Determination Of Serum Cortisol Research Articles

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.

Determination of serum cortisol by reversed-phase liquid chromatography using precolumn sulphuric acid—ethanol fluorescence derivatization and column switching

An assay method for serum cortisol, using precolumn sulphuric acid—ethanol fluorescence derivatization and reversed-phase liquid chromatography with a column-switching technique, has been developed. The crude precolumn fluorescence cortisol derivative was prepared by the addition of sulphuric acid to serum deproteinized with ethanol, and directly injected onto an octadecylsilane-bonded silica gel (ODS) precolumn for concentration and purification. After switching columns the samples were separated using an ODS analytical column and monitored fluorimetrically. When the pH of the mobile phase in the analytical separator decreased to 1.85, the emission wavelength of the cortisol derivative changed to 520 nm (excitation of 365 nm) and the fluorescence itensity increased. Among the sulphuric acid—ethanol derivatives of various steroids, cortisol, corticosterone and testosterone emitted fluorescence. However, their retention times differed from those of the cortisol derivatives (12.5 min). The detection limit of cortisol was 0.3 μg/dl (signal-to-noise ratio of 3). Use of the fully automated column-switching system contributed to good reproducibility and recovery.

Multicentre Evaluation of an Enzyme-Immunoassay for Cortisol Determination

The present paper describes a multicentre evaluation of a one step enzyme-immunoassay for the determination of cortisol in serum or plasma. Data from the investigation were analysed in terms of imprecision, detection limit, and correlation with other test methods. Within-run and between-run imprecisions (coefficient of variation) of Enzymun-Test Cortisol were less than 8% and 12%, respectively. The detection limit was 30 nmol/l (11 micrograms/l). With the exception of prednisolone, only low interference was found with other endogenous steroids. A good correlation between Enzymun-Test Cortisol and HPLC, LIA, FPIA and RIA was registered, although the latter two methods showed a scattering of regression lines from the different evaluators. The results show that Enzymun-Test Cortisol can be recommended as an alternative for the measurement of cortisol. As the method is calibrated against isotope dilution-mass spectrometry, results obtained with Enzymun-Test Cortisol are in agreement with the reference method.

液体クロマトグラフィー (血清直接注入法) による血中Cortisolの測定

液体クロマトグラフィー (血清直接注入法) による血中Cortisolの測定

Mass-spectrometric determination of serum cortisol: comparison of data from two independent laboratories.

Isotope dilution and mass spectrometry were used in two independent laboratories to determine cortisol in 15 plasma and serum pools used in the British and German national schemes for the external quality assessment of routine assays. For the concentration range 240-700 nmol/L, differences between the data obtained by the two laboratories were generally less than 4% but were approximately 7% in two instances. The discrepancies are nevertheless small in comparison with the bias observed for many routine assays.

Direct radioimmunoassay of cortisol in saliva and its application to the dexamethasone suppression test in affective disorders

In order to perform the dexamethasone suppression test (DST) with saliva as an alternative to serum, we assayed directly the cortisol concentrations in 25 μl saliva samples, using a commercial radioimmunoassay kit for serum cortisol with minor modifications. Cortisol in saliva showed a diurnal rhythm parallel to that of cortisol in serum samples collected simultaneously. Saliva cortisol levels increased significantly after ACTH injection, but with a 60 min delay in reaching their peak compared to peak serum cortisol levels. The increase in saliva cortisol was five-fold, while that in serum was two-fold. Saliva cortisol levels continued to increase in some subjects while serum total cortisol levels already had begun to decline. In those subjects, the correlation of saliva with serum cortisol was greater when a quadratic curve was fitted than when calculated for a linear correlation. Considerable variation was observed for within-subject correlations, ranging from +0.48. to 0.999. The DST with saliva sample collection was performed on 43 inpatients with affective disorders. Sensitivity, specificity and diagnostic confidence of the DST for major depressive episode with melancholia were 33%, 91%, and 78%, respectively, at the criterion value of 0.3 μg/100 ml for saliva cortisol, which are similar to those most often reported for the DST with serum cortisol determination. These results indicate that saliva cortisol levels do not always parallel serum cortisol levels and thus are not an unequivocal substitute. The findings for the DST in psychiatric patients, however, support the practical clinical usefulness of saliva cortisol measurements.

