Erythropoietic Protoporphyria Patients Research Articles

The pro- and antioxidant properties of protoporphyrin IX

The effect of protoporphyrin on lipid peroxidation in rat liver microsomes was investigated in the presence and absence of light. Protoporphyrin and light stimulated Fe 3+-ADP/ascorbate-induced lipid peroxidation, whereas Fe 3+-ADP/NADPH-mediated lipid peroxidation was inhibited. In the dark, on the other hand, protoporphyrin inhibited lipid peroxidation in both systems in a concentration-dependent manner. Protoporphyrin did not affect the reduction of the Fe 3+-ADP complex by ascorbate, nor did it affect the activity of the NADPH-cytochrome P−450 reductase under these conditions. Uroporphyrin, a hydrophilic porphyrin and not localized in the membrane, did not inhibit lipid peroxidation in the dark in these systems, whereas bilirubin, a well-known radical scavenger and degradation product of protoporphyrin, inhibited lipid peroxidation in both systems. When peroxyl radicals were generated in solution by the azocompound 2,2'-azobis (2-amidino-propane)dihydrochloride lipid peroxidation was inhibited by both porphyrins and bilirubin. Reaction of bilirubin or protoporphryin, in the presence of microsomes or human serum albumin, with these peroxyl radicals resulted in degradation of both compounds, which could be determined by a decrease in their absorbance at 460 or 407 nm, respectively. These results indicate that the inhibition of lipid peroxidation is most likely caused by scavenging of peroxyl radicals by protoporphyrin. Massive accumulation of protoporphyrin occurs in livers of erythropoietic protoporphyria patients. The fact that protoporphyrin was able to inhibit lipid peroxidation completely at micromolar concentrations also indicates that the deleterious effects of protoporphyrin, observed in these patients, are most likely not mediated by oxidation of lipids.

Determination of porphyrins in bile using high performance liquid chromatography and some clinical applications.

A simple and fast HPLC method for the determination of porphyrins in bile without extraction is described. Porphyrins were determined in bile from control subjects and from patients after orthotopic liver transplantation, including three patients with erythropoietic protoporphyria. It was found that: 1) coproporphyrin I is the predominant porphyrin in bile of controls, accompanied by some coproporphyrin III and protoporphyrin, whereas protoporphyrin mostly but not always is the predominant porphyrin in the bile of erythropoietic protoporphyria patients. In two of the three erythropoietic protoporphyria patients, the bile contained a hundred times more protoporphyrin than that of non-porphyric orthotopic liver transplantation patients. The third erythropoietic protoporphyria patient remained cholestatic and was unable to excrete sufficient amounts of protoporphyrin. 2) All investigated bile samples contained no secondary porphyrins derived from protoporphyrin, i.e. no deutero-, pempto-, or mesoporphyrin. Even when extracts of bile and serum were concentrated fifty to a hundred times, no traces of deutero-, pempto- and mesoporphyrin were detected. This complete absence of secondary porphyrins suggests that an enterohepatic circulation of dicarboxylic porphyrins from the distal gastrointestinal tract does not exist. 3) The HPLC chromatograms contain peaks from unknown compounds. No correlation between porphyrins and these compounds was found. Porphyrin profiles were followed in the bile of some orthotopic liver transplantation patients. Three episodes are recognizable. During the first three days after orthotopic liver transplantation there is a very high coproporphyrin excretion. There is then a lag of one to three weeks, in which no or very low porphyrin concentrations are detectable, followed by the restoration of normal biliary porphyrin patterns.

Screening for ferrochelatase mutations: molecular heterogeneity of erythropoietic protoporphyria

The DNA of 21 patients from 19 unrelated families with erythropoietic protoporphyria (EPP) were screened for the 6 ferrochelatase point mutations so far described. The mutation previously described by us (A ⪢ t transversion at position −3 of the donor site of intron 10, causing exon 10 skipping) was detected in two additional unrelated EPP patients: in these patients, cDNA lacking exon 10 was also detected. The mutation described by Nakahashi et al. as responsible for exon 2 skipping (C ⪢ T transition at position −23 of the acceptor site of intron 1), although also observed in some normal individuals, was invariably observed in all EPP patients tested and may thus play some role in the pathognesis of EPP. Thus, it does not appear that this mutation is the primary mechanism underlying exon 2 skipping. None of the other four previously described mutations were detected. These data demonstrate the heterogeneity of the ferrochelatase locus and of the genetic defect in EPP.

