Testosterone Glucuronide Research Articles

Plasma and urinary markers of oral testosterone undecanoate misuse.

Orally administered testosterone undecanoate (TU), an anabolic, androgenic steroid, can potentially be abused by athletes. Indirect evidence for detecting oral TU intake could be deduced from the changes in steroid profile post-administration. Direct evidence could be obtained by detection of unchanged TU in plasma. To this end, both urinary and plasma steroid profiles of six healthy male subjects given a single oral dose of 120 mg of TU were studied by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The increased concentration of glucuronidated testosterone in plasma appears to be the most characteristic sign of oral TU intake. The testosterone glucuronide (TG)/nonconjugated testosterone (T) ratio, TG/17-hydroxyprogesterone (17OHP) ratio, and TG/luteinizing hormone (LH) ratio were observed to be significantly elevated above their basal levels for 10 h, 10 h, and 6 h, respectively. Urinary ratios of TG/epitestosterone glucuronide (EG) were found to be higher than the cut-off value of 6 for the period 4 approximately 8 h post-administration, but only in three subjects. One subject failed to respond with respect to all of the above-mentioned indirect markers, as TG was not significantly increased in either plasma or urine. Unchanged TU was directly detected in plasma of all six subjects from 1 approximately 1.5 h to 4 approximately 6 h after oral TU intake by GC/MS/MS, providing unequivocal proof of exogenous testosterone intake. Distinct and complementary markers for detecting oral TU intake could be obtained from plasma and urine, respectively.

Mass spectrometry of steroid glucuronide conjugates. II-Electron impact fragmentation of 3-keto-4-en- and 3-keto-5alpha-steroid-17-O-beta glucuronides and 5alpha-steroid-3alpha,17beta-diol 3- and 17-glucuronides.

The steroid glucuronide conjugates of 16,16,17-d(3)-testosterone, epitestosterone, nandrolone (19-nortestosterone), 16,16,17-d(3)-nortestosterone, methyltestosterone, metenolone, mesterolone, 5alpha-androstane-3alpha,17beta-diol, 2,2,3,4,4-d(5)-5alpha-androstane-3alpha,17beta-diol, 19-nor-5alpha-androstane-3alpha,17beta-diol, 2,2,4,4-d(4)-19-nor-5alpha-androstane-3alpha,17beta-diol and 1alpha-methyl-5alpha-androstane-3alpha/beta,17beta-diol were synthesized by means of the Koenigs-Knorr reaction. Selective 3- or 17-O-conjugation of bis-hydroxylated steroids was performed either by glucuronidation of the corresponding steroid ketole and subsequent reduction of the keto group or via a four-step synthesis starting from a mono-hydroxylated steroid including (a) protection of the hydroxy group, (b) reduction of the keto group, (c) conjugation reaction and (d) removal of protecting groups. The mass spectra and fragmentation patterns of all glucuronide conjugates were compared with those of the commercially available testosterone glucuronide and their characterization was performed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. For mass spectrometry the substances were derivatized to methyl esters followed by trimethylsilylation of hydroxy groups and to pertrimethylsilylated products using labelled and unlabelled trimethylsilylating agents. The resulting electron ionization mass spectra obtained by GC/MS quadrupole and ion trap instruments, full scan and selected reaction monitoring experiments are discussed, common and individual fragment ions are described and their origins are proposed.

Effects of prochloraz and nonylphenol diethoxylate on hepatic biotransformation enzymes in trout: a comparative in vitro/in vivo-assessment using cultured hepatocytes

