JP2526599B2 - Method and apparatus for measuring lipid peroxide - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring a hydroperoxide type lipid peroxide on a lipid class level in the pmol order and a measuring apparatus therefor.

PRIOR ART The production of lipid peroxides generally requires the presence of fatty acids, especially unsaturated fatty acids, and the presence of molecular oxygen, active oxygen, free radicals, or enzymes such as lipoxynase. Oxidation of unsaturated fatty acids is carried out by introducing oxygen molecules into the double bond part, which is called autoxidation.When this peroxidation reaction occurs, the cis-type double bond structure part forms a conjugated double bond structure. The shape of the bond is changed to form a hydroperoxide type lipid peroxide having a conjugated double bond structure.

Further, in the photosensitized oxidation reaction, oxygen molecules are directly introduced into saturated fatty acids and unsaturated fatty acids, and hydroperoxide-type lipid peroxides having a conjugated double bond and hydroperoxide-type lipid peroxides having no conjugated double bond are produced. It is formed. In the living body, unsaturated fatty acids are oxidized by enzymes such as lipoxynase. The hydroperoxide type lipid peroxide, which is a primary product produced by such oxidation, further produces a secondary oxidation product in the process of decomposition and polymerization reaction.

Generally, the analysis of lipid peroxides in foods and biological samples is based on the colorimetric determination of hydroperoxides, or oxidized secondary products, in total lipids. However, some lipids have different properties and functions, and when investigating the existing form and physiological significance of lipids in foods and living bodies, or when studying the mechanism of lipid peroxidation in detail, individual lipids Lipid peroxide levels need to be analyzed by class (eg, phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide in the phospholipid fraction). In particular, phospholipids are constituents of biological membranes, form micelles with proteins, and are very important as functional lipids. They are rich in polyunsaturated fatty acids such as arachidonic acid and eicosapentaenoic acid, and are therefore extremely susceptible to oxidation. Arachidonic acid and eicosapentaenoic acid are precursors of many physiologically active substances, for example, precursors of prostanoids having a strong hormone-like action. Analyzing such hydroperoxides of phospholipids rich in arachidonic acid and eicosapentaenoic acid is important not only for understanding their physiological significance but also for studying various diseases and adult diseases. . Therefore, it is strongly desired to establish a fractional quantification method for hydryl peroxide of glycerophospholipids including phosphatidylcholine which is a major phospholipid.

However, in the conventional high-performance liquid chromatography (hereinafter, abbreviated as HPLC) ultraviolet absorption method, the hydroperoxide form and its reduced product, the hydroxy form, often have the same retention time, and both have the same conjugated diene absorption ( twenty three
4 nm), it was difficult to separate and analyze both, and it was not possible to reliably measure the hydroperoxide form. Further, the recently developed photodetection method by HPLC-enzyme reaction-chemylmi requires an expensive enzyme and a special weak photodetector, and is not simple and cannot be said to be a general method.

(Problems to be Solved by the Invention) As described above, since the hydroperoxide form and the hydroxy form have the same retention time and the same absorption wavelength,
Since it is difficult to measure only hydroperoxides, the object of the present invention is to separate lipids in a sample into lipid class levels and to determine the hydroperoxide-type lipid peroxides contained in the lipid class levels. It is an object of the present invention to provide a method for measuring lipid peroxide and a measuring apparatus therefor capable of reliably and accurately measuring with high sensitivity.

(Means for Solving the Problems) In view of these problems of the prior art, the inventors of the present invention have made extensive studies on the method for analyzing lipid peroxides. It has been found that lipid peroxide can be measured with extremely high sensitivity by measuring light.

That is, the present invention is to separate the lipids in a sample into individual lipid classes, and then mix the separated individual lipid classes with a diphenyl aromatic phosphine compound as a fluorescent reagent, thereby coexisting in the lipid classes. Method for measuring lipid peroxide by liquid chromatography characterized by reacting with peroxide-type lipid peroxide and measuring fluorescence produced thereby, and eluent pump and sample for feeding eluent For injection into the eluent column channel, a sample injection injector, an eluent delivery pump, and a filler that separates the lipids in the sample connected to the sample injection injector into individual lipid classes. Separated column, a pump for the fluorescent reagent for mixing the fluorescent reagent into the column flow path between the separated column and the reaction column, and dissolution from the separated column By liquid chromatography, characterized in that it comprises a reaction column for reacting the hydroperoxide-type lipid peroxide and a fluorescent reagent, and a fluorescence detector for detecting the fluorescence of the reaction product An apparatus for measuring lipid peroxide is provided.

(Operation) The present invention will be described in more detail below.

