Preparation of specific antiserum to estriol 3-sulfate 16-glucuronide

The preparation and antigenic properties of estriol 3-sulfate 16-glucuronide-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Coupling of 6-oxoestriol 3-sulfate 16-glucuronide acetate-methyl ester 6-(O-carboxymethyl)oxime with BSA by the activated ester method followed by removal of the protecting groups with alkali provided the desired conjugate. The antisera raised against the conjugate in rabbits were highly specific to the double conjugate, estriol 3-sulfate 16-glucuronide, discriminating from ring A or D monoconjugated and unconjugated estrogens. The specificity of antisera elicited has been discussed on the basis of stereochemistry of the hapten-[C-6]-BSA conjugate.     

Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates in which the hapten is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.     

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.     

Determination of 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol in human plasma by radioimmunoassay

5 alpha-Androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) were measured in human peripheral plasma by radioimmunoassay using celite microcolumn purification. The antisera used for the assay were obtained by immunization of rabbits with 3 alpha,17 beta-dihydroxy-5 alpha-androstane-6-(O-carboxymethyl) oxime: BSA for 3 alpha-diol and 3 beta,17 beta-dihydroxy-5 alpha-androstane-15 alpha-carboxymethyl: BSA for 3 beta-diol. The concentrations (pg/ml +/- SD) of the two diols in normal male and female plasma are respectively: 216 +/- 51 and 49 +/- 32 for 3 alpha-diol, 239 +/- 76 and 82 +/- 45 for 3 beta-diol. Comparison of these results with published ones shows that 3 beta diol concentrations were significantly lower. The high specificity of the assay is due to chromatography on celite microcolumns, allowing elimination of 5-androstene-3 beta,17 beta-diol from the plasma sample.     

Selective 3-(O-carboxymethyl)oxime formation in steroidal 3,20-diones for hapten immunospecificity

The direct one-step synthesis of 3-(O-carboxymethyl)oximes of representative C₂₁-4-pregnen-3,20-diones is reported. The method requires the preparation of a 3-enamine derivative which, serving as an intermediate product, is readily converted to the 3-(O-carboxymethyl)oxime upon addition of one molar equivalent of O-(carboxymethyl)hydroxylamine hemihydrochloride. The reaction appears to be generally applicable for selective 3-(O-carboxymethyl)oxime formation in steroids possessing multi-carbonyl groups, thus facilitating the coupling of steroidal haptens to protein at the C-3 position of the steroid molecule for enhanced immunospecificity. In this manner, antisera to 16α-hydroxyprogesterone and 17α-hydroxy-progesterone were obtained from immunized rabbits and specificity was established by radioimmunoassay.     

Synthesis and stereochemistry of C-3- and C-7-linked (O-carboxymethyl)-oximino- and hemisuccinamido derivatives of 5 alpha-dihydrotestosterone.

The 3-(O-carboxymethyl)oximino derivative of 17β-hydroxy-5α-androstan-3-one (5α-dihydrotestosterone) was prepared. Thin-layer chromatography of the corresponding methyl ester showed the presence of two syn (60%) and anti (40%) geometrical isomers of the oxime chain to the C-4 position, which were characterized by ¹³C nmr. The 3β-hemisuccinami-do-5α-androstan-17β-ol was obtained after selective saponification with potassium carbonate of the 17β-hemisuccinate group of the 3,17-dihemi-succinoylated derivative of the previously described 3β-amino-5α-androstan-17β-ol. This 3β-hemisuccinamide was purified as the corresponding methyl ester-17β-acetate and was regenerated after saponification. The 3,3'-ethylenedioxy-7-oxo-5α-androstan-17β-yl acetate was obtained in quantitative yield by catalytic hydrogenation over 10% palladium-oncharcoal of the Δ⁵-7-oxo precursor in a dioxane-ethanol mixture containing traces of pyridine. The exclusive 5α-configuration of this hydrogenated product was established from nmr data and was confirmed by the synthesis of methyl 3,3'-ethylenedioxy-7-oxo-5β-cholan-24-oate as 5β-H-reference compound. The preceding 5α-H-7-ketone was converted into the 7-(O-carboxymethyl)oximino derivative (syn isomer to the C-6 position, exclusively) which was esterified into the corresponding methyl ester. The selective hydrolysis of the 3-ethyleneketal group was achieved by a short treatment with a formic acid-ether 1:1 (v/v) mixture at 20°C. Saponification of the latter reaction product with ethanolic potassium hydroxide gave the 7-(O-carboxymethyl)oximino-17β-hydroxy-5α-androstan-3-one derivative, which was characterized as the corresponding methyl ester. The reduction of the oxime of the 5α-H-7-ketone with sodium in ethanol or with lithium-aluminium hydride gave respectively the 7β-amine or the 7α-amine as the major product. The 7β- and 7α-configurations were established from nmr spectra of the corresponding 7-acetamido derivatives. The 7β- and 7α-hemisuccinamido derivatives were prepared from the mixture of 7β- and 7α-amines, as described above for 3-derivatives and were isolated after thin-layer chromatography of the methyl esters, followed by saponification of the corresponding 17β-acetates.     