Clinical applications of the rapid high-performance liquid chromatographic determination of serum cortisol

We report a rapid and specific analytical method for the measurement of serum cortisol by reversed-phase high-performance liquid chromatography (HPLC). The method allows the diagnostic measurement of cortisol in emergency clinical circumstances of glucocorticoid deficiency or excess. Results obtained by HPLC are comparable to those by radioimmunoassay. The lower limit of sensitivity is 6 ng cortisol per ml of serum.

Replacement of serum cortisol determination by measurements of cortisol in saliva

Journal Article Replacement of serum cortisol determination by measurements of cortisol in saliva Get access U Deuss, U Deuss 2. Medizinische Universitätsklinik, Köln-Merheim Search for other works by this author on: Oxford Academic Google Scholar B Allolio, B Allolio 2. Medizinische Universitätsklinik, Köln-Merheim Search for other works by this author on: Oxford Academic Google Scholar G Feltes, G Feltes 2. Medizinische Universitätsklinik, Köln-Merheim Search for other works by this author on: Oxford Academic Google Scholar D Kaulen D Kaulen 2. Medizinische Universitätsklinik, Köln-Merheim Search for other works by this author on: Oxford Academic Google Scholar Acta Endocrinologica (Norway), Volume 104, Issue 4_Supplement_b, Dec 1984, Page S109, https://doi.org/10.1530/acta.0.107S109 Published: 01 December 1984

Homogeneous enzyme immunoassay for cortisol with a centrifugal analyzer.

We determine serum cortisol by a homogeneous enzyme immunoassay in the Cobas Bio centrifugal analyzer. To unbind cortisol from its protein complex, serum is treated for 15 min with an acidic solution. The reaction then proceeds automatically in the analyzer at 37 degrees C. To 50 microL of sample mixture is added 125 microL of reagent (cortisol antibodies, glucose 6-phosphate, and NAD+). This mixture is incubated for 60 s, after which 25 microL of a cortisol derivative labeled with glucose 6-phosphate is added; the increase in absorbance is monitored at 340 nm. The standard curve was linear from 10 to 500 micrograms of cortisol per liter. Within-assay precision (CV) varied from 0.2 to 0.6%, between-assay precision from 6.2 to 10.6%. Analytical recovery ranged from 100 to 103%. Results for control samples deviated from target values by 1.4 to 7.8%. Results compared well with those by radioimmunoassay. The method is reliable and practicable and will usefully replace previous routine methods for serum cortisol.

The Effects of a Pacifying Stimulus on Behavioral and Adrenocortical Responses to Circumcision in the Newborn

Eighteen, healthy male newborns, 2–5 days old, were subjects in this study of the effects of a pacifier on the newborn's behavioral and adrenocortical responses to circumcision. Half of the subjects were randomly assigned to a condition in which they were encouraged to suck on a pacifier during circumcision, while half served in a no pacifier, control condition. Behavioral observations were made for 1/2 hour before, during, and after circumcision; while blood samples for serum cortisol determination were obtained immediately before circumcision and 30 min later. The results showed that stimulating the newborn with the pacifier reduced crying by about 40%. Reducing crying, however, had no significant effect on the adrenocortical response. Elevations of serum cortisol predicted average behavioral state following circumcision, whereas crying during circumcision did not. Furthermore, there was evidence that the neonatal adrenocortical system was sensitive to variations in surgical procedures. The results indicate the importance of obtaining data on both behavioral and hormonal systems in studies of stress and coping in human newborns.

