title: Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates coupled with immunoinhibition.
abstract: 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 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.
authors.affiliation: Department of Molecular and Pharmaceutical Biotechnology, Graduate School of Pharmaceutical Sciences, University of Tokushima, Tokushima, Japan.
authors.forename: Eiki
authors.initials: E
authors.lastname: Maeda
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Kataoka
authors.forename: Shouki
authors.initials: S
authors.lastname: Yatsushiro
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Kajimoto
authors.forename: Mami
authors.initials: M
authors.lastname: Hino
authors.forename: Noritada
authors.initials: N
authors.lastname: Kaji
authors.forename: Manabu
authors.initials: M
authors.lastname: Tokeshi
authors.forename: Mika
authors.initials: M
authors.lastname: Bando
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Kido
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Ishikawa
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Baba
mesh_terms: D000911:Antibodies, Monoclonal; D003124:Colorimetry; D046209:Electrophoresis, Microchip; D005240:Feasibility Studies; D005456:Fluorescent Dyes; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000097:blood / Q000276:immunology; D009844:Oligosaccharides; D010179:Pancreas / Q000201:enzymology*; D010949:Plasma; D010969:Plastics; D011721:Pyrenes; D012463:Saliva / Q000201:enzymology*; D012680:Sensitivity and Specificity; D000516:alpha-Amylases / Q000097:blood* / Q000276:immunology
chemical_list: C098210:1-aminopyrene-3,6,8-trisulfonic acid; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007527:Isoenzymes; D009844:Oligosaccharides; D010969:Plastics; D011721:Pyrenes; C021705:maltooligosaccharides; D000516:alpha-Amylases