JP2001017197A - Measurement and measuring reagent for lipoprotein cholesterol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately measure the subject compound which is a diagnostic index to arteriosclerosis, etc. by making an enzyme act on a sample containing a lipoprotein in the coexistence of a specific polymer and determining a compound consumed or produced by the enzyme reaction. SOLUTION: An enzyme is made to act on a lipoprotein cholesterol in the coexistence of a polymer having a recurring unit represented by formula I (R1 is a 4-30C alkyl group; R2 and R3 are each H or methyl) and a recurring unit represented by formula II [X is H or COOH; Y is a group having COOH, SO3H or PO(OH2) or a group derived therefrom] in a weight ratio of formula I:formula II within the range of (1:99) to (99:1) to thereby accurately measure the objective lipoprotein cholesterol considered as important as a diagnostic index deeply related to arteriosclerosis or cardiac infarction in a method for determining a lipoprotein cholesterol comprising making the enzyme act on a sample containing a lipoprotein and determining a compound consumed or produced in an enzyme reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is mainly intended for use in the field of clinical examination, and a method for quantifying cholesterol in a specific lipoprotein fraction in a sample containing lipoprotein and a sample containing lipoprotein. The present invention relates to a reagent for quantifying cholesterol in a specific lipoprotein fraction therein.

[0002]

2. Description of the Related Art Generally, blood cholesterol is contained in lipoproteins in blood and is regarded as an important diagnostic index closely related to arteriosclerosis and myocardial infarction.
Lipoprotein is a complex of apolipoprotein and lipid, and depending on the specific gravity of each, high density lipoprotein (HD
L), low density lipoprotein (LDL), very low density lipoprotein
(VLDL) and chylomicron (CM).

The measurement of total cholesterol in blood, which has been performed in clinical tests for a long time, measures cholesterol contained in all lipoproteins in blood, and is important as a predictor of the risk of the above-mentioned diseases. Have been. However, in recent clinical studies, cholesterol contained in LDL is considered to be a more accurate risk factor for the above-mentioned diseases, whereas cholesterol contained in HDL is a negative risk factor, that is, the amount of HDL cholesterol and the frequency of occurrence of the above-mentioned diseases. Since there is a negative correlation between HDL and LD,
It is more important to measure the cholesterol contained in L individually (arteriosclerosis 25 (1.2):
1-34, 1997).

[0004] As a method for analyzing these lipoproteins, besides an ultracentrifugation method using a difference in specific gravity, a method for detecting lipoproteins separated by electrophoresis by lipid staining, and a method for HPLC analysis
There is a reaction liquid chromatography method or immunochemical method, etc., in which the peak containing cholesterol is detected with an enzyme reagent after the separation by liposome separation, but all of these procedures are complicated and a large number of samples cannot be measured quickly. Due to problems, it was rarely used for routine testing.

[0005] In the past, a method of measuring HDL cholesterol using a lipoprotein precipitant such as dextran sulfate or phosphotungstic acid has been used for routine clinical examinations. First, a lipoprotein precipitant and an alkaline earth metal ion are added to a sample such as serum to aggregate lipoproteins other than HDL, which is removed by centrifugation, and cholesterol in HDL remaining in the supernatant is measured. Further, the blood total cholesterol level and triglyceride level are measured, and these values are applied to an empirically derived Friedewald equation to calculate the LDL cholesterol level.

However, although this method is simpler than ultracentrifugation or the like, it involves an operation of adding a precipitant and centrifuging, so that a relatively large amount of sample is required, and a large number of samples can be prepared in a short time. Was difficult to process. Furthermore, Fri
The edewald equation is derived from an empirical rule that the ratio of the amount of cholesterol to the amount of triglyceride in VLDL is almost constant, so that a large error occurs in diseases in which lipoprotein metabolism or lipid metabolism is abnormal. There was a problem.

[0007] Under such circumstances, a differential quantification method has been developed in which lipoprotein cholesterol in plasma or serum can be mounted on an automatic analyzer without pretreatment such as centrifugation. Among them, as a method for directly measuring HDL cholesterol, the above-mentioned precipitation method is applied to HDL cholesterol.
Lipoproteins other than lipoproteins are aggregated so that the enzyme does not act, and HDL cholesterol is selectively reacted (JP-A-8-131197, JP-A-6-242).
110, JP-A-11-56395, JP-A-9-96637, JP-A-9-285298), and the enzyme reaction of lipoprotein cholesterol using a substance other than a precipitant such as a surfactant. Control method (JP-A-63-126498, JP-A-62-128498)
No. 69999 and Japanese Patent Application Laid-Open No. 9-299), but none of them is sufficiently satisfactory in terms of versatility and accuracy.

