Rapid, specific assay for plasma cortisol by competitive protein binding.

We describe a modified competitive protein-binding method for assay of plasma cortisol. Plasma samples are deproteinized by dilution with an ethanol/phosphate buffer, followed by heating at 100 degrees C for 2 min. Horse serum is used as the source of transcortin. Free radioactivity is separated from the protein-bound component by partition into liquid scintillation counting within 60 min. The assay has better specificity and precision than a competitive protein-binding assay in which ethanol extraction and Florisil adsorbent are used, and results correlate well with those of a specific radioimmunoassay method.     

Corticosteroid-binding globulin and free cortisol in the early postoperative period after cardiac surgery.

OBJECTIVES: To characterize concentrations of corticosteroid-binding globulin (CBG), total and free serum cortisol, and free urinary cortisol in patients during the postoperative period of cardiac surgery. DESIGN AND METHODS: In 24 patients serum was sampled on the first and second postoperative day after cardiac surgery (21 procedures with thoracotomy, 3 thoracoscopic procedures); urine was collected for two 10-h periods (8 P.M. until 6 A.M.) on the respective postoperative days. Total serum cortisol and free urinary cortisol were measured with an automated chemiluminescence assay (analysis of urine after extraction with dichloromethane), and CBG using a coated-tube RIA. Free serum cortisol was calculated from the concentrations of total serum cortisol and CBG as described previously. Thirty healthy volunteers were studied as controls. RESULTS: CBG was reduced to about one-half of the normal concentration on both postoperative days. Whereas total cortisol was about two-fold increased on the first postoperative day compared to controls extremely high concentrations of free serum cortisol were calculated from CBG and total cortisol [median 136 nmol/L (interquartile range 100-185); controls 21.8 nmol/L (interquartile range 16.9-29.8)]. On the second postoperative day, median total serum cortisol was within the interquartile range of the controls, free serum cortisol in contrast was still two-fold increased. Free serum cortisol and free urinary cortisol were significantly correlated (r = 0.60). CONCLUSIONS: Extremely high concentrations of free serum cortisol are typically found in the postoperative period of cardiac surgery; under these conditions the mere consideration of total cortisol does not appropriately display the activation of the adrenal cortex.     

LEUKOCYTE LOCOMOTION AND CHEMOTAXIS : NEW METHODS FOR EVALUATION, AND DEMONSTRATION OF A CELL-DERIVED CHEMOTACTIC FACTOR

Polymorphonuclear leukocyte (PMN) locomotion and chemotaxis have been evaluated by direct microscopic observation of individual cells in thin slide-cover slip preparations, and also by observations on populations of cells migrating into a Millipore filter. The direct microscopic method used the polarity of the locomoting PMNs (broad, advancing lamellipodium and knoblike constriction at the rear) to record the direction of movement. The Boyden chamber Millipore assay was made more reliable by following the front of cells advancing into the filter, rather than counting the number of cells on the lower filter surface. Special modifications of the Millipore assay were necessary in order to distinguish between influences on rate of locomotion and true chemotaxis. In both systems the results indicate that under certain conditions leukocytes, and in particular PMNs, release into the medium a factor stimulating locomotion and exerting chemotactic action on PMNs in the vicinity. This cell-derived factor appears not to require serum factors for its release or action.     

Evaluation of a cytocentrifuge method for measuring neutrophil granulocyte chemotaxis.

An in vitro method for the assessment of neutrophil granulocyte chemotaxis employing a cytocentrifuge has been evaluated. The cytocentrifuge is used to directly sediment leukocytes on a Millipore filter, and thus (1) eliminates errors due to abnormal or unequal settling of the cells and (2) eliminates the need for serum, plasma, or albumin on the cell side of the chemotactic chamber to obtain optimal chemotaxis. In addition, other advantages of the method are that it requires smaller volumes of blood, less handling of the cells, and less time is required for visual counting of the neutrophils. The method has been utilized to assess chemotactic activity of neutrophils from 80 control subjects and over 100 patients with various diseases and to study the effects of various pharmacologic agents on neutrophil chemotactic responsiveness.     

