ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 19, No. 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 of Laboratory Medicine, Department o f Pathology, University o f North Carolina, Chapel Hill, NC 27514 ABSTRACT The pre-clinical diagnosis of acute intermittent porphyria (AIP) is important because acute attacks can be brought about by drugs, liver toxins, hormonal changes and diet. There also may be no obvious precipi­ tating agent. The discovery that the activity of uroporphyrinogen I syn­ thase (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 normal urinary delta-aminolevulinic acid and porphobilinogen excre­ tion. It also appears to distinguish other types of porphyria from acute intermittent porphyria. It must also be recognized that some red blood cells URO-S determinations will yield indeterminate results; therefore, repeat assays, including examination of kinship, will improve discrimina­ tion and confidence in the final diagnosis. Introduction porphyria is the most common form of porphyria, with an incidence of one in Acute intermittent porphyria (AIP) is 1 0 0 ,0 0 0 , occurring more often in women frequently familial and is inherited as an than in men. The age of onset is usually autosomal dominant trait with variable after puberty, suggesting a steroid-linked penetrance, therefore the variability of or hormonal initiated pathobiological symptoms. 18 The disease is characterized process.20 During specific phases of the by three identified enzyme abnormali­ menstrual cycle, acute attacks may be ties: increased activity of delta-amino- exacerbated, again pointing to possible levulinate synthase (EC 2.3.137), endocrine mediating factors. In patients decreased activity of steroid 4-5a-reduc- with latent AIP, attacks of this poten­ tase, and decreased activity of uropor­ tially fatal disease may be precipitated by phyrinogen I synthase (EC 4.3.1.8)17 drugs such as barbiturates, hydantoins, (table I). There is evidence of the hetero­ and estrogens as well as other factors geneity of the genetic defect as uropor­ such as liver toxins, hormonal changes, phyrinogen I synthase (URO-S) forms and diet. 15 There also may be no obvious four intermediates during synthesis of precipitating agent. The diagnostic assay uroporphyrinogen I . 2 Acute intermittent and biochemical usefulness of URO-S as 128 0091-7370/89/0300-0128 $00.90 © Institute for Clinical Science, Inc. ERYTHROCYTE UROPORPHYRINOGEN I SYNTHASE ACTIVITY 129 TABLE I m al. 19,21 Since carriers of AIP have nor­ Acute Intermittent Porphyria mal ALA and PBG excretion, the most appropriate test appears to be the mea­ Familial autosomal dominant trait surement of erythrocyte uroporphyrino­ Characterized by: gen I synthase activity. The latter is reduced in latent and as well manifest 1. Erythrocyte uroporphyrinogen I synthase + 2. Delta-aminolevulinate synthase + forms of AIP5 thus providing enhanced 3. Steroid A 4-5a-reductase + specificity. Other acute forms of por­ phyria may have an identical clinical and chemical picture during the acute phase, but erythrocyte uroporphyrinogen I an indicator of AIP are discussed in the synthase activity will be normal in these following sections along with proposed porphyrin disorders. An approximately guidelines for using the assay for opti­ 50 percent reduction in activity of mally evaluating individuals and kindred URO-S has been reported to occur in suspected of carrying the AIP trait. erythrocytes, leukocytes, cultured skiri fibroblasts and amniotic cells. Thus, the Assay of Erythrocyte Uroporphyrinogen URO-S assay also enables the intrauter­ I Synthase Activity ine diagnosis of AIP. Patients with AIP (overt or latent) Principle of Method for URO-S may have decreased uroporphyrinogen I synthase (URO-S) activity, which ac­ Two features of this enzymatic reac­ counts for the several laboratory assays tion8,12 have allowed for easy adaptability that have been developed to detect this of the assay to the clinical laboratory. biochemical defect. 5,7 First, the enzyme is cytoplasmic and, The designation of the disease as acute therefore, is retained within mature intermittent porphyria is not an entirely erythrocytes, which provide an ideal appropriate nomenclature because the specimen for assay. Second, the sub­ porphyrins are not involved but rather strate for the reaction is non-fluorescent, the porphyrin precursors, amino-levu- while the resulting product of the enzy­ linic acid (ALA) and porphobilinogen matic reaction (uroporphyrin) is highly (PBG). The enzymatic lesion is a defi­ fluorescent. This permits its measure­ ciency of URO-S in red cells and other ment in picomolar quantities. The tissues, and this synthase partially blocks URO-S enzyme catalyzes the formation the conversion of porphobilinogen into of uroporphyrinogen from the monopyr­ the porphyrinogens (figure l ) . 