JPH0359460A - Detection of lipid peroxide with high sensitivity and composition for detection - Google Patents

Abstract

PURPOSE:To detect lipid peroxide with high sensitivity by subjecting a sample to liquid chromatography, separating the lipids to individual lipid classes, bringing the respective lipid classes into specific reaction with the lipid peroxide in the presence of a surfactant and bringing the same into contact with a detecting medium contg. a light emitting reagent which generates the light responding to the presence quantity thereof. CONSTITUTION:An elution medium 1 delivered by a liquid delivery pump 2 is intimately mixed with the sample 3 contg. the lipids in an injector 4 and the mixture is supplied to a column 5. The lipids contained uin the sample 3 are separated to the lipid classes according to the adsorbing power of an adsorbent from the column 5 and are eluted into a UV absorption detector 6. The detecting medium 8 contg. the light emitting reagent is injected by a liquid delivery pump 7 into the respective lipid class components past the detector 6 and is introduced to a flow cell 9. The light generated from the respective components past the inside of a flow cell 9 is detected by a slight light detector 10 and the results of the detection are recorded by a recorder 11.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a detection method capable of highly sensitive detection of hydroperoxide-type lipid peroxides at the lipid class level, and a detection method used in this method. Regarding the composition.

[Prior Art] Peroxides of lipids such as phospholipids are generally produced by the action of molecular oxygen, active oxygen, or free radicals on fatty acids, particularly unsaturated fatty acids. Hydroperoxide-type lipid peroxides are produced by the autoxidation reaction of unsaturated fatty acids. Lipid hydroperoxides are also produced by photosensitized oxidation reactions.

The importance of detecting such lipid hydroperoxides at the lipid class level is described in JP-A-63-233374 filed by the present applicant. This publication discloses a method that involves subjecting a sample containing lipids to liquid chromatography to separate the lipids into individual lipid classes, and allowing a specific luminescent reagent to act on the separated lipid classes. has been done. The luminescent reagent reacts specifically with lipid hydroperoxides included in the lipid class to generate light responsive to their abundance.

This generated light is detected by a light detection means. thus,
Hydroperoxide type lipid peroxide can be detected quantitatively.

[Problems to be Solved by the Invention] The method described in the above publication is capable of sensitively detecting i lff1-formed lipids on the nanomole order contained in a sample of several μm to several tens of μl at the lipid class level. However, since the intensity of the generated light is still weak, it is desired to develop a method that can detect lipid peroxide with even higher sensitivity.

This invention aims to improve the above-mentioned conventional technology,
The purpose is to provide a method for detecting hydroperoxide-type lipid peroxides with higher sensitivity at the lipid class level without sacrificing the advantages of the above-mentioned conventional techniques, and a detection composition used therein. .

[Means to solve the problem]

A method for detecting hydroperoxide-type lipid peroxides in a sample containing lipids according to the first aspect of the invention includes: (a) subjecting the sample to liquid chromatography to separate the lipids into individual lipid classes; (b) separating each lipid class with a detection medium comprising a luminescent reagent that reacts specifically with the lipid peroxide to generate light responsive to its abundance in the presence of a surfactant; and (c) optically detecting the wave light with a light detection means.

A method for detecting hydroperoxide-type lipid peroxides in a sample containing lipids according to the second aspect of the present invention includes: (a) using an eluent containing an amphiphilic solvent as an eluent for the sample; (b) reacting each lipid class specifically with the lipid peroxide to generate light responsive to its abundance; The method includes a step of contacting a detection medium containing a luminescent reagent, and (c) a step of optically detecting the wave light with a light detection means.

Sensitivity is further improved by using an eluent to which an amphipathic solvent is added and by bringing the lipid class into contact with the luminescent reagent in the presence of a surfactant.

Further, according to the present invention, there is provided a composition for detecting lipid peroxide, which acts on hydroperoxide-type lipid peroxide in a sample containing lipids to generate light corresponding to the amount of the lipid peroxide. A detection composition is provided that includes a surfactant and a luminescent reagent that reacts specifically with the luminescent reagent to generate light responsive to its abundance.

