CA1192613A - Electrochemical determination of orthophosphoric monoester phosphohydrolase activity (ec 3.1.1.1 and ec 3.1.3.2: alkaline and acid phosphatases) - Google Patents

Abstract

ELECTROCHEMICAL DETERMINATION OF ORTHOPHOSPHORIC MONOESTER
PHOSPHOHYDROLASE ACTIVITY (EC 3.1.3.1 AND EC 3.1.3.2:
ALKALINE AND ACID PHOSPHATASES) ABSTRACT OF THE DISCLOSURE
A method is disclosed for the measurement of phosphate ester substrate containing a nitro group is re-acted with acid or alkaline phosphatase under appropriate reaction conditions and the hydrolytic process is monitor-ed at electrodes which measure the current produced by the reduction of the nitro groups of the product and/or sub-strate. Alternatively, an aromatic phosphate ester sub-strate containing an amino group is reacted with acid or alkaline phosphatase under appropriate reaction conditions with the hydrolytic process being monitored at solid elec-trode which measure the current produced by the oxida-tion of the amino groups of the product and/or substrate.

Description

ELECTROCHEMICAL DETERMINATION OF ORTHOPHOSPHORIC MONOESTER
PHOSPHOIIYDROLASE ACTIVITY (EC 3.1.3.1 AND EC 3.1.3.2:ALKALINE
AND ACID PHOSPHATASES) BACKGROUND OF THE INVENTION
This inve~ion relates to a new and useful electrochemical process for the detection and measurement of orthophosphoric monoester phosphohydrolase activity (EC3.1.3.1 and EC3.1.3.2). These enzymes are commonly known as alkaline and acid phosphatase, depending upon whether they prefer reaction conditions of about pH 10.0 or pH 5.0, respectively.
The phosphatases have a low substrate specificity and the general chemical reaction involves the hydrolysis of a monophosphoric ester substrate to its corresponding alcohol and phosphate ion. For example, alkaline phosphatase (ALP) hydrolyzes p-nitrophenyl phosphate (PNPP) to p-nitro-phenol (PNP) and phosphate ion (Ref. 1).

, The chemical reaction is depicted as follows:

" N~ N

+ HOH ~ Rearron9es ~ + O=p_ o ~J M92 ~ at olkaline pH

o=P--o p-Ni~rophenyl-phosphate p-Nitropheno~dde p-Nilropheno~dde (colorlessi (colorless benzenoid torm) (yellow, quinoid form) In the presence of excess substrate, under appropriate reaction conditions, the rate limiting factor i5 the concentration and activity of the ALP.
A similar hydrolysis reaction occurs under acidic conditions in the presence of acid phosphatase (ACP).
The measurement of serum ALP activity is of primary importance for the diagnosis of two groups of conditions: hepatobiliary disease, and bone disease associated with increased osteoblastic activity (Refs.

2,4). Moderate elevations of serum ALP have been reported for parenchymal liver disease e.g. infectious hepatitis, infectious mononucleosis, portal cirrhosis, and the like. Elevated serum ALP has also been reported for the following bone assoicated diseases: Paget's disease, Fanconi ~L9~ 3 syndrome, osteomalacia, rickets, hyperparathyroidism, and bone cancer (Refs. 2,~).
Similar to ALP, ACP is widely clistributed throughout the body tissues. However, the major diagnostic application of serum ACP
measurement is for males with prostatic cancer with metastases (Refs.

3,5). More specific testing of the prostatic ACP fraction may be accomplished by employing a tartrate inhibition test procedure (Ref. 3).
SUMMARY OF THE INVENTION

