Ragnar Södergård

Calculation of free and bound fractions of testosterone and estradiol-17β to human plasma proteins at body temperature

A mathematical model for the calculation of free and protein bound concentrations of testosterone and estradiol in plasma is presented. The method is based on the knowledge of the total concentrations of all steroids competing for the same binding site on testosterone-estradiol-binding globulin (TeBG), the concentration of albumin, the binding capacity of TeBG, and the association constants of the steroids to the two binding proteins. For the calculations we have determined the total concentrations of testosterone and estradiol. TeBG binding capacity, albumin concentration and the association constants for the binding of testosterone, estradiol and 5 alpha-dihydrotestosterone (DHT) to TeBG and albumin at 37 degrees C. Physiological concentrations of some androgen metabolites reported in the literature were also included in the calculations, namely: DHT, 5-androstene-3 beta, 17 beta-diol (Ae) and 5 alpha-androstane-3 alpha, 17 beta-diol (Aa). The binding constants for Ae and Aa to TeBG and albumin were also from the literature. Mean values of testosterone were calculated for 11 normal men and expressed as percentages of total: 2.0% was unbound, 53--55% bound to albumin and 43--45% bound to TeBG. For 16 normal women of a fertile age the corresponding values were 1.5%, 36--37% and 62%. For estradiol they were 2.4% 68--70% and 28--30% in the men and 2.0%, 52% and 45--46% in the women. Variations in the concentrations of DHT. Ae and Aa did not influence the free concentrations of testosterone and estradiol to any significant extent. It was furthermore concluded that the androgen metabolites could be omitted from the calculations without affecting the calculated concentrations.

A new method for the determination of the binding capacity of testosterone-estradiol-binding-globulin in human plasma

Abstract A new method for the quantitative determination of the testosterone-estradiol-binding-globulin, TeBG, in human plasma is described. The concentration of the globulin is measured in terms of the specific binding capacity in plasma for 5α-dihydrotestosterone, DHT. The method is based on equilibrium partition in an aqueous two-phase system containing 10% (w/w) dextran (Mw = 4 × 105), 7% (w/w) poly-(ethylene glycol) (Mn = 6 × 103), 0.1M KSCN in 0.005 M phosphate buffer. In this two-phase system more than 99% of the total plasma proteins partition into the lower phase and the partition coefficients for DHT and testosterone are 1.66 and 1.73 respectively. The concentration of the bound and unbound DHT or testosterone in equilibrium with the plasma proteins was determined from the concentration of the steroid in the upper phase. The method is rapid and simple and requires only small quantities of plasma. In contrast to those for testosterone, the results for the binding of DHT are not affected by the presence of transcortin and endogenous steroids in plasma. The intrinsic association constant for the binding of DHT and testosterone to TeBG and the apparent association constant for the binding to albumin were determined from Scatchard-type binding plots. The constants for DHT were 2.2 × 109 L mol−1 for TeBG and 8.6 × 104 L mol−1 for albumin. The affinity of TeBG for DHT was found to be about 2.4 times that for testosterone. The specific binding capacity values obtained, expressed as μg DHT bound/100 ml plasma, were: men, 1.76, women: 3.00; pregnant women, 12.7.

Danazol and gestagen displacement of testosterone and influence on sex-hormone-binding globulin capacity*

The mechanism of action of danazol is poorly understood, but this testosterone (T) derivate is frequently used in the clinical treatment of endometriosis, and its tendency to androgenic/anabolic side effects is well known. The interaction of danazol with T binding to sex-hormone-binding globulin (SHBG) was studied with the use of an aqueous two-phase system with polyethylene glycol (PEG) and dextran for equilibrium partition. Competitive binding studies were also performed with norethisterone (NET), d-norgestrel (d-Ng), medroxyprogesterone acetate (MPA), and tamoxifen (TMX). Danazol, d-Ng, and NET were found to exert a marked T displacing activity, while MPA and TMX had no significant effect. The low values for SHBG binding capacity that were found during danazol therapy mainly reflect occupation of binding sites by danazol and to a lesser degree a real decrease in protein concentration. It was calculated that during treatment the total SHBG capacity in serum is approximately 20 times exceeded. Therapeutic danazol serum levels are 1000 times those of normal female total T levels; and since the affinity to SHBG for danazol was found to be 1/20 that to T one should conclude an almost total occupation of binding sites. The endocrine effects of danazol might be interpreted in terms of T displacement and as a consequence of increased levels of free T during therapy.

Free testosterone levels during danazol therapy

Danazol is a testosterone (T) derivative widely used in the clinical treatment of endometriosis. Its mechanism of action is poorly understood, but is side effects are mainly androgenic. Previously it was demonstrated that danazol can displace T from sex-hormone-binding globulin (SHBG). The binding properties of danazol to SHBG and albumin were studied with the use of labeled danazol in an aqueous two-phase equilibrium partition system. Levels of total T, SHBG, and albumin were measured in 16 women undergoing danazol treatment for endometriosis. Thereafter, free and protein-bound T levels were calculated. A marked rise in free T was found during danazol therapy as compared with pretreatment levels. The data suggest that many of the effects of danazol could be explained by increased levels of free T during treatment.

