Uroporphyrinogen Decarboxylase Activity Research Articles

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture.

Uroporphyrinogen decarboxylase (UROG-D) activity in the 10,000g supernatant of 17-day-old chick embryo liver homogenates was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I. The optimum pH of the enzyme was found to be approximately 6.0 and enzyme activity was found to be linear with protein concentrations ranging from 0.3 to 2.0 mg/mL. At a protein concentration of 1.2 mg/mL and pH 6.0, the activity was found to be linear for a reaction time of 50 min and to be approximately 10 pmol/(mg protein.min). This enzyme assay was used to demonstrate that a UROG-D inhibitor, previously reported to accumulate in rodent liver, also accumulates in 3,3'4,4'-tretrachlorobiphenyl (TCBP) and sodium phenobarbital (PB) treated chick embryo hepatocytes in culture. This results accords with the previous demonstration of a TCBP- and PB-induced decrease in UROG-D activity in this system. Uroporphyrin accumulation in chick embryo hepatocyte culture is interpreted as resulting from a combination of two mechanisms, viz., inhibition of UROG-D activity and uroporphyrinogen oxidation to uroporphyrin catalyzed by a cytochrome P-450 isozyme.

A Point Mutation in the Coding Region of Uroporphyrinogen Decarboxylase Associated With Familial Porphyria Cutanea Tarda

Familial porphyria cutanea tarda (PCT) is inherited as an autosomal dominant trait caused by decreased activity of uroporphyrinogen decarboxylase (URO-D). In most families with PCT, URO-D mRNA levels are normal but both catalytic activity and immunologic reactivity of URO-D are half normal. We have cloned and sequenced 8 URO-D cDNA transcripts derived from a pedigree member with familial PCT. Three of the cDNAs had sequences encoding normal URO-D but five cDNA's contained a point mutation resulting in a gly----val substitution at amino acid position 281. An oligonucleotide probe complementary to the mutant sequence hybridized to DNA from affected individuals within the pedigree, but not to DNA from normal individuals. Measurements of pulse labeled URO-D in Epstein-Barr virus transformed lymphocytes indicated that the mutant protein has a half-life in vivo of less than four hours. In vitro measurements utilizing labeled URO-Ds generated in a reticulocyte lysate system revealed a 12-hour half-life for the mutant protein compared with a 102-hour half-life for normal URO-D. This is the first URO-D mutation to be characterized in a pedigree with familial PCT. This mutation was not detected in affected individuals from seven other PCT pedigrees, suggesting that PCT can result from different mutations.

A point mutation in the coding region of uroporphyrinogen decarboxylase associated with familial porphyria cutanea tarda

Familial porphyria cutanea tarda (PCT) is inherited as an autosomal dominant trait caused by decreased activity of uroporphyrinogen decarboxylase (URO-D). In most families with PCT, URO-D mRNA levels are normal but both catalytic activity and immunologic reactivity of URO-D are half normal. We have cloned and sequenced 8 URO-D cDNA transcripts derived from a pedigree member with familial PCT. Three of the cDNAs had sequences encoding normal URO-D but five cDNA's contained a point mutation resulting in a gly----val substitution at amino acid position 281. An oligonucleotide probe complementary to the mutant sequence hybridized to DNA from affected individuals within the pedigree, but not to DNA from normal individuals. Measurements of pulse labeled URO-D in Epstein-Barr virus transformed lymphocytes indicated that the mutant protein has a half-life in vivo of less than four hours. In vitro measurements utilizing labeled URO-Ds generated in a reticulocyte lysate system revealed a 12-hour half-life for the mutant protein compared with a 102-hour half-life for normal URO-D. This is the first URO-D mutation to be characterized in a pedigree with familial PCT. This mutation was not detected in affected individuals from seven other PCT pedigrees, suggesting that PCT can result from different mutations.

