JP6779776B2 - Oxidized HDL measurement method and oxidized HDL measurement kit - Google Patents

Description

The present invention relates to a method for measuring HDL oxide and a kit for measuring HDL oxide.

High-density lipoprotein (HDL) is known to have functions such as antioxidant ability, cholesterol recovery, and removal of oxidized lipids, and has a function of suppressing arteriosclerosis, and is also called good cholesterol. On the other hand, low-density lipoprotein (LDL) is also called bad cholesterol because when it increases, cholesterol accumulates in blood vessels and causes arteriosclerosis and myocardial infarction. Therefore, it is considered that lowering LDL and increasing HDL leads to prevention of arteriosclerosis. However, since oxidized HDL (oxidized HDL) has a decrease in functions such as cholesterol recovery ability, it is important not only to increase the amount of HDL but also to reduce oxidized HDL to prevent arteriosclerosis. It is considered.

As described above, oxidized HDL is considered to be useful for predicting the risk of arteriosclerosis / myocardial infarction and determining the risk reduction due to lifestyle improvement. Therefore, a method for measuring oxidized HDL has been studied. There is. For example, Patent Document 1 proposes a method of measuring oxidized HDL by an ELISA method using an antibody against an oxidized lipid and an antibody against an apolipoprotein. Further, for example, in Non-Patent Documents 1 and 2, a method of extracting lipids from a blood sample using an organic solvent, fractionating HDL by high performance liquid chromatography, and quantifying oxidized HDL by ultraviolet absorption or chemiluminescence is used. Proposed.

However, the above-mentioned method has a drawback that the operation is complicated because the oxidized HDL is measured by the ELISA method, and when the plasma component is isolated, purification is required and the measurement takes time. Therefore, a simple and low-cost method for selectively measuring oxidized HDL using a small amount of blood sample has been desired, but such a technique has not yet been reported.

Japanese Unexamined Patent Publication No. 9-335525

Arterioscler Thrombo Vasc Biol. , 2009, 29, 2169-2175 Journal of Japan Society for Bioscience and Biotechnology, 1988, 62, No. 10,1491-1494

The present invention has been made in view of the above-mentioned problems of the prior art, and an object of the present invention is to provide a method for selectively measuring oxidized HDL by a simple method.

As a result of diligent research to achieve the above-mentioned object, the present inventor surprisingly includes a blood sample collected from a subject and a specific solution by a simple operation of mixing the blood sample. We have found that it is possible to measure the oxidized HDL with high accuracy, and further found a method for selectively measuring only the oxidized HDL. Based on such findings, the present inventor has completed the present invention by repeating further research.

That is, the present invention includes the method for measuring HDL oxide and the kit for measuring HDL oxide described in the following sections.
Item 1.
(1) A method for measuring HDL oxide, which comprises a step of mixing a blood sample collected from a subject or a separated product thereof with a solution containing a transition metal compound and an acidic buffer solution.
Item 2.
Item 2. The method according to Item 1, wherein the acidic buffer is an acetate buffer.
Item 3.
Item 2. The method according to Item 1 or 2, wherein the pH of the acidic buffer is 2 to 6.9.
Item 4.
Item 6. The method according to any one of Items 1 to 3, wherein the transition metal compound is an iron compound.
Item 5.
Item 4. The method according to Item 4, wherein the iron compound is a divalent iron compound.
Item 6.
further,
(2) The method according to any one of Items 1 to 5, which comprises a step of measuring free radicals produced in the step (1).
Item 7.
A kit for measuring oxidized HDL, which comprises a transition metal compound and an acidic buffer solution.
Item 8.
Item 7. The kit according to Item 7, wherein the acidic buffer is an acetate buffer.
Item 9.
Item 4. The kit according to Item 7 or 8, wherein the pH of the acidic buffer solution is 2 to 6.9.
Item 10.
The kit according to any one of Items 7 to 9, wherein the transition metal compound is an iron compound.
Item 11.
Item 5. The kit according to Item 10, wherein the iron compound is a divalent iron compound.

According to the present invention, the oxidized HDL contained in a blood sample can be accurately measured by a simple operation.