. . .
The process described herein may be employed to measure acid and alkaline phosphatase activity. An aromatic phosphate ester sub-strate containing a nitro group is reacted with acid or alkaline phosphatase under appropriated reaction conditions and the hydrolytic process is monitored at electrodes which measure the current produced by the reduction of the nitro groups of the product and/or substrate.
Alternatively, an aromatic phosphate ester substrate containing an amino group is reacted with acid or alkaline phosphatase under appropriate reaction conditions with the hydrolytic process being monitored at solid electrodes which measure the current produced by the oxidation of the amino groups of the product and/or substrate.
The process may be adapted to measure phophatase activities in:
animal body fluids or tissues; plants; and, microorganisms. The process, with or without modiFication, may be adapted to polarographic and other electrochemical apparatus currently available, or specific analyzers may More economically be built to monitor the electrochemical reactiv;ty of n;tro or am;no groups of the substrate and/or product.
The electrochemical detection procedure described herein for the measurement of alkaline and acid phosphatase activity is highly speciFic and sensitive. Chromogenic and turbidimetric in-terferences are eliminated due to the nature of the detection system.
In accordance with the invention there is provided a process for the measurement of phosphatase activity in serum, other fluids or tissues brought into solution; whereby, the sample is allowed to react with an aromatic phosphate ester substrate containing a nitro or amino group, with the hydrolysis process being monitored at electrodes. The phosphatase activity is established by conventional kinetic techniques, end-point techniques, and the like.
In the analyses included herein by way o-f examples, the following chemicals were obtained from Sigma Chemical Co., St. Louis, Missouri: ALP enzyme (from chicken intestine), p-nitrophenylphosphate hexahydrate (PNPP), and sodium nitrite. Normal human pooled serum was obtained from the Regina General Hospital, Regina, Saskatchewan, Canada.
Certified A.C.S, grade ethylenediaminetetraacetic acid (EDTA), p-nitrophenol (PNP), sodium chloride, and sodium hydroxide were obtained from Fisher Scientific Co., Fair Lawn, New Jersey. Reagent grade magnesium chloride hexahydrate and sodium bicarbonate were purchased from J.T, Baker Chemical Co., Phillipsburg, New Jersey. The activating buffer, 2-amino-2-methyl-1-propanol (AMP) was obtained from Eastman Kodak Co., Rochester, New York. Concentrated hydrochloric acid was supplied by Canadian Industries Ltd., St. Boniface, Manitoba, Canada.
However, other sources of chemicals can of cour~e be used.
For polarographic analyses, a Sargent Model XVI polarograph from Sargent-Welch Scientific Co., Torontol Ontario, Canada, and a Model 170 Electrochemistry System from Princeton Applied Research, Princeton, New Jersey, were employed. Titration vessels (polarography cells), saturated calomel electrodes, and related accessories were from Brlnkmann Instruments, Rexdale, Ontario, Canada. Triple distilled mercury was supplied by Engelhard Industries, Toronto, Ontario, Canada.
Nitrogen gas (99.9% purity) from CanadianLiquid Air Ltd., Regina, Saskatchewan, Canada, was used to displace dissolved oxygen in the test solutions throughout this project. A constant temperature water bath was maintained by either a Thermomix-148 water pump from B. Braun, Melsungen AG, West Germany, or a Haake circulator from Fisher Scientific Co., Fair Lawn, New Jersey. A Gilford automatic dispenser was obtained From the Gilford Instrument Laboratory Inc., Oberlin, Ohio. The above instrumentat.ion is listed for reference purposes only. Other instrumentation can of course be employed.
With the foregoing in view, and other advantages as will become apparent to those skilled in the art to which this invention relates as this specification proceeds, the invention is herein de-scribed by reference to the accompanying drawings forming a part thereof, which includes a description of a typical embodiment of the principles of the present invention in which:

~9Z~13 DESCRIPTION OF ~HE DRAWINGS
FIG. 1 contains plots of diffusion current versus voltage for a buffer blank solution, a buffer solution containing p-nitro-phenylphosphate, and a buffer solution containing p-nitrophenol.
FIG. 2 contains plots of diffusion current versus p-nitrophenylphosphate concentration at pH 10.0 for nitro group reduction waves I and II, at E~ values of -0.82 and -1.28 volts, respectively.
FIG. 3 contains plots of diffusion current versus p-nitrophenol concentration in the presence of 15xlO-2mM p-nitro-phenylphosphate at pH 10Ø Nitro group reduction waves I and II
were at -0.83 and -1.23 volts, respectively.
FIG. 4 contains plots of diffusion current versus time, showing the effect of alkaline phosphatase activity upon nitro group redu~tionwaves I and II in pH 10.0 supporting electrolyte nnedia.
FIG. 5 contains characteristic plots of diffusion current versus voltage for an AMP buffer blank, p-nitrophenylphosphate in AMP buffer, and p-nitrophenol in AMP buffer, each at pH 12Ø
FIG. 6 contains plots of diffusion current versus p-nitrophenylphosphate concentration in AMP buffer at pH 12.0 for nitro group reduction waves I and II, at E, values of -0.25 and -0.76 volts, respectively.
FIG. 7 contains plots of diffusion current versus p-nitroDhenolin AMP buffer at pH 12.0 for nitro group reduction waves I and II, at E~ values of -0.35 and -0.85 volts, respectively.
FIG. 8 is a plot/di$Fusion current versus voltage, showing the separation of the first reduction waves of p~nitro-phenylphosphate and p-nitrophenol in AMP buffer at pH 12Ø
FIG. 9 is a plot of diffusion current versus p-ni-tro-phenol concentration in the presence of 75mM of p-nitrophenylphosphate, for nitro group reduction wave I at an El value of -0.37 volts.
FIG. 10 contains plots of diffusion current versus p-nitrophenylphosphate concentration in the presence of denatured serum and AMP buffer at pH 12.0 for nitro group reduction waves I and II, at E, values of -0.26 and -0.81 volts, respectively.
In the drawings like characters of reference indicate corresponding parts in the different figures.
_ETAILED_DESCRIPTION
Proceeding therefore to describe the invention in detail, the following methods were used in preparing the necessary standards:
Polarographic Reduction of PNPP and PNP at Varied pH
A 7.5 mM stock solution of PNPP was prepared by adding 0.06959 g of PNPP to a 25-ml volumetric flask which was filled to volume with distilled water. A supporting electrolyte medium was prepared by adding 1.050 g of anhydrous sodium carbonate~ 0.05088 9 of magneslum chloride hexahydrate, and 2.250 9 of sodium chloride to a beaker containing 225 ml of distilled water. The solution was adjusted to pH 8.50 with 0.1 M sodium hydroxide, and transferred tO d .

6~

250-ml volumetric -flask which was filled tu volume with distilled water.
Polarographic analyses were performed with a Sargent Model XVI Polarograph. A dropping mercury electrode (DME) was employed as the working electrode. A saturated calomel electrode was employed as the reference electrode. Analyses were performed in a water-jacketed polarography cell maintained at 25C. Twenty-five milli-liters of supporting electrolyte medium was transferred to the polarography cell. The solution was deaerated for 10 min prior to polarographic analysis. The above procedure was simiarly performed in duplicat~. A typical supporting electrolyte polarùgram (Blank) is presented in Figure 1.
A Gilford automatic dispenser was employed to dispense reagent solutions. A PNPP test solution was prepared by dispensing 0.1 ml of PNPP stock solution into a 25-ml volumetric flask which was filled to volume with supporting electrolyte medium. Polaro-~raphic analysis was performed as previously described. The above procedure was similarly performed in duplicat~. Half-wave potentials (E,) and diffusion currents (Id) were calculated from the polarograms by the "box technique" (Ref. 6).
A series of supporting electrolyte media were similarly prepared as described above, however, increasing amounts of 0.1 M
sodium hydroxide were added to produce solutions of pH 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, and 10.50. Corresponding PNPP test solutions were similarly prepared as previously described at pH 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, and lO.S0. Polarographic analysis of blank and test solutions was performed as previously described. Average E~

and Id values have been calculated for each of the duplicate test solutions (see Table 1).
A 7.5mM stock solution of PNP was prepared by adding 0.10435 9 of PNP to a 100-ml volumetric flask which was filled to volume with distilled water. A PNP test solution was prepared by dispensing 0.1 ml of PNP stock solution into a 25--ml volumetric flask which was filled to volume with pH ~.50 supporting electrolyte medium. Polarographic analysis was per-formed as previously described.
PNP test solutions were similarly prepared and analyzed using supporting electrolyte media of pH 9.00, 9.25,/g 75, 10.00, 10.25, and 10.50.Average E~ and Id values have been calculated for each of the duplicate test solutions(see Table 2).
Characteristic polarograms of a supporting electrolyte medium (Blank), a PNPP test solution, and a PNP test solution, each at pH 10.00, are depicted in Figure 1.

