JP2002202314A - Method for determinating cholesterol in lipoprotein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cholesterol measuring method excellent in correlativity as compared with conventional technique and simple in additive compounding system, especially, excellent in the correlativity with a CDC method being a standard and capable of obtaining a measured value excellent in reliability, in a method for simply and directly determinating and measuring cholesterol in lipoprotein in a specimen without performing fractional operation. SOLUTION: In the method for directly and selectively measuring the amount of cholesterol in the specimen containing at least either one of chylomicron, high density lipoprotein, low density lipoprotein and ultralow density lipoprotein, phospholipid or a phospholipid like group-containing compound is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for individually quantifying cholesterol in each lipoprotein, for example, each of high-density lipoprotein, low-density lipoprotein, and very-low-density lipoprotein without fractionation. About the method. In particular, the present invention relates to a method for quantifying cholesterol in lipoprotein, which is important in lipid metabolism in the field of clinical diagnosis.

[0002]

2. Description of the Related Art High-density lipoprotein (HDL) has attracted attention as a lipoprotein having an anti-atherosclerotic effect, and low-density lipoprotein (LDL) has a role of supplying cholesterol to peripheral cells. It is a direct factor of various arteriosclerosis including coronary atherosclerosis, and its blood level is known to be an indicator of arteriosclerotic disease. Very low density lipoprotein (VLDL) has also received attention for its association with arteriosclerosis. Conventional methods for quantifying HDL cholesterol include ultracentrifugation, electrophoresis, and conversion, and methods for quantifying VLDL cholesterol include ultracentrifugation and electrophoresis. The ultracentrifugation method is known as a basic quantification method.
DL, LDL or VLDL is separated and its cholesterol level is measured (Advanced Lipid Research, Vol. 6, page 1, 1968). However, there are drawbacks in terms of quantitative method, simplicity, economy and the like. When the electrophoresis method is used, cholesterol is quantified by separation using a cellulose acetate membrane or agarose gel as a support. However, these quantification methods are unsuitable for automatic analyzers that are frequently used in the fields of multi-sample processing, rapid quantification, and clinical tests.

[0003] The ultracentrifugation method is the source method for HDL-cholesterol measurement, and measurement at the Welfare and Medical Technology Promotion Association SR Center or the like is the standard method. However, in general, as a standard method, in connection with NCEP in the United States, the CDC method (a method using a combination of ultracentrifugation and sedimentation, HDL and LDL-
Cholesterol measurement reference method) has become the standard for such measurement items in clinical diagnostic tests. However, since the CDC method is a method using both the ultracentrifugation method and the precipitation method, it is not a practical method using an automatic analyzer. Various methods that combine the above-described simplicity with the CDC method have been studied. As is required by NCEP to be within ± 5% bias, it is required to have a high correlation with the CDC method and, in practice, to easily obtain measured values by measurement with an automatic analyzer. Have been. (For details on the CDC method, see “Clinical Testing Methods Proposal (Revised 31st Edition), p. 567, Kanai, Kanehara Publishing (19
1998) ". )

Recently, as a method of directly measuring cholesterol of lipoprotein, a method using a polycation such as dextran sulfate alone or a flocculant in combination with a divalent cation (JP-A-6-242110, JP-A-8-242110) -1
No. 31197), a method using a surfactant (Japanese Patent Application Laid-Open Nos. 62-6999 and 58-165800), and a method of combining a protein solubilizing agent and a sulfated polysaccharide (WO 96/29599). JP-A-7-30
No. 1636). In any of the above methods, a specific lipoprotein in the lipoprotein, substantially only a lipoprotein other than HDL is mainly removed from the reaction system, and the HD method is used.
Methods have been devised for selectively quantifying cholesterol in L. However, there is a problem that none of the above-mentioned systems can accurately measure the amount of cholesterol in lipoproteins in a sample high in triglyceride (TG) and M protein. Therefore, a method using a phosphorus compound (JP-A-2
No. 000-116400), it is possible to increase the measurement sensitivity with higher accuracy by solubilizing the M protein by adding an inorganic phosphate salt to the above-mentioned conventional measurement system and suppressing the turbidity of the solution. Have been done. Further, in the disclosed technique, as a method for improving the sulfated inorganic salt of the polysaccharide, a method of combining a phosphate inorganic salt of an organic substance such as a polysaccharide with a surfactant which is a protein solubilizer is disclosed. However, the correlation with a reference method such as the CDC method is not sufficient. For example, in the above-described technique, when a reproduction test is performed, the correlation is 0.98 or less and is not necessarily sufficient. Further, the system is a mixed system of a larger number of components, which is becoming a problem both in terms of simplicity and reproducibility, and it is presently required to simplify the components.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for directly and quantitatively measuring cholesterol in each lipoprotein in a sample more easily and directly than the above-mentioned conventional technique without performing a fractionation operation. It is an object of the present invention to provide a method for measuring cholesterol, which is excellent in the cholesterol content and is simple in the additive compounding system.
In particular, it is an object of the present invention to provide a method for measuring cholesterol, which has excellent correlation with the standard CDC method and can obtain a highly reliable measurement value.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in view of the above problems, and have found that HDL, LDL, VL
In the measurement of cholesterol of a sample containing DL and chylomicron (CM), HDL, LDL, VLDL and CM fractionated by ultracentrifugation in the presence of a specific phospholipid or a compound having a phospholipid-like group in the presence of a measurement reagent
As a result, the reactivity of lipoprotein differs depending on the combination of the type and concentration of the compound, and as a result, HDL, LDL, VLDL and CM
The inventors have found that the reactivity of each cholesterol contained therein is different, and completed the present invention. That is, the present invention includes the following [1] to [11].

