Urinary excretion of probenecid and its metabolites in humans as a function of dose.

A GLC assay was used to study the excretion of probenecid and its metabolites in the urine of human subjects following oral doses of 0.5, 1, and 2 g. From 75 to 88% of the dose was found in the urine. The major metabolite, probenecid acyl glucuronide, accounted for 34-47% of the dose. Approximately equal amounts (10-15%) of the mono-N-propyl, secondary alcohol, and carboxylic acid metabolites were excreted in the unconjugated from with only traces in the conjugated form. The primary alcohol metabolite was not found in measurable amounts. The terminal half-lives for excretion of all metabolites were in the range of 4-6 hr, were independent of dose, and were limited by their rates of formation. A prolonged time course of excretion of the metabolites, particularly at higher doses, suggests that probenecid, being poorly soluble in water, precipitates from solution in the GI tract, forming a depot of drug from which absorption is dissolution rate limited. The urinary excretion of unchanged probenecid, which accounts for 4-13% of the dose, is dependent on both the pH and flow rate of urine.     

Urinary metabolites of French maritime pine bark extract in humans

After oral administration of 5.28 g and 1.06 g of French maritime pine bark extract to a human volunteer, metabolites of some of the components of the extract could be detected. Ferulic acid and taxifolin, conjugated as glucuronide/sulphate, were excreted within 18 h. The peak urinary excretion was observed approximately 2-3 h after intake. Recovery of ferulic acid in urine was 36-43% and 7-8% for taxifolin. Two further metabolites could be identified as delta-(3,4-dihydroxy-phenyl)-gamma-valerolactone and delta-(3-methoxy-4-hydroxyphenyl)-gamma-valerolactone conjugated with glucuronic acid/sulphate. These metabolites could also be detected after intake of 960 mg of a procyanidin fraction of French maritime pine bark extract. Thus, it was shown that procyanidins are metabolised by humans. Both metabolites show maximal urinary excretion 8-12 h after intake and are excreted within 28-34 h.     

Fatty-acid conjugation with cyclamate metabolites as a possible mechanism for ultimate retention

In an in vitro rat-liver microsomal system fortified with coenzyme. A palmitic acid was found to conjugate at the nitrogen moiety of the cyclamate metabolite cyclohexylamine (CHA) and at both the nitrogen and oxygen moieties of its metabolite N-cyclohexylhydroxylamine (CHHA). The fatty acid was preferentially conjugated at or preferentially retained to the nitrogen moiety of CHHA. Stearic acid was also shown to conjugate with CHA. Amines may thus be another class of compounds that, like hydroxylated compounds, are retained in vivo as fatty-acid conjugates in lipid-containing tissues of animals.     

Urinary metabolites of DX-8951, a novel camptothecin analog, in rats and humans

Urinary metabolites of DX-8951 ((1S,9S)-1-amino-9-ethyl-5-fluoro- 1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione, CAS 171335-80-1, exatecan) in rats and humans were identified. Rats were dosed with the drug, and two major metabolites (UM-1 and UM-2) in the urine were isolated and purified by using ion-exchange column and HPLC. From NMR and mass spectra, they are suggested to be 4-hydroxymethyl metabolite (UM-1) and 3-hydroxy metabolite (UM-2) of the drug. Their chemical structures were confirmed by comparing their NMR spectra with those of chemically synthesized metabolites. Two major metabolites were found in human urine obtained in phase I trial. They were also confirmed to be UM-1 and UM-2 by LC/MS/MS by comparing their mass fragment patterns with those of synthetic metabolites.     

Urinary metabolites of dodecylcyclohexane in Salmo gairdneri: evidence of aromatization and taurine conjugation in trout

1. The urinary metabolites of ³H-dodecylcyclohexane were investigated in rainbow trout, Salmo gairdneri R. after a single intragastric dose. In 72?h, 14% of the ingested radioactivity was excreted in urine.

2. Cyclohexylacetic acid, 1-hydroxy-, 3-hydroxy- and 4-hydroxy-cyclohexylacetic acids were present in the unconjugated fraction.

3. In the glucuronide fraction (1.2% dose) labelled aglycones were cyclohexylacetic acid and phenylacetic acid.

4. More than 30% of the urinary ³H was present as phenylacetic and cyclohexylacetic acids conjugated with taurine.     

Urinary metabolites of dodecylcyclohexane in Salmo gairdneri: evidence of aromatization and taurine conjugation in trout

1. The urinary metabolites of 3H-dodecylcyclohexane were investigated in rainbow trout, Salmo gairdneri R. after a single intragastric dose. In 72 h, 14% of the ingested radioactivity was excreted in urine. 2. Cyclohexylacetic acid, 1-hydroxy-, 3-hydroxy- and 4-hydroxy-cyclohexylacetic acids were present in the unconjugated fraction. 3. In the glucuronide fraction (1.2% dose) labelled aglycones were cyclohexylacetic acid and phenylacetic acid. 4. More than 30% of the urinary 3H was present as phenylacetic and cyclohexylacetic acids conjugated with taurine.     

