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Placental alkaline phosphatase determination by inhibition with ethylendiaminetetracetic acid
331
Clinica Chimica Acta, 145 (1985) 331-336 Elsevier
CCA 03076
Brief technical note
Placental alkaline phosphatase determination by inhibition with ethylendiaminetetracetic acid G.A.D. Miggiano *, M. Pileri, A. Mordente, G.E. Martorana and A. Castelli Isriruro di Chimica Biologica, UniversiLi Cattolica de1 Sacro Cuore, L.argo Francesco Vito I, 00168 Rome (Ira!H (Received
May 15th, 1984; revision September
18th, 1984)
Key words: EDTA; Inactivation; Placenfal; Alkalinephosphatase
Introduction
Alkaline phosphatase (AP; orthophosphoric-monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1) is one of the most complex metalloenzymes yet known because the metal-binding sites are critical to the catalytic process and essential to structure stabilization [l]. In normal conditions native placental AP, a dimer of molecular mass 140000 [2], is an expression of the placental tissue but an AP with similar properties has been found in various malignant tumors [3]. Several methods have been used to characterize the placental subform [4-61, but they are rather complex or suffer from lack of discrimination. The heat stability test is one of the most widely used assay procedures for placental AP determination, about which Moss illustrated the difficulties regarding the accuracy of this kind of approach [7]. Since alkaline phosphatases have been already reported to be variously affected by the action of chelating agents [8-lo], a selective chemical inactivation procedure employing ethylendiaminetetracetate (EDTA) is here investigated in order to provide an accurate, rapid and simple method for the determination of the placental AP activity in sera or tissues. Materials and methods
Alkaline phosphatase from bovine kidney, calf intestine and human placenta, used without further purification, were purchased from Calbiochem (La Jolla, CA, USA), diethanolamine (DEA), 2-amine-2-methyl-l-propanol (AMP), p-nitrophenyl phosphate (p-NPP) were obtained from Carlo Erba (Milan, Italy); ethylendiaminetetracetate disodium salt (EDTA), magnesium chloride and zinc chloride from Merck (Darmstadt, FRG). Buffers were prepared in reagent grade water obtained from MilliQ (Millipore, Bedford, MA, USA). * To whom correspondence
0009-8981/85/$03.30
should be addressed.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
332
Alkaline phosphatase assay Enzyme activity was determined by hydrolysis at 37°C of 10.0 mmol/l p-NPP in 0.5 mol/l DEA (pH 9.8) [ll] or 1.0 mmol AMP (pH 10.5) [12], both buffers containing 0.5 mmol/l magnesium and 1.0 mmol/l zinc ions. Units are expressed as ~mol/min-’ of p-NP released at 37°C. Inactivation studies Experiments (final: 5.0 mmol/l in DEA and ranging from O-9 min. Moreover, 0.5 mol/l DEA buffer (pH 9.8) or were preincubated in the EDTA stopped by the saturating amounts activity was measured.
were performed at a fixed EDTA concentration 40 mmol/l in AMP) with preincubation times various concentrations of EDTA were tested in in 1.0 mol/l AMP buffer (pH 10.5). The samples buffer solution for 5 min. The inactivation was of p-NPP as starter of the reaction, and the AP
Thermostability AP was tested by preincubating duplicate serum at 63°C for 30 min. The percentage of residual activity controls kept at 4°C.
