Metabolic Evaluation of Urine from Patients Diagnosed with High Grade (HG) Bladder Cancer by SPME-LC-MS Method.

Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study's results may support a better understanding of bladder cancer development and progression mechanisms.

Metabolomic biomarkers in urine of rats following long-term low-dose exposure of cadmium and/or chlorpyrifos.

Heavy metals and pesticides can be easily enriched in food chains and accumulated in organisms, thus pose significant threat to human health. However, their combined effects for long-term exposure at low dose has not been thoroughly investigated; especially there was no biofluid biomarker available to noninvasively diagnose the toxicosis of the combined exposure of the two chemicals at their low levels. In this study, we investigated the change of urine metabolites of rats with 90-day exposure to heavy metal cadmium (Cd) and/or organophosphorus pesticide chlorpyrifos (CPF) using gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach. Our results showed that the interaction of Cd and CPF mainly displayed an antagonistic effect. We identified the panels of metabolite biomarkers in urine: benzoic acid and mannose were unique biomarkers for Cd exposure; creatinine and N-phenylacetyl glycine were unique biomarkers for CPF exposure; anthranilic acid, ribitol, and glucose were unique biomarkers for Cd plus CPF exposure. Our results suggest that 90-day exposure to Cd and/or CPF could cause a disturbance in energy and amino acid metabolism. And urine metabolomics analysis can help understand the toxicity of low dose exposure to mixed environmental chemicals.

Urinary NMR-based metabolomic analysis of rats possessing variable sperm count following orally administered Eurycoma longifolia extracts of different quassinoid levels.

ETHNOPHARMACOLOGICAL RELEVANCE: Eurycoma longifolia (Tongkat Ali, TA) roots have been ethnically used as a remedy to boost male sexual desire, libido, energy and fertility. AIM OF THE STUDY: The study evaluated the effect of TA extracts with different quassinoid levels on rats sperm count and examined corresponding post-treatment urinary metabolic changes. MATERIALS AND METHODS: Twenty-four male Sprague-Dawley rats, categorized into 4 groups of 6 rats each, were orally administered for 48 days with water for the control (group 1), 125mg/kg of TA water extract (TAW, group 2), 125mg/kg of TA quassinoid-poor extract (TAQP, group 3) and 21mg/kg of TA quassinoid-rich extract (TAQR, group 4). Upon completion of the 48-day treatment, the urine samples were analyzed by NMR and the animals were subsequently sacrificed for sperm count analysis. The urine profiles were categorized according to sperm count level. RESULTS: The results showed that the sperm count in TAW- and TAQR-treated groups was significantly higher compared to the TAQP-administered and control groups. The orthogonal partial least squares discriminant analysis (OPLS-DA) model indicated a clear separation among the urine profiles with respect to sperm count level. Urine (1)H-NMR profiles of the high-sperm count group contained higher concentrations of trigonelline, alanine, benzoic acid and higher intensity of a signal at 3.42ppm, while ethanol was at higher concentration in the normal-sperm count group. CONCLUSIONS: The results proved the efficacy of quassinoids on sperm count increase in rats and provided quantitative markers in urine suitable for analysis of sperm profile and male fertility status.

Systemic exposure to benzoic acid and hippuric acid following topical application of clindamycin 1%/benzoyl peroxide 3% fixed-dose combination gel in Japanese patients with acne vulgaris.

Clindamycin 1%/benzoyl peroxide 3% fixed-dose combination gel (CLDM/BPO3%) is a topical product for the treatment of acne vulgaris. In this study, plasma and urine concentrations of benzoic acid (BA) and hippuric acid (HA) were analyzed to estimate the pharmacokinetics (PK) of BPO after application of CLDM/BPO3% twice-daily for 7 days in Japanese patients with acne vulgaris. Seven-day repeated application of CLDM/BPO3% appears to be safe in this patient population. Concentrations of plasma and urine BA were below the limit of quantification before and after repeated application in most of the 12 adult male patients. Mean difference in Cmax and AUC0-last for plasma HA indicated increased exposures after repeated application, but with wide 90% confidence intervals. Mean Ae0-12 for urine HA was similar before and after repeated application. Repeated application of CLDM/BPO3% is thus unlikely to result in accumulation of BA and HA. The study suggests negligible systemic exposure to BPO metabolites from CLDM/BPO3% after 7-day repeated application in male patients with acne vulgaris.

