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Physicians should be aware of porphyrias, which could be responsible for unexplained gastrointestinal, neurologic, or skin disorders. Despite their relative rarity and complexity, most porphyrias can be easily defined and diagnosed. They are caused by well-characterized enzyme defects in the complex heme biosynthetic pathway and are divided into categories of acute vs non-acute or hepatic vs erythropoietic porphyrias. Acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria, hereditary coproporphyria, and aminolevulinic acid dehydratase deficient porphyria) manifest in attacks and are characterized by overproduction of porphyrin precursors, producing often serious abdominal, psychiatric, neurologic, or cardiovascular symptoms. Patients with variegate porphyria and hereditary coproporphyria can present with skin photosensitivity. Diagnosis relies on measurement of increased urinary 5-aminolevulinic acid (in patients with aminolevulinic acid dehydratase deficient porphyria) or increased 5-aminolevulinic acid and porphobilinogen (in patients with other acute porphyrias). Management of attacks requires intensive care, strict avoidance of porphyrinogenic drugs and other precipitating factors, caloric support, and often heme therapy. The non-acute porphyrias are porphyria cutanea tarda, erythropoietic protoporphyria, X-linked protoporphyria, and the rare congenital erythropoietic porphyria. They lead to the accumulation of porphyrins that cause skin photosensitivity and occasionally severe liver damage. Secondary elevated urinary or blood porphyrins can occur in patients without porphyria, for example, in liver diseases, or iron deficiency. Increases in porphyrin precursors and porphyrins are also found in patients with lead intoxication. Patients with porphyria cutanea tarda benefit from iron depletion, hydroxychloroquine therapy, and, if applicable, elimination of the hepatitis C virus. An α-melanocyte-stimulating hormone analogue can reduce sunlight sensitivity in patients with erythropoietic protoporphyria or X-linked protoporphyria. Strategies to address dysregulated or dysfunctional steps within the heme biosynthetic pathway are in development., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Porphyrias are metabolic disorders of the heme biosynthesis. Clinically, they can be differentiated into acute and non-acute porphyrias. The symptomatic phase of acute hepatic porphyrias is characterized by overproduction of neurotoxic porphyrin precursors and porphyrins. Acute intermittent porphyria, Variegate porphyria, Hereditary coproporphyria and Doss porphyria belong to this group of metabolic disorders. The clinical presentation of the acute hepatic porphyria syndrome includes abdominal, psychiatric, neurological and cardiovascular symptoms. The diagnosis is based on a tenfold increased urinary excretion of porphobilinogen (apart from Doss porphyria). Besides symptomatic therapy with non-porphyrinogenic drugs, electrolyte compensation and intensive monitoring, intravenous administration of glucose and heme arginate is established for treatment. Among the non-acute types like Porphyria cutanea tarda, Erythropoietic protoporphyria and Congenital erythropoietic porphyria, the accumulated porphyrins cause photosensitivity of the skin up to severe liver damage. The location of the deficient enzyme within the heme biosynthesic pathway determines the pattern of the accumulated porphyrins. Besides light protection, there are different therapies depending on the type of non-acute porphyria. Ultimately, liver transplantation may be considered in therapy-resistant cases of acute hepatic porphyrias and bone marrow transplantation in severe cases of erythropoietic porphyrias.

Acute porphyrias are caused by enzyme defects along the heme synthesis pathway. Patients usually present with abdominal pain, impaired intestinal motility, neurological and psychiatric symptoms, hypertension, tachycardia, hyponatriemia and reddish urine. This article gives an overview over drugs that are recommended in patients with acute hepatic porphyrias and represents a compilation of four so far existing lists.

The biochemical and the molecular diagnoses of an inherited porphyria require experience. False positive or negative screening tests and the low penetrance of the disease make a correct diagnosis difficult.The biochemical and the molecular procedures for the diagnosis of acute intermittent porphyria were applied to five unrelated patients suffering from acute intermittent porphyria. All patients were shown to be gene carriers of acute intermittent porphyria by both methods. The two different possibilities of the diagnosis corresponded well. In a family definitively identified by molecular diagnosis of one of the patients and his relatives, the patient's two children were asymptomatic. His son was shown to be a gene carrier of the father's deficiency by biochemical as well as molecular analysis, whereas his daughter was not affected by acute intermittent porphyria.

Delta-aminolevulinic acid dehydratase (ALAD) deficiency porphyria, or Doss porphyria, was first reported in Germany in 1979. Only four bona fide cases of Doss porphyria have been reported to date that were confirmed by immunological and molecular analyses of their ALAD mutations. Here we describe the fifth case of Doss porphyria. A 17-year-old German male suffered from colicky abdominal pain and severe polyneuropathy for 2 years. Urinary delta-aminolevulinic acid (ALA) was increased 32-fold, and coproporphyrin 76-fold compared with the upper limit of their respective normal ranges. Urinary excretion of porphobilinogen (PBG) and uroporphyrin was only slightly increased. Faecal porphyrins were within the normal range. Erythrocyte zinc protoporphyrin concentrations were elevated 5.4-fold. ALAD activity in erythrocytes was decreased to 10% of the normal value, and was not activated by zinc and by dithiothreitol. Blood lead levels were within the normal range, excluding lead poisoning in the proband. Erythrocyte ALAD activity was about one-half of the normal value in both parents, whereas it was normal in the proband's brother. Urinary excretion of ALA, PBG and total porphyrins was within the normal range in both parents and the brother. Molecular genetic studies of the ALAD gene in the proband revealed two base changes, C to A and C to T, both in intron 3 at -11 bp upstream of the exon 3 start site. In addition to the proband, the father carried the (-11)C-to-T, while the mother carried the ALAD gene in the proband's brother. These findings suggest that the observed compound heterozygosity of the ALAD gene may be responsible for Doss porphyria in the proband. The proband was successfully treated with haem arginate infusion. The clinical condition improved, and urinary excretion of ALA and coproporphyrin fell to levels of approximately 50% compared with their pretreatment levels during acute relapses. The haem therapy was continued once weekly for 1 year. At the end of 1 year, urinary ALA and porphyrin levels were significantly lowered, and the proband is now almost free of clinical symptoms.

