Tier 4 — mechanistic

Assessment of bioavailability after in vitro digestion and first pass metabolism of bioactive peptides from collagen hydrolysates

Christina E. Larder, Michèle M. Iskandar, Stan Kubow
Current Issues in Molecular Biology 2021 43(3):1592–1605

Bibliography

PubMed
PMID 34698092
PubMed Central
PMC8928955
DOI
10.3390/cimb43030113
Funding
Funding statement (verbatim): "The present study was supported by a MITACS Accelerate Program PhD studentship (IT10556) collaboration between McGill University and Genacol Canada Corporation and the Collaborative Research Development Grant Program from the Natural Sciences and Engineering Council of Canada to S.K. (535744-18)."
Competing interests
Competing interests statement (verbatim): "S.K. has received consultant honoraria and travel support from Genacol Canada Corporation. C.E.L. has received travel support from Genacol Canada Corporation. M.M.I. declares no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; nor in the writing of the manuscript. The funders partook in the decision to publish the results." Genacol Canada Corporation is the manufacturer of one of the two tested hydrolysates (CH-GL) and is a commercial marketer of collagen supplements. Substantial commercial ties across authorship and funding despite the formal declaration.

Study snapshot

DesignStandardized in-vitro gastrointestinal digestion followed by HIEC-6 human intestinal epithelial transwell transport and HepG2 hepatic first-pass metabolism, with LC-MS/MS analysis of collagen-derived peptides.
ModelGenacol Original Formula® (CH-GL) and Selection/Uniprix collagen hydrolysate (CH-OPT) subjected to simulated gastrointestinal digestion, then applied to an HIEC-6 intestinal/HepG2 hepatic transwell co-culture.
SampleNot applicable — in vitro study. Peptide analysis performed on triplicate biological samples per condition.
InterventionTwo bovine collagen hydrolysates—Genacol Original Formula® (CH-GL) and Selection/Uniprix (CH-OPT)—were subjected to simulated gastric digestion (pepsin 4% w/w, pH 2, 30 min) followed by intestinal digestion (pancreatin 4% w/w, pH 8, 2 h), then applied to HIEC-6 intestinal cells in transwell co-culture with HepG2 hepatic cells.
DurationSimulated gastric digestion: 30 min; simulated intestinal digestion: 2 h; subsequent transwell intestinal transport and hepatic metabolism were assessed over the experimental sampling interval.
EndpointsPeptide transport across intestinal cell layer for 5 target bioactive peptides (Gly-Pro, Hyp-Gly, Ala-Hyp, Pro-Hyp, Gly-Pro-Hyp); Hepatic first pass production of same peptides; Overall bioavailability percentage per peptide per hydrolysate

What the study showed, in plain terms

By 2021, decades of research had established that some collagen peptides survive digestion and reach the human bloodstream. What was still poorly understood was how much of what ends up in blood depends on the liver — the first organ blood passes through after leaving the gut — and whether the liver actively creates additional peptides or removes the ones that make it through the gut. Answering that in humans is technically impossible without invasive sampling of the hepatic portal vein, so researchers turned to a laboratory model.

Larder and colleagues at McGill University built a two-chamber laboratory setup in which human intestinal cells sat on top of human liver cells, separated by a thin membrane. They took two commercial collagen hydrolysate products, put them through a simulated stomach-and-intestine digestion, and then applied the resulting soup to the intestinal side of the model. Over the next few hours they measured five specific bioactive peptides — Gly-Pro, Hyp-Gly, Ala-Hyp, Pro-Hyp, and Gly-Pro-Hyp — appearing on the liver side, and separately measured whether the liver cells generated additional peptides of their own.

All five peptides made it across the intestinal cell layer, but at different rates depending on which of the two hydrolysates was tested. The tripeptide Gly-Pro-Hyp behaved differently between the two products — it crossed the intestine with one hydrolysate but not the other, matching an earlier human observation from the Yazaki 2017 study. The liver actively produced Ala-Hyp with both hydrolysates and produced additional Pro-Hyp and Gly-Pro from one of them, meaning the peptides showing up in human blood are partly gut-absorbed and partly liver-generated. Except for Gly-Pro-Hyp with one hydrolysate, all peptides were bioavailable at over 10% — a mechanistically important benchmark for a food-derived bioactive.

Key findings

  • Four target peptides—Gly-Pro, Hyp-Gly, Ala-Hyp and Pro-Hyp—were transported across the intestinal epithelial cell layer from both hydrolysates; Gly-Pro-Hyp transport was detected from CH-GL but was negligible from CH-OPT.
  • The contrasting Gly-Pro-Hyp result indicates that peptide transport in this in-vitro model depended on hydrolysate composition rather than being uniform across collagen products.
  • The model showed net hepatic production of Ala-Hyp with both hydrolysates.
  • Pro-Hyp and Gly-Pro also showed net hepatic production under some conditions with CH-GL, while peptide-specific hepatic handling varied between the tested hydrolysates.
  • Estimated bioavailability exceeded 10% for the measured peptides except Gly-Pro-Hyp with CH-OPT, where it was negligible.
  • The direction of the hepatic effect varied by peptide, with some peptides showing net production and others net removal in the in-vitro first-pass model.
  • These are simulated-digestion and cell-culture findings; they do not establish oral bioavailability or clinical effects in humans.

What this study can and cannot tell us

  • In vitro model — HIEC-6 intestinal cell line is a physiologically relevant human line but does not replicate the full complexity of intact intestinal architecture (villus/crypt structure, mucus layer, immune interactions, microbiome).
  • HepG2 hepatic line is a cancer-derived cell line — its enzymatic profile approximates but does not perfectly replicate primary hepatocytes.
  • No human validation of the specific bioavailability percentages reported — the model produces predictions, not clinical measurements.
  • Only 5 target peptides quantified, though CH digesta likely contains dozens of additional bioactive peptides not captured.
  • Only two hydrolysates compared (CH-GL Genacol vs CH-OPT); generalisability to marine, bovine, chicken, or other collagen sources not established.
  • Substantial commercial conflict of interest — study funded by MITACS partnership with Genacol Canada, senior and first authors received financial support from Genacol, and one of the two test hydrolysates is a Genacol commercial product.
  • Reported paired-t p-values without formal multiple-comparison correction across the peptide panel — findings for the largest effect (Gly-Pro-Hyp) are robust, but smaller-effect findings should be treated as exploratory.

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