Measurement Of Urinary Free Cortisol Research Articles

Comparison of Direct and Extraction Immunoassay Methods With Liquid Chromatography-Tandem Mass Spectrometry Measurement of Urinary Free Cortisol for the Diagnosis of Cushing's Syndrome.

Twenty-four-hour urinary free cortisol (UFC) measurement is the initial diagnostic test for Cushing's syndrome (CS). We compared UFC determination by both direct and extraction immunoassays using Abbott Architect, Siemens Atellica Solution, and Beckman DxI800 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, we evaluated the value of 24-hr UFC measured by six methods for diagnosing CS. Residual 24-hr urine samples of 94 CS and 246 non-CS patients were collected. A laboratory-developed LC-MS/MS method was used as reference. UFC was measured by direct assays (D) using Abbott, Siemens, and Beckman platforms and by extraction assays (E) using Siemens and Beckman platforms. Method was compared using Passing-Bablok regression and Bland-Altman plot analyses. Cut-off values for the six assays and corresponding sensitivities and specificities were calculated by ROC analysis. Abbott-D, Beckman-E, Siemens-E, and Siemens-D showed strong correlations with LC-MS/MS (Spearman coefficient r=0.965, 0.922, 0.922, and 0.897, respectively), while Beckman-D showed weaker correlation (r=0.755). All immunoassays showed proportionally positive bias. The areas under the curve were 0.975 for Abbott-D, 0.972 for LC-MS/MS, 0.966 for Siemens-E, 0.948 for Siemens-D, 0.955 for Beckman-E, and 0.877 for Beckman-D. The cut-off values varied significantly (154.8-1,321.5 nmol/24 hrs). Assay sensitivity and specificity ranged from 76.1% to 93.2% and from 93.0% to 97.1%, respectively. Commercially available immunoassays for measuring UFC show different levels of analytical consistency compared to LC-MS/MS. Abbott-D, Siemens-E, and Beckman-E have high diagnostic accuracy for CS.

Evidence of persistent mild hypercortisolism in patients medically treated for Cushing's disease: the Haircush study.

Cortisol-lowering drugs may not restore a normal cortisol secretion in Cushing's disease (CD). Assess the long-term cortisol exposure in medically treated CD patients using hair-cortisol (HF) and -cortisone (HE) measurement. Multicenter prospective study. Three groups of female patients: CushMed=16 treated with a stable cortisol-lowering drug dosage and normal UFCs; CushSurg=13 cured by pituitary surgery; CushBla=15 receiving stable recommended doses of hydrocortisone following bilateral adrenalectomy. Patients were evaluated during 3 months with their usual treatments. Two late-night saliva and 24h urine samples were monthly collected in CushMed, and at study end in CushSurg and CushBla patients. A 3-cm hair sample was collected at study end in all patients. Clinical score and centralized measurement of UFC, late-night salivary cortisol (LNSF) and -cortisone (LNSE), HE, HF. Despite having almost all UFCs normalized, CushMed patients exhibited increased HE as compared to CushSurg controls (p=0.003). CushMed patients also had increased clinical score (p=0.001), UFC (p=0.03), LNSF, LNSE (p=0.0001) and variability in the latter parameters (p=0.004). CushBla patients had increased HF and HE contrasting with similar LNSE to CushSurg patients. Six of 15 CushMed patients exhibited increased HE concentrations and had increased antihypertensive drug dosage compared to CushMed patients with normal HE (p=0.05). Despite normalized UFCs, a subset of medically-treated CD patients displays an altered circadian rhythm of serum cortisol. A single HE measurement identifies chronic mild persistent hypercortisolism and could replace multiple saliva analyzes to monitor medical treatments in CD patients once UFC is normalized.

Cushing's syndrome in the elderly: data from the European Registry on Cushing's syndrome.

