Reduction mechanism of tetrazolium salt XTT by a glucosamine derivative

Article abstract of DOI:10.1271/bbb.67.295

XTT (3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(methoxy-6- nitro)benzenesulfonic acid hydrate) was reduced by incubated glucosamine hydrochloride. The XTT reducibility by incubated glucosamine was linearly related with the DNA-breaking activity. In order to elucidate the reaction mechanism, the glucosamine derivatives formed during the incubation process were separated by HPLC, and the compound responsible for the reduction was analyzed. Among the incubated products, fructosazine and deoxyfructosazine were identified by LC-MS, FAB-MS, and ¹H- and ¹³C-NMR. These products showed no XTT reducibility, but an unstable intermediate with a molecular weight of 322 displayed reducibility. Since the intermediate gave fructosazine by oxidation with XTT and was a precursor of deoxyfructosazine, we conclude that the intermediate could have been dihydrofructosazine. Therefore, the XTT reducibility by incubated glucosamine was based on dihydrofructosazine formed by the condensation of two molecules of glucosamine.

Full text of DOI:10.1271/bbb.67.295

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Reduction Mechanism of Tetrazolium Salt XTT by a
Glucosamine Derivative
ab
a
a
a
Tomoko SHIMAMURA , Atsuko TAKAMORI , Hiroyuki UKEDA & Masayoshi SAWAMURA
a
Department of Bioresources Science, Faculty of Agriculture, Kochi UniversityMonobe
B-200, Nankoku, Kochi 783-8502, Japan
b
Department of Food Science and Technology, Nippon Veterinary and Animal Science
University
Published online: 22 May 2014.
To cite this article: Tomoko SHIMAMURA, Atsuko TAKAMORI, Hiroyuki UKEDA & Masayoshi SAWAMURA (2003) Reduction
Mechanism of Tetrazolium Salt XTT by a Glucosamine Derivative, Bioscience, Biotechnology, and Biochemistry, 67:2,
295-299, DOI: 10.1271/bbb.67.295
To link to this article: http://dx.doi.org/10.1271/bbb.67.295
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Biosci. Biotechnol. Biochem., 67 (2), 295–299, 2003
Reduction Mechanism of Tetrazolium Salt XTT by a Glucosamine Derivative
Tomoko SHIMAMURA,* Atsuko TAKAMORI, Hiroyuki UKEDA,† and Masayoshi SAWAMURA
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku,
Kochi 783-8502, Japan
Received July 11, 2002; Accepted October 1, 2002
XTT (3
?
-
s
1-[(phenylamino)-carbonyl]-3,4-tetrazoli-
generated a carbon-centered radical. These inter-
mediate compounds were regarded as DNA-breaking
substances. Therefore, monitoring those biologically
active intermediates is important for the further ap-
plication of glucosamine as a drug or supplement.
We have recently found a good relationship
between the DNA-breaking activity of incubated
um -bis(methoxy-6-nitro)benzenesulfonic acid hydrate)
t
was reduced by incubated glucosamine hydrochloride.
The XTT reducibility by incubated glucosamine was
linearly related with the DNA-breaking activity. In or-
der to elucidate the reaction mechanism, the glucosa-
mine derivatives formed during the incubation process
were separated by HPLC, and the compound responsi-
ble for the reduction was analyzed. Among the incubat-
ed products, fructosazine and deoxyfructosazine were
identiˆed by LC-MS, FAB-MS, and H- and C-NMR.
