JP4708531B2 - Method for measuring cholesterol in HDL subfractions - Google Patents

Description

【００４１】
【実施例２】
以下の試薬Ｃ及びＤを調製した。検体は、ボランティアによるヒト血清２２例を用いた。測定は、日立７１７０型自動分析装置で実施した。操作法は、先ず検体 ４μＬに試薬Ｃ １８０μＬを加え３７℃で５分間恒温し、この時点で主波長３４０ｎｍ及び副波長５７０ｎｍで吸光度１を測定した。更に、試薬Ｄ ６０μＬを加え３７℃で５分間恒温し、この時点で主波長３４０ｎｍ及び副波長５７０ｎｍで吸光度２を測定した。吸光度１と吸光度２の差を求めて既知ＨＤＬ_３−コレステロール濃度のコントロールを標準液として検体の値を換算した。対照法は、実施例１同様の操作を行った。自動分析法のＨＤＬ_２−Ｃ値は、ホモジニアスＨＤＬ−Ｃ値から試薬Ｃ及びＤを用いて求めたＨＤ_３−Ｃ値を差し引いて求めた。
【００４２】
図２に超遠心操作で得られたＶＬＤＬ、ＬＤＬ、（ＬＤＬ＋ＨＤＬ_２）、ＨＤＬ_３及びＨＤＬを測定したときの３４０ｎｍにおける反応タイムコースを示した。この図から、本法はＶＬＤＬ、ＬＤＬ、（ＬＤＬ＋ＨＤＬ_２）画分にはほとんど反応せず、ＨＤＬ_３及びＨＤＬのみ反応していることが分かる。
【００４３】
各画分中の総コレステロール濃度は、ＶＬＤＬ＝９９．９ｍｇ／ｄＬ、ＬＤＬ＝１９９．９ｍｇ／ｄＬ、（ＬＤＬ＋ＨＤＬ_２）＝１７５．２＋３１．７ｍｇ／ｄＬ、ＨＤＬ_３＝２８．５ｍｇ／ｄＬ及びＨＤＬ＝８４．２ｍｇ／ｄＬであった。
【００４４】
本法による測定結果は、ＶＬＤＬ＝０．２ｍｇ／ｄＬ、ＬＤＬ＝３．５ｍｇ／ｄＬ、（ＬＤＬ＋ＨＤＬ_２）＝０．２ｍｇ／ｄＬ、ＨＤＬ_３＝２７．８ｍｇ／ｄＬ及びＨＤＬ＝５９．２ｍｇ／ｄＬであった。この結果からもＨＤＬ_３に高い特異性を有することが分かる。
【００４５】
また、ヒト血清２２例を用いた測定結果を表２に示した。本法のＨＤＬ_３−Ｃ値は、対照法との相関係数が、０．９５２（ｐ＜０．０５）となり、よく一致した結果となった。また、本法のＨＤＬ２−Ｃ値は、対照法との相関係数が、０．９４２（ｐ＜０．０５）となった。
【００４６】
試薬Ｃ
ＨＥＰＥＳ緩衝液 （ｐＨ７．０） １０ ｍｍｏｌ／Ｌ
二塩化ヒドラジニウム １００ ｍｍｏｌ／Ｌ
β−ニコチンアミドアデニンジヌクレオチド酸化型 ６．０ ｍｍｏｌ／Ｌ
コール酸ナトリウム（活性化剤） ０．０６ ％
ノニオンＫ−２３０（ＨＬＢ値１７．３） ０．３ ｗ／ｖ％
Ｂｒｉｊ９８（ＨＬＢ値１９） １．６ ｗ／ｖ％
硫酸カリクス［８］アレン ２．０ ｍｍｏｌ／Ｌ
【００４７】
試薬Ｄ
ＴＡＰＳ緩衝液 （ｐＨ８．５） ５００ ｍｍｏｌ／Ｌ
コレステロール脱水素酵素 ２０ Ｕ／ｍＬ
ＬＰＬ ６ Ｕ／ｍＬ
（Chromobacterium viscosum由来）
コール酸ナトリウム（活性化剤） ０．１ ％
【００４８】
【表２】

【００４９】
【発明の効果】
本発明の高密度リポ蛋白亜画分（ＨＤＬ_２及びＨＤＬ_３）中の成分、特にコレステロールを測定する方法は、ＨＬＢ値が１７以上の非イオン性界面活性剤の存在下でＨＤＬ_３中の成分、特にコレステロール（ＨＤＬ_３−Ｃ）に選択的に酵素を作用をさせて、ＨＤＬ_３−Ｃ量を測定し、総ＨＤＬ−ＣからＨＤＬ_３−Ｃ量を減算してＨＤＬ_２−Ｃ量を求めることにより、極めて容易にＨＤＬ亜画分のＨＤＬ−Ｃを測定する方法である。本発明は、汎用の自動分析装置を用いて、遠心操作による分離をせずに、また、反応液中に複合体や凝集体による濁りを形成することなく、生体試料中、特に血清中のＨＤＬ亜画分中の成分を定量することができるため、高脂血症等のリポ蛋白に関連する疾病の臨床検査や研究等に有用である。
【図面の簡単な説明】
【図１】 超遠心操作で得た各リポ蛋白画分の反応タイムコースを示す。測光ポイントは１７ポイントで５分、３４ポイントで１０分を表す。
【図２】 超遠心操作で得た各リポ蛋白画分の反応タイムコースを示す。測光ポイントは１７ポイントで５分、３４ポイントで１０分を表す。[0001]
[Technical field to which the invention belongs]
The present invention relates to a method for measuring a component in a high-density lipoprotein (HDL) subfraction in blood. Specifically, HDL is a sub-fraction of HDL ₂ And HDL ₃ In cholesterol and HDL ₂ -C, HDL ₃ It relates to a method for measuring C.
[0002]
[Prior art]
