Chemistry: analytical and immunological testing – Lipids – triglycerides – cholesterol – or lipoproteins
Patent
1995-11-02
1998-04-07
Snay, Jeffrey
Chemistry: analytical and immunological testing
Lipids, triglycerides, cholesterol, or lipoproteins
436166, 435 11, 435 19, G01N 3350
Patent
active
057364060
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 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 determined the amount of cholesterol in lipoproteins such as HDL, LDL, VLDL and CM, each of which has been fractionated through ultracentrifugation, using a reagent containing a sugar compound and/or a protein solubilizing agent, and found that the reactivity of each of the lipoproteins with the reagent differs depending on the combination of the sugar compound and/or the protein solubilizing agent in the reagent, which leads to the difference in the reactivity of HDL cholesterol, LDL cholesterol, VLDL cholesterol and CM cholesterol. This finding has led to the completion of the present inv
REFERENCES:
patent: 4647532 (1987-03-01), Watanabe et al.
patent: 4778753 (1988-10-01), Yamanishi et al.
patent: 4851335 (1989-07-01), Kerscher et al.
patent: 4892815 (1990-01-01), Kerscher et al.
Original by Izumi Kanai, Edited by Masamitsu Kanai, "A Summary of Clinical Test" (No. 29 Revised Edition) Jun. 30, 1983 (30.06.83) pp. 471-474.
Irie Tetsumi
Kayahara Norihiko
Miyauchi Kazuhito
Ohsawa Susumu
Shutoh Eiko
Kyowa Medex Co., Ltd.
Snay Jeffrey
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