JP2001346598A - Method for analyzing hdl subfraction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for fractionating a component contained in a high density lipoprotein subfraction (HDL2 and HDL3) without centrifugal operation, or the like, and conveniently and accurately measuring the component. SOLUTION: This method comprises selectively acting an enzyme on the component, particularly cholesterol (HDL3-C), contained in HDL3 in the presence of a nonionic surfactant with an HLB value of more than 17 to measure the amount of HDL3-C and substracting the amount of HDL3-C from the whole HDL-C to calculate the amount of HDL2-C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring a component in a high-density lipoprotein (HDL) subfraction in blood. Specifically, cholesterol contained in HDL ₂ and HDL ₃ is a subfraction of _HDL, HDL 2 -C, relates to a method for measuring _HDL 3 -C.

[0002]

2. Description of the Related Art Serum is classified as lipoproteins into four types of lipoproteins, namely, high density lipoproteins (HD).
L), low density lipoprotein (LDL), very low density lipoprotein (VLDL) and chylomicron (CM). It is known that various types of diseases such as hyperlipidemia are caused by an increase in the amount of one or two of these lipoprotein fractions, and a method for fractionating and quantifying each lipoprotein has been reported. . Among them, HDL has been examined and measured as a marker for diagnosis or analysis of various arteriosclerosis such as coronary arteriosclerosis. HDL is also HDL ₂ and HDL
₃ subfractions (hereinafter referred to as HDL subfractions).

[0003] The clinical significance of HDL subfractions has been little studied due to the difficulty of fractionation. Conventionally, fractionation of HDL has been performed by ultracentrifugation. This method requires skill and the centrifugation is carried out for several days with a separate ultracentrifuge. Therefore, it was not possible to process multiple samples. Instead, LDL is obtained by mixing a divalent cation such as magnesium or calcium with a polyanion such as polyethylene glycol and dextran sulfate, or mixing a serum and a fractionating agent having a divalent cation with phosphotungstic acid. , VLDL, and CM are precipitated, and only HDL remaining in the supernatant after centrifugation is fractionated, but the HDL subfraction cannot be fractionated.

[0004] Further, a precipitation method in which HDL remaining in the supernatant is further separated into subfractions by applying this method is disclosed in Lewis I. Gidez, et al.
al [Journal of Lipid Research, 23, 1206-1223 (198
2)], Kurt Widhalm and Renate Pakosta [Clinical Che
mistry, 37 (2), 238-241 (1992)] et al., but these methods have not been widely used due to doubts about their accuracy.

[0005] In recent years, studies have been made on the fractionation of HDL subfractions by electrophoresis, and abnormally high HDL
Genetic deficiencies (CETP deficiency, decreased hepatic lipase activity, etc.) have been pointed out in bloodemia. High HDL cholesterol (HDL-C) is also referred to as longevity syndrome, but when it is high due to genetic abnormalities, clinical observation is still necessary. Since a decrease in HDL-C increases arteriosclerosis, it is necessary to maintain a certain concentration. However, there is a report that an extremely high HDL-C tends to cause cerebrovascular disorder.

[0006] Regarding these phenomena, HDL ₂ and HDL ₃
Analysis may provide clinically important information at an early stage. In recent years, reports have also been made on the clinical significance of the HDL subfraction cholesterol. Less than,
For reference, literatures with descriptions on hyper-HDL blood disease and HDL subfraction are listed.