[0008] In addition, as a method of combining a lipoprotein flocculant and a surfactant, HDL is preferentially used in the presence of a surfactant that acts preferentially on HDL and a substance that inhibits the enzymatic reaction of lipoprotein cholesterol. Reaction method and detection (JP-A-11-56395), lipoprotein cholesterol other than HDL is quenched in the presence of a surfactant and a sugar compound for controlling lipoprotein cholesterol reactivity to quantify HDL cholesterol A method (JP-A-7-301636) and the like are disclosed.

On the other hand, a method for measuring LDL cholesterol by the same method as the reagent for directly measuring HDL cholesterol has been disclosed, and a method for measuring LDL cholesterol using a lipoprotein aggregating agent is disclosed in 6-213899, JP-A-7-2
No. 80812, WO 96/28734, JP-A-10-31833, JP-A-11-30617
No. publication. As a method for measuring LDL cholesterol using a surfactant, Japanese Patent Application Laid-Open No. 58-16580
0, JP-A-10-84997, JP-A-10-84997
JP-A-38888 is disclosed. Furthermore, L
Method for selectively reacting and detecting LDL cholesterol using a chemical modifying enzyme acting only on DL
No. -80300) is also disclosed.

Many of these selective methods for measuring lipoprotein cholesterol control the reaction selectivity of lipoprotein cholesterol by combining a surfactant or a lipoprotein aggregating agent. However, there is a problem that the effect of the surfactant is easily affected by the blood component due to the presence of the component having For example, lecithin, which is a phospholipid contained in lipoproteins, has a strong surfactant activity, and a higher fatty acid having a surfactant activity is generated in the course of hydrolysis of cholesterol ester. Most of cholesterol in blood is present as an ester with higher fatty acids, and the measurement of cholesterol requires the process of hydrolysis of ester cholesterol. Therefore, generation of higher fatty acids is inevitable. Furthermore, cholesterol esterases used for the hydrolysis of ester-type cholesterol, except for cholesterol esterases obtained from the pancreas of mammals, mostly have the activity of hydrolyzing neutral fats, and neutral glycerol esters of higher fatty acids. Higher fatty acids are also produced by the hydrolysis of fats.

In addition, serum albumin having an action of adsorbing a surfactant is present in blood, and the concentration of free surfactant in a reagent to which serum is added may fluctuate.
The action of a surfactant greatly changes depending on its concentration or the coexistence of another substance having a surfactant. Therefore,
In serum with abnormalities in serum albumin concentration, cholesterol concentration, neutral fat concentration, etc., it becomes difficult to maintain the effect of surfactant designed to selectively enzymatically react cholesterol of specific lipoprotein, The measurement accuracy of the target lipoprotein cholesterol tends to decrease.

The lipoprotein aggregating agent has little problem of being affected by blood components, while the lipoprotein aggregating agent contains lipoprotein as disclosed in Japanese Patent Application Laid-Open No. 6-213899. There is a problem of increasing the turbidity of the reagent. Most current methods for measuring cholesterol use an optical method to quantify hydrogen peroxide or reduced coenzyme generated by the oxidation-reduction reaction of cholesterol, and lipoprotein aggregation increases the turbidity of the reagent. This causes a measurement error. In addition, there is a risk that lipoprotein aggregates may block the lines of the automatic analyzer. Therefore, in order to accurately measure a specific lipoprotein cholesterol, Japanese Patent Application Laid-Open No.
A step of dissolving a lipoprotein aggregate as disclosed in JP-A-42110 and a step of measuring a reaction rate as disclosed in JP-A-8-131195 are required.

[0013]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and has been developed to provide a high-precision method that is not affected by blood components having a surfactant effect and does not generate a factor that interferes with optical measurement. The present invention relates to a method for measuring lipoprotein cholesterol which is highly versatile and provides a measuring reagent.

[0014]

Means for Solving the Problems As a result of intensive studies, the present inventors have selected a reaction between LDL cholesterol and an enzyme for measuring cholesterol without forming lipoprotein aggregates that interfere with optical measurement. Completed the present invention by finding that the above problem can be achieved by coexistence of a substance having a characteristic that is not affected by blood components having a surface-active effect such as higher fatty acids and the effect of suppressing the reaction and its reaction suppressing effect. I came to.

