The athletes were provided with a CP supplement, the porcine-derived commercial collagen peptide (Wellnex SCP-5200) from Nitta Gelatin (Osaka, Japan). turnover2. Among the metabolites produced during this process are bioactive collagen peptides (CP) resulting from its enzymatic degradation3,4. These collagen peptides are efficiently assimilated in the digestive tract, appearing in the blood within a few hours of the ingestion of collagen-containing substances5. Oral intake of commercially available CP is usually reported to have physiological effects such as promoting skin pressure ulcer recovery6, stimulating bone formation and inhibiting bone resorption7, inhibiting arthritis8, increasing excess fat free body mass and grip strength9, and preventing atherosclerosis10. Recent studies report that prolylhydroxyproline (Pro-Hyp), one of the final collagen Bronopol metabolites, stimulates the proliferation and differentiation of skin fibroblasts11, chondrocytes12, and osteocytes13. The detailed mechanism underlying the bioactivity of CP is usually unclear. However, evidence indicates that Pro-Hyp is usually taken up by mesenchymal cells via peptide transporters14 and subsequently plays a role in regulating cell function as a binding factor for nuclear receptors15. During biosynthesis, collagen undergoes post-translational modifications including hydroxylation, glycation, and the formation of intra- Bronopol and intermolecular crosslinks16. Collagen turnover, involving biosynthesis and degradation, is altered by the effects of aging17, physical activity18, and nutritional status19. These factors may also affect the status of collagen metabolites in the body. Therefore, the investigation of CP within the body may be very important to understanding its bioactivity. Serum and urinary levels of collagen-derived peptides can be used to monitor collagen metabolism. Collagen-derived amino acids and di- and tripeptides are identified and quantified using liquid chromatography20, gas chromatography21, and mass spectrometry22, a highly selective technique known as LCCMS/MS. A recently developed, highly sensitive ELISA also detects bioactive CP. This assay does not require dedicated equipment Bronopol and can be used to analyze many samples simultaneously in a short time. In this study, we determine the specific active Bronopol collagen oligopeptides (ACOP) detected by this ELISA for use in monitoring collagen metabolism. We then investigate the effect of CP ingestion Bronopol and Rabbit polyclonal to ALDH1L2 exercise on urinary ACOP concentrations in a cohort of university student athletes using colorimetric, LCCMS/MS, and ELISA. Results Cross-reactivity of anti-ACOP antibody ELISA was used to investigate the cross-reactivity of anti-ACOP antibody with six different peptides: Pro-Hyp, Gly-Pro-Hyp, Pro-Hyp-Gly, Pro-Pro, Hyp-Gly, and commercial CP. In this ELISA, the value measured per weight was calculated for each sample relative to that of Pro-Hyp. As shown in Fig.?1, the cross-reactivity was approximately fivefold for Gly-Pro-Hyp and approximately 0.1 to 0.001-fold for Pro-Hyp-Gly, Pro-Pro, and Hyp-Gly. The cross-reactivity of commercial CP, the hydrolysis product of collagen, was approximately 0.1-fold. These results show that this anti-ACOP antibody had particularly strong cross-reactivity with Pro-Hyp and Gly-Pro-Hyp, binding not only to dipeptides and tripeptides but also to the collagen-derived oligopeptide CP. Open in a separate window Physique 1 Cross-reactivity of anti-ACOP antibody to five collagen-derived peptides. Data are presented as relative values per weight normalized to that of Pro-Hyp. Urinary level of ACOP after collagen peptide (CP) ingestion We observed that this urine levels of seven out of eight collagen degradants (ACOP, Peptide forms of Hyp, Pro-Hyp, Hyp-Gly, Pro-Pro, Pro-Hyp-Gly, and Gly-Pro-Hyp) increased after CP ingestion, with no change in Free Hyp (Fig.?2A). In the group of control, free Hyp in the first early morning urine 4?h before CP intake was only significantly higher, and other.