Skatole metabolites in urine as a biological marker of pigs with enhanced hepatic metabolism.

Boar taint is a quality defect in meat, related to accumulation of skatole and androstenone in male pigs. The levels of skatole and its main metabolites in plasma and urine samples were measured with a validated liquid chromatography-MS method and related to activity of hepatic cytochrome P450 (CYP450) in order to identify 'fast metabolizing' pigs. Urine (n=46), blood (n=12), liver (n=25) and adipose tissue (n=46) were sampled from a total of 46 entire male pigs. Skatole levels in fat were negatively correlated to CYP2E1 activity and positively to 3-hydroxy-3-methyloxindole (HMOI), indole-3-carboxylic acid (ICA) and 2-aminoacetophenone in urine. HMOI and ICA levels in urine were the best predictors of high skatole levels in fat. In summary, the present study provided further evidence for the key role of CYP2E1 in skatole metabolism and suggested that measurement of HMOI and/or ICA in urine might provide information about skatole levels in live pigs.

Isolation and identification of the metabolites of paeonol in human urine.

Paeonol, a major component of Paeonia suffruticosa Andrews, is used in clinical situations in China as a natural anti-inflammatory agent. The aim of the present study is to investigate the metabolism of paeonol in humans. Six metabolites were isolated from human urine after oral administration of paeonol, and their structures were elucidated as resacetophenone (M1), resacetophenone-2-O-sulfate (M2), 2-hydroxy-4-methoxyacetophenone-5-O-sulfate(M3), 2-hydroxy-4-methoxyacetophenone-5-O-glucopyranuronoside (M4), 2-hydroxyacetophenone-4-O-glucopyranuronoside (M5) and 2,5-dihydroxy-4-methoxyacetophenone(M6) by a series of analyses involving mass spectrometry, (1)H NMR, (13)C NMR and NOESY spectra. In addition, three more metabolites 2,4-dihydroxyacetophenone-5-O-sulfate (M7), paeonol-2-O-glucopyranuronoside (M8) and paeonol-2-O-sulfate (M9), were identified in human urine by using a UPLC/Q-TOF-MS/MS method. This is the first study of paeonol metabolism in humans. Based on the identified metabolites, possible metabolic pathways of paeonol in humans are proposed. Paeonol is metabolized mainly by hydroxylation and demethylation to give the corresponding phase I metabolites, M1, M6 and 2,4,5-trihydroxyacetophenone, and which then underwent conjugation with glucuronic acid or sulfuric acid to form phase II metabolites.

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.

Pharmacokinetics, disposition, and metabolism of [14C]-nebicapone in humans.

OBJECTIVE: This study investigated the absorption, distribution, metabolism and excretion (ADME) of nebicapone [BIA 3-202; 1-(3,4-dihydroxy-5-nitrophenyl)-2-phenyl-ethanone], a reversible catechol-O-methyltransferase (COMT) inhibitor, in 4 healthy male subjects. METHODS: This was a single center, open, non-placebo-controlled, single-group, and a single 200 mg dose study of [(14)C]-nebicapone (2.5 MBq). Blood, urine and faeces were collected up to 264 hours post-dose. RESULTS: Collectively more than 22 metabolites were identified in plasma, urine and faeces, with 3-O-nebicapone-glucuronide (BIA 3-476) identified as the major metabolite. Plasma concentration-time profiles of [(14)C]-nebicapone demonstrated T(max) (h) 1.25+/-0.65, t(1/2) (h) 134.55+/-25.67, C(max) (ng-eq/g) 19647.02+/-4930.20, AUC(0-t) (h.ng-eq/g) 161735.51+/-9224.66, AUC(0-infinity) (h.ng-eq/g) 199603.30+/-16854.08, and for whole blood T(max) 1.00+/-0.41, t(1/2) 32.98+/-22.82, AUC(0-t) 35539.23+/-13664.87, AUC(0-infinity) 36970.64+/-14559.17. Plasma pharmacokinetics of nebicapone demonstrated T(max) (h) 1.00+/-0.41, t(1/2) (h) 2.34+/-0.51; C(max) (ng-eq/g) 12650.00+/-2898.85, AUC(0-t) (h.ng-eq/g) 18719.96+/-734.18, AUC(0-infinity) (h.ng-eq/g) 18392.12+/-753.81; BIA 3-476 demonstrated T(max) 1.25+/-0.50, t(1/2) 3.47+/-0.68; C(max) 15250+/-2563.20, AUC(0-t) 53810.61+/-7358.81, AUC(0-infinity) 54541.21+/-7135.70; 3-O-methyl-nebicapone (BIA 3-270) demonstrated T(max) 21.01+/-6.01, t(1/2) 103.43+/-6.01; C(max) 286.25+/-20.48, AUC(0-t) 27641.89+/-4569.99, AUC(0-infinity) 36968.12+/-4294.42. CONCLUSIONS: Nebicapone and BIA 3-476 accounted for most early phase circulating nebicapone-derived moieties, have limited circulating cell association, peak concentrations shortly after dosing, and short body residence. In longer terminal half-life phases low concentrations of BIA 3-270 predominate. While about 70% of the dose was eliminated in the urine as BIA 3-476, < 1% of the dose was excreted as unchanged nebicapone. Faecal excretion accounted for 17.3% administered dose. On average, the total recovery of 88.6% of the radioactivity suggested no worrisome retention of drug derived material following a single 200 mg administration of nebicapone to healthy volunteers. The treatment was very well tolerated with no reported adverse events.

