Measurement of Plasma Free Cortisol, Estradiol, Testosterone and Prednisolone by Direct Radioimmunoassay of Ultrafiltrate and Its Clinical Application

Abstract

The authors aimed at developing a simplified method for the measurement of plasma free (unbound) steroids with ultrafiltration using Grace MPS device, and its clinical application. In this method, the movement of free steroids from plasma into ultrafiltrate was monitored with [14C]glucose added to the plasma. Plasma free steroids (cortisol, testosterone, estradiol and prednisolone) were measured as follows: Plasma was incubated with [14C]glucose (1.2 X 10(4) dpm/5 microliters) for 30 min at 37 degrees C. A 0.5 ml of aliquot was transferred to MPS device containing a single YMT membrane, and centrifuged at 1100 x g, at 37 degrees C, for 30 min in a 45 degree fixed angle head with the special temperature controller. After centrifugation, [14C] in 30 microliters of plasma and ultrafiltrate were counted. For the calculation of plasma free level, steroid concentration of ultrafiltrate measured directly by radioimmunoassay (RIA) was multiplied by the ratio of [14C]glucose (dpm) in plasma to [14C]glucose (dpm) in ultrafiltrate. For RIA of cortisol, testosterone and estradiol, commercially available kits were used, and for prednisolone, anti-prednisolone antibody developed in our laboratory was utilized. Since plasma free cortisol level showed a parallel increase with rise in temperature, strict temperature control during ultracentrifugation was required. On the other hand, the duration of centrifugation and the sample volume applied to MPS did not show any significant effect on the estimated values. Intraassay and interassay variations were 4.4% and 6.1% in free cortisol, 6.3% and 8.7% in free testosterone, 8.5% and 9.2% in free estradiol, and 8.8% and 9.9% in free prednisolone, respectively. The correlations between the plasma free steroid levels obtained by ultrafiltration (y) and equilibrium dialysis (x) were as follows, respectively: free cortisol; y = 1.16x + 0.017 (r = 0.95, n = 10), free testosterone; y = 1.17x-0.027 (r = 0.92, n = 10), free estradiol; y = 1.33x + 8.55 (r = 0.98, n = 12), free prednisolone; y = 1.03x + 1.00 (r = 0.98, n = 7). The mean plasma free cortisol levels and percent free fractions (%FF) were 1.16 +/- 0.40 (+/- SD) micrograms/dl and 10.9 +/- 3.0% in 10 control patients, 4.4 +/- 1.6 micrograms/dl and 15.6 +/- 3.3% in 8 patients with Cushing's syndrome, and 1.45 +/- 0.48 micrograms/dl and 5.9 +/- 2.4% in 11 normal pregnant women (10-40 weeks of pregnancy), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantification of unbound prednisolone, prednisone, cortisol and cortisone in human plasma by ultrafiltration and direct injection into liquid chromatrography tandem mass spectrometry

“…Dialysis involves an equilibration step of approximately 6-18 h 7,20 while ultrafiltration requires 30 -60 min equilibration and a 10-30 min centrifugation step. 18,19 Centrifree w ultrafiltration devices (Millipore, Bedford, MA, USA) have been employed for separating corticosteroids; 16,17,21,22 however, the small diameter of the sample reservoir makes the device difficult to load and therefore it is not amenable to automation. Microcon w centrifugal filter devices (Millipore, Bedford, MA, USA) are similar separation devices which can hold up to 0.5 mL of a biological specimen such as plasma.…”

“…Samples can be easily loaded onto Microcon w centrifugal filter devices using automated liquid handlers and they have been used previously for isolating the free fraction of corticosteroids from plasma. 23 Previously published methods using liquid -liquid extraction, 22,24 protein precipitation, 23 radioimmunoassay 19 and detection by high-performance liquid chromatography (HPLC)-ultraviolet (UV) 22 or gas chromatography -MS 12 have high limits of quantification and involve laborious extraction and derivatization steps. On-line extraction methods for measuring cortisol in serum have been previously reported, but either no separation between cortisol and prednisolone was achieved with cortisol eluting at 28 min, 15 or a protein precipitation step preceded the on-line extraction.…”

“…Ultrafiltration and equilibrium dialysis are the most commonly used separation methods for the measurement of unbound concentration. 14,15 Trace amounts of the radioactive isotope of the compound under investigation [ 3 H]-cortisol or [ 3 H]-prednisolone 4,16–18 or of a surrogate chemical moiety such as [ 14 C]-glucose 19 have been employed in the past. Dialysis involves an equilibration step of approximately 6–18 h 7,20 while ultrafiltration requires 30–60 min equilibration and a 10–30 min centrifugation step.…”