Identification of the metabolites of gigantol in rat urine by ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Gigantol is a typical bibenzyl compound isolated from Dendrobii Caulis that has been widely used as a medicinal herb in China for the treatment of diabetic cataract, cancer and arteriosclerosis obliterans and as a tonic for stomach nourishment, saliva secretion promotion and fever reduction. However, few studies have been carried out on its in vivo metabolism. In the present study, a rapid and sensitive method based on ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) in positive ion mode was developed and applied to identify the metabolites of gigantol in rat urine after a single oral dose (100 mg/kg). Chromatographic separation was performed on an Acquity UPLC HSS T3 column (100 × 2.1 mm i. d., 1.8 µm) using acetonitrile and 0.1% aqueous formic acid as mobile phases. A total of 11 metabolites were detected and identified as all phase II metabolites. The structures of the metabolites were identified based on the characteristics of their MS, MS(2) data and chromatographic retention times. The results showed that glucuronidation is the principal metabolic pathway of gigantol in rats. The newly identified metabolites are useful to understand the mechanism of elimination of gigantol and, in turn, its effectiveness and toxicity. As far as we know, this is the first attempt to investigate the metabolic fate of gigantol in vivo.

[10]-Gingerdiols as the major metabolites of [10]-gingerol in zebrafish embryos and in humans and their hematopoietic effects in zebrafish embryos.

Gingerols are a series of major constituents in fresh ginger with the most abundant being [6]-, [8]-, and [10]-gingerols (6G, 8G, and 10G). We previously found that ginger extract and its purified components, especially 10G, potentially stimulate both the primitive and definitive waves of hematopoiesis (blood cell formation) in zebrafish embryos. However, it is still unclear if the metabolites of 10G retain the efficacy of the parent compound toward pathological anemia treatment. In the present study, we first investigated the metabolism of 10G in zebrafish embryos and then explored the biotransformation of 10G in humans. Our results show that 10G was extensively metabolized in both zebrafish embryos and humans, in which two major metabolites, (3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol, were identified by analysis of the MS(n) spectra and comparison to authentic standards that we synthesized. After 24 h of treatment of zebrafish embryos, 10G was mostly converted to its metabolites. Our results clearly indicate that the reductive pathway is a major metabolic route for 10G in both zebrafish embryos and humans. Furthermore, we investigated the hematopoietic effect of 10G and its two metabolites, which show similar hematopoietic effects as 10G in zebrafish embryos.

Characterization of thiol-conjugated metabolites of ginger components shogaols in mouse and human urine and modulation of the glutathione levels in cancer cells by [6]-shogaol.

SCOPE: Shogaols, a series of major constituents in dried ginger with the most abundant being [6]-, [8]-, and [10]-shogaols, show much higher anticancer potencies than gingerols. Previously, we reported the mercapturic acid pathway as a major metabolic route for [6]-shogaol in mice. However, it is still unclear how the side chain length affects the metabolism of shogaols and how shogaols are metabolized in humans. METHODS AND RESULTS: We first investigate the metabolism of [10]-shogaol in mouse urine, and then investigate the biotransformation of shogaols in human urine. Our results show that eight major thiol-conjugated metabolites of [10]-shogaol were detected in mouse urine, while six major thiol-conjugated metabolites of [6]-shogaol, two thiol-conjugated metabolites of [8]-shogaol, and two thiol-conjugated metabolites of [10]-shogaol were detected in urine collected from human after drinking ginger tea, using LC/ESI-MS/MS. Our results clearly indicate the mercapturic acid pathway is a major metabolic route for [10]-shogaol in mice and for shogaols in human. Furthermore, we also investigated the regulation of glutathione (GSH) by [6]-shogaol in human colon cancer cells HCT-116. Our results show [6]-shogaol, after initially depleting glutathione levels, can subsequently restore and increase GSH levels over time. CONCLUSION: Shogaols are metabolized extensively in mouse and human to form thiol-conjugated metabolites and GSH might play an important role in the cancer-preventive activity of ginger.

Occupational exposure to phenolic compounds at coke plants--urinary excretion of methoxyphenols as an indicator of exposure to methoxyphenols.

OBJECTIVES: This study describes the exposure of coke plant workers to methoxyphenols. The relationship between exposure to methoxyphenols and urinary excretion of metabolites was examined. METHODS: We determined concentrations of 2-methoxyphenol, 2-methoxy-4-methylphenol and 1-(4-hydroxy-3-methoxyphenyl)ethanone in the breathing-zone air and in the urine of workers, collected after the workshift. Urine metabolites were extracted after enzymatic hydrolysis by solid-phase extraction. Concentrations of methoxyphenols in air and urine were determined by gas chromatography with flame-ionization. RESULTS: The time-weighted average concentrations (median) of methoxyphenols in the breathing zone air were as follows: 9.9 ng/m(3), 15.4 ng/m(3) and 92.5 ng/m(3) for 2-methoxyphenol, 2-methoxy-4-methylphenol and 1-(4-hydroxy-3-methoxyphenyl)ethanone, respectively. The median values of urinary concentrations were as follows: 582.5, 190.1, 235.0 and 21.8 µmol/mol creatinine for 2-methoxyphenol, 2-methoxy-4 methylphenol, 1-(4-hydroxy-3-methoxyphenyl)ethanone and 2,6-dimethoxyphenol, respectively. A statistically significant correlation between the exposure level and the urinary level was found for 2-methoxyphenol (r=0.573, p<0.01). CONCLUSION: We found that the presence of 2-methoxyphenol in urine can be used as a biomarker for 2-methoxyphenol exposure. The analysis performed at the coke plant showed that the workers were exposed to relatively low concentrations of methoxyphenols.

