JP2006288399A - Method for determining cholesterol in lipoprotein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining cholesterols in a fraction of lipoproteins, such as HDL (high-density lipoprotein) and LDL (low-density lipoprotein) in a biological sample, with a general automatic analyzer without separating by a remote operation and without causing turbidity in the reaction. <P>SOLUTION: This method for determining the cholesterols in the lipoproteins is characterized by reacting and decomposing cholesterols causing measurement errors except the target cholesterols in a fraction of the lipoproteins with a cholesterol-oxidizing enzyme in the reaction solution in the first reaction and then reacting and measuring the target cholesterols in the lipoprotein fraction with a cholesterol-dehydrogenating enzyme in the second reaction. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for quantifying cholesterol in a lipoprotein fraction in a biological sample in the field of clinical diagnosis.

  Since ancient times, lipoproteins have been obtained by ultracentrifugation, such as high-density lipoprotein (HDL, specific gravity 1.063 to 1.21), low-density lipoprotein (LDL, specific gravity 1.019 to 1.063), and ultra-low density lipoprotein. (VLDL, specific gravity 1.006 to 1.019), chylomicron (CM, specific gravity <1.006). This fractionation operation requires an ultracentrifuge, and the centrifugation has to be performed for several days, so that a large number of samples cannot be processed. Instead, LDL is mixed with a polyanion such as polyethylene glycol or dextran sulfate in the presence of a divalent cation such as magnesium or calcium, or a solution containing a divalent cation coexisting with phosphotungstic acid and serum. The method of precipitating VLDL and CM and fractionating only the HDL remaining in the supernatant after centrifugation was the mainstream. This method could use an automatic analyzer widely used in the field of clinical examination. That is, as for cholesterol in the fractionated HDL fraction, the measurement of total cholesterol by an enzymatic method has been established by an automatic analyzer, so that it was possible to determine the cholesterol concentration in the HDL fraction by applying it.

  However, although this method is also low speed, a centrifugal operation is necessary, and there have been problems such as an artificial quantification error when mixing the fractionating agent and serum, and a mistake in the specimen. In addition, it could not be measured simultaneously with other general biochemical items using an automatic analyzer. Clinical tests are required to respond promptly, and this has made it a challenge to reduce testing time.

  On the other hand, a report that emphasizes the cholesterol level in the LDL fraction, which is a risk factor for arteriosclerosis clinically (Non-patent Document 1) [reference value and setting basis of total cholesterol, arteriosclerosis, 24 (6), 260 ( 1996)]. The cholesterol value in the current LDL fraction is determined by substituting empirical factors from the measurement results of total cholesterol (T-CHO), neutral fat (TG) and cholesterol in the HDL fraction. The formula is shown below (Non-Patent Document 2; Friedewald WT, et al, Clin. Chem., 18, 499-502 (1972)).

  Cholesterol value in LDL fraction = (T-CHO value) − (cholesterol value in HDL fraction) − (TG value) / 5

  This method does not hold unless all three items to be measured are accurately measured. Further, it is said that when the TG value exceeds 400 mg / dl or the cholesterol concentration in the LDL fraction becomes 100 mg / dl or less, the calculated value does not reflect the cholesterol concentration in the LDL fraction (Non-patent Document 3). Warnick GR, et al, Clin. Chem. 36 (1), 15-19 (1990), Non-Patent Document 4; McNamara JR, et al, Clin. Chem. 36 (1), 36-42 (1990)) . Therefore, this is a method in which an important abnormal value cannot be obtained. In addition, there are a method for separating lipoproteins by electrophoresis and measuring the amount of protein, and a method for measuring cholesterol by lipoprotein by HPLC. However, these methods lack sample processing ability and require expensive equipment.

  In recent years, in order to solve the above-described problems relating to the measurement of cholesterol in the HDL fraction, kits for measuring cholesterol in the fully automatic HDL fraction have been developed and are becoming popular. In the techniques disclosed in Patent Documents 1 to 3 (Japanese Patent No. 2600065, JP-A-8-201393 and JP-A-8-131195), a fractionating agent is used in combination. A metal used as a valent cation forms an insoluble precipitate by a detergent generally used in an automatic analyzer, which accumulates in a waste liquid mechanism and causes a failure. In addition, insoluble aggregates are formed in the reaction solution, causing turbidity that affects the measurement results and causing measurement errors. In addition, the reaction cell is contaminated by aggregates, and measurement is performed simultaneously. It has a considerable influence on the measurement results of biochemical items.

