Testosterone Glucuronide Research Articles

Meet-in-metabonomics: Insights into associations between hair heavy metal and adverse child growth in e-waste recycling area

Heavy metal pollution from informal e-waste recycling may adversely affect child growth. However, the potential toxic mechanisms from a population perspective remain unknown. Herein, 18 hair heavy metals, urinary metabolomics, and three child growth indices [i.e., weight-for-age Z-score (WAZ), height-for-age Z-score (HAZ), and BMI Z-score (BMIZ)] were measured in children from e-waste recycling (ER, N = 426) and control areas (CR, N = 247). We examined longitudinal changes in heavy metal exposure and child growth after e-waste control to further elucidate causal relationships. Results showed that children in regulated ER site were still exposed to higher levels of several heavy metals and experienced poorer growth compared to those in control areas. Elevated exposure to heavy metals like tin, antimony, lead, cadmium, and cobalt correlated with poor child growth, particularly affecting girls and younger children. Tin, rather than traditionally concerning heavy metals, exhibited the most crucial role in driving the adverse effects of metal mixtures on child growth. Reducing heavy metal exposure through e-waste control could notably improve child growth, confirming the causal relationship between heavy metal exposure and poor child growth and underscoring the health benefits of e-waste regulation. Our research identified the roles of steroid biosynthesis, folate biosynthesis, amino acid metabolism, and purine metabolism in mediating the effects of metal exposure on child growth. Testosterone glucuronide, riboflavin, folic acid, xanthosine, and xanthine emerged as key mediators, potentially serving as metabolic signatures of heavy metal exposure. These findings illuminate the toxic mechanisms underlying poor child growth resulted from heavy metal exposure, offering important insights from a population-based perspective. In addition to lead and cadmium, monitoring and regulating tin and antimony are crucial to mitigate their negative impact on child growth in e-waste recycling areas.

Effectiveness of blood steroidal passport markers for detecting testosterone abuse in Asians.

The World Anti-Doping Agency (WADA) has introduced an Athlete Biological Passport (ABP) with a steroidal module, which is intended for the monitoring of longitudinal profiles of an athlete's steroid variables in urine to identify endogenous anabolic androgenic steroids that are administered exogenously. It has been in use since 2014. The prevalence of UGT2B17 gene deletion with relatively low levels of testosterone (T) glucuronide in urine is high in the Asian region. There are cases in which urinary T is below the detection limit in specific urine samples, for example, diluted urine, urine collected from females, or urine collected from UGT2B17 deletion individuals. Additional steroid markers T, 4-androstenedione (A4), and the T/A4 ratio in serum were newly added to the ABP steroidal module by WADA in 2023 to compensate for the urine steroid profile. In this study, populations of blood steroid markers in Asians (n = 510) were investigated and classified according to UGT2B17 polymorphism to confirm the effectiveness of blood steroid markers in monitoring Asian athletes. No significant difference in the T/A4 ratio was observed between the genotypes. Furthermore, an administration study of T enanthate in females (n = 10) who were classified according to UGT2B17 genotypes was performed. The concentration of T and the T/A4 ratio were found to be significantly increased in all post-administration samples until 15 days after administration (p < 0.01). The overall results supported the high effectiveness of subject-based monitoring for serum T and T/A4 ratio for recently identified shortcomings in the detection of T abuse in Asians.

Urine and serum metabolomics study of wild ginseng in rats with spleen-qi deficiency using rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.

Patients with a spleen-qi deficiency often exhibit dysfunction in the metabolic system. Metabolites are considered the most direct reflection of individual physiological and pathological conditions and represent attractive candidates to provide deep insights into disease phenotypes. This study examines the potential therapeutic mechanism of wild ginseng on spleen-qi deficiency through the analysis of serum and urine metabolomics using rapid-resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry. The reasons for the superiority of wild ginseng treatment over cultivated ginseng were also analyzed in depth. After wild ginseng intervention, anandamide, urobilinogen, aldosterone, and testosterone glucuronide were significantly reduced in serum. Meanwhile, argininosuccinic acid, L-cysteine, and seven other metabolites were significantly elevated in serum. Nine metabolites, including L-acetylcarnitine, and citrulline were elevated in the urine, and trimethylamine N-oxide, adrenic acid, and 10 other metabolites were reduced. Arginine biosynthesis, pantothenate and CoA biosynthesis, thiamin metabolism, taurine, and tryptophan metabolism pathways were mainly improved. Further analysis was conducted on the relationship between Lactobacillus and Akkermansia bacteria with metabolites, and it was found that they are mainly related to amino acid metabolites. This study provides strong theoretical support and direction for further explanation of the immune mechanism of wild ginseng and lays the foundation for future studies.