Studies on uroporphyrinogen decarboxylase of etiolated Euglena gracilis Z

1. A 423-fold purified fraction of uroporphyrinogen decarboxylase (EC 4.1.1.37) showing a specific activity of 770 units/mg protein has been employed in order to study some properties in etiolated Euglena gracilis Z. 2. Uroporphyrinogen decarboxylase has a relative molecular mass of 54,000, an optimum pH of 7.2 and exhibits Michaelis-Menten kinetics, employing both uroporphyrinogen I and uroporphyrinogen III as substrates. 3. Anaerobic conditions seem not to be necessary for uroporphyrinogen decarboxylase activity. Neither EDTA nor cysteine affected enzyme activity, whereas dithiothreitol produced a remarkable activation of coproporphyrinogen formation. 4. Kinetic data employing both substrates showed an accumulation of porphyrinogen (i.e. hexa- and hepta-porphyrin) containing six or seven COOH groups, depending on the uroporphyrinogen concentration used. 5. An unusual elution profile of the intermediates on Sephacryl S-200 was found.

Development of hexachlorobenzene porphyria in rats: Time sequence and relationship with lipid peroxidation

The relationship between the development of porphyria and free-radical formation induced by hexachlorobenzene was studied in iron-overloaded rats. The first sign of porphyria, an increase in porphyrins in the liver, was detected at day 22. Liver malondialdehyde was also increased at day 22. During the following weeks, liver porphyrins and malondialdehyde increased simultaneously, accompanied by a decrease in uroporphyrinogen decarboxylase activity and glucose-6-phosphate activity in liver, and a high excretion of porphyrins in the urine. In the rats given hexachlorobenzene, changes were detected in the pattern of lipids in the liver microsomes. In comparison with the controls, there were decreases in C20:4 and C22:5 fatty acids, whereas the fatty acid C20:3w6 was increased. In this study of hexachlorobenzene-induced liver damage there was no difference in the time course of the development of porphyria and that of lipid peroxidation.

Uroporphyrin accumulation in cultured chick embryo hepatocytes: Comparison of 2,3,7,8-tetrachlorodibenzo- p-dioxin and 3,4,3′,4′-tetrachlorobiphenyl

Uroporphyrin (URO) accumulation caused by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and 3,4,3′,4′-tetrachlorobiphenyl (TCB) in cultured chick embryo hepatocytes was found to depend on the concentration of the added polyhalogenated aromatic compound, and on either the addition of 5-aminolevulinic acid or the induction of 5-aminolevulinic acid synthase. TCDD alone did not cause more than a slight increase in uroporphyrin, whereas TCB alone caused considerable uroporphyrin accumulation associated with increased 5-aminolevulinic acid synthase activity. However, in the presence of exogenous 5-aminolevulinic acid, TCDD was more potent than TCB in causing uroporphyrin accumulation. The concentrations of TCDD or TCB which maximally induced ethoxyresorufin deethylase activity, an indicator of induced cytochrome P450 activity, were lower than those required for maximal uroporphyrin accumulation. Furthermore, ethoxyresorufin deethylase activity was found to decline at concentrations of TCDD or TCB which caused maximum uroporphyrin accumulation. Pretreatment with 3-methylcholanthrene enhanced uroporphyrin accumulation, whereas addition of inhibitors of cytochrome P450 decreased uroporphyrin accumulation. Uroporphyrin accumulation occurred without a decrease in uroporphyrinogen decarboxylase activity, and was unrelated to the degree of conversion of the polyhalogenated aromatic compounds to water-soluble metabolites. Our results indicate that URO accumulation caused by TCDD and TCB requires two separate actions; (1) induction of cytochrome P450 which occurs at low concentrations of the halogenated chemicals, and (2) increased uroporphyrinogen oxidation which is catalyzed by the induced cytochrome P450 and which occurs at higher concentrations of the halogenated chemicals.

An assay of uroporphyrinogen decarboxylase in erythrocytes.

