WO2019177093A1 - Procédé pour quantifier le cholestérol dans la lipoprotéine de haute densité - Google Patents

Procédé pour quantifier le cholestérol dans la lipoprotéine de haute densité Download PDF

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WO2019177093A1
WO2019177093A1 PCT/JP2019/010525 JP2019010525W WO2019177093A1 WO 2019177093 A1 WO2019177093 A1 WO 2019177093A1 JP 2019010525 W JP2019010525 W JP 2019010525W WO 2019177093 A1 WO2019177093 A1 WO 2019177093A1
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cholesterol
fraction
hdl
reagent
phospholipase
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成則 内田
裕子 平尾
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デンカ生研株式会社
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention relates to a method for quantifying cholesterol in high-density lipoprotein (hereinafter sometimes referred to as “HDL cholesterol”) and a reagent kit therefor.
  • HDL cholesterol high-density lipoprotein
  • High density lipoprotein receives cholesterol from each tissue including the arteriosclerotic wall, and is therefore related to the removal action of cholesterol accumulated in the cells. Therefore, it is also called reverse cholesterol transfer system. It is known that there is a negative correlation with arteriosclerotic diseases such as coronary arteriosclerosis, and the low value of HDL is set as a low value limit as one of dyslipidemia and is useful as an index of arteriosclerosis It is known that
  • HDL is composed of apoprotein, phospholipid, cholesterol, and neutral fat.
  • Methods for measuring cholesterol in HDL include, for example, a method in which HDL is separated from other lipoproteins by ultracentrifugation and then subjected to cholesterol measurement, or the color intensity is measured by staining lipids after separation by electrophoresis.
  • the method of doing is known.
  • these methods have problems such as complicated operations and inability to process a large number of samples, and are rarely used on a daily basis.
  • a method for measuring cholesterol in HDL As a method for measuring cholesterol in HDL, a method generally used in the field of clinical examination at present is that a precipitant is added to a sample to aggregate lipoproteins other than HDL, and this is removed by centrifugation, and only the separated HDL is separated. Is a method for measuring cholesterol in a supernatant containing. Although this method is simpler than ultracentrifugation or electrophoresis, it involves an operation of adding a precipitant and separating it, so it is not satisfactory in terms of simplicity, and a relatively large amount of sample is required. I need.
  • an enzyme reaction is carried out in the presence of a bile salt or a nonionic surfactant (Patent Document 2). More recently, cholesterol esterase and cholesterol oxidase are chemically modified to include cyclodextrin and the like.
  • a method of specifically capturing cholesterol in HDL in the presence of a contact compound (Patent Document 3) or a method of forming an aggregate or complex with lipoproteins other than HDL, and then capturing cholesterol in HDL by an enzymatic reaction Patent Document 4 and Patent Document 5 are known, but both are problematic in terms of specificity, such as a deviation from the precipitation method being observed in some of the clinical specimens.
  • JP-A-6-242110 Japanese Patent Laid-Open No. 62-126498 JP-A-7-301636 JP-A-8-131197 JP-A-8-201393 Patent No. 5706418 publication
  • An object of the present invention is to selectively, easily and accurately quantify HDL cholesterol in a test sample containing HDL and other lipoproteins such as LDL, without requiring a complicated fraction separation operation. It is to provide a method for quantifying cholesterol in HDL.
  • the inventors of the present application select cholesterol in lipoproteins other than HDL lipoproteins by causing phospholipase and / or sphingomyelinase and a nonionic surfactant to act on the test sample. And the present invention has been reached.
  • the present invention provides the following.
  • a lipoprotein other than the high-density lipoprotein in the test sample comprising (i) phospholipase and / or sphingomyelinase and (ii) a nonionic surfactant acting on the test sample To remove cholesterol from the reaction system.
  • the phospholipase is phospholipase C and / or phospholipase D.
  • the nonionic surfactant is a polyoxyethylene polyoxypropylene block copolymer.
