Studies on the In Vitro Resin Uptake of Radioactive Corticosteroids from Serum

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The in vitro resin uptake of radioactive corticosteroids from serum has been studied to establish a simple method for the determination of cortisol in serum (or plasma).
1) On the in vitro resin uptake of 14C-4-Cortisol (obtained from The Radiochemical Centre in England) from serum : Two ml of sample serum was pipetted to a small test tube containing a known amount of 14C-4-Cortisol. After vigorous shaking, 1 ml of it was pipetted to another test tube containing 600 mg of Amberlite IRA 400 formate. Then, it was placed in a refrigerator for 90 min at 4°C, shaking it every 10 min. The radioactivity was counted in a liquid scintillation spectrometer. The resin uptake of 14C-4-Cortisol was calculated by the follwing formula :
1-the radioactivity of the supernatant/the initial radioactivity×100.
The results indicate that the resin uptake of 14C-4-Cortisol is consistent with the change of CBG (Corticosteroids-Binding Globulin) rather than cortisol level in the sample serum.
2) On the in vitro resin uptake of 3H-Prednisolone (obtained from The Radiochemical Centre in England) from serum : The procedure was the same as that of 14C-4-Cortisol except for the use of Amberlite CG 400 Type 1 instead of Amberlite IRA 400 formate. The results indicate that in vitro resin uptake of 3H-Prednisolane well reflects the cortisol levels in sera.
3) On the method for the determination of cortisol in serum (or plasma) by using the in vitro resin uptake of 3H-Prednisolone : The standard curve was determined by adding known amounts of nonradioactive cortisol to the standard serum with the same procedure as for the determination of cortisol in the sample serum (as described below).
Two ml of 95% ethanol was added to 2 ml of sample serum (or plasma) in a small test tube, mixed by shaking and centrifuged for 10 min at 3000 rpm. Two 1 mls of supernatant were evaporated to dryness in similar test tubes with a stream of N₂ gas. One ml of standard serum was added to each, and the tubes were incubated for 30 min at 4°C. Then one half ml of 3H-Prednisolone solution was added to each, and the tubes were incubated again for 30 min at 4°C. Two hundreds mg of Amberlite GC 400 Tyre 1 was added to each tube. After that, the procedure was the same as that of 14C-4-Cortisol resin uptake described above. The amount of cortisol in the supernatant was calculated from the standard curve according to the resin uptake and multiplied by 100/25.6 x 50 to express it in pg cortisol per 100 ml of serum (or plasma). The figure of 25.6 means the recovery of cortisol from the supernatant.
4) The variations of cortisol levels in sera of 4 normal subjects after the administration of ACTH were determined by the present method. On the limited evidence it would appear that a good correlation is obtained with the cortisol level expected. Further investigations of the clinical applicability of this method are in progress.