Hyperphosphatasemia related to three intestinal alkaline phosphatase isoforms: Biochemical study

145

Ciinicu Chimicu Acta, 189 (1990) 145-152

Elsevier

CCA 04740

Hyperphosphatasemia related to three intestinal alkaline phosphatase isoforms: biochemical study L. Bentouati, M. Samadi Baboli, H. Hachem, M. Hamza, P. Canal

and G. Soula DPpartement de Biologie Clinique, Centre Claudius Regaucl, and Laboratoire de Biochimie, Fact&P des Sciences Pharmaceutiques, Toulouse (France) (Received

Key work

19 August 1989; revision received 7 March 1990; accepted 16 March 1990)

Alkaline phosphatase;

Intestinal isoform; Preparative isoelectric characteristic

focusing; Physicochemical

An unexplained hyperphosphatasemia was noted in a healthy 47-year-old woman. The electrophoretic pattern on agarose gel with and without wheat germ lectin of the serum of this patient showed the presence of three isoforms which have been character&d as being of intestinal origin: their biochemical (neuraminidase, phenylalanine) and thermodenaturation properties are similar to those of intestinal tissue extract. The pI and the pH optima of these isoforms agree with an intestinal origin. Our results suggest the existence of different allelozymes of intestinal alkaline phosphatase.

Introduction

Human serum alkaline phosphatase activity (orthophosphoric-monoester phospho-hydrolase, alkaline optimum EC 3.1.3.1) (ALP) is commonly measured in clinical chemistry laboratories. The enzyme is heterogeneous with more than five isoforms depending on the tissue source [l-3]. The liver, bone and intestinal isoforms, are most commonly found in serum in both healthy and diseased persons [4,5]. We report a case with raised serum alkaline phosphatase activity related to intestinal isoenzyme activity without any other recognised biochemical or clinical disorder. A biochemical study

Correspondence to: G. Soula, D&arteme.nt de Biologie Pont Saint Pierre, 31052, Toulouse Cedex, France.

0009-8981/90/$03.50

0 1990 Elsevier Science Publishers

Clinique,

Centre Claudius

B.V. (Biomedical

Division)

Regaud, 20 Rue du

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of the properties of these isoforms has been performed purified bone, liver and intestinal isoforms.

through comparison

with

Materials and methods Apparatus

Immunoelectrophoresis (IEF) was performed on an LKB 2117 multiphor and an LKB 2203 generator (LKB Instrument, Les Ulis France). Agarose electrophoresis was carried out on Sebia material (France), and electrophoretic profiles were performed on a ‘preference’ Ecran densitometer (Sebia Laboratories, Issy les Moulineaux, France). Products

The determination of ALP levels was performed according to Hausamen’s method at 30 ’ C [6] using a commercial kit (Biomerieux, France). Agarose gel, agar wheat germ lectin gel, staining solutions were obtained from SEBIA. Ultrodex, SDS, polyacrylamide, Sepharose 6B, ampholines were purchased from Pharmacia produktor (Pharmacia France, Saint-Quentin en Yvelines, France). Neuraminidase, L-phenylalanine and diethanolamine were from Sigma, St Louis, MO, USA. Other reagents of analytical grade were obtained from Prolabo, Paris (France). Samples Case report (C.R.). During a routine medical check up, hyperphosphatasemia (570 mu/ml; normal range: 130-260 mU/ml) was noted in a 47-year-old healthy woman (blood group A; Rh - ; weight: 50 kg). All the other biochemical and hematological variables were normal, as were the immunoglobulin levels. The clinical status of this patient was normal as were her abdominal echography and tomodensitometry or colonoscopy. Renal and hepatic functions were normal and no gastrointestinal and gynecological disorders were found in her medical history.

