Joint Health of Athletes and the Chondroprotective Action of Glucosamine

In endurance athletes with intense joint loading, cartilage metabolism (degradation of type II collagen) is enhanced compared with non-athletes and non-endurance athletes. Recently, we have revealed that glucosamine, a functional food, exerts a protective action on cartilage metabolism in not only osteoarthritis patients but also endurance athletes (such as soccer players and rugby players) by suppressing the degradation of type II collagen. In this review, to demonstrate these findings, the following topics will be explained: 1. Biomarkers for cartilage metabolism; 2. Evaluation of osteoarthritis and endurance sports by using biomarkers of cartilage metabolism; 3. Chondroprotective action of glucosamine on osteoarthritis patients and endurance sports athletes; 4. Glucosamine as a“Food with Function Claim”.


Introduction
The frequency and severity of joint loading are critical factors for the development of joint destruction, characterized by the damage of articular cartilage.In fact, excessive loading on the joint with motion and exposure causes the damage of articular cartilage 1)-4) .Thus, sports with repetitive impact and torsional loading on the joints increase the risk of articular cartilage degeneration, and results in the clinical symptoms of osteoarthritis 4) .
The disease process of osteoarthritis is related to the degradation and functional loss of articular cartilage.Importantly, the early changes in the metabolic and biochemical properties of cartilage matrix can be detected before the appearance of morphological changes of cartilage 2) .Thus, various biomarkers have been developed as indicators of cartilage and bone metabolism in subjects with joint and bone disorders 5) .In this context, it is interest-ing to note that sports-related mechanical loading on the joints affects the turnover rate of cartilage as well as bone in humans, and these changes can be detected by the assays with biomarkers 1)-4) .
Nutritional supplements, including glucosamine, chondroitin and collagen, are sometimes used for joint health to treat or prevent sports-related cartilage injuries (i.e., osteoarthritis) in athletes 6) -8) .Among these, glucosamine, a naturally occurring amino monosaccharide, has been widely used to treat osteoarthritis in humans 9) -11) .Recently, we have revealed that glucosamine exhibits a protective action on cartilage metabolism in not only osteoarthritis patients but also endurance athletes by suppressing the degradation of type II collagen, as evidenced by the reduced level of type II collagen degradation markers 12)-14) .Thus, in this review, to demonstrate these findings, I will present the data on the cartilage metabolism of athletes as well as osteoarthritis patients, and the chondroprotective action of glucosamine on osteoarthritis patients and athletes, by analyzing type II collagen degradation and synthesis markers.

Biomarkers for cartilage and bone metabolism
Type II collagen is one of the major components of cartilage 15) , and the fragments of type II collagen are utilized as biomarkers for cartilage metabolism (Figure -1).A C-terminal telopeptide (CTX-II) is cleaved during degradation of type II collagen 16) , whereas a neo-epitope (C2C) is cleaved at the C terminus of the 3/4 piece of degraded type II collagen 17) .Thus, both CTX-II and C2C are used as markers for type II collagen degradation.In contrast, a C-terminal type II procollagen peptide (CPII) is present in newly formed type II procollagen and cleaved during processing of synthesized type II procollagen; thus, CPII can be used as a marker for type II collagen synthesis 18) .In addition, deoxypyridinoline (Dpyr), a crosslink product of type I collagen and cross-linked N-terminal telo-peptides of type I collagen (NTx) are used as markers for type I collagen degradation in bone (bone resorption) 5) .

Evaluation of osteoarthritis by using biomarkers
First, to evaluate the cartilage and bone metabolism in osteoarthritis, we measured the levels of biomarkers in knee osteoarthritis patients (16 subjects; 74.3±7.8 years old, mean±SD), and compared with those in healthy control (17 subjects; 70.5±5.2year old) 19) .In contrast to healthy control, osteoarthritis patient have pain at rest, and radiological findings of Kellgren and Lawrence grades 20) of II-IV (minimal-severe).The patients were performing muscle training, and treated with NSAIDs (non-steroidal anti-inflammatory drugs) and intra-articular injection of hyaluronic acid.
Figures-2A, B and C show the levels of CTX-II, NTx and hyaluronic acid in healthy control and knee osteoarthritis patients, respectively.In osteoarthritis patients, the levels of type II collagen degradation marker CTX-II, type I collagen degradation marker NTx and synovitis marker hyaluronic acid were significantly increased compared with healthy control.These results indicate that type II collagen degradation and type I collagen degradation are increased in osteoarthritis, accompanied with synovitis.Furthermore, we examined the levels of type II collagen synthesis marker CPII in healthy control and knee osteoarthritis patients (Figure -2D).Interestingly, CPII level was significantly decreased in osteoarthritis.These observations suggest that type II collagen degradation is increased, whereas type II collagen synthesis is decreased; thus, the imbalance between degradation and synthesis of type II collagen may be involved the cartilage damage in osteoarthritis.Actually, the imbalance between the degradation and synthesis of type II collagen is reported to be important for the progression of cartilage damage in osteoarthritis 21) 22) .Thus, we can evaluate the cartilage damage by measuring the levels of biomarkers, such as type II collagen degradation markers CTX-II and C2C and synthesis marker CPII.

