JPS63283596A - Determination of free fatty acid and determinating reagent composition used therein - Google Patents

Abstract

PURPOSE:To enable to stably determine free fatty acid in a specimen, especially free fatty acid in blood on clinical inspection in good precision, by using a specific fatty acid or water soluble salt thereof as a part of reagents. CONSTITUTION:The first reagent containing at least ATP, CoA and acyl CoA synthesizing enzyme is reacted with a free fatty acid in a specimen to afford CoA. Then the second reagent containing a fatty acid expressed by formula I (R is 1-20C aliphatic group; n is integer of 0, or 1-2; A is -CH=CH- or -CHY-CHZ-; Y and Z are H, hydroxyl group or lower alkyl group) or water soluble salt thereof and acyl CoA oxidation enzyme and as necessary further reagent for measuring hydrogen peroxide by colorimetric method is reacted with the above-mentioned acyl CoA and change caused by oxidation of acyl CoA, e.g. produced hydrogen peroxide is measured.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for quantifying free fatty acids, and more specifically, a method for quantifying free fatty acids suitable for quantifying free fatty acids in blood in clinical tests, and a method for quantifying free fatty acids used therein. This invention relates to a quantitative reagent composition.

[Conventional technology]

Quantification of free fatty acids (hereinafter abbreviated as NEFA) in blood is as follows:
It is said to be clinically significant because it reflects the energy status based on in vivo lipid metabolism.

Regarding the quantitative determination of NEFA, the titration method reported by Dole [V, P, Dole, Journal of Observatory Investigations (J, Cl1n.

Invest, ) 35.150. 1!358], many quantitative methods have been reported since then. For example, N
Cobalt in EFA [Novak, M.; Journal
of Lipid Research (Novak, M.; J.
Lipid, Res, ) 6 4311985]
Or copper [Dancombe W, Clinica]
Kimika Acta (Dun, combine, W, G-, Cl
1n, Chin, Acta) 9122, IH
A method of colorimetric determination as a cobalt salt or copper salt of NEFA by reacting NEFA with acyl-coenzyme A in the presence of adenosine triphosphate (hereinafter abbreviated as ATP) and coenzyme A (hereinafter abbreviated as CoA). 10&
Acyl coenzyme A lt lower acyl Co produced by reacting with M element (hereinafter abbreviated as AC5) and producing the following reaction.
A) and adenosine monophosphate (hereinafter abbreviated as AMP)
Or consumed C.

A method for measuring A - the so-called enzymatic method - etc. have been reported.

Reaction formula I: NEFA + ATP + CoA CS Acyl CoA + AMP + pyrophosphoric acid (I) Among these quantitative methods, the colorimetric method using cobalt or copper is complicated to operate and has poor measurement accuracy, so it is based on reaction formula I using AC3. Quantitative methods became popular.

Among them, acyl C produced in reaction formula I.

The method of oxidizing A using acyl coenzyme A oxidase (hereinafter abbreviated as ACOD) according to the following reaction formula H is a method for measuring AMP that has a problem with measurement accuracy,
This method is currently frequently used because it is superior in terms of operability, accuracy, sensitivity, etc. compared to the method of measuring CoA consumption, which has a limited quantitative range for NEFA.

Reaction formula ■: ACOD acyl-CoA + 02 2.3-1 lansenoylconzaeim A + hydrogen peroxide (■) By the way, the oxidation reaction of acyl-CoA by ACOD is inhibited due to the reducing effect of CoA added to the reaction system as a reagent. receive. Therefore AC
In order to put a measurement system using OD into practical use, it was necessary to suppress the influence of unreacted CoA in Reaction Formula I by adding an SH reagent such as N-ethylmaleimide (hereinafter abbreviated as NEM) (Japanese Patent Publication No. 57 -33955 publication).

However, since ACOD is an enzyme having an SH group in its active center, if it is allowed to coexist with an SH reagent for a long time, it will cause structural destruction, leading to a decrease in the stability of the reagent itself.

