JP2001286297A - Method for pre-treating specimen for cholesterol determination and method for cholesterol determination in specific lipoprotein by utilizing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method, capable of determining cholesterol in a specific fraction accurately and in a good efficiency by a simple operation without requiring basically a polyanion, etc., as indispensable and suitably being used in various automatic analyzing apparatuses. SOLUTION: This method for pre-treating a specimen for a determination of cholesterol presenting in a specific lipoprotein is characterized by acting an enzyme taking free type cholesterol as its substrate and as necessary a reaction promoting substance on the specimen containing the lipoprotein in an advance to the measurement of the cholesterol in the specimen. The method for determining cholesterol in the specific lipoprotein by utilizing the above pre-treating method and the cholesterol determination kit for the cholesterol in the specific lipoprotein and used for the above determining method are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pretreatment method for separating and quantifying cholesterol present in a specific fraction accurately and efficiently by a simple operation with a small number of samples, and a cholesterol in the specific fraction using the same. Related to the measurement method.

[0002]

2. Description of the Related Art Lipids such as cholesterol form lipoproteins together with apoproteins in blood. This lipoprotein has different chylomicrons,
Very low density lipoprotein (VLDL), low density lipoprotein (LD
L), high-density lipoprotein (HDL) and the like. Among these lipoproteins, LDL is one of the causative substances causing arteriosclerosis, while HDL is known to exhibit an anti-atherosclerotic effect.

In other words, epidemiologically, the cholesterol level in LDL shows a positive correlation with the frequency of atherosclerotic disease, while the cholesterol level in HDL has an inverse correlation with the frequency of atherosclerotic disease. It is known to show. Therefore, measurement of cholesterol in LDL or HDL is widely performed today for the purpose of preventing or diagnosing ischemic heart disease.

As a method of measuring cholesterol in LDL or HDL, for example, a method of separating LDL or HDL from other lipoproteins by ultracentrifugation and then subjecting it to cholesterol measurement, or a method of separating lipid by electrophoresis. There is known a method of performing dyeing and measuring the coloring intensity. However, each of these methods
There are problems such as complicated operation and inability to process a large number of samples, and they are hardly used on a daily basis.

[0005] As a method for measuring cholesterol in HDL at present, methods used in the field of clinical tests include:
A precipitant was added to the sample to aggregate lipoproteins other than HDL, and this was removed by centrifugation.
There is a precipitation method for measuring cholesterol in a supernatant containing only L. Although this method is simpler than ultracentrifugation or electrophoresis, it requires a relatively large amount of sample because it involves the operation of adding a precipitant and separating, and may cause analysis errors. Therefore, the entire analysis process could not be fully automated.

On the other hand, a method for enzymatically separating and quantifying cholesterol in HDL is also being studied. For example, a method of performing an enzymatic reaction in the presence of a bile salt and a nonionic surfactant (JP-A-63-126498) is known. This method utilizes the fact that the enzyme reaction at the beginning of the reaction is proportional to the cholesterol concentration in LDL, and the subsequent reaction rate is proportional to the cholesterol concentration in HDL. However, cholesterol in HDL and other lipoproteins The reaction of cholesterol in it could not be completely separated, and there was a problem with accuracy.

[0007] A method in which lipoproteins other than HDL are aggregated in advance and only cholesterol in HDL is enzymatically reacted, and then the enzyme is inactivated and the aggregate is redissolved to measure the absorbance. (JP-A-6-2
No. 42110) is known. However, since this method requires at least three operations of adding a reagent, it can be applied only to a limited automatic analyzer and has a problem in versatility. Further, upon re-dissolving the precipitate, it was not satisfactory in terms of damage to the analyzer and disposal of reagents, such as use of a high-concentration salt.

Further, during the first reaction, cholesterol oxidase and cholesterol esterase are allowed to act on lipoproteins other than HDL in the presence of a special surfactant, and cholesterol contained therein is caused to act preferentially. A method of measuring cholesterol in HDL while suppressing the reaction of HDL with cholesterol is also known (JP-A-9-299).
The present invention is greatly different from the present invention in that a special surfactant and cholesterol oxidase and cholesterol esterase for simultaneously introducing both free form and ester form cholesterol of lipoproteins other than L to the outside of the reaction system are required.

Further, in Japanese Patent No. 2606005,
HD which does not precipitate using a combination of a precipitation reagent for precipitating lipoproteins other than HDL and a cholesterol measurement reagent
Although a method for measuring cholesterol (HDL-C) in L is disclosed, it is a method that can be practiced when a modified enzyme and sulfated α-cyclodextrin are used as a precipitating agent, The measurement method was problematic in terms of accuracy, for example, turbidity caused by the effect of the agent interfered with the measurement system.

In "Biological Sample Analysis," published in a paper on the above patent method, Vol. 19, No. 5, pp. 305 to 320, the measurement of HDL-C using a modified enzyme is described in detail. Since it was found that HDL-C in the serum of a hyperlipidemic patient could not be measured because introduction into the reaction system alone would give a plus error (a higher value than in the precipitation method), It reports that HDL-C was measured using sulfated cyclodextrin and magnesium chloride, one of the polyanions, as precipitants.

In order to reduce the influence of turbidity caused by the precipitant of the above patent method, a technique of coexisting with a surfactant (Japanese Patent Application Laid-Open No. Hei 8-116996), the use of an antibody (Japanese Patent Application Laid-Open No. 9-96637), and the use of sugar Using a compound (JP-A-7
No. 316636), but all of these techniques are based on the premise that a reagent that causes agglomeration is included, and the use of a precipitant such as a polyanion is basically unavoidable. .