That is, the present invention relates to the following matters. [1] In a method for measuring lipoprotein cholesterol, in which an enzyme is allowed to act on a sample containing lipoprotein and a compound consumed or generated in the enzyme reaction is quantified, the formula (a)

[0016]

Embedded image And equation (b)

[0017]

Embedded image Wherein R ¹ is an alkyl group having 4 to 30 carbon atoms, R ² and R ³ are each independently a hydrogen atom or a methyl group, X is a hydrogen atom or COOH, and Y is COO
H, SO ₃ H, a group having PO (OH) ₂ or a group derived therefrom. Characterized in that lipoprotein cholesterol is reacted with an enzyme in the presence of a high molecular compound having a weight ratio of (a) :( b) in the range of 1:99 to 99: 1. Measurement method. [2] The high molecular compound has a molecular weight of 5,000 to 50.
The method for measuring lipoprotein cholesterol according to the above [1], wherein the lipoprotein cholesterol is 000 daltons. [3] The method for measuring lipoprotein cholesterol according to the above [1] or [2], wherein the concentration of the polymer compound is in the range of 0.001 to 1 w / v%.

[4] The method for measuring lipoprotein cholesterol according to any one of [1] to [3] above, wherein the pH when reacting cholesterol in the presence of the polymer compound is in the range of 5 to 9. [5] The polymer compound is at least one compound selected from the group consisting of a copolymer of a 1-olefin having 6 to 32 carbon atoms and maleic acid or acrylic acid or methacrylic acid, and an acid amide thereof. Characterized by the above
The method for measuring lipoprotein cholesterol according to any one of [1] to [4]. [6] The above-mentioned [1] to [5], wherein the lipoprotein cholesterol to be measured is high density lipoprotein (HDL) cholesterol or low density lipoprotein (LDL) cholesterol.
The method for measuring lipoprotein cholesterol according to any one of the above. [7] Cholesterol esterase, cholesterol oxidase or cholesterol dehydrogenase and formula (a)

[0019]

Embedded image And equation (b)

[0020]

Embedded image Wherein R ¹ is an alkyl group having 4 to 30 carbon atoms, R ² and R ³ are each independently a hydrogen atom or a methyl group, X is a hydrogen atom or COOH, and Y is COO
H, SO ₃ H, a group having PO (OH) ₂ or a group derived therefrom. And a weight ratio of (a) :( b) in the range of 1:99 to 99: 1. [8] The reagent for measuring lipoprotein cholesterol according to the above [7], which measures high density lipoprotein (HDL) cholesterol and / or low density lipoprotein (LDL) cholesterol.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for measuring LDL cholesterol and cholesterol without forming a lipoprotein aggregate which prevents a polymer compound having the above repeating units (a) and (b) from interfering with optical measurement. It is based on the new finding that the reaction with an enzyme is selectively suppressed, and the effect of suppressing the reaction is not affected by blood components having a surface-active action such as higher fatty acids. Many compounds having similar effects have already been disclosed. For example, the water-soluble comb polymer disclosed in JP-A-6-213899 selectively acts on LDL but forms LDL aggregates to increase the turbidity of the reaction solution. Different from molecular compounds. Also, Japanese Patent Application Laid-Open No. 9-121895
The copolymer of acrylic acid / methacrylic acid and lauryl acrylate disclosed in US Pat.
It differs from the polymer compound of the present invention in that it also acts on DL to inhibit its enzymatic reaction. Furthermore, lipoprotein aggregating agents such as heparin, dextran sulfate, phosphotungstic acid and the like can be used only in the presence of alkaline earth metals in LDL and VLD.
The polymer compound of the present invention is clearly different from the polymer compound of the present invention in that the aggregation activity of L and the enzyme reaction inhibitory activity are expressed.

Since the polymer compound having the repeating units (a) and (b) has a feature of selectively inhibiting the enzyme reaction of LDL cholesterol, it can be used in the presence of a compound which inhibits the enzyme reaction of LDL cholesterol. When used in a method for measuring LDL cholesterol based on the principle of selectively reacting and quantifying LDL cholesterol after partially or completely eliminating cholesterol of lipoproteins other than LDL, its characteristics are more effectively exhibited, but it is not HDL. The present invention can also be applied to a method for measuring HDL cholesterol based on the principle of selectively reacting HDL in the presence of a substance that inhibits the reaction of lipoprotein. That is, due to a component having a surfactant activity generated when HDL cholesterol reacts, it is possible to prevent LDL cholesterol from reacting due to a decrease in the inhibitory effect of a surfactant or the like which is suppressing the reaction of LDL cholesterol, HDL cholesterol can be determined with high accuracy.