1) Yuji Matsuzawa, Shizuya Yamashita, Seiichiro Tarui, H
DL abnormality, metabolism, 25 (4), 351-358, 1988. 2) Karl H. Weisgraber and Robert W. Mahley, Subfra
ction of human highdensity lipoproteins by heparin
-Sepharose affinity chromatography., J. Lipid Res.,
21, 316-325, 1980. 3) Lewis I. Gidez, et al, Separation and quantitat.
ion of subclasses ofhuman plasma high density lipo
proteins by a simple precipitationprocedure., J. L
ipid Res., 23, 1206-1223, 1982. 4) Kurt Widhalm and Renate Pakosta, Precipitation
with polyethyleneglycol and density-gradient ultra
centrifugation compared fordetermining high-densit
y lipoprotein subclasses HDL ₂ and HDL _3. , Clinical C
hemistry, 37 (2), 238-241, 1992. 5) Hidaka Hiroya et al., Study of LDL fractionation in patients with hyper-HDLemia with normal CETP, clinicopathology, 42 (11), 1172-11
76, 1994. 6) Koji Aosaki Others, a simple method for measuring the genotype of cholesteryl ester transfer protein (CETP) and high HDL-C
Of abnormal CETP in patients with hypertension, analysis of biological samples, 20 (2), 119-125, 1997. 7) Akihiro Inazu et al., CETP deficiency and high HDL cholesterol, clinical pathology, 44 (4), 322- 326, 1996. 8) Naoi Sakai et al., HDL abnormalities and arteriosclerosis, Clinical Science, 27 (4), 416-424, 1991. 9) Tadashi Hoshino et al., High lipoprotein electrophoretic pattern Case of HDL-C blood disease, clinical pathology, 37
(7), 835-839, 1989. 10) Umemori S. Others, A case of hyperHDLemia caused by deficiency of cholesterol ester transfer activity, clinical pathology, 40 (9), 999-1003, 1989. 11) Shizuya Yamashita Other, characterized by Small Heterogeneous LDL and giant particle HDL, Cholesteryl ester transf
study of two families with hyper-HDLemia caused by complete deficiency of er activity, arteriosclerosis, 17 (2), 371-379, 1989. 12) Shizuya Yamashita and others, high HDL ₂ with juvenile corneal rings
-C plasma clinical and biochemical studies, atherosclerosis, 13 (4),
981-990, 1985. 13) Ken-ischi Hirono, et al, Frequency of intron 1
4 splicing defect ofcholesterol ester transfer pro
tein gene in the Japanese generalpopulation-relati
on between the mutation and hyperalphalipoproteine
mia., Atherosclerosis, 100, 85-90, 1993.

[0008] As described above, it is clinically significant to fractionate the HDL subfractions and measure the cholesterol contained in each fraction. However, currently known methods for fractionating HDL subfractions involve mixing the fractionating agent with serum as described above,
Although it is a low-speed method, it is a method of performing a centrifugal operation, and there have been problems such as artificial quantification errors and mixing of samples when mixing the fractionating agent and serum. Moreover, automatic analyzers could not measure simultaneously with other common biochemical parameters. In clinical tests, it is required to obtain test results quickly, and reducing the test time is an issue.

In recent years, fully automatic kits for measuring cholesterol in HDL have been developed and are becoming popular. Patent No. 2
No. 600605 (Heisei 6/11/30), JP-A-8-20
No. 1393 (Hei 7/1/31) and JP-A-8-13
The technique disclosed in Japanese Patent No. 1195 (Hei 6/12/21) uses a fractionation agent in combination, and a metal used as a divalent cation contained in the fractionation agent is generally used in an automatic analyzer. Insoluble precipitates are formed in the wastewater and accumulate in the wastewater mechanism, causing a failure.

Furthermore, insoluble aggregates are formed during the reaction and turbidity which affects the measurement result is caused to cause a measurement error. In addition, the reaction cells are contaminated by the aggregates and the measurement is performed at the same time. Has had a considerable effect on the measurement results of biochemical items. Most of the popular automatic analyzers often complete the reaction in 10 minutes. In addition, known methods such as the two-point end method, rate method, and fixed time method can be selected.
Measurement is possible even in a cloudy state.

However, in the measurement in the turbid state, if the turbidity changes during the reaction, an error occurs in the measured value,
Accuracy matters. In addition, when the reaction solution becomes turbid, reproducibility decreases. Therefore, the sample to be measured is restricted, and it is not possible to cope with a wide range of measurement wavelengths and a wide variety of patient samples. For example, in the vicinity of 340 nm (UV region), there is a disadvantage that the absorbance becomes 2 or more or more due to the phenomenon of turbidity due to the aggregates and often exceeds the allowable range of the analyzer. The technique disclosed in Japanese Patent Application Laid-Open No. 9-96637 (Hei 8/7/19) without using divalent cations is a method of including an antiserum that aggregates with lipoproteins. As a result, the reaction cell is contaminated. Therefore,
It has a considerable effect on the measurement results of other biochemical items measured at the same time. In addition, since the turbidity in the reaction solution becomes strong, it is impossible to accurately measure cholesterol in HDL particularly in the UV region due to the same reason as described above.

