KineticMethodfor DeterminingAcidPhosphatase
Activityin Serumwith Useof the “CentrifiChem”
Diane L. Fabiny-Byrd and Gerhard Ertingshausen
Acid phosphatase activity is determined by splitting 1-naphthyl phosphate, concurrently diazotizing the released 1-naphthol with Fast Red TR,
and measuring the resulting color. The test is performed in the presence and absence of tartrate.
Reaction rates can be continuously monitored,
and their difference is proportional to acid phosphatase activity that is inhibited by tartrate. Results for sera with normal and increased acid
phosphatase activities are presented and three
different methods for acid phosphatase are compared. The kinetic blank used in the reaction
eliminates all nonenzymatic contributions to substrate splitting.
The ideal test for the determination of a serum
enzyme should involve a zero-order reaction permitting continuous and direct measurement of one
of the released reaction products. In all of the existing methods for measuring acid phosphatase activity in which the substrate is phenyl phosphate
(1), 1-naphthyl phosphate (2), phenolphthalein
phosphate (3), p-nitrophenyl phosphate (4), 2glycerophosphate (5), or thymolphthalein
phosphate (6), a second step is required to generate a
chromophore after the enzyme reaction has proceeded under controlled conditions of temperature
and time. The rate of the reaction cannot be
continuously followed.
2,4-Dinitrophenyl
phosphate is hydrolyzed by
Escherichia
ccli phosphatases at pH 5.5-6.8, and
the released 2,4-dinitrophenol can be directly measured at 400 nm because it is dissociated in this pH
range (7). 2,4-Dinitrophenyl phosphate reacts with
serum acid phosphatase, but we found the substrate to be too unstable for clinical use. Hillmann
(8) recently suggested a method with 1-naphthyl
phosphate as substrate and concurrent diazotation
of the released 1-naphthol. This reaction is possible
because the diazo reagent, which is based on 2amino-5-chloro-toluene, does not react with bilirubin under the conditions of the test. In addition,
effects of side reactions are eliminated by process-
From
10591.
the Union
Carbide
Research
Institute,
Tarrytown,
N.Y.
ing a sample in the presenceand absence of tartrate
and determining the differential
rate.
This preliminary
report shows that the method
was found to be adaptable to the “CentrifiChem.”
We investigated
the constancy of the absorbance
change up to absorbances greater than 2.0, established the linearity
of the differential
reaction
rate vs. various enzyme concentrations,
and established precision and correlation
data for the
method. An in-depth clinical evaluation
is under
way.
Materials and Methods
Instrumentation
A CentrifiChem
system (Biomedical
Products
and Research, Union Carbide Corp., Tarrytown,
N.Y. 10591) as described previously (9) is used for
the test, at 405 nm.
The reference methods were performed
on a
Model 300-N spectrophotometer
(Gilford Instrument Labs., Oberlin, Ohio 44074).
Reagents
“Tween-80”
solution.
Stir 30 ml of Tween-80
(Polyoxyethylene
[20]sorbitanmonooleate, Fisher
Chemical
Co., Fair Lawn, N.J. 07410) with 70 ml
of water until the solution is homogeneous.
i,-Tartaric
acid (Sigma Chemical
Co., St. Louis,
Mo. 63178.)
Citrate buffer 1, 0.25 mol/liter,
pH 5.2. Dissolve
52.5 g citric acid monohydrate
(Fisher Chemical
Co.) in 800 ml of distilled water. Add 16.7 ml of the
above Tween-80 solution and adjust the pH to
5.2 with NaOH solution (30 g/100 ml). Add water
to 1 liter.
Citrate buffer 2, 0.25 mol/liter,
pH 5.2. Dissolve
52.5 g of citric acid monohydrate
and 7.5 g of Ltartaric acid in 800 ml of 1120 and proceed as for
citrate buffer 1.
1-Naphthyl
phosphate,
sodium
salt (Sigma).
Purify by dissolving 5 g in 50 ml of distilled water
and extract three times with 50 ml of benzene.
Discard the benzene layers, filter the water layer,
and evaporate the water on a rotary evaporator.
