Urinary Metabolic Pattern Research Articles

Urine and serum metabolomic analysis of endometrial cancer diagnosis and classification based on ultra-performance liquid chromatography mass spectrometry.

This study aimed to reveal the urinary and serum metabolic pattern of endometrial cancer (EC) and establish diagnostic models to identify EC from controls, high-risk from low-risk EC, and type II from type I EC. This study included 146 EC patients (comprising 79 low-risk and 67 high-risk patients, including 124 type I and 22 type II) and 59 controls. The serum and urine samples were analyzed using ultraperformance liquid chromatography mass spectrometry. Analysis was used to elucidate the distinct metabolites and altered metabolic pathways. Receiver operating characteristic (ROC) analyses were employed to discover and validate the potential biomarker models. Serum and urine metabolomes displayed significant differences between EC and controls, with metabolites related to amino acid and nicotinamide metabolisms. The serum and urine panels distinguished these two groups with Area Under the Curve (AUC) of 0.821 and 0.902, respectively. The panel consisting of serum and urine metabolites demonstrated the best predictive ability (AUC = 0.953 and 0.976 in discovering and validation group). In comparing high-risk and low risk EC, differential metabolites were enriched in purine and glutamine metabolism. The AUC values for serum and urine panels were 0.818, and 0.843, respectively. The combined panel exhibited better predictive accuracy (0.881 in discovering group and 0.936 in external validation). In the comparison between type I and type II group, altered folic acid metabolism was identified. The serum, urine and combined panels discriminated these two groups with the AUC of 0.829, 0.913 and 0.922, respectively. The combined urine and serum metabolome effectively revealed the metabolic patterns in EC patients, offering valuable diagnostic models for EC diagnosis and classification.

Investigation of Urinary Exosome Metabolic Patterns in Membranous Nephropathy by Titania‐Assisted Intact Exosome Mass Spectrometry

Exosomes are regarded as the emerging potential targets for liquid biopsy and bioprocess study owing to their abundant inclusive cargos that carry significant disease information. In addition, metabolites have been promising biomarkers for diagnosis. However, little metabolic research on exosomes is carried out by now. Herein, the mix‐crystal titania‐assisted laser desorption/ionization mass spectrometry (LDI‐MS) method is established, which features fast speed, high throughput, and efficiency, to directly extract urinary exosome metabolic patterns of healthy controls (HC) and membrane nephropathy (MN) patients. Besides, this method is also adopted to acquire the primitive urinary metabolic patterns from the same samples for comparison. By taking advantage of principal component analysis, unpaired parametric t‐test, and orthogonal partial least squares discriminant analysis on the exosome metabolic patterns, 27 significant m/z signals are filtrated, which possess more prominent differentiation capacity toward HC and MN patients (AUC = 0.942), and hold greater potential in MN diagnosis, compared to primitive urine (AUC = 0.801). The work reveals the important clinical value of exosome metabolic analysis, and paves a way to exosome‐based diagnosis at metabolomic level toward large‐scale clinical use.

Noninvasive monitoring of evolving urinary metabolic patterns in neonatal encephalopathy.

Infants with moderate and severe neonatal encephalopathy (NE) frequently suffer from long-term adverse outcomes. We hypothesize that the urinary metabolome of newborns with NE reflects the evolution of injury patterns observed with magnetic resonance imaging (MRI). Eligible patients were newborn infants with perinatal asphyxia evolving to NE and qualifying for therapeutic hypothermia (TH) included in the HYPOTOP trial. MRI was employed for characterizing brain injury. Urine samples of 55 infants were collected before, during, and after TH. Metabolic profiles of samples were recorded employing three complementary mass spectrometry-based assays, and the alteration of detected metabolic features between groups was assessed. The longitudinal assessment revealed significant perturbations of the urinary metabolome. After 24 h of TH, a stable disease pattern evolved characterized by the alterations of 4-8% of metabolic features related to lipid metabolism, metabolism of cofactors and vitamins, glycan biosynthesis and metabolism, amino acid metabolism, and nucleotide metabolism. Characteristic metabolomic fingerprints were observed for different MRI injury patterns. This study shows the potential of urinary metabolic profiles for the noninvasive monitoring of brain injury of infants with NE during TH. A comprehensive approach for the study of the urinary metabolome was employed involving a semi-targeted capillary electrophoresis-time-of-flight mass spectrometry (TOFMS) assay, an untargeted ultra-performance liquid chromatography (UPLC)-quadrupole TOFMS assay, and a targeted UPLC-tandem MS-based method for the quantification of amino acids. The longitudinal study of the urinary metabolome identified dynamic metabolic changes between birth and until 96 h after the initiation of TH. The identification of altered metabolic pathways in newborns with pathologic MRI outcomes might offer the possibility of developing noninvasive monitoring approaches for personalized adjustment of the treatment and for supporting early outcome prediction.