-ln-Table 1 Polarographic reduction of PNPP at varied pH

Wave I Wave II
p~ E~ (volts~* Id (~A~ E~ (volts)* Id (,uA) .
.50 -0.773 0.080 -1.307 0.140 9.00 -0.783 0.080 -1.305 0.142 9.25 -0.790 0.084-1.307 0.144 9.50 -Q.783 0.088-1.302 0.150 9.75 -0.779 0.086-1.304 0.142 10.00 -0.785 0.080-1.303 0.140 10.25 -0.792 0.084-1.306 0.140 10.50 --0.782 0.084~ -1.300 0.140 *Each value reported represents an ayerage of duplicate test results.

Table 2 Polarcgraphic reduction of PNP a~varied pH

Wave I
p~l E~ (volts)* Id (~A)*

8.50 -0.740 0.609 9.00 -0.772 0.606 9.25 -0.800 0.582 9.50 -0.817 0.555 9.75 -0.838 0.552 o . oo -b . 849 0.540 10.25 -0.865 0.5~6 10.50 -0.879 0.540 _ .
*Each value reported represents an average of duplicate test results.

._ ..

Quantitation of PNPP and PNP

Stock solutions of PNPP and PNP were prepared as described under the DETAILED DESCRIPTION. A series of PNPP standards was prepared by pipetting 0, 0.5, 1.0, 1.53 and 2.0 ml of PNPP stock solution into five 25-ml volume-tric Flasks. A 0.3 ml volume of PNP stock solution was ad-ded to each flask. The volumetric flasks were Filled to volume with pH 10.00 supporting electrolyte medium. This produced standard solutions containing 0, 15 x 10-2, 30 x 10-23 45 x 10-2, and 60 x 10 2 mM of PNPP. The PNP concentration in each flask was 9 x 10-2 mM. Polarographic analysis and subsequent measurements of E~ and Id were performed as des-cribed under the DETAILED DESCRIPTION. Two polarographic reduction waves were observed at approximately E~ values of -0.82 volts and -1.28 volts for waves I and II, respec-tively. The Id values for waves I and II were each plot-ted versus PNPP concentration (see Table 3 and Figure 2).
A series of PNP standards was prepared by pipet-ting 0, 0.1, 0.2,0.3 and 0.4 ml of PNP stock solution into five 25-ml volumetric flasks. A 0.5 ml volume of PNPP solu-tion was added to each flask. The volumetric flasks were filled to volume with pH 10.00 supporting electrolyte me-edium. This produced standard solutions containing 0, 3 x 10-2, 6 x 10 2, 9 x 10 , and 12 x 10 mM of PNP. The PNPP concentration in each flask was 15 x 10-2 mM. Pola-rographic analysis and subsequent measurements of E~ and Id were performed as described under the DETAILED DESCRIP-TIONo Only one reduction wave (wave I, approximate E~ of -0.83 volts) was observed for PNP at pH 10.00. The con-stant concentration of PNPP produced wave II with an ap-proximate E~ of -1.28 volts and a reproducible average Id value of 0.38,uA. A linear response up to a concentration of 12 x 10 2 mM of PNP was obtained when the Id values of wave I were plotted versus PNP concentration (see Table and Figure 3).

Table 3 Quantitation of PNPP in the presence of 9 x 10 2 mM PNP at pH 10.00 Concentration Wave I Wave II
(X10-2 M) El, (volts)* Id (~uA)* E~ (volts)* Id (yA)*
.
0 -0.832 1.63 -0.832 1.81 -1.274 0.44 -0.825 1.97 -1.292 0.71 ~5 -0.821 2.24 -1.279 1.00 -0.81~ 2.26 -1.276 1.27 . . . _ . . ~ .
*Each value reported represents an average of duplicate test results.

Table 4 Quantitation of PNP in the presence of 15 x 10 2 mM PNPP at pEi 10.00 -Concentration Wave I Wave II
(X10-2 mM) E~ (volts)* Id (~)* E~ (volts)~ Id (~)*

0 -0.787 0.18 -1.295 0.37 3 -0.829 0.80 -1.280 0.38 6 -0.83~ 1 15 -1.280 0.37 9 -0.832 1.81 -1.274 0.41 12 -0.833 2.17 -1.269 0.38 .
*Each value reported reprcsents an average of duplicate test res~llts.