Toxicological determination and in vitro metabolism of the designer drug methylenedioxypyrovalerone (MDPV) by gas chromatography/mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry.

A method for the toxicological screening of the new designer drug methylenedioxypyrovalerone (MDPV) is described; with an emphasis on its application for anti-doping analysis. The metabolism of MDPV was evaluated in vitro using human liver microsomes and S9 cellular fractions for CYP450 phase I and uridine 5'-diphosphoglucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II metabolism studies. The resulting metabolites were subsequently liquid/liquid extracted and analyzed using gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) derivatives. The structures of the metabolites were further confirmed by accurate mass measurement using a liquid chromatography/quadrupole time-of-flight (LC/QTOF) mass spectrometer. The studies demonstrated that the main metabolites of MDPV are catechol and methyl catechol pyrovalerone, which are in turn sulfated and glucuronated. The method for the determination of MDPV in urine has been fully validated by assessing the limits of detection and quantification, linearity, repeatability, and accuracy. This validation demonstrates the suitability for screening of this stimulant substance for anti-doping and forensic toxicology purposes.

GC profiles of volatile constituents from human urine obtained by closed loop stripping, purge and trap technique and simultaneous stem distillation-extraction.

Different techniques like "closed loop stripping" [CLSA], "purge and trap" [PTI], and continuous steam distillation extraction [SDE] were used to establish GC profiles of major histocompatibility complex-associated volatile constituents of human urine and statistically evaluated for reliability. Of the three methods investigated, PTI appeared to be superior for the detection of very volatile substances, whereas SDE was the most efficient one with respect to yield. A number of short to medium-chain ketones, 4-hydroxy-3-methoxy-styrene, menthol and nicotine were identified in preliminary analyses.

Metabolism of 1-(4-hydroxy-3-methoxyphenyl)-2-propanone, a smoke flavour ketone, in rat.

1. Metabolites of 1-(4-hydroxy-3-methoxyphenyl)-2-propanone (HMP-one), a smoke flavour compound, were isolated from rat urine using hydrolysis, ether extraction, t.l.c. and g.l.c. 2. Three metabolites were identified by mass spectrometry and independent synthesis, namely: 1-(3, 4-dihydroxyphenyl)-2-propanone (Met I), 1-(3, 4-dihydroxyphenyl)-2-propanol (Met II), and 1-(4-hydroxy-3-methoxyphenyl)-2-propanol (Met III). 3. A g.l.c. method for the quantitative determination of the parent compound and metabolites in urine was devised. Unchanged HMP-one accounted for about 74% dose, with Met I 11%, Met II 5%, and Met III 9%. All compounds were excreted both as sulphate and glucuronide conjugates.

Determination of catechol and guaiacol estrogens in urine by capillary gas chromatography/mass spectrometry.

A gas chromatographic (GC)/mass spectrometric method for the simultaneous determination in urine of 2- and 4-hydroxyestrones and hydroxyestradiols, and their monomethyl ethers, is described. Separation of these catechol and guaiacol estrogens was achieved by derivatization into their trimethylsilyl and tert-butyldimethylsilyl ethers, followed by capillary GC on a DB-1 column. The calibration graphs were satisfactorily constructed for these estrogen metabolites by selected ion monitoring at the respective molecular ions using 2-bromoestrone and 4-hydroxyestradiol-d3 3-methyl ether as internal standards. The extraction and purification of the desired estrogens in biological fluids were effected by the combined use of Extrelut-3 and ion exchange columns. The sensitivity and reliability obtained by the newly developed method has proved to be satisfactory for the quantitation of catechol and guaiacol estrogens in human urine.

[Initial trial of a new test for the evaluation of hepatocellular function: the TMB test]. / Premiers essais d'un nouveau test d'évaluation du fonctionnement hépatocellulaire: le test au TMB.

The oral administration of 400 mg of trimethoxy 1-3-5-benzene or T.M.B. is normally followed by a high urinary excretion of dimethoxy, 1, 3, hydroxy 5 benzene and other metabolites mainly in the form of conjugates. Urinary excretion during the first 3 hours is strongly reduced in liver disease. By a simple blind test we showed that the test is positive in subclinical and laboratory-negative liver failure.

The metabolism of vanillin and isovanillin in the rat.

1. The metabolism of vanillin, isovanillin and the corresponding alcohols and acids in rats was investigated using t.l.c., g.l.c. and combined g.l.c.-mass spectrometry. 2. Oral dosage (100 mg/kg) of the aldehyde resulted in urinary excretion of most metabolites within 24 h, mainly as glucuronide and/or sulphate conjugates although the acids formed were also excreted free and as their glycine conjugates. In 48 h 94% of the dose of vanillin was accounted for as follows (%) : vanillin (7), vanillyl alcohol (19), vanillic acid (47), vanilloylglycine (10), catechol (8), 4-methylcatechol (2), guaiacol (0-5) and 4-methylguaiacol (0-6). Similarly, 89% of the dose of isovanillin was accounted for as follows: isovanillin (19), isovanillyl alcohol (10), isovanillic acid (22), vanillic acid (11), isovanilloylglycine (19), catechol(7) and 4-methylcatechol (1). Protocatechuic acid was also formed from both aldehydes. 3. By means of (a) investigation of biliary metabolites, (b) prevention of biliary excretion, (c) suppression of intestinal bacteria with neomycin sulphate and (d) inhibition of intestinal beta-glucuronidase with saccharo-1,4-lactone, it was found that glucuronides of the aldehydes and their respective alcohol and acid derivatives are excreted in the bile and that the conjugates are metabolized by the intestinal bacteria to toluene derivatives and decarboxylated products.