JPH082318B2 - Transparent triglyceride substrate solution for measuring colipase and method for measuring colipase activity using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial field of application> The present invention is a transparent triglyceride for measuring colipase for highly sensitive and efficient measurement of a small amount of colipase in a biological fluid, particularly human pancreatic juice, serum and the like. The present invention relates to a substrate solution and a method for measuring colipase activity using the substrate solution.

<Problems to be Solved by Conventional Techniques and Inventions> Colipase is an essential substance (coenzyme) for expressing lipase activity, and lipase has the property of being activated proportionally by the amount of lipase.

The necessity of measuring colipase will be described as follows, together with the properties of colipase, using colipase secreted from human pancreas as an example.

(B) A protein factor having a molecular weight of about 11,000, which contributes to the expression and enhancement of lipase activity in vivo alone or in cooperation with calcium ions, bile acids and the like. Deficiency of colipase causes indigestion and malabsorption.

(B) As an indicator of pancreatic diseases such as acute pancreatitis and pancreatic cancer, a method for measuring amylase and lipase has already been adopted, but colipase is used as an indicator of exocrine pancreatic function and for the detection of mild pancreatic dysfunction. It is useful as an index (Furukawa et al .: Nissho, 83, (7) 1367-1375, 1986).

(C) The ratio of the amount of colipase / lipase in the urine and serum of patients with pancreatic disease is determined, and it is being studied to apply this to diagnosis (Yunge et al .: Clinica.Chimica.Acta.123 (1982) 2
93-302).

As described above, although the measurement of colipase is useful from a clinical point of view, there is currently no active use of colipase as an indicator of pancreatic disease. The reason is that the conventional method for measuring colipase is complicated and not satisfactory in accuracy as described in (1) to (3) below.

(1) Titration method As a substrate for a suspension of triglyceride, mainly olive oil, or a glycerol ester of higher fatty acid,
The fatty acid liberated by the following enzyme reaction is titrated with an alkali.

However, this method has a long reaction time, requires a large amount of sample, and cannot be said to have good accuracy. For this reason, free fatty acids are
Improvements such as using a stat and quantifying as a copper salt have been made, but considerable time and labor are required.

(2) Nephelometry This is a method of measuring the decrease in turbidity of the substrate due to the enzymatic reaction of (1) by absorbance.

Although this method is simple, if there is a sample in which the decrease in turbidity and the colipase activity do not always match and the absorbance in the emulsified sample rises conversely, the decrease in turbidity can be measured to calculate the colipase activity. However, when the activity of the lipase is low, the sensitivity is poor and the reproducibility is not good.

(3) Method for colorimetric determination using a color former using an artificial (synthetic) substrate other than triglyceride For example, BALB (2,3-dimercaptopropan-1-ol tributyrate), α-naphthyl palmitate , A monoglyceride, 1,2-diglyceride, p-nitrophenol lauric acid ester, a fatty acid derivative such as dimethylcaprol tributyrate, or an ester with an alcohol derivative is used as a substrate.

However, in addition to the disadvantages (1) and (2) described above, the water-insoluble substances in these artificial (synthetic) substrates are hydrolyzed by esterases other than the lipase-colipase system. However, it is necessary to add an inhibitor and the like, and the water-soluble substance has solved the drawback (2), but it is still an esterase other than the lipase-colipase system. The problem of substrate specificity is that it is also hydrolyzed by.

Therefore, the inventors attach importance to the fact that the definition of the lipase-colipase system firstly hydrolyzes long-chain triglycerides, use triglyceride as a substrate, and measure the decrease in turbidity (substrate). Instead, the result of extensive studies in consideration of direct measurement of fatty acid or glycerol produced by hydrolysis of triglyceride by lipase-colipase system, simple measurement method that can be used in clinical tests, etc. Was completed.

<Means for Solving the Problem> The present application comprises the following claims (I) to (IX).

(I) A uniform and soluble (clear) aqueous solution of triglyceride is allowed to contain lipase, and bile acid,
A transparent triglyceride substrate solution for measuring lipase, which contains a lipase function promoting substance such as calcium chloride.