The present invention is characterized in that a high molecular compound having the repeating units (a) and (b) is used, and there is no particular limitation on the cholesterol measuring enzyme to be used. The cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase used to do so can be used. In particular, Pseudomonas
Cholesterol esterases derived from microorganisms such as the genus Nocardia and the genus Nocardia have a high activity of hydrolyzing neutral fats, so that a great effect can be obtained by coexisting the polymer compound of the present invention.

The concentration of the enzyme to be used is set according to the concentration of cholesterol in the sample, the reaction temperature and the reaction time.
For example, when the sample containing lipoprotein is serum or plasma and is reacted for about 10 minutes, the concentration of cholesterol esterase is 0.1 U / ml to 50 U / ml, and the concentration of cholesterol oxidase is 0.1 U / ml to 10 U / ml.
ml is preferred.

In the present invention, a sample containing lipoprotein such as serum or plasma is provided with an enzyme for measuring cholesterol and the above-mentioned enzyme.
A polymer compound having the repeating units (a) and (b) is brought into contact. The polymer compound having the repeating units (a) and (b) is a polymer compound composed of a monomer unit having a higher alkyl group and a monomer unit having an anionic group. The alkyl group (R ¹ ) preferably has 4 or more carbon atoms, and examples thereof include butyl, cyclohexyl, octyl, dodecyl, tetradecyl, and hexadecyl groups. Of these, dodecyl,
Tetradecyl and hexadecyl groups are preferred. Alkyl group
Those having no (R ¹ ) or those having 3 or less carbon atoms are not preferable because they have a low effect of suppressing the reaction of LDL cholesterol. Further, as Y, COOH, CONH _{2 ,} C
ONHC ₂ H ₂ OH, CONHNH ₂ , CH ₂ COOH, C
OOC ₂ H ₄ COOH, COOC ₂ H ₄ SO ₃ H, COOC ₂
H ₂ PO (OH) ₂ , CONHC ₂ H ₂ SO ₃ H, CH ₂ OC ₆
H ₆ SO ₃ H, etc., among which COOH, C
_{ONH 2, CONHC 2 H 2 OH} , CONHNH 2 is preferred.

Examples of the compound having a repeating unit of the above (a) and (b) include a copolymer of a 1-olefin having 6 to 32 carbon atoms with acrylic acid, methacrylic acid, or maleic acid, and acid amides thereof. , Esters and the like. Among them, poly (1-eicosene-c) is particularly preferable.
o-maleic acid), poly (1-nonadecene-co-maleic acid), poly (1-octadecene-co-maleic acid), poly (1-heptadecene-co-maleic acid),
Poly (1-hexadecene-co-maleic acid), poly (1-pentadecene-co-maleic acid), poly (1-
Tetradecene-co-maleic acid), poly (1-tridecene-co-maleic acid), poly (1-dodecene-co
-Maleic acid) and their acid amides, poly (1-
Eicosene-co-acrylate), poly (1-
Nonadecene-co-acrylate), poly (1-
Octadecene-co-acrylate), poly (1
-Heptadecene-co-acrylate), poly (1-hexadecene-co-acrylate), poly (1-pentadecene-co-acrylate),
Poly (1-tetradecene-co-acrylate), poly (1-tridecene-co-acrylate), poly (1-dodecene-co-acrylate) poly (1-eicosene-co-methacrylate),
Poly (1-nonadecene-co-methacrylate), poly (1-octadecene-co-methacrylate), poly (1-heptadecene-co-methacrylate), poly (1-hexadecene-co-methacrylate), poly (1-pentadecene-) co-methacrylate), poly (1-tetradecene-co-methacrylate), poly (1-tridecene-co-methacrylate), poly (1
-Dodecene-co-methacrylate) is preferred.

The ratio of the above (a) and (b) is 1: 9
9 to 99: 1, and may include repeating units other than the repeating units (a) and (b). The average molecular weight (weight) of the polymer having a repeating unit of the above (a) and (b) measured by liquid chromatography is 5.0
Preferably it is between 00 and 500,000 daltons, more preferably between 10,000 and 100,000 daltons.

