The clinical management of porphyria cutanea tarda: An update.

Porphyria cutanea tarda (PCT) is the commonest of the porphyrias (Semin Liver Dis 1998;18:67). It often occurs secondary to an underlying internal disorder, has significant impacts on liver health and longevity, and is a treatable disease. Thus, for the clinician, recognising the disease to make the correct diagnosis, identifying causative underlying diseases, and treating the porphyria and its complications, are crucial. Although reviews on the management of PCT have been written, there have recently been significant advances in the understanding of the factors predisposing to the disease, and of its wider health impacts. This review aims to help the clinician to diagnose and manage patients with PCT, with an emphasis on the impact of recent advances on clinical management.

Harderoporphyria: Case of lifelong photosensitivity associated with compound heterozygous coproporphyrinogen oxidase (CPOX) mutations.

A 78-year-old man with a history of neonatal anemia and jaundice and life-long photosensitivity was found to have harderoporphyria, as evidenced by increased porphyrins in urine, plasma, erythrocytes and feces including large amounts of harderoporphyrin in feces and erythrocytes. Two previously undescribed coproporphyrinogen oxidase (CPOX) mutations were identified, including a deletion of four amino acids in a region of the enzyme mutated in 7 of the 8 previously reported cases. This case increases the molecular heterogeneity of this rare porphyria, and illustrates that it should be considered as a cause of chronic photosensitivity and porphyrin elevation at any age.

Hepatoerythropoietic porphyria due to a novel mutation in the uroporphyrinogen decarboxylase gene.

BACKGROUND: Hepatoerythropoietic porphyria (HEP) is a rare form of porphyria that results from a deficiency of uroporphyrinogen decarboxylase (UROD). The disease is caused by homoallelism or heteroallelism for mutations in the UROD gene. OBJECTIVE: To study a 19-year-old woman from Equatorial Guinea, one of the few cases of HEP of African descent and to characterize a new mutation causing HEP. METHODS: Excretion of porphyrins and residual UROD activity in erythrocytes were measured and compared with those of other patients with HEP. The UROD gene of the proband was sequenced and a new mutation identified. The recombinant UROD protein was purified and assayed for enzymatic activity. The change of amino acid mapped to the UROD protein and the functional consequences were predicted. RESULTS: The patient presented a novel homozygous G170D missense mutation. Porphyrin excretion showed an atypical pattern in stool with a high pentaporphyrin III to isocoproporphyrin ratio. Erythrocyte UROD activity was 42% of normal and higher than the activity found in patients with HEP with a G281E mutation. The recombinant UROD protein showed a relative activity of 17% and 60% of wild-type to uroporphyrinogen I and III respectively. Molecular modelling showed that glycine 170 is located on the dimer interface of UROD, in a loop containing residues 167-172 that are critical for optimal enzymatic activity and that the carboxyl side chain from aspartic acid is predicted to cause negative interactions between the protein and the substrate. CONCLUSIONS: The results emphasize the complex relationship between the genetic defects and the biochemical phenotype in homozygous porphyria.

Structural and kinetic characterization of mutant human uroporphyrinogen decarboxylases.

Porphyria cutanea tarda (PCT) is caused by inhibition of uroporphyrinogen decarboxylase (URO-D) activity in hepatocytes. Subnormal URO-D activity results in accumulation and urinary excretion of uroporphyrin and heptacarboxyl porphyrin. Heterozygosity for mutations in the URO-D gene is found in the familial form of PCT (F-PCT). Over 70 mutations of URO-D have been described but very few have been characterized structurally. Here we characterize 3 mutations in the URO-D gene found in patients with F-PCT, G318R, K297N, and D306Y. Expression of the D306Y mutation results in an insoluble recombinant protein. G318R and K297N have little effect on the structure or activity of recombinant URO-D, but the proteins display reduced stability in vitro.

Longitudinal study of a mouse model of familial porphyria cutanea tarda.

