Porphobilinogen deaminase in acute intermittent porphyria: activity and concentration in erythrocytes and lymphocytes

77

Clinica Chimica Acta, 197 (1991) 77-84 Elsevier Science Publishers B.V. ADONIS 000989819100083N

CCA 04940

Short Communication

Porphobilinogen deaminase in acute intermittent porphyria: activity and concentration in erythrocytes and lymphocytes Lena Lilius ‘, Lars Lannfelt 2, Lennart Wetterberg 2, Ylva Floderus I, Ann Henrichson’ and Stig Thunell ’ ’ Departments of Clinical Chemistry and ‘Psychiatry, Karolinska Institute, St. G&an’s Hospital, Stockholm

(Sweden)

(Received 9 July 1990; revision received 12 November 1990; accepted 15 November 1990) Key wora!s: Acute intermittent porphyria; ELISA; Erythrocyte; deaminase.

Lymphocyte; Porphobilinogen

Introduction

Porphobilinogen deaminase (PBGD) (hydroxymethylbilane synthetase EC 4.3.1.8) is the third enzyme in the heme synthetic pathway. It catalyses the condensation of four molecules of porphobilinogen (PBG) to form the straight chain tetrapyrrole hydroxymethylbilane, which is converted to uroporphyrinogen III [l]. Acute intermittent porphyria (AIP) is a dominantly autosomal inherited disease [2]. Manifest symptoms may be precipitated by exogenous factors such as drugs, alcohol, stress and hormones [3]. The attacks are characterized by abdominal and neuropsychiatric symptoms [4]). Previous studies have demonstrated a decreased PBGD activity in carriers of the AIP trait in liver cells [5,6], in cultured skin fibroblasts [7,8], in amniotic cells [8], and in mitogen-stimulated lymphocytes [9]. Measurement of PBGD enzyme activity in erythrocytes is of great value in the detection of latent AIP gene carriers. Unfortunately, there is an overlap zone of enzyme activity between some AIP carriers and non-carriers [lo-131. To further investigate these ‘uncertain’ cases, an ELISA was developed, to determine the PBGD concentration in erythrocyte lysate [14,15]. Genetic heterogeneity in AIP has been revealed by immunological determination of PBGD, measuring the amount of cross-reacting immunological material (CRIM) in erythrocyte lysates [16-191. Two main types of AIP were found: one with decreased CRIM ((RIM-negative), and another with increased CRIM (CRIM-posiCorrespondence to: Dr. Lars Lannfelt, Department of Clinical Genetics, Karolinska Hospital, S-104 01 Stockholm, Sweden.

78

tive). These mutations could as well be distinguished by ELISA measurement of PBGD in red blood cells [20]. The PBGD gene encodes two enzymes [21]. One form (44 kDa) is present in all tissues, and another version (42 kDa) is present in e~t~opoietic cells. The enzyme in red blood cells differs from the ubiquitous moiety by the absence of a 17-amino acid residue at the N-terminus 122,231. The effect on the catalytic activity of the enzyme of this truncation has not been elucidated. Furthermore, Mustajoki [24] and Wilson et al. [19] have reported AIP families with a normal PBGD activity in red blood cells. It was presumed that the enzyme defect in these AIP gene carriers was restricted to non-erythropoietic tissue. However, this type of AIP has so far not been detected in Swedish AIP families [15]. The existence of AIP with normal erythrocyte PBGD act&ity implies that symptomatolo~ in the disease is caused by a deficiency of the non-e~t~oid form of PBGD. It might be assumed that the activity of the non-erythroid enzyme should be a better marker for the AIP trait than the erythrocyte enzyme activity. In this study AIP gene carriers of the CRIM-negative and the CRIM-positive type and controls were investigated by PBGD activity and PBGD concentration measured by ELISA. The aim was to determine the level of the PBGD gene expression in erythrocytes and lymphocytes and to investigate if determination of the non-erythroid PBGD enzyme was a better diagnostic instrument than the erythroid enzyme. Materials and methods Blood samples

