Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving cholesterol
Patent
1995-11-02
1997-11-25
Leary, Louise
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving cholesterol
435 19, 435 25, 435 26, 435 28, 435 12, 435 4, 435 10, C12Q 160, C12Q 144, C12Q 126, C12Q 100
Patent
active
056911599
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method of determining the amount of as "HDL cholesterol"!. HDL is important in lipid metabolism in the field of clinical diagnosis.
BACKGROUND ART
It is known that HDL is related to the removal of cholesterol accumulated in cells for receiving cholesterol from tissues including arterial walls, that HDL is a negative risk factor of various types of arteriosclerosis such as coronary arteriosclerosis, and that HDL level in blood is an index useful for the precognition of arteriosclerosis. The conventional method of determining the amount of HDL cholesterol consists of two steps, a fractionation step and a step of determining the amount of cholesterol. Examples of the fractionation include an ultracentrifugation method, an immunochemical method, an electrophoretic method and a precipitation method. In the ultracentrifugation method, HDL is separated through specific gravity using an ultracentrifuge to determine the amount of HDL cholesterol. However, this method is defective in precision in determination, complexity and economical efficiency. The immunochemical method includes an immunoelectrophoretic method, a single radial immunodiffusion (SRID) method, and an Ouchterlony diffusion method. However, these methods are defective in that an apoprotein is recognized but a lipoprotein is not exactly recognized. In the electrophoretic method, a cellulose acetate film or an agarose gel is separated as a support, and the amount of cholesterol is enzymatically determined. This method is defective in simplicity and economical efficiency. In the precipitation method, polyethylene glycol or a polyanion such as heparin, phosphotungstic acid and dextran sulfuric acid, and a divalent cation are bound to an apoprotein B, which is present on surfaces of low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL) and chylomicron (CM) to form an insoluble precipitate, and this insoluble precipitate is removed by centrifugation to determine the amount of HDL cholesterol in the supernatant (Summary of Clinical Investigation Method, 29th edition, Kanai I., Kanehara Shuppan, p. 471, 1983). This method is the simplest. However, this method is not suitable in case of using an autoanalyzer which is often used in the measuring a large number of specimens, for rapid measurement and in clinical investigation, since this method involves centrifugation step by a centrifuge. Further, in the fractionation, a mannual error tends to occur, for example, when the amount of the HDL fraction separated is determined using a measuring pipet. Thus, the complexity of the determination of the amount of HDL cholesterol lies in the fractionation procedure. However, if a serum specimen is directly added to a reagent containing a cholesterol esterase and a cholesterol oxidase without fractionating HDL, this method is not different from a system of determining the total amount of cholesterol, and the amount of HDL cholesterol cannot be specifically determined by this method. Japanese Published Unexamined Patent Application No. 126,498 (1988) describes that a cholic acid is added to increase the specificity. However, in this prior art method, not only HDL but also LDL, VLDL and the like gradually react, and it is difficult to obtain a clear terminal point of the reaction, and thus, the specificity of HDL by the use of this prior art method is not satisfactory.
DISCLOSURE OF THE INVENTION
The present inventors have found that amount of HDL cholesterol in an HDL-containing sample can be specifically determined without separating an aggregated substance by conducting an enzymatic reaction for determining the amount of cholesterol using a cholesterol reagent in the presence of a reagent for aggregating lipoproteins except HDL, such as LDL, VLDL and CM and completed the present invention based on this finding.
The present invention relates to a method of determining the amount of HDL cholesterol, which comprises reacting a HDL-containing sample with cholesterol hydrolase and cholesterol oxidase or c
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Trinder et al; "Ann Clin Biochem"; vol. 21, pp. 430-433, 1984 month not available.
Irie Tetsumi
Miike Akira
Miyauchi Kazuhito
Ohsawa Susumu
Shutoh Eiko
Kyowa Medex Co., Ltd.
Leary Louise
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