LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.

INTRODUCTION: Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs. METHODS: The present study aimed to validate a simple routine LC-HRMS method to quantify cortisol, cortisone, 6ß-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism. RESULTS: The LC-HRMS method involved liquid-liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6ß-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity). CONCLUSION: A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism.

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.

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.

Effect of Vasopressin on the Hypothalamic-Pituitary-Adrenal Axis in ADPKD Patients during V2 Receptor Antagonism.

BACKGROUND: Patients with autosomal dominant polycystic kidney disease (ADPKD) are treated with a vasopressin V2 receptor antagonist (V2RA) to slow disease progression. This drug increases vasopressin considerably in these patients with already elevated baseline levels. Vasopressin is known to stimulate the hypothalamic-pituitary-adrenal (HPA) axis through V1 and V3 receptor activation. It is unknown whether this increase in vasopressin during V2RA treatment affects glucocorticoid production. METHODS: Twenty-seven ADPKD patients were studied on and off treatment with a V2RA and compared to age- and sex-matched healthy controls and IgA nephropathy patients, the latter also matched for kidney function. Vasopressin was measured by its surrogate copeptin. Twenty-four-hour urinary excretions of cortisol, cortisone, tetrahydrocortisone, tetrahydrocortisol, allotetrahydrocortisol, and the total glucocorticoid pool were measured. RESULTS: At baseline, ADPKD patients demonstrated a higher copeptin concentration in comparison with healthy controls, while urinary excretion of cortisol and cortisone was lower (medians of 0.23 vs. 0.34 µmol/24 h, p = 0.007, and 0.29 vs. 0.53 µmol/24 h, p < 0.001, respectively). There were no differences in cortisol and cortisone excretion compared to IgA nephropathy patients. Cortisol, cortisone, and total glucocorticoid excretions correlated with kidney function (R = 0.37, 0.58, and 0.19, respectively; all p < 0.05). Despite that V2RA treatment resulted in a 3-fold increase in copeptin, only cortisone excretion increased (median of 0.44 vs. baseline 0.29 µmol/24 h, p < 0.001), whereas no changes in cortisol or total glucocorticoid excretion were observed. CONCLUSIONS: Increased concentration of vasopressin in ADPKD patients at baseline and during V2RA treatment does not result in activation of the HPA axis. The impaired glucocorticoid production in these patients is related to their degree of kidney function impairment.

Acupuncture in persons with an increased stress level-Results from a randomized-controlled pilot trial.

BACKGROUND: In today's Western societies a high percentage of people experience increased or chronic stress. Acupuncture could serve as treatment for persons affected adversely by the increased stress. METHODS: The AkuRest study was a two-centre randomized controlled pilot study in adult persons with increased stress levels. Participants were randomly allocated to one of three groups: verum acupuncture treatment, sham acupuncture, and a waiting control group. The feasibility of the study was assessed. In addition, effects on stress level (measured by the Perceived Stress Questionnaire (PSQ-20)) and other variables were assessed at the end of treatment and a 3-month follow-up. RESULTS: Altogether, N = 70 persons were included in the study. At the end of the treatment 15.7% were lost to follow-up. The adherence to the protocol was good: 82.9% of the participants completed 100% of their treatment. The stress level of the participants was high at baseline (mean PSQ-20 score 75.5, SD = 8.2). Effect sizes (ES) at T1 showed that verum and sham acupuncture were superior to the waiting condition in reducing stress (ES (verum) = -1.39, 95%-CI = [-2.11; -0.67]: ES (sham) = -1.12, CI = [-1.78;-0.44]). At follow-up, effect sizes were in favour of the verum group (as compared to sham). However, confidence intervals and t-tests showed that these differences were not significant. CONCLUSION: The pilot study demonstrated the feasibility of the acupuncture RCT in persons with increased stress levels. Estimated parameters can be used to design a larger RCT to prove the-here indicated-efficacy of verum acupuncture to decrease stress. TRIAL REGISTRATION NUMBER: ISRCTN15259166.

Glucocorticoid metabolism and the action of 11 beta-hydroxysteroid dehydrogenase 2 in canine congestive heart failure.

