TY - JOUR
T1 - Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates couples with immunoinhibition
AU - Maeda, Eiki
AU - Kataoka, Masatoshi
AU - Yatsushiro, Shouki
AU - Kajimoto, Kazuaki
AU - Hino, Mami
AU - Kaji, Noritada
AU - Tokeshi, Manabu
AU - Bando, Mika
AU - Kido, Jun Ichi
AU - Ishikawa, Mitsuru
AU - Shinohara, Yasuo
AU - Baba, Yoshinobu
PY - 2008/5/1
Y1 - 2008/5/1
N2 - 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 relatioship between amylase activity and fluorescence intensity in the range of 5-500 U/L of amylase activity (r2 = 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.
AB - 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 relatioship between amylase activity and fluorescence intensity in the range of 5-500 U/L of amylase activity (r2 = 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.
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U2 - 10.1002/elps.200700688
DO - 10.1002/elps.200700688
M3 - Article
C2 - 18393344
AN - SCOPUS:43449118082
VL - 29
SP - 1902
EP - 1909
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 9
ER -