Automated Determination of Serum Alkaline Phosphatase Using a Modified Bodansky Technic H. Keay and J. A. Trew* An automated procedure for the measurement of alkaline phosphatase activity by a modification of the Bodansky method is described. It has been possible to adapt the Fiske-SubbaRow method for phosphate so that alkaline phosphatase is determined by a second run, immediately following the determination of inorganic phosphate. Studies of the effect of time, ph, and concentration of barbital on the enzyme activity are discussed, and the advantages of the method are listed. A METHODOLOGY for the clinical determination of inorganic phosphate and alkaline phosphatase in serum has been distributed to users of automated equipment. The method for phosphate is an adaptation of the method of Fiske and SubbaRow (1). The method for alka1ine phosphatase is a modification of the King-Armstrong method (2). The Bodansky method, as adapted by Mahoney et al. (3), has been used in this laboratory for several years. Since this procedure employs: sodium $-glycerophosphate as the substrate, rather than phenol-phosphate as in the King-Armstrong procedure, it was desirable to adapt: the former method to the automated technic and continue to obtain results in terms of Bodansky units. In the procedure for inorganic phosphate, the phosphate ion couples with molybdic acid to form phosphomolybdic acid, which is reduced with 1-amino-2-naphthol-4-sulfonic acid. The product has a stable bluecolor and is suitable for photometric determination. To adapt this procedure to the determination of alkaline phosphatase activity, it seemed From the Department of Laboratories, Regina General Hospital, Regina, Sask. Received for publication, Mar. 30, 1962. *Present address: Department of Biochemistry, University of Saskatchewan, Saskatoon,. Sask., Canada. 75
76 KEAY & TREW Clinical Chemistry that all that was necessary would be the incubation of the serum with the substrate, followed by the automated phosphate determination. Development of the Method The automated phosphate determination was modified to include the following changes: (1) replacement of the diluent with a buffered substrate; (2) directing the flow of the serum-substrate mixture through the 37#{176} incubator module; and (3) adjustment of the solution proportions to give a satisfactory colorimeter-recorder response. For satisfactory dialysis it was desirable to have solutions on either side of tile dialysis membrane more or less balanced with respect to ionic strength. Estimations of the ionic concentrations of the solutions were made and adjustments were calculated to approximate the balance. Table 1 shows the composition of the buffered solutions. A simple modification of the established procedure to direct the flow through the incubator module is shown by comparison of the flow diagram for phosphate as originally described (Fig. 1) with the flow diagram that was used for this procedure (Fig. 2). Table 1. COMPOSITION* OF BUFFERED SOLUTIONS Buffered di1uent Buffered subst rate Sodium diethyl barbiturate 4.24 gm. 4.24 gm. NaOH (0.1N solution) 28 ml. 28 ml. Di-sodium-3-glycerophosphate - #{149} 5.0 gm. NaCl 7.0 gm. 5.15 gm. Calculated ionic strength 0.143 0.160 Calculated ionic strength of recipient solution: 0.175. *Per liter. Effect of Time on Enzyme Reaction Bodansky and Bodansky (4) showed data with their original system which suggested a nonlinear response with time. Shinowara et al. (5) concluded that, under optimum conditions, hydrolysis of $-glycerophosphate by serum alkaline phosphatase was linear up to at least 2 hr. A further investigation during the present study confirmed this linear relationship up to 60 mm. These data are presented in Fig. 3. The confirmation of the linear time relationship allowed the use of an incubalion period of less than 60 mm., and when the time factor was used in the calculations, the enzyme activity was the same as one based on a
Vol. 10, No. I. 1964 ALKALINE PHOSPHATASE 77 flow dla.ram PHOSPHATE 0 1.20 I. to Al, 0. SO O0C,D Fig. 1. Flow diagram for inorganic phosphate as described iii standard automated procedure (i). flow diagram PH OS PH ATASE Manifold No fl liicubotion 37c Dialyser 3Sc 0.S0 1.20 1.20 1.60 0. eo H(ATIG BATH Fig. 2. Flow diagram for phosphatase determination by modified procedure.
78 KEAY & TREW Clinical Chemistry 60-mm. incubation. To establish the correct time of incubation it was only necessary to measure the time required for the solution to flow through the incubation coil and the dialyzer. This was the length of time during which the mixture was held at 37#{176}C. S F 6 Fig. 3. Relationship of hydrolysis of p.glycerophos. phate by serum alkaline phosphatase with time. (Phosphatase activity equals modi- 4 fled Bodansky units.) U 3 0 0 2 2 0. 10 20 30 40 50 60 Time of Incubation ( Minutes) Effect of ph on Phosphatase Activity An investigation of the effect of ph of the mixture revealed the optimum range to be from ph 9.25 to 9.75. This is comparable to the optima defined by Mahoney et al. (3), who reported ph 9.3, and Shinowara and coworkers (5), who reported ph 9.3 ± 0.15. Figure 4 shows the data for enzyme activity in the presence of buffers of different ph. It also demonstrates the relationship between the ph of the final enzyme reaction mixture and the phosphatase activity. It may be seen from these data, that in order to have the final ph in the optimum range it is necessary to start with buffer in the range of ph from 10.0 to 11.0. This was defined for the manual determination in which there is a dilution of 1 volume of serum with 9 volumes of substrate solution. It was obviously not practical to use a 1 :10 dilution of serum in the automated procedure because of the limitation of tube sizes and low colorimeter-recorder response.
