Inactivation of protoporphyrin IX in erythrocytes in patients with erythropoietic protoporphyria: A new treatment modality.

BACKGROUND: Erythropoietic protoporphyria (EPP) is a rare, genetic disease with reduced ferrochelatase activity causing protoporphyrine IX (PpIX) to accumulate in erythrocytes. PpIX activation by daylight causes skin erythema, edema, burning, and stinging. No treatment exists to reduce PpIX. AIM: To introduce a method that reduces PpIX in erythrocytes to relieve skin symptoms in patients with EPP. METHOD: A case series of 7 patients with EPP constituted this explorative study. Erythrocyte PpIX was inactivated by illuminating the patients' heparinized blood outside their body, then returning it to the patient. About 3 litres of blood was illuminated with 630 nm light, 20 J/cm2. The effect was measured as a reduction in erythrocyte PpIX. The patients reported the number of minutes in daylight tolerated before and after intervention. RESULTS: This procedure reduced PpIX by about 30 % and daylight tolerance was, on average, increased by 14 times. The subsequently excreted photoproducts resulted in some liver toxicity. Three treatments during spring and early summer were sufficient to reduce the patients' symptoms throughout the year in Northern Europe. CONCLUSION: Extracorporeal erythrocyte photodynamic therapy is the first treatment to successfully reduce the amount of PpIX in the blood of EPP patients, thus "normalizing" their daylight tolerance.

Inflammatory involvement into phototoxic reaction in erythropoietic protoporphyria (EPP) patients.

Phototoxic reaction is a known feature of EPP at least in part triggered by the oxidative status, complement system activation, and mast cell response. The aim of this study was to verify some aspects involved in phototoxic reaction during a season. The complement system was evaluated by C3 assay, alternative pathway by factor-B, and classical pathway by C1q; oxidative status was tested with malondialdehyde (MDA) and mast cell by IL-10 assay. The serum samples were collected in winter and summer from 19 EPP patients and 13 controls. The reaction to sun exposure within each group was monitored without any invasive treatment. In summer, C3 and factor B were higher in patients than in controls (p = 0.002 and < 0.0001 respectively), while no change was detected for C1q. The oxidative stress was increased in summer in comparison with the control group (p = 0.04), and IL-10 an assay was normal in both seasons. The correlation between the C3 and factor-B in summer was significant. This study shows that the phototoxic reaction is not limited to the dermis but can also exert a systemic response, which could affect the general health of a patient. The knowledge of the pathophysiology of phototoxic reaction is essential for identifying new disease markers useful for improving clinical studies of known and future drugs.

Prevention of photosensitivity with action spectrum adjusted protection for erythropoietic protoporphyria.

Erythropoietic protoporphyria is a genetic disease characterized by sensitivity to sunlight caused by the accumulation of protoporphyrin IX. Photoprotection against ultraviolet A and visible light is necessary for erythropoietic porphyria patients because the absorption spectrum of protoporphyrin IX lies in both ultraviolet A and visible light region. We developed a novel index, in vitro porphyrin protection factor, based on the protoporphyrin IX absorbance spectrum. We also selected appropriate photoprotective products designed according to protoporphyrin IX absorbance. The porphyrin protection factors of a combination of make-up base with a powder as well as with a liquid foundation were significantly higher than those of a conventional sunscreen product, even at a small application dose. An in-use test carried out for 6 months showed that the efficacy of these products was 78.3%, and no adverse reactions were observed. Male subjects preferred liquid foundation, whereas all female subjects used powder foundation. The preference of the subjects could lead to the long-term use of the tested products. In conclusion, this study provided a new approach to improve photoprotection in erythropoietic protoporphyria patients.

Modeling the ferrochelatase c.315-48C modifier mutation for erythropoietic protoporphyria (EPP) in mice.

Erythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe FECH mutation (Mut) in trans to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3' splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized Fech gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe Fech mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing.

Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients.

