4-Hydroxyestrone: a new metabolite of estradiol-17 beta from humans

4-Hydroxyestrone has been identified, by reverse isotope dilution, as a urinary metabolite after injection of a mixture of 4-³H- and 4-¹⁴C-estradiol-17β into a 23 year old woman and a 39 year old man. This new metabolite accounted for 1.1% of the -¹⁴C dose administered to the woman and 0.51% of the -¹⁴C dose administered to the man. 7.3% Of the ³H dose was liberated into the body water pool of the woman and 6.1% into that of the man. The yields of radioactive estrone, estradiol-17β, estriol, 2-hydroxyestrone, 2-methoxyestrone and 2-hydroxyestrone 3-methyl ether were also measured in both the Ketodase and Glusulase hydrolyzed urinary fractions from both subjects.     

The effect of oral contraceptive steroid therapy on the urinary metabolites of radioactive estrone and estradiol-17beta

Eight urinary metabolites of radioactive estrone and estradiol-17β (estrone, estradiol-17β, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone 3-methyl ether, 16α-hydroxyestrone, 2-hydroxyestradiol and estriol) have been measured by reverse isotope dilution from young women on oral contraceptive therapy. There was a decrease in the sum of the 16α-hydroxy1ated metabolites in the Ketodase liberated fraction from the subjects taking ethynylestradiol containing preparations as compared to those taking preparations containing mestranol and those subjects who were taking no oral contraceptives. This report is also the first to document and measure 2-hydroxyestradiol as a urinary metabolite of radioactive estrone and estradiol.     

Metabolism of radioactive 17alpha-ethynylestradiol by women

Data on the urinary excretion and subsequent fractionation of radioactivity derived from 6,7-tritiated-17alpha-ethinyl estradiol (tritiated EE) are presented. Tritiated EE was administered orally to 9 women. 17alpha-ethinyl estradiol 2-methoxy-17alpha-ethinyl estradiol, 2 -hydroxy-17alpha-ethinyl estradiol 3-methyl ether, and D-homoestradiol-1 7abeta were identified as urinary metabolites by reverse isotope dilution. The extent of D-homoannulation was much less than that reported previously with rabbits.     

Metabolism of 2-3H- and 4-14C-17alpha-ethynylestradiol 3-methyl ether (mestranol) by women.

A mixture of 2-3H and 4-14C-mestranol was administered orally to five women and 2-3H-mestranol alone to one woman. Reactions involving position 2 were extensive as judged by liberation of 3H into body water (14-45% of the dose). 17alpha-Ethynylestradiol, 2-hydroxy-17alpha-ethynylestradiol, 2-methoxy-17alpha-ethynylestradiol, 2-hydroxy-17alpha-ethynylestradiol 3-methyl ether and 16geta-hydroxy-17alpha-ethynylestradiol were measured in the "glucuronide" and pH1 fractions and mestranol, D-homoestrone-17a and D-homoestradiol-17abeta were also measured in the "glucuronide" fraction frum the urine to two of the women by reverse isotope dilution. Radioactive 2-methoxyestradiol accounted for less than 0.011% of the 14C dose in the "glucuronide" fraction of one of the women, consistent with the extent of de-ethynylation previously reported (Steroids, 25, 343 (1975).     

Catechol estrogen methylene ethers

We have prepared 2-hydroxyestrone 2,3-methylene ether and 2-hydroxy-estradiol 2,3-methylene ether; these compounds were not found as urinary metabolites of radioactive estrone, estradiol or 2-methoxyestrone in humans.     

Metabolism of 4-3-H- and 4-14-C-17alpha-ethynylestradiol 3-methyl ether (mestranol) by women.

A mixture of 4-3-H and 4-14-C-mestranol was administered orally to four women. Reactions involving position 4 were no greater than 1.7-3% of the dose as measured by liberation of 3-H into body water. The extent of de-ethynylation in vivo was no greater than 1-2% of the dose as measured by urinary estrone metabolites. Mestranol (0.7 and 0.32% of the dose), 17alpha-ethynylestradiol (6.6 and 11.3%) and 2-hydroxy-17alpha-ethynylestradiol (0.64 and 0.7%) were identified as metabolite aglycons by reverse isotope dilution after Ketodase hydrolysis of the urine from two of the women.     