[Purpose of the invention]

The present invention provides a method for quantifying NEFA using AC3 and ACOD, which can improve the stability of the reagent without impairing the operability, accuracy, sensitivity, etc. of conventional methods, and a reagent composition therefor. is intended to provide.

[Structure of the invention]

In the present invention, free fatty acids in a sample are treated with AC3 in the presence of ATP and CoA, and the resulting acyl-CoA is treated with A
A method for quantifying NEFA characterized by measuring changes caused by oxidizing acyl-CoA by applying COD, and a first reagent containing at least ATP, CoA and AC3, and at least a fatty acid represented by the following general formula I or an aqueous solution thereof and a second reagent containing an ACOD salt.
This invention relates to a reagent composition for quantifying FA.

General formula I: R-A-(CH2)n-COOH (wherein, R represents an aliphatic group having 1 to 20 carbon atoms, and n is O
or represents an integer of 1 to 2, A is -CH=CH- or Z Y and Z represent a hydrogen atom, a hydroxyl group or a lower alkyl group, provided that Y and Z do not represent a hydrogen atom at the same time) In the present invention General formula above! The fatty acid represented by is subjected to the action of AC3 in the presence of CoA and ATP added as reagents according to the reaction represented by the following reaction formula m, and is converted to
Produces a substance that cannot be a substrate for D.

Reaction formula ■: FA + COA + ATP C3 - - - → FA -Co A + A M P + pyrophosphoric acid (m) (wherein, FA represents the fatty acid represented by the above general formula 1,
FA-CoA indicates a substance that cannot be a substrate for ACOD (7)) The above general formula! If we enumerate the relationship between the fatty acid represented by and substances that cannot be substrates for ACOD, which are produced based on this fatty acid according to reaction formula ①, for example, those shown in Table 1 are listed as specific substances. It will be done.

Table 1 The use of the fatty acid represented by the above general formula I is determined based on the expected degree of NEFA in the sample and the amount of Co present in the reaction solution.
Although it depends on the absolute amount of A, the molar ratio to CoA is preferably 1 or more, preferably about 5 to 50.

Furthermore, ACOD is preferably allowed to act within the range of pH 8.0 to 8.5.

In the method for quantifying NEFA according to the present invention, methods for detecting changes caused by the action of ACOD include the following.

(2) Hydrogen peroxide produced by the action of ACOD is measured by a known method.

(2) Measure the enzyme consumed by the action of ACOD using a known method.

Among them, the colorimetric method for measuring hydrogen peroxide, which is currently frequently used in the field of clinical testing, is the same method used in the NEFA quantitative method, so it does not require special equipment or skill. Therefore, this is the preferred method.

Substances other than the fatty acid of general formula 1 in the composition for quantitative determination of NEFA according to the present invention can be the same as those used in compositions for quantitative determination based on conventional enzyme methods. That is, an appropriate buffer such as phosphoric acid, boric acid, citric acid, etc., and if necessary, a preservative, a known stabilizer, etc. can be added to the first reagent. Similarly, for the second reagent, buffers, preservatives, stabilizers, etc. can be added. Furthermore, the first reagent or the second reagent contains ACO
It is also possible to coexist a reagent system that can detect changes caused by the action of D.

A widely known example of such a reagent system is a reagent system for measuring hydrogen peroxide produced by the action of ACOD. Specifically, examples include those using peroxidase (hereinafter abbreviated as POD) and catalase. In these methods, a pigment is produced by the action of POD or catalase, based on, for example, the following reaction formulas 〇 to 〇, and this is colorimetrically quantified.

■ Hydrogen peroxide + 4-7 minoantipyrine + aniline compound or phenol compound OD Quinone dye H20 t7 Hydrogen peroxide benzidine compound OD Quinone dye H20 The reagent composition according to the present invention can be used not only in a dissolved state but also in a lyophilized state It is also possible to provide it in a dry state depending on the method.

