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Epitestosterone is a stereoisomer of the active androgen testosterone and its circulating concentrations are similar to those of testosterone in women and children. However, its biological function and pathways of metabolism remain unknown. The structural similarity to testosterone suggests a potential function in the modulation of androgen receptor signalling. It is well established that the conversion of testosterone to 5α-dihydrotestosterone enhances local androgen receptor signalling. In this study, we show that epitestosterone is metabolized to 5α-dihydroepitestosterone by both human steroid 5α-reductase isoforms, SRD5A1 and SRD5A2. Using two different variations of a reporter assay for transactivation of the human androgen receptor, we show that epitestosterone is a partial AR agonist and that the 5α-reduction of epitestosterone increases its androgenic activity. In line with this, we show that 5α-reduction of epitestosterone reduces its ability to antagonize 5α-dihydrotestosterone-induced androgen receptor transactivation. In conclusion, we provide evidence that steroid 5α-reductases regulate the modulatory effect of epitestosterone on androgen receptor signalling., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Polycystic ovary syndrome (PCOS) is a common infertility disorder which affects reproductive-aged women. However, metabolic change profiles of follicular fluid (FF) in lean and obese women diagnosed with and without PCOS remains unclear., Methods: 95 infertile women were divided into four subgroups: LC (lean control), OC (overweight control), LP (lean PCOS), and OP (overweight PCOS). The FF samples were collected during oocyte retrieval and assayed by ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) metabolomics., Results: A total of 236 metabolites were identified by metabolic analysis. The pathway enrichment analysis revealed that the glycerophospholipid metabolism (impact = 0.11182), ether lipid metabolism (impact = 0.14458), and primary bile acid biosynthesis (impact = 0.03267) were related to metabolic pathway between PCOS and control. Correlation analyses showed that epitestosterone sulfate was found positively correlated with fertilization rate in PCOS, while falcarindione, lucidone C. and notoginsenoside I was found to be negatively correlated. The combined four biomarkers including lucidone C, epitestosterone sulfate, falcarindione, and notoginsenoside I was better in predicting live birth rate, with AUC of 0.779., Conclusion: The follicular fluid of women with PCOS showed unique metabolic characteristics. Our study provides better identification of PCOS follicular fluid metabolic dynamics, which may serve as potential biomarkers of live birth., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 K m values, between 0.4 and 4 μM, while the K m values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 μM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by K m 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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The introduction of alternative markers to the steroid profile can be an effective approach to improving the screening capabilities for the detection of testosterone (T) misuse. In this work, endogenous steroid sulfates were evaluated as potential markers to detect intramuscular (IM) T administration. Fourteen sulfate metabolites were quantified using mixed-mode solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples after a single IM injection (100 mg) of T cypionate to six Caucasian and six Asian healthy male volunteers were analyzed. Principal component analysis (PCA) was used to characterize the sample cohort and to obtain the most useful markers for discrimination between pre- and post-administration samples. For Caucasian volunteers, a separation between pre- and post-administration samples was observed in PCA, whereas for Asian volunteers no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. Detection times (DTs) of each marker were evaluated using individual thresholds for each volunteer. The best results were obtained using ratios involving T and epitestosterone (E) sulfates in the denominator. The best marker was the ratio androsterone sulfate/testosterone sulfate (A-S/T-S) which prolonged the DT 1.2-2.1 times in respect to those obtained using T/E ratio in all Caucasian volunteers and 1.3-1.5 times in two Asian volunteers. Other ratios between A-S or etiocholanolone sulfate and E-S, and sulfates of etiocholanolone, dehydroandrosterone or epiandrosterone, and T-S were also found adequate. These ratios improve the DT after IM T administration and their incorporation to complement the current steroid profile is recommended., (© 2019 John Wiley & Sons, Ltd.)

Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17β-estradiol (17β-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3β, 17β-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low μM concentrations of celecoxib strikingly enhanced the formation of the 17-sulfates of 6-dehydroestradiol (6D-E2), 17β-dihydroequilenin (17β-Eqn), 17β-dihydroequilin (17β-Eq), and 9-dehydroestradiol (9D-E2) as well as the overall rate of sulfonation. For 6D-E2, 9D-E2 and 17β-Eqn, celecoxib inhibited 3-sulfonation, however 3-sulfonation of 17β-Eq was stimulated at celecoxib concentrations below 40 μM. Ligand docking studies in silico suggest that celecoxib binds in the substrate-binding site of SULT2A1 in a manner that prohibits the usual binding of substrates but facilitates, for appropriately shaped substrates, a binding mode that favors 17-sulfonation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The large variation in disposition known for most drugs is also true for anabolic androgenic steroids. Genetic factors are probably the single most important cause of this variation. Further, there are reasons to believe that there is a corresponding variation in efficacy of doping agents. Doped individuals employ a large variety of doping strategies in respect of choice of substance, dose, dose interval, duration of treatment and use of other drugs for enforcement of effects or correction of side effects. Metabolic steps up-stream and down-stream of testosterone are genetically variable and contribute substantially to the variation in disposition of testosterone, the most common doping agent in sports and in society. Large inter- and intra-ethnic variation in testosterone glucuronidation and excretion is described as well as the pit-falls in evaluation of testosterone doping test results. The hydrolysis and bioactivation of testosterone enanthate is also genetically variable yielding a 2-3 fold variation in excretion rate and serum concentration, thereby implicating a substantial variation in 'efficacy' of testosterone. Given this situation it is logical to adopt the new findings in the doping control programme. The population based cut-off level for the testosterone : epitestosterone ratio should be replaced by a Bayesian interpretation of consecutive tests in the same individual. When combined with the above genetic information the sensitivity of the test is considerably improved. The combination of the three approaches should reduce the rate of falsely negative or positive results and the number of expensive follow-up tests, stipulated by the World Anti-Doping Agency., (© 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.)

The study of the metabolism of drugs, in particular steroids, by both in vitro and in vivo methods has been carried out in the authors' laboratory for many years. For in vitro metabolic studies, the microsomal fraction isolated from horse liver is often used. However, the process of isolating liver microsomes is cumbersome and tedious. In addition, centrifugation at high speeds (over 100 000 g) may lead to loss of enzymes involved in phase I metabolism, which may account for the difference often observed between in vivo and in vitro results. We have therefore investigated the feasibility of using homogenized horse liver instead of liver microsomes with the aim of saving preparation time and improving the correlation between in vitro and in vivo results. Indeed, the preparation of the homogenized horse liver was very simple, needing only to homogenize the required amount of liver. Even though no further purification steps were performed before the homogenized liver was used, the cleanliness of the extracts obtained, based on gas chromatography-mass spectrometry (GC-MS) analysis, was similar to that for liver microsomes. Herein, the results of the in vitro experiments carried out using homogenized horse liver for five anabolic steroids-turinabol, methenolone acetate, androst-4-ene-3,6,17-trione, testosterone, and epitestosterone-are discussed. In addition to the previously reported in vitro metabolites, some additional known in vivo metabolites in the equine could also be detected. As far as we know, this is the first report of the successful use of homogenized liver in the horse for carrying out in vitro metabolism experiments. Copyright © 2011 John Wiley & Sons, Ltd., (Copyright © 2011 John Wiley & Sons, Ltd.)

The degradation processes in deficiently stored urine samples are well investigated regarding steroid concentrations and diagnostic ratios, such as the quotient of testosterone divided by epitestosterone. In contrast, nothing is known about the influence on carbon isotope ratios (CIR) by inappropriate storage conditions. In general, it is assumed that degradation, i.e. deconjugation or dehydrogenation, does not change CIR and thus CIR can be used in cases where the steroid profile turns out to be invalid. Therefore, the CIR of urinary steroids was investigated in different urine samples during the course of degradation over a time period of six months. Several steroids excreted as glucuronides (androsterone (A), etiocholanolone (E), testosterone, pregnanediol (PD) and 5α- and 5β-androstane-3α,17β-diol) or sulfo-conjugated (A, E and androst-5-ene-3β,17β-diol (5EN17b)) were investigated together with their unconjugated correspondents (A, E, PD and 5EN17b) and the main dehydrogenation products (5α- and 5β-androstane-3,17-dion and androst-4-ene-3,17-dion). For this purpose, the exiting methods for CIR determination were extended and validated. In addition, the urinary concentrations of all investigated steroids were monitored. Particular attention was paid to dehydroepiandrosterone conjugated and unconjugated together with its degradation product 3α,5-cyclo-5α-androstan-6β-ol-17-one as here the strongest influence on CIR was expected., (Copyright © 2010 John Wiley & Sons, Ltd.)