The hydroperoxide type lipid peroxide to be analyzed in the present invention includes hydroperoxide compounds of saturated fatty acids such as palmitic acid hydroperoxide, stearic acid hydroperoxide and ester type derivatives thereof, oleic acid hydroperoxide, linoleic acid hydroperoxide, Hydroperoxide compounds of unsaturated fatty acids such as linolenic acid hydroperoxide, arachidonic acid hydroperoxide, eicosapentaenoic acid hydroperoxide and their ester-type derivatives, triacylglycerol hydroperoxide, glycerophospholipid hydroperoxide, glyceroglycolipid hydroperoxide, etc. Derived from hydroperoxide compounds of glycero-type lipids, serum lipids, edible oils and fats, lipoproteins, foods such as biological tissues and fish meat, and in vivo components Hydroperoxide compounds, and the like benzoyl peroxide further food additive.

In order to separate the lipids in the sample into individual lipid classes, chemically bonded silica gel, hydrophilic polymer gel, silica gel, polysaccharide gel, polystyrene gel, polystyrene gel derivative, polysaccharide gel derivative, heparin, etc. as a ligand A packing material for so-called liquid chromatography such as an affinity gel to which is bound is used, and an ODS-based reverse phase chromatography-based packing or a normal phase silica gel-based packing treated with octadecylsilane is preferable.

As the eluent, methanol, methanol: water (80-100: 20-0, volume ratio) mixed solution, 0.01% triethylamine / methanol, acetonitrile: butanol: water (55:30:15, volume ratio) mixed solution, It can be appropriately selected and used according to a separation column using a mixed solution of chloroform: methanol: water (5:85:10, volume ratio).

The fluorescent reagent that emits fluorescence by reacting with the hydroperoxide type lipid peroxide used in the present invention is a diphenyl aromatic phosphine compound having a fluorescent aromatic group. Examples thereof include naphthyldiphenylphosphine, anthryldiphenylphosphine, pyrenyldiphenylphosphine, 9-acridinyldiphenylphosphine, and 3,4-dimethoxyphenyl-N-methyl-2-imidazoyldiphenylphosphine. A preferred compound is pyrenyldiphenylphosphine, which is the strongest fluorescent reagent.

The concentration of the fluorescent reagent to be used is selected in the range of 1 to 1000 μg / ml, but it is usually used in the range of 100 μg / ml or less. For example, in the case of pyrenyldiphenylphosphine, a working concentration of several μg / ml is sufficient.

The fluorescent reagent is used by dissolving it in a chloroform-methanol mixed solution, a dichloromethane-methanol mixed solution, a dichloroethane-methanol mixed solution, an acetone-methanol mixed solution, or the like.

When analyzing a very small amount of hydroperoxide-type lipid peroxide, the dissolved oxygen in the fluorescent reagent solution may change other lipids to hydroperoxide-type lipid peroxide, which may interfere with the analysis. Degas the light reagent solution,
Substitution with an inert gas is preferable for obtaining stable measured values. Nitrogen gas, argon gas or the like is used as the inert gas.

The reaction between the hydroperoxide type lipid peroxide and the fluorescent reagent is represented by the following formula, and the non-fluorescent trivalent diphenyl aromatic phosphine becomes a fluorescent pentavalent diphenyl aromatic phosphine oxide. .

As a fluorescence measuring method of the present invention, a fluorescence detector which is usually used has no problem. The excitation wavelength of 352 nm and the fluorescence wavelength of 380 nm are preferably selected as the fluorescence detection wavelength.

For the preparation of a normal calibration curve, linoleic acid methyl hydroperoxide, arachidonic acid hydroperoxide, phosphatidylcholine hydroperoxide, phosphatidylethanolamine hydroperoxide, triacylglycerol hydroperoxide, etc. are selected and used.

Next, the outline of the liquid chromatography measuring device for hydroperoxide type lipid peroxide of the present invention is shown in FIG. 1. The eluent 1 is introduced into the flow channel by the eluent liquid feeding pump 2, and the sample injection injector is used. The sample is introduced into the eluent column flow path by 3. Next, pump 2 for sending eluent
And a separation column 5 filled with a filler that communicates with the injector 3 for sample injection separates lipids in a sample into individual lipid classes, and protects the separation column 5 for protection such as deterioration prevention. It is also possible to provide a protective column 4 in front. Further, an ultraviolet absorption detector 6 for detecting a lipid class component eluted from the separation column 5
The fluorescent reagent 8 is mixed into the lipid class component that has passed through the ultraviolet absorption detector 6 by the fluorescent reagent delivery pump 7, and the hydroperoxide type lipid peroxide and the fluorescent reagent are mixed in the reaction column 9. After reaction, the fluorescence of the reaction product is detected by the fluorescence detector 10. Then, the measurement result is displayed on the recorder 13 such as a pen recorder.

The structure of the reaction column 9 is not particularly limited as long as the lipid component eluted from the separation column does not diffuse more than necessary. For example, a Teflon tube (0.25 mmid × 10
m) is wound in a cylindrical shape, and it suffices if the reaction liquid can be heated from the outside to complete the reaction. The reaction temperature should be about 70 ° C., and the reaction time should be
2 minutes is enough.

