Department of Nutrition, Dietetics and Food, School of Clinical Sciences at Monash Health, Faculty of Medicine, Nursing and Health Sciences, Monash University, BASE Facility, Notting Hill, Victoria, Australia.
Nat Protoc. 2022 Dec;17(12):2882-2919. doi: 10.1038/s41596-022-00736-0. Epub 2022 Sep 30.
Carbohydrate digestion in the mammalian gastrointestinal tract is catalyzed by α-amylases and α-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 α-amylases and α-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 α-amylases and α-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 anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Multiple enzyme sources can be used, but here we present the protocol using commercially available human α-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 ~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.
哺乳动物胃肠道中的碳水化合物消化由α-淀粉酶和α-葡萄糖苷酶催化,产生用于吸收的单糖。这些酶的抑制作用是阿卡波糖和米格列醇等药物的主要活性,这些药物用于治疗糖尿病。此外,通过抑制α-淀粉酶和α-葡萄糖苷酶来延迟碳水化合物消化是缓冲血糖飙升的有效策略,血糖飙升是导致代谢疾病的主要风险因素。在这里,我们提出了一种体外方案,用于准确和专门评估α-淀粉酶和α-葡萄糖苷酶(包括蔗糖酶、麦芽糖酶和异麦芽糖酶)的活性。该测定法特别适合于测量化合物、药物和提取物的抑制作用,因为酶活性测定中的底物和消化产物通过高效阴离子交换色谱与脉冲安培检测(HPAE-PAD)直接定量,因此最小程度地受到杂质或内源性成分的干扰。可以使用多种酶源,但这里我们使用市售的人α-淀粉酶来评估淀粉的水解,以麦芽七糖作为底物,并使用源自分化的人肠 Caco-2(/TC7)细胞的刷状缘蔗糖酶-异麦芽糖酶(具有麦芽糖酶、蔗糖酶和异麦芽糖酶活性)来评估二糖的水解。湿实验室测定的时间取决于样品的数量,约为 2-5 小时,而每个样品的 HPAE-PAD 分析时间为 35 分钟。因此,完整的数据集需要 1-3 天,并且可以以高灵敏度和可靠性检测到酶活性的细微变化。