A rapid and accurate spectrofluorometric method for quatification and screening of urinary porphyrins

Abstract

We describe a fluorescence method for screening and quantifying urinary porphyrins. New and effective approaches are used to oxidize porphyrinogens, correct the baseline, and ensure that uroporphyrin (uro) and coproporphyrin (copro) are equally detected, mole for mole. No preliminary purification is required. A 45-μL aliquot of urine is oxidized with 3 mmol/L iodine in 3 mmol/L HCI to convert porphyrinogens to porphyrins, and then decolorized with 5 mL of 0.45 mmol/L sodium thiosulfate. An excitation scan is done from 350 nm to 440 nm, monitoring emission at 650 nm. Total porphyrin content is determined at the isosbestic point for uro and copro, and the mole fractions of uro and copro are estimated from the wavelength of the signal maximum. There is no interference from protein, glucose bilirubin, or hemoglobin in high concentration. The limit of detection is < 30 nmol/L and linearity is maintained up to 3200 nmol/L. Recoveries and precision are excellent. This is a rapid, sensitive screen for porphyrinuria as well as an accurate and precise quantitative method. We compared the method with existing methods and discuss some shortcomings common to many of them.