Synthesis of 7 alpha- and beta-carboxymethyl derivatives of cortisol, corticosterone, deoxycorticosterone and cortisone. Immunogenic properties of cortisol, corticosterone and deoxycorticosterone derivatives

7 alpha- and 7 beta-Carboxymethylderivatives of cortisol, corticosterone and deoxycorticosterone have been synthetized. After coupling to bovine serum albumin, they were used to elicit antibodies in rabbits. Highly specific antisera were obtained which may possibly be used for a direct radioimmunoassay of these steroids in human and rodent plasma. In the case of the derivatives of cortisol and corticosterone and stereoisomery of the coupling had an effect on the affinity and the specificity of the antisera. In all immunized rabbits the antisera obtained with the 7 alpha-derivative had a higher affinity and a narrower specificity than the antiserum obtained with the 7 beta-derivative.     

Simultaneous determination of serum cortisol and cortisone by reversed-phase liquid chromatography with ultraviolet detection

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cortisol and cortisone in a single extract of 1 ml of serum is described. The method employs meprednisone as the internal standard. The steroids were analysed isocratically by reversed-phase HPLC with an octadecylsilane-bonded (ODS) column using ultraviolet detection. The matrix effect was reduced by lowering the sample pH by adding glacial acetic acid to the sera. The samples were then filtered through regenerated cellulose membranes at 4°C and extracted with diethyl ether. The dried eluates were redissolved in the mobile phase and injected into the column. The detection limit of the assay for both steroids was 500 ng/l. Cortisol was determined in twenty serum samples by both HPLC and radioimmunoassay (RIA). The results were similar. Interference by other steroids and certain steroid analogue drugs was also studied. The HPLC method yielded no cross-reactivity between the different steroids as may occur with the RIA technique. The HPLC method was technically easy to perform and it allowed us to quantify both cortisol and cortisone in a single serum extract with high specificity.     

Simultaneous determination of cortisol and cortisone in urine by reversed-phase high-performance liquid chromatography clinical and doping control applications

A reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous determination of cortisol and cortisone in human urine samples using methylprednisolone as the internal standard is decribed. The method involves the systematic use of isocratic mobile phases of water and methanol, acetonitrile or tetrahydrofuran and a reversed-phase Hypersil C₁₈ column. A water-acetonitrile mixture used as the mobile phase proved to be the most adequate one for analyzing urine samples purified by solvent extraction. The proposed method is sensitive, reproducible and selective. It was applied to the determination of cortisol and cortisone in several human urine samples: healthy subjects, sportsmen before and/or after stress for doping control purposes, and patients with Cushing's syndrome.     

Radioimmunoassay of corticosterone,cortisol and cortisone: Their application to human cord and maternal plasma

A critical analysis of the applicability of corticosterone, cortisone and cortisol radioimmunoassays in human plasma is presented. Rabbits immunized with corticosteroid-3-CMO-BSA conjugates yielded specific antisera. Yet, their specificity may become insufficient when the concentrations of weakly cross-reacting steroids are much higher than that of the steroid to be assayed. In these particular instances, appropriate and simple purification steps, such as hexane extraction or celite chromatography, must be added. Normal values for pregnant women at delivery and their babies are presented.     

Simultaneous determination of prednisolone, prednisone, cortisol, and cortisone in plasma by GC-MS: estimating unbound prednisolone concentration in patients with nephrotic syndrome during oral prednisolone therapy

Individual variability of the pharmacokinetics of prednisolone based on the unbound concentration in plasma is of significant clinical consideration. The unbound concentrations of prednisolone were measured in 10 patients with nephrotic syndrome, two patients with systemic lupus erythematosus, and one patient with dermatomyositis by examining protein bindings of prednisolone on one or more occasions during prednisolone treatment. In this study, plasma concentrations of prednisolone, prednisone, cortisol, and cortisone were simultaneously analyzed by GC-MS by using stable isotope-labeled internal standards. Equilibrium dialysis was employed to accurately estimate the unbound fractions of prednisolone in plasma. The unbound fraction of prednisolone changed depending on plasma total prednisolone concentration and plasma albumin concentration. The unbound fraction of prednisolone (Y) is calculated: Y=(-0.0101x' + 0.0736) x + 10.23, where x' is the plasma albumin concentration and x is the total prednisolone concentration. The estimated concentrations of unbound prednisolone by using the above equation were in good agreement with the measured concentrations of unbound prednisolone. Since the protein binding of prednisolone did not change in the presence of prednisone (114.0 ng/ml), it appeared that prednisone produced from the therapeutic dose of prednisolone did not affect the unbound fraction of prednisolone.     

Simultaneous determination of urinary CEA, ferritin and TPA in Egyptian bladder cancer patients.

Urinary carcinoembryonic antigen (CEA), ferritin (Fer) and tissue polypeptide antigen (TPA) were determined in 328 cases (106 with bladder cancer, 152 with non-malignant urinary tract disease and 70 healthy controls). CEA was determined by the kit supplied by Roche Diagnostica (CEA EIA Doumab 60), ferritin by the Tandem-E Fer kit supplied by Hybritech and TPA by the Prolifigen TPA-IRMA kit supplied by Sangtec Medical. The results of this work revealed that combined determination of urine CEA and Fer, CEA and TPA or Fer and TPA showed higher sensitivity than determination of the individual markers. There was no significant difference between combined and individual marker determination with respect to false positivity in non-malignant urinary tract diseases. At 97% specificity, the sensitivities of urine CEA, Fer and TPA were 82.1%, 71.7% and 90.6%, respectively, while combined urine CEA & Fer, CEA & TPA and Fer & TPA showed sensitivities of 92.5%, 99.1% and 98.1%, respectively. When the specificity was related to the entire non-cancer group (patients with benign urinary tract diseases and normal controls), some reduction in the sensitivities of the combined markers was noted compared to the normal group only. In conclusion, combined determination of urine markers is superior to determination of individual markers in the diagnosis of bladder cancer.     

