MXPA96005693A - Method for the determination of low-density cholesterol in a low-density lipoprotein or a very low-densi lipoprotein enuna - Google Patents

Abstract

The present invention relates to a method for the determination of cholesterol in a sample of low density lipoprotein (LDL) or very low density lipoprotein (VLDL), which comprises the determination of the amount of cholesterol in LDL or VLDL in the sample in the presence of a sugar compound and / or a protein solubilizing agent

Description

TECHNICAL FIELD The present invention relates to a method for the determination of low-density lipoprotein (LDL) cholesterol or to a low-density lipoprotein (LDL) cholesterol determination method in a low-density lipoprotein (LDL) cholesterol or a low-density lipoprotein. very low density lipoprotein (VLDL) (hereinafter referred to as LDL cholesterol or VLDL soterol), which is important in relation to lipid metabolism in the field of clinical diagnosis. BACKGROUND OF THE INVENTION LDL is considered to have a function to provide cholesterol to peripheral cells and to be a direct factor in the occurrence of various types of arteriosclerosis, such as coronary arteriosclerosis. It is known that the level of LDL in the blood is useful as an indicator of arteriosclerosis. The relationship between VLDL, which is rich in triglycerides (TG) and arteriosclerosis, is also observed. Currently, the determination of LDL cholesterol is carried out by the ultracentrifugation method, the electrophoretic method, the conversion method, etc., and the determination of the VLDL cholesterol is carried out by the ultracentrifugation method, the electrophoretic method, etc. . In the ultracentrifugation method, which is used as a basic method, the LDL or VLDL is separated by the difference in a specific gravity using an ultracentrifuge for separation and the amount of cholesterol in it is determined (Adv. Lipid, Res. , 6, 1 1968). However, this method is imperfect in accuracy, simplicity, economic efficiency, etc. In the electrophoretic method, the LDL or VLDL is separated by using an agarose gel or a cellulose acetate membrane as support, and the amount of cholesterol in it is enzymatically determined (Clinical Tests (Rinsho Kensa) 29, 1344, 1985). This method is imperfect in simplicity, in economic efficiency. Etc. In the conversion method, the amount of LDL cholesterol is calculated according to the following equation (Clin.Chem., 18, 499, 1972). (Amount of LDL cholesterol) = (Amount of total cholesterol) - (Amount of HDL cholesterol) - (Amount of triglycerides) / 5 However, the use of this method is restricted by the content of TG in the serum, the type of hyperlipaemia, etc. , so this method is imperfect in simplicity, accuracy, applicability in the analysis of a large number of samples, etc. As described above, the methods for the determination of LDL cholesterol or VLDL cholesterol are not suitable for the analysis of a large number of samples, and a rapid analysis, and an analysis with an automatic analyzer that is widely used in the field of the clinical tests. In addition, in these methods, manual errors may occur, for example, when the amount of the separated LDL fraction is determined using a measuring pipette. However, in case the blood serum sample is added directly to a reagent containing cholesterol esterase and cholesterol oxidase without the LDL or VLDL fraction, the resulting test system is not different from a system for the determination of Total cholesterol, LDL cholesterol or VLDL cholesterol can not be determined specifically. Disclosure of the Invention The present invention has determined the amount of cholesterol in the high density lipoprotein (HDL), LDL, VLDL and chylomicron (CM), each of which has been fractionated through ultracentrifugation, using a reagent for the determination of the cholesterol containing a sugar compound and / or a solubilizing agent of the protein, and it was found that these lipoproteins differ in reactivity relative to the reagent based on