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Quantitation of endogenous steroids and their precursors is essential for diagnosis of a wide range of endocrine disorders. Usually, these analyses have been carried out using immunoassays. However, immunoassays often overestimate concentrations due to assay interference by other endogenous steroids, especially for low concentrations. Mass spectrometry based methods offer superior specificity, accuracy, and sensitivity. We therefore present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with automated sample preparation for determination of 17α-hydroxyprogesterone (17OHP), cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione (A

Individuals with pulmonary arterial hypertension experience debilitating symptoms and psychological distress which may influence their cortisol regulation. We describe associations between diurnal salivary cortisol/cortisone levels and quality of life in adults with pulmonary arterial hypertension. Findings suggest potential clinical utility of cortisol/cortisone assessment as applied to a pulmonary arterial hypertension population.

Background: Lung cancer is the most common cause of cancer-related deaths in men and women worldwide. Novel diagnostic biomarkers are urgently required to enable the early detection and treatment of lung cancer, and using novel methods to explore tumor-related biomarkers is a hot topic in lung cancer research. The purpose of this study was to use ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) metabolomics analysis technology combined with multivariate data processing methods to identify potential plasma biomarkers for non-small cell lung cancer (NSCLC). Methods: Plasma samples from 99 NSCLC patients and 112 healthy controls were randomly divided into the screening group and the validation group, respectively. UPLC-MS metabolomics analysis technology combined with multivariate data processing methods were used to identify potential plasma biomarkers for NSCLC. Results: A total of 254 metabolites were detected and validated in plasma. Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) modeling indicated that 28 endogenous metabolites were present at significantly different levels in patients with NSCLC than healthy controls (variable importance in projection (VIP)>1 and PConclusions: Metabolomics has the potential to distinguish between NSCLC patients and healthy controls, and may reveal new plasma biomarkers for the early detection of NSCLC.

Two sensitive assays based on AlphaLISA technology were developed and validated for the measurement of cortisol and cortisone in hair of pigs, that also enabled estimation of 11β-hydroxysteroid dehydrogenase type 2 activity. These assays were applied to hair samples from sows (n = 32) collected at 5 days before, and at 23 and 59 after farrowing, in reproductive cycles in two different periods: spring-summer (n = 16) and winter-spring (n = 16). The assays were precise (imprecision

Context Cortisol in blood has a robust circadian rhythm and exerts potent effects on energy balance that are mediated in part by central mechanisms. These interactions involve orexigenic agouti-related protein (AgRP) neurons that are stimulated by glucocorticoids. However, diurnal changes in brain or cerebrospinal fluid (CSF) cortisol and cortisone, which are interconverted by 11ß-HSD1, have not been characterized in humans. Objective To conduct a secondary analysis of existing samples to characterize diurnal changes in cortisol and cortisone in CSF and examine their relationships to changes in AgRP. Methods Stored CSF and plasma samples were obtained from 8 healthy subjects who served as controls for a sleep study. CSF was collected every 2h for 36h via indwelling lumbar catheter; plasma was collected every 2h. Results There was a diurnal rhythm for cortisol and cortisone in CSF that closely followed the plasma rhythm by 2 h with peak and nadir levels at 0900h and 0100h. The ratio of cortisol (active) to cortisone (inactive) in CSF was 48% higher at the peak versus nadir. There was a diurnal rhythm for AgRP in plasma that was out of phase with the cortisol rhythm. There was a less distinct diurnal rhythm for AgRP in CSF that oscillated with a similar phase as cortisol. Conclusions There is a robust diurnal rhythm for cortisol and cortisone in CSF. Diurnal changes were noted for AgRP that are related to the cortisol changes. It remains to be determined if AgRP mediates adverse metabolic effects associated with disruption of the cortisol circadian rhythm.

The effectiveness of selected physiological and perceptual measures for monitoring training load and fatigue was studied in 16 male elite rugby sevens players during a 6-week intense training block (IT) and 2-week tapering (TAP). Daily training load (TL) and strain (TS) as well as weekly total score of fatigue (TSF) were quantified respectively by the session-rating of perceived exertion (RPE) method and an 8-item questionnaire. Also, testing was performed and 24 h urinary cortisol (C), cortisone (Cn), adrenaline (A) and noradrenalin (NA) excretion was measured before (T0) and after the IT (T1) and after the TAP (T2). The TL, TS and TSF increased during the IT and decreased during the TAP, in conjunction with a significant drop and improvement, respectively, of performance standards during the two periods. At T1, C and Cn levels increased while A and NA levels decreased, resulting in a higher C/Cn ratio and lower A/NA ratio, respectively. At T2, both C/Cn and A/NA returned to baseline values. The changes in C/Cn ratio, after the 6-week IT, were more closely related to mean TL, TS and TSF (r=0.75-0.76 vs. r=0.48-0.58, p

