Decavol DSHEA Information

We believe fully that 19Nor DHEA is legal to sell under US law. It is a naturally occurring product that is legal under DSHEA. It is a naturally occurring metabolite of standard DHEA due to incomplete aromatisation and is found in both humans and pigs in fairly large quantities.

To make dietary supplements compliant under current DSHEA regulation, one of two premises must be completed. First a supplement must be naturally occurring and part of the food supply where the food has not been chemically altered. Second it must have been sold as a dietary supplement prior to 1994 or have a New Dietary Ingredient filing and approval. 19NorAndrosterones are proven in literature to naturally occur in Pig testicles along with other 19Nor compounds. Under DSHEA, 19Nor-4-DHEA is a natural metabolite of 19NorEtiochollane-3,17-dione that is proven to occur in pig flesh and pig testicles (23,24,25). Pig testicles under the natural tissue “orchic” have been sold as a means to increase hormonal response for over 30 years and used medicinally for over 100 years. Similar to standardizing other dietary supplements for active ingredients, we have shown that 19NorAndrosterones including 19NorDHEA are naturally occurring in pig testicles which have been sold as a dietary supplement for bodybuilders since the early 80’s. Orchic Extract can be standardized for 19NorDHEA and provides 100% legal supplementation as the original 80’s supplement had intended. This product is not intended to treat or cure any disease, but it can be used to augment hormone levels in men wishing to increase their levels. Additionally, this supplement is part of the food supply and the ingredient, though standardized has not been chemically altered from the pork tissue where it is found.

Product Description

  1. The dietary supplement that contains a mix of 19-norDHEA will consist of one hundred (100) mg of 19-norDHEA in a tablet or capsule that will be suggested to be taken up to three times per day.
  2. The label will contain the following instructions for use: “DIRECTIONS FOR USE: This product is for adults over the age of 21 only. Do not exceed recommended dosage. This product is not intended to diagnose, treat, cure or prevent any disease”. KEEP OUT OF REACH OF CHILDREN

Background

Dehydroepiandrosterone (DHEA) is a dietary ingredient that was marketed in the United States before October 15, 1994. DHEA.

The endogenous formation of norandrostanes has been well documented with norandrosterone, norepiandrosterone, noretiocholanolone detected in healthy human and animal tissues (7,8,9,10,11,12,13). The formation of 19-nor-metabolites has been suggested to be a byproduct of the incomplete aromatization of androgens to estrogens (12). DHEA is known to undergo aromatization to estrogens (1) therefore it is also subject to conversion to 19Nordehydroepiandrosterone.

The presence of endogenous norandrostane metabolites in urine and tissues strongly suggests the formation of 19-norDHEA through the incomplete action of aromatase in vivo.

19-norandrostanes are also NATURALLY present in the food chain (8,11,13). Consumption of a meal consisting of boar meat or testes resulted in high plasma levels of both norandrosterone and noretiocholanolone (8). Boar meat and testes contain high levels of norandrostenedione which has been shown in the literature to also convert into 19NorDHEA and when consumed has been shown to result in the excretion of 19-norDHEA in human urine (22).

Structure

Routes of Metabolic Formation

There are three major metabolic routes to the formation of norDHEA. One route is through the reduction of the 3-keto group with concomitant isomeric switching of the 4 double bond to the 5-position. This reaction is catalyzed by the 3-beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase enzyme (3), which is present in both the adrenals and testes. Norandrostenedione has been shown to be present in porcine testes and is created through the actions of 17-beta hydroxysteroid dehydrogenase (2) on the 17-hydroxyl group of nortestosterone, which has also been shown to be present in porcine testes. Both nortestosterone and norandrostenedione are created through the incomplete action of aromatase (1) through 19-hydroxylation. NorDHEA can be created through the incomplete aromatization (1) of DHEA as well. Pregnenolone is a direct precursor to DHEA and is converted through the actions of 17-beta hydroxylase (5). Pregnenolone is also subject to incomplete aromatization (1) forming norpregnenolone which can then be converted directly to norDHEA through the action of 17-beta hydroxylase (5). Norpregnenolone is also converted by 3-beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase to form norprogesterone which has be shown to be naturally occurring in many mammalian species.