HEMOGLOBIN OXYGEN AFFINITY IS INCREASED IN ERYTHROPOIETIC PROTOPORPHYRIA

Whole blood and hemolysates from seven normal and three erythropoietic protoporphyria patients were compared in terms of their hemoglobin function. The oxygen affinity (P50) of the erythropoietic protoporphyria hemolysates compared to normals (13.1 +/- 0.2 vs 17.5 +/- 0.3 mmHg; P < 0.001) and erythropoietic protoporphyria erythrocytes compared to normals (23.4 +/- 0.6 vs 27.1 +/- 0.5 mmHg; P < 0.001) were increased while oxygen-binding cooperativity (n-value of the Hill equation) were similar. We conclude that hemoglobin function in erythropoietic protoporphyria patients is altered, but without pathophysiologic consequences. Because hemoglobin in which protoporphyrin IX substitutes for heme has a low oxygen affinity, our findings of a higher than normal affinity in erythropoietic protoporphyria red cells and hemolysates may indirectly support the findings by others that protoporphyrin IX binds to hemoglobin at non-heme-binding sites. In addition, based on the effect of other abnormal hemoglobins, this shift in P50 will decrease any tendency for anemia in erythropoietic protoporphyria patients.

Accumulation of iron in erythroblasts of patients with erythropoietic protoporphyria.

We have studied the iron metabolism in nine patients with erythropoietic protoporphyria (EPP) and three patients with sideroblastic anaemia (SA). All, except one EPP patient were iron deficient. The SA patients had a secondary haemochromatosis. The bone marrow aspirates of patients with SA and also three patients with EPP had a high incidence of ring sideroblasts. Ultrastructural examination of the bone marrow consistently showed finely dispersed electron-dense deposits localized in mitochondria of erythroblasts in all patients with EPP and SA. Mitochondrial electron energy-loss spectroscopy (EELS) indicated identical iron compounds in erythroblasts of all EPP and SA patients. These findings indicate that the mitochondrial iron utilization is disturbed in EPP and SA. The observation of mitochondrial iron deposition in erythroblasts in EPP and SA suggests that this failure is not of pathognomonic value for diagnosis of SA, but is apparently the result of an inefficient haem synthesis, in EPP due to a defective ferrochelatase. The mitochondrial iron deposition does not depend on the iron status (iron overload or iron deficiency) of the EPP patient.

Human Erythropoietic Protoporphyria: Two point mutations in the ferrochelatase gene

The molecular basis of the ferrochelatse defect responsible for human Erythropoietic Protoporphyria (EPP), a usually autosomal dominant disease, was investigated in a family with an apparently homozygous patient. Two mutations of the ferrochelatase gene were identified by sequencing the proband's cDNA after in vitro amplification of the mRNA and subcloning of the amplified products. One mutation results from a G to T transition at nucleotide 163 which produces a glycine to cysteine substitution at amino-acid residue 55 (G-55-C) The other one was a G to A change at nucleotide 801, leading to a methionine to isoleucine substitution at amino-acid residue 267 (M-267-I). This EPP patient was then double heterozygous and as expected each of his parents carried one of the mutations. A second similar EPP patient was screened for these mutations with negative results, showing a genetic heterogeneity in EPP.

Genetic Heterogeneity in Erythropoietic Protoporphyria: A Study of the Enzymatic Defect in Nine Affected Families

Erythropoietic protoporphyria (EPP) is associated with a deficiency of protohaem ferrolyase. We have used a novel assay for this enzyme based on its ability to utilize zinc as a substrate to investigate the inheritance of EPP in nine affected families. Zinc chelatase activity was markedly reduced in peripheral blood mononuclear cells from 14 EPP patients (mean, 3.3 nmol Zn protohaem/h/mg protein; range, 0.3-8.0) when compared with 41 controls (16.8 +/- 3.6) p less than 0.01. In three families with parent-to-child transmission of disease, the asymptomatic parent had an enzymatic activity within the normal range. In three pedigrees where the parents were asymptomatic, enzymatic activities were below the 95% confidence limits in both. Zinc chelatase activity was below the mean control value in 17 of the 18 parents in nine affected pedigrees, and six of seven asymptomatic offspring of patients with protoporphyria. The findings suggest that EPP is not transmitted as a simple dominant trait and that inheritance of more than one gene may be required for disease expression.

