JPH05123194A - Composition for measuring lipase activity and method for measuring lipase activity using the same composition - Google Patents

Abstract

PURPOSE:To obtain the subject composition homogeneously containing glyceride and a fatty acid or aliphatic amine in water, capable of measuring by colorimetric technique because of aqueous transparency when used as a substrate liquid for lipase activity measurement. CONSTITUTION:The objective composition homogeneously containing glyceride and fatty acid (preferably capric acid or myristic acid) or aliphatic acid amine (e.g. lauryl amine or stearylamine) in water. The composition is prepared e.g. by a method giving physical disturbance by adding the fatty acid or aliphatic amine to an aqueous dispersion of glyceride. The physical disturbance is preferably carried out by ultrasonic wave treatment under heating to <=100 deg.C.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring lipase activity contained in a biological component, and a composition used for the measurement.

[0002]

2. Description of the Related Art Lipase is an enzyme widely distributed in the natural world regardless of whether it is an animal or a plant, and its purpose is to measure its activity for various purposes. From a clinical point of view, it is particularly important as a diagnostic index for pancreatic diseases such as acute pancreatitis and pancreatic cancer. Therefore, lipase activity has been clinically measured by various methods. Triglyceride has long been used as a substrate for measuring the activity of pancreatic lipase in serum.

For example, a method of alkali titrating the acidity of fatty acid liberated by the action of lipase using as an substrate an emulsion obtained by adding gum arabic or polyvinyl alcohol to olive oil containing triglyceride as a main component and using the emulsion as a substrate, The copper complex salt extraction method [Clinical Examination 15 (2), 191, 1971] has been used. However, since this composition exhibits strong turbidity as an emulsion, not only is it prone to phase separation after preparation, but also it could not be used for spectroscopic measurements. In recent years, this method has also been improved, and a nephelometric method has been used in which emulsified olive oil is used as a substrate and the turbidity reduced by the action of lipase is measured spectroscopically. However, although the measurement procedure was simplified, the sensitivity was low, and factors other than lipase activity sometimes caused not only a decrease in turbidity but also an increase in turbidity. Further, since it is difficult to prepare a uniform and stable substrate, the lot difference becomes large, and many problems remain in terms of reproducibility.

Compositions using various surfactants have been reported to solve these problems. For example, it is a solubilized aqueous solution of a water-insoluble substance (Japanese Patent Publication No. 59-391693) or a composition for measuring lipase activity (Japanese Patent Publication No. 59-91898), both of which have higher fatty acids as constituents. A composition obtained by solubilizing triglyceride, monoglyceride, or 1,2-diglyceride in water in the presence of 0.1% or more of a nonionic surfactant, which depends on the use of the surfactant. There were problems such as the existence of lot differences, destabilization of reagent components, and the influence of impurities in the surfactant depending on the type and detection system. On the other hand, the lipase substrate, which is a color development method using a synthetic substrate, and a visual measuring method of lipase and a measuring reagent (JP-A-61-254197) have insufficient specificity.

[0005]

As described above, the conventional composition for measuring lipase activity still has various problems due to the substances constituting the composition. As a result of various researches conducted by the present inventor to solve these problems,
It has been found that an aqueous clear solution obtained by micronizing glyceride using a fatty acid and / or an aliphatic amine is a useful substrate composition that can be used in any lipase activity measurement system. Finely pulverizing various substances such as metals, metal oxides, other metal compounds (carbides, nitrides, etc.), ceramics, organic compounds, etc. significantly increases the surface area. As a result, stable water of, for example, water-insoluble or sparingly water-soluble compounds is obtained. It is known to obtain a dispersion (JP 62-106833 A). However, according to the present invention, an aqueous transparent liquid obtained by mixing (a) glyceride, (b) fatty acid and / or aliphatic amine and (c) water, and performing ultrasonic treatment while heating, for example. Surprisingly, although it is composed of only water-insoluble glyceride (a), water-insoluble fatty acid and / or aliphatic amine (b) and water (c) as a solvent, without using a surfactant or the like, A clear, stable solution is formed. This aqueous clear liquid can be distinguished from conventional compositions in that no other additives (stabilizers, solubilizers, etc.) are used, and at the same time, it is extremely good as a substrate for detecting or measuring lipase activity. Do you get it. The present invention is based on these findings.