  • the final concentration of the phospholipase is 0.1 to 200 U / mL, and the final concentration of the sphingomyelinase is 0.01 to 50 U / mL, according to any one of (1) to (3) the method of.
  • the final concentration of the phospholipase is 0.1 to 3 U / mL, the final concentration of the sphingomyelinase is 0.02 to 2 U / mL, and the final concentration of the nonionic surfactant is 0.01 to 6.
  • the method according to any one of (1) to (4) which is 1% by weight.
  • the elimination of cholesterol in lipoproteins other than high-density lipoproteins from the reaction system is carried out by eliminating cholesterol with an elimination system containing cholesterol esterase and cholesterol oxidase.
  • the method according to any one of the above.
  • a first step of erasing cholesterol in lipoproteins other than the high-density lipoprotein in the test sample out of the reaction system, and a second step of quantifying cholesterol in the high-density lipoprotein remaining in the reaction system A method for quantifying cholesterol in high-density lipoprotein in a test sample, wherein the first step is performed by the method according to any one of (1) to (6).
  • a kit for quantifying cholesterol in high-density lipoprotein comprising (i) phospholipase and / or sphingomyelinase and (ii) a nonionic surfactant.
  • HDL cholesterol in a test sample can be quantified more selectively and accurately.
  • phospholipase and / or sphingomyelinase (hereinafter sometimes abbreviated as “phospholipase etc.”) and (ii) a nonionic surfactant are included in the test sample.
  • phospholipase etc. phospholipase and / or sphingomyelinase
  • a nonionic surfactant is included in the test sample.
  • a first step in a method for quantifying HDL cholesterol in a test sample comprising a first step of eliminating cholesterol other than HDL in the test sample and a second step of quantifying HDL cholesterol remaining in the reaction system
  • HDL cholesterol in the test sample can be quantified by applying the method of the present invention.
  • this two-step method will be described.
  • the first step of the two-step method is the method of the present invention.
  • the following 2nd processes are not essential, It is possible to quantify HDL cholesterol in a test sample also by applying another well-known method.
  • the method for quantifying HDL cholesterol including the first step and the second step is also the method of the present invention.
  • test sample to be subjected to the method of the present invention is not particularly limited as long as HDL cholesterol in the sample is to be quantified, but is preferably serum or plasma or a dilution thereof, particularly serum. Or a dilution thereof is preferred.
  • HDL to be measured in the present invention is composed of apoprotein, phospholipid, cholesterol, and neutral fat.
  • phospholipase or the like and a nonionic surfactant are allowed to act on the test sample. Further, a cholesterol-reactive enzyme such as cholesterol esterase, cholesterol oxidase or cholesterol dehydrogenase, or lipoprotein lipase as required is reacted. Each of these enzymes may be added alone or in combination of two or more.
  • the phospholipase used in the first step only needs to act on at least glycerophospholipid, and it is preferable if it has at least activity on phosphatidylcholine, but lysophosphaethanolamine other than phosphatidylcholine, or glycerophospholipid It may have activity against other sphingomyelin and ceramide. Phospholipase etc.
  • Phospholipase A2 Phospholipase A2
  • PLC Phospholipase C
  • PPD Phospholipase D
  • LYPL lysophospholipase
  • sphingomyelinase Sigma Aldrich
  • the phospholipase concentration at that time is 0. It is preferably 1 to 200 U / mL, more preferably 0.05 to 100 U / mL, and further preferably 0.1 to 3 U / mL.
  • the final concentration of sphingomyelinase is preferably about 0.01 to 50 U / mL, and more preferably 0.02 to 2 U / mL.
  • Sphingomyelinase is not particularly limited as long as it acts on at least sphingomyelin, and may have activity against phosphatidylinositol, which is a component constituting phospholipids other than sphingomyelin.
  • the nonionic surfactant used in the present invention is preferably added at a final concentration of 0.001 to 5% by weight (a total final concentration when a plurality of types of nonionic surfactants are used in combination). It is more preferably about 002 to 3.0% by weight, and further preferably about 0.01 to 1% by weight.
