This report describes a model of steroid transport in human plasma. The binding affinities of 21 endogenous steroids for both testosterone-binding globulin (TeBG) and corticosteroid-binding globulin (CBG) were determined under equilibrium conditions using a solid phase method at physiological pH and temperature. A computer program was used to solve the complex equilibrium interactions between these steroids and TeBG, CBG, and albumin. In this manner, we calculated the plasma distribution of each steroid into TeBG-bound, CBG-bound, albumin-bound, and unbound fractions in normal men, normal women during both the follicular and luteal phases of the ovarian cycle, and women during the third trimester of a normal pregnancy.

Transport of Steroid Hormones: Binding of 21 Endogenous Steroids to Both Testosterone-Binding Globulin and Corticosteroid-Binding Globulin in Human Plasma

Chronic fatigue syndrome is characterized by persistent or relapsing debilitating fatigue for at least 6 months in the absence of a medical diagnosis that would explain the clinical presentation. Because primary glucocorticoid deficiency states and affective disorders putatively associated with a deficiency of the arousal-producing neuropeptide CRH can be associated with similar symptoms, we report here a study of the functional integrity of the various components of the hypothalamic-pituitary-adrenal axis in patients meeting research case criteria for chronic fatigue syndrome. Thirty patients and 72 normal volunteers were studied. Basal activity of the hypothalamic-pituitary-adrenal axis was estimated by determinations of 24-h urinary free cortisol-excretion, evening basal plasma total and free cortisol concentrations, and the cortisol binding globulin-binding capacity. The adrenal cortex was evaluated indirectly by cortisol responses during ovine CRH (oCRH) stimulation testing and directly by cortisol responses to graded submaximal doses of ACTH. Plasma ACTH and cortisol responses to oCRH were employed as a direct measure of the functional integrity of the pituitary corticotroph cell. Central CRH secretion was assessed by measuring its level in cerebrospinal fluid. Compared to normal subjects, patients demonstrated significantly reduced basal evening glucocorticoid levels (89.0 +/- 8.7 vs. 148.4 +/- 20.3 nmol/L; P less than 0.01) and low 24-h urinary free cortisol excretion (122.7 +/- 8.9 vs. 203.1 +/- 10.7 nmol/24 h; P less than 0.0002), but elevated basal evening ACTH concentrations. There was increased adrenocortical sensitivity to ACTH, but a reduced maximal response [F(3.26, 65.16) = 5.50; P = 0.0015). Patients showed attenuated net integrated ACTH responses to oCRH (128.0 +/- 26.4 vs. 225.4 +/- 34.5 pmol/L.min, P less than 0.04). Cerebrospinal fluid CRH levels in patients were no different from control values (8.4 +/- 0.6 vs. 7.7 +/- 0.5 pmol/L; P = NS). Although we cannot definitively account for the etiology of the mild glucocorticoid deficiency seen in chronic fatigue syndrome patients, the enhanced adrenocortical sensitivity to exogenous ACTH and blunted ACTH responses to oCRH are incompatible with a primary adrenal insufficiency. A pituitary source is also unlikely, since basal evening plasma ACTH concentrations were elevated. Hence, the data are most compatible with a mild central adrenal insufficiency secondary to either a deficiency of CRH or some other central stimulus to the pituitary-adrenal axis. Whether a mild glucocorticoid deficiency or a putative deficiency of an arousal-producing neuropeptide such as CRH is related to the clinical symptomatology of the chronic fatigue syndrome remains to be determined.

Evidence for impaired activation of the hypothalamic-pituitary-adrenal axis in patients with chronic fatigue syndrome.

