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Method of pretreatment of sample for quantitating cholesterol and method for quantitating cholesterol in specific lipoproteins by using the same

a pretreatment method and a technology for specific lipoproteins, applied in biochemistry equipment and processes, instruments, oxidoreductases, etc., can solve the problems of not being practicable, many samples cannot be handled, and the sample is a relatively large quantity, so as to achieve efficient quantitation of cholesterol, simple procedures, and superior

Inactive Publication Date: 2005-01-13
SEKISUI MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for measuring cholesterol in a specific lipoprotein in a sample. This is done by using an enzyme that acts on free cholesterol as a substrate. The method also includes a pretreatment step where the enzyme acts on the sample to remove any cholesterol from non-specific lipoproteins. This results in a more accurate measurement of cholesterol in the specific lipoprotein.

Problems solved by technology

These methods are however not used practically, because they involve one or more problems in that procedures are intricate and many samples cannot be handled.
This method is simpler compared with ultracentrifugation or electrophoresis, but due to the inclusion of the procedures to add the precipitation reagent and to perform the separation, requires each sample is a relatively large quantity, and involves a potential problem of causing an analytical error.
Furthermore, the entire analysis steps of this method can not be fully automated.
A problem however exists in accuracy because the reaction with the cholesterol in HDL and the reaction with cholesterol in other lipoproteins cannot be fully distinguished.
This method, however, requires at least three procedures to add reagents so that it can be applied only to particular automated analyzers, leading to a problem in a wide applicability.
Further, this method is not satisfactory from the standpoint of damages to analytical equipment and disposal of the reagents because of the use of a salt at a high concentration upon redissolution of an agglutinate.
This method, however, also involves a problem in accuracy in that turbidity, which occurs as a result of the use of the precipitation reagent, interferes with the measurement system.
Further, it is also economically unreasonable to use a polyanion or the like for making the resulting data consistent with those obtained by the precipitation method although the polyanion or the like is not needed from the standpoint of the principle of the measurement.

Method used

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  • Method of pretreatment of sample for quantitating cholesterol and method for quantitating cholesterol in specific lipoproteins by using the same
  • Method of pretreatment of sample for quantitating cholesterol and method for quantitating cholesterol in specific lipoproteins by using the same
  • Method of pretreatment of sample for quantitating cholesterol and method for quantitating cholesterol in specific lipoproteins by using the same

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0103] With respect to each of 30 serum samples containing lipoproteins, the cholesterol in HDL was quantitated by the below-described method according to the present invention and the precipitation method, and the measurement values were compared.

[0104] (Invention Method)

[0105] 10 mM phosphate buffer (First Reagent; pH 8.5) (300 μL), which contained 0.1 U / mL cholesterol dehydrogenase (product of Amano Pharmaceutical Co., Ltd.), 2.5 mM NAD and 0.03% 4-aminoantipyrine, was added to each sample (3 μL) (pretreatment). About 5 minutes later, a cholesterol quantitation reagent (Second Reagent) (100 μL)—which was composed of 100 mM MES buffer (pH6) containing 1% “EmulgenB-66”, 1.3 U / mL cholesterol esterase (product of Asahi Chemical Industry Co., Ltd.), 2 U / mL cholesterol oxidase (product of Asahi Chemical Industry Co., Ltd.), 5 U / mL peroxidase (product of Toyobo Co., Ltd.) and 0.04% disulfobutylmetatoluidine—was added.

[0106] Just before the addition of the Second Reagent and upon an e...

example 2

[0112] Measurements were conducted by another method of the present invention, which was similar to the invention method conducted in Example 1 except that in the first reagent, cholesterol dehydrogenase, NAD and the phosphate buffer were replaced by 5 U / mL cholesterol oxidase (product of Toyobo Co., Ltd.), 5 U / mL peroxidase (product of Toyobo Co., Ltd.) and 100 mM MES buffer (pH 6). The measurement values were compared with those obtained by the precipitation method in Example 1.

[0113] (Results)

[0114] The results are shown in Table 2 and FIG. 2.

TABLE 2PrecipitationInventionSample No.method (mg / dL)method (mg / dL)173732393835352454565575767574751528525094344105858115957124951134445147069153537165454174547186061195050205855213837225656233535242929256361265152273333285252296566304746

[0115] As is readily envisaged from the results, the invention method, despite the omission of a polyanion or the like, showed an extremely good correlation with the conventional precipitation method.

example 3

[0116] Using the reagents of Example 1 and Example 2, five serum samples of different triglyceride levels were measured. The measurement values were then compared with those obtained by the precipitation method. The results are shown in Table 3.

TABLE 3InventionInventionmethod inmethod inPrecipitationExample 1Example 2 Triglyceridemethod (mg / dL)(mg / dL)(mg / dL)level(mg / dL)Sample A474949198Sample B495049301Sample C262724742Sample D606161517Sample E374036428

[0117] As is shown in Table 3, measurement values of comparable levels with those obtained by the conventional method were also obtained by the present invention with respect to the samples of the high triglyceride levels.

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Abstract

A method of a pretreatment of a sample for quantitating cholesterol characterized by, before measuring cholesterol contained in specific lipoproteins, treating the sample containing lipoproteins with an enzyme, the substrate of which is free cholesterol, optionally together with a reaction accelerator; a method for quantitating cholesterol in specific lipoproteins by using the above method; and a kit for quantitating cholesterol in specific lipoproteins to be used in the above quantification method. By using this quantification method, cholesterol in a specific fraction can be conveniently, accurately and efficiently quantitated fundamentally without resort to polyanion, etc. Thus, this method is appropriately usable in various automatic analyzers.

Description

TECHNICAL FIELD [0001] This invention relates to a pretreatment method for accurately and efficiently discriminating and quantitating cholesterol, which exists in the specific lipoprotein fraction, by simple procedures while using a small amount of a sample, and also to a method for measuring cholesterol in the specific lipoprotein fraction by using the pretreatment method. BACKGROUND ART [0002] Lipids such as cholesterol are complexed with apoproteins in blood to form lipoproteins. Depending on differences in physical properties, lipoproteins are classified into chylomicron, very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and so on. Among these lipoproteins, LDL is known to be one of causative substances which induce arteriosclerosis, while HDL is known to show anti-arteriosclerotic activity. [0003] Epidemiologically, the level of cholesterol in LDL is known to exhibit a positive correlation with the frequency of onset of arterios...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/04C12Q1/26C12Q1/60G01N33/92
CPCC12Q1/60G01N2800/044G01N33/92
Inventor NAKAMURA, MITSUHIROTANIGUCHI, YURIKOMANABE, MITSUHISAYAMAMOTO, MITSUAKI
Owner SEKISUI MEDICAL CO LTD
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