JPS58126798A - Novel method for determination of unsaturated fatty acid - Google Patents

Abstract

PURPOSE:To determine an unsaturated fatty acid liberated in measuring the lipase activity with the unsaturated fatty acid, particularly glyceride of the unsaturated fatty acid, as a substrate easily and accurately, by using lipoxygenase and peroxidase. CONSTITUTION:A test solution containing an unsaturated fatty acid, e.g. a test solution, containing the unsaturated fatty acid liberated and produced by the lipase activity from a glyceride of the unsaturated fatty acid, is reacted with a lipoxygenase in the presence of oxygen to produce a peroxide of the unsaturated fatty acid, which is then reacted with peroxidase and a hydrogen donor, e.g. an oxidizable compound which is a color reagent capable of producing the visual change in hue by the oxidation. After completing the reaction, the degree of the coloring is measured by the absorbance at a specific absorption wavelength, and the amount of the unsaturated fatty acid in the test solution is calculated and determined from the absorbance.

Description

[Detailed Description of the Invention] The present invention relates to a novel unsaturated fatty acid,
It is also particularly useful for quantifying unsaturated fatty acids liberated in lipase activity measurements using unsaturated fatty acid cricephids as substrates.

Lipase converts glycephyte into fatty acids and glycerin 10
It is an enzyme that has the action of degrading fatty acids, and is distinguished from esterases, which act on glycephyte, which has a short carbon chain of fatty acids. This lipase is widely distributed in animal tissues, and is highly concentrated in the secretion fluid of spleen. Furthermore, in the case of bond disease, lipase flows into the blood, so measuring serum lipase activity is useful for detecting bond disease.

As a conventional method for measuring lipase activity in serum, for example, an excess of triglyceride, which is a substrate for IJbase, is added to serum tc as a test solution and allowed to act, and then the amount of fatty acids liberated from the lipase activity is measured using a thymol cap. It was measured by alkaline titration using an indicator such as rhein or by extraction with copper salt (DeuLs).
oh MedizinischWochenschri
fL, Tao, i'yyo<iri65), Ana
Lytical Biochemistry, b +
'I j / (/ri63) CIin, Cheto
12/, /4'69~/1I73 (/ri75)] is,
In this tail measurement, the substrate used causes turbidity, so photometer measurements based on transparency are inappropriate, and the amount of fatty acids to be measured is extremely high, such as serum/w.
Even if the reaction was carried out for 10 minutes using llf, the amount of fatty acids liberated was only 0,000 millimoles, which was extremely difficult to quantify. In addition, in recent years, another method for measuring serum lipase activity has been developed, in which triglycephyte of lylunic acid is used as a substrate, and the lylunic acid released and produced by the interaction with blood is oxidized with ribooxygenase, and the oxidation reaction is consumed. A method of electrically measuring the amount of oxygen produced using an oxygen electrode (C11nical Che-mitry,
, LZ, /63~/65 (/7 times)], but in this measurement method, the amount of oxygen consumed by the oxygen electrode is determined, so the amount of oxygen! It has the disadvantage that the stability and sensitivity for polar detection are significantly affected, and it is also unsuitable for photometric measurements because it produces turbidity due to the use of triglycephyte as a substrate.

As a result of various studies regarding the measurement of lipase activity, the present inventors used monoglycephyte, diglyceride, or triglyceride of unsaturated fatty acids as a substrate, and released unsaturated fatty acids from the substrate due to the lipase activity in the test solution. In the presence of oxygen, unsaturated fatty acids are converted into peroxides of unsaturated fatty acids using ribooxygenase, and this peroxide is reacted with peroxidase and a hydrogen donor to form a reaction system based on the hydrogen donor used in the reaction system. It has been completed that this can be accurately and easily quantified as a specific spectroscopic optical change.

The present invention was completed based on the above findings. Ribooxygenase is made to act on a test solution containing unsaturated fatty acids in the presence of oxygen to form peroxides of unsaturated fatty acids, and peroxidase and Let a hydrogen donor act,
A method for quantifying unsaturated fatty acids, which is characterized in that a detectable change in the reaction system Q is then measured by spectroscopic means, and in particular, it does not require the conventional complicated steps for measuring lipase activity. It is useful as a simple measurement method.

Furthermore, when measuring trace amounts of lipase activity using glycephyte as a substrate, the present invention does not cause unsuitability of measurement due to turbidity of the reaction solution (also, the electrode used
This provides a useful measuring method that allows positive H to be measured very simply using spectroscopic optical means in a short time.

