JP5866944B2 - Method for measuring cholesterol and vitamin E in lipoprotein, and measuring apparatus using the method - Google Patents

Description

  The present invention relates to lipoproteins (high density lipoprotein (HDL), low density lipoprotein (LDL) and intermediate lipoprotein (IDL), very low density lipoprotein (VLDL), chylomicron (CM) contained in a sample. In addition, the present invention relates to a method for simultaneously measuring cholesterol and vitamin E (α-tocopherol, γ-tocopherol) in lipoprotein (a) (Lp (a)), and a measuring apparatus using the measuring method.

  Oxidized lipoproteins have attracted attention as a risk factor for arteriosclerosis, and many studies have been conducted. On the other hand, vitamin Es (mainly α-tocopherol and γ-tocopherol) are known as main antioxidants for preventing lipoproteins from becoming oxidized lipoproteins. Therefore, analyzing the amount of vitamin E in each lipoprotein contained in the blood sample is important for understanding the mechanism of arteriosclerosis.

  Several academic studies have been conducted on α-tocopherol, one of vitamin E contained in a large amount in serum lipoprotein. It has been reported that the amount of α-tocopherol relative to the total cholesterol level in serum LDL of patients with coronary artery disease is significantly lower than that of healthy subjects, and this is one of the causes of arteriosclerosis by reducing the antioxidant effect of LDL. (Non-patent Documents 1 and 2). There have been no reports on γ-tocopherol in LDL and vitamin Es in lipoproteins other than LDL, but vitamin Es are also contained in lipoproteins other than LDL. By analyzing and comparing the amount of vitamin E, it is possible to know details of the mechanism of arteriosclerosis progression, and is expected to be linked to the prevention and treatment of arteriosclerotic diseases.

  Conventionally, as a method for measuring vitamin E in lipoproteins, after separating each lipoprotein in the sample, vitamin E in the lipoprotein is extracted with hexane, the extract solution is dried and concentrated, and then methanol is used. A method of re-dissolving and using for reverse phase chromatography is known (Non-Patent Documents 1 to 4). In the measurement method, the lipoproteins are separated by ultracentrifugation (Non-Patent Documents 1 to 4), acrylamide electrophoresis, gel filtration chromatography, and ion exchange chromatography (non Patent documents 5 and 6) are known.

On the other hand, the applicant,
Separation of lipoproteins by ion exchange chromatography
Vitamin E is liberated by reacting with a pretreatment liquid containing an organic solvent and a surfactant,
Free vitamin Es are separated by reverse phase chromatography
Measure the amount of vitamin E separated,
A method for measuring vitamin E in lipoproteins has been established (Japanese Patent Laid-Open No. 2008-003082: Patent Document 1). The measurement method is a method that can easily measure vitamin E in each lipoprotein contained in a sample, and can also be automated.

JP 2008-003082 A JP 2002-296261 A

Haidari M et al., Clinical Chemistry, 47, p. 1234 (2001) Feki M et al., Clinical Chemistry, 46, p. 1401 (2000) Yolanda B, et al., Arteriosclerosis Thrombosis and Vascular Biology, 17, p. 127 (1997) Teissier E et al., Clinical Chemistry, 42, p. 430 (1996) Hirowatari Y et al. Lipid Research, 44, p. 1404 (2003) Hirowatari Y et al., Anal. Biochem. 308, p. 336 (2002)

  The method for measuring vitamin E in lipoprotein disclosed in Patent Document 1 described above is a method by which vitamin E in each lipoprotein contained in a sample can be easily measured. However, the measured value of vitamin E in the lipoprotein varies depending on the concentration of each lipoprotein contained in the sample. Therefore, for example, when the patients are different, when the same patient is at different times, or when comparing between patient populations having a specific disease state, the measured value of vitamin E in each lipoprotein, It is necessary to calculate and compare the ratio of the amount of cholesterol, the amount of protein, the amount of neutral fat, the total amount of fat, etc. in the lipoprotein. However, in order to make the comparison, a method for measuring vitamin E in lipoprotein disclosed in Patent Document 1 and a method for measuring cholesterol, protein, neutral fat or total fat in each lipoprotein (for example, non-patent) After performing the method described in Document 5 separately, it was necessary to combine the results obtained by the respective methods, so the operation was complicated. In addition, there is a problem that an error becomes large when calculating the ratio.

  Therefore, an object of the present invention is to provide a method capable of simultaneously measuring cholesterol in each lipoprotein in a method for measuring vitamin E in each lipoprotein contained in a sample.

The present invention made to achieve the above object includes the following embodiments:
That is, the first aspect of the present invention is:
Samples containing lipoproteins are subjected to ion exchange chromatography to separate lipoproteins,
The separated lipoprotein is reacted with a pretreatment liquid containing an organic solvent and a surfactant to release cholesterol and vitamin E,
Separating the released cholesterol and vitamin E using a reverse phase chromatograph,
Measure the amount of separated cholesterol and vitamin E,
This is a method for simultaneously measuring cholesterol and vitamin E in lipoproteins.

  The second aspect of the present invention is the method according to the first aspect, wherein the measurement of the separated cholesterol amount and the measurement of the separated vitamin E amount are performed with different detectors.

  Moreover, the third aspect of the present invention is to calculate the ratio of the amount of cholesterol and the amount of vitamin E in each lipoprotein obtained by the measurement method according to the first or second invention, This is a method for measuring the pathology of diabetes.

  Moreover, the 4th aspect of this invention is calculating the ratio of the amount of cholesterol in each lipoprotein and the amount of vitamin E obtained by the measuring method as described in said 1st or 2nd invention, This is a method for measuring the pathology of myocardial infarction.

The fifth aspect of the present invention provides
A sample introduction part for introducing a certain amount of a sample containing lipoprotein,
An ion exchange chromatograph section having an ion exchange column for separating lipoproteins;
A reagent mixing part for mixing a part or all of the liquid eluted from the ion exchange chromatograph part with a pretreatment liquid containing an organic solvent and a surfactant;
A reverse phase chromatograph having a reverse phase column for separating cholesterol and vitamin E released by the pretreatment solution;
A detector for measuring the amount of cholesterol contained in the liquid eluted from the reverse phase chromatograph part, and a detector for measuring the amount of vitamin E contained in the liquid eluted from the reverse phase chromatograph part. Having a detection unit;
A first liquid feeding section for feeding a sample introduced in the sample introduction section and an eluent to be introduced into the ion exchange column;
A second liquid feeding section for feeding the pretreatment liquid;
A third liquid feeding section for feeding an eluent to be introduced into the reverse phase column;
An apparatus for simultaneously measuring cholesterol and vitamin E in lipoprotein.

