JP4884012B2 - Reagent kit and method for measuring cholesterol in high density lipoprotein - Google Patents

Description

  The present invention relates to a reagent kit for measuring cholesterol in high-density lipoprotein contained in a sample (hereinafter referred to as HDL-C) and a method for measuring HDL-C in the field of clinical diagnosis.

  Lipoprotein is a general term for complexes of lipids such as cholesterol and apoproteins, and is included in blood and the like. Cholesterol is present in lipoproteins mainly as ester-type cholesterol esterified with fatty acids. Lipoproteins are in descending order of specific gravity: chylomicron (hereinafter referred to as CM), very low density lipoprotein (hereinafter referred to as VLDL), low density lipoprotein (hereinafter referred to as LDL), and high density lipoprotein (hereinafter referred to as LIDL). And HDL). Among these, HDL is attracting attention as an anti-arteriosclerotic factor, and the measurement of HDL-C is one of important clinical examination items for cardiovascular diseases including various arteriosclerosis.

  As a reagent for quantifying HDL-C in a sample, for example, a reagent described in Patent Document 1 is known. This reagent uses an inhibition method to release cholesterol from HDL. In the inhibition method, an inhibitor that inhibits lipoproteins other than HDL from reacting with an enzyme is first added to the sample. Next, cholesterol is released from HDL by the action of an enzyme that reacts with cholesterol ester of HDL. HDL-C is quantified by reacting the enzyme with the released HDL-C and optically measuring the resulting reaction product. However, when a sample containing a large amount of HDL is used as a specimen, the enzyme may not completely react with all HDL within a predetermined measurement time, and HDL-C may not be measured accurately.

JP-A-5-176797

  An object of the present invention is to provide a reagent kit capable of accurately measuring HDL-C even in a specimen containing a large amount of HDL, and a method for measuring HDL-C.

The present invention comprises a first reagent containing inhibitor inhibits the reaction of lipoproteins and enzymes other than HDL, and a first enzyme which reacts with HDL cholesterol esters, second enzyme which reacts with HDL cholesterol esters and a second reagent containing cholesterol dehydrogenase, only containing at least one enzyme which first enzyme and second enzyme, respectively, are selected from the group consisting of cholesterol esterase, lipoprotein lipase and lipase, A reagent kit is provided.

Further, the present invention includes a sample, comprising the steps of mixing the first reagent containing the first enzyme that reacts inhibitor suppresses the reaction between lipoproteins and enzymes other than HDL, and HDL cholesterol esters, second enzyme reactive to sample and HDL cholesterol ester in a mixture of the first reagent, and a step of adding the second reagent containing cholesterol dehydrogenase, seen including a is first enzyme and second enzyme Provided is a method for measuring HDL-C , each of which is at least one enzyme selected from the group consisting of cholesterol esterase, lipoprotein lipase and lipase .

  According to the present invention, there are provided a reagent kit and a measurement method capable of accurately measuring HDL-C even in a specimen containing a large amount of HDL.

  The HDL-C measurement reagent kit of this embodiment includes a first reagent and a second reagent. The first reagent contains an inhibitor that inhibits lipoproteins other than HDL from reacting with the enzyme, and a first enzyme that reacts with HDL cholesterol ester, and the second reagent reacts with HDL cholesterol ester. Contains a second reagent containing a second enzyme.

  In HDL-C measurement, cholesterol is liberated from HDL, and measurement is performed by the UV method or dye method described later. For this reason, it is necessary to inhibit lipoproteins other than HDL from reacting with the enzyme and releasing cholesterol.

  Examples of the inhibitor include calixarene, surfactant, polyanion, water-soluble polymer, antibody and the like. Among these inhibitors, one or a combination of two or more can be used. These inhibitors form complexes or aggregates with lipoproteins other than HDL in the sample. When a complex or an aggregate is formed, an enzymatic reaction with a substance (for example, ester-type cholesterol) contained therein is hindered, so that release of cholesterol from a cholesterol ester in the complex or aggregate is suppressed. .