Different characteristics of ferrochelatase in cultured fibroblasts of erythropoietic protoporphyria patients and normal controls

Ferrochelatase activity was measured in crude extracts of fibroblasts, obtained from erythropoietic protoporphyria patients and healthy controls. The enzyme activity in erythropoietic protoporphyria fibroblasts was about 50% lower, compared to the controls. The sulfhydryl-oxiding reagent diamide inhibited the normal enzyme by about 50%, whereas ferrochelatase from erythropoietic protoporphyria fibroblasts was completely insensitive to the reagent. Pb 2+ inhibits ferrochelatase activity by reacting with essential sulfhydryl groups. Low concentrations of Pb 2+ inhibited the normal enzyme by 56%, but the mutant enzyme by only 8%. The photodynamic activity of bound mesoporphyrin substrate caused a biphasic inactivation of the normal enzyme.During the first 5 min of illumination a fast decrease of enzyme activity occured to about 60% of the initial value. Experimental evidence indicates that this first phase of inactivation is caused by photooxidation of sulfhydryl groups. Durings further illumination inactivation continued at a much slower rate. With ferrochelatase from erythropoitic protoporphyria fibroblasts only the second, slow phase of photodynamic inactivation was observed. These observations suggest a mutation of ferrochelatase in erythropoietic proporphyria, affecting the reactivity of sulfhydryl groups, involved in the catalytic of activity of the enzyme.

ERYTHROPOIETIC PROTOPORPHYRIA: PHOTODYNAMIC TRANSFER OF PROTOPORPHYRIN FROM INTACT ERYTHROCYTES TO OTHER CELLS

Erythrocytes in patients with erythropoietic protoporphyria (EPP) contain large amounts of protoporphyrin and are regarded as the main source of protoporphyrin in this disease. Cells in the skin of EPP patients accumulate protoporphyrin released from the erythrocytes and upon sun exposure endothelial cells are photodamaged. In the present study a light-induced transfer of protoporphyrin directly from EPP erythrocytes to cultured cells is demonstrated. Erythrocytes were layered upon cultured cells and irradiated. The nearness of erythrocyte and cultured cell membranes potentiated the transfer of protoporphyrin between these cells. This transfer was rapid and preceded the release of protoporphyrin to proteins in the medium. Further irradiation of the protoporphyrin-enriched cultured cells, after removal of the erythrocytes, caused severe photodamage to the cells and survival was dependent on both the amount of protoporphyrin transferred and on the light fluence. Clinical observations and the results of this study indicate that light energy may be involved in two steps in the pathophysiology of EPP: (A) light-induced release of protoporphyrin from erythrocytes to endothelial cells and (B) photodynamic damage to protoporphyrin-enriched endothelial cells.

Hepatic disease in erythropoietic protoporphyria.

A standardized hepatological investigation was performed in 9 unselected patients with erythropoietic protoporphyria (EPP). The aim of this study was to detect early liver involvement due to EPP and to determine the significance of several diagnostic procedures. Scintigraphy revealed slight enlargement of liver and spleen in all cases. Light microscopic examination of liver tissue in 7 patients showed protoporphyrin deposition in 4 and signs of fibrosis in 3 cases. Cirrhosis was not found. Electron microscopical examination (EM) of all 7 cases was negative and needle-shaped crystals were not found. Therefore we regard EM of little diagnostic value in the detection of early liver involvement. The literature on fatal and asymptomatic cases of liver involvement in EPP is discussed with emphasis on possible predictive and provocative factors. A proposal for EPP patients intending to reduce possible risk factors is made.