The enzyme 11-beta-hydroxysteroid dehydrogenase isoenzyme 2 (11BHSD2) is responsible for converting the active glucocorticoid cortisol to inactive cortisone and in the renal medulla protects the mineralocorticoid receptor (MR) from activation by cortisol. Derangements in 11BHSD2 activity can result in reduced conversion of cortisol to cortisone, activation of the MR by cortisol and, consequently, sodium and water retention. The objective of this study was to examine glucocorticoid metabolism in canine congestive heart failure (CHF), specifically to evaluate whether renal 11BHSD2 activity and expression were altered. Dogs were prospectively recruited into one of two phases; the first phase (n=56) utilized gas chromatography-tandem mass spectrometry to examine steroid hormone metabolites normalised to creatinine in home-caught urine samples. Total serum cortisol was also evaluated. The second phase consisted of dogs (n=18) euthanased for refractory CHF or for behavioural reasons. Tissue was collected from the renal medulla for examination by quantitative reverse transcription polymerase chain reaction, immunohistochemistry and protein immune-blotting. Heart failure did not change urinary cortisol:cortisone ratio (P=0.388), or modify renal expression (P=0.303), translation (P=0.427) or distribution of 11BHSD2 (P=0.325). However, CHF did increase excretion of 5α-tetrahydrocortisone (P=0.004), α-cortol (P=0.002) and α-cortolone (P=0.009). Congestive heart failure modifies glucocorticoid metabolism in dogs by increasing 5α-reductase and 20α-hydroxysteroid dehydrogenase activity. Differences between groups in age, sex and underlying disease processes may have influenced these results. However, 11BHSD2 does not appear to be a potential therapeutic target in canine CHF.

Long-Term Stability of Cortisol Production and Metabolism Throughout Adolescence: Longitudinal Twin Study.

Life-course experiences have been postulated to program hypothalamus-pituitary-adrenal (HPA) axis activity, suggesting that HPA axis activity is, at least partially, stable over time. Yet, there is paucity of data on the long-term stability of cortisol production and metabolism. We performed a prospective follow-up study in twins recruited from a nationwide register to estimate the stability of cortisol production and metabolism over time, and the contribution of genetic and environmental factors to this stability. In total, 218 healthy mono- and dizygotic twins were included. At the ages of 9, 12 and 17 years, morning urine samples were collected for assessment (by gas chromatography-tandem mass spectrometry) of cortisol metabolites, enabling the calculation of cortisol metabolite excretion rate and cortisol metabolism activity. Our results showed a low stability for both cortisol metabolite excretion rate (with correlations <.20) and cortisol metabolism activity indices (with correlations of .25 to .46 between 9 and 12 years, -.02 to .15 between 12 and 17 years and .09 to .28 between 9 and 17 years). Because of the low stability over time, genetic and environmental contributions to this stability were difficult to assess, although it seemed to be mostly determined by genetic factors. The low stability in both cortisol production and metabolism between ages 9 and 17 years reflects the dynamic nature of the HPA axis.

Effects of truck transportation and slaughtering on the occurrence of prednisolone and its metabolites in cow urine, liver, and adrenal glands.

