Improved Urinary Cortisol Metabolome in Addison Disease: A Prospective Trial of Dual-Release Hydrocortisone.

CONTEXT: Oral once-daily dual-release hydrocortisone (DR-HC) replacement therapy has demonstrated an improved metabolic profile compared to conventional 3-times-daily (TID-HC) therapy among patients with primary adrenal insufficiency. This effect might be related to a more physiological cortisol profile, but also to a modified pattern of cortisol metabolism. OBJECTIVE: This work aimed to study cortisol metabolism during DR-HC and TID-HC. DESIGN: A randomized, 12-week, crossover study was conducted. INTERVENTION AND PARTICIPANTS: DC-HC and same daily dose of TID-HC were administered to patients with primary adrenal insufficiency (n = 50) vs healthy individuals (n = 124) as controls. MAIN OUTCOME MEASURES: Urinary corticosteroid metabolites were measured by gas chromatography/mass spectrometry at 24-hour urinary collections. RESULTS: Total cortisol metabolites decreased during DR-HC compared to TID-HC (P < .001) and reached control values (P = .089). During DR-HC, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity measured by tetrahydrocortisol + 5α-tetrahydrocortisol/tetrahydrocortisone ratio was reduced compared to TID-HC (P < .05), but remained increased vs controls (P < .001). 11ß-HSD2 activity measured by urinary free cortisone/free cortisol ratio was decreased with TID-HC vs controls (P < .01) but normalized with DR-HC (P = .358). 5α- and 5ß-reduced metabolites were decreased with DR-HC compared to TID-HC. Tetrahydrocortisol/5α-tetrahydrocortisol ratio was increased during both treatments, suggesting increased 5ß-reductase activity. CONCLUSIONS: The urinary cortisol metabolome shows striking abnormalities in patients receiving conventional TID-HC replacement therapy, with increased 11ß-HSD1 activity that may account for the unfavorable metabolic phenotype in primary adrenal insufficiency. Its change toward normalization with DR-HC may mediate beneficial metabolic effects. The urinary cortisol metabolome may serve as a tool to assess optimal cortisol replacement therapy.

Measuring cortisol in serum, urine and saliva - are our assays good enough?

Cortisol is a steroid hormone produced in response to stress. It is essential for maintaining health and wellbeing and leads to significant morbidity when deficient or present in excess. It is lipophilic and is transported bound to cortisol-binding globulin (CBG) and albumin; a small fraction (∼10%) of total serum cortisol is unbound and biologically active. Serum cortisol assays measure total cortisol and their results can be misleading in patients with altered serum protein concentrations. Automated immunoassays are used to measure cortisol but lack specificity and show significant inter-assay differences. Liquid chromatography - tandem mass spectrometry (LC-MS/MS) offers improved specificity and sensitivity; however, cortisol cut-offs used in the short Synacthen and Dexamethasone suppression tests are yet to be validated for these assays. Urine free cortisol is used to screen for Cushing's syndrome. Unbound cortisol is excreted unchanged in the urine and 24-h urine free cortisol correlates well with mean serum-free cortisol in conditions of cortisol excess. Urine free cortisol is measured predominantly by immunoassay or LC-MS/MS. Salivary cortisol also reflects changes in unbound serum cortisol and offers a reliable alternative to measuring free cortisol in serum. LC-MS/MS is the method of choice for measuring salivary cortisol; however, its use is limited by the lack of a single, validated reference range and poorly standardized assays. This review examines the methods available for measuring cortisol in serum, urine and saliva, explores cortisol in disease and considers the difficulties of measuring cortisol in acutely unwell patients and in neonates.

Determination of cortisol in serum, saliva and urine.

Cortisol is quantitatively the major glucocorticoid product of the adrenal cortex. The main reason to measure cortisol is to diagnose human diseases characterised by deficiency of adrenal steroid excretion in Addison's disease or overproduction in Cushing's syndrome (CS). In both cases a sensitive, accurate and reproducible assay of cortisol is required. Several methods have been described for the quantitative measurement of cortisol in both serum and urine. The most widely used methods in routine clinical laboratories are immunoassays (IA) and enzyme immunoassays (EIA), luminescence and fluorescence assays, which are available in numerous commercial kits and on automated platforms. However, there remains a number of problems in the so-called direct immunoassays if extraction and prepurification are not carried out before the assay. Recently, more specific chromatographic methods have been introduced, such as high pressure liquid chromatographic (HPLC) or liquid chromatography tandem mass spectrometric assays (LC-MS/MS). The high specificity especially of LC-MS/MS facilitates reliable measurement of cortisol both in plasma, urine and saliva samples.