  According to a sixth aspect of the present invention, the detector for measuring the amount of cholesterol contained in the liquid eluted from the reverse phase chromatograph is an ultraviolet detector, and vitamin E contained in the liquid eluted from the reverse phase chromatograph. The device according to the fifth aspect, wherein the detector for measuring the amount of the species is a fluorescence detector.

  In addition, a seventh aspect of the present invention is the apparatus according to the fifth or sixth aspect, wherein there are two or more reverse phase columns in the reverse phase chromatograph section.

  The present invention is described in detail below.

  Examples of the sample containing lipoprotein, which is a measurement target of the measurement method of the present invention, include blood samples such as whole blood, serum, plasma, and serum, and suspension solutions obtained by suspending the sample in an appropriate solution. .

  Cholesterol measured by the measurement method of the present invention is free cholesterol released by a pretreatment liquid containing an organic solvent and a surfactant. There are two types of cholesterol in the blood: free cholesterol and ester cholesterol, which is usually used for cholesterol measurement. After converting ester cholesterol into free cholesterol by cholesterol esterase, it is released by cholesterol oxidase. In the method of measuring the amount of hydrogen peroxide produced by decomposing type cholesterol by the colorimetric method, the total (total cholesterol amount) of free cholesterol and ester type cholesterol is measured. However, in the measurement method of the present invention, if the reaction for converting ester-type cholesterol to free cholesterol is incorporated, the measurement apparatus becomes complicated, and therefore only free cholesterol is measured. However, since there is a good correlation between the total amount of cholesterol and the amount of free cholesterol in each lipoprotein (see Example 1 described later), free cholesterol in each lipoprotein obtained by the measurement method of the present invention. The ratio between the amount and vitamin E may be used as the ratio between the total cholesterol amount in each lipoprotein and vitamin E used when measuring the pathological condition.

  In the measurement method of the present invention, a sample containing lipoprotein is first subjected to ion exchange chromatography to separate each lipoprotein. The ion exchange chromatograph has a high resolution and can accurately separate various lipoproteins. The ion exchange chromatograph is preferably carried out in a mode in which a separation agent is packed in a column, and the separation agent (ion exchanger) to be used is a separation agent for anion exchange chromatography having high lipoprotein separation ability ( Anion exchangers) are preferred. In the separation of lipoproteins by ion exchange chromatography, the lipoproteins in a sample are once adsorbed to a separating agent (for example, anion exchanger), and then the eluents with different salt concentrations are applied in a step gradient or linear gradient. Each lipoprotein is separated and eluted based on the difference in charge and the difference in hydrophobicity. It is preferable to add chaotropic ions to the eluent used in the ion exchange chromatograph (Patent Document 1). Various conditions such as the amount of the separating agent (ion exchange capacity), the composition of the eluent, the flow rate of the eluent, etc. used in the ion exchange chromatograph are the conditions adopted in the examples described later depending on the type and amount of the sample to be provided. Or, by preliminary studies, conditions may be appropriately determined so that each lipoprotein can be separated and recovered as a different fraction. What is necessary is just to set suitably the elution conditions of a step gradient or a linear gradient according to which vitamin E in which lipoprotein is measured. For example, when the purpose is to measure vitamin E in high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL), which are the main lipoproteins in blood First, after lipoproteins in the sample are once adsorbed on the surface of the ion exchanger, HDL is eluted under the first elution condition (step), and LDL and intermediate lipoprotein (IDL) are eluted in the second step. The second step is to elute VLDL, and the fourth step is to elute chylomicron (CM) and lipoprotein (a) (Lp (a). In the second step, LDL and IDL are eluted simultaneously. However, this is due to the fact that LDL in the broad sense also includes IDL, and the pathological condition in which IDL shows a high value is rare, so that it is eluted simultaneously. Collection of the eluate from the ion exchange chromatography is, for example, each step of the step gradient, or may be collected every constant volume.

  The recovered eluate from the ion exchange chromatograph is prepared by adding a pretreatment liquid at a constant volume ratio to all of the eluate or a fraction containing lipoprotein in the eluate. Liberates cholesterol and vitamin E. The pretreatment liquid only needs to contain at least an organic solvent for dissolving cholesterol and vitamin E, and a surfactant for breaking the lipoprotein particles to release cholesterol and vitamin E, and further, ascorbic acid. It may contain a reducing agent for preventing oxidation of isocholesterol and vitamin E and a chaotropic ion having an effect of breaking lipoprotein particles by a protein denaturing action together with a surfactant. In particular, when chaotropic ions are not added to the eluent in the ion exchange chromatograph, the pretreatment liquid preferably contains chaotropic ions. Since cholesterol and vitamin E are both contained in lipoprotein and the hydrophobicity of both is similar, the composition of the pretreatment liquid is the vitamin in lipoprotein described in Patent Document 1. It is good also as a composition similar to the composition of the pretreatment liquid which liberates E.

  Cholesterol and vitamin E released from each lipoprotein by the reaction with the pretreatment liquid are subjected to reverse phase chromatography as a mixture of the lipoprotein and the pretreatment liquid. When each lipoprotein fraction is separated and eluted by ion exchange chromatography, an eluent containing a salt is used. Therefore, in the measurement method of the present invention, a sample containing salts is used for separating free cholesterol and vitamin Es. A reverse-phase chromatograph that can be separated with high accuracy. As with the ion exchange chromatograph, the reverse phase chromatograph is preferably implemented in a mode in which a separation agent is packed in a column. A sample containing cholesterol and vitamin E released by the pretreatment liquid is subjected to reverse phase chromatography to separate and elute cholesterol and vitamin E, and the amount of cholesterol and vitamin E contained in the eluate. Cholesterol and vitamin E in the lipoprotein can be measured simultaneously by measuring the amount of. Various conditions such as the amount of separating agent used, the composition of the eluent, the flow rate of the eluent, etc. in the reverse phase chromatograph are the same as those used in the examples described later or preliminary depending on the type and amount of the sample to be analyzed. What is necessary is just to determine suitably by examination. For the purpose of improving analysis accuracy, δ-tocopherol may be added as an internal control after separation of lipoprotein by ion exchange chromatography and before use in reverse phase chromatography. δ-tocopherol is one of vitamin E, but vitamin E contained in lipoprotein is mainly α-tocopherol and γ-tocopherol, and δ-tocopherol exists only about 1/100 of α-tocopherol. Ideal as a null control.