Identification of trimoprostil metabolites excreted in rat bile formed by oxidation and taurine conjugation

14C-Trimoprostil was administered iv and orally to male rats. Radioactivity was excreted mainly via the feces after both routes of drug administration. Bile duct-cannulated rats excreted an average of 76% of an iv dose in the bile within 6 hr after dosing. Four biliary metabolites were isolated by HPLC and identified by proton NMR spectroscopy and mass spectrometry. These metabolites were taurine conjugates of: trimoprostil (approximately 11% of dose), 5,6-dihydro-2,3-dinor trimoprostil (approximately 5% of dose), 3,4-dehydro trimoprostil (less than 5% of dose) and 5,6-dihydro-2,3,4,5-tetranor trimoprostil (less than 5% of dose).     

Urinary metabolites of 2-bromoethanamine identified by stable isotope labelling: evidence for carbamoylation and glutathione conjugation

2-Bromoethanamine (BEA) causes renal papillary necrosis (RPN) in rats after a single dose and has been widely used as a model compound for studying the lesion. Although the metabolism of BEA may be an important determinant of toxicity, the metabolic fate of the compound has not been fully elucidated. To date, the only identified BEA metabolites are aziridine, 2-oxazolidone and 5-hydroxy-2-oxazolidone. In this study, stable isotope labelling (SIL) of BEA analogs ((13C and 2H) were used to differentiate generated BEA metabolites from endogenous molecules which enabled the accurate liquid chromatography mass spectrometry detection of more than 180 novel metabolites. BEA metabolism was evaluated in rats after acute administration of a non-toxic dose (50 mg/kg) and a toxic dose (250 mg/kg) that caused frank RPN and polyuria. Newly identified metabolites include three carbamoylation products, two mercapturic acids and a group of amino acid conjugates. Overall, the results indicate that BEA metabolism is very complex, suggest the potential formation of reactive intermediates and establish that BEA is subject to conjugation with glutathione. The results also demonstrate the utility and sensitivity of the SIL approach for identification of metabolites from small, reactive compounds.     

Urinary metabolites of 2-bromoethanamine identified by stable isotope labelling: evidence for carbamoylation and glutathione conjugation

1. 2-Bromoethanamine (BEA) causes renal papillary necrosis (RPN) in rats after a single dose and has been widely used as a model compound for studying the lesion. Although the metabolism of BEA may be an important determinant of toxicity, the metabolic fate of the compound has not been fully elucidated. To date, the only identified BEA metabolites are aziridine, 2-oxazolidone and 5-hydroxy-2-oxazolidone.

2. In this study, stable isotope labelling (SIL) of BEA analogs (¹³C and ²H) were used to differentiate generated BEA metabolites from endogenous molecules which enabled the accurate liquid chromatography mass spectrometry detection of more than 180 novel metabolites. BEA metabolism was evaluated in rats after acute administration of a non-toxic dose (50?mg/kg) and a toxic dose (250?mg/kg) that caused frank RPN and polyuria. Newly identified metabolites include three carbamoylation products, two mercapturic acids and a group of amino acid conjugates.

3. Overall, the results indicate that BEA metabolism is very complex, suggest the potential formation of reactive intermediates and establish that BEA is subject to conjugation with glutathione. The results also demonstrate the utility and sensitivity of the SIL approach for identification of metabolites from small, reactive compounds.

     

Urinary oxidative metabolites of di(2-ethylhexyl) phthalate in humans

Di(2-ethylhexyl) phthalate (DEHP) is added to polyvinyl chloride (PVC) plastics used widely in medical devices and toys to impart flexibility and durability. DEHP produces reproductive and development toxicities in rodents. Initial metabolism of DEHP in animals and humans results in mono(2-ethylhexyl) phthalate (MEHP), which subsequently metabolizes to a wide range of oxidative metabolites before being excreted in urine and feces. We investigated the metabolism of DEHP in humans by identifying urinary oxidative metabolites of DEHP from individuals with urinary MEHP concentrations about 100 times higher than the median concentration in the general US population. In addition to the previously identified DEHP metabolites MEHP, mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), and mono(2-carboxymethylhexyl) phthalate (MCMHP), we also identified for the first time in humans three additional oxidative metabolites, mono(2-ethyl-3-carboxypropyl) phthalate (MECPrP), mono(2-ethyl-4-carboxybutyl) phthalate (MECBP), and mono(2-(1-oxoethyl)hexyl) phthalate (MOEHP) based on their chromatographic behavior and mass spectrometric fragmentation patterns. We also tentatively identified metabolites with two functional groups in the side alkyl chain as isomers of mono(2-hydroxyethyl-4-carboxybutyl) phthalate (MHECBP), mono(2-ethyl-4-oxo-5-carboxypentyl) phthalate (MEOCPP), and mono(2-ethyl-4-hydroxy-5-carboxypentyl) phthalate (MEHCPP). We report the presence of urinary DEHP metabolites in humans that have fewer than eight carbons in the alkyl chain. These metabolites were previously identified in rodents. Although quantitative information is not available, our findings suggest that, despite potential differences among species, the oxidative metabolism of DEHP in humans and rodents results in similar urinary metabolic products.     