Evaluation of two replacement regimens in primary adrenal insufficiency patients. effect on clinical symptoms, health-related quality of life and biochemical parameters.

OBJECTIVE: To evaluate clinical symptoms, health-related quality of life (HRQL) and biochemical parameters in patients with primary adrenal insufficiency under treatment with two different hydrocortisone regimens (20 mg-0 mg-10 mg/day and 10 mg-5 mg-5 mg/day), each maintained for 3 months and compare results obtained with those in healthy controls. DESIGN, PATIENTS AND METHODS: Twelve patients with primary adrenal insufficiency were studied. Clinical symptoms and HRQL with the Nottingham Health Profile (NHP) were evaluated and Na, K and serum cortisol determined at 09:00 h, 12:30 h and 17:30 h and urinary free cortisol (UFC) throughout the day. Control group comprised 19 healthy subjects. RESULTS: No differences in specific adrenal insufficiency symptoms were detected between the two regimens. HRQL was worse in energy dimension assessed by the NHP compared to the general population, regardless of 20 mg-0 mg-10 mg/day or 10 mg-5 mg-5 mg/day treatment (p=0.03 and p=0.013). The total NHP score was only adversely affected when patients were on the 10 mg-5 mg-5 mg/day hydrocortisone replacement regimen (p=0.008). Serum cortisol concentrations were higher than controls at 09:00 h, and lower at 17:30 h with both regimens, whereas serum cortisol at 12:30 h and UFC were within the 5th-95th percentile normal range only with the 10 mg-5 mg-5 mg/day regimen. CONCLUSIONS: Patients with primary adrenal insufficiency had worse HRQL in the NHP energy dimension compared with the general population, regardless of the hydrocortisone regimen although total score for HRQL was worse only with the 10 mg-5 mg-5 mg/day regimen. Patients on the thrice-daily hydrocortisone regimen showed a more physiological cortisol profile, leading us to recommend initially treating patients with this dose and increasing it in the case of impaired HRQL.

[Evaluation of corticosteroid replacement therapy and its effect on bones in Addison's disease]. / Evaluation de la substitution par corticostéroïdes et de ses répercussions osseuses dans la maladie d'Addison.

Assessment of patients on steroid replacement therapy is important to avoid the consequences of overtreatment such as osteoporosis. The aim of this prospective study is to evaluate the severity and the etiology of osteopenia in 24 patients (15 women, 9 men) with Addison's disease receiving 30 mg hydrocortisone. Mean age of patients was 55 15 years. Osteoporosis, diagnosed by the measurement of bone mineral density (BMD) at the level of lumbar spine and right hip, was found in 58% of patients, i.e. in 10 women and 4 men. The latter had normal testosterone levels while seven women had an early menopause, the etiology of their Addison's disease being autoimmune. Three were on hormonotherapy. Correlations were found between BMD in the femoral neck and hip and the dose of hydrocortisone (mg/m(2)/day; mg/kg/day), the duration of treatment and 24 hr-cortisoluria/g creatinine. Multivariate analysis shows that 24-hr cortisoluria/g creatinine is a good predictor of BMD values. Thus, osteoporosis is frequent in Addison's disease and cortisoluria could be a useful tool to predict this complication.

Glucocorticoid replacement therapy: are patients over treated and does it matter?

BACKGROUND AND OBJECTIVES: Adequate assessment of patients on glucocorticoid replacement therapy is of great importance to avoid the consequences of under or over treatment, but no simple test is available for this. The aims of this study were (1) to assess adequacy of glucocorticoid replacement in hypoadrenal patients, (2) to correlate serum cortisol levels (cortisol day curve) with 24-hour urine free cortisol excretion and (3) to assess the impact of glucocorticoid dose optimization on markers of bone formation and bone resorption. DESIGN: Cross-sectional study of current replacement therapy and a prospective study of the effect of dose alteration on bone turnover markers. PATIENTS: Thirty-two consecutive patients on replacement glucocorticoid therapy (12 Addison's disease, 20 hypopituitarism) from a University teaching hospital out-patient department. MEASUREMENTS: Serum and urinary cortisol, osteocalcin, N-telopeptide of type I collagen (NTX) and bone mineral density. RESULTS: 28/32 (88%) patients required a change of therapy; 24/32 (75%) a total reduction in dose, 18/32 (56%) a change in replacement therapy regimen or drug and 14/32 (44%) both changes. The mean daily dose of hydrocortisone was reduced from 29.5 +/- 1.2 to 20.8 +/- 1.0 mg. A significant correlation was found between peak cortisol and 24-hour urine free cortisol/ creatinine (Spearman correlation r = 0.60, P < 0.0001; n = 51). Following hydrocortisone dose reduction, median osteocalcin increased from 16.7 micrograms/l (range 8.2-65.7) to 19.9 micrograms/l (8.2-56.3); P < 0.01, with no change in the NTX/creatinine ratio. CONCLUSIONS: A high proportion of patients on conventional corticosteroid replacement therapy are over treated or on inappropriate replacement regimens. To reduce the long term risk of osteoporosis, corticosteroid replacement therapy should be individually assessed and over replacement avoided.

