Erythrocyte Uroporphyrinogen I Synthase Activity as an Indicator of Acute Porphyria

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1 A N N A L S O F C L IN IC A L A N D L A B O R A T O R Y S C IE N C E, Vol. 19, N o. 2 Copyright 1989, Institute for Clinical Science, Inc. Erythrocyte Uroporphyrinogen I Synthase Activity as an Indicator of Acute Porphyria DONALD T. FORMAN, Ph.D. Division o f Laboratory Medicine, Department o f Pathology, University o f North Carolina, Chapel Hill, NC ABSTRACT T he pre-clinical diagnosis of acute in te rm itte n t porphyria (AIP) is im portant because acute attacks can be brought about by drugs, liver toxins, hormonal changes and diet. There also may be no obvious precipitating agent. T he discovery that the activity of uroporphyrinogen I synthase (URO-S) activity in the red blood cells of patients with AIP is half that found in normal persons is of great value in diagnosing this disorder and also appears useful in detecting patients with the latent disease who have norm al urinary delta-am inolevulinic acid and porphobilinogen excretion. It also appears to distinguish other types of porphyria from acute interm ittent porphyria. It m ust also be recognized that some red blood cells URO-S determ inations will yield indeterm inate results; therefore, rep eat assays, including examination of kinship, will im prove discrim ination and confidence in the final diagnosis. Introduction Acute interm ittent porphyria (AIP) is frequently familial and is inherited as an autosom al dom inant trait with variable penetrance, therefore the variability of symptoms. 18 The disease is characterized by th re e id entified enzym e abnorm alities: increased activity of delta-am inole v u lin a te s y n th a s e (E C ), decreased activity of steroid 4-5a-reductase, and decreased activity of uro p o r phyrinogen I synthase (EC )17 (table I). There is evidence of the heterogeneity of th e genetic defect as uroporphyrinogen I synthase (URO-S) forms four interm ediates during synthesis of uroporphyrinogen I. 2 Acute interm ittent porphyria is the most common form of porphyria, w ith an incidence of one in 1 0 0,0 0 0, occurring more often in women than in men. The age of onset is usually after puberty, suggesting a steroid-linked or horm onal initiated pathobiological process.20 During specific phases of the m enstrual cycle, acute attacks may be exacerbated, again pointing to possible endocrine mediating factors. In patients w ith latent AIP, attacks of this potentially fatal disease may be precipitated by drugs such as barbiturates, hydantoins, and estrogens as well as o th er factors such as liver toxins, hormonal changes, and diet. 15 There also may be no obvious precipitating agent. The diagnostic assay and biochem ical usefulness of URO-S as /89/ $00.90 Institute for Clinical Science, Inc.

2 ERYTHROCYTE UROPORPHYRINOGEN I SYNTHASE ACTIVITY 129 TABLE I Acute Intermittent Porphyria Familial autosomal dominant trait Characterized by: 1. Erythrocyte uroporphyrinogen I synthase + 2. Delta-aminolevulinate synthase + 3. Steroid A 4-5a-reductase + an indicator of AIP are discussed in the following sections along w ith proposed guidelines for using th e assay for optimally evaluating individuals and kindred suspected of carrying the AIP trait. Assay o f E rythrocyte U roporphyrinogen I Synthase Activity P atients w ith AIP (overt or latent) may have decreased uroporphyrinogen I synthase (URO-S) activity, w hich accounts for the several laboratory assays that have been developed to detect this biochem ical defect. 5,7 The designation of the disease as acute interm ittent porphyria is not an entirely appropriate nom enclature because the porphyrins are not involved but rather the porphyrin precursors, amino-levulinic acid (ALA) and porphobilinogen (PBG). T he enzym atic lesion is a deficiency of URO-S in red cells and other tissues, and this synthase partially blocks the conversion of porphobilinogen into the porphyrinogens (figure l ) The deficiency o f uroporphyrinogen I synthase leads to an increase in urinary PBG excretion, which is often coupled with an increased ALA excretion. The latter is the result of stim ulated activity of ALA synthase secondary to dim inished feedback inhibition by h em e. 9 D uring the attack, increased am ounts of PBG and ALA are excreted in the urine. During the latent periods, the urinary PBG and ALA levels are still elevated, although they are m uch lower than acute attack concentrations and may even be norm al. 19,21 Since carriers of AIP have normal ALA and PBG excretion, the most appropriate test appears to be th e m easurem ent of erythrocyte uroporphyrinogen I synthase activity. T he la tte r is reduced in latent and as well manifest forms of AIP5 thus providing enhanced specificity. O th er acute forms of p o r phyria may have an identical clinical and chemical picture during the acute phase, b u t ery th ro cy te uroporp h y rin o g en I synthase activity will be normal in these porphyrin disorders. An approximately 50 p e rc e n t re d u c tio n in a c tiv ity of URO-S has been reported to occur in erythrocytes, leukocytes, cultured skiri fibroblasts and amniotic cells. Thus, the URO-S assay also enables the in trau terine diagnosis of AIP. Principle of M ethod for URO-S Two features of this enzym atic reaction8,12 have allowed for easy adaptability of the assay to the clinical laboratory. First, the enzym e is cytoplasmic and, th erefo re, is reta in e d w ithin m ature erythrocytes, which provide an ideal specim en for assay. Second, th e su b strate for the reaction is non-fluorescent, w hile the resulting product of the enzymatic reaction (uroporphyrin) is highly fluorescent. This p erm its its m easu rem e n t in p ico m o la r q u a n titie s. T he URO-S enzyme catalyzes the formation of uroporphyrinogen from the m onopyrrole precursor substrate, porphobilinogen. U nder the assay conditions, the reaction forms uroporphyrinogen, which is rapidly oxidized to uroporphyrin d u r ing the acid deproteinization of th e reaction mixture. Uroporphyrin fluorescence at wavelengths greater than 600 nm is then m easured directly after excitation at 405 nm. The rate of uroporphyrin production is calculated and expressed in enzym e units of activity p e r gram of hemoglobin. The reference range in a healthy population is 1.27 to 2.01 m A per g hem oglobin. 11