Mutations in the C-terminus of human erythroid 5-aminolevulinate synthase (hALAS2), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are associated with two different blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA-causing mutations yield hALAS2 variants with decreased activity, while XLPP-causing mutations result in a gain-of-function of hALAS2. There are no specific treatments for XLPP. Isonicotinic acid hydrazide (isoniazid, INH), an antituberculosis agent, can cause sideroblastic anemia as a side-effect, by limiting PLP availability to hALAS2, via inhibition of pyridoxal kinase or reaction with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We hypothesized that INH also binds and directly inhibits hALAS2. Using fluorescence-activated cell sorting and confocal fluorescence microscopy, we demonstrate that INH reduces protoporphyrin IX levels in HeLa cells expressing either wild-type hALAS2 or XLPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) reversed the cellular inhibition of hALAS2 activity by INH. Steady-state kinetic analyses with purified hALAS2 indicated that INH directly inhibits the enzyme, noncompetitively or uncompetitively, with an apparent Ki of 1.2µM. Circular dichroism spectroscopy revealed that INH triggered tertiary structural changes in hALAS2 that altered the microenvironment of the PLP cofactor and hampered the association of PLP with apo-hALAS2. Treatment of four XLPP patients with INH (5mg·kg-1·day-1) over a six-month period was well tolerated but without statistically significant modification of PPIX levels. These results, taken together, permit us to further an INH inhibition kinetic mechanism for ALAS, which suggests the possible use of INH-derived drugs in treating patients with XLPP and potentially other protoporphyrin-accumulating porphyrias.

Pitfalls in Erythrocyte Protoporphyrin Measurement for Diagnosis and Monitoring of Protoporphyrias.

BACKGROUND: Laboratory diagnosis of erythropoietic protoporphyria (EPP) requires a marked increase in total erythrocyte protoporphyrin (300-5000 µg/dL erythrocytes, reference interval <80 µg/dL) and a predominance (85%-100%) of metal-free protoporphyrin [normal, mostly zinc protoporphyrin (reference intervals for the zinc protoporphyrin proportion have not been established)]; plasma porphyrins are not always increased. X-linked protoporphyria (XLP) causes a similar increase in total erythrocyte protoporphyrin with a lower fraction of metal-free protoporphyrin (50%-85% of the total). CONTENT: In studying more than 180 patients with EPP and XLP, the Porphyrias Consortium found that erythrocyte protoporphyrin concentrations for some patients were much higher (4.3- to 46.7-fold) than indicated by previous reports provided by these patients. The discrepant earlier reports, which sometimes caused the diagnosis to be missed initially, were from laboratories that measure protoporphyrin only by hematofluorometry, which is intended primarily to screen for lead poisoning. However, the instrument can calculate results on the basis of assumed hematocrits and reports results as "free" and "zinc" protoporphyrin (with different reference intervals), implying separate measurements of metal-free and zinc protoporphyrin. Such misleading reports impair diagnosis and monitoring of patients with protoporphyria. SUMMARY: We suggest that laboratories should prioritize testing for EPP and XLP, because accurate measurement of erythrocyte total and metal-free protoporphyrin is essential for diagnosis and monitoring of these conditions, but less important for other disorders. Terms and abbreviations used in reporting erythrocyte protoporphyrin results should be accurately defined.

A Novel Mutation in the FECH Gene in a Czech Family with Erythropoietic Protoporphyria and a Population Study of IVS3-48C Variant Contributing to the Disease.

Erythropoietic protoporphyria (EPP), a chronic erythropoietic porphyria, is characterized by excess accumulation of protoporphyrin, particularly in erythroid cells. EPP inheritance is complex, almost always associated with two molecular defects. In most EPP patients, clinical expression requires coinheritance of a private ferrochelatase (FECH) mutation trans- to a hypomorphic FECH*IVS3-48C allele. This leads to a decrease of FECH activity below the critical threshold. This is characterized by cutaneous photosensitivity in early childhood such as itching, burning, swelling and redness in sun-exposed areas. Hepatic failure occurs in some patients (about 1-10 % of EPP patients), which may necessitate liver transplantation. We investigated a Czech family with two patients with manifested EPP in four generations. We found a novel mutation, c.84G >A, in the FECH gene in four individuals including proband and his mother (G84A transition in exon 2; p.W28*). Both clinically manifested probands inherited the hypomorphic IVS3-48C allele as well, while two clinically latent individuals with FECH mutation did not. To address the question whether the relatively low incidence of EPP in the Czech Republic might be due to lower frequency of the IVS3-48C allele, we screened for the frequency of the low expression allele in a control Czech (West Slaves) Caucasian population. Such study has not been performed in any Slavic population. Among 312 control individuals, there were no IVS3-48C/C (c.68-23C-T) homozygotes; 35 IVS3-48C/T heterozygous individuals were detected. The frequency of IVS3-48C allele was thus found to be 5.5 % in the Czech population, comparable to most West Caucasian populations.