First, the unsaturated fatty acid-containing test solution used in the present invention may be any one containing unsaturated fatty acids, and preferably, the unsaturated fatty acid-containing test solution used in the lipase activity measurement can be obtained from a substrate that is glycephyte of unsaturated fatty acids. The test solution is a test solution containing unsaturated fatty acids released and produced by the process, and is usually a fixed amount of a solution with lipase activity such as serum lipase or lipase reagent solution, and a fixed concentration of unsaturated fatty acid glyceride. Mix a certain amount of substrate solution containing 37
A solution is used that is reacted at around 0.degree. C. for more than S minutes to release unsaturated fatty acids from the substrate based on its lipase activity. The unsaturated fatty acid glyceride used as a substrate for lipase activity measurement may be any of monoglyceride, diglyceride, and triglyceride (preferably monoglycephyt and diglycetoid). The unsaturated fatty acids in the test liquid containing unsaturated fatty acids are preferably V-s type unsaturated fatty acids having 12 to 20 carbon atoms and having two or more double bonds, such as 2,'I-dodecadienoic acid, Ri, /2-hexadecadienoic acid, linoleic acid (sinuta, nsou, -/2-octadecadienoic acid), lylunic acid (ri, /2゜lj-octadecatrienoic acid), otsu, ri, 12-octadecadienoic acid trienoic acid, ii, 1t
i-eicozadiconic acid %j+♂, l/-eicof)
I) xonic acid, ♂, ii, i4-mono-eicosatrienoic acid, afchidonic acid (j, f, Il, /'I-eicosatrienoic acid), and the like.

In addition, ribooxygenase is an enzyme (E
, C, /, /3. /, /3. Linoleat
e:oxygen oxidoreducLase)
If the enzyme is responsible for this reaction, the crude product,
Any purified product may be used, and it may be a commercially available product, or one isolated and collected from leguminous plants such as soybeans and pushups, barley, wheat, etc. [Yari, Tappel, Food Re5]
earch.

/I, 1011 (/ta 3; 3),
H, TI+eorel, etal.

AcLaChem, 5cand, /, j7/ (
/9!7)). In addition, as a cap for ribooxygenase, it is usually sufficient to use 1000 units or more per 1 test, preferably about 100 to 5000 units.

Furthermore, as the reoxidase, it is convenient to use commercially available horseradish-derived berokindase (normally, it is sufficient to use more than one unit per tenuto, preferably about 2 to 20 units). .

As the hydrogen donor, there may be used a color reagent that is a compound that can be oxidized and causes a visible change in color tone upon oxidation, a fluorescent reagent that emits fluorescence upon irradiation with ultraviolet light, or a luminescent reagent that develops color. In addition, a combination of an electron acceptor and a phenolic compound is often used as a coloring reagent.
Examples of electron acceptors include guaminoantipyrine,
Guamino-3-human fusino-5-mercapto-1,2
゜+-triazole, 3-methyl-1-benzoti7zolinonehydrofuzone, 2-aminobenzothiazole, etc. are used, and as the phenolic compound, for example, pheno-lv%P-chloropheno-〃, λ, Hiro -cyclophenol, λ, 4! -Sialompheno/l/, P-
Sodium hydroxybenzoate, +,6-dichloro〇-cregur, 3. Sodium j-dichloro-λ-hydroxybenzenesulfonate, 3. j-xylenol, N
, N-diethylv-m-treizine, N,N-diethanolam-)isoidine, N-ethyl-N-sulfopropyl-m-)luidine, N,N-dimethyaniline,
N,N-diethylaniline, N,N-dimethyl-m-methoxyaniline, 3-acetamino-N,N-diethylaniline, 3-methyl-N-ethyl-N-(β-hydroquinethyl)aniline, N -Ethyl-N-(3-methylphenyl>%/-leacetylethiene diamine, etc.).Furthermore, as the fluorescent substrate in the fluorescent reagent or luminescent reagent 1, various known ones can be mentioned, such as −(λ, 11.1.−
+) chlorophenol) oxalate, Fe = 7 rethiohydantoin, homovanillic acid, +-hydroxyphenylacetic acid, vanillin\amine, 3-
Examples include methoxyaniline, phloretic acid, hordenine, luminol monoanion, lucikenin, and waffin. These reagents are illustrative and do not limit the hydrogen donor of the present invention in any way, and these reagents can be used for unsaturated fatty acids or unsaturated fatty acids contained in the test liquid. The peroxide derived from the unsaturated fatty acid
1Usually, 5 times the mole or more is used for 10 unsaturated fatty acids, but the amount of test solution to be used, the degree of unsaturated bonding and content of unsaturated fatty acids in the test solution, etc. should be determined accordingly. All you have to do is change it.

