Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving cholesterol
Patent
1996-11-13
1998-09-15
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving cholesterol
435 4, 435 19, 435 25, 435 20, 435975, 435 18, 436 71, 424 941, 424 944, C12Q 160, C12Q 100, C12Q 144, G01N 3353
Patent
active
058076964
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for the determination of cholesterol in low-density lipoprotein (LDL) (hereinafter referred to as LDL cholesterol) which is important for the diagnosis of arteriosclerosis in the field of clinical diagnosis.
BACKGROUND ART
The conventional method for the determination of LDL cholesterol comprises first determining the total amount of cholesterol in a portion of a given sample, then adding a precipitant for LDL and very low-density lipoprotein (VLDL) to a separate portion of the sample, centrifuging the resultant mixture to obtain a supernatant, determining the amount of cholesterol in high-density lipoprotein (HDL) (hereinafter referred to as HDL cholesterol) of the supernatant, and calculating the amount of LDL cholesterol in accordance with the Friedewald formula of conversion Urine, Immunological Test, Volume 1, p. 615, published by Nippon Rinshosha, 1995!. This method requires determination of two quantities, i.e. the total amount of cholesterol and the amount of HDL cholesterol, and also requires the step of centrifugation, etc., which makes the process complicated. However, if a blood serum sample is directly added to a reagent containing cholesterol esterase and cholesterol oxidase, the resultant test system is not different from a system for the determination of total cholesterol and has no specificity to LDL cholesterol.
Japanese Published Unexamined Patent Application No. 165800/83 discloses a method for directly determining the amount of LDL cholesterol in the presence of a special surfactant without the step of separation. However, this method, which allows both of HDL cholesterol and LDL cholesterol to undergo the relevant reaction, has low LDL specificity and requires complicated setting of reaction conditions; thus it is not readily applicable to a variety of samples.
Japanese Published Unexamined Patent Application No. 280812/95 discloses a method which comprises aggregating LDL, eliminating cholesterol in lipoproteins other than LDL, resolving the LDL aggregation, and subjecting LDL cholesterol to an enzyme reaction to determine the amount of LDL cholesterol.
DISCLOSURE OF THE INVENTION
The present inventors have found a) that LDL cholesterol in a sample containing LDL can be specifically determined without particular separation thereof by specifically eliminating HDL cholesterol by a reaction with a reagent for the reaction of cholesterol in the presence of a reagent which inhibits the reaction of lipoproteins other than HDL, i.e. LDL, VLDL and chylomicron (CM), and then, if necessary in the presence of a reagent which enables the reaction of LDL cholesterol, subjecting LDL cholesterol to an enzyme reaction for the determination of cholesterol by the use of a reagent for the reaction of cholesterol, and b) that LDL cholesterol in a sample containing LDL can be specifically determined without particular separation thereof by inhibiting only the reaction of LDL, eliminating cholesterol in lipoproteins other than LDL by a reaction with a reagent for the reaction of cholesterol, and thereafter subjecting LDL cholesterol to an enzyme reaction for the determination of cholesterol by the use of a reagent for the reaction of cholesterol. The present invention has thus been completed.
The expression "inhibiting the reaction of lipoproteins other than HDL" as used herein means enabling selectively cholesterol in HDL to undergo an enzyme reaction by aggregating lipoproteins other than HDL or lowering the reactivity of the outer walls of lipoproteins other than HDL so as to render selectively unbreakable the outer walls of lipoproteins other than HDL. The expression "enabling the reaction of LDL cholesterol" means enabling LDL cholesterol to undergo an enzyme reaction by breaking the outer walls of LDL. The expression "inhibiting only the reaction of LDL" conversely means enabling selectively cholesterol in lipoproteins other than LDL to undergo an enzyme reaction by aggregating LDL or lowering the reactivity of the outer walls of LDL s
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patent: 5213964 (1993-05-01), Jones
patent: 5286626 (1994-02-01), Law et al.
Miike Akira
Miyauchi Kazuhito
Kyowa Medex Co., Ltd.
Leary Louise N.
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