Measurement of uroporphyrinogen decarboxylase (UROD; EC 4.1.1.37) activity in erythrocytes is useful in distinguishing between familial porphyria cutanea tarda (PCT), in which UROD activities are low, and acquired PCT, in which UROD activity is normal. In this method for measuring UROD, pentacarboxylic acid porphyrinogen I (PPI) is used as substrate. A sample of the patient's whole blood is incubated with PPI at 37 degrees C for 30 min at pH 6.0. The reaction is stopped by adding trichloroacetic acid/dimethyl sulfoxide containing mesoporphyrin (internal standard). The coproporphyrin so produced is measured directly by high-performance liquid chromatography, with fluorescence detection. Our values by this method for healthy subjects and non-PCT patients ranged from 1.8 to 4.0 U/L. The CV for the assay was 10% at 1.1 U/L and 9% at 2.4 U/L. Twelve of 42 patients with PCT had low erythrocyte UROD activities. In each of six families of patients with low UROD activity we found at least one other family member with a low UROD activity in erythrocytes.

A simplified method for determination of uroporphyrinogen decarboxylase activity in human blood

The determination of erythrocyte uroporphyrinogen decarboxylase activity is essential for differentiating familial (type II) porphyria cutanea tarda from the sporadic (type I) form of the disease. A new technique for the determination of uroporphyrinogen decarboxylase activity in human blood is described. Haemolysate is incubated with uroporphyrinogen III as substrate. Uroporphyrinogen unconverted during the reaction is oxidised to uroporphyrin and measured directly as free acid by HPLC. The enzyme activity is then calculated from the amount of substrate consumed. The technique is simple, rapid and highly reproducible. It is recommended as a clinical assay.

Development of porphyria cutanea tarda after treatment with cyclophosphamide

Porphyria cutanea tarda, a metabolic disorder of heme biosynthesis, is characterized by cutaneous hyperpigmentation, facial hypertrichosis, dark urine, and a distinctive pattern of excess porphyrin production. Hepatic uroporphyrinogen decarboxylase activity is markedly reduced in patients with this disorder. Although porphyria cutanea tarda may be familial, it is more often sporadic in occurrence, and has been associated with excess alcohol ingestion, estrogen administration, iron overload, and several environmental hepatotoxins. It has also been associated on occasion with malignancy. We report a 46-yr-old woman with ovarian carcinoma who developed porphyria cutanea tarda while undergoing treatment with cisplatin and cyclophosphamide. The temporal course of the porphyrin abnormality suggested that cyclophosphamide was the pathogenic agent, and symptoms regressed after cessation of this drug with continued administration of cisplatin. The pathogenesis of the porphyria is not clear; however, cyclophosphamide is a substrate for cytochrome P450, and may produce metabolites that destroy this protein. The resulting increased turnover of heme might then result in overproduction of porphyrin precursors, resulting in the clinical syndrome. Studies of porphyrin metabolism in patients treated with cyclophosphamide may help to elucidate this possibility.

The role of the Ah locus in hexachlorobenzene-induced porphyria. Studies in congenic C57BL/6J mice.

The role of the Ah locus in hexachlorobenzene (HCB)-induced porphyria and the possible involvement of P-450 cytochromes P(1)450 and P(3)450 in the pathogenesis of this disease were investigated in two congenic strains of C57BL/6J mice that differ only at this locus. Female B6-Ahb mice (Ah receptor: approximately 30-70 fmol/mg of cytosolic protein) and B6-Ahd mice (Ah receptor: undetectable) were pretreated with iron (500 mg/kg) and then fed a diet containing 0 or 200 p.p.m. of HCB for up to 17 weeks. Mice from the two strains consumed similar amounts of HCB. Urinary excretion of porphyrins was increased after 7 weeks of HCB treatment in B6-Ahb mice, and after 15 weeks was over 200 times greater than that of mice given iron only. In B6-Ahd mice, porphyrin excretion did not begin to increase until after 13 weeks, and after 15 weeks was only six times greater than that of controls. Similar differences were seen in the 15-week hepatic porphyrin concentrations (B6-Ahb: 1110 +/- 393; B6-Ahd: 17.6 +/- 14.5; controls: approximately 0.20 nmol/g). Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was diminished by 70 and 20% in B6-Ahb B6-Ahd mice respectively after 15 weeks of treatment with HCB. Cytochromes P(1)450 and P(3)450 were measured in hepatic microsomes (microsomal fractions) by radioimmunoassay and immunoblotting, using antisera raised against the orthologous rat isoenzymes P450c and P450d. HCB induced small amounts of a protein recognized by anti-P450c (P(1)450) in B6-Ahd mice, but not in B6-Ahd mice. Relatively large amounts of a protein recognized by anti-P450d (P(3)450) were induced in both strains, but to a somewhat greater extent in the B6-Ahb mice. The hepatic accumulation of HCB at 15 weeks was greater in B6-Ahb than in B6-Ahd mice, in association with elevated hepatic lipid levels in the former strain. The results of this experiment indicate that the Ah locus influences the susceptibility of C57BL/6J mice to HCB-induced porphyria and are consistent with the suggestion that the sustained induction of P(3)450 and/or P(1)450 may be a causative factor in the development of this disease.