An insight into gut microbiota and metabolites in the mice with adenomyosis.

Adenomyosis (AM) is a benign uterine disease characterized pathologically by the invasion of endometrial tissue into the myometrium. The pathogenesis of AM is still far from clear. Although the gut microbiome and metabolomics are thought to contribute to a variety of diseases, the role of them in AM has not been revealed. To investigate changes in the gut microbiota and derived metabolites in AM mice. Female ICR mice were randomly assigned to AM and control groups, and pituitary transplantation was employed to perform AM modeling. Then, the fecal samples were obtained for microbial (16S rRNA gene sequencing) and metabolomic (liquid chromatography mass spectrometry, LC-MS) analysis. The results of gut microbiota analysis showed that the intestinal microbiota composition of AM mice was altered. The ratio of Firmicutes/Bacteroidetes and the relative abundance of Lactobacillus in AM group increased compared with the control group. Sixty differential expressed metabolites were identified in intestinal metabolites, mainly involved in steroid hormone biosynthesis, cysteine and methionine metabolism, and alanine, aspartate, and glutamate metabolism. Further, correlation analysis verified that L-methionine and L-cystine were negatively correlated with Bacteroides and positively correlated with Desulfovibrio. The Pregnenolone, Androsterone glucuronide, and Testosterone glucuronide were negatively correlated with Unidentified_Ruminococcaceae and Alistipes, whereas they positively correlated with Bacteroides. AM mice have a unique gut microbiome and intestinal metabolites.

Metabolomic study combined with the low-level light therapy of Chinese acupuncture points and combined oral contraceptives in treatment of primary dysmenorrhea: A prospective, multicenter, randomized controlled study

ObjectiveTo compare the changes of metabolites between Low-level light therapy (LLLT) and combined oral contraceptive (COC) after treatment of primary dysmenorrhea (PD), and to compare and analyze the biological and biochemical effects of the two treatments by analyzing the differences in metabolite profiles. MethodsA multicenter, double-blind, prospective, parallel, randomized controlled study was conducted on 69 women aged 16–35 years old with PD who were randomly divided into COC treatment group or LLLT treatment group. Low-level light therapy with light-emitting diodes (LED) was applied on two acupoints named “Guanyuan” (CV4) and “Qihai” (CV6). After 12 weeks of treatment intervention, blood samples were collected before and after treatment for metabolomic analysis. We used UPLC-MS/MS analysis to compare the differences in metabolite changes between LLLT and COC before and after treatment. Results76 differential metabolites were detected in the LLLT group, and 92 differential metabolites were detected in the COC group, which were up-regulated or down-regulated (p < 0.001). Prostaglandin D2 (PG D2) was down-regulated and biliverdin was up-regulated after LLLT treatment, 4a-Hydroxytetrahydrobiopterin, Prostaglandin D2, 5-Hydroxy-l-tryptophan, Cholic acid were down-regulated and cortisol was up-regulated after COC treatment, and the differences were statistically significant. Cortisol and testosterone glucuronide in LLLT group were significantly lower than those in COC group. The metabolic pathways affected were glycerophospholipid metabolism, linoleic acid metabolism and arachidonic acid metabolism in the LLLT group, and glycerophospholipid metabolism, folate biosynthesis, arachidonic-acid-metabolism, and tryptophan metabolism in the COC group. The differential metabolic pathway were linoleic acid metabolism, steroid hormone biosynthesis, and alpha-Linolenic acid metabolism after the comparison of LLLT with COC. ConclusionLLLT and COC might relieve dysmenorrhea by down-regulating PGD2, and LLLT might also relieve dysmenorrhea by up-regulating biliverdin. The level of cortisol and testosterone glucuronide after LLLT treatment was lower than that after COC treatment, which might lead to the difference in the clinical efficacy of the two treatments for dysmenorrhea.