Serum includes lipoproteins roughly classified into four types of lipoproteins: high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), and chylomicron (CM). ing. Diseases such as various hyperlipidemias are known to be derived from 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 is inspected and measured as a marker for diagnosis of various arteriosclerosis such as coronary atherosclerosis or analysis of pathological condition. HDL is further HDL ₂ And HDL ₃ Are subdivided into two subfractions (hereinafter referred to as HDL subfractions).
[0003]
The clinical significance of HDL subfractions has been hardly studied due to the difficulty of fractionation. Conventionally, HDL fractionation has been performed by ultracentrifugation. This method requires skill, and is centrifuged for several days with a separate ultracentrifuge. Therefore, it was not possible to process many samples. Instead of this, LDL is mixed with a serum that is a fractionating agent in which divalent cations such as magnesium and calcium are coexisted with polyanions such as polyethylene glycol and dextran sulfate, or divalent cations are coexisted with phosphotungstic acid. , VLDL, and CM are precipitated, and the method of fractionating only HDL remaining in the supernatant after centrifuging them is the mainstream, but the HDL subfraction cannot be fractionated.
[0004]
Further, by applying this method, a precipitation method in which HDL remaining in the supernatant is further separated into subfractions is described by Lewis I. Gidez, et al [Journal of Lipid Research, 23, 1206-1223 (1982)], Kurt Widhalm and Although reported by Renate Pakosta [Clinical Chemistry, 37 (2), 238-241 (1992)], these methods are not popular because of their accuracy.
[0005]
In recent years, the HDL subfraction has been studied by fractionation by electrophoresis, and genetic defects (CETP deficiency, decreased hepatic lipase activity, etc.) have been pointed out due to abnormally high HDL blood. . High HDL cholesterol (HDL-C) is said to be a longevity syndrome, but clinical observation is still necessary when it is high due to a genetic abnormality. When HDL-C decreases, arteriosclerosis increases, so it is necessary to maintain a certain concentration. However, there is a report that an extremely high value of HDL-C tends to cause cerebrovascular disorder.