These products showed no XTT reducibility, but an un-
stable intermediate with a molecular weight of 322 dis-
played reducibility. Since the intermediate gave fruc-
tosazine by oxidation with XTT and was a precursor of
deoxyfructosazine, we conclude that the intermediate
could have been dihydrofructosazine. Therefore, the
XTT reducibility by incubated glucosamine was based
on dihydrofructosazine formed by the condensation of
two molecules of glucosamine.
glucosamine and the XTT (3
s
?- 1-[(phenylamino)-
carbonyl]-3,4-tetrazolium -bis(methoxy-6-nitro)ben-
t
zenesulfonic acid hydrate) reducibility and suggested
that the XTT assay based on this reducibility may be
applicable for monitoring those biologically active
1
13
8
)
products. In order to conˆrm the usefulness of the
XTT assay to monitor the biologically active
products derived from glucosamine, we elucidate in
the present investigation the reaction mechanism for
XTT reduction by glucosamine.
Materials and Methods
Reagents.
chased from Nacalai Tesque (Kyoto, Japan). XTT
t
(3 1-[(phenylamino)-carbonyl]-3,4-tetrazolium -
D
-Glucosamine hydrochloride was pur-
Key words: glucosamine; DNA-breaking activity;
fructosazine; XTT
?
-
s
bis(methoxy-6-nitro)benzenesulfonic acid hydrate)
and diethylenetriaminepentaacetic acid (DTPA) were
obtained from Sigma Chemical Co. (St. Louis, MO,
U.S.A.). All other chemicals were of the highest
grade available and were used without further puriˆ-
cation. All solutions were prepared with water puri-
ˆed by a Milli-Q system (Millipore, Tokyo, Japan).
Glucosamine hydrochloride is currently used for
the treatment of degenerative joint disease in small
animals and presents a novel approach for the treat-
ment of degenerative conditions. Glucosamine, an
amino monosaccharide, is a natural component of
glycoproteins found in connective tissues and gas-
trointestinal mucosal membranes. It is a precursor of
the disaccharide unit of glycosaminoglycans which
are the building blocks of the articular cartilage, the
proteoglycans. Numerous reports have recently fo-
cused on the utility of glucosamine for the treatment
Incubation of glucosamine. Glucosamine (0.5
dissolved in a 50 m sodium phosphate buŠer (pH
7.4) containing 5 DTPA was incubated at 37 C.
M
)
M
m
M
9
After incubating for the indicated period, the reac-
tion mixture was used in the subsequent experiment.
1
–4)
of osteoarthritis.
Glucosamine has also been shown to have high
DNA-breaking activity, and this activity is promoted
6,7)
XTT assay procedure. The assay was performed in
2
+ 5)
more by the presence of Cu . Yamaguchi et al
.
a 96-well microtiter plate according to the method
8
)
have suggested that glucosamine was unstable in the
neutral pH range, being converted into certain inter-
mediates such as a dihydropyrazine compound which
previously described. With the standard procedure,
each well contained 60
m
l of 0.5 m
M
XTT prepared
with a 0.2 potassium phosphate buŠer (pH 7.0)
M
†
To whom all correspondence should be addressed. Tel: +81-88-864-5189; Fax: +81-88-864-5200; E-mail: hukeda
＠
cc.kochi-u.ac.jp
*
Present address: Department of Food Science and Technology, Nippon Veterinary and Animal Science University, 1-7-1 Kyonan-cho,
Musashino, Tokyo 180-8602, Japan

