JP3146692B2 - Alkaline phosphatase assay reagent and assay method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for measuring alkaline phosphatase (hereinafter abbreviated as ALP) in a biological sample such as blood or urine, and a measuring method using the same.

[0002]

BACKGROUND OF THE INVENTION ALP activity measurement has a long history, and several types of substrates have been proposed so far. Phenylphosphate (Kind-King method and the like) and p-nitrophenylphosphate (hereinafter referred to as N
Abbreviated as PP. ) (Bessey Lowry method, etc.) is generally widely used.

[0003] It is said that the most average activity value is obtained when phenyl phosphate is used as a substrate. Is often used as a substrate, for example, as recommended by the Japanese Society of Clinical Chemistry (hereinafter referred to as JSC).
Abbreviated as C's recommendation law. ), The International Clinical Chemistry Union Recommendation Act (hereinafter abbreviated as the IFCC Recommendation Act), the German Clinical Chemistry Recommendation Act (hereinafter abbreviated as the GSCC Recommendation Act).
NPP is also used as a substrate in the standard recommendation method of each country and international organizations.

[0004] However, the stability of this NPP as an aqueous solution is not always good. That is, when an aqueous solution of NPP is stored at 10 ° C., the absorbance of the solution after 6 months is NP
0.15 per 10 mM of P (OD410nm, absorption of decomposition product P-nitrophenol), 0.25% when stored at 15 ° C
Extend to Therefore, the solution containing NPP had a shelf life of about 6 months even when strictly refrigerated.

On the other hand, as an attempt to improve the instability, JP-A-54-12796 discloses a method in which imidazole or the like is added as a substrate stabilizer. However, imidazole has the effect of inhibiting ALP activity and decreasing the measured value, so that this method is hardly preferable.

[0006] Therefore, there is a long-awaited need for a new ALP test solution that can ensure long-term stability in a solution state, measure a rate, and obtain good correlation with various recommended methods. is there.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and has as its object to provide a reagent solution for ALP measurement capable of measuring a rate and having excellent stability, and a measuring method using the same. I do.

[0008]

The present invention relates to a compound represented by the general formula [I]:

Embedded image (Wherein, R represents an alkyl group). A reagent solution for measuring alkaline phosphatase, characterized by using p-acylphenyl phosphate or / and a salt thereof represented by the formula: It is an invention of a method for measuring alkaline phosphatase, which is characterized by the following.

That is, the present inventors have conducted intensive studies in order to find a substrate agent which can secure long-term stability in a solution state, can measure a rate, and can obtain a good correlation with various recommended methods. The present inventors have found that the p-acylphenyl phosphate represented by the general formula [I] is suitable for the purpose, and have completed the present invention.

Examples of R in the general formula [I] include an alkyl group such as a methyl group, an ethyl group, a propyl group, and a butyl group (which may be linear or branched). . Specific examples of the p-acylphenyl phosphate represented by the general formula [I] include, for example, p-acetylphenyl phosphate,
Examples include propionyl phenyl phosphate, p-butyryl phenyl phosphate, p-isobutyryl phenyl phosphate, p-valeryl phenyl phosphate, and p-isovaleryl phenyl phosphate. These can be easily synthesized by a conventional method (for example, the method described in J. Chem. Soc 1966, 227.).

The p-acylphenyl phosphate represented by the general formula [I] includes, for example, a primary sodium salt and a secondary sodium salt.
Examples thereof include alkali metal salts such as sodium salt, primary potassium salt and secondary potassium salt, ammonium salts such as primary ammonium salt and secondary ammonium salt, and organic amine salts such as p-toluidine salt and cyclohexylamine salt. These are preferred over the acid state because they are stable for long periods in the powder state.

The reagent solution for ALP measurement of the present invention can be prepared either as a single solution containing all the necessary components or as a combined reagent solution in which the necessary components are dispersed in two or more solutions. However, considering the stability of the test solution and the adaptability to an automatic analyzer, it is desirable to use a combined test solution consisting of two solutions.

