Predominant Urinary Metabolite Research Articles

Identification of etazene (etodesnitazene) metabolites in human urine by LC-HRMS.

Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of "nitazenes." Etazene metabolites were identified through analysis of a human urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking a new psychoactive substances (NPS) cocktail purchased online and was analyzed by ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Etazene metabolites were predicted with BioTransformer 3.0, and the exact masses were added to the inclusion list. Eight possible metabolites were identified in the urine sample. N- and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene), and M3 (N,O-dideethylated etazene) metabolites. Less abundant hydroxylated products of these deethylated metabolites and etazene were also found. Additionally, in the analysis without β-glucuronidase treatment, M1- and M3-glucuronide phase II metabolites were found. As N- and O-deethylated products seem to be the predominant urinary metabolites, the detection of these metabolites in urine can be useful to demonstrate etazene exposure.

Identification of etazene metabolites in human urine by LC-HRMS

Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of ‘nitazenes’. We examined the in-vivo metabolism of etazene through analysis of a human (overdose) urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking an NPS cocktail purchased online. Next to etazene, N-ethylpentedrone, deschloroketamine and flubromazepam were also taken by the patient. The urine sample was analyzed on a Q-Exactive high resolution mass spectrometer (HRMS) operating in full scan mode (range 70-700 m/z) with data-dependent acquisition based on an inclusion list. Etazene metabolites were predicted with the BioTransformer 3.0 software and the exact masses were added into the inclusion list. The urine sample was analyzed both with simple dilution of the sample (addition of internal standard and dilution with aqueous mobile phase) and after β-glucuronidase treatment (addition of internal standard and Abalonase® to the sample with subsequent incubation at 37 °C for 30 min). Six possible metabolites were identified in the urine sample of the patient. The parent compound was less abundant than some metabolites. In line with previous findings for other nitazene opioids, N- and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene) and M3 (N,O-dideethylated etazene) metabolites. Less abundant hydroxylated products of these deethylated metabolites (M5: hydroxylated M2 and M6: hydroxylated M1) and etazene (M4) were also found. Hydroxylation is presumed at the benzimidazole ring. For M5 and M6, different chromatographic peaks with the same MS 2 spectra were detected, suggesting the presence of positional isomers of the hydroxyl-group at the benzimidazole ring. Additionally, in the analysis without β-glucuronidase treatment, M1- and M3-glucuronide phase II metabolites were found. A rapid and straightforward HRMS method was developed to study the in-vivo metabolites of etazene. N- and O-deethylated products were the predominant urinary metabolites. Detection of these metabolites in urine can be useful to demonstrate etazene exposure. Our findings are comparable to results obtained in a metabolism study performed in rats.

Tailored sensitivity reduction improves pattern recognition and information recovery with a higher tolerance to varied sample concentration for targeted urinary metabolomics

Variation in total metabolite concentration among different samples has been a major challenge for urinary metabolomics. Here we investigated the potential of tailored sensitivity reduction of high abundance metabolites for improved targeted urinary metabolomics. Two levels of sensitivity reduction of the 21 predominant urinary metabolites were assessed by employing less sensitive transition or collision energy with level 1 (reduced 1) and 2 (reduced 2) exhibiting 30–90% and 2–20% of the optimal sensitivity, respectively. Five postacquisition normalization methods were compared including no normalization, probabilistic quotient normalization, and normalization to sample median, creatinine intensity, and total intensity. Normalization to total intensity with reduced 2 gave the best pattern recognition and information recovery with a higher tolerance to varied sample concentration. Pareto scaling could improve the performance of tailored sensitivity reduction (reduced 2) for targeted urinary metabolomics while data transformation and autoscaling were susceptible to varied sample concentration. Using controlled spike-in experiments, we demonstrated that tailored sensitivity reduction revealed more differentially expressed markers with higher accuracy than did the conventional optimal sensitivity. This was particularly true when the differences between the sample groups are small. This work also served as an introductory guideline for handling targeted metabolomics data using the open-source software MetaboAnalyst.

Human metabolism and excretion kinetics of aniline after a single oral dose.