The Chianina, one of the oldest and most important cattle breeds of Italy, is now reared all over the world. The Chianina has been known and appreciated since ancient times because, from a nutritional point of view, its meat has no proper rivals. To date, studies have been performed to evaluate the genetic profile of the breed, but knowledge about the chemical profile is generally lacking. Due to the increased interest from farmers regarding breeding of the Chianina, this study proposes a preliminary evaluation of main endogenous urinary corticosteroids (cortisol and cortisone) and most commonly used synthetic one (dexamethasone). Moreover, after recent findings regarding the presence of endogenous prednisolone in the urine of more popular breeds, particular attention was given to analysis of the presence of prednisolone and prednisone, as well. For this aim, the urine samples of 12 young cows and 30 young bulls was collected at the farms and analysed using a fit-for-purpose LC-MS/MS method. The preliminary results of this study show that prednisolone was found only in Chianina females (3 out of 12). Cortisol and cortisone were found at concentrations that showed a high inter-individual variability, and that were higher in female urine compared to that of males.

In the present studies, a simple rapid ultra performance liquid chromatography (UPLC) method with UV detection for the simultaneous determination of cortisol, cortisone and 6β-hydroxycortisol in human urine was developed. The three analytes and the internal standard dexamethasone were separated on a Waters Acquity UPLC-Tunable UV system with an Acquity UPLC BEH C18 column (50 × 2.1 mm ID, 1.7 μm) using a gradient elution of methanol and water (containing 0.01% formic acid) with a run time of 7 min. The method was accurate and precise, over the ranges of 5–200 ng mL−1 for cortisol, and 10–1,000 ng mL−1 for both cortisone and 6β-hydroxycortisol, and showed good linearity (r 2 > 0.999). This method was applied for the measurement of cortisol, cortisone and 6β-hydroxycortisol in samples collected over different periods as a tool to assess the activity of CYP3A and 11β-hydroxysteroid dehydrogenase type 2 enzymes.

We developed a high sensitivity method for simultaneous measurement of cortisol, cortisone and dexamethasone. Using this method, we compared concentrations of cortisol, cortisone and their ratios in samples from intensive care unit (ICU) and non-ICU patients, and cortisol and dexamethasone concentrations in patients with Cushing's and suspected Cushing's syndrome.Two hundred microliters of human serum aliquots were extracted using solid phase extraction and analyzed using liquid chromatography-tandem mass spectrometry. Primary mass transitions monitored for cortisol, cortisone and dexamethasone were m/z 363/121, 361/163 and 393/373 respectively.The limits of quantification for cortisol and cortisone were 0.3 μg/l (0.8 nmol/l) and for dexamethasone it was 0.5 μg/l (1.2 nmol/l). Total imprecision was10.9%. Median cortisol to cortisone ratio of ICU patient samples was found to be 2 times higher than non-ICU samples. 54.2% of patients after 1mg dose of overnight dexamethasone could be categorized as consistent with Cushing's syndrome.The method has high sensitivity and specificity. High cortisol to cortisone ratios in samples from ICU patients suggest change in activity of 11β-hydroxysteroid dehydrogenase in modulation of systemically available cortisol. Simultaneous measurement of dexamethasone and cortisol can be used to differentially diagnose diseases causing increased concentrations of cortisol.

The effect of training variations on 24-h urinary cortisol/cortisone (C/Cn) ratio was investigated in highly trained swimmers to determine whether it could be a good marker of training stress and performance.Fourteen swimmers (five female and nine male) were tested after 4 wk of intense training (IT), 3 wk of reduced training (RT), and 5 wk of moderate training (MT). At the end of each period, the swimmers performed in their best event at an official competition. Individual performances were expressed as a percentage of the previous season's best performance. The fatigue state was evaluated with a questionnaire.The C/Cn ratio was statistically different for the three periods (IT: 1.10 +/- 0.7, RT: 0.64 +/- 0.3, and MT: 0.57 +/- 0.2). The differences in the C/Cn ratio between two consecutive performances were related to the differences in performance (r = -0.52, P0.01), and the C/Cn ratio was significantly related to the total training (r = 0.32, P0.05) and total score of fatigue (TSF) (r = 0.35, P0.03) over the follow-up period. Cn levels were related to the dryland training (r = -0.46; P0.01) and TSF (r = -0.40; P0.02). During IT, variations in the C/Cn ratio were related to the changes in the mean intensity (r = -0.67; P0.02) and to TSF (r = 0.69; P0.01).The 24-h C/Cn ratio was moderately related to both training and performance whereas Cn levels were only related to training. The C/Cn ratio could be a useful indicator for monitoring the overreaching state in elite swimmers.

