JP2002296261A - Analytical method for lipoprotein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which makes a special device unnecessary, and to quickly and easily separate and analyze a lipoprotein. SOLUTION: In a lipoprotein separating and analyzing method using a column filled with an anion exchange gel, an eluent containing anions having a chaotropic effect of perchlorate ion or more is used when the adsorbed lipoprotein is eluted from the column. The method is suitable when the lipoprotein contains a high-density lipoprotein(HDL), a low-density lipoprotein(LDL), an intermediate density lipoprotein(IDL), a very low-density lipoprotein(VLDL) and/or a chylomicron(CM).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a tissue extract, serum,
The present invention relates to a method for analyzing or separating lipoprotein contained in a sample such as plasma.

[0002]

2. Description of the Related Art Lipoprotein is a complex in which cholesterol, triglyceride (neutral fat), phospholipid and apoprotein are associated. Depending on the specific gravity of the lipoprotein, high-density lipoprotein (hereinafter referred to as “HDL”), low-density lipoprotein (hereinafter referred to as “LDL”), intermediate lipoprotein (hereinafter referred to as “IDL”), It is classified into low-density lipoprotein (hereinafter referred to as “VLDL”), chylomicron (hereinafter referred to as “CM”), and the like.

[0003] CM, VLDL, IDL, LDL, HDL
Is 0.930 g / mL or less, 0.93 g / mL or less, respectively.
0 to 1.006 g / mL, 1.006 to 1.019
g / mL, 1.019 to 1.063 g / mL, 1.0
63 to 1.210 g / mL and their diameters are 75 to 1200 nm, 30 to 80 nm, 25 to 35 nm, 18 to 25 nm, 12 to 9 nm, respectively (Methods of Enzymology, 12
8, p10, 1986).

[0004] Here, IDL is considered to be composed mainly of VLDL remnants and other intermediates generated in the process of metabolizing VLDL to LDL, and CM is hydrolyzed by the action of lipoprotein lipase to cause specific gravity. It has also been reported that an increase in the amount of liposome is included in the IDL fraction (Norio Tada, Hyperlipidemia (Nankodo), p209, 1991).
On the other hand, it has been reported that IDL is a lipoprotein not found in healthy people and reflects the progress of coronary artery disease.
Its separation and analysis is becoming important in clinical diagnosis.

[0005]

Conventional methods for separating and analyzing lipoproteins include ultracentrifugation, agarose gel disk electrophoresis, cellulose acetate membrane electrophoresis, acrylamide gel electrophoresis, separation using a gel filtration column, A separation method using an ion exchange column is known.

In the ultracentrifugation method, a sample is extracted from the bottom of a tube containing a sample after centrifugation with a peristaltic pump, and a cholesterol enzyme solution is mixed, reacted and detected on-line. IDL, V
LDL can be separated and detected (Byung et al., M
methods of Enzymology, 128,
p181, 1986). However, this method requires an ultracentrifuge and the separation itself takes time,
Naturally, there is a problem that rapid analysis cannot be performed.

In agarose gel electrophoresis and cellulose acetate membrane electrophoresis, HDL, LDL and VLDL are used.
Are separable, but LDL and IDL, IDL and VLD
There is a problem that L cannot be separated, and as a result, IDL analysis cannot be performed.

[0008] For the separation method using a gel filtration column,
Reports that CM, VLDL, LDL and HDL can be separated and analyzed (Okazaki et al., Handbook of
Lipoprotein Testing (AAC
C), p531, 1997), but no method capable of separating and analyzing IDL has been reported.

In the separation method using an anion exchange column, a report using a column in which a diethylaminoethyl group is introduced into a gel carrier in which damage to lipoproteins having a hydrophilic surface is minimized (Yamaguchi et al., J. Am. .
Chromatography B, 716, p57,
1998), but there is no report on the separation and analysis of IDL.

As a result of various studies, the applicant of the present invention has concluded that the present invention, which uses a column packed with an anion-exchange gel, does not require a special apparatus, and can quickly and easily separate and analyze lipoproteins. Reached.

[0011]

Means for Solving the Problems Chaotropic anions have an effect of disturbing the structure of water in an aqueous solution and weakening a hydrophobic bond (chaotropic effect). The effect is thiocyanate ion> iodine ion> perchloric acid Ion> nitrate ion> bromide ion> chloride ion> acetate ion> fluorine ion> sulfate ion
Edition), Tokyo Kagaku Dojin, p250, 1990; Biochemistry Experiment Course 1 Chemical I-separation and purification of proteins, Tokyo Kagaku Dojin, p27, 1976). In separation / analysis using an ion exchange column, a sample is introduced into a column equilibrated with an aqueous solution with a low salt concentration, multiple types of target substances are adsorbed on the gel, and the salt concentration of the eluent is increased to increase the column concentration. The ionic strength inside is increased, and substances adsorbed in the order of ionic strength are separated and eluted. When the target substance is a protein, it is common to use an ion having a weak chaotropic effect such as sodium chloride or sodium sulfate as the eluent. This is for avoiding that the structure of water is disturbed by the chaotropic effect and the higher-order structure of the protein surface is distorted. When a substance having a strong chaotropic effect is used, for example, in the case of an enzyme, the activity may be reduced due to the distortion of the higher-order structure.

Lipoprotein is a complex in which cholesterol, triglyceride (neutral fat), phospholipid and apoprotein are associated, and the higher-order structure is formed by the association. The report on the separation / analysis used (not the report on the separation / analysis of lipoproteins including IDL) merely uses sodium chloride having a weak chaotropic effect to increase the ionic strength of the eluent ( For example, Yama
aguchi, et al. Chromatography
B, 716, p57, 1998; JP-A-9-1262
No. 9, JP-A-7-89984). According to the study of the present inventor, even if the concentration of sodium chloride having a weak chaotropic effect in the eluent used for eluting the adsorbed lipoprotein is gradually increased using an anion exchange column, good separation of lipoprotein can be achieved. Could not be realized. However, when the ionic strength of the eluent was increased using sodium perchlorate having a strong chaotropic effect,
Lipoproteins including DL and VLDL were well separated. This is presumably because non-specific adsorption of lipoprotein to the gel surface was suppressed by the eluent containing an anion having a strong chaotropic effect.

[0013] Claim 1 of the present invention completed as described above.
The invention relates to a lipoprotein analysis method using a column packed with an anion exchange gel, wherein an eluent containing an anion having a chaotropic effect equal to or more than perchlorate ion is used when eluting the adsorbed lipoprotein from the column. It is an analysis method.

According to a second aspect of the present invention, there is provided a method for separating a lipoprotein using a column packed with an anion exchange gel, wherein the adsorbed lipoprotein is eluted from the column with a chaotropic effect more than that of perchlorate ions. This is a separation method using an eluent containing anions.