Aniline is an important source material in the chemical industry (e.g., rubber, pesticides, and pharmaceuticals). The general population is known to be ubiquitously exposed to aniline. Thus, assessment of aniline exposure is of both occupational and environmental relevance. Knowledge on human metabolism of aniline is scarce. We orally dosed four healthy male volunteers (two fast and two slow acetylators) with 5mg isotope-labeled aniline, consecutively collected all urine samples over a period of 2days, and investigated the renal excretion of aniline and its metabolites by LS-MS/MS and GC-MS. After enzymatic hydrolysis of glucuronide and sulfate conjugates, N-acetyl-4-aminophenol was the predominant urinary aniline metabolite representing 55.7-68.9% of the oral dose, followed by the mercapturic acid conjugate of N-acetyl-4-aminophenol accounting for 2.5-6.1%. Acetanilide and free aniline were found only in minor amounts accounting for 0.14-0.36% of the dose. Overall, these four biomarkers excreted in urine over 48h post-dose represented 62.4-72.1% of the oral aniline dose. Elimination half-times were 3.4-4.3h for N-acetyl-4-aminophenol, 4.1-5.5h for the mercapturic acid conjugate, and 1.3-1.6 and 0.6-1.2h for acetanilide and free aniline, respectively. Urinary maximum concentrations of N-acetyl-4-aminophenol were reached after about 4h and maximum concentrations of the mercapturic acid conjugate after about 6h, whereas concentrations of acetanilide and free aniline peaked after about 1h. The present study is one of the first to provide reliable urinary excretion factors for aniline and its metabolites in humans after oral dosage, including data on the predominant urinary metabolite N-acetyl-4-aminophenol, also known as an analgesic under the name paracetamol/acetaminophen.

Abstract 114: Mass balance study of 14C- belinostat in patients with recurrent or progressive malignancy

Abstract Background: Belinostat is a hydroxamic acid based pan-histone deacetylase (HDAC) inhibitor that inhibits Class I, II, and IV HDAC enzymes at micromolar concentrations; it (Beleodaq) was recently approved in the US for the treatment of relapsed/refractory PTCL. The excretion of belinostat and its metabolites has been studied using radiolabeled belinostat in dogs and rats (∼70% and 50% excreted fecally and 30% and 50% excreted renally, respectively). The renal excretion of parent belinostat was <1% in both species. Objective: To determine the excretion route of radiolabeled belinostat and its metabolites following single IV administration of 14C-labeled belinostat in patients with advanced cancers. Secondary objectives included an assessment of safety. Methods: Patients with progressive or recurrent malignancies who failed standard therapies were eligible for the study. Patients with severe constipation, urinary incontinence, primary hepatic or renal carcinomas were excluded. Six eligible patients received a single dose of 14 C-labeled belinostat (approximately 90 to 105 μCi, 1500 mg) as a 30-minute IV infusion. Blood, urine, and fecal samples were collected at pre-determined time points (up to 168 hours) for measurement of total radioactivity. Results: Six patients with a median age of 66 yrs (range: 55 to 75) were enrolled. Total recoveries of radiolabeled material ranged from 87.6% to 97.9% (mean 94.5% ± 4.0%). The majority of the excreted belinostat dose in urine was recovered within 48 hours (83.8%) with only 9.3% in the feces within 96 hours. Renal elimination was the principal route of excretion of radioactivity in all patients, representing a mean of 84.8% ± 9.8% of the administered dose. Fecal excretion accounted for only 9.7% ± 6.5% of the administered dose. Belinostat was shown to be extensively metabolized. The parent compound accounted for only ∼10% of the total drug exposure and was below the level of detection in plasma samples taken 4.5 hours post-dose. Belinostat glucuronide was the major circulating plasma metabolite accounting for ∼65% of the total drug exposure with urinary excretion as the major route of elimination. Belinostat glucuronide was the predominant urinary metabolite accounting for 36% of the total administered dose. Belinostat amide was the most abundant metabolite in feces, accounting for 6% of the total administered radioactivity. The most commonly treatment-related adverse events were Grade 1-2 nausea (50%), infusion site pain (50%) and fatigue (33%). Conclusion: In this study, mass balance was achieved with a mean recovery of 94% radiolabeled belinostat. Belinostat was shown to be extensively metabolized with parent compound accounting for only about 10% of the excreted radiolabeled material and radiolabeled material was primarily excreted by the renal route. Belinostat glucuronide is the primary circulating metabolite and the primary metabolite excreted in the urine. Citation Format: Emiliano Calvo, Valentian Boni, Lina Garcia Canamaque, Guru Reddy, Jette Tjornelund, Tao Song, Mi Rim Choi, Lee F. Allen. Mass balance study of 14C- belinostat in patients with recurrent or progressive malignancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 114. doi:10.1158/1538-7445.AM2015-114

