Variegate Porphyria Pathology

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European Journal of Clinical Investigation (1986) 16 257-261

Protoporphyrinogen oxidase and porphobilinogendeaminase in variegate porphyria

P. N . MEISSNER, R. S. DAY*, M. R . MOORE?, P. B. DISLERS & E. HARLEY, MRC Liver ResearchGroup, Department of Medicine, University of Cape Town, Observatory, 7925, South Africa, *MRCInstitute for Biostatistics, P.O. Box 70 , Tygerberg, 7505, Cape Town, ?University of Glasgow, 'Department ofMedicine, Gardiner Institute, Western Infirmary, Glasgow G11 6NT, Scotland, Departments of $CommunityHealth and Chemical Pathology, University of Cape Town, South Africa

Received 25 July 1985 and in revised form 24 January 1986

Abstract. Two enzymes of the haem biosynthetic

pathway were investigated in patients with variegateporphyria. Protoporphyrinogen oxidase in cultures ofEpstein-Barr virus transformed lymphoblasts fromtwenty-seven patients showed a mean maximal velo-city ( Vmax) f 0.39 k 0.08+ nmol of protoporphyrin mgprotein-' h-',a52%reduction (P 0.05)from control values in any of the patients. The meanVmaX f porphobilinogen deaminase in the cultures was1.50k 0.1 8 nmol of uroporphyrin mg protein-' min-I,a 24% reduction (P


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258 P. N . MEISSNER et a1.

lymphoblasts from patients with VP, including akindred of three generations. The erythrocyte PBGdeaminase activities in most of the same VP popula-tion were also measured. The aim was to confirm thesite of the primary block in VP and to determinewhether the suspected deficiency in PBG deaminasewas present. The data obtained from the VP kindredstudied were used to analyse any relationship betweenthe two enzymes.

Methods

Establishment and maintenance of transformedlymphohlasts

20 ml of blood were taken into heparin or EDTAfrom twenty-seven VP patients and thirty normalsubjects (age and sex matched). Although it is gener-ally believed that all the VP patients theoretically havea common ancestor [l-31, in twenty-one patients noblood relationship was apparent for at least threegenerations. The remaining six VP patients belongedto three generations of a well established local VPkindred. The diagnosis of VP was based on the clinicalfeatures and family histories, and biochemically on thedemonstration of the characteristic pattern of por-phyrins in faeces, urine and plasma by using aquantitative thin-layer chromatographic technique [4].Epstein-Barr virus (EBV) transformed lymphoblastcell lines were established as described elsewhere [12,131. When required for assay, the lymphoblast suspen-sion was centrifuged at 850 x g for 15 min, washedtwice in Hanks Balanced Salt Solution (HBSS) (Gibco,Europe) and resuspended in a final volume of 1.2 ml of

HBSS. The cells were counted on a Coulter counter(Coulter Electronics Inc., Model ZF) and their v'iabi-lity measured using a trypan blue exclusion test. Thecells were sonicated at full power (Heat SystemsUltrasonic Inc., Model W-10) for three periods of 20sec each, the preparations being cooled intermittentlyon ice. The protein concentration was determined bythe Lowry method [14].

Measurement of PO oxidase activity

A fluorimetric assay modified from previously de-scribed methods was used [ I5-17]. Protoporphyrino-gen as substrate was prepared as previously described[16, 181 and used at five different concentrations,between 0.75 and 2.5 pmol being added together with0.2 ml of lymphoblast sonicate and correspondingvolumes of buffer concentrate to give a final composi-tion of SO mM Tris-HC1 (pH 8.7) , 1 mM EDTA, 3 mMdithiothreitol and 1% Tween-20 when the volume wasmade up to 0.375 ml with 10 mM KO H in 20% vjvethanol. Five control tubes contained 1 rng/ml bovineserum albumin fraction V (BSA) instead of thepreviously described heated tissue preparation toavoid turbidity experienced with the latter. Non-enzymatic oxidation of protoporphyrinogen to proto-porphyrin was similar whether the controls contained

BSA, water or preparation of lymphoblasts heated to90C for 15 min. The reaction was allowed to proceedin the dark for 1 h at 37C. Protoporphyrin concentra-tions were measured at zero time and at 1 h using aspectrofluorimeter (Perkin Elmer-Model 204A) with aflow cell of 60 p1 volume and set at an excitationwavelength of 402 nm and an emission wavelength of625 nm. Use of a single measurement of protopor-phyrin produced after 1 h was validated in a time-course experiment where the PO activity was shown tobe linear for up to 90 min. After correcting for non-enzymatic production of protoporphyrin, apparentV,,, and K,,, values for PO were read from double-reciprocal Lineweaver-Burke plots.

