Uroporphyrinogen Synthetase Research Articles

Congenital Erythropoietic Porphyria about a succcessful evolution case

BACKGROUND: Congenital erythropoietic porphyria is an extremely rare autosomal recessive disease. Caused by a deficiency of the enzymatic activity of uroporphyrinogen synthetase III (UROS), collect accumulation of porphyrins of the type I isomer and alterations in the synthesis of the heme group, responsible for the clinical manifestations, mainly cutaneous, hematological, ophthalmic and bone. Diagnosis is based on elevated levels of type I uroporphyrinogen in urine and fecal coproporphyrinogen and clinical findings. CLINICAL CASE: This is a 31-year-old female patient who presented since childhood, multiple hyper and hypopigmented macules both on the back of the hands and on the face, slight destruction of the nasal cartilage, areas of scarring and perioral retraction, sclerodermiform changes, erythrodontia and marked retraction of the phalanges, who was diagnosed with congenital hematopoietic porphyria, who began conservative treatment, achieving an adequate evolution and clinical stability. CONCLUSIONS: It is important to know the different manifestations of this disease to show a prompt diagnosis and avoid known progression, since despite being a rare disease, it can have high morbidity in untreated patients.

Sodium butyrate increases the effect of the photodynamic therapy: a mechanism that involves modulation of gene expression and differentiation in astrocytoma cells

In order to evaluate the improvement of the photodynamic therapy (PDT) due to sodium butyrate (NaBu), its effectiveness in U373-MG and D54-MG astrocytoma cell lines was evaluated. Cells were exposed to delta-aminolevulinic acid (δ-ALA) as a precursor to endogenous photosensitizer protoporphyrin IX (PpIX). In both astrocytoma cells, an important increase by ALA was observed in uroporphyrinogen synthetase gene expression: 1.8- and 52-fold for D54-MG and U373-MG cells, respectively. After irradiation, they showed 16.67 and 28.9% of mortality in U373-MG and D54-MG, respectively. These mortalities increased to 70.62 and 96.7% when U373-MG and D54-MG cells, respectively, were exposed 24 h to 8 mM NaBu, before to PpIX induction. NaBu induced expression of caspase-3, caspase-9, and Bcl-2 and increased Bax in U373-MG cells. ALA-induced morphological changes are compatible to differentiation. Genes and differentiation induced mainly by NaBu improve cell death performed by PDT in astrocytoma cells. These facts prove the synergistic effect of NaBu on cytotoxic damage induced by PDT.

Effect of Zinc or S‐Adenosyl‐l‐methionine on Long Term Administration of Low Doses of Lead to Rats

Two alternatives for the treatment of lead intoxication, administration of zinc or a thiol donor, S-adenosyl-L-methionine (SAM), were analysed. Rats were exposed to lead (Pb)-acetate (60 mg/l) in drinking water during 90 days; one group also received SO4Zn in water (40 mg/l), while another received both Pb and SAM (5 mg/24 hr intraperitoneally. Erythrocytic delta-aminolaevulinic dehydratase (ALA-D) activity was significantly reduced (P < 0.001) both in rats receiving Pb alone and in rats receiving Pb and each of the other two treatments. The high erythrocytic uroporphyrinogen synthetase (URO-S) activity noticed in Pb administered rats, was significantly (P < 0.001) reduced in animals treated either with zinc or with SAM. Hepatic ALA-D activity tended to decrease while renal enzyme activity was not modified by the low level Pb exposure used in this work. Interestingly, SAM treated rats in both tissues exhibited significantly (P < 0.01) higher activities of the enzyme. It is argued that SAM treatment causes a surplus of thiols that allows the full expression of ALA-D catalytic activity.

A new method for the synthesis of glutamate 1-semialdehyde. Characterization of its structure in solution by NMR spectroscopy

Glutamate 1-semialdehyde is formed in high yields by ozonolysis of 4-vinyl-4-aminobutyric acid and purification of the reaction products by cation exchange chromatography. The structure of the product and its pyrrole derivative have been confirmed by1H-NMR and13C-NMR spectroscopy, and by its conversion to σ-aminolevulinate by purified glutamate 1-semialdehyde aminotransferase, and to uroporphyrinogen with porphobilinogen and uroporphyrinogen synthetase. The NMR spectra provide evidence for the presence in solution of a hydrate of glutamate 1-semialdehyde. Concentration dependent formation of dimers of the hydrate was also shown. It is suggested that chemical conversion of glutamate 1-semialdehyde to σ-aminolevulinate proceeds through dimeric intermediate stages.

