CS212729B2 - Method of determining glycerol ester and fatty acid contents in aqueous media,and an enzymatic agent for carrying out the method - Google Patents

Abstract

1373106 Determination of triglyceride CALBIOCHEM 30 Nov 1971 [16 Dec 1970] 55609/71 Heading G1B A glycerol ester of a fatty acid in an aqueous liquid, for example serum or milk is assayed by hydrolyzing the ester by adding a lipase and a protease to the liquid, then determining the amount of glycerol present in the resulting solution. The lipase may be from Chromobacterium viscosum or Rhizopus delemar; the protease may be chymotrypsin (exemplified), trypsin, a streptomyces griseus protease, elastase, papain or bromelin. The assay may be carried out (a) using a reagent comprising the lipase and protease, glycerol kinase, ATP, phosphoenol pyruvate (PEP), a magnesium ion source, pyruvate kinase (PK), lactate dehydrogenase (LDH) and NADH and measuring the decrease in optical density resulting from the oxidation of the NADH; (b) using the reagent as in (a), without the NADH and LDH, and reacting the pyruvate ion formed with dinitrophenyl hydrazine; (c) using the reagent as in (a), without the PEP, PK NADH and LDH, adding glycerol phosphate dehydrogenase and NAD, and measuring increase in fluorescence produced by formation of NADH, or optionally also including in the reaction mixture dinitrophenyl hydrazine which forms a coloured product with dihydroxyacetone phosphate; or (d) using a reaction mixture which comprises the lipase, protease, NAD and glycerol dehydrogenase and measuring the increase in optical density, resulting from formation of NADH, or alternatively adding dinitrophenylhydrazine to form a coloured product with the dihydroxyacetone in the reaction mixture. In other methods the NADH formed may be determined by reaction with a tetrazolium salt in the presence of a hydrogen transfer agent such as diaphorase or phenazine methosulphate to form a coloured dye.

Description

Glyeerol and fatty acid esters, such as serum triglycerides and milk fat, in aqueous media are determined by complete enzymatic hydrolysis using lipase and protease by determining glycerol released in this way. The glycerol assay method has many alternatives that can be carried out mostly in the original aqueous environment, such as: conversion to glycerol-1-phosphate by glycerol kinase, conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by the same reaction followed by conversion of ADP and phosphoenolpyruvate the use of pyruvate kinase on ATP and the pyruvate ion, which is then determined. Alternatively, the pyruvate ion so formed with nicotinamide adenine dinucleotide (oxidized form, NAD) using lactate dehydrogenase can be converted to a lactate ion and a reduced form of nicotinamide adenine nucleotide (NAA), which is then determined by optical measurement.

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The invention relates to a rapid method for determining the glycerol ester and fatty acid content of various aqueous media such as serum, milk and the like.

In many fields, especially in clinical biochemistry, it is important to determine the content of fatty acid esters and glycerol in liquid samples. This assay is particularly important in human serum, where elevated levels of these esters have a high diagnostic value. Methods for determining fatty acid residues and methods in which glycerin released from other sources contributes to the result of the assay have various disadvantages. Since the known methods are generally not entirely satisfactory, there is an intensive effort to find for clinical purposes a rapid and accurate method of determination by which only glycerin and fatty acid esters are determined exclusively but in their entirety.

The purpose of the invention is to find a rapid and accurate method of releasing glycerin from its fatty acid esters, present, for example, in aqueous media, such as serum, which allows glycerin to be determined by various alternative methods without isolating it from the test medium.

Other objects of the invention will become apparent from the following description.

Method for the determination of glycerol esters and fatty acids. in aqueous liquid by hydrolysis of the esters according to the invention consists essentially in that the hydrolysis is carried out as enzymatic hydrolysis by a lipase, preferably a microbial lipase from Rhizopus delemar or from Chromobacterium viscosum, as well as proteases selected from the group consisting of chymotrypsin, trypsin , protease from Streptomyces griseus, elestase, papin and bromelin as essential agents, the protease to lipase ratio preferably being 5 to 500 International Units per 1000 International Units of Lipase, after which hydrolysis of the glycerol released is determined and derived therefrom content of said esters.

The process according to the invention can advantageously be carried out by carrying out enzymatic hydrolysis following hydrolytic liberation of glycerol in the presence of adenosine triphosphate and glycerol kinase to convert glycerol to glycerol-1-phosphate and adenosine triphosphate to adenosine diphosphate, then determining the amount of adenosine diphosphate or glycerol-1- phosphate, which is proportional to the original content of glycerol esters and fatty acids.

The enzymatic hydrolysis of the aqueous liquid medium containing glycerol-1-phosphate is then preferably continued in the presence of nicotinamide adenine dinucleotide and glycerol phosphate dehydrogenase to convert glycerol-1 phosphate to dihydroxyacetone phosphate, while converting nicotinamide adenine dinucleotide from its oxidized form to its reduced form. detecting the originally present esters. glycerol with fatty acids determines at least one of the products of said conversions.