Measurement of PBG deaminase activity

The rate of uroporphyrin production from PBG(substrate) was measured by a fluorimetric method [19,201 with assay volumes scaled down such that only 10

pl of either transformed lymphoblast preparation orsaline-washed erythrocytes were required. Lymphob-last PO and PBG deaminase activities were determinedin twenty-seven patients of which twenty-one also haderythrocyte PBG deaminase measured.

Statistical analysis

Where appropriate, P values were determined byusing Student's t-test for comparing the means of twosamples.

Results

Biochemical diagnosis o V P

The results of the porphyrin and urinary porphyrinprecursor analyses of the twenty-seven VP patientsused in the study are summarized in Table 1. Theelevated levels of faecal protoporphyr in and copropo-phyrin together with the normal levels of urinaryporphyrin precursors show that all patients were in thequiescent phase of VP, the criteria for which have beendefined previously [3].

Lympnhlast cultures

Twenty-seven EBV transformed lymphoblast linesfrom VP patients were established together with thirtylines from age and sex matched controls. In allexperiments where V P lymphoblast cultures werecompared to control cultures, the protein concentra-tion, cell number and cell viability of both groups ofcells were measured. None of these parameters differedbetween the groups by more than 12% (P>O.OS).

Enzyme activities in E B V lymphohlasts

The individual results for PO and PBG deaminaseactivities of each lymphoblast line are given in Fig. 1.The PO mean apparent V,,,, value for the control


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ENZYME STUDIES IN VARIEGATE PORPHYRIA 259

2 . 2 -

2 . 0 -

1.8.

1.6

1.4

1210

0.8

0.6

0. 4

0.2

Table 1. Mean (and range) of levelsof porphyrins and precursors insubjects investigated

Faeces Urine

(nmol g dry weight) (nrnolI I) (pmol 1 I)

P R O TO * C O P R O U R O C O P R O AL A PB G

~

Variegate 361 8 6 3 7 2 6 8 2 6 11 11 8 8 9porphyria (211-1053) ( 1 6 1 1 2 ) (10-41) (98-313) (5-17) (3-14)(1 8 female,9 male)

Nor m a l i 2 0 0


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260 P. N. MEISSNER et al.

VP Family

2.*

0.851.49

*3.

0.19 0.36 0.57 0.79 0.74 1.05 0 . 3 9 0.81 0.841.36 1.58 1.98 2.00 1.84 1.95 1.37 2.19 1 .75

0 ale0 emale

CombinedDecreased PO

Decreased PB G - D

Figure 2. Pedigree of the V P family studied. Their activit ies are given in nmo l protop orphyrin mg protein' h an d P BCdeaminase activit ies in nmo l uroporphyrin mg proteinI mi n I. Asterisks denote those subjects who were diagnosed as having VPaccording to their porphyrin excretory profiles.

* Results are quotedas mean f tandard deviation.

activities given in a simplified family tree in Fig. 2. Inall but one of the subjects where the PO activity wasmore than two standard deviations below the controlmean activity, the PBG deaminase activity was alsogreater than two standard deviations below the controlmean activity. The one subject had a PO activity of0.57 nmol protoporphyrin mg pro tein-' h-' whichcould not be unequivocally classified in either theporphyric nor the control group.

