Biochemical Differentiation of the Porphyrias

Total Page:16

File Type:pdf, Size:1020Kb

Biochemical Differentiation of the Porphyrias Clinical Biochemistry, Vol. 32, No. 8, 609–619, 1999 Copyright © 1999 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/99/$–see front matter PII S0009-9120(99)00067-3 Biochemical Differentiation of the Porphyrias J. THOMAS HINDMARSH,1,2 LINDA OLIVERAS,1 and DONALD C. GREENWAY1,2 1Division of Biochemistry, The Ottawa Hospital, and the 2Department of Pathology and Laboratory Medicine, University of Ottawa, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada Objectives: To differentiate the porphyrias by clinical and biochem- vals for urine, fecal, and blood porphyrins and their ical methods. precursors in the various porphyrias and in normal Design and methods: We describe levels of blood, urine, and fecal porphyrins and their precursors in the porphyrias and present an subjects and have devised an algorithm for investi- algorithm for their biochemical differentiation. Diagnoses were es- gation of these diseases. Except for Porphyria Cuta- tablished using clinical and biochemical data. Porphyrin analyses nea Tarda (PCT), our numbers of patients in each were performed by high performance liquid chromatography. category of porphyria are small and therefore our Results and conclusions: Plasma and urine porphyrin patterns reference ranges for these should be considered were useful for diagnosis of porphyria cutanea tarda, but not the acute porphyrias. Erythropoietic protoporphyria was confirmed by approximate. erythrocyte protoporphyrin assay and erythrocyte fluorescence. Acute intermittent porphyria was diagnosed by increases in urine Materials and methods delta-aminolevulinic acid and porphobilinogen and confirmed by reduced erythrocyte porphobilinogen deaminase activity and nor- REAGENTS AND CHEMICALS mal or near-normal stool porphyrins. Variegate porphyria and he- reditary coproporphyria were diagnosed by their characteristic stool porphyrin patterns. This appears to be the most convenient diag- Porphyrin standards were obtained from Porphy- nostic approach until molecular abnormalities become more exten- rin Products Inc. (Logan, UT). All solvents were of sively defined and more widely available. Copyright © 1999 The high performance liquid chromatography (HPLC) Canadian Society of Clinical Chemists grade. KEY WORDS: Porphyria; porphyrins; feces; urine; EQUIPMENT blood; plasma; erythrocytes; renal failure; abdominal pain; neuropathy; erythema; urticaria; photodermatitis. HPLC was performed using a Varian 5500 (Var- ian Canada Inc., Mississauga, ON) equipped with a Introduction Shimadzu RF-1501 spectrofluorometer (Shimadzu Scientific Instruments, Inc., Columbia, MD). The he biochemical differentiation of the porphyrias column was a Perkin-Elmer (Norwalk, CT) reversed Tresides, at present, with the measurement of phase Percospher 3 C18 Brownlee, 33 ϫ 4.6 mm. porphyrins and their metabolites in urine, feces and Data analysis was performed using Dionex A1-450 blood (1). Enzyme tests are often technically difficult chromatography software (Dionex Canada Ltd., and require tissues such as cultured fibroblasts, Oakville, ON). lymphocytes, or liver biopsy material and therefore, except for the use of erythrocyte porphobilinogen PLASMA PORPHYRINS deaminase in acute intermittent porphyria, they are rarely used (2). Genetic analysis produces a precise Plasma porphyrin analysis was performed accord- diagnosis in some of the porphyrias provided the ing to the method of Hindmarsh et al. (4). Heparin- patient has one of the previously described point ized plasma was used, samples being centrifuged mutations, but this technique is currently confined immediately after collection and frozen at Ϫ20° C. to a few research laboratories (3). As a consequence Stock standards were prepared using