Study of factors causing excess protoporphyrin accumulation in cultured skin fibroblasts from patients with protoporphyria.

Abstract

A B S T R A C T The activity of heme synthetase, which catalyzes the chelation offerrous iron to protoporphyrin to form heme, is deficient in sonicates of skin fibroblasts cultured from patients with protoporphyria. During culture in Eagle's medium supplemented with fetal calf serum, these cells do not accumulate protoporphyrin, however. This may be due to a minimal re quirement for heme synthesis, since glycine is incorporated into heme at a low rate which is similar to that in normal fibroblasts. In addition, the activity of 8-aminolevulinic acid (ALA) synthetase, the first and rate-limiting enzyme of heme biosynthesis which catalyzes the formation of ALA from glycine, is normal in lysates of the fibroblasts.Cultured fibroblasts were therefore incubated with ALA in order to bypass the rate-limiting step of heme biosynthesis. In the presence of 25 ,M iron, protoporphyrin was detected in protoporphyria cell lines when the concentration of ALA in the medium reached 50 ,M, but not in normal lines. As the concentration of ALA was increased above 50 ,uM, all lines accumulated protoporphyrin. However, the amount was 2-3 times more in cultured fibroblasts from patients with protoporphyria, reflecting their deficiency ofheme synthetase activity. When iron was not added to the medium, protoporphyrin accumulated to a similar degree in normal and protoporphyria fibroblasts; this was significantly more than that in the presence of iron. These studies indicate that excessive protoporphyrin accumulation in protoporphyria, which is due principally to deficient heme synthetase activity, may be modified by the rate of ALA formation in heme-producing tissues, and by the availability of iron.

Studies in porphyria: functional evidence for a partial deficiency of ferrochelatase activity in mitogen-stimulated lymphocytes from patients with erythropoietic protoporphyria.

“…SC). A similar effect of added iron has been reported in cultured skin fibroblasts from EPP patients (10). However, when EPP lymphocytes were treated with iron in addition to ALA there was no detectable difference in protoporphyrin IX concentrations (Fig.…”

“…This finding strongly suggests that ferrochelatase activity in lymphocytes from patients with EPP as determined in the intact cell is functionally deficient and is only -50% that of normal cells. Similar findings of excess protoporphyrin IX accumulation from added ALA have been reported in skin fibroblast cultures from patients with EPP (10) and from cattle with an autosomal recessive form of EPP (6). Our previous studies have shown that the iron chelator CaMgEDTA is an inhibitor of ferrochelatase in cultured chick embryo hepatocytes (7), chick embryo dorsal root ganglion cells (11,12), and bovine skin fibroblasts (6).…”

“…Finally, only 4 d are required for the technique described in this method, a time which is much shorter than that required for assays using cultured skin fibroblasts (6,10,26). The lymphocyte culture system is thus considerably more convenient for detecting EPP gene carriers than the fibroblast system.…”

Protoporphyrin-induced Cholestasis in the Isolated In Situ Perfused Rat Liver

“…Protoporphyria is an inherited disorder of porphyrin metabolism in which deficiency of heme synthase leads to overproduction of protoporphyrin (1)(2)(3)(4)(5). The initial manifestation and dominant symptom of the disorder is a photodermatitis characterized by erythema, edema and pain (1,6,7).…”

The molecular defect of ferrochelatase in a patient with erythropoietic protoporphyria.

“…In humans, EPP is thought to be an autosomal dominant disease, but the frequency of its clinical expression is highly variable (2). Previous studies have shown that, in patients with EPP, the activity of ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) is decreased to -50% compared with normal levels, in all tissues and isolated cell preparations so far examined: e.g., bone marrow (3), liver (4), cultured skin fibroblasts (5), and lymphoblasts (6). A deficiency of ferrochelatase activity is consistent with the marked increase in protoporphyrin IX, a substrate for the enzyme, observed in erythrocytes, plasma, and liver of patients with EPP, and accounts for cutaneous photosensitivity in this disorder.…”