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.

Microbiota and Malodor-Etiology and Management.

Accumulating evidence indicates that microbiota plays a critical role in physiological processes in humans. However, it might also contribute to body malodor by producing numerous odorous molecules such as ammonia, volatile sulfur compounds or trimethylamine. Although malodor is commonly overlooked by physicians, it constitutes a major problem for many otherwise healthy people. Thus, this review aims to investigate most common causes of malodor and describe potential therapeutic options. We searched PUBMED and Google Scholar databases to identify the clinical and pre-clinical studies on bad body smell, malodor, halitosis and microbiota. Unpleasant smell might originate from the mouth, skin, urine or reproductive fluids and is usually caused by odorants that are produced by resident bacterial flora. The accumulation of odorous compounds might result from diet, specific composition of microbiota, as well as compromised function of the liver, intestines and kidneys. Evidence-based guidelines for management of body malodor are lacking and no universal treatment exists. However, the alleviation of the symptoms may be achieved by controlling the diet and physical elimination of bacteria and/or accumulated odorants.

Higher Levels of Low Molecular Weight Sulfur Compounds and Homocysteine Thiolactone in the Urine of Autistic Children.

In this study, the levels of concentration of homocysteine thiolactone (HTL), cysteine (Cys), and cysteinylglycine (CysGly) in the urine of autistic and non-autistic children were investigated and compared. HTL has never been analyzed in autistic children. The levels of low molecular weight sulfur compounds in the urine of both groups were determined by validated methods based on high-performance liquid chromatography with spectrofluorometric and diode-array detectors. The statistical data show a significant difference between the examined groups. Children with autism were characterized by a significantly higher level of HTL (p = 5.86 × 10-8), Cys (p = 1.49 × 10-10) and CysGly (p = 1.06 × 10-8) in urine compared with the control group. A difference in the p-value of <0.05 is statistically significant. Higher levels of HTL, Cys, and CysGly in the urine of 41 children with autism, aged 3 to 17, were observed. The obtained results may indicate disturbances in the metabolism of methionine, Cys, and glutathione in some autistic patients. These preliminary results suggest that further research with more rigorous designs and a large number of subjects is needed.

Metabolism of sulfur compounds in homocystinurias.

BACKGROUND AND PURPOSE: Homocystinurias are rare genetic defects characterized by altered fluxes of sulfur compounds including homocysteine and cysteine. We explored whether the severely perturbed sulfur amino acid metabolism in patients with homocystinurias affects the metabolism of hydrogen sulfide. EXPERIMENTAL APPROACH: We studied 10 treated patients with a block in the conversion of homocysteine to cysteine due to cystathionine ß-synthase deficiency (CBSD) and six treated patients with remethylation defects (RMD) and an enhanced flux of sulfur metabolites via transsulfuration. Control groups for CBSD and RMD patients consisted of 22 patients with phenylketonuria on a low-protein diet and of 12 healthy controls respectively. Plasma and urine concentrations of selected sulfur compounds were analysed by HPLC and LC-MS/MS. KEY RESULTS: Patients with CBSD exhibited plasma concentrations of monobromobimane-detected sulfide similar to appropriate controls. Urinary homolanthionine and thiosulfate in CBSD were increased significantly 1.9 and 3 times suggesting higher hydrogen sulfide synthesis by γ-cystathionase and detoxification respectively. Surprisingly, patients with RMD had significantly lower plasma sulfide levels (53 and 64% of controls) with lower sulfite concentrations, and higher taurine and thiosulfate levels suggesting enhanced cysteine oxidation and hydrogen sulfide catabolism respectively. CONCLUSION AND IMPLICATIONS: The results from this study suggest that severe inherited defects in sulfur amino acid metabolism may be accompanied by only moderately perturbed hydrogen sulfide metabolism and lends support to the hypothesis that enzymes in the transsulfuration pathway may not be the major contributors to the endogenous hydrogen sulfide pool. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Applications of silver nanoparticles capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight and atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry for rapid analysis of sulfur drugs and biothiols in human urine.

A strategy is presented for the analysis of sulfur drugs and biothiols using silver nanoparticles (AgNPs) capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) and atmospheric pressure-matrix assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI-ITMS). Biothiols adsorbed on the surface of AgNPs through covalent bonding were subjected to ultraviolet (UV) radiation that enabled desorption and ionization due to the excellent photochemical property of NPs. The proposed method has been successfully applied for the determination of cysteine and homocysteine in human urine samples using an internal standard. The limit of detection (LOD) and limit of quantification (LOQ) for cysteine and homocysteine in urine sample are 7 and 22 nM, respectively, with a relative standard deviation (RSD) of <10%. The advantages of the present method compared with the methods reported in the literature for biothiol analysis are simplicity, rapidity and sensitivity without the need for time-consuming separation and tedious preconcentration processes. Additionally, we also found that the bare AgNPs can be directly used as the matrix in MALDI-TOF MS for the analysis of sulfur drugs without the addition of an extra proton source.

