Free Plasma Cortisol Concentrations Research Articles

Genetic variation of the glucocorticoid receptor from a steroid-resistant primate

The neotropical cotton-top marmoset (Saguinus oedipus) is a New World primate known to have markedly increased total and free plasma cortisol concentrations when compared with Old World primates including man. The relative end-organ 'resistance' to glucocorticoids found in various New World primates has been attributed to a glucocorticoid receptor (GR) with diminished affinity for glucocorticoids. It has been demonstrated that the marmoset GR has approximately tenfold lower binding affinity for dexamethasone when compared with the human GR. We have examined the primary structure of the marmoset GR by molecular cloning and sequencing of GR functional domains. A library of cDNA clones was constructed in the phage vector gamma gt10 using poly(A)+ RNA from a marmoset-derived lymphoid cell line, and screened using the human GR cDNA. DNA sequencing determined 76 individual nucleotide substitutions in the coding region of the marmoset GR. Comparison of the marmoset GR nucleotide sequence with the human GR cDNA coding region indicated an overall sequence homology of about 97%. Thirty of the nucleotide substitutions lead to alterations in the predicted amino acid sequence (28 amino acid substitutions) of the marmoset GR. The size of the marmoset GR predicted from the 778 amino acids is approximately 90,000 which is in agreement with previous size estimates of the human and marmoset GRs. Alterations of amino acid sequence in the marmoset GR were greatest towards the amino terminus, including the tau 1 domain putatively involved in transcriptional activation. The DNA-binding domain contained an additional codon (arginine).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of design of individual cage-stalls on the behavioural and physiological responses related to the welfare of pregnant pigs

This experiment describes the effects of two designs of individual cage-stalls on the behavioural and physiological responses of pregnant pigs. There was a total of 32 experimental pigs housed in two designs of cage-stalls, vertical or horizontal bars, and two control treatments, tether-stalls (vertical bars) and group housing. Video recordings taken of individually housed pigs (over 3 consecutive days) for 60 min day −1 (commencing 10 min after the start of feeding) after 14 and 49 days in treatments were used to determine the time pigs spent in three areas of the stalls (0–31, 32–63 and more than 63 cm from the front of the stall), number of movements between these areas, the duration that four neighbouring (within a treatment) pigs were concurrently in the front area of the stall and the number and duration of aggressive interactions. The duration of head-to-head contact of four focal pigs per individual housing treatment was also determined. Plasma-free cortisol concentrations were determined after 30 and 57 days in the treatments. The design of the stall division in the individual cage-stalls affected both the spatial use of the stall, aggressive behaviour and cortisol concentrations. Pigs in the horizontal stall treatment spent more time in the back area of the stall (618 s vs. 388 s; P < 0.01) and spent less time concurrently (four neighbouring pigs) in the front area of the stall (55 s vs. 193 s; P < 0.05) than animals in the vertical stall treatment. Duration of aggressive interactions was less in the horizontal stall treatment (11.1 s v. 20.3 s; P < 0.05) and there were fewer pigs (2 vs. 8; P < 0.05) involved in aggressive interactions than in the vertical stall treatment. Head-to-head contact (of focal pigs) while standing was also less in the horizontal stall treatment (171 s vs. 374 s; P < 0.05). There was evidence of a chronic stress response in the horizontal stall treatment (mean free cortisol concentrations in the horizontal and vertical stall treatments were 6.6 nmol l −1, respectively; P < 0.05). The behavioural data suggest that pigs in the horizontal stall treatment were actively avoiding their neighbours. In conclusion, this experiment has shown that the design of cage-stalls can affect the welfare of pigs. Pigs housed in stalls comprised of horizontal bars showed evidence of a chronic stress response of a magnitude sufficient to adversely affect welfare, and active avoidance by neighbouring. Pigs housed in stalls comprised of vertical bars showed cortisol concentrations similar to group-housed pigs (and lower than pigs in tether-stalls) and they also showed the highest levels of aggression of all treatments. Across the individual housing treatments, the hormone data were not correlated with measures of aggressive behaviour and thus the relationship (s), if any, between aggressive behaviour and physiological responses are unclear and remain a subject for further research.

