Dietary collagen peptides alleviate exercise-induced muscle soreness in healthy middle-aged males: a randomized double-blinded crossover clinical trial.

BACKGROUND: Post-exercise muscle soreness and fatigue can negatively affect exercise performance. Thus, it is desirable to attenuate muscle soreness and fatigue and promote recovery even for daily exercise habits aimed at maintaining or improving health. METHODS: This study investigated the effects of dietary collagen peptides (CPs) on post-exercise physical condition and fitness in healthy middle-aged adults unfamiliar with exercise. Middle-aged males (n = 20, 52.6 ± 5.8 years) received the active food (10 g of CPs per day) or the placebo food for 33 days in each period of the randomized crossover trial (registered at the University Hospital Medical Information Network Clinical Trials Registry with UMIN-CTR ID of UMIN000041441). On the 29th day, participants performed a maximum of five sets of 40 bodyweight squats. Muscle soreness as the primary outcome, fatigue, the maximum knee extension force during isometric muscle contraction of both legs, the range of motion (ROM), and the blood level of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) were assessed before and after the exercise load. RESULTS: The analysis set was the per-protocol set (n = 18, 52.6 ± 6.0 years) for efficacy and the full analysis set (n = 19, 52.8 ± 5.9 years) for safety. The visual analog scale (VAS) of muscle soreness immediately after the exercise load was significantly lower in the active group than in the placebo group (32.0 ± 25.0 mm versus 45.8 ± 27.6 mm, p < 0.001). The VAS of fatigue immediately after the exercise load was also significantly lower in the active group than in the placebo group (47.3 ± 25.0 mm versus 59.0 ± 22.3 mm, p < 0.001). Two days (48 hours) afterthe exercise load, muscle strength was significantly higher in the active group than in the placebo group (85.2 ± 27.8 kg versus 80.5 ± 25.3 kg, p = 0.035). The level of CPK did not change over time. The level of LDH increased slightly but was not different between the groups. No safety-related issues were observed. CONCLUSIONS: These results showed that dietary CPs alleviated muscle soreness and fatigue and affected muscle strength after exercise load in healthy middle-aged males.

Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen.

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)10, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2' positions, which may be attributed to the larger space available for substrate binding at the S2 and S2' sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.

Identification of a highly stable bioactive 3-hydroxyproline-containing tripeptide in human blood after collagen hydrolysate ingestion.

There are increasing reports demonstrating high bioavailability of 4-hydroxyproline (4Hyp)-containing oligopeptides after oral ingestion of collagen hydrolysate and their bioactivity. In contrast, no study investigates the fate of another collagen-specific but minor amino acid, 3Hyp. Here, we identified Gly-3Hyp-4Hyp tripeptide in human blood at high concentrations, comparable to other 4Hyp-containing oligopeptides, after ingesting porcine skin collagen hydrolysate. Additionally, Gly-3Hyp-4Hyp uniquely maintained the maximum concentration until 4 h after the ingestion due to its exceptionally high resistance to peptidase/protease demonstrated by incubation with mouse plasma. In mice, oral administration of collagen hydrolysate prepared from bovine tendon, which contains a higher amount of 3Hyp, further increased blood Gly-3Hyp-4Hyp levels compared to that from bovine skin. Furthermore, Gly-3Hyp-4Hyp showed chemotactic activity on skin fibroblasts and promoted osteoblast differentiation. These results highlight the specific nature of the Gly-3Hyp-4Hyp tripeptide and its potential for health promotion and disease treatment.

Preservation of collagen in the soft tissues of frozen mammoths.

We investigated the characteristics of extracellular matrix (ECM) in the soft tissue of two frozen baby woolly mammoths (Mammuthus primigenius) that died and were buried in Siberian permafrost approximately 40,000 years ago. Morphological and biochemical analyses of mammoth lung and liver demonstrated that those soft tissues were preserved at the gross anatomical and histological levels. The ultrastructure of ECM components, namely a fibrillar structure with a collagen-characteristic pattern of cross-striation, was clearly visible with transmission and scanning electron microscopy. Type I and type IV collagens were detected by immunohistochemical observation. Quantitative amino acid analysis of liver and lung tissues of the baby mammoths indicated that collagenous protein is selectively preserved in these tissues as a main protein. Type I and type III collagens were detected as major components by means of liquid chromatography-mass spectrometry analysis after digestion with trypsin. These results indicate that the triple helical collagen molecule, which is resistant to proteinase digestion, has been preserved in the soft tissues of these frozen mammoths for 40,000 years.

