WO2004087945A1 - Methode de determination du contenu en triglycerides d'une lipoproteine de faible densite - Google Patents

Methode de determination du contenu en triglycerides d'une lipoproteine de faible densite Download PDF

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Publication number
WO2004087945A1
WO2004087945A1 PCT/JP2004/004394 JP2004004394W WO2004087945A1 WO 2004087945 A1 WO2004087945 A1 WO 2004087945A1 JP 2004004394 W JP2004004394 W JP 2004004394W WO 2004087945 A1 WO2004087945 A1 WO 2004087945A1
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Prior art keywords
low
density lipoprotein
ldl
lipoproteins
triglyceride
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PCT/JP2004/004394
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English (en)
Japanese (ja)
Inventor
Hiroshi Matsui
Junko Hirashima
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Denka Seiken Co., Ltd.
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Application filed by Denka Seiken Co., Ltd. filed Critical Denka Seiken Co., Ltd.
Publication of WO2004087945A1 publication Critical patent/WO2004087945A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention relates to a method for quantifying tridaliceride in low-density lipoprotein in a test sample.
  • LDL Low-density lipoprotein
  • Non-patent document 1 As the fractionation operation, there are an ultracentrifugation method, a precipitation method, a method utilizing an immunoreaction, and the like. In any case, the sample must be subjected to a centrifugal treatment or a filter treatment. There is a method for quantification using the LDL obtained by fractionation as a sample, and quantification using an automatic analyzer and TG reagent used in the clinical laboratory.However, it is simple and economical. It is a method that is difficult to spread in terms of sex.
  • Non-patent document 1 Non-patent document 1
  • An object of the present invention is to provide a method for simply quantifying triglyceride in LDL in a test sample. Specifically, an object of the present invention is to provide a method for eliminating triglycerides in lipoproteins other than LDL in the first step and quantifying triglycerides in LDL in the second step.
  • CM chylomicron
  • VLDL high density lipoprotein
  • HDL high density lipoprotein
  • the present invention comprises a first step of eliminating triglycerides in lipoproteins other than LDL in a test sample, and a second step of quantifying triglycerides in LDL remaining in the test sample.
  • the method of the present invention comprises a first step and a second step.
  • triglycerides in lipoproteins other than LDL in the test sample are eliminated, and in the subsequent second step, residual triglycerides in the test sample are removed.
  • Lipoproteins other than LDL include HDL, VL DL and CM. Since triglycerides in HDL, VLDL and CM are eliminated in the first step, the triglycerides quantified in the second step are mainly triglycerides in LDL in the test sample.
  • test sample used in the method of the present invention may be any sample that may contain lipoproteins such as HDL, LDL, ⁇ 1 ⁇ 01 ⁇ and ⁇ M, such as blood and serum. Examples thereof include body fluids such as plasma and dilutions thereof, but are not limited thereto.
  • lipoproteins such as HDL, LDL, ⁇ 1 ⁇ 01 ⁇ and ⁇ M
  • body fluids such as plasma and dilutions thereof, but are not limited thereto.
  • “Eliminating triglycerides” in the first step means degrading tridaliceride and preventing its degradation product from being detected in the next second step.
  • the lipoprotein lipase concentration in the reaction solution of the first step is 50 to 500 I UZm
  • the concentration of hydrogen peroxide in the case of decomposing hydrogen peroxide into water and oxygen is preferably about 40 to OU / mL.
  • the concentration of peroxidase is preferably 0.4 to 1.0 UZmL, and the concentration of the phenol- or aniline-based hydrogen donor compound to be added at this time is different from that of the phenol- or aniline-based hydrogen donor compound. Therefore, it is preferably from 0.4 to 0.8 mmo1 / L.
  • Preferred examples of the surfactant used in the first step that acts on lipoproteins other than LDL include a polyalkylene oxide derivative having a ⁇ [8 value of 13 or more and 14 or less.
  • the derivatives include higher alcohol condensates, higher fatty acid condensates, higher fatty acid amide condensates, higher alkylamine condensates, higher alkyl mercaptan condensates, and alkylphenol condensates.
  • the method for calculating the HLB of a surfactant is well known, and is described in, for example, “New Surfactant”, written by Hiroshi Horiuchi, 1986, Sankyo Shuppan.
  • Preferred examples of the polyalkylene oxide derivative having an HLB value of 13 or more include polyoxyethylene lauryl ether, polyoxyethylene acetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, and polyoxyethylene.
  • Compounds having an HLB value of 13 or more and 14 or less such as ethylene octyl phenyl ether, polyoxyethylene nonyl phenyl ether, and polyoxyethylene benzyl phenyl ether can be mentioned, but are not limited thereto.
  • the concentration of the surfactant used in the first step is preferably from about 0.1 to about 0.1 O gZL, and more preferably from about 0.5 to 5. O gZL.
