Reference ranges of late-night salivary cortisol and cortisone measured by LC-MS/MS and accuracy for the diagnosis of Cushing's syndrome.

PURPOSE: International guidelines recommend salivary cortisol for the diagnosis of Cushing's syndrome. Despite mass spectrometry-based assays are considered the analytical gold-standard, there is still the need to define reference intervals and diagnostic accuracy of such methodology. METHODS: 100 healthy volunteers and 50 consecutive patients were enrolled to compare LC-MS/MS and electrochemiluminescence assay for the determination of late-night salivary cortisol and cortisone. Moreover, we aimed to determine reference intervals of salivary steroids in a population of healthy individuals and diagnostic accuracy in patients with suspected hypercortisolism and in a population including also healthy individuals. RESULTS: Method comparison highlighted a positive bias (51.8%) of immunoassay over LC-MS/MS. Reference intervals of salivary cortisol (0.17-0.97 µg/L), cortisone (0.84-4.85 µg/L) and ratio (0.08-0.30) were obtained. The most accurate thresholds of salivary cortisol for the diagnosis of hypercortisolism were 1.15 µg/L in the population with suspected hypercortisolism (AUC 1) and 1.30 µg/L in the population including also healthy individuals (AUC 1). Cut-off values of salivary cortisone (7.23 µg/L; Se 92.9%, Sp 97.2%, AUC 0.960 and Se 92.9%, Sp 99.1%, AUC 0.985 in suspected hypercortisolism and in overall population, respectively) and cortisol-to-cortisone ratio (0.20; Se 85.7%, Sp 80.6%, AUC 0.820 and Se 85.7%, Sp 85.5%, AUC 0.855 in suspected hypercortisolism and in overall population, respectively) were accurate and similar in both populations. CONCLUSION: LC-MS/MS is the most accurate analytical platform for measuring salivary steroids. Obtained reference intervals are coherent with previously published data and diagnostic accuracy for diagnosis of overt hypercortisolism proved highly satisfactory.

Pharmacological and analytical interference in hormone assays for diagnosis of adrenal incidentaloma.

Adrenal incidentaloma refers to an asymptomatic adrenal mass detected through an imaging procedure performed for reasons unrelated to adrenal dysfunction or suspected dysfunction. In general, adrenal incidentalomas are non-functioning adrenal adenomas, but in some cases, may require therapeutic intervention: eg., adrenocortical carcinoma, pheochromocytoma, primary aldosteronism, cortisol hypersecretion, or adrenal insufficiency. Hormone assessment is crucial to characterize adrenal incidentaloma. Nowadays, various hormone assay methods are available, such as immunoassay and mass spectrometry. However, there are several pitfalls that should be considered: e.g., circadian rhythm, gender/age dependency, preanalytical and analytical issues, and drug interactions. Pharmacological or analytical interference can lead to false serum concentrations and may result in misinterpretation of results and thus inappropriate treatment. The purpose of this review was to study the main interferences that may be observed in the different tumor types of adrenal incidentalomas in order to help physicians in their clinical decision-making and for the overall benefit of patients.

Plasma Free Cortisol in States of Normal and Altered Binding Globulins: Implications for Adrenal Insufficiency Diagnosis.

