MXPA99009358A - Method for quantitating cholesterol present in low density lipoproteins - Google Patents

Abstract

A method whereby LDL cholesterol can be conveniently fractionated and quantitated without resort to any complicated centrifuging procedure. This method comprises the first step of eliminating from a specimen high density lipoproteins, very low density lipoproteins and cholesterol present in chylomicrons and the second step of quantitating the cholesterol remaining in the specimen.

Description

METHOD FOR QUANTIFYING CHOLESTEROL IN LOW DENSITY LIPOPROTEINS FIELD OF THE INVENTION The present invention relates to a method for quantifying cholesterol in low density lipoproteins (LDL, the cholesterol in low density lipoproteins will also be referred to herein as "LDL cholesterol." In the present specification, the term "Cholesterol" includes both ester cholesterol and free cholesterol), which is important for the diagnosis of arteriosclerosis.

BACKGROUND OF THE INVENTION LDL plays an important role in the transport of cholesterol in the blood and the majority of the cholesterol deposited in the walls of the blood vessels in arteriosclerosis pultácea originates from the LDL. The increase in the amount of LDL in the plasma is one of the main risk factors in the pultácea sclerosis as an ischemic heart disease, so the separate quantification of LDL cholesterol is clinically important. Conventional methods for quantifying LDL cholesterol include a method comprising two steps, i.e., a fractionation operation and another operation to quantify cholesterol and a method in which blood levels are determined.

P1557 / 99MX of total cholesterol, HDL cholesterol and triglycerides, and the amount of LDL cholesterol is determined according to the Friedewald equation. The fractionation operation includes the ultracentrifugation method, the precipitation method, the immunochemical method and the like. In the ultracentrifugation method, the LDL are separated taking advantage of the specific gravity difference by means of an ultracentrifugation and the amount of cholesterol is measured in it. In the precipitation method, the anti-HDL antibody, the polyanion and the divalent cation are added to form an insoluble precipitate and the LDL cholesterol is quantified in the supernatant after centrifugation (WPI Acc No. 85-116848 / 20). In the immunochemical method, the anti-HDL antibody, the anti-VLDL antibody and the anti-CM antibody bind to latex particles and the latex particles are removed by centrifugation or by passing through a filter after agglutination , followed by the quantification of LDL cholesterol (WPI Acc No. 84-301275 / 49). However, these conventional methods are not simple and are very expensive. According to the Friedewald equation, the amount of LDL cholesterol is calculated by subtracting the amount of HDL cholesterol from the total amount of cholesterol and then subtracting 1/5 of the amount of triglycerides again. However, since this P1557 / 99MX method is not influenced by diet and individual difference, this one is not very accurate. Recently, a method for the quantification of LDL cholesterol has been reported, which does not require the fractionation operation (WPI Acc.

No. 83-766269 / 38). However, in this method, the LDL specification is not enough.

SUMMARY OF THE INVENTION The objective of the present invention is to provide a method for quantifying LDL cholesterol, by which LDL cholesterol is quantified separately and simply without requiring the complicated operation of centrifugation. The inventors of the present discovered that the amount of cholesterol in LDL can be quantified by canceling the cholesterol other than cholesterol in the low density lipoproteins in the first step and by measuring the remaining cholesterol in the subsequent second step, so it is complemented the present invention. That is, the present invention provides a method for quantifying cholesterol in low density lipoproteins in a test sample that may contain low density lipoproteins, high density lipoproteins, lipoproteins and / or very low density chylomicron; The method comprises a first step of canceling cholesterol in the P1557 / 99MX high density lipoproteins, very low density lipoproteins and chylomicron in a test sample and a second step to quantify the remaining cholesterol in the test sample. By the present invention, a method is provided for quantifying LDL cholesterol, by which LDL cholesterol is easily and separately quantified without requiring the complicated operation of centrifugation.BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the correlation between the results of the LDL cholesterol measurements in Example 1 and the amount calculated by the Friedewald equation. Figure 2 shows the correlation between the results of the measurement of LDL cholesterol in Example 2 and the amount calculated by the Friedewald equation. Figure 3 shows the correlation between the concentration of LDL cholesterol in the absence or presence of HDL, VLDL and CM and the absorbance measured by the present invention in Example 4.

