Urinary glycosaminoglycans in horse osteoarthritis. Effects of chondroitin
sulfate and glucosamine
Raquel Y.A. Baccarinb,⇑, Thaís S.L. Machadob, Ana P. Lopes-Moraesb, Fabiana A.C. Vieiraa,
Yara M. Michelaccia
aDepartamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, UNIFESP, São Paulo, SP, Brazil
bDepartamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, USP, São Paulo, SP, Brazil
a r t i c l ei n f o
Received 13 May 2011
Accepted 19 August 2011
a b s t r a c t
Our objectives were to characterize the urinary excretion of glycosaminoglycans (GAGs) in horse osteo-
arthritis, and to investigate the effects of chondroitin sulfate (CS) and glucosamine (GlcN) upon the dis-
ease. Urinary GAGs were measured in 47 athletic horses, 20 healthy and 27 with osteoarthritis. The
effects of CS and GlcN were investigated in mild osteoarthritis. In comparison to normal, urinary GAGs
were increased in osteoarthritis, including mild osteoarthritis affecting only one joint. Treatment with
CS + GlcN led to a long lasting increase in the urinary CS and keratan sulfate (KS), and significant improve-
ment in flexion test of tarsocrural and metacarpophalangeal joints was observed. In conclusion, urinary
CS and KS seems to reflect the turnover rates of cartilage matrix proteoglycans, and the measurement of
these compounds could provide objective means of evaluating and monitoring joint diseases.
? 2011 Elsevier Ltd. All rights reserved.
Osteoarthritis (OA) is a chronic condition characterized by pro-
gressive degeneration of articular cartilage accompanied by sclero-
sis of the subchondral bone (Reginster, 2002). Clinical features
include joint effusion, pain, instability, limitation of motion and
breakdown of articular cartilage, and its presence and severity is
usually documented by X-ray imaging. Breakdown of the cartilage
components – collagen, proteoglycans and other proteins – results
in the generation of fragments of these macromolecules, which can
eventually be detected in the blood, synovial fluid, or urine.
Systemic biomarkers (serum or urine) offer a potential method
for quantifying the disease status, and these indicators in serum
or urine would provide objective means of evaluating and monitor-
ing OA. Furthermore, the identification of biomarkers is one way to
accelerate drug discovery and trials.
Cartilage is the mammalian tissue that contains the highest
concentration of glycosaminoglycans (GAGs)1, especially chondroi-
in the destructionand
tin sulfate (CS) and keratan sulfate (KS), which occur in the tissue as
proteoglycans. There are evidences indicating that most of the uri-
nary GAGs are of systemic origin: CS is the main urinary and plas-
matic GAG in many mammalian species, but it is not present
either in the urinary tract (Pereira et al., 2004; de Lima et al.,
2005) or kidney (Cadaval et al., 2000); CS given to rats was rapidly
excreted in the urine, part as polymeric CS and part as degradation
products (Michelacci et al., 1992); cartilage and cornea are the only
tissues that contain significant amounts of KS, and trace amounts of
KS are present in mammalian urine (Pereira et al., 2004; Vieira et al.,
2005). Consequently, these compounds could be systemic biochem-
ical markers of joint diseases.
We have previously shown that when growth and calcification
processes take place in human cartilages, changes occur in the
structure and composition of cartilage proteoglycans, especially
aggrecan. These changes were observed with age, in osteoarthritis
(Michelacci et al., 1979), and in cartilage tumors, both chondrosar-
coma (Mourão et al., 1979) and enchondromatosis (Michelacci
et al., 1981).
Because horses have naturally occurring OA (Richardson and
Loinaz, 2007), which is similar to that of humans, the horse was
chosen as a species to investigate a possible correlation between
urinary excretion of GAGs and OA. The main urinary GAGs in
horses are CS, dermatan sulfate (DS) and heparan sulfate (HS), with
small amounts of KS. A marked decrease in urinary GAGs occurred
with age for healthy horses, both sedentary and athletes, and the
concentration of KS increased with age. Athletic horses excreted
0034-5288/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
⇑Corresponding author. Address: Departamento de Clínica Médica, Faculdade de
Medicina Veterinária e Zootecnia, Universidade de São Paulo, USP, Av. Prof. Dr.