3~ 3 Kinetic Determination of ALP Activity A standard solution of ALP was prepared by adding 10 mg of ALP to a 10-ml volumetric flask which was filled to volume with distilled water. The ALP standard solution was mixed by gentle inversion and equilibrated in a 30C water bath prior to use. A stock solution of PNPP was prepared as described on page

4. A 2.n ml volume of PNPP stock solution was pipetted into a 25-ml volumetric flask which was filled to volume with pH 10.00 supporting electrolyte medium. This produced a PNPP substrate solution containing 60 x lo 2 mM of PNPP. The substrate was transferred to a water-jacketed polarography cell mainta;ned at 30C. Polarographic analysis was performed as described on page 4. Thereafter, 0.2 ml of the ALP standard solution was added to the contents of the polarography cell. The solution was simultaneously mixed and deaerated by purging wit'n nitrogen gas for 3 min. Polarograms were recorded at 5-min intervals for 1 hour. The Id values of the reduction waves were measured as described on page 5 . Test results have been tabulated in Table 5 and graphically depicted versus t-ime in Figure 4 2~3 Table S
Effect of ALP activity upon reduction waves I and II

Id (~A) Time (min) Wave 1 Wave 0 1.36 2.46 3 1.60 2.32 8 1.76 2.36 13 1.94 2.20 18 2.06 2.02 23 2.20 2.08 28 2.40 1.96 33 2.52 1.96 38 2.68 1.90 43 2.82 1.84 48 2.94 ~ 1.76 53 3.04 1.70 58 3.14 1.64 63 3.30 1.64 . . . _ _ . . _ _ _ _ .. .. _ ..

~26i:~3 Polarographic Behavior of PNPP and PNP in the Presence of AMP
Buffers at Oifferent pH

Reagent Grade AMP was warmed to 35C until it was completely liquified. A total of 17.83 9 of AMP was transferred to a 500-ml beaker. Two hundred ml of distilled water, 2.50 9 of sodium chloride, and 0.00093 9 of EDTA were added to the beaker. The result;ng solution was adjusted to pH 10.00 with concentrated HCl. The AMP buffer solution was transferred to a 250-ml volumetric flask which was filled to volume with distilled water.
A 3 x lo-2 mM PNPP test solution was prepared by dispensing 0.1 ml of PNPP stock solution into a 25-ml volumetric flas~
which was filled to volume with AMP buffer solution. The -test solution was transferred to a polarography cell and deaerated as described on page 4 . Polarographic studies were performed with a Princeton Applied Research Model 170 Electrochemistry System. Polarograms were recorded in duplicate. El/2 and Id measurements were made as described on page 5 . The above procedure was similarly performed for a 3 x 10 2 mM PNP test solution. AMP buffer solutions of pH 7.00, 8.00, 9.00, 11.00, and 12.00 were similarly prepared as described above. PNPP and PNP test solutions were prepared for each of the AMP buffer solutions. Polarographic analysis and subsequent measurement of El/2 and Id values were performed as described above.
Average El/2 and Id values have been calculated from duplicate ~9~

~ g analyses of PNPP and AMP buffers of varying pH (see Table 6).
Average El/2 and Id values have similarly been calculated for PNP in AMP buffers of varying pH (see Table 7). Characteristic polarograms of an AMP buffer (Blank), a PNPP test solution, and a PNP test solution, each at pH 12.00, are depicted in Figure 5.

-~o-Table 6 Polarographic reduction of PNPP in AMP buffers of different pH

__ Wave I Wave II
pH
E~ (volts)* Id (yA) E~ (volts)* Id (~A)*

7.00 -0.765 0.189-1.320 0.106 8.00 ~0.732 0.183 1.283 0.136 9.00 -0.615 0.142-1.125 0.154 10.00 -0.440 0.100-0.942 0.154 11.00 -0.356 0.073-0.891 0.150 12.00 -0.259 0.0~7 -0.792 0.146 *Each value reported represents an average of duplicate test results.

~26~

Table 7 Polarographic reduction of PNP in AMP buffers of different pH

_ Wave I Wave II
pH E~ (volts)* Id (~A) E~ (volts)* Id (yA)*

7.00 -0.669 0.679 8.00 -0.608 0.683 9.00 -0~580 0.689 lO.00 -0.525 0.677 ll.00 -0.448 0.496 12.00 -0.40& 0.189 -0.873 0.339 *Each value reported represents an average of duplicate test results.