Metabolic profiling of phenolic acids and oxidative stress markers after consumption of Lonicera caerulea L. fruit.

This study investigated the effect of one-week consumption of 165 g/day fresh blue honeysuckle berries (208 mg/day anthocyanins) in 10 healthy volunteers. At the end of intervention, levels of benzoic (median 1782 vs 4156), protocatechuic (709 vs 2417), vanillic (2779 vs 4753), 3-hydroxycinnamic (143 vs 351), p-coumaric (182 vs 271), isoferulic (805 vs 1570), ferulic (1086 vs 2395), and hippuric (194833 vs 398711 µg/mg creatinine) acids by LC/MS were significantly increased in the urine. Clinical chemistry safety markers were not altered. Oxidative stress markers, erythrocyte glutathione peroxidase (0.73 vs 0.88 U/g Hb) and catalase (2.5 vs 2.8 µkat/g Hb) activities, and erythrocyte/plasma thiobarbituric acid reactive substance (522 vs 612/33 vs 38 µmol/g Hb/protein) levels were significantly increased, without change in plasma antioxidant status. Nonsignificant changes of advanced oxidation protein products and oxidized LDL were observed. The results provide a solid base for further study of metabolite excretion and antioxidant parameters after ingestion of anthocyanins.

[Exploring the mechanism of rhizoma coptidis in treating type II diabetes mellitus based on metabolomics by gas chromatography-mass spectrometry].

Metabolomics was used to explore the mechanism of Rhizoma coptidis in treating type II diabetes mellitus. The rat model of type II diabetes mellitus was constructed by an injection of streptozocin (40 mg/kg), along with diets of fat emulsion. The rats were divided into four groups, the control group, the model group, the Rhizoma coptidis group (10 g/kg) and the metformin group (0.08 g/kg). After the treatment for 30 d, blood samples were collected to test biomedical indexes, and 24 h urine samples were collected for the metabolomics experiment. In the Rhizoma coptidis group, fasting blood glucose (FBG), total cholesterol (TC) and total plasma triglycerides (TG) were significantly decreased by 59.26%, 58.66% and 42.18%, respectively, compared with those in the model group. Based on gas chromatography-mass spectrometry, a urinary metabolomics method was used to study the mechanism of Rhizoma coptidis in treating diabetes mellitus. Based on the principal component analysis, it was found that the model group and control group were separated into two different clusters. The Rhizoma coptidis group was located between the model group and the control group, closer to the control group. Twelve significantly changed metabolites of diabetes mellitus were detected and identified, including 4-methyl phenol, benzoic acid, aminomalonic acid, and so on. After diabetic rats were administered with Rhizoma coptidis, 7 metabolites were significantly changed, and L-ascorbic acid and aminomalonic acid which related with the oxidative stress were significantly regulated to normal. The pharmacological results showed that Rhizoma coptidis could display anti-hyperglycemic and anti-hyperlipidemic effects. The Rhizoma coptidis had antioxidation function in preventing the occurrence of complications with diabetes mellitus to some extent. The work illustrates that the metabolomics method is a useful tool to study the treatment mechanism of traditional Chinese medicine.

Simultaneous determination of aromatic acid metabolites of styrene and styrene-oxide in rat urine by gas chromatography-flame ionization detection.