Uroporphyria produced in mice by 20-methylcholanthrene and 5-aminolaevulinic acid.

Iron-loaded male C57BL/6 mice allowed free access to an aqueous solution of 5-aminolaevulinic acid (ALA) (2 mg/ml) as their only drink, develop severe uroporphyria within 9 days of a single intraperitoneal dose of 20-methylcholanthrene (MC) (125 mg/kg). At 21 days, uroporphyrinogen decarboxylase (EC 4.1.1.37) activities are less than 10% of control activities. The porphyria is not dependent on pretreatment with iron and persists for at least 21 days after withdrawal of ALA. The same intraperitoneal dose of MC does not produce porphyria within 21 days when given without ALA. Continuous administration of ALA markedly accelerates the onset of porphyria in iron-loaded male C57BL/6 mice after a single intraperitoneal dose of hexachlorobenzene (200 mg/kg); mice given phenobarbitone and ALA do not become porphyric. MC with ALA does not produce porphyria in iron-loaded male DBA/2 mice. At least two separate events are needed to produce uroporphyria in mammals: induction of a specific form of cytochrome P-450 and stimulation of the formation of intermediates of haem biosynthesis in the liver. These results show that severe, persistent porphyria can be produced in mammals by compounds other than polyhalogenated aromatic hydrocarbons and suggest that a similar mechanism underlies the porphyrogenic action of halogenated and non-halogenated compounds.

Hepatic uroporphyrin accumulation and uroporphyrinogen decarboxylase activity in cultured chick-embryo hepatocytes and in Japanese quail (Coturnix coturnix japonica) and mice treated with polyhalogenated aromatic compounds.

The relationship between hepatic uroporphyrin accumulation and uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was investigated in cultured chick-embryo hepatocytes, Japanese quail (Coturnix coturnix japonica) and mice that had been treated with polyhalogenated aromatic compounds. Chick-embryo hepatocytes treated with 3,3',4,4'-tetrachlorobiphenyl accumulated uroporphyrin in a dose-dependent fashion without a detectable decrease in uroporphyrinogen decarboxylase activity when either pentacarboxyporphyrinogen III or uroporphyrinogen III were used as substrates in the assay. Other compounds, such as hexachlorobenzene, parathion, carbamazepine and nifedipine, which have been shown previously to cause uroporphyrin accumulation in these cells, did not decrease uroporphyrinogen decarboxylase activity. Japanese quail treated with hexachlorobenzene for 7-10 days also accumulated hepatic uroporphyrin without any decrease in uroporphyrinogen decarboxylase activity. In contrast, hepatic uroporphyrin accumulation in male C57BL/6 mice treated with iron and hexachlorobenzene was accompanied by a 20-80% decrease in uroporphyrinogen decarboxylase activity, demonstrating that the assay used for uroporphyrinogen decarboxylase, using pentacarboxyporphyrinogen III as substrate, could detect decreased enzyme activity. Our results with chick hepatocytes and quail, showing uroporphyrin accumulation without a decrease in uroporphyrinogen decarboxylase activity, are consistent with a new two-stage model of the uroporphyria: initially uroporphyrinogen is oxidized by a cytochrome P-450-mediated reaction, followed in rodents by a progressive decrease in uroporphyrinogen decarboxylase activity.

