MXPA96005627A - Method for the determination of cholesterol in low-densi lipoprotein - Google Patents

Abstract

The present invention relates to a method for the determination of cholesterol in a low density lipoprotein (LDL) in a sample containing LDL, which comprises removing the high density lipoprotein cholesterol in the sample, subjecting the sample to a reaction using the action of an enzyme-cholesterol-hydrolyzing enzyme and the action of a cholesterol oxidant enzyme or cholesterol oxidoreductase, and determining the amount of hydrogen peroxide or a reduced-type coenzyme generated by the reaction

Description

METHOD FOR THE DETERMINATION OF CHOLESTEROL IN LOW DENSITY IPOPROTEIN 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 arteriesclerosis in the field of clinical diagnosis. Prior 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, centrifugate the resulting mixture to obtain a supernatant, determine the amount of cholesterol in the high density lipoprotein (HDL) (hereinafter referred to as HDL cholesterol) of the supernatant and determine the amount of LDL cholesterol according to the formula Friede ald of conversion (Japanese Clinic (Nippon Rinsho), extensive chemical tests of blood and urine, Immunological test, Volume 1, page 615, published by Nippon Rinshosha, 1995). This method requires the determination of two quantities, that is, the total amount of cholesterol and the amount of HDL cholesterol, and also requires the centrifugation stage, etc., which makes the process complicated.

However, if the blood serum sample is added directly to a rectifier containing cholesterol esterase and cholesterol oxidase, the resulting test system does not differ from a system for the determination of total cholesterol and has no specificity for LDL cholesterol. Unexamined and unexamined Japanese 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 separation step. However, this method, which allows both HDL cholesterol and LDL cholesterol to undergo the relevant reaction, has low LDL specificity and requires complicated sedimentation of reaction conditions; thus, a variety of samples are not easily applicable. Unexamined and unexamined Japanese patent application No. 280812/95 discloses a method comprising adding LDL, removing cholesterol from lipoproteins other than LDL, resolving LDL aggregation, and subjecting LDL cholesterol to an enzyme reaction to determine the amount of LDL cholesterol. Disclosure of the Invention The present invention has found that a) LDL cholesterol in a sample containing LDL can be specifically determined without particular separation thereof by specifically removing HDL cholesterol by a reaction with a reagent for the cholesterol reaction. in the presence of a reagent that inhibits the reaction of lipoproteins other than HDL, ie, LDL, VLDL and cilomicron (CM), and then, if necessary in the presence of a reagent that enables the LDL cholesterol reaction, subject LDL cholesterol to an enzyme reaction for the determination of cholesterol through the use of a reagent for the cholesterol reaction, and b) that the LDL cholesterol in a sample containing LDL can be determined specifically without the particular separation thereof by inhibiting only the LDL reaction, removing the cholesterol in lipoproteins different from LDL by a reaction with a reagent for the cholesterol reaction, and after that subjecting the LDL cholesterol to an enzyme reaction for the determination of cholesterol. The present invention has been completed in this way. The expression "inhibit the reaction of lipoproteins other than HDL" as used herein means selectively enable HDL cholesterol to undergo a enzymatic reaction by adding lipoproteins other than HDL or decreasing the reactivity of the external walls of lipoproteins other than HDL to selectively render the external walls of lipoproteins other than HDL unbreakable. The expression "enable "LDL Cholesterol Reaction" means enabling LDL cholesterol to undergo an enzymatic reaction by breaking down the outer walls of LDL The expression "inhibit only the LDL reaction" similarly means selectively enabling cholesterol in lipoproteins other than LDL to experience a enzymatic reaction by LDL aggregation or decrease the reactivity of the outer walls of LDL to selectively render the outer walls of LDL unbreakable The present invention relates to a method for the determination of LDL cholesterol in a sample containing LDL, which it comprises removing HDL cholesterol in the sample, then subjecting the sample to a reaction using the action of a cholesterol ester-hydrolyzing enzyme and the action of a cholesterol oxidant enzyme or cholesterol oxidoreductase, if necessary in the presence of a reagent that enables the LDL cholesterol reaction, and determine the nity of hydrogen peroxide or a reduced type enzyme generated by the reaction. The present invention also provides a reagent for the determination of cholesterol in LDL, which contains a reagent that inhibits the reaction of lipoproteins other than HDL and a reagent that enables the reaction of cholesterol in LDL; and a reagent for the determination of cholesterol in LDL, which is a set composed of a reagent that inhibits the reaction of lipoproteins other than HDL and a reagent that enables the reaction of cholesterol in LDL. In addition, the present invention provides a reagent for the determination of cholesterol in LDL, which contains a reagent that inhibits only the LDL reaction; a reagent for the determination of cholesterol in LDL, which contains a reagent that inhibits only the reaction of LDL and a reagent that enables the reaction of cholesterol in LDL; and a reagent for the determination of cholesterol in LDL, which is a set composed of a reagent that inhibits only the reaction of LDL and a reagent that enables the reaction of cholesterol in LDL. According to the present invention, the determination of LDL cholesterol can be carried out as follows: a) HDL cholesterol is specifically removed by reaction with a reagent for the reaction of cholesterol in the presence of a reagent that inhibits the reaction of lipoproteins other than HDL, ie, LDL, VLDL, and CM, then, if necessary in the presence of a reagent that enables the LDL cholesterol reaction, the sample is subjected to a reaction using the action of a sterile enzyme. cholesterol hydrolyzate and the reaction of a cholesterol oxidant enzyme or cholesterol oxidoreductase, and the amount of hydrogen peroxide or a reduced type coenzyme generated by the reaction is determined; or b) the cholesterol in lipoproteins other than LDL is eliminated by a reaction with a reagent for the cholesterol reaction in the presence of a reagent that inhibits only the LDL reaction, the sample is subjected to a reaction using the action of an enzyme ester. - cholesterol hydrolyzate and the action of a cholesterol oxidant enzyme or cholesterol oxidoreductase, and the amount of hydrogen peroxide or a reduced type coenzyme generated by the reaction is determined. For example, LDL cholesterol in a sample containing HDL and LDL can be determined by subjecting the sample to a reaction using the action of a cholesterol ester-hydrolyzing enzyme and the action of a cholesterol oxidizing enzyme in the presence of a reagent which inhibits the reaction of lipoproteins other than HDL to form hydrogen peroxide, subsequently or simultaneously adding catalase, peroxidase and an aniline compound, peroxidase and a phenol compound or peroxidase and 4-aminoantipyridine to remove the hydrogen peroxide, and then add a chromogen (in combination with peroxidase when catalase is used), and an appropriate surfactant, cyclodextrin or a cholesterol ester-hydrolyzing enzyme capable of acting in LDL for the sample to develop color. The term "acting on LDL" as used herein means enabling LDL cholesterol to undergo an enzymatic reaction by breaking down