The detection of the administration of an androgen such as testosterone that could be present normally in human bodily fluids is based upon the methodical evaluation of key parameters of the urinary profile of steroids, precisely measured by GC/MS. Over the years, the markers of utilization were identified, the reference ranges of diagnostic metabolites and ratios were established in volunteers and in populations of athletes, and their stability in individual subjects was studied. The direct confirmation comes from the measurement of delta (13)C values reflecting their synthetic origin, ruling out a potential physiological anomaly. Several factors may alter the individual GC/MS steroid profile besides the administration of a testosterone-related steroid, the nonexhaustive list ranging from the microbial degradation of the specimen, the utilization of inhibitors of 5alpha-reductase or other anabolic steroids, masking agents such as probenecid, to inebriating alcohol drinking. The limitation of the testing strategy comes from the potentially elevated rate of false negatives, since only the values exceeding those of the reference populations are picked up by the GC/MS screening analyses performed by the laboratories on blind samples, excluding individual particularities and subtle doping. Since the ranges of normal values are often described from samples collected in Western countries, extrapolating data to all athletes appears inefficient. Furthermore, with short half-life and topical formulations, the alterations of the steroid profile are less pronounced and disappear rapidly. GC/C/IRMS analyses are too delicate and fastidious to be considered for screening routine samples. An approach based upon the individual athlete's steroid profiling is necessary to pick up variations that would trigger further IRMS analysis and investigations.

Testosterone and epitestosterone are endogenous steroids that differ in the configuration of the hydroxyl-bearing carbon at C-17. Testosterone is the predominant male sex hormone, whereas the role of epitestosterone is largely unclear. In humans, both androgens are excreted mainly as glucuronide conjugates and the urinary ratio of testosterone to epitestosterone (T/E), used to expose illicit testosterone abuse by male athletes, indicates the relative concentrations of the respective glucuronides. Some male athletes have T/E values greater than the accepted threshold value (4.0), even without testosterone abuse. We have analyzed athletes' urine samples and found that the main reason for such false-positive results in doping tests was a low epitestosterone glucuronide concentration not a high level of testosterone glucuronide. Sulfate conjugates of both testosterone and epitestosterone were also detected in the different urine samples. Glucuronidation assays with the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies UGT1A, UGT2A, and UGT2B revealed that UGT2B17 is the most active enzyme in testosterone glucuronidation. UGT2B17 does not glucuronidate epitestosterone, but inhibition studies revealed that it binds epitestosterone with affinity similar to that of testosterone. Epitestosterone glucuronidation is catalyzed mainly by UGT2B7, and the K(m) of this reaction is significantly lower than the K(m) of UGT2B17 for testosterone. Although UGT2B7 and UGT2B17 exhibited high, although converse, stereoselectivity in testosterone and epitestosterone glucuronidation, UGT2A1, an extrahepatic enzyme that is expressed mainly in the nasal epithelium, catalyzed the glucuronidation of both steroids at considerable rates and similar kinetics. The results shed new light on the substrate specificity and stereoselectivity of human UGTs.

Glucuronidation is one of the most important metabolic processes which eliminates androgens from the human organism. Three enzymes (UDP-glucuronosyltransferases) are responsible for transfering the glucuronic group from glucuronic acid to androgens: UGT2B7, UGT2B15 and UGT2B17. Glucuronide products are more polar, water soluble, less toxic and easily extracted form the body. Testosterone is a common androgen abused in sport. The norm for urinary testosterone/epitestosterone ratio is below 4.0. Large testosterone excretion is associated with a deletion polymorphism of the UGT2B17 gene. This polymorphism decreases T/E ratio level.