the combinations of the sugar compound and / or the solubilization agent of the protein, which leads to the difference in cholesterol reactivity in HDL, LDL cholesterol, VLDL cholesterol, and cholesterol in CM. This finding has led to the termination of the present invention. The present invention relates to a method for the determination of LDL cholesterol or VLDL cholesterol in a sample, which comprises determining the amount of LDL cholesterol or VLDl cholesterol in the sample in the presence of a sugar compound and / or a solubilizing agent of the protein. In this method, a bivalent metal salt can be added to the determination system in order to improve its specific character. The present invention also provides a reagent for the determination of cholesterol in LDL or VLDL, which contains a sugar compound and / or a protein solubilizing agent.; and a reagent for the determination of cholesterol in LDL or VLDL, which is a team composed of a sugar compound and a solubilizing agent of the protein. As the sugar compound, it is preferred to use them derived from glucose. Examples of the glucose derivatives include the compounds represented by the general formula (I): wherein R1, R2 and R3 independently represent hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkanoyl, SO3M2 (wherein M2 is hydrogen or a metal), - (glucosyl) pH (where p is 1 or 2), or - (maltosil) qH (in which q is 1 or 2); R4 and R5 independently represent hydrogen, a metal or S03M3 (wherein M3 is hydrogen or a metal); and m is an integer of 6 to 8 / and the compounds represented by the general formula (II): wherein Rβ, R7 and R8 independently represent hydrogen or SO3M4 (wherein M4 is hydrogen or a metal); R9 represents hydrogen, OM5 (in which M5 is hydrogen or a metal), or 0S03Ms (in which Mβ is hydrogen or a metal); R10 represents hydrogen, a metal, or S03M7 (wherein M7 is hydrogen or a metal); and n is an integer from 4 to 8000. Preferable examples of the protein solubilizing agents are compounds represented by the general formula (III): Rll (C2H4?) a- (C3H60) bR12 (III) wherein each a and b represent a whole from 0 to 200; R11 represents R20-X-0 (wherein R20 is an alkyl or alkenyl, and X is a single bond or CO) or H- (CH2CH20) cN (R21) - (wherein c is an integer from 1 to 200 , and R21 is alkyl or alkenyl); and R-12 represents C2H4COOR, C3HßCOOR, 24 2 H4CH (COOR-,) 2, or C2H4CH (COOR "6 6 * C2) (COOR") (wherein R, R, R24, R25 and R26 independently represent hydrogen, a metal, alkyl or alkenyl), provided that at least one of a and b is 0, and the two elements can be located in the random compounds represented by the general formula (IV): wherein R 13, R 14, R 15, R 16, R 17 and R 1 independently represent alkanoyl; and the compounds represented by the general formula (V): R19-Y-S03Mx (V) wherein R19 represents an alkyl, alkenyl or a substituted or unsubstituted aryl; And it represents a simple link; -0-. -CH (R27) - (wherein R27 is alkyl or alkenyl), -CH2CH (OH) (CH2) d- (wherein d is an integer from 1 to 22), -CH = CH (CH2) ß ( wherein e is an integer from 1 to 22), -OCOCH (CH2COOR2ß) - (wherein R28 is alkyl or alkenyl) or a mixture thereof; and M1 represents hydrogen or a metal. The compounds represented by the formula (I) to (V) in the present are respectively referred to below as Compounds (I) to (V). In the definitions of the groups in formulas (I) to (V), the alkyl and the alkyl portion of the alkanoyl mean a straight chain or branched chain group having from 1 to 22 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, ses-butyl, tere-butyl, pentyl isopentyl, neopentyl, hexyl, heptyl, decyl, pentadecyl, icosanyl and docosanyl. Alkenyl means a straight or branched chain alkenyl group having 2 to 22 carbon atoms, such as vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl decenyl, pentadecenyl, icosenyl and docosenyl. Aryl means phenyl or naphthyl. The metal includes lithium, sodium and potassium. Examples of the substituents of the substituted alkyl and the substituted alkanoyl are