[1] A method for directly and selectively measuring the amount of cholesterol in a sample containing at least one of chylomicron, high-density lipoprotein, low-density lipoprotein, and ultra-low-density lipoprotein, the method comprising: Alternatively, a method for determining cholesterol, comprising using a compound containing a phospholipid-like group.

[2] A method for directly and selectively measuring the amount of cholesterol in a sample containing at least one of chylomicron, high-density lipoprotein, low-density lipoprotein, and ultra-low-density lipoprotein, comprising: Alternatively, a method for determining cholesterol, comprising using a phospholipid-like group-containing compound and a protein solubilizing agent.

[3] The aforementioned phospholipid or phospholipid-like group-containing compound is a compound having at least one or more of phosphorylcholine, phosphorylinositol and phosphorylserine groups. The method for quantifying cholesterol according to [1] or [2].

[4] The method for quantifying cholesterol according to the above [1] to [3], wherein the phospholipid-like group-containing compound is a compound containing a phosphorylcholine-like group represented by the formula (1).

[0011]

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(Wherein, R ¹ , R ² and R ³ are the same or different and represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a hydroxyalkyl group, and n represents an integer of 1 to 4). )

[5] The method for quantifying cholesterol according to any one of [1] to [4], wherein the phospholipid-like group-containing compound is a polymer containing a monomer represented by the formula (2) as a constituent.

[0014]

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Wherein X is a divalent organic residue, and Y is carbon number
1 to 6 alkylene oxide groups, Z is a hydrogen atom or
Is R^Five-O- (C = O)-, where R^FiveHas 1 to 1 carbon atoms
10 alkyl groups or hydroxy groups having 1 to 10 carbon atoms
A alkyl group). Also R ^FourIs a hydrogen atom or
Indicates a methyl group. Also, R¹, R^Two, R^ThreeAre the same or different
Such as a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
Or m is 0 or 1, n
Represents an integer of 1 to 4. ｝

[6] The method for quantifying cholesterol according to the above [2] to [5], wherein the protein solubilizing agent is an anionic surfactant, a cationic surfactant or a nonionic surfactant.

[7] A method for measuring the amount of cholesterol, which comprises activating cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase on a sample to determine the amount of hydrogen peroxide or reduced coenzyme produced. The above-mentioned [1] to [6], wherein each enzyme used is not chemically modified.
The method for quantifying cholesterol according to the above.

[8] A method for measuring the amount of cholesterol, which comprises activating cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase on a sample to determine the amount of hydrogen peroxide or reduced coenzyme produced. The method for quantifying cholesterol according to any one of the above [1] to [6], wherein each enzyme to be used is chemically modified.

[9] The method for quantifying cholesterol according to the above [1] to [7], wherein a metal salt of a valence of 1 to 3 is present when measuring the amount of cholesterol.

[10] The above-mentioned [1], wherein a sugar compound is present when measuring the amount of cholesterol.
-The method for quantifying cholesterol according to [9].

[11] The method for quantifying cholesterol according to the above [6] to [9], wherein the nonionic surfactant is a polyethylene oxide derivative, a polypropylene oxide derivative, or a poly (ethylene oxide-propylene oxide) block derivative.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides a method for directly and selectively measuring the amount of cholesterol in a sample containing at least one of chylomicron, high-density lipoprotein, low-density lipoprotein, and ultra-low-density lipoprotein. Wherein the method for determining cholesterol comprises using a phospholipid or a compound containing a phospholipid-like group. In particular, it is a method for quantifying HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol or CM-cholesterol in a sample in the presence of a phospholipid or a compound containing a phospholipid-like group. As a sample containing cholesterol containing at least one or more of chylomicron (CM), high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) used in the present invention Examples include serum, urine and the like. As the sample containing lipoprotein, since the lipoprotein is present in the serum, a sample obtained by partially deriving the above-mentioned serum or blood components is preferably used.

Examples of the phospholipid used in the present invention include, for example, phosphatidylcholine, phosphatidylinositol and phosphatidylserine. Examples of the phospholipid-like group-containing compound used in the present invention include compounds having a polar group of the above-described phospholipid, and particularly include compounds having a phosphorylcholine group, a phosphorylinositol group, a phosphorylserine group, and the like. Further, as the compound containing a phospholipid-like group (hereinafter abbreviated as PC polymer) used in the present invention, the following formula (1)

[0024]

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Compounds containing a group represented by the following are preferred. Here, R ¹ , R ² and R ³ are the same or different and represent a hydrogen atom or an alkyl or hydroxyalkyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 4. When R ¹ , R ² , and R ³ are an alkyl group or hydroxyalkyl group having 7 or more carbon atoms and n is an integer of 5 or more, it is not preferable because synthesis is difficult.

