Emako Suzuki

Assay of enzymic O-methylation of catechol oestrogens by high-performance liquid chromatography with coulometric detection.

A simple and sensitive method for the determination of guaiaicol oestrogens enzymatically formed from 2- or 4-hydroxyoestradiol, by means of high-performance liquid chromatography with coulometric detection, has been developed. Catechol and guaiacol oestrogens were efficiently separated on a reversed-phase column, using 0.5% ammonium phosphate buffer (pH 3.0)-acetonitrile (59:41, v/v) as the mobile phase, and detected coulometrically in a screening-oxidation mode at +0.10 V and +0.35 V, respectively. The method was applied to the assay of in vitro enzymic O-methylation of catechol oestrogens. After 2- or 4-hydroxyoestradiol had been incubated with rat red blood cells in the presence of S-adenosylmethionine, the resulting guaiacols and unchanged substrate were percolated through an Extrelut-3 cartridge. The dried eluate was redissolved and directly injected. This simple procedure was as sensitive as the previously reported method using gas chromatography-mass spectrometry in a selected ion monitoring mode.

Synthesis of 15α-hydroxyestrogen 15-N-acetylglucosaminides

The synthesis of 15-N-acetylglucosaminides of 15 alpha-hydroxyesterone, 15 alpha-hydroxyestradiol, and 15 alpha-hydroxyestriol (estetrol) is described. The latter two were prepared by condensation of 2-acetamido-1 alpha-chloro-1,2-dideoxy-3,4,6-trio-O-acetyl-D-glucopyranose with appropriately protected 15 alpha-hydroxyestrogens by the Koenigs-Knorr reaction employing cadmium carbonate as a catalyst. Subsequent removal of protecting groups with methanolic potassium hydroxide provided the desired conjugates. 15 alpha-Hydroxyestrone 15-N-acetylglucosaminide was synthesized from the corresponding 15 alpha-hydroxyestradiol derivative by Jones oxidation followed by brief alkaline hydrolysis. These conjugates underwent enzymatic hydrolysis with beta-N-acetylglucosaminidase from Jack beans to produce 15 alpha-hydroxyestrogens.

Enzymatic O-methylation of catechol estrogens in red blood cells: differences in animal species and strains

Enzymatic O-methylation of catechol estrogens in red blood cells has been investigated with respect to species difference. In the presence of S-adenosylmethionine, 2- or 4-hydroxyestradiol (2-OHE2 or 4-OHE2) was incubated with blood lysate obtained from rats (five strains), guinea pigs, mice, rabbits, dogs, monkeys, and humans, respectively. The yielded guaiacols and unchanged substrate were determined by gas chromatography/mass spectrometry in a selected ion monitoring mode employing the corresponding 2H4-labeled compounds as internal standards. The total amounts of guaiacols formed from 2-OHE2 and 4-OHE2 were different, being the highest (79.6% and 38.1%) in monkeys and the lowest (5.1% and 1.9%) in humans. The ratios of isomeric guaiacols formed from 4-OHE2 (4Me/3Me) were 7.6-71, while those from 2-OHE2 (2Me/3Me) were 1.4-3.2. Thus, marked differences in O-methylation of catechol estrogens were observed among animal species, but no significant strain difference was detected in rats.

Preparation of specific antisera to 15α-hydroxyestrogens

Abstract The synthesis of haptens of 15α-hydroxyestrone, 15α-hydroxyestradiol, and 15α-hydroxyestriol (estetrol) was undertaken, to obtain specific antisera required for enzyme immunoassay. 3-(1-Carboxypropyl) ethers of these 15α-hydroxyestrogens were prepared and conjugated with bovine serum albumin and horseradish peroxidase. The specificity of antisera elicited against bovine serum albumin conjugates was checked by the enzyme immunoassay by using horseradish peroxidase-labeled antigen, and proved to be satisfactory in terms of cross-reactivities to related compounds.

Preparation of specific antisera to 15α-hydroxyestrogen 15-N-acetylglucosaminides ☆

3-(1-Carboxypropyl) ether derivatives of 15alpha-hydroxyestradiol 15-N-acetylglucosaminide (15alpha-OHE2 15NAG) and 15alpha-hydroxyestriol (E4) 15NAG were synthesized and conjugated with bovine serum albumin. Antisera elicited in rabbits possessed high affinity and specificity for the 15alpha-hydroxyestrogen (15alpha-OHEs) 15NAG, exhibiting no significant cross-reactivity with 15alpha-OHEs and their positional isomers such as 16NAG and 17NAG. Enzyme immunoassay methods developed by using the purified antisera and horseradish peroxidase-labeled antigens were applied to the measurement of 15alpha-OHEs 15NAG and E4 15NAG in normal pregnancy urine. We demonstrated for the first time that the conjugation of N-acetylglucosamine to E4 occurs at the C-15alpha position.

Synthesis of N-acetylcysteine conjugates of catechol estrogens.