hydroxy, carboxy and sulfo. An example of the substituent of the substituted aryl is alkyl, and the alkyl has the same meaning as defined above. As the sugar compound, cyclodextrin derivatives are preferred between Compounds (I) and (II), and methylated cyclodextrin is especially preferred. Examples of the preferred compounds are α-cyclodextrin, β-cyclodextrin, β-cyclodextrin, dimethyl-β-cyclodextrin, trimethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β- cyclodextrin, carboxymethyl-β-cyclodextrin, glucosyl-β-cyclodextrin, maltosyl-α-cyclodextrin, maltosyl-β-cyclodextrin, partially methyl-β-siclodextrin, α-cyclodextrin sulfate and β-cyclodextrin sulfate. As the solubilizing agent of the protein, nonionic and anionic surfactants are especially preferred among surfactants such as Compounds (III), (IV) and (V). Examples of the nonionic surfactants are polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene sterile ether, polyoxyethylene oleyl ether, polyoxyethylene behenyl ether, polyoxyethylene monolaurate, polyoxyethylene mono-stearate, polyoxyethylene monoolate, polyoxyethylene-laurylamine, polyoxyethylene-sterilelamine, and sucrose fatty acid ester. Examples of the nonionic surfactants are sodium dodecylbenzenesulfonate, sodium n-dodesylbenzenesulfonate, lauryl sodium sulfate, and a sodium salt of a high alcohol content sulfuric acid ester. As the bivalent metal salt, 0.01-20 mM of a magnesium salt, a calcium salt, a manganese salt, a nickel salt or a cobalt salt is used. Preferably 0.01-20 mM of a magnesium salt is used. The present invention is characterized by the presence of the sugar compound and / or the solubilizing agent of the protein in the reagent system for the determination of cholesterol. The system for the determination of cholesterol, follows a general method based on the principle of the following reaction, provided that chromogen and the wavelength of measurement is not limited to those shown below. Ester cholesterol type * H2? cholesterol ester cholesterol hydrolyzing enzyme free cholesterol + fatty acid cholesterol oxidation enzyme free cholesterol +? 2 - cholestenone + H2? 2H2? 2 + 4-aminoantipyrine + EMSE + H3 +? peroxidase? ._- px.g en-t-o d-, e q? inona + SH2? (^ M-x = 555 nm) EMSE: cholesterol type N-ethyl-N- (3-methyl-eneyl) -N '-s-cinylethylenediamine ester * H2? cholesterol-free cholesterol ester hydrolyzing enzyme ^ cholesterol-free fatty acid + NAD (P) + cholestenone cholesterol dehydrogenase + NAD (P) H + H + (? aax = 340 nm) Like the chromogen, can be used combinations of 4-aminoanti-irin and a Trinder reagent (Dojin Kagaky Kenkyusho General Catalog, 19th of., 1994), as well as the generally used combinations of 4-aminoantipyrine and phenols such as phenol, 4-chlorophenol, m -cresol and 3-hydroxy-2, 6-triiodobenzoic acid (HTIB). Examples of Trinder reagents are anilines such as N-sulfopropylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-toluidine (TOOS), N-ethyl-N- (2-hydroxy) -3-sulfopropyl) -dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-m-toluidine (TOPS) , 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-sulfopropylaminine, 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' -acetylethylenediamine . as the high sensitivity chromogen, the 10- (N-methylcabamoyl) -3,7-bis (dimethylamino) phenothiadine (MCDP) disclosed in the Japanese Patent Application examined and published No. 33479/85, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophene-niljamine (BCMA) disclosed in Japanese Patent Application Examined and Published No. 27839/92, the chromogens disclosed herein can be used in the Japanese Patent Application examined and published 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 cholesterol ester hydrolyzing enzyme, commercially available enzymes such as the cholesterol oxidation enzyme and cholesterol dehydrogenase can be used. For example, cholesterol esterase and lipoprotein lipase derived from microorganisms or animals that have the ability to hydrolyze the cholesterol ester, the cholesterol