The Transcription Factor HAND1 Is Involved in Cortical Bone Mass through the Regulation of Collagen Expression.

Temporal and/or spatial alteration of collagen family gene expression results in bone defects. However, how collagen expression controls bone size remains largely unknown. The basic helix-loop-helix transcription factor HAND1 is expressed in developing long bones and is involved in their morphogenesis. To understand the functional role of HAND1 and collagen in the postnatal development of long bones, we overexpressed Hand1 in the osteochondroprogenitors of model mice and found that the bone volumes of cortical bones decreased in Hand1Tg/+;Twist2-Cre mice. Continuous Hand1 expression downregulated the gene expression of type I, V, and XI collagen in the diaphyses of long bones and was associated with decreased expression of Runx2 and Sp7/Osterix, encoding transcription factors involved in the transactivation of fibril-forming collagen genes. Members of the microRNA-196 family, which target the 3' untranslated regions of COL1A1 and COL1A2, were significantly upregulated in Hand1Tg/+;Twist2-Cre mice. Mass spectrometry revealed that the expression ratios of alpha 1(XI), alpha 2(XI), and alpha 2(V) in the diaphysis increased during postnatal development in wild-type mice, which was delayed in Hand1Tg/+;Twist2-Cre mice. Our results demonstrate that HAND1 regulates bone size and morphology through osteochondroprogenitors, at least partially by suppressing postnatal expression of collagen fibrils in the cortical bones.

Quantitative Analysis of the Positional Distribution of Hydroxyproline in Collagenous Gly-Xaa-Yaa Sequences by LC-MS with Partial Acid Hydrolysis and Precolumn Derivatization.

Collagen is extensively modified by various enzymes, including prolyl hydroxylases. Pro residues at the Yaa position of repeating Gly-Xaa-Yaa amino acid sequences are mostly hydroxylated to 4-hydroxyproline (4Hyp), which is essential for the thermal stability of collagen triple helix. In contrast, Pro residues at the Xaa position are rarely modified to 3Hyp and 4Hyp, the biological function of which is poorly understood. Overall estimation of prolyl hydroxylation with discrimination of the position (Xaa or Yaa) and hydroxylation type (4Hyp or 3Hyp) has been difficult to perform using traditional methods. In the present study, we developed a novel position-specific analytical method featuring LC-MS detection of collagenous Gly-containing dipeptides, including Gly-Pro, Pro-Gly, Gly-4Hyp, Gly-3Hyp, and 4Hyp-Gly, after partial acid hydrolysis and precolumn derivatization using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS). We performed acid hydrolysis at 55 °C with HCl/trifluoroacetic acid/water (2:1:1, v/v) to avoid peptide inversion and imbalanced peptide generation observed for collagenous model peptides. The positional distribution of Pro, 4Hyp, and 3Hyp can be calculated from the relative concentrations of the APDS-derivatized dipeptides, and in combination with amino acid analysis, we can determine their absolute contents at the Xaa and Yaa positions. Bovine type I, III, and V collagens were analyzed by the established method, and the amount of 4Hyp was higher than that of 3Hyp at the Xaa position in type I and III collagens. In addition, we clearly showed that collagen extracted from earthworm cuticles has an extremely high content of Xaa position 4Hyp, reaching over 10% of the total amino acids.

Prolyl-hydroxyproline, a collagen-derived dipeptide, enhances hippocampal cell proliferation, which leads to antidepressant-like effects in mice.

Depression has been a mental health issue worldwide. We previously reported that ginger-degraded collagen hydrolysate (GDCH) suppressed depression-like behavior in mice. Furthermore, prolyl-hydroxyproline (PO) and hydroxyprolyl-glycine (OG) were detected in the circulating blood after the oral administration of GDCH. In the present study, PO, but not OG, was detected in the cerebrospinal fluid of rats after the oral administration of GDCH, suggesting that PO is transported from blood to the brain. We then investigated the effects of PO and OG on the depression-like behavior of mice. The oral administration of PO significantly decreased depression-like behavior in the forced swim test. OG had no antidepressant-like effect. In addition, proline and hydroxyproline, components of PO, also had no antidepressant-like effect after their oral administration. PO significantly increased the gene expression of brain-derived neurotrophic factor and nerve growth factor in the hippocampus, and promoted the proliferation of neural progenitor cells in vivo and in vitro. PO also increased the dopamine concentration in the prefrontal cortex. Thus, PO-dependent regulation of neurotrophic function and neurotransmitter may be the mechanism for antidepressant-like behavior. Together, these results demonstrate that PO is an antidepressant bioactive peptide accompanying the proliferation of hippocampal neural progenitor cells.