Further, the compound containing a phospholipid analogous group is represented by the formula (2)

[0027]

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A monomer (abbreviated as PC monomer) represented by
Polymers as constituent components are more preferred. here
X is a divalent organic residue, and Y is an alkyl group having 1 to 6 carbon atoms.
An oxygen group, Z is a hydrogen atom or R^Five-O- (C =
O)-, (where R^FiveRepresents an alkyl group having 1 to 10 carbon atoms.
Or a hydroxyalkyl group having 1 to 10 carbon atoms)
Show. Also R ^FourIs a hydrogen atom or a methyl group, R¹,
R^Two, R^ThreeAre the same or different groups and each represents a hydrogen atom or carbon
Represents an alkyl group of 1 to 6 or a hydroxyalkyl group
And m represents 0 or 1, n represents an integer of 1 to 4,

Examples of the divalent organic residue X in the formula (1), for _{example, -C 6 H 4 -, -} C 6 H 10 -, - (C = O)
-O -, - O -, - CH 2 -O -, - (C = O) NH
-, -O- (C = O)-, -O- (C = O) -O-,-
_{C 6 H 4 -O -, -} C 6 H 4 -CH 2 -O -, - C 6 H 4 -
And a group such as (C = O) -O-.

Y in the formula (1) is an alkyleneoxy group having 1 to 6 carbon atoms, for example, a methyleneoxy group, an ethyleneoxy group, a propyleneoxy group, a butyleneoxy group,
Examples include groups such as a pentyleneoxy group and a hexyleneoxy group.

Z in the formula (1) is a hydrogen atom or R ⁵
It represents a -O- (C = O)-group. However, R ⁵ has 1 carbon atom
Represents an alkyl group having 10 to 10 or a hydroxyalkyl group having 1 to 10 carbon atoms. Here, examples of the alkyl group having 1 to 10 carbon atoms include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group,
Examples include an octyl group, a nonyl group, and a decyl group.

The hydroxyalkyl group having 1 to 10 carbon atoms includes, for example, hydroxymethyl group, 2-hydroxyethyl group, 3-hydroxypropyl group, 2-hydroxypropyl group, 4-hydroxybutyl group, 2-hydroxybutyl group. Butyl group, 5-hydroxypentyl group, 2-hydroxypentyl group, 6-hydroxyhexyl group, 2-hydroxyhexyl group, 7-hydroxyheptyl group, 2-
Hydroxyheptyl group, 8-hydroxyoctyl group, 2
-Hydroxyoctyl group, 9-hydroxynonyl group, 2
-Hydroxynonyl group, 10-hydroxydecyl group, 2
-Hydroxydecyl group and the like.

Specific examples of the PC monomer represented by the above formula (2) include, for example, 2-((meth) acryloyloxy) ethyl-2 ′-(trimethylammonio) ethyl phosphate and 3-(( (Meth) acryloyloxy) propyl-2 '-(trimethylammonio) ethyl phosphate, 4-((meth) acryloyloxy) butyl-
2 ′-(trimethylammonio) ethyl phosphate,
5-((meth) acryloyloxy) pentyl-2'-
(Trimethylammonio) ethyl phosphate, 6-
((Meth) acryloyloxy) hexyl-2 ′-(trimethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2 ′-(triethylammonio) ethylphosphate, 2-((meth) (Acryloyloxy) ethyl-2 ′-(tripropylammonio) ethylphosphate, 2-((meth) acryloyloxy) ethyl-2 ′-(tributylammonio) ethylphosphate, 2-((meth) acryloyloxy) ) Ethyl-2 '-(tricyclohexylammonio)
Ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2 '-(triphenylammonio) ethylphosphate, 2-((meth) acryloyloxy) ethyl-2'-(trimethanolammonio) ethylphosphate 2-((meth) acryloyloxy) propyl-2 '-(trimethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) butyl-2'
-(Trimethylammonio) ethyl phosphate, 2-
((Meth) acryloyloxy) pentyl-2 '(trimethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) hexyl-2'-(trimethylammonio) ethylphosphate, 2- (vinyloxy) ethyl- 2 '-(trimethylammonio) ethyl phosphate, 2- (allyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate, 2- (p-
Vinylbenzyloxy) ethyl-2 '-(trimethylammonio) ethylphosphate, 2- (p-vinylbenzoyloxy) ethyl-2'-(trimethylammonio) ethylphosphate, 2- (styryloxy) ethyl-2'- (Trimethylammonio) ethyl phosphate, 2- (p-vinylbenzyl) ethyl-2 ′-(trimethylammonio) ethylphosphate, 2- (vinyloxycarbonyl) ethyl-2 ′-(trimethylammonio) ethylphosphate, 2 -(Allyloxycarbonyl) ethyl-2 '-(trimethylammonio) ethyl phosphate, 2- (acryloylamino) ethyl-
2 ′-(trimethylammonio) ethyl phosphate,
2- (vinylcarbonylamino) ethyl-2 '-(trimethylammonio) ethyl phosphate, ethyl-
(2′-trimethylammonioethyl phosphorylethyl) fumarate, butyl- (2′-trimethylammonioethyl phosphorylethyl) fumarate, hydroxyethyl- (2′-trimethylammonioethyl phosphorylethyl) fumarate, ethyl- (2′-) Trimethylammonioethylphosphorylethyl) malate, butyl-
(2'-trimethylammonioethyl phosphorylethyl) malate, hydroxyethyl- (2'-trimethylammonioethyl phosphorylethyl) malate and the like can be mentioned.

