JPH11318494A - Lipase substrate solution for measuring enzymatic activity and reagent kit for measuring lipase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject solution not inhibiting lipase activity, having high transparency and safety, useful for measuring enzyme activity, etc., by including a specific lipase substrate and 1,2-diphthaloyl-sn-glycero-3- phosphocholine. SOLUTION: This lipase substrate solution for measuring enzyme activity contains at least a lipase substrate of the formula [A is methylene, a 2-16C alkylene or 2-16C alkenylene; R<1> and R<2> are each a 1-20C alkyl, an acyl, a 2-20C alkenyl, a (substituted) aryl or an aralkyl with the proviso that either of R<1> and R<2> may be H; X is a group derived from an aromatic hydroxy compound or a group derived from an aromatic thiol compound; Y and Z are S or O; Z may be methylene] and 1,2-diphthaloyl-sn-glycero-3-phosphocholine, does not inhibiting lipase activity, has high transparency and high stability.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a lipase substrate solution for measuring an enzyme activity in which a specific lipase substrate is solubilized by a novel lipase substrate solubilizing agent, and a lipase activity measurement using the lipase substrate solution for measuring the enzyme activity. It relates to a reagent kit. More specifically, the present invention relates to a lipase substrate solution for measuring enzyme activity, in which a specific lipase substrate is solubilized using 1,2-diphthanoyl-sn-glycero-3-phosphocholine as a lipase substrate solubilizing agent. The present invention relates to a reagent kit for measuring lipase activity using a lipase substrate solution for measurement.

[0002]

Lipase is an enzyme that hydrolyzes glycerol esters of glycerol and fatty acids, and is widely distributed in the living world. In mammals,
In particular, large quantities are present in the pancreas to digest fats consumed as food. Therefore, in humans, this lipase is released into the blood from the pancreas due to the destruction of pancreatic cells at the time of pancreatic disease, and the lipase activity in the blood becomes higher than in normal. Therefore, by accurately measuring the lipase activity in each subject such as plasma and serum, it is possible to accurately diagnose whether or not a patient has pancreatic disease.

For example, in order to measure lipase activity in each specimen such as blood and serum, a fixed amount of the specimen whose lipase activity is to be measured is determined in the same manner as in other enzyme activity measurements. A fixed amount of lipase substrate is mixed to cause an enzymatic reaction, and the amount of change and / or residual amount of the lipase substrate based on the enzymatic reaction is determined by a colorimetric method and U
It is measured by an analysis means such as the V method (ultraviolet absorption method). Thus, in these assays, the lipase substrate must be solubilized to form a substantially clear lipase substrate solution.

However, a lipase substrate solution containing a lipase substrate solubilized by a conventional lipase substrate solubilizing agent does not provide practically satisfactory high transparency, and suppose that the lipase substrate has high transparency. Even if a substrate solution was obtained, its stability was low and often clouded or precipitated, so that it had to be used for measurement within a short time after it was prepared. Therefore, conventionally, for each of these reasons, a lipase substrate solution had to be prepared every time the lipase activity was measured, which was extremely complicated.

The inventors of the present invention have found that, when the lipase activity is measured, even if the lipase activity is contained in the lipase-containing solution, the lipase substrate is not substantially solubilized. A lipase substrate solution with high transparency is obtained, and this lipase substrate solution is highly stable, and intensive studies are conducted to find a lipase substrate solubilizing agent that can maintain the high transparency over a long period of time. As a result of the superposition, a new finding was obtained that 1,2-diphthanoyl-sn-glycero-3-phosphocholine achieves this object and can completely overcome the above-mentioned disadvantages of the prior art. And reached the present invention.

[0006]

According to the first aspect of the present invention, there is provided an enzyme activity comprising at least a lipase substrate represented by the following general formula and 1,2-diphthanoyl-sn-glycero-3-phosphocholine. It is a lipase substrate solution for measurement.