CONTEXT: Accurate diagnosis of adrenal insufficiency is critical because there are risks associated with overdiagnosis and underdiagnosis. Data using liquid chromatography tandem mass spectrometry (LC/MS/MS) free cortisol (FC) assays in states of high or low cortisol-binding globulin (CBG) levels, including cirrhosis, critical illness, and oral estrogen use, are needed. DESIGN: Cross-sectional. OBJECTIVE: Determine the relationship between CBG and albumin as well as total cortisol (TC) and FC in states of normal and abnormal CBG. Establish the FC level by LC/MS/MS that best predicts TC of <18 µg/dL (497 nmol/L) (standard adrenal insufficiency diagnostic cutoff) in healthy individuals. SUBJECTS: This study included a total of 338 subjects in four groups: healthy control (HC) subjects (n = 243), patients with cirrhosis (n = 38), intensive care unit patients (ICU) (n = 26), and oral contraceptive (OCP) users (n = 31). MAIN OUTCOME MEASURE(S): FC and TC by LC/MS/MS, albumin by spectrophotometry, and CBG by ELISA. RESULTS: TC correlated with FC in the ICU (R = 0.91), HC (R = 0.90), cirrhosis (R = 0.86), and OCP (R = 0.70) groups (all P < 0.0001). In receiver operator curve analysis in the HC group, FC of 0.9 µg/dL (24.8 nmol/L) predicted TC of <18 µg/dL (497 nmol/L; 98% sensitivity, 91% specificity; AUC, 0.98; P < 0.0001). Decreasing the cutoff to 0.7 µg/dL led to a small decrease in sensitivity (92%) with similar specificity (91%). CONCLUSIONS: A cutoff FC of <0.9 µg/dL (25 nmol/L) in this LC/MS/MS assay predicts TC of <18 µg/dL (497 nmol/L) with excellent sensitivity and specificity. This FC cutoff may be helpful in ruling out adrenal insufficiency in patients with binding globulin derangements.

Pitfalls in the interpretation of the cosyntropin stimulation test for the diagnosis of adrenal insufficiency.

PURPOSE OF REVIEW: Adrenal insufficiency is a rare disease characterized by cortisol deficiency. The evaluation of patients suspected of having adrenal insufficiency can be challenging because of the rarity of the disease and limitations in the biochemical assessment of the cortisol status by either basal or dynamic testing [adrenocorticotropic hormone (ACTH) stimulation test]. Prompt and adequate diagnosis is of paramount importance to avoid adverse outcomes. We aimed to summarize the recent developments in the conduction and interpretation of the ACTH stimulation test for the diagnosis of adrenal insufficiency. RECENT FINDINGS: The ACTH stimulation test is commonly performed in patients suspected of having adrenal insufficiency when the basal serum cortisol levels are inconclusive. Recent literature has evaluated the impact of technical aspects such as time of the day the test is performed, type of assay and sample source used for cortisol measurement on the clinical value of this test, as well as the feasibility of reliable low dose ACTH testing. SUMMARY: Clinicians evaluating patients with suspected adrenal insufficiency should take into consideration the clinical presentation (likelihood of adrenal insufficiency before testing) when interpreting the results of the ACTH stimulation test and be aware of clinical and technical factors that can affect cortisol values and diagnostic accuracy of this test.

Cortisol cut-points for the glucagon stimulation test in the evaluation of hypothalamic pituitary adrenal axis.

Diagnosis of adrenal insufficiency requires evaluation by dynamic stimulation tests. The insulin tolerance test (ITT) is accepted as the gold-standard test for the evaluation of hypothalamo-pituitary-adrenal (HPA) axis but the test is unpleasant and dangerous. Although it takes more time, glucagon stimulation test (GST) is a good alternative to ITT. The primary aim of this study was to compare the ITT and GSTs in the evaluation of HPA axe in patients with pituitary disorders. We conducted a prospective study in which ITT and GST were performed within 7 days in 81 patients. Serum cortisol was measured. We divided our population in Group 1 (G1): Adrenal Insufficiency (Peak cortisol under ITT <200 ng/mL) and Group 2 (G2): normal response (Peak cortisol under ITT >200 ng/mL). Receiver-operating characteristic (ROC) analysis was performed to identify the thresholds for GST. The mean peak of cortisol under GST was not significantly different from that obtained after ITT in the whole cohort (182.67 ± 89.07 ng/mL vs. 179.75 ± 79.01 ng/mL), and it was significantly reduced in patients of G1 (p < 10-3). ROC curve analysis showed that the best diagnostic accuracy was obtained with a peak cortisol cut-off to GST of 167 ng/mL (sensitivity, 89%; specificity, 79%). Using this cut-off, 86.4% of the patients were correctly classified. In our prospective series, GST is a potential accurate and safe alternative test for the assessment HPA. Test-specific cut-offs should be applied to avoid misinterpretation.

SFE/SFEDP adrenal insufficiency French consensus: Introduction and handbook.