The above-mentioned (a) and (a) were added to the sample containing lipoprotein.
The concentration at which the high molecular compound having the repeating unit (b) is brought into contact depends on the LDL content in the sample containing lipoprotein, and when the sample containing lipoprotein is serum or plasma, 0.001 to 1 w / v% is good, preferably 0.01 to
It is in the range of 0.3 w / v%. When the concentration is less than 0.001 w / v%, a sufficient effect cannot be obtained, so that it is not preferable.
If it exceeds v%, the activity of the enzyme may be affected, which is not preferable. The polymer compound having the repeating units (a) and (b) does not require the coexistence of an alkaline earth metal for its effect. Therefore, it is not necessary to add an alkaline earth metal, but there is no particular limitation.

In the present invention, the pH at which the high molecular compound having the repeating units (a) and (b) is brought into contact with the sample containing the lipoprotein is determined in the range of 5 to 9. In order to further enhance the effect of the polymer compound of the present invention, the pH is preferably in the range of 6.0 to 8.0.

There is no particular limitation on the pH buffer used, and a pH of 1 to 200 mM having a pKa corresponding to the set pH is used.
It can be adjusted by a buffer. N- (2-2 hydroxyethyl) piperazine-N '-(2
-Ethanesulfonate) (HEPES), piperazine-N, N'-bis (2-ethanesulfonate) (PIP
ES) and 3- (N-morpholino) propanesulfonate (MOPS).

In the present invention, the above (a) and
Any substance that does not affect the action of the polymer compound having the repeating unit (b) can be coexisted. Examples include salts such as sodium chloride and potassium phosphate, proteins such as serum albumin, enzymes such as ascorbate oxidase, peroxidase and catalase, and chromogens for quantifying enzymatic reaction products of cholesterol. Furthermore, as long as it does not interfere with the action of the high molecular compound having the repeating units (a) and (b), a surfactant or the like that promotes the reaction between the lipoprotein cholesterol and the enzyme for measuring cholesterol may be coexisted. It is possible.

When a cholesterol measuring enzyme is allowed to act on a sample containing lipoprotein such as serum or plasma in the presence of a polymer compound having a repeating unit of the above (a) and (b), it is contained in lipoproteins other than LDL. Cholesterol reacts selectively, leaving LDL cholesterol. The time required for the reaction is determined by the amount of cholesterol contained in the sample, the concentration of the enzyme, the reaction temperature, and the like, and can be known by a general method for quantifying the substance produced or consumed by the cholesterol reaction. For example,
When cholesterol esterase and cholesterol oxidase are used as enzymes for measuring cholesterol,
Hydrogen peroxide produced by the reaction of cholesterol is reacted with 4-aminoantipyrine and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (hereinafter referred to as “TOOS”) in the presence of peroxidase. ), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAO
S) Absorbance as the concentration of a dye formed by an oxidative condensation reaction of a Triander reagent such as N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline (FDAOS) When peroxidase, 4-aminoantipyrine and TOOS are previously added to the reaction solution and the absorbance during the reaction is measured over time, the time required for lipoprotein cholesterol other than LDL to be consumed by the reaction is measured. Can be known by the time when the absorbance does not change. Also, by measuring the oxygen consumed when cholesterol reacts with an oxygen electrode, it is possible to know the time during which lipoprotein cholesterol other than LDL is consumed by reaction.

When cholesterol esterase and cholesterol dehydrogenase are used as enzymes for measuring cholesterol, NAD (P) H produced by the reaction of cholesterol is measured by, for example, ultraviolet absorption at an absorbance of 340 nm, or by forming a formazan dye. Color can be determined.

Cholesterol contained in a lipoprotein other than LDL is partially or completely reacted in the presence of a polymer compound having the repeating units (a) and (b), and then LDL cholesterol is reacted. LDL cholesterol can be measured by quantifying the compound consumed or produced. L
To react DL cholesterol, a surfactant or the like is used within a concentration range that does not inactivate the cholesterol measuring enzyme. Examples of the surfactant to be used include octylphenoxypolyethoxyethanol, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether and the like. There is no particular limitation on the method for quantifying the substance consumed or produced by the cholesterol enzymatic reaction, and a known method can be used.

