doi:10.1017/S175525400999016X

Comparative Exercise Physiology 6(3); 121–128 q Cambridge University Press 2009

Neither age nor osteoarthritis is associated with synovial fluid antioxidant disturbance or depletion in the horse RC Murray1,*, CM Deaton1, NC Smith1, WE Henley1,† and DJ Marlin2 1

Centre for Equine Studies, Animal Health Trust, Lanwades Park, Kentford, Suffolk CB8 7UU, UK 2 Hartpury College, Hartpury, Gloucester, UK * Corresponding author: [email protected] Submitted 13 May 2009: Accepted 18 September 2009: First published online 22 October 2009

Research Paper

Abstract Studies investigating the role of oxidative stress in both the ageing process and osteoarthritis (OA) in human beings are limited by the unavailability of samples from healthy subjects. OA occurs naturally in the horse and has been used as a model of human OA. The objective of this study was to determine the effect of ageing and OA on the non-enzymatic synovial fluid antioxidant status of the horse. The concentrations of ascorbic acid, dehydroascorbate (DHA, oxidized ascorbic acid), uric acid, glutathione, a-tocopherol and thiobarbituric acid reactive substances (TBARS) were determined in paired synovial fluid and plasma samples from 25 horses aged between 3 and 25 years. Osteoarthritic lesions were scored from 0 (healthy) to 4 (severe OA). Glutathione was not detectable in synovial fluid. Neither plasma nor synovial fluid antioxidant concentrations were affected by age. Ascorbic acid concentrations in plasma correlated strongly with those in synovial fluid from both healthy (P , 0.001) and diseased joints (P ¼ 0.003). Synovial fluid concentrations of ascorbic acid and uric acid were not influenced by OA compared with healthy joints. However, the concentration of DHA was slightly, but significantly, elevated in synovial fluid from joints with severe OA (95% CI: [2.2, 11.8] mmol l21; P , 0.001). OA is associated with only a mild oxidative burden, which does not appear to overwhelm the synovial fluid antioxidant capacity. Consequently, antioxidant supplementation is unlikely to have a beneficial effect in the treatment of OA. Keywords: joint; ageing; oxidative stress; pathology

Introduction Osteoarthritis (OA) is an important disease with high prevalence1 and economic impact2. Despite this, understanding of the mechanisms of development and progression remains unclear and methods for prevention and management are limited. It appears that a variety of mechanisms can be involved in progression to OA, manifesting as a disease of the whole joint3. While mechanical loading has been shown to be an † Present address: School of Mathematics and Statistics, University of Plymouth, Drake Circus, Plymouth, Devon PL4 8AA, UK.

important factor in OA development, evidence from studies in a variety of species suggests that oxidative stress may also be involved. Reactive oxygen species (ROS) are an integral part of many biological processes, and controlled production is essential for maintenance of life. However, excess production can result in tissue damage and inflammation; therefore, ROS levels are balanced by enzymatic and non-enzymatic antioxidant pathways for deactivation or removal of ROS. When ROS production exceeds the antioxidant capacity, oxidative stress results. Oxidative stress has been demonstrated to induce cartilage senescence and chondrocyte dysfunction