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The activities of δ-aminolevulinic acid (ALA) synthetase,1 ALA dehydratase, uroporphyrinogen synthetase, and the quantities of the metabolites, porphobilinogen (PBG), coproporphyrin, and protoporphyrin were serially assayed in the livers of inbred mice which had received repeated injections of 3,5-diethoxycarbonyl-1,4-dihydro-2, 4,6-trimethylpyridine (DDC). During 48 hr of drug treatment, ALA synthetase activity increased 5- to 20-fold above a basal activity of 30–100 nmoles ALA formed/ hr/g liver. Hepatic uroporphyrinogen synthetase activity did not increase above a basal level of 35–60 nmoles PBG consumed/hr/g liver and appeared to represent the rate-limiting step during most of the course of induction of porphyria. ALA dehydratase activity was dependent upon genotype at the Lv locus and was strain specific, but in all strains this enzyme was present in excess (700–4800 nmoles/hr/g liver). The activity of the dehydratase increased 30% during 48 hr of drug treatment. PBG, proto- and, to a lesser extent, coproporphyrins accumulated, but much less porphyrin was found in livers of C57L/J mice than in those of A/HeJ, C57BL/6J, DBA/2J, SWR/J or C3H/HeJ. Hepatic protoporphyrin accumulation was directly correlated with ALA synthetase activity provided that the protoporphyrin level was less than 50 nmoles/g liver. When hepatic protoporphyrin exceeded 100 nmoles/g liver, there was an inverse correlation between ALA synthetase activity and protoporphyrin content suggesting that either feedback inhibition or feedback repression of the synthetase had occurred.

38 Erythrocyte uroporphyrinogen synthetase in children

A micellar electrokinetic capillary chromatographic (MECC) method has been developed and optimised for the separation of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG). The running buffer consisted of a mixture of 20 mM sodium phosphate and 20 mM sodium borate containing 50 mM sodium dodecyl sulphate (SDS) adjusted to pH 9.5 with 1 M NaOH. The running voltage and temperature were 20–25 kV and 30°C, respectively. The MECC method for the analysis of PBG is fast and simple and is useful for the screening of PBG in the urine of patients suspected to have acute intermittent porphyria (AIP), and for the confirmation of lead exposure by measuring red-cell ALA-dehydratase (ALA-D) activity with ALA as the enzyme substrate.

582 Δ-AMINOLEVULINIC ACID (ALA) SYNTHETASE DEFECT IN A FEMALE WITH CONGENITAL SIDEROBLASTIC ANEMIA

Since birth a 3-year old black girl has had severe refractory hypochromic microcytic anemia. Her usual red blood cell (RBC) values are Hgb 6.0 gm/dl, Hct 20%, Retic. 0.5%, MCV 52 fl, MCH 15 pg. Iron deficiency and clinically significant thalassemia were excluded by: lack of response to oral or parenteral iron, serum Fe/TIBC 231/276 μg/dl and serum ferritin 407 ng/ml; normal hemoglobin electrophoresis, absence of erythroid inclusions in the peripheral blood and bone marrow (BM), α/β globin synthetic ratio of 0.85 in peripheral blood reticulocytes and negative family studies. Intensive erythroid hyperplasia and numerous ring sideroblasts were present in the BM aspirate, and electron microscopy confirmed intramitochondrial deposits of iron. Free RBC protoporphyrin, urine porphyrins and porphyrin precursors, and BM ferrochelatase activity were normal. RBC ALA dehydrase, uroporphyrinogen synthetase and pyridoxal kinase activities were increased. Activity of BM ALA synthetase was markedly reduced to 5.5 pmoles ALA/106 erythroblasts/30 min (normal 127±29) but was enhanced 5-fold by pyridoxal phosphate (normal 0-25% increase). Therapy with oral pyridoxine has thus are not noticeably increased effective RBC production. The sideroblastic anemia in this patient appears to be related to an inherited defect in the initial step of heme biosynthesis.