[0012] These techniques are formed by devising a common technique for inhibiting an enzymatic reaction by forming a complex or an aggregate and devising a photometric method, which has an adverse effect on the inherent measurement of turbidity. Has not been completely eliminated.

[0013]

SUMMARY OF THE INVENTION An object of the present invention is to form a turbidity due to a complex or an aggregate in a reaction solution without using a general-purpose automatic analyzer to perform separation by centrifugation. , A method for quantifying components in a biological sample, particularly in a HDL subfraction (HDL ₂ and HDL ₃ ) in serum, more specifically, cholesterol (HDL ₂ -C
And to provide a method for measuring _HDL 3 -C).

[0014]

Means for Solving the Problems The present inventors have proposed lipoprotein lipase (LPL) or cholesterol esterase (CE), which are cholesterol esterases that directly act on HDL preferentially, with non-ionic having an HLB value of 17 or more. are reacted in the presence of sexual surfactant, without forming a complex or aggregate causing errors in spectrophotometric measurements, HDL 3 _- cholesterol (HDL
A method for measuring _3- C) was found, and the present invention was completed.

That is, the present invention relates to (1) a method for measuring a component in an HDL subfraction by an enzymatic reaction, the method comprising the steps of: releasing an ingredient from the HDL subfraction; A method for measuring a component in an HDL subfraction, which comprises using a nonionic surfactant having an HLB value of 17 or more. (2) An enzyme showing high specificity for HDL is lipoprotein lipase ( LPL) or cholesterol esterase (CE) (1) the method for measuring the components in the HDL subfraction, (3) the enzymatic reaction for releasing the components from the HDL subfraction, and measuring the released components (1) or (2), wherein the enzymatic reaction is carried out in a pH range of 6 to 9.
The component in the subfraction is cholesterol, and the enzymatic reaction for measuring cholesterol is a reaction by cholesterol dehydrogenase and oxidized coenzyme. (1) the method for measuring a component in the HDL subfraction according to any one of (1) to (3), wherein the component in the HDL subfraction to be measured is cholesterol, and the cholesterol is measured; The HDL subfraction according to any one of the above (1) to (3), wherein the enzymatic reaction is a reaction by cholesterol oxidase, and the method comprises measuring hydrogen peroxide, which is a product of the reaction, using peroxidase. Method for measuring components in water, (6) HDL
Wherein the subfractions is characterized in that it is a HDL ₃ (1) ~
(5) a method for measuring a component in any of the HDL subfractions,
(7) Total HDL- cholesterol (HDL-C) HDL from weight ₃ - HDL by subtracting cholesterol _(HDL 3 -C) of ₂ - Cholesterol (HDL ₂ -
C) The above (1) to (6), wherein the amount is also obtained.
And (8) a method for measuring a component in the HDL subfraction according to any one of (1) to (7). Reagent kit.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The method for measuring the components in the HDL subfraction of the present invention is based on the method used for measuring the total HDL-C in the HDL fraction, and the following means are introduced. This makes it possible to measure components in the HDL subfraction, particularly cholesterol. In the measurement of the components in the HDL fraction, in particular, cholesterol (HDL-C), an enzyme acts on the HDL fraction to hydrolyze and release the components contained in the HDL. The released component is then measured using a method compatible with the measurement of the component. When the component to be measured is cholesterol, cholesterol released by the action of cholesterol dehydrogenase (CDH) or cholesterol oxidase (COD) is measured.

One means of measuring the components in the HDL subfraction, especially cholesterol, is to select an enzyme that reacts preferentially to HDL. Examples of enzymes that act on lipoproteins to hydrolyze and release components contained in lipoproteins include cholesterol esterases such as LPL and CE. Among them, for example, LPL or CE derived from Chromobacterium viscosum is preferably used because it preferentially reacts with HDL as compared with other lipoproteins. In addition, an enzyme derived from Pseudomonas can be appropriately selected in consideration of compatibility with other measurement components. Enzymes modified for stabilization or the like of the enzyme can be used as long as they are enzymes that react preferentially with HDL and can achieve the object of the present invention. do not do.

