Transepithelial flux of early and advanced glycation compounds across Caco-2 cell monolayers and their interaction with intestinal amino acid and peptide transport systems.

Maillard products arise from condensation reactions between amino acids or proteins with reducing sugars during food processing. As ubiquitous components of human food, these early or advanced glycation products may be subject to intestinal absorption. The present study was performed to investigate the intestinal uptake of Maillard products and to determine whether they are substrates for peptide and amino acid transporters expressed at the apical membrane of Caco-2 cells. At a concentration of 10 mM, N(epsilon)-(carboxymethyl)-L-lysine, N(alpha)-hippuryl-N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine, N(alpha)-hippuryl-N(epsilon)-(carboxymethyl)-L-lysine and N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine inhibited the [(14)C]glycylsarcosine uptake mediated by the H(+)-peptide co-transporter PEPT1 by 13 to 45%. For N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine, an inhibitory constant of 8.7 mM was determined, reflecting a low affinity to PEPT1 in comparison with natural dipeptides. Uptake of L-[(3)H]lysine was weakly affected by N(epsilon)-(carboxymethyl)-L-lysine, N(alpha)-hippuryl-L-lysine and N(alpha)-hippuryl-N(epsilon)-(carboxymethyl)-L-lysine but strongly inhibited by N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine (81%). None of the Maillard products was able to inhibit the uptake of L-[(3)H]leucine by more than 15%. We also studied the transepithelial flux of Maillard products across Caco-2 cell monolayers cultured on permeable filters. The flux rates of Maillard products ranged from 0.01 to 0.3%/cm(2) per h and were shown to be much lower than those of carrier substrates such as glycylsarcosine, L-proline and the space marker [(14)C]mannitol. We conclude that the Maillard products investigated in the present study are neither transported by PEPT1 nor by carriers for neutral amino acids. The low transepithelial flux measured for these compounds most probably occurs by simple diffusion.

Transport of the phosphonodipeptide alafosfalin by the H+/peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells.

The interaction of the antibacterial phosphonodipeptide alafosfalin with mammalian H(+)/peptide cotransporters was studied in Caco-2 cells, expressing the low-affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high-affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [(14)C]glycylsarcosine with K(i) values of 0.19 +/- 0.01 mm and 0.07 +/- 0.01 mm for PEPT1 and PEPT2, respectively. Saturation kinetic studies revealed that in both cell types alafosfalin affected only the affinity constant (K(t)) but not the maximal velocity (V(max)) of glycylsarcosine (Gly-Sar) uptake. The inhibition constants and the competitive nature of inhibition were confirmed in Dixon-type experiments. Caco-2 cells and SKPT cells were also cultured on permeable filters: apical uptake and transepithelial apical to basolateral flux of [(14)C]Gly-Sar across Caco-2 cell monolayers were reduced by alafosfalin (3 mm) by 73%. In SKPT cells, uptake of [(14)C]Gly-Sar but not flux was inhibited by 61%. We found no evidence for an inhibition of the basolateral to apical uptake or flux of [(14)C]Gly-Sar by alafosfalin. Alafosfalin (3 mm) did not affect the apical to basolateral [(14)C]mannitol flux. Determined in an Ussing-type experiment with Caco-2 cells cultured in Snapwells trade mark, alafosfalin increased the short-circuit current through Caco-2 cell monolayers. We conclude that alafosfalin interacts with both H(+)/peptide symporters and that alafosfalin is actively transported across the intestinal epithelium in a H(+)-symport, explaining its oral availability. The results also demonstrate that dipeptides where the C-terminal carboxyl group is substituted by a phosphonic function represent high-affinity substrates for mammalian H(+)/peptide cotransporters.