The French endocrinology society (SFE) and the French pediatric endocrinology society (DFSDP) have drawn up recommendations for the management of primary and secondary adrenal insufficiency in the adult and child, based on an analysis of the literature by 19 experts in 6 work-groups. A diagnosis of adrenal insufficiency should be suspected in the presence of a number of non-specific symptoms except hyperpigmentation which is observed in primary adrenal insufficiency. Diagnosis rely on plasma cortisol and ACTH measurement at 8am and/or the cortisol increase after synacthen administration. When there is a persistant doubt of secondary adrenal insufficiency, insulin hypoglycemia test should be carried out in adults, adolescents and children older than 2 years. For determining the cause of primary adrenal insufficiency, measurement of anti-21-hydroxylase antibodies is the initial testing. An adrenal CT scan should be performed if auto-antibody tests are negative, then assay for very long chain fatty acids is recommended in young males. In children, a genetic anomaly is generally found, most often congenital adrenal hyperplasia. In the case of isolated corticotropin (ACTH) insufficiency, it is recommended to first eliminate corticosteroid-induced adrenal insufficiency, then perform an hypothalamic-pituitary MRI. Acute adrenal insufficiency is a serious condition, a gastrointestinal infection being the most frequently reported initiating factor. After blood sampling for cortisol and ACTH assay, treatment should be commenced by parenteral hydrocortisone hemisuccinate together with the correction of hypoglycemia and hypovolemia. Prevention of acute adrenal crisis requires an education of the patient and/or parent in the case of pediatric patients and the development of educational programs. Treatment of adrenal insufficiency is based on the use of hydrocortisone given at the lowest possible dose, administered several times per day. Mineralocorticoid replacement is often necessary for primary adrenal insufficiency but not for corticotroph deficiency. Androgen replacement by DHEA may be offered in certain conditions. Monitoring is based on the detection of signs of under- and over-dosage and on the diagnosis of associated auto-immune disorders.

Can Screening and Confirmatory Testing in the Management of Patients with Primary Aldosteronism be Improved?

Widespread application of the plasma aldosterone/renin ratio (ARR) as a screening test has led to the recognition that primary aldosteronism (PA) is the most common specifically treatable and potentially curable form of hypertension, accounting for 5-10% of patients. Maximal detection requires accurate diagnostic approaches and awareness and control of factors that confound results, including most antihypertensives, posture, time of day, dietary salt, and plasma potassium. Recent studies have revealed potential for false positives in patients on beta-adrenoceptor blockers, and, when direct renin concentration (but not plasma renin activity) is used to measure renin, in women during the luteal phase of the menstrual cycle or receiving estrogen-containing contraceptives or hormonal replacement therapy. In addition to verapamil slow release, hydralazine and prazosin, moxonidine has minimal effects on the ARR and can be used to control hypertension during work-up. Fludrocortisone suppression testing, while probably the most reliable means of definitively confirming or excluding PA, is time consuming and expensive, requiring a five day inpatient stay. A novel approach, upright (seated) saline infusion suppression testing (SST), has shown excellent reliability with much greater sensitivity than conventional recumbent SST in a recent pilot study, and requires only a day visit. Accurate measurement of aldosterone is essential for each step of PA workup: introduction of new, highly reliable high-throughput mass spectrometric methods into clinical practice has represented a major advance. In response to concerns raised about accuracy of renin assays, new mass spectrometric methods for measuring angiotensin II are currently being assessed in the clinical setting.

Is Primary Aldosteronism Still Largely Unrecognized?

Primary aldosteronism (PA) was first reported by Jerome W. Conn in 1954 when it was considered a rare disorder, only suspected in cases of hypertension and spontaneous hypokalemia. Over the last 30 years, with the wide application of the plasma aldosterone to plasma renin activity ratio as screening test, the clinical spectrum of PA has dramatically changed. Different studies displayed significant differences in term of patients investigated, diagnostic criteria and hormonal assays; however, large prospective studies with robust diagnostic criteria indicated that the prevalence of PA is around 6% of the general hypertensive population and 11% of the patients referred to hypertension centers. In light of these epidemiological studies, the Endocrine Society Guideline recommends the screening for PA of around 50% of patients with hypertension, and identifies the categories of patients at high risk for the disease. However, clinical data obtained from "real-life" show that the screening rate is much lower and PA remains an under-diagnosed and under-treated cause of secondary hypertension with an associated increased risk of cardio- and cerebrovascular mortality and morbidity.

