Cortisol levels in central adrenal insufficiency: light and shade.

Evaluating children or adolescents with central adrenal insufficiency (CAI) is a difficult task in clinical practice, especially in subjects with hypothalamic-pituitary diseases and partial ACTH deficiency, or in those with recent pituitary surgery or brain irradiation when the adrenal cortex may still be responsive to stress. In 2008, a meta-analysis reported a three-step approach for evaluating patients at risk for CAI with no acute illness. In particular, the authors recommended the evaluation of morning cortisol, a low dose ACTH test (LDST) and the "gold standard" insulin tolerance test or metyrapone test if the low LDCT was not diagnostic. Cortisol and ACTH secretion exhibit significant fluctuation throughout the day. The reference ranges supplied by labs are so wide that they only flag up extremely low cortisol levels. Interpreting the results correctly can be difficult for a physician without an experience in adrenal dysfunctions. The lack of uniformity in these cut-off levels could in part be attributed to differences in study populations, variability of dynainic tests, the use of different serum cortisol assays and dissimilar cut-off peak serum cortisol response indicative of a normal axis response and the difference in the clinical context in which the studies were done. Therefore, Laboratories have to advertise the need to establish reference values for given populations, both for basal or stimulated hormone levels. Failure to apply this rule may elicit false-positive and more critically, false-negative results. LDST (1 pg synthetic ACTH as iv bolus with measurement of serum cortisol) has been proposed as a sensitive test for the diagnosis of CAl. However, the advantage of LDST compared with the high dose test may be offset by the technical difficulties inherent to dilution of 250 pg ampoules. Clinical judgment remains imperative especially regarding the use of glucocorticoid supplementation during extreme stress.

Medición de cortisol y sus fracciones. Una puesta al día / [A critical analysis of cortisol measurements: an update].

Serum cortisol measurement is a very useful tool in the biochemical evaluation of adrenocortical function. Since this hormone circulates in blood mainly linked to binding globulins but is also partially free, it can be measured not only in the blood but also in urine, saliva and other biological fluids and tissues. Basal determinations as well as dynamic testing may be performed to evaluate the circadian variations, to estimate the diurnal cortisol secretion and to analyze its relations with other components of the hypothalamic-pituitary-adrenal axis. Measurements of cortisol in blood, saliva and urine may reflect the cortisol secretion at the time of sample collection or during a 24 h span. Recently, it has been proposed the determination of cortisol in tissues such as hair and nails like a means of evaluating the hormonal status during prolonged periods. The aim of this paper is to update the methodology for measuring cortisol and its usefulness for the clinical diagnosis of troubles of the hypothalamic-pituitary-adrenal axis.

N-glycans modulate the function of human corticosteroid-binding globulin.

Human corticosteroid-binding globulin (CBG), a heavily glycosylated protein containing six N-linked glycosylation sites, transports cortisol and other corticosteroids in blood circulation. Here, we investigate the biological importance of the N-glycans of CBG derived from human serum by performing a structural and functional characterization of CBG N-glycosylation. Liquid chromatography-tandem MS-based glycoproteomics and glycomics combined with exoglycosidase treatment revealed 26 complex type N-glycoforms, all of which were terminated with α2,3-linked neuraminic acid (NeuAc) residues. The CBG N-glycans showed predominantly bi- and tri-antennary branching, but higher branching was also observed. N-glycans from all six N-glycosylation sites were identified with high site occupancies (70.5-99.5%) and glycoforms from all sites contained a relatively low degree of core-fucosylation (0-34.9%). CBG showed site-specific glycosylation and the site-to-site differences in core-fucosylation and branching could be in silico correlated with the accessibility to the individual glycosylation sites on the maturely folded protein. Deglycosylated and desialylated CBG analogs were generated to investigate the biological importance of CBG N-glycans. As a functional assay, MCF-7 cells were challenged with native and glycan-modified CBG and the amount of cAMP, which is produced as a quantitative response upon CBG binding to its cell surface receptor, was used to evaluate the CBG:receptor interaction. The removal of both CBG N-glycans and NeuAc residues increased the production of cAMP significantly. This confirms that N-glycans are involved in the CBG:receptor interaction and indicates that the modulation is performed by steric and/or electrostatic means through the terminal NeuAc residues.

Progesterone sperm chemoattraction may be modulated by its corticosteroid-binding globulin carrier protein.