To compare the biochemical properties of the ALP Samples used as references. isoforms of this case report, sera were obtained from fasting subjects, and two kinds of volunteer or donor were studied: - Serum from 25 children, 2 to 5 years old, were used as bone isoenzyme control. These samples were obtained during a routine medical check-up. - Serum from patients with microscopically proven metastatic hepatic involvement of digestive adenocarcinoma. These samples were characterized by the presence of an abnormal ALP isoform called, according to Viot et al. [7], biliary isoenzyme, the high molecular mass isoenzyme or ALP1 of H2. For IEF, H2 was partially purified by chromatography on a Sepharose 6B (100 X 2.5 cm) column eluted with Tris-HCl 100 mmol/l, NaCl 50 mmol/l, MgCI 1 mmol/l pH 7.7, at 4°C [8]. Tissue extract

for intestinal

control. A sample of macroscopically from autopsy materials [9] was washed in 140

isoenzyme

normal human mucosa obtained

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mmol/l NaCl solutions, homogenized in 20 ml distilled water and then sonicated. The homogenate was centrifuged at 105 000 x g (4 h at 4” C) the supematant (Sl) was discarded and the pellet homogenized again as mentioned above in the presence of 0.5% Triton X-100. The supematant, S2, was obtained by centrifugation at 200 000 X g (30 min at 4” C). Triton X-100 was eliminated by dialysis for 24 h at 4O C, against 140 mmol/l NaCl containing 1 mmol/l sodium azide and 0.3 mmol/l PMSF (phenylmethylsulfonyl fluoride). The enzyme activity of S2 was adjusted to 800 U/l. Methods

Electrophoretic technics Electrophoresis. Samples containing alkaline phosphatase were subjected to electrophoresis in 0.9% (w/v) agarose gel or agarose gel containing 0.05 g/l wheat germ lectin at 100 V for 45 min at room temperature [l]. 10 ~1 of the sample were used. After separation, isoenzyme bands were visualized by incubating the gel for 50 min at 37 o C with a chromogenic substrate: 5-bromo-3-indolylphosphate p-toluidine salt in ethanolamine buffer (1 mmol/l, pH 9.8). Then, the gel was washed for 4 h in distilled water in the dark, and dried out at a temperature of less than 60 o C and used for profile determinations. Preparative isoelectric focusing (I. E.F.) [2,10]. A 4% Ultrodex slurry gel 3 mmol/l thickness was used containing 5% ampholines with a pH range of 3.5 to 9. The gel support was treated with 0.5% Triton X-100, flowed and dried afterwards by using a fun max air at 60 o C. Three ml of sample were applied in the middle of the gel, and the focusing was carried out in a refrigerated LKB 2117 model at 1400 V for 14 to 16 h at 4°C with strips containing 1 mmol/l H,PO, at the anode and 1 mmol/l NaOH at the cathode. At the end of focusing, the gel was fractionated, then eluated by 2 ml distilled water for enzyme assay, isoenzyme electrophoresis, pl and pH optimum determinations and physicochemical characterization. Isoenzyme pH optimum and pI determinhtions In order to determine the optimum pH of each isoenzyme, fractions obtained by IEF were incubated with 10 mmol/l nitro4-phenyl phosphate in solution in a diethanolamine buffer (1 mol/l) with a pH range of 7.5-12 performed with a HCl solution (1 mol/l). Physico-chemical characteristics Sialic acid residues. Five volumes sample were desialylated by incubation with 1 vol. neuraminidase (10 mmol/l) for 10 min at 25 o C. The result of the neuraminidase pretreatment was evaluated by electrophoresis in agarose lectin gel [ll].

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Chemical inhibition. For the chemical inhibition study, samples were incubated for 5 min in ethanolamine buffer (1 mol/l, pH 9.8) containing L-phenylalanine at various concentrations (range 5-30 mmol/l). Residual activities were expressed as a percentage of initial rate [12]. Thermostability [13]. Alkaline phosphatase preparations were incubated at 56 o C for various time intervals. After incubation, samples were immediately put in melting ice in order to the thermodenaturation. Then, the residual activity was assayed. Determination of molecular mass The apparent molecular mass values of the isoforms obtained after IEF were determined by sodium dodecyl sulfate-polyacrylamide polyacrylamide gels.

electrophoresis

in 6% (w/v)

Electrophoresis The electrophoresis

performed on the serum of C.R. showed four bands on an agarose gel and five bands on an agarose lectin gel (Fig. 1): - a normal bone isoenzyme retarded in buffer containing wheat-germ lectin (7%), - a normal liver isoenzyme (Hl) (I%), - and three ‘supposed intestinal’ forms called: 11 (67%), 12 (20%) and 13 (5%). This profile differed from that obtained with children serum and with patients suffering from metastatic liver involvement characterized by the presence of H2. When comparing this profile with that obtained from intestinal tissue (Fig. lC), no difference in the migrating rate was noticed.