the outer walls of LDL from which the reaction has been inhibited. The LDL cholesterol in an LDL-containing sample can be determined by adding a cholesterol ester-hydrolyzing enzyme, and a chromogen to the sample in the presence of a reagent that only inhibits the LDL reaction to develop a color, and measuring the change in the absorption of the sample subsequent to the reaction of the cholesterol in lipoproteins other than LDL. LDL cholesterol in a sample containing LDL can also be determined by subjecting the sample to a reaction using the action of a cholesterol oxidizing enzyme in the presence of a reagent that inhibits only the LDL reaction (which is not required when the enzyme cholesterol ester-hydrolyzant mentioned above inhibits only the LDL reaction) to form hydrogen peroxide, subsequently or simultaneously add catalase, peroxidase and an aniline compound, peroxidase and a phenol compound, or peroxidase and 4-aminoantipyrine to remove the peroxide of hydrogen, and then adding a chromogen (in combination with peroxide when catalase is used), and a reagent that enables the LDL cholesterol reaction (which is not required when the ester-cholesterol hydrolyzing enzyme mentioned below enables the reaction of the LDL cholesterol) and a cholesterol ester-hydrolyzing enzyme (which is not required when the enzyme ester-hydrolyzing co initially added lesterol is enabled to react with LDL cholesterol by the reagent that enables the reaction of LDL cholesterol) to the sample for color development. The method of the present invention can be applied to body fluid samples containing LDL such as blood and urine. Representative procedures for the determination according to the present invention are described below. Procedure 1 The determination was carried out by (1) adding a neutral buffer solution containing a reagent that inhibits the reaction of lipoproteins other than HDL to a prescribed amount of a sample, followed by, for example, heating at 37 ° C for several hours. minutes, to inhibit the reaction of LDL, VLDL and CM; (2) add to the sample a cholesterol ester-hydrolyzing enzyme which is not reactive to LDL (preferably a chemically modified ester-cholesterol hydrolyzing enzyme), a cholesterol oxidant enzyme which is not reactive to LDL (preferably an enzyme chemically modified cholesterol oxidant) [or cholesterol oxidoreductase (preferably chemically modified cholesterol oxidoreductase)], and catalase, peroxidase and an aniline compound, peroxidase and a phenol compound, or peroxidase and 4-aminoantipyrine [or NAD (P) ] to remove HDL cholesterol by reaction; (3) add to the sample a surfactant, cyclodextrin, a chelating agent, a cholesterol ester-hydrolyzing enzyme capable of acting in LDL (preferably a cholesterol-esterifying enzyme hydrolyzing chemically unmodified), a cholesterol oxidizing enzyme capable of acting in LDL (preferably a chemically modified cholesterol oxidant enzyme) or cholesterol oxidoreductase capable of acting on LDL (preferably cholesterol oxidoreductase without chemically modifying), and a chromogen [which is not added or replaced with NAD (P) when uses cholesterol oxidoreductase] for the reaction of LDL cholesterol to form hydrogen peroxide and develop a color [or to form NAD (P) H], - and (4) measure the absorbance of the pigment formed at the maximum wavelength with a spectrophotometer [measuring the increase of NAD (P) H in terms of absorbance at 300-500 nm, preferably at 330-400 nm when cholesterol oxidoreductase is used, in case A, forming a formazan pigment by the addition of diaphorase and a tetrazolium salt, followed by the calorimetric determination of the formazan pigment)]. The expression "not reactive to LDL" as used herein means failure to break down the outer walls of LDL to enable LDL cholesterol to undergo an enzymatic reaction. The amount of LDL cholesterol is calculated based on the absobance determined separately by using a standard solution containing LDL cholesterol at a known concentration under the same conditions. Steps (1) and (2) can be carried out at the same time. An example of the reagent that inhibits the reaction of lipoproteins other than HDL is a combination of an aggregating agent and a divalent metal salt. Examples of the aggregation agent are heparin and its salts, phosphotungstatic acid and its salts, dextran sulfuric acid and its salts, polyethylene glycol, sulphated cyclodextrin and its salts, sulphated oligosaccharide and its salts, and mixtures thereof. Examples of cyclodextrin are oi-cyclodextrin, 0-cyclodextrin, and β-cyclodextrin. Examples of oligosaccharide are maltotriose, maltotetraose, maltopentaose, maltohexatoase, and maltoheptaose. Examples of the salt are sodium salt, potassium salt, lithium salt, ammonium salt, and magnesium salt. Examples of the divalent metal salt are magnesium salt, calcium salt, manganese salt, nickel salt, and cobalt salt. Specifically, as the aggregation agent, 0.02-10 mM of heparin having a molecular weight of 5,000-20,000 or one of its salts, 0.1-10 mM of phosphotungstenic acid having a molecular weight of 4,000-8,000 or one of its salts, 0.01-5 mM dextran sulfuric acid having a molecular weight of 10,000-500,000 or a salt thereof, 0.1-20 mM dextran sulfuric acid having a molecular weight of 1,000-10,000 or a salt thereof, 0.3- 100 mM polyethylene glycol (PEG) having a molecular weight of 4,000-25,000, 0.1-50 mM sulfated cyclodextrin having a molecular weight of 1,000-3,000 or a salt thereof, 0.1-50 mM sulfated oligosaccharide having a molecular weight of 400-3,000 or a salt thereof, or any of the mixtures thereof is used. Preferably, 0.03-1 mM of heparin having a molecular weight of 14,000-16,000 or one of its salts, 0.1-3 mM of phosphotungstenic acid having a molecular weight of 5,000-7, 000 or a salt thereof, 0.01-5 mM dextran sulfuric acid having a molecular weight of 150,000-250,000 or one of its salts, 0.1-10 mM dextran sulfuric acid having a molecular weight of 1,000-5,000 or one of its salts, 1.0-50 mM PEG having a molecular weight of 5,000-22,000, 0.1-10 mM of sulphated cyclodextrin having a molecular weight of 1,000-2,000 or one of its salts, 0.1-10 mM of sulfated oligosaccharide having a molecular weight of 400-2,000 or one of its salts, or any of the mixtures thereof is used. As the divalent metal salt, 0.1-50 mM magnesium salt, calcium salt, manganese salt, nickel salt, cobalt salt, etc. are used. Preferably 0.1-50 mM magnesium salt is used. As the reagent that inhibits the reaction of lipoproteins other than HDL, an anti-Apo-B antibody, an anti-Apo-C antibody, etc. can also be used. Examples of antiapo-B antibody and antiapo-C antibody are: an IgG fraction which is obtained by immunizing rabbits against apoprotein-B or purified apoprotein-C from human blood serum, collecting the antiapo-B antiserum or antiapora antiapo -C of the immunized rabbits, and subject the antiapo-B antiserum or antiapo-C antiserum with precipitation of ammonium sulfate and precipitate by adding salt; and an antiapo-B monoclonal antibody or an antiapo-C monoclonal antibody that is obtained by immunizing mice against the aforementioned apoprotein B or apoprotein C [Introduction to the experimental procedure for monoclonal antibodies, written by Tamie Ando, Kodansha Scientific, 21 (1991) ] Like the enzymes, commercially available enzymes can be used. For example, cholesterol esterase and liprotein lipase derived from animals, plants, or microorganisms can be used which has the ability to hydrolyse cholesterol ester, cholesterol oxidase derived from animals, plants or microorganisms that has the ability to oxidize cholesterol to form hydrogen peroxide, and cholesterol dehydrogenase derived from animals, plants or microorganisms. To improve the specificity and stability of these enzymes, they can be chemically modified with a group having polyethylene glycol as the main