[0006]

Therefore, the present invention relates to a composition for measuring lipase activity, characterized in that glyceride and fatty acid and / or aliphatic amine are homogeneously contained in water. The present invention also relates to a powdery composition for measuring lipase activity, characterized in that it contains glyceride, a fatty acid and / or an aliphatic amine and an excipient uniformly. Further, the present invention provides that the glyceride and the fatty acid and / or the aliphatic amine are homogeneously contained in water in the presence of a reagent capable of acting on the compound produced by the action of lipase from glyceride to produce a detectable change. The present invention also relates to a method for measuring lipase activity, which comprises contacting the composition with a test sample and detecting the detectable change.

Glyceride, which is a substrate for measuring lipase activity in the composition of the present invention, is an ester of a fatty acid and glycerol which is a trihydric alcohol, and has three or two glycerol hydroxyl groups (1,2- or 1,3-position). Position) or one (1st position, 2nd position or 3rd position) is esterified with a fatty acid and is represented by the following general formula (I).

[0008]

[Chemical 1]

The higher fatty acid residues in R ₁ , R ₂ and R ₃ of the general formula (I) are, for example, the following fatty acid residues. (1) Saturated fatty acid Lauric acid C ₁₁ H ₂₃ COOH; Myristic acid C ₁₃ H ₂₇ C
OOH; palmitic acid C ₁₅ H ₃₁ COOH; stearic acid C ₁₇ H ₃₅ COOH; arachidic acid C ₁₉ H ₃₉ COOH; behenic acid C ₂₁ H ₄₃ COOH. (2) unsaturated fatty acids linderic acid C ₁₂ H ₂₂ O _2; lauroleic C ₁₂ H
₂₂ O _2; tsuzuic acid C ₁₄ H ₂₆ O _2; Fisetorein acid C ₁₄ H
₂₆ O ₂ ; myristoleic acid C ₁₄ H ₂₆ O ₂ ; palmitooleic acid C ₁₆ H ₃₀ O ₂ ; petroselinic acid C ₁₈ H ₃₄ O ₂ ; oleic acid C ₁₈ H ₃₄ O ₂ ; elaidic acid C ₁₈ H ₃₄ O ₂ ; vaccenic acid C ₁₈ H ₃₄ O ₂ ; linoleic acid C ₁₈ H ₃₂ O ₂ ; linolenic acid C ₁₈ H ₃₀ O ₂ ; α-eleostearic acid C ₁₈ H ₃₀
O ₂ ; β-eleostearic acid C ₁₈ H ₃₀ O ₂ ; punicic acid C ₁₈ H ₃₀ O ₂ ; parinaric acid C ₁₈ H ₂₈ O ₂ ; cadreic acid C ₂₀ H ₃₈ O ₂ ; cetrain acid C ₂₂ H ₄₂ O ₂ ; erucic acid C ₂₂ H ₄₂ O ₂ ; arachidonic acid C ₂₀ H ₃₂ O ₂ .

In the present invention, fatty acids and / or aliphatic amines are used as additives in order to mix these glycerides with water and keep them stable. The fatty acid used as an additive in the present invention is preferably a saturated or unsaturated fatty acid having 8 to 30 carbon atoms (more preferably 8 to 24 carbon atoms), and the glyceride-constituting fatty acids described above can also be used. However, particularly preferably, capric acid, myristic acid, palmitic acid, oleic acid, linolenic acid and the like can be mentioned.