  Cholesterol and vitamin E in lipoproteins separated by reverse phase chromatography can be detected by a method suitable for each measurement target, whereby the amount of cholesterol and the amount of vitamin E can be measured. However, if cholesterol in lipoprotein and vitamin E in lipoprotein are detected by the same method, it is difficult to detect when the peaks overlap in the chromatogram. Detection and detection of vitamin E in lipoprotein are preferably detected by different methods (detectors).

By calculating the ratio between the amount of cholesterol in lipoprotein and the amount of vitamin E from the amount of cholesterol in lipoprotein and the amount of vitamin E in lipoprotein obtained by the measurement method of the present invention, The pathological condition and the pathological condition of myocardial infarction can be measured. In particular,
(1) If the γ-tocopherol / cholesterol value in VLDL is lower than the average value of healthy individuals, it has a diabetic condition, and if it is not lower than the average value of healthy persons, it does not have a diabetic condition. Measuring the pathology of diabetes,
(2) When the α-tocopherol / cholesterol value in LDL is higher than the average value of healthy individuals, it has a pathological condition of diabetes, and when it is not higher than the average value of healthy persons, it has no pathological condition of diabetes. Measuring the pathology of diabetes,
(3) If the α-tocopherol / cholesterol level in LDL is higher than the average value of healthy persons, it has a pathological condition of myocardial infarction, and if it is not higher than the average value of healthy persons, it has no pathological condition of myocardial infarction. Measuring the pathology of myocardial infarction,
(4) If the α-tocopherol / cholesterol value in VLDL is lower than the average value of healthy individuals, it has a pathological condition of diabetes, and if not lower than the average value of healthy persons, it does not have a pathological condition of diabetes. Measuring the pathology of diabetes,
(Patent Document 1).

  In the apparatus for simultaneously measuring the amount of cholesterol and the amount of vitamin E in lipoprotein using the measurement method of the present invention (hereinafter simply referred to as the measurement apparatus of the present invention), the sample introduction part is a predetermined place of the sample introduction part There is no particular limitation as long as a certain amount of sample can be automatically collected from a sample container carried into the container, and a commonly used auto sampler such as AS-8020 (trade name, manufactured by Tosoh Corporation) is used. can do. The sample introduction unit may further include a device that automatically carries the sample container into the predetermined place.

  The ion exchange column in the measuring apparatus of the present invention is not limited as long as each lipoprotein can be separated, and specific examples include DEAE-NPR and DEAE-5PW (both are trade names, manufactured by Tosoh Corporation). In particular, a nonporous anion exchange column having a high separation capability such as DEAE-NPR (trade name, manufactured by Tosoh Corporation) is preferable.

  The reverse phase column in the measuring apparatus of the present invention is not limited as long as it can adsorb cholesterol and vitamin E, and specific examples thereof include ODS-80Ts (trade name, manufactured by Tosoh Corporation). In the measuring apparatus of the present invention, a certain time is required for one analysis by the reverse phase chromatograph unit. As in Example 2 described later, when about 15 minutes are required for one analysis, before the analysis on the reverse phase column for each lipoprotein eluted from the ion exchange chromatograph portion is completed as it is, Other lipoproteins are eluted from the ion exchange chromatograph. For this reason, after the target lipoprotein is eluted, the flow rate of the eluent introduced into the ion exchange column is once lowered, the eluent is stopped, or an eluent having a low salt concentration is flowed. After the analysis of the lipoprotein by the reverse phase chromatograph, the flow rate of the eluent introduced into the ion exchange column is returned (when the flow rate of the eluent is reduced or the eluent is stopped), or the salt concentration of the eluent (When an eluent having a low salt concentration is flowed), the next lipoprotein is eluted from the ion exchange chromatograph and introduced into the reverse phase chromatograph. Therefore, it can be said that it is preferable to use a column that can shorten the time required for analysis, such as a microcolumn, as the reverse phase column provided in the measurement apparatus of the present invention.

  The reagent mixing part in the measuring apparatus of the present invention may be configured so that a part or all of the eluate from the ion exchange chromatograph part can be mixed with a pretreatment liquid containing an organic solvent and a surfactant. In Example 2 described later, the entire eluate from the ion exchange chromatograph part is mixed with the pretreatment liquid, and a part thereof is introduced into the reverse phase chromatograph part. However, if the chromatographic conditions are constant, the elution time of each lipoprotein from the ion exchange chromatograph part can be predicted, so the pretreatment reagent is fed in accordance with the expected time, and other times A mode of stopping the liquid feeding may be adopted, and consumption of the pretreatment reagent can be saved by this mode. The elution from the ion exchange chromatograph part and the pretreatment reagent may be mixed, for example, by simply joining the pipe for feeding the pretreatment reagent and the pipe for feeding the eluate from the ion exchange chromatograph part. However, after merging, for example, using a mixing device such as Static Mixer C (trade name, manufactured by Tosoh Corporation), after merging, mixing by reducing the flow velocity by providing a pipe with a large pipe inner diameter, or unevenness on the inner wall It is preferable to mix by using the formed pipe or the pipe whose inner diameter changes.

  The detection unit in the measuring apparatus of the present invention includes a detector for measuring the amount of cholesterol contained in the liquid eluted from the reverse phase chromatograph part, and the amount of vitamin E contained in the liquid eluted from the reverse phase chromatograph part. And a detector for measuring. Examples of the detector capable of measuring the amount of cholesterol include an ultraviolet detector, a mass detector, a fluorescence detector, and an electrochemical detector. Among these, an ultraviolet detector having a sufficient detection sensitivity and easy maintenance work such as maintenance is particularly preferable. Examples of the detector for measuring the amount of vitamin E include an ultraviolet detector, a mass detector, a fluorescence detector, and an electrochemical detector. Among these, a fluorescence detector is particularly preferable because it has sufficient detection sensitivity and can be easily maintained such as maintenance.