Of these, 2-((meth) acryloyloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate is preferred, and 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate (= 2- (methacryloyloxy) ethylphosphorylcholine (hereinafter, abbreviated as MPC) is more preferable in terms of availability and the like.

The above-mentioned PC polymer is preferably composed mainly of a polymer obtained by polymerizing a monomer composition containing a PC monomer represented by the above formula (2). The monomer composition containing a PC monomer may contain only the PC monomer or may contain other polymerizable monomers. further,
Specifically, the PC polymer is obtained by polymerizing a monomer composition comprising 20 to 100 mol% of a PC monomer as the component A and 0 to 80 mol% of a hydrophobic monomer as the component B. Polymers can preferably be mentioned. More preferably, the PC monomer is 40 to 80 mol% as the component A.
And 20 to 60 mol% of a hydrophobic monomer as the B component.

When the content of the hydrophobic monomer of the component B is more than 80 mol%, a phosphorylcholine-like group (hereinafter abbreviated as PC group) is used.
It is not preferable because it is difficult to exert the effect of (1).
Furthermore, the PC polymer contains 20 to 100 mol% of a PC monomer as an A component, 0 to 40 mol% of a hydrophobic monomer as a B component, and 0 hydrophilic monomer as a C component.
A polymer obtained by polymerizing the monomer composition of about 70 mol% can be preferably exemplified. More preferably, it is a monomer composition comprising 40 to 80 mol% of a PC monomer as the component A, 10 to 30 mol% of a hydrophobic monomer as the component B, and 10 to 60 mol% of a hydrophilic monomer as the component C.
If the amount of the PC monomer of the component A is less than 20 mol%, it is difficult to exert the effect of the PC group, which is not preferable.

When polymerizing the PC monomer, the above-mentioned PC
One of the monomers can be used alone, or a mixture of two or more can be used. The PC monomer can be produced by a known method. For example, it can be produced according to a known method disclosed in JP-A-54-63025, JP-A-58-155491, and the like. The hydrophobic monomer of the B component copolymerized with the PC monomer is represented by the formula (3)

[0038]

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Where L ¹ is —C ₆ H ₄ —,
_{C 6 H 10 -, - (} C = O) -O -, - O -, - (C =
O) NH-, -O- (C = O)-and -O- (C =
O) represents a group selected from the group consisting of -O-, L ² is
Hydrogen _{atom, - (CH 2) g -L} 3 and - ((CH _{2) p} -
O) shows the hydrophobic functional group selected from _h -L ^3. (G, h
Is 1 to 24, p is an integer of 3 to 5, L ³ represents a hydrogen atom, a functional group selected from a methyl group, -C ₆ H ₅ and -O-C ₆ H _5. ) A monomer represented by｝.

Specific examples of the hydrophobic monomer of component B include methyl (meth) acrylate and ethyl (meth) acrylate.
Linear or branched alkyl (meth) acrylates such as acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) acrylate, and stearyl (meth) acrylate; cyclic alkyl (meth) such as cyclohexyl (meth) acrylate ) Acrylates; aromatic ring (meth) acrylates such as benzyl (meth) acrylate and phenoxyethyl (meth) acrylate; polyalkylene glycol (meth) acrylates such as polypropylene glycol (meth) acrylate; styrene, methylstyrene, chloromethylstyrene and the like Styrene monomers; vinyl ether monomers such as methyl vinyl ether and butyl vinyl ether; and vinyl ester monomers such as vinyl acetate and vinyl propionate. One or more of these are used.

The hydrophilic monomer of component C includes, for example, hydroxy group, carboxyl group, histone group, sulfone group, amide group, amino group, dialkylamino group, trialkylamino group, trialkylphosphonium base and polyoxyethylene. It is a monomer having a hydrophilic group selected from the group consisting of groups. More specifically, for example, hydroxyl-containing (meth) acrylates such as 2-hydroxyethyl (meth) acrylate, 2-hydroxybutyl (meth) acrylate, and 4-hydroxybutyl (meth) acrylate; acrylic acid and methacrylic acid; Carboxylic acid; styrene group sulfonic acid, (meth) acryloyloxyphosphonic acid, ionic group-containing monomer such as 2-hydroxy-3- (meth) acryloxypropyltrimethylammonium chloride; (meth) acrylamide, aminoethyl methacrylate, dimethyl Nitrogen-containing monomers such as aminoethyl (meth) acrylate; and polyethylene glycol (meth) acrylate. One or more of these are used.

The PC polymer is a monomer composition of the PC monomer of the component A and the hydrophobic monomer of the component B, or the hydrophobic monomer of the PC monomer of the component A and the hydrophobic component of the component B. And C
What is necessary is just to polymerize the monomer composition with the hydrophilic monomer of the component, and it can be produced by ordinary radical copolymerization.

The molecular weight of the PC polymer is 50 by weight on average.
The range is preferably from 0 to 5,000,000, more preferably from 1,000 to 1,000,000, and still more preferably from 10,000 to 500,000. If the molecular weight of the polymer is less than 500, it is difficult to sufficiently promote a biochemical reaction, and if the molecular weight of the polymer is
If it is larger than 0,000,000, the viscosity of the aqueous solution of the PC polymer becomes too high, which may hinder the biochemical reaction, which is not preferable.