Alternatively, enzymatic hydrolysis of an aqueous liquid medium containing glycerol-1-phosphate and adenosine diphosphate is continued in the presence of phosphoenolpyruvate and pyruvate kinase to convert phosphoenolpyruvate to pyruvate ion, and then determine the amount of pyruvate corresponding to the amount of pyruvate. of the glycerol fatty acid esters present.

Alternatively, the process according to the invention can be advantageously carried out by carrying out enzymatic hydrolysis of the aqueous liquid medium in the presence of nicotinamide adenine dinucleotide with glycerol dehydrogenase to convert glycerol to dihydroxyacetone and nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide. of fatty acids.

It preferably uses glycerol dehydrogenases obtained from the microorganism of Enerobacter aerogenes.

The invention also provides an enzymatic reagent for carrying out said assay comprising (a) a protease selected from the group consisting of chymotrypsin, trypsin, a protease from Streptomyces griseus, an elastase, papain and bromelin, and (b) a llpase from Rhizopus delemar or from Chomobacterium viscosum.

The enzymatic agent of the invention preferably comprises 5 to 500 international units of α-chymotrypsin per 1000 international units of lipase from the microorganism Rh ^. zopus delemar.

The lipase used may be of plant or animal origin, for example a lipase from Chromobacterium viscosum, various paralipolytics. crude or purified, lipase. from Rhizopus delemar, purified, for example, by Fukumoto et al. Appl. Microbiol., 10, 257-265 (1964), and lipase with a similar effect. Examples of non-limiting proteases that may be used include chymotrypsin, trypsin, Streptomyces griseus protease (commercially available under the trademark "Pronase"), elastase, papain and bromelin. Mixtures thereof may also be used. Usually, the use of this simple enzyme mixture is sufficient, although hydrolysis can be somewhat accelerated by the simultaneous addition of a simple protein such as serum albumin, egg albumin, globullin, and the like.

As will be described in detail below, the glycerin released by the above enzyme mixture can be determined by various methods, but preferably the method described below is used.

The enzymatic hydrolysis by the combination of the lipase and protease according to the invention is preferably carried out in the presence of the components of three additional enzymatic transformation systems by simultaneously converting the liberated glycerin by the action of glycerine kinase to α-glycerine phosphate while converting adenosine triphosphate to adenosine diphosphate. adenosine diphosphate back to adenosine triphosphate with simultaneous conversion of phosphoenolpyruvic acid to pyruvate ion by pyruvate kinase; and third, pyruvate ion conversion. to the lactase ion at the same time. conversion of nicotinamide adenine disinleide from its reduced form to its oxidized form by the action of lactate dehydrogenase. All components of these systems are present in the mixture simultaneously, so as the triglyceride hydrolysis proceeds, the optical density of the solution decreases at 340 nanometers due to the oxidation of the dinucleotide. The order of enzymatic conversions can be schematically represented as follows (^ see below for the abbreviations used):

enzymatic triglycerides

.glycerol + VMK hydrolysis

GK glycerin + ATP

ADP. + PEP

o-GP + ADP

ATP + pyruvate pyruvate + NADH

lactate + NAD

· z 's colored * colorless at 340 nm at 340 nm of

Explanation of abbreviations:

VMK = free fatty acids

ATP = adenosine triphosphate α-GP = a · glycerol phosphate ADP = adenosine diphosphate

PEP =. phosphonoenolpyruvic acid LDH = lactate dehydrogenase

GK = glycerol kinase

PK = pyruvate kinase

NAD and NADH = nicotinamide adenine dinucleotide, oxidized and reduced u

ie absorbs strongly at 340 nm; molar extinction coefficient α _M = 6.22 X 10 3.

** ie transparent at 340 nm.

A suitable embodiment of the method according to the invention comprises a reagent (mixture) for the determination, containing the preferred components, for carrying out the determination of triglycerides individually. When the preferred embodiment is carried out, i.e. the method comprising the enzymatic hydrolysis described above using a combination of enzymes according to the invention, followed by the conversion of the liberated glycerol described above, it is expedient to separate the reagent for. determination in two separate containers, such as glass containers, one of which for simplicity, will be referred to as container A; it contains all the components required for the assay, with the exception of glycerol kinase, which is placed in container B. Of course, the components can also be separated in different ways; if desired, any of the ingredients applicable only to the conversion of glycerol and other steps may be added to the vessel (B). Preferably, however, the following reaction mixtures are used:

Nádobka A.