BACKGROUND: The recognition of illegal administration of synthetic corticosteroids in animal husbandry has been recently challenged by the case of prednisolone, whose occasional presence in the urine of bovines under strong stressful conditions was attributed to endogenous biosynthesis, not to exogenous administration. The study of the natural stress sources possibly inducing endogenous prednisolone production represents a stimulating investigation subject. The biochemical effects of transportation and slaughtering were verified in untreated cows by studying the possible occurrence of prednisolone and its metabolites in urine, liver and adrenal glands, and the cortisol/cortisone quantification. RESULTS: Cortisol, cortisone, prednisolone and its metabolites were measured in urine, collected at farm under natural micturition and then at the slaughterhouse. The study was performed on 15 untreated cows reared in different farms at the end of their productive cycle. 2-3 days after the first urine collection, the animals were transported by trucks to the abattoir, slaughtered, and subjected to a second urine sampling from the bladder. Specimens of liver and adrenal gland were also collected and analysed by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated method. The stressful conditions of transportation and slaughtering proved to increase considerably the urinary levels of cortisol and cortisone as compared to those collected at farm. Prednisolone was detected in the urine collected at the slaughterhouse of two cows only, at a concentration level (≈0.6 µg L- 1) largely below the official cut off (5.0 µg L- 1) established to avoid false non-compliances. These two animals exhibited the highest urinary cortisol levels of the series. Prednisolone and prednisone were also detected in the adrenal glands of a different cow. Prednisolone metabolites were not detected in any urine, liver, and adrenal gland sample. CONCLUSION: Within the constraints of the condition adopted, this study confirms the sporadic presence of prednisolone traces (2 samples out of 15) and the consistently increased concentration of cortisone and cortisol in the urines collected from cows subjected to truck transportation and subsequent slaughtering. No prednisolone metabolites were detected in any liver and adrenal gland samples, nor in urine specimens, unlike what was previously reported for cows artificially stressed by pharmacological treatment.

Performance of LC-MS/MS and immunoassay based 24-h urine free cortisol in the diagnosis of Cushing's syndrome.

24-h urine free cortisol (UFC) is an indicator of integrated cortisol secretion and established screening tool for Cushing's syndrome (CS). Doubts have been raised regarding specificity of immunoassays, and mass spectrometric techniques have been proposed as an alternative. In the present study we compared diagnostic accuracy of UFC measured with LC-MS/MS vs. immunoassay in patient with CS and patients where CS has been excluded. We examined 24-h urine samples from patients with surgically confirmed CS (n = 77; Cushing's disease (n = 44), ectopic CS (n = 5), adrenal CS (n = 28)) and patients in whom Cushing's syndrome was excluded (n = 97) by long-term follow up. UFC was first measured by automated chemiluminescence immunoassays (ADVIA Centaur, Siemens; LIAISON, DiaSorin). Aliquots of all samples were also analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Statistics: Passing-Bablok Regression, Receiver operating characteristic (ROC) analysis with Youden's index calculation. UFC of CS patients were higher with both immunoassays compared to LC-MS/MS (913 +/- 235 vs. 303 +/- 155 µg/24 h (ADVIA) and 898 +/-216 vs. 399 +/- 196 µg/24 h (LIAISON)). Similarly, UFC were higher with immunoassays than with LC-MS/MS in the control group (223 +/- 10 vs. 23 +/- 2 µg/24 h (ADVIA) and 105 +/- 6 vs. 27 +/- 4 ug/24 h for (LIAISON)). Passing-Bablok regression showed good correlation between LC-MS/MS and ADVIA as well as between LCMS/MS and LIAISON (r = 0.96 and r = 0.99, p < 000.1) but less correlation in controls (r = 0.83 and r = 0.74, respectively, p < 000.1). ROC calculation revealed the highest ROC AUC (0.89) for the LIAISON immunoassay, followed by LC-MS/MS (0.82) and the ADVIA (0.80). In direct comparison, AUCs from LC-MS/MS and immunoassays in the same patient were not statistically different (p < 0,001). Best cut-off concentration to identify patients with CS was 234 µg/24 h (LIAISON), 51 µg/24 h for LC-MS/MS and 330 µg/24 h (ADVIA Centaur). In summary, UFC values were measured substantially higher by both immunoassays compared to LC-MS/MS. This is most likely due to cross-reactivity from interfering glucocorticoid metabolites. Nevertheless, all three methods correlated well. ROC analysis revealed the highest AUC for one of the immunoassays, although differences between the three methods were not significant. Direct comparison with LC-MS/MS indicates that high diagnostic accuracy can be obtained with suitable immunoassays.

Prenatal maternal stress is associated with lower cortisol and cortisone levels in the first morning urine of 45-month-old children.

