Determination Of Cortisol In Plasma Research Articles

Cushing's disease with intermittent hypercortisolism

In a patient with proved pituitary-dependent Cushing's syndrome (Cushing's disease), 24-hour urinary excretion of free cortisol fluctuated between normal (69 percent of the time, often in the low range and for several days in sequence) and high values. Increased urinary free cortisol excretion occurred unpredictably within the context of a persistent, progressive clinical picture. This case stands in contrast with previous reports of urinary steroid levels varying in a periodic infradiem pattern. Even with normal baseline cortisol indexes, control of the hypothalamic-pituitary axis (as indicated by the suppression test and by the circadian cortisol pattern in plasma) remained abnormal. This patient emphasizes the fact that abnormal control regulation, more than cortisol hypersecretion, is at times indicative of Cushing's disease. Hence, sensitive accurate screening requires not only urinary free cortisol measurement (the usefulness of which may be improved by assay of more than one, possibly nonsequential, 24-hour urine sample), but also dexamethasone suppression testing and late-evening plasma cortisol determination, even if baseline indexes are within the range of normal.

Salivary cortisol for the evaluation of Cushing's syndrome

Cortisol concentrations were measured in matched plasma and salivary samples from 8 healthy controls, 8 patients with Cushing's syndrome and 4 patients suspected of having spontaneous hypercortisolism. In healthy subjects, the circadian rhythm in salivary cortisol paralleled that in plasma. Absence of the diurnal rhythm in Cushing's syndrome was seen in saliva as well as in plasma. After ACTH stimulation, mean peak cortisol in saliva showed a 3-fold increase while in plasma there was a 2.5-fold increment above baseline. Cushing's syndrome, due to pituitary or adrenal adenoma was diagnosed equally well by measuring the cortisol response to cosyntropin in either plasma or saliva. Finally, the low- and high-dose dexamethasone suppression test was reflected equally well in both plasma and saliva. In patients suspected of having Cushing's syndrome dynamic tests can be performed in both plasma and saliva. However, in some samples, the salivary cortisol measurement appears advantageous over plasma cortisol determination.

Adrenocortical and behavioral responses to limb restraint in human neonates.

Two experiments were conducted on human neonates examining their behavior and adrenocortical activity in response to physical restraint. In both experiments, newborns (9 per condition in Experiment I; 10 per condition in Experiment II) were tested in one of three conditions: Restraint, which involved strapping newborns to a circumstraint board for 20 min; Strapping Control, which involved strapping newborns to the board and then immediately taking them off the board and returning them to their cribs; and Control, which did not involve any restraining manipulation. In Experiment I, blood samples for plasma cortisol determination were obtained via heel-stick immediately prior to the assigned manipulations and 30 min later. In Experiment II, only the postmanipulation blood sample was taken. Two behavioral states, Active Awake and Crying, were examined as indices of behavioral distress. In Experiment I, the effects of the blood sampling procedure on behavioral distress and plasma cortisol were significant; however, no significant effects of restraint were observed. In Experiment II, restraint was associated with small but significant increases in behavioral distress and in plasma cortisol levels.

Coping with Aversive Stimulation in the Neonatal Period: Quiet Sleep and Plasma Cortisol Levels during Recovery from Circumcision

Measures of behavioral state and plasma cortisol were obtained on 80 healthy, full-term, 2-3-day-old, male newborns who were scheduled to be circumcised. To establish baseline or precircumcision levels, the newborns were observed, and behavioral state was recorded for the half hour prior to circumcision. Blood was sampled via heelstick for plasma cortisol determination at the end of this observation period. The newborns were then circumcised and assigned randomly to one of 4 postcircumcision, blood-sampling time-point groups. The time points were 30, 90, 120, and 240 min following the beginning of circumcision. Behavioral state was observed during circumcision and for the half hour prior to taking the second blood sample. The results showed a return to baseline cortisol levels sometime prior to 240 min, with data from an additional group of 10 newborns indicating that the return occurred by 150 min. Behavioral distress during circumcision was associated with elevations in plasma cortisol at 30 and 90 min. Quiet sleep was correlated negatively with plasma cortisol prior to circumcision, and significant increases in quiet sleep followed circumcision, with the greatest increase corresponding to the period of most rapid reductions in cortisol.