oxidase derived from microorganisms that have the ability to oxidize cholesterol to form the cholesterol esterase, can be used. hydrogen peroxide, and cholesterol dehydrogenase derived from microorganisms. In order to improve the specific character and stability of these enzymes, they can be chemically modified with a group having polyethylene glycol as the main component, a water-soluble oligosaccharide residue or a sulfopropyl group. In addition, enzymes obtained by introducing genes of the enzymes mentioned above into other microorganisms and their subsequent expression, optionally followed by chemical modification, and enzymes obtained by the modification of genes of the aforementioned enzymes can also be used. above and their subsequent expression thereof, optionally followed by the chemical modification Examples of the reagent to modify the enzymes (chemical modifier) are the compounds where the polyethylene glycol is connected and a group that can be linked to an amino group [by example, Sunbright WEM 101 (from NOF Corporation), where the polyethylene glycol is connected and a group that can be linked to an amino group, such as N-hydroxysuccinimido], Sunbright series AKM, ADM series, and ACM series [NOF Corporation: Chemical Engineering Monographs (Kagaku Kogaku Ronbunshu), 20 (3), 459, (1994)], which are compounds that have the structure of polyalkylene glycol and the structure of an acid anhydride, the compounds where a polyethylene glycol copolymer and the polypropylene glycol are connected and a group that can be linked to an amino group, the polyethylene glycol monomethacryl monomethylether and maleic anhydride copolymers, etc. In addition, activated P4000 polyurethane (Boehringer Mannheim, Addresses for the Enzyme Modification Set) which is a chemical modifier for polyurethane, Dextran T40, activated TCT (the same as above), which is a chemical modifier for dextran, 1,3-propanesultone. A method for the reaction of an enzyme with a chemical modifier is described below. However, it will be noted that the reaction is not limited to this method. First, the enzyme is dissolved in a stabilizer such as a phosphate stabilizer with a pH of 8 or more, for example, Sunbright (0.01-500 times the molar amount of the enzyme), then added to the solution at a temperature of 0-50 ° C, followed by stirring for 5 minutes at 24 hours. The resulting reaction mixture is used as is, or is used after removing the compounds with low molecular weight by utfiltration, if necessary. The cholesterol ester hydrolyzing enzyme, the cholesterol oxidation enzyme and the cholesterol dehydrogenase are advantageously used in concentrations of 0.1-100 u / ml. The method of the present invention can be applied to body fluid samples containing LDL or VLDL such as blood and urine. Representative procedures for the determination according to the present invention are described below. PROCEDURE 1 To carry out the method of the present invention, first a solution of the sugar compound and / or a solution of the solubilizing agent of the protein is prepared by dissolving the sugar compound in a suitable stabilizer, for example 50 mM Tris HCl Stabilizer (pH 7.4), so that the concentration of the sugar compound, for example, becomes 100 mM or less, preferably between 3 to 80 mM at the time of reaction. The sugar compound can be added initially to the reagent for cholesterol determination. The solution of the protein solubilizing agent is prepared by dissolving the solubilizing agent of the protein in a suitable stabilizer, for example, 50 mM Tris-stabilizer of HCl (pH 7.4) and is added to the reagent for cholesterol determination, so that the concentration of the solubilizing agent of the protein, for example, becomes 50 g / 1 or less, preferably 0.1 to 20 g / 1 at the time of the reaction. The reagent of the present invention is prepared from the solution of the sugar compound and / or the solution of the solubilization agent of the protein containing the reagent for the determination of cholesterol (when