Radioimmunoassay of β-MSH in Human Plasma and Tissues

A radioimmunoassay method for beta-melanocyte-stimulating hormone (beta-MSH) has been developed and utilized in the identification and quantification of this hormone in human plasma and tissues. The concentration of beta-MSH in two human pituitary glands was found to be approximately 350 mug/g. beta-MSH was identified in the tumor tissue of all 11 patients with the ectopic ACTH syndrome who were studied; concentrations in individual cases ranged from 3 to 1600 ng/g. In plasma of chronically hyperpigmented patients with Addison's disease, Cushing's disease (after bilateral adrenalectomy), and the ectopic ACTH syndrome, beta-MSH concentrations of 0.5-6 ng/ml were found. The degree of clinical hyperpigmentation was well correlated with the quantity of beta-MSH in the plasma. beta-MSH concentrations in the plasma of normal subjects were less than 0.09 ng/ml. In all of these circumstances, bioassays for MSH were also performed, and it was found that most of the biologic MSH activity of the plasma and tissues could be accounted for by beta-MSH.     

Technical notes and clinical use of radioimmunologic determination of plasma ACTH]

Particular aspects of plasma ACTH radioimmunoassay are examinated. The results obtained with this method in adrenocortical diseases (Addison's disease, Cushing's syndrome, congenital adrenal hyperplasia) are reported and clinical value in etiological diagnosis of Cushing's syndrome is outlined. Moreover the results obtained in other syndromes of endocrinologic interest (massive obesity, Turner's and Klinefelter's syndromes, Laurence-Moon-Biedl syndrome, true precocious puberty, primary hypothyroidism, hypopituitary dwarphism) are reported and discussed.     

Corticotropin releasing factor as an aid in the diagnosis of Cushing syndrome

6 patients with Cushing's syndrome were investigated with regard to the effect of synthetic ovine corticotropin-releasing factor (o-CRF), administered as an intravenous bolus of 100 micrograms, on peripheral plasma concentrations of ACTH and cortisol. The purpose of this study was to evaluate the usefulness of this "CRF test" in the differential diagnosis of Cushing's syndrome as compared with conventional diagnostic procedures. 100 micrograms CRF caused a rise in plasma ACTH and cortisol in patients with bilateral adrenal hyperplasia (n = 3). However, in patients with cortisol-producing adrenal adenoma (n = 2) and ectopic ACTH overproduction (n = 1), no increase in plasma cortisol and ACTH was induced by exogenous CRF. We conclude from these findings that the CRF test will prove a valuable diagnostic tool to differentiate pituitary from extrapituitary forms of endogenous hypercortisolism in patients with Cushing's syndrome.     

Corticotropin-releasing hormone, proopiomelanocortin, and glucocorticoid receptor gene expression in adrenocorticotropin-producing tumors in vitro.

To differentiate between ectopic ACTH syndrome and Cushing's disease, gene expression of corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), and glucocorticoid receptor was examined in 10 pituitary adenomas (Cushing's disease) and in 10 ectopic ACTH-producing tumors. CRH increased plasma ACTH levels in all patients with Cushing's disease and in five patients with ectopic ACTH syndrome whose tumors contained CRH and CRH mRNA. In five CRH nonresponders, CRH was not detected in tumors that contained no CRH mRNA or that contained only long-size CRH mRNA. Dexamethasone (Dex) decreased plasma ACTH levels in all patients with Cushing's disease and in three patients with ectopic ACTH-producing bronchial carcinoid. These tumors contained glucocorticoid receptor mRNA. CRH increased and Dex decreased ACTH release and POMC mRNA levels in pituitary adenoma and bronchial carcinoid cells. PMA increased POMC mRNA levels only in carcinoid cells. These results reveal characteristics of ectopic ACTH-producing tumors: long-size CRH mRNA and PMA-induced POMC gene expression. In addition, there are two ectopic ACTH syndrome subtypes: tumors containing ACTH with CRH (CRH responder) and tumors without CRH. Dex decreases ACTH release and POMC mRNA levels in some bronchial carcinoids. Therefore, CRH and Dex tests have limited usefulness in differentiating between Cushing's disease and ectopic ACTH syndrome.     