Gut microbiota-mediated xanthine metabolism is associated with resistance to high-fat diet-induced obesity

Resistance to high-fat diet-induced obesity (DIR) has been observed in mice fed a high-fat diet and may provide a potential approach for anti-obesity drug discovery. However, the metabolic status, gut microbiota composition, and its associations with DIR are still unclear. Here, ultraperformance liquid chromatography-tandem mass spectrometry-based urinary metabolomic and 16S rRNA gene sequencing-based fecal microbiome analyses were conducted to investigate the relationship between metabolic profile, gut microbiota composition, and body weight of C57BL/6J mice on chow or a high-fat diet for 8 weeks. PICRUSt analysis of 16S rRNA gene sequences predicted the functional metagenomes of gut bacteria. The results demonstrated that feeding a high-fat diet increased body weight and fasting blood glucose of high-fat diet-induced obesity (DIO) mice and altered the host-microbial co-metabolism and gut microbiota composition. In DIR mice, high-fat diet did not increase body weight while fasting blood glucose was increased significantly compared to chow fed mice. In DIR mice, the urinary metabolic pattern was shifted to a distinct direction compared to DIO mice, which was mainly contributed by xanthine. Moreover, high-fat diet caused gut microbiota dysbiosis in both DIO and DIR mice, but in DIR mice, the abundance of Bifidobacteriaceae, Roseburia, and Escherichia was not affected compared to mice fed a chow diet, which played an important role in the pathway coverage of FormylTHF biosynthesis I. Meanwhile, xanthine and pathway coverage of FormylTHF biosynthesis I showed significant positive correlations with mouse body weight. These findings suggest that gut microbiota-mediated xanthine metabolism correlates with resistance to high-fat DIO.

An NMR-Based Approach to Identify Urinary Metabolites Associated with Acute Physical Exercise and Cardiorespiratory Fitness in Healthy Humans-Results of the KarMeN Study.

Knowledge on metabolites distinguishing the metabolic response to acute physical exercise between fit and less fit individuals could clarify mechanisms and metabolic pathways contributing to the beneficial adaptations to exercise. By analyzing data from the cross-sectional KarMeN (Karlsruhe Metabolomics and Nutrition) study, we characterized the acute effects of a standardized exercise tolerance test on urinary metabolites of 255 healthy women and men. In a second step, we aimed to detect a urinary metabolite pattern associated with the cardiorespiratory fitness (CRF), which was determined by measuring the peak oxygen uptake (VO2peak) during incremental exercise. Spot urine samples were collected pre- and post-exercise and 47 urinary metabolites were identified by nuclear magnetic resonance (NMR) spectroscopy. While the univariate analysis of pre-to-post-exercise differences revealed significant alterations in 37 urinary metabolites, principal component analysis (PCA) did not show a clear separation of the pre- and post-exercise urine samples. Moreover, both bivariate correlation and multiple linear regression analyses revealed only weak relationships between the VO2peak and single urinary metabolites or urinary metabolic pattern, when adjusting for covariates like age, sex, menopausal status, and lean body mass (LBM). Taken as a whole, our results show that several urinary metabolites (e.g., lactate, pyruvate, alanine, and acetate) reflect acute exercise-induced alterations in the human metabolism. However, as neither pre- and post-exercise levels nor the fold changes of urinary metabolites substantially accounted for the variation of the covariate-adjusted VO2peak, our results furthermore indicate that the urinary metabolites identified in this study do not allow to draw conclusions on the individual’s physical fitness status. Studies investigating the relationship between the human metabolome and functional variables like the CRF should adjust for confounders like age, sex, menopausal status, and LBM.

