JPS6121099A - Reagent for measuring lipase activity - Google Patents

Abstract

PURPOSE:The titled water-soluble reagent, consisting of an ester compound of a water-soluble compound with a hydroxycarboxylic acid and 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 an ester compound having carboxyl group of a 5-22C fatty acid, e.g. lauric acid, palmitic acid or coconut oil fatty acid, linked to hydroxyl group of a hydroxycarboxylic acid, e.g. lactic acid or alpha-hydroxybutyric acid, and hydroxyl group of a water- soluble compound, e.g. polyethyleneglycol having about 200-20,000mol.wt. linked to the carboxyl group of the above-mentioned hydroxycarboxylic acid. In measurement, covered lipase is brought into contact with the above-mentioned reagent, which is decomposed into the water-soluble compound n-hydroxycarboxylic acid ester and the fatty acid. The above-mentioned ester is then decomposed with esterase, and the formed hydroxycarboxylic acid is determined by the colorimetry, etc. to obtain the lipase activity.

Description

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

(Prior art and problems to be solved by the invention) The conventional method for measuring rehase activity involves suspending olive oil or triolein in an appropriate buffer solution, allowing lipase to act on the suspension, and measuring the change in turbidity. However, this method has problems in sensitivity and reproducibility, such as the activity of the expressed linocuse differing depending on the suspension method or suspension state. In addition, in the past, there was a method by Cherry-Grandall et al. that involved alkaline titration of free fatty acids, but there was a problem in that the titration operation was difficult and was likely to cause individual differences. In addition, methods using synthetic substrates such as α-naphthol derivatives have been developed, but these problems tend to cause errors because the substrates are insoluble in water and other esterases such as allyl esterases also act on them. there were.

In order to solve these problems and develop a method to accurately and easily measure the activity of linosose, the present inventors conducted various studies and found that fatty acid esters of alcohol polymers ( Patent application 1972-272
No. 96) and sugar fatty acid esters (Patent Application No. 59-8858)
No. 9) was developed, and the inventors discovered that using this substrate could solve all of the above-mentioned problems and accurately and easily measure iR-se activity, and have already filed a patent application for this discovery.

(Means to Solve the Problem) The present inventors further investigated and found a new substrate that solved the above problem, a compound in which a water-soluble compound, an oxycarboxylic acid, and a fatty acid are ester bonded. As a result, the present invention was completed.

That is, the present invention relates to a reagent for measuring lipase activity, which is a compound in which a fatty acid carboxyl group having 5 to 22 carbon atoms is bonded to the hydroxyl group of an oxycarboxylic acid, and a hydroxyl group of a water-soluble compound is bonded to the cardexyl group. It is.

The oxycarboxylic acid is preferably one that is easy to quantify colorimetrically and that allows lipase to easily act on the fatty acid bound to the hydroxyl group, such as lactic acid, α-oxybutyric acid, glyceric acid, glycolic acid, phosphoric acid, and tartaric acid. , citric acid, etc. are suitable. Among these, lactic acid and α-oxybutyric acid are particularly preferred.

Suitable fatty acids have a carbon number of about 5 to 22, such as lauric acid, palmitic acid, stearic acid, behenic acid, oleic acid, lylunic acid, arachidonic acid, coconut oil fatty acid, and hydrogenated beef fatty acid.

Water-soluble compounds have hydroxyl groups, such as alcohol polymers and sugars. Alcohol polymers include polyethylene glycol (PEG), polyvinyl alcohol (
PVA), etc., and sugars include glycose, maltose, sucrose, xylose, chitosan, starch, etc. The molecular weight is preferably about 200 to 20,000, and 10
About 00 to 2000 is particularly suitable.

The method of ester bonding these may be carried out according to a known method, and any method such as a method using an acid catalyst, a method using an alkali catalyst, an acid chloride method, an acid amidation method, etc. can be used. There are no particular restrictions on the esterification site. Some oxycarboxylic acids have one hydroxyl group and one carboxyl group, but for example, glyceric acid and tartaric acid have two hydroxyl groups, and malic acid, tartaric acid, and citric acid have two carboxyl groups.
There are two or three locations. Furthermore, some fatty acids have two or more carboxyl groups, such as dicarboxylic acid and tricardic acid. As for water-soluble compounds, polyethylene glycol has hydroxyl groups at both ends, and polyvinyl alcohol has hydroxyl groups for each polymerized unit. Therefore,
The compounds of the reagent of the present invention include oxycarboxylic acids, fatty acids,
and a water-soluble compound may be combined in various molar ratios, but usually they may be combined in a molar ratio of 1:1:1.

However, this molar ratio may be changed as appropriate depending on measurement sensitivity and the like.

As a method for measuring lipase activity using the reagent of the present invention, first, a lipase to be measured is allowed to act on a compound of this reagent, and this compound is decomposed into a water-soluble compound-oxycarboxylic acid ester pv and fatty acid. Subsequently, esterase is allowed to act on this ester to decompose it into a water-soluble compound and oxycarboxylic acid, and this oxycarboxylic acid may be quantified by a known method.

The conditions under which IJ ie-ase and esterase are allowed to act may be determined by taking into consideration the activity of the enzymes, the optimum PH1, the optimum temperature, etc. Esterase may be added after the lipase reaction, but it is more convenient to add it together with lipase.

