Abstract
Hepatic iron content is increased in patients with PCT and phlebotomy-induced iron depletion corrects the clinical and biochemical phenotype. Approximately 20 percent of patients with PCT are homozygous for mutations of the hemochromatosis gene (HFE) but the cause of iron overload in most patients is unknown. Hepatic URO-D activity is markedly reduced when PCT is manifest and URO-D activity improves following iron depletion. Most patients with PCT have no mutations of the URO-D gene (sporadic PCT) but approximately 1/3 of cases are heterozygous for URO-D mutations (familial PCT) (
BLOOD. 2000; 95:1565–71
). To determine the mechanism by which iron overload causes PCT we created 3 murine models: Mice with one null allele of Uro-d (Uro-d+/−) and 2 null Hfe alleles (Hfe−/ −) (PNAS. 2001; 98:259–64
); Uro-d+/− mice treated with iron-dextran, aminolevulinic acid (ALA) and polychlorinated biphenyls (PCB) and; wild type mice treated with iron, ALA and PCB (J. Biochem. Mol. Toxicol. 2001; 15:287–93
). All models accumulate uroporphyrin in the liver and all have hepatic URO-D activity of 25% or less but Western blots revealed no change in URO-D protein. An iron deficient diet prevented the PCT phenotype in Uro-d+/−, Hfe−/ − animals and greatly attenuated the phenotype in animals treated with ALA and PCB (Env. Toxicol. Pharmacol. 2005; 417–23
). Liver homogenates from all porphyric models were heat denatured and clarified by centrifugation. The supernatants inhibited the activity of purified recombinant human URO-D (rhURO-D) by approximately 60%. The inhibitory activity was further purified by solid phase extraction and HPLC. The fraction containing the inhibitory activity did not fluoresce. Mass spectrometry of this fraction demonstrated a dominant peak with a mass of 835 Da and an absorption maximum of approximately 500 nM, the optical signature of a porphomethene. An inhibitor with identical properties was generated by partially oxidizing the uroporphyrinogen (837 Da) substrate of URO-D under UV light. Full oxidation of either the inhibitor purified from porphyric mouse liver or from partially oxidized, enzymatically generated uroporphyrinogen (either isomer I or III) yielded a compound with a mass of 831 Da and an absorption maximum of approximately 400 nM, indicating that the fully oxidized inhibitor was uroporphyrin. Tandem mass spectrometry of the 835 Da inhibitor indicated that the inhibitor was a tetrapyrrole. Collectively these data indicate that the inhibitor is a porphomethene derived form uroporphyrinogen through oxidation of a single bridge carbon between adjacent pyrrole rings. An inhibitor of rhURO-D was also identified in heat-denatured cytosol from liver biopsy samples obtained from four humans with PCT. We conclude that clinical expression of PCT requires an iron dependant oxidation reaction that generates a porphomethene inhibitor of URO-D.Disclosure: No relevant conflicts of interest to declare.
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2006, The American Society of Hematology
2006
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