Bile acids. 39. Metabolism of 5 -cholestane-3 ,26-diol and 5 -cholestane-3 ,7 ,26-triol in the rat with a bile fistula

25r-5alpha-[5alpha,6alpha-(3)H(2)]Cholestane-3beta,7alpha,26-triol was prepared from 3beta,26-diacetoxy-5alpha[5alpha,6alpha-(3)H(2)]cholestan-7-one that was obtained from kryptogenin. Huang-Minlon reduction of the ketone provided 25r-5alpha-[5alpha-(3)H]cholestane-3beta,26-diol. Results from mass spectrometry, molecular rotation, and several types of chromatography are consonant with the assigned structures. Bile was collected for 8 days from adult male rats, with cannulated bile ducts, that had received approximately 0.8 mg of the triol or diol intraperitoneally. Bile from the first 12 hr was hydrolyzed, and the bile acids were separated by partition chromatography. The chromatographic pattern of separated bile acids was much simpler for the triol than the diol. Approximately 50% of the bile acids derived from the triol were trihydroxy allo acids (allocholic acid, 44%, and its 3beta isomer, 5.3%); only 16.4% allocholic acid was obtained from the diol. Comparable amounts of allochenodeoxycholic acid were derived from the diol and triol (21.2% and 28.2%, respectively). Unidentified metabolites in the dihydroxy acid fraction derived from the diol constitute 15.8% of chromatographed material.     

Cannulation in situ of equine umbilicus. Identification by gas chromatography-mass spectrometry (GC-MS) of differences in steroid content between arterial and venous supplies to and from the placental surface

Equine umbilicus was cannulated in utero and a series of cord plasma samples removed for analysis. After steroid extraction and derivatisation, gas chromatographic-mass spectrometric (GC-MS) analysis demonstrated large differences in steroid content between the plasma samples obtained from the umbilical artery and vein, the blood supplies leading to and from the placental surface, respectively. 3β-Hydroxy-5,7-androstadien-17-one, dehydroepiandrosterone, pregnenolone, 3β-hydroxy-5-pregnan-20-one, 5-pregnene-3β,20β-diol and 5β-pregnane-3β,20β-diol were identified as major constituents in extracts from umbilical arterial plasma samples, mostly as unconjugated steroids. Together with 5-pregnane-3,20-dione, these steroids were identified in extracts from umbilical venous plasma samples but at significantly reduced levels to those determined in arterial plasma samples. Oestradiol-17, dihydroequilin-17 and dihydroequilenin-17 were identified in extracts (mostly sulphate-conjugated) from both umbilical arterial and venous plasma samples, much larger amounts being detected in the plasma sampled from, rather than to, the placental surface. Equilin, equilenin, oestrone, oestradiol-17β, dihydroequilin-17β and dihydroequilenin-17β were not detected in the present studies. Isomers of 5(10)-oestrene-3,17β-diol together with 5(10),7-oestradiene-3,17β-diol and its possible oxidative artifact, 5(10),7,9-oestratriene-3,17β-diol, were tentatively identified only in sulphate-conjugated extracts from umbilical venous plasma samples. No glucuronic acid-conjugated steroids could be detected. The implications of this work in the elucidation of the biosynthetic pathways leading to both the formation of oestrogens and C₁₈ neutral steroids at the placental surface are discussed.     

Steroid glucuronides: Human circulatory levels and formation by LNCaP cells

We studied the relationship between circulating androsterone glucuronide, androstane-3,17β-diol glucuronide and androstane-3β,17β-diol glucuronide concentrations and adrenal as well as testicular C-19 steroids in men. Among the three 5-reduced steroid glucuronides, androsterone glucuronide is the predominant C-19 steroid measured in plasma and its levels are markedly elevated compared to those of the non-conjugated steroid. The marked rise in testosterone during puberty was strongly correlated with the increase in both androsterone glucuronide and androstane-3,17β-diol glucuronide, thus suggesting that testicular C-19 steroids are the main precursors of the steroid glucuronides. We also found that the presence of testicular androgen in plasma contributes to approx. 70% of plasma androsterone glucuronide and androstane-3,17β-diol glucuronide. Our data suggest that the adrenal C-19 steroids remaining in circulation after castration in men are converted into potent androgen which are then glucuronidated by UDP-glucuronyltransferase. We also demonstrated that the human prostate cell line LNCaP is capable of converting to a large extent androstenedione into androsterone glucuronide. Our data further confirm that glucuronidation is a major pathway of steroid metabolism in steroid target tissues.     