Objective: To examine the role of hemochromatosis (HFE) gene mutations, which are associated with porphyria cutanea tarda (PCT), in the therapeutic response to chloroquine., Design: We retrospectively analyzed a database (Excel version 2001 [Microsoft Excel, Redmond, Wash]; date range of search, 1985-1999) of chloroquine-treated patients with PCT on whether HFE mutations (C282Y and H63D) might have influenced the clinical response, urinary porphyrin excretion, liver enzyme activities, and serum iron markers. Serum samples and corresponding complete sets of data before and after therapy were available in 62 of 207 patients with PCT who were treated exclusively with chloroquine., Settings: Academic teaching hospital., Intervention: For treatment, low-dose chloroquine diphosphate, 125 to 250 mg twice weekly, was used during a median time of 16 months (range, 12-26 months)., Results: Of the 62 German patients with PCT, 37 (60%) carries HFE mutations. Chloroquine therapy was accompanied by clinical remission and reduced urinary porphyrin excretion (P<.001) in the 24 patients (39%) with HFE wild type as well as in 35 HFE heterozygous patients with PCT (56%). Decreases of serum iron markers following chloroquine therapy were limited to patients with PCT and HFE wild type. All patients homozygous for the C282Y mutation (3 [5%] of 62) had high serum iron, ferritin, and transferrin saturation and failed to respond to chloroquine treatment., Conclusions: The therapeutic response to chloroquine was not compromised by C282Y heterozygosity and compound heterozygosity of HFE mutations. Because HFE C282Y homozygotes (+/+) did not respond to chloroquine and a decrease in serum iron concentration was limited to patients with PCT and HFE wild type, phlebotomy should be first-line therapy in patients with PCT and HFE mutations.

5-Aminolevulinic acid (5-ALA) is a useful agent to enhance the detection of early epithelial lesions in head and neck cancers. It is applied either topically or systemically and converted intracellular into photosensitive protoporphyrin IX (PpIX). By ultraviolet light illumination malignant and fast proliferating lesions are detected by a characteristic red fluorescence and delineated by the bluish fluorescence of healthy tissue. To assess the elimination patterns 5-ALA, porphobilinogen (PBG) and porphyrin were measured 12h and 36h after administration in urine, 12h and 24h after examination in blood and in feces 12h after endoscopy. 5-ALA was applied either by inhalation (250 mg) or mouth rinse (200 mg). After both administration routes, excretion levels in urine returned to background levels within 12 hours after administration and only in feces values are slightly increased for PpIX and total porphyrin. Concentrations in erythrocytes were elevated, but not in plasma. No side effects were observed. According to our results the topical administration of 5-ALA is a useful method with satisfying fluorescence imaging results. Levels of metabolites in urine and plasma return to normal within 12 hours so that skin photosensitization can be neglected.

Coproporphyrinogen III oxidase is deficient in hereditary coproporphyria. An activity assay for this enzyme in mononuclear cells, besides the preparation of the substrate, are presented. The separation conditions for the product of the test protoporphyrin IX by gradient, reversed-phase high-performance liquid chromatography are given. The normal value from mononuclear cells of healthy volunteers was 138 +/- 21 pkat/g total soluble protein (mean +/- SD). The enzyme activity of a family with hereditary coproporphyria was measured. The gene carriers exhibit a specific coproporphyrinogen III oxidase activity of 61-90 pkat/g total soluble protein.

A 30-year-old woman suffered from acute crises with abdominal, neurological and psychiatric complaints. Urinary haem precursors and faecal porphyrins were excessively elevated compared to the upper level of the normal range. Urinary coproporphyrin isomer III was increased and faecal coproporphyrin isomers I and III showed a complete inversion of the normal ratio. Thus, hereditary coproporphyria was diagnosed in this woman. The father, one brother and a sister were shown to be gene carriers of hereditary coproporphyria by their urinary and faecal excretory constellations. The excretory patterns of the mother and a second brother were normal. Coproporphyrinogen oxidase activity was decreased to 49% and 58%, in the patient and her father, respectively. The mother's enzyme activity was normal (98%). Coproporphyrinogen oxidase concentration was enhanced 1.8-fold and 2.7-fold in the patient and her father, respectively. Mutation analysis revealed the insertion of an adenine at position 857 in exon 4 of the coproporphyrinogen oxidase gene. The gene defect was confirmed by denaturing gradient gel electrophoresis in the patient and her father. The patient was treated by intravenous interval therapy with haem arginate for 10 months, with good clinical and metabolic response.

Autoimmunity and high rates of autoantibodies have been implicated in the pathogenesis of porphyria cutanea tarda. These abnormalities could be in part virus-induced, since porphyria cutanea tarda in most geographical regions is highly associated with hepatitis C virus infection. We analyzed the link of autoantibodies, autoimmune hepatitis and systemic lupus erythematosus in 111 patients with porphyria cutanea tarda and sex- and age-matched controls (mean age 58+/-13 years) in Germany, a region with a low prevalence of hepatitis C virus infection. Patients with porphyria cutanea tarda displayed lower rates of anti-nuclear antibodies (16/111, 14% vs 28/111, 25%, p<0,05) and of antibodies against smooth muscle (25/111, 23% vs 48/111, 43%, p<0,01), than controls. The percentage of patients with porphyria cutanea tarda with positive anti-HCV was low but significantly higher than in our controls (9/111, 8% vs 0/111, 0%, respectively), (p<0,05). Two patients with porphyria cutanea tarda (2/111, 2%) fulfilled the criteria for systemic lupus erythematosus and not one of 65 patients was found to have clinical autoimmune hepatitis. In the first controlled study of a large cohort of patients with porphyria cutanea tarda no increased prevalence of selected autoantibodies and autoimmune hepatitis was found. However, a higher prevalence of HCV infection and systemic lupus erythematosus in patients with porphyria cutanea tarda was confirmed.

A 27-year-old woman who had recurrent pain in renal bed since 1998 with increasing character, was stationary admitted. The patient showed dark urine, complained of hair loss and took since 1994 a hormonal oral contraceptive. No photosensitivity was observed. Determinations of urinary porphyrin metabolites in 1998 revealed a porphyria cutanea tarda like excretion pattern with elevations of uro- (1767 nmol/24 hr, normal <29 nmol/24 hr) and heptacarboxyporphyrin (568 nmol/24 hr; normal <4 nmol/24 hr). Follow-up studies in feces showed the characteristics of a hereditary coproporphyria with dominance of coproporphyrin isomer III (total= 1470 nmol/g, isomer III= 93%), (normal: <37 nmol/g, isomer III = 25-35%). The excretion of porphyrin precursors (delta-aminolevulinic acid and porphobilinogen) was increased by taking an ethinylestradiol-cyproteronacetate-preparation, but acute and/or chronic manifestations were not observed. Coproporphyrinogen oxidase activity was decreased to 35% in the patient (normal=138+/-21 pkat/g protein; x+/-s), whereas the activity of red cell uroporphyrinogen decarboxylase was normal. Her mother and both sisters could be verified as heterozygous gene carriers of hereditary coproporphyria by their urinary and fecal excretion parameters and because of reduced coproporphyrinogen oxidase activity up to 50%. The father was normal with respect to his genotype. Molecular analysis revealed a hitherto unknown mutation with the transversion of a cytosine to thymine at nucleotide position 854 in exon 4 of the coproporphyrinogen oxidase gene. The gene defect was confirmed by DGGE in the mother and her three daughters. The investigation of the immunological nature of the defective coproporphyrinogen oxidase gene from the whole family revealed decreased concentrations of coproporphyrinogen oxidase protein in the patient, her mother and her two sisters.

