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The Role of Iron in the Pathogenesis of Porphyria Cutanea Tarda. II

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The Role of Iron in the Pathogenesis of Porphyria Cutanea Tarda. II The role of iron in the pathogenesis of porphyria cutanea tarda. II. Inhibition of uroporphyrinogen decarboxylase. J P Kushner, … , D P Steinmuller, G R Lee J Clin Invest. 1975;56(3):661-667. https://doi.org/10.1172/JCI108136. Research Article Porphria cutanea tarda is characterized biochemically by excessive hepatic synthesis and urinary excretion of uroporphyrin I and 7-carboxylporphyrins. This pattern of excretion suggest an impaired ability to decarboxylate uroporphyrinogen to the paired ability to decarboxylate uroporphyringen to the 4-carboxyl porphyrinogen, coproporphyrinogen, a reaction catalyzed by the enzyme uroporphyringen decarboxylase. Because clinical evidence has implicated iron in the pathogenesis of porphyria cutanea tarda, these experiments were designed to study the effect of iron on uroporphyrinogen decarboxylase in procine crude liver extracts. Mitochondria-free crude liver extracts were preincubated with ferrous ion and aliquots were assayed for uroporphyrinogen decarboxylase activity. Uroporphyrinogens I and III, the substrates for the decarboxylase assay, were prepared enzymatically from (3H)porphobilinogen. The products of the decarboxylase reaction were identified and quantitated by three methods: (a) extraction into 1.5 N HCl and spectrophotometric quantitation; (b) adsorption onto talc, esterification, paper chromatographic identification, and quantitation by liquid scintillation counting; and (c) adsorption onto talc, esterification, thin-layer chromatographic identification on silica gel, and quantitation by liquid scintillation counting. The thin-layer scinllation method proved most sensitive as it was the only method which accurately identified and quantitated the 7-carboxyl porphyrin reaction product. Uroporphyrinogens I and III were decarboxylated at the same rate by porcine hepatic uroporphyrinogen decarboxylase, and the addition of iron induced marked inhibition of the decarboxylase activity. Ortholpehanthroline blocked the inhibitory […] Find the latest version: https://jci.me/108136/pdf The Role of Iron in the Pathogenesis of Porphyria Cutanea Tarda II. INHIBITION OF UROPORPHYRINOGEN DECARBOXYLASE J. P. KusmqNi, D. P. STEiNmuuim, and G. R. LEE From the Departments of Internal Medicine of the Veterans Administration Hospital, and The University of Utah College of Medicine, Salt Lake City, Utah 84132 A B S T R A C T Porphyria cutanea tarda is character- Uroporphyrinogens I and III were decarboxylated at ized biochemically by excessive hepatic synthesis and the same rate by porcine hepatic uroporphyrinogen de- urinary excretion of uroporphyrin I and 7-carboxyl carboxylase, and the addition of iron induced marked porphyrins. This pattern of excretion suggests an im- inhibition of the decarboxylase activity. Orthophenan- paired ability to decarboxylate uroporphyrinogen to the throline blocked the inhibitory effect of iron. 4-carboxyl porphyrinogen, coproporphyrinogen, a re- The inhibition of uroporphyrinogen decarboxylase by action catalyzed by the enzyme uroporphyrinogen de- ferrous ion, coupled with its previously reported in- carboxylase. hibitory effect on uroporphyrinogen III cosynthetase, Because clinical evidence has implicated iron in the provides a possible biochemical explanation for the pathogenesis of porphyria cutanea tarda, these experi- pattern of urinary porphyrin excretion observed in pa- ments were designed to study the effect of iron on tients with porphyria cutanea tarda and the clinical uroporphyrinogen decarboxylase in porcine crude liver association with disordered iron metabolism. extracts. Mitochondria-free crude liver extracts were preincu- INTRODUCTION bated with ferrous ion and aliquots were assayed for uroporphyrinogen decarboxylase activity. Uroporphyri- Porphyria cutanea tarda (PCT),' the most common of nogens I and III, the substrates for the decarboxylase the clinically recognized disorders of porphyrin me- assay, were prepared enzymatically from [3H]porpho- tabolism (1), is characterized biochemically by the bilinogen. The products of the decarboxylase reaction hepatic synthesis and urinary excretion of excessive were identified and quantitated by three methods: (a) amounts of porphyrins, particularly uroporphyrin I extraction into ethyl acetate at pH 4.0, back extraction (URO I) and smaller amounts of 7-carboxyl porphyrin into 1.5 N HCO and spectrophotometric quantitation; (2-4). The pattern of porphyrin excretion is similar to (b) adsorption onto talc, esterification, paper chromato- that found in congential erythropoietic porphyria graphic identification, and quantitation by liquid scin- (Gunther's disease), but in PCT the erythrocyte does tillation