Glucocorticoid receptors in lymphocytes in anorexia nervosa

The aim was to explore the down-regulation of the glucocorticoid receptors during hypercortisolaemia in anorexia nervosa. Urine and plasma samples were obtained for cortisol determination and blood lymphocytes were isolated for receptor binding studies. Sixteen anorexic patients, aged 16-27 years, with a mean +/- SEM body mass index of 14.2 +/- 2.0 (ranging from 11.1 to 17.4), and 15 normal women were studied. Six patients were reinvestigated after a significant weight gain. The binding capacity and affinity of the glucocorticoid receptors were measured with dexamethasone as ligand on lymphocytes. In patients, both total and free plasma cortisol concentrations were higher than in the normal women, as was their urinary free cortisol; the number of glucocorticoid receptors per cell (Ro) and the binding affinity (Kd) for dexamethasone were, however, not significantly different (Ro: 7687 +/- 1750 vs 7347 +/- 1285 sites/cell; Kd: 7.7 +/- 2.4 vs 7.4 +/- 1.7 nM at 24 degrees C). After weight gain (14 +/- 2 to 16 +/- 2 kg/m2), receptor numbers were 8421 +/- 2126 (pre) and 9011 +/- 500 (post) sites/cell, which are not significantly different (P greater than 0.2); the Kd was unchanged (9.3 +/- 2.6 vs 9.2 +/- 2.4 nM). CONCLUSIONS Hypercortisolaemia does not down-regulate the lymphocyte glucocorticoid receptors in anorexia nervosa and a post-receptor defect might be involved in peripheral tissue resistance to the effects of glucocorticoid hormones in undernutrition.

Plasma free cortisol concentrations in dogs with hyperadrenocorticism

SUMMARY Unbound or free cortisol constitutes a small fraction of total plasma cortisol, but is believed to represent the biologically active portion of this circulating glucocorticoid. We tested the hypothesis that the percentage free cortisol was altered in plasma from dogs with hyperadrenocorticism, which could account for a greater target tissue response to this circulating hormone. The percentage free cortisol in plasma samples from human beings, healthy dogs, and dogs with hyperadrenocorticism was estimated, using centrifugal ultrafiltration-dialysis. Total cortisol concentrations were determined by use of radioimmunoassay. Total cortisol concentrations appeared greater in plasma from human beings than in plasma from either group of dogs. However, the percentage free cortisol was lower in plasma from human beings, resulting in a calculated concentration of free cortisol that was quite similar between plasma from human beings and healthy dogs. Total plasma cortisol concentrations were greater (P &lt; 0.01) in samples from dogs with hyperadrenocorticism (190 ±113 nmol/L; mean ± SD) than in healthy dogs (102 ± 85 nmol/L), but the percentage free cortisol was not different between these 2 groups (dogs with hyperadrenocorticism, 16 ± 9%; healthy dogs, 13 ± 6%). However, plasma free cortisol concentrations (product of total and the percentage of free cortisol) were greater (P &lt; 0.01) in samples from dogs with hyperadrenocorticism (36 ± 41 nmol/L) than in those from healthy dogs (16 ± 9 nmol/ L). Significant (P &lt; 0.001) positive linear relationships were found between total cortisol concentrations and percentage free cortisol in plasma samples from healthy dogs and dogs with hyperadrenocorticism. Furthermore, the slope of these lines was not different between the 2 groups, providing no evidence for alterations in cortisol binding associated with hyperadrenocorticism. The higher total cortisol concentrations in dogs affected with this disease do, however, result in greater concentrations of free cortisol in circulation, contributing to the development of clinical signs observed in this disease.

Metabolic effects of cortisol, ACTH, adrenalin and insulin in the marsupial sugar glider, Petaurus breviceps

The effects of cortisol, ACTH, adrenalin and insulin on indices of carbohydrate, fat and protein metabolism were investigated in the conscious marsupial sugar glider Petaurus breviceps. Short-term i.v. infusion of cortisol at dose rates of 0.02, 0.2 and 1.0 mg/kg per h caused the plasma glucose concentration to rise sharply from the normal range of 3.3-4.4 to 8.1-8.7 mmol/l at the end of the infusion period without significant alteration in plasma free fatty acid (FFA), amino acid or urea concentrations. Infusions of ACTH at dose rates of 0.02, 0.06 and 0.45 IU/kg per h caused a similar rise in plasma glucose concentration; however, this was now accompanied by an elevation in plasma FFA concentration, but again without significant changes in either plasma amino acid or urea concentrations. Infusion of adrenalin at 10 micrograms/kg per h caused an increase in the plasma concentrations of both glucose and FFA. Intravenous injections of 0.15 IU insulin/kg caused a rapid and marked decrease in the plasma glucose concentration within 30 min and an increase in the plasma free cortisol concentration. Associated with this change was a marked rise in the plasma concentration of both FFA and free cortisol. The rise in free cortisol was, however, significantly reduced by infusion of glucose. Pretreatment with five daily i.m. injections of 1 mg cortisol acetate/kg, which produced an increase in plasma free cortisol concentration to near the maximum of the physiological range, caused a marked reduction in insulin sensitivity. Cortisol pretreatment caused an increase in the plasma FFA and amino acid concentrations. Petaurus breviceps is highly sensitive to the metabolic effects of glucocorticoids and is similar in this respect to the brush-tailed possum Trichosurus vulpecula. The interactive effects between insulin and glucocorticoids on carbohydrate, fat and protein metabolism in Petaurus breviceps are similar to those shown by Trichosurus vulpecula and some eutherian mammals but contrast with the pattern described for two macropodid marsupials, the red kangaroo Macropus rufus and the quokka Setonix brachyurus.

