JP3262846B2 - Confirmation and measurement method of oxidized lipid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for confirming / measuring an oxidized lipid for easily confirming that a test object is an oxidized lipid.

[0002]

2. Description of the Related Art Oxidized lipids are deeply involved in important diseases such as arterial effects and aging and physiological changes due to nonspecific oxidation.

On the other hand, oxidized lipids such as arachidonic acid oxidized by lipoxygenase and cyclooxygenase, which are enzymes in the living body, are physiologically important and are converted into prostaglandins and leukotrienes having various activities. Is done.

Various methods have been developed for measuring stabilized oxidized lipids or final metabolites. For example, iodine is oxidized and ionized, and the color is observed. The iodine oxidation method is used. The pigment is produced by reacting with malondialdehyde which is a lipid peroxide metabolism. Conjugated diene is produced by the method of peroxidation of unsaturated fatty acids, and the conjugated diene is measured by an absorptiometer. The lipid peroxide sample is analyzed by HPLC (HIGH PERFORMAN
CE LIQUID CHROMATOGRAPHY)
High-performance liquid chromatography method in which the lipid peroxide sample is esterified and then separated and measured by gas chromatography.

[0005]

However, the iodine oxidation method and the conjugated diene measurement method have a drawback that they are not suitable for a sample containing contaminants. In addition, the iodine oxidation method, the TBARS measurement method, and the conjugated diene measurement method do not directly measure the oxidized lipid itself, but indirectly measure by-products generated when the oxidized lipid is generated or destroyed. This is a non-specific measurement method for quantitatively measuring oxidized lipid.

On the other hand, high-performance liquid chromatography and gas chromatography are specific methods for directly measuring lipid oxide itself, but high-performance liquid chromatography requires a long time to perform measurement, May be denatured. In addition, the oxidized lipid itself is easily changed by a solvent such as ether or ethanol. In the gas chromatography method, the sample is easily denatured by a treatment such as esterification.

As described above, there has been no method for accurately measuring and analyzing oxidized lipids having a physiological action and unstable oxidized lipids which adversely affect the body.

Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an important and simple activity to easily and accurately measure that the test object is an oxidized lipid.
It is intended to provide a method for confirming and measuring a water-soluble oxidized lipid which is considered to have an action .

[0009]

SUMMARY OF THE INVENTION In order to achieve the above object, the present invention provides a method of oxidizing soybean lipoxygenase.
Dyspro for linoleic acid or arachidonic acid
Unstable oxidation such as indium, europium, thulium, etc.
Adds lansanide shift reagent that has little effect on lipids
By NMR spectroscopy, the water
Based on the ton peak (0 ppm), C
Observe proton peaks such as H ₂ and CH ₃ ,
Appears when oxidized and non-oxidized
Based on the difference in proton signal,
It is characterized by confirming whether it is a oxidized lipid .

The object to be inspected is a hydroperoxyta
Glutathione, glutathione to make it possible to identify
Peroxidase was added.

Further, the object to be inspected is a hydroperoxyta
To make it possible to confirm that the stabilizing group has stabilized.
Quixed dismutase was added.

[0002]

According to the present invention, a lanthanide shift reagent is added to a test object, and the reagent is subjected to spectroscopic analysis to confirm and measure that the test object is an oxidized lipid. This is because, when a lanthanide shift reagent is added to an analyte, the proton signal differs between when the analyte is an oxidized lipid and when it is not, so the difference in the signal is measured by spectroscopic analysis. It confirms and measures that the specimen is oxidized lipid.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, a lansanide shift reagent is added to a test object, and the reagent is subjected to spectroscopic analysis to confirm and measure that the test object is an oxidized lipid. Then, linoleic acid or arachidonic acid oxidized by soybean lipoxygenase is used as the test object. In addition, disprosium is used as a lansanide shift reagent.

[0014] When lanthanide shift reagent was used as well as dysprosium, europium (Eu) or Tm gave the same results as dysprosium. As the chelating agent, ethylenediaminetetraacetic acid or the like may be used in addition to the tripolyphosphate.

