ABSTRACT
AIP is an acute liver disorder caused by a deficiency of porphobilinogen deaminase (PBGD) characterized by neuroabdominal symptoms. It is an autosomal dominant disease. However, homozygous dominant AIP (HD-AIP) have been described. In some cases erythrodontia was observed. CEP is an autosomal recessive disease produced by mutations in the uroporphyrinogen III synthase gene (UROS), characterized by severe cutaneous lesions and erythrodontia. The aim of the work was to establish the differential diagnosis of porphyria in a patient with abdominal pain, neurological attacks, skin symptoms and erythrodontia. The PBGD activity was reduced 50% and the genetic analysis indicated the presence of two genetic variants in the PBGD gene, p.G111R and p.E258G, a new genetic variant, revealing a case of heteroallelic HD-AIP. The patient, first diagnosed as a carrier of a dual porphyria: AIP / CEP based on the excretion profile of porphyrins, precursors and her clinical symptoms, would be an atypical case of human HD-AIP. These results would also suggest the presence of a phenocopy of the CEP, induced by an endogenous or exogenous factor. Our findings highlight the importance of genetic studies for a proper diagnosis of porphyria, prevention of its manifestation and its treatment.
Subject(s)
Genetic Variation , Hydroxymethylbilane Synthase/genetics , Liver/pathology , Porphyria, Acute Intermittent/diagnosis , Porphyria, Acute Intermittent/genetics , Acute Disease , Adult , Base Sequence , DNA Mutational Analysis , Female , Heterozygote , Humans , Hydroxymethylbilane Synthase/metabolism , Liver/metabolism , Molecular Sequence Data , Mutation , Porphyria, Acute Intermittent/blood , Porphyria, Acute Intermittent/urine , Porphyrins/blood , Porphyrins/urine , Uroporphyrinogen III Synthetase/genetics , Uroporphyrinogen III Synthetase/metabolismABSTRACT
Acute intermittent porphyria (AIP) caused by mutations in the hydroxymethylbilane synthase gene (HMBS), has been reported in almost all human populations, with varying frequencies. A founder effect for a few specific mutations in geographic regions where prevalence is high (Sweden, The Netherlands, Switzerland) has been established through haplotype analyses, while some other mutations (R26H, R26C) have been repeatedly reported in many populations with different genetic backgrounds. Epidemiological, biochemical and molecular data on AIP in Venezuela were gathered during the last two decades; 24 independent families with AIP were ascertained, based on a deficient HMBS activity and increased porphobilinogen (PBG) urinary excretion. Molecular analyses of coding and splicing regions were performed in 23 families, to establish disease-causing changes, and haplotype analyses were used to assess ancestral kinships between them. Changes were detected in 16 out of 23 families, 9 of them being different: R26H, R26C, c.87+5G>A, c.267-54_61delgaaggggt, R116W, Q180X, c.825+1G>A, c.913-1delG, and 3' UTR *277G>A. Seven mutations were found, each one in a single family; one mutation was present in two unrelated families, whereas mutation Q180X was shared by 7 independent kindreds, all of which had the same haplotype (-);T;A;T;G;T;A;G (3167delG; 3530T>C; 3581A>G; 3982T>C; 6479G>T; 7052T>C; 7064A>C; 7779G>A). Six out of seven different Q180X carrier families came from the same geographic focus (Santa Lucía, Miranda State). Dense geographic aggregation with one identical haplotype strongly suggests a remote founder phenomenon for these Venezuelan AIP families, carrying an unreported but most frequent mutation.