Most rodent models of porphyria cutanea tarda (PCT) share in common the administration of iron and agents that induce transcription of cytochrome P450s. Dissection of changes related to porphyrin accumulation required generation of a genetic model free from exogenous precipitants. Mice heterozygous for a null Urod mutation and homozygous for null Hfe alleles spontaneously develop major increases in hepatic and urinary porphyrins several months after weaning but the high % uroporphyrin signature of PCT is established earlier, before total hepatic and urinary porphyrins rise. Total porphyrin levels eventually plateau at higher levels in females than in males. Porphyrinogens were the dominant tetrapyrroles accumulating in hepatocytes. Hepatic Urod activity is markedly reduced but total hepatic heme content does not diminish. Microsomal heme, however, is reduced and in vitro metabolism of prototype substrates showed that some but not all cytochrome P450 activities are reduced. High hepatic levels of uroporphyrinogen are also associated with increased glutathione S-transferase activity and elevated mRNA of 2 transporters, Abcc1 and Abcc4. This murine model of familial PCT affords the opportunity to study changes in porphyrinogen and porphyrin accumulation and transport in the absence of exogenous factors that alter P450 activity and transmembrane transporters.

Identification and characterization of novel uroporphyrinogen decarboxylase gene mutations in a large series of porphyria cutanea tarda patients and relatives.

Porphyria cutanea tarda (PCT) arises from decreased hepatic activity of uroporphyrinogen decarboxylase (UROD). Both genetic and environmental factors interplay in the precipitation of clinically overt PCT, but these factors may vary between different geographic areas. Decreased activity of UROD in erythrocytes was used to identify patients with UROD mutations among a group of 130 Spanish PCT patients. Nineteen patients (14.6%) were found to harbor a mutation in the UROD gene. Eight mutations were novel: M1I, 5del10, A22V, D79N, F84I, Q116X, T141I and Y182C. Five others were previously described: F46L, V134Q, R142Q, P150L and E218G. The new missense mutations and P150L were expressed in Escherichia coli. D79N and P150L resulted in proteins that were localized to inclusion bodies. The other mutations produced recombinant proteins that were purified and showed reduced activity (range: 2.3-73.2% of wild type). These single amino acid changes were predicted to produce complex structural alterations and/or reduced stability of the enzyme. Screening of relatives of the probands showed that 37.5% of mutation carriers demonstrated increased urinary porphyrins. This study emphasizes the role of UROD mutations as a strong risk factor for PCT even in areas where environmental factors (hepatitis C virus) have been shown to be highly associated with the disease.

Prenatal detection and evaluation of an extralobar pulmonary sequestration in the posterior mediastinum.

Extralobar pulmonary sequestration (EPS) is a rare developmental anomaly with aberrant nonfunctioning parenchymal tissue, associated with an increased risk of perinatal morbidity and, rarely, mortality owing to possible neonatal respiratory distress. In most cases supernumerary lobes are detected as isolated intra- or extrapleural lesions with independent systemic arterial blood supply. We report an atypical case of prenatal detection and perinatal outcome of a mediastinal EPS.

Structural diversity in metal ion chelation and the structure of uroporphyrinogen III synthase.

All tetrapyrroles are synthesized through a branched pathway, and although each tetrapyrrole receives unique modifications around the ring periphery, they all share the unifying feature of a central metal ion. Each pathway maintains a unique metal ion chelatase, and several tertiary structures have been determined, including those of the protoporphyrin ferrochelatase from both human and Bacillus subtilus, and the cobalt chelatase CbiK. These enzymes exhibit strong structural similarity and appear to function by a similar mechanism. Met8p, from Saccharomyces cerevisiae, catalyses ferrochelation during the synthesis of sirohaem, and the structure reveals a novel chelatase architecture whereby both ferrochelation and NAD(+)-dependent dehydrogenation take place in a single bifunctional active site. Asp-141 appears to participate in both catalytic reactions. The final common biosynthetic step in tetrapyrrole biosynthesis is the generation of uroporphyrinogen by uroporphyrinogen III synthase, whereby the D ring of hydroxymethylbilane is flipped during ring closure to generate the asymmetrical structure of uroporphyrinogen III. The recently derived structure of uroporphyrinogen III synthase reveals a bi-lobed structure in which the active site lies between the domains.