  • Nonionic surfactants include nonionic interfaces such as polyoxyethylene-polyoxypropylene copolymers, amide nonions, polyoxyethylene nonyl phenyl ethers, and polyoxyethylene polycyclic phenyl ethers having an HLB value of 14-17. Although an activator can be mentioned, it is not limited to these.
  • a polyoxyethylene polyoxypropylene block copolymer particularly a block copolymer in which a polyoxypropylene portion is sandwiched between polyoxyethylene portions is preferable.
  • Particularly preferred are those having a weight average molecular weight of 10,000 to 17,000 and an ethylene oxide content of 75 to 95 mol% in the molecule.
  • Pluronic P123 (Adeka), Pluronic F68 (Adeka), Pluronic F88 (Adeka), Pluronic P85 (Adeka), Pluronic 17R-3 (Adeka), Pluronic 17R-4 (Adeka), Pluronic TR-704 (Adeka), Pluronic PE6100 (Adeka), Pluronic PE6400 (Adeka), Pluronic PE6800 (Adeka), Adeka Carpole GH5 (Adeka), Adeka Carpole GH10 (Adeka), Adeka Carpole GH200 (Adeka), Adeka Carpole MH150 (Adeka), Adeka Carpol MH10000 (Adeka), Rebenol WX (Kao), Nonion HS220 (Nippon) Kashiwa, Nymid MT-215 (Nippon), Pronon 208 (Nippon), Newcol-723 (Nippon Emulsifier), Newcol -2614 (Japan emulsifier), Newcol
  • nonionic surfactants such as alkyl ether type nonionic surfactants such as Adecatol LB-83 (Adeka) and Adekatol LB-103 (Adeka), polyoxyethylene alkyl ether type nonionic surfactants Emulgen 109P (Kao), Emulgen TW0106 (Kao) and Emulgen 1108 (Kao), Neugen EA-207D (Daiichi Kogyo Seiyaku), polyoxyethylene alkylphenyl ether, Nonion HS- 240 (Nippon Oil), Newcol-CMP-1 (Nippon Emulsifier) and Newcol-CMP-11 (Nippon Emulsifier) which are polyoxyethylene p-cumylphenyl ether, Polystar OMP (Nippon Oil) which is sodium polycarboxylate, Fatty acid ester type nonionic surfactants, Rheodor TW-L120 (Kao), Rheodor 460 (Kao),
  • lipoprotein cholesterol other than HDL is eliminated by the action of phospholipase, cholesterol esterase and the like.
  • erasing means degrading lipoprotein cholesterol in a test sample so that it does not affect the reaction of cholesterol measurement in the subsequent steps.
  • Examples of a method for eliminating lipoprotein cholesterol include a method in which hydrogen peroxide generated by the action of cholesterol esterase and cholesterol oxidase is decomposed into water and oxygen using catalase.
  • the hydrogen donor and the generated hydrogen peroxide may be reacted with peroxidase to convert to colorless quinone, but is not limited thereto.
  • the method of cholesterol elimination itself is well known in the art.
  • the first step is to add phospholipase etc. and nonionic surfactant together with phospholipase etc. and nonionic surfactant by adding enzyme and surfactant to move out of the reaction system.
  • Decomposition of lipoproteins other than HDL by the agent and elimination of cholesterol contained therein can be simultaneously performed as a single step.
  • the cholesterol esterase concentration (in this specification, the concentration means the final concentration unless otherwise specified) is preferably about 0.1 to 10.0 U / mL. 0.2 to 2.0 U / mL is more preferable.
  • the concentration of cholesterol oxidase is preferably about 0.05 to 10.0 U / mL, more preferably about 0.1 to 1.0 U / mL.
  • the cholesterol esterase is not particularly limited as long as it acts on ester-type cholesterol.
  • cholesterol esterase (CEBP, CEN) manufactured by Asahi Kasei Co., Ltd.
  • cholesterol esterase COE-311, manufactured by Toyobo Co., Ltd.
