Note: Descriptions are shown in the official language in which they were submitted.
~()70597
The present invention relates to a method for the
determination of acid phosphatase. More particularly, the
invention is concerned with an improved method for the
~ determination of acid phosphatase of human origin using a
reagent for activating the acid phosphatase, with said reagent
per se and with a reagent kit containing said reagent.
Acid phosphatase is an enzyme which can optimally split
phospho-monoesters in the acid pH range. Acid phosphatase of
human origin is found in various organs (e.g. in the prostate
gland, the liver and the spleen) and in ~lood cells, and is
transferred to the serum when cells are damaged. The
determination of acid phosphatase in body fluids, ln particular
ln serum or plasma, thus plays an important part ln the
diagnosls of dlseases of the organs concerned.
The activity of acid phosphatase in serum or plasma
increases considerably if a carcinoma is present in the
prostate gland and in the case of hypertrophy of the prostate
gland and the determination of prostate gland phosphatase
activity in these body fluids enables the diagnosis of these
specific diseases to be made.
It has now been found in accordance with the present
invention that the activity of acid phosphatase of human origin
can be considerably increased by adding a straight-chain
alcohol containing 4 to 6 carbon atoms to the body fluid.
~en/14.10.1976 - 2 -
1070597
Since the activity of acid phosphatase of human origin in body
fluids is relatively low, increasing this activity is a definite
advance in the state of the art with respect to the determination
of this important enzyme.
The present invention is based on the foregoing finding
and accordingly relates to an improved metho~ for the
determination of acid phosphatase using an activator consisting
of a straight-chain alcohol containing 4 to 6 carbon atoms.
More precisely, the present invention is concerned with
a method for the determination of acid phosphatase of human
origin in a body fluid, which method comprises subjecting a
solution containing, as the substrate, 50 ~mols/litres to
50 mmols/litres of a phospho-monoester, 50 to 300 mmols/litres
of a buffer substance to adjust the pH value to between 4.5
and 6.5 and 10 to 300 mmols/litres of an activator consisting of
at least one straight-chain alcohol containing 4 to 6 carbon
atoms to the phosphatase action of the body fluid under
lnvestigation at an incubation temperature between 20C and
45C and measuring the substrate conversion.
The phospho-monoester used as the substrate in the
enzymatic reaction has the general formula
R- 0 -P03H2
in which R represents any desired organic
group.
R preferably represents a chromophoric group (e.g. the
4-nitrophenyl, thymolphthalein, phenolphthalein or 2-chloro-4-
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-nitrophenyl group) or a fluorophoric group (e.g. the l-naphthyl
- group). R can also represent the adenosine, glycerine or
phenyl group.
The substrate is preferably employed at an optimum
concentration which is different for each individual substrate
and is also influenced by the pH value. The optimum substrate
concentration which effects the maximum enzyme activity possible
- at a given pH value can be determined from a substrate activity
curve.
The substrate activity curve for a given substrate is
obtained by measuring the enzyme activity at various substrate
concentrations under otherwise identical conditions.
In general, the optimum substrate concentration for acid
phosphatase is between 50 ~mols/litresand 50 mmols/litres,
preferably between 60 ~mols/litresand 25 mmols/litres. Thus,
at a pH value of 5.5 the optimum substrate concentration is
approximately 25 mmols/litresfor ~-glycerine phosphate, 2.5
mmols/litres for 4-nitrophenyl phosphate and 100 ~mols/litres
for phenolphthalein diphosphate.
According to the present method, the p~ value is
adjusted to between 4.5 and 6.5, preferably between 5 and 6,
using a buffer substance. Examples of suitable buffer
substances are citrate buffer and acetate buffer. The buffer
substance concentration is generally between 50 mmols/litres
and 300 mmols/litres and is preferably 100 mmols/litres.
1070597
According to the present method, the activator used
consists of a straight-chain alcohol containing 4 to 6 carbon
atoms, preferably n-butanol, n-pentanol or 1,5-pentanediol.
l-Pentanol and 2-pentanol are particularly preferred.
The concentration of the activator is generally 10
mmols/litresto 300 mmols/litres,preferably leO mmols/litres to
200 mmols/litres.