The aliphatic amine used in the present invention is preferably a primary amine having a saturated or unsaturated aliphatic hydrocarbon having 8 to 24 carbon atoms (more preferably 10 to 22 carbon atoms), A secondary amine or a tertiary amine,
For example, laurylamine or stearylamine.

The substrate solution of the present invention contains the above-mentioned fatty acid and / or aliphatic amine in an amount of preferably 0.5 to 200% by weight, more preferably 1 to 50%, based on the amount of glyceride.
By including the weight%, the glyceride can be contained in water in an amount of 0.005 to 0.5% by weight.

The substrate solution of the present invention can be prepared, for example, by the following method. (1) A method in which a fatty acid and / or an aliphatic amine is added to an aqueous dispersion of glyceride to give physical perturbation. (2) A method in which an aqueous dispersion of glyceride and an aqueous dispersion of fatty acid and / or aliphatic amine are mixed to give a physical disturbance. (3) A method of imparting physical disturbance to an aqueous dispersion of glyceride and fatty acid and / or aliphatic amine. (4) A method in which glyceride is added to an aqueous dispersion of a fatty acid and / or an aliphatic amine to give a physical disturbance.

The aqueous dispersion of glyceride or the aqueous dispersion of fatty acid and / or aliphatic amine is prepared by purifying glyceride or fatty acid and / or aliphatic amine with purified water, physiological saline or various buffers (for example, phosphoric acid). Buffer or borate buffer), as well as preliminary physical disturbance.

The physical disturbance includes stirring by an ultra-high speed homogenizer, a method by ultrasonic treatment, a high-pressure homogenizer,
It can be given by a French press, a colloid mill or the like. Physical disturbance due to ultrasonic treatment is preferable, the ultrasonic wave is 10 to 30 KHz, and the strength in water is 1
0 to 100 W is sufficient. Further, high-pressure homogenizer in the range of 100~1,000kg / cm ^2, the French press range of 1,000 ~10,000kg / cm ^2, and a colloid mill is sufficient each be in the range of 1,000 ~20,000rpm.

It is preferable to perform a heating treatment when applying the physical disturbance. The heating treatment is carried out at a temperature at which the glyceride, fatty acid and / or aliphatic amine contained in the aqueous dispersion do not deteriorate. It is preferably performed at 100 ° C. or lower.

When an aqueous clear liquid having a relatively high concentration of glyceride is prepared, the desired aqueous clear liquid may be prepared from the beginning by adding a desired concentration of glyceride to the aqueous dispersion. It is also possible to first prepare an aqueous clear liquid containing glyceride at a concentration lower than the desired concentration, and then distill off the water under reduced pressure.

To mix triglycerides (eg triolein) with water by conventional methods, see, for example, JP-A-58.
-156330, that is, a special and complicated method that applies a change in physical action that appears at the cloud point of an aqueous solution of a nonionic surfactant can be used. Other than that, it was extremely difficult. However, the present inventors have found that triolein was added to 0.005
Aqueous clear liquid containing ~ 0.5 wt% has a fatty acid (for example, palmitic acid) of 1-2 with respect to the amount of triolein.
It can be obtained only by coexisting with 0 wt%. That is, purified water, physiological saline, or various buffer solutions are added to triolein to a desired concentration, and ultrasonication is performed as a physical disturbance for 5 to 60 seconds, and the amount of triolein is 1 to the amount of triolein. An aqueous clear solution of triolein can be prepared by simply adding ˜20% by weight of palmitic acid and dispersing it, and then performing ultrasonic treatment while heating at about 70 ° C. The thus-prepared triolein aqueous transparent liquid has extremely excellent transparency, and its transmittance at a wavelength of 550 nm (visible wavelength of 400 to 700 nm) is 95% or more. In addition, the transparency does not change even after 6 months from the preparation.