Androgens are considered to play a substantial role in pathogenesis of both benign prostatic hyperplasia (BPH) and prostate cancer. The importance of determination of androgen levels in tissue and serum for cancer progression and prognosis has been poorly understood. The aim of study was to find out hormonal differences in both diseases, their correlations between intraprostatic and serum levels and predicted value of their investigation. Testosterone, dihydrotestosterone, androstenedione and also epitestosterone were determined in prostate tissue from 57 patients who underwent transvesical prostatectomy for BPH and 121 patients after radical prostatectomy for prostate cancer. In 75 subjects with cancer and 51 with BPH the serum samples were analyzed for testosterone, dihydrotestosterone and SHBG. Significantly higher intraprostatic androgen concentrations, i.e. 8.85+/-6.77 versus 6.44+/-6.43 pmol/g, p<0.01 for dihydrotestosterone, and 4.61+/-7.02 versus 3.44+/-4.53 pmol/g, p<0.05 for testosterone, respectively, were found in patients with prostate cancer than in BPH. Higher levels in cancer tissue were found also for epitestosterone. However, no differences were found in serum levels. Highly significant correlations occurred between all pairs of intraprostatic androgens and also epitestosterone as well as between serum testosterone and dihydrotestosterone (p<0.001) in both BPH and cancer groups. Correlation was not found between corresponding tissue and serum testosterone and dihydrotestosterone, either in benign or cancer samples. The results point to importance of intraprostatic hormone levels for evaluation of androgen status of patients, contrasting to a low value of serum hormone measurement.

Amateau and McCarthy's findings published in Nature Neuroscience (June 2004) are noteworthy for suggesting a role for prostaglandins in sexual development. However, evidence suggests that in manipulating PGE2, they unknowingly implicated 3alpha-hydroxysteroid dehydrogenase [E.C. 1.1.1.50], 3(or 17)alpha-hydroxysteroid dehydrogenase [E.C. 1.1.1.209] and their respective products, androsterone (ADT) and epitestosterone (EpiT), in the developmental masculinization of sex behavior. EpiT is generally regarded as a hormonally inactive 17alpha-epimer of testosterone (T). In rats, the kidney is the primary site of EpiT formation, whereas in humans it originates from the gonads, with only a small contribution secreted by the adrenals. Because the ratio of T to EpiT is nearly constant, it is presently used for assessing steroid abuse in competitive sports, where the World Anti-Doping Agency (WADA) considers a T/EpiT ratio >4 evidence of T doping. Despite its central role in the detection of illict anabolic steroid use, our knowledge of factors effecting EpiT production is poor. Clues in the literature, however, reveal that prostaglandin-mediated processes, such as LHRH release, may influence its production. Antimycotics, NSAIDs, and opioid analgesics used in sports medicine are all known to effect prostaglandin E2 synthesis. Primary PGs are potent inhibitors of ADT oxidation, while indomethacin, a prostaglandin blocker, powerfully inhibits 3alpha-HSD reduction and ADT oxidation. This is significant because ADT inhibits the oxidation of EpiT, and may modulate its antiandrogenic and neuroprotective effects. It is hypothesized that the T/EpiT ratio is increased by COX-2 inhibitors and opiod analgesics, and decreased by antimycotics that do not impair testosterone biosynthesis. Given the devastating personal and career consequences that may result from false positive drug tests, substantive research on the effects of PGE2 manipulations on EpiT is warranted.

A series of 4- and/or 17alpha-substituted testosterone analogues has been incubated with the hydroxylating fungus Fusarium culmorum AM282. It was found that 19-norandrostenedione, 19-nortestosterone, 4-methoxytestosterone, 4-methyltestosterone, and 4-chloro-17alpha-methyltestosterone were hydroxylated exclusively or mainly at the 6beta-position. The mixtures of 6beta-, 15alpha-, and 12beta- or 11alpha-monohydroxy derivatives were obtained from 17alpha-methyltestosterone and 17alpha-ethyl-19-nortestosterone--the substrates with alkyl group at C-17alpha. 4-Chlorotestosterone was predominantly hydroxylated at 15alpha-position, but the reaction was accompanied by the reduction of 4-en-3-one system, which proceeded in the sequence: reduction of ketone to 3beta-alcohol and then reduction of the double 4,5 bond. The results obtained indicate an influence of stereoelectronic and steric effects of substitutes on regioselectivity of the hydroxylation of 4-en-3-one steroids by F. culmorum.