  Most popular automated analyzers often complete the reaction in 10 minutes. At this time, if the turbidity changes during the reaction, there is a problem in the accuracy of the measured value. In addition, there is another problem that the reproducibility decreases when the reaction solution becomes cloudy. Therefore, there is a limitation on the sample to be measured, and it is not possible to deal with a wide range of measurement wavelengths and various patient samples. For example, in the vicinity of 340 nm (UV region), due to the turbidity caused by aggregates, there is a drawback that the absorbance becomes 2-3 or more and often exceeds the allowable range of the analyzer.

  The technique disclosed in Patent Document 4 (Japanese Patent Laid-Open No. 9-96637) that does not use a divalent cation is a method of including an antiserum that aggregates with a lipoprotein. As a result, the reaction cell is contaminated. Therefore, the measurement results of other biochemical items that are measured at the same time have a considerable influence. In addition, since the turbidity in the reaction solution becomes strong, accurate measurement is impossible for the same reason as described above, particularly for cholesterol measurement in the HDL fraction in the UV region. Even in the high wavelength range, the measurement value is not accurate due to turbidity.

  Further, a technique for finally eliminating such turbidity is disclosed in Patent Document 5 (Japanese Patent Laid-Open No. 6-242110), but this method requires at least 3 to 4 steps of reagent dispensing operation. In general, automatic analyzers for biochemical items that are in widespread use cannot be applied because they are mainly in two stages. On the other hand, for the measurement of cholesterol in the LDL fraction, it is still a situation that a calculation method must be employed.

  Measurement reagents used in clinical tests emphasize the versatility of the reagents, and have been devised to maintain and stabilize buffer capacity, liquefaction of reagents, antiseptic effects, and activation of enzymes for short-time measurements. .

  The measurement of cholesterol using cholesterol dehydrogenase (CDH) requires the addition of hydrazines for the purpose of shortening the reaction time (Patent Document 6; JP-A-5-176797). However, in this method, since hydrazines are present in the first reaction, the reaction solution is under high ionic strength, and in the second reaction, the reaction solution is changed to around 8.0, which is the optimum pH of CDH, that is, to the alkaline side. When a surfactant that is an activator of cholesterol dehydrogenase or cholesterol esterase is added, substances other than the target substance in the form of a soluble complex are decomposed, and the decomposition products are involved in this reaction, resulting in variations in measured values. Cause.

  In the condition for measuring cholesterol in the HDL fraction, examples of the substance that is easily involved in this reaction include free cholesterol present on the surface in the LDL fraction. As a method for suppressing these degradations, polyanions, divalent metal ions, water-soluble polymer compounds, or antibodies against substances other than the target substance are added alone or in combination of two or more to form aggregates or immune complexes. Therefore, it is possible to suppress the participation in this reaction.

However, the generation of turbidity due to these aggregates and immune complexes is a major obstacle to the above-mentioned various problems in the ultraviolet measurement method (UV method) at a wavelength of 340 nm using CDH.
Atherosclerosis, 24 (6), 260 (1996) Clin. Chem. , 18, 499-502 (1972) Clin. Chem. 36 (1), 15-19 (1990) Clin. Chem. 36 (1), 36-42 (1990) Japanese Patent No. 2600065 JP-A-8-201393 JP-A-8-131195 JP-A-9-96637 JP-A-6-242110 Japanese Patent Laid-Open No. 5-176797

  In view of such a situation, the present inventors do not perform separation by centrifugation using a general-purpose automatic analyzer, and do not cause turbidity during the reaction, such as HDL and LDL in a biological sample. Intensive research was conducted on the method for quantifying cholesterol in the lipoprotein fraction.

  The inventors previously decomposed cholesterol in a lipoprotein fraction other than the target, which causes measurement errors in the reaction solution, with cholesterol oxidase, and then decomposed the cholesterol in the target lipoprotein fraction. A method of measuring using cholesterol dehydrogenase was found. Furthermore, the present inventors formed a water-soluble complex with lipoprotein cholesterol such as calixarenes and derivatives thereof, polyanions, water-soluble polymers and polysaccharides during the reaction of the biological sample with cholesterol oxidase and cholesterol dehydrogenase. It was found that the presence of a possible compound (that is, addition) enables various fractional determinations of cholesterol in the lipoprotein fraction without causing turbidity in the reaction solution.

