METHOD OF DETERMINING CONJUGATED URINAL HYDROCORTISONE FRACTION Russian patent published in 1998 - IPC

FIELD: analytical methods in medicine. SUBSTANCE: sample (0.5-1 ml) of daily urine is boiled for 1 min, cooled, and equal volume of β- glucuronidase solution in acetate buffer at pH 4.5 is added on the basis of 500 IU enzyme activity per 1 ml of urine. Mixture is hydrolyzed at 37 C for 18-20 hr. Hydrolyzate supernatant is poured out and mixed, as well as native urine, with ether in ratio from 1:10 to 1:30. Resulting mixture is extracted with water for 30 to 120 s. After removal of aqueous fraction and ether, dry residue is taken up in phosphate buffer at pH 7.2-7.4 and original volume is restored. Solution is heated for 5 min at temperature not exceeding 60 C, cooled, and γ-labeled hydrocortisone and solution of hydrocortisone- specific antibodies are added. Resulting sample is incubated at 18-25 C for 1 to 4 hr and precipitating reagent containing second hydrocortisone-specific antibodies is added. Resulting mixture is centrifuged for 10-30 min at 1500-2000 g and 18-25 C and supernatant is removed. Precipitate is analyzed for gamma-radiation level using calibration curve. Taking into account dilution and daily diuresis in hydrolyzed urine sample, index of daily excretion of total hydrocortisone is calculated. Further, index of daily excretion of free hydrocortisone in native urine sample is calculated. Index of conjugated hydrocortisone fraction is found by subtracting index of daily excretion of free hydrocortisone from index of daily excretion of total hydrocortisone. Method allows determining free, conjugated, and total hydrocortisone excretion and can be utilized for differential diagnostics of adrenal disease when administering glucocorticoid treatment. EFFECT: simplified procedure and improved specificity. 1 dwg, 1 tbl, 3 ex