Structural characterization of chondroitin sulfate from sturgeon bone

Article abstract of DOI:10.1016/j.carres.2010.05.016

Chondroitin sulfate (CS) was purified for the first time from the bones of sturgeon and analyzed to evaluate its structure and properties. A single polysaccharide was extracted from sturgeon bone in a concentration of 0.28-0.34% for dry tissue and characterized as CS. By means of specific chondroitinases and HPLC separation of generated unsaturated repeating disaccharides, this polymer was found to be composed of ～55% of disaccharide monosulfated in position 6 of the GalNAc, ～38% of disaccharide monosulfated in position 4 of the GalNAc, and ～7% of nonsulfated disaccharide. The charge density was 0.93 and the ratio of 4:6 sulfated residues was equal to 0.69, a value confirmed by ¹³C NMR experiments. Chondroitinase B confirmed that the purified sturgeon CS contained mainly GlcA (>99.5%) as uronic acid. PAGE analysis showed a CS having a high molecular mass with an average value of 39,880 according to HPSEC values producing a weight average molecular weight (Mw) of 37,500. On the basis of the data collected, it is reasonable to assume that CS isolated from sturgeon bone might be potentially useful for scientific and pharmacological applications, making this bony fish, which is generally discarded after ovary collection, a useful source of this polymer. Finally, this newly identified source of CS would enable the production of this macromolecule having a particular repeating disaccharide composition, structure, and biological properties.

Full text of DOI:10.1016/j.carres.2010.05.016

Carbohydrate Research 345 (2010) 1575–1580
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Structural characterization of chondroitin sulfate from sturgeon bone
Francesca Maccari a, Fabrizio Ferrarini b, Nicola Volpi a,
*
a Department of Biology, University of Modena and Reggio Emilia, Modena, Italy
b Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 April 2010
Received in revised form 11 May 2010
Accepted 16 May 2010
Available online 24 May 2010
Chondroitin sulfate (CS) was purified for the first time from the bones of sturgeon and analyzed to eval-
uate its structure and properties. A single polysaccharide was extracted from sturgeon bone in a concen-
tration of 0.28–0.34% for dry tissue and characterized as CS. By means of specific chondroitinases and
HPLC separation of generated unsaturated repeating disaccharides, this polymer was found to be com-
posed of ꢀ55% of disaccharide monosulfated in position 6 of the GalNAc, ꢀ38% of disaccharide monosulf-
ated in position 4 of the GalNAc, and ꢀ7% of nonsulfated disaccharide. The charge density was 0.93 and
the ratio of 4:6 sulfated residues was equal to 0.69, a value confirmed by 13C NMR experiments. Chondroi-
tinase B confirmed that the purified sturgeon CS contained mainly GlcA (>99.5%) as uronic acid. PAGE
analysis showed a CS having a high molecular mass with an average value of 39,880 according to HPSEC
values producing a weight average molecular weight (Mw) of 37,500. On the basis of the data collected, it
is reasonable to assume that CS isolated from sturgeon bone might be potentially useful for scientific and
pharmacological applications, making this bony fish, which is generally discarded after ovary collection, a
useful source of this polymer. Finally, this newly identified source of CS would enable the production of
this macromolecule having a particular repeating disaccharide composition, structure, and biological
properties.