Monosodium Glutamate (MSG) Renders Alkalinizing Properties and Its Urinary Metabolic Markers of MSG Consumption in Rats.

Monosodium glutamate (MSG) is widely used as a flavor enhancer and its effects on human health are still debated. We aimed to investigate whether MSG can act as alkalinizing agent in murine models and if its metabolites are biomarkers of MSG consumption. For this purpose, adult male Wistar rats were given water added with 1 g% MSG or three types of control water, including sodium chloride (NaCl) and sodium bicarbonate (NaHCO3). At 14 days, urinary pH, electrolytes, urinary metabolites and ion-exchanger gene expression were determined. The results revealed that MSG-treated rats had significantly more alkaline urine and higher levels of urinary sodium and bicarbonate similar to NaHCO3 controls. These changes correlated with a lower expression of ion-exchanger genes, namely, CAII, NBC1, and AE1, which are involved in bicarbonate kidney reabsorption. The urinary metabolic profiles also revealed similar patterns for the MSG and NaHCO3 groups. In conclusion, MSG exhibits similar properties to NaHCO3, an alkalinizing agent, with regard to inducing alkaline urine, reducing bicarbonate kidney reabsorption, and generating a specific urinary metabolic pattern. We believe that these observations will be useful to further study the MSG effects in humans.

The concomitant lower concentrations of vitamins B6, B9 and B12 may cause methylation deficiency in autistic children

Autism spectrum disorder (ASD) is characterized by severe and persistent difficulties in social communication and social interaction at multiple levels. Recently, metabolic disorders have been associated with most cases of patients with ASD. The aim of this study was to investigate, through a new and more sophisticated mass technique, such as UHPLC-mass spectrometry (Q-exactive analyzer), alteration in metabolisms analyzing ASD children urine samples from children showing simultaneous vitamin B6, B9 and B12 deficiencies. This in order to study how these concurrent deficiencies may influence some phenotypic aspects of autistic disorder. Thus, urinary metabolic patterns specific to ASD were explored at an early age in 60 children with ASD, showing lower three vitamins levels, and 60 corresponding controls (age group 3–8, M: F=42:18). The results showed significant block of cystathionine formation with consequent accumulation of homocysteine. A lower glutathione levels (GSH), with reduction of essential intracellular reducing environment required for normal immune function, detoxification capacity and redox-sensitive enzyme activity. Increased concentration of 5-methyltetrahydrofolate, which leads to a lower availability of methyl group and significant decrease in urinary methionine and S-adenosyl-L-methionine (SAM) concentrations, the major methyl donor. The latter justify the well-known reduction in protein and DNA methylation reported in autistic children. As a final consideration, the concomitant deficiencies of all three B vitamins, recorded in a significant number of autistic children, suggests that intestinal dysbiosis in these patients may be the main cause of a reduction in their absorption, in addition to the genetic mutation of a specific gene.

Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography-high resolution mass spectrometry.

Over the past ~8years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC-HRMS/MS system consisted of a YMC-UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β-glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono-hydroxy metabolites, and mono-hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono-hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2-19-fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites.

Metabolomic Analysis Using Liquid Chromatography/Mass Spectrometry for Gastric Cancer.