Progesterone, the main steroidal component secreted by the cumulus cells that surround the egg, chemotactically guides human spermatozoa. The aim of this work was to evaluate whether the carrier protein corticosteroid-binding globulin also participates in the sperm P chemotactic response. By means of videomicroscopy and image analysis, we observed that corticosteroid-binding globulin modulates the chemotactic activity of P, when a solution of corticosteroid-binding globulin + P is at the nanomolar range.

Corticosteroid-binding globulin status at the fetomaternal interface during human term pregnancy.

The status of the corticosteroid-binding globulin (CBG) at the fetomaternal interface, especially in the maternal intervillous blood space (I), was investigated and compared to that of CBG in the maternal (M) and fetal (umbilical arteries [A] and vein [V]) peripheral circulations at term. Immunoquantitation of plasma CBG showed that the CBG concentration in I was 30% less than that in M (P < 0.001) and threefold higher than that in umbilical cord blood (P < 0.001). The microheterogeneity of CBG studied by immunoaffinoelectrophoresis in the presence of concanavalin A and Western blotting indicated that the CBG in I was mainly of maternal origin and different from fetal CBG. A CBG mRNA, but no classic 50- to 59-kDa CBG, was found in isolated term trophoblastic cells. The steroid environment of the CBG in I differed greatly from that in the peripheral maternal and fetal circulations, because the progesterone:cortisol molar ratio in I was 75-fold higher than that in M and 7- to 10-fold higher than that in the fetal circulation. Binding studies revealed that the affinity constants of CBG for cortisol in I, A, and V were significantly lower than that in M plasma (P < 0.02) in their respective hormonal contexts. The binding parameters for I-CBG stripped of endogenous steroids and lipids were close to those for M-CBG but different from those of fetal CBG (P < 0.001). These data reflect the physiological relevance of the CBG-steroid interaction, especially with very CBG-loaded progesterone at the fetomaternal interface during late pregnancy.

Levels of sex hormone-binding globulin and corticosteroid-binding globulin mRNAs in corpus luteum of human subjects: correlation with serum steroid hormone levels.

To understand regulation of the function of human ovarian corpus luteum by sex steroid-binding proteins, the levels of luteal intracellular sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) mRNAs and serum steroid hormones were simultaneously determined. The expression of SHBG and CBG mRNAs was detected in all samples analyzed. SHBG mRNA level was positively correlated with serum estradiol-17 beta level (p < 0.05), but not with serum progesterone level. There was a positive correlation between SHBG mRNA level and serum estradiol-17 beta/progesterone ratio (p < 0.01). On the other hand, CBG mRNA level was positively correlated with serum estradiol-17 beta and progesterone level (p < 0.01 and p < 0.01, respectively). There was no correlation between CBG mRNA level and serum estradiol-17 beta/progesterone ratio. SHBG and CBG mRNA levels were not correlated with the levels of serum testosterone, free testosterone or cortisol. These findings suggest that the synthesis of luteal SHBG and CBG is complexly regulated by estrogen and progesterone, and that SHBG and CBG interact with estrogen and progesterone, respectively, for luteal steroidal activity.

Biochemical and immunological characterization of the acrosome reaction-inducing substance (ARIS) of hFF.

Gel filtration experiments with 3H-progesterone labeled hFF demonstrated that the AR-inducing activity of hFF might be mediated by a progesterone-binding protein. Further immunological investigations added evidence in support to ARIS being identical with the SERPIN CBG. Moreover, only the protein-progesterone-complex was able to induce AR. It is suggested that the CBG-progesterone-complex is proteolytically cleaved at the plasma membrane of spermatozoa, releasing a high local concentration of progesterone which leads to induction of the AR.

Plasma steroid-binding proteins.

Two steroid-binding proteins circulate in plasma, corticosteroid-binding globulin and sex hormone-binding globulin. They both have several different but connected, physiologic functions. Each is the major determinant of the concentration of the physiologically important hormones that they bind. CBG regulates the concentration of free cortisol and progesterone, and SHBG regulates the concentration of free testosterone, dihydrotestosterone, and, to a lesser extent, estradiol. It is this small free fraction of the appropriate hormone that is the active principal in affecting hormone action. In the past few years, it has been shown that both of these proteins have high affinity, specific receptors on the plasma membranes of a variety of cells. It has also been shown that when SHBG's binding sites are occupied it cannot bind to its receptor; only unliganded SHBG can. There are, as yet, no published reports on the control of CBG binding by steroids. For both SHBG and CBG, if an appropriate steroid is present when the binding protein is itself bound to its receptor, rapid induction of adenylate cyclase activity and the accumulation of intracellular cAMP occur. Finally, CBG has been shown to be a member of the superfamily of serine proteinase inhibitors. When it is exposed to a serine protease, it is cleaved and release all, or most, of its bound cortisol.