That is, this invention consists of the following.
1. In quantifying cholesterol in specific lipoproteins in biological samples,
In the first reaction, cholesterol other than cholesterol in the specific lipoprotein is reacted with cholesterol oxidase,
Cholesterol quantification method characterized by reacting cholesterol in said specific lipoprotein with cholesterol dehydrogenase in the second reaction.
2. 2. The quantification method according to item 1, wherein cholesterol in the specific lipoprotein is quantified based on a difference between the absorbance after the reaction with the cholesterol oxidase and the absorbance after the reaction with the cholesterol dehydrogenase.
3. The specific lipoprotein is a high-density lipoprotein;
3. The method according to item 1 or 2, wherein cholesterol other than cholesterol in the specific lipoprotein is free cholesterol.
4). In the first reaction, calixarene sulfate having 4 to 8 molecules of phenol as a basic skeleton, sodium salt or potassium salt of dextran sulfate, sodium salt or potassium salt of phosphotungstic acid in low-density lipoprotein and ultra-low density lipoprotein , Polyacrylic acid, carrageenan, Apo-B antiserum, concanavalin A, partially hydroxypropylated cyclodextrin, partially methylated cyclodextrin, potassium or sodium salt of heparin, sodium alginate, and polyethylene glycol having an average molecular weight of 200-6000 By allowing at least one compound selected from the group to act to form a soluble complex, the low-density lipoprotein and the ultra-low-density lipoprotein, the cholesterol oxidase, and the co-oxidant Inhibit the reaction of the sterol dehydrogenase, quantitative method described in the preceding paragraph 3.
5). The specific lipoprotein is a low density lipoprotein;
3. The quantification method according to item 1 or 2, wherein the cholesterol other than cholesterol in the specific lipoprotein is cholesterol other than cholesterol in the low-density lipoprotein.
6). In the first reaction, the low-density lipoprotein is calixarene sulfate having 4 to 8 molecules of phenol as a basic skeleton, sodium or potassium salt of dextran sulfate, sodium or potassium salt of phosphotungstic acid, polyacrylic acid, Selected from the group consisting of carrageenan, Apo-B antiserum, concanavalin A, partially hydroxypropylated cyclodextrin, partially methylated cyclodextrin, potassium or sodium salt of heparin, sodium alginate, and polyethylene glycol having an average molecular weight of 200-6000 The reaction of the low density lipoprotein with the cholesterol oxidase and the cholesterol dehydrogenase is inhibited by allowing at least one compound to act to form a soluble complex, Quantification method of mounting.
7). 7. The quantification method according to item 5 or 6, wherein the reaction between the low-density lipoprotein and the compound in the first reaction is performed under conditions of pH 6 to 8 and ionic strength 200 to 500 mmol / L.
8). The specific lipoprotein is an ultra-low density lipoprotein;
3. The method according to item 1 or 2, wherein the cholesterol other than cholesterol in the specific lipoprotein is cholesterol contained in a lipoprotein other than the ultra-low density lipoprotein.

  According to the present invention, by reacting with cholesterol oxidase in the first reaction and reacting with cholesterol dehydrogenase in the second reaction, the influence of cholesterol derived from fractions other than the target lipoprotein fraction is eliminated. It becomes possible.

  Furthermore, according to the present invention, cholesterol in a specific lipoprotein fraction can be quantified without aggregating the lipoprotein in the biological sample. That is, by including calixarenes and derivatives thereof, polyanions, polysaccharides (cyclodextrins, etc.), water-soluble polymers, etc. in the reaction solution, the agglutination of lipoproteins in biological samples can be achieved. In addition, cholesterol in a specific lipoprotein fraction can be selectively quantified without being affected by other lipoproteins.

  Hereinafter, various conditions for carrying out the present invention will be described in detail, but the present invention is not limited to the following description.

  In the present invention, the biological sample is serum or plasma. For example, the above-mentioned HDL, LDL, VLDL, CM, and lipoprotein fraction such as remnant-like lipoprotein produced by the action of VLDL and CM by lipoprotein lipase are used. Is included. Lipoproteins are those in which lipids containing cholesterol and apoproteins are combined to form lipids inside and proteins outward, and are solubilized in the living body.