0.05 ml portions of was computed from
Optimal assay conditions 50 ~1 of the sample were preincubated at 37°C for 5 min in 0.5 mmol/l DEA (pH 9.8) or 1.0 mmol/l AMP (10.5) containing 5.0 mmol/l and 40.0 mmol/l EDTA, respectively. Then 100 ~1 of p-NPP (final concentration 10.0 mmol/l) were added. The absorbance at 404 pm was recorded every 15 s for 3 min and the rate was calculated. In these conditions, the AP activity is due to the placental isoenzyme only which is inhibited to 60% (in DEA) or 55% (in AMP) of the initial level (see below). For this reason, the observed activity value was multiplied by a correction factor (lOO/% of residual activity) to obtain the ‘true’ placental AP concentration. Apparatus Absorbance measurements were carried out with a HP 8450A UV-Vis spectrophotometer (Hewlett-Packard, Palo Alto, CA, USA). Data processing and statistical analysis were performed with a M 40 computer (Olivetti, Ivrea, Italy). Results EDTA inhibits alkaline phosphatase activity (Table I). The residual activity is zero for the bone, kidney, liver and intestine forms, whereas it is reduced to about
TABLE
I
Inactivation
by EDTA of alkaline
phosphatase
from different
tissues
Buffer
EDTA (mmol/l)
Residual Kidney
Bone
Liver
Intestine
Placenta
DEA AMP
5.0 40.0
0 0
0 Cl
0 0
Cl il
60 55
a % Residual
activity = (observed
activity
activity (W) a
with EDTA)/(activity
without
EDTA)XlOO.
333
60% for the placental isoenzyme in DEA and to 55% in AMP buffer when the sample is preincubated for 5 min at 37’C. The same percentages are displayed by the commercial enzyme and by samples whose placental fraction had been previously determined by the heat test (data not shown). The activity decay of the placental and non-placental forms depends on the chelating agent concentration. In fact the residual activity of the latter subforms is practically absent with inhibitor concentrations above 2.0 mmol/l EDTA in DEA and 40.0 mmol/l EDTA in AMP buffer (Fig. 1). Moreover, when the length of the preincubation time is considered, a much faster decay is observed for all the non-placental forms treated with 40.0 mmol/l EDTA in AMP buffer, while the placentally derived one shows an almost steady course as a function of time after an initial immediate fall (Fig. 2). The same behavior is shown by experiments performed in DEA buffer (data not reported). Precision Variability was determined at two different levels of AP activity with pooled sera from samples collected from pregnant women. The 34 U/l pool showed a within run CV of 3.0% and the 10 U/l pool of 4.0% (n = 20 in both cases); day-to-day variability (n = 15) was < 5% for both levels. Accuracy Recovery tests (n = 20) were performed by adding serum whose placental activity ranged from O-50 U/l, to a serum pool previously kept at 56°C overnight in order to destroy completely the EDTA resistant activity. The mean recovery was 98% ranging from 94-106%.
9
19
ELITA(mmM)
27
36 T~melm~nl
Fig. 1. Inactivation of placental and non-placental AP by different concentrations of EDTA. Samples were assayed after 5 min of preincubation with EDTA at 37°C. The placental (A A) and non-placental (0 0) forms were determined in 0.5 mol/l DEA (empty symbols) or in 1.0 mol/l AMP (filled in symbols). The residual activity is expressed as a percentage of the control (samples without EDTA). Fig. 2. Time course of the inactivation of AP by EDTA. The placental (0) and non-placental (0) AP were preincubated with 40.0 mmol/l EDTA in AMP buffer. The residual activity is expressed as a percentage of the control (samples without EDTA).
334
A sample with 50 U/l activity underwent a serial dilution. titration are presented in Fig. 3, which shows a good correlation and expected values.
The results of the between the found
Spec’ifcify The EDTA resistant fraction was assayed in sera from normal subjects (n = 65), patients with hepato-biliary diseases (n = 20) and pregnant women (n = 35). The distribution of the placental activity in the three groups is reported in Fig. 4 which clearly shows no false positive results for the normal group, and values not significantly different from zero in the hepato-biliar~ group. On the other hand, the sera from pregnant women show an evident EDTA resistant fraction, which is absent only in a small number of cases (12%), presenting a total AP activity at the lower limits of the normal range and for which the heat test also yielded no resistant activity. ~~~~~~~u~~y Zero activity level was controlled in a sample with no EDTA resistant fraction which had been previously destroyed by heat denaturation. The results are not significantly different from 0 U/l. The threshold of sensitivity was found to be 3.0 U/l in DEA and 4.0 U/l in AMP buffer by means of repetitive assays (n = IS) of a sample, whose activity had been predetermined and had been suitably diluted with the above-mentioned zero activity sample. was performed on these sera Comparison with the heat test Thermal denaturation by heating the samples at 63°C for 30 min. Results from the heat treated samples were compared with the one treated with 40.0 mmol/l EDTA (Fig. 5). The correlation coefficient was found to be 0.995 ( y = 2.48 -t 0.98x, n = 65).
