A porphomethene inhibitor of uroporphyrinogen decarboxylase causes porphyria cutanea tarda

Abstract

Porphyria cutanea tarda (PCT), the most common form of porphyria in humans, is due to reduced activity of uroporphyrinogen decarboxylase (URO-D) in the liver. Previous studies have demonstrated that protein levels of URO-D do not change when catalytic activity is reduced, suggesting that an inhibitor of URO-D is generated in hepatocytes. Here, we describe the identification and characterization of an inhibitor of URO-D in liver cytosolic extracts from two murine models of PCT: wild-type mice treated with iron, delta-aminolevulinic acid, and polychlorinated biphenyls; and mice with one null allele of Uro-d and two null alleles of the hemochromatosis gene (Uro-d(+/-), Hfe(-/-)) that develop PCT with no treatments. In both models, we identified an inhibitor of recombinant human URO-D (rhURO-D). The inhibitor was characterized by solid-phase extraction, chromatography, UV-visible spectroscopy, and mass spectroscopy and proved to be uroporphomethene, a compound in which one bridge carbon in the uroporphyrinogen macrocycle is oxidized. We synthesized uroporphomethene by photooxidation of enzymatically generated uroporphyrinogen I or III. Both uroporphomethenes inhibited rhURO-D, but the III isomer porphomethene was a more potent inhibitor. Finally, we detected an inhibitor of rhURO-D in cytosolic extracts of liver biopsy samples of patients with PCT. These studies define the mechanism underlying clinical expression of the PCT phenotype, namely oxidation of uroporphyrinogen to uroporphomethene, a competitive inhibitor of URO-D. The oxidation reaction is iron-dependent.

Fig. 1.

The specific activity of URO-D is reduced in cytosolic extracts of porphyric mouse liver. Both wild-type (gray bars) and Uro-d^+/− (black bars) mice were treated with iron dextran (Fe), drinking water supplemented with ALA, and a mixture of polychlorinated biphenyls (Aroclor 1254) and killed after 21 days, when porphyria was fully developed. (A) Western blotting of 30 μg of total cytosolic protein from each animal by using a 1:200 dilution of a polyclonal rabbit anti-human URO-D antiserum as a primary antibody and a 1:2,000 dilution of a horseradish-peroxidase-labeled goat anti-rabbit IgG as a secondary antibody. The blot was developed by using ECL reagents (Amersham Biosciences/GE Healthcare, Piscataway, NJ). URO-D protein in Uro-d^+/− animals was approximately half of the wild-type value. Protein content did not change when URO-D catalytic activity fell in animals of either genotype. (B) URO-D catalytic activity before and after treatment. Two animals of each genotype were analyzed per group. URO-D activity for each animal is aligned below the corresponding band on the Western blot.

Fig. 2.

Structures of the I isomers of uroporphyrinogen and uroporphomethene. (Left) Uroporphyrinogen, a fully reduced substrate of URO-D. (Right) Uroporphomethene, the partially oxidized inhibitor of URO-D, has lost two hydrogens and gained a double bond on one bridge carbon, as indicated in the green oval. The molecular mass of each compound is indicated. Switching of the acetate and propionate groups (shown in red) on the D ring produces the III isomer compounds. Uroporphyrin, the fully oxidized fluorescent compound, is not a substrate of URO-D.

Fig. 3.

Visible absorbance and HPLC/MS base peak chromatogram overlays obtained from the analysis of partially purified inhibitor. (A) Base peak HPLC/MS chromatogram of substances detected between m/z 200 and 1,200. The base peak ions for peaks 1, 2, and 3 were m/z 835 (uroporphomethene), 837 (uroporphyrinogen), and 831 (uroporphyrin, visible absorbance at 400 nm), respectively. (B) Base peak HPLC/MS chromatogram representing ions of m/z 835 ± 1.0 and visible absorbance at 500 nm.

Fig. 4.

Tandem mass analysis of uroporphomethene I. The 835-Da base peak at 30 min was further analyzed by tandem mass spectroscopy (SI Methods). Product ions with masses of 791, 747, 703, and 659 Da represent the loss of one or more carboxyl groups from the octacarboxylic uroporphomethene. The smaller product ions represent additional breakdown products of the tetrapyrrole macrocycle that are consistent with tri- and dipyrroles. Neutral loss of 197 or 209 Da from m/z 791 via elimination of one pyrrole ring by cleavage of bonds at one of two positions yields m/z 594 and 582 (tripyrroles) with additional breakdown via loss of CO₂. Neutral loss of 195 or 209 from m/z 582 yields m/z 387 and 373, which also decompose by loss of CO₂. Products of heterolytic cleavage of m/z 835 are also detectable at m/z 419, 431, and 448/387.

Fig. 5.

Inhibition of rhURO-D by denatured human cytosolic extracts. Biopsy samples ranging in weight from 8 to 22 mg were processed as described (see Materials and Methods) and assayed for inhibition of rhURO-D activity. Samples H1–H4 were obtained from untreated patients with hemochromatosis. Samples P1–P4 were obtained from untreated patients with PCT. Sample P2 was from a patient with F-PCT. All other porphyric samples were from patients with S-PCT.