Ginger-Degraded Collagen Hydrolysate Exhibits Antidepressant Activity in Mice.

Collagen is the most abundant protein in animals. Collagen hydrolysate has been found to have multiple functions in the skin, bones, joints, muscles, and blood vessels. Recently, it has been reported that the low molecular weight fraction of collagen hydrolysate exhibited anxiolytic activity, suggesting that collagen peptides affect brain functions. In the present study, we found that oral administration of ginger-degraded collagen hydrolysate (GDCH) significantly decreased depression-like behavior in a forced swim test, suggesting that GDCH exhibited antidepressant activity in mice. The antidepressant activity of GDCH was abolished by pre-treatment with an antagonist of the dopamine receptor, but not treatment with a serotonin receptor antagonist. GDCH significantly increased gene expression of glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) in the hippocampus, molecules that affect the differentiation and survival of neurons, relative to that in the control condition. Meanwhile, there were no changes in the gene expression of brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3, major factors related to depression-like behavior. We also found that GDCH exhibited antidepressant activity in corticosterone-administered mice in a model of stress. In addition, GDCH increased GDNF and CNTF expression in the stressed condition, suggesting that mechanisms of the antidepressant activity of GDCH were the same in unstressed and stressed conditions. These results imply that GDCH exhibits antidepressant activity in unstressed and stressed conditions in mice. The upregulation of neurotrophic genes in the hippocampus may contribute to the reduction of depression-like behavior via a dopamine signal pathway modulated by GDCH.

Characterization of Angiotensin-Converting Enzyme Inhibitory Activity of X-Hyp-Gly-Type Tripeptides: Importance of Collagen-Specific Prolyl Hydroxylation.

Hydroxyproline (Hyp) is a collagen-specific amino acid formed by post-translational hydroxylation of Pro residues. Various Hyp-containing oligopeptides are transported into the blood at high concentrations after oral ingestion of collagen hydrolysate. Here we investigated the angiotensin-converting enzyme (ACE) inhibitory activity of X-Hyp-Gly-type tripeptides. In an in vitro assay, ginger-degraded collagen hydrolysate enriched with X-Hyp-Gly-type tripeptides dose-dependently inhibited ACE and various synthetic X-Hyp-Gly-type tripeptides showed ACE-inhibitory activity. In particular, strong inhibition was observed for Leu-Hyp-Gly, Ile-Hyp-Gly, and Val-Hyp-Gly with IC50 values of 5.5, 9.4, and 12.8 µM, respectively. Surprisingly, substitution of Hyp with Pro dramatically decreased inhibitory activity of X-Hyp-Gly, indicating that Hyp is important for ACE inhibition. This finding was supported by molecular docking experiments using Leu-Hyp-Gly/Leu-Pro-Gly. We further demonstrated that prolyl hydroxylation significantly enhanced resistance to enzymatic degradation by incubation with mouse plasma. The strong ACE-inhibitory activity and high stability of X-Hyp-Gly-type tripeptides highlight their potential for hypertension control.

Collagen-derived peptides modulate CD4+ T-cell differentiation and suppress allergic responses in mice.

INTRODUCTION: Collagen peptides have been widely used as a food supplement. After ingestion of collagen peptides, oligopeptides containing hydroxyproline (Hyp), which are known to have some physiological activities, are detected in peripheral blood. However, the effects of collagen-peptide administration on immune response are unclear. In the present study, we tested the effects of collagen-peptide ingestion on allergic response and the effects of collagen-derived oligopeptides on CD4+ T-cell differentiation. METHODS: BALB/c mice fed a collagen-peptide diet were immunized with ovalbumin (OVA), and their serum IgE and IgG levels, active cutaneous anaphylaxis, and cytokine secretion by splenocytes were examined. Naive CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of collagen-derived oligopeptides, and the expression of IFN-γ, IL-4, and Foxp3 was analyzed. RESULTS: In an active anaphylaxis model, oral administration of collagen peptides suppressed serum OVA-specific immunoglobulin E (IgE) production and diminished anaphylaxis responses. In this model, the ingestion of collagen peptides skewed the pattern of cytokine production by splenocytes toward T-helper (Th) type 1 and regulatory T (Treg) cells. In vitro T-helper cell differentiation assays showed that Hyp-containing oligopeptides promoted Th1 differentiation by upregulating IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) signaling. These oligopeptides also promoted the development of Foxp3+ Treg cells in response to antigen stimulation in the presence of TGF-ß. CONCLUSIONS: Collagen-peptide ingestion suppresses allergic responses by skewing the balance of CD4+ T cells toward Th1 and Treg cells and seems to be a promising agent for preventing allergies and inflammatory diseases.

Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating.

Cyclic dipeptides (2,5-diketopiperazines) are present in a variety of foods and are reported to demonstrate antioxidant, antidepressant, and other beneficial effects. We recently developed a novel collagen hydrolysate characterized by a high content of X-hydroxyproline (Hyp)-Gly-type tripeptides using ginger protease. In the present study, we found that, through heating, X-Hyp-Gly can be easily converted into Hyp-containing cyclic dipeptides. After heating for 3 h at 85 °C and pH 4.8, Ala-Hyp-Gly was almost completely cyclized to cyclo(Ala-Hyp), in contrast to a slight cyclization of Ala-Hyp. The contents of cyclo(Ala-Hyp) and cyclo(Leu-Hyp) reached 0.5-1% (w/w) each in the ginger-degraded collagen hydrolysate under the heating conditions. Oral administration experiments using mice revealed that cyclo(Ala-Hyp) and cyclo(Leu-Hyp) were absorbed into the blood at markedly higher efficiencies compared to collagenous oligopeptides, including Pro-Hyp. The high productivity and oral bioavailability of the collagen-specific cyclic dipeptides suggest significant health benefits of the heat-treated ginger-degraded collagen hydrolysate.

Hydroxyhomocitrulline Is a Collagen-Specific Carbamylation Mark that Affects Cross-link Formation.

Carbamylation is a non-enzymatic post-translational modification that physiologically occurs during aging and is a risk factor for various diseases. The most common product of carbamylation is homocitrulline (HCit), where a lysine (Lys) amino group has reacted with urea-derived cyanate. HCit has recently been detected in collagen; however, given that 15%-90% of total Lys in collagen is hydroxylated, it is unclear how hydroxylation affects collagen carbamylation. Here, we identified a collagen-specific carbamylation product, hydroxyhomocitrulline (HHCit), and showed that high levels of HHCit are correlated with age in rat tissue collagen and in vivo carbamylation in mice, as well as with the decline of kidney function in the serum of dialysis patients. Proteomic analysis of the carbamylated collagens identified α2(I) Lys933, a major cross-linking site, as a preferential HHCit site. Furthermore, our results suggest that hydroxylysine carbamylation affects the mechanical properties of connective tissue by competitively inhibiting collagen cross-link formation.

Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease.

Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P2 position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of <600 Da. In addition, the gluten hydrolysate contained substantial amounts of tripeptides, including Gln-Pro-Gln, Gln-Pro-Gly, Gln-Pro-Phe, Leu-Pro-Gln, and Ser-Pro-Gln (e.g. 40.7 mg/g at pH 5.2). These gluten-derived tripeptides showed high inhibitory activity on dipeptidyl peptidase-IV with IC50 values of 79.8, 70.9, 71.7, 56.7, and 78.9 µM, respectively, suggesting that the novel gluten hydrolysate prepared using ginger protease can be used as a functional food for patients with type 2 diabetes.

Efficient Absorption of X-Hydroxyproline (Hyp)-Gly after Oral Administration of a Novel Gelatin Hydrolysate Prepared Using Ginger Protease.

Recent studies have reported that oral intake of gelatin hydrolysate has various beneficial effects, such as reduction of joint pain and lowering of blood sugar levels. In this study, we produced a novel gelatin hydrolysate using a cysteine-type ginger protease having unique substrate specificity with preferential peptide cleavage with Pro at the P2 position. Substantial amounts of X-hydroxyproline (Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The in vivo absorption of the ginger-degraded gelatin hydrolysate was estimated using mice. The plasma levels of collagen-derived oligopeptides, especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for Glu-Hyp-Gly, p < 0.05) compared with those of the control gelatin hydrolysate, which was prepared using gastrointestinal proteases and did not contain detectable X-Hyp-Gly. This study demonstrated that orally administered X-Hyp-Gly was effectively absorbed into the blood, probably due to the high protease resistance of this type of tripeptide.

Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen.

3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5-18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro(707) and newly identified sites at Pro(716) and Pro(719), at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.

Oral ingestion of collagen peptide causes change in width of the perimysium of the chicken iliotibialis lateralis muscle.