[0015] The invention of claim 3 of the present application is directed to the invention of claim 1 or 2, wherein the aqueous solution used for adsorbing the lipoprotein contains an anion having a chaotropic effect more than perchlorate ion. The ionic strength of the anion contained in the eluent used for eluting the adsorbed lipoprotein from the column is higher than that of the anion in the aqueous solution. The invention of claim 4 of the present application is directed to any one of claims 1 to 3, wherein the lipoprotein is a high density lipoprotein (HDL),
Low density lipoprotein (LDL), intermediate lipoprotein (ID
L), very low density lipoprotein (VLDL), and / or chylomicron (CM).

The invention of claim 5 of the present application is directed to any one of the inventions of claims 1 to 3, wherein the anions having a chaotropic effect more than the perchlorate ion are perchlorate ion, iodine ion and thiocyanate ion. Characterized in that it is one kind of anion selected from the group consisting of:
The invention of claim 6 of the present application is directed to the invention of claim 1 or 2, wherein the gel has pores of 500 nm or more, 18 nm or less, or no pores on its surface. It is characterized by the following. The invention of claim 7 of the present application is directed to the invention of claim 4, wherein the salt concentration of the eluent is changed stepwise to remove the high-density lipoprotein (HD) from the column.
L), low density lipoprotein (LDL), intermediate lipoprotein (IDL), very low density lipoprotein (VLDL), and / or
Alternatively, it is characterized in that chylomicron (CM) is separated and eluted. Hereinafter, the present invention will be described in detail.

In the present invention, gels such as silica-based and polymer-based gels can be used without any particular limitation, but their diameters should be about 1 to 100 microns in order to realize good separation and analysis of lipoproteins. preferable. The gel is
For the purpose of anion exchange, the surface of the gel has anion exchange groups, but in the present invention, a gel having positively charged anion exchange groups such as diethylaminoethyl group, trimethylaminoethyl group, and amino group is separated and analyzed. The amount may be sufficient to adsorb the estimated amount of the lipoprotein to be introduced into the lipoprotein.

According to the present invention, the lipoprotein is separated and analyzed based on the principle of ion exchange chromatography.
Lipoprotein elutes in the order of LDL, IDL, VLDL, and CM. However, when a column packed with a gel having a pore diameter of about 100 nm is used, the lipoprotein is converted into CM or VLD due to size exclusion chromatography.
It elutes in the order of L, IDL, LDL, and HDL. Here, LDL, IDL, VLDL, and CM having similar molecular weights cannot be separated by size exclusion chromatography, so that the separation according to the present invention based on the principle of ion exchange chromatography may be affected. Therefore, in the present invention, the diameter is 500 nm.
It is preferable to use a gel having pores of not less than or equal to or less than 18 nm in order to eliminate the influence of the size exclusion chromatography factor, but it is more preferable to use a so-called non-porous pore. Use a gel that has not been used. This is because a non-porous gel does not have the above-described size exclusion chromatography factors.
However, the present invention does not mean that a gel having a pore diameter of about 100 nm cannot be used. When such a gel is used, the number (amount) of anion exchange groups in the column is increased and the volume of the column is reduced so as to reduce the influence of the above-described size exclusion chromatography factors. You should do the same.

As an example of a commercially available gel that satisfies the above conditions, a polymer having a diethylaminoethyl group (TSKgel DEAE-NPR,
Trade name, manufactured by Tosoh Corporation, non-porous; TSKgel D
EAE-5PW, trade name, manufactured by Tosoh Corporation, pore size is 1
ProtEx-DEAE, trade name, manufactured by Mitsubishi Chemical Corporation, pore diameter is about 30 nm).

The above-mentioned gel is used by packing it in a usual column. For the column, for example, an ordinary column made of glass, metal such as stainless steel, or resin can be used. With regard to the volume, special care is required when using the above-mentioned gel having a pore diameter of about 100 nm, but there is no particular limitation.

The anion having a strong chaotropic effect used in the present invention has an effect more than that of perchlorate ion. Specifically, perchlorate ion, iodine ion,
Thiocyanate ion can be exemplified. Actually, eluent contains 50 to 200 mM perchloric acid, iodine, thiocyanic acid, etc.
It may be added so that it is on the order. These may be used alone or in combination of two or more.

The present invention relates to, for example, HDL and LD in a sample.
L, IDL, VLDL, and / or CM are separated and eluted from the column. Therefore, if each of the separated and eluted lipoproteins is detected, quantitative analysis of each lipoprotein becomes possible. The present invention is not necessarily used only for separating and analyzing each of the above five types of lipoproteins. For example, when a sample contains only two types of the above lipoproteins, It can be used for separation and analysis of these two types of lipoproteins, and for separating and analyzing the IDL from other components even if, for example, the sample contains only IDL.

The separation / analysis of the lipoprotein according to the present invention is carried out, for example, as follows. First, a column filled with an anion exchange gel is equilibrated with an aqueous solution having a low salt concentration. The aqueous solution for this equilibrium
Preferably has a buffering action for keeping lipoprotein in a stable pH range of 6.5 to 8.5. To provide a buffering action, tris (hydroxyl) aminomethane, N-2-hydroxylethylpiperazine-N-2-
An appropriate buffer such as ethanesulfonic acid may be used.

Next, a sample containing lipoprotein is introduced into the equilibrated column, and the lipoprotein in the sample is adsorbed on the gel. The amount of the sample to be introduced may be, for example, about 1 to 30 μL if the sample is a serum sample, for example. When adsorbing lipoproteins, the same aqueous solution as used for the equilibration is used.

Next, each lipoprotein adsorbed on the gel is separated from the gel and eluted from the column. The separation and elution of the lipoprotein is carried out by introducing an eluent into the column to increase the salt concentration of the aqueous solution in the column (to increase the ionic strength). For example, preferably adjusted to the same pH using the same buffer as the aqueous solution used for equilibration, having a higher ionic strength than the aqueous solution, and containing an anion having a chaotropic effect equal to or higher than perchlorate ion. The eluate to which about 50 to 200 mM of the agent has been added is introduced into the column. Here, the anion having a strong chaotropic effect added to the eluent plays a role of enabling lipoprotein separation analysis and also a role of increasing the ionic strength of the eluate. Therefore, the salt concentration of the eluent may be increased only by the anion itself having a chaotropic effect mainly of, for example, perchlorate ion, or may be increased mainly by anion having a weak chaotropic effect by using sodium nitrate or the like. Alternatively, the concentration may be increased by both anions having a chaotropic effect and anions having a weak chaotropic effect. As described above, the present invention is characterized only in that the eluent contains an anion having a high chaotropic effect, preferably at about 50 to 200 mM.