An example of the measuring apparatus of the present invention is shown in FIG. The measuring device shown in FIG.
An autosampler 4 for introducing a certain amount of a sample containing lipoprotein,
An ion exchange chromatograph section having an anion exchange column 5 for separating lipoproteins;
A reagent mixing section having a three-way mixing channel 7 and a mixing device 3b for mixing a part or all of the liquid eluted from the ion exchange chromatography section with a pretreatment liquid 6 containing an organic solvent and a surfactant. When,
A reverse phase chromatograph section having reverse phase columns 10a and 10b for separating cholesterol and vitamin E released by the pretreatment liquid;
Ultraviolet detector 11 for measuring the amount of free cholesterol contained in the liquid eluted from the reverse phase chromatograph part, and fluorescence for measuring the amount of vitamin E contained in the liquid eluted from the reverse phase chromatograph part A detector having a detector 12;
Sample introduced at the sample introduction section, eluents 1a and 1b introduced into the ion exchange column 5, pumps 2a and 2b for feeding the eluents 1a and 1b, and mixing for mixing the eluents 1a and 1b A first liquid feeding section having the device 3a;
A pretreatment liquid 6; a pump 2e for feeding the pretreatment liquid 6;
Eluents 1c and 1d to be introduced into the reverse phase column 10, pumps 2c and 2d for feeding the eluents 1c and 1d, and a third liquid feed section having
A state (OFF state) in which the mixed solution of the eluate from the ion exchange chromatograph and the pretreatment liquid can be filled in the sample loop 9 (OFF state), and the mixed liquid filled in the sample loop 9 is fed to the reverse phase column 10a by the eluent 1c. A flow path switching valve 8a capable of switching between a liquid feedable state (ON state);
When the mixed liquid filled in the sample loop 9 can be filled in the reverse phase column 10a (OFF state), cholesterol and vitamin E in the lipoprotein adsorbed on the reverse phase column 10a are fed to the reverse phase column 10b by the eluent 1d. A flow path switching valve 8b capable of switching between a liquid feedable state (ON state);
It is an apparatus provided with.

  In the measuring apparatus shown in FIG. 2, by controlling the drive of the pumps 2a and 2b with a computer or the like and controlling the amount of the eluents 1a and 1b having different salt concentrations, different compositions (salts) to be introduced into the ion exchange column 5 can be obtained. Concentration) eluent can be introduced in either step gradient or linear gradient mode. Specific examples of the pumps 2a and 2b include DP-8020 (trade name, manufactured by Tosoh Corporation). In the measuring apparatus shown in FIG. 2, the eluents 1 a and 1 b sent by the pumps 2 a and 2 b are mixed by the mixing device 3 a before being introduced into the ion exchange column 5. A specific example of the mixing device 3a is a static mixer C (trade name, manufactured by Tosoh Corporation).

  In the measuring apparatus shown in FIG. 2, the pump 2e for feeding the pretreatment reagent and the pumps 2c and 2d for feeding the eluent to be introduced into the reverse phase column are DP-8020 (product) as in the pumps 2a and 2b described above. A pump usually used in a liquid chromatograph, such as a name manufactured by Tosoh Corporation, can be used. In addition, in the measuring apparatus shown in FIG. 2, by providing the six-way switching valve 8a, the mixture of the eluate from the ion exchange chromatograph and the pretreatment reagent and the eluent 1c to be introduced into the reverse phase column are sent. This simplifies the liquid feeding part.

  Further, the measuring apparatus shown in FIG. 2 has two reversed phase columns in the reversed phase chromatograph section. This suppresses the occurrence of tailing (a phenomenon in which the tail is pulled after the original peak) and reading (a phenomenon in which the tail is pulled before the original peak) in the chromatogram of cholesterol and vitamin E in the lipoprotein. The shape is improved. As a result, improvement in separation performance and improvement in measurement reproducibility and measurement accuracy can be expected. Further, in the measuring apparatus shown in FIG. 2, one of the two reverse phase columns 10a is provided on the sample loop of the flow path switching valve 8b, and the reverse phase column 10a is composed of the liquid eluted from the ion exchange chromatograph section. While adsorbing cholesterol and vitamin E contained in the mixed solution with the pretreatment solution, the impurities contained in the mixed solution serve to remove to some extent. Thereby, for example, when the sample containing lipoprotein is patient blood, it is less likely to be disturbed by the administered drug contained in the sample. Further, the durability of the subsequent reverse phase column 10b is also improved. Furthermore, since the adsorption process and the separation process of cholesterol and vitamin E are performed separately, the analysis time can be shortened.

The measurement of the amount of cholesterol and the amount of vitamin E in lipoprotein by the measuring apparatus shown in FIG. 2 is specifically performed by the following method.
(A) A sample containing lipoproteins collected using the autosampler 4 is fed together with the eluents 1a and 1b by the pumps 2a and 2b and supplied to the ion exchange column 5 to hold the lipoproteins in the sample on the columns. .
(B) After the eluent of the same composition is sent in a certain amount, the amount of the eluent introduced into the ion exchange column 5 is increased by controlling the amount of the pumps 2a and 2b sent to the ion exchange column 5. Separate and elute each retained lipoprotein.
(C) Cholesterol and vitamin E are liberated from the lipoprotein by mixing the pretreatment liquid 6 fed by the pump 2e with each lipoprotein eluted. It should be noted that the amount of the pretreatment liquid 6 to be fed may decrease the detection sensitivity in the subsequent reverse phase chromatograph if the lipoprotein eluted from the ion exchange column 5 is excessively diluted, and is included in the pretreatment liquid. Depending on the concentration of the surfactant to be used, if the amount of liquid to be fed is increased, bubbles may be generated and the liquid may be poorly fed. Therefore, 0.2 to 5 times the amount of liquid eluted from the ion exchange column It is preferable that the amount is 0.5 to 2 times.
(D) The flow path switching valve 8 a is switched to the OFF state, and the liquid containing the released cholesterol and vitamin E is introduced into the sample loop 9. The volume of the sample loop 9 may be set to the amount of liquid to be introduced into the subsequent reverse phase column 10a.
(E) The flow path switching valve 8a is switched to the ON state and the flow path switching valve 8b is switched to the OFF state, and the liquid containing cholesterol and vitamin E introduced into the sample loop 9 is introduced into the reverse phase column 10a by the pump 2c. As a result, the reverse phase column 10a adsorbs cholesterol and vitamin E, while removing other impurities.
(F) The flow path switching valve 8b is switched to the ON state, and the eluent 1d is introduced into the reverse phase column 10a by the pump 2d, so that adsorbed cholesterol and vitamin E are eluted and introduced into the reverse phase column 10b.
(G) The reverse phase column 10b separates and elutes cholesterol and vitamin E, and the amount of separated and eluted cholesterol is measured by the ultraviolet detector 11, and the amount of separated and eluted vitamin E is measured by the fluorescence detector 12.

