MXPA01009548A - Method for quantitating cholesterol - Google Patents

Abstract

A method for quantitating cholesterol wherein cholesterol contained in a lipoprotein fraction to be measured is quantitated in the presence of a compound having a relatively strong affinity for one of lipoproteins contained in a sample, a surfactant acting relatively strongly on the other lipoprotein and a cholesterol reagent;and a quantification reagent to be used in this method.

Description

METHOD FOR QUANTIFYING CHOLESTEROL TECHNICAL FIELD The present invention relates to a method for the quantitative determination of cholesterol that can be fractionated and to quantitatively determine the amount of cholesterol present in the specific fractions of lipoproteins efficiently by a simple procedure using a small amount of sample.

TECHNICAL BACKGROUND Lipids, like cholesterol, are combined with an apoprotein and form a lipoprotein in the blood serum. Lipoproteins are grouped into chylomicron, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) according to their physical properties. Among these lipoproteins, LDL is one of the substances that cause arteriosclerosis, while HDL is known to have an antiarteriosclerosis effect. From the point of view of epidemiology, the cholesterol value in LDL has a positive correlation with the frequency of the occurrence of arteriosclerosis, while the HDL cholesterol value is known to have an inverse correlation with the frequency of the occurrence of the arteriosclerosis. Currently, cholesterol values, in HDL and LDL, are frequently measured in order to prevent and diagnose ischemic heart diseases. As a method of measuring cholesterols in HDL and LDL, a method for measuring cholesterol after separating HDL and LDL among other lipoproteins by ultracentrifugation, for example methods for separating HDL and LDL are known by electrophoresis, lipid staining, and measurement of the intensity of color. However, none of these known methods are used in daily practice due to problems such as complicated procedures, inability to treat a large number of samples, and the like. A method for measuring HDL cholesterol that is widely used in the field of clinical examination is a precipitation method that involves the addition of a precipitating agent to a sample to cause proteins other than HDL to aggregate, removing the aggregate by centrifugation, and measuring the cholesterol in the supernatant fluid containing only HDL. Although this method is simple and easy compared to other methods of precipitation and electrophoretic analysis, the method not only requires a large amount of sample in comparison due to the separation procedure by the addition of a precipitating agent, it also involves a large risk of errors analytical Therefore, it has become possible to fully automate the entire analytical procedure.

A method for the determination of enzymatic fractions of HDL cholesterol has been studied. For example, an enzymatic reaction is known in the presence of a bile salt and a nonionic surfactant (Japanese Laid-open Patent Application No. 126498/1988). This method uses the nature of the enzymatic reaction proceeding in proportion to the concentration of LDL in the cholesterol in the initial stage, but in proportion to the concentration of HDL in the cholesterol at a later stage. However, the analytical accuracy of the method is questionable because it is impossible to completely separate the reaction of cholesterols in HDL and the reaction of cholesterols in other lipoproteins. Another method known in the art comprises previously adding different lipoproteins to HDL, enzymatically reacting only cholesterols in HDL, deactivating the enzyme and, at the same time, re-dissolving the aggregate, and measuring the absorbency (Japanese patent application no. 242110/1994). However, this method can be applied only to limited automatic analyzers, since reagent addition is required at least 3 times. In this way, the generality of the method is limited. The method is also not satisfactory from the point of view of the damage to the analytical instrument and the disposal of the reagents, because the redissolution of the precipitate requires the use of salts in high concentrations, and the like. The Japanese patent of no. 2600065 describes a method for using a precipitation reagent for lipoproteins other than HDL used in a conventional precipitation method in a reagent that is commonly used for the measurement of cholesterols in HDL that have not yet been precipitated. A specific example described is a combination of a modification enzyme and a-cyclodextrin sulfate. The other methods include a method for using a surfactant to reduce the effect of the precipitating agents (Japanese Patent Application Laid-open No. 116996/1996), a method for using an antibody, in addition to the conventional reagent, to precipitate lipoproteins other than HDL. (Japanese open patent application No. 96637/1997), a method for using carrageenan (Japanese open patent application No. 121895/1997), and a method for using a sugar compound (Japanese open patent application No. 301636 / nineteen ninety five). These methods present problems such as the formation of turbidity due to aggregation, even in the case where normal serum is mixed, the requirement of adding lipoproteins other than HDL (LDL, VLDL, etc.) of said method is unnecessary, and the like. A method for measuring LDL cholesterols is widely accepted in the field of chemical testing, the Freedwald method (Clinical Chemistry, vol.18, pages 459-502 (1972)). According to this method, the amount of LDL cholesterol is determined by the use of amounts of total cholesterol, cholesterols in HDL, and neutral fats determined by enzymatic methods. However, this method can not be applied when the concentration of neutral fats is greater than 400 mg / dl.