Calixarene is a cyclic oligomer in which phenol is a basic skeleton and 4 to 8 molecules of phenol are cyclically polymerized with a methylene group.
Any calixarene can be used as long as it suppresses the release of cholesterol from lipoproteins other than HDL. Specifically, calix (4) allene, calix (6) allene, calix (8) allene, calix sulfate (4) allene, calix sulfate (6) allene, calix sulfate (8) allene, calix acetate (4) allene Calix acetate (6) allene, calix acetate (8) allene, carboxycalix (4) allene, carboxycalix (6) allene, carboxycalix (8) allene, calix (4) allenamine, calix (6) Allenamine, calix (8) allenamine and the like can be mentioned. One or two or more of these calixarenes can be used as an inhibitor. Among the calixarenes described above, calixarene sulfate is preferable because it is excellent in water solubility and easy to handle. The final concentration of calixarene (concentration in the reaction solution obtained by mixing the first reagent, the second reagent, and the sample) is preferably 0.05 to 20 mM, more preferably 0.1 to 5 mM.

  Any surfactant can be used as long as it suppresses the release of cholesterol from lipoproteins other than HDL. Among the surfactants, those having an HLB value (Hydrophile-Lipophile Balance value: a value indicating the ratio of hydrophilicity to hydrophobicity of the surfactant) of 16 or more are preferable, and those having an HLB value of 17 or more are more preferable. Specifically, polyoxyethylene cetyl ether (C16) (hexadecyl ether) (trade name: Nikko Chemicals Co., Ltd .: BC-25TX, BC-30TX, BC-40TX), polyoxyethylene lauryl ether (C12) ( Dodecyl ether) (trade name: Nikko Chemicals Co., Ltd .: BL-21, BL-25), polyoxyethylene oleyl ether (trade name: Nikko Chemicals Co., Ltd .: BO-50), polyoxyethylene behenyl ether (C22) (Trade name: Nikko Chemicals Co., Ltd .: BB-30), polyoxyethylene lauryl ether (trade name: Nippon Oil & Fat Co., Ltd .: Nonion K-230), polyoxyethylene monolaurate (trade name: Nippon Oil & Fat Co., Ltd.) ): Nonion S-40), polyoxyethylene alkyl ethers (trade name: Sigma: rij98, Brij721, Brij78, Brij99) and the like. One or more of these surfactants can be used as an inhibitor.

  Any polyanion can be used as long as it suppresses the release of cholesterol from lipoproteins other than HDL. Specific examples include dextran sulfate, phosphotungstic acid, heparin and the like. One or more of these polyanions can be used as an inhibitor. These inhibitors are preferably used with divalent cations. Examples of the divalent cation include magnesium ion, calcium ion, manganese ion, nickel ion and the like.

  Any water-soluble polymer can be used as long as it suppresses the release of cholesterol from lipoproteins other than HDL. Specific examples include polyacrylamide, polyacrylic acid, polymethacrylic acid, polyethylene glycol (hereinafter referred to as PEG) and the like. One or two or more of these water-soluble polymers can be used as an inhibitor. These water-soluble polymers can also be used with divalent cations. Examples of the divalent cation include magnesium ion, calcium ion, manganese ion, nickel ion and the like.

The antibody may be a polyclonal antibody or a monoclonal antibody as long as it suppresses the release of cholesterol from lipoproteins other than HDL, and these may be used in combination. Antibody fragments and derivatives thereof can also be used. As used herein, “antibody” includes antibody fragments and derivatives thereof. Examples of antibody fragments and their derivatives include Fab, Fab ′, F (ab) ₂ and sFv fragments (Blazar et al., 1997, J. Immunol., 159: 5821-5833 and Bird et al., 1988, Science). , 242: 423-426). The subclass of the antibody is not limited to IgG, and may be IgM.

  Specific examples of antibodies used in the reagent kit of the present embodiment include anti-apolipoprotein antibodies (for example, anti-apolipoprotein B antibody, anti-apolipoprotein C antibody, anti-apolipoprotein E antibody, etc.), anti-lipoprotein antibodies (for example, And anti-β lipoprotein antibody). One or more of these antibodies can be used as an inhibitor.

Of the above-mentioned inhibitors, in this embodiment, calixarene that binds to lipoproteins other than HDL to form a complex is preferably used.

  If the concentration of the inhibitor in the first reagent is too high, not only the lipoprotein other than HDL but also the inhibitor binds to HDL and the reaction between the enzyme and HDL is inhibited. It is preferable that the concentration be such that the reaction between HDL and HDL is not inhibited. In addition, if the concentration of the inhibitor in the first reagent is too low, lipoproteins other than HDL cannot be sufficiently inhibited, and not only HDL but also lipoproteins other than HDL and enzymes react. The concentration of the inhibitor is preferably a concentration that can sufficiently inhibit lipoproteins other than HDL.

