ORIGINAL RESEARCH published: 11 October 2021 doi: 10.3389/fphys.2021.722718 Marathon Running Increases Synthesis and Decreases Catabolism of Joint Cartilage Type II Collagen Accompanied by High-Energy Demands and an Inflamatory Reaction Edited by: Beat Knechtle, José A. Hernández-Hermoso 1,2*† , Lexa Nescolarde 3† , Emma Roca 4 , Universitätsklinikum Elena Revuelta-López 5 , Jordi Ara 6,7 and Antoni Bayes-Genis 5,6,8 Zürich, Switzerland 1 Department of Orthopedic Surgery and Traumatology, Hospital Universitari Germans Trias i Pujol, Barcelona, Spain, Reviewed by: 2 Department of Surgery, Faculty of Medicine, Universitat Autònoma Barcelona, Bellaterra, Spain, 3 Department of Electronic Anna-Maria Liphardt, Engineering, Universitat Politècnica de Catalunya, Barcelona, Spain, 4 Summit 2014 SL, Barcelona, Spain, 5 Research University of Erlangen Program, Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Barcelona, Spain, 6 Departament of Nuremberg, Germany Medicine, Faculty of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain, 7 Department of Nephrology, Hospital Canan Yilmaz, Universitari Germans Trias i Pujol, Barcelona, Spain, 8 Department of Cardiology, Hospital Universitari Germans Trias i Pujol, Gazi University, Turkey Barcelona, Spain Heidi Munk, Odense University Hospital, Denmark *Correspondence: Objective: To determine the effect of marathon running on serum levels of inflammatory, José A. Hernández-Hermoso high energy, and cartilage matrix biomarkers and to ascertain whether these biomarkers jahernandezh.germanstrias@ gencat.cat levels correlate. orcid.org/0000-0002-9167-3782 Design: Blood samples from 17 Caucasian male recreational athletes at the Barcelona † These authors have contributed Marathon 2017 were collected at the baseline, immediately and 48 h post-race. Serum equally to this work and share first authorship C reactive protein (CRP), creatin kinase (CK), and lactate dehydrogenase (LDH) were determined using an AU-5800 chemistry analyser. Serum levels of hyaluronan (HA), Specialty section: cartilage oligomeric matrix protein (COMP), aggrecan chondroitin sulphate 846 (CS846), This article was submitted to glycoprotein YKL-40, human procollagen II N-terminal propeptide (PIINP), human type Exercise Physiology, a section of the journal IIA collagen N-propeptide (PIIANP), and collagen type II cleavage (C2C) were measured Frontiers in Physiology by sandwich enzyme-linked immune-sorbent assay (ELISA). Received: 09 June 2021 Accepted: 06 September 2021 Results: Medians CK and sLDH levels increased (three-fold, two-fold) post-race Published: 11 October 2021 [429 (332) U/L, 323 (69) U/L] (p < 0.0001; p < 0.0001) and (six-fold, 1.2-fold) 48 h Citation: post-race [658 (1,073) U/L, 218 (45) U/L] (p < 0.0001; p < 0.0001). Medians CRP Hernández-Hermoso JA, increased (ten-fold) after 48 h post-race [6.8 (4.1) mg/L] (p < 0.0001). Mean sHA levels Nescolarde L, Roca E, Revuelta-López E, Ara J and increased (four-fold) post-race (89.54 ± 53.14 ng/ml) (p < 0.0001). Means PIINP (9.05 Bayes-Genis A (2021) Marathon ± 2.15 ng/ml) levels increased post-race (10.82 ± 3.44 ng/ml) (p = 0.053) and 48 h Running Increases Synthesis and Decreases Catabolism of Joint post-race (11.00 ± 2.96 ng/ml) (p = 0.001). Mean sC2C levels (220.83 ± 39.50 ng/ml) Cartilage Type II Collagen decreased post-race (188.67 ± 38.52 ng/ml) (p = 0.002). In contrast, means COMP, Accompanied by High-Energy sCS846, sPIIANP, and median sYKL-40 were relatively stable. We found a positive Demands and an Inflamatory Reaction. Front. Physiol. 12:722718. association between sCK levels with sLDH pre-race (r = 0.758, p < 0.0001), post-race doi: 10.3389/fphys.2021.722718 (r = 0.623, p = 0.008) and 48-h post-race (r = 0.842, p < 0.0001); sHA with sCRP Frontiers in Physiology | www.frontiersin.org 1 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers post-race vs. 48 h post-race (r = 0.563, p = 0.019) and sPIINP with sCK pre-race vs. 48- h post-race (r = 0.499, p = 0.044) and with sLDH 48-h pre-race vs. post-race (r = 0.610, p = 0.009) and a negative correlation of sPIIANP with sCRP 48-h post-race (r = −0.570, p = 0.017). Conclusion: Marathon running is an exercise with high-energy demands (sCK and sLDH increase) that provokes a high and durable general inflammatory reaction (sCRP increase) and an immediately post-marathon mechanism to protect inflammation and cartilage (sHA increase). Accompanied by an increase in type II collagen cartilage fibrils synthesis (sPIINP increase) and a decrease in its catabolism (sC2C decrease), without changes in non-collagenous cartilage metabolism (sCOMP, sC846, and sYKL-40). Metabolic changes on sPIINP and sHA synthesis may be related to energy consumption (sCK, sLDH) and the inflammatory reaction (sCRP) produced. Keywords: biomarker, marathon runners, articular cartilage, PIINP, C2C, hyaluronic acid INTRODUCTION new therapeutic alternatives to maintain cartilage thickness as vibration training (Liphardt et al., 2009). Despite the substantial increase in marathon runners, still, Inflammatory biomarkers as C-reactive protein (CRP), an it is not known what is a physiological or expected acute acute-phase inflammatory protein, and high-energy enzyme inflammatory or cartilage metabolic response to an increase in biomarkers as creatin kinase (CK), a high-energy phosphate physical activity (Cattano et al., 2017b; Driban et al., 2017). enzyme, reportedly have a dose-dependent behaviour with a Long-distance running imposes high levels of stress on the transient increase after a 24-h marathon and ultra-marathon musculoskeletal system, causing changes in cartilage volume (Kim et al., 2009). Lactate dehydrogenase (LDH) is a key (Boocock et al., 2009; Doré et al., 2013) and numerous cellular glycolytic enzyme that increases with increased training loads and molecular changes (Cattano et al., 2017b). The intensive (Radzimiński et al., 2020). training and the