  In the present invention, in order to decompose cholesterol derived from a lipoprotein fraction other than the target fraction, the cholesterol and cholesterol oxidase are first allowed to act (first reaction). As a cholesterol oxidase, as long as it can oxidize cholesterol, there are no particular restrictions on its origin. The first reaction is carried out in the range of pH 6.0 to 8.5, preferably pH 6.5 to 7.5, 25 to 40 ° C, preferably 30 to 37 ° C, 1 to 10 minutes, preferably 3 to 5 minutes. . After completion of the first reaction, the absorbance of the reaction solution is measured. Absorbance measurement conditions vary depending on the reaction used to measure cholesterol in the target fraction and the type of enzyme. For example, as a coenzyme used in the second reaction described below, cholesterol dehydrogenase is used. In the case of utilizing conversion of oxidized form of β-nicotinamide adenine dinucleotide (NAD) to reduced form, absorbance at a wavelength around 340 nm may be measured.

  The second reaction for causing cholesterol in a specific lipoprotein fraction in a biological sample to act on cholesterol dehydrogenase is pH 7.0 to 10.0, preferably pH 7.5 to 8.5, and 25 to 40. ° C., preferably 30-37 ° C., 1-10 minutes, preferably 3-5 minutes. After completion of the second reaction, the absorbance is measured according to the conditions of the first reaction.

  The cholesterol dehydrogenase is not particularly limited in its origin and the like, and any substance that catalyzes a cholesterol dehydrogenation reaction may be used.

  The coenzyme when using cholesterol dehydrogenase in the second reaction includes β-nicotinamide adenine dinucleotide oxidized form (NAD), Thio-nicotinamide adenine dinucleotide oxidized form (t-NAD), β-nicotinamide adenine Dinucleotide phosphate oxidation type (NADP), Thio-nicotinamide adenine dinucleotide phosphate oxidation type (t-NADP) and the like can be mentioned.

  By changing pH 6.0-8.5 in the first reaction to pH 7.5 or higher, preferably pH 8.0 or higher in the second reaction, cholesterol oxidase used in the first reaction can be obtained from pH conditions optimal for the reaction. Come off. Cholesterol dehydrogenase acts effectively in the second reaction because it has an optimum pH above 8.0. This difference in pH conditions makes it possible to quantitate cholesterol in a specific lipoprotein fraction of interest, but a nonionic surfactant or ionic interface of HLB17 or higher which is a specific inhibitor for cholesterol oxidase An activator, sodium fluoride, or the like may be added in the second reaction.

  In the present invention, compounds such as calixarenes and calixarene derivatives, polyanions, water-soluble polymers, and polysaccharides are added in order to prevent agglomerates from being generated during the reaction between the biological sample and the enzyme and the reaction solution becoming cloudy. The These compounds form a complex but do not aggregate due to ionic bonds with the lipoprotein surface or coordinate bonds that recognize the protein sugar chains on the lipoprotein surface. That is, these complexes are soluble complexes that do not cause turbidity in the reaction solution. These compounds mainly form soluble complexes with LDL and VLDL fractions and inhibit the release of components in lipoproteins.

  In these compounds, it is necessary to set the concentration of the metal ion so that aggregation does not occur or the concentration of the metal ion is not added, because aggregation occurs due to the addition of a certain concentration of metal ions. There is. When adding metal ions, it is suppressed to a level not exceeding about 1 mM, but it is usually preferable not to add metal ions.

  Calixarenes are cyclic oligomers in which phenol is a basic skeleton and 4 to 8 molecules of phenol are cyclically polymerized with a methylene group. A calixarene derivative in which a water-soluble (hydrophilic) group is introduced into these calixarenes has also been developed. The calixarenes and their derivatives include calix (4) allene [Calix (4) arene], calix (6) allene, calix (8) allene, calix sulfate (4) allene, calix sulfate (6) allene, calix sulfate ( 8) Allene, calix acetate (4) allene, calix acetate (6) allene, calix acetate (8) allene, carboxycalix (4) allene, carboxycalix (6) allene, carboxycalix (8) allene, calix (4) Allenamine, calix (6) allenamine, calix (8) allenamine and the like can be mentioned, but the use is not particularly limited. Calixarene sulfate, which has been successfully commercialized, has excellent water solubility and is easy to handle. When calixarenes or derivatives thereof are applied to cholesterol measurement, 0.05-50 mmol / L, preferably 0.1-10 mmol / L, is added to the reaction solution under conditions where cholesterol is enzymatically measured. do it. One or two or more calixarenes and their derivatives can be used, and the amount of each calixarene and its derivatives when two or more are used can be appropriately changed within the above range.

  Examples of polyanions include sodium salts and potassium salts such as dextran sulfate, phosphotungstic acid, and heparin, and 0.01 to 1.0 w / v%, preferably 0.05 to 0.2 w / v% in the reaction solution. What is necessary is just to add so that it may become.