Primary Aldosteronism: The Next Five Years.

The management of primary aldosteronism is widely varied within various published guidelines, with very little in the way of data supporting the choice of one variation over others. Current estimates of prevalence are probably accurate for aldosterone producing adenoma, but fall very short of that for bilateral adrenal hyperplasia. Discovery at the level of basic science has proven illuminating over the past 6 years in terms of unilateral disease and both somatic and germline mutations, with much less focus on the much more common bilateral disease; Attempts at harmonization have begun - for example, criteria for complete/partial/absent cure after adrenalectomy for unilateral disease; again focus on bilateral disease is muted. A number of possibilities are suggested as agenda for active consideration and change, across a wide range of areas - referral patterns, screening, confirmation and lateralization, what will be needed is discussion and agreement, to fill the lacunae within the current guidelines. Those involved will want to change to make such an agenda possible.

Calibration and evaluation of routine methods by serum certified reference material for aldosterone measurement in blood.

We attempted to study the standardization of aldosterone measurement in blood. The serum certified reference material (serum CRM) was established by spiking healthy human serum with pure aldosterone. ID-LC/MS/MS as a reference measurement procedure was performed by using the serum CRM. LC-MS/MS as a comparison method (CM) was routinely used for clinical samples, and the values with and without calibration by the serum CRM were compared. The serum CRM demonstrated similar reactivity with peripheral blood plasma as clinical samples in routine methods (RM) of RIA, ELISA, and CLEIA. In comparison between RM and CM, the results in regression analysis indicated that the range of the correlation coefficient (r) was 0.913 - 0.991, the range of y intercept was 0.9 - 67.3 pg/mL and the range of slope was 0.869 - 1.174. The values by RM in 100 - 150 pg/mL for the diagnostic level, had a significant calibration effect, and the relative difference between calibrated value in RM and result by CM was within ±20%. Furthermore, the calibrated value using the serum CRM was 10,187 pg/mL, which corresponds to measured value of 14,000 pg/mL using RIA for the adrenal venous sampling. Measured values between plasma and serum as a sample for the aldosterone measurement from clinical samples showed no significant differences. In conclusion, we succeeded to prepare the certified reference material of aldosterone for RM. Then, we can accurately calculate corrected values by using our equation for four RMs of determination of aldosterone.

Diagnosis and treatment of adrenal insufficiency including adrenal crisis: a Japan Endocrine Society clinical practice guideline [Opinion].

This clinical practice guideline of the diagnosis and treatment of adrenal insufficiency (AI) including adrenal crisis was produced on behalf of the Japan Endocrine Society. This evidence-based guideline was developed by a committee including all authors, and was reviewed by a subcommittee of the Japan Endocrine Society. The Japanese version has already been published, and the essential points have been summarized in this English language version. We recommend diagnostic tests, including measurement of basal cortisol and ACTH levels in combination with a rapid ACTH (250 µg corticotropin) test, the CRH test, and for particular situations the insulin tolerance test. Cut-off values in basal and peak cortisol levels after the rapid ACTH or CRH tests are proposed based on the assumption that a peak cortisol level ≥18 µg/dL in the insulin tolerance test indicates normal adrenal function. In adult AI patients, 15-25 mg hydrocortisone (HC) in 2-3 daily doses, depending on adrenal reserve and body weight, is a basic replacement regime for AI. In special situations such as sickness, operations, pregnancy and drug interactions, cautious HC dosing or the correct choice of glucocorticoids is necessary. From long-term treatment, optimal diurnal rhythm and concentration of serum cortisol are important for the prevention of cardiovascular disease and osteoporosis. In maintenance therapy during the growth period of patients with 21-hydroxylase deficiency, proper doses of HC should be used, and long-acting glucocorticoids should not be used. Education and carrying an emergency card are essential for the prevention and rapid treatment of adrenal crisis.

