ACTH sensitivity of isolated human pathological adrenocortical cells: variability of responses in aldosterone, corticosterone, deoxycorticosterone and cortisol production

In vitro aldosterone, deoxycorticosterone, corticosterone and cortisol production of human adrenocortical cells derived from adenomas (Conn's syndrome, Cushing's syndrome), from hyperplastic adrenals (Cushing's syndrome) and from adrenals surrounding aldosteronoma are described. Cells from adenomas causing either Cushing's syndrome or Conn's syndrome harboured the highest basal and ACTH-stimulated corticosteroid production. Adrenocortical cells derived from micronodular hyperplasia causing Cushing's syndrome and cells from cortisol producing adenoma displayed predominantly cortisol and corticosterone secretion both under basal conditions and following stimulation with ACTH. Aldosteronoma cells showed highly variable aldosterone, deoxycorticosterone, corticosterone and cortisol response to ACTH. However, in aldosteronoma cell suspensions, the basal and ACTH-stimulated ratios of aldosterone to cortisol were increased when compared to ratios of steroids produced by cells from other adrenal tissues. Chronic treatment with spironolactone of patients with Conn's syndrome before surgery was associated with a decreased ratio of aldosterone to corticosterone, revealing that 18-hydroxylase in aldosteronoma cells may be inhibited during long-term therapy. Non-tumorous cells isolated from adrenals surrounding aldosteronoma displayed less aldosterone prior to and after stimulation with ACTH than aldosteronoma cells.     

Trend analysis of serum progesterone, deoxycorticosterone, deoxycorticosterone sulfate, cortisol, corticosterone, 18-hydroxydeoxycorticosterone and estradiol in early neonates

To elucidate the origin and regulatory mechanism of deoxycorticosterone (DOC) and deoxycorticosterone sulfate during fetal life, the levels of serum DOC, DOC sulfate, progesterone, cortisol, corticosterone and 18-hydroxydeoxycorticosterone (18OH-DOC) were determined in the fraction separated on high performance liquid chromatogram (HPLC) by radioimmunoassay (RIA) using the serum from normal newborn. Elimination curves both of serum DOC and DOC sulfate showed two phases: rapidly decreasing and slowly decreasing ones. Both serum DOC and DOC sulfate correlated with progesterone (r = 0.340, p less than 0.01; r = 0.737, p less than 0.01, respectively). They also correlated with cortisol (DOC, r = 0.467, p less than 0.01; DOC sulfate, r = 0.549, p less than 0.01, respectively). Serum DOC reached normal adult levels by 16 hrs after birth. However serum DOC sulfate concentration was maintained high throughout the entire early neonatal period. On the contrary, the changes in serum cortisol, corticosterone and 18OH-DOC showed a peak surge in the initial phase after delivery. Both serum corticosterone and 18OH-DOC correlated with cortisol (r = 0.518, p less than 0.01; r = 0.410, p less than 0.01, respectively). These findings suggest that, in the fetus, serum DOC and DOC sulfate are mainly produced at extraadrenal sites isolated from normal mineralocorticoids synthesis and after birth they begin to be formed at adrenal glands.     

Plasma cortisol and corticosterone concentrations in the golden hamster, (Mesocricetus auratus)

Because some recent studies of hamster adrenocortical function have depended on older studies that may have been inadequate or misinterpreted, the present study re-examined plasma corticosterone and cortisol concentrations in hamsters under several conditions to determine which plasma glucocorticoid predominated in this animal. Sensitive radioimmunoassays were used to measure separately the two glucocorticoids in the basal condition, after adrenocorticotropin (ACTH) treatment, after acute stress, and after chronic stress. In the basal condition, corticosterone concentrations were 3-4 times higher than those of cortisol. After stimulation, this difference disappeared, but rarely were any hamster's cortisol levels higher than their corticosterone levels. Both ACTH and acute stress elevated plasma corticosterone and cortisol concentrations, but only plasma cortisol concentrations were elevated following chronic stress. The dissociation between cortisol and corticosterone concentrations after chronic stress suggests that the two glucocorticoid hormones in the hamster may be regulated independently. The data also indicate that both corticosterone and cortisol should be measured when assessing adrenocortical function in the hamster.     