  • COE-3113 Commercially available products such as COE-313) and cholesterol esterase (CHE-XE) manufactured by Kikkoman
  • the cholesterol oxidase is not particularly limited as long as it acts on free cholesterol.
  • cholesterol oxidase CONII
  • cholesterol oxidase COO-311, COO- 321 and COO-331
  • cholesterol oxidase CHO-CE, CHO-PEWL, CHO-BS
  • Kikkoman Corporation can be used.
  • cholesterol dehydrogenase When cholesterol dehydrogenase is used, it is preferably 0.01 to 200 U / mL, more preferably 0.1 to 100 U / mL. Cholesterol dehydrogenase is not particularly limited as long as it has the ability to oxidize cholesterol and reduce oxidized coenzyme.
  • cholesterol dehydrogenase (CHDH-5) manufactured by Amano Enzyme Co., Ltd. Commercial products can be used.
  • lipoprotein lipase can be further added, and one or two or more kinds may be used in combination, and the final concentration (the final concentration when two or more types are combined) is 0.01 to 0. About 5 U / mL is preferable, and 0.02 to 0.5 U / mL is more preferable.
  • Commercially available products can be used for lipoprotein lipase and phospholipase, such as lipoprotein lipase (LPL-311 and LPL-314) manufactured by Toyobo Co., Ltd., lipoprotein lipase (LPL-3 manufactured by Amano Enzyme, etc.), etc. ), Lipoprotein lipase (LPBP, LP, etc.) manufactured by Asahi Kasei Corporation can be used.
  • reaction solution used in the first step various buffer solutions used in normal biochemical reactions can be used, and the pH is preferably between 5 and 8.
  • a buffer solution of Good, Tris, phosphate, and glycine is preferable.
  • Good buffer solutions such as bis (2-hydroxyethyl) iminotris (hydroxyethyl) methane (Bis-Tris), piperazine-1, 4-bis ( 2-ethanesulfonic acid (PIPES), piperazine-1,4-bis (2-ethanesulfonic acid), 1.5 sodium salt, monohydrate (PIPES1.5Na), 2-hydroxy-3-morpholinopropanesulfone Acid (MOPSO), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) and Piperazine-1,4-bis (2-hydroxy-3-propanesulf
  • a monovalent or divalent cation or a salt thereof can be added for the purpose of easily distinguishing lipoproteins other than HDL.
  • sodium chloride, potassium chloride, manganese chloride, calcium chloride, ammonium chloride, magnesium sulfate, potassium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, and the like can be used.
  • the concentration is preferably 1 to 50.0 g / L, more preferably 5 to 30 g / L.
  • the reaction temperature in the first step is preferably about 25 to 40 ° C, more preferably 35 to 38 ° C, and most preferably 37 ° C.
  • the reaction time is not particularly limited, and is usually about 2 to 10 minutes.
  • HDL cholesterol in the HDL remaining in the reaction system is quantified.
  • the cholesterol quantification method itself is well known, and any known method can be adopted, and is specifically described in the following examples.
  • ester cholesterol in lipoproteins is hydrolyzed using cholesterol esterase to produce free cholesterol and fatty acids, and the resulting free cholesterol and free cholesterol originally present in lipoproteins using cholesterol oxidase Cholesteinone and hydrogen peroxide are generated and quantified by forming a quinone dye in the presence of peroxidase.
  • Examples of compounds that generate quinone dyes include HDAOS (N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline), DAOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl)) -3,5-dimethoxyaniline) or TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline) and 4-aminoantipyrine, which can generate quinone dyes. It is not limited to these as long as it is a possible combination.
  • cholesterol esterase and cholesterol oxidase are used in the first step
  • the cholesterol esterase and cholesterol oxidase used in the first step can be used as they are in the second step, and it is not necessary to add them newly.
  • NAD (P) H is generated from NAD (P) by the enzymatic reaction.
  • the generated NAD (P) H can quantitate cholesterol in HDL by measuring the absorbance at a wavelength of 330 to 400 nm.
