The reaction of hyaluronic acid and its monomers, glucuronic acid and N- acetylglucosamine, with reactive oxygen species

Article abstract of DOI:10.1016/S0008-6215(99)00186-X

Synovial fluid is a ~0.15% (w/v) aqueous solution of hyaluronic acid (HA), a polysaccharide consisting of alternating units of GlcA and GlcNAc. In synovial fluid of patients suffering from rheumatoid arthritis, HA is thought to be degraded either by radicals generated by Fenton chemistry (Fe²⁺/H₂O₂) or by NaOCl generated by myeloperoxidase. We investigated the course of model reactions of these two reactants in physiological buffer with HA, and with the corresponding monomers GlcA and GlcNAc. meso-Tartaric acid, arabinuronic acid, arabinaric acid and glucaric acid were identified by GC-MS as oxidation products of glucuronic acid. When GlcNAc was oxidised, erythronic acid, arabinonic acid, 2-acetamido-2-deoxy-gluconic acid, glyceric acid, erythrose and arabinose were formed. NaOCl oxidation of HA yielded meso-tartaric acid; in addition, arabinaric acid and glucaric acid were obtained by oxidation with Fe²⁺/H₂O₂. These results indicate that oxidative degradation of HA proceeds primarily at glucuronic acid residues. meso-Tartaric acid may be a useful biomarker of hyaluronate oxidation since it is produced by both NaOCl and Fenton chemistry.

Full text of DOI:10.1016/S0008-6215(99)00186-X

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Carbohydrate Research 321 (1999) 228–234
The reaction of hyaluronic acid and its monomers,
glucuronic acid and N-acetylglucosamine, with reactive
oxygen species
Michael Jahn a, John W. Baynes b, Gerhard Spiteller a,*
a Lehrstuhl Organische Chemie I, Uni6ersita¨t Bayreuth, Uni6ersita¨tsstraße 30, D-95440 Bayreuth, Germany
b Department of Chemistry and Biochemistry, Uni6ersity of South Carolina, Columbia, SC 29208, USA
Received 15 March 1999; accepted in revised form 24 July 1999
Abstract
Synovial fluid is a ꢀ0.15 % (w/v) aqueous solution of hyaluronic acid (HA), a polysaccharide consisting of
alternating units of GlcA and GlcNAc. In synovial fluid of patients suffering from rheumatoid arthritis, HA is
thought to be degraded either by radicals generated by Fenton chemistry (Fe2+/H2O2) or by NaOCl generated by
myeloperoxidase. We investigated the course of model reactions of these two reactants in physiological buffer with
HA, and with the corresponding monomers GlcA and GlcNAc. meso-Tartaric acid, arabinuronic acid, arabinaric
acid and glucaric acid were identified by GC–MS as oxidation products of glucuronic acid. When GlcNAc was
oxidised, erythronic acid, arabinonic acid, 2-acetamido-2-deoxy-gluconic acid, glyceric acid, erythrose and arabinose
were formed. NaOCl oxidation of HA yielded meso-tartaric acid; in addition, arabinaric acid and glucaric acid were
obtained by oxidation with Fe2+/H2O2. These results indicate that oxidative degradation of HA proceeds primarily
at glucuronic acid residues. meso-Tartaric acid may be a useful biomarker of hyaluronate oxidation since it is
produced by both NaOCl and Fenton chemistry. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Hyaluronic acid; Reactive oxygen species; meso-Tartaric acid; Arabinaric acid; Glucaric acid
the aqueous solutions have gel-like properties
and show molecular-weight-dependent viscos-
ity [1].
