2.7. Determination of Amylase Activity in Blood Serum

The Amylase Assay Kit (Colorimetric) (Abcam, ab102523) was used to quantify α-amylase activity through a two-step reaction. In the protocol, the substrate ethylidene-pNP-G7 was cleaved by α-amylase to produce smaller fragments, which were then modified by α-glucosidase. This resulted in the release of a chromophore with an OD 405 nm. Accordingly, blood serum samples prepared according to manufacturer’s protocol, as well as standards and reaction mix, were added to the wells. Then, the samples were analyzed with a microplate reader every 2–3 min for 30–60 min at OD 405 nm. All measurements were performed in triplicate. Amylase activity in the test samples was calculated as:

where:

B = nitrophenol amount from the standard curve (in nmol);

ΔT = reaction time (T2–T1) (min);

V = pretreated sample volume added to the reaction well (in mL);

D = sample dilution factor.

Amylase = nmol/min/mL = mU/mL; 1 Unit Amylase = amount of amylase that cleaves ethylidene-pNP-G7 to generate 1.0 µmol of nitrophenol per min at pH 7.2 at 25 °C.