The use of the intravenous anesthetic etomidate for prolonged sedation has been associated with low levels of plasma cortisol and increased mortality. We measured the cortisol and aldosterone responses to ACTH stimulation in five patients receiving etomidate, and we also studied the direct effects of etomidate on enzymes in the rat steroidogenic pathway. One patient who was receiving a 20-hour infusion of etomidate (1.3 to 1.5 mg per kilogram of body weight per hour) had marked adrenocortical suppression that was still evident four days after etomidate was discontinued. Four surgical patients receiving etomidate during their operations were all found to have adrenal suppression four hours after the operation; mean (+/- S.D.) increases in cortisol and aldosterone after ACTH stimulation were only 1.8 +/- 0.5 micrograms per deciliter and 0.5 +/- 1.1 ng per deciliter, respectively. In rat adrenal cells, etomidate produced a concentration-dependent blockade of the two mitochondrial cytochrome P-450-dependent enzymes, cholesterol-side-chain cleavage enzyme, and 11 beta-hydroxylase, without evident inhibition of the microsomal enzymes in the glucocorticoid pathway. Physicians should be aware that etomidate inhibits adrenal steroidogenesis, and they should consider treating selected patients with corticosteroids if etomidate is used.

Inhibition of Adrenal Steroidogenesis by the Anesthetic Etomidate

The regulation of the onset of maternal behavior in the rat is under hormonal control. This study reports a new endocrine model for the study of the hormonal regulation of maternal responsiveness. The model employs the administration of physiological amounts of the steroids estradiol (E2) and progesterone (P) via Silastic implants to inexperienced nulliparous rats and measurement of the effects of these implants on maternal behavior. In the first two experiments, the levels of E2 and P in the sera of pregnant and hormone-treated rats were measured by RIA. Using known physiological treatments of E2 given in combination with P, the effects of E2 and P on maternal behavior were measured. Treatment with a combination of E2 at all dosages plus P for 2 weeks before P removal and behavioral testing stimulated a fast onset of maternal behavior in ovariectomized nulliparous rats. Exposure for 2 weeks to small E2 implants (1 or 2 mm; approximately 20-30 pg/ml serum) did not affect maternal responsiveness, whereas large E2 implants (10 mm; approximately 110 pg/ml serum) stimulated maternal behavior. P treatment alone had no behavioral effect. Simultaneous removal of E2 plus P before exposure to foster young also resulted in a stimulation of behavioral responsiveness, indicating that the presence of elevated titers of circulating E2 is not a requirement for stimulation to occur. In addition to facilitating a rapid onset of behavior, the quality of the response in steroid-primed rats was similar to that measured in lactating rats in a T-maze test. In another experiment, when female rats were treated with P before E2 administration, maternal behavior was rapidly induced. Thus, P itself can sensitize the female to the behavioral effects of E2. Finally, the duration of steroid-exposure before testing was found to influence maternal behavior. Increased durations of E2 plus P exposure before testing were accompanied by decreased latencies to respond maternally to foster young. These data indicate that during pregnancy, E2 and P prime the female to respond to her young at birth. The intensity of the steroidal priming increases as pregnancy progresses, and this primed potential is subsequently unmasked by the decline in P and the maintenance of E2 secretion around parturition. These findings demonstrate that behavioral processes can be modified in the adult animal as a result of long term changes in endocrine state, i.e. pregnancy.

A quantitative analysis of the roles of dosage, sequence, and duration of estradiol and progesterone exposure in the regulation of maternal behavior in the rat.

SOME difficulties in clinical studies of endocrine function based on plasma sampling regimens include time-consuming venipuncture and measurement of the “total” rather than the “free” biologically active fraction in plasma. Simple methods for determining plasma free steroids have not yet been developed, and most current procedures involve technically demanding ultrafiltration or equilibrium dialysis. In this context measurement of steroids in saliva is attractive. Steroid concentrations in saliva are independent of flow rate and reflect those in the free fraction in plasma. Recent improvements in immunoassay techniques have allowed development of simple, high output assays for salivary steroids which are well suited for routine use. Since saliva samples can be collected at frequent intervals by both adults and children, they facilitate short term dynamic tests, pharmacokinetic analyses, and studies of chronobiological changes. Problems of viscosity, which restrict processing of freshly collected saliva, m...

Steroids in Saliva for Assessing Endocrine Function

This report describes the separation and purification of various steroids using partition chromatography on celite microcolumns. The mobile phase consists of a mixture of ethyl acetate in isooctane with increasing polarity. The stationary phases for three systems are described, using propylene glycol, ethylene glycol, and water as solvents. Elution is carried out in a stepwise fashion. Combining the above systems of chromatography with partially specific antisera, specific radioimmunoassays for a variety of steroids have been developed.

