JP2001103998A - Reagent for determining cholesterol in high-density lipoprotein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a reagent for determining a cholesterol in a high-density lipoprotein useful for a method for determining a cholesterol in HDL, not requiring a complicated fraction and separation operation, capable of selectively, simply and precisely determining a HDL cholesterol in a sample to be tested containing other lipoproteins such as HDL and low-density lipoprotein(LDL), very low density lipoprotein(VLDL), chylomicron(CM), etc. SOLUTION: This reagent for determining a cholesterol in a high-density lipoprotein comprises a combination of at least cholesterol esterase, cholesterol oxidase and a surfactant which specifically acts on cholesterol in the high- density lipoprotein and has 13-14 HLB.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a reagent for quantifying cholesterol in high density lipoprotein (hereinafter sometimes referred to as “HDL”).

HDL, which receives cholesterol from various tissues including atherosclerotic wall, is related to the action of removing cholesterol accumulated in cells, and is a risk prevention factor for various arteriosclerosis including coronary atherosclerosis, It is known that the blood level serves as a useful guide for predicting the onset of atherosclerotic disease.

[0003] As a method of measuring cholesterol in HDL, for example, HDL is separated from other lipoproteins by ultracentrifugation, and then subjected to cholesterol measurement. Methods for measuring the strength and the like are known. However, all of these methods require complicated operations,
There are problems such as the inability to process a large number of samples, and they are hardly used on a daily basis.

As a method for measuring cholesterol in HDL, a method generally used in the field of clinical tests at present is as follows.
A precipitant is added to the sample to aggregate lipoproteins other than HDL, and this is removed by centrifugation.
This is a method for measuring cholesterol in a supernatant containing only L. Although this method is simpler than ultracentrifugation and electrophoresis, it involves an operation of adding a precipitant and separating it. I need.

On the other hand, methods for enzymatically separating and quantifying cholesterol in HDL have already been studied. For example, H
A method in which lipoproteins other than DL are aggregated in advance with an antibody and a polyanion, only cholesterol in HDL is enzymatically reacted, and then the enzyme is inactivated and the aggregate is redissolved to measure the absorbance. (JP-A-6-242
No. 110 publication). However, since this method requires at least three operations of adding a reagent, it can be applied only to a limited number of analyzers, and has a problem in versatility.

Another method is to carry out an enzymatic reaction in the presence of a bile salt or a nonionic surfactant (Japanese Patent Application Laid-Open No. 63-128498). More recently, cholesterol esterase or cholesterol oxidase enzyme has been used. A method of specifically capturing cholesterol in HDL in the presence of an inclusion compound such as cyclodextrin (JP-A-7-301636) or forming an aggregate or complex with a lipoprotein other than HDL; To H
Method for capturing cholesterol in DL by an enzymatic reaction (JP-A-8-131197 and JP-A-8-20139)
No. 3) is known, but all of them have a problem in terms of specificity, for example, a difference from the precipitation method is observed in some of clinical samples.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to eliminate the need for complicated separation and separation procedures, and to provide HDL and low density lipoprotein (LDL), very low density lipoprotein (VLDL) and chylomicron (CM). Cholesterol in high-density lipoprotein used in a method for quantifying cholesterol in HDL, which can selectively, simply and accurately quantify HDL cholesterol in a test sample containing other lipoproteins such as It is to provide.

[0008]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that some surfactants act on HDL but hardly act on other lipoproteins. Then, first, cholesterol in lipoproteins other than the high-density lipoprotein in the test sample is selectively eliminated, and then cholesterol derived from HDL is enzymatically quantified in the presence of the above-mentioned surfactant, thereby obtaining HDL. The present inventors have found that HDL cholesterol in a test sample containing lipoproteins and other lipoproteins can be selectively, simply and accurately determined, and completed the present invention.