Failure of glucocorticoid feedback during breeding in the male red-tailed phascogale Phascogale calura (Marsupialia: Dasyuridae)

An investigation was made into the factors which lead to an elevation in plasma free cortisol concentration during the last weeks of life of males in natural populations of the red-tailed phascogale Phascogale calura. The dexamethasone suppression-test was employed to examine the glucocorticoid feedback control of plasma cortisol both before and during the breeding season. In both sexes ACTH alone or in combination with dexamethasone caused an elevation in the plasma concentration of cortisol, corticosterone and free cortisol. Dexamethasone administration in both males and females resulted in significant decreases in the plasma concentration of each of the glucocorticoid groups both before and during the first week of the breeding season (June and early July), however during the last week of breeding (late July) dexamethasone decreased the plasma glucocorticoid concentration of females but not of males. Administration of ACTH caused a significant elevation in the plasma cortisol concentration in all groups. However, the magnitude of this response diminished with time in both sexes. Dexamethasone treatment resulted in a decrease in the plasma testosterone concentration in males before and early in the breeding season however toward the end of breeding this effect was abolished. It is apparent that towards the end of the breeding season and during the last week of life of the males, glucocorticoid feedback control of ACTH is almost abolished. These changes, which occur only in the males late in the breeding season and near the time of their disappearance from the population, are consistent with a condition known as end organ resistance to steroid hormones.

Salivary cortisol measurement: a practical approach to assess pituitary-adrenal function.

The salivary cortisol concentration is an excellent indicator of the plasma free cortisol concentration. To establish its normal and pathological ranges, salivary cortisol concentrations were measured in 101 normal adults, 18 patients with Cushing's syndrome, and 21 patients with adrenal insufficiency. The normal subjects had a mean (+/- SEM) salivary cortisol concentration of 15.5 +/- 0.8 nmol/L (range, 10.2-27.3) at 0800 h and 3.9 +/- 0.2 nmol/L (range, 2.2-4.1) at 2000 h (n = 20). The mean value 60 min after ACTH administration in 58 normal subjects was 52.2 +/- 2.2 nmol/L (range, 23.5-99.4), and it was 1.4 +/- 1.1 nmol/L (range, 1.6-3) at 0800 h in 23 normal subjects given 1 mg dexamethasone 8 h earlier. In patients with primary or secondary adrenal insufficiency (n = 21) the mean salivary cortisol level was 7.5 +/- 0.4 nmol/L (range, 1.9-21.8) 60 min after ACTH. In patients with Cushing's syndrome (n = 7), the mean value after the 1-mg dexamethasone suppression test was 16.1 +/- 7.8 nmol/L (range, 5.8-66.8). No overlap was found between the values in the normal subjects and those in the patients during the dynamic tests. Discrepancies between salivary and total plasma cortisol were found in 8 patients with adrenal insufficiency, which may be explained by the effects of drugs such as thyroid hormones, Op'-dichlorodiphenyldichloroethane, and psychotropic agents. We conclude that salivary cortisol measurements are an excellent index of plasma free cortisol concentrations. They circumvent the physiological, pathological, and pharmacological changes due to corticosteroid-binding globulin alterations and offer a practical approach to assess pituitary-adrenal function.

Regulation of the pituitary-adrenal axis and corticosteroid-binding globulin-cortisol interaction in the guinea pig.