Cushing's syndrome due to primary multinodular corticotrope hyperplasia

In this report, a case of Cushing's syndrome due to primary multinodular corticotrope hyperplasia is described. The patient had typical features of Cushing's syndrome and dynamic pituitary-adrenal testing, which suggested an ectopic adrenocorticotropic hormone (ACTH) syndrome. Results of petrosal sinus catheterization indicated that the pituitary gland was the source of excess ACTH. Total hypophysectomy resulted in complete remission of Cushing's syndrome. Light microscopic and immunohistochemical studies revealed multinodular corticotrope hyperplasia. Plasma corticotropin releasing hormone (CRH) was undetectable, and computed tomography of the chest and abdomen disclosed no neoplastic source of CRH. We speculate that either an abnormality in hypothalamic CRH secretion or corticotrope hypersensitivity to CRH might have been responsible for Cushing's syndrome in this patient.     

An unusual cause of Cushing's syndrome: primary pigmented nodular adrenal dysplasia

We report a case of Cushing's syndrome due to primary pigmented nodular adrenal dysplasia (PPNAD) and discuss the diagnostic process and management of this rare case. The diagnosis of PPNAD is discussed in the context of other causes of Cushing's syndrome. Eighty-five per cent of cases of Cushing's syndrome are due to a pituitary corticotrophic tumour (Cushing's disease). Rarer causes include cortisol secreting adrenal adenoma and ectopic ACTH secretion. In the routine investigation of Cushing's disease it is not unusual to find bilateral adrenal nodules on the CT scan. We present a case of Cushing's syndrome in which this radiographic finding was present and yet the biochemical diagnosis was one of ACTH independent disease. Histology revealed PPNAD.     

Medical treatment for Cushing's syndrome

Systemic cortisol plays an important role in the metabolism of glucose, lipids and proteins, as well as in the regulation of electrolyte balance. It is well known that the development of the microvascular disease of various organs such as the heart and kidney, in patients with diabetes mellitus, hyperlipidemia and hypertension of which disorders are frequently associated with Cushing's syndrome. Thus, we should treat Cushing's syndrome as soon as possible, since many complications, including cardiovascular diseases and infections, will soon occur when the definite diagnosis is delayed. Adrenalectomy is essential for treatment for Cushing's syndrome even in the patients with pituitary or ectopic ACTH-producing tumor. Some case can not be treated with surgical procedures because of worsened conditions with several complications of infection and diabetes. Then we choose medical treatment. Medical adrenalectomy is achieved by using with mitotane which is usually used for adrenocortical cancer. We commonly treat the patients with Cushing's syndrome due to adrenal tumor and pituitary or ectopic ACTH producing tumor by using metyrapone which mainly inhibits 11-hydroxylase. Metyrapone is also recommended to treat the patients who are not well differentiated Cushing's disease from ectopic ACTH syndrome. We rarely use trilostane which is an inhibitor against 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Replacement therapy with hydrocortisone should be considered if adrenal failure will occur during treatment with those drugs.     

Parallel assays of beta-endorphin and ACTH in Cushing's patients undergoing petrosal sinus sampling

This study explores the possibility of improving endocrinologic testing during petrosal sinus catheterization by determining both beta-endorphin and corticotropin (ACTH). We studied 14 patients with Cushing's disease, two with adrenal tumor, and three with ectopic tumors secreting ACTH. In patients with Cushing's disease, beta-endorphin concentrations paralleled those of ACTH in all basal plasma samples collected either from petrosal sinuses or peripheral veins. Individual responses of beta-endorphin and ACTH to corticotropin releasing hormone (CRH) were closely related to the presence of a corticotroph adenoma. In such patients, a consistently higher concentration of beta-endorphin over ACTH was observed in all samples collected either from petrosal sinuses or peripheral veins; the ratios were unchanged after the administration of CRH. In patients with ectopic ACTH secretion, the mean ratio of beta-endorphin over ACTH (with both values expressed in pmol/L) was significantly higher (3.5) than that of patients with Cushing's disease (2.9) or Cushing's syndrome due to adrenal tumor (2.7).     

An evaluation of the distinction of ectopic and pituitary ACTH dependent Cushing's syndrome by clinical features, biochemical tests and radiological findings