Chondroprotective action of glucosamine on osteoarthritis
Next, we evaluated the chondroprotective action of glucosamine.Glucosamine is an amino monosaccharide with an amino group, and functions as a component of glycosaminoglycans (such as hyaluronic acid and chondroitin sulfate) 23) (Figure -3). Figure -4 shows the synthetic pathway of glycosaminoglycans in our body 24) ; glucose is converted into glucosamine with the addition of amino group from glutamine, and further converted into N-acetyl-glucosamine and N-acetyl-galactosamine.Then, these glucosamine-derivatives are coupled with uronic acid to form glycosaminoglycans (such as hyaluronic acid, keratin sulfate and chondroitin sulfate) present in the articular cartilage, skin and other tissues.Importantly, glucosamine supplement is efficiently absorbed from the intestine, distributed to various tissues and used as a constituent of glycosaminoglycans.Thus, glucosamine is widely utilized as a functional food with a chondroprotective action to treat human diseases such as osteoarthritis as a precursor of glycosaminoglycans 9) -11) .Furthermore, we demonstrated that glucosamine suppresses the activation of neutrophils 25) , synovial cells 26) and intestinal epithelial cells 27) , endothelial cells 28) , and inhibits adjuvant arthritis 29) , colitis 30) and atherosclerosis 31) in animal models.Thus, glucosamine expectantly exhibits anti-inflammatory actions.
As a functional food, glucosamine is mostly manufactured from chitin, a polymer of N-acetylglucosamine present in the shells of shrimps and crabs (Figure -5).Chitosan, a polymer of glucosamine is produced from the chitin by deacetylation under alkaline condition.In contrast, glucosamine, a chitosan monomer is produced as glucosamine hydrochloride by hydrolysis and deacetylation of chitin under acidic condition.
To evaluate the chondroprotective action of glucosamine, we utilized the anterior cruciate ligament transection model as an osteoarthritis model 32) .A knee joint is stabilized by several ligaments such as collateral, posterior cruciate and anterior cruciate ligaments.By anterior cruciate ligament transection, osteoarthritis is developed due to instability of the knee joint, accompanied with the degeneration of cartilage 33) .Actually, anterior cruciate ligament transection clearly induced the erosion in the cartilage; interestingly, however, glucosamine administration markedly suppressed the erosive change of the cartilage 32) .Furthermore, anterior cruciate ligament transection apparently induced the surface depletion and reduced toluidine blue staining of proteoglycans in the cartilage 32) .Notably, glucosamine administration suppressed the surface depletion and proteoglycan degeneration in the cartilage.
Moreover, the effects of glucosamine on the type II collagen-degradation and type II collagen-synthesis markers were evaluated (Figure -6).The level of CTX-II, a type II collagen-degradation marker, was significantly elevated by anterior cruciate ligament transection.Of importance, glucosamine administration suppressed the increase of CTX-II.
In addition, the level of CPII, a type II collagensynthesis marker, was substantially increased by glucosamine administration.Thus, the results suggest that glucosamine exerts a chondroprotective action by inhibiting type II collagen degradation but enhancing type II collagen synthesis in the articular cartilage in this osteoarthritis model.
Next, to evaluate the chondroprotective action of glucosamine on knee osteoarthritis patients, we performed a randomized double-blind placebocontrolled study 12) .In this study, patients with knee joint pain and Kellgren and Lawrence grades 20) of 0-III were recruited, and glucosamine-containing diet (16 subjects; 54.5±9.1 years old) or placebo diet (16 subjects; 56.4±7.7 years old) was administered for 16 weeks.Glucosamine diet mainly contained glucosamine (1,200 mg) but also chondroitin sulfate (80 mg) and other functional substances.Importantly, the administration of glucosamine-containing diet significantly improved the symptoms of osteoarthritis, based on JKOM (Japanese Osteoarthritis Measure) total score 34) (Figure -7A).Similarly, the administration of glucosamine-containing diet significantly reduced the level of type II collagen degradation marker C2C (Figure -7B), indicating the suppression of type II collagen degradation.Moreover, the administration of glucosamine-containing diet significantly reduced the serum level of hyaluronic acid, a synovial inflammation marker (Figure -7C), indicating the suppression of synovial inflammation.These observations suggest that the administration of glucosamine-containing diet exhibits a chondroprotective action on knee osteoarthritis by inhibiting type II collagen degradation and synovial inflammation, and improves the symptoms.

