A reagent and assay conditions for the determination of the catalytic concentration of alpha-amylase (E.C. 3.2.1.1) in serum with 2-chloro-4-nitrophenyl-alpha-D-maltotrioside as substrate are described. The selected reaction mixture contains 50 mmol/l 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.1 (37 degrees C), 300 mmol/l sodium chloride, 5 mmol/l calcium chloride and 450 mmol/l potassium thiocyanate. The described method is suitable for the measurement of total as well as pancreatic alpha-amylase by including antibodies against the salivary isoenzyme. The method shows the absence of a lag phase period, is sensitive and precise, has a large analytical range and is free from interference by hemoglobin, bilirubin and triglycerides. Comparative studies showed good correlation with other well established methods.

We describe the preparation of a lyophilized material containing purified human pancreatic alpha-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was > 99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic alpha-amylase had a molar mass of 57,500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified alpha-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/l and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20 degrees C. The certified value for alpha-amylase catalytic concentration in the reconstituted reference material is 555 U/l +/- 11 U/l when measured by the specified method at 37 degrees C. The material can be used to verify the comparability of results from laboratories, for intra-laboratory quality control or for calibration of alpha-amylase catalytic concentration measurements.

Six different methods for alpha-amylase determination were compared by assaying human serum samples covering a wide range of alpha-amylase values. All the methods studied use as substrate a maltooligosaccharide with a chromophore group at the reducing end; some are chemically blocked at the nonreducing end. Intermethod comparison by regression and correspondence analyses showed significant differences for two methods. The commutability of 12 commercial control materials containing alpha-amylase was also assessed by the different methods in comparison with human serum specimens containing the pancreatic and salivary isoenzymes. We also studied the behavior of pancreatic and salivary materials prepared in our laboratory. Control materials with alpha-amylase of nonhuman origin were not commutable with the enzyme in human sera and should not be used for intermethod calibration.

The cytosolic isoenzyme of human liver alanine aminotransferase exhibited a progressive change in its chromatographic behavior on DEAE-Sepharose when partially purified preparations were stored for up to 8 days at 4 degrees C. This change was characterized by the appearance of an additional chromatographic variant and was avoided by addition of 2-mercaptoethanol. The experimental evidence presented indicates that the progressive oxidation of free sulfhydryl groups of the enzyme is responsible for the charge modifications and heterogeneity observed.