JP2598233B2 - Determination of cholesterol in high density lipoprotein - Google Patents
Determination of cholesterol in high density lipoproteinInfo
- Publication number
- JP2598233B2 JP2598233B2 JP20682994A JP20682994A JP2598233B2 JP 2598233 B2 JP2598233 B2 JP 2598233B2 JP 20682994 A JP20682994 A JP 20682994A JP 20682994 A JP20682994 A JP 20682994A JP 2598233 B2 JP2598233 B2 JP 2598233B2
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- JP
- Japan
- Prior art keywords
- cholesterol
- density lipoprotein
- hdl
- present
- determination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- Investigating Or Analysing Materials By Optical Means (AREA)
Description
【0001】[0001]
【産業上の利用分野】本発明は、高密度リポ蛋白(HD
L)に含まれるコレステロール(以下、HDLコレステ
ロールという)の定量法に関する。The present invention relates to a high-density lipoprotein (HD)
The present invention relates to a method for determining cholesterol contained in L) (hereinafter referred to as HDL cholesterol).
【0002】[0002]
【従来の技術】HDLは、動脈壁を含めた各組織からコ
レステロールを受け取るため細胞内に蓄積したコレステ
ロールの除去作用に関係し、冠動脈硬化症をはじめとす
る各種動脈硬化症の危険予防因子であり、その血中レベ
ルは動脈硬化性疾患の発症予知に有用な指針となること
が知られている。従来のHDLコレステロールの定量法
は、大きく分けて分画操作とコレステロール定量操作の
2段階からなる。分画操作法には、超遠心法、免疫化学
的方法、電気泳動法、沈殿法などがある。超遠心法を用
いる場合には、分離用超遠心器で比重の差によってHD
Lを分離し、そのコレステロール量を測定する。しかし
ながら、定量性、簡便性、経済性などの面で欠点があ
る。免疫化学的方法には、免疫電気泳動法、一元免疫拡
散法(SRID法)、オクタロニー法などがあるが、こ
れらの方法を用いる場合にはアポ蛋白を認識しており、
正確にはリポ蛋白を認識していないという問題がある。
電気泳動法を用いる場合には、セルロースアセテート膜
やアガロースゲルなどを支持体として分離し、酵素法に
よりコレステロールを定量する。この方法は、簡便性、
経済性などの面で問題がある。沈殿法を用いる場合に
は、低密度リポ蛋白(LDL)、超低密度リポ蛋白(V
LDL)およびカイロミクロン(CM)の表面に存在す
るアポ蛋白Bにポリエチレングリコール、ヘパリン、リ
ンタングステン酸、デキストラン硫酸などのポリアニオ
ンと2価の陽イオンを結合させ、不溶性沈殿物を形成さ
せ、これを遠心分離操作によって除去し、上清中のHD
Lコレステロールを定量する(臨床検査法提要、第29
版、金井泉著、金原出版、471頁、1983年)。こ
の方法は最も簡便であるが、遠心分離器による遠心分離
操作を行うため、多数検体処理、迅速測定および臨床検
査の分野で多く使用されている自動分析装置には不向き
である。2. Description of the Related Art HDL is related to the action of removing cholesterol accumulated in cells in order to receive cholesterol from various tissues including the arterial wall, and is a risk preventive factor for various arteriosclerosis including coronary atherosclerosis. It is known that its blood level can be a useful guide for predicting the onset of arteriosclerotic disease. The conventional method for quantifying HDL cholesterol is roughly divided into two steps, a fractionation operation and a cholesterol quantification operation. The fractionation method includes ultracentrifugation, immunochemical methods, electrophoresis, and precipitation. When using the ultracentrifugation method, the difference in specific gravity of the ultracentrifuge for separation
L is separated and its cholesterol level is measured. However, there are drawbacks in terms of quantitativeness, simplicity, economy, and the like. Examples of immunochemical methods include immunoelectrophoresis, one-way immunodiffusion (SRID), and Ouchterlony. When these methods are used, the apoprotein is recognized.
There is a problem that lipoproteins are not accurately recognized.
When the electrophoresis method is used, separation is performed using a cellulose acetate membrane, agarose gel or the like as a support, and cholesterol is quantified by an enzymatic method. This method is simple,
There is a problem in terms of economy. When the precipitation method is used, low density lipoprotein (LDL), very low density lipoprotein (V
LDL) and apoprotein B present on the surface of chylomicron (CM) are combined with a polyanion such as polyethylene glycol, heparin, phosphotungstic acid, dextran sulfate and a divalent cation to form an insoluble precipitate, Removed by centrifugation
Quantify L cholesterol (Procedure of clinical test method, No. 29
Edition, Izumi Kanai, Kanehara Publishing, p. 471, 1983). Although this method is the simplest, it is not suitable for an automatic analyzer that is frequently used in the fields of processing a large number of samples, rapid measurement, and clinical tests because of performing centrifugation operation using a centrifuge.
