Free cortisol measurement

Urinary free cortisol obtained from a 24-hour urine collection is an integrated measure of plasma free cortisol and eliminates the circadian influence on cortisol secretion. Urine free cortisol measurements are therefore considered to be the best screening test for hyperadrenocorticism. The urinary free cortisol excretion rate in normal subjects falls between 20 and 80[lg/day (Figure 51-9), The measurement of urinary 17-hydroxycorticosteroid excretion rates for determining adrenocortical hyperfunction is no longer recommended because of problems with method sensitivity and specificity. 

Figure 5 -9 Urinary free cortisol excretion in normal individuals with Cushing s syndrome. (From Bur/ce CW, Beardwell CG. Cushing s syndrome. An evaluation of the clinical usefulness of urinary free cortisol measurements in screening for Cushing s syndrome. QJ Med I973 42 l 175.)...

Adrenal Tumours The assay-method is entirely based on the Schwartz-Mann Kit. According to this method, cortisol is first extracted from the plasma using CH2C12 (methylene chloride). In the actual radioimmunoassay the cortisol present in the extract competes with Cortisol-H3 i.e., the radioactive tracer) for the common binding sites on transcortin, which is incidently not an antibody but a cortisol-binding protein. Now, the free cortisol is quantitatively removed by adsorption on dextran-coated charcoal from the one bound to the transcortin. Finally, the bound radioactivity (due to Cortisol-H3) is measured which is then employed to calculate exactly the amount of cortisol present in the sample by the help of a Standard Curve (or Calibration Curve). 

Kanter et al. reported an increase concentration of the corticosteroid binding globulin (CBG) (Kanter et al. 2001). Most cortisol is bound to CBG, and is biologically inactive. A greater concentration of CBG is consistent with low levels of measurable free cortisol, and provides a putative explanation for how cortisol levels could be measurably low even though other aspects of HPA axis functioning do not seem hypoactive. However, the extent to which CBG levels are a contributing cause of low cortisol requires further examination. 

Many centers have reported specific abnormalities on measures of hypo-thalamic-pituitary-adrenal axis activity in patients with psychotic depression. Patients with psychotic major depression (PMD) are among those with the highest rates of nonsuppression on the dexamethasone suppression test, and many of them have markedly elevated posttest levels (Carroll et al. 1976c, 1980 Coryell et al. 1982). Significant differences have been observed in 24-hour measures of urinary free cortisol between patients with psychotic and those with nonpsychotic major depression (NPMD) (R. F. Anton 1987). 

The method measures cortisone (urinary free cortisone, UFE), cortisol (urinary free cortisol, UFF), 6/1-hydroxycortisol, and 18-OHF using deuterated internal standards [62]. Commercial tetradeuterocortisol was used as an internal standard for cortisol, and the remaining dideutero homologs prepared in the laboratory by deuteration of A1 analogues. UFF and UFE are considered better indicators of hormone availability and hypersecretion than the F and E (free plus conjugated) quantified in the comprehensive profile. Typically the values of total F and E are about three times that of... 

Concentrations of Drug XYZ, 6-fS-hydroxy-cortisol and free cortisol in urine collected over the profiling period were measured. Volumes of urine collected over each profiling period were recorded. 

No abnormalities were found in Brian s urine and his blood cell count was normal. However, his blood glucose was 8.1 mmol l-1 (normal value 3.5-6.7 mmol l-1) and a glucose tolerance test later indicated impaired glucose tolerance. Tests for plasma insulin and thyroid hormones (T3, T4 and TSH) showed normal levels. Two further tests were then performed. A 24-hour urine sample was collected and Brian s free cortisol excretion was found to be considerably higher than normal. A second test, the dexamethasone suppression test, was also carried out. In this test, the patient is given a dose of dexamethasone at 11 -12 p.m. and plasma cortisol is measured early next morning. 

The free component of plasma cortisol is biologically active and is filtered and excreted in the urine, where it can be measured. It would appear that urinary free cortisol estimations may give a more accurate picture of glucocorticoid activity at a tissue level than most other measurements. 

The different methods of assessing hypothalamic-pituitary-adrenal axis activity in patients using inhaled glucocorticoids have been compared (35). The AUC of serum cortisol concentrations was the most reliable method. There were significant positive correlations between AUC and the 8 a.m. serum and salivary cortisol concentrations. The authors favored the non-invasive method of salivary concentration measurement. However, 24-hour urine collection is not recommended, as it correlated only moderately well. This finding is consistent with the results of other studies. Urinary free-cortisol estimation based on immunoassay after inhaled glucocorticoids may be an unreliable surrogate marker of... 

Procedure ACTH gel, 80U/day, is injected for 3 days. This is followed by a standard 8-hour infusion of ACTH (250 p.g of Synacthen over 8 hours). Urinary free cortisol and serum cortisol are measured daily. 