porphyrin of our role as a major reference laboratory for markers in porphyrin-free plasma obtained from our porphyrin analysis performing approximately 2500 hospital blood transfusion service; 2-vinyl 4-hy- tests per year, we have assembled reference inter- droxymethyl-deuteroporphyrin IX was used as an internal standard. The recovery of individual por- Correspondence: Dr. J.T. Hindmarsh, The Ottawa Hos- phyrins added to porphyrin-free plasma varied from pital, General Campus, 501 Smyth Road, Ottawa, Ontario 89% to 114% except for coproporphyrin-III, whose K1H 8L6, Canada. recovery varied from 71% to 99%. Recoveries of Manuscript received July 13, 1999; revised August 13, protoporphyrin were low, therefore, no attempt was 1999; accepted August 17, 1999. made to quantify this fraction. Between-run preci- CLINICAL BIOCHEMISTRY, VOLUME 32, NOVEMBER 1999 609 HINDMARSH, OLIVERAS, AND GREENWAY sion varied from 5.4% to 13.2%, coproporphyrin-III Urine ALA and porphobilinogen (PBG) were mea- being the least precise. sured by the method of Mauzerall and Granick (9) using a BioRad kit (BioRad Laboratories, Missis- URINE PORPHYRINS sauga, Ontario, Canada). Urine porphyrins were measured by a reversed SCREENING TESTS phase HPLC method based upon that of Johnson et al. (5). To achieve separation of the I and III isomers Urine was screened for increased porphyrin con- of uroporphyrin and coproporphyrin, we substituted tent by spectrophotometric scanning of an acidified their mobile phase with a gradient ofa1mol/L sample between 350 and 415 nm and measuring the ammonium acetate solution (pH 5.16) and metha- peak absorption at 405 nm. The molar absorptivity nol, varying from 27% (starting) to 90% (ending) of was then used to obtain the total porphyrin content methanol V/V. Direct standardization was used with (10) and a “cut off” of 150 nmol/d was used to ensure uroporphyrin-I, uroporphyrin-III, heptacarboxyl-I, a maximal detection of normal subjects. Currently hexacarboxyl-I, pentacarboxyl-I, coproporphyrin-I, in our service laboratory we use a “cut off” of 110 and coproporphyrin-III. Recoveries of standard por- nmol/d to ensure maximal detection of abnormal phyrins added to urine samples varied from 92% to subjects (11). Fecal screening was performed by 115%. Between-run precision for the various frac- extracting interfering substances from an acidified tions varied between 7% and 10%. Samples were sample with diethylether and scanning the remain- preserved at 4° C with 5-g sodium carbonate per ing aqueous fraction in a spectrophotometer. Peak daily collection in brown glass bottles. absorption was measured and a “cut off” of 35 nmol/g wet feces was used (12). We prefer spectrophotomet- FECAL PORPHYRINS ric absorption screening techniques rather than flu- orometric screening methods because we have en- Our fecal porphyrin method that was based upon countered too many false positive results with the the reversed phase HPLC method of Pudek et al. (6), latter. but using direct standardization with uroporphyrin-I, uroporphyrin-III, heptacarboxyl-I, hexacarboxyl-I, Results pentacarboxyl-I, coproporphyrin-I and copropor- phyrin-III, deuteroporphyrin, mesoporphyrin, and Tables 1, 2, and 3 report plasma, urine, and fecal protoporphyrin. We used a 3 ϫ 0.43 cm (ID) cartridge porphyrin and precursor results in a variety of analytical column containing 3-␮m octadecylsilyl par- porphyrias and include our normal reference inter- ticles. The solvent system was a gradient ofa1mol/L vals. The data on Table 1 have been published solution of ammonium acetate and methanol varying previously (4). All data were derived from samples from 27% (starting) to 90% (ending) of methanol V/V. we received in our role as a major porphyrin refer- Recovery of standards added to samples varied from ence laboratory and were checked for proper preser- 75% to 102%. Random