Prenatal stress (PS) has been related to altered hypothalamic-pituitary-adrenal (HPA) axis activity later in life. So far, studies in children assessing HPA axis functioning have focused on salivary cortisol, reflecting daytime activity. The present work is part of a prospective study and aims to extend knowledge about the association between PS and HPA axis regulation in children. To do so, we investigated cortisol, cortisone, and the ratio cortisone/(cortisone + cortisol) in the first morning urine of 45-month-old children in relation to several measures of maternal stress during pregnancy. Urinary cortisol and cortisone were measured by online turbulent flow chromatography coupled with high performance liquid chromatography-tandem mass spectrometry. PS was defined as: perceived stress for aim 1 (Perceived Stress Scale; n = 280); presence of self-reported (n = 371) and expert-rated psychopathology for aim 2 (Mini International Neuropsychiatric Interview; n = 281); continuous measures of anxiety and depression for exploratory aim 3 (State-Trait Anxiety Inventory and Edinburgh Postnatal Depression Scale; n = 280). Aim 1: Perceived maternal PS showed negative associations with cortisol and cortisone levels. Aim 2: The presence of expert-rated maternal psychopathology was associated with reduced morning cortisone. Aim 3: Continuous measures of anxiety and depression showed negative associations with cortisol and cortisone levels. After correcting for multiple testing, perceived maternal PS (aim 1) and prenatal level of anxiety (aim 3) were significant predictors of children's urinary cortisol and cortisone in the morning (and, in the case of cortisone, also prenatal level of depression). The ratio cortisone/(cortisone + cortisol) as a global marker for the balance between the enzymes metabolizing cortisol to cortisone and vice versa (11ß-hydroxysteroid dehydrogenases type 1 and 2; 11ß-HSD1 and 2) was not associated with any measure of maternal PS (aims 1-3). The present study provides insight into possible programming effects of PS on nocturnal HPA axis activity and a proxy of 11ß-HSD in a large sample. The results suggest that the nocturnal rate of cortisol production is lower in children exposed to PS, but do not support the hypothesis of divergent 11ß-HSD activity.

High-performance liquid chromatography methods for the analysis of endogenous cortisol and cortisone in human urine: comparison of mass spectrometry and fluorescence detection.

BACKGROUND: The analysis of steroids in biological matrices is challenging. One can apply immunoassay as well as gas and liquid chromatography with various types of detection, depending on the available equipment and the experience of the analyst. The question is how the methods are interchangeable between themselves. Doubts were reported having compared immunoassays and chromatography-mass spectrometry, but there are scarce data on chromatographic methods with detection types other than mass spectrometry. METHODS: Here, we present the detailed comparison of two liquid chromatographic methods for the determination of free urinary cortisol and cortisone: one with fluorescence detection (high-performance liquid chromatography [HPLC-FLD]) and the other with tandem mass spectrometry (HPLC-MS/MS). The comparison was made with 199 human urine samples. The data analysis included Passing-Bablok and Deming regression, Bland-Altman test, Wilcoxon test, mountain plot and Lin's concordance correlation coefficient. RESULTS: The validation data indicated that both methods met the requirements of the European Medicines Agency. However, the statistical analysis revealed the systematic bias between the two assays. The Passing-Bablok and the Deming tests showed that the HPLC-FLD method overestimated results for cortisol and underestimated measurements for cortisone. The Bland-Altman analysis estimated the mean differences between the methods: 18.8 nmol/L for cortisol and -16.9 nmol/L for cortisone measurement. CONCLUSIONS: Both methods' results led to the same conclusion in observational studies, but the techniques are not interchangeable. The literature data, the observations from the clinical setting and our experience clearly indicate that the future of steroid measurements will belong to chromatography coupled with mass spectrometry.

DIFFERENTIAL REGULATION OF 11ß-HYDROXYSTEROID DEHYDROGENASE TYPE 1 ACTIVITY IN PATIENTS WITH DIFFERING ETIOLOGIES OF HYPOPITUITARISM.