Antisera for radioimmunoassay of 17alpha-ethynylestradiol and mestranol

Antisera for 17α-ethynylestradiol and mestranol have been prepared by immunizing rabbits with 6-(O-carboxymethyl) oxime-bovine serum albumin conjugates prepared from 6-oxo-17α-ethynylestradiol and 6-oxomestranol, respectively. These antisera showed little cross-reaction with known metabolites of these steroids. A comparison is made between our antisera and some prepared by others, where coupling to the steroid is effected through the C-7 position.     

Comparative specificity of antisera raised against estrone, estradiol-17 and estriol using 6-0-carboxy-methyloxime bovine serum albumin derivatives

Antisera for the radioimmunoassay of estrone and estradiol-17β in plasma are usually raised against estradiol-17β coupled to a protein through a derivative at carbon 17. Such antisera cross react with other naturally occurring estrogens, necessitating preliminary chromatographic separation. This difficulty could be overcome by the use of more specific antisera. We have raised antisera against the 6-0-carboxymethyloxime-bovine serum albumin (BSA) derivatives of estrone, estradiol-17β and estriol respectively. We have determined cross reactions with a number of estrogens and other naturally occurring steroids, and have compared the cross reactivity with that of an antiserum raised against estradiol-17β-17-succinyl-BSA. The former antisera show greatly reduced cross reaction with naturally occurring estrogens known or thought to be in relatively high concentration in plasma, as compared with the latter antiserum, but at the expense of greatly increased cross reaction with estrogens substituted at carbon 6. However, since these latter estrogens are thought to be in low concentration in plasma, the use of antisera raised against the 6-0-carboxymethyloxime-BSA derivatives should result in a net gain in specificity. The antisera raised against the estrone and estriol 6-0-carboxymethyloxime-BSA derivatives should be particularly useful.     

Isolation of testosterone-binding globulin from bovine serum by affinity chromatography and its molecular characterization.

The testosterone-binding globulin (TeBG) from bovine serum was purified by affinity chromatography and hydroxylapatite chromatography. The affinity column used was prepared by coupling 17 alpha-carboxyethynyl-17-hydroxy-4-androsten-3-one to aminoethyl-Sepharose. The compound was replaceable by 17alpha-carboxyethynyl-17-hydroxy-5alpha-androstan-3-one, but not by testosterone 17-hemisuccinate, estradiol 17-hemisuccinate, or testosterone 3-(O-carboxymethyl)oxime. The TeBG isolated was homogeneous on analytical polyacrylamide gel electrophoresis and equilibrium centrifugation. The protein was a glycoprotein having a molecular weight of 89,500 and a carbohydrate content of 17%. The association constant (M-1) at 4 degrees C was 1.1 X 10(8) and the number of binding sites per molecule was 0.8. Treatment with guanidine-HCl dissociated the protein into subunits having a molecular weight of 28,400 (about one-third of that of the original molecule). SDS-gel electrophoresis showed that two of the three subunits were slightly larger than the other. The dissociation into subunits could also be accomplished by GEDTA treatment with concomitant loss of testosterone-binding activity. The activity and molecular size were reversibly restored by incubation with excess Ca2+.     

