Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving cholesterol
Reexamination Certificate
1998-08-06
2002-11-12
Wilson, James O. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving cholesterol
C435S025000, C435S028000, C436S071000, C436S169000, C436S170000, C436S824000
Reexamination Certificate
active
06479249
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for quantifying cholesterol in high density lipoprotein (hereinafter also referred to as “HDL”).
BACKGROUND ART
It is known that HDL acts to remove cholesterol accumulated in cells because it receives cholesterol from various tissues including walls of blood vessels with arterial sclerosis, so that HDL is useful for estimating the risk for various arterial sclerosises including coronary artery sclerosis, and that its blood level is an indicator for the risk of onset of arterial sclerosis.
Methods for measuring cholesterol in HDL include a method in which HDL is separated from other lipoproteins by ultracentrifugation and then the HDL is measured; and a method in which the cholesterol in HDL is separated by electrophoresis, then the lipid is stained, and the intensity of the generated color is measured. However, these methods are either complex or the number of possible samples to be assayed is limited, so that these methods are not commonly used.
The method for measuring the cholesterol in HDL, which is generally used in the clinical field involves the addition of a precipitating agent to the sample so as to coagulate the lipoproteins other than HDL, removing the coagulated lipoproteins by centrifugation, and the cholesterol in the resulting supernatant containing HDL alone is measured. Although this method is simpler than the ultracentrifugation method and the electrophoresis method, it is not satisfactorily simple because it comprises the addition of a precipitating agent and subsequent separation, and a relatively large amount of sample is needed.
On the other hand, methods in which the cholesterol in HDL is separately quantified by using enzymes have been proposed. For example, a method is known, which comprises the steps of preliminarily coagulating the lipoproteins other than HDL by an antibody and polyanion, enzymatically reacting the cholesterol in HDL alone, inactivating the enzyme and simultaneously re-dissolving the coagulated mass, and measuring the absorbance of the resulting solution (Japanese Laid-open Patent Application (Kokai) No. 6-242110). However, this method has the disadvantage in that it is necessary to add reagents at least three times, so that this method can be practiced only by analyzing apparatuses which have limited availability. Therefore, this method is not widely used.
Other methods include a method in which an enzyme reaction is carried out in the presence of a bile salt or a nonionic surfactant (Japanese Laid-open Patent Application (Kokai) No. 63-126498); a more recently developed method in which the cholesterol in HDL is specifically trapped by chemically modified cholesterol esterase and/or cholesterol oxidase in the presence of a clathrate compound such as cyclodextrin (Japanese Laid-open Patent Application (Kokai) No. 7-301636); and a method in which the lipoproteins other than HDL are made into aggregates or complexes and then the cholesterol in HDL is trapped by an enzyme reaction (Japanese Laid-open Patent Application (Kokai) Nos. 8-131197 and 8-201393). However, with these methods, the results for certain samples are different from the results by the precipitation method, so that their specificities are problematic.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a method for quantifying cholesterol in HDL, which does not require complex fragmentation and separation operations, and by which the HDL cholesterol in test samples containing HDL and other lipoproteins such as low density lipoprotein (LDL), very low density lipoprotein (VLDL) and chylomicron (CM) may be quantified selectively, simply and accurately.
The present inventors intensively studied to discover that surfactants which act on HDL but substantially do not act on other lipoproteins exist. The present inventors further discovered that HDL cholesterol in test samples containing HDL and other lipoproteins may be selectively, simply and accurately quantified by selectively erasing the cholesterol in the lipoproteins other than the high density lipoprotein in the test sample and then enzymatically quantifying the cholesterol originated from HDL in the presence of the above-mentioned surfactant, thereby completing the present invention.
That is, the present invention provides a method for quantifying cholesterol in high density lipoprotein, comprising a first step of erasing cholesterol in lipoproteins other than high density lipoprotein in a test sample, and a second step of adding a surfactant which specifically acts on high density lipoprotein to the product of the first step and enzymatically quantifying cholesterol in high density lipoprotein.
By the method of the present invention, the cholesterol in HDL in a test sample containing HDL and other lipoproteins such as LDL, VLDL and CM may be quantified selectively, simply and accurately, without complex fragmentation and separation operations.
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Fujiwara Akira
Ito Yasuki
Matsui Hiroshi
Ohara Shuichi
Birch & Stewart Kolasch & Birch, LLP
Denka Seiken Co., Ltd.
Owens Howard V.
Wilson James O.
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