The temperature dependence of the binding of 5α- dihydrotestosterone, testosterone and estradiol to the sex hormone globulin (SHBG) of human plasma

The binding of 5 alpha-dihydrotestosterone (DHT), testosterone and estradiol to the sex hormone binding globulin (SHBG) and albumin in human plasma has been studied at 4, 20 and 37 degrees C using the method of equilibrium partition in an aqueous two-phase system based on dextran, poly(ethylene glycol) and water. The intrinsic association constants for the binding to SHBG and the apparent association constant for the binding to albumin have been determined from Scatchard-type binding plots. The affinity of SHBG for DHT is 1.2-1.3 times higher than that for testosterone and 4 times higher than that for estradiol. The affinity of SHBG for the steroids decreases with increasing temperature. The mean values of the free energy of binding, delta G degree, in the temperature range used are -52.3, -51.7 and -48.9 kJ X mol-1 for the binding of DHT, testosterone and estradiol, respectively, to SHBG. The corresponding values of the enthalpy change, delta H degree, are 73.7, 70.0 and 99.0 J X mol-1 X K-1. These values are discussed in terms of the difference in the structure of the steroids. The affinity of albumin for testosterone and estradiol is almost equal and is lower than that for DHT. The delta G degree for the binding to albumin is about 55% lower than that for the binding to SHBG.

A Comparative Longitudinal Study on Sex Hormone Binding Globulin Capacity During Estrogen Replacement Therapy

Abstract. An aqueous two‐phase equilibrium partition system was used to assay SHBG‐binding capacity. Sera from groups of postmenopausal women before and during unopposed estrogen replacement therapy were analyzed. The induction of SHBG showed considerable differences between different estrogens. Ethinyl‐estradiol in a daily dose of 0.05 mg gave a 70% increase in the serum concentration of this liver derived protein. Estradiol‐17β, 2 mg daily and estrone sulphate 1.25 mg gave moderate changes, whereas estriol in different doses had no effect. SHBG induction may reflect estrogen overtreatment.

Specific binding of 17β-estradiol in the human thymus

Abstract Many observations suggest that estrogenic preparations can depress cell-mediated immune reactions. Since cell-mediated immunity is thymus-dependent, the estrogen-binding properties of human and mouse thymus tissue were studied with the use of an aqueous two-phase separation system. A high-affinity and low-capactiy estrogen binding was found in human thymus tissue from six prepubertal children of both sexes and two grown women. A similar specific binding was found in the mouse thymus and in the human, uterus. The association constant (Ka) for the binding in the human thymus was 16.01 ± 8.98 × 109M−1. The human uterus, which is known to possess estrogen receptors, had Ka values in the same range. The binding seems to be located in the reticuloepithelial cells of the thymus. Data suggest that the human thymus is a target organ for estrogens, which may influence lymphocyte function during therapy.

Lack of steroid-binding by “pregnancy zone” protein

Abstract The “pregnancy zone” protein (PZ) is a serum factor of unknown origin and function. The formation of this α 2 -globulin is stimulated by steroids and a carrier function has been postulated by most authors. The purified PZ protein was investigated in vitro using a new two-phase system for equilibrium partition. The association constant for PZ to six different steroids (oestriol, oestradiol, oestrone, progesterone, testosterone, cortisol) was low and there was no evidence for steroid-binding properties.

Characterization and Localization of Specific Oestrogen Binding in the Human Thymus

Clinical and experimental observations suggest that oestrogens may influence immune reactivity. The oestrogen-binding properties of the human thymus were studied by the use of an aqueous two-phase separation technique. A specific, high-affinity and low-capacity binding was found in 9 out of 10 thymic tissue samples obtained from children and adults of both sexes, their ages ranging from 1 to 42 years. The association constant (+/- SD) for the oestradiol binding in human thymus was 3.7 +/- 1.4 X 10(9) M-1 and was in the same range as the receptor binding in the human uterus. The steroid specificity was found to differ from the classical oestrogen receptor. The concentration of oestrogen-binding sites (+/- SD) was 182 +/- 181 fmol/mg protein and was higher than the receptor concentration of the uterus. According to histochemical analyses the binding is located in the reticulo-epithelial stroma rather than the thymocytes. Data suggest that the human thymus is a target organ for oestrogens which may influence lymphocyte function during therapy.

Estrogen receptor and aromatase activity in the testes of the unilateral cryptorchid rat.

The intratesticular concentration of estradiol-17 beta (E2) is increased in the abdominal testis of rats made unilaterally cryptorchid at birth. To elucidate this observation testicular aromatase activity and testicular estrogen receptor concentration were studied. Aromatase activity was lower while the concentration of estrogen receptor was higher in the abdominal testis. Treatment of cryptorchid rats with the anti-estrogen Tamoxifen did not change testicular concentration of testosterone. However, Tamoxifen induced a significant reduction of E2 in the abdominal testis. The main factor responsible for the increased E2 concentration in the abdominal testis appears to be increased binding of estrogen.