BEST MODE FOR CARRYING OUT THE INVENTION The cholesterol content of lipoproteins includes an ester-type cholesterol (ester of cholesterol ester) and free cholesterol. In this specification, the P1557 / 99MX term "cholesterol" includes both unless otherwise specified. The test sample subjected to the method of the present invention can be any sample containing lipoproteins such as HDL; LDL, VLDL and CM. Examples of the test samples include body fluids such as blood, serum and plasma as well as dilutions thereof, although the test samples are not restricted thereto. The method of the present invention comprises a first step and a second step. In the first step, cholesterol is canceled in HDL, VLDL and CM in the test sample and in the second step, the remaining cholesterol in the test sample is quantified. Because the cholesterol in HDL, VLDL and CM is canceled in the first step, the cholesterol quantified in the second step is mainly the LDL cholesterol in the test sample. The term "cancel" in the first step, in the sense in which it is used herein, means to decompose the cholesterol and render the decomposed products undetectable in the subsequent second step. Methods for selectively terminating cholesterol in lipoproteins other than LDL, ie in HDL, VLDL, CM and the like include the following methods. That is, cholesterol esterase and cholesterol oxidase are made to act on the P1557 / 99MX test sample in the presence of a surfactant that acts on lipoproteins other than low density lipoproteins and cancels the generated hydrogen peroxide. Methods for canceling hydrogen peroxide include a method in which hydrogen peroxide is broken down into water and oxygen by catalysis; and a method in which a phenol-based or an aniline-based hydrogen donor compound is reacted with hydrogen peroxide to convert hydrogen peroxide to a colorless quinone, although methods for removing hydrogen peroxide do not they are limited to these methods. The concentration of cholesterol esterase in the reaction mixture in the first step can preferably be about 0.2 to 1.0 U / ml and the cholesterol esterase can be originated, preferably, from a bacterium belonging to the genus Pseudomonas. The concentration of the cholesterol oxidase may preferably be from about 0.1 to 0.7 U / ml and the cholesterol oxidase may be originated, preferably, from a bacterium or yeast. The catalase concentration may preferably be from about 40 to 100 U / ml and the concentration of peroxidase by which the hydrogen peroxide is converted to a colorless quinone may preferably be from about 0.4 to 1.0 U / ml. The concentration of the hydrogen donor compound with P1557 / 99MX phenol base or aniline base may preferably be from about 0.4 to 0.8 mmol / 1. Preferred surfactants that act on lipoproteins other than LDL, which are used in the first step, include polyalkylene oxide derivatives with HLB values of not less than 13 and not greater than 15, preferably not less than 13 and not greater than 15. 14. Examples of these derivatives include condensation products with higher alcohols, condensation products with higher fatty acids, condensation products with higher fatty acid amides, condensation products with higher alkylamines, condensation products with higher alkylmercaptan and condensation products with alkylphenols. The method for calculating the HLB of surfactants is well known, and is described, for example, in Hiroshi HORIUCHI, "New Surfactants", 1986, Sa yo, Shuppan. Preferred specific examples of polyalkylene oxide derivatives with HLB values of not less than 13 and not greater than 15, include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene octylphenyl ether, nonylphenyl ether polyoxyethylene and the like, of which the HLB value is not less than 13 and not more than 15, although the surfactant is not limited to them. As the surfactant used in the first step, P1557 / 99MX can also be used a cationic surfactant. In this case, as the cationic surfactant, those with quaternary ammonium salt as a hydrophilic group, represented by the following formula (I) are preferred: wherein R independently represents the C? -C8 linear alkyl group and R1 represents the C3-C or C3 alkenyl group. The concentration of the aforementioned surfactants used in the first step may preferably be from about 0.1 to 10 g / l, more preferably from about 0.5 to 5.0 g / l. The reaction in the first step can be carried out, preferably in a regulator with a pH of 5 to 8 and the regulator can preferably be one containing an amine such as, for example, Tris, triethanolamine or Good buffer. Especially, Bis-Tris, PIPES, MOPSO, BES, HEPES and POPSO which are Good's regulators are preferred. The concentration of the regulator can preferably be from about 10 to 500 mM. To inhibit the LDL reaction and to increase the degree of cancellation of the other lipoproteins, a divalent metal ion may be P1557 / 99MX contained in the reaction mixture. Preferred examples of the divalent metal ion include copper ion, iron ion and magnesium ion. Among these, the magnesium ion is especially preferred. The divalent metal ion concentration may preferably be from about 5 to 200 mM. Optionally, a lipoproteinase can be added to the reaction mixture during the first step. The addition of this enzyme is preferred because, especially the cholesterol in VLDL reacts easily. The concentration of this enzyme in the reaction mixture can be preferably from about 5.0 to 10.0 U / ml. Preferably, the reaction temperature in the first step can be from about 25 ° C to 40 ° C, 37 ° C is the most preferred temperature. The reaction time may be from about 2 to 10 minutes. In the second subsequent step, the remaining cholesterol in the test sample is quantified. This can be carried out, for example, by adding a surfactant that acts on at least LDL and quantifying hydrogen peroxide by the action of cholesterol esterase and cholesterol oxidase added in the first step. At this point, the surfactant that acts on at least LDL may be a surfactant that selectively acts only on LDL or may be a surfactant that acts on all P1557 / 99MX lipoproteins. Preferred examples of the surfactant acting on all lipoproteins include polyalkylene oxide derivatives with HLB values of not less than 11 and not greater than 13, preferably not less than 12 and not more than 13. Examples of the derivatives include products condensation with higher alcohols, condensation products with high fatty acids, condensation products with high fatty acid amides, condensation products with high alkylamines, condensation products with high alkylmercaptan and condensation products with alkylphenols. Preferred specific examples of polyalkylene oxide derivatives with HLB values of not less than 11 and not greater than 13 include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenyl ether and the like, of which the HLB value is not less than 11 and not greater than 13, although the surfactant is not limited to these. Examples of surfactants that selectively act only in LDL include anionic surfactants. Although the cationic surfactants used herein are not limited, those with one or more aromatic rings to which one or more linear or branched C -C18 alkyl groups are attached are preferred. Here, the aromatic ring can P1557 / 99MX is, preferably, that which consists of carbon atoms and hydrogen atoms, such as benzene, naphthalene and diphenyl. Also preferred are those in which one or more hydrophilic groups such as for example sulfonate groups are attached to the aforementioned aromatic ring. Preferred examples of these anionic surfactants include those represented by the following formulas (II) to (VI): P1557 / 99MX In formulas (II) to (VI), R independently represents a linear or branched alkyl group of C -C 8. Preferred examples of anionic surfactants that can be used in the second step also include sodium sulfate of higher alcohol and the like. The concentration of the surfactant used in the second step may preferably be from about 0.1 to 100 g / 1, more preferably from 1 to 50 g / 1. Other preferred reaction conditions in the second step are the same preferred reaction conditions for the first step. The present invention will now be described more specifically by way of examples thereof. However, it should be noted that the present invention is not limited to the following examples.