(Method of oxidizing linoleic acid and arachidonic acid by soybean lipoxygenase)

Linoleic acid and arachidonic acid to be used as test objects are prepared by the following method.

First, linoleic acid and arachidonic acid are dissolved in deuterated methyl alcohol at a concentration of 0.1M.
The solution was further emulsified in 20 mM deuterated phosphate buffer (pH 7.0 ) saturated with oxygen to prepare a 4 mM linoleic acid and 2 μm arachidonic acid emulsion. After that, one with superoxide dismutase (SOD) added at 0.1 U / ml and one without superoxide dismutase (SOD) were prepared. Soybean lipoxygenase was added at a final concentration of 2000 / ml for fatty acid oxidation. And the final capacity is 1m
Oxidation was performed at 0 ° C. at 1 ° C.

After a certain period of time, pH 4 with deuterated hydrochloric acid
The activity of soybean lipoxygenase was stopped by oxidation. Thereafter, addition of ethylenediamine tetraacetic acid at a concentration of 1 m M, pH 7 with sodium hydroxide dissolved by heavy water.
Adjusted to zero. Finally, the volume was adjusted to 1.5 ml with 20 mM deuterated phosphate buffer.

The oxidized linoleic acid sample contains various concentrations
Of glutathione and glutathione peroxidase at 0.2 U / ml to a final concentration of 1.5 ml.
After reacting at 30 ° C. for 10 minutes, the sample
Dysprosium polyphosphate at a concentration of 40mM in Le [D
y (ppp)] was added as a lansanide shift reagent.

(Method for producing disprosium tripolyphosphate)

Disprosium chloride 2 added to the specimen
00mM and pentasodium tripolyphosphate 480m
M was sufficiently stirred with heavy water. The solution had a precipitate, but was adjusted to pH 7.4 with deuterated hydrochloric acid. This makes the precipitate more soluble. Then 3000g30
The remaining precipitate was removed by centrifugation. Add this solution to 20
0 mM disprosium polyphosphate [Dy (ppp)]
The solution was used.

(1H-NMR SPECTROSPOP)
Y measurement method)

According to the present invention, a lansanide shift reagent is added to a test object, and the lanthanide shift reagent is subjected to spectroscopic analysis to confirm and measure that the test object is an oxidized lipid. 1H-NMR SPECTROCOPY measurement method is used. This means that at a probe temperature of 35 ° C., the spectrum is FREE INDUCTION D
This is the result of accumulating ACAY 512 times. In addition, H2O
Peak is 4s, 200mW GATED, CONTIN
UOUS WAVE, SINGLE FREQUENC
Pre-saturated with Y RADIO WAVE.

The parameters of the spectrum were 8192, the data points and the width of the spectrum were 7246 Hz, and the acquisition time was 0.565 s. The sample rotation speed is 15
Hz. The frequency was locked with heavy water before the measurement.

(Effect of disprosium (Dy) concentration on proton signal of linoleic acid)

According to the present invention, when dysprosium (Dy) is added at various concentrations to an oxidized linoleic acid sample, the sample becomes
Focusing on the exit difference in proton signals in the case and where water soluble oxidized lipid otherwise may test sample water
It confirms and measures that it is a soluble oxidized lipid.

Therefore, the effect of the concentration of disprosium (Dy) on the proton signal of linoleic acid was examined by adding disprosium (Dy) at various concentrations to the oxidized linoleic acid sample (see FIG. 1). .

The numerical values in FIG. 1 indicate the concentration (mM) of disprosium (Dy) added to the sample. Also,
CH3, (CH2) n, CH2-C = C, CH2 in FIG.
CO indicates the proton signal in the previously identified fatty acid molecule. That is, it indicates a proton signal at the above position in the fatty acid.

The scale of the shift is based on the H2O peak (0
PPM). Peak 1 is dysprosium (D
the influence of y), a peak has been separated from the C H3 peak. This peak corresponds to the hydroperoxide group (-
(COOH). It is considered that peak 3 was also affected by the hydroperoxide group, but the origin of peak 5 is unknown.