In the case where the reagent solution for ALP measurement of the present invention is a combined reagent solution comprising two solutions, for example, a substrate solution containing p-acylphenylphosphate represented by the general formula [I] or / and a salt thereof, For example, a buffer containing an activator of ALP such as magnesium chloride or the like, for example, 2-ethylaminoethanol (hereinafter abbreviated as EAE), diethanolamine (hereinafter abbreviated as DEA), N-methyl-D-glucamine (hereinafter abbreviated) , MEG)), 2-amino-2-methyl-1-
It is desirable to use two solutions, such as a buffer solution such as propanol (hereinafter abbreviated as AMP).

The substrate solution may contain a suitable buffer (eg, carbonate, AMP, etc.) in addition to the p-acylphenyl phosphate represented by the general formula [I]. When the concentration is increased, the stability of the substrate solution may be reduced. Therefore, it is desirable that the working concentration be 100 mM or less.

In the test solution for ALP measurement of the present invention, the concentration of p-acylphenyl phosphate represented by the general formula [I] is usually from 3 to 10 mM as a final concentration at the time of ALP activity measurement. Selected.

The pH of the substrate solution is usually between pH 8 and 12.
Is adjusted to the range.

Since the activity of ALP varies greatly depending on the type of buffer, the activity value obtained using the reagent solution for ALP measurement of the present invention has a good correlation with that obtained by a specific recommended method. In order to achieve this, it is desirable that the buffer of the buffer containing the above-mentioned activator be the same as the buffer of the intended recommendation method. By the way, JS
According to the CC's recommended method, EAE, which has the highest activity and the highest correlation with the assay using phenylphosphate as a substrate,
According to CC's recommended method, AMP with little change in activity at molar concentration
However, in the GSCC recommendation method, DEA having high stability and high activity is used, so that the buffer of the buffer containing the above-described activator may be appropriately selected according to these.

Also, it has been reported that the correlation with a measurement method using phenylphosphate as a substrate is also better than that of the JSCC recommended method when a 0.35 M MEG buffer is used [Ochi et al. Method (5th Summer Seminar Program of the Japanese Society of Clinical Chemistry, Collection of Materials)], when it is desired to obtain an activity value showing a good correlation with the ALP measurement method using MEG as a buffer, use MEG as a buffer. May be used.

In the buffer containing the above-mentioned activator, the concentration of the buffer used is, for example, from 0.5 to 1.5 M when EAE, DEA or the like is used.
When MP or the like is used, it is appropriately selected from the range of 0.1 to 0.5 M in accordance with the above-mentioned recommendation method.

The pH of the buffer containing the above-mentioned activator is usually adjusted appropriately from pH 9 to 11 in accordance with the above-mentioned recommendation method.

If necessary, the above-mentioned substrate solution or buffer may contain, if necessary, a preservative, a surfactant such as a stabilizer such as β-thiodiglycol or thiodiglycolic acid, which is generally used in this field. And the like, and the use concentration thereof may be appropriately selected from the concentration range usually used in this field.

Using the reagent solution for ALP measurement of the present invention, AL
When measuring P, use the ALP of the present invention as a measurement reagent solution.
GSCC recommendation method, IFCC recommendation method, except that the measurement wavelength (main wavelength in the case of two-wavelength measurement) is set near the maximum wavelength of p-acylphenol released by the enzymatic action of ALP using a test solution for measurement. It is sufficient to carry out the measurement in accordance with the operation method such as the method recommended by JSCC, the method of Ochi et al.