component, a group having polypropylene glycol as the main component, a group having a saccharide in the structure such as a water-soluble oligosaccharide residue, a sulfopropyl group, a polyurethane group, etc. In addition, enzymes that are obtained by introducing genes from the aforementioned enzymes into other microorganisms and the subsequent expression thereof, optionally followed by chemical modification, and enzymes obtained by gene modification of the enzymes above can also be used. mentioned and their subsequent expression of these, optionally followed by the chemical modification. Examples of the reagent for chemically modifying the enzymes (chemical modifier) are compounds wherein the polyethylene glycol and a group that can be linked to an amino group are connected [eg, Sunbright VFM4101 (NOF Corporation) where the polyethylene glycol and a group that can be attached to an amino group such as the N-hydroxysuccinimido group are connected, Sunbright AKM series, ADM series, and ACM series [NOF Corporation: (Chemical Engineering Monographs ) Chemical Engineering Monographs (Kagaku Kogaku Ronbunshu), 20, (3), 459 (1994)], which are compounds having the polyalkylene glycol structure and the acid anhydride structure, compounds in which a copolymer of polyethylene glycol and polypropylene glycol and a group that can be bonded to an amino group, monomethyl ether, polyethylene glycol monomethyl ether and maleic anhydride copolymers, etc. In addition, activated P4000 polyurethane (Boehringer Mannheim, Directions for Enzyme modification Set, which is a chemical modifier for polyurethane, Dextran T40, TCT-activated (same as the previous one) which is A chemical modifier for dextran, 1, 3-propanosulton, etc. are also usable.With the use of these chemical modifiers, the enzymes can be modified with a group having polyethylene glycol as the main component, a group having polypropylene glycol as main component, a group having a copolymer of polypropylene glycol and polyethylene glycol, a group having a saccharide in the structure, a sulfopropyl group, a polyurethane group, etc. A method for the reaction of an enzyme with a chemical modifier However, it should be noted that the method is not limited to this method.First, the enzyme is dissolved in such a regulator. as a HEPES regulator of pH 8 or higher, and then, for example, Sunbright (0.01-500 times the molar amount of the enzyme) is added to the solution at 0-50 ° C, followed by stirring for 5-6 minutes. The resulting reaction mixture is used as is, or is used after removal of low molecular weight compounds, by ultrafiltration, if necessary. The cholesterol ester-hydrolyzing enzyme, cholesterol oxidant enzyme, and cholesterol oxidoreductase are advantageously used at a concentration of 0.1-100 u / ml. It is preferred that the cholesterol ester-hydrolyzing enzyme, the cholesterol oxidizing enzyme and the cholesterol oxidoreductase which are not reactive to LDL be chemically modified with a group having polyethylene glycol as a main component, a group having glycol of polypropylene as a main component, a group having a saccharide as in the structure such as a water-soluble oligosaccharide residue, a sulfopropyl group, a polyurethane group, etc. As the cholesterol ester-hydrolyzing enzyme, the cholesterol oxidizing enzyme and the cholesterol reductase which are capable of acting in LDL, the enzymes without chemical modification are preferred. However, enzymes slightly modified for the purpose of stabilization can be used as long as they are able to act only in LDL. An example of the modifier to be used is Sunbright VFM4101 (NOF Corporation) mentioned above. The amount of enzyme to be used is preferably 0.5-100 u / ml. As the surfactant that was used for the purpose of producing reactive LDL, nonionic surfactants such as Triton X-100, cationic surfactants and anionic surfactants are used in an amount of 0.02-10%. The cyclodextrin that was used for the purpose of making the LDL reactive includes α-cyclodextrin, β-cyclodextrin, α-cyclodextrin, dimethyl-α-cyclodextrin, dimethyl-j-β-cyclodextrin, dimethyl-β-cyclodextrin, hydroxypropyl α- cyclodextrin, hydroxypropyl- / 8-cyclodextrin, hydroxy-β-cyclodextrin, 2,3,6-0-methyl-α-cyclodextrin, and poly-β-cyclodextrin. Cyclodextrin is used in an amount of 0.1-10%. As chelating agents that are used for the purpose of producing reactive LDL, compounds that are capable of complexing with magnesium are advantageously used. For example, ethylenediaminetetraacetic acid monohydrate (EDTA), triethylenetetramine-N, N, N ', N ", N'", N '"-hexaacetic acid (TTHA), and trans-1,2-cyclohexanediamine-N, N, N acid ', N' -tetraacetic (CyDTA) are used in an amount of 0.005-2%. As the chromogen which serves as a substrate for cholesterol oxidant enzymes for the detection of hydrogen peroxide, combinations of 4-aminoantipyrine and Trinder reagents [General Catalog of Dojin Kagaku Kenkusho, 19th ed. (1994)], as well as the combinations generally employed of 4-aminoantipyrine and phenols such as phenol, 4-chlorophenol, m-cresol and 3-hydroxy-2 acid, 4,6-triiodobenzoic acid (HTIB) Examples of the Trinder reagent are anilines such as N-sulfopropylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl- N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl- N-sulfopropyl-m-toluidine (TOP S), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-m-toluidine, N, N-disulfopropyl-3,5-dimethoxyaniline, N-ethyl- N-sulfopropyl-m-anisidine, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3, 5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline and N-ethyl-N- (2- hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-methylphenyl) - N '-succinylethylenediamine (EMSE), and N-ethyl-N- (3-methylphenyl) -N' -acetylenediamine . As the high sensitivity chromogen, 10 - (N-methemocarbamoyl) -3,7-bis (dimethylamino) phenothiadine (MCDP) disclosed in Japanese Patent Application Published and Examined No. 33479/85, bis [3] can be used. bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA) disclosed in Japanese Published and Examined Patent Application No. 27839/92, the chromogens disclosed in published and unexamined Japanese Patent Application No. 296 / 87, etc. These high sensitivity chromogens can be used in combination with 4-aminoantipyrine or with the Trinder reagents listed above. The concentration of the chromogen is preferably 0.01-10 mg / ml, and is limited by solubility. As the phenol compound and the aniline compound to be used in combination with peroxidase in the removal of HDL, the phenols and anilines listed above are similarly useful. As the regulator, the regulator Tris, the regulator Good, they are used advantageously, as well as the phosphate regulator. The concentration of the regulator is preferably 5-500 mM. The pH value thereof is preferably 5-9. Procedure 2 The determination is carried out by (1) adding a sample to a regulator containing ascorbic acid oxidase, and adding to the resulting mixture a reagent containing a cholesterol ester-hydrolyzing enzyme having low reactivity to LDL (inhibiting only the LDL reaction), a cholesterol oxidizing enzyme that has low reactivity to LDL (or cholesterol oxidoreductase having low reactivity to LDL), peroxidase and a chromogen [or NAD (P)]; and (2) subsequent to the end of the reaction of cholesterol in lipoproteins other than LDL, measure the change in absorbance with a spectrophotometer, and calculate the amount of LDL cholesterol based on the absorbance determined separately by using a standard solution containing cholesterol LDL at a known concentration under the same conditions. As the cholesterol ester-hydrolyzing enzyme has low reactivity to LDL, a chemically modified cholesterol ester-hydrolyzing enzyme is preferably used. Since the cholesterol oxidizing enzyme or cholesterol oxidoreductase has low reactivity to LDL, a chemically modified or