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This article is a revision of the previous edition article by James L. Wittliff, Sarah A. Andres, and D. Alan Kerr II, volume 2, pp 248–251, © 2005, Elsevier Inc.

The intestinal bacterial metabolism of 2-methoxyestrone was studied by incubation in the isolated coecum from rats. Following isolation of estrogens by a combination of ion-exchange and ligand-exchange chromatography, the metabolites were identified by gas chromatography-mass spectrometry. The two main reactions were oxidoreduction at C-17 and extensive demethylation at C-2. Thus, the demethylation of 2-methoxyestrogens known to occur in vivo may be due to the action of microbial enzymes. The study also shows that the intestinal microflora is capable of converting biologically inactive into active steroid hormones.

The acid ionization constants of estrone (10.77), 17 beta-estradiol (10.71) and 2-methoxyestrone (10.81) have been determined spectrophotometrically and shown to be consistent with the additivity of substituent effects of the phenol ring. Previously published values for estrone (10.914) and 17 beta-estradiol (10.078) are shown to be incorrect, at variance with the established trend for phenols, and inconcsistent with the similarity of the compounds.

The plasma and tissue concentrations of 2-methoxyestrone (2-MeOE1) and 2-hydroxyestrone (2-OHE1) were measured in immature rats. The plasma levels of 2-MeOE1 were found to be high at birth and to decrease through puberty, when the low levels found in the adult rats were achieved. 2-OHE1 was undetectable in the plasma and brain, and barely detectable in the uterus and liver. 2-MeOE1 was undetectable in the brain and uterus, but high in the liver. The affinity of 2-MeOE1 and 2-OHE1 for rat alpha-fetoprotein was found to be low, while the affinity of estradiol, estrone, 4-hydroxyestrone, and 4-fluoroestradiol was high. This data suggests that 2-OHE1 and 2-MeOE1 would be available to estrogen target tissues in the fetal and neonatal rat. Although these metabolites lack uterotropic activity they are capable of acting in the liver. It is suggested that the plasma 2-MeOE1 of neonatal rats acts as a prohormone capable of stimulating the liver and other estrogen target tissues which possess demethylating enzymes. It is pointed out that unlike estradiol the non-steroidal estrogens such as diethylstibestrol (DES) lack the ability to form two sets of catechol and guaicol metabolites, i.e. "C-2" and "C-4" metabolites with their different biological characteristics are not formed by DES.

The synthesis, purification and structural confirmation of the 6-(carboxymethoxyimino) derivatives of 2-methoxyestrone and 2-methoxyestradiol-17 beta are described. These derivatives were coupled to bovine serum albumin by the mixed anhydride method, and rabbits were immunized with the product. The resulting antisera showed high affinity and specificity for 2-methoxyestrone and 2-methoxyestradiol-17 beta, respectively, with low cross reactivities to structurally related estrogens.

We studied, in four normal men, the metabolism of 2-methoxyestrone (2-MeOE1) using pulse injections of either [3H]2MeOE1 (two men) or [14C]methoxy-2-MeOE1 plus [3H]2-MeOE1 (two men) by analysis of blood samples drawn at increasing time intervals after the pulse and of urine collected for 5 days. The disappearance from the blood of radioactivity as 2-MeOE1 could be characterized as a function that was the sum of three exponentials. The mean +/- SE value for the initial volume of distribution was 32 +/- 9 liters, and the mean MCR was 2470 +/- 770 liters/day. The disappearance of total 3H radioactivity from the blood was considerably slower, with a mean MCR of 290 +/- 30 liters/day, indicating the presence of a slowly turning over pool of 2-MeOE1 metabolites, probably including the 2-MeOE1 3-sulfate conjugate. The disappearance of total 14C radioactivity was slower than that of total 3H, indicating considerable demethylation of 2-MeOE1 with a very slow excretion of 14C from the released methyl group. In none of the subjects could we find in the blood radioactivity as unconjugated [3H]2-hydroxyestrone ( [3H]2-OHE1). However, examination of the urine indicated that considerable demethylation of [3H]2-MeOE1 had occurred. At least 64% of the urinary 3H-containing metabolites from the mixed dose had lost the 14C-bearing methoxylcarbon atom. The fractionated metabolites were qualitatively and quantitatively similar to those found earlier for [3H]2-OHE1. We conclude that 2-MeOE1, which of itself has little biological activity, can act as a pool of potentially active 2-OHE1 in the tissues.

During the last 20–30 weeks of pregnancy, the level of 2-methoxyestrone in the urine of 4 subjects ranged from less than 25 μg/24 hr to about 1 mg/24 hr. The levels fluctuated widely both in any one pregnancy and from one subject to another. There was a great difference in the trend of excretion of this metabolite and that of total estrogens in 2 of the subjects. The behavior of 2-methoxyestrone may be partly due to demethylation to form 2-hydroxy steroids. Some evidence in favor of this was obtained by studying the metabolism of exogenous 2-methoxyestrone in male subjects.

The nonuterotropic metabolite of estradiol, 2-hydroxyestrone, administered at noon of proestrus to four-day cycling rats, abolishes the preovulatory prolactin rise in a large percentage of animals tested. In animals synchronized with exogenous estradiol, 2-hydroxyestrone universally induced a long delay in the prolactin surge. The principal metabolite of 2-hydroxyestrone, 2-methoxyestrone, given at noon of proestrus, significantly augments the magnitude of the preovulatory prolactin rise possibly by inhibiting the formation of endogenous 2-hydroxyestrogens in the brain. The results obtained are consistent with the concept of a physiological function for 2-hydroxyestrogens as estrogen antagonists in the CNS.

A metabolite of 2,3-dihydroxyestra-1,3,5(10)-trien-17-one-6, 7- 3 H isolated from rat bile, was partially characterized by mass spectrometry as a methyl ether of 2,3,16-trihydroxyestra-1,3,5(10)-trien-17-one. The α configuration of the 16-hydroxy function was established by chromatographic comparison of the sodium borohydride reduced metabolite with synthetic 2-methoxy-estra-1, 3,5(10)-triene-3,16α,17β-triol and 2-methoxy-estra-1, 3,5(10)-triene-3,16β,17β-triol. The methyl group was located on the C-2 position by comparison with authentic 2- and 3- monomethyl ethers of 2,3-dihydroxy-estra-1, 3,5(10)-trien-17-one following pyrolytic removal of the 16α-hydroxyl group. 3,16α-dihydroxy-2-methoxyestra-1,3,5(10)-trien-17-one was found to constitute 2% and 15% of the biliary radioactivity following administration of estrone-6,7- 3 H and 2,3-dihydroxyestra-1,3,5(10)-trien-17-one-6,7- 3 H respectively.