The study investigates the bioavailability and bioactivity of 3-hydroxyproline (3Hyp), a minor collagen-specific amino acid, in contrast to the well-studied 4-hydroxyproline (4Hyp). Previous research has shown high bioavailability and bioactivity of 4Hyp-containing oligopeptides from collagen hydrolysate. These peptides reach micromolar levels in blood, significantly higher than other food-derived peptides, and exhibit various biological activities, including stimulating fibroblast growth, promoting osteoblast differentiation, and exhibiting chemotactic activity. The high bioavailability is attributed to the presence of 4Hyp, which confers resistance to peptidase/protease digestion. 3Hyp, a less prevalent modification of proline in collagen, is found only adjacent to 4Hyp. While 4Hyp-containing oligopeptides are well-studied, research on 3Hyp-containing peptides is limited. This study aims to analyze the kinetics, stability, and biological activity of 3Hyp-containing peptides, specifically focusing on Gly-3Hyp-4Hyp, found in high concentrations in human blood after collagen hydrolysate ingestion.

Existing literature extensively documents the high bioavailability and bioactivity of 4-hydroxyproline (4Hyp)-containing oligopeptides following collagen hydrolysate ingestion. Key peptides identified include Pro-4Hyp and 4Hyp-Gly, along with various other di- and tri-peptides containing 4Hyp. These peptides have been shown to stimulate the growth of skin fibroblasts, promote osteoblast differentiation, and exhibit chemotactic activity on various cell types. These bioactivities are linked to the beneficial effects of collagen hydrolysate on bone, joint, skin, and other tissues. The resistance of these peptides to digestion is largely due to the presence of 4Hyp. However, studies on 3Hyp-containing oligopeptides, despite 3Hyp's presence in collagen, remain scarce.

The study employed several methodologies to investigate the fate and bioactivity of 3Hyp-containing peptides. First, using LC-MS in multiple reaction monitoring (MRM) mode with APDS derivatization, they analyzed changes in free and peptide-form 4Hyp/3Hyp in human blood after ingestion of porcine skin collagen hydrolysate. This allowed differentiation of 4Hyp and 3Hyp, otherwise difficult in biological fluids. Subsequently, they identified the specific 3Hyp-containing oligopeptide in the blood using synthetic peptide standards as references. The kinetic profile of Gly-3Hyp-4Hyp and other major 4Hyp-containing oligopeptides were analyzed in time-course human plasma samples using MRM analysis with an internal standard. A peptidase/protease resistance assay, involving incubation of synthetic peptides with mouse plasma, assessed the stability of Gly-3Hyp-4Hyp compared to other peptides. To investigate the effect of collagen source on Gly-3Hyp-4Hyp levels, mice were orally administered collagen hydrolysates from bovine skin and tendon (tendon having higher 3Hyp content). Blood samples were analyzed to compare Gly-3Hyp-4Hyp levels. Finally, in vitro experiments assessed Gly-3Hyp-4Hyp's chemotactic activity on skin fibroblasts using a transwell system and its effect on osteoblast differentiation using ALP activity and mineralization assays.

The study's key findings include: 1. After collagen hydrolysate ingestion, a significant portion of absorbed 3Hyp was present as Gly-3Hyp-4Hyp in human plasma, with levels comparable to other 4Hyp-containing oligopeptides. 2. Gly-3Hyp-4Hyp showed exceptionally high resistance to enzymatic degradation in mouse plasma, significantly higher than other peptides like Pro-4Hyp and 4Hyp-Gly. This was attributed to the Gly-3Hyp sequence. 3. The maximum plasma concentration of Gly-3Hyp-4Hyp was delayed compared to other oligopeptides, and it remained elevated for a longer duration. 4. Oral administration of tendon collagen hydrolysate (higher 3Hyp content) resulted in substantially higher blood levels of Gly-3Hyp-4Hyp in mice compared to skin collagen hydrolysate. 5. Gly-3Hyp-4Hyp demonstrated significant chemotactic activity on skin fibroblasts and promoted osteoblast differentiation in vitro, effects comparable to or surpassing that of other known bioactive 4Hyp-containing peptides. The prolonged presence of Gly-3Hyp-4Hyp in the blood, coupled with its bioactivity, suggests a significant contribution to the reported health benefits of collagen hydrolysate ingestion.

This study's findings demonstrate the remarkable bioavailability and bioactivity of Gly-3Hyp-4Hyp, a previously understudied 3Hyp-containing tripeptide. The high stability of Gly-3Hyp-4Hyp due to its unique sequence is crucial for its prolonged presence in the blood, potentially contributing significantly to the reported health benefits of collagen hydrolysate ingestion. The observed chemotactic activity on fibroblasts and the promotion of osteoblast differentiation further support its potential therapeutic applications for skin and bone health. The increased Gly-3Hyp-4Hyp levels following tendon collagen hydrolysate administration highlight the importance of collagen source selection for maximizing the absorption of this beneficial tripeptide. Future studies should investigate the tissue distribution of Gly-3Hyp-4Hyp, its cellular uptake mechanisms, and potential interactions with specific intracellular targets.

This research reveals Gly-3Hyp-4Hyp as a highly stable and bioactive tripeptide with significant oral bioavailability. Its unique resistance to enzymatic degradation and its potent effects on fibroblast migration and osteoblast differentiation suggest a substantial contribution to the beneficial effects of collagen hydrolysate. Future research should focus on exploring potential therapeutic applications of Gly-3Hyp-4Hyp and investigating its precise mechanisms of action in vivo.

The study utilized a relatively small sample size (n=8) for the human study. Furthermore, the in vitro bioactivity assays were conducted at a specific peptide concentration (200 nmol/mL), and further studies are needed to assess the dose-response relationship. Additionally, the study primarily focused on Gly-3Hyp-4Hyp; future work could explore other 3Hyp-containing peptides and their potential bioactivities.