marathon itself induce inflammation and Mechanosensitive non-collagenous joint homeostasis muscle fibre necrosis (Neidhart et al., 2000). Blood-circulating biomarkers as the hyaluronic acid (HA) have shown that their biomarkers used to monitor inflammation and joint homeostasis serum level decreases within 1 h of walking (Pruksakorn et al., change after joint loading with different types of activity level or 2013) on healthy individuals. COMP seems to have a temporary sports, and offer a method to analyse cartilage metabolic response dose-dependent increase in response to physical activity with a to physical activity, although data available give contrasting gradual post-activity return to normal (Mündermann et al., 2005; results (Cattano et al., 2017b). The discrepancy of these results Erhart-Hledik et al., 2012; Celik et al., 2013; Pruksakorn et al., may be attributable to differences in the study methods and 2013; Matt Denning et al., 2015; Liphardt et al., 2018). CS846, an to many confounding factors such as ethnicity (Jordan et al., aggrecan synthesis marker, after a 30-min walk, correlates with 2003), gender (Jordan et al., 2003; Niehoff et al., 2010), body cartilage thickening of the lateral femur at 5-year follow-up (Chu weight (Matt Denning et al., 2015), activity time or level (Cattano et al., 2018). Human chitinase-3-like-1 protein, YKL-40 synthesis et al., 2017a), loading protocols (Niehoff et al., 2010), vibration is related to alteration of cartilage in osteoarthritis (Di Rosa et al., (Cattano et al., 2017b), food intake (Gordon et al., 2008), 2013) and to altered joint mechanical conditions after anterior diurnal variation (Andersson et al., 2006), and osteoarthritis cruciate transection in dogs (Lorenz et al., 2005). (Chu et al., 2018); further, biomarkers may adapt to training over Mechanosensitive cartilage collagen biomarkers as the time (Cattano et al., 2017b). To date, the correlation between Procollagen type II N terminal pro-peptide (PIINP) is a inflammatory, energy enzymes, and collagenous and non- precursor of type II collagen, a major structural protein of collagenous cartilage markers after a marathon run is unknown. cartilage that exists in two splice variants termed PIIANP and Inflammatory and joint homeostasis (anabolic and catabolic) PIIBNP; the concentration of these peptides assesses biosynthetic biomarkers can be informative about the overall physiological activity (Munk et al., 2014). The cleavage of type II collagen state of a patient but are less informative for determining if a by collagenases yields fragments, such as the C2C epitope, specific joint of a patient is failing to adapt to a given loading that correlate with cartilage degeneration (King et al., 2004). stimulus (Cattano et al., 2017b). Since metabolic changes of Well-rounded exercise may have beneficial effects on type II joint tissues begin long before the onset of structural alterations, collagen metabolism (Azukizawa et al., 2019); however, the biomarker responses to physical activity may serve as indicators behaviour of metabolites, such as PIINP, PIIANP, and C2C, after of tolerance (or intolerance) to physical activity and could load or sports has not been investigated. potentially be utilised to help in prediction of cartilage loss The aim of the study was to extend knowledge about the from post-walking (Erhart-Hledik et al., 2012) or in developing response of serum levels of inflammatory proteins, high-energy Frontiers in Physiology | www.frontiersin.org 2 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers enzymes, and synthesis and degradation of non-collagenous each participant was the official time from the race organisation and type II collagenous cartilage biomarkers pre- and post- (Zurich Barcelona Marathon). marathon running in healthy non-elite athletes and to ascertain Blood samples were collected by a certified phlebotomist from whether they correlate. We hypothesised marathon running an antecubital vein immediately after arrival to the laboratory. would increase the metabolism of CRP (inflammatory), CK, The area was cleaned with isopropyl alcohol, then the needle LDH (high-energy), COMP, CS846, YKL40 (non-collagenous), was inserted, and blood was drawn into an 8.5 ml, 16 × PIINP, and PIIANP (type II collagen) biomarkers and decrease 100-mm Vacutainer R BD R tube with SSTTM II Advance gel HA (non-collagenous) and C2C (cartilage degradation) (reference 5T02.367953) at the baseline 24–48 h before the race, biomarkers. We further hypothesised that inflammatory and immediately post-race and 48 h post-race. Non-fasting blood energy metabolic biomarkers increase may be related with samples were collected at the same time of the day (from 11:30 h non-collagenous and type II collagenous cartilage biomarkers to 13 h am). Before the pre and 48-h post-race blood sample draw, metabolic changes. the subjects were recommended to have breakfast, after overnight fasting, with sports drinks, fruits, and nuts from 9 to 10 am. The blood samples were centrifuged at 3,000 rpm at 4◦ C for 10 min MATERIALS AND METHODS in a bench-top centrifuge. The supernatant serum was aliquoted and stored in dry ice until samples were frozen at −80◦ C in We examined the dynamics of a panel of inflammatory, sealed Eppendorf tubes to avoid evaporation until analysis. The high-energy, and cartilage biomarkers in non-elite athletes laboratory analysis was done in the IGTP laboratories that is completing the Barcelona Marathon in 2017. These new data adhered to standards in biosecurity and safety procedures. were recorded as part of the SUMMIT Project, whose main goal is analysing health risk in the population engaged in Measurements of Inflammatory and high-intensity physical activities (Maqueda et al., 2017; Roca et al., 2017, 2019; Nescolarde et al., 2020). The procedures High-Energy Enzymes Biomarkers Serum C reactive protein (CRP), CK, and LDH were determined