Routine alpha-amylase assay using protected 4-nitrophenyl-1, 4-alpha-D-maltoheptaoside and a novel alpha-glucosidase

Background: In contrast to numerous methods for measuring alpha-amylase activity, the approved IFCC reference method offers an invariable time-independent constant product pattern, thus avoiding possibly changing stoichiometric calculations. However, reference methods do not lend themselves to routine use, so that such methods need to be modified.

Methods: Ethylidene-blocked 4-nitrophenylmaltoheptaoside (EPS-G7) is degraded to glucose and 4-nitrophenol in a coupled assay with a Bacterial alpha-glucosidase under the following measurement conditions: 3.5 mmol/L EPS-G7, 7.1 kU/L alpha-glucosidase, 70 mmol/L sodium chloride, 1 mmol/L calcium chloride, 50 mmol/L HEPES, pH 7.15, at 37 degrees C. The increase of absorbance is continuously monitored for 3 min at 405 nm after a lag phase of 2 min.

Results: Catalytic concentrations up to 15-fold higher than the upper reference limit can be determined without dilution. Precision studies in manual performance show CVs of 1.4-2. 6% (within-run) and 1.9-2.8% (day-to-day). There was no interference from 100 mmol/L glucose, 30 mmol/L triacylglycerols, 610 micromol/L bilirubin, and 2.95 g/L hemoglobin. The method closely correlates with other chromogenic assays. The preliminary 0.95 reference interval for adults, not dependent on age and sex, is 33.6-96.2 U/L.

Conclusion: The procedure is a robust adaptation of the reference method to routine use at 37 degrees C with increased sensitivity, fewer interferences, and reduced cost.