Inactivation of delta 5-3-oxo steroid isomerase with active-site-directed acetylenic steroids.

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 x 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 x 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.     

Simultaneous radioimmunoassay of 5α-amdrostane-3α, 17β-diol and 5α-androstane-3β, 17β-diol unconjugated and conjugated in human serum

The simultaneous determinations of both 3α and 3β epimers of 5α-androstane-3, l7β-diol as their glucuronides, sulfates and in their unconjugated forms are described. The diol estimation is carried out by radioimmunoassay with two specific immune sera after purification of the serum by use of chromatography on Sephadex LH-20. The values obtained (mean ± S.D.) in pg/ml for the unconjugated 3α and 3β epimers were, respectively, 267 ± 67 and 816 ± 76 for men; 114 ± 33 and 515 ± 177 for women; 142 ± 77 and 779 ± 200 for hirsute women. Among the conjugates, the most important were the sulfoconjugates, their rates being, respectively, (men ± S.D. in ng/ml) 41.3 ± 9.5 and 103 ± 40 for men; 12.4 ± 3.1 and 51.2 ± 14.9 for women and 36 ± 22 and 72 ± 36 for hirsute women. Differences in the conjugation of both epimers were also noticed.     

Synthesis of 5 beta,17 alpha-19-norpregn-20-yne-3 beta,17-diol and of 5 beta,17 alpha-19-norpregn-20-yne-3 alpha,17-diol, human metabolites of norethindrone

A convenient synthesis of both 5 beta,17 alpha-19-norpregn-20-yne-3 beta,17-diol (1) and 5 beta,17 alpha-19-norpregn-20-yne-3 alpha,17-diol (2) in multigram quantities from estr-4-ene-3,17-dione is reported. Full characterization of these often-cited human metabolites of norethindrone is presented for the first time.     

Olfactory responses to steroids in an African mouth-brooding cichlid, Haplochromis burtoni(Günther)

Underwater electro‐olfactogram (EOG) recordings involving 150 steroids and eight prostaglandins were used to determine which of these potential odorants are detected by the olfactory organ of an African cichlid, Haplochromis burtoni. In initial EOG tests at 10^?9 M, H. burtoni did not respond to unconjugated steroids or prostaglandins, but did respond to 17 conjugated steroids, 11 of which (17β‐oestradiol‐17β‐glucuronide; 17β‐oestradiol‐3‐sulphate; 17β‐oestradiol‐3,17β‐disulphate; epiandrosteron‐3β‐sulphate; etiocholanolone‐3α‐glucuronide; testosterone‐17β‐sulphate; dehydroepiandrosterone‐3β‐sulphate; 5α‐pregnan‐3β‐ol‐20‐one‐3β‐sulphate; 5β‐pregnan‐3α,17‐diol‐20‐one‐3α‐glucuronide; 5β‐pregnan‐3α,17,21‐triol‐11,20‐dione‐3α‐glucuronide; pregnenolone‐3β‐sulphate) were selected for EOG concentration‐response, cross‐adaptation and binary mixture tests. The EOG detection thresholds ranged from 10^?11 to 10^?9 M in all but one instance (female threshold to pregnenolone‐3β‐sulphate; 10^?8 M), and males and females exhibited only minor differences in EOG threshold or response magnitude. Results of EOG cross‐adaptation tests, which were supported by results of binary mixture tests, indicated that the response to the 11 steroid conjugates is mediated by five putative olfactory receptor mechanisms characterized by specificity for conjugate position and type: 3‐sulphate, 17‐sulphate, 3,17‐disulphate, 3‐glucuronide, 17‐glucuronide. Although there is no evidence that H. burtoni releases, or exhibits biological response to, the steroids shown to be detected in this study, the present results are suggestive of a complex pheromone system utilizing steroid conjugates.     

5 alpha-androstane-3,17-dione in peripheral plasma of men and women.

An antibody to androstanedione obtained in a rabbit by immunization with androstenedione-7 alpha-carboxymethyl-thioether conjugated to bovine serum albumin was found to cross-react 100% with 5 alpha-androstane-3,17-dione, a property that was used to develop a radioimmunoassay for this steroid. Plasma 5 alpha-androstane-3,17-dione concentrations were determined in young men, and in women throughout an ovulatory cycle. In the men (n = 6), plasma 5 alpha-androstane-3,17-dione concentrations were in the range of 84 to 273 pg/ml with a mean (+/- SD) value of 164 +/- 57 pg/ml. The plasma levels in the women (n = 5) were in the ranges of 35 +/- 14 to 145 +/- 75 pg/ml during the follicular phase, and 109 +/- 50 to 151 +/- 44 pg/ml during the luteal phase. The tissue sites of origin of 5 alpha-androstane-3,17-dione have not been defined, however, some extraglandular tissues are known to contain enzymes that convert C19-steroids to 5 alpha-androstane-3,17-dione. It is possible that 5 alpha-androstane-3,17-dione in circulation serves as a substrate for peripheral synthesis of 5 alpha-dihydrotestosterone.     