Orlando Marques de Paiva, 87, CEP 05508-270, São Paulo, SP, Brazil. Tel.: +55 11
3091 1323; fax: +55 11 3091 1283.
E-mail address: firstname.lastname@example.org (R.Y.A. Baccarin).
1Abbreviations: CS, chondroitin sulfate; GAG, glycosaminoglycan; GlcN, glucosa-
mine; GLUT, glucose transporter protein; HAS, hyaluronan synthase; i.p., intraperi-
toneal; i.m., intramuscular; KS, keratan sulfate; p.o., per oral.
Research in Veterinary Science 93 (2012) 88–96
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less GAGs in the urine than age-matched sedentary horses (includ-
ing KS) (Vieira et al., 2005).
Horses are also a good model to investigate the potential of
‘‘nutraceutical’’ agents, such as glucosamine (GlcN) and CS, for
the treatment of OA. From the treatment standpoint, they are easy
to administer and generally received as benign (Trumble, 2005),
and both the dosing and the patients’ adherence are easy to
The molecular mechanisms mediating anti-arthritic activities of
GlcN and CS are not completely understood. The transport of GlcN
through plasma membrane is facilitated by glucose transporter
proteins (GLUTs) (Uldry et al., 2002). Chondrocytes express several
GLUTs and actively import and metabolize GlcN, but not N-acetyl-
glucosamine. GlcN inhibits glucose transport in a non-competitive
fashion (Shikhman et al., 2009), and in equine chondrocytes, GlcN
reduced the interleukin-1b-induced expression of inflammatory
reaction-related enzymes (Neil et al., 2005b).
Concerning CS, many authors have shown anti-inflammatory
activities for CS in cultured chondrocytes or cartilage explants from
different mammalian species (reviews in du Souich et al., 2009;
Iovu et al., 2008). Also, there are evidences suggesting that the
combination of GlcN and CS may be more effective in preventing
cartilage degradation (Dechant et al., 2005). However, most of
the above cited studies were performed in cultured cells or
cartilage explants. There are few in vivo studies concerning the
biochemical effects of CS and/or GlcN in horses (Forsyth et al.,
2006; Neil et al., 2005a). So, the objectives of the present study
were to characterize the urinary excretion of GAGs in athletic
horses, both healthy and with OA, and to investigate the effects
of CS and GlcN upon clinical and biochemical markers of OA in
2. Materials and methods
Standard chondroitin 4-sulfate(fromwhale cartilage),chondroi-
tin 6-sulfate (from shark cartilage) and DS (from hog skin) were
purchased from Sigma Chemical Co. HS (from bovine pancreas)
and chondroitinases AC and B (from Flavobacterium heparinum)
were prepared by methods previously described (Aguiar et al.,
2003; Dietrich and Nader, 1974). Agarose (standard, low Mr) was
purchased from Bio-Rad Laboratories. Q-Sepharose Fast Flow was
purchased from Amersham Pharmacia Biotech do Brasil Ltda.
MST1 anti-KS monoclonal antibody was obtained as previously de-
scribed (Alves et al., 1994). Two commercial preparations of CS and
GlcN from Vetnil Ind. e Com. de Produtos Veterinários Ltda. were
used: (A) an isotonic solution of CS (75 g/l) and GlcN (75 g/l),
administered by intramuscular route (i.m.), (10 ml = 750 mg
CS + 750 mg GlcN); and (B), a mixture of GlcN (800 mg), selenium
(4 mg), vitamine E (600 mg), and amino acids (L-glutamine,
800 mg, L-arginine, 200 mg, proline, 400 mg, DL-methionine,
800 mg, L-lysine, 800 mg), given by oral route (p.o.). Before use,
these preparations were submitted to analysis for CS and GlcN
(Dietrich et al., 1977; Giusti et al., 1988), which have shown that
the concentrations stated by the manufacturer were correct.