Effects and Pharmacokinetic Properties of the Rat/Human Corticotropin-Releasing Factor in Rhesus Monkeys

The biological effects and pharmacokinetic properties of the recently sequenced rat/human corticotropin-releasing factor (r/hCRF) were evaluated in the rhesus monkey and compared to those of the previously studied ovine corticotropin-releasing factor (oCRF). An iv bolus of 0, 0.1, 1, 10, and 100 micrograms/kg r/hCRF and 1 microgram/kg oCRF were given to rhesus monkeys (four to five tests per dose). Serial blood samples were drawn before and up to 180 min after the injection for determination of plasma immunoreactive (IR) ACTH, cortisol, and IR-r/hCRF or IR-oCRF concentrations. Mean arterial blood pressure and heart rate were monitored. r/hCRF stimulated ACTH and cortisol secretion in a dose-dependent fashion. Its potency was similar to that of oCRF. r/hCRF, however, had a shorter half-life and a 3-fold higher MCR than oCRF. A dose-dependent decrease in the MCR of r/hCRF was observed, which may indicate a saturation of the clearance mechanisms. Significant decreases in mean arterial blood pressure, increases in heart rate, and a profound facial flush occurred at the dose of 100 micrograms/kg r/hCRF. We conclude that r/hCRF stimulates ACTH and cortisol secretion in a nonhuman primate with a potency similar to that of oCRF. The peptide has marked hypotensive effects at high doses and is cleared 3 times more rapidly than oCRF.

The effects of ACTH on the renin-aldosterone system in normotensive man.

The present study examined the effects of low dose ACTH administration (0.1 mg/day for 2 days) on plasma renin concentration, (PRC), activity (PRA) and substrate (PRS), cortisol and aldosterone in man. Six healthy male volunteers on a diet calculated to contain 150 mmol Na/day received an infusion of 5% dextrose (6 ml/h) for 24 hours, then ACTH (Synacthen, Ciba-Geigy) was added to the infusion at the rate of 100 micrograms per day, for 48 h. Blood samples were taken four hourly for determination of plasma cortisol, aldosterone, PRC, PRA and PRS. There was a highly significant increase in plasma cortisol and aldosterone concentrations during ACTH infusion compared with dextrose infusion, but no significant increase in active or inactive PRC, PRA or PRS. In a separate study of 15 healthy male volunteers, dexamethasone (1 mg at 2300 h) suppressed plasma cortisol but had no effect on PRC. These results do not support the view that stimulation of aldosterone by ACTH is mediated through the renin angiotensin system.

Determination of Corticosteroids in Fish Plasma by High Performance Liquid Chromatography: Evaluation of the Method Using Striped Bass (Morone saxatilis)

A sensitive, specific, and reproducible technique for the simultaneous determination of cortisol, cortisone, and corticosterone in fish plasma was developed using high performance liquid chromatography (HPLC). Corticosteroids were extracted from plasma by methylene chloride and Sep-Pak cartridges for comparative purposes. The Sep-Pak method proved more convenient and efficient. Chromatography was accomplished on a nitrile bonded phase using a methanol–water mobile phase. Steroids were identified and quantified by monitoring absorbance at 254 and 280 nm simultaneously. Sensitivity is approximately 5 ng for each steroid. An evaluation of the technique using subadult striped bass (Morone saxatilis) revealed that cortisol was the only corticosteroid detected. Plasma cortisol concentrations increased in response to brief confinement and crowding, supporting the efficacy of this measurement as an evaluator of stress in striped bass. The HPLC technique provides specific determination of each corticosteroid (thus eliminating cross-reactivity problems inherent in radioimmunoassay methods), resolves corticosteroids from androgens and estrogens, and permits the isolation and purification of individual steroids.

Normal and abnormal function of the hypothalamic-pituitary-adrenocortical system in man.