Effect of ketoconazole on the 11-hydroxylation step of adrenal steroidogenesis

We studied the effect of ketoconazole on glucocorticoid metabolism in three patients before and after a continuous four-hour infusion of ACTH. A single 400 mg oral dose of ketoconazole caused a decrease in serum cortisol concentration, while the concentration of 11-deoxycortisol and its ratio to cortisol increased. The decrease in serum cortisol levels was not accompanied by an increase in plasma ACTH concentration. A four-hour continuous infusion of ACTH resulted in an appropriate elevation of serum cortisol, but with a marked increase in serum concentration of 11-deoxycortisol and its ratio to serum cortisol. We conclude that 400 mg of ketoconazole can decrease cortisol synthesis apparently through a partial block of the 11-beta-hydroxylation step; the observed degree of inhibition may not be sufficient to stimulate the hypothalamic-pituitary-adrenal axis or to cause overt symptoms or signs of adrenal insufficiency; and this partial block of the 11-beta-hydroxylation step becomes more evident during a continuous infusion of ACTH, which can stimulate a normal response of cortisol.     

Free serum cortisol: quantification applying equilibrium dialysis or ultrafiltration and an automated immunoassay system.

BACKGROUND: Quantification of bioactive, free serum cortisol concentrations can characterize adrenocortical function more appropriately compared to total serum cortisol measurement. Ultrafiltration or equilibrium dialysis of serum samples allow direct measurement of free serum cortisol concentrations but respective methods have poorly been validated so far. The aim of our study was to investigate the analytical performance of free serum cortisol measurement employing equilibrium dialysis and ultrafiltration. METHODS: Two commercially available ultrafiltration devices and self-assembled dialysis cells, respectively, were studied. Cortisol was quantified in filtrate or dialysate using an automated immunoassay system. Using two serum pools, the inter-assay coefficient of variation was determined for the three methods and a method comparison was performed. RESULTS: Inter-assay coefficients of variation (n=10) between 3.2% and 14.8% were observed in the imprecision study. Method comparison demonstrated close agreement between free serum cortisol results obtained by ultrafiltration and equilibrium dialysis, respectively (equilibrium dialysis=1.2xultrafiltration+3.9 nmol/L; r=0.99; n=35). CONCLUSIONS: Direct quantification of free serum cortisol after equilibrium dialysis or ultrafiltration of the samples offers acceptable reproducibility and results in close agreement can be obtained. Both methods can potentially be introduced into a routine laboratory setting.     

A combined adsorption-gel filtration technique for the determination of the cortisol-binding capacity of transcortin.

A combined adsorption-gel filtration technique has been developed for the quantitation of the cortisol-binding capacity of transcortin: Endogenous steroids are removed from plasma by adsorption on uncoated charcoal. Saturation of the "stripped" binding sites of transcortin is accomplished by equilibrating the sample with a definite amount of labeled cortisol of low specific activity (0.1 muCi/mug). Transcortin-bound [4-14C]cortisol is isolated by gel filtration over Sephadex G-50 at 4 degrees C and measured by liquid scintillation counting. The cortisol-binding capacity of transcortin is calculated directly on the basis of the known specific activity of cortisol. The modification described eliminates methodological disadvantages associated with the original gel filtration procedures, i.e. the possible interference of various endogenous steroids with cortisol binding to transcortin, and the necessity of fluorometric or colorimetric determination of protein-bound cortisol. The values of the cortisol-binding capacity of transcortin in plasma obteined by this simplified assay are in close agreement with results reported in the literature (mean +/- S.D.): healthy males 261 +/- 23 mug/l) of transcortin-bound cortisol (n = 13), healthy nonpregnant females 255 +/- 31 mug/l (n = 15), and pregnant females prior to delivery 560 +/- 82 mug/l (n = 12).     

Clinical value of serum cortisol determination (a literature review)

The generalization of the results of a considerable body of papers on the use of cortisol as an adaptation hormone allows one to conclude that the negative moments of life and health in human beings, including children, are followed by a reduction in its serum values and the moments of stressful situations of a healthy organism are attended by an increase in its serum levels. The data available in the literature on serum cortisol determination in diseases, including infectious diseases, suggest that the mild, uncomplicated, and acute forms of the disease without a history of prior premorbid conditions run mainly with the elevated serum levels of cortisol and the severe, complicated, lingering, and chronic ones in the presence of comorbidity with lowered cortisol values. It is concluded that serum cortisol measurement is an informative test for the health of a human being, his/her adaptation to the environment and, in case of disease, a test to predict its exacerbation and outcome, which allows therapy to be corrected in proper time.     