Characterization of hepatic mitochondrial injury induced by fatty acid oxidation inhibitors.

Impairment of liver mitochondrial beta-oxidation is an important mechanism of drug-induced liver injury. Four inhibitors of fatty acid oxidation were compared in short-term rat in vivo studies in which the rats were administered one or four doses. The hepatocellular vacuolation represented ultra-structural mitochondrial changes. Urine nuclear magnetic resonance (NMR) spectroscopy revealed that both FOX988 and SDZ51-641 induced a persistent dicarboxylic aciduria, suggesting an inhibition of mitochondrial beta-oxidation and incomplete fatty acid metabolism. Etomoxir caused minimal mitochondrial ultrastructural changes and induced only transient dicarboxylic aciduria. CPI975 served as a negative control, in that there were no significant perturbations to the mitochondrial ultrastructural morphology or in the urine NMR composition; however, compound exposure was confirmed by the up-regulation of liver gene expression compared to vehicle control. The liver gene expression changes that were altered by the compounds were indicative of mitochondria, general and oxidative stress, and peroxisomal enzymes involved in beta-oxidation, suggestive of a compensatory response to the inhibition in the mitochondria. In addition, both FOX988 and SDZ51-641 up-regulated ribosomal genes associated with apoptosis, as well as p53 pathways linked with apoptosis. In summary, metabonomics and liver gene expression provided mechanistic information on mitochondrial dysfunction and impaired fatty acid oxidation to further define the clinical pathology and histopathology findings of hepatotoxicity.

Dynamic liquid phase nanoextraction coupled to GC/MS for rapid analysis of methoxyacetophenone and anisaldehyde isomers in urine.

This study introduces a novel extraction technique in the nanoscale and challenges the limits of solvent extraction in the GC/MS using electronic ionization (EI) method for quantitative determination of six methoxyacetophenone (MAP) and anisaldehye (AAH) isomers in one drop of water and urine. This technique is termed as dynamic liquid phase nanoextraction (DLPNE). The optimum parameters for the DLPNE technique were: selection of solvent, toluene; sampling volume, 0.44 microL; dwell time, 2 s; number of sampling, 15; extraction time, 1.5 min; volume of extraction solvent, 60 nL; and no salt addition. The LODs for this technique were 5-20 ng/mL. The RSDs were in the range of 9.7-12.6% (n = 6). The linear dynamic range of the calibration curve of DLPNE is from 0.02 to 0.5 microg/mL with correlation coefficient (r(2)) >0.9705. The advantages of the DLPNE technique are rapidity, ease of operation, simple device, and extremely little solvent and sample consumption. This technique was also compared with the static liquid phase nanoextraction (SLPNE) while the SLPNE failed to detect any signal for the six isomers. We believe that this technique can be very useful for the detection of volatile organic compounds in environmental science from microscale of water or it can be applied to clinical or pharmaceutical application such as diagnosis of microamount of urine or blood samples by GC/MS.