Bone mineral density and vitamin D levels in erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare disease with painful cutaneous photosensitivity, in which patients are recommended to avoid sun exposure, and wear sunscreen and adequate clothing. Our aim was to study bone mineral density (BMD) and other mineral parameters, including serum 25(OH)D levels, to evaluate the impact of these measures in the follow-up of EPP patients. A cross-sectional study of ten EPP patients (median age 25; range 22-55, four males and six females), was performed evaluating clinical features, biochemical values (bone markers and serum 25 hydroxyvitamin D), and BMD. Median serum 25(OH)D level was 19.65 ng/ml [17.50; 24.80]. Four patients had 25(OH)D in insufficiency range (20-30 ng/ml) and five patients in the deficiency range (<20 ng/ml). Lumbar T-score median levels were in the osteopenia range in both females (-1.50 [-2.30; -1.0]) and males (-1.90 [-2.40; -0.70]). Also, in the female group median femoral neck T-score were in the osteopenia range (-1.20 [-1.60; -0.60]). This is the first study reporting low BMD in EPP patients. Osteoporosis, osteopenia, and vitamin D deficiency are frequent findings in EPP patients. The contribution of sunlight avoidance measures to these results remains to be clarified. Serum levels of protoporphyrins were not related to these alterations and other factors should be investigated. We suggest that the monitoring of serum vitamin D levels in EPP patients should be mandatory, as well as vitamin D and calcium supplementation.

Electrospray ionization on porous spraying tips for direct sample analysis by mass spectrometry: enhanced detection sensitivity and selectivity using hydrophobic/hydrophilic materials as spraying tips.

RATIONALE: Despite various porous materials having been widely adopted as spraying tips for direct sample analysis using electrospray ionization mass spectrometry (ESI-MS), the effect of surface property and porosity of spraying tip materials on their analytical performances is not clear. Investigation of their relationships could provide insight into the proper choice and/or design of spraying tip materials for direct sample analysis. METHODS: The effect of spraying tip materials with different polarities, including polyester and polyethylene (hydrophobic) and wood (hydrophilic), on the detection sensitivity for a variety of compounds, and on the ESI onset voltage, were studied using ESI-MS. The porosity of each type of spraying tip was characterized by scanning electron microscopy (SEM). Factors governing the detection sensitivity were determined based on the correlation of the detection sensitivity to the ESI onset voltage, the polarity, and the porosity of the spraying tip materials. RESULTS: Hydrophobic tips (i.e., polyester and polyethylene) show better detection sensitivity for polar compounds but not for non-polar compounds, while hydrophilic tips (wooden tips) show the opposite effect. This phenomenon could be due to the difference in interaction between the analytes and the tips, causing the analytes to adsorb on the tip to different extents. In addition, the micro-porous nature of the tips could facilitate solvent diffusion for transporting analytes to the tip and maintain a stable spray for recording MS data. With the proper choice of spraying tip materials, trace amount of analytes at the picomole level can be detected with minimal sample pretreatment. CONCLUSIONS: Both the polarity and the porosity of the spraying tip materials could significantly affect detection sensitivity for a wide variety of analytes. With proper choice of spraying tip material, ESI on a porous spraying tip could be a sensitive method for the direct analysis of daily life samples.

Genetic study in a Singaporean patient with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare autosomal dominant disorder of haem biosynthesis resulting from a partial decrease in ferrochelatase (FECH) activity which leads to the excessive accumulation of protoporphyrin in blood, erythrocytes and tissues. Cutaneous manifestations of photosensitivity usually appear in early infancy upon the first sun exposures. This normally requires the co-inheritance of a common hypomorphic FECH allele and a deleterious FECH mutation. Here, we report the first Singaporean Chinese patient with EPP characterized at the molecular level.

Erythropoietic protoporphyria: spectrum of three cases.