Chromatographic Separation of Steroid Hormones for Use in Radioimmunoassay

A radioimmunoassay method for the direct measurement of cortisol in plasma after precipitation of the proteins with ethanol is described. Utilizing a specific antiserum against cortisol, with minimal or no cross reaction with other steroids, cortisol was meas-red accurately in a volume of 0.001 ml of plasma. The range of levels that could be measured per ml of plasma varied from 10 to 500 ng. The mean value of plasma cortisol at 8 a. m. obtained in twelve subjects by this method was about half the mean levels reported by another competitive protein binding assay.

Radioimmunoassay of Plasma Cortisol

Combining a simple chromatographic system on celite microcolumns with specific antisera as binding reagents, simultaneous radioimmunoassay of pregnenolone (5Δ-P), 17-hydroxypregnenolone (17-5Δ-P), and dehydroepiandrosterone (DHEA) could be performed on the same aliquot of plasma. These steroids have been measured in plasma samples obtained from subjects of both sexes under various conditions. The plasma levels of 5Δ-P, 17-5Δ-P, and DHEA were found to be within the same range when 12 prepubertal boys and 15 men were studied. The mean levels of these steroids were respectively: 1.57, 0.95, and 1.2 ng/ml in prepubertal boys and 0.88, 0.73, and 1.82 in adult men. Six premenopausal women were studied during a complete menstrual cycle. Comparing the levels of 5Δ-P, 17-5Δ-P, and DHEA during the follicular and luteal phases showed no significant difference between these two phases of the cycle. However, plasma 5Δ-P was found to be significantly elevated in the luteal phase when the mean follicular and lu...

Radioimmunoassay of Plasma Pregnenolone, 17-Hydroxypregnenolone and Dehydroepiandrosterone Under Various Physiological Conditions

A radioimmunoassay for the measurement of plasma unconjugated pregnenolone (5Δ-P) is described. The 3-hemisuccinate of 5Δ-P was coupled to human serum albumin using carbodiimide. One adult ewe was immunized with 2 mg of this conjugate. The antiserum obtained after 7 months of immunization was used in the assay. One to two ml of plasma or serum, with 3H-5Δ-P added for recovery was extracted with ether and chromatographed on a microcolumn of celite. Incubation of the dried 5Δ-P fraction was carried out at 4 C in the presence of 3H-5Δ-P and diluted antiserum at 4 C in phosphate buffer with 0.1% gelatin. The mean recovery of 3H-5AΔ-P after extraction and chromatography was 62%. The sensitivity varied between 0.05 and 0.1 ng 5Δ-P. Recovery experiments of 5Δ-P added to plasma confirmed accuracy. Concentrations of 5Δ-P in plasma from men averaged 0.75 ± 0.55 (SD) ng/ml. Using single determinations during each phase of the menstrual cycle, premenopausal women had levels of 0.71 ± 0.45 (sd) and 1.38 ± 0.6...

Radioimmunoassay of plasma pregnenolone.

This report describes a radioimmunoassay method for the measurement of unconjugated 17-hydroxypregnenolone (17-5δ-P) in plasma. Utilizing a highly specific 17-5δ-P antiserum combined with a one step celite microcolumn chromatographic system, we have measured this steroid accurately in small aliquots of plasma from both male and female subjects. The plasma levels of 17-5δ-P obtained by this method agreed well with levels observed in adult men and women, using two other techniques. Compared with the latters, however, the present method offered a tenfold greater sensitivity and a better precision.

Ronald C. Teller

Papers

Chromatographic Separation of Steroid Hormones for Use in Radioimmunoassay

Radioimmunoassay of Plasma Cortisol

Radioimmunoassay of Plasma Pregnenolone, 17-Hydroxypregnenolone and Dehydroepiandrosterone Under Various Physiological Conditions

Radioimmunoassay of plasma pregnenolone.

Radioimmunoassay of Plasma 17-Hydroxypregnenolone