In the present invention, when a cholesterol measuring enzyme is allowed to act on a sample containing lipoprotein such as serum or plasma, a high molecular compound having the above repeating units (a) and (b) is allowed to coexist with LDL cholesterol. HDL cholesterol can be measured with higher precision by coexisting with a surfactant that selectively inhibits the enzyme reaction between HDL and VLDL cholesterol. Examples of surfactants that selectively inhibit the enzymatic reaction between LDL cholesterol and VLDL cholesterol include polyoxyethylene alkyl ether and bile acid derivatives, and these surfactants are allowed to coexist at a concentration of 0.01 to 1%. . The action of this surfactant suppresses the reaction between LDL and VLDL cholesterol, and HDL cholesterol selectively reacts with an enzyme to produce a component having a surfactant action such as a higher fatty acid produced at that time. And the reaction of LDL cholesterol with a polymer compound having the repeating units (a) and (b) is suppressed, and highly accurate measurement of HDL cholesterol becomes possible.

[0036]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. (Production Example) 0.1 g of a copolymer of linear 1-olefin and maleic anhydride was suspended in 100 ml of a 1N sodium hydroxide solution, heated at 70 ° C. for 2 hours, and then p-hydrogenated with 1N hydrochloric acid.
The polymer was neutralized to H7.0 and desalted with a dialysis membrane for 12 hours to obtain a polymer compound having the above repeating units (a) and (b).

Example 1 The effect of suppressing the cholesterol reaction due to the difference in the group of the polymer compound having the repeating units (a) and (b) in the present invention was examined. 150m
M sodium chloride and 30 mM MO as pH buffer
The pH of the aqueous solution containing PS was adjusted to 7.0, and Pseudo
A cholesterol esterase (1 U / ml) derived from a microorganism belonging to the genus monas and a cholesterol oxidase (1 U / ml) derived from a microorganism belonging to the genus Streptomyces are added, and a peroxidase for quantifying hydrogen peroxide, which is an enzyme reaction product of cholesterol ( 3U / m
l), 4-aminoantipyrine (1 mM), TOOS
(1 mM) was added to prepare an enzyme solution for measuring cholesterol. The lipoprotein fraction separated by ultracentrifugation was added at a ratio of 1 volume to 100 volumes of the enzyme solution for cholesterol measurement and reacted at 37 ° C. for 10 minutes. The absorbance of the reaction solution proportional to the amount of cholesterol reacted (main Wavelength 550
nm / subwavelength 700 nm). The cholesterol concentration in each lipoprotein fraction was about 120 mg for the HDL fraction.
/ dl, the VLDL fraction is about 50 mg / dl, and the LDL fraction is about 240 mg / dl. Next, the compound prepared in Example 1 was added to the enzyme solution for measuring cholesterol, the lipoprotein fraction was reacted in the same manner, and the absorbance (main wavelength: 550 nm / sub wavelength: 700 nm) of the reaction solution was measured.
Based on the absorbance obtained with the enzyme solution for measuring cholesterol to which the compound was not added, the enzyme reaction inhibition rate of the compound against lipoprotein cholesterol was calculated.

Table 1 shows the Y and alkyl groups (R ¹ ) of the compound.
(C1 to C26) and the average molecular weight. still,
X and Y are COOH or its sodium salt.
In addition, the reaction inhibitory effect of lipoprotein cholesterol is 0 to 5% for the reaction inhibitory rate, -5 to 30% for +, and 30 to 80% for +.
+, 80 to 100% are indicated by +++.

[0039]

[Table 1] As shown in Table 1, compounds having 4 or more carbon atoms in the alkyl group (R ¹ ) selectively suppress the enzymatic reaction of LDL cholesterol.

Example 2 Comparison between the method of the present invention and the HPLC method in measuring LDL cholesterol was performed. The compound having 16 carbon atoms of the alkyl group (R ¹ ) prepared according to the production example was added to the enzyme solution for measuring cholesterol prepared in Example 1 at 0.05%, and 1 volume of serum was added to 100 volumes thereof. After reacting at 37 ° C. for 5 minutes, the absorbance of the reaction solution (main wavelength 550 nm / sub wavelength 700 nm) is measured, and then octylphenoxypolyethoxyethanol having a final concentration of 0.5% is added to the reaction solution. After the reaction was performed for 5 minutes, the absorbance (main wavelength 550 nm / sub wavelength 700 nm) of the reaction solution was measured. Separately, a standard solution was prepared by suspending cholesterol palmitate in a 1% aqueous solution of octylphenoxypolyethoxyethanol at a concentration equivalent to 50 mg / dl in terms of cholesterol, and one volume thereof was used as a cholesterol measuring reagent prepared in Example 1. 100
After adding to the vessel and reacting at 37 ° C. for 5 minutes, the absorbance (main wavelength 550 nm / sub-wavelength 700 nm) was measured, and a calibration curve between the absorbance and the reacted cholesterol amount was prepared. Using this calibration curve, the amount of cholesterol in the serum reacted after addition of octylphenoxypolyethoxyethanol was calculated.