the solution of the solubilizing agent of the protein is not used , the sugar compound is initially added to the reagent for the determination of the cholesterol), and is maintained at a temperature of 20 to 50 ° C, preferably 30 to 40 ° C for about 5 minutes. Subsequently, the sample as such or the sample that has been diluted with water or a physiological saline solution is added to the reagent mentioned above, and the reaction is carried out for 5 to 30 minutes. After finishing the reaction, the absorbance of the reaction mixture is measured at 340 to 900 nm, for example at 555 nm in the case of the measurement of a single wavelength, and at 600 nm (main wavelength) and 700 nm (underlying wavelength) in the case of the measurement in two wavelengths, to calculate the amount of cholesterol (in the case of measurement in two wavelengths) the amount of cholesterol is calculated from the difference between the absorbencies in two wavelengths). The amount of cholesterol in each HDL, LDL, VLDL, and CM fractions were determined using the reagent described above. As a result, it was confirmed that HDL cholesterol, LDL cholesterol, VLDL cholesterol and CM cholesterol differ in reactivity with the reagent in case in the combinations of the sugar compound and the solubilizing agent of the protein, and that these lipoproteins differ in reactivity with the reagent based on the combinations of the sugar compound and the solubilizing agent of the protein. Table 1 shows the difference between lipoproteins in reactivity with a cholesterol (unmodified) cholesterol reagent containing a combination of 5 mM of the sugar compound and 5 g / 1 of polyoxyethylene monolaurate, which is an agent of solubilization of a protein. TABLE 1 -. +, ++, and +++ indicate the degree of reaction the order of the reactivity is - < + < ++ < +++ Table 2 shows the difference between lipoproteins in the reactivity of the reagent for the determination of cholesterol (unmodified) containing a combination of 5 mM of trimethyl-β-siclodextrin, which is a sugar compound, and 5 g / 1 of the solubilizing agent of the protein. TABLE 2 Solubilization Agent of LDL HDL Protein VLDL CM Polyoxyethylene lauryl ether - +++ +++ +++ Polyoxyethylene cetyl ether + +++ + Polyoxyethylene stearyl ether + +++ + Polyoxyethylene oleyl ether + +++ + Polyoxyethylene behenyl ether - »- +++ Polyoxyethylene monolaurate - +++ + + Polyoxyethylene monostearate - +++ Polyoxyethylene monooleate - +++ .. + Polyoxyethylene laurylamine ----- +++ Polyoxyethylene stearylamine - +++ Sucrose fatty acid ester + ++ ++ + Sodium dodecylbenzenesulfonate "~ +++ + Sodium N-dodecylbenzenesulfonate ~ ++ ++ ++ Sodium lauryl sulfate - ++ ++ ++ Sodium salt of sulfuric acid ester with high alcohol + ++ -. +, ++, and +++ indicate the degree of reaction the order of the reactivity is - < + < ++ < +++ Procedure 2 A solution of the sugar compound was prepared by dissolving the sugar compound in a suitable stabilizer, for example, 50 mM of a Tris-HCl stabilizer (with a pH of 7.4) so that the concentration of the compound of sugar reaches, for example, 100 mM or less, preferably 3 to 80 mM at the time of the reaction. A solution of the protein solubilizing agent was prepared by dissolving the protein stabilizing agent in a suitable stabilizer, for example, 50 mM Tis-HCl stabilizer (with a pH of 7.4) so that the concentration of the agent of protein stabilization becomes, for example 50 g / 1 or less, preferably 0.1 to 20 g / 1 at the time of the reaction. The solution of the sugar compound and / or the solution of the protein stabilizing agent was preheated to a temperature of 20 to 50 ° C, preferably between 30 to 40 ° C, for example, to 37 ° C, it was added the sample as such or the sample that was diluted with water or physiological saline. After the mixture was heated, for example at 37 ° C for 5 minutes, the absorbance of the mixture was measured at 55 nm (El). Subsequently, the reagent for the determination of the cholesterol previously heated at a temperature between 