Diuron metabolites and urothelial cytotoxicity: In vivo, in vitro and molecular approaches

Diuron is carcinogenic to the rat urinary bladder at high dietary levels. The proposed mode of action (MOA) for diuron is urothelial cytotoxicity and necrosis followed by regenerative urothelial hyperplasia. Diuron-induced urothelial cytotoxicity is not due to urinary solids. Diuron is extensively metabolized, and in rats, N-(3,4-dichlorophenyl)urea (DCPU) and 4,5-dichloro-2-hydroxyphenyl urea (2-OH-DCPU) were the predominant urinary metabolites; lesser metabolites included N-(3,4-dichlorophenyl)-3-methylurea (DCPMU) and trace levels of 3,4-dichloroaniline (DCA). In humans, DCPMU and DCPU have been found in the urine after a case of product abuse. To aid in elucidating the MOA of diuron and to evaluate the metabolites that are responsible for the diuron toxicity in the bladder epithelium, we investigated the urinary concentrations of metabolites in male Wistar rats treated with 2500ppm of diuron, the urothelial cytotoxicity in vitro of the metabolites and their gene expression profiles. DCPU was found in rat urine at concentrations substantially greater than the in vitro IC50 and induced more gene expression alterations than the other metabolites tested. 2-OH-DCPU was present in urine at a concentration approximately half of the in vitro IC50, whereas DCPMU and DCA were present in urine at concentrations well below the IC50. For the diuron-induced MOA for the rat bladder, we suggest that DCPU is the primary metabolite responsible for the urothelial cytotoxicity with some contribution also by 2-OH-DCPU. This study supports a MOA for diuron-induced bladder effects in rats consisting of metabolism to DCPU (and 2-OH-DCPU to a lesser extent), concentration and excretion in urine, urothelial cytotoxicity, and regenerative proliferation.

Simulation of urinary excretion of 1-hydroxypyrene in various scenarios of exposure to polycyclic aromatic hydrocarbons with a generic, cross-chemical predictive PBTK-model

A physiologically based toxicokinetic (PBTK) model can predict blood and urine concentrations, given a certain exposure scenario of inhalation, dermal and/or oral exposure. The recently developed PBTK-model IndusChemFate is a unified model that mimics the uptake, distribution, metabolism and elimination of a chemical in a reference human of 70 kg. Prediction of the uptake by inhalation is governed by pulmonary exchange to blood. Oral uptake is simulated as a bolus dose that is taken up at a first-order rate. Dermal uptake is estimated by the use of a novel dermal physiologically based module that considers dermal deposition rate and duration of deposition. Moreover, evaporation during skin contact is fully accounted for and related to the volatility of the substance. Partitioning of the chemical and metabolite(s) over blood and tissues is estimated by a Quantitative Structure-Property Relationship (QSPR) algorithm. The aim of this study was to test the generic PBTK-model by comparing measured urinary levels of 1-hydroxypyrene in various inhalation and dermal exposure scenarios with the result of model simulations. In the last three decades, numerous biomonitoring studies of PAH-exposed humans were published that used the bioindicator 1-hydroxypyrene (1-OH-pyrene) in urine. Longitudinal studies that encompass both dosimetry and biomonitoring with repeated sampling in time were selected to test the accuracy of the PBTK-model by comparing the reported concentrations of 1-OHP in urine with the model-predicted values. Two controlled human volunteer studies and three field studies of workers exposed to polycyclic aromatic hydrocarbons (PAH) were included. The urinary pyrene-metabolite levels of a controlled human inhalation study, a transdermal uptake study of bitumen fume, efficacy of respirator use in electrode paste workers, cokery workers in shale oil industry and a longitudinal study of five coke liquefaction workers were compared to the PBTK-predicted values. The simulations showed that the model-predicted concentrations of urinary pyrene and metabolites over time, as well as peak-concentrations and total excreted amount in different exposure scenarios of inhalation and transdermal exposure were in all comparisons within an order of magnitude. The model predicts that only a very small fraction is excreted in urine as parent pyrene and as free 1-OH-pyrene. The predominant urinary metabolite is 1-OH-pyrene-glucuronide. Enterohepatic circulation of 1-OH-pyrene-glucuronide seems the reason of the delayed release from the body. It appeared that urinary excretion of pyrene and pyrene-metabolites in humans is predictable with the PBTK-model. The model outcomes have a satisfying accuracy for early testing, in so-called 1st tier simulations and in range finding. This newly developed generic PBTK-model IndusChemFate is a tool that can be used to do early explorations of the significance of uptake of pyrene in the human body following industrial or environmental exposure scenarios. And it can be used to optimize the sampling time and urine sampling frequency of a biomonitoring program.