unmodified cholesterol oxidant enzyme or chemically modified or unmodified cholesterol oxidoreductase can be used. An example of the modifier is Sunbright VFM4101 (NOF Corporation) mentioned above. The amount of the enzyme to be used is preferably 0.5-100 u / ml. As the chromogen and the regulator, the chromogens and regulators listed in Procedure 1 are used in a similar manner. Procedure 3 The determination is carried out by (1) adding a sample to a regulator containing a reagent that inhibits only the LDL reaction (which is not required when the cholesterol ester-hydrolyzing enzyme mentioned above inhibits only the reaction of LDL), a cholesterol ester-hydrolyzing enzyme, a cholesterol oxidant enzyme (or cholesterol oxidoreductase), and catalase, peroxidase and an aniline compound, peroxidase and a phenol compound, or peroxidase and 4-aminoantipyrine [or NAD ( P)] to eliminate cholesterol in lipoproteins other than LDL by means of the reaction; (2) add a reagent that enables the LDL cholesterol reaction (which is not required when the ester-cholesterol hydrolyzing enzyme mentioned below enables the LDL cholesterol reaction), an ester-cholesterol hydrolyzing enzyme (which is not requires when the cholesterol ester-hydrolyzing enzyme added is initially enabled to react with LDL cholesterol by the reagent that enables the LDL cholesterol reaction) and a chromogen [which may be unnecessary or may be replaced with NAD (P)] (in combination with peroxidase when catalase is used) for the reaction of LDL cholesterol to form hydrogen peroxide and develop a color [or to form NAD (P) H]; and (3) measuring the absorbance of the pigment formed at the maximum wavelength with a spectrophotometer [measuring the increase of NAD (P) H in terms of the absorbance at 300-500 nm, preferably at 330-400 nm, for example at 340 nm when cholesterol oxidoreductase is used (otherwise forming a formazan pigment by the addition of diaphorase and a tetrazolium salt, followed by the calorimetric determination of the formazan pigment)]. The amount of LDL cholesterol is calculated based on a standard solution containing cholesterol LDL at a known concentration under the same conditions. As the reagent which inhibits the LDL reaction, an ester-cholesterol hydrolyzing enzyme capable of inhibiting only the LDL reaction, etc. can be used. As the reagent that enables the LDL cholesterol reaction, an ester-cholesterol hydrolyzing enzyme can be used to enable the reaction of LDL cholesterol, a surfactant, a chelating agent, etc. As the surfactant and the chelating agent, the surfactants and chelating agents listed in Process 1 are similarly useful.

As enzymes, the enzymes listed in Process 1 are similarly useful. As the ester-cholesterol hydrolyzing enzyme capable of inhibiting only the LDL reaction, a cholesterol ester-hydrolyzing enzyme prepared by adding to an ester-hydrolyzing enzyme can be preferably used. of cholesterol a chemical modification derived from an animal, a plant or a microorganism (not less than 10 times the molar amount of the enzymes), and a cholesterol ester-hydrolyzing enzyme derived from an animal, plant or microorganism having similar specificity, or which is endowed with similar specificity by gene modification of the aforementioned enzymes and their subsequent expression. Specifically, the product of the cholesterol esterase reaction derived from a microorganism belonging to the genus Pseudomonas. Chromobacterium, etc. in an aqueous solution with the chemical modifier mentioned in Procedure 1 (not less than 10 times the molar amount of the enzyme) can be cited as an example. The molar ratio of the chemical modifier to the enzyme is preferably 10-500: 1 and is determined by considering the specificity endowment effect and the degradation of activity by the modification. As the chemically modified cholesterol ester-hydrolysing enzyme, for example, an enzyme obtained by the following steps can be used: randomly altering a part of the lipase DNA sequence derived from a microorganism belonging to the genus Brevibacterium, introducing the altered gene in another microorganism such as E. Coli, let it be expressed there, select by screening a strain that produces cholesterol esterase exhibiting enzymatic activity and possessing the ability to inhibit only the LDL reaction, and mass culture the strain. The ester-cholesterol hydrolyzing enzyme is advantageously used in a concentration of 0.1-100 u / ml. For the purpose of improving the aforementioned specificity, heparin, phosphotungstinic acid, dextran sulfuric acid, sulfated cyclodextrin, sulphated oligosaccharide, or a salt thereof, or polyethylene glycol together with a divalent metal salt such as magnesium salt may be added. , calcium salt, manganese salt, nickel salt and cobalt salt during the step (1) mentioned above in an amount not so great as to induce the aggregation of LDL. As the cyclodextrin, oligosaccharide, and salt, the cyclodextrins, oligosaccharides and salts listed in Procedure 1 are similarly used. As the ester-cholesterol hydrolyzing enzyme that enables the reaction of the LDL cholesterol, unmodified cholesterol esterase is preferred. It is preferable to use the enzyme at a concentration of 0.5-100 u / ml. As the cholesterol oxidant enzyme or cholesterol oxidoreductase, cholesterol oxidase derived from microorganisms that have the ability to oxidize cholesterol to form hydrogen peroxide, and cholesterol dehydrogenase derived from an animal or microorganism are advantageously used. The aforementioned enzymes can be chemically modified with a group having polyethylene glycol as the main component or a water-soluble oligosaccharide residue for the purpose of improving its specificity and stability. The molar ratio of the chemical modifier to the enzyme is preferably 0.1-500: 1 and is determined by considering the stabilization effect and the degradation of activity by means of modification. The cholesterol oxidizing enzyme and the cholesterol oxidoreductase are advantageously used at a concentration of 0.1-100 u / ml. A method for the reaction of an enzyme with a chemical modifier is described below. It should be noted, however, that the method is not limited to this method. First, the enzyme is dissolved in a regulator such as HEPES buffer of pH 8 or higher and then, a prescribed molar amount of Sunbright at 0-50 ° C is added to the solution, followed by stirring for 1-24 hours. The resulting reaction mixture is used as is, or is used after removal of low molecular weight compounds by filtration, if necessary. As the phenol compound and aniline compound to be used in combination with peroxidase in the elimination of cholesterol in lipoproteins other than LDL, the phenols and anilines listed above are similarly used. Since the systems of the present invention described above each include an ordinary system for the determination of cholesterol, a surfactant or cholic acid which is often used to activate a cholesterol oxidant enzyme can also be employed. In addition, various salts can also be used to solubilize proteins such as globulin. As the surfactant, nonionic, anionic, and cationic surfactants are used in an amount of 0-1%. Examples of cholic acid are cholic acid, deoxycholic acid, taurocholic acid, and cenodeoxycholic acid. Colic acid is used in an amount of 0-5%. Examples of the salt are sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium nitrate, lithium chloride, lithium sulfate, ammonium chloride, sulfate of ammonium, calcium chloride, calcium nitrate, calcium acetate, nickel chloride, nickel nitrate, nickel acetate, cobalt chloride, and cobalt nitrate. The salt is used at a concentration of 0-100 mM. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the correlation between the blood serum solution containing 228.4 mg / dL of LDL cholesterol and the absorbance as determined by the method of Example 1.

