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エレクトロスプレーイオン化質量分析法を用いたオリゴペプチドの腸管吸収に関する研究

申, 偉琳 WEILIN, SHEN シン, イリン 九州大学

2022.03.23

概要

Dietary intake of food derived short peptides (2 – 20 amino acid residues) have beendemonstrated to be potentially involved in the prevention of various life-style related diseasesincluding cardiovascular disease, diabetes, hyperlipidemia, etc. Upon oral intake, such bioactiveoligopeptides have to be absorbed into blood circulation across the small intestinal barrier to reachtargeted tissues and organs to elicit their physiological functions. Therefore, elucidation of theintestinal absorption of oligopeptide is of vital importance. Di-/tripeptides have been demonstratedto be absorbed via intestinal peptide transporter PepT1. In contrast, large remains unknownregarding the absorption of longer peptides of more than three amino acid resides, presumably dueto a lack of highly sensitive and selective assay. Electrospray ionization (ESI)-mass spectrometry(MS) is considered as an essential tool for the quantitative analysis of oligopeptides in complexbiofluids. Therefore, firstly, the ESI-MS detection characteristics of oligopeptides with chemicalderivatization was investigated. Then liquid chromatography (LC)-ESI-time-of-flight (TOF)/MS wasapplied to investigate the intestinal absorption of bioactive oligopeptides.

Chemical derivatization has been used to enhance ESI-MS detection of small amines anddipeptides, but its effect on longer oligopeptides remains unclarified. Therefore, the ESI-MSdetection of a series of synthetic di- to pentapeptides consisting of glycine and sarcosine wascharacterized by four amine derivatization methods. All the standard and chemically modifiedoligopeptides were detected by LC-ESI-TOF/MS. A chain length-dependent trend was observedsince chemical derivatization induced greater MS signal intensity increase to di-/tripeptides than tolonger oligopeptides. A moderate correlation was observed between MS signal intensity andhydrophobicity (log P) on the whole but increase in hydrophobicity did not necessarily lead toincrease in MS signal intensity for individual peptides, especially for tetra-/pentapeptides.

Meanwhile, a bell-shaped trend between MS signal intensity and molecular surface area wasobserved, with an optimum of 250 – 300 Å2. Consequently, it was concluded that the size of amolecule might be related with its ESI-MS detection characteristics and that addition of hydrophobictags might be less advantageous for longer oligopeptides. Indeed, chemical derivatization did notfurtherly enhance the detection of Cblin pentapeptides.

Then intestinal absorption of muscle atrophy-preventive Cbl-b inhibitory (Cblin) pentapeptidesDGpYMP and DGYMP. In vitro Caco-2 cell monolayer transport experiments demonstrated that bothpeptides were transportable across Caco-2 cell monolayers in their intact pentapeptide forms. ThePapp was 3.5 ± 1.2 cm/s for DGpYMP and 7.0 ± 0.8 cm/s for DGYMP. A screening of peptidemetabolites showed that DGpYMP was partly dephosphorylated to generate DGYMP. Meanwhile,no other peptide fragments were detected from the transport of DGpYMP and DGYMP. PepT1 wasnot involved in the transport of DGpYMP and DGYMP, while passive diffusion via paracellular tightjunction could be the major route involved. In vivo single oral administration experiments toSprague-Dawley (SD) rats (100 mg/kg-body weight) revealed that DGYMP, despite its pentapeptidelength, could be absorbed in its intact peptide form into SD rat blood circulation (Cmax: 2.78 ± 0.17pmol/mL-plasma, tmax: 15 min, AUC0 – 60 min: 100.35 ± 23.40 pmol·min/mL-plasma, t1/2: 28 min).Peptide fragments GYMP and MP were also detected in rat plasma, suggesting that part of DGYMPexperienced degradation during the absorption process.

In conclusion, using ESI-MS as a quantitative tool, this study demonstrated that Cblinpentapeptides could cross small intestinal barrier and reach blood circulation. These resultssuggested that longer oligopeptides with more than tripeptide lengths could be absorbed andpotentially elicit bioactivity upon oral intake.

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