【0003】[0003]
【発明が解決しようとする課題】本発明の目的は、煩雑
な分画分離操作の不要な簡便なHDLコレステロールの
定量法を提供することにある。SUMMARY OF THE INVENTION An object of the present invention is to provide a simple method for quantifying HDL cholesterol which does not require complicated fractionation and separation operations.
【0004】[0004]
【課題を解決するための手段】本発明者らは、リポ蛋白
含有試料とジメチル−β−シクロデキストリン(DM−
β−CyD)とを水性液中適当な条件下で混合すること
によりDM−β−CyDと試料中のHDLとが特異的に
作用して濁りを形成し、このときの該混合液の濁度がH
DLコレステロール濃度とよい相関性を示すことを見い
出し、本発明に至った。Means for Solving the Problems The present inventors have prepared a sample containing lipoprotein and dimethyl-β-cyclodextrin (DM-
and β-CyD) in an aqueous liquid under appropriate conditions, whereby DM-β-CyD and HDL in the sample specifically act to form turbidity. Is H
The present inventors have found that they show a good correlation with the DL cholesterol concentration, leading to the present invention.
【0005】本発明は、HDLとDM−β−CyDとを
水性液中で混合して濁りを生成させ、該混合液の濁度を
測定することを特徴とするHDLコレステロールの定量
法に関する。次に、本発明の定量法について説明する。
本発明を実施するに際しては、まず、DM−β−CyD
を適当な緩衝液、例えば50mM Tris−HCl緩
衝液(pH7.4)に溶解して試薬を調製する。DM−
β−CyDの濃度は、試料と共存した場合に例えば10
mM未満、好ましくは5mMの濃度になるように調整す
る。次いで、試料そのものもしくは必要に応じて水ある
いは生理食塩水で希釈した試料を上記試薬で、10〜5
0℃、好ましくは30〜40℃で1〜30分間処理す
る。その後、混合液の濁度を300〜500nm、好ま
しくは330〜400nm、例えば340nmでの吸光
度により測定し、吸光度の値からHDLコレステロール
量を算出する。[0005] The present invention relates to a method for determining HDL cholesterol, comprising mixing HDL and DM-β-CyD in an aqueous liquid to form turbidity, and measuring the turbidity of the mixture. Next, the quantification method of the present invention will be described.
In practicing the present invention, first, DM-β-CyD
Is dissolved in an appropriate buffer, for example, a 50 mM Tris-HCl buffer (pH 7.4) to prepare a reagent. DM-
The concentration of β-CyD is, for example, 10 when coexisting with the sample.
Adjust to a concentration of less than mM, preferably 5 mM. Then, the sample itself or, if necessary, a sample diluted with water or physiological saline is used for 10 to 5
The treatment is carried out at 0 ° C, preferably 30 to 40 ° C for 1 to 30 minutes. Then, the turbidity of the mixture is measured by the absorbance at 300 to 500 nm, preferably 330 to 400 nm, for example, 340 nm, and the HDL cholesterol amount is calculated from the absorbance value.
【0006】試料として血清から超遠心により分画され
たHDL、LDL、VLDLおよびCMの各フラクショ
ンを使用し、上記定量法に従って吸光度を測定した。5
mMの各種糖化合物含有試薬を調製して測定した結果を
図1に示す。(図中、CyDはシクロデキストリンを表
し、HEはヒドロキシエチルを表し、HPはヒドロキシ
プロピルを表し、DMはジメチルを表し、TMはトリメ
チルを表し、CMはカルボキシメチルを表し、Gluは
グルコシルを表し、Malはマルトシルを表し、PMは
パーシャリーメチルを表す) 図1に示すように、糖化合物としてDM−β−CyDを
用いた場合にHDLの存在下でのみ濁りが形成されるこ
とが確認された。Using HDL, LDL, VLDL and CM fractions fractionated from serum by ultracentrifugation as samples, the absorbance was measured according to the above-mentioned quantitative method. 5
FIG. 1 shows the results obtained by preparing and measuring mM various sugar compound-containing reagents. (In the figure, CyD represents cyclodextrin, HE represents hydroxyethyl, HP represents hydroxypropyl, DM represents dimethyl, TM represents trimethyl, CM represents carboxymethyl, Glu represents glucosyl, (Mal represents maltosyl and PM represents partially methyl). As shown in FIG. 1, it was confirmed that turbidity was formed only in the presence of HDL when DM-β-CyD was used as the sugar compound. .
【0007】次に、実施例によって本発明の態様を説明
する。Next, embodiments of the present invention will be described with reference to examples.
