Biology Reference
In-Depth Information
2.6.2 Study of ROS production
in neutrophils, monocytes
and other cell lines
are challenged with oxidizing agents such
as HOCl, TBHP, H
2
O
2
or AAPH, which pro-
mote membrane damage leading to haemoly-
sis. Thus, determining the percentage of
haemolysis in the presence of increasing
concentrations of antioxidants is a good
indicator of the antioxidant potential of the
sample. Recently, Gonzalez and coworkers
developed a new method called erythrocyte
cellular antioxidant activity (ERYCA). They
proposed a modification of the assay, mak-
ing turbidity readings at 660 nm, instead of
haemolysis (Gonzalez
et al.
, 2010). This
allows the conversion of an end-point assay
to a kinetic one, where decreases in absorb-
ance (light scattering) versus time plots gen-
erated decay in which the area under the
curve is calculated in the same manner as in
the ORAC assay (Fig. 2.4 a, b). Although
this method is not widely used, hypotheti-
cally it could be adapted to include other
ROS-generating systems. The method is also
visually more interesting than the ORAC
assay, particularly for the
in vivo
evaluation
of antioxidant capacity, lacking only valid-
ity in its usefulness, reproducibility and
repeatability with more studies.
Neutrophils are phagocytic cells able to
assemble a highly efficient ROS-producing
machine to deal with different stimuli.
Neutrophils are easily purified from blood
collected from laboratory animals or human
volunteers. Typically, using double gradient
centrifugation with Histopaque 1119 and
1077 it is possible to obtain both neutrophils
and mononuclear cells with high purity and
viability. Mononuclear cells could be cul-
tured and used to study the production of
certain radicals such as NO. Neutrophils
have a lifetime of ~4 h and can be stimu-
lated with various agents such as PMA,
fMLP, opsonized zymosan, ionophores (ion-
omycin) and bacteria, among others (Pastene
et al
., 2009a). Initially, ROS production is
governed by the activity of NADPH oxidase.
This complex, under normal conditions, is
assembled inside the phagosome, where it
directs the production of O
2
•−
. This species
can subsequently lead to H
2
O
2
, which in
turn is the substrate of another enzyme,
(a)
Haemolysis = Absorbance (540 nm)
Other determinations:
-Membrane lipid peroxidation
-Measurement of K
+
ion loss
-Glutathione estimation
Oxidants:
AAPH
HOCI
t-BHP, etc
Haemolysis = Tu rbidity (700 nm)
(b)
1.5
1.0
24
36
RBC + PBS
RBC + AAPH
RBC + AAPH + rutin 6 µM
RBC + AAPH + rutin 12 µM
RBC + AAPH + rutin 24 µM
RBC + AAPH + rutin 36 µM
6
0
12
0.5
0.0
0
50
100
150
200
Time (min)
Fig. 2.4.
(a) Illustration of the ERYCA assay. AAPH, 2,2´ azobis (2-amidinopropane) hydrochloride;
t-BHP, tert-butyl hydroperoxide. (b) Antioxidant effect of rutin (0-36
m
M) on AAPH-induced peroxidation
on human erythrocytes (RBC) (Aguayo, 2011).
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