  The first reagent of this embodiment contains an enzyme (first enzyme) that reacts with HDL cholesterol ester, and the second reagent also has an activity similar to that of the first enzyme (enzyme that reacts with HDL cholesterol ester: A second enzyme). The first enzyme and the second enzyme may be the same type of enzyme or different types of enzymes. Any enzyme capable of reacting with HDL cholesterol ester can be used as the first enzyme and the second enzyme. Moreover, you may have the activity which reacts not only with HDL but with the cholesterol ester of lipoproteins other than HDL. Specific examples of the first enzyme and the second enzyme include cholesterol esterase (hereinafter referred to as CE), lipase, lipoprotein lipase and the like. As these enzymes, those chemically modified by binding PEG or the like or those not chemically modified can be used. The origin of these enzymes is not particularly limited, such as those derived from bacteria, protists, animals, plants, fungi and the like. These enzymes may be obtained by genetic manipulation.

  In the inhibition method, first, a first reagent containing an inhibitor and a sample are mixed to bind a lipoprotein other than HDL and the inhibitor. Next, a second reagent containing an enzyme that reacts with the cholesterol ester of lipoprotein is added to guide only HDL to the enzymatic reaction. For this reason, when the enzyme which reacts with a cholesterol ester is contained in the 1st reagent conventionally, lipoproteins other than HDL will react with this enzyme, and it was thought that HDL-C cannot be measured correctly. . However, when the first reagent of this embodiment and the sample are mixed, an inhibitor binds to lipoproteins other than HDL, and cholesterol is released from HDL by the action of the first enzyme. The reason why the first enzyme does not act on lipoproteins other than HDL is thought to be because the inhibitor binds to these lipoproteins before the first enzyme acts. By starting the release of cholesterol from HDL from the time when the first reagent is added, it is considered that even a sample containing a large amount of HDL can release cholesterol from all HDL in the sample within a predetermined measurement time. It is done. For this reason, when the reagent kit of this embodiment is used, HDL-C in a sample can be accurately measured.

  The measured HDL-C value measured using this reagent kit correlates very well with the measured HDL-C value by the precipitation method. In the precipitation method, first, a polyanion such as PEG and a divalent cation are added to a specimen to precipitate VLDL, LDL, etc. having high hydrophobicity, and centrifuged to collect HDL remaining in the supernatant. Next, a reagent for measuring cholesterol (for example, T-CHO reagent / KL “Kokusai”) or the like is added to the HDL using an automatic analyzer, and HDL-C is measured. The precipitation method can measure HDL-C very accurately, but it is necessary to fractionate HDL and lipoproteins other than HDL before cholesterol measurement. When the reagent kit of this embodiment is used, fractionation before measurement is unnecessary, and therefore, the measurement operation is simpler than the precipitation method.

As a sample to be subjected to HDL-C measurement, a sample collected from a living body such as blood, plasma, serum, urine, spinal fluid, saliva, semen can be used.
Any of the known HDL-C measurement methods may be used as a method for measuring the released cholesterol. For example, it can be measured by a UV method or a dye method. Hereinafter, the UV method and the dye method will be described.

  In the UV method, cholesterol dehydrogenase (hereinafter referred to as CDH) is allowed to act on cholesterol released from HDL in the presence of an oxidized coenzyme to produce cholesterol, and the absorbance of the reduced coenzyme generated at the same time is measured. Is the method. As shown in the following chemical reaction formula, the amount (mole) of HDL-C produced by the action of CDH and the amount (mole) of reduced coenzyme produced in the reaction stoichiometrically. Since they are equal, the concentration of HDL-C can be determined by measuring the concentration of reduced coenzyme.

  As the CDH used in the UV method, an enzyme chemically modified by binding PEG or the like or an enzyme not chemically modified can be used. The origin of CDH is not particularly limited, such as those derived from microorganisms, animals, plants, and fungi. Alternatively, CDH obtained by genetic manipulation may be used. CDH may be contained in either the first reagent or the second reagent, or may be contained in both reagents.