Fractionation of cortisol antisera by immunoadsorption chromatography: characterisation and use in an enzyme-linked immunosorbent assay (ELISA)

The use of affinity chromatography in the presence of 20% acetonitrile combined with a decreasing pH gradient has allowed the fractionation of two cortisol antisera into components of varying affinity. The high affinity fractions generate considerably improved ELISA standard curves compared to the intact sera but do not grossly alter the specificity of the antisera. Accordingly, the high affinity fraction of the least cross-reactive antiserum was used for a plasma cortisol ELISA. The cortisol ELISA, although returning slightly higher values than RIA, was comparable in the ability to distinguish dexamethasone suppression and cortisol response to synacthen and should thus prove of value in the assay of plasma cortisol.     

Site of conjugation of bovine serum albumin to corticosteroid hormones and specificity of antibodies

The influence of the site of attachment of bovine serum albumin (BSA) to corticosteroids on the specificity of the antisera obtained in rabbits was investigated. The steroids and positions studied were cortisol and 11-desoxycortisol at C-3, C-6α, C-6β and C-21 and 21-desoxycortisol and C-21-desoxycortisone at C-6α and C-6β. None of the antisera to cortisol showed high specificity. Similar cross reactions with antisera derived from cortisol coupled at C-6β, C-3 and C-21 to BSA were observed. 11-Desoxycortisol coupled at C-6α to BSA yielded the most specific antisera to this adrenal hormone. Cross reactions of antisera derived from coupling the protein to the extreme ends (C-3 and C-21) of 11-desoxycortisol were similar. The orientation of the conjugate at C-6 in 21-desoxycortisol and in 21-desoxycortisone did not influence the relative specificity of the antisera derived from the epimers. Highly specific antibodies were obtained against 21-desoxy-cortisone. Except tor 15% cross reaction with 17-hydroxyprogesterone, the antibodies to 21-desoxycortisol were relatively specific. It was concluded that the site on the steroid molecule to which BSA is attached influences the specificity of the antisera produced but there are also other factors operative.     

A specific, non-chromatographic radioimmunoassay for human plasma cortisol.

A radioimmunoassay technique has been developed for the measurement of cortisol in a single methylene chloride extract of human plasma without chromatography. The antiserum, obtained by immunizing rabbits with cortisol-3-carboxymethyl-oxime conjugated to bovine serum albumin, had a high affinity (KA = 1.8 X 10(9) 1/mole) and capacity (2.3 X 10(-6) moles/L undiluted serum) for cortisol. The minimum detectable amount determined at the lower 95% confidence limit of the buffer control tubes was 8.3 +/- 4.7 pg/tube and a log dose - logit response standard curve was linear between 20 pg and 20 ng/tube. The antiserum was highly specific for cortisol with only corticosterone, cortisone, 11-deoxycortisol and 21-deoxycortisol showing significant cross-reaction (12.4, 6.6, 3.8 and 3.7%, respectively). The cross-reaction for the other tested naturally occurring and synthetic steroids did not exceed 1%. Regression analysis of cortisol concentration estimates obtained on 20 samples before and after Sephadex LH-20 column chromatography gave a coefficient of correlation (r) of 0.995 and a regression coefficient (b) of 1.04. Recovery of cortisol added to plasma samples was quantitative. The intra-assay error was 8.5% and the inter-assay error averaged 5.7%. The method is simple requiring a single solvent extraction of plasma, therefore permitting large numbers of samples to be handled efficiently by a single technician.     

The effect of oxytocin on cortisol and corticosterone secretion in cyclic gilts--in vivo and in vitro studies

Oxytocin (OT) may be implicated in the modulation of hypothalamo-pituitary-adrenal axis (HPA) at each level. In mature females the influence of OT on the HPA axis appeared to be dependent on ovarian steroid milieu and stress. In cyclic sows, the role of OT in the regulation of corticoid secretion is unknown. In the present study changes in plasma cortisol and corticosterone concentrations in response to exogenous OT (in vivo experiment) and a direct influence of OT on adrenocortical steroidogenesis (in vitro experiment) were determined in luteal- and follicular-phase gilts. In the luteal-phase gilts (n=5), OT injections increased both cortisol (p<0.01) and corticosterone (p<0.05) plasma concentrations, but in the follicular-phase gilts (n=5) only the concentration of cortisol (p<0.05) was elevated in response to the treatment. Areas under the cortisol and corticosterone curves calculated for 30 min period after the OT injection were statistically higher (p<0.05) during the luteal than the follicular phase. In the in vitro experiment, two doses of OT (10(-7) and 10(-6) M) increased (p<0.05) secretion of cortisol by porcine adrenocortical cells representing the luteal phase, but not the follicular phase. However, OT did not affect the release of corticosterone by the cells. Incubation of the cells with the OT-antagonist (10(-5) M) abolished the effects of OT on cortisol secretion. Thus, in the present study, stimulatory effects of OT on the hypothalamo-pituitary-adrenal axis were demonstrated in cyclic gilts. The changes in plasma corticoid concentrations in response to exogenous OT were more prominent during the luteal than the follicular phase of the estrous cycle. Moreover, the in vitro experiment revealed a possibility of direct action of OT on adrenocortical cells isolated from luteal phase gilts. These results suggest that OT may participate in the modulation of HPA axis activity in pigs.     

