Total Erythrocyte Protoporphyrin Research Articles

When the diagnosis is written in the DNA: a case of erythropoietic protoporphyria in a patient with a chromosome-18 deletion.

In this case study, we describe a 21-year-old man with erythropoietic protoporphyria who sought medical attention in April 2022 for diffuse edema and erythema of the hands. These symptoms had been present since childhood and usually occurred soon after sun exposure. The patient's medical history showed that chromosome 18's long arm had partially deleted. We performed a number of tests, including measuring total erythrocyte protoporphyrin levels and utilizing a spectrofluorometer to assess the fluorometric emission peak of plasma porphyrins, based on the patient's medical history and clinical symptoms. Furthermore, a genetic analysis identified an intronic variant on one allele, c.315-48T>C (IVS3-48T>C), which is categorized as a susceptibility polymorphism, and a complete deletion of the ferrochelatase gene on the other allele. The patient's clinical condition improved following the June 2022 afamelanotide implant procedure.

Clinical, Biochemical, and Genetic Characterization of North American Patients With Erythropoietic Protoporphyria and X-linked Protoporphyria

ImportanceAutosomal recessive erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are rare photodermatoses presenting with variable degrees of painful phototoxicity that markedly affects quality of life. The clinical variability, determinants of severity, and genotype/phenotype correlations of these diseases are not well characterized.ObjectiveTo describe the baseline clinical characteristics, genotypes, and determinants of disease severity in a large patient cohort with EPP or XLP.Design, Setting, and ParticipantsA prospective observational study was conducted among patients with confirmed diagnoses of EPP or XLP from November 1, 2010, to December 6, 2015, at 6 academic medical centers of the Porphyrias Consortium of the National Institutes of Health Rare Diseases Clinical Research Network. Detailed medical histories, including history of phototoxicity and treatment, were collected on standardized case report forms. Patients underwent baseline laboratory testing, total erythrocyte protoporphyrin (ePPIX) testing, and molecular genetic testing. Data were entered into a centralized database.Main Outcomes and MeasuresResults of biochemical and genetic tests were explored for association with clinical phenotype in patients with EPP or XLP.ResultsOf the 226 patients in the study (113 female and 113 male patients; mean [SD] age, 36.7 [17.0] years), 186 (82.3%) had EPP with a FECH (OMIM 612386) mutation and the common low-expression FECH allele IVS3–48T>C, and only 1 patient had 2 FECH mutations. Twenty-two patients had XLP (9.7%; 10 male and 12 female patients), and 9 patients (4.0%) had elevated ePPIX levels and symptoms consistent with protoporphyria but no detectable mutation in the FECH or ALAS2 (OMIM 301300) gene. Samples of DNA could not be obtained from 8 patients. Patients’ mean (SD) age at symptom onset was 4.4 (4.4) years. Anemia (107 [47.3%]), history of liver dysfunction (62 [27.4%]), and gallstones (53 [23.5%]) were commonly reported. Higher ePPIX levels were associated with earlier age of symptom onset (median ePPIX levels for those who developed symptoms before vs after 1 year of age, 1744 vs 1567 µg/dL; P = .02), less sun tolerance (median ePPIX levels for those reporting symptoms before vs after 10 minutes of sun exposure, 2233 vs 1524 µg/dL; P ≤ .001), and increased risk of liver dysfunction (median ePPIX levels for those with liver dysfunction vs normal liver function, 2016 vs 1510 µg/dL; P = .003). Patients with EPP and FECH missense mutations had significantly lower ePPIX levels than those with other mutations (1462 vs 1702 µg/dL; P = .01). Male patients with XLP had significantly higher ePPIX levels, on average, than did patients with EPP (3574 vs 1669 µg/dL; P < .001). Marked clinical variability was seen in female patients with XLP owing to random X-chromosomal inactivation.Conclusions and RelevanceThese data suggest that higher ePPIX levels are a major determinant of disease severity and risk of liver dysfunction in patients with EPP or XLP. These findings provide a framework for clinical monitoring and management of these disorders.