Subject(s)
Hydroxymethylbilane Synthase/genetics , Mutation , Polymorphism, Single Nucleotide , Porphyria, Acute Intermittent/genetics , Adolescent , Adult , Biomarkers/urine , Child , Child, Preschool , DNA Mutational Analysis , Female , Founder Effect , Gene Frequency , Genetic Predisposition to Disease , Haplotypes , Heredity , Heterozygote , Humans , Hydroxymethylbilane Synthase/metabolism , Male , Middle Aged , Pedigree , Phenotype , Porphobilinogen/urine , Porphyria, Acute Intermittent/diagnosis , Porphyria, Acute Intermittent/enzymology , Porphyria, Acute Intermittent/epidemiology , Prevalence , Time Factors , Venezuela/epidemiology , Young AdultABSTRACT
Acute attacks of porphyria are most commonly precipitated by events that decrease heme concentrations. Enzyme inducing-drugs are the most important triggering factors, particularly in relation to anaesthesia. We have reported previously that Enflurane and Isoflurane produced significant heme metabolism alterations, indicating that the use of these anaesthetics in porphyric patients should be avoided. The aim of this work was to evaluate the effect of the anaesthetic Sevoflurane on heme pathway and drug metabolizing Phase I system in mice. To this end, animals received different doses of the anaesthetic (1-2 ml/kg) and were sacrificed at different times (5-60 min). Data revealed important alterations in the enzymes involved in Acute Intermittent Porphyria, such as an induction in hepatic 5-Aminolevulinic acid synthetase activity and a diminished Porphobilinogen deaminase activity in liver and blood 20 minutes after Sevoflurane administration to mice in a dose of 1.5 ml/kg. Heme oxygenase activity was also induced, indicating the onset of oxidative stress. Total CYP levels and CYP2E1 expression were enhanced. As a consequence of these events, heme free pool would be depleted. In conclusion, our results in mice would suggest that Sevoflurane should be used with caution and very careful control in porphyric patients.
Subject(s)
Anesthetics, Inhalation/toxicity , Heme/metabolism , Liver/drug effects , Methyl Ethers/toxicity , 5-Aminolevulinate Synthetase/metabolism , Animals , Cytochrome P-450 CYP2E1/metabolism , Heme Oxygenase (Decyclizing)/metabolism , Hydroxymethylbilane Synthase/metabolism , Liver/enzymology , Male , Mice , Oxidative Stress/drug effects , SevofluraneABSTRACT
Erythropoietic Protoporphyria (EPP) is an inherited deficiency of ferrochelatase, the last enzyme of the heme pathway. Under general anaesthesia, some patients develop neurological dysfunction suggesting upregulation in heme biosynthesis similar to that described for acute porphyrias after xenobiotic administration. Our aim has been to evaluate whether Isoflurane induces alterations in the heme pathway in a mouse model for EPP. Administration of Isoflurane (a single dose of 2 ml/kg, i.p) to wild-type (+/+), heterozygous (+/Fechm1Pas) and homozygous (Fechm1Pas/Fechm1Pas) mice, was evaluated by measuring the activity of delta-aminolevulinic acid synthetase (ALA-S) and Porphobilinogen-deaminase (PBG-D) in different tissues, as well as Heme oxygenase (HO), cytochrome P-450, CYP2E1 and glutathione levels in liver. Porphyrin precursors were measured in 24 h-urine samples. Fechm1Pas/Fechm1Pas mice receiving anaesthesia show enhanced ALA-S and CYP2E1 activities in the liver and increased urinary excretion of porphyrin precursors. No alterations were found in either PBG-D or HO activities. Diminished glutathione levels suggest that anaesthesia may produce oxidative stress in these animals. In conclusion, Isoflurane induces ALA-S activity and increased excretion of porphyrin precursors in EPP mice. These findings appear to confirm our previous hypothesis and indicate that Isoflurane may be an unsafe anaesthetic not only for patients with acute porphyrias but also for individuals with non acute porphyrias.
