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Method for quantitatively determining LDL cholesterols

a quantitative and ldl technology, applied in the field of quantitatively determining ldl cholesterol, can solve the problems of unsuitable methods for handling a large number of specimens, difficult automation of assay procedures, and most of these methods are not used routinely, so as to achieve preferential reactions of cholesterols, induce cholesterol preferential reactions, and strengthen bonding affinity

Inactive Publication Date: 2006-08-17
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 quantitatively determining LDL cholesterol by adding a specific surfactant to serum and measuring the amount of cholesterol that reacts with the enzyme reagent. This method allows for the selective measurement of LDL cholesterol without interference from other lipoproteins. The invention also provides a kit for quantitatively determining LDL cholesterol, including the enzyme reagent and the surfactant. The technical effect of this invention is the ability to accurately measure LDL cholesterol levels, which is important for the diagnosis and treatment of cardiovascular disease.

Problems solved by technology

However, most of these methods are not used routinely, due to their intricate operations and limitations in handling a number of specimens.
Although this method is more suited I for routine assay as compared with the previous two methods, the assay procedure involves manual steps, which makes automation of the assay procedures difficult.
Thus, the method is still unsuited for handling a large number of specimens.
However, these methods have limitations in that they can quantitatively determine only HDL fractionated from lipoproteins other than HDL, and have no further ability to determine LDL quantitatively and fractionally from a mixture of VLDL and chylomicron.
Therefore, these methods cannot meet an objective to measure LDL cholesterol without using separation means.
However, similar to the methods described in the above two publications, Japanese Patent Application Laid-Open (kokai) 7-280812 proposes no resolution to quantitative and fractional determination of LDL and VLDL and / or chylomicron, which is absolutely essential for determining LDL cholesterol.
There is also a problem with this method; it cannot be applied to commonly-used automatic analyzers due to a large ! number of steps required for the assay, making this method of very limited use.
Thus, with conventional techniques, LDL cholesterol can never be assayed effectively without performance of an operation for separation, and, moreover, there has been no information indicating possibility of the above measurement.

Method used

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  • Method for quantitatively determining LDL cholesterols
  • Method for quantitatively determining LDL cholesterols
  • Method for quantitatively determining LDL cholesterols

Examples

Experimental program
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example 1

[0023] Normal-lipid serum specimens were assayed for LDL cholesterol through a method of the present invention by use of a Hitachi model 7070 automatic analyzer, and the measurements were compared with those obtained through ultracentrifugation. The results are shown in FIG. 1.

[0024] Briefly, to a specimen (4[1), a reagent (300˜L1) containing sodium phosphotungstate (0.02 wt.%) and MgC12—6H20 (0.2 wt.%) was added. Approximately five minutes later, there was added a cholesterol-assaying reagent (100 μl) containing Emulgen A60 (product of Kao Corporation) (0.5 wt.%), cholesterol esterase (1 U / ml), cholesterol oxidase (1 U / ml), peroxidase (1 U / ml), 4-aminoantipyrine (0.005 wt.%), and N,N-dimethyl-m-toluidine (0.04 wt.%), and the changes in absorbance at 545 nm during the period of one minute to five minutes after the addition of the second reagent were measured.

[0025] For ultracentrifugation, the serum was subjected to centrifugation at 100,0008 for two hours by use of an ultracentri...

example 2

[0027] A specimen that contains chylomicron-containg serum having a high triglyceride level was assayed for LDL cholesterol through a method of the present invention by use of a Hitachi model 7070 automatic analyzer, and the measurements were compared with those obtained through ultracentrifugation. The results are shown in FIG. 2.

[0028] Briefly, to a specimen (4 μ1), a reagent (300 μl) containing Emulgen B66 (product of Kao Corporation) (0.5 wt.%), cholesterol esterase (0.3 U / ml), cholesterol oxidase (0.3 U / ml), peroxidase (0.3 U / ml), and 4-aminoantipyrine (0.002 wt.%) was added. Approximately five minutes later, there was added a reagent (100μl) containing Triton X-100 (1 wt.%) and N,N-dimethyl-m-toluidine (0.04 wt.%), and the changes in absorbance were measured by subtracting the absorbance measured at 545 nm before the addition of the second reagent from that measured five minutes after the addition thereof (correction in consideration of the change in amount of the reagents). ...

example 3

[0031] The procedure of Example 2 was repeated by use of the same specimen and reagents except that phosphotungstic acid (0.3 wt.%) was further incorporated in the first reagent, and the measurements were compared with those obtained through ultracentrifugation. The results are shown in FIG. 3.

[0032] As shown in FIG. 3, similar to the case of Example 1, in Example 3 measurements of LDL cholesterol having excellent correlation to those obtained through conventional centrifugation were obtained, even though a serum specimen containing chylomicron-containing serum was used.

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Abstract

A method for quantitatively determining LDL cholesterol, including the steps of adding to serum a surfactant selected from among polyoxyethylenealkylene phenyl ethers and polyoxyethylenealkylene tribenzylphenyl ethers and a cholesterol-assaying enzyme reagent so as to preferentially react cholestrols in high density- and very low density-cholesterols among lipoproteins, and subsequently determining the amount of cholesterol that reacts thereafter. This method can eliminate the necessity for pretreatments such as centrifugation and electrophoresis, enables the quantitative determination to be conducted in an efficient, simple manner, and can be applied to various automatic analyzers.

Description

CONTINUATION DATA [0001] This application is a Continuation of U.S. application Ser. No. 10 / 859,999, filed Jun. 4, 2004, pending, which is a Continuation of U.S. application Ser. No. 09 / 971,673, filed on Oct. 9, 2001, now allowed, which is a Continuation of U.S. application Ser. No. 09 / 510,170, filed on Feb. 22, 2000, now U.S. Pat. No. 6,333,166, which is a Continuation of U.S. Ser. No. 09 / 147,296, filed Nov. 23, 1998, now U.S. Pat. No. 6,057,118, which is a 371 of PCT / JP97 / 01232, filed Apr. 10, 1997.TECHNICAL FIELD [0002] The present invention relates to a-method for quantitatively and fractionally determining LDL (Low Density Lipoprotein) cholesterol and cholesterol in lipoproteins other than LDL in an efficient, simple manner which requires a small amount of samples and requires no treatment for separation, such as centrifugation or electrophoresis. BACKGROUND ART [0003] Lipids such as cholesterols bind to apoprotein in serum to form lipoprotein. Lipoprotein is typically classifi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/54G01N33/92C12Q1/60
CPCC12Q1/60
Inventor NAKAMURA, MITSUHIRONAKANISHI, KAZUOHINO, KOICHIMANABE, MITSUHISA
Owner SEKISUI MEDICAL CO LTD