Recently, the present inventors have found that cholesterol in a specific fraction can be accurately determined without using a precipitant by using a substance that acts only on a specific lipoprotein. Filed (Japanese Patent Application No. 9-24482)
No. 1). Although this method has a very high correlation with the conventional precipitation method, the same tendency as that reported in the above "Biological Sample Analysis" has been observed in relation to measured values with the precipitation method. In order to obtain continuous data using conventional precipitation methods in medical institutions, polyanions have been added.

However, it is not desirable to add a polyanion or the like to cause precipitation in the measurement system due to problems such as cell contamination and the dispersion of measured values.
Expulsion of sediment from the system was strongly required. Furthermore,
It is economically irrational to use a polyanion or the like that is unnecessary on the measurement principle only for matching data, and a solution has been demanded.

[0014]

Accordingly, the present invention basically does not require a polyanion or the like, and can accurately and efficiently quantify cholesterol in a specific fraction by a simple operation. It is an object to provide a method suitably used for an analyzer.

[0015]

Means for Solving the Problems The present inventors have determined the quantification of cholesterol in a specific fraction using a substance which acts only on a specific lipoprotein such as HDL, which was reported in the above "Biological Sample Analysis". When pursuing the cause of the higher cholesterol value than the value of the precipitation method, a small amount of free cholesterol present on the surface or in the immediate vicinity of lipoproteins (LDL, VLDL, etc.) other than the HDL fraction which should not be measured Was drawn, and it was concluded that this was the cause of the positive error. Based on this finding, if the cholesterol level is measured after consuming only free cholesterol under the condition that the lipoprotein does not substantially change, the value of the quantification method using a substance acting only on the specific lipoprotein can be obtained. And found that the values obtained by the precipitation method coincided with each other, and completed the present invention.

That is, the present invention provides a cholesterol quantification method comprising, prior to measuring cholesterol present in a specific lipoprotein in a sample, reacting a sample containing lipoprotein with an enzyme using free cholesterol as a substrate. The present invention provides a method for pretreating a sample for use.

The present invention also relates to a method for producing a cholesterol present in a specific lipoprotein by using a substance which acts only on a specific lipoprotein after allowing an enzyme using free cholesterol as a substrate to act on a sample containing the lipoprotein. The present invention provides a specific lipoprotein cholesterol quantification method characterized by measuring lipoprotein cholesterol.

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, prior to the measurement of cholesterol present in a specific lipoprotein in a sample, first, an enzyme using free cholesterol as a substrate is allowed to act on the sample as pretreatment. Consumes cholesterol.

As the enzyme using free cholesterol as a substrate, any enzyme having free cholesterol as a substrate, such as cholesterol dehydronase or cholesterol oxidase, may be used. These may be of microbial origin, animal origin, plant origin, etc., or may be those produced by genetic manipulation. Also, the presence or absence of chemical modification does not matter. The above enzyme is generally used in an amount of 0.001 unit to 100 units / ml, preferably 0.1 unit.
Used in units-100 units / ml.

The conditions for allowing the enzyme using free cholesterol as a substrate to act on the sample are not particularly limited.
The conditions recommended for the enzyme may be used. However, in the step of allowing an enzyme using free cholesterol as a substrate to act on a sample, care must be taken so that a reaction for converting ester cholesterol to free cholesterol does not occur. That is, the presence or absence of cholesterol esterase does not matter, but it is necessary to maintain conditions that do not substantially make it work.

As the enzyme using free cholesterol as a substrate, a coenzyme can be used if necessary, and nicotinamide adenine dinucleotide or the like can be used as the coenzyme. These enzymes can be used alone or in combination of two or more kinds. The amount of use varies depending on the enzyme and is not particularly limited, but is 0.001 unit to 100 units / ml, preferably 0.1
Used in units-100 units / ml.

As described above, the enzyme using free cholesterol as a substrate used in the present invention has no restriction on its origin, and the concentration and the like can be appropriately selected so as to satisfy desired performance and operability. Therefore, for example, in order to complete the pretreatment within a certain period of time, the amount of the enzyme may be increased, and conversely, if it is desired to save the enzyme, the time of the pretreatment may be extended.

However, in the case of a test drug which is premised on measurement by an automatic analyzer, it is required to satisfy both at the same time. That is, it is required to complete the pretreatment in a short time by using a small amount of the enzyme. In such a case, by coexisting a reaction promoting substance selected from the following group, in a pretreatment using an enzyme using free cholesterol as a substrate, the amount of the enzyme can be reduced without extending the pretreatment time. , It is possible to obtain desired performance.

Examples of the reaction promoting substance used for the above purpose include flufenamic acid, mefenamic acid,
2,2 ', 6', 2 "-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin, mevinolin, etc. (Salts of reaction promoting substances or those in which metal derivatives of reaction promoting substances (such as aluminum derivatives) are present) Among them, flufenamic acid and mefenamic acid are non-steroidal anti-inflammatory drugs, and fusidic acid and monensin are substances known as antibiotics.

In using these compounds as a reaction accelerating substance, it is necessary to appropriately select the concentration and the like in consideration of the physical properties of each, the pH and ionic strength of the measurement system, the type and concentration of the inclusions, and the like. .