  As the oxidized coenzyme, nicotinamide adenine dinucleotide oxidized form (hereinafter referred to as NAD) or nicotinamide adenine dinucleotide phosphate oxidized form (hereinafter referred to as NADP) can be used. Examples of NAD include β-nicotinamide adenine dinucleotide oxidized form (hereinafter referred to as βNAD), Thio-nicotinamide adenine dinucleotide oxidized form (hereinafter referred to as t-NAD), and the like. Examples of NADP include β-nicotinamide adenine dinucleotide phosphate oxidation type (hereinafter referred to as βNADP), Thio-nicotinamide adenine dinucleotide phosphate oxidation type (hereinafter referred to as t-NADP), and the like. The coenzyme may be contained in either the first reagent or the second reagent, or may be contained in both reagents. One or more of these coenzymes can be used.

  In the presence of cholesterol, the oxidized coenzyme is converted to a reduced form (NADH, NADPH, etc.) by the action of CDH. βNADH and βNADPH can be measured by absorbance at 340 nm, and t-NADH and t-NADPH can be measured by absorbance at 405 nm.

Next, the dye method will be described.
In the dye method, cholesterol released from HDL is first produced by the action of cholesterol oxidase (hereinafter referred to as COD) to produce cholesterol. At this time, peroxidase (hereinafter referred to as PO) is allowed to act from hydrogen peroxide and chromogen generated simultaneously to produce a quinone dye. The color of the produced quinone dye is measured at an absorbance of 450 nm or more (depending on the type of chromogen) to determine the concentration of hydrogen peroxide, which is converted to the concentration of HDL-C.

  As COD and PO, those chemically modified by binding PEG or the like or those not chemically modified can be used. The origin of these enzymes is not particularly limited, such as those derived from bacteria, protists, animals, plants, fungi and the like. These enzymes may be obtained by genetic manipulation.

  Any chromogen can be used as long as it reacts with hydrogen peroxide to develop color. For example, a combination of a coupler such as 4-aminoantipyrine (hereinafter referred to as 4-AA) and a developer (a substance that produces a dye by oxidative condensation with the coupler) is used. For example, a combination of 4-AA and a phenol compound, a naphthol compound or an aniline compound, a combination of 3-methyl-2-benzothiazolinone hydrazone and an aniline compound, or the like, for example, 2,2′-azinobis (3 -Ethylbenzothiazoline-6-sulfonic acid), triphenylmethane leuco dye, diphenylamine derivative, benzidine derivative, triallylimidazole derivative, leucomethylene blue derivative, o-phenylenediamine derivative, etc. Is mentioned. Specific examples of the phenolic compound as a developer include phenol, p-chlorophenol, 2,4-dichlorophenol, and the like. Specific examples of the naphtholic compound include, for example, 1-naphthol and 1-naphthol-2. -Sulfonic acid, 1-naphthol-2-carboxylic acid and the like, and specific examples of aniline compounds include, for example, N, N-diethylaniline, N-ethyl-N- (β-hydroxyethyl) -m. -Toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5 -Dimethoxy-4-fluoroaniline (FDAOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy Aniline (HDAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N- (3-methylphenyl) -N′-succinyl-ethylenediamine (EMSE) ) And the like. When a combination of a coupler and a developer is used, the amount of coupler used varies depending on the type of coupler to be used and the type of developer to be combined, so it cannot be generally stated, but the final concentration is preferably 0.01 to 100 mM, more preferably Is 0.1 to 10 mM, and the amount used when 4-AA is used as the coupler is preferably 0.01 to 50 mM, more preferably 0.1 to 5 mM as the final concentration. In addition, the amount of developer used varies depending on the type of developer used, the type of coupler to be combined, etc., and thus cannot be generally stated, but the final concentration is preferably 0.01 to 50 mM, more preferably 0.1 to 5 mM. . Specific examples of the triphenylmethane leuco dye include leucomalachite green, bis (p-diethylaminophenyl) -2-sulfophenylmethane, bis (p-diethylaminophenyl) -3,4-disulfopropoxyphenylmethane di Specific examples of diphenylamine derivatives include bis [4-di (2-butoxyethyl) amino-2-methylphenyl] amine, N, N-bis (4-diethylamino-2-methylphenyl), and the like. ) -N′-p-toluenesulfonylurea and the like, and specific examples of the leucomethylene blue derivative include, for example, 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine / sodium salt, 10- [3- (methoxycarbonylaminomethyl) ) Phenyl-methylamino carbonyl] -3,7-bis (dimethylamino) such as phenothiazine and the like. Furthermore, specific examples of the benzidine derivative include, for example, benzidine, o-tolidine, o-dianisidine, 3,3′-diaminobenzidine, 3,3 ′, 5,5′-tetraaminobenzidine, and triallylimidazole. Specific examples of the derivative include, for example, 2- (4-carboxyphenyl) -3-N-methylcarbamoyl-4,5-bis (4-diethylaminophenyl) imidazole, 2- (3-methoxy-4-diethylaminophenyl)- Examples include 3-N-methylcarbamoyl-4,5-bis (2-methyl-4-diethylaminophenyl) imidazole. The amount of these chromogens used is usually the concentration used in this field.