1. Introduction
Hyaluronic acid (HA) is a linear acidic
polysaccharide consisting of alternating units
In synovial fluid of patients with rheuma-
toid arthritis, the viscosity is reduced in com-
parison with healthy subjects, due to a
reduction in molecular weight of HA [2]. The
alteration of the molecular-weight distribution
is assumed to be a result of the action of
reactive oxygen species (ROS) on HA. Poly-
morphonuclear leukocytes invade inflamed
joints [3] and, upon stimulation, release
myeloperoxidase, which catalyses the forma-
tion of the powerful oxidant OCl− from H2O2
and Cl− [4]. H2O2 is produced by stimu-
lated leukocytes, and the concentration of
of
D
-GlcA and
D
-GlcNAc: [“4)-b- -GlcpA-
D
(1“3)-b-
D
-GlcpNAc-(1“]n. It is a major
component of the extracellular matrix of con-
nective tissue and is found in high concentra-
tions in umbilical cord, vitreous body of the
eye, and in synovial fluid. Usually, HA has a
high molecular weight (105–107 g/mol), and
* Corresponding author. Tel.: +49-921-552-680; fax: +
49-921-552-671.
E-mail address: gerhard.spiteller@uni-bayreuth.de (G.
Spiteller)
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00186-X

M. Jahn et al. / Carbohydrate Research 321 (1999) 228–234
229
7.4) and incubated with FeSO4·7H2O (40 mM)
[5] and H2O2 (500 mM) for 6 h at 37 °C.
Chelex 100 (1 g dry weight/mL solution) was
added for removing iron, vortexed for 2 min,
centrifuged and the supernatant was taken to
dryness by centrifugal evaporation. Alterna-
tively, HA or oxidised HA was precipitated
with 4 volumes of EtOH, the solution stored
for 12 h at −4 °C, the precipitate centrifuged,
washed with 80% (v/v) aq EtOH, with EtOH
and with Et2O. After drying by centrifugal
evaporation, methanolic HCl (2 M, 1 mL/mg
carbohydrate) containing MeOAc (2 M) [17]
was added, oxygen was removed under a mild
flow of argon for 20 s and the vial was stop-
pered with a Teflon-coated cap and placed in
an oven at 100 °C for 24 h. After adding 2
volumes of 4:1 tert-BuOH–MeOH [17], the
mixture was taken to dryness by centrifugal
evaporation. MSTFA (50 mL/mg carbohy-
drate) and TFA (5 mL/mg carbohydrate) [18]
were added, and the turbid solution was
heated for 2 h at 35 °C. The centrifuged, clear
solution (1 mL) was analysed by GC–MS. For
comparison, the same work-up and derivatisa-
tion procedure was done without incubation
with Fe2+/H2O2.
bleomycin-detectable iron in the synovial fluid
of patients with rheumatoid arthritis is ele-
vated in comparison with healthy controls [5].
The reaction of Fe2+ with H2O2 leads to the
formation of hydroxyl radicals, which react
with and damage biomolecules [6].
Based on size-exclusion chromatography,
HA is oxidatively depolymerised by Fenton
reagent Fe2+/H2O2 [7]. Frati et al. [8] demon-
strated, using viscosity measurements, that
photosensitive molecules are able to induce
the degradation of HA by UV irradiation.
Chromogenic assays indicated that the num-
ber of reducing GlcA residues was increased
after exposure of HA to oxygen-derived free
radicals [9]. Electron spin resonance was used
as a tool to get insight into the reaction mech-
anism of OH-radicals with HA, and the inter-
mediates were characterised [10]. In a detailed
investigation of the products obtained by reac-
tion of ROS with HA, Uchiyama et al. [11]
detected arabinuronic acid and 2-acetamido-2-
deoxy-gluconic acid. Schiller et al. [12] con-
ducted 1H NMR studies on the reaction
mixtures of HA and Fe2+/H2O2 and found
mobile, i.e., not polymer-bound, N-acetyl
groups and formiate.
Oxidation with NaOCl.—
D
-GlcA or
D-Glc-
In this work, we studied the products of the
action of Fe2+/H2O2 and NaOCl on HA un-
der physiological conditions, and have applied
GC–MS for their characterisation. Our re-
sults indicate that these reagents cleave HA
primarily at GlcA residues.