20 to 50 ° C, preferably between 30 to 40 ° C, for example, at 37 ° C, was added to the mixture, followed by stirring. After 5 minutes, the absorbance of the mixture was measured at the same wavelength [E2 (value after adjustment based on concentration)]. The amount of cholesterol was calculated by separately submitting a standard cholesterol solution to a known concentration in the same procedure and comparing the respective values of (E2-El). Certain embodiments of the present invention are illustrated in the following examples. BEST MODE FOR CARRYING OUT THE INVENTION The determination of LDL cholesterol was carried out by the method of the present invention in which the amount of LDL cholesterol was determined directly and for comparison was determined by the electrophoretic method with agarose [Clinical Trials ( Rinsho Kensa), 29, 1344 (1985)]. Composition of reagents in the method of the present invention: First Reagent 5 mM Trimethyl-β-cyclodextrin Polyoxyethylene monolaurate 5 g / 1 EMSE 1.1 mM Tris stabilizer (pH 7.0) 30 mM Second Reagent Cholesterol esterase (unmodified) 1.0 U / ml Cholesterol oxidase (unmodified) 5.0 U / ml 25 U / ml peroxidase 4-aminoantipyrine 2.2 mM Tris stabilizer (pH 7.0) 30 mM In the method of the present invention, 50 μl of a blood serum sample was added to 2.25 ml of the first reagent pre-heated to 37 ° C . The mixture was heated at 37 ° C for 5 minutes, and then the absorbance of the mixture at 55 nm (El) was measured. Subsequently, 0.75 ml of the second reagent preheated to 37 ° C was added to the mixture, followed by stirring. After 5 minutes, the absorbance of the mixture was measured at the time of the wavelength [E2 (value after adjustment based on concentration)]. The amount of LDL cholesterol was calculated by separately submitting a cholesterol solution at a concentration of 200 mg / dl with the same procedure and comparing the respective values of (E2-E1). In the electrophoretic agarose method, after electrophoresis, the cholesterol in the lipoprotein fraction in the support was enzymatically colored, and the amount of LDL cholesterol was determined by densitometry (Cliniscan 2; Helena Institute). The results are shown in Table 3. TABLE 3 Concentration of LDL Cholesterol (mg / dL) Sample Method of the present method Electrophoretic invention 1 62 55 2 85 81 3 77 72 4 148 138 5 122 116 6 156 151 7 150 139 8 133 121 9 133 123 10 140 129 As shown in Table 3, the results obtained by the method of the present invention are closely correlated with the results obtained by the electrophoretic method. EXAMPLE 2 The determination of cholesterol was carried out by the same procedure as in the method of the present invention in Example 1, except to use the combinations of a sugar compound and a solubilizing agent of the protein shown below in the first reagent. The correlation of the results obtained for the 20 serum samples with those obtained by the electrophoretic agarose method was expressed in the terms of the correlation coefficient. Composition of the first reagent: A. Trimethyl-β-cyclodextrin 5 M Polyoxyethylene monolaurate 5 g / 1 EMSE 1.1 mM Tris stabilizer (pH 7.0) 30 mM B. Trimethyl-β-cyclodextrin 5 mM Sodium dodecylbenzenesulfonate 5 g / 1 EMSE 1.1 mM Tris stabilizer (pH 7.0) 30 mM C. Dimetil-β-siclodextrin 5 m Polyoxyethylene monolaurate 5 g / 1 EMSE 1.1 mM Tris stabilizer (pH 7.0) 30 mM D. Dimethyl-β-cyclodextrin 5 mM Sodium dodecylbenzenesulfonate 5 g / 1 EMSE 1.1 mM Tris Stabilizer (pH 7.0) 30 mM In this method, measurements were made when using an automatic analyzer (Hitachi 7070) under the following conditions. Sample: 4 μl First reagent: 300 μl Second reagent: 100 μl Measurement wavelength: Main wavelength: 600 nm Underlying wavelength: 700 nm The results are shown in Table 4. TABLE 4 First Reagent Coefficient of Correlation A 0.9324 B 0.8227 C 0.8523 D 0.7876 As shown in Table 4, the results obtained by the method of the present invention are closely correlated with the results obtained by the electrophoretic method.