  • the first step is preferably carried out in a buffer having a pH of 5 to 9, and a buffer containing amine, such as Tris, triethanolamine or Dat's buffer, is preferable.
  • a buffer containing amine such as Tris, triethanolamine or Dat's buffer
  • Biss-Tris, PIPES, MOPS0, BES, HEPES and POPSO which are good buffer solutions are preferable, and the concentration of the buffer solution is preferably about 10 to 500 mM.
  • a divalent metal ion may be included in the reaction solution in order to suppress the reaction with LDL and further enhance the elimination of other lipoproteins.
  • the divalent metal ion copper ion, iron ion and magnesium ion can be used, and magnesium ion is particularly preferable.
  • the concentration of the divalent metal ion is preferably about 5 to 20 OmM.
  • the reaction temperature in the first step is suitably about 30 to 40 ° C, and most preferably 37 ° C.
  • the reaction time may be about 2 to 10 minutes.
  • the first step is performed in the presence of albumin.
  • albumin By performing the reaction in the lower stage, debris resulting from the reaction in the first step is adsorbed on albumin, and it is possible to prevent the debris from interfering with the reaction in the second step.
  • the albumin is not particularly limited as long as it is albumin, and commercially available albumin such as serum albumin can be suitably used.
  • the origin of albumin is not limited at all, and it may be any animal such as human, mouse, bush, dog, etc., and preferably use widely used serum albumin. Can be.
  • the concentration of the albumin in the reaction solution in the first step is preferably 0.1 to 5.0 g ZdL, more preferably 0.3 to 3.0 Og / dL.
  • enzymes that act on lipoproteins other than LDL consisting of surfactants, lipoprotein kinases, glycerol quinases and glycerol to degrade these lipoproteins, and hydrogen peroxide such as catalase Enzymes for eliminating the enzymes may be added simultaneously or separately.
  • a reagent containing all of these reagents is prepared and mixed with a test sample.
  • triglyceride in residual LDL in the test sample is quantified.
  • at least a surfactant that acts on LDL is added, and peroxidation caused by the action of lipoprotein lipase, glycerol kinase and glycerol triphosphate oxidase remaining in the reaction system in the first step This can be done by quantifying hydrogen.
  • the surfactant that acts on at least LDL may be a surfactant that selectively acts only on LDL, or may be a surfactant that acts on all lipoproteins.
  • Preferred examples of the surfactant that acts on all lipoproteins include polyalkylene oxide derivatives having an HLB value of 11 or more and less than 13 and preferably 12 or more and less than 13.
  • Examples of derivatives include higher alcohol condensates, higher fatty acid condensates, higher fatty acid amide condensates, higher alkylamine condensates, higher alkyl mercapnone condensates, and alkylphenol condensates.
  • Preferred examples of the polyalkylene oxide derivative having an HLB value of 11 or more and less than 13 include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, and polyoxyethylene higher alcohol ether.
  • Compounds with an HLB value of 11 or more and less than 13 such as oxyethylene octyl phenyl ether and polyoxyethylene nonyl phenyl ether
  • the present invention is not limited thereto.
  • the concentration of the surfactant used in the second step is preferably about 0.1'00 gZL, more preferably about 1 to 50 gZL.
  • the produced hydrogen peroxide can be quantified by oxidative condensation of 4-aminoantipyrine and a phenolic or aniline-based hydrogen donor compound by the action of peroxidase in the presence of hydrogen peroxide, and the resulting dye can be quantified.
  • the phenol-based or aniline-based hydrogen donor compounds include 5-dimethoxyaniline (HDAOS), N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine (EMSE), and the like.
  • the LDL fraction was collected by ultracentrifugation, and the concentration of triglyceride in LDL was determined using a commercially available triglyceride reagent (using FG-TG (S) reagent manufactured by Denrikseiken Co., Ltd.).
  • the ultracentrifugation method was carried out according to the method described in “Shinsei Kagaku Jikkenshosho 4 Lipid I Neutral fat and lipoprotein”, Tokyo Chemical Dojin Press.
  • triglycerides in LDL can be easily quantified without performing a centrifugation operation or the like.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
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  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne une méthode de détermination du contenu en triglycérides d'une lipoprotéine de faible densité contenue dans un échantillon d'essai, se caractérisant en ce qu'elle consiste à éliminer les triglycérides des lipoprotéines autres que la lipoprotéine contenue dans l'échantillon d'essai, puis à mesurer le contenu en triglycérides de la lipoprotéine de faible densité restant dans l'échantillon d'essai.
PCT/JP2004/004394 2003-03-28 2004-03-29 Methode de determination du contenu en triglycerides d'une lipoproteine de faible densite WO2004087945A1 (fr)