It is a figure which shows the result of Test Example 1. It is a figure which shows the result of Test Example 2-3. It is a figure which shows the result of Test Example 4. It is a figure which shows the result of Test Example 5. It is a figure which shows the result of Test Example 6.

Hereinafter, the present invention will be described in detail.

1. 1. Method for Measuring Oxidized HDL The present invention includes a method for measuring oxidized HDL. In the present specification, the method may be referred to as "the method of the present invention".

The method of the present invention includes (1) mixing a blood sample or a separated product thereof collected from a subject with a solution containing a transition metal compound and an acidic buffer solution. In the present specification, the process may be referred to as "process (1)". In step (1), a blood sample or peroxy radicals (R-OO ^·) by the hydroperoxide in the oxidation HDL contained in the isolates and (R-OOH) and the transition metal ions are reacted alkoxy radical (R- free radicals of O ^·), and the like are produced.

Examples of the blood sample collected from the subject used in the step (1) include whole blood, serum, plasma and the like. Further, as the blood sample, a blood sample obtained by separating HDL (also referred to as "separated product of blood sample" in the present specification) can also be used. The method for separating HDL from a blood sample is not particularly limited, and a known method can be adopted. For example, a method such as a dextran sulfate method using dextran sulfate and magnesium ions can be adopted.

The subject for which the blood sample is collected is not particularly limited, and may be a non-human mammal as well as a human. The human being to be a subject is not particularly limited, and examples thereof include healthy subjects, arteriosclerosis patients, diabetic patients, cardiovascular patients, and humans suspected of having high oxidized HDL and oxidized LDL levels. The non-human mammal is not particularly limited, and a mammal bred as a pet, livestock, experimental animal or the like is preferable. Examples of such non-human mammals include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas and the like.

The method for collecting a blood sample from a subject is not particularly limited, and the method can be performed according to a conventional method using a known instrument, device, or the like. When serum or plasma is used as the blood sample, it is preferable to use a vacuum blood collection tube containing serum or plasma separating agent from the viewpoint of ease of handling, infection prevention, and the like. Further, the blood sample collected from the subject may be subjected to the step (1) as it is, or may be freeze-dried and stored, and then the freeze-dried product may be dissolved in an appropriate solvent described later and used. Good. Further, the blood sample collected from the subject may be frozen and stored as it is, or may be frozen and stored by dissolving it in an appropriate solvent, and then thawed at the time of use. The method and conditions for storing the blood sample collected from the subject are not particularly limited, and the method can be performed according to a conventional method.

The acidic buffer solution is not particularly limited as long as it has an acidic pH and has a buffering action, and a known buffer solution can be used. For example, acetic acid buffer solution and the like can be mentioned.

The pH of the acidic buffer solution is not particularly limited as long as it is acidic, and is preferably about 2 to 6.9, more preferably about 3 to 6.5, and more preferably about 4.5 to 6. Is even more preferable. When the color development method is adopted as the method for measuring free radicals in the step (2) described below, the pH of the acidic buffer solution is particularly preferably about 3 to 6.9.

The concentration of the acidic buffer solution is not particularly limited, and is, for example, 0.005 to 0.5 M, preferably 0.01 to 0.2 M.

The transition metal compound is not particularly limited as long as it can be ionized into a metal ion in the mixed solution and can react with hydroperoxide (R-OOH) to produce a free radical, for example. , Copper (II) compound, iron (II) compound (divalent iron compound), iron (III) compound (trivalent iron compound) and the like. Among these, iron compounds such as iron (II) compounds and iron (III) compounds are preferable. Examples of the iron compound include ammonium iron (II) sulfate hexahydrate, iron (III) chloride hexahydrate and the like. By using the transition metal compound in step (1), 1) the presence of a sufficient amount of transition metal ions that react with hydroxyperoxide makes it possible to measure oxidized HDL with a small amount of blood sample, and 2) it is contained in blood. Advantageous effects such as the ability to measure oxidized HDL without being affected by the amount of iron are exhibited.

It should be noted that the divalent iron compound can measure the oxidized HDL in a smaller amount than the trivalent iron compound. In other words, the divalent iron compound has a higher measurement sensitivity of oxidized HDL than the trivalent iron compound. Therefore, among the above-mentioned iron compounds, a divalent iron compound is particularly preferable.