Simultaneous determination of 2-methoxyphenol, 2-methoxy-4-methylphenol, 2,6-dimethoxyphenol and 4'-hydroxy-3'-methoxyacetophenone in urine by capillary gas chromatography.

A method for the simultaneous determination of 2-methoxyphenol, 2-methoxy-4-methylphenol, 2,6-dimethoxyphenol and 4'-hydroxy-3'-methoxyacetophenone in urine has been described. The metabolites were analyzed after enzymatic hydrolysis and extraction on octyl (C8) cartridges by using gas chromatography with flame ionization detection and a 5/95% copolymer of diphenyl-poly(dimethylsiloxane) capillary column. Methoxyphenols were well separated within 12 min. Recovery was over 90% in the range from 0.5 to 20 microg/ml; the detection limit was varying in the range of 0.05-0.11 microg/ml. The relative standard deviations and the accuracy were in the range of 3.1-15.5 and 2.4-16.0%, respectively.

Pharmacokinetics and pharmacodynamics of BIA 3-202, a novel COMT inhibitor, during first administration to humans.

OBJECTIVE: To determine the tolerability, pharmacodynamics and pharmacokinetics of single oral doses of BIA 3-202, a novel catechol-O-methyltransferase (COMT) inhibitor, in healthy male volunteers. METHODS: Single increasing oral doses of BIA 3-202 (10, 30, 50, 100, 200, 400 and 800mg) were administered under fasting conditions to seven sequential groups of nine subjects, under a double-blind, randomised, placebo-controlled design. In an additional group of eight subjects (group 8), a single dose of BIA 3-202 400mg was administered on two occasions, once under fasting conditions and once with a high-fat meal, under an open-label, two-way crossover design. RESULTS: BIA 3-202 was well tolerated at all doses tested. Most adverse events were mild in severity and their incidence was similar between BIA 3-202 and placebo. Maximum plasma concentrations (C(max)) of BIA 3-202 were attained at 0.5-2.5h (t(max)) and thereafter declined with an apparent terminal half-life (t(1/2)) of 1.5-5h. Over the dose range of 10-800mg, there was an approximately dose-proportional increase in the area under the plasma concentration-time curve (AUC) values of BIA 3-202: for a dose level increase in the ratio 3.0:1.7:2.0:2.0:2.0:2.0, AUC increased in the ratio 3.1:1.7:1.9:2.2:2.1:1.7. Plasma concentrations of the O-methylated derivative, BIA 3-270, increased markedly less than predicted from a proportional relationship: for a dose level increase in the ratio 1:80, AUC(0-t )increased in the ratio 1:5. In most subjects, the t(max) of BIA 3-270 was attained at the last sampling time and, therefore, t(1/2 )could not be estimated. Urine assays showed that less than 1% of the total dose administered was excreted in urine as BIA 3-202. Urine concentrations of BIA 3-270 were below the limit of quantification. In group 8, the rate and extent of systemic availability (t(max), AUC and C(max)) of BIA 3-202 and BIA 3-270 after a high-fat meal were similar to those under fasting conditions. Inhibition of COMT activity in erythrocytes reached maximum levels at 2-2.5h post dose, with sustained inhibition up to approximately 4-6 hours, returning to baseline by about 16 hours. CONCLUSION: BIA 3-202 was well tolerated at single 10-800mg oral doses and presented dose-proportional kinetics. It effectively inhibited COMT activity and the presence of food did not affect its pharmacokinetics or COMT inhibitory activity. The results provide a basis for further clinical studies with BIA 3-202.

Metabolism of paeonol in rats.

As a part of our studies on the metabolism of active components from traditional Chinese medicines, paeonol was orally administered to rats. The urinary metabolites were analyzed by 3D HPLC, and their structures were determined to be 2, 4-dihydroxyacetophenone-5-O-sulfate (P1), resacetophenone-2-O-sulfate (P2), 2-hydroxy-4-methoxyacetophenone-5-O-sulfate (P3), paeonol-2-O-sulfate(P4), resacetophenone (P5), and unchanged paeonol, on the basis of their chemical and spectral data. Among these metabolites, P2-P4 and paeonol were detected in the plasma after the oral administration of paeonol. Furthermore, the bile of rats given paeonol orally was found to contain P3, suggesting the enterohepatic circulation of paeonol.

