Human hereditary hepatic porphyrias

The human hereditary hepatic porphyrias are diseases due to marked deficiencies of enzymes in the heme biosynthetic pathway. Porphyrias can be classified as either hepatic or erythroid, depending on the major production site of porphyrins or their precursors. The pathogenesis of inherited hepatic porphyrias has now been defined at the molecular level. Some gene carriers are vulnerable to a range of exogenous and endogenous factors, which may trigger neuropsychiatric and/or cutaneous symptoms. Early diagnosis is of prime importance since it makes way for counselling. In this article we present an overview of recent advances on hepatic porphyrias: 5-aminolevulinic acid dehydratase deficiency porphyria, acute intermittent porphyria (AIP), porphyria cutanea tarda (PCT), hereditary coproporphyria (HC), and variegate porphyria (VP).     

The porphyrias

The importance of recognizing these rare metabolic disorders in the Emergency Department patient is discussed. The pathophysiologic mechanisms behind the neurologic and cutaneous manifestations of these disorders is reviewed; presentation and management of the acute hepatic porphyrias are described; and features of the erythropoietic porphyrias and porphyria cutanea tarda are presented.     

Porphobilinogen oxygenase from human erythrocytes.

Porphobilinogen oxygenase was isolated from red cells of healthy persons and of patients with disturbances in porphyrin metabolism. The hemolysates were purified by DEAE-cellulose chromatography and the oxygenase was eluted with either 3 mmol/l phosphate buffer (pH 7.4) or with 10 mmol/l Tris-HCl buffer (pH 7.6). The oxygenase can also be isolated by filtration of the hemolysate through Sephadex G-100. In healthy persons a mean value of 84.1 +/- 29.7 nmol of porphobilinogen consumed in 30 min per ml of blood was obtained. In patients with hepatic porphyrias (acute intermittent porphyria and porphyria cutanea tarda) the activity of porphobilinogen oxygenase was very low. In the case with acute intermittent porphyria the activity was increased when measured after storage at 4 degrees C but never reached normal values. In the cases of porphyria cutanea tarda, oxygenase activity increased after recovery and reached normal values. In patients with erythropoietic porphyria and in anemias the activity of porphobilinogen oxygenase gave high values. The erythrocyte enzyme was found to be heterogeneous when compared with the enzyme of other sources. It was only partially succinylated and was inactivated after storage for a few days at 4 degrees C. Some preparations showed the usual allosteric kinetics (n = 1.6--2.0), although Michaelian kinetics were also often observed. The enzyme was inhibited by alpha, alpha'-dipyridyl and EDTA as well as by several metals.     

An inherited enzymatic defect in porphyria cutanea tarda: decreased uroporphyrinogen decarboxylase activity.

Uroporphyrinogen decarboxylase activity was measured in liver and erythrocytes of normal subjects and in patients with porphyria cutanea tarda and their relatives. In patients with porphyria cutanea tarda, hepatic uroporphyrinogen decarboxylase activity was significantly reduced (mean 0.43 U/mg protein; range 0.25-0.99) as compared to normal subjects (mean 1.61 U/mg protein; range 1.27-2.42). Erythrocyte uroporphyrinogen decarboxylase was also decreased in patients with porphyria cutanea tarda. The mean erythrocyte enzymatic activity in male patients was 0.23 U/mg Hb (range 0.16-0.30) and in female patients was 0.17 U/mg Hb (range 0.15-0.18) as compared with mean values in normal subjects of 0.38 U/mg Hb (range 0.33-0.45) in men and 0.26 U/mg Hb (range 0.18-0.36) in women. With the erythrocyte assay, multiple examples of decreased uroporphyrinogen decarboxylase activity were detected in members of three families of patients with porphyria cutanea tarda. In two of these families subclinical porphyria was also recognized. The inheritance pattern was consistant with an autosomal dominant trait. The difference in erythrocyte enzymatic activity between men and women was not explained but could have been due to estrogens. This possibility was supported by the observation that men under therapy with estrogens for carcinoma of the prostate had values in the normal female range. It is proposed that porphyria cutanea tarda results from the combination of an inherited defect in uroporphyrinogen decarboxylase and an acquired factor, usually siderosis associated with alcoholic liver disease.     