Microbiological hydroxylation of estradiol: formation of 2- and 4-hydroxyestradiol by Aspergillus alliaceus

Microorganisms known to hydroxylate alkaloids, amino acids, and aromatic substrates were examined for their potential to hydroxylate 17 beta-estradiol and estrone. Thin-layer chromatography of fermentation extracts revealed a wide range of steroid products. Aspergillus alliaceus (UI 315) was the only culture capable of producing good yields of catechol estrogens with 17 beta-estradiol. The organism also transformed estrone but not to catechol products. Analytical experiments with high-performance liquid chromatography revealed that A. alliaceus formed 4- and 2-hydroxyestradiol with yields of 45 and 16%, respectively. A preparative-scale incubation was conducted in 2 liters of medium containing 1 g of 17 beta-estradiol as substrate. 4-Hydroxyestradiol was isolated and identified by proton nuclear magnetic resonance and high-resolution mass spectrometry. Ascorbic acid was added to microbial reaction mixtures as an antioxidant to prevent the decomposition of unstable catechol estrogen metabolites. The microbial transformation of 17 beta-estradiol by A. alliaceus provides an efficient one-step method for the preparation of catechol estrogens.     

Microbiological hydroxylation of estradiol: formation of 2- and 4-hydroxyestradiol by Aspergillus alliaceus.

Microorganisms known to hydroxylate alkaloids, amino acids, and aromatic substrates were examined for their potential to hydroxylate 17 beta-estradiol and estrone. Thin-layer chromatography of fermentation extracts revealed a wide range of steroid products. Aspergillus alliaceus (UI 315) was the only culture capable of producing good yields of catechol estrogens with 17 beta-estradiol. The organism also transformed estrone but not to catechol products. Analytical experiments with high-performance liquid chromatography revealed that A. alliaceus formed 4- and 2-hydroxyestradiol with yields of 45 and 16%, respectively. A preparative-scale incubation was conducted in 2 liters of medium containing 1 g of 17 beta-estradiol as substrate. 4-Hydroxyestradiol was isolated and identified by proton nuclear magnetic resonance and high-resolution mass spectrometry. Ascorbic acid was added to microbial reaction mixtures as an antioxidant to prevent the decomposition of unstable catechol estrogen metabolites. The microbial transformation of 17 beta-estradiol by A. alliaceus provides an efficient one-step method for the preparation of catechol estrogens.     

Isolation of antibodies of improved specificity after fractionation on Antigen-Sepharose affinity columns of antisera to bovine serum albumin conjugates of C-3- and C-7-linked (O-carboxymethyl)oximino- and hemisuccinamido derivatives of 5 alpha-dihydrotestosterone.

Rabbit antisera to bovine serum albumin (BSA) conjugates of 3-(O-carboxymethyl)oximino-, 7-(O-carboxymethyl)oximino- and 7β-hemi-succinamido derivatives of 5α-dihydrotestosterone (DHT) were applied to four affinity columns bearing respectively these three antigens and a fourth 3β-hemisuccinamido-5α-androstan-17β-ol-BSA antigen as ligands.The antibodies retained on the columns were totally desorbed by an excess of DHT, but in DHT-bound form, whereas 1M mh₄oh and electrophoretic elution allowed a recovery of 60% of the retained antibodies in unbound form. The antibody fractions (40%) remaining on the columns after NH₄OH or electrophoretic elution were totally recovered by addition of DHT following the electrophoretic elution only. All the DHT-bound fractions were dissociated by dialysis but with a 70% loss of binding activity.The association constants for DHT of most of the antibody fractions were similar to those of the crude antisera (Ka ~ 10¹⁰M^?1), with the exception of the antibodies recovered from the antibody fractions resistant to electrophoretic elution which had higher affinities (Ka ~ 2.0 to 30 × 10¹⁰M^?1).The specificity charts of the antisera were in some cases considerably modified after fractionation, according to the choice of the ligand employed in the affinity columns as well as of the elution methods. The lowest cross-reactions with testosterone were observed after elution with 1M NH₄OH (17–20%) or electrophoresis (23–25%) of the anti-7-(O-carboxymethyl)oximino-DHT antisera fractions retained on 3β-hemisuccinamido-5α-androstan-17β-ol-BSA-Sepharose columns.     