Metabolomics is a post-genomics research field for analysis of low molecular weight compounds in biological samples and has shown great potentials for elucidating complex mechanisms associated with diseases. However, metabolomics studies on gastric cancer (GC), which is the second leading cause of cancer death worldwide, remain scarce, and the molecular mechanisms to metabolomics phenotypes are also still not fully understood. This study reports that the metabolic pathways can be exploited as biomarkers for diagnosis and treatment of GC progression as a case study. Importantly, the urinary metabolites and metabolic patterns were analyzed by high-throughput liquid chromatography mass spectrometry (LC-MS) metabolomics strategy coupled with chemometric evaluation. Sixteen metabolites (nine upregulated and seven downregulated) were differentially expressed and may thus serve as potential urinary biomarkers for human GC. These metabolites were mainly involved in multiple metabolic pathways, including citrate cycle (malic acid, succinic acid, 2-oxoglutarate, citric acid), cyanoamino acid metabolism (glycine, alanine), primary bile acid biosynthesis (glycine, taurine, glycocholic acid), arginine and proline metabolism (urea, L-proline), and fatty acid metabolism (hexadecanoic acid), among others. Network analysis validated close association between these identified metabolites and altered metabolic pathways in a variety of biological processes. These results suggest that urine metabolic profiles have great potential in detecting GC and may aid in understanding its underlying mechanisms. It provides insight into disease pathophysiology and can serve as the basis for developing disease biomarkers and therapeutic interventions for GC diseases.

Urine and plasma metabonomics coupled with UHPLC-QTOF/MS and multivariate data analysis on potential biomarkers in anemia and hematinic effects of herb pair Gui-Hong

The compatibility of Angelicae Sinensis Radix (Danggui) and Carthami Flos (Honghua), a famous herb pair Gui-Hong, can produce synergistic and complementary hematinic effects. Our previous studies have indicated that Gui-Hong has therapeutic potential treatment in hemolytic and aplastic anemia (HAA). The present study aimed to investigate the hematinic effects of Danggui, Honghua and Gui-Hong on HAA rats induced by acetyl phenylhydrazine (APH) and cyclophosphamide (CP) and to explore the underlying hematinic regulation mechanisms. Rats were divided into 5 groups, and drugs were administered by oral gavage one time each day for continuous 7 days from the experiment began. Urine and plasma were analyzed by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). Partial least-squares discriminate analysis (PLS-DA) models were built to evaluate the therapeutic effects of Danggui, Honghua and Gui-Hong. Pearson correlation matrix analysis method was used to discover the correlations between potential biomarkers and biochemical indicators of HAA rats. Seven potential biomarkers contribute to the separation of model group and control group were tentatively identified. The levels of l-kynurenine, phenylalanine, nicotinic acid and sphingosine increased significantly (P<0.05) in HAA rats, while the levels of l-isoleucine, l-tyrosine and serotonin decreased significantly (P<0.05) in comparison with control rats. Those endogenous metabolites were chiefly involved in phenylalanine, tyrosine and tryptophan biosynthesis, valine, leucine and isoleucine biosynthesis, tryptophan metabolism and tyrosine metabolism. The metabolic deviations could be regulated closer to normal level after Danggui, Honghua and Gui-Hong intervention. In term of hematinic effects, Gui-Hong was the most effective as shown by the relative distance in PLS-DA score plots and relative intensity of potential biomarkers. The result reflected the synergic action between Danggui and Honghua. The above results were found to be reasonable in explaining the hematinic effects mechanism of Gui-Hong. The results of routine blood, urinary metabolic pattern and plasma metabolic pattern show the Danggui, Honghua and Gui-Hong groups are moving toward the control group and the HAA was being prevented and alleviated. The effect of Gui-Hong group is more remarkable than Danggui and Honghua groups. Some potential biomarkers like l-kynurenine, phenylalanine, l-isoleucine, l-tyrosine, serotonin, nicotinic acid and sphingosine have been found and identified. The work shows that the metabonomics method is a promising tool in the efficacy and mechanism research of traditional Chinese medicines.