[The hormonal mechanisms of the programming and regulation of liver function differentiation by sex]. / Gormonal'nye mekhanizmy programmirovaniia i reguliatsii differentsirovki funktsii pecheni po polu.

Crucial role of androgens and estrogens in programming and regulation of the sex-dependent expression of a specific estrogen-binding protein, androgenous and estrogenous receptors and corticosteroid-binding globulin, was shown. Both types of the sex steroids effects were shown to be realized in the hepatocytes through respective hormonal receptors. The realization of physiological effects of the sex hormones demands the participation of STH as a permissive factor. Some theoretical and practical aspects of the sex differentiation of the liver functions, are discussed.

[Effect of transcortin on the corticosterone-transforming activity of cytosol from the rat liver]. / Vliianie transkortina na kortikosterontransformiruiushchuiu aktivnost' tsitozolia pecheni krys.

The study of transcortin role in 3H-corticosterone metabolism has shown that transcortin of blood plasma from rats bearing Walker carcinosarcoma preserves the hormone conversion to dihydrocompounds 4 time less intensively than transcortin taken from healthy rats. Inactivated transcortin exerts no effect on the rate of formation of 5 beta-metabolites. Under the influence of homogeneous transcortin samples, a decrease in the content of 5 beta-reduced corticosterone metabolites is revealed to occur depending on transcortin concentration in the system. It is shown that in incubation systems where hormone is in the bound state the metabolism preserving capacity of transcortin depends on the temperature degree. The transcortin activity on corticosterone metabolism is supposed to be closely related to the intensity of its complexing with transcortin.

Are corticosteroid-binding globulin and sex hormone-binding globulin hormones?

Because it no longer seemed reasonable to us that the sole function of the steroid-binding proteins in plasma was to serve as a buffer reservoir for steroid hormones, we conducted experiments which sought out other possibilities. Both CBG and SHBG bind to cell membranes, and this interaction partakes of the general characteristics of peptide hormone-membrane receptor systems. Additionally, human CBG has the ability to cause an increase in the activity of membrane-bound adenylate cyclase in MCF-7 cells, and this, in turn, results in an increase in cellular cAMP content. Thus, CBG appears to be a protein hormone. As a first consideration, one might presume that because CBG's half-life is measured in days, it would be counted among the hormones which, for the most part, are tonic in their effects, e.g., thyroid hormone. However, two important considerations tend to believe this presumption: (1) CBG which is unoccupied by steroid is not hormonally active (Figure 5): (2) Depending upon the time of day, circulating CBG is approximately 0-60% occupied in normal humans. These observations result in a circumstance in which a substantial portion of circulating CBG is available for activation by bursts of cortisol secretion. It seems prudent to speculate that, because steroids are essential for CBG's activity, the hormonal role of CBG may be entwined with, or complementary to the steroids which it binds. Finally, we should comment on the impact that our model of CBG as a hormone has on the view that only unbound steroid can be hormonally active. First, it should be stated that we have not addressed this question experimentally. Although there is evidence that CBG may be required for cortisol action, we feel that an obligate role for it is not documented adequately. At this time, we believe that CBG's hormonal role is compatible with a hypothesis that encompasses the view that unbound steroid hormones can diffuse into cells in some tissues and that both free and bound steroid can enter cells in others. Obviously, the final word on these important topics, as always, awaits the proper experiments.

Inhibition by cortisol of human natural killer (NK) cell activity.

The effects of cortisol on the natural killer (NK) activity of human peripheral blood mononuclear (PBM) cells were studied in vitro using a direct 4-h 51Cr-release assay and K 562 cell line as a target. Preincubation for 20 h of PBM cells drawn from healthy donors with 1 X 10(-8) to 1 X 10(-5) M cortisol resulted in a significant decrease of NK cell activity. The magnitude of the suppression was directly related to the steroid concentration and inversely related to the number of effector cells. Cortisol was able to minimize the enhancement of NK cytotoxicity obtainable in the presence of immune interferon (IFN-gamma). A significantly higher suppression was achieved after sequential exposure of PBM cells to cortisol and equimolar levels of prostaglandin E2 (PgE2). The concomitant incubation with theophylline and isobutyl-methylxanthine failed to enhance the cortisol-induced suppression, whereas PgE2-dependent inhibition significantly increased after exposure of PBM cells to methyl-xanthines. The inhibitory effect of cortisol was partially or totally prevented by the concomitant incubation with equimolar amounts of 11-deoxycortisol and RU 486 but not of progesterone. Treatment of NK effectors with a monoclonal anti-human corticosteroid-binding globulin (CBG) antibody produced an enhancement of the spontaneous NK activity and a partial suppression of cortisol-mediated effects. Our results suggest that endogenous glucocorticoids play a role in the regulation of NK cell-mediated cytotoxicity. Since the effect of cortisol was additive to that of PgE2 and was not changed by phosphodiesterase inhibitors, it is conceivable that the hormone acts at a level different from the adenylate cyclase-phosphodiesterase system. Data obtained with the use of antiglucocorticoids and the anti-CBG antibody are compatible with a role both of high-affinity glucocorticoid receptors and of CBG in mediating cortisol action on the human NK cell activity.