2
96
T. SHIMAMURA et al.
Fig. 1. HPLC Chromatogram of the D-Glucosamine Derivatives
and XTT Reducibility of the Fractionated Eluate (
at 492 nm).

absorbance
Fig. 2. EŠect of Incubation Time on the Area of Peak A (

), B
(

) and C (
-Glucosamine (0.5
pH 7.4) containing 5
$
).
D
-Glucosamine (0.5
M
) in a 50 m
M
M
sodium phosphate buŠer
C for 4 h.
D
M
) in a 50 m
DTPA was incubated at 37
l) was injected
M
sodium phosphate buŠer
(
pH 7.4) containing 5
m
DTPA was incubated at 37
9
(
m
M
9
C for 24 h.
HPLC conditions are described in the Materials and Methods
section. The XTT reducibility was measured after the eluate had
been fractionated from 3.00 to 10.00 min at intervals of 5 s.
At the indicated time, the incubated solution (5
m
for an HPLC analysis under the conditions described in the
Materials and Methods section.
saturated with menadione. A 40-
m
l amount of the
Results
sample was dropped into a well. After it had been
mixed by a microplate shaker at a speed of 500 rpm
for 15 s, the diŠerence in the absorbance between
When 0.5
M
9
glucosamine was incubated at 37 C,
the solution showed XTT reducibility depending on
the incubation time. The maximum reducibility ap-
peared after 4 h of incubation and thereafter gradual-
ly decreased in accordance with the results in our
4
92 nm and 600 nm (as the reference) was read by an
MPR A4i microplate reader (Tosoh, Tokyo, Japan)
as the absorbance at 0 min. After 20 min at room
temperature, the absorbance diŠerence was again
read, and the increase in absorbance diŠerence was
recorded as the ability of the sample to reduce XTT
8
)
previous paper. The glucosamine solution that had
been incubated for 4 h was analyzed by HPLC
(Fig. 1). Three major peaks were detected in the chro-
matogram when monitoring at 274 nm that were
characteristic of fructosazine derived from glucosa-
(
XTT reducibility). When the XTT reducibility of a
sample solution was too high, it was subjected to the
assay after appropriate dilution.
9
)
mine. These peaks are designated as A, B and C in
order of their elution time. Peaks A, B and C showed
absorption maxima at 272, 272 and 276 nm, respec-
tively. In order to know the time-course characteris-
tics of these peaks during the incubation process,
glucosamine was incubated for 24 h and each peak
area was plotted against the incubation time (Fig. 2).
Peaks A and C progressively increased with incuba-
tion time, whereas peak B reached a maximum after
5 h and thereafter decreased. The eluate corre-
sponding to each peak was collected and incubated
Separation of incubated glucosamine by HPLC.
The HPLC system consisted of a Shimadzu (Kyoto,
Japan) LC-10A pump combined with Shimadzu
SPD-M10A photodiode array detector and a Cosmo-
sil HPLC column (5C18-MS; 4.6 mm i.d.
The separation conditions were as follows: detection,
74 nm (200–400 nm); eluent, Milli-Q water; ‰ow
×
150 mm).
2
rate, 0.5 ml min; injection volume, 5 ml. The data
W
were analyzed by Shimadzu CLASS VP-5 software.
again at 37 9C . When the incubated solution was ap-
Analytical methods. LC-(ESI)MS analyses were
run on a Jasco (Tokyo, Japan) PV-1580 HPLC sys-
tem coupled with a Finnigan LCQ Duo mass spec-
trophotometer equipped with an electrospray (ESI)
interface (Thermo Quest, Tokyo, Japan). The HPLC
conditions were the same as those just described.
FAB mass spectra were obtained with a JEOL
plied to the HPLC analysis, the areas of both peaks
A and C did not change in spite of the re-incubation.
Peak B decreased with incubation time, and no new
peaks appeared in the chromatogram. This result in-
dicates that both peaks A and C were stable end-
products derived from glucosamine and that peak B
was an intermediate during the incubation process.
In order to identify which peak was related to the
XTT reduction, the eluate from 3.00 to 10.00 min
was collected every 5 s, and the XTT reducibility of
each fraction was measured (Fig. 1). Unexpectedly,
(
Tokyo, Japan) MS route JMS-600 W instrument,
using glycerol as the matrix. NMR spectra were
measured with a JEOL LNM-LA400 spectrometer in
D O.
2

Reduction of XTT by a Glucosamine Derivative
297
Fig. 4. HPLC Chromatogram of the Reaction Mixture of Col-
lected Fraction D and the XTT Solution.
Collected fraction D was reacted with XTT for 20 min and
then applied to an HPLC analysis under the conditions de-
scribed in the Materials and Method section.
Fig. 3. EŠect of Re-incubation Time on the XTT Reducibility of
Fraction D ( ) and on the Area of Peak A ( ), B ( ) and C
).
Collected fraction D was re-incubated at 37
indicated time, re-incubated fraction D was subjected to the
XTT assay and HPLC analysis. HPLC conditions are described
in the Materials and Methods section.