Samples were obtained from 17 apparently healthy volunteers and 18 AIP heterozygotes, of whom 14 were CRIM-negative and four were GRIM-positive. The AIP gene carriers were diagnosed through evaluating excretion of &rninolevulinic acid (ALA) and PBG in the urine, PBGD activity and PBGD concentration in red cells and clinical and genealogical information. Blood was drawn into evacuated heparinized test tubes, and lymphocytes were isolated by a Ficoll-gradient, according to the recommendations of the manufacturer (Ficoll-Paque, Pharmacia LKB, Uppsala, Sweden). The lymphocyte pellets were frozen and stored at - 80°C. To isolate erythrocytes, heparinized blood was centrifuged at 2000 x g for 10 min. Plasma was removed and the packed red cells were stored at - 20 o C. Porphobilinogen deaminase activity in erythrocytes

The PBGD activity was measured according to the method of Sassa et al. [9,25] with determination of hemoglobin concentration of the hemolysate as well [26]. The PBGD activity was related to the hemoglobin concentration of the samples and expressed as pkat/g Hb.

19

Porphobilinogen deaminase concentration by ELISA

in erythrocytes

Concentration of PBGD in human erythrocytes was determined by an ELISA method previously developed [14]. Absorbance was read at 450 nm in an MR 700 Microplate reader, Dynatech Laboratories (Guernsey, UK). The concentration of PBGD was calculated and related to the hemoglobin concentration of the samples, as estimated by the absorbance at 410 nm, and the PBGD concentration was expressed as pg/g Hb. Protein concentration in lymphocytes Lymphocyte pellets were thawed and diluted in 600 ~1 0.1 mol/l sodium phosphate buffer, pH 7.4. The pellets were sonicated 3 X 10 s while kept on ice. The protein concentration was determined according to the method of Peterson [27] in duplicate samples of homogenized lymphocytes. Porphobilinogen deaminase activity in lymphocytes The PBGD activity in lymphocytes was measured by the same method as in erythrocytes [9,25]. Duplicate samples of sonicated lymphocytes, containing 60-120 pg of protein, were incubated for 1 h at 37” C with 1 mmol/l PBG in 0.1 mol/l sodium phosphate buffer, with constant shaking in darkness. The PBGD activity was related to the protein concentration in lymphocytes and expressed as pkat/g protein. Porphobilinogen deaminase concentration in lymphocytes by ELISA Sonicated lymphocyte pellets were diluted in 0.1 mol/l sodium phosphate buffer, pH 7.4, to a protein concentration of 0.75 mg/ml. One hundred ~1 (75 pg) of the lymphocyte dilution was added to the microtiter plate wells. The ELISA was performed according to Lannfelt et al. [14]. The PBGD concentration in lymphocytes was related to the protein concentration and expressed as pg/g protein. Porphobilinogen deaminase specific activity The PBGD specific activity in erythrocytes and lymphocytes was calculated through dividing the PBGD activity with the PBGD concentration, and expressed as r&at/g [14]. Results and discussion

The PBGD activity in erythrocytes of controls (n = 17) was 69 + 15 SD (pkat/g Hb), in the CRIM-negative AIP heterozygotes (n = 14) 36 f 7 SD (pkat/g Hb) and in the (RIM-positive (n = 4) AIP heterozygotes 33 f 5 SD (pkat/g Hb) (Table I, Fig. 1). In this study, investigating a limited number of AIP heterozygotes and

80 TABLE 1 Erythrocyte and Lymphocyte porhobihnogen deaminase (PBGD) activity, concentration activity in controls and in acute intermittent porphyria heterozygotes Controls n=17 Mean PBGD act in erythrocytes (pkat/gHb) PBGD cone in erythrocytes (~g/g~) PBGD spec act in erythrocytes (r&at/g PBGD) PBGD act in lymphocytes (pkat/gprot~n) PBGD cone in lymphocytes (pg/gprotein) PBGD spec act in lymphocytes (r&at/g PBGD)