(II) The transparent triglyceride substrate solution for measuring colipase according to claim 1, wherein the surfactant used for preparing a homogeneous and soluble (clear) aqueous solution of triglyceride is a nonionic surfactant. .

(III) The transparent triglyceride substrate solution for measuring colipase according to claim 2, wherein the nonionic surfactant is a polyoxyethylene derivative. The transparent triglyceride substrate solution for measuring colipase according to claim 2, wherein the nonionic surfactant (IV) is HLB 10 to 16.

(V) The transparent triglyceride substrate solution for measuring colipase according to claim 1, wherein the fatty acid constituting the triglyceride has 12 to 22 carbon atoms.

(VI) The transparent lipase measurement according to any one of claims 1 to 5, wherein the content of lipase in the uniform and soluble (clear) aqueous solution of triglyceride is larger than the lipase content of the biological sample to be measured. Substrate solution for triglyceride.

(VII) A transparent triglyceride substrate solution for lipase measurement containing a lipase-containing sample containing a colipase with unknown colipase activity is used to liberate fatty acids or glycerol, which are quantified by a conventionally known absorbance measurement method. A method for measuring colipase activity using a transparent triglyceride substrate solution for measuring colipase, which comprises measuring colipase activity.

(VIII) Adenosine triphosphate (AMP) produced by reacting free fatty acid with adenosine triphosphate (ATP) and arsicoenzyme A synthetase (ACS) in the presence of coenzyme A (CoA). ), (2) acyl CoA, or (3) one component of the CoA remaining in the reaction is quantified by a conventionally known absorbance measurement method,
The method for measuring colipase activity using the transparent triglyceride substrate solution for measuring colipase according to claim (VII), wherein the colipase activity is measured from this quantitative value.

(IX) The method for measuring colipase activity using the transparent triglyceride substrate solution for measuring lipase according to claim (VII), wherein monoglyceride lipase is further present in the reaction system when detecting free glycerol.

The above-mentioned transparent triglyceride substrate solution for measuring colipase of the present application is a uniform and soluble (transparent) water solution of triglyceride that does not hinder colorimetric determination, and lipase (free glycerol In the case of measurement, if necessary, monoglyceride lipase is included) was added in advance.

In addition, the method for measuring colipase activity using this solution was to add colipase of unknown concentration to the solution, and triglyceride was converted to lipase-lipase in proportion to the content of colipase.
Free fatty acids generated by hydrolysis by colipase system,
Or it measures glycerol.

Conventionally, a method of uniformly and solubilizing (clearing) triglyceride is disclosed in Japanese Patent Publication No. 59-39168 (Patent No. 127160) as "Method for producing solubilized aqueous solution of water-insoluble substance".
No. 63,91136), but the present application is the first to utilize the transparent aqueous solution of triglyceride obtained by this method as a substrate for measuring colipase in which all the above-mentioned drawbacks are solved. Further, the present invention is the first to directly measure the amount of fatty acid or glycerol liberated by the lipase-colipase system from triglyceride in this clear aqueous solution to measure the colipase activity.

The above-mentioned transparent triglyceride substrate solution for measuring colipase of the present application is a uniform and solubilized (clear) aqueous solution of triglyceride (5 mM to 1 M pH 6.0 to 9.0), which does not hinder the colorimetric determination. This solution contains lipase (excess amount) and, if necessary, bile acid (salts such as cholic acid, deoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid 1 mM-100 mM), calcium chloride (1 m
M to 150 mM) etc. are added beforehand.

In addition, the method for measuring colipase activity using this solution was to add colipase of unknown concentration to the solution, and triglyceride was converted to lipase-lipase in proportion to the content of colipase.
Free fatty acids generated by hydrolysis by colipase system,
Or it measures glycerol.

As the fatty acid used in the present invention, the following fatty acid having 12 to 22 carbon atoms is suitable.

(1) Saturated fatty acid lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid (2) Unsaturated fatty acid Lindelic acid, lauroleic acid, tudzuic acid, ficetrain acid, myristoleic acid, palmitooleic acid, petrothelin Acid, oleic acid, elaidic acid vaccenic acid, linoleic acid, linolenic acid, α-eleostearic acid, β-eleostearic acid, bunic acid, parinaric acid, caderic acid, cetoleic acid, erucic acid, arachidonic acid and the like. To be Examples of triglycerides in which R, R ′, R ″ of triglyceride are esterified with these fatty acids include the following.