(
$
+
9C for 24 h. At the
MS, A, B, C and D showed (M+H) at m z of 321,
W
3
05, 305, and 323, respectively. Stable peak A and C
fractions were collected by preparative HPLC and
lyophilized. The FAB-MS spectrum of isolated peak
+
A showed (M+H) at m z
＝
321.0, in accordance
W
1
XTT reducibility was only observed in the eluate with
a retention time of 3.50–4.25 min, diŠerent from
peaks A, B and C. The fraction from 3.58 to 3.91
min having high XTT reducibility is designated as
fraction D. The XTT reducibility of fraction D de-
pended on the incubation time of glucosamine and
reached a maximum value at 5 h. In spite of the ap-
pearance of XTT reducibility, no peak was recogniz-
able in fraction D. Thus, the substance responsible
for the XTT reducibility seemed to have a relatively
low absorption at 274 nm.
with that obtained by LC-MS. The H-NMR spec-
trum exhibited the following signals [ (D O)]: 3.53
(q, 2H, 6.4 Hz, H-3 , H-3 ), 3.71 (m, 6H, H-2
H-2 , H-4 , H-4 ), 5.04 (s, 2H, H-1 , H-1 ) and
8.60 (s, 2H, H-3, H-6). The C-NMR spectrum
showed following signals [ (D O)]: 65.8 (C-4
C-4 ), 73.9 (C-3 , C-3 ), 74.3 (C-1 , C-1 ), 76.2
(C-2 , C-2 ), 144.8 (C-3, C-6) and 157.9 (C-2, C-5).
These data coincide with those of fructosazine shown
d
H
2
J
＝
?
?
!
?,
!
!
?
!
1
3
d
c
2
?,
!
?
?
!
?
!
!
1
0)
in Fig. 5. The FAB-MS spectrum of isolated peak C
+
showed (M+H) at m z 305.0, in accordance with
W
1
Although the formation of glucosamine derivative
that obtained by LC-MS. The H-NMR spectrum
5
,9,10)
has already been reported,
no systematic investi-
exhibited the following signals [
1H, 13.9 and 10.9 Hz, H-1
14.1 Hz, H-1 a), 3.52 (m, 3H, H-2
3.70 (m, 4H, H-4 , H-4 ), 3.88 (m, 1H, H-2
(s, 1H, H-1 ), 8.38 (s, 1H, H-6) and 8.56 (s, 1H,
d
H
(D
b), 3.07 (d, 1H,
, H-3 , H-3
), 5.00
2
O)]: 2.82 (dd,
gation on the formation mechanism has previously
been made. To clarify the relationship between frac-
tion D and other intermediate products, fraction D
J
＝
!
J
＝
!
!
?
!),
?
!
?
was again incubated at 37
9
C, and the XTT reducibili-
?
13
ty and HPLC pattern of the incubated solution were
examined (Fig. 3). The XTT reducibility decreased
with incubation time and, at the same time, peaks A,
B and C appeared in the chromatogram. Peak B was
formed via fraction D and changed to other sub-
stances. After the XTT reducibility of fraction D had
been measured, the reaction mixture was applied to
an HPLC analysis. Multiple peaks were apparent in
the chromatogram but the major one was peak A
whose absorption maximum was 272 nm (Fig. 4),
while no peak appeared with a retention time corre-
sponding to that of peak B or C. It is clear, therefore,
that fraction D was mainly oxidized to peak A with
XTT and that peaks B and C were not the oxidized
form of fraction D.
H-3). The C-NMR spectrum showed the following
signals [ (D O)]: 40.4 (C-1 ), 65.3 (C-4 ), 65.8
(C-4 ), 73.9 (C-3 ), 74.15 (C-2 ), 74.20 (C-1 ), 76.3
(C-2 ), 77.3 (C-3 ), 145.0 (C-3), 147.0 (C-6), 156.1
d
c
2
!
!
!
?
?
?
!
?
(C-5) and 156.9 (C-2). These data coincide with those
of deoxyfructosazine (Fig. 5) described by Sumoto
1
0)
et al., except for the assignment of H-2
? and H-2!.
Since H-2 has fewer neighboring hydrogens and an
?
excess hydroxyl group instead of one hydrogen, the
proton should appear in a lower magnetic ˆeld and
give a more complex signal. Therefore, peak C could
be identiˆed as deoxyfructosazine as shown in Fig. 5.
Similarly, peak B and fraction D were separated and
lyophilized. Due to the instability of these com-
+
pounds, (M+H) obtained by FAB-MS failed to
We next tried to elucidate the structures of peaks
A, B and C and fraction D after separating from the
reaction mixture by HPLC. By monitoring with LC-
agree with that by LC-MS. Therefore, it was impossi-
1
13
ble to identify these compounds by H- and C-
NMR. Fraction D had a molecular weight of 322

