A Method for the Determination of O-Glucuronide in Urine

Determination of conjugated glucuronic acid by combining enzymatic hydrolysis with lucigenin chemiluminescence

“…Since the range of glucuronic acid in urine is usually 0.8 mM to 2.4 mM and in blood is usually 0.10 mM to 0.23 mM, this method is not sensitive or selective enough to obtain accurate values in these biological fluids. The results obtained for a working curve for p-nitrophenyl glucuronide in this HPLC system using CL detection gave a linear plot with a detection limit of [10][11][12][13][14][15] µ , which is low enough for the analysis of glucuronic acid in either urine or blood serum. Interference studies with ascorbic acid, creatinine, and uric acid showed no effect on the CL signal using this chromatographic system.…”

“…In particular, luminescence spectroscopists require the relative output of their excitation sources in order to generate corrected excitation spectra; these measurements are crucial in many areas of analytical chemistry, physical chemistry, and biochemistry (1,2). However, in spite of advances in thermal detectors (4, 5, 8) and absolute calibration of silicon photodiodes (4-7), luminescence quantum counters (QC's) have remained one of the most popular methods of spectrofluorimeter calibration (1,2,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). This continuing popularity of QC's rests on their simplicity, versatility, and low cost.…”

“…Our earlier manual QC comparator possessed many of the attributes of the ideal system (11,12) but was slow and tedious to use. We wished to develop an instrument that preserved its good features while delegating the tedious and time-con-…”

“…Because most of these conjugation reactions occur in the liver, an accurate measure of the total amount of glucuronic acid in biological fluids would be a good indication of the proper functioning of the liver. In the past, the predominant technique used for the determination of free and conjugated glucuronic acid has been a colorimetric reaction with naphthoresorcinol that was developed by Fishman and Green (7); several modifications of this technique also have been reported (8)(9)(10)(11). The major disadvantages of these techniques are (a) the long times necessary for the acid hydrolysis step and for the reaction with naphthoresorcinol, (b) the necessity of an extraction step for the removal of the pigment before a measurement can be made, and (c) the unsuitably of these techniques for automation in a clinical laboratory.…”