The Uroporphomethene Inhibitor Causitive for Porphyria Cutanea Tarda (PCT) Is Generated by Oxidation of Hydroxymethylbilane (HMB)

Porphyria cutanea tarda (PCT) is caused by a uroporphomethene (UPN) inhibitor of uroporphyrinogen decarboxylase (Phillips, JD et al, Proc Natl Acad Sci U S A. 2007 104(12):5079–84). In the absence of the inhibitor very little uroporphyrin (URO) is secreted in the urine and the excreted URO is predominantly the isomer-III (URO-III). In contrast, in patients with PCT and mice with experimental PCT the predominant porphyrin excreted in urine is uroporphyrin-I (URO-I). Direct measurements of uroporphyrinogen III synthase activity (UIIIS) in mice with PCT have shown no reduction in activity indicating that an alternate mechanism is responsible. We hypothesized that oxidation of a single bridge carbon of HMB, the product of the porphobilinogen deaminase reaction, is the initial event leading to the porphomethene. Our structural data indicate that oxidized HMB cannot serve as a substrate for UIIIS (Schubert, HL et al, Biochem J. 2008 47(33):8648–55) but can non-enzymatically cyclize to form UPN I. UPN I would then serve as the inhibitor of uroporphyrinogen decarboxylase but and then be fully oxidized to yield URO-I. To test this hypothesis we generated authentic UPN’s I and III. URO’s I and III were reduced with palladium on carbon to yield uroporphyrinogens (Uro’gen) I and III. UPN’s I and III were then generated by controlled photo-oxidation of the corresponding Uro’gens. We then utilized LC-MS to separate and characterize the synthesized UPN’s. Retention times of the two compounds differed by approximately 1 min (UPN III elutes prior to UPN I) but the profile of the daughter ions derived from the UPN’s (molecular mass 835 Da) was identical. The same approach was applied to clarified liver cytosol from mice with experimental PCT. The retention time of the 835 Da UPN identified in mouse liver cytosol corresponded to that of the synthesized UPN I and yielded identical daughter ions. These data support the hypothesis that oxidation of HMB leads to formation of UPN I in vivo. This is concordant with our view that UPN I serves both as the inhibitor of URO-D and as the source of the excess URO-I found in PCT.