2
98
T. SHIMAMURA et al.
Discussion
The results of our previous study show that the in-
cubated glucosamine had XTT reducibility that was
8
)
related to its DNA-breaking activity. However, the
XTT-reducing substance which was generated during
the incubation of glucosamine was not clariˆed. In
this study, the mechanism for the reduction of XTT
with incubated glucosamine was investigated by us-
ing an HPLC analysis with an ODS column. When
the incubated glucosamine was subjected to HPLC
and monitored at 274 nm, which is the characteristic
absorption maximum of a glucosamine derivative,
three peaks (A, B and C) could be detected on the
chromatogram. These peaks, however, had no ability
to reduce XTT. Contrary to our expectation, XTT
reducibility was only observed in the eluate with a
retention time of 3.50–4.25 min. The fraction of
3
.58–3.91 min showing high XTT reducibility was
designated as fraction D. The molecular weight of
fraction D was determined as 322 by the LC-MS
analysis. Although an NMR analysis of fraction D
could not be performed because of its instability, it
was revealed that fraction D was a precursor of peak
A (fructosazine), peak B (an unknown product) and
peak C (deoxyfructosazine). It was also found that
fraction D was oxidized by XTT to fructosazine.
Judging from these results, we conclude that fraction
D was dihydrofructosazine and that dihydrofructosa-
zine was the XTT-reducing substance in the incubat-
ed glucosamine. It has been reported that dihydro-
fructosazine showed DNA-breaking activity by the
generation of active oxygen and a carbon-centered
Fig. 5. Reduction Mechanism for XTT by
D-Glucosamine
Derivatives.
according to the LC-MS analysis. Oxidation of the
fraction with XTT clearly gave fructosazine. It was
also a precursor of deoxyfructosazine. These results
strongly suggest that fraction D could have been di-
hydrofructosazine as shown in Fig. 5. The possibility
of generating dihydrofructosazine in a dehydro-
chlorinated glucosamine solution has been reported
5
,7,10)
radical.
Therefore, the XTT assay, which can
detect dihydrofructosazine, can be employed for
evaluating the DNA-breaking activity of glucosa-
mine. Since amino sugars such as mannosamine and
galactosamine, apart from glucosamine described
here, have shown DNA-breaking activity in the same
5
)
by Kashige et al. The results of this study conˆrmed
this by an analysis of the product separated by
HPLC. Since there is no information on a glucosa-
mine derivative having a molecular weight of 304
other than deoxyfructosazine, the structural elucida-
tion of peak B remains for a future investigation.
These ˆndings show that the reaction mechanism for
the reduction of XTT with glucosamine can be ex-
plained as indicated in Fig. 5; glucosamine generated
dihydrofructosazine by the condensation of two
molecules during the incubation process. Dihydro-
fructosazine could be oxidized to fructosazine with
dissolved oxygen, but when XTT was present, it
could be more quickly oxidized; at the same time,
XTT was reduced to the corresponding formazan.
Although deoxyfructosazine and peak B (unknown
structure) were also generated from dihydrofructosa-
zine, their formation did not result from the oxida-
tion reaction.
8
,10–12)
manner and the XTT reducibility,
the dihydro-
fructosazine type of compound could also be in-
volved in the reduction of XTT with those amino su-
gars. An investigation of these amino sugars is now
in progress. The DNA-breaking activity of amino su-
gars has recently been suggested to be related to the
13)
apoptosis of cancer cells. Based on this ˆnding, the
XTT assay might be of value to present the diverse
biological activities that amino sugars show.
Acknowledgment
We are grateful to Dr. S. Tebayashi of the Faculty
of Agriculture at Kochi University for the NMR
analysis. We also thank Mr. H. Moriyama of Kochi
Prefectural Industrial Technology Center for the LC-
MS analysis.

Reduction of XTT by a Glucosamine Derivative
299
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