The activity value obtained by the ALP measurement method of the present invention can be used as an activity value obtained by a specific recommendation method by multiplying by an appropriate coefficient.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0025]

EXAMPLES Example 1 p-Acetylphenylphosphate (hereinafter abbreviated as APP)
Was filtered through a 0.45 μm membrane filter with a 30 mM AMP buffer solution of various pHs containing 25 mM of each as substrate solutions. Immediately after preparation and after storage at 15 ° C. for 6 and 12 months, 2.0 ml of 0.1 M carbonate buffer (p
A mixture of H10) and 0.5 ml of each substrate solution was used as a sample, and its absorbance (340 nm) was measured. The absorbance difference was obtained by subtracting the absorbance obtained for each of the substrate solutions immediately after preparation from the measured values obtained for the substrate solutions after storage at 15 ° C. for 6 months and 12 months. The result is shown in FIG.
In FIG. 1,-●-indicates the results obtained for each substrate solution stored for 6 months, and-○-indicates the results obtained for each substrate solution stored for 12 months.

[0026]

FIG.

As is clear from FIG. 1, the substrate solution according to the present invention is extremely stable in the range of pH 8 to 12, especially pH 9 to 11, and the absorbance after storage at 15 ° C. for 12 months is increased at pH 10.
It turns out that it is about 0.02. This is equivalent to about 8% of the degree of decomposition of NPP under the same conditions. In addition, a 1.25 mM EAE buffer (pH 9.9, 0.7
5mM MgCl ₂ containing. ) Was used as the first reagent (R1) and the substrate solution stored at pH 10 for 12 months was used as the second reagent (R2) to determine the ALP activity of 10 human serum samples from Hitachi 7050.
The measurement was performed using an automatic shape analyzer (Hitachi, Ltd., analysis conditions are as described in Table 1). The activity value obtained is
The newly prepared 30 mM AMP buffer (pH 10, containing 25 mM APP) was designated as R2, and the activity was the same as that obtained by performing the same operation as above using the same R1 and sample as above.
As is clear from the results, the substrate solution according to the present invention comprises:
It can be seen that even when stored at 15 ° C. for 12 months, it can be used as a test solution for ALP measurement without any problem.

[0028]

[Table 1]

Example 2 An aqueous solution (substrate solution) containing 1.25 M DEA buffer (pH 9.8, containing 0.625 mM MgCl ₂ ) prepared according to the method recommended by GSCC and containing 25 mM APP disodium salt (R1)
Was used as a second reagent (R2), and the ALP activity value of 38 human serum samples was determined using a Hitachi 7050 automatic analyzer (Hitachi, Ltd.) according to the analysis conditions in Table 1.

Reference Example 1 Measurement by GSCC Recommended Method An aqueous solution (substrate solution) containing 50 mM NPP disodium salt was used as a second reagent solution (R2), and the measurement wavelength in Table 1 was a main wavelength: 415 nm.
(Sub wavelength: 505 nm) except that the first
The activity value of each sample was determined by the same operation as in Example 2 using the test solution and the sample. FIG. 2 shows a correlation diagram obtained from the measured values of Example 2 and the measured values of Reference Example 1.

[0031]

FIG. 2

The results obtained by statistically processing the measured values obtained in Example 2 and the measured values obtained in Reference Example 1 are shown below. Regression linear equation: Y = 0.7723X-0.4998 Correlation coefficient (r): 0.9991 From the above results, the activity value obtained by the measurement method of the present invention shows a good correlation with that obtained by the GSCC recommended method. I understand.

Example 3 A first reagent (0.438 M AMP buffer (pH 10.4, containing 2.5 mM MgCl _2, 2.5 mM hydroxyethylethylenediamine triacetic acid (HEDTA), and 1.25 mM ZnSO ₄ ) prepared according to the method recommended by the IFCC) was used. R1), and an aqueous solution (substrate solution) containing 25 mM of APP sodium salt was used as a second reagent (R2).
The ALP activity values of 38 human sera were determined using a type automatic analyzer (Hitachi, Ltd.) according to the analysis conditions in Table 1.

