in the case oflNO) is used for spin trapping as this more stable
adduct formation allows measurement oflNO at a specific point
in time.
Oxidative stress is the most important pathological modulator
of endothelial function aslNO may react with superoxide anions
under formation of peroxynitrite (ONOO)[ 2]. Peroxynitrite and
its derived free radicals in turn are highly reactive compounds that
may cause protein modifications such as nitration of tyrosine resi-
dues [3, 4]. Therefore, tyrosine nitration can be used as a surrogate
for ONOOlevels in the vessel and as an estimate whether oxida-
tive stress plays a functional role with respect tolNO scavenging.
Here we describe a method that utilizes the dot blot technique for a
quantitative analysis of this interaction. As soon as endothelial
dysfunction is present and increased 3-nitrotyrosine levels are
observed, the level of oxidative stress in the vasculature should be
assessed for a thorough understanding of vascular pathology. Since
ROS assays usually have limitations with respect to their sensitivity
(e.g., EPR-based assays) or specificity (e.g., chemiluminescence
compounds such as lucigenin), it is important to combine at least
two different methods.
In this chapter, we will therefore describe two assays (dihy-
droethidium cryo-staining and Amplex red assay) that can be used
for this purpose. Dihydroethidium is a compound that reacts with
lO 2 to form 2-hydroxyethidium, which intercalates with endoge-
nous DNA and causes red fluorescence [5]. By fluorescence micros-
copy, the locallO 2 production can be visualized and analyzed
within the different vascular layers (endothelium, media, adventi-
tia) [ 6]. Of note, without the additional use of superoxide dismu-
tase and successful suppression of the fluorescence signal, a part of
dihydroethidium cryo-staining can originate from “unspecific” oxi-
dation and formation of ethidium. Therefore, it is more accurate to
define the dihydroethidium cryo-staining signal as overall vascular
ROS formation. The strengths of the assay are a good sensitivity
and spatial resolution of vascular ROS production, while the semi-
quantitative analysis is a weakness.
The Amplex red assay utilizes a horseradish peroxidase cata-
lyzed oxidation of the non-fluorescent substrate by H 2 O 2 to form
resorufin that can be detected by fluorescence plate reader or
HPLC assays as described in detail below [7]. This assay has a
good sensitivity and specificity for H 2 O 2 but is not useful if ROS
other than H 2 O 2 play a role in vascular pathology (e.g., the signal is
even diminished when high superoxide formation rates are
obtained as in activated granulocytes).2 Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water, to attain a sensitivity of 18 MΩ/cm at 25C) and496 Swenja Kro ̈ller-Scho ̈n et al.