As the protein solubilizing agent used in the present invention, an anionic surfactant, a nonionic surfactant, a cationic surfactant and the like can be used. HD in sample
The type and concentration of the protein solubilizing agent, or a combination of several types of protein solubilizing agent and each concentration may be selected depending on which amount of cholesterol in L, LDL, VLDL or CM is to be measured. For example, H in the sample
When measuring cholesterol in DL, a poly (ethylene oxide-propylene oxide) block derivative of a nonionic surfactant is preferable, and when measuring cholesterol in LDL in a sample, a polyethylene oxide derivative of a nonionic surfactant is used. Poly (ethylene oxide-propylene oxide) derivatives of nonionic surfactants are preferred.

The metal salt used in the present invention includes, for example,
Magnesium salts, calcium salts, manganese salts, cobalt salts and the like. The amount of the salt used is, for example, 0.001 to 50 mol.

The method for quantifying cholesterol according to the present invention relates to a compound having a phospholipid or a phospholipid polar group or a phospholipid polar analog group, that is, particularly a phosphorylcholine group,
One or more compounds having at least one phosphorylinositol group, phosphorylserine group or each similar group, and / or a protein solubilizing agent coexist in a reagent for measuring cholesterol in lipoproteins and directly This is a method for separating and quantifying cholesterol, and any cholesterol measuring system can be used as long as it is an enzymatic measuring system. In the present invention, the cholesterol measurement system itself follows a general method based on the reaction principle of the following formula (4) which is already known.

[0047]

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Examples of the dye source include, in addition to the commonly used combinations of 4-aminoantipyrine and phenols, 4-aminoantipyrine and the following reagents known as Trinder's reagents: N-sulfopropylaniline , N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N- (2
-Hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS),
N-ethyl-N-sulfopropyl-m-toluidine (TOP
Anilines such as S) or N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine (E
MSE) can be used. The dye source is not limited to the above compounds. In addition, it is assumed that an optimum measurement wavelength is used for the dye source to be used.

Here, as the enzyme to be used, for example,
Examples include microorganism- or animal-derived cholesterol esterase and lipoprotein lipase, microorganism-derived cholesterol oxidase, and microorganism- or animal-derived cholesterol dehydrogenase, which are commercially available as cholesterol esterase, cholesterol oxidase, or cholesterol dehydrogenase.

Further, in order to further enhance the specificity and stability of the above enzyme, the above enzyme is chemically modified with a group mainly composed of polyethylene glycol, a water-soluble oligosaccharide residue, a sulfopropyl group, or the like. May be used. As a modifying agent for modifying an enzyme, a compound in which a functional group or a molecular group capable of binding to an amino group is bound to polyethylene glycol, for example, N-
Sunbright VFM4101 (manufactured by NOF Corporation) having a hydroxysuccinimide group bonded thereto, or Sunbright AK having an acid anhydride structure bonded to polyalkylene glycol.
Various compounds of a series such as M (manufactured by NOF CORPORATION) are exemplified.

In the above measurement, a protein solubilizer, a divalent metal salt, a polysaccharide phosphate, or a polysaccharide sulfate can be used in combination to further increase the specificity. Said protein solubilizer,
One or more divalent metal salts, polysaccharide phosphates, and polysaccharide sulfates may be used. Further, according to the present invention, by using a compound having a phospholipid or a phospholipid-like group alone or in combination with a protein solubilizing agent, a reagent for quantifying each cholesterol in HDL, LDL, VLDL or CM containing these components can be prepared. Can be.

According to the cholesterol quantification method of the present invention, cholesterol in lipoproteins in samples such as blood and urine can be directly measured without separation, so that automatic analysis is possible, and the samples can be analyzed at once. Suitable for multiple inspections.

[0053]

The method for quantifying cholesterol of the present invention comprises:
By using a phosphorus compound, particularly a phosphorylcholine group, which is a polar group derived from a phospholipid, a phosphorylinositol group, a compound containing any of a phosphorylserine group, or a compound containing a similar group of a phospholipid polar group, blood, This is an excellent method for quantitatively determining cholesterol in lipoprotein contained in a body fluid sample such as urine, which can be directly measured without fractionation. In particular, it is excellent in quantitativeness having a correlation comparable to that of the conventional ultracentrifugation method, fractionation method, and CDC method. The cholesterol quantification method of the present invention comprises simultaneously adding these phospholipids or a compound having a phospholipid polar group or a phospholipid polar analog group to a measurement container, a measurement analyzer, an enzyme,
It has a synergistic effect because it prevents adsorption of proteins and the like, and also has the effect of stabilizing enzymes and proteins.