The suitability of cultured rainbow trout hepatocytes as a model system for the assessment of xenobiotic effects on hepatic biotransformation enzymes in fish was examined. Two model water pollutants, the imidazole fungicide prochloraz and the alkylphenolic compound nonylphenol diethoxylate (NP2EO), were investigated in a comparative in vitro/in vivo approach. Biotransformation enzymes were measured in cultured rainbow trout hepatocytes following exposure to xenobiotics in vitro, or in the liver of juvenile rainbow trout ( Oncorhynchus mykiss) exposed in vivo. The patterns of biochemical responses to the model pollutants were generally similar between in vitro and in vivo investigations. Levels of cytochrome P4501A (CYP1A) protein and the catalytic activity of the CYP1A-dependent enzyme 7-ethoxyresorufin- O-deethylase (EROD) were induced in vitro after 24 h of exposure to 1.0 μM prochloraz. In vitro, higher prochloraz concentrations induced only the levels of CYP1A above control levels, but not EROD activity. In vivo exposure of juvenile trout to 0.27 μM prochloraz resulted in an induction of CYP1A and EROD after 7 and 14 days, while 0.027 μM prochloraz had no effects. In vitro, the 6β- and 16β-hydroxylation of testosterone was significantly decreased by 1.0–3.0 μM prochloraz, while in vivo these variables were significantly inhibited after exposure to 0.27 μM prochloraz for 7 and 14 days. NP2EO did not affect EROD activity in vitro. In vivo, EROD activity and CYP1A remained unchanged following 7 days of exposure to 0.32 or 1.30 μM NP2EO. NP2EO (15–50 μM) inhibited the 16β-hydroxylation and glucuronidation of testosterone in vitro. In vivo, 7 days of exposure to 0.32 or 1.30 μM NP2EO resulted in a significant inhibition of the 6β- and 16β-hydroxylation of testosterone. The good qualitative correspondence between in vitro and in vivo results indicates that studies using trout hepatocytes allow the identification of biochemical targets of xenobiotic effects in fish liver. However, more research is needed before quantitative predictions, e.g. of effective concentrations, can be made from in vitro investigations.

Sex steroids in plasma of lake whitefish Coregonus clupeaformis during spawning in Lake Erie

Lake whitefish, Coregonus clupeaformis, were collected from the Western basin of Lake Erie during spawning. Free and conjugated (sulfated and glucuronidated) steroids including testosterone (T), 11-ketotestosterone (11-kT), estradiol-17β (E2) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) were measured in the plasma by radioimmunoassay. In males, the progression of spermiation was characterized by a significant decrease in plasma free steroids, whereas the levels of conjugated steroids remained similar and low, except for sulfated and glucuronidated testosterone. Plasma sex steroids did not correlate with the density or the motility of the spermatozoa. In females, the concentration of plasma T was significantly higher in preovulating than in ovulating females. The levels of E2 and 17,20βP in ovulating lake whitefish exhibited large variations ranging from below detection limit to 0.9 ng ml −1 and from 0.2 to 13 ng ml −1, respectively. Analysis of conjugated steroids revealed high levels of glucuronidated and sulfated 17,20βP and glucuronidated T in females ovulating in December. However, no significant differences in the proportion of the conjugated steroids were observed.

Oral Testosterone Administration Detected by Testosterone Glucuronidation Measured in Blood Spots Dried on Filter Paper

Blood sampling is not a common practice for sports drug testing. Our aim was to investigate whether dried blood spots on filter paper could be an alternative to plasma samples for monitoring steroid profiles in dope testing. We collected dried blood spots and plasma from six healthy Caucasian subjects after an oral 120-mg dose of testosterone undecanoate (TU). Nonconjugated testosterone, testosterone glucuronide (TG), androsterone glucuronide (AG), and etiocholanolone glucuronide (EtG) were measured by gas chromatography-mass spectrometry in both matrices. 17alpha-Hydroxyprogesterone (17alphaOHP) and luteinizing hormone (LH) also were measured in the plasma samples. For comparison, similar measurements were done on samples obtained from the same subjects given 25 mg of testosterone propionate (TP) plus 110 mg of testosterone enanthate (TE) intramuscularly after a wash-out period. After oral TU intake, TG, AG, and EtG increased sharply, whereas nonconjugated testosterone did not change significantly. Results on dried blood spots correlated well with those on plasma. The TG/testosterone ratio in blood or plasma was verified to be a sensitive and specific marker (significantly increased for up to 8 h after intake; P <0.05) for oral TU intake but not for intramuscular administration of TP plus TE. Little suppression of plasma LH and 17alphaOHP was observed after a single oral dose of TU. One subject did not show a significant increase of blood TG after oral TU intake. The measurement of glucuronide conjugates in blood and plasma samples is relevant for sports drug testing when analyzing the steroid profile. Dried blood spots collected on filter paper are a suitable alternative to plasma for detecting testosterone abuse.