The concentration of the transition metal compound in the mixed solution is not particularly limited, and can be, for example, about 0.1 to 150 μM, preferably 1 to 100 μM.

The reaction temperature in the step (1) is not particularly limited, and can be, for example, about 20 to 40 ° C.

As described above, in step (1), a blood sample or a hydroperoxide in the oxidation HDL contained in the isolate (R-OOH) with a transition metal peroxy radicals by ions and react (R-OO ^·) and alkoxy free radicals such as the radical (R-O ^·) is produced. Oxidized HDL can be measured by measuring the free radicals produced in step (1).

Therefore, it is preferable that the method of the present invention further includes (2) a step of measuring the free radicals produced in the above step (1). In this specification, the process may be referred to as "process (2)".

The method for measuring the free radical is not particularly limited, and a known method can be adopted. For example, a color development method, a chemiluminescence method, an electron spin resonance method (ESR method), and the like can be mentioned.

The color-developing method is a method of measuring the absorbance of a color-developed substance using a spectrophotometer or the like using a substance (color-developing substance) having an action of reacting with free radicals to develop color.

The color-developing substance is not particularly limited as long as it is a compound having an action of reacting with a free radical or an alkoxy radical to develop a color, and a known compound can be used. For example, a compound represented by the following general formula (1) or a salt thereof and the like can be mentioned.

In the above general formula (1), each R represents the same or different hydrogen atom, halogen atom, methyl, or ethyl. In particular, it is preferable that at least two of each R are methyl or ethyl, and it is more preferable that both Rs substituted with the same nitrogen atom are methyl or ethyl.

As the compound represented by the general formula (1), N, N-dimethyl-p-phenylenediamine (DMPD) and N, N-diethyl-p-phenylenediamine (DPD) are preferable.

Examples of the salt of the compound represented by the general formula (1) include sulfate, oxalate, diacetate and the like.

The compound represented by the general formula (1) or a salt thereof used in the reaction step may be used alone or in combination of two or more.

Further, the compound represented by the general formula (1) can be used by dissolving it in a solvent, if necessary. The solvent is not particularly limited, and examples thereof include pure water, a buffer solution, and dimethyl sulfoxide (DMSO). The concentration of the solution is not particularly limited, and can be, for example, about 0.1 to 50 mM, preferably about 1 to 20 mM.

The method for measuring the absorbance of the developed substance is not particularly limited, and can be carried out by a conventional method. For example, since radical cations exhibiting reddish purple are generated by the reaction of free radicals with a color-developing substance, radical cations can be measured by measuring the absorbance using a known device. The wavelength for measuring the absorbance can be, for example, 460 to 570 nm, preferably 500 to 560 nm.

Further, when measuring the absorbance, it is preferable to measure the time course of the absorbance between the measurement start time and the measurement end time in order to measure quantitatively. For example, it is preferable to measure the rate of increase in absorbance starting from the time point 2 minutes after the start of the reaction between the free radical and the color-developing substance and ending point 5 minutes after the start of the reaction.

The chemiluminescent method uses a substance (luminescent substance) that reacts with free radicals to emit light, and measures the light emitted when the excited substance returns to the ground state using a luminescence photometer or the like. is there.

The luminescent substance is not particularly limited as long as it is a compound having an action of reacting with free radicals to emit light, and a known compound can be used. For example, luminol, Dansyl-TEMPO, luciferinlu, 2-methyl-6-p-methoxyphenylethylimidazopyrazinone (MPEC), Hydroxyphenyl Fluorescein (HPF), Aminophenyl Fluorescein (HPF), Aminophenyl Fluorescein (HPF), Aminophenyl Fluorescein (HPF)

The method for measuring the light of the luminescent substance is not particularly limited, and can be appropriately determined according to the type of the luminescent substance to be used and the like.