After the reaction, the produced oxycarboxylic acid or water-soluble compound may be quantitatively determined. Generally, a colorimetric method is convenient for quantitative determination. Colorimetric assay methods are known for many oxycarboxylic acids, and can be used. In the case of water-soluble compounds, for example polyethylene glycol oxidase (PEGO)
) and colorimetrically quantify the decrease in absorption of dichlorophenolindophenol, a hydrogen acceptor. The reaction in this method proceeds as follows.

At the time of reaction (II), hydrogen is transferred to dichlorophenolindophenol, and dichlorophenolindophenol is reduced from blue to colorless leuco form. Polyethylene glycol is produced by the action of PEGO (■)
, (2), (g), and (V) proceed, and dichlorophenolindophenol is reduced in each reaction. Therefore, the reduction of dichlorophenol indophenol is amplified according to the degree of polymerization of polyethylene glycol, and for example, when the degree of polymerization (n) of polyethylene glycol is about 20, the reduction is about seven times. The type and origin of PEGO used in this method does not matter, but for example, how many PEGO units (A
pplied andF: nvironmental
Microbio1ogyL978. April, 679-
684) can be used. In the case of polyvinyl alcohol, the existence of bobbinyl alcohol oxidase is known. This method, which was newly developed by the present inventors, has the advantage of being extremely easy to measure and also greatly improving the measurement sensitivity due to the effect of amplifying the reaction.

There are no particular restrictions on the types of shirururi and yase that can be measured using the method of the present invention. It can measure any type of lipase, including lipase derived from various microorganisms such as lipase, mold, yeast, and bacteria.

(Action and Effect of the Invention) The reagent of the present invention is water-soluble and is easily acted on by lipase.
, I) i+-ase activity can be measured with high sensitivity and high specificity. The method for measuring IJ 9-ase activity using the reagent of the present invention is simple and can be determined by both the rate assay method and the end point method.

(Example) Synthesis Example I PEG-lactic acid-palmitic acid ester pore e
1.8.9 (0.02 mol) and normitic acid chloride 5.5.9 (0.02 mol) were added in dimethylformamide and reacted at room temperature for 5 hours. After the reaction was completed, unreacted substances were extracted and removed with petroleum ether, and dimethylformamide was removed by evaporation. The residue was subjected to silica gel chromatography to obtain lactic acid-palmitic acid ester.

Esther with 3. ! ? (0,01 mol) and PEG 10
00 (average molecular weight 11,000) was added to dimethylformamide 100-, 2.1 g of DCC was added thereto, and the mixture was reacted at room temperature overnight. The reaction solution was filtered through a G-3 glass filter, and the solvent was removed from the tear fluid using an evaporator. Ether was added to the remaining viscous liquid and the precipitate was collected to obtain the desired PEG-lactic acid-palmitic acid ester. FIG. 1 shows an infrared absorbing slit of the obtained ester obtained by the coating method.

Synthesis Example 2 α-oxybutyric acid 2.1, p (0.02 mol) and hyoleic acid chloride 6,1, @ (0.02 mol) were added to 100 mIV of trimethylformamide and reacted at room temperature for 5 hours. After the reaction was completed, unreacted substances were extracted and removed with petroleum ether, and dimethylformamide was removed using an evaporator. The residue was purified by silica gel chromatography to obtain α-oxybutyric acid-oleic acid ester.

3.7 g (0.0 t mol) of this ester and 50 PVA
0 (average molecular weight 500.) 1.5. ! 7 (0,003 mol) was added to 100 ml of trimethylformamide, 2.7 g of DCC was added thereto, and the mixture was reacted at room temperature overnight. The reaction solution was filtered through a G-3 glass filter, and the solvent was removed from the p solution using an evaporator. Ether was added to the remaining viscous liquid and the precipitate was collected to obtain the desired PVA-α-oxybutyric acid-oleic acid ester.

Use example 1 10 mM PEG-lactic acid-palmitic acid ester,
A square shaped solution containing 3000 units of microorganism-derived esterase, 2000 units of yeast-derived lactate dehydrogenase, and 2.4 mM NAD+ (pH 8.2, 50mM) Lys-HC4 buffer 2- was heated to 37°C in advance. It was placed in a cell and prewarmed for 3 minutes. Next, 50 serum samples containing rhinocuses were collected.
- Rate assay was performed by adding μl and measuring the absorbance at 340 nm.

Su-zo-ze activity was calculated using the following formula.

The results obtained are shown in the table below.

Serum Invention method Nephelometry A 2.8 U/l O, 81 B
0.51# 0.13 /IC20
, 3#6.1 Usage example 2 5mM PVA-α-oxybutyric acid-oleic acid ester)
P) I8 containing 3000 units of esterase, 2000 units of α-oxybutyrate dehydrogenase, 2.4 mM NAD+
．． 50mM of IJ-HCt buffer 2- was placed in a square cell preheated to 37°C and prewarmed for 3 minutes. Next, Su/? - Add 50 μl of serum sample containing enzyme,
Rate assay was performed by measuring absorbance at 340 nm.

Rhino-yase activity was calculated using the following formula.

The results obtained are shown in the table below.

Serum Invention method Nephelometry D 3.4 UAO, 8U/AE2.1
〃0,53// F 5.31 1.41#

[Brief explanation of drawings]

The drawing shows an infrared absorption spectrum obtained by measuring PEG-lactic acid-glumitic acid ester using the yeast method.

Claims (1)

[Claims] A reagent for measuring lipase activity comprising a compound in which a carboxyl group of a fatty acid having 5 to 22 carbon atoms is bonded to the hydroxyl group of an oxycarboxylic acid, and a hydroxyl group of a water-soluble compound is bonded to the carboxyl group.