Keywords:
Glycosaminoglycans
Chondroitin sulfate
Sturgeon
Bones
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
and other GAGs), are not only structural components but also par-
ticipate in and regulate many cellular events and physiological
Chondroitin sulfate (CS) is a linear, complex, sulfated, polydis-
perse natural polysaccharide belonging to the class of macromole-
cules known as glycosaminoglycans (GAGs).1,2 It is composed of
alternate sequences of GlcA and differently sulfated residues of
GalNAc linked by b-(1?3) bonds. Depending on the repeating
disaccharide nature, CS with different structures is known to have
different degrees of charge density and sulfate groups linked in
various positions (see Fig. 1 for the structural nature of various
CS disaccharides). Furthermore, it is a very heterogeneous polysac-
charide in terms of relative molecular mass, chemical properties,
biological and pharmacological activities.1,3,4
processes.5 As a consequence, GAGs are a class of macromolecules
of great importance in the fields of biochemistry, pathology, and
pharmacology. In fact, CS is currently recommended by EULAR6
Recent evidence from glycobiology studies suggests that prote-
oglycans, and their complex polysaccharidic macromolecules (CS
Abbreviations: CS, chondroitin sulfate; DS, dermatan sulfate; EULAR, The
European League Against Rheumatism; GAG(s), glycosaminoglycan(s); IdoA,
a-L-
idopyranosyluronic acid; Mn, number-average molecular weight; Mw, weight-
average molecular weight; Mz, Z average molecular weight; OA, osteoarthritis; SAX,
strong anion exchange; SYSADOA, Symptomatic Slow Acting Drug for OA;
4-deoxy- -threo-hex-4-enopyranosyluronic acid.
DHexA,
a-L
* Corresponding author. Address: Department of Biology, University of Modena
and Reggio Emilia, Via Campi 213/D, 41100 Modena, Italy. Tel.: +39 59 2055543;
fax: 0039 59 2055548.
Figure 1. Structures of repeating disaccharide units forming chondroitin sulfate.
Minor disaccharides may be present, such as that characterized by a sulfate group
in position 3 of glucuronic acid.
E-mail address: volpi@unimo.it (N. Volpi).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.05.016

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F. Maccari et al. / Carbohydrate Research 345 (2010) 1575–1580
as a SYSADOA (Symptomatic Slow Acting Drug for OA) drug in Eur-
ope in the treatment of knee osteoarthritis (OA) based on meta-
analysis of numerous clinical studies.7 Moreover, CS alone or in
combination with glucosamine is utilized as a dietary supplement
based on meta-analysis of studies confirming its safe and effective
options for the treatment of symptoms of OA.8
CS, like other natural polysaccharides, is derived from animal
sources by extraction and purification processes.1 Commercial
manufacture of CS relies at present on bovine,9 porcine,9 chicken,10
or cartilaginous fish such as sharks11 and skate12,13 by-products, in
particular, cartilage as raw material.
D
UA-2s-[1?3]-GalNAc-4s),
GalNAc-6s), di4,6dis ( Di-dis E,
Di2,4,6tris ( Di-tris, UA-2s-[1?3]-GalNAc-4s,6s)] were from
D
Di2,6dis (
DDi-dis D, DUA-2s-[1?3]-
D
D
D
D
D
UA-[1?3]-GalNAc-4,6dis), and
D
Seikagaku Corporation (Tokyo City, Japan). Stains-All (3,30-di-
methyl-9-methyl-4,5,40,50-dibenzothiacarbocyanine) was from Sig-
ma. QAE SephadexÒ A-25 anion-exchange resin was from Pharmacia
Biotech (Uppsala, Sweden). Spectrapore dialysis tubing (Mr 1000
daltons cut off) was from Spectrum (Rancho Dominguez, CA, USA).
All other reagents were of analytical grade.
2.2. Purification of sturgeon CS
As previously illustrated, due to its very complex heterogeneous
structure, CS from different sources may possess repeating disac-
charides having various sulfate groups located, in different per-
centages, inside the polysaccharide chains (see Fig. 1). These
repeating disaccharide units are generally monosulfated but,
depending on the origin, various disulfated disaccharides (and pos-
sibly also a trisulfated one) may be present in the polysaccharide
backbone. As a consequence, CS with different charge densities
may be produced from various sources. Furthermore, as a result
of the biosynthetic processes related to specific tissues and species,
CSs with different grades of polymerization may be biosynthetized
producing macromolecules having various molecular masses and
polydispersity. Due to these structural variations, CS from different
sources may have different properties and capacities.
