Determination of Serum Alkaline Phosphatase By Amperometric Measurement of Rate of Oxygen Depletion in a Polyphenol Oxidase Coupled Reaction Anand Kumar‘ and Gary D. Christian* Deparfment of Chemistry, University of Washington, Seattle, Wash. 98 795
Alkallne phosphatase catalyzes the hydrolysis of the substrate phenolphosphate at pH above 7. The phenol produced is coupled with another enzyme, polyphenol oxldase, which catalyzes the oxldation of phenol to o-qulnone In the presence of oxygen. The rate of oxygen depletion is measured amperometrically with a Clark electrode in a Beckman Glucose Analyzer. This is directly related to the amount of phenol and, hence, to the alkaline phosphatase activity. The method does not require Incubation or protein removal, and it takes about 2-3 mln to run a sample.
Serum alkaline phosphatase (ALP) activity is determined by measuring the rate of hydrolysis of a phosphate ester at a p H above 7. A number of procedures and a variety of substrates have been developed since the original work of Martland and Robison in 1926 (1-9). Most procedures involve the spectrophotometric measurement of one of the hydrolysis products following their conversion to a suitable chromogen. More recently, some chromogenic substrates (10-13) which themselves are colorless but give a colored hydrolysis product have also been introduced. Several procedures have been adapted to AutoAnalyzers or automated otherwise (14-16). Traditionally, clinical chemists have shied away from using electrochemical techniques, and little work has been done in the area regarding the measurement of alkaline phosphatase activity. Brooks and Purdy (17) have described a coulometric procedure in which phenol is titrated with electrogenerated bromine. Although precise, the procedure requires preincubation and is not amenable to rapid measurements. In the present procedure, the rate of hydrolysis of t h e substrate phenolphosphate is determined by coupling the reaction with another enzyme, polyphenoloxidase (tyrosinase; EC 1.14.18.1), which catalyzes the oxidation of phenol t o o quinone in the presence of oxygen ( I 8). monophenol
+ 0 2 Tyrosinase o-quinone + HzO ---+
Thus, a measurement of the rate of oxygen consumption is directly related to the amount of phenol produced and, hence, to the alkaline phosphatase activity. T h e rate of oxygen depletion is measured amperometrically with a Clark electrode in a Beckman Glucose Analyzer. T h e principle of the measurement is the same as described by Kadish e t al. (19) for glucose determination. The procedure for blood serum does not involve deproteinization nor incubation, and takes 2-3 minutes to run a sample. Guilbault and Nanjo (20) have recently described a similar two-step enzymatic reaction as the basis of a phosphate-selective electrode. Alkaline phosphatase and glucose oxidase are immobilized on a platinum electrode. T h e alkaline phosphatase hydrolyzes glucose-6-phosphate added t o the test solution to give glucose, which is oxidized by molecular oxygen Present address, Technicon Instruments, Inc., Tarrytown, N.Y.
10591.
in the presence of the glucose oxidase. The initial reaction is inhibited by the presence of phosphate which causes a smaller and slower oxygen consumption, as detected amperometrically b y the electrode. T h e most commonly used alkaline phosphatase methods employ p-nitrophenolphosphate as substrate which upon hydrolysis gives an intense yellow color in alkaline medium with maximum absorbance between 400-420 n m (4). The main drawback of these methods is that both bilirubin and hemoglobin also absorb around 400 n m (21, 22) and serum blanks have to be run to account for the interference. Since t h e present method is based on electrochemical rather than colorimetric measurement, these interferences do not occur, which is a distinct advantage over the existing methods.