The conversion process that occurs in mammals via Hydroxysteroid-dehydrogenases to verify that indeed 19Nor-Androstenedione and 19Nor-Androstenediol can convert into Norandrostene-3b-ol,17-one (better known as NorDHEA). Hydroxysteroid dehydrogenases are known to be bi-directional and this paper shows just one example (which exactly parallels the 19Nor system). This process is known to happen in C19 isomers and utilizing the same enzymatic systems in humans and pigs to bi-directionally convert adrenal steroids to active androgens via this pathway:

This bi-directional nature of 17bHSD type 4 can be verified in the attached paper from which this excerpt was taken.
"The activity of 1713-HSD is in fact responsible for the interconversion of 17-ketosteroids (e.g., dehydroepiandrosterone, androstenedione, and estrone) with the corresponding 17[3-hydroxysteroids (e.g., androst-5-ene-313,1713-diol, testosterone, and 1713-estradiol). The reduction step catalyzed by the various 17[3-HSDs is thus essential for the formation of the active estrogens, 17[3-estradiol (E2) and androst-ene-3[3,17[3-diol (5-diol), as well as for the biosynthesis of the active androgen testosterone, and the oxidative reaction catalyzed by other 17[3-HSDs inactivates the potent sex steroids into compounds having no or low biological activity. 17[3-HSD is thus the key enzyme involved in the development, growth, and function of all reproductive tissues in both males and females."
"and type 4 17f3-HSD mainly degrades 17[3-estradiol into estrone and androst-5-ene-3~, 17~-diol into dehydroepiandrosterone."

The human type 4 17[3-HSD is a 736-amino acid protein of M r 80 kDa that shares 84% identity with the corresponding porcine enzyme and transforms E 2 into E l and 5-diol into DHEA.

Name of Ingredient:

19-nordehydroepiandrosterone, 19NorAndrosterone, 19NorEtiochollanolone

Safety of the ingredient

DHEA has a long history of use in healthy and dieased humans and has been shown to be safe in doses up to 200 mg per day for 24 weeks (20) and 2250 mg for 16 weeks (18) with minimal side effects. The side effects that are encountered are due, in large part to the formation of estrogen and potent 5-alpha reduced metabolites (4,20,21). DHEA has been shown in the literature to convert via the aromatase enzyme to estrogens (1). In addition, DHEA has been shown to convert to more potent 5-alpha reduced metabolites. 19-norandrostanes, including 19-norDHEA, are known to convert to less potent metabolites through 5-alpha reduction. In addition, the lack of a carbon in the 19-position reduces affinity for the aromatase enzyme and results in reduce aromatization to estrogens (14,15). 19-norDHEA would therefore be expected to have a better safety profile than DHEA. Numerous opinions have been written showing that comparing an ingredient to one that is already on the market is a valid and defensible form of showing safety. The numerous benefits of 19NorDHEA over standard 5-DHEA make this certainly more safe and effective as a means of increasing adrenal hormones.

Conclusion

Porcine orchic raw tissues and extracts have been used as nutritional supplements for decades in the US. The presence of precursors and metabolites of norDHEA in porcine testes (the source of orchic raw tissue and extracts) as well as the necessary metabolic enzyme systems protects norDHEA as a DSHEA compliant nutritional supplement. Additionally, being part of the food supply, NorDHEA could satisfy either aspect of DSHEA’s compliance.