Sodium
nitrite (Fisher).
2-Amino-5-chloro-toluene
(4-chloro-2-methyl
aniCLINICAL CHEMISTRY, Vol. 18, No. 8, 1972 841
line) (Aldrich Chemical Co., Cedar Knolls, N.J.
07927). Purify by dissolving
100 g in 20 ml of
methylene
chloride, treating
with Norit A, filtering, and evaporating
the methylene chloride on a
rotary evaporator.
“Ultravon
JG”
(CIBA-Geigy,
Summit,
N.J.
07901). Dispersing agent.
Naphthalene-1
,5-disulfonic
acid
(Pfaltz
and
Bauer, Flushing, N.Y. 11368). Purify by dissolving
22 g in 45 ml of water, treating with Norit A decolonizing carbon (Fisher) and filtering.
Use the
filtrate in the preparation of Fast Red TR.
Fast Red TR (diazo-2-amino-5-chloro-toluene1,5-naphthalene
disulfonate).
Combine 1.8 g (0.013
mol) of purified 2-amino-5-chlorotoluene,
2 ml of
water, 0.1 ml of Ultravon,
and 5 ml of coned
HC1, and stir in an ice bath to obtain a finely
divided slurry. Then add 10 g of ice. When the
temperature
has decreased to 0#{176}C,
rapidly
add
(under the surface of the reaction mixture) a solution of 1 g of NaNO2 (0.014 mol) in 2 ml of water.
The temperature
will increase to 5#{176}-7#{176}C.
After
stirring for 20 mm, filter the reaction mixture. To
the filtrate, add dropwise a filtered solution of 3.8
g (13 mmol) of 1,5-naphthalene
disulfonic acid in
8 ml of water. Chill in an ice bath. Filter to remove
the fluffy needle-shaped crystals which precipitate,
wash them successively with 25 ml of acetone and
of methanol, and air dry. Fast Red TR is available
commercially,
but most preparations are not pure
enough.
Working reagents (a) For total acid phosphatase,
dissolve 45 mg (0.18 mmol) of purified 1-naphthyl
phosphate and 30 mg of Fast Red TR (0.063 mmol)
in 13.1 ml of citrate buffer.
(b) For tartrate stable acid phosphatase,
use the
same concentration
of substrate but dissolve it in
buffer containing 50 mmol of tartrate per liter.
the prostatic
acid phosphatase
activity
of the
sample.
The reference method is that of Roy et al. (6),
who used sodium thymolphthalein
monophosphate.
Measurements were made at 590 nm on the Model
300-N
spectrophotometer
(Gilford
Instrument
Labs, Oberlin, Ohio 44074).
Results and Discussion
We first investigated
the linearity
of a plot of
absorbance vs. reaction time for the total reaction
at 30#{176}C
(Figure 1). After about 8 mm, the reaction
was linear, and the reaction rate remained constant for at least 30 mm when normal sera were
chosen.
A blank rate for the reagent alone was, however,
observed and had to be eliminated.
Following
Hillmann’s
(8) suggestion, we decided to design a
test that is specific for the fraction of the various
acid phosphatase isoenzymes that is inhibited
by
tartrate.
This approach not only enhances the
specificity
of the method for prostatic acid phosphatase but at the same time provides a blank rate,
which is subtracted from the total rate.
Typical rate curves for a number of samples with
normal
acid phosphatase
activity
(Figure
2).
clearly demonstrate
the need for a blank rate.
Whereas both the total and tartrate-inhibited
rate
for serum C are higher than for serum D, the difference between the rates is smaller for serum C.
The reverse is true for sera A and B. The total and
refractory
(i.e., tartrate-uninhibited)
rates for
2.0
Procedure
E
Each sample is analyzed with both working solutions. For both tests, 40 el of sample and 50 cl diluent are placed in the sample cavities and 350 cl
0
C.,
z
.0
0
0
0
reagent in the reagent cavities of the transfer disk.
The reference position (0) contains 350 il of the
respective reagent and 50 cl of diluent. For the
tests, the wavelength is 405 nm and the temperature 30#{176}C.