Subject(s)
5-Aminolevulinate Synthetase/metabolism , Isoflurane/pharmacology , Liver/drug effects , Liver/enzymology , Protoporphyria, Erythropoietic/metabolism , Animals , Enzyme Activation/drug effects , Enzyme Induction/drug effects , Glutathione/metabolism , Heme Oxygenase (Decyclizing) , Hydroxymethylbilane Synthase/metabolism , Mice , Mice, Mutant Strains , Oxidative Stress/drug effectsSubject(s)
Enzymes, Immobilized/metabolism , Hydroxymethylbilane Synthase/metabolism , Pyrroles/chemistry , Animals , Cattle , Enzymes, Immobilized/chemistry , Hydroxymethylbilane Synthase/chemistry , Liver/enzymology , Molecular Structure , Pyrroles/metabolism , Tetrapyrroles , Uroporphyrinogen III Synthetase/metabolism , Uroporphyrinogens/biosynthesisABSTRACT
BACKGROUND AND AIMS: Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of the enzyme porphobilinogen deaminase (PBG-D). The kidney is an important target for numerous porphyrinogenic drugs and it may contribute to the clinical manifestations of porphyric attacks. An evaluation of kidney PBG-D role in the AIP pathophysiology requires detailed information on kidney PBG-D properties, under normal conditions. METHODS: Rat kidney PBG-D was purified to homogeneity and initial reaction velocities were calculated by measuring uroporphyrinogen I formation at pH 8.2 for different incubation times (0-20 min) and over a wide range of substrate concentrations (0.8-66 microM). RESULTS: Purified rat kidney PBG-D is a monomeric enzyme showing only a single protein band after SDS-PAGE, Western blot and isoelectric focusing (pI 4.9). Its molecular mass is 40 +/- 2.3 kDa, determined by SDS-PAGE and 39.8 +/- 2 kDa by gel filtration chromatography. Rat kidney PBG-D has an unusual kinetic behaviour, exhibiting a deviation from the Michaelis-Menten hyperbola. PBG-D kinetic data required a fitting to an equation of higher degree, leading to the following apparent kinetic constants: K(1) = 2.08 +/- 0.01 microM and K(2) = 0.102 +/- 0.003 microM. CONCLUSION: The values of these constants fulfil the restriction 4K(2) < or = K(1)(2), necessary for the occurrence of isoenzymes, interpreted in this work as enzyme-substrate intermediates. The initial reaction velocity expression here defined, correlates with an enzyme carrying only one active site but allowing, through conformational changes, the detection of at least two enzyme-substrate intermediates formed during PBG-D reaction.
Subject(s)
Hydroxymethylbilane Synthase/metabolism , Kidney/enzymology , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Kinetics , Male , Rats , Substrate SpecificityABSTRACT
1. Chick embryos of 7, 9, 11, 12, 13, 14, 15, 17 and 19 d of embryonic development were examined to determine the activities of 5-aminolevulinic dehydratase (ALA-D, EC 4.2.1.24) and porphobilinogen deaminase (PBG-D, EC 4.3.1.8). 2. Liver and yolk sac membrane ALA-D specific activities showed a maximum between 12 and 13 d of embryonic development, yolk sac membrane PBG-ase activity a maximum at 9 d and at 7 d in liver. Total activities of ALA-D and PBG-D were not constant during the course of embryonic development but probably related to the changes of intensity of haem synthesis. 3. ALA-D and PBG-ase activities were higher in yolk sac membrane than in liver, showing the importance of the yolk sac membrane as erythropoietic tissue. PBG-D catalysed the rate-limiting reaction of the cytosolic steps in the biosynthetic pathway in both tissues.
Subject(s)
Chick Embryo/enzymology , Hydroxymethylbilane Synthase/metabolism , Liver/enzymology , Porphobilinogen Synthase/metabolism , Yolk Sac/enzymology , Age Factors , Animals , Chick Embryo/growth & development , Liver/embryology , Spectrophotometry , Yolk Sac/embryologyABSTRACT
1. The effect of the fluorinated ether anaesthetics enflurane and isoflurane in mice on haem metabolism and regulation in different metabolic states, such as depression and induction of cytochrome P450 produced by allylisopropylacetamide (AIA) and imidazole, respectively, was investigated. 2. Mice previously treated with AIA (350 mg/kg, i.p.) or imidazole (400 mg/kg, i.p.) received a single dose (1 mL/kg, i.p.) of enflurane or isoflurane and were killed 20 min after anaesthetic administration. 3. Induction of delta-aminolevulinic acid synthetase (ALA-S) activity was found, as expected, in animals receiving AIA and also in animals treated with AIA plus anaesthesia, but no change in the activity of either porphobilinogenase (PBGase) or porphobilinogen deaminase (PBG-D) activities was detected in these two groups of animals. An additional increase in haem destruction was observed in the AIA plus isoflurane-treated group. When mice were injected with imidazol alone or in combination with the anaesthetics, ALA-S activity was increased 50-90% in all groups, but again no change in PBGase or PBG-D activity was observed. Haem oxygenase was diminished in mice receiving imidazole and anaesthesia. 4. In conclusion, neither enflurane nor isoflurane caused additional disturbances in haem metabolism to those produced by AIA or imidazole alone.