The first half of this manuscript is devoted to a review of the methods used and the results obtained in the published measurements of the normal responses to tests of the three main types of hypothalamic-pituitary-adrenocortical (HPA) activity in man. These are, I, basal, unstressed activity leading to appropriate levels of total daily production of cortisol in the characteristic circadian pattern; II, responses to feedback stimulation of HPA activity by metyrapone administration; and III, responses to tests of the effects of stress on the HPA system including the effects of hypoglycemia, induced fever, vasopressin administration, and ACTH injections and infusions. The advantages and shortcomings of each type of procedure are discussed. The second half of this paper describes the authors' attempts to establish the limits of normality of standard and modified methods of evaluating the HPA system. The defined limits of normality have been used to assess the HPA function in 158 patients with known or suspected disorders of the HPA system. In normal controls, halfhourly plasma cortisol determinations established the normality of circadian and postprandial fluctuations and of mean plasma cortisol concentration, 6.2 +/- 0.3 (SEM) micrograms/dl, which were closely approximated by determinations every 6 h. Metyrapone, given in a dose of 500 mg every 2 h for 24 h increased urinary 17-OHCS excretion to 10.5-32.6 mg/day or to 1.7-7.8 times basal excretion rate. Increasing rates of insulin infusion disclosed significant relationships between resulting plasma glucose and cortisol concentrations. The slopes of the delta cortisol/delta glucose responses were similar after insulin infusions (0.46 +/- 0.05) and after insulin injections, 0.15 U/kg (0.43 +/- 0.09), and were always greater than 0.20 micrograms/mg. This index provides a useful objective measure of the normality of responses to hypoglycemic stress, 0.20-0.87 micrograms/mg. Adrenocortical responses to iv infusions of ACTH (cosyntropin 0.25 mg) may be equivocal at 2 h but are clear cut at 4, 6 and 8 h. Of 158 patients in whom hypopituitarism was known or suspected because of the presence of a pituitary tumor, acromegaly, hyperprolactinemia, or clinical features, HPA function was found to be entirely normal in 88 patients and partially or severely abnormal in the remaining 70 patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of bromocriptine on the circadian rhythm of 18-hydroxycorticosterone and cortisol secretion in essential hypertensives.

This study examines the influence of bromocriptine, a dopamine agonist, on circadian secretory patterns of plasma 18-hydroxycorticosterone (18-OHB) and cortisol in essential hypertension. Patients with sustained essential hypertension were studied after they had reached equilibrium on a constant 150 mmol sodium and 80 mmol potassium intake. Plasma 18-OHB and cortisol determinations were made at 30-min intervals over 24 h during a control and bromocriptine treatment period (bromocriptine, 2.5 mg t.i.d. for five days). Circadian patterns for plasma 18-OHB and cortisol were observed in all patients before and after bromocriptine. Although bromocriptine did not affect the circadian rhythm of 18-OHB and cortisol it did decrease mean 24-h recumbent 18-OHB from 23 +/- 42.2 to 14.3 +/- 1.4 ng/dl. These results suggest that there is a circadian rhythm of both 18-OHB and cortisol secretion in patients with essential hypertension as in normotensives. Dopaminergic mechanisms exert an effect on the quantitative secretion of 18-OHB. However, the circadian rhythm for 18-OHB and cortisol does not appear to be dependent on dopaminergic mechanisms.

Cortisol Determination and the Dexamethasone Suppression Test

The possibility that unreliability of plasma cortisol level determinations might interfere with the use of the dexamethasone suppression test (DST) in clinical practice was evaluated by examining published data on the reliability of plasma cortisol determinations as well as a comparison of two radioimmunoassay (RIA) methods with a competitive protein binding assay (CPBA). Significant discrepancies between various methods of plasma cortisol level determinations have been reported in most, but not all, studies. The most widely used method, RIA kits, may not produce levels comparable with those produced by the CPBA, used in a recent effort to standardize the DST for use in psychiatry. The use of the DST by physicians should be preceded by an assessment of the validity of the cortisol level determinations in the critical range for the DST by the laboratory they use.