OBJECTIVE Trophoblast cells synthesise CRH and ACTH, which are peptide hormones. On the strength of special enzymes they are capable of catalyzing the reaction cortisol cortisone. In vitro experiments should give a proof of influence to ACTH- and cortisol secretion by CRH, ACTH and prednisolon. The basal rate of cortisol secretion was examined in a long term experiment. MATERIAL AND METHODS Trophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step. After adjusting the cell suspension to a defined cell concentration of 1 x 10(6) cells/ml the cells were cultivated. The addition of CRH, ACTH or prednisolon followed every eight hours. The samples collected 20 or 30 minutes later, also from unstimulated cultures, were assayed for ACTH and cortisol by enzyme-immunometric methods. RESULTS The concentration of cortisol shows a rhythmical course in long term cell cultures. The addition of CRH (500 ng/ml, 1 microg/ml) stimulates the concentration of ACTH- and cortisol in a time-depending manner. The addition of ACTH (500 ng/ml-2 microg/ml) stimulates the concentration of cortisol in a time-depending manner. The addition of prednisolon stimulates the concentration of ACTH. CONCLUSIONS The trophoblast cell shows a rhythmical course in the concentration of cortisol. For the first time a CRH-ACTH-cortisol feedback loop could be demonstrated in cultured trophoblast cells.

Patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD) have an impaired cortisol synthesis, but it is unknown whether the metabolism of glucocorticoids differs between neonates and infants with and without 21OHD.The objective of this study was to compare the glucocorticoid metabolism between neonates and infants with and without 21OHD.We analyzed 14 urinary glucocorticoid metabolites, 7 metabolites each of cortisol and cortisone, by gas chromatography-mass spectrometry of 89 untreated 21OHD neonates and infants and 161 neonates and infants without 21OHD.Neonates with 21OHD exhibit elevated relative amounts of cortisol metabolites in total glucocorticoid metabolism and an increased ratio of cortisol to cortisone metabolites (p0.0001). This reflects a shift toward cortisol in the relative balance of the interconversion between cortisol and cortisone. The ratio of cortisol to cortisone metabolites correlated significantly with low urinary glucocorticoid concentrations (p0.03), with low 21-hydroxylase activity (p0.001) and high urinary sodium and chloride concentrations (p0.05) in neonates with 21OHD.Our results demonstrate substantial changes in the relative cortisone to cortisol interconversion in neonates with 21OHD. The shift of glucocorticoid metabolism toward active cortisol in neonates with 21OHD seems to be related to the severity of 21OHD and adrenal dysfunction. Our data provide new insights into the regulation of glucocorticoid homeostasis in 21OHD.