counting; and (c) adsorption onto talc, esteri- not appear to contribute to the excess porphyrin pro- fication, thin-layer chromatographic identification on duction. silica gel, and quantitation by liquid scintillation Defects in porphyrin biosynthetic enzymes have not counting. The thin-layer scintillation method proved been clearly identified in PCT. However, the pattern of most sensitive as it was the only method which accu- 1Abbreviations used in this paper: COPRO, copropor- rately identified and quantitated the 7-carboxyl porphyrin phyrin; COPROGEN, coproporphyrinogen; COSYN, uro- reaction product. porphyrinogen III cosynthetase; PBG, porphobilinogen; PCT, porphyria cutanea tarda; SYN, uroporphyrinogen I Received for publication 16 December 1974 and in revised synthetase; URO, uroporphyrin; UROGEN, uroporphy- form 2 May 1975. rinogen; URODECARB, uroporphyrinogen decarboxylase. The Journal of Clinical Investigation Volume 56 September 1975* 661-667 661 Sticcinyl-CoA + Glycine nate was prepared for each experiment. The homogenate ALA was centrifuged at 37,000 g for 30 min at 4VC. This pro- cedure yielded a mitochondria-free solution which will be referred to as a "crude liver extract." Protein determina- __________PBI tions were performed by the method of Lowry, Rosebrough, UROGENI UROGENIII Farr, and Randall (13). Crudc liver extracts were incu- bated in 25-ml flasks for 2 h at 370C under an 4 J 4 Erlenmeyer 7,6,5-carboxyl porphyrinogens 0@ 7,6,5-carboxyl porphyrinogens air atmosphere in a Dubnoff shaker. Control flasks con- s ~I I 4 tained 4.0 ml of crude liver extract, cysteine (6.7 mM), COPROGENI J 1 COPROGENIII and sufficient 0.1 M Tris-HCl buffer, pH 6.8, to make a 40~ final volume of 8.0 ml. Experimental flasks contained, in PROTOIX addition, ferrous ammonium sulfate (0.7 mM Fe). 0.1-0.4- 40( ml aliquots of the preincubation mixtures were then uti- Heme lized as the source of URODECARB in the assay system. FIGURE 1 The enzymatic pathway for the biosynthesis of URODECARB was assayed by a modification of the heme. (1) =8-aminolaevulinic acid synthetase; (2) = 6- method of Romeo and Levin (11). Unlabeled and tritium- aminolaevulinic acid dehydratase; (3) = SYN; (4) = CO- labeled UROGEN I and III for use as substrates were SYN; (5) = URODECARB; (6) = COPROGEN oxi- prepared enzymatically from PBG. SYN was partially puri- dase; (7) = heme synthetase; ALA - &aminolaevulinic fied from mouse spleen (14) and COSYN was extracted acid; PROTO = protoporphyrin. from the same tissue (15). Tritium-labeled PBG was pre- pared enzymatically from [3,5-3H] aminolaevulinic acid porphyrin excretion suggests the presence of more than (New England Nuclear, Boston, Mass.) by Dr. Ephraim one such defect (Fig. 1). In the normal metabolic path- Y. Levin. Dr. Levin also kindly supplied the SYN and the COSYN. PBG and cysteine were freshly prepared just way leading to the synthesis of heme, two enzymes are before use by dissolving them in 0.1 M Tris-HCl at pH required for the synthesis of uroporphyrinogen III 7.65 for UROGEN I generation or at pH 7.9 for URO- (UROGEN III) from porphobilinogen (PBG): uro- GEN III generation, to optimize COSYN activity.2 porphyrinogen I synthetase (SYN) and uroporphyrino- Aliquots of the incubation mixtures were then added to the tubes in which the UROGEN substrates had been gen III cosynthetase (COSYN) (5). The excretion of generated and the volume in each tube was adjusted to URO I in patients with PCT implies the presence of 1.0 ml with 0.1 M Tris-HCl, pH 6.8. The assay tubes at least a partial deficiency of hepatic COSYN activity. were then incubated for 30 min at 370C in the dark. The In addition, however, the predominance of URO I and reactions were then stopped in one of two ways, depending upon the method to be used in determining the products 7-carboxyl porphyrin in the urine, rather than copro- of the reactions. porphyrin I (COPRO I), suggests the presence of a When reaction products were to be determined by a second enzymatic defect, namely decreased hepatic uro- solvent extraction method, the reactions were stopped with porphyrinogen decarboxylase (URODECARB) activity. 8 ml of 2 M acetate buffer, pH 4.4 and the tubes were Iron overload has been implicated in the pathogenesis allowed to stand in the light for 30 min to oxidize por- phyrinogens to porphyrins (16). From the clear supernate of PCT on the basis of clinical observations (6-9), obtained by centrifuging at 37,000 g for 15 min, decar- and a previous report from our laboratory demonstrated boxylated porphyrins were extracted into 8 ml of ethyl that ferrous ion was a potent inhibitor of hepatic acetate. A 5-ml aliquot of this extract was back extracted COSYN activity (10). The present report describes into 5 ml of 1.5 N HCl. Porphyrin concentration was the inhibitory effect of ferrous ion on hepatic URODE- measured spectrophotometrically
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