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 porphyrin metabolism were analyzed chromatographically. The activities of coproporphyrinogen oxidase and uroporphyrinogen decarboxylase were determined in blood cells. Molecular analysis was carried out by denaturing 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 (854C-->T), 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 co-existence 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.

Variegate porphyria is a rare disease caused by a deficiency of protoporphyrinogen oxidase. In most cases, the clinical findings are a combination of systemic symptoms similar to those occurring in acute intermittent porphyria and cutaneous lesions indistinguishable from those of porphyria cutanea tarda. We report on a 24-year-old woman with variegate porphyria who, after intake of lynestrenol, developed typical cutaneous lesions but no viscero-neurological symptoms. The diagnosis was based on the characteristic urinary coproporphyrin and faecal protoporphyrin excretion patterns, and the specific peak of plasma fluorescence at 626 nm in spectrofluorometry. Biochemical analysis revealed that most of the family members, though free of clinical symptoms, excrete porphyrin metabolites in urine and stool similar to variegate porphyria, accompanied by a significant decrease of porphobilinogen deaminase activity of a range which is ordinarily found in patients with acute intermittent porphyria only (approximately 50%). These data first led to the assumption of two separate and independently inherited genetic defects, similar to the dual porphyria of Chester. Molecular analysis, however, revealed only a missense mutation of the protoporphyrinogen oxidase gene, but not of the porphobilinogen deaminase gene. Thus, in the family presented, porphobilinogen deaminase deficiency is likely to be a phenomenon secondary to the genetic defect of protoporphyrinogen oxidase.

The properties of 9 delta-aminolevulinate dehydratase (ALAD) mutants from patients with ALAD porphyria (ADP) were examined by bacterial expression of their complementary DNAs and by enzymologic and immunologic assays. ALADs were expressed as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and purified by glutathione-affinity column chromatography. The GST-ALAD fusion proteins were recognized by anti-ALAD antibodies and were enzymatically active as ALAD. The enzymatic activities of 3 ALAD mutants, K59N, A274T, and V153M, were 69.9%, 19.3%, and 41.0% of that of the wild-type ALAD, respectively, whereas 6 mutants, G133R, K59N/G133R, F12L, R240W, V275M, and delTC, showed little activity (< 8%). These variations generally reflect the phenotype of ALAD in vivo in patients with ADP and indicate that GST-ALAD fusion protein is indeed useful for predicting of the phenotype of ALAD mutants. The location of F12L mutation in the enzyme's molecular structure indicates that its disturbance of the quaternary contact of the ALAD dimer appears to have a significant influence on the enzymatic activity. Mouse monoclonal antibodies to human ALAD were developed that specifically recognized a carboxy terminal portion of ALAD, or other regions in the enzyme. This study represents the first complete analysis of 9 mutants of ALAD identified in ADP and indicates the highly heterogeneous nature of mutations in this disorder.

Activated phagocytes, particularly polymorphonuclear leukocytes (neutrophils), by means of oxidative photonic burst, i.e., the combined activation of NADPH-oxidase and myeloperoxidase, generate large amounts of oxidants of the hypochlorite/chloramine type that are an important physiologic source for the nonradical, photon-emitting oxidant singlet oxygen (1O2), which (in the dark blood stream) is both a signal and an agent of defense against bacteria or fibrin. 1O2-oxidized fibrinogen or oxidized fibrin monomer has previously been shown to be unpolymerizable, and methionine to methionine sulfoxide-oxidized fibrinogen occurs in circulating blood. The present study demonstrates that thrombin converts oxidized fibrinogen into a soluble stimulator of tissue-type plasminogen activator (t-PA). After addition of 0.1 IU thrombin to 25 microl oxidized normal human plasma and an incubation time of 10 min (room temperature), t-PA activity increases about 20-fold when compared with oxidized plasma without the addition of thrombin. Thus, since oxidized fibrin monomer is a t-PA cofactor, thrombin-degraded oxidized fibrinogen can be used as a stimulator in functional t-PA assays.

The course of delta-aminolaevulinic acid dehydratase activity was studied over the 23 years in erythrocytes of two male patients. The enzyme activity was originally 1-2%, which then increased to approximately 8%, of normal levels several years after clinical manifestation of the acute hepatic porphyria syndrome. Urinary excretions of delta-aminolaevulinic acid and coproporphyrin III were excessively increased in the two patients with compound-heterozygous delta-aminolaevulinic acid dehydratase deficiency porphyria.

Porphyrias are divided into erythropoietic and hepatic manifestations. Erythropoietic porphyrias are characterized by cutaneous symptoms and appear in early childhood. Erythropoietic protoporphyria is complicated by cholestatic liver cirrhosis and progressive hepatic failure in 10%, of patients. Acute hepatic porphyrias (delta-aminolaevulinic acid dehydratase deficiency porphyria, acute intermittent porphyria, hereditary coproporphyria and variegate porphyria) are characterized by variable extrahepatic gastrointestinal, neurological-psychiatric and cardiovascular manifestations requiring early diagnosis to avoid life-threatening complications. Acute hepatic porphyrias are pharmacogenetic and molecular regulatory diseases (without porphyrin accumulation) mainly induced by drugs, sex hormones, fasting or alcohol. The disease process depends on the derepression of hepatic delta-aminolaevulinic acid synthase following haem depletion. In contrast to the acute porphyrias, nonacute, chronic hepatic porphyrias such as porphyria cutanea tarda are porphyrin accumulation disorders leading to cutaneous symptoms associated with liver disease, especially caused by alcohol or viral hepatitis. Alcohol, oestrogens, haemodialysis, hepatitis C and AIDS are triggering factors. Porphyria cutanea tarda is the most common porphyria, followed by acute intermittent porphyria and erythropoietic protoporphyria. The molecular genetics of the porphyrias is very heterogenous. Nearly every family has its own mutation. The mutations identified account for the corresponding enzymatic deficiencies, which may remain clinically silent throughout life. Thus, the recognition of the overt disorder with extrahepatic manifestations depends on the demonstration of biochemical abnormalities due to these primary defects and compensatory hepatic overexpression of hepatic delta-aminolaevulinic acid synthase in the acute porphyrias. Consequently, haem precursors are synthesized in excess. The increased metabolites upstream of the enzymatic defect are excreted into urine and faeces. The diagnosis is based on their evaluation. Primary enzymatic or molecular analyses are noncontributary and may be misleading. Acute polysymptomatic exacerbations accompany a high excretory constellation of porphyrin precursors delta-aminolaevulinic acid and porphobilinogen. Homozygous or compound heterozygous variants of acute hepatic porphyrias may already manifest in childhood.

