KR20000015925A - Method for quantitatively determining ldl cholesterols - Google Patents

Abstract

PURPOSE: Methods for quantitatively determining LDL (Low Density Lipoprotein) cholesterol are provided, which can eliminate the necessity for pretreatment such as centrifugation and electrophoresis. CONSTITUTION: The method comprises the steps of adding surfactants solubilizing lipoproteins, such as polyoxyethylenealkylene phenyl ethers and polyoxyethylenealkylene tribenzylphenyl ethers, and cholesterol assaying enzyme reagents to the serum; preferentially reacting HDL (High Density Lipoprotein) and VLDL (Very Low Density Lipoprotein) cholesterol among lipoproteins; and determining the amounts of the cholesterol reacted thereafter, that is, of the LDL cholesterol. Preferably the materials whose bonding affinity to VLDL is stronger than that of LDL are employed further.

Description

METHOD FOR QUANTITATIVELY DETERMINING LDL CHOLESTEROLS

Lipids such as cholesterol bind to apoproteins in the serum to form lipoproteins. Lipoproteins are classified into chiromicrons, ultra low specific lipoproteins (VLDL), low specific lipoproteins (LDL), and high specific lipoproteins (HDL) according to physical properties. Among these lipoproteins, LDL is known as one of the causative agents of atherosclerosis.

Epidemiologically, LDL cholesterol levels have been shown to correlate strongly with the incidence of atherosclerotic disease. Clinically, LDL cholesterol levels are very useful if they can be measured by routine methods.

As a conventional method for measuring LDL cholesterol, for example, after separating LDL from other lipoproteins by ultracentrifugation, cholesterol is measured, and lipids are stained after separation by electrophoresis. Methods of measurement have been known. However, none of these methods have problems such as complicated operation and inability to process a large number of specimens, and are rarely used on a daily basis. In addition, a method of sensitizing an antibody that binds to a lipoprotein other than LDL on a carrier, and after mixing with a sample, separates and removes the fraction that does not bind to the carrier, and measures cholesterol in the fraction. Although this method is more suitable for routine measurement than the previous two methods, it is difficult to automate because a manual process is included in the measurement operation, and thus, processing of a large number of samples is still not suitable.

On the other hand, as a method of fractionating and quantifying lipoproteins in a sample without using separation means such as ultracentrifugation or electrophoresis, enzymes used to measure cholesterol for HDL and other lipoproteins (ie, chiromicron, VLDL, LDL) (Mainly, cholesterol oxidase and cholesterol esterase) are known and methods for quantifying by inducing only HDL cholesterol by enzymatic reaction are known. For example, Japanese Patent Application Laid-Open No. 7-301636 discloses a method for measuring only HDL cholesterol by using a surfactant and a sugar compound, and Japanese Patent Application Laid-Open No. 6-242110 should be measured. A method of inhibiting reactivity with enzymes by aggregating lipoproteins other than proteins and detecting only cholesterol in lipoproteins to be measured is disclosed. These methods are extremely useful because they can be applied to automated analyzers, allowing the entire process to be automated, but these methods are only for the quantitative determination of lipoproteins other than HDL and HDL, and for the quantitative determination of LDL, VLDL and chiromicrons. Can not. Therefore, these techniques did not support the purpose of measuring LDL cholesterol without using a separation means.

In addition, Japanese Patent Application Laid-Open No. 7-280812 discloses quantification by inducing LDL aggregation once and then inducing cholesterol in other lipoproteins into a system that does not participate in the LDL determination system and reacting LDL cholesterol after dissolving LDL aggregation. A method of doing this is disclosed. However, even in this Japanese Patent Application Laid-Open No. 7-280812, the solution to the fractional quantification of LDL and VLDL and / or chiromicron, which is absolutely indispensable for the measurement of LDL cholesterol, like the two patent publications described above, Not presented at all. In addition, there is a problem that can not be applied to a general automatic analysis device because of the large number of reaction processes required for the measurement, its use value is also extremely limited.