[0007]

Embedded image

Wherein A represents a methylene group, an alkylene group having 2 to 16 carbon atoms or an alkenylene group having 2 to 16 carbon atoms; R ¹ and R ² are the same or different from each other and each has a carbon atom An alkyl group or acyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an aryl group optionally substituted with an alkyl group having 1 to 8 carbon atoms, or an alkyl group having 1 to 8 carbon atoms Represents an aralkyl group derived from Where R ¹
One of R and R ² may be a hydrogen atom; X
Represents a group derived from an aromatic hydroxy compound or a group derived from an aromatic thiol compound; Y and Z each independently represent a sulfur atom or an oxygen atom; and Z may be a methylene group. ]

The second invention provides a first reagent obtained by dissolving a lipase activator in a buffer, and a second reagent a solution which is a lipase substrate solution for enzyme activity measurement of the first invention and a second solution comprising a colipase-containing solution. This is a lipase activity measurement reagent kit obtained by combining a second reagent which is a mixed solution with two reagents b liquid.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The lipase substrate solution for measuring enzyme activity according to the first aspect of the present invention comprises a lipase substrate represented by the above general formula (hereinafter sometimes referred to as a specific lipase substrate) and 1,2-diphthanoyl-sn- It is a solution in which glycero-3-phosphocholine is substantially completely dissolved in an organic solvent. This lipase substrate solution for measuring enzyme activity is used as the second reagent a solution of the reagent kit for measuring lipase activity of the second invention. The above specific lipase substrates are known per se,
For example, a lipase substrate described in JP-B-6-87800 is preferable.

That is, the specific lipase substrate is a compound represented by the above general formula. In this general formula, the alkylene group represented by A is defined as a divalent atomic group generated by removing two hydrogen atoms, one of which is bonded to each of the different carbon atoms in the aliphatic saturated hydrocarbon, and An alkenylene group is defined as a divalent atomic group formed by removing two hydrogen atoms, one bonded to each of two different carbon atoms in an aliphatic hydrocarbon having a double bond. A
Is preferably an alkylene group or alkenylene group having 3 to 7 carbon atoms. Examples of the alkylene group include a propylene group, an α-butylene group, a β-butylene group, a γ-butylene group, and a tetramethylene group. Examples of the alkenylene group include a propenylene group and a 2-butenylene group.

R ¹ and R ² each preferably have 6 to 18 carbon atoms, and particularly preferably have 8 to 12 carbon atoms. R ¹ and R ² are each preferably an alkyl group. Further, the group X derived from the aromatic hydroxy compound or the group X derived from the aromatic thiol compound was formed by removing a hydrogen atom from each of the hydroxyl group of the aromatic hydroxy compound and the mercapto group of the aromatic thiol compound. Group. The aromatic hydroxy compound and the aromatic thiol compound are those which form a chromophore from the beginning or can be converted into a chromophore by reaction with lipase, as long as they do not inhibit lipase activity. However, preferred representative examples include phenol, naphthol, thiophenol and thionaphthol and derivatives thereof such as resorufin, methylresorufin, fluorescein and thiofluorescein.

As representative examples of specific lipase substrates, 1,2
-O-dilauryl-rac-glycero-3-glutaric acid-
(6′-methylresorufin) -ester or 1,2-
o-Dilauryl-rac-glycero-3-glutaric acid-resorufin ester and the like can be mentioned. As these lipase substrates, commercially available products can be used as they are. Commercially available products include, for example, commercially available products from Boehringer Manheim Biochemica.

1,2-diphthanoyl-sn-glycero-3
-Phosphocholine is a compound represented by the following structural formula, and a commercially available product can be used. A typical example of a commercially available product is AVANTI POL
AR LIPIDS, Inc.), which is a product of L-α-lecithin diphthanoyl.

[0015]

Embedded image

The organic solvent of the second reagent a does not inhibit the activity of lipase, dissolves a specific lipase substrate and 1,2-diphthanoyl-sn-glycero-3-phosphocholine, and has a high solubility in water. There is no particular limitation as long as it is an organic solvent, but practically preferred are aliphatic lower alcohols having 1 to 4 carbon atoms and acetone, and isopropyl alcohol is most preferred as the aliphatic lower alcohol having 1 to 4 carbon atoms. .

The amount of the organic solvent used depends on the type of the organic solvent and the type of the lipase substrate, and cannot be specified unconditionally. However, the specific amount of the lipase substrate and 1,2-diphthanoyl-sn-glycero-3-phosphocholine Should be the minimum amount that can dissolve both. When the organic solvent is the above-mentioned aliphatic lower alcohol or acetone,
The second reagent a solution is usually preferably about 10 to 250 ml per 1 mmol of the specific lipase substrate,
Particularly preferred is about 150 ml.