Determination of the kinetics of rat UDP-glucuronosyltransferases (UGTs) in liver and intestine using HPLC

Uridinediphosphoglucuronosyl transferases (UGTs) are a group of membrane bound proteins which catalyze the transfer of glucuronic acid from UDP-glucuronic acid to a wide variety of xenobiotics and drug molecules enabling them to be eliminated. The major UGT isoforms found in the rat are 1A1, 1A6, 2B1 and 2B12. Conventional methods for the assay of glucuronides (GLs) include TLC, extraction and colorimetry or quantification of the aglycone, liberated after hydrolyzing the GL with β-glucuronidase. However these techniques cannot distinguish between isomeric GLs or GLs of multiple acceptor site substrates. Therefore the purpose of this study was to develop simple and sensitive HPLC methods for the direct and simultaneous analysis of the GL(s) and their aglycones without the drawbacks of the conventional methods. The three classical substrates we chose were 4-methylumbelliferone (4MU), testosterone (TES) and 8-hydroxyquinoline (8HOQ) representing UGT isoforms 1A6, 2B1 and 2B12 of the rat family, respectively. Here we report the validated HPLC conditions, for the detection and separation of 4-methylumbelliferone glucuronide (4MUG), testosterone glucuronide (TESG) and 8-hydroxyquinoline glucuronide (8HOQG) and their aglycones in incubation media containing male Sprague–Dawley rat liver and intestinal microsomal preparations. The separations were achieved on a Zorbax SB-CN column (150×4.6 mm, 5 μ). The analysis time for the separation of TES, 8HOQ and 4MU and their glucuronides were 17, 12 and 30 min, respectively. The methods showed excellent linearity ( r 2>0.99) over the concentration ranges tested (0.25–5.0 nmoles of TESG; 0.125–18.75 nmoles of 8HOQG and 0.125–12.5 nmoles of 4MUG), good precision and accuracy (RSD<2.5%). Inter-day variability studies ( n=3) showed no significant difference between the regression lines obtained on the three days. Recoveries were good (>90%) at all three points (low, mid-point, high) of the standard curve. The limits of detection were 0.125, 0.1 and 0.1 nmole for TESG, 8HOQG and 4MUG, respectively. The above methods were used to estimate kinetic parameters such as V max and K m for the GLs of the three substrates in both liver and intestinal tissue preparations and the values were comparable with previously reported results. UGT2B1 was found primarily in the liver while UGTs 1A6 and 2B12 were present in comparable amounts in both tissues.

Evaluation of Testosterone/Epitestosterone Ratio Influential Factors as Determined in Doping Analysis

The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in doping analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair analysis, and high-performance liquid chromatography-mass spectrometry, are discussed.

Quantification of testosterone and epitestosterone in human urine by capillary liquid chromatography

A capillary-liquid chromatography (LC) method was developed for the quantification of the endogenous steroids testosterone and epitestosterone in human urine. One milliliter of urine was used for the overall method. Free testosterone was first separated by liquid–liquid extraction with n-pentane at pH 7. Glucuronides of testosterone and epitestosterone were enzymatically hydrolyzed and the free compounds were extracted with n-pentane at pH 11. A capillary column switching system with a low back pressure precolumn (PC) was used for fast loading of large sample volumes (20 μL). Chromatographic separation was carried out on a 15 cm×300 μm inner diameter (i.d.) column, packed with 3 μm Hypersil BDS-C18 at a flow rate of 4 μL/min with isocratic elution and UV absorbance detection (240 nm). Limit of detection for free testosterone was established at 0.5 ng/mL. Limits of detection were established at 1.5 and 3.2 ng/mL for testosterone and epitestosterone, respectively, after being hydrolysed from their glucuronides. Good reproducibility and robustness were observed through the entire calibration range (up to 250 ng/mL). © 2001 John Wiley & Sons, Inc. J Micro Sep 12: 623–629, 2000