To evaluate whether age-related differences exist in clinical characteristics, diagnostic approach, and management strategies in patients with Cushing's syndrome (CS) included in the European Registry on Cushing's Syndrome (ERCUSYN). Cohort study. We analyzed 1791 patients with CS, of whom 1234 (69%) had pituitary-dependent CS (PIT-CS), 450 (25%) adrenal-dependent CS (ADR-CS), and 107 (6%) had an ectopic source (ECT-CS). According to the WHO criteria, 1616 patients (90.2%) were classified as younger (<65 years old) and 175 (9.8%) as older (≥65 years old). Older patients were more frequently males and had a lower Body Mass Index (BMI) and waist circumference when compared with the younger. Older patients also had a lower prevalence of skin alterations, depression, hair loss, hirsutism, and reduced libido, but a higher prevalence of muscle weakness, diabetes, hypertension, cardiovascular disease, venous thromboembolism, and bone fractures than younger patients, regardless of sex (P < .01 for all comparisons). Measurement of urinary free cortisol supported the diagnosis of CS less frequently in older patients when compared with the younger (P < .05). An extrasellar macroadenoma (macrocorticotropinoma with extrasellar extension) was more common in older PIT-CS patients than in the younger (P < .01). Older PIT-CS patients more frequently received cortisol-lowering medications and radiotherapy as a first-line treatment, whereas surgery was the preferred approach in the younger (P < .01 for all comparisons). When transsphenoidal surgery was performed, the remission rate was lower in the elderly when compared with their younger counterpart (P < .05). Older CS patients lack several typical symptoms of hypercortisolism, present with more comorbidities regardless of sex, and are more often conservatively treated.

Diagnostic workup of Cushing's syndrome.

Cushing's syndrome (CS) is a rare but detrimental endocrine disorder. Early diagnosis and prompt treatment are essential since the duration of hypercortisolism has an adverse impact on the extent of comorbidities and overall survival. The diagnostic approach involves a stepwise process that includes (1) screening and confirming the diagnosis and (2) establishing the aetiology of CS. The tests currently used to confirm the diagnosis of CS include urinary free cortisol measurements, the dexamethasone suppression test and late- night salivary cortisol or midnight serum cortisol measurements. None of these tests are ideal; all have pitfalls and require careful interpretation. Following confirmation of CS, measurement of ACTH discriminates between ACTH-dependent and non-ACTH dependent causes of CS. Adrenal imaging provides clues for the aetiology of non-ACTH dependent forms. Differentiation between the ACTH-dependent forms that involve pituitary corticotroph adenomas and ectopic ACTH sources is more complex and include pituitary MRI imaging, the high dose dexamethasone suppression test, the CRH test, bilateral inferior petrosal sinus sampling and, when required imaging modalities to detect ectopic ACTH secreting lesions. This review, which is part of a special issue on "Update of Cushing's syndrome: 100years after Minnie G" will provide an update on our current diagnostic workup for the confirmation and differential diagnosis of CS.

0379 At-home Sample Collection for Identification of Alterations in HPA-axis Activity and Cortisol Area Under the Curve

Abstract Introduction Sleep problems are associated with alterations in diurnal cortisol patterns, reflective of altered hypothalamic-pituitary-axis (HPA) activity. At-home collection of dried samples for measurement of urinary free cortisol are useful for assessment of diurnal rhythms because they do not impose the stress or sleep disruption associated with inpatient studies. The purpose of this analysis was to assess urinary cortisol responses among a large real-world dataset of at-home collection kit users and the potential for utility in identifying alterations in HPA-axis activity. Methods This retrospective analysis evaluated data from 3,966 individuals who used a Sleep and Stress Test (Everlywell, Inc.) between September 2017 and July 2021. Four dried urine spot samples were collected according to habitual sleep patterns: upon waking (T1), two hours after waking (T2), prior to the evening meal (T3), and at bedtime (T4). Urinary free cortisol normalized to creatinine, indicative of HPA-axis activity was assessed (area under the curve (AUC), wake to bedtime diurnal cortisol slope (DCS), mid-morning (T2) and bedtime (T4) cortisol levels). Sample reference ranges were established from two standard deviations above and below the log-transformed mean at each time point, values were reverted to original units for reporting. Results The sample was predominantly female (n=2,832, 71%). Mean age was 42.8 (sd=12.0) years. Seventy-eight individuals had mid-morning (T2) cortisol levels that were higher than the sample reference range (7.0-170.7 ug/g Cr), and 88 individuals below. Bedtime (T4) cortisol levels were elevated in 145 individuals (reference range 1.1-25.0 ug/g Cr). Total AUC for cortisol was high in 93 and low in 81 of individuals. The slope of the diurnal cortisol response was flattened in 20.6% (n=816) of the sample. Conclusion This real-world study demonstrates that at-home dried urine sample collection can be used to characterize normal and atypical HPA-axis activity. Convenient assessment of alterations in diurnal cortisol patterns are critical in identifying and understanding the mechanisms of HPA-axis activity common in sleep disruptions/disorders. Future studies utilizing this methodology may prove useful in identifying subpopulations with altered HPA-axis activity, sleep issues, and potential relationships to cardiometabolic health. Support (If Any)