[Action of the invention]

The fatty acid represented by the general formula 1 in the method and reagent composition for quantifying NEFA according to the present invention has the effect of suppressing CoA from interfering with the reaction represented by the reaction formula (2) that proceeds due to the action of ACOD. That is, in the reaction formula I, C that did not participate in the reaction with NEFA
oA and ATP, a fatty acid represented by the general formula 1 and A
By reacting in the presence of C3, it becomes a substance that does not act on ACOD, and it is taken out of the reaction system.

〔Example〕

The present invention will be explained below using examples.

Example 1 NEFA was quantified using the following first and second reagents.

01st reagent/phosphate buffer (PH8,8) 50■A・A
TP 2■N・Magnesium chloride・Hexahydrate 5mM・4-7 minoantipyrine 2mM・CoA lithium salt 0.2P・AC3(
Specific activity 2U/Peng Protein) 0.3Peng/s Bun・) IJ To 7X -1000, 1g/d
JlO second reagent/phosphate buffer (pH 8,8) 50 ml/N-
Ethyl-N-(0-methylsulfonamidoethyl) m-)luidine 2. Fatty acid represented by the above general formula I (see Table 2) 2. Tribute. POD (specific activity 100 U/mg protein) 0.04 Simple g/■ 交・
ACOD (specific activity 20U/sg protein) 0.25g/■mi・
)! Jton
) 7X -1005g/di solution 10ml 1k:
After dissolving, the total volume was made up to 100% using the same solution.

The measurement operation is as follows.

■ Mix 50 samples and the first reagent IS, and react at 37°C for 5 minutes.

(2) Add two more layers of the second reagent and react at 37°C for 5 minutes, then measure the absorbance at 555 nm.

As a conventional method, NEMlm was used instead of the fatty acid in the second reagent.
Quantification was performed in the same manner using a sample containing M, and the obtained results were compared.

The results are shown in Table 2.

Table 2 Effect of suppressing the influence of CoA by the fatty acid represented by the general formula I. The optical density when NEM was used was about 0.26.

Example 2 The relationship between the results obtained by the NEFA quantitative method according to the present invention using 3-octenoic acid as the fatty acid represented by the general formula (1) and the conventional method using NEM was investigated.

The method for quantifying NEFA according to the present invention followed the same procedure as in Example 1, and the conventional method used a commercially available reagent for quantifying NEFA containing NEM, and the procedure was performed according to the instruction manual.

In addition, for each quantitative method, 501L is required separately from the sample determination.
The same operation was performed using jL of purified water to determine the optical density of the reagent blank, and the NEFA value (
JLE q/jL) was calculated. The samples were 15 human serum samples, and the standard sample was too much oleic acid.

A JLE q/l (30.45 mg/dll, same as that prepared in Example 1) solution was used.

Calculation formula As shown in Table 3, there was a good correlation between the method for quantifying NEFA according to the present invention and the conventional method.

Table 3 Incidentally, the calibration curve in the method for quantifying NEFA according to the present invention had good linearity passing through the origin O, as shown in FIG.

Example 3 The stability of ACOD was investigated when 3-octenoic acid (invention) or NEM (conventional method) was added to the second reagent.

A second reagent containing two portions of 3-octenoic acid or one layer of NEM is left in a dissolved state under the following two conditions;
The reactivity of the reagents was compared.

0 condition 1: The sample was left at 25° C. for 4 days, a quantitative operation was actually performed using serum, and the time course of the second reagent was observed.

The results are shown in FIGS. 2 and 3.

O condition 2: The sample was left for 24 days at 2-1θ°C, and a quantitative operation was actually performed using serum to observe the time course of the second reagent. The results are shown in FIGS. 4 and 5.

In conditions 1 and 2, in order to confirm the stability of the reagent system for measuring peroxide water J in the second reagent, a hydrogen peroxide solution was added instead of the standard sample and the state of color development was observed. There was almost no difference in reactivity between the reagents in Figures 2 to 5, which was caused by the addition of NEM.
This was thought to be due to a decrease in COD activity.