BACKGROUND: Erythropoietic protoporphyria is a rare photodermatosis of childhood, and the diagnosis can be delayed. A deficient ferrochelatase enzyme leads to accumulation of protoporphyrins in the dermis, causing phototoxic burning. OBJECTIVE: To report three cases with great variability in severity of symptoms and age at diagnosis. We discuss clinical and biochemical findings, mutation analysis, and therapeutic options. METHODS: We report three cases with different degrees of photosensitivity, laboratory results, psychosocial impact, and preventive and therapeutic treatments. RESULTS: The diagnosis of erythropoietic protoporphyria was confirmed by both typical elevation of plasma porphyrins and the discovery of a mutated FECH gene. CONCLUSION: Erythropoietic protoporphyria should be suspected in any cases of childhood photosensitivity. Systemic complications are unusual. Mutation analysis confirms the diagnosis. Photoprotection is the cornerstone of treatment.

Plasma and red cell exchange transfusions for erythropoietic protoporphyria: a case report and review of the literature.

Erythropoietic protoporphyria (EPP) is a rare and usually autosomal dominant disorder characterized by ferrochelatase deficiency and accumulation of protoporphyrin in red blood cells (RBCs), skin, and liver. A small minority of patients develop severe liver dysfunction for which optimum treatment is lacking. Therapeutic plasma exchange (TPE) and RBC exchange (RCE) have been anecdotally reported to benefit patients with EPP and liver failure. A 50-year-old female with EPP developed severe liver dysfunction after knee replacement surgery and high-dose acetaminophen use. Liver biopsy showed cholestatic liver injury without fibrosis. A total of 20 TPE procedures, six RCE procedures, and then 14 more TPE procedures were performed as adjunctive therapy with the purpose of preventing progression to end-stage liver failure. After initial TPE, the plasma and RBC protoporphyrin levels decreased from 834.9 to 180.4 µg/dL (normal, ≤1 µg/dL), and from 3,905 to 2,879 µg/dL (normal, ≤80 µg/dL), respectively, without liver function improvement. RCE decreased RBC protoporphyrin levels from 2,879 to 1,225 µg/dL but plasma protoporphyrin increased from 180.4 to 1,044.1 µg/dL, and liver function failed to improve. Additional TPE again stabilized plasma protoporphyrin and improved RBC protoporphyrin levels but the patient ultimately died owing to end-stage liver disease complications. This case illustrates that TPE and RCE may improve the plasma and RBC biochemical markers of EPP activity but liver function abnormalities may persist and patients may still progress to liver failure either because of irreversible liver injury or independent pathobiological factors unrelated to EPP-induced hepatotoxicity.

A novel rapid method for simultaneous determination of three diagnostically important porphyrins in erythrocytes using hyphenated synchronous fluorescence techniques.

The species and concentrations of porphyrins in erythrocytes are of great importance in the clinical screening and diagnosis of porphyrias. However, it is difficult to analyze them simultaneously by conventional spectrofluorimetry. In this paper, we proposed a novel, simple and rapid method for the simultaneous determination of three diagnostically important porphyrins in human erythrocytes, protoporphyrin IX (PP), coproporphyrin III (CP) and zinc protoporphyrin IX (ZnPP), using hyphenated techniques based on derivative matrix isopotential synchronous fluorescence spectrometry and nonlinear variable-angle synchronous fluorescence spectrometry (DMI-NLVASFS). The spectral overlapping problems were well resolved and these three components were determined in one scanning without spectral compensation factors and chromatographic separation. The detection limits were 0.58, 0.21, 0.05 nmol L(-1) for PP, CP and ZnPP, respectively. Only 30s was needed for a single scanning and the recoveries were from 73% to 105% in erythrocytes. The Bland-Altman analysis indicates no significant difference between the proposed DMI-NLVASFS method and conventional fluorimetry method. The PP level of the erythropoietic protoporphyria (EPP) patients was significantly higher than that of healthy volunteers. This method can determine PP, CP and ZnPP simultaneously in a single scanning, thus providing a potential tool for the clinical analysis of porphyrins in human erythrocytes and the differential diagnosis of porphyrias.

Erythropoietic protoporphyria in Sweden: demographic, clinical, biochemical and genetic characteristics.