The above-mentioned considerations have motivated us to look
for alternative sources of this complex polysaccharide also consid-
ering the possibility of producing CS with a particular repeating
disaccharide composition, structure, and activity. In this regard,
the sturgeon belongs to one of the oldest families of bony fish in
existence. It is a native of subtropical, temperate, and sub-Arctic
rivers, lakes, and coastlines of Eurasia and North America but it
is also found along the European Atlantic coast, including the Med-
iterranean Sea.14 The common name is used for some 26 species of
fish in the Acipenseridae family, including over 20 species com-
monly referred to as sturgeon and several closely related species
that have distinct common names, notably sterlet, kaluga, and be-
luga. Collectively, the family is also known as the true sturgeons.14
Where sturgeons are caught in large quantities, as in the rivers of
southern Russia and in the great lakes of North America, their flesh
is dried, smoked, or salted. The ovaries, which are large in size, are
prepared for caviar, and the air bladder is used to produce one of
the best kinds of gelatine,15 while the rest of the animals is usually
discarded. In this study, CS was extracted and purified from stur-
geon bones and its structure characterized along with its impor-
tant physico-chemical properties, thus demonstrating that
another part of these animals might be exploited for commercial
preparations.
Sturgeon bones (ꢀ50 g) were defatted by grinding with 100 mL
of acetone, followed by filtration and drying at 60 °C for 24 h. The
pellet was solubilized (1 g/10 mL) in 100 mM Na–acetate buffer pH
5.5 containing 5 mM EDTA and 5 mM cysteine. 50 mg of papain
were added per g of tissue and the solution incubated for 24 h at
60 °C in a stirrer. After boiling for 10 min, the mixture was centri-
fuged at 5000g for 15 min, and three volumes of ethanol saturated
with sodium acetate were added to the supernatant and left at
+4 °C for 24 h. The precipitate was recovered by centrifugation at
5000g for 15 min and dried at 60 °C for 6 h. The dried precipitate
was dissolved in 50 mL of 50 mM NaCl. After centrifugation at
10,000g for 10 min, the supernatant was applied to a column
(2 cm ꢁ 40 cm) packed with QAE SephadexÒ A-25 anion-exchange
resin equilibrated with the same NaCl solution. GAGs were eluted
with a linear gradient of NaCl from 50 mM to 1.2 M in 150 min
using low-pressure liquid chromatography (Biological LP chroma-
tography system from BioRad) at a flow of 1 mL/min. Two volumes
of ethanol were added to the collected fractions corresponding to
fractionated species of polysaccharides evaluated by uronic acid
assay16 and agarose gel electrophoresis.17,18 After precipitation at
4 °C and centrifugation at 10,000g for 10 min, the pellet was dried
at 60 °C and solubilized in 20 mM Tris-Cl buffer pH 8.0 containing
2 mM MgCl2 and treated with DNAse I (750 mg) at 37 °C for 12 h.
After boiling for 5 min, NaCl concentration was brought to 16%
and the GAGs were precipitated by adding 80% methanol. The
recovered precipitate (ꢀ0.1 g) was solubilized in 20 mL doubly dis-
tilled water, dialyzed overnight at 4 °C, and freeze-dried for further
characterization.
2.3. Agarose gel electrophoresis
Agarose gel electrophoresis in barium acetate–1,2-diaminopro-
pane was performed as reported elsewhere17,18 with minor modi-
fications.
A Pharmacia Multiphor II (from Pharmacia LKB
Biotechnology, Uppsala, Sweden) electrophoretic cell instrument
was used. Agarose gel was prepared at a concentration of 0.5% in
0.04 M barium acetate buffer pH 5.8. The run was in 0.05 M 1,2-
diaminopropane (buffered at pH 9.0 with acetic acid) for 150 min
at 50 mA. After migration, the plate was soaked in cetyltrimethyl-
ammonium bromide 0.1% solution for at least 6 h, dried and
stained with toluidine blue.18 Extracted CS was also evaluated by
agarose gel electrophoresis after treatment with various lyases.