The concentration of the reaction accelerator used can be determined experimentally according to the conditions of the measurement system. In general, about 0.01 to 100 mM for flufenamic acid and about 100 to 100 mM for fusidic acid. Is about 0.01 to 10 mM, about 0.01 to 5 mM for mefenamic acid, 2,2 ', 6', 2 "-terpyridine and betamethasone acetate, and about 0.01 to 1 mM for monensin and mevinolin. In the case of tiglic acid, it may be used at about 1 to 500 mM.

By utilizing the above reaction accelerator,
It is possible to reduce the amount of the enzyme using free cholesterol as a substrate to several tenths to several tenths. Also,
It is also possible to shorten the reaction time with the same amount of enzyme.

In the above-mentioned pretreatment with an enzyme using free cholesterol as a substrate (and, if necessary, a reaction accelerator), in order to adjust the action of the enzyme without further impairing the specificity of the measurement, a specific treatment may be carried out. To the extent that lipoprotein aggregation does not occur, other enzymes (excluding those that substantially affect lipoprotein), salts, buffers for adjusting pH, surfactants (substantially affect lipoprotein) Reagents having an affinity for specific lipoproteins such as preservatives, proteins such as albumin, antibodies, antibiotics, saponins, lectins, and polyanions can be used.

Therefore, in the present invention, as a pretreatment agent for measuring cholesterol present in a specific lipoprotein in a sample, one containing the following components can be used.

(Essential components) Enzymes using free cholesterol as a substrate, for example, cholesterol dehydronagease and cholesterol oxidase. (Optional ingredients) For example, flufenamic acid, mefenamic acid,
Reaction promoting substances such as 2,2 ', 6', 2 "-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin, mevinolin, etc. (Other components) Coenzymes, such as NAD, peroxidase, catalase, diaphorase, ascorbin Other enzymes such as acid oxidase, acids such as pyruvate, salts, buffers for pH adjustment, surfactants that do not substantially affect lipoproteins, preservatives, proteins such as albumin, antibodies, Antibiotics, saponins, lectins, polyanions, 4
-Oxidative color reagents such as couplers such as aminoantipyrine, hydrogen donors such as Trinder reagents, electron acceptors such as phenazine methosulfate, and reducing color reagents such as nitroblue tetrazolium.

In the present invention, after the free cholesterol in the lipoprotein is consumed by the above pretreatment, the cholesterol present in the specific lipoprotein in the sample is measured.

As a method for measuring cholesterol present in a specific lipoprotein in a sample, a method capable of measuring cholesterol present in the specific lipoprotein using a substance which acts only on the specific lipoprotein is used. Then, any method can be used.

As an example of the method, for example, a surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzyl phenyl ether disclosed in JP-A-11-56395 is applied only to a specific lipoprotein. Cholesterol measurement enzyme reagent is added in the presence of cholesterol, and the cholesterol in the high-density lipoprotein among the lipoproteins is preferentially reacted with the cholesterol measurement enzyme reagent. There is a method of measuring.

Among these, Emulgen A-60 (manufactured by Kao Corporation) is an example of the former commercially available polyoxyethylene alkylene phenyl ether, and Emulgen A-60 is an example of the latter commercially available polyoxyethylene alkylene tribenzyl phenyl ether. B66 (manufactured by Kao Corporation) and the like.

Further, as another method, a biological sample analysis,
Vol. 19, No. 5, pp. 305 to 320, a method using the modified enzyme disclosed as a substance acting only on a specific lipoprotein. In the method of this document, HDL
Sulfated α- to suppress the reaction of lipoprotein fractions other than
Although cyclodextrin and magnesium chloride are used, the pretreatment method of the present invention eliminates the need for these.

The method for measuring cholesterol present in a specific lipoprotein can be carried out by using a reagent used in a usual method for measuring cholesterol, except that a substance which acts only on a specific lipoprotein is used. Examples of the components contained in the reagents used include enzymes such as cholesterol esterase, cholesterol oxidase, cholesterol dehydrogenase, isocitrate dehydrogenase, diaphorase, peroxidase, color formers, coenzymes, electron acceptors, proteins (such as albumin),
Examples include preservatives, surfactants, salts, acids, and buffers for adjusting pH.

As the surfactant, any of ionic and nonionic surfactants can be used. For example, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene-poly Oxypropylene condensates, polyoxyethylene alkyl ether sulfates, alkylbenzene sulfonates, bile acids and the like can be mentioned. The amount of the surfactant used varies depending on the compound and is not particularly limited, but is 0.0001% to 5%, preferably 0.001%.
% To 5%.

The buffer is not particularly limited, and general buffers such as Good's buffer, phosphoric acid, Tris and phthalate can be used. The amount of use is not particularly limited, but is 0.005M to 2M, preferably 0.05M to 2M.
Used at 1-1M.

The method of quantifying cholesterol in a specific lipoprotein fraction according to the present invention is typically carried out by first adding a pretreatment agent that acts only on free cholesterol to a measurement sample, followed by adding a specific agent. A method for measuring the amount of cholesterol in a specific lipoprotein fraction by adding and mixing a substance that acts only on lipoprotein and a cholesterol quantitative reagent (hereinafter referred to as “quantitative reagent”) containing a reagent used in a usual method for measuring cholesterol. is there.

As a specific example, a sample is mixed with cholesterol dehydrogenase and coenzyme (NAD),
Then, a method of adding a cholesterol measuring reagent containing cholesterol esterase, cholesterol oxidase, etc., a method of mixing a sample with cholesterol dehydrogenase and NAD, and then adding a cholesterol measuring reagent containing cholesterol esterase and the like,
A method in which a sample and cholesterol oxidase are mixed together with peroxidase, 4-aminoantipyrine or catalase, and then a cholesterol measuring reagent containing cholesterol esterase or the like is added.
After mixing with aminoantipyrine, etc., cholesterol esterase, cholesterol dehydrogenase,
A method of adding a cholesterol measuring reagent containing NAD or the like can be exemplified, but the method is not limited thereto.