The efficiency of various laboratory and radiological investigations in the differentiation of ectopic from pituitary dependent Cushing's syndrome was studied, based on findings in 23 patients with verified Cushing's disease and seven patients with the ectopic ACTH syndrome. Clinical features strongly favouring the ectopic type were male sex and history for less than 18 months. Basal biochemical features strongly indicating the ectopic syndrome included plasma K+ less than 3.0 mmol/l and HCO3 greater than 30 mmol/l; serum cortisol at 9 a.m. or midnight of greater than 800 nmol/l; urine free cortisol greater than 1300 nmol/24 hours; plasma ACTH greater than 100 ng/l. In the high-dose dexamethasone suppression test, suppression by less than 50 per cent of 9 a.m. serum cortisol, urine free cortisol or 17-oxogenic steroids was usually indicative of an ectopic source of ACTH. A mean suppressed value of greater than 450 nmol/l for the 9 a.m. and midnight cortisol combined occurred in all of those with the ectopic syndrome, but in none of the 23 patients with Cushing's disease. For urine free cortisol, a mean suppressed value of less than 1000 nmol/24 hours was found in all patients with Cushing's disease, but in none of those in the ectopic group. In the metyrapone test, there was an increase of less than or equal to 3-fold in 11-deoxycortisol at 24 hours in patients with ectopic ACTH; the increase was greater than 3-fold in all but one of the patients with Cushing's disease. Failure to respond to either dexamethasone or metyrapone was found in only one of the patients with Cushing's disease (Patient 16); in the ectopic group, all patients except Patient D failed to respond to either test. It is concluded that patients presenting with clinically obvious Cushing's syndrome along with measurable plasma ACTH can be reliably divided by conventional tests into those that are driven from the pituitary and those driven by ectopic ACTH.     

Primary cortisol resistance in man. A glucocorticoid receptor-mediated disease.

We have studied a man suspected of having primary cortisol resistance on the basis of high 24-h mean plasma cortisol levels (27.4 micrograms/dl) and no stigmata of Cushing's syndrome. His son had slightly elevated 24-h mean plasma cortisol levels (9.9 micrograms/dl; normal 7.52 micrograms/dl). Both had high plasma protein unbound cortisol and increased urinary free cortisol. Plasma ACTH concentration was high, and both were resistant to adrenal suppression by dexamethasone. The father appeared to have mineralocorticoid excess resulting in hypertension, hypokalemia, and metabolic alkalosis. This was found to be due to markedly elevated plasma levels of deoxycorticosterone and corticosterone. The son, who was normotensive, had mildly increased plasma corticosterone and normal deoxycorticosterone levels. To study the apparent end-organ resistance to cortisol, we examined the glucocorticoid receptor in the white cells and fibroblasts of these patients. In both tissues, using both whole cell and cytosol assays, the glucocorticoid receptor was found to have reduced affinity for dexamethasone. In the cytoxol assays, a reduced receptor number was found as well. We conclude that cortisol resistance is a rare familial syndrome owing to an abnormal glucocorticoid receptor with a decreased affinity for cortisol.     

Diagnosis and pathophysiology of Cushing's syndrome

Cushing's syndrome is the consequence of a sustained overproduction of cortisol (hydrocortisone) by the adrenal cortex. This may be due to excessive secretion of cortisol by functioning adrenocortical tumors or to "nontumorous" adrenocortical hyperfunction. The latter may be a result of stimulation of the adrenal cortex by increased release of corticotropin (ACTH) from a small pituitary tumor or from nonpituitary nonadrenal tumor. Carcinoids or carcinomas of the lung or pancreas, and even pheochromocytomas have caused the syndrome of ectopic ACTH production. The problems involved in the diagnosis of Cushing's syndrome are establishing its presence and determining the underlying cause. Treatment is then dependent upon the underlying pathogenetic lesion.     

Salivary cortisol for the evaluation of Cushing's syndrome

Cortisol concentrations were measured in matched plasma and salivary samples from 8 healthy controls, 8 patients with Cushing's syndrome and 4 patients suspected of having spontaneous hypercortisolism. In healthy subjects, the circadian rhythm in salivary cortisol paralleled that in plasma. Absence of the diurnal rhythm in Cushing's syndrome was seen in saliva as well as in plasma. After ACTH stimulation, mean peak cortisol in saliva showed a 3-fold increase while in plasma there was a 2.5-fold increment above baseline. Cushing's syndrome, due to pituitary or adrenal adenoma was diagnosed equally well by measuring the cortisol response to cosyntropin in either plasma or saliva. Finally, the low- and high-dose dexamethasone suppression test was reflected equally well in both plasma and saliva. In patients suspected of having Cushing's syndrome dynamic tests can be performed in both plasma and saliva. However, in some samples, the salivary cortisol measurement appears advantageous over plasma cortisol determination.     

Immunoreactive corticotropin-releasing factor in human plasma.

Plasma immunoreactive corticotropin-releasing factor (I-CRF) levels were determined by using a human CRF radioimmunoassay and an immunoaffinity procedure. The basal plasma I-CRF level in normal subjects was 6 +/- 0.5 pg/ml (mean +/- SD). We found that most plasma I-CRF levels were affected by stress, negative feedback, and circadian rhythm. Basal I-CRF levels were high in patients with Addison's disease, Nelson's syndrome, hypopituitarism stemming from pituitary macroadenoma, and CRF- and adrenocorticotropic hormone-producing tumors. A very low, but significant, amount of I-CRF was detected (1-3 pg/ml) in patients with Cushing's syndrome, in corticosteroid-treated patients, and in a patient with hypothalamic hypopituitarism. These results suggest that a major component of plasma I-CRF is of hypothalamic origin, however, other extrahypothalamic tissues cannot be ruled out as a minor source of plasma I-CRF.     

