Metabolism of [6,7-3H, 35S] estradiol 17-sulfate in rats
Introduction
Previously, we reported the presence of estradiol 17-sulfate (ES, Fig. 1) in the urine of non-pregnant [1] and pregnant [2] women, and in the blood of pregnant women [3]. This was the first discovery of ES in mammals. Later, a similar estrogen sulfate, 2-hydroxyestradiol 17-sulfate (2-OH-ES, Fig. 1), was detected in the blood [4] and urine [5] of pregnant women, and it was surmised that 2-OH-ES during pregnancy is a feto-placental metabolite of ES [6].
Both estrogen sulfates were found to be endogenous also in rats [7]. In addition, the hepatic microsomes of rats were shown to possess the ability to convert ES into 2-OH-ES [8]. Thus, it became evident that the Phase I reaction of ES occurs not only in human but also in rat.
Recently, we found a big sex difference in the metabolic profiles of rat hepatic microsomal hydroxylation of ES [9]. In male rats, 2-OH-ES was produced accompanied by two minor products 4-hydroxyestradiol 17-sulfate (4-OH-ES, Fig. 1) and a metabolite the structure of which is still unknown. In female rats, 6α-, 6β-, 7β-, and 15β-hydroxyestradiol 17-sulfates (6α-, 6β-, 7β-, and 15β-OH-ES, respectively, Fig. 1) were produced, accompanied by the above two minor catechols.
Judging from the experimental conditions, these products are considered to be formed via the direct hydroxylation of ES. However, a different metabolic pathway is predicted to occur in the in vivo metabolism of ES. For example, such a pathway as “deconjugation of ES at first, followed by the hydroxylation of estradiol (E) produced, and finally a reconjugation at C17 of monohydroxylated products” cannot be excluded.
To confirm whether or not the in vivo Phase I reaction of ES occurs without the removal of the conjugate group at C17, the doubly labeled steroid [6,7-, ] ES was injected into rats, and the urinary and biliary products were analyzed.
Section snippets
Materials
Estradiol was purchased from Steraloids (Wilton, NH, USA), and their 17-sulfates were prepared in this laboratory using the method described previously [8], [10]. The preparation of 2-methoxyestradiol-17-sulfate (2-MeO-ES, Fig. 1) was carried out by a known method [11]. Monohydroxylated ES derivatives, 4-OH-ES [12], and 6α-, 6β-, 7α-, and 7β-OH-ES (Fig. 1) [13], were prepared in this laboratory according to known methods. The new sulfate compound, 15β-OH-ES, was prepared from 15β-OH-E [14] by
Excretion profiles of radioactivity
After injection of the doubly labeled ES into rats, bile, and urine samples were collected for 24 h. In Table 1, the cumulative radioactivities of and in bile and urine, and their ratios (/) are summarized. Over three-quarters of the isotopes administered were excreted in 24 h in both male and female rats, and a big sex difference in the excretion profile was recognized.
In male rats, most of the radioactivity excreted was present in the biliary fraction. In contrast, biliary
Discussion
In the metabolism of ES by rat hepatic microsomes, hydroxylation occurs in the A, B, and D rings while retaining the conjugated groups [9]. In order to investigate whether or not this Phase I reaction occurs in vivo as well, a method that was frequently employed in metabolic research on steroid sulfates more than 30 years ago was used, which involves the use of two types of labeled radioisotopes. As a few examples of the “direct metabolism”, the conversion of androstene-3β, 17β-diol 3-sulfate
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