Age-related decrease in serum dihydrotestosterone concentration is accompanied by impaired vascular status

IntroductionThe effect of androgens on the cardiovascular system in humans is ambiguous. Moreover, still little is known about the effects of the most potent androgen, dihydrotestosterone, on arterial stiffness and endothelial function. The aim of this study was to evaluate whether age-dependent alterations in serum concentration of dihydrotestosterone and its circulating metabolite are accompanied by changes in endothelial function and arterial stiffness. MethodsIn 12 young and 11 older men, basal serum concentrations of testosterone, dehydroepiandrosterone sulfate (DHAE-S), androstenedione (AE), dihydrotestosterone (DHT) and androstanediol glucuronide (ADG) were analyzed in relation to vascular status including cIMT — carotid intima media thickness, cAI — central augmentation index, crPWV — carotid radial pulse wave velocity, SI — stiffness index, endothelial and inflammatory markers. ResultsAlthough concentration of testosterone was not different between young and older group, it was demonstrated that DHT, DHEA-S, AE and ADG were significantly lower in older men in comparison to young men (p < 0.01). Interestingly the most surprising difference was found for DHT concentration, that was as much as 61 % lower in aged men that displayed significantly higher values of cIMT, AI, crPWV and SI (p < 10−4), suggestive of arterial stiffness. Furthermore, DHT was negatively correlated to all arterial wall parameters (cAI, crPWV, SI and cIMT), c-reactive protein (CRP) and hyaluronic acid (HA) concentration, as well as positively correlated to markers of endothelial function (MNA and 6-keto-PGF1α) in all studied individuals (n = 23). ConclusionsWe have shown that ageing leads to a significant decrease in DHT concentration that is accompanied by impaired arterial wall characteristics and worsened endothelial function. Therefore more attention should be paid to the DHT, DHEA-S and ADG concentrations as a biomarkers for vascular dysfunction in ageing men.

Relevance of Human Aldoketoreductases and Microbial β-Glucuronidases in Testosterone Disposition.

Testosterone exhibits high variability in pharmacokinetics and glucuronidation after oral administration. Although testosterone metabolism has been studied for decades, the impact of UGT2B17 gene deletion and the role of gut bacterial β-glucuronidases on its disposition are not well characterized. We first performed an exploratory study to investigate the effect of UGT2B17 gene deletion on the global liver proteome, which revealed significant increases in proteins from multiple biological pathways. The most upregulated liver proteins were aldoketoreductases [AKR1D1, AKR1C4, AKR7A3, AKR1A1, and 7-dehydrocholesterol reductase (DHCR7)] and alcohol or aldehyde dehydrogenases (ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A). In vitro assays revealed that AKR1D1 and AKR1C4 inactivate testosterone to 5β-dihydrotestosterone (5β-DHT) and 3α,5β-tetrahydrotestosterone (3α,5β-THT), respectively. These metabolites also appeared in human hepatocytes treated with testosterone and in human serum collected after oral testosterone dosing in men. Our data also suggest that 5β-DHT and 3α, 5β-THT are then eliminated through glucuronidation by UGT2B7 in UGT2B17 deletion individuals. Second, we evaluated the potential reactivation of testosterone glucuronide (TG) after its secretion into the intestinal lumen. Incubation of TG with purified gut microbial β-glucuronidase enzymes and with human fecal extracts confirmed testosterone reactivation into testosterone by gut bacterial enzymes. Both testosterone metabolic switching and variable testosterone activation by gut microbial enzymes are important mechanisms for explaining the disposition of orally administered testosterone and appear essential to unraveling the molecular mechanisms underlying UGT2B17-associated pathophysiological conditions. SIGNIFICANCE STATEMENT: This study investigated the association of UGT2B17 gene deletion and gut bacterial β-glucuronidases with testosterone disposition in vitro. The experiments revealed upregulation of AKR1D1 and AKR1C4 in UGT2B17 deletion individuals, and the role of these enzymes to inactivate testosterone to 5β-dihydrotestosterone and 3α, 5β-tetrahydrotestosterone, respectively. Key gut bacterial species responsible for testosterone glucuronide activation were identified. These data are important for explaining the disposition of exogenously administered testosterone and appear essential to unraveling the molecular mechanisms underlying UGT2B17-associated pathophysiological conditions.

Androgen Glucuronidation in Mice: When, Where, and How.

