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Comparison of alkaline phosphatase isoenzymes determined by an inhibition method and by electrophoresis
111
Clinica CIzimica Acta, 85 (1978) 111-114 0 Elsevier/North-Holland Biomedical Press
CCA 9166
COMPARISON OF ALKALINE PHOSPHATASE ISOENZYMES DETERMINED BY AN INHIBITION METHOD AND BY ELECTROPHORESIS
RAIJA
PUUKKA
Department of Clinicat Chemistry, Oulu University Central Hospital, Central Laboratory, SF-90220 0th 22 (Finland) (Received
September
9th, 1977)
Summary
A chemical inhibition procedure suitable for the routine determination of alkaline phosphat~e (AP) isoenzymes in serum has been adapted for use with a fast kinetic analyzer, System Olli 3000. The results of this procedure are compared with the electrophoretic separation of alkaline phosphatase isoenzymes. The comparison of the results obtained indicates that the AP-urea/AP ratio can be used to differentiate between patients with bone and liver disease and that it is possible to estimate the relative bone and liver isoenzyme activities from this ratio quickly using two simple equations.
Introduction
A practical way for separating the most common alkaline phosphatase isoenzymes in serum is to assay the enzyme inhibition with L-phenyl~~~e and urea. This method was introduced by Statland et al. [l] for use with the centrifugal analyzer, Rotochem. More recently other workers have applied the same approach to the AutoAnalyzer II or SMA 12/60 System [ 21, to the LKB 8600 Reaction Rate Analyzer [3,4] and to the Beckman DSA564B Discrete Sample Analyzer [ 51. In these procedures the relative inhibitions of bone, liver and intestinal isoenzymes were determined first and then the resulting figures were put either on a computer program for the calculation of the isoenzyme activities [1,3,5] or on a three-isoenzyme diagram with which the isoenzyme profile can be both visualized and calculated quickly [2]. In addition, the primary data were also used without any calculations [4]. In the present study alkaline phosphate isoenzyme analysis is performed using the chemical inhibition method, adapted for a System Olli 3000 analyzer, and an electrophoretic procedure, and the results are compared. A simple way for the calculation of relative bone and liver isoenzyme activities is described.
112
Materials
and methods
Four groups of patients were investigated: 16 patients with hepato-b~lia~ disorders, 11 patients with bone disease, 14 women in the 38-4-f week of pregnancy and 12 normal children (2-10 years). In addition, the alkaline phosphatase isoenzymes were determined from 56 medical in-patients with elevated total alkaline phosphatase, for whom alkaline phosphatase isoenzymes were requested. The alkaline phos~batase isoenzymes were separated by electrophoresis on cellulose acetate strips (Sepraphore III, Gelman Instruments Co., Ann Arbor, Mich., U.S.A.). Relative activities were determined by incubation on an agar gel containing an indoxyl substrate followed by densitometric determination of the resulting color. In the presence of magnesium, alkaline phosphatase catalyzes the hydrolysis of ~-toluidi~~um 5-bromo-4-chloro-3-iIldoxy1 phosphate (Dade Division, American Hospital Supply Co., Miami, Fla., U.S.A.) to the corresponding indole, which is then oxidized by air to indigo blue, which precipitates on the membrane. Total serum alkaline phosphatase activity (AP) was determined according to the method recommended by the Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology 161. Serum alkaline phosphatase activity in the presence of L-phenylalanine (Fluka AG, Buchs, Switzerland) (AP-Phe) and in the presence of urea (E. Merck AG, Darmstadt, G.F.R.) (AP-urea) were determined as AP, but the samples were preincubated first in the presence of an inhibitor without substrate for 10 min. In the incubation mixture the final concentrations of L-Phe and urea were 9.9 mmol/l and 3.8 mol/l, respectively. The procedure was adapted for a fast kinetic analyzer, System Ofli 3000 (Ollituote Oy, Espoo, Finland) (see ref. 7). Results
Alkaline phosphat~e isoenzymes were first studied from diagnosed cases. The patients with hepato-bili~ disease (n = 16) had AP-urea/Al? values of 0.10-0.22 (mean 2 2 S.D.), while the corresponding ratio for patients with bone disease (n = 11) was 0.02-0.09 and for children (n = 12) below 0.06. The ratio AP-Phe/AP was 0.73-0.81 in all the groups, but for pregnant women (n = 14) it was 0.38-0.62. The alkaline phos~hat~e isoenzymes were also studied by both electrophoresis and the inhibition methods from a total of 56 medical in-patients with elevated total alkaline phosphatase activity, for whom alkaline phosphatase isoenzymes were requested; the results were then compared. 6 patients had an AP-Phe/AP ratio clearly below 0.73 and in electrophoresis the relative activity of bone and placental isoenzymes (these isoenzymes could not be separated from each other} was 0.56-0.84. There was a good correlation for the other 50 serum samples between the results of the electrophoresis (y = AP-bone/AP) and the inhibition method (x = AP-urea/AP) (Fig. 1). Similarly, the relative liver isoenzyme activities correlated well with the AP-urea/AP ratio (r = 0.91, y = 6.22x + 0.02). With electrophoresis the intestinal isoenzyme was found to have a relative
113
Fig. 1. Comparison of alkaline phosphatase electrophoresis and by chemical inhibition.