The U.K. erythropoietic protoporphyria register: a progress report

A national register was set up in 1979 to establish the prevalence and natural history of erythropoietic protoporphyria (EPP). Detailed information on EPP patients has been received from dermatologists throughout the U.K. Three hundred and eighty-two patients with confirmed EPP have been identified including two patients in clinical remission and 18 latent cases. A further 83 biochemically unconfirmed cases have been found from family studies. Seventy-eight families have more than one affected member. The mean age of onset is 2.6 years; the mean age of patients at the time of registration was 29.5 years. Patients' symptoms occur mainly between March and September. Discomfort of the skin usually begins within 30 mins of exposure to sunlight followed sometimes by swelling and occasionally crusting and petechiae. Symptoms settle in hours to days. Shallow waxy linear, punctate or irregular scars often develop on the nose and cheeks with thickening and accentuated markings of the skin over the knuckles. This study confirms the rarity of acute liver failure in EPP. Three patients have been affected; two have died and one survives with severe liver impairment. Long-term follow-up of this cohort of relatively young EPP patients will aid in identification of risk factors for liver disease.

Protoporphyrin accumulation by mitogen stimulated lymphocytes and protoporphyrinogen oxidase activity in patients with porphyria variegata and erythropoietic protoporphyria: evidence for deficiency of protoporphyrinogen oxidase and ferrochelatase in both diseases.

In erythropoietic protoporphyria (EPP) and porphyria variegata (PV) excess protoporphyrin is excreted in the stool, suggesting one or more enzyme defects in the terminal steps of the haem biosynthetic pathway. We measured protoporphyrinogen oxidase (PPO), which catalyses the oxidation of protoporphyrinogen to protoporphyrin, in both EPP and PV patients and in the offspring of PV patients. In the same subjects we measured protoporphyrin formation by mitogen stimulated lymphocytes, with delta aminolaevulinic acid (ALA) as substrate and with the addition of chelators or iron, an indirect measure of ferrochelatase activity. PPO activity was reduced by 41% (P less than 0.001) in PV patients and in 50% of their offspring, and by 36% (P less than 0.001) in EPP patients. Protoporphyrin accumulation in stimulated lymphocytes was increased by 1.3-fold (P less than 0.001) in EPP and 1.5-fold (P less than 0.001) in PV patients compared to normal subjects. There was a significant difference in protoporphyrin accumulation between iron deficient and iron replete cells from PV patients as compared to normals but not as marked as for EPP cells treated similarly. Stimulated lymphocytes from prepubertal PV offspring with reduced PPO activity accumulated normal amounts of protoporphyrin. We have interpreted our findings as follows: PPO is significantly reduced in both diseases. Ferrochelatase becomes defective in PV patients after puberty. This could explain why PV is clinically and biochemically manifest only after puberty. As it has been repeatedly shown that ferrochelatase is markedly reduced in EPP, it would appear that both enzymes are deficient in these two porphyrias.

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

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, gamma-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.

Inhibitory effect of plasma on photohaemolysis in erythropoietic protoporphyria.

The effect of plasma and serum on the photohaemolysis of red cells from erythropoietic protoporphyria patients was studied. Low concentrations (up to 50-100 ml/l) markedly inhibited the rate of photohaemolysis, this effect being partially reversed at higher plasma concentrations. Most of the lysis-inhibiting material co-eluted with serum albumin on gel filtration columns, although a considerable inhibiting effect was also associated with higher molecular weight material. A significant part of the albumin effect can be ascribed to its protoporphyrin-binding capacity. However, most of the effect seems due to an as yet unidentified property of this protein. Evidence is presented that the effect of albumin must be somehow specific to the human protein, as albumins from other sources were much less effective.

The detection of porphyria in photosensitive patients

The biochemical diversity of the various porphyris often leads to incomplete investigation of photosensitive patients and porphyria may be excluded wrongly on the basis of normal urinary porphyrins alone. Establishing a biochemical referral centre for photosensitive patients suspected of having porphyria led to the diagnosis of 5 cases of porphyria cutanea tarda (PCT) and 2 cases of erythropoietic protoporphyria (EPP) among 34 patients referred by dermatologists over a period of 12 months. Iron overload was conformed in 3 the PCT patients by plasma ferritin assay. Studies on the available families of the two EPP patients revealed elevated red cell protoporphyrin levels in several clinically asymptomatic relatives.