3 130 FORMAN SUCCINYL GOA + GLYCINE ^ Ala Synthase AMINOLEVULINIC ACID Ala Dehydrase PORPHOBILINOGEN ^ Porphobilinogen Deaminase NEGATIVE FEEDBACK INHIBITION HYDROXYMETHYLBILANE Uroporphyr III Synthas^ ^Uroporphyr I Synthase UROPORPHYRINOGEN III UROPORPHYRINOGEN I ^ Decarboxylase ^ Decarboxylase F ig u r e 1. T h e path w ay for h e m e sy n th e sis in c lu d in g sites o f en zy m e insufficiency in th e p o rp h y rin s. COPROPORPHYRINOGEN III COPROPORPHYRINOGEN I ^ Oxidase PROTOPORPHYRINE Fe+Z ^ Heme Synthase HEME ^ + Protein HEMOGLOBIN Since e ry th ro c y te URO-S activity varies with cell age and differentiation, a shift in th e erythrocyte population tow ard younger cells (also observed in the new born) may lead to an enzym e activity that does not accurately re p re sent AIR Usually an elevated reticulocyte percentage would indicate that such a condition could be present.5 Clinical Biochemistry of AIP A partial block in hepatic hem e biosy n th e sis at th e en z y m a tic ste p of URO-S which catalyzes the conversion of PBG to uroporphyrinogen I, coupled with increased activity of ALA synthase, explains the long known increased urinary excretion of the porphyrin p recu r sors PBG and ALA in AIP (figure l ). 14 Although decreased URO-S activity has b een dem onstrated, the precise m olecular basis for this deficiency has not been e s ta b lis h e d. N e v e r th e le s s, in th e absence of increased urinary excretion of PBG, th e assay of URO-S activity in e ry th ro c y te lysates has p ro v id e d an im proved m eans for detectin g th e genetic defect. 13 There are, however, limitations of the erythrocyte URO-S assay. In some cases, enzyme activity in patients with AIP is just below the range of normal controls, and other patients with definitive AIP m ay have URO-S values w ith in th e reference range of norm al controls. 22 O thers have reported that if nonaffected persons w ithin families are used as controls, affected persons are more readily separable. 10 Doss6 reported that erythrocyte URO-S increased to normal levels in a patient during the recovery phase of an acute attack, suggesting that URO-S may correlate in some m anner with the clinical course and perhaps urinary findings. A d d itio n a l factors th a t in flu e n c e erythrocyte URO-S activity are erythrocyte age and specim en storage. The U R O -S a c tiv ity has b e e n fo u n d to decrease as erythrocytes age. 1 It also appears th at storage of ery th ro cy tes enhances variability of m easured en zyme activity. If blood is stored at 4 C, there is an inconsistent loss of activity, as m uch as 10 p e rc e n t by day 3 and as much as 20 percent by day 5; at room tem p eratu re th e losses can be 25 to 35

