JPH0915225A - Method for analyzing serum lipoprotein - Google Patents

Abstract

(57) Abstract: CM, VLDL, LDL and H in serum are improved by improving the complexity of the measuring operation, accurately and reproducibly in a short time.
Provided is a method for analyzing serum lipoprotein, which can collectively quantify DL and facilitates automation of analysis. SOLUTION: Chylomicron, ultra-low-density lipoprotein, low-density lipoprotein and high-density lipoprotein in a serum sample are separated by liquid chromatography, and the resulting chromatogram includes a Gaussian distribution curve approximation method. A chylomicron, ultra-low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein, which are characterized by being subjected to a waveform treatment to fractionate the chylomicrons, ultra-low-density lipoprotein and low-density lipoprotein in the serum sample. A method for analyzing lipoproteins and high-density lipoproteins.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for analyzing serum lipoprotein.

[0002]

2. Description of the Related Art Lipoprotein in blood is a complex of lipid and protein in which lipids such as cholesterol, neutral fat (triglyceride) and phospholipid are associated with apolipoprotein. There are several types depending on the ratio. Therefore, since lipoproteins in blood have different functions depending on the kind of lipoproteins, not only single lipoproteins in blood, but not only measuring total lipoproteins in blood when analyzing serum lipoproteins but also separating lipoproteins Quantifying each separated lipoprotein is important for elucidating the correlation between diseases such as ischemic heart disease, liver disease and diabetes and abnormal lipid metabolism.

Conventionally, as a method for analyzing lipoproteins, an ultracentrifugation method in which the difference in specific gravity of lipoproteins is used for separation, an electrophoresis method in which a difference in surface charge of lipoproteins is used for separation, and colloidal stability And heparin and CaC
There are a binding precipitation method utilizing formation of an insoluble precipitate with a lipoprotein having l2 or dextran sulfate, and a liquid chromatographic method separating by utilizing a difference in size of lipoprotein molecules.

[0004]

The ultracentrifugation method has problems that it takes a long time for analysis, the apparatus is expensive, and the operation is complicated. The electrophoresis method is widely used for daily inspection and is excellent in qualitative quality, but it has a problem that it is non-quantitative and chylomicrons cannot be analyzed. The binding precipitation method is most widely used in routine tests, but high-density lipoproteins are mainly measured and other lipoproteins cannot be measured. Furthermore, the measured values differ depending on the precipitation conditions, and the operation is complicated. There are challenges.

On the other hand, the liquid chromatographic method has the advantages that it does not require a pretreatment operation, has good reproducibility, and can analyze several kinds of lipoproteins at the same time, but it has a low resolution and a large molecular size. There is a problem that low specific gravity lipoproteins and low specific gravity lipoproteins cannot be accurately quantified.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors separated lipoproteins in serum by high performance liquid chromatography and then separated lipoproteins. By performing waveform processing including a Gaussian distribution curve approximation method on a chromatogram of serum lipoprotein obtained by reacting a reagent containing an enzyme that reacts with cholesterol and / or triglyceride of protein and cholesterol and / or triglyceride of lipoprotein, Micron (hereinafter, CM), ultra-low-density lipoprotein (hereinafter, VLDL), low-density lipoprotein (hereinafter, LDL) and high-density lipoprotein (hereinafter, HD
The present invention has been completed by finding that each component of L) can be simply and accurately reproducibly quantified in a short time.

That is, the present invention is directed to CM, VLD in serum samples.
It is characterized in that L, LDL and HDL are separated by liquid chromatography, and the resulting chromatogram is fractionated into CM, VLDL, LDL and HDL by waveform processing including a Gaussian distribution curve approximation method. In particular, CM, VLDL, LDL and HDL in a serum sample are separated by liquid chromatography using a column packed with a packing material for gel filtration, and the separated CM, VLDL, It is an analytical method characterized in that cholesterol and / or triglyceride of LDL and HDL are measured, and the obtained chromatogram is subjected to waveform processing including a Gaussian distribution curve approximation method. Hereinafter, the present invention will be described in detail.

As shown in Example 1, samples prepared by changing the mixing ratio of VLDL, LDL and HDL standard lipoproteins of human serum separated by ultracentrifugation were separated by high performance liquid chromatography. Then, the ratio (%) of the VLDL, LDL, and HDL cholesterol levels and triglyceride levels in each sample obtained by performing waveform processing on the obtained chromatogram including the Gaussian distribution curve approximation There is a high correlation with the theoretical value. Therefore, according to the present invention, LDL and VL in serum are
By measuring the cholesterol amount and / or triglyceride amount of DL and HDL, LDL and the like can be quantified.