Trilaurein (12,12,12 carbon atoms in bound fatty acids), Tridecanoin (13,13,13), Trimyristin, (14,14,14), Tripentadecanoin (15,15,15), Tripalmitin (16 , 16,16), trimergarin (17,17,17), tristearin (18,18,18), trino-ondecanoin (19,19,19), triolein (18,18,18), trileiden (18,18,18), Trilinolein (18,18,18), Trilinolenin (18,18,18,), Triercine (22,22,22), Tribrazidine (22,22,22), 1-lauro Dimyristin (12,14,14), 1-myristodipalmitin (14,16,16), 1-palmitodistearin (16,18,18), 1-stearodipalmitin (16,16,18), 2-stearodipalmitin (16,18,16), 2-palmitodistearin (18,16,18), 1-oleodiestearin (18,18,18), 1-linoleode Stearin (18,18,18), 2-Oleodistearin (18,18,18), 1-Stearodiolein (18,18,18), 1-Stearodilinolein (18,18,18), 1 -Stearo-2 myristo-3 palmitin (18,14,1
6), 1-stearo-2 palmito-3 myristin (18,16,1
4), 1 lauro-2 myristo-3 palmitin (12,14,16).

Of these, as a substrate when trying to measure the human lipase-colipase system, triglyceride containing fatty acids contained in a large amount in the living body is desirable, and examples thereof include myristic acid, palmitic acid, stearic acid, palmitooleic acid, Among them, oleic acid, linoleic acid, etc., among them, the fatty acid which is the most widespread and most abundant in nature, and triglyceride containing oleic acid, which is said to be the most abundant in human body, is a preferable substrate.

Further, the surfactant used to obtain the transparent substrate solution of triglyceride of the present invention is a nonionic surfactant, such as polyoxyethylene lauryl ether, polyooxyethylene cetyl ether, polyoxyethylene stearyl ether, and polyoxyethylene stearyl ether. Oxyethylene olein ether, polyoxyethylene octyl phenyl ether,
Derivatives of polyoxyethylene such as polyoxyethylene nonyl phenyl ether, polyoxyethylene polyoxypropylene block polymer, and polyoxyethylene higher alcohol are mentioned, and those having an HLB of 10 to 16 are suitable. Among them, polyoxyethylene higher alcohols (HLB10 to 16) are selected as the most suitable for the lipase reaction.

In the present invention, in order to obtain a transparent substrate solution of triglyceride, first, a nonionic surfactant is dissolved in distilled water or a buffer solution and heated above the cloud point of the nonionic surfactant, and triglyceride is added and dissolved while continuing stirring. Take the method of doing.

In some cases, a nonionic surfactant is dissolved in distilled water or a buffer solution, a builder that does not affect the lipase-colipase reaction is added, triglyceride is added, and stirring is continued at room temperature. Finally, distilled water or a buffer solution is added. It is also possible to obtain a transparent liquid by diluting with.

At this time, since an excessive amount of nonionic surfactant may inhibit the lipase-colipase system reaction, 5 to 20% by weight of nonionic surfactant and 0.05 to 0.5% by weight of triolein relative to 100% by weight of distilled water. It is desirable to use it in the ratio of.

The lipase-colipase system reaction of the solubilized triglyceride of the present invention is preferably carried out in the range of pH 6.5 to 9.0, and examples of the buffer include citric acid-sodium phosphate buffer, imidazole hydrochloric acid buffer, triethanolamine hydrochloric acid. -Sodium hydroxide buffer, Tris buffer,
Glycylglycine-sodium hydroxide buffer, diethanolamine-hydrochloric acid buffer, borate buffer, 2,4,6-trimethylpyridine-hydrochloric acid buffer, phosphate buffer, Good's buffer, etc. It is preferred to use it in a concentration.