  Examples of the water-soluble polymer include polyacrylamide, polyacrylic acid, polymethacrylic acid, polyethylene glycol and the like, and 0.01 to 5.0 w / v%, preferably 0.05 to 0.2 w / v in the reaction solution. It may be added so as to be%.

  Examples of the polysaccharide include lectins (such as concanavalin A and castor lectin), λ-carrageenan, κ-carrageenan, sodium alginate, Apo-B antiserum, and cyclodextrins, and 0.01-2. What is necessary is just to add so that it may become 0 w / v%, Preferably 0.05-0.5 w / v%.

  Any cyclodextrin (CD) can be used as long as the object of the present invention can be achieved. Particularly, partially hydroxypropylated α-CD, partially hydroxypropylated β-CD, and partially methylated α-CD. Partially methylated β-CD, partially polymerized β-CD, and the like are preferable. These CDs are used alone or in combination. Partial hydroxypropylation means that in at least one glucose residue of 6 or 7 glucose residues constituting α or β-CD, the hydrogen atom of the hydroxyl group at 2-position and / or 6-position is a hydroxypropyl group [for example, 2-hydroxypropyl group “—O—CH 2 CH (OH) CH 3”]. Partial methylation is the substitution of a hydrogen atom at the 2-position and / or 6-position hydroxyl group with a methyl group in at least one glucose residue of 6 or 7 glucose residues constituting α or β-CD. It means being done. Furthermore, partial polymerization means that at least one of the 7 glucose residues constituting one β-CD has at least one hydroxyl group out of the 2nd, 3rd and 6th hydroxyl groups. That at least one of the hydroxyl groups at the 2-position, 3-position and 6-position of the glucose residue constituting the β-CD of the β-CD is mutually cross-linked by a cross-linking agent, and a plurality of β-CD molecules form a polymer. Say.

  These compounds that form a soluble complex with the LDL fraction and the like are more effective when used in combination according to the purpose.

  Next, quantification of cholesterol in each fraction will be described. When quantifying cholesterol in the HDL fraction, in the first reaction, the lipoprotein fraction other than the HDL fraction, particularly free cholesterol in the LDL fraction, is easily released due to the salt concentration and pH. Examples of a method for suppressing the decomposition of LDL include addition of calixarenes, calixarene derivatives, polyanions, water-soluble polymers (water-soluble polymer compounds), and polysaccharides that form a soluble complex. However, since a wide variety of lipoproteins exist in the biological sample to be measured, it is difficult to completely suppress the release of cholesterol. Therefore, preferably, the free cholesterol is decomposed by the first reaction with cholesterol oxidase, and the cholesterol in the HDL fraction by the second reaction, if necessary, in the presence of lipoprotein lipase or cholesterol hydrolase, A method of quantifying by the reaction of cholesterol dehydrogenase is used in combination.

  The amount of compound such as calixarene used to form the soluble complex is as described above.

  When quantifying cholesterol in the LDL fraction, the above-mentioned conditions are combined, that is, soluble in LDL and soluble in the first reaction under conditions of pH 6.0 to 8.0 and ionic strength 200 to 500 mmol / L. While forming and stabilizing the complex, cholesterol oxidase is allowed to act on cholesterol in the HDL fraction and VLDL fraction in the presence of lipoprotein lipase and cholesterol hydrolase as necessary. The remaining LDL fraction is decomposed by increasing the salt concentration or adding a surfactant in the second reaction, and cholesterol in the LDL fraction can coexist with lipoprotein lipase and cholesterol hydrolase as necessary. Below, it quantifies by reaction of cholesterol dehydrogenase. In the first reaction, a compound that forms a soluble complex with LDL can be used by appropriately adjusting the combination and concentration of the types.

  When quantifying cholesterol in the VLDL fraction, the conditions described above are combined, that is, under the conditions of pH 6.5 to 8.5 and ionic strength of 300 to 600 mmol / L, the VLDL fraction is reacted in the first reaction. It forms and stabilizes soluble complexes with cholesterol, while degrading cholesterol in the HDL and LDL fractions. In the first reaction, a compound that forms a soluble complex with VLDL can be used by appropriately adjusting the combination and concentration of the types. The means for degrading the soluble complex of VLDL in the second reaction is not limited as long as it does not interfere with the enzyme reaction in the second reaction, and a surfactant, cholic acid or enzyme is used, or the salt concentration is adjusted appropriately. do it.

