JPH0649000B2 - Reagent for measuring lipase activity - Google Patents

Description

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

(Problems to be Solved by Conventional Techniques and Inventions) A conventional method for measuring lipase activity is to suspend olive oil or triolein in an appropriate buffer solution, and allow lipase to act on the suspension to measure changes in turbidity. However, this method has problems in sensitivity and reproducibility, such as the suspension method or the activity of lipase expressed depending on the suspension state. In addition to this, there was a problem that the method of Cherry-Grandall et al. For alkali titrating free fatty acids used to be difficult to carry out the titration operation, and thus individual differences tend to occur. Further, although a method using a synthetic substrate such as an α-naphthol derivative has been developed, there is a problem that the substrate is insoluble in water, and an error is likely to occur due to other esterases such as allyl esterase also acting. It was.

The present inventors have conducted various studies in order to solve these problems and develop a method for accurately and simply measuring the activity of lipase, and as a result, as a novel lipase substrate having water solubility, a fatty acid ester of an alcohol polymer ( Japanese Patent Application No. 59-27296) and sugar fatty acid ester (Japanese Patent Application No. 59-88589) were developed. By using this substrate, all the above problems can be solved and the lipase activity can be measured accurately and simply. I found out that I had applied for a patent for the contents.

(Means for Solving Problems) The present inventors have further studied 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 for solving the above problems. The present invention has been completed.

As the water-soluble thiol, a thiol group may be introduced into a known water-soluble compound. An alcohol polymer can be used as the water-soluble compound. The alcohol polymer is polyethylene glycol (PEG), polyvinyl alcohol (PVA) or the like. The molecular weight is preferably about 200 to 20000, particularly about 1000 to 2000.

The method of introducing a thiol group into the water-soluble compound may be selected from known methods depending on the functional group, for example, a method of halogenating thiourea to change a hydroxyl group into a thiol group, sodium oxide, etc. The method of reacting hydrogen sulfide in the presence of the catalyst can be used.

The fatty acid preferably has about 5 to 22 carbon atoms, and examples thereof include lauric acid, palmitic acid, stearic acid, behenic acid, oleic acid, linolenic acid, coconut oil fatty acid, and hardened beef tallow fatty acid.

The method for binding the thiol group of the water-soluble thiol and the carboxyl group of the fatty acid may be carried out according to a known method, a method of using an acid catalyst, a method of acid chloride formation,
Any method such as acid amidation can be used.

Since two or more thiol groups can be introduced into one molecule of the water-soluble compound, 2 mol or more of fatty acid can be introduced per mol of the water-soluble thiol. For example,
In the case of PEG, one or both of the hydroxyl groups at both ends can be changed to a thiol group and a fatty acid can be bound to it, and in the case of PVA, about 1/1000 to 1/3 of all hydroxyl groups can be thiolated to form a fatty acid. Can be combined. In the case of other water-soluble thiols, the binding molar ratio may be determined appropriately in consideration of measurement sensitivity and the like.

Examples of such compounds include PEG-mercapto palmitate ester, PEG-dimercapto dipalmitate ester, PVA-mercapto oleate ester and the like.

As a method for measuring lipase activity using the reagent of the present invention, first, a lipase which is a measurement target is allowed to act on a compound of this reagent, and this compound is decomposed into a water-soluble thiol and a fatty acid, and the produced SH group is known. Method, for example, a colorimetric method using Ehlmann's reagent.

The conditions for lipase action are the activity of this enzyme, optimum pH,
It may be set in consideration of the optimum temperature and the like.

The type of lipase that can be measured by the method of the present invention is not particularly limited, pancreatic juice, gastric juice, serum, lipase derived from various body fluids such as urine, castor, lipase derived from seeds of rapeseed, mold, yeast, bacteria. For example, lipase derived from various microorganisms such as lipase can be measured.

(Operation and Effect of the Invention) The reagent of the present invention is water-soluble, and lipase easily acts on it, 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 simple and can be quantified by both the rate assay method and the end point method.

(Example) Synthetic Example 100 g (0.1 M) of PEG1000 was dissolved in about 200 ml of benzene with heating, and 1 ml of pyridine and 14.6 ml of thionyl chloride (0.
2M) was added and reacted at room temperature for 15 hours. Benzene and unreacted thionyl chloride were removed from the reaction solution by an evaporator, the residue was thoroughly washed with ether, and then dried.

50 g (0.05 M) of PEG chloride thus obtained was dissolved in 200 ml of ethanol, 7.6 g (0.1 M) of thiourea was added, and the mixture was reacted for 3 hours under reflux. 1M NaOH 100
After adding ml, the mixture was refluxed for 1 hour, and then the solvent was removed by an evaporator. 100 ml of absolute ethanol was added to the residue to dissolve it, and the insoluble matter was removed by filtration. 300 ml of ether was added to 100 ml of the liquid, and the mixture was allowed to stand at 4 ° C. for 1 hour to collect a precipitate, which was thoroughly dried to obtain the desired PEG-mercaptan.

To 50 g (0.05 M) of this PEG-mercaptan was added 27.4 g (0.1 M) of palmitic acid chloride. To this mixture, 300 ml of dimethylformamide was added and dissolved, 1 ml of pyridine was further added, and the mixture was reacted at room temperature for 6 hours. The solvent was removed with an evaporator, ether was added to precipitate the desired product, and the product was thoroughly washed with ether on the filter.

The infrared absorption spectrum of the PEG-mercapto-palmitin thus obtained was measured by the paste method using liquid paraffin, and the results are shown in the drawing.

Example of Use 1 ml of 50 mM Tris-hydrochloric acid buffer pH 7.8 containing 10 mM polyethylene glycol mercapto palmitate and 1 mM DTNB was preheated to 37 ° C. and placed in a rectangular cell. After further heating this cell to 37 ° C, 50 µl of a serum sample containing lipase was added, and the cells were heated at 37 ° C to 412-4
A rate assay was performed by measuring the absorbance at 05 nm.

The lipase activity was calculated by the following formula.

The measurement results are shown in the table below.

[Brief description of drawings]

The drawing shows an infrared absorption spectrum obtained by measuring polyethylene glycol mercapto palmitate by a paste method.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Biochim Biophys Acta, 795 (2), 1984, p. 212-220 J Clin Chem Clin Biochem, 20 (8), 1982, p. 537-552 Biochim Biophys Acta, 572 (3), 1979, p. 519-530 J Lipid Res, 22 (3), 1981, p. 496-505

Claims (1)

[Claims] 1. A reagent for measuring lipase activity, which comprises an ester in which a thiol group of a water-soluble thiol compound selected from a thiol group-introduced polyethylene glycol or polyvinyl alcohol is bound to a thiol group having a fatty acid having 5 to 22 carbon atoms.