A Medlock, R Meldau, A Corrigall, A Josias, R Hift, Pete Meissner

Lennox Eales Porphyria Laboratories, UCT/MRC Liver Research Centre and IIDMM, K-floor Old GSH Main Building, Observatory, South Africa

Variegate porphyria (VP) is inherited as an autosomal dominant disorder with variable penetrance. The prevalent mutation underlying the disease in South Africa is the R59W mutation in protoporphyrinogen oxidase (PPOX). The clinical features of the disease are photosensitivity and acute neurovisceral attack. Drugs, chemicals and infections can precipitate acute attacks. There is no truly satisfactory lab. or animal model of VP. The R59W mouse model is established in our lab. However, pilot experiments in our labs suggest that the R59W defect alone is not sufficient to induce typical VP biochemistry in mice and further inhibition by chemical means (such as feeding the mice a known PPOX inhibitor, acifluorfen) may be necessary to provoke a useful porphyrin phenotype. In this study we establish the R59W VP mouse model further by the measurement of ALA synthase (ALAS), PBG deaminase (PBGD) and PPOX enzyme activities in the haem biosynthetic pathway. The affect of further inhibition of PPOX on the R59W heterozygous mice and wild type mice (controls) was also studied by adding aciflurofen (AF) to the mice diet as appropriate.

In this study we have developed assays for measuring mouse liver ALAS, PBGD and PPOX activities from a single liver. We have used groups of control mice, R59W heterozygous mice, with and without AF.

PPOX activity was decreased by 50% in the R59W mouse model, as expected. AF (0.125%) resulted in a 42% decrease in PPOX in control mice, and reduced PPOX activity to 29% of control mice when added to R59W VP mice. ALAS was uninduced in VP mice, induced by 170% in control mice on AF, and induced by 530% in R59W VP mice. PBGD gave a similar pattern of induction as ALAS in the various mice groups, but to a lesser extent.

Interestingly baseline hepatic ALAS activity was not routinely upegulated in “normal” VP mice, without added AF. ALAS was induced when AF added to both control and VP mice. As both R59W and WT+AF have 50% PPOX activity, AF appears porphyrinogenic in it’s own right. In the VP mice with added AF the PPOX deficiency appears to be severe enough to engender a haem deficiency, which leads to further ALAS up-regulation, in addition to independent porphyrinogenic effect of AF. As there was a smaller relative increase in PBGD activity, compared to induction of ALAS, either there is PBGD molar insufficiency, or PBGD is being (relatively) inhibited by protoporphyrinogen when PPOX activity is severely diminished, such as occurred in the VP mice with added AF.