Figure 2 is a graph showing the course of absorbance time as determined with an autoanalyzer (Hitachi 7259) by the method of Example 9 using HDL, LDL and VLDL separated by ultracentrifugation method and human blood serum. Figure 3 is a graph showing the correlation between the blood serum solution containing 228.4 mg / ml of LDL cholesterol and the absorbance as determined by the method of Example 11. The examples of the present invention are shown below. BEST MODE FOR CARRYING OUT THE INVENTION Example 1 Determination of LDL Cholesterol (1) Chemical Modification of Enzyme Cholesterol esterase derived from a microorganism belonging to the genus Pseudomonas (lg) was dissolved in 100 ml of a 20 nM phosphate buffer (pH (), followed by cooling to 5 ° C. To the solution was added 15 g of Sunbrigth VFM4101 (NOF Corporation), and the mixture was reacted for 4 hours.The resulting reaction mixture was used as cholesterol esterase modified with PEG in Reagent B (molecular weight of the PEG grouping = 6000) .Separately, 1 g of cholesterol oxidase derived from a microorganism belonging to the genus Brevibacterium and 0.1 g of Sunbrigth VFM4101 were subjected to reaction in the same manner as The resulting reaction mixture was used as the cholesterol oxidase modified with PEG in Reagent B (molecular weight of the PEG = 6000 grouping). (2) Determination of LDL cholesterol Reagent A 20 mM 3-Morpholinophoric Sulfonic Acid (MOPS) Regulator (pH 7) dextran sulfuric acid 0.7 g / 1 Mg Sulfate Heptahydrate 7.5 g / 1 Sodium Azide 0.1 g / 1 Ascorbic Acid Oxide 3 U / ml Reagent B 20 mM MOPS regulator (pH 7) Peroxidase 30 u / ml Cholesterol esterase modified with PEG 1 u / ml Modified cholesterol oxidase with PEG 3 u / ml Sodium cholate 5 g / ml EMSE 0.3 g / 1 Reagent C Regulator 20 mM MOPS (pH 7) Unmodified cholesterol esterase 2 u / ml 4-Aminoantipyrine 0.4 g / 1 The following examples were used: (l) blood serum containing 228.4 mg / dl of LDL cholesterol, (2) a dilution 8 : 10 the serum of (1) with physiological saline, (3) a 6:10 dilution of the serum of (1) with physiological saline, (4) a 4:10 dilution of the serum of (1) with physiological saline, (5) ) a 2:10 dilution of the serum of (1) with physiological saline, and (6) physiological saline. To 2.25 ml of Reagent A was added 20 μl of a sample, and the mixture was incubated at 37 ° C for 5 minutes. Then, 0.75 ml of Reagent B was added to it and the mixture was incubated at 37 ° C for 5 minutes to remove HDL cholesterol, and the absorbance (El) was measured at 555 nm. After the addition of 0.75 ml of Reagent C, the mixture was further incubated at 37 ° C for 5 minutes and the absorbance (E2) was measured at a wavelength of 555 nm. The LDL cholesterol concentration was calculated by separately submitting a standard cholesterol solution at a concentration of 200 mg / dl to the same procedure and comparing the respective values of (E2-E1) x dilution ratio. The term "dilution ratio" used herein means the volume ratio of (Reagent A + Reagent B) / (Reagent A + Reagent B + Reagent C). The results obtained by using the blood serum containing 228.4 mg / dL of LDL cholesterol are shown in Figure 1. Example 2 The blood serum samples were subjected to the determination of LDL cholesterol using Reagent B and Reagent C described in Example 1 (2) and the combination of an aggregating agent and a divalent metal salt shown below in Reagent A with an autoanalyzer (Hitachi 7070) (4 μl of sample, 270 μl of Reagent A, 90 μl of Reagent B , and 90 μl of Reagent C). Separately, the sample was subjected to LDL cholesterol determination according to the method described in "Igaku no Ayumi", 94 (8), 359 (1975) (ultracentrifugation method) using a rotor (Hitachi RPL 42 T). As is evident from Table 1, the results obtained when using Reagents A, B and C showed to be in agreement with the results obtained by the centrifugation method. <; A > Phosphotungstenic acid 10 mg / ml Mg sulfate heptahydrate 7.5 mg / ml < B > Sodium dextran sulfate 1 mg / ml Mg sulfate heptahydrate 10 mg / ml < C > Heparin sodium salt 10 mg / ml Ca chloride dihydrate 10 mg / ml < D > PEG 20000 50 mg / ml Mg sulfate heptahydrate 5 mg / ml < E > Phosphotungstic acid 10 mg / ml Sodium dextran sulfate (M: 200000) 7.5 mg / ml Mg sulfate heptahydrate 7.5 mg / ml < F > Phosphotungstic acid 10 mg / ml Heparin sodium salt 7.5 mg / ml Mg Sulfate Heptahydrate 7.5 mg / ml < G > Phosphotungsic acid 10 mg / ml PEG 6000 7.5 mg / ml Mg Sulfate Heptahydrate 7.5 mg / ml Table 1 Example 3 Chemical modification of the enzymes was carried out by the same procedure as in Example 1 (1) except that Sunbright AKM1511 (NOF Corporation), activated P4000 polyurethane (Boehringer Mannheim), and Dextran T40, TCT- were used. activated (Boehringer Mannheim), respectively instead of Sunbright VFM4101. The same blood samples that were used in Example 2 were subjected to LDL cholesterol determination in a similar manner as in Example 2 using Reagent A and Reagent C described in Example 1 (2) and using the enzymes chemically Modifications obtained above in place of cholesterol esterase modified with PEG and cholesterol oxidase modified with PEG in Reagent B. The concentration of LDL cholesterol was determined to be 178.0 mg / dl, 179.1 mg / dl, and 179.8 mg / dl, respectively, which proved to be in agreement with the result obtained by the centrifugation method. EXAMPLE 4 The same blood serum sample that was used in Example 2 was subjected to LDL cholesterol determination in a similar manner as in Example 2 using Reagent A described in Example 1 (2), using catalase at a concentration of 300 u / ml in place of peroxidase in Reagent B, and using peroxidase additionally at a concentration of 30 u / ml in Reagent C. The concentration of LDL cholesterol was determined to be 178.6 mg / dl, which showed be consistent with the result obtained by the ultracentrifugation method. Example 5 The same blood serum samples that were used in Example 2 were subjected to cholesterol determination LDL in a similar manner as in Example 2, using the Reagent A and Reagent C described in Example 1 (2) and using TOOS (measurement at 555 nm), DAOS (measurement at 593 nm), MAOS (measurement at 630 nm), and TOPS (measurement at 550 nm), respectively, at a concentration of 0.3 g / 1 in place of EMSE in Reagent B. LDL cholesterol concentrations were determined to be 177.9 mg / dL, 177.8 mg / dl, 179.2 mg / dl and 178.8 mg / dl, respectively, which showed to be in agreement with the result obtained by means of the centrifugation method.