Androstenedione is a steroid hormone sold over-the-counter to individuals who expect that it will enhance strength and athletic performance. Endogenous androstenedione is the immediate precursor of testosterone. To evaluate the metabolism of oral androstenedione, we randomly assigned 37 healthy men to receive 0 (group 1), 100 mg (group 2), or 300 mg (group 3) of androstenedione in a single daily dose for 7 days. Eight-hour urines were collected 1 day before the start of androstenedione, and on days 1 and 7. Using gas chromatography-mass spectrometry, we measured excretion rates of glucuronide-conjugated epitestosterone, its putative precursor (E-precursor), and metabolites (EM-1 and EM-2), and we evaluated possible markers of androstenedione administration. Day 1 and 7 rates were not different: the means were averaged. The means (microg/h) for groups 1, 2, and 3, respectively were, for epitestosterone 2.27, 7.74, and 18.0; for E-precursor, 2.9, 2.0, and 1.5; for EM-1/E-precursor 0.31, 1.25, and 2.88; for EM-2/E-precursor 0.14, 0.15, and 1.15; for testosterone/epitestosterone (T/E) 1.1, 3.5, and 3.2. Epitestosterone, EM-1, and EM-2 excretion was greater in groups 2 and 3 versus group 1 (0.0001 < P < 0.03), as were EM-1/E-precursor, EM-2/E-precursor, and T/E. E-precursor excretion was lower in groups 2 (P = 0.08) and 3 (P = 0.047) versus group 1. Androstenedione increases excretion of epitestosterone and its two metabolites, while decreasing that of its precursor. Elevated ratios of EM-1- and EM-2/E-precursor, and the presence of 6alpha-hydroxyandrostenedione are androstenedione administration markers.

The risk of breast cancer is 2 to 5 times higher in patients suffering from gross cystic disease. Breast cysts are categorized into two groups (type I and type II) according to the concentration of electrolytes in the cyst fluid. The two types also differ with respect to accumulation of steroids and steroidogenic enzyme activity. In type I cysts a higher risk of breast carcinoma could be expected. Here, we studied a possible relationship between the type of cyst and levels of epitestosterone (an endogenous antiandrogen), allopregnanolone (a product of 5alpha-reductase activity), and pregnenolone-sulfate (an activator of N-methyl-D-asparate receptors). We have found five times higher levels of epitestosterone in BCF in comparison with the circulation. Allopregnanolone levels were similar to those in plasma of women in the luteal phase of the menstrual cycle. Pregnenolone-sulfate levels in BCF were about two orders of magnitude higher when compared with the circulation. No differences were found in concentrations of the steroids studied between the types of cysts.

In establishing a theory to predict male-pattern baldness, we investigated the correlation of testosterone, epitestosterone, and dihydrotestosterone with 5alpha-reductase in hair using gas chromatography-mass spectrometry. One hundred milligram hair samples were obtained from a group of balding subjects and their sons, as well as from a corresponding aged-matched, nonbalding group. The ratio of testosterone to epitestosterone was significantly greater (mean 46.41, p < 0.001; mean 35.83, p < 0.001, respectively) in the hair of balding fathers (n = 19, age 28-50 y) and their sons (n = 16, age 8-16 y) than in the hair of the nonbalding control subjects (mean 9.17 and 10.47, respectively). These findings demonstrate that analysis of terminal hair may not only provide a basis for predicting baldness when the subject is still young, but also for preventing and treating male-pattern baldness by controlling the steroid hormone balance.

Administration of supraphysiological doses of testosterone to normal men causes inhibition of spermatogenesis, but while most become azoospermic, 30-55% maintain a low rate of spermatogenesis. We have investigated whether there are differences in endogenous androgen production, of testicular and adrenal origin, which may be related to the degree of suppression of spermatogenesis. Thirty-three healthy Caucasian men were given weekly i.m. injections of 200 mg testosterone oenanthate (TE), 18 became azoospermic, while 15 remained oligozoospermic. Urinary excretion of epitestosterone, a specific testicular product, was reduced to <10% of pretreatment values, with no differences between the groups. Similar results were obtained for other markers of testicular steroidogenesis. Urinary and plasma adrenal androgens were also reduced during TE treatment: a statistically significant decrease in both (P < 0.001 and P < 0.05 respectively) was seen in the azoospermic but not oligozoospermic responders. These results suggest that testicular steroidogenesis is decreased to <10% by the administration of supraphysiological doses of exogenous testosterone. Differences in the degree of ongoing steroidogenesis in the testis do not appear to account for incomplete suppression of spermatogenesis, thus differences in androgen metabolism may underlie this heterogeneous response. A small but significant reduction in secretion of adrenal androgens was also detectable, the relevance of which is unclear.

