Biology Reference
In-Depth Information
myeloperoxidase (MPO). The latter gener-
ates the species HOCl, whose bactericide
and oxidant activity is important for the
destruction of bacterial agents. Therefore,
the neutrophil is a complex but reliable sys-
tem for the study of antioxidants in biologi-
cal environments, particularly where it is
developing an inflammatory process. For a
proper interpretation of the results, we must
keep in mind the use of inhibitors and
amplifiers of the response. For example, the
systems luminol / superoxide (SOD) / cata-
lase (CAT) and isoluminol / horseradish
peroxidase (HRP) allow us to obtain infor-
mation from the production of ROS both
within and outside the neutrophil. In the
first case, luminol acts as a permeable
chemiluminescent probe that is distributed
evenly inside and outside the neutrophil. If
using the mixture of SOD / CAT, the extra-
cellular production of ROS may be sup-
pressed, leaving only the intracellular
production. The latter can be amplified in
the presence of luminol. On the other hand,
isoluminol is impermeable and therefore
can be used to detect extracellular produc-
tion of ROS. Because such production is
strongly linked with MPO released by neu-
trophils, often it must be reinforced with
HRP, because in the first steps of the respira-
tory burst the amount of MPO in the extra-
cellular environment is minimal. As
mentioned below, it must be remembered
that the probes used are not always selec-
tive enough. Thus, both luminol and isolu-
minol preferably detected HOCl, showing
less sensitivity for the case of superoxide
anion and hydrogen peroxide. When look-
ing for a detailed study of the effect on
NADPH oxidase activity, and therefore on
superoxide anion production, the alterna-
tive use of lucigenin is more recommend-
able. As discussed in the next section, new
probes have been developed for the study of
specific cellular levels of certain radicals of
biological relevance. The use of fluorescent
probes derived from fluorescein is a fine
approach to detect visually where ROS pro-
duction occurs. Mononuclear cells are
obtained together with neutrophils after the
double gradient separation. Usually, these
cells require a special culture medium such
as RPMI 1640. The production of ROS by
monocytes has a certain delay compared
with neutrophils when different activators
are used. Although monocytes have intra-
cellular granules with peroxidases, they
possess less MPO than neutrophils and
therefore produce less HOCl. Monocytes
could be stimulated to promote differentia-
tion to macrophages, in which it is possible
to study the production of NO , generated
by the enzyme iNOS. Traditionally nitric
oxide can be measured indirectly by the
Griess reaction for the oxidation product
(NO 2 ), and ultimately by specific electrodes
(Gobert et al. , 2001; Rocha et al ., 2009).
Overall, the production of ROS and
RNS can be measured in various cellular
contexts and the choice depends on the
research problem and the resources of each
laboratory. Because of their importance in
cardiovascular pathology, human umbilical
vein endothelial cells (HUVECs) are an
interesting model, but their preparation
requires more expertise, which increases
the cost of any analysis. For those who study
the antioxidant capacity in cell lines it is
highly recommended to assess the permea-
bility of the tested molecules in such a con-
text. Many of the effects (including the
production of ROS) of polyphenols do not
require penetration into a cell.
2.7 Every ROS has its Thorn:
New Probes under Investigation
2.7.1 Common probes used
to detect ROS in cells
So far, there are few 'gold standard' probes
universally employed and specific enough
to measure certain free radicals. The
reader who wishes to delve into this area
can see some good reviews published
recently that cover the advantages and
limitations of various reagents for the
detection of ROS (Freitas et al. , 2009; Niki
2010a,b; Rhee et al. , 2010). Thus, this sec-
tion only summarizes some of the most
widely used reagents for cellular and non-
cellular systems.
 
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