Direct assay of enzymes in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry. Uroporphyrinogen decarboxylase and coproporphyrinogen III oxidase

Abstract

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, respectively. Electrospray ionization tandem mass spectrometry is used to monitor the decarboxylation 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 Km value for pentaporphyrinogen I was measured as 0.17 +/- 0.03 microM. 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 (Km = 0.066 +/- 0.009 microM). The assays show good reproducibility, use simple workup by liquid-liquid extraction of enzymatic products, and employ commercially available substrates and internal standards.

Figure 1

Electrospray MS/MS spectrum of the (M + H)⁺ ion at m/z 655 from coproporphyrin I (8P).

Figure 2

UROD activities in the formation of 3 (hepta), 4 (hexa), 5 (penta), and 6 (copro) measured in blood samples from four individuals. The bars are means of triplicate measurements for each patient and porphyrinogen intermediate.

Figure 3

Electrospray MS/MS spectra of (top) the (M + H)⁺ ion at m/z 563 from protoporphyrin IX (11P) and (bottom) the (M + H)⁺ ion at m/z 567 from mesoporphyrin (9P).

Figure 4

CPO activity measured under N₂ (left bar) and under aerobic conditions (right bar). Both assays were carried out at 37 °C using 4 μM coproporphyrinogen III as substrate.

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