2.2. Animals and urine samples
This work was approved by the Ethical Committees of Universid-
ade Federal de São Paulo – UNIFESP (CEP 0800/07) and Universidade
de São Paulo – USP (946/2006), and was carried out in accordance
with UNIFESP and USP guidelines, and also in accordance with EC
Directive 86/609/EEC for animal experiments http://ec.europa.eu/
All urine samples were collected via urethral catheterization
always at 10:00 a.m. The samples were stored at ?20 ?C until use.
To evaluate the variations in the concentration of urinary GAGs
in horses, urine samples were collected from three healthy athletic
horses for six consecutive days.
For the determination of horse urinary excretion pattern and to
identify possible changes that occur in OA, urine samples were col-
lected from 47 warmblood athletic horses, 20 healthy and 27 with
To evaluate the urinary excretion of exogenous CS, three athletic
horseswithmildOA wereselectedbyphysicalandradiographic cri-
teria. Urine samples were collected on days 1 and 2, at 10:00 a.m.,
and soon after the urine sample collection of day 2, CS + GlcN was
i.m. administered. Urine samples were collected on the subsequent
five days, always at 10:00 a.m. On day 7, CS + GlcN was again i.m.
administered soon after the urine sample collection, and again ur-
ine samples were collected on the subsequent five days. Creatinine
and GAG concentrations were measured.
The effects of CS and GlcN administration were investigated in
six athletic horses with mild OA affecting only one joint, selected
by radiographic criteria. These animals were dressage and jumping
warmblood horses, 8–12 years old. The inclusion criteria were:
1. Lameness due to either metacarpophalangeal or tarsocrural
joints for more than one month, increased articular volume or
history of intermittent joint lameness;
2. Radiographic image of either metacarpophalangeal or tarsocru-
ral joints compatible with osteoarthritis types 1 or 2, according
to McIlwraith (2002);
3. Absence of treatment with GAGs and/or anti-inflammatory
After the first urine sample collection, these animals were trea-
ted every five days, for 25 days, with 10 ml of i.m. CS + GlcN (prep-
aration (A), Section 2.1). Urine samples were collected immediately
before each dosing on days 1, 6, 11, 16, and 21. Subsequently, urine
samples were collected every seven days, up to the 60th day. Two
month later (day 115), the same animals received daily p.o. GlcN
doses (800 mg/day, preparation (B), Section 2.1) for 25 days, and
again urine samples were collected every five days during the
treatment, and every seven days after the end of treatment, up to
the 184th day.
2.4. Glycosaminoglycan identification and quantification
Urinary GAGs were analyzed by two different methods: (1)
Dialysis: Urine samples (500 ll) were dialyzed against pure water,
freeze-dried in Speed Vac (VR-1 Heto Lab), and resuspended in
20 ll of water; (2) Ion-exchange chromatography: Urine samples
(10–40 ml) were diluted with three volumes of water, and then ap-
plied to an ion-exchange column of Q-Sepharose Fast Flow (5 ml
bed volume, 1 ? 6.5 cm), previously equilibrated with water. The
column was eluted in a stepwise fashion with 0.3 M NaCl (20 ml)
and 2 M NaCl (20 ml).Three ml fractions were collected. Three vol-
umes of methanol were added to each fraction, slowly and under
agitation. After standing for 24 h at ?20 ?C, the precipitates formed
were collected by centrifugation, vacuum dried and resuspended in
100 ll of water.