Example 3 LDL cholesterol determination was carried out with the same procedure as in Example 2, except that a metal salt was additionally used in the compositions of B and D. The correlation of the results obtained for the 20 serum samples with those obtained by the electrophoretic method of agarose was expressed in terms of the correlation coefficient. Composition of the first reagent: E. Trimetil-β-cyclodextrin 5.mM Sodium dodecylbenzenesulfonate 5 g / 1 Mg chloride hexahydrate 6 mg / ml EMSE 1.1 M Tris stabilizer (pH 7.0) 30 mM F. Dimethyl-β-cyclodextrin 5 mM Sodium dodecylbenzenesulfonate 5 g / 1 Mg chloride hexahydrate 6 mg / ml EMSE 1.1 mM Tris Stabilizer (pH 7.0) 30 rnM The results are shown in Table 5. TABLE 5 First Reagent Correlation Coefficient E 0.9302 F 0.9298 As shown in Table 5, the results obtained by the method of the present invention are closely correlated with the results obtained by the electrophoretic method. Example 4 Determination of VLDL cholesterol was carried out by the method of the present invention, in which the amount of VLDL cholesterol was determined directly and for comparison was determined by the agarose electrophoretic method [Clinical Trial (Rinsho Kensa), 29 , 1344 (1985)] according to the same procedures as in Example 1. Composition of reagents in the method of the present invention: First Reagent 5 mM 2-Hydroxypropyl-β-sislodextrin Polyoxyethylene lauryl ether 5 g / 1 EMSE 1.1 mM Tris Stabilizer (pH 7.0) 30 mM Second Reagent Cholesterol Estearase Modified 1.0 U / ml Modified Cholesterol Oxidase 5.0 U / ml Peroxidase 25 U / ml 4-Aminoantipyrine 2.2 mM Tris Stabilizer (pH 7.0) 30 mM The modification of the enzymes was carried out in the following manner. Cholesterol esterase or cholesterol oxidase was dissolved in 20 mM of a phosphate stabilizer (pH 8) (10 mg / ml), followed by cooling to 5 ° C. To the solution was added Sunbright 4001 (NOF Corporation) (20 times the molar amount of the enzyme) followed by the solution, and the mixture was subjected to the reaction at 5 ° C for 4 hours to modify the enzyme with polyethylene glycol. . The resulting reaction mixture was used as the modified cholesterol esterase or the cholesterol oxidase (molecular weight of the polyethylene glycol portion = 6000). The results are shown in Table 6. TABLE 6 Concentration of LDL Cholesterol (mg / dL) Sample Method of a present Method Rhodian electrofoinvention 1 24 19 2 2 2 299 22 3 17 15 4 19 23 5 12 15 6 25 23 7 46 49 8 44 39 9 33 27 10 31 34 As shown in Table 6, the results obtained by the method of the present invention correlate closely with the results obtained by the electrophoretic method. Industrial Applicability The present invention provides a simple method for the determination of LDL cholesterol or VLDL cholesterol, which does not need complicated fractional and separation steps, and which is applicable to the analysis with the automatic analyzer.

Claims (21)

1. NOVELTY OF THE INVENTION Having described the invention, the contents of the following are considered to be our property: CLAIMS 1. A method for the determination of cholesterol in a low density lipoprotein (LDL) or a very low density lipoprotein (VLDL) , which comprises the determination of the amount of cholesterol in LDL or VLDL in the sample in the presence of a sugar compound and / or the solubilizing agent of the protein.
2. 2. A method for determining LDL cholesterol in a sample, which comprises determining the amount of LDL cholesterol in the sample in the presence of a sugar compound and / or a solubilizing agent of the protein.
3. 3. A method for determining cholesterol in VLDL in a sample, which comprises determining the amount of cholesterol in VLDL in the sample in the presence of a sugar compound and / or a solubilizing agent of the protein.
4. 4. The method according to any of claims 1-3, wherein the sugar compound is a derivative of glucose.
5. 5. The method according to claim 4, wherein the sugar compound is a compound represented by the formula (I) wherein R1, R2 and R3 independently represent hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkanoyl, SO3M2 (wherein M2 is hydrogen or a metal), - (glucosyl) PH (where p is 1 or 2), or - (maltosil) qH (in which q is 1 or 2); R4 and R5 independently represent hydrogen, a metal or SOsM3 (wherein M3 is hydrogen or a metal); and m is an integer of 6 to 8 (hereinafter referred to as Compound (I), the same applies to compounds of other numbers in the formula), or a compound represented by general formula (II): wherein Rβ, R7 and R8 independently represent hydrogen or SO3M4 (wherein M4 is hydrogen or a metal); R9 represents hydrogen, OM5 (in which M5 is hydrogen or a metal), or OSO3M6 (in which Mβ is hydrogen or a metal); R10 represents hydrogen, a metal, or SO3M7 (wherein M7 is hydrogen or a metal); and n is an integer from 4 to 8000.