samples of feces were preserved vation and transportation (acid pH for urine ALA at Ϫ20° C in opaque containers. Between-run CVs and PBG measurements, alkaline pH for urine por- varied from 10% to 15%. phyrin samples, samples frozen and transported in dry-ice for plasma porphyrins, samples frozen and ERYTHROCYTE PROTOPORPHYRIN wrapped in aluminum foil for fecal porphyrins). All abnormal data include the complete range of results Erythrocyte protoporphyrin was measured by the encountered by our laboratory for a particular dis- method of Piomelli (7). Blood samples were collected ease classification. Disease stratification was made in EDTA tubes (Becton Dickinson Canada, Inc., by an experienced clinician and porphyrin chemist Mississauga, ON) wrapped in aluminum foil and (J.T.H.) after discussion with the referring physician preserved at 4° C. Our between-run CV for this and perusal of porphyrin and precursor patterns in method was 10%. When defining our normal refer- urine and feces and erythrocyte porphobilinogen ence interval, all results from samples with hemat- deaminase activities. ocrit Յ 0.35 were excluded. NORMAL REFERENCE INTERVALS ERYTHROCYTE FLUORESCENCE “Normal” samples were chosen by visual inspec- Erythrocyte fluorescence was demonstrated by tion of the results of all specimens processed. In the briefly viewing an erythrocyte smear (diluted with case of urine and fecal samples, those designated 154 mmol/L saline, if necessary) using an Olympus “normal” had all tested negative by our screening fluorescence microscope with a BG3 (405 nm) exci- tests (10,12). The reference intervals were deter- tation filter. mined as a central 95% interval of ranked data Erythrocyte porphobilinogen deaminase activity using a nonparametric method (13). Modest eleva- was measured in duplicate using delta-aminolevu- tions above the apparently normal range were occa- linic acid (ALA) as substrate (8). Between
Recommended publications
  • Hyperbilirubinemia
    Porphyrins Porphyrins (Porphins) are cyclic tetrapyrol compounds formed by the linkage )). of four pyrrole rings through methenyl bridges (( HC In the reduced porphyrins (Porphyrinogens) the linkage of four pyrrole rings (tetrapyrol) through methylene bridges (( CH2 )) The characteristic property of porphyrins is the formation of complexes with the metal ion bound to nitrogen atoms of the pyrrole rings. e.g. Heme (iron porphyrin). Proteins which contain heme ((hemoproteins)) are widely distributed e.g. Hemoglobin, Myoglobin, Cytochromes, Catalase & Tryptophan pyrrolase. Natural porphyrins have substituent side chains on the eight hydrogen atoms numbered on the pyrrole rings. These side chains are: CH 1-Methyl-group (M)… (( 3 )) 2-Acetate-group (A)… (( CH2COOH )) 3-Propionate-group (P)… (( CH2CH2COOH )) 4-Vinyl-group (V)… (( CH CH2 )) Porphyrins with asymmetric arrangement of the side chains are classified as type III porphyrins while those with symmetric arrangement of the side chains are classified as type I porphyrins. Only types I & III are present in nature & type III series is more important because it includes heme. 1 Heme Biosynthesis Heme biosynthesis occurs through the following steps: 1-The starting reaction is the condensation between succinyl-CoA ((derived from citric acid cycle in the mitochondria)) & glycine, this reaction is a rate limiting reaction in the hepatic heme synthesis, it occurs in the mitochondria & is catalyzed by ALA synthase (Aminolevulinate synthase) enzyme in the presence of pyridoxal phosphate as a cofactor. The product of this reaction is α-amino-β-ketoadipate which is rapidly decarboxylated to form δ-aminolevulinate (ALA). 2-In the cytoplasm condensation reaction between two molecules of ALA is catalyzed by ALA dehydratase enzyme to form two molecules of water & one 2 molecule of porphobilinogen (PBG) which is a precursor of pyrrole.