Example 6 The same blood serum samples that were used in Example 2 were subjected to the determination of LDL cholesterol in a similar manner as in Example 2, using Reagent A and Reagent B described in Example 1 (2) and using MCDP (measurement at 666 nm) and BCMA (measurement at 755 nm), respectively, at a concentration of 0.1 mg / ml instead of 4-aminoantipyrine in Reagent C. LDL cholesterol concentrations were determined to be 178.3 mg / dl and 179.0 mg / dl, respectively, which showed to be in agreement with the result obtained by the centrifugation method. Example 7 The same blood serum samples that were used in Example 2 were subjected to the determination of LDL cholesterol in a similar manner as in Example 2, using Reagent A and Reagent B described in Example 1 (2) and using dimethyl-0-cyclodextrin at a concentration of 20 mg / ml in place of the unchanged cholesterol esterase in reagent C. The concentration of LDL cholesterol was determined to be 177.4 mg / dl, which showed to be in accordance with the result obtained by the centrifugation method. EXAMPLE 8 Reagent A and Reagent B described in Example 1 (2) were mixed at a ratio of 3: 1 to prepare Reagent D. To 3 ml of Reagent D was added 20 μl of the same serum sample. blood that was used in Example 2, and after incubation at 37 ° C for 5 minutes, the absobance (El) was measured at 555 nm. After the addition of 0.75 ml of reagent C, the mixture was further incubated at 37 ° C for 5 minutes, and the absorbance (E2) was measured at a wavelength of 555 nm. The concentration of LDL cholesterol was calculated by separately submitting a standard cholesterol solution at a concentration of 200 mg / dl with the same procedure and comparing the respective values of (E2-E1) x dilution ratio. The term "dilution ratio" used herein means the volume ratio of (Reagent A + Reagent B) / (Reagent A + Reagent B + Reagent C). The concentration of LDL cholesterol was determined to be 177.6 mg / dl, which showed to be in agreement with the result obtained by means of the ultracentrifugation method. Example 9 Reagent A 10 mM MOPS regulator (pH 7) Na sulfate 2 mg / ml EMSE 0.3 mg / ml Ascorbic acid oxidase 3 u / ml Reagent B 10 mM MOPS regulator (pH 7) 4-Aminoantipyrine 0.5 mg / ml Collato sodium 3 mg / ml cholesterol esterase modified with PEG 5 u / ml unmodified cholesterol oxidase 7 u / ml 10 u / ml peroxidase The same cholesterol esterase modified with PEG used in Example 1 was used in this Example . The same blood serum sample that was used in Example 2 was subjected to the determination of LDL cholesterol with an autoanalyzer (Hitachi 7250) using the aforementioned reagents. The absorbance change (E3) was measured between 3.5 minutes and 5 minutes after the addition of Reagent B. The LDL cholesterol concentration was calculated by separately submitting a standard cholesterol solution at a concentration of 200 mg / dl to the same procedure for measure the change in absorbance (E4) and compare the values of E3 and E4. The concentration of LDL cholesterol was determined to be 178.6 mg / dl, which showed to be consistent with the result obtained by ultracentrifugation method. Figure 2 shows the course of the absorbance time as determined using HDL, LDL and VLDL separated by ultracentrifugation method and human blood serum. Example 10 Reagent A 10 mM MOPS regulator (pH 7) Sodium dextran sulfate (MW: 500000) 0.5 mg / ml Mg sulfate heptahydrate 5 mg / ml EMSE 0.3 mg / ml 10 u / ml peroxidase 10% cholesterol esterase PEG 2 u / ml Unmodified cholesterol oxidase 3 u / ml Ascorbic acid oxidase 3 u / ml Reagent B 10 mM MOPS regulator (pH 7) 4-Aminoantipyrine 0.5 mg / ml Triton X-100 3 mg / ml Tetrasodium salt of EDTA 5 u / ml The same esterase of cholesterol modified with PEG that was used in Example 1 was used in this Example. The same blood serum sample that was used in Example 2 was subjected to the determination of LDL cholesterol with an autoanalyzer (Hitachi 7250) using the aforementioned reagents, (absorbance: E5). The concentration of LDL cholesterol was calculated by separately submitting a standard cholesterol solution at a concentration of 200 mg / dl to the same procedure (absorbance: E6) and comparing the values of E5 and E6. The concentration of LDL cholesterol was determined to be 177.3 mg / dl, which showed to be consistent with the result obtained by ultracentrifugation method. Absorbance measurements were made with the autoanalyzer 5 minutes after the addition of Reagent B. Example 11 (1) Chemical modification of enzyme The cholesterol esterase derived from a microorganism belonging to the genus of Pseudomonas (lg) was dissolved in 100 ml of a 20 mM phosphate buffer (pH 8), followed by cooling at 15 ° C. To the solution was added 25 g of Sunbright VFM4101 (NOF Corporation), and the mixture was reacted for 4 hours. The resulting reaction mixture was used as the cholesterol esterase PEG-modified in Reagent B (molecular weight of the PEG 6000 pool). Separately, 1 g of cholesterol oxidase derived from a microorganism belonging to the genus Brevibacterium and 0.5 g of Sunbrigth VFM4101 were subjected to reaction in the same manner as the previous one. The resulting reaction mixture was used as the cholesterol oxidase modified with PEG in Reagent A (molecular weight of the PEG = 6000 grouping). (2) Determination of LDL cholesterol Reagent A Regulator (MOPS) 20 mM (pH 7) Mg sulfate heptahydrate 2 g / 1 Peroxidase 30 u / ml cholesterol esterase modified with PEG 2 u / ml cholesterol oxidase modified with PEG 5 u / ml Sodium colato 1 g / 1 EMSE 0.3 g / 1 Ascorbic acid oxidase 3 u / ml Reagent B 20 mM MOPS regulator (pH 7) Unmodified cholesterol esterase 3 u / ml 4-Aminoantipyrine 0.4 g / 1 The following examples were used: (1) blood serum containing 228.4 mg / dl LDL cholesterol, as determined by the ultracentrifugation method, (2) an 8:10 dilution of the serum of (1) with physiological saline, (3) a 6:10 dilution of the serum of (l) with physiological saline, (4) a 4:10 dilution of the serum of (1) with physiological saline, (5) a 2:10 dilution of the serum of (1) with physiological saline, and (6) physiological saline. To 2.25 ml of reagent A was added 20 μl of a sample, and the mixture was incubated at 37 ° C for 5 minutes to remove cholesterol in lipoproteins other than LDL. After the addition of 0.75 ml of Reagent B, the mixture was further incubated at 37 ° C for 5 minutes, and the absorbance was measured at a wavelength of 600 nm. The results are shown in Figure 3. Separately, to a mixture of Reagent A and Reagent B was added 20 μl of a standard cholesterol solution at a concentration of 200 mg / dl, the mixture was incubated at 37 ° C during 5 minutes, and the absorbance at the wavelength of 600 nm was measured. The concentration of LDL cholesterol was calculated based on the absorbance thus obtained. The concentration