Aliquots of these preparations (5 ll) were submitted to agarose
gel electrophoresis in 0.05 M 1,3-diaminopropane-acetate buffer,
pH 9 (PDA) (Dietrich et al., 1977). After fixation with cetyltrimeth-
ylammonium bromide and Toluidine Blue staining, the urinary
GAGs were quantified by densitometry of the gel slabs (Scanner
R.Y.A. Baccarin et al./Research in Veterinary Science 93 (2012) 88–96
CS-9000, Shimadzu). These compounds were further characterized
by enzymatic degradation with bacterial GAG lyases (chondroitin-
ase AC, chondroitinase B and heparitinase II from Flavobacterium
heparinum), as previously described (Petricevich and Michelacci,
1990). Only the fractions 2–3 eluted with 2 M NaCl contained
2.5. Identification and quantification of keratan sulfate
Urinary KS was detected by immunoblotting probed with MST1,
a monoclonal antibody that recognizes KS both as free chains and
as proteoglycans (Alves et al., 1994; Pereira et al., 2004). After aga-
rose gel electrophoresis, GAGs were transferred to nitrocellulose
and Zeta-Probe nylon membranes (Bio-Rad Laboratories, Rich-
mond, CA, USA). After blocking, membranes were probed with
MST1. Then, the membranes were incubated with peroxidase-con-
jugated rabbit anti-mouse IgG secondary antibody. The antibody
binding was visualized through diaminobenzidine (DAB), as previ-
ously described (Sambrook et al., 1989). Quantitative analysis were
performed by ELISA (Alves et al., 1994), using KS purified from bo-
vine nucleus pulposus as standard. In brief, ELISA plates were
coated with 0.5% poly-Lys. After 1 h incubation at room tempera-
ture, the plates were washed with water and incubated with anti-
gen (either standard KS, 10–1000 ng, or urine samples, 50–200 ll,
1 h, room temperature). After washing to remove unbound anti-
gen, 1% bovine serum albumin in phosphate buffered saline (PBS)
was added (200 ll/well), and after 2 h incubation at room temper-
ature, and washing with PBS, MST1 monoclonal antibody, was
added (100 ll/well). The plates were washed to remove unbound
MST1 antibody, and rabbit immunoglobulin-peroxidase conjugate
against mouse IgG was used as secondary antibody. After washing,
the peroxidase activity was measured with OPD/H2O2.
2.6. Quantification of creatinine
Creatinine was measured by the alkaline picrate method,
adapted to micro-scale as previously described (de Lima et al.,
2.7. Physical and radiographic examination
The joint examination was performed on the selected animals
the day before the beginning of CS + GlcN and GlcN administration
(days 1 and 115) and 60 days later (days 60 and 180). The proce-
dures for examination were: visual examination at rest, joint
palpation and manipulation, grading of the lameness (1–5) accord-
ing to Stashak (2002), and joint flexion tests.
Both metacarpophalangeal and tarsocrural joints were radio-
graphically examinedduringthe selectionphaseinorder to identify
Three independent extractions and quantifications of glycosaminoglycans from the
same sample of horse urine.
CS (mg/l)DS (mg/l) HS (mg/l) Total GAGs (mg/l)
Mean3.15 0.41 1.74 5.30
St. dev. 0.006 0.021 0.0310.0
Fig. 1. Agarose gel electrophoresis of horse urinary GAGs isolated from urine samples collected on six consecutive days (A), and quantitative data (B) expressed both as
concentration (mg/l) and GAG/creatinine ratios (mg/g). Urinary GAGs were extracted by ion-exchange chromatography on Q-Sepharose from urine samples (40 ml) collected
on six consecutive days, always at 10:00 a.m., from three healthy athletic horses. Aliquots (5 ll) of the GAG-containing fractions were submitted to agarose gel
electrophoresis in PDA buffer, as described in Section 2.4, and the GAGs were quantified by densitometry of the agarose gel slabs. Quantitative data are mean ± standard
deviation of three independent determinations for each animals. CS, chondroitin sulfate; DS, dermatan sulfate, HS, heparan sulfate; S, mixture of standard
glycosaminoglycans; Total, total urinary GAGs.
90R.Y.A. Baccarin et al./Research in Veterinary Science 93 (2012) 88–96
OA. The radiographic images of the joints served as a baseline for
radiographic post-treatment examination on days 60 and 180.
2.8. Statistical analysis
The Bartlett’s test was used to test if groups are homoscedastic.
One-way ANOVA with Dunnett’ spot test and Student’s t-test were
performed using GraphPad Prism, GraphPad Software, San Diego
California USA. Significance was defined by P values less than 0.05.
3.1. Horse urinary glycosaminoglycans
To evaluate the reproducibility of the extraction and quantifica-
tion procedures here used, and also to analyze the variations in uri-
nary GAGs in healthy athletic horses, urine samples were collected
from three animals, always at 10:00 a.m., for six consecutive days.