The waveform processing by the Gaussian distribution curve approximation method used in the present invention is a method of separating insufficiently separated peaks into each peak by approximating with a Gaussian distribution curve. In this case, the higher the separation by liquid chromatography, the higher the approximation accuracy. The amount of cholesterol in CM and the amount of triglyceride can be quantified by subtracting the sum of the amounts of cholesterol and the amount of triglycerides of VLDL, LDL, and HDL which are quantified from the total amount of cholesterol and the amount of triglyceride.

FIG. 1A shows VLDL: LDL: HDL.
Cholesterol ratio of 15.6: 57.0: 27.4
It is a chromatogram obtained by separating a mixed sample (%) by high performance liquid chromatography, but it is difficult to accurately quantify VLDL and LDL because the separation of VLDL and LDL is insufficient. On the other hand, FIG. 1B shows the result of waveform processing by the Gaussian distribution curve approximation method for the chromatogram of FIG. 1A (the chromatogram of the broken line is the chromatogram of VLDL and LDL after waveform processing). However, the area ratio is 15.5: 57.2, which is almost equal to the cholesterol amount ratio of the mixed sample, which enables precise quantification.

On the other hand, FIG. 2A shows VLDL: LDL:
HDL triglyceride amount ratio is 32.4: 44.3: 2
It is a chromatogram obtained by separating a 3.3 (%) mixed sample by high performance liquid chromatography. In the measurement of triglyceride, peaks of CM and free glycerol are recognized in addition to the three components, but it is difficult to quantify each component due to insufficient separation. On the other hand, FIG. 2B is a chromatogram after waveform processing. VLDL after waveform processing: LDL
The area ratio of 32.9: 43.5 is almost equal to the triglyceride amount ratio of the mixed sample.

As the serum sample, for example, human blood from which blood cell components have been removed is used, but no special pretreatment is required.

Separation of serum lipoproteins by liquid chromatography is preferably performed by high performance liquid chromatography from the viewpoint of improvement in operability and rapidity. Of these, high performance liquid chromatography performed using a column packed with a gel filtration filler is particularly preferable. Separation conditions are not particularly limited, but when CM is present in the sample, it is preferable that the peak can be separated at least as an independent peak from VLDL, and as a particularly preferable example, a difference in molecular size between these substances is considered. An example is high-performance liquid chromatography performed using a column packed with a gel filtration packing material to be used.

Particularly, when the present invention is carried out by high performance liquid chromatography using a column packed with a packing material for gel filtration, CM, VLDL, LDL and HDL in serum can be well separated. CM, VLDL, LDL
Examples of the gel filtration filler capable of separating HDL and HDL include those having an average pore diameter of 800 to 1200 angstroms. With a filler having an average pore size of less than 800 Å, it is difficult for lipoproteins having a large molecular size such as CM and VLDL to enter the pores,
On the other hand, with a filler having an average pore diameter of more than 1200 Å, separation of lipoproteins having a small molecular size such as LDL and HDL is deteriorated, so that the average pore diameter of 800 to 1200 Å is preferable as described above. Among them, the packing material having an average pore size of 900 to 1100 angstroms can satisfactorily separate CM, VLDL, LDL and HDL in serum, and finally enables more accurate analysis of lipoprotein.

That is, the separation by high performance liquid chromatography using the column packed with the packing material as described above,
To facilitate data processing by the Gaussian curve fitting method,
It improves the accuracy of lipoprotein analysis. It is also preferable from the viewpoints of easy operation, reproducibility of analysis, rapidity, and easy automation of analysis. When used as routine analysis, high-performance liquid chromatography is preferable because analysis time is important, but in this case, the packing material must have sufficient mechanical strength to withstand high-performance liquid chromatography. You need to choose what you have. As such a base material, for example,
Examples of the filler include silica gel, polyvinyl alcohol, polyhydroxymethacrylate, and other hydrophilic resin-based fillers (for example, TSKgel Lipopropak, trade name, manufactured by Tosoh Corporation).