Porphyria cutanea tarda. Don't forget to look at the urine

Diagnosis of porphyria cutanea tarda is usually fairly straightforward because of the characteristic clinical findings. Blisters and erosions develop acutely on sun-exposed skin, sometimes accompanied by hypertrichosis, abnormal pigmentation, and milia formation. Iron stores are usually elevated, and liver transaminases and blood glucose levels are often above normal as well. Gross examination of the urine can provide a valuable clue, since urine of porphyria cutanea tarda patients is red to brown in natural light and pink to red in fluorescent light. Biopsy of a bullous lesion is useful to rule out other diseases. Confirmation of porphyria cutanea tarda requires measurement of porphyrin levels in a 24-hour urine collection. Once the diagnosis is confirmed, it appears reasonable to screen all patients with porphyria cutanea tarda for hepatitis C and possibly B, but especially those less than 30 years old who have extremely high liver transaminase levels. Therapeutic measures for porphyria cutanea tarda include avoidance of exacerbating factors, especially ultraviolet light, ethanol, and certain medications. Phlebotomy or chloroquine therapy is reserved for patients in whom conservative measures fail.     

Erythrocyte porphyrinogen carboxy-lyase activity in porphyria cutanea tarda and certain other human porphyrias

Red cell porphyrinogen carboxy-lyase activity was measured using uroporphyrinogen III as substrate in 18 normal persons, 7 male patients with porphyria cutanea tarda, 3 female patients with erythropoietic protoporphyria and 2 female patients with variegate porphyria.The mean values obtained in normal subjects were 0.151 nmol of uroporphyrinogen disappeared in 30 min per mg of protein, and 0.038 nmol of coproporphyrinogen formed in 30 min per mg of protein. We have not been able to detect significant differences between males and females.In porphyria cutanea tarda the enzyme activity was the same as in normal subjects considering either substrate disappearance or end product formation. The differences were not significant at the p < 0.05 level. Patients with variegate porphyria also exhibited normal erythrocyte porphyrinogen carboxy-lyase activity.The enzyme activity of erythrocytes from patients with erythropoietic protoporphyria was higher than in normals; mean values for specific activities being 0.204 nmol of uroporphyrinogen disappeared, and 0.071 nmol of coproporphyrinogen formed.The significance of the results with respect to the chemical picture of different porphyrias is discussed.     

The porphyrias: recent advances

Recent research has elucidated several of the hitherto poorly understood steps in heme synthesis. This review describes this metabolic pathway and pinpoints the enzymatic blockages in the various porphyrias. Recent advances in the understanding of the etiology of porphyria cutanea tarda are discussed, as are the abnormalities of porphyrin metabolism seen in chronic renal failure and in lead poisoning. An outline is given of the clinical and biochemical abnormalities seen in the porphyrias. Included is an algorithm to aid in the differential diagnosis of these diseases, and a brief review of the new analytical techniques available for the identification and quantification of porphyrins and their precursors in body fluids.     

Uroporphyrinogen decarboxylase: a splice site mutation causes the deletion of exon 6 in multiple families with porphyria cutanea tarda.

Uroporphyrinogen decarboxylase (URO-D) is a cytosolic heme-biosynthetic enzyme that converts uroporphyrinogen to coproporphyrinogen. Defects at the uroporphyrinogen decarboxylase locus cause the human genetic disease familial porphyria cutanea tarda. A splice site mutation has been found in a pedigree with familial porphyria cutanea tarda that causes exon 6 to be deleted from the mRNA. The intron/exon junctions on either side of exon 6 fall between codons, so the resulting protein is shorter than the normal protein, missing only the amino acids coded by exon 6. The shortened protein lacks catalytic activity, is rapidly degraded when exposed to human lymphocyte lysates, and is not detectable by Western blot analysis in lymphocyte lysates derived from affected individuals. The mutation was detected in five of 22 unrelated familial porphyria cutanea tarda pedigrees tested, so it appears to be common. This is the first splice site mutation to be found at the URO-D locus, and the first mutation that causes familial porphyria cutanea tarda to be found in more than one pedigree.     

