Upon arrival of the kit, please remove standards, positive control, and enzyme conjugates and store at -20°C until use. Store other vials anf microtiter plates at 2-8°C until use (Do not freeze plates).
The sensitivity of the 16α-OHE1 assay is about 0.05 ng/ml urinary 16α-OHE1 (after urine dilution of 1: 40 in assay). The sensitivity of the 2OHE1 assay is about 0.15 ng/ml urinary 2OHE1 (after diluted 1: 40 for assay).
The within-assay C.V.s for the metabolite ratio areless than 4% for the EIAs for 16α-OHE1 and 2OHE1 assays, the between-assay C.V.s are less than 10%.
Both EIAs have been shown to demonstrate 100% recovery of metabolites with serial dilution and "spiking" of exogenous estrogens into urine samples.
Assay incubation time is 3 hoursat room temperature, with about 30 to 60 minutes for development of alkaline phosphatase activity (color). Kinetic reading and analysis (slopes) of the assay is prefered to endpoints. The entire assay requires about 6 hours to perform.
The important role played by estrogen oxidative metabolism in hormonesensitive diseases has come from studies of estrogen-dependent neoplasia of reproductive organs. With respect to breast cell malignancies, estradiol is believed to act primarily as a promotional factor, causing increased growth rates in breast cells already transformed to a cancerous state. However, estradiol is not the only active estrogen in the human body; other metabolism formed estradiol andestrone have the capacity to act as estrogens, and in some cases, as anti-estrogens. Considerable work has shown thatmajor metabolites of estradiol are those hydroxylated at either the C-2 or the C-16α positions. There exists a complete divergence in the biological properties of the 2- and 16α-hydroxylated metabolites of estradiol. C-2 metabolites are essentially devoid of peripheral biological activity, as shown in studies on uterine weight, gonadotropin secretion, and cell proliferation. 2-Hydroxyestrogen has even been found to exert a modest antiestrogenic effect. The 16α-hydroxylated metabolite, 16α-hydroxyestrone (16OHE1) and estriol (E3), are estrogen agonists.
16OHE1 has several unique properties: it is capable of binding covalently to the estrogen receptor, to nuclearhistone proteins, and to DNA. Because of this covalent linkage to thereceptor, 16OHE1 shows persistent biological responses. The formation of 16OHE1 is elevated in women with breast cancer, women at high risk for breast cancer, and in strains of mice with a high incidence of spontaneous mammary tumors. Estradiol 16α-hydroxylation has been shown to take place in murine and human breast epithelial cell lines, both normal and transformed, and has also been demonstrated in terminal duct lobular units (TDLU), the functional units of the breast.
In addition to the persistence in estrogenic activity described above, it was recently established that 16OHE1, unlike E2 or E3, possesses initiator and promoter activities in normal (non-transformed) mammary epithelial cells. In proliferation assays 16OHE1 had activity comparable to that observed for DMBA, unlike E2 andE3, both of which showed only minimal responses. In a mutagenic assay measuring unscheduled DNA repair, 16OHE1 was likewise considerably more potent than estrone (E1), E2 or E3. Measurements of anchorageindependent colony formation of mammary epithelial cells grown in soft agar showed that 16OHE1 was far more potent than E1, E2 or E3 at increasing growth. Because the 16-hydroxylated compounds are more potent estrogens, these significant changes may have important hyperestrogenic consequences that could have a bearing upon the etiology of gynecologic cancers, especially breast cancer. Were this association proven, the ratio 2OHE1/16αOHE1 could serve as an innovative intermediate biomarker for breast cancer risk. Thisbiomarker could be used to identify women at high risk, and provide ananalytic framework for the development of new pharmaceutical and dietary intervention strategies.