Assessing the Metabolic Effects of Calcineurin Inhibitors in Renal Transplant Recipients by Urine Metabolic Profiling

Biomarkers that can predict graft function and/or renal side effects of calcineurin inhibitors (CNI) at each stage of treatment in kidney transplantation are still lacking. We report the first untargeted GC-MS-based metabolomic study on urines of renal transplant patients. This approach would bring insight in biomarkers useable for graft function monitoring. All consecutive patients receiving a kidney allograft in our transplantation department over a 6-month period were prospectively included and followed up for 12 months. We collected urine samples on the seventh day (D7) after transplantation, then at month 3 (M3) and month 12 (M12), and obtained mass-spectrometry-based urinary metabolic profiles. Multivariate analyses were conducted to compare metabolic profiles at the 3 different periods and to assess potential differences between cyclosporine and tacrolimus. Differences in metabolic signatures were also assessed according to graft function at D7 and renal function at M3 and M12. The urinary metabolic patterns varied over time in cyclosporine- and tacrolimus-treated patients and were somewhat different at D7, M3, and M12 between the 2 treatment groups. Principal metabolites that differed, regardless of the treatment used, were mainly sugars, inositol, and hippuric acid. Interestingly, among tacrolimus-treated patients, different metabolic signatures were found between patients with immediate or delayed graft function at D7. Urinary metabolomics represents a noninvasive way of monitoring immunosuppressive therapy in renal transplant patients. Although it is too early to consider it as a biomarker of CNI-induced injury or graft function, metabolomics appears a promising evaluation tool in this area.

Urinary metabolome identifies signatures of oligozoospermic infertile men

To identify the differential urinary metabolic pattern of oligozoospermic infertile men and to determine the potential biomarkers indicative of infertility. Observational study. University hospital. Totals of 158 fertile volunteers and 135 oligozoospermic infertile men. None. Urinary metabolic profiles were acquired with the use of liquid chromatography/mass spectrometry. Potential oligozoospermic biomarkers were screened from orthogonal projections to latent structures discriminant analysis and further evaluated by receiver operating characteristic analysis. The Spearman correlations between the individual sets of biomarkers and between biomarkers and sperm parameters were investigated. The disrupted biologic pathways which the biomarkers were involved in also were analyzed. Oligozoospermic infertile men could be differentiated from fertile control subjects based on altered urinary metabolic profiles. A total of ten potential biomarkers were screened and tentatively identified. Among those, decreased acylcarnitines, aspartic acid, and leucylproline and increased adenine and methylxanthine were strongly associated with oligozoospermic risk. Many biomarkers were associated with sperm concentration and amplitude of lateral head displacement. The combined pattern of acetylcarnitine, carnitine C3:1, and aspartic acid provided moderate diagnostic power. Urinary metabolomics identified unique metabolic pattern of oligozoospermic infertility. The potential biomarkers suggested that oligozoospermia may be tightly associated with energy consumption and antioxidant defenses in spermatogenesis.

Urinary metabonomic study of the surface layer of Poria cocos as an effective treatment for chronic renal injury in rats

Ethnopharmacological relevancePoria cocos Wolf (Polyporaceae) is a well-known medicinal fungus. The epidermis of the sclerotia (“Fu-Ling-Pi” in Chinese) is used as a diuretic and traditionally used for promoting urination and reduce edema. Aim of the studyTraditional Chinese medicines (TCM) treat many diseases through multi-components, multi-ways and multi-targets. However, the molecular mechanisms of TCM are not yet well understood. In the present work, ultra performance liquid chromatography-based metabonomics analysis was applied to investigate the urinary metabolite profiling of the renoprotective effect of FLP on adenine-induced chronic kidney disease (CKD) rat model and involved possible mechanism. Material and methodsA metabonomic approach based on ultra performance liquid chromatography coupled with quadrupole time-of-flight high-sensitivity mass spectrometry and a novel mass spectrometryElevated Energy data collection technique was developed. The resulting dataset was analyzed by principal component analysis and partial least squares discriminant analysis. The identification of all potential biomarkers was performed using reference standard by comparing their mass spectra, MSE fragments information, isotopic pattern and MassLynx i-FIT algorithm. ResultsBy partial least squares-discriminate analysis, 15 biomarkers in rat urine were identified and 11 of them were related to the pathway of adenine metabolism and amino acid metabolism. Among these biomarkers, eight biomarkers like adenine, l-acetylcarnitine, 8-hydroxyadenine, hypoxanthine, creatine, methionine, phytosphingosine and phenylalanine were reversed to the control level in FLP-treated group and six biomarkers like 2,8-dihydroxyadenine, indole-3-carboxylic acid, 3-methyldioxyindole, ethyl-N2-acetyl-l-argininate, 3-O-methyldopa and xanthurenic acid were reversed to high control group by FLP, which indicates that the urinary metabolic pattern significantly changed after FLP treatment. ConclusionsOur study indicates that FLP treatment can ameliorate CKD by intervening in some dominating metabolic pathways, such as adenine metabolism and amino acid metabolism. The metabonomic results not only supplied a systematic view of the development and progression of CKD and mechanism studies of FLP but also provided the theoretical basis for the prevention or treatment of CKD.