Objectives: To describe the biochemical and clinical features in hereditary coproporphyria (HCP)., Design and Method: Within the last 20 years, we investigated 53 patients (male:female = 1:2.5; age = 8-86 years) suffering from HCP. We describe the characteristic levels of urine, and fecal porphyrins and their precursors in hereditary coproporphyria and present the clinical features. Especially, we measured the coproporphyrin isomers I and III., Results and Conclusion: The group of hereditary coproporphyria patients exhibited a significantly higher (p<0.0001) excretion of urinary porphyrin precursors, delta-aminolevulinic acid (median = 84 micromol/24 h) and porphobilinogen (median = 39 micromol/24 h), as compared to controls (delta-aminolevulinic acid: 22 micromol/24 h, porphobilinogen: 3 micromol/24 h; median, n = 20). The median of coproporphyrin in urine (1315 nmol/24 h) and feces (1855 nmol/g) were enhanced 12- and 168-fold, as compared to healthy subjects (urinary coproporphyrin: 106 nmol/24 h, fecal coproporphyrin: 11 nmol/g; median, n = 20). During therapy on one female patient, with IV application of heme arginate, a considerable decline of porphyrin precursors and porphyrin excretion was observed. The examination of urinary and fecal coproporphyrin isomers I and III revealed an excessive elevation of the coproporphyrin isomer III of 87% in urine and 94% in feces, respectively (normal: urinary isomer III = 69-83% and fecal isomer III = 25-40%). In feces the increase of isomer III caused an inversion of the physiologic coproporphyrin isomer III:I ratio that could be recognized in all various stages in hereditary coproporphyria and in children. Acute attacks of hereditary coproporphyria are accompanied by an acute polysymptomatic clinical syndrome, and this is associated with high levels of urinary porphyrin precursors. On review of our patients, the highest percentage had abdominal pain (89%), followed by neurologic (33%), psychiatric (28%), cardiovascular (25%), and skin symptoms (14%).

alpha2-macroglobulin (alpha2M) is a broad-spectrum proteinase inhibitor and one of the major plasma proteins in humans. Activated phagocytes (especially granulocytes) generate large amounts of oxidants of the HOCI- and chloramine-type that release the mild nonradical, excited (light-emitting) oxidant singlet oxygen ((1)O2). These oxidants have been shown to inactivate several specific serine protease inhibitors in human blood [e.g., alpha1-antitrypsin or alpha2-antiplasmin (plasmin inhibitor)]. The studies reported here demonstrate that nonradical oxidants also inactivate plasmatic alpha2M. The effective dose for 50% inactivation (ED50) of plasmatic alpha2M is similar to that for plasmatic alpha2-antiplasmin. Chloramines are about 1,000-fold more effective than hydrogen peroxide (ED50)=0.75 micromol chloramine T/50 microl plasma). Serine protease-serine protease inhibitor complexes are resistant to oxidants. In contrast, here it is shown that alpha2-macroglobulin, even after binding to serine proteases is sensitive to oxidation, the captured protease is released from the protease/alpha2M complex. This is the first time that oxidative inactivation of a complexed (i.e., bound to a target protease) human protease inhibitor has be shown. The (1)O2 inhibitors methionine, cysteine, cystine, or ascorbate-in contrast to the oxy-radical scavengers mannitol, superoxide dismutase, or catalase-antagonize the chloramine/NaOCl-mediated inactivation of both uncomplexed and complexed alpha2M. Thus, the oxidant involved here is of nonradicalic nature and has reaction characteristics of (1)O2. For the inhibitory function, critical oxidizable methionines or the internal thiol-ester might be targets for (1)O2. Consequently, alpha2M can also be considered a carrier for proteases, since the alpha2M-complexed proteases regain full activity in an oxidative environment. In local areas of inflammation or thrombolysis, activated phagocytes could create microenvironments of uncontrolled protease activity by generation of (1)O2.

Activated polymorphonuclear leukocytes participate in hemostasis. These phagocytes generate up to 5 mmol/l of oxidants of the HOCl- and chloramine-type. The present study shows, for the first time, that physiological concentrations of NaOCl or chloramines act as anticoagulants in human plasma. Prothrombin time, activated partial thromboplastin time, and thrombin time at chloramine concentrations greater than 1 mmol/l are prolonged proportional to the oxidant concentration. Plasmatic coagulation factors sensible to oxidation are fibrinogen, factor V, factor VIII, and factor X with a 50% effective dose of 2-3 mmol/l NaOCl or taurine-chloramine. Chloramines or chloramine-like agents (e.g., chloramine T(R) or vancomycin) also inactivate platelet aggregation (in whole blood or platelet-rich plasma) at an 50% effective dose of about 1.0 mmol. This irreversible oxidation of the hemostasis components is inhibited by addition of methionine, cysteine, ascorbic acid, or azide in 10-fold molar excess prior to oxidation. The oxy-radical inhibitors mannitol, superoxide dismutase, or catalase do not antagonize the action of NaOCl or chloramines. Therefore, the oxidant here involved has reaction characteristics of singlet oxygen (1O(2)), a nonradical, excited (i.e., light-emitting) oxidant. The hemostasis factors sensible to oxidation might dispose of oxidizable, for their function critical, methionine or cysteine residues. In conclusion, blood coagulation factors I, V, VIII, X and thrombocytes are sensible to nonradical oxidants of activated phagocytes. Via 1O(2) generation, polymorphonuclear leukocytes can generate a local pericellular zone of anticoagulation. The data suggest that the cell signal 1O(2) in physiological amounts is an antithrombotic agent.

