Free Cortisol Fraction Research Articles

Effect of temperature and pH on the magnitude of the free fraction of cortisol in serum.

Concomitant effects of temperature and pH on changes in the magnitude of the free, unbound fraction of cortisol in human serum were studied at temperatures and pH ranging from 36 to 39 degrees C and from 6.9 to 7.4, respectively. Free fraction increased with increasing temperature and decreasing pH, the two factors acting synergically: a rise in temperature from 36 to 39 degrees C caused an about 30% increase in the free fraction at pH = 7.4, and at pH 6.9--over 2.2-fold. At highest temperature-acidity combination, i.e. at 39 degrees C, pH = 6.9, the free fraction was as much as 2.5-fold higher, relative to standard conditions (ph 7.4, 37 degrees C). This implies that in vivo the free, biologically active fraction of cortisol may increase due to e.g. hyperthermia and acidosis at a constant total concentration of this hormone.

Personality dimensions harm avoidance and self-directedness predict the cortisol awakening response in military men

To account for individual differences in vulnerability for stress-related disorders, studies have examined the relationship between hypothalamic–pituitary–adrenal (HPA) axis functioning and personality. The present study examined the relationship between the free fraction of cortisol in saliva after awakening and personality as measured with Cloninger's Temperament and Character Inventory [Cloninger, C.R., Przybeck, T.R., Svrakic, D.M., Wetzel, R.D., 1994. The Temperament and Character Inventory (TCI): A Guide to its Development and Use. Washington University, Center for Psychobiology of Personality, St. Louis, MO] in 107 healthy male soldiers. Harm avoidance explained 9% of variance in cortisol levels after awakening (AUCG), and harm avoidance and self-directedness predicted 10% of variance in mean cortisol increase. The cortisol awakening response (CAR) was lower in participants with low scores on harm avoidance, and mean cortisol increase after awakening was higher in soldiers high on self-directedness and harm avoidance. These results show that the CAR is related to personality and that it can be used to examine individual differences in HPA (re)activity.

Free Cortisol in Sepsis and Septic Shock

Severe sepsis activates the hypothalamopituitary axis, increasing cortisol production. In some studies, hydrocortisone substitution based on an adrenocorticotropic hormone-stimulation test or baseline cortisol measurement has improved outcome. Because only the free fraction of cortisol is active, measurement of free cortisol may be more important than total cortisol in critically ill patients. We measured total and free cortisol in patients with severe sepsis and related the concentrations to outcome. In a prospective study, severe sepsis was defined according the American College of Chest Physicians/Society of Critical Care Medicine criteria. Blood samples were drawn within 24 h of study entry. Serum cortisol was analyzed by electrochemiluminescence immunoassay. The Coolens method was used for calculating serum free cortisol concentrations. Blood samples were collected from 125 patients, of whom 62 had severe sepsis and 63 septic shock. Hospital mortality was 21%. Calculated free serum cortisol correlated well with serum total cortisol (r = 0.90, P < 0.001). There was no difference in the total cortisol concentrations in patients with sepsis and septic shock (728 +/- 386 nmol/L vs 793 +/- 439 nmol/L, P = 0.44). Nonsurvivors had higher calculated serum free (209 +/- 151 nmol/L) and total (980 +/- 458 nmol/L) cortisol concentrations than survivors (119 +/- 111 nmol/L, P = 0.002, and 704 +/- 383 nmol/L, P = 0.002). Depending on the definition, the incidence of adrenal insufficiency varied from 8% to 54%. Clinically, calculation of free cortisol does not provide essential information for identification of patients who would benefit from corticoid treatment in severe sepsis and septic shock.

Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic–pituitary–adrenal axis activity

The aim of this study was to determine whether salivary cortisol measured by a simple enzyme immunoassay (EIA) could be used as a surrogate for serum total cortisol in response to rapid changes and across a wide range of concentrations. Comparisons of matched salivary and serum samples in response to dynamic hypothalamic-pituitary-adrenal (HPA) axis testing. Subjects Healthy women (n=10; three taking oral oestrogens) and men (n=2), aged 23--65 years, were recruited from the community. Measurements Paired saliva and serum samples were obtained during three protocols: 10 min of exercise at 90% of maximal heart rate (n=8), intravenous administration of corticotrophin-releasing hormone (CRH; n=4), and dexamethasone suppression (n=7). Cortisol was measured in saliva using a commercial high-sensitivity EIA and total cortisol was measured in serum with a commercial radioimmunoassay (RIA). Results The time course of the salivary cortisol response to both the exercise and CRH tests paralleled that of total serum cortisol. Salivary cortisol demonstrated a significantly greater relative increase in response to the exercise and CRH stimuli (697+/- 826%vs. 209+/- 150%, P=0.04 saliva vs. serum). A disproportionately larger increase in free cortisol, compared with total, would be expected when the binding capacity of cortisol-binding globulin (CBG) is exceeded. In response to dexamethasone suppression, relative decreases in cortisol were not significantly different between the two media (-47+/- 56%vs.-84+/- 8%, P=0.13 saliva vs. serum). Although a significant linear correlation was found for all paired salivary and serum total cortisol samples (n=183 pairs, r=0.60, P<0.001), an exponential model provided a better fit (r=0.81, P<0.001). The linear correlations were strengthened when data from subjects on oral oestrogens (n=52 pairs, r=0.75, P < 0.001) were separated from those not taking oestrogens (n=131 pairs, r=0.67, P<0.001). Conclusions Salivary cortisol measured with a simple EIA can be used in place of serum total cortisol in physiological research protocols. Evidence that salivary measures represent the biologically active, free fraction of cortisol includes: (1) the greater relative increase in salivary cortisol in response to tests that raise the absolute cortisol concentration above the saturation point of CBG; (2) the strong exponential relationship between cortisol assessed in the two media; and (3) the improved linear correlations when subjects known to have increased CBG were analysed separately. Thus, an advantage of measuring salivary cortisol rather than total serum cortisol is that it eliminates the need to account for within-subject changes or between-subject differences in CBG.

Plasma free cortisol fraction reflects levels of functioning corticosteroid-binding globulin

Background In normal plasma free cortisol accounts for less than 6% of the total with 80–90% bound to corticosteroid-binding globulin (CBG) and the remainder associated albumin. However little is known about the distribution of free cortisol in plasma where CBG is inactivated or in congenital CBG deficiency. Methods and results Here we describe ligand binding experiments revealing that while free cortisol in unstressed individuals is less than 6% of total cortisol this rises markedly to 25% when CBG is totally inactivated by heat. Similar elevations of the free cortisol fraction were noted in a patient with a rare genetically determined complete lack of CBG (mean 32% on frequent circadian sampling). Following heat inactivation of CBG or in the congenital absence of CBG, there is a shift in cortisol binding from CBG to albumin. That this shift occurs is further supported by experiments adding [ 3H]-cortisol to physiological human serum albumin solutions, where 25% of cortisol remained in the free fraction. Conclusion Taken together the data provide strong evidence that when CBG is inactivated or congenitally absent then more than 25% of the total cortisol appears in the free fraction with the remainder associated with albumin. The proportion of free cortisol measured in plasma thus reflects a simple measure of functional corticosteroid-binding globulin.

Naturally occurring scrapie is associated with a lower CBG binding capacity in ewes.