Measurement of Urinary Free Cortisol and Cortisone by LC-MS/MS.

Monitoring urinary free cortisol (UFC) excretion helps assess adrenal function and is used to screen for endogenous Cushing's syndrome caused by an adrenal or pituitary tumor. While serum cortisol levels fluctuate in response to time of day, stress, and concentrations of cortisol-binding globulin (CBG), a 24-h urine collection measures the cortisol produced over the entire day and does not suffer from as much variability as a serum measurement.We describe here a method of measurement of urinary free cortisol (UFC) and cortisone using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples, combined with stable isotope-labeled internal standards, are extracted by liquid-liquid extraction using ethyl acetate and hexane. An API 5500 mass spectrometer operated in positive atmospheric pressure chemical ionization (APCI) mode is used for detection.

Two-day low-dose dexamethasone suppression test more accurate than overnight 1-mg in women taking oral contraceptives.

IntroductionLate‐night salivary cortisol (LSaC) and 24‐h urinary free cortisol measurement, and overnight 1‐mg dexamethasone suppression test (1 mg‐DST) are the first‐line screening tests recommended for Cushing's syndrome. Through elevations in the level of cortisol‐binding globulin, oral contraceptive agents lead to increases in the total plasma cortisol concentration, yielding false‐positive 1 mg‐DST results.ObjectiveTo compare the accuracy of the overnight 1‐mg DST and two‐day low‐dose DST (2d‐DST) in female volunteers taking combined oestrogen‐progestin oral contraceptives (COCs).MethodsThis prospective study enrolled 30 healthy participants. Their plasma cortisol response levels were compared after the 1‐mg DST and 2d‐DST and classified into three categories: normal (≤50 nmol/L), doubtful (51–138 nmol/L) and abnormal (>138 nmol/L). Salivary cortisol was also measured at late night and after the DSTs.ResultsFollowing the 1‐mg DST and 2d‐DST, the plasma cortisol concentrations decreased to a median of 69 nmol/L and 37 nmol/L, respectively (p < 0.001). A statistically significant higher proportion of unclear or abnormal results were observed after the 1‐mg DST (63%) than after the 2d‐DST (27%) (p = 0.004). None of the values were >138 nmol/L after the 2d‐DST, while 11% of them were abnormal after the 1‐mg DST (p = 0.25). No LSaC value was abnormal.ConclusionOur results suggest that, when late‐night salivary cortisol is not available, the 2d‐DST could be a better screening option than the 1‐mg DST for women taking oral contraceptive agents who are reluctant to stop them. This finding requires confirmation in those with a suspicion of hypercortisolism.

