Total and pancreatic amylase measured with 2-chloro-4-nitrophenyl-4-O- β-D-galactopyranosylmaltoside

Abstract

Background: Many different methods have been used to assay amylase activity, using nitrophenylated oligosaccharides as substrate; however, the hydrolysis steps in these methods are complex. Methods: We developed a new continuously monitoring assay for amylase activity in biological fluids, using 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside (GalG2CNP) as the substrate; this assay was used with anti-human salivary amylase monoclonal antibodies for specific determination of the pancreatic isoenzyme. Amylase converted GalG2CNP into β-D-galactopyranosylmaltose and 2-chloro-4- nitrophenol, which was measured at 405 nm. Results: GalG2CNP was cleaved between 2-chloro-4-nitrophenol and β-D-galactopyranosylmaltose and did not undergo transfer reactions. The within-assay CVs (n = 20) for total amylase (T-AMY) and pancreatic amylase (P-AMY) were 0.6-1.6% and 0.5-2.5%, respectively; and day-to-day CVs (n = 10) for T-AMY and P-AMY were 0.8-3.7% and 0.6-4.1%, respectively. TAMY and P-AMY activities in serum or urine obtained by the proposed method correlated well with those detenmined by the 2-chloro-4-nitrophenyl 4-O-β-D-galactopyranosyl-β-maltotetraoside method or the modified IFCC method. Conclusions: This novel assay for T-AMY and P-AMY measures both activities stoichiometrically, directly, and easily, and may be suitable for routine procedures. (C) 2000 American Association for Clinical Chemistry.