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leading to the development of OA in vitro . ROS may also act as intra-cellular messengers. Inflammatory cytokines stimulate chondrocytes to produce ROS5 and infiltrating inflammatory cells also release ROS. Non-enzymatic antioxidants include ascorbic acid, uric acid, a-tocopherol and glutathione. Ascorbic acid is an important antioxidant in synovial fluid as it is essential for collagen synthesis and cartilage function. On reacting with oxidants, ascorbic acid is oxidized to dehydroascorbate (DHA) by two one-electron oxidation reactions. Uric acid is produced from purine metabolism and functions as an antioxidant by preventing ascorbic acid oxidation and scavenging ROS6. Synovial fluid uric acid concentrations are similar to those in serum in non-inflammatory arthropathies and gout7. In gout, high concentrations of uric acid in synovial fluid compared with serum have also been reported7. In human inflammatory joint diseases other than gout, uric acid concentrations were lower in synovial fluid compared with serum7. a-Tocopherol is a lipophilic antioxidant. In human patients with inflammatory joint disease, the concentration of a-tocopherol is lower in synovial fluid compared with plasma8. The antioxidant function of glutathione is regulated by the activity of glutathione peroxidase and glutathione reductase. The activities of these two antioxidant enzymes have been demonstrated to be elevated in patients with OA of the knee joint, but the activities were negatively associated with disease duration9. There is increasing interest in the role of nutrition and dietary supplementation in the development and management of OA10. If there is depletion or reduced production of synovial antioxidants in chronic pathology, dietary supplementation with antioxidants could provide a method of modification of OA. The risk of OA increases with age11,12. In rats, chondrocyte ROS production increased with age, and chondrocytes from aged rats were more susceptible to oxidantinduced cytotoxicity13. If there is alteration in joint antioxidant status with age, and there is an association between antioxidant levels and OA, then dietary manipulation will provide potential for reducing this risk. However, in order to investigate this possibility, synovial fluid antioxidant levels in normal and osteoarthritic joints and the effect of age need to be evaluated. The horse is a long-lived species with naturally occurring OA, which has been used as an acceptable model for development of OA in humans14–19. The potential for collection of synovial samples from both normal joints and joints from horses with known OA lesions allows study in a way that may not be possible for human joints. We have previously shown that mechanical loading can induce osteoarthritic lesions in the equine carpus19, and that there are potential mechanisms for increased OA risk with age in the

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horse , but the role of antioxidants and ROS in OA has not been investigated. A higher level of ROS in joints from young horses with osteochondritis dissecans or traumatic osteochondral lesions compared with joints from healthy horses indicates the potential importance of ROS in acute joint pathology20. However, to our knowledge there is no information on the role of oxidative stress and antioxidant depletion in chronic joint pathology. The aims of the present study are to compare the concentrations of non-enzymatic antioxidants in equine synovial fluid and plasma and determine the effects of age and OA on the antioxidant status. We hypothesized that (1) synovial fluid antioxidant status from joints with no pathology would correlate with circulating antioxidant status; (2) antioxidant status in synovial fluid from joints with no pathology would decrease with age of the horse after maturity; (3) synovial fluid antioxidant status would be decreased in joints with OA. Methods Animals Synovial fluid and plasma were collected from 25 horses (mean age 10 years, range 3–21 years; 19 geldings and six mares; nine Warmbloods; four native ponies; four Irish breeds; two Thoroughbreds and six Thoroughbredcross breeds). Horses were included if they had no known systemic medical problems and had undergone an orthopaedic evaluation in the 5 days prior to euthanasia. Venous blood samples and synovial fluid samples were used from horses euthanized for clinical reasons unconnected with this study. Collection and processing of blood and synovial fluid Samples were collected from the jugular vein via a catheter and placed into lithium heparin and EDTA tubes. Synovial fluid samples were obtained from the middle carpal joint bilaterally, metacarpophalangeal joint bilaterally and any other joints with known pathology diagnosed using intra-articular analgesia, radiography and/or magnetic resonance imaging. Samples were collected in the following order: right metacarpophalangeal (RF); right carpus (RC); left metacarpophalangeal (LF); left carpus (LC); other joints. Synovial fluid was placed into tubes containing EDTA and no additives. Synovial fluid from LC was analysed for ascorbic acid (n ¼ 25 horses), uric acid (n ¼ 24), a-tocopherol (n ¼ 3) and thiobarbituric acid reactive substances (TBARS; n ¼ 8). RC fluid was analysed for ascorbic acid (n ¼ 23), uric acid (n ¼ 22), a-tocopherol (n ¼ 3) and TBARS (n ¼ 4). LF fluid was analysed for ascorbic acid (n ¼ 22), uric acid (n ¼ 21), a-tocopherol