Primary cortisol resistance associated with a thermolabile glucocorticoid receptor in a patient with fatigue as the only symptom.

We have studied a woman with an apparent receptor-mediated resistance to cortisol on the basis of elevated 24-h mean plasma cortisol levels and increased urinary free cortisol. Plasma ACTH concentrations were normal but she was resistant to adrenal suppression by dexamethasone. No stigmata of Cushing's syndrome were seen. To study the proposed end-organ resistance to cortisol, we examined the glucocorticoid receptor (GR) in lymphocytes and in fibroblasts from this patient and from her son. Several molecular properties of the GR of lymphocytes from the patient were indistinguishable from that of normal control subjects. In thermolability assays, however, the patient's GR as well as her son's GR showed a striking heat sensitivity at 40 degrees and 45 degrees C when compared with GR from normal persons. In addition, data from the thermolability assays correlated well with the lack at 45 degrees C of dexamethasone-induced decrease in in vitro [3H]thymidine incorporation into lymphocytes derived from both patients.     

Plasma Progesterone and 17-hydroxyprogesterone in normal men and children with congenital adrenal hyperplasia

Plasma 17-hydroxyprogesterone (17-OHP) concentrations in normal men averaged 0.094 mug/100 ml. Studies using suppressive doses of androgens and glucocorticoids showed that 90% of the 17-OHP originated from the Leydig cell. The 17-OHP production rate was 1.8 mg/24 hr. Plasma 17-OHP has a marked circadian variation, the 8 p.m. values being only 40% of the 8 a.m. values. Plasma luteinizing hormone measured in the same samples did not vary.The adrenal cortex has the capacity to synthesize and secrete 17-OHP and progesterone since adrenocorticotrophic hormone (ACTH) caused a fourfold increase in these plasma steroids. In children with congenital adrenal hyperplasia, plasma 17-OHP levels were 50-200 times those of normal men and plasma progesterone was increased 6- to 10-fold over normal men.     

Is whole-lung CT scanning still necessary in all cases of ACTH-dependent Cushing's syndrome in the era of petrosal sinus sampling?

We reviewed 31 patients in whom both bilateral inferior petrosal sinus sampling without CRH stimulation, and a CT scan of the lungs were done. Twenty-five had normal lung CT scans, of whom 23 had a higher inferior petrosal sinus: peripheral ACTH ratio > or = 1.5. After careful follow-up, none was subsequently shown to have ectopic ACTH syndrome. Six had abnormal lung CT scans, of whom two had ratios > or = 1.5. In these two patients, other investigations suggested pituitary disease, and pituitary surgery led to apparent cure. Of the remaining four patients, who had ratios < 1.5, two had incidental lung findings, and pituitary abnormalities were demonstrated at pituitary surgery. The third underwent bilateral adrenalectomy, and no evidence of ectopic ACTH syndrome has emerged as yet after 4 years follow-up. The fourth had a small-cell carcinoma of the lung, confirmed histologically. Our series suggests that whole-lung CT scanning is only necessary in cases of ACTH-dependent Cushing's syndrome where bilateral inferior petrosal sinus sampling has not demonstrated a significant increase in petrosal sinus ACTH levels as compared with the peripheral level. Thus, in our experience the test is now only necessary in those patients (approximately 25%) where the ratio is < or = 1.5.     

Nelson's syndrome: case report and review of the literature.

A patient who developed a pituitary tumor after adrenalectomy for Cushing's disease (Nelson's syndrome) is presented. The literature reviewed shows less than a 10 percent incidence of this disorder for which extremely elevated plasma ACTH levels are diagnostic. Special radiologic technics may be required to detect small pituitary tumors, but pituitary hormone levels may be elevated in the cerebrospinal fluid if there is suprasellar tumor extension. The different modalities available for treatment of Nelson's syndrome are discussed.     