Furthermore, during the reaction, an appropriate reaction medium such as dimethyl gluglulic acid-sodium hydroxide buffer, phosphate Mr# solution, Tris-clump acid buffer, boric acid M buffer, and other commonly used A Hi solution is used, and the pH is usually within the range of 1 to 2 to achieve the purpose.
Just select H.

For example, 0.7 to 0.2 electron acceptors such as 0.3% deraminoantibilin, and 0.2% phenol! ,
Phenolic compounds such as goojichlor a7 el/- 0.
/ -0,2d hydrogen donor, peroxidase 5~
20 units, ribooxygenase 10,000-30,000
A composition for measuring unsaturated fatty acids having a composition of 2 ml of 0.7 M white acid buffer (pHL?0) or 02 M Tris-hydrochloric acid buffer containing the unit is prepared. Next, if you are unable to fully quantify the unsaturated fatty acids, for example, first preheat the prepared composition for measurement to 37°C, add jOμL of Saka test solution containing unsaturated fatty acids, and add 37°C. Incubate for 20 minutes at 10 °C.
After the reaction is completed, the degree of coloration is determined by measuring the absorbance using a spectrophotometer (1 trowel) and 1 specific absorption wavelength (1 trowel). From the absorbance, jt of unsaturated fatty acids in the test liquid is calculated and quantified. In addition, to quantify unsaturated fatty acids without measuring lipase activity, use a substrate for measuring lipase activity in advance. The unsaturated fatty acids liberated by the action of a test solution having koribase activity may be quantified, and preferably a composition prepared by adding a substrate for measuring lipase activity to the aforementioned composition for measuring unsaturated fatty acids. ℃, and a test solution for measuring lipase activity is applied to this stage, and the amount is determined using a general trowel. In addition, as a substrate used in lipase activity measurement, for example, prepare a solution of grisephyte in ethanol at a concentration of about 0/M, and add this to the composition/~2m
It is sufficient to use an amount of about 20 to JOμ per liter, and when measuring lipase activity, it is preferable to use a surfactant such as sodium dequincholate in the composition.

Furthermore, in the present invention, the reaction pH 9 time, measurement wavelength, etc. vary depending on the reagents used, and suitable conditions may be set as appropriate.

The present invention will be described in detail with reference to the following example, but the present invention is not limited thereto in any way.

Example 1 O,/M borate buffer (pH 9,0) 0.2
d03% Guaminoantipyrine O5/102
%phenol 0. /WLl
Peroxidase (jO units/,,) 0.2
d-ribooxygenase < soo, ooo units/,)2
0μ distilled water 0.3♂
1 total/, □ tgl Composition for measuring unsaturated fatty acids having the above composition/, Od
was preheated to 37°C, and 0 to jOuL of a 7mM linoleic acid-containing ethanol solution or 0 to jOμL of a 7mM linoleic acid-containing ethanol solution was added thereto as a test solution, and the mixture was heated to 37°C.
After reaction for 10 minutes, add distilled water 2-
The absorbance of the color development was measured using a spectrophotometer at a wavelength of 00 nm. The result is as shown in Fig. 10.

In the figure, - indicates the results when linoleic acid was used as the test solution, and 〇-〇 indicates the results when linoleic acid was used as the test solution, which showed extremely good quantitative results. It was something.

Example λ 0.2M Tris-HCl buffer (PHg,!; ) 0.
1m 12% Sodium Deoquincholate Q,
/rRlo, /M silyl twin-containing ethanol solution
Hiro 0 μL lipoxygenase (300 pθθ units/m
l) 60 μt peroxidase <so units/, 4)
0.2wd! 03% Daraminoantipyrine
Q.2 ml 10.2% Goodichlorophenol 0.21 distilled water
Og/m/total, 2.0 at Reaction composition containing the above composition 2.0 Tllfc37
Prewarm it at ℃ and use a commercially available lipase reagent (reagent derived from Chromobacterium viscosum:
(manufactured by Toyo Jozo Co., Ltd.) 0. /U/Ill was added to θ ~ 50 μt and reacted at 37°C for 10 minutes. After one reaction, the color development was measured using a spectrophotometer with a wavelength of 50 nmk and the absorbance (Δ
A505nm) tMIL was determined.

As a result, as shown in FIG. 2, extremely good results were obtained in the measurement of Koripase activity.