4 ERYTHROCYTE UROPORPHYRINOGEN I SYNTHASE ACTIVITY 131 percent. 5 Therefore, heparinized blood sam ples should be cen trifu g ed, and, after rem oval of the plasma and buffy coat, the red cells washed thrice in cold isotonic saline. After packing by centrifugation and freezing in dry ice-acetone, the samples should be stored at 20 C. Conclusion On the basis of clinical and biochem i cal findings, the following guidelines are suggested in the evaluation of patients and kindred for AIP: Patients who are ill and suspected of having this disorder because of abdominal pain, neuropathy, or other symptoms or signs, should have their urine assayed for PBG and ALA. If qualitative and quantitative studies for PBG and ALA are normal, the patients symptoms are probably not due to AIP. If the urine PBG or ALA are increased, the differential diagnosis includes AIP, variegate porphyria, hereditary coproporphyria, and lead poisoning. A single determination of erythrocyte URO-S activity that is clearly normal is usually sufficient to rule out the diagnosis of AIP. A repeat URO-S determination should be performed when clinical suspicion is high or if urine results are abnormal and characteristic of AIP. A reticulocyte count should be obtained on the blood sample and the URO-S assay interpreted in light of the count. A reticulocyte count of five percent or more may lead to falsely elevated URO-S activity. If urine results are characteristic and repeat erythrocyte URO-S values are low, the diagnosis of AIP is clearly suggested and the patient may be considered a carrier of the AIP trait. Finally, it must be recognized that some erythrocyte URO-S determ inations will yield indeterminate results, even when the samples are meticulously handled. Repeat assays and examination of the kinship, will improve discrimination and confidence in the final diagnosis. References 1. An d e r s o n, K. E., Sassa, S., P e t e r s o n, C. M., et al.: Increased erythrocyte uroporphyrinogen- I-synthase, delta-amino-levulinic acid dehydratase and protoporphyrin in hemolytic anemias. A m. J. Med. 63: , A n d e r s o n, P. M., R e d d y, R. M., An d e r s o n, K. E., and D e s n ic k, R. J.: Characterization of the porphobilinogen deaminase deficiency in acute interm ittent porphyria. J. Clin. Invest. 68:1 12, Astru p, E. G.: Family studies on the activity of uroporphyrinogen I synthase in diagnosis of acute interm ittent porphyria. Clin. Sci. Mol. Med. 54: , B i c k e r s, D. R.: P orphyria: Basic science aspects. Dermatologic Clinics 4: , B o t o m l e y, S. S., B o n k o w s k y, H. L., and K r e im e r -Bir m b a u m, M.: The diagnosis of acute interm ittent porphyria, usefulness and limitations of erythrocyte uroporphyrinogen I synthase assay. Am. J. Clin. Pathol. 76: , Doss, M., S C H E H M U L Y, E., V O N T le P E R M A N N, R., et al.: Biochemical course in acute interm ittent porphyria. Supplement to the Proceedings of the First International Meeting on Porphyrins in Human Diseases. Doss, M. and Nawrocki, P., eds. Basel, Karger, 1976, pp E ld er, G. H.: Recent advances in the identification of enzyme deficiencies in the porphyrias. Brit. J. Dermatol. J0S: , F o r d, R. E., O u, C-N., and E l l e f s o n, R. D.: Assay for erythrocyte uroporphyrinogen I synthase activity, w ith porphobilinogen as substrate. Clin. Chem. 26: , H ln D M A R S H, J. T.: The porphyrias: R ecent advances. Clin. Chem. 32: , K r e im e r -B ir n b a u m, M. and T o r n io, M. J.: Studies on uroporphyrinogen synthase from human erythrocytes. Porphyrins in Human Diseases. Doss, M. and Nawrocki, P., eds. Basel, Karger, 1976, pp L a b be, R. F. and L a m o n, J. M.: Porphyrins and disorders of porphyrin metabolism. Textbook of Clinical Chemistry. Tietz, N. W., ed. Philadelphia, W.B. Saunders Co., pp M a g n u s s e n, C. R., L e v in e, J. B., D o h er ty, J. M., C h e e s m a n, J. O., andtschudy, D. P.: A red cell enzyme method for the diagnosis of acute interm ittent porphyria. Blood 44: , M ey e r, U. A.: Interm ittent acute porphyria. Clinical and biochemical studies of disordered heme biosynthesis. Enzyme i6: , M iy a g i, K., C a r d in a l, R., Bo sse n m a ie r, I., et al.: The serum porphobilinogen and hepatic porphobilinogen deam inase in norm al and porphyric individuals. J. Lab. Clin. Med. 75: , M o o r e, M. R. and D is l e r, P. B.: Drug-induction of the porphyrias. Adv. D rug React. Ac. Pois. Rev. 2: , 1983.

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