The activator can consist of one or more straight-chain
alcohol containing 4 to 6 carbon atoms, but preferably consists
of a single, straight-chain alcohol of this type.
A sample of the body fluid under investigation is mixed
with the test solution and the enzymatic reaction is then
carried out by incubating the solution at a temperature between
20C and 40~C, preferably between 30C and 37C. The
lncubation period is generally 5 to 60 minutes, but the
reaction is preferably stopped a~ter 30 minutes by adding a
suitable substance (e.g. sodium hydroxide solution or trisodium
phosphate solution).
By choosing a suitable substrate (e.g. 2-chloro-4-nitro-
phenyl phosphate) the enzymatic reaction can also be measured
kinetically.
The substrate conversion is a measure of the enzyme
activity. It is measured by determining the decrease in the
phospho-monoester concentration or the increase in the
concentration of the organic radical liberated. Since the
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activation of acid phosphatase is effected by a transphos-
phorylation reaction, i.e. a phosphate transfer from the
phospho-monoester used to the alcohol used as the activator,
the amount of free phosphate after the enzymatic reaction does
not correspond to the substrate conversion and accordingly
cannot be ta~en as a measure thereof.
The type of measurement depends on the substrate used.
If 4-nitro-phenyl phosphate, thymolphthalein phosphate,
phenolphthalein phosphate or naphthyl phosphate is used as the
substrate, the ~ncrease in the concentration of the organic
radical liberated can be determ~ned directly photometrically
or fluorimetrically after suitably stopping the enzymatic
reaction.
If other substrates (e.g. B-glycerine phosphate, phenyl
lS phosphate or adenosine monophosphate) are used, the measurement
of the substrate conversion is carried out by chemically or
enzymatically converting the organic radical liberated into a
substance, the concentration of which can be determined
photometrically. For example, if phenyl phosphate $s used,
the phenol liberated is converted into a blue dyestuff using a
phenol reagent ~Folin-Ciocalteus~ Merc~ 90013.
The present method ls suitable for both manual and
automatic determinations of acid phosphatase. Examples of
substances on which thé determination can be carried out are
serum, plasma, blood, fluid and urine.
Additives suitable for diagnostic purposes ~e.~. detergents
such as Brij-3S (polyoxyethylene lauryl ether), or stabilisers,
~Trade Mar~
B - 6 - -
1070597
such as magnesium ions] can be added to the phospho-monoesters,
buffers and activators.
The reagents are preferably packed in a reagent kit. A
reagent kit according to the present invention preferably
comprises, in one or more containers,
a) a phospho-monoester,
b) a buffer substance to adjust the pH value to
between 4.5 and 6.5 and
c) a straight-chain alcohol containing 4 to 6
carbon atoms as an activator.
The reagent kit can additionally comprise, in a container,
a control solution or standard solution and/or, in a container,
a reagent for stopping the reaction and/or, in a container a
reagent for the conversion of the organic radical liberated
and/or, in a container, the additives mentioned hereinbefore
whlch are suitable for dlagnostic purposes (e.g. detergents
and stabilisers).
However, it is not necessary to store the reagents and
additlves in separate containers. The present invention i5
also concerned with a reagent kit in which some of the
components necessary for carrying out the method of the
invention are stored together in the same container. ~or
e~ample the buffer substance and the activator can be stored
together in the dissolved form in a single container.
With the exception of the activator which is always in
the liquid form, the various reagents and additives can be
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packed both in the liquid and in the solid form (e.g. as a
. powder, granules, tablets or a lyophilisate).
....
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1070597
The following Examples illustrate the present invention:
ExamPle 1
0.1 ml of acid prostate gland phosphatase solution
(isolated by the method described by Lam et al., Clin. Chem.
19 483, 1~73) is mixed with 2.0 ml of a substrate buffer
solution consisting of 0 1 mol/litre of citrate huffer (pH
5.25), 5 g/litresof Brij-35, 5 mmols/litresof 4-nitro-phenyl
phosphate and various concentrations of l-pentanol and the
mixture is incubated at 37C for 30 minutes. The reaction is
stopped by adding 1.O ml of l-N sodium hydroxide solution and
the 4-nitro-phenolate liberated is determined photometrically
at a wave-length o 405 nm.
