JPS6121100A - Reagent for measuring lipase activity - Google Patents

Abstract

PURPOSE:The titled water-soluble reagent, consisting of a water-soluble thioester compound of a water-soluble thiol with a fatty acid, reactive easily with lipase, and capable of measuring lipase activity with high sensitivity and specificity. CONSTITUTION:A reagent for measuring lipase activity consisting of a thioester compound having carboxyl group of an about 5-22C fatty acid, e.g. lauric acid, palmitic acid or coconut oil fatty acid, to thiol group of a water-soluble thiol, e.g. synthesized by introducing thiol group into polyethylene glycol having 200- 20,000mol.wt. In measurement, a test lipase is brought into contact with the above-mentioned reagent, which is decomposed into the water-soluble thiol and fatty acid, and the formed SH group is determined by the colorimetry, etc. using Ellman's reagent to obtain the lipase activity.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a reagent for measuring lipase activity.

(Prior Art and Problems to be Solved by the Invention) The conventional method for measuring IJ/IP-ase activity involves suspending olive oil or triolein in an appropriate buffer solution and adding IJ
This method is determined by measuring the change in turbidity after the action of Ze-ze, but this method has problems with sensitivity and reproducibility, such as the activity of the Su-A'-ase expressed depending on the suspension method or suspension state. There was a problem with sexuality. In addition, Cherry-Grandal, which is an alkaline titration of free fatty acids, has been used since ancient times.
There was a method by et al., but there was a problem that the titration operation was difficult and individual differences were likely to occur. In addition, methods using synthetic substrates such as α-naphthol derivatives have been developed, but they are prone to errors because the substrates are insoluble in water and other esterases such as allyl esterases also act on them. There was a problem.

In order to solve these problems and develop a method to accurately and easily measure lipase activity, the present inventors conducted various studies and found that an alcohol polymer was used as a new water-soluble substrate for lipase. fatty acid ester (patent application 1982-27)
No. 296) and sugar fatty acid esters (Japanese Patent Application No. 1988-885)
No. 89), they found that using this substrate could solve all of the above problems and accurately and easily measure lipase activity, and they have already filed a patent application for the content.

(Means for solving the problem) The present inventors further investigated and developed a compound in which a carboxyl group of a fatty acid is bonded to a thiol group of a water-soluble thiol as a new substrate that solves the above problems. As a result, the present invention was completed.

Water-soluble thiol can be obtained by introducing a thiol group into a known water-soluble compound. Alcohol polymers, sugars, etc. can be used as this water-soluble compound. Alcohol polymers include polyethylene glycol (PEG), polyvinyl alcohol (P
VA), and sb, m are glucose, maltose, sucrose, xylose, chitosan, starch, etc. The molecular weight is preferably about 200 to 20,000, and about 1,000
A value of about 2,000 to 2,000 is particularly suitable.

The method for introducing a thiol group into a water-soluble compound may be selected from among known methods depending on the functional group. For example, when converting a hydroxyl group to a thiol group, a method of halogenation and the action of thiourea, sodium oxide, etc. Methods such as allowing hydrogen sulfide to act in the presence of a catalyst can be used.

The fatty acid preferably has about 5 to 22 carbon atoms, such as lauric acid, palmitic acid, stearic acid, behenic acid, oleic acid, rillic acid, coconut oil fatty acid, and hardened tallow fatty acid.

The method for bonding the thiol group of water-soluble thiol and the caroxyl group of fatty acid can be carried out according to known methods, such as a method using an acid catalyst, a method of acid chloride, a method of acid amidation, etc. Both are available.

Since two or more thiol groups can be introduced into one molecule of a water-soluble compound, two or more moles of fatty acid can be introduced per mole of water-soluble thiol. For example, in the case of PEG, one or both of the hydroxyl groups at both ends may be converted into a thiol group and a fatty acid may be bonded to it;
In the case of VA, it is the heat of all hydroxyl groups. The fatty acid may be bound by thionylating approximately oo''!'a. In the case of other water-soluble thiols, the binding molar ratio may be appropriately determined in consideration of measurement sensitivity.

Examples of such compounds include PEG-mercap)
Examples include iR lumitate ester, PEG-dimercazotodipalmitate ester, PvA-mercagtooleate ester, and the like.

As a method for measuring lipase activity using the reagent of the present invention, first, lipase, which is the target to be measured, is allowed to act on the compound of this reagent, and this compound is decomposed into water-soluble thiol and fatty acid. The SH group may be determined by a known method, such as a colorimetric quantitative method using Ehlmann's reagent.

The conditions for lipase to work are the activity of this enzyme, the optimal pH
1. It may be determined by considering the optimum temperature, etc.

The types of urinary tracts that can be measured by the method of the present invention are not particularly limited; It can be measured regardless of the type, such as IJA-ase, di-4-ase derived from various microorganisms such as mold, yeast, and bacteria.

(Action and Effect of the Invention) The reagent of the present invention is water-soluble and easily acts on lipase, and lipase activity can be measured with high sensitivity and high specificity.

The method for measuring lipase activity using the reagent of the present invention is fecal testing, and it can be quantified by both the rate assay method and the end point method.

(Example) Synthesis Example 100 g (0, IM) of PEG 1000 was dissolved in about 200 ml of benzene under heating, and 14.6 ml (0,2 M') of pyridine l al and thiochloride tv was added to this.
) was added and reacted at room temperature for 15 hours. Benzene and unreacted thionyl chloride were removed from the reaction solution using an evaporator, and the residue was thoroughly washed with ether and then dried.

The thus obtained PEG chloride 50.9 (0.05M
) was dissolved in 200 ml of ethanol, and 7.6 ml of thiourea was added.
g (0.1 M) was added thereto, and the mixture was reacted for 3 hours under reflux. Furthermore, add 10' Oml of I M NaOH and
After refluxing for an hour, the solvent was removed using an evaporator.

100 mJ of absolute ethanol was added to the residue to dissolve it, and insoluble matter was removed by filtration. Add 3C of ether to IQQml of filtrate.
) was added, and the mixture was left to stand at 4° C. for 1 hour to remove the precipitate, which was thoroughly dried to obtain the desired PEG-mercaptan.

To this PEG-mercaptan 5(19 (0.05M)) was added 27.41 (·0.1M) of chloropalmitic acid.

To this mixture was added dimethylformamide 300 + nA! was added to dissolve the mixture, 1 ml of bilicin was further added, and the mixture was reacted at room temperature for 6 hours. Remove the solvent with an evaporator,
Ether was added to precipitate the target product, which was thoroughly washed with ether on the filter.

The infrared absorption depth of the thus obtained PEG-mercazotonoglumitin was measured by the wave strike method using liquid paraffin, and the results are shown in the drawing.

Usage example 10mM polyethylene glycol mercap) 50mM containing i9 lumitate and 1mM DTNB
) 81 ml of lithium-hydrochloric acid buffer (pH 7) was placed in a square cell that had been preheated to 37°C. Add this cell to 37 more
Warm to ℃ and then add 50μ of serum sample containing IJ tR-ase.
A rate assay was performed by adding t and measuring the absorbance at 412 to 405 nm at 37°C.

Sui4-ase activity was calculated using the following formula.

The measurement results are shown in the table below.

Serum Invention method Conventional method A 12.5 mU/m1 1.3 m
U/mJB 21.3 2.1C
5,40,5

[Brief explanation of drawings]

The drawing shows an infrared absorption spectrum obtained by measuring polyethylene glycol mercaptopalmitic acid ester using a paste method.

Claims (1)

[Claims] A reagent for measuring lipase activity consisting of a compound in which the carboxyl group of a fatty acid is bonded to the thiol group of a water-soluble thiol.