Bromo and chlorobiphenyl metabolism: gas chromatography mass spectrometric identification of urinary metabolites and the effects of structure on their rates of excretion

The identification of the hydroxylated rat urinary metabolites of the 2-, 3- and 4-chlorobiphenyls and 2-, 3- and 4-bromobiphenyls has been determined by gas chromatographic mass spectrometric analysis of their corresponding methyl ether derivatives. The electron impact fragmentation patterns of the bromotheoxybiphenyls and chloromethoxybiphenyls were used to assign the position of the methyoxyl group (ortho, meta or para to the biphenyl bond); the mass spectra of the corresponding [2H5]halobiphenyls confirmed the sites of the hydroxylation by distinguishing between the halophenyl and phenyl rings. The results illustrated that ring hydroxylation occurs predominantly at the para positions of the biphenyl nucleus and at sites which are ortho and para to the halogen substituents. 4,4'-Dimethoxyhalobiphenyls are major urinary metabolites of the 2- and 3-halobiphenyls and the rapid formation of these metabolites is illustrated in a time course study which monitors the urinary metabolites formed after the separate coadministration of the isomeric chlorobiphenyl and bromobiphenyl substrates to rats.     

Metabolism of exogenous 4- and 2-hydroxyestradiol in the human male

The metabolic fate of the isomeric catecholestrogens 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) was studied to elucidate possible differences in their metabolism as an explanation for their different bioactivities. Healthy young men (n = 3 each) were infused (90 min) with 4-OHE2 (60 micrograms/h) or 2-OHE2 (100 micrograms/h). The main metabolites were determined in plasma and urine before, during and after infusion. Unconjugated and conjugated steroids, the latter after hot acid hydrolysis, were subjected to chromatography on LH-20 columns and measured by specific RIAs. During the infusion 4-OHE2 reached significant plasma concentrations whereas 2-OHE2 was so rapidly metabolised that its plasma levels remained virtually undetectable in spite of a higher infusion rate. The metabolism of 4-OHE2 was dominated by direct conjugation, that of 2-OHE2 by methyl ether formation. These findings were corroborated by the urinary excretion rates: during the infusion and the first hours afterwards, 4-OHE2 was mainly excreted as 4-OHE2 and 4-hydroxyestrone, while 2-OHE2 was predominantly excreted as 2-hydroxyestradiol 2-methyl ether and 2-hydroxyestrone 2-methyl ether.     

In vitro metabolic conjugation of catechol estrogens

In vitro metabolic conjugation of the catechol estrogens, 2-hydroxyestrone and 4-hydroxyestrone, has been investigated by means of HPLC with electrochemical detection. Sulfation of 2-hydroxyestrone and 4-hydroxyestrone with the rat liver 105 000 g supernatant fortified with 3'-phosphoadenosine-5'-phosphosulfate provided the 2- and 4-monosulfates, respectively. Glucuronidation of the two catechols with the rat and human liver 1500 g supernatant in the presence of uridine-5'- phosphoglucuronic acid gave the 2- and 4-glucuronides, respectively. In contrast, incubation with the guinea pig liver 1500 g supernatant yielded both isomeric monoglucuronides . When 2'-hydroxyestrone was incubated with rat liver 1500 g supernatant and S-adenosyl-L-methionine, the 2- and 3-monomethyl ethers were formed in an equal amount, while 4-hydroxyestrone was transformed into the 4-methyl ether in 12 times greater yield than the 3-methyl ether. The participation of sulfation and glucuronidation in the formation of guaiacol estrogens is discussed.     

Synthesis of 16alpha-bromoacetoxyestradiol 3-methyl ether and study of the steroid binding site of human placental estradiol 17beta-dehydrogenase.

Homogeneous estradiol 17beta-dehydrogenase (EC 1.1.1.62) was prepared from human placenta by affinity chromatography and the steroid binding site was studied with affinity-labeling techniques. 16alpha-Bromoacetoxyestradiol 3-methyl ether and the tritated compound were synthesized by condensation of estriol 3-methyl ether with bromoacetic acid or [2-3H]bromoacetic acid in the presence of dicyclohexylcarbodiimide. 16alpha-Bromoacetoxyestradiol 3-methyl ether is stable in 0.01 M phosphate buffer at pH 7.0, 25 degrees, for at least 24 hours. It alkylates cysteine, histidine, methionine, lysine, and tryptophan under physiological conditions. The steroid is a substrate of estradiol 17beta-dehydrogenase, thus it must bind at the steroid binding site. The inactivation of estradiol 17beta-dehydrogenase by 150-fold molar concentrations of 16alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo-first order kinetics with a half-time of 1.5 hours. Estradiol-17beta, NADH, and NADPH slow the rate of inactivation. 2-Mercaptoethanol in molar concentrations 50-fold that of 16alpha-bromoacetoxyestradiol 3-methyl ether stops the inactivation, but does not reverse it. 16alpha-Bromoacetoxyestradiol 3-methyl ether alkylates both NADH and NADPH; the presence of small amounts of enzyme markedly increases the rate of this alkylation. When the enzyme is inactivated with 16alpha-[2-3H]bromoacetoxyestradiol 3-methyl ether, amino acid analysis of acid hydrolysates reveals 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine. Comparison of 28 and 51% inactivated samples indicates that, as inactivation proceeds, the total amount of 3-carboxymethylhistidine decreases, while 1,3-dicarboxymethylhistidine increases, suggesting that the former is converted to the latter by a second alkylation step. When the enzyme is inactivated in the presence of a large excess of NADPH, only 1,3-dicarboxymethylhistidine is found. From the present study it is concluded that estradiol 17beta-dehydrogenase has a histidyl residue present in the catalytic region of the active site as does the previously studied 20beta-hydroxysteroid dehydrogenase.     

