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Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates coupled with immunoinhibition
Author(s) -
Maeda Eiki,
Kataoka Masatoshi,
Yatsushiro Shouki,
Kajimoto Kazuaki,
Hino Mami,
Kaji Noritada,
Tokeshi Manabu,
Bando Mika,
Kido Junichi,
Ishikawa Mitsuru,
Shinohara Yasuo,
Baba Yoshinobu
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700688
Subject(s) - amylase , chemistry , chromatography , substrate (aquarium) , quantitative analysis (chemistry) , biochemistry , enzyme , biology , ecology
A high‐performance determination system for α‐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 amylase activity ( r 2 = 0.9995, p <0.01), and the LOD was 4.38 U/L. Amylase activities in 13 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.