bone isoenzyme
from 60 medical in-patients
determined
by
activity below 0.14 in 14 of the above 50 cases, while with the inhibition method these cases had quite a normal AP-Phe/AP ratio. The intestinal isoenzyme was also found by electrophoresis in some patients (n = 12) without recognizable liver or bone disease and with a normal total alkaline phosphatase. In these case the AP-Phe/AP ratio was below 0.73 if the relative intestinal isoenzyme activity was over 0.14, but over 0,73 if the relative intestinal isoenzyme activity was below 0.14 in the electrophoresis. Discussion The chemical inhibition method for the dete~ination of alkaline phosphatase isoenzymes was adapted for the System Olli 3000 analyzer. With this procedure it was possible to control strictly both the incubation time and the temperature, since the analyzer allows for simultaneous pipetting of 24 samples and reagents, simultaneous preincubation of samples in the presence of an inhibitor and simultaneous kinetic measurements of 24 cuvettes after addition of the substrate. Mathematical three-isoenzyme models have been described for the calculation of alkaline phosphatase isoenzyme activities [ 1,3,5], where the activities of bone, liver and intestinal isoenzymes have been calculated from the AP, AP-Phe and AP-urea activities. However, as Bergstrom and Lefvert [4] have shown, these calculations do not improve the clinical value of the primary data. In these models the influence of an analytical error in the three activities obtained primarily has been disregarded and this relatively small error may thus cause dispropo~ionately large errors in the final results. Furthermore, the models evaluate only the intestinal isoenzyme, but they exclude other L-phenyl-
114
alanine-sensitive isoenzymes, placental and Began (tumor) isoenzymes. The results obtained in this work support Bergstrom and Lefvert’s conclusion [4] that one way to discriminate between patients with bone liver disease is to use the AP-urea/AP ratio. Elevated intestinal (over 14%) or placental isoenzymes can be differentiated from bone and liver isoenzymes by their AP-Phe/ AP ratio. In addition, the AP-urea/AP ratio can also be used for the estimation of the relative bone and liver isoenzyme activities, because the enzymatic activity remaining after urea inhibition is inversely proportional to the relative bone isoenzyme activity and directly proportional to the relative liver isoenzyme activities found by electrophoresis. Thus, it is possible to estimate the relative bone isoenzyme activity quickly with the equation, AP-bone/AP = -6.34 (AP-urea/AP) + 0.98 (Fig. 1) and the relative liver isoenzyme activities with the equation, AP-liver/AP = 6.22 (AP-urea/AP) + 0.02 (not shown). The use of these equations, too, causes a rather large error in the final results; for example, if the AP-urea/AP value is 0.10, the relative bone isoenzyme activity is 0.35, but it is 0.41 or 0.28, if the AP-urea/AP value is 0.09 or 0.11, respectively. In any case the error is much smaller than when the three-isoenzyme models described are used (cf. ref. 4). Acknowledgements I should like to thank help during this work.