Potassium phosphate stand-by solution, 0 · M, pH 7 magnesium aspartate 1,6mg disodium salt ATP 0, 9μΜ phosphoenolpyruvate 0 ·, 9μΜ bovine serum albumin 5,0mg

NADH · up to final absorption 0,8 (optical density at 340 nm)

LDH 2 International jedn.

pyruvate kinase 6 inter. jedn.

α-chymotrypsin 1100 N. F. lipase units from the microorganism

Rhizopus delemar 1200 lipase units

(Total volume: 3 ml)

Explanatory notes:.

1) One lipase unit is the amount of enzyme that liberates fatty acids from the triolein substrate in such an amount that their neutralization after 30 minutes incubation at 37 ° C requires 1 ml of 0.05 N potassium hydroxide.

2) One N.F. α-chymotrypsin unit is the amount of enzyme that causes a change in absorption of 0.0075 per minute at 237 nm when incubated with N-acetyl-L-tyrosine ethyl ester as substrate under assay conditions. [

4¾. !

Container B _ · J

Glycerol kinase: 2 international units

The assay is performed according to the invention by adding an aliquot of liquid containing the assay triglyceride (for example 50 μ · serum) to 3 ml of the mixture in vial A. This is then incubated at 25-37 ° C for approximately 10 10 minutes. The optical density at 340 nm is then determined. 2 internationals are added. One glycerol kinase from vessel B, the mixture is allowed to stand for an additional 10 minutes at the same temperature, after which the optical density is again determined. The difference is proportional to the triglyceride content of the aliquot.

Thus, the basic components of the reaction mixture according to the invention, especially in the individual assay packages, are: microbial lipase, protease, pyruvate kinase, lactate dehydrogenase, NADH, ATP, phosphonolpyruvate, magnesium ion source, buffer and glycerol kinase.

An alternative procedure is to omit NADH and lactate dehydrogenase from the above mixture so that only the first three reaction steps of the above reaction sequence are shown and a sufficient amount of di-nitrophenylhydrazine is added to react with the pyrivate ion formed in said third reaction. degree. The reaction product is colored after alkalization and can be conveniently detected by colorimetry.

According to another alternative, phosphonenolpyruvate, pyruvate kinase and lactate dehydrogenase can be omitted from the reaction mixture described above, instead glycerol phosphate dehydrogenase is added, whereupon the α-glycerol phosphate is converted to dihydroxyacetone phosphate and NAD to. NADH. The amount of NADH formed, which is proportional to the amount of triglyceride initially present, can suitably be determined by increasing the fluorescence. According to one alternative sub-procedure, a sufficient amount of dinitrophenylhydrazine is added to the system, resulting in the formation of a colored reaction product with liberated dihydroxyacetone phosphate. The amount of reaction product can then be determined colorimetrically and is also proportional to the amount of triglyceride initially present.

Yet another alternative method is that the reaction mixture utilizes only an enzymatic conversion to glycerol, which is the first reaction step shown in the above scheme. However, the reaction mixture also contains NAD and glycerol dehydrogenase. As a result, the glycerol released in the first step is converted to dihydroxyacetone while forming NADH. The measure of the amount of triglyceride initially present is then, as already mentioned, the increase in optical density at 340 nm. A suitable glycerol dehydrogenase is that dehydrogenase obtainable from the microorganism Enterobacter aerogenes; This enzyme can be obtained from the store. In one alternative process, dinitrophenylhydrazine is added to form a colored reaction product with dihydroxyacetone, the amount of which can be determined colorimetrically.

Two of the alternative processes just described, firstly, the process using glycerol phosphate dehydrogenase, and secondly, the process using glycerol dehydrogenase, as mentioned above, give an equivalent amount

NADH. Another sub-process applicable to both of these processes consists in utilizing the known behavior of various tetrazolium salts, which are reduced from colorless water-soluble compounds to colored dyes by reduction. NADH, which is quantitatively derived from triglyceride, can be used to transfer its hydrogen to the tetrazole salt (being oxidized during the process), again quantitatively, by any of several known substances, among which diaphorases are particularly preferred or alternatively phenazine methosulfate. The amount of dye thus formed can be readily determined colorimetrically, i.e. by measuring the change in optical density in the visible region.

It is not necessary to give all the details of the particular procedures just mentioned, as they are fully documented in the literature. Representative sources in which reference is also made to other literature are the following articles: American Journal of Clinical Pathology, Volume 45, No. 5, May 1966: "Rapid Colorimetric (Tetrazolium Salt) Assay for Lactate Dehydrogenase" by RO Briere, JA Preston and JG Batsakis; Wethods of Enzyme Analysis ”, by H. U. Bergmeyer, Academic Press, New York, 1965; pp. 953 to 955.

With respect to the ratio of the selected lipase (or mixture of selected lipases) to the selected protease (or mixture thereof), it is preferably used per 1000 units of lipase present in the mixture for determination, 5 to about 500 international units of protease. a range of 20 to 100 international protease units was found.

