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PHOSPHATIDYLINOSITOL QUANTIFICATION METHOD AND QUANTIFICATION KIT

Foreign code F190009758
File No. (S2017-0751-N0),S2019-0091-N0
Posted date May 7, 2019
Country WIPO
International application number 2018JP020027
International publication number WO 2018216776
Date of international filing May 24, 2018
Date of international publication Nov 29, 2018
Priority data
  • P2017-103714 (May 25, 2017) JP
Title PHOSPHATIDYLINOSITOL QUANTIFICATION METHOD AND QUANTIFICATION KIT
Abstract Disclosed is a method for quantifying the phosphatidylinositol in a sample, wherein the method has the following step: (1) a step for causing the action of phospholipase D and inositol dehydrogenase on the sample. Also disclosed is a phosphatidylinositol quantification kit that contains phospholipase D and inositol dehydrogenase.
Outline of related art and contending technology BACKGROUND ART
Phosphatidylinositol(PI) is a kind of phospholipids, fatty acids and glycerol backbone of the inositol phosphate and 2 coupled to a structure. PI is, in mammalian cells in a cell plasma membrane, endoplasmic reticulum and Golgi in a cell containing a film and film is present, in a cell occupy 5-10% of the phospholipid. PI is, in addition to the role of the structure forming the cell membrane, various membrane protein (channel, a transporter, receptor, enzyme or the like) of the activity and adjust the localization, an extremely important role in signal transduction in the cell and is, in recent years has been and increasingly apparent.
Conventional, the quantification of the PI, thin layer chromatography (TLC) /phosphorus has been carried out by the quantification method, the quantitative accuracy and detection sensitivity is low, much time and labor is required. In order to accurately quantify, various coloring method the beam spot on TLC obtained by carefully scraping with a spatula, and the like must be performed for a predetermined amount of phosphorus, and requires skilled techniques.
High performance liquid chromatography (HPLC) was quantified by using the PI, the double bond in the molecule in the acyl chains for detecting the ultraviolet absorber, strongly affected by the kind of the fatty acid chain. That is, the PI, only chain saturated fatty acids with a molecular species is not detected, a polyvalent unsaturated acyl chains in the molecular species having the peak is increased, leads to a consistent amount of liquid.
(MS) mass spectrometry is, PI of the kinds of the fatty acid chains and therefore for each molecular species, as PI is difficult to quantify. For example, a mammalian cell with respect to the PI, two acyl chains 50 by a combination of one or more molecular species is present, each having a different ionization mass analysis are detected as a peak.
PI is a wide variety of important functions of the body cannot be an essential component, the study has been made in the world is very even though, the current methods of analysis also results in very poor PI. Therefore, the blood in the PI associated with the disease and the role and not at all known.
The present inventors have, to this series of phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidyl serine, sphingomyelin, and cardiolipin) and quantified to develop the enzyme (such as Patent Document 1, 2, 3).
In Patent Document 1, phospholipase D, L- amino acid oxidase and peroxidase or not a sample, measuring the fluorescence intensity of the compound that generated by the enzyme assay Dimyristoyl have been reported.
In addition, in Patent Document 2, sphingomyelinase, alkaline phosphatase, peroxidase and choline or not a sample, measuring the fluorescence intensity of the compound that generated by the quantification method of the sphingomyelin enzyme have been reported.
Further, in Patent Document 3, phospholipase D, glycerol, glycerol phosphate oxidase and peroxidase-3-acts on the sample, thereby generating the fluorescence intensity of the quantified by measuring the enzyme cardiolipin have been reported.
However, for the quantified PI of the enzyme has not been developed, by falling through the PI, the overall profile of the phospholipid is not know what problem.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 以下の工程を有する試料中のホスファチジルイノシトールの定量方法:
(1)試料にホスホリパーゼD及びイノシトールデヒドロゲナーゼを作用させる工程。

[請求項2]
 前記工程(1)において、NADHオキシダーゼ及びペルオキシダーゼを更に作用させる、請求項1に記載の方法。

[請求項3]
 更に以下の工程を有する、請求項1又は2に記載の方法:
(2)前記工程(1)で生成する化合物の蛍光強度、吸光度又は発光量を測定し、予め求めた検量線からホスファチジルイノシトールを定量する工程。

[請求項4]
 前記工程(1)において、ホスホリパーゼDを作用させた後に60℃以上での加熱処理を行い、次いでイノシトールデヒドロゲナーゼを作用させる、請求項1~3のいずれか一項に記載の方法。

[請求項5]
 一連の酵素処理を中性領域のpHで行う、請求項1~4のいずれか一項に記載の方法。

[請求項6]
 ホスホリパーゼD及びイノシトールデヒドロゲナーゼを含むホスファチジルイノシトールの定量用キット。

[請求項7]
 NADHオキシダーゼ及びペルオキシダーゼを更に含む、請求項6に記載のキット。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION SHIGA UNIVERSITY OF MEDICAL SCIENCE
  • Inventor
  • MORITA, Shin-ya
IPC(International Patent Classification)

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