Plasma Transcortin Research Articles

Evaluation of adrenal function in mice by measurement of urinary excretion of free corticoids

Methods for the determination of urinary total free corticoids and urinary free corticosterone in small laboratory rodents are described. Total free eorticoids and free corticosterone were determined in samples of 50–200 μl urine by a competitive protein binding method using human plasma transcortin as the source of binding protein. For both methods employed the reliability criteria were satisfactory. The validity of both methods was tested by measuring both total free corticoids and free corticosterone in urine samples of mice with the obese-hyperglycemic syndrome (gene symbol C57BL/6J-ob/ob) and their lean controls (gene symbol C57B1/6J). Measurements were taken from urine samples under basal conditions and after adrenal stimulation or suppression. Urinary corticoid levels were significantly different between the two mouse lines, 15 ng/day for the lean and 75 ng/day for the obese mice, reflecting plasma corticosterone levels. After stimulation by ACTH urinary corticoids rose 3–5-fold whereas suppression by dexamethasone was followed by a decrease to very low levels. The contribution of corticosterone to total corticoids averaged 50% in lean and 30% in obese mice. It is concluded that urinary total excretion of free corticoids as well as urinary excretion of free corticosterone offer a valuable parameter of adrenal function in mice. Furthermore, when small laboratory rodents are used, under many circumstances measurements in urine are more advantageous than measurements in plasma.

A Radioreceptor Assay for Evaluation of the Plasma Glucocorticoid Activity of Natural and Synthetic Steroids in Man1

An assay for plasma glucocorticoid activity has been developed using specific glucocorticoid receptors. Unlike other assays for cortisol and certain synthetic corticosteroids, this radioreceptor assay measures the glucocorticoid activity of all natural an synthetic steroids. Steroids extracted from as little as 0.05 ml of plasma are incubated with 3H-dexamethasone and cytosol receptors from cultured rat hepatome cells. From 0.5 to 50 ng of cortisol are accurately detected. Glucocorticoid activities of adult determined by the assay correlate closely with corticoid levels obtained in the CBG-isotope and fluorometric assays. Other steroids are measured in proportion to both concentration and potency as glucocorticoids. Relative activities include: cortisol 100, dexamethasone 940, prednisolone 230, prednisone 3, estradiol 1 and androstenedione 1. A similar ranking of steroids was found using receptors from a human source (fetal lung). The assay has been useful in detecting glucocorticoid activity in unidentified medications and in measuring plasma glucocorticoid levels after administration of synthetic corticosteroids. A modification of the assay is described for also estimating the level of free (unbound) glucocorticoid activity in plasma. Assays are performed before and after removal by charcoal of steroids not bound to plasma transcortin. This consideration is shown to be particularly important with prednisolone where the unbound steroid level is much less than the total. Thus, the radioreceptor assay provides a relatively simple technique for measuring the total of free plasma of glucocorticoid activity in man due to any natural or synthetic steroid or combination of steroids.

Prostatic cancer Transcortin levels during treatment with estramustine phosphate

Plasma transcortin and cortisol levels, which are significantly elevated by estrogens, were determined in a group of patients with prostatic cancer who were on estramustine phosphate (Estracyt) therapy. In a series of 44 male patients studied transcortin and cortisol levels became elevated above normal and were maintained at these high levels as long as the patients were on estramustine phosphate therapy. The levels observed in this study may serve as reliable indices to monitor patients who are on this drug.

Feedback Inhibition of Adrenocorticotropin Release by Corticosterone Infusions in the Adrenalectomized Rat

Advantage was taken of a specific and sensitive bioassay for rat plasma adrenocorticotropin (ACTH) based on the dispersion of rat adrenal cells with trysin, to investigate the relationship between plasma corticosterone concentration and inhibition of ACTH release under steady-state conditions achieved by graded rates (0-5.12 mug/min per 100 g body weight) of intravenous infusion of the steroid for 45 min in 28-day adrenalectomized rats. In contrast to prior reports involving suppression of stress-induced ACTH release, the inhibitory effect of corticosterone was shown, under our experimental conditions, to be exerted also on the basal rate of ACTH secretion. Indeed, a slight though not significant decrease of plasma ACTH concentration was observed with the corticosterone infusion rate of 0.64 mg/min per 100 g body weight, and further progressive and highly significant drops in concentration were recorded for infusion rates of 2.56 and 5.12 mg/min per 100 g body weight. An increase of the metabolic clearance rate of corticosterone, observed as a function of the infusion rate, was ascribed to saturation by the steroid of the plasma transcortin binding sites.

