Measuring key human carbohydrate digestive enzyme activities using high-performance anion-exchange chromatography with pulsed amperometric detection

Carbohydrate digestion in the mammalian gastrointestinal tract is catalyzed by alpha-amylases and alpha-glucosidases to produce monosaccharides for absorption. Inhibition of these enzymes is the major activity of the drugs acarbose and miglitol, which are used to manage diabetes. Furthermore, delaying carbohydrate digestion via inhibition of alpha-amylases and alpha-glucosidases is an effective strategy to blunt blood glucose spikes, a major risk factor for developing metabolic diseases. Here, we present an in vitro protocol developed to accurately and specifically assess the activity of alpha-amylases and alpha-glucosidases, including sucrase, maltase and isomaltase. The assay is especially suitable for measuring inhibition by compounds, drugs and extracts, with minimal interference from impurities or endogenous components, because the substrates and digestive products in the enzyme activity assays are quantified directly by high-performance anionexchange chromatography with pulsed amperometric detection (HPAE-PAD). Multiple enzyme sources can be used, but here we present the protocol using commercially available human alpha-amylase to assess starch hydrolysis with maltoheptaose as the substrate, and with brush border sucrase-isomaltase (with maltase, sucrase and isomaltase activities) derived from differentiated human intestinal Caco-2(/TC7) cells to assess hydrolysis of disaccharides. The wet-lab assay takes similar to 2-5 h depending on the number of samples, and the HPAE-PAD analysis takes 35 min per sample. A full dataset therefore takes 1-3 d and allows detection of subtle changes in enzyme activity with high sensitivity and reliability.