(Change in proton peak of oxidized linoleic acid by addition of glutathione and glutathione peroxidase)

Next, the effect of various concentrations of glutathione and glutathione peroxidase to catalyze the reduction was examined for the influence of linoleic acid on the proton peak (see FIG. 2).

The numerical values in FIG. 2 indicate the concentration of glutathione added to the sample in nanomolar (nM) units. It can be seen that the height of peak 1 has decreased due to the concentration dependency. This indicates that peak 1 is a proton signal affected by the hydroperoxide group (—OOH). Contrary to peak 1, peak 2 is clearly recognized by addition of glutathione. In other words, this peak corresponds to the hydroxide group (-O) generated by reducing the hydroperoxide group with glutathione.
H) is presumed to have been affected.

The peaks 4 and 5 are not affected by glutathione and their origin is unknown, but they are caused by the oxidation of linoleic acid by lipoxygenase and are affected by oxidized groups such as endoperoxide groups. Probably a proton signal.

(Time course of oxidation of linoleic acid by soybean lipoxygenase)

In the present invention, the process in which linoleic acid is oxidized by soybean lipoxygenase is described by 1H-NMR.
Measured by SPECTROCOPY measurement method (Fig. 3
reference).

The time entered in FIG. 3 (min)
Indicates the time of reaction with soybean lipoxygenase. None of the peaks is seen in the state where the lipoxygenase is not affected by oxidation. This is because linoleic acid is insoluble in water and is not affected by disprosium (Dy). On the other hand, the oxidized linoleic acid becomes soluble and is affected by disprosium (Dy), so that there is a great difference in the resonance frequency of the protons.

Therefore, when the spectrum WINDOW is adjusted to the proton signal of the oxidized linoleic acid, the proton signal of the linoleic acid which has not been oxidized departs from the WINDOW. From this, it was proved that the present invention is effective as a method for confirming and measuring whether or not the specimen is an oxidized lipid.

(Superoxide dismutase (S
OD) Changes over time in the oxidation of linoleic acid by soybean lipoxygenase in the presence)

Furthermore, linoleic acid oxidized by soybean lipoxygenase under the same conditions as described above in the presence of 0.1 U of superoxide dismutase (SOD) was measured over time by 1H-NMR SPECTROCOPY measurement. (See FIG. 4).

Superoxide dismutase (SO
D) is present (see FIG. 4) and superoxide dismutase (SOD) is not present (see FIG. 3).
It showed a distinctly different pattern compared to.

That is, when superoxide dismutase (SOD) is present, peaks 1 and 3 indicating the presence of a hydroperoxide group (—OOH) in the fatty acid appear even after 20 minutes from the start of oxidation. It is that you are. These results indicate that superoxide dismutase (SOD) has a hydroperoxide group (—OOH)
Must be considered as stabilizing. Conventionally, superoxide dismutase (SOD) has been considered to have a main effect of disproportionating superoxide, but the above results indicate that superoxide dismutase (SOD) directly acts on oxidized lipids. It was proved that it was exerting.

(Change over time of oxidation of arachidonic acid by soybean lipoxygenase)

On the other hand, arachidonic acid was also oxidized by soybean lipoxygenase in the same manner as in the above-mentioned oxidation of linoleic acid, and measured by 1H-NMR SPECTROCOPY measurement (see FIG. 5).

Time (min) entered in FIG.
Indicates the time of reaction with soybean lipoxygenase. Unlike the case of linoleic acid, the peak has already been affected by dysprosium (Dy) before being oxidized by soybean lipoxygenase. This indicates that some arachidonic acid has been oxidized.
This is probably because arachidonic acid has already undergone autoxidation by oxygen in the air. The oxidation pattern was also different from that of linoleic acid, and no clear peaks seemed to be affected by the hydroperoxide group (-OOH).

(Superoxide dismutase (S
OD) Time-dependent change of oxidation of arachidonic acid by soybean lipoxygenase in the presence)

In the presence of 0.1 U of superoxide dismutase (SOD), arachidonic acid oxidized with soybean lipoxygenase under the same conditions as above was subjected to 1H-NMR SPECTROCOPY.
It was measured by a measurement method (see FIG. 6).