Furthermore, in the present invention, bile acid is essential, and cholic acid, deoxycholic acid, lithocholic acid, glycocholleic acid, taurocholic acid and the like as a bile salt are contained in the substrate solution at 1 mM to 100 mM.
It is effective to add in the range of mM. If necessary, calcium chloride 1 mM to 80 mM and sodium chloride 5 mM to
It is more effective if it is added to the substrate reaction solution at a ratio of 500 mM.

The thus obtained reagent for measuring colipase according to the present invention is a very effective and stable composition as a reagent for measuring colipase activity.

According to the present invention, lipase is added (contained) to a uniform and solubilized (clear) aqueous solution of triglyceride.
To the produced transparent triglyceride substrate solution for measuring colipase, a lipase of unknown activity was added to produce a fatty acid produced by hydrolysis by a lipase-colipase system reaction, and adenosine triphosphate (ATP) coenzyme A (Co
A) Based on the reaction of reacting fatty acid with acyl CoA synthetase (ACS) in the presence, 1) produced adenosine monophosphate (AMP) is measured. 2) Acyl C produced
Measure oA. 3) A method of measuring the residual CoA can be adopted.

Examples of these measurement methods include 1) adenosine monophosphate (AMP) in the presence of adenosine triphosphate (ATP) and phosphoenolpyruvate (PEP), myokinase (MK) and pyruvate kinase ( PK) is caused to act to generate pyruvic acid, and the resulting pyruvic acid is measured for reduction of nicotinamide adenine dinucleotide-reduced form (NADH) using lactate dehydrogenase (LDH). Alternatively, it is measured by a colorimetric method using pyruvate oxidase (POP).

Or In 1), a method of measuring ammonia using ADP deaminase, or a method of measuring ammonia using AMP deaminase, or AMP or cleotidase,
Examples thereof include a method of measuring ammonia by causing adenine deaminase to act, and a method of measuring ammonia by causing AMP pyrophosphorylase and adenine deaminase to act.

2) Examples of the method for measuring acyl CoA include an example in which the hydrogen peroxide produced by reacting acyl CoA with acyl CoA oxidase (ACO) is transferred to a system of peroxidase or catalase, and the like.

The produced hydrogen peroxide is measured using catalase, peroxidase, or the like.

The generated hydrogen peroxide is oxidatively condensed with 4-aminoantipyrine and various couplers. Examples of such couplers include aniline type and toluidine type in addition to phenol, such as N-ethyl-N-sulfopropyl-m-toluidine, N, N-diethyl-m-toluidine, 3-methyl-N-ethyl-N. -(Β-hydroxyethyl) -aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, 3,5-dimethoxy-N-
(3-Sulfopropyl) aniline, etc. Others 3-methyl-2-benzothiazolinone hydrazone is used in place of 4-aminoantipyrine, or 4-aminoantipyrine is not used 10- (3-methoxycarboxyl-aminomethyl -Benzoyl-carbamoyl) -3.7
-Bis (dimethyl-3nk) -10H-phenothiazine, 3-
A coupler such as bis (4-chlorophenyl) methyl-4-dimethyl-aminophenylamine may be used. In addition, the β-oxidation system, which is the main pathway of fatty acid metabolism, directly converts acyl CoA into 2-3 trans-enoyl CoA with acyl CoA oxidase, and then enoyl CoA hydratase, 3-hydroxyacyl CoA dehydrogenase, 3-
The increase of NADH may be measured by acting with ketoacyl-CoA thiolase.

Remaining CoA as a method for measuring the remaining CoA in 3)
Can be measured with an SH group quantification reagent such as 5,5′-dithiobis-2-nitrobenzoic acid (DTNB).

In addition to the system for measuring these fatty acids, glycerol produced by the lipase reaction may be measured. In this case, the lipase reaction of triglyceride is carried out at the 1- or 3-position to give 1,2-diglyceride, which is then 2-monoglyceride. And finally glycerol, but 2-
Since reaction of glycerol from monoglyceride is unlikely to occur, it is effective to use monoglyceride lipase together.

 Next, the present invention will be described with reference to examples.