As described above, the conventionally known technique cannot efficiently measure LDL cholesterol without a separate operation, and there is no information suggesting its possibility.

Accordingly, an object of the present invention is to provide a fractional quantitative determination method of LDL cholesterol which can be efficiently measured by a simple operation without the need for pretreatment such as centrifugation or electrophoresis and can be applied to various automatic analyzers.

The present invention eliminates the need for separation operations such as centrifugation and electrophoresis, and efficiently quantifies cholesterol in low density lipoprotein (LDL) and cholesterol in lipoproteins other than LDL efficiently by simple operation with a small amount of samples. It is about how to.

1 is a diagram showing the correlation between the measured value of LDL cholesterol obtained in Example 1 by the method of the present invention and the measured value of LDL cholesterol obtained by ultracentrifugation.

Figure 2 is a diagram showing the correlation between the measurement value of LDL cholesterol obtained in Example 2 by the method of the present invention and the LDL cholesterol measurement obtained by ultracentrifugation.

Figure 3 is a diagram showing the correlation between the measurement value of LDL cholesterol obtained in Example 3 by the method of the present invention and the LDL cholesterol measurement obtained by ultracentrifugation.

Best Mode for Carrying Out the Invention

The surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzylphenyl ether used in the present invention is a surfactant that dissolves lipoprotein, and commercially available emalen A-60 (Emulgen) A-60, the product made by Kao company), The example of the latter is Emalgen B66 (Emulgen B66, the product made by Kao company). These surfactant can be used individually or in combination of 2 or more types. The amount of use varies depending on the compound, and there is no particular limitation, but it is preferable to use it in a concentration of 0.01 to 2% by weight so that LDL cholesterol can be detected within a desired measurement time for each analytical device to which a reagent is applied.

In addition, the cholesterol measurement method according to the present invention is preferably carried out again in the presence of a substance exhibiting a stronger binding affinity for VLDL than for LDL, and in particular, when the sample is chiromicron containing serum, the substance The result obtained by measuring by adding is obtained. As such a substance, the substance which produces | generates a polyanion and a divalent metal ion, etc. are mentioned, Specifically, a polyanion includes phosphotungstic acid and its salt, dextran sulfate, and heparin; 2 is the substance for generating the metal ion in a 2 _{MgCl 2, CaCl 2, MnCl 2} , NiCl 2 , etc. can be mentioned a metal chloride and a hydrate thereof. These substances may be used alone or in combination of two or more thereof, and the amount of use thereof depends on the type of the substance, and there is no particular limitation, but as the reaction termination concentration, polyanion is 0.002 to 10% by weight, and divalent metal ions are generated. In the case of materials, the range of 0.01 to 1% by weight is preferred.

When a substance showing stronger binding affinity for VLDL than for surfactant and LDL is added to the sample serum, a mixture of the former, the latter and the enzyme reagent for cholesterol measurement may be added, respectively; Moreover, you may mix three and add as an identical reagent.

As a measuring method of cholesterol, any of known enzyme measuring methods may be used. For example, a method using a combination of cholesterol esterase and cholesterol oxidase as an enzyme reagent may be used. Among these, a method using a combination of cholesterol esterases and cholesterol oxidases is preferred. In addition, the method of finally detecting cholesterol after adding these cholesterol measuring enzyme reagents is not particularly limited. For example, absorbance analysis performed by combining peroxidase and chromogen, and directly detecting coenzyme or hydrogen peroxide. Etc. can be mentioned.