Cloning and expression of the defective gene for delta-aminolevulinate dehydratase (ALAD) from the second of 2 German patients with ALAD deficiency porphyria (ADP), who had been originally reported by Doss et al. in 1979, were performed. Cloning of cDNAs for the defective ALAD were performed using Epstein-Barr virus (EBV)-transformed lymphoblastoid cells of the proband, and nucleotide sequences of cloned cDNA were determined. Two separate mutations of ALAD cDNA were identified in each ALAD allele. One was G457A, termed "H1," resulting in V153M substitution, while the other was a deletion of 2 sequential bases at T(818) and C(819), termed "H2," resulting in a frame shift with a premature stop codon at the amino acid position of 294. Using allele-specific oligonucleotide hybridization, the mother of the proband was shown to have an H1 defect, while using genomic DNA analysis, the father was shown to have an H2 defect. Expression of H1 cDNA in Chinese hamster ovary cells produced an ALAD protein with only a partial activity (10.65% +/- 1.80% of the normal), while H2 cDNA encoded no significant protein. These data thus demonstrate that the proband was associated with 2 novel molecular defects of the ALAD gene, 1 in each allele, and account for the extremely low ALAD activity in his erythrocytes ( approximately 1% of normal).

Alcohol is a porphyrinogenic agent which may cause disturbances in porphyrin metabolism in healthy persons as well as biochemical and clinical manifestations of acute and chronic hepatic porphyrias. After excessive consumption of alcohol, a temporary, clinically asymptomatic secondary hepatic coproporphyrinuria is observable, which can become persistent in cases of alcohol-induced liver damage. Nowadays, the alcohol-liver-porphyrinuria syndrome is the first to be mentioned in secondary hepatic disturbances of porphyrin metabolism. Acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria and hereditary coproporphyria) are considered to be molecular regulatory diseases, in contrast to non-acute, chronic hepatic porphyria, clinically appearing as porphyria cutanea tarda (PCT). Porphyrins do not accumulate in the liver in acute porphyrias, whereas in chronic hepatic porphyrias they do. Thus, chronic hepatic porphyria is a porphyrin-accumulation disease, whereas acute hepatic porphyrias are haem-pathway-dysregulation diseases, characterized in general by induction of delta-aminolevulinic acid synthase in the liver and excessive stimulation of the pathway without storage of porphyrins in the liver. The clinical expression of acute hepatic porphyrias can be triggered by alcohol, because alcohol augments the inducibility of delta-aminolevulinic acid synthase. In chronic hepatic porphyrias, however, which are already associated with liver damage, alcohol potentiates the disturbance of the decarboxylation of uro- and heptacarboxyporphyrinogen, which is followed by a hepatic accumulation of uro- and heptacarboxyporphyrin and their sometimes extreme urinary excretion. Especially in persons with a genetic deficiency of uroporphyrinogen decarboxylase, but also in patients with the so-called sporadic variety of PCT, alcohol is able to transform an asymptomatic coproporphyrinuria into PCT. Alcohol has many biochemical and clinical effects on porphyrin and haem synthesis both in humans and laboratory animals. Ethanol suppresses the activity of porphobilinogen synthase (synonym: delta-aminolevulinic acid dehydratase), uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase, whereas it induces the first and rate-limiting enzyme in the pathway, delta-aminolevulinic acid synthase and also porphobilinogen deaminase. Therefore, teetotalism is a therapeutically and prophylactically important measure in all types of hepatic porphyrias.

Hemostasis, the system of generation and degradation of thrombi, consists of coagulation and fibrinolysis. Whereas global assays to study coagulation have existed for many years, there has been no simple, rapid, and economic routine test for the plasmatic fibrinolysis parameters plasminogen activator inhibitor-1, alpha2-antiplasmin, plasminogen, and aprotinin. Here a fast functional global assay for these plasmatic fibrinolytic parameters is presented. However, the present assay is not sensitive to physiological concentrations of prourokinase or tissue-type plasminogen activator. The following assay conditions have been found to be optimal: 50 microL of citrated plasma is incubated with 50 microL of 10 IU urinary-type plasminogen activator (urokinase)/mL, 1.1 mmol/L tranexamic acid, 1% polygelin, 0.1% Triton X-100, phosphate-buffered saline, pH 7.4, for 20 min at 37 degrees C (plasmin generation phase). Then 50 microL of 3 mmol/L HD-Nva-CHA-Lys-pNA, 1.05 mol/L KCl is added, and deltaA (405 nm)/10 min (37 degrees C) is determined, by using a microtiterplate reader (plasmin detection phase). The results are calibrated against pooled normal plasma (100% plasmatic fibrinolytic parameters activity). The intra- and interassay coefficients of variation have been found to be less than 5%. The detection limit (sensitivity) of the functional fibrinolysis assay is 5 % of the normal plasmatic fibrinolysis parameters activity. The normal plasmatic fibrinolysis parameters activity is 100%, sigma = 25%. The plasmatic fibrinolysis parameters activity correlates negatively (r = -0.684) with the plasminogen activator inhibitor-1 activity of patient samples. The plasmatic fibrinolysis parameters assay is a simple, rapid, and economic functional test for several clinical relevant fibrinolysis parameters.

Acute intermittent porphyria (AIP) is a low-penetrant, autosomal dominant disorder caused by decreased activity of hydroxymethylbilane synthase (HMBS; MIM 176 000), the third enzyme in the heme biosynthetic pathway. We report the first molecular analysis of HMBS gene mutations in classical AIP patients of German origin. The HMBS gene of 5 German AIP patients was analysed by DGGE-screening and direct sequencing of amplified genomic DNA. Five different mutations including four novel mutations were found. Three of them are single base substitutions that affected exon 3 (R16C), exon 10 (V202L), and intron 13 (T to A, IVS13+2) The two remaining mutations are frameshifts which produce a stop codon (del GA in exon 6 and insA in exon 14). These mutations are likely to be responsible for the decrease in HMBS activity found in both erythrocytes and non-erythroid cell lines (lymphocytes). Our results demonstrate the allelic heterogeneity of HMBS mutations in AIP patients of German origin., (Copyright 1999 Academic Press.)