Selective Serotonin Reuptake Inhibitors Increase Urinary Free Cortisol in Patients with Carney Complex and Primary Pigmented Nodular Adrenocortical Disease

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent Cushing syndrome predominantly associated with Carney Complex (CNC), a multiple endocrine neoplasia syndrome primarily caused by inactivating defects in PRKAR1A. PPNAD, which has neuroendocrine features, demonstrates cortisol production in response to serotonin as well as increased expression of tryptophan hydroxylase type 2, a serotonin synthesizing enzyme, and the serotonin (5-HT) receptors types 4, 6, and 7. This creates an autocrine/paracrine serotonergic regulatory loop that activates cortisol production. PPNAD can be diagnosed through a 6-day Liddle test (LT) showing a paradoxical increase of >50% from baseline in 24-h urinary free cortisol (UFC) on the 2nd day of high-dose dexamethasone administration (Day 6). Selective serotonin reuptake inhibitors (SSRIs) inhibit the reuptake of serotonin and are widely used for the treatment of depression. We performed a retrospective cohort study of patients with CNC and PPNAD that underwent a LT to evaluate the effect of SSRIs on UFCs, with the hypothesis that SSRI use leads to an exaggerated increase in UFC through presumed activation of the described serotonergic regulatory loop. Of the 34 patients (4–65 y) with CNC and PPNAD that underwent a LT at our institution between 2004 and 2018, 4 took an SSRI during testing. No differences were observed between the SSRI (S) group and the non-SSRI (NS) group in baseline UFCs and the percent increase in UFC on D6. Specifically, the median (IQR) baseline UFC in the S group was 36 (13–252) mcg/24h (nl 4–56) vs 35 (13–98) mcg/24h in the NS group (P=0.95). The percent change in UFC was 208 (93–683)% in the S group and 185 (28–364)% in the NS group (P=0.89). However, there was a difference for overall UFC measurement (across days 1–6 of the LT) in the S group vs the NS group (P=0.03). Age <18 vs 18+ and sex did not have an effect on the outcomes (P=0.17 and P=0.74, respectively). Thus, we conclude that though the percent change in UFC during the LT was similar in both groups, there was a significant difference in overall UFC in the S group when compared to the NS group. These data support an effect of SSRIs and serotonin on UFC and consequently cortisol production in PPNAD. This interesting observation has to be confirmed in more patients with CNC and PPNAD to further elucidate the effects of SSRIs on cortisol production in patents with PPNAD-caused Cushing syndrome, as this may have significant diagnostic and therapeutic implications.

SAT-221 False-Positive Urinary Free Cortisol with Oral Contraceptive Use

Introduction It is well-established that estrogen-containing oral contraceptives can raise serum cortisol levels and yield false-positive results on dexamethasone suppression testing, however it is less known that they can raise urinary free cortisol levels by an unknown mechanism. Case presentation A 47-year old woman taking microgestin 1/20 oral contraceptive (OC) was found incidentally to have osteopenia on chest X-ray. Subsequent DEXA scan demonstrated osteoporosis. During the work-up to determine the underlying etiology of her osteoporosis, she underwent a 24-hour urinary free cortisol, which was 207 ug/24 hours (0-50 ug/24 hours). Serum cortisol was 40.1 (6.2-19.4 ug/dL) and overnight 1mg dexamethasone suppression test yielded a cortisol of 2.7. After one week off her OC, serum cortisol was 24.6. After five weeks off her OC, urinary free cortisol was 19 and 1mg dexamethasone suppression test yielded a cortisol of 0.8. On exam, the patient did not have any cushingoid features. MRI of the brain showed a small hypoenhancing lesion, which was determined by neurosurgery to be insignificant. Ultimately, the cause of her osteoporosis was thought to be likely secondary to vitamin D deficiency and calcium malabsorption due to celiac disease. Discussion The biochemical diagnosis of hypercortisolism during treatment with estrogen-containing OCs remains a challenge. Prior studies have demonstrated that serum cortisol levels are typically elevated during OC use and that 1mg dexamethasone suppression testing may yield false-positive results in more than 50% of cases. This is thought to be due to an increase in the major binding protein for cortisol, corticosteroid-binding globulin. Although it is commonly thought that OCs do not affect urinary free cortisol measurements, a recent study showed that more than one-third of women taking OCs had a mild elevation of urinary free cortisol. There are a few hypotheses for the mechanism by which OC use leads to elevated urinary free cortisol, including direct effect on steroidogenesis in the adrenal glands. Our patient had a significantly elevated 24-hour urinary free cortisol and an equivocal 1mg dexamethasone suppression test while on OCs that normalized with OC discontinuation. Further, serum cortisol decreased with OC discontinuation. Conclusion The 24-hour urinary free cortisol level may be altered by oral contraceptive use and is therefore not a valid tool to evaluate for hypercortisolism in women on OCs. References Nickelsen T, et al. (1989). The dexamethasone suppression test and long-term contraceptive treatment. Experimental and Clinical Endocrinology, 94, 275–280. Paschali M, et al. (2013). False positives on both dexamethasone testing and urinary free cortisol in women on oral contraception. Clin Endocrinol, 79, 443-445. Trainer PJ. (2002). Corticosteroids and pregnancy. Seminars in Reproductive Medicine, 20, 375–280.

