Highly Accurate Quantification of Hydroxyproline-Containing Peptides in Blood Using a Protease Digest of Stable Isotope-Labeled Collagen

Collagen-derived hydroxyproline (Hyp)-containing dipeptides and tripeptides, which are known to possess physiological functions, appear in blood at high concentrations after oral ingestion of gelatin hydrolysate. However, highly accurate and sensitive quantification of the Hyp-containing peptides in blood has been challenging because of the analytical interference from numerous other blood components. We recently developed a stable isotope-labeled collagen named "SI-collagen" that can be used as an internal standard in various types of collagen analyses employing liquid chromatography-mass spectrometry (LC-MS). Here we prepared stable isotope-labeled Hyp-containing peptides from SI-collagen using trypsin/chymotrypsin and plasma proteases by mimicking the protein degradation pathways in the body. With the protease digest of SI-collagen used as an internal standard mixture, we achieved highly accurate simultaneous quantification of Hyp and 13 Hyp-containing peptides in human blood by LC-MS. The area under the plasma concentration-time curve of Hyp-containing peptides ranged from 0.663 +/- 0.022 nmol/mL(.)h for Pro-Hyp-Gly to 163 +/- 1 nmol/mL(.)h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was <7% for each peptide except for Glu-Hyp-Gly, which was near the detection limit. Our method is useful for absorption/metabolism studies of the Hyp-containing peptides and development of functionally characterized gelatin hydrolysate.