Direct spectrofluorophotometric determination of porphyrin in diluted urine

Direct dilution of freshly passed urine with 19 to 99 volumes of an acid-alcohol solution of chloranil (2, 3, 5, 6-tetrachloro-1, 4-benzoquinone) results in rapid and apparently quantitative conversion of porphyrinogen to fluorescing porphyrin. Dilution must be sufficient to yield an essentially colorless solution. Under these conditions, excitation of fluorescence by 401--405 nm light and fluorescence analysis at 652 nm results in almost complete elimination of quenching and of non-specific fluorescence under the conditions described. Fluorescence intensity is relatively stable for up to several weeks. Alternatively, urine is added to NaHCO3 to permit quantitative conversion of porphyrinogen to porphyrin in 1 to 2 days, and assayed similarly following dilution with the same acid-alcohol diluent (with or without chloranil) or with dilute HCl solution alone. Quantitative results are shown to agree well with those obtained following purification by other, more tedious, procedures. The method has been tested successfully with urines from humans, cows, and rats.