The electron spin resonance method (ESR method) is a spectroscopic analysis that observes the transition between levels that occurs when an unpaired electron is placed in a magnetic field. The electron spin resonance method is not particularly limited, and can be carried out by using a known method and equipment. Either a direct method for directly measuring free radicals or an indirect method for reacting a spin trapping agent with free radicals is performed. May be. When the indirect method is adopted, the spin trapping agent is not particularly limited, and a known spin trapping agent can be used. Examples of the spin trap agent include 5,5-dimethyl-1-pyrrolin-N-oxide (DMPO), 2,5,5-triethyl-1-pyrrolin-N-oxide (M ₃ PO), 3,3,5. Nitron spin trapping agents such as 5-tetramethyl-1-pyrrolin-N-oxide (TMPO), N-tert-α-phenylnitron (PBN); 2-methyl-2-nitrosopropane (MNP), nitrosobenzene ( Examples thereof include nitroso-based spin trapping agents such as ND).

2. Oxidized HDL Measurement Kit The present invention also includes a kit for measuring HDL oxide. In the present specification, the kit may be referred to as "the kit of the present invention".

The kit of the present invention contains the above-mentioned transition metal compounds and acidic buffers. The transition metal compound and the acidic buffer solution contained in the kit of the present invention are the same as those in the above-mentioned method of the present invention.

In addition, the kits of the invention can include reagents for measuring free radicals. For example, when the above-mentioned coloring method is adopted for measuring free radicals, the above-mentioned coloring substance can be included.

In addition to the above-mentioned kits, the kit of the present invention may contain other reagents, instruments and the like, if necessary. For example, a serum or plasma separating agent, a blood sampling device, and the like can be mentioned.

Further, the kit of the present invention may include an instrument for measuring free radicals, if necessary, in addition to the above. For example, when the above-mentioned color-developing method is adopted for measuring free radicals, a spectrophotometer or the like may be provided.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Test Example 1
In this test example 1, the oxidized HDL was measured according to the following procedure.
(1) Preparation of blood sample Blood is collected from the vein of the subject using a vacuum blood collection tube and centrifuged at 4 ° C. and 3500 rpm (1100 g) for 15 minutes to obtain a supernatant (plasma) as a blood sample. After that, it was stored at −80 ° C.

(2) Measurement of HDL oxide A mixture of 0.1 M acetate buffer (pH 4.8) and 100 μM ammonium iron (II) sulfate hexahydrate (Fe 2 ⁺ addition group), 0.1 M acetate buffer (pH 4.8). ) And 100 μM iron (III) chloride (Fe ³⁺ added group) and 0.1 M acetate buffer (pH 4.8) (Fe not added group). The mixed solution of each group is micro. It was added to each well of the plate and kept warm at 37 ° C. Next, an N, N-diethyl-p-phenylenediamine sulfate solution (solvent: DMSO) was added to each well to which the mixed solution of each group was added, and then the plasma obtained in (1) above was added (plasma). Addition group). As a control, only N, N-diethyl-p-phenylenediamine sulfate solution (DMSO) was added to each well to which the mixed solution of Fe ²⁺ added group and Fe ³⁺ added group was added (plasma-free group). The absorbance at a wavelength of 505 nm was measured using a microplate reader (manufactured by Bio-Rad Laboratories, Inc.) set at 37 ° C. The absorbance was set to Kinetics, measured 30 times every 10 seconds (total 5 minutes), and the absorbance (unit: mOD / min) was calculated from each value 2 to 5 minutes after the start of measurement. I went by doing. The results are shown in FIG.

From the comparison of each group of the Fe ²⁺ added group, the Fe ³⁺ added group, and the Fe non-added group, it was confirmed that the absorbance value was larger when the iron compound was added. Furthermore, since it was confirmed that the Fe ²⁺ -added group had a larger absorbance value than the Fe ³⁺ -added group, the divalent iron compound could measure the oxidized HDL with higher sensitivity than the trivalent iron compound. Do you get it.

Test Example 2
In this Test Example 2, various tests were performed to demonstrate that the test result of Test Example 1 measures oxidized HDL.

<Test Example 2-1>
0.3 ml of plasma was placed in a centrifugal column with an ultrafiltration membrane (manufactured by Millipore, trade name: Amicon Ultra-0.5 100 kDa), centrifuged at 20 ° C. and 14000 g for 10 minutes, and remained on the filtrate and membrane. The liquids (concentrated liquids) were collected. Then, the absorbance was measured in the same manner as in the Fe ²⁺ addition group of Test Example 1 except that the blood sample was replaced with the filtrate and the concentrate. The test was conducted three times. As a result, when the measurement result of the absorbance of the sample (plasma) not subjected to centrifugation was 100%, the filtrate was 0.4% and the concentrate was 99.3% (the average value of all three times was taken). Show.).