Urinary Metabolic Evaluation of Stone Formers—A Malaysian Perspective

To investigate the urinary metabolic excretion pattern among local stone formers given the great differences in the intrinsic and extrinsic risk factors as well as the urinary metabolic excretions compared with other populations. Thirty urinary stone formers out of an initial 62 recruited provided a complete 24-hour urine sample for metabolic evaluation. Student's t-test and Pearson correlation test were used for the statistical analysis. Urinary volume (1719 ± 712 vs 1215 ± 575, P < .05) and oxalate excretion (0.386 ± 0.111 vs 0.306 ± 0.104, P < .05) were significantly higher among stone formers than controls. Other commonly studied urinary parameters and urinary melamine did not differ significantly between the 2 groups. Similarly, the calcium/citrate ratio was unable to discriminate the stone formers from their controls. Hypocitraturia was the most prevalent urinary abnormality found in stone formers and low urinary citrate excretion was a general phenomenon in both stone formers and controls. Comparing within the stone formers cohort, the recurrent stone formers had a significantly higher urinary saturation and calcium excretion than their first-time stone former counterparts. Elevated urinary oxalate level was the most important urinary risk factor among the local stone formers. A low urinary citrate excretion appeared to be a general phenomenon among the studied cohorts.

Effect of liver diseases on the urinary metabolic pattern and salivary clearance of caffeine in childre

Effect of liver diseases on the urinary metabolic pattern and salivary clearance of caffeine in childre

Evidence of the Regulatory Effect ofGinkgo bilobaExtract on Skin Blood Flow and Study of its Effects on Urinary Metabolites in Healthy humans

Ginkgo biloba extract has been advocated for the improvement of blood circulation in circulatory disorders. This study investigated the effect of the Gingko biloba extract EGb 761 on skin blood flow in healthy volunteers and accompanying changes in urinary metabolites. Twenty-seven healthy middle-aged subjects participated in a randomized, double-blind, placebo-controlled, crossover study. Subjects received 240 mg/d EGb 761 or placebo for periods of 3 weeks. Skin blood flow was measured on the forefoot using laser Doppler flowmetry and changes in urinary metabolites were identified by a combination of nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis (MVDA). These measurements were performed on 24-h urine samples collected at the end of the intervention periods. Following EGb 761 treatment, overall mean skin blood flow was significantly reduced as compared with placebo. Remarkably, the change of skin blood flow after EGb 761 intervention was proportionally related to blood flow after placebo treatment: subjects showed either an increased, decreased or unaltered skin blood flow. NMR/MDVA analyses showed that urinary metabolic patterns differed depending on the change in baseline blood flow after treatment with EGb 761. The present findings substantiate that EGb 761 has a multi-directional modulating action on blood flow in healthy subjects and support findings of a vasoregulatory role of this extract. Moreover, the results indicate that metabolic fingerprinting provides a powerful means to identify biochemical markers that are associated with functional changes.