Epitestosterone, a C19-steroid with anti-androgenic activity, was determined in the plasma of 234 boys and men from the ages of 6-86 years, and in the prostate tissue of 15 men 55-82 years of age. It was documented that, while in adulthood the concentration of epitestosterone is about ten times lower than the concentration of testosterone, in the pre-pubertal period the level of epitestosterone is similar or even higher than that of testosterone. In the hyperplastic prostate tissue the content of epitestosterone is comparable to that of androstenedione, it is about twice as high as the content of testosterone and approximately half that of the content of dihydrotestosterone. At least in the case of pre-pubertal boys and in the prostatic tissue it is therefore possible to include epitestosterone into consideration as a regulatory factor for the androgen-dependent events.

Sexual differences on thyroxin 5'-deiodinase (5'-D) in the Harderian gland of Syrian hamsters were investigated. We compared the 24-h profile of 5'-D activity in male and female hamsters, observing a clear rhythm in males but not in females. Female values were always significantly higher than male ones. After pinealectomy day/night variations in male 5'-D activity at the time points studied were abolished, results that are in correlation with serum thyroid hormones. We also studied the regulation by androgen of the enzyme activity. Basal 5'-D activity increased in castrated males and levels fell when animals were implanted with testosterone or its product 5 alpha-dihydrotestosterone (DHT). Female 5'-D activity was also inhibited by androgens. As only the addition of DHT in the presence of epitestosterone, an inhibitor of the conversion of testosterone on DHT, in castrated males was able to decrease 5'-D activity to the control animal levels, we suggest a probable direct effect of DHT by itself.

The authors assessed in the plasma of 156 boys and men aged 6 - 65 years and in prostatic tissues of 15 men aged 55 - 82 years epitestosterone, C19-steroid with anti-androgenic properties. It was revealed that during the prepubertal period the epitestosterone level is higher than that of testosterone, in adult age the epitestosterone is about ten times lower than the testosterone concentration. In tissue of hyperplastic prostates the epitestosterone level is comparable with the androstendione level; it is about twice as high as the testosterone level and approximately half as high as the dihydrotestosterone level. Thus at least during the prepubertal period in boys and in the androgen-dependent prostate tissue a more significant participation of epitestosterone in the regulation of androgen-dependent processes must be taken into consideration.

Testosterone administration to male athletes can be safely detected in the vast majority of cases by the urinary excretion ratio of testosterone to epitestosterone glucuronides (TG/EG), which may not exceed 6. Some rare cases of physiologically high TG/EG ratios (between 6 and 12) are encountered; these may be attributed to a dysregulation of the testicular secretions of epitestosterone which is decreased, and of epitestosterone sulphate (ES) which is normal or increased. Impaired hydrolysis of circulating epitestosterone sulphate by deficiency of a specific sulphatase acting on 17 alpha-sulphates must also be considered as a possible reason for the decreased availability of epitestosterone for hepatic glucuronidation. Urinary excretions of conjugates and metabolites of testosterone and epitestosterone (expressed in nmol/mmol creatinine) have been determined by gas chromatography-mass spectrometry associated with stable isotope dilution, in a reference population of 90 healthy male subjects and in 12 subjects with chronic TG/EG > 4. Urinary excretion ratios such as TG/(EG+ES), EG/ES and TG/5-androstene-3 beta,17 alpha-diol glucuronide are shown to be efficient criteria which allow discrimination between physiologically high and pharmacologically high TG/EG ratios. A simple oral loading test with deuterium-labelled epitestosterone demonstrates the difference between hepatic and total epitestosterone metabolism clearly, particularly in subjects with physiologically high TG/EG in comparison with subjects with normal TG/EG.

In this cross-sectional study on 140 subjects, several testosterone and epitestosterone metabolites have been analyzed by gas chromatography-mass spectrometry associated with stable isotope dilution, a technique requested for doping analysis in general, and detection of exogenous testosterone supply in particular. Urinary excretions of luteinizing hormone, testosterone and epitestosterone glucuronides and sulfates, as well as glucuronides of 5-androstene-3 beta, 17 alpha-diol, 5 alpha- and 5 beta-androstane-3 alpha, 17 alpha-diol and the corresponding 17 beta-isomers, present similar patterns of increase throughout pubertal development, from stage 1 up to stage 5. Excretion levels are significantly different in general between stages 1, 2, 3 and 4, the highest percentage increase being observed between stages 3 and 4. None of the ratios of testosterone to epitestosterone glucuronides are beyond the threshold value of 6, where testosterone abuse by athletes is suspected. No particular pubertal stage exceeded this critical value with a probability higher than p = 0.006, a value that was determined on the whole population. This is consistent with the non-significant differences between correlation slopes of regression curves, relating either testosterone or epitestosterone glucuronide to chronological age. The ratio of testosterone glucuronide to luteinizing hormone increases significantly throughout puberty and this might be a limitation to the widespread use of this ratio for the detection of testosterone misuse.(ABSTRACT TRUNCATED AT 250 WORDS)