Osteoarthritis, ageing and antioxidants

(n ¼ 3) and TBARS (n ¼ 5). RF fluid was analysed for ascorbic acid (n ¼ 22), uric acid (n ¼ 20), a-tocopherol (n ¼ 3) and TBARS (n ¼ 1). The low numbers for a-tocopherol and TBARS were due to technical issues at the start of the study. In addition, due to the instability of TBARS, only samples collected immediately after euthanasia were considered suitable for this analysis. Antioxidant concentrations were also determined in synovial fluid from the right antebrachiocarpal joint (n ¼ 1), left elbow (n ¼ 1), left shoulder (n ¼ 1) and left stifle joint (n ¼ 1). Blood samples were processed as described previously26. Blood and synovial fluid samples were centrifuged at 400 £ g for 10 min and 100 £ g for 5 min, respectively, at 48C within 10 min of collection. For ascorbic acid analysis, EDTA plasma and synovial fluid were deproteinized by the addition of an equal volume of 10% metaphosphoric acid containing 1 mmol l21 Na2EDTA. Samples were snap frozen in liquid nitrogen and stored at 2 808C. In order to reduce the oxidized form of vitamin C (dehydroascorbic acid, DHA) and to measure the total concentration of vitamin C, 0.2 ml of 10 mmol l21 dithiothreitol was added to 0.3 ml EDTA sample, and incubated at room temperature for 10 min. Following which, 0.5 ml of 10% metaphosphoric acid containing 1 mmol l21 Na2EDTA was added, and the sample was snap frozen and stored at 2 808C. For synovial fluid glutathione analysis, an aliquot of synovial fluid from the EDTA tube was stored at 2 1968C. Lithium heparin plasma and EDTA synovial fluid aliquots were stored at 2 808C for a-tocopherol and uric acid analysis. For TBARS analysis, 1 ml lithium heparin plasma or EDTA synovial fluid was added to the evaporated residue of 100 ml butylated hydroxy toluene (100 mg ml21 in ethanol) and 100 ml desferal (100 mg ml21 in water) and stored at 2 808C. Blood and synovial fluid sample analyses Concentrations of reduced and oxidized glutathione in red blood cell haemolysate and synovial fluid were determined by HPLC with electrochemical detection21. Concentrations of reduced and total ascorbic acid22 and a-tocopherol23 in plasma and synovial fluid were determined by HPLC with UV detection. The concentration of DHA was calculated by subtracting the concentration of reduced ascorbic acid from the concentration of total ascorbic acid. The ascorbic acid redox ratio was determined by dividing the concentration of DHA by the total ascorbic acid concentration. Plasma and synovial fluid uric acid concentrations were determined using a commercial kit (Sigma kit 685-10, Sigma Chemical Co., Dorset, UK). Plasma and synovial fluid TBARS concentration were analysed using a fluorometric assay24. Intraassay coefficients of variation for the various assays based on a minimum of six paired measurements for

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each analyte were: AA, 0.8%; DHA, 9.0%; total AA, 0.8%; UA, 0.7%; TBARS, 8.3%. Scoring of osteoarthritic lesions All joints were opened, photographed and examined by the same observer, blinded to the antioxidant status, for the presence/absence of osteoarthritic lesions, and graded as 0 (no lesions); 1, very mild cartilage erosion (, 1 mm at periphery of joint) with no synovial proliferation and no lameness; 2, mild cartilage erosion 1–5 mm not full depth with no synovial proliferation and no lameness; 3, moderate cartilage erosion full depth or . 5 mm with osteophytes present but no synovial proliferation and no lameness; 4, severe OA with full-depth cartilage erosion and more than 50% cartilage loss, osteophytes, subchondral bone erosion, synovial proliferation, radiographic evidence of severe OA and marked lameness. Statistical analyses Statistical analyses were performed to test the following hypotheses: H1

Antioxidant status in synovial fluid from joints with no pathology is associated with plasma antioxidant status. H2a Antioxidant status in synovial fluid from joints with no pathology reduces with age of the horse. H2b Antioxidant status in plasma reduces with age of the horse. H3 Antioxidant status in synovial fluid from joints with pathology (OA score ¼ 1, 2, 3 or 4) is associated with plasma antioxidant status. H4 Pathology is associated with altered antioxidant status in synovial fluid from joints: a Grade 0 versus 4 b Grade 0 versus 3–4 c Grade 0 versus 1–4 d Grade 0 versus 1–3 e Grade 1–3 versus 4 H5 Antioxidant status in synovial fluid from joints with no pathology varies according to the type of joint There were insufficient numbers to allow inclusion of sex or breed as variables. Linear mixed-effects models25 were used to investigate the relationship between antioxidant status in synovial fluid and plasma. Separate models were developed for each antioxidant with the antioxidant status in the joint as the dependent variable. Correlations between synovial antioxidant levels in different joints for the same horse were accounted for using two-level models with a random effect for each animal. In the preliminary analysis, separate models were developed for joints with no pathology and for osteoarthritic joints. The base models contained a categorical

