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

Eiki Maeda, Masatoshi Kataoka, Shouki Yatsushiro, Kazuaki Kajimoto, Mami Hino, Noritada Kaji, Manabu Tokeshi, Mika Bando, Jun Ichi Kido, Mitsuru Ishikawa, Yasuo Shinohara, Yoshinobu Baba

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1902-1909
Number of pages8
JournalELECTROPHORESIS
Volume29
Issue number9
DOIs
Publication statusPublished - May 1 2008
Externally publishedYes

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Clinical Biochemistry

Cite this