Fig. 1. Separation of plasma samples by gel agarose electrophoresis without wheat-germ lectin (A, C) and with wheat-germ lectin (B). Al-Bl-C2, !krum of case report; A-B2, serum of subject suffering from hepatic metastases; A-B3, normal sexum; A-B4, serum of children; Cl, extract of intestinal tissue. 1, Bone and liver isoforms; 2, intestinal 1 isoform (11); 3, intestinal 2 isoform (12); 4, intestinal 3 isoform (13); 5, normal liver isoform (Hl); 6, biiary isoform (HZ); 7, bone isoform (B).

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Fig. 2. EWtrophoretic profile of samples before and after neuraminidase pretreatment. 1-2, serum of case report; 3-4, sernm of subject suffering from hepatic metastases; 5-6, partially purified biliary &enzyme. 1, intestinal 1 isoform (11); 2, intestinal 2 isoform (i2); 3, intestinal 3 isoform (13); 4, bone isofonn (B); 5, normal liver isoform (Hl); 6, biliary isoform (H2).

IEF assay: p1 and pH optimum IEF was performed to determine the pI of the different isoforms of CR. The results are summarized in Table I which compares the pl of the intestinal preparation, H2 hepatic fraction and children serum. No significant difference exists between the pI of the same isoenzyme, whatever the tissue source. The pl of serum C ALP isoforms 11, 12 and 13 have the same pl values as the intestinal tissue isoforms: 4.30 for. 11, 4.70 for 12 and 5.50 for 13. Table II shows the pII optimum values of the isoforms obtained from IEF. 11,12,13 have the same pH optimum: pH 9.8. Physicochemicai characteristics Effect of neuraminidase pretreatment (Fig. 2). After pretreatment of serum of CR. by neuraminidase, on agarose electrophoresis, no modification in the migrating rate of the 11,12, 13 isoforms, with regard to the intestinal tissue extract, was noted. When analyzing the serum of the children or of a patient with hepatic metastases, we noticed that the H2 and the Hl or the bone isoform migrating rate were strongly modified. Phenylalanine inhibition. To define isoforms of the serum of CR, with regard to the chemical inhibition, we used L-phenylalanine at concentrations ranging from 5 to 30 mmol/l. The results are summarized in Fig. 3 (Fig. 3). The isoforms from intestine tissue and the serum of CR. are strongly ~bit~ (75%) with a high concentration of phenylalanine (30 mmol/l). Bone and liver isoforms were less inhibited in the same conditions. Thermostability. Fig. 4 shows the thermostability of the different isoforms at 56°C as function of time. The serum of CR was more inhibited than intestinal tissue extract.

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Molecular mass. The molecular masses of 11, 12 and 13 were determined using an SDS-polyacrylamide gel previously calibrated with known molecular mass proteins. A similar apparent value of 70 kDa was observed for the three isoforms. Results concerning the other fractions corroborate with those of literature.