In Vitro Glucuronidation of the Antibacterial Triclocarban and Its Oxidative Metabolites

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is widely used as an antibacterial in bar soaps. During use of these soaps, a significant portion of TCC is absorbed by humans. For the elimination from the body, glucuronidation plays a key role in both biliary and renal clearance. To investigate this metabolic pathway, we performed microsomal incubations of TCC and its hydroxylated metabolites 2'-OH-TCC, 3'-OH-TCC, and 6-OH-TCC. Using a new liquid chromatography-UV-mass spectrometry method, we could show a rapid glucuronidation for all OH-TCCs by the uridine-5'-diphosphate-glucuronosyltransferases (UGT) present in liver microsomes of humans (HLM), cynomolgus monkeys (CLM), rats (RLM), and mice (MLM). Among the tested human UGT isoforms, UGT1A7, UGT1A8, and UGT1A9 showed the highest activity for the conjugation of hydroxylated TCC metabolites followed by UGT1A1, UGT1A3, and UGT1A10. Due to this broad pattern of active UGTs, OH-TCCs can be efficiently glucuronidated in various tissues, as shown for microsomes from human kidney (HKM) and intestine (HIM). The major renal metabolites in humans, TCC-N-glucuronide and TCC-N'-glucuronide, were formed at very low conversion rates (<1%) by microsomal incubations. Low amounts of N-glucuronides were generated by HLM, HIM, and HKM, as well as by MLM and CLM, but not by RLM, according to the observed species specificity of this metabolic pathway. Among the human UGT isoforms, only UGT1A9 had activity for the N-glucuronidation of TCC. These results present an anomaly where in vivo the predominant urinary metabolites of TCC are N and N'-glucuronides, but these compounds are slowly produced in vitro.

Urinary and serum metabolites of di- n-pentyl phthalate in rats

Di- n-pentyl phthalate (DPP) is used mainly as a plasticizer in nitrocellulose. At high doses, DPP acts as a potent testicular toxicant in rats. We administered a single oral dose of 500 mg kg −1 bw of DPP to adult female Sprague–Dawley rats ( N = 9) and collected 24-h urine samples 1 d before and 24- and 48-h after DPP was administered to tentatively identify DPP metabolites that could be used as exposure biomarkers. At necropsy, 48 h after dosing, we also collected serum. The metabolites were extracted from urine or serum, resolved with high performance liquid chromatography, and detected by mass spectrometry. Two DPP metabolites, phthalic acid (PA) and mono(3-carboxypropyl) phthalate (MCPP), were identified by using authentic standards, whereas mono- n-pentyl phthalate (MPP), mono(4-oxopentyl) phthalate (MOPP), mono(4-hydroxypentyl) phthalate (MHPP), mono(4-carboxybutyl) phthalate (MCBP), mono(2-carboxyethyl) phthalate (MCEP), and mono- n-pentenyl phthalate (MPeP) were identified based on their full scan mass spectrometric fragmentation pattern. The ω − 1 oxidation product, MHPP, was the predominant urinary metabolite of DPP. The median urinary concentrations (μg mL −1) of the metabolites in the first 24 h urine collection after DPP administration were 993 (MHPP), 168 (MCBP), 0.2 (MCEP), 222 (MPP), 47 (MOPP), 26 (PA), 16 (MPeP), and 9 (MCPP); the concentrations of metabolites in the second 24 h urine collection after DPP administration were significantly lower than in the first collection. We identified some urinary metabolic products in the serum, but at much lower levels than in urine. Because of the similarities in metabolism of phthalates between rats and humans, based on our results and the fact that MHPP can only be formed from the metabolism of DPP, MHPP would be the most adequate DPP exposure biomarker for human exposure assessment. Nonetheless, based on the urinary levels of MHPP, our preliminary data suggest that human exposure to DPP in the United States is rather limited.

