Diseases of Metabolism (Porphyrias) Nuala A. McCamll Department of Biochemistry, St. James’ Hospital, Dublin 8, Ireland
The porphyrias are a group of diseases that are each associated with hereditary or acquired deficiency of one of seven of the eight enzymes involved in heme biosynthesis. Several reviews provide in-depth coverage of the biochemical pathway, the properties of the enzymes and substrates, and the prevalence and clinical expression of the genetic defects (Hl-H3). Diagnosis of porphyria in symptomatic individuals depends on the identification of patterns of accumulation of heme precursors which are associated with specific enzyme deficiencies (H2) . This involves analysis of Saminolevulinate (ALA) and porphobilinogen (PBG) in urine or the porphyrin oxidation products of the intermediate porphyrinogens in blood, urine, or feces. Analytical techniques have ranged from simple qualitative screening tests for PBG and porphyrins to more specific chromatography-basedanalyses which allow accurate separation and quantitation of ALA, PBG, and porphyrins. Identification of many asymptomatic gene carriers has been achieved by demonstration of reduced activity or concentration of the specific enzyme. These enzyme measurements and more recent applications of molecular biology techniques have identified significant heterogeneity within the individual porphyrias. This review includes developments in the understanding of the clinical expression and diagnosis of porphyrias and analysis of substrates, enzymes, and genetic defects of heme biosynthesis which were identified by a search through Chemical Abstracts from October 1989 to October 1994. Only articles in the English language that described studies in human subjects were included. Additional references were identified from these sources. Publications prior to 1989 are included when relevant. CLINICAL EXPRESSION OF THE GENETIC DEFECTS With the exception of sporadic porphyria cutanea tarda (PCT type I), all of the porphyrias are inherited in autosomal recessive or autosomal dominant mode but with variable clinical expression. Homozygous variants of some of the dominantly inherited porphyrias, and mixed porphyrias where two types of porphyria coexist, have been described (H4-H6). Clinical expression is common in individuals who are homozygous for the genetic defect and rare in other gene carriers (H7). Up to 90% of acute intermittent porphyria gene carriers may never experience symp toms. Overt porphyria in prepubertal children is suggestive of a homozygous condition, although protoporphyria often presents in childhood. A group of case presentations of porphyria in childhood are instructive (H8). Two well-recognized groups of symptoms are associated with accumulation and excretion of heme precursor compounds (HlH3, H9). Severe, intermittent, and potentially life-threatening episodes of acute illness that are associated with excretion in urine of excessive amounts of ALA and frequently PBG are the most important clinical feature of the acute porphyrias. Such attacks occur in porphobilinogen synthase (PBGS) deficiency porphyria, acute intermittent porphyria (AIP), hereditary coproporphyria (HC), and porphyria variegata 0. The most common symptom is abdominal pain; other features include nausea, vomiting, constipation or diarrhea, hypertension, peripheral neuropathy, or
psychiatric symptoms. Overproduction of porphyrins and their accumulation in skin leads to the development of skin lesions following exposure to sunlight. The effects range from severe mutilating lesions in porphyria cutanea tarda, congenital erythropoietic porphyria (CEP), HC and PV, to burning and itching sensations in erythropoietic porphyria (EPP). Liver damage, which may progress to liver failure, occurs in some cases of EPP and is thought to be related to the hepatic accumulation of protoporphyrin which has been observed in this condition. Many questions remain to be answered about the factors that determine selection of the small proportion of carriers of defective genes that will develop clinically manifest disease within groups with comparable enzyme deficiency and about the mechanisms underlying symptoms and signs (H6, H9-Hll). These questions are relevant to prevention and management of the disease process. The major sites of heme biosynthesis are the liver and bone marrow, and different regulatory mechanisms are in operation (H3, HE?). Negative feedback by heme regulates the activity of ALA-synthase (ALA-S) in the liver (Hl-H3). Reduction of the regulatory heme pool will result in increased activity of ALA-S. Many factors including drugs, alcohol, hormones, fasting, infection, and toxins have been identified as precipitants of overt disease through either a direct inhibitory effect on enzyme activity or reduction of the regulatory heme pool (H1-H3, H7). In many cases, the precipitating factor or mechanism is unknown. Preventive strategies are directed primarily at avoidance