followed were in accordance with the ethical standards of the using an AU-5800 Chemistry Analyzer (Beckman Coulter Inc, responsible committee on human experimentation (institutional CA, United States) before freezing the sample. and national) and with the Helsinki Declaration of 1975, as revised in 2000. The local ethics committee approved the study (approval No. PI-17-037), and all the participants provided Measurements of Non-collagenous and written informed consent. Type II Collagenous Cartilage Biomarkers A total of 35 runners, 29 male and 6 females, responded The quantitative detection of serum cartilage biomarkers to a call for volunteers. The participants were excluded if time was carried out by sandwich enzyme-linked immune-sorbent race was slower than the third pacemaker (3:15 h) in Barcelona assay technology (ELISA). Biomarker analysis was performed Marathon 2016 or if they had lower limb joints symptoms 6 according to the manuals of the manufacturer. Singlet data months previous to marathon, any history of previous lower analysis was carried out using duplicate standards; the acceptance limb injury or surgery, or if they have taken any medication, criteria were that the coefficient of variance of mean calculated including nonsteroidal anti-inflammatory drugs, in the previous concentrations between single result, and duplicate mean should month and during the 48-h post-marathon. We recommended be <15%. Duplicate standards were loaded at the beginning and no intense physical exercise for the 48-h period before and after the end of the plate. First, the samples, standards, and controls the marathon race. A checklist was used to supervise medication, were loaded on an inert plate and then with a multi-channel food intake, and exercise constraints, which were compiled by pippete, in less than a minute; the samples were passed to the all the runners. The study sample included 17 healthy Caucasian ELISA plate. In addition, all analyses were carried out by the male recreational athletes (mean age 41 ± 4 years old; 77.4 ± same technical person and on ELISA plates from the same batch 7.1 kg; 1.80 ± 0.1 m; 24. ± 2.1 kg/m2 ) that ran 2.7 ± 1.6 h/of of each biomarker to reduce intra- and inter- assay variation, intensive training per week and had a running history of 8.2 ± respectively. All samples were analysed in the first or second 5.1 years, on average, prior to the race. thaw-defrost cycle. Biomarker analysis staff were blinded to any The Barcelona Marathon 2017 (42.195 km on asphalt at the data related to participating subjects. sea level) began at 8:30 at a temperature of 11–14◦ C and Measurements of hyaluronic acid (HA) were performed with 68–79% humidity. During the race, the runners maintained the HA ELISA Kit (Abbexa Ltd., Cambridge Science Park, adequate levels of hydration by eating and drinking at aid stations Cambridge, UK; code No. abx257152, batch No. E1904223U). every 5 km, according to pre-established guidelines, with such Serum aliquots were diluted 1:4 in a dilution buffer provided sustenance as mineral water, sports drinks, fruits, and nuts. by the manufacturer before incubation. The standard range and The first liquid intake was programmed at 60 min of race: sensitivity are between 1.56–100 and <0.938 ng/ml, respectively. 400 ml for lighter/slower runners and 800 ml for heavier/faster Intra and inter-assay precisions were <8 and <10%, respectively. runners, drinking 100–150 ml every 15–20 min. Commercialised Human COMP was analysed by COMP ELISA Kit (Abbexa beverages were provided for runners with an average of 480 mg/l Ltd., Cambridge Science Park, Cambridge, UK; code No. for Na+ , 85 mg/l for K+ , and 45 mg/l for Mg2+ . Race time abx151136, batch No. E1904400E). Serum aliquots were diluted of our runners was 03:29:36 ± 00:17:06. Race time recorded of 1:20 in PBS (pH 7.0–7.2). Assay range is between 3.12 and Frontiers in Physiology | www.frontiersin.org 3 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers 200 ng/ml. Sensitivity in this assay is <1.21 ng/ml. Intra and RESULTS inter-assay precisions were <10 and 12%, respectively. Aggrecan chondroitin sulphate 846 (CS846) was measured by The 17 study participants completed all data collection the CS846 epitope ELISA (IBEX Pharmaceuticals Inc., Quebec, time points. Canada; code No. #60-1004; batch No. #2019-01). This assay is a competitive enzyme immunoassay. Serum samples were Inflammatory and High-Energy Enzymes diluted 1:5 in a dilution buffer provided by the manufacturer. The Biomarkers standard range is 20–1,000 ng/ml. Pre-race median sCK and sLDH levels increased immediately Human chitinase-3-like protein 1 (CHI3L1) levels were post-race, up to three-fold and two-fold increase, respectively analysed by the Glycoprotein YKL-40 ELISA kit (Blue Gene, (p < 0.0001) [10 (59%) runners over three-fold and 12 (71%) Shanghai, China; code No. E01C0072, batch No. BG190510). over two-fold, respectively]. The level of sCK remained elevated Samples were loaded undiluted. The standard range and 48-h post-race compared to immediately post (two-fold increase) sensitivity are 2.5–50 and 1 ng/ml, respectively. (p < 0.0001) and pre-race (four-fold increase) (p < 0.0001) Measurements of human procollagen II N-terminal [8 (47%) runners over four-fold]. In contrast, sLDH median propeptide (PIINP) were performed with the PIINP ELISA levels decreased 48 h post-race relative to immediately post-race Kit (Abbexa Ltd., Cambridge Science Park, Cambridge, UK; code (p < 0.0001) but remained elevated with respect to pre-race No. abx152741, batch No. E1904136K). Samples were diluted concentration (1.2-fold increase) (p = 0.003) [14 (82%) runners 1:10 in PBS (pH 7–7.2). The standard range is 93.75–6,000 over one-fold]. pg/ml, and the sensitivity is <37.55 pg/ml. Intra and inter-assay The median sCRP increased up to ten-fold 48 h post-race precisions were <10 and 12%, respectively. compared to