Chemistry Reference
In-Depth Information
L-band EPR spectroscopy (1.2 GHz) using spin pH probes has been shown to be a valuable tool for
in
vivo
monitoring of microacidity in small animals such as rodents.
2,18,115 - 117
The first
in vivo
applications
of spin pH probes were performed using L-band technique in mice.
115,116
Mader
et al
. demonstrated
that L-band EPR spectroscopy is a valuable tool for
in vivo
monitoring of pH-induced degradation of
an implanted polymer in mice.
115
Gallez
et al
.
116
took advantage of the oral administration of the HMI
(pK
a
=
7, Scheme 16.5) probe to monitor the pH value inside the stomach of mice after administration
of different antacids. The HMI spin pH probe has subsequently been applied to
in vivo
studies of stomach
acidity in rats using L-band EPR, longitudinally detected EPR (LODEPR), and PEDRI.
117
The structure of
the nitroxide API with properties optimized for monitoring stomach acidity is shown in Scheme 16.6.
18
The
introduction of bulky groups in the vicinity of the NO fragment enhanced API stability towards reduction,
resulting in about a fourfold increase of its lifetime
in vivo
compared with that for HMI. In addition,
the presence of two ionizable groups in the API structure - the imino nitrogen N3 and the pyridine
nitrogen - significantly extended its range of pH sensitivity (Figure 16.9b). Moreover, the hydrophilic
character of pyridine, hydroxy, and amino groups of the API probe prevents its penetration through
biomembranes and redistribution from the stomach. Figure 16.9 demonstrates the 300 MHz EPR spectrum
of the API probe, a calibration curve for hyperfine splitting (a
N
), and a typical example of real-time
measurements of the stomach acidity of living rats.
18
The long lifetime of the API probe
in vivo
allows
for monitoring of drug-induced perturbation of stomach acidity and its normalization afterwards for one
hour or longer periods, therefore demonstrating the applicability of pH-sensitive NRs to the studies of drug
pharmacology and disease in living animals.
L-band EPR spectroscopy using pH-sensitive NRs is also a useful tool to monitor myocardial pH in
isolated rat hearts. The ATI probe (pK
a
4
.
=
.
1, Scheme 16.5) has a convenient range of pH sensitiv-
ity (Figure 16.10a) to study ischemia-induced acidosis and has been applied in isolated rat hearts
11,118
(Figure 16.10b). Interestingly, L-band spectral measurement at modulation amplitude, 2G, significantly
larger than the line width, 0.8 G, is strongly recommended. The latter results in an increased signal-to-
noise ratio, a symmetric EPR spectrum, and a smooth dependence of nitrogen hyperfine splitting on pH
in the range from 5.2 to 7.2 (Figure 16.10a). Figure 16.10b shows the typical decrease in myocardial pH
measured by EPR and ATI probe. The hydrophilic ATI probe apparently measures extracellular pH mostly
from the aorta, as confirmed by the spatial image of the radical using L-band imager (data not shown).
Nevertheless, the EPR data are in reasonable agreement with the data observed by the
31
P-NMR tech-
nique for ischemia-induced intracellular pH changes.
119,120
It seems to be very attractive to use recently
developed EPR/NMR co-imaging
121
to monitor simultaneously the values of extra- and intracellular pH.
Intracellular and extracellular targeting is an important direction in the development of pH spin probes.
The penetration depth for L-band frequency, 1.2 GHz, in aqueous samples is somewhat greater than
1 cm and, in general, allows pH mapping by L-band EPR spectroscopy
in vivo
in small animals such as
6
OH
N
HO
N
N
O
N
Scheme 16.6
The chemical structure of API probe, 4-[bis(2-hydroxyethyl)amino]-2-pyridine-4-yl-2,5,5-triethyl-
2,5-dihydro-1H-imidazol-oxyl, synthesized to optimize NR properties for the measurement of stomach acidity
invivo
18
Search WWH ::
Custom Search