OBJECTIVE: Pituitary patients with different etiologies of hypopituitarism exhibit differing phenotypes, despite similar replacement therapy strategies. We hypothesized that differential regulation of the isoenzyme 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), which mediates the net autocrine conversion of cortisone to cortisol in adipose tissues and liver, may play a role. METHODS: We studied 11ß-HSD1 activity (using urine cortisol/cortisone metabolites ratio) in 36 hypopituitary patients with treated craniopharyngiomas, treated remitted Cushing disease, and treated nonfunctioning pituitary adenomas + prolactinomas on and off growth hormone (GH) replacement. RESULTS: 11ß-HSD1 activity was higher in subjects with craniopharyngioma both on and off GH, as evidenced by increased tetrahydrocortisol to tetrahydrocortisone metabolite ratios compared to other diagnostic groups, but there was no difference in body mass index, insulin levels, serum hormone measurements, or hydrocortisone dose between groups. CONCLUSION: Craniopharyngiomas are associated with enhanced 11ß-HSD1 activity compared to other diagnostic hypopituitary groups, and this may contribute to the adverse phenotypic and metabolic features seen in this condition. ABBREVIATIONS: BMI = body mass index; Em = cortisone metabolites; Fm = cortisol metabolites; GH = growth hormone; 11ß-HSD1 = 11ß-hydroxysteroid dehydrogenase type 1; IGF-1 = insulin-like growth factor 1; NFPA = nonfunctioning pituitary adenoma; THE = tetrahydrocortisone; THF = tetrahydrocortisol.

Assessment of Hepatic Cytochrome P450 3A Activity Using Metabolic Markers in Patients with Renal Impairment.

BACKGROUND: The renal function of individuals is one of the reasons for the variations in therapeutic response to various drugs. Patients with renal impairment are often exposed to drug toxicity, even with drugs that are usually eliminated by hepatic metabolism. Previous study has reported an increased plasma concentration of indoxyl sulfate and decreased plasma concentration of 4ß-hydroxy (OH)-cholesterol in stable kidney transplant recipients, implicating indoxyl sulfate as a cytochrome P450 (CYP) inhibiting factor. In this study, we aimed to evaluate the impact of renal impairment severity-dependent accumulation of indoxyl sulfate on hepatic CYP3A activity using metabolic markers. METHODS: Sixty-six subjects were enrolled in this study; based on estimated glomerular filtration rate (eGFR), they were classified as having mild, moderate, or severe renal impairment. The plasma concentration of indoxyl sulfate was quantified using liquid chromatography-mass spectrometry (LC-MS). Urinary and plasma markers (6ß-OH-cortisol/cortisol, 6ß-OH-cortisone/cortisone, 4ß-OH-cholesterol) for hepatic CYP3A activity were quantified using gas chromatography-mass spectrometry (GC-MS). The total plasma concentration of cholesterol was measured using the enzymatic colorimetric assay to calculate the 4ß-OH-cholesterol/cholesterol ratio. The correlation between variables was assessed using Pearson's correlation test. RESULTS: There was a significant negative correlation between MDRD eGFR and indoxyl sulfate levels. The levels of urinary 6ß-OH-cortisol/cortisol and 6ß-OH-cortisone/cortisone as well as plasma 4ß-OH-cholesterol and 4ß-OH-cholesterol/cholesterol were not correlated with MDRD eGFR and the plasma concentration of indoxyl sulfate. CONCLUSION: Hepatic CYP3A activity may not be affected by renal impairment-induced accumulation of plasma indoxyl sulfate.

Determination of endogenous and exogenous corticosteroids in bovine urine and effect of fighting stress during the "Batailles des Reines" on their biosynthesis.

Natural corticosteroids include two families of substances: mineralocorticoids and glucocorticoids. Several drugs of similar structure and biological activity have been synthesized and are currently used in the clinical practice. Beside legal pharmacological treatments, these drugs have been consistently misused in animal breeding. One of the most abused corticosteroids is prednisolone. For many years, prednisolone has been considered of exclusive synthetic origin, but nowadays a debate about its possible endogenous production is under way. Several studies have been addressed to ascertain the potential relationship between stressful conditions, such as transportation and slaughtering, and endogenous production of prednisolone. In order to verify further the effect of stressful conditions, our laboratory analysed urine samples collected from the cows participating to the "Batailles des Reines" (a traditional contest based on ritual and spontaneous fights of pregnant cows), to verify if an endogenous prednisolone production may occur in these animals. We developed and validated a LC-MS/MS method for the simultaneous determination of cortisol, cortisone, prednisolone and five of its metabolites. The method was applied to the analysis of urine samples collected from "Batailles des Reines" competitions in 2012 and 2013. All these samples had been previously analysed within an anti-doping control program and tested compliant to all screenings.

