Liquid-chromatographic assay of urinary porphobilinogen
Jamani, A.; Pudek, M.; Schreiber, W.E.
Clinical Chemistry 35(3): 471-475
1989
ISSN/ISBN: 0009-9147 PMID: 2920415 Document Number: 341034
This is a rapid (10 min per sample), highly sensitive procedure for quantifying urinary porphobilinogen (PBG). Interfering substances are removed by selectively adsorbing PBG onto an ion-exchange resin. After PBG is eluted with 0.5 mol/L formic acid, Ehrlich's reagent is added to produce the chromophore, which is then injected into a liquid chromatograph equipped with a diode-array detector. PBG is separated by a linear gradient (10% to 100%) of methanol in 10 mmol/L phosphate buffer, pH 3.0. Absorbance is monitored at 555 nm. Assay response varies linearly with PBG concentration over the range 0-110 mumol/L (0-25 mg/L). As little as 1.5 mumol/L (0.3 mg/L) can be detected. In prepared urine samples with known PBG concentrations, the within-run coefficient of variation (CV) ranged from 1.7% to 3.2%, the day-to-day CV from 3.5% to 6.1%. PBG concentrations in 24-h urine collected from 25 healthy subjects were all below the detection limit of the assay (less than 1.5 mumol/L). We used the new assay to measure PBG concentrations in the urine of two patients with latent porphyria. This method is more sensitive than spectrophotometric techniques currently used for measuring urinary PBG.