Pros and cons of dexamethasone suppression test for screening of subclinical Cushing's syndrome in patients with adrenal incidentalomas.

The results of dexamethasone suppression tests (DST) in the screening of subclinical hypercortisolism are not readily comparable. Aim of the present study was to review the effectiveness of overnight 1-mg DST and 8-mg DST to look for functional autonomy of clinically inapparent adrenal adenomas. Sixty-eight consecutive patients with clinically inapparent adrenal adenomas were enrolled. All patients underwent 1-mg DST. The 8-mg DST was performed in the 11 patients who had post 1-mg DST cortisol >138 nmol/l and in 11 patients who had post 1-mg DST cortisol between 50 and 138 nmol/l. The a priori probability to have autonomous cortisol secretion was defined by the presence of at least two alterations of the hypothalamic-pituitary-adrenal axis among reduced ACTH concentrations, elevated urinary free cortisol (UFC) or elevated midnight serum cortisol. Cortisol levels >138 nmol/l after the 1-mg DST increases the post-test probability of adrenal functional autonomy to 55%, whereas cortisol levels <50 nmol/l reduce the post-test probability to 8%. Cortisol levels recorded after the 8-mg DST were nonsignificantly lower than after the 1-mg DST and all the patients with cortisol >138 nmol/l after the 1-mg DST maintained cortisol above this cut-point. The 1-mg DST should be considered as the more effective test to detect autonomous cortisol secretion by a clinically inapparent adrenal adenoma when cortisol levels are >138 nmol/l, while cortisol levels <50 nmol/l reduce remarkably the post-test probability of this event. The 8-mg DST seems to replicate by large the results of the 1-mg DST.

Salivary cortisol for assessment of hypothalamic-pituitary-adrenal axis function.

OBJECTIVE: Cortisol is an important hormone/steroid in the regulation of intermediate metabolism and stress. It exists in free (unbound) and protein-bound forms in serum but only in a free form in saliva. The free form is the biologically active one. There is an advanced biofeedback system regulating the cortisol secretion/concentration by the hypothalamic-pituitary-adrenal axis. METHODS: There are many different methods to collect saliva, either directly from the mouth as an expectorate or indirectly by using swabs, capillary tubes or paper foams. The swab may be cotton-based, synthetic, with or without a plastic sheath. There are different methods for analysis - immunological, radioisotopic, mass fragmentographic and chromatographic. RESULTS: The diurnal variation in salivary cortisol concentrations in healthy school-aged boys and girls are described and show dependence to different degrees of age and sex. CONCLUSION: Sampling for salivary cortisol determination is a sensitive, specific and pain-free procedure and it is therefore very useful for following healthy and sick children before, during and after pharmacological interventions. Different conditions as time for sampling, interaction with other endogenous and exogenous steroids, and physical and psychological activity interact with the salivary cortisol concentration.

Evaluation of the hypothalamic-pituitary-adrenal axis function in childhood and adolescence.

The hypothalamic-pituitary-adrenal (HPA) axis plays an important role in the maintenance of basal and stress-related homeostasis. The hypothalamus controls the secretion of adrenocorticotropic hormone (ACTH) from the anterior pituitary, which in turn stimulates the secretion of glucocorticoids from the adrenal cortex. Glucocorticoids, the final effectors of the HPA axis, regulate a broad spectrum of physiologic functions essential for life and exert their effects through their ubiquitously distributed intracellular receptors. Alterations in the activity of the HPA axis may present with symptoms and signs of glucocorticoid deficiency or excess. Detailed endocrinologic evaluation is of primary importance in determining the diagnosis and/or etiology of the underlying condition. We review the most common endocrinologic investigations used in the evaluation of the HPA axis integrity and function.