[0009] That is, the present invention is characterized by comprising a combination of at least cholesterol esterase, cholesterol oxidase and a surfactant having an HLB of 13 to 14, which specifically acts on cholesterol in high-density lipoprotein. Provided is a reagent for determining cholesterol in density lipoprotein.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Cholesterol contained in lipoproteins includes ester cholesterol (cholesterol ester) and free cholesterol. In this specification, the term “cholesterol” includes both of these. The test samples used in the method of the present invention include HDL, LDL, VLDL and CM
Any sample may be used as long as it contains a lipoprotein such as, for example, a body fluid such as serum or a dilution thereof, but is not limited thereto.

[0011] In the first step of the method of the present invention, cholesterol in lipoproteins other than HDL in the test sample is selectively eliminated. Here, “elimination” means decomposing cholesterol and preventing the decomposed product from being detected in the next second step. As a method for selectively eliminating lipoproteins other than HDL, that is, cholesterol contained in LDL, VLDL, CM and the like, the following methods can be mentioned.

That is, in the first method, cholesterol esterase and cholesterol oxidase are allowed to act on a test sample in the absence of a surfactant that acts on HDL, and the generated hydrogen peroxide is removed. Ester cholesterol in lipoproteins is hydrolyzed by the action of cholesterol esterase to produce free cholesterol and fatty acids. Next, the resulting free cholesterol and free cholesterol originally present in the lipoprotein are oxidized by the action of cholesterol oxidase to produce cholesterone and hydrogen peroxide. The generated hydrogen peroxide is removed. As a method to remove hydrogen peroxide,
By the method of decomposing into water and oxygen by the action of catalase, and the action of peroxidase, for example, DAOS (N-
Ethyl-N- (2-hydroxysulfopropyl) -3,
A method of converting hydrogen peroxide to colorless quinone by reacting with a phenol-based or aniline-based hydrogen donor compound which reacts with hydrogen peroxide to produce colorless quinone, such as 5-dimethyoxyaniline). Is not limited to these.

The first step is performed in the absence of a surfactant that acts on HDL, so that cholesterol in HDL hardly reacts, and LDL, VLDL,
Cholesterol in other lipoproteins such as CM reacts and is eliminated. Thereby, cholesterol in HDL is selectively quantified in the next second step.

The concentration of cholesterol esterase in the reaction solution of the first step is preferably about 0.2 to 1.0 U / ml, and the concentration of cholesterol oxidase is about 0.1 U / ml.
It is preferably about 0.7 U unit / ml. Further, the concentration of catalase is preferably about 40 to 100 U / ml, and the concentration of peroxidase is preferably about 0.4 to 1.0 U / ml. Further, the concentration of the compound which produces colorless quinone by reacting with hydrogen peroxide is preferably about 0.4 to 0.8 mmol / l.

The reaction of the first step is preferably carried out in a buffer having a pH of 5 to 8, and the buffer is preferably a buffer of phosphate, glycine, Tris and Good. Bis-Tris, PIPES, MO especially good buffers
PSO, BES, HEPES and POPSO are preferable, and the concentration of the buffer is preferably about 10 to 500 mM.

In the first step, a divalent metal ion may be contained in the reaction solution in order to further enhance elimination of lipoproteins other than HDL. Copper ions as divalent metal ions,
Although iron ions and magnesium ions can be preferably used, magnesium ions are particularly preferable. 2
The concentration of the valent metal ion is preferably about 5 to 200 mM.

The reaction solution of the first step may optionally contain a lipoprotein hydrolase. The addition of this enzyme is preferred because cholesterol in VLDL is particularly easy to react. The concentration of this enzyme in the reaction solution is preferably about 5.0 to 10.0 U / ml.

The reaction temperature in the first step is suitably about 25 ° C. to 40 ° C., and most preferably 37 ° C. The reaction time may be about 2 to 10 minutes.