Steroid structure and function. Molecular conformation of 4-hydroxyestradiol and its relation to other catechol estrogens

Hydroxylation of estrogens at C(2) or C(4) effects differentially their binding affinity to and dissociation rate from the estrogen receptor. The X-ray crystal structure of 4-hydroxyestradiol (4-OH-E2) is reported here and compared with that of 2-hydroxyestradiol (2-OH-E2), the 2- and 4-hydroxylated derivatives of estrone (E1) and with that of the parent estrogens, E1 and E2. The overall molecular shape and hydrogen bonding patterns of each were examined for their possible relevance to their binding to the estrogen receptor and their biological activity. A shift in the B-ring conformation away from the symmetrical 7 alpha,8 beta-half-chair form toward the 8 beta-sofa form is induced by both 2- and 4-hydroxy substitution. This shift appears to be larger in the case of E2 than E1 derivatives and to be correlated with an observed change in the hydrogen bonding potential of the C(3) hydroxyl. In 4-OH-E2, as in E2 and 4-OH-E1, the C(3) hydroxyl functions both as a hydrogen bond donor and acceptor. In contrast in 2-OH-E2 the hydroxyl functions only as a donor. The markedly reduced affinity of 2-hydroxylated estrogens for the estrogen receptor could be due to a combination of steric interactions, competition between O(2) and O(3) for hydrogen bonds for a common site on the receptor, and to general interference with hydrogen bond formation of O(3). The C(4) hydroxyl participates in the formation of a chain of hydrogen bonds in the solid state that is similar to a chain seen in single crystals of E2. The presence of a similar chain of hydrogen bonds involving O(3) in the receptor site could account for the decreased dissociation rate of the 4-OH-E2 receptor complex.     

Preparation and antigenic property of 3-dehydrolithocholylglycine 3-(O-carboxymethyl) oxime-bovine serum albumin conjugate

The preparation and antigenic property of 3-dehydrolithocholyglycine-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-3 position on the steroid nucleus is described. Antibody raised against antigen in the rabbit possessed high titer and specificity to lithocholylglycine, exhibiting no significant cross-reaction with free lithocholic acid or lithocholyltaurine.     

Synthesis of compounds of potential value in the radioimmunoassay of 17 alpha-ethynylestradiol and mestranol

The 6-(0-carboxymethyl)oxime derivatives of 6-oxomestranol and 6-oxo-17α-ethynylestradiol were prepared from 3-methoxy-6-oxoestrone. These haptens were then coupled to bovine serum albumin by the mixed anhydride technique to yield the steroid-protein conjugates required for the production of specific antisera for the radioimmunoassay of mestranol and 17a-ethynylestradiol. In addition we have described the preparation of certain expected metabolites of mestranol and 17α-ethynylestradiol which are also required for studying cross-reactivity in the radioimmunoassay.     

Preparation and antigenic properties of 5alpha-dihydrotestosterone-11-(O-carboxymethyl) oxime-BSA conjugate.

The C-11 (O-carboxymethyl) oxime derivative of 5-alphadihydrotestosterone (5alphaDHT) has been prepared. Due to steric hindrance at C-11, a novel two step procedure was used to introduce the (O-carboxymethyl) oxime at this position. Condensation of this oxime to bovine serum albumin afforded a conjugate which produced anti-5alphaDHT sera inoculated rabbits. Apart from a 30% cross reaction with testosterone, the antisera was reasonably specific for 5alphaDHT.     