44
Fig. 3. Analytical
recovery
66
88 Expected
of the placental
I%)
isoenzyme.
The experiments are performed as described in the
Fig. 4. Distribution of the EDTA resistant subform. AP activity with hepatobiliary diseases (2) and pregnant women (3).
of sera from normal
subjects (I), patients
335
Fig. 5. Split sample comparison described in the text.
between
EDTA
and heat
treatment.
Experiments
are performed
as
Discussion
The isoenzymatic fractionation of human alkaline phosphatase has received much consideration because of its clinical usefulness [13]. Although the issue is mainly the separation of the bone from the hepato-biliary form, the contribution of the placental to the total AP activity is important in assaying sera from pregnant women and from tumor bearing individuals, in which a placental-like form may eventually be present [3,14]. On the other hand, along with the electrophoretic procedures which alone do not afford a clear identification of the placental enzyme, the methods employing selective chemical inhibition do not completely discriminate between the various isoenzymatic fractions [4-61. More recently McLaughlin et al [IS] have estimated the placental-type AP using a monoclonal antibody in a solid phase enzyme immunoassay. This method appears highly sensitive and specific for placental AP, but could not recognize some allelic variants with respect to conventional enzymatic assays. EDTA has the advantage of clearly distinguishing between the placental and the non-placental forms, including the intestinal one, because in the latter inactivation is very rapid and complete. In fact, the incubation of commercial enzymes (from bovine kidney and calf intestine) and of AP present in sera of patients with hepatobiliary diseases results in a complete loss of activity, while the AP purified from human placenta or present in sera of pregnant women is EDTA resistant. The validity of the selective inactivation procedure is confirmed by the results obtained from hepatobiliary disease patients and normal subjects, in whom any EDTA resistant activity has not hitherto been found. On the other hand, the capacity to titrate the placental form is clearly evident on assaying a mixture where this isoenzyme is surely present, and the sensitivity shown is moreover very high with a detection limit of about 3 U/l in DEA and 4 U/l in the AMP buffer. The high discrimination capacity of the procedure also can be employed, by subtracting the placental from the total activity, to evaluate the bone-liver fraction which has been
336
reported to display a significant correlation with the gestational age [4]. Other advantages also pertain to the method. The inhibition can be investigated in each sample under well-defined, controlled and homogeneous physico-chemical conditions with all the reactants, temperature and pH maintained constant throu~ou& the assay without all the precautions and devices necessary for the best performance of the heat test 1131. The procedure, moreover, does not need the adoption of a thermostatting apparatus for denaturation temperatures above that of the assay reaction and an execution time much longer than that required for routine total activity measurements. Lastly, the procedure employs a largely used and inexpensive inhibitor and offers a valid, selective and reproducible assay for the placental isoenzym~. References 1 Trotman CNA, Greenwood C. Effects of zinc and other metal ions on the stability and activity of Escherichia coii alkaline phosphatase. Biochem J 1971; 124: 25-30. 2 Ezra E, Blather R. Udenfriend S. Purifjcat~on and partial sequencing of human placental alkahne phosphatase. B&hem Biophys Res Commun 1983; 116: 3. 1076-1083. 3 Usategin-Gornez M, Yeager FM, Fernandez de Castro A. Regan isoenzyme of alkaline phosphatase in cancer patients. Cancer 1974; 27: 2545. 4 Pledge DR. Steele CJ, Belfield A, Hutchinson RS, McLelland AS. A routine method for alkaline phosphatase assay in pregnancy serum. Ciin Chim Acta 1982; 122: 71-74. 5 Ansari A, Salem FA. Determination of human serum placental alkaline phosphatase. using theophylline inhibition. Clin Chim Acta 1982; 118: 135-139. 6 Van Belle H, De Broe RJ, Wieme RJ. L,p-Bromotetramisole, a new reagent of use in measuring placental or intestinal isoenzymes of alkaline phosphatase in human serum. Clin Chem 1977; 23: 454-459. 