The method for increasing the salt concentration in the column may be any of a method for increasing the salt concentration in a stepwise manner and a method for continuously increasing the salt concentration, as is carried out by ordinary liquid chromatography. . Linear gradient method to increase salt concentration linearly,
The convex gradient method of gradually reducing the width of the increase in the salt concentration in a certain period of time, and the cone-cave gradient method of gradually increasing the width of the increase of the salt concentration in a certain period of time are known. The methods may be used, or these methods may be combined. Specifically, for example, when the salt concentration is increased in the range of 10 mM to 500 mM, a gradient method in which the salt concentration is increased stepwise in about 30 minutes can be exemplified. As a preferable method of increasing the salt concentration for rapid and accurate separation and analysis of lipoproteins, gradually increase the salt concentration until HDL, LDL and VLDL are eluted, and then elute CM by a step gradient method. However, in order to achieve good separation of LDL and VLDL that elute closely, it is necessary to linearly increase the concentration of anions having a strong chaotropic effect, thereby increasing the ionic strength of the eluate to increase the lipoprotein content. It is particularly preferable to employ a linear gradient method for separating and eluting the compound.

In addition, increasing the salt concentration of the eluent stepwise improves the separation of IDL. Therefore, when separating CM from other lipoproteins, especially CM and LDL, VL
This is a preferred method for performing separation from DL. Specifically, when the exchange group capacity per column is about 0.1 meq, HDL is an eluent having a sodium perchlorate concentration of about 100 mM, and LDL is a sodium perchlorate concentration of about 120 mM.
mM eluent, IDL is an eluent with a sodium perchlorate concentration of about 140 mM, VLDL is an eluent with a sodium perchlorate concentration of about 160 mM, and CM is an eluent with a sodium perchlorate concentration of about 500 mM. Can be eluted.

Equilibration of the column in the above-described example can also be performed with an aqueous solution containing an anion having a strong chaotropic effect. In this case, it is preferable to use an aqueous solution for equilibration, to which sodium perchlorate, sodium iodide, sodium thiocyanate, or the like is added so as to have a concentration of about 10 to 100 mM. Thus, when the aqueous solution used for equilibration contains anions with strong chaotropic effects, the adsorbed lipoproteins can be separated and eluted by continuing to introduce the same aqueous solution into the column as an eluent. Can also. However, as far as the inventor knows, in this case, it takes a long time to separate and elute lipoproteins, and the separation of each lipoprotein may be insufficient. It is preferable to increase the ionic strength of the liquid compared to that of the aqueous solution used for equilibration. For this reason, when equilibrating the column with an aqueous solution containing an anion having a strong chaotropic effect, it is preferable to equilibrate the column at about 10 to 100 mM.

The anion exchange gel packed column used in the present invention preferably has an ion exchange capacity per column of 0.1 to 1.0 meq. With such a column, it is possible to separate and elute each lipoprotein from the column with an eluent having a salt concentration of about 200 to 600 mM, and to separate and elute the lipoprotein adsorbed only with an anion having a strong chaotropic effect. Because it can be. Of course, it is possible to use a column having an ion exchange capacity higher than the above. For example, in the case of a column of about 10 milliequivalents, in order to separate and elute LDL, a salt concentration of about 2,000 mM is required in an eluent, which is chaotropic. This is because, if only strong anions are used, the higher order structure of the lipoprotein may be distorted by too strong a chaotropic effect during separation and elution from the column. For this reason, the upper limit of the anion having a strong chaotropic effect in the eluent is preferably about 200 mM. However, if the lipoprotein can be separated and eluted within this range, other salts (such as those having a strong chaotropic effect) may be added to the eluate. Need not be added, and the adjustment of the eluent can be simplified.

By the above operation, the lipoprotein is separated and eluted from the column. That is, lipoprotein separation is achieved at this stage. Therefore, it is possible to obtain each separated lipoprotein by collecting the eluate from the column.

The analysis of lipoproteins can be carried out by applying a conventional lipoprotein analysis method to each lipoprotein separated and eluted as described above. For example, focusing on lipoprotein constituents such as cholesterol, triglyceride (neutral fat), and phospholipid, it is possible to analyze each lipoprotein separated by using an enzyme specific to any of these. Can be illustrated. Specifically, cholesterol can be analyzed by allowing cholesterol esterase and cholesterol oxidase to act and measuring the amount of hydrogen peroxide generated as a result. Triglyceride (neutral fat) can be analyzed by Can be analyzed by measuring the amount of hydrogen peroxide produced as a result, and phospholipids can be analyzed by acting phospholipase and choline oxidase and measuring the amount of hydrogen peroxide produced as a result. . These analyzes can be easily performed by using a commercially available enzyme reagent. As such commercially available reagents, for example,
Determiner LTC for cholesterol analysis
(Trade name, manufactured by Kyowa Medix Co., Ltd.), Colles Color Liquid (trade name, manufactured by Toyobo Co., Ltd.), Pure Auto S
CHO-N (trade name, manufactured by Daiichi Pure Chemicals Co., Ltd.), Cholesterol E Test Wako (trade name, manufactured by Wako Pure Chemical Industries, Ltd.)
Are commercially available. For example, if it is a reagent for triglyceride analysis, Determiner TG (trade name, manufactured by Kyowa Medix Co., Ltd.), Pure Auto S TG-N (trade name, manufactured by Daiichi Pure Chemicals Co., Ltd.), triglyceride E test Wako (trade name) And Wako Pure Chemical Industries, Ltd.) are commercially available. For example, if the reagent is a phospholipid analysis reagent, the determiner PL
(Trade name, manufactured by Kyowa Medix Co., Ltd.) and phospholipid test Wako (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) are commercially available.

As described above, when an enzyme reaction is used for analysis of lipoproteins, all or a part of the eluate from the column may be mixed with an enzyme solution, reacted, and analyzed. However, it is preferable to provide only a part of the eluate for the enzyme reaction to reduce the amount of the enzyme used. In particular,
In the case of performing on-line analysis without separating the eluate from the column at a detection unit in a liquid chromatography device as described below, it is significant to reduce the amount of enzyme used.

In addition to using the above-described analysis reagents, it is also possible to use an enzyme sensor in which a membrane on which the above-mentioned enzyme is immobilized is placed on a hydrogen peroxide electrode. Still further, for example, cis-parinaric acid that emits fluorescence in the presence of a lipid component can be used.

The separation / analysis method of the present invention comprises a sample injection part for introducing a sample, an anion exchange column filled with an anion exchange gel, a container containing an eluate containing anions having a strong chaotropic effect, and an eluent. Can be carried out by a device comprising a pump for sending lipoprotein to a column, a detection unit having a detection system for measuring lipoproteins separated and eluted from the column, and the like. Of course, in addition to these, if control means for controlling the sample injection unit and pump, and calculation means for analyzing and digitizing the chromatographic pattern obtained by the detection unit are added, lipoprotein Can be provided. When the separation and analysis of lipoproteins is performed continuously, an autosampler capable of placing a plurality of samples and automatically introducing a fixed amount of the sample to the column at regular intervals for a fixed time is used as the sample injection unit. You can use it.