Koexistenz von heredit?rer Koproporphyrie und Porphyria cutanea tarda: Eine neue Form einer dualen Porphyrie

Zusammenfassung Hintergrund: Duale Porphyrien sind durch die Koexistenz zweier unabhängiger Porphyrinstoffwechselstörungen charakterisiert. Patient und Methoden: Bei einer 26-jährigen Patientin mit Rückenschmerzen und rotem Urin wurde zunächst eine Porphyria cutanea tarda und durch weitere Untersuchungen eine hereditäre Koproporphyrie diagnostiziert. Die Metaboliten des Porphyrinstoffwechsels wurden chromatographisch analysiert. Die Aktivitäten der Koproporphyrinogenoxidase und der Uroporphyrinogendecarboxylase wurden in Blutzellen bestimmt. Die molekulare Analyse erfolgte mittels denaturierender Gradientengelelektrophorese, gefolgt von direkter Sequenzierung. Ergebnisse: Eine exzessiv hohe Porphyrinurie von 3 128 nmol/24 h (normal < 165 nmol/24 h) mit Dominanz von Uro- und Heptacarboxyporphyrin (75% der Gesamtporphyrine) wies bei der Patientin auf eine Porphyria cutanea tarda hin. Verlaufsuntersuchungen zeigten eine Konstellationsänderung mit Dominanz von Koproporphyrinisomer III in Urin und Stuhl, die für eine hereditäre Koproporphyrie charakteristisch ist. Prophyrinvorläufer und Porphyrine stiegen unter Einnahme von Ethinylestradiol-Cyproteronacetat an. Neben erhöhtem Uro- und Heptacarboxyporphyrin im Urin blieb die Dominanz von Koproporphyrinisomer III im Stuhl konstant. Die Aktivität der Koproporphyrinogendecarboxydase war um 35% vermindert. Die erythrozytäre Uroporphyrinogendecarboxylase war normal. Die Mutter und beide Schwestern wurden als heterozygote Genträger einer hereditären Koproporphyrie in der Latenzphase aufgrund eines Anstiegs des Koproporphyrins mit Isomer-I/III-Inversion im Stuhl und einer Minderung der Koproporphyrinogenoxidase-Aktivität um etwa 50% erkannt. Molekulargenetische Analysen ergaben eine Punktmutation im Exon 4 (854CMT) des Koproporphyrinogenoxidase-Gens, die einen Aminosäurenaustausch (P258L) im Koproporphyrinogenoxidase-Protein zur Folge hat. Schlussfolgerung: Bei einer dualen Porphyrie mit Koexistenz von Porphyria cutanea tarda und hereditärer Koproporphyrie ist die hereditäre Koproporphyrie durch eine neue Mutation im Koproporphyrinogenoxidase-Gen bedingt. Die Porphyria cutanea tarda, als sporadische hepatische Form Typ I, ist östrogeninduziert. Die großen exkretorischen Variationen reflektieren den Einfluss hormonaler Faktoren auf den hepatischen Porphyrieprozess von hereditärer Koproporphyrie und Porphyria cutanea tarda. Abstract Background: Dual porphyrias are characterized by two independent disturbances of porphyrin metabolism. Patient and Methods: At first a porphyria cutanea tarda was diagnosed in a 26-year-old female with back pain and red urine. Later a hereditary coproporphyria was ascertained by additional examinations. The metabolites of porphyria metabolism were analyzed chromatographically. The activities of coproporphyrinogen oxidase and uroporphyrinogen decarboxylase were determined in blood cells. Molecular analysis was carried out by denaturating gradient gel electrophoresis followed by direct sequencing. Results: Excessive porphyrinuria of 3 128 nmol/24 h (normal < 165 nmol/24 h) with dominance of uro- and heptacarboxyporphyrin (75% of total porphyrins) indicated that the patient suffered from porphyria cutanea tarda. The course of the examination showed an alteration of the constellation with dominance of urinary and fecal coproporphyrin isomer III, which is characteristic for hereditary coproporphyria. Porphyrin precursors and porphyrins increased under the application of ethinylestradiol-cyproteronacetat. The dominance of coproporphyrin III stayed constant in feces besides enhanced urinary uro- and heptacarboxyporphyrin. The activity of the coproporphyrinogen oxidase was diminished to 35%. The uroporphyrinogen decarboxylase in erythrocytes was normal. The mother and both sisters were recognized as heterozygous gene carriers of hereditary coproporphyria in the latent phase by enhanced coproporphyrin with isomer I/III inversion in feces and decrease of the coproporphyrinogen oxidase activity to about 50%. Molecular analyses resulted in a point mutation at exon 4 (854CMT), which revealed in an amino acid exchange (P258L) in the coproporphyrinogen oxidase protein. Conclusion: The hereditary coproporphyria is caused by a new mutation in the coproporphyrinogen oxidase gene in the case of a dual porphyria with coexistence of porphyria cutanea tarda and hereditary coproporphyria. The sporadic, hepatic porphyria cutanea tarda Type I is induced by estrogens. The large excretory variations reflect the influence of hormonal factors on the porphyria process of hereditary coproporphyria and porphyria cutanea tarda.     