Procedure Twenty-four-hour urine samples are collected daily for 4 consecutive days. Dexamethasone, 0.5 mg, is given orally every 6 hours starting at 0800 hours on day 2 (for a total of 8 doses). Free cortisol, 17-hydroxycorticosteroids, and creatinine are measured in each 24-hour urine sample. Other measurements include serum cortisol determinations on day 1 (at 0800 hours and 2000 hours) to look for diurnal variation and on day 5 (at 0800 hours). 

The measurement of steroid hormones in a free state distinct from a protein-bound state may be advantageous under certain situations. When alterations in the binding proteins that carry the bulk of steroids in the circulation occur, the interpretation of the total circulating concentration of that particular steroid may be significantly influenced. In addition, it is the free hormone that binds to the steroid receptor and elicits the biochemical effect thus knowledge of the free hormone concentration is desirable in a number of clinical situations. The concept of measuring free hormones has been in the literature for a number of years with the documented clinical utility of urinary free cortisol. Other applications include the measurement of free and weakly bound testosterone in the clinical work-up of the patient presenting with hirsutism. ... 

Altliough methods involving organic solvent extraction or protein precipitation wfil estimate both free and protein-bound cortisol in tlie circulation, immunoassay-based methods that determine cortisol directly from serum have essentially replaced all extraction-based methods for routine cortisol measurements save for urine free cortisol methods. Flistorically four general methods have been used for the quantitative estimation of total blood cortisol concentrations in the clinical laboratory. These are the Porter-Silber color reaction method modified for plasma,the use of sulfuric acid-induced fluorescence, HPLC methods, or immunoassay. In general, HPLC and immunoassay are now used in both clinical laboratories and reference laboratories for the quantitative determination of cortisol in serum or urine. 

Measurement of Free Cortisol in Blood, Urine, and Saliva... 

Free or unbound cortisol represents the biologically active form of the circulating hormone, and its concentration is practically independent of alterations of its transport proteins. Various methods have been developed for estimating the free fraction in serum, but these assays are technically demanding, expensive, and not in general use. The measurement of urine free cortisol comes closest to providing an estimate of the free hormone concentration. As mentioned previously, approximately 2% of cortisol is excreted into the urine in a free form, and its measurement has been shown to be of use as a screening test for cortisol hypersecretion. However, P-hydroxycortisol has been reported to interfere with the immunoassay of free cortisol in urine. ... 

Most immunoassays for total serum cortisol can be used for the measurement of urinary free cortisol after appropriate... 

Urine Specimens. A complete 24-hour urine specimen is collected with 10 g of boric acid to maintain the urine pH below 7.5. Urine should be refrigerated during the collection period. After measuring the total volume, a thoroughly mixed aliquot ( 10mL) is stored frozen at -20 °C. Care should be taken to ensure an appropriately timed, complete 24-hour collection because an incorrectly timed sample is the largest source of error with this method. Free cortisol determinations on randomly collected urines are discouraged because of the variation and pulsatile characteristic of cortisol secretion. 

Varelis P, Smythe GA, Hodgson D, Lazarus L. Measurement of urinary free cortisol by stable isotope dilution mass spectrometry using a new cortisol derivative. J Chromatogr B Biomed Appl 1994 660 151-9. 

Lykkesfeldt, J. Loft, S. Poulsen, H.E. Simultaneous determination of urinary free cortisol and 6fl-hydrojgrcortisol by high-performance liquid chromatography to measure human CYP3A activity. J.Chromatogr.B, 1994, 660, 23—29 [urine extracted 6p-hydroxycortisol dexamethasone (IS) SPE gradient LOQ 1 ng/mL]... 

A comparison of the therapeutic efficacy of the 800-pg/day HFAand 1500-pg/day CFC formulations of beclomethasone has shown that the two preparations have similar clinical effects (62). In that study, patients with asthma were randomized to use the HFA or CFC preparation for 12 weeks. Peak flow and FEVj were measured at regular intervals. Statistical analysis showed that the two formulations were equivalent in terms of improvement in these two parameters. Another study showed equivalence for the HFA formulation compared to the CFC formulation in terms of possible adverse effects— measured as acute tolerability, urinary free cortisol, and adrenal responsiveness (63). 

McCann, S.J. Gillingwater, S. Keevil, B.G. Measurement of urinary free cortisol using liquid chromatography-tandem mass spectrometry Comparison with the urine adapted ACS 180 serum cortisol chemiluminescent immunoassay and development of a new reference range. Ann. Clin. Biochem. 2005, 42 (2), 112-118. 

In conclusion, only obesity and hypertension are consistent findings. Among the clinical findings, the most reliable seems to be that of free cortisol excreted in the urine. The results of the administration of dexa-methasone (which suppresses urinary 17-ketogenetic steroid excretion) may be suggestive, but not always conclusive. Measurements of 17-ketosteroid excretion or of 11-hydroxycorticosteroid excretion are of little or limited value. 

The majority of the plasma cortisol is protein bound, only a small proportion being in the free state. This free cortisol is filtered at the glomerulus and passes into the urine where it can be measured, for example by radioimmunoassay. Urinary free cortisol determinations therefore correlate with the plasma free cortisol and the cortisol secretion rate. Increased urinary free cortisol levels are found in cases of Cushing s syndrome. 

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