Transport of Free and Peptide‐Bound Glycated Amino Acids: Synthesis, Transepithelial Flux at Caco‐2 Cell Monolayers, and Interaction with Apical Membrane Transport Proteins

Abstract

In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.

Quantification of the Maillard reaction product 6-(2-formyl-1-pyrrolyl)-l-norleucine (formyline) in food

“…Formyline, a recently identified MRP with a pyrrole core, has only been measured in model incubations until now [8,22]. It was therefore the aim of this project to determine formyline in a broad range of more complex food samples.…”

“…Based on these data, the daily intake of pyrraline from milk and bakery products has been estimated to approximately 25 mg [27]. No quantitative data, however, are available for formyline, although the very high transport rates measured at intestinal cells in vitro point to the fact that it might be taken up into the body from food proteins [22]. It was, therefore, the principle aim of this study to establish an analytical strategy for the quantification of the new glycation product formyline in a variety of foodstuffs and to estimate its daily intake.…”

“…This membrane-bound multi-substrate carrier protein is able to translocate dipeptide-bound pyrraline into the cell, from where free pyrraline can pass into the circulation after peptidolysis [20,21]. Recent data on the transport properties of a variety of other dipeptidebound AGEs indicate that products such as formyline will also show high bioavailability [22].…”

Urinary excretion of dietary Maillard reaction products in healthy adult female cats1,2

“…Therefore, LAL is suggested to be poorly available for absorption, and most LAL will be transported into the large intestine. Protein-bound FL, although digested into small dipeptides, is not fully transported by the intestinal peptide transporter PEPT1 and might also flow into the large intestine (Hellwig et al, 2011). These mechanisms could explain part of the rather low urinary recovery of both dietary FL (between 8.1% and 22.9%) and LAL (between 3.4% and 18.7%) compared with CML (between 24.9% and 72.6%) in the current study as well as in the study of Somoza et al (2006) and suggest that the foods in the present study contained protein-bound MRP rather than free MRP.…”

DETERMINATION OF ADVANCED GLYCATION (AGEs) AND LIPOXIDATION (ALEs) END PRODUCTS IN FOODS AND BIOLOGICAL SYSTEMS: ADVANCES, CHALLENGES AND PERSPECTIVES

“…88 Sugere-se que a CML seja absorvida no epitélio intestinal através de difusão simples, mas verificou-se que CML, CEL e MG-H1 (imidazolona derivada de MG) em dipeptídeos são carreados por transportadores de peptídeos, em especial pelo PEPT1 (peptide transporter 1), sendo esse também o sistema de absorção da pirralina. [89][90][91][92] Havendo absorção intestinal, a distribuição e o destino metabólico dos AGEs são também amplamente desconhecidos. Devido às propriedades anfotéricas e de solubilidade em água, infere-se que os AGEs de baixo peso molecular possam ser mais prontamente distribuídos aos compartimentos extra e intracelulares do que aqueles que apresentam alto peso molecular.…”