Applications Claiming Priority (2)

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JP2003-091651 2003-03-28
JP2003091651A JP2006180707A (ja) 2003-03-28 2003-03-28 低密度リポ蛋白中のトリグリセリドの定量方法

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WO2004087945A1 true WO2004087945A1 (fr) 2004-10-14

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007052646A1 (fr) 2005-10-31 2007-05-10 Kyowa Medex Co., Ltd. Procede de mesure de triglyceride dans une lipoproteine de basse densite et kit de mesure
EP1739189A4 (fr) * 2004-03-31 2009-07-08 Denka Seiken Kk Procédé de multiquantification pour le cholestérol de lipoprotéine de faible densité
WO2013068572A2 (fr) 2011-11-11 2013-05-16 Axis-Shield Asa Procédé de dosage d'échantillon de sang
US8518659B2 (en) 2009-10-14 2013-08-27 Natonal University Corporation Hamamatsu University School of Medicine Method for determination of degree of risk of onset of high-functioning autism

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013157642A1 (fr) 2012-04-20 2013-10-24 デンカ生研株式会社 Procédé pour l'élimination de triglycérides dans des lipoprotéines autres que les lipoprotéines de basse densité
JP6967857B2 (ja) * 2017-02-03 2021-11-17 デンカ株式会社 循環器疾患等のリスクの判断を補助する方法
WO2019175962A1 (fr) * 2018-03-13 2019-09-19 デンカ生研株式会社 Procédé d'aide à la détermination du risque de maladie cardiovasculaire ou similaire

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS57137858A (en) * 1981-02-20 1982-08-25 Wako Pure Chem Ind Ltd Quantitative measuring method for triglyceride
WO1997045553A1 (fr) * 1996-05-29 1997-12-04 Daiichi Pure Chemicals Co., Ltd. Procede de determination quantitative des cholesterols des lipoproteines de basse densite
WO1998047005A1 (fr) * 1997-04-14 1998-10-22 Denka Seiken Co., Ltd. Procede pour determiner le taux de cholesterol present dans des lipoproteines de basse densite
WO1999031512A1 (fr) * 1997-12-17 1999-06-24 Heinrich Wieland Determination de la presence de triglyceride contenu dans une lipoproteine
WO2000043537A1 (fr) * 1999-01-20 2000-07-27 Kyowa Medex Co., Ltd. Methode pour mesurer un triglyceride dans une lipoproteine
WO2000060112A1 (fr) * 1999-04-01 2000-10-12 Masahiko Okada Procede de quantification de triglycerides contenus dans des lipoproteines a tres faible densite et dans des lipoproteines a densite moyenne
JP2001252096A (ja) * 2000-03-13 2001-09-18 Internatl Reagents Corp トリグリセリド測定試薬

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS57137858A (en) * 1981-02-20 1982-08-25 Wako Pure Chem Ind Ltd Quantitative measuring method for triglyceride
WO1997045553A1 (fr) * 1996-05-29 1997-12-04 Daiichi Pure Chemicals Co., Ltd. Procede de determination quantitative des cholesterols des lipoproteines de basse densite
WO1998047005A1 (fr) * 1997-04-14 1998-10-22 Denka Seiken Co., Ltd. Procede pour determiner le taux de cholesterol present dans des lipoproteines de basse densite
WO1999031512A1 (fr) * 1997-12-17 1999-06-24 Heinrich Wieland Determination de la presence de triglyceride contenu dans une lipoproteine
WO2000043537A1 (fr) * 1999-01-20 2000-07-27 Kyowa Medex Co., Ltd. Methode pour mesurer un triglyceride dans une lipoproteine
WO2000060112A1 (fr) * 1999-04-01 2000-10-12 Masahiko Okada Procede de quantification de triglycerides contenus dans des lipoproteines a tres faible densite et dans des lipoproteines a densite moyenne
JP2001252096A (ja) * 2000-03-13 2001-09-18 Internatl Reagents Corp トリグリセリド測定試薬

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIRANO T. ET AL.: "A novel and simple method for qualification of small, dense LDL", JOURNAL OF LIPID RESEARCH, vol. 44, November 2003 (2003-11-01), pages 2193 - 2201, XP002986861 *
TSUSHIMA MOTOO: "Shinkekkan kiken inshi no shinten, cholesterol igai no shishitsu", JOURNAL OF BLOOD PRESSURE, vol. 10, no. 8, August 2003 (2003-08-01), pages 857 - 862, XP002986862 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1739189A4 (fr) * 2004-03-31 2009-07-08 Denka Seiken Kk Procédé de multiquantification pour le cholestérol de lipoprotéine de faible densité
US7811779B2 (en) 2004-03-31 2010-10-12 Denka Seiken Co., Ltd. Method of multiquantification for cholesterol of low-density lipoprotein
WO2007052646A1 (fr) 2005-10-31 2007-05-10 Kyowa Medex Co., Ltd. Procede de mesure de triglyceride dans une lipoproteine de basse densite et kit de mesure
JP5191236B2 (ja) * 2005-10-31 2013-05-08 協和メデックス株式会社 低密度リポ蛋白中トリグリセリドの測定方法及び測定用キット
US9360432B2 (en) 2005-10-31 2016-06-07 Kyowa Medex Co., Ltd. Method for measuring triglyceride in low-density lipoprotein
US8518659B2 (en) 2009-10-14 2013-08-27 Natonal University Corporation Hamamatsu University School of Medicine Method for determination of degree of risk of onset of high-functioning autism
WO2013068572A2 (fr) 2011-11-11 2013-05-16 Axis-Shield Asa Procédé de dosage d'échantillon de sang

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