The above-mentioned aqueous transparent liquid can be stored as a powdery composition (for example, a freeze-dried product) for a long period of time, and can be easily reproduced as the original aqueous transparent liquid by adding water. Excipients are generally required to produce freeze-dried products. Excipients include sugars (eg glucose, saccharose, lactose, etc.), polysaccharides (eg,
Dextran, cellulose, carboxymethylcellulose, ficoll, etc.), polysaccharide derivatives, amino acids (glycine, etc.), proteins (albumin, etc.), synthetic polymer compounds (polyvinylpyrrolidone, polyethylene glycol, etc.), inorganic salts (potassium chloride, etc.), etc. Used. It can be produced by adding about 1 to 10% of these excipients (one or more kinds) to an aqueous transparent liquid and freeze-drying by a known method. The transparency of the aqueous transparent liquid obtained by adding water to the powder composition thus prepared is equal to or improved as compared with that before lyophilization. this is,
This is due to the auxiliary effect of the excipient.

The above-mentioned aqueous clear liquid and powdery composition may be, if desired, conventionally known in addition to the essential components (that is, the above-mentioned glyceride, fatty acid and / or aliphatic amine, and excipient). It may contain optional additives such as surfactants, stabilizers, preservatives and the like. Moreover, the desired components such as these synthetic surfactants can achieve the intended effects even if the amount of use thereof is greatly reduced.

The lipase detecting composition of the present invention produces fatty acid or glycerol from its substrate, glyceride, by the action of lipase present in a test sample,
It is based on detecting those products by means known in the art. The concentration of the substrate glyceride during the reaction is 0.01 to 0.5%, preferably 0.02 to 0.
It is adjusted to 4%, more preferably 0.04 to 0.2%. The concentration of the cofactor colipase during the reaction is 50 to 300 μg / ml, preferably 75 to 300 μg / ml.
It is 150 μg / ml.

If necessary, bile acid can be present as a reaction accelerator. As bile acids, colanic acids,
For example, cholic acid, deoxycholic acid, lithocholic acid, and the like, preferably salts thereof, can be used. The concentration of bile acid at the time of reaction is 0.1 to 10 mM, preferably 0.5 to 2 mM. Similarly, calcium chloride can be present if desired. The concentration during the reaction is 0.2 to 5 mM, preferably 0.5 to 2 mM. The pH range for lipase activity expression is 7-9, and the buffer for maintaining the pH value is 5 mM to 1 M, preferably 10 mM to 500 mM Tris buffer, Good's buffer, phosphate buffer, etc. Known buffer solutions can be used. When triglyceride or diglyceride is used as a substrate, diglyceride lipase and / or monoglyceride lipase can be present. That is, diglyceride is produced from the substrate triglyceride, in order to promptly proceed the reaction until monoglyceride or glycerol is produced from this,
Preference is given to using these enzymes. Each enzyme can be present alone or both. These enzymes may be present in 0.1-20 units, preferably 0.5-5 units, during the reaction. Each of the above components,
Each can be used individually or in combination or as one reagent. Furthermore, there is no problem even if a conventionally known stabilizer or preservative is made to coexist.

The fatty acid or glycerol produced from the substrate is detected by a conventionally known means to measure the lipase activity.
The reagent for lipase measurement of the present invention thus obtained,
The composition is very effective and stable as a reagent for measuring lipase activity.

The composition of the present invention can be directly applied to various conventionally known lipase activity measuring methods. The method for detecting or measuring the lipase activity is carried out by measuring the production amount of various products obtained through various enzyme reactions from a known amount of substrate (that is, glyceride) or the residual amount of intermediates. As an example of the reaction system when using triglyceride or diglyceride as a substrate, the following formula (1) is used.
And Formula (2) can be mentioned.

[0025]

[Chemical 2]

From the substrate triglyceride or diglyceride, fatty acid and monoglyceride are produced by the lipase reaction (1). Subsequently, the obtained fatty acids were coenzyme A (CoA) and adenosine triphosphate (A
In the presence of TP), the action of acyl coenzyme A synthetase (ACS) produces acyl CoA, AMP and pyrophosphate (2). Then, the above equation (2)
In (a) a method for measuring adenosine monophosphate (AMP) produced, (b) a method for measuring acyl CoA produced,
Alternatively, (c) a method of measuring the remaining CoA can be adopted. To give specific examples of these measuring methods, the measuring method (a) utilizes the reaction formulas shown by the following reaction formulas (3) to (5).