140

AIP GRIM-negative n=14 SD

Range

Mean

SD

and specific

AIP GRIM-positive n=4 Range

Mean

SD

Range

69

1.5

47-104

36

7

24-45

33

S

186

52

127-332

97

20

57-127

354

47

309-416

373

40

298-471

374

64

265-473

94

22

65-116

26

7

18-45

17

6

11-31

13

3

10-17

13

4

8-19

10

3

6-19

16

2

14-19

2086

349

1727

395

824

288

1316-2727

1308-2700

27-38

526-l 214

-

120 100.

+

80 .

6o 40

‘:’ .. *::

L

i .

20

Controls n=17

AIP, GRIM-cove n-l4 Erythrocytas

AtP, GRIM-positive n=4

t:: .:g

Controls n=17

.

%$I&? AIP, GRIM-naqatlvs n=14

AIP, GRIM-sieve n=4

Lymphocytes

Pig. 1. Erythrocyte (+) and lymphocyte (A) PBGD activity in controls, GRIM-negative and CRIM-positive AIP heteroxygotes, expressed as pkat/g Hb and pkat/g protein, respectively.

81

controls, there was only a small overlap in enzyme activity between controls and AIP heterozygotes. The PBGD activity in lymphocytes was 26 f 7 SD (pkat/g protein) in controls, in the CRIM-negative group 17 f 6 SD (pkat/g protein) and in the CRIM-positive group 13 f 3 SD (pkat/g protein) (Table I, Fig. 1). The PBGD activity in lymphocytes reflected the situation in red cells, although the overlap of enzyme activity between controls and AIP heterozygotes was more pronounced in lymphocytes. A

a.

600 I 500 400

-

300

-

200

-

100

-

l

.

;;; :*:

Controls n=l7

l.. .3? .

AIP, GRIM-negative n=14

AIP, CRIM-positive n-4

b.

. . .

* . . .

.?a-. ..L . .

Controls n.17

AIP, GRIM-negative n=l4

AIP, GRIM-positive n=4

Fig. 2. a. Erythrocyte PBGD concentration (+) as measured by ELISA, expressed as pg/g Hb, in controls, CRIM-negative and CRIM-positive AIP heterozygotes. b. Lymphocyte PBGD concentration (A) as measured by ELISA in controls, GRIM-negative and CRIM-positive AIP heterozygotes, expressed as pg/g protein.

82

straight line, passing origo, was achieved when measuring PBGD activity in lymphocyte suspensions, at different concentrations of protein (not shown). The controls had a PBGD concentration measured by ELISA in erythrocytes of 186 &-52 SD (pg/g Hb), the CRIM-negative a decreased concentration of 97 f 20 SD @g/g Hb), while the CRIM-positive group had a highly significant increase of the PBGD concentration, 354 f 47 SD @g/g Hb) (Table I, Fig. 2a). Concentration of PBGD measured by ELISA in lymphocyte suspension, at different protein dilutions, gave rise to a hyperbolar curve (not shown). Therefore the ELISA was standardized, and 75 pg of protein was added into the assay to avoid biases. The PBGD concentration in lymphocytes measured by the ELISA was in controls 13 & 4 SD (/.tg/g protein), in the CRIM-negative group 10 * 3 SD (pg/g protein) and in the CRIM-positive group 16 + 2 SD (pg/g protein) (Table I, Fig. 2b). This was a result similar to that found in erythrocytes, although the overlap was more pronounced in lymphocytes. Approximately 40% of the PBGD activity, and 7% of the PBGD concentration was found in lymphocytes compared to red cells, in controls. It could be argued that the antibody used was raised against PBGD purified from

.

. _A

A

L

.

. .

:

.

.

.

.* A

: :.:.>:. ;. *

‘.’ .: .:... . .