A convenient and reliable gas chromatographic method was developed for the simultaneous determination of six aromatic acid metabolites of styrene and styrene-oxide in rat urine; i.e., benzoic (BA), phenylacetic (PAA), mandelic (MA), phenylglyoxylic (PGA), hippuric (HA) and phenylaceturic (PAUA) acids. The method involves a one-pot esterification-extraction procedure, performed directly on urine without prior treatment. Analyses were performed on a RTX-1701 capillary column and the recovered isopropyl esters derivatives were detected by flame ionization detection. The analytical method was validated for selectivity, linearity, detection and quantification limits, recovery and intra-day and inter-day precisions. Calibration curves showed linearity in the range of 8-800 mg/L, except for HA and PAUA (40-800 mg/L). Limits of detection were between 0.2 (PPA) and 7.0 (PAUA) mg/L. The intra-day precisions determined at three concentrations levels were less than 5% for BA, PAA, MA and PGA and 9% for HA and PAUA, respectively. The corresponding mean inter-day precisions for these two groups were 8 and 16%, respectively. The method was successfully applied to quantitatively analyze styrene, styrene-oxide, ethylbenzene and toluene metabolites in urine samples from rats exposed by inhalation to these compounds at levels close to the occupational threshold limit values. Provided that this method can be transposed to human urine, it could have applications as part of biological monitoring for workers exposed to styrene or related compounds.

Detection of carboxylic acids and inhibition of hippuric acid formation in rats treated with 3-butene-1,2-diol, a major metabolite of 1,3-butadiene.

Epidemiological studies have indicated that 1,3-butadiene exposure is associated with an increased risk of leukemia. In human liver microsomes, 1,3-butadiene is rapidly oxidized to butadiene monoxide, which can then be hydrolyzed to 3-butene-1,2-diol (BDD). In this study, BDD and several potential metabolites were characterized in the urine of male B6C3F1 mice and Sprague-Dawley rats after BDD administration (i.p.). Rats given 1420 micromol kg(-1) BDD excreted significantly greater amounts of BDD relative to rats administered 710 micromol kg(-1) BDD. Rats administered 1420 or 2840 micromol kg(-1) BDD excreted significantly greater amounts of BDD per kilogram of body weight than mice given an equivalent dose. Trace amounts of 1-hydroxy-2-butanone and the carboxylic acid metabolites, crotonic acid, propionic acid, and 2-ketobutyric acid, were detected in mouse and rat urine after BDD administration. Because of the identification of the carboxylic acid metabolites and because of the known ability of carboxylic acids to conjugate coenzyme A, which is critical for hippuric acid formation, the effect of BDD treatment on hippuric acid concentrations was investigated. Rats given 1420 or 2272 micromol kg(-1) BDD had significantly elevated ratios of benzoic acid to hippuric acid in the urine after treatment compared with control urine. However, this effect was not observed in mice administered 1420 or 2840 micromol kg(-1) BDD. Collectively, the results demonstrate species differences in the urinary excretion of BDD and show that BDD administration in rats inhibits hippuric acid formation. The detection of 1-hydroxy-2-butanone and the carboxylic acids also provides insight regarding pathways of BDD metabolism in vivo.

Hippuric acid as a modifier of calcium oxalate crystallisation.

Hippuric acid (HA) originating from the conjugation of benzoic acid with glycine is a physiological component of human urine. Findings suggest that HA inhibits calcium oxalate (CaOx) growth and considerably enhances the CaOx solubility in artificial urine. Thus, it is assumed that HA is a major modifier of CaOx formation. However, only a slight CaOx growth inhibition of 1-8% was also reported. These values were also derived from artificial urine. The key mechanism, which led HA to be of interest in urolithiasis research is the fact that in presence of Ca2+ ions HA can form a hippurate complex. By forming such a complex, Ca2+ concentration in urine decreases, and as a consequence, CaOx formation is inhibited. This study was performed in order to clarify the role of HA in native and artificial urine. Biochemical analyses to calculate the relative CaOx supersaturations and crystallisation experiments using an in-line laser probe were examined. BONN Risk Indices indicating the risk of CaOx crystallisation were calculated from the results of the crystallisation experiments. The results obtained from artificial as well as from native urines showed that HA has no significant effects on CaOx formation. We suggest that HA plays only a minor role as a crystallisation modifier in human urine.