As a method for measuring cholesterol in a specific lipoprotein fraction, any known enzymatic assay can be used. For example, a method using a combination of cholesterol esterase and cholesterol oxidase as an enzyme reagent, A method using an esterase and a cholesterol dehydrogenase in combination is exemplified.

In the present invention, the enzyme used in the first reaction as the pretreatment reaction and the enzyme used in the cholesterol measurement in the second reaction as the quantification method may be the same or different. In the first reaction, an excess amount of enzyme may be used and used in the second reaction. The point is that a small amount of free cholesterol present on the surface of the lipoprotein is consumed (first reaction / pretreatment reaction), and then the large amount of the lipoprotein constituting the lipoprotein in a state where the enzyme acts only on the specific lipoprotein to be measured. Partial cholesterol (free cholesterol + ester cholesterol)
What is necessary is just to be able to quantify.

The method of finally detecting cholesterol after adding these enzyme reagents for measuring cholesterol is not particularly limited. For example, peroxidase and chromogen, or diaphorase or electron acceptor and reducing chromogenic reagent can be used. And a method of directly detecting a coenzyme or hydrogen peroxide, which is performed by further combining the above methods. The coenzyme may be amplified by a coenzyme cycling system.

In order to easily carry out the method of the present invention, it is preferable to use a quantification kit for measuring cholesterol in a specific lipoprotein suitable for the method. Such a kit can be easily designed based on the above description, and examples thereof are divided into a case where cholesterol oxidase is used as a representative enzyme using free cholesterol as a substrate and a case where cholesterol dehydrogenase is used. It is as follows if shown.

[Kit for cholesterol oxidase] (a) The following reagents (1) and (2) (1) a first reagent containing cholesterol oxidase and a hydrogen peroxide consuming substance (and optionally a reaction promoting substance); 2) A kit for determining cholesterol in a specific lipoprotein, comprising a second reagent containing a substance acting only on a specific lipoprotein, cholesterol esterase and a color former. (B) the following reagents (1) and (2): (1) a first reagent containing cholesterol oxidase, cholesterol esterase and a hydrogen peroxide consuming substance (and optionally a reaction promoting substance); (2) only a specific lipoprotein; A kit for determining cholesterol in a specific lipoprotein, comprising a second reagent containing a substance that acts and a color former. (C) the following reagents (1), (2) and (3) (1) a first reagent containing cholesterol oxidase and a hydrogen peroxide consuming substance (and optionally a reaction promoting substance), (2) only a specific lipoprotein A kit for quantifying cholesterol in a specific lipoprotein, comprising a second reagent containing a substance acting on lipoprotein and (3) a third reagent containing cholesterol esterase and a color former.

In the above-mentioned kit, the term “hydrogen peroxide consuming substance” refers to a substance that consumes and eliminates hydrogen peroxide generated by the reaction between cholesterol oxidase and cholesterol, and examples thereof include couplers such as catalase and 4-aminoantipyrine. And a redox coloring reagent containing a hydrogen donor such as a Trinder reagent.

Among them, a coupler such as 4-aminoantipyrine and a hydrogen donor such as a Trinder's reagent are combined to react with hydrogen peroxide to form a color, and the above (2) or (3) It is used as a color former. However, it is preferable that only one of a coupler and a hydrogen donor is used as the reagent (1) used in the pretreatment step of the present invention, and hydrogen peroxide is consumed in a non-color-forming reaction. Of course, it is also possible to make a color reaction and adjust the measured value (it can be adjusted by subtracting the color value of the reagent (1) from the color value of the reagent (2) or the reagent (3)).

[Kit for cholesterol dehydrogenase] (d) The following reagents (1) and (2) (1) a first reagent containing cholesterol dehydrogenase and a coenzyme (and optionally a reaction promoting substance),
(2) A kit for quantifying cholesterol in a specific lipoprotein, comprising a substance acting only on a specific lipoprotein and a second reagent containing cholesterol esterase. (E) the following reagents (1) and (2) (1) a first reagent containing cholesterol dehydrogenase and a coenzyme (and optionally a reaction promoting substance),
(2) A kit for quantifying cholesterol in a specific lipoprotein, comprising a second reagent containing a substance acting only on a specific lipoprotein, cholesterol oxidase, cholesterol esterase, peroxidase, and a coloring agent. (F) the following reagents (1) and (2): (1) a first reagent containing cholesterol dehydrogenase, coenzyme and cholesterol esterase (furthermore, a reaction promoting substance in some cases); (2) a substance acting only on a specific lipoprotein A kit for quantifying cholesterol in a specific lipoprotein, comprising a second reagent comprising: (G) the following reagents (1) and (2) (1) a first reagent containing cholesterol dehydrogenase, a coenzyme and a cholesterol esterase (in some cases, a reaction promoting substance), (2) a substance that acts only on a specific lipoprotein A kit for quantifying cholesterol in a specific lipoprotein, comprising a second reagent containing cholesterol oxidase, peroxidase and a color former. (H) the following reagents (1), (2) and (3) (1) a first reagent containing cholesterol dehydrogenase and a coenzyme (and optionally a reaction promoting substance),
(2) A kit for quantifying cholesterol in a specific lipoprotein, comprising a second reagent containing a substance acting only on a specific lipoprotein, and (3) a third reagent containing a cholesterol esterase. (I) the following reagents (1), (2) and (3): (1) a first reagent containing cholesterol dehydrogenase and a coenzyme (and optionally a reaction promoting substance),
(2) A kit for quantifying cholesterol in a specific lipoprotein, comprising a second reagent containing a substance acting only on a specific lipoprotein, and (3) a third reagent containing cholesterol oxidase, cholesterol esterase, peroxidase and a coloring agent. (1) The following reagents (1) and (2): (1) a first reagent containing cholesterol dehydrogenase, a coenzyme, and a coenzyme reaction product consuming substance (and optionally a reaction promoting substance); (2) a specific lipoprotein A kit for determining cholesterol in a specific lipoprotein, comprising a substance acting only on cholesterol and a second reagent containing cholesterol esterase. (1) the following reagents (1) and (2): (1) a first reagent containing cholesterol dehydrogenase, a coenzyme and a coenzyme reaction product consuming substance (and optionally a reaction promoting substance); (2) a specific lipoprotein A kit for determining cholesterol in a specific lipoprotein, comprising a second reagent containing a substance acting only on cholesterol esterase and a color former.