In peripheral tissues, corticosteroid hormone action is determined, in part, through the activity of 11beta-hydroxysteroid dehydrogenases (11beta-HSD), two isozymes of which interconvert hormonally active cortisol (F) and inactive cortisone (E). 11beta-HSD type 2 (11beta-HSD2) inactivates F to E in the kidney, whilst 11beta-HSD type 1 (11beta-HSD1) principally performs the reverse reaction activating F from E in the liver and adipose tissue. Alteration in expression of these 11beta-HSD isozymes in peripheral tissues modifies corticosteroid action: loss of 11beta-HSD2 activity in the kidney results in cortisol-induced mineralocorticoid excess, and loss of hepatic 11beta-HSD1 activity improves insulin sensitivity through a reduction in cortisol-induced gluconeogenesis and hepatic glucose output. Conversely, overexpression of 11beta-HSD1 in omental adipose tissue can stimulate glucocorticoid-induced adipocyte differentiation which may lead to central obesity. Patients with hypopituitarism have many clinical features in common with patients with Cushing's syndrome--notably visceral obesity, insulin resistance, osteoporosis and increased vascular mortality. Our hypothesis was that many of these features may be explained by an effect of growth hormone (GH) on the 11beta-HSD isozymes. As assessed by urinary free cortisol/urinary free cortisone ratios and endorsed through in vitro studies, neither GH nor insulin-like growth factor (IGF)-I affect 11beta-HSD2 activity. Patients with acromegaly show a reduction in hepatic-derived metabolites of cortisol/cortisone - levels return to normal when GH concentrations are normalized. Conversely, patients with GH deficiency in the setting of hypopituitarism demonstrate an increased cortisol/cortisone metabolite ratio and reduction in circulating cortisol concentrations in patients on hydrocortisone replacement. Treatment with low-dose GH replacement reverses these abnormalities. These clinical data suggest that GH (and/or IGF-I) inhibits 11beta-HSD1 (i.e. E to F conversion) (parallel in vitro studies suggest that IGF-I and not GH inhibits 11beta-HSD1). These findings have important clinical ramifications. Firstly, the GH-mediated increase in cortisol metabolism (mediated via reduced E to F conversion) may precipitate adrenal insufficiency in hypopituitary patients with partial adrenocorticotropic hormone deficiency commencing GH therapy. Secondly, many of the phenotypic features of hypopituitarism can be explained by an alteration in 11beta-HSD1 activity: GH deficiency effectively increases cortisol production in key target tissues including liver and adipose tissue, promoting insulin resistance and visceral adiposity. Thirdly, the reported beneficial effects of GH on cardiovascular risk factors in patients with hypopituitarism may be an indirect effect via alterations in cortisol metabolism. Finally, the GH/IGF-I modulation of cortisol metabolism may underpin the pathogenesis of common diseases such as central obesity and idiopathic osteoporosis. Patients with central obesity but with no evidence of hypopituitarism have relative GH deficiency and it is exciting to speculate that low-dose GH treatment in this group, by inhibiting cortisol generation within omental fat, may offer a novel therapeutic approach.

Summary Levels of unconjugated cortisol, cortisone and 11-deoxycortisol were measured in umbilical arterial and venous plasma, and in maternal antecubital and uterine venous plasma by a method specific for each steroid, utilizing competitive proteinbinding radioassay following steroid fractionation by column chromatography. Samples were obtained (1) at elective Caesarean section, (2) at vaginal delivery or emergency Caesarean section after induced labour, and (3) at vaginal delivery or emergency Caesarean section after labour of spontaneous onset. In the umbilical circulation there was no significant arteriovenous difference for any of the steroids measured. There were significantly higher (p

To evaluate the effect of thyroid function on the in vivo cortisol ⇌ cortisone (F ⇌ E) equilibrium, double-labeled cortisol tracer techniques were used to measure separately the rates of the F → E and E → F reactions in 4 euthyroid, 2 hypothyroid, and 2 hyperthyroid subjects. The rate of the F → E reaction was calculated from the appearance rate of 3H in body water after the i.v. injection of [11α-3H]-cortisol; the rate of the E → F reaction was calculated from the difference in the plasma turn-over rates of [11α-3H]-cortisol and [4-14C]-cortisol after simultaneous i.v. injection of both tracers; the F ⇌ E set-point was calculated by dividing the F → E rate by the E → F rate. In euthyroid subjects the F → E reaction rate averaged 1.6%/min, the E → F reaction rate averaged 1.0%/min, and the F ⇌ E set-point averaged 1.6. In hyperthyroid subjects the two reaction rates were supranormal: the F → E rate averaged ⩾ 3.9%/min and the E → F rate averaged 1.54%/min; since the increase in the F → E rate was proportionally greater, the F ⇌ E set-point (average ⩾ 2.6) was displaced towards cortisone. In hypothyroid subjects both reaction rates were equally slowed, to an average of 1.0%/min (F → E) and 0.68%/min (E → F); the F ⇌ E set-point averaged 1.65, the same as in euthyroid subjects. Displacement of the F ⇌ E equilibrium towards cortisone in hyperthyroid subjects appears to account for their elevated urinary THE/THF ratios, but the normal F ⇌ E set-point in hypothyroid subjects makes it necessary to invoke a different mechanism for their depressed THE/THF ratio; it is suggested that increased conversion of THE to cortoic acids may be the responsible factor.

Plasma levels of cortisol, cortisone and aldosterone were measured during the first 20 days of life in 10 healthy calves. Cortisolemia, high at birth, decreased from birth to day 15. Changes in plasma cortisone were similar to those of cortisol. The value of the cortisol/cortisone plasma ratio increased from birth to day 8 and remained steady until day 20. A positive correlation was found between plasma levels of cortisol and cortisone, during the 6- to 20-day period; no correlation was observed before that. These results raise the question of whether, as is assumed in the human, maternal cortisol would cross the placenta with conversion to cortisone in the bovine. Plasma aldosterone increased from day 1 to day 3, and decreased steadily until day 20 when its level was very low. Negative correlation found between the values of aldosterone and sodium led us to think that natremia regulation by this hormone could be functional in the neonatal calf.