Chemical synthesis of 7- and 8-dehydro derivatives of pregnane-3,17alpha,20-triols,potential steroid metabolites in Smith-Lemli-Opitz syndrome

Pregnane-3,17 alpha,20-triols bearing unsaturation at delta(7), delta(8), delta(5,7), or delta(5,8) have been tentatively identified as steroid metabolites in Smith-Lemli-Opitz syndrome (SLOS). Starting with 17 alpha-hydroxypregnenolone diacetate, we have synthesized 13 unsaturated C(21) triols by four different routes in one to four steps. These multifunctional steroids were prepared by a series of regio- and stereoselective transformations chosen to minimize facile olefin isomerization and 17-deoxygenation. The results include a study of stereoselectivity in the reduction of 17 alpha-hydroxy-20-ketosteroids, an alternative method for reducing diethyl azodicarboxylate adducts of delta(5,7) steroids, and an efficient oxidation-isomerization of a delta(5,7) steroid using cholesterol oxidase. The 13 triols and their synthetic precursors were fully characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The NMR data, together with molecular modeling, indicated unanticipated conformational heterogeneity for two synthetic intermediates, 17 alpha-hydroxypregna-4,7-diene-3,20-dione and 17 alpha-hydroxy-5 beta-pregn-7-ene-3,20-dione. The unsaturated C(21) triols are useful as reference standards to study adrenal steroid production in SLOS and to develop methods for pre- and postnatal diagnosis of this congenital disorder.     

Conjugate hydrocyanation of 17-acetyl-11-carbomethoxygona-1,3,5(10),13(17)-tetraenes

The conjugate hydrocyanation of 17-acetylgona-11-carbomethoxy-1,3,5(10),13(17)-tetraenes using diethylaluminum cyanide (Nagata reaction) is reported. This methodology has allowed the introduction of an angular cyano group at the C-13 position of the steroid skeleton. Subsequent reduction of the nitrile group yielded various functionalized steroids. One of them, 22 bears the natural trans/anti/trans stereochemistry and possesses an hydroxyl and aminomethyl functionalities in the positions 11beta and 13beta, respectively. The characteristic (1)H and (13)C NMR spectroscopic features of the synthesized steroids are reported.     

Profiling of urinary steroids by gas chromatography-mass spectrometry detection and confirmation of androstenedione administration using isotope ratio mass spectrometry

Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2-22 h post-administration. However, IRMS could provide positive determinations for up to 55 h post-administration. This study demonstrated that, 5β-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2-36 h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS.     

A new radioimmunoassay for serum 16 alpha-hydroxyandrost-4-ene-3,17-dione with specific antiserum

A radioimmunoassay system for serum 16 alpha-hydroxyandrost-4-ene-3,17-dione was developed with the use of rabbit antiserum against 16 alpha-hydroxyandrost-4-ene-3,17-dione-3-(O-carboxymethyl)oxime which was conjugated with bovine serum albumin. The antiserum was highly specific for 16 alpha-hydroxyandrost-4-ene-3,17-dione, with cross reactions to other steroids being less than 0.8% except for androst-4-ene-3,17-dione(3.4% cross reaction). Use of LH-20 column chromatography, however, clearly separated these two steroids. Pregnancy sera were measured with this assay system after an addition of labelled internal standard, extraction and separation by column chromatography. The lower limit of detection for 16 alpha-hydroxyandrost-4-ene-3,17-dione was 2 pg/tube. The mean recovery rate of the added standard was 98.3 +/- 8.8% (mean +/- SE). Intra- and inter-assay coefficients of variation were 8.6% (n = 6) and 12.1% (n = 7), respectively.     

Hypocholesterolemic activity of 17-alpha-methyl-5-alpha-androstane-3-beta, 17-beta-diol, a metabolite of 17-alpha-methyltestosterone

The hypocholesterolemic activities of 17alpha-methyltestosterone and its major identified fecal metabolite, 17alpha-methyl-5alpha-androstane-3,17-diol, were compared in dogs. The dose-response curves indicated that the two compounds had similar effects, although at doses below 1 mg/kg per day the diol appeared to be more active than the parent compound.     

