Concentrations of 2-hydroxyoestrogens in human sera measured by a heterologous immunoassay with an 125I-labelled ligand

Abstract

A heterologous immunoassay for 2-hydroxyoestrogens1 has been established in which antibodies raised against 2-hydroxyoestradiol-17-succinyl-BSA serve as binding protein and 2-hydroxyoestrone-17-cmo\x=req-\[125I]iodohistamine as radioligand. Lipophilic serum components competing for binding sites in this system were defined as 'total 2-hydroxyoestrogens'. The underlying assumption of specificity was supported by the pattern of cross-reactivity evaluated with structural related steroids and o-diphenols and by the fact, that an additional chromatography of the serum extracts preceding the competing reaction had little if any effect. Sensitivity: 2.8 \m=+-\1 pg/tube; accuracy: Y = 0.91x + 2.2; r = 0.989; precision: 5.8% intra-assay; 6.5% inter-assay. The following concentrations ( \ m=+-\ standard deviation) were found in the sera of healthy subjects.Young men: 29 \m=+-\5 pg/ml (n = 11); women follicular phase: 32 \m=+-\8 pg/ml (n = 25); luteal phase: 53 \m=+-\13 pg/ml (n = 23); postmenopausal women: 13 \m=+-\4 pg/ml (n = 10); pregnant women 11th\p=n-\20th week: 70 \m=+-\16 mg/ml (n = 64); 36th\p=n-\40thweek: 240 \m=+-\23 pg/ml (n = 40); newborn cord blood: 604 \m=+-\43 pg/ml (n = 48).2-Hydroxyoestrogens have been recognized as major metabolites of oestrone and oestradiol in both laboratory animals and humans. The opinions on their physiological significance are still at vari¬ ance (Ball & Knuppen 1980). The assay of cate¬ choloestrogens in biological fluids and tissue by the classical methods of steroid separation and detec¬ tion is hampered by their chemical instability. They are highly susceptible to attack by oxidants, especi¬ ally in alcaline media. In addition, the classical methods are time consuming, expensive and not applicable for routing determinations. These pro¬ blems have been mastered by the development of suitable radioimmunoassays (Chattoraj et al. 1978;Ball et al. 1978). Methods currently available utilise tritium labelled radioligands, which are not avail¬ able commercially. Their syntheses are lengthy and tedious with generally small yields. In establishing a sensitive and specific radioimmunoassay of serum catecholestrogens we preferred the use of 125I-labelled radioligands, which offer the advan¬ tages of higher specific activities and cost-saving counting for at least 3 months after preparation. In order to obtain a very high sensitivity of the assay we utilized a heterologous bridge system employing anti-2-hydroxyoestradiol-17ß-succinyl-BSA-serum and 2-hydroxyoestrone-17-cmo-[125I]-iodohistamine. Preliminary results have been pub¬ lished previously (Berg et al. 1981).1 The following trivial names and abbreviations were used: 2-hydroxyoestrone = 2,3-dihydroxy-l,3,5(10)-oestratrien-17-one, 2-hydroxyoestradiol = l,3,5(10)-oestratriene-2,3,17ß-triol, 2-hydroxyoestriol = l,3,5(10)-oestratriene-2,3,16a,17ß-tetrol, oestradiol-17ß = 1,3,5(10)-oestratriene-3,17ß-diol, oestrone = 3-hydroxy-1,3,5(10)-oestratrien-17-one, oestriol = l,3,5(10)-oestratriene-3-16a, 17ß-triol, 2-methoxyoestrone = 2-methoxy-3-h...

Pharmacologic Effects of 2-Methoxyestradiol on Angiotensin Type 1 Receptor Down-Regulation in Rat Liver Epithelial and Aortic Smooth Muscle Cells

“…The relative quantities of this estradiol metabolite vary according to gender differences as well as hormonal fluctuations during menses. In males, 2ME2 median levels are below 10 pg/ml, whereas levels in females may vary anywhere from 46 pg/ml to 70 pg/ml, excluding pregnancy in which levels may increase to over 3,500 pg/ml [13]. In postmenopause, the levels of 2ME2 drop to a median concentration of 33 pg/ml, with very little fluctuation reflecting static estradiol titers [13].…”

“…In males, 2ME2 median levels are below 10 pg/ml, whereas levels in females may vary anywhere from 46 pg/ml to 70 pg/ml, excluding pregnancy in which levels may increase to over 3,500 pg/ml [13]. In postmenopause, the levels of 2ME2 drop to a median concentration of 33 pg/ml, with very little fluctuation reflecting static estradiol titers [13]. However, plasma concentrations of this metabolite postmenopause reflect a reduction of circulating estrogen and estrogen metabolites, though studies have shown that certain tissue concentrations may be significantly higher than in the plasma [14], and thus concentration dependent effects are difficult to interpret in vivo .…”

Urinary excretion of catecholestrogens, 2-methoxyestrogens and “classical estrogens” throughout the normal menstrual cycle

“…The 17-hemisuceinate-125I-histamine derivatives of 2-hydroxyestradiol, 4-hydroxyestradiol, and 2-methoxyestradiol -used as heterologous indicator-haptens -were synthesized and purified as previously described (Berg et al 1982;Berg and Kuss 1980;Berg et al 1983). …”

“…The methods were essentially the same as described previously (Berg and Kuss 1980;Berg et al 1982;Berg et ai. 1983).…”

2-Hydroxylation and O-methylation of oestrogens by human placenta in vivo

“…Catecholoestrogens and their methylethers were expressed throughout as immunochemical equiva¬ lents of 2-hydroxyoestrone and 2-methoxyoestrone, re¬ spectively. The technical details, accuracy, and specific¬ ity of the analytical methods have been described previ¬ ously (Berg et al 1982(Berg et al , 1983Goebel et al 1975). Hydrolysis of the conjugates was performed in boiling HCl(Balletal.…”