Safety References

  1. Longcope C, Bourget C, Flood C. The production and aromatization of dehydroepiandrosterone in post-menopausal women. Maturitas. Dec;4(4):325-32, 1982
  2. Callies F, Arlt W, Siekmann L, Hubler D, Bidlingmaier F, Allolio B. Influence of oral dehydroepiandrosterone (DHEA) on urinary steroid metabolites in males and females. Steroids. Feb;65(2):98-102, 2000
  3. Dehennin L, Ferry M, Lafarge P, Peres G, Lafarge JP. Oral administration of dehydroepiandrosterone to healthy men: alteration of the urinary androgen profile and consequences for the detection of abuse in sport by gas chromatography-mass spectrometry. Steroids. Feb;63(2):80-7, 1998
  4. Gilad S, Chayen R, Tordjman K, Kisch E, Stern N. Assessment of 5 alpha-reductase activity in hirsute women: comparison of serum androstanediol glucuronide with urinary androsterone and aetiocholanolone excretion. Clin Endocrinol (Oxf). Apr;40(4):459-64, 1994
  5. Rao MS, Ide H, Alvares K, Subbarao V, Reddy JK, Hechter O, Yeldandi AV. Comparative effects of dehydroepiandrosterone and related steroids on peroxisome proliferation in rat liver. Life Sci. 52(21):1709-16, 1993
  6. Milewich L, Hendricks TS, Johnson AR. Metabolism of dehydroisoandrosterone and androstenedione in human pulmonary endothelial cells in culture. J Clin Endocrinol Metab. May;56(5):930-5, 1983
  7. Reznik Y, Dehennin L, Coffin C, Mahoudeau J, Leymarie P. Urinary nandrolone metabolites of endogenous origin in man: a confirmation by output regulation under human chorionic gonadotropin stimulation. J Clin Endocrinol Metab. Jan;86(1):146-50, 2001
  8. Le Bizec B, Gaudin I, Monteau F, Andre F, Impens S, De Wasch K, De Brabander H. Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19-norandrosterone and 19-noretiocholanolone in human urine. Rapid Commun Mass Spectrom. 14(12):1058-65, 2000
  9. Le Bizec B, Monteau F, Gaudin I, Andre F. Evidence for the presence of endogenous 19-norandrosterone in human urine. J Chromatogr B Biomed Sci Appl. Feb 19;723(1-2):157-72, 1999
  10. Dehennin L, Bonnaire Y, Plou P. Urinary excretion of 19-norandrosterone of endogenous origin in man: quantitative analysis by gas chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl. Jan 22;721(2):301-7, 1999
  11. Debruyckere G, Van Peteghem C. Detection of 19-nortestosterone and its urinary metabolites in miniature pigs by gas chromatography-mass spectrometry. J Chromatogr. Apr 5;564(2):393-403, 1991
  12. Van Eenoo P, Delbeke FT, de Jong FH, De Backer P. Endogenous origin of norandrosterone in female urine: indirect evidence for the production of 19-norsteroids as by-products in the conversion from androgen to estrogen. J Steroid Biochem Mol Biol. Oct;78(4):351-7, 2001
  13. De Wasch K, Le Bizec B, De Brabander H, Andre F, Impens S. Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. II. Identification and quantification of 19-norsteroids responsible for 19-norandrosterone and 19-noretiocholanolone excretion in human urine. Rapid Commun Mass Spectrom. 15(16):1442-7, 2001
  14. Numazawa M, Nagaoka M, Sohtome N. Aromatase reaction of 3-deoxyandrogens: steric mode of the C-19 oxygenation and cleavage of the C10-C19 bond by human placental aromatase. Biochemistry. Aug 16;44(32):10839-45, 2005
  15. Fishman J. Biochemical mechanism of aromatization. Cancer Res. Aug;42(8 Suppl):3277s-3280s, 1982
  16. Rabkin JG, McElhiney MC, Rabkin R, McGrath PJ, Ferrando SJ. Placebo-controlled trial of dehydroepiandrosterone (DHEA) for treatment of nonmajor depression in patients with HIV/AIDS. Am J Psychiatry. Jan;163(1):59-66, 2006
  17. Piketty C, Jayle D, Leplege A, Castiel P, Ecosse E, Gonzalez-Canali G, Sabatier B, Boulle N, Debuire B, Le Bouc Y, Baulieu EE, Kazatchkine MD. Double-blind placebo-controlled trial of oral dehydroepiandrosterone in patients with advanced HIV disease. Clin Endocrinol (Oxf). Sep;55(3):325-30, 2001
  18. Dyner TS, Lang W, Geaga J, Golub A, Stites D, Winger E, Galmarini M, Masterson J, Jacobson MA. An open-label dose-escalation trial of oral dehydroepiandrosterone tolerance and pharmacokinetics in patients with HIV disease. J Acquir Immune Defic Syndr. May;6(5):459-65, 1993
  19. Alhaj HA, Massey AE, McAllister-Williams RH. Effects of DHEA administration on episodic memory, cortisol and mood in healthy young men: a double-blind, placebo-controlled study. Psychopharmacology (Berl). Oct 18;:1-11, 2005
  20. Chang DM, Lan JL, Lin HY, Luo SF. Dehydroepiandrosterone treatment of women with mild-to-moderate systemic lupus erythematosus: a multicenter randomized, double-blind, placebo-controlled trial. Arthritis Rheum. Nov;46(11):2924-7, 2002
  21. Acacio BD, Stanczyk FZ, Mullin P, Saadat P, Jafarian N, Sokol RZ. Pharmacokinetics of dehydroepiandrosterone and its metabolites after long-term daily oral administration to healthy young men. Fertil Steril. Mar;81(3):595-604, 2004
  22. Uralets VP, Gillette PA. Over-the-counter delta5 anabolic steroids 5-androsen-3,17-dione; 5-androsten-3beta, 17beta-diol; dehydroepiandrosterone; and 19-nor-5-androsten-3,17-dione: excretion studies in men. J Anal Toxicol. Apr;24(3):188-93, 2000
  23. J Steroid Biochem. 1989 May;32(5):729-35.Links Aromatization of 19-norandrogens by porcine Leydig cells. Raeside JI, Renaud RL, Friendship RM. Department of Biomedical Sciences, University of Guelph, Ontario, Canada.
  24. J Steroid Biochem Mol Biol. 1992 Jul;42(6):637-41.Links Hydroxylation of 19-norandrogens by porcine Leydig cells. Raeside JI, Renaud RL, Marshall DE. Department of Biomedical Sciences, University of Guelph, Ontario, Canada.
  25. J Endocrinol. 1993 May;137(2):281-9.Links Secretion of 19-hydroxyandrostenedione and 19-hydroxytestosterone by porcine Leydig cells in vitro and in vivo. Raeside JI, Renaud RL, Friendship RM, Khalil MW. Department of Biomedical Sciences, University of Guelph, Ontario, Canada.