of cholesterol was determined to be 229.7 mg / dl, which showed to be consistent with the result obtained by the centrifugation method. Example 12 The same blood serum samples that were used in Example 11 (2) were subjected to the LDL cholesterol determination using Reagent B described in Example 11 (2), and additionally using combinations of an aggregation agent and a divalent metal salt shown below in Reagent A with an autoanalyzer (Hitachi 2070) (4 μl of sample, 270 μml of Reagent A, and 90 μml of Reagent B. Separately, the sample was subjected to cholesterol determination LDL according to the method described in Modern Medical Treatment (Gendai Iryo), 23 (1), 113 (1991) (ultracentrifugation method) using a rotor (Hitachi PRL 42 T). Table 2, the results obtained using Reagents A and B showed good congruence with the result obtained by the ultracentrifugation method. <A> 0.1 mg / ml phosphotungsten acid Mg sulfate heptahydrate 1 mg / ml < B > Sodium dextran sulfate MW: 20000) 0.1 mg / ml Mg sulfate heptahydrate 2 mg / ml < C > Heparin sodium salt 0.3 mg / ml Ca chloride dihydrate 3 mg / ml < D > PEG 20000 20 mg / ml Mg sulphate heptahydrate 3 mg / ml < E > Phosphotungstenic acid 0.1 mg / ml Dextran sodium sulphate (MW: 200000) 0.1 mg / ml < F > Phosphotungstic acid 0.1 mg / ml Heparin sodium salt 0.1 mg / ml Mg sulfate heptahydrate 2 mg / ml < G > Dextran sulfuric acid (MW: 500,000) 0.1 mg / ml PEG 6000 5 mg / ml Mg sulfate heptahydrate 2 mg / ml Table 2 Example 13 Chemical modification of the enzymes was carried out by the same procedure as in Example 11 (l) except that Sunbright AKM1511 (NOF Corporation), activated P4000 polyurethane (Boehringer Mannheim), and Dextran T40, TCT- were used. activated (Boehringer Mannheim), respectively instead of Sunbright VFM4101. The same blood samples that were used in Example 11 (2) were subjected to the determination of LDL cholesterol in a similar manner as in Example 11 (2) using Reagent B described in Example 11 (2) and using the chemically modified enzymes obtained above in place of cholesterol esterase modified with PEG and cholesterol oxidase modified with PEG in Reagent A. The concentration of LDL cholesterol was determined to be 228.0 mg / dl, 229.1 mg / dl, and 226.8 mg / dl, respectively, which proved to be consistent with the result obtained by the centrifugation method. Example 14 The same blood serum sample that was used in Example 11 (2) was subjected to the determination of LDL cholesterol in a similar manner as in Example 11 (2) using catalase at a concentration of 300 u / ml in Place of peroxidase in Reagent A described in Example 11 (2), and using additionally 300 u / ml of peroxidase and sodium azide in Reagent B. The concentration of LDL cholesterol was determined to be 228.6 mg / dl, which showed to be in agreement with the result obtained by the ultracentrifugation method. Example 15 The same blood serum samples that were used in Example 11 (2) were subjected to the determination of LDL cholesterol in a similar manner as in Example 11 (2), using Reagent B described in Example 11 ( 2) and using TOOS (measurement at 555 nm), DAOS (measurement at 593 nm), MAOS (measurement at 630 nm), and TOPS (measurement at 550 nm), respectively, at a concentration of 0.3 g / 1 in place of EMSE in Reagent A. LDL cholesterol concentrations were determined to be 277.9 mg / dL, 277.4 mg / dl, 225.2 mg / dl and 224.8 mg / dl, respectively, which showed to be in agreement with the result obtained by means of the centrifugation method.

Example 16 The same blood serum samples that were used in Example 11 (2) were subjected to LDL cholesterol determination in a similar manner as in Example 11 (2), using EMSA-free Reagent A and using MCDP (measurement at 666 nm) and BCMA sulfate (measurement at 755 nm), respectively, at a concentration of 0.1 mg / ml instead of 4-aminoantipyrine in Reagent B. LDL cholesterol concentrations were determined to be 228.3 mg / dl and 229.0 mg / dl, respectively, which showed to be in agreement with the result obtained by means of the centrifugation method. Example 17 The same blood serum samples that were used in Example 11 (2) were subjected to the determination of LDL cholesterol in a similar manner as in Example 11 (2), using Reagent A described in Example 11 ( 2) and additionally using 5 mg / ml of polyoxyethylene monolaurate, 5 mg / ml of Triton X-100, and 1 mg / ml of sodium dodecylbenzenesulfornate, respectively, in Reagent B. The reactions were completed within 3 minutes . The concentrations of LDL cholesterol were determined to be 228.6 mg / dl, 226.1 mg / dl and 227.0 mg / dl, respectively, which showed to be in agreement with the result obtained by means of the centrifugation method. Example 18 The same blood serum samples that were used in Example 11 (2) were subjected to the determination of LDL cholesterol in a similar manner as in Example 11 (2), using Reagent A described in Example 11 ( 2) and using 5 mg / ml of polyoxyethylene monolaurate, 5 mg / ml of Triton X-100, and 1 mg / ml of sodium dodecylbenzenesulfonate, respectively, in Reagent B. The reactions were completed within 3 minutes. The concentrations of LDL cholesterol were determined to be 227.9 mg / dl, 229.2 mg / dl and 226.0 mg / dl, respectively, which showed to be in agreement with the result obtained by means of the centrifugation method. Example 19 The same blood serum samples that were used in Example 11 (2) were subjected to the determination of LDL cholesterol in a similar manner as in Example 11 (2), using Reagent B described in Example 11 ( 2) and using deoxycholic acid and taurocholic acid, respectively, at a concentration of 1 g / 1 in place of the cholic acid in Reagent A. The concentrations of LDL cholesterol were determined to be 229.9 mg / dl and 225.7 mg / dl, respectively, which showed to be in agreement with the obtained result by means of the centrifugation method. Industrial Applicability The present invention provides a simple method for the determination of LDL cholesterol which does not require complicated separation steps.

Claims (27)

1. CLAIMS 1. - A method for the determination of low density lipoprotein (LDL) cholesterol in a sample containing LDL, which comprises eliminating high density lipoprotein (HDL) cholesterol in the sample, subjecting the sample to a reaction which uses the action of a cholesterol ester-hydrolyzing enzyme and the action of a cholesterol oxidant enzyme or cholesterol oxidoreductase, and determines the amount of hydrogen peroxide of a reduced-type coenzyme generated by the reaction.
2. 2. The method according to claim 1, wherein the reaction using the action of a cholesterol ester-hydrolyzing enzyme and the action of a cholesterol oxidant enzyme or cholesterol oxidoreductase is carried out in the presence of a reagent that enables the reaction of cholesterol in LDL.