Another means of measuring components in the HDL subfraction, especially cholesterol, is the choice of pH,
It is preferable to measure the enzyme reaction for releasing the components in the DL subfraction and the reaction for measuring the released components at pH 6 to 9. When the pH is 6 or less, the lipoprotein itself becomes cloudy due to the isoelectric point effect regardless of the aggregate. The measurement conditions may be adjusted by selecting around pH 7 where the lipoprotein is relatively stable, but LPL or CE has a pH of 8.0 to 9.0.
Reacts very strongly with HDL.

Further, the pH at which enzymes such as COD, CDH, LPL and CE can react is also taken into consideration. Preferably a CD
The reaction of H, LPL and CE is preferably at pH 7-9,
When COD is used, pH 6 to 8 is preferable.

The pH is adjusted with a buffer. The buffer is
Various buffers commonly used for biochemical reactions can be used.
EPES buffer, TAPS buffer, PIPES buffer,
BES-BisTris buffer, MOPS buffer, Tr
is-HCl buffer, MES buffer, ADA buffer, AC
ES buffer, MOPSO buffer, BES buffer, TES
Buffer, DIPSO buffer, TAPSO buffer, POP
SO buffer, HEPPS buffer, HEPPSO buffer,
Tricine buffer, Bicine buffer, phosphate buffer and the like.

The components in the HDL ₃ is HDL subfractions, the means for particularly measuring cholesterol _(HDL 3 -C),
The use of a nonionic surfactant having an HLB value of 17 or more, preferably 19 or more, particularly preferably 20 or more. The effects are LDL, VLDL, CM, and HD
LPL and C for HDL ₃ other lipoproteins L ₂ such as
It inhibits the reactivity of E. Accordingly, components in the HDL _3, can be particularly measured _HDL 3 -C. Also, HLB
The higher the value of the nonionic surfactant, the more selectively the components in HDL ₃ can be measured with a smaller amount of addition. Although it illustrated actually surfactants which can be used in the following, without being limited thereto, as long as it inhibits the reactivity of LPL and CE for lipoproteins other than HDL _3.

The surfactant used in this method is cetyl ether (C16) (hexadecyl ether) (trade name: Nikko Chemical Co., Ltd .: BC-25TX, BC-30TX, B
C-40TX), lauryl ether (C12) (dodecyl ether) (trade name: Nikko Chemical Co., Ltd .: BL-2)
1, BL-25), oleyl ether (trade name: Nikko Chemical Co., Ltd .: BO-50), behenyl ether (C2
2) (trade name: Nikko Chemical Co., Ltd .: BB-30), polyoxyethylene lauryl ether (trade name: Nippon Yushi Co., Ltd .: Nonion K-230), polyoxyethylene monolaurate (trade name: Nippon Yushi) Co., Ltd .: Nonion S-
40), polyoxyethylene ethers (trade names: Sigma: Brij98, Brij721, Brij78, B
ri99) and the like. Preferably, the HLB value is 2
Polyoxyethylene ethers near 0 are selected. These surfactants may be used alone or in combination of two or more as appropriate. The amount of addition varies depending on the amount of lipoprotein to be measured and the HLB value of the surfactant used, but the optimum amount can be determined by simple experimental repetition. For example, when a surfactant having an HLB value of around 17 is used, the reactivity of LPL and CE with respect to LDL and VLDL is inhibited.
Since HDL ₂ may be measured in addition to L ₃ , in such a case, it is preferable to increase the amount of addition as compared with the case where a surfactant having a higher HLB value is used. Specifically, in the range of 0.001 w / v% to 10 w / v%,
Preferably, it is used in the range of 1 w / v% to 5 w / v%.

The amount of components in HDL ₂ , which is an HDL subfraction, is determined by subtracting the amount of components in HDL ₃ from the amount of components in HDL. HDL ₂ to be measured
If components in is cholesterol, _HDL 2 -C amount can be obtained by subtracting the _HDL 3 -C amount from the total HDL-C content.

In the present invention, the above three means, pH
, An enzyme, and a surfactant, either alone or as appropriate. More preferably, all these means are introduced at the same time, but it is not necessary to introduce all of them at the same time.

