Direct assay of enzymes in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry. Porphobilinogen deaminase.

We report a new assay of human porphobilinogen deaminase (PBGD). Deficiency in this enzyme activity causes acute intermittent porphyria, the most common disorder of heme biosynthesis. The assay involves incubation of blood erythrocyte lysate with porphobilinogen, the natural PBGD substrate. Two subsequent enzymes in the heme biosynthetic pathway, uroporphyrinogen III synthase and uroporphyrinogen decarboxylase, are deactivated by heating so that their activity does not interfere with the PBGD assay. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) is used to monitor the production of uroporphyrinogen I and thus measure the PGBD activity. A simple and efficient workup using liquid-liquid extraction with >90% product recovery was employed to avoid separation by liquid chromatography. The assays show good reproducibility (+/-3.3%) and linear dependence of the uroporphyrinogen I formation on incubation time and protein amount. The Km of PGBD for porphobilinogen was measured as 11.2 +/- 0.5 microM with Vmax of 0.0041 +/- 0.0002 microM/(min.mg of hemoglobin). The coefficient of variation of PBGD activity among several unaffected individuals (12%) is significantly lower than the decrease due to acute intermittent porphyria (50%).