  The operations (A) to (G) can be automatically controlled by connecting each device to a personal computer. Further, the measured data can be taken with the same personal computer, and the ratio of the amount of cholesterol and the amount of vitamin E in each lipoprotein can be automatically calculated.

  When the lipoprotein is fractionated into four (HDL, LDL + IDL, VLDL, CM + Lp (a)) by ion exchange chromatography using the measuring apparatus of FIG. 2, and cholesterol and vitamin E in each lipoprotein are measured, The analysis time per sample is about 1 hour.

  The measurement method of the present invention is a method capable of simultaneously measuring the amount of cholesterol and the amount of vitamin E in lipoprotein. When calculating the ratio between the amount of cholesterol in lipoproteins and the amount of vitamin E, which is used for pathologic conditions and risk assessment, the conventional method uses a method for measuring the amount of cholesterol in lipoproteins and vitamin E in lipoproteins. It was necessary to perform the same measurement method with separate devices and calculate the ratio separately from the results obtained with the two devices. Therefore, when calculating the ratio between the amount of cholesterol in lipoproteins and the amount of vitamin E, there is a problem that errors in sample injection amounts due to two measurement methods (measuring devices) are added. On the other hand, in the measuring method of the present invention, since the amount of cholesterol and the amount of vitamin E in lipoprotein can be measured simultaneously by a single measurement with a single device, compared with the conventional method, It has the effect that the ratio between the amount of cholesterol and the amount of vitamin E can be easily calculated. In addition, when calculating the ratio, there is an effect that an error relating to the sample injection amount does not occur.

  In the apparatus using the measurement method of the present invention, when the reverse phase chromatograph section has two or more reverse phase columns and at least one of them is for impurity removal, the subsequent cholesterol and vitamins Occurrence of tailing (a phenomenon in which the tail is pulled after the original peak) and reading (a phenomenon in which the tail is pulled before the original peak) can be suppressed in the chromatogram obtained by the reverse phase column for separating E species. . Therefore, measurement accuracy and sensitivity can be improved.

The figure which shows the correlation with the total amount of cholesterol in each lipoprotein, and the amount of free cholesterol. (A) in HDL, (b) in LDL, (c) in IDL, (d) in VLDL, and (e) in chylomicron. The figure which shows an example of the measuring apparatus of this invention. The result (chromatogram) measured by the method of Example 2. (A) is a result (chromatogram) of detecting vitamin E in each lipoprotein with a fluorescence detector, and (b) is a result (chromatogram) of detecting cholesterol in each lipoprotein with an ultraviolet detector. is there. The result (chromatogram) measured by the method of Example 3. (A) is a result (chromatogram) of measuring vitamin E in the HDL fraction with a fluorescence detector, and (b) is a result (chromatogram) of measuring cholesterol in the HDL fraction with an ultraviolet detector. is there. The result (chromatogram) measured by the method of Example 3. (A) is the result (chromatogram) of measuring vitamin E in the (LDL + IDL) fraction with a fluorescence detector, and (b) is the result of measuring cholesterol in the (LDL + IDL) fraction with an ultraviolet detector ( Chromatogram). The result (chromatogram) measured by the method of Example 5. (A) is a result (chromatogram) of measuring vitamin E in each lipoprotein with a fluorescence detector, and (b) is a result (chromatogram) of measuring cholesterol in each lipoprotein with an ultraviolet detector. is there. The figure which shows the result of the dilution linearity test of vitamin E in each lipoprotein. (A) is γ tocopherol in the HDL fraction, (b) is α tocopherol in the HDL fraction, (c) is γ tocopherol in the (LDL + IDL) fraction, and (d) is α in the (LDL + IDL) fraction. Tocopherol, (e) is the result of γ tocopherol in the VLDL fraction, and (f) is the result of α tocopherol in the VLDL fraction. The figure which shows the result of the dilution linearity test of the cholesterol in each lipoprotein. (A) is the result of cholesterol in the HDL fraction, (b) is the result of cholesterol in the (LDL + IDL) fraction, and (c) is the result of cholesterol in the VLDL fraction. The figure which shows the correlation of the quantity of vitamin E in each lipoprotein measured by the HPLC method and the ultracentrifugation method. (A) is the amount of γ-tocopherol in the HDL fraction, (b) is the amount of α-tocopherol in the HDL fraction, (c) is the amount of γ-tocopherol in the (LDL + IDL) fraction, and (d) is the (LDL + IDL) fraction. (A) is the result of the amount of γ-tocopherol in the VLDL fraction, and (f) is the result of the amount of α-tocopherol in the VLDL fraction. The figure which shows the correlation of the amount of cholesterol in each lipoprotein measured by the HPLC method and the ultracentrifugation method. (A) is the amount of cholesterol in the HDL fraction, (b) is the result of the amount of cholesterol in the (LDL + IDL) fraction, and (c) is the result of the amount of cholesterol in the VLDL fraction.

  EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, an Example shows an example of this invention and does not limit this invention.