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explanatory variable for the joint type and a covariate for the antioxidant level in plasma. Hypotheses H1 and H3 were tested by assessing the statistical significance of the plasma antioxidant effects in the models fitted to subsets of joints with appropriate pathology. The statistical significance of the categorical joint effect, in the model for joints with no pathology, was used to test hypothesis H5. Interactions between joint type and plasma antioxidant level were tested to assess whether the relationship between antioxidant status in synovial fluid and plasma varied by joint. The effect of age on antioxidant status in synovial fluid from joints with no pathology was tested by adding age as an explanatory variable to the base model (hypothesis H2a). Hypothesis H2b was tested by fitting a simple linear regression model with antioxidant status in plasma as the dependent variable and age as the explanatory variable. The association between pathology and synovial antioxidant levels in joints (H4) was assessed by fitting linear mixed models to the combined data for joints with and without pathology (for different definitions of pathology, see 4a–4e). Each model consisted of, as before, explanatory variables for the joint and the antioxidant status in plasma, with the addition of a binary contrast for the effect of pathology. Model parameters were estimated using residual maximum likelihood estimation. Statistical inference for the fixed effects was carried out using Wald tests. Likelihood ratio tests were used to make inferences about the random horse effects. Statistical analyses were performed using SAS version 8.2. Results All joints were sampled and provided sufficient volume of synovial fluid for analysis. Neither reduced nor oxidized glutathione was detectable in synovial fluid from joints with or without evidence of disease (limit of detection of 16.3 nmol l21).

Antioxidant status of synovial fluid from joints without evidence of disease The concentrations of uric acid, reduced and total ascorbic acid (AA), DHA and ascorbic acid redox ratio in plasma and synovial fluid from joints without pathological evidence of disease (OA score ¼ 0) were not influenced by the age of the horse or the type of joint (Table 1; H5 marginally significant for tAA). The concentrations of reduced ascorbic acid (Fig. 1a) and total ascorbic acid (P , 0.001) were significantly correlated with the concentrations in plasma. There were no associations between the concentrations of uric acid, DHA and ascorbic acid redox ratio in plasma and synovial fluid. The mean concentrations of a-tocopherol and TBARS from healthy joints were 0.19 ^ 0.33 and 0.16 ^ 0.09 mmol l21, respectively, in synovial fluid and 4.7 ^ 2.8 and 0.88 ^ 0.24 mmol l21, respectively, in plasma.

Antioxidant status of synovial fluid from joints with evidence of disease In agreement with the findings from joints without disease, significant correlations were determined between the concentrations in plasma and synovial fluid from joints with evidence of disease (OA score ¼ 1, 2, 3 or 4) for reduced (P ¼ 0.003) ascorbic acid (Fig. 1b) and total ascorbic acid (P ¼ 0.004), but not for DHA, ascorbic acid redox ratio or uric acid. The concentrations of reduced and total ascorbic acid (Fig. 2) and uric acid (Fig. 3) in synovial fluid were not influenced by joint OA score. Although the absence of evidence for an effect cannot be taken as evidence of equivalence, our results demonstrate that any possible decrease in concentration of synovial fluid AA in joints with pathology will not be of clinical significance (95% CI: [2 2.81, 0.65] mmol l21). Joints with an OA score of 4 had significantly higher synovial fluid DHA concentrations and ascorbic acid redox ratios compared with joints with an OA score of 0 or 1–3 (95% CI: [2.2, 11.8] mmol l21; P , 0.001; Fig. 2).

Table 1 Summary of analysis of the effects of ageing and osteoarthritis on plasma and synovial fluid antioxidant concentrations H1 AA tAA DHA ARR Uric acid