<Example 1> (A) Preparation of reagent Reagent (1) Emulgen 70 as a nonionic surfactant in 90 ml of distilled water
When 9 g of 9 (trade name of Kao Atlas, polyoxyethylene higher alcohol cloud point 56 ° C) is dissolved and heated to 56 ° C or higher, the liquid reaches the cloud point and becomes cloudy. Add 2 g of triolein there and stir for about 30 minutes while maintaining at 56 ° C or higher, then stop heating and leave while stirring. The liquid becomes transparent as it approaches room temperature, so use distilled water when it becomes transparent.
Dilute 10 times to obtain a clear aqueous solution (triolein solubilized substrate solution). To this, 4,400 U of swine-derived purified lipase and 700 mg of calcium chloride are added (transparent triglyceride substrate solution for measuring colipase).

Reagent (2) 100 mM of the following substances with 50 mM Tris-HCl buffer (pH 7.9)
ml.

Triolein-solubilized substrate solution: 20 ml Adenosine-5'-triphosphate (ATP): 8.6 ml Phosphoenolpyruvate (PEP): 200 mg Reduced nicotine adenine nucleotide (NADH): 450 mg Myokinase (MK): 214 mg Pyruvate Kinase (PK): 15000U Lactate dehydrogenase (LDH): 20000U Coenzyme A (CoA): 1900mg Deoxycholate Na (SDC): 2000mg Acyl coenzyme A synthetase (ACS): 160U Magnesium chloride: 100mg Reagent (3) (colipase) Standard solution) Dilute 30000U / mg colipase in physiological saline to obtain 5,10,1
It was diluted to 5,20,25,30,35,40 ng / ml.

(B) Measuring method 350 μl of the reagent (1) prepared in the previous section in a 1.1 ml test tube,
Collect 20 μl of reagent (3) or purified water and heat at 37 ° C. for 5 minutes. Next, 50 μl of the reagent (2) is added, and the decrease in the absorbance at 3 minutes to 5 minutes is measured at λ = 550 nm using a reagent blank (the above distilled water) as a control.

When a calibration curve was prepared by plotting the absorbance per minute on the vertical axis, a graph showing good linearity was obtained (see the drawing).

Next, instead of the reagent (1), serum (as it was) and pancreatic juice (100-fold diluted solution) were treated in the same manner, and colipase was measured by the above calibration curve. The results are shown below.

* Serum Reagent A: 3.1ng / ml Reagent B: 8.5ng / ml Reagent C: 23.2ng / ml Reagent D: 10.4ng / ml Reagent E: 0.5ng / ml (healthy person) * Pancreatic juice Reagent A: 18.3ng / ml Reagent B: 32.7 ng / ml Reagent C: 29.7 ng / ml Reagent D: 1.8 ng / ml (healthy person) Reagent E: 0.4 ng / ml (healthy person) <Example 2> (A) Reagent Reagent (1) Distillation Emulgen 70 as a nonionic surfactant in 90 ml of water
7 g (polyoxyethylene higher alcohol, trade name of Kao Atlas) is dissolved, 5 g of sodium chloride is added as a builder, and the mixture is stirred at room temperature (25 ° C). Although this solution becomes cloudy, 2 g of trilinolein is added as it is and stirred vigorously for about 2 hours (trilinolein-solubilized substrate solution). This solution is 100 mM with 10 mM Tris-HCl buffer (pH 7.9).
Diluted twice to obtain a transparent aqueous solution. To this, 5000 U / dl of lipase purified product from pig, 10 mM sodium deoxycholate,
Add 70 mM sodium chloride and 5 mM calcium chloride to obtain reagent (1) (clear triglyceride substrate solution for colipase measurement).

Reagent (2) Acyl CoA synthetase: 5.0U / 5ml adenosine-5'-triphosphate: 33.0μmol CoA lithium salt: 7.8μmol Trishydroxymethylaminomethane (pH7.85) containing 5mM magnesium chloride hexahydrate ) Dissolve in solution to give reagent (2).

Reagent (3) Acyl CoA oxidase: 10.0 μ / 20 ml Peroxidase: 20.000 U / 20 ml 4-Aminoantipyrine: 24 μmol The above reagent was added to N-ethyl-N-sulfopropyl-m-
It is dissolved in a 20 mM N, N-bis (2-hydroxyethyl) 2-aminoethanesulfonic acid (pH 7.30) solution containing 0.75 mM toluidine to obtain a reagent (3).

