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insensitive (Braker and Mossman 1975; Archer 1993). For example, the use of moist iodide
paper to detect the oxidation products of NO in air has a sensitivity of 300ppm and has a
response time of 5 minutes. When collecting samples to measure the NO concentration, the
presence of oxygen will continue to degrade the sample. This means the sample needs to be
measured quickly, oxygen contamination must be avoided (which is difficult in biological
specimens), or the loss of NO with these reactions needs to be compensated for by converting
the other compounds back to releasing NO just prior to measurement.
It is possible to use collected expirate which can be bubbled through degassed water and the
NO can be trapped by nitroso compounds or reduced haemoglobin to form stable adducts that
can then be detected by electron paftrmagnetic resonance (detection threshold = I nmol).
Electron paramagnetic resonance spectroscopy allows a specific assessment of molecules
whose energy levels are altered in the presence of a magnetic field and this property is typical
of molecules with an uneven number of electrons (Henry, Ducrocq et al. l99l; Henry,
Irpoivre et al. L993; Singel and Lancaster 1996). Usually a continuous wave of
electromagnetic radiation in the microwave frequency is applied to the sample. When the
frequency of this is equivalent to the energy difference of the electron spin energy levels,
which is known as the 'resonance', the radiation is absorbed and the absorption leads to the
generation of a signal which is measured. This