Seized designer supplement named "1-Androsterone": identification as 3β-hydroxy-5α-androst-1-en-17-one and its urinary elimination

New analogues of androgens that had never been available as approved drugs are marketed as “dietary supplement” recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids.In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product “1-Androsterone” of the brand name “Advanced Muscle Science” was labeled to contain 100 mg of “1-Androstene-3b-ol,17-one” per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3β-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3β-hydroxy-5α-androst-1-en-17-one in the capsules as labeled.Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17β-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17β-diol, and 5α-androst-1-ene-3β,17β-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of “1-Androsterone”. Especially the ratios of androsterone/etiocholanolone and 5α-/5β-androstane-3α,17β-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.     

The identification of C-18 neutral steroids in normal stallion urine

As part of a continuing research program associated with the detection of anabolic steroid residues in horse urine, normal samples from entire male horses have now been investigated. Isomers of three C-18 neutral steroids; 4-estren-17-ol-3-one (1), estrane-3,17-diol (2) and an unsaturated estranediol having a possible structure (3), have been identified in urine samples from two male horses aged 8 and 14 years. Of these three steroids, compound (2) was not detected in the urine of a 2.5 yr old entire male nor in the majority of post-race urine samples from entire male horses average age 3.8 yrs (n = 34). Ten of these samples showed tentative indications of this compound. Although the isolation of isomers of estrane-3,17-diol from human non-pregnancy urine has been reported previously, analysis of non-pregnancy urine samples in the present study did not reveal the presence of these compounds.     

CYP7B1-mediated metabolism of 5α-androstane-3α,17β-diol (3α-Adiol): A novel pathway for potential regulation of the cellular levels of androgens and neurosteroids

The current study presents data indicating that 5α-androstane-3α,17β-diol (3α-Adiol) undergoes a previously unknown metabolism into hydroxymetabolites, catalyzed by CYP7B1. 3α-Adiol is an androgenic steroid which serves as a source for the potent androgen dihydrotestosterone and also can modulate gamma-amino butyric acid A (GABA_A) receptor function in the brain. The steroid hydroxylase CYP7B1 is known to metabolize cholesterol derivatives, sex hormone precursors and certain estrogens, but has previously not been thought to act on androgens or 3α-hydroxylated steroids. 3α-Adiol was found to undergo NADPH-dependent metabolism into 6- and 7-hydroxymetabolites in incubations with porcine microsomes and human kidney-derived HEK293 cells, which are high in CYP7B1 content. This metabolism was suppressed by addition of steroids known to be metabolized by CYP7B1. In addition, 3α-Adiol significantly suppressed CYP7B1-mediated catalytic reactions, in a way as would be expected for substrates that compete for the same enzyme. Recombinant expression of human CYP7B1 in HEK293 cells significantly increased the rate of 3α-Adiol hydroxylation. Furthermore, the observed hydroxylase activity towards 3α-Adiol was very low or undetectable in livers of Cyp7b1(?/?) knockout mice. The present results indicate that CYP7B1-mediated catalysis may play a role for control of the cellular levels of androgens, not only of estrogens. These findings suggest a previously unknown mechanism for metabolic elimination of 3α-Adiol which may impact intracellular levels of dihydrotestosterone and GABA_A-modulating steroids.     

Effect of 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 4-acetoxy-4-androstene-3,17-dione (4-Ac-A) on the 5 alpha-reduction of androgens in the rat prostate

The present study reports the effects exerted by 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 4-acetoxy-4-androstene-3,17-dione (4-Ac-A), three steroids known to inhibit the aromatization of androgens to estrogens, on the in vitro metabolism of labelled testosterone (T), dihydrotestosterone (DHT) and androstenedione (delta-4-A) in the ventral prostate of adult male rats. It has been found that ATD, in the concentration tested, does not influence the conversion of labelled T into DHT, but decreases the formation of 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol (diols). On the contrary, 4-OH-A and 4-Ac-A simultaneously decrease the formation of DHT and the diols. When T is used as the substrate, the presence in the medium of these three steroids enhances the formation of delta-4-A and of 5 alpha-androstanedione (5 alpha-A). ATD, but not 4-OH-A and 4-Ac-A inhibits the conversion of labelled DHT into the diols. The transformation of labelled delta-4-A into 5 alpha-A is not modified by either ATD or 4-OH-A, while 4-Ac-A exerts only a small inhibition. These results suggest that the three aromatase inhibitors tested are able to profoundly modify the metabolism of T in the ventral prostate of the rat. In particular: 4-OH-A and 4-Ac-A are able to inhibit the conversion of T into DHT; ATD is able to inhibit the conversion of DHT into the diols; ATD and 4-OH-A do not inhibit the process of 5 alpha-reduction of delta-4-A into 5 alpha-A, while 4-Ac-A exerts only a minor effect. It is suggested that in the ventral prostate of the rat there are two different 5 alpha-reductase isoenzymes, one sensitive to the inhibitory effect of the steroid tested and which is responsible for the conversion of T into the 5 alpha-reduced metabolites of the 17-OH series (DHT and the diols), and a second one, insensitive to the effects of the three steroids, which affects the conversion of delta-4-A into 5 alpha-A.     