The GAGs were isolated and analyzed by the two procedures de-
scribed in Methods, Section 2.4, which gave very similar results
for total GAGs. Nevertheless, due to the presence of pigments in
the samples submitted to dialysis, it was difficult to quantify indi-
vidual GAGs. For this reason, the quantitative analyses of individ-
ual GAGs were always performed on the samples submitted to
ion exchange chromatography. The dialysis method was used to
check the total amounts of urinary GAGs.
Table 1 shows that very small differences in the GAG concentra-
tion were obtained in three independent extractions (ion exchange
chromatography) and quantifications (agarose gel electrophoresis
and densitometry) for the same urine sample (2–3%). The daily
variations in GAG concentration for the same animal, expressed
both as concentration (mg/l of urine) and as GAG/creatinine ratios
(mg/g of creatinine) were also very small (Fig. 1).
3.2. Urinary glycosaminoglycans in osteoarthritis
Using these methods, urine samples from 20 healthy athletic
horses and 27 athletic horses with OA were analyzed (Fig. 2). Ani-
mals with OA excreted more GAGs, specifically CS, than healthy
athletic horses (expressed as GAG/creatinine ratios, P < 0.05). The
identification of each GAG was confirmed by enzymatic degrada-
tion with specific GAG lyases (not shown).
Fig. 2. Urinary GAGs from 47 athletic horses, 20 healthy and 27 with osteoarthritis. Urinary GAGs were isolated by ion exchange chromatography, identified and quantified as
described in Methods, Section 2.4. The quantitative results, expressed both as concentration (mg/l) and GAG/creatinine ratios (mg/g), are mean ± standard deviation of three
independent determinations for each sample. CS, chondroitin sulfate; DS, dermatan sulfate, HS, heparan sulfate; Total, total urinary GAGs.⁄Differences statistically significant
as compared to healthy athletic horses (P < 0.05).
R.Y.A. Baccarin et al./Research in Veterinary Science 93 (2012) 88–96
Nine athletic horses were selected among the 27 with OA to
investigate the effects of CS and GlcN. These selected animals pre-
sented mild OA affecting only one joint, which did not prevent ath-
letic activities. Fig. 3 shows that the mean GAG/creatinine ratios of
these animals were similar to the OA group and higher than the
healthy group (see Fig. 2 for comparison).
3.3. Urinary excretion of exogenous chondroitin sulfate given to
athletic horses with mild osteoarthritis
To check if the exogenous CS does appear in the horse urine and
to assess its elimination rate, two doses of CS + GlcN were given to
three of the selected athletic horses with mild OA, and urine sam-
ples were collected for 12 days, always at 10:00 a.m. Fig. 4 shows a
remarkable increase in the CS concentration in the urine samples
collected 24 h after the administration of CS + GlcN (days 3 and
8), that returned to the basal levels 24–48 h later (days 4 and 10).
3.4. Excretion of glycosaminoglycans in the urine before, during and
Six of the selected athletic horses with mild OA were treated
with CS + GlcN (i.m., every 5 days, for 25 days) and GlcN (p.o.,
for 25 days), according to the protocol described in Methods, Sec-
tion 2.3. Urine samples were always collected before each dosing
of CS + GlcN, in order to avoid exogenous CS in the urine (see Fig
4 for excretion rate of CS). GAGs and creatinine were measured in
allurine samples,and the
(mean ± standard deviation). Each point is the mean of three
determinations for each animal. During the treatment with
CS + GlcN (horizontal bars) and afterward, an increase in the uri-
nary excretion of GAGs, especially CS, was observed (expressed as
GAG/creatinine ratios). This increase was not due to the exoge-
nous CS, since urine samples were collected five days after each
administration, when all exogenous CS had already been excreted
(see Fig. 4), and before the new dosing. Furthermore, the CS con-
centration remained high for months after the end of CS + GlcN
treatment (last dose on day 21). Three months after the last
CS + GlcN dose (day 115), the urinary CS was still high, and re-
mained so until day ?130, when treatment with GlcN was al-
ready going on (second horizontal bar). The effects of treatment
with GlcN upon urinary GAGs were not clear, since the urinary
excretion of CS was still higher than the basal levels when this
treatment was initiated (Fig. 6). Nevertheless, a new increase oc-
curred during the first days of treatment, decreasing afterwards
to the basal levels (Fig. 5).
