[Show abstract][Hide abstract] ABSTRACT: Recent studies have demonstrated the presence of food-derived peptides in human blood after ingestion of enzymatic hydrolysates of food proteins, while most peptides in food are degraded into amino acids during digestion and absorption. To capture and clarify the food-derived peptides in blood, solid-phase extraction (SPE) using a mini-spin column packed with a strong cation exchanger was developed. This technique allows the use of a nonvolatile acid such as trichloroacetic acid, a strong protein denaturant, for the deproteinizing procedure. To improve resolution of hydrophilic peptide and increase specificity and sensitivity in the detection of peptide by reversed-phase high-performance liquid chromatography (RP-HPLC) after subfractionation by size-exclusion chromatography (SEC), peptides are derivatized with phenyl isothiocyanate. The resultant phenyl thiocarbamyl (PTC)-peptides can be resolved with high resolution and sensitivity by RP-HPLC. By comparing chromatograms of PTC derivatives from blood before and after ingestion of a peptide sample, food-derived peptide can be detected. The isolated PTC-peptide can be applied to a peptide sequencer based on the Edman degradation reaction.
Preview · Article · Jul 2008 · Journal of AOAC International
[Show abstract][Hide abstract] ABSTRACT: The transcellular transport of oligopeptides across intestinal epithelial cells has attracted considerable interest in investigations into how biologically active peptides express diverse physiological functions in the body. It has been postulated that the tripeptide, Gly-Pro-Hyp, which is frequently found in collagen sequences, exhibits bioactivity. However, the mechanism of uptake of dietary di- and tripeptides by intestinal epithelial cells is not well understood. In this study, we used porcine brush-border membrane (BBM) vesicles to assess Gly-Pro-Hyp uptake, because these vesicles can structurally and functionally mimic in vivo conditions of human intestinal apical membranes. The present study demonstrated the time-dependent degradation of this tripeptide into the free-form Gly and a dipeptide, Pro-Hyp, on the apical side of the BBM vesicles. In parallel with the hydrolysis of the tripeptide, the dipeptide Pro-Hyp was identified in the BBM intravesicular space environment. We found that the transcellular transport of Pro-Hyp across the BBM was inhibited by the addition of a competitive substrate (Gly-Pro) for peptide transporter (PEPT1) and was pH-dependent. These results indicate that Gly-Pro-Hyp can be partially hydrolyzed by the brush-border membrane-bound aminopeptidase N to remove Gly, and that the resulting Pro-Hyp is, in part, transported into the small intestinal epithelial cells via the H+-coupled PEPT1. Gly-Pro-Hyp cannot cross the epithelial apical membrane in an intact form, and Pro-Hyp is highly resistant to hydrolysis by intestinal mucosal apical proteases.
No preview · Article · Jul 2007 · Journal of Peptide Science
[Show abstract][Hide abstract] ABSTRACT: For the isolation and detection of food-derived peptides in blood, an approach based on the derivatization of peptides with phenyl isothiocyanate (PITC) was developed. This approach allows hydrophilic peptides to be resolved and specifically detected by reversed-phase (RP) HPLC. For the rapid capturing and clarification of peptides in human plasma, solid-phase extraction by using a mini spin column (5 mmx5 mm) packed with a strong cation exchanger was used. The clarified peptide fraction was further fractionated by size-exclusion chromatography (SEC). The peptides in the SEC fractions were derivatized with PITC, and the derivatives were resolved by RP-HPLC by using an ammonium acetate buffer or a trifluoroacetic acid system. An automatic peptide sequencer based on Edman degradation with a modified program can directly analyze the resolved derivatives. Some synthetic peptides and food-derived peptides in human plasma were successfully isolated and identified by this approach.
No preview · Article · Aug 2006 · Journal of Agricultural and Food Chemistry