Skeletal muscle is mainly composed of myofibers and intramuscular connective tissue. Bundles composed of many myofibers, with each myofiber sheathed in connective tissue called the endomysium, are packed in the perimysium, which occupies the vast bulk of the intramuscular connective tissue. The perimysium is a major determination factor for muscle texture. Some studies have reported that collagen peptide (Col-Pep) ingestion improves the connective tissue architecture, such as the tendon and dermis. The present study evaluated the effects of Col-Pep ingestion on the chicken iliotibialis lateralis (ITL) muscle. Chicks were allocated to three groups: the 0.15% or 0.3% Col-Pep groups and a control group. Col-Pep was administered by mixing in with commercial food. On day 49, the ITL muscles were analyzed by morphological observation and the textural property test. The width of the perimysium in the 0.3% Col-Pep group was significantly larger than other two groups. Although scanning electron microscopic observations did not reveal any differences in the architecture of the endomysium, elastic improvement of the ITL muscle was observed as suggested by an increase of the width of perimysium and improved rheological properties. Our results indicate that ingestion of Col-Pep improves the textural property of ITL muscle of chickens by changing structure of the perimysium.

Detection of endogenous and food-derived collagen dipeptide prolylhydroxyproline (Pro-Hyp) in allergic contact dermatitis-affected mouse ear.

Generation of collagen dipeptides and deposition of orally administered prolylhydroxyproline (Pro-Hyp) in local inflammatory sites were examined in mice with hapten (2,4-dinitrofluorobenzene)-induced dermatitis in the ear. Pro-Hyp content in the hapten-treated ear was significantly higher in the chronic phase of contact dermatitis than the vehicle control. In contrast, hydroxyprolylglycine contents remained at lower levels in all cases compared to Pro-Hyp. Four hours after the ingestion of [(13)C5,(15)N]Pro and [(13)C5,(15)N]Pro-Hyp, labeled-Pro-Hyp and Pro, respectively, appeared only in the ear with dermatitis. Thus, Pro-Hyp is generated and degraded as part of the rapid synthesis and degradation of collagen in the ear with dermatitis. In addition to the endogenously generated Pro-Hyp, the orally administered Pro-Hyp was deposited in the ears.

Medical-grade collagen peptide in injectables provides antioxidant protection.

Medical-grade collagen peptide is used as an additive agent in pharmaceutical formulations; however, it is unknown as to whether the compound exerts antioxidant effects in vitro. In this study, we evaluated the antioxidant effects of medical-grade collagen peptide on reactive oxygen species such as hydroxyl radical, superoxide anion radical and singlet oxygen using electron spin resonance and spin trapping. We confirmed that medical-grade collagen peptide directly inhibited hydroxyl radical generated by the Fenton reaction or by ultraviolet irradiation of hydrogen peroxide, and singlet oxygen. In addition, an antioxidant effect of medical-grade collagen peptide on singlet oxygen was observed in peptide fractions 12-22. The total amount of antioxidant amino acids (Gly, Hyp, Glu, Ala, Cys, Met and His) constituted more than half of the total amino acids in these fractions. These results suggest that the observed antioxidant properties of medical-grade collagen peptide are due to the compound containing antioxidant amino acids. Medical-grade collagen peptide, which is used in pharmaceuticals, and especially in injectables, could provide useful antioxidant properties to protect the active ingredient from oxidation.

Highly accurate quantification of hydroxyproline-containing peptides in blood using a protease digest of stable isotope-labeled collagen.

Collagen-derived hydroxyproline (Hyp)-containing dipeptides and tripeptides, which are known to possess physiological functions, appear in blood at high concentrations after oral ingestion of gelatin hydrolysate. However, highly accurate and sensitive quantification of the Hyp-containing peptides in blood has been challenging because of the analytical interference from numerous other blood components. We recently developed a stable isotope-labeled collagen named "SI-collagen" that can be used as an internal standard in various types of collagen analyses employing liquid chromatography-mass spectrometry (LC-MS). Here we prepared stable isotope-labeled Hyp-containing peptides from SI-collagen using trypsin/chymotrypsin and plasma proteases by mimicking the protein degradation pathways in the body. With the protease digest of SI-collagen used as an internal standard mixture, we achieved highly accurate simultaneous quantification of Hyp and 13 Hyp-containing peptides in human blood by LC-MS. The area under the plasma concentration-time curve of Hyp-containing peptides ranged from 0.663 ± 0.022 nmol/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was <7% for each peptide except for Glu-Hyp-Gly, which was near the detection limit. Our method is useful for absorption/metabolism studies of the Hyp-containing peptides and development of functionally characterized gelatin hydrolysate.