[0006]
HDL about these phenomena ₂ And HDL ₃ Analyzing this information may provide early clinically important information. In recent years, the clinical significance of HDL subfraction cholesterol has been reported. For reference, documents with descriptions on hyperHDLemia and HDL subfractions are listed below.
[0007]
1) Shinji Matsuzawa, Shizuya Yamashita, Kiyoichiro Tarui, HDL Abnormalities, Metabolism, 25 (4), 351-358, 1988.
2) Karl H. Weisgraber and Robert W. Mahley, Subfraction of human high
density lipoproteins by heparin-Sepharose affinity chromatography., J. Lipid Res., 21, 316-325, 1980.
3) Lewis I. Gidez, et al, Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation
procedure., J. Lipid Res., 23, 1206-1223, 1982.
4) Kurt Widhalm and Renate Pakosta, Precipitation with polyethylene
glycol and density-gradient ultracentrifugation compared for
determining high-density lipoprotein subclasses HDL ₂ and HDL _Three .,
Clinical Chemistry, 37 (2), 238-241, 1992.
5) Hiroya Hidaka et al., Examination of LDL fraction in patients with hyperHDLemia with normal CETP, Clinicopathology, 42 (11), 1172-1176, 1994.
6) Aozaki, K. Others, Simple determination of cholesteryl ester transfer protein (CETP) genotype and frequency of abnormal CETP in patients with high HDL-C blood, biological sample analysis, 20 (2), 119-125, 1997.
7) Akihiro Inazu et al., CETP deficiency and high HDL cholesterolemia, Clinicopathology, 44 (4), 322-326, 1996.
8) Naohiko Sakai Others, HDL abnormalities and arteriosclerosis, Clinical Science, 27 (4), 416-424, 1991.
9) Tadashi Hoshino and others, A case of hyper-HDL-C blood symptom with a specific pattern of lipoprotein electrophoresis, Clinical Pathology, 37 (7), 835-839, 1989.
10) Yoshio Umemori Others, A case of hyperHDLemia caused by deficiency in cholesterol ester transfer activity, Clinical Pathology, 40 (9), 999-1003, 1989.
11) Shizuya Yamashita and others, Small Heterogeneous LDL and giant particle HDL, characterized by two families with hyperHDLemia due to complete deficiency of cholesterol ester transfer activity, arteriosclerosis, 17 (2), 371-379, 1989 .
12) Shizuya Yamashita Other, High HDL with juvenile corneal ring ₂ -C plasma clinical and biochemical studies, arteriosclerosis, 13 (4), 981-990, 1985.
13) Ken-ischi Hirono, et al, Frequency of intron 14 splicing defect of cholesterol ester transfer protein gene in the Japanese general
population-relation between the mutation and hyperalphalipoproteinemia., Atherosclerosis, 100, 85-90, 1993.
[0008]
As described above, it is clinically significant to fractionate HDL subfractions and measure the cholesterol contained in each. However, the currently known method for fractionating HDL subfractions is a method in which a fractionating agent and serum are mixed as described above, and centrifugation is performed at a low speed, and the fractionating agent and serum are mixed. At times, artificial quantitative errors and sample mix-ups were problems. In addition, it could not be measured simultaneously with other general biochemical items with an automatic analyzer. In clinical tests, it is required to obtain test results quickly, and shortening the test time is a problem.
[0009]
In recent years, fully automatic kits for measuring cholesterol in HDL have been developed and are becoming popular. The techniques found in Japanese Patent No. 2600065 (Hei 6/11/30), Japanese Patent Application Laid-Open No. 8-201393 (Hei 7/1/31) and Japanese Patent Application Laid-Open No. 8-131195 (Hei 6/12/21) are: In combination with a fractionation agent, the metal used as a divalent cation contained in the fractionation agent forms an insoluble precipitate with detergents commonly used in automatic analyzers, and it accumulates in the waste liquid mechanism. This causes a failure.
