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INFOGEST inter-laboratory recommendations for assaying gastric and pancreatic lipases activities prior to in vitro digestion studies

Myriam M.-L. Grundy 1 Evan Abrahamse 2, 3 Annette Almgren 4 Marie Alminger 4 Ana Andres 5 Claire Bourlieu-Lacanal 6, 7 Renata M.C. Ariëns 3 Shanna Bastiaan-Net 3 André Brodkorb 8 Maria Bronze 9, 10, 11 Irene Comi 12 Leslie Couëdelo 13 Didier Dupont 6 Annie Durand 14 Sedef El 15 Tara Grauwet 16 Christine Heerup 17 Ana Heredia 5 Marcos Infantes Garcia 16 Christian Jungnickel 18 Ilona Kłosowska-Chomiczewska 18 Marion Létisse 14 Adam Macierzanka 18 Alan Mackie 19 David Mcclements 20 Olivia Ménard 6 Anne Meynier 21 Marie-Caroline Michalski 14 Ana-Isabel Mulet-Cabero 11, 22 Anette Mullertz 17 Francina Payeras Perelló 23 Irene Peinado 5 Mélina Robert 7 Sébastien Secouard 23 Ana Serra 9, 10 Sandra Silva 9 Gabriel Thomassen 3 Cecilia Tullberg 4 Ingrid Undeland 4 Carole Vaysse 13 Gerd Vegarud 12 Sarah H.E. Verkempinck 16 Michelle Viau 21 Mostafa Zahir 3 Ruojie Zhang 20 Frédéric Carrière 24
Abstract : In vitro digestion studies often use animal digestive enzyme extracts as substitutes of human gastric and pancreatic secretions. Pancreatin from porcine origin is thus commonly used to provide relevant pancreatic enzymes such as proteases, amylase and lipase. Rabbit gastric extracts (RGE) have been recently introduced to provide gastric lipase in addition to pepsin. Before preparing simulated gastric and pancreatic extracts with targeted enzyme activities as described in in vitro digestion protocols, it is important to determine the activities of enzyme preparations using validated methods. The purpose of this inter-laboratory study within the INFOGEST network was to test the repeatability and reproducibility of lipase assays using the pH-stat technique for measuring the activities of gastric and pancreatic lipases from various sources. Twenty-one laboratories having different pH-stat devices received the same protocol with identical batches of RGE and two pancreatin sources. Lipase assays were performed using tributyrin as a substrate and three different amounts (50, 100 and 200 µg) of each enzyme preparation. The repeatability results within individual laboratories were satisfactory with coefficients of variation (CVs) ranging from 4 to 8% regardless of the enzyme amount tested. However, the inter-laboratory variability was high (CV > 15%) compared to existing standards for bioanalytical assays. We identified and weighted the contributions to inter-laboratory variability of several parameters associated with the various pH-stat equipment used in this study (e.g. reaction vessel volume and shape, stirring mode and rate, burette volume for the automated delivery of sodium hydroxide). Based on this, we established recommendations for improving the reproducibility of lipase assays using the pH-stat technique. Defining accurate and complete recommendations on how to correctly quantify activity levels of enzyme preparations is a gateway to promising comparison of in vitro data obtained from different laboratories following the same in vitro digestion protocol.
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https://hal.archives-ouvertes.fr/hal-03224818
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Submitted on : Wednesday, May 12, 2021 - 2:14:31 AM
Last modification on : Thursday, May 27, 2021 - 10:28:10 AM

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Myriam M.-L. Grundy, Evan Abrahamse, Annette Almgren, Marie Alminger, Ana Andres, et al.. INFOGEST inter-laboratory recommendations for assaying gastric and pancreatic lipases activities prior to in vitro digestion studies. Journal of Functional Foods, Elsevier, 2021, 82, pp.104497. ⟨10.1016/j.jff.2021.104497⟩. ⟨hal-03224818⟩

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