WO1997000971A1

WO1997000971A1 - Method for assaying cholesterol in high-density lipoprotein fraction and assay reagent kit

Info

Publication number: WO1997000971A1
Application number: PCT/JP1996/001602
Authority: WO
Inventors: Masafumi Ikeda; Hiromasa Tabata; Tsutomu Kakuyama; Yoichi Hashiguchi
Original assignee: International Reagents Corporation
Priority date: 1995-06-21
Filing date: 1996-06-12
Publication date: 1997-01-09
Also published as: JP3614514B2; JPH09299A

Abstract

A method for assaying cholesterol in a high-density lipoprotein (HDL) fraction which comprises the following steps. By using an acylpolyoxyethylene sorbitan ester, cholesterol in the fractions of lipoproteins in a sample other than HDL is preferentially treated with enzymes such as cholesterol esterase and cholesterol oxidase to thereby form hydrogen peroxide. In the presence of peroxidases, colorless complexes are formed to thereby eliminate the hydrogen peroxide from the reaction system. Next, the actions of these enzymes on the cholesterol remaining in the fractions of lipoproteins other than HDL are suppressed by alkyl polyoxyethylene ethers. At the same time, the cholesterol contained in the HDL fraction is treated by these enzymes to thereby form hydrogen peroxide. The quinone coloring matter formed by the hydrogen peroxide is measured by the colorimetry to thereby determine the cholesterol content of the HDL fraction. By using this simplified method, a number of samples can be processed in a short time and even samples in a very small amount can be continuously assayed with an automatic analyzer. Thus it contributes to the improvement in the efficiency of routine work in clinical examinations.

Description

W 7

Specification

Method for quantifying cholesterol in high-density lipoprotein fraction and reagent kit for quantification

The present invention relates to a method for quantifying cholesterol in a high-density lipoprotein (HDL) fraction (hereinafter also referred to as “HDL cholesterol”) and a reagent kit for quantification. The present invention relates to a method for quantifying HDL cholesterol in a sample, and a reagent kit for quantifying HDL cholesterol.

Background art

Lipoproteins in blood are classified into chylomicron (specific gravity 1,006), ultra-low-density lipoprotein (¥ 101 ^, specific gravity 1.006-1.019), low-density lipoprotein (LDL, specific gravity) 1.019-1.063), high-density lipoprotein (HDL, specific gravity 1.063-1.21), etc., and research on diseases that affect the metabolism of these lipoproteins is ongoing. Have been. Among them, HDL, in 1977, stated that cholesterol, a component in the fraction, was closely related to ischemic heart disease.

Research has progressed rapidly since Framingham's report.

Conventionally, the quantification of HDL cholesterol has been measured by a known method for cholesterol, a component of the HDL fraction separated by a precipitation fractionation method, an ultracentrifugation method, an electrophoresis method, or the like. Precipitation fractionation is commonly used in clinical laboratory measurements. This involves precipitating a lipoprotein fraction other than HDL using a precipitant, and centrifuging it to obtain an HDL fraction. As a precipitant at this time, a combination of polyadione and divalent thione is often used. Such polyadiones include polyethylene glycol, phosphotungstic acid, dextran sulfate, and the like, and divalent cations such as Mg, Mn, Ca, Li, and Ni are known.

Next, as a known method for quantifying cholesterol in the HDL fraction obtained by centrifugation, measurement by an enzyme reaction is often used. Above all, cholesterol-esterase (CE) and cholesterol oxidase (CO) are used, and peroxidase (POD) is combined with a chromogen to measure absorbance in the visible region. Methods for measuring are well known.

However, centrifugation is required to separate the HDL fraction using such a conventional method using a precipitant, so an automatic analyzer is used to improve efficiency in daily clinical testing. In some cases, there was a restriction that it could not be used directly. Therefore, it was difficult to measure HDL cholesterol in a multichannel manner with other test items.

Disclosure of the invention

An object of the present invention is to solve the above-mentioned problems and to provide a useful method for efficiently measuring HD L cholesterol, particularly for a clinical test using an automatic analyzer, and a reagent kit therefor. .

The present inventors have conducted intensive studies and found that a surfactant that causes an enzyme to preferentially act on cholesterol in a lipoprotein fraction other than HDL, and an enzyme action on cholesterol in a lipoprotein fraction other than HDL. It has been found that cholesterol in the HDL fraction can be measured by using a surfactant that suppresses cholesterol. As a result of further studies, they found that the above-mentioned object was achieved, and completed the present invention.

That is, the present invention provides a reaction product obtained by causing an enzyme to preferentially act on cholesterol in a lipoprotein fraction other than HDL by using an acylpolyoxyethylene sorbitan ester to guide the reaction product out of the reaction system. The polyoxyethylene ester suppresses the action of enzymes on cholesterol remaining in lipoprotein fractions other than HDL, and allows the enzyme to act on HD L cholesterol to promote the reaction. It relates to a method for quantifying HD L cholesterol.

Furthermore, the present invention relates to a reagent kit for quantifying HD L cholesterol, comprising: a first reagent containing an acyl polyoxyethylene sorbitan ester and an enzyme; and a second reagent containing an alkyl polyoxyethylene ether. is there Detailed description of the invention

In the present invention, examples of the enzyme acting on cholesterol include cholesterol esterase (CE) and cholesterol oxidase (CO). Since lipoprotein fractions contain cholesterol esters in addition to cholesterol, cholesterol esterase (CE) is usually converted from cholesterol oxidase (CO) to hydrolyze cholesterol esters and convert them to cholesterol. ) And coexist.

For example, in the case of CE, the enzyme in the first reagent in the reagent kit of the present invention is 1 unit / m1 to 100 units / m1, preferably 1 unit Zm1 to 50 units In the case of C0, 0.5 unit Zml to l00 unit Zm, preferably 1 unit / ml to 50 units Zml, but it is appropriately determined according to the type of sample, etc. .

An acylboroxethylene ethylene sorbitan ester that causes the above enzyme to preferentially act on cholesterol contained in riboprotein fractions other than HDL, such as LDL, VLDL, and chylomicron, is usually abbreviated as C _n sorbitan E. Any non-ionic surfactant having polyoxyethylene in the hydrophilic portion and which meets the purpose of the present invention can be used. Specific trade names, Tween 2 1 (〇 ₁₂ sorbitan _{E 4), Tween 8 1 (} C! 2 Sorbitan _{E 5), Tween 20 ((} 12 sorbitan _{E 2.), Tween 40 (} C 16 sorbitan E ₂ ), Tween 60 (C ₁₈ sorbitan E ₂₀ ), Tween 80 (C ₁₈ sorbitan E ₂₀ ), Emasol 4130 ((: ₁₈ sorbitan Ex), Tween 85 (C _17: norbitan E ₂₀ ), etc. An example is shown.

The acylpolyoxyethylene sorbin ester in the first reagent in the reagent kit of the present invention is usually contained in the reaction solution at a concentration of from 0.001 ^ ¥% to 0.1 lw / v%, preferably 0.001%. It is blended so as to be 1 w / v% to 0.05 w / v%, but it is appropriately determined according to the type of the sample.

Inhibits the action of the above enzymes on cholesterol in riboprotein fractions other than HDL 9 71 T /

The alkyl polyoxyethylene ether to be used is a nonionic surfactant having a polyoxyethylene in the hydrophilic portion, usually abbreviated as C _n Ex, and should be used if it is compatible with the purpose of the present invention. Can be. Specific trade names, Atlas G 2 1 27 (C 12 E 8), Brij 36 T (C 12 E 10), Nopalcal 6- L (C12E14). Brij 35 (C12E23). Emulogphene BC 720 (C 12 _{_{E 9. 8) Sterox AJ 1}} 0 0 (C13E9. 5)> Brij 5 6 (CieE, 0)> Brij 5 8 (C, 6 E 20), Brij 76 (C 18 E 10), Brij 96 (C 18 _{_{: 1 E 10), Brij 78}} (C ] _{8 E20), Brij 98 (C} 18: 1 E 23) and the like are exemplified.

The alkylpolyoxyethylene ether in the second reagent in the reagent kit of the present invention is usually 0.001 wZv to 5 wZv%, preferably 0.05 w / v to 2 w / v in the reaction solution. %, And it is blended so as to have a higher concentration than that of the acylpolyoxyethylene sorbitan ester blended in the first reagent, but it is appropriately determined according to the type of the sample and the like.

In the quantification method of the present invention, first, an enzyme acting on cholesterol, the above-mentioned acylpolyoxyethylene sorbitan ester, and a sample are mixed. Then, due to the presence of acyl polyoxyethylene sorbitan ester, the enzyme contained in the reaction solution acts preferentially on cholesterol in the riboprotein fraction other than HDL.

When the enzyme is composed of a combination of CE and C 0, hydrogen peroxide is produced as a cholesterol reaction product by the action of the enzyme. In the quantification method of the present invention, before the alkylpolyoxyethylene ether in the second reagent is mixed in the reaction system, it is necessary to guide the generated reaction product out of the reaction system. For example, a chromogen is used. A chromogen is an organic compound containing a chromophore such as a nitro group, an azo group, a carbonyl group, an ethylene bond, and a cyan group, and not containing an auxiliary chromophore such as a hydroxyl group or an amino group. As a chromogen that can be used in the present invention, a chromogen having a low color-forming ability by itself, but having an enhanced color-forming ability due to the coexistence of the organic compound containing the auxiliary chromophore is preferable. When the reaction product is hydrogen peroxide, N- (2-hydroxy-3-sulfopropyl) -1,3,5-dimethoxya in the presence of peroxidase (POD) A colorless complex is formed by reacting chromogens such as sodium diphosphate (HDAOS) and N-ethyl (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS) with hydrogen peroxide. Hydrogen peroxide can be led out of the reaction system. Usually, the POD is from 0.01 U / ml to 10 UZm1, preferably 0.5 UZm1 to 5 U / ml, and the concentration of the chromogen is 0.001 wZv% to lwZv%, preferably 0. 0.1 wZv% to 0.5 wZv%, which is determined as appropriate according to the type of sample.

Next, by mixing the alkylpolyoxyethylene ether in the second reagent into the reaction solution, the action of the enzyme on the remaining cholesterol in the lipoprotein fraction other than HDL is suppressed, and Acts on HDL cholesterol, and the cholesterol reaction proceeds by the action of enzymes.

If the enzyme consists of a combination of CE and CO, this reaction produces hydrogen peroxide. In the presence of POD, this hydrogen peroxide can be converted to an organic compound containing an auxiliary chromophore such as, for example, 4-aminoantipyrine (0.001 wZv% to 0.1 wZv%, preferably 0.001 wZv% to 0.001 wZv% to 0.001 wZv% to 0.001 wZv% to 0.001 wZv%). 05 wZv%) and a chromogen to form a quinone dye. The chromogen includes HDAOS and TOOS described above. HDL cholesterol can be measured by colorimetric measurement of this quinone dye. The colorimetric measurement can be performed by a known method, for example, by using an automatic analyzer.

Each reaction in the method of the present invention, that is, each reaction before or after mixing the second reagent in the reaction system, is usually performed at room temperature for 1 minute to 10 minutes, preferably 3 minutes to 7 minutes. However, it is not particularly limited to this condition.

The reagent kit for quantifying HDL cholesterol according to the present invention comprises a first reagent containing the above-mentioned acylpolyoxyethylene sorbin ester and an enzyme, and a second reagent containing an alkylpolyoxyethylene ether.

Examples of the enzyme in the first reagent include CE, CO, and the like. Also the

In one reagent, besides the enzyme, usually, for example, the enzyme POD, HDAOS, TO Chromogens such as OS can be blended, but those that react with these chromogens to produce quinone dyes, such as 4-aminoantipyrine, are not blended.

On the other hand, the second reagent usually contains a substance which reacts with the chromogen to form a quinone dye, for example, 4-aminoantipyrine.

The reagent kit of the present invention may be divided into the first and second reagents at the latest at the time of quantitative measurement of HDL cholesterol at the latest. Therefore, in general, the form of a reagent kit should be divided into three or more types of reagents in consideration of stability, etc., and prepared into the above-mentioned first and second types of reagents at the time of use for measurement. May be something.

Example

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

(Example 1)

The following first and second reagents were prepared using Tween 85 (trade name) as an acylpolyoxyethylene sorbitan ester and Brij 98 (trade name) as an alkylpolyoxyethylene ether.

1 OmM phosphate buffer (pH 7.0), 0.12 mg gZm 1 HD AOS, 0.6 unit Zml POD, 10 unit 1 0 11 of 0 !, 10 unit 11 1 〇 £, 0.0 1 w / v% Tween 8 5

1 OmM phosphate buffer (pH 7.0), 0.15 mg / m 1 4-aminoantipyrine, 1.5 w / v% Brij 98

Using a Hitachi 7170 automatic analyzer, add the first reagent 250/1 to sample 41, add the second reagent 501 after 5 minutes, and then 600 nm / 70 minutes after 5 minutes. Measured at 0 nm. When a serum sample with an HDL cholesterol concentration of 150 mg / d1 diluted in 10 steps was used, good linearity was obtained as shown in Table 1. <

table 1

In this example, after adding the first reagent, complicated operations such as centrifugal separation are not required, and the amount of HDL cholesterol in the sample can be adjusted simply by adding the second reagent to the sample. The quinone dye thus produced is subjected to colorimetric measurement.

(Example 2)

Using the same reagents as in Example 1, 20 human serum samples were similarly measured with a Hitachi 7170 automatic analyzer, and the HDL cholesterol level was determined from the measurement of the standard solution. The same sample was compared with a value measured using a commercially available product (trade name: HDL-choles (PG), manufactured by International Reagents). As a result, as shown in Table 2, good correlation was obtained. Table 2 Samples Νοι Method of the present invention Conventional method (commercial product)

1 5 7. 3 5 5. 8

2 5 5. 6 5 8. 6

3 28. 5 2 1. 0

4 75, 4 74. 4

5 5 5.1 1 5 0.8

D 6 9.0 65.5

7 6 7. 0 6 1.5

8 3 7. 3 3 3.2

9 4 3.8 4 0.1

1 0 77.8 7 8.4

1 1 6 5. 6 5 6. 7

1 2 8 3.5 8 2.0

1 3 54. 4 5 2. 1

1 4 4 9. 4 4 7.5

1 5 57. 9 5 5.5

1 6 4 8. 3 44.0

1 7 5 6. 1 5 5.1

1 8 52. 9 4 9. 7

1 9 6 0. 3 5 2. 9

2 0 1 27. 4 1 24.5

Unit: / d £

Industrial applicability

According to the method for quantifying HDL cholesterol of the present invention, the operation of centrifugation conventionally required in the precipitation fractionation method is not required, so that the operation is simplified, and a large number of samples can be prepared in a short time. Can be easily processed. Moreover, since it can be applied to a method using two types of reagents, continuous measurement can be performed using a general-purpose automatic analyzer, and HDL cholesterol can be measured in a multichannel manner with other test items. In addition, the risk of virus infection is reduced because the chances of directly handling the sample in handling the sample are significantly reduced. In addition, it can measure even small amounts of sample Because of its ability, it can be measured even when blood collection is limited, such as in children and the elderly. Therefore, the method of the present invention is extremely useful in the field of clinical testing, and can contribute to improvement of work efficiency in daily clinical testing.

Further, the reagent kit for quantifying HDL cholesterol of the present invention has an effect as a reagent kit for performing the method of the present invention.

Claims

The scope of the claims

1. The reaction product obtained by preferentially acting the enzyme on cholesterol in the lipoprotein fraction other than the high-density riboprotein is guided to the outside of the reaction system by the acylpolyoxyethylene sorbin ester. Polyoxyethylene ether suppresses the enzyme's effect on cholesterol remaining in riboprotein fractions other than high-density riboprotein, and allows the enzyme to act on cholesterol in high-density lipoprotein fraction to proceed with the reaction. And a method for quantifying cholesterol in a high-density lipoprotein fraction.

2. The method according to claim 1, wherein the enzymes are cholesterol esterase and cholesterol oxidase.

3. Determination of cholesterol in a high-density riboprotein fraction characterized by comprising a first reagent containing an acylpolyoxyxylene sorbin ester and an enzyme, and a second reagent containing an alkylpolyoxyethylene ether Reagent kit.

4. The reagent kit according to claim 3, wherein the enzymes are cholesterol esterase and cholesterol oxidase.