Absorption of Hydroxyproline-Containing Peptides in Vascularly Perfused Rat Small Intestine in Situ

To assess the digestion and assimilation of gelatin and gelatin hydrolysates, the in situ absorption of typical hydroxyproline-containing dipeptides, Pro-Hyp, Hyp-Gly, Ser-Hyp Ala-Hyp, and pentadecapeptide, (ProHyp-Gly)(5), was investigated in the rat small intestine. During vascular perfusion after the injection of HypGly, Pro-Hyp and (Pro-Hyp-Gly)(5) into the jejunum, peptide-form Hyp but not free-Hyp gradually increased in the perfusate. In contrast, in the case of Ser-Hyp and Ala-Hyp, both free- and peptide-form Hyp rapidly increased. The presence of these dipeptides and the pentadecapeptide in the perfusates was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using multiple reaction monitoring (MRM). Some digestive and absorbed forms from (Pro-Hyp-Gly)(5) were identified as Gly-(Pro-Hyp-Gly)(4), (Pro-Hyp-Gly)(4), Gly-(Pro-Hyp-Gly)(3), (Pro-Hyp-Gly)(3), Gly-(Pro-Hyp-Gly)(2), and (Pro-Hyp-Gly)(2) by MALDI-TOF/MS. The dipeptide hydrolase activity in intestinal mucosa toward Pro-Hyp and Hyp-Gly was extremely low, while Ser-Hyp and Ala-Hyp were substantially hydrolyzed in the cytosol. These results suggest that Hyp-peptides were resistant to intracellular hydrolysis and that a significant amount of these peptides was transported across the intestinal wall and may enter the portal circulation in an intact form.