[0010]
In addition, the formation of insoluble aggregates during the reaction causes turbidity that affects the measurement results, causing measurement errors, as well as other biochemistry that is measuring simultaneously when the reaction cell is contaminated by aggregates. It has a considerable influence on the measurement results of items. Most popular automated analyzers often complete the reaction in 10 minutes. Furthermore, since a two-point end method, a rate method, a fix time method, or the like, which is a known method, can be selected, measurement can be performed even in a cloudy state.
[0011]
However, in the measurement in the turbid state, if there is a change in turbidity during the reaction, an error occurs in the measured value, and accuracy is a problem. In addition, reproducibility decreases when the reaction solution becomes cloudy. Therefore, the sample to be measured is limited, and it is not possible to deal with a wide range of measurement wavelengths and a wide variety of patient specimens. For example, in the vicinity of 340 nm (UV region), there is a drawback that the absorbance becomes 2-3 or more due to the turbidity phenomenon due to the aggregates and often exceeds the allowable range of the analyzer. Japanese Patent Laid-Open No. 9-96637 (Hei 8/7/19), which does not use a divalent cation, is a method of including an antiserum that aggregates with a lipoprotein, which is also a hardly soluble antigen-antibody aggregate. 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. Moreover, since the turbidity in the reaction solution becomes strong, it is impossible to measure accurately for the same reason as described above, particularly for cholesterol measurement in HDL in the UV region.
[0012]
These technologies are formed by devising common techniques and photometric methods that inhibit enzyme reactions by forming complexes and aggregates. It has not been resolved.
[0013]
[Problems to be solved by the invention]
An object of the present invention is to use a general-purpose automatic analyzer, without separation by centrifugation, and without forming turbidity due to complexes or aggregates in a reaction solution, in a biological sample, particularly in serum. HDL sub-fraction (HDL ₂ And HDL ₃ ) For quantifying the components in the HDL, in detail cholesterol in HDL subfractions (HDL ₂ -C and HDL ₃ -C) to provide a method for measuring.
[0014]
[Means for solving the problems]
The present inventors have made lipoprotein lipase (LPL) or cholesterol esterase (CE), which is a cholesterol ester hydrolase preferentially acting on HDL, in the presence of a nonionic surfactant having an HLB value of 17 or more. Without the formation of complexes or aggregates that cause errors in spectrophotometer measurements. ₃ -Cholesterol (HDL ₃ A method for measuring -C) was found and the present invention was completed.
[0015]
That is, this invention consists of the following.
1. Cholesterol ester hydrolase selected from lipoprotein lipase (LPL) and cholesterol esterase (CE), two different nonionic surfactants having an HLB value of 17-20, hydrazine salts, and calixare The Use HDL ₃ HDL, characterized in that cholesterol is released from a fraction and the released cholesterol is measured by an enzymatic reaction with cholesterol dehydrogenase or cholesterol oxidase ₃ -Determination of cholesterol.
2. Two different types of nonionic surfactants, hydrazine salts, and calixare The HDL by adding a reagent containing cholesterol esterase after mixing the reagent containing and serum sample ₃ 2. HDL according to item 1 above, wherein cholesterol is released from the fraction ₃ -Determination of cholesterol.
3. HDL ₃ 3. The HDL according to item 1 or 2, wherein an enzyme reaction for releasing cholesterol from the fraction and an enzyme reaction for measuring the released cholesterol are performed in a pH range of 6-9. ₃ -Determination of cholesterol.
4). Any of the preceding items 1 to 3, wherein the enzyme reaction for measuring the released cholesterol is a reaction with cholesterol dehydrogenase and oxidized coenzyme, and the reduced coenzyme which is the product of the reaction is detected spectroscopically HDL according to item 1 ₃ -Determination of cholesterol.
5. The HDL according to any one of items 1 to 3, wherein the enzyme reaction for measuring the released cholesterol is a reaction by cholesterol oxidase, and hydrogen peroxide as a product of the reaction is measured using peroxidase. ₃ -Determination of cholesterol.
6 . in front The HDL is measured by the measurement method according to any one of Items 1 to 4. ₃ -Measure cholesterol level and calculate HDL from total HDL-cholesterol (HDL-C) level ₃ -Cholesterol (HDL ₃ -C) HDL characterized by obtaining by subtracting quantity ₂ -Determination of cholesterol.