[0054]

Now, the present invention will be described in further detail with reference to specific examples. [Example 1] The results of the method for directly quantifying HDL cholesterol using the PC polymer of the present invention, as compared with an ultracentrifugation method as a standard method and a fractionation method as a conventional method (phosphotungstic acid method), are shown. . <Reagent composition> Second reagent Cholesterol esterase 1.1 U / mL cholesterol oxidase 7.0 U / mL 4-aminoantipyrine 2.3 mM peroxidase 23 U / mL Polyoxyethylene monolaurate 0.7 g / L Good buffer (pH 6.9) 25 mM

In this method, 6 μL of a serum sample was added to 300 μL of the first reagent preliminarily heated at 37 ° C., and 5 μl at 37 ° C.
After heating for 1 minute, the absorbance of the obtained solution at 585 nm was measured (E1). Next, 100 μL of the second reagent preliminarily heated to 37 ° C. was added and stirred, and after 5 minutes, the absorbance at the same wavelength was measured (E2: value after concentration correction).
The amount of HDL cholesterol is
The same operation was performed using a standard solution of 50 mg / dL,
It was calculated by comparing the values of (E2-E1). The measurement operation was performed on 10 serum samples, and for the obtained cholesterol value, a correlation coefficient of a linear relationship between the value of the method of the present invention and the value of the ultracentrifugation method was obtained. Was compared with the correlation coefficient. Table 1 shows the results.

As shown in Table 1, the measurement results of Example 1, which is the method of the present invention, show that the measurement of only cholesterol in the target HDL was possible only by adding the MPC polymer. Compared to the fractionation method, which requires no fractionation operation, it is simple and easy to apply to automatic analyzers, so it is economical and has a high correlation coefficient with the ultracentrifugation method, which is the source method. Precise diagnostic judgment is now possible. In particular, for the ultracentrifugation method, the correlation coefficient in the fractionation method was 0.1.
It can be seen that the correlation coefficient of Example 1 is 0.988, which is even better than that of Example 952.

Example 2 The compound having a phospholipid polar analogous group used in the first reagent of Example 1 was used as an inositol phosphate and a protein solubilizing agent, and the enzyme used in the second reagent was used as a modifying enzyme. Quantitative measurement of cholesterol was performed in the same manner as in Example 1 except that the following first and second reagents that were changed were used.

<Reagent composition> Second reagent β-glucan disodium phosphate 2.2 mM disodium phosphate 26 mM peroxidase 20 U / mL modified cholesterol esterase 1 U / mL modified cholesterol oxidase 5.5 U / mL 4-aminoantipyrine 0.5 g / mL Good Buffer 20 mM Vitamin E disodium phosphate 0.1 μM

In Example 2, the measured values obtained by the automatic analyzer are shown in Table 2.

As shown in Table 2, the measurement results of Example 2 which is the method of the present invention are based on the measurement of phosphoric acid-containing inorganic salts of organic compounds such as phosphorylated polysaccharide which is a method described in a conventional patent. By adding the compound defined by the present invention to the system, the measurement of only cholesterol in the target HDL requires no fractionation operation as compared with the conventional fractionation method, and is simple and applicable to an automatic analyzer. It can be seen that the method is easy, so that it is economical and has a high correlation coefficient with the ultracentrifugation method, which is the source method, as compared with the method described in the conventional patent. In particular, as compared with the ultracentrifugation method, the correlation coefficient in the fractionation method is 0.972, and the correlation coefficient in Example 2 is 0.983, which indicates that the correlation is even better.

Example 3 A method for directly quantifying LDL cholesterol using phosphatidylserine as a phospholipid was compared with a CDC method. <Reagent composition> Second reagent Cholesterol esterase 1.1 U / mL cholesterol oxidase 5.0 U / mL peroxidase 27.0 U / mL 4-aminoantipyrine 2.3 mM disodium phosphate 21.9 mM phosphatidylserine 3 mg / mL β-glucan phosphate 2 Sodium 7.0 mM Tris buffer (pH 7.0) 25 mM

In Example 3, 6 μL of the serum sample was added to 300 μL of the first reagent preliminarily heated at 37 ° C.
For 5 minutes, and the absorbance at 585 nm of the resulting solution was measured (E1). Next, 100 μL of the second reagent preliminarily heated to 37 ° C. was added and stirred, and after 5 minutes, the absorbance at the same wavelength was measured (E2: value after concentration correction). The amount of LDL-cholesterol was calculated by performing the same operation using a standard solution of LDL-cholesterol 200 μg / dL and comparing the value of (E2-E1). Table 3 shows the results.

As shown in Table 3, the measurement result of Example 3 which is the method of the present invention is based on the measurement including phosphoric inorganic salt of an organic compound such as phosphorylated polysaccharide which is a method described in a conventional patent. By adding the compound defined by the present invention to the system, the measurement of only cholesterol in the target LDL requires no fractionation operation as compared with the conventional fractionation method, and is simple and applicable to an automatic analyzer. It is easy and economical, and has a high correlation coefficient with the CDC method, which is the standard method, as compared with the method described in the conventional patent. In particular, a comparative test was performed on the ultracentrifugation method according to a known method (for example, JP-A-2000-116400). As a result, it can be seen that the correlation coefficient of Example 3 is 0.989, which is even better than the correlation coefficient of 0.979.

Example 4 Instead of the polymer of Reference Example 1 used in the first reagent of Example 1, Reference Example 3 and Second Example
The same operation as in Example 3 was performed except that a polyoxyethylene derivative (polyoxyethylene alkyl ether) was used instead of the protein solubilizing agent polyoxyethylene monolaurate used in the reagent, and the measurement was performed on five serum samples. Values were compared. Table 4 shows the results. <Reagent composition> Second reagent Polyoxyethylene derivative (Note 1) 2 g / L cholesterol esterase 1.1 U / mL cholesterol oxidase 5.1 U / mL peroxidase 28 U / mL 4-aminoantipyrine 2.5 mM Disodium phosphate 14 mM Tris buffer Solution (pH 7.0) 28 mM Note 1) Emulgen L-40 (trade name of Kao, polyoxyethylene alkyl ether) was used.