An objective of the present invention, therefore, is to provide a method for the quantitative determination of cholesterols in specific fractions that does not require a prior treatment such as centrifugation, can be carried out efficiently by a simple procedure, and can be applied to Various types of automatic analyzers.

DESCRIPTION OF THE INVENTION As a result of extensive studies, the inventors of the present invention have discovered that if the reaction of an enzymatic reagent for cholesterol determination is carried out in the presence of a compound having a relatively strong affinity with one of the lipoproteins in a sample and of a surfactant that exhibits a relatively strong action on other lipoproteins in the sample, it is possible to provide an important difference between the reaction of cholesterols present in a specific lipoprotein in the sample and the reaction of cholesterols included in the other lipoproteins, this way cholesterols in the target lipoprotein can be divided and determined with substantially sufficient precision. Specifically, the present invention provides a method for selectively quantifying cholesterols, which comprises determining the amount of cholesterols in a measurement lipoprotein fraction in a sample in the presence of a compound having a relatively strong affinity for lipoproteins. which are not being measured in the sample, a surfactant that exhibits a relatively strong action on the measurement lipoproteins, and a cholesterol determination reagent. The present invention further provides a method for selectively determining the amount of cholesterol, which comprises reacting cholesterols present in lipoproteins that are not being measured in a sample in the presence of a compound having a relatively strong affinity for measuring lipoprotein in the sample, a surfactant that exhibits a relatively strong action on lipoproteins that are not being measured, and a cholesterol determination reagent, and determining the amount of cholesterol in the remaining lipoprotein measurement. The present invention further provides a method for determining the concentration of cholesterols in various lipoproteins, which comprises reacting cholesterols present in a second measurement lipoprotein in a sample in the presence of a compound having a relatively strong affinity with a first lipoprotein. lipoprotein in the sample, a surfactant that shows a relatively stronger action on the second lipoprotein than the first lipoprotein, and a cholesterol determination reagent, which determines the amount of cholesterol in the first remaining lipoprotein, and determine the concentration of cholesterols in each lipoprotein of the resulting amount of cholesterol in the first remaining lipoprotein and the total cholesterol concentration. The present invention also provides a reagent for the quantitative determination of cholesterols comprising, separately or as a mixture, a compound having a relatively strong affinity with one of the lipoproteins in the sample to carry out the above methods, an agent surfactant that exhibits a relatively strong action on the other lipoproteins, and a cholesterol determining reagent.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the correlation between the method of Example 4 and a conventional precipitation method. Figure 2 is a graph showing the correlation between the method of example 5 and a conventional precipitation method. Figure 3 is a graph showing the correlation between the method of Example 6 and a conventional precipitation method. Figure 4 is a graph showing the correlation between the method of Example 7 and a conventional precipitation method.