In addition, under condition 1, the specific activity of ACOD was determined by O8UMI et al.
al, Biochem, Biophys, Res
, Goms, 83.2.479-485 (1978
)) Measurements were made according to the method described, and changes in activity during storage were observed. As a result, the ACOD activity immediately after reagent preparation was 1
00, the ACOD activity after 4 days was 18.8% with the conventional method using NEM and 80.1% with the present method using 3-octenoic acid, so it is important to avoid adding NEM. It was clear that the decrease in ACOD activity was significantly suppressed.

〔Effect of the invention〕

According to the present invention, NEFA does not use an SR reagent such as NEM, which is considered to have an adverse effect on the storage stability of ACOD.
and a reagent composition for the same. Improving the stability of reagent components is considered to have a particularly great effect on reagents for automatic analyzers, which are often supplied in the form of solutions.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the calibration curve in the NEFA quantitative method according to the present invention, FIGS. 2 and 4 are graphs showing the calibration curve according to the conventional method, and FIGS.
The figure is a graph showing the stability of ACOD according to the method of the present invention.

Claims (7)

[Claims] (1) Acyl-coenzyme A synthetase is allowed to act on free fatty acids in a sample in the presence of adenosine triphosphate and coenzyme A, and the resulting acyl-coenzyme A is expressed by the following general formula:
1. A method for quantifying free fatty acids, which comprises allowing acyl-coenzyme A oxidase to act in the presence of a fatty acid represented by I or a water-soluble salt thereof, and measuring changes caused by oxidation of acyl-coenzyme A. General formula I: R-A-(CH_2)_n-COOH (wherein, R represents an aliphatic group having 1 to 20 carbon atoms, and n is 0
or represents an integer from 1 to 2, A represents -CH=CH- or ▲There are mathematical formulas, chemical formulas, tables, etc.▼, Y and Z represent hydrogen atoms, hydroxyl groups, or lower alkyl groups, provided that Y and Z are At the same time, it does not represent a hydrogen atom. ) (2) The fatty acid represented by the general formula I is 2-hexenoic acid, 3-hexenoic acid, 2-heptenoic acid, 3-heptenoic acid,
2-octenoic acid, 3-octenoic acid, 2-nonenoic acid, 3-
selected from the group consisting of nonenoic acid, 2-decenoic acid, 3-decenoic acid, 2-undecenoic acid, 3-undecenoic acid, 2-dodecenoic acid, 2-tridecenoic acid and 2-hexadenoic acid. A method for quantifying free fatty acids according to claim 1. (3) The fatty acid represented by the general formula I is 2-hydroxymyristic acid, 3-hydroxymyristic acid, 2-hydroxypalmitic acid, 3-hydroxypalmitic acid, 2-hydroxymyristic acid,
2. The method for quantifying free fatty acids according to claim 1, wherein the fatty acid is selected from the group consisting of -hydroxystearic acid and 3-hydroxystearic acid. (4) The method for quantifying free fatty acids according to claim 1, which comprises measuring hydrogen peroxide produced by oxidizing acyl coenzyme A. (5) The method for quantifying free fatty acids according to claim 5, characterized in that hydrogen peroxide is determined by a colorimetric method. (6) At least adenosine triphosphate, coenzyme A
and a first reagent comprising acyl-coenzyme A synthase;
A second compound containing at least a fatty acid represented by the following general formula I or a water-soluble salt thereof and an acyl coenzyme A oxidase.
A reagent composition for quantifying free fatty acids, comprising a reagent. General formula I: R-A-(CH_2)_n-COOH (wherein, R represents an aliphatic group having 1 to 20 carbon atoms, and n is 0
or represents an integer from 1 to 2, A represents -CH=CH- or ▲There are mathematical formulas, chemical formulas, tables, etc.▼, Y and Z represent hydrogen atoms, hydroxyl groups, or lower alkyl groups, provided that Y and Z are At the same time, it does not represent a hydrogen atom. ) (7) The reagent composition for quantifying free fatty acids according to claim 6, wherein the second reagent further contains a reagent composition for hydrogen peroxide colorimetric measurement.