Subject(s)
Anesthetics, Inhalation/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Enflurane/pharmacology , Heme/metabolism , Isoflurane/pharmacology , Liver/drug effects , Microsomes, Liver/drug effects , 5-Aminolevulinate Synthetase/metabolism , Allylisopropylacetamide/pharmacology , Aminolevulinic Acid/urine , Ammonia-Lyases/metabolism , Animals , Heme Oxygenase (Decyclizing)/metabolism , Hydroxymethylbilane Synthase/metabolism , Imidazoles/pharmacology , Liver/enzymology , Mice , Microsomes, Liver/enzymologyABSTRACT
Some late complications of diabetes are associated with alterations in the structure and function of proteins due to glycation and free radicals generation. Aspirin inhibits protein glycation by acetylation of free amino groups. In the diabetic status, it was demonstrated that several enzymes of heme pathway were diminished. The aim of this work has been to investigate the in vivo effect of short and long term treatment with acetylsalicylic acid in streptozotocin induced diabetic mice. In both treatments, the acetylsalicylic acid prevented delta-aminolevulinic dehydratase and porphobilinogen deaminase inactivation in diabetic mice and blocked the accumulation of lipoperoxidative aldehydes. Catalase activity was significantly augmented in diabetic mice and the long term treatment with aspirin partially reverted it. We propose that oxidative stress might play an important role in streptozotocin induced diabetes. Our results suggest that aspirin can prevent some of the late complications of diabetes, lowering glucose concentration and probably inhibiting glycation by acetylation of protein amino groups.
Subject(s)
Aspirin/therapeutic use , Catalase/antagonists & inhibitors , Diabetes Mellitus, Experimental/prevention & control , Hydroxymethylbilane Synthase/metabolism , Oxidative Stress/drug effects , Porphobilinogen Synthase/metabolism , Animals , Aspirin/pharmacology , Blood Glucose , Catalase/metabolism , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/enzymology , Diet , Eating/drug effects , Enzyme Inhibitors/pharmacology , Glycated Hemoglobin/analysis , Hydroxymethylbilane Synthase/antagonists & inhibitors , Lipid Peroxidation/drug effects , Male , Mice , Porphobilinogen Synthase/antagonists & inhibitors , StreptozocinABSTRACT
A partial deficiency of Porphobilinogen deaminase (PBG-D) is responsible for acute intermittent porphyria (AIP). AIP is inherited in an autosomal dominant fashion, and the prevalence in the Argentinean population is about 1:125,000. Here, two new mutations and three previously reported were found in the PBG-D gene in 12 Argentinean AIP patients corresponding to 5 different families. To screen for AIP mutations in symptomatic patients, genomic DNA isolated was amplified in 2 Multiplex PCR reactions, then all coding exons and flanking intronic regions were sequenced. The new mutations are 453-455delAGC in exon 9 which results in the loss of an alanine residue at position 152, and one new point mutation in the splicing aceptor site in the last position of intron 8 (IVS8-1G>T) which leds to a 15 bp deletion because a cryptic site (first AG upstream) is used. Both mutations produce amino acid deletion without frameshift effect. To further characterize the 453-455delAGC mutation, the pKK-PBGD construct for the mutant allele was expressed in E. coli, the enzymatic activity of the recombinant protein was 1.3% of the mean level expressed by the normal allele. Finally, three missense mutations, previously reported, were identified in three unrelated families.