Quantitation of PNPP and PNP in the Presence of AMP Buffer Twenty-five milliliters of PNPP stock substrate solution was prepared to contain PNPP and magnesium chloride at concentrations of 225 m~1 and 1.5 mM, respectively. A pH 12.00 AMP buffer solution was prepared as described on page l9 . A
supporting electrolyte medium was prepared by adding 0.8 9 of sodium hydroxide, lO.00 9 of sodium chloride, and 0.00372 9 of EDTA to a one-liter volumetric flask which was filled to volume with distilled water.
A 0.2 ml volume of PNPP stock solution, 2.7 ml of AMP
buffer, and O.l ml of distilled water were pipetted into a test tube. The solution was mixed and the test tube was suspended in a 30C water bath for 15 min. A 0.5 ml aliquot was pipetted into a 50~ml volumetric flask which was filled to volume with supporting electrolyte medium. Twenty-five milliliters were transferred to a polarography cell. Polarographic analysis and subsequent El/2 and Id measurements were made as described on page l9. A series of four addi-tional PNPP standard solutlons were prepared by pipetting l.0, 2.0, 3.0, and 4.0 ml of PNPP
stock substrate solution into four 5-ml volumetric flasks which were filled to volume with 1.5 mM magnesium chloride solution.
This produced a series of standards containing 45, 90, 135, and 180 mM of PNPP. Polarographic analysis of each test solution was performed as described above. The procedure was similarly performed in duplicate. Two reduction waves, designated as waves I and II, were observed at approximate El/2 values of -0.25 volts and -0.76 volts, respectively. The average Id values of each wave have been plotted versus PNPP concentration (see Table 8 and Figure 6).
A 225 mM PNP stock solution was prepared by dissolving 0.78249 9 of PNP in a 25-ml volumetric flask which was filled to volume with l.S mM magnesium chloride solution. A series of P~P standards was prepared by pipetting 0.50, 1.0, l.S, 2.0, and 2.5 ml of PNP stock solution into each of S-ml volumetric flasks which were filled to volume with 1.5 mM magnesium chloride solution. This produced a seriës of standards containing 22.5, 45.0, 67.5, 90.0 and 112.5 mM of PNP. Polarographic analysis and subsequent measurements of El/2 and Id were performed as described above. Two reduction waves, designated as wave I
and II, were observed at approximate Elt2 values of -0.35 volts and -0.85 volts, respectively. The average Id values of each wave have been plotted versus PNP concentration (see Table 9 and Figure 7).
A standard solution containing PNPP and PNP was prepared by mixing O.S ml of a 90 mM PNPP standard solution with O.S ml of a 45 mM PNP standard. Polarograohic analysis was performed as described above, however, to optimize separation of the first nitro reduction waves of PNPP and PNP, a more rapid scan rate was employed to expand the X-axis. !~lave I of PNPP has been separated from wave I of PNP in the presence of AMP buffer at pH 12.00 (see Figure 8).

Tab~e 8 Quant.itation of PNPP in A~P buffer of pH 12.00 _ _ Wave I Wave II
Concentration (mM) E~2 (volts)* Id (juA)* E~2 (volts~* Id (~A)*

-0.241 0.110 -0.750 0.213 -0.239 0.209 -0.751 0.472 135 -0.217 0.331 -0.738 0.732 180 -0.25l. 0.441 -0.780 0.988 225 -0.2~8 0.561 -0.820 1.201 _ _ _ _ _ . _ _ *Each value reported represents an average of duplicate test res~ s.

Table 9 Quantitation of PNP in AMP buffer of p~l 12.00 Wave I ~ave II
Concentration (mM) E~2 (volts)* Id (,uA)* E~ (volts)* Id (~uA)*

22~ 5 ~0~ 366 0~ 165~0~ 901 0~ 124 4 5 ~ 00 ~ 3 5 0 0 ~ 2 4 00 ~ 8 4 9 0 r 3 3 5 67 ~ 5~0~ 344 0~ 398~0~ 858 0 ~ 555 90 ~ 0~0 ~ 34 3 0 ~ 502-0. 848 0 ~ 670 112 ~ 5~0 ~ 3 11 0 ~ 620-0. 830 0 ~ 916 __ _ _ _ __ _ _ _ *Each value reported represents an average of duplicate test results .

