Chemicals
Hydroxyproline (Hyp), 8-hydroxy-2’-deoxyguanosine (8-OHdG), 3-methylhistidine (3-MH) and creatinine were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Pro-Hyp, Hyp-Gly, Pro-Pro, Gly-Pro-Hyp, Pro-Hyp-Gly, were purchased from GL Biochem (Shanghai, China). The fish-derived low-molecular weight commercial collagen peptide (CP, Wellnex TYPE-S) was purchased from Nitta Gelatin (Osaka, Japan). The Pierce BCA Protein Assay Kit was purchased from Bio-Rad (Hercules, CA, USA). The ELISA kit was purchased from Air Plants Bio (Tokyo, Japan). The other analytical grade chemicals were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan).
Study approval
This study was approved by the Ethics Committee of Juntendo University (No. 2018133) and conducted in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects published by the Ministry of Health, Labour and Welfare of Japan and the Declaration of Helsinki. Written informed consent was obtained from all participants.
Cross-reactivity confirmation
Five collagen-derived peptides (Pro-Hyp, Hyp-Gly, Gly-Pro-Hyp, Pro-Hyp-Gly, Pro-Pro) and CP were mixed with purified water to a concentration of 20 mg/mL. These samples were analyzed by ELISA for cross-reactivity with the anti-ACOP antibody.
Urine ACOP concentration before and after CP ingestion
One healthy, non-athlete female subject in her thirties was limited to a low-protein diet for dinner and a 12-hour fast before the start of the experiment. During this time, the subject was restricted from taking supplements and medications, and liquid intake was limited to water only. The subject was then orally administered 20 g of commercial CP. Urine samples were collected before and 1, 2, and 4 hours after ingestion, stored at −20°C, and analyzed for the content of ACOP, collagen-derived di- and tripeptides, Hyp, and creatinine. The concentration of all analyzed urine constituents was normalized to that of creatinine.
Urine collection before and after physical activity
Forty-six healthy male university students in their teens and twenties were recruited from a running club at Josai University. All subjects received no intervention regarding diet, dietary supplements, medications, hydration intake, or exercise regimen during the study. Results of a dietary questionnaire confirmed that all subjects had eaten the same dormitory food the day before the study. The subjects were divided into two groups according to whether they did (n = 24) or did not (n = 22) take CP routinely. While the subjects were not asked about their specific CP intake, we did confirm that they had not taken any CP-containing supplements after 20:00 the day before. Physical exercise among the cohort consisted of running for a mean duration of 1 hour and a distance of approximately 13 km. Urine samples were collected from the subjects before and after physical activity, stored at −20°C, and analyzed for the content of ACOP, collagen-derived di- and tripeptides, Hyp, 8-OHdG, 3-MH, total protein, and creatinine. The concentration of all analyzed urine constituents was normalized to that of creatinine. Concentrations before and after physical activity were compared between the two groups.
ELISA
Active collagen oligopeptide concentration was determined using the ELISA kit according to the manufacturer’s instructions. The concentrations were calculated in terms of Pro-Hyp equivalents using a Pro-Hyp standard.
LC-MS/MS analysis
LC-MS/MS was used to measure Pro-Hyp, Hyp-Gly, Gly-Pro-Hyp, Pro-Hyp-Gly, and 3-MH concentrations. An aliquot of urine diluted 500-fold in 50 mM ammonium bicarbonate solution was added to a 3-fold volume of ethanol. Samples were centrifuged at 3000 rpm for 5 min followed by filtration through a 0.22 µm filter. An aliquot of each sample was used for mass spectrometry. Chromatography analysis was performed using an ACQUI UPLC H-Class Bio system (Waters, MA, USA) equipped with a Hypersil GOLD PFP column (2.1 × 150 mm, 5 µm; Thermo Fisher Scientific, Waltham, MA, USA). The isocratic elution was performed with 2% methanol/0.2% formic acid/2 mM ammonium acetate in water under the following conditions: sample injection volume, 0.5 µL; flow rate, 0.4 mL/min; elution time, 3.5 min; column temperature, 40°C. Mass spectrometry was performed using a Xevo TQ-XS quadrupole mass spectrometer (Waters, Milford, MA, USA) under the following conditions: capillary voltage, 1000 V (positive ionization mode); desorption temperature, 500°C; source temperature, 150°C. Data were acquired using selective reaction monitoring with m/z transition of 229 > 132 for Pro-Hyp, 189 > 86 for Hyp-Gly, 286 > 189 for Pro-Hyp-Gly, 286 > 155, and 170 > 124 for 3-MH. Data were acquired and quantified using Mass Lynx software (version 4.2, Waters).
To quantify 8-OHdG, an aliquot of each urine sample was diluted 10-fold in pure water, centrifuged at 3000 rpm (1000 × g) for 5 min, and filtered using a 0.22 µm filter. An aliquot of each filtrate was analyzed by mass spectrometry. Chromatography analysis was performed with an UPLC proteomics Series (Shimadzu, Kyoto, Japan) equipped with an Inertsustain AQ-C18 (2.1 x 150 mm, 3 µm; GL Sciences, Tokyo, Japan). The. The isocratic elution was performed with 8% acetonitrile/0.1% acetic acid in water under the following conditions: sample injection volume, 10 µL; flow rate, 0.3 mL/min; elution time, 3 min; column temperature, 40°C. Mass spectrometry was performed using a 4000 TRAP quadrupole mass spectrometer (AB Sciex, Tokyo, Japan) under the following conditions: ion spray voltage, 5500 V (positive ionization mode); source temperature, 500°C. Data were acquired using selective reaction monitoring with m/z transition of 284 > 168 for 8-OHdG. Data were acquired and quantified using Analyst software (version 1.5, AB Sciex).
Colorimetry
Hyp concentrations were determined by colorimetric analysis using the Ehrlich reagent after oxidation with chloramine T.30 An aliquot of the urine sample was hydrolyzed by treatment with 6 N hydrochloric acid at 110°C for 24 hours and then neutralized w sodium hydroxide. The hydrolyzed sample analyzed for Hyp content. Free and total Hyp were determined before and after hydrolysis. The peptide form of Hyp was estimated by subtracting the concentration of free Hyp from that of total Hyp. Total protein concentration was determined using the colorimetric assay kit according to the manufacturer’s instructions. Creatinine concentrations were determined using the Jaffe reaction.31
Statistical analysis
Statistical analysis was performed using EZR (version 1.40, Saitama Medical Center, JICHI Medical University, Saitama, Japan).32 The coefficient of determinations was calculated to determine whether ACOP concentration correlated with other that of collagen metabolites. Two-way analysis of variance was used to examine the effect of routine CP intake and physical activity on urinary components. The Smirnov-Grubbs test was used to detect outliers. P < 0.05 was considered statistically significant.