature, odours are the least well understood. The worldwide distribution of pyrazines in plants, insects, terrestrial vertebrates, marine organisms, fungi and bacteria suggests that they are of special significance. We speculate that these molecules served as natural points of convergence in the evolution of widespread alerting signals, which are used for differing but related intraspecific purposes by various species. In aposematic, self-advertising toxic insects and their mimics, for example, pyrazines function as additional warning signals; preliminary data indicates that their odour can potentiate taste aversion learning in rats and the associative learning of immune suppression in mice. The latter suggests that in addition to their alerting properties, pyrazine odours may act as ectohormones which interact with predator physiology.