[0027]

[Chemical 3]

That is, in AMP, in the presence of ATP and phosphoenolpyruvate (PEP), myokinase (M
K) and pyruvate kinase (PK) [(3) and (4)], and lactate dehydrogenase (LDH) acts on the resulting pyruvate to reduce reduced nicotinamide adenine dinucleotide (NADH) Measure the quantity (5). Alternatively, as shown in the following reaction formula (6), the amount of H ₂ O ₂ produced is measured using pyruvate oxidase (POP) by a colorimetric method known to those skilled in the art.

[0029]

[Chemical 4]

In this way, the activity of lipase present in the test sample can be measured as the amount of fatty acid. In addition,
When a fatty acid is used to prepare an aqueous transparent liquid used as a substrate, the fatty acid may be added in advance to the aqueous transparent liquid, but the amount added is low when the relative concentration with respect to glyceride is about several% (% by weight). Therefore, there is no particular problem with the conventional operation of simply subtracting the reagent blank. Further, in the case of measuring the reduced amount of reduced nicotinamide adenine dinucleotide (NADH) [the above reaction formula (5)], if the amount of fatty acid contained in the test sample is large, the measurement range is narrowed accordingly. If necessary, the elimination reaction of the fatty acid contained in the test sample may be performed before the reaction of the reaction formula (5). Alternatively, a substrate aqueous clear solution prepared by using an aliphatic amine in place of the fatty acid may be used.

As an example of the above-mentioned measuring method (a), a method of measuring ammonia using ADP deaminase, AM
Method for measuring ammonia using P deaminase,
Alternatively, a method for measuring ammonia by acting AMP nucleotidase and adenine deaminase, and further AM
A method in which P-pyrophosphorylase and adenine deaminase are allowed to act to measure ammonia can also be mentioned.

In the above measuring method (b), for example, as shown in the following reaction formula (7), hydrogen peroxide produced by the action of acyl CoA oxidase (ACO) is used in a reaction system of peroxidase or catalase. Examples of measurement and the like can be given.

[0033]

[Chemical 5]

When measuring the produced hydrogen peroxide,
4-aminoantipyrine is oxidatively condensed with various couplers shown below in the presence of peroxidase. Examples of the coupler include phenol-based, aniline-based, and toluidine-based compounds such as N-ethyl-N-sulfopropyl-m-toluidine, N, N-diethyl-m-toluidine, and 3-methyl-N-ethyl-. N- (β-hydroxyethyl) -aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, 3,5-
Examples include dimethoxy-N- (3-sulfopropyl) aniline. In addition, instead of 4-aminoantipyrine, 3-
Methyl-2-benzothiazolinone hydrazone is used, or in the absence of 4-aminoantipyrine, 1
0- (3-methoxycarboxyl-aminomethyl-benzoyl-carbamoyl) -3,7-bis (dimethyl-3)
A coupler such as nk) -10H-phenothiazine or 3-bis (4-chlorophenyl) methyl-4-dimethyl-aminophenylamine may be used. Also, direct acyl C
oA is a β-oxidation system that is the main pathway for fatty acid metabolism, that is, acyl CoA is converted into 2,3-
After converting to trans-enoyl CoA, enoyl CoA hydratase, 3-hydroxyacyl CoA dehydrogenase, and 3-ketoacyl CoA thiolase are allowed to act on NA.
The amount of increase in DH may be measured.

As the above measuring method (c) [measurement of residual CoA], as shown in the following reaction formula (8), residual C
5,5'-dithiobis-2 which is a reagent for quantifying SH group
-It can be measured with nitrobenzoic acid (DTNB) or the like.

