Accurate method to measure carbohydrate digestion in humans using HPAE-PAD

Accurate method to measure carbohydrate digestion in humans using HPAE-PAD
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The carbohydrates (starch and sugars) we eat are rapidly digested by α-amylases in the saliva and pancreas and by α-glucosidases (sucrase, maltase and isomaltase) in the intestine, to produce absorbable sugars. α-Amylases hydrolyse the linear linkages in starch (amylose and amylopectin) to yield dextrins and maltose, while the branched α-limit dextrins (from amylopectin) are digested by isomaltase to produce maltose and isomaltose. Sucrose and maltose are abundant sugars in foods and are digested by sucrase and maltase, respectively. Isomaltose, which is produced industrially and ubiquitously present in processed foods, is also digested by isomaltase. Complete digestion of carbohydrates eventually produces glucose and fructose that are absorbed rapidly across the intestinal absorptive cells (enterocytes) 1. However, the uncontrolled continuous appearance of glucose in the blood can ultimately lead to the development of metabolic diseases, particularly type 2 diabetes.

Carbohydrate digestion and blood glucose spikes can be blunted by inhibiting α-amylase and α-glucosidase activities, as proven by several pharmaceutical (e.g., acarbose) and plant-based (e.g., polyphenols) anti-diabetic inhibitors 2-4. Pharmaceutical inhibitors are highly effective but can lead to the accumulation of undigested starch in the gut contributing to uncomfortable side effects, such as bloating and diarrhoea. Natural inhibitors may offer a compromise without the associated side effects since they are milder inhibitors and therefore may prove effective at blunting postprandial blood glucose spikes, although precise measurement of their enzyme inhibitory potential is needed. A reliable and sensitive method is warranted to accurately measure small changes in enzyme activity.

Human α-amylases are commercially available to easily assess enzyme inhibition, unlike human intestinal α-glucosidases which are inaccessible. Often, non-human α-glucosidases (from other mammals, bacteria and yeast) are used instead to predict the inhibitory potential of a compound in humans. However, very different enzyme inhibitory patterns were observed between species 1. For example, acarbose, a drug designed for humans, strongly inhibits human digestive enzymes but poorly inhibits non-human enzymes 2,5-7. Since it is unsafe to assume the same enzyme inhibition between species, it is crucial to conduct assays using human enzymes when exploring anti-diabetic agents for human consumption. In addition, the potential inhibition of isomaltase is rarely assessed, albeit important to provide a complete picture of digestive processes.

Most commonly, enzyme inhibition is estimated by colourimetric assays, but inaccuracy can occur when naturally-coloured compounds, such as polyphenols, interfere with product detection by forming polyphenol-sugar-reagent complexes 8-10. Alternatively, the chromatographic method does not have the same interference problems, while directly measuring the concentrations of the sugar products without the use of coloured reagents to assess the inhibitory potentials of compounds or extracts of interest 8,11.

Here, a highly sensitive and precise in vitro protocol has been developed to measure the inhibition of human α-amylases and α-glucosidases using high-performance anion-exchange chromatography with pulsed amperometric detection  (HPAE-PAD), without interference from impurities, or endogenous or coloured compounds (e.g., polyphenols). The method comprises three parts: 1) extraction of α-glucosidases from cultured human Caco-2/TC7 intestinal cells, 2) α-glucosidase and α-amylase enzyme assays, and 3) chromatographic analysis of the enzyme assay products to measure enzyme activity and inhibition. Caco-2/TC7 cells express an abundance of sucrase-isomaltase, which has sucrase, maltase and isomaltase activities 12,13 and are therefore a suitable source of human α-glucosidase activities. We used commercially available human α-amylase, but any appropriate source could be assayed (e.g., human saliva). The complete protocol can be used to efficiently screen any potential inhibitors of human carbohydrate digestion and show subtle changes in digestive enzyme activities. The HPAE-PAD chromatography can accurately and rapidly quantify the substrate consumption and sugar production in the enzyme assays simultaneously, as low as ≤ 0.7 µM for smaller sugars and < 2.7 µM for larger sugars (CVprecision < 3.7%), with no matrix effects or interferences from impurities 1,8,9.

The complete method includes information on the culture of Caco-2/TC7 cells and, for the enzyme assays and analyses, the appropriate blanks, buffers, assay temperatures, concentrations of assay components (i.e., substrates, enzymes and inhibitors), a calibration curve of sugar standards, the number of replicates and the associated specialised software used to analyse data. The velocity of the reaction is calculated from the amount of product formed, or substrate converted, determined after accurate chromatographic separation. Michaelis-Menten and Lineweaver-Burk plots were used to obtain the kinetic parameters of the digestive enzymes for the varying enzyme and substrate amounts tested. Half- or quarter-maximal inhibition concentrations (IC50 or IC25) of test compounds were determined using specialised software (e.g., GraphPad) to assess the efficacy of the potential inhibitors. A complete assessment of the digestive enzyme inhibitory potentials can be made by conducting both α-amylase and α-glucosidase assays to show any potential combined effects.