Discussion

In this study, lymphoblast PO levels were found to besignificantly decreased by approximately 50% intwenty-seven quiescent cases of VP. The PO deficiencywas found in six members covering three generationsof the family in Fig. 2, each of whom showed clinicalsymptoms and/or biochemical signs of VP. Thisdecrease in the apparent V,,, of PO supports previousreports that a decrease in its activity is the heterozy-gous autosomal dominant factor contributing to VP[6-81. The lack of any significant variation of theapparent K , of the enzyme in lymphoblasts fromcontrol and VP subjects suggests that only activityfrom the enzyme produced by the normal allele is beingmeasured. The abnormal allele is either producing noenzyme or an enzyme with a markedly increased K,.

The use of EBV transformed lymphoblasts offeredseveral advantages. Cells can be obtained with littleinconvenience to patients (only 10-20 ml of blood isrequired) as compared with more invasive proceduressuch as skin biopsy [8, 151. This is a particularadvantage where large surveys are to be undertaken. Along-lived cell line is produced which can be storedwith minimal loss of cell viability [13]. Thus repeatassays, further experimentation, transport and rapid

cell population expansion are facilitated. The use of akinetic as opposed to an end-point assay for POactivity enable comparisons to be made for bothapparent V,,, and apparent K,,, whilst ensuring thatthe enzyme was working in substrate excess.

The mean PBG deaminase activity in the group ofpatients with quiescent VP, including the six pre-viously established cases in the VP family, was signifi-cantly reduced in both lymphoblasts and erythrocytes.

There have been other studies where erythrocyte PBGdeaminase levels in patients with VP have beenmeasured. In some of these, the number of cases weresmall, ranging from 1 to 6 , [I I , 21-23] and statisticallysignificant changes from normal were not apparent . Intwo larger studies (sixteen and seventeen VP patientsrespectively [7-241) significant reductions in PBGdeaminase activity could be observed; either 10%( P < .01, our calculations from their da ta [24]) or 20%(P < 0.005) [7]. Similar reductions on PBG deaminaseactivity have been reported in subjects with hereditarycoproporphyria, another of the acute porphyrias [25,261.

The coexistence of a low PBG deaminase activitytogether with the PO deficiency in the cases reportedhere may explain some of the features characteristic ofVP. The raised levels of faecal protoporphyrin foundin quiescent VP may be explained by the decreased P Oactivity, although the site of excess production is notnecessarily the liver [27]. It is possible that the synthesisof ALA synthase is not sufficiently derepressed [10,291in the quiescent phase of VP to give to biochemicaldisturbances secondary to the diminished PBG deami-nase activity. Additional endogenous or exogeneousporphyrogenic agents appear to be necessary to initiatethe acute phase of VP by further inducing the synthesisof ALA synthase, thereby causing any rate-limiting


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E N Z Y M E S T U D IE S I N VA R I E G AT E P O R P H Y R I A 261

properties of PBG deaminase to be exacerbated by thesecondary deficiency and resulting in elevated levels ofurinary porphyrin precursors [ 2 ] . Further factorsremain to be identified to explain the other phases ofVP. T he identification of casesof silent VP remains amajor diagnostic problem since presumably they aresusceptible to the life-threatening acute attack onexposure to porphyrogenic agents. Since these silentVP cases may only be detectable using PO assay, itshould be adapted for routine diagnostic use.

The cause of the P BG deaminase deficiency found inVP is not known. It it were a dominantly inheritedmutation (as in acute intermittent porphyria[28, 291)independent of the primary PO mutation then thefamily study should have revealed some subjects withthe PBG deaminase deficiency alone, yet none weredetected. It would seem, however, that the PBGdeaminase is inherited in some manner since it isretained in culture. It is apparently a secondaryphenomenon which is expressed by an unknownproportion of subjects inheriting the PO deficiency.

Acknowledgments

This work was supported by the South AfricanMedical Research Coun cil. We are indebted to Profes-sors L. Eales, R. E. Kirsch, Drs G. Blekkenhorst, B.Kehoe, L. Steyn, E. du Toit, Sr D. Meissner and E.Sturrock for advice and enthusiastic co-operation; toR. Schadle, B. Davidson, L. Pieters, N . Ally and E.Campbell for technical assistance; and to the Univer-sity of Cape Town and Groote Schuur Hospital forproviding facilities.

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Variegate Porphyria Pathology

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