Subject(s)
Hydroxymethylbilane Synthase/genetics , Porphyrias/genetics , Adolescent , Adult , Escherichia coli/enzymology , Female , Humans , Hydroxymethylbilane Synthase/biosynthesis , Hydroxymethylbilane Synthase/metabolism , Male , Middle Aged , Mutation , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND AND AIMS: Uroporphyrin and protoporphyrin produce alterations on 5-aminolevulinic acid dehydratase and porphobilinogen deaminase, as a result of a direct effect of porphyrins on the protein structure. With the aim of assessing the possible protection from the porphyrins effect on the proteins, some chemicals and the enzyme substrates were assayed. METHODS: Enzymes were pre-incubated with the protecting agents (beta-mercaptoethanol, dithiotreitol, hydroxylamine, succinic anhydride) or the corresponding substrates (delta-aminolevulinic acid and porphobilinogen), and then exposed to the porphyrins. All experiments were performed in the enzyme solutions after removing the porphyrins. RESULTS: The presence of sulfhydryl reagents partially protected both the enzyme activities and the content of total SH and free amino groups, but they did not prevent the appearance of molecular aggregates in the electrophoresis. Similar results were obtained in the presence of the corresponding substrates. Nucleophilic addition of hydroxylamine to the aromatic amino acids on the enzymes and blockage of their free amino groups did not prevent the direct effect of porphyrins, but these agents protected the enzyme activities from the photodynamic action of the tetrapyrroles, and also prevented the formation of molecular aggregates. However, an increased amount of free amino groups was observed, probably due to protein fragmentation. CONCLUSIONS: Porphyrins mainly affected the SH groups at or near the active site of the enzymes. Most of the free amino groups on the treated enzymes were involved in the formation of cross-links among the protein molecules. Protein fragmentation induced by porphyrins under UV light, and the consequent increased amount of free amino groups, were observed.
Subject(s)
Heme/chemistry , Hydroxymethylbilane Synthase/chemistry , Porphobilinogen Synthase/chemistry , Protoporphyrins/chemistry , Uroporphyrins/chemistry , Animals , Cattle , Heme/metabolism , Hydroxymethylbilane Synthase/metabolism , Porphobilinogen Synthase/metabolism , Protein Conformation , Protoporphyrins/metabolism , Substrate Specificity , Uroporphyrins/metabolismABSTRACT
Accumulation of delta-aminolevulinic acid (ALA), as it occurs in acute intermittent porphyria, is a potential endogenous source of reactive oxygen species (ROS) which can then produce oxidative damage to cell structures and macromolecules. This in vivo study investigated whether melatonin could prevent the deleterious effects of ALA. Rats were injected i.p. for 2 weeks with ALA (40 mg/kg on alternate days) and/or with melatonin (50 microg/kg or 500 microg/kg daily). Administration of pharmacological doses of melatonin reduced and/or prevented ALA-induced lipid peroxidation (LPO) in both cerebral cortex and cerebellum, providing further evidence of melatonin's action as a ROS scavenger. Administration of pharmacological concentrations of melatonin to ALA-injected rats showed the protective properties of melatonin on the activities of both porphobilinogen-deaminase and delta-aminolevulinate dehydratase (ALA-D) in the cerebral cortex; the effect on ALA-D activity was unexpectedly high (at least 6-fold), indicating that, besides acting as a scavenger of hydroxyl radicals, melatonin may exert its protection on ALA-D through other mechanisms, such as increasing mRNA levels of antioxidant enzymes or/and inducing glutathione peroxidase activity. The possibility that changes in the expression of antioxidant enzymes could affect the expression of other proteins, even those not related to the cellular ROS homeostasis, should also not be discarded. The potential use of melatonin as an antioxidant and for its reactivating properties in the treatment of acute porphyrias is considered.