Naturally scrapie-affected ewes present a syndrome of hypercortisolism as evaluated by measuring total plasma cortisol concentrations. The objective of this study was to investigate the plasma protein binding of cortisol and to evaluate the concentration of the biologically active free fraction of cortisol in scrapie-affected ewes. Corticosteroid binding globulin (CBG) binding parameters were evaluated by equilibrium dialysis in 13 naturally scrapie-affected ewes and nine healthy ewes, during two periods of the clinical evolution of the disease. The hypercortisolism of the scrapie-affected ewes was confirmed by a significant increase of the plasma 20 beta-dihydrocortisol and cortisone concentrations, while total cortisol concentrations, obtained from an isolated sample, did not differ between scrapie-affected and control ewes. The scrapie diagnosis was confirmed by histopathology. The CBG maximal capacity (B(max)) was two times lower in scrapie-affected ewes than in healthy ewes (37+/-32 nM and 73+/-28 nM respectively). The dissociation constant K(d) (8.8+/-3.7 nM and 9.8+/-3.0 nM respectively) and the non-specific constant value of binding to albumin (1.13+/-0.18 and 1.14+/-0.23 respectively) did not differ significantly between diseased and control ewes. The significant increased concentrations of CBG-free cortisol (i.e. both albumin-bound and free cortisol fractions) in scrapie-affected ewes indicates that total plasma cortisol concentration is not an appropriate index of pituitary-adrenocortical hyperactivity. In conclusion, ewes with naturally occurring scrapie display a syndrome of hypercortisolism associated with a lower CBG binding capacity which leads to an overexposure of glucocorticoid-sensitive targets to CBG-free cortisol. The physiopathological consequences of this overexposure on the development of the neurodegenerative process in prion disease are discussed.

Corticosteroid-Binding Globulin during Inflammation and Burn Injury: Nutritional Modulation and Clinical Implications

Corticosteroid-binding globulin (CBG) is the main carrier of glucocorticoids in mammals. Serum CBG shows little physiological variation with the exception of pregnancy. Experimental inflammation and burn injury decrease serum CBG in rats and while the mechanism of this effect is unknown, in vitro experiments suggest that interleukin-6 may be involved. In severely burned patients, we have found that CBG was markedly decreased within a few hours postinjury. This decrease lasted about 2 weeks and was accompanied by an increase in the free fraction of serum cortisol. In addition, serum CBG responded to dietary manipulation in these patients, with low fat feeding resulting in higher serum CBG concentrations and lower serum-free cortisol values. This feeding suggests that during severe stress, CBG may be important in regulating the amount of cortisol reaching target tissues such as the immune system and wounds.

Interspecies variations of corticosteroid-binding globulin parameters

In mammalian plasma, cortisol binds to a specific α 1-glycoprotein: corticosteroid-binding globulin (CBG). In this study, we measured the protein binding of cortisol by equilibrium dialysis in seven species in which plasma cortisol concentrations varied from 0.02 to 0.05 (ewe, dog, cow) to 0.1 to 0.6 (horse, human, cynomolgus monkey) to reach 1.6 μM (squirrel monkey). No binding of cortisol to CBG was discernible in plasma from squirrel monkey. In all other species examined, we showed that the CBG maximal capacity ( B max) was 3 (1.7 to 5.2) times more than the plasma cortisol levels, with cow, dog, ewe exhibiting the lowest and cynomolgus monkey exhibiting the highest values. We also noted the existence of a linear relationship between B max and the corresponding dissociation constant ( K d ), B max being systematically 10 (8.5 to 11.8) times more than K d . The low binding affinity of cortisol assigned to albumin did not differ between species. The free (6 to 14%), CBG-bound (67 to 87%), and albumin-bound (7 to 19%) cortisol fractions calculated from the estimated binding parameters and measured plasma cortisol concentrations were similar within species, except for squirrel monkey, in which half of the cortisol was albumin bound, and the other half remained protein free. Our most appealing finding was that in most species, as much as 68% of plasma CBG remained free of cortisol under physiologic conditions. These results are discussed with respect to the theories concerning the role of CBG in plasma transport and the local delivery of cortisol and free CBG as a proper hormone.

Increased free cortisol in plasma of dogs with portosystemic encephalopathy (PSE)