As a control method, HDL cholesterol and LDL cholesterol in the same serum were quantified by a reaction liquid chromatography method. Using a Shodex (Showa Denko KK) KW-804 column (manufactured by Showa Denko KK), the lipoprotein in the serum was separated using 150 mM phosphate buffer (pH 7.0) as an eluent. A reaction coil for mixing a cholesterol detection solution was connected, and the mixture of the column eluate and the cholesterol detection solution was reacted at 45 ° C. for 3 minutes in the reaction coil.
The cholesterol content of each lipoprotein fraction was determined by measuring the absorbance at 0 nm.

The cholesterol detection solution was Pseudo.
cholesterol esterase (1)
0% octylphenoxypolyethoxyethanol solution containing cholesterol oxidase (10 U / ml) and peroxidase (20 U / ml), 4-aminoantipyrine (2 mM) and TOOS (2 mM) derived from Streptomyces sp. And mixed 1: 1 with the eluate in the reaction coil. The peaks in the chromatogram are HDL separated by ultracentrifugation,
Identification was performed using LDL and VLDL fractions. FIG. 1 shows a dispersion diagram in which the LDL cholesterol concentration in serum determined by the measurement method according to the present invention is plotted on the ordinate and the LDL cholesterol concentration in serum determined by reaction liquid chromatography is plotted on the abscissa.

FIG. 1 shows that the serum cholesterol concentration determined by the LDL measurement method according to the present invention shows a high correlation with the serum LDL cholesterol concentration determined by reaction liquid chromatography.

Example 3 The effect of adding a polymer compound having the above repeating units (a) and (b) to lipoprotein cholesterol by a change in absorbance was examined. A surfactant [3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (hereinafter referred to as a surfactant) that selectively inhibits the enzymatic reaction between LDL cholesterol and VLDL cholesterol is added to the cholesterol measurement enzyme solution prepared according to the production example. , "CHAPS") at 0.2%.
An HDL cholesterol measuring reagent for selectively reacting and quantifying L cholesterol was prepared. The HDL fraction and LDL used in Example 1 were used for this HDL cholesterol measurement reagent.
The fractions are added singly or as a mixture, and reacted in the same manner as in Example 1 to absorb light (main wavelength 550 nm / sub wavelength 700 nm).
Was measured over time. Table 2 shows the results.

[0045]

[Table 2] As shown in Table 2, the enzyme reaction of LDL cholesterol is suppressed by the coexistence of the surfactant CHAPS.
When DL is present, the effect of CHAPS, which suppresses the reaction of LDL cholesterol, is reduced by the enzyme reaction product, and LDL cholesterol also reacts.

Next, 0.05% of the high molecular compound having the repeating units (a) and (b) used in Example 2 was added to the reagent for measuring HDL cholesterol, and
The DL fraction and the LDL fraction are reacted singly or as a mixture, and the absorbance of the reaction solution (main wavelength 550 nm / sub wavelength 700 nm)
Was measured over time. Table 3 shows the results.

[0047]

[Table 3] As shown in Table 3, in the HDL cholesterol measuring reagent to which the polymer compound having the repeating units (a) and (b) was added, the effect of suppressing the reaction of LDL cholesterol was not reduced even in the presence of HDL, and Reacted selectively.

Example 4 The effect of adding a polymer compound having the repeating units (a) and (b) in measuring HDL cholesterol in serum was examined. One volume of serum was added to 100 volumes of the reagent for measuring HDL cholesterol used in Example 3, reacted at 37 ° C. for 10 minutes, and the absorbance (main wavelength: 550 nm / sub wavelength: 700 nm) of the reaction solution was measured.
Further, to the HDL cholesterol measurement reagent, 0.05% of the polymer compound having the repeating units (a) and (b) used in Example 3 was added, and the same serum was reacted in the same manner, and the absorbance of the reaction solution was measured. It was measured. In the same manner as in Example 2, a calibration curve was prepared to calculate the amount of cholesterol in the serum reacted with both HDL cholesterol measurement reagents,
In addition, the amount of HDL cholesterol in the serum was quantified by reaction liquid chromatography (HPLC). Table 4 shows the measurement results.