Padronizaçäo e aplicaçäo dos testes rápido e prolongado de estímulo com ACTH em pacientes com insuficiência adrenocortical primária e secundária / Standardization and clinical applications of the rapid and prolonged ACTH stimulation tests in patients with primary and secondary adrenal insufficiency

Apesar de utilizados há muito tempo na avaliaçäo funcional do córtex adrenal, os testes de estímulo rápido e prolongado com ACTH apresentaram aspectos ainda näo devidamente padronizados como: via de administraçäo do produto, duraçäo e horário de realizaçäo, critérios de interpretaçäo dos testes e aplicabilidade clínica. Assim, procuramos uniformizar o procedimento e os critérios de interpretaçäo de ambos os estímulos, além de estabelecer os padröes de resposta obtidos em portadores de insuficiência adrenocortical primária e secundária. Para isto, utilizamos 16 indivíduos controles normais nos quais os testes rápido e prolongado foram realizados empregando-se, respectivamente, as vias intravenosa (250 mcg erm bolo) e intramuscular (20 UI de uma preparaçäo de depósito, a cada 8 horas); o primeiro teve duraçäo de 60 minutos e o segundo de três dias. Dentre os vários critérios analisados para a interpretaçäo dos testes, optamos pelo valor isolado de cortisol após ACTH. Os limites críticos inferiores deste parâmetro (média menos 1,64 desvios-padräo, com 95% de confiança foram de 19,1 e 49,0 mcg/dl, respectivamente, para os testes rápido e prolongado. Uma vez padronizados, ambos os testes foram aplicados em 16 pacientes comprovadamente addisonianos, 14 com hipopituitarismo e 13 em uso crônico de corticosteróides. Em resposta ao teste rápido, os pacientes com doença de Addison näo alteraram os níveis de cortisol (1,1 ñ 1,2 para 1,2 ñ 1,1 mcg/dl), enquanto nos pacientes com hipopituitarismo e naqueles sob uso crônico de corticosteróides houve uma elevaçäo discreta, mas significante (1;6 ñ 5,6 ñ 1,0 mcg/dl). Durante o teste prolongado, 15 dos 16 pacientes addisonianos näo alteraram seus níveis circulantes de cortisol (1,7 ñ 1,3 mcg/dl), enquanto 11 dos 14 pacientes com hipopituitarismo (20,3 ñ 9,5 mcg/dl) e 11 dos 13 sob uso crônico de corticosteróides (19,8 ñ 8,8 mcg/dl) apresentaram uma elevaçäo signficante, logo no primeiro dia, que se acentuou com a manutençäo do estímulo...

[Standardization and clinical applications of the rapid and prolonged ACTH stimulation tests in patients with primary and secondary adrenal insufficiency]. / Padronização e aplicação dos testes rápido e prolongado de estímulo com ACTH em pacientes com insuficiência adrenocortical primária e secundária.

Some methodological aspects of the rapid ACTH stimulation test (RST) and the prolonged ACTH stimulation test (PST) remain heterogeneous, e.g. administration routes, time of the day, duration, interpretation criteria and clinical indications. To standardize both tests we studied the serum cortisol responses to ACTH in 16 normal subjects, 16 patients with Addison's disease (AD), 14 with hypopituitarism (HP) of different etiologies and 13 patients on chronic glucocorticoid (CG) treatment (doses equivalent to 5-20 mg/d of prednisone for at least 6 months). For the RST, 250mcg of Cortrosina Organon were injected as an IV bolus and blood collected before and 60 minutes later, whereas for the PST, 250mcg of Cortrosina-Depot Organon were injected IM, every 8 hours for 3 days, and blood drawn daily between 8 and 9 o'clock AM. The post-ACTH cortisol value was the single parameter chosen to interpret both tests (the absolute or percent increase was of no further value). The 95% lower confidence limits (mean minus 1.64 SD) for the RST and the 3rd day of PST were 19.1 and 49.0mcg/dL, respectively. AD patients did not increase cortisol in response to both the RST (1.1 +/- 1.2 to 1.2 +/- 1.1mcg/dL) or the PST (1.2 +/- 1.3 to 2.0 +/- 1.7mcg/dL). However, whereas 22 out of 27 patients with HP and CG showed a modest cortisol increase to the RST (1.6 +/- 2.3 to 5.6 +/- 4.7 mcg/dL), all had a gradual and nearly normal response on the 3rd day of the PST (1.6 +/- 2.1 to 29.6 +/- 16.6mcg/dL).(ABSTRACT TRUNCATED AT 250 WORDS)