JP4058828B2 - Lipoprotein analyzer - Google Patents

Description

[0001]
[Technology to which the invention belongs]
The present invention relates to an apparatus for analyzing lipoproteins contained in samples such as serum, plasma, and cell culture supernatant.
[0002]
[Prior art]
Lipoprotein is a substance in which cholesterol, triglyceride (neutral fat), phospholipid and apoprotein are associated, and is a complex complex in which vitamin E and the like are sometimes associated. Lipoprotein is based on its particle size and the type of apoprotein contained, chylomicron (hereinafter referred to as “CM”), very low density lipoprotein (hereinafter referred to as “VLDL”), low density lipoprotein ( Hereinafter, it is classified as “LDL”), high specific gravity lipoprotein (hereinafter referred to as “HDL”), and the like. And, for example, analyzing the quantitative ratio for the content of cholesterol, triglyceride (neutral fat) and phospholipid contained in CM, VLDL, LDL, etc. in human blood samples ischemic heart disease, liver disease, diabetes, etc. It is important to elucidate the relationship between diseases and abnormal 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, and the lipoprotein is reduced in particle size, charge or After separation into CM, VLDL, LDL, etc. based on hydrophobicity, etc., the cholesterol, triglyceride (neutral fat) or phospholipid, etc. in each separated fraction is detected and measured and analyzed with good reproducibility. It is common to use liquid chromatography. In the liquid chromatographic method, 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 and measured with a detector to obtain a chromatogram. Perform analysis from gram.
[0004]
[Problems to be solved by the invention]
As described above, at present, the liquid chromatographic method is used to analyze the quantitative ratio of cholesterol, triglyceride (neutral fat), phospholipid, and the like for lipoproteins such as CM, VLDL, and LDL contained in the liquid sample. Therefore, elucidation of the relationship between diseases such as ischemic heart disease, liver disease and diabetes and abnormal lipid metabolism has been promoted. However, it is easy to separate lipoproteins into CM, VLDL, LDL, etc. based on particle size, charge or hydrophobicity by a single liquid chromatographic operation. However, the amount of cholesterol, triglycerides (medium If you try to detect and measure the amount of sexual lipids) and the amount of phospholipids at the same time, for example, the reaction reagent for detecting and measuring cholesterol will affect the detection and measurement of triglycerides (neutral fats) and phospholipids. There is.
[0005]
For this reason, in order to measure cholesterol, phospholipid, and triglyceride (neutral fat) for each separated fraction separated by liquid chromatography, the same sample is subjected to liquid chromatography at least three times, and the first operation is performed. Then, in the second operation, triglyceride (neutral fat) is mixed, and in the third operation, a reaction reagent for measuring phospholipid is mixed and reacted. Had to get.
[0006]
As a result, prior to each chromatographic operation, prepare or replace reaction reagents to be mixed and reacted with the components eluted from the separation column, or prepare a plurality of (for example, three) liquid chromatograph devices. It was necessary to set different reaction reagents in the sample and perform analysis using all the devices in sequence or at a time.
[0007]
When the analysis of lipoproteins is performed as described above, the sample must be subjected to multiple liquid chromatograph operations in addition to the problem that it takes time to prepare for the analysis or the number of devices used for the analysis increases. Therefore, there is a problem that a relatively large amount of sample is required. Furthermore, when a plurality of operations are performed using a single 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 apparatuses are used, the same problem arises if the degradation state of the separation column, the amount of liquid transport, the detection sensitivity of the detector, and the like are not kept uniform.
[0008]
Accordingly, an object of the present invention is to eliminate the need for exchanging reaction reagents, and to perform lipoproteins such as CM, VLDL, and LDL contained in a sample by a single liquid chromatograph operation using a single liquid chromatograph apparatus. An object of the present invention is to provide a liquid chromatography apparatus capable of analyzing 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 lipoproteins in a sample, wherein the sample is introduced into the separation column using a single channel, and the components eluted from the separation column Are each led to a plurality of flow paths provided with detectors, and the detection and measurement of lipoproteins are performed using the detector for each component led to each flow path. Hereinafter, a liquid chromatography apparatus (hereinafter simply referred to as “the apparatus of the present invention”) for analyzing lipoproteins in a sample of the present invention will be described in detail.
