Food-Derived Collagen Peptides, Prolyl-Hydroxyproline (Pro-Hyp), and Hydroxyprolyl-Glycine (Hyp-Gly) Enhance Growth of Primary Cultured Mouse Skin Fibroblast Using Fetal Bovine Serum Free from Hydroxyprolyl Peptide.

Tomoko T Asai,Kenji Sato,Kazunobu Yoshikawa,Fumi Oikawa,Naoki Inoue

doi:10.3390/ijms21010229

Abstract

Prolyl-hydroxyproline (Pro-Hyp) and hydroxyprolyl-glycine (Hyp-Gly) appear in human blood after ingestion of collagen hydrolysate and trigger growth of fibroblasts attached on collagen gel, which has been associated with beneficial effects upon ingestion of collagen hydrolysate, such as improvement of skin and joint conditions. In the present study, inconsistent results were obtained by using different lots of fetal bovine serum (FBS). Fibroblasts proliferated in collagen gel without adding Pro-Hyp and Hyp-Gly and did not respond to addition of Pro-Hyp and Hyp-Gly, which raises doubts about conclusions from prior research. Unexpectedly high levels of hydroxyprolyl peptides, including Pro-Hyp, however, were present in the FBS (approximately 100 µM), and also in other commercially available forms of FBS (70–80 µM). After removal of low molecular weight (LMW, < 6000 Da) compounds from the FBS by size exclusion chromatography, Pro-Hyp and Hyp-Gly again triggered growth of fibroblasts attached on collagen and increased the number of fibroblasts migrated from mouse skin. These results indicate the presence of bioactive hydroxyprolyl peptides in commercially available FBS, which can mask effects of Pro-Hyp and Hyp-Gly supplementation; our work confirms that Pro-Hyp and Hyp-Gly do play crucial roles in proliferation of fibroblasts.

Highlights

Collagen is the main protein in the extracellular matrix and has a triple-helical structure
We previously reported that Pro-Hyp and Hyp-Gly triggered the growth fibroblasts attached on collagen gel [11,17], which has been associated with biological responses upon of fibroblasts attached on collagen gel [11,17], which has been associated with biological responses ingestion of collagen hydrolysate
The present study demonstrates that some commercially available fetal bovine serum (FBS) contain high levels of low molecular weight (LMW) hydroxyprolyl peptides (70–100 μM), including Pro-Hyp

Summary

Introduction

Collagen is the main protein in the extracellular matrix and has a triple-helical structure. Collagen has two specific post-translationally modified amino acids: hydroxyproline (Hyp) and hydroxylysine (Hyl). Heat treatment converts the triple-helical structure of collagen into a globular structure, which is referred to as gelatin. The protease digest of gelatin is referred to as collagen hydrolysate, gelatin hydrolysate, or collagen peptide. Collagen hydrolysate is prepared from skin, bones, and tendons of animals, or the skin and scales of fish. In human trials with placebo controls, ingestion of collagen hydrolysate (2.5–10 g/day) suppresses transepidermal water loss, reduces wrinkle volume, and increases elasticity of skin [1,2,3,4]. Ingestion of collagen hydrolysate moderates the symptoms of

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