  • the concentration of the compound that generates a quinone dye is preferably about 0.5 to 2.0 mmol / L for HDAOS, and preferably 0.1 to 2.0 mmol / L for 4-aminoantipyrine,
  • the concentration of peroxidase is preferably 0.4 to 5.0 U / mL.
  • sodium azide that is an inhibitor of catalase is used and added to the reaction solution in the second step.
  • the concentration of sodium azide is usually about 0.1 g / L to 1.0 g / L.
  • the concentration of peroxidase is preferably about 2.0 to 5.0 U / mL, more preferably about 3.0 to 4.0 U / mL.
  • the concentration is preferably about 0.4 to 0.8 mmol / L.
  • the presence of the surfactant is not essential and may or may not be added.
  • the surfactant that can be used in the first step is added within the same concentration or lower range in the first step.
  • reaction conditions reaction temperature, time, buffer solution, pH, etc.
  • reaction temperature, time, buffer solution, pH, etc. reaction temperature, time, buffer solution, pH, etc.
  • Example 1 Collect the CM, VLDL, IDL (CVI) fraction and LDL fraction using the ultracentrifugation method, and react by combining the following preparation reagent A or B as the first step and preparation reagent X as the second step.
  • PLDP phospholipase D
  • the ultracentrifugation method is a method of preparing each fraction of CM, VLDL, IDL, LDL, HDL, etc. by using a difference in lipoprotein density of a test sample such as serum by using a sodium bromide solution or the like. .
  • the CVI fraction has a density d ⁇ 1.019 g / mL
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 1. In this example, the prepared reagent C was used as the HDL reagent.
  • Preparation reagent A BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer (trade name Pluronic F68, the same shall apply hereinafter) 0.25g / L Phospholipase D (PLDP) 0.5 U / mL
  • reaction rate of each preparation reagent with respect to the CVI fraction and LDL fraction was lower than the reaction rate measured with Preparation Reagent C.
  • Phospholipase D and a nonionic surfactant By using Phospholipase D and a nonionic surfactant, CVI fraction was obtained in the first step. As a result, the erased amount of the image and LDL fraction was increased.
  • Example 2 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagent D or E as the first step and preparation reagent X as the second step. By using phospholipase C in one step, it was confirmed whether the amount of CVI and LDL erased in the first step increased and the reaction of these fractions in the second step decreased.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 1. In this example, the prepared reagent C was used as the HDL reagent.
  • reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction was lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction was measured in the first step. As a result, the erased amount of the image and LDL fraction was increased.
  • Example 3 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents F to J as the first step and preparation reagent X as the second step. It was confirmed that the use of sphingomyelinase in one step increased the amount of CVI and LDL erased in the first step and reduced the reaction of these fractions in the second step.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 3. In this example, the prepared reagent C was used as the HDL reagent.
  • Preparation reagent F BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L Sphingomyelinase 0.05U / mL
  • Preparation reagent G BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L Sphingomyelinase 0.10 U / mL
  • reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction is used in the first step. As a result, the amount of erasure of the LDL fraction was increased.
  • Example 4 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents K to O as the first step and preparation reagent X as the second step. It was confirmed that the use of sphingomyelinase in one step increased the amount of CVI and LDL erased in the first step and reduced the reaction of these fractions in the second step.
  • reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction is used in the first step. As a result, the amount of erasure of the LDL fraction was increased.
  • Example 5 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents P to T as the first step and preparation reagent X as the second step. By using PLC in one step, it was confirmed whether the amount of CVI and LDL erased in the first step increased and the reaction of these fractions in the second step decreased.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 5.
  • the prepared reagent C was used as the HDL reagent.
  • the reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction and LDL are used in the first step. As a result, the amount of elimination of the fraction was increased.
  • Example 6 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using an ultracentrifugation method, and the following preparation reagents U to Y were combined as a first step and prepared reagent X was combined as a second step. It was confirmed that the use of PLDP in one step increased the amount of CVI and LDL erased in the first step, and reduced the reaction of these fractions in the second step.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 6. In this example, the prepared reagent C was used as the HDL reagent.