7 Moss DW, W~tby LG. A simplified heat-inactivation method for investigating alkaline phosphatase isoenzymes in serum, Clin Chim Acta 1975; 61: 63-71. 8 Conyers RAJ, Birkett DJ, Neale FC, Posen S, Brudenellwoods J. The action of EDTA on human -alkaline phosphatases. Biochim Biophys Acta 1967; 139: 363-371. 9 Ackermann BP, Ahlers J. Kinetics of alkaline phosphatase from pig kidney. Influence of complexing agents on stability and activity. Biochem J 1976: 153: 151-157. 10 Bramley TA. Ethylendiamine-NN’-tetra-acetate-dependent amino acid-stimulated inactivation of mouse ovarian alkaline phosphatase activity. Biochem 3 1975; 147: 259-265. 11 Bowers Jr GN, McCorub RB. Total alkaline phosphatase activity in human serum. Clin Chem 1975; 2%: 1988-1995. 12 Hausamen TU, H&&et R. Rick W. Gross W. Optimal conditions for the deter~nat~on of serum alkaline phosphatase by a new kinetic method. Chn Chim Acta 1967; 15: 241. 13 Moss DW. Alkaline phosphatase isoenzymes. Clin Chem 1982; 28: 2007-2016. 14 Aleem FA. Total and heat stable serum alkaline phosphatase in normal and abnormal pregnancy. Obstet Gynecol 1972; 40: 163-172. 15 McLaughlin PJ, Gee H, Johnson PM. Placental-type alkaline phosphatase in pregnancy and mahg nancy plasma:specific estimation using a monoclonal antibody in a solid phase enzyme immunoassay. Clin Chim Acta 1983; 130: 199-209.
Clinica Chimica Acta, 145 (1985) 331-336 Elsevier
CCA 03076
Brief technical note
Placental alkaline phosphatase determination by inhibition with ethylendiaminetetracetic acid G.A.D. Miggiano *, M. Pileri, A. Mordente, G.E. Martorana and A. Castelli Isriruro di Chimica Biologica, UniversiLi Cattolica de1 Sacro Cuore, L.argo Francesco Vito I, 00168 Rome (Ira!H (Received
May 15th, 1984; revision September
18th, 1984)
Key words: EDTA; Inactivation; Placenfal; Alkalinephosphatase
Introduction
Alkaline phosphatase (AP; orthophosphoric-monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1) is one of the most complex metalloenzymes yet known because the metal-binding sites are critical to the catalytic process and essential to structure stabilization [l]. In normal conditions native placental AP, a dimer of molecular mass 140000 [2], is an expression of the placental tissue but an AP with similar properties has been found in various malignant tumors [3]. Several methods have been used to characterize the placental subform [4-61, but they are rather complex or suffer from lack of discrimination. The heat stability test is one of the most widely used assay procedures for placental AP determination, about which Moss illustrated the difficulties regarding the accuracy of this kind of approach [7]. Since alkaline phosphatases have been already reported to be variously affected by the action of chelating agents [8-lo], a selective chemical inactivation procedure employing ethylendiaminetetracetate (EDTA) is here investigated in order to provide an accurate, rapid and simple method for the determination of the placental AP activity in sera or tissues. Materials and methods
Alkaline phosphatase from bovine kidney, calf intestine and human placenta, used without further purification, were purchased from Calbiochem (La Jolla, CA, USA), diethanolamine (DEA), 2-amine-2-methyl-l-propanol (AMP), p-nitrophenyl phosphate (p-NPP) were obtained from Carlo Erba (Milan, Italy); ethylendiaminetetracetate disodium salt (EDTA), magnesium chloride and zinc chloride from Merck (Darmstadt, FRG). Buffers were prepared in reagent grade water obtained from MilliQ (Millipore, Bedford, MA, USA). * To whom correspondence
0009-8981/85/$03.30
should be addressed.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
332
Alkaline phosphatase assay Enzyme activity was determined by hydrolysis at 37°C of 10.0 mmol/l p-NPP in 0.5 mol/l DEA (pH 9.8) [ll] or 1.0 mmol AMP (pH 10.5) [12], both buffers containing 0.5 mmol/l magnesium and 1.0 mmol/l zinc ions. Units are expressed as ~mol/min-’ of p-NP released at 37°C. Inactivation studies Experiments (final: 5.0 mmol/l in DEA and ranging from O-9 min. Moreover, 0.5 mol/l DEA buffer (pH 9.8) or were preincubated in the EDTA stopped by the saturating amounts activity was measured.