Alcohol has an porphyrinogenic action and can cause a disturbance of porphyrin metabolism in healthy people as well as lead to a biochemical and clinical manifestation of acute and chronic hepatic porphyrias, especially acute intermittent porphyria and porphyria cutanea tarda. After excessive consumption of alcohol a temporary, clinically asymptomatic secondary hepatic coproporphyrinuria in man can be observed, which can become persistent in cases of alcohol-induced liver damage. Nowadays alcohol-liver-porphyrinuria syndrome is the first to be mentioned in secondary hepatic disturbances of porphyrin metabolism. In people with a genetic lack of uroporphyrinogen-decarboxylase alcohol is able to transform an asymptomatic coproporphyrinuria into a chronic hepatic porphyria or porphyria cutanea tarda. From experimental and clinical studies the conclusion can be drawn that alcohol inhibits the enzymes delta-aminolevulinic-acid-dehydratase (synonym: porphobilinogen-synthase), uroporphyrinogen-decarboxylase and coproporphyrinogen-oxidase and induces delta-aminolevulinic-acid-synthase in the liver. Abstinence of alcohol is a therapeutically and prophylactically important measurement in all types of hepatic porphyrias. For clinical experience follows that in cases with chronic consumption of alcohol, fatty liver, alcohol induced hepatitis and liver cirrhosis porphyrin studies in urine should be made to notice a hepatic porphyria in the latent phase very early. When dealing with abdominal and cutaneous symptoms in clinical context with consumption of alcohol one has to exclude hepatic porphyria differential diagnostically.

The urinary and fecal distribution and the relative proportions of the four coproporphyrin (copro) isomers I-IV were analysed in 20 healthy subjects and in patients suffering from one of the seven common types of hepatic or erythropoietic hereditary porphyrias. The ratios of copro isomers I-IV were analyzed by ion-pair high-performance liquid chromatography (HPLC). Observations showed significantly increased proportions of fecal copro isomer I and decreased proportions of copro isomers III, II and IV in erythropoietic porphyrias. In acute hepatic porphyrias the excretion of fecal copro isomer III is dominant (isomer III = 58.4+/-24.0%; x+/-S.D.) and significantly higher (P < 0.001) than in erythropoietic porphyrias (isomer III = 15.3+/-7.7%; x+/-S.D.) and chronic hepatic porphyrias (isomer III = 25.8+/-7.6%; x+/-S.D.). The increased proportions of fecal copro isomer III proved to be important for the diagnosis of hereditary coproporphyria and porphyria variegata independent of the clinical phase existing. These last two acute hepatic porphyrias also showed markedly elevated percentages of the fecal atypical isomers II and IV. In urine significantly decreased proportions of copro isomer I in acute hepatic porphyrias (isomer I = 12.3+/-6.0%; x+/-S.D.) could be observed as compared with non-acute porphyrias (isomer I = 53.7+/-15.2%; x+/-S.D.). Conversely, the proportion of urinary copro isomer III was significantly higher in acute hepatic porphyrias (isomer III= 81.4+/-6.4%; x+/-S.D.). As expected, the greatest amounts of urinary copro isomer I were found in congenital erythropoietic porphyria (isomer I =92.0+/-3.3; x+/-S.D.) and protoporphyria with hepatobiliary complications (isomer I = 81.3+/-10.7; x+/-S.D.). The atypical urinary copro isomers I1 and IV were detected in all types of porphyrias ranging from 0.1 to 11.5%. The combined amounts of copro isomers II and IV show a significantly decreased percentage in congenital erythropoietic porphyria as compared with all other types of hereditary porphyrias. In conclusion, we demonstrate that the characteristic pattern of the copro isomer constellations I-IV in the various types of porphyrias are of differential diagnostic importance. The inversion of the I to III ratio in feces in hereditary coproporphyria and porphyria variegata allows the recognition of gene carriers.

Objectives: Investigation of the metabolism of the four urinary coproporphyrin isomers I-IV in the extremely rare 5-aminolevulinic acid dehydratase (ALAD) deficiency porphyria (syn.: Doss porphyria), in acute lead intoxication, and after oral 5-aminolevulinic acid (ALA) loading., Design and Methods: We analyzed the excretion of total urinary coproporphyrins and the composition of the respective isomers I-IV with ion-pair HPLC methods in these conditions., Results: The concentration of total coproporphyrins was about 30-fold increased in patients with ALAD deficiency porphyria and acute lead intoxication as compared with controls. In addition, the proportion of coproporphyrin III as well as that of the atypical isomers II and IV were significantly elevated at the expense of isomer I. After oral ALA administration to normal volunteers, a 10- to 15-fold increase in the maximal concentration of total urinary coproporphyrins was observed within 12 to 24 h. Urinary levels were back to normal after another 24 h. The excretion pattern of the individual urinary coproporphyrin isomers I-IV after ALA ingestion revealed a dynamic process: initially isomer III was preferentially formed, followed by a 3-fold increase of isomers II and IV via non-enzymatic rearrangement of isomer III, and finally normalization of all four isomers occurred within 48 h., Conclusions: These results demonstrate that oral ALA loading can be used as an in vivo model to study the metabolism of the four urinary coproporphyrin isomers I-IV especially in ALAD deficiency porphyria and in acute lead poisoning.

Congenital erythropoietic porphyria (CEP), a rare autosomal recessive inborn error of heme biosynthesis, results from reduced activity of uroporphyrinogen III synthase (URO-III-S) leading to an excessive production and accumulation of porphyrins. Various clinical and biochemical observations point to a relationship between degree of disease expression and metabolic disturbance. We investigated 20 patients with early onset of clinical symptoms of CEP and, additionally, the relatives of six patients. CEP was confirmed by porphyrinemia and porphyrinuria with dominance of uroporphyrin and its isomer I. The investigation of the immunological nature of the defective URO-III-S gene from unrelated patients with unknown mutations was possible thanks to an antibody against the human enzyme. URO-III-S concentration in erythrocytes was determined by ELISA. No signal was achieved when assaying nonimmune serum by ELISA, whereas there was a positive reaction with the serum after immunisation. Furthermore, specificity of immune sera is demonstrated by immunoprecipitation of URO-III-S activity which caused a 33% reduction of enzyme activity. Normal levels of immunoreactive enzyme protein 100+/-10% of control (x +/- SD, n = 12) with a reduced specific activity 15+/-8.5% (x +/- SD, n = 12) were found in erythrocytes from all patients, with the exception of a girl with a remarkably high enzyme concentration of 149% of controls and a very low specific activity of 4%. In consequence, all patients had cross-reacting immunological material (CRIM)-positive mutations. CRIM-ratios varied between 3.2 and 24.5. The CRIM-positive nature of the gene defect indicated that the mutations altered the activity of URO-III-S. The different CRIM ratios implied the presence of various mutations, which is further evidence for the heterogeneity in the genetic defect found in CEP. URO-III-S activity was determined in erythrocyte lysates by a coupled enzyme assay. Erythrocyte URO-III-S activities of patients were reduced to 4-33% of the normal value (1.72+/-0.14 pkat/mg protein). An increase of urinary coproporphyrin isomer I (40-61%, norm = 17-31%) and a halved URO-III-S activity can serve as a biochemical test for asymptomatic heterozygous gene carriers of CEP.