Radioimmunoassay of human plasma corticosterone: method, measurement of episodic secretion and adrenal suppression and stimulation

A radioimmunoassay for human plasma corticosterone has been developed. Antisera were obtained by immunizing rabbits with corticosterone-21-hemisuccinate-BSA. An antiserum titer of 1:4000 was used for standard curves ranging from 0–1000 pg. Interfering steroids were removed from plasma extracts by paper chromatography. Plasma blanks obtained from adrenalectomized or Addisonian patients ranged from 29 to 42 ng/dl. Recovery of radioactive corticosterone through the entire method was 67.6 ± 5.2%. The coefficient of variation within assays was 19% and between assays 13%. The average 8 a.m. value in males was 396 ± 228 ng/dl and in females it was 655 ± 271 ng/dl. Corticosterone was found to be secreted episodically, in parallel with cortisol. Secretion of this steroid was suppressed by dexamethasone and stimulated by ACTH infusion.     

Characterization of antisera to 2-hydroxyestradiol and 4-hydroxyestradiol using 6-(O-carboxymethyl)oxime- and 17-hemisuccinate-bovine serum albumin conjugates and radioimmunoassay

For radioimmunoassay of the catechol estrogens, four hapten-bovine serum albumin (BSA) conjugates were prepared from 6-oxo-2-hydroxyestradiol 6-(O-carboxymethyl)oxime, 2-hydroxyestradiol 17-hemisuccinate, 6-oxo-4-hydroxyestradiol 6-(O-carboxymethyl)oxime and 4-hydroxyestradiol 17-hemisuccinate by coupling with BSA, employing the mixed anhydride method. The antisera elicited in rabbits by immunization with these antigens showed high affinity and specificity for 2-hydroxyestradiol or 4-hydroxyestradiol with cross-reactivities to a few structurally related estrogens. The specificity of antisera obtained is discussed in relation to the site of attachment of the hapten to BSA.     

Non-breeding feather concentrations of testosterone, corticosterone and cortisol are associated with subsequent survival in wild house sparrows

Potential mechanistic mediators of Darwinian fitness, such as stress hormones or sex hormones, have been the focus of many studies. An inverse relationship between fitness and stress or sex hormone concentrations has been widely assumed, although empirical evidence is scarce. Feathers gradually accumulate hormones during their growth and provide a novel way to measure hormone concentrations integrated over time. Using liquid chromatography-tandem mass spectrometry, we measured testosterone, corticosterone and cortisol in the feathers of house sparrows (Passer domesticus) in a wild population which is the subject of a long-term study. Although corticosterone is considered the dominant avian glucocorticoid, we unambiguously identified cortisol in feathers. In addition, we found that feathers grown during the post-nuptial moult in autumn contained testosterone, corticosterone and cortisol levels that were significantly higher in birds that subsequently died over the following winter than in birds that survived. Thus, feather steroids are candidate prospective biomarkers to predict the future survival of individuals in the wild.     

Endogenous inhibitors (GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2: derivatives of adrenally produced corticosterone and cortisol

Two isoforms of 11β-HSD exist; 11β-HSD1 is bi-directional (the reductase usually being predominant) and 11β-HSD2 functions as a dehydrogenase, conferring kidney mineralocorticoid specificity. We have previously described endogenous substances in human urine, “glycyrrhetinic acid-like factors (GALFs)”, which like licorice, inhibit the bi-directional 11β-HSD1 enzyme as well as the dehydrogenase reaction of 11β-HSD2.

Many of the more potent GALFs are derived from two major families of adrenal steroids, corticosterone and cortisol. For example, 35-tetrahydro-corticosterone, its derivative, 35-tetrahydro-11β-hydroxy-progesterone (produced by 21-deoxygenation of corticosterone in intestinal flora); 35-tetrahydro-11β-hydroxy-testosterone (produced by side chain cleavage of cortisol); are potent inhibitors of 11β-HSD1 and 11β-HSD2-dehydrogenase, with IC₅₀'s in range 0.26–3.0 μM, whereas their 11-keto-35-tetrahydro-derivatives inhibit 11β-HSD1 reductase, with IC₅₀'s in range 0.7–0.8 μM (their 35β-derivatives being completely inactive).