The lipase unit has already been defined above. The international unit of proteolytic efficacy is the amount of protease which causes, per minute, the conversion of 1 micromole of the substrate specific for the particular enzyme under consideration, under conditions approaching the optimum for the system under consideration. Thus, for chymotrypsin, the substrate is ethyl tyrosine ester and the rate of conversion can be determined in various ways, such as by changing the optical density at 237 nm or by determining the liberated amino acid, such as tyrosine equivalents of the phenolic reagent or formol titration.

The N.F. (National Formulary) unit is occasionally used for proteases; this unit is shown in the above example. One International Unit is equivalent to 28 N.F. units, so that 1100 NF of chymotrypsin units in the example is equal to 39 International Units. Since 1200 lipase units were present in the blend of the Example, it is apparent that for every 1000 lipase units in the Example there are about 32 International Protease Units.

From the foregoing, it can be seen that the distribution of the components of the preferred assay reagent mixture between containers A and B has one container, for example container A, containing at least lipase and a protease, while the other container, for example B, should contain at least glycerol kinase. The other ingredients can be divided into both vials at will. However, the above distribution is preferred.

In the process according to the invention, the glycerol esters are completely hydrolyzed so that, as already explained, a stoichiometric amount of glycerin is released. There is no explanation for the particular reaction mechanism that occurs, but it is clear that this is due to the co-presence of lipase and protease as described and explained above.

For example, when following the above detailed embodiment and using human serum as the test aqueous medium, it is found that:

Claims (8)

SUBJECT Method for determining the content of glycerol and fatty acid esters in an aqueous liquid medium by hydrolysis of said esters, characterized in that the hydrolysis is carried out as enzymatic hydrolysis by lipase, preferably microbial lipase from Rhizopus delemar or Chromobacterium viscosum, as well as proteases selected from the group comprising chymotrypsin, trypsin, a protease from Streptomyces griseus, elastase, papain and bromelin as essential agents, the protease to lipase ratio preferably being 5 to 500 international units per 1000 international lipase units, after which the glycerol released is determined after hydrolysis; from which the content of said esters is derived. 2. Process according to claim 1, characterized in that after hydrolytic release of glycerol, enzymatic hydrolysis is continued in the presence of adenosine triphosphate. and glycerol kinases for converting glycerol to glycerol-1-phosphate and adenosine triphosphate to adenosine diphosphate, then determining the amount of adenosine phosphate or glycerol-1-phosphate present, which is proportional to the original glycerol ester and fatty acid content. 3. The method of claim 2, wherein the enzymatic hydrolysis of the aqueous liquid medium containing glycerol-1-phosphate is continued in the presence of nicotinamide adenine dinucleotide and glycerol phosphate dehydrogenase to convert glycerol-1 phosphate to dihydroxyacetone phosphate, and simultaneously converting nicotinamide adenine dinucleotide. of the oxidized form to the reduced form, whereupon the amount of glycerol liberated to detect the initially present glycerol fatty acid esters is the amount that can be expected based on complete hydrolysis of the triglycerides present, as would also be determined by well known standard procedures. The term "glycerol phosphate" refers to a compound also referred to as glycerol-1-phosphate, serum glycerin and fatty acid esters may and are generally referred to simply as triglycerides. By one lipase unit is meant an amount of lipase equivalent to one lipase unit as defined above. The precise details of the ingredients and processes as described above do not limit the invention; it is intended that other modifications thereof be within the scope of the invention, of course to those skilled in the art. OF THE INVENTION new at least one of the products of said transformations. 4. The method of claim 2, wherein the enzymatic hydrolysis of the aqueous liquid medium containing glycerol-1-phosphate and adenosine diphosphate is continued in the presence of phosphoenolpyruvate and pyruvate kinase to convert phosphoenolpyruvate to a pyruvate ion and determining the amount of pyruvate corresponding to the amount of glycerol fatty acid esters initially present. 5. The method of claim 1, wherein the enzymatic hydrolysis of the aqueous liquid medium is continued in the presence of nicotinamide adenine dinucleotide and glycerol dehydrogenase to convert glycerol to dihydroxyacetone and nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide, glycerol and fatty acids. 6. The method of claim 5, wherein glycerol dehydrogenase derived from Enterobacter aerogenes is used. 7. An enzyme reagent for carrying out the method of claim 1 comprising: a) a protease selected from the group consisting of chymotrypsin, trypsin, a protease from Streptomyces griseus, dastase, papain, and bromelin, and bj a lipase from Rhizopus delemar; from Chromobacterium viscosum. 8. The enzymatic agent of claim 7 comprising from about 5 to about 500 International Units of [alpha] -cytmotrpsin per 1000 International Units of Rhizopus delemar lipase.