Epitestosterone (ET) and testosterone (T), free and sulfoconjugated, as well as 5-androstene-3 beta,17 alpha-diol (5AD3 beta 17 alpha) and its 17 beta-epimer have been analyzed, by gas chromatography-mass spectrometry with stable isotope dilution, in peripheral and spermatic venous plasma of patients with left varicocele. All these androgens are secreted by the testis as evidenced by the significant concentration gradients between peripheral and spermatic venous plasma. Half of the daily ET production is ascribed to the testis, while 95% of T sulfate and roughly 70% of ET sulfate are also of testicular origin. Significant correlations between ET and 5AD3 beta 17 alpha are an indication that the 5-ene pathway is also operative for ET biosynthesis. High ratios of spermatic to peripheral venous plasma levels of ET and 5AD3 beta 17 alpha are also related to the high clearance rates of 17 alpha-hydroxy-androgens.

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.

Multiple forms of the soluble 17 alpha-hydroxysteroid dehydrogenases of female rabbit liver and kidney having similar purification characteristics and isoelectric points were compared with regard to their relative rates of oxidation and reduction of estrogen and androgen substrates, their kinetic parameters and their primary structures. All of the enzyme forms exhibited both 3 alpha- and 17 alpha-enzyme activity toward androgen substrates and the oxidation of the 17 alpha-hydroxysteroid epitestosterone was competitively inhibited by the 3 alpha-hydroxysteroid androsterone. The most basic enzyme forms from liver and kidney had similar relative activities toward estrogen and androgen substrates in the oxidative direction but differed in their activities in the reductive direction. Major differences in the peptide maps of these enzymes were observed. The less basic enzyme forms from the two tissues had similar activities toward estrogen substrates but differed considerably in their relative activities toward androgens. Only minor differences were observed in the peptide maps of these enzymes.

Androgen production during the annual reproductive cycle was investigated in the male scincid lizard Tiliqua (Trachydosaurus) rugosa. Concentrations of testosterone and epitestosterone were measured in plasma and testis (incubated and nonincubated) using radioimmunoassay. Morphological and histological techniques were used to determine the anatomical changes in the testis. Mating behavior was observed during spring, and sperm were most numerous in the testis at this time. Histological evidence and changes in testicular weight indicated that spermatogenesis begins in autumn and culminates in spring. Testicular regression occurred soon after mating. Plasma concentrations of both androgens were maximal during spring and minimal during summer. The maximal concentrations were approximately 500 and 150 nmol/liter (epitestosterone and testosterone, respectively). The minimal plasma concentrations were 250 and 15 nmol/liter, respectively. Plasma and testicular androgen cycles followed a pattern similar to that of the spermatogenetic cycle, suggesting the possible involvement of one or both of these androgens in the control of spermatogenesis and mating behavior in these lizards.

The aromatization of epitestosterone (17 alpha-hydroxy-4-androsten-3-one) and testosterone by lyophilized human placental microsomes was studied. Upon incubation of epitestosterone, 12% was converted to 17 alpha-estradiol, 15% to 19-keto-epitestosterone (17 alpha-hydroxy-4-oxo-4-androsten-19-al), 10% to 19-hydroxyepitestosterone (17 alpha, 19-dihydroxy-4-androsten-3-one), and about 10% to several unidentified products. A similar incubation with testosterone resulted in 60% conversion to 17 beta-estradiol; 30% was unchanged. At increasing substrate concentrations (0.1-50 microM), the aromatization rate of epitestosterone increased gradually and did not reach a plateau, whereas aromatization rate of testosterone plateaued at about 3 microM. The presence of either testosterone or 17 beta-estradiol in concentrations 0.1-10 times the concentration of epitestosterone inhibited the aromatization of epitestosterone by about 70%, while the aromatization of testosterone was not inhibited by either epitestosterone or 17 alpha-estradiol. Lyophilization of fresh microsomes or storage of the lyophilized microsomes at -20 C greatly reduced the aromatizing activity upon epitestosterone but not upon testosterone. These results suggest that the aromatizing system for epitestosterone is different from that for testosterone.

