Chemistry Reference
In-Depth Information
oxygen homeostasis, at least locally, may be compromised. The effectiveness of cancer therapy is affected
by the oxygenation status of normal and tumor tissues.
48,49
Oxygenation status of ischemic tissue in stroke
and myocardial infarction is critically important but is measured only with difficulty and invasiveness that
frustrates clinical application.
16.4.1 Magnetic resonance approaches for
in vivo
oximetry
A reasonable depth of penetration of magnetic field in the living tissues makes NMR and low field EPR
techniques the most appropriate approaches for non-invasive
in vivo
oxygen measurements.
50
Magnetic
resonance methods rely on the effect of paramagnetic oxygen on the relaxation times of the molecules
excited by electromagnetic radiation. The effect of oxygen on NMR relaxation times was described when
NMR was first discovered by Bloch
et al
.
51
. After inventing MRI, oxygen was the primary candidate to
be considered as potential contrast agent.
52,53
However, this possibility was ruled out due to the small
effect of oxygen on the T
1
and T
2
values of the protons in blood and loss of its paramagnetism upon
complexation by carrier molecules.
54
When the other parameters are kept constant, MRI oximetry is
possible via water T
2
measurements, due to the difference in magnetic properties of oxyhaemoglobin
(diamagnetic) and deoxyhaemoglobin (paramagnetic). The blood oxygen level dependent (BOLD) effect
55
has been extensively used for functional MRI of the brain despite the relatively low sensitivity of effective
T
2
to tissue oxygenation and dispersion of the data. Several NMR techniques for oxygen measurement
were developed relying on exogenous fluorinated probes, including
19
F NMR spectroscopy/imaging using
perfluorocarbon (PFC) emulsions
56,57
and fluorinated nitroimidazoles.
58
However, spin lattice relaxation
rates of fluorinated probes may also depend on other physiological or histological parameters.
57
EPR oximetry is one of the most promising and rapidly developing techniques for measurement of
oxygen in living tissues.
9
While oxygen in its triplet ground state demonstrates a strong X-band EPR signal
in the gas phase of 120 lines at low pressure,
59
no EPR spectra have ever been reported for dissolved
oxygen due to line broadening. Hence, biological EPR oximetry relies on exogenous paramagnetic probes.
16.4.2 Nitroxide probes for EPR oximetry
Nitroxyl radicals were the first paramagnetic probes used for EPR oximetry.
10,31,60,61
The physical basis
of the spin label EPR oximetry method relies on Heisenberg exchange between NR and dioxygen, a stable
diradical. Both the longitudinal (T
1
)
relaxation time of the NRs can be rather close
to the bimolecular collision rate of dissolved oxygen in aqueous solutions and be significantly affected
by the encounter rate. The encounter rate (w) is governed by the Smoluchowski equation, w
and transverse (T
2
)
D
[O
2
], where r is the interaction distance and D is the diffusion constant of oxygen, which is much greater
than the diffusion constant of the NR. According to the early work of Pake and Tuttle (1959)
62
,the
exchange-induced EPR line broadening is proportional to the radical - radical collision rate and, therefore,
to oxygen concentration. The observation of line width broadening of the TEMPO radical by dissolved
oxygen in various solvents was reported by Povich in 1975.
63
Two years later, Backer
et al
.
31
were the
first to apply the T
2
oximetry method based on oxygen-induced line broadening of the NR to follow
mitochondrial respiration in samples containing about 100 liver cells. The T
1
-sensitive EPR oximetry
methods were introduced by Hyde
et al
.
64,65
T
1
-sensitive experiments include pulsed saturation recovery,
continuous wave saturation and rapid passage displays.
66
T
1
-sensitive methods might have an advantage
for highly viscous environments or spin labeled macromolecules because for the nitroxides T
2
=
4
π
·
r
·
T
1
,and
for the optimal sensitivity the collision rate needs to be comparable to the relaxation time. However, for
most biological applications in low viscosity solutions, T
2
-sensitive methods are preferred because they are
instrumentally easy. An advantage of EPR oximetry is that it is based on pure physical interaction between
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