Detection of non-typical porphyrin isomers in human urines by ion-pair reversed-phase high-performance liquid chromatography.

An improved ion-pair reversed-phase high-performance liquid chromatographic system has been developed for the separation of uroporphyrin isomers I, II and III, whereas the isomers III and IV could not be resolved. Application of this method to the analysis of urines from porphyric patients indicated the presence of small amounts of the non-typical uroporphyrin isomer II. The questionable presence of the isomer IV was confirmed by acid-catalyzed decarboxylation to the corresponding coproporphyrin isomers, which were completely separated by a modified ion-pair method at elevated column temperatures. These procedures enabled the detection of small fractions of the atypical isomers II (1-3%) and IV (8-15%) besides the normal isomers I and III in urines of patients suffering from attacks of acute intermittent porphyria. Because such urines contain large amounts of porphobilinogen, the nonenzymatic self-condensation of porphobilinogen to uroporphyrinogens was studied under mild reaction conditions. In these experiments quite similar isomeric compositions were observed as compared to those in urines of patients with acute intermittent porphyria. Thus the non-typical uroporphyrin isomers II and IV present in human urines originate from a simple non-enzymatic condensation of porphobilinogen.