::

Contro’t n.17

AIP. GRIM-negative n=14 Erythrocytes

AIP, GRIM-positive n-4

Controls n-17

AIP. GRIM-negative n=14

- _ . . AIP, Call-l-pWU”e n=4

Lymphocyte3

Fig. 3. Erythrocyte (+) and lymphocyte (A) PBGD specific activity in controls, GRIM-negative GRIM-positive AIP heterozygotes, expresssed as nkat/g PBGD protein.

and

83

erythrocytes, and thus did not fully recognize the non-erythroid PBGD in lymphocytes. However, the antiserum used was polyclonal [14]. As the difference between the two forms is only 17 amino acids at the N-terminal end of the protein, it could be presumed that the antiserum will cross-react with both PBGD forms. The PBGD specific activity was approximately seven times higher in lymphocytes than in erythrocytes, of controls (Fig. 3) which leads to the conclusion that the ubiquitous PBGD is more active than the erythroid PBGD form. According to studies on protein and DNA levels two forms of the PBGD enzyme exists [21,23]. One form (44 kDa) is present in all tissues, and a shorter version (42 kDa) is present only in erythroid tissue. The finding of a lower PBGD specific activity in erythrocytes than in lymphocytes implies that the erythroid form of the enzyme has a lower catalytic activity. Thus it can be hypothesized that the 17-amino acid residue found in the non-erythropoietic form is essential for enzyme activity. The present investigation has demonstrated that the PBGD activity and PBGD concentration in lymphocytes by large reflects the situation in red blood cells, both for CRIM-negative and CRIM-positive AIP heterozygotes. However, the overlap in PBGD enzyme activity and concentration between AIP gene carriers and controls was more pronounced in lymphocytes. Considering the overlap and that the lymphocyte assays are more time consuming, it is not suitable to measure the PBGD enzyme in lymphocytes with the aim to identify latent AIP heterozygotes. Acknowledgements

This investigation was supported by grants from the Swedish Medical Research Council (project 13X-7483), the Professor Gadelius Memorial Foundation, the Siiderstrbm-Konigska Foundation, and the Swedish Society of Medicine. The authors wish to thank associate professor Jan Satif, the Karolinska Institute, Department of Psychiatry, St. G&an’s Hospital, for cooperation on various aspects of this study. References 1 Battersby AR, Fookes CJR, Matcham GWJ, McDonald EJ. Order to assembly of the four pyrrole rings during biosynthesis of natural porphyrins. Chem Sot Chem Cornmun 1979;12:539-541. 2 Waldenstrom J. Studien iiber Potphyrie. Acta Med Stand 1937;Suppl 82. 3 Wetterberg L. Report on an international survey of safe and unsafe drugs in acute intermittent porphyria. In: Doss M, Nowrocki P, eds. Porphyrins in human disease. Report of the discussions. Marburg an der Lahn, 1976;191-202. 4 Kappas A, Sassa S, Anderson KE. The porphyrias. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS, eds. The metabolic basis of inherited disease. New York: McGraw-Hill Inc., 1983;1301-1384. 5 Strand LJ, Felsher BF, Redeker AG, Marver HS. Heme biosynthesis in intermittent acute porphyria: Decreased hepatic conversion of porphobihnogen to porphyrins and increased d-amino levulinic acid synthetase activity. Proc Nat1 Acad Sci USA 1970;67:1315-1320. 6 Miyagi K, Cardinal R, Bossenmaier 1, Watson CJ. The serum porphobihnogen and hepatic porphobihnogen deaminase in normal and porphyric individuals. J Lab Clin Med 1971;78:683-695.