In the subsequent second step, a surfactant that specifically acts on HDL is added to the product of the first step, and cholesterol in the high-density lipoprotein is enzymatically quantified. Here, the “surfactant specifically acting on HDL” means
When enzymes such as cholesterol esterase and cholesterol oxidase are allowed to act in the presence of the surfactant, cholesterol in HDL reacts (reaction rate 70% or more, preferably 90% or more), but HD
Cholesterol in lipoproteins other than L hardly reacts (reaction rate 30% or less, preferably 20% or less). Examples of such a surfactant include surfactants having a hydrophilic / lipophilic balance (HLB) of 13 to 14, and among them, an HLB of 13 to 14 is preferable.
14 nonionic surfactants, especially polyalkylene oxide derivatives. Here,
Examples of the derivatives include higher alcohol condensates, higher fatty acid condensates, higher fatty acid amide condensates, higher alkylamine condensates, higher alkylmercaptan condensates, and alkylphenol condensates. Further, among the polyalkylene oxide derivatives, a polyethylene oxide derivative is most preferable. Further, the HLB can be adjusted within the above range by mixing a plurality of surfactants, and a mixture of such a plurality of surfactants can be used. The method for calculating the HLB of a surfactant is well known, and is described in, for example, "New Surfactant", written by Hiroshi Horiguchi, published in 1986, Sankyo Publishing.

Specific examples of preferred surfactants include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, and polyoxyethylene higher alcohol (having 4 to 3 carbon atoms).
5) Ether, polyoxyethylene octyl phenyl ether, polyoxyethylene nonyl phenyl ether and the like can be mentioned, but not limited thereto.

[0021] The concentration of the surfactant in the second step is not particularly limited, but is preferably 0.05 to 10% based on the whole reaction mixture.
It is preferably 3% by weight, more preferably 0.1 to 1.5% by weight.

In the presence of the above surfactant, HDL cholesterol in the test sample can be enzymatically determined. That is, in the first step, cholesterol in lipoproteins other than HDL is mostly eliminated, but only the cholesterol in HDL is quantified by a synergistic effect with the reaction in the second step.

The method of enzymatic determination of cholesterol itself is well known in the art, for example, as in the first step,
It can be carried out by generating hydrogen peroxide from cholesterol ester and free cholesterol by the action of cholesterol esterase and cholesterol oxidase, and quantifying the generated hydrogen peroxide. The quantification of hydrogen peroxide can be performed, for example, by reacting with a compound that reacts with hydrogen peroxide to form a quinone dye in the presence of peroxidase, and measuring the amount of the resulting quinone dye by absorbance measurement or the like. it can. Quinone dyes include, for example, hydrogen peroxide and 4-aminoantipyrine and D
It is formed by reacting AOS or HDAOS (N- (2-hydroxysulfopropyl) -3,5-dimethyoxyaniline). The quinone dye thus formed has a maximum absorption at a wavelength of 593 nm when DAOS is used, and has a wavelength of 583 nm when HDAOS is used.
It has a maximum absorption at nm. The concentration of the compound that forms the quinone dye is not particularly limited, but preferably, for example, 4-aminoantipyrine is 0.1% with respect to the entire reaction mixture.
1 to 2.0 mM, more preferably 0.5 to 1.5 mM
And preferably 0.1 for DAOS or HDAOS.
-1.5 mM, more preferably 0.4-1.0 mM. The concentration of peroxidase is not particularly limited, but is preferably 0.4 to 5 U / ml based on the whole reaction mixture. The preferred reaction conditions (reaction temperature, reaction time, buffer, pH) of the second step are the same as the preferred reaction conditions of the first step.

When the produced hydrogen peroxide is decomposed with catalase in the first step, it is necessary to inhibit the catalase in the second step. Therefore, in the second step, for example, catalase such as sodium azide is used. Catalase is inhibited using an inhibitor.

[0025] Example Hereinafter, now be described by way of the present invention embodiment.
However, the present invention is not limited to the following examples. In the following examples, "%" indicates "% by weight" unless otherwise specified.

Reference Example 1 Using a sample containing purified HDL, LDL, VLDL or CM at a known concentration, Kao Corporation's nonionic surfactant Emulgen 911 (polyoxyethylene nonyl ether, H
LB 13.7), Emulgen B66 (polyoxyethylene derivative, HLB 13.2), or Emulgen B66 and Emulgen A90 (polyoxyethylene derivative, HLB)
Cholesterol in each lipoprotein was enzymatically quantified in the presence of the mixture of 14.5). This operation was specifically performed as follows.