The detection of porphyria in photosensitive patients

The exclusion of porphyria as a possible diagnosis requires the examination of urine, faecal and blood specimens. We find that urinary porphyrins are frequently the only request received and it is not realized that urinary porphyrin excretion is normal in some of the porphyrias. This problem was approached by producing a circular for the clinicians asking that they request spot urinary porphobilinogen, 24-hr urine porphyrins, faecal porphyrins and red cell porphyrins when the clinical signs were suggestive of porphyria. One of the results was that patients with photosensitivity have been sent to the biochemistry laboratory on an outpatient basis for the exclusion of porphyria. Since August last year we have assessed urine, faecal and red cell porphyrins on about 30 patients with photosensitivity symptoms. Five of these were found to have porphyria cutanea tarda and 2 had erythropoietic protoporphyria (EPP). Studies were undertaken on the families of the 2 EPP patients with the aim of detecting further cases and investigating biochemically abnormal carriers. Several clinically asymptomatic relatives were found with elevated red cell protoporphyrin levels.

Letter: Sunlight protection for erythropoietic protoporphyria patients.

To the Editor.— In the treatment of any disorder, the simultaneous use of two different therapies usually does little more than either individually. It is only occasionally that the effect of two different modes of treatment are additive. Yet, such an occurrence may be available in erythropoietic protoporphyria (EPP). Just recently, Mathews-Roth et al (228:1004, 1974) reported sevenfold (median) photoprotection in sunlight in EPP patients using orally administered β-carotene. In 1970 ( Br Med J 1:730, 1970), a 15-fold (median) increase in sunlight protection was reported in seven EPP patients using a topically administered dihydroxyacetone and lawsone preparation. Since then, we have treated six additional EPP patients with a stable preparation of dihydroxyacetone and lawsone (Duoshield). The Table summarizes the results. Since β-carotene is taken orally and the dihydroxyacetone and lawsonepreparation is used topically, the total photoprotective effect should be additive. Thus, we can approximately double the time these patients

Possible use of vitamins C and-or E in erythropoietic protoporphyria.

To the Editor.— The current interest in therapeutic vitamin supplementation has produced a confused picture with respect to porphyria. As noted by Mustajoki inThe Journal(221:714, 1972), the results of vitamin E therapy are contradictory. Thus, his group observed no clinical improvement in four patients with acute intermittent porphyria and one with variegate porphyria; whereas Nair et al 1 reported the successful use of vitamin E in porphyria. Ludwig 2 detected low tocopherol levels in persons with erythropoietic protoporphyria (EPP) and observed that the vitamin inhibited the in vitro photohemolysis of their red cells. This disease (EPP) is a good model for evaluating therapeutic agents, because it provides a sensitive in vivo criterion of efficacy (prevention of sunlight photosensitivity) as well as an in vitro standard (inhibition of erythrocyte photohemolysis). Provitamin A (β-carotene) has been employed successfully in EPP patients by Mathews-Roth et al. 3 Although the rationale for

EP Study Indicates Two Sites For Abnormal Protoporphyrin

A Los Angeles investigator's study of families with erythropoietic protoporphyria (EP), an inherited photocutaneous disorder first described in 1961, suggests there are two sites for abnormal protoporphyrin formation; one is erythropoietic and the other probably hepatic. The two sites apparently can function independently of each other. "The hepatic site appears to be the source of the increased plasma protoporphyrin which seems requisite for photosensitivity to occur," Allan G. Redeker, MD, Department of Medicine, University of Southern California Medical School, told the American College of Physicians. Redeker said that protoporphyrin has been found in the plasma of all his EP patients "and seems clearly to be the photosensitizing agent." In one EP family, in which fraternal twin boys have the disease, the mother, who has never had a cutaneous lesion, does, however, have a large increase in her fecal protoporphyrin excretion. "This apparent evidence of a latent trait suggests a dissociation