[0010]
The apparatus of the present invention is equipped with a separation column for separating lipoproteins in a sample into CM, VLDL, LDL or the like based on particle size, charge or hydrophobicity. The separation column is, for example, a gel filtration column when separating the lipoprotein based on the particle size of the lipoprotein, an ion exchange column when separating the lipoprotein based on the charge of the lipoprotein, and A reverse phase column may be used when reseparating based on the hydrophobicity of the lipoprotein. Examples of the various columns include those commercially available (for example, if it is a gel filtration column, manufactured by Tosoh Corporation, trade name TSKgel Lipopropak XL).
[0011]
In the apparatus of the present invention, samples such as serum, plasma, and cell culture supernatant are introduced into a separation column using a single channel. Here, a normal autosampler or the like is used for introducing the sample into the liquid chromatograph, a normal pump is used for feeding the sample or the eluent, and a normal liquid chromatography is used for the tube forming the single flow path. Among them, those inactive with respect to lipoproteins and reaction reagents described later can be used. The eluent is appropriately determined according to the type of separation column used. For example, if a column for gel filtration is used, adding 100 mM or more of salt components such as sodium sulfate, sodium sulfate, sodium nitrate, sodium acetate, sodium perchlorate, ammonium nitrate for the purpose of reducing ionic interactions is an example. it can.
[0012]
In the apparatus of the present invention, the component eluted from the separation column is divided into two or more, each led to a plurality of flow paths equipped with detectors, and this detector is used for each component guided to each flow path. It is characterized in that it detects and measures proteins. 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]
In order to divide the components eluted from the separation column and guide them to a plurality of flow paths each equipped with a detector, a splitter is disposed behind (downstream) the separation column. The splitter is not particularly limited as long as it can divide the elution components from the separation column into two or more, but it is preferable to use a splitter (for example, product name: Microsplitter P-450, manufactured by UPCHURCH SCIENTIFIC INC.) Capable of adjusting the split ratio. . If the splitter is capable of adjusting the division ratio, for example, for those that can be detected and measured with relatively high sensitivity, the flow rate to the flow path for detecting and measuring is small, and conversely, detection and measurement with relatively low sensitivity is performed This is because the flow rate to the flow path can be increased. Further, when the flow path is divided into three in the present invention, two splitting splitters can be connected and used in addition to using the splitting splitter.
[0014]
As the flow path connected to the rear (downstream) side of the splitter, a tube used in normal liquid chromatography can be used as described above. The number of channels may be two or more, but depending on how many types of measurements are performed later on CM, VLDL, LDL, etc. separated on the separation column based on lipoprotein particle size, charge or hydrophobicity. It can be determined as appropriate. At present, knowledge about the quantitative ratio of cholesterol, triglyceride (neutral fat) and phospholipid is considered to be important in elucidating the relationship between diseases such as ischemic heart disease, liver disease and diabetes and abnormal lipid metabolism. Therefore, the component eluted from the separation column is divided into three parts and led to three flow paths, so that cholesterol, triglyceride (neutral fat) or phospholipid can be measured for each component led to each dew. It is particularly preferable to connect three flow paths to the splitter. Of course, in addition to the above, for example, when detection and measurement of vitamin E or the like is necessary, the number of channels connected to the splitter may be increased to four or more.
[0015]
Cholesterol, triglyceride (neutral fat) or phospholipid is measured for each component led to each channel. After splitting, the cholesterol reaction reagent and triglyceride (neutral fat) are measured for the components led to each channel. It can be exemplified that a reaction reagent, a phospholipid reaction reagent or the like is mixed and reacted and then introduced into a detector, and the state of reaction with each reaction reagent is measured. That is, for example, the component eluted from the separation column is divided into three parts and led to the first, second or third flow path, and the first flow path is mixed and reacted with the cholesterol reaction reagent to perform the measurement. Specifically, the triglyceride (neutral fat) reaction reagent is mixed and reacted for the flow path of, and the measurement is performed by mixing and reacting the phospholipid reaction reagent for the third flow path. it can.
[0016]
In order to implement the above specific example, for example, a reaction coil is arranged in a part of each flow path before reaching the detector, and a flow path from a liquid reservoir for storing each reaction reagent is provided immediately before this reaction coil. What is necessary is just to connect via a liquid feeding pump.