Detection and quantification of glucuro- and sulfoconjugated metabolites in human urine following oral administration of xenobiotic 19-norsteroids.

Recently, the endogenous origin of nandrolone (19-nortestosterone) and other 19-norsteroids has been a focus of research in the field of drug testing in sport. In the present study, we investigated metabolites conjugated to a glucuronic acid and to a sulfuric acid in urine following administration of four xenobiotic 19-norsteroids. Adult male volunteers administered a single oral dose (10 mg) of each of four 19-norsteroids. Urinary samples collected from 0 to 120 h were subjected to methanolysis and beta-glucuronidase hydrolysis and were derivatized by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) before gas chromatography-mass spectrometry analysis. We confirmed that 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) were present in both glucuronide (g) and sulfate (s) conjugates and 19-norepiandrosterone (19-NEA) was excreted exclusively as a sulfate fraction in urine of all 19-norsteroids tested. The overall levels of the three metabolites can be ranked as follows: 19-NA(g+s)>19-NE(g+s)>19-NEA(s). The concentration profiles of these three metabolites in urine peaked between 2 to 12h post-administration and declined thereafter until approximately 72-96 h. 19-NA was most prominent throughout the first 24 h post-administration, except for a case in which an inverse relationship was found after 6h post-administration of nandrolone. Furthermore, we found that sulfate conjugates were present in both 19-NA and 19-NE metabolites in urine of all 19-norsteroids tested. The averaged total amounts of metabolites (i.e. 19-NA(s+g)+19-NE(s+g)+19-NEA(s)) excreted in urine were 38.6, 42.9, 48.3 and 21.6% for nandrolone, 19-nor-4-androsten-3,17-dione, 19-nor-4-androsten-3beta,17beta-diol and 19-nor-5-androstene-3beta,17beta-diol, respectively. Results from the excretion studies demonstrate significance of sulfate-conjugated metabolites on interpretation of misuse of the 19-norsteroids.

Sulfur-containing arsenical mistaken for dimethylarsinous acid [DMA(III)] and identified as a natural metabolite in urine: major implications for studies on arsenic metabolism and toxicity.

It is vital that methylated trivalent arsenicals [MA(III) and DMA(III)] are described and characterized unequivocally due to their high toxicity. Two different ways of generating the methylated trivalent arsenicals have been practiced-reduction of the methylated pentavalent arsenical either by the sodium-metabisulfite (Na(2)S(2)O(5))/sodium thiosulfate (Na(2)S(2)O(3)) reagent (method A) or by KI, H(2)SO(4), and SO(2) (method B). The shared identity between the products of the two synthetic methods has never been questioned or proven. Here, we characterize and identify the arsenic species formed when reducing DMA(V) by method A or B. Dimethylarsinous acid [DMA(III)] was formed when reducing DMA(V) by method B, but DMA(III) was not the main product of the reaction by method A. The product was revealed by HPLC-ICP-MS coupled simultaneously to HPLC-ES-MS and ES-Q-TOF-MS to have the molecular formula C(2)H(7)OSAs. The structure was further confirmed by (1)H NMR, and ab initio tautomeric energy calculations showed it to be present as Me(2)As(=S)OH (dimethylarsinothioic acid). Dimethylarsinothioic acid was also identified as a metabolite in urine and in wool extract from sheep naturally consuming large amounts of arsenosugars (35 mg of As daily) through their major food source, seaweed.

Profiling of sulfoconjugates in urine by using precursor ion and neutral loss scans in tandem mass spectrometry. Application to the investigation of heavy metal toxicity in rats.

This paper reports a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method for profiling a wide range of structurally different sulfoconjugated compounds in urine and its application to the characterization of biomarkers for heavy metal toxicity in rat urine. Sulfoconjugates were first isolated by solid-phase extraction and the LC separation was performed on a reversed-phase column. Sulfoconjugates were detected in a triple-quadrupole mass spectrometer by simultaneously monitoring constant losses of 80 u (or 80 Th for doubly charged ions), precursors of m/z 80 (SO(3) (-*)) and precursors of m/z 97 (HSO4-). The ESI-MS detection conditions were optimized on dehydroepiandrosterone sulfate and estradiol sulfate and tested on other sulfoconjugates. The analysis of urine samples from humans and rats by using the developed method allowed the detection of about 15 peaks in each mode of detection. It was then applied to the investigation of heavy metal toxicity in rats. Comparative analysis of the chromatographic fingerprints of urine from control and uranium- and cadmium-treated rats showed several variations in the chromatographic pattern of the sulfoconjugates. Diagnostic m/z ratios were confirmed by analyzing individual urine samples and one of the observed variations seemed to be specific to uranium toxicity. The ion responsible for this variation has been identified as 4-ethylphenol sulfate by comparison of its chromatographic retention time and collision-induced dissociation mass spectra (MS(2) and MS(3) performed on a quadrupole ion trap instrument) with those of the synthesized compound.