Additional Supporting Abstracts

  1. J Anal Toxicol. 2002 Jan-Feb;26(1):43-7.
    Endogenous nandrolone metabolites in human urine. Two-year monitoring of male professional soccer players.

    • Le BB,
    • Bryand F,
    • Gaudin I,
    • Monteau F,
    • Poulain F,
    • Andre F.

    LABERCA, Ecole Nationale Veterinaire, Nantes, France. lebizec@vet-nantes.fr
    19-Norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) are the two main indicators used to prove the illegal use of nandrolone by humans. Recent studies showed that 19-NA and 19-NE can be endogenously produced in some individuals. The mediated cases observed over the last three years generated some questions about the appropriateness of the official International Olympic Committee cutoff level, which is 2 ng/mL of 19-NA in male urine samples. In the present study, professional soccer players belonging to the French First League were studied over a period of 19 months. In total, 385 urine samples were taken immediately before and after soccer competitions and were coupled with 200 blood samples for testosterone and LH determination. Results of the study showed that the mean values for 19-NA and 19-NE were 0.097 ng/mL and 0.033 ng/mL, respectively. For 19-NA, 70% of the samples proved to be below 0.1 ng/mL, whereas less than 20% were found to be between 0.1 and 0.2 ng/mL, and 7% were between 0.2 and 0.3 ng/mL. Only four urine samples were above 1.0 ng/mL; the maximal value was 1.79 ng/mL. For 19-NE, only one sample was above 1.0 ng/mL; the value was 1.42 ng/mL. Concentrations of these compounds after games were generally significantly higher than those before games.

  2. Rapid Commun Mass Spectrom. 2001;15(16):1442-7.
    Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. II. Identification and quantification of 19-norsteroids responsible for 19-norandrosterone and 19-noretiocholanolone excretion in human urine.

    • De Wasch K,
    • Le Bizec B,
    • De Brabander H,
    • Andre F,
    • Impens S.

    Laboratory of Chemical Analysis, Ghent University, Faculty of Veterinary Medicine, Salisburylaan 133, B-9820 Merelbeke, Belgium. Katia.DeWasch@rug.ac.be
    In previous work (Le Bizec et al., Rapid Commun. Mass Spectrom. 2000; 14: 1058), it was demonstrated that a boar meal intake could lead to possible false accusations of abuse of 17beta-nortestosterone in antidoping control. The aim of the present study was to identify and quantify endogenous 19-norsteroids in boar edible tissue at concentrations that can alter the steroid urinary profile in humans, and lead to excretion of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). The samples were analysed in two laboratories. The methodologies used for extraction and detection (GC/MS(EI) and LC/MS/MS(APCI+)) are compared and discussed. 19-Norandrostenedione (NAED), 17beta- and 17alpha-nortestosterone (bNT, aNT), and 17beta- and 17alpha-testosterone (bT, aT) were quantified. The largest concentrations of NAED and bNT were observed in testicles (83 and 172 microg/kg), liver (17 and 63 microg/kg) and kidney (45 and 38 microg/kg). A correlation between the bNT and NAED content of a typical meal prepared with boar parts and the excreted concentrations of 19-NA and 19-NE in human urine was demonstrated. Copyright 2001 John Wiley & Sons, Ltd.