pre-race (p < 0.0001) [6 (35%) runners over ten- Human type IIA collagen N-propeptide (PIIANP) levels fold] and immediately post (p < 0.0001). Data is shown in Table 1 were analysed by Human PIIANP Kit (Creative Diagnostics R , and Figure 1. New York, EEUU; code No. DEIA7442, batch No. 3175077). Samples were diluted 1:2 in an assay buffer. The sensitivity is Non-collagenous Cartilage Biomarkers 39 ng/ml. Intra and inter-assay variations were 3.37–6.60 and Mean sHA levels increased immediately after post-race compared 4.78–7.77%, respectively. with pre-race (four-fold increase) (p < 0.0001) [10 (59%) runners Collagen Type II Cleavage (C2C) was analysed by a over four-fold] and returned to pre-race levels 48-h post-race. competitive assay, C2C ELISA Kit (IBEX Pharmaceuticals Inc., In contrast, sCOMP, sCS846 means, and sYKL-40 median Quebec, Canada; code No. #60-1001-001; batch No. #2019-02). levels were relatively stable immediately post and 48-h Samples were diluted 1:2 in a dilution buffer provided by the post-race with respect to pre-race, although mean sCOMP manufacturer. The calibration range is 10–1,000 ng/ml. showed a tendency to increase (n.s.) and mean sCS846 to decrease (n.s.), immediately and 48-h post-race (Table 2; Statistical Analysis Figure 2). The normality of distribution of the variables was checked by the Shapiro-Wilk test and homogeneity of variances by Levene’s test. The variables normally distributed are shown as mean ± Type II Collagenous Cartilage Biomarkers SD, 95% confidence interval for mean (lower and upper bound), Mean sPIINP levels increased immediately (p = 0.053) [13 (76%) while those non-normally distributed data are shown as median, runners over one-fold] and 48-h post-race (p = 0.001) relative to interquartile range (IQR) (minimum–maximum). The serum pre-race [17 (100%) runners over one-fold]. biomarker levels were normally distributed, except for CK, LDH, Mean levels of sPIIANP showed a trend to decrease CRP, and YKL-40. immediately post-race relative to pre-race, and the trend was still Repeated measures ANOVA test was used to evaluate the present 48-h post-race (p = 0.154). effect of inflammation proteins, high-energy enzymes, non- Mean-circulating levels sC2C was lower immediately post- collagenous and collagenous cartilage proteins biomarkers race compared to pre-race (p = 0.002) [6 (35%) runners over one- measured 24-h pre-race (1), immediately post-race (2), and 48- fold] and returned to the pre-race level 48-h post-race (Table 3; h post-race (3), with multiple comparison test by Bonferroni. Figure 3). The Friedman test was used for the repeated measure of data non-normally distributed with the Wilcoxon test. Correlation Between Biomarkers In addition, we made Spearman Rho correlations between Median sCK levels pre-race were positively correlated with sLDH immediately post-race (2) with respect to 24-h pre-race (1); 48-h pre-race (r = 0.758, p < 0.0001). Immediately post and 48- post-race (3) with respect to 24-h pre-race (1); and 48-h post- h post-race sCK median-circulating levels positively correlated race (3) with respect to immediately post-race (2) for all the with median sLDH immediately post (r = 0.623, p = 0.008) biomarkers and time points and presented the significant ones (r = 0.520, p = 0.033) and 48-h post-race (r = 0.730, p = 0.001) for the biomarkers that showed a statistically significant change. (r = 0.842, p < 0.0001). Data were analysed using SPSS for windows version 26.0 Pre-race mean sPIINP-circulating levels positively correlated statistical program (SPSS, Inc., Chicago, IL). The statistical with median sCK 48-h post-race (r = 0.0494, p = 0.044) and significance was set at p < 0.05. with median sLDH immediately post-race (r = 0.616, p = 0.009). Frontiers in Physiology | www.frontiersin.org 4 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers TABLE 1 | Serum energy enzymes and inflammation biomarkers levels of 17 non-elite marathon runners pre- and post-marathon. Parameters 24 h Immediately 48 h Friedman Z Z Z Fold-change Fold-change pre-race post-race post-race p valuea p value p value p value (2–1) (3-1) (1) (2) (3) (2–1)b (3–1)b (3–2)b CK (U/L) Median (IQR) 152 (134) 429 (332) 658 (1073) 23.412 −3.574 −3.527 −3.006 ∼3 ∼4 (Min–Max) (76–400) (125–946) (95–2999) 0.000 0.000 0.000 0.000 LDH (U/L) Median (IQR) 187 (44) 323 (69) 218 (45) 29.059 −3.621 −3.006 −3.621 ∼2 ∼1 (Min–Max) (142–237) (195–493) (148–425) 0.000 0.000 0.003 0.000 CRP (mg/L) Median (IQR) 0.7 (0.6) 0.5 (0.6) 6.8 (4.1) 24.364 −1.851 −3.575 −3.621 ∼(–)1 ∼10 (Min–Max) (0.2–3.4) (0.3–1.7) (1.8–15.2) 0.000 0.064 0.000 0.000 a Friedman test; b Wilcoxon test. CK, Creatine kinase; LDH, Lactate dehydrogenase; CRP, C-reactive protein. FIGURE 1 | Serum energy enzymes and inflammation biomarkers levels of 17 non-elite marathon runners, pre, immediately-post, and 48-h-post marathon. CK, creatine kinase; LDH, lactate dehydrogenase; CRP, C-reactive protein. Boxplot (median, lower fence, and upper fence). Results of Wilcoxon test [Z-statistic based on negative rank and statistical significance, p < 0.0001 (***)]. Mean sPIINP 48-h post-race levels positively correlated with DISCUSSION median sLDH immediately post-race (r = 0.610, p = 0.009) and 48-h post-race (r = 0.488, p = 0.047). Long-distance running has become very popular and as an Median sCRP 48-h post-race was positively correlated with exercise imposes high levels of stress on the musculoskeletal mean sHA immediately post-race (r = 0.563, p = 0.019) system (Neidhart et al., 2000). Nowadays, little is known on what and a negative correlation with mean sPIIANP 48-h post- is a physiological or expected acute inflammatory or cartilage race (r = −0.570, p = 0.017). Data is shown in Table 4 and metabolic response to an increase in physical activity (Cattano Figure 4. et al., 2017b; Driban et al., 2017). Frontiers in Physiology | www.frontiersin.org 5 