JP2000116400A - Quantitative analysis of cholesterol in lipoprotein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for individually and quantitatively analyzing cholesterol in each lipoprotein (e.g. respectively in high-density lipoprotein, in low-density one and in very low-density one) without performing a fractionation procedure, which is important for lipid metabolism in the field of clinical diagnosis. SOLUTION: The method of this invention is to directly and selectively measure cholesterol quantity in each lipoprotein (i.e., chylomicron, HDL, LDL, or VLDL) in a specimen in the presence of a phosphorus compound, a surfactant and a solubilizer for dissolving protein. It is possible to impart selectivity to the reaction of each lipoprotein and enzyme by selecting the kind of the phosphorous compound and by selecting the kinds and concentrations of the surfactant and the solubilizer, and to selectively measure cholesterol in each lipoprotein even in a state that various lipoproteins coexist in a specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for individually quantifying cholesterol in each lipoprotein, for example, high-density lipoprotein, low-density lipoprotein, and ultra-low-density lipoprotein without fractionation operation. Quantification of cholesterol in lipoprotein which is important in lipid metabolism in the field.

[0002]

BACKGROUND ART HDL has been attracting attention as a lipoprotein having an anti-atherosclerotic effect, and LDL is said to have a role of supplying cholesterol to peripheral cells, and is directly used for various arteriosclerosis including coronary atherosclerosis. It is known that the blood level is an indicator of atherosclerotic disease. VLDL has also been attracting attention for its association with arteriosclerosis. Conventional methods for quantifying HDL cholesterol include ultracentrifugation and electrophoresis, and methods for quantifying LDL cholesterol include ultracentrifugation, electrophoresis, and conversion. Examples of VLDL cholesterol quantification include:
There are ultracentrifugation and electrophoresis. Ultracentrifugation is used as a basic quantification method. HDL, LDL or VLDL is separated by the difference in specific gravity using an ultracentrifuge for separation, and the amount of cholesterol is measured. (Advanced Rapid Research. Vol. 6, p. 1, 1968). However, it has disadvantages in terms of quantitativeness, simplicity, economy, etc. When using electrophoresis, cholesterol is quantified by separation using a cellulose acetate membrane or agarose gel as a support. However, these quantification methods are not suitable for automated analyzers that are frequently used in the fields of multi-sample processing, rapid quantification, and clinical tests. Recently, as a method for directly measuring cholesterol in lipoproteins, a method using a flocculant such as dextran sulfate (JP-A-6-24211)
No. 2,600,065), a method using a surfactant or the like (Japanese Patent Publication No. 6-16720, Japanese Patent Application Laid-Open No. 58-1).
65800), a method using a protein solubilizing agent and a sugar compound (PCT / JP96 / 00665, EP069879).
1A1), etc., but none of these methods can accurately measure cholesterol in lipoproteins in samples high in triglyceride (TG) and M protein.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have proposed a system of a cholesterol measuring reagent in which a phosphorus compound, a surfactant and a protein solubilizing agent are present, and HDL, LDL and VL fractionated by ultracentrifugation.
When measured using DL and chylomicron (CM) lipoproteins, the reactivity of lipoproteins varies depending on the combination of the phosphorus compound, surfactant and protein solubilizer. As a result, cholesterol in HDL and cholesterol in LDL The present inventors have found that the reactivity of cholesterol, cholesterol in VLDL and cholesterol in CM is different, and completed the present invention. In the present invention, the amount of HDL cholesterol in a sample in the presence of a phosphorus compound, a surfactant, and a protein solubilizing agent, L
The present invention relates to a method for determining the amount of DL cholesterol, VLDL cholesterol, or CM cholesterol. In the above measurement, in order to further increase the specificity, a divalent metal salt and also a sugar phosphate compound or a sugar phosphate compound can be used in combination. Further, according to the present invention, a reagent for quantifying cholesterol in HDL, cholesterol in LDL, cholesterol in VLDL or cholesterol in CM containing a phosphorus compound, a surfactant and a protein solubilizer can be produced. As the phosphorus compound, an inorganic phosphate compound, an organic phosphorus compound, an organic phosphate compound, and / or a glucan phosphate compound are used. As surfactants and protein solubilizers, use anionic surfactants, nonionic surfactants, cationic surfactants, amphoteric (betaine-type) surfactants, and function-providing surfactants be able to. Cholesterol in HDL, cholesterol in LDL, cholesterol in VLDL and CM in sample
Depending on which part of cholesterol in the sample is measured, the type and concentration of surfactant and protein solubilizer, or the combination of several types of surfactant and protein solubilizer and the combination of each concentration must be selected. I have to. For example, when measuring cholesterol in HDL in a sample, a polyoxyethylene oxypropylene block polymer system of a nonionic surfactant is selected. When measuring cholesterol in LDL in a sample, polyoxyethylene oxypropylene block polymer is used. Ethylene derivatives and polyoxyethylene polyoxypropylene derivatives as nonionic surfactants can be selected. As the metal salt, a magnesium salt, a calcium salt, a manganese salt, a cobalt salt or the like of 0.00150M is used. The present invention relates to a phosphorus compound and a surfactant,
It is a method for directly separating and quantifying cholesterol in lipoprotein by coexisting a protein solubilizing agent in a reagent for measuring cholesterol in lipoprotein, and any method can be used as long as the cholesterol measuring system is an enzymatic measuring system. is there. According to the method of the present invention, cholesterol in lipoprotein in a sample such as blood or urine can be directly fractionated and measured. Next, the method of the present invention will be described with reference to examples.