Biochemical differentiation of the porphyrias

Objectives: To differentiate the porphyrias by clinical and biochemical methods.

Design and methods: We describe levels of blood, urine, and fecal porphyrins and their precursors in the porphyrias and present an algorithm for their biochemical differentiation. Diagnoses were established using clinical and biochemical data. Porphyrin analyses were performed by high performance liquid chromatography.

Results and conclusions: Plasma and urine porphyrin patterns were useful for diagnosis of porphyria cutanea tarda, but not the acute porphyrias. Erythropoietic protoporphyria was confirmed by erythrocyte protoporphyrin assay and erythrocyte fluorescence. Acute intermittent porphyria was diagnosed by increases in urine delta-aminolevulinic acid and porphobilinogen and confirmed by reduced erythrocyte porphobilinogen deaminase activity and normal or near-normal stool porphyrins. Variegate porphyria and hereditary coproporphyria were diagnosed by their characteristic stool porphyrin patterns. This appears to be the most convenient diagnostic approach until molecular abnormalities become more extensively defined and more widely available.     

Uroporphyrinogen decarboxylase gene mutations in Danish patients with porphyria cutanea tarda

Decreased uroporphyrinogen decarboxylase (UROD) activity is a characteristic feature of the most common of the porphyrias, porphyria cutanea tarda (PCT). A subgroup of the clinically overt PCT cases is associated with mutations in the gene encoding UROD and inherited as an autosomal-dominant trait. In this study, DNAs from 53 Danish PCT patients were subjected to genetic analysis for UROD mutations using denaturing gradient gel electrophoresis. Eleven genetic variations, seven of which are possible disease causing, were identified. All but one of these mutations were previously unknown, lending further support to the assumption that PCT is a heteroallelic disease. Only 11% of the examined patients were previously recognized as familial PCT cases. However, possible disease-related UROD mutations were identified in 24% of the examined patients, indicating that genetic analysis of PCT patients may improve differentiation between familial and sporadic PCT cases.     

Uroporphyrinogen decarboxylase gene mutations in Danish patients with porphyria cutanea tarda

Decreased uroporphyrinogen decarboxylase (UROD) activity is a characteristic feature of the most common of the porphyrias, porphyria cutanea tarda (PCT). A subgroup of the clinically overt PCT cases is associated with mutations in the gene encoding UROD and inherited as an autosomal-dominant trait. In this study, DNAs from 53 Danish PCT patients were subjected to genetic analysis for UROD mutations using denaturing gradient gel electrophoresis. Eleven genetic variations, seven of which are possible disease causing, were identified. All but one of these mutations were previously unknown, lending further support to the assumption that PCT is a heteroallelic disease. Only 11% of the examined patients were previously recognized as familial PCT cases. However, possible disease-related UROD mutations were identified in 24% of the examined patients, indicating that genetic analysis of PCT patients may improve differentiation between familial and sporadic PCT cases.     