Morning and afternoon serum levels of cortisone and cortisol in asthmatic patients

While the diurnal change of serum cortisol in asthmatics has been suggested to influence the occurrence of attacks, this relation remains controversial. Serum concentrations of cortisol, cortisone, and cortisol precursors were measured in the morning and in the afternoon in asthmatic patients by reversed-phase high-performance liquid chromatography. Cortisol and cortisone were detected but their precursors were not. In remission or during attacks, the serum cortisone level in the morning was significantly lower than that in the afternoon, and the serum cortisol level in the morning did not significantly exceed that in the afternoon, which differs from the normal diurnal patterns. In patients with severe asthma in remission, the diurnal pattern of serum cortisone and cortisol was opposite to that seen in normal controls. During the attack, the serum cortisol level showed a lesser increase in the afternoon than in the morning. In patients with persistent asthmatic attacks, the serum cortisone level in the afternoon showed a significant increase as compared with that in remission while their serum cortisol level in the afternoon was equal to that in remission. The diurnal pattern and the duration of asthmatic attacks might be taken into consideration for steroid treatment of asthma.     

运动应激试验前后血清皮质醇和IL-6水平与白大衣性高血压的关系

目的探讨运动应激试验前后血清皮质醇和IL-6水平的变化及其与白大衣性高血压的关系。方法选取2014年1月至2016年8月该院收治的白大衣性高血压患者48例作为研究组,选取同期普通高血压患者30例作为普通组和30例健康体检者作为对照组。3组均在空腹状态,在功率200 W的自行车上以蹬车2min后间歇5min再重复运动直至极度疲劳,完成运动应激试验。检测比较3组运动前、运动后即刻及运动后3h的血清皮质醇和IL-6水平及平均动脉压(MAP),分析血清皮质醇和IL-6水平对白大衣性高血压的诊断价值及其与患者MAP的关系。结果与对照组比较,研究组和普通组运动前后的血清皮质醇和IL-6水平及MAP均升高;与普通组比较,研究组运动前后的血清皮质醇和IL-6水平及MAP均升高(P0.05)。研究组运动后即刻及运动后3h的血清皮质醇和IL-6水平及MAP均较运动前升高(P0.05)。ROC曲线分析结果显示,运动应激试验前后血清皮质醇和IL-6水平对白大衣性高血压的诊断价值良好,其中以运动后即刻血清皮质醇和IL-6水平联合诊断白大衣性高血压的价值最优。Pearson线性相关分析结果显示,运动应激试验前后血清皮质醇和IL-6水平与白大衣性高血压MAP均呈正相关(r分别为0.844、0.802,P0.05)。结论运动应激试验前后血清皮质醇和IL-6水平对白大衣性高血压的诊断价值良好且与其血压水平相关,可能作为白大衣性高血压诊断和病情评估的参考指标。     

External quality assessment of assays for cortisol in plasma: use of target data obtained by gas chromatography/mass spectrometry

We describe procedures for measuring cortisol in plasma and serum by isotope dilution and mass spectrometry. A method that incorporated solvent extraction, derivatization, and gas chromatography/high-resolution mass spectrometry provided data of good precision; interassay CVs were generally 3 to 4% for the concentration range of 100-650 nmol/L. Replacing solvent extraction with extraction on a column of Lipidex 1000 or extraction by immunoadsorption yielded data in excellent agreement with the first method. Plasma and serum pools were analyzed to provide target data for use in the U.K. National External Quality Assessment Scheme for cortisol assays. Routine laboratory assays, as judged by comparison with mass-spectrometric data, were generally positively biased except for analysis of a charcoal-stripped plasma supplemented with cortisol. The results emphasize the importance of using unadulterated plasma or serum pools in assessments of steroid assay procedures.     

Use of saliva for monitoring unbound free cortisol levels in serum

In order to verify the clinical usefulness of saliva in assessing the adrenocortical function, we measured saliva cortisol levels by a radioimmunoassay after extraction with dichloromethane, and compared the results with the levels of serum unbound cortisol determined by the method of equilibrium dialysis. Paired samples of saliva and serum were obtained from 10 healthy male volunteers.

Morning levels of saliva cortisol and serum unbound cortisol were 0.99 ± 0.42 and 1.56 ± 0.54 μg/100 ml, respectively, where serum total cortisol was 19.28 ± 3.56 μg/100 ml. A well-defined diurnal rhythm and a greater response to ACTH stimulation were observed in saliva cortisol than in serum total cortisol. Kinetic study of injected cortisol revealed almost identical values for the compartments of saliva cortisol and serum unbound cortisol. The correlation coefficient (r) between the levels of saliva cortisol and serum unbound cortisol was 0.893 (p < 0.001, n = 150).

From these results we concluded that the measurement of saliva cortisol can be used sufficiently to monitor its unbound free concentrations in serum.     