Examples of the eluent used include phosphate buffer, Tris buffer, Bis-Tris buffer and the like, but there is no particular limitation as long as lipoprotein can be separated. The concentration of the buffer solution is 20 to 200 mM, particularly preferably 50 to 100 mM. When the concentration of the buffer solution is less than 20 mM, the buffer capacity is low, and when it exceeds 200 mM, the enzyme reagent described below and CM, VLDL, LDL and HDL are used.
This is because the reaction of cholesterol or triglyceride may be inhibited. The pH of the buffer solution is 5-9,
Particularly preferably, it is 7-8. PH of buffer is less than 5,
Alternatively, if the pH exceeds 9, the reaction with the enzyme reagent may be inhibited as in the above case. However, this is not the case when cholesterol and / or triglyceride is measured without using an enzyme.

CM and VLD separated as described above
Cholesterol and / or triglycerides of L, LDL and HDL are reacted with a reagent consisting of an enzyme which reacts with cholesterol and / or triglyceride to give an optically measurable signal and a chromogenic dye. The optically measurable signal here means a signal that can be measured by, for example, a fluorescence detector or an ultraviolet-visible detector.

Examples of the reagent that reacts with cholesterol used in the present invention include enzymes such as ascorbate oxidase, cholesterol esterase, cholesterol oxidase and peroxidase. it can. As the quinone-based coloring dye, oxidative condensation of N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine or N-ethyl-N- (3-sulfopropyl) -m-anisidine and 4-antiaminopyrine The body. More specifically, commercially available Determiner LTC (trade name, manufactured by Kyowa Medex Co., Ltd.), Choles Color Liquid (trade name, manufactured by Toyobo Co., Ltd.) and the like can be exemplified.

As a reagent that reacts with triglyceride, an enzyme such as ascorbate oxidase,
Examples of the coloring dyes that combine glycerol kinase, glycerol-3-phosphate oxidase, lipoprotein lipase, and peroxidase with these enzymes include quinone-based coloring dyes. As the quinone-based coloring dye, N-ethyl-N- (3-methylphenyl)-
N'-succinylethylenediamine or N-ethyl-N
An oxidative condensate of-(3-sulfopropyl) -m-anisidine and 4-antiaminopyrine can be mentioned. More specifically, commercially available Determiner LTG (trade name, manufactured by Kyowa Medex Co., Ltd.), Lipidos Liquid (trade name, manufactured by Toyobo Co., Ltd.) and the like can be illustrated.

The reaction temperature of these reagents with CM, VLDL, LDL and HDL cholesterol and / or triglyceride is 35 to 50 ° C., preferably 45 to 50 ° C.
Is good. If the reaction temperature is lower than 35 ° C, the reaction tends to be insufficient, and if it exceeds 50 ° C, the enzyme may deteriorate during the reaction. The amount of cholesterol and / or triglyceride is determined by measuring the absorbance using, for example, an ultraviolet-visible detector, and determining the result when the same operation (measurement) is performed on a standard serum having a known cholesterol concentration and / or triglyceride concentration. It can be carried out by comparing the calibration curve created based on the absorbance actually measured. The measurement wavelength of the UV-visible detector when the above-mentioned quinone-based coloring dye reagent is used is 540 to 560 nm.
It is good.

An apparatus for carrying out the present invention will be specifically described based on an example of the analyzing apparatus shown in FIG.

The sample is set in the sample injection device 4 capable of setting a large number of samples at one time. The sample injected from 4 is introduced into the separation column 5 together with the eluent 1 sent by the high-performance liquid chromatography solution sending pump 3. The sample introduced into the separation column 5 is CM in serum,
Separation into VLDL, LDL and HDL within about 15 minutes. The separated CM, VLDL, LDL and HDL are mixed with the cholesterol or triglyceride reaction reagent 2 delivered by the delivery pump 3 at the outlet of the separation column 5, and introduced into the reaction coil 7 in the reaction oven 7 and inside the coil. And react for about 1 to 3 minutes. The absorbance of the reaction product is measured by the UV-visible detector 8. The measured absorption curve of the reaction product, that is, the chromatogram, is subjected to data processing by the system controller 9 having a data processing function by the Gaussian distribution curve approximation method, and CM, VLDL, LDL and HDL.
After being fractionated into four components, the amount of cholesterol or triglyceride in CM, VLDL, LDL and HDL is output as the measurement result. Further, in the case of automatically analyzing a large amount of sample, the flow rate of the liquid feed pump 3, the sample injection amount and the sample injection order of the sample injection device 4, the reaction temperature of the reaction oven 6,
The system controller 9 is used for measuring the detection wavelength and the absorbance of the UV-visible detector 8 and for controlling each device such as data processing and output of measurement results.