The contributions of various chloroquine salts to the biliary and urinary execretion of hepatic paracetamol conjugation metabolites in the rat

As an approach to explain the possible in vivo interaction of paracetamol (acetaminophen) with various chloroquine salts that are often administered during malaria tropica, the effects of these salts (chloroquine sulphate, chloroquine phosphate, chloroquine hydrochloride and ferrous sulphate) were examined in male rats. The coadministration of chloroquine salts with paracetamol for 7 days showed varied effects on urinary and biliary excretion of paracetamol sulphate and paracetamol glucuronide conjugates--the major metabolites of paracetamol metabolism. These findings suggest that chloroquine sulphate and ferrous sulphate may enhance the sulphation pathway in paracetamol metabolism and influence detoxification of paracetamol in the liver and thus protect the liver. Chloroquine sulphate is therefore a better choice compared to other chloroquine salts in the treatment of malaria with paracetamol as an antipyretic and analgesic.     

Urinary atrazine metabolites as indicators for rat and human exposure to atrazine

Rats were given atrazine (2-chloro-4-ethylamino-6-(isopropylamino)-s-triazine) in drinking water for 1 or 3 weeks at 0.1 (0.45 mM), 0.2 (0.9 mM) or 0.5 g/l (2.3 mM) concentrations of the commercial agent. They excreted at both time points as the principal metabolite 2-chloro-4-ethylamino-6-amino-s-trazine in a dose-dependent fashion. The same urine test was applied on 6 railway men engaged in the weeding operation of railway lines with known atrazine exposure measured with hygienic techniques in their breathing zone. The spectrum of their atrazine metabolites was comprised of fully N-dealkylated atrazine and 2-chloro-4-ethylamino-6-amino-s-triazine. The sum of the two urinary metabolites reflected quantitatively the exposure.     

Urinary metabolites of phenobarbitone, primidone, and their N-methyl and N-ethyl derivatives in humans.

1. Phenobarbitone (1) and three of its N-alkyl derivatives, and primidone (10) and four of its N-alkyl derivatives, were orally administered separately to two human volunteers. Total urine was collected for approximately 2 weeks following each dose, and the drugs and their metabolites were assayed by g.l.c.-mass spectrometry. 2. Recoveries in the phenobarbitone series increased from approximately 40% to approximately 50% as alkylation of (1) increased. There was a linear relationship between the extent of p-hydroxylation and the lipophilicity (log P) of the substrates. The increased total recovery was largely attributable to increased p-hydroxylation. 3. Urinary recoveries in the primidone series decreased from approximately 80% for (10) to approximately 30% for its diakyl derivatives, despite a slight increase in p-hydroxylation with increasing alkylation (and increasing lipophilicity). The decreased recovery was mainly the result of decreased urinary excretion of the drug.     

Urinary metabolites of timolol from humans and laboratory animals. Syntheses and beta-adrenergic blocking activities

Syntheses are reported for three metabolites (2-4) of timolol (1) formed by oxidative metabolism of the morpholine ring. GLC-MS comparisons are presented which establish that the two metabolites whose structures were previously in question are identical with their synthetic counterparts 2 and 3. In 2, metabolic oxidation of the 4-morpholinyl group of 1 had occurred at the carbon next to oxygen to give the 2-hydroxy-4-morpholinyl moiety, whereas in 3, the morpholine of 1 has been oxidized one step further and then ring opened to produce the N-(2-hydroxyethyl)glycine substituent. Biological testing of synthetic samples of the three major metabolites from human urine (3, 4, and 6) indicated that only 4, in which the morpholine moiety has been degraded to a 2-hydroxyethylamino group, had significant beta-adrenergic blocking activity (one-seventh that of timolol in anesthetized dogs).     