Reagent (4) N-ethylmaleimide 10 mM was dissolved in hydrochloric acid solution to pH 3.0.
This is designated as reagent (4).

(B) Measurement results In order to investigate the simultaneous reproducibility of this measurement method, collect the reagent (1) prepared in the previous section in a 1.1 ml test tube, 50 μl of purified water as a reagent blank, and 10 ng / ml of colipase solution as a sample.
And 20 ng / ml of 50 μl each are added and heated at 37 ° C. for 10 minutes. Next, add 1 ml of reagent (2), react for 30 minutes, add 1 ml of reagent (4), add reagent (3) after 2 minutes, and leave at room temperature, then measure the absorbance at λ550 nm using the reagent blank as a control. did. Also, the serum sample was measured in the same manner.

 The results were as follows.

* Simultaneous reproducibility 10ng / ml 20ng / ml (I) 10.1 20.1 (II) 9.9 20.2 (III) 9.9 20.2 (IV) 10.0 20.0 (V) 10.2 19.8 (VI) 9.9 20.1 (VII) 9.7 20.0 (VIII) 10.0 20.1 (IX) 10.1 19.9 (X) 10.1 19.9 * Serum Reagent A: 18.9ng / ml Reagent B: 31.3ng / ml Reagent C: 9.9ng / ml Reagent D: 24.8ng / ml Reagent E: 0.2ng / ml (healthy person) ) Example 3 (A) Reagent Reagent (1) Emulgen 70 as a nonionic surfactant in 90 ml of distilled water.
When 9 g of 9 (trade name of Kao Atlas, polyoxyethylene higher alcohol cloud point 56 ° C) is dissolved and heated to 56 ° C or higher, the liquid reaches the cloud point and becomes cloudy. Add 2 g of triolein there and stir for about 30 minutes while maintaining at 56 ° C or higher, then stop heating and leave while stirring. Since the solution becomes transparent as it approaches room temperature, when it becomes transparent, it is diluted 200-fold with 10 mM Tris-HCl buffer (pH 7.9), 2.5 mM sodium deoxycholate, 35 mM sodium chloride, pig-derived pancreatic lipase. Purified product 4000U / dl, 3.5mM calcium chloride is added to make reagent (1).

Reagent (2) Acyl-CoA synthetase: 66U / dl Acyl-CoA oxidase: 90U / dl Peroxidase: 16000U / dl ATP: 140μmol Coenzyme A: 16μmol 10- (3-methoxycarboxylaminomethylbenzoylcarbamoyl) -3.7- Bis (dimethyl-amino) -10H-phenothiazine): 2.2 μmol The above reagent is dissolved in 20 mM Good's buffer (pH 6.75).

Reagent (3) As a reagent for sample blank, reagent (3) is prepared by removing treolein, purified product of pancreatic lipase from pig and calcium chloride from reagent (1).

(B) Measurement results 1 ml each of the reagents (1) and (3) prepared in the examples
Collect in a test tube and add 20 μl of human serum to (1) and (3).
After reacting at 37 ° C for 20 minutes, 1.5 ml of each reagent (2) was dispensed, and after 5 minutes at 37 ° C, the wavelength was measured at 666 mm, and the absorbance of the sample dispensed with reagent (1) was subtracted from the lipase activity. And the activity of human serum colipase determined by preparing a standard solution of 20 ng / ml colipase as a standard solution were as follows.

* Serum Reagent A: 2.2ng / ml Reagent B: 12.2ng / ml Reagent C: 20.1ng / ml Reagent D: 13.1ng / ml Reagent E: 0.6ng / ml (healthy person) <Example 4> Reagent (1) The following reagents were dissolved in 15 ml of 0.1 M trishydroxymethylaminomethane solution (pH 8.3).

4-Aminoantipyrine: 5.7 mg Monoglyceride lipase: 1000 U Triolein solubilizing substrate (Example 3): 1.7 ml Calcium chloride: 100 mg Magnesium chloride: 8 mg Reagent (2) Next, the reagents of group (a) were added to group (b). 36 ml of the reagent prepared in
Dissolved in.