[0023]

EFFECTS OF THE INVENTION Conventionally, ultracentrifugation method, electrophoresis method, binding precipitation method and the like have been used for the analysis of lipoproteins. However, CM, VLDL, LDL and HDL can be easily analyzed in a short time with good reproducibility. And simultaneous analysis of these four components was difficult. On the other hand, the liquid chromatographic method has many advantages over other conventional methods, such as simultaneous analysis in a short time, and the like, but CM, VLDL, LDL and H
It was difficult to completely fractionate DL, and precise analysis was difficult.

On the other hand, in the present invention, the low resolution of the liquid chromatographic method is solved by performing data processing by the Gaussian distribution curve approximation method, and serum lipoprotein analysis is performed by column separation and data processing. By combining with, CM, VLDL, LD in serum
It becomes possible to precisely quantify the amount of cholesterol and / or the amount of triglyceride which reflects the amount of L and HDL. Therefore, according to the present invention, pretreatment of a sample is not required, and particularly preferably when serum is analyzed by high performance liquid chromatography, the analysis time for one sample is about 15 minutes for cholesterol analysis and about 26 for triglyceride analysis. In this way, a method capable of simultaneously analyzing CM, VLDL, LDL and HDL is provided, which is superior in operability, speed and quantitativeness to the conventional analysis method.

Furthermore, the present invention can be easily automated by using a system controller capable of controlling the setting conditions of each component of the analysis system.

[0026]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not necessarily limited to the examples.

Example 1 Serum lipoproteins were analyzed by the apparatus shown in FIG. A commercially available column (TSK
gel Lipopropak trade name, Tosoh Corporation
Manufactured by Toso Co., Ltd., and a commercially available device (UV) for the UV-visible detector 8.
-8020 (trade name, manufactured by Tosoh Corporation) was used, and the detection wavelength was set to 550 nm.

The reaction oven 6 is a commercially available device (CO-
8020 product name, manufactured by Tosoh Corporation, and a commercially available device for reaction coil (reaction coil K, product name, Tosoh Corporation)
(Made of 0.4 mm in inner diameter and 7.5 m in length), and the reaction temperature was 45 ° C.

A commercially available device (CC
PM, trade name, manufactured by Tosoh Corporation, was used, the flow rate of the liquid feed pump was 0.7 ml / min for the eluent, and 0. 0 for the cholesterol reaction reagent or the triglyceride reaction reagent.
It was set to 35 ml / min.

As the eluent 1, 75 mM Tris buffer (pH 7.5) was used. In the case of cholesterol analysis, a commercially available reagent (Determiner LTC, trade name, manufactured by Kyowa Medex Co., Ltd.) was used as the reaction reagent 2. In the case of triglyceride analysis, commercially available reagents (determiner LTG,
The product name, Kyowa Medex Co., Ltd. was used. As a sample, VLD of human serum separated by ultracentrifugation
Samples prepared by changing the mixing ratio (protein amount ratio) of standard lipoproteins of L, LDL and HDL were used at 10 μl each for cholesterol analysis and 20 μl each for triglyceride analysis.

The results of cholesterol analysis are shown in Table 1, FIG.
4 and 5 show the results of triglyceride analysis in Table 2, FIG. 6, FIG. 7 and FIG.

[0032]

[Table 1]

Table 1 shows VLDL, L in the above mixed sample.
Regarding the cholesterol amount ratio (%) of DL and HDL,
The theoretical value obtained from the amount of cholesterol obtained by measuring each standard alone and the same sample were separated under the above analysis conditions, and the ratio (%) of the amount of cholesterol obtained by data processing by the Gaussian distribution curve approximation method It is a comparison of measured values.

FIGS. 3, 4 and 5 show the correlation between the theoretical value and the measured value of the amount of VLDL, LDL and HDL cholesterol in each mixed sample prepared based on the results of Table 1. As is clear from the figure, VLDL, LDL
And HDL, there is a high correlation between the theoretical value and the measured value. In addition, total cholesterol, VLDL, LD in the sample
The cholesterol level of CM can also be calculated by measuring the cholesterol levels of L and HDL and subtracting the sum of the cholesterol levels of VLDL, LDL and HDL from the total cholesterol.

[0035]

[Table 2]

Table 2 also shows VLD in each mixed sample.
The theoretical value and the measured value are compared for the triglyceride amount ratio (%) of L, LDL and HDL. FIG.
7 and 8 show the correlation between the theoretical value and the measured value of the amount of VLDL, LDL and HDL triglyceride in each sample prepared based on the results of Table 2. As is clear from the figure, there is a high correlation between the theoretical value and the measured value for VLDL, LDL and HDL, and similarly, it is possible to quantify each fraction of triglyceride.