Simple SummaryHormone metabolism can vary from one species to another. In humans, specific UDP-glucuronosyltransferase (UGT) enzymes transform androgens (the male hormones) into glucuronide derivatives, which are easier to eliminate. Whether a similar mechanism also takes place in mice has never been ascertained. This study aimed at addressing this question. Organs and pure Ugt2b enzymes from mice were assayed for their ability to transform several androgens into their glucuronide derivatives. Results show that, as in humans, both murine organs and enzymes are reactive with androgen molecules, and glucuronide derivatives are formed with substrate-, organ- and enzyme-specific manner. In conclusion, these observations revealed that glucuronosyltransferase enzymes from mice works in a similar manner as their human counterparts.Glucuronidation, catalyzed by UDP-glucuronosyltransferase UGT2B enzymes, is a major inactivating and elimination pathway for androgen hormones in humans. Whether Ugt2b enzymes from mice are also reactive with these hormones have never been investigated. The present study aimed at evaluating the capability of murine tissues and Ugt2b enzymes to glucuronidated androgens. The 7 murine Ugt2b (Ugt2b1, 2b5, 2b34, 2b35, 2b36, 2b37 and 2b38) enzymes were cloned and stably expressed into HEK293 cells. In vitro glucuronidation assays were performed with microsomal proteins or homogenates from mice tissues (liver, kidney, intestine, adipose, testis, prostate, epididymis, bulbo, seminal vesicle, mammary glands, uterus, and ovary) and from Ugt2b-HEK293 cells. Male and female livers, as well as male kidneys, are the major sites for androgen glucuronidation in mice. The male liver is highly efficient at glucuronidation of dihydrotestosterone (DHT) and testosterone and is enriched in Ugt2b1 and 2b5 enzymes. Androsterone and 3α-Diol are conjugated in the male kidney through an Ugt2b37-dependent process. Interestingly, castration partially abolished hepatic Ugt2b1 expression and activity, while Ugt2b37 was totally repressed. DHT injection partially corrected these changes. In conclusion, these observations revealed the substrate- and tissue-specific manner in which murine Ugt2b enzymes conjugate androgens. They also evidence how androgens modulate their own glucuronide conjugation in mice.

Potential Assessment of UGT2B17 Inhibition by Salicylic Acid in Human Supersomes In Vitro.

Glucuronidation is a Phase 2 metabolic pathway responsible for the metabolism and excretion of testosterone to a conjugate testosterone glucuronide. Bioavailability and the rate of anabolic steroid testosterone metabolism can be affected upon UGT glucuronidation enzyme alteration. However, there is a lack of information about the in vitro potential assessment of UGT2B17 inhibition by salicylic acid. The purpose of this study is to investigate if UGT2B17 enzyme activity is inhibited by salicylic acid. A UGT2B17 assay was developed and validated by HPLC using a C18 reversed phase column (SUPELCO 25 cm × 4.6 mm, 5 μm) at 246 nm using a gradient elution mobile phase system: (A) phosphate buffer (0.01 M) at pH = 3.8, (B) HPLC grade acetonitrile and (C) HPLC grade methanol. The UGT2B17 metabolite (testosterone glucuronide) was quantified using human UGT2B17 supersomes by a validated HPLC method. The type of inhibition was determined by Lineweaver–Burk plots. These were constructed from the in vitro inhibition of salicylic acid at different concentration levels. The UGT2B17 assay showed good linearity (R2 > 0.99), acceptable recovery and accuracy (80–120%), good reproducibility and acceptable inter and intra-assay precision (<15%), low detection (6.42 and 2.76 μM) and quantitation limit values (19.46 and 8.38 μM) for testosterone and testosterone glucuronide respectively, according to ICH guidelines. Testosterone and testosterone glucuronide were found to be stable up to 72 h in normal laboratory conditions. Our investigational study showed that salicylic acid uncompetitively inhibited UGT2B17 enzyme activity. Thus, drugs that are substrates for the UGT2B17 enzyme have negligible potential effect of causing interaction with salicylic acid in humans.