The synthesis of N-acetylcysteine conjugates of 2-hydroxyestrone (2-OHE1) and 4-hydroxyestrone (4-OHE1) is described. The reaction of estrone 2,3-quinone with N-acetylcysteine provided 2-OHE1 and its C-4 and C-1 thioether conjugates in a ratio of 1:1, while estrone 3,4-quinone with N-acetylcysteine gave 4-OHE1 and its C-2 thioether conjugate as a sole product. Their structures were characterized by inspection of NMR spectra, chemical derivatization (methylation and acetylation), and comparison with the reactivity of 4-bromoestrone 2,3-quinone or 2-bromoestrone 3,4-quinone toward N-acetylcysteine.

Determination of urinary 15α-hydroxyestrogen levels via immunoaffinity extraction

15α-Hydroxyestrogens (15α-OHEs) are metabolites of the female hormone estradiol. In this study, to discover physiological markers that can be utilized for monitoring fetal conditions and estrogen-induced cancers, we established a method for quantifying 15α-OHEs in rat urine via immunoaffinity column extraction and HPLC-electrochemical detection, and detected 15α-OHEs in urine obtained male rats treated with estradiol. Notably, the standard curves for quantification obtained using the column were linear over a range of 0.5-50ng 15α-OHEs. The accuracy of the analytical method with cleanup was 97-109% for the three kinds of 15α-OHEs examined, and the intra-assay precision of the measured values had a coefficient of variation of ≤20.6%. Therefore, the theoretical limit of quantification was 0.5ng. However, the actual measured values obtained from the urine of male rats indicated that the detection limits were 0.425, 0.103, and 0.047ng for estetrol, 15α-hydroxyestradiol, and 15α-hydroxyestrone, respectively. Our newly established method for measuring 15α-OHE concentrations in urine could facilitate characterization of the in vivo metabolic profile of 15α-OHEs in mammals under various physiological conditions, which could comprise clinical markers for monitoring human fetal health conditions in mammals.

Enzymic and chemical O-methylation of a 4-hydroxyestrone N-acetylcysteine conjugate

4-Hydroxyestrone N-acetylcysteine conjugate (4-OHE1-2SR) is considered to be an important compound for monitoring the in vivo formation of catechol estrogen quinones, an intermediary in estrogen carcinogenicity. This article describes the selective synthesis of isomeric monomethyl ethers of 4-OHE1-2SR utilizing the formation of a seven-membered ring lactone by dehydration with acetic anhydride. Using these authentic specimens, enzymic and chemical O-methylation were examined. Enzymic O-methylation, using a rat liver cytosolic fraction, of 4-OHE1-2SR gave its 3-methyl ether as the sole product, while preferential O-methylation of 4-hydroxyestrone (4-OHE1) at the C-4 position was confirmed under the same conditions. Methylation of 4-OHE1-2SR with diazomethane gave initially carboxylate methylation, then the corresponding 3-methyl ether almost exclusively, while methylation of 4-OHE1 also gave its 3-methyl ether preferentially. However, much more rapid formation of the 3-methyl ether was observed with 4-OHE1-2SR than with 4-OHE1 itself. These results show that the hydroxy group at the C-3 position of 4-OHE1-2SR is more reactive than that at the C-4 position, both chemically and enzymatically.

Endocrine disrupting chemicals, 4-nonylphenol, bisphenol A and butyl benzyl phthalate, impair metabolism of estradiol in male and female rats as assessed by levels of 15α-hydroxyestrogens and catechol estrogens in urine: ENDOCRINE DISRUPTING CHEMICALS ALTER ESTRADIOL METABOLITES IN URINE

Bisphenol A (BPA), 4‐nonylphenol (NP) and butyl benzyl phthalate (BBP), termed endocrine‐disrupting chemicals, are known to mimic estrogen activity. The effects of these chemicals on 17β‐estradiol (E2) metabolism in vivo in rats were examined. Male and female rats were given NP (250 mg kg–1 day–1), BPA (250 μg kg–1 day–1) or BBP (500 mg kg–1 day–1) by gavage for 14 days, followed by a single intraperitoneal injection of E2 (5 mg kg–1) on the final day. The urinary excretion over 72 hours of 2‐hydroxyestrone 1‐N‐acetylcysteine thioether, 2‐hydroxyestrone 4‐N‐acetylcysteine thioether, 4‐hydroxyestrone 2‐N‐acetylcysteine thioether, 2‐hydroxy‐17β‐estradiol (2‐OHE2), 2‐hydroxyestrone (2‐OHE1), 4‐hydroxy‐17β‐estradiol, 4‐hydroxyestrone, 15α‐hydroxyestriol (E4), 15α‐hydroxy‐17β‐estradiol and 15α‐hydroxyestrone was measured. Increases in urinary excretion of 2‐OHE1 and decreases in E4 were observed in males treated with NP or BBP. Decreases in urinary excretion of 2‐OHE2 and E4 were observed in males treated with BPA. Decreases in urinary excretion of 2‐OHE1 and 2‐OHE2 were observed in females treated with BBP. Normalized liver and weights were increased in both sexes treated with NP or BBP. Histologic observations revealed marked changes in the distal tubules and collecting ducts in the kidneys of rats exposed to NP and BBP, and hypertrophy in the hepatocytes of the centrilobular zone of the liver. No BPA‐related effects on organ weight and on liver or kidney histopathology were found. These results suggest that the 14 day oral dosing of NP and BBP disrupted E2 metabolism, resulting from marked morphological and functional alterations in the liver and kidneys. In addition, BPA could induce metabolic and endocrine disruption.