Subject(s)
Aminolevulinic Acid/toxicity , Antioxidants/pharmacology , Hydroxymethylbilane Synthase/metabolism , Melatonin/pharmacology , Oxidative Stress/drug effects , Porphobilinogen Synthase/metabolism , Animals , Cerebellum/drug effects , Cerebellum/enzymology , Cerebral Cortex/drug effects , Cerebral Cortex/enzymology , Free Radical Scavengers/pharmacology , Injections, Intraperitoneal , Lipid Peroxidation/drug effects , Male , Malondialdehyde/metabolism , Rats , Reactive Oxygen Species/metabolismABSTRACT
Properties of purified porphobilinogen deaminase (PBG-D; EC 4.3.1.8) from rat harderian gland are here presented. The enzyme behaves as a monomer of Mr 38 +/- 2 kDa and is optimally active at pH 8.0-8.2. Its activation energy, determined by an Arrhenius plot, is 76.1 kJ/mol. Initial velocity studies showed a linear progress curve for uroporphyringen I formation and a hyperbolic dependence of the initial rate on substrate concentration, indicating the existence of a sequential displacement mechanism. Apparent kinetic constants, Km and Vm, calculated at 37 degrees C and pH 8.0 were 1.1 microM and 170 pmol/min mg, respectively. The pH dependence of the apparent kinetic parameters revealed the ionization of residues with pKAES and pKBES of 7.4 +/- 0.1 and 8.6 +/- 0.1, respectively, and a pKE value of 8.0 +/- 0.1. Incubation of PBG-D with 5.0 mM N-ethylmaleimide and 5.0 mM 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 led to inhibitions of 70 and 50%, respectively. The effect of pH, as well as the effect of thiol reagents, on enzyme activity strongly suggests the involvement of cysteine residue(s) in the mechanism of uroporphyrinogen I biosynthesis, in both the catalytic reaction and the substrate binding. Rat harderian gland PBG-D activity decreased with increasing concentrations of protoporphyrin IX, reaching a 40% inhibition at the in vivo concentration of the porphyrin and 7 microM PBG. Even at saturating concentrations of substrate, inhibition by protoporphyrin was not completely reversed. So, accumulated porphyrin may act as an regulator of PBG-D activity in rat harderian gland.
Subject(s)
Harderian Gland/enzymology , Hydroxymethylbilane Synthase/metabolism , Protoporphyrins/pharmacology , Animals , Binding Sites , Chromatography, High Pressure Liquid , Enzyme Inhibitors/pharmacology , Enzyme Stability , Ethylmaleimide/pharmacology , Hydrogen-Ion Concentration , Hydroxymethylbilane Synthase/chemistry , Kinetics , Male , Porphyrins/analysis , Porphyrins/isolation & purification , Rats , Rats, Inbred Strains , Sulfhydryl Compounds/metabolism , Uroporphyrinogens/biosynthesisABSTRACT
In all the cutaneous porphyrias, alterations in the heme pathway lead to an excessive production and accumulation of porphyrins. Absorption of light energy by circulating porphyrins induces reactive oxygen species generation, which provoke enzyme inactivation and protein structure changes. Protein structure alterations induced by porphyrins with different physico-chemical properties on delta-aminolevulinic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D) were examined. The action of uroporphyrin (URO), a highly hydrophilic porphyrin, and protoporphyrin (PROTO), most hydrophobic, was tested. ALA-D and PBG-D were partially purified from bovine liver and exposed to URO or PROTO, both in the dark and under UV light. All experiments were performed in solution after removing the porphyrins. Treatment with 10 microM URO I or 10 microM PROTO IX reduced the activity of ALA-D and PBG-D. This effect increased with increasing time of exposure to porphyrins. Solubility of the enzymes in buffer containing 3 M KCl decreased with increasing time of porphyrin treatment; this may be because of exposure of hydrophobic residues that are normally shielded in the native protein structure. Tryptic digestion of ALA-D and PBG-D exposed to URO I or PROTO IX resulted in an increase of protein degradation products, indicating an enhanced susceptibility to proteolysis. Fluorescence emission of several enzymes aminoacids was greatly modified. The structural changes described were observed when the enzymes were exposed to porphyrins both in the dark or under UV light. However, they were more noticeable with UV light. These results suggest that porphyrins per se can act directly on protein structure and that this action may be enhanced by UV irradiation.