Group (a): Glycerokinase: 5.38U Peroxidase: 135000U Adenosine triphosphate: 42.4mg L-α Glycerophosphate oxidase: 161U Ascorbate oxidase: 53.8U Group (b): The following reagents were made up to 100 ml with distilled water and pH 6 Prepared to .65.

N, N bis (2-hydroxyethyl) 2-aminoethane sulfonic acid: 4.27 g N-ethyl-N-sulfopropyl-m-toluidine:
0.14g Magnesium chloride: 3.05g Triton X-100 (trade name of Rohm and Haas Company,
Polyoxyethylene octyl phenyl ether): 0.1 g (B) measurement results colipase standard solution (20 ng / ml) prepared in Example 1,
Or 50 μl of serum sample and 1.5 ml of reagent (2) 37
Reaction is performed at 5 ° C for 5 minutes to eliminate glycerol in the sample.

1.5 ml of the following reagent (1) was dispensed and allowed to react at 37 ° C for 5 minutes. After 15 minutes, the absorbance was measured at a wavelength of 550 nm using the reagent blank as a control, and the value of colipase was calculated from ΔEAbs. It was as follows.

* Serum Reagent A: 8.8ng / ml Reagent B: 9.8ng / ml Reagent C: 19.6ng / ml Reagent D: 14.9ng / ml Reagent E: 0.3ng / ml (healthy person) <Effect of invention> The transparent triglyceride substrate solution for colipase measurement and the method for measuring colipase activity using this solution are configured as described above, so that trace amounts of colipase such as human pancreatic juice and serum, which have been difficult to measure in the past, can be measured with high sensitivity and high efficiency. This has the effect of contributing to early detection of pancreatic diseases and the like.

[Brief description of drawings]

 The drawing is a graph showing a calibration curve of colipase activity.

Claims (9)

[Claims] 1. A uniform and soluble triglyceride (transparent).
A transparent triglyceride substrate solution for colipase measurement, which comprises adding lipase to a chlorinated aqueous solution and further containing a lipase function-promoting substance such as bile acid and calcium chloride. 2. A uniform and soluble triglyceride (transparent).
The transparent triglyceride substrate solution for measuring colipase according to claim 1, wherein the surfactant used for preparing the cationized aqueous solution is a nonionic surfactant. 3. The transparent triglyceride substrate solution for measuring colipase according to claim 2, wherein the nonionic surfactant is a polyoxyethylene derivative. 4. The transparent triglyceride substrate solution for measuring colipase according to claim 2, wherein the nonionic surfactant is HLB10-16. 5. The transparent triglyceride substrate solution for measuring colipase according to claim 1, wherein the fatty acid constituting the triglyceride has 12 to 22 carbon atoms. 6. A uniform and soluble triglyceride (transparent)
The lipase content of the modified aqueous solution is larger than the lipase content of the biological sample to be measured.
5. A transparent triglyceride substrate solution for measuring colipase according to item 5. 7. A transparent triglyceride substrate solution for lipase measurement containing a lipase-containing triglyceride substrate solution, and a fatty acid or glycerol liberated by allowing a sample containing a lipase-containing colipase to act thereon is quantified by a conventionally known absorbance measurement method. A method for measuring colipase activity using a transparent triglyceride substrate solution for measuring colipase, which comprises measuring colipase activity from a quantitative value. 8. (1) Adenosine monophosphate, which is produced by reacting liberated fatty acid with Arsicoenzyme A synthetase (ACS) in the presence of adenosine triphosphate (ATP) and coenzyme A (CoA). Claims: (AMP), (2) acyl CoA, or (3) one component of CoA remaining in the above reaction is quantified by a conventionally known absorbance measurement method, and colipase activity is measured from this quantified value. A method for measuring colipase activity using the transparent triglyceride substrate solution for measuring colipase according to item (VII). 9. When detecting free glycerol,
The method for measuring colipase activity using the transparent triglyceride substrate solution for measuring colipase according to claim (VII), wherein monoglyceride lipase is further present in the reaction system.