93
Clinica Chimica ACM, 193 (1990) 93-102 Elsevier
CCA 04740
Erratum
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 Regaud and Loboratoire de Biochimie, FacultP des Sciences Pharmaceutiques, Toulouse (France) (Received
19 August
Key worakt Alkaline
1989; revision received 7 March
phosphatase; Intestinal Physicochemical
1990; accepted
isoform; Preparative characteristic
16 March
isoelectric
1990)
focusing;
was noted in a healthy 47-year-old woAn unexplained hyperphosphatasemia 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 characterised as being of intestinal origin: their biochemical (neuraminidase, phenylalanine) and thermodenaturation properties are similar to those of intestinal tissue extract. The p1 and the pH optima of these isoforms agree with an intestinal origin. Our results suggest the existence of different allelozymes of intestinal alkaline phosphatase. man.
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
* Please note: due to the omission of Tables I and II and Figures 3 and 4, the completed version of this article has been run again. The reader is advised to void the original which appeared in Volume 189, number 2, pages 143-152. Correspondence to: G. Soula, Departement de Biologie Pont Saint Pierre, 31052, Toulouse Cedex, France.
Clinique,
0009-8981/90/$03.50
B.V. (Biomedical
0 1990 Elsevier Science Publishers
Centre
Claudius
Division)
Regaud,
20 Rue du
94
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 recognized biochemical or clinical disorder. A biochemical study of the properties of these isoforms has been performed through comparison with purified bone, liver and intestinal isoforms. 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 o 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 donors 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 or 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, MgCl 1 mmol/l pH 7.7, at 4°C [8].
95
Tissue extract for intestinal isoenzyme control. A sample of macroscopically normal human mucosa obtained from autopsy materials [9] was washed in 140 mmol/l NaCl solutions, homogenized in 20 ml distilled water and then sonicated. The homogenate was centrifuged at 105000 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 200000 X g (30 min at 4“ C). Triton X-100 was eliminated by dialysis for 24 h at 4” 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” C with a chromogenic substrate: 5-bromo-3-indolylphosphate p-toluidine salt in eth~ola~ne 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 0 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 ’ 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, pf and pH optimum dete~nations and physi~he~~~ ~haracte~ation. Isoenzyme pH optimum and pI determinations In order to determine the optimum pH of each isoenzyme, fractions obtained by IEF were incubated with 10 mmol/l nitro-4-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 of sample were -desialylated by incubation with 1 vol. neur~~dase (10 mmol/l) for 10 min at 25O C. The result of the neura~~dase pre~eatment was evaluated by el~trophoresis in agarose lectin gel WI.
96
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 1121. Thermostability 1131. Alkaline phosphatase preparations were incubated at 56 * C for various time intervals. After incubation, samples were immediately put in melting ice in order to stop 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 electrophoresis in 6% (w/v) polyacrylamide gels.
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) (f%), _ and three ‘supposed intestinal’ forms called: If (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.
Fig. 1. Separation of plasma with wheat-germ lectin (B). hepatic metastases; A3-B3, Bone and liver isoforms; 2, (13); 5, normal
samples by gel agarose electrophoresis without wheat-germ lectin (A, C) and Al-Bl-C2, Serum of case report; A2-B2, serum of subject suffering from normal serum; A4-B4, sermn of children; Cl, extract of intestinal tissue. 1, intestinal 1 isoform (11); 3, intestinal 2 isoform (12); 4, intestinal 3 isoform liver isoform (Hl); 6, biliary isoform (H2); 7, bone isoform (B).
