Atle Brun

Porphyrins, porphyrin metabolism and porphyrias. IV. Pathophysiology of erythyropoietic protoporphyria - diagnosis, care and monitoring of the patient

An extremely painful cutaneous condition with no or only slight visible skin changes, presenting in a child or an adult as an acute reaction to sun light, is probably a manifestation of the porphyrin metabolic disorder erythropoietic protoporphyria (EPP). The disease is the result of a genetically determined condition where a mutation in the gene for the final enzyme in the haem synthetic chain, ferrochelatase, results in impaired activity of the enzyme. In some predisposed individuals, the condition is accompanied by heavy accumulation of the substrate for the deficient enzyme, i.e. of protoporphyrin. Distributing to the skin, and there absorbing light of certain wavelengths, the metabolite generates free radicals that give rise to photodynamic cell injury. The primary event takes place in the endothelial cells of the superficial skin capillaries, but complement activation and mast cell degranulation in the surrounding tissue follow in the process. Even if the disease is primarily dermatological the hepatic and psychosocial complications are features requiring close attention by the physician. In order to provide a basis for suggestions regarding lege artis protocols for the diagnosis, treatment and monitoring of the patient with EPP, the pathophysiology of the cutaneous and hepatic manifestations are discussed in some detail in the article.An extremely painful cutaneous condition with no or only slight visible skin changes, presenting in a child or an adult as an acute reaction to sun light, is probably a manifestation of the porphyrin metabolic disorder erythropoietic protoporphyria (EPP). The disease is the result of a genetically determined condition where a mutation in the gene for the final enzyme in the haem synthetic chain, ferrochelatase, results in impaired activity of the enzyme. In some predisposed individuals, the condition is accompanied by heavy accumulation of the substrate for the deficient enzyme, i.e. of protoporphyrin. Distributing to the skin, and there absorbing light of certain wavelengths, the metabolite generates free radicals that give rise to photodynamic cell injury. The primary event takes place in the endothelial cells of the superficial skin capillaries, but complement activation and mast cell degranulation in the surrounding tissue follow in the process. Even if the disease is primarily dermatological the hepatic and psychosocial complications are features requiring close attention by the physician. In order to provide a basis for suggestions regarding lege artis protocols for the diagnosis, treatment and monitoring of the patient with EPP, the pathophysiology of the cutaneous and hepatic manifestations are discussed in some detail in the article.

Patterns of Azathioprine Metabolites in Neutrophils, Lymphocytes, Reticulocytes, and Erythrocytes: Relevance to Toxicity and Monitoring in Recipients of Renal Allografts

Monitoring of azathioprine (AZA) therapy by the measurement of 6-thioguanine nucleotides (6-TGN) concentrations in red blood cells (RBC) may improve safety and ensure optimal immunosuppressive effects of AZA in organ transplantation. The authors explored the rationale for such monitoring by measuring thiopurine metabolites in peripheral blood cell types that are more relevant to the effects and kinetics of AZA and its active metabolites. Neutrophil granulocytes were isolated by density gradient centrifugation, and CD4+ lymphocytes and reticulocytes by using specific immunomagnetic beads. In neutrophils, 6-TGN concentrations had median measurements 31 times higher than in RBCs. In contrast to the high methylated mercaptopurine (me-MP) concentrations in RBCs, these metabolites were not detected in the neutrophils. Thiopurine metabolite levels were lower than the analytic limit of detection in all the CD4+ samples. The concentrations of 6-TGN and me-MPs were lower in reticulocytes than in RBCs in general, indicating that thiopurine metabolites are taken up by RBCs in the circulation. This studys findings, that 6-TGN concentrations are very high in neutrophils, whereas me-MPs are undetectable, many explain the specific neutropenic adverse effect of AZA. The results also add support to monitoring AZA through measurements of 6-TGN and me-MPs in RBCs.