As described above, the operation of increasing the ionic strength in the column upon separation and elution of lipoproteins includes, for example, a solution containing a low concentration of an anion having a strong chaotropic effect and a solution containing a high concentration of an anion. Use a pump that can introduce the solution into the column at the specified flow rate, or configure a flow path so that each of these solutions is introduced into the column via the pump, and install an electromagnetic valve in each flow path By controlling the opening and closing of the solenoid valve after the arrangement, the concentration can be automatically controlled by controlling the concentration of chaotropic ions in the eluent introduced into the column.

In the examples described later, examination was performed on a patient sample in which MIDAND was confirmed and a healthy subject sample in which MIDAND was not confirmed in acrylamide disk electrophoresis. In acrylamide disk electrophoresis, it is known that, in a patient sample, a peak that is not confirmed in a healthy person is found between the VLDL peak and the LDL peak in the serum of a healthy person, and this peak is called MIDBAND.

Although no detailed analysis has been performed on MIDAND, Zhao S. et al. In lipoprotein analysis by polyacrylamide gel electrophoresis. -P. (J.
Lab. Clin. Med. 125, p. 641-64
9, 1995), VLDL is divided into a fraction VLDL1 having a low specific gravity and a fraction VLDL2 having a high specific gravity. According to the result of analyzing the properties, VLDL2 is detected between VLDL1 and LDL. VLDL2 is a lipoprotein having a high cholesterol content, and its amount is small in healthy individuals.
Considered LDL2. VLDL1 is a VLDL fraction with a low cholesterol content.

In the embodiment of the present invention, since cholesterol enzyme solution is used in the detection system, VLDL1 has low detection sensitivity, and VLDL2, ie, MIDBAND is easy to detect.
Actually, in Example 1, VLDL was confirmed in the present invention in a patient sample containing MIDBAND, and MIDBAND was confirmed.
The VLDL peak was not confirmed in a healthy subject sample containing no D.

A commercially available column (TSKgel DEAE-
NPR (trade name, manufactured by Tosoh Corporation) was used to carry out the separation / analysis method of the present invention. As a result, sodium perchlorate or sodium thiocyanate was added as compared with the comparative example using an eluent to which sodium nitrate was added. In the method of the present invention using an eluent, good lipoprotein separation was achieved. The method of the present invention using an eluent to which sodium perchlorate was added was compared with a comparative example using an eluent to which sodium chloride was added using another column (ProtEx-DEAE; trade name, manufactured by Mitsubishi Chemical Corporation). And good lipoprotein separation was achieved.

In Examples, a porous gel having a pore diameter of about 30 nm (ProtEx-DEAE; trade name,
Separation of lipoproteins could also be achieved with Mitsubishi Chemical Corporation), but non-porous gel (TSKgel DEAE-N)
PR; trade name, manufactured by Tosoh Corporation) could achieve better lipoprotein separation.

[0041]

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

Examples 1 to 3 and a comparative example were performed using the apparatus shown in FIG. In FIG. 1, enzyme solution 3 is a commercially available cholesterol analysis reagent (Cholesterol E Test Wako; trade name, manufactured by Wako Pure Chemical Industries, Ltd.), and eluents A and B (1, 2 respectively) are pumps (CCPM-II). Degasser 4 (SD-8) before being sent to Tosoh Corporation 6
022; trade name, manufactured by Tosoh Corporation) to remove dissolved gas components. The eluents A and B are uniformly mixed by a mixer (Static Mixer A; trade name, manufactured by Tosoh Corporation) and sent to the column. The above enzyme solution was used in an air trap 5 (Air trap G; trade name, manufactured by Tosoh Corporation).
Through the pump 7 (DP-8020; trade name, manufactured by Tosoh Corporation). The pump 7 has a diameter of 0.2 mm.
I. D. Two × 2 m resistance tubes were connected in series.

As the column 10, two types of columns (TS
Kgel SP-NPR; trade name, manufactured by Tosoh Corporation, column size 4.6 mmI. D. × 35 mm (hereinafter referred to as column 1) or ProtEx-DEAE; trade name, manufactured by Mitsubishi Chemical Corporation, column size 4.6 mmI. D. × 50
mm (hereinafter referred to as column 2) and a filter 9 (filter S (HLC-723GHbI)
II filter); trade name, manufactured by Tosoh Corporation). The autosampler 17 is commercially available (AS-80).
20; trade name, manufactured by Tosoh Corporation, and the column oven 11 (CO-8020; trade name, manufactured by Tosoh Corporation) was set at a set temperature of 25 ° C.

The eluate from the column is a splitter (Micro Splitter P-450; trade name, UPCHURC)
H SCIENTIFIC INC. And the enzyme solution is added online to only half the amount,
Reaction coil 13 (size 0.4 mm ID × 20 m)
Led to. The temperature of the reactor thermostat (CO-8020; trade name, manufactured by Tosoh Corporation) was set at 37 ° C. The detector 15 (UV-8020; trade name, manufactured by Tosoh Corporation)
The measurement wavelength is 600 nm.

The samples used in this experiment were serum (1 sample) obtained from a patient with lipid metabolism disorder with consent and serum (1 sample) obtained from a healthy person. The sample introduction amount was 10 μL in the experiment using column 1 and 7 μL in the experiment using column 2. The eluent flow rate was such that the eluate from the column was constant at 0.6 mL / min.

Reference Example The above-mentioned serum of a healthy individual and the serum of a patient were analyzed using a commercially available acrylamide disk electrophoresis kit for lipoprotein analysis (Lipophor; trade name, manufactured by Tokomitsu Co., Ltd.). This commercial reagent kit is based on Sudan Black B,
This is a lipid pre-staining kit. The analysis was performed according to the manual attached to the kit, and after performing acrylamide disk electrophoresis, the gel was graphed with a densitometer. FIGS. 6 and 7 show the results regarding the serum of a healthy individual and the results regarding the serum of a patient.

In healthy human serum, VLDL, LDL and HD
The three peaks of L can be confirmed, and VLDL,
Four peaks of MIDBAND, LDL and HDL were confirmed.

As described above, MIDBAND is mainly composed of a VLDL fraction (VLDL2) having a high cholesterol content. VLD in acrylamide disk electrophoresis
The major component of L is considered to be a VLDL fraction with low cholesterol content (VLDL1).

From these facts and Example 1, VLDL is detected by the present invention in a patient sample in which MIDAND is detected in polyacrylamide electrophoresis, and only VLDL having a low cholesterol content without MIDAND in polyacrylamide electrophoresis is detected. It is consistent with the fact that the peak of VLDL cannot be clearly confirmed in the present invention in the sample of a healthy human being confirmed.