Fig. 3. Urinary GAGs from nine athletic horses with mild OA. Nine animals with mild OA, which affected only one joint and did not prevent athletic activities, were selected.
The experiment was performed as described in Fig. 1. The quantitative results, expressed both as concentration (mg/l) and GAG/creatinine ratios (mg/g), are mean ± standard
deviation of three independent determinations for each sample. CS, chondroitin sulfate; DS, dermatan sulfate, HS, heparan sulfate; Total, total urinary GAGs.
92 R.Y.A. Baccarin et al./Research in Veterinary Science 93 (2012) 88–96
3.5. Urinary keratan sulfate
Fig. 7 shows that the urinary KS also increased upon treatment
with CS + GlcN and GlcN. This increase also appeared in immuno-
blotting (Fig. 7A).
3.6. Clinical assessments
Upon treatment with CS + GlcN, there was a significant
improvement in flexion test of tarsocrural and metacarpophalan-
geal joints (P < 0.05). There was also a decrease in joint volume,
and the scores assigned to lameness and pain in different joints
also improved, although the differences were not statistically sig-
nificant. The clinical signs of osteoarthritis did not change after
treatment with GlcN (day 150, Table 2).
The analysis of the radiographic images of each animal has
shown that the initial radiographic scores of horse 1 were lower
than those of horses 2–6. Although improvement was observed,
the differences in radiographic scores upon treatment with
CS + GlcN and GlcN were not statistically significant.
4. Discussion and conclusions
Osteoarthritis (OA) is the end result of a variety of disorders
leading to the structural and functional failure of one or more
joints. The disease progresses through several phases, and involves
an advancing loss of articular cartilage. The cartilage tries to repair
itself, the bone remodels, the subchondral bone hardens, and bone
cysts form. The stationary phase of OA involves the formation of
osteophytes or joint space narrowing. OA progresses further with
obliteration of the joint space. The appearance of subchondral cysts
indicates the erosive phase of disease, and the last phase involves
bone repair and remodeling.
Among the most abundant components of cartilage extracellu-
lar matrix are the proteoglycans, especially aggrecan. We have
Fig. 4. Excretion of GAGs in the urine following i.m. CS + GlcN administration.
Urine samples from three athletic horses with mild OA were collected on days 1
and 2. On day 2 and on day 7 (arrows), after the urine sample collection, a
solution containing CS + GlcN (10 ml) was i.m. administered. Urine samples were
collected every day, at 10:00 a.m., and urinary GAGs were isolated, analyzed, and
quantified as described in Fig. 1. Quantitative data are mean ± standard deviation
of three independent determinations for each sample.
Fig. 5. Urinary GAGs of six athletic horses with mild OA treated with CS + GlcN (i.m.) and GlcN (p.o.). Six athletic horses with mild OA in metacarpophalangeal or tarsocrural
joints were selected by physical and radiographic examinations, urine samples were collected (day 1), and then the animals were treated every five days, by 25 days, with a
preparation of CS + GlcN (i.m.). Urine samples were collected immediately before each administration (days 1–21, horizontal bars), and every seven days for 35 days after the
end of the treatment (days 34–60). Three months after the last CS + GlcN dose, urine samples were again collected (day 115), and the same animals received daily p.o. GlcN
doses (800 mg/day), for 25 days, and again urine samples were collected every five days during the treatment (days 115–140, horizontal bars), and every seven days after the
end of the treatment, for 35 days (days 147–180). Urinary GAGs were isolated, analyzed, and quantified as described in Fig. 1. The graph shows mean ± standard deviation of
GAG/creatinine ratios from 132 urine samples, 22 from each animal (three independent determinations for each sample). CS, chondroitin sulfate; DS, dermatan sulfate; HS,
heparan sulfate; Total GAG, total urinary glycosaminoglycans.⁄Differences statistically significant as compared to basal levels (day 1) (P < 0.05).