Novel mechanisms of testosterone detoxification in UGT2B17 gene deletion carriers and androgen activation by gut microbiome

Background and objectives Testosterone metabolism by the highly polymorphic UDP-glucuronosyltransferase 2B17 (UGT2B17) is widely recognized, but the metabolic fate of testosterone in the carriers of UGT2B17 gene deletion is not well understood. Similarly, testosterone glucuronide (TG) is prone to secretion into intestinal lumen (1); however, the role of microbiome in activation of testosterone is not studied. Therefore, the aims of this study were: i) to elucidate the alteration in the liver proteome and its effect on steroid metabolism pathways (i.e., metabolic switching) in the UGT2B17 gene deletion, and ii) to assess the TG deconjugation by gut β-glucuronidases. Methods Human liver S9 fraction with gene deletion and high expression (n=3) were used. Untargeted proteomics data was acquired using Easy nano-LC 1200 coupled with the high-resolution QE-HF Orbitrap. The proteomic data were analyzed, processed and pathway mapping was performed using MaxQuant, Perseus and STRING, respectively. In vitro studies in liver S9 fraction of UGT2B17 gene deletion were carried out to investigate testosterone metabolism by aldo-keto reductases (AKRs). The glucuronidation pathway of 5β-dihydrotestosterone (5β-DHT) and 5β-tetrahydrotestosterone (5β-THT) were identified using recombinant UGT2B isoforms. These data were verified using testosterone incubations in human hepatocyte and serum levels of 5β-THTG after oral testosterone dosing (800 mg) in human. Activation mechanism TG to testosterone in the human gut was investigated by incubating TG with recombinant human gut microbial β-glucuronidases and human fecal extracts. Results A total of 1002 liver proteins were quantified in the S9 fraction, of which 14 and 39 proteins were exclusively from the deletion and high-expressor groups, respectively, whereas 949 proteins were common. The following pathways were upregulated in UGT2B17 deletion group: oxidation-reduction, drug metabolic, steroid metabolic, and fatty acid metabolic process. Notable proteins that were upregulated in the gene deletion groups were: AKR1D1, AKR1C4, AKR7DA3, AKR1A1, DHCR7, ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A1 A deeper molecular pathway analysis revealed that the AKR1D1 and AKR1C4 that metabolize testosterone to inactive 5β-DHT and 5β-THT, respectively (2) (Figure 1) were significantly upregulated in UGT2B17 deletion group. UGT2B7 is primarily responsible for glucuronidation of 5β-THT (Figure 1). Finally, incubation with purified bacterial β-glucuronidases and human fecal extracts confirmed facile but variable deconjugation of TG to testosterone in human gut. Conclusion Novel mechanisms of testosterone metabolic switching in UGT2B17 gene deletion and testosterone activation by gut microbiome have been identified. These findings have a potential to provide deeper insights into the mechanisms of testosterone associated pathophysiological conditions, such obesity, insulin sensitivity, and cancer. References Li, C. Y. et al. J Steroid Biochem Mol Biol (2019). Rižner et al. Steroids (2014).

Can Cannabinoids Precipitate UGT‐mediated Drug Interactions?