The amount of the specific lipase substrate used depends on the type of the lipase substrate, the type of the organic solvent, the amount of 1,2-diphthanoyl-sn-glycero-3-phosphocholine, and cannot be specified unconditionally. The organic solvent is the above-mentioned aliphatic lower alcohol or acetone, and the specific lipase substrate is the above-mentioned 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin)-
In the case of an ester or 1,2-o-dilauryl-rac-glycero-3-glutaric acid-resorufin ester, the concentration in the second reagent a solution is preferably 4 to 4
The amount is adjusted to be 0 mM, particularly preferably 10 to 30 mM.

On the other hand, the amount of 1,2-diphthanoyl-sn-glycero-3-phosphocholine used depends on the type of lipase substrate and the type of organic solvent, and cannot be specified unconditionally. In the case of a lower alcohol or acetone, the concentration in the second reagent a solution is preferably 0.3 to 5 mM, particularly preferably 2 to 4 mM.
The amount is set to be M.

In the reagent kit for measuring lipase activity of the second invention, the second reagent a solution, which is the lipase substrate solution for enzyme activity measurement of the first invention, and the second reagent b solution containing colipase are used. Are mixed to form a second reagent.
That is, the reagent kit for measuring lipase activity of the second invention comprises a first reagent obtained by dissolving a lipase activator such as the above deoxycholic acid in a buffer solution and a second reagent a which is a lipase substrate solution. The second reagent, which is a mixture of the liquid and the second reagent b liquid containing colipase, is combined. Each of the buffer and the lipase activator used in the preparation of the buffer constituting the first reagent may be known per se, and commercially available products can be used as they are.

As described above, the first reagent is a solution in which a lipase activator such as deoxycholic acid is dissolved in a buffer, but as a buffer used for preparing the buffer, usually, for example, The zwitterionic buffer used for the preparation of the Good buffer is preferably used. Above all,
N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (hereinafter referred to as BES) or tris (hydroxymethyl) aminomethane is most preferred.

The concentration of the buffer in the buffer used for the preparation of the buffer varies depending on the type of the buffer, but is usually preferably from 5 to 150 mM, particularly preferably from 15 to 100 mM. Deoxycholic acid is most preferred as the lipase activator of the first reagent, but other lipase activators can also be used. The concentration of the lipase activator in the first reagent depends on, for example, the type of the lipase activator and cannot be specified unconditionally. However, when the lipase activator is deoxycholic acid, the concentration is usually 10 to 60 mM. Degree is preferable, and about 20 to 40 mM is particularly preferable.

The second reagent b solution is an aqueous solution containing, if desired, a lipase activator such as tartaric acid and a water-soluble calcium salt and an anionic surfactant together with colipase. As the water-soluble calcium salt as the lipase activator, usually, a water-soluble calcium salt of an organic acid or an inorganic acid is suitably used, and calcium acetate is most preferable. In addition, it does not prevent using a lipase activator other than a water-soluble calcium salt. As the anionic surfactant, an alkali metal salt of dodecyl sulfonic acid is preferred, and among them, lithium dodecyl sulfonate or sodium dodecyl sulfonate is most preferred. The use of an anionic surfactant other than dodecylsulfonic acid alkali metal salt is not prevented.

The concentration of colipase in the second reagent (b) is so-called colipase, which is usually determined from the lipase activity using 1,2,3-tributylglyceride as a substrate in the coexistence of lipase and excess colipase. For example, when a commercially available colipase having an activity of 112,000 units per mg (a product of Boehringer Mannheim) is used, 0.1 to 50 μg / ml is used. Degree is preferable, and about 1 to 20 μg / ml is particularly preferable.

The concentration of tartaric acid in the second reagent b solution is 1 to 10
About 0 mM is preferable, and about 5 to 50 mM is particularly preferable.
The concentration of the lipase activator in the second reagent b solution varies depending on the type of the lipase activator, but when the lipase activator is a water-soluble calcium salt, the concentration is 0.1 to 10 mM.
Degree is preferable, and about 0.2 to 5 mM is particularly preferable. The concentration of the anionic surfactant in the second reagent b solution differs depending on the type of the anionic surfactant and cannot be specified unconditionally. However, the second reagent b solution usually has a concentration of about 0.1 to 50 mM. Preferably, about 0.5 to 10 mM is particularly preferable.