BEST MODE FOR DEVELOPING THE INVENTION In the present invention, before the reaction between cholesterols in the lipoproteins and a cholesterol determining reagent, a compound having a relatively strong affinity with one of the lipoproteins in the sample (hereinafter referred to as "affinity agent") "selective") and a surfactant that exhibits a relatively strong reaction on the other lipoproteins (hereinafter referred to as "selective activator") should be added. Of these, the selective affinity agent exhibits a mutual action with lipoproteins for which the reaction or determination is not desired, and inhibits or suppresses the reaction between those lipoproteins and a cholesterol determining reagent. On the other hand, the selective activator exhibits a strong action on the lipoproteins that will react or be determined when the lipoproteins that will react or will be determined and the lipoprotein for which the reaction or determination is not desired are present in the same system, and accelerates the reaction between those lipoproteins that will react or be determined and the cholesterol determination reagent. The affinity of the selective affinity agent with a lipoprotein and the action of the selective activator on other lipoproteins in the present invention must be relatively strong, but it is necessary that it is not absolutely strong. The reason for this is that due to relative errors, which can be important when only one of these is used, it can be reduced to a level where there are no problems in actual practice when both are used. Since the selective affinity agent used in the present invention, a compound that exhibits affinity with the lipoprotein surface layer forming components of the lipoproteins for which the reaction or determination is not desired can be provided. Cholesterols, phospholipids and apoproteins and the like can occur as the components that form a lipoprotein surface layer. Examples of said selective affinity agent may be derivatives of saponins, polyenes, cholesterol derivatives, peptides, lectins and phospholipids. As saponins that exhibit affinity with cholesterols, for example, digitonin, tomatin and the like may be given; As a polyene, nystatin, philippine, pimacillin, pentamycin, trichomycin, fungicromine, perimycin, amphotericin, etholuscomycin, primicin, cadigine, and the like; As cholesterol derivatives, [N- [2-cholesterylcarboxylamino) ethyl] carbamoylmethyl] -pulline (abbreviated as "Col-AECM-Pullulan") and the like can be provided; Bacitracin, polymyxin, suzucasilin, gramicidin, and the like may be provided as peptides; as lectin concanavalin A, castin lectin, peanut lectin, and the like may be provided; and L-a-phosphatidyl glycerol dipalmitoyl and the like can be provided as phospholipid derivatives. A reaction mixture may become cloudy when a lipoprotein-containing sample is mixed with any of the aforementioned selective affinity agents, in accordance with conditions such as the reagent composition. This may be due to the production of flocculants in lipoproteins. However, the aggregation of lipoprotein that are not measuring is not indispensable in the present invention. For example, turbidity is not observed when digitonin is mixed (a saponin derivative), Col-AECM-pulunano (a cholesterol derivative), filipin (a polyene compound), La-phosphatidyl glycerol dipalmitoyl (a phospholipid derivative), or the like with a lipoprotein-containing sample under conditions where the effect of the present invention can be exhibited. It is important for the selective affinity agent of the present invention to absorb or bind to the components by forming lipoprotein surface layers so that the reaction between cholesterols in lipoproteins and an enzyme is inhibited or suppressed. It is absolutely unnecessary for lipoproteins to be added. These selective affinity agents can be used, either individually or in combination of two or more. Although not specifically limited, the amount of selective affinity agents used differs in accordance with the compounds in the usual scale from about 1 nM to 0.1 M (1x10-7% to 10%), and preferably from 10 nM to 0.1 M (1x10-6% to 10%). An organic solvent such as alcohol, a surfactant, and a phospholipid can be used to dissolve these compounds. These solvents and the like can be used individually or in combination with two or more. The amount used differs according to the type of compound that will be dissolved, but is not specifically limited. On the other hand, either a selective ionic or non-ionic activator can be used in view of the fact that such a selective activator exhibits an action on the lipoproteins to be reacted or they will be determined to a different degree, wherein the selective activator exhibits an action on the lipoproteins for which the reaction or determination is not desired. As examples, polyoxyethylene octylphenyl ether (10), polyoxyethylene higher alcohol ether, polyoxyethylene alkylene phenyl ether, and polyoxyethylene alkylenetribenzylphenyl ether may be provided. Particularly preferred selective activators are polyoxyethylene alkylenediphenyl ether and polyoxyethylene alkylenetribenzyl ether, which are known as surfactants which exhibit strong reactivity with specific lipoproteins when they react alone with these lipoproteins (Japanese Patent Application Laid-open No. 313200/1997) . As examples of commercially available products of these selective activators, Triton X-100, Emulgen 709, Emulgen A-60, Emulgen B-66, heptanesulfonic acid and octane sulfonic acid may be provided.