R.Y.A. Baccarin et al./Research in Veterinary Science 93 (2012) 88–96
previously shown that the structure of aggrecan changes when
growth and calcification processes occur in human articular carti-
lage: the young cartilage proteoglycan contains lower amounts of
KS, and the CS is hybrid, composed by both 4-sulfated and 6-sul-
fated disaccharide units, in contrast to the adult articular cartilage
CS that is almost exclusively 6-sulfated. Such structural changes in
composition could lead to alterations in the structure and size of
the aggregates, providing the conditions for the growth and calci-
fication processes to occur. The aggrecan from OA cartilages is very
similar to the young normal cartilages, suggesting that the carti-
lage is actively synthesizing extracellular matrix components of
the ‘‘growth and calcification’’ type, in an effort to regenerate itself
(Michelacci et al., 1979).
Increased turnover of cartilage components would generate
fragments of its constituents, which could appear in blood and ur-
ine, and offer potential methods to evaluate the disease status. This
evaluation could contribute to the early diagnosis and monitoring
of OA. Our results show that an increase in urinary CS and KS does
occur in OA, indicating higher turnover rates of cartilage matrix
For the analysis of urinary GAGs, different methods have been
described in the literature. Measurement of urinary GAGs by color-
imetric methods, such as determination of uronic acid, or binding
of alcian blue or 1,9-dimethylmethylene blue to GAGs, is unreli-
able, since other urinary components such as salts, nucleic acids
and neutral sugars, do interfere (Michelacci et al., 1989; de Lima
et al., 2007). We have previously shown that the gold standard
method for identification and quantification of urinary GAGs is
agarose gel electrophoresis and densitometry (Michelacci et al.,
1989). Urine samples can be desalted, concentrated and directly
submitted to agarose gel electrophoresis or, alternatively, urinary
GAGs can be isolated by ion exchange chromatography, precipi-
tated by methanol, resuspended in water and analyzed. Similar re-
sults were obtained for horse urinary GAGs by both procedures.
The desalting-concentration method was very good for small urine
samples (0.5–2 ml), while the ion exchange chromatography per-
mitted isolation of urinary GAGs from larger samples. The identifi-
cation of the urinary GAGs was confirmed by incubation with
specific GAG lyases (Petricevich and Michelacci, 1990). In the pres-
ent paper we have shown that the error of the method was very
It was previously shown that athletic horses excreted less GAGs
in the urine than age-matched sedentary horses (Vieira et al.,
2005). According to Murray et al. (1999, 2001), articular cartilages
from strenuously trained horses is less stiff, presents fibrillation,
and has reduced superficial Toluidine Blue staining, in comparison
to gently exercised animals. Thus, the reduced proteoglycan con-
centration in athletic horse cartilage, particularly in the superficial
layers, may explain the reduced urinary excretion of CS. In con-
trast, athletic horses with OA excreted more GAGs, especially CS,
Fig. 6. Effect of treatment with CS and GlcN upon the urinary excretion of GAGs in
athletic horses with mild OA. The experiment was performed as described in Fig. 5,
except that mean ± standard deviation of all animals and samples before, during
and after each treatment are shown. CS, chondroitin sulfate; DS, dermatan sulfate;
HS, heparan sulfate.⁄Differences statistically significant as compared to basal levels
(day 1) (P < 0.05).
Fig. 7. Effect of treatment with CS and GlcN upon the urinary excretion of KS in
athletic horses with mild OA. Immunoblotting of KS (A) and mean ± standard
deviation (B) of urinary KS measured by ELISA in all animals and samples before,
during and after each treatment.⁄Differences statistically significant as compared
to basal levels (day 1) (P < 0.05).
94R.Y.A. Baccarin et al./Research in Veterinary Science 93 (2012) 88–96
than healthy age-matched animals, possibly indicating higher
turnover rates of CS-rich cartilage proteoglycans.
CS and GlcN were introduced as safer alternatives, in compari-
son to anti-inflammatory agents, originally considered to serve
only as exogenous sources of building blocks for proteoglycans.