Improved liquid chromatographic determination of serum cortisol with double internal standardization compared to radioimmunoassay and fluorometry, and evaluated by isotope dilution/mass spectrometry

A sensitive and specific high-performance liquid chromatographic (HPLC) method for the determination of cortisol in only 200 μl of serum is described. Cortisol and two internal standards, 19-nortestosterone (IS 1) and 6α-methylprednisolone (IS 2) are extracted with dichloromethane and analyzed on a C 18 reversed-phase column eluted with a mobile phase of methanol:water at a flow rate of 0.75 ml/min. Ultraviolet absorption at 254 nm is used for detection and quantitation is performed by peak height ratio measurement. Using 200 μl of serum, the lower limit of detection for cortisol is 10 ng/ml, the analytical recovery is 104 ± 3.6% ( n = 8), and the day-to-day precision was 1.69% at a level of 90 ng/ml ( n = 16). Cortisol values obtained by this method were generally lower than those obtained by radioimmunoassay or by fluorometry. A serum pool was analyzed both by HPLC and by isotope dilution/mass spectrometry ( ID MS ). A mean value of 90.1 ng/ml was obtained by HPLC ( n = 16, CV = 1.7%), whereas ID MS yielded a mean of 90.8 ng/ml ( n = 28, CV = 0.4%). These results clearly demonstrate the high specificity and the accuracy of the HPLC procedure. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of medication by either of these two products.

The simultaneous determination of serum cortisol and cortisone by high-performance liquid chromatography with fluorimetric detection

A new method for the simultaneous determination of cortisol and cortisone in serum by high-performance liquid chromatography with fluorimetric detection has been developed. The two steroids were derivatized by treatment with 9-anthroyl nitrile in triethylamine-acetonitrile to give the fluorescent esters through the 21-hydroxyl group. These derivatives were efficiently separated on a straight-phase Cosmosil 5SL column with hexane-ethyl acetate (6:5) as eluent. The detection limit was 20 fmol, using the 9-anthroyl derivative of prednisolone as an internal standard. The use of Sep-Pak C18 and Clin-Elut cartridges proved to be efficient for the clean-up of cortisol and cortisone in serum samples.

Dissociation of cortisol and adrenal androgens (AA) in selected patients with hypopituitarism

Dissociation of secretion of cortisol and AA, observed in several physiologic and pathologic conditions, has led to the hypothesis that secretion of AA may be controlled by another hormone in addition to ACTH. An alternate hypothesis attempts to explain all instances of such dissociation by changes in ACTH alone, wherein ACTH controls synthesis of AA by changing the intraadrenal concentration of cortisol. This hypothesis does not allow for dissociation of cortisol and AA in a subject with normal ACTH secretion and intact adrenals. To test this hypothesis, we studied cortisol secretion in 4 patients (20-26 years) with idiopathic hypopituitarism (who never received cortisol replacement therapy) and deficiency of AA as reflected by lack of adrenarche and low levels of serum DHEAS. Determination of serum cortisol and 17-OH progesterone (17 OHP) at 2 h intervals for 2 days and of cortisol and 17 OHP responses to insulin induced hypoglycemia and glucagon infusion revealed normal cortisol and 17 OHP secretion in 2 of the 4 patients. Although intraadrenal concentrations of cortisol and serum levels of ACTH were not measured, there is no reason to suspect that these levels should be abnormal in the 2 patients who had normal cortisol secretion. Low serum levels of DHEAS in these patients cannot be explained by deficiency of ACTH secretion. Our data provide further evidence that secretion of AA may be controlled by another hormone in addition to ACTH.

Improved liquid-chromatographic method for determination of serum cortisol.

We describe a specific and precise method for measuring concentrations of cortisol in serum or plasma by liquid chromatography. Cortisol, together with an internal standard, equilenin, is extracted from 1 mL of serum or plasma and analyzed isocratically on a reversed-phase column with a mobile phase of acetonitrile/phosphate buffer (30/70, by vol.), at a flow rate of 2.0 mL/min. The eluted cortisol is detected by its absorption at 254 nm and quantitated by peak height measurements. Each analysis requires no longer than 15 min at the optimum column temperature of 50 degrees C. The lower limit of detection for cortisol is about 2 ng/sample for a standard solution; sensitivity is routinely 5 micrograms/L of serum. Analytical recoveries exceeded 95%, with good day-to-day precision (coefficients of variation between 4 and 7%). Of more than 50 drugs and steroids tested for possible interference, only the steroids cortisone, prednisone, and prednisolone may interfere with the analysis of cortisol.

