抄録
A new method for the determination of urinary 17-Hydroxycorticosteroids (Porter-Silber chromogen) is described. The conjugated steroid in urine is hydrolyzed with bovine liver β-glucuronidase in acetate buffer (pH 5.0) containing a Na2SO4-NaHSO3 mixture (300: 1), which not only interferes almost entirely with the formation of an electrostatic complex from the enzyme and its urinary endogenous high molecular inhibitors, but inactivates unknown low molecular inhibitors often present in alkaline or putrefying urine. The yield of urinary steroids obtained by hydrolysis in presence of the above mixture was 7±4.8 % (S. D.) higher than that in its absence.
The addition of Na2SO4 to urine (1 volume) is highly effective for the extraction of the steroids with methylene chloride (6 volume): THE and Allo-THF, two of the three major cortisol metabolites containing dihydroxyacetone side chain, which are relatively insoluble in the solvent, show 96±1.1, 95±1.1% (S. D.) recoveries in its presence respectively after one extraction only, while 63±1.9, 62±2.0 in its absence. In urine the addition of Na2So4 at the extraction step increased the yields by 7±3.9% (S. D.).
After hydrolysis with the enzyme the urine is saturated with NaHSO3, followed by extraction with methylene chloride, where NaHSO3 removes largely non-tetrahydro compounds of steroids and non-steroid impurities (eg. ketone compounds) interfering with the subsequent Porter-Silber reaction. As a result not only does this blank show two-thirds of that in the control, but the spectrum presents a symmetrical curve with an absorption peak at 410 mμ. For increasing specificity in a routine methods, the procedure based on a study by Allen is often followed, but in our presnt method it was considered unnecessary.
The extract is washed with N/2 Na2CO3, N/20 NaOH and N/10 H2SO4 respectively, all of which containing Na2SO4.
The Porter-Silber reagent was improved in composition(sulfuric acid+ethano1)12-14: the details are given in the foot note of the method.
