Accumulation of protoporphyrin IX from delta-aminolevulinic acid in bovine skin fibroblasts with hereditary erythropoietic protoporphyria. A gene-dosage effect

Bovine skin fibroblasts accumulated protoporphyrin IX when incubated in culture with the porphyrin-heme precursor, delta-aminolevulinic acid (ALA). Fibroblasts from cattle homozygous for erythropoietic protoporphyria (EPP) and with the clinical symptoms of the disease accumulated approximately sixfold greater amounts of protoporphyrin IX than cells from normal control animals. Cells from obligatory heterozygous animals, which are clinically normal, accumulated an intermediate level of protoporphyrin IX. When these cells were incubated with ALA and CaMg EDTA, all types of cells accumulated approximately the same amount of protoporphyrin IX (approximately 500 nmol/mg protein), suggesting that ferrochelatase activity was equally low after inhibition by treatment with CaMg EDTA in all cells. Thus the ratio of protoporphyrin IX accumulation from ALA in cultures treated with CaMg EDTA compared with controls treated with ALA alone was greatest in normal cells, least in EPP cells, and intermediate in the heterozygote cells. These findings suggest that the amount of protoporphyrin IX accumulation from ALA reflects the extent of deficiency of ferrochelatase and is proportional to the dosage of abnormal EPP gene in cultured fibroblasts. Similarly, stimulation of porphyrin accumulation by CaMg EDTA reflects diminished ferrochelatase activity in these cells. Thus, the results of this study demonstrate the usefulness of estimating protoporphyrin IX formation from ALA for the detection of an EPP gene defect in cultured bovine skin fibroblasts.