These selective activators can be used either individually or in combination of two or more. Although not specifically limited, the amount of selective activators used differs in accordance with the compounds on a scale from 0.0001 to 5%, and preferably from 0.001 to 5%. These selective affinity agents and selective activators can be added to a sample serum, either separately or concurrently as a mixture. Any known enzymatic methods can be used for the determination of cholesterols. Examples include a method of using cholesterol esterase and cholesterol oxidase in combination as enzyme reagents, a method for using cholesterol esterase and cholesterol dehydrogenase in combination, and the like. Among these, a method using cholesterol esterase and cholesterol oxidase in combination is preferred. There are no specific limitations to the final cholesterol detection method after the addition of these cholesterol determination enzyme reagents. For example, an absorbance analysis may additionally be provided using a combination of a peroxidase and a coloring substance, a method for directly detecting coenzymes and acid peroxide, and the like. Specifically preferred embodiments of the present invention are those listed below: (1) A method for selectively determining the amount of cholesterol in a measurement lipoprotein in a sample in the presence of a compound having a relatively high affinity strong with lipoproteins that are not measured in the sample, a surfactant that exhibits a relatively strong action on the lipoproteins of measurement, and a reagent for determining cholesterol. (2) A method for selectively determining the amount of cholesterol comprising preferably reacting cholesterols present in the lipoproteins that are not measured in a sample in the presence of a compound having a relatively strong affinity with the lipoprotein in the sample, a surfactant that exhibits a relatively strong action on the lipoproteins that are not measured, and a reagent for determining cholesterol, and determining the amount of cholesterol in the remaining lipoprotein. (3) A method for determining the concentration of cholesterols in various lipoproteins which preferably comprises reacting cholesterol cholesterols present in a second lipoprotein in a sample in the presence of a compound having a relatively strong affinity with a first lipoprotein in the sample. shows, a surfactant that exhibits a relatively strong action on the second lipoprotein that the first lipoprotein, and a cholesterol determination reagent, which determines the amount of cholesterol in the first remaining lipoprotein, and determine the concentration of cholesterols in each lipoprotein the resulting amount of cholesterol in the first remaining lipoprotein measurement, and the total cholesterol concentration. In performing the above methods, a reagent is conveniently used for the quantitative determination of cholesterols comprising, separately or as a mixture, a selective affinity agent, a selective activator, and a cholesterol determining reagent. The reagents for the quantitative determination of cholesterols used in the above methods are those indicated below: Reagent used in the above method (1): A reagent for the quantitative determination of cholesterols comprising, separately or as a mixture, a compound having a relatively strong affinity with lipoproteins that are not measured in the sample, a surfactant that exhibits a relatively strong action on measuring lipoproteins, and a cholesterol determination reagent.

Reagent used in the above method (2): A reagent for the quantitative determination of cholesterols comprising, separately or as a mixture, a compound having a relatively strong affinity with measuring lipoproteins in the sample. A surfactant that exhibits a relatively strong ratio to lipoproteins that are not measured, and a cholesterol determination reagent.

Reagent used in the above method (3): A reagent for the quantitative determination of cholesterols comprising, separately or as a mixture, a compound having a relatively strong affinity with a first measurement protein in the sample. A surfactant that exhibits a stronger action on a second measurement lipoprotein than on the first measurement lipoprotein, and a cholesterol determination reagent. A pH regulation solution which is commonly used as a phosphate pH regulator, a pH Good regulator and the like can be added to the aforementioned reagents for the quantitative determination of cholesterols. There are no specific limitations to the pH value of a solution where these reagents for quantitative determination dissolve to the extent that the enzyme reagents are not affected. In addition, an inorganic salt such as sodium chloride, additives such as albumin used to stabilize enzymatic activity, a dibasic metal salt, a compound acting as a preservative, and the like may also be used. Cholesterols can be quantitatively determined efficiently by a simple procedure without prior treatment, such as centrifugation, being necessary using the method of the present invention described above. In addition, because the method allows for specific determination by a simple method using a small amount of sample, the method can be applied to several types of automatic analyzers. The method in this way is extremely useful in the field of diagnosis.

EXAMPLES The present invention will be described in greater detail with the following examples, which should not be construed as limiting the present invention.

EXAMPLE 1 Samples containing lipoproteins were prepared according to the following method. The amount of cholesterol in each of the lipoprotein fractions was determined by the following method to compare the reactivity. The results are shown in table 1.

Sample preparation Samples were prepared by fractionating human serum in VLDL, LDL, and HDL by centrifugation.

Measurement method 30 μl of a first reagent, a 50 mM phosphate pH buffer (pH 6.5) containing 0.005% digitonin, was added to 3 μl of the sample.

After 5 minutes, 100 μl of a cholesterol determination reagent (second reagent), which is a 50 mM phosphate buffer (pH 6.5) containing 0.2% Triton X-100, 1 U / ml cholesterol was added. esterase, 1 U / ml of cholesterol oxidase, 5 U / ml of peroxidase, 0.004% of disulfobutyl m-toluidine, and 0.004% of 4-aminoantipyrine. Absorbance at 600 nm and 700 nm was measured in the sample immediately before and 5 minutes after the addition of the cholesterol determination reagent to compare the difference in reactivity between the lipoprotein fractions (two-point method). The mentioned procedure was carried out using a Hitachi 7150 automatic analyzer (manufactured by Hitachi Ltd.).