    [Show full text]
  • UROPORPHYRINOGEN HII COSYNTHETASE in HUMAN Hemolysates from Five Patientswith Congenital Erythropoietic Porphyriawas Much Lower
    UROPORPHYRINOGEN HII COSYNTHETASE IN HUMAN CONGENITAL ERYTHROPOIETIC PORPHYRIA * BY GIOVANNI ROMEO AND EPHRAIM Y. LEVIN DEPARTMENT OF PEDIATRICS, THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE Communicated by William L. Straus, Jr., April 24, 1969 Abstract.-Activity of the enzyme uroporphyrinogen III cosynthetase in hemolysates from five patients with congenital erythropoietic porphyria was much lower than the activity in control samples. The low cosynthetase activity in patients was not due to the presence of a free inhibitor or some competing en- zymatic activity, because hemolysates from porphyric subjects did not interfere either with the cosynthetase activity of hemolysates from normal subjects or with cosynthetase prepared from hematopoietic mouse spleen. This partial deficiency of cosynthetase in congenital erythropoietic porphyria corresponds to that shown previously in the clinically similar erythropoietic porphyria of cattle and explains the overproduction of uroporphyrin I in the human disease. Erythropoietic porphyria is a rare congenital disorder of man and cattle, characterized by photosensitivity, erythrodontia, hemolytic anemia, and por- phyrinuria.1 Many of the clinical manifestations of the disease can be explained by the production in marrow, deposition in tissues, and excretion in the urine and feces, of large amounts of uroporphyrin I and coproporphyrin I, which are products of the spontaneous oxidation of uroporphyrinogen I and its decarboxyl- ated derivative, coproporphyrinogen I. In cattle, the condition is inherited
    [Show full text]
  • Clinical and Biochemical Characteristics and Genotype – Phenotype Correlation in Finnish Variegate Porphyria Patients
    European Journal of Human Genetics (2002) 10, 649 – 657 ª 2002 Nature Publishing Group All rights reserved 1018 – 4813/02 $25.00 www.nature.com/ejhg ARTICLE Clinical and biochemical characteristics and genotype – phenotype correlation in Finnish variegate porphyria patients Mikael von und zu Fraunberg*,1, Kaisa Timonen2, Pertti Mustajoki1 and Raili Kauppinen1 1Department of Medicine, Division of Endocrinology, University Central Hospital of Helsinki, Biomedicum Helsinki, Helsinki, Finland; 2Department of Dermatology, University Central Hospital of Helsinki, Biomedicum Helsinki, Helsinki, Finland Variegate porphyria (VP) is an inherited metabolic disease resulting from the partial deficiency of protoporphyrinogen oxidase, the penultimate enzyme in the heme biosynthetic pathway. We have evaluated the clinical and biochemical outcome of 103 Finnish VP patients diagnosed between 1966 and 2001. Fifty-two per cent of patients had experienced clinical symptoms: 40% had photosensitivity, 27% acute attacks and 14% both manifestations. The proportion of patients with acute attacks has decreased dramatically from 38 to 14% in patients diagnosed before and after 1980, whereas the prevalence of skin symptoms had decreased only subtly from 45 to 34%. We have studied the correlation between PPOX genotype and clinical outcome of 90 patients with the three most common Finnish mutations I12T, R152C and 338G?C. The patients with the I12T mutation experienced no photosensitivity and acute attacks were rare (8%). Therefore, the occurrence of photosensitivity was lower in the I12T group compared to the R152C group (P=0.001), whereas no significant differences between the R152C and 338G?C groups could be observed. Biochemical abnormalities were significantly milder suggesting a milder form of the disease in patients with the I12T mutation.
    [Show full text]
  • Phlebotomy As an Efficient Long-Term Treatment of Congenital
    Letters to the Editor after chronic gastrointestinal bleeding that resulted in Phlebotomy as an efficient long-term treatment of iron deficiency.4 They treated her with an iron chelator, congenital erythropoietic porphyria resulting in correction of the hemolysis, decreased por- phyrin levels and improved quality of life with reduced Congenital erythropoietic porphyria (CEP, MIM photosensitivity. We recently identified three CEP sib- 263700) is a rare autosomal recessive disease caused by lings with phenotypes ranging from moderate to asymp- impaired activity of uroporphyrinogen III synthase tomatic which were modulated by iron availability, high- (UROS), the fourth enzyme of the heme biosynthetic lighting the importance of iron metabolism in the disease. 1 pathway. Accumulation of porphyrins in red blood cells, Based on these data, we prospectively treated a CEP mainly uroporphyrinogen I (URO I) and copropor- patient with phlebotomies to investigate the feasibility, phyrinogen I (COPRO I), leads to ineffective erythro- safety and efficacy of this treatment. We observed dis- poiesis and chronic hemolysis. Porphyrin deposition in continuation of hemolysis and a marked decrease in plas- the skin is responsible for severe photosensitivity result- ma and urine porphyrins. The patient reported a major ing in bullous lesions and progressive photomutilation. improvement in photosensitivity. Finally, erythroid cul- Treatment options are scarce, relying mainly on support- tures were performed, demonstrating the role of iron in ive measures and, for severe cases, on bone marrow the rate of porphyrin production. transplantation (BMT). Increased activation of the heme The study was conducted in accordance with the biosynthetic pathway by gain-of-function mutations in World Medical Association Declaration of Helsinki ethi- ALAS2, the first and rate-limiting enzyme, results in a 2 cal principles for medical research involving human sub- more severe phenotype.