Prolonged‐release gonadotrophin‐releasing hormone analogue implants enhance oocyte final maturation and ovulation, and increase plasma concentrations of sulphated C21 steroids in North Sea plaice

Reproductively mature female plaice were implanted with or without 50 μg of gonadotrophin‐releasing hormone analogue (GnRHa), suspended in either coconut oil or methacrylate resin. The weight of the GnRHa‐treated fish increased significantly (due to hydration of the oocytes) and reached a peak between 10 and 14 days. The fish produced several batches of eggs, which were consistently bigger than those produced by control fish. Plasma concentrations of free 17β‐oestradiol and glucuronidated testosterone rose briefly (4 days) in response to the GnRHa, but then fell continuously till the end of the experiment (20 days). Plasma concentrations of sulphated 5β‐pregnane‐3α,17,20β‐triol and 5β‐pregnane‐3β,17,20β‐triol (which are putative metabolites of 17,20β‐dihydroxy‐4‐pregnen‐3‐one, the oocyte maturation‐inducing steroid) increased significantly at 4 days and reached a peak between 12 and 16 days. Concentrations were still very elevated on day 20. Plasma concentrations of sulphated 3α,17,21‐trihydroxy‐5β‐pregnan‐20‐one showed a slight increase on day 4 but did not change thereafter. There was a highly significant difference in the amounts of GnRHa released into the bloodstream by the two methods of administration on day 4. However, this was not matched by significant differences in the concentrations of any of the steroids.

Optimisation of UDP-glucuronyl transferase-catalysed synthesis of testosterone-β-D-glucuronide by inhibition of contaminating β-glucuronidase

Attempted synthesis of testosterone glucuronide using commercial UDP-glucuronyl transferase from either bovine or ovine sources was unsuccessful, giving 2–5% yields. This low yield was due to contaminating β-glucuronidase which hydrolysed the product. Limitation of glucuronidase activity using improved pH control and inhibitors, plus optimisation of other reaction conditions, increased the yield to 20–30% with the ovine enzyme and allowed practical preparative synthesis.

Isolated Absence of Adrenarche in A Postpubertal Female

Background The development of pubic and axillary hair (adrenarche) is part of the process of normal puberty. We report a case of isolated absence of adrenarche in a post-pubertal female. A review of the literature reveals no previously reported case of isolated absence of adrenarche in a postpubertal female. An investigation was understaken to evaluate the etiology of the lack of sexual hair growth in this patient. Case A 14 year-old postpubertal female presented complaining of heavy menstrual bleeding approximately four months after her first menstrual period. The patient had been previously diagnosed with a developmental delay and speech impediment by her referring physician. Further workup revealed a normal menstrual pattern, yet the physical examination was remarkable for the absence of axillary and pubic hair, as well as short stature. A complete evaluation was performed which included chromosome analysis, serum dehydroepiandrosterone sulfate (DHEA-S), androstenedione, testosterone, dihydrotestosterone (DHT), and 3-alpha androstanediol glucuronide. The serum androstenedione, testosterone, DHT, and 3-alpha androstanediol glucuronide levels were within normal limits. The DHEA-S levels were low yet adrenal function as analyzed by the Adrenocorticotropic hormone (ACTH) stimulation test was normal. Radiologic studies included pelvic ultrasound and bone age assessment with a bone age of 16 years. Skin biopsy of the mons pubis documents the presence of hair follicles. No previous or similar history has been noted in the patient's family history. Also of note, the patient is the product of a consanguineous relationship. Conclusion After review of our investigational findings, it is felt that this unique case likely represents an androgen receptor or post-receptor defect in the hair follicle.