Subject(s)
Hydroxymethylbilane Synthase/chemistry , Porphobilinogen Synthase/chemistry , Protoporphyrins/pharmacology , Uroporphyrins/pharmacology , Amino Acids/chemistry , Amino Acids/drug effects , Animals , Cattle , Enzyme Stability , Fluorescence , Hydroxymethylbilane Synthase/drug effects , Hydroxymethylbilane Synthase/metabolism , Porphobilinogen Synthase/drug effects , Porphobilinogen Synthase/metabolism , Protein Folding , Structure-Activity Relationship , Trypsin/metabolism , Ultraviolet RaysABSTRACT
In previous work we found a 30% increase in the effectiveness of the photodynamic treatment of cancer when combined with the administration of cyclophosphamide (CPM). Here we have tried to elucidate the mechanism responsible for such potentiation. Male Balb/C mice bearing a transplantable adenocarcinoma were given 2 or 3 doses of 150 mg of CPM/kg weight intraperitoneally. At 16 and 40 hrs. after the last injection the animals were sacrificed. Tumor and liver were excised and 5-aminolevulinic acid dehydratase and porphobilinogen deaminase activities were determined. Intracellular levels of glutathione and cytochrome P450 were also measured. A 15 to 30% decrease in liver 5-aminolevulinic acid dehydratase activity was observed 40 hrs. after the last injection. The tumor enzyme was 30 to 40% inhibited. The activity of liver porphobilinogen deaminase in CPM treated mice decreased to a minimum (15% below the control) at 16 hrs. after administration of the drug and in tumors a decrease of 20% was shown 40 hrs. post CPM injection. The greater the number of CPM doses administered the higher the decrease in the enzymatic activities. CPM treatment did not change total tumor glutathione levels but the reduced/oxidized glutathione ratio was significantly modified in the tumoral tissue. Cytochrome P450 levels were not increased. These data indicate that CPM-induced potentiation of the photodynamic damage of tumoral tissue is mediated by a mechanism other than that of increased porphyrin synthesis.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Cyclophosphamide/pharmacology , Heme/metabolism , Animals , Cytochrome P-450 Enzyme System/metabolism , Glutathione/metabolism , Hydroxymethylbilane Synthase/metabolism , Male , Mice , Mice, Inbred BALB C , Porphobilinogen Synthase/metabolismABSTRACT
1. The influence of strain and sex on the effect of enflurane and isoflurane and administration on heme metabolism was investigated to identify the animal model which could best reproduce the biochemical signs of acute intermittent porphyria. 2. Enflurane produced 35% and 80% increases in ALA-S activity only in CF1 male and female mice, respectively, whereas isoflurane induced 40% enzyme activity in CF1 male. 3. CF1 males showed around 35% decrease in blood PBGase and PBG-deaminase after administration of enflurane, whereas isoflurane provoked a striking inhibition (70%) in males of the C57 strain. 4. Enflurane produced alterations in heme synthesis, which would fit a model of acute porphyria in CF1 male mice. On the other hand, isoflurane would mimic biochemical alterations of this porphyria in C57 males.
Subject(s)
Anesthetics, Inhalation/pharmacology , Enflurane/pharmacology , Heme/metabolism , Isoflurane/pharmacology , 5-Aminolevulinate Synthetase/metabolism , Ammonia-Lyases/metabolism , Animals , Female , Hydroxymethylbilane Synthase/metabolism , Male , Mice , Mice, Inbred C57BL , Sex Factors , Species SpecificityABSTRACT
In the last decades several authors have observed a frequent association between diabetes mellitus and porphyria, mainly porphyria cutanea tarda. In previous studies, it has been demonstrated that both d delta d-aminolevulic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D), enzymes of the heme pathway, are inhibited by high concentrations of glucose in vitro in crude preparations of erythrocytes. The activity of these same enzymes was diminished in different tissues obtained from streptozotocin induced diabetic mice. Therefore, we decided to investigate the incidence of heme metabolism alterations in diabetes mellitus in a population of 100 non selected adult patients. The activities of erythrocytic ALA-D and PBG-D were measured. Rhodanese, an enzyme of the sulfocompounds pathway closely related to the regulation of heme biosynthesis, was also studied. Urine porphyrin content as well as the chromatographic pattern of esterified porphyrins were determined. ALA-D and PBG-D activities were diminished in diabetic patients (40 and 20 respectively), while rhodanese was only slightly increased (Fig. 1). ALA-D activity was subnormal in a 92 of the complete diabetic population, while PBG-D activity was less than normal in a 79 of the same population. No significative differences between enzymic activities were observed in the groups insulin and non-insulin dependent (Fig. 3). Urine porphyrin content was increased in 5 of the diabetic population. Chromatographic pattern of urinary porphyrins was notably altered in diabetic patients irrespectively of their porphyrin content (Fig. 4), suggesting an alteration in the enzyme uroporphyrinogen decarboxylase resembling the primary enzymic defect observed in porphyria cutanea tarda.(ABSTRACT TRUNCATED AT 250 WORDS)(Au)
Subject(s)
Humans , Male , Female , Adult , Middle Aged , RESEARCH SUPPORT, NON-U.S. GOVT , Diabetes Mellitus/metabolism , Heme/metabolism , Blood Glucose/analysis , Diabetes Mellitus/complications , Diabetes Mellitus/enzymology , Hydroxymethylbilane Synthase/metabolism , Porphobilinogen Synthase/metabolism , Porphyria Cutanea Tarda/etiology , Porphyrins/urine , Uroporphyrinogen Decarboxylase/metabolismABSTRACT
Uroporphyrinogen I Synthase (URO-S) activity was measured in erythrocytes of female and male rats which had received diethylnitrosamine (DENA) as an inducer of hepatic tumors. Twenty-two weeks after the last dose of the carcinogen, the rats showed statistically significant increases in the URO-S activity. Differences in the body weight, erythrocyte porphyrin content or the hematocrit between treated and control rats were not found. Fifty percent of female rats and thirty percent of male rats treated with DENA were found to have hepatic tumors but there was no correlation between blood URO-S activity and tumoral development in spite of the increase in URO-S activity observed in DENA treated rats. This was observed both in male and female rats.