In the kit using cholesterol dehydrogenase described above, the coenzyme reaction product consuming substance refers to a reduced coenzyme (eg, NADH) produced by the reaction of cholesterol, cholesterol dehydrogenase and a coenzyme (eg, NAD), and re-adds the original coenzyme. It refers to a substance returned to the enzyme, examples of which include lactate dehydrogenase and pyruvate (substrate). In the above kits, the reaction product of the coenzyme is produced by the addition of the reagent (1). Among them, (d), (f), (h) and (nu)
In the above, when light having the same wavelength as the color developed by the addition of the reagent (1) is measured in the measurement stage without consuming the reaction product, the color of the reagent (2) or (3) The cholesterol in a specific lipoprotein can be quantified by subtracting the color development value in the pretreatment stage to which (1) is added. The reagent (1)
A substance that consumes a reaction product may be added in advance, and after this is consumed, the reagent (2) or the reagent (3) may be added to form a color. At this time, the reagent (2) or the reagent (3)
It is better to incorporate a substance that reduces the effect of the substance consuming the reaction product. On the other hand, kits (e), (g),
In (1) and (2), the color development at a different wavelength from the color development in the pre-processing is measured, so that it is not always necessary to subtract the color development value in the pre-processing from the measured value.

The above-mentioned flufenamic acid, mefenamic acid, 2,2 ′, 6 ′, 2 ″ -terpyridine, tiglic acid,
Uses of the reaction promoting substances such as fusidic acid, beta-methasone acetate, monensin, mevinolin and the like are limited to the pretreatment method (agent) of the present invention and the method (kit) for quantifying cholesterol in a specific lipoprotein of the present invention using the same. It is not something to be done.

A method for quantifying cholesterol using an enzyme using free cholesterol as a substrate, for example, a method for quantifying free cholesterol assembled using cholesterol oxidase, peroxidase, a coloring agent, or cholesterol oxidase, cholesterol esterase, If a reaction-promoting substance such as flufenamic acid coexists in the method for determining total cholesterol assembled using oxidase, a coloring agent, etc., the amount of enzyme (here, cholesterol oxidase) using free cholesterol as a substrate can be reduced. Naturally, it is possible to obtain effects such as shortening of the reaction time of the enzyme reaction.

Further, referring to the description in this specification and the like relating to the cholesterol quantification method (for example, selecting a specific surfactant and measuring a specific lipoprotein), a more specific quantification method can be obtained. Can be assembled.

[0053]

According to the present invention, not only mechanical pretreatment such as centrifugation but also polyanion is not used.
Cholesterol in the specific fraction can be efficiently quantified with a simple operation. Since no precipitation due to polyanion or the like occurs, the measuring device (particularly, the cell) does not become dirty, and there is no variation in measured values. is there.

Further, as shown in Examples described later, even for a sample having a high neutral fat level, a measured value having a high correlation with the conventional precipitation method can be obtained, which is excellent in that a sample is not selected. .

Further, when a reaction accelerator is used, the amount of the enzyme using free cholesterol as a substrate in the pretreatment can be reduced.

As described above, the method of the present invention uses a small number of samples,
Since accurate and specific measurement is possible for a wide range of samples by a simple operation, it can be applied to various automatic analyzers and is extremely useful in the field of clinical examination.

[0057]

Next, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.

Example 1 Cholesterol in HDL was quantified as described below for 30 serum samples containing lipoproteins by the method of the present invention and the precipitation method described below, and the measured values were compared.

(The present invention) 10 mM phosphate buffer containing 0.1 unit / ml cholesterol dehydrogenase (Amano Pharmaceutical), 2.5 mM NAD and 0.03% of 4-aminoantipyrine (1st reagent; pH 8.5) 300 μl was added (pretreatment). After about 5 minutes, Emulgen B-66 1%, cholesterol esterase (Asahi Kasei) 1.3 units / ml, cholesterol oxidase (Asahi Kasei) 2 units / ml, peroxidase (Toyobo) 5 units / ml, and disulfobutyl methtoluidine 0 100 μl of a cholesterol measurement reagent (second reagent) consisting of 100 mM MES buffer (pH 6) containing 0.04% was added.