STUDIES IN PORPHYRIA

Porphyrin biosynthesis in mammalian skin and in skin obtained from patients with selected types of porphyria has been studied. Cutaneous porphyrinogenesis required the precursor delta-aminolevulinic acid (ALA) which, when added to murine, rat, and human skin in vitro, was rapidly converted to porphyrins. Total porphyrin content was quantitated by fluorescence assay, and spectral studies indicated that more than 80% of the porphyrin produced was protoporphyrin. The majority of skin porphyrinogenesis occurrred in epidermis or in epidermal derivatives such as hair roots. Known inducers of hepatic delta-aminolevulinic acid synthetase (ALAS), the rate-limiting enzyme for heme biosynthesis, were not inducers when added to skin in vitro. Skin from patients with acute intermittent porphyria demonstrated a 43% decrease in cutaneous porphyrin production as compared to unaffected normals. This is consistent with the known deficiency of uroporphyrinogen synthetase that has been previously demonstrated in the liver and red blood cells of these patients. Porphyrinogenesis in skin of patients with porphyria cutanea tarda was not different from controls. These studies demonstrate that skin has the enzymatic capacity to synthesize porphyrins from added ALA and that cutaneous porphyrinogenesis from ALA is deficient in patients with acute intermittent porphyria.

The activities of uroporphyrinogen synthetase and cosynthetase in congenital erythropoietic porphyria (CEP).

Normal or increased amounts of series III porphyrins with greater amounts of series I were observed on incubation of PBG in hemolysates of congenital erythropoietic porphyria vs. normal erythrocytes, human or bovine. Correlation with reticulocyte percentage was poor, in the aggregate a general trend toward increased values of both isomers I and III was noted with increasing reticulocytes. When the percent of type III was low the net amount was increased as compared with normal. Hemolysates of non-porphyric, reticulocyte-rich red cells (hemolytic or posthemorrhagic anemia) formed only minute amounts of type I porphyrin but at the same time no more, or even less type III than the porphyric hemolysates, although representing red cells of greater reticulocyte content. No evidence of deficient heme synthesis was observed in porphyric hemolysates incubayed with [14C]-porphobilinogen or 59Fe. Other studies of porphyric hemolysates incubated with and without added mouse spleen synthetase failed to reveal evidence of an absolute UPG-III cosynthetase (Co-S) deficiency. The large increases of type I porphyrin with normal or increased formation of type III, both in the disease and in the hemolysates, are believed due to a primary increase of ALA-S or UPG-S activity rather than a decrease of Co-S. Possible mutations which might be responsible for this increase are considered.

Uroporphyrin-accumulating mutant of Escherichia coli K-12.

An uroporphyrin III-accumulating mutant of Escherichia coli K-12 was isolated by neomycin. The mutant, designated SASQ85, was catalase deficient and formed dwarf colonies on usual media. Comparative extraction by cyclohexanone and ethyl acetate showed the superiority of the former for the extraction of the uroporphyrin accumulated by the mutant. Cell-free extracts of SASQ85 were able to convert 5-aminolevulinic acid and porphobilinogen to uroporphyrinogen, but not to copro- or protoporphyrinogen. Under the same conditions cell-free extracts of the parent strain converted 5-aminolevulinic to uroporphyringen, coproporphyrinogen, and protoporphyrinogen. The conversion of porphobilinogen to uroporphyrinogen by cell-free extracts of the mutant was inhibited 98 and 95%, respectively, by p-chloromercuribenzoate and p-chloromercuriphenyl-sulfonate, indicating the presence of uroporphyrinogen synthetase activity in the extracts. Spontaneous transformation of porphobilinogen to uroporphyrin was not detectable under the experimental conditions used [4 h at 37 C in tris(hydroxymethyl)aminomethane-potassium phosphate buffer, pH 8.2]. The results indicate a deficient uroporphyrinogen decarboxylase activity of SASQ85 which is thus the first uroporphyrinogen decarboxylase-deficient mutant isolated in E. coli K-12. Mapping of the corresponding locus by P1-mediated transduction revealed the frequent joint transduction of hemE and thiA markers (frequency of co-transduction, 41 to 44%). The results of the genetic analysis suggest the gene order rif, hemE, thiA, metA; however, they do not totally exclude the gene order rif, thiA, hemE, metA.

Editorial: Hematin and porphyria.

The discovery that hematin represses induction of δ-aminolevulinic acid synthetase (ALA-S), the rate-limiting enzyme for hepatic porphyrin and heme synthesis,1 is central to recent studies seeking to provide a means of terminating or ameliorating the acute attack of acute intermittent porphyria, porphyria variegata or hepatic coproporphyria. The induction of ALA-S in the acute attack of AIP, acute intermittent porphyria, porphyria variegata or hepatic coproporphyria markedly increases the porphyrin precursors, ALA and porphobilinogen (PBG), in liver, blood plasma and urine. The enzyme is induced because of heme deficiency in the liver secondary to the genetic partial lack of uroporphyrinogen synthetase (UPG-S) . . .