In addition, free cholesterol in LDL, VLDL and chylomicron is involved in the reaction during HDL-C measurement and often causes an error. There is also a method in which cholesterol is reacted and converted into cholesterone hydrazone in the presence of hydrazine, and is converted into a non-substrate when measuring HDL-C. The technique of non-substrate conversion is disclosed in Japanese Patent Laid-Open No. 5-17 / 1993.
No. 6797 (Hei 3/12/27) may be referred to.

On the other hand, calixarenes can be used to suppress the reactivity of CE or LPL to LDL. Calix allenes include calix [4] allene,
Calix [6] arene, calix [8] arene, calix sulfate [4] arene, calix sulfate [6] arene, calix sulfate [8] arene, calix acetate [4] arene, calix acetate [6] arene, calix acetate [ 8] Allen,
Carboxy calix [4] arene, carboxy calix [6] arene, carboxy calix [8] arene, calix [4] arene amine, calix [6] arene amine, calix [8] arene amine. One or more of them are appropriately selected and used. The working concentration can be determined by simple experimental repetitions.

When the component in the HDL subfraction to be measured is cholesterol, the basic reaction for measuring cholesterol is a method for measuring total cholesterol,
The components may be appropriately prepared according to the desired fraction.

For example, the measurement of cholesterol is carried out by performing a reaction between cholesterol and cholesterol dehydrogenase (CDH) in the presence of an oxidized coenzyme, and optically measuring the coenzyme reduced as a result of the reaction. Examples of coenzymes when using CDH include β-nicotinamide adenine dinucleotide oxidized form (NAD), Thio-nicotinamide adenine dinucleotide oxidized form (t-NAD), and β-nicotinamide adenine dinucleotide oxidized form (t-NAD).
-Nicotinamide adenine dinucleotide phosphorylated (NADP), and Thio-nicotinamide adenine dinucleotide phosphorylated (t-NADP).

Cholesterol oxidase (COD) can also be used for cholesterol measurement.
The measurement is performed by combining peroxidase with a known hydrogen peroxide determination method.

Furthermore, the activating agent such as cholic acid and the stabilizing agent required for the measurement of cholesterol may be appropriately selected according to existing techniques, and the concentration used may be adjusted by simple experimental repetition.

The present invention also provides a reagent kit including reagents necessary for performing the method for measuring a component in the HDL subfraction of the present invention. The reagent kit of the present invention is a reagent kit used for measuring a component in an HDL subfraction containing a nonionic surfactant having an HLB value of at least 17 or more.

[0032]

The following is a specific example of the measurement of cholesterol in HDL ₃ .

Example 1 The following reagents A and B were prepared. The sample is
22 human sera from volunteers were used. The measurement is
Performed on a Hitachi 7170 automatic analyzer. First, sample 4μ
Add 180 μL of reagent A to L and incubate at 37 ° C for 5 minutes.
At this point, absorbance 1 was measured at a main wavelength of 340 nm and a sub wavelength of 570 nm. Further, 60 μL of reagent B was added and 37
The solution was kept at a constant temperature of 5 ° C. for 5 minutes. At this time, absorbance 2 was measured at a main wavelength of 340 nm and a sub wavelength of 570 nm. Known HDL 3 _-C concentration control by converting the value of the specimen as a standard solution determines the difference in absorbance 1 and absorbance 2.

As a control method, measurement was carried out using the method of Kurt Widhalm et al. Using polyethylene glycol. The cholesterol concentration of the supernatant fractionated by centrifugation using this method was determined using T-CHO Reagent L "Kokusai" manufactured by Kokusai Reagents. In addition, the measurement of homogeneous HDL-C using an automatic analyzer is performed by HD
It performed using LC reagent and KL "Kokusai". _HDL 2 -C values for the control method was determined by subtracting the _HDL 3 -C value by a manual method from HDL-C by a manual method. _HDL 2 -C value for automatic analysis was determined by subtracting the _HDL 3 -C value obtained using reagents A and B from the homogeneous HDL-C value.

FIG. 1 shows an ultracentrifugation operation [Havel RJ, Eder HA, B
ragdon JH: The distribution and chemical compositi
on of ultracentrifugally separated lipoproteinsin
human serum., J. Clin. Invest., 34, 1345-1353 (19
55)], the VLDL, LDL, (LDL + HDL
₂ ), 340 nm when HDL ₃ and HDL are measured
The reaction time course was shown.