  • Preparation reagent X BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L PLDP 2.5U / mL
  • Preparation reagent Y BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L PLDP 5.0U / mL
  • the reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction and LDL are used in the first step. As a result, the amount of elimination of the fraction was increased.
  • Example 7 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents Z to AD as the first step and preparation reagent X as the second step. It was confirmed that the use of PLDPV in one step increased the amount of CVI and LDL erased in the first step and reduced the reaction of these fractions in the second step.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 7. In this example, the prepared reagent C was used as the HDL reagent.
  • Preparation reagent Z BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L PLDPV 0.1U / mL
  • Preparation reagent AB BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L PLDPV 1.0U / mL
  • the reaction rate of each preparation reagent with respect to the CVI fraction and the LDL fraction is lower than the reaction rate measured with the preparation reagent C.
  • the CVI fraction and LDL are used in the first step. As a result, the amount of elimination of the fraction was increased.
  • Example 8 Reagent (i) -1, reagent (i) -2 and reagent X having the following reagent composition were prepared, and various surfactants were added to reagent (i) -1 and reagent (i) -2 at 0.025% (W / V) Reagents with added concentrations were prepared.
  • all the various surfactants described in Table 8 below are the above-described commercially available nonionic surfactants.
  • reaction rate of each preparation reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with Preparation Reagent C.
  • SPC and a nonionic surfactant CVI fractionation and LDL are performed in the first step. As a result, the amount of elimination of the fraction was increased.

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Abstract

L'invention concerne : un procédé pour quantifier le cholestérol dans une lipoprotéine de haute densité, moyennant quoi il devient possible de quantifier de manière sélective, simple et correcte le cholestérol dans la lipoprotéine de haute densité dans un échantillon d'intérêt contenant une lipoprotéine de haute densité ainsi qu'une autre lipoprotéine telle qu'une lipoprotéine de basse densité sans nécessiter aucune procédure compliquée de fractionnement/séparation ; et un procédé d'élimination sélective du cholestérol, qui peut être utilisé dans le procédé susmentionné. Un procédé d'élimination du cholestérol dans une lipoprotéine autre qu'une lipoprotéine de haute densité dans un échantillon d'intérêt hors d'un système de réaction consiste à permettre (i) la phospholipase et/ou la sphingomyélinase et (ii) à un tensioactif non ionique d'agir sur l'échantillon.
PCT/JP2019/010525 2018-03-15 2019-03-14 Procédé pour quantifier le cholestérol dans la lipoprotéine de haute densité WO2019177093A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09299A (ja) * 1995-06-21 1997-01-07 Internatl Reagents Corp 高比重リポ蛋白分画中のコレステロールの定量方法及び定量用試薬キット
WO1998026090A1 (fr) * 1996-12-09 1998-06-18 Denka Seiken Co., Ltd. Procede pour determiner la teneur en cholesterol des lipoproteines de haute densite
WO2012011554A1 (fr) * 2010-07-23 2012-01-26 デンカ生研株式会社 Procédé de quantification de la quantité de cholestérol dans une lipoprotéine haute densité 3

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09299A (ja) * 1995-06-21 1997-01-07 Internatl Reagents Corp 高比重リポ蛋白分画中のコレステロールの定量方法及び定量用試薬キット
WO1998026090A1 (fr) * 1996-12-09 1998-06-18 Denka Seiken Co., Ltd. Procede pour determiner la teneur en cholesterol des lipoproteines de haute densite
WO2012011554A1 (fr) * 2010-07-23 2012-01-26 デンカ生研株式会社 Procédé de quantification de la quantité de cholestérol dans une lipoprotéine haute densité 3

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NORIAKI ET AL.: "Development of a direct method of new HDL subfraction cholesterol", JAPANESE JOURNAL OF OF CLINICAL LABORATORY AUTOMATION, vol. 40, no. 3, 2015, SATOH, pages 198 - 204, ISSN: 0286-1607 *

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