Fluorescence polarization immunoassay for cortisol

A fluorescence polarization immunoassay for serum cortisol was established using cortisol 21-amine which was readily coupled with fluorescein isothiocyanate. The proposed method is sufficiently sensitive, reliable, specific and simple for routine determination of serum cortisol. The assay is rapid, without separation of antibody-bound and free ligands. The minimal amount of cortisol detected was 1.5 ng/tube and the measurable range was from 1.5 to 100 μg/dl. There was a good correlation between values obtained from radioimmunoassay and the proposed method.

EVALUATION OF STEROID LABORATORY TESTS AND ADRENAL GLAND IMAGING WITH RADIOCHOLESTEROL IN THE AETIOLOGICAL DIAGNOSIS OF CUSHING'S SYNDROME

Basal values of the urinary excretion of 17-oxogenic steroids and serum levels of cortisol were not satisfactory in the differentiation of 'suspected' subjects from patients with true Cushing's syndrome. With an RIA method for serum cortisol determination, the overnight dexamethasone suppression test provided the most reliable single test in establishing adrenocortical hyperfunction. Thirty-five normal subjects, fifty-nine obese patients, thirteen 'suspected' patients, and thirteen patients with disease states other than Cushing's syndrome had suppressed values below 4.0 microgram/100 ml. None of the ten patients with Cushing's syndrome had a cortisol concentration less than 16.3 microgram/100 ml. Adrenal gland scintigraphy after radiocholesterol injection is a more valuable tool than the metyrapone test and the high-dose dexamethasone suppression test in the localization and differential diagnosis of adrenocortical lesions causing Cushing's syndrome. It obviates the need for angiographic procedure in the localization of adenomas. It is a reliable technique for identifying functioning adrenal remnants. We therefore propose a schedule for studying patients with suspected adrenocortical hyperfunction.

Determination of serum cortisol and 11-deoxycortisol by liquid chromatography.

Determination of serum cortisol and 11-deoxycortisol by liquid chromatography.

Enzyme immunoassay for cortisol in serum using cortisol 21-amine

Cortisol 21-amine (21-amino-11β,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 μg/d1, using 10 μ1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme Immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.

Comparison of competitive protein binding analysis and radioimmunoassay for the determination of cortisol in serum and urine

The determination of serum and urinary free cortisol by competitive protein binding and radioimmunoassay were compared on patient samples using two commercially available kits. In addition, the Beckman RIA kit reagents were examined for precision and accuracy through the performance of reproducibility, recovery, cross reactivity and parallelism studies on serum and urine. Analytical recovery experiments with cortisol added to urine and serum showed 90% and 97% recovery respectively with the RIA reagents. Good reproducibility and parallelism criteria were also obtained using Beckman kit reagents. Antiserum specificity was documented and demonstrated no cross reactivity with structurally related steroids at clinically normal serum concentrations.

An integrated direct functional test of the adenohypophysis

A sequential stimulation by arginine infusion and LRF plus TRF injection for the release of five pituitary hormones (LH, FSH, GH, PRL, and TSH) as an integrated functional reserve test of the adenohypophysis was devised. The serial hormonal release elicited by sequential stimuli in normal male (N = 11) and normal female subjects (N = 11) was characterized; concomitant with a rise in GH, arginine induced a consistent increase in PRL levels; a simultaneous rise in serum TSH and PRL, and in LH and FSH occurred in response to TRF and LRF, respectively. The usefulness of this integrated reserve test was evaluated in patients with abnormal hypothalamic-pituitary axis; differentiation between hypothalamic vs. pituitary defect with single or multiple hormone involvement may be revealed in patients with hypopituitarism, amenorrhea-galactorrhea syndrome, and “hypothalamic amenorrhea.” Thus, together with serum cortisol determinations, all trophic hormones of the adenohypophysis may be accurately assessed in this simple six-hour integrated test.