Example 1 FIGS. 8 and 9 show column 1, eluent A (50 mM tris (hydroxyl) aminomethane (hereinafter referred to as “Tris”) + 1 mM disodium ethylenediaminetetraacetate (hereinafter referred to as “EDTA2Na”). PH7.
5) and eluent B (50 mM Tris + 1 mM EDTA)
2Na + 1000 mM sodium perchlorate, pH7.
Results for healthy human serum when 5) was used (FIG. 8)
And results for patient sera (FIG. 9).

The column was equilibrated with a mixture of eluent A 88% and eluent B 12%, and the gradient of the eluent was set at 88% from the sample introduction to 20 minutes.
Eluent A 80% and Eluent B from the ratio of Eluent B 12%
Linear gradient up to 20% ratio, 80% eluent A and 20% eluent B from 20 minutes to 25 minutes
(Fixed). From 25 minutes to 30 minutes, the ratio of eluent B is 100% (fixed), and after 30 minutes,
The column was equilibrated with a ratio (fixed) of eluent A 88% and eluent B 12%, and samples were continuously introduced every 45 minutes.

In the separation and analysis using the column 1 and an eluent to which sodium perchlorate was added, that is, an anion having a strong chaotropic effect, the same results as those obtained in the reference example were obtained. . That is, although three distinct peaks of HDL, LDL, and CM could be confirmed in the serum of a healthy subject, LDL, especially,
It can be seen that the separation of the lipoprotein is improved, such as a sharp peak. In patient serum, HDL,
Clear peaks of LDL, VLDL and CM were confirmed.
It can be seen that the separation is greatly improved as compared with a comparative example described later.

Example 2 FIG. 10 shows column 1, eluent A (50 mM Tris +
1 mM EDTA2Na + 100 mM sodium thiocyanate, pH 7.5 and eluent B (50 mM Tris
+1 mM EDTA2Na + 100 mM sodium thiocyanate + 500 mM sodium nitrate, pH 7.5).

The column was equilibrated with eluent A 100%, and the gradient of the eluent was set from eluent A 100% to eluent A 75% from the time of sample introduction to 20 minutes.
And a linear gradient up to a ratio of eluent B 25%, and a ratio (fixed) of eluent A 75% and eluent B 25% from 20 minutes to 25 minutes. And 3 from 25 minutes
Until 0 minute, the ratio of eluent B 100% (fixed), 30
After that, the eluent A was 100%.

Even in column 1 and separation / analysis using an eluent containing sodium thiocyanate added, that is, an anion having a strong chaotropic effect, peaks of HDL, LDL, VLDL and CM can be confirmed in patient serum. I understand.

Example 3 FIGS. 11 and 12 show column 2, eluent A (50 mM).
Tris + 1 mM EDTA2Na, pH 7.5) and Eluent B (50 mM Tris + 1 mM EDTA2N)
(a + 1000 mM sodium perchlorate, pH 7.5) when using a healthy human serum (FIG. 11) and a patient serum (FIG. 12).

The column was equilibrated with a mixture of 88% of eluent A and 12% of eluent B. The gradient of the eluent was set at 88% of eluent A from the time of sample introduction to 20 minutes.
Eluent A 80% and Eluent B from the ratio of Eluent B 12%
A linear gradient up to a ratio of 20% was used. From 20 minutes to 25 minutes, eluent A 55% and eluent B 45%
(Fixed). From 25 minutes to 30 minutes, the ratio of eluent B is 100% (fixed), and after 30 minutes,
The column was equilibrated with a ratio (fixed) of eluent A 88% and eluent B 12%, and samples were continuously introduced every 45 minutes.

In the separation and analysis using column 2 and an eluent to which sodium perchlorate was added, that is, an anion having a strong chaotropic effect, the same results as those obtained in the above reference example were obtained. . That is, although three peaks of HDL, LDL, and CM could be confirmed in the serum of a healthy individual, it was found that the separation of lipoproteins was improved as compared with the results of Comparative Examples described later, particularly, the LDL peak was sharper. . In patient sera, HDL, LD
Four peaks of L, VLDL and CM were confirmed. It can be seen that the separation is improved as compared with the comparative example described later.

Comparative Example 1 FIGS. 2 and 3 show column 1, eluent A (50 mM Tr).
is + 1 mM EDTA2Na, pH 7.5) and eluent B (50 mM Tris + 1 mM EDTA2Na +
FIG. 2 shows the results for healthy human serum (FIG. 2) and the results for patient serum (FIG. 3) when using 500 mM sodium nitrate (pH 7.5). The column was equilibrated with a mixture of eluent A 85% and eluent B 15%. The gradient of the eluent was set based on the ratio of eluent A 85% and eluent B 15% from the time of sample introduction to 20 minutes. Liquid A
A linear gradient of a ratio of 60% to eluent B 40% was used. From 20 minutes to 25 minutes, eluent A 60%
And eluent B 40% (fixed). From 25 minutes to 30 minutes, the ratio of the eluent B was 100% (fixed), and after 30 minutes, the ratio of the eluent A was 85% and the eluent B was 15% (fixed), and the column was equilibrated. The sample was introduced.

From the results of this comparative example, even when the same column (column 1) was used, when the eluent contained no anion having a strong chaotropic effect, it was compared with the result of the above-mentioned example. HDL, LDL in serum of healthy human
Although CM and CM could be separated and analyzed, the chromatographic pattern of patient serum was similar to that of healthy human serum, and the peak of VLDL in patient serum could not be identified.

Comparative Example 2 FIGS. 4 and 5 show column 2, eluent A (20 mM disodium hydrogen phosphate + 3 mM EDTA2Na, pH 7.0).
0) and eluent B (50 mM disodium hydrogen phosphate +
3 mM EDTA2Na + 500 mM sodium chloride,
FIG. 4 shows the results for healthy human serum (FIG. 4) and the results for patient serum (FIG. 5) when (pH 7.0) was used. The column was equilibrated with a mixture of 75% eluent A and 25% eluent B. The gradient of the eluent was set from 75% of eluent A to eluent B over 20 minutes from the sample introduction.
The linear gradient was from the ratio of 25% to the ratio of eluent A 55% and eluent B 45%, and the ratio (fixed) of eluent A 55% and eluent B 45% from 20 minutes to 25 minutes. From 25 minutes to 30 minutes, eluent B1
Eluent A7 after 30 minutes
The column was equilibrated with a ratio (fixed) of 5% and eluent B 25%, and samples were continuously introduced every 45 minutes.

In this comparative example, the separation of lipoprotein was inadequate even in the case of the serum of a healthy individual and the serum of a patient as compared with the case of comparative example 1, and it was difficult to identify the lipoprotein peak. Was.