Subject(s)
Carcinoma, Hepatocellular/enzymology , Diethylnitrosamine/therapeutic use , Erythrocytes/metabolism , Hydroxymethylbilane Synthase/metabolism , Liver Neoplasms/enzymology , Animals , Female , Hydroxymethylbilane Synthase/blood , Male , RatsABSTRACT
In the last decades several authors have observed a frequent association between diabetes mellitus and porphyria, mainly porphyria cutanea tarda. In previous studies, it has been demonstrated that both d delta d-aminolevulic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D), enzymes of the heme pathway, are inhibited by high concentrations of glucose in vitro in crude preparations of erythrocytes. The activity of these same enzymes was diminished in different tissues obtained from streptozotocin induced diabetic mice. Therefore, we decided to investigate the incidence of heme metabolism alterations in diabetes mellitus in a population of 100 non selected adult patients. The activities of erythrocytic ALA-D and PBG-D were measured. Rhodanese, an enzyme of the sulfocompounds pathway closely related to the regulation of heme biosynthesis, was also studied. Urine porphyrin content as well as the chromatographic pattern of esterified porphyrins were determined. ALA-D and PBG-D activities were diminished in diabetic patients (40% and 20% respectively), while rhodanese was only slightly increased (Fig. 1). ALA-D activity was subnormal in a 92% of the complete diabetic population, while PBG-D activity was less than normal in a 79% of the same population. No significative differences between enzymic activities were observed in the groups insulin and non-insulin dependent (Fig. 3). Urine porphyrin content was increased in 5% of the diabetic population. Chromatographic pattern of urinary porphyrins was notably altered in diabetic patients irrespectively of their porphyrin content (Fig. 4), suggesting an alteration in the enzyme uroporphyrinogen decarboxylase resembling the primary enzymic defect observed in porphyria cutanea tarda.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Diabetes Mellitus/metabolism , Heme/metabolism , Adult , Blood Glucose/analysis , Diabetes Complications , Diabetes Mellitus/enzymology , Female , Humans , Hydroxymethylbilane Synthase/metabolism , Male , Middle Aged , Porphobilinogen Synthase/metabolism , Porphyria Cutanea Tarda/etiology , Porphyrins/urine , Uroporphyrinogen Decarboxylase/metabolismABSTRACT
Se midió la actividad de Uroporfirinógeno I sintasa (URO-S) en eritrocitos de ratas hembras y machos que habían recibido dietilnitrosamina (DENA) como inductor de tumores hepáticos. Veintidós semanas después de la última dosis del carcinógeno, las ratas mostraron incrementos estadísticamente, significativos en la actividad de URO-S. No se encontraron diferencias en el peso de los animales, en el contenido de porfirinas eritrocitarias ni en el hematocrito entre las ratas tratadas y los animales control. Se observó que el cincuenta por ciento de las ratas hembras y el treinta por ciento de las ratas machos tratadas con DENA habían desarrollado tumores hepáticos pero no hubo correlación, ni en machos ni en hembras, entre la actividad de URO-S y el desarrollo tumoral a pesar del incremento obtenido en los animales tratados con DENA en la actividad de esta enzima