Biomedical Engineering Reference
In-Depth Information
Superoxide production:
XOD
H
2
O
2
O
2
(two-electron pathway)
(one-electron pathway)
(hypo)xanthine
O
2
H
2
O
uric acid
Superoxide detection:
Superoxide decomposition:
Spontaneous dismutation
2O
2
→
s
s
s
s
s
s
2H
O
2
H
2
O
2
Reaction with antioxidant
→
O
2
A
ox
Products
coo
COOH
Cyt. C
O
2
Heme(Fe3
)
COO
COOH
COO
Cyt. C
O
2
Heme(Fe2
)
COOH
FIGURE 17.3
Scheme of the detection principle of antioxidant activity using a cytochrome
c
functional-
ized gold electrode.
A
ox
is the antioxidant under investigation. (From [213], with permission.)
Overproduction of ROS can be harmful. Thus, cells have developed a complex antioxi-
dant defense system to counteract the biological potential of ROS formation [213].
Many methods including photometric, fl uorimetric, chromatographic, and electro-
chemical methods have been used to detect the antioxidants so far. Recently, electro-
chemical methods have intensively been used for antioxidant detection. Among the
electrochemical methods, the detection of antioxidant based on the direct redox trans-
formation of cyt
c
has been studied over the decade. Since cyt
c
can act as an oxidant
of superoxide, the superoxide level in solution can be detected as an oxidation cur-
rent at the sensor electrode due to electron transfer from the radical via cyt
c
to the
electrode.
The principle of antioxidant detection is shown in Fig. 17.3. Superoxide was enzy-
matically produced and dismutated spontaneously to oxygen and H
2
O
2
. Under control-
led conditions of superoxide generation such as air saturation of the buffer, optimal
hypoxanthine concentration (100
M) and XOD activity (50 mU ml
1
) a steady-state
superoxide level could be obtained for several min (580-680 s). Since these steady-
state superoxide concentrations can be detected by the cyt
c
-modifi ed gold electrode,
the antioxidate activity can be quantifi ed from the response of the sensor electrode by
the percentage of the current decrease.
O
2
also can be detected by cyt
c
-modifi ed electrode [214]. When cyt
c
was covalently
attached to the modifi ed electrode, the immobilized cyt
c
was used as an integral part of
an amperometric O
2
sensor. Superoxide generated by xanthine/XOD caused the one-
electron reduction of cytochrome
c
3
to cytochrome
c
2
. The reduced protein was then
reoxidized at the electrode surface. McNeil used this immobilization procedure to detect
O
2
production by stimulated human neutrophils [215]. The neutrophils that stimulated
with phorbol-12 myristate-13 acetate (PMA) produced current changes that were cell
number dependent. Fabian [216] used a platinized activated carbon electrode (PACE) to
µ
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