The complete in vitro protocol presented here for the accurate assessment of the digestive enzyme activities of human α-amylases and α-glucosidases will benefit researchers involved in the discovery and development of anti-diabetic agents from natural sources, foods or candidate drugs. Further, the assay is fundamentally versatile to include both human and non-human enzyme sources, or other chromatographic methods as long as appropriate preliminary assay optimisation and validation experiments are performed before test compounds are assessed for enzyme inhibition. Here, the combination of multiple assays using appropriate human enzyme sources with highly sensitive and reliable chromatographic analyses is the most prominent and promising tool currently known to detect subtle changes in digestive enzyme activities by potential inhibitors. Therefore, this method can be used to screen almost any compound or mixture for improved and correct translation into human intervention studies.

References

1          Barber, E., Houghton, M. J. & Williamson, G. Flavonoids as Human Intestinal alpha-Glucosidase Inhibitors. Foods 10, doi:10.3390/foods10081939 (2021).

2          McIver, L. A. & Tripp, J. in Acarbose     (StatPearls [Internet] StatPearls Publishing, 2020).

3          Aryaeian, N., Sedehi, S. K. & Arablou, T. Polyphenols and their effects on diabetes management: A review. Med J Islam Repub Iran 31, 134, doi:10.14196/mjiri.31.134 (2017).

4          Al-Duhaidahawi, D., Hasan, S. A. & Al-Zubaidy, H. F. S. Flavonoids in the Treatment of Diabetes Clinical Outcomes and Mechanism to ameliorate Blood Glucose levels. Curr Diabetes Rev, doi:10.2174/1573399817666201207200346 (2020).

5          Priscilla, D. H., Roy, D., Suresh, A., Kumar, V. & Thirumurugan, K. Naringenin inhibits alpha-glucosidase activity: a promising strategy for the regulation of postprandial hyperglycemia in high fat diet fed streptozotocin induced diabetic rats. Chem Biol Interact 210, 77-85, doi:10.1016/j.cbi.2013.12.014 (2014).

6          Pyner, A., Chan, S. Y., Tumova, S., Kerimi, A. & Williamson, G. Indirect Chronic Effects of an Oleuropein-Rich Olive Leaf Extract on Sucrase-Isomaltase In Vitro and In Vivo. Nutrients 11, doi:10.3390/nu11071505 (2019).

7          Da Silva, D. et al. Antidiabetic activity of Sedum dendroideum: metabolic enzymes as putative targets for the bioactive flavonoid kaempferitrin. IUBMB Life 66, 361-370, doi:10.1002/iub.1270 (2014).

8          Visvanathan, R., Houghton, M. J. & Williamson, G. Maltoheptaoside hydrolysis with chromatographic detection and starch hydrolysis with reducing sugar analysis: Comparison of assays allows assessment of the roles of direct alpha-amylase inhibition and starch complexation. Food Chem 343, 128423, doi:10.1016/j.foodchem.2020.128423 (2021).

9          Visvanathan, R. et al. Critical review on conventional spectroscopic alpha-amylase activity detection methods: merits, demerits, and future prospects. J Sci Food Agric 100, 2836-2847, doi:10.1002/jsfa.10315 (2020).

10        Robyt, J. F. & Whelan, W. J. Reducing value methods for maltodextrins. I. Chain-length dependence of alkaline 3,5-dinitrosalicylate and chain-length independence of alkaline copper. Anal Biochem 45, 510-516, doi:10.1016/0003-2697(72)90213-8 (1972).

11        Lim, J., Zhang, X., Ferruzzi, M. G. & Hamaker, B. R. Starch digested product analysis by HPAEC reveals structural specificity of flavonoids in the inhibition of mammalian alpha-amylase and alpha-glucosidases. Food Chem 288, 413-421, doi:10.1016/j.foodchem.2019.02.117 (2019).

12        Van Beers, E. H. et al. Lactase and sucrase-isomaltase gene expression during Caco-2 cell differentiation. Biochem J 308, 769-775, doi:https://doi.org/10.1042/bj3080769 (1995).

13        Cheng, M. W. et al. Different sucrose-isomaltase response of Caco-2 cells to glucose and maltose suggests dietary maltose sensing. J Clin Biochem Nutr 54, 55-60, doi:10.3164/jcbn.13-59 (2014).

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