Urinary free cortisol and cortisone excretion in healthy individuals: influence of water loading

The influence of water loading on urinary excretion of free cortisol and cortisone was investigated in healthy men. The results were as follows: water loading tests (intake of 0.25-1.5 L) in a single individual showed that a water load of 1.5 L reliably increased the excretion of urine, free cortisol and cortisone (p < 0.01). Regression analyses gave significant correlations of urine volume with free cortisol and free cortisone, and of free cortisol and free cortisone. Corresponding results were obtained when water loading tests were performed in males who ingested 1.5 L of water (n = 8): the excretion of urine, free cortisol and free cortisone were significantly augmented; correlated was urine volume with free cortisol and free cortisone, and free cortisol with free cortisone. In a third set of tests, volunteers collected one 5 h urine (10:00-15:00 h) after the intake of 3 x 0.1 or 0.5 L at 11:00, 12:00 and 14:00 h. Excretion of urine, free cortisol and free cortisone in males of the low water loading group (3 x 0.1 L) was 0.59 mL/min, and 8.2 or 15.0 microg/5 h; corresponding values in individuals ingesting 3 x 0.5 L of water were 1.5 mL/min (p < 0.01), 12.3 microg/5 h (p > 0.05) and 26.3 microg/5 h (p < 0.02). In summary, urinary free cortisol and cortisone excretion in healthy men depends on urine volume, especially during water diuresis. Thus, interpretation of free cortisol and especially of free cortisone excretion is only possible if subjects strictly control their fluid intake and if urine volume is considered an important pre-analytical parameter-otherwise, interpretation of urinary free cortisol results is difficult and of urinary free cortisone data remains tenuous at best.     

Determination of urinary free cortisol and cortisone by sequential thin layer and high-performance liquid chromatography

A specific, rapid and sensitive method for urinary free cortisol and cortisone utilizing sequential thin layer (TLC) and high-performance liquid chromatography (HPLC) was developed. After addition of prednisone as the internal standard to 1 ml of urine, extraction of the steroids was accomplished by automated sorption on and desorption from a styrenedivinylbenzene copolymeric resin cartridge. Further purification was carried out by TLC. Quantitation of the recovered steroids at 254 nm was achieved during HPLC in a reverse phase system with 21-deoxycortisone as the external standard. When the values of urinary free cortisol both from normal subjects and patients were compared with those obtained by a current RIA procedure, the greater specificity of the new method was clearly demonstrated. Simultaneous measurement of both urinary free cortisol and cortisone in various clinical states appears to offer a more complete index of adrenocortical function than urinary free cortisol alone.     

HPLC method for determination of fluorescence derivatives of cortisol,cortisone and their tetrahydro- and allo-tetrahydro-metabolites in biological fluids

11β-Hydroxysteroid dehydrogenase isoform 2 (11β-HSD2) is responsible for conversion of cortisol (F) to inactive cortisone (E). Disturbance of its activity can cause hypertension. To estimate 11β-HSD2 activity, besides F and E, their tetrahydro- (THF, THE) as well allo-tetrahydro- (allo-THF, allo-THE) metabolites should be determined. This study describes HPLC-FLD method for the quantitative determination of endogenous glucocorticoids (GCs) in plasma and urine (total and free) and their metabolites in urine. Following extraction at pH 7.4 using dichloromethane, GCs (F, E, THF, allo-THF, THE, allo-THE and internal standard – prednisolone) were derivatized with 9-anthroyl nitrile and purified by SPE using C₁₈ cartridges. The enzymatic hydrolysis of conjugated steroids was provided using β-glucuronidase. The influence of organic bases on 9-AN derivatization of steroids was investigated. The best yield of the derivatization was obtained in presence of the mixture of 10.0% triethylamine (TEA) and 0.1% quinuclidine (Q). Chromatographic separation was accomplished in the Chromolith RP-18e monolithic column. The elaborated method was validated. Calibration curves were linear in the ranges: for F, E and THF 5.0–1000.0 ng mL^?1, for allo-THF and THE + allo-THE 10.0–1000.0 ng mL^?1. LOD (S/N = 3:1) for all analytes amounted 3.0 ng mL^?1. Recoveries of GCs exceeded 90%. The method was precise and accurate, intra- and inter-day precision were 3.0–12.1% and 9.2–14.0%, respectively. Accuracy ranged from 0.2 to 15.1%. The method was applied for estimating endogenous GCs in plasma and urine. Plasma levels of F and E were in the ranges: 133.0–174.5 ng mL^?1 and 17.4–35.9 ng mL^?1, respectively. Free urinary steroids were in the ranges: 12.0–54.1 μg/24 h (UFF) and 37.8–76.2 μg/24 h (UFE). The ratio of (THF + allo-THF)/(THE + allo-THE) amounted from 1.01 to 1.23. The obtained results confirmed utility of the elaborated method in the assessment of 11β-HSD2 activity in man.     

EEG spectral coherence data distinguish chronic fatigue syndrome patients from healthy controls and depressed patients-A case control study

Background  

Previous studies suggest central nervous system involvement in chronic fatigue syndrome (CFS), yet there are no established diagnostic criteria. CFS may be difficult to differentiate from clinical depression. The study's objective was to determine if spectral coherence, a computational derivative of spectral analysis of the electroencephalogram (EEG), could distinguish patients with CFS from healthy control subjects and not erroneously classify depressed patients as having CFS.