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers TABLE 2 | Serum non-collagenous cartilage biomarkers levels of 17 non-elite marathon runners pre- and post-marathon. Paramaters 24 h Immediately 48 h F SE SE SE Fold-change Fold-change Pre-race Post-race Post-race p valuec p value p value p value (2–1) (3–1) (1) (2) (3) (1–2)d (1–3)d (2–3)d HA (ng/ml) Mean ± SD 20.34 ± 7.88 89.54 ± 53.14 20.99 ± 8.04 15.002 12.268 2.377 12.564 ∼4 ∼1 95% CI Lowerbound 16.29 62.22 16.86 0.000 0.000 1.000 0.000 Upperbound 24.4 116.86 25.13 COMP (ng/ml) Mean ± SD 222.50 ± 56.86 233.43 ± 54.95 231.65 ± 49.83 0.879 8.665 12.672 13.051 ∼1 ∼1 95% CI Lowerbound 192.8 205.17 206.04 0.435 0.622 1.000 1.000 Upperbound 251.27 261.68 257.27 CS846 (ng/ml) Mean ± SD 80.25 ± 37.29 78.76 ± 34.98 78.41 ± 30.55 0.019 7.784 9.771 6.067 ∼(–)1 ∼(–)1 95% CI Lowerbound 61.08 60.77 62.7 0.981 1.000 1.000 1.000 Upperbound 99.42 96.74 94.12 Parameters 24 h Immediately 48 h Friedman Z Z Z Pre-race Post-race Post-race p valuea p value p value p value (1) (2) (3) (2–1)b (3–1)b (3–2)b YKL40 (pg/ml) Median (IQR) 3.54 (1.47) 3.90 (2.02) 3.49 (2.30) 0.925 −0.308 −0.497 −0.207 ∼1 ∼(–)1 (Min–Max) (2.37–12.95) (2.42–11.50) (2.39–20.59) 0.63 0.758 0.619 0.836 a Friedman test; b Wilcoxon test; c Repeated measures ANOVA test; d Bonferroni test. HA, hyaluronic acid; COMP, cartilage oligomeric matrix protein; CS846, aggrecan chondroitin sulphate 846; YKL40, Chitinase 3-like protein 1 glycoprotein YKL-40. This study broadens the set of serum cartilage biomarkers physiological function of CRP is still not completely understood investigated (HA, CS846, YKL-40, PIINP, PIIANP, and C2C) (Peisajovich et al., 2008; Rhodes et al., 2011). Because CRP can for response to an intense exercise (Barcelona Marathon) function as an opsonin by coating particles and dying cells to in non-elite runners, with a special focus on the relation facilitate their uptake by phagocytic cells (Gershov et al., 2000; with inflammation and high-energy consumption. The major Newling et al., 2019), we suggest that runners with higher-coating finding of this study is that marathon running modifies particles and cell damage produced during the intense exercise type II cartilage collagenous biomarkers serum levels with (Neidhart et al., 2000) will have higher levels of sCRP. an increase in collagen synthesis (sPIINP increase) during We agree with Kim et al. (2009) who described a temporary 48 h and a transient decrease in collagen catabolism (sC2C three-fold increase of CK in plasma during and after marathon decrease) immediately after race. Levels of sPIIANP and non- running with further increase after day 1 that returns to pre- collagenous cartilage biomarkers (sCOMP, sCS846, and sYKL- race levels after day 6. These authors demonstrated a dose- 40) remained relatively stable during the 48-h studied period. We dependent behaviour of CK with a higher increase in ultra- found an increase in high-energy consumption (sCK and sLDH marathon. We observed an even higher increase after day 2 increase) and, in general, inflammatory reaction (sCRP increase) (six-fold). These differences in behaviour may be due to distinct during 48 h correlated with collagen synthesis (sPIINP, sPIIANP) endurance training of runners (Wyss and Kaddurah-Daouk, and an immediate post-marathon cartilage inflammatory and 2000). Marathon running is an intense sport with high-energy degradation protection mechanism (sHA increase). demands, especially on musculoskeletal cells. Creatin kinase A 3.4-fold increase in plasma hs-CRP has also been observed reaction is involved in the high-energy phosphate metabolism of after day 1 in marathon runners (Kim et al., 2009); the increase cell in tissues with high-energy demands as muscle, brain, heart, returns to the pre-race level on day 4, with a dose-dependent and kidney. All CK isoenzymes catalyse the reversible transfer of behaviour reflected by a higher increase in ultra-marathon the gamma-phosphate group of adenosine triphosphate (ATP) (200 km) runners. We observed even a higher median ten-fold to the guanidino group of creatine (Cr) to yield adenosine increase of sCRP after day 2. CRP is a prototypic acute-phase diphosphate (ADP) and phosphoryl creatine (PCr) (Wyss and protein produced in high quantities in the liver; however, the Kaddurah-Daouk, 2000). Frontiers in Physiology | www.frontiersin.org 6 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers FIGURE 2 | Serum non-collagenous cartilage biomarker levels of 17 non-elite runners, pre, immediately-post, and 48 h-post marathon. HA, hyaluronic acid; COMP, cartilage oligomeric matrix protein; CS846, aggrecan chondroitin sulphate 846; YKL40, Chitinase 3-like protein 1 Glycoprotein YKL-40. Boxplot (median, lower fence, and upper fence). Bonferroni pairwise comparison test (standard error, SE, and statistical significance) for HA, COMP, and CS846. Wilcoxon test (Z statistic based on negative rank and statistical significance) for YKL40. p < 0.0001 (***). Lactate dehydrogenase is a key glycolytic enzyme and is a proposed protective effect on reduction of inflammation and believed to be the major enzyme responsible for pyruvate to on cartilage enhancement of proteoglycan synthesis (Moreland, lactate conversion. It is released from many tissues into the 2003). Moreover, HA, lubricin, and phospholipid species endow circulation and indicates an increase in energy demands and synovial fluid with its viscoelastic properties and contribute in muscle cell membrane permeability, which occurs as the cell either independently or together to the lubrication of the becomes energy depleted and by muscle cell damage, which articular surfaces. Although a decrease in sHA concentration has occurs after prolonged exercise (Rumley et al., 1985). The been reported after 1-h walking (Pruksakorn et al., 2013), we increase in total serum LDH activity that occurs after marathon observed a transient increase of sHA immediately after marathon training can be modified by training (Rumley et al., 1985). running. Gordon et al. (2008) observed a transient increase We observed that this increase also occurs immediately after after 1 h of monitored activity. These results suggest a different marathon running (two-fold increase) and stays elevated until behaviour depending on intensity and duration of applied 48-h post-race. LDH- and CK serum-level elevation correlated, cyclic loads. especially at 48-h post-race; this suggests activated energy Cartilage oligomeric matrix protein is a component of the pathways or cell damage remains at least 48 h after intense cartilage matrix synthesised by chondrocytes (Hedbom et al., physical activity. 