The diagnosis of adrenal insufficiency in the critically ill patient: does it really matter?

The definition of what constitutes a 'normal' adrenal response to critical illness is unclear. Consequently, published studies have used a variety of biochemical criteria to define 'adrenal insufficiency'. These criteria have been based on the baseline cortisol level or the increment in cortisol following corticotropin administration. However, in critically ill patients there are a number of confounding factors that make interpretation of these tests difficult. Furthermore, in those patients who are most likely to benefit from treatment with low-dose glucocorticoids, there is no evidence that treatment should be based on adrenal function testing. In those patients in whom the diagnosis of adrenal insufficiency may be important, this diagnosis may best be made based on the free cortisol level or the total cortisol level stratified by serum albumin.     

Cortisol directly determined in serum by fluoroimmunoassay with magnetizable solid phase

We developed a direct fluoroimmunoassay for cortisol in serum. This method involves cortisol labeled at the 3 position with fluorescein, and antibodies to cortisol coupled to magnetizable cellulose/iron oxide particles. Sodium salicylate is used as a blocking agent to prevent interference from endogenous binding proteins in serum. Serum sample and labeled cortisol are incubated with the antibody-coupled solid phase for 30 min. The solid phase is then separated and washed to remove free labeled cortisol and endogenous fluorophores of the sample. Finally we elute the antibody-bound fraction of the labeled cortisol into an alkaline methanolic medium and measure its fluorescence. The separation, wash, and elution steps are facilitated by magnetic sedimentation. The assay is sufficiently sensitive, specific, and reliable for routine use and correlates acceptably (r = 0.92) with an established radioimmunoassay. Precision (CV) ranged from 4 to 10% in experiments on three pooled sera; analytical recovery for sera supplemented with as much as 360 microgram of cortisol per liter was 91 to 109%.     

The steroid resistance of Crohn's disease.

BACKGROUND: A partial or complete steroid resistance, whose cause is not yet clarified, has been documented in many patients with long-standing Crohn's disease (CD). The primary aim of this study was to evaluate the number and affinity of serum protein steroid-binding sites in steroid-resistant patients with Crohn's disease. A secondary goal was to measure insulin sensitivity, an indirect index of steroid effectiveness, in these patients. METHODS: The study included 8 male steroid-resistant patients with active ileal CD and 6 healthy male volunteers. Corticosteroid binding globulin (CBG), binding capacity and affinity for cortisol were measured. The binding of cortisol to normal human serum and to serum of patients with CD was also determined. Whole body glucose uptake and oxidation were assessed by euglycemic hyperinsulinemic clamp and indirect calorimetry. RESULTS: Crohn's patients showed a significantly greater capacity of serum albumin for cortisol than controls (by about 40%, or about 0.15 moles per mole). Conversely, the binding of cortisol to CBG did not show significant differences between groups. Glucose uptake was higher in Crohn's patients than in normal controls (8.82 +/- 2.50 vs 7.01 +/- 2.24 mg/kgFFM/min; P = 0.036). Basal serum nonesterified fatty acid (NEFA) levels were lower in patients than in controls (459.64 +/- 69.95 vs 1026.48 +/- 112.58 mumol/L; P = 0.002). CONCLUSIONS: The observed increase in albumin binding might limit the bioactivity of cortisol in patients with Crohn's disease and contribute to the decreased effectiveness and weaker side effects of glucocorticoid therapy in these patients. The increased number of cortisol binding sites on albumin from patients with CD might be correlated with the significant decrease in serum NEFA, which may compete with steroids for the same sites.     

Leucodepletion process performance variation: is it dependent on the batch of filters, the processing centre or the counting technology?

The aim of this retrospective study was to determine whether variations in the enumerated white cell contamination of leucocyte-depleted products was caused by the filter batch, the processing centre or by counting technology related issues. The influence of donor variation is also considered. The results suggest that for some red cell processes, variation is mainly the result of counting technology differences. Other products do not display similar trends though all leucodepletion processes may give rare high white count failures due to donor related issues, though defective filter batch cannot be excluded requiring continual review.     