EXPERIMENTAL Apparatus. A Beckman Glucose Analyzer (Beckman Instruments, Inc., Fullerton, Calif. 92634) was used in conjunctionwith a strip-chart recorder (“OmniScribe”, Houston Instrument Corp., P.O. Box 22234, Houston, Texas 77027) as described before (23).This instrument was used without any modifications. The effects of “Air Adjust” and “Sensitivity” controls on the signal and the response of the electrode have been discussed previously (23). Reagents. Alkaline Phosphatase. Pooled human sera spiked with chicken intestine alkaline phosphatase (-350 U/IJ was used in optimizing conditions for the present method. Buffer. To prepare 100 ml of 1.5molh. 2-ethylaminoethanol (EAE) buffer, dissolve 13.4 g EAE (Aldrich Chemical Co.) and 15.7g KCl in about 80 ml deionized water and adjust the pH between 9.6-9.7 by adding concd HC1. The reaction with HCI is exothermic and the beaker should be cooled to room temperature before measuring the pH. Add 0.25 ml of 1.0 mol/l. of MgClz solution and make up the volume to 100 ml. Store in refrigerator. Substrate. Dissolve 0.15 g-0.20 g disodium phenylphosphate (Pierce Chemical Co.) per ml of solution in deionized water. Store in a refrigerator. Polyphenol Oxidase. Dissolve 30-40 mg (45-60 U) of a lyophilized preparation of mushroom tyrosinase (Aldrich Chemical Co.) per ml of deionized water. The activity of this enzyme is stated to be ap proximately 1.5 U/mg dry powder and varies from batch to batch. However, it can be conveniently estimated by an amperometric procedure using the Glucose Analyzer as described previously (23).Tke solution is stable for the day at room temperature in deionized water but stable only for 1-2 h in the buffer at higher pH (Table I). It is, therefore, preferable to prepare the solution separately in water and mix it with the buffer in the cell just before making the measurement. Procedure. 1)Place 0.90 ml (0.85 ml) of EAE buffer and 50 11 of tyrosinase solution in the cell. Pipet 50 p1 (100 pl) of serum sample or standard control and equilibrate with atmospheric oxygen. Initially, there is a rapid change in the numbers on the digital output display (with the selector switch on “check”). When there is no change or when the number changes by only 1unit every 2 or 3 s (this happens sometimes when the electrode performance is not optimum or when there is insufficient intial saturation of the buffer reagent with oxygen), the equilibrium with atmospheric oxygen is then sufficient to measure rates of current change due to the enzyme reaction. This takes 30-60 s depending upon the sample size as well as the amount of phenolic compounds already present in serum; the addition of the serum sample prior to the addition of the substrate, removes interference from any phenolic compounds already present in serum which are oxidized in the presence of tyrosinase. 2) Finally, pipet 10 bl of the substrate solution and record the derivative of the rate of oxygen depletion. The peak (i.e., the maximum ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
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Table I. Stability of Tyrosinase in Solution at Room Temperature Relative alkaline phosphatase activity" in Time, DEA b'(l.O mol/l.; EAEb (1.35 mol/l.; min pH 9.91) pH 9.60) WaterC 0 100 100 100 54 98 101 104 81 83 100 120 162 80 67 102 242 ... ... 102 ... ... 103 304 362 ... ... 100 a Assuming initial activity to be 100 U/1. Contains -1.5 mg tyrosinase/ml of solution. 50 111of tyrosinase solution (-1.5 mg tyrosinase) in water was added to 0.9 ml of a DEA buffer (1.0 mol/l.; pH 9.91) immediately before making the measurement.
A
0
0
TIME
pH of the buffer EAE
55 -0.
60.01
; ; 50.0. Ti
45.0.
?
30 -0.
24.0.
.
-
AMP
15.0-
12.0. 6.0.
#.EO
9.16
9.52
9.88
10.24
10.60
PH
Figure 1. Effect of buffer composition and pH on alkaline phosphatase activity Buffer concentrationwas 0.9 mol/l.;tyrosinase 2.0 mg/ml and DSPP 1.6 mg/ml. 100 pI of serum (-350 UII.) was used rate) is obtained within 1-1%min and is directly related to the serum alkaline phosphatase activity. It is not necessary to continue recording the peak after the maximum point is reached. The measurement cell should be flushed with about a 2-ml portion of deionized water 2-3 times after each determination. Blank. A blank is determined by substituting the serum sample with 50 pl of 0.85%saline solution.