UDP-glucuronosyltransferases in human intestinal mucosa

While UDP-glucuronosyltransferases (UGTs) are known to be expressed at high levels in human liver, relatively little is known about extrahepatic expression. In the present study, UGT2B family isoforms involved in the glucuronidation of steroid hormones and bile acids have been characterized in microsomes prepared from jejunum, ileum and colon from six human subjects. Glucuronidation of androsterone and testosterone was highly significant and increased from proximal to distal intestine. In contrast, hyodeoxycholic acid was glucuronidated at a low level in jejunum and ileum and activity was barely detectable in colon. No significant glucuronidation of lithocholic acid was found. Small phenols were glucuronidated with much lower activity than found in liver. High levels of UGT protein were detected with polyclonal anti-rat androsterone- and testosterone-UGT antibodies, whereas UGT2B4, a major hepatic hyodeoxycholic acid-specific UGT, was undetectable using a highly specific anti-human UGT2B4 antibody. Screening for RNA expression by RT-PCR confirmed the absence of UGT2B4 and UGT1A6 and showed expression of UGT2B7, a hepatic isoform shown to glucuronidate androsterone, in all intestinal segments. To our knowledge, the presence of functional androsterone and testosterone directed isoforms in human intestine is a novel finding which supports the idea that the intestinal tract functions as a steroid-metabolizing organ and plays a significant role in steroid hormone biotransformation.

Regulation of uridine diphosphate glucuronosyltransferase during the acute-phase response.

The acute-phase response is associated with profound effects on oxidative drug metabolism. However, the effects on glucuronidation are poorly characterized. The aim of the present study was to determine the role of mediators of the acute-phase response in the regulation of hepatic uridine diphosphate glucuronosyltransferase (UGT) expression. Family 1 and family 2 UGT isoforms were studied in turpentine-injected rats and in primary hepatocyte cultures exposed to cytokines and/or dexamethasone. In the in vivo model, glucuronidation of p-nitrophenol was unaffected, while testosterone glucuronidation was reduced to 65% of control (P<0.01). In contrast, the mRNA level of UGT1*1 (which metabolizes bilirubin, not phenols) was depressed to 16% of control (P<0.002), while the mRNA level of UGT2B3 (which metabolizes testosterone) was reduced to 53% (P<0.05). In primary hepatocyte culture, dexamethasone treatment resulted in a 3.4-fold induction of UGT1*1 mRNA levels (P<0.001) but only a 1.5-fold induction of UGT2B3 (P=0.1). Interleukin-6 in the presence of dexamethasone resulted in a marked dose-dependent suppression of both UGT1*1 and UGT2B3, although to different degrees. Interleukin-1 had no effect on UGT mRNA levels. Thus, inflammatory mediators, such as cytokines and glucocorticoids, may be important determinants of both oxidative and conjugative drug metabolism by the liver.

Plasma concentrations of ovarian steroids in relation to oocyte final maturation and ovulation in female plaice sampled at sea

Blood plasma concentrations of free 17β‐oestradiol, free testosterone and glucuronidated testosterone were strongly positively related to the percentage of vitellogenic oocytes remaining in the ovaries of plaice Pleuronectes platessa caught at sea–being at their highest in pre‐spawning (stage IV) females (i.e. those in which the oocytes were close to fully grown, but had not yet entered the stage of final maturation). In contrast, the concentrations of free and sulphated 17,20β‐P, 3αaL,17, 20β‐P‐5β, and 3α,17,21‐P‐5β were at their lowest in stage IV females. Free 17,20β‐P (the putative maturation‐inducing steroid) became only slightly elevated (less than twofold) during spawning (i.e. in stage V and VI females with hydrated and/or ovulated eggs). Sulphated 17,20β‐P and 3α,17,21‐P‐5β became slightly more elevated (three‐ to fourfold). However, sulphated 3α, 17,20β‐P‐5β concentrations increased 30‐fold and were at their highest in fish in which only 40% of vitellogenic oocytes remained in the ovaries. Sulphated 17,20β‐P, 3α, 17, 20β‐P‐5β and 3α,17,21‐P‐5β concentrations were significantly positively related to hyaline oocyte batch size; and sulphated 17,20β‐P and sulphated 3α, 17,20β‐P‐5β were significantly negatively related to the degree of hydration of the hyaline oocytes. None of the steroid concentrations, however, was related to the time of capture. More ovulated females were found in the afternoon than at any other time of the day.