More recently it was shown that CS and GlcN can reduce inflam-
mation and the extent of cartilage degradation (Dingle, 1999),
and some authors have observed that the combination of GlcN
and CS may enhance this efficacy (Lippiello et al., 2000).
The pharmacokinetics of GlcN and CS has been studied in ani-
mals and man (Adebowale et al., 2002; Michelacci et al., 1992),
and it was shown that most of the GlcN (given either p.o. or i.v.)
is readily absorbed and rapidly diffuses into most tissues and or-
gans. Several organs and tissues have shown capacity to concen-
trate GlcN from plasma, and incorporate into glycoproteins and
proteoglycans. CS was also shown to be bioavailable (Lippiello
et al., 2000). In dogs, the absorption of CS appears to be rapid after
oral administration (2–3 h), with an elimination half-life of 10–
12 h. Katta et al. (2009) have shown that, in vitro, CS was able to
diffuse into the cartilage, at higher rate in GAG-deficient tissues.
Thus, it is likely that also in vivo the exogenous CS could diffuse
into cartilage and reach chondrocytes.
In the present paper, the appearance in the urine of exogenous
CS given to horses indicates that this compound was systemically
distributed, possibly reaching all tissues, including cartilages. After
CS administration, urinary CS increased and then returned to basal
levels in 24–48 h, indicating that most of the exogenous polymeric
CS had already been excreted. Nonetheless, the concentration of
endogenous CS and KS gradually increased and remained high in
the urine for about three months after the end of treatment. This
finding suggests even higher turnover rates, perhaps in an attempt
of the cartilage to regenerate itself.
There are evidences indicating that GlcN can inhibit chondro-
cyte-mediated catabolism of aggrecan (Sandy et al., 1998), has
immunosuppressive (Ma et al., 2002) and anti-inflammatory
(Largo et al., 2009) effects. CS also seems to have anti-inflammatory
activities. For example, it was shown that in lipopolysaccharide-
treated chondrocytes, CS and HS reduced the apoptotic process
and the expression of inflammation mediators, while DS and KS
had no effect (Campo et al., 2009). CS also inhibited lipid peroxida-
tion and protected cells from reactive oxygen species (Campo
et al., 2008). Treatment of cartilage explants with a combination of
GlcN and CS downregulated the expression of inflammatory media-
tors, such as inducible nitric oxide synthase, cyclooxygenase-2 and
prostaglandin E synthase 1, and also decreased matrix metallopro-
teinase-3 and aggrecanase-2, while increased the expression of
TIMP-3 (Chan et al., 2007).
In conclusion, it seems that urinary CS and KS reflect the turn-
over rates of cartilages and could be used in evaluating and moni-
toring joint diseases. Exogenous CS and GlcN had beneficial effects
on mild OA, since improvement was observed in both biochemical
and clinical parameters.
Conflict of interest statement
None of the authors of this paper has any actual or potential
conflict of interest, including financial or personal relationship
with other people or organizations that could inappropriately
influence, or be perceived to influence, this work.
This manuscript was prepared in accordance to the Uniform
Requirements for Manuscripts Submitted to Biomedical Journals of
International Committee of Medical Journal Editors. This research
was supported by Conselho Nacional de Desenvolvimento Científ-
ico e Tecnológico (CNPq), Brasília, DF, Brazil; Fundação Coorde-
nação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Brasília, SP, Brazil; Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP), São Paulo, SP, Brazil; and Sociedade Paulista
para o Desenvolvimento da Medicina (SPDM-FADA), São Paulo,
SP, Brazil. These funding sources had no involvement in the collec-
Physical scores of athletic horses with mild OA and treated with CS and GlcN.
# JointFlexion test Increased joint volume LamenessPain
Day:1 60 150 1801 60150 1801 60 150 1801 60 150 180
1 Right metacarpophalangeal
2. Right metacarpophalangeal
4. Right metacarpophalangeal
5. Right metacarpophalangeal
Flexion test, increased articular volume and pain were classified as presence (+) or absence (?), lameness was classified as scores 1–5, according to Stashak (2002).
R.Y.A. Baccarin et al./Research in Veterinary Science 93 (2012) 88–96
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