~3~ L3 Quantitation of PNP in the Presence of AMP Buffer and a Constant Amount of PNPP

Stock solutions of PNPP and PNP, each at 225 mM, were prepared as descrlbed on pages 24 and 25. A series of PNP
standards was prepared by pipetting 1.0, 2.0, 3.0, and 4.0 ml of PNP stock solution into four 10-ml volumetric flasks. The volumetric flasks were filled to volume with aqueous 1.5 mM
magnesium chloride solution. This produced standard solutions containing 22.5, 45.0, 67.S, and 90.0 mM of PNP with a constant PNPP concentration of 75 mM. Polarographic analysis of each standard solution was performed as described on page 19.
The approximate El/2 value for reduction wave I was -0.37 volts.
The Id values have been plotted versus PNP concentration (see Table 10 and Figure 9).

2~ 3 Table 10 Quantitation of PNP in the presence of 75 mM of PNPP

_ PNP
Concentration PNP reduction wave (mM) El/2 (volts) Id (~A) 22.5 -0.334 0.136 45.0 -0.362 0.232 67.5 -0.366 0.374 90.0 -0.406 0.480 Quantitation of PNPP in the Presence of AMP Buffer and Denatured Serum Denatured serum was prepared by incubating a poolecl serum at 56~C for 2 hours. A series of PNPP standards in AMP buffer of pH 12.00 were prepared as described on page 19 , however, a 0.1 ml volume of denatured pooled serum was added to each of the standards prior to being brought to volume. Polarographic analysis and subsequent El/2 and Id measurements were performed as described on page 19. Two reduction waves, designated as waves I and II, were observed at approximate El/2 values of -0.26 volts and -0.81 volts, respectively. The Id values for waves I and II have been plotted versus PNPP concentration (see Table 11 and FigurelO).

~9~L3 Table 11 Polarographic reduction of PNPP in the presence of AMP buffer and denatured serum PNPP
Concentration Wave I Wave II
(mM) El/2 (volts) Id (~A) El/2 (volts) Id (~A) -0.262 0.093 -0.805 0.213 -0.266 0.165 -0.812 0.476 135 -0.242 0.291 -0.796 0.772 180 -0.282 0.378 -0.835 1.028 225 -0.239 0.531 -0.795 1.260 -REFERENCES

1. Kachmar, J.F., and Moss, D.W., In fundamentals of Clinical Chemistry, Ed. by N.W. Tietz, I~.B. Saunders Company, Philadelphia, London, Toronto, 2nd Ed., p. 607 (1976).
2 Ibid. pp 602-613.
.
3. Ibid. pp 613-618.
4. Eastham, R.D., In 8iochemical Values in Clinical Medicine, John Wright and Sons Ltd., Bristol, 5th Ed., pp 146-149 (1975).

5. Ibid. pp 144-146.

6. Willard, H.H., Merritt, L.L. ~r., and Dean, J.A., In Instrumental Methods of Analysis, D. Van Nostrand Co., Toronto, London, Melbourne, 4thEd., p 692 (1968).

Claims

CLAIMS:
(1) An electrochemical process for measuring alkaline and/or acid phosphatase activity in biological fluid samples such as serum or other fluids or tissues brought into solution; consisting of allowing the enzyme to react under appropriate conditions i.e. alkaline pH with optimum near pH 10, with magnesium ion concentration near 1 x 10-4 moles/liter, and at a temperature between 20 to 40 degrees centrigrade, with 25, 27, 30, 35, or 37 C most often selected ... with an aromatic phosphate ester reagent containing a group selected from the group consisting of nitro groups or amino groups and monitoring the chemical reaction at electrodes which measure the current produced by the reduction or oxidation respectively of the selected functional group attached to the product and/or substrate.
(2) An electrochemical process to measure alkaline and/or acid phosphatase activity in biological fluid samples like serum, or other fluids or tissues brought into solution, whereby the enzyme is allowed to react under appropriate conditions with an aromatic phosphate ester reagent con-taining a nitro group, and the chemical reaction is monitored at electrodes which measure the current produced by the reduction of the nitro group attached to the product and/or substrate.
(3) An electrochemical process to measure alkaline and/or acid phosphatase activity in biological fluid samples like serum, or other fluids or tissues brought into solution, whereby the enzyme is allowed to react under appropriate conditions with an aromatic phosphate ester reagent con-taining an amino group, and the chemical reaction is monitored at electrodes which measure the current produced by the oxidation of the amino group attached to the product and/or substrate.