of known precipitating factors. It is therefore essential to detect and counsel gene carriers among relatives of patients with the acute porphyrias (H6). Successful prevention and treatment of acute attacks has been achieved by employing the known suppressive effect of glucose on ALA-S by maintaining a high carbohydrate intake. Direct manipulation of the regulatory heme pool by administration of hematin or inhibition of heme breakdown using inhibitors of heme oxygenase has been used successfullyin treatment of acute attacks ( H l l , H13-Hl5). Several studies indicate a pathogenic role for iron in PCT, which is the most common of the porphyrias with cutaneous manifestations (H16). Complete clinical remission is possible in both type I and familial or type I1 PCT when iron stores are reduced, by phlebotomy (H16, H17),or by erythropoietin therapy in patients with renal failure where phlebotomy is contraindicated (H18). Accumulation of substrate proximal to the enzyme deficiency explains most of the abnormalities of heme precursors seen in the porphyrias. However, in addition to the expected abnormalities of coproporphyrin and protoporphyrin in HC and PV, respectively, increased excretion of ALA and PBG are frequently observed. This anomaly has recently been clarified by the observation that protoporphyrinogen and coproporphyrinogen inhibit porphobilinogen deaminase (PBGD) activity (H19). Both acute attacks and photosensitivity occur in these conditions although not necessarily coincidentally. Possible mechanisms underlying the symptoms of acute attacks of porphyria and evidence for ALA toxicity have been reviewed (H6,H11, H20). Similar syndromes occur in lead poisoning and in hereditary tyrosinosis where inhibition of PBGS Analytical Chemistry, Vol. 67, No. 12,June 15, 1995
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by lead and succinylacetone, respectively, is associated with increased excretion of ALA The toxic effects of porphyrins are generally related to freeradical-induced damage to membrane lipids following excitation of the porphyrins by ultraviolet light. This mechanism is the basis of the therapeutic administration of free-radical scavengers such as carotenoids,which has achieved some success in the amelioration of symptoms in protoporphyria (H21). DIAGNOSIS
Background. Symptoms exhibited in the porphyrias are nonspecific and common to many other disorders with a higher prevalence. Correct diagnosis is dependent primarily on the clinician having a high index of suspicion and requesting laboratory conlirmation. It is essential that analyses appropriate to the clinical presentation are performed. Patients with current symp toms of an acute attack will have elevated concentrations of ALA and usually PBG in their urine if their symptoms are due to porphyria. Patients with photosensitivity will have increased plasma porphyrin concentration if the photosensitivity is caused by porphyria. The type of porphyria can be identified by determination of porphyrin profiles in urine and feces. These analyses are inappropriate for detection of asymptomatic gene camers of the acute porphyrias as results are usually normal. Genetic analysis is the most accurate method for carrier detection, but a high proportion of carriers can be detected by quantitation of the activity or mass of the appropriate enzyme. Heme Precursor Analysis. Appropriate first-line analyses and their application have been clearly presented (H22-HZ4). Patterns of abnormalitiesassociated with symptomatic porphyrias are described in each of these references. The Watson-Schwartz and Hoesch tests are qualitative or semiquantitative screening tests for excess PBG in urine which depend on the formation of a colored product with &dimethylamino)benzaldehyde in acid solution (Ehrlich's reagent). Although they are considered useful as rapid tests for emergency situations, followup quantitative analysis is mandatory because of the occurrence of both false negative and false positive results. A rapid and sensitive quantitative technique for PBG, which employs a chromatography step, has been described (H25). In the investigation of suspected acute porphyria quantitation of ALA is also useful as this may be the only abnormality in the very rare PBG synthase deficiency. The ion-exchange chromatographic extraction of ALA and PBG followed by reaction with (4 dimethylamino) benzaldehyde and colorimetricquantitation,which was developed by Mauzerall and Granick in 1956 (H26') and is available in kit form from Bio-Rad Laboratories (Hercules, CA), has stood the test of time. Quantification of ALA in plasma by a modfication of the colorimetric technique (H27) and in plasma (H28) and urine (H29, H30) by HPLC has been described. However, the finding of elevated ALA without knowledge of PBG concentration is less specific for diagnosis of porphyria. Qualitative screening tests for excess porphyrins in blood urine and feces previously involved visual detection of fluorescence on exposure to ultraviolet light of porphyrins extracted into organic solvent and then back-extracted into acid. Interpretation of results was highly subjective and false negative and false positive results were obtained. False negative results were reported in 9 of 10 patients with EPP who were subsequently identified by fluorescence microscopy of erythrocytes (H31). These qualitative 426R
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screening tests should now be replaced by any of the very simple quantitative tests for total porphyrins (HZZ-HZ4, H32, H 3 3 ) . It has been proposed that in view of the relative simplicity of porphyrin analysis in urine by direct injection into a HPLC system that urine porphyrin screening tests could be abandoned (H24). Analysis of plasma porphyrins by fluorescence emission spectrometry has been proposed as a first-line test in the evaluation of patients presenting with photosensitivity (H23). Porphyria may be ruled out as a cause of the problem or the spectrum may be diagnostic of W (H34) or suggestive of one of PCT, CEP, or PP, which may then be distinguished by chromatographic analysis of urine or feces. Plasma porphyrin fluorescence spectra in 109 patients with porphyria and 45 controls allowed clear distinction of patients with AIP and normal controls from patients with EPP, CEP, PV, and HC (H35). Detection of the plasma porphyrin peak associated with PV had a diagnostic sensitivity of 86%,compared with 36%by analysis of porphyrins in feces, in the detection of asymptomatic carriers of PV (H36). Bile porphyrin concentrations in 10 patients with W were significantly different from controls (H37). This measurement proved superior to plasma porphyrin fluorescence and fecal porphyrin concentrations, which were diagnostic in eight and six patients, respectively. Plasma porphyrin analysis has been proposed as an alternative to urine porphyrin quantitation in monitoring disease activity and response to therapy in patients with PCT (H38). A procedure has been described for separating and quantitating erythrocyte protoporphyrin, which is elevated in EPP, and zincprotoporphyrin,which is elevated in lead poisoning, iron deficiency anemia, and homozygous porphyrias, by HPLC using threedimensional analysis of absorption spectra (H39). Fractionation of porphyrins by solvent extraction was used for many years despite the fact that variable mixtures of porphyrins were obtained. These procedures have been abandoned in favor of relatively simple HPLC or TLC procedures for separation of individual porphyrins and the type I and 111 isomers. Separation of porphyrin isomers is important, particularly in suspected cases of CEP where type I isomers predominate over the type 111 isomers, which are usually normally more abundant. Techniques have been described that do not require derivatization of porphyrins (H40, H41). Inclusion of internal standards will allow both quantitation and pattern analysis (H41). A robotic system for analysis of porphyrins in feces by HPLC has been developed (H42). The coproporphyrin I isomer was present in excess of the coproporphyrin 111 isomer in patients with AIP during an acute attack whereas the type I11 isomer predominated in PV (H43). This observation could be useful in distinguishing AIP from HC and PV if elevated fecal porphyrin concentrations accompany the acute attack of AIP. The ratio of coproporphyrin 111to coproporphyrin I in feces may be more sensitive than quantitative total porphyrins in detecting carriers of HC (H44). The distribution of coproporphyrin I and 111isomers as well as the more unusual and possibly artifactual type IT and IV isomers in urine from patients with porphyria and controls has been described (H45). Analysis of porphyrins in bile by HPLC has been applied to the study of hepatic excretion of protoporphyrin in patients with EPP following liver transplantation (H46).Bile from normal controls contained predominantly coproporphyrin I with some coproporphyrin I11 whereas protoporphyrin predominated in the
patients with EPP. Failure to detect deutero-, pempto-, and mesoporphyrin, which are products of intestinal bacterial metabolism of protoporphyrin, suggested that enterohepatic circulation of porphyrins does not occur. Heme Precursor Abnormalities in Other Diseases. It is important to be aware of abnormalities of heme biosynthesis or heme precursor excretion that occur secondary to other diseases to avoid misdiagnosis and inappropriate therapy. Lead poisoning and h e r e d i m tyrosinosis have already been mentioned as causes of abnormal urinary ALA concentrations. Other causes have been H2,H23, discussed in the review articles listed previously (HI, H24, Of particular note is the mild coproporphyrinuria that can occur secondary to alcoholism, liver disease, and lead poisoning. Elevated erythrocyte zinc-protoporophyrin is a feature of lead poisoning and iron deficiency anemia but also occurs in homozygous forms of porphyria. Abnormal concentrations of stool porphyrins may be due to intestinal bacterial activity (H47), gastrointestinal bleeding or porphyrin intake in food. Analysis of the porphyrin profile by HPLC will eliminate porphyria from consideration. Two reports describe interference by drugs in porphyrin analyses which could lead to misdiagnosis (H48, H49). Enzymes and Molecular Biology. Quantitation of activity or mass of deficient enzymes and the more recent application of molecular biology techniques have significantly increased the detection rate of gene carriers and demonstrated signiiicant heterogeneity within each type of porphyria. Activity measurements of several of the enzymes are complex because of instability of the porphyrinogen substrates, the formation of multiple products, and the requirement for leucocyte or fibroblast preparations for detection of coproporphyrinogen oxidase, protoporphyrin oxidase, and ferrochelatase, which are located within the mitochondrion. A compilation of methods for determining the activity of all of the enzymes of heme biosynthesis has been published (H50). In the autosomal dominant porphyrias, residual enzyme activity of 50%of normal is anticipated. However, results from gene carriers show sigdicant overlap with those from noncarriers. This problem was recently illustrated in results for both mass and activity of PBG-deaminase obtained in erythrocytes from 845 members of a Swedish family with AIP (H51).In the majority of patients, the enzyme activity and mass concentrations were in agreement. However, additional heterogeneity in PBGD mutations, which result in the presence of cross-reacting immunological material (CRIM) in excess of the enzyme activity, were also illustrated in this report. Tissuespecific expression of enzyme deficiency has been described in AIP, where deficiency of the ubiquitous isoenzyme is associated with normal erythrocyte enzyme activity, and in PCT, where deficiency of uroporphyrinogen decarboxylase (UROD) is present in all cells including erythrocytes from patients with the familial variant but only in the liver in patients with sporadic PCT. The activity of erythrocyte UROD was reduced to 50% of normal in 50%of 471 cases of PCT in a German study (H52)but in only 22%of 80 patients in Hungary
(H53). The heterogeneity of the enzyme defects and the underlying genetic mutations in AIP, familial PCT, HEP, CEP, and PBGS deficiency porphyria have been reviewed (H54-H57).In view of the genetic heterogeneity, definitive identification of carriers is best achieved by identification of the mutation in the index case followed by hybridization of genomic or cDNA fragments with allele-specific oligonucleotides (ASO). Denaturing gradient gel
electrophoresis (H58)and singlestrand conformation polymorphism (H59)have been used to screen ampwed DNA segments for mutations prior to sequencing. In the latter case, hair roots were used as a source of DNA with the advantages of ease of sample handling and transport and minimal sample preparation. Numerous reports have described the identification of mutations in the genes for which DNA sequences were known. The recent cloning of the cDNA encoding coproporphyrinogen oxidase (H60) will allow detection of defects responsible for HC. Cloning of the gene for protoporphyrinogen oxidase has not yet been reported. CONCLUSIONS Identification of typical patterns of heme precursors in plasma, blood, urine, and feces is a fundamental requirement for the diagnosis of porphyria in symptomatic patients. Qualitative screening tests for porphobilinogen and porphyrins should be abandoned. Simple techniques for quantitating total porphyrin concentrations in blood urine and feces together with fluorometric scanning of plasma samples are suitable first-line tests. Urine and feces samples with elevated porphyrin concentrations should have their porphyrin profile examined following chromatographic separation. Enzyme activity measurements and immunoassays contribute to the identification of gene carriers. These analyses cannot be used to exclude carrier status. Molecular genetics studies, although labor intensive because of genetic heterogeneity, will allow definitive identification of asymptomatic gene camers. Nuala MeCarroll is a Clinical Biochemist in St. lames’ Hospital, Dublin, Ireland. She received her BSc. degree in baochemisty from Universit Colle e Dublin, her MSc. degree in CliniCaJbiochem+tyfrom Tnnzt &lege Sublzn, and her Ph.D. de ree an clznrcal chemzst from CleveJnd State University. She is a A m b e r of the Royal C o z g e of Pathologists (Chemacal Pathology) and a Da lomate of the American Board of Clinical Chemisty. From 1991 to 1892she was a Postdoctoral Fellow in the Department of Biochemist of the Cleveland Clanac Foundation. Her research interests inch% bone biochemisty, drug metabolism, and biochemical aspects of nutrition.
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