Metabolites of Testosterone Enanthate: how the body breaks down the drug

After an injection of testosterone enanthate, a whole series of enzymatic reactions is triggered in the body: cleavage of the ester, formation of active metabolites, hepatic inactivation and excretion in the urine. The editorial team explains which compounds exactly are formed, why some of them act more strongly than the parent hormone, and how knowledge of metabolism is used by doctors and anti-doping laboratories.

From ester to free hormone

Testosterone enanthate itself does not interact with androgen receptors: it is a prodrug, a "packaged" form of the hormone. After intramuscular administration the oil solution forms a depot, from which ester molecules slowly diffuse into tissue fluid and blood. The rate of this process is determined by the lipophilicity of the seven-carbon ester "tail" and the properties of the oil base.

In the blood and tissues the ester bond is cleaved by nonspecific enzymes — esterases. The hydrolysis reaction yields two molecules: free testosterone, identical to endogenous, and enanthic (heptanoic) acid. Cleavage occurs fairly quickly, so what circulates in the plasma is predominantly free and protein-bound testosterone, not the ester.

Enanthic acid is an ordinary medium-chain fatty acid. The body utilizes it through β-oxidation in the mitochondria, just like dietary fatty acids. From a toxicological standpoint it has no independent significance, and its amount is negligible compared with the daily intake of fats.

Thus, everything that happens next is the metabolism of testosterone itself. The difference of enanthate from other esters lies only in how long and with what amplitude the hormone enters the blood, not in the set of end products. That is why the metabolites of enanthate, cypionate or propionate in the urine are practically the same.

In the blood, testosterone is predominantly bound to sex hormone-binding globulin (SHBG) and albumin. Only a small free fraction is directly available to cells and enzymes. The level of SHBG determines how quickly the hormone reaches the liver and other tissues, where further transformations take place.

Active metabolites: DHT and estradiol

Part of the testosterone in peripheral tissues is converted into compounds biologically more active than the parent molecule or with a different spectrum of action. The most important of these are dihydrotestosterone (DHT) and estradiol. Quantitatively these pathways account for a small share of the hormone's total metabolism, but it is precisely they that explain a significant part of the clinical effects.

DHT is formed under the action of the enzyme 5α-reductase. There are two main isoenzymes: type 1 predominates in the skin and sebaceous glands, type 2 — in the prostate, genitals and hair follicles. DHT binds to the androgen receptor more tightly than testosterone and does not aromatize, so it is a "pure" potent androgen.

Estradiol is formed thanks to aromatase (CYP19A1), which is present in adipose tissue, the brain, bones, liver and testes. This pathway is physiologically important: estradiol in men is needed for bone mineralization, closure of growth plates, regulation of libido and feedback with the pituitary. With an excess of testosterone, the formation of estrogens also increases.

It is precisely the ratio of these two pathways that explains why supraphysiological concentrations of testosterone can simultaneously enhance androgenic effects (acne, baldness, prostate growth) and estrogenic ones (gynecomastia, fluid retention). The activity of the enzymes is individual and depends on genetics, age and the amount of adipose tissue.

• 5α-reductase→ DHT: skin, follicles, prostate.
• Aromatase→ estradiol: adipose tissue, brain, bones.
• 17β-hydroxysteroid dehydrogenase→ androstenedione: liver and other tissues.

Hepatic catabolism and conjugation

The main part of testosterone is inactivated in the liver. First, 17β-hydroxysteroid dehydrogenase oxidizes it to androstenedione, then 5α- and 5β-reductases and 3α-hydroxysteroid dehydrogenase reduce the steroid nucleus. The end products of this phase are androsterone and etiocholanolone — the main 17-ketosteroids of urine.

The second phase of metabolism is conjugation. Glucuronic acid is attached to the molecule (the reaction is catalyzed by enzymes of the UDP-glucuronosyltransferase family) or a sulfate group. Conjugates are much more soluble in water, so the kidneys easily excrete them in the urine. A small part of the metabolites passes with bile into the intestine.

The key enzyme for the glucuronidation of testosterone itself is UGT2B17. Its gene is characterized by a common polymorphism — a deletion in which the enzyme is not synthesized. In people with such a deletion there is significantly less testosterone glucuronide in the urine, although the hormone level in the blood may be normal. The frequency of the deletion differs substantially between populations.