The 600 immediately before the addition of the second reagent and 5 minutes after the addition
The absorbance at nm was measured, and the HDL cholesterol concentration in the serum sample was determined from the difference (two-point method).
A control serum with a known concentration was used as a calibration substance. The above operations were performed using a Hitachi 7150 automatic analyzer.

(Precipitation method) 200 μl of HDLC2 “Daiichi” fractionation reagent (Daiichi Pure Chemicals Co., Ltd.) was
, and centrifuged at 3000 rpm for 10 minutes. 50 μl of the supernatant was collected, and Triton X-1 was used.
00 1%, cholesterol esterase 1 unit / m
1. Cholesterol measurement consisting of 100 mM MES buffer (pH 6.5) containing 1 unit / ml of cholesterol oxidase, 5 units / ml of peroxidase, 0.04% of disulfobutylmethtoluidine and 0.04% of 4-aminoantipyrine After mixing with 3 ml of the reagent and incubating at 37 ° C. for 10 minutes, the absorbance at 600 nm was measured to determine the cholesterol concentration in HDL.

(Results) The results are shown in Table 1 and FIG.

[Table 1]

As is evident from the results, the method of the present invention showed an extremely good correlation with the conventional precipitation method, although no polyanion or the like was used.

Example 2 Cholesterol dehydrogenase, NAD and phosphate buffer of the first reagent of Example 1 were combined with 5 units / ml of cholesterol oxidase (Toyobo), 5 units / ml of peroxidase (Toyobo) and 100 mM MES.
The measured values were compared in place of (pH 6).

(Results) The results are shown in Table 2 and FIG.

[Table 2]

As is evident from the results, the method of the present invention showed an extremely good correlation with the conventional precipitation method, although no polyanion was used.

Example 3 Using the reagents of Examples 1 and 2, five serum samples having different neutral fat levels were measured, and the measured values were compared with those of the conventional method. Table 3 shows the results.

[0068]

[Table 3]

As shown in Table 3, according to the present invention, the same level of measured value as in the conventional method was obtained for a sample having a high neutral fat level.

Example 4 The same measurement as in Example 1 was carried out except that 5 units / ml of cholesterol oxidase in the first reagent of Example 2 was changed to the composition and concentration shown in Table 4 below, and the measurement was carried out. The values were compared with the precipitation method and the method of Example 2 (standard test system). The same second reagent as in Example 1 was used.
Table 5 shows the results.

(Test Reagent Composition)

[Table 4]

(Result)

[Table 5]

From these results, when cholesterol oxidase was reduced to 1/5 (test system A) as compared with the reference test system (Example 2), the correlation coefficient was slightly lowered and the intercept value was slightly increased. When a reaction promoting substance was used, almost the same results as in the standard test system were obtained even when cholesterol oxidase was 1/5, and it was clear that the use of the reaction promoting substance could reduce the amount of cholesterol oxidase used. It became.

Example 5 1.25 units / ml peroxidase (Toyobo), 0.2
Table 6 which contains in common 01% 4-aminoantipyrine, 0.02% disulfobutylmeta-toluidine, 50 mM NaCl and different buffer types and pH, cholesterol oxidase (Toyobo) and flufenamic acid (Sigma) concentrations (J to L) were prepared.

[Table 6]

To 3 μl of serum sample, add 300 μl of reagents J to L
After adding 1 l of the mixture and reacting at 37 ° C. for 5 minutes, the absorbance at 600 nm was measured. The above operation is performed by Hitachi 7150
This was performed using a mold automatic analyzer. Four serum samples were prepared using reagent J
~ L. 5.0 for each of reagents J-L
The relative absorbance was calculated for each cholesterol oxidase and flufenamic acid concentration reagent, taking the absorbance obtained with the unit / ml cholesterol oxidase and 0 mM flufenamic acid reagents as 100.

(Results) The results of averaging the relative absorbances of the four samples are shown in FIG. In the figures, cholesterol oxidase is described as COD.

As a result, at any pH, the relative absorbance increased depending on the concentration of flufenamic acid. It was confirmed that the use of the reaction accelerator can reduce the amount of cholesterol oxidase used.

It was also found that the reaction promoting substance can be used in a method for measuring free cholesterol using an enzyme using free cholesterol as a substrate or a method for measuring total cholesterol.

[Brief description of the drawings]

FIG. 1 is a drawing showing the correlation between the present invention and the precipitation method in Example 1.

FIG. 2 is a drawing showing the correlation between the present invention and the precipitation method in Example 2.

FIG. 3 is a view showing the effect of a reaction promoting substance in Example 5. that's all

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) G01N 33/92 G01N 33/92 B (72) Inventor Mitsuhisa Manabe 3-3-1 Koyodai, Ryugasaki City, Ibaraki Prefecture Daiichi Chemicals Co., Ltd. Diagnostics Research Laboratory (72) Inventor Mitsuaki Yamamoto 3-3-1 Koyodai, Ryugasaki-city, Ibaraki Pref. Daiichi Chemicals Co., Ltd. Diagnostics Research Laboratory F-term (reference) 2G045 AA13 BA11 BB60 CA25 CA26 DA63 DA69 FA26 FA29 FB01 GC10 4B063 QA01 QA18 QA19 QQ02 QQ76 QR03 QR04 QR12 QR65 QR66 QS07 QS12 QS36 QX01

Claims (37)