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

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). 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. 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.

Vitamin D deficiency in patients with erythropoietic protoporphyria

The main symptom of patients with erythropoietic protoporphyria (EPP) is painful photosensitivity, starting within minutes of sun exposure and leading to sun-avoidance. As 80-100% of vitamin D is synthesized under the influence of sunlight, we investigated whether the avoidance of sunlight exposure in the Dutch EPP patient population causes vitamin D deficiency. Furthermore, we studied the relation between vitamin D levels, total erythrocyte protoporphyrin and quality of life. In a cross-sectional study of 48 Dutch EPP patients (mean age 41.4 years; range 16-77; 23 male, 25 female), we assessed serum 25-hydroxyvitamin D (25(OH)D) levels between June and November 2007, as well as total erythrocyte protoporphyrin (TEP) levels and Dermatology Life Quality Index (DLQI) scores. Mean serum 25(OH)D was 66 nmol/L (range 18-140, quartiles 36, 87). Twenty-two patients (46%; 15 male, 7 female) were vitamin D deficient. There was a significant difference (p = 0.029) in mean serum 25(OH)D between female (mean 75 nmol/L, range 18-140) and male patients (mean 55 nmol/L, range 18-115). The level of serum 25(OH)D showed a negative correlation with total erythrocyte protoporphyrin (TEP) (Pearson rank correlation (r(p)) = -0.337; p = 0.034). Serum 25(OH)D was inversely associated with scores of the Dermatology Life Quality Index (DLQI) (Spearman's rho correlation (r(s)) = -0.486; p = 0.001). The prevalence of vitamin D deficiency is high in the Dutch EPP population, especially in male patients, and correlates with the severity of EPP. Screening for and treatment of vitamin D deficiency should therefore be implemented in the care of these patients.

Serum 25-hydroxyvitamin D in erythropoietic protoporphyria

Vitamin D, produced by the action of sunlight on skin, is an important hormone for calcium homeostasis and has been implicated as tumour-protective agent. Some previous studies of photosensitive patients who actively avoid sunlight have failed to show convincing evidence of vitamin D insufficiency. The aim of this study was to characterize the vitamin D status of a large cohort of patients with erythropoietic protoporphyria (EPP). U.K. patients with EPP were recruited prospectively and seen locally by a single study investigator. A blood sample was taken for vitamin D assay. All blood analyses were performed in the same laboratory. A cohort of 201 patients with known EPP was seen over a 7-month period between January and July. Thirty-four patients (17%) were deficient in vitamin D and 126 (63%) had insufficient vitamin D. Both insufficiency and deficiency were significantly associated with the total erythrocyte protoporphyrin concentration and inversely with the time in minutes to the onset of symptoms following sunlight exposure. This is the first report of significant levels of vitamin D deficiency and insufficiency in a large cohort of patients with a photodermatosis. Such individuals are at risk of associated adverse events. In future, clinicians should consider monitoring 25-hydroxyvitamin D levels and instigating oral supplementation or dietary advice if appropriate.

Comparison of unitages for total free erythrocyte protoporphyrin (total EP) in anemia screening

The presence of free protoporphyrin in erythrocytes was previously reported by Hymans van den Bergh and Hyman [l]. In addition, increased free erythrocyte protoporphyrin is often observed in some diseases [2]. In iron-deficiency anemia, free erythrocyte protoporphyrin also increases, because heme synthesis cannot be completed and a relative excess of the unused ‘free’ protoporphyrin accumulates in the erythrocytes [3]. Lamola and Yamane, however, reported that the fluorescent porphyrin in the erythrocytes of patients with lead intoxication or with iron-deficiency anemia is zinc protoporphyrin that easily dissociates into free protoporphyrin and zinc ions in acidic solvents [4]. In another report, Piomelli [5] developed a micromethod for free erythrocyte protoporphyrin determination in which protoporphyrin in erythrocytes is extracted with an acidic solvent then determined as a free type of protoporphyrin, therefore the method determines free and zinc protoporphyrin, the so-called total erythrocyte protoporphyrin (total EP). Since then, the measurement of total EP has been recognized as a sensitive indicator for lead intoxication in children and is a simple procedure for any clinical laboratory. The total EP can be determined and calculated as four different units: pg/dl-blood, pg/dl-packed cell volume (pcv), pg/g-heme and pg/g-hemoglobin (Hb). The aim of the present study is to detect which unit is the most useful to evaluate or screen for the presence anemia.