RESULTS AND DISCUSSION Buffer Choice and Optimum pH. It has been shown by several workers (24-26) that EAE and diethanolamine (DEA) result in significantly higher alkaline phosphatase (ALP) activity compared to the widely used 2-amino-2 methyl-lpropanol (AMP) because of the greater transphosphorylation in these two buffers. We compared the ALP activity in these three buffers and our results (Figure 1) show that the ALP activity in EAE and DEA is 3-4 times as high as in AMP. Since the signals are significantly larger in EAE and DEA these two buffers were used in further studies. The optimum pH range in our procedure is narrow (9.6-9.7) for EAE but broad (9.3-10.0) for DEA similar to previously reported results (26). However, the present optimum p H ranges are different from the reported values of 10.2-10.3 for EAE and 9.8-10.3 for DEA; this is so because in our system there are two enzymes and these ranges reflect a compromise between the optimum activities of the two enzymes. [The optimum pH for tyrosinase activity in phosphate buffer is about 8.0 (23)].At higher p H values than the above ranges, 1284
ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
0
Figure 2. Effect of pH on time required to reach the peak Conditions were the same as in Figure 1. The numbers on the peak indicate the 60.0-
18 .o
-
10.10
1
Table 11. Effect of Ionic Strength on Alkaline Phosphatase Activity
27 -0
FAF
Relative activitya Relative activitya KCl 1.35 mol/l. 1.0 mol/l. NaC1- 1.35 mol/l. 1.0 mol/l. (mol/l.) EAE DEA (mol/l.) EAE DEA 0.00 100 100 0.00 100 100 0.50 106 104 0.50 100 100 1.05 124 112 1.05 126 112 1.47 135 127 1.47 135 119 1.89 153 146 1.89 156 138 a Assuming an activity of 100 U/1. when no KCl or NaCl was added to the buffers. Table 111. Effect of Mg2+on Alkaline Phosphatase Activity Relative activitya 1.0 mol/l. DEA 1.35 mol/l. EAE 0.0 100 100 1.0 120 134 2.0 125 137 5.0 125 137 10.0 123 137 a Assuming an activity of 100 U/l. when no Mg2+ was added to the buffers.
0
1.00
2.00
Mg2+ (mmol/l.)
EAE
45 -0 50-0~
3.00
4.00
5.00
DSPP (rng/ml)
FIgure 5. Effect of substrate (DSPP) concentration on alkaline phos-
phatase activity
t -
2 rnin. 570
A
l-ii:E
-
Figure 6. Some actual recorded signals using the recommended procedure. (Sample size 100 yl, recorder sensitivity 10 V full scale). The numbers on the peak represent ALP activity (U/l.)
0
.50
1.00
1.50
TyrOSlnaSQ
2.00
2.50
(mg/ml)
Figure 4. Effect of tyrosinase concentration on signals
effect of tyrosinase concentration on the peak heights in EAE (1.35 mol/l.; pH 9.62) and DEA (1.0 mol/l.; pH 9.80). The optimum,concentration of tyrosinase is about 1.5 mg [2.5 U, as determined amperometrically under the conditions stated in our earlier work (23)]of dry powder per ml of buffer solution. Effect of S u b s t r a t e ( D S P P ) Concentration. In both EAE (1.35 mol/l.; pH 9.60) and DEA (1.10 mol/l.; pH 9.87), the optimum DSPP concentration appears to be about 1.5 mg/ml (8 mmol/l.) of solution (Figure 5 ) . Calibration of t h e Instrument. Since the enzyme activity depends upon several experimental conditions, such as pH, temperature, buffer composition, buffer concentration, ionic strength, and choice of substrate, in a new procedure i t becomes important to establish a range of the enzyme activity in apparently healthy subjects as well as for various disease conditions in order for the results to be meaningful for interpretative purposes. With numerous methods and substrates proposed for alkaline phosphatase, it becomes difficult to correlate interlaboratory results. This has been a current topic of discussion and a solution to the problem is still eluding clinical chemists.