Role of glucocorticoid in excretion of an acute potassium load in patients with Addison's disease and panhypopituitarism.

Glucocorticoid (GC) has been shown to stimulate potassium (K) excretion in various conditions, but it is still incompletely resolved whether its presence is essential for the normal K homeostasis. We addressed this question in patients with selective GC deficiency (panhypopituitarism) and with combined GC and mineralocorticoid deficiency (Addison's disease), studied 24 hours after withdrawal of their regular substitution therapy. Compared to data in healthy subjects, both basal K excretion and the kaliuresis after a KCl load (1 mmol/kg body wt orally) were impaired in either patient group (P < 0.05). Physiological cortisol supplementation (20 mg 3 hr prior to test, and 1 mg/hr during test) increased basal K excretion (from 10.6 +/- 1.8 to 19.2 +/- 1.9 mmol/5 hr, P < 0.01) and KCl stimulated kaliuresis (from 47.9 +/- 6.1 to 54.8 +/- 4.7 mmol/5 hr, P = 0.06) to normal levels in panhypopituitarism. Cortisol also improved basal K excretion (from 10.2 +/- 1.5 to 16.9 +/- 3.5 mmol/5 hr, P < 0.05) and KCl-stimulated K excretion (from 31.6 +/- 2.5 to 45.2 +/- 3.8 mmol/5 hr, P < 0.05) in Addison's disease, although KCl-stimulated K excretion remained below normal (P < 0.01). The effects of cortisol on sodium excretion differed between the two patient groups (P < 0.05) in that only in Addison's disease the improved K excretion was associated with sodium retention. Additional experiments with the purely GC compound dexamethasone (0.5 mg 3 hr prior to test, and 0.03 mg/hr during test) in the patients with Addison's disease also improved K excretion (P < 0.05), but without the concomitant sodium retention observed after cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of daily urinary excretion of met-enkephalin-like immunoreactivity and epinephrine in men.

To investigate the source of urinary Met-enkephalin-like immunoreactivity (MELI), 24-h urinary excretion of MELI and catecholamines (CAs) were examined in normal subjects and patients with tuberculous Addison's disease. MELI was present in urine and 24-h urinary excretion of MELI averaged 813.8 +/- 446.9 ng/day in normal subjects (N = 33, Mean +/- SD). 24-h urinary excretion of MELI in normal subjects significantly showed positive correlation with 24-h urinary epinephrine (E) (R = 0.392, P less than 0.05) but no correlation with that of norepinephrine (NE) or dopamine (DA). In two patients with tuberculous Addison's disease, 24-h urinary excretion of MELI and that of E were significantly lower than those of normal subjects. These results indicate that the main source of urinary MELI may be adrenal medulla.

[Acid labile aldosteronuria. Comparison of radio-immunoassay and colorimetry (author's transl)]. / L'adostéronurie acido-labile. Comparaison des méthodes radio-immunologique et colorimétrique.

The daily excretion of aldosteronuria was studied in 87 normal or pathological subjects by 3 methods : one, classical, by thin layer chromatography (TLC), based on a modification of Neher's technic, the others, by radio-immunoassay with or without prior extraction of urinary aldosterone (RIA). The normal subjects receiving a daily sodium intake of 150 nmol excrete 24,9 +/- 3,4 nmol/24 hours of aldosterone (TLC) and 23,0 +/- 4,2, 26,3 +/- 3,7 nmol/24 hours (RIA) respectively. These values are not statistically different, RIA of aldosteronuria appears to be a reliable and simple technic and permits one to carry out a large number of examinations within a short period of time with only one technician.

Free urinary cortisol radioimmunoassay (application in the diagnosis of adrenal diseases).