  When performing HDL-C measurement using a dye method, COD, PO, and chromogen may be contained in either the first reagent or the second reagent.

  The first reagent and the second reagent preferably contain a buffer. The kind of buffer is not particularly limited. The pH of the first reagent is preferably 6 to 10, and more preferably 6.5 to 7.5. The pH of the second reagent is preferably 6-10, and more preferably 8-9.

  The reagent preferably contains a substance that stabilizes the enzyme (enzyme stabilizing substance). Examples of the enzyme stabilizing substance include alkaline earth metal salts, glycine compounds, cholic acid, glycosides, crystallin, and derivatives thereof. Among these, one kind or two or more kinds can be used.

  The alkaline earth metal salt is not particularly limited as long as it has an effect of stabilizing the first enzyme and / or the second enzyme. For example, calcium salt, magnesium salt, radium salt, beryllium salt, strontium salt and barium salt can be used. These alkaline earth metal lactates, acetates, nitrates, sulfates and the like can be added to the reagents. Of these, one or more can be added to the reagent.

The glycine compound is represented by the following chemical formula (1).
R— (NH—CH ₂ —CO) _n —NH—CH ₂ —COOH (1)
(In the formula, R represents hydrogen or / and an optionally substituted alkyl group, an optionally substituted phenyl group, or an optionally substituted carbonyl group, and n is 0 or 1) Indicate)
Here, R represents an alkyl group which may have a substituent, a phenyl group which may have a substituent, a carbonyl group which may have a substituent, and examples of the alkyl group include a methyl group and an ethyl group. Examples of the phenyl group include a hydroxyphenyl group. Examples of the substituent include a hydroxymethyl group, a hydroxyl group, an amino group, a carbonyl group, a nitro group, a methoxy group, and a thiol group. When a glycine compound is added to the reagent, one or more of the above can be added to the reagent. In addition, glycine, glycylglycine, and tricine are preferably used as the glycine compound represented by the chemical formula (1). The concentration of the glycine compound in the reagent is preferably 0.01 to 2M, more preferably 0.1 to 1.5M.

  Examples of cholic acid and derivatives thereof include cholic acid salts (for example, sodium salt), deoxycholic acid or salts thereof, 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate ( CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) coleamide (deoxy) -BIGCHAP). One or more of these can be added to the reagent.

  Examples of glycosides and derivatives thereof include n-dodecyl-β-D-maltoside (dodecyl maltose), n-heptyl-β-D-thioglucoside, digitonin, sucrose monocaprate, sucrose monolaurate, 2 -Ethyl-hexylglucoside, n-octanoyl-N-methylglucamide, n-methylglucamide, n-nonanoyl-N-methylglucamide, n-decanoyl-N-methylglucamide and the like. One or more of these can be added to the reagent.

  Examples of crystallin and derivatives thereof include α-crystallin, β-crystallin, γ-crystallin, and δ-crystallin. One or more of these can be added to the reagent.

  Hereinafter, it demonstrates more concretely by an Example. The HDL-C measurement in the following examples was performed by the above-described UV method.

(Example)
A first reagent A and a second reagent having the following composition were prepared.
First reagent A (pH 7.3)
PIPES 10 mM
Hydrazinium dichloride 100 mM
Calix sulfate (8) allene 1.5 mM
βNAD 5.5 mM
Second reagent (pH 8.7)
TAPS 0.2 mM
Sodium cholate 0.1%
Calcium lactate 0.5 mM
Dodecyl maltose 4.0 mM
Glycine 0.4M
CE 14KU
CDH 24KU

  Furthermore, various concentrations of CE were added to the first reagent A to prepare first reagents B to H. The CE addition concentration to the first reagent B is 0.25 KU / L, the CE addition concentration to the first reagent C is 0.5 KU / L, the CE addition concentration to the first reagent D is 0.75 KU / L, the first The CE addition concentration to Reagent E is 1 KU / L, the CE addition concentration to First Reagent F is 1.25 KU / L, the CE addition concentration to First Reagent G is 1.5 KU / L, and to First Reagent H The concentration of CE added was 2 KU / L.