Mechanisms of photosensitivity in porphyric patients with special emphasis on erythropoietic protoporphyria

In erythropoietic protoporphyria, protoporphyrin overproduction occurs mainly in erythroid tissue. Protoporphyrin can be released from erythrocytes in the dark, but the release is greatly increased if the erythrocytes are exposed to small amounts of light. Protoporphyrin can be bound in plasma either to albumin or to low density or high density lipoprotein. The cutaneous symptoms in erythropoietic protoporphyria are primarily elicited by protoporphyrin-sensitized photodamage of endothelial cells due to the presence of protoporphyrin in lipid structures. Which structures are damaged first in endothelial cells is unknown. Endothelial cells probably accumulate protoporphyrin from albumin or lipoproteins present in the plasma. A direct transfer from the erythrocyte membrane to the endothelial cell membrane can also occur. The transfer processes are probably facilitated by light exposure. Degranulation of mast cells, invasion of neutrophils into interstitial tissue and complement activation seem to be of less importance than endothelial cell injury in the pathogenesis of erythropoietic protoporphyria. These processes may, however, participate in the final expression of the cutaneous symptoms. Uroporphyrin and coproporphyrin are hydrophilic and are probably unbound in plasma, although weak binding to plasma proteins cannot be excluded. In the hepatic porphyrias and in erythropoietic porphyria, the clinical symptoms are probably evoked by uroporphyrin and coproporphyrin present in the interstitial tissue. Very little is known about the primary targets of uroporphyrin and coproporphyrin photodamage in these disorders, but photodamage to intercellular structures probably represents the initial event. Activation of complement may contribute to the final expression of the cutaneous symptoms.

Protection from phototoxic injury during surgery and endoscopy in erythropoietic protoporphyria.

Erythropoietic protoporphyria is an inherited condition characterized by pronounced solar photosensitivity and in a minority of patients severe liver disease that necessitates liver transplantation for survival. Phototoxic injury to abdominal organs and skin has been reported in several cases of liver transplantation surgery, including a few transplants in which protective light filters were used. This study discusses the optimal characteristics of light filters used during liver transplantation surgery. An experimental model is used to evaluate the relative protection of different filters, and the results are compared with theoretical calculations regarding the risk for phototoxic injury from light sources in health‐care procedures. Whether protective measures are warranted in other illuminated procedures besides liver transplantation has been discussed often but never studied. This study elucidates the risk for phototoxic injury in endoscopy, laparoscopy, and non–liver transplant surgery. A theoretical model and epidemiological data are considered. Our findings indicate that endoscopy, laparoscopy, and surgical procedures other than liver transplantation are safe in the noncholestatic protoporphyria patient and that general recommendations for using filters in these situations are not warranted. Among the tested filters, a flexible yellow filter omitting wavelengths below 470 nm is recommended for liver transplant surgery. This filter has been readily accepted by surgeons and offers a good balance between protection and altered visual color perception. The experimental model, using hemolysis of protoporphyrin‐loaded erythrocytes as a measure of phototoxicity, has substantiated theoretical findings on relative filter protection. Liver Transpl 14:1340–1346, 2008.

Photodynamic release of protoporphyrin from intact erythrocytes in erythropoietic protoporphyria: the effect of small repetitive light doses

Abstract— Erythrocytes from patients with erythropoietic protoporphyria contain large amounts of protoporphyrin bound to (hemo)globin. Irradiation of these cells causes a shift in fluorescence emission maximum and a decreased fluorescence intensity which is consistent with transfer of protoporphyrin from (hemo)globin to the cell membrane. When the erythrocytes were irradiated intermittently, nearly 70% of the protoporphyrin was released and the hemolysis was less than 3%. Giving the total light dose as a single pulse, resulted in 84% protoporphyrin release and 16% hemolysis.

Drugs in porphyria: From observation to a modern algorithm-based system for the prediction of porphyrogenicity.

The acute porphyrias are a group of disorders which result from inherited defects in the enzymes of the heme biosynthetic pathway. Affected patients are prone to potentially fatal acute attacks. These attacks are frequently precipitated by exposure to commonly used drugs. Correctly identifying the safety or otherwise of drugs in porphyria is therefore important. In this review we describe how clinical experience and the findings of experimental systems using whole animal or cell culture models have been interpreted to determine porphyrogenicity, that is the potential of a drug to induce an acute attack in a patient carrying a gene for acute porphyria. It is now well established that induction of delta-aminolevulinic acid synthase, the rate controlling enzyme of the heme biosynthetic pathway, is fundamental to porphyrogenicity, and that drug-induced hepatic heme depletion via induction or suicidal inactivation of cytochrome P450 is central to this process. The process is now sufficiently well understood that prediction of porphyrogenicity from structural and functional information alone would appear to be justified.