[0049]

[Table 4] It can be seen that the coexistence of the high molecular compound having the repeating units (a) and (b) improves the measurement accuracy of HDL cholesterol.

(Example 5) The HDL cholesterol reagent which does not form a pigment even when cholesterol reacts to generate hydrogen peroxide except for TOOS which is a chromogen is removed from the HDL cholesterol measuring reagent used in Example 3. Prepared and further added with 0.05% of a polymer compound having a repeating unit of the above (a) and (b) used in Example 2
A DL cholesterol reaction reagent and a HDL cholesterol reaction reagent to which a lipoprotein aggregating agent, 0.1% of phosphotungstic acid and 100 mM of magnesium chloride were added as a control method were prepared. Serum cholesterol was reacted with each HDL cholesterol reagent in the same manner as in Example 4, and the absorbance of the reaction solution before and after serum addition (main wavelength 550 nm)
The turbidity change was measured by the difference between the turbidity and the sub wavelength 700 nm.
Table 5 shows the results.

[0051]

[Table 5] It can be seen that the polymer compound having the repeating units (a) and (b) does not increase the turbidity of the reaction solution, and enables highly accurate optical measurement.

[0052]

EFFECT OF THE INVENTION A method for measuring lipoprotein cholesterol in a sample containing lipoprotein such as serum or plasma by a simple operation with high accuracy by coexisting the compound of the present invention with a sample containing lipoprotein and A reagent can be provided, and a more accurate diagnosis can be made particularly in the field of clinical examination such as arteriosclerosis.

[0053]

[Brief description of the drawings]

FIG. 1 is a dispersion diagram showing an example of a correlation between a measurement value and an HPLC value by the method for measuring LDL cholesterol of Example 2 of the present invention.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Toyoji Sawayanagi 5-1 Ogimachi, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture F-term (reference) 2G045 AA13 CA25 DA62 DA63 DA64 DA69 FB01 4B063 QA01 QA19 QQ03 QQ76 QR03 QR04 QR12 QR41 QS02 QS20 QX01

Claims (8)

[Claims] 1. A method for measuring lipoprotein cholesterol, which comprises reacting an enzyme on a sample containing lipoprotein and quantifying a compound consumed or produced in the enzymatic reaction. And formula (b): Wherein R ¹ is an alkyl group having 4 to 30 carbon atoms, R ² and R ³ are each independently a hydrogen atom or a methyl group, X is a hydrogen atom or COOH, and Y is COO
H, SO ₃ H, a group having PO (OH) ₂ or a group derived therefrom. Characterized in that lipoprotein cholesterol is reacted with an enzyme in the presence of a high molecular compound having a weight ratio of (a) :( b) in the range of 1:99 to 99: 1. Measurement method. 2. The polymer compound having a molecular weight of 5,000.
The method for measuring lipoprotein cholesterol according to claim 1, wherein the lipoprotein cholesterol is in a range of from 500,000 daltons. 3. The method according to claim 1, wherein the concentration of the polymer compound is 0.001 to
The method for measuring lipoprotein cholesterol according to claim 1 or 2, which is in a range of 1 w / v%. 4. The method for measuring lipoprotein cholesterol according to claim 1, wherein the pH when cholesterol is reacted in the presence of the polymer compound is in the range of 5 to 9. 5. The at least one compound selected from the group consisting of a copolymer of a 1-olefin having 6 to 32 carbon atoms and maleic acid or acrylic acid or methacrylic acid, and an acid amide thereof. The method for measuring lipoprotein cholesterol according to any one of claims 1 to 4, characterized in that: 6. The method according to claim 1, wherein high density lipoprotein (HDL) cholesterol and / or low density lipoprotein (LDL) cholesterol in the lipoprotein cholesterol is measured. Method for measuring lipoprotein cholesterol. 7. A cholesterol esterase, cholesterol oxidase or cholesterol dehydrogenase and a compound represented by the formula (a): And formula (b): Wherein R ¹ is an alkyl group having 4 to 30 carbon atoms, R ² and R ³ are each independently a hydrogen atom or a methyl group, X is a hydrogen atom or COOH, and Y is COO
H, SO ₃ H, a group having PO (OH) ₂ or a group derived therefrom. And a weight ratio of (a) :( b) in the range of 1:99 to 99: 1. 8. The reagent for measuring lipoprotein cholesterol according to claim 7, which measures high density lipoprotein (HDL) cholesterol and / or low density lipoprotein (LDL) cholesterol.