  The cholesterol concentration in a specific lipoprotein fraction is determined by measuring the absorbance in the first reaction and the second reaction, respectively, and subtracting the absorbance. For example, when quantifying cholesterol in the HDL fraction, after measuring the absorbance in a state where the cholesterol of the lipoprotein fraction other than the HDL fraction is decomposed in the first reaction, the absorbance in the second reaction is measured. Thus, cholesterol in the unreacted HDL fraction in the first reaction can be quantified.

  This quantification method can be applied not only to the above-mentioned HDL fraction, LDL fraction and VLDL fraction, but also to other lipoprotein fractions such as a remnant-like lipoprotein fraction.

  The quantification method of the present invention can be preferably carried out by the reagent kit of the present invention. The reagent kit of the present invention is preferably formulated so that cholesterol oxidase is present in at least the reaction solution in the first reaction and cholesterol dehydrogenase is present in the reaction solution in the second reaction. is there. The form of the reagent is not particularly limited, such as dry or liquid. The reagent kit may contain an enzyme activator or the like, and the reagent kit is added to the reaction solution, for example, a combination of the first reaction reagent and the second reaction reagent. A combination of a plurality of types of reagents with different times may be used.

  The reagent kit of the present invention is a reagent kit for measuring cholesterol in a specific lipoprotein fraction with cholesterol dehydrogenase, contains cholesterol oxidase and cholesterol dehydrogenase, and is specified as other components. It is suitably prepared so as to contain a reagent such as an enzyme used for quantifying cholesterol in the lipoprotein fraction. For example, cholesterol in the HDL fraction and LDL fraction is measured by a method of measuring total cholesterol. In this reagent kit, in addition to cholesterol oxidase and cholesterol dehydrogenase, enzymes such as lipoprotein lipase and cholesterol esterase, activators added under conditions to complete the reaction to quantify cholesterol, stable An agent, a coenzyme and the like are appropriately selected and can be blended in a reagent kit. Examples of the cholesterol dehydrogenase included in the kit of the present invention include nicotinamide adenine dinucleotide-dependent cholesterol dehydrogenase, nicotinamide adenine dinucleotide phosphate-dependent cholesterol dehydrogenase and the like as described above.

  Hereinafter, in order to describe the present invention in more detail, examples of quantification of cholesterol in the HDL fraction, the LDL fraction, and the VLDL fraction will be described, but the present invention is not limited thereto.

[Example 1] Confirmation result of turbidity The compounds shown in Table 1 were added to a buffer solution to obtain a reagent. Specimens used were VLDL, LDL, and HDL fractions obtained by ultracentrifugation (100,000 G), and normal human serum and chyle specimens. In the measurement, 180 μl of a reagent was added to 5 μl of a sample and incubated at 37 ° C. for 5 minutes. At this point, the absorbance and turbidity at a main wavelength of 340 nm were confirmed. Kits commercially available as control methods, Determiner L HDL-C (manufactured by Kyowa Medex Co., Ltd.), Correstest HDL (manufactured by Daiichi Chemicals Co., Ltd.), HDL-C Direct Wako (Wako Pure Chemical ( The 1st reagent of the company make) was used. Reagents were also prepared with reference to the examples described in Japanese Patent No. 2600065, Japanese Patent Application Laid-Open No. 8-201393, and Japanese Patent Application Laid-Open No. 9-96637. The operation followed the instruction manual attached to the kit and the examples described in each publication. The results are shown in Table 1. It can be seen that the turbidity does not occur in the reagent to which the compound used in the present invention is added.

  Note 1) Upper row of each compound: Shows the strength of turbidity. Lower panel: shows the change in absorbance due to complex formation by subtracting the absorbance of the specimen itself at 340 nm.

  Note 2) The symbols in the table indicate the following turbidity. -: No turbidity, +: Low turbidity, ++: Medium turbidity, +++: High turbidity, +++++: Strong turbidity (absorbance of 2.0 or more)

  Note 3) HP-γ-CD in the table is 2-hydroxypropyl-γ-cyclodextrin, DM-β-CD is the hydrogen atom of the hydroxyl group at the 2nd and 6th positions of the glucose residue constituting β-CD. Each represents γ-cyclodextrin substituted with a methyl group, and PEG represents polyethylene glycol.