The peak 1 in FIG. 6 was clearly recognized 5 minutes after oxidation compared to the absence of superoxide dismutase SOD, which is considered to be a peak affected by the hydroperoxide group (—OOH). This indicates that superoxide dismutase (S
OD) is considered to stabilize the hydroperoxide group (-OOH). In addition, it is considered that the presence of superoxide dismutase (SOD) promotes the decomposition of CH2 and CH3 serving as the skeleton of the fatty acid in the oxidation process. This fact is proved by the fact that the proton peaks of CH2 and CH3 almost disappeared at an oxidation time of 15 minutes by soybean lipoxygenase.

[0048]

According to the present invention, when a lansanide shift reagent is added to a test object, the proton signal differs between the case where the test object is a water-soluble oxidized lipid and the case where the test substance is not. the spectrally analyzed object to be inspected is water
It confirms and measures that it is a soluble oxidized lipid.
Therefore, it is easy to determine that the specimen is a water-soluble oxidized lipid,
And it can measure accurately.

That is, according to the present invention, it is possible to directly confirm and measure that the test object is a water-soluble oxidized lipid by performing a spectroscopic analysis of the difference in the proton signal.

[0050] In addition, without the affected even if the contaminant is mixed in the inspection object, the object to be inspected is water
It can be confirmed and measured as a oxidized lipid.

Furthermore, the spectroscopic analysis of the proton signal of the test object makes the measurement method extremely simple, and it is possible to confirm and measure that the test object is a water-soluble oxidized lipid in a short time. Further, since no treatment such as esterification is required for the inspection object, there is no possibility that the inspection object is denatured at all.

On the other hand, a lanthanide shift reagent such as disprosium (Dy) was added to the oxidized lipid, and 1H-NMR S
The presence of a hydroxide group (—OH) in the oxidized lipid could be confirmed for the first time by measuring with the PECTROSCOPY measurement method.

It was also possible to identify certain oxidized groups in oxidized lipids that had not yet been identified.

The present invention is completely different from the method for confirming and measuring oxidized lipids that has been developed so far. The present invention confirms a hydroperoxide group in a fatty acid and has a very short lifetime in a phosphate buffer or the like. I found it. In addition, it was confirmed by this method that superoxide dismutase (SOD) has an action of stabilizing its hydroperoxide group (—OOH).

In the future, it will be possible to use this method to measure the dynamics of lipid metabolism, and furthermore, to measure the oxidized lipid contained therein using a biological sample.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of disprosium (Dy) concentration on the proton signal of linoleic acid.

FIG. 2 is a graph showing changes in linoleic acid proton peaks due to the addition of glutathione and glutathione peroxidase.

FIG. 3 is a graph showing the time course of oxidation of linoleic acid by soybean lipoxygenase.

FIG. 4 Superoxide dismutase (SOD)
FIG. 2 is a graph showing the change over time of the oxidation of linoleic acid by soybean lipoxygenase in the presence.

FIG. 5 is a graph showing the time course of oxidation of arachidonic acid by soybean lipoxygenase.

FIG. 6: Superoxide dismutase (SOD)
FIG. 4 is a graph showing the time course of oxidation of arachidonic acid by soybean lipoxygenase in the presence.

Claims (3)

(57) [Claims] (1) oxidation by soybean lipoxygenase;
Linoleic acid or arachidonic acid
Unstable acids such as rhodium, europium, thulium, etc.
Lanthanide shift reagent with little effect on immobilized lipid
By NMR spectroscopy, the water
Based on the ton peak (0 ppm), C
Observe proton peaks such as H ₂ and CH ₃ ,
Appears when oxidized and non-oxidized
Based on the difference in proton signal,
A method for confirming / measuring oxidized lipids, which comprises confirming whether or not the lipid is oxidized lipid. 2. The test object is hydroperoxidase.
Glutathione, glutathione pero so that the group can be confirmed
Confirmation of the oxidized lipid according to claim 1 , wherein the oxidase is added.
Measuring method. 3. The test object is hydroperoxidase.
The super oxa group is used to confirm that the group has stabilized.
The method for confirming / measuring oxidized lipid according to claim 1, further comprising addition of id dismutase .