An inherited enzymatic defect in porphyria cutanea tarda: decreased uroporphyrinogen decarboxylase activity. J P Kushner, … , A J Barbuto, G R Lee J Clin Invest. 1976;58(5):1089-1097. https://doi.org/10.1172/JCI108560. Research Article Uroporphyrinogen decarboxylase activity was measured in liver and erythrocytes of normal subjects and in patients with porphyria cutanea tarda and their relatives. In patients with porphyria cutanea tarda, hepatic uroporphyrinogen decarboxylase activity was significantly reduced (mean 0.43 U/mg protein; range 0.25-0.99) as compared to normal subjects (mean 1.61 U/mg protein; range 1.27-2.42). Erythrocyte uroporphyrinogen decarboxylase was also decreased in patients with porphyria cutanea tarda. The mean erythrocyte enzymatic activity in male patients was 0.23 U/mg Hb (range 0.16-0.30) and in female patients was 0.17 U/mg Hb (range 0.15-0.18) as compared with mean values in normal subjects of 0.38 U/mg Hb (range 0.33-0.45) in men and 0.26 U/mg Hb (range 0.18-0.36) in women. With the erythrocyte assay, multiple examples of decreased uroporphyrinogen decarboxylase activity were detected in members of three families of patients with porphyria cutanea tarda. In two of these families subclinical porphyria was also recognized. The inheritance pattern was consistant with an autosomal dominant trait. The difference in erythrocyte enzymatic activity between men and women was not explained but could have been due to estrogens. This possibility was supported by the observation that men under therapy with estrogens for carcinoma of the prostate had values in the normal female range. It is proposed that porphyria cutanea tarda results from the combination of an inherited defect in uroporphyrinogen decarboxylase and […] Find the latest version: https://jci.me/108560/pdf An Inherited Enzymatic Defect in Porphyria Cutanea Tarda DECREASED UROPORPHYRINOGEN DECARBOXYLASE ACTIVITY J. P. KUSHNER, A. J. BARBUTO, and G. R. LEE From the Department of Internal Medicine of The Veterans Administration Hospital and The Unriversity of Utah College of Medicine, Salt Lake City, Utah 84132 A B S T R A C T Uroporphyrinogen decarboxylase INTRODUCTION activity was measured in liver and erythrocytes of normal subjects and in patients with porphyria cutanea Specific enzymatic defects in the porphyrin bio- tarda and their relatives. In patients with porphyria synthetic pathway have been defined in several cutanea tarda, hepatic uroporphyrinogen decar- types of inherited porphyria (acute intermittent por- boxylase activity was significantly reduced (mean 0.43 phyria, congenital erythropoietic porphyria, and U/mg protein; range 0.25-0.99) as compared to nor- protoporphyria) (1-5). Assays for these enzymes mal subjects (mean 1.61 U/mg protein; range 1.27- have made family studies possible and the genetic 2.42). Erythrocyte uroporphyrinogen decarboxylase characteristics of the diseases have been documented was also decreased in patients with porphyria cutanea (6, 7). tarda. The mean erythrocyte enzymatic activity in In contrast, a specific enzymatic defect has not male patients was 0.23 U/mg Hb (range 0.16-0.30) been defined in porphyria cutanea tarda (PCT).' and in female patients was 0.17 U/mg Hb (range This disease is usually considered to be an acquired 0.15-0.18) as compared with mean values in normal disorder of porphyrin metabolism because of its subjects of 0.38 U/mg Hb (range 0.33-0.45) in men onset late in life and common association with al- and 0.26 U/mg Hb (range 0.18-0.36) in women. coholic liver disease (8, 9). With the erythrocyte assay, multiple examples of Biochemically PCT is characterized by hepatic decreased uroporphyrinogen decarboxylase ac- synthesis and urinary excretion of excessive amounts tivity were detected in members of three families of porphyrins, particularly uroporphyrin I and smaller of patients with porphyria cutanea tarda. In two amounts of 7-carboxyl porphyrin (10-12). The pre- of these families subclinical porphyria was also ponderance of uroporphyrin I and 7-carboxyl por- recognized. The inheritance pattern was consistant phyrin in both the liver and urine suggests that the with an autosomal dominant trait. enzymatic decarboxylation of the reduced forms of The difference in erythrocyte enzymatic activity these compounds (porphyrinogens) to coproporphy- between men and women was not explained but rinogen I is impaired. This decarboxylation is catalyzed could have been due to estrogens. This possibility by the enzyme tiroporphyrinogen decarboxylase was supported by the observation that men under (URODECARB) (13) (Fig. 1). therapy with estrogens for carcinoma of the prostate The finding of hepatic siderosis in virtually all had values in the normal female range. patients with PCT and the clinical benefits of phle- It is proposed that porphyria cutanea tarda results botomy therapy (14-16) have raised the possibility from the combination of an inherited defect in uro- that iron might play an important part in the etiology porphyrinogen decarboxylase and an acquired fac- of the disease. In support of this hypothesis it has tor, usually siderosis associated with alcoholic liver been shown, in an in vitro model system, that iron disease. 'Abbreviations used in this paper: ALA, 8-aminolevulinic Receivedfor publication 22 August 1975 and in revisedform acid; PCT, porphyria cutanea tarda; URODECARB, uro- 12 July 1976. porphyrinogen decarboxylase. The Journal of Clinical Investigation Volume 58 November 1976 -1089-1097 1089 Succinyl CoA + Glycine prior transfusion therapy. Liver was obtained by needle biopsy from one nonporphyric patient with hemochroma- 1D tosis. Porphyric human liver was obtained by open biopsy ALA in two instances and by needle biopsy in the remaining 1) cases. In all cases the informed consent of the patient was 10 obtained. Fresh or frozen liver was first carefully rinsed PBG 11) in saline and then homogenized in 10 vol of 0.1 M Tris- HCI buffer, pH 6.8, in a 2-ml TenBroeck glass-glass ipo'r.PFs(T homogenizer. The homogenate was centrifuged at 37,000g for 30 min at 4°C yielding a mitochondria-free supernate. 7,6,5- carboxyl porphyrinogens | 6,5-carboxyl porphyrinogens Aliquots of 0.2 ml were then utilized as the source of URODECARB in the assay system. Protein determinations were performed by the method of Lowry et al. (23). A COPRO GEN I COPRO 'GENIl portion of each liver biopsy sample was processed by routine histologic methods and stained for iron by the usual PROTO IX Perls' Prussian blue method. The stainable iron content of the parenchymal cells was graded according to the criteria 10 of Scheuer et al. (24). These criteria assign a value of Heme grade 0 to normal hepatic iron content and increasingly FIGURE 1 The enzymatic pathway for the biosynthe- severe hepatic siderosis is graded 1 through 4. sis of heme. (1) = ALA synthetase, (2) = ALA dehy- Venous blood samples were processed by centrifuging dratase, (3) = uroporphyrinogen I synthetase, (4) = uro- the blood, removing the buffy coat, and washing the packed porphyrinogen III cosynthetase, (5) = uroporphyrinogen erythrocytes twice with chilled saline. The washed cells decarboxylase, (6) = coproporphyrinogen oxidase, (7) were then resuspended in an equal volume of 0.1 M = heme synthetase, ALA = 8-aminolevulinic acid, PBG Tris pH 6.8. Cell lysis was accomplished by sonication = porphobilinogen; URO'GEN = uroporphyrinogen, followed by freeze-thawing twice. The lysate was then centri- COPRO'GEN = coproporphvrinogen, PROTO = proto- fuged at 32,000 g for 30 min at 4°C and aliquots of 0.4 porphyrin. ml of the supernate were utilized as the source of URODE- CARB in the assay system. The hemoglobin content of the supemate was determined with a Coulter counter model alters hepatic porphyrin metabolism in a way that S (Coulter Electronics Inc., Hialeah, Fla.). parallels the abnormalities observed in PCT (17). URODECARB was assayed by a previously published method by utilizing enzymatically generated, tritiated However, the vast majority of patients with alcoholic uroporphyrinogen as the substrate and thin layer chromato- liver disease and hepatic siderosis do not develop graphic identification and liquid scintillation quantitation PCT. This clinical observation has led to specula- of the reaction products (25). tion that patients with PCT have an occult genetic Hepatic porphyrin content was quantitated by analysis of the pellet and the remaining supemate from the centri- abnormality expressed only when the liver cell is fuged homogenates. These were both extracted with 2.0- injured in a specific way (18). The concept that ml aliquots of 3.0 N HC1 until no fluorescence was ob- there may be a genetically determined contribution to served in the eluates. The HC1 concentration was then the pathogenesis of PCT has been strengthened by adjusted to 1.5 N, and the porphyrin content was measured occasional reports describing a familial occurrence spectrophotometrically by scanning the spectrum from 370 to 440 nm with a Cary spectrophotometer (Cary Instru- of the disease (19, 20). In addition, several reports ments, Fairfield, N. J.) and applying the correction factors have described abnormalities of porphyrin metab- of With (26). The porphyrins were then adsorbed on talc olism in apparently unaffected relatives of patients and esterified in H,SO4-methanol (27). Porphyrins were with PCT (21, 22).2 then identified chromatographically by the method of Doss (28). This study was designed to evaluate the hypothesis Urine porphyrin concentrations were determined by a that PCT is associated with an inherited defect in previously published method (29). Chromatographic identifi- the enzyme URODECARB. To evaluate the hy- cation of the urinary porphyrins was done as described above pothesis, a method for assaying URODECARB ac- for hepatic porphyrins. Isomers of uroporphyrin were tivity was applied to both crude liver extracts and identified by decarboxylation to coproporphyrin (30) and erythrocyte hemolysates of six patients with PCT.