84 7 Bortkowsky HL, Tshudy DP, Weinbach EC, Ebert PS, Doherty JM. Porphyrin synthesis and mitochondrial respiration in acute intermittent porphyria. Studies using cultured human fibroblasts. J Lab Clin Med 1975;85:93-102. 8 Sassa S, Solish G, Levere RD, Kappas A. Expression of the gene defect of acute intermittent porphyria in cultured human skin fibroblasts and amniotic cells; prenatal diagnosis of the porphyric trait. J Exp Med 1975;142:722-731. 9 Sassa S, Zalar GL, Kappas A. Induction of uroporphyrinogen-l-synthase and expression of the gene defect of acute intermittent porphyria in mitogen-stimulated human lymphocytes. J Clin Invest 1978;61:499-508. 10 Formgren B, Wetterberg L. Uroporphyrinogen-I-synthetase activity in erythrocytes as a diagnostic index in acute intermittent porphyria. Laartidningen 1978;75:1921-1924. 11 Lamon JM, Frykholm BC, Tschudy DP. Family evaluation in acute intermittent porphyria using red cell uroporphyrinogen-I-synthase. J Med Genet 1979;16:134-139. 12 Thunell S. Diagnosis of disturbances of porphyrin metabolism. Liikartidningen 1986;83:32423245,3248-3251. 13 Pierach CA, Weimer MK, Cardinal RA, Bossenmaier IC, Bloomer JR. Red blood cell porphobilinogen deaminase in the evaluation of acute intermittent porphyria. JAMA 1987;257:60-61. 14 Lannfelt L, Wetterberg L, Lilius L, Thunell S, Gellerfors P. ELISA for measuring porphobilinogen deaminase in human erythrocytes. Clin Chim Acta 1989; 83:227-238. 15 Lamrfelt L. Immunological determination of porphobilinogen deaminase as a diagnostic measure in acute intermittent porphyria. J Clin Chem Clin Biochem 1990;28:273-278. 16 Anderson PM, Reddy RM, Anderson KE, Desnick RJ. Characterization of the porphobilinogen deaaminase deficiency in acute intermittent porphyria. J Clin Invest 1981;68:1-12. 17 Desnick RJ, Ostasiewicz LT, Tishler PA, Mustajoki P. Acute intermittent porphyria: characterization of a novel mutation in the structural gene for porphobilinogen deaminase. J Clin Invest 1985:76;865874. 18 Mustajoki P, Desnick RJ. Genetic heterogeneity in acute intermittent porphyria: characterization and frequency of porphobilinogen deaminase mutation in Finland. Br Med J 1985;291:505-509. 19 Wilson JHP, de Rooy FWM, te Wilde K. Acute intermittent porphyria in The Netherlands. Heterogeneity of the enzyme porphobilinogen deaminase. Neth J Med 1986;29:393-399. 20 Latmfelt L, Wetterberg L, Gellerfors P, Lilius L, Floderus Y, Thunell S. Mutations in acute intermittent porphyria detected by ELISA measurement of porphobilinogen deaminase. J Clin Chem Clin Biochem 1989;27:857-862. 21 Grandchamp B, de Vemeuil H, Beaumont C, Chretien S, Walter 0, Nordmann Y. Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene. Eur J Biochem 1987;162:105-110. 22 Raich N, Romeo PH, Dubart A, Beaupain D, Cohen-Solal M, Goossens M. Molecular cloning and complete primary sequence of human erythrocyte porphobilinogen deaminase. Nucleic Acid Res 1986;14:5955-5968. 23 Chretien S, Dubart A, Beaupain D, Raich N, Grandchamp B, Rosa J, Goossens M, Romeo PH. Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression. Proc Nat1 Acad Sci USA 1988;85:6-10. 24 Mustajoki P. Normal erythrocyte uroporphyrinogen I synthase in a kindred with acute intermittent porphyria. Ann Intern Med 1981;95:162-166. 25 Sassa S, Granick S, Bickers DR, Bradlow HL, Kappas A. A microassay for uroporphyrinogen-l-synthase, one of three abnormal enzyme activities in acute intermittent porphyria, and its application to the study of the genetics of this disease. Proc Nat1 Acad Sci USA 1974;71:732-736. 26 Johansson L, Thunell S, Wetterberg L. A filter paper dry blood spot procedure for acute intermittent porphyria population screening by use of whole blood uroporphyrinogen-I-synthase assay. Clin Chim Acta 1984;137:317-331. 27 Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 1977;83:346-356.