[Function] As in the prior art described above, basically in the present invention, a sample containing lipids is separated into individual lipid classes by liquid chromatography, and the separated lipid classes are used as a luminescent reagent. This causes a specific reaction with the hydroperoxide-type peroxide lipid coexisting in the lipid class to emit light, and this light is detected by a photodetector. In this invention, the contact with the lipid class luminescent reagent is carried out in the presence of a surfactant, or
And/or by performing separation by liquid chromatography using an eluent containing an amphipathic solvent, the luminescence intensity can be significantly increased, thereby making detection sensitivity even higher. A surfactant can be mixed in advance with a luminescent reagent and used as a detection composition.

[Example] Hereinafter, the present invention will be explained in detail with reference to the drawings.

FIG. 1 shows a specific example of a detection device used to carry out the present invention, and its basic configuration is the same as the device described in Japanese Patent Application Laid-Open No. 63-233374.

That is, the apparatus is equipped with liquid chromatography, preferably high performance liquid chromatography (HPLC). This chromatography HPLC consists of a liquid delivery pump 2 that delivers a column mobile phase, that is, an elution medium (described in detail below) 1;
13, and a column 5 that receives the elution medium mixed with the sample delivered by the injector 4.

Column 5 is filled with an adsorbent (not shown).

From column 5, lipids contained in sample 3 are separated into lipid classes and eluted according to the adsorption capacity of the adsorbent.

The elution section of the column 5 is provided with an ultraviolet absorption detector 6 for detecting the ultraviolet absorption of each lipid class component separated and eluted from the column 5 (this ultraviolet absorption detector 6 is not particularly provided). (optional).

A detection line 8 (described in detail below) containing a luminescent reagent is injected into each lipid class component that has passed through the detector 6 by a liquid delivery pump 7.

Each component injected with a luminescent reagent is led to a flow cell 9. In the vicinity of this flow cell 9, for example, a weak photodetector (chsml-Iuaine) using a small-photoelectron counting method is installed.
10 is installed so that its photomultiplier tube faces the flow cell 9. Light generated from each component passing through the flow cell 9 is detected by a weak light detector 10. The detection results of the weak light detector 10 and the ultraviolet absorption detector 6 are recorded by a recorder 11 consisting of, for example, a pen recorder.

As the adsorbent packed in the column 5 of high performance liquid chromatography HPLC, those described in JP-A-63-233374 can be used. Preferable column 5 in the present invention is a reverse phase column filled with octadecylsilane-treated silica gel or a normal phase silica gel column.

Luminescent reagents usually consist of a combination of a catalyst that acts on hydroperoxide-type lipid peroxides to generate active oxygen species such as active oxygen and oxygen radicals, and a luminescent agent that reacts with the generated active oxygen species to emit light. .

Examples of catalysts include salts of transition metals that produce divalent cations, complexes of transition metals in a divalent state, heme proteins, heme peptides, and heme enzymes, which are described in JP-A-63-233374. etc.

These catalysts are used in the concentrations described in JP-A-63-233374.

Examples of luminescent agents include polyhydroxyphenols, phthalazine derivatives, indole derivatives, thiazoline derivatives, which are described in JP-A No. 63-233374,
These include acridine derivatives, oxalic acid derivatives, 1,2-dioxa-4,5-azine derivatives, etc., and these are used in the concentrations described in JP-A-63-233374.

As the luminescent reagent, a combination of cytochrome C and luminol is particularly preferred.

Others, il? JP-A No. 63-2333 also describes the liquid properties of the luminescent reagent solution, measurement atmosphere, hydroperoxide type peroxide lipid to be analyzed, preparation of a calibration curve, etc. for J determination.
As described in Publication No. 74.