    [Show full text]
  • AOP 131: Aryl Hydrocarbon Receptor Activation Leading to Uroporphyria
    Organisation for Economic Co-operation and Development DOCUMENT CODE For Official Use English - Or. English 1 January 1990 AOP 131: Aryl hydrocarbon receptor activation leading to uroporphyria Short Title: AHR activation-uroporphyria This document was approved by the Extended Advisory Group on Molecular Screening and Toxicogenomics in June 2018. The Working Group of the National Coordinators of the Test Guidelines Programme and the Working Party on Hazard Assessment are invited to review and endorse the AOP by 29 March 2019. Magdalini Sachana, Administrator, Hazard Assessment, [email protected], +(33- 1) 85 55 64 23 Nathalie Delrue, Administrator, Test Guidelines, [email protected], +(33-1) 45 24 98 44 This document, as well as any data and map included herein, are without prejudice to the status of or sovereignty over any territory, to the delimitation of international frontiers and boundaries and to the name of any territory, city or area. 2 │ Foreword This Adverse Outcome Pathway (AOP) on Aryl hydrocarbon receptor activation leading to uroporphyria, has been developed under the auspices of the OECD AOP Development Programme, overseen by the Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST), which is an advisory group under the Working Group of the National Coordinators for the Test Guidelines Programme (WNT). The AOP has been reviewed internally by the EAGMST, externally by experts nominated by the WNT, and has been endorsed by the WNT and the Working Party on Hazard Assessment (WPHA) in xxxxx. Through endorsement of this AOP, the WNT and the WPHA express confidence in the scientific review process that the AOP has undergone and accept the recommendation of the EAGMST that the AOP be disseminated publicly.
    [Show full text]
  • Ex Vivo Gene Therapy: a “Cultured” Surgical Approach to Curing Inherited Liver Disease
    Mini Review Open Access J Surg Volume 10 Issue 3 - March 2019 Copyright © All rights are reserved by Joseph B Lillegard DOI: 10.19080/OAJS.2019.10.555788 Ex Vivo Gene Therapy: A “Cultured” Surgical Approach to Curing Inherited Liver Disease Caitlin J VanLith1, Robert A Kaiser1,2, Clara T Nicolas1 and Joseph B Lillegard1,2,3* 1Department of Surgery, Mayo Clinic, Rochester, MN, USA 2Midwest Fetal Care Center, Children’s Hospital of Minnesota, Minneapolis, MN, USA 3Pediatric Surgical Associates, Minneapolis, MN, USA Received: February 22, 2019; Published: March 21, 2019 *Corresponding author: Joseph B Lillegard, Midwest Fetal Care Center, Children’s Hospital of Minnesota, Minneapolis, Minnesota, USA and Mayo Clinic, Rochester, Minnesota, USA Introduction Inborn errors of metabolism (IEMs) are a group of inherited diseases caused by mutations in a single gene [1], many of which transplant remains the only curative option. Between 1988 and 2018, 12.8% of 17,009 pediatric liver transplants in the United States(see were primarily due to an inherited liver). disease. are identified in Table 1. Though individually rare, combined incidence is about 1 in 1,000 live births [2]. While maintenance www.optn.transplant.hrsa.gov/data/ Table 1: List of 35 of the most common Inborn Errors of Metabolism. therapies exist for some of these liver-related diseases, Inborn Error of Metabolism Abbreviation Hereditary Tyrosinemia type 1 HT1 Wilson Disease Wilson Glycogen Storage Disease 1 GSD1 Carnitine Palmitoyl Transferase Deficiency Type 2 CPT2 Glycogen Storage
    [Show full text]
  • Module 01: Classification of Porphyria