Estrogen and the metabolism of progesterone in vivo

The effect of estrogen administration on the metabolism of progesterone was studied in ovariectomized, hysterectomized women. Estrogen was withheld from each subject for at least 4 weeks, then 200 μg of ethinyl estradiol were administered orally each day for 3 weeks and then no estrogen was given for 3 weeks. The subjects were studied before estrogen was started, on the third day of estrogen, after 3 weeks of daily estrogen and after the estrogen was withdrawn for 3 weeks. In 5 women plasma transcortin concentrations, measured by equilibrium dialysis, were 0.82 ± 0.06, 1.5 ± 0.16, 2.1 ± 0.13, and 0.90 ± 0.09 (S.E.) μM before starting estrogen (control), on the third day of estrogen treatment, after 3 weeks of estrogen, and after having stopped estrogen for 3 weeks, respectively. Corresponding values for the metabolic clearance rates (MCR) of cortisol (by the continuous infusion method) were 306 ± 33, 172 ± 18, 136 ± 14, and 258 ± 23 (S.E.) L. per day. Although estrogen administration caused a significant elevation of the plasma transcortin concentrations and a significant decrease in the MCR of cortisol, it had no significant effect on the MCR of progesterone or of cortisone. The estrogen administration did cause a decrease in the peripheral conversion of progesterone to 20α-hydroxy-pregn-4-en-3-one (20α-OHP) and of cortisol to cortisone but an increase in the conversion of 20α-OHP to progesterone and of cortisone to cortisol.

Evaluation of three constants involved in the binding of corticosterone to plasma proteins in the rat.

Equilibrium dialysis was used to evaluate three constants involved in the binding of corticosterone to transcortin and albumin in rat plasma, namely ST (concentration of binding sites of transcortin), KT (association constant of transcortin), and SAKA (binding constant of albumin). Undiluted plasma from rats adrenalectomized 24 h earlier was enriched with increasing amounts of corticosterone (0–5.0 μg/ml) and dialyzed for 4.3 h at 37 °C.Assuming that the law of mass action applies to the interaction between corticosterone and its two binding proteins, the values of ST, KT, and SAKA were estimated by means of a new method of calculation. Derived from the maximum likelihood principle, this method allows computation of a confidence region for the parameters. The following values and statistical bounds were obtained:[Formula: see text]

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

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 (M w = 4 × 10 5), 7% (w/w) poly-(ethylene glycol) (M n = 6 × 10 3), 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 × 10 9 L mol −1 for TeBG and 8.6 × 10 4 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.

Identification and Partial Purification of “Transcortin”-like Protein within Human Lymphocytes

Abstract We have detected and localized in situ lymphocyte proteins with antigenic determinants similar to those of the cortisol-binding globulin of human plasma. All lymphocyte fractions, obtained by differential centrifugation and ammonium sulfate fractionations exhibited cortisol-binding capacities and were recognized by transcortin-specific antibody. Inasmuch as the 40 to 65% ammonium sulfate fraction of the 105,000 x g lymphocyte cytosol accounted for over 90% of the total binding capacity of the cell, this fraction was further purified by the procedure employed for the purification of plasma transcortin. These procedures yielded a protein with about 20% the cortisol-binding capacity of transcortin, migrated more rapidly than transcortin in disc acrylamide electrophoretic systems, and formed a line of identity with transcortin when treated with transcortin-specific antibody. Fluorescein-labeled transcortin antibody applied to sectioned lymphocytes showed almost exclusive cytoplasmic localization of transcortin-like protein. These results show for the first time the existence of cortisol-binding proteins within the human lymphocyte antigenically identical with transcortin.

The apparent affinity of plasma transcortin (CBA) in inbred Tamilian Hindus belonging either to a diabetes-prone community or to a group without family history of diabetes.

The apparent cortisol-binding affinity of plasma transcortin (CBA) was studied in two groups of inbred Tamilian Hindus. The first group was part of a small community living in Cape Town and presenting a very high prevalence of diabetes mellitus; the second belonged to a much larger community, originally living in Durban, and consisted of individuals without known family history of diabetes. In the diabetes-prone group, the CBA of the nondiabetics with two diabetic parents was significantly higher than the CBA of the nondiabetics with only one diabetic parent and nearly as high as the CBA of diabetics. On the other hand, the men, but not the women, of the nondiabetic group with one diabetic parent had a CBA significantly lower than the nondiabetic Tamils living in a similar environment but belonging to families apparently free of diabetes. The fact that the CBA in children of connubial diabetics is not significantly different from the CBA of diabetics, suggests that the high transcortin activity, already n...