Thus, the second reagent and the first reagent, which are a mixed solution in which the mixing ratio of the second reagent a solution and the second reagent b solution is a predetermined ratio, are combined, This is a lipase activity measurement reagent kit. The mixing ratio of the second reagent a solution and the second reagent b solution when preparing the second reagent is as follows:
It depends on the type of the lipase substrate, the type of the organic solvent, the amount of 1,2-diphthanoyl-sn-glycero-3-phosphocholine, the type of the lipase activator and the type of the anionic surfactant optionally contained, and the like. Although it cannot be specified, the second reagent b liquid is used in an amount of preferably 4 to 18 parts by volume, particularly preferably 6 to 12 parts by volume per 1 part by volume of the second reagent a solution.

The measurement of lipase activity in a test sample using this reagent kit for measuring lipase activity is carried out in a conventional manner. That is, in the measurement of the lipase activity of an analyte such as serum, a predetermined amount of the analyte is added to a predetermined amount of the first reagent of the lipase activity measurement reagent kit, and pre-incubation is performed at a predetermined temperature for a predetermined time. A second reagent a solution and a second reagent b solution are mixed in advance at a predetermined mixing ratio with a predetermined amount of a second reagent of a reagent kit for measuring lipase activity to cause an enzyme reaction, Enzyme activity is measured by a colorimetric method.

Each of the first reagent and the second reagent is filled with a required amount in a container such as a bottle and stored in one packaging container to form a set, which is a reagent kit for measuring lipase activity. You. The first and second reagents of the reagent kit for measuring lipase activity of the second invention are both highly stable and can withstand long-term storage.

[0029]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples. Example 1 Analysis method: Analysis was performed as follows using a Hitachi 7050 automatic analyzer. The same applies to the second and subsequent embodiments.

A mixed solution obtained by adding 4 μl of each test serum (1 to n) to 300 μl of each first reagent was preincubated at 37 ° C. for 5 minutes. After the preincubation, 100 μl of the second reagent was added to each of these mixed solutions to start the enzyme reaction.

The main wavelength 600 nm (sub wavelength 700 nm) at about 3 minutes 20 seconds (26 measurement points) and about 4 minutes 20 seconds (29 measurement points) from the start of the enzymatic reaction of each test serum (1 to n). ) Was measured, and these were
_{1 (1-n)} mAbs and A _{2 (1-n)} mAbs. Each test serum (1 to n) in the examples is commercially available from Boehringer Mannheim and has a lipase activity of 485.
Pletipas, 6 IU / l control serum for control
U® and a series of multiple dilutions thereof were used.

On the other hand, measurement was performed at about 3 minutes and 20 seconds (26 measurement points) and at about 4 minutes and 20 seconds (29 measurement points) in the same manner as above, except that physiological saline was used instead of the test serum. The measured absorbances were defined as A ₃ mAbs and A ₄ mAbs, respectively. Absorbances A _{1 (1-n)} mAbs and A _{2 (1-n)} mAbs in each of the test sera (1 to n) obtained in this manner and absorbances A ₃ and A ₄ mAbs in physiological saline. Then, the respective absorbances A _(1-n) mAbs showing the lipase activity in the test sera ( _{1 to n)} were calculated by the following formulas, and a calibration curve was prepared. _{A (1-n) mAbs =} [A 2 (1-n) mAbs-A 1 (1-n) mAbs] - (A 4
mAbs-A ₃ mAbs)

Preparation of the first reagent: BES (a product of Dojindo Laboratories, Inc.) and deoxycholic acid (Boehringer
Was dissolved in purified water to obtain a first reagent which was a mixed solution of 60 mM BES-26.7 mM deoxycholic acid.

Preparation of second reagent: (a) Preparation of second reagent a solution 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6'-methylresorufin) -ester (Boehringer Mannheim) 1,2-diphthanoyl-sn-glycero-3-phosphocholine (Avanti), a novel lipase substrate solubilizer of the present invention, was dissolved in a solution of 12 mg of isopropyl alcohol (commercial product of Katayama Chemical Industry Co., Ltd.) in 1 ml of isopropyl alcohol. Polar Lipids products) 2
1 mg was dissolved to obtain a second reagent a solution.

(B) Preparation of solution b of the second reagent Tartaric acid (product of Katayama Chemical Co., Ltd.) 6.8 mg, calcium acetate (product of Katayama Chemical Co., Ltd.) 0.35 m
g, 2.5 mg of lithium dodecylsulfonate (a product of Wako Pure Chemical Industries, Ltd.) and 20 μg of colipase (a product of Boehringer Mannheim) were dissolved in 4.5 ml of purified water to obtain a second reagent b solution.

