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, 21 (1)

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

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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 et al. Int J Mol Sci.

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.

Keywords: collagen peptide; fetal bovine serum (FBS); fibroblasts; hydroxyprolyl-glycine (Hyp-Gly); prolyl-hydroxyproline (Pro-Hyp).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of a mixture of Pro-Hyp and Hyp-Gly on the growth of fibroblasts on collagen gel in the presence of 10% FBS-1; (□), control; (■), medium containing Pro-Hyp and Hyp-Gly at 100 µM, respectively. Data are shown as mean ± standard deviation (SD) (n = 5). Asterisks indicate significant differences (p < 0.05; Tukey’s test). N.S. indicates results that are not significantly different.
Figure 2
Figure 2
Contents of hydroxyprolyl peptide in commercially available fetal bovine serum (FBS) and adult bovine serum (ABS): (A) Hyp-containing peptide; (B) content of Pro-Hyp () and Hyp-Gly (■); a peptide form of Hyp.
Figure 3
Figure 3
Elution of protein and hydroxyprolyl peptide in FBS-1 from the Econo-Pac 10DG column; (■), protein; (), Hyp-containing peptide; a peptide form of Hyp.
Figure 4
Figure 4
Effect of FBS-1 and its fractions on fibroblast growth on plastic plates. FBS-1 free from LMW, SEC Fr. 4–7; LMW of FBS-1, SEC Fr. 8–13. Data are shown as the mean ± SD (n = 5). Different letters indicate significant differences (p < 0.05, Tukey’s test).
Figure 5
Figure 5
Effect of mixture of Pro-Hyp and Hyp-Gly on the growth of fibroblasts on collagen gel in the presence of 10% FBS-1 free from LMW hydroxyprolyl peptides; (□), control; (■), medium containing Pro-Hyp and Hyp-Gly at 100 µM. Data are shown as mean ± SD (n = 5). Asterisks indicate significant differences (p < 0.05, Tukey’s test). N.S. indicates results that are not significantly different.
Figure 6
Figure 6
Effect of Pro-Hyp on the number of fibroblasts migrated from mouse skin in the absence or presence of FBS-1 free from LMW hydroxyprolyl peptides. Open column, control; closed column, medium containing Pro-Hyp at 200 µM. Data are shown as the mean ± SD (n = 6). Asterisks indicate significant differences (p < 0.05, Student’s t-test).

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