Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving cholesterol
Patent
1998-11-23
2000-05-02
Woodward, Michael P.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving cholesterol
435 19, 435 25, 435 26, 436 71, C12Q 160, C12Q 144, C12Q 126, C12Q 132, G01N 3392
Patent
active
060571187
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
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
Lipids such as cholesterols bind to apoprotein in serum to form lipoprotein. Lipoprotein is typically classified as chylomicron, very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), etc. according to physical properties. Among them, LDL is known to be a casual substance inducing arteriosclerosis.
Several epidemiological studies have clarified that the LDL cholesterol level is strongly correlated to onset frequency of arteriosclerotic disease. Therefore, realization of measurement of LDL cholesterol through a simple routine method might be very useful clinically.
With regard to conventional methods for measuring LDL cholesterol, there have been known, for example, a method in which LDL is separated from other lipoproteins by ultracentrifugation to measure cholesterol and a method in which lipid is stained after separation through electrophoresis so as to measure the intensity of developed color. However, most of these methods are not used routinely, due to their intricate operations and limitations in handling a number of specimens. There is also known a method in which a carrier is sensitized with an antibody which binds a lipoprotein other than LDL, then mixed with a sample, and a fraction not bound to he carrier is fractionated to measure cholesterols therein. Although this method is more suited 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.
Meanwhile, with regard to a method for quantitatively and fractionally determining lipoproteins in a sample without using means for separation such as ultracentrifugation or electrophoresis, there has been known a method in which, upon fractional determination of cholesterols in HDL and other lipoproteins (i.e., chylomicron, VLDL, and LDL), reactivity of enzymes employed (typically cholesterol oxidase and cholesterol esterase) is controlled to induce exclusively HDL cholesterol to enzyme reaction. For example. Japanese Patent Application Laid-Open (kokai) No. 7-301636 discloses a method for exclusively measuring HDL cholesterol by use of a surfactant and a sugar compound, and Japanese Patent Application Laid-Open (kokai) No. 6-242110 discloses a method for exclusively measuring cholesterol in a target lipoprotein by agglutinating lipoproteins other than the lipoprotein to be measured so as to control reactivity with an enzyme. These methods are significantly useful in view of applicability thereof to automatic analyzers which realize automation of all steps. 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.
Japanese Patent Application Laid-Open (kokai) No. 7-280812 discloses a method for determining LDL cholesterol comprising the steps of agglutinating LDL; removing cholesterols in other lipoproteins by a system which differs from a system for determining LDL; dissolving the agglutination of LDL; and reacting the LDL cholesterol. However, similar to the method described in the above two publications, Japanese Patent Application Laid-Open (kokai) No. 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 metho
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Hino Koichi
Manabe Mitsuhisa
Nakamura Mitsuhiro
Nakanishi Kazuo
Daiichi Pure Chemicals Co. Ltd.
Moran Marjorie A.
Woodward Michael P.
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