SUMMARY A method is described for the resolution and individual quantitation of cortisol, cortisone, 11-deoxycortisol and corticosterone in foetal sheep plasma. The steroids were extracted by solvent partition and separated by LH-20 Sephadex column chromatography. Radioimmunoassay was used for the measurement of 11-deoxycortisol and cortisone and competitive protein-binding for corticosterone and cortisol. The relative levels of these steroids in the plasma of chronically catheterized sheep foetuses from 12 days before birth to term and then in the newborn lamb until 2 days of age are recorded. Cortisol gradually increased from a basal concentration of between 0·5 and 3·0 μg/100 ml plasma between days 12 and 5 pre partum, and then rose rapidly to 10 μg/100 ml plasma during the last 5 days of pregnancy to reach a maximum during or just after birth. Two days post partum the levels had fallen to approximately 3 μg/100 ml plasma. The mean value for 11-deoxycortisol between days 8 and 3 pre partum was 0·4 μg/100 ml plasma and increased in the final days before delivery to 1·0 μg/100 ml. Corticosterone initially showed slightly higher levels (∼ 1·5 μg/100 ml) in the earlier period of investigation but then fell during the immediate pre-partum period to 0·8 μg/100 ml. Cortisone was not detected at any stage of the investigations. The relationship between levels of cortisol and 11-deoxycortisol in foetal plasma and myometrial contractility is shown. An increase in uterine activity was seen to occur at the time that cortisol levels were at their maximum. The 11-deoxycortisol values throughout this particular study remained low. The results are discussed in relation to recorded levels in the adult and to previous studies in vitro with regard to changing steroid biosynthetic enzyme activity.

Trace doses of one or more of the following radioactive steroids were given to 2 normal and 4 rheumatoid arthritic men: cortisol, cortisone, 20α-dihydrocortisol, 20β-dihydrocortisol and tetrahydrocortisol. The specific activities of the given steroids and some of their unconjugated and conjugated metabolites in urine were measured. The secretion rates of cortisol and cortisone for the 2 normal men, deduced from their specific activities in urine, were found to be approximately equal and in sum approximately equal to the secretion rate of cortisol deduced from the specific activities of their conjugated metabolites. Evidence of the speed of interconversion of cortisol and cortisone was obtained by giving [4-14C] cortisol and [1,2-3H] cortisone simultaneously to the 2 normal subjects and measuring the specific activities of the cortisol and cortisone in urine; also by measuring, in one subject, the proportion of 14C and 3H in plasma cortisol and cortisone at intervals after administration. The specific activities were similar, the plasma cortisone was approximately 50 % derived from cortisol at 30 min and the plasma cortisol 40 % derived from cortisone at 40 min. For both the normal subjects given 14C-cortisol and 3H-cortisone the specific activities of the urinary unconjugated metabolites were consistently and markedly higher than those of the hydrolysed conjugated metabolites. The combined secretion rate of 20α- and 20β-dihydrocortisol and tetrahydrocortisol, for one normal subject, was estimated to be approximately 4 mg/24 h. It is suggested that the findings are consistent with the hypotheses that under basal unstressed conditions, 1, cortisol and cortisone are secreted by the adrenal in approximately equal amount; 2, cortisol and cortisone are interconverted at a speed that precludes the liver from being the main site of the interconversion; 3, the major fractions of the unconjugated and conjugated cortisol and cortisone metabolites are formed at different sites.

A mixture of cortisol-3H and cortisone-14C was administered to guinea pig foetusesin situ at mid-term and full-term gestation and to adult guinea pigs. After 30 min the radioactivity recovered in the liver accounted for 3–11% of the administered dose. In the foetal liver at midterm a significant percentage of cortisol was converted to cortisone, but the reverse conversion did not occur. The latter conversion took place to a small extent at the end of gestation. In the liver of the adult animals conversion occurred in both directions. In the foetal liver the greater part of the radioactivity was unconjugated. On the other hand, in adult liver tissues conjugated radioactive material accounted for 27–30%. Five to fourteen per cent of the total radioactivity in the hepatic cells was associated with the nuclear fraction and 60–80% of the tritiated radio-active material in this fraction consisted of unchanged cortisol; unchanged cortisone accounted for 30–40%.