[0017]
If an example is described about each said reaction reagent, the reagent liquid containing cholesterol oxidase and cholesterol esterase will be used for the cholesterol reaction reagent, and the reagent liquid containing lipoprotein lipase and glycerol oxidase will be used for the triglyceride (neutral fat) reaction reagent. Examples of the reagent include a reagent solution containing phospholipase and choline oxidase. Here, the reaction reagent preferably contains a surfactant for decomposing lipoproteins so that cholesterol, triglyceride or phospholipid can be reacted with the corresponding reaction reagent. As such preferable reaction reagents, commercially available reagents (for cholesterol reaction reagents, Kyowa Medix Co., Ltd., trade name Determiner LTC, Toyobo Co., Ltd., trade name Chorus Color Liquid, Daiichi Chemicals Co., Ltd. Name Pure Auto S CHO-N; Triglyceride (neutral fat) reaction reagent may be Kyowa Medics Co., Ltd., trade name Determiner TG, Daiichi Chemical Co., Ltd., Pure Auto S TG-N) it can.
[0018]
The detector equipped in each flow path is not particularly limited as long as it can detect components such as cholesterol, triglyceride (neutral fat), phospholipid and the like and measure the amount thereof. For example, as described above, if detection / measurement is performed after mixing and reacting a cholesterol reaction reagent, a triglyceride (neutral fat) reaction reagent, or a phospholipid reaction reagent, two or more of the same absorbance detector are used. It is also possible.
[0019]
In the apparatus of the present invention, the number of the elution components from the separation column (that is, the number of dew connected to the splitter), the detection / measurement method of the cholesterol after the division, the detection sensitivity of the detector provided in each flow path, etc. In consideration of the above, it is preferable to determine the amount of the sample to be subjected to the analysis. For example, in the case where the reaction reagent as described above is used and the flow path is divided into two, it is preferable to use a sample of at least 15 μl, preferably about 20 μl. In this way, if the eluted components from the separation column are evenly divided, it is possible to exemplify using a sample of the number of channels × 10 μl so that 10 μl of the components are guided to each channel. .
[0020]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, although this invention is demonstrated in detail using embodiment and Example described in drawing, this invention is not limited to these.
[0021]
FIG. 1 is a diagram for explaining the outline of the device of the present invention. 1 is an eluent reservoir, 2 is a degassing device (degasser), 3 is a liquid feed pump, 4 is an autosampler, 5 is a filter, and 6 is a separation column (in this example, two columns are arranged in series for separation performance) 7 is a column oven, 8 is a splitter for dividing the flow path, 9 is a resistance tube, 10 and 11 are reaction coils, 13 and 14 are detectors, 15 is a reaction reagent feed pump, and 16 is An air trap, 17 represents a cholesterol reaction reagent reservoir, and 18 represents a triglyceride reaction reagent reservoir.
[0022]
The sample introduced into the single flow path leading to the separation column by the autosampler is separated and eluted on the separation column based on the particle size, charge or hydrophobicity of the lipoprotein. The eluted component is divided into two by a splitter and introduced into different flow paths. In one channel, a cholesterol reaction reagent is mixed with the divided components, reacted in a reaction coil, and then guided to a detector to detect and measure cholesterol. In the other channel, the triglyceride reaction reagent is mixed with the divided components, reacted in the reaction coil, and then guided to the detector to detect and measure triglyceride.
[0023]
Example 1
Lipoprotein analysis in human serum was performed using the apparatus shown in FIG. In addition, as a liquid feeding pump 3 in FIG. 1, a commercial product (product name CCPS, manufactured by Tosoh Corporation), and as a liquid reagent pump 15 for a reaction reagent, a commercially available product (trade name CCPM-2, manufactured by Tosoh Corporation). ), A commercially available product (manufactured by Tosoh Corporation, trade name AS-8020) as the autosampler 4, a commercially available product (manufactured by Tosoh Corporation, filter K) as the filter 5, and a commercially available product (as column 6). A Tosoh Co., Ltd. product, TSKgel Lipopropak XL, column size; 7.8 mm ID × 30 cm) was used.
[0024]
The resistance tube 9 is 0.2 mmI. D. A stainless steel tube having a size of 2 m is used as the reaction coils 10 and 11, respectively. D. × 7.5 m, 0.4 mm I.D. D. A Teflon tube having a size of 40 m was used.
[0025]
As the column oven 7 and the reactor 12, commercially available products (trade name CO-8020, manufactured by Tosoh Corporation) were used, and the set temperatures were 25 ° C. and 37 ° C., respectively. As the detectors 13 and 14, commercially available products (trade names UV-8020 and UV-8000, manufactured by Tosoh Corporation) were used, and the detection wavelengths were both 550 nm.