Evaluation of urinary dihydrocodeine excretion in human by gas chromatography–mass spectrometry

Urinary metabolic pattern after the therapeutic peroral dose of dihydrocodeine tartrate to six human volunteers has been explored. Using the GC–MS analytical method, we have found that the major part of the dose administered is eliminated via urine within the first 24 h. However, the analytical monitoring of dihydrocodeine and its metabolites in urine was still possible 72 h after the dose was administered. The dihydrocodeine equivalent amounts excreted in urine in 72 h ranged between 32 and 108% of the dose, on average 62% in all individuals. The major metabolite excreted into urine was a 6-conjugate of dihydrocodeine, then in a lesser amount a 6-conjugate of nordihydrocodeine (both conjugated to approximately 65%). The O-demethylated metabolite dihydromorphine was of a minor amount and was 3,6-conjugated in 85%. Traces of nordihydromorphine and hydrocodone were confirmed as other metabolites of dihydrocodeine in our study. This information can be useful in interpretation of toxicological findings in forensic practice.

Fate of the Mushroom Hydrazine Agaritine in the Rat and Mouse

The fate of the mushroom hydrazine [14C]agaritine was investigated in the mouse and rat strains previously employed in carcinogenicity studies with the edible mushroom Agaricus bisporus. Agaritine was rapidly absorbed in both species, achieving higher blood levels in the mouse, but with similar area under the curve. Covalent binding of agaritine material to proteins was detected only in the liver and kidney, but the extent of binding was the same in the rat and mouse. Most of the radioactivity was excreted during the first 24 hours in both animal species: in the rat it was distributed equally between urine and feces, whereas in the mouse more of the radioactivity was excreted in the urine. No qualitative differences in the metabolic profile were evident, but quantitative differences were observed. Treatment of the urine with deconjugating enzymes did not reveal the presence of any conjugates. Agaritine, N'-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-(hydroxymethyl)benzene diazonium ion were not detected in the urine or in the plasma of either species. No mutagens or promutagens were detected by the Ames mutagenicity assay in the urine of either species after exposure to agaritine. Repeated administration of agaritine to rats and mice did not alter the urinary metabolic profile and excretion of radioactivity. Similarly, feeding mice a raw mushroom diet, according to the protocol employed in the carcinogenicity studies, did not modulate the excretion of radioactivity or the urinary metabolic pattern. No major species differences in the fate of agaritine in rat and mouse were noted that could provide a rationale for the carcinogenicity of A. bisporus in the mouse, but not in the rat.

Isolation and Identification of a Major Urinary Canthaxanthin Metabolite in Rats

The urinary metabolic pattern after administration of the radiolabeled non-provitamin A carotenoid canthaxanthin was investigated in rats. In the rather complex HPLC urinary metabolic pattern a fraction was found which was conjugated. Deconjugation of the polar conjugates with glusulase, purification of the metabolite with HPLC and identification with GC-MS and NMR revealed that it was 3-hydroxy-4-oxo-7,8-dihydro-beta-ionone. This structure was confirmed by comparisons with HPLC retention times, UV/VIS- and NMR-spectroscopy and GC-MS of the synthesized compound.

Urinary and Biliary Metabolic Patterns of Chlorothalonil in Germ-Free and Conventional Rats

The metabolic fate of chlorothalonil, a broad spectrum fungicide that is known to be metabolized via glutathione conjugation, was examined through the analysis of urine and bile metabolites. The role of digestive microflora in the metabolism of chlorothalonil was assessed by comparing the metabolic patterns in germ-free and conventional rats. Low urinary and biliary excretion of radioactivity was observed in both conventional and germ-free rats. However, the urinary excretion of radioactivity was higher in conventional than in germ-free rats. Radio-HPLC analysis of urine and bile showed a complex metabolic profile in both conventional and germ-free rats. Methylthio metabolites of chlorothalonil were determined in ethyl acetate extracts of urine and bile of conventional and germ-free rats. These metabolites were excreted in a higher amount in the urine of conventional rats than in the urine of germ-free rats. This study shows the complexity of chlorothalonil metabolism and the role of the digestive microflora in chlorothalonil metabolism.