Inhibitors of 11β-HSD2 increase local cortisol levels, permitting it to act as a mineralocorticoid in kidney. Inhibitors of 11β-HSD1 dehydrogenase/11β-HSD1 reductase serve to adjust the set point of local deactivation/reactivation of cortisol in vascular and other glucocorticoid target tissues, including adipose, vascular, adrenal tissue, and the eye. These adrenally derived 11-oxygenated C₂₁- and C₁₉-steroidal substances may serve as 11β-HSD1- or 11β-HSD2-GALFs. We conclude that adrenally derived products are likely regulators of local cortisol bioactivity in humans.     

Changes in plasma levels of cortisol and corticosterone after acute ACTH stimulation in rusa deer (Cervus rusa timorensis)

Resting cortisol and corticosterone levels in immobilized mature rusa stags (Cervus rusa timorensis) and the influence of synthetic ACTH on the cortisol/corticosterone ratio (F/B ratio) were investigated. The basal concentration of cortisol was found to be 14.07 nmol/l (SD = 9.3, N = 15) and corticosterone was 3.79 nmol/l (SD = 2.3, N = 15). The cortisol/corticosterone ratio for the basal level was 5.31 (SD = 3.9, N = 15). After ACTH administration the cortisol/corticosterone ratio increased to 11.41 (SD = 5.4, N = 147) regardless of doses of ACTH administered to individual stags. The adrenal response to ACTH administration has a potential application for selection of deer most suitable for deer farming.     

A proton magnetic resonance study of the 18-hydroxy derivatives of cortisol and corticosterone

Proton magnetic resonance spectroscopy played a crucial role in the identification of a new corticosteroid, 18-hydroxycortisol, recently isolated from the urine of a patient with an aldosterone-producing adenoma. Mass spectrometric analysis and chemical degradative studies demonstrated an empirical formula of C21 H30 O6 corresponding to a diketopregnenetetrol and placed three of the four hydroxyl groups at the 11 beta, 18 and 21 positions. The first suggestion that the locus of the fourth hydroxyl was 17 alpha came from the n.m.r. spectrum in the form of negative evidence for proton linked to the carbon atom bearing this fourth hydroxyl. The n.m.r. spectral features of the 18-hydroxy derivatives of cortisol and corticosterone are compared. Both were found to exist in the 20, 18 cyclic hemiketal form.     

Adrenal steroids and extinction behavior: Antagonism by progesterone,deoxycorticosterone and dexamethasone of a specific effect of corticosterone

Extinction behavior was nearly absent in rats adrenalectomized one hour prior to forced extinction of a passive avoidance response. A low dose of corticosterone administered immediately after adrenalectomy normalized extinction behavior. Progesterone, 11-deoxycorticosterone and the synthetic glucocorticoid dexamethasone were not effective at the same or ten times higher doses. Instead, pre-treatment with these steroids prevented the normalizing effect of corticosterone on extinction behavior. These characteristics of steroid effects on behavior correspond to the strict specificity of the corticosterone receptor system in hippocampal neurons. The agonist or antagonist interaction of naturally occurring adrenal steroids with brain cells may serve behavioral adaptation.     

A radioimmunoassay for human plasma corticosterone.

A radioimmunoassay for human plasma corticosterone has been developed. Antiserum against corticosterone was produced in rabbits immunized with corticosterone-21-hemisuccinate conjugated to bovine serum albumin. The antiserum cross-reacted with progesterone, DOC and dehydrocorticosterone more than 20%. After the extraction with ether, and the separation by Sephadex LH-20 microcolumn chromatography, recovery was 51.2 +/- 12.1% in 50 assays. The mean coefficient of variation between assays was 7.7% and within assays was 8.6%. Human plasma corticosterone is measured readily by assaying aliquots of an ether extract of 0.05 to 0.1 ml of plasma after microcolumn chromatography. The mean plasma corticosterone concentration at 9 a.m. was 7.1 +/- 3.2 ng/ml in 45 normal subjects. Plasma corticosterone increased 5.2 times as much as basal values after ACTH injection, whereas radioimmunoassayed cortisol increased 2.4 times. On the other hand, plasma corticosterone decreased to 22.6% of basal values at four hours after 1 mg dexamethasone, whereas radioimmunoassayed cortisol decreased to 12.3% of basal values.     

Validation of a specific radioimmunoassay to measure plasma cortisol in the fetal sheep.

