Technical Report

Abstract

The determination of -amylase activity using an ethylidene-blocked 4-nitrophenyl-maltoheptaoside (EPS) has been evaluated in five laboratories on eight diffefent analysers at 25 °C, 30 °C and 37 °C. The protecting ethylidene group inhibits hydrolysis at the non-reducing end of the Substrate molecule by the auxiliary enzyme, -glucosidase. The combined reagerit is therefore stable for at least 10 days at 2 -8 °C. HEPES is used, because the molar absorbance of 4-nitrophenpl is indeperident of temperature in the presence of this buffer.Compared with the method using unprotected Substrate 4-riitrophenyl-a-D-maltoheptaoside (4NP-G7), the present method is equal or better with respect to the imprecision, linearity and interlaboratory transferability of results in human and control sera. Since the protected and unprotected Substrates differ in their turnover rate, the new assay yields activities which differ from those of the 4-nitrophenyl-a-/)-maltoheptaoside method. Based on the homogeneous results obtained in method comparisons between EPS and 4-nitrophenyl-a-Dmaltoheptaoside, and in order to maintain the 4-nitrophenyl-a-£>-maltoheptaoside reference values, a conversion factor was derived to eliminate the above differences: activity E ps x 2.50 = activity 4N p. G 7. The temperature and Instrument independence of this relationship was demonstrated in a total of 720 human sera and plasmas.