[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 by introducing the means shown below, It is possible to measure the components, particularly cholesterol. In the measurement of components in the HDL fraction, particularly cholesterol (HDL-C), an enzyme is allowed to act on the HDL fraction to hydrolyze it to release components contained in the HDL. The liberated component is then measured using a method compatible with the measurement of that component. When the component to be measured is cholesterol, cholesterol liberated by acting on cholesterol dehydrogenase (CDH) or cholesterol oxidase (COD) is measured.
[0017]
One means of measuring components in HDL subfractions, particularly cholesterol, is to select an enzyme that preferentially reacts with HDL. Examples of enzymes that act on lipoproteins and hydrolyze them to release components included in lipoproteins include cholesterol ester hydrolases such as LPL and CE. Among them, for example, LPL or CE derived from Chromobacterium viscosum is preferably used because it reacts preferentially with HDL as compared with other lipoproteins. In addition, an enzyme derived from the genus Pseudomonas can be appropriately selected in consideration of compatibility with other measurement components. Even if the enzyme is modified for stabilization of the enzyme or the like, it can be used as long as it is an enzyme that reacts preferentially with HDL and can achieve the object of the present invention. do not do.
[0018]
Another means of measuring components, particularly cholesterol, in HDL subfractions is the choice of pH for the enzymatic reaction that liberates the components in HDL subfractions and the measurement of the released components. It is preferable to measure the reaction at pH 6-9. 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 lipoprotein is relatively stable, but LPL or CE reacts very strongly to HDL at pH 8.0 to 9.0.
[0019]
Furthermore, the pH at which enzymes such as COD, CDH, LPL, and CE can react is also considered. Suitably, the reaction of CDH, LPL and CE is preferably pH 7-9, and pH 6-8 is preferred when COD is used.
[0020]
The pH is adjusted with a buffer solution. Various buffers commonly used in biochemical reactions can be used as the buffer, such as HEPES buffer, TAPS buffer, PIPES buffer, BES-BisTris buffer, MOPS buffer, Tris-hydrochloric acid buffer, MES buffer, ADA buffer, ACES buffer, MOPSO buffer, BES buffer, TES buffer, DIPSO buffer, TAPSO buffer, POPSO buffer, HEPPS buffer, HEPPSO buffer, Tricine buffer, Bicine buffer, phosphate buffer Liquid and the like.
[0021]
HDL, a sub-fraction of HDL ₃ Ingredients, especially cholesterol (HDL ₃ A means for measuring -C) is to use 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 HDL ₂ HDL etc. ₃ It inhibits the reactivity of LPL and CE to other lipoproteins. Therefore, HDL ₃ Ingredients, especially HDL ₃ -C can be measured. In addition, a nonionic surfactant having a high HLB value can be selectively added with a small amount of HDL. ₃ The components in it can be measured. Examples of surfactants that can actually be used are shown below, but are not limited thereto. ₃ Anything that inhibits the reactivity of LPL and CE to other lipoproteins may be used.
[0022]
The surfactant used in this method is cetyl ether (C16) (hexadecyl ether) (trade name: Nikko Chemical Co., Ltd .: BC-25TX, BC-30TX, BC-40TX), lauryl ether (C12) (dodecyl ether). (Product name: Nikko Chemical Co., Ltd .: BL-21, BL-25), Oleyl ether (Product name: Nikko Chemical Co., Ltd .: BO-50), Behenyl ether (C22) (Product name: Nikko Chemical Co., Ltd.) ): BB-30), polyoxyethylene lauryl ether (trade name: Nippon Oil & Fat Co., Ltd .: Nonion K-230), polyoxyethylene monolaurate (trade name: Nippon Oil & Fat Co., Ltd .: Nonion S-40), And polyoxyethylene ethers (trade names: Sigma: Brij98, Brij721, Brij78, Brij99). Preferably, polyoxyethylene ethers having an HLB value of around 20 are selected. These surfactants may be used alone or in appropriate combination of two or more. The addition amount varies depending on the amount of lipoprotein to be measured and the HLB value of the surfactant to be used, but the optimum addition amount can be determined by simple experimental repetition. For example, when using a surfactant with an HLB value of around 17, the reactivity of LPL and CE to LDL and VLDL is inhibited, but depending on the amount added, HDL ₃ HDL in addition to ₂ In such a case, it is preferable to increase the amount of addition as compared with the case of using a surfactant having a higher HLB value. Specifically, it is used in the range of 0.001 w / v% to 10 w / v%, preferably in the range of 1 w / v% to 5 w / v%.