Discussion

This study concerns the characterisation of the ALP isoforms inducing an hyperphosphatasemia in a healthy 47-year-old woman without any clinical recognized disorder. The electrophoresis carried out in agarose gel with and without a wheat germ lectin showed that this hyperphosphatasemia is due to the presence of three isoforms. Their electrophoretic properties are identical to those of an intestinal tissue extract. As Rosalki and Koo [l] have shown, the use of wheat germ lectin in agarose gel allows a better resolution between isoforms, particularly the differentiation of liver and bone isoforms. The neuraminidase pretreatment of serum in the case report has confirmed the intestinal origin of 11, 12, 13 isoforms: the electrophoretic migration rates were not modified [15,16]. These isoforms were inhibited by r_-phenylalanine [17]. However, the results obtained from thermodenaturation of the case report isoforms and of intestinal extract highly differ: as Moos and Shakespeare suggest [18], some factors may influence the behaviour of tissuelar isoforms with regard to heat sensitivity (pH, buffer) and consequently modify their characteristics. The results obtained for the pl after IEF of the different isoforms are in agreement with those previously published [2,10,19]. The pl of 11, 12 and 13 of the case report and from intestinal tissue extract slightly varied from 4.35 to 5.6. Our results showed no difference in the pH optimum and molecular mass between the three intestinal isoforms for serum of CR. and from intestinal tissue extract (pH 9.8 is in agreement with Fishman [20], molecular mass is 70 kDa). The obtained molecular mass differs from that obtained by Stinson and Seargeant [21] (92.4 kDa for intestinal isoform and 74 kDa for the placental isoform). All the results confirm the intestinal origin of the hyperphosphatasemia of the case report. The two children of this woman have been tested: they have normal serum ALP activity and the electrophoretic profile did not reveal any intestinal isoforms. This fact should indicate that the unusual properties of the intestinal isoforms in the patient’s serum are derived from an allelic variation at the genetic locus. Other authors have shown an increase in intestinal isoenzyme linked with immunoglobulin G of the kappa type [22] which appeared with a very low frequency in healthy blood donors (0.003%) [23]. In our case report, no increase in immunoglobulin levels has been noticed, and the obtained molecular mass data for intestinal isoforms confirmed a lack of linkage with immunoglobulins. It has been demonstrated that a fatty meal could be related to a low increase in intestinal isoforms [24]. However, all the subjects sampled had been fasted overnight. On the other hand, intestinal AP is more often found in the serum of persons

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with blood group 0 and B than in persons with blood group A [25]. Finally, Fishman et al, [26] have demonstrated that elevated levels of ALP in serum might be from intestinal origin in patients with cancer (lung, breast or other) or liver cirrhosis. It has been clearly established that ALP exhibits tissue-specific characteristics which are derived from the existence of distinct structural genes for placental and intestinal ALP, but which in the other tissues (bone, liver and kidney) probably result from modification processes operating differentially on a single gene product common to them all [27-291. The placental and intestinal isoforms are expressed predominantly in their eponymous tissues. The placental ALP exists as numerous allelozymes [30]. Our findings reporting an hyperphosphatasemia due to the presence of intestinal isoforms suggest the existence of different allelozymes of intestinal ALP. Acknowledgements

The technical assistance of MT. Despeyroux thank Mrs J. Leon for secretarial aid.

is greatly appreciated.

The authors

References 1 Sidney B, Rosa&, Yng Foo A. Two new methods for separating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma. CIin Chem 1984;30:1182-1186. 2 Griffiths J, Biack J. Separation and identification of a&dine phosphatase isoenzymes and isoforms in serum of heahhy persons by isoeiecttic focusing. CIin Chem 1987;33:2172-2177. 3 Sir&a PK, Bianchi-Bosisio A, Meyer-SabelIek W, Righetti PG. Resolution of alkaline phosphatase isoenzymes in serum by isoelectric focusing in immobohzed pH gradients. CIin Chem 1986;32:12641268. 4 Kuwana T, Sugita 0, Yakata M. Reference limits of bone and liver alkaline phosphatase isoenzymes in the serum of healthy subjects according to age and sex as determined by wheat germ-Pectin affinity eleetrophoresis. Clin Chim Acta 1988;173:273-280. 5 Onica ,D, Sundblad L, WaIdenIind L, Shanweli A. Characterization of serum alkaline phosphatase isoenzymes by affinity electrophoresis in agarose gel containing lectin combined with agar gel electrophoresis. Stand J Clin Lab Invest 1987;47:239-245. 6 Hausament TU, Helgerrick W, Gross W. Optimal conditions for the determination of serum alkaline phosphatase by a new kinetic method. CIin Chim Acta 1%7;15:241-244. 7 Viot M, Thyss A, Schneider M, Viot G, Ramaioli A, Cambon P, Lalanne C. cd Isoenzyme of alkaline phosphatase. Cancer 1983;52:140-145. 8 Canal P, Yao M, Soula G. Associations e~~es-~~prot~~es dam le &rum de sujets atteints de mttastases hepatiques. Acta Pharm Biol CIin 1984;3:111-114. 9 Fishman WH, Green S, IngIis NI. Organ-specific behavior exhibited by rat intestine and liver alkaline phosphatase. Biochim Biophys Acta 1%2;62:363-375. 10 Blum-SkoInik J, Pace F, Munst G, Mmder W. Isoelectric focusing of serum alkaline phosphatase isoenzymes. Chn Chim Acta 1983;129:157-164. 11 Moss DW, Edwards RK improved electrophoretic resolution of bone and liver a&a&e phosphatases resulting from partial digestion with neuraminidase. CIin Chim Acts 1984;143:177-182. 12 Fishman WH, Shinowara GY. I-Phenylalanine: an organ specific, stereo-specific inhibitor of human intestinal aIkaIine phosphatase. Nature 1963;198:685-686. 13 Wbitby LG, Moss DW. Analysis of heat inactivation on curves of alkaline phosphatase isoenzymes in serum. Chn Chim Acta 1975;59:361.