For both tests, the total absorbance
change between 8 mm and 16 mm is used to determine the acid phosphatase activity of the samples
(in U/liter).
The absorptivity
of the reaction product is 12.9 cm2/cmol and the conversion factor,
corrected for an 8-mm time interval, is 107.
The acid phosphatase
activity
inhibited
by
tartrate is the difference between the two results.
Since prostatic
acid phosphatase is inhibited
by
tartrate while acid phosphatases from other organs
are much less affected, this difference represents
842 CLINICAL CHEMISTRY, Vol. 18, No. 8, 1972
1.0
0
#{149}0
0
4
16
TINE, mm
Fig. 1. Total acid phosphatase activity.
changes with time for dilutions of Enzatrol
-0---
AbsorbancE
31.8 U/liter, undiluted;
-023.4 U/liter, 3/4 dilution
16.4 U/liter, 1/2 dilution; -#{149}- 8.2 U/liter, 1/4 dIlution
-#{149}3.2 U/liter, 1/10 dilution. The Enzatrol was reconstituted
with 2 ml of water instead of 3 ml
.5
Table 1. Precision of the Method
.4
WIthin-run
precIsion, 25
samples
.3
.2
SERUM C
__TOTAL
Mean ±
SD, U/liter
CV, %
SERUMC TARTRATE
.1
r
Serum pool
Enzatrol’
15.8
0.873
1.40
0.109
7.80
5.52
SERUMDTARTRATE
Day-to-day precision
Enzatrol
.5
Mean ±
SD, U/liter
.4
.3
CV, %
TOTAL SERUM A
SERUM A
.2
IARTRATE
SERUM B
ERUM
0
Lot No. ET236D, 21.5 U/liter by the Bodansky method. Values
vary considerably
B
.1
14.1
0.843
5.98
from method to method.
TA RT BAT E
4
B
12
16
20
24
28
.090
32
TIME, mm
E
Fig. 2. Total and refractory acid phosphatase
rates for
A,
normal sera
0
E
.0
.060
I
a
I0
SERUM
z
F
NO TARTRATE
w
I-
ADDED
.030
.0
0
I-
.0
ISERUM
TARTRATE
ADDED
6
0
SERUM
11
C
L
1
ND TARTRATE
ADDED
C
0
2
SERUM
m
TARTRATE
C
2
F
1
4
DILUTION (ACTIVITY)
ADDED
Fig. 4. Differential acid phosphatase. Linearity of rates
and activity for dilutions of Enzatrol and a serum from a
patient
.5
-0-
with elevated
=
4
8
12
16
lIME.
20
24
28
32
mm
Fig. 3. Total and refractory acid phosphatase rates for
high-activity sera
some samples with high acid phosphatase activity
are shown in Figure 3; some of them showed constant reaction
rates until absorbances
slightly
greater than 2.0 were reached.
The differential
rates per minute (total minus
efractory
rate) for serial dilutions
of the control
erum, “Enzatrol,”
and a serum from a patient
vith elevated prostatic acid phosphatase are plotted
n Figure 4, and demonstrate
the linearity
of the
ates with respect to enzyme concentration.
The within-run
precision of the method was
[etermined by simultaneous analysis of 25 aliquots
Day-to-day
acid phosphatase
Serum with elevated activity; --
of a normal
0
prostatic
sample
and of Enzatrol
=
activity
Enzatrol
(Table
1).
precision was studied over two weeks
with Enzatrol (Table 1).
The normal range was found to be 0-.9 U/liter
when fasting samples from 25 healthy males between the ages of 30 and 60 years were analyzed.
Samples with high activity were compared (Table
2). Good correlation
was obtained between
the
CentnifiChem
method and both the 2-glycerophosphate and the thymolphthalein
monophosphate
methods.
