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Under these conditions HOONO may undergo cleavage to a hydroxyl free
radical (OH') a nitrogen dioxide free radical (NO^) or nitronium cation
(N02 ) and hydroxide anion (OH "). Three of these cleavage products (OH',
NO 2 , NO 2) are among the most reactive and damaging species in
biological systems, and may be major contributors to the severe damage of
the endothelium and the cardiovascular system occurring in hypertension.
A similar increase in O2 production by dysfunctional endothelium is also
observed during endotoxemia.
7. In Vivo Measurements of Nitric Oxide in the Lung
Figure 16a shows the amperometric curve measured in vivo during
endotoxemia with a porphyrinic sensor placed in the
Figure 16. In vivo measurement of nitric oxide release with porphyrinic
sensor in lung (rat) during the first 100 min after administration of LPS
(20 mg/kg) (a); NO2 and NOJ measured in blood (UV-Visible spectroscopy,
Griess reagent) after administration of LPS (b).
44/Porphyrin-Based Electrochemical Sensors
rat lung parenchyma. During administration of lipopoly-saccharide (LPS),
an increase of NO production from its basal concentration was observed.
The concentration of NO reached a peak of 140 nM after 180 seconds,
persisting for 12 minutes before decaying at a rate of 0.20 nMs - 1.
After 45 minutes, the NO concentration started to rise again, but at a
much slower rate. A plateau of NO release was established 80 ± 15 minutes
after LPS administration.
UV-Vis spectroscopy (Griess reagent) was used to determine a change of
NO2 /NO3 concentration in blood during endotoxemia.91 Both NO2 and N03
are end products of NO oxidation in biological systems (Figure 16b). The
concentration of NO 2 and NO3 significantly increased from basal 1.0 to
6.4 öò after 6 h of endotoxemia. The most dramatic rate of increase was
observed after 2 h of endotoxemia. The concentration of O2 (measured in
vitro during stimulation with A23187 using chemiluminescence method) also
increased significantly during endotoxemia.69 During the first hour of
endotoxemia, the O2 concentration increased threefold relative to the
Induction of iNOS by bacterial toxins directly or through cytokines
leads to the generation of NO in arterial walls.
Figure 17. Response of the catheter-protected porphyrinic sensor to
changing fluid pressure (a), to mechanically bending the active tip of
the sensor at a frequency of 3Hz (b), and to the ECG signal (c). The
vertical bars represent the equivalent current that would be generated by
the porphyrinic sensor in the 100 nM NO ((a), lower tracing) or 50 nM NO
Administration of toxin (LPS) also generated NO by calcium-dependent eNOS
in the endothelium during endotoxemia. The high production of NO by the
endothelium is observed only in the early acute phase of endotoxemia
(first 25-40 minutes); however, it has a profound effect on the late
chronic phase of endotoxemia when iNOS becomes the main generator of NO.
The porphyrinic NO sensor measured only free NO, that is, the net NO
concentration not consumed in the extremely fast chemical reaction with
O2 and other redox centers in the tissue. This free NO, measured
intermittently, remained approximately the same during the second chronic
phase of endotoxemia, in spite of high NO production by iNOS (after 45
minutes from LPS infusion), measured indirectly by assaying the
accumulation of the NO decay products NO2 / NO3 in blood plasma by the
Griess method. NO measurement with the porphyrinic sensor clearly
indicates that the net concentration of NO is only two times higher
during late chronic phase of endotoxemia than the observed pre-
endotoxemic basal concentration. This net NO concentration is much lower
than the total NO produced during this period, but still sufficient to
account for the coincident chronic hypotension observed. However, low net
NO concentration cannot be accountable for the sudden death of animals
after about 6h of endotoxemia. Direct electrochemical measurements
suggest that the dysfunction of endothelial cells eventually leading to
the death of the animal is triggered by 02 /OONO- rather than by NO
itself during endotoxemia.
8. In Vivo Measurements of Nitric Oxide in the Heart
Measurement of NO amidst the dynamic in vivo conditions of cyclic
breathing and heart beating is a challenging task. In order to overcome
these potential interferences and to record a reliable NO signal, the
catheter-protected porphyrinic sensor is slightly modified in two ways.
First, the active sensor tip is shortened to 50-60 öò from 0.3-0.5 mm
usually used for in vivo measurements. Also, the truncated needle from
which the active sensor tip emerges is cut 50 to 60 öò shorter than its
protective catheter so that the tip of the sensor is completely recessed
within the ventilated catheter tip rather than protruding from an