2. Experimental
2.1. Materials and methods
Heparin from bovine intestinal mucosa, heparan sulfate from bo-
vine kidney, CS from bovine trachea, DS from porcine intestinal mu-
cosa, and HA from rooster comb were from Sigma–Aldrich (St. Louis,
MO, USA). Papain from papaya latex (EC 3.4.22.2), specific activity of
16–40 units/mg protein, and deoxyribonuclease I, DNase I (EC
3.1.21.1) from bovine pancreas, specific activity of 10,000 units/
mL, were from Sigma–Aldrich. Chondroitinase ABC from Proteus
vulgaris (EC 4.2.2.4), specific activity of 0.5–2 units/mg, and
chondroitinase B from Flavobacterium heparinum (EC 4.2.2.), specific
activity of 100–300 units/mg, were from Sigma–Aldrich. Unsatu-
2.4. Enzymatic treatments and disaccharide evaluation
After treatment of purified CS with chondroitinase ABC or B, the
generated unsaturated disaccharides were separated and quanti-
fied by strong anion-exchange (SAX)-HPLC using an HPLC equip-
ment from Jasco equipped with a 150 ꢁ 4.6-mm stainless-steel
column spherisorb 5-SAX (5 lm, trimethylammoniopropyl groups
Si–CH2–CH2–CH2–N+(CH3)3 in Clꢂ form, from Phase Separations
Limited, Deeside Industrial Park, Deeside Clwyd, U.K.) and detec-
tion at 232 nm. Isocratic separation was performed using 50 mM
NaCl pH 4.00 for 5 min followed by a linear gradient from 5 to
rated chondro/dermato disaccharides
[
D
D
D
Di0s
Di6s (
Di2,4dis (D
(
D
UA-[1?3]-
UA-[1?3]-Gal-
Di-dis B,
GalNAc),
NAc-6s),
D
D
Di4s (
Di2s (
D
D
UA-[1?3]-GalNAc-4s),
UA-2s-[1?3]-GalNAc),
D

F. Maccari et al. / Carbohydrate Research 345 (2010) 1575–1580
1577
60 min of 50 mM NaCl to 1.2 M NaCl pH 4.00, at a flow rate of
1.2 mL/min. Authentic unsaturated standard disaccharides were
used for qualitative and quantitative purposes.
2.5. CS molecular mass determination
The molecular mass of sturgeon CS was determined by PAGE
according to Edens et al.19 20
lg of the purified CS were layered
on the gel and the calibration curve was constructed by using oli-
gosaccharide standards of known molecular mass prepared from
CS. The gel was stained with toluidine blue (0.1% in acetic acid
1%) for 30 min. After decoloration with acetic acid 1%, molecular
mass evaluation was performed after densitometric acquisition.
The molecular mass and polydispersity of CS were also deter-
mined by HPSEC using CS fractions of known molecular mass.20
50 lg of the extracted CS were injected into the column. The mo-
bile phase was composed of 125 mM Na2SO4 and 2 mM NaH2PO4
adjusted to pH 6.0 with 0.1 N NaOH. Flow rate was 0.9 mL/min.
Standards were solubilized in the mobile phase at a concentration
of 10 mg/mL and 10 lL (100 lg) were injected into HPLC. Columns
were Protein Pak 125 (Waters, cod. 84601, 7.8 mm ꢁ 30 cm) and
Protein Pak 300 (Waters, cod. T72711, 7.5 mm ꢁ 30 cm) assembled
in series. The retention times were plotted against the logarithm of
molecular mass for standard heparins. The curve that fits the
experimental data is a third grade polynomial with the formula
y(fx) = ꢂax3 + bx2 ꢂ cx + d performed by the Jasco Borwin program.