The role of transcortin in the distribution of corticosterone in the rat.

In rats treated with ACTH for 14 days, 24 hours after the last injection the plasma transcortin level is low. The biological half-life of corticosterone is of the same duration as in the controls; transcortin leaves it unaffected. The distribution volume of corticosterone is significantly higher in the treated than in the control animals and transcortin decreases it in both groups.

Progesterone-binding Components of Chick Oviduct: II. NUCLEAR COMPONENTS

Abstract Specific progesterone-binding components are shown to be present in oviduct nuclei of estrogen-treated chicks. The macromolecular complex containing 3H-progesterone can be detected by sucrose gradient centrifugation and distinguished from chick plasma transcortin by Agarose gel filtration. The nuclear binding components behave identically to the cytoplasmic components previously described in our ultracentrifugal, chromatographic, and inactivation studies. The macromolecular-progesterone complex formed in vivo or in vitro can be extracted from nuclei by 0.3 m KCl. Little or no salt-extractable progesterone-binding activity can be detected in oviduct nuclei prior to progesterone administration. The binding molecules appear to exist initially in oviduct cytoplasm, where they presumably function as specific target tissue receptors. Following injection of 3H-progesterone in vivo or incubation of oviduct slices in vitro with 3H-progesterone, a progressive increase in nuclear binding occurs. The data indicate an absolute prerequisite for an initial interaction of the steroid with the cytoplasm, and a concomitant depletion of cytoplasmic receptor as nuclear binding increases. These results, and the apparent identity of the cytoplasmic and nuclear components, support the hypothesis that the nuclear macromolecular-steroid complex arises by a hormone-dependent transfer of the cytoplasmic receptor complex into the nucleus.

Binding characteristics of transcortin in human plasma in normal individuals, pregnancy and liver disease.

SUMMARY The binding characteristics of transcortin have been measured in 16 normal individuals, 10 pregnant women, five subjects taking oral contraceptives or on oestrogen therapy and in six patients with advanced liver disease. The affinity of transcortin for cortisol as measured by its equilibrium constant was found not to differ significantly between the normal individuals and the rest. The transcortin capacity was greatly increased in pregnancy and by oestrogen therapy but was normal in the cirrhotics studied. Despite the increase in total plasma cortisol in pregnancy, the unbound plasma cortisol concentration was only slightly raised and no increase was noted in the oestrogen-treated group. One of the 'normal' group was found to be transcortin deficient with low total plasma cortisol but the concentration of unbound cortisol and the rate of cortisol secretion were normal.

Binding of cortisol and 21-dehydrocortisol with transcortin.

The interaction of human plasma transcortin (corticosteroid-binding globulin) with cortisol and 21-dehydrocortisol was studied under various experimental conditions. The amounts of protein-bound steroids were determined by gel filtration on Sephadex G-25. Both steroids interacted with bovine serum albumin and transcortin. The binding increased with increasing alkalinity and was low at pH 4. The patterns of binding in relation to pH values with the 2 steroids were different. When unlabeled cortisol was added to the assay mixture, the binding of labeled cortisol was blocked and little or no effect was observed with the binding of 21-dehydrocortisol. Upon heating, acidification or extraction with methylene chloride, practically all of the cortisol bound with transcortin was liberated, whereas significant amounts of 21-dehydrocortisol remained bound. The maximum binding of transcortin with cortisol occurred at 0–4 C and with 21-dehydrocortisol at 37 C. The results of this study indicate that cortisol and 21-d...

Factors related to the age at discovery of diabetes mellitus.

ABSTRACT The possible relation between age at discovery on the one hand and body weight, body height, plasma cholesterol, cortisol binding capacity of plasma transcortin (CBC) and menarchial age on the other hand, was studied in 108 diabetic men and 111 diabetic women with onset of diabetes after age 19. Age at the time of study was included in the multiple regression analysis and only partial correlation coefficients of highest order were taken into account. By doing so, secular changes in certain parameters and changes with age in others could be eliminated. In adult men and women the age at discovery of diabetes mellitus correlated significantly but negatively with body height. In men, but not in women, the age at discovery also correlated with body weight (positively) and with the cortisol binding capacity of plasma transcortin (negatively). Similar significant correlations were recorded when the age at discovery in older diabetics was corrected (up to 10 yr) for a possible delay in diagnosis.

Cortisol binding affinity of plasma transcortin (CBA) as studied by competitive adsorption.