Background: Cannabidiol (CBD) and tetrahydrocannabinol (THC) are cannabinoids used predominately for medicinal and recreational purposes, respectively. Because these cannabinoids are often co-consumed with conventional drugs, there is concern for deleterious cannabinoid-drug interactions. Both cannabinoids and their major circulating metabolites (7-OH CBD or 11-OH THC) are potent inhibitors of CYPs, but their potency to inhibit UGTs has not been investigated. Objective The objective of the study was to predict the potential for CBD and THC to precipitate UGT-mediated drug interactions using an IVIVE approach. The aims were to determine the following for CBD, THC, and their metabolites: 1) fraction unbound in incubations (fu,inc) containing recombinant UGT (rUGTs) and 0.2% BSA and 2) unbound UGT inhibitory potency (IC50,u). These data were used to predict the potential for interactions between each cannabinoid and UGT-metabolized drugs after typical doses used orally or by inhalation. Methods Fraction unbound (fu,inc) of CBD, THC, or metabolite in incubations containing rUGTs and 0.2% BSA was determined by ultracentrifugation. The inhibition extent (at 10 μM) or potency (IC50,u) of CBD, 7-OH CBD, 7-COOH CBD, THC, 11-OH THC, or 11-COOH THC towards the glucuronidation of estradiol (UGT1A1), chenodeoxycholic acid (UGT1A3), trifluoperazine (UGT1A4), 4-OH indole (UGT1A6), propofol (UGT1A9), gemfibrozil (UGT2B4), naloxone (UGT2B7), amitriptyline (UGT2B10), oxazepam (UGT2B15), or testosterone (UGT2B17) was determined using rUGTs. Estimated IC50,u and unbound hepatic inlet (oral dose) or systemic plasma (inhaled dose) concentrations were used to predict the potential for interactions between CBD (700 mg) or THC (20 mg) and drugs metabolized by UGT with fm values of 0.25, 0.5, or 0.75. Results fu,inc of CBD, 7-OH CBD, and THC was 0.05, 0.20, and 0.04, respectively. CBD, 7-OH CBD, THC, and 11-OH THC at 10 μM inhibited all UGTs tested (by 30-98%), whereas 7-COOH CBD inhibited only UGT2B7 (by 25%) and 11-COOH THC inhibited only UGT1A1, 1A4, and 1A9 (by 20-35%). IC50,u of CBD, 7-OH CBD, and THC against UGT1A9, 2B4, and 2B7 ranged from 0.002-0.02 μM, 0.6-1.6 μM, and 0.07-0.13 μM, respectively. IC50,u of CBD against UGT1A4, 1A6, 2B10, and 2B15 was < 1.3 μM. A mechanistic static model predicted strong in vivo interactions (AUC ratio > 3.3) between CBD and drugs extensively metabolized (fm = 0.75) by UGT1A9, 2B4, or 2B7. CBD was also predicted to result in moderate or weak interactions (1.3 ≥ AUC ratio ≤ 2.0) with drugs not extensively metabolized by these UGTs (fm = 0.25 or 0.5). In contrast, oral or inhaled THC was not predicted to precipitate UGT-mediated drug interactions (AUC ratio < 1.2). UGT inhibition by 7-OH CBD did not change the predicted potential of CBD-drug interactions. Conclusion This study is the first to determine the UGT inhibition potencies of cannabinoids. CBD was a potent inhibitor of UGT1A9, 2B4, and 2B7 and was predicted to interact with drugs metabolized by these UGTs. Further investigation via dynamic PBPK modeling and simulation and clinical evaluation is warranted to verify these predictions.

Targeted LC–MS/MS analysis of steroid glucuronides in human urine

Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1−3 G), 17β-estradiol 17-glucuronide (E2−17 G), 17β-estradiol 3-glucuronide (E2−3 G), estriol 16-glucuronide (E3−16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F–G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R2≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F–G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC–MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC–MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC–MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15−20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0−1 month of age: 184.5 nmol/L; 15−20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime.

Equine uridine diphospho-glucuronosyltransferase 1A1, 2A1, 2B4, 2B31: cDNA cloning, expression and initial characterization of morphine metabolism

ObjectiveUridine diphospho-glucuronosyltransferases (UGTs) are membrane-bound enzymes that catalyze the conjugation of glucuronic acid onto a diverse set of xenobiotics. Horses efficiently and extensively glucuronidate a number of xenobiotics, including opioids, making UGTs an important group of drug-metabolizing enzymes for the clearance of drugs. Recombinant enzymes have allowed researchers to characterize the metabolism of a variety of drugs. The primary objective was to clone, express and characterize equine UGTs using drugs characterized as UGT substrates in other species. A secondary objective was to characterize the in vitro metabolism of morphine in horses. Study designIn vitro drug metabolism study using liver microsomes and recombinant enzyme systems. AnimalsLiver microsomes and RNA from tissue collected from two Thoroughbred mares euthanized for other reasons. MethodsBased on homology to the human UGT2B7, four equine UGT variants were expressed: UGT1A1, UGT2A1, UGT2B31 and UGT2B4. cDNA sequences were cloned and resulting protein expressed in a baculovirus expression system. Functionality of the enzymes was assessed using 4-methylumbelliferone, testosterone, diclofenac and ketoprofen. Recombinant enzyme, control cells, equine liver microsomes and human UGT2B7 supersomes were then incubated with morphine. Concentrations of metabolites were measured using liquid chromatography–tandem mass spectrometry and enzyme kinetics determined. Results4-Methylumbelliferone was glucuronidated by all expressed equine UGTs. Testosterone glucuronide was not produced by any of the expressed enzymes, and diclofenac glucuronide and ketoprofen glucuronide were produced by UG2A1 and UGT1A1, respectively. UGT2B31 metabolized morphine to morphine-3-glucuronide and low concentrations of morphine-6-glucuronide. Conclusions and clinical relevanceThis is the first successful expression of functional recombinant equine UGTs. UGT2B31 contributes to the glucuronidation of morphine; however, it is probably not the main metabolizing enzyme. These results warrant further investigation of equine UGTs, including expression of additional enzymes and further characterization of UGT2B31 as a contributor to morphine metabolism.