Clinical utility of an ultrasensitive urinary free cortisol assay by tandem mass spectrometry

Background24 h urinary free cortisol measurement is a clinically important first-line screening test for Cushing’s syndrome (CS). Tandem mass spectrometry (LC-MS/MS) assays have superior sensitivity and specificity compared to immunoassays. Our goal was to improve and validate a LC-MS/MS method to measure urinary free cortisol in both adult and pediatric patients and to characterize its clinical diagnostic performance of CS by chart review. MethodsWe improved a LC-MS/MS method previously reported for urinary free cortisol to be able to measure urinary and salivary cortisol in the same batch for increased efficiency. The sample preparation was by liquid-liquid extraction using dichloromethane followed by stepwise washing with acidic, basic and neutral solutions. The assay’s analytical performance was characterized, and a retrospective patient chart review was conducted to evaluate the assay’s clinical performance in diagnosing CS. ResultsThe LC-MS/MS assay demonstrated enhanced sensitivity and was linear within an analytical measurement range of 10–10,000 ng/dL. Assay accuracy was satisfactory as determined by spike and recovery studies and highly correlated with a reference LC-MS/MS method. The assay’s clinical diagnostic sensitivity and specificity in detecting CS was 96% and 91%, respectively, when compared to a urinary cortisol excretion of at least 50 µg/24 h. ConclusionsThe improved LC-MS/MS method is both sensitive and specific with enhanced analytical performance and clinical diagnostic utility to screen for CS. The clinical diagnostic sensitivity and specificity were superior based on retrospective patient chart review.

Diagnostic accuracy of increased urinary cortisol/cortisone ratio to differentiate ACTH-dependent Cushing's syndrome.

Differential diagnosis between Cushing's Disease (CD) and Ectopic ACTH Syndrome (EAS) may be a pitfall for endocrinologists. The increasing use in clinical practice of chromatography and mass spectrometry improves the measurement of urinary free cortisol (UFF) and cortisone (UFE). We have recently observed that cortisol to cortisone ratio (FEr) was higher in a small series of EAS; in this study we collected a larger number of ACTH-dependent Cushing's Syndrome (CS) to study the role of FEr to characterize the source of corticotropin secretion. High-pressure liquid chromatography with UV detection (HPLC-UV, n=35) or liquid chromatography-tandem mass spectrometry (LC-MS/MS, n=72) were used to measure UFF, UFE and FEr in 83 patients with CD and 24 with EAS. UFF, UFE and FEr levels were higher in EAS than in CD (UFF: 6671 vs 549nmol/24hours; UFE: 2069 vs 464nmol/24hours; FEr: 4.13 vs 0.97; all P<.001). FEr >1.15 (the best ROC-based threshold) was able to distinguish CD from EAS with 75% sensitivity (SE) and 75% specificity (SP), AUC 0.811; results were similar between HPLC-UV (SE 73%, SP 79%, AUC 0.708) and LC-MS/MS (SE 77%, SP 73%, AUC 0.834; P=.727). The diagnostic accuracy of FEr was similar to that of CRH test or high-dose dexamethasone suppression test (respectively P=.171 and P=.683), also combined. Finally, FEr was able to increase the number of correct diagnosis in patients with discordant dynamic tests. Urinary FEr >1.15 was able to suggest EAS, with a diagnostic accuracy similar to that of other dynamic tests proposed to study ACTH-dependent CS.