[0036]

[Chemical 6]

In addition to the above-mentioned method of measuring fatty acid, there is also a method of changing monoglyceride produced together with fatty acid in the above reaction formula (1) into glycerol as shown in the following reaction formula (9) and measuring the glycerol. ..

[0038]

[Chemical 7]

That is, glycerol and fatty acids are produced from monoglyceride by the action of monoglyceride lipase (9). This glycerol produces glycerophosphate and ADP by glycerokinase in the presence of ATP (10), and glycerophosphate produces dihydroxyacetone phosphate by glycerophosphate oxidase, and simultaneously produces H ₂ O ₂ (11). ). Generated H ₂
O ₂ can be measured by a known colorimetric method as in the case of the above fatty acids. Thus, not only as the amount of fatty acid, but also the amount of monoglyceride (Japanese Patent Laid-Open No. 63-245672) and the amount of glycerol which is a metabolite thereof (Japanese Patent Laid-Open No.
265899), the composition of the present invention may be used as it is as a substrate solution for lipase measurement, but it is preferable to add colipase which is a lipase function promoter or bile salt. Furthermore, reagents necessary for the constitution of each detection system may be added together with the composition of the present invention.

When monoglyceride is used as the substrate, the reactions represented by the above formulas (9) to (11) can be used. At this time, it is not necessary to add monoglyceride lipase. Due to the action of human-derived lipase (substrate specificity), 1-monoglyceride is preferably used as a substrate. Whether to use mono-, di- or triglyceride as a substrate can be selected depending on the kind of lipase expected to be contained in the test sample.

The method for measuring lipase activity according to the present invention is different from the conventional method for measuring lipase activity only in that the above-mentioned aqueous transparent liquid is used as the substrate solution, and in other respects, the operations and reagents of the conventional method are applied as they are. Therefore, the “reagent capable of producing a detectable change by acting on the compound produced by the action of lipase from glyceride” used in the method of the present invention depends on the type of test sample or the type of measurement system. It can be appropriately selected by those skilled in the art. The test sample is not particularly different from that of the conventional method, and can be easily prepared and adjusted by those skilled in the art. Therefore, the test sample used in the method of the present invention is not particularly limited as long as it may contain lipase, but in particular, biological samples such as blood, serum, plasma, urine, pancreatic juice, etc. Is.

[0042]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1 100 mg of tripalmitin was placed in a beaker, 5.0% by weight of palmitic acid was added to the amount, and 100 parts of purified water was added.
ml was added. The mixture was heated to 70 ° C. in a warm bath and sonicated (50 W) for 5 minutes. Further, the same operation was performed using 100 mg each of 1,2-dipalmitin and 1,3-dipalmitin in place of tripalmitin, and the obtained substrate solution was used in the following examples. Further, a substrate solution was prepared in the same manner by using stearylamine instead of palmitic acid.

Example 2 Fatty acids as reaction products were measured by an enzymatic method. That is, 0.1% prepared in Example 1 as a substrate solution in a test tube.
50 μl of tripalmitin solution (containing palmitic acid) was added, and further 5 μl of 1 mg / ml colipase (derived from porcine pancreas) dissolved in 20 mM Tris-HCl buffer solution (pH 8.0) as a cofactor, 0.1 M Tris-HCl buffer solution (pH 7) ．
10 μl of 6.7 mM deoxycholic acid dissolved in 8) and 1 μl of 50 mM calcium chloride aqueous solution were added and mixed well. After pre-warming at 37 ° C, 1 µl of a lipase solution (5 types of human pancreatic juice dilution series for which lipase activity values had been determined in advance) was added and reacted at 37 ° C. 5 minutes later,
500 μl of a 20 mM Good buffer (pH 7.0) containing 3.3 mM 4-aminoantipyrine, 0.2 mM coenzyme A and 0.3 U / ml of acylcoenzyme A synthetase
Was added and the heating was continued for 5 minutes, then 1 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-
1 ml of 50 mM Good's buffer (pH 7.0) containing sodium toluidine, 0.1 mg / ml peroxidase and 1.5 U / ml acylcoenzyme A oxidase was added. After reacting at 37 ° C. for 5 minutes, the absorbance at 550 nm was measured using purified water as a control. Good results were obtained as shown in FIG. Similarly, good results were obtained when a tripalmitin substrate solution obtained by using stearylamine was used. In addition, in the lipase dilution series of FIG. 1, "1.0"
Corresponds to an activity value of the used lipase solution of 51.5 U / liter (during reaction).