  3. Rapid Commun Mass Spectrom. 2000;14(12):1058-65.
    Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19-norandrosterone and 19-noretiocholanolone in human urine.

    • Le Bizec B,
    • Gaudin I,
    • Monteau F,
    • Andre F,
    • Impens S,
    • De Wasch K,
    • De Brabander H.

    LDH-LNR, Ecole Nationale Veterinaire, BP 50707, F-44087 Nantes Cedex 03, France.
    For the first time in the field of steroid residues in humans, demonstration of 19-norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone (19-NE: 3alpha-hydroxy-5beta-estran-17-one) excretion in urine subsequent to boar consumption is reported. Three male volunteers agreed to consume 310 g of tissues from the edible parts (meat, liver, heart and kidney) of a boar. The three individuals delivered urine samples before and during 24 h after meal intake. After deconjugation of phase II metabolites, purification and specific derivatisation of target metabolites, the urinary extracts were analysed by mass spectrometry. Identification was carried out using measurements obtained by gas chromatography/high resolution mass spectrometry (GC/HRMS) (R = 7000) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (positive electrospray ionisation (ESI+)). Quantification was realised using a quadrupole mass filter. 19-NA and 19-NE concentrations in urine reached 3.1 to 7.5 microg/L nearby 10 hours after boar tissue consumption. Levels returned to endogenous values 24 hours after. These two steroids are usually exploited to confirm the exogenous administration of 19-nortestosterone (19-NT: 17beta-hydroxyestr-4-en-3-one), especially in the antidoping field. We have thus proved that eating tissues of non-castrated male pork (in which 17beta-nandrolone is present) might induce some false accusations of the abuse of nandrolone in antidoping. Copyright 2000 John Wiley & Sons, Ltd.

  4. Anal Chim Acta. 2007 Mar 14;586(1-2):184-95. Epub 2006 Aug 24.
    Quantitation of 17beta-nandrolone metabolites in boar and horse urine by gas chromatography-mass spectrometry.

    • Roig M,
    • Segura J,
    • Ventura R.

    Unitat de Recerca en Farmacologia, Institut Municipal d’Investigacuo Medica, Dr. Aiguader, 80, 08003 Barcelona, Spain.
    A method to quantify metabolites of 17beta-nandrolone (17betaN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C18 cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with beta-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-butylmethylether. The dried organic extracts were derivatized with MSTFA:NH4I:2-mercaptoethanol to obtain the TMS derivatives, and were subjected to analysis by gas chromatography mass spectrometry (GC/MS). The procedure was validated in boar and horse urine for the following metabolites: norandrosterone, noretiocholanolone, norepiandrosterone, 5beta-estran-3alpha, 17beta-diol, 5alpha-estran-3beta, 17beta-diol, 5alpha-estran-3beta, 17alpha-diol, 17alpha-nandrolone, 17betaN, 5(10)-estrene-3alpha, 17alpha-diol, 17alpha-estradiol and 17beta-estradiol in the different metabolic fractions. Extraction recoveries were higher than 90% for all analytes in the free fraction, and better than 80% in the glucuronide and sulphate fractions, except for 17alpha-estradiol in the glucuronide fraction (74%), and 5alpha-estran-3beta, 17alpha-diol and 17betaN in the sulphate fraction (close to 70%). Limits of quantitation ranged from 0.05 to 2.1 ng mL(-1) in the free fraction, from 0.3 to 1.7 ng mL(-1) in the glucuronide fraction, and from 0.2 to 2.6 ng mL(-1) in the sulphate fraction. Intra- and inter-assay values for precision, measured as relative standard deviation, and accuracy, measured as relative standard error, were below 15% for most of the analytes and below 25%, for the rest of analytes. The method was applied to the analysis of urine samples collected after administration of 17betaN laureate to boars and horses, and its suitability for the quantitation of the metabolites in the three fractions has been demonstrated.