Results TABLE 1 (amount of absorbance) It can be seen from Table 1 that cholesterols in HDL can preferably be reacted with an enzyme on cholesterols in LDL and cholesterols in VLDL if digitonin is present.

EXAMPLE 2 The amount of cholesterol was determined and compared in the same manner as in Example 1, with the exception that Col-AEMC-Pullulan at 0.005% was used instead of digitonin as a first reagent and the surfactant (Triton X- 100) in the second reagent was replaced with Emulgen B-66 at 1%. The results are shown in table 2.

Results TABLE 2 (amount of absorbance) It can be seen from Table 2 that cholesterols in HDL can be preferably reacted with an enzyme on cholesterols in LDL and cholesterols in VLDL when Col-AECM-Pullulan is present.

EXAMPLE 3 Samples were prepared in the same manner as in the example 1. The amount of cholesterol in each of the lipoprotein fractions was determined by the same method as in Example 1 using reagents of the composition shown below to compare the reactivity. The results are shown in table 3.

USED REAGENTS First reagent: A 50 mM PIPES pH regulator solution (pH 6.5) containing 5 mM L-a-phosphatidylglycerol dipalmitoyl and 0.5% Triton X-100 Cholesterol determination reagent: A 50 mM PIPES pH regulator solution (pH 6.5) containing 1 U / ml of cholesterol esterase 1, 1 U / ml of cholesterol oxidase, 5 U / ml of peroxidase, 0.04% of m- disulfobutyl toluidine, and 0.004% 4-aminoantipyrine.

Results TABLE 3 (amount of absorbance) It can be seen from Table 3 that cholesterols in HDL can preferably be reacted with an enzyme on cholesterols in LDL and cholesterols in VLDL if L-a-phosphatidylglycerol dipalmitoyl is present.

EXAMPLE 4 Cholesterols in HDL were determined with the method of the present invention and the conventional precipitation method using serum samples containing lipoproteins. Specifically, 300 μl of a first reagent, a 50 mM MES pH buffer (pH 6.5) containing 0.005% digitonin, was added to 3 μl of the samples. After 5 minutes, 100 μl of a cholesterol determination reagent (a second reagent), which is a pH buffer of 50 mM phosphate (pH 6.5) containing 1% Emulgen B-66, 1 U / ml was added. of cholesterol esterase, 1 U / ml of cholesterol oxidase, 5 U / ml of peroxidase, 0.04% of disulfobutyl m-toluidine, and 0.004% of 4-aminoantipyrine. The absorbance was measured at 600 nm and 700 nm in the sample immediately before and five minutes after the addition of the cholesterol determination reagent, to determine the HDL cholesterol concentration of the difference in absorbance values (two point method) . A control serum with a known concentration was used as a calibration standard. The above procedure was carried out using a Hitachi 7150 automatic analyzer (manufactured by Hitachi Ltd.). On the other hand, the determination of cholesterol in HDL by the precipitation method (comparative method) was carried out as indicated below. 200 μl of an aqueous solution containing 0.3% sodium phosphotungstate and 2% magnesium chloride was mixed with 200 μl of the sample. The mixture was centrifuged at 3000 rpm for 10 minutes. 50 μl of the supernatant solution was mixed with 3 ml of a cholesterol determination reagent, which is a pH regulation solution MES 100 mM (pH 6.5) containing 1% Triton X-100, 1 U / ml cholesterol esterase, 1 U / ml of cholesterol oxidase, 5 U / ml of peroxidase, 0.04% of disulfobutyl m-toluidine, and 0.004% of 4-aminoantipyrine. The mixture was incubated for 10 minutes at 37 ° C to measure absorbance at 600 nm, based on cholesterols determined in HDL. The results are shown in table 4 and figure 1.

Results TABLE 4 As can be seen from Table 4 and Figure 1, the method of the present invention shows a very good correlation with the conventional precipitation method despite the simple procedure.

EXAMPLE 5 Cholesterols in HDL were determined with the method of the present invention and the conventional precipitation method using serum samples containing lipoproteins. The method of the present invention that was used in Example 4 was followed, except that the digitonin in the first reagent was replaced with 0.1% polymyxin B and 0.005% concanavalin A. The results are shown in table 5 and figure 2.

(Results) TABLE 5 As can be seen from Table 5 and Figure 2, the method of the present invention shows a very good correlation with the conventional precipitation method despite the simple procedure.