Biotransformation of 3-(3′,5′ -Dichlorophenyl)-5,5-dimethyl oxazolidine-2,4-dione

Using 3-(3′,5′-dichlorophenyl)-5,5-dimethyloxazolidine-2,4-dione labeled with ¹⁴C or ³H, absorption, excretion, and tissue distribution in male Wistar rats were studied, and metabolites excreted were identified. At the dosage rates of 100, 300, 1000 and 3000 mg/kg, the maximum excretion of orally administered radioactivity occurred within 24 hr. Increase in the dosage rate was paralleled by decrease in the proportion of urinary elimination. Essentially all the radioactivity was excreted in 2 weeks. DDOD level was generally low in most tissues. Adipose tissue contained higher radioactivity compared with others. Most of the urinary metabolites identified were characterized by hydroxylation at the 4′ position of the benzene ring moiety, and hydrolytic or oxidative modification of the oxazolidine ring portion.     

Studies on anabolic steroids--6. Identification of urinary metabolites of stenbolone acetate (17 beta-acetoxy-2-methyl-5 alpha-androst-1-en-3-one) in human by gas chromatography/mass spectrometry

The metabolism of stenbolone acetate (17 beta-acetoxy-2-methyl-5 alpha-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. Nine metabolites were detected in urine either as glucuronic or sulfuric acid aglycones after oral administration of a single 50 mg dose to a male volunteer. Stenbolone, the parent compound, was detected for more than 120 h after administration and its cumulative excretion accounted for 6.6% of the ingested dose. Most of the stenbolone acetate metabolites were isolated from the glucuronic acid fraction, namely: stenbolone, 3 alpha-hydroxy-2-methyl-5 alpha-androst-1-en- 17-one, 3 alpha-hydroxy-2 xi-methyl-5 alpha-androst-17-one; 3 isomers of 3 xi, 16 xi-dihydroxy-2-methyl-5 alpha-androst-1-en-17-one; 16 alpha and 16 beta-hydroxy-2-methyl-5 alpha-androst-1-ene-3, 17-dione; and 16 xi, 17 beta-dihydroxy-2-methyl-5 alpha-androst-1-en-3-one. Only isomeric metabolites bearing a 16 alpha or a 16 beta-hydroxyl group were detected in the sulfate fraction. Interestingly, no metabolite was detected in the unconjugated steroid fraction. The steroids identities were assigned on the basis of their TMS ether, TMS enol-TMS ether, MO-TMS and d9-TMS ether derivatives and by comparison with reference and structurally related steroids. Data indicated that stenbolone acetate was metabolized into several compounds resulting from oxidation of the 17 beta-hydroxyl group and/or reduction of A-ring delta-1 and/or 3-keto functions with or without hydroxylation at the C16 position. Finally, comparison of stenbolone acetate urinary metabolites with that of methenolone acetate shows similar biotransformation pathways for both delta-1-3-keto anabolic steroids. This indicates that the position of the methyl group at the C1 or C2 position in these steroids has little effect on their major biotransformation routes in human, to the exception that stenbolone cannot give rise to metabolites bearing a 2-methylene group since its 2-methyl group cannot isomerize into a 2-methylene function through enolization of the 3-keto group as previously observed for methenolone.     