166. Determination of free plasma cortisol by means of an immunoextraction method

166. Determination of free plasma cortisol by means of an immunoextraction method

The ovine fetoplacental endocrine response to placental damage

In the chronic pregnant ewe preparation, daily blood samples, for the determination of plasma cortisol and progesterone levels by specific radioimmunoassay, were obtained from the fetal distal aorta, uterine vein, and uterine artery prior to, during, and after repetitive embolization of the uterine circulation with 15 μ microspheres. A significant increase in fetal cortisol was observed in all animals during the first 3 days of embolization. In those which subsequently entered premature labor, this increase was progressive and was associated with a fall in the uterine venous progesterone level but was unrelated to changes in either the maternal cortisol level or fetal physiologic parameters. In animals which did not enter premature labor, progesterone levels in the uterine vein and estimated progesterone release rates were elevated with an accentuated peak-trough pattern during the postembolization period. In these animals fetal cortisol returned to control levels. The data suggest that cortisol and progesterone biosynthesis within the fetoplacental unit is altered by microembolic placental damage.

Determination of cortisol in human plasma by isotope dilution-mass spectrometry. Definitive methods in clinical chemistry, I.

A definitive method is described for the determination of cortisol in human plasma. The method is based on the principle of isotope dilution-mass spectrometry. The analytical procedure comprises the following steps: (1) Addition of [4-14C]cortisol to the plasma sample; (2) extraction of the 14C-labelled and of the non-labelled cortisol by dichloromethane; (3) purification of the cortisol fraction by column chromatography on Sephadex LH-20; (4) formation of the trimethylsilyl ether of cortisol methoxime; (5) selected ion monitoring at m/z-values 605, 607, 636 and 638 during gas liquid chromatography. The accuracy of the method is based on the high specificity of mass spectrometry and on the exact control of recovery employing the principle of isotope dilution. The precision of the method was evaluated by calculation of the coefficient of variation from day to day; this varied from 0.7-2.3% in the range of 256-845 nmol/l. The lower limit of detection (ratio of signal to noise 3:1) is 200 pg cortisol per sample.

Radioimmunoassay and chemical ionization/mass spectrometry compared for plasma cortisol determination.

We describe a method for determination of cortisol in plasma and urine, based on chemical ionization/mass spectrometry with deuterium-labeled cortisol as the internal standard. The within-run precision (CV) was 2.5-5.7%, the between-run precision 4.6%. Results by this method were compared with those by a radioimmunological method (RIANEN Cortisol, New England Nuclear) for 395 plasma samples. The latter method gave significantly higher (approx. 25%) cortisol values.

Luminescent immunoassay (lia) of cortisol—2. development and validation of the immunoassay monitored by chemiluminescence

An immunoassay procedure for determination of cortisol in human plasma and in urine is described, which utilizes chcmiluminescence as the end point. The assay utilizes cortisol-21-hemisuccinate-aminobutyl-ethyl-isoluminol (C-21-HS-ABEI) as the chemiluminescent marker, and dextran coated charcoal is used for separation of bound and free forms of the ligand. The chemiluminescent assay for cortisol was validated in terms of sensitivity, specificity, linear range and precision. An assay procedure for measuring of plasma and urinary free cortisol was established and assay results were compared with radioimmunoassay, using the same antiserum with tritiated cortisol as the label. The two methods agreed well ( r = 0.95 for plasma cortisol and r = 0.97 for urinary free cortisol). The proposed method takes full advantage of specificity and sensitivity that result from the use of an antibody, without employing an isotopically labelled ligand.

383 CORTICOSTEROID BINDING GLOBULIN (CBG) IN FETAL AND NEWBORN SHEEP

To examine the ontogeny and regulation of CBG in the perinatal period, we determined plasma cortisol binding capacity using a charcoal absorption assay. CBG capacity increased progressively from 1.6 μg/dl at 75 days to 7.1 μg/dl at 141 days (n=249), with the greatest increase from 121 days to term. There was a similar increase in CBG (+41.1%) and total proteins (+36.7%) in 6 fetuses during the 4-6 days before spontaneous delivery. After birth, both CBG and proteins decreased during the first half day; thereafter CBG decreased (t½=5 days) to 1.0 μg/dl at 14 days while proteins did not change. In 7 fetuses with loss of pituitary function there was no increase in CBG during the 22-35 days after surgery. Infusions of hydrocortisone for 2 days, estradiol for 5 days, prolactin for 5-8 days and ACTH for 3 days to intact fetuses did not affect CBG levels. We conclude that the pituitary controls the major increase in CBG after 121 days; there is an additional prepartum increase in all serum proteins with labor. The pituitary hormone(s) which stimulate CBG production are not identified, but this hormonal influence apparently ceases with birth.

