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Clinical Chemistry 46: 644-649, 2000;
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Right arrow Proteomics and Protein Markers
(Clinical Chemistry. 2000;46:644-649.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Routine {alpha}-Amylase Assay Using Protected 4-Nitrophenyl-1,4-{alpha}-D-maltoheptaoside and a Novel {alpha}-Glucosidase

Klaus Lorentza,1

1 Institut für Klinische Chemie, Medizinische Universität Lübeck, D-23538 Lübeck, Germany.
a Address for correspondence: Hugo-Kauffmann-Strasse 7, D-83209 Prien, Germany. Fax 49-8051-969032.

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 °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 µmol/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 °C with increased sensitivity, fewer interferences, and reduced cost.




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Clin. Chem., January 1, 2001; 47(1): 149 - 149.
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