were performed at a fixed EDTA concentration 40 mmol/l in AMP) with preincubation times various concentrations of EDTA were tested in in 1.0 mol/l AMP buffer (pH 10.5). The samples buffer solution for 5 min. The inactivation was of p-NPP as starter of the reaction, and the AP
Thermostability AP was tested by preincubating duplicate serum at 63°C for 30 min. The percentage of residual activity controls kept at 4°C.
0.05 ml portions of was computed from
Optimal assay conditions 50 ~1 of the sample were preincubated at 37°C for 5 min in 0.5 mmol/l DEA (pH 9.8) or 1.0 mmol/l AMP (10.5) containing 5.0 mmol/l and 40.0 mmol/l EDTA, respectively. Then 100 ~1 of p-NPP (final concentration 10.0 mmol/l) were added. The absorbance at 404 pm was recorded every 15 s for 3 min and the rate was calculated. In these conditions, the AP activity is due to the placental isoenzyme only which is inhibited to 60% (in DEA) or 55% (in AMP) of the initial level (see below). For this reason, the observed activity value was multiplied by a correction factor (lOO/% of residual activity) to obtain the ‘true’ placental AP concentration. Apparatus Absorbance measurements were carried out with a HP 8450A UV-Vis spectrophotometer (Hewlett-Packard, Palo Alto, CA, USA). Data processing and statistical analysis were performed with a M 40 computer (Olivetti, Ivrea, Italy). Results EDTA inhibits alkaline phosphatase activity (Table I). The residual activity is zero for the bone, kidney, liver and intestine forms, whereas it is reduced to about
TABLE
I
Inactivation
by EDTA of alkaline
phosphatase
from different
tissues
Buffer
EDTA (mmol/l)
Residual Kidney
Bone
Liver
Intestine
Placenta
DEA AMP
5.0 40.0
0 0
0 Cl
0 0
Cl il
60 55
a % Residual
activity = (observed
activity
activity (W) a
with EDTA)/(activity
without
EDTA)XlOO.
333
60% for the placental isoenzyme in DEA and to 55% in AMP buffer when the sample is preincubated for 5 min at 37’C. The same percentages are displayed by the commercial enzyme and by samples whose placental fraction had been previously determined by the heat test (data not shown). The activity decay of the placental and non-placental forms depends on the chelating agent concentration. In fact the residual activity of the latter subforms is practically absent with inhibitor concentrations above 2.0 mmol/l EDTA in DEA and 40.0 mmol/l EDTA in AMP buffer (Fig. 1). Moreover, when the length of the preincubation time is considered, a much faster decay is observed for all the non-placental forms treated with 40.0 mmol/l EDTA in AMP buffer, while the placentally derived one shows an almost steady course as a function of time after an initial immediate fall (Fig. 2). The same behavior is shown by experiments performed in DEA buffer (data not reported). Precision Variability was determined at two different levels of AP activity with pooled sera from samples collected from pregnant women. The 34 U/l pool showed a within run CV of 3.0% and the 10 U/l pool of 4.0% (n = 20 in both cases); day-to-day variability (n = 15) was < 5% for both levels. Accuracy Recovery tests (n = 20) were performed by adding serum whose placental activity ranged from O-50 U/l, to a serum pool previously kept at 56°C overnight in order to destroy completely the EDTA resistant activity. The mean recovery was 98% ranging from 94-106%.