Simultaneous Assay of Immunoreactive β-Lipotropin, γ-Lipotropin, and β-Endorphin in Plasma of Normal Human Subjects, Patients with ACTH/Lipotropin Hypersecretory Syndromes, and Patients undergoing Chronic Hemodialysis

We have studied the relative concentrations of the human immunoreactive (IR) peptides gamma-lipotropin (hgammaLPH, [1-58]hbetaLPH), beta-lipotropin (hbetaLPH), and beta-endorphin (hbetaEND, [61-91]hbetaLPH) using gel exclusion chromatography together with a specific radio-immunoassay (RIA) for hgammaLPH and a RIA that (because hbetaEND is the COOH-terminus of the hbetaLPH molecule) measures both hbetaEND and hbetaLPH on an equimolar basis. In normal subjects, basal plasma IR-hgammaLPH was often undetectable (<12.5 fmol/ml), but ranged up to 21 fmol/ml, and IR-hbetaEND/hbetaLPH was 10.8+/-0.7 fmol/ml; previous studies by others suggest that most of the IR-hbetaEND/hbetaLPH was probably hbetaLPH. Both IR-hgammaLPH and IR-hbetaEND/hbetaLPH were significantly elevated (P < 0.001) in patients undergoing chronic hemodialysis (101.5+/-12.7 and 23.8+/-2.0 fmol/ml, respectively). Their IR-hgammaLPH coeluted with standard hgammaLPH as a single peak, and IR-hbetaEND/hbetaLPH coeluted with hbetaLPH; no distinct peak of IR-hbetaEND was observed. In patients with ACTH/LPH hypersecretion due to Addison's disease, Nelson's syndrome, or ectopic ACTH syndrome, IR-hgammaLPH and IR-hbetaEND/hbetaLPH were both elevated, and IR-hbetaEND/hbetaLPH eluted as two peaks, one coeluting with hbetaLPH and the other with hbetaEND. The molar concentrations of all three peptides were significantly correlated with one another. The lower concentrations of endogenous IR-hbetaEND observed may be due in part to its apparent shorter plasma half-life, as estimated in an Addison's patient given a cortisol infusion. The biologic significance of these three peptides in circulating blood is still unknown. The increased levels of hbetaLPH and hgammaLPH in plasma of patients with chronic renal failure suggest that the kidney may be an important organ for their metabolism.     

Ectopic ACTH Production in Carcinoma of the Lung

Immunoreactive ACTH was found in almost all tissue extracts of lung carcinoma from patients without clinical evidence of Cushing's syndrome; i.e. 14 of 15 primary tumors, nine of nine metastatic lymph nodes, and four of four metastatic liver nodules contained immunoreactive ACTH. The incidence of ACTH in extracts of other tumor types was much lower. Comparable normal tissues contained no detectable ACTH. Immunoreactive growth hormone, parathyroid hormone, or gastrin was not found in the same carcinoma tissue. The predominant form of ACTH in the tumor extracts was big ACTH. In pituitary extracts little ACTH predominated.53% of 83 patients with lung carcinoma had afternoon plasma ACTH levels greater than 150 pg/ml; more than 90% of plasmas containing less than 150 pg/ml were obtained from patients who had received radiation therapy or chemotherapy. 31% of 45 patients with chronic obstructive pulmonary disease (COPD), 28% of 25 patients with other severe lung disease, and 6% of 33 controls had elevated values. Big ACTH predominated in the plasma of patients with lung carcinoma or COPD having elevated ACTH levels. Tissue from the lung of a smoking dog with atypical histologic changes contained immunoreactive ACTH, almost exclusively in the big form, while tissue from another smoking dog that was histologically normal contained no ACTH. Thus ACTH may be present even in precancerous lung lesions. These studies suggest that serial plasma ACTH levels may be of value in screening for, and/or management of, patients with carcinoma of the lung.     

RENIN, ANGIOTENSIN, CORTICOSTEROIDS, AND ELECTROLYTE BALANCE IN ADDISON'S DISEASE

Two patients with previously untreated Addison's disease werestudied. Both showed defective adrenal responsiveness to infusionsof corticotrophin and angiotensin, and both had abnormally lowplasma sodium and high plasma renin concentrations. Plasma potassiumwas elevated in one and normal in the other. Steroid replacement therapy was accompanied by an increase inNa_E, correction of plasma electrolyte concentrations, and areduction of plasma renin concentration to normal. K_E was virtuallyunchanged in the first weeks of treatment in either patient. In Case 1 treatment caused a movement of water and sodium fromICF to ECF, and probably of potassium from ECF to ICF. ECF volumeincreased more than TBW. Angiotensin infusion caused abdominal pain in this man, suggestingthat the elevated plasma renin may contribute to the characteristicabdominal pain of Addison's disease. In Case 2 treatment restored the normal nyothemeral rhythm ofurine flow, and caused a slight initial loss of TBW and weight.