(Experimental Example) The apparatus used in the experiment is shown in FIG. Eluent A (18) is 50 mM Tris-HCl + 1
mM EDTA2Na pH 7.5, eluent B (19)
Is 50 mM Tris-HCl + 1 mM EDTA2N
a + 500 mM sodium perchlorate pH 7.5. The pump (21) for flowing the eluents A and B is CCPM2
(Manufactured by Tosoh Corporation) was used. CCPM2 is a pump having two pump heads and capable of performing a gradient of two eluents. Mixer for mixing eluents A and B (2
For 2), a static mixer C (manufactured by Tosoh Corporation) was used. AS-8021 (manufactured by Tosoh Corporation) was used for the autosampler (23), and CO-8 was used for the column oven (26).
021 (manufactured by Tosoh Corporation) was used. Column (25) has a DEAE-NPR column size of 4.6 mmI. D. x2
0 mm (manufactured by Tosoh Corporation), filter (24) is H
A column filter S type (manufactured by Tosoh Corporation) for LC-723GHb3 type was used. The cholesterol reaction liquid (27) used was Cholesterol E Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), and the cleaning liquid (28) for the reaction liquid line was pure water. A three-way solenoid valve (29) was installed for switching between the cholesterol reaction solution and the washing solution, and an air trap (30) was installed immediately before the pump. As a pump (31) for the cholesterol reaction solution, DP-8020 (manufactured by Tosoh Corporation) was used. A degasser (20) was provided for the eluents A and B and the cholesterol reaction solution. A 0.1 mm I.I. D.
× 2m were connected in series and installed. Reaction coil (3
3) is 0.5 mmI. D. × 60 m. UV-8020 (manufactured by Tosoh Corporation) was used as the detector (34). The flow rate of the eluent was 1.4 mL / min for both A and B, and the flow rate of the cholesterol reaction solution was 0.7 mL / m
in. The temperature of the column oven (26) was 25 ° C, and the temperature of the reaction coil (33) was maintained at 37 ° C. Detector (34) detected at 600 nm. The gradient pattern of the eluent shows that the composition of B is 15% from 0 minutes to 4.9 minutes.
From 4.9 to 5.9 minutes, the composition of B is fixed at 32%, and from 5.9 to 6.3 minutes, the composition of B is fixed at 100%, and after 6.3 minutes Fixed the composition of B at 15%. Samples were injected with an autosampler (23) in a 8.3 minute cycle. The sample injection volume was 3 μL. During the measurement, the three-way solenoid valve (29) was turned on, the cholesterol enzyme solution (27) was allowed to flow, and after the measurement was completed, the three-way solenoid valve (29) was turned off and washing water was allowed to flow, and then the apparatus was stopped.

Example 4 Patient samples 1, 2, and 3 were measured. The patient sample 3 was stored at 4 ° C. overnight, and the CM layer was clearly confirmed in the upper layer. The measured results are shown in FIGS.

Example 5 The patient specimens 1 and 2 were subjected to ultra-centrifugation at a specific gravity of 1.
Fraction containing VLDL and CM of 006 g / mL or less, fraction containing IDL of specific gravity 1.006 to 1.019 g / mL, specific gravity 1.019 to 1.063 g / mL
Fraction containing mL LDL, specific gravity 1.063g
Each fraction was analyzed by dividing into fractions containing HDL / mL or more.

The results of the fractions obtained from the patient sample 1 are shown in FIGS. The VLDL and CM fractions (FIG. 17) had a main peak at 10.40 minutes, while the IDL fractions (FIG. 18) had a main peak at 9.40 minutes.
The main peak was the LDL fraction at 37 minutes (Fig. 1).
In 9), a peak was found at 8.73 minutes, and in the HDL fraction (FIG. 20), a peak was found at 6.86 minutes. From these facts, the peak at 6.76 minutes of patient sample 1 (FIG. 14) is HDL, and the peak at 8.61 minutes is LDL, 10.1 minutes.
It can be seen that the peak at 7 minutes is VLDL. IDL elutes between LDL and VLDL.

The results of the fractions obtained from the patient sample 2 are shown in FIGS. In the VLDL and CM fractions (FIG. 21), the main peak was at 10.16 minutes, and in the IDL fraction (FIG. 22), 9.1.
The main peak is the LDL fraction at 50 minutes (Fig. 2
In 3), a peak was observed at 8.99 minutes, and in the HDL fraction (FIG. 24), a peak was observed at 7.98 minutes. From these facts, the peak of 6.43 minutes and 7.90 minutes of patient sample 1 (FIG. 15) is HDL, and the peak of 9.06 minutes is L.
It can be seen that the peak at DL 10.16 minutes is VLDL. IDL elutes between LDL and VLDL.

Example 6 A patient sample 3 was subjected to ultracentrifugation at a specific gravity of 1.06.
CM with a levitation coefficient of 400 or more was obtained at 3 g / mL.
This CM fraction was analyzed by the present invention (FIG. 2).
5). A peak was detected at 11.36 minutes. From this, it was confirmed that the peak at 11.33 minutes in the chromatogram (FIG. 16) of the patient sample 3 was CM. Considering the results of Example 5, 5.47 minutes and 7.17 minutes for patient sample 3 (FIG. 16).
The peak at 13 minutes is HDL and the peak at 8, 24 minutes is LD
L, the peak at 10.12 minutes can be said to be VLDL. Also,
The peak of 12.06 minutes of the patient sample 2 (FIG. 15) can be said to be CM from the result of the present embodiment.

(Experimental Example) The apparatus used in the experiment is shown in FIG. Eluent A (18) was 50 mM Tris-HCl p
H7.5, eluent B (19) was 50 mM Tris-H
Cl + 500 mM sodium perchlorate, pH 7.5. The pump (21) for flowing the eluents A and B is CCPM2
(Manufactured by Tosoh Corporation) was used. CCPM2 is a pump having two pump heads and capable of performing a gradient of two eluents. Mixer for mixing eluents A and B (2
For 2), a static mixer B (manufactured by Tosoh Corporation) was used. AS-8021 (manufactured by Tosoh Corporation) was used for the autosampler (23), and CO-8 was used for the column oven (26).
021 (manufactured by Tosoh Corporation) was used. Column (25) has a DEAE-NPR column size of 4.6 mmI. D. x2
0 mm (manufactured by Tosoh Corporation), filter (24) is H
A column filter S type (manufactured by Tosoh Corporation) for LC-723GHb3 type was used. The cholesterol reaction liquid (27) used was Cholesterol E Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), and the cleaning liquid (28) for the reaction liquid line was pure water. A three-way solenoid valve (29) was installed for switching between the cholesterol reaction solution and the washing solution, and an air trap (30) was installed immediately before the pump. As a pump (31) for the cholesterol reaction solution, DP-8020 (manufactured by Tosoh Corporation) was used. A degasser (20) was provided for the eluents A and B and the cholesterol reaction solution. A 0.1 mm I.I. resistance tube (32) was connected to the cholesterol reaction solution line. D.
× 2m were connected in series and installed. Reaction coil (3
3) is 0.5 mmI. D. × 31 m. UV-8020 (manufactured by Tosoh Corporation) was used as the detector (34). The flow rate of the eluent was 0.8 mL / min for both A and B, and the flow rate of the cholesterol reaction solution was 0.4 mL / m
in. The temperature of the column oven (26) was 25 ° C, and the temperature of the reaction coil (33) was maintained at 37 ° C. Detector (34) detected at 600 nm. The step gradient pattern of the eluent is such that the composition of B is fixed at 20.5% from 0 minutes to 3.5 minutes, and the composition of B is 20.5% from 3.5 minutes to 7 minutes.
Fixed at 4.5%, 7 to 9 minutes B composition 27.5%
The composition of B is fixed at 32.5% for 9 to 12 minutes, and the composition of B is fixed at 100% for 12 to 13 minutes.
From 3 minutes to 20 minutes, the composition of B was fixed at 20.5%. 2
Samples were injected every 0 minutes. The sample was diluted 2-fold with a 0.05% bovine serum albumin solution, and 7 μL was injected. During the measurement, the three-way solenoid valve (29) was turned on, the cholesterol enzyme solution (27) was allowed to flow, and after the measurement was completed, the three-way solenoid valve (2) was turned on.
9) was turned off and the washing water was allowed to flow, and then the apparatus was stopped.