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14 Ghosh NK. Purification and molecular properties of placental and intestinal alkaline phosphatases. AM NY Acad Sci 1969;166:604-608. 15 Robinson JC, Pierce. JE. Differential action of neuraminidase on human serum aIkaline phosphatase. Nature 1964;204:472-475. 16 Fishman WH, Inglis NR, Ghosh NK. Distinctions between intestinal and placental isoenzymes of alkaline phosphatase. Clin Chim Acta 1968;19:71-77. 17 VanbeIIe H. Alkaline phosphatase kinetics and inhibition by levamisole of purified &enzymes from humans. Clin Chem 1976;22:972-975. 18 Moss DW, Shakespeare SH. Observations on the heat stability of alkaline phosphatase isoenzymes in serum. Clin Chim Acta 1975;40:35-40. 19 Angelis D, Inglis N, Fishman WH. Isoelectric focusing of alkaline phosphatase isoenzymes in polyacrylamide gels. Use of Triton X-100 and improved staining technic. Ann J Clin Path 1976;66:929-933. 20 Fishman WH. Perspectives on alkaline phosphatase isoenzymes. Am J Med 1974;56:617-650. 21 Stinson RA, Seargeant LE. Comparative studies of pure aIkaline phosphatase from five human tissues. CIin Chim Acta 1980;110:261-272. 22 Kohno H, Sudo K, Kanno T. Intestinal alkaline phosphatase linked to immunoglobulin G of the kappa type. CIin Chim Acta 1983;135:41-48. 23 Hattori Y, Yamamoto K, Urabe C, et al. Frequency of alkaline phosphatase-immunoglobuIin complex among diseased and healthy populations. Clin Chim Acta 1985;147:155-158. 24 Langman MJS, Levthold E. Influence of diet on the “intestinal” component of serum alkaline phosphatase in people of different ABO blood groups ans secretory status. Nature 1966;212:41-48. 25 Bayer PM, Hotschek H, Knoth E. Intestinal alkaline phosphatase and the ABO blood group system a new aspect. CIin Chim Acta 1980;108:81-87. 26 Fishman WH, Inglis NI, Krant MJ. Serum alkaline phosphatase of intestinal origin in patients with cancer and with cirrhosis of the liver. Clin Chim Acta 1%5;12:298-303. 27 Moss DW. Alkaline phosphatase isoenzymes Clin Chem 1982;28:2007-2016. 28 McKenna MJ, Hamilton TA, Sussman HH. Comparison of alkaline phosphatase isoenzymes. Structural evidence for three protein classes. Biochem J 1979;181:67-73. 29 Seargeant LE, Stinson RA. Evidence that three structural genes code for human alkahne phosphatase. Nature 1979;281:152-154. 30 Donald LJ, Robson EB. Rare variants of placental alkaline phosphatase. AM Hum Genet 1975;31:303-313.