A double isotope ratio technique was used to estimate the specific binding of testosterone (T), as opposed to its biologically nonactive stereoisomer, epitestosterone (Epi T). The mouse erythropoietic spleen formed in response to a phenylhydrazine-induced hemolytic anemia was used as the target organ. Spleen minces from preanemic mice, as well as those in the early and late phases of erythropoietic spleen development, were incubated with 10(-9) M of 14C-T and 3H-EpiT, and the selective uptake of T was calculated from the 14C/3H ratio measured in the media before and after incubation, as well as in the subcellular fractions of the minces. Preferential uptake of T was seen in the early phase of development, but not in spleens obtained from preanemic animals or those in the late phase. There was no evidence of metabolic conversion of T or EpiT. The selective uptake of T by early phase spleens was reflected in a preferential nuclear accumulation of T. These data represent the first demonstration of a specific binding of T in vitro to a developing erythroid tissue.

Human placental microsomes converted epitestosterone to estradiol-17 alpha at rates of 23-48 pmol/min X mg protein with a Km of 113 microM. Activity was inhibited 70-90% by concentrations of CO, metyrapone, n-octylamine, 7,8-benzoflavone and 7-ethoxycoumarin which had no effect on the aromatization of 4-androstene-3, 17-dione. Conversely, cyanide and azide were more effective inhibitors of the conversion of the latter androgen. A variety of neutral steroids inhibited the aromatization of epitestosterone with 19-norsteroids being particularly effective, but competitive effects could not be demonstrated. Both 17 beta-hydroxy-4-estren-3-one and 16 alpha-hydroxy-4-androstene-3,17-dione caused a mixed inhibition. A number of phenolic steroids were also inhibitory with 16-oxo compounds being particularly effective. Inhibition by estrone was non-competitive (Ki = 16 microM). The aromatization of epitestosterone resembles placental microsomal oxidase activities against estrone and benzo [a]pyrene in its inhibitor specificity and epitestosterone may be the native substrate for an oxidase also active in the metabolism of aromatic xenobiotic chemicals.

5 alpha-reductase activity was investigated in scalp hair roots in 5 patients with 5 alpha-reductase deficiency and in 10 normal volunteers (5 men and 5 women) who served as controls. Metabolism of [3H]testosterone and [3H]epitestosterone was measured under standardized conditions in 1-4 growing (anagen) hair roots. When the mean values of the 5 alpha-reduced metabolites from testosterone (androstanedione and dihydrotestosterone) or from epitestosterone (epidihydrotesterone) were compared in both groups, similar values were observed. However, since most of the hair roots were incubated singly, it could be demonstrated that in two patients 20 respectively 24% of the hair roots showed neither dihydrotestosterone nor epidihydrotestestosterone formation. This did not influence the mean values determined for the whole group perceptibly. Our results support the concept that 5 alpha-reductase deficiency is a genetically heterogenous disorder by demonstrating that the enzyme defect is variously expressed in hair roots of affected individuals.

19-Nortestosterone was identified as a product of testosterone conversion in vitro in male mouse kidney. After incubation of [14C]testosterone with kidney slices of castrated male mice, 64% of the radioactivity was recovered as testosterone, about 6% as epitestosterone and 2.5% was identified as 19-nortestosterone. The renotrophic effect of 19-nortestosterone was at least as high as that of testosterone. Possible role of 19-nortestosterone in mouse kidney is discussed.

The action of the endogenous steroid epitestosterone administered to castrated male mice substituted with testosterone propionate is manifested by reduced weight increments and a reduced relative weight of their seminal vesicles and kidneys. Epitestosterone in vitro displaces androgens from their bond with receptors in cytosol from rat prostates and markedly inhibits the testosterone transformation to the more effective androgen dihydrotestosterone. Epitestosterone can be thus defined as a true endogenous antiandrogen; to its action at the receptor level a potent inhibitory effect on 5 alpha-reductase must be added.