4 1 6 The role precursor substrate, porphobilino­ deficiency of uroporphyrinogen I syn­ gen. Under the assay conditions, the thase leads to an increase in urinary PBG reaction forms uroporphyrinogen, which excretion, which is often coupled with an is rapidly oxidized to uroporphyrin dur­ increased ALA excretion. The latter is ing the acid deproteinization of the reac­ the result of stimulated activity of ALA tion mixture. Uroporphyrin fluorescence synthase secondary to diminished feed­ at wavelengths greater than 600 nm is back inhibition by heme . 9 During the then measured directly after excitation at attack, increased amounts of PBG and 405 nm. The rate of uroporphyrin pro­ ALA are excreted in the urine. During duction is calculated and expressed in the latent periods, the urinary PBG and enzyme units of activity per gram of ALA levels are still elevated, although hemoglobin. The reference range in a they are much lower than acute attack healthy population is 1.27 to 2.01 m|A concentrations and may even be nor­ per g hemoglobin. 11 130 FORMAN SUCCINYL GOA + GLYCINE ^ Ala Synthase AMINOLEVULINIC ACID | Ala Dehydrase NEGATIVE FEEDBACK INHIBITION PORPHOBILINOGEN ^ Porphobilinogen Deaminase HYDROXYMETHYLBILANE F ig u r e 1. The pathway Uroporphyr III Synthas^ ^Uroporphyr I Synthase for heme synthesis includ­ ing sites of enzyme insuffi­ UROPORPHYRINOGEN III UROPORPHYRINOGEN I ciency in the porphyrins. ^ Decarboxylase ^ Decarboxylase COPROPORPHYRINOGEN III COPROPORPHYRINOGEN I ^ Oxidase PROTOPORPHYRINE Fe+Z ^ Heme Synthase HEME ------------------— ^ + Protein HEMOGLOBIN Since erythrocyte URO-S activity There are, however, limitations of the varies with cell age and differentiation, a erythrocyte URO-S assay. In some cases, shift in the erythrocyte population to­ enzyme activity in patients with AIP is ward younger cells (also observed in just below the range of normal controls, the newborn) may lead to an enzyme and other patients with definitive AIP activity that does not accurately repre­ may have URO-S values within the sent AIR Usually an elevated reticulo­ reference range of normal controls. 22 cyte percentage would indicate that such Others have reported that if nonaffected a condition could be present.5 persons within families are used as con­ trols, affected persons are more readily Clinical Biochemistry of AIP separable. 10 Doss6 reported that erythro­ cyte URO-S increased to normal levels A partial block in hepatic heme bio­ in a patient during the recovery phase of synthesis at the enzymatic step of an acute attack, suggesting that URO-S URO-S which catalyzes the conversion of may correlate in some manner with the PBG to uroporphyrinogen I, coupled clinical course and perhaps urinary find­ with increased activity of ALA synthase, ings. explains the long known increased uri­ Additional factors that influence nary excretion of the porphyrin precur­ erythrocyte URO-S activity are erythro­ sors PBG and ALA in AIP (figure l ) . 14 cyte age and specimen storage. The Although decreased URO-S activity has URO-S activity has been found to been demonstrated, the precise molecu­ decrease as erythrocytes age . 1 It also lar basis for this deficiency has not been appears that storage of erythrocytes established. Nevertheless, in the enhances variability of measured en­ absence of increased urinary excretion of zyme activity. If blood is stored at 4°C, PBG, the assay of URO-S activity in there is an inconsistent loss of activity, as erythrocyte lysates has provided an much as 10 percent by day 3 and as improved means for detecting the ge­ much as 20 percent by day 5; at room netic defect. 13 temperature the losses can be 25 to 35 ERYTHROCYTE UROPORPHYRINOGEN I SYNTHASE ACTIVITY 131 percent. 5 Therefore, heparinized blood References samples should be centrifuged, and, 1. An d e r s o n , K. E ., Sassa, S., P e t e r s o n , C . M., after removal of the plasma and buffy et al.: Increased erythrocyte uroporphyrinogen- coat, the red cells washed thrice in cold I-synthase, delta-amino-levulinic acid dehydra­ isotonic saline. After packing by centrifu­ tase and protoporphyrin in hemolytic anemias. A m . J. Med. 63:359-364, 1977. gation and freezing in dry ice-acetone, 2. A n d e r s o n , P. M., R e d d y , R. M., An d e r s o n , the samples should be stored at — 20°C. K. E ., and D e s n ic k , R. J.: Characterization of the porphobilinogen deaminase deficiency in Conclusion acute intermittent porphyria. J. Clin. Invest. 68:1—12, 1981. On the basis of clinical and biochemi­ 3. Ast ru p, E . G.: Family studies on the activity of cal findings, the following guidelines are uroporphyrinogen I synthase in diagnosis of acute intermittent porphyria. Clin. Sci. Mol. suggested in the evaluation of patients Med. 54:251-256, 1978. and kindred for AIP: 4. B i c k e r s , D.