Hans Carstensen

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.

Estrogen and progesterone in plasma in relation to premenstrual tension.

Abstract Plasma concentration of estrogen and progesterone were measured during the last 6 days of the menstrual cycle in women with premenstrual tension, and compared with a group of healthy women. Those women with anxiety as the main symptom (PMT-a) had significantly higher estrogen levels on days 5-2 before the onset of menstruation. On days 6-4 they had lower levels of progesterone. Estrogen-progesterone ratios were significantly higher on days 6-3 before menstruation. The PMT-a group also showed increases in body wt. during the last days of the menstrual cycle.

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.

Concentration of estradiol, testosterone and progesterone in cerebrospinal fluid compared to plasma unbound and total concentrations.

Abstract Estradiol, progesterone and testosterone are measured in plasma and CSF in 17 women and 11 men. The results show a transfer from plasma to CSF of about 4% for estradiol, 10% for progesterone and 2.5% for testosterone. There was found to be a clear correlation between the plasma and CSF levels of these steroids. A comparison is also made between the calculated levels of unbound estradiol and testosterone in plasma and the levels in the CSF. The results show approximately the same concentrations of these steroids in the CSF as the calculated levels of unbound steroids in the plasma.

FSH, LH, TeBG-capacity, estrogen and progesterone in women with premenstrual tension during the luteal phase.

Levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone-estradiol binding globulin (TeBG), estrogen, and progesterone were measured in 15 women with premenstrual tension (PMT) syndrome and compared with a control group of 17 women. Levels of plasma immunoreactive estrogen were markedly (p less than .005) lower on Days 9 and 8 before menstruation in the PMT group compared with controls. However, by Day 5 before mestruation, estrogen levels were significantly (p less than .005) higher in the PMT group compared with controls, and this difference was sustained until Day 1 before menstruation. The PMT group showed consistently lower progesterone levels than controls. On Days 9 through 6 before menstruation, PMT patients showed significantly (p less than .05) higher levels of FSH than controls. However, no marked differences were observed between the groups in levels of LH, serum albumin, and the binding capacity of TeBG. The possible influence of FSH in PMT syndrome is discussed in relation to the observed increases in estrogen secretion.

The postoperative decrease of plasma testosterone in man, after major surgery, in relation to plasma FSH and LH.

Abstract Confirmation was obtained of the pronounced postoperative decrease of plasma testosterone in the human male. Testosterone levels were determined using a competitive protein binding assay after one paper Chromatographie step. After different types of major surgical operation, in 8 patients, a nadir in the testosterone concentration (sample taken at 9 a.m.) was found on the first postoperative day (127±84 S.D. ng/100ml, compared to controls of 782 ±259 ng/100 ml) and on the second postoperative day (136 ±73 ng/100 ml). The decrease was still significant on days 5 and 6 (p No change in immunoreactive plasma LH, as measured in the same blood samples used for testosterone determinations, occurred after surgery, nor was any change noted in FSH levels, except in the cross-sectional study where a decrease occurred on day 2. It was concluded that neither gonadotropic hormone was responsible for the decreased level of testosterone.

Compensatory testosterone secretion in unilaterally orchidectomized rats

Abstract Blood and testis samples were taken from rats 3 weeks after unilateral (sinistral) orchidectomy or sham operation to study the regulation of circulating testosterone. Although plasma testosterone concentrations did not differ, the concentration of testicular testosterone was twofold greater in orchidectomized rats than in sham operated controls. At autopsy, weights of right testes as well as Leydig cell number of orchidectomized and control rats were similar. These observations indicate that, after unilateral orchidectomy, compensatory hypersecretion is not related to compensatory testicular hypertrophy.

A paper chromatographic saturation analysis method for measuring estradiol, testosterone and 5α-dihydrotestosterone from the same sample

Abstract A new method permitting the estimation of estradiol, testosterone and 5α-dihydrotestosterone in the same plasma sample is presented. This is made possible by a 20 h overflow paper chromatography system used to purify the plasma extract prior to steroid analysis. Estradiol is well separated from estrone as is testosterone from 5α-dihydrotestosterone. The method has low blank values, good agreement between duplicates, acceptable recovery through the extraction and purification and a good recovery of steroids added to plasma prior to extraction. Plasma levels of steroids measured using this method agree well with the values obtained by other methods.

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.

Testosterone binding capacity in relation to the production and metabolism of testosterone in dogs, experiences of a new method

Abstract Testosterone dynamics were studied in vivo in five male dogs using constant infusion techniques, for the first time comparing conscious dogs with anesthetized ones. Barbiturate narcosis was shown to inhibit testosterone production drastically so that plasma levels were near zero for at least 24 h. This causes a 50% reduction of the specific testosterone binding globulin demonstrated in the male dog using a new two-phase equilibrium partition method[5]. An increased association constant was observed at the same time. This protein was shown not to be identical with transcortin. It bound testosterone but not estradiol-17β. The testosterone-protein complex was efficiently extracted by the liver (85–100%). The hepatic metabolic clearance rate was however only 13–20% of the total MCR. It was unaffected by barbiturate anesthesia but showed a small decrease during halothane + oxygen anesthesia, while the extrahepatic MCR decreased to a greater extent during halothane narcosis or laparotomy.