Example 7 Serum from healthy subjects and patient samples were measured. 26 and 27 show the measurement results.

Example 8 A healthy human serum and a patient sample were subjected to ultracentrifugation using a fraction containing VLDL and CM having a specific gravity of 1.006 g / mL or less, specific gravity of 1.006 to 1.019 g / m 2.
Fraction containing L IDL, specific gravity 1.019-
It was divided into a fraction containing 1.063 g / mL LDL and a fraction containing HDL having a specific gravity of 1.063 g / mL or more. The fraction containing VLDL and CM was further divided into a CM fraction having a levitation coefficient Sf of 400 or more and a VLDL fraction of less than 400 at a specific gravity of 1.063 g / mL. Each fraction was analyzed.

The results of the fractions obtained from the sera of healthy individuals are shown in FIGS. 28, 29, 30, 31, and 32. HDL
The main peak at 6.28 minutes in the fraction (FIG. 28), the main peak at 10.48 minutes in the LDL fraction (FIG. 29), and 1 in the IDL fraction (FIG. 30).
A main peak was observed at 3.51 minutes, and a main peak was observed at 15.43 minutes in the VLDL fraction (FIG. 31). No peak was detected in the CM fraction (FIG. 32).

From these results, it was found that the serum of a healthy subject (FIG. 2)
6.) The peak at 6.40 minutes is HDL, the peak at 10.26 minutes is LDL, the peak at 13.64 minutes is IDL,
It can be seen that the peak at 41 minutes is VLDL.

The results of the fractions obtained from the patient sera are shown in FIGS. 33, 34, 35, 36 and 37. The HDL fraction (FIG. 33) had a main peak at 6.20 minutes, the LDL fraction (FIG. 34) had a main peak at 10.31 minutes, and the IDL fraction (FIG. 35) had a main peak at 1 minute.
Main peaks at VLDL at 2.16 min and 13.48 min
In the fraction (FIG. 36), a main peak was observed at 15.39 minutes, and in the CM fraction (FIG. 37), a peak was observed at 18.27 minutes.

From these results, it was found that the serum of the patient (FIG. 27)
The peak at 6.24 minutes is HDL, the peak at 10.42 minutes is LDL, the peak at 13.31 minutes is IDL, and 15.50.
It can be seen that the minute peak is VLDL and the 18.23 minute peak is CM.

[0076]

According to the present invention, there is provided a method for analyzing lipoproteins using an anion exchange column, wherein an eluent containing an anion having the same or stronger chaotropic effect as perchlorate ion is used. Lipoproteins (HDL, LDL, IDL, VL) important in diagnosing sclerosis
DL, CM).

In the present invention, a normal liquid chromatography apparatus can be applied in the practice, so that it is not necessary to introduce a new special apparatus, and the same apparatus and column can be repeatedly used for a large number. Can be successively subjected to separation and analysis.

As described above, according to the separation / analysis method of the present invention, separation / analysis of lipoproteins, in which the separation / analysis has recently become increasingly important, can be performed quickly and accurately. Becomes

[Brief description of the drawings]

FIG. 1 shows a configuration of a measuring device used in Examples and Comparative Examples.

FIG. 2 is a measurement result (chromatogram) of healthy human serum in Comparative Example 1, and the horizontal axis shows the time (minute) since the sample was introduced. HDL, LDL, and CM in the figure indicate high-density lipoprotein, low-density lipoprotein, and chylomicron, respectively.

FIG. 3 is a measurement result (chromatogram) of a patient serum in Comparative Example 1, and the horizontal axis indicates a time (minute) since the sample was introduced. HDL, LDL, and CM in the figure indicate high-density lipoprotein, low-density lipoprotein, and chylomicron, respectively.

FIG. 4 is a measurement result (chromatogram) of healthy human serum in Comparative Example 2, in which the horizontal axis indicates the time (minute) since the sample was introduced.

FIG. 5 is a measurement result (chromatogram) of a patient's serum in Comparative Example 2, and the horizontal axis indicates the time (minute) since the sample was introduced.

FIG. 6 shows the results of acrylamide disk electrophoresis of healthy human serum in Reference Example, and the horizontal axis shows the migration distance in the sample migration. HDL, LDL, and CM in the figure indicate high-density lipoprotein, low-density lipoprotein, and chylomicron, respectively.

FIG. 7 is a measurement result (chromatogram) of a patient serum in a reference example, and the horizontal axis indicates time (minutes) since the sample was introduced. HDL, LDL, and VLDL in the figure indicate a high-density lipoprotein, a low-density lipoprotein, and an ultra-low-density lipoprotein, respectively.

FIG. 8 is a measurement result (chromatogram) of healthy human serum in Example 1, and the horizontal axis represents time (minute) since the sample was introduced. HDL, LDL, and CM in the figure indicate high-density lipoprotein, low-density lipoprotein, and chylomicron, respectively.

FIG. 9 is a measurement result (chromatogram) of patient serum in Example 1, and the horizontal axis indicates time (minutes) after the sample was introduced. HDL, LDL, VLDL, CM in the figure
Indicates high density lipoprotein, low density lipoprotein, ultra-low density lipoprotein, and chylomicron, respectively.

FIG. 10 is a measurement result (chromatogram) of a patient's serum in Example 2, and the horizontal axis represents time (minutes) since the sample was introduced. HDL, LDL, VLDL, CM in the figure
Indicates high density lipoprotein, low density lipoprotein, ultra-low density lipoprotein, and chylomicron, respectively.