In order to detect the reaction of LDL cholesterol, it is necessary to measure the reaction amount after terminating the reaction of cholesterol in lipoproteins other than LDL, and after the reaction of cholesterol in lipoproteins other than LDL is almost completed , A method of dynamically monitoring the reaction in progress, or a method of adding a separate reaction promoter for the purpose of promoting the reaction of the LDL, and measuring the reaction at the end point of the reaction and correcting it as a blank value (2 point method). Can be used. In addition, in the two-point method, it is also possible to induce cholesterol in the reaction of cholesterol in lipoproteins other than LDL in a separate reaction system that does not participate in the quantification of LDL cholesterol in the next step, and detect only the reaction of LDL cholesterol.

In addition, other lipoproteins present in serum include chiromicron, which usually appears only after ingestion of food, but is almost as reactive as VLDL. For this reason, even in the presence of chiromicron, the reactivity of the chiromicron, which is almost the same as that of VLDL, is promoted by adding a polyanion, a substance which generates a divalent metal ion, and the reaction of the chiromicron is terminated at the end of the reaction of the VLDL. In addition, fractional quantification of LDL cholesterol is possible by measuring the reaction amount of cholesterol.

In this situation, the present inventors have diligently studied and reacted with an enzyme reagent for measuring cholesterol in the presence of a specific surfactant that dissolves lipoproteins, while accelerating the reaction of cholesterol in HDL and VLDL, while suppressing the reaction of cholesterol in LDL. Significantly delayed, and the response of cholesterol in HDL and VLDL terminates prior to the cholesterol response in LDL, and therefore, it is found that LDL cholesterol can be fractionated and further applied to an automatic analysis device by selecting the measuring point appropriately. The present invention has been completed.

Accordingly, the present invention provides a method for quantifying LDL cholesterol, by adding a surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzylphenyl ether and enzyme reagent for measuring cholesterol to serum, The present invention provides a method for quantifying LDL cholesterol characterized by preferentially reacting cholesterol in high and ultra low specific lipoproteins, and then measuring the reaction amount of cholesterol thereafter.

In addition, the present invention is a surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzyl phenyl ether, a substance having a strong binding affinity for a low specific gravity lipoprotein, and cholesterol determination for a very low specific gravity lipoprotein. A method for quantifying low specific lipoprotein cholesterol, characterized in that the enzyme reagent is added, the high lipoprotein in the lipoprotein and the cholesterol in the ultra low specific lipoprotein are preferentially reacted, and then the reaction amount of the cholesterol is measured. To provide.

Furthermore, the present invention provides a kit for quantifying low specific gravity lipoprotein cholesterol, comprising a surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzylphenyl ether and an enzyme reagent for cholesterol measurement. will be.

In addition, the kit for quantification of low specific weight lipoprotein cholesterol is further included in the kit for quantification of low specific weight lipoprotein cholesterol, further comprising a substance having a stronger affinity for ultra low specific weight lipoprotein. To provide.

Hereinafter, although an Example is given and this invention is demonstrated in detail, the scope of the present invention is not limited to these Examples.

Example 1

Normal lipid serum was used as a sample, and LDL cholesterol was measured by Hitachi 7070 type automatic analyzer by the method of the present invention, and the measurement was compared with the measurement obtained by centrifugation. This result is shown in FIG.

That is, a reagent (300 µl) containing sodium phosphotungate (0.02 wt%) and MgCl ₂ · 6H ₂ O (0.2 wt%) were added to the sample (4 µl). After about 5 minutes, Emalgen A-60 (from Kao) (0.5 wt.%), Cholesterol esterase (1 U / ml), cholesterol oxidase (1 U / ml) and N, N-dimethyl-m- Cholesterol assay reagent (100 μl) containing toluidine (0.04 wt%) was added and the absorbance change was measured at 545 nm from 1 minute to 5 minutes after the addition of the second reagent.