Adult male guinea pigs were used to examine pituitary-adrenal function and corticosteroid-binding globulin (CBG)-cortisol interaction. A biphasic pattern in the circadian rhythm of plasma cortisol was observed, with nadirs occurring at 0800 and 2400 h and peaks and 1600 and 0400 h. An excellent correlation was noted between total and free plasma cortisol levels. In contrast, no correlation was noted between CBG binding capacity and either the total or free plasma cortisol level. The free plasma cortisol concentration ranged from 0.6-5.8 micrograms/dl, representing 6.1-14.5% of the total cortisol concentration. The CBG binding capacity ranged from 12.2-161.7 micrograms/dl, and the binding affinity was 1.3-2.2 x 10(7) M-1 at 22 C, which is 20-fold lower than that of human CBG. These results suggest that CBG in the guinea pig has a relatively minor effect on the plasma distribution of cortisol, and that free plasma cortisol is dependent on cortisol binding to nonspecific plasma proteins as well as on the total plasma cortisol concentration. It was found that the guinea pig was relatively resistant to dexamethasone suppression, requiring at least 1 mg/kg BW to obtain essentially complete suppression of the pituitary-adrenal axis. In addition, it was found that administering dexamethasone 6 h before the time that the animals were killed led to suppression, whereas giving the steroid 12 h before killing the animals was totally ineffective. Stress induced by both ether vapor and histamine injection significantly increased plasma cortisol levels. When the guinea pigs were treated with a 1 mg/kg BW dose of dexamethasone 6 h before the stimulus, however, a marked suppression of the plasma cortisol increment secondary to application of the stressful stimulus occurred.

Progressively elevated levels of biologically active (free) cortisol during pregnancy by a direct radioimmunological assay of diffusible cortisol in an equilibrium dialysis system.

The measurement of free cortisol would be preferable with respect to the total hormone content, since it yields more reliable information about the plasma levels of the biologically active cortisol. We have developed a new method for the determination of the apparent free plasma cortisol concentration (AFCC) by means of direct radioimmunological measurement of dialyzed cortisol. The AFCC was measured in 23 plasma pools obtained from normal pregnant women at various gestational times and in 18 nonpregnant women. The mean AFCC was 19.5 +/- 7.1 ng/ml in pregnant women and 9.0 +/- 6.2 ng/ml in nonpregnant women (p less than 0.005). The percent of free cortisol (%FC) showed a progressive increase during pregnancy reaching the lower limits of the normal range at the third trimester. However, the mean of %FC was significantly lower (p less than 0.005) in pregnant women than in nonpregnant women. Our data show a progressive increase of biologically active cortisol during pregnancy.

Radioimmunological determination of apparent free cortisol concentration: Some physiological and clinical applications

The measurement of free cortisol would be preferable with respect to the total hormone content, since it yields more reliable information about the plasma levels of the biologically active steroid. The methods so far described for the measurement of free cortisol in plasma use radiolabelled cortisol for determination of the steroid free fraction, and are generally unsuitable for routine use. We have developed a new method for the determination of the apparent free plasma cortisol concentration by means of direct radioimmunological measurement of dialyzed cortisol. This method is characterized by a sufficient degree of reproducibility and high sensitivity. Apparent free cortisol concentration in 40 control subjects of both sexes (blood drawn at 8 a.m.) was 9.00 ± 4.6 ng/ml. The mean value of free cortisol concentration in blood samples drawn at 11--12 p.m. from 21 of these subjects was highly significantly different (2.3 ± 1.6 ng/ml, p < 0.001). In addition, in 13 of these subjects circadian variation of the apparent free cortisol concentration showed a pattern similar to that of total cortisol concentration. The mean free cortisol concentration found in a group of women during normal pregnancy was significant higher than in non-pregnant women. Patients with renal insufficiency do not show a significant difference in free cortisol plasma levels, whereas higher values were found in hepatic cyrrhosis.

The cortisol response during heart-lung bypass.

The response patterns of plasma cortisol and plasma free cortisol have been studied in 20 consecutive patients undergoing cardiac surgery involving the use of heart-lung bypass. Sixteen consecutive patients undergoing closed mitral valvotomy have been used as controls. Total plasma cortisol levels fell at the onset of extracorporeal circulation (ECC) and did not rise significantly throughout the period of ECC. The peak cortisol level in the bypass patients occurred at 24 hours postoperatively with elevated levels still present at 48 hours. This pattern was significantly different from the control patients (P less than 0.001) who exhibited the typical cortisol stress response pattern. Synthetic ACTH stimulation of the adrenal cortex during heart-lung bypass produced a positive response (i.e., a rise in plasma cortisol greater than 7.5 mug/100 ml) in all six patients studied while the six mitral valvotomy patients had no response to synthetic ACTH administration during operation (P less than 0.01). Plasma free cortisol estimations indicate that a rapid and significant rise in % free cortisol occurred at the onset of ECC, but that despite the rise in percentage free, the overall plasma free cortisol concentration (i.e., total plasma cortisol X % free) was significantly lower during ECC in the bypass patients when compared with control (P less than 0.01). Twenty-four hours postoperatively these concentrations were significantly higher in the bypass patients (P less than 0.001).