9
19
ELITA(mmM)
27
36 T~melm~nl
Fig. 1. Inactivation of placental and non-placental AP by different concentrations of EDTA. Samples were assayed after 5 min of preincubation with EDTA at 37°C. The placental (A A) and non-placental (0 0) forms were determined in 0.5 mol/l DEA (empty symbols) or in 1.0 mol/l AMP (filled in symbols). The residual activity is expressed as a percentage of the control (samples without EDTA). Fig. 2. Time course of the inactivation of AP by EDTA. The placental (0) and non-placental (0) AP were preincubated with 40.0 mmol/l EDTA in AMP buffer. The residual activity is expressed as a percentage of the control (samples without EDTA).
334
A sample with 50 U/l activity underwent a serial dilution. titration are presented in Fig. 3, which shows a good correlation and expected values.
The results of the between the found
Spec’ifcify The EDTA resistant fraction was assayed in sera from normal subjects (n = 65), patients with hepato-biliary diseases (n = 20) and pregnant women (n = 35). The distribution of the placental activity in the three groups is reported in Fig. 4 which clearly shows no false positive results for the normal group, and values not significantly different from zero in the hepato-biliar~ group. On the other hand, the sera from pregnant women show an evident EDTA resistant fraction, which is absent only in a small number of cases (12%), presenting a total AP activity at the lower limits of the normal range and for which the heat test also yielded no resistant activity. ~~~~~~~u~~y Zero activity level was controlled in a sample with no EDTA resistant fraction which had been previously destroyed by heat denaturation. The results are not significantly different from 0 U/l. The threshold of sensitivity was found to be 3.0 U/l in DEA and 4.0 U/l in AMP buffer by means of repetitive assays (n = IS) of a sample, whose activity had been predetermined and had been suitably diluted with the above-mentioned zero activity sample. was performed on these sera Comparison with the heat test Thermal denaturation by heating the samples at 63°C for 30 min. Results from the heat treated samples were compared with the one treated with 40.0 mmol/l EDTA (Fig. 5). The correlation coefficient was found to be 0.995 ( y = 2.48 -t 0.98x, n = 65).
44
Fig. 3. Analytical
recovery
66
88 Expected
of the placental
I%)
isoenzyme.
The experiments are performed as described in the
Fig. 4. Distribution of the EDTA resistant subform. AP activity with hepatobiliary diseases (2) and pregnant women (3).
of sera from normal
subjects (I), patients
335
Fig. 5. Split sample comparison described in the text.
between
EDTA
and heat
treatment.
Experiments
are performed
as
Discussion
The isoenzymatic fractionation of human alkaline phosphatase has received much consideration because of its clinical usefulness [13]. Although the issue is mainly the separation of the bone from the hepato-biliary form, the contribution of the placental to the total AP activity is important in assaying sera from pregnant women and from tumor bearing individuals, in which a placental-like form may eventually be present [3,14]. On the other hand, along with the electrophoretic procedures which alone do not afford a clear identification of the placental enzyme, the methods employing selective chemical inhibition do not completely discriminate between the various isoenzymatic fractions [4-61. More recently McLaughlin et al [IS] have estimated the placental-type AP using a monoclonal antibody in a solid phase enzyme immunoassay. This method appears highly sensitive and specific for placental AP, but could not recognize some allelic variants with respect to conventional enzymatic assays. EDTA has the advantage of clearly distinguishing between the placental and the non-placental forms, including the intestinal one, because in the latter inactivation is very rapid and complete. In fact, the incubation of commercial enzymes (from bovine kidney and calf intestine) and of AP