Doctors
Inkeri Lehtinen
and Matti Eloranta
for their
References 1 Statland, B.E., Nishi, H.H. and Young, D.S. (1972) Clin. Chem. 18, 1468-1474 2 Kim. J.C., Stastny, M. and Manning, J.P. (1974) Clin. Chem. 20. 816419 3 Gerhardt. W., Nielsen, M.L., Nielsen. O.V., Olsen, J.S. and Statland, B.E. (1974) Clin. Chim. Acta 53. 281-290 4 BergstrBm, K. and Lefvert, A.K. (1975) Clin. Chim. Acta 64. 95-100 5 O’Carroll, D., Statland, B.E.. Steele, B.W. and Burke. M.D. (1975) Am. J. Clin. Path& 63, 564-572 6 The Committee on Enzymes of the Scandinavian Society for Clinical Chemxstry and Clinical Physiology (1974) &and. J. Clin. Lab. Invest. 33,292-306 7 Adlercreutz, H., Peltonen, V. and Voipio, T. (1975) Clin. Chem. 21.676-684
Clinica CIzimica Acta, 85 (1978) 111-114 0 Elsevier/North-Holland Biomedical Press
CCA 9166
COMPARISON OF ALKALINE PHOSPHATASE ISOENZYMES DETERMINED BY AN INHIBITION METHOD AND BY ELECTROPHORESIS
RAIJA
PUUKKA
Department of Clinicat Chemistry, Oulu University Central Hospital, Central Laboratory, SF-90220 0th 22 (Finland) (Received
September
9th, 1977)
Summary
A chemical inhibition procedure suitable for the routine determination of alkaline phosphat~e (AP) isoenzymes in serum has been adapted for use with a fast kinetic analyzer, System Olli 3000. The results of this procedure are compared with the electrophoretic separation of alkaline phosphatase isoenzymes. The comparison of the results obtained indicates that the AP-urea/AP ratio can be used to differentiate between patients with bone and liver disease and that it is possible to estimate the relative bone and liver isoenzyme activities from this ratio quickly using two simple equations.
Introduction
A practical way for separating the most common alkaline phosphatase isoenzymes in serum is to assay the enzyme inhibition with L-phenyl~~~e and urea. This method was introduced by Statland et al. [l] for use with the centrifugal analyzer, Rotochem. More recently other workers have applied the same approach to the AutoAnalyzer II or SMA 12/60 System [ 21, to the LKB 8600 Reaction Rate Analyzer [3,4] and to the Beckman DSA564B Discrete Sample Analyzer [ 51. In these procedures the relative inhibitions of bone, liver and intestinal isoenzymes were determined first and then the resulting figures were put either on a computer program for the calculation of the isoenzyme activities [1,3,5] or on a three-isoenzyme diagram with which the isoenzyme profile can be both visualized and calculated quickly [2]. In addition, the primary data were also used without any calculations [4]. In the present study alkaline phosphate isoenzyme analysis is performed using the chemical inhibition method, adapted for a System Olli 3000 analyzer, and an electrophoretic procedure, and the results are compared. A simple way for the calculation of relative bone and liver isoenzyme activities is described.
112
Materials
and methods
Four groups of patients were investigated: 16 patients with hepato-b~lia~ disorders, 11 patients with bone disease, 14 women in the 38-4-f week of pregnancy and 12 normal children (2-10 years). In addition, the alkaline phosphatase isoenzymes were determined from 56 medical in-patients with elevated total alkaline phosphatase, for whom alkaline phosphatase isoenzymes were requested. The alkaline phos~batase isoenzymes were separated by electrophoresis on cellulose acetate strips (Sepraphore III, Gelman Instruments Co., Ann Arbor, Mich., U.S.A.). Relative activities were determined by incubation on an agar gel containing an indoxyl substrate followed by densitometric determination of the resulting color. In the presence of magnesium, alkaline phosphatase catalyzes the hydrolysis of ~-toluidi~~um 5-bromo-4-chloro-3-iIldoxy1 phosphate (Dade Division, American Hospital Supply Co., Miami, Fla., U.S.A.) to the corresponding indole, which is then oxidized by air to indigo blue, which precipitates on the membrane. Total serum alkaline phosphatase activity (AP) was determined according to the method recommended by the Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology 161. Serum alkaline phosphatase activity in the presence of L-phenylalanine (Fluka AG, Buchs, Switzerland) (AP-Phe) and in the presence of urea (E. Merck AG, Darmstadt, G.F.R.) (AP-urea) were determined as AP, but the samples were preincubated first in the presence of an inhibitor without substrate for 10 min. In the incubation mixture the final concentrations of L-Phe and urea were 9.9 mmol/l and 3.8 mol/l, respectively. The procedure was adapted for a fast kinetic analyzer, System Ofli 3000 (Ollituote Oy, Espoo, Finland) (see ref. 7). Results
Alkaline phosphat~e isoenzymes were first studied from diagnosed cases. The patients with hepato-bili~ disease (n = 16) had AP-urea/Al? values of 0.10-0.22 (mean 2 2 S.D.), while the corresponding ratio for patients with bone disease (n = 11) was 0.02-0.09 and for children (n = 12) below 0.06. The ratio AP-Phe/AP was 0.73-0.81 in all the groups, but for pregnant women (n = 14) it was 0.38-0.62. The alkaline phos~hat~e isoenzymes were also studied by both electrophoresis and the inhibition methods from a total of 56 medical in-patients with elevated total alkaline phosphatase activity, for whom alkaline phosphatase isoenzymes were requested; the results were then compared. 6 patients had an AP-Phe/AP ratio clearly below 0.73 and in electrophoresis the relative activity of bone and placental isoenzymes (these isoenzymes could not be separated from each other} was 0.56-0.84. There was a good correlation for the other 50 serum samples between the results of the electrophoresis (y = AP-bone/AP) and the inhibition method (x = AP-urea/AP) (Fig. 1). Similarly, the relative liver isoenzyme activities correlated well with the AP-urea/AP ratio (r = 0.91, y = 6.22x + 0.02). With electrophoresis the intestinal isoenzyme was found to have a relative
113
Fig. 1. Comparison of alkaline phosphatase electrophoresis and by chemical inhibition.
