一种使用燕麦幼苗多胺氧化酶对红细胞中的多胺进行灵敏的比色测定法。

A sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase.

作者信息

Takagi Kenji, Tatsumi Yasuaki, Kitaichi Kiyoyuki, Iwase Mitsunori, Shibata Eiji, Nakao Makoto, Matsumoto Takatoshi, Takagi Kenzo, Hasegawa Takaaki

机构信息

Department of Medical Technology, Nagoya University School of Health Sciences, 1-1-20 Daikominami, Nagoya 461-8673, Higashi, Japan.

出版信息

Clin Chim Acta. 2004 Feb;340(1-2):219-27. doi: 10.1016/j.cccn.2003.11.002.

DOI:10.1016/j.cccn.2003.11.002

PMID:14734216

Abstract

BACKGROUND

Most of the polyamines circulating in blood are spermidine (Spd) and spermine (Spm) with only trace amounts of putrescine (Put), and they are mainly localized in erythrocytes. We developed a simple and sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase (OSPO). The method is based on the unique substrate specificity of OSPO, which is active toward Spd and Spm, but not toward diamines such as Put and cadaverine and monoamines such as histamine.

METHODS

The polyamines, which were purified from packed erythrocytes by weak cation-exchange chromatography, were incubated with OSPO at 37 degrees C for 15 min. In the presence of the H(2)O(2) produced by this polyamine oxidase reaction and a new chromogen, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine sodium salt (DA-64), peroxidase (POD) catalyzes the formation of N-[4-[[4-(dimethylamino)phenyl]imino]-2,5-cyclohexadien-1-ylidene]-N-methylmethanaminium chloride (Bindschedler's Green) having an absorption maximum at 727 nm.

RESULTS

The detection limit was 0.2 microM/l for packed erythrocytes. The within-run and between-run precisions (coefficient of variation, CVs) were 5.6-15.2% and 6.5-16.4%, respectively. Analytical recoveries were 93.3-97.4%. Polyamine values obtained by this assay correlated well with those obtained by an HPLC (y=0.948x + 1.912; r=0.944; n=46).

CONCLUSIONS

This colorimetric assay is simple and highly sensitive and practical for clinical use.

摘要

背景

血液中循环的多胺大多是亚精胺（Spd）和精胺（Spm），腐胺（Put）含量仅痕量，且它们主要定位于红细胞中。我们利用燕麦幼苗多胺氧化酶（OSPO）开发了一种用于检测红细胞中多胺的简单且灵敏的比色测定法。该方法基于OSPO独特的底物特异性，其对Spd和Spm有活性，但对二胺如Put和尸胺以及单胺如组胺无活性。

方法

通过弱阳离子交换色谱从压积红细胞中纯化得到的多胺，在37℃下与OSPO孵育15分钟。在该多胺氧化酶反应产生的H₂O₂和一种新型显色剂N -（羧甲基氨基羰基）-4,4'-双（二甲基氨基）-二苯胺钠盐（DA - 64）存在的情况下，过氧化物酶（POD）催化形成在727nm处有最大吸收的N - [4 - [[4 -（二甲基氨基）苯基]亚氨基]-2,5 - 环己二烯 - 1 - 亚基]-N - 甲基甲铵氯化物（宾氏绿）。

结果

压积红细胞的检测限为0.2μM/l。批内和批间精密度（变异系数，CV）分别为5.6 - 15.2%和6.5 - 16.4%。分析回收率为93.3 - 97.4%。通过该测定法获得的多胺值与通过高效液相色谱法获得的值相关性良好（y = 0.948x + 1.912；r = 0.944；n = 46）。