present in sera of patients with hepatobiliary diseases results in a complete loss of activity, while the AP purified from human placenta or present in sera of pregnant women is EDTA resistant. The validity of the selective inactivation procedure is confirmed by the results obtained from hepatobiliary disease patients and normal subjects, in whom any EDTA resistant activity has not hitherto been found. On the other hand, the capacity to titrate the placental form is clearly evident on assaying a mixture where this isoenzyme is surely present, and the sensitivity shown is moreover very high with a detection limit of about 3 U/l in DEA and 4 U/l in the AMP buffer. The high discrimination capacity of the procedure also can be employed, by subtracting the placental from the total activity, to evaluate the bone-liver fraction which has been
336
reported to display a significant correlation with the gestational age [4]. Other advantages also pertain to the method. The inhibition can be investigated in each sample under well-defined, controlled and homogeneous physico-chemical conditions with all the reactants, temperature and pH maintained constant throu~ou& the assay without all the precautions and devices necessary for the best performance of the heat test 1131. The procedure, moreover, does not need the adoption of a thermostatting apparatus for denaturation temperatures above that of the assay reaction and an execution time much longer than that required for routine total activity measurements. Lastly, the procedure employs a largely used and inexpensive inhibitor and offers a valid, selective and reproducible assay for the placental isoenzym~. References 1 Trotman CNA, Greenwood C. Effects of zinc and other metal ions on the stability and activity of Escherichia coii alkaline phosphatase. Biochem J 1971; 124: 25-30. 2 Ezra E, Blather R. Udenfriend S. Purifjcat~on and partial sequencing of human placental alkahne phosphatase. B&hem Biophys Res Commun 1983; 116: 3. 1076-1083. 3 Usategin-Gornez M, Yeager FM, Fernandez de Castro A. Regan isoenzyme of alkaline phosphatase in cancer patients. Cancer 1974; 27: 2545. 4 Pledge DR. Steele CJ, Belfield A, Hutchinson RS, McLelland AS. A routine method for alkaline phosphatase assay in pregnancy serum. Ciin Chim Acta 1982; 122: 71-74. 5 Ansari A, Salem FA. Determination of human serum placental alkaline phosphatase. using theophylline inhibition. Clin Chim Acta 1982; 118: 135-139. 6 Van Belle H, De Broe RJ, Wieme RJ. L,p-Bromotetramisole, a new reagent of use in measuring placental or intestinal isoenzymes of alkaline phosphatase in human serum. Clin Chem 1977; 23: 454-459. 7 Moss DW, W~tby LG. A simplified heat-inactivation method for investigating alkaline phosphatase isoenzymes in serum, Clin Chim Acta 1975; 61: 63-71. 8 Conyers RAJ, Birkett DJ, Neale FC, Posen S, Brudenellwoods J. The action of EDTA on human -alkaline phosphatases. Biochim Biophys Acta 1967; 139: 363-371. 9 Ackermann BP, Ahlers J. Kinetics of alkaline phosphatase from pig kidney. Influence of complexing agents on stability and activity. Biochem J 1976: 153: 151-157. 10 Bramley TA. Ethylendiamine-NN’-tetra-acetate-dependent amino acid-stimulated inactivation of mouse ovarian alkaline phosphatase activity. Biochem 3 1975; 147: 259-265. 11 Bowers Jr GN, McCorub RB. Total alkaline phosphatase activity in human serum. Clin Chem 1975; 2%: 1988-1995. 12 Hausamen TU, H&&et R. Rick W. Gross W. Optimal conditions for the deter~nat~on of serum alkaline phosphatase by a new kinetic method. Chn Chim Acta 1967; 15: 241. 13 Moss DW. Alkaline phosphatase isoenzymes. Clin Chem 1982; 28: 2007-2016. 14 Aleem FA. Total and heat stable serum alkaline phosphatase in normal and abnormal pregnancy. Obstet Gynecol 1972; 40: 163-172. 15 McLaughlin PJ, Gee H, Johnson PM. Placental-type alkaline phosphatase in pregnancy and mahg nancy plasma:specific estimation using a monoclonal antibody in a solid phase enzyme immunoassay. Clin Chim Acta 1983; 130: 199-209.