Simultaneous quantification of cortisol and cortisone in urines from infants with packed-fiber solid-phase extraction coupled to HPLC-MS/MS.

Cortisol and cortisone are two important glucocorticoids in human body, their interconversion is controlled by two isotypes of 11ß-hydroxy steroid dehydrogenase (11ß-HSD1 and 11ß-HSD2). The ratio of urinary cortisol to cortisone can be used to assess the activity of 11ß-HSDs. An analytical method to quantify urinary cortisol and cortisone using high performance liquid chromatographic tandem mass spectrometry following a packed-fiber solid-phase extraction (PFSPE) was developed. The proposed method was validated and applied to determine the urinary cortisol and cortisone concentrations in infants. Linearity was observed in the range of 0.6-150ng/mL for cortisol and 0.8-200ng/mL for cortisone. The intra-day RSD was 2.4-4.5% for cortisol and 3.3-6.2% for cortisone. Inter-day RSD was 3.7-6.6% for cortisol and 4.3-8.2% for cortisone. The recovery was 97.8±4.6% for cortisol and 98.9±4.4% for cortisone. The established method is simple and efficient for the quantification of urinary cortisol and cortisone and for indirectly assessing the activity of 11ß-hydroxy steroid dehydrogenase.

Is There an Association Between Cortisol and Hypertension in Overweight or Obese Children?

OBJECTIVE: The precise mechanisms behind the development of hypertension in overweight or obese children are not yet completely understood. Alterations in hypothalamic-pituitary-adrenal axis activity may play a role. We aimed to investigate the association between cortisol parameters and hypertension in overweight or obese children. METHODS: Random urine (n=180) and early-morning saliva samples (n=126) for assessment of cortisol and cortisone were collected from 1) hypertensive overweight children (n=50), 2) normotensive overweight children (n=145), and 3) normotensive non-overweight children (n=75). RESULTS: The age of participants was 10.4±3.3 years and 53% were boys. The urinary cortisol-to-cortisone ratio [ß 1.11, 95% confidence interval (CI) 1.05-1.19] as well as urinary cortisol/creatinine (ß 1.38, 95% CI 1.09-1.54), and cortisone/creatinine ratios (ß 1.26, 95% CI 1.17-1.36) were significantly higher in overweight or obese than in non-overweight children. After adjusting for body mass index-standard deviation score and urinary cortisone/creatinine ratio, but not cortisol/creatinine ratio, was significantly associated with presence of hypertension (ß 1.12, 95% CI 1.02-1.23). Salivary cortisol and cortisone levels were significantly lower in overweight or obese than in non-overweight children (ß -4.67, 95% CI -8.19- -1.15, and ß 0.89, 95% CI 0.80-0.97 respectively). There were no significant differences in cortisol parameters between hypertensive and normotensive overweight or obese children. CONCLUSION: This study provided further evidence for an increased cortisol production rate with decreased renal 11ß-hydroxysteroid dehydrogenase 2 activity and flattening of early-morning peak cortisol and cortisone in overweight or obese children. However, there were no significant differences in cortisol parameters between hypertensive and normotensive overweight and obese children.

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

BACKGROUND AND AIM: 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. MATERIALS AND METHODS: 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. RESULTS: UFF, UFE and FEr levels were higher in EAS than in CD (UFF: 6671 vs 549 nmol/24 hours; UFE: 2069 vs 464 nmol/24 hours; 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. CONCLUSIONS: 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.

Development of suitable method for large-scale urinary glucocorticoid analysis by liquid chromatography-mass spectrometry.