As shown in FIG. 1, the present method uses VLDL, L
It can be seen that it hardly reacted with the DL and (LDL + HDL ₂ ) fractions, but only HDL ₃ and HDL. The total cholesterol concentration in each fraction was VLDL = 9
9.9 mg / dL, LDL = 199.9 mg / dL,
(LDL + HDL ₂ ) = 175.2 + 31.7 mg / d
L, HDL ₃ = 28.5 mg / dL and HDL = 84.
It was 2 mg / dL.

The measurement result by this method is as follows: VLDL = 0.7
mg / dL, LDL = 2.9 mg / dL, (LDL + H
DL ₂ ) = 4.0 mg / dL, HDL ₃ = 28.0 mg
/ DL and HDL = 63.2 mg / dL. These results also show that HDL ₃ has high specificity.

Table 1 shows the measurement results using 22 human sera. _HDL 3 -C value of this method is the correlation coefficient of the control method, 0.957 (p <0.05), and the became well consistent results. Moreover, _HDL 2 -C value of this method,
The correlation coefficient with the control method was 0.939 (p <0.05).

Reagent A HEPES buffer (pH 7.0) 10 mmol / L hydrazinium dichloride 100 mmol / L β-nicotinamide adenine dinucleotide oxidized form 6.0 mmol / L Sodium cholate (activator) 0.06 % Nonionic K-230 (HLB value: 17.3) 0.3 w / v% BC-40TX (HLB value: 20) 1.0 w / v% Calix [8] arene sulfate 2.0 mmol / L

Reagent B TAPS buffer (pH 8.5) 500 mmol / L cholesterol dehydrogenase 20 U / mL LPL 6 U / mL (from Chromobacterium viscosum) Sodium cholate (activator) 0.1%

[0040]

[Table 1]

[0041]

Example 2 The following reagents C and D were prepared. The sample is
22 human sera from volunteers were used. The measurement is
Performed on a Hitachi 7170 automatic analyzer. The procedure is as follows: First, add 180 μL of reagent C to 4 μL of the sample, and add
At a constant wavelength of 340 nm and a sub wavelength of 5
Absorbance 1 was measured at 70 nm. In addition, reagent D 60μ
L was added and the temperature was kept constant at 37 ° C. for 5 minutes.
Absorbance 2 was measured at 0 nm and sub wavelength 570 nm. Absorbance 1 and by obtaining a difference in absorbance 2 known HDL 3 _- by converting the value of the sample control of cholesterol concentration as a standard solution. In the control method, the same operation as in Example 1 was performed. _HDL 2 -C value for automatic analysis was determined by subtracting the _HD 3 -C value obtained using reagents C and D from homogeneous HDL-C value.

FIG. 2 shows VLDL and L obtained by ultracentrifugation.
The reaction time course at 340 nm when measuring DL, (LDL + HDL ₂ ), HDL ₃ and HDL is shown. From this figure, this method can be applied to VLDL, LDL, (LDL
+ HDL ₂ ) It can be seen that the fraction hardly reacted with the fraction, and only HDL ₃ and HDL reacted.

The total cholesterol concentration in each fraction was VL
DL = 99.9 mg / dL, LDL = 199.9 mg /
dL, (LDL + HDL ₂ ) = 175.2 + 31.7 m
g / dL, HDL ₃ = 28.5 mg / dL and HDL =
It was 84.2 mg / dL.

The result of measurement by this method is as follows: VLDL = 0.2
mg / dL, LDL = 3.5 mg / dL, (LDL + H
DL ₂ ) = 0.2 mg / dL, HDL ₃ = 27.8 mg
/ DL and HDL = 59.2 mg / dL. These results also show that HDL ₃ has high specificity.

Table 2 shows the measurement results using 22 human sera. _HDL 3 -C value of this method is the correlation coefficient of the control method, 0.952 (p <0.05), and the became well consistent results. Also, the HDL2-C value of the present method is:
The correlation coefficient with the control method was 0.942 (p <0.05).