  5. Food Addit Contam. 2005 Sep;22(9):808-15.
    Endogenous occurrence of some anabolic steroids in swine matrices.

    • Poelmans S,
    • De Wasch K,
    • Noppe H,
    • Van Hoof N,
    • Van Cruchten S,
    • Le Bizec B,
    • Deceuninck Y,
    • Sterk S,
    • Van Rossum HJ,
    • Hoffman MK,
    • De Brabander HF.

    Laboratory of Chemical Analysis, Research Group of Veterinary Public Health and Zoonoses, Department of Veterinary Public Health and Food Safety, Salisburylaan, 133 B-9820 Merelbeke, Belgium.
    Following findings of 17beta-19-nortestosterone (150-200 microg kg(-1)) in pigs of unspecified gender imported into the European Union, a study to determine steroid and hormone levels in swine from six age/gender categories (uncastrated ‘old’ boars, cryptorchids, one intersex, barrows, gilts and sows) was initiated. Indeed, for some hormones there has been a discussion about their being endo- or exogenous. Tissue and urine samples from swine from each of the six categories were obtained in Belgium, France, the Netherlands and the USA. Samples were analysed in three laboratories. Quantitation was obtained for norandrostenedione, 19-nortestosterone and boldenone. The results give a well-documented overview of the status of the presence of these hormones in swine. The data illustrate that uncastrated ‘old’ boars produce the highest percentage of ‘positive’ matrices, followed by the cryptorchids. Concentrations in the matrices of the barrows and the gilts are lower. Also, sow matrices contain low amounts of nor-steroids. Furthermore, urine samples from an intersex pig contains a higher concentration of nortestosterone than sows and can therefore be suspected for illegal use of these hormones. Veterinarians taking samples in pig farms for the analysis of hormones need to be aware of the presence and concentrations of these substances in the different categories.

  6. Biochem Biophys Res Commun. 1988 Aug 30;155(1):144-50.
    Formation and metabolism of 5(10)-estrene-3 beta,17 beta-diol, a novel 19-norandrogen produced by porcine granulosa cells from C19 aromatizable androgens.

    • Khalil MW,
    • Chung N,
    • Morley P,
    • Glasier MA,
    • Armstrong DT.

    Dept. of Obstetrics and Gynecology, University of Western Ontario, London, Canada.

    The biosynthesis of non-aromatic 19-norsteroids has been studied using primary cultures of porcine granulosa cells. Formation of 5(10)-estrene-3 beta,17 beta-diol, a novel 19-norsteroid, from androstenedione and 19-hydroxyandrostenedione by porcine granulosa cells is reported for the first time. The structure was deduced from (i) comparison of its elution times on C18 reverse phase HPLC with authentic 5(10)-estrene-3 beta,17 beta-diol (ii) identification with 5(10)-estrene-3 beta,17 beta-diol-diacetate after acetylation (iii) oxidation/acid catalysed isomerization to 19-norandrostenedione. Serum or serum plus FSH significantly stimulated (seven fold increase) formation of 5(10)-estrene-3 beta,17 beta-diol from androstenedione and 19-hydroxyandrostenedione. Formation of 5(10)-estrene-3 beta,17 beta-diol from both substrates was significantly (p less than 0.01) reduced by the aromatase inhibitors 4-hydroxyandrostenedione (15 microM) and aminoglutethimide phosphate (10(-4)M). These results suggest that 5(10)-estrene-3 beta,17 beta-diol (and 19-norandrostenedione) may be formed by enzymes similar to the aromatase complex required for estradiol-17 beta biosynthesis. 5(10)-Estrene-3 beta,17 beta-diol is converted by granulosa cells to four metabolites. 19-Norandrostenedione was identified by crystallization to constant specific activity; 19-nortestosterone is a minor product. Production of 19-norandrostenedione and 19-nortestosterone indicates that granulosa cells possess the enzymes necessary for the transformation of 5(10)-estrene-3 beta,17 beta-diol and other 3-hydroxy-5(10)-estrenes to 19-nor-4-ene-3-ketosteroids. The formation of 5(10)-estrene-3 beta,17 beta-diol and 19-norandrostenedione as substantial metabolites of androstenedione suggest a physiological role for these 19-norsteroids in ovarian follicular development.