6. 6. The method according to claim 5, wherein the sugar compound is a cyclodextrin derivative. The method according to any of claims 1-6, wherein the solubilizing agent of the protein is a compound represented by the general formula (III): Rll (C2H40) a- (C3H60) bR12 (III) in where each a and b represent an integer from 0 to 200; R11 represents R20-X-0 (wherein R20 is an alkyl or alkenyl, and X is a single bond or CO) or H- (CH2CH20) CN (R21) - (wherein c is an integer from 1 to 200 , and R21 is alkyl or alkenyl); and R12 represents C2H4COOR22, C3H6COOR23, C2H4CH (COOR24) 2, or C2H4CH (COOR25) (COOR26) (wherein R22, R23, R24, R25 and R26 independently represent hydrogen, a metal, alkyl or alkenyl), provided that at least one of a and b is not 0, and the two elements can be located in the random compounds represented by the general formula (IV): wherein R13, R14, R15, R16, R17 and R18 independently represent alkanoyl; and a compound represented by the general formula (V): R ^ -Y-SOaM1 (V) wherein R19 represents an alkyl, alkenyl or a substituted or unsubstituted aryl; And it represents a simple link; -0-. -CH (R27) - (wherein R27 is alkyl or alkenyl), -CH2CH (OH) (CH2) d- (wherein d is an integer from 1 to 22), -CH = CH (CH2) e ( wherein e is an integer from 1 to 22),, 28 OCOCH (CH2COOR ") - (wherein R is alkyl or alkenyl) or a mixture thereof, and M1 represents hydrogen or a metal. according to claim 7, wherein the solubilizing agent of the protein is a nonionic surfactant or an anionic surfactant 9. The method according to any of claims 1.8, wherein the determination of the amount of cholesterol is carried out. performed in the presence of a bivalent metal salt 10. The method according to any of claims 1-9, which comprises subjecting the sample to a reaction employing the action of a cholesterol ester hydrolyzing enzyme and the action of cholesterol oxidation enzyme or cholesterol dehydrogenase, and determining the amount of hydrogen peroxide or a reduced type coenzyme generated by the reaction, wherein the cholesterol ester hydrolyzing enzyme, the cholesterol oxidation enzyme, or the cholesterol dehydrogenase is chemically modified or unmodified cholesterol esterase, a chemically modified or non-modified cholesterol oxides modify, or chemically modified or unmodified cholesterol dehydrogenase. 11. A reagent for the determination of cholesterol in LDL or VLDL, which contains a sugar compound? / Or a solubilizing agent of the protein. 12. A reagent for the determination of cholesterol in LDL, containing a sugar compound and / or a solubilizing agent of the protein. 13. A reagent for the determination of cholesterol in VLDL, which contains a sugar compound and / or a solubilizing agent of the protein. 14. A reagent for the determination of cholesterol in LDL or VLDL, which is a set composed of a sugar compound and a solubilizing agent of the protein. 15. A reagent for the determination of cholesterol in LDL, which is a set composed of a sugar compound and a solubilizing agent of the protein. 16. A reagent for the determination of cholesterol in VLDL, which is a set composed of a sugar compound and a solubilizing agent of the protein. 1
7. 7. The reagent according to any of claims 11-16, wherein the sugar compound is a glucose derivative. 1
8. 8. The reagent according to claim 17, wherein the sugar compound is Compound (I) or a Compound (II). 1
9. 9. The reagent according to the claim 18, wherein the sugar compound is a derivative of the cyclodextrin. The reagent according to any of claims 11-19 wherein the solubilizing agent of the protein is a Compound (III), Compound (IV) or a Compound (V). 21. The reagent according to the claim 20, wherein the solubilizing agent of the protein is a nonionic surfactant or an anionic surfactant.