Chondroprotective action of glucosamine on endurance athletes
Finally, we looked at the chondroprotective So, we evaluated the effect of glucosamine on cartilage metabolism using collegiate soccer players with intense joint loading 13) .In this study, 10 non-athletes (23.5±2.5 years old) and 21 soccer players (20.3±0.9 years old) were recruited.Non-athletes experienced no hard exercise in the past year.In contrast, soccer players performed the training 5 days per week, and played the official match almost every weekend, during the test period.
Figures -8A and B show the levels of CTX-II and NTx in non-athlete control and soccer players.In soccer players, the levels of CTX-II and NTx were significantly increased compared with non-athlete control, indicating that cartilage and bone metabolism (type II collagen degradation and bone resorption) is increased in soccer players, as reported in other endurance athletes 3) .Moreover, a type II collagen synthesis marker CPII was evaluated in soccer players.Interestingly, the level of CPII was substantially increased in soccer players compared with non-athlete control (Figure -8C), suggesting that cartilage metabolism as evaluated by type II collagen synthesis is also increased in soccer players.
In addition, we evaluated the type II collagen degradation and synthesis balance in the cartilage of soccer players by calculating CTX-II/CPII ratio.As shown in Figure -9A, CTX-II/CPII ratio was significantly higher in soccer players than nonathlete control, suggesting that type II collagen degradation is relatively increased compared with type II collagen synthesis in soccer players.
Next, we examined the effect of glucosamine administration on type II collagen degradation and synthesis markers.Importantly, CTX-II level was significantly decreased after the glucosamine administration for 3 months at both 1.5 g and 3 g/day (Figure -10A).Interestingly, however, the CTX-II level returned to almost the pre-administration level after withdrawal of glucosamine administration in 1.5 g/day-group for 3 months, although the CTX-II level was still reduced in 3 g/day-group.In contrast, CPII level was not essentially changed even after the glucosamine administration and withdrawal of glucosamine administration (Figure -10B), suggesting that the increased level of type II collagen synthesis in soccer players is maintained during the test period.
Furthermore, we evaluated the effect of glucosamine on type II collagen degradation and synthesis balance in soccer players by using CTX-II/CPII ratio.Importantly, the ratio was reduced by glucosamine administration especially at 3 g/day, and returned to the pre-administration level after withdrawal of glucosamine (Figure -9B).
These observations suggest that glucosamine exhibits a chondroprotective action in soccer players by preventing type II collagen degradation but maintaining type II collagen synthesis; however, its effect on type II collagen degradation is transient and disappears after withdrawal of administration.
Further, we evaluated the effect of glucosamine on cartilage metabolism using professional rugby players with intense joint loading.In this study, 19 rugby players (29.4±3.7 years old) and 19 non-athletes (29.4±3.7 years old) were recruited 14) .Rugby players were administered with a jelly-type diet containing 3 g glucosamine for 16 weeks.
Figures -11A and B show the levels of CTX-II and NTx in non-athletes and rugby players.In rugby players, the levels of CTX-II and NTx were significantly increased compared with non-athletes, indicating that cartilage and bone metabolism (type II collagen degradation and bone resorption) is increased in rugby players, as reported in soccer players and other endurance athletes 3) 13) .Next, we evaluated a type II collagen synthesis marker CPII

Figure-10 Effect of glucosamine administration on urine levels of CTX-II and CPII in soccer players
Soccer players were orally administered with glucosamine hydrochloride (GlcN; 1.5 or 3 g/day for 3 months, as indicated by an arrow).Urine levels of CTX-II (A) and CPII (B) were measured before (0 month), after the glucosamine administration (3 months) and after the withdrawal of glucosamine administration for 3 months (6 months).These levels are expressed as a ratio relative to those before glucosamine administration (0 month).Data represent the mean±SEM of 9 subjects (1.5 g GlcN/day) and 10 subjects (3 g GlcN/day).Values are compared between before (0 month) and after the glucosamine administration (3 months) or after the withdrawal of glucosamine administration (6 months).＊ p<0.05, ＊＊ p<0.01. in rugby players; however, CPII level in rugby players was almost the same as in non-athletes (Figure -11C).Based on these findings, CTX-II/ CPII ratio was slightly higher in rugby players than non-athletes (Figure -11D), suggesting that type II collagen degradation is relatively increased compared with type II collagen synthesis in rugby players.
Next, we examined the effect of glucosamine administration on type II collagen degradation and synthesis markers.Importantly, CTX-II level was significantly decreased after the glucosamine administration (Figure -12A).Interestingly, however, the CTX-II level returned to almost the preadministration level after withdrawal of glucosamine administration.In contrast, CPII level was not essentially changed even after the glucosamine administration and withdrawal of glucosamine administration (Figure -12B).Finally, we evaluated the effect of glucosamine on type II collagen degradation and synthesis balance in rugby players by using CTX-II/CPII ratio.Importantly, the ratio was significantly reduced by glucosamine administration, and returned to the pre-administration level after withdrawal of glucosamine (Figure -12C).
These observations suggest that glucosamine exhibits a chondroprotective action also in rugby players by preventing type II collagen degradation but maintaining type II collagen synthesis.However, the effect is transient and disappears after withdrawal of administration.