The cortisol binding affinity (CBA) of plasma transcortin was measured in more than 400 individual plasmas by means of competitive adsorption with fuller's earth. Within the limits of precision of the method no within-person variation in CBA was detected. More specifically, the CBA did not change during pregnancy or estrogen treatment. Between-person variations in CBA were looked for in both a normal Belgian and a normal Sardinian population; again no differences were found. The apparent association constant obtained here in whole plasma is the same as that described for isolated transcortin.

Plasma cortisol binding globulin levels in gastro-intestinal bleeding of stress origin.

ABSTRACT Plasma cortisol levels, transcortin binding capacities and percentages of total plasma cortisol bound by protein were determined in patients diagnosed as having gastro-intestinal bleeding of stress origin. These patients all had elevated cortisol levels and abnormally low plasma transcortin binding capacities. It could be assumed then that the amount of free (or active) cortisol in their plasma was unusually high. Thus, whatever the effect corticosteroids have upon the gastro-intestinal mucosa resulting in erosion and bleeding, in all probability it is potentiated by a low plasma content of transcortin.

Study of steroid-protein binding by means of competitive adsorption: Application to cortisol binding in plasma

The distribution of a steroid between a solution of steroid-binding proteins and a steroid-adsorbing solid added to this solution, depends not only on the properties of the adsorbent but also on the properties of the binding proteins and thus may be used for the study of steroid-protein interactions. Different types of distribution can be distinguished and were applied to the study of cortisol binding in plasma. 1. (1) Endogenous steroids were removed from plasma by incubation with a rather large amount of adsorbent. 2. (2) The cortisol-binding capacity of plasma transcortin was measured after incubation with a balanced amount of adsorbent and cortisol. 3. (3) A relative index of cortisol-protein binding proportional to the ratio of bound to unbound cortisol, was determined by incubation of charcoal-treated plasma with a trace of [1,2- 3H]cortisol and appropriate amounts of adsorbent. 4. (4) From this relative index of cortisol—protein binding in plasma an estimate of the cortisol-binding affinity of plasma transcortin was derived.

Cortisol-binding capacity of plasma transcortin: a sex-linked trait?

In men, but not in women, the distribution of CBC values (cortisol-binding capacity of plasma transcortin) has a bimodal aspect, both subgroups being of about equal size. This bimodal distribution is also present within pairs of brothers. A statistical excess of low CBC values is seen in the plasma of relatives of subjects with a CBC below 20 μg/100 ml. Fathers of sons, but not of daughters, with a CBC below 20 μg/100 ml have a normal mean CBC. These facts suggest that an x-linked gene determines, at least partly, the cortisol-binding capacity of plasma transcortin. The high incidence of low CBC values in patients with hemophilia or other conditions in which sex-linked inheritance may be of major importance is compatible with this hypothesis.

Cortisol binding capacity of plasma transcortin in diabetic subjects.

ABSTRACT Diabetic men and women have a higher mean cortisol binding capacity of plasma transcortin (CBC) and a larger between-person variation of CBC than normal. Diabetics with a CBC below 20 μg/100 ml are heavier than diabetics with a CBC above 28 μg/100 ml. The latter are more insulin dependent and, at least in women, have higher plasma cholesterol levels. Diabetic men with a CBC above 28.0 μg/100 ml have the onset of their disease at an earlier age than men with a CBC <20.0 μg/100 ml. Women who before they became diabetic gave birth to heavy (≥4.5 kg) babies have a higher mean CBC than women who had babies of normal birth weight.

The binding of cortisol by ovine plasma proteins.

SUMMARY Albumin was isolated from ovine plasma and its affinity for cortisol was determined by equilibrium dialysis at 37°. The value of Ka[σpa] for a 1 % (w/v) albumin solution was 0·275 which is similar to the value for human plasma albumin. The affinity constant of transcortin in ovine plasma was determined by equilibrium dialysis of diluted plasma at several concentrations of cortisol. The value found, Kt (37°) = 0·87 x 108 l./mole, is close to that found for human plasma transcortin by Mills (1962). The concentration of transcortin in ovine plasma, expressed as cortisolbinding capacity, was 6–49 μg. (mean 24 μg.) cortisol/l. These concentrations are much lower than those found in human plasma. The observation of Lindner (1964) that cortisol binding capacity did not increase during pregnancy in sheep has been confirmed. In sheep which were accustomed to handling, the mean concentration of cortisol in plasma was 17·8 μg./l. and of this amount 59% was bound to transcortin, 19 % to albumin and 22 % was not bound to protein.