Example 1 First reagents BES regulator, pH 6.0 100 mmol / l HDAOS: N-2 (2-hydroxysulfopropyl) -3,5-dimethioxianiline 0.7 mmol / l Cholesterol Esterase from a bacterium belonging to the genus Pseudonomas (registered trademark "CEN" available commercially from Asahi Chemical Industry Co., Ltd.) 0.8 U / ml P1557 / 99MX Cholesterol oxidase originated from a bacterium belonging to the genus Streptomyces (registered trademark "C00" available commercially from Toyobo Co. Ltd.) 0.5 U / ml Catalase 80 U / ml Magnesium chloride 10 mmol / l Emulgen B66 available commercially from KAO CORPORATION (derived from polyoxyethylene (HLB = 13.2)) 0.2% Seconds Reagents Regulator BES, pH 7.0 50 mmo1 / 1 4-aminoantipyrine 4.0 mmol / l Peroxidase 2.4 U / ml Sodium Azide 0.1% Emulgen A60 commercially available from KAO CORPORATION (derived from polyoxyethylene (HLB = 12.8)) 5.0% To each of the 4 samples with a volume of 4 μl containing HDL, LDL, VLDL and CM purified at a concentration of 100 mg / dl in terms of cholesterol, respectively, 300 μl of the first reagents described above, previously heated to 37 ° C, were added and each of the resulting mixtures were allowed to react at 37 ° C for 5 minutes. Then, 100 μl of the second reagents were added to each mixture and each of the mixtures P1557 / 99MX were allowed to react for 5 minutes, followed by the absorbance measurement of each reaction mixture at 600 nm. Based on the measured absorbances, the cholesterol amounts were calculated and the proportion of the quantities thus calculated was calculated with the amount of cholesterol in the sample, which is defined as the capture rate. The results are shown in Table 1 below.

Table 1 Capture Rate CM VLDL LDL HDL < 1.0% < 5.0% 70.0% < 1.0% As shown in Table 2, by the method described above, the majority of the LDL cholesterol was captured while the cholesterol in other lipoproteins was practically not captured, so the LDL cholesterol can be quantified selectively.

Example 2 First Reagents Regulator PIPES, pH 7.0 50 mmo1 / 1 HDAOS 0.7 mmol / l Cholesterol esterase from a bacterium belonging to the genus Pseudonomas (trademark "CEN" P1557 / 99MX available commercially from Asahi Chemical Industry Co. Ltd.) 0.8 U / ml Cholesterol oxidase originating from a bacterium belonging to the genus Streptomyces (trademark "C00" available commercially from Toyobo Co. Ltd.) 0.5 U / ml Catalase 80 U / ml Magnesium chloride 10 mmol / 1 Emulgen B66 commercially available from KAO CORPORATION 0.2% Seconds Reagents Regulator PIPES, pH 7.0 50 mmo1 / 1 4-aminoantiprine 4.0 mmo1 / 1 Peroxidase 2.4 U / ml Sodium Azide 0.1% Triton X100 3.0% The same procedures as in Example 1 were repeated and the reactivity was measured with each lipoprotein. The results are shown in Table 2 below: Table 2 Capture Rate CM VLDL LDL HDL < 1.0% < 5.0% 71.0% < 1.0% P1557 / 99MX Example 3 Using serum from normal persons as test samples, the procedures in Examples 1 or 2 were repeated to measure LDL cholesterol concentrations. As controls, LDL cholesterol concentrations in the serum were measured using the Friedewald equation (CLIN, CHEM., 41, 1414, 1995). The results are shown in Figures 1 and 2 and indicate the correlation between them. As shown in Figures 1 and 2, the results of the measurements by both methods are congruent, so it was proved that the LDL cholesterol can be quantified accurately by the method of the present invention.