Increased urinary catechol estrogen excretion in female smokers

Premenopausal female smokers show significantly increased estrogen 2-hydroxylation, which may account in part for the anti-estrogenic effects of cigarette smoking. We have measured five major urinary estrogens, including estradiol (E2), estrone (E1), 16 alpha-hydroxyestrone (16 alpha OHE1), estriol (E3), and 2-hydroxyestrone (2OHE1), in premenopausal female smokers and non-smokers, to determine whether increased C-2 hydroxylation affected the urinary excretory patterns in these subjects. While total measured estrogen excretion in the follicular phase did not differ significantly between the two groups, urinary 2OHE1 among the smokers constituted a significantly greater proportion of the total (31.1 vs 18.2%, P less than 0.02). This difference was largely caused by significantly increased urinary 2OHE1 and decreased E3 observed in smokers. A urinary catechol estrogen index, defined by [2OHE1]/[E3], was significantly elevated in smokers compared with non-smokers (1.67 +/- 0.21 vs 0.56 +/- 0.08, P less than 0.001), and this urinary index correlated strongly with radiometrically determined estrogen 2-hydroxylation (r = 0.84, P less than 0.01). Ratios of the various estrogen metabolites did not vary substantially throughout the menstrual cycle. Urinary estrogen indices as described here may therefore be useful in demonstrating differences in estrogen metabolism, specifically 2-hydroxylation vs 16 alpha-hydroxylation, in selected populations.     

Effects of exogenous thyroxine on C-2 and C-16 alpha hydroxylations of estradiol in humans

Thyroid dysfunction in humans is known to alter the excretory pattern of estrogen metabolites, suggesting that thyroid hormone directly influences the oxidative metabolism of estradiol. We examined the extent to which a brief period of hyperthyroidism specifically affected estradiol hydroxylation at C-2 and C-16 alpha, the two primary and competing sites of estrogen oxidation, using an in vivo radiometric assay in healthy male volunteers. Hydroxylation at C-2 was increased by a 2-week course of thyroxine (4.3 micrograms/kg/d) from 29.9% +/- 2.6% to 35.9% +/- 3.1% (P = 0.04), while 16 alpha-hydroxylation was unchanged (10.3% +/- 0.8% versus 9.3% +/- 0.5%). The greater extent of oxidation at C-2 was evidenced by a twofold increase in the urinary excretion of 2-hydroxyestrone (2.88 +/- 0.32 versus 5.30 +/- 0.85 micrograms/g creatinine), while the excreted products of 16 alpha-hydroxylation were unchanged. At the same time, significant reductions in total cholesterol (173.8 +/- 7.9 versus 139.4 +/- 8.9 mg/dl), low-density lipoprotein cholesterol (110.0 +/- 5.3 versus 83.8 +/- 7.7 mg/dl), and apolipoprotein B (68.2 +/- 3.3 versus 53.1 +/- 3.6 mg/dl) were observed. Serum levels of estrone, estradiol, sex hormone-binding globulin, high-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, triglycerides, and apolipoprotein A-I were not significantly affected. This study adds to the growing evidence that catechol estrogen production in humans is more readily regulated than 16 alpha-hydroxylation, which is relatively refractory to treatment.     

Species differences in biotransformation of 17α-cyanomethylestradiol 3-methyl ether

Following oral administration of 9,11- ³H-17α-cyano-methylestra-1,3,5(10)-triene-3,17-diol 3-methyl ether, urinary metabolites were studied in man, baboon, beagle dog, minipig and rat. The metabolite pattern revealed remarkable species differences, especially in quantitative respects. 17α-Cyanomethylestra-1,3,5(10)-triene-3,17-diol, 17α-cyanomethylestra-1,3,5(10)-triene-2,3,17-triol 2-methyl ether, 17α-hydroxymethylestra-1,3,5(10)-triene-3,17-diol and 17α-cyanomethylestra-1,3,5(10)-triene-3,16$\text{6}\text{5}$,17-triol were isolated as principal metabolites. In rat bile, a metabolite was tentatively identified as aγ-lactone of a 17α-carbozymethyl-16α-hydroxy compound.     

Cortisol production rate in children by gas chromatography/mass spectrometry using [1,2,3,4-13C]cortisol