    MODULE 01 Classification of Porphyria Do not reprint, reproduce, modify or distribute this material without the prior written permission of Alnylam Pharmaceuticals. © 20120199 Alnylam PharmaceuticalsPharmaceuticals,, Inc.Inc. All rights reserved. -USA-00001-092018 1 Porphyria—A Rare Disease of Clinical Consequence • Porphyria is a group of at least 8 metabolic disorders1,2 – Each subtype of porphyria involves a genetic defect in a heme biosynthesis pathway enzyme1,2 – The subtypes of porphyria are associated with distinct signs and symptoms in patient populations that can differ by gender and age1,3 • Prevalence of some subtypes of porphyria may be higher than generally assumed3 Estimated Prevalence of Most Common Subtypes of Porphyria1,4 Estimated Prevalence Based on European Subtype of Porphyria and US Data Porphyria cutanea tarda (PCT) 1/10,000 (EU)1 0.118-1/20,000 (EU)1,4 Acute intermittent porphyria (AIP) 5/100,000 (US)1 Erythropoietic protoporphyria (EPP) 1/50,000-75,000 (EU)1 1. Ramanujam V-MS, Anderson KE. Curr Protoc Hum Genet. 2015;86:17.20.1-17.20.26. 2. Puy H et al. Lancet. 2010;375:924-937. 3. Bissell DM et al. N Engl J Med. 2017;377:862-872. 4. Elder G et al. J Inherit Metab Dis. 2013;36:848-857. Do not reprint, reproduce, modify or distribute this material without the prior written permission of Alnylam Pharmaceuticals. © 2019 Alnylam Pharmaceuticals, Inc. All rights reserved. -USA-00001-092018 2 Classification of Porphyria Porphyria can be classified in 2 major ways1,2: 1 According to major physiological sites: liver or 2 According to major clinical manifestations1,2 bone marrow1,2 • Heme precursors originate in either the liver or Acute Versus Photocutaneous Porphyria bone marrow, which are the tissues most • Major clinical manifestations are either neurovisceral active in heme biosynthesis1,2 symptoms (eg, severe, diffuse abdominal pain) associated with acute exacerbations or cutaneous lesions resulting from phototoxicity1,2 • Acute hepatic porphyria may be somewhat of a misnomer since the clinical features may be prolonged and chronic3 1.
    [Show full text]
  • Variegate Porphyria with Coexistent Decrease in Porphobilinogen Deaminase Activity
    Acta Derm Venereol 2001; 81: 356–359 CLINICAL REPORT Variegate Porphyria with Coexistent Decrease in Porphobilinogen Deaminase Activity GEORG WEINLICH1, MANFRED O. DOSS2, NORBERT SEPP1 and PETER FRITSCH1 1Department of Dermatology, University of Innsbruck, Innsbruck, Austria and 2Department of Clinical Biochemistry, University of Marburg, Marburg, Germany Variegate porphyria is a rare disease caused by a de ciency of deaminase. Its activity is reduced by about 50%, resulting in protoporphyrinogen oxidase. In most cases, the clinical ndings are varying degrees of overproduction and increased urinary excre- a combination of systemic symptoms similar to those occurring in tion of delta-aminolaevulinic acid (ALA) and PBG. In AIP, acute intermittent porphyria and cutaneous lesions indistinguishable skin changes are absent, but patients suVer from episodic from those of porphyria cutanea tarda. We report on a 24-year- central or peripheral nervous system and/or psychiatric symp- old woman with variegate porphyria who, after intake of lynestrenol, toms, or acute attacks of abdominal pain (2, 4). developed typical cutaneous lesions but no viscero-neurological Variegate porphyria (VP), a rare autosomal-dominant hep- symptoms. The diagnosis was based on the characteristic urinary atic porphyria due to a de ciency of protoporphyrinogen coproporphyrin and faecal protoporphyrin excretion patterns, and (PROTO) oxidase, the related gene (PPOX ) for which has the speci c peak of plasma uorescence at 626 nm in spectro uor- been located to chromosome 1q22-23 (5, 6), is characterized ometry. Biochemical analysis revealed that most of the family by both acute neurological symptoms as in AIP and skin members, though free of clinical symptoms, excrete porphyrin lesions indistinguishable from PCT; it is thus also termed metabolites in urine and stool similar to variegate porphyria, mixed porphyria (2, 7, 8).