Increased non-transferrin bound iron in plasma-depleted SAG-M red blood cell units.

BACKGROUND AND OBJECTIVES: Non-transferrin bound iron (NTBI) is associated with increased morbidity in a number of transfusion-dependent disease states such as the severe haemoglobinopathies. We hypothesized that this may be related to excess NTBI present in plasma-depleted red blood cell units that are free of clear haemolysis. MATERIALS AND METHODS: The level of NTBI was determined using the bleomycin assay in samples from 20 stored plasma-depleted red cell units, at approximate 5-day intervals up to day 33 after donation. Forty units of fresh-frozen plasma (FFP) and 40 units of platelet concentrates were used as negative controls, and samples from 12 units of FFP were also serially assessed. RESULTS: Median [interquartile range (IQR)] NTBI was 0 microm (0-0.35) in samples taken from units 3-10 days after donation. Thereafter, the levels of NTBI increased, becoming significant (median 3.05; IQR: 0.05-6.7 microm) 17-22 days after donation. After 30 days, NTBI was detectable in all red cell units. NTBI was undetectable in platelet concentrates and FFP. CONCLUSIONS: Increased levels of NTBI become detectable 17-22 days after donation and increase further with storage time. This excess NTBI may promote bacterial infection in iron-loaded individuals.

Reduced vitamin E antioxidant capacity in sickle cell disease is related to transfusion status but not to sickle crisis.

In homozygous sickle cell disease (SCD), decreased serum Vitamin E is present. Excessive transfusions may lead to iron overload. We hypothesised a relationship between the two and found that Vitamin E type antioxidant capacity was significantly lower in 30 SCD patients than in 30 age- and sex-matched controls (P < 0.001). Antioxidant capacity was lower in 10 transfused patients compared with 20 non-transfused patients (P < 0.001). Transfusional iron overload in SCD may increase the potential for oxidative damage, and low antioxidant capacity may compound this effect.

Short-term treatment with novel ribonucleotide reductase inhibitors Trimidox and Didox reverses late-stage murine retrovirus-induced lymphoproliferative disease with less bone marrow toxicity than hydroxyurea.

We evaluated the ability of a short course of treatment with the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU) and two novel RR inhibitors Trimidox (TX) and Didox (DX) to influence late-stage murine retrovirus-induced lymphoproliferative disease. LPBM5 murine leukaemia virus retrovirus-infected mice were treated daily with HU, TX or DX for 4 weeks, beginning 9 weeks post-infection, after development of immunodeficiency and lymphoproliferative disease. Drug effects on disease progression were determined by evaluating spleen weight and histology. Effects on haematopoiesis were determined by measuring peripheral blood indices (white blood cells and haematocrit) and assay of femur cellularity and femoral and splenic content of colony-forming units granulocyte-macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E). HU, TX and DX partially reversed late-stage retrovirus-induced disease, resulting in spleen weights significantly below pre-treatment values. Spleen histology was also improved by RR inhibitor treatment (DX>TX>HU). However, as expected, HU was significantly myelosuppressive, inducing a reduction in peripheral indices associated with depletion of femoral CFU-GM and BFU-E. In contrast, although TX and DX were moderately myelosuppressive, both drugs were significantly better tolerated than HU. In summary, short-term treatment in late-stage murine retroviral disease with HU, TX or DX induced dramatic reversal of disease pathophysiology. However, the novel RR inhibitors TX and DX had more effective activity and significantly less bone marrow toxicity than HU.

Crystal structure of human uroporphyrinogen III synthase.

Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F(430) and vitamin B(12). U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 A resolution. The protein folds into two alpha/beta domains connected by a beta-ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.

Functional consequences of naturally occurring mutations in human uroporphyrinogen decarboxylase.