Longitudinal evolution of HIV-1-associated lipodystrophy is correlated to serum cortisol:DHEA ratio and IFN-alpha

BACKGROUND: We have previously shown that lipid alterations in HIV-1-associated lipodystrophy (LD) are correlated with decreased serum dehydroepiandosterone (DHEA) and increased cortisol:DHEA ratio and IFN-alpha levels. OBJECTIVE: To evaluate in a longitudinal study whether steroid and cytokine modifications are associated with the evolution of physical changes and lipid alterations associated with LD. METHODS: Thirty-four HIV-1-positive men were followed during 32.5 +/- 4.0 months and tested at four time-points. The patients were subdivided into five groups according to physical changes and anthropometric measurements: LD-negative, initially LD-negative becoming LD-positive, LD-positive unchanged, aggravated or improved. Serum lipids, apolipoproteins, adrenal steroids and cytokines were measured and compared with baseline values. RESULTS: (1) LD aggravation is associated with persistent elevated lipids, a decrease in serum DHEA, an increase in cortisol:DHEA ratio and persistent high levels of IFN-alpha. (2) LD improvement is associated with normalization of serum lipids, an increase in serum DHEA leading to normalization in cortisol:DHEA ratio, and normalization of IFN-alpha levels. (3) In LD-positive men evolution of VLDL cholesterol is negatively correlated with DHEA (r = -0.56, P < 0.01) and positively with cortisol:DHEA ratio (r = 0.62, P < 0.004) and with IFN-alpha (r = 0.57, P < 0.01). (4) The switch to LD is associated with a decrease in serum DHEA. (5) Patients who remained LD-negative maintained normal lipids, elevated cortisol and DHEA, and normal cortisol:DHEA ratio and normal levels of IFN-alpha. CONCLUSIONS: This study indicates that cortisol:DHEA ratio and serum IFN-alpha levels are closely associated with clinical evolution and atherogenic lipid alterations in LD.     

Serum cortisol/cortisone ratio after Synacthen stimulation

OBJECTIVES: 11beta-Hydroxysteroid dehydrogenase (11beta-HSD) enzymes interconvert active cortisol and inactive cortisone. There is growing evidence that local tissue concentrations of cortisol are generally modulated by site specific different 11beta-HSD actions. While 11beta-HSD type 2 unidirectionally inactivates cortisol, type 1 isoform acts bidirectionally. 11beta-HSD type 1 is mainly localized in the liver and may thus restore circulating biologically inactive cortisone to active cortisol thereby modulating systemic glucocorticoid action; such a mechanism might be of importance in stressful situations. To study this hypothesis we investigated the influence of exogenous ACTH on serum cortisol/cortisone ratio. DESIGN AND METHODS: Paired serum samples that were submitted for routine analysis of cortisol before and 1 h after stimulation with 250 microg ACTH (1-24) (Synacthen) were collected prospectively if the routine tests indicated normal adrenal function; 40 patients were included in the study, 29 patients were female, 11 male, median age was 31 yr (range 14-70). Serum cortisol and cortisone were determined using LC-ESI/MS/MS with an online sample extraction system and tri-deuterated cortisol as the internal standard. RESULTS: While mean serum cortisol increased by 109% (mean basal concentration 373 nmol/L (SD 151 nmol/L), stimulated 781 nmol/L (SD 194 nmol/L)), mean serum cortisone significantly decreased after ACTH administration by 31% (p < 0.001, paired t-test for differences). Mean serum cortisone was 70 nmol/L (SD 16 nmol/L) before and 48 nmol (SD 16 nmol/L) after ACTH administration; decrease in serum cortisone was observed in 34 (85%) of the patients. The ratio of serum cortisol/cortisone increased in all subjects (mean 5.4 (SD 1.9) before ACTH, and 16.2 (SD 6.2) after ACTH; p < 0.001). CONCLUSIONS: The data of our observational study suggest a modulation of peripheral metabolism of cortisol by ACTH with a stimulation of systemic 11beta-HSD type 1 activity, leading to restoration of inactive cortisone to biologically active cortisol.     

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.     

Effects of ciprofloxacin on testosterone and cortisol concentrations in healthy males.

Several inhibitors of oxidative drug metabolism inhibit the synthesis of endogenous compounds such as testosterone and cortisol. Since ciprofloxacin is a potent inhibitor of the metabolism of a number of drugs, we studied its effect on serum testosterone and cortisol concentrations in eight healthy male subjects. Blood samples were collected over a 12-h period under baseline conditions and following the first and final doses of ciprofloxacin (500 mg orally every 12 h for 4 days). No significant differences in concentrations or area under the concentration-time curve were found when baseline values were compared with those observed for either testosterone or cortisol after ciprofloxacin administration. These results suggest that ciprofloxacin is unlikely to have either antiandrogenic side effects or clinical utility in lowering testosterone or cortisol concentration.