[0023]
HDL, a sub-fraction of HDL ₂ The amount of ingredients in HDL is calculated from the amount of ingredients in HDL. ₃ It is obtained by subtracting the amount of components in it. HDL to be measured ₂ When the component in the sample is cholesterol, the total amount of HDL-C ₃ HDL by subtracting -C amount ₂ -C amount is obtained.
[0024]
In the present invention, the above three means, selection of pH, selection of enzyme, and selection of surfactant, are introduced alone or appropriately selected and combined. More preferably, all these means are introduced at the same time, but it is not always necessary to introduce all of them at the same time.
[0025]
In addition, free cholesterol in LDL, VLDL and chylomicron is involved in the reaction at the time of HDL-C measurement and often causes an error. However, these free cholesterol is reacted with COD and CDH in advance. There is also a method of converting to cholesteinone hydrazone in the presence of hydrazine and making it non-substrate when measuring HDL-C. A technique for forming a non-substrate may be referred to known JP-A-5-176797 (Hei 3/12/27).
[0026]
On the other hand, calixarenes can also be used to suppress the reactivity of CE or LPL to LDL. The calix arenes include calix [4] allene, calix [6] allene, calix [8] allene, calix sulfate [4] allene, calix sulfate [6] allene, calix sulfate [8] allene, calix acetate [4] allene, Calix [6] arene acetate, calix [8] allene acetate, carboxycalix [4] allene, carboxycalix [6] allene, carboxycalix [8] allene, calix [4] allenamine, calix [6] allene Examples include amines and calix [8] areneamine. One or more of these are appropriately selected and used. The working concentration can be determined by simple experimental repetition.
[0027]
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, and the components may be appropriately prepared according to the target fraction.
[0028]
For example, cholesterol is measured 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. As coenzymes when using CDH, β-nicotinamide adenine dinucleotide oxidized form (NAD), Thio-nicotinamide adenine dinucleotide oxidized form (t-NAD), β-nicotinamide adenine dinucleotide phosphate oxidized form ( NADP), Thio-nicotinamide adenine dinucleotide phosphate oxidation type (t-NADP) and the like.
[0029]
Cholesterol oxidase (COD) can also be used for cholesterol measurement. In this case, measurement is performed by combining peroxidase and a known hydrogen peroxide assay method.
[0030]
Furthermore, the conditions for the activator and stabilizer such as cholic acid, which are necessary for cholesterol measurement, may be appropriately selected according to existing techniques, and the concentration used may be adjusted by simple experimental repetition.
[0031]
The present invention also provides a reagent kit provided with reagents necessary for carrying out 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 measurement of a component in an HDL subfraction comprising a nonionic surfactant having an HLB value of 17 or more.
[0032]
【Example】
HDL ₃ An example of measurement of cholesterol in the inside will be specifically described.
[Example 1]
The following reagents A and B were prepared. As specimens, 22 cases of human serum by volunteers were used. The measurement was carried out with a Hitachi 7170 automatic analyzer. First, 180 μL of Reagent A was added to 4 μL of specimen, and the temperature was kept constant at 37 ° C. for 5 minutes. At this time, absorbance 1 was measured at a main wavelength of 340 nm and a subwavelength of 570 nm. Furthermore, 60 μL of reagent B was added, and the temperature was kept constant at 37 ° C. for 5 minutes. At this point, absorbance 2 was measured at a main wavelength of 340 nm and a sub wavelength of 570 nm. Find the difference between absorbance 1 and absorbance 2 to find the known HDL ₃ The value of the specimen was converted using the -C concentration control as a standard solution.