Functional consequences of 12 mutations-10 missense, 1 splicing defect, and 1 frameshift mutation-were characterized in the uroporphyrinogen decarboxylase (URO-D) gene found in Utah pedigrees with familial porphyria cutanea tarda (F-PCT). All but one mutation altered a restriction site in the URO-D gene, permitting identification of affected relatives using a combination of polymerase chain reaction and restriction enzyme digestion. In a bacterial expression system, 3 of the missense mutants were found in inclusion bodies, but 7 were expressed as soluble proteins. Enzymatic activity of soluble, recombinant mutant URO-D genes ranged from 29% to 94% of normal. URO-D mRNA levels in Epstein-Barr-virus transformed cells derived from patients were normal (with the exception of the frameshift mutation) even though protein levels were lower than normal, suggesting that missense mutations generally cause unstable URO-Ds in vivo. The crystal structures of 3 mutant URO-Ds were solved, and the structural consequences of the mutations were defined. All missense mutations reported here and by others were mapped to the crystal structure of URO-D, and structural effects were predicted. These studies define structural and functional consequences of URO-D mutations occurring in patients with F-PCT.

Severe hypothermia with cardiac arrest: complete neurologic recovery in a 4-year-old child.

A 4-year-old girl was lost for 17 hours in a snowstorm. Upon discovery, her core temperature was 72 degrees F (22 degrees C). While undergoing endotracheal intubation in the emergency department, she experienced sudden ventricular fibrillation and cardiac arrest. Closed chest cardiopulmonary resuscitation (CPR) was instituted, and standard rewarming measures were begun. Despite this, neither core temperature, nor the patient's arrhythmia, changed. An infraumbilical "mini-laparotomy" incision was made, with placement of a large silicone peritoneal dialysis catheter into the abdomen. This was then connected to a rapid infuser device, with the delivery of 1L of warmed, normal saline every 90 seconds. The core temperature reached 29 degrees C in 15 minutes, and a palpable pulse was detected. Lavage was continued until core temperature reached 34 degrees C, at which time transport to the pediatric intensive care unit was arranged. She was extubated the next day and discharged home, on the fourth hospital day, with apparent complete neurologic recovery. This is the first report of the successful use of rapid high-volume peritoneal lavage for the treatment of accidental severe hypothermia in a pediatric patient.

Vitamin E correlates inversely with non-transferrin-bound iron in sickle cell disease.

Decreased serum vitamin E levels are found in homozygous sickle cell disease (SCD). Excessive transfusions may lead high non-transferrin-bound iron (NTBI). Hypothesizing a relationship between the two, vitamin E (measured using high performance liquid chromatography) was significantly lower in 30 SCD patients than in 30 age-/sex-matched controls (P < 0.001), but NTBI (bleomycin assay) was higher (P < 0.001). Vitamin E was lower in 10 transfused patients than in 20 non-transfused patients (P < 0.001) with a significant inverse correlation between the NTBI and vitamin E (r = -0.58, P < 0.001). NTBI associated with iron overload in SCD may increase the potential for oxidative damage and low vitamin E activity may compound this effect.

A mouse model of familial porphyria cutanea tarda.

Approximately one-third of patients with porphyria cutanea tarda (PCT), the most common porphyria in humans, inherit a single mutant allele of the uroporphyrinogen decarboxylase (URO-D) gene. PCT associated with URO-D mutations is designated familial PCT. The phenotype is characterized by a photosensitive dermatosis with hepatic accumulation and urinary excretion of uroporphyrin and hepta-carboxylic porphyrins. Most heterozygotes for URO-D mutations do not express a porphyric phenotype unless hepatic siderosis is present. Hemochromatosis gene (HFE) mutations are frequently found when the phenotype is expressed. We used homologous recombination to disrupt one allele of murine URO-D. URO-D(+/-) mice had half-wild type (wt) URO-D protein and enzymatic activity in all tissues but did not accumulate hepatic porphyrins, indicating that half-normal URO-D activity is not rate limiting. When URO-D(+/-) mice were injected with iron-dextran and given drinking water containing delta-aminolevulinic acid for 21 days, hepatic porphyrins accumulated, and hepatic URO-D activity was reduced to 20% of wt. We bred mice homozygous for an HFE gene disruption (HFE(-/-)) to URO-D(+/-) mice, generating mice with the URO-D(+/-)/HFE(-/-) genotype. These animals developed a porphyric phenotype by 14 weeks of age without ALA supplementation, and URO-D activity was reduced to 14% of wt. These data indicate that iron overload alone is sufficient to reduce URO-D activity to rate-limiting levels in URO-D(+/-) mice. The URO-D(+/-) mouse serves as an excellent model of familial PCT and affords the opportunity to define the mechanism by which iron influences URO-D activity.