The effects in vivo of mutationally modified uroporphyrinogen decarboxylase in different hem12 mutants of baker's yeast (Saccharomyces cerevisiae).

Nine new hem12 haploid mutants of baker's yeast (Saccharomyces cerevisiae), totally or partially deficient in uroporphyrinogen decarboxylase activity, were subjected to both genetic and biochemical analysis. The mutations sites studied are situated far apart within the HEM12 gene located on chromosome IV. Uroporphyrinogen decarboxylase activity in the cell-free extracts of the mutants was decreased by 50-100%. This correlated well with the decrease of haem formation and the increased accumulation and excretion of porphyrins observed in vivo. The pattern of porphyrins (uroporphyrin and its decarboxylation products) accumulated in the cells of mutants partially deficient in uroporphyrinogen decarboxylase activity did not differ significantly, although differences in vitro were found in the relative activity of the mutant enzyme at the four decarboxylation steps. The excreted porphyrins comprised mainly dehydroisocoproporphyrin or pentacarboxyporphyrin. In heterozygous hem12-1/HEM12 diploid cells, a 50% decrease in decarboxylase activity led to an increased accumulation of porphyrins as compared with the wild-type HEM12/HEM12 diploid, which points to the semi-dominant character of the hem12-1 mutation. The biochemical phenotypes of both the haploid and the heterozygous diploid resembles closely the situation encountered in porphyria cutanea tarda, the most common human form of porphyria.

Porphyria Cutanea Tarda Associated With the Acquired Immunodeficiency Syndrome

To the Editor.— The porphyrias are a group of disorders caused by biosynthetic abnormalities in heme synthesis, which result in an accumulation of precursor substrates. Porphyria cutanea tarda (PCT) is the most common form of porphyria, and is caused by a reduced activity of the enzyme uroporphyrinogen decarboxylase. 1 As the number of acquired immunodeficiency syndrome (AIDS) cases have increased, so too have the legion presentations, manifestations, and complications associated with infection by the human immunodeficiency virus (HIV). Report of a Case.— A 40-year-old adopted bisexual man, with a history of chronic anemia, intravenous drug abuse, and alcoholic binge drinking for more than ten years, presented for evaluation of fatigue, malaise, diarrhea of a few weeks' duration, pruritus, and skin lesions that had been present for several years. He had been transfusion dependent, having received at least 30 transfusions over the past three years. Physical examination revealed a cachectic, middle-aged,

Xenobiotic mediated inhibition of hepatic uroporphyrinogen decarboxylase activity in 17-day-old chick embryo liver cells in culture

Uroporphyrin, heptacarboxylic acid porphyrin and coproporphyrin were the major porphyrins to accumulate when phenobarbital, nifedipine, 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (Ox-DDC) and 3,3',4,4'-tetrachorobiphenyl (TCBP) were added to chick embryo hepatocyte culture. This pattern of porphyrin accumulation is consistent with the demonstration that these chemicals inhibit uroporphyrinogen decarboxylase (UROG-D). The degree of UROG-D inhibition observed was: TCBP (39%), Ox-DDC (39%), nifedipine (25%) and phenobarbital (50%). Since significant UROG-D inhibition was observed when the bulk of the porphyrins in the crude enzyme preparation was removed by gel filtration, it is unlikely that porphyrins produce the enzyme inhibition. When succinylacetone, a potent inhibitor of δ-aminolevulinic acid dehydratase, was coadministered with Ox-DDC, phenobarbital, TCBP and nifedipine, UROG-D inhibition was not observed. These results suggest that heme biosynthesis must proceed in order for xenobiotic mediated UROG-D inhibition to occur.