NAc (0.8 mg/mL) or HA (1.5 mg/mL) was
dissolved in phosphate-buffered saline (pH
7.4) and incubated with NaOCl (500 mM) for
6 h at 37 °C. One volume of iso-propanol was
added [19], and the solvent was evaporated.
Alternatively, HA or oxidised HA were iso-
lated from the solutions by EtOH precipita-
tion as described above. Methanolysis and
derivatisation were performed as described
above. Again, controls without oxidising
agent were done.
Viscosimetry.—The kinetics of decrease in
viscosity of HA solutions (mixtures as de-
scribed above) were monitored with a Physica,
Rheolab MC10-viscosimeter (concentric cylin-
ders, double-gap-measuring system according
to DIN 54453 with a cylinder MS-Z1 DIN) at
2000 rpm and 37 °C.
2. Materials and methods
Materials.—N - Methyl - N - trimethylsilyltri-
fluoroacetamide (MSTFA) was obtained from
Macherey and Nagel, Chelex 100 was from
Sigma, HA sodium salt from human umbilical
cords and all other chemicals were obtained
from Fluka. 2-Acetamido-2-deoxy-gluconic
acid was synthesised according to Hoffmann
et al. [13] and Pravdic et al. [14]. Arabinuronic
acid was synthesised according to Wu and
Serianni [15], arabinaric and arabinonic acid
according to Hardegger et al. [16].
GC–MS.—GC separation was performed
on a HP 5890 Series II gas-chromatograph (30
m DB-5MS column, 0.25 mm ID, temperature
program: from 100 to 300 °C with 3 °C/min);
MS analysis was done by a Finnigan MAT 95,
Oxidation with Fe2+/H2O2.—
D
-GlcA or
D-
GlcNAc (0.8 mg/mL) or HA (1.5 mg/mL) was
dissolved in phosphate-buffered saline (pH

230
M. Jahn et al. / Carbohydrate Research 321 (1999) 228–234
(9): Ri 1545, 1560. MS 73(100), 75(50), 147(30),
204(35), 217(100). Arabinonic acid g-lactone
(3TMS) (10a): Ri 1630. MS 73(100), 75(85),
117(10), 147(25), 217(10). Methyl arabinonate
(4TMS) (10b): Ri 1710. MS 73(100), 75(50),
103(50), 147(50), 217(45). Methyl 2-amino-2-
deoxy-glucopyranoside (2TMS) (11a): Ri 1830,
1835. MS 73(50), 75(45), 131(100), 147(20),
equipped with an ICIS data system. EI mass
spectra were recorded at an ionisation energy
of 70 eV.
For determination of Ri values, an appropri-
ate amount of a mixture of saturated, non-
branched hydrocarbons was coinjected and the
values were calculated by linear interpolation
[20].
217(10).
Methyl
2-deoxy-2-trimethylsily-
GC–MS data for standard compounds are
indicated below. The fi6e most prominent MS
fragments are listed with relati6e intensity in
parentheses.—Dimethyl meso-tartrate (2TMS)
(1a): Ri 1420. MS 73(100), 89(35), 147(65),
234(45), 307(20). Methyl trimethylsilyl meso-
tartrate (2TMS) (1b): Ri 1500. MS 73(100),
147(70), 219(35), 234(40), 247(35). Methyl
(methyl arabinofuranosid)-uronate (2TMS)
(2a): Ri 1540, 1555. MS 73(60), 75(50), 89(20),
159(15), 217(100). Methyl (trimethylsilyl ara-
binofuranosid)uronate (2TMS) (2b): Ri 1615,
1625. MS 73(45), 75(40), 147(20), 191(10),
217(100). Dimethyl arabinarate (3TMS) (3): Ri
1690. MS 73(100), 75(50), 103(70), 147(50),
234(85). Methyl glucofuranosidurono-6,3-lac-
tone (2TMS) (4a): Ri 1750, 1770. MS 73(100),
75(40), 147(20), 217(30), 230(65). Methyl
(methyl glucofuranosid)uronate (3TMS) (4b):
Ri 1785, 1795. MS 73(50), 75(30), 133(20),
217(100), 277(15). Trimethylsilyl glucofuran-
osidurono-6,3-lactone (2TMS) (4c): Ri 1815,
1830. MS 73(60), 75(50), 147(20), 217(15),
laminoglucofuranoside (3TMS) (11b): Ri 1845,
1865. MS 73(35), 75(40), 147(20), 216(100),
217(40).