[0033]
Measurement was performed using the method of Kurt Widhalm et al. Using polyethylene glycol as a control method. The cholesterol concentration of the supernatant obtained by centrifugal fractionation using this method was determined using T-CHO Reagent · L “Kokusai” manufactured by Kokusai Reagent Co., Ltd. Moreover, the measurement of homogeneous HDL-C using an automatic analyzer was performed using HDL-C reagent KL “Kokusai” manufactured by Kokusai Reagent Co., Ltd. Control method HDL ₂ The -C value is calculated from the HDL-C according to the usage method to the HDL according to the usage method. ₃ Obtained by subtracting -C value. HDL for automated analysis ₂ -C value is HDL determined using reagents A and B from homogeneous HDL-C value ₃ Obtained by subtracting -C value.
[0034]
Fig. 1 shows VLDL obtained by ultracentrifugation [Havel RJ, Eder HA, Bragdon JH: The distribution and chemical composition of ultracentrifugally separated lipoproteinsin human serum., J. Clin. Invest., 34, 1375-1353 (1955)] , LDL, (LDL + HDL ₂ ), HDL ₃ The reaction time course at 340 nm when HDL was measured was shown.
[0035]
As shown in FIG. 1, this method uses VLDL, LDL, (LDL + HDL ₂ ) Almost no reaction to fractions, HDL ₃ It can be seen that only HDL reacts. The total cholesterol concentration in each fraction was VLDL = 99.9 mg / dL, LDL = 199.9 mg / dL, (LDL + HDL ₂ ) = 175.2 + 31.7 mg / dL, HDL ₃ = 28.5 mg / dL and HDL = 84.2 mg / dL.
[0036]
The measurement results by this method are VLDL = 0.7 mg / dL, LDL = 2.9 mg / dL, (LDL + HDL ₂ ) = 4.0 mg / dL, HDL ₃ = 28.0 mg / dL and HDL = 63.2 mg / dL. From this result, HDL ₃ It can be seen that it has high specificity.
[0037]
The measurement results using 22 human sera are shown in Table 1. HDL of this method ₃ The -C value was 0.957 (p <0.05) as a correlation coefficient with the control method. In addition, HDL of this method ₂ As for -C value, the correlation coefficient with the control method was 0.939 (p <0.05).
[0038]
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%
Nonion K-230 (HLB value 17.3) 0.3 w / v%
BC-40TX (HLB value 20) 1.0 w / v%
Calix sulfate [8] allene 2.0 mmol / L
[0039]
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. As specimens, 22 cases of human serum by volunteers were used. The measurement was carried out with a Hitachi 7170 automatic analyzer. In the operation method, first, 180 μL of reagent C was added to 4 μL of the sample, and the temperature was kept constant at 37 ° C. for 5 minutes. At this point, absorbance 1 was measured at a main wavelength of 340 nm and a subwavelength of 570 nm. Further, 60 μL of Reagent D was added, and the temperature was kept constant at 37 ° C. for 5 minutes. At this point, absorbance 2 was measured at a main wavelength of 340 nm and a sub wavelength of 570 nm. Find the difference between absorbance 1 and absorbance 2 to find the known HDL ₃ -The sample value was converted using the control of cholesterol concentration as a standard solution. For the control method, the same operation as in Example 1 was performed. HDL for automated analysis ₂ -C value is HD determined using reagents C and D from homogeneous HDL-C value ₃ Obtained by subtracting -C value.
[0042]
Figure 2 shows VLDL, LDL, (LDL + HDL obtained by ultracentrifugation. ₂ ), HDL ₃ The reaction time course at 340 nm when HDL was measured was shown. From this figure, the present method uses VLDL, LDL, (LDL + HDL ₂ ) Almost no reaction to fractions, HDL ₃ It can be seen that only HDL reacts.