Exacerbation of hereditary hepatic porphyria by surreptitious ingestion of an unusual provocative agent--a mouthwash preparation.

EXACERBATIONS of acute intermittent porphyria are provoked by many commonly used drugs known to stimulate hepatic porphyrin-heme synthesis. In the patient described below, surreptitious ingestion of a seemingly innocuous agent — a mouthwash preparation — appeared to precipitate acute attacks of this disease.Case ReportA 48-year-old woman known to have acute intermittent porphyria was admitted to the Rockefeller University Hospital in severe clinical crisis in 1970. Urinary δ-aminolevulinic acid and porphobilinogen levels were highly elevated, and erythrocyte uroporphyrinogen synthetase activity was diagnostic for this genetic disease.1,2 She responded to therapy and was discharged within a month. A series of . . .

Anesthesia and the porphyrias.

A simplified classification of the porphyrias is given which is thought to be advantageous to the anesthesiologist in determining those patients who are predisposed to acute attacks. These acute attacks may be precipitated by the administration of barbiturates, but may also be spontaneous. The current theory for the precipitation of the acute attack is described, with the probable mechanism being a decrease in uroporphyrinogen synthetase levels and the resultant interference in heme production. Increased formation of cytochrome P-450 with barbiturates also produces increased levels of delta aminolevulinic acid, which may be a cause of the acute attack. The significance in anesthesia and suggested means of anesthetic management are discussed.

Polypyrroles formed from porphobilinogen and amines by uroporphyrinogen synthetase of Rhodopseudomonas spheroides.

1. Uroporphyrinogen I synthetase of Rhodopseudomonas spheroides was purified more than 200-fold from the soluble protein of broken bacterial cells. The enzyme had molecular weight 36000, an isoelectric point of 4.46 and migrated as a single active protein band on disc-gel electrophoresis at pH7.5 and 8.9. 2. The enzyme consumed porphobilinogen and formed uroporphyrinogen at pH8.2 without the accumulation of intermediates. In the presence of hydroxylamine, ammonia or methoxyamine the production of porphyrinogen was inhibited and the enzyme formed open-chain polypyrroles instead. 3. These polypyrroles behaved like uroporphyrinogen on Sephadex G-25; they were colourless and had unsubstituted alpha-pyrrolic positions. The inhibitory amines were incorporated into the molecules. 4. The polypyrroles formed porphyrins non-enzymically and the cyclization reaction was accompanied by the release of the inhibitory amine. Exchange of the amino function of the original porphobilinogen in the polypyrrole was complete with hydroxylamine and almost complete with methoxyamine, both ammonia and methoxyamine being present in the polypyrrolic material. 5. The behaviour, properties and composition of the radioactive hydroxylamine derivative were consistent with a tetrapyrrolic structure, probably a pyrrylmethane, that was not cyclized, rather than with di-, tri- or penta-pyrrolic structures. No monopyrrolic or dipyrrolic Ehrlich-positive material was released on cyclization. The ammonia and methoxyamine derivatives had properties similar to the hydroxylamine derivative. 6. Another modified pyrrole was detected only in experiments with hydroxylamine. It differed from both porphobilinogen and known dipyrroles and appeared to be a monopyrrole. 7. The participation of positively charged reaction centres in the enzymic mechanism, particularly in the cyclization step, is discussed.

Congenital erythropoietic porphyria: a family study.

We present the results of a study of the porphyrin‐forming enzymes in the erythrocytes of a recently detected case of CBP, in the immediate family, and in the family of a second cousin who died in infancy with a clinical picture similar lo that seen in the present patient. Except for a moderate increase in the activity of the uroporphyrinogen synthetase in the patient's lysates no patological changes have been found either in the patient or in any of the other persons studied.Our findings do not exclude the possibility that impaired activity of the isomerase (uroporphyrinogen‐III‐cosynthetase) plays a role, but they do indicate that this is not the only or even the most important metabolic error in the present type of erythropoictic porphyria.A study of the kinship between the two families seems to make a simple autosomal recessive in. heritance unlikely, hut does not exclude dominant inheritance by a rare single gene with low penetrance carried by the fathers. It is possible that the present case may represent a type of porphyria variegata in which the metabolic defect in porphyrin biosynthesis is located not in the liver hut in the bone marrow.

