Note: Descriptions are shown in the official language in which they were submitted.
--1--
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
--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
--4--
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
--5--
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
-7--
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.
L3
-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.
._ ..
- 12
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
- 13
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).
-14-
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.
L3
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
-16-
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~
-21-
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
-23-
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
L3
-24-
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).
-25-
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.
-26-
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
-27-
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
-28-
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
-29-
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
-30-
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).