[0004]

Example 1 A method for directly determining HDL cholesterol and an ultracentrifugation method were compared. Reagent composition of this method. In this method, 6 μl of a serum sample was added to 300 μl of the first reagent preliminarily heated at 37 ° C., heated at 37 ° C. for 5 minutes, and the absorbance of the obtained solution at 585 nm was measured (E).
1). Next, the second reagent 1 previously heated to 37 ° C.
After adding and stirring 00 μl, the absorbance at the same wavelength was measured 5 minutes later (E2, value after concentration correction). The amount of HDL cholesterol was calculated by performing the same operation using a standard solution of HDL cholesterol 50 mg / dl and comparing the value of (E2-E1). Ultracentrifugation was measured according to the CDC method. Table 1 shows the results.

[0005] As shown in Table 1, the measurement results of the method of the present invention agreed well with the results of the ultracentrifugation method.

[0006]

Example 2 A phosphorus compound, a surfactant and a protein solubilizer used in the first and second reagents were changed, except that
The same operation as in the present method of Example 1 was performed to compare three serum samples with the ultracentrifugation method. In this method, each measured value was obtained by an automatic analyzer. Table 2 shows the results.

[0007]

[0008]

Example 3 The present method for directly measuring LDL cholesterol and the CDC method were compared. Reagent composition of this method In this method, 6 μl of a serum sample was added to 300 μl of the first reagent preliminarily heated to 37 ° C., and after heating at 37 ° C. for 5 minutes, the absorbance at 585 nm was measured (E1). Next, 100 μl of the second reagent preheated at 37 ° C. was added and stirred, and after 5 minutes, the absorbance at the same wavelength was measured (E2, value after concentration correction). The LDL cholesterol amount was calculated by performing the same operation using a standard solution having an LDL cholesterol concentration of 200 mg / dl and comparing the values. Table 3 shows the results.

[0009] As is apparent from Table 3, the results obtained by the method of the present invention are as follows.
Good agreement with the CDC method.

[0010]

Example 4 The same operation as in Example 3 was carried out, except that the combination of the phosphorus compound, the surfactant, and the protein solubilizing agent used in the first and second reagents was changed. The values were compared. Table 4 shows the results.

[0011] As shown in Table 4, the measurement result by the method of the present invention is C
It was in good agreement with the measurement result by DC method.

[0012]

Example 5 The same measurement as in the present method of Example 3 was carried out except that the combination of the color former, the phosphorus compound, the surfactant, the protein solubilizing agent, etc. was changed. Was compared with. Table 5 shows the measurement results.

[0013] As shown in Table 5, the measurement result by the method of the present invention was C
It was in good agreement with the measurement result by DC method.

Claims (10)

[Claims] 1. A lipoprotein (chylomicron, HD) in a sample in the presence of a phosphorus compound, a surfactant, and a protein solubilizing agent.
L, LDL, VLDL), wherein the cholesterol content in chylomicron, HDL, LDL, or VLDL is determined. 2. A method for determining cholesterol in HDL, comprising measuring the amount of cholesterol in HDL in a sample in the presence of a phosphorus compound, a surfactant, and a protein solubilizing agent. 3. A method for quantifying cholesterol in LDL, which comprises measuring the amount of cholesterol in LDL in a sample in the presence of a phosphorus compound, a surfactant, and a protein solubilizing agent. 4. A method for quantifying cholesterol in VLDL, which comprises measuring the amount of cholesterol in VLDL in the presence of a phosphorus compound, a surfactant, and a protein solubilizing agent. 5. The method according to claim 1, wherein the phosphorus compound is an inorganic phosphoric acid, an inorganic phosphate, an organic phosphate, or an organic phosphorus compound.
4. Quantitative method described in 4. 6. The protein solubilizing agent is an anionic surfactant,
The method according to any one of claims 1 to 5, which is a cationic surfactant or a nonionic surfactant. 7. A method for measuring the amount of cholesterol, which comprises determining the amount of hydrogen peroxide or reduced lipogen produced by the action of cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase on a sample. Cholesterol esterase, cholesterol oxidase or cholesterol dehydrogenase chemically modified or unmodified cholesterol esterase, chemically modified or unmodified cholesterol oxidase, or chemically modified or unmodified cholesterol Claims 1 to 6 which are dehydrogenases
Quantitative method described. 8. When measuring the amount of cholesterol, 1 to
8. The method according to claim 1, wherein a trivalent metal salt is present. Determination of 8 wherein ^- 9. claim 1, wherein the presence of a sugar compound when measuring the amount of cholesterol. 10. The method according to claim 1, wherein the surfactant is a polyoxyethylene polyoxypropylene copolymer compound, a polyoxyethylene polymer compound and / or a polypropylene polymer compound.