EXAMPLE 6 Cholesterols in HDL were determined with the method of the present invention and the conventional precipitation method using serum samples containing lipoproteins. The method of the present invention used in Example 4 was followed, with the exception that the digitonin in the first reagent was replaced with 0.005% filipin (76 μM). the results are shown in table 6 and figure 3.

(Results) TABLE 6 As can be seen from Table 6 and Figure 3, the method of the present invention shows a very good correlation with the conventional precipitation method despite the simple procedure.

EXAMPLE 7 Cholesterols in HDL were determined with the method of the present invention and the conventional precipitation method using serum samples containing lipoproteins. 260 μl of a cholesterol-determining reagent containing a selective affinity agent, consisting of a 50 mM PIPES pH regulator solution (pH 6.5) containing 0.0075% digitonin (60 μNM), 0.25 Emulgen B-66 %, 0.25 U / ml of cholesterol esterase, 0.25 U / ml of cholesterol oxidase, 1.25 U / ml of peroxidase, 0.01% of disulfobutol m-toluidine, and 0.005% of 4-aminoantipyrine, was added to 2 μl of the sample. The absorbance was measured at 600 nm and 700 nm in the sample 2 minutes and 7 minutes after the addition of the selective affinity agent, to determine the cholesterol in HLD from the difference in the absorbance values (two point method) . A control serum with a known concentration was used as a calibration standard. The above procedure was carried out using the Hitachi 7170 automatic analyzer (developed by Hitachi Ltd). The same method as in Example 3 was used for the determination of HDL cholesterol according to the precipitation method (the comparative method). The results are shown in table 7 and figure 4. results TABLE 7 As can be seen from Table 7 and Figure 4, the method of the present invention shows a very good correlation with the conventional precipitation method despite the simple procedure.

Claims (30)

NOVELTY OF THE INVENTION CLAIMS

1. -A method for selectively quantifying cholesterols, which comprises determining the amount of cholesterols in a lipoprotein fraction measured in a sample in the presence of a compound having a relatively strong affinity with non-measuring lipoproteins in the sample , a surfactant that exhibits a relatively strong action on the measurement lipoproteins, and a cholesterol determination reagent.

2. The method according to claim 1, further characterized in that the compound having a relatively strong affinity with non-measuring lipoproteins in the sample has an affinity with a lipoprotein surface layer component of said lipoproteins.

3. The method according to claim 2, further characterized in that the component that forms the lipoprotein surface layer of non-measuring lipoproteins is cholesterol.

4. The method according to claim 2, further characterized in that the component that forms the lipoprotein surface layer of non-measuring lipoproteins is phospholipid.

5. The method according to claim 2, further characterized in that the component that forms the lipoprotein surface layer of non-measuring lipoproteins is apoprotein.

6. The method according to claim 1, further characterized in that the compound having a relatively strong activity with non-measuring lipoproteins in the sample is at least one compound selected from the group consisting of saponins, polyenes, cholesterol derivatives, peptides, lectins and phospholipid derivatives.

7. The method according to claim 1 or 6, further characterized in that the compound having a relatively strong affinity with lipoproteins that are not measurable in the sample is a saponin.

8. The method according to claim 7, further characterized in that the saponin is a spheroid saponin.

9. The method according to claim 7 or 8, further characterized in that the saponin is digitonin or tomatin.

10. The method according to claim 1 or 6, further characterized in that the compound having a relatively strong affinity with non-measuring lipoproteins in the sample is a polyene

11. The method according to claim 10 , further characterized in that the polyene is an antibiotic.

12. The method according to claim 10 or 11, further characterized in that the polyene is selected from the group consisting of nystatin, philipin, pimacillin, pentamycin, tricomycin, fungicromin, perimycin, amphotericin, etoluscomycin, primicin and candigin.

13. The method according to claim 1 or 6, further characterized in that the compound having a relatively strong affinity with non-measuring lipoproteins in the sample is a peptide.

14. The method according to claim 13, further characterized in that the peptide is bacitracin, polymyxin, suzucasilin, or gramicidin.

15. The method according to claim 1 or 6, further characterized in that the compound having a relatively strong affinity with the non-measuring lipoproteins in the sample is a lectin.

16. The method according to claim 15, further characterized in that the lectin is concanavalin A, castorine lectin, or peanut lectin.