3. 3. The method according to claim 2, wherein the removal of cholesterol in HDL is carried out by a reaction using the action of a chemically modified or unmodified cholesterol ester-hydrolyzing enzyme and the action of an enzyme chemically modified or unmodified cholesterol oxidant or cholesterol oxidoreductase chemically modified or unmodified in the presence of a reagent that inhibits the reaction of lipoproteins other than HDL.
4. 4. The method according to claim 3, wherein the reagent that inhibits the reaction of lipoproteins other than HDL is a combination of a divalent metal salt, and heparin or a salt thereof, phosphotungstenic acid or a salt thereof, acid dextran sulfuric acid or a salt thereof, polyethylene glycol, sulphated cyclodextrin or a salt thereof, sulfated oligosaccharide or a salt thereof, or a mixture thereof.
5. 5. The method according to claim 3, wherein said reagent that inhibits the reaction of lipoproteins other than HDL is an anti-Apo-B antibody or an anti-Apo-C antibody.
6. 6. - The method according to any of claims 3-5, wherein the modified grouping of said chemically modified cholesterol ester-hydrolyzing enzyme, chemically modified cholesterol oxidant enzyme, and chemically modified cholesterol oxidoreductase is a group having polyethylene glycol as the main component, a group having polypropylene glycol as the main component, a group having a copolymer of polypropylene glycol and polyethylene glycol, a group having a saccharide in the structure, a sulfopropyl group, or a group polyurethane.
7. 7. - The method according to any of claims 3-6, wherein said reagent that enables the reaction of the cholesterol in LDL is a nonionic, cationic or anionic surfactant, cyclodextrin, a chemically modified cholesterol ester-hydrolyzing enzyme or unmodified capable of acting in LDL, a chemically modified or unmodified cholesterol oxidizing enzyme capable of acting exclusively in LDL, or chemically modified or unmodified cholesterol oxidoreductase capable of acting in LDL.
8. 8. - The method according to claim 1, wherein the removal of cholesterol in HDL is carried out by eliminating cholesterol in lipoproteins other than LDL in the presence of a reagent that only inhibits the LDL reaction.
9. 9. - The method according to claim 8, wherein said elimination of cholesterol in lipoproteins other than LDL is followed by the addition of a reagent that enables the reaction of the cholesterol in LDL.
10. 10. The method according to claim 9, wherein said reagent that inhibits only the LDL reaction is a chemically modified or unmodified cholesterol ester-hydrolyzing enzyme capable of inhibiting only the LDL reaction.
11. 11. The method according to claim 10, wherein the modified grouping of said chemically modified cholesterol esterase is a group having polyethylene glycol as the main component., a group having polypropylene glycol as the main component, a group having a copolymer of polypropylene glycol and polyethylene glycol, a group having a saccharide in the structure, a sulfopropyl group, or a polyurethane group.
12. 12. - The method according to any of claims 9-11, wherein said reagent that enables the cholesterol reaction in LDL is a cholesterol ester-hydrolyzing enzyme that enables the reaction of cholesterol in LDL.
13. 13. - The method according to any of claims 9-11, wherein the reagent that enables the cholesterol reaction in LDL is a surfactant or a chelating agent.
14. 14. - The method according to any of claims 1-13, wherein the enzyme cholesterol-hydrolyzing cholesterol, cholesterol oxidant enzyme or cholesterol oxidoreductase is an enzyme derived from an animal, a plant or a microorganism, an enzyme obtained by introducing the gene of such an enzyme into another microorganism and subsequently expressed, or an enzyme obtained by modifying the gene of such an enzyme and subsequently expressed.
15. 15. - The method according to any of claims 1-14, wherein the determination of hydrogen peroxide is carried out by converting the hydrogen peroxide into a pigment by the action of a chromogen, and determining the pigment.
16. 16. - A reagent for the determination of cholesterol in LDL, which contains a reagent that inhibits the reaction of lipoproteins other than HDL and a reagent that enables the reaction of cholesterol in LDL.
17. 17.- A reagent for the determination of cholesterol in LDL, which is a set composed of a reagent that inhibits the reaction of lipoproteins other than HDL and a reagent that enables the reaction of cholesterol in LDL.
18. 18. The reagent according to any of claims 16 or 17, wherein the reagent that inhibits the reaction of lipoproteins other than HDL is a combination of a divalent metal salt, and heparin or a salt thereof, phosphotungstinic acid or a salt thereof, dextran sulfuric acid or a salt thereof, polyethylene glycol, sulfated cyclodextrin or a salt thereof, sulphated oligosaccharide or a salt thereof, or a mixture thereof.
19. 19. The reagent according to any of claims 16 or 17, wherein the reagent that inhibits the reaction of lipoproteins other than HDL is an anti-B-antibody or an anti-AAP-C antibody.
20. 20. The reagent according to any of claims 16-19, wherein the reagent that enables the cholesterol reaction in LDL is a nonionic, cationic or anionic surfactant, cyclodextrin, a chemically modified cholesterol ester-hydrolyzing enzyme or unmodified capable of acting in LDL, a chemically modified or unmodified cholesterol oxidizing enzyme capable of acting exclusively in LDL, or chemically modified or unmodified cholesterol oxidoreductase capable of acting in LDL.
21. 21.- A reagent for the determination of cholesterol in LDL, which contains a reagent that inhibits only the LDL reaction.
22. 22. A reagent for the determination of cholesterol in LDL, which contains a reagent that inhibits only the LDL reaction and a reagent that enables the reaction of cholesterol in LDL.
23. 23.- A reagent for determining cholesterol in LDL, which is a set composed of a reagent that inhibits only the reaction of LDL and a reagent that enables the reaction of cholesterol in LDL.
24. 24. The reagent according to any of claims 21-23, wherein said reagent that inhibits only the LDL reaction is a chemically modified or unmodified cholesterol hydrolyzing enzyme capable of inhibiting only the LDL reaction.
25. 25. The reagent according to claim 24, wherein the modified grouping of said chemically modified cholesterol esterase is a group having polyethylene glycol as the main component, a group having polypropylene glycol as the main component, a group which it has a copolymer of polypropylene glycol and polyethylene glycol, a group having a saccharide in the structure, a sulfopropyl group, or a polyurethane group.
26. 26. The reagent according to any of claims 21-25, wherein said reagent that enables the cholesterol reaction in LDL is a ester-cholesterol hydrolyzing enzyme that enables the reaction of cholesterol in LDL.
27. 27. The reagent according to any of claims 21-25, wherein the reagent that enables the cholesterol reaction in LDL is a surfactant or a chelating agent EXTRACT The present invention relates to a method for the determination of cholesterol in a low density lipoprotein (LDL) in a sample containing LDL, which comprises removing the high density lipoprotein cholesterol in the sample, subjecting the sample to a reaction using the action of an ester-cholesterol hydrolyzing enzyme and the action of a cholesterol oxidizing enzyme or cholesterol oxidoreductase, and determining the amount of hydrogen peroxide or a reduced type coenzyme generated by the reaction.