H2a i

,0.001 ,0.001 0.985 0.861 0.982

0.501 0.093 0.395 0.621 0.137

H2b 0.591 0.418 0.188 0.460 0.366

H3 i

0.003 0.004i 0.631 0.408 0.332

H4a

H4b

H4c

H4d

H4e

H5

0.322 0.007ii ,0.001ii ,0.001 0.508

0.238 0.119 0.017 0.005ii 0.868

0.216 0.541 0.250 0.127 0.993

0.426 0.754 0.794 0.803 0.492

0.407 0.051ii ,0.001ii ,0.001 0.447

0.203i 0.050 0.264 0.351 0.532

AA, ascorbic acid; tAA, total ascorbic acid; DHA, dehydroascorbate; ARR, ascorbic acid redox ratio; TBARS, thiobarbituric acid reactive substances. H1 antioxidant status in synovial fluid from joints with no pathology is associated with plasma antioxidant status; H2a antioxidant status in synovial fluid from joints with no pathology reduces with age of the horse; H2b antioxidant status in plasma reduces with age of the horse; H3 antioxidant status in synovial fluid from joints with pathology (OA score 1, 2, 3 or 4) is associated with plasma antioxidant status; H4 pathology is associated with altered antioxidant status in synovial fluid from joints: a, Grade 0 versus 4; b, Grade 0 versus 3–4; c, Grade 0 versus 1–4; d, Grade 0 versus 1–3; e, Grade 1–3 versus 4; H5 antioxidant status in synovial fluid from joints with no pathology varies according to the type of joint. The table shows the P-Values for testing the null versions of hypotheses H1–H5.i Effect of plasma varies with joint. ii Effect of pathology varies with joint.

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Synovial fluid AA (µmol l–1)

a) 40

30

20

10

0

Synovial fluid AA (µmol l–1)

b)

0

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20 30 Plasma AA (µmol l–1)

40

0

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20 30 Plasma AA (µmol l–1)

40

40

30

20

10

0

FIG . 1 Concentrations of reduced ascorbic acid (AA) in plasma and synovial fluid from (a) joints without evidence of disease (OA score ¼ 0) and (b) joints with evidence of disease (OA score . 0). W, left carpus; K, right carpus; , left metacarpophalangeal; O, right metacarpophalangeal; A, other; ––, line of equality

Discussion Ascorbic acid and uric acid were the non-enzymatic antioxidants identified in the highest concentrations in equine synovial fluid. Severe OA was associated with a small but significant increase in the oxidation of ascorbic acid and no change in the profiles of the other non-enzymatic antioxidants. Ascorbic acid therefore appears to be the principal buffer of oxidants produced in joints with severe OA disease. However, based on the degree of oxidation of ascorbic acid, the magnitude of oxidative stress appears to be low in chronic equine OA. Determining the role of a-tocopherol in the synovial fluid antioxidant defence was limited by the small number of samples analysed for a-tocopherol in the present study. However, the

concentration of a-tocopherol was considerably lower in synovial fluid compared with plasma and, therefore, the contribution of a-tocopherol to oxidant consumption is probably minimal in equine synovial fluid. The absence of detectable concentrations of reduced and oxidized glutathione in synovial fluid probably reflects their absence in plasma21. In the present study, the concentration of ascorbic acid did not decline with age in either plasma or synovial fluid. Similarly, we have previously found no difference in the plasma antioxidant concentrations of aged horses (n ¼ 20, mean age 30 years) compared with young horses (n ¼ 20, mean age ¼ 5l; Marlin and Deaton, unpublished observations), and plasma ascorbic acid concentrations were not associated with age in either healthy horses or horses in remission from recurrent airway obstruction26. In contrast, plasma ascorbic acid concentration has been demonstrated to decrease with age27. Differences between studies may reflect the presence of underlying disease. In the study of Ralston et al.27, only three out of the eight aged horses investigated were categorized as clinically normal. Concentrations of ascorbic acid in synovial fluid were higher than those in plasma in both healthy and diseased horses. This may reflect local production of ascorbic acid, as horses are able to synthesize ascorbic acid, in contrast to human beings. However, ascorbic acid concentrations have previously been demonstrated to be higher in synovial fluid compared with serum in human patients with rheumatoid arthritis and OA and in healthy guinea pigs28–30. Furthermore, in the present study, there was a significant correlation between plasma and synovial fluid ascorbic acid concentrations. Therefore, it is more probable that the ascorbic acid accumulation in synovial fluid reflects active transport of ascorbic acid into the synovial fluid. Joints with severe OA (score ¼ 4) had significantly greater DHA concentration than joints with less severe or no disease. This suggests that severe OA is associated with an increase in ROS production. However, the increase in DHA, although statistically significant, was small and the concentration of ascorbic acid was not decreased, inferring that the oxidant load did not overwhelm ascorbic acid. This may reflect the relatively low level of ROS production, rapid recycling of ascorbic acid, repletion of ascorbic acid from the circulation or ascorbic acid synthesis. In equine joints with acute lesions, overall antioxidant status tended to be higher than synovial fluid from healthy horses20, indicating upregulation of antioxidant production or accumulation. In human OA patients, the degree of ascorbic acid oxidation is similar to that measured in the present study29. However, in human patients with rheumatoid arthritis, approximately

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Synovial fluid total AA (µmol l–1)

Synovial fluid AA (µmol l–1)

40

30

20

10

40

30

20

10

0

0 0

1

2 3 OA score (/4)

4

0

1

P < 0.001

4

P < 0.001 0.8

P = 0.017

P = 0.005 P < 0.001

P < 0.001

15

0.6 Synovial fluid ARR

Synovial fluid DHA (µmol l–1)

20

2 3 OA score (/4)

10

5

0.4

0.2

0

0.0 0

1

2

3

4

OA score (/4)

0

1

2 3 OA score (/4)

4

FIG . 2 Concentrations of reduced ascorbic acid (AA), total ascorbic acid (total AA), dehydroascorbate (DHA) and ascorbic acid redox ratio (ARR) in synovial fluid from joints with OA scores from 0 to 4. W, left carpus; K, right carpus; , left metacarpophalangeal; O, right metacarpophalangeal; A, other

80% of the ascorbic acid is in the form of DHA in both plasma and synovial fluid28, suggesting that the oxidant load is far greater in this disease or the rate of recycling of DHA to ascorbic acid is reduced. The presence of enzymes with DHA reductase activity has not been investigated in the joint. The antioxidant composition is not only important extracellularly but also intracellularly. Uptake of ascorbic acid and DHA into chondrocytes by sodium-dependent vitamin C transporters31 and glucose transporters29 results in intracellular accumulation of ascorbic acid, which is required to prevent oxidative damage and maintain cartilage production. The study of intracellular antioxidant concentrations therefore warrants investigation. In order to ascertain the importance of antioxidants and oxidative stress in human joint disease, antioxidant supplementation has been investigated. Administration of dietary vitamins (vitamins E, C, A, B6

and B2) and selenium in mice decreased the incidence of mechanically induced OA lesions32. In rats, intraarticular injection of hydrogen peroxide in combination with a running load resulted in OA that was partially inhibited by vitamin E administration33. However, supplementation with vitamin E did not improve symptomatic knee OA34. High intake of ascorbic acid has been demonstrated to decrease OA progression in guinea pigs35. Similarly in human patients, high intake of ascorbic acid in particular but also vitamin E and b-carotene were associated with a reduction in the risk of OA disease progression, but there was no association with the incidence of disease and antioxidant intake36. High doses of ascorbic acid supplemented for prolonged periods have even been demonstrated to enhance spontaneous OA in guinea pigs37. Furthermore, ascorbic acid has the potential to act as a pro-oxidant by reducing ferric ion (Fe3þ)

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Synovial fluid uric acid (µmol l–1)

60

5

35

6

30 25

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20 15

8 10 5 0 0

1

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3

4

9

OA score (/4) FIG . 3 Concentrations of uric acid in synovial fluid from joints with OA scores from 0 to 4. W, left carpus; K, right carpus; , left metacarpophalangeal; O, right metacarpophalangeal; A, other

to ferrous ion (Fe2þ). Ferrous ion can subsequently reduce hydrogen peroxide to the more reactive hydroxyl radicals, resulting in lipid peroxidation38,39. However, this reaction does not occur if iron is sequestered; therefore, the pro-oxidant effect of ascorbic acid in vivo is unclear. As antioxidant concentrations were not lower in synovial fluid from diseased joints compared with healthy ones, the present study does not support the supplementation of these antioxidants in OA disease. In conclusion, ascorbic acid appears to be the principal non-enzymatic antioxidant in equine synovial fluid. Neither age nor OA induces a marked oxidative burden on the equine joint; therefore, these findings do not support the use of dietary antioxidant supplements for the treatment of equine OA.

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Acknowledgements The study was funded by the Petplan Charitable Trust. 18

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