5-Aminolevulinic acid dehydratase (ALAD) activity in two patients with compound heterozygous 5-aminolevulinic acid dehydratase deficiency porphyria was studied over the last 20 years. The patients' enzyme activity was <10% from 1977 to 1997. An acute crisis in each patient was successfully treated by infusion of glucose and heme arginate. After this therapy both urinary 5-aminolevulinic acid (ALA) and total porphyrins were diminished to 65% in patient B. In patient H, ALA was decreased to 80%, and total porphyrins were reduced to 15% after treatment with heme arginate and glucose. The patients remained free of symptoms after this therapy. Family studies of patient B showed cross-reactive immunological material (CRIM), in which the maternal mutation is CRIM(+), whereas the paternal mutation is CRIM(-). Incubation of erythrocyte lysates with ALA decreased porphyrin formation, whereas incubation with porphobilinogen produced porphyrin concentrations within reference values in both patients, confirming that ALAD activity is rate-limiting in these cells.

The oxidative decarboxylation of coproporphyrinogen III catalysed by an oxygen-dependent oxidase (HemF) and an oxygen-independent dehydrogenase (HemN) is one of the key regulatory points of haem biosynthesis in Pseudomonas aeruginosa. To investigate the oxygen-dependent regulation of hemF and hemN, the corresponding genes were cloned from the P. aeruginosa chromosome. Recognition sequences for the Fnr-type transcriptional regulator Anr were detected -44.5 bp from the 5' end of the hemF mRNA transcript and at an optimal distance of -41.5 bp with respect to the transcriptional start of hemN. An approximately 10-fold anaerobic induction of hemN gene expression was mediated by the dual action of Anr and a second Fnr-type regulator, Dnr. Regulation by both proteins required the Anr recognition sequence. Surprisingly, aerobic expression of hemN was dependent only on Anr. An anr mutant did not contain detectable amounts of hemN mRNA and accumulated coproporphyrin III both aerobically and anaerobically, indicating the importance of HemN for aerobic and anaerobic haem formation. Mutation of hemN and hemF did not abolish aerobic or anaerobic growth, indicating the existence of an additional HemN-type enzyme, which was termed HemZ. Expression of hemF was induced approximately 20-fold during anaerobic growth and, as was found for hemN, both Anr and Dnr were required for anaerobic induction. Paradoxically, oxygen is necessary for HemF catalysis, suggesting the existence of an additional physiological function for the P. aeruginosa HemF protein.

A quarter of patients with erythropoietic protoporphyria develop mild to severe cholestatic liver disease. The determination of early indicators of hepatobiliary involvement are of pivotal importance to select patients for choleretic therapy. Porphyrin parameters were studied during ursodeoxycholic acid treatment in eight patients with protoporphyrin-associated liver disease and eight patients with liver failure before and after liver transplantation. The patients with intrahepatic cholestasis exhibited excessive protoporphyrinemia (27 mumol/l) compared with controls (normal < 0.64 mumol/l). Fecal protoporphyrin excretion decreased in patients with deterioration of liver function, whereas urinary coproporphyrin increased up to 2290 nmol/24 h (normal < 119 nmol/24 h). Coproporphyrin isomer I proportion increased to 71 +/- 10% (mean +/- SD, n = 8) in patients with terminal liver failure (normal < 31%). During therapy with ursodeoxycholic acid biochemical improvement occurred but without clinical remission in most cases. Eight patients underwent liver transplantation between 1987 and 1997. One patient died of liver failure. Two transplant recipients are in a good condition since 8 and 9 years, respectively. All explanted livers revealed micronodular cirrhosis and high protoporphyrin levels of about 25,000-fold (mean, n = 3). Immediately after liver transplantation protoporphyrin in erythrocytes decreased to 46-96% of pre-operative values. Coproporphyrin remained moderately elevated due to post-operative cholestasis. A post-operative rise in fecal protoporphyrin elimination reflected sufficient biliary clearence of protoporphyrin by the transplant. In conclusion, moderate coproporphyrinuria with isomer I is the earliest sign of liver complications in erythropoietic protoporphyria. Progression of protoporphyrin induced toxic liver injury is indicated by excessive protoporphyrinemia and coproporphyrinuria with an isomer I proportion > 71 +/- 10%, and reduction of fecal protoporphyrin excretion. Results suggest that therapy of intrahepatic cholestasis with ursodeoxycholic acid is only effective in the initial stages of liver disease in erythropoietic protoporphyria. In patients with severe cholestatic hepatic failure, liver transplantation is the treatment of choice.

Various clinical and biochemical observations point to a relationship between degree of disease expression and metabolic disturbance in autosomal recessive congenital erythropoietic porphyria (Günther's disease). Although the clinical manifestations have been well described since Günther's fundamental observations, an interdependence between disease severity and porphyrin excess has yet to be elucidated. We investigated porphyrin metabolism in nine Indian patients suffering from the characteristic clinical symptoms: skin photosensitivity, red-colored urine as a sign of extremely elevated porphyrinuria and mild to severe hemolytic anemia. Porphyrins in urine, feces and blood were analysed by HPTLC and HPLC in conjunction with spectrophotometry and spectrofluorometry. Uroporphyrinogen III synthase activities in red blood cells were determined using a coupled-enzyme assay. Biochemical studies revealed varying degrees of porphyrinuria with total urinary porphyrins between 23 and 102 mumol/24 h (normal < 0.2 mumol/24 h) and uroporphyrin predominance. Urinary and fecal coproporphyrin isomer I were markedly elevated to 87-97% and 81-93% (normal < 31%, < 75%), respectively. Overproduction of porphyrins led to a considerable porphyrinemia with mainly copro- and protoporphyrin. A hitherto undescribed fecal porphyrin pattern with increased protoporphyrin levels was found in three patients. This atypical finding was probably related to severe hemolysis since protoporphyrin can be excreted only via the liver with bile in the feces. High porphyrin levels in urine, feces and blood were associated with worse cutaneous symptoms. Activities of uroporphyrinogen III synthase in red blood cell lysates were decreased to between 9% and 30% of controls. Patients showed increased porphobilinogen deaminase activities, up to 190% of control. Deficiency of uroporphyrinogen III synthase activity was reflected by inversion of the relationship between and isomer III leading to dominance of isomer I. Elevation of porphobilinogen deaminase activities is related to hemolysis and, additionally, to regulatory compensation for the enzyme deficiency. Variations in both the severity of photosensitivity and the enhancement of porphyrin production and excretion indicate the molecular heterogeneity of this disease. These findings suggest a close relationship between the metabolic disturbance reflected by porphyrin excess and the severity of disease expression.