    [Show full text]
  • A Primitive Pathway of Porphyrin Biosynthesis and Enzymology in Desulfovibrio Vulgaris
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 4853–4858, April 1998 Biochemistry A primitive pathway of porphyrin biosynthesis and enzymology in Desulfovibrio vulgaris TETSUO ISHIDA*, LING YU*, HIDEO AKUTSU†,KIYOSHI OZAWA†,SHOSUKE KAWANISHI‡,AKIRA SETO§, i TOSHIRO INUBUSHI¶, AND SEIYO SANO* Departments of *Biochemistry and §Microbiology and ¶Division of Biophysics, Molecular Neurobiology Research Center, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-21, Japan; †Department of Bioengineering, Faculty of Engineering, Yokohama National University, 156 Tokiwadai, Hodogaya-ku, Yokohama 240, Japan; and ‡Department of Public Health, Graduate School of Medicine, Kyoto University, Sakyou-ku, Kyoto 606, Japan Communicated by Rudi Schmid, University of California, San Francisco, CA, February 23, 1998 (received for review March 15, 1998) ABSTRACT Culture of Desulfovibrio vulgaris in a medium billion years ago (3). Therefore, it is important to establish the supplemented with 5-aminolevulinic acid and L-methionine- biosynthetic pathway of porphyrins in D. vulgaris, not only methyl-d3 resulted in the formation of porphyrins (sirohydro- from the biochemical point of view, but also from the view- chlorin, coproporphyrin III, and protoporphyrin IX) in which point of molecular evolution. In this paper, we describe a the methyl groups at the C-2 and C-7 positions were deuter- sequence of intermediates in the conversion of uroporphy- ated. A previously unknown hexacarboxylic acid was also rinogen III to coproporphyrinogen III and their stepwise isolated, and its structure was determined to be 12,18- enzymic conversion. didecarboxysirohydrochlorin by mass spectrometry and 1H NMR. These results indicate a primitive pathway of heme biosynthesis in D. vulgaris consisting of the following enzy- MATERIALS AND METHODS matic steps: (i) methylation of the C-2 and C-7 positions of Materials.
    [Show full text]
  • Demystification of Chester Porphyria: a Nonsense Mutation in the Porphobilinogen Deaminase Gene
    Physiol. Res. 55 (Suppl. 2): S137-S144, 2006 Demystification of Chester Porphyria: A Nonsense Mutation in the Porphobilinogen Deaminase Gene *P. POBLETE-GUTIÉRREZ1, *T. WIEDERHOLT2, 3, A. MARTINEZ-MIR4, H. F. MERK2, J. M. CONNOR5, A. M. CHRISTIANO4, 6, J. FRANK1,2,3 1Department of Dermatology, University Hospital Maastricht, The Netherlands, 2Department of Dermatology and Allergology and 3Porphyria Center, University Hospital of the RWTH Aachen, Germany, 4Department of Dermatology, Columbia University, New York, NY, USA, 5Institute of Medical Genetics, Yorkhill Hospitals, Glasgow, Scotland, 6Department of Genetics and Develop- ment, Columbia University, New York, NY, USA Received October 4, 2005 Accepted March 24, 2006 Summary The porphyrias arise from predominantly inherited catalytic deficiencies of specific enzymes in heme biosynthesis. All genes encoding these enzymes have been cloned and several mutations underlying the different types of porphyrias have been reported. Traditionally, the diagnosis of porphyria is made on the basis of clinical symptoms, characteristic biochemical findings, and specific enzyme assays. In some cases however, these diagnostic tools reveal overlapping findings, indicating the existence of dual porphyrias with two enzymes of heme biosynthesis being deficient simultane- ously. Recently, it was reported that the so-called Chester porphyria shows features of both variegate porphyria and acute intermittent porphyria. Linkage analysis revealed a novel chromosomal locus on chromosome 11 for the underly- ing genetic defect in this disease, suggesting that a gene that does not encode one of the enzymes of heme biosynthesis might be involved in the pathogenesis of the porphyrias. After excluding candidate genes within the linkage interval, we identified a nonsense mutation in the porphobilinogen deaminase gene on chromosome 11q23.3, which harbors the mutations causing acute intermittent porphyria, as the underlying genetic defect in Chester porphyria.