Testosterone sulphation and glucuronidation in the human liver: interindividual variability.

Presystemic sulphation and glucuronidation at OH-C17 limits the bioavailability of testosterone; the aim of this investigation was to describe the variability in testosterone sulphation and glucuronidation rates in the human liver. Liver samples were obtained from 61 women and 40 men of similar age (mean 53 and 55 years, respectively) submitted to surgery. The mean rate of testosterone sulphation was significantly (P = 0.002) higher in men (22.4 pmol/min/mg) than in women (17.5 pmol/min/mg), was not age-dependent, followed bimodal distribution and varied over 7-fold in men and women. There was a weak, but significant negative correlation (r = -0.380; P = 0.003), between the rate of testosterone glucuronidation and age in the liver of women but not in that of men. The mean rate (pmol/min/mg) of testosterone glucuronidation was 155 (men) and 105 (women) (NS) and varied over 20-fold. When the rate of testosterone glucuronidation was expressed on the basis of g liver equivalent, the mean estimates were significantly (P = 0.003) greater in men (3323 pmol/min/g) than in women (1841 pmol/min/g). The present findings are consistent with the view that the hepatic activities of sulphotransferase and glucuronosyltransferase are higher in men than in women and that they vary in the human liver.

Differential effect of hypophysectomy and growth hormone treatment on hepatic glucuronosyltransferases in male rats: Evidence for an action at a pretranslational level for isoforms glucuronidating bilirubin

The influence of growth hormone (GH) on 4-nitrophenol, bilirubin, testosterone, androsterone and estrone glucuronidation activities was studied in fully activated male rat hepatic microsomes. Sham-operated and hypophysectomized animals were injected with two different dosages of GH, mimicking either the male or female GH secretion pattern. Half the animals received thyroxine and cortisol in concentrations chosen to compensate for the lack of thyroid hormones and glucocorticoids in hypophysectomized rats. GH induced a decrease in several glucuronidation activities: bilirubin glucuronidation in both sham-operated and cortisol/ thyroxine-treated hypophysectomized rats in a dose-dependent manner, testosterone glucuronidation in hypophysectomized animals, and androsterone and estrone glucuronidation in cortisol/thyroxin-treated hypophysectomized rats. 4-nitrophenol glucuronidation was not affected by GH treatment. A hypothetical “feminizing” effect of GH (due to an almost continuous secretion) could not be invoked to explain these results, contrary to what has been observed elsewhere for other hepatic enzyme activities. Hypophysectomy altered all the activities tested, with bilirubin the most modified (a 200% enhancement). Restoration of control values was achieved in hypophysectomized animals with cortisol/thyroxine replacement together with a low dosage of GH (mimicking a male GH secretion pattern), except for androsterone glucuronidation activity where both GH and cortisol/thyroxine treatments reinforced the decreasing effect of hypophysectomy. Variations in protein amounts were correlated to variations in bilirubin, testosterone and androsterone conjugation activities induced by hypo-physectomy and GH treatment. Reverse transcription-polymerase chain reaction (RT-PCR) mRNA analysis of bilirubin cluster isoforms or uridine diphosphate glucuronosyltransferase 1B1 (UGT1B1), UGT1B2 and UGT1B5 showed that GH controlled the different isoforms involved in bilirubin glucuronidation differentially at a pretranslational level.

Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes.