[Claims] 1. A method for determining a cholesterol sample, comprising the step of allowing a sample containing lipoprotein to act on an enzyme using free cholesterol as a substrate before measuring the cholesterol present in a specific lipoprotein in the sample. Preprocessing method. 2. Prior to measuring cholesterol present in a specific lipoprotein in a sample, an enzyme using free cholesterol as a substrate and flufenamic acid, mefenamic acid, 2,2 ′, 6 ', 2 "-A pretreatment method for a cholesterol quantification sample, which comprises reacting a reaction promoting substance selected from terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin and mevinolin. 3. The pretreatment method according to claim 1, wherein the enzyme using free cholesterol as a substrate is cholesterol oxidase or cholesterol dehydrogenase. 4. A sample containing lipoprotein is reacted with an enzyme using free cholesterol as a substrate, and then a cholesterol present in the specific lipoprotein is measured using a substance which acts only on a specific lipoprotein. A method for determining cholesterol in a specific lipoprotein, characterized in that: 5. An enzyme using free cholesterol as a substrate and flufenamic acid, mefenamic acid, 2,2 ', 6', 2 "-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin, and a lipoprotein-containing sample. Cholesterol in a specific lipoprotein characterized by measuring a cholesterol present in a specific lipoprotein by using a substance that acts only on a specific lipoprotein after allowing a reaction promoting substance selected from mevinolin to act Assay method. 6. The method according to claim 4, wherein the enzyme using free cholesterol as a substrate is cholesterol oxidase and / or cholesterol dehydrogenase. 7. The method according to claim 4, wherein the specific lipoprotein is a high-density lipoprotein. 8. A pretreatment agent for a cholesterol measurement sample containing an enzyme using free cholesterol as a substrate and substantially free of a substance acting on lipoprotein. 9. An enzyme using free cholesterol as a substrate and flufenamic acid, mefenamic acid, 2,2 ′, 6 ′, 2 ″-
A pretreatment agent for a cholesterol measurement sample containing terpyridine, tiglic acid, fusidic acid, beta-methasone acetate, monensin, and mevinolin, which is substantially free of a substance acting on lipoprotein. 10. A pretreatment agent for a cholesterol measurement sample containing an enzyme using free cholesterol as a substrate and substantially free of cholesterol esterase. 11. An enzyme using free cholesterol as a substrate and flufenamic acid, mefenamic acid, 2,2 ′, 6 ′, 2 ″.
A pretreatment agent for a cholesterol measurement sample containing terpyridine, tiglic acid, fusidic acid, beta-methasone acetate, monensin and mevinolin, and substantially free of cholesterol esterase; 12. The pretreatment agent for a cholesterol measurement sample according to any one of claims 8 to 10, wherein the enzyme using free cholesterol as a substrate is cholesterol dehydronase or cholesterol oxidase. 13. The following reagents: (1) a first reagent containing cholesterol oxidase and a hydrogen peroxide consuming substance; and (2) a second reagent containing a substance acting only on a specific lipoprotein, cholesterol esterase and a coloring agent. A kit for quantifying cholesterol in specific lipoproteins. 14. A specific lipoprotein comprising the following reagents: (1) a first reagent containing cholesterol dehydrogenase and a coenzyme; (2) a substance acting only on a specific lipoprotein and a second reagent containing cholesterol esterase Cholesterol determination kit. 15. The following reagents: (1) a first reagent containing cholesterol dehydrogenase and a coenzyme; and (2) a first reagent containing a substance acting only on a specific lipoprotein, cholesterol oxidase, cholesterol esterase, peroxidase and a coloring agent. A kit for quantifying cholesterol in a specific lipoprotein comprising two reagents. 16. The following reagents: (1) a first reagent containing cholesterol oxidase, cholesterol esterase and a hydrogen peroxide consuming substance; and (2) a second reagent containing a substance acting only on a specific lipoprotein and a color former. A kit for quantifying cholesterol in specific lipoproteins. 17. A specific lipoprotein comprising the following reagents: (1) a first reagent containing cholesterol dehydrogenase, a coenzyme and cholesterol esterase; and (2) a second reagent containing a substance acting only on a specific lipoprotein. Cholesterol determination kit. 18. The following reagents: (1) a first reagent containing cholesterol dehydrogenase, coenzyme and cholesterol esterase; and (2) a first reagent containing a substance acting only on a specific lipoprotein, cholesterol oxidase, peroxidase and a coloring agent. A kit for quantifying cholesterol in a specific lipoprotein comprising two reagents. 19. The following reagents: (1) a first reagent containing cholesterol oxidase and a hydrogen peroxide consuming substance; (2) a second reagent containing a substance acting only on a specific lipoprotein; (3) cholesterol esterase; A kit for quantifying cholesterol in a specific lipoprotein, comprising a third reagent containing a color former. 20. The following reagents: (1) a first reagent containing cholesterol dehydrogenase and a coenzyme; (2) a second reagent containing a substance acting only on a specific lipoprotein; and (3) a third reagent containing cholesterol esterase. A kit for determining cholesterol in a specific lipoprotein comprising reagents. 21. The following reagents: (1) a first reagent containing cholesterol dehydrogenase and a coenzyme; (2) a second reagent containing a substance acting only on a specific lipoprotein; (3) cholesterol oxidase, cholesterol esterase; Third containing peroxidase and color former
A kit for determining cholesterol in a specific lipoprotein comprising reagents. 22. The following reagents: (1) a first reagent containing cholesterol dehydrogenase, a coenzyme and a coenzyme reaction product consuming substance; and (2) a second reagent containing a substance acting only on a specific lipoprotein and a cholesterol esterase. A kit for determining cholesterol in a specific lipoprotein comprising reagents. 23. The following reagents: (1) a first reagent containing cholesterol dehydrogenase, a coenzyme, and a coenzyme reaction product consuming substance; and (2) a substance acting only on a specific lipoprotein, cholesterol esterase, and a coloring agent. A kit for determining cholesterol in a specific lipoprotein, comprising a second reagent. 24. The following reagents: (1) (a) cholesterol oxidase, (b) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′, 2 ″ -terpyridine,