Epitestosterone is a natural isomer of testosterone that is not formed from it but synthesized in parallel in the testes. It is glucuronidated predominantly by a different enzyme, so its excretion is almost independent of the UGT2B17 genotype. This fact is important for interpreting the urinary steroid profile.

Scheme of transformations

To clearly show how these reactions are related to each other, the editorial team reduced the main pathways to a simplified scheme. It does not reflect quantitative proportions and does not account for the dozens of minor metabolites described in the analytical literature, but it gives an idea of the logic of the process.

It is important that metabolism is not a one-way process. Androstenedione can be converted back into testosterone, and in the tissues there is local regulation: the same hormone in one cell becomes DHT, and in a neighboring one — estradiol. Therefore the level of testosterone in the blood does not always accurately describe its action in a specific organ.

The kinetics of the whole chain are determined primarily by two stages: the rate of ester release from the depot and the rate of hepatic clearance. The half-life of free testosterone is only hours, whereas the "effective" period for enanthate is several days precisely because of the slow absorption.

The activity of the enzymes is affected by concomitant medications, alcohol, the state of the liver, the mass of adipose tissue and age. For example, in people with obesity the proportion of aromatization is higher, and in liver diseases both catabolism and SHBG synthesis are impaired.

Metabolites in the laboratory and anti-doping control

Knowledge of metabolism underlies both clinical laboratory diagnostics and anti-doping analysis. In the clinic, total testosterone, SHBG, estradiol and hematocrit are usually measured, and if needed — LH and FSH, to assess suppression of one's own axis. Urinary metabolites are not tested for routine treatment.

In anti-doping practice, the urinary steroid profile is analyzed: the ratio of testosterone to epitestosterone (T/E), the concentrations of androsterone, etiocholanolone and other markers. These indicators are tracked over time within the steroidal module of the Athlete Biological Passport, which makes it possible to detect deviations from the individual norm.

Since synthetic and natural testosterone are chemically identical, isotope ratio mass spectrometry (IRMS) is used to confirm exogenous origin. It assesses the ratio of carbon isotopes, which differs in a hormone synthesized from plant raw material and in a hormone produced by the body.

The UGT2B17 polymorphism is a vivid example of why a one-time T/E measurement without accounting for genetics and dynamics is interpreted with caution. That is precisely why the modern system relies on individual profiles and confirmatory methods rather than on a single number. Testosterone and all its esters are on the WADA Prohibited List at all times — both in-competition and out-of-competition.

Editorial conclusions

Testosterone enanthate is quickly converted in the body into ordinary testosterone, and enanthic acid is utilized like a fatty acid. The hormone's further fate is classic steroid metabolism: activation into DHT and estradiol, hepatic inactivation and excretion of conjugates in the urine.

It is precisely the active metabolites that explain a significant part of the side effects of supraphysiological levels: the androgenic action on the skin, hair and prostate and the estrogenic one — on the mammary gland and water-salt balance.

Individual differences in enzymes (5α-reductase, aromatase, UGT2B17) make the body's response to the same drug very different, so generalized advice from the internet does not replace an examination by a doctor.

To complete the picture, we recommend reading our materials on the tests for monitoring the hormonal background, on the role of estradiol in the male body, and on how the steroidal module of the Athlete Biological Passport works.

References

1. Kicman AT. Pharmacology of anabolic steroids. Br J Pharmacol. 2008;154(3):502–521.
2. Schänzer W. Metabolism of anabolic androgenic steroids. Clin Chem. 1996;42(7):1001–1020.
3. Schulze JJ, Lundmark J, Garle M, et al. Doping test results dependent on genotype of uridine diphospho-glucuronosyl transferase 2B17, the major enzyme for testosterone glucuronidation. J Clin Endocrinol Metab. 2008;93(7):2500–2506.
4. Nieschlag E, Behre HM, Nieschlag S (eds). Testosterone: Action, Deficiency, Substitution. 4th ed. Cambridge University Press; 2012.
5. Bhasin S, Brito JP, Cunningham GR, et al. Testosterone therapy in men with hypogonadism: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2018;103(5):1715–1744.
6. World Anti-Doping Agency. The World Anti-Doping Code International Standard: Prohibited List. Montreal: WADA; чинна редакція.