A urinary method of determining the cortisol production rate (CPR) in children was studied under physiologic conditions by administration of low amounts of [1,2,3,4-13C]cortisol. The CPR in three patients with multiple pituitary deficiency ranged from 7 to 16 mumoles d-1 m-2, and the CPR in three patients with congenital adrenal hyperplasia (CAH) due to 11 beta-hydroxylase deficiency (11 beta OHD) and 17 alpha-hydroxylase deficiency (17 alpha OHD) from 0.1 to 2.11 mumoles d-1 m-2. Results showed that with this method, very low CPRs can be reliably measured. The metabolism of [13C4]cortisol or [9,12,12-2H]cortisol was compared with that of native cortisol in adrenalectomized piglets. For the urinary cortisol metabolites, small to substantial differences in isotope dilution were noted relative to that in the original cortisol mixture. With [13C4]cortisol, the so-called secondary isotope effects were approximately 2% to 3% for tetrahydrocortisone (THE) and tetrahydrocortisol (THF), and about 10% for the cortolones, relative to the cortisol mixture. When [2H3]cortisol was used, the cortisol metabolites THE and THF contained only two deuterium atoms. Together with this apparent loss of one deuterium atom, the secondary isotope effects in these steroids amounted to 5% to 10%. It was concluded that [13C4]cortisol was the better tracer to use for the measurement of urinary CPR.     

Microsomal hydroxylation of specifically deuterated monosubstituted benzenes. Evidence for direct aromatic hydroxylation

The aromatic hydroxylation of six pairs of selectively deuterated monosubstituted benzenes was investigated with rat liver microsomes of various induction states. The substrates studied included 3,5-D2C6H3X (1a-6a) and 2,4,6-D3C6H2X (1b-6b), where X = Br, CN, NO2, OCH3, CH3, or Ph, respectively. The deuterium content of the ortho, meta, and para hydroxylated metabolites, as well as side chain oxidation products from 4 and 5, was determined by capillary gas chromatography-mass spectroscopy. These data were analyzed according to a hypothetical model in which a molecule of substrate can undergo either direct aromatic hydroxylation (defined as obligatory and complete loss of deuterium from the site of hydroxylation) or indirect aromatic hydroxylation (defined as the obligatory and complete shift of deuterium to an adjacent position, followed by its partial loss as governed by a kinetic deuterium isotope effect). From this and other analyses of the data the following conclusions were reached. (1) The relative extent of meta hydroxylation increased and the total yield of metabolites decreased as the substituents X became more electron withdrawing. (2) The induction state of the microsomes altered the regioselectivity of hydroxylation (2, 3, 4, or side chain) noticeably and predictably but had little or no effect on the retention or loss of deuterium during each hydroxylation. (3) With each substrate and at each ring position hydroxylation was found to occur by a combination of direct and indirect mechanisms. (4) The relative importance of direct vs. indirect mechanisms did not vary in a simple manner with either the position of hydroxylation or the nature of the substituent X.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrone and estradiol metabolism in vivo in human breast cysts

Fibrocystic disease of the breast manifesting palpable cysts express breast cyst fluids frequently containing estrogen sulfates at concentrations far exceeding those found in sera of the patient. The study explored the potential of the breast cyst to synthesize some of these estrogen sulfates. Deuterated estrone and estradiol were synthesized and either (estradiol, 4 cases or estrone, 2 cases) was injected into a cyst. The cyst was aspirated at approximately 0, 4 and 8 h, the target being 1 ml, 50% and complete aspiration respectively. Metabolites were purified sequentially by ether extraction, enzymatic hydrolysis of estrogen conjugates, chromatography on Sephadex LH 20 and identified by gas chromatography linked to mass spectrometry. The unconjugated fraction isolated from the ether extract was subjected to the same purification and detection scheme. Among the conjugates, deuterated estrone sulfate was the major metabolite of either precursor in all studies, while estradiol sulfate was not detected in any of the 6 experiments. The sulfate fractions also yielded traces of 16alpha-hydroxyestrone (2 studies), 4-hydroxyestrone (4 studies) and 2-hydroxyestrone (1 study). In the unconjugated fraction, one study with deuterated estradiol, 4- hydroxyestrone was obtained. In one study with deuterated estrone, traces of 2-hydroxyestrone and 16alpha- hydroxyestrone were obtained. These novel data are significant because patients with fibrocystic disease are at slightly elevated risk for developing breast cancer and 16alpha-hydroxyestrone and 4- hydroxyestrone are reported carcinogens.     

Identification of 6α- and 7α-hydroxyestrone as major metabolites of estrone and estradiol in porcine uterus.

Polar metabolites extracted from the effluents of viable porcine uterine strips superfused with either 6,7-³H-estrone or 6,7-³H-estradiol were identified as a 1:1 mixture of 6α-hydroxyestrone and 7α-hydroxyestrone by paper chromatography in various systems, derivatization and crystallizations to a constant specific activity. The hydroxylated compounds are the only derivatives detected after estrone superfusion. The major metabolite of estradiol released in short-time experiments is estrone followed by its 6α- and 7α-hydroxylated derivatives.