Methyl
2-deoxy-2-trimethylsily-
lamino-glucopyranoside (3TMS) (11c): Ri
1900. MS 73(40), 75(20), 147(15), 203(100),
204(25). Trimethylsilyl 2-deoxy-2-trimethylsi-
lylamino-glucopyranoside (3TMS) (11d): Ri
1915, 1960. MS 73(30), 75(50), 147(15),
203(100), 204(25). Methyl 2-acetamido-2-de-
oxy-glucopyranoside (3TMS) (11e): Ri 2085.
MS 73(60), 75(100), 147(30), 173(100), 217(25).
Methyl
2-deoxy-2-trimethylsilylamino-glu-
conate (4TMS) (12): Ri 1960. MS 73(100),
75(50), 147(65), 205(70), 319(50).
3. Results
Oxidation of GlcA.—A comparison of the
chromatograms of a GlcA solution (A) incu-
bated with Fe2+/H2O2 (B) or with NaOCl (C)
is given in Fig. 1 (the numbers of the peaks in
the chromatograms correspond to the num-
bers of the compounds). The methanolysis
products of GlcA are methyl glucofura-
nosidurono-6,3-lactone (4a), methyl (methyl
glucofuranosid)uronate (4b), trimethylsilyl
glucofuranosidurono-6,3-lactone (4c), methyl
(methyl glucopyranosid)uronate (4d), methyl
(trimethylsilyl glucopyranosid)uronate (4e)
and trimethylsilyl (methyl glucopyranosid)-
uronate (4f) (all as pertrimethylsilylated com-
pounds; a and b anomers for each glycoside).
Oxidation products with both oxidising agents
are meso-tartaric acid as dimethyl ester (1a)
and mixed methyl trimethylsilyl ester (1b),
methyl (methyl arabinofuranosid)uronate (2a),
230(100).
Methyl
(methyl
glucopyran-
osid)uronate (3TMS) 4d: R: 1905, 1920. MS
73(70), 75(50), 147(30), 204(70), 217(100).
Methyl (trimethylsilyl glucopyranosid)uronate
(3TMS) (4e): Ri 1935, 1945. MS 73(45), 75(25),
147(30), 204(80), 217(100). Trimethylsilyl
(methyl glucopyranosid)uronate (3TMS) (4f):
Ri 1955, 1960. MS 73(50), 147(35), 204(70),
217(100), 292(20). Glucaric acid lactone
methyl ester (3TMS) (5a,c): Ri 1850, 1880. MS
73(100), 75(35), 147(40), 217(35), 407(20).
Dimethyl glucarate (4TMS) (5b): Ri 1865. MS
73(95), 75(35), 103(40), 147(35), 275(100).
Methyl glycerate (2TMS) (6): Ri 1225. MS
73(100), 89(20), 103(25), 117(25), 147(35).
Methyl erythrofuranoside (2TMS) (7): Ri 1300,
1305. MS 73(60), 75(35), 147(100), 191(20),
217(30). Methyl erythronate (3TMS) (8): Ri
1460. MS 44(100), 73(80), 75(60), 147(50),
234(45). Methyl arabinopyranoside (3TMS)
methyl
(trimethylsilyl
arabinofuranosid)-
uronate (2b), dimethyl arabinarate (3), glu-
caric acid lactone methyl ester (5a,c), and
dimethyl glucarate (5b). As above, the gly-

M. Jahn et al. / Carbohydrate Research 321 (1999) 228–234
231
cosides give two peaks corresponding to a and
b anomers.
oxidising agent, and in addition, methyl glyc-
erate 6, methyl erythrofuranoside 7 and
methyl arabinopyranoside 9 are formed.