FIG. 11 is a measurement result (chromatogram) of healthy human serum in Example 3, and the horizontal axis shows the time (minute) since the sample was introduced. HDL, LDL, and CM in the figure indicate high-density lipoprotein, low-density lipoprotein, and chylomicron, respectively.

FIG. 12 is a measurement result (chromatogram) of patient serum in Example 3, and the horizontal axis indicates time (minute) since the sample was introduced. HDL, LDL, VLDL, CM in the figure
Indicates high density lipoprotein, low density lipoprotein, ultra-low density lipoprotein, and chylomicron, respectively.

FIG. 13 is a diagram showing a configuration of a measuring device used in Example 4-6.

FIG. 14 is a measurement result (chromatogram) of the patient sample 1 of Example 4, and the horizontal axis represents the time (minute) since the sample was introduced.

FIG. 15 is a measurement result (chromatogram) of the patient sample 2 of Example 4, and the horizontal axis indicates the time (minute) since the sample was introduced.

FIG. 16 is a measurement result (chromatogram) of the patient sample 3 of Example 4, and the horizontal axis indicates a time (minute) since the sample was introduced.

FIG. 17 shows the measurement results (chromatogram) of the VLDL and CM fraction of patient sample 1, and the horizontal axis shows the time (minutes) after the sample was introduced.

FIG. 18 shows the measurement result (chromatogram) of the IDL fraction of patient sample 1, and the horizontal axis shows the time (minute) since the sample was introduced.

FIG. 19 shows the measurement result (chromatogram) of the LDL fraction of patient sample 1, and the horizontal axis shows the time (minute) since the sample was introduced.

FIG. 20 is a measurement result (chromatogram) of the HDL fraction of patient sample 1, and the horizontal axis represents the time (minute) since the sample was introduced.

FIG. 21 shows the measurement results (chromatogram) of the VLDL and CM fraction of patient sample 2, and the horizontal axis shows the time (minute) since the sample was introduced.

FIG. 22 shows the measurement result (chromatogram) of the IDL fraction of patient sample 2, and the horizontal axis shows the time (minute) since the sample was introduced.

FIG. 23 shows the measurement result (chromatogram) of the LDL fraction of patient sample 2, and the horizontal axis shows the time (minute) since the sample was introduced.

FIG. 24 shows the measurement result (chromatogram) of the HDL fraction of patient sample 2, and the horizontal axis shows the time (minute) since the sample was introduced.

FIG. 25 shows the measurement results (chromatogram) of the CM fraction of patient sample 3, with the horizontal axis representing the time (minutes) since the sample was introduced.

FIG. 26 is a measurement result (chromatogram) of healthy human serum of Example 7, and the horizontal axis indicates a time (minute) since the sample was introduced.

FIG. 27 is a measurement result (chromatogram) of patient serum in Example 7, and the horizontal axis is the time (minute) since the sample was introduced.
Is shown.

FIG. 28 is a measurement result (chromatogram) of a serum HDL fraction of a healthy subject in Example 8, and the horizontal axis indicates a time (minute) since the sample was introduced.

FIG. 29 is a measurement result (chromatogram) of a healthy human serum LDL fraction of Example 8, and the horizontal axis indicates a time (minute) since the sample was introduced.

FIG. 30 is a measurement result (chromatogram) of a serum IDL fraction of a healthy individual in Example 8, and the horizontal axis indicates a time (minute) since the sample was introduced.

FIG. 31 shows the measurement results (chromatogram) of the serum VLDL fraction of healthy subjects of Example 8, with the horizontal axis representing the time (minutes) after the sample was introduced.

FIG. 32 is a measurement result (chromatogram) of a healthy human serum CM fraction of Example 8, with the horizontal axis representing the time (minute) since the sample was introduced.

FIG. 33 shows the measurement results (chromatogram) of the serum HDL fraction of the patient in Example 8, with the horizontal axis representing the time (minute) since the sample was introduced.

FIG. 34 shows the measurement results (chromatogram) of the serum LDL fraction of the patient in Example 8, with the horizontal axis representing the time (minutes) since the sample was introduced.

FIG. 35 shows the measurement results (chromatogram) of the IDL fraction of the patient's serum in Example 8, with the horizontal axis representing the time (minute) since the sample was introduced.

FIG. 36 shows the measurement results (chromatogram) of the serum VLDL fraction of the patient in Example 8, with the horizontal axis representing the time (minute) since the sample was introduced.

FIG. 37 is a measurement result (chromatogram) of a patient serum CM fraction of Example 8, with the horizontal axis representing the time (minute) since the sample was introduced.

[Brief description of reference numerals]

1; Eluent A, 2; Eluent B, 3; Enzyme solution, 4; Degasser, 5; Air trap, 6.7;
Mixer, 9; Filter, 10; Column, 11; Column oven (incubator), 12; Resistance tube, 13; Reaction coil, 14; Reactor (Incubator), 15;
6; Splitter, 17; Autosampler, 18; Eluent A, 19; Eluent B, 20; Degasser, 21; Pump for eluent, 22; Mixer, 23; Autosampler, 24; Filter, 25; Column oven, 27; cholesterol reaction liquid, 28;
29; three-way solenoid valve, 30; air trap, 31; pump for reaction liquid, 32; resistance tube, 33; reaction coil, 34;
Detector

Claims (7)

[Claims] In a method for analyzing lipoproteins using a column packed with an anion exchange gel, an eluent containing an anion having a chaotropic effect equal to or higher than perchlorate ion when eluting the adsorbed lipoprotein from the column. Analytical method to use. 2. A method for separating a lipoprotein using a column packed with an anion exchange gel, wherein an eluent containing an anion having a chaotropic effect higher than that of perchlorate ion when the adsorbed lipoprotein is eluted from the column. Using separation method. An aqueous solution used for adsorbing a lipoprotein contains an anion having a chaotropic effect higher than that of perchlorate ion, and an elution used for eluting the adsorbed lipoprotein from a column. 3. The method according to claim 1, wherein the ionic strength of the anion contained in the liquid is higher than the ionic strength of the anion in the aqueous solution. 4. The high density lipoprotein (H)
DL), low density lipoprotein (LDL), intermediate lipoprotein (IDL), very low density lipoprotein (VLDL), and / or chylomicron (CM). That way. 5. The anion having a chaotropic effect equal to or higher than that of perchlorate ion is one kind of anion selected from the group consisting of perchlorate ion, iodine ion and thiocyanate ion. Claims 1 to 3
Either way. 6. The method according to claim 1, wherein the gel has pores of 500 nm or more, 18 nm or less, or no pores on its surface. 7. The method according to claim 7, wherein the salt concentration of the eluate is changed stepwise to remove high density lipoprotein (HDL), low density lipoprotein (LDL), intermediate lipoprotein (IDL), ultra low density lipoprotein (IDL) from the column. The method according to claim 4, wherein VLDL) and / or chylomicron (CM) are separated and eluted.