Dogs with portosystemic encephalopathy (PSE) are known to develop pituitary-dependent hyperadrenocorticism, but there have been no reports on the plasma protein binding of cortisol in these dogs. Since the liver is involved in the synthesis of corticosteroid-binding globulin (CBG) and other transport proteins for cortisol, the binding characteristics of these proteins and thus the biologically-active free fraction of cortisol might be altered in dogs with PSE. We investigated the total concentration of cortisol and the free fraction and the free cortisol concentration in plasma of thirty-two dogs with PSE due to inherited portosystemic shunts or chronic active hepatitis with cirrhosis. We found a significantly higher free fraction (14.7 ± 5.8%, P<0.0001) and free cortisol concentration (26.3 ± 23.1 nM, P<0.001) in these dogs than in healthy controls (8.2 ± 2.3% and 9.2 ± 7.2 nM, respectively). Moreover, basal concentrations of total cortisol in the dogs with PSE were higher than in the healthy controls (190 ± 146 nM v. 107 ± 65, P<0.01). The per cent free cortisol in plasma was not significantly correlated with the concentration of albumin or the total cortisol in plasma. We conclude that there is decreased binding of cortisol in plasma of dogs with PSE due to decreased hepatic synthesis of cortisol binding proteins. The presence of increased concentrations of free cortisol in these dogs indicates that their basal pituitary-adrenocortical activity was increased, probably due to aberrant neurotransmission in brain centers associated with pituitary function, as a result of hepatic encephalopathy.

Uptake of Cortisol by the Perfused Rat Liver: Validity of the Free Hormone Hypothesis Applied to Cortisol*

The mechanism by which cortisol in plasma enters hepatic cells was investigated using the isolated perfused rat liver. To determine whether hepatic uptake of cortisol from serum can be accounted for entirely by the pool of unbound (free) cortisol, we compared observed uptake rates with the equilibrium-free fraction of cortisol in serum and the rates of dissociation of cortisol from its serum binding proteins (determined using a rapid filtration assay based on transfer of [3H] cortisol to dextran-coated charcoal). More than 95% of the cortisol in both human and rat serum dissociated spontaneously from its binding proteins within 5 sec at 37 C. The fractional unidirectional hepatic uptakes of cortisol from pooled human serum and pooled rat serum were 59.4 +/- 5.4% and 59.5 +/- 1.0% (mean +/- SE), respectively, at the physiological flow rate of 1 ml/min.g liver. The corresponding free cortisol fractions in these sera were 4.53 +/- 0.15% and 8.16 +/- 0.23%, respectively. The fractional unidirectional hepatic uptake of cortisol from protein-free buffer averaged 99.9% (n = 5) at a flow rate of 3 ml/min.g liver. By calculating the appropriate rate constants and applying the Kety-Renkin-Crone equation to the above data, it can be shown that all of the cortisol taken up from serum by the perfused rat liver can be accounted for by the pool of free cortisol, which turns over very rapidly. The physiological significance of this finding is discussed in terms of a general mathematical model of hormone transport that delineates the conditions under which the free hormone hypothesis is and is not valid.

Cortisol and loss of malaria immunity in human pregnancy.

The concentration of both total (bound to corticoid-binding-globulin plus free) and free cortisol was determined in sera from two groups of pregnant women in Tanzania. One group consisted of 152 pregnant women (57 nulliparas and 95 multiparas) exhibiting clinical malaria during pregnancy, and the other group comprised 527 pregnant women (105 nulliparas and 422 multiparas), who did not have a record of malaria during pregnancy. The serum concentration of total cortisol was significantly higher in women with clinical malaria than in women without recorded malaria, and this is true for both nulliparae and multiparae. Free cortisol fractions did not differ significantly between these groups. Indications were obtained that higher total cortisol levels cause loss of malaria immunity rather than being concomitant with malaria infection only. The data are compatible with those of a murine malaria model.

Plasma free cortisol during secretory episodes.

Plasma free cortisol fractions were determined in normal men during spontaneous cortisol secretory episodes and ACTH- or exercise-provoked cortisol peaks in the physiological range. The percentage of free cortisol was measured using ultrafiltration by centrifugation at 37 C, and total cortisol concentrations were measured by RIA. The percent free cortisol varied with the time of day, as did total cortisol. It increased parallel to total cortisol increases, whether they were spontaneous, as occurring in the early morning or postprandially, or provoked by external stimulation. A highly significant positive correlation between relative increases in free and total cortisol was found throughout the experiment. As reported previously, total cortisol increases, according to their origin, did or did not reduce the effects of subsequent stimuli. The present results indicate that the variations in the percent free cortisol are not responsible for the differences in the cortisol responses to these stimuli.