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.

Erythropoietic protoporphyria: A quantitative determination of erythrocyte protoporphyrin in individual cells by flow cytometry

A fraction of the erythrocytes from patients with erythropoietic protoporphyria contains large amounts of protoporphyrin. By extraction methods, only a mean value of the protoporphyrin content in erythrocytes is obtained. In this study, flow cytometry was used to determine the protoporphyrin content in each individual erythrocyte. The mean erythrocyte protoporphyrin fluorescence, calculated from flow cytometric data, correlated linearly to the mean cellular protoporphyrin concentration measured by ethyl acetate/acetic acid extraction. This indicates that flow cytometry gives a reliable quantitative determination of protoporphyrin in individual cells. Erythrocytes from patients with erythropoietic protoporphyria had a markedly skewed distribution of protoporphyrin. In the most fluorescent erythrocytes (protoporphyrin concentration 3.3 mM), there was about one protoporphyrin molecule for every sixth haeme molecule. This might explain the slight anaemia in some of these patients.

The MDRD equation may mask decline of glomerular filtration rate in Fabry patients with normal or nearly normal kidney function.

AIMS The renal prognosis in Fabry disease is better when enzyme replacement therapy (ERT) is initiated before glomerular filtration rate (GFR) deteriorates. Current studies evaluating kidney function in Fabry disease are mainly based on the MDRD equation. The aim of this cross sectional study was to compare estimated and measured GFR in adult Fabry patients with normal or near normal kidney function. METHODS Iohexol GFR (mGFR) was compared to estimated GFR (eGFR) (MDRD, Cockcroft-Gault and Counahan-Barratt equations) in 8 male and 13 female Fabry patients with minimal albuminuria and mean mGFR of 94 ml/min/1.73 m2 for both genders. RESULTS A significant overestimation of eGFR-MDRD by 24 ml/min/1.73 m2 was seen in male Fabry patients. The performance of the MDRD equation was similar to mGFR in female Fabry patients. GFR was significantly overestimated by the Cockcroft-Gault equation, whilst Counahan-Barratt equation gave results in agreement with mGFR for both male and female Fabry patients. CONCLUSIONS Overestimation of eGFR-MDRD in Fabry patients with CKD stage 1 - 2 and minimal albuminuria may prevent recognition of early progressive renal failure and delayed ERT initiation may be the consequence. Exact GFR markers should be part of the routine evaluation of GFR in Fabry patients.

Glomerular filtration rate measured by iohexol clearance: A comparison of venous samples and capillary blood spots.

Background. Glomerular filtration rate (GFR) measured by iohexol clearance using venous samples is widely used. Capillary sampling on filter paper is easier to perform, may be less painful and spares the blood volume. The purpose of the study was to validate a blood spot method for measuring GFR in children aged 6 years or younger suffering from chronic kidney disease (CKD). Methods. We examined 32 children with CKD, median age (range) 3.0 (0.3–6.2) years. Seven venous samples (10, 30/60, 120, 180, 210, 240, 300 min) were collected and GFR based on all samples was calculated for reference. Following injection of iohexol, blood spots were collected at 120, 180, 210 and 240 min and compared to the reference iohexol clearance. Results. Median (range) reference GFR was 65 (6–122) mL/min/1.73 m2. The 2, 3 and 4-point blood spot GFR were highly correlated to the reference GFR (r = 0.947, 0.945, 0.937). The mean relative bias between 2-point blood spot and reference GFR was 7.2%, and only 2.3% in the patients with reference GFR < 60 mL/min/1.73 m2. The diagnostic accuracy for 2-point blood spot was: 87.5% and 96.9% within ± 15% (P15) and ± 30% (P30) of the reference GFR respectively. In patients with GFR < 60 mL/min/1.73 m2, both P15 and P30 were 100%. Conclusions. GFR calculation based on blood spot iohexol measurement is an alternative method to traditional venous iohexol measurement in children. Our study demonstrates strong agreement between the blood spot and the venous GFR with acceptable bias, precision and diagnostic accuracy, especially in patients with GFR < 60 mL/min/1.73 m2.