    [Show full text]
  • Enzymatic Synthesis of Dihydrosirohydrochlorin (Precorrin-2) and of a Novel Pyrrocorphin by Uroporphyrinogen III Methylase
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 261, number 1, 76-80 FEBS 08106 February 1990 Enzymatic synthesis of dihydrosirohydrochlorin (precorrin-2) and of a novel pyrrocorphin by uroporphyrinogen III methylase Martin J. Warren, Neal J. Stolowich, Patricia J. Santander, Charles A. Roessner, Blair A. SowaN and A. Ian Scott Centre for Biological NMR, Department of Chemistry and #Department of Veterinary Pathology, Texas A & M University, College Station, TX 77843, USA Received 28 November 1989 Uroporphyrinogen III methylase was purified from a recombinant hemB- strain of E. coli harbouring a plasmid containing the cysG gene. N-termi- nal analysis of this purified protein gave an amino acid sequence corresponding to that predicted from the genetic code. From the u.v./visible spec- trum of the reaction catalysed by this SAM dependent methylase it was possible to observe the sequential appearance of the chromophores of a dipyrrocorphin and subsequently of a pyrrocorphin. Confirmation of this transformation was obtained from W-NMR studies when it was dem- onstrated, for the first time directly, that uroporphyrinogen is initially converted into dihydrosirohydrochlorin (precorrin-2) and then, by further methylation, into a novel trimethylpyrrocorphin. Uroporphyrinogen III methylase; Dihydrosirohydrochlorin; Precorrin-2; Dipyrrocorphin; Pyrrocorphin; NMR, W- 1. INTRODUCTION reaction, were able to isolate sirohydrochlorin octa- methyl ester. However, since no spectral changes were The structure proposed for precorrin-2 (1; R = H), observed during the course of the enzymic reaction an intermediate in vitamin Brz and sirohaem synthesis (conducted under anaerobic conditions) is was assumed [l-4], is that of a dipyrrocorphin formed by C- that the initial enzymatic product was the dipyrrocor- methylation of uro’gen III at positions 2 and 7 by the phin, dihydrosirohydrochlorin (1).
    [Show full text]
  • A High Urinary Urobilinogen / Serum Total Bilirubin Ratio Reported in Abdominal Pain Patients Can Indicate Acute Hepatic Porphyria
    A High Urinary Urobilinogen / Serum Total Bilirubin Ratio Reported in Abdominal Pain Patients Can Indicate Acute Hepatic Porphyria Chengyuan Song Shandong University Qilu Hospital Shaowei Sang Shandong University Qilu Hospital Yuan Liu ( [email protected] ) Shandong University Qilu Hospital https://orcid.org/0000-0003-4991-552X Research Keywords: acute hepatic porphyria, urinary urobilinogen, serum total bilirubin Posted Date: June 14th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-587707/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/10 Abstract Background: Due to its variable symptoms and nonspecic laboratory test results during routine examinations, acute hepatic porphyria (AHP) has always been a diagnostic dilemma for physicians. Misdiagnoses, missed diagnoses, and inappropriate treatments are very common. Correct diagnosis mainly depends on the detection of a high urinary porphobilinogen (PBG) level, which is not a routine test performed in the clinic and highly relies on the physician’s awareness of AHP. Therefore, identifying a more convenient indicator for use during routine examinations is required to improve the diagnosis of AHP. Results: In the present study, we retrospectively analyzed laboratory examinations in 12 AHP patients and 100 patients with abdominal pain of other causes as the control groups between 2015 and 2021. Compared with the control groups, AHP patients showed a signicantly higher urinary urobilinogen level during the urinalysis (P < 0.05). However, we showed that the higher urobilinogen level was caused by a false- positive result due to a higher level of urine PBG in the AHP patients. Hence, we used serum total bilirubin, an upstream substance of urinary urobilinogen synthesis, for calibration.
    [Show full text]