[0026]
A commercially available product (trade name Air Trap G, manufactured by Tosoh Corporation) was used as the air trap 16, and a commercially available product (trade name SD-8022, manufactured by Tosoh Corporation) was used as the degasser 2.
[0027]
As eluent 1, a pH 8.0 solution containing 50 mM Tris and 300 mM sodium acetate was used, and the flow rate was 0.60 ml / min. A commercial product (manufactured by UPCHURCH SCIENTIFIC INC., Trade name: Microsplitter P-450) was used as the splitter 8, and the eluate after column separation was adjusted so as to be divided into equal parts.
[0028]
For the cholesterol reaction reagent 17, a commercially available reagent (Daiichi Chemical Co., Ltd., trade name Pure Auto S CHO-N Reagents 1 and 2 previously mixed in a ratio of 2 to 1) was used. The flow rate was 0.15 ml / min. For the triglyceride (neutral fat) reaction reagent 18 (Daiichi Chemical Co., Ltd., trade name Pure Auto S TG-N Reagents 1 and 2 previously mixed in a ratio of 2 to 1), The flow rate was 0.30 ml / min.
[0029]
Normal human serum was subjected to a 15 μl apparatus using an autosampler 4 and separated by a column 6 and then divided into equal parts by a splitter 8. One of them was mixed with a cholesterol reaction reagent, reacted in 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 in the reaction coil 11, and detected and measured by the detector 14 at a wavelength of 550 nm. The results are shown in FIGS.
[0030]
FIG. 2 shows the detection / measurement results (chromatogram) for cholesterol. The peaks of LDL and HDL could be confirmed well, and although the sample used for analysis was as small as 15 μl, the peaks were confirmed even though VLDL was incompletely separated in the part before the peak of LDL. .
[0031]
FIG. 3 shows the results of detection and measurement (chromatogram) of triglycerides (neutral fat). The peaks of CM, LDL, HDL, and FG (free glycerol) can be confirmed well. Even though the sample used for analysis was as small as 15 μl, the separation of VLDL was not complete before the peak of LDL. There was a peak.
[0032]
【The invention's effect】
According to the present invention, there is no need to exchange reaction reagents, and cholesterol, such as CM, VLDL, and LDL, contained in a sample by a single liquid chromatographic operation using a single liquid chromatograph, A liquid chromatography apparatus capable of detecting, measuring and analyzing the amount of triglyceride (neutral fat) or phospholipid is provided.
[0033]
As a result, after separating the lipoprotein by the separation column, the eluted component is guided to a plurality of flow paths, and the analysis apparatus of the present invention performs detection / measurement using a detector provided for each flow path. For example, even when only one device is used, the amount of cholesterol, phospholipids, triglycerides (neutral fats) of lipoproteins contained in a sample in one operation without exchanging reaction reagents. ) Detect and measure the amount, and finally analyze the amount of cholesterol, triglyceride (neutral fat) or phospholipid present in each sample for each lipoprotein such as CM, VLDL or LDL Can be.
[0034]
Therefore, according to the apparatus of the present invention, compared to the conventional method in which a plurality of chromatographic operations or a plurality of liquid chromatograph apparatuses must be used in order to obtain a similar analysis result, in a shorter time, An analysis result can be obtained by using a smaller number of samples.
[Brief description of the drawings]
FIG. 1 is a diagram for explaining the 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 the time (minutes) after the sample was applied to the apparatus. In the figure, VLDL, LDL, and HDL indicate ultra low density lipoprotein, low density lipoprotein, and high density lipoprotein, respectively.
FIG. 3 is a result (chromatogram) of detection and measurement of triglyceride (neutral fat) in Example 1, and the horizontal axis indicates the time (minutes) after the sample was applied to the apparatus. CM, VLDL, LDL, HDL, and FG in the figure indicate chylomicron, ultra-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 reactor (incubator) , 13.14 detector, 15 reaction reagent feed pump, 16 air trap, 17 cholesterol reaction reagent, 18 triglyceride reaction reagent

Claims (1)

A liquid chromatography apparatus for analyzing lipoproteins in a sample, wherein the sample is introduced into the separation column using a single flow path, and the elution component from the separation column is divided into n (n is 2 or 3). Each led to n flow paths equipped with detectors, and n kinds of components selected from cholesterol reaction reagent, triglyceride (neutral fat) reaction reagent or phospholipid reaction reagent for the components led to each flow path Liquid chromatography device that detects and measures lipoproteins using a detector for each flow channel after selecting and mixing one of the reaction reagents for each flow channel .

Images