OBJECTIVE: To investigate the demographic, clinical, biochemical and genotypic features of patients with erythropoietic protoporphyria (EPP) in a Swedish cohort. DESIGN: Cross-sectional questionnaire, biochemical and genetic study. SETTING: Sweden. SUBJECTS: Fifty-one Swedish individuals known in 2008 to have EPP confirmed by molecular diagnosis. There were no exclusion criteria; all patients were included in the demographic and genetic study. A total of 92% participants completed the questionnaire study and 82% the biochemical study. RESULTS: The prevalence of EPP was 1 : 180,000. Nine novel ferrochelatase gene mutations were found. The most commonly reported age at onset of symptoms was the first year of life and the mean age at diagnosis was 22 years. Painful photosensitivity was the main symptom. Exogenous factors other than sunlight were frequently reported to cause cutaneous symptoms. One in five patients reported a positive effect of beta-carotene therapy. A marked impact of EPP on quality of life was reported. Women had a significantly lower mean erythrocyte protoporphyrin concentration than men. Of all participants, 84% had insufficient vitamin D concentrations, 44% had below normal serum ferritin or transferrin saturation levels and red cell abnormalities were common. CONCLUSIONS: The notably delayed diagnosis suggests the need for an increased awareness of EPP. Disturbed erythropoiesis, biochemical signs of iron deficiency and low vitamin D levels are frequent findings in this disease. New and better treatments are needed as current treatment options for symptom amelioration are limited. Vitamin D supplementation should be considered.

The prognostic value of cord blood analysis in erythropoietic protoporphyria: the 'Duesseldorf Cord Blood Study'.

BACKGROUND: Erythropoietic protoporphyria (EPP) is the most common porphyria in childhood, presenting with painful and burning skin sensations as well as erythema and edema after sun exposure. It represents an inherited disorder of heme metabolism that is due to a reduced ferrochelatase enzyme activity. The diagnosis is usually established when symptoms start by measuring elevated levels of protoporphyrin in erythrocytes. The aim of our study was to question the predictive value of cord blood analysis in newborn relatives of EPP patients as this may offer the earliest possible diagnosis of EPP in newborn relatives of affected patients. METHODS: Erythrocyte porphyrin (EP) was measured immediately after birth in 18 newborn relatives of EPP patients. EP was correlated to the subsequent clinical follow-up of mean 9 years after birth. RESULTS: We found EP to be within reference values in all 18 newborn relatives of EPP patients at birth. Out of 14 patients who were included in the follow-up period of median 9 years, 13 remained asymptomatic whereas one boy developed the typical symptoms of EPP at the age of three in combination with elevated EP. CONCLUSION: Based on the findings of our study, we assume that cord blood analysis is not a reliable prognostic tool in EPP from the actual point of view.

Excessive erythrocyte PPIX influences the hematologic status and iron metabolism in patients with dominant erythropoietic protoporphyria.

Partial deficiency of the last enzyme of the heme biosynthetic pathway (namely ferrochelatase, FECH) in humans is responsible for erythropoietic protoporphyria (EPP). This disorder is characterised by painful photosensitivity, due to excessive production of protoporphyrin IX (PPIX) by erythrocytes. Controversial hypotheses have been proposed to explain the hematologic and iron status of EPP patients. In the present work, we explored these parameters in 55 patients with dominant EPP recruited at the French Center of Porphyrias (Colombes, France) and confirmed by molecular analysis. Our data show that erythrocyte accumulation of PPIX in EPP patients influences hematologic and iron status. Patients studied had a mild anemia and thrombocytopenia, as shown by the downward shift of hematologic parameters, which positively correlated with the amount of erythrocyte PPIX. Interestingly, erythropoiesis did not seem to be limited by iron supply in patients, since serum iron and soluble transferring (Tf) receptor (sTfR) were normal. However, iron and Tf saturation negatively correlated with erythrocyte PPIX. Moreover, and as previously described in a mouse model of EPP, we noted a positive correlation between erythrocyte PPIX and Tf levels. Altogether, these results suggest a positive effect of PPIX on the synthesis on Tf, which could facilitate the mobilization of tissue iron stores to meet erythropoiesis requirement. Based on these observations and previous results in EPP mouse model, we propose that the PPIX-liver transferrin pathway plays a role in the orchestration of iron distribution between peripheral iron stores, the spleen and the bone marrow.