TY - JOUR

T1 - INFOGEST inter-laboratory recommendations for assaying gastric and pancreatic lipases activities prior to in vitro digestion studies

AU - Grundy, Myriam M.L.

AU - Abrahamse, Evan

AU - Almgren, Annette

AU - Alminger, Marie

AU - Andres, Ana

AU - Ariëns, Renata M.C.

AU - Bastiaan-Net, Shanna

AU - Bourlieu-Lacanal, Claire

AU - Brodkorb, André

AU - Bronze, Maria R.

AU - Comi, Irene

AU - Couëdelo, Leslie

AU - Dupont, Didier

AU - Durand, Annie

AU - El, Sedef N.

AU - Grauwet, Tara

AU - Heerup, Christine

AU - Heredia, Ana

AU - Infantes Garcia, Marcos R.

AU - Jungnickel, Christian

AU - Kłosowska-Chomiczewska, Ilona E.

AU - Létisse, Marion

AU - Macierzanka, Adam

AU - Mackie, Alan R.

AU - McClements, David J.

AU - Menard, Olivia

AU - Meynier, Anne

AU - Michalski, Marie Caroline

AU - Mulet-Cabero, Ana Isabel

AU - Mullertz, Anette

AU - Payeras Perelló, Francina M.

AU - Peinado, Irene

AU - Robert, Mélina

AU - Secouard, Sébastien

AU - Serra, Ana T.

AU - Silva, Sandra D.

AU - Thomassen, Gabriel

AU - Tullberg, Cecilia

AU - Undeland, Ingrid

AU - Vaysse, Carole

AU - Vegarud, Gerd E.

AU - Verkempinck, Sarah H.E.

AU - Viau, Michelle

AU - Zahir, Mostafa

AU - Zhang, Ruojie

AU - Carrière, Frédéric

N1 - Publisher Copyright: © 2021

PY - 2021

Y1 - 2021

N2 - 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.

AB - 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.

KW - Enzyme activity

KW - INFOGEST

KW - Inhibitor

KW - Lipases

KW - Lipolysis

KW - Titration method

U2 - 10.1016/j.jff.2021.104497

DO - 10.1016/j.jff.2021.104497

M3 - Journal article

AN - SCOPUS:85105584594

VL - 82

JO - Journal of Functional Foods

JF - Journal of Functional Foods

SN - 1756-4646

M1 - 104497

ER -