Diurnal Patterns and Regulation of Cortisol Secretion in Pregnancy*

Plasma concentrations of cortisol and corticosteroid-binding globulin are elevated substantially in pregnancy; only the free cortisol fraction is biologically active. Previous suggestions that plasma free cortisol increased above nongravid levels in pregnancy were difficult to reconcile with the absence of manifestations of hypercortisolism. Furthermore, any such elevation should be associated with some alteration in the regulation of cortisol secretion. Therefore, the physiology of cortisol secretion was studied by measuring plasma cortisol at 20-min intervals for 24 h. Free cortisol levels were evaluated by determining the plasma free cortisol index and the rate of urinary cortisol excretion. Circadian cortisol patterns in pregnant and nonpregnant subjects were virtually identical, but total plasma cortisol was significantly higher in pregnancy. Calculated plasma biological half-lives and production rates of cortisol were increased significantly in gravidas (t½: pregnant, 104.6 min; nonpregnant, 69.5 min; daily production rate: pregnant, 28.0 mg; nonpregnant, 11.2 mg). Such increases should produce rises in plasma free cortisol, an assumption supported by finding elevations of the free cortisol index in gravidas at 0800 and 2400 h (pregnant, 14.9 ± 1.9 and 3.3 ± 0.2; nonpregnant, 4.0 ± 0.6 and 1.2 ± 0.3) and elevations of the 24-h urinary cortisol excretion rate. Normal circadian rhythmicity despite elevated free cortisol suggests that maternal hypothalamic-pituitary regulation is altered. Resetting of feedback and lack of manifestations of hypercortisolism could be explained by tissue refractoriness to the effects of cortisol in pregnancy.

The effect of hemodilution on plasma levels of cortisol and free cortisol

The effect of hemodilution on plasma levels of total cortisol and free cortisol was studied in 12 patients undergoing open-heart surgery. In each case, at the onset of extracorporeal circulation (ECC) the hematocrit value was rapidly reduced to 25 to 30 per cent and was maintained around this level during perfusion. Total plasma cortisol levels fell significantly with the hemodilution (p less than 0.0001), but the plasma concentration of free, biologically active cortisol was maintained at its pre-dilution level because of an increase in the percentage of plasma cortisol existing in the free form. There was a highly significant correlation between the degree of hemodilution and the percentage rise in the free cortisol fraction (r = 0.79; p less than 0.001). These results indicate the presence of a compensatory mechanism associated with cortisol binding which maintains the plasma level of free, biologically active cortisol when hemodilution has reduced the plasma level of total cortisol.

Possible placental origin of ACTH in normal human pregnancy

IT is well known that the free fraction of plasma cortisol is increased in pregnancy1, but it has not been established whether this is a result of maternal pituitary or placental adrenocorticotrophic hormone (ACTH) secretion. Maternal plasma ACTH levels in human pregnancy have been variously reported as elevated2, or depressed3; but there is no information on the relationship between ACTH levels and the stage of gestation. We now report that maternal ACTH levels increase progressively throughout pregnancy, that urinary free cortisol levels are raised and show resistance to suppression by dexamethasone and we present evidence suggesting that ACTH may be produced by the placenta.

Effects of oral contraceptive therapy on the circadian patterns of cortisol and thyrotropin (TSH).

The daily variation of serum cortisol and thyrotropin (TSH) has been simultaneously recorded every 30-min. in 4 women taking the same oral contraceptive containing oestrogens and progestogens and in 4 control women. The circadian rhythm of cortisol persisted under contraceptive therapy with about a 2.5 fold elevation of the mean level and amplitude of the basal rhythm. Theoretical equilibrium calculations of the circadian variations of the free, transcortin-bound and albumin-bound cortisol fractions showed that these elevations are explained qualitatively and quantitatively by an oestrogen-induced increase of the same order of the transcortin cortisol-binding sites. As a consequence of the already high saturation of transcortin in normal conditions, the magnitude of the variation of free cortisol level resulting from a burst in cortisol secretion varies with the time of day. The role of albumin as a buffer is thereby emphasized. The early morning maximum, characterizing the normal TSH daily pattern, appeared to be considerably enhanced in women under contraceptive therapy. If the circadian variations of TSH are driven by thyrotropin releasing hormone (TRH), these higher morning peaks probably reflect a higher burst of TRH secretion rather than an increased responsiveness of the pituitary to TRH secretion induced by contraceptive therapy. Finally these results do not support the hypothesis of a regulation of TSH circadian variations by an inhibiotry action of cortisol. Contraceptive therapy does not appear to play a role at the level of the central clock or on the resetting mechanism.