Metabolism of trans-cinnamic acid in the rat and the mouse and its variation with dose.

The metabolism of [3-14C/phenyl-2H5] cinnamic acid was investigated in rats and mice at a dose level of 2.5 mmol/kg body weight. Recoveries of the 14C dose were between 92 and 98% with most (82-90%) present in the 0-24 hr urine samples. Urinary metabolites were identified by their chromatographic properties and mass spectra. In both species the major metabolite was hippuric acid, which is also an endogenous urinary component. Several minor metabolites, 3-hydroxy-3-phenylpropionic acid, benzoic acid and benzoyl glucuronide, were found in both species. Two, acetophenone and cinnamoylglycine, the glycine conjugate of cinnamic acid, could be positively identified only in mouse urine. The effect of dose size on the urinary excretion of 14C-cinnamic acid metabolites was studied over the dose range 0.0005 to 2.5 mmol/kg. In the rat the pattern of metabolite excretion was very similar over the whole dose range with slight increases in the proportion of the dose excreted as minor metabolites as dose size increased. In the mouse the excretion of cinnamoylglycine was more important at the lowest dose level and decreased in relative importance as dose size increased. Changes in the other metabolites were similar to those seen in the rat.

Determination of an acetylcholinesterase inhibitor, MDL 73745, in dog plasma and urine by combined gas chromatography/negative ion chemical ionization mass spectrometry.

A highly sensitive and specific assay has been developed for the determination of MDL 73745 [2,2,2-trifluoro-1-(3-trimethylsilyl-phenyl) ethanone] (I) and the internal standard (MDL 74398) at the nanomolar level in dog plasma and urine by gas chromatography/mass spectrometry. After a single-step extraction process, an aliquot was directly injected onto the gas chromatograph column. The mass spectrometer was run in the negative ion chemical ionization mode with ammonia as reagent gas, and was set to monitor the abundant M-. ion at m/z 246 of both compounds. The method yielded a linear response over the concentration range 0.1-10 pmol 100 microliters -1 plasma or urine. Within-day reproducibility at a concentration of 0.25, 1 and 5 pmol 100 microliters -1 plasma was 8.6%, 1.0% and 1.0%, respectively. The method was applied to the determination of I in plasma and urine after administration of 1 mg kg-1 i.v. and 10 mg kg-1 p.o. to dogs.

Possible involvement of 1,2-diacetylbenzene in diethylbenzene-induced neuropathy in rats.

The role of 1,2-diacetylbenzene (1,2-DAB) in the peripheral nerve toxicity of 1,2-diethylbenzene (1,2-DEB) was investigated in rats. Gas chromatography-mass spectrometry identified 1,2-DAB in the urine samples of rats given 165 mg kg-1 1,2-DEB orally on four consecutive days. 1,2-DAB shared not only the ability of 1,2-DEB to cause bluish discoloration of skin, internal organs and urine, but unlike 1,2-DEB it turned hair blue at the site of intraperitoneal injection. Intraperitoneal administration of 10 mg kg-1 and 20 mg kg-1 1,2-DAB to groups of 12 rats, 4 days a week for 11 and 6 weeks, caused a dose- and time-dependent decrease in mean sensory and motor conduction velocities. Recovery in a 5-week post-exposure period was gradual but consistent. The effect of 1,2-DAB on the amplitude of the sensory action potential was ambiguous. The findings support the hypothesis that the formation of 1,2-diacetylbenzene derivatives contributes to the neurotoxicity of 1,2-DEB.

Urinalysis of minor metabolites of ethylbenzene and m-xylene.

Gas chromatographic methods have been developed for the urinalysis of metabolic products of ethylbenzene. "Minor" metabolites were emphasized in this process. The methods were worked out so that simultaneous determination of several compounds could be achieved with one method. On the whole, four assays are described, which together allow 12 different compounds to be measured. Since one of the methods presented can be applied to determine "minor" metabolites of xylene as well, methodological data for these are also reported. The methods described have successfully been used in metabolic studies of ethylbenzene in rat and man.