TABLE
I
The values of the pHi of different
alkaline
phosphatase
Preoarations
Isoenzymes
Serum of the case report
HI I, I2
isoenzymes
determined pHi
3.6 4.35 4.75-5 5.5-5.6 3.95-4
13
bone Isoenzymes from intestinal tissue extract
4.25-4.35 4.55-4.6 5.5
4 I2 I3
Biliary isoenzyme partially purified
Clinica Chimica ACM, 193 (1990) 93-102 Elsevier
CCA 04740
Erratum
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 Regaud and Loboratoire de Biochimie, FacultP des Sciences Pharmaceutiques, Toulouse (France) (Received
19 August
Key worakt Alkaline
1989; revision received 7 March
phosphatase; Intestinal Physicochemical
1990; accepted
isoform; Preparative characteristic
16 March
isoelectric
1990)
focusing;
was noted in a healthy 47-year-old woAn unexplained hyperphosphatasemia 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 characterised as being of intestinal origin: their biochemical (neuraminidase, phenylalanine) and thermodenaturation properties are similar to those of intestinal tissue extract. The p1 and the pH optima of these isoforms agree with an intestinal origin. Our results suggest the existence of different allelozymes of intestinal alkaline phosphatase. man.
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
* Please note: due to the omission of Tables I and II and Figures 3 and 4, the completed version of this article has been run again. The reader is advised to void the original which appeared in Volume 189, number 2, pages 143-152. Correspondence to: G. Soula, Departement de Biologie Pont Saint Pierre, 31052, Toulouse Cedex, France.
Clinique,
0009-8981/90/$03.50
B.V. (Biomedical
0 1990 Elsevier Science Publishers
Centre
Claudius
Division)
Regaud,
20 Rue du
94
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 recognized biochemical or clinical disorder. A biochemical study of the properties of these isoforms has been performed through comparison with purified bone, liver and intestinal isoforms. 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 o 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 donors 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 or 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, MgCl 1 mmol/l pH 7.7, at 4°C [8].
95
Tissue extract for intestinal isoenzyme control. A sample of macroscopically normal human mucosa obtained from autopsy materials [9] was washed in 140 mmol/l NaCl solutions, homogenized in 20 ml distilled water and then sonicated. The homogenate was centrifuged at 105000 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 200000 X g (30 min at 4“ C). Triton X-100 was eliminated by dialysis for 24 h at 4” 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” C with a chromogenic substrate: 5-bromo-3-indolylphosphate p-toluidine salt in eth~ola~ne 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 0 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 ’ 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, pf and pH optimum dete~nations and physi~he~~~ ~haracte~ation. Isoenzyme pH optimum and pI determinations In order to determine the optimum pH of each isoenzyme, fractions obtained by IEF were incubated with 10 mmol/l nitro-4-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 of sample were -desialylated by incubation with 1 vol. neur~~dase (10 mmol/l) for 10 min at 25O C. The result of the neura~~dase pre~eatment was evaluated by el~trophoresis in agarose lectin gel WI.
96
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 1121. Thermostability 1131. Alkaline phosphatase preparations were incubated at 56 * C for various time intervals. After incubation, samples were immediately put in melting ice in order to stop 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 electrophoresis in 6% (w/v) polyacrylamide gels.
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) (f%), _ and three ‘supposed intestinal’ forms called: If (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.
Fig. 1. Separation of plasma with wheat-germ lectin (B). hepatic metastases; A3-B3, Bone and liver isoforms; 2, (13); 5, normal
samples by gel agarose electrophoresis without wheat-germ lectin (A, C) and Al-Bl-C2, Serum of case report; A2-B2, serum of subject suffering from normal serum; A4-B4, sermn of children; Cl, extract of intestinal tissue. 1, intestinal 1 isoform (11); 3, intestinal 2 isoform (12); 4, intestinal 3 isoform liver isoform (Hl); 6, biliary isoform (H2); 7, bone isoform (B).
TABLE
I
The values of the pHi of different
alkaline
phosphatase
Preoarations
Isoenzymes
Serum of the case report
HI I, I2
isoenzymes
determined pHi
3.6 4.35 4.75-5 5.5-5.6 3.95-4
13
bone Isoenzymes from intestinal tissue extract
4.25-4.35 4.55-4.6 5.5
4 I2 I3
Biliary isoenzyme partially purified