Assessing Exposure to Atrazine and Its Metabolites Using Biomonitoring

BackgroundAtrazine (ATZ) is the second most abundantly applied pesticide in the United States. When we assessed exposure to ATZ by measuring its urinary mercapturic acid metabolite, general population data indicated that < 5% of the population was exposed to ATZ-related chemicals (limit of detection < 0.8 ng/mL).ObjectivesThe aim of our study was to determine if we were underestimating ATZ exposure by measuring its urinary mercapturic acid metabolite and if the urinary metabole profile changed with the exposure scenario.MethodsWe conducted a small-scale study involving 24 persons classified as high- (n = 8), low(n = 5), and environmental- (n = 11) exposed to ATZ. Using online solid phase extraction high performance liquid chromatography–tandem mass spectrometry, we measured nine ATZ-related metabolites in urine that included dealkylated, hydroxylated, and mercapturic acid metabolites.ResultsWe found that the urinary metabolite profiles varied greatly among exposure scenarios and among persons within each exposure scenario. Although diaminochlorotriazine (DACT) appeared to be the predominant urinary metabolite detected in each exposure category, the variation in proportion of total ATZ metabolites among persons was consistently large, suggesting that one metabolite alone could not be measured as a surrogate for ATZ exposure.ConclusionsWe have likely been underestimating population-based exposures by measuring only one urinary ATZ metabolite. Multiple urinary metabolites must be measured to accurately classify exposure to ATZ and its environmental degradates. Regardless, DACT and desethylatrazine appear to be the most important metabolites to measure to evaluate exposures to ATZ-related chemicals.

Evaluation of the genotoxic potential of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites, glycidol and β-chlorolactic acid, using the single cell gel/comet assay

3-Monochloropropane-1,2-diol (3-MCPD) is a member of a group of chemicals known as chloropropanols. It is found in many foods and food ingredients as a result of food processing. 3-MCPD is regarded as a rat carcinogen known to induce Leydig-cell and mammary gland tumours in males and kidney tumours in both genders. The aim of our study was to clarify the possible involvement of genotoxic mechanisms in 3-MCPD induced carcinogenicity at the target organ level. For that purpose, we evaluated DNA damages in selected target (kidneys and testes) and non-target (blood leukocytes, liver and bone marrow) male rat organs by the in vivo alkaline single cell gel electrophoresis (comet) assay, 3 and 24 h after 3-MCPD oral administration to Sprague-Dawley and Fisher 344 adult rats. 3-MCPD may be metabolised to a genotoxic intermediate, glycidol, whereas the predominant urinary metabolite in rats following 3-MCPD administration is β-chlorolactic acid. Therefore, we also studied the DNA damaging effects of 3-MCPD and its metabolites, glycidol and β-chlorolactic acid, in the in vitro comet assay on CHO cells. Our results show the absence of genotoxic potential of 3-MCPD in vivo in the target as well as in the non-target organs. Glycidol, the epoxide metabolite, induced DNA damages in CHO cells. β-Chlorolactic acid, the main metabolite of 3-MCPD in rats, was shown to be devoid of DNA-damaging effects in vitro in mammalian cells.

Identification of the 1-cyano-3,4-epithiobutane-derived urinary mercapturic acid N-acetyl-S-(4-cyano-2-thio-1-butyl)-cysteine in male Fischer 344 rats.