A procedure utilizing co-chromatography and complementary antiserum comparisons was employed to assess the specificity of a cortisol radioimmunoassay for use in the chronically catheterized fetal sheep preparation. Complementary antiserum comparisons is a technique by which two different cortisol antisera, prepared from conjugates attached at opposite ends of the cortisol molecule, were used to determine cortisol concentrations in the same ovine fetal plasma specimens. Results were not significantly different between the two groups, each measured by a different antiserum. This procedure may be used to assess assay specificity in any species in which steroid radioimmunoassays are being adapted.     

斑马鱼cdk7和cyclin H基因的原核表达及多克隆抗体的制备与鉴定

构建了两个表达斑马鱼cdk7(基因片段)和cyclinH(基因全长)的重组质粒,分别转化大肠杆菌BL21进行原核表达,所得的CDK7和cyclinH两个融合蛋白均以包涵体形式存在。经SDS-PAGE分离,切下含有目的蛋白条带的凝胶冻成干粉,分别免疫新西兰大耳兔,制备并纯化了分别抗CDK7和抗cyclinH的两个多克隆抗体。经酶联免疫吸附测定、蛋白质印迹分析检测,确定获得了两种具有较高效价的特异性多克隆抗体。免疫荧光组织化学结果显示,CDK7和cyclinH这两个蛋白质均普遍存在于斑马鱼胚胎动物极的各个细胞中。     

The use of multivariable standard curves in the radioimmunoassay of testosterone and 5alpha-dihydrotestosterone

Multivariable calibration curves have been used to enable testosterone and 5alpha-dihydrotestosterone to be assayed directly in plasma extracts without further pre-purification of the sample. Two antisera were used, both with relatively high, but different affinities for the substances measured, and with relatively low affinity towards all other substances tested. The antisera were obtained from rabbits immunized against testosterone-3-BSA and 5alpha-dihydrotestosterone-3-BSA. The technique was of adequate precision, accuracy and specificity. The last was examined by comparison of values obtained by the present method and those obtained following pre-purification by thin layer chromatography.     

Physiological and pathophysiological applications of sensitive ELISA methods for urinary deoxycorticosterone and corticosterone in rodents

Deoxycorticosterone (DOC: a weak mineralocorticoid) is the precursor to corticosterone (B: the major glucocorticoid in rodents) and aldosterone (the major mineralocorticoid). The genes Cyp11b1 and Cyp11b2 that encode the enzymes responsible for DOC to B (11β-hydroxylase) and DOC to aldosterone (aldosterone synthase) conversions are located on the same chromosome. The aim of this study was to develop sensitive and specific ELISA methods to quantify urinary DOC and B concentrations to assess the physiological and genetic control of the Cyp11b1/b2 locus. Antibodies raised in rabbits against DOC and B and horse radish peroxidase-goat anti-rabbit IgG enzyme tracer were used to develop the assays. Urine samples collected from mice held in metabolic cages were extracted with dichloromethane and reconstituted in assay buffer. The assays were validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivities with major interfering steroids were minimal: DOC assay (progesterone = 0.735% and corticosterone = 0.045%), and for B assay (aldosterone = 0.14%, 11-dehydro-B = 0.006%, cortisol = 0.016% and DOC = 0.04%) and minimum detection limit for DOC ELISA was 2.2 pg/mL (6.6 pmol/L), and for B ELISA was 6.2 pg/mL (17.9 pmol/L). The validity of urinary DOC and B ELISAs was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC (DOC ELISA: Y = 1.092X − 0.054, R² = 0.988; B ELISA: Y = 1.047X − 0.226, R² = 0.996). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. The methods were used in a series of metabolic cage studies which demonstrated that (i) females produce more DOC and corticosterone than males; (ii) DOC and corticosterone respond to ACTH treatment but not dietary sodium restriction; (iii) DOC:B ratios in Cyp11b1 null mice were >200-fold greater than wild type.     

Production and specificity of antisera raised against 25-hydroxyvitamin D3-[C-3]-bovine serum albumin conjugates.

In order to obtain specific antisera for use in the enzyme immunoassay of 25-hydroxyvitamin D3, three hapten-carrier conjugates having different lengths of bridges at the C-3 position were prepared from 25-hydroxyvitamin D3 by coupling with bovine serum albumin using the active ester method. The specificity of anti-25-hydroxyvitamin D3 antisera elicited in rabbits was tested by a cross-reaction study with closely related secosterols and by measuring the plasma levels of 25-hydroxyvitamin D3 by means of radioimmunoassay using tritium-labeled antigen. The results indicated that the specificity of the antisera obtained is higher than that of vitamin D-binding protein, and that some of these antisera are suitable for enzyme immunoassay.