(C) Preparation of Second Reagent While stirring 4.5 ml of second reagent b solution, add second reagent a
0.5 ml of the liquid was added dropwise little by little and mixed to obtain a second reagent.

(D) Concentration of each component in the second reagent The concentration of each component of the second reagent prepared as described above is shown below. Tartaric acid 9.0 mM Calcium acetate 0.4 mM Lithium dodecyl sulfate 1.8 mM colipase 4 μg / ml 1,2-o-Dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester 1 2.6 mM isopropyl alcohol 10% by weight 1,2-diphthanoyl-sn-glycero-3-phosphocholine 2.5 mM

Results: The lipase activity A _(1-n) mAbs of each test serum _(1-n) was determined using the first reagent and the second reagent immediately after the preparation as described above. The results are shown by the straight line (a) in FIG. After preparing each of the first and second reagents described above, store them at 8 ° C. for 12 months.
Using the reagents, the lipase activity A _{(1-n )} mAbs of each test serum _{(1-n )} was determined. The results are shown by the straight line (b) in FIG.

For comparison, L-α-phosphatidylcholine (a product of Avanti Polar Lipds) was used in place of 1,2-diphthanoyl-sn-glycero-3-phosphocholine as a lipase substrate solubilizing agent. Lipase activity A _(1-n) mAbs was determined for each of the test sera (1 to n) in the same manner using the first reagent and the second reagent immediately after preparation in the same manner as described above. The result is shown by the straight line (c) in FIG.
Shown in After preparing the first reagent and the second reagent using L-α-phosphatidylcholine as the lipase substrate solubilizing agent, respectively, store at 8 ° C. for 12 months, and use the first reagent and the second reagent. Then, the lipase activity A _(1-n) mAbs of each test serum _(1-n) was determined. The result is shown by the straight line in Fig. 1 (d).
Shown in

FIG. 1 shows the respective dilution rates of the test sera (1 to n) and the absorbance A indicating the lipase activity value in each of the test sera (1 to n) measured as described above.
_(1-n) Indicates a calibration curve (hereinafter referred to as a calibration curve) that is a relationship curve with mAbs. As shown in FIG. 1, the calibration curve was a straight line, regardless of whether any of the first and second reagents was used, and the respective dilution rates of the test sera (1 to n) and the dilution rates thereof. Each of the test sera corresponding to
Absorbance A _(1-n) mAbs indicating lipase activity value in _n)
And a linear relationship is established.

As shown in each of the straight line (a) and the straight line (b), a second reagent in which the lipase substrate solubilizing agent is 1,2-diphthanoyl-sn-glycero-3-phosphocholine is used. In this case, each straight line passed through the origin, regardless of whether the first and second reagents were prepared immediately or stored at 8 ° C. for 12 months, and the slopes of these straight lines were almost equal.

On the other hand, as shown by the straight line (c) and the straight line (d), the lipase substrate solubilizing agent
When the second reagent as -α-phosphatidylcholine was used, the gradients of the respective straight lines were substantially equal immediately after the preparation of the first and second reagents and after storage at 8 ° C. for 12 months. However, when the first reagent and the second reagent were prepared immediately after the preparation, the straight line as the calibration curve passed through the origin, but when the first reagent and the second reagent were stored at 8 ° C. for 12 months, the calibration curve was not obtained. One straight line had a large upward parallel displacement of about 10 mAbs compared to that immediately after preparation. This replaces the lipase substrate solubilizing agent with 1,2-diphthanoyl-sn-glycero-3-phosphocholine and L-
The second reagent, which was α-phosphatidylcholine, lacked storage stability, indicating that it was not able to withstand long-term storage.

Example 2 The buffer used for the preparation of the buffer in the first reagent was B
Example 1 except that ES was replaced with tris (hydroxymethyl) aminomethane (a product of Katayama Chemical Co., Ltd.)
And the same results as in Example 1 were obtained.

Example 3 A specific lipase substrate in the second reagent solution a was 1,2-o
-Dilauryl-rac-glycero-3-glutaric acid- (6 '
-Methylresorufin) -ester was replaced with 1,2-o-dilauryl-rac-glycero-3-glutaric acid-resorufin ester (a product of Boehringer Mannheim) except that the procedure was as in Example 1. The same result as in Example 1 was obtained.