Levels of urinary glucocorticoids and their concentration ratios have been analyzed as potential markers for various pathological statuses. Large-scale studies may possibly accelerate the investigations; however, a suitable method needs to be established. Analytical conditions for measurement of urinary glucocorticoids with LCMS were examined. Electrospray ionization in the positive ion mode was applied for detection of cortisol (precursor>product ion: 363.3>121.0), cortisol-d4 (internal standard, IS, 367.4>121.1), and cortisone (361.2>163.2). To maximize ionization, acetic acid-ammonium acetate buffer (18mM) at pH 5.3 was employed as eluent A. A C18 column (100mm×2.1mm, 2.7µm) at 50°C was used for the 9.5min binary gradient separation starting with 60% eluent A with methanol being eluent B. Linear correlations were observed between the concentrations and the peak areas in the concentration range of 1-300ng/mL with correlation coefficients (r) of 0.998 and 0.997 for cortisol and cortisone, respectively, without IS adjustment, and 0.999 with IS adjustment for both cortisol and cortisone. Solid-phase extraction (SPE) using a 2mL centrifuge column was performed for the urine samples, with the original and final volumes being 100µL. The SPE of 12 urine specimens could be performed within 30min. The effect of the sample matrix on the quantification of endogenous compounds present in the urine extract was limited (coefficient of variation (CV) of IS-adjusted matrix factor: 4.4-8.1%; urine extracts of 8 individuals); however, substantial peak reduction of cortisol was observed at low concentrations. Exogenous contaminants originating from the SPE centrifuge column seemed to be a main cause for this phenomenon because the pure-water extract showed similar peak reduction. A recovery of ∼50% was obtained for both cortisol and cortisone. Adjustment with the IS improved the apparent recovery, with ∼100% being obtained for both cortisol and cortisone. The recovery rate decreased when the urine samples were concentrated in the SPE step; the reduction was greater for cortisol than for cortisone. The lower limit of quantification (LLOQ) was set at 2.5ng/mL when the injection volume was 10µL, based on the reproducibility of the standards which were measured (CV of 12 repetitions: 10.1% for 0.5ng/mL cortisol and 19.6% for 1ng/mL cortisone), the matrix effect (-55% at 2ng/mL concentrations of cortisol), and the recovery rate (∼50%). Furthermore an alternative approach for preparation of the cortisol standards was required for low concentration range (2.5-20ng/mL) because of the effect of the matrix. Degradation of original urine specimens at room temperature was minimal during the first 24h. The extracted urine samples degraded over time; however, their concentrations were corrected with the IS, allowing for analysis up to 5days after extraction. In conclusion, an analytical method for urinary glucocorticoids was established, which is fast, sensitive, and well suited for practical application to large-scale study.

Increased Cortisol and Cortisone Levels in Overweight Children.

BACKGROUND It has been unclear whether relatively high cortisol and cortisone levels are related to overweight in childhood, parental body mass index (BMI), and family dietary habits. The aim of this study was to compare cortisol and cortisone levels in urine and saliva from overweight and normal children, as well as correlations between children's BMI, parental BMI and family dietary behavior questionnaire score (QS). MATERIAL AND METHODS We analyzed the data from 52 overweight children and 53 age- and sex-matched normal-weight children aged 4-5 years. The concentrations of salivary cortisol (SF), salivary cortisone (SE), urinary cortisol (UF) and urinary cortisone (UE) were measured using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The family dietary behavior QS was answered by the parent mainly responsible for the family diet. RESULTS Average cortisol and cortisone levels were significantly higher in overweight children. There was no significant difference in the ratio of cortisol to cortisone (Rcc) and the marker of 11b-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) activities. The results displayed correlations among cortisol, cortisone, and Rcc. Positive correlations were weak-to-moderate between BMI and SF, SE, UF, and UE. There were correlations between BMI and maternal BMI (mBMI), and BMI was significantly associated with QS. CONCLUSIONS Our results suggest that cortisol and cortisone levels are associated with overweight in children, but the 11ß-HSD2 activities showed no significant differences. Unhealthy family diet was associated with higher BMI, UF, and UE, and families with maternal overweight or obesity had a higher prevalence of children's overweight or obesity.