Example 1
The total cholesterol level and free cholesterol level in each lipoprotein were measured by the same method as the lipoprotein measurement method disclosed in the examples of JP-A No. 2002-296261 (Patent Document 2). However, the size of the column (TSKgel DEAE-NPR, manufactured by Tosoh) is 3.0 mmI. D. × 15 mm, the flow rate was 0.5 mL / min in total for eluent A and eluent B, and the flow rate of the reaction solution was 0.2 mL / min. The mixer for mixing the eluent A and the eluent B is a static mixer C (manufactured by Tosoh Corporation), and the size of the reaction coil is 0.25 mmI. D. X30 m and the reaction temperature were 45 ° C. The injection volume of serum was 1.9 μL. TCHO-CL (manufactured by Cellotech) was used as a reaction liquid for measuring total cholesterol, and free cholesterol E test Wako (manufactured by Wako Pure Chemical Industries) was used as a reaction liquid for measuring free cholesterol. The step gradient pattern of the eluent is 17.2% for eluent B from 0 to 2 minutes, 21.7% for eluent B from 2 minutes to 4 minutes, and eluent B from 4 minutes to 5.5 minutes. 23.6%, 5.5 to 7 minutes is 32.6% of eluent B, 7 to 8 minutes is 100% of eluent B, and 8 to 10 minutes is eluent B Was set to 10%, and the measurement of one sample was completed in 10 minutes.

  Serum samples collected from 20 volunteers with consent from Tosoh Corporation's Tokyo Research Laboratories were used to separate lipoproteins (HDL, LDL, IDL, VLDL, chylomicron (CM)) contained in the serum samples by the method described above. Then, the total cholesterol amount and the free cholesterol amount in each lipoprotein were measured. The results of plotting the measurement results for each serum sample are shown in FIG. In any lipoprotein, since the correlation between the total cholesterol level and the free cholesterol level is good, when calculating the amount of vitamin E per cholesterol level in each lipoprotein, the total cholesterol level is calculated as the cholesterol level. Even if the amount of free cholesterol is used, the result can be said to be equivalent. In the following examples, free cholesterol is simply referred to as cholesterol.

Example 2
Using the measuring apparatus of the present invention shown in FIG. 2, the amount of cholesterol and the amount of vitamin E in each lipoprotein were measured. The apparatus configuration and measurement conditions are shown below.

Apparatus configuration of FIG. 2 Eluent A (1a):
50 mM Tris + 1 mM EDTA2Na (pH 7.5)
Eluent B (1b):
50 mM Tris + 1 mM EDTA2Na + 500 mM sodium perchlorate (pH 7.5)
Eluent C (1c):
25 mM sodium perchlorate + 30% ethanol Eluent D (1d):
25 mM sodium perchlorate + 86.5% ethanol + 0.025 mM perchloric acid Feed pump 2a, 2b, 2c, 2d, 2e: DP-8020 (manufactured by Tosoh Corporation)
Mixing devices 3a and 3b: Static mixer C (manufactured by Tosoh Corporation)
Autosampler 4: AS-8020 (manufactured by Tosoh)
Anion exchange column 5:
TSKgel DEAE-NPR (manufactured by Tosoh Corporation), 3.0 mmI. D. × 15mm
Pretreatment liquid 6:
1% Triton X-100 (trade name) + 70% ethanol Sample loop 9:
Stainless steel tube with a capacity of 2.5 mL (0.8 mm ID × 497.5 cm)
Reversed phase column 10a:
TSKgel ODS-80Ts (manufactured by Tosoh Corp.), 4.6 mmI. D. × 3.5
cm
Reversed phase column 10b:
TSKgel ODS-80Ts (manufactured by Tosoh Corp.), 4.6 mmI. D. × 7.5
cm
Measurement conditions Sample introduction:
100 μL (Introduced sample is a sample obtained by diluting a serum sample 15 times with a solution of (eluent A: eluent B = 9: 1))
Amount of eluent introduced into the anion exchange column 5:
0.25 mL / min (total of eluent A and eluent B)
Gradient pattern of anion exchange column 5:
From 0 to 2 minutes, Eluent B 19% + Eluent A 81%
From 2 to 5 minutes, eluent B 19% + eluent A 81% to eluent B 10% + eluent A
Linear gradient to 90% Eluent B 10% + Eluent A 90% from 5 to 10 minutes
10 to 12 minutes: 26% eluent B + 74% eluent A
From 12 to 15 minutes, eluent B26% + eluent A 74% to eluent B 10% + eluent A 90% linear gradient From 15 to 20 minutes eluent B 10% + eluent A 90%
From 20 to 22 minutes, eluent B33% + eluent A67%
Linear gradient from eluent B33% + eluent A67% to eluent B10% + eluent A90% from 22 minutes to 25 minutes From 25 minutes to 30 minutes eluent B10% + eluent A90%
Eluent B 100% for 30 to 32 minutes
From 32 minutes to 35 minutes, linear gradient from 100% eluent B to 10% eluent B + 90% eluent A From 35 minutes to 50 minutes eluent B 10% + eluent A 90%
Flow rate of pretreatment liquid 6: 0.25 mL / min
Flow rate of eluent C (1c) and eluent D (1d): 1.5 mL / min
Switching of the flow path switching valves 8a and 8b The valve 8a is turned off at 0.3 minutes and the valve 8b is turned off at 2.3 minutes.
Valve 8a is turned on at 6.3 minutes, valve 8b is turned on at 8.3 minutes
Valve 8a is turned off at 10.8 minutes, valve 8b is turned off at 12.8 minutes
The valve 8a is turned on at 16.8 minutes, and the valve 8b is turned on at 18.8 minutes.
Valve 8a is turned off at 20.8 minutes, and valve 8b is turned off at 23.3 minutes
Valve 8a is turned on at 26.8 minutes, and valve 8b is turned on at 28.8 minutes.
The valve 8a is turned off at 30.8 minutes, and the valve 8b is turned off at 33.3 minutes.
Valve 8a is turned on at 36.8 minutes, and valve 8b is turned on at 38.8 minutes.
Wavelength of the ultraviolet detector 11: 205 nm
Wavelength of fluorescence detector 12: excitation wavelength 298 nm, fluorescence wavelength 325 nm
Measurement time per sample: 50 minutes FIG. 3 shows the results (chromatogram) of the serum sample collected from one volunteer who obtained consent from Tosoh Corporation Tokyo Research Laboratory under the above conditions. Fluorescence detector 11 allows gamma and alpha tocopherol in the HDL fraction, gamma and alpha tocopherol in the (LDL + IDL) fraction, gamma tocopherol and alpha tocopherol in the VLDL fraction, and (chylomicron (CM) + liposome Α-tocopherol in the protein (a) (Lp (a)) fraction was confirmed (FIG. 3 (a)), and cholesterol in the HDL fraction and cholesterol in the (LDL + IDL) fraction were detected by the ultraviolet detector 12. Cholesterol in the VLDL fraction and cholesterol in the (CM + Lp (a)) fraction were confirmed (FIG. 3 (b)).