[Example 2] Quantification of cholesterol in HDL fraction The following reagent 1-A, reagent 1-B and reagent 2 were prepared, and the HDL-cholesterol concentration was measured. The specimen used was 10 cases of general human serum. The measurement was carried out with a Hitachi 7170 automatic analyzer. In the operation method, first, 180 μl of Reagent 1-A or Reagent 1-B was added to 5 μl of the sample, and the temperature was kept constant at 37 ° C. for 5 minutes.

  Furthermore, 60 μl of Reagent 2 was added, and the temperature was kept constant at 37 ° C. for 5 minutes.

  The difference between absorbance 1 and absorbance 2 was determined, and the value of the sample was converted using a known HDL-cholesterol concentration control as a standard solution. A polyethylene glycol (PG) method was used as a control method. In the PG method, PG pole manufactured by Kokusai Reagent Co., Ltd. was used. The cholesterol concentration in the supernatant after centrifugation was determined using T-CHO Reagent · A manufactured by International Reagents Co., Ltd. As a measurement result, a comparison with the control method is shown in Table 2. The reagent 1-A to which no cholesterol oxidase is added shows a higher value than the control method, but the reagent 1-B to which the cholesterol oxidase is added gives good results that are in good agreement with the results of the control method. It was.

Preparation of reagent 1-A
Buffer pH 6.5
Hydrazinium dichloride 80 mmol / L
Calix sulfate (6) allene 1.0 mmol / L
β-nicotinamide adenine dinucleotide oxidized form [NAD] 5.0 mmol / L

Preparation of Reagent 1-B Buffer pH 6.5
Hydrazinium dichloride 80 mmol / L
Calix sulfate (6) allene 1.0 mmol / L
β-nicotinamide adenine dinucleotide oxidized form [NAD] 5.0 mmol / L
Cholesterol oxidase 10 U / ml

Preparation of Reagent 2 Buffer pH 8.5
Cholesterol dehydrogenase 20 U / ml
Cholesterol hydrolase 6 U / ml

[Example 3] Quantification of cholesterol in LDL fraction The following Reagent 1 and Reagent 2 were prepared, and the LDL-cholesterol concentration was measured. The specimen used was 10 cases of general human serum. The measurement was carried out with a Hitachi 7170 automatic analyzer. In the operation method, 210 μl of reagent 1 was first added to 3 μl of sample, and the temperature was kept constant at 37 ° C. for 5 minutes.

  Furthermore, 70 μl of Reagent 2 was added, and the temperature was kept constant at 37 ° C. for 5 minutes.

  The difference between absorbance 1 and absorbance 2 was determined, and the value of the sample was converted using a known LDL-cholesterol concentration control as a standard solution. In the control method, the LDL-cholesterol concentration was determined by the Free Dewald equation. For the HDL cholesterol level, PG pole manufactured by Kokusai Reagent Co., Ltd. was used. The total cholesterol value was determined using T-CHO Reagent · A manufactured by International Reagents Co., Ltd. The TG value was determined using TG Reagent • A manufactured by Kokusai Reagent Co., Ltd. As a measurement result, comparison with the control method is shown in Table 3. This method was a good result that was in good agreement with the result of the control method.

Preparation of Reagent 1 Buffer pH 7.0
Calix sulfate (6) allene 1.0 mmol / L
Cholesterol dehydrogenase 20 U / ml
β-nicotinamide adenine dinucleotide oxidized form [NAD] 5.0 mmol / L
Cholesterol oxidase 10 U / ml
Cholesterol hydrolase 1 U / ml

Preparation of Reagent 2 Buffer pH 8.5
Hydrazinium dichloride 300 mmol / L
Cholesterol hydrolase 3 U / ml

[Example 4] Quantification of cholesterol in VLDL fraction The following reagent 1 and reagent 2 were prepared, and the VLDL-cholesterol concentration was measured. As specimens, 5 cases of general human serum were used. The measurement was carried out with a Hitachi 7170 automatic analyzer. The operating method was as follows. First, 180 μl of reagent 1 was added to 5 μl of sample, and the temperature was kept constant at 37 ° C. for 5 minutes.

  Furthermore, 60 μl of Reagent 2 was added, and the temperature was kept constant at 37 ° C. for 5 minutes. The difference between absorbance 1 and absorbance 2 was determined, and the value of the specimen was converted using serum with a known VLDL-cholesterol concentration as a standard solution. The ultracentrifugation method was used as a control method. As a measurement result, comparison with the control method is shown in Table 4. This method was a good result that was in good agreement with the result of the control method.