Accumulation of porphobilinogen and other pyrroles by mutant and wild type Rhodopseudomonas spheroides: Regulation by heme

Certain mutant strains of Rhodopseudomonas spheroides accumulate coproporphyrin(ogen) and porphobilinogen when incubated with low aeration in malate-glutamate medium supplemented with glycine and succinate. Strains 6-6 and 6-6R have barely detectable levels of uroporphyrinogen synthetase and accumulate porphobilinogen. Strain 6-6R is more active than 6-6 in porphobilinogen formation but is less active in heme synthesis. Production of porphobilinogen by strain 6-6 is stimulated by addition of δ-aminolevulinate or o-phenanthroline but neither additive affects strain 6-6R. Strain 2–33 accumulates porphobilinogen and coproporphyrin(ogen) and is exceptionally low in heme synthesis. Inhibition of bacteriochlorophyll synthesis by puromycin in the wild type and strain 6-6R is accompanied by an acceleration in heme synthesis. The wild type accumulates coproporphyrin(ogen) upon addition of o-phenanthroline but this is prevented by the prior addition of puromycin. It is concluded that the excess production of pyrroles by the mutants and by the wild type in response to o-phenanthroline is attributable to failure of feedback control of δ-aminolevulinate synthetase by heme. The mutants are convenient sources of porphobilinogen and coproporphyrin.

Regulation of porphyrin biosynthesis in the Harderian gland of inbred mouse strains

The porphyrin biosynthetic pathway has been evaluated in Harderian glands of several inbred mouse strains. In female mice porphyrin content in a pair of Harderian glands varies from 0.6 μg in C57BL/6J to 73.4 μg in AKR/J. This latter is equivalent to nearly 3000 μg/g tissue. The quantity of porphyrin and δALA 1 1 The following abbreviations are used in the text: δALA, 3-aminolevulinic acid; DDC, 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethyl pyridine; PBG, porphobilinogen; SD, standard deviation of the mean; Uro, uroporphyrinogen. synthetase is different in males and females of the same strain. Compounds that induce hepatic porphyria are without effect on Harderian gland porphyrin content. Evaluation of the activities of three enzymes in the pathway suggests that the porphyrin level is correlated with the activity of δALA synthetase but not of uroporphyrinogen synthetase or δALA dehydratase. The genetic locus (loci) regulating this system is complex and distinct from that which determines the level of δALA dehydratase in mouse liver (Lv). It is also apparently unrelated to the inducibility of hepatic δALA synthetase in mice and, therefore, probably represents a new aspect of regulation of the porphyrin pathway in mice.

Chemical induction of hepatic porphyria in inbred strains of mice

The activities of δ-aminolevulinic acid (ALA) synthetase, 1 1 The following ABBREVIATIONS are used in the text: ALA, δ-aminolevulinic acid; PBG, porphobilinogen; Uro, uroporphyrinogen; Copro, coproporphyrinogen; Proto, protoporphyrin; DDC, 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine; Tween 80, polyoxyethylene sorbitan monooleate; SEM, standard error of the mean. ALA dehydratase, uroporphyrinogen synthetase, and the quantities of the metabolites, porphobilinogen (PBG), coproporphyrin, and protoporphyrin were serially assayed in the livers of inbred mice which had received repeated injections of 3,5-diethoxycarbonyl-1,4-dihydro-2, 4,6-trimethylpyridine (DDC). During 48 hr of drug treatment, ALA synthetase activity increased 5- to 20-fold above a basal activity of 30–100 nmoles ALA formed/ hr/g liver. Hepatic uroporphyrinogen synthetase activity did not increase above a basal level of 35–60 nmoles PBG consumed/hr/g liver and appeared to represent the rate-limiting step during most of the course of induction of porphyria. ALA dehydratase activity was dependent upon genotype at the Lv locus and was strain specific, but in all strains this enzyme was present in excess (700–4800 nmoles/hr/g liver). The activity of the dehydratase increased 30% during 48 hr of drug treatment. PBG, proto- and, to a lesser extent, coproporphyrins accumulated, but much less porphyrin was found in livers of C57L/J mice than in those of A/HeJ, C57BL/6J, DBA/2J, SWR/J or C3H/HeJ. Hepatic protoporphyrin accumulation was directly correlated with ALA synthetase activity provided that the protoporphyrin level was less than 50 nmoles/g liver. When hepatic protoporphyrin exceeded 100 nmoles/g liver, there was an inverse correlation between ALA synthetase activity and protoporphyrin content suggesting that either feedback inhibition or feedback repression of the synthetase had occurred.