Familial porphyria cutanea tarda: hybridization analysis of the uroporphyrinogen decarboxylase locus.

Familial porphyria cutanea tarda (PCT) results from a deficiency of uroporphyrinogen decarboxylase (URO-D) activity. Hybridization analysis of genomic DNA from unrelated normal individuals and PCT pedigree members failed to detect any major deletions, rearrangements or restriction fragment length polymorphisms at the URO-D locus.

Comparison of Uroporphyrinogen Decarboxylase Activities and Uroporphyrin Accumulation in Mice, Japanese Quail, and Cultured Chick Embryo Hepatocytes Treated with Polyhalogenated Aromatic Compoundsa

Comparison of Uroporphyrinogen Decarboxylase Activities and Uroporphyrin Accumulation in Mice, Japanese Quail, and Cultured Chick Embryo Hepatocytes Treated with Polyhalogenated Aromatic Compoundsa

Inhibitory effect of tetrachloro-p-hydroquinone and other metabolites of hexachlorobenzene on hepatic uroporphyrinogen decarboxylase activity with reference to the role of glutathione.

Exposure of rats to HCB caused a dose-dependent depletion of GSH. Chlorophenolic and sulfur-containing metabolites of HCB incubated with GSH-free rat liver cytosolic protein drastically diminished the UROD activity. In addition, HCB also exhibited inhibitory potency. The most effective compounds studied were TCH and its oxidation product, chloranil. Incubation of liver cytosolic protein and of GSH with HCB and its metabolites yielded results that suggested interaction between the compounds and cell constituents--an interaction that may cause inhibition of the hepatic UROD activity in the HCB-exposed organism.

Pseudoporphyria Cutanea Tarda and Non-A Non-S Hepatitis in a CAPD Patient

A 47-year-old man was treated by continuous ambulatory peritoneal dialysis (CAPD) because of chronic glomerulonephritis. One year later he developed skin friability and bullous dermatosis resembling porphyria cutanea tarda. The skin lesions were associated with an elevation of serum aminotransferases, most probably due to a blood transfusion-induced, non-A non-B hepatitis. Urinary, fecal and blood porphyrins including the activity of red cell uroporphyrinogen decarboxylase were normal. Thus we accepted the diagnosis of pseudoporphyria cutanea tarda in view of the relatively frequent association of cutaneous disease with maintenance dialysis.

Role of inhibition of uroporphyrinogen decarboxylase in PCB-induced porphyria in mice

The oral administration of 3,4,5,3′,4′,5′-hexachlorobiphenyl for 3 weeks to mice caused a marked accumulation of porphyrins in the liver of C57BL 6 and C57BL 10 mice but not in the liver of ddY mice. The time course of induction of δ-aminolevulinic acid synthetase (ALA-S), cytochrome P-450, and mixed function oxidases and inhibition of uroporphyrinogen decarboxylase (URO-D) in the liver of C57BL 6 mice and ddY mice fed a diet containing 500 ppm of a commercial PCB (Kanechlor-500) were investigated to clarify the sole factor in inducing porphyria. The activity of URO-D in the liver of C57BL 6 mice was depressed approximately 80% at 3 weeks when a large amount of uroporphyrin accumulated. Male ddY mice showed only a slight increase in uroporphyrin accumulation in the liver and a moderate decrease of URO-D activity even at the 10th week. ALA-S, cytochrome P-450, and mixed function oxidases were induced in both strains of mice, although the magnitude of these inductions in C57BL 6 mice was greater than that in ddY mice. No differences were detected between the two strains in the content and gas chromatographic pattern of PCB remaining in liver cytosol (6 weeks). In addition there was no relationship between the time of onset of porphyria and that of the maximal induction of drug-metabolizing function in C57BL 6 mice. These results indicate that the development of porphyria is causally related to the inhibition of URO-D rather than the induction of drug-metabolizing function. The hypothesis that porphyria first develops when the ratio of hepatic URO-D and ALA-S activities decreases to less than 1.0 is presented.