Patients with AIP can be subdivided into three different groups concerning their PBGD activity in erythrocytes: The first of which has lowered, the second overlapping and the third normal PBGD activity. Out of 385 AIP patients 87% had lowered, 8% had overlapping and 5% had normal PBGD activity. Gene carriers of AIP having slight, moderate or high metabolic aberrations of excretion parameters are recognized by analysis of urinary haem precursors and faecal porphyrins. The haem precursor excretion of the groups with lowered, overlapping and normal PBGD activity in erythrocytes compared to each other is not significantly different but differs significantly (p < 0.001) from the normal values. One individual suffering from AIP was detected in a family with normal PBGD. Lymphocytes can be stored in liquid nitrogen for 3 months without loss of PBGD activity. Specific PBGD activity in lymphocytes is 5% from specific PBGD activity in erythrocytes. In AIP patients with lowered specific PBGD activity in erythrocytes specific PBGD activity is lowered to the same extent in their lymphocytes.

A hitherto undescribed dual deficiency of uroporphyrinogen III synthase and uroporphyrinogen decarboxylase was observed in the erythrocytes in a 14 year-old patient who had presented with congenital erythropoietic porphyria since early childhood. Whereas congenital erythropoietic porphyria was metabolically and clinically overt, a hereditary deficiency of uroporphyrinogen decarboxylase was confirmed by family study. The uroporphyrinogen III synthase activity of the propositus was decreased to 26% of the control while his asymptomatic family members had activities between 53-65% of the control. Additionally, the uroporphyrinogen decarboxylase activity was 55-66% of the control in the patient and his family. Family investigations have shown that the two disorders do not consistently segregate together. Although urinary porphyrin excretions of relatives were in the physiological range, the proportion of coproporphyrin isomer I showed a relative increase, which can serve as a biochemical indicator for heterozygous uroporphyrinogen III synthase gene carriers.

Patients with acute intermittent porphyria can be subdivided into three groups, according to the porphobilinogen deaminase activity in their erythrocytes. The first group has lowered, the second overlapping and the third normal porphobilinogen deaminase activity. Of 385 acute intermittent porphyria patients 5% had normal porphobilinogen deaminase activity. Gene carriers of acute intermittent porphyria, which have normal porphobilinogen deaminase activity but display slight, moderate or high aberrations of excretion, are recognized by analysis of urinary haem precursors and faecal porphyrins. Six individuals suffering from acute intermittent porphyria were detected in three families with normal porphobilinogen deaminase. There were no differences in the latent and clinical phases of acute intermittent porphyria between patients with lowered and those with normal porphobilinogen deaminase. One female with normal activity in erythrocytes, in which the porphyria disease process is triggered by barbiturates and carbamazepine, is presented. After therapy with high doses of glucose and omission of inducing agents, this woman was free of symptoms, and the excretion of different urinary porphyrin precursors and porphyrins decreased by between 65 and 93%.

In a 58-year-old woman with erythropoietic protoporphyria, asymptomatic liver involvement had been diagnosed 12 years earlier. For more than 20 years the patient had been known to have symptomatic gallstones. A mild polyneuropathy of the lower limbs had been diagnosed several years ago. In December 1992, she presented with colicky upper abdominal pain, dyspepsia and mild jaundice. Diagnosis of beginning cholestasis in erythrohepatic protoporphyria and coincidental choledocholithiasis was made. A causal relation between choledocholithiasis and deterioration of liver function was assumed. Endoscopic extraction of the bile duct stones, however, could not prevent the development of terminal hepatic failure. Biochemically, an excessive protoporphyrinemia and coproporphyrinuria were found. Five weeks after presentation, the patient underwent orthotopic liver transplantation. Immediately after the operation she developed a severe axonal neuropathy with cranial nerve involvement. One year after transplantation, her general condition has markedly improved, but there is still a disabling polyneuropathy. Recently, there were single reports on patients with very similar neurological symptoms following liver transplantation in erythropoietic protoporphyria. This case supports the assumption of a distinct protoporphyrin-induced neural damage in severe hepatic failure.

The relative proportions of the four coproporphyrin isomers I-IV were analysed in faeces of 20 healthy subjects and 60 patients suffering from one of the seven common types of hepatic or erythropoietic hereditary porphyrias. A newly developed, reliable method for sample preparation was applied, using reversed-phase thin layer chromatography for the isolation of naturally occurring coproporphyrin free carboxylic acids. Accurate separation and quantitation of the individual isomers I-IV were achieved with the help of ion-pair high-performance liquid chromatography. The four coproporphyrin isomers I-IV were positively identified by on-line scanning of their fluorescence spectra in the emission and excitation modes. Recovery rates with this new analytical procedure were between 90 and 100%, and coefficients of variation varied between 0.8 and 5.7% (N = 7). Diagnostically important findings were greatly increased proportions of isomer I and decreased proportions of isomers III, II and IV in erythropoietic porphyrias, such as congenital erythropoietic porphyria and protoporphyria. Significantly increased proportions of isomers III, II and IV, on the other hand, were observed in acute hepatic porphyrias, e.g. acute intermittent porphyria and porphobilinogen synthase deficiency porphyria, as compared with porphyria cutanea tarda (p < 0.005 and p < 0.03, respectively). Inversion of the faecal coproporphyrin III to I ratios and markedly elevated percentages of the atypical isomers II and IV were important diagnostic markers for variegate porphyria and hereditary coproporphyria. The highest proportions of isomer III were found in hereditary coproporphyria, where the amount of the isomers II and IV exceeded that of isomer I. Asymptomatic carriers of the relevant gene defect in families with hereditary coproporphyria could be detected by an increased faecal coproporphyrin III to I ratio. Our results clearly demonstrate the potential of faecal coproporphyrin I-IV isomer ratios for the diagnosis and differential diagnosis of hereditary porphyrias.