[0065]

As shown in Table 4, the measurement results of Example 4 which is the method of the present invention are based on the measurement results of the method described in the conventional patent, which includes the phosphorylated inorganic salt of an organic compound such as phosphorylated polysaccharide. By adding the compound defined by the present invention to the system, the measurement of only cholesterol in the target LDL requires no fractionation operation as compared with the conventional fractionation method, and is simple and applicable to an automatic analyzer. It is easy and economical, and has a high correlation coefficient with the CDC method, which is a standard method, as compared with the method described in a known patent, thereby enabling more precise diagnosis and judgment. In particular, as compared with the ultracentrifugation method, the correlation coefficient in a known method (for example, the method disclosed in Japanese Patent Application Laid-Open No. 2000-116400) is 0.976, and in Example 4, the correlation coefficient is 0.992. Is good.

[Embodiment 5] In the fifth embodiment, the P
The polymer produced in Reference Example 4 described below was used in place of the C polymer, and the protein solubilizing agent was changed to a poly (ethylene oxide-propylene oxide) block derivative (manufactured by BASF, trade name: Pluronic L-122) to produce color. Agent 4
The same operation as in Example 3 was carried out except that the aminoantipyrine was used, and the measurement values of the five serum samples were compared with the CDC method. Table 5 shows the results. <Reagent composition> First reagent monopotassium phosphate 20 mM β-cyclodextrin-lithium phosphate 3 mM HDAOS 1.1 mM magnesium sulfate 4 mg / mL Good buffer (pH 6.9) 25 mM Second reagent monopotassium phosphate 10 mM cholesterol esterase 5.0 U / mL cholesterol oxidase 1.1 U / mL peroxidase 25 U / mL 4-aminoantipyrine 2.2 mM Pluronic L-122 2.2 g / L Emulgen L-40 1.5 g / L Nimine S- 220 1.0 g / L Tocoherol phosphate 0.2 g / L Good buffer (pH 6.9) 25 mM phosphatidylinositol phosphate 1.1 g / L For PC polymer produced in Reference Example 4 0.7 g / L Note) The compounds shown are as follows: Pluronic L-122; (PEO-PPO block polymer, HL
B = 4.0), BASF Wyandotte Corp., Emulgen L-40; Emulgen (PEO-alkyl ether), Kao
Corp., Nimin S-220; diethylene glycol stearylamine, manufactured by NOF Corporation, trade name.

[0068]

As shown in Table 5, the measurement result of Example 5 which is the method of the present invention is based on the measurement method which includes a phosphorylated inorganic salt of an organic compound such as phosphorylated polysaccharide which is a method described in a conventional patent. By adding the compound defined by the present invention to the system, the measurement of only cholesterol in the target LDL requires no fractionation operation as compared with the conventional fractionation method, and is simple and applicable to an automatic analyzer. It is easy and economical, and has a high correlation coefficient with the CDC method, which is the standard method, as compared with the method described in the conventional patent. In particular, as compared with the ultracentrifugation method, the correlation coefficient of the method of the conventional patent is 0.974, and the correlation coefficient of Example 5 is 0.991, which indicates that the correlation is even better.

The sample used above was obtained by the following method. Reference Example 1: Synthesis of PC polymer used in Example 1; 5.4 g of MPC, 3.71 g of methacrylic acid, 0.351 g of perroyl-SA (manufactured by NOF Corporation) as an initiator, and 42.919 g of a polymerization solvent. Distilled water was taken into a polymerization tube and uniformly dissolved. After argon gas was blown into this solution for 10 minutes and the tube was sealed, polymerization was carried out at 70 ° C. for 6 hours. After completion of the reaction, the mixture was cooled to room temperature, the polymerization tube was opened, and the solution was then dialyzed with a dialysis membrane ("Spectrum" manufactured by Spectrum Medical Industries).
/ Por membranes Mw CO, 6000-8000 "), and dialysis was performed using 10 times the amount of distilled water of the polymerization solution.
Exchange of distilled water was carried out at an interval of once / day for 7 days to remove unreacted monomers and the like, and purification was performed. The results of the molecular weight of the copolymer obtained by GPC measurement and the copolymer composition ratio determined by H-NMR measurement are shown below. MPC / methacrylic acid = 3.8 / 6.1 (molar ratio)

Reference Example 2 Preparation of Modification Enzymes Used in Example 2 The modification enzymes used in Example 2 were obtained by modifying cholesterol esterase and cholesterol oxidase enzymes. 0.1M (p
H8.0) with a Good buffer solution to 50 g / L, and then added 10 g of Sunbright VFM4101 (manufactured by NOF Corporation) to 0.15 g / g of enzyme.
The mixture was allowed to stand at 20 ° C for 12 hours to prepare each enzyme modified with polyethylene glycol. This reaction solution was used as it was.