Reaction promoting substances selected from tiglic acid, fusidic acid, beta methasone acetate, monensin and mevinolin; and
(c) a first reagent containing a hydrogen peroxide consuming substance; and (2) a kit for quantifying cholesterol in a specific lipoprotein, comprising a substance acting only on a specific lipoprotein, a second reagent containing cholesterol esterase and a coloring agent. 25. The following reagents: (1) (a) cholesterol dehydrogenase, (b) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′, 2 ″ -terpyridine, tiglic acid, fusidic acid, beta methasone acetate, monensin And a reaction promoting substance selected from mevinolin and (c) a first reagent containing a coenzyme, (2) a cholesterol determination in a specific lipoprotein comprising a substance acting only on a specific lipoprotein and a second reagent containing cholesterol esterase Kit. 26. The following reagents: (1) (a) cholesterol oxidase, (b) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′, 2 ″ -terpyridine,
Reaction promoting substance selected from tiglic acid, fusidic acid, beta methasone acetate, monensin and mevinolin, (c)
A kit for quantifying cholesterol in a specific lipoprotein, comprising: a first reagent containing cholesterol esterase and (d) a hydrogen peroxide consuming substance; and (2) a second reagent containing a substance acting only on a specific lipoprotein and a coloring agent. 27. The following reagents: (1) (a) cholesterol dehydrogenase, (b) coenzyme, (c) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′,
2 "-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin and mevinolin, a reaction promoting substance selected from the group consisting of: (d) a first reagent containing cholesterol esterase; and (2) a first reagent containing a substance acting only on a specific lipoprotein. A kit for quantifying cholesterol in a specific lipoprotein comprising two reagents. 28. The following reagents: (1) (a) cholesterol dehydrogenase, (b) coenzyme, (c) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′,
2 "-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin and mevinolin, a reaction promoting substance selected from the group consisting of: (d) a first reagent containing cholesterol esterase; (2) a substance acting only on a specific lipoprotein; cholesterol oxidase Kit for quantifying cholesterol in a specific lipoprotein, comprising a second reagent containing lipase, peroxidase and a coloring agent. 29. The following reagents: (1) (a) cholesterol oxidase, (b) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′, 2 ″ -terpyridine,
Reaction promoting substances selected from tiglic acid, fusidic acid, beta methasone acetate, monensin and mevinolin;
(c) a first reagent containing a hydrogen peroxide consuming substance; (2) a second reagent containing a substance acting only on a specific lipoprotein; and (3) a specific liposome comprising a third reagent containing cholesterol esterase and a color former. Kit for determination of cholesterol in protein. 30. The following reagents: (1) (a) cholesterol dehydrogenase, (b) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′, 2 ″ -terpyridine, tiglic acid, fusidic acid, beta methasone acetate, monensin And (c) a first reagent containing a coenzyme, (2) a second reagent containing a substance acting only on a specific lipoprotein, and (3) a third reagent containing cholesterol esterase. Kit for determination of cholesterol in specific lipoproteins. 31. The following reagents: (1) (a) cholesterol dehydrogenase, (b) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′, 2 ″ -terpyridine, tiglic acid, fusidic acid, beta methasone acetate, monensin And (c) a first reagent containing a coenzyme, (2) a second reagent containing a substance acting only on a specific lipoprotein, (3) cholesterol oxidase, cholesterol esterase, peroxidase and Third containing a color former
A kit for determining cholesterol in a specific lipoprotein comprising reagents. 32. The following reagents: (1) (a) cholesterol dehydrogenase, (b) coenzyme, (c) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′,
2 "-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin and mevinolin, a first reagent containing a reaction promoting substance and (d) a coenzyme reaction product consuming substance, (2) acting only on a specific lipoprotein A kit for quantifying cholesterol in a specific lipoprotein, comprising a second substance containing a substance to be modified and cholesterol esterase. 33. The following reagents: (1) (a) cholesterol dehydrogenase, (b) coenzyme, (c) flufenamic acid, mefenamic acid, 2,2 ′, 6 ′,
2 "-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin and mevinolin, a first reagent containing a reaction promoting substance and (d) a coenzyme reaction product consuming substance, (2) acting only on a specific lipoprotein A kit for quantifying cholesterol in a specific lipoprotein, comprising a second reagent containing a substance, a cholesterol esterase and a coloring agent. 34. Flufenamic acid, mefenamic acid, 2,
2 ', 6', 2 "-A reaction promoting substance of an enzyme using free cholesterol as a substrate selected from terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin and mevinolin. 35. The reaction promoting substance according to claim 34, wherein the enzyme using free cholesterol as a substrate is cholesterol oxidase or cholesterol dehydrogenase. 36. An enzyme using free cholesterol as a substrate and flufenamic acid, mefenamic acid, 2,2 ′,
A method for quantifying free cholesterol, which comprises reacting a reaction promoting substance selected from 6 ', 2 "-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin and mevinolin. 37. At least an enzyme using free cholesterol as a substrate and flufenamic acid, mefenamic acid,
A method for quantifying total cholesterol, which comprises reacting a reaction promoting substance selected from 2,2 ', 6', 2 "-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin, and mevinolin.