Reference Example 2 Measurement by IFCC Recommended Method An aqueous solution (substrate solution) containing 80 mM NPP disodium salt was used as the second reagent (R2), and the measurement wavelength in Table 1 was the main wavelength: 415 nm.
(Sub wavelength: 505 nm) except that the first
The activity value of each sample was determined by the same operation as in Example 3 using the test solution and the sample. FIG. 3 shows a correlation diagram obtained from the measured values of Example 3 and the measured values of Reference Example 2.

[0035]

FIG. 3

The results obtained by statistically processing the measured values obtained in Example 3 and the measured values obtained in Reference Example 2 are shown below. Regression linear equation: Y = 0.7284X + 0.2502 Correlation coefficient (r): 0.9973 From the above results, the activity value obtained by the measurement method of the present invention shows a good correlation with that obtained by the IFCC recommended method. I understand.

Example 4 An aqueous solution containing 25 mM of APP disodium salt (substrate solution) was prepared using a 1.25 M EAE buffer (pH 9.9, containing 0.75 mM MgCl ₂ ) prepared according to the method recommended by JSCC as a first test solution (R1).
Was used as a second reagent (R2), and the ALP activity value of 38 human serum samples was determined using a Hitachi 7050 automatic analyzer (Hitachi, Ltd.) according to the analysis conditions in Table 1.

Reference Example 3 Measurement by JSCC's Recommended Method An aqueous solution (substrate solution) containing 75 mM of NPP disodium salt was used as the second reagent (R2), and the measurement wavelength in Table 1 was the main wavelength: 415 nm.
(Sub wavelength: 505 nm), except that the first
The activity value of each sample was determined by the same operation as in Example 4 using the test solution and the sample. FIG. 4 shows a correlation diagram obtained from the measured values of Example 4 and the measured values of Reference Example 3.

[0039]

FIG. 4

The results obtained by statistically processing the measured values obtained in Example 4 and the measured values obtained in Reference Example 3 are shown below. Regression linear equation: Y = 0.5931X + 1.4577 Correlation coefficient (r): 0.9967 From the above results, the activity value obtained by the measurement method of the present invention shows good correlation with that obtained by the recommended method of JSCC. I understand.

Example 5 A 0.438 M MEG buffer (p) prepared according to the method of Ochi et al.
H10.4, MgCl ₂ 0.625 mM, NaCl 100 mM) as the first reagent solution (R1), and an aqueous solution (substrate solution) containing 25 mM APP 2 sodium salt as the second reagent solution (R2). The ALP activity value of 38 human serum samples was determined using Hitachi, Ltd.) according to the analysis conditions in Table 1.

Reference Example 4 Measurement by the method of Ochi et al. An aqueous solution (substrate solution) containing 85 mM NPP disodium salt was used as the second reagent (R2), and the measurement wavelength in Table 1 was the main wavelength: 415 nm.
(Sub wavelength: 505 nm) except that the first
The activity value of each sample was determined by the same operation as in Example 5 using the test solution and the sample. FIG. 5 shows a correlation diagram obtained from the measured values of Example 5 and the measured values of Reference Example 4.

[0043]

FIG. 5

The results obtained by statistically processing the measured values obtained in Example 5 and the measured values obtained in Reference Example 4 are shown below. Regression linear equation: Y = 0.6753X-0.3947 Correlation coefficient (r): 0.9992 From the above results, the activity value obtained by the measurement method of the present invention shows a good correlation with that obtained by the method of Ochi et al. You can see that.

Reference Example 5 The ALP activity of 38 human serum samples was measured using a commercial ALP measurement reagent [alkaline phosphatase-TR (manufactured by Wako Pure Chemical Industries, Ltd.)] based on the Kind-King method (substrate: phenyl phosphate). Each was measured by the method used and the method recommended by the JSCC (substrate: NPP). FIG. 6 shows a correlation diagram based on the measured values obtained by these two methods.