Sleep Hypoxemia and Urinary Free Cortisol in Patients with Obstructive Sleep Apnea

Objective: This study evaluated the influence of obstructive sleep apnea (OSA) on hypothalamic–pituitary–adrenal (HPA) axis aiming to identify the polysomnographic data associated with cortisol abnormalities. Methods: Forty-one men referred for suspected OSA underwent polysomnography and were classified according to the apneahypopnea index (AHI) into control group (n=16) and OSA group (n=25). The assessment of the HPA axis function included low-dose (0.25 mg) dexamethasone suppression test, the circadian rhythm of salivary cortisol secretion and the measurement of urinary free cortisol (UFC) excretion in a 24-hour urine collection. Results: No differences were found between groups for age (p=0.112), body mass index (BMI) (p=0.617), waist circumference (WC) (p=0.358). UFC was higher in the OSA group compared with the control group (p=0.013). There was no correlation between the UFC and BMI. There was a negative correlation between UFC and minimum oxygen saturation (p=0.006) and positive correlations between UFC and rapid-eye-movement (REM) oxygen desaturation index (p=0.004), AHI (p=0.024) and arousal index (p=0.010). In a multivariable linear regression analysis, including, arousal index and minimum O2 saturation or REM ODI as independent variables and UFC as the dependent variable, only minimum O2 saturation or REM ODI were associated with high UFC levels. Conclusion: Our results demonstrated that OSA is associated with increased UFC. The severity of hypoxemia is the major factor of this neuroendocrine dysregulation.

Prolactine and urinary free cortisol measurement improve inferior petrosal sinus sampling results in ACTH dependent Cushing's Syndrome

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Characterization of persistent and recurrent Cushing’s disease

A case of possible recurrent Cushing's disease (CD) is presented and data on current definitions of CD remission, persistence, and recurrence are reviewed. While prevailing guidelines recommend the use of serum cortisol in the first post-operative week for defining initial remission and predicting sustained remission, with the use of 24h urinary free cortisol measurements if serum cortisol values are equivocal, there is debate as to which methods and thresholds best define CD remission and predict successful outcomes. Other methods used to define remission (and hence persistence and recurrence) include restoration of cortisol suppression after dexamethasone and physiologic diurnal cortisol rhythm as measured by midnight salivary free cortisol. However, the number and degree of abnormal test results needed to define recurrence, and the determination of which biochemical test has more significance when there are discrepancies between markers is inconsistent among studies. Further inquiry is warranted to examine if patients in apparent CD remission who have subtle hypothalamic pituitary adrenal (HPA) axis abnormalities represent distinctive remission subtypes versus mild or early recurrence. Additional investigation could also explore the degree to which these HPA axis abnormalities, such as alterations in cortisol circadian rhythm or partial resistance to dexamethasone, are associated with persistence of CD morbidities, including neuropsychiatric impairments, alterations in body composition, and cardiovascular risk.

High variability in baseline urinary free cortisol values in patients with Cushing's disease