The number average molecular weight (Mn), the weight average
molecular weight (Mw), the Z average molecular weight (Mz),
and the polydispersity index (Mw/Mn) were calculated by the Jas-
co Borwin GPC software ver 4.1.
2.6. NMR analysis
The 13C NMR spectrum of purified CS was recorded by a Bruker
AMX400 WB spectrometer operating at 100.61 MHz. The sample
was prepared by dissolving 50 mg in 2.0 mL of D2O at a high level
of deuteration (99.997%) to avoid the presence of a relatively high
percentage of water. The spectra were recorded at a temperature of
33 °C and pH 6.5, unless specified. 13C chemical shifts (d, ppm) are
quoted with respect to external sodium 4,4-dimethyl-4-silapen-
tane-1-sulfonate (0.0 ppm).
Figure 3. SAX-HPLC of the unsaturated repeating disaccharides produced by
treating CS isolated from sturgeon bone with chondroitinase ABC (Lyase ABC) or
chondroitinase
B
(Lyase B).
DDi0s, DUA-[1?3]-GalNAc. DDi6s, DUA-[1?3]-
GalNAc-6s. Di4s,
D
DUA-[1?3]-GalNAc-4s.
3. Results and discussion
After defatting with organic solvents, extraction by proteolytic
treatment, and degradation of DNA by using DNAse, GAGs from
the sturgeon bones were fractionated on an anion-exchange resin.
The carbazole test for uronic acids16 and agarose gel electrophore-
sis17,18 confirmed the presence of a single polysaccharide, that is,
CS (Fig. 2), in the recovered fractions. Quantitative analyses yielded
ꢀ0.34% CS for dry tissue by carbazole test16 and ꢀ0.28% by agarose
gel electrophoresis.
To characterize the structure of sturgeon bone CS, the purified
CS was subjected to treatment with two lyases, chondroitinase
ABC and B, and the unsaturated disaccharides produced were ana-
lyzed by SAX-HPLC (Fig. 3) for a full characterization of the constit-
uent repeating disaccharides. The chondroitinase ABC was able to
produce three unsaturated disaccharides, the nonsulfated
UA1?3GalNAc], the monosulfated di6s UA1?3Gal-
NAc6(SO4)], and the di4s [ UA1?3GalNAc4(SO4)]. No disulfated
Ddi0s
[D
D
[D
D
D
or trisulfated disaccharides were generated, thus confirming the
absence of these species inside the CS carbohydrate backbone. This
polymer was found to be composed of ꢀ55% of disaccharide mono-
sulfated in position 6 of the GalNAc, ꢀ38% of disaccharide mono-
sulfated in position 4 of the GalNAc, and ꢀ7% on nonsulfated
disaccharide, with a charge density of ꢀ0.93 and a 4/6-sulfated
ratio of 0.69. Due to the incapacity of chondroitinase ABC to distin-
guish between GlcA and iduronic acid (IdoA),1 chondroitinase B
Figure 2. Agarose gel electrophoresis stained with toluidine blue of the chondroitin
sulfate purified from sturgeon bones (CS) before and after treatment with
chondroitinase ABC (+ABC) or B (+B). CS, chondroitin sulfate standard (also marked
St). DS, dermatan sulfate. HS, heparan sulfate. FM, fast-moving heparin. SM, slow-
moving heparin. o = origin.

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F. Maccari et al. / Carbohydrate Research 345 (2010) 1575–1580
Figure 4. 13C NMR spectrum of the purified sturgeon CS. Chemical shift is reported as ppm. Inset illustrates the expanded spectrum in the region 50–110 ppm. The spectra
were recorded by a Bruker AMX400 WB spectrometer operating at 100.61 MHz at a temperature of 33 °C and pH 6.5.