Oxidation of GlcNAc.—The course of oxi-
dation of GlcNAc (A) with Fe2+/H2O2 (B) or
with NaOCl (C) is shown in Fig. 2 (the num-
bers of the peaks in the chromatograms corre-
spond to the numbers of the compounds).
Methanolysis and derivatisation of GlcNAc
yields methyl 2-amino-2-deoxy-glucopyran-
oside (11a), methyl 2-deoxy-2-trimethylsilyl-
amino-glucofuranoside (11b), methyl 2-
deoxy-2-trimethylsilylamino-glucopyranoside
(11c), trimethylsilyl 2-deoxy-2-trimethylsilyl-
amino-glucopyranoside (11d) and methyl 2-
acetamido - 2 - deoxy - glucopyranoside (11e).
The identified products of oxidation with
Fe2+/H2O2 are methyl erythronate (8), ara-
binonic acid g-lactone (10a), methyl ara-
binonate (10b) and methyl 2-deoxy-2-
trimethylsilylamino-gluconate (12). The same
products are obtained when NaOCl is the
Oxidation of HA.—The time-course of the
oxidation of HA with Fe2+/H2O2 and NaOCl
is illustrated in Fig. 3 by means of viscosime-
try. Within 14 h, the viscosity of the pure HA
solution is reduced by ꢀ15%, the oxidised
solution with NaOCl by ꢀ35% and by oxida-
tion with Fe2+/H2O2 by ꢀ75%. The degrada-
tion process seems to be completed within 6 h.
The GC–MS chromatograms of a methano-
lised and derivatised HA solution (A), oxi-
dised with Fe2+/H2O2 (B) or NaOCl (C) are
shown in Fig. 4 (the numbers of the peaks in
the chromatograms correspond to the num-
bers of the compounds). When HA is
methanolised and trimethylsilylated, the
derivatives of GlcA (4a–4f) and of GlcNAc
(11b,c,d,e) can be observed. Mixtures incu-
bated with Fe2+/H2O2 contain, in addition,
Fig. 1. Reconstructed ion current of a 0.08% (w/v)
(B) or with 500 mM NaOCl (C) at 37 °C for 6 h. Preparation of samples for GC–MS is described in Section 2. Designation of
peaks is given in the text.
D
-GlcA solution (A) either incubated with 40 mM Fe2+ and 500 mM H2O2
Fig. 2. Reconstructed ion current of a 0.08% (w/v)
-GlcNAc solution (A) either incubated with 40 mM Fe2+ and 500 mM H2O2
D
(B) or with 500 mM NaOCl (C) at 37 °C for 6 h. Preparation of samples for GC–MS is described in Section 2. Designation of
peaks is given in the text.

232
M. Jahn et al. / Carbohydrate Research 321 (1999) 228–234
Fig. 3. Kinetics of the decrease in viscosity of a 0.15% (w/v) HA solution either incubated with 40 mM Fe2+ and 500 mM H2O2
or with 500 mM NaOCl. As a control, the loss of viscosity of a pure HA solution is shown. Conditions are given in Section 2.
Fig. 4. Reconstructed ion current of a 0.15% (w/v) HA solution (A) either incubated with 40 mM Fe2+ and 500 mM H2O2 (B)
or with 500 mM NaOCl (C) at 37 °C for 6 h. Preparation of samples for GC–MS is described in Section 2. Designation of peaks
is given in the text.
the derivatives of meso-tartaric acid (1a,b),
arabinaric acid (3) and glucaric acid (5a,b,c).