In our procedure, the instrument can be calibrated by measuring the oxygen tension of a solution with known oxygen concentration, although there are some problems in working with an open cell where the oxygen concentration in solution can change when the equilibrium with the atmosphere is disturbed. This would allow direct calculation of micromoles of oxygen consumed per minute t o obtain a new unit of alkaline phosphatase activity under our conditions. However, the values would be of limited use until a normal range for ALP activity is established under the conditions of our method, either by direct analysis or by establishing an empirical relationship with presently accepted procedure. In view of these difficulties, we have decided for the moment to use one of the more widely accepted procedures (12),which is a modification of the method of Bessey et al. ( 4 ) ,as a reference method for calibration of our instrument. Lyophilized pooled sera are generally used as control material (28-30) and can be obtained commercially. For our purpose, control pooled sera (Hyland, Division of Travenol Laboratories, Inc., Costa Mesa, Calif. 92626) was analyzed by the reference procedure and then used as a primary standard. The ALP activity of the control sera was 90 U/1. In a recent publication, Hafkenschied and Jansen (31)have reported that human placental alkaline phosphatase is stable up to a year at room temperature and can be an excellent source of alkaline phosphatase primary standard. This, however, is not yet available commercially. Precision. The coefficient of variation was f4.12% for 18 determinations with a 50-wl serum sample a t two different levels of alkaline phosphatase activity, viz., 36 U/1. and 370 U/l. The precision of the reference method (22) is reported to ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
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800.0r
A
4'
720 -0 U
5O
-b 2
640.0
t
I
was 0.999. T h e student's t-test (assuming similar standard deviations for the two methods) gave a value o f t = 1.62, which indicates no significant difference between the two methods a t the 90% confidence level for 19 determinations.
ACKNOWLEDGMENT
560.0
We are grateful to Karen Hobson, Shirley Krehbiel, and other members of the staff of the Chemistry Division, Department of Laboratory Medicine, University Hospital, for providing analyzed serum samples.
480.0
P
a
LITERATURE CITED
y = I . O 4 X - 1.30
r = 0.999 I
0'
7
160.0
320.0
480.0
A L P ( U / l i t e r ) ; Bessey -e
640.0 800.0 Method
Figure 7. Correlation of the results of the present method with those of the reference method. (0) loo-pl serum sample, (A)50-pl serum sample
be 4~2.3%for 10 determinations. The precision of an automated procedure (14)used by the University of Washington Hospital for the analysis of samples used in comparison studies (below) is found t o be about f5-7%. Linearity. Nineteen individual serum samples were obtained from the University of Washington Hospital, with alkaline phosphatase activities ranging from 25-768 U/1. T h e samples with activities up to 400 U/1. were analyzed by the Morgenstern et al. (14) procedure on an AutoAnalyzer and others by a manual procedure (12) with a Gilford 300-N spectrophotometer. Some of the actual recorded peaks using our recommended procedure (with EAE buffer) are shown in Figure 6. With 50-pl samples, signals were recorded a t two different recorder sensitivities: 1V full scale for activities up to 90 U/1. and 10 V full scale for activities above 90 U/1. The signals were linear up to 768 U/l. with 50-pl sample size (Figure 7). With 100-pl sample size, linearity was observed up to only 578 U/1. (also shown in Figure 7); 100 pl is the recommended size for serum samples with alkaline phosphatase activity up t o about 100 U/1. The signals, using 100-pl samples with activities below approximately 50-60 U/l., may be recorded a t 1V full scale and others a t 10 V full scale recorder sensitivity. Correlation. A correlation curve of the results between our method and the reference method is shown in Figure 7. T h e correlation coefficient, calculated by the least-squares method,
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RECEIVEDfor review March 5, 1976. Accepted May 3,1976. Presented a t the 30th Northwest Regional Meeting of the American Chemical Society, June 12-13, 1975, Honolulu, Hawaii.