50 mM PIPES buffer pH 7.0,
Cholesterol esterase 0.5 U / ml, cholesterol oxidase 0.4 U / ml, peroxidase 0.5 U / ml, 4-aminoantipyrine 1.0 mmol
1 / l, HDAOS 0.5 mmol / l, 0.1% by weight of Emulgen 911 or Emulgen B66, or a mixture of Emulgen B66 / Emulgen A90 (9 /
1) A reagent containing 1.3% by weight was prepared, 2.0 ml of the reagent was mixed with 20 μl of the sample, reacted at 37 ° C. for 10 minutes, and the absorbance at 600 nm was measured.

As a result, the reaction rate of cholesterol (the ratio of cholesterol determined in cholesterol) is
Cholesterol in HDL was about 95% and cholesterol in other lipoproteins was about 18-22%.

From this, it can be seen that Emulgen 911, Emulgen B66, and Emulgen B66 / Emulgen A90
The mixture corresponds to the “surfactant specifically acting on high-density lipoprotein” in the present invention.

Example 1 First and second reagents having the following compositions were prepared. First reagent PIPES buffer, pH 7.0 100 mmol / l HDAOS 0.7 mmol / l cholesterol esterase 0.8 U / ml cholesterol oxidase 0.5 U / ml catalase 80 U / ml magnesium chloride 10 mmol / l Second reagent PIPES buffer, pH 7 0.0 100 mmol / l 4-aminoantipyrine 4.0 mmol / l peroxidase 2.4 U / ml sodium azide 0.1% Kao Corporation Emulgen B66 (HLB 13.2) 0.3%

Purified HDL and LD at a concentration of 100 mg / dl
L, VLDL or CM each containing 4 types of samples, each containing 4
μl, 300 μl of the first reagent previously heated at 37 ° C.
, And reacted at 37 ° C. for 5 minutes.
Reaction was carried out at 37 ° C. for 5 minutes, and 600 n
The absorbance at m was measured. The amount of cholesterol was calculated from the measured absorbance, and the ratio to the amount of cholesterol in the sample was calculated to obtain the capture rate.

In this method, the hydrogen peroxide produced in the first step is decomposed by catalase, while the hydrogen peroxide produced in the second step reacts with HDAOS and 4-aminoantipyrine to produce a quinone dye. The results are shown in Table 1 below.

[0033]

[Table 1] Table 1

As shown in Table 1, according to the above method, cholesterol in HDL is mostly determined.
Cholesterol in other lipoproteins was hardly or not quantified, indicating that cholesterol in HDL can be selectively quantified by the method of the present invention.

Example 2 HDL cholesterol in a test sample was quantified in the same manner as in Example 1 except that healthy human serum was used as the test sample. On the other hand, HDL cholesterol in the same test sample was quantified by the precipitation method described in “Clinical Examination”, 23, 121 (1979). FIG. 1 shows a correlation diagram of the obtained measurement results.
Shown in

As shown in FIG. 1, the quantification results obtained by the two methods agree very well.
It was revealed that cholesterol in HDL can be accurately determined.

Example 3 The same operation as in Example 1 was performed except that the first and second reagents had the following compositions.

First reagent HEPES buffer, pH 7.0 50 mmol / l DAOS 1.5 mmol / l cholesterol esterase 0.8 U / ml cholesterol oxidase 0.5 U / ml peroxidase 1.0 U / ml Second reagent HEPES buffer, pH 7.0 50 mmol / l 4-aminoantipyrine 4.0 mmol / l Kao Corporation Emulgen 911 (HLB13.7) 0.3%

In this method, the hydrogen peroxide generated in the first step reacts with DAOS under the action of peroxidase to produce colorless quinone, and the hydrogen peroxide generated in the second step also undergoes the action of peroxidase in the first step. Reacts with residual DAOS and 4-aminoantipyrine added in the second step to form a quinone dye. The results are shown in Table 2 below.

[0040]

[Table 2] Table 2

As shown in Table 2, according to the above method, cholesterol in HDL is mostly determined.
Cholesterol in other lipoproteins was hardly or not quantified, indicating that cholesterol in HDL can be selectively quantified by the method of the present invention.