The interactions between UDP-glucuronosyltransferase (UGT) isozymes, UGT1s and UGT2B1, in rat hepatic microsomes were investigated using an immunopurification technique with anti-peptide antibodies and a chemical cross-linking strategy. A 50 kDa protein coimmunopurified with UGT1s was identified as UGT2B1 by amino-terminal sequencing and immunodetection with anti-peptide antibody against UGT2B1. Evidence for direct interaction of UGT2B1 with UGT1s was obtained by the loss of UGT2B1 adsorption to immunoaffinity column in Gunn rat hepatic microsomes, which lack all UGT1 isozymes. When the microsomes were treated with the chemical cross-linking reagent 1,6-bis(maleimido)-hexane, a cross-linked product with an apparent molecular mass of 120-130 kDa was obtained that immunostained with antibodies against UGT1s and UGT2B1, indicating the formation of a heterodimer containing one of the UGT1 isozymes and UGT2B1. The effects of UGT complex formation on the stimulation of glucuronidation of testosterone and uptake of UDP-glucuronic acid (UDP-GlcUA) by UDP-N-acetylglucosamine (UDP-GlcNAc) were examined. Alkaline pH-induced dissociation of the complexes was associated with the loss of UDP-GlcNAc-dependent stimulation of glucuronidation, suggesting that two functional states of UGTs with different kinetic parameters correspond to the monomer and oligomer form of UGTs in the membranes. The UDP-GlcNAc-dependent stimulation of UDP-GlcUA uptake into the microsomal vesicles also was affected by the extent of complex formation. These results suggest that complex formation of the UGT isozymes affects the UDP-GlcNAc-dependent stimulation of glucuronidation via stimulation of UDP-GlcUA uptake.

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry.

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated from normal human urine was achieved by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urine.

Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography-mass spectrometry

Direct detection of several steroid glucuronide and sulfate conjugates was achieved with electrospray reversed-phase HPLC-mass spectrometry. Separation of steroid 17-OH or 5-H epimers conjugated with glucuronide or sulfate could be achieved using gradient elution. Testosterone glucuronide, testosterone sulfate, epitestosterone sulfate and epitestosterone glucuronide were chromatographically resolved, although significant variation in solvent strength was observed between methanol and acetonitrile. Positive ionization mode MS and MS-MS spectra were employed to obtain both quantitative and structural information. Some differences were noted with respect to steroid structure and adduct formation, including significant differences in the stability of epimers in the declustering region of the interface. Negative ionization mode, although having lower limits of detection, did not provide useful structural information in either the MS or MS-MS mode. Using a packed capillary column (300 microns I.D.), a detection limit of 25 pg was achieved for epitestosterone glucuronide.

ATP-dependent transport of beta-estradiol 17-(beta-D-glucuronide) in rat canalicular membrane vesicles.

The ATP-dependent transport of beta-estradiol 17-(beta-D-glucuronide) (E217G), a cholestatic metabolite of estradiol, was investigated in rat liver canalicular membrane vesicles. ATP-dependent transport was dependent on time and temperature and occurred into an osmotically sensitive space; kinetic analysis indicated a saturable transport system (Michaelis-Menten constant value, 75 microM; maximum transport rate, 598 pmol.min-1.mg protein-1). The steroid conjugates estradiol glucuronide, estriol 3-glucuronide, estriol 16 alpha-glucuronide, testosterone glucuronide, and the three-sulfate conjugate of 17G were effective inhibitors of transport. Bromosulfophthalein, S-(2,4-dinitrophenyl)glutathione, and glutathione disulfide, all substrates of the canalicular ATP-dependent non-bile acid organic anion transport system, were also effective inhibitors, whereas taurocholate had no effect on transport. Conversely, E217G inhibited the ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione. Daunorubicin, vinblastine, etoposide, cyclosporin, and PSC-833, substrates/modulators of P-glycoprotein, were also potent inhibitors of E217G transport, and E217G competitively inhibited the ATP-dependent transport of daunorubicin. C219, a monoclonal antibody against P-glycoprotein, inhibited ATP-dependent transport of E217G and daunorubicin but not of taurocholate or S-(2,4-dinitrophenyl)glutathione. These data indicate that E217G is substrate of both the non-bile acid organic anion transport system and P-glycoprotein but not of the ATP-dependent bile acid transport system in canalicular membranes.