Peripheral hormonal responses to D-fenfluramine as a probe of central serotonergic function in humans

We tested the hypothesis that D-fenfluramine (DFEN)-elicited cortisol (CORT) release in humans may be mediated by a direct effect on the adrenal gland by pretreating subjects with dexamethasone (DEX), to prevent release of ACTH from the pituitary, followed by a DFEN challenge test. Eight healthy subjects (four males; four female) (mean age = 38.1 ± 8.4 years (SD)] were studied > 1 week apart (same phase of menstrual cycle) and testing order was randomised. On the with-DEX day (DEX+), subjects took 2 mg DEX orally at 10 p.m.; 30 mg DFEN was then given orally at 9 a.m. and samples were taken at 0–5 h for PRL and CORT. Peak hormone responses (Δ values) were calculated by subtracting baseline values from the maximum levels post-DFEN administration. Peak and baseline hormonal values were compared between the two test conditions; DFEN-induced CORT and PRL responses were compared across all time points, with and without DEX. There was no significant difference in ΔPRL between the two test conditions (DEX−and DEX+), but Δ CORT values were significantly reduced after DEX: mean Δ CORT DEX− = 68.4 ± 26.3 nmol/l; DEX+ = 0.0 nmol/l (all blunted) (df 7,1; P = 0.03). The completely blunted peripheral cortisol response indicates that DFEN cortisol responses are of central origin only. Received: 20 March 1998/Final version: 15 July 1998     

Urinary acrylamide metabolites as biomarkers for short-term dietary exposure to acrylamide.

It has previously been reported that heat-treated carbohydrate rich foods may contain high levels of acrylamide resulting in consumers being inadvertently exposed to acrylamide. Acrylamide is mainly excreted in the urine as mercapturic acid derivatives of acrylamide and glycidamide. In a clinical study comprising of 53 subjects, the urinary excretion of these metabolites was determined using solid-phase extraction and liquid chromatography with positive electrospray MS/MS detection. The median (range) total excretion of acrylamide in urine during 24 h was 16 (7-47) microg acrylamide for non-smokers and 74 (38-106) microg acrylamide for smokers, respectively. It was found that the median intake estimate in the study based on 24 h dietary recall was 21 (13-178) and 26 (12-67) for non-smokers and smokers, respectively. The median dietary exposure to acrylamide was estimated to be 0.47 (range 0.17-1.16) microg/kg body weight per day. In a multiple linear regression analysis, the urinary excretion of acrylamide metabolites correlated statistically significant with intake of aspartic acid, protein, starch and coffee. Consumption of citrus fruits correlated negatively with excretion of acrylamide metabolites.     

Urinary excretion of the main metabolites of methamphetamine,including p-hydroxymethamphetamine-sulfate and p-hydroxymethamphetamine-glucuronide,in humans and rats

The urinary concentrations of the main metabolites of methamphetamine (MA), specifically p-hydroxymethamphetamine-sulfate (?p-OHMA-Sul) and p-hydroxymethamphetamine-glucuronide (?p-OHMA-Glu), were directly measured in MA users and rats using an optimized LC-ESI MS method. The concentrations of the two conjugates in 50 MA human users’ urine ranged from 0.09 to 88.6?µM (0.02–21.7?µg?ml^?1) for p-OHMA-Sul and from <0.05 to 7.13?µM (<0.02–2.43?µg?ml^?1) for p-OHMA-Glu; the ratios of sulfate to glucuronide (S/G ratios) ranged from 2.2 to 37.1 (13.8?±?8.1). The results demonstrate that the sulfation is quantitatively more important than glucuronidation for the conjugation of p-OHMA in humans. The urinary concentration time-dependency in two MA users also revealed that the conjugates were mostly excreted in urine within 3 days post-intake. In contrast, in rat, almost all of the conjugated p-OHMA (>99%) was excreted as the glucuronide in urine. These findings confirm that a large species variation exists in the conjugation of p-OHMA between humans and rats.     

Urinary excretion of the main metabolites of methamphetamine, including p-hydroxymethamphetamine-sulfate and p-hydroxymethamphetamine-glucuronide, in humans and rats

The urinary concentrations of the main metabolites of methamphetamine (MA), specifically p-hydroxymethamphetamine-sulfate (p-OHMA-Sul) and p-hydroxymethamphetamine-glucuronide (p-OHMA-Glu), were directly measured in MA users and rats using an optimized LC-ESI MS method. The concentrations of the two conjugates in 50 MA human users' urine ranged from 0.09 to 88.6 microM (0.02-21.7 microg ml-1) for p-OHMA-Sul and from <0.05 to 7.13 microM (<0.02-2.43 microg ml-1) for p-OHMA-Glu; the ratios of sulfate to glucuronide (S/G ratios) ranged from 2.2 to 37.1 (13.8+/-8.1). The results demonstrate that the sulfation is quantitatively more important than glucuronidation for the conjugation of p-OHMA in humans. The urinary concentration time-dependency in two MA users also revealed that the conjugates were mostly excreted in urine within 3 days post-intake. In contrast, in rat, almost all of the conjugated p-OHMA (>99%) was excreted as the glucuronide in urine. These findings confirm that a large species variation exists in the conjugation of p-OHMA between humans and rats.