Example 2 As in Example 1, the total cholesterol amount was 152 mg /
dl serum samples were analyzed. FIG. 9 shows a chromatogram separated into CM, VLDL, LDL and HDL by the conventional data processing method.

The total cholesterol, CM, VLDL, LDL and HDL cholesterol levels measured by the present invention were 151.0 mg / dl and 1.2 mg / dl, respectively.
dl, 44.1 mg / dl, 65.3 mg / dl and 4
It was 0.4 mg / dl. On the other hand, for the same sample, total cholesterol, CM, VLDL, and LDL measured by the peak splitting method used in the data processing method of the conventional liquid chromatography without performing the waveform processing by the Gaussian distribution curve approximation method. And cholesterol levels of HDL are 151.2 mg / dl and 1.3 mg / dl, respectively.
dl, 29.0 mg / dl, 80.6 mg / dl and 4
It was 0.3 mg / dl.

Triglyceride in the same sample was measured according to the manual using a commercially available reagent kit for measuring triglyceride (Determiner LTG, trade name, manufactured by Kyowa Medex Co., Ltd.), and the amount of triglyceride in the sample was 2
It was 24 mg / dl. Currently, the amount of cholesterol in LDL is calculated from an empirical formula (Friedewald's formula) of (LDL cholesterol) = (total cholesterol) − (HDL cholesterol) − (0.2 × triglyceride). Is obtained from the measurement result of triglyceride and the empirical formula, 6
It was 6.1 mg / dl, which is in good agreement with the measurement result of the method of the present invention, but a measurement error of about 20% was recognized with respect to the cholesterol level of LDL obtained by the conventional data processing method.

Example 3 As in Example 1, cholesterol in two types of serum samples (Samples A and B) was analyzed. As a result, the total cholesterol of sample A, the amount of cholesterol in CM, VLDL, LDL, and HDL were 153.0, 1.1, 73.3, and 4 respectively.
0.8 and 37.3 mg / dl. In addition, total cholesterol, CM, VLDL, LDL and H of sample B
DL cholesterol levels are 280.2, 1.9, 7
It was 5.4, 125.5 and 77.4 mg / dl.

Example 4 As in Example 1, triglycerides in two types of serum samples (Samples C and D) were analyzed. As a result, the total triglyceride amount of sample C, the triglyceride amount of CM, VLDL, LDL, and HDL was 309.9, 0.8, 214.7,
57.1 and 37.3 mg / dl. Further, in the sample D, the total triglyceride, CM, VLDL, LDL, and HDL triglyceride amount were 148.3, 7.9, and 7.
It was 7.4, 52.2 and 10.7 mg / dl.

[Brief description of the drawings]

FIG. 1 shows that the cholesterol content ratio of VLDL, LDL, and HDL is 15.6: 57.0: 27.4 (%).
2 shows a chromatogram (A) obtained by separating the mixed sample of No. 1 by high performance liquid chromatography and a chromatogram (B) obtained by performing waveform processing by the Gaussian distribution curve approximation method.

FIG. 2 shows that the triglyceride amount ratio of VLDL, LDL, and HDL is 32.4: 44.3: 23.3 (%).
The chromatogram (A) obtained by separating the mixed sample of (1) by high performance liquid chromatography and the chromatogram (B) obtained by performing the waveform processing by the Gaussian curve approximation method are shown.

FIG. 3 is a graph in which the vertical axis represents the measured value of the cholesterol content ratio (%) of VLDL in each sample, and the horizontal axis represents V in the same sample.
It is the correlation diagram which plotted the theoretical value of the ratio (%) of the amount of cholesterol of LDL.

FIG. 4 is a graph in which the vertical axis represents the measured value of the cholesterol content ratio (%) of LDL in each sample, and the horizontal axis represents LD in the same sample.
It is the correlation diagram which plotted the theoretical value of the ratio (%) of the amount of cholesterol of L.

FIG. 5 is a graph in which the vertical axis represents the measured value of the percentage (%) of HDL cholesterol in each sample, and the horizontal axis represents HD in the same sample.
It is the correlation diagram which plotted the theoretical value of the ratio (%) of the amount of cholesterol of L.

FIG. 6 is a graph in which the vertical axis indicates the measured value of the triglyceride content ratio (%) of VLDL in each sample, and the horizontal axis indicates V in the same sample.
It is the correlation diagram which plotted the theoretical value of the ratio (%) of the amount of triglycerides of LDL.