Example 4 HDL cholesterol in a test sample was quantified in the same manner as in Example 3, except that serum from a healthy person was used as the test sample. As in Example 2, HDL cholesterol in the same test sample was quantified by the precipitation method. FIG. 2 shows a correlation diagram of the obtained measurement results.

As shown in FIG. 2, the results of the quantification by the two methods agree very well, and the method of the present invention
It was revealed that cholesterol in HDL can be accurately determined.

Example 5 First and second reagents having the following compositions were prepared. 1st reagent BES buffer, pH 7.0 100 mmol / l HDAOS 0.7 mmol / l cholesterol esterase 0.8 U / ml cholesterol oxidase 0.5 U / ml catalase 100 U / ml magnesium chloride 18 mmol / l 2nd reagent BES buffer, pH7 4.0 100 mmol / l 4-aminoantipyrine 4.0 mmol / l peroxidase 2.4 U / ml sodium azide 0.1% Kao Corporation Emulgen B66 (HLB 13.2) 1.17% Kao Corporation Emulgen A90 (HLB 14.5) 0.13%

Purified HDL and LD at a concentration of 100 mg / dl
L, VLDL or CM each containing 4 types of samples, each containing 4
μl, 300 μl of the first reagent previously heated at 37 ° C.
, And reacted at 37 ° C. for 5 minutes.
00 µl was reacted for 5 minutes, and the absorbance of the reaction solution at 600 nm was measured. The amount of cholesterol was calculated from the measured absorbance, and the ratio to the amount of cholesterol in the sample was calculated to obtain the capture rate.

In this method, the hydrogen peroxide produced in the first step is decomposed by catalase, while the hydrogen peroxide produced in the second step reacts with HDAOS and 4-aminoantipyrine to form a quinone dye. The results are shown in Table 3 below.

[0047]

[Table 3] Table 3

As shown in Table 3, according to the above method, cholesterol in HDL is mostly determined.
Cholesterol in other lipoproteins was hardly or not quantified, indicating that cholesterol in HDL can be selectively quantified by the method of the present invention.

Example 6 HDL cholesterol in a test sample was quantified in the same manner as in Example 5 except that healthy human serum was used as the test sample. On the other hand, HDL cholesterol in the same test sample was quantified by the precipitation method described in “Clinical Examination”, 23, 121 (1979). FIG. 3 shows a correlation diagram of the obtained measurement results.
Shown in

As shown in FIG. 3, the results of the quantification by the two methods agree very well, and the method of the present invention
It was revealed that cholesterol in HDL can be accurately determined.

[0051]

According to the method of the present invention, HDL and HDL in a test sample containing other lipoproteins such as LDL, VLDL and CM can be selectively obtained without the need for complicated fractionation and separation operations. Quantification can be performed simply and accurately.

[Brief description of the drawings]

FIG. 1 shows HDL measured by the method of one embodiment of the present invention.
FIG. 4 is a correlation diagram showing the relationship between the amount of cholesterol in HDL and the amount of cholesterol in HDL measured by a conventional precipitation method.

FIG. 2 shows H measured by the method of another embodiment of the present invention.
It is a correlation diagram which shows the relationship between the cholesterol amount in DL and the cholesterol amount in HDL measured by the conventional precipitation method.

FIG. 3 is a correlation diagram showing the relationship between the amount of cholesterol in HDL measured by the method of still another embodiment of the present invention and the amount of cholesterol in HDL measured by a conventional precipitation method.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shuichi Ohara 135-1 Kagita, Kigoshi, Goizumi-city, Niigata Prefecture Inside the Kadenda Plant of Denka Seikaken Co., Ltd. (72) Inventor Akira Fujiwara, Kagita, Kiyoshi, Goizumi-shi, Niigata 1359-1 Denka Seiken Co., Ltd. Kagamida Factory

Claims (1)

[Claims] An HLB which specifically acts on at least cholesterol esterase, cholesterol oxidase, and cholesterol in high-density lipoprotein is 13%.
A reagent for quantifying cholesterol in high-density lipoprotein, which comprises a combination of the surfactants of Nos. 1 to 14.