INFOGEST inter-laboratory recommendations for assaying gastric and pancreatic lipases activities prior to in vitro digestion studies
Journal article, 2021
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.
Enzyme activity
Inhibitor
Lipolysis
Lipases
INFOGEST
Titration method
Author
Myriam M.L. Grundy
Physiologie, Environnement et Génétique pour l'Animal et les Systèmes d'Elevage (PEGASE)
Evan Abrahamse
Nutricia Research
Wageningen University and Research
Annette Almgren
Chalmers, Biology and Biological Engineering, Food and Nutrition Science
Marie Alminger
Chalmers, Biology and Biological Engineering
Ana Andres
Polytechnic University of Valencia (UPV)
Renata M.C. Ariëns
Wageningen University and Research
Shanna Bastiaan-Net
Wageningen University and Research
Claire Bourlieu-Lacanal
University of Montpellier
National Research Institute for Agriculture, Food and the Environment (INRAE)
A. Brodkorb
Moorepark Food Research Centre
Maria Bronze
University of Lisbon
Nova University of Lisbon
Instituto de Biologia Experimental e Tecnologica
Irene Comi
Norwegian University of Life Sciences
Leslie Couëdelo
University of Bordeaux
D. Dupont
National Research Institute for Agriculture, Food and the Environment (INRAE)
Annie Durand
Lyon Sud Hospital Center
Sedef N. El
Ege University
Tara Grauwet
KU Leuven
Christine Heerup
University of Copenhagen
Ana Heredia
Polytechnic University of Valencia (UPV)
Marcos R. Infantes Garcia
KU Leuven
Christian Jungnickel
Gdansk University of Technology
Ilona E. Kłosowska-Chomiczewska
Gdansk University of Technology
Marion Létisse
Lyon Sud Hospital Center
A. Macierzanka
Gdansk University of Technology
A. Mackie
University of Leeds
D. J. McClements
University of Massachusetts
O. Menard
National Research Institute for Agriculture, Food and the Environment (INRAE)
Anne Meynier
National Research Institute for Agriculture, Food and the Environment (INRAE)
Marie Caroline Michalski
Lyon Sud Hospital Center
Ana Isabel Mulet-Cabero
Institute of Food Research
Moorepark Food Research Centre
Anette Mullertz
University of Copenhagen
Francina M. Payeras Perelló
Fresenius Kabi AG
Irene Peinado
Polytechnic University of Valencia (UPV)
Mélina Robert
University of Montpellier
Sébastien Secouard
Fresenius Kabi AG
Ana T. Serra
Instituto de Biologia Experimental e Tecnologica
Nova University of Lisbon
Sandra D. Silva
Instituto de Biologia Experimental e Tecnologica
Gabriel Thomassen
Wageningen University and Research
Cecilia Tullberg
Chalmers, Biology and Biological Engineering, Food and Nutrition Science
Ingrid Undeland
Chalmers, Biology and Biological Engineering, Food and Nutrition Science
Carole Vaysse
University of Bordeaux
G. E. Vegarud
Norwegian University of Life Sciences
Sarah H.E. Verkempinck
KU Leuven
Michelle Viau
National Research Institute for Agriculture, Food and the Environment (INRAE)
Mostafa Zahir
Wageningen University and Research
Ruojie Zhang
University of Massachusetts
F. Carriere
Aix Marseille University
Journal of Functional Foods
1756-4646 (ISSN)
Vol. 82 104497Subject Categories (SSIF 2011)
Biochemistry and Molecular Biology
Analytical Chemistry
Other Veterinary Science
DOI
10.1016/j.jff.2021.104497