Accelerated development of uroporphyria in mice heterozygous for a deletion at the uroporphyrinogen decarboxylase locus.

Three weeks after a single dose of iron-dextran and Aroclor 1254, mice maintained continuously on delta-aminolevulinic acid supplemented drinking water showed significantly elevated levels of hepatic uroporphyrin and depressed (25% of normal) uroporphyrinogen decarboxylase (URO-D) activity. Depressed URO-D activity was paralleled by the ability of heat denatured cytosol to inhibit rhURO-D activity. Mice heterozygous for a targeted disruption at the URO-D locus (URO-D+/-) exhibited half the URO-D activity of homozygous controls prior to treatment. After treatment, these animals showed URO-D activity and rhURO-D inhibitory activity comparable to similarly treated wild type (URO-D +/+) mice but with significantly greater uroporphyrin accumulation. With only 10 days of treatment, URO-D +/- but not URO-D +/+ mice showed changes similar in magnitude to those seen after 21 days. Prior to treatment, URO-D genotype did not influence overall hepatic P450 concentration in either sex and there was no significant difference between sexes. The treatment regimen significantly elevated P450 in animals of either URO-D genotype and in both sexes, although the induction response at the 10-day point was attenuated in URO-D +/- mice. From differences in the CO absorbance maximum, and by P450 activity analysis, this attenuated induction response resulted from an attenuation of the CYP2B not the CYP1A induction.

The heme biosynthesis pathway and clinical manifestations of abnormal function.

Biosynthesis of heme is important for both prokaryotes and eukaryotes. The enzymes for this multistep process are distributed between the cytosol and mitochondria in eukaryotes. In humans there are inherited and acquired disorders characterized by over synthesis of one or more enzymes or absence of an enzyme. This overview discusses each enzyme in the heme biosynthesis pathway.

Measurement of uroporphyrinogen decarboxylase activity.

Uroporphyrinogen decarboxylase (UROD) catalyzes decarboxylation of the four acetate side chains of urophyrinogen to form coproporphyrinogen. Activity of UROD can be measured using an enzymatically prepared substrate or a chemically prepared one. For the former, bacterial porphobilinogen deaminase is prepared and used to prepare the porphyrinogen substrate for the enzymatic assay. Erythrocyte lysates can be used to measure hemoglobin content as an indicator of UROD activity.

Transdermal estrogen replacement therapy in postmenopausal women previously treated for porphyria cutanea tarda.

Oral contraceptives and postmenopausal estrogen replacement therapy are recognized as risk factors for the development of porphyria cutanea tarda (PCT) in women. The recommended clinical practice is to withhold estrogen therapy in women who have had phlebotomy therapy for PCT and are clinically and biochemically normal. We tested the safety and efficacy of transdermal estrogen replacement therapy in 7 women previously treated for PCT and compared them with 19 non-porphyric control subjects treated with transdermal or oral estrogens. Gonadotrophic hormone levels, estrogen levels, liver function studies, body iron stores, urine porphyrin excretion, and cytochrome P4501A2 (CYP1A2) activity were monitored for 1 year. Four of the women previously treated for PCT completed the study. None had evidence of a porphyric relapse. CYP1A2 activity, measured by three different methods, did not differ between study subjects receiving estrogens, patients with active PCT, and non-porphyric control subjects, nor did CYP1A2 activity change during the study period. Gonadotrophic hormone levels fell and estrogen levels rose in all women receiving estrogens. The administration of estrogens by the transdermal route appeared to be safe in the small number of subjects we studied and should be considered for women previously treated for PCT.