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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 ([+ or -]3.3%) and linear dependence of the uroporphyrinogen I formation on incubation time and protein amount. The [K.sub.m] of PGBD for porphobilinogen was measured as 11.2 [+ or -] 0.5 [micro]M with [V.sub.max] of 0.0041 [+ or -] 0.0002 [micro]M/(min x 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%).

We report new assays of enzymes uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen III oxidase (CPO) in the heme biosynthetic pathway. The assays were developed for use in clinical diagnostics of inherited disorders porphyria cutanea tarda and hereditary coproporphyria, respectvely. Electrospray ionization tandem mass spectrometry is used to monitor the decarboxylafion of pentaporphyrinogen I or uroporphyrinogen III catalyzed by UROD and to determine the enzyme activity in human erythrocytes by measuring the production of coproporphyrinogen I or III. The [K.sub.m] value for pentaporphyrinogen I was measured as 0.17 [+ or -] 0.03 [micro]M. A mass spectrometric assay was also developed for the two-step decarboxylative oxidation of coproporphyrinogen III to protoporphyrinogen IX catalyzed by CPO in mitochondria from human lymphocytes ([K.sub.m] = 0.066 [+ or -] 0.009 [micro]M). The assays show good reproducibility, use simple workup by liquid-liquid extraction of enzymatic products, and employ commercially available substrates and internal standards.