The results are given in Table I in % of the prostate
gland phosphatase activity at a l-pentanol concentration of
zero.
Ta~le I
.
l-Pentanol concentration Activity of the prostate
in the test solution gland phosphatase
0 mmol/litre 100~
2050 mmols/litres 137%
100 mmols/litres 173%
150 mmolstlitres 189%
200 mmols/ll~res 139%
1~7~)597
Example 2
The method described in Example 1 hereinbefore is
repeated using 2-pentanol in place of l-pentanol. The results
are given analogously in Table II.
Table II
2-Pentanol concentration Activity of the prostate
in the test solution gland phosphatase
_ . _ . .
0 mmol/litre 100%
_ 50 mmols/litres 138%
100 mmols/litres 172%
150 mmols/litres 190%
200 mmols/litres 139
Exam~le 3
The method described in Example 1 hereinbefore is
repeated using 1,5-pentaned~ol in place of l-pentanol. The
results are given analogously in Table III.
Table III
l,S-Pentanediol concentration Activity of the prostate
in the test solution gland phosphatase
o mmolJlitre 100%
50 mmols/litres 120%
100 mmols/litres 131~
150 mmols/litres 140%
200 mmols/litres 148%
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1~'70597
Example 4
The method described in Example l hereinbefore is
repeated using l-butanol in place of l-pentanol. The results
are given analogously in Table IV.
Table IV
l-Butanol concentration Activity of the prostate
in the test solution gland phosphatase
_ __
0 mmol/litre lO0~
50 mmols/litres 119%
10100 mmols/litres 131%
150 mmols/litres 131%
200 mmols/litres 116%
-
Example 5
The method described in Example 1 hereinbefore is
repeated using l-hexanol in place of l-pentanol. The results
are given analogously in Table V.
Table V
l-Hexanol concentration Activity of the prostate
in the test solution gland phosphatase
_ .__
O mmol/litre 100%
25 mmols/litres 107%
50 mmols/litres 114%
lO0 mmols/litres 120%
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1070597
Example 6
.
0.05 ml of prostate gland phosphatase solution is mixed
with 2.0 ml of a substrate buffer solution consisting of
0.1 mol/litre of acetate buffer (pH 5.5), 62 ~mols/litres of
S phenolphthalein diphosphate and various concentrations of
l-pentanol and the mixture is incubated at 37C for 15 minutes.
In order to stop the reaction and to develope the colour,
2.0 ml of 0.4 mol/litre sod~um phosphate buffer (pH-10) are
admixed and the colour intensity is measured at a wave-length
of 546 nm.
The results are given in Table VI in % of the prostate
gland phosphatase activity at a l-pentanol concentration of
zero.
i
Table VI
. __
l-Pentanol concentration Activity of the prostate
in the test solution gland phosphatase
.
0 mmol/litre 100%
So mmols/litres 154%
100 mmols/litres 174%
20150 mmols/litres 177%
200 mmols/litres 131%
Example 7
0.1 ml of erythrocyte phosphatase is mixed with 2.0 ml
of a substrate buffer solution consisting of 0.1 mol/litre of
acetate buffer (pH 5.5), 5 mmols/litresof 4-nitrophenyl
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1070597
phosphate, 5 g/litres ofBrij-35 and various concentrations of
l-pentanol. After 30 minutes incubation at 37C, the reaction
is stopped by adding 1.O ml of l-N sodium hydroxide solution
and the 4-nitrophenolate liberated is determined photometrically
S at a wavelength of 405 nm.
The results are given in Table VII in ~ of the erythrocyte
phosphatase activity at a l-pentanol concentration of ~ero.
Table VII
. ~_ _ _ . _ . . _
l-Pentanol concentration Activity of the erythrocyte
10in the test solution phosphatase
.