[0043]
The total cholesterol concentration in each fraction was VLDL = 99.9 mg / dL, LDL = 199.9 mg / dL, (LDL + HDL ₂ ) = 175.2 + 31.7 mg / dL, HDL ₃ = 28.5 mg / dL and HDL = 84.2 mg / dL.
[0044]
The measurement results by this method are VLDL = 0.2 mg / dL, LDL = 3.5 mg / dL, (LDL + HDL ₂ ) = 0.2 mg / dL, HDL ₃ = 27.8 mg / dL and HDL = 59.2 mg / dL. From this result, HDL ₃ It can be seen that it has high specificity.
[0045]
The measurement results using 22 human sera are shown in Table 2. HDL of this method ₃ The -C value was 0.952 (p <0.05) as a correlation coefficient with the control method, and the results were in good agreement. Further, the HDL2-C value of this method had a correlation coefficient with the control method of 0.942 (p <0.05).
[0046]
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%
Nonion K-230 (HLB value 17.3) 0.3 w / v%
Brij98 (HLB value 19) 1.6 w / v%
Calix sulfate [8] allene 2.0 mmol / L
[0047]
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 invention's effect】
High density lipoprotein subfraction (HDL) of the present invention ₂ And HDL ₃ ), In particular, cholesterol is measured by HDL in the presence of a nonionic surfactant having an HLB value of 17 or more. ₃ Ingredients, especially cholesterol (HDL ₃ -C) selectively reacts with an enzyme to generate HDL ₃ -Measure the amount of C, total HDL-C to HDL ₃ HDL by subtracting -C amount ₂ -It is a method of measuring HDL-C of the HDL sub-fraction very easily by determining the amount of -C. The present invention uses a general-purpose automatic analyzer, does not perform separation by centrifugation, and does not form turbidity due to complexes or aggregates in a reaction solution, and does not form turbidity in a biological sample, particularly serum. Since the components in the subfraction can be quantified, it is useful for clinical examination and research of diseases related to lipoproteins such as hyperlipidemia.
[Brief description of the drawings]
FIG. 1 shows the reaction time course of each lipoprotein fraction obtained by ultracentrifugation. The metering point is 17 points for 5 minutes and 34 points for 10 minutes.
FIG. 2 shows the reaction time course of each lipoprotein fraction obtained by ultracentrifugation. The metering point is 17 points for 5 minutes and 34 points for 10 minutes.

Claims (6)

Lipoprotein lipase (LPL) and cholesterol esterase (CE) cholesterol esterase selected from, HLB value is 17 to 20 two different nonionic surfactants, hydrazine salts, and used Karikusuare down A method of measuring HDL ₃ -cholesterol, wherein cholesterol is released from the HDL ₃ fraction and the released cholesterol is measured by an enzymatic reaction with cholesterol dehydrogenase or cholesterol oxidase. Two different nonionic surfactants, hydrazine salts, and after mixing the reagent and serum sample containing Karikusuare down, according to liberate cholesterol from HDL ₃ fraction by adding a reagent containing a cholesterol esterase measurement of cholesterol - HDL ₃ according to claim 1. HDL according to claim 1 or 2, characterized in that in a range of pH6~9 the enzyme reaction to measure the enzyme reaction and the free released cholesterol for releasing cholesterol from HDL ₃ fraction 3 _- Cholesterol Measurement method. The enzyme reaction for measuring the released cholesterol is a reaction by cholesterol dehydrogenase and oxidized coenzyme, and the reduced coenzyme which is a product of the reaction is detected spectroscopically. The method for measuring HDL ₃ -cholesterol according to any one of the above. The enzyme reaction for measuring released cholesterol is a reaction by cholesterol oxidase, and hydrogen peroxide as a product of the reaction is measured using peroxidase. HDL ₃ - measurement of cholesterol. Claim HDL, measurement method according to any one of 1 to 4 ₃ - measured amount of cholesterol, total HDL- cholesterol HDL ₃ from (HDL-C) content - subtracting cholesterol _(HDL 3 -C) weight A method for measuring HDL ₂ -cholesterol, characterized by

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