Preparation of Reagent 1 Buffer pH 8.0
Cholesterol dehydrogenase 20 U / ml
β-nicotinamide adenine dinucleotide oxidized form [NAD] 5.0 mmol / L
Sodium chloride 100 mmol / L
Sodium cholate 0.1%
Cholesterol oxidase 10 U / ml
Cholesterol hydrolase 1 U / ml

Preparation of Reagent 2 Buffer pH 8.5
Hydrazinium dichloride 300 mmol / L
Triton X-100 0.4%
Cholesterol hydrolase 3 U / ml

  According to the present invention, by reacting with cholesterol oxidase in the first reaction and reacting with cholesterol dehydrogenase in the second reaction, the influence of cholesterol derived from fractions other than the target lipoprotein fraction is eliminated. It becomes possible.

  Furthermore, according to the present invention, cholesterol in a specific lipoprotein fraction can be quantified without aggregating the lipoprotein in the biological sample. That is, by including calixarenes and derivatives thereof, polyanions, polysaccharides (cyclodextrins, etc.), water-soluble polymers, etc. in the reaction solution, the agglutination of lipoproteins in biological samples can be achieved. In addition, cholesterol in a specific lipoprotein fraction can be selectively quantified without being affected by other lipoproteins.

  From the above, the method of the present invention and the method of using the reagent kit of the present invention do not require separation fraction such as centrifugal fractionation, so that the operation is simple, and there is a problem due to measurement errors and human factors. Can be reduced.

In addition, since it can be applied to a method using two types of reagents, continuous measurement using a general-purpose automatic analyzer is possible, and measurement can be performed in multichannel with other test items.

Claims (8)

In quantifying cholesterol in specific lipoproteins in biological samples,
In the first reaction, cholesterol other than cholesterol in the specific lipoprotein is reacted with cholesterol oxidase,
Cholesterol quantification method characterized by reacting cholesterol in said specific lipoprotein with cholesterol dehydrogenase in the second reaction. The quantification method according to claim 1, wherein cholesterol in the specific lipoprotein is quantified based on a difference between the absorbance after the reaction with the cholesterol oxidase and the absorbance after the reaction with the cholesterol dehydrogenase. The specific lipoprotein is a high-density lipoprotein;
The quantification method according to claim 1 or 2, wherein cholesterol other than cholesterol in the specific lipoprotein is free cholesterol. In the first reaction, low-density lipoprotein and ultra-low density lipoprotein are calixarene sulfate having 4 to 8 molecules of phenol as a basic skeleton, sodium salt or potassium salt of dextran sulfate, sodium salt or potassium salt of phosphotungstic acid. , Polyacrylic acid, carrageenan, Apo-B antiserum, concanavalin A, partially hydroxypropylated cyclodextrin, partially methylated cyclodextrin, potassium or sodium salt of heparin, sodium alginate, and polyethylene glycol having an average molecular weight of 200-6000 By allowing at least one compound selected from the group to act to form a soluble complex, the low-density lipoprotein and the ultra-low-density lipoprotein, the cholesterol oxidase, and the co-oxidant Inhibit the reaction of the sterol dehydrogenase, quantitative method of claim 3. The specific lipoprotein is a low density lipoprotein;
The quantification method according to claim 1 or 2, wherein cholesterol other than cholesterol in the specific lipoprotein is cholesterol other than cholesterol in the low-density lipoprotein. In the first reaction, the low-density lipoprotein is calixarene sulfate having 4 to 8 molecules of phenol as a basic skeleton, sodium salt or potassium salt of dextran sulfate, sodium salt or potassium salt of phosphotungstic acid, polyacrylic acid, Selected from the group consisting of carrageenan, Apo-B antiserum, concanavalin A, partially hydroxypropylated cyclodextrin, partially methylated cyclodextrin, potassium or sodium salt of heparin, sodium alginate, and polyethylene glycol having an average molecular weight of 200-6000 6. The reaction of the low density lipoprotein with the cholesterol oxidase and the cholesterol dehydrogenase is inhibited by allowing at least one compound to act to form a soluble complex. The method of quantifying described. The method according to claim 5 or 6, wherein the reaction between the low-density lipoprotein and the compound in the first reaction is performed under conditions of pH 6 to 8 and ionic strength 200 to 500 mmol / L. The specific lipoprotein is an ultra-low density lipoprotein;
The quantification method according to claim 1 or 2, wherein cholesterol other than cholesterol in the specific lipoprotein is cholesterol contained in a lipoprotein other than the ultra-low density lipoprotein.