Example 3 The change with time in the lipase reaction was measured as an amount of fatty acid by an enzymatic method. 0.1% 1,2-dipalmitin solution (containing palmitic acid) prepared in Example 1 as a substrate solution in a test tube
50 μl of 1 mg / ml colipase dissolved in 20 mM Tris-HCl buffer (pH 8.0) as a cofactor, and 0.1 M Tris-HCl buffer (pH 7.8) were added.
100 μl and 6.7 mM deoxycholic acid dissolved in
10 μl of 0 mM calcium chloride aqueous solution was added and mixed. Further, 10 μl of the lipase solution was added, the mixture was heated to 37 ° C., and 50 μl of the reaction solution was sampled with time. After reacting this sample in the same manner as in Example 2, the absorbance at 550 nm was measured using purified water as a control.
Further, the same measurement was carried out using 1,3-dipalmitin or tripalmitin instead of 1,2-dipalmitin. The results are shown in Fig. 2. The activity value of the lipase solution used was 77.4 U / liter (during reaction).
In FIG. 2, line 1 shows the results of 1,2-dipalmitin, line 2 shows the results of tripalmitin, and line 3 shows the results of 1,3-dipalmitin.

Example 4 In this example, comparison with esterase was performed. That is, a lipase solution having various concentrations and an esterase solution having various concentrations were used as samples, and freeze-dried tripalmitin powder was redissolved in 1 ml of purified water as a substrate and used. Others were measured similarly to Example 2. The results are shown in Fig. 3. In FIG. 3, line 4 is lipase and line 5 is esterase. In contrast to the lipase that showed good reactivity (51.8 U / liter at the time of the reaction of 1.0 in FIG. 3), 1392 U
It was clear that even an esterase up to / ml (enzyme solution stock solution: equivalent to 1.0 in FIG. 4) did not react at all. The freeze-dried tripalmitin powder used as a substrate was prepared as follows. 30% Ficoll 400 [water-soluble synthetic high molecular compound of sucrose and epichlorohydrin (molecular weight: about 400,000, manufactured by Pharmacia)] and 20 as excipients for 9 parts by volume of 0.1% aqueous clear liquid 1 volume part of an aqueous solution containing 10% sucrose was mixed, dispensed in 1 ml aliquots in 10 ml vials, cooled and frozen at -60 ° C, and vacuum dried.

Example 5 In this example, a comparison was made with a conventional method using a surfactant (JP-A-2-243617). That is, in the preparation of the substrate by the conventional method, Emulgen 709 is used as the nonionic surfactant.
(Polyoxyethylene higher alcohol; cloud point 56 ° C)
When 3.5 g was dissolved in 32 ml of purified water and heated to 56 ° C or higher, the solution reached a cloud point and became cloudy. 0.35 g of triolein was added thereto, and the mixture was stirred for about 30 minutes while maintaining the temperature at 56 ° C. or higher, then heating was stopped, and the mixture was allowed to stand while continuing stirring. When the liquid became clear, Ficoll 400 (3
%) And sucrose (2%). 5 ml of the obtained solution was dispensed into a vial, lyophilized and stored. Dissolve 1 bottle of the stored vial with 5 ml of purified water, and add 0.1M Tris-HCl buffer (pH
It was diluted 3.3 times with 8.0). Add this substrate solution 5 to a test tube.
0 μl and 0.18 mg / ml colipase 5 dissolved in 20 mM Tris-HCl buffer (pH 8.0) as a cofactor
μl, 10 μl of 52 mM deoxycholic acid dissolved in 0.1 M Tris-HCl buffer (pH 7.8) and 1 μl of 74 mM calcium chloride aqueous solution were added and mixed well. After prewarming to 37 ° C, 2 µl of the lipase solution was added, and the mixture was reacted at 37 ° C. After 5 minutes, 200 μl of 20 mM Good buffer solution (pH 7.0) containing 3.3 mM 4-aminoantipyrine and 0.2 U of coenzyme A 0.3 U / ml of acylcoenzyme A synthetase was added, and heating was continued for 5 minutes. Later 1 mM N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) -m-toluidine sodium, 0.3
400 μl of 50 mM Good's buffer (pH 7.0) containing mg / ml peroxidase and 1.5 U / ml acylcoenzyme A oxidase was added. After reacting at 37 ° C. for 5 minutes, the absorbance at 550 nm was measured using purified water as a control.