From the results, it was strongly suggested that the absorbance value measured in Test Example 1 was derived from a polymer such as lipoprotein contained in plasma.

<Test Example 2-2>
In Test Example 2-1 it was found that macromolecules such as lipoprotein contained in plasma were involved. Therefore, in Test Example 2-2, low-density lipoprotein (LDL) and very low-density lipoprotein The test was conducted with an eye on (VLDL).

In the experiment, an LDL / VLDL purification kit (manufactured by Cell Biolab, trade name: STA-606) was used, and LDL / VLDL was purified according to the manual attached to the kit. Specifically, the dextran solution and precipitation solution A attached to the kit were added to plasma, reacted on ice for 5 minutes, and then centrifuged under the condition of 6000 g for 10 minutes. After centrifugation, the supernatant was collected and stored at −80 ° C., and the pellets were purified by LDL / VLDL according to the manual. Then, the absorbance was measured in the same manner as in the Fe ²⁺ addition group of Test Example 1 except that the blood sample was replaced with the supernatant and purified LDL / VLDL. As a result, when the measurement result of the absorbance of the unpurified sample (plasma) was 100%, the supernatant was 105% and the purified LDL / VLDL was less than 3%.

From the results, it was suggested that the absorbance value measured in Test Example 1 was not derived from LDL and VLDL among the lipoproteins contained in plasma.

<Test Example 2-3>
Since it was found that LDL and VLDL contained in plasma were not involved in Test Example 2-2, in Test Example 2-3, the test was conducted focusing on high-density lipoprotein (HDL).

Plasma is filtered through a filtration membrane with a pore size of 0.45 μm (manufactured by Millipore, trade name: Milex-HV), and a solution of sodium dextran sulfate (molecular weight: 36000 to 50000) (pH 7.3) is added to the obtained filtrate (filtrate A). (Containing 0.5 M magnesium chloride) was added, and the mixture was stirred and allowed to stand at room temperature. Then, centrifugation was performed for 30 minutes under the condition of 1500 g, the supernatant was collected, and the filtrate (filter B) was collected by filtering using the same filtration membrane as above. Then, the absorbance was measured in the same manner as in the Fe ²⁺ addition group of Test Example 1 except that the blood sample was replaced with the filtrate A, the supernatant and the filtrate B. As a result, when the measurement result of the absorbance of the unfiltered sample (plasma) was 100%, the filtrate A was 99%, the supernatant was 104%, and the filtrate B was 115%.

Further, the lipoprotein contained in the filtrate A and the filtrate B was analyzed by gel filtration HPLC. The chromatogram by gel filtration HPLC is shown in FIG. 2, and the quantification results of each lipoprotein are shown in Table 1 below. In Table 1 below, "CM" indicates chylomicrons, "VLDL" indicates very low density lipoprotein, "LDL" indicates low density lipoprotein, and "HDL" indicates high density lipoprotein, which are attached to each term. The numbers in parentheses indicate the particle size of each lipoprotein.

From the results shown in FIG. 2, in both the filtrate A and the filtrate B, a peak derived from HDL-C was observed near 26.0 min. Further, in the filtrate A, a peak derived from LDL-C was observed in the vicinity of 23.2 min, whereas in the filtrate B, a peak derived from LDL-C was not observed. From FIG. 2, Table 1 and the results of the absorbance measurement test, it was strongly suggested that the absorbance value measured in Test Example 1 was derived from HDL among the lipoproteins contained in plasma.

Test Example 3
In this Test Example 3, plasma and HDL separated from plasma were used as samples, and it was examined whether or not the measured values of both were correlated. Specifically, the absorbance was the same as in the Fe ²⁺ addition group of Test Example 1 except that the plasma (plasma group) of three healthy subjects and the filtrate B (HDL separation group) of Test Example 2-3 were used as samples. Was measured. After that, the correlation of the results by each group of 3 people was analyzed.

From the analysis results, it was found that there is a high positive correlation (R = 0.966) between the HDL separation group and the plasma group. Therefore, it was found that not only blood samples such as plasma but also HDL separated from blood samples can be used as samples.