0 mmol/litre 100%
50 mmols/litres 132
100 mmols/litres 153~
150 mmols/litres 162%
lS 200 mmols/litres 136
Example 8
0.1 ml of serum is mixed with 0.5 ml of a substrate
buffer solution con~isting of 0.1 mol/litre of citrate buffer
(pH 5.5) and 5 mmols/litres of 4-nitrophenyl phosphate. After
30 minutes incubation at 37C, the reaction is stopped by
add~ng 2.5 ml of 0.1-N sodium hydroxide solution and the 4-
-nitrophenolate libera,ted is determined photometrically at a
wavelength of 405 nm. This determination of the activity of
acid phosphatase is ~arried out once in the absence of l-pentanol
and once in the presence of 150 mmols/litres of 1-pentanol.
9 r ~ K
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107059~
The results are given in Table VIII.
Table VIII
_ .
Sam le Activity without Activity with 150 mmols/litres
S P l-pentanol (U/l) of l-pentanol (U/l)
.
Serum 1 1.12 2.10
Serum 2 16.63 24.55
Serum 3 18.41 31.53
Serum 4 20.65 33.5~
.
Example 9
0.1 ml of prostate gland phosphatase solution is mixed
with 2.0 ml of a substrate buffer solutio~ consisting of
0.1 mol/lltre of acetate buffer (pH 5.5) and various concen-
trations of phenyl phosphate. After 5 minutes incubation at
37C, 0.5 ml of phenol reagent [Folin-Ciocalteus] and 1.0 ml
of 20% strength sodium carbonate solution are p~petted in and
the mixture is lncubated for a further 10 minutes at 37~C.
The intensity of the blue dye, which corresponds to the
concentration of phenol liberated, is measured at a wavelength
of 578 nm. These determinations are carried out once in the
absence of l-pentanol and once,;in the presence of 150 mmols/
litres of l-pentanol.
The results are given in Table IX.
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1070597
Table IX
_ ~ E
Phenyl phosphate 578 nm/5 Min. ~E578 nm/5 min. in the
concentration in in the absence of presence of 150 mmols/litres
the test solution l-pentanolof l-pentanol
_ _ _ . _ _ _
O.078 mmol/litre 0.1050.169
0.155 mmol/litre 0.1500.280
0.312 mmol/litre 0.1980.390
0.625 mmol/litre 0.2450.490
1.25 mmols/litres 0.2750.550
2.50 mmols/litres 0.280O.S90
5.00 mmols/litres 0.2800.590
ExamPle 10
0.05 ml of purified prostate gland phosphatase solution
is mixed with 1.0 ml of a substrate buffer solution consisting
of 0.1 mol/litre of acetate buffer (p~ 5.5), 0.5 mmol/litre of
phenolphthalein monophosphate and various concentrations of
l-pentanol. After 10 minutes incubation at 37~C, the reaction
is stopped by adding 2.0 ml of a 0.4 mol/litre sodium phosphate
buffer solution (pH 10~ and the phenolphthalein liberated is
determined photometrically at a wavelength of 546 nm.
The results are given in Table X in % of the prostate
gland phosphatase activity at a l-pentanol concentration of
zero.
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1070597
~ Table X
.~ .
l-Pentanol concentration ~ctivity of the prostate
in the test solution gland phosphatase
o mmol/litre 100%
50 mmols/litres 125%
100 mmols/litres 135%
150 mmols/litres 1~0%
Example 11
0.05 ml of purified prostate gland phosphatase solution
is mixed with 1.0 ml of a substrate buffer solution consisting
of O.l~mol/litre of citrate buffer (pH 5.75), 1.2 mmols/litres
of thymolphthalein phosphate, S g/litres of Bri~-35 and
various concentrations of l-pentanol. After 10 minutes
incubation at 37~C, the react~on is stopped by adding 2.0 ml
of a 0.2 mol/litre bicarbonate buffer solution ~pH 10) and the
thymolphthalein liberated is determined photometrically at a
wavelength of 578 nm.
The results are given in Table XI in ~ of the prostate
gland phosphatase activity at a l-pentanol concentration of
zero.
Table XI
.~ _ .
1-Pentanol concentration Activity of the prostate
in the test solution gland phosphatase
O mmol/litre 100%
50 mmols/litres 117%
100 mmols/litres 125%
150 mmols/litres 140%