bone isoenzyme
from 60 medical in-patients
determined
by
activity below 0.14 in 14 of the above 50 cases, while with the inhibition method these cases had quite a normal AP-Phe/AP ratio. The intestinal isoenzyme was also found by electrophoresis in some patients (n = 12) without recognizable liver or bone disease and with a normal total alkaline phosphatase. In these case the AP-Phe/AP ratio was below 0.73 if the relative intestinal isoenzyme activity was over 0.14, but over 0,73 if the relative intestinal isoenzyme activity was below 0.14 in the electrophoresis. Discussion The chemical inhibition method for the dete~ination of alkaline phosphatase isoenzymes was adapted for the System Olli 3000 analyzer. With this procedure it was possible to control strictly both the incubation time and the temperature, since the analyzer allows for simultaneous pipetting of 24 samples and reagents, simultaneous preincubation of samples in the presence of an inhibitor and simultaneous kinetic measurements of 24 cuvettes after addition of the substrate. Mathematical three-isoenzyme models have been described for the calculation of alkaline phosphatase isoenzyme activities [ 1,3,5], where the activities of bone, liver and intestinal isoenzymes have been calculated from the AP, AP-Phe and AP-urea activities. However, as Bergstrom and Lefvert [4] have shown, these calculations do not improve the clinical value of the primary data. In these models the influence of an analytical error in the three activities obtained primarily has been disregarded and this relatively small error may thus cause dispropo~ionately large errors in the final results. Furthermore, the models evaluate only the intestinal isoenzyme, but they exclude other L-phenyl-
114
alanine-sensitive isoenzymes, placental and Began (tumor) isoenzymes. The results obtained in this work support Bergstrom and Lefvert’s conclusion [4] that one way to discriminate between patients with bone liver disease is to use the AP-urea/AP ratio. Elevated intestinal (over 14%) or placental isoenzymes can be differentiated from bone and liver isoenzymes by their AP-Phe/ AP ratio. In addition, the AP-urea/AP ratio can also be used for the estimation of the relative bone and liver isoenzyme activities, because the enzymatic activity remaining after urea inhibition is inversely proportional to the relative bone isoenzyme activity and directly proportional to the relative liver isoenzyme activities found by electrophoresis. Thus, it is possible to estimate the relative bone isoenzyme activity quickly with the equation, AP-bone/AP = -6.34 (AP-urea/AP) + 0.98 (Fig. 1) and the relative liver isoenzyme activities with the equation, AP-liver/AP = 6.22 (AP-urea/AP) + 0.02 (not shown). The use of these equations, too, causes a rather large error in the final results; for example, if the AP-urea/AP value is 0.10, the relative bone isoenzyme activity is 0.35, but it is 0.41 or 0.28, if the AP-urea/AP value is 0.09 or 0.11, respectively. In any case the error is much smaller than when the three-isoenzyme models described are used (cf. ref. 4). Acknowledgements I should like to thank help during this work.
Doctors
Inkeri Lehtinen
and Matti Eloranta
for their
References 1 Statland, B.E., Nishi, H.H. and Young, D.S. (1972) Clin. Chem. 18, 1468-1474 2 Kim. J.C., Stastny, M. and Manning, J.P. (1974) Clin. Chem. 20. 816419 3 Gerhardt. W., Nielsen, M.L., Nielsen. O.V., Olsen, J.S. and Statland, B.E. (1974) Clin. Chim. Acta 53. 281-290 4 BergstrBm, K. and Lefvert, A.K. (1975) Clin. Chim. Acta 64. 95-100 5 O’Carroll, D., Statland, B.E.. Steele, B.W. and Burke. M.D. (1975) Am. J. Clin. Path& 63, 564-572 6 The Committee on Enzymes of the Scandinavian Society for Clinical Chemxstry and Clinical Physiology (1974) &and. J. Clin. Lab. Invest. 33,292-306 7 Adlercreutz, H., Peltonen, V. and Voipio, T. (1975) Clin. Chem. 21.676-684