(Fig. 3), specific for IdoA, was used. This last lyase produced <0.5%
of the expected repeating disaccharides, in particular di4s (Fig. 3),
confirming that the purified sturgeon CS mainly contained GlcA
(>99.5%) as uronic acid. These data were also in agreement with
the results of agarose gel electrophoresis of the sample treated
with both chondroitinases (Fig. 2).
the origin, various disulfated disaccharides (and possibly also a tri-
sulfated one) may be present in the carbohydrate backbone9,21–24
producing CS having different charge densities. Furthermore, CS
samples may also possess various molecular masses and polydis-
persities depending on the source. To date, considering these struc-
tural aspects, CS from sturgeon shows distinctive properties and
characteristics. In fact, CS purified from ‘terrestrial’ sources, that
is, avian, porcine, and bovine cartilages, generally shows Mw val-
ues between 13,000 and 26,000,22,23 lower than those observed
for sturgeon CS, ꢀ37,000–40,000. On the contrary, CS from carti-
laginous fishes, shark and raja, has greater MW values, ꢀ50,000–
70,000.22,23 As a consequence, sturgeon bone CS possesses a molec-
ular mass value intermediate between cartilaginous fishes and
pure terrestrial cartilages. Furthermore, we should bear in mind
that, for the first time, we have been able to give a full character-
ization of CS from bones as opposed to previous common CS carti-
lage sources.
The repeating disaccharide pattern determination gives us more
information on the various CS samples and related origins22,23 as it
is possible to determine the charge density values (as sulfate group
number per disaccharide unit) and the 4-sulfated/6-sulfated ratio
(4s/6s ratio as the ratio between the sulfated groups located in po-
sition 4 and 6 on GalNAc). In fact, CS produced from various species
shows different qualitative and quantitative repeating disaccharide
patterns and values. In particular, ‘terrestrial’ CS samples from bo-
vine, porcine, and avian cartilages shows the same charge density
values, from 0.90 to 0.96 (0.93 for sturgeon CS), due to the absence
of disulfated (and trisulfated) disaccharides and to the presence of
6–8% of the nonsulfated disaccharide, but different 4s/6s ratios. In
fact, the 4-sulfated disaccharide content in bovine CS is almost
double that of the monosulfated disaccharide in position 6 produc-
ing a 4s/6s ratio in the range of 1.5–2.5. The percentage of the 4-
sulfated disaccharide increases in comparison with the 6-sulfated
one in avian CS causing a 4s/6s ratio in the range of 3.0–4.0, and
D
The 13C NMR spectrum of the sturgeon CS is shown in Figure 4.
All signals were found in the region 50-110 ppm21 except for those
of carbonyl (around 177–178 ppm) and acetamido methyl carbons
(all at 25.6 ppm). Inspection of the 50–70 and 100–110 ppm re-
gions (Fig. 4 inset) revealed a relatively high amount of CS sulfated
in position 4 or 6 of the GalNAc confirming the 4/6-sulfated ratio
obtained by HPLC of repeating disaccharides. The signals at 107.3
and 104.6 were assigned to the C1 of the GlcA and to the C1 of Gal-
NAc-6SO4, respectively, and signals at 106.3 and 103.6 were
assigned to the C1 of the GlcA and to the C1 of GalNAc-4SO4
(Fig. 4).21 Finally, the signal at 70.5 was related to C6 of GalNAc-6SO4
and the signal at 64.1 was assigned to C6 of GalNAc-4SO4.
Figure 5A reports the PAGE analysis (densitometric acquisition
is illustrated in Fig. 5B) of sturgeon CS showing an average molec-
ular mass of 39,880 calculated on a calibration curve of CS fractions
of known molecular masses. Figure 5C illustrates the HPSEC profile
of the same extract obtained by UV detection at 214 nm and the
third grade polynomial calibration curve used to determine the
size-exclusion chromatography parameters. The number average
molecular weight (Mn) was 28,030, the weight average molecular
weight (Mw) was 37,500, in agreement with the PAGE result, the Z
average molecular weight (Mz) was 48,250, and the dispersity in-
dex (Mw/Mn) was 1.3378.