The metabolism of cis- and trans-indane-1,2-diol

1. The metabolism of cis-indane-1,2-diol, trans-indane-1,2-diol, indene epoxide and 2-hydroxyindan-1-one in rats has been studied. The substances were administered to the animals by subcutaneous injection. 2. The urine of the dosed animals was examined for the presence of free and conjugated cis- and trans-dihydrodiols, and for each compound it was possible to isolate both cis and trans forms of indane-1,2-diol from the urine. 3. The urines were also examined by paper chromatography for ketones and two ketonic metabolites were detected in the urine of rats dosed separately with cis-indane-1,2-diol, trans-indane-1,2-diol, 2-hydroxyindan-1-one and indene epoxide. The ketones were provisionally identified as (1-oxoindan-2-yl glucosid)uronic acid and 1-oxoindan-2-yl sulphuric acid. 4. (1-Oxoindan-2-yl glucosid)uronic acid was isolated as the 2,4-dinitrophenylhydrazone from the urine of rats dosed separately with cis-indane-1,2-diol and trans-indane-1,2-diol. 5. Possible mechanisms for the interconversion of cis- and trans-indane-1,2-diol are discussed.     

Excretion of estrone,estradiol, and progesterone in the urine and feces of the female cotton-top tamarin (Saguinus oedipus oedipus)

The excretion of three gonadal steroids was studied in the urine and feces of female cotton-top tamarins (Saguinus oedipus oedipus). Each steroid, ¹⁴C-estrone, ¹⁴C-estradiol, and ¹⁴C-progesterone, was injected into a separate female cotton-top tamarin. Urine and feces were collected at 8 hr intervals for 5 days on the three tamarins. Samples were analyzed to determine the proportion of free and conjugated steroids. Steroid excretion patterns were determined by sequential ether extraction, enzyme hydrolysis, and chromatography. Labeled estrone was excreted in a slow and continuous manner into the urine (57%) and feces (43%) with 90% of the steroid conjugated. The nonconjugated form had an elution profile identical to ³H estrone, but the conjugated portion was not completely hydrolyzed by enzyme. Labeled estradiol was excreted primarily in the urine (87%) and was released rapidly. Over 90% of the injected ¹⁴C-estradiol was excreted in urine as a conjugate, of which 41% was converted to an estrone conjugate and the remaining 59% was excreted as a polar estradiol conjugate. Labeled progesterone was excreted primarily in the feces (95%), 61% of which was free steroid. Four to six individual peaks of radioactivity were found when using celite chromatography and high performance liquid chromatography (HPLC), indicating that progesterone is metabolized into several urinary and fecal metabolites. One of these peaks matched ³H-progesterone and others may be pregnanediols, pregnanetriols, and 17-hydroxyprogesterone. These steroidal excretion patterns help explain the atypical hormonal patterns seen during the tamarin ovarian cycle.     

Enzymic and nonenzymic reduction of (+)-dihydroquercetin to its 3,4,-diol

A NADPH-dependent reductase activity, capable of converting (+)-dihydroquercetin (2,3-trans) to its 3,4-diol (a leucocyanidin), has been demonstrated in crude, soluble protein extracts derived from cell suspension cultures of Douglas fir (Pseudotsuga menziessi). Neither NADH nor ascorbate substituted as the H-donor. Quantitative analyses were based on the production of cyanidin, the formation of an adduct with vanillin, and on absorbance at 280 nanometers. Nonenzymic reduction of (+)-dihydroquercetin with NaBH₄ produced two presumably isomeric flavan-3,4,-diols. One of these was similar to the enzymically produced diol, based on products isolated by chromatography on paper, on thin-layer cellulose and on C₁₈ reversed-phase columns (high performance liquid chromatography), and on the conversion of the diol to the all-trans dimer of (+)-catechin upon the addition of (+)-catechin.