Coupling 16S rDNA Sequencing and Untargeted Mass Spectrometry for Milk Microbial Composition and Metabolites from Dairy Cows with Clinical and Subclinical Mastitis.

The internal environment of the cow's udder directly affects the udder health and milk quality. 16S rDNA sequencing and liquid chromatography-mass spectrometry (LC-MS) methods were used to investigate the significant differences in milk microbial diversity and metabolites among cows that are healthy (H) and those suffering from subclinical mastitis (SM) and clinical mastitis (CM). Results uncovered more than 16 and 192 differently abundant microbiota at the phylum and genus levels, respectively, and 673 different levels of metabolites enriched in 20 pathways in milk among the 3 groups. This study revealed the positive relevance between Staphylococcus and Streptococcus and ceramide in milk from CM cows. Similarly, Acinetobacter and Corynebacterium were positively associated with testosterone glucuronide and 5-methyl-tetrahydrofolate, in milk from SM cows. On the basis of the combined analysis of microbiome and metabolome, this study indicated that, apart from the exogenous pathogens, some beneficial symbiotic bacteria, such as Dietzia, Aeromicrobium, Alistipes, and Sphingobacterium, rarely reported in milk have been found to be significantly reduced during mastitis.

Organic Anion Transporting Polypeptide-Mediated Hepatic Uptake of Glucuronide Metabolites of Androgens.

We previously established that androgen glucuronides are effluxed by multidrug resistance-associated proteins 2 and 3. However, no data exist on the mechanism of hepatic uptake of these metabolites. The first goal of this study was to explore the role of hepatic uptake transporters and characterize transport kinetics of glucuronides of testosterone (TG), dihydrotestosterone (DHTG), androsterone (AG), and etiocholanolone (EtioG) using cell lines overexpressing organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1). Using a quantitative proteomics-guided approach, we then estimated the fractional contribution of individual OATPs in hepatic uptake of these glucuronides. The transport screening assays revealed that the glucuronides were primarily taken up by OATP1B1 and OATP1B3. The K m values for OATP1B1-mediated uptake were low for EtioG (6.2 µM) as compared with AG, TG, and DHTG (46.2, 56.7, and 71.3 µM, respectively), whereas the K m value for OATP1B3-mediated uptake for EtioG, AG, DHTG, and TG were 19.8, 29.3, 69.6, and 110.4 µM, respectively. Both OATP1B1 and OATP1B3 exhibited the highest transport rate toward AG as compared with other glucuronides. When adjusted for the transporter abundance in human livers, EtioG and DHTG were predicted to be transported by both OATP1B1 and OATP1B3, whereas TG and AG were preferentially (>68%) transported by OATP1B3. Collectively, this report elucidates the mechanisms of hepatic uptake of androgen glucuronides. Perturbation of these processes by genetic polymorphisms, disease conditions, or drug interactions can lead to changes in enterohepatic recycling of androgens. TG and AG can be further investigated as potential biomarkers of OATP1B3 inhibition. SIGNIFICANCE STATEMENT: This is the first study to elucidate the mechanism of hepatic uptake of androgen glucuronides and estimate the fractional contribution of individual OATPs using quantitative proteomics. Our results show that both OATP1B1 and OATP1B3 are responsible for the hepatic uptake of major circulating testosterone glucuronides. The apparent higher selectivity of OATP1B3 toward testosterone glucuronide and androsterone glucuronide can be leveraged for establishing these metabolites as clinical biomarkers of OATP1B3 activity.

Digit Ratio (2D:4D) and Physical Performance in Female Olympic Athletes.