Studies on the percutaneous absorption of paeonol in experimental animals by a radioactive tracer technique.

Studies on in vivo percutaneous absorption of paeonol (2-hydroxy-4-methoxyacetophenone: I) in rabbits, guinea pigs, and rats were carried out by a radioactive tracer technique. Hydrophilic ointment (specific activity: 376 Bq (0.01016 microCi)/mg) containing I[carbonyl-14C] was applied for 24 h by occlusive dressing treatment on the shaven back of experimental animals. Then, the 14C-activity in urine, which is the dominant excretion route, was determined for 8 days. Percentage excreted within 24 h after application to rabbits, guinea pigs, and rats were 42.8%, 46.5% and 52.0%, respectively. Urinary metabolites were identified as 2,5-dihydroxy-4-methoxyacetophenone, resacetophenone, and substrate I in all the case.

Inhibitors of hepatic mixed function oxidases. I. The metabolism of 2,6-dihydroxy-,2-hydroxy-6-methoxy- and 2,6-dimethoxyacetophenones.

1. 2,6-Dihydroxyacetophenone, its mono- and di-methyl ethers are inhibitors of hepatic mixed function oxidases. The dimethyl ether is a competitive inhibitor of aminopyrine demethylase with the others displaying mixed kinetics. The metabolism of all three ketones has been studied. 2. 2,6-Dihydroxyacetophenone is excreted unchanged and as conjugates. 3. 2-Hydroxy-6-methoxyacetophenone is largely excreted unchanged and conjugated but small amounts of the 3- and 5-hydroxylated derivatives are formed. 4. 2,6-Dimethoxyacetophenone is demethylated to 2-hydroxy-6-methoxy-acetophenone. In addition 3-hydroxy-2,5-dimethoxyacetophenone and 2,3-dihydroxy-6-methoxyacetophenone were identified as metabolites. 5. Quantitative data on the excretion of metabolites were obtained with 14C-labelled ketones.

[Studies on tryptophan metabolism in untreated phenylketonuric patients]. / Tryptophan-Stoffwechseluntersuchungen bei unbehandelten Phenylketonurikern

The products of the oxidative degradation of tryptophan via the kynurenine pathway were quantitatively determined in the urine of ten untreated patients with phenylketonuria, aged 4--35 years. All the patients were sevrely mentally retarded. The results of the analysis suggest a division of the patients into two groups, A and B. The patients of group A showed a basal urinary excretion of kynurenine, kynurenic acid, 3-hydroxykynurenine and xanthurenic acid which lies in the lower part of the normal range. The increase in excretion of tryptophan metabolites under tryptophan loading was, however, significantly less than in controls. On the average, only 0.63 % of the load was excreted in the form of these assayed metabolites; in contrast, the control value is 1,13 %. In group B, the rate of excretion was higher than normal under basal and loading conditions. The post-tryptophan excretion was four times greater than that of controls (4.64 %). 3-hydroxyanthranilic acid could only be detected in group B after loading. The metabolite 8-hydroxyquinaldic acid, which is supposed to be an abnormal metabolic product of tryptophan, was excreted in milligram amounts. The analysis of the metabolites of 3-hydroxyanthranilic acid showed that the excretion of N1-methylnicotinamide and N1-methyl-2-pyridone-5-carboxamide was within the normal range. The excretion of nicotinic acid and its amide was sporadic in both the patients and controls. Other theoretically possible metabolites in the pathway could not be found. A number of unidentified metabolites could be detected by thin-layer chromatography in the basal state. The excretion of these metabolites was greatly augmented after tryptophan loading. Other substances which were not detectable in the basal state became evident on loading. A number of these metabolites are characteristic either of group A or B. The structural identification of one of the new products has been hindered by its instability. A stable cleavage product was identified as omicron-aminoacetophenon by mass-spectroscopy. This metabolite its typical for group B. The possible influence of the blood phenylalanine on the metabolism of tryptophan in phenylketonuria is discussed.