Example 4 First Reagents Good Regulator, pH 7.0 50 mmo1 / 1 HDAOS 0.7 mmol / l Cholesterol estearase 0.8 U / ml Cholesterol oxidase 0.5 U / ml Catalase 80 U / ml Cationic surfactant (lauryltrimethylammonium chloride) 0.1% Seconds Reagents 4-aminoantipyrine 4.0 mmol / 1 Peroxidase 2.4 U / ml Sodium Azide 0.1% P1557 / 99MX Nonionic Surfactant (Polyoxyethylene Lauryl Ether) 0.1% (The non-ionic surfactant was used in the second reaction) Twenty microliters of a sample were mixed with 180 μl of the first reagents, previously heated to 37 ° C and the resulting mixture it was allowed to react at 37 ° C for 5 minutes. Then, 60 μl of the second reagents were added and the resulting mixture was allowed to react for 5 minutes, followed by measurement of the absorbance at 600 nm. Figure 3 shows the relationship between LDL cholesterol concentration and absorbance. As can be seen from Figure 3, LDL cholesterol can be measured in a specific way and even depending on the concentration, with the presence of HDL, VLDL and CM.

Example 5 Using serum as test samples, the same procedures as in Example 4 were repeated to determine the LDL cholesterol concentration. As controls, LDL cholesterol concentrations in serum were measured using the Friedewald equation (CLIN, CHEM., 41, 1414, 1995). The results are shown in Table 3. As shown in Table 3, the results obtained by the method of the present invention are consistent with the P1557 / 99MX results calculated according to the Friedewald equation. Table 3 P1557 / 99MX

Claims (14)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property. 1. A method for quantifying low density lipoprotein cholesterol in a test sample that may contain low density lipoproteins, high density lipoproteins, lipoproteins and / or very low density lipoprotein, the method comprises a first step of canceling cholesterol in high density lipoproteins, very low density lipoproteins and chylomicron in a test sample, and a second step of quantifying the remaining cholesterol in the test sample. The method according to claim 1, wherein the first step is carried out by the actions of cholesterol esterase and cholesterol oxidase in the presence of a surfactant acting on lipoproteins other than low density lipoproteins and cancel hydrogen peroxide generated. 3. The method according to claim 2, wherein the second step comprises adding a surfactant that acts on at least the low density lipoproteins and quantifying the hydrogen peroxide generated by the actions of cholesterol esterase and cholesterol oxidase. P1557 / 99MX 4. The method according to claim 3, wherein the surfactant acts on at least low density lipoproteins in a surfactant that acts on all lipoproteins. The method according to any of claims 1 to 4, wherein the surfactant acting on lipoproteins other than low density lipoproteins, which is used in the first step, is a polyalkylene oxide derivative with a value HLB not less than 13 and not more than 15. 6. The method according to claim 4, wherein the surfactant acting on all the lipoproteins used in the second step is a polyalkylene oxide derivative with a HLB value not lower to 11 and not greater than 13. The method according to claim 2, wherein the surfactant acting on lipoproteins other than low density lipoproteins, used in the first step, is a cationic surfactant. 8. The method according to claim 7, wherein the cationic surfactant has a quaternary ammonium salt. The method according to claim 3, wherein the surfactant acting on at least the low density lipoproteins, used in the second step, is an anionic surfactant. 10. The method of compliance with any P1557 / 99MX of claims 2 to 9, wherein the first step is carried out at a surfactant concentration of 0.1 to 10 g / 1. The method according to any of claims 6 or 9, wherein the polyoxyalkylene derivative having an HLB value of not less than 11 and not less than 13 or the anionic surfactant used in the second step has a concentration of 1 to 100 g / 1. The method according to any of claims 1 to 11, wherein the first and second steps are carried out in a regulator with a pH of 5 to 8. The method according to claim 12, wherein the regulator contains an amine. The method according to any of claims 1 to 13, wherein the first and second steps are carried out at a temperature of 25 to 40 ° C. P1557 / 99MX