Example 3 0.2M Tris-hydrochloric acid buffer (pH o) o, g Goco% sodium dequincholate 0. / dO
,/M silyl twin-containing ethanol solution qoμt
Lipoxygenase C300ρ00 units/I++7ri5
0μt peroxidase <SO unit 7ml)
0.211L 103% Daraminoantipyrine
Q, 2JLt0.2%λ, dichlorophenol
0.2Ll distilled water
Ohiro/at total 2.0 - 2.0 N of the reaction composition having the above composition, prewarmed to 37°C ~, 1 sample, serum diluted solution 508
ml was added and reacted at 37°C for 20 minutes, and after the reaction was completed, the color was measured at wavelength 60! Absorbance (ΔA505 nm) was measured using a spectrophotometer using nm.

As a result, as shown in FIG. 3, very good quantitative results were obtained.

Reaction composition 2.01R1 having the same composition as Higashi Seikihiro Example 3
' was prewarmed to 37°C, and diluted serum j0μ was added thereto to make it diluted to 11c at 37°C. The change in absorbance for 5 minutes from 5 minutes after reaction to 70 minutes after reaction is measured at wavelength JQ, f
Measured using a nml trowel spectrophotometer needle.

The results were as shown in FIG.

Example 5 The same method as in Example D and the known method for measuring serum lipase activity (copper salt method + CI in, Chezr4, .
Serum lipase activity was measured in 37 samples at 2/, /Da69~/11.73C/Li7j)].

As a result, the correlation obtained by both methods is shown in Figure 5, and the correlation coefficient is given by the regression formula y= /, 2 f
10g and 6, showing a good correlation.

[Brief explanation of drawings]

Figure 1 shows the quantitative curves of linoleic acid and linuric acid;
Figure 2 shows the lipase activity measurement curve using lipase solution (&), Figures 3 and 4 show the serum lipase activity measurement curve, and Figure 5 shows the correlation between the method of the present invention and the known copper salt method. Patent applicant: Toyo Jozo Co., Ltd. Representative: Fujima Ito / Figure H) 20 30 40 50. ] Treatment liquid (linoleic acid, lylu/acid) Fig. 2 11J 20 30 40
5+)1+1 9... -Se゛(,Hg (0,+LLz
ml) Figure 3 Blood 7 #4 Interpretation 1 - No. q Figure 1 River (^ Interpretation / Indication of Mutual Procedural Amendment 7S Case 1966 Patent Application No. G390 Co) Title of Invention Novel unsaturated fatty acid Quantification method 3 - Relationship with the case of the person making the amendment Patent applicant address 632 Mifuku, Ohito-cho, Tagata-gun, Shizuoka Prefecture
, 2 diglyceride,” he corrected. "3. s-xylenol s N%" on page 1, line 1 of the same page
"N-di" is corrected to "N)N-di" as a 3,5-xylenol-1 aniline compound.

Claims (1)

[Scope of Claims] (1) The unsaturated fatty acid-containing test rtt is treated with ribooxygenase in the presence of oxygen to form an unsaturated fatty acid peroxide, which is then treated with verokindase and a hydrogen donor, A method for quantifying unsaturated fatty acids, characterized in that a detectable change in the reaction system temperature is then measured by spectrophotometric means. (2) Claim 7, which is a test solution containing unsaturated fatty acids or a test solution containing unsaturated fatty acids released and produced from glycerides of unsaturated fatty acids through lipase activity.
Quantitative method described in section. (3) The assay method according to claim 2, wherein the unsaturated fatty acid glycephyte is an unsaturated fatty acid monoglycephyte, diglyceride, or triglycephyte. (4) The quantitative method of Claims 1, 2, or 3, wherein the unsaturated fatty acid is an unsaturated fatty acid having 72 to 20 carbon atoms. (5) Carbon number 72~. 20 unsaturated fatty acids, 2. '
/'-dodecadienoic acid, ri/λ-hexatecadienoic acid. 9. InoA catecatecatrienoic acid, / /, / +-eicozanenoic acid, left side. Claim 4 which is l/-eicosatrienoic acid, gl/, /9-eicosatrienoic acid or afquidone
Quantitative method described in section. (6) The quantitative method according to claim 1, wherein the hydrogen donor is a color-forming reagent, a fluorescent reagent, or a photoreagent. Special features that are spectroscopic optical means that determine the absorption wavelength! l-l-Claim 1
The law stated in the section. (8) The quantitative method according to claim 7 or 2, wherein the unsaturated fatty acid sample solution is a test solution for measuring lipase activity.