Vol. 10, No. I, 1964 ALKALINE PHOSPHATASE 79 When a 1:3 dilution of serum (as used in the automated method for phosphate, but with the buffered substrate substituted) was tried, the results did not agree with the manual method. Using a buffered substrate at ph 11.0 three different dilutions of se- Fig. 4. Effect of ph on 6 /,,/ phosphatase activity. Opti-,, *- 10,0 - u.o mum activity was observed 4 /, at ph 9.25-9.75, using sub- #{149} 4 / I strate solution at ph 10.0-.1 11.0. Dashed line indicates 3 - ph of buffered substrate; solid line, ph of serum di 2 luted with 9 volumes of buf- 4 fered substrate. I I I I I I I 8 9 10 II ph rum were made and the resulting ph measured. The dilutions of 1:5 gave a ph of 9.5, which is the middle of the optimal range. A manifold was constructed to give this dilution and the details are shown in Fig. 2. Effect of Increasing Barbital Buffer Strength An alternative method of control of ph of the enzyme-substrate mixture might have been to increase the concentration of the buffer. It was thought that by this change it might be possible to avoid making a greater dilution of serum, thereby reducing the colorimetric response. A study of the variation of the ph of the mixture by increasing the barbital concentration to 150, 200, and 300% of the original demonstrated that the ph of the final mixture could be modified in this way. Further investigation confirmed the suspicion that high concentrations of barbital would affect the enzyme activity. This observation is illustrated in Fig. 5. It may be seen that the activity of alkaline phosphatase was greatly reduced with increasing barbital concentration. It was then obvious that control of the ph of the final enzyme-substrate mixture had to be achieved by using the 1 :5 dilution. This then became the requirement in selecting the tubing for the manifold. In the final manifold, tubing which delivers 0.32 ml./min. was used for the
80 KEAY & TREW Clinical Chemistry 7, 6- a. B Fig. 5. Effect of concen- 4. tration of barbital on activio ty of serum alkaline phos- 3 phatase. (Activity equals modified Bodansky units.), 2. 0. 4.24 6.36 8.48 12.72 Concentration of Barbital in Buffer (Q Na Barbital Ii... sample and tubing which delivers 1.20 ml./min. was used for the buffered substrate or buffered diluent (Fig. 2). The Method Reagents The composition of the buffered diluent and the buffered substrate solutions are given in Table 1. The ph of each solution is checked and adjusted, if necessary, to ph 10.9. The buffered substrate was overlayed with petroleum ether and both solutions stored at 4#{176}. The sulfuric-molybdic acid and the amino-naphthol reagents were those used for the inorganic phosphate determination by the automated method. Phosphate Standard Stock standard solution was prepared from monobasic potassium phosphate (K H9P04) and working standards contained from 1 to 13 mg. of phosphorus per 100 ml. Procedure The procedure has two parts: measurement of inorganic phosphate, and measurement of alkaline phosphatase activity. Run A: Inorganic Phosphate Settings on the instrument modules were as follows: rate, 20 specimeiis per hour; Manifold, modified for phosphatase (Fig. 2); Refer-
Vol. 10, No. I, 1964 ALKALINE PHOSPHATASE 81 ence orifice, No. 8; Zero setting, with zero orifice on sample side; and 100% setting, with reagent being pumped. Using the buffered diluent, the reagents were pumped for a period of 20 miii. or longer if necessary for a steady base line. Standards of concentration of 1-7 mg./100 ml. were analyzed and followed by the required samples. The setting on the shut-off marker was placed two spaces after the last sample. At the end Run A all the samples were not yet recorded but Run B was stai ted directly. Run B: Alkaline Phosphatase The diluent was replaced with buffered substrate and the sampler plate turned back to begin sampling the standards once again. The standards used in Run B were from 3 to 13 mg./100 ml. The shut-off marker was set 8 spaces after the last sample. (This time was required to allow for the incubation of the last sample.) The base line was checked while the substrate was being pumped and before the first standard was recorded. Calculation A calibration graph was drawn using the standards for Run A, and the inorganic phosphorus was calculated. A second calibration was drawn from the Standards used in Run B, and the total phosphorus was calculated for each specimen. The value in Run A was subtracted from that of Run B to obtain the amount of phosphorus liberated by the enzyme. Multiplication by the time correction factor gave the activity of alkaline phosphatase in modified Bodansky units (based on 60-mm. incubation). Discussion The time of passage of the mixture through the part of the apparatus which was controlled at 37#{176} needs to be carefully recorded so that, when the enzyme activity is calculated, it is actually based on a 60-mm. time interval. It should be noted that this may change slightly during the life of a manifold and should be checked periodically. The method, as described here, has been used in this laboratory for 12 months and has been found satisfactory. The advantages of this modification include the following. 1. Mechanical changes and adjustments during the series of analyses are minimal.
82 KEAY & TREW Clinical Chemistry 2. Reliability that accompanies automation is increased as compared to manual procedures. 3. With the reduced incubation time it is possible to record accurate results for very high enzyme activities, as compared to the limit of about 25 units in the manual procedure, without repeating the assay with a smaller sample. 4. A smaller sample is required. The determinations may be done on 1.3 ml. as compared to 2.5 ml. for the manual method. 5. Calculation is easy. References 1. Fiske, C. H., and SubbaRow, Y., J. Biol. Chem. 66, 375 (1925). 2. Technicon Methodology Procedure Manual, Methods 5 and 6. Technicon Instruments Corp., Chauncey, N. Y. 3. Mahoney, N., Draycott, M., and Robertson, H., Canad. J. Med. Technol. 9, 107 (1947). 4. Bodausky, M., and Bodansky, 0., J. Biol. Cheat. 99, 197 (1932). 5. Shinowara, G. Y., Jones, L. M., and Reinhart, H. L., J. Biol. Chem. 142, 921 (1942).