Test Example 4
In Test Example 4, it was examined whether or not the measured values obtained by the method for measuring oxidized HDL using the conventional ELISA method and the method of the present invention are correlated. Specifically, plasma of three healthy subjects was used to measure the absorbance in the same manner as in Test Example 2-3 (method group of the present invention). Further, as the ELISA method, an oxidized HDL measurement kit (manufactured by Cell Biolab, trade name: STA-869) was used, and the oxidized HDL was measured according to the manual attached to the kit (ELISA method group). Then, the correlation between the results of the three method groups of the present invention and the ELISA method group was analyzed. The results are shown in FIG.

From the results, it was found that there is a high positive correlation (R = 0.987) between the test results in the method group of the present invention and the test results in the ELISA method group.

Test Example 5
In Test Example 5, oxidized HDL was measured using whole blood and plasma as blood samples. Blood was collected in a heparin tube, and a part of it was stored at 4 ° C. for whole blood measurement. The rest was centrifuged at 4 ° C. and 1000 g for 10 minutes to obtain plasma. After that, the blood samples were replaced with whole blood (10 μl and 20 μl) and plasma (10 μl), and the sample using whole blood was added with Fe ^{2+ in} Test Example 1 except that glucose was added to prevent hemolysis. The absorbance was measured in the same manner as in the above. The results are shown in FIG.

From the results, it was found that not only plasma but also whole blood can be used as a blood sample.

Test Example 6
In this Test Example 6, in the result of Test Example 1 (FIG. 1), it was confirmed that the Fe ²⁺ added group had a larger absorbance value when the Fe ²⁺ added group and the Fe ³⁺ added group were compared. Therefore, the effect of the concentration of the divalent iron compound on the measurement results was examined.

(1) Preparation of blood sample In the same manner as in Test Example 1, blood was collected from the vein of the subject using a vacuum blood collection tube, centrifuged at 4 ° C. and 3500 rpm (1100 g) for 15 minutes, and the supernatant was obtained. (Plasma) was obtained as a blood sample and then stored at −80 ° C. The serum iron (complex of transferrin and trivalent iron) contained in the blood samples obtained from the subjects 1 and 2 was measured by the nitroso-PASP method and found to be 76.8 μg / dL. there were.

(2) Measurement of Oxidized HDL A mixed solution of 0.1 M acetate buffer (pH 4.8) and 1 μM, 10 μM, or 100 μM ammonium iron (II) sulfate hexahydrate was prepared.

Each of the mixed solutions prepared above was added to each well of the microplate, and the temperature was kept at 37 ° C. Next, an N, N-diethyl-p-phenylenediamine sulfate solution (solvent: DMSO) was added to each well to which the mixed solution of each group was added, and then the blood sample obtained in (1) above was added. As a control, N, N-diethyl-p-phenylenediamine sulfate solution (solvent: DMSO) and blood sample were added without adding the mixed solution of each group to each well. Then, the absorbance at a wavelength of 505 nm was measured using a microplate reader (manufactured by Bio-Rad) set at 37 ° C. The absorbance was set to Kinetics, measured 30 times every 10 seconds (total 5 minutes), and the absorbance (unit: mOD / min) was calculated from each value 2 to 5 minutes after the start of measurement. I went by doing. The measurement results are shown in FIG.

From the results of FIG. 5, it was confirmed that the absorbance value increased as the concentration of the divalent iron compound increased.

Claims (5)

(1) A step of mixing a blood sample collected from a subject or a separated product thereof with a solution containing a transition metal compound and an acidic buffer solution , and
(2) A method for measuring oxidized HDL, which comprises a step of measuring free radicals produced in the step (1) .
The method, wherein the pH of the acidic buffer is 4.5-6 . The method according to claim 1, wherein the acidic buffer solution is an acetate buffer solution. The method according to claim 1 or 2 , wherein the transition metal compound is an iron compound. The method according to claim 3 , wherein the iron compound is a divalent iron compound. A kit for measuring oxidized HDL, which contains a transition metal compound and an acidic buffer solution .
A kit for measuring oxidized HDL, wherein the pH of the acidic buffer solution is 4.5 to 6 .