On the other hand, in the method of the present invention, 50 μl of the 0.1% 1,2-dipalmitin solution (containing palmitic acid) prepared in Example 1 was dissolved in 20 mM Tris-HCl buffer (pH 8.0) as a cofactor. 1 mg / ml colipase 5
10 μl of 6.7 mM deoxycholic acid dissolved in 0.1 M Tris-HCl buffer (pH 7.8) and 1 μl of 50 mM calcium chloride aqueous solution were added to a test tube and mixed. To this, 2 μl of lipase solution was added and reacted at 37 ° C. Then, measurement was performed in the same manner as in the case of the above-mentioned conventional method, and the obtained results are shown in Table 1. The method of the present invention showed better results in measurement sensitivity and reproducibility than the conventional method.

Example 6 The reaction product, monoglyceride, was measured by an enzymatic method. That is, by the same operation as in Example 2, the reaction was carried out at 37 ° C. using tripalmitin as a substrate in the presence of a cofactor. 5 minutes later 1 U / ml monoglyceride lipase,
0.5 U / ml glycerokinase, 20 U / ml glycerophosphate oxidase, 0.5 mM adenosine triphosphate (ATP), 0.7 mM magnesium chloride, 10 mM ammonium chloride, 1.1 mM 4-aminoantipyrine, 0.7
20 mM Good's buffer (pH 7.7) containing mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium, 0.1 mg / ml peroxidase.
1.5 ml was added. The mixture was reacted at 37 ° C. for 5 minutes, and the absorbance at 550 nm was measured using purified water as a control. The results are shown in Fig. 4.

[0049]

[Table 1]

[0050]

According to the present invention, a stable aqueous clear solution of water-insoluble glyceride prepared using a small amount of fatty acid and / or aliphatic amine is used as a substrate solution for detecting or measuring lipase activity. Colorimetric techniques can be used. Further, since the substrate solution does not contain a surfactant, the lot difference due to the surfactant is eliminated, and the stability of the reagent component is also improved.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the amount of fatty acid produced from triglyceride and the concentration of lipase.

FIG. 2 is a graph showing the relationship between various glycerides and lipase reaction time.

FIG. 3 is a graph showing the effects of lipase and esterase on the substrate solution of the present invention.

FIG. 4 is a graph showing the relationship between the amount of monoglyceride produced from triglyceride and the concentration of lipase.

Claims (3)

[Claims] 1. A composition for measuring lipase activity, characterized in that glyceride and a fatty acid or an aliphatic amine are uniformly contained in water. 2. A powdery composition for measuring lipase activity, which contains glyceride, a fatty acid or an aliphatic amine and an excipient uniformly. 3. A composition containing glyceride and a fatty acid or an aliphatic amine homogeneously in water in the presence of a reagent capable of acting on a compound produced from glyceride by the action of lipase to produce a detectable change. And a test sample are brought into contact with each other to detect the above-mentioned detectable change, and a method for measuring lipase activity.