Meanwhile, in the ultracentrifugation method, the serum was centrifuged at 100,000 g for 2 hours in the ultracentrifuge to remove the upper layer. 1 ml of the obtained lower layer was taken, a heparin solution (40 µl; heparin = 5000 usp units / ml) and a 1 M MgCl ₂ (50 µl) solution were added, and centrifuged at 5000 rpm for 30 minutes to obtain a supernatant. The supernatant fraction obtained by ultracentrifugation (containing LDL and HDL) and the fraction supernatant (containing HDL) obtained by adding heparin and MgCl ₂ solutions were measured for cholesterol, and the value obtained by subtracting the latter value from the former value was calculated as LDL cholesterol. (Ref. Paul S. Bachorik et al., Clin. Chem. 41/10, 1414-1420, 1955).

As shown in Fig. 1, the method of the present invention obtains the LDL cholesterol measurement value having a good correlation with the conventional ultracentrifugation method, although the sample to be used is extremely small and can be easily operated.

Example 2

Samples containing serum-containing chiromicrons with a high level of triglycerides were used as samples, and LDL cholesterol was measured by a Hitachi 7070 type automatic analyzer according to the present invention, and the measured values were compared with those obtained through ultracentrifugation. The results are shown in FIG.

In other words, Emalgen B66 (from Kao) (0.5 wt%), cholesterol esterase (0.3 U / ml), cholesterol oxidase (0.3 U / ml), peroxidase (0.3 U / Ml) and a cholesterol measuring reagent (300 μl) containing 4-aminoantipyrine (0.002 wt.%) Were added. After about 5 minutes, a reagent (100 μl) containing Triton X-100 (1 wt.%) And N, N-dimethyl-m-toluidine (0.04 wt.%) Was added and 545 nm 5 minutes after the addition of the second reagent. The absorbance was subtracted from the absorbance at 545 nm immediately before addition of the second reagent (correction was made in consideration of the change in reagent), and the absorbance change thereof was measured.

On the other hand, ultracentrifugation was performed in the same manner as in Example 1.

As shown in FIG. 2, as in Example 1, a measurement having a good correlation with the conventional LDL cholesterol measurement by ultracentrifugation was obtained.

Example 3

In Example 2, the same reagents and reagents were used as in Example 2 except that the first reagent contained phosphotungstic acid (0.3 wt%), and the measured values according to the method of the present invention were compared with those obtained by ultracentrifugation. . The results are shown in FIG.

As shown in Fig. 3, a serum sample containing a serum-containing chiromicron was used in the same manner as in Example 1, and a measurement value having a very good correlation with the LDL cholesterol measurement by the conventional ultracentrifugation method was obtained.

According to the present invention, there is no need for a separation operation such as centrifugation and electrophoresis, and it is possible to efficiently quantify LDL cholesterol in other lipoproteins efficiently, and can be widely applied to an automatic analysis device used in clinical tests. Clinically extremely useful.

Claims (5)

A surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzylphenyl ether and an enzyme reagent for measuring cholesterol are added to the serum, and the cholesterol in the high-weight lipoprotein and the ultra-low-weight lipoprotein is preferentially in the lipoprotein. After the reaction, the method for quantifying low specific gravity lipoprotein cholesterol, characterized in that the reaction amount of cholesterol is then measured. Surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzylphenyl ether, a substance having a strong binding affinity for a low specific lipoprotein, and an enzyme reagent for measuring cholesterol And preferentially reacting the cholesterol in the lipoprotein and the high-weight lipoprotein in the lipoprotein, and then measuring the reaction amount of the cholesterol in the lipoprotein. 3. The method for quantifying low specific gravity lipoprotein cholesterol according to claim 2, wherein the substance which shows stronger binding affinity for ultra low specific weight cholesterol for the low specific gravity lipoprotein is a substance which produces polyanion or divalent metal ions. . A kit for quantifying low specific gravity lipoprotein cholesterol, comprising a surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzylphenyl ether and an enzyme reagent for measuring cholesterol. The kit for quantifying low specific weight lipoprotein cholesterol according to claim 4, wherein the kit is made of a substance having a stronger affinity for ultra low specific weight lipoprotein than the low specific weight lipoprotein.