FIG. 7 is a graph in which the vertical axis represents the measurement value of the ratio (%) of the amount of triglyceride of LDL in each sample, and the horizontal axis represents the LD in the same sample.
It is the correlation diagram which plotted the theoretical value of the ratio (%) of the amount of triglycerides of L.

FIG. 8 is a graph in which the vertical axis represents the measurement value of the ratio (%) of the triglyceride amount of HDL in each sample, and the horizontal axis represents the HD in the same sample.
It is the correlation diagram which plotted the theoretical value of the ratio (%) of the amount of triglycerides of L.

FIG. 9 shows the chromatogram obtained in Example 2.

FIG. 10 is a schematic view showing an example of an apparatus using the method of the present invention.

[Explanation of symbols]

 1 Eluent 2 Reaction solution 3 Liquid feed pump 4 Sample injection device 5 Separation column 6 Reaction oven 7 Reaction coil 8 Ultraviolet-visible detector 9 System controller

Claims (12)

[Claims] 1. Chylomicron, ultra-low-density lipoprotein, low-density lipoprotein and high-density lipoprotein in a serum sample are separated by liquid chromatography, and the resulting chromatogram is analyzed by a Gaussian distribution curve approximation method. A chylomicron, ultra-low-density lipoprotein, a low-density lipoprotein, and a high-density lipoprotein are fractionated into a chylomicron, a low-density lipoprotein, and a high-density lipoprotein by a waveform treatment including Method for analyzing high density lipoprotein and high density lipoprotein. 2. Chylomicron, ultra-low-density lipoprotein, low-density lipoprotein and high-density lipoprotein in a serum sample are separated by liquid chromatography using a column packed with a packing material for gel filtration. Cholesterol and / or triglyceride of separated chylomicron, ultra-low-density lipoprotein, low-density lipoprotein and high-density lipoprotein were measured, and the obtained chromatogram was subjected to waveform processing including Gaussian distribution curve fitting method. The method for analyzing lipoprotein according to claim 1, which is characterized in that. 3. The sample according to claim 1, which is a human serum sample.
Method for analysis of lipoproteins of. 4. An enzyme that produces an optically measurable signal by reacting with cholesterol to measure the amount of cholesterol in separated chylomicron, ultra-low-density lipoprotein, low-density lipoprotein and high-density lipoprotein, and color development. The method for analyzing lipoprotein according to claim 2, wherein the method is performed using a combination of dyes. 5. An enzyme that produces an optically measurable signal by reacting with triglyceride in the measurement of the triglyceride amount of separated chylomicron, ultra-low-density lipoprotein, low-density lipoprotein and high-density lipoprotein, and color development. The method for analyzing lipoprotein according to claim 2, wherein the method is performed using a combination of dyes. 6. A combination of an enzyme and a coloring dye is a combination of at least an enzyme selected from the group consisting of ascorbate oxidase, cholesterol esterase, cholesterol oxidase and peroxidase and a quinone coloring dye. Method for analysis of lipoproteins of. 7. A combination of an enzyme and a coloring dye is a combination of at least an enzyme selected from the group consisting of ascorbate oxidase, glycerol kinase, glycerol-3 phosphate oxidase, lipoprotein lipase and peroxidase and a quinone coloring dye. The method for analyzing lipoprotein according to claim 5, characterized in that 8. A quinone color-forming dye is N-ethyl-N-
(3-Methylphenyl) -N'-succinylethylenediamine or N-ethyl-N- (3-sulfopropyl)-
The method for analyzing lipoprotein according to claim 6 or 7, which is an oxidative condensate of m-anisidine and 4-aminoantipyrine. 9. The method for analyzing lipoprotein according to claim 2, wherein the packing material for gel filtration is a packing material having an average pore diameter of 800 to 1200 angstroms. 10. The method for analyzing lipoprotein according to claim 2, wherein the packing material for gel filtration is a packing material having an average pore size of 900 to 1100 angstroms. 11. An enzyme and a coloring dye are reacted with chylomicron, ultra-low-density lipoprotein, low-density lipoprotein and high-density lipoprotein cholesterol and / or triglyceride under a temperature condition of 35 to 50 ° C. 6. The method for analyzing lipoprotein according to claim 4 or 5. 12. An enzyme and a dye are allowed to react with cholesterol and / or triglyceride of chylomicron, ultra-low-density lipoprotein, low-density lipoprotein and high-density lipoprotein under a temperature condition of 45 to 50 ° C. The method for analyzing lipoprotein according to claim 4 or 5.