17. The method according to claim 1 or 6, further characterized in that the compound having a relatively strong affinity with non-measuring lipoproteins in the sample is a spheroid binding compound.

18. - A reagent for the quantitative determination of cholesterols comprising, separately or as a mixture, a compound having a relatively strong affinity with lipoproteins that are not measurable in the sample, a surfactant that exhibits a relatively strong action on the measurement lipoproteins, and a cholesterol determination reagent.

19. A method for selectively quantifying cholesterols which preferably comprises reacting cholesterols present in non-measuring lipoproteins in a sample in the presence of a compound having a relatively strong affinity with the lipoprotein in the sample, an agent surfactant that exhibits a relatively strong action on non-measuring lipoproteins, and a cholesterol determination reagent, and determine the amount of cholesterols in the remaining measuring lipoprotein.

20. The method according to claim 19, further characterized in that the method having a relatively strong affinity with the measurement lipoproteins and the sample has an affinity with the components that form a lipoprotein surface layer of said lipoproteins.

21. The method according to claim 20, further characterized in that the components that form a lipoprotein surface layer of the measurement lipoproteins are cholesterols.

22. The method according to claim 20, further characterized in that the components that form a lipoprotein surface layer of the measurement lipoiproteins is phospholipid.

23. The method according to claim 20, further characterized in that the components that form a lipoprotein surface layer of the measuring lipoproteins are apoprotein.

24. A reagent for the quantitative determination of cholesterols comprising, separately or as a mixture, a compound having a relatively strong affinity with the lipoproteins of measurement in the sample, a surfactant that exhibits a relatively strong action on the lipoproteins that they are not measuring, and a cholesterol determination reagent.

25. A method for determining the concentration of cholesterols in several lipoproteins which preferably comprises reacting cholesterols present in a second measurement lipoprotein in a sample in the presence of a compound having a relatively strong affinity with a first measurement lipoprotein in the sample, a surfactant that exhibits a relatively stronger action on the second lipoprotein than the first lipoprotein, and a cholesterol determination reagent, which determines the amount of cholesterol in the first remaining lipoprotein, and determine the concentration of cholesterols in the each lipoprotein from the resulting amount of cholesterol in the first remaining lipoprotein and the total cholesterol concentration.

26. - The method according to claim 25, further characterized in that the compound having a relatively strong affinity with the first measurement lipoproteins in the sample has an affinity with the components that form a lipoprotein surface layer of said lipoproteins.

27. The method according to claim 26, further characterized in that the components that form a lipoprotein surface layer of the first measurement lipoproteins are cholesterols.

28. The method according to claim 26, further characterized in that the components that form a lipoprotein surface layer of the first measurement lipoproteins is phospholipid.

29. The method according to claim 26, further characterized in that the components that form a lipoprotein surface layer of the first measuring lipoproteins is apoprotein.

30. A reagent for the qualitative determination of cholesterols comprising, separately or as a mixture, a compound having a relatively strong affinity with a first lipoprotein in the sample, a surfactant that exhibits a stronger action on a second lipoprotein measuring it on the first lipoprotein measurement, and a cholesterol determination reagent. SUMMARY OF THE INVENTION A method for selectively quantifying cholesterols is described, which comprises determining the amount of cholesterols in a lipoprotein measured in a sample in the presence of a compound having a relatively strong affinity with non-measuring lipoproteins in the sample, a surfactant that exhibits a relatively strong action on the measurement lipoproteins, and a cholesterol determination reagent; a method for selectively determining the amount of cholesterols, which preferably comprises reacting cholesterols present in the non-measuring lipoproteins in a sample in the presence of a compound having a relatively strong affinity with the lipoprotein in the sample , a surfactant that exhibits a relatively strong action on non-measuring lipoproteins, and a cholesterol determination reagent, and determine the amount of cholesterols in the remaining lipoprotein measurement; and a reagent for quantifying cholesterol determination comprising, separately or as a mixture, a compound having a relatively strong affinity with one of the lipoproteins in the sample to carry out the methods mentioned, a surfactant that exhibits a relatively high action strong on the other lipoproteins, and a cholesterol determination reagent; the methods and the reagent ensure efficient quantitative determination of cholesterols in specific lipoprotein fractions without the need for prior treatment, such as centrifugation by a simple procedure; The methods and reagents can be applied to various types of automatic analyzers. HL / jtc * tpr * aom * ald * jtr * yac * flu "P01 / 1322F