Reagent C HEPES buffer (pH 7.0) 10 mmol / L hydrazinium dichloride 100 mmol / L β-nicotinamide adenine dinucleotide oxidized form 6.0 mmol / L Sodium cholate (activator) 0.06 % Nonionic K-230 (HLB value: 17.3) 0.3 w / v% Brij98 (HLB value: 19) 1.6 w / v% Calix sulfate [8] arene 2.0 mmol / L

Reagent D TAPS buffer (pH 8.5) 500 mmol / L cholesterol dehydrogenase 20 U / mL LPL 6 U / mL (from Chromobacterium viscosum) Sodium cholate (activator) 0.1%

[0048]

[Table 2]

[0049]

The high-density lipoprotein subfraction of the present invention (HDL
Components ₂ and HDL ₃₎ in, how particular measuring cholesterol, components in HDL ₃ in the presence of an HLB value of 17 or more non-ionic surfactants, optionally in particular cholesterol _(HDL 3 -C) Enzyme acts on the HD
Measured L ₃ -C amount, by determining the _HDL 2 -C amount from the total HDL-C by subtracting the _HDL 3 -C amount is a method of measuring the HDL-C of HDL subfractions very easily . The present invention uses a general-purpose automatic analyzer, without separation by centrifugation, without forming turbidity due to complexes or aggregates in the reaction solution, and HDL in biological samples, particularly in serum. Since the components in the sub-fraction can be quantified, it is useful for clinical tests and research of lipoprotein-related diseases such as hyperlipidemia.

[Brief description of the drawings]

FIG. 1 shows the reaction time course of each lipoprotein fraction obtained by ultracentrifugation. The photometry point is 17 points for 5 minutes,
34 points represent 10 minutes.

FIG. 2 shows a reaction time course of each lipoprotein fraction obtained by ultracentrifugation. The photometry point is 17 points for 5 minutes,
34 points represent 10 minutes.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12Q 1/44 C12Q 1/44 G01N 33/92 G01N 33/92 BZ (72) Inventor Koji Ochiai Nishi-ku, Kobe-shi, Hyogo 1-1-2 Murotani F-term in R & D Center of International Reagents Co., Ltd. (Reference) 2G045 AA13 BB29 BB60 CA25 CA26 DA63 DA69 FA26 FA27 FA29 FB01 GC10 4B063 QA01 QA19 QQ03 QQ76 QQ89 QR02 QR03 QR04 QR12 QR42 QR52 QS28 QS36 QX01

Claims (8)

[Claims] In a method for measuring a component in an HDL subfraction by an enzymatic reaction, an enzyme and HL exhibiting high specificity for HDL in order to release the component from the HDL subfraction.
A method for measuring a component in an HDL subfraction, comprising using a nonionic surfactant having a B value of 17 or more. 2. The HD according to claim 1, wherein the enzyme having high specificity for HDL is lipoprotein lipase (LPL) or cholesterol esterase (CE).
Method for measuring components in L subfraction. 3. An enzyme reaction for releasing components from the HDL subfraction and an enzyme reaction for measuring the released components are carried out in a pH range of 6 to 9.
Or the method for measuring a component in the HDL subfraction described in 2. 4. The component in the HDL subfraction to be measured is cholesterol, and the enzymatic reaction for measuring cholesterol is a reaction by cholesterol dehydrogenase and oxidized coenzyme. The method for measuring a component in an HDL subfraction according to any one of claims 1 to 3, which is a method for spectroscopically detecting a reduced coenzyme. 5. The component in the HDL subfraction to be measured is cholesterol, the enzyme reaction for measuring cholesterol is a reaction by cholesterol oxidase, and hydrogen peroxide which is a product of the reaction is converted to peroxidase. The method for measuring a component in an HDL subfraction according to any one of claims 1 to 3, which is a method for measuring by using the method. 6. A method for measuring components in HDL subfractions according to any one of claims 1 to 5, wherein the HDL subfractions is HDL _3. 7. Total HDL-cholesterol (HDL-cholesterol)
HDL ₃ from C) weight - Cholesterol _(HDL 3 -C)
Measurement of components in HDL subfractions according to any one of claims 1 to 6, characterized in that finding also cholesterol _(HDL 2 -C) amount - HDL ₂ by subtracting the amount. 8. The H according to claim 1, wherein
A reagent kit comprising reagents necessary for performing a method for measuring a component in a DL subfraction.