It is well known that CS from different sources may have vari-
able structures and properties. In particular, the disaccharide
repeating units may differ for the number and position of substitu-
tion of sulfate groups, as well as for their amount. These repeating
disaccharide units are generally monosulfated but, depending on

F. Maccari et al. / Carbohydrate Research 345 (2010) 1575–1580
1579
Figure 5. (A) PAGE analysis of sturgeon CS at two different concentrations with the related densitometric scanning (B). The calibration curve was constructed using
saccharide standards of known molecular mass prepared from CS and having masses of 29,880, 25,780, 16,750, and 4460. Gels were stained with 0.1% toluidine blue in 1%
acetic acid for 30 min. After decoloration with 1% acetic acid, molecular mass evaluation was performed through densitometric acquisition (B). (C) HPSEC analysis of sturgeon
CS. The calibration curve was constructed using saccharide standards having mass values of 29,880, 8700, 4460, 3700, and 2130 (see the third grade polynomial curve inside
the HPSEC profile).
porcine CS has the lowest amount of the 6-sulfated disaccharide
with a high 4s/6s ratio in the range of 4.5–7.0. On the contrary,
CS samples from cartilaginous fishes, shark,22,23 dogfish,25 and
skate22,23 have particular charge density values greater than ap-
prox. 1.0 due to the presence of disulfated disaccharides, and a
4s/6s ratio lower than 0.7 in the case of shark CS due to the pres-
ence of a higher percentage of 4-sulfated groups with respect to 6-
sulfated ones, and between 1.0 and 1.4 in the case of dogfish and
skate polysaccharides due to a similar percentage of the two
monosulfated disaccharides. Also in this context, sturgeon CS
shows intermediate characteristics between cartilaginous fishes
and pure terrestrial cartilages. In fact, this CS from the bony fish

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F. Maccari et al. / Carbohydrate Research 345 (2010) 1575–1580
sturgeon has no disulfates (or trisulfated) disaccharides typical of
cartilaginous fishes but it shows a greater percentage of 6-sulfated
disaccharide than 4-sulfated disaccharide, with an overall 4s/6s ra-
tio of ꢀ0.7 very close to that of shark cartilages.
References
1. Chondroitin Sulfate: Structure, Role and Pharmacological Activity; Volpi, N., Ed.;
Academic Press: Amsterdam, Boston, Heidelberg, London, New York, Oxford,
Paris, San Diego, San Francisco, Singapore, Sydney, Tokyo, 2006.
2. Development of Non-heparin Glycosaminoglycans as Therapeutic Agents;
Mammen, E. F., Ed.; Theme Medical Pub: New York, 1991; Vol. 17, (Suppl. 1–2).
3. Volpi, N. Curr. Drug Targets Immune Endocr. Metabol. Disord. 2004, 4, 119–127.
4. Volpi, N. Curr. Med. Chem. 2006, 13, 1799–1810.
GAGs constitute a considerable fraction of the glycoconjugates
located on cellular membranes and in the extracellular matrix of
virtually all mammalian tissues and also other vertebrates and
many invertebrates. CS is particularly interesting for further stud-
ies and applications because it is expressed in many tissues and
organs. In fact, growing evidence suggests that this GAG is an
important cofactor in a variety of cell behaviors, in the develop-
ment of the CNS, and it also acts as a receptor for various patho-
gens.1,5 Biological activities of CS chains possibly involve various
growth factors and chemokines, and these functions are closely
associated with the sulfation patterns and characteristics of the
polysaccharide chains.5 As a consequence, CS functions and possi-
ble therapeutic applications are closely related to its specific
structure and properties. With this aim, new bioactive sources
of CS, such as from sturgeon bone, may represent potential drugs
for future research and development and would enable the pro-
duction of this polysaccharide having a distinctive repeating
disaccharide composition, structure, and possible biological
properties.