When HA was incubated with NaOCl, only
meso-tartaric (1a,b) could be identified as an
oxidation product. Again, the glycosides show
two peaks for a and b anomers. We did not
detect any significant differences in the chro-
matograms when HA or oxidised HA were
isolated from the solution by ethanol precipi-
tation. This indicates that the oxidised
residues are situated on the polymer.
lutions. Therefore, we first examined the oxi-
dation behaviour of the monomers.
The product spectrum of GlcA indicates
oxidation at C-1 to yield glucaric acid, which
is the most prominent product formed with
both oxidants; the C-1–C-2-bond is cleaved
and C-2 is oxidised to give arabinuronic and
arabinaric acid. When C-2–C-3 is cleaved,
and C-3 is oxidised, meso-tartaric acid is the
product. It is not clear why the oxidation does
not proceed on C-3 to an aldehydic product
(erythruronic acid), or why we could not find
products of oxidation at C-4 and C-5. Al-
though the kinetics and yield of products dif-
fered, similar products were obtained on
oxidation of GlcA by Fe2+/H2O2 and NaOCl.
In contrast, the products found by oxida-
tion of GlcNAc differ according to the oxida-
tion reagent: C-1 is oxidised to give
2-amino-2-deoxy-gluconic acid, which is a ma-
jor product formed by the action of the two
oxidants. Experiments where the methanolysis
4. Discussion
Preliminary experiments with LC–MS (data
not shown) indicated that the fragmentation
products formed during oxidation of HA un-
der physiological conditions are present at
only trace concentrations in the reaction mix-
ture, in spite of a visible loss in viscosity of the
solutions compared with non-oxidised HA so-

M. Jahn et al. / Carbohydrate Research 321 (1999) 228–234
233
carbohydrates that are subjected to free radi-
cals yield a broad product spectrum [10]. In
contrast, in our investigation the polysaccha-
ride chain is cleaved by hydroxyl radicals
formed by the Fenton reaction. Our model
reactions have biological relevance, because
we obtain a limited degradation of HA under
conditions and with reagents that might be
encountered in vivo as it is postulated in the
literature.
We also examined urine and synovial fluid
of patients with rheumatoid arthritis [21]. In
spite of a very low detection limit of the
oxidised products of HA, meso-tartaric acid,
arabinaric acid and glucaric acid (ꢀ100 ng/
mg HA), we were unable to detect the
expected degradation products. We used spik-
ing experiments and found that we can re-
cover the three aldaric acids when we add
oxidised HA to the biological samples. This
would mean that the long postulated process
of oxidative degradation of HA takes place
— if at all — only in a very limited way.
Therefore, we consider that HA in the joints
of patients with rheumatoid arthritis might
suffer degradation by hydrolysis rather then
by oxidation.
step was omitted showed that 2-acetamido-2-
deoxy-gluconic acid is formed as well; during
methanolysis, the amide group is hydrolysed
completely. Cleavage of C-1–C-2 and oxida-
tion at C-2 yields arabinonic acid when Fe2+
/
H2O2 is used as oxidant and also arabinose,
when NaOCl is used. Arabinonic acid is the
other major product that is obtained by oxi-
dation with NaOCl. Cleavage of C-2–C-3
and oxidation of C-3 is observed by applica-
tion of both oxidants; but again, only NaOCl
gives both the aldehyde (erythrose) and the
acid (erythronic acid), whereas only the acid
is detected by oxidation with Fe2+/H2O2.
Cleavage of C-4–C-5 with oxidation at C-4 to
glyceric acid is observed only with NaOCl.
Both monomers seem to be oxidised in a
manner, that C-6 remains in the products.
HA oxidation yielded only the products of
oxidation of GlcA, but none from the Glc-
NAc part of the polymer. We propose that
Fe2+ is bound to the carboxylic groups of
GlcA. The Fenton reaction takes place in the
proximity of GlcA, and as the reaction of the
hydroxyl-radical is almost diffusion con-
trolled, oxidised GlcA residues are formed in
high yield. It is not clear why only meso-tar-
taric acid is formed by NaOCl oxidation.