Background: The second to fourth digit ratio (2D:4D ratio) is suggested to be a negative correlate of prenatal testosterone. Little is known about the role of the 2D:4D ratio in relation to serum and urinary androgens for physical performance in female athletes. We aimed to compare the 2D:4D ratio in female Olympic athletes with sedentary controls, and to investigate the 2D:4D ratio in relation to serum and urinary androgens and physical performance in the athletes.Methods: This cross-sectional study included 104 Swedish female Olympic athletes participating in power, endurance and technical sports and 117 sedentary controls. The 2D:4D ratio was calculated using direct digit measurements. Serum androgens and urinary androgen metabolites were analyzed by liquid chromatography-tandem mass spectrometry. The athletes performed standardized physical performance tests and body composition was established by dual-energy X-ray absorptiometry.Results: The 2D:4D ratio was significantly lower in the athletes compared with controls although serum testosterone levels were comparable between groups and within normal reference values. The 2D:4D ratio correlated negatively with urinary levels of testosterone glucuronide and 5α- and 5βAdiol-17G, whereas there were no correlations to serum androgen levels. Furthermore, the 2D:4D ratio correlated negatively with strength tests and positively with 3,000-meter running in the athletes.Conclusion: Female Olympic athletes had a lower 2D:4D ratio, possibly reflecting a higher prenatal androgen exposure, than sedentary controls. Furthermore, the 2D:4D ratio was related to urinary levels of androgen metabolites and physical performance in the athletes but not to serum androgen levels. It is suggested that the 2D:4D ratio could reflect androgen metabolism and may be of importance for sporting success in female athletes.

Normalized Testosterone Glucuronide as a Potential Urinary Biomarker for Highly Variable UGT2B17 in Children 7-18 Years.

UDP-glucuronosyltransferase 2B17 (UGT2B17) is a highly variable androgen-metabolizing and drug-metabolizing enzyme. UGT2B17 exhibits a unique ontogeny profile characterized by a dramatic increase in hepatic protein expression from prepubertal age to adulthood. Age, sex, copy number variation (CNV), and single nucleotide polymorphisms only explain 26% of variability in protein expression, highlighting the need for a phenotypic biomarker for predicting interindividual variability in glucuronidation of UGT2B17 substrates. Here, we propose testosterone glucuronide (TG) normalized by androsterone glucuronide (TG/AG) as a urinary UGT2B17 biomarker, and examine the associations among urinary TG/AG and age, sex, and CNV. We performed targeted metabolomics of 12 androgen conjugates with liquid-chromatography tandem mass spectrometry in 63 pediatric subjects ages 7-18years followed over 7 visits in 3years. Consistent with the reported developmental trajectory of UGT2B17 protein expression, urinary TG/AG is significantly associated with age, sex, and CNV. In conclusion, TG/AG shows promise as a phenotypic urinary UGT2B17 biomarker.

A1 - Is urinary testosterone glucuronide a potential biomarker for UGT2B17 ontogeny in ages 7 to 18 years?

A1 - Is urinary testosterone glucuronide a potential biomarker for UGT2B17 ontogeny in ages 7 to 18 years?

Human efflux transport of testosterone, epitestosterone and other androgen glucuronides

Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen glucuronides investigated. In kinetic analyses, MRP3 transported glucuronides of androsterone, epitestosterone and etiocholanolone at low Km values, between 0.4 and 4 μM, while the Km values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 μM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by Km values over 100 μM. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.

The gut microbiota is a major regulator of androgen metabolism in intestinal contents.

Androgens exert important effects both in androgen-responsive tissues and in the intestinal tract. To determine the impact of the gut microbiota (GM) on intestinal androgen metabolism, we measured unconjugated (free) and glucuronidated androgen levels in intestinal contents from the small intestine, with a low bacterial density, and from cecum and colon, with a high bacterial density. Using a specific, sensitive gas chromatography-tandem mass spectrometry method, we detected high levels of glucuronidated testosterone (T) and dihydrotestosterone (DHT) in small intestinal content of mice of both sexes, whereas in the distal intestine we observed remarkably high levels of free DHT, exceeding serum levels by >20-fold. Similarly, in young adult men high levels of unconjugated DHT, >70-fold higher than in serum, were detected in feces. In contrast to mice with a normal GM composition, germ-free mice had high levels of glucuronidated T and DHT, but very low free DHT levels, in the distal intestine. These findings demonstrate that the GM is involved in intestinal metabolism and deglucuronidation of DHT and T, resulting in extremely high free levels of the most potent androgen, DHT, in the colonic content of young and healthy mice and men.