Abstract
The role of PEPT1 in the uptake of intact peptides as compared to hydrolysis prior to uptake of their constituents is unknown. Here, dipeptides, tripeptides, and amino acids are quantified to study the fate of selected peptides in different intestinal models. An LC-MS/MS-based method is applied for the simultaneous assessment of rates of hydrolysis and transport of a peptide panel in Caco-2 transwell cell culture, in vitro and in vivo in mice expressing or lacking PEPT1, and in hydrolysis studies in vitro using human intestinal samples. It is shown that susceptibility to hydrolysis of peptides at the brush border membrane or within epithelial cells is practically identical in all tested models and strictly structure-dependent. Peptides with high luminal disappearance show substantial hydrolysis and low basolateral appearance, while peptides with low disappearance show strong PEPT1 dependency and high basolateral appearance in intact form in Caco-2 transwell culture. Hydrolysis and transport of intact peptides are highly variable and structure-dependent. For peptides possessing less polar N-terminal residues, hydrolysis usually dominates over transport via PEPT1. For other peptides with high intrinsic hydrolysis resistance, including anserine, carnosine, ɣ-glutamyl-dipeptides, and aminocephalosporins, PEPT1 is the main determinant for appearance in peripheral blood.
Highlights
Scope: The role of PEPT1 in the uptake of intact peptides as compared to and proteases
The pep- plasma after ingestion and their higher resistance to hydrolysis tide fraction would be highly diverse in chain length and com- when compared to dipeptides with, e.g., leucine or other bulky position depending on the nature of the ingested protein, its residues in N-terminal position.[1,8,9]
In order to assess the roles in intestinal protein assimilation of PEPT1 on the one hand and brush border peptidases on the other, PEPT1 knockout and control Caco-2 cells were incubated with a panel of 20 peptides in the apical compartment of transwell cell culture (V = 0.5 mL, 500 μm each, corresponding to 250 nmol per peptide per apical compartment)
Summary
Acetonitrile (LC–MS grade), ammonium acetate, formic acid (LC–MS grade), phenyl isothiocyanate (PITC), and pyridine were purchased from Sigma–Aldrich (Taufkirchen, Germany). LC–MS grade water was purchased from J. Masschrom internal standard from ChromSystems (München, Germany) was used and expanded by glutamine-D5 and asparagine15N2 (20.0 μmol L–1 each) from Cambridge Isotope Laboratories, Inc. (Andover, USA), and tryptophan-D5 (2.0 μmol L–1) from Santa Cruz Biotechnology, Inc. Analytes for the external standard solution comprised glycine, l-alanine, l-arginine, l-asparagine, l-aspartic acid, l-glutamic acid, l-glutamine, l-histidine, l-isoleucine, l-leucine, l-lysine, l-methionine, l-phenylalanine, l-proline, l-serine, l-threonine, l-tryptophan, l-tyrosine, and l-valine, purchased from Sigma– Aldrich (Taufkirchen, Germany). The di- and tripeptides for the external standard Ala-Gly, Ala-His, Ala-Phe, Arg-Gly, ɣ-Glu-Glu, ɣ-Glu-Gly, ɣ-Glu-Leu, Gly-Asn, Gly-Asp, Gly-Gln, Gly-Gly-Ile, Gly-Pro, Gly-Sar, Gly-Val, Lys-Glu, Phe-Ala, Pro-Gly, Trp-Glu, and Val-Pro-Pro were purchased from Bachem (Bubendorf, Schweiz), Phe-Gly and Trp-Leu from Serva (Heidelberg, Germany), and anserine, carnosine, cefadroxil, cefalexin, cefradine, and PenStrep 100× from Sigma–Aldrich (Taufkirchen, Germany)
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