The differentiation of the porphyrias by means of high pressure liquid chromatography

The analysis of faecal and urinary porphyrins by high pressure liquid chromatography (H.P.L.C.) provides characteristic profiles and facilitates rapid diagnosis of variegate (porphyria cutanea tarda hereditaria), symptomatic porphyria (porphyria cutanea tarda symptomatica), hereditary coproporphyria, acute intermittent porphyria, erythro-hepatic protoporphyria and congenital porphyria (erythropoietic porphyria).     

Genetic haemochromatosis presenting as porphyria cutanea tarda

Porphyria cutanea tarda (PCT) is the commonest form of porphyria. It can be inherited or acquired. We present a case of genetic haemochromatosis with associated PCT. Venesection led to improvement in both conditions. We highlight the need for the awareness of PCT and its associated conditions.     

The role of iron in the pathogenesis of porphyria cutanea tarda. II. Inhibition of uroporphyrinogen decarboxylase.

Porphria cutanea tarda is characterized biochemically by excessive hepatic synthesis and urinary excretion of uroporphyrin I and 7-carboxylporphyrins. This pattern of excretion suggest an impaired ability to decarboxylate uroporphyrinogen to the paired ability to decarboxylate uroporphyringen to the 4-carboxyl porphyrinogen, coproporphyrinogen, a reaction catalyzed by the enzyme uroporphyringen decarboxylase. Because clinical evidence has implicated iron in the pathogenesis of porphyria cutanea tarda, these experiments were designed to study the effect of iron on uroporphyrinogen decarboxylase in procine crude liver extracts. Mitochondria-free crude liver extracts were preincubated with ferrous ion and aliquots were assayed for uroporphyrinogen decarboxylase activity. Uroporphyrinogens I and III, the substrates for the decarboxylase assay, were prepared enzymatically from (3H)porphobilinogen. The products of the decarboxylase reaction were identified and quantitated by three methods: (a) extraction into 1.5 N HCl and spectrophotometric quantitation; (b) adsorption onto talc, esterification, paper chromatographic identification, and quantitation by liquid scintillation counting; and (c) adsorption onto talc, esterification, thin-layer chromatographic identification on silica gel, and quantitation by liquid scintillation counting. The thin-layer scinllation method proved most sensitive as it was the only method which accurately identified and quantitated the 7-carboxyl porphyrin reaction product. Uroporphyrinogens I and III were decarboxylated at the same rate by porcine hepatic uroporphyrinogen decarboxylase, and the addition of iron induced marked inhibition of the decarboxylase activity. Ortholpehanthroline blocked the inhibitory effect of iron. The inhibition of uroporphyrinogen decarboxylase by ferrous ion, coupled with its previously reported inhibitory effect on uroporphyrinogen III cosynthetase, provides a possible biochemical explanation for the pattern of urinary porphyrin excretion observed in patients with porphyria cutanea tarda and the clinical association with disordered iron metabolism.     

Enzyme heterogeneity in the porphyrias

The use of immunological methods for measuring enzyme mass has identified several varieties of porphyria in which the reduction of porphyrin enzyme activity is not accompanied by a corresponding change in the enzyme mass. Currently, acute intermittent porphyria and hepatoerythropoietic porphyria have exhibited this phenomenon. In porphyria cutanea tarda, it has recently been shown that the pattern of enzyme deficiency in erythrocytic and nonerythrocytic tissues does not strictly follow the inheritance pattern (familial and sporadic) previously described. Also, contrary to previous dogma, some cases of type 1 porphyria cutanea tarda appear to be positive for cross reactive immunological material (CRIM).     

Plasma porphyrins in the porphyrias.