1992) and by fibroblast in tendon, meniscus, and synovial HA is a biomarker specific to synovial joints and is the tissue (Hummel et al., 1998). It has been widely studied after main component of articular cartilage. There is evidence that exercise (Neidhart et al., 2000; Kersting et al., 2005; Mündermann training (Cattano et al., 2017b) protocols may mitigate the et al., 2005; Kim et al., 2009; Niehoff et al., 2011; Celik et al., HA biomarker response. HA binds to a cluster of different 2013), with various studies showing discrepancies in results, CD44 receptors, and this binding inhibits interleukin-1 (IL-1) depending on varying study conditions. Serum COMP levels expression and leads to a decline in matrix metalloproteinase respond to mechanical stimulus, reportedly increase temporarily (MMP) 1, 2, 3, 9, and 13 production (Karna et al., 2008). We in a dose-dependent fashion in response to physical activity observed a positive correlation with CRP that is consistent with in highly trained marathon individuals and gradually return Frontiers in Physiology | www.frontiersin.org 7 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers TABLE 3 | Serum type II collagenous cartilage biomarkers levels of 17 non-elite marathon runners pre- and post-marathon. Parameters 24 h Immediately 48 h F SE SE SE Fold-change Fold-change Pre-race Post-race Post-race p valuea p value p value p value (2–1) (3–1) (1) (2) (3) (1–2)b (1–3)b (2–3)b PIINP (ng/ml) Mean ± SD 9.05 ± 2.15 10.82 ± 3.44 11.00 ± 2.96 10.482 0.67 0.423 0.464 ∼1 ∼1 95% CI 0.001 0.053 0.001 1.000 Lowerbound 7.94 9.05 9.48 Upperbound 10.15 12.59 12.52 PIIANP (ng/ml) Mean ± SD 2042.04 ± 567.25 1819.94 ± 548.34 1769.56 ± 419.67 3.151 101.824 129.449 132.059 ∼(–)1 ∼(–)1 95% CI 0.072 0.133 0.154 1.000 Lowerbound 1750.38 1538.01 1553.79 Upperbound 2333.69 2101.87 1985.33 C2C (ng/mL) Mean ± SD 220.83 ± 39.50 188.67 ± 38.52 221.68 ± 45.64 8.989 7.778 6.713 8.604 ∼(–)1 ∼1 95% CI 0.003 0.002 1.000 0.004 Lowerbound 200.52 168.87 198.21 Upperbound 241.14 208.47 245.15 a Repeated measures ANOVA test; b Bonferroni test; PIIANP, procollagen type II N terminal pro-peptide; C2C, cleavage type II collagen; PIINP, procollagen type II N terminal pro-peptide. FIGURE 3 | Serum type II collagenous cartilage biomarker levels of 17 non-elite runners, pre, immediately post, and 48 h-post marathon. PIIANP, Procollagen type IIA N terminal pro-peptide; C2C, cleavage type II collagen; PIINP, procollagen type II N-terminal pro-peptide. Boxplot [(median, lower fence, and upper fence). Bonferroni pairwise comparison test (standard error, SE, and statistical significance p < 0.01 (***)]. Frontiers in Physiology | www.frontiersin.org 8 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers TABLE 4 | Spearman’s rho correlations [correlation coefficient and sig. (2-tailed)] between biomarkers. CK LDH CRP CK-1 vs. LDH-1 CK-2 vs. LDH-2 CK-3 vs. LDH-2 LDH-2 vs. PIINP-1 LDH-3 vs. PIINP-3 CRP-3 vs. HA-2 CRP-3 vs. PIIANP-3 0.758** 0.623** 0.520* 0.616** 0.488* 0.563* −0.570* 0.000 0.008 0.033 0.009 0.047 0.019 0.017 CK-2 vs. LDH-3 CK-3 vs. LDH-3 LDH-2 vs. PIINP-3 0.730** 0.842** 0.610** 0.001 0.000 0.009 CK-3 vs. PIINP-1 0.494* 0.044 1 pre-race; 2 post-race; 3 48-h post-race; CK, creatine kinase; LDH, lactate dehydrogenase; CRP, C-reactive protein; PIINP, procollagen type II N terminal pro-peptide; PIIANP, procollagen type II N terminal pro-peptide. *p < 0.05; **p < 0.01. to baseline levels over 24 h (Neidhart et al., 2000; Kim et al., in patients with medial knee OA and correlates with an increase 2009); these increases are probably adaptive and mitigated by a in collagen degradation biomarker C1, C2 (Chu et al., 2018). running training protocol (Hoch et al., 2012; Celik et al., 2013). We observed a similar behaviour of sCOMP, sCS846, andYKL- We find a trend for increased (n.s.) sCOMP levels immediately 40 with no significant changes in serum levels after marathon post-race, which was maintained 48 h after marathon running running, but with a tendency of sCOMP to increase and of CS846 in well-trained non-elite runners. It has been suggested that to decrease. The increase of sYKL-40 and sCS846, reportedly, serum COMP levels after physical activity that reflects enhanced protects against cartilage degradation (Johansen et al., 2001; Chu diffusion of COMP fragments form cartilage to blood due to et al., 2018). The absence of changes in sYKL-40 levels and the joint loading (Mündermann et al., 2005) or increase in lymphatic tendency to decrease of CS846 are consistent with the absence drainage (Neidhart et al., 2000; Kersting et al., 2005). The of an increase of collagen degradation (sC2C decrease) that we higher baseline serum levels observed in marathon runners, in observed after marathon running and may indicate no increase osteoarthritis, and rheumatoid arthritis than in healthy controls in diffusion, or cartilage matrix turnover or damage (Otterness (Neidhart et al., 2000; Garnero et al., 2001; Sharif et al., 2007) et al., 2000). and the larger lag time to return to basal levels after a marathon Collagen is produced and secreted as a precursor molecule (Neidhart et al., 2000; Kim et al., 2009) than after a moderate with C- and N-terminal extensions termed procollagen peptides physical activity (Mündermann et al., 2005) suggest a maintained (PIICP and PIINP). Since procollagen peptides are released from increase in synthesis or diffusion due to the rapid elimination the parent molecule in exact proportions, the concentration of of COMP once it has reached the circulation (Andersson et al., these peptides assesses the biosynthetic activity (Munk et al., 2006). 