The tartrate-inhibited
rate is not completely
specific for prostatic
acid phosphatase
activity
with any of the suggested substrates as Roy et al.
demonstrated
(6). 1-Naphthyl
phosphate, which
has been strongly criticized
(10) as not being specific for prostatic acid phosphatase as was claimed,
showed by far the lowest relative sensitivity
to
erythrocytic
and platelet acid phosphatases of six
CLINICAL CHEMISTRY, Vol. 18, No. 8, 1972 843
substrates when compared with thymolphthalein
Table 2. Comparison of Results with
Three Acid Phosphatase Methods
Serum
1
2
3
Centrif IChem
substrate:
1-naphthyl
phosphate
U/liter
30C
14
15
16
17
18
23.1
19.9
10.2
25.2
10.5
43.6
38.7
9.6
403
21.4
5.3
247
480
10.0
98.0
6.1
39.5
15.0
19
20
3.1
14.5
21
22
23
Enzatrol
(control
4.3
3.9
2.2
12.4
4
5
6
7
8
9
10
11
12
13
Substrate:D
2-glycerophosphate
U/liter 37C
7.4
6.3
2.8
8.0
3.9
11.3
12.1
2.9
43.3
7.2
2.3
36.0
63.1
3.0
25.3
2.1
11.4
nac
na
na
na
na
na
Ratio
CC/2-GP
3.11
3.16
3.66
Substrate:
(ref. 6)
thymolphthaieln
monophosphate
U/liter 37’C
2.4
6.8
2.4
Ratio
CC/TM P
9.63
2.93
4.25
8.2
3.08
4.1
14.9
12.8
2.4
256
82.0
4.91
2.90
6.3
4.7
53.4
75.0
3.3
35.1
2.2
340
1.13
4.63
6.40
3.03
2.79
2.77
3.46
12.6
3.13
3.8
.9
3.95
3.44
5.6
2.59
1.4
1.7
.7
3.7
3.07
3.15
2.69
3.86
3.21
3.35
9.31
2.97
2.30
6.86
7.61
3.33
3.87
2.93
3.02
4.03
2.29
3.14
serum)
For CentrifiChem substrate (X) compared to 2-glycerophosphate (V):5 r = .995, Y = .253X + io
For CentrifiChem
substrate (X) compared to thymolphthalein
monophosphate (V):5 r = .975, V = .337XResults obtained at Memorial Hospital for Cancer and Allied
Diseases, New York, N.Y.
Enzatrol and samples 9, 12, and 13 were not Included in the
correlation data.
na = not applicable.
phosphate.
The presented method is the only one providing
a “kinetic
blank,”
thus eliminating
all contnibutions to substrate splitting
that are not of enzymatic origin.
This should further
decrease the
incidence of false-positive
results that will continue to occur as long as no substrate specific for
prostatic acid phosphatase is available.
We thank Dr. Morton K. Schwartz
pathologic sera.
CLINICAL CHEMISTRY, Vol. 18, No. 8, 1972
providing
analyzed
References
1. Gutman, E. B., and Gutman, A. B., Estimation of “acid”
phosphatase activityof blood serum. J. Biol. Chem. 136, 201
(1970).
2. Babson, A. L., and Read, P. A., A new assay for prostatic
acidphosphataseinserum. Amer. J. Clin. Pathol. 32,88 (1959).
3. Huggins, C., and Tallay, P., Sodium phenolphthalein
phosphate
as a substratefor phosphatase tests.
J. Biol. Chem. 159,
399 (1945).
4. Huson,
P. B., Brendler,
H., and Scott, W. W., A simple
method forthe determinationofserum acidphosphatase.J. Urol.
58,89(1947).
5. Bodansky,
A., Phosphatase
studies.
II. Determination
of
serum phosphatase.
Factors influencing the accuracy of the determination..1. Biol. Chem. 101, 93 (1933).
6. Roy, A. V., Brower, M. E., and Hayden, J. E., Sodium thymolphthalein
monophosphate:
A new acid phosphatase substratewith greaterspecificity
forthe prostaticenzyme in serum.
CLIN. CHEM. 17, 1093 (1971).
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with alkaline phosphatase
from
Escherichia coti. Biochem. J. 106, 455 (1968).
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G., Fortlaufende
photometrische
Messung der
sauren Phosphatase-Aktivitht.
Z. Kim. Chem. Kim. Biochem.
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creatinine
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CentrifiChem. CLUE. CHEM. 17, 696 (1971).
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844
for