According to the method of the present invention, since the hydroperoxide type lipid peroxide can be reliably detected by the fluorescence detector 10, the ultraviolet absorption detector 6 is not always necessary, but it is necessary to confirm the separated components. Also good to use. In this case, the detection output signals of the ultraviolet absorption detector 6 and the fluorescence detector 10 are supplied to the coincidence circuit 11, and the coincidence circuit 11 compares the detection results of both detectors, that is, the peak positions. Then, when this result is supplied to the data processing device 12 (for example, a microcomputer) and the coincidence output is supplied from the coincidence circuit 11 with respect to the detection results of both detectors, the hydroperoxide type lipid peroxide is detected. Then, if the result is displayed on the recorder 13, the measurement result can be easily recognized.

Further, the protection column 4 is not always necessary, but it can be conveniently used in terms of prolonging the life of the separation column.

Of course, various modifications can be made without departing from the scope of the present invention.

(Effect of the Invention) As described above, according to the measuring method and the apparatus thereof of the present invention, lipids in a sample can be separated into individual lipid classes, and the separated lipid classes can be fluoresced. By reacting a diphenyl aromatic phosphine compound as a reagent, the hydroperoxide type lipid peroxide can be measured with extremely high sensitivity and accuracy.

(Examples) The present invention will be further described with reference to Examples below, but the present invention is not limited to these Examples.

Example 1 A mixture of phosphatidylcholine hydroperoxide (PCOOH) obtained by photosensitizing phosphatidylcholine obtained from egg yolk with methylene blue and phosphatidylethanolamine hydroperoxide (PEOOH) was used as a measurement sample, and acetonitrile: butanol was used as an eluent. : Flow water (55:30:15, volume ratio) at a flow rate of 1 ml / min,
As the fluorescent reagent, pyrenyldiphenylphosphine (10 μg / ml, dichloromethane-methanol (1: 9, volume ratio) mixed solution) was used at a flow rate of 1 ml / min, and the measurement was performed by the apparatus shown in FIG. . The reaction between the hydroperoxide type lipid peroxide and the fluorescent reagent was carried out at 70 ° C. The measurement conditions of high performance liquid chromatography are as follows.

Liquid sending pump: CCPM (Toyo Soda Kogyo Co., Ltd.) Sample injector: Injection valve unit A (Toyo Soda Kogyo Co., Ltd.) Separation column: TSKgel Silica-60 (Toyo Soda Kogyo Co., Ltd.) UV absorption detector: UV-8000 (Toyo Soda Kogyo) Fluorescence detector: FS-8000 (Toyo Soda Kogyo Co., Ltd.) Column oven: CO-8000 (Toyo Soda Kogyo Co., Ltd.) Recorder: FBR-2 (Toyo Soda Kogyo Co., Ltd.) Chromatogram showing the results of both UV absorption and fluorescence absorption obtained is shown in PCOOH (peak A, A ')
And PEOOH (peak B) are well separated. This indicates that it is possible to measure the concentration in the state of being separated into individual lipid classes, even if analysis is performed using a sample of the same phospholipid fraction.

Example 2 In the same manner as in Example 1, a calibration curve for PCOOH was prepared. P
The COOH concentration was prepared by diluting with unoxidized phosphatidylethanolamine. The results are shown in FIG. 3, in which the vertical axis represents the amount of fluorescence obtained by integrating the peak areas and the horizontal axis represents the concentration of PCOOH. As you can see, PCOOH is 2 pmol to 1 nmol
Good linearity was obtained between the concentration and the amount of fluorescence in the range of. When the S / N ratio is 3, the detection limit of PCOOH is 0.8p.
mol.

[Brief description of drawings]

FIG. 1 is a diagram showing an outline of the measuring apparatus of the present invention, and FIG. 2 is a chromatogram obtained in Example 1, and FIG.
The figure is an example of a calibration curve according to the present invention. 2 …… Eluent delivery pump 3 …… Sample injection injector 5 …… Separation column 7 …… Fluorescent reagent delivery pump 9 …… Reaction column 10 …… Fluorescence detector

Claims (2)

(57) [Claims] 1. A hydroperoxide coexisting in a lipid class obtained by separating lipids in a sample into individual lipid classes, and then mixing the separated individual lipid classes with a diphenyl aromatic phosphine compound as a fluorescent reagent. A method for measuring lipid peroxide by liquid chromatography, which comprises reacting with a lipid-type lipid peroxide and measuring the resulting fluorescence. 2. An eluent liquid feed pump for feeding an eluent liquid, a sample injection injector for introducing a sample into an eluent column channel, an eluent liquid feed pump and a sample injection injector. Separation column packed with packing material for separating lipids in individual samples into individual lipid classes, and fluorogenic reagent for mixing fluorogenic reagent into column flow path between separation column and reaction column Consists of a liquid delivery pump, a reaction column for reacting the hydroperoxide-type lipid peroxide eluted from the separation column with a fluorescent reagent, and a fluorescence detector for detecting the fluorescence of the reaction product An apparatus for measuring lipid peroxides by liquid chromatography, comprising: