JP2000214150A - Analyzer for lipoprotein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an analyzer in which replacement of a reaction reagent is not required and in which the quantity ratio of cholesterol or the like can be analyzed regarding a lipoprotein contained in a sample by one chromatographic operation by using one liquid chromatograph by a method wherein the sample is introduced to an isolation column by using a single flow passage and a component which is eluted from the isolation column is divided so as to be guided to a plurality of flow passages equipped with detectors. SOLUTION: A sample which is introduced to a single flow passage reaching respective isolation columns 6 by using an autosampler 4 is isolated so as to be eluted by the isolation columns 6 on the basis of the particle size, the electric charge or the hydrophobility of a lipoprotein. An eluted component is divided into two components by a splitter 8 so as to be introduced to respectively different flow passages. In the flow passage on one side, a cholesterol reaction reagent is mixed with the divied component, this mixture is reacted by a reaction coil so as to be guided to a detector, and cholesterol is detected and measured, In the flow passage on the other side, a triglyceride reaction reagent is mixed with the divided component, this mixture is reacted by a reaction coil so as to be guided to a detector, and a triglyceride is detected and measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an apparatus for analyzing lipoproteins contained in samples such as serum, plasma, cell culture supernatant and the like.

[0002]

2. Description of the Related Art Lipoprotein is a complex substance in which cholesterol, triglyceride (neutral fat), phospholipid and apoprotein are associated, and sometimes vitamin E and the like are also associated. Lipoproteins can be classified into chylomicrons (hereinafter referred to as "C") based on their particle size and the type of apoprotein contained.
M), very low density lipoprotein (hereinafter referred to as “VLD”).
L "), low density lipoprotein (hereinafter" LDL ")
HDL) and high-density lipoprotein (hereinafter referred to as “HDL”). For example, analyzing the ratio of cholesterol, triglyceride (neutral fat) and phospholipid contained in CM, VLDL, LDL, etc. in a human blood sample can be performed by analyzing ischemic heart disease, liver disease, diabetes, etc. It has become important in elucidating the relationship between diseases of the stomach and abnormalities in lipid metabolism.

[0003] In order to analyze the quantitative ratio of CM, VLDL, LDL, etc., a blood sample or the like is subjected to liquid chromatography such as gel filtration chromatography, ion exchange chromatography or reverse phase chromatography to reduce the lipoprotein particle size. CM, VLDL and L based on charge, hydrophobicity, etc.
After separation into DL, etc., cholesterol in each separated fraction,
In general, a simple and reproducible liquid chromatographic method of detecting, measuring and analyzing triglyceride (neutral fat) or phospholipid is used. In liquid chromatography, after separation by a separation column, a reaction reagent that reacts with cholesterol, phospholipid or triglyceride is mixed, reacted with a reaction coil, and then detected with a detector.
The chromatogram is obtained by the measurement, and the analysis is performed from the chromatogram.

[0004]

As described above, at present, lipoproteins such as CM, VLDL, and LDL contained in a liquid sample are determined by cholesterol, triglyceride (neutral fat) or lipoprotein contained in a liquid sample by liquid chromatography. By analyzing the ratio of phospholipids etc., ischemic heart disease, liver disease,
Elucidation of the relationship between diseases such as diabetes and abnormalities in lipid metabolism has been promoted. However, lipoproteins can be converted into CM,
Although separation into VLDL, LDL and the like is easy, if it is attempted to simultaneously detect and measure the cholesterol amount, triglyceride (neutral fat) amount and phospholipid amount in each of these separated fractions, for example, detection and measurement of cholesterol However, there is a problem that the reaction reagent for the reaction affects the detection and measurement of triglyceride (neutral fat) and phospholipid.

Therefore, in order to measure cholesterol, phospholipid, and triglyceride (neutral fat) for each of the separated fractions separated by liquid chromatography, the same sample is subjected to liquid chromatography at least three times. In the second operation, cholesterol, in the second operation, triglyceride (neutral fat), and in the third operation, a reaction reagent for measuring phospholipid is mixed and reacted, and detected and measured by a detector. Needed to obtain a chromatogram.

As a result, prior to each chromatographic operation, a reaction reagent to be mixed and reacted with a component eluted from the separation column is prepared and exchanged, or a plurality of liquid chromatograph devices (for example, three) are prepared. In addition, it is necessary to set different reaction reagents in each device and perform analysis using all the devices sequentially or at once.

[0007] When the lipoprotein is analyzed as described above, the sample is subjected to a plurality of liquid chromatograph operations, in addition to the problem that it takes time to prepare the analysis or increases the number of apparatuses used for the analysis. There is also a problem that a relatively large amount of sample is required because of the necessity. Furthermore, when a plurality of operations are performed using one liquid chromatograph, there is a possibility that the obtained chromatograms cannot be compared unless each operation is performed uniformly. Even when a plurality of liquid chromatograph devices are used, the same problem occurs unless uniformity is maintained between the devices, for example, the state of deterioration of the separation column, the amount of liquid sent, the detection sensitivity of the detector, and the like.

Accordingly, an object of the present invention is to eliminate the need for exchanging reaction reagents, and to carry out CM, VLDL, LDL, etc. contained in a sample by one liquid chromatograph operation using one liquid chromatograph. An object of the present invention is to provide a liquid chromatography apparatus capable of analyzing the lipoprotein of the present invention for the quantitative ratio of cholesterol, triglyceride (neutral fat), phospholipid and the like.

[0009]

Means for Solving the Problems The present invention made to achieve the above object is a liquid chromatography apparatus for analyzing a lipoprotein in a sample, wherein the sample is applied to a separation column using a single flow path. The components introduced and eluted from the separation column are divided and led to a plurality of flow paths each having a detector, and the lipoprotein is detected and measured using the detector for each of the components guided to each flow path. It is the device which did in this way. Hereinafter, a liquid chromatography apparatus for analyzing lipoprotein in a sample of the present invention (hereinafter simply referred to as “the apparatus of the present invention”) will be described in detail.

[0010] The apparatus of the present invention uses a lipoprotein in a sample based on CM, VLDL or L based on particle size, charge or hydrophobicity.
A separation column for separation into DL or the like is provided. Separation columns are, for example, gel filtration columns when trying to separate lipoproteins based on lipoprotein particle size, ion exchange columns when trying to separate lipoproteins based on lipoprotein charge, and A reverse phase column may be used for re-separation based on the hydrophobicity of the lipoprotein. Various types of columns are commercially available (for example, gel filtration columns manufactured by Tosoh Corporation)
Product name TSKgel Lipopropak XL, etc.)
Can be used.

In the apparatus of the present invention, a sample such as serum, plasma, cell culture supernatant, etc. is introduced into a separation column using a single channel. Here, a normal autosampler or the like is used to introduce the sample into the liquid chromatograph, a normal pump is used for sending the sample and the eluent, and a normal liquid chromatography is used for the tube forming the single flow path. Among them, those inactive against lipoproteins and reaction reagents described below can be used. The eluent is appropriately determined according to the type of separation column used. For example, if a gel filtration column is used, a salt component of 100 mM or more, such as sodium chloride, sodium sulfate, sodium nitrate, sodium acetate, sodium perchlorate, and ammonium nitrate, may be added for the purpose of reducing ionic interactions. it can.

In the apparatus of the present invention, the component eluted from the separation column is divided into two or more parts, each of which is led to a plurality of flow paths provided with a detector, and this detector is provided for each of the components led to each flow path. It is characterized in that lipoprotein is detected and measured by using the method. Here, the division in the apparatus of the present invention means that the lipoprotein contained in the sample is divided into two or more in a state of being separated based on the particle size, charge or hydrophobicity. Therefore, the divided components have completely the same composition, and the ratio of each composition is completely the same.

[0013] A splitter is arranged behind (downstream) the separation column in order to divide the components eluted from the separation column and guide them to a plurality of flow paths each having a detector. The splitter may be any as long as it can split the eluted component from the separation column into two or more. A splitter (for example, UPCHURCH SC) capable of adjusting the splitting ratio is used.
IENTIFIC INC. It is preferable to use a micro splitter (manufactured and trade name: Micro Splitter P-450). If the splitter can adjust the splitting ratio, for example, for those that can be detected and measured with relatively high sensitivity, the flow rate to the channel for performing detection and measurement is small, and on the contrary, detection and measurement with relatively low sensitivity are performed. This is because the flow rate to the flow path can be increased. In the present invention, when the flow path is divided into three parts, two splitters for two divisions can be connected and used instead of using the splitter for three divisions.

As described above, a tube used in ordinary liquid chromatography can be used for the flow path connected to the back (downstream) side of the splitter. The number of channels may be two or more, but CM, VLDL or L separated on a separation column based on the particle size, charge or hydrophobicity of lipoprotein.
DL and the like can be appropriately determined depending on how many types of measurements are performed later. At present, knowledge on the ratio of cholesterol, triglyceride (triglyceride) and phospholipid is considered to be important for elucidating the relationship between diseases such as ischemic heart disease, liver disease and diabetes and abnormal lipid metabolism. Therefore, the components eluted from the separation column are divided into three and led to three flow paths, and cholesterol, triglyceride (neutral fat) or phospholipid can be measured for each component led to each flow. It is particularly preferable to connect three flow paths to the splitter. Of course, in addition to the above, when it is necessary to detect and measure, for example, vitamin E, the number of channels connected to the splitter may be increased to four or more.

In the measurement of cholesterol, triglyceride (neutral fat) or phospholipid performed for each component guided to each flow channel, the cholesterol reaction reagent, triglyceride (medium) For example, after mixing and reacting a reactive reagent or a phospholipid reaction reagent, the mixture is introduced into a detector, and the state of the reaction with each reaction reagent is measured. That is, for example, the component eluted from the separation column is divided into three and guided to the first, second or third flow path, and the first flow path is mixed and reacted with a cholesterol reaction reagent to perform measurement. For the flow path, triglyceride (neutral fat) reagent is mixed, reacted and measured.
Then, for the third channel, a phospholipid reaction reagent is mixed,
A specific example is that measurement is performed by reacting.

In order to implement the above example, for example,
A reaction coil is placed in the part of each flow path before reaching the detector,
Immediately before this reaction coil, a flow path from a reservoir for storing each reaction reagent may be connected via a liquid sending pump.

As an example of each of the above-mentioned reaction reagents, a cholesterol reaction reagent contains a reagent solution containing cholesterol oxidase and cholesterol esterase.
A reagent solution containing lipoprotein lipase and glycerol oxidase can be exemplified as the triglyceride (neutral fat) reaction reagent, and a reagent solution containing phospholipase and choline oxidase can be exemplified as the phospholipid reaction reagent. Here, the reaction reagent preferably contains a surfactant for decomposing the lipoprotein so that cholesterol, triglyceride or phospholipid can react with the corresponding reaction reagent. As each of such preferable reaction reagents, commercially available reagents (for cholesterol reaction reagents, Determiner LTC manufactured by Kyowa Medix Co., Ltd., Collas Color Liquid manufactured by Toyobo Co., Ltd., manufactured by Daiichi Kagaku Co., Ltd.) Name Pure Auto S
CHO-N; a triglyceride (neutral fat) reaction reagent manufactured by Kyowa Medix Co., Ltd.
Pure Auto S TG-N manufactured by Daiichi Pure Chemicals Co., Ltd. can also be used.

The detector provided in each channel is not particularly limited as long as it can detect components such as cholesterol, triglyceride (neutral fat) and phospholipid and measure the amount. For example, as described above, if detection and measurement are performed after mixing and reacting a cholesterol reagent, a triglyceride (neutral fat) reagent or a phospholipid reagent, two or more identical absorbance detectors are used. It is also possible.

In the apparatus of the present invention, the number of components to be eluted from the separation column is divided (that is, the number of dewdrops connected to the splitter), the method of detecting and measuring cholesterol and the like after division,
Then, it is preferable to determine the amount of the sample to be analyzed in consideration of the detection sensitivity and the like of the detector provided in each channel. For example, when the above-mentioned reaction reagent is used and the flow path is divided into two, at least 15 μm is used.
It is preferable to use a sample of about 1, preferably about 20 μl. As described above, if the eluted components from the separation column are equally divided, the use of a sample of the number of flow channels × 10 μl can be exemplified as a tentative guide so that 10 μl of the components is guided to each flow channel. .

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to embodiments and examples shown in the drawings, but the present invention is not limited to these.

FIG. 1 is a diagram for explaining the outline of the apparatus of the present invention. 1 is an eluent reservoir, 2 is a degasser (degasser), 3 is a liquid sending pump, 4 is an autosampler, 5 is a filter, and 6 is a separation column (in this example, two columns are arranged in series and the separation capacity is , 7 is a column oven, 8 is a splitter for dividing the flow path into two, 9 is a resistance tube, 1
0 and 11 are reaction coils, 13 and 14 are detectors, 15
Is a liquid sending pump for a reaction reagent, 16 is an air trap, 17
Denotes a cholesterol reaction reagent reservoir, and 18 denotes a triglyceride reaction reagent reservoir.

The sample introduced into the single channel up to the separation column by the autosampler is separated and eluted by the separation column based on the particle size, charge or hydrophobicity of the lipoprotein. The eluted components are split into two by a splitter,
Each is introduced into a different flow path. In one flow path, a cholesterol reaction reagent is mixed with the divided components and reacted by a reaction coil, and then guided to a detector to detect and measure cholesterol. In the other flow path, a triglyceride reaction reagent is mixed with the divided components and reacted by a reaction coil, and then guided to a detector to detect and measure triglyceride.

Example 1 Using the apparatus shown in FIG. 1, lipoprotein analysis in human serum was performed. A commercially available product (manufactured by Tosoh Corporation, trade name: CCPS) is used as the liquid sending pump 3 in FIG. 1, and a commercially available product (manufactured by Tosoh Corporation, trade name: CCPM-2) is used as the liquid sending pump 15 for the reaction reagent. ), Autosampler 4
Are commercially available products (manufactured by Tosoh Corporation, trade name AS-8)
020), a commercially available product (Filter K, manufactured by Tosoh Corporation) as the filter 5, and a commercially available product (TSKgel Lipoprop, manufactured by Tosoh Corporation) as the column 6.
ak XL, column size; D. × 30
cm) was used.

The resistance tube 9 has a diameter of 0.2 mmI. D. × 2
m-sized stainless steel tubes were used as the reaction coils 10 and 11 at 0.4 mmI. D. × 7.5m, 0.4
mmI. D. A Teflon tube having a size of × 40 m was used.

Both the column oven 7 and the reactor 12 are commercially available products (manufactured by Tosoh Corporation, trade name CO-80).
20), and the set temperatures were 25 ° C. and 37 ° C., respectively. As the detectors 13 and 14, commercially available products (trade names UV-8020, UV-80, manufactured by Tosoh Corporation)
00), and the respective detection wavelengths were set to 550 nm.

The air trap 16 is a commercially available product (trade name: Air Trap G, manufactured by Tosoh Corporation), and the degasser 2 is a commercially available product (trade name: S, manufactured by Tosoh Corporation)
D-8022) was used.

Eluent 1 was 50 mM Tris and 3
A pH 8.0 solution containing 00 mM sodium acetate was used, and the flow rate was 0.60 ml / min. As the splitter 8, a commercially available product (UPCHURCH SCIENTI)
FIC INC. Product name, micro splitter P-
450), the eluate after column separation was adjusted to be divided into equal volumes.

As for the cholesterol reaction reagent 17,
A commercially available reagent (a reaction reagent obtained by mixing reagents 1 and 2 of Pure Auto S CHO-N (trade name, manufactured by Daiichi Pure Chemicals Co., Ltd.) at a ratio of 2: 1 in advance) was used at a flow rate of 0.15 m.
1 / min. Triglyceride (neutral fat) reaction reagent 1
For No. 8, (reaction reagent prepared by mixing the reagents 1 and 2 of Pure Auto S TG-N (trade name, manufactured by Daiichi Pure Chemical Co., Ltd.) at a ratio of 2: 1 in advance) was used, and the flow rate was 0.30 m.
1 / min.

[0029] Serum from a normal human was analyzed by an autosampler 4 for 15 minutes.
The mixture was applied to a μl apparatus, separated by a column 6, and divided into equal parts by a splitter 8. One of them was mixed with a cholesterol reaction reagent, reacted with the reaction coil 10, and detected and measured by the detector 13 at a wavelength of 550 nm. The other part was mixed with a triglyceride (neutral fat) reaction reagent, reacted with the reaction coil 11, and detected and measured by the detector 14 at a wavelength of 550 nm. The results are shown in FIGS.

FIG. 2 shows the results of detection and measurement (chromatogram) of cholesterol. LDL and H
The DL peak can be confirmed well, and the sample subjected to analysis is 1
Despite the small volume of 5 μl, VLDL is not completely separated at the LDL peak,
A peak was confirmed.

FIG. 3 shows the results of detection and measurement (chromatogram) of triglyceride (neutral fat). C
The peaks of M, LDL, HDL, and FG (free glycerol) could be confirmed well, and even though the amount of the sample used for analysis was as small as 15 μl, the VLDL was also LDL.
Although the separation was incomplete before the peak of, a peak was confirmed.

[0032]

According to the present invention, there is no need to exchange reaction reagents, and C contained in a sample can be obtained by one liquid chromatograph operation using one liquid chromatograph.
Provided is a liquid chromatography apparatus capable of detecting, measuring, and analyzing the amount of cholesterol, triglyceride (neutral fat) or phospholipid for lipoproteins such as M, VLDL, and LDL.

As a result, after the lipoproteins are separated by the separation column, the eluted components are guided to a plurality of flow paths, and the detection and measurement are performed using the detector provided for each flow path. According to the apparatus, even if only one apparatus is used, the cholesterol amount, phospholipid amount, triglyceride ( Triglyceride) is detected and measured, and finally CM, VLDL or LD
For each lipoprotein such as L, it is possible to analyze what amount of cholesterol, triglyceride (neutral fat) or phospholipid is present.

Therefore, according to the apparatus of the present invention, a shorter period of time is required as compared with the conventional method in which a plurality of chromatographic operations must be performed or a plurality of liquid chromatograph apparatuses must be used in order to obtain the same analysis results. It is possible to obtain an analysis result by using less sample in time.

[Brief description of the drawings]

FIG. 1 is a diagram for explaining an outline of an apparatus of the present invention.

FIG. 2 is a result (chromatogram) of cholesterol detection / measurement in Example 1, and the horizontal axis represents a time (minute) after the sample was supplied to the apparatus. VLD in the figure
L, LDL, and HDL are very low density lipoproteins,
Shows low density lipoprotein and high density lipoprotein.

FIG. 3 is a result (chromatogram) of detection and measurement of triglyceride (neutral fat) in Example 1.
The horizontal axis indicates the time (minutes) after the sample was supplied to the apparatus. CM, VLDL, LDL, HDL, and FG in the figure represent chylomicron, very low density lipoprotein, low density lipoprotein, high density lipoprotein, and free glycerol, respectively.

[Explanation of symbols]

1 eluent, 2 degasser, 3 feed pump, 4 autosampler, 5 filter, 6 separation column, 7 column oven (incubator), 8 splitter, 9 resistance tube, 10.11
Reaction coil, 12 reactors (constant temperature chamber), 13/14 detector, 15 liquid supply pump for reaction reagent, 16 air trap, 17 cholesterol reaction reagent, 18 triglyceride reaction reagent

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 G01N 33/50 U 33/68 33/68 33/92 33/92 A CZ // C12M 1/34 C12M 1/34 E

Claims (2)

[Claims] 1. A liquid chromatography apparatus for analyzing a lipoprotein in a sample, wherein the sample is introduced into a separation column by using a single flow path, components eluted from the separation column are divided, and a detector is used for each. An apparatus configured to guide to a plurality of flow paths provided and to detect and measure lipoprotein using the detector for each component guided to each flow path. 2. The method according to claim 1, wherein the components eluted from the separation column are n (n is 2).
Or 3) is divided into n flow paths each having a detector, and cholesterol reaction reagent, triglyceride (neutral fat) reaction reagent or phospholipid reaction is performed on the components guided to each flow path. 2. The apparatus according to claim 1, wherein n kinds of reaction reagents selected from the reagents are mixed and reacted, and then a lipoprotein is detected and measured using a detector for each channel.