Serum markers of lipid peroxidation, antioxidant

enzymatic defense, and collagen degradation in

an experimental (Pond-Nuki) canine model
of osteoarthritis
Nikolay V. Goranov

Background: Chondrocyte lipid peroxidation is strongly suggested to mediate collagen degradation and thus, to be involved in
the pathogenesis of cartilage degradation and osteoarthritis (OA). Objectives: The objective of this study was to evaluate early
changes in serum biochemical indicators of oxidative stress during the development of OA in a canine model. Methods: Exper-
imental OA was induced in 7 dogs by transection of the anterior cruciate ligament (Pond-Nuki model). Venous blood samples
were obtained prior to the operation and on postoperative days 30, 60, and 105. The activity of serum catalase (an antioxidant
enzyme), malondialdehyde (MDA) concentration (a marker of lipid peroxidation), and serum C2C neoepitope concentration (a
marker of collagen type II degradation) were measured. Results: A statistically significant increase in all parameters as early as
the 30th postoperative day was observed, compared with preoperative values. Serum catalase activity peaked on day 60,
whereas MDA and C2C concentrations increased continuously until the end of the experimental period. A significant positive
correlation was found between MDA and C2C concentrations, but not between catalase activity and MDA or C2C
concentrations. Conclusions: The results support the hypothesis that oxidative stress is involved in the pathogenesis of OA in the
dog based on the Pond-Nuki model. The correlation between MDA and C2C concentrations suggests a possible association
between oxidative stress and cartilage degeneration. (Vet Clin Pathol. 2007;36:192–195)

2007 American Society for Veterinary Clinical Pathology

Key Words: C2C neoepitope, catalase, dog, malondialdehyde, Pond-Nuki osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis
encountered in dogs, affecting weightbearing joints. It is
a significant problem in middle-aged and geriatric animals.
OA is characterized by cartilage degradation and abrasions,
subchondral bone remodeling, and low-grade inflammation.1
Causes of OA vary.2
Serum and urinary assays of bone and cartilage markers
are noninvasive alternatives to bone biopsy for assessing the
response of the skeleton to disease and injury. Other non-
invasive diagnostic methods for canine OA (eg, radiography,3
magnetic resonance imaging,4 bone densitometry) provide
a snapshot of the body’s net bone balance at a given moment,
whereas serum and urinary biomarker assays provide real-
time quantification of the formative and resorptive activities of
bone.5 For this reason, the in vivo utilization of biomarkers to
signal the process of degenerative change prior to clinical or
radiographic changes is a promising diagnostic strategy in OA
patients.6 There are, however, few publications about clinically
useful biochemical indices of the effects of isolated joint injury,
the progression of joint changes, and the response to therapy
in companion animals.7
According to an increasing number of reports, reactive
oxygen species released by chondrocytes and activated
neutrophils are among the principal factors causing damage
of collagen, proteoglycans, and hyaluronic acid. Lipid
peroxidation is one of the main pathways of cellular damage
in OA.8,9 Age-related changes in the lipid composition of
cartilage could push the normal lipid peroxidation process
into a state of uncontrolled oxidative stress, leading to the
oxidation of cartilage collagen. Oxidation of collagen could
cause fragmentation, which alters the material properties of
collagen fibrils, thereby making them more brittle and prone
to mechanical fatigue and failure. Such failure could initiate
OA.10 Plasma and tissue thiobarbituric acid reactive substan-
ces (TBARS) have been widely used as an indirect biomarker
of lipid peroxidation. The lipid soluble breakdown product,
malondialdehyde (MDA) is a major TBARS, and can be easily
quantitated colorimetrically. The simplicity of the analytical
procedure makes it advantageous for screening the effects of
lipid peroxidation in many human and animal disorders and
in in vitro and in vivo investigations.11 Cellular defense
mechanisms against reactive oxygen species include a series of

From the Department of Veterinary Surgery, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria (nickgoranov@yahoo.com). ª2007 American Society for
Veterinary Clinical Pathology

Page 192 Veterinary Clinical Pathology Vol. 36 / No. 2 / 2007

 Goranov

linked enzyme reactions that remove the toxic radicals and

repair radical–induced damage. The first of these enzymes is
superoxide dismutase (EC 1.15.1.1), which converts superox-
ide anion to hydrogen peroxide. The latter, also toxic to cells, is
removed by catalase (EC 1.11.1.6).11
Studying the mechanism of collagen degradation is
important, because several studies indicate that damage to
cartilage collagen is a central event in the pathogenesis of
cartilage aging and osteoarthritis.12 The C-terminal neoepitope
/
of the 3 4 length collagen type II cleavage product (C2C) is one
of biomarkers of articular cartilage degradation caused by
collagenases. C2C is a specific marker for cleavage of collagen
type II, primarily present in hyaline cartilage and interverte-
bral disks with very small amounts present in other tissues.
Moreover, in some studies, serum C2C concentration is shown
to predict specifically both short-term and long-term pro-
gression of joint damage in rheumatoid arthritis.13 In OA, the
ability to predict outcome is perhaps of even greater impor-
tance. The utility of using C2C has been reported in various
animal species, including mice,14 rats,15 and dogs.7
The aim of the present study was to monitor the dynamics
of early changes in blood biomarkers of oxidative stress
throughout the development of post-traumatic canine OA,
and preceding clinical or radiologic signs. To achieve this aim,
the activity of catalase and the concentrations of malondial-
dehyde and C2C were measured at different time intervals,
and correlations among them were determined.
The experiment was performed on 7 clinically healthy
adult mixed-breed dogs of both sexes, between 2 and 2.5 years
of age, weighing 15 6 2 kg. The animals were placed in 3 m2
individual boxes and given appropriate amounts of commer-
cial dry canine food and access to drinking water. After a 10-
day period of adaptation, experimental OA was induced by
the method of Pond-Nuki16 by unilateral surgical transection
of the anterior cruciate ligament. The experiment was ap-
proved by the Committee on Animal Experimentation at the
Trakia University, Stara Zagora, Bulgaria and was performed
according to current animal welfare regulations.
Blood samples (4–5 mL) without anticoagulant were
obtained from the cephalic vein prior to the operation (day 0)
and on postoperative days 30, 60, and 105. The blood was
allowed to clot and serum was separated by centrifugation.
Serum catalase activity and MDA concentration were assayed
immediately after serum collection while C2C levels were
assayed on samples frozen at 20 8C and stored for 5 months.
The MDA assay is based on the formation of a 1:2 red adduct
between MDA and 2-thiobarbituric acid in acid medium that is
quantitated spectrophotometrically at 532 nm after extraction
with n-butanol.17,18 As a MDA standard, 1,1,3,3 tetraethoxy- Figure 1. Serum catalase activity, malondialdehyde (MDA) concentra-
propane (Sigma Aldrich Chemie GmbH, Munich, Germany) tion, and C2C neoepitope concentration in 7 dogs with experimentally
was used. Serum catalase activity was assayed by the method induced osteoarthritis prior to surgery and on selected postoperative
of Goth19 based on the formation of a stable yellow complex days. Solid lines indicate median values.
between the substrate (H2O2 in sodium-potassium phosphate
buffer, pH 7.4) and ammonium molybdate, and quantified
spectrophotometrically at 405 nm. Serum C2C concentration This is a competitive immunoassay in which a specific mouse
was assayed with a commercial ELISA kit (Ibex Technologies IgG antibody (C2C antibody) binds either to the C2C coating
Inc, Montréal, Canada) that measures a neoepitope created by conjugate in the plate, to C2C standards, or to the neoepitope
the cleavage of collagen type II by collagenases at the C- in the samples. According to the manufacturer, the antibody
/
terminus of the 3 4 length collagen type II cleavage product. has broad cross reactivity and recognizes canine C2C. Data are

Vol. 36 / No. 2 / 2007 Veterinary Clinical Pathology Page 193

 Oxidative Stress and Collagen Degradation in Canine Osteoarthritis
reported as mean 6 SEM and were analyzed by Mann-
Whitney U tests (as no assumptions about the normal dis-
tribution of data were made) and Pearson’s correlation
analysis between individual measurement points.
Serum catalase activity was increased significantly on day
30 (118.29 6 19.84 U/mL, P 5 .013) and day 60 (158.71 6
28.60 U/mL, P 5 .025), compared with the baseline value
(53.57 6 10.86 U/mL) (Figure 1). On day 105, catalase activity
decreased to 81.00 6 14.77 U/mL and did not differ
statistically from initial levels (P 5 .250). MDA and C2C
concentrations increased gradually during the entire 105-day
period. The MDA concentration at all time intervals (day 30,
14.94 6 0.82 lmol/L, P 5 .004; day 60, 15.96 6 3.09 lmol/L,
P 5 .018; day 105, 21.70 6 2.63 lmol/L, P 5 .002) was
statistically higher than the baseline value (8.82 6 0.91 lmol/
L). Similar significant increases were observed in serum C2C
neoepitope concentrations on day 30 (42.28 6 9.67 ng/mL,
P 5 .041), day 60 (74.82 6 11.38 ng/mL, P 5 .003), and day
105 (77.68 6 12.41 ng/mL, P 5 .006) compared with the
baseline value (23.50 6 6.57 ng/mL). The correlation analysis
revealed a significant positive correlation between MDA and
C2C concentrations (r 5 .530; P 5 .037). The correlation
between the other biomarkers was weak and insignificant
(r 5 0.203 for C2C vs catalase and 0.197 for MDA vs catalase).
Clinical and radiologic signs of canine OA in the Pond-
Nuki model appear by the 12th week of its induction.16 The
results for the serum biomarkers evaluated in this study
changed considerably as early as the fourth week (day 30). In
human patients with OA of the knee, abnormal antioxidant
status with increased antioxidant enzyme activities in synovial
fluid was found.20 In the present experiment, serum catalase
activity was statistically higher by day 30, probably represent-
ing a compensatory increase due to increased substrate
concentration.21 The production of catalase may have de-
creased later (day 105) because the increased free radical load
had exhausted the body’s antioxidant defenses.
Increased blood MDA levels in arthritis are reported to
correlate with disease severity9. Significantly higher articular
cartilage MDA concentrations were observed in rats with
collagen-induced arthritis as early as the 21st day (third week)
vs normal control levels.8 In our study, serum MDA con-
centration also was higher at a similar time interval (fourth
week). Considerably elevated C2C concentrations were
reported in animal models of OA and rheumatoid arthritis
within 3–4 weeks7,22,23 compared to healthy controls, with
further increases at weeks 4, 12, and 16. These observations are
completely in agreement with the present data.
We did not find published data linking serum catalase,
MDA, and C2C levels with the development of Pond-Nuki
osteoarthritis in dogs. The correlation between serum C2C and
MDA concentrations suggests that articular cartilage degra-
dation may be related to lipid peroxidation. Therefore, the
present data confirmed the hypothesis of other authors about
the key role of lipid peroxidation in the pathogenesis of
secondary OA.9,10
In conclusion, the experimental canine Pond-Nuki OA,
observed over a period of 105 days, was accompanied by an
impaired oxidative balance. Serum MDA concentration, being
correlated to C2C concentration, suggests a relationship of
Page 194 Veterinary Clinical Pathology
lipid peroxidation and cartilage degeneration in the Pond-
Nuki model.
Acknowledgments
Many thanks to Associate Professor L Sotirov for his assistance in
performing the C2C ELISA.
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