According to the first aspect of the improvement of the present invention, a surfactant is allowed to coexist when each lipid class component separated and eluted from column 5 of high performance liquid chromatography HPLC is brought into contact with a luminescent reagent. The eluent 1 can be a conventional eluent such as methanol, chloroform, methanol-chloroform, etc. The surfactant may be injected into sample 3 separately from the luminescent reagent, but it is also possible to prepare a detection composition by separately mixing it with the luminescent reagent and dissolve it in a buffer before use. It is convenient to dissolve it in a buffer form beforehand and use it as the detector 8.

The surfactant used in this invention may be one that falls within the range of ordinary surfactants, such as anionic surfactants such as sulfate ester salts and sulfonates such as sodium dodecyl sulfate, alkyl amines, etc. cationic surfactants such as salts, alkylammonium salts, pyridinium salts,
Nonionic surfactants such as polyhydric alcohol fatty acid esters, amphoteric surfactants such as betaine type and amino acid type surfactants, etc. can be used. Sodium dodecyl sulfate is preferred. Surfactants are used at concentrations of 0.1-10 μM. That is, the detection composition described above has 0
．． Catalyst in a proportion of 1 to 1000 μg/m+ (preferably 1 to 200 μg/m 1 ), 0.1 μg
/ m Contains a luminescent agent in a proportion of 1 or more, and 0.1μ
M to 10 μM surfactant. In this first embodiment, the other procedures are the same as those described with respect to FIG. 1, except that the contact between each lipid class and the luminescent reagent is in the presence of the surfactant described above.

In a second aspect of the improvement of the invention, when separating a sample into lipid classes by high performance liquid chromatography HPLC, an amphiphilic solvent is added to a conventional eluent such as methanol, chloroform, methanol-chloroform, etc. Elution and separation are performed using this as elution medium 1. In this case, there is no particular need to mix a surfactant with the luminescent reagent, and the luminescent reagent may be used as the detector 8 as it is.

In addition to pyridine, amphipathic solvents include ketones such as acetone, ethers such as tetrahydrofuran and 1,4-dioxane, esters such as glycol carbonate, ether alcohols such as diethylene glycol, polyhydric alcohols such as glycerin, other acetonitrile, and formamide. , dimethylformamide, carbon disulfide, aniline, etc.

The amphipathic solvent is preferably 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, relative to the eluent.
used at a rate of In this second embodiment, the other operations are the same as those described with respect to FIG. 1, except that a mixture of an eluent and an amphipathic solvent is used as the eluent.

According to the present invention, the detection sensitivity of hydroperoxide-type lipid peroxides can be improved without impairing the advantages of the conventional methods described above. Note that this invention is carried out by combining the first aspect and the second aspect (i.e., using an eluent to which an amphipathic solvent is added as an eluent, and contacting the lipid class with the luminescent reagent). (in the presence of surfactants). Thereby, detection sensitivity can be further improved.

Examples of this invention will be described below.

Example 1 Hydroperoxide type lipid peroxide was detected using an apparatus having the configuration shown in FIG. As a sample, phosphatidylcholine hydroperoxide (PCOOH) was prepared by photosensitizing and oxidizing egg yolk phosphatidylcholine using methylene blue.
20 μl of a sample containing 497 pmol of ) was used.

Eluent 1 was chloroform-methanol (volume ratio 1; 9
) and flowed at a flow rate of 1 ml/min.

Additionally, the specifications of the device were as follows.

Delivery pump 2: 880-PU type pump manufactured by JASCO Corporation Sample injector 4: VMD-EIE sample injector manufactured by Shimamura Keiki Co., Ltd. Column 5: Nucleosil 10 C, a column (ODS treated silica 5 μm 50 x 4.6 mm) Ultraviolet absorption detector 6: UV IDEC-100-mUV detector manufactured by E3hon Bunkosha (however, no measurement data was taken with this detector) Delivery pump 7 KHD-52 type pump weak light detector manufactured by Kyowa Seimitsu Co., Ltd. 10: Tohoku Denshi Sangyo Chemiluminescence analyzer 0X-
Type 7 recorder 11: 5EKONIK 5S-250F type 2 pen recorder As the luminescent reagent, cytochrome C (100 μg/
A 50 mM borate buffer (pH 9,3) solution containing m I ) and luminol (1 μg/ml) was used, and the surfactant sodium dodecyl sulfate (SDS) was added to this at the concentration shown in Table 1 below. The blended composition was used as the detection line 8 and flowed at a flow rate of 1.0 ml/min. pco under these conditions, respectively.