What are"Foods with Function Claims" ?
The system of"Foods with Function Claims 機能 性表示食品制度"has been launched in April 2015 35) ."Foods with Function Claims 機能性表示食品"are foods submitted to the Secretary-General of the Consumer Affairs Agency 消費者庁長官 as products whose labels bear function claims based on scientific evidence, under the responsibility of food business operators.Based on our findings, glucosamine has been submitted to the Secretary-General of the Consumer Affairs Agency as a"Food with Function Claim"of chondroprotective action with a submission number A147.Glucosamine is expected to be helpful for maintaining joint health during exercise and walking, because it suppresses the degradation of articular cartilage components such as type II collagen in endurance athletes with intense joint loading.

Conclusions
Our recent studies revealed the following findings.1. Glucosamine exhibits a chondroprotective action in a rat osteoarthritis model by inhibiting type II collagen degradation and increasing type II collagen synthesis.2. Glucosamine improves the symptom of osteo-arthritis patients by suppressing type II collagen degradation and synovial inflammation (synovitis).3. Type II collagen degradation is relatively increased compared with type II collagen synthesis in endurance athletes (soccer and rugby players).Glucosamine exhibits a chondroprotective action in athletes by preventing type II collagen degradation but maintaining type II collagen synthesis.However, the effect is transient and disappears after withdrawal of administration.Thus, glucosamine should be continuously administered for expecting joint health.
Based on these findings, glucosamine has been submitted to the Secretary-General of the Consumer Affairs Agency as a"Food with Function Claim" , which is helpful for maintaining joint health.

Figure- 1
Figure-1 Biomarkers for type II collagen degradation and synthesisCTX-II and C2C are used as markers for type II collagen degradation.In contrast, CPII is used as a marker for type II collagen synthesis.

Figure- 7
Figure-7 Effects of the administration of glucosamine-containing diet on JKOM total score, serum levels of C2C and hyaluronic acid in individuals with knee pain

Figure- 9
Figure-9 Evaluation of type II collagen degradation and synthesis balanceThe ratio of type II collagen degradation to synthesis (CTX-II/CPII) of non-athlete control and soccer players are calculated using the levels of CTX-II and CPII shown in Figure-8A and B. Data represent the mean±SEM of non-athlete control (10 subjects) and soccer players (18 subjects), and values are compared between non-athlete control and soccer players.＊ p<0.05.Furthermore, soccer players were orally administered with glucosamine hydrochloride (GlcN; 1.5 or 3 g/day for 3 months, as indicated by an arrow), and CTX-II/CPII ratios were calculated before (0 month), after the glucosamine administration (3 months) and after the withdrawal of glucosamine administration for 3 months (6 months)(B).Data represent the mean±SEM of 9 subjects (1.5 g GlcN/day) and 10 subjects (3 g GlcN/day).(Yoshimura M, et al: Int J Mol Med, 2009; 24: 487-49413) )

Nagaoka:Figure- 11 Figure- 12
Figure-11 Comparison of the urine levels of CTX-II, NTx and CPII, CTX-II/CPII ratio between non-athlete control and rugby players The urine levels of CTX-II (A), NTx (B) and CPII (C) were measured by ELISA and corrected by urinary creatinine (Cr).Moreover, the ratio of type II collagen degradation to synthesis (CTX-II/CPII) of non-athlete control and rugby players are calculated (D) using the levels of CTX-II and CPII shown in A and C. Data represent the mean±SD and are compared between non-athlete control (19 subjects) and rugby players (19 subjects).＊ p<0.05, ＊＊ p<0.01 (Tsuruta A, et al: Functional Food Research, 2016; 12: 39-41 14) )