Influence of indomethacin on the distribution of cortisol in man

Simultaneous determinations of free and protein bound plasma cortisol and of the concentrations of cortisol in skin biopsies were performed after treatment with indomethacin. Neither after a single dose nor in patients on continuous treatment, were any consistent changes found in the protein binding of plasma cortisol. However, in patients treated for more than 3 weeks a significantly greater number of skin biopsies were observed with very low concentrations of cortisol. No relation between the free fraction of plasma cortisol and the tissue cortisol could be demonstrated.In vitro experiments showed no alteration of the protein binding of cortisol after the addition of indomethacin to plasma. It is concluded that indomethacin does not have antirheumatic activity because of displacement of the protein bound plasma cortisol as proposed by other workers. However, long term treatment with indomethacin does seem to influence the tissue distribution of cortisol. The possible relationship of this observation is discussed in view of reported fatalities after long continued indomethacin treatment.

Clinical Studies on the Secretion and Metabolism of Cortisol and Aldosterone

The tracer doses of tritiated aldosterone and tritiated or carbon-14 cortisol were injected intravenously in 10 cases with liver cirrhosis, 2 cases with acute hepatitis, a case with chronic hepatitis, 4 cases with hyperthyroidism, 3 cases with hypothyroidism and a case with cancer of the prostate. Daily secretion rate, mean plasma concentration, speed of metabolism, metabolic clearance rate, volume of distribution space and miscible pool of aldosterone and cortisol were calculated in these patients using two compartmental models of human body, discribed by Tait et al. Urinary metabolites of these radioactive steroid hormones were also estimated.1) In all patients with liver cirrhosis, the delayed metabolism of cortisol and aldosterone was shown. Increased daily secretion rate, mean plasma concentration and miscible pool were found in most but not all patients with liver cirrhosis. The secretion rate of cortisol decreased in all cases with lirver cirrhosis. There were some changes in the metabolism of aldosterone and cortisol in patients with liver cirrhosis compared with normal subjects. Increased urinary excretion of free fraction of cortisol and aldosterone, and 3-oxo-conjugate of aldosterone and elevated tetrahydrocortisol/ tetrahydrocortisone ratio were found, while glucuronide fractions of these hormones in urine were decreased in patients with liver cirrhosis. These results suggest that there is decreased conversion to metabolites conjugated with grucuronic acid and reduced reaction of 11, β-hydroxylation in patients with liver cirrhosis.2) In acute hepatitis, slightly delayed metabolism of cortisol and aldosterone was observed, but in chronic hepatitis, no significant changes in metabolism of these hormones compared with that in normal control, were obtained.3) In hyperthyroidism, the speed of metabolism of cortisol was higher than normal controls. However, these values for aldosterone were slightly elevated. Urinary free fraction of cortisol was reduced, but metabolites conjugated with glucuronic acid were increased. The ratio of tetrahydrocortisol/tetrahydrocortisone in urine was decreased.4) In hypothyroidism, the daily secretion rate of cortisol and aldosterone was decreased, but mean plasma concentration of these hormones was within normal range or slightly elevated, while metabolic clearance rate or speed of metabolism of these hormones was markedly decreased. Urinary free faction of these hormones was increased. Tetrahydrocortisol/tetrahydrocortisone ratio in urine was elevated. These results suggest that thyroid hormone accelarates the metabolism of these steroid hormones, and that it may play some role in the activation of 11 β-hydroxysteroid dehydrogenase in liver.5) On the administration of estrogens, delayed metabolism of cortisol and aldosterone was observed. Mean plasma concentration of these steroid hormones was markedly elevated.