Urinary sulfur-containing metabolite produced by intestinal bacteria following oral administration of dimethylarsinic acid to rats.

Our long-term oral administration of dimethylarsinic acid (DMAV) in rats revealed that three unidentified metabolites, M-1, M-2, and M-3, were detected in urine and feces. DMAV and trimethylarsine oxide (TMAO) were converted to M-2 and M-3 and M-1 by Escherichia coli strain A3-6 isolated from the ceca of DMAV-administered rats, respectively. In this study, we report on the mechanism of production and the chemical properties of these unknown metabolites. To investigate the pattern of conversion of DMAV or TMAO by A3-6 in the presence of cysteine (Cys), arsenic metabolites of DMAV or TMAO in medium after incubation with A3-6 and Cys were analyzed by liquid chromatography with inductively coupled plasma mass spectrometry (LC-ICP-MS). DMAV was reduced to dimethylarsinous acid (DMAIII) to form M-2 in the presence of Cys and A3-6, and M-2 was further converted to M-3. TMAO was rapidly converted to M-1 by A3-6. The cytotoxicity of the unidentified metabolites was investigated. M-2 was more cytotoxic than DMAV, M-1, and M-3 in V79 cells. The cytotoxicity of M-2 in HL-60 cells was decreased by the addition of superoxide dismutase, suggesting that the cytotoxicity of M-2 might be due to the production of reactive oxygen species. In addition, we examined the chemical properties of M-2 by LC-ICP-MS and LC-MS. M-2 was oxidized to DMAV by hydrogen peroxide, suggesting that M-2 may be a reduced form of DMAV. M-2 was consistent with the reactant of DMAV with metabisulfite-thiosulfate reagent but not DMAIII by analyses of LC-ICP-MS and LC-MS. The molecular weight of M-2 was 154, and M-2 was a sulfur-containing metabolite.

Molybdenum cofactor deficiency associated with Dandy-Walker complex.

Molybdenum cofactor deficiency is a rare and devastating disease leading to intractable seizures in the neonatal period. Severe loss of neocortical neurons, gliosis, and cystic necrosis of cerebral white matter resulting in significant cerebral volume loss are the neuropathological findings. The mechanism of cerebral injury is unknown, but sulphite excess, and sulphate or uric acid deficiencies are possible factors. We present here a new case of Molybdenum cofactor deficiency associated with Dandy-Walker complex with a history of three dead siblings, the latter also having Dandy-Walker malformation. We speculate that severe cerebral volume loss due to the above mentioned mechanisms may lead to an appearance resembling Dandy-Walker malformation.

The fate of diphenyl sulphide, diphenyl sulphoxide and diphenyl sulphone in the rat.

Radiolabelled [UL-14C]-diphenyl sulphide, [UL-14C]-diphenyl sulphoxide and [UL-14C]-diphenyl sulphone were administered by gavage (1.0 mmol/kg body weight) to adult male Wistar rats following an overnight fast. For all compounds, faeces were the major route of excretion of radioactivity (50%). Urinary elimination (40%) was similar during the first (19%) and second (16%) days and a small amount of radioactivity (6%) was found within the carcass after four days. From urinary and faecal data, metabolism occurred via ring hydroxylation with subsequent conjugate formation. Oxidation of the sulphur to form the sulphoxide and sulphone also took place; a small amount of sulphoxide reduction was apparent but no sulphone reduction was found. No evidence for exclusion of the sulphur was obtained, and it appeared unlikely that extensive cleavage of the ring structures occurred.

Differentiation of mouth versus gut as site of origin of odoriferous breath gases after garlic ingestion.

Utilizing the sulfur-containing gases of garlic as probes, we investigated the gut versus mouth origin of odoriferous breath gases. Five individuals ingested 6 g of garlic, and sulfur gases in mouth, alveolar air, and urine samples were measured. The mouth normally contained low concentrations of hydrogen sulfide, methanethiol, and dimethyl sulfide. Immediately after garlic ingestion, transient high concentrations of methanethiol and allyl mercaptan and lesser concentrations of allyl methyl sulfide (AMS), allyl methyl disulfide, and allyl disulfide were observed. With the exception of AMS, all gases were present in far greater concentrations in mouth than alveolar air, indicating an oral origin. Only AMS was of gut origin as evidenced by similar partial pressures in mouth, alveolar air, and urine. After 3 h, AMS was the predominant breath sulfur gas. The unique derivation of AMS from the gut is attributable to the lack of gut and liver metabolism of this gas versus the rapid metabolism of the other gases. Breath odor after garlic ingestion initially originates from the mouth and subsequently from the gut.