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

Abstract

A B S T R A C T In this paper we show that the ferrochelatase defect in erythropoietic protoporphyria (EPP) can readily be identified in mitogen-stimulated lymphocytes since such cells from patients with EPP accumulate approximately twice as much protoporphyrin IX as cells from normal subjects when incubated with a porphyrin precursor, 6-aminolevulinic acid (ALA). Treatment of cultures with ALA and with the iron chelator, CaMgEDTA significantly increased the level of protoporphyrin IX in mitogen-stimulated lymphocytes from normal subjects, while the same treatment failed to produce an increase in protoporphyrin IX in cell preparations from EPP patients. In contrast to the results with the chelator treatment, supplementation of the cultures with iron and ALA reduced the level of protoporphyrin IX in normal cells, but not in EPP cells. These findings are compatible with a partial deficiency of ferrochelatase in EPP lymphocytes. The gene defects of acute intermittent porphyria and hereditary coproporphyria have previously been identified using lymphocyte preparations from the gene carriers of these diseases. The present study demonstrates that EPP represents another form of human porphyria in which the gene defect of the disease can now be identified in lymphocyte preparations.

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

“…Although the parents were available for ferrochelatase assays in an early study, they and several other members of this family were no longer available for the molecular studies on the ferrochelatase defect, for reasons beyond our control. Partial ferrochelatase deficiency (-50%) is also known to occur in both asymptomatic carriers and clinically affected patients in other EPP families (6,14). Thus the occurrence of '50% ferrochelatase deficiency both in the father and in the daughter is similar to that in other EPP families and is compatible with an autosomal dominant inheritance of EPP in this family.…”

“…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.…”

“…Protoporphyrin concentrations in her erythrocytes, plasma, and stool were markedly elevated (Table 1), and erythrocyte porphyrins consisted almost exclusively of free, not Zn-chelated, protoporphyrin (13). Ferrochelatase activity, as determined by an assay based on the ability of lymphocytes to form protoporphyrin from its precursor, 6-aminolevulinic acid (6), indicated that the proband's enzyme activity was about one-half (46.5%) the normal level (Table 1). These findings in the proband's cells are compatible with the diagnosis of EPP.…”

“…(mean + SEM, n = 8) (mean ± SEM, n = 9) As an indirect assay for ferrochelatase, protoporphyrin IX formation by lymphocytes was determined as described previously (6). Lymphocytes were incubated with phytohemagglutinin (PHA) and pokeweed mitogen (PWM) for 3 days and then with 0.6 mM 8-aminolevulinic acid (ALA) for 24 h. Protoporphyrin IX accumulation found by this assay has been shown to be inversely correlated with ferrochelatase activity (6). Both PHA/PWM-stimulated lymphocytes and Epstein-Barr virus-transformed lymphoblastoid cells give similar results with this assay (unpublished observation).…”

Porphyrin Synthesis by Murine Epidermal Cells

“…The former is a spe cific inhibitor of ferrochelatase in various types of cultured cells [7,9,11], while the lat-ter is one of the two substrates of ferrochelatase; the other substrate of the enzyme is pro toporphyrin. Our results indicate that iron decreased porphyrin levels, and this decrease was partially reversed by CaMg EDTA (fig.…”