  Serum samples 1 to 11 collected from 11 living bodies were prepared as samples.

  Using a Hitachi 7180S type automatic analyzer, 4 μL of the sample, 180 μL of the first reagent and 60 μL of the second reagent were mixed to prepare a measurement sample, and the HDL-C concentration of each sample was measured. The temperature during the measurement was set to be maintained at 37 ° C. from the start of the reaction to the end of the measurement.

  As a control, HDL-C contained in the specimens 1 to 11 used in the above examples was measured using the precipitation method described above. Lipoproteins other than HDL in the sample were precipitated using PG pole (manufactured by Sysmex), and centrifuged to collect a supernatant containing HDL. This supernatant was mixed with T-CHO reagent / KL “Kokusai” (both manufactured by Sysmex) using an automatic analyzer, and the HDL-C concentration of each specimen was measured.

  The measurement results are shown in Table 1 below. Moreover, the correlation graph of the HDL-C measured value calculated in the Example and the HDL-C measured value calculated in the comparative example and the linear equation (y = ax + b) are shown in FIGS. FIGS. 1-8 are the correlation diagrams and correlation equations between the HDL-C measurement values and the HDL-C measurement values obtained by the precipitation method when the first reagents A to H are used, respectively.

  From Table 1 and FIGS. 1-8, when CE was added to the first reagent, the slope of the graph approximated 1 and the intercept approximated 0. That is, it was found that when CE was contained in the first reagent, the graph equation approached y = x and correlated well with the PEG method. From the above, it was confirmed that HDL-C in a sample can be accurately measured by using a reagent kit containing a first reagent containing CE.

It is a correlation diagram of the HDL-C measurement value at the time of using the 1st reagent A, and the HDL-C measurement value by a precipitation method. It is a correlation diagram of the HDL-C measurement value at the time of using the 1st reagent B, and the HDL-C measurement value by a precipitation method. It is a correlation diagram of the HDL-C measurement value at the time of using the 1st reagent C, and the HDL-C measurement value by a precipitation method. It is a correlation diagram of the HDL-C measurement value at the time of using the 1st reagent D, and the HDL-C measurement value by a precipitation method. It is a correlation diagram of the HDL-C measurement value at the time of using the 1st reagent E, and the HDL-C measurement value by a precipitation method. It is a correlation diagram of the HDL-C measurement value at the time of using the 1st reagent F, and the HDL-C measurement value by a precipitation method. It is a correlation diagram of the HDL-C measurement value at the time of using the 1st reagent G, and the HDL-C measurement value by a precipitation method. It is a correlation diagram of the HDL-C measurement value at the time of using the 1st reagent H, and the HDL-C measurement value by a precipitation method.

Claims (4)

A reagent kit for quantifying cholesterol in high density lipoprotein in a sample,
A first reagent containing the first enzyme to react reaction suppressing inhibitors, and cholesterol ester in high-density lipoprotein with lipoproteins and enzymes other than high density lipoprotein,
I viewed including the second enzyme reacts with cholesterol ester of high density lipoprotein, and a second reagent containing cholesterol dehydrogenase, a,
The reagent kit , wherein the first enzyme and the second enzyme are at least one enzyme selected from the group consisting of cholesterol esterase, lipoprotein lipase and lipase, respectively . The reagent kit according to claim 1, wherein NAD or NADP is contained in at least one reagent selected from the group consisting of the first reagent and the second reagent. The reagent kit according to claim 1 or 2 , wherein the inhibitor is at least one selected from the group consisting of calixarene, a surfactant, a polyanion, a water-soluble polymer, and an antibody. A sample, comprising the steps of mixing the first reagent, a containing a first enzyme that reacts inhibitors inhibit the reaction, and cholesterol esters of high density lipoprotein in the lipoprotein and enzymes other than high density lipoprotein ,
Adding a second enzyme that reacts with cholesterol ester of high-density lipoprotein to the mixed solution of the sample and the first reagent, and a second reagent containing cholesterol dehydrogenase ;
Only including,
The method for measuring cholesterol in high-density lipoprotein , wherein the first enzyme and the second enzyme are at least one enzyme selected from the group consisting of cholesterol esterase, lipoprotein lipase and lipase, respectively .

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