Reference Example 3 Synthesis of PC Polymer Used in Example 4 MPC 318.6 g, stearyl methacrylate 41.4
g, 7.81 g of t-butyl peroxyneodecaate as an initiator, and 1440 g of ethanol as a polymerization solvent were placed in a four-necked flask and uniformly dissolved. After nitrogen gas was blown into this solution for 30 minutes, polymerization was carried out at 50 ° C. for 3 hours, followed by heating to 60 ° C. for further 2 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, and the obtained polymerization liquid was collected and the polymerization reaction rate and the molecular weight were measured by GPC. As a result, the polymerization reaction rate was 9
8.7%, weight average molecular weight was 140000. The polymerization solution was added dropwise to 8 L of diethyl ether while stirring, and the deposited precipitate was filtered and further vacuum-dried at room temperature for 48 hours to prepare a polymer powder. The obtained polymer powder was dissolved in 1 L of distilled water, charged into a dialysis membrane having a molecular weight cutoff of 3500 (manufactured by Spectrapore), and immersed in 10 L of distilled water. The dialysate was changed 3 times / day for a total of 96
After time treatment, unreacted monomers and the like were removed and purification was performed. The results of the copolymer composition ratio determined from the H-NMR measurement are shown below. MPC / methacrylic acid = 9.0 / 1.0
(Molar ratio)

Reference Example 4 Synthesis of PC Polymer Used in Example 5 600 g of MPC and 900 g of distilled water as a polymerization solvent were placed in a four-necked flask and uniformly dissolved. After nitrogen gas was blown into this solution for 30 minutes, 11.7 g of succinyl peroxide as an initiator was added while maintaining the temperature at 60 ° C., and polymerization was performed for 6 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, and the obtained polymerization liquid was collected and the polymerization reaction rate and the molecular weight were measured by GPC. As a result, the polymerization reaction rate was 95.3%, and the weight average molecular weight was 1030.
000. The polymerization solution was diluted with 1500 mL of ion-exchanged water, charged into a dialysis membrane having a molecular weight cutoff of 12,000 (manufactured by Spectrapore), and immersed in 15 L of distilled water. The dialysate was exchanged at an interval of 3 times / day, and the treatment was performed for a total of 96 hours.
Unreacted monomers were removed and purification was performed.

Claims (11)

[Claims] 1. A method for directly and selectively measuring the amount of cholesterol in a sample containing at least one of chylomicron, high-density lipoprotein, low-density lipoprotein, and ultra-low-density lipoprotein, comprising: A method for quantifying cholesterol, comprising using a phospholipid-like group-containing compound. 2. A method for directly and selectively measuring the amount of cholesterol in a sample containing at least one of chylomicron, high-density lipoprotein, low-density lipoprotein, and ultra-low-density lipoprotein, the method comprising: A method for quantifying cholesterol, comprising using a phospholipid-like group-containing compound and a protein solubilizing agent. 3. The method of claim 1, wherein the phospholipid or the compound containing a phospholipid-like group is at least one of a phosphorylcholine group, a phosphorylinositol group and a phosphorylserine group.
3. The method for quantifying cholesterol according to claim 1, which is a compound having one or more kinds of groups. 4. The method for quantifying cholesterol according to claim 1, wherein the phospholipid-like group-containing compound is a compound containing a phosphorylcholine-like group represented by the formula (1). Embedded image (In the formula, R ¹ , R ² , and R ³ are the same or different groups, each representing a hydrogen atom or an alkyl group or a hydroxyalkyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 4.) 5. The compound having a phospholipid analogous group according to the formula (2)
2. A polymer comprising the indicated monomer as a constituent component.
Method for quantifying cholesterol according to any one of claims 4 to 4
Law. Embedded image中 In the formula, X is a divalent organic residue, and Y is an alkyl group having 1 to 6 carbon atoms.
A kylene oxide group, Z is a hydrogen atom or R^Five-O-
(C = O)-, where R^FiveIs alk having 1 to 10 carbon atoms
Or a hydroxyalkyl group having 1 to 10 carbon atoms.
). Also R ^FourRepresents a hydrogen atom or a methyl group
You. Also, R¹, R^Two, R^ThreeAre the same or different groups, water
A hydrogen atom or an alkyl group having 1 to 6 carbon atoms or hydroxy
M represents 0 or 1; n represents an integer of 1 to 4;
Indicates a number. ｝ 6. The method according to claim 2, wherein the protein solubilizing agent is an anionic surfactant, a cationic surfactant or a nonionic surfactant. 7. A method for measuring the amount of cholesterol which comprises reacting cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase on a sample to determine the amount of hydrogen peroxide or reduced coenzyme produced. The method for quantifying cholesterol according to any one of claims 1 to 6, wherein each of the enzymes is not chemically modified. 8. A method for measuring the amount of cholesterol which comprises reacting a sample with cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase to determine the amount of hydrogen peroxide or reduced coenzyme produced. The method for quantifying cholesterol according to any one of claims 1 to 6, wherein each of the enzymes is chemically modified. 9. The method for quantifying cholesterol according to any one of claims 1 to 8, wherein when measuring the amount of cholesterol, a monovalent to trivalent metal salt is present. 10. The method for quantifying cholesterol according to claim 1, wherein a sugar compound is present when measuring the amount of cholesterol. 11. The method for quantifying cholesterol according to claim 6, wherein the nonionic surfactant is a polyethylene oxide derivative, a polypropylene oxide derivative, or a poly (ethylene oxide-propylene oxide) block derivative.