[0046]

FIG. 6

The results obtained by statistically processing the measurement results obtained in Reference Example 5 are shown below. Regression linear equation: Y = 0.0302X + 1.1100 Correlation coefficient (r): 0.9673 As is clear from the above results, both phenyl phosphate and NPP are generally widely used substrates, but because of their different chemical structures, each isozyme It can be understood that the activity ratio differs, and that the correlation when measuring the ALP activity of human serum is not good. As is clear from the results of FIGS. 2 to 5, the p-acylphenylphosphate represented by the general formula [I] according to the present invention as a substrate and the type of buffer used in the buffer were determined according to the specific recommendation method. If they are the same, the obtained activity values show a very good correlation with the activity values obtained by the specific recommendation method, and the regression linear equations for these activity values almost pass through the origin. I understand. Also,
These facts show that the activity value obtained by the ALP measurement method of the present invention can be converted to a specific recommended activity value by multiplying the activity value by a certain coefficient. When the conversion coefficient is obtained from the regression line equation obtained this time, the conversion coefficient is as shown in Table 2, for example.

[0048]

[Table 2]

[0049]

As described above, the present invention provides an ALP measurement reagent which has solved the problem of poor stability over time of a measurement reagent in various recommended methods using conventional NPP as a substrate. According to the present invention, it is not necessary to prepare a substrate solution at the time of use, so that there is no variation in a lot such as a reagent which has been provided in the form of a conventional solid preparation, and it is obtained by the measurement method of the present invention. Multiplying the obtained activity value by a specific coefficient, it is possible to convert it to the activity value of various recommended methods using NPP as a substrate. It is.

[0050]

[Brief description of the drawings]

FIG. 1 shows the results obtained in Example 1, in which the horizontal axis represents the pH of the substrate solution, and the vertical axis represents the difference in absorbance at 340 nm.

FIG. 2 shows measured values obtained in Example 2 and Reference Example 1.
FIG. 4 shows a correlation diagram with the measured values obtained in FIG.

FIG. 3 shows measured values obtained in Example 3 and Reference Example 2.
FIG. 4 shows a correlation diagram with the measured values obtained in FIG.

FIG. 4 shows measured values obtained in Example 4 and Reference Example 3.
FIG. 4 shows a correlation diagram with the measured values obtained in FIG.

FIG. 5 shows measured values obtained in Example 5 and Reference Example 4.
FIG. 4 shows a correlation diagram with the measured values obtained in FIG.

FIG. 6 shows measured values obtained by the Kind-King method and JS.
FIG. 4 shows a correlation diagram with measured values obtained by the CC recommendation method.

[Explanation of symbols]

 In FIG. 1, ● and ○ indicate the following, respectively. ● ・ ・ ・ When the substrate solution stored at 15 ℃ for 6 months is used.・ ・ ・: When a substrate solution stored at 15 ° C. for 12 months is used.

Claims (5)

(57) [Claims] 1. A compound of the general formula [I] (In the formula, R represents an alkyl group.) A reagent solution for measuring alkaline phosphatase, comprising using p-acylphenylphosphate or / and a salt thereof represented by the formula: 2. The alkaline phosphatase assay reagent according to claim 1, comprising a substrate solution containing p-acylphenyl phosphate represented by the general formula [I] and a buffer solution containing an activator. 3. The reagent solution for measuring alkaline phosphatase according to claim 2, wherein the pH of the substrate solution is in the range of 8 to 12. 4. A buffer comprising 0.5 to 1.5 M of 2-M as a buffer.
Ethylaminoethanol, 0.5-1.5 M diethanolamine, 0.1-1.5 M N-methyl-D-glucamine, or 0.1-M
PH 9 containing 1.5 M 2-amino-2-methyl-1-propanol
The alkaline phosphatase measurement reagent according to claim 2 or 3, wherein the buffer is a buffer of (11) to (11). 5. A method for measuring alkaline phosphatase, comprising using the reagent solution for measuring alkaline phosphatase according to claim 1 or 2. [0001]