Differences in excretion of hippurate, as a metabolite of benzoate and as an administered species, in the single-pass isolated perfused rat kidney explained.

The extents of excretion of [14C]hippurate and [3H]hippurate were compared in the single-pass, constant flow (8 ml/min) isolated rat kidney which was perfused simultaneously with tracer concentrations of [14C]benzoate and [3H]hippurate. The steady-state renal extraction ratio of [14C]benzoate was 0.26 +/- 0.04 and was associated with a renal clearance of 1.13 +/- 0.17 ml/min/g. The urinary clearance of [14C]benzoic acid was low (0.011 +/- 0.01 ml/min/g), yielding a low fractional excretion [unbound urinary clearance/glomerular filtration rate (GFR)] value of 0.27 +/- 0.19 and suggesting that glycination of [14C]benzoate to [14C]hippurate accounted almost completely for the total renal clearance. Fractional excretion for preformed [3H]hippurate was eight times that of GFR, but the steady-state renal extraction ratio of preformed [3H]hippurate, E(pmi) (0.24 +/- 0.05) was much lower than the apparent extraction ratio of the renally formed [14C]hippuric acid [E(mi) = 0.39 +/- 0.09] (p <.05). The theoretical basis for the discrepancy was explored with mathematical formulations developed from a physiologically based model of the kidney. It was found that parent drug kinetic parameters (transport and metabolic intrinsic clearance of benzoate) were unimportant for E(mi) or E(pmi). Rather, the value of EK(mi) exceeded EK(pmi) because of the ratio of efflux clearances at the basolateral and luminal membranes for hippurate [corrected] was less than 26.089, a value determined by the GFR, plasma renal flow, and the unbound fraction of hippurate of the system that would render E(mi) to equal E(pmi) in the system. The influx clearance for hippurate to enter from plasma to cell at the basolateral membrane and the reabsorption clearance of hippurate to enter from tubular urine to cell at the luminal membrane failed to alter the ratio of EK(pmi)/EK(mi).

Valores de referência do ácido hipúrico urinário na regiäo metropolitana de Belo Horizonte / Urinary hippuric acid reference values in Belo Horizonte metropolitan area, Brazil

Objetivando estabelecer a faixa de valor referência para o ácido hipúrico na regiäo metropolitana de Belo Horizonte, urinas de indivíduos näo expostos ocupacionalmente ao tolueno foram analisadas por cromatografia em fase gasosa, utilizando-se detector de ionizaçäo de chama. Os valores encontrados experimentalmente (n=281) variaram de <0,1 a 2,79 g/L, mas a aplicaçäo de estudo estatístico delimita como faixa de referência,a nível de significância de 95 porcento, os valores de 0,361 a 0,481 g/L. Säo descritas, também, as condiçöes analíticas padronizadas para a determinaçäo cromatográfica do ácido hipúrico urinário

Effect of p-nitro-substitution on the metabolism of benzoic acid in the Indian fruit bat.

The introduction of a nitro-group at the 4-position of benzoic acid does not alter remarkably the metabolic pattern of benzoic acids in the fruit bat. It is shown here that the Indian fruit bat does not form hippuric acid with nitro benzoic acid, the main conjugate being the glucuronide in addition to other conjugates suspected to be p-amino benzoylglutamic acid and p-nitro benzoylglutamic acid respectively. More unmetabolized nitrobenzoic acid is present in the 24hr. urine than reported for unsubstituted benzoic acid. The Indian fruit bat exhibits an appreciable ability to reduce the nitrogroup in nitrobenzoic acid to the amino compound and to acetylate the amino group to the acetamido compound.