Example 3
The amount of cholesterol and vitamin E in the HDL fraction and (LDL + IDL) fraction under the same conditions as in Example 2 except that the following contents were changed among the apparatus configuration and measurement conditions shown in Example 2. It was measured.

Details of change Reversed phase column 10a:
TSKgel ODS-80Ts (manufactured by Tosoh Corp.), 4.6 mmI. D. × 12cm
Reversed phase column 10b: Not provided Switching of the flow path switching valves 8a and 8b (when measuring the HDL fraction)
Valve 8a is turned off at 0.3 minutes and valve 8b is turned off at 2.3 minutes
Valve 8a is turned on at 6.3 minutes, valve 8b is turned on at 8.3 minutes
Switching of flow path switching valves 8a and 8b (when measuring (LDL + IDL) fraction)
Valve 8a is turned off at 10.8 minutes, valve 8b is turned off at 12.8 minutes
The valve 8a is turned on at 16.8 minutes, and the valve 8b is turned on at 18.8 minutes.
FIG. 4 (HDL fraction) and FIG. 5 ((LDL + IDL) fraction) show the results (chromatogram) of serum samples collected from one volunteer who obtained consent from Tosoh Corporation's Tokyo Laboratory. . In the α-tocopherol peak in the HDL fraction (FIG. 4 (a)) and the α-tocopherol peak in the (LDL + IDL) fraction (FIG. 5 (a)), two reversed-phase columns were placed in the reverse-phase chromatograph section. Reading (a phenomenon in which a tail is pulled before the original peak), which could not be confirmed by the measurement apparatus (Example 2), occurred. For this reason, it turns out that it is more preferable to have two or more reversed phase columns in the reversed phase chromatograph part with which the measuring apparatus of this invention is equipped.

Example 4
Serum samples collected from 3 volunteers who obtained consent from Tosoh Corporation Tokyo Research Laboratory were measured by the method of Example 2. As a comparative control method, the plasma sample is fractionated into HDL, LDL + IDL, VLDL, and chylomicron (CM) by ultracentrifugation, and then extracted with hexane to measure vitamin E (α tocopherol, γ tocopherol). In addition, an ultracentrifugation method was also performed in which free cholesterol in a sample was measured by free cholesterol E test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The measurement conditions in the ultracentrifugation method are shown below.

Measurement conditions of ultracentrifugation Fractionation conditions of each lipoprotein by ultracentrifugation HDL fraction: specific gravity 1.063 g / mL or more (LDL + IDL) fraction: specific gravity 1.006 to 1.063 g / mL
VLDL fraction:
Specific gravity of 1.006 g / mL or less and a floating coefficient (specific gravity liquid: 1.063 g / mL)
20 to 400
CM fraction:
Specific gravity of 1.006 g / mL or less and a floating coefficient (specific gravity liquid: 1.063 g / mL)
More than 400 Extraction of vitamin E from each lipoprotein i) Each fraction of lipoprotein (HDL fraction and (LDL + IDL) fraction is
75 μL, VLDL fraction and CM fraction are 100 μL)
Control solution (ethanol containing 10 μg / mL δ tocopherol)
Solution) Add 100 μL and mix ii) Add 600 μL ethanol and mix iii) Add 2 mL hexane and mix iv) Take 1 mL hexane layer (upper layer) and blow to dryness with nitrogen gas v ) Add 500 μL of ethanol and re-dissolve Liquid chromatographic conditions Analysis column:
TSKgel ODS-80Ts (manufactured by Tosoh Corp.), 4.6 mmI. D. × 15
cm
Eluent:
25 mM sodium perchlorate + 0.025 mM perchloric acid + 86.5%
Tanol Flow rate: 1.0 mL / min
Wavelength of fluorescence detector: excitation wavelength 298 nm, fluorescence wavelength 325 nm
Sample injection volume: 10 μL
Tables 1 to 3 show the results of comparison between the measurement values of the measurement method of Example 2 (HPLC method) and the measurement values of the comparative control method (ultracentrifugation method). Of the lipoproteins, Lp (a) is contained in the CM fraction in the HPLC method and in the (LDL + IDL) fraction and the HDL fraction in the ultracentrifugation method. In addition, the amount of γ-tocopherol and cholesterol in the CM fraction could not be measured due to insufficient sensitivity.

一部を除き、試料中に含まれる各リポタンパク中のビタミンＥ類（αトコフェロール量、γトコフェロール）の量およびコレステロール量の、測定値の比率（ＨＰＬＣ法での測定値に対する超遠心法での測定値の比率）は、１００±４０％の範囲に収まった。

Except for a part, the ratio of the amount of vitamin E (α tocopherol amount, γ tocopherol) and cholesterol amount in each lipoprotein contained in the sample (measured by the ultracentrifugation method relative to the measured value by the HPLC method) The ratio of measured values) was in the range of 100 ± 40%.

Example 5
Serum samples collected from one volunteer who obtained consent from Tosoh Corporation Tokyo Research Laboratories (different from the serum samples used in Example 2) were collected using the measurement apparatus of the present invention shown in FIG. Fractions were divided into three (LDL + IDL) and VLDL fractions, and the amounts of γ-tocopherol, α-tocopherol, and cholesterol in each fraction were measured. The measurement conditions are the same as in Example 2 except for the following changes.