Interlaboratory evaluation of four RIA kits for determination of plasma cortisol, with special reference to accuracy: influence of matrix in calibration standards.

We used four RIA kits for determination of plasma cortisol to assay 12 plasma samples in four laboratories, and compared the results with those obtained with a highly accurate method based on isotope dilution/mass spectrometry. All kits (Farmos, NEN, Sorin, and Clinical Assays) showed 10-50% deviation from the comparison method in the normal range (200-700 nmol/L). The inter- and intralaboratory imprecision (CV) ranged between 8-27% and 5-11%, respectively. Possible explanations for this low accuracy are discussed. Retention of an intact biological matrix in the calibration standards seems to be one prerequisite for accuracy.

Plasma Cortisol and Behaviour in Early Weaned Piglets

Comparisons were made of Cortisol concentration and behavioural activity between piglets weaned at 3 weeks of age (early weaned piglets), and piglets weaned at 8 weeks of age (control piglets). Early weaned littermates were placed in cages with a floor area of either 0.20 m2 per piglet or 0.15 m2 per piglet. At 8 weeks of age, the piglets in the control litters were moved from the sow and early weaned piglets were moved from the cages. All litters were placed in pens, 1 pen for each litter. Radioimmunoassay was used for plasma Cortisol determination. Behavioural activity included determination of play, aggressive behaviour and non-nutritive oral activity. Statistical analysis for possible association between plasma Cortisol concentration and age, housing conditions, litter and sequence of capture of littermates for blood collection revealed a complex pattern. Generally, higher plasma Cortisol concentrations and more aggressive and non-nutritive oral activity, but less playing activity, were associated with early weaning. Plasma Cortisol concentration in individual piglets was positively correlated to both aggressive and non-nutritive oral behaviour, but was negatively correlated to playing behaviour. It is concluded that early weaning is a stressor.

Determination of prednisolone, prednisone, and cortisol in human plasma by high-performance liquid chromatography

A method is described for the measurement of prednisone and prednisolone in human plasma by high-performance liquid chromatography (hplc). An ether/dichloromethane extract (60:40, v v ) of plasma is evaporated to dryness and chromatographed on a 250 × 4.5-mm Whatman Partisil column with uv detection at 239 nm. Prednisone, prednisolone cortisol, and the internal standard dexamethasone are satisfactorily resolved with the elution system of water-saturated dichloromethane/ethanol (95:4, v v ). The hplc method can be used for plasma prednisolone concentrations as low as 25 ng/ml. The values correlate well with those obtained by a radioimmunoassay procedure for prednisolone.

An immunoassay for plasma cortisol based on chemiluminescence

An immunoassay procedure for the determination of cortisol in human plasma is described, which utilizes chemiluminescence as the end point. A cortisol-isoluminol conjugate serves as the chemiluminescent marker. The light emission by this conjugate upon oxidation is delayed by prior incubation with anti-cortisol IgG, but not by unrelated γ-globulin. This delayed light emission was inhibited by cortisol in a dose-dependent manner, with a linear range of 20–1000 pg steroid/assay tube. A competitive protein binding assay based on this procedure was applied to methylene chloride extracts of cortisol from normal and pathological human plasma (2–40 μg/100 ml). Cortisol values obtained by this procedure agreed well with those obtained by radioimmunoassay, using the same antiserum with tritiated cortisol as the label ( r = 0.98). The chemiluminescence immunoassay is comparable to radioimmunoassay with regard to sensitivity, specificity, precision and accuracy. The advantage of the new assay procedure is that it obviates the need for counting radioactivity and for separation of bound and free ligand.