  7. J Endocrinol. 1989 Feb;120(2):251-60.
    Formation of 4-oestrene-3,17-dione (19-norandrostenedione) by porcine granulosa cells in vitro is inhibited by the aromatase inhibitor 4-hydroxyandrostenedione and the cytochrome P-450 inhibitors aminoglutethimide phosphate and ketoconazole.

    • Khalil MW,
    • Morley P,
    • Glasier MA,
    • Armstrong DT,
    • Lang T.

    MRC Group in Reproductive Biology, University of Western Ontario, London, Canada.
    The origin and biosynthesis of 4-oestrene-3,17-dione (19-norandrostenedione), a major steroid in porcine ovarian follicular fluid, was investigated by culturing granulosa cells from 4-6 mm follicles of prepubertal gilts with radiolabelled androstenedione and 19-hydroxyandrostenedione. Steroid metabolites were purified by solvent extraction and lipophilic column chromatography, and analysed by C18 reverse-phase high-performance liquid chromatography. 19-Hydroxyandrostenedione, 19-norandrostenedione and oestradiol-17 beta were obtained as major metabolites from androstenedione, while 19-norandrostenedione and oestradiol-17 beta were the major products from 19-hydroxyandrostenedione. Serum alone or serum plus FSH significantly enhanced formation of 19-norandrostenedione and oestradiol-17 beta from each substrate, compared with controls. Micromolar concentrations (1 mumol/l) of 4-hydroxyandrostenedione, an aromatase inhibitor, significantly reduced formation of 19-norandrostenedione and oestradiol-17 beta by granulosa cells cultured with serum and FSH. Formation of 19-norandrostenedione and oestradiol-17 beta from androstenedione and 19-hydroxyandrostenedione was also significantly inhibited by aminoglutethimide phosphate, a cytochrome P-450 inhibitor known to block the conversion of androstenedione to oestrogens. Ketoconazole, an inhibitor of the cytochrome P-450 dependent 17,20-lysase, blocked formation of 19-norandrostenedione and oestradiol-17 beta only at millimolar concentrations. These results suggest that 19-norsteroid and oestrogen formation from C19 aromatizable androgens may share a common or overlapping pathway, and imply that 19-norsteroid and oestrogen synthesis is mediated by cytochrome P-450 dependent enzymes.

Supporting References

  1. Raeside JI, Renaud RL, Friendship RM, Khalil MW. Secretion of 19-hydroxyandrostenedione and 19-hydroxytestosterone by porcine Leydig cells in vitro and in vivo. J Endocrinol. May;137(2):281-9, 1993
  2. Raeside JI, Renaud RL, Marshall DE. Hydroxylation of 19-norandrogens by porcine Leydig cells. J Steroid Biochem Mol Biol. Jul;42(6):637-41, 1992
  3. Mauduit C, Chauvin MA, de Peretti E, Morera AM, Benahmed M. Effect of activin A on dehydroepiandrosterone and testosterone secretion by primary immature porcine Leydig cells. Biol Reprod. Jul;45(1):101-9, 1991
  4. Raeside JI, Renaud RL, Friendship RM. Aromatization of 19-norandrogens by porcine Leydig cells. J Steroid Biochem. May;32(5):729-35, 1989
  5. Kwan TK, Taylor NF, Watson D, Gower DB. Gas chromatographic-mass spectrometric study of metabolites of C21 and C19 steroids in neonatal porcine testicular microsomes. Biochem J. May 1;227(3):909-16, 1985
  6. Hurden EL, Gower DB, Harrison FA. Comparative rates of formation, in vivo, of 16-androstenes, testosterone and androstenedione in boar testis. J Endocrinol. Nov;103(2):179-86, 1984
  7. Bernier M, Gibb W, Saez JM, Collu R, Ducharme JR. The 3 beta-hydroxysteroid dehydrogenase activity of cultured porcine Leydig cells in primary culture. Can J Physiol Pharmacol. Oct;62(10):1300-3, 1984
  8. Raeside JI, Lobb DK. Metabolism of androstenedione by Sertoli cell enriched preparations and purified Leydig cells from boar testes in relation to estrogen formation. J Steroid Biochem. Jun;20(6A):1267-72, 1984
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