Example 4 The buffer used for the preparation of the buffer in the first reagent was B
The ES was replaced with tris (hydroxymethyl) aminomethane, and the specific lipase substrate in the second reagent a solution was 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -Ester to 1,2-o-dilauryl-rac-glycero-3-glutaric acid-resorufin ester, and 1,2-diphthalanoyl-sn-glycero-3 which is a lipase substrate solubilizer.
-Except that the concentration of phosphocholine in the second reagent was changed from 2.5 mM to either 2.0 mM or 3.8 mM
Performed in the same manner as in Example 1.

FIG. 2 shows the results. In FIG. 2,
Each straight line of (e) to (h) is a second straight line prepared as follows.
It is a calibration curve when a reagent is used. (E) Immediately after preparation, the concentration of 1,2-diphthanoyl-sn-glycero-3-phosphocholine was 2.0 mM. (F) After storage at 8 ° C. for 12 months at a concentration of 1,2-diphthanoyl-sn-glycero-3-phosphocholine of 2.0 mM. (G) Immediately after preparation, the concentration of 1,2-diphthanoyl-sn-glycero-3-phosphocholine was 3.8 mM. (H) 1,2-diphthanoyl-sn-glycero-3-phosphocholine at 3.8 mM after storage at 8 ° C for 12 months.

As shown in FIG. 2, the lipase substrate solubilizer 1,2-diphthanoyl-sn-glycero-
The concentration of 3-phosphocholine was 2.0 mM and 3.8 mM, respectively, immediately after the preparation of each concentration and 12 mM at 8 ° C.
After each month, each test serum (1 to
When the lipase activity A _(1-n) mAbs in _n) was determined,
With the second reagent having the same concentration of 1,2-diphthanoyl-sn-glycero-3-phosphocholine, almost the same calibration curve was obtained despite long-term storage. Note that 1, 2,
The slopes of the calibration curves when the second reagents having different concentrations of -diphthanoyl-sn-glycero-3-phosphocholine were respectively different, and 1,2-diphthanoyl-sn-
When the second reagent having a glycero-3-phosphocholine concentration of 2.0 mM was used, the gradient of the calibration curve was as follows: 1,2-diphthanoyl-sn-glycero-3-phosphocholine having a 3.8 mM second concentration.
It was larger than that when the reagent was used.

Example 5 The same procedure as in Example 1 was carried out except that the anionic surfactant of the second reagent was changed from lithium dodecylsulfonate to sodium dodecylsulfonate (a product of Wako Pure Chemical Industries, Ltd.). Similar results as in Example 1 were obtained.

Example 6 The organic solvent of the second reagent was changed from isopropyl alcohol to acetone (a product of Katayama Chemical Co., Ltd.).
The same operation as in Example 1 was performed, and the same result as in Example 1 was obtained.

Example 7 The anionic surfactant of the second reagent was changed from lithium dodecylsulfonate to sodium dodecylsulfonate (a product of Wako Pure Chemical Industries, Ltd.). From acetone to the specific lipase substrate 1,2-o-dilauryl-rac
The concentration of glycero-3-glutaric acid- (6′-methylresorufin) -ester in the second reagent from 1.6 mM,
The procedure was performed in the same manner as in Example 1 except that the concentration was changed to either 0.4 mM or 3.2 mM.

FIG. 3 shows the results. In FIG. 3,
Each of the straight lines (i) to (e) is a second straight line prepared as follows.
It is a calibration curve when a reagent is used. That is, (ii) 1,2-o-dilauryl-rac-glycero-3-
Glutaric acid- (6'-methylresorufin) -ester concentration: 0.4 mM, immediately after preparation. (Nu) 1,2-o-dilauryl-rac-glycero-3-
Glutaric acid- (6′-methylresorufin) -ester at a concentration of 0.4 mM after storage at 8 ° C. for 12 months. (R) 1,2-o-dilauryl-rac-glycero-3-
Glutaric acid- (6'-methylresorufin) -ester concentration was 3.2 mM, immediately after preparation. (E) 1,2-o-dilauryl-rac-glycero-3-
Glutaric acid- (6′-methylresorufin) -ester at a concentration of 3.2 mM after storage at 8 ° C. for 12 months.

As shown in FIG. 3, the concentration of the specific lipase substrate 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6'-methylresorufin) -ester was 0%. 0.4 mM and 3.2 mM, respectively.
When the lipase activity A _(1-n) mAbs in each test serum _(1-n) was determined using each second reagent immediately after the preparation of each concentration and after storage at 8 ° C. for 12 months, 1 With the second reagent having the same concentration of 2-o-dilauryl-rac-glycero-3-glutaric acid- (6'-methylresorufin) -ester, almost the same calibration curve was obtained despite long-term storage. Was done. In addition, 1,2-o-dilauryl-rac-
The gradients of the calibration curves for the respective second reagents having different concentrations of glycero-3-glutaric acid- (6′-methylresorufin) -ester were respectively different, and 1,2-o-dilauryl-rac-glycero-3-glutaryl was different. When the second reagent having an acid- (6′-methylresorufin) -ester concentration of 3.2 mM was used, the gradient of the calibration curve was 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6 '-Methylresorufin) -ester was larger than that when the second reagent having a concentration of 0.4 mM was used.

[0053]

The lipase substrate solution for enzyme activity measurement of the present invention has high transparency by using 1,2-diphthanoyl-sn-glycero-3-phosphocholine as a lipase substrate solubilizing agent. Lipase activity can be measured accurately, and the storage stability is large.
It also made it possible to maintain the high clarity of the solution over a long period of time. Based on such excellent properties of the enzyme activity measurement lipase substrate solution of the present invention, the enzyme activity measurement lipase substrate solution prepared in advance is used as one of the lipase activity measurement reagents, and further, separately. By combining a lipase activity measurement reagent that is a lipase activator solution prepared in advance and a lipase activity measurement reagent that is a colipase-containing solution, it can be distributed on the market as a lipase activity measurement reagent kit. It goes without saying that the accuracy of the lipase activity measurement reagent such as the enzyme activity measurement lipase substrate solution need not be prepared each time the lipase activity is measured. Can contribute.

[Brief description of the drawings]

FIG. 1 shows a calibration curve for lipase activity A _(1-n) mAbs in each test serum _(1-n) obtained using the lipase activity measurement reagent kit of the present invention.

FIG. 2 shows a calibration curve for lipase activity A _(1-n) mAbs in each test serum _(1-n) obtained using the lipase activity measurement reagent kit of the present invention.

FIG. 3 shows a calibration curve for lipase activity A _(1-n) mAbs in each test serum _(1-n) obtained using the lipase activity measurement reagent kit of the present invention.

Claims (7)

[Claims] 1. A lipase substrate solution for measuring enzyme activity, comprising at least a lipase substrate represented by the following general formula and 1,2-diphthanoyl-sn-glycero-3-phosphocholine. Embedded image [Wherein A represents a methylene group or an alkylene group having 2 to 16 carbon atoms or an alkenylene group having 2 to 16 carbon atoms; R ¹ and R ² are the same or different from each other;
An alkyl group or acyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an aryl group optionally substituted with an alkyl group having 1 to 8 carbon atoms, or an alkyl group having 1 to 8 carbon atoms. Represents an aralkyl group derived from the above alkyl group. Provided that ^one of R ¹ and R ² may be a hydrogen atom; X represents a group derived from an aromatic hydroxy compound or a group derived from an aromatic thiol compound; Independently represents a sulfur atom or an oxygen atom; Z may be a methylene group. ] 2. The method according to claim 1, wherein the lipase substrate is 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6′-methylresorufin) -ester or 1,2-o-dilauryl-r.
The lipase substrate solution for enzyme activity measurement according to claim 1, which is ac-glycero-3-glutaric acid-resorufin ester. 3. A first reagent obtained by dissolving a lipase activator in a buffer, a second reagent a which is a lipase substrate solution for enzyme activity measurement according to claim 1 or 2, and a second reagent which is a colipase-containing liquid. The second liquid which is a mixed liquid with the liquid b
A reagent kit for measuring lipase activity comprising a combination of reagents. 4. The reagent kit for measuring lipase activity according to claim 3, wherein the lipase activator is deoxycholic acid. 5. The reagent kit for measuring lipase activity according to claim 3, wherein the second reagent b solution is an aqueous solution containing tartaric acid, a lipase activator, an anionic surfactant and colipase. 6. The reagent kit for measuring lipase activity according to claim 5, wherein the lipase activator is a water-soluble calcium salt. 7. The reagent kit for measuring lipase activity according to claim 5, wherein the anionic surfactant is an alkali metal salt of dodecylsulfonic acid.