Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates coupled with immunoinhibition

被引：13

作者：

Maeda, Eiki ^{[2
]}

Kataoka, Masatoshi ^{[1
]}

Yatsushiro, Shouki ^{[1
]}

Kajimoto, Kazuaki ^{[1
]}

Hino, Mami ^{[1
]}

Kaji, Noritada ^{[3
,4
]}

Tokeshi, Manabu ^{[3
,4
]}

Bando, Mika ^{[5
]}

Kido, Jun-Ichi ^{[5
]}

Ishilkawa, Mitsuru ^{[1
]}

Shinohara, Yasuo ^{[2
,6
]}

Baba, Yoshinobu ^{[1
,3
,4
]}

机构：

[1] Natl Inst Adv Ind Sci & Technol, Hlth Technol Res Ctr, Takamatsu, Kagawa 7610395, Japan

[2] Univ Tokushima, Grad Sch Pharmaceut Sci, Dept Mol & Pharmaceut Biotechnol, Tokushima 770, Japan

[3] Nagoya Univ, Grad Sch Engn, Dept Appl Chem, Nagoya, Aichi 4648601, Japan

[4] Nagoya Univ, MEXT Innovat Res Ctr Prevent Med Engn, Nagoya, Aichi 4648601, Japan

[5] Univ Tokushima, Dept Periodontol & Endodontol, Inst Hlth Biosci, Tokushima 770, Japan

[6] Univ Tokushima, Inst Genome Res, Tokushima 770, Japan

来源：

ELECTROPHORESIS | 2008年 / 29卷 / 09期

关键词：

amylase; borate ion; clinical diagnosis; conformation; microchip electrophoresis;

D O I：

10.1002/elps.200700688

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A high-performance determination system for alpha-amylase isoenzyme activities in human plasma involving microchip electrophoresis with a plastic chip was developed. The combination of microchip electrophoresis for substrate and hydrolysate separation and an immunoinhibition method for the differentiation of isoenzyme activities using antihuman salivary amylase (S-AMY) mAb allowed the highly selective determination of amylase isoenzyme (S-AMY and pancreatic amylase (P-AMY)) activities even in a complex matrix such as a crude plasma sample. We used 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled maltohexaose (G6) as a substrate. Amylase in a human plasma sample hydrolyzed APTS-G6 into APTS-maltotriose (G3) and G3, which was measured as the fluorescence intensity of APTS-G3 on microchip electrophoresis. A double logarithm plot revealed a linear relationship between amylase activity and fluorescence intensity in the range of 5-500 U/L of and the LOD was 4.38 U/L. Amylase activities in 13 amylase activity (r(2) = 0.9995 p < 0.01), subjects determined by the present method were compared with the results obtained by conventional methods with nitrophenylated oligosaccharides as substrates, respectively. Good correlations were observed for each method on simple linear regression analysis (both p < 0.01). The reproducibilities of within-days for total amylase and P-AMY were 2.98-6.27 and 3.83-6.39%, respectively, and these between-days were 2.88-5.66 and 3.64-5.63%, respectively. This system enables us to determine amylase isoenzyme activities in human plasma with high sensitivity and accuracy, and thus will be applicable to clinical diagnosis.

引用

页码：1902 / 1909

页数：8