2014). In the extracellular space, the procollagen terminal Kersting et al. (2005) found no COMP increase 25 min or extensions are cleaved off by specific C- and N-pro-peptidases; 2.5 h after a 1-h run at a self-selected pace in physically trained after which, the type II collagen through a collagen-proteoglycan individuals. The different behaviours of serum COMP levels may interaction can participate in fibril formation (Graham et al., be explained by their sensitivity to the loading mode, for instance, 2000). N-terminal pro-peptide of type II collagen (PIINP) is slow and deep knee bends do not affect COMP levels (Niehoff a biomarker-reflecting cartilage formation. PIINP exists in two et al., 2010) and by the adaptations that may be experiment within main splice variants termed as type IIA and type IIB collagen the time frame of load application (Liphardt et al., 2018) and with NH2-propeptide (PIIANP, PIIBNP) (Nah and Upholt, 1991). training (Hoch et al., 2012; Celik et al., 2013). PIIANP has been recognised as a cartilage formation biomarker, Serum YKL-40 is a glycoprotein secreted by chondrocyte probably restricted to embryogenesis and fracture healing but (Einarsson et al., 2013) and also expressed in osteoblast and may be reexpressed in osteoarthritis (Aigner et al., 1999). On osteocytes present in osteophytes (Živanović et al., 2009). It the other hand, PIIBNP is mainly expressed during type II has been hypothesised that YKL-40 production is a part of collagen formation in healthy adult cartilage (Sandell et al., 1991). the inflammatory response in chondrocytes, acting to limit There is, reportedly, no correlation between PIIBNP and PIIANP connective tissue degradation (Johansen et al., 2001) and linked levels in knee osteoarthritis, rheumatoid arthritis, and paediatric with tissue remodelling, joint injury, and “in situ” inflammatory serum samples (Luo et al., 2018). Serum-PIIANP is, reportedly, macrophages (Lorenz et al., 2005). Our findings also suggest that decreased in patients with knee osteoarthritis (Sharif et al., 2007) sYKL-40 is not associated with sCRP (Karalilova et al., 2018). and decreased in rheumatoid arthritis of long duration (Rousseau Serum CS846, an aggrecan synthesis marker, was not significantly et al., 2004). changed measures of 0.5 and 5.5 h after 30 min walk, whereas an The increase of sPIINP we observed after marathon running increase in the synthesis correlates with MRI cartilage thickening was maintained over 48 h. This may reflect type II collagen Frontiers in Physiology | www.frontiersin.org 9 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers FIGURE 4 | Rho Spearman’s correlations and coefficient of linear regression R2 of the biomarkers that showed statistical significance variations. (A) CK Spearman’s correlations. (B) LDH Spearman’s correlations. (C) CRP Spearman’s correlations. 1: pre-race; 2: post-race; 3: 48-h post-race. CK, creatine kinase; LDH, lactate dehydrogenase; CRP, C-reactive protein; PIINP, procollagen type II N terminal pro-peptide; PIIANP, procollagen type II N terminal pro-peptide. *p < 0.05, **p < 0.01. Frontiers in Physiology | www.frontiersin.org 10 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers formation in healthy cartilage (PIIBNP) because the splice but we believe studied biomarkers well represent cartilage variant PIIANP showed a trend to decrease after marathon metabolism. Fifth, a singlet data analysis of the biomarkers may running. Healthy cartilage is characterised by a low turnover of be considered a limitation as duplicate or triplicate analysis collagens (Tchetina, 2011), but upregulation of col II and col I has been the standard practise in research but not in clinical synthesis in chondrocytes has been reported in response to cyclic diagnostic labs. Nowadays, it is questionable if duplicate analysis compression in vitro with a time constant of 2.9 h (Bonassar improves variability, and individual analysis could be adopted et al., 2001), a finding consistent with our in vivo observations as standard practise in modern ligand-binding assays (Ye et al., in marathon running. 2018). Sixth, corrections for plasma volume change that can The cleavage of type II collagen by collagenases yields occur following an acute bout of exercise has been recommended fragments, such as the C2C epitope, and serum levels of when studying serum biomarkers (Kargotich et al., 1997); C2C correlate with cartilage degeneration in patients with however, this does not apply to all published studies (Neidhart symptomatic knee OA (King et al., 2004). We observed a et al., 2000; Kersting et al., 2005; Mündermann et al., 2005; transient decrease in sC2C levels immediately after marathon Niehoff et al., 2011). Immediate and 48-h post-marathon plasma running that returns to normal 48 h later, possibly indicating volume change determined with haemoglobin and haematocrit cyclic loads transiently reduce collagen catabolism by data of a previous study of our group (Roca et al., 2019) collagenases. Our results on collagen cartilage biomarkers in 41 runners of 2016 Barcelona Marathon was −1.2 and metabolism (sPIINP increase and sC2C decrease) agree with −1.7%, respectively. The percentage of biomarker change when those of Azukizawa (Azukizawa et al., 2019) who observed correcting for plasma volume is small, and we believe it would well-rounded exercise may have beneficial effects on type II apply to our study. collagen metabolism, especially in people without