Urinary urobilinogen determined by a mercuric chloride procedure

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Urinary urobilinogen is a key biomarker for liver function and hemolytic disorders.
• Accurate quantification of urobilinogen is essential for clinical diagnosis.
• Existing methods often rely on artificial standards, limiting accuracy.

Purpose of the Study:

• To develop and validate a novel quantitative assay for urinary urobilinogen.
• To replace artificial standards with a more relevant urobilin standard.
• To assess the method's reproducibility, recovery, and specificity.

Main Methods:

• Urobilinogen is oxidized to urobilin using iodate in an acidic medium.
• pH adjustment with sodium acetate, followed by reaction with alcoholic HgCl2 solution.
• Extraction with CHCl3 and spectrophotometric measurement at 513 nm using crystalline stercobilin IX standards.

Main Results:

• The assay demonstrated high reproducibility with a coefficient of variation of 3.9% from 10 replicates.
• Analytical recovery of added urobilinogen was 90.4% (SD 14.5%).
• No interference was observed from bilirubin, biliverdin, mesobilirubin, coproporphyrin I, uroporphyrin I, or porphobilinogen.

Conclusions:

• The developed assay provides a reliable and accurate method for quantifying urinary urobilinogen.
• The use of crystalline stercobilin IX enhances the clinical relevance of the assay.
• This method offers improved accuracy and specificity compared to previous urobilinogen assays.