Simultaneous quantitation of zinc protoporphyrin and free protoporphyrin in erythrocytes by acetone extraction.

Erythrocyte protoporphyrin content is increased in several human and veterinary disorders in which heme synthesis is disrupted. The nature of the defect determines whether zinc protoporphyrin, unchelated (free) protoporphyrin, or both are present in the erythrocytes. Extraction of the protoporphyrin into acetone/water (80/20 by vol) is a simple, rapid technique by which each protoporphyrin species is delivered into solution without removing zinc from zinc protoporphyrin. The ratio of zinc protoporphyrin to total protoporphyrin concentration correlated significantly with the ratio of fluorescence emission intensities at the zinc protoporphyrin peak and at an isosbestic point. This concentration ratio may be used with a quantitative measure of total erythrocyte protoporphyrin to define the absolute zinc protoporphyrin and free protoporphyrin content of erythrocytes.

Quantitative assay of erythrocyte “free” and zinc-protoporphyrin: Clinical and genetic studies

1. 1. Two new procedures are described for the quantitative assay of “free” and zinc-protoporphyrin in red blood cells. 2. 2. Major clinical findings have included the following: (i) An average of about 10% of the total erythrocyte protoporphyrin is “free” in normal humans, but about one-third or more is usually “free” in blood of normal cows, rats and rabbits, (ii) In addition to human and bovine protoporphyria, “free” protoporphyrin was increased in (a) genetic carriers (clinically normal) of bovine protoporphyria, (b) acute Pb poisoning, and (c) some patients undergoing dialysis, (iii) Significant amounts of zinc-coproporphyrin were found in the blood of several dialyzed patients and in some “normal” cows.

Comparative study of protoporphyrins in erythropoietic protoporphyria and griseofulvin-induced murine protoporphyria. Binding affinities, distribution, and fluorescence spectra in various blood fractions.

Excess erythrocyte protoporphyrins of human congenital erythropoietic protoporphyria and of griseofulvin-induced murine hepatic protoporphyria were found to be associated with hemoglobin and stroma fractions in similar relationships. More than 99.5% of total erythrocyte protoporphyrin was bound to hemoglobin in each case. However, profound differences were found when protoporphyrin concentration was measured in erythrocytes that had been segregated into populations of progressive age on discontinuous density gradients. In erythropoietic protoporphyria, porphyrin content diminished rapidly with age; in murine protoporphyria, the aging erythrocyte populations became progressively more porphyrin rich. In vitro diffusion of protoporphyrin from plasma across the intact erythrocyte membrane was demonstrated. The equimolar binding affinity of protoporphyrin to hemoglobin was shown to be 40 times that of protoporphyrin to serum albumin. This strong affinity provides the driving force for the observed transmembrane diffusion, and explains the high erythrocyte/plasma porphyrin ratio in murine hepatic protoporphyria. The opposite rapid efflux of intra-erythrocytic protoporphyrin into plasma previously shown in uncomplicated erythropoietic protoporphyria occurs despite this strong hemoglobin affinity, implying continuous efficient clearance of protoporphyrin from plasma by the liver. Furthermore, these and other data suggest that a hepatic synthetic source for any significant fraction of the blood protoporphyrin in erythropoietic protoporphyria is highly improbable.