OA increasing In conclusion, we confirmed previous studies suggesting sPIICP, secreted in exact proportions than PIINP, and decreasing marathon running is an exercise with high-energy demands urine-C2C and uCTX-II (urine carboxy-terminal crosslinked (sCK and sLDH levels increase) that provokes a high and telopeptide of type II collagen). durable general inflammatory reaction (sCRP levels increase). The positive correlation between sPIINP and sCK and We observed a transient increase in sHA levels immediately post- sLDH levels and the negative correlation between sCRP and marathon that may be a mechanism to protect inflammation and sPIIANP may indicate an interaction of type II collagen cartilage, accompanied by an upregulation of type II collagen synthesis in adult cartilage with the high-energy demands cartilage metabolism increasing fibrils synthesis (sPIINP levels required and the inflammatory reaction produced after marathon increase) and decreasing its catabolism (sC2C levels decrease), running. The observed changes in the studied biomarkers without changes in non-collagenous cartilage metabolism appear to be caused by altered metabolic activity rather than (sCOMP, sCS846, and sYKL-40). The duration of marathon diffusion due to marathon because of the large duration stimulus and the lag time that serum levels changes persist (3–4 h) of marathon stimulus enough for cell metabolism suggest that changes in the studied biomarkers are caused activation (Bonassar et al., 2001) and the time lag (48 h) that by metabolic activity rather than diffusion. Metabolic changes biomarkers changes persist after the stimulus (Erhart-Hledik on sHA and sPIINP synthesis may be related to energy et al., 2012). consumption (sCK, sLDH) and the inflammatory reaction This study has the following limitations. First, we had a (sCRP) produced. limited sample size with no control group, but, even with a small sample size, we found statistically significant differences. The aim of the study was to know whether there are changes DATA AVAILABILITY STATEMENT in the biomarker profile in healthy subjects after undergoing high-intensity exercise, a marathon. We considered that the The raw data supporting the conclusions of this article will be control group would be made of a sample of healthy individuals made available by the authors, without undue reservation. who do not run the marathon, which, in this case, will be the baseline or pre-race point. Second, we did document the diet and exercise constraints by the participants in the 48- ETHICS STATEMENT h period pre and post-marathon, but we did not have the The studies involving human participants were reviewed runners controlled in a sport centre under strict supervision and approved by Germans Trias i Pujol University 48 h before and after the marathon, and that can be a limitation Hospital Ethics Committee. The patients/participants for the strict follow-up of diet and exercise constraints by provided their written informed consent to participate in runners. Third, imaging exams on joint status (knee, hips, and this study. ankles) were not evaluated before or after the study; presence of osteoarthritis can alter biomarkers levels and behaviour, although no participants had symptoms or previous surgery on lower AUTHOR CONTRIBUTIONS limb joints. Fourth, we selected specific inflammatory, energy enzymes, non-collagenous, and type II collagenous cartilage JH-H participated in the conception and design of the biomarkers; other biomarkers may show different patterns, study, analysis, interpretation of data, and drafting the Frontiers in Physiology | www.frontiersin.org 11 October 2021 | Volume 12 | Article 722718 Hernández-Hermoso et al. Marathon Running and Cartilage Biomarkers article. LN participated in acquisition of data, statistical ACKNOWLEDGEMENTS analysis, and interpretation of data. ER participated in the provision of study patients, in technical and logistic We thank A. Cserkóová for the technical processing in support, in acquisition of data, and in collection and the laboratory. assembly of data. ER-L participated in technical and logistic support, in acquisition of data, and in collection SUPPLEMENTARY MATERIAL and assembly of data. JA and AB-G participated in technical and logistic support and critical revision of the The Supplementary Material for this article can be found article. All authors read and approved the final version to online at: https://www.frontiersin.org/articles/10.3389/fphys. be submitted. 2021.722718/full#supplementary-material REFERENCES A systematic review. J. Athl. Train. 52, 497–506. doi: 10.4085/1062-6050- 50.2.08 Aigner, T., Zhu, Y., Chansky, H. H., Matsen, F. A., Maloney, W. J., and Sandell, L. Einarsson, J. M., Bahrke, S., Sigurdsson, B. T., Ng, C. H., Petersen, P. H., J. (1999). Reexpression of type IIA procollagen by adult articular chondrocytes Sigurjonsson, O. E., et al. (2013). 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Orthop. this article, or claim that may be made by its manufacturer, is not guaranteed or 33, 1165–1170. doi: 10.1007/s00264-009-0747-8 endorsed by the publisher. Conflict of Interest: The authors declare that the research was conducted in the Copyright © 2021 Hernández-Hermoso, Nescolarde, Roca, Revuelta-López, Ara and absence of any commercial or financial relationships that could be construed as a Bayes-Genis. This is an open-access article distributed under the terms of the Creative potential conflict of interest. Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance Publisher’s Note: All claims expressed in this article are solely those of the authors with accepted academic practice. No use, distribution or reproduction is permitted and do not necessarily represent those of their affiliated organizations, or those of which does not comply with these terms. Frontiers in Physiology | www.frontiersin.org 14 October 2021 | Volume 12 | Article 722718