The availability of a specific antiserum has made it possible to develop in our laboratory a radioimmunoassay for free urinary cortisol which is quite simple and rapid to perform. No preliminary procedures of chromatographic purification are necessary, and no correction for losses is required. Precision and accuracy tests are satisfactory. Seventeen normal subjects, 10 obese subjects, 18 patients with Cushing's syndrome, 4 hypopituitaric patients and 4 with Addison's disease were studied. The values of free urinary cortisol were compared with those of urinary 17-OHCS, plasma cortisol and, in some cases, the cortisol secretion rate. In normal subjects, the mean free urinary cortisol was 77.22 +/- 7.74 microgram/24 h, in obese subjects it was 112.05 +/- 13.64 microgram/24 h, and in patients with Cushing's syndrome it was at significantly higher levels with a mean value of 488.06 +/- 64.39 microgram/24 h. There was no overlap between the values obtained in the first two groups and those of subjects with adrenal hyperfunction, and the results were similar to those shown by the cortisol secretion rate. The same was not true for urinary 17-OHCS and plasma cortisol. Thus, the assay of free urinary cortisol, that is the free biologically active circulating fraction, may provide a relatively simple alternative to measurement of the cortisol secretion rate, as a single differential test for the diagnosis of conditions with adrenal hyperfunction. Less satisfactory were the results obtained in patients with primary and secondary adrenal hypofunction. Another limitation of the method is its relative non-specificity, since the results of 16 urinary samples measured without preliminary chromatography were 29.6% higher than those after previous chromatographic purification. However, in our experience, this overestimation does not affect the value of the method as a screening test for the diagnosis of Cushing's syndrome.

Renal hemodynamics and water excretion in Addison's disease.

Patients with Addison's disease have impaired ability to excrete free water. The mechanism of this defect is still not clear. These experiments were designed to help clarify the role of altered renal hemodynamics in the genesis of the defect. Six patients with Addison's disease were studied. Enough salt and water was administered to maintain adequate hydration throughout the study but no hormonal replacement was instituted. After a water load, the urine output (V), free water clearance (CH2O), sodium excretion (UNaV), total solute clearance (COsm), inulin clearance (CIn), and para-aminohippurate clearance (CPAH) were measured by standard clearance techniques. In the control studies V and CH2O were abnormally low. The studies were repeated after administration of DOCA, 2.5 mg daily, for a week. This medication decreased UNaV and improved the plasma electrolyte profile but did not cause a significant change in V, CH2O, GFR, or RPF. The same studies were repeated once more after administration of prednisone, 5 mg daily in divided doses, for a week. This drug induced a marked water diuresis while GFR and RPF remained unchanged. It is concluded that hypovolemia and altered renal hemodynamics are not the only mechanisms of the impaired water excretion in Addison's disease. A severe defect remains after normalization of hypovolemia and this is corrected by prednisone but not by DOCA, through mechanisms which do not involve changes in GFR or RPF.

A direct radioimmunoassay for free aldosterone in urine without extraction and chromatography.

A direct radioimmunoassay for the determination of free (unconjugated) aldosterone in urine has been developed for the clinical laboratory. The method is based on a determination without extraction and chromatography. In comparison with a reference method including extraction and column chromatography on Sephadex LH 20 a highly significant correlation was found. The coefficient of variation was 5.6% for intraassay variability and 10.4% for interassay determinations. In normal adults the average value of the urinary free aldosterone was 190 ng/24 h (range: 60--320 ng/24 h). During sodium restriction, a 3 fold increase in free aldosterone was observed in normal subjects. In patients with adrenal insufficiency, the values were below 60 ng/24 h, and in two patients with Conn's syndrome, the excretion of free aldosterone was 890 and 1040 ng/24 h. The method was found suitable for routine clinical use.

Application of adrenocorticotropin assays in a routine clinical laboratory.

The radioimmunoassay of ACTH was used in a routine laboratory to localize the site of the lesion in 20 patients with Cushing's syndrome. Eight of the patients had no detectable circulating ACTH and had adrenal tumors removed, 12 had high levels and were diagnosed as having pituitary Cushing's syndrome. Very high levels of plasma ACTH were found in eight patients who had primary adrenal insufficiency, while ACTH was undetectable in ten patients with secondary hypoadrenalism. The routine use of this assay in endocrinology should reduce the hospitalization of patients under investigation for disorders of the pituitary--adrenal axis. Eight patients who had the ectopic ACTH syndrome and carcinoma of the lung were found to have very high levels of ACTH with no diurnal variation. Forty-seven patients with oat-cell carcinoma but without evidence of the ectopic ACTH syndrome had normal ACTH levels. A possible role of ACTH and other peptide hormones as tumor markers is mentioned.

Plasma corticotrophin levels in addison-schilder's disease.

Raised plasma immunoreactive corticotrophin (ACTH) levels were found in five boys with the sex-linked disorder progressive leucodystrophy associated with adrenal insufficiency (Addison-Schilder's disease) and in a symptom-free brother of one of them. Similar ACTH concentrations were found using two antisera, one against the N-terminal part of the ACTH molecule and the other against the C-terminal part. In one patient the circulating ACTH had normal biological activity as measured using the cytochemical ACTH bioassay. Immunoreactive beta/-melanocyte-stimulating hormone was also determined in one patient and found to be raised.