Details of Change Eluent D (1d):
Gradient pattern of 25 mM sodium perchlorate + 0.025 mM perchloric acid + 84% ethanol anion exchange column 5:
From 0 to 2 minutes, Eluent B 19% + Eluent A 81%
From 2 to 5 minutes, eluent B 19% + eluent A 81% to eluent B 10% + eluent A
Linear gradient to 90% Eluent B 10% + Eluent A 90% from 5 to 15 minutes
From 15 minutes to 17 minutes, eluent B26% + eluent A74%
From 17 to 20 minutes eluent B26% + eluent A74% to eluent B10% + eluent A90% linear gradient From 20 to 30 minutes eluent B10% + eluent A90%
From 30 to 32 minutes, eluent B33% + eluent A67%
From 32 to 35 minutes, eluent B33% + eluent A67% to eluent B10% + eluent A90% linear gradient From 35 to 45 minutes eluent B10% + eluent A90%
Eluent B 100% from 45 to 47 minutes
From 47 to 55 minutes, eluent B 10% + eluent A 90%
Switching of the flow path switching valves 8a and 8b The valve 8a is turned off at 0.3 minutes and the valve 8b is turned off at 2.3 minutes.
Valve 8a is turned on at 6.3 minutes, valve 8b is turned on at 8.3 minutes
Valve 8a is turned off at 15.3 minutes and valve 8b is turned off at 17.3 minutes
Turn valve 8a on at 21.3 minutes, turn valve 8b on at 23.3 minutes
The valve 8a is turned off at 30.3 minutes, and the valve 8b is turned off at 32.3 minutes.
Valve 8a is turned on at 36.3 minutes, and valve 8b is turned on at 38.3 minutes.
The measurement results (chromatogram) are shown in FIG. The fluorescence detector 11 confirmed γ tocopherol and α tocopherol in the HDL fraction, γ tocopherol and α tocopherol in the (LDL + IDL) fraction, and γ tocopherol and α tocopherol in the VLDL fraction (FIG. 6 (a)). . Further, cholesterol in the HDL fraction, cholesterol in the (LDL + IDL) fraction, and cholesterol in the VLDL fraction were confirmed by the ultraviolet detector 12 (FIG. 6 (b)).

Example 6
A simultaneous reproducibility test (n = 10) was performed using the same sample as the serum sample used in Example 5. The measurement method is the same as in Example 5. The results are shown in Table 4.

各リポタンパク画分中のビタミンＥ類およびコレステロールのＣＶ（％）は、全て１０％以内であった。

The CV (%) of vitamin Es and cholesterol in each lipoprotein fraction were all within 10%.

Example 7
A dilution linearity test was conducted using the same sample as the serum sample used in Example 5. Specifically, a serum sample is diluted into 6 stages (eluent A: B = 9: 1 solution, diluted 7.5 times, 10 times, 15 times, 20 times, 30 times, or 60 times) And measured under the same conditions as in Example 5. When each diluted sample is converted into the amount of introduced serum sample, it becomes 13.3 μL, 10 μL, 6.7 μL, 5.0 μL, 3.3 μL, and 1.7 μL, respectively. The results are shown in FIG. 7 and FIG. The correlation coefficient between the measured value of the amount of γ-tocopherol and the amount of α-tocopherol in each lipoprotein fraction and the amount of diluted sample introduced was 0.9 or more (FIG. 7), and the amount of cholesterol in each lipoprotein fraction The correlation coefficient between the measured value and the amount of diluted sample introduced was 0.9 or more (FIG. 8).

Example 8
Serum samples collected from 10 volunteers who have obtained consent from Tosoh Corporation's Tokyo Research Laboratory, using the measurement apparatus of the present invention shown in FIG. 2, are divided into HDL fraction, (LDL + IDL) fraction, and VLDL fraction. The amount of γ-tocopherol, the amount of α-tocopherol, and the amount of cholesterol in each fraction were measured (HPLC method). The measurement conditions are the same as in Example 5. As a comparative control method, the ultracentrifugation method shown in Example 4 (but not using the CM fraction) was also performed.

  FIG. 9 and FIG. 10 show the results of comparison between the measurement values of the measurement method (HPLC method) of Example 5 and the measurement values of the comparative control method (ultracentrifugation method). Regarding the HDL fraction, the (LDL + IDL) fraction and the VLDL fraction, and the γ-tocopherol amount, the α-tocopherol amount and the cholesterol amount contained in each, the correlation coefficient between the measured value by the HPLC method and the measured value by the ultracentrifugation method is All were 0.8 or more.

1: Eluent 2: Pump 3: Mixing device 4: Autosampler 5: Anion exchange column 6: Pretreatment liquid 7: Three-way mixing channel 8: Channel switching valve (hexagonal solenoid valve)
9: Sample loop 10: Reversed phase column 11: Ultraviolet detector 12: Fluorescence detector

Claims (5)

Samples containing lipoproteins are subjected to ion exchange chromatography to separate lipoproteins,
The separated lipoprotein is reacted with a pretreatment liquid containing an organic solvent and a surfactant to release cholesterol and vitamin E,
Separating the released cholesterol and vitamin E using a reverse phase chromatograph,
Measure the amount of separated cholesterol and vitamin E,
A method for measuring cholesterol and vitamin E in lipoproteins , comprising:
A measurement of the amount of vitamin E that is separate from the measurement of the separated cholesterol levels, in different detector rows that have, the measurement of cholesterol levels is performed in the ultraviolet detector, fluorescence detector measuring the amount of vitamin E Performing said method. A method for measuring the pathological condition of diabetes by calculating the ratio between the amount of cholesterol in each lipoprotein and the amount of vitamin E obtained by the measurement method according to claim 1 . A method for measuring a disease state of myocardial infarction by calculating a ratio between the amount of cholesterol in each lipoprotein and the amount of vitamin E obtained by the measurement method according to claim 1 . A sample introduction part for introducing a certain amount of a sample containing lipoprotein,
An ion exchange chromatograph section having an ion exchange column for separating lipoproteins;
A reagent mixing part for mixing a part or all of the liquid eluted from the ion exchange chromatograph part with a pretreatment liquid containing an organic solvent and a surfactant;
A reverse phase chromatograph having a reverse phase column for separating cholesterol and vitamin E released by the pretreatment solution;
A detector for measuring the amount of cholesterol contained in the liquid eluted from the reverse phase chromatograph part, and a detector for measuring the amount of vitamin E contained in the liquid eluted from the reverse phase chromatograph part. Having a detection unit;
A first liquid feeding section for feeding a sample introduced in the sample introduction section and an eluent to be introduced into the ion exchange column;
A second liquid feeding section for feeding the pretreatment liquid;
A third liquid feeding section for feeding an eluent to be introduced into the reverse phase column;
A device for measuring cholesterol and vitamin E in lipoproteins , comprising:
The detector for detecting cholesterol contained in the liquid eluted from the reverse phase chromatograph is an ultraviolet detector, and the detector for detecting vitamin E contained in the liquid eluted from the reverse phase chromatograph is a fluorescence detector. there, the device. The apparatus according to claim 4 , wherein there are two or more reversed-phase columns in the reversed-phase chromatograph section.

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