【0008】[0008]
実施例1 DM−β−CyD(5mM)および50mM Tris
−HCl緩衝液(pH7.4)からなる試薬を調製し
た。血清から超遠心で分画し各種濃度に希釈したHDL
のサンプル50μl(HDLコレステロール濃度は酵素
法により別途測定)を上記試薬3mlで37℃で15分
間処理し、得られた溶液の吸光度を測定した。Example 1 DM-β-CyD (5 mM) and 50 mM Tris
A reagent consisting of -HCl buffer (pH 7.4) was prepared. HDL fractionated from serum by ultracentrifugation and diluted to various concentrations
Was treated with 3 ml of the above-mentioned reagent at 37 ° C. for 15 minutes, and the absorbance of the obtained solution was measured.
【0009】その結果を第2図に示す。第2図は、HD
Lコレステロール濃度と吸光度との相関関係を示すもの
で、HDLコレステロール濃度は吸光度とよい相関を示
した。FIG. 2 shows the results. Figure 2 shows the HD
The figure shows the correlation between the L cholesterol concentration and the absorbance, and the HDL cholesterol concentration showed a good correlation with the absorbance.
【0010】実施例2 血清から超遠心で分画し各種濃度に希釈したHDLのサ
ンプルの代わりに血清サンプルを用いる以外は実施例1
と同様の操作を行って吸光度を測定し、第1図を基準に
血清サンプル中のHDLコレステロール濃度を求めた。
対照法として、リンタングステン酸−デキストラン硫酸
−Mg沈殿法〔デタミナーHDL(協和メデックス社
製)で沈殿〕(臨床化学、初版、荻三男著、医典社、1
10頁、1987年)を用いて、血清サンプル中のHD
Lコレステロール濃度を求めた。Example 2 Example 1 was repeated except that serum samples were used instead of HDL samples fractionated from serum by ultracentrifugation and diluted to various concentrations.
The absorbance was measured in the same manner as described above, and the HDL cholesterol concentration in the serum sample was determined based on FIG.
As a control method, phosphotungstic acid-dextran sulfate-Mg precipitation method (precipitated by Determiner HDL (manufactured by Kyowa Medex)) (Clinical Chemistry, first edition, written by Ogumi Mitsuo, Ikkisha,
10, 1987) using HD in serum samples.
L cholesterol levels were determined.
【0011】その結果、本発明の方法による結果は沈殿
法による結果と良好な相関を示した〔本発明の方法での
平均値(y)=49.8mg/dl、沈殿法での平均値
(x)=48.1mg/dl、相関係数r=0.850
6、回帰式y=1.006x+1.406(n=6
2)〕。As a result, the results obtained by the method of the present invention showed a good correlation with the results obtained by the precipitation method (mean value (y) in the method of the present invention = 49.8 mg / dl, mean value in the precipitation method ( x) = 48.1 mg / dl, correlation coefficient r = 0.850
6, regression equation y = 1.006x + 1.406 (n = 6
2)].
【0012】[0012]
【発明の効果】本発明により、煩雑な分画分離操作の不
要な簡便なHDLコレステロールの定量法が提供され
る。Industrial Applicability According to the present invention, a simple method for quantifying HDL cholesterol which does not require complicated fractionation and separation operations is provided.
【図1】各リポ蛋白分画と各種CyDとを共存させたと
きの吸光度を示すものである。FIG. 1 shows the absorbance when each lipoprotein fraction and various CyDs coexist.
【図2】HDLコレステロール濃度と本発明の方法によ
り測定された吸光度との相関関係を示すものである。FIG. 2 shows a correlation between HDL cholesterol concentration and absorbance measured by the method of the present invention.
Claims (1)
デキストリンとを水性液中で混合して濁りを生成させ、
該混合液の濁度を測定することを特徴とする高密度リポ
蛋白中のコレステロールの定量法。1. A high-density lipoprotein and dimethyl-β-cyclodextrin are mixed in an aqueous liquid to form turbidity.
A method for determining cholesterol in high-density lipoprotein, which comprises measuring the turbidity of the mixture.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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JP20682994A JP2598233B2 (en) | 1994-08-31 | 1994-08-31 | Determination of cholesterol in high density lipoprotein |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP20682994A JP2598233B2 (en) | 1994-08-31 | 1994-08-31 | Determination of cholesterol in high density lipoprotein |
Publications (2)
Publication Number | Publication Date |
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JPH0868795A JPH0868795A (en) | 1996-03-12 |
JP2598233B2 true JP2598233B2 (en) | 1997-04-09 |
Family
ID=16529769
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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JP20682994A Expired - Lifetime JP2598233B2 (en) | 1994-08-31 | 1994-08-31 | Determination of cholesterol in high density lipoprotein |
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JP (1) | JP2598233B2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2165947T3 (en) | 1995-07-21 | 2002-04-01 | Wako Pure Chem Ind Ltd | METHOD FOR MEASURING THE AMOUNT OF A CONSTITUENT CONTAINED IN A SPECIFIC LIPOPROTEIN. |
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1994
- 1994-08-31 JP JP20682994A patent/JP2598233B2/en not_active Expired - Lifetime
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JPH0868795A (en) | 1996-03-12 |
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