BACKGROUND: As an aid in the diagnosis and management of porphyria we have developed a method to fractionate and quantify plasma porphyrins and have evaluated its use in various porphyrias. METHODS: We used HPLC with fluorometric detection to measure plasma concentrations of uroporphyrin I and III, heptacarboxyl III, hexacarboxyl III, pentacarboxyl III, and coproporphyrin I and III. We studied 245 healthy subjects, 32 patients with classical porphyria cutanea tarda (PCT), 12 patients with PCT of renal failure, 13 patients with renal failure, 8 patients with pseudoporphyria of renal failure, 3 patients with acute intermittent porphyria, 5 patients with variegate porphyria, 5 patients with hereditary coproporphyria, and 4 patients with erythropoietic protoporphyria. RESULTS: Between-run CVs were 5.4-13%. The recoveries of porphyrins added to plasma were 71-114% except for protoporphyrin, which could not be reliably measured with this technique. Plasma porphyrin patterns clearly identified PCT, and its clinical sensitivity equaled that of urine porphyrin fractionation. The patterns also allowed differentiation of PCT of renal failure from pseudoporphyria of renal failure. CONCLUSIONS: The assay of plasma porphyrins identifies patients with PCT and appears particularly useful for differentiating PCT of renal failure from pseudoporphyria of renal failure.     

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.     

Management of acute and cutaneous porphyrias

The porphyrias comprise a group of disorders of the haem biosynthesis pathway that can present with acute neurovisceral symptoms, skin lesions or both. Acute porphyrias present with severe abdominal pain, confusion and seizures which may be life-threatening. Specific treatment with haem preparations should be instituted as soon as possible following confirmation of increased excretion of porphobilinogen in the urine. Supportive treatment includes opiate analgesia, monitoring for and treating complications such as hypertension and hyponatraemia. Follow-up should include counselling on lifestyle modification involving avoidance of alcohol, smoking and known porphyrogenic drugs and diet. Identification and counselling of at risk relatives is essential. The cutaneous porphyrias result from porphyrin-induced photosensitivity and can present with either acute photosensitivity or skin fragility and blisters. All cutaneous porphyrias can be alleviated by avoidance of sunlight. Treatment of erythropoietic protoporphyria involves administering large doses of beta-carotene, which may improve tolerance to sunlight. Porphyria cutanea tarda can be effectively treated by phlebotomy or low dose chloroquine. Congenital erythropoietic porphyria is a rare, early onset, severe, photomutilating condition for which bone marrow transplantation has been shown to be successful.     

Urinary porphyrin excretion in hepatitis C infection.

A high prevalence of hepatitis C virus infection in porphyria cutanea tarda in some populations suggests a close link between viral hepatitis and alteration of porphyrin metabolism. Moreover, there is evidence of a role of porphyrinopathies in hepatocarcinogenesis. The aim of our study was to obtain data on the prevalence and patterns of heme metabolism alterations in patients with chronic hepatitis C virus infection. Urinary porphyrin excretion was prospectively studied in 100 consecutive outpatients with chronic hepatitis C infection without signs of photosensitivity, using an ion-pair high-performance liquid chromatography method. Increased total porphyrin excretion was found in 41 patients, with predominant excretion of coproporphyrins (whole study group: mean 146 microg/g creatinine, interquartile range 76-186; normal < 150), in 10 patients excretion exceeded 300 microg/g creatinine. In the majority of all patients studied (75/100) an increased ratio of the relatively hydrophobic coproporphyrin isomer I to isomer III was found. In just one case, urinary porphyrin pattern characteristic for chronic hepatic porphyria was present (uroporphyrin > coproporphyrin, heptacarboxyporphyrin III increased) but the total porphyrin excretion was only slightly elevated in this case. In the whole group, total urinary porphyrin excretion correlated well with serum bilirubin and was inversely correlated with albumin and thrombin time. In conclusion, secondary coproporphyrinuria occurs frequently in heptatitis C infection, whereas in Germany, preclinical porphyria cutanea tarda seems to be rare in these patients.