Absence of plasma protein binding of aldosterone in normal and estrogen-treated rabbit.

In experiments on direct effects of prolonged administration of estrogen on In experiments on direct effects of prolonged administration of estrogen on mean arterial pressure (MAP) and plasma corticosteroid-binding variables in the rabbit the following observations were made. Estrogen had no effect on MAP but resulted in a nonsignificant stimulation of total plasma corticosteroids and a marked increase in corticosteroid-binding globulin (CBG) binding capacity which increased from a control value of 18.8 +/- (SD) 1.2 micrograms/100 ml to 28.1 +/- 2.3 micrograms/100 ml (p less than 0.001) following the administration of estrogen for the first 21 days (approx. 10 micrograms/day) and then further to 31.4 +/- 2.8 (p less than 0.001 vs. control values) after a higher estrogen dose of approximately 30 micrograms/day for the next 30 days, respectively. Plasma aldosterone concentration was not affected by estrogen treatment. In contrast to CBG, binding of aldosterone to plasma aldosterone-binding globulin was totally absent before and following the estrogen treatment. The striking difference between the rabbit showing an absence of plasma protein binding of aldosterone and several other animal species is perhaps of great importance for the blood pressure regulation and for understanding of the particular resistance of blood pressure to salt or mineralocorticoids reported in this species.

A phylogenetic study of the structural and functional characteristics of corticosteroid binding globulin in primates.

A monospecific antiserum against human corticosteroid binding globulin (hCBG) has been used to identify structural similarities between hCBG and CBG in the blood of other primates and representative species of different vertebrate classes. Double immunodiffusion analysis indicated that only CBG in Old World monkeys and apes cross-react with the hCBG antiserum. This was confirmed by a solid-phase radioimmunoassay for hCBG which also demonstrated that CBG in apes is immunologically identical to hCBG and that Old World monkey CBG comprises most, but not all, of the hCBG epitopes. The electrophoretic mobilities of human, gorilla and gibbon CBG were similar (RF 0.50-0.51), but differed from Old World monkey CBG (RF 0.44-0.49) and chimpanzee CBG (RF 0.47). Although serum/plasma cortisol binding capacities were similar in Old World primates, the dissociation half-times (t 1/2) of cortisol were higher from human and ape CBG (18-25 min) than from Old World monkey CBG (14-18 min). The steroid binding specificities of human and ape (CBG corticosterone greater than cortisol greater than progesterone greater than or equal to testosterone) were also different from those of Old World monkey CBG (corticosterone much greater than cortisol approximately equal to progesterone greater than testosterone). Lemur plasma cortisol binding capacity and CBG dissociation t 1/2 of cortisol were similar to hCBG, but its steroid binding specificity was different (cortisol greater than corticosterone greater than progesterone greater than or equal to testosterone) and it did not cross-react with the hCBG antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)

[Study of the characteristics of transcortin and the progesterone binding protein of the plasma of pregnant guinea pig]. / Izuchenie kharakteristik transkortina i progesteronsviazyvaiushchego belka plazmy beremennoi morskoi svinki

Experiments were conducted with the plasma of pregnant guinea pigs; it was shown that the corticosteroid-binding protein (CBP) of the plasma in a pregnant guinea pig (in difference from the CBP of other species) bound cortisol only, and not progesterone. Progesterone-binding protein (PBP) of these animals bound progesterone, but not glucocorticoids and their metabolites. The binding capacity of CBP proved to be several hundred microgram per cent of cortizol and of PBP--about 100 microgram per cent of progesterone. The association constant of the CBP with cortisol constituted 10(6) M-1, and of the PBP-10(-8) M-1. These proteins are also analysed according to a number of other parameters.