1-Cyano-3,4-epithiobutane (CEB), a naturally occurring nitrile derived from cruciferous plants, causes nephrotoxicity in male Fischer 344 rats. Nephrotoxicity induced by CEB is dependent on glutathione (GSH) conjugation and bioactivation. Conjugation with GSH and subsequent metabolism leads to the formation of specific urinary metabolites. The objectives of the present study were to identify CEB-derived urinary metabolites and quantify urinary non-protein thiols and thioethers in male Fischer 344 rats. Animals received 125 mg kg(-1) of CEB alone or following pretreatment with one of three selective inhibitors of GSH metabolism: acivicin, probenecid or aminooxyacetic acid. Total non-protein urinary thiol and urinary thioether concentrations were elevated in all treated groups at 12 and 24 h; however, elevations in non-protein thiols were not significantly greater in rats administered CEB alone as compared to negative controls. A single predominant urinary metabolite was identified as the CEB-derived mercapturic acid N-acetyl-S-(4-cyano-thio-1-butyl)-cysteine. Evidence for other CEB-derived metabolites was also demonstrated. These findings represent the identification of a unique compound and provide further evidence for the importance of GSH conjugation as a significant pathway in CEB metabolism.

Potency of monomethyl-, dimethylformamide and some of their metabolites to induce abnormal development in a limb Bud organ culture

DMF, NMF and their major metabolites were investigated for their developmental toxicity in the mouse limb bud assay. We found that neither DMF, NMF nor the predominant urinary metabolite HMFF exhibited developmental activity. In contrast, all metabolites resulting from the glutathione binding pathway, SMG, SMC and AMCC showed potent developmental activity. Under the chosen exposure conditions, the developmental toxicity of DMF in different species appears to be related to the magnitude of glutathione binding. The results further show the value of using an in vitro system which is incapable of metabolic transformation of exogenous compounds for the identification of ultimate teratogenic species.

Metabolic fate of endogenously synthesized prostaglandin D 2 in a human female with mastocytosis

Increased production of prostaglandin D 2 was recently demonstrated in patients with systemic mastocytosis. One female patient investigated with mastocytosis was found to have overproduction of prostaglandin D 2 of such magnitude (150-fold above normal) that it provided the unique opportunity to delineate the metabolic fate of endogenously synthesized prostaglandin D 2. A five percent aliquot of a twenty-four hour urine collection from the patient was extracted, purified by silicic acid chromatography, methylated, and finally subjected to high pressure liquid chromatography. Column fractions collected were derivatized and analyzed by gas chromatography-mass spectrometry. Increased quantities of sixteen urinary metabolites were identified and included a series of metabolites retaining the PGD-ring as well as series of metabolites with a PGF-ring. PGF-ring metabolites were excreted in approximately 4-fold greater relative abundance than PGD-ring metabolites. More than one apparent isomeric form of some PGF-ring metabolites were found. The predominant urinary metabolite was 2,3-dinor-prostaglandin F 2. This study provides evidence that endogenously synthesized prostaglandin D 2 is converted in substantial part to prostaglandin F 2 metabolites in vivo in humans.

Lymphatic absorption and metabolism of orally administered testosterone undecanoate in man.

[3H]-testosterone undecanoate ([3H]TU) was administered orally to 4 patients with a thoracic duct catheter after neck dissection surgery. Appearance of radioactivity in lymph, plasma and urine was measured at different times. Metabolites of TU in these fluids were investigated. Peak levels of radioactivity appeared simultaneously in lymph and plasma (2.5-5 h after administration) while the excretion in urine was highest approximately 2 h after the plasma and lymph peak. The main compounds appearing in the lymph were TU and 5alpha-dihydrotestosterone undecanoate (5alpha-DHTU), but 5beta-DHTU could not be detected. In plasma almost all metabolites were probably conjugated. During the first 24 h approximately 40% of the administered radioactivity was excreted in the urine. The total amount of radioactivity excreted in the urine during the first week was 45-48%. The predominant urinary metabolites were testosterone- and androsterone-glucuronide. The results indicate that TU is metabolized partly in the intestinal wall. The remaining TU and newly-formed 5alpha-DHTU, at least partly, are absorbed via the lymphatic system.