The Determination of 17-OHCS in Urine-with Regard to the Collection and Storage of Urine Samples, the Removal of Endogenous β-Glucuronidase Inhibitors, Contaminants Induced by Medication and the Composition of the Porter-Silber Reagent

Abstract

This is a summary of our studies on the determination of urinary 17-hydroxycorticosteroids (Porter-Silber Chromogens).
The conjugated steroid is hydrolysed with bovine liver β-glucuronidase in the presence of Na₂SO₄ and NaHSO₃. Na₂SO₄ almost entirely inhibits the formation of electrostatic complexes between the enzyme and its urinary endogenous high molecular inhibitors, which play the main role in inhibiting the activity of β-glucuronidase. NaHSO₃ maximizes the enzyme hydrolysis in the presence of Na₂SO₄. Probably NaHSO₃ reduces some urinary endogenous thermostable low molecular inhibitors to inactive substances.
The addition of Na₂SO₄ to urine is highly effective for the extraction of the steroids with methylene chloride: Na₂SO₄ increases the recoveries of THF_K and Allo-THF_K, two of the three major cortisol metabolites possessing the dihydroxyacetone side chain, which are relatively insoluble in the solvent.
After the enzyme hydrolysis the urine isisaturated with NaHSO₃, followed by extraction with methylene chloride. The NaHSO₃ forms addition compounds with Δ⁴-3-keto-steroids, ketonic and ketogenic medications interfering with the Porter-Silber reaction: These addition compounds are hardly soluble in methylene chloride. With NaHSO₃ the blank values are two thirds of those without NaHSO₃.
In order to increase the specificity in routine analysis, a procedure based on a study by Allen is often conducted. But this procedure can not always be used with urines from patients receiving drugs, because the absorption curves of the drugs and their metabolites in the Porter-Silber reaction are often strongly convex or concave over the range of wave lengths studied.
The Porter-Silber reagent has been improved in order to obtain satisfactory colour intensities for THF_K and Allo-THF_K.
Twenty four hr urine samples are collected with NaHSO₄, resulting in the urine being kept acidic to inhibit the growth of urinary microorganisms including bacteria: Thus the steroids remain unchanged at 20-30° in a dark place for five days.