ObjectiveTwenty-four-hour urinary free cortisol (UFC) sampling is commonly used to evaluate Cushing’s syndrome. Because there are few data on UFC variability in patients with active Cushing’s disease, we analysed baseline UFC in a large patient cohort with moderate-to-severe Cushing’s disease and assessed whether variability correlates with hypercortisolism severity. These data will help clinicians establish the minimum number of UFC samples required to obtain reliable data.DesignObservational study (enrolment phase of Phase III study).MethodsPatients (n = 152) with persistent/recurrent or de novo Cushing’s disease and mean UFC (mUFC) ≥1·5×ULN (normal: 30–145 nmol/24 h) were included. Mean UFC level was calculated from four 24-h urine samples collected over 2 weeks.ResultsOver 600 24-h UFC samples were analysed. The mUFC levels of samples 1 and 2 and samples 3 and 4 were 1000 nmol/24 h (SD 1872) and 940 nmol/24 h (SD 2148), respectively; intrapatient coefficient of variation (CV) was 38% for mUFC. The intrapatient CV using all four samples was 52% (95% CI: 48–56). The intrapatient CV was 51% (95% CI: 44–58) for samples 1 and 2, 49% (95% CI: 43–56) for samples 3 and 4 and 54% (95% CI: 49–59) for samples 1, 2 and 3. Variability in mUFC increased as UFC levels increased. There were no correlations between UFC and clinical features of hypercortisolism.ConclusionsThere is intrapatient variability of approximately 50% in 24-h UFC measurements, which is relevant to targets set to estimate any treatment effect. Analysing more than two 24-h collection periods in individual patients does not result in a relevant decrease in variability. Interestingly, UFC levels did not correlate with hypercortisolism severity.

Clozapine Use Presenting with Pseudopheochromocytoma in a Schizophrenic Patient: A Case Report

Introduction. There have been six previous cases that reported pseudopheochromocytoma in patients taking clozapine. Our case showed the direct link of clozapine to serum levels of certain markers. Case. This is a case of a 49-year-old obese Caucasian female who was referred to endocrinology for investigation of Cushing's syndrome, based on raised blood pressure and Cushingoid facies. The patient had underlying schizophrenia and was stable on clozapine. Her blood pressure was 150/99 mmHg on bendroflumethiazide and candesartan. We measured her 24-hour urinary-free cortisol, which was normal but 24-hour urinary-free noradrenaline was elevated at 835 nmol (76–561) with normal adrenaline 36 nmol (7–82) and dopamine 2679 nmol (366–2879), as the patient had history of palpitations and sweating. Two sets of 24-hour urinary-free cortisol measurements were normal and serum cortisol suppressed to <50 nmol/l after a 1 mg overnight dexamethasone. Two further 24-hour urinary-free catecholamines showed a raised level of noradrenaline. MRI demonstrated normal adrenals and MIBG scan did not show any abnormal uptake at adrenal glands. Conclusion. Pseudopheochromocytoma has been reported in patients taking clozapine. A number of different mechanisms for raised plasma noradrenaline levels with clozapine have been postulated. The above case highlights an unusual but known side effect of clozapine.

Measurement of Urinary Free Cortisol by Current Immunoassays: Need for Sex-dependent Reference Ranges to Define Hypercortisolism

Urinary free cortisol (UFC) is used to assess disease activity in hypercortisolemic patients. However, reference ranges are often lacking, especially with respect to potential confounding variables. This study analyzed upper limits of normal (ULN, mean + 2 SD) for 2 newer immunoassays, using gas chromatography-mass spectrometry (GC-MS) as reference method. Each 10 healthy subjects were grouped by age (18-29; 30-49; ≥ 50 years), BMI (< 25; ≥ 25 kg/m2), and sex, resulting in a total of 120 controls (60 males; age: 39.3±1.3 years; BMI: 25.9±0.4 kg/m2). ULN were calculated for a radioimmunoassay (RIA, Immunotech) and an electrochemiluminescence immunoassay (ECLIA, Roche) and applied to 12 hypercortisolemic patients (4 males; age: 53.1±3.1 years; BMI: 29.1±1.8 kg/m2). To determine degradation, samples were stored at 4°C (without light) or 22°C (with and without light) for 0, 24, and 72 h. Cortisol concentrations were significantly correlated: r=0.88 for RIA vs. ECLIA, r=0.75 for RIA vs. GC-MS, and r=0.77 for ECLIA vs. GC-MS (always p<0.0001). For each procedure, multiple stepwise regression analysis identified sex as the only significant predictor, resulting in sex-dependent ULN (males vs. females): 294 vs. 208 nmol/24 h (RIA), and 379 vs. 277 nmol/24 h (ECLIA). These ULN classified samples from patients as hypercortisolemic in 100% (RIA) and 95% (ECLIA). Different storage conditions over 72 h did not alter UFC levels significantly. Results of the 3 procedures were well correlated, and the use of assay- and sex-specific ULN allowed excellent identification of hypercortisolic states. UFC is stable over 72 h irrespective of the storage conditions applied.

