Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving cholesterol
Reexamination Certificate
2000-02-22
2001-12-25
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving cholesterol
C436S071000
Reexamination Certificate
active
06333166
ABSTRACT:
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 causal 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 the 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 methods 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 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.
Accordingly, an object of the present invention is to provide a method for quantitatively and fractionally determining LDL cholesterol efficiently in a simple manner while eliminating necessity for pretreatments such as centrifugation or electrophoresis and which can be applied to a variety of automatic analyzers.
DISCLOSURE OF THE INVENTION
In view of the foregoing, the present inventors have conducted earnest studies, and have found that reaction with a cholesterol-assaying enzyme reagent performed in the presence of a specific surfactant which dissolves lipoproteins accelerates reaction of HDL cholesterol and VLDL cholesterol and remarkably retards reaction of LDL cholesterol; that reaction of HDL cholesterol and VLDL cholesterol are terminated prior to reaction of LDL cholesterol; and that LDL cholesterol can be measured quantitatively and fractionally by appropriate selection of a point of measurement, allowing for application to automated analyzers. The present invention was accomplished based on these findings.
Accordingly, the present invention provides a method for quantitatively determining LDL cholesterol, comprising the steps of adding to serum a surfactant selected from among polyoxyethylenealkylene phenyl ethers and polyoxyethylenealkylene tribenzylphenyl ethers and a cholesterol-assaying enzyme reagent, to thereby induce preferential reactions of cholesterols in high density- and very low density-lipoproteins among lipoproteins, and subsequently determining the amount of cholesterol which reacts thereafter.
The present invention also provides a method for quantitatively determining LDL cholesterol, characterized by comprising the steps of adding to serum a surfactant selected from among polyoxyethylenealkylene phenyl ethers and polyoxyethylenealkylene tribenzylphenyl ethers, a substance exhibiting stronger bonding affinity to VLDL than to LDL, and a cholesterol-assaying enzyme reagent, to thereby induce preferential reactions of cholesterols in high density- and very low density-lipoproteins among lipoproteins, and subsequently determining the amount of cholesterol which reacts thereafter.
Furthermore, the present invention provides a kit for quantitatively determining LDL cholesterol, comprising a cholesterol-assaying enzyme reagent and a surfactant selected from among polyoxyethylenealkylene phenyl ethers and polyoxyethylenealkylene tribenzylphenyl ethers.
Furthermore, the present invention provides a kit for quantitatively determining LDL cholesterol as described above, further comprising a substance which exhibits stronger bonding affinity to VLDL than to LDL.
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Hino Koichi
Manabe Mitsuhisa
Nakamura Mitsuhiro
Nakanishi Kazuo
Daiichi Pure Chemicals Co. Ltd.
Gitomer Ralph
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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