Measurement of viscosity during oxidation in-
dicates that the breakdown of HA proceeds
more drastically in the case where the Fenton
reactant is used as oxidant. We could find the
same oxidised products from HA when
we isolated the degraded polymer by ethanol
precipitation. Therefore, it seems likely that
the oxidised residues are situated on the poly-
mer.
Acknowledgements
M.J. was supported by a DAAD-fellowship
HSP III, financed by the German Federal
Ministry of Education, Science, Research and
Technology.
The reported products of HA oxidation
(ꢀ30 mM Fe2+, oxygen atmosphere) ob-
tained by Uchiyama et al. [11], arabinuronic
acid and 2-acetamido-2-deoxy-gluconic acid,
were found in our investigations only by oxi-
dation of the monomers. As ferric ions, which
are formed immediately under these condi-
tions, are known to be very strong Lewis
acids, we suppose, that degradation of HA in
this investigation is a hydrolysis step and not
an oxidative cleavage of the polymer. The
newly formed reducing ends might then be
oxidatively modified by ferric ions. This sug-
gestion is supported by the observation that
References
[1] H. Bothner, O. Wik, Acta Otolaryngol. (Stockholm)
Suppl., 442 (1987) 25–30.
[2] J.M. McCord, Science, 185 (1974) 529–531.
[3] K.A. Brown, Br. J. Rheumatol., 27 (1988) 150–155.
[4] J.M. Albrich, M.C. McCarthy, J.K. Hurst, Proc. Natl.
Acad. Sci. USA, 78 (1981) 210–214.
[5] S.F. Wong, B. Halliwell, R. Richmond, W. Skowroneck,
J. Inorg. Biochem., 14 (1981) 127–134.
[6] S. Goldstein, D. Meyerstein, G. Czapski, Free Radic.
Biol. Med., 15 (1993) 435–445.
[7] R. Moseley, R. Waddington, P. Evans, B. Halliwell, G.
Embery, Biochim. Biophys. Acta, 1244 (1995) 245–252.
[8] E. Frati, A.M. Khatib, P. Front, A. Panasyuk, F. Aprile,
D.R. Mitrovic, Free Radic. Biol. Med., 22 (1997) 1139–
1144.

234
M. Jahn et al. / Carbohydrate Research 321 (1999) 228–234
[9] J.D. McNeil, O.W. Wiebkin, L.G. Cleland, B. Vernon-
[15] J. Wu, A.S. Serianni, Carbohydr. Res., 210 (1991) 51–
70.
[16] E. Hardegger, K. Kreis, H.E. Khadem, Hel6. Chim. Acta,
35 (1952) 618–623.
[17] N. Jentoft, Anal. Biochem., 148 (1985) 424–433.
[18] M.J. Donike, J. Chromatogr., 85 (1973) 1–7.
[19] R.V. Stevens, K.T. Chapman, C.A. Stubbs, W.W. Tam,
K.F. Albizati, Tetrahedron Lett., 23 (1982) 4647–
4651.
Roberts, Agents Actions Suppl., 18 (1986) 95–101.
[10] C.L. Hawkins, M.J. Davies, Free Radic. Biol. Med., 21
(1996) 275–290.
[11] H. Uchiyama, Y. Dobashi, K. Ohkouchi, K. Nagasawa,
J. Biol. Chem., 265 (1990) 7753–7759.
[12] J. Schiller, J. Arnhold, J. Schwinn, H. Sprinz, O. Brede,
K. Arnold, Free Radic. Res., 28 (1998) 215–228.
[13] M. Hoffmann, F. Burkhart, G. Hessler, H. Kessler, Hel6.
Chim. Acta, 79 (1996) 1519–1532.
[14] N. Pravdic, H.G. Fletcher, Carbohydr. Res., 19 (1971)
335–364.
[20] E. Kovats, Hel6. Chim. Acta, 41 (1958) 1915–1932.
[21] M. Jahn, Dissertation, Bayreuth, 1999.
.