膜性増殖性糸球体腎炎の治療中,尿中遊離コルチゾール測定によりステロイド薬の吸収を評価し得た短腸症候群の1例

膜性増殖性糸球体腎炎の治療中,尿中遊離コルチゾール測定によりステロイド薬の吸収を評価し得た短腸症候群の1例

Peri-operative management of Cushing’s disease

Management of patients with ACTH producing pituitary adenoma remains to be challenging. Removal of the pituitary adenoma through transsphenoidal surgery is the main stay of treatment. Complete resection of the adenoma is followed by the development of ACTH deficiency since the normal corticotrophs are suppressed by the pre-existing hypercortisolemia. The concern for ACTH deficiency has led many centers to advocate the use glucocorticoids before, during and after surgery. We provide evidence that such coverage with glucocorticoids is unnecessary until clinical or biochemical documentation of need is established. Given that patients are closely monitored, they are immediately treated with glucocorticoids once they exhibit any clinical and/or biochemical evidence of adrenal insufficiency. Defining remission in the immediate postoperative period has been rather difficult despite using different biochemical markers. Serum cortisol continues to be the best determinant of disease activity after surgical adenomectomy. However it needs to be interpreted with caution as a biochemical marker of remission in patients given glucocorticoids during and after surgery. Other biochemical markers are also used in the peri-operative period to determine the possibility of remission. These include the dexamethasone suppression test, CRH stimulation without dexamethasone, urinary free cortisol measurements, desmopressin stimulation test, the determination of salivary cortisol and / or plasma ACTH concentrations. Each test has its own advantages and limitations. The simplest and most informative approach is to measure serum cortisol levels repeatedly after surgery without the administration of exogenous glucocorticoids. Low serum cortisol levels (less than 2 μg/dL) in the peri-operative period are highly indicative of surgical success and a high likelihood for clinical remission. Higher serum cortisol levels require careful interpretation and further planning and discussions between the patient and the management team.

Measurement of urinary free cortisol by tandem mass spectrometry and comparison with results obtained by gas chromatography-mass spectrometry and two commercial immunoassays

Determination of urinary free cortisol (UFC) is an important adjunct for the assessment of adrenal function. In this study, we have analysed cortisol concentrations in urine samples by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and two immunoassays. The results were compared with GC-MS. The interference of cortisol ring-A metabolites in immunoassays was also assessed. The GC-MS technique involved solvent extraction, LH-20 clean-up and derivatization. Only solid-phase extraction procedure was used for LC-MS/MS. The samples were analysed in positive electro-spray ionization mode, monitoring the transitions for cortisol and deuterated-cortisol at m/z 363.3 > 121.2 and m/z 365.3 > 122.2, respectively. Immunoassays were performed according to the manufacturer's instructions. When compared with GC-MS results both immunoassays (Coat-A-Count; approximately 1.9-fold, Centaur; approximately 1.6-fold) overestimated UFC concentrations. Cortisol ring-A dihydro- and tetrahydrometabolites contribute significantly to this overestimation. There was no interference by these metabolites in either GC-MS or LC-MS/MS methods. The sensitivity of the LC-MS/MS procedure was 2 nmol/L and the intra- and inter-assay variations were <5% in each quality-control sample. The comparison of the UFC results achieved by assaying the study samples with GC-MS and LC-MS/MS indicated that the agreement between the two methods was excellent (LC-MS/MS = 1.0036GC-MS - 0.0841; r2 = 0.9937). The interference of cortisol ring-A metabolites in immunoassays contribute to overestimation of UFC concentrations. The LC-MS/MS procedure had the sensitivity, specificity, linearity, precision and accuracy for the determination of UFC concentrations. The method is suitable for routine use provided that method-dependant reference values are established.