The catalytic effect of tyrosinase upon oxidation of 2-hydroxyestradiol in presence of catechol

The hydroxylating activity of mushroom tyrosinase has been utilized for over a decade in the preparation of 2-hydroxyestradiol from estradiol, yet this same enzyme is known to function as an oxidant of o-dihydric compounds to the corresponding o-quinones. It was questioned why catechol estrogens do not react further, particularly since the tyrosinase activity (hydroxylating) is exceeded many fold by the diphenol oxidase activity of the enzyme. This report describes that the catechol estrogen will react in presence of enzyme but only if catechol is also present. Diphenol oxidase activity was measured either by the polarographic oxygen-utilization technique or by changes in the absorption spectrum at 206 and 256 nm. The enzyme activity was standardized with catechol (Km = 5.2 X 10(-4) M). The steroid did not react with the enzyme if catechol was absent. With catechol, the steroid reacted rapidly and completely (Km = 4.2 X 10(-4) M). The consumption of oxygen with catechol and 2-hydroxyestradiol was additive and stoichiometric, 1 g-atom oxygen/mol of either substrate. Kinetic analysis shows that catechol functions as an activator of the tyrosinase.     

Plasma estradiol 17-sulfate and 2-hydroxyestradiol 17-sulfate levels and their metabolic clearance rates in rats

By using highly specific antisera against estradiol 17-sulfate (E2-17-S) and against 2-hydroxyestradiol 17-sulfate (2-OH-E2-17-S), plasma concentrations of these sulfates in Wistar rats were determined. The plasma levels of E2-17-S and 2-OH-E2-17-S in the male were 23.5 +/- 5.3 and 21.6 +/- 6.2 pg/ml, respectively. During the estrus cycle of the female, the plasma concentration of E2-17-S reached its highest level 69.0 +/- 11.8 pg/ml, during the diestrus stage, and its lowest level 36.9 +/- 6.6 pg/ml, during the proestrus stage. Similar tendencies were observed in the case of 2-OH-E2-17-S. To examine the dynamic behavior of both sulfates, the plasma metabolic clearance rate (MCRp) of E2-17-S and 2-OH-E2-17-S were determined by infusion experiments. MCRp of E2-17-S and 2-OH-E2-17-S in male rats were 102 and 653 ml/h (means), respectively, and in female rats were 115 and 644 ml/h (means), respectively. The low MCRp values of both sulfates imply their slow metabolic turn-over.     

Preparation and antigenic property of methyl 3 beta-hydroxy-19-oxo-5-cholen-24-oate 19-(O-carboxymethyl) oxime-bovine serum albumin conjugate

The preparation and antigenic property of 3 beta-hydroxy-5-cholen-24-oic acid-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-19 position on the steroid nucleus is described. Antibody raised against antigen in the rabbit possessed high titer and specificity to 3 beta-hydroxy-5-cholen-24-oic acid, exhibiting no significant cross-reactions with various bile acids.     

Antioxidant activities of estrogens against aqueous and lipophilic radicals; differences between phenol and catechol estrogens

Natural estrogens have much greater radical-scavenging antioxidant activity than has previously been demonstrated, with activities up to 2.5 times those of vitamin C and vitamin E. The biological significance of this finding remains to be elucidated. In this work the antioxidant activity of a range of estrogens (phenolic, catecholic and stilbene-derived) has been studied. The activity of these substances as hydrogen-donating scavengers of free radicals in an aqueous solution has been determined by monitoring their relative abilities to quench the chromogenic radical cation 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS*+). The results show that the order of reactivity in scavenging this radical in the aqueous phase is dependent on the precise estrogenic structure, with phenolic estrogens being more potent antioxidants than catecholestrogens or diethylstilbestrol. The ability of the same estrogens to scavenge lipid phase radicals has also been assessed, determined by the ex vivo enhancement of the resistance of low-density lipoprotein (LDL) to oxidation; the order of efficacy is different from that in the aqueous phase, with the phenolic estrogens estriol, estrone and 17beta-estradiol being less potent than 2-hydroxyestradiol, 4-hydroxyestradiol, or diethylstilbestrol. In this lipid-based system, phenolic estrogens were found to be unable to regenerate alpha-tocopherol from LDL subjected to oxidative stress, while at the same time 2- and 4-hydroxyestradiol significantly delayed alpha-tocopherol loss. These results indicate that the various estrogens are good scavengers of free radicals generated in both the aqueous and the lipophilic phases. The antioxidant activity of an estrogen depends not only on the hydrophilic or lipophilic nature of the scavenged radical, but also on the phenol and catechol structures of the estrogen compound.