H detection operation was performed. The results (PCOOH counts) are also listed in Table 1.

Table 1 SDS addition concentration Number of counts (unit: 10,000) No addition
82±3 0.01HM 93±4 0.18M 102±3 1.08M 124±3 10.08M 138±4 As can be seen from the results shown in Table 1, surfactant was not added during contact between the sample and the luminescent reagent. By allowing them to coexist, the detection sensitivity of lipid peroxide is greatly improved.

Example 2 Lipid peroxide was detected using an apparatus with the same specifications as the apparatus used in Example 1. The sample was a methanol solution containing 500 pmol of tocopherol hydroperoxydiene (Toe-00H) produced by generating singlet oxygen through photosensitized oxidation using methylene blue and reacting it with α-tocopherol, and 20 μl of the solution was used. .

Moreover, the detection line 8 is the luminescence reagent used in Example 1 that does not contain a surfactant, and this was flowed at a flow rate of 1.1 ml/min. Under these conditions, methanol containing 1% by weight of pyridine and methanol alone were used as the eluent, and 1. Toc-00II was detected by flowing at a flow rate of 0 ml/min. The detection results are shown in Figure 2. In the figure, curve a is the result when methanol alone was used as the eluent, and curve a is the result when methanol added with pyridine was used as the eluent. From these results, when an amphiphilic solvent is mixed with the eluent, the amount of luminescence is significantly greater (about 9.5 times in this example) than when the eluent is used alone.
I can see that it will improve.

Although the present invention has been described above with reference to specific embodiments, the present invention is not limited thereto. for example,
In carrying out the present invention, a device having the configuration shown in FIG. 9 of Japanese Unexamined Patent Publication No. 63-233374 can be used instead of the device shown in FIG. In addition, various modifications can be made without departing from the gist of the invention.

[Effects of the Invention] As detailed above, according to the present invention, hydroperoxide type lipid peroxides can be detected at the lipid class level with extremely high sensitivity.

[Brief explanation of drawings]

FIG. 1 is a configuration diagram showing an example of an apparatus suitable for use in carrying out the present invention, and FIG. 2 is a graph diagram showing the measurement results of lipid peroxide according to the present invention together with a comparative example. HPLC・・High performance liquid chromatography・Eluent, 3・・Sample, 5・・Column, 8・・Detection line, 10・・Weak light detector

Claims (4)

[Claims] (1) A method for detecting hydroperoxide-type lipid peroxides in a sample containing lipids, comprising: (a) subjecting the sample to liquid chromatography to separate the lipids into individual lipid classes; (b) contacting each lipid class in the presence of a surfactant with a detection medium comprising a luminescent reagent that reacts specifically with the lipid peroxide to generate light responsive to its abundance; and (c) a method for detecting lipid peroxide, which includes the step of optically detecting the light with a light detection means. (2) A method for detecting hydroperoxide-type lipid peroxides in a sample containing lipids, the method comprising: (a) subjecting the sample to liquid chromatography using an eluent containing an amphipathic solvent; (b) separating the lipids into individual lipid classes by subjecting each lipid class to a luminescent reagent that reacts specifically with the lipid peroxide to generate light responsive to its abundance; and (c) optically detecting the light with a light detection means. (3) The detection method according to claim 2, wherein the lipid class and the luminescent reagent are brought into contact in the presence of a surfactant. (4) A lipid peroxide detection composition that interacts with hydroperoxide-type lipid peroxides in a sample containing lipids to generate light corresponding to the amount of the lipid peroxides, and which reacts specifically with the lipid peroxides. A detection composition comprising a surfactant and a luminescent reagent that generates light in response to its abundance.