5. Sugahara, K.; Mikami, T.; Uyama, T.; Mizuguchi, S.; Nomura, K.; Kitagawa, H.
Curr. Opin. Struct. Biol. 2003, 13, 612–620.
6. Jordan, K. M.; Arden, N. K.; Doherty, M.; Bannwarth, B.; Bijlsma, J. W.; Dieppe,
P.; Gunther, K.; Hauselmann, H.; Herrero-Beaumont, G.; Kaklamanis, P.;
Lohmander, S.; Leeb, B.; Lequesne, M.; Mazieres, B.; Martin-Mola, E.; Pavelka,
K.; Pendleton, A.; Punzi, L.; Serni, U.; Swoboda, B.; Verbruggen, G.;
Zimmerman-Gorska, I.; Dougados, M. Ann. Rheum. Dis. 2003, 62, 1145–1155.
7. Sarzi-Puttini, P.; Cimmino, M. A.; Scarpa, R.; Caporali, R.; Parazzini, F.; Zaninelli,
A.; Atzeni, F.; Canesi, B. Semin. Arthritis Rheum. 2005, 35, 1–10.
8. McAlindon, T. E.; LaValley, M. P.; Gulin, J. P.; Felson, D. T. JAMA 2000, 283, 1469–
1475.
9. Fuentes, E. P.; Diaz, V. B. Acta Farm. Bonaerense 1998, 17, 135–142.
10. Luo, X. M.; Fosmire, G. J.; Leach, R. M., Jr. Poult. Sci. 2002, 81, 1086–1089.
11. Sugahara, K.; Nadanaka, S.; Takeda, K.; Kojima, T. Eur. J. Biochem. 1996, 239,
871–880.
12. Lignot, B.; Lahogue, V.; Bourseau, P. J. Biotechnol. 2003, 103, 281–284.
13. Jo, J. H.; Park, D. C.; Do, J. R.; Kim, Y. M.; Kim, D. S.; Park, Y. K.; Lee, T. K.; Cho, S.
M. Food Sci. Biotecnol. 2004, 13, 622–626.
14. Bemis, W. E.; Findeis, E. K.; Grande, L. Envir. Biol. Fish. 1997, 48, 25–71.
15. Burtzev, L. A. J. Appl. Ichthyol. 1999, 15, 325.
16. Cesaretti, M.; Luppi, E.; Maccari, F.; Volpi, N. Carbohydr. Polym. 2003, 54, 59–61.
17. Volpi, N. Carbohydr. Res. 1993, 247, 263–278.
18. Volpi, N.; Maccari, F. Electrophoresis 2002, 23, 4060–4066.
19. Edens, R. E.; al-Hakim, A.; Weiler, J. M.; Rethwisch, D. G.; Fareed, J.; Linhardt, R.
J. J. Pharm. Sci. 1882, 81, 823–827.
In conclusion, CS was purified for the first time from a bony fish
and analyzed to evaluate its structure and properties also in rela-
tionship to similar polysaccharides extracted from cartilaginous
fish and terrestrial avian and mammalian species. Furthermore,
on the basis of the data collected, it is reasonable to assume that
CS isolated from sturgeon might be potentially useful for scientific
and pharmacological applications, making this bony fish, which is
generally discarded after ovary collection, a useful source of this
polymer.
20. Volpi, N.; Bolognani, L. J. Chromatogr. 1993, 630, 390–396.
21. Mucci, A.; Schenetti, L.; Volpi, N. Carbohydr. Polym. 2000, 41, 37–45.
22. Volpi, N. J. Pharm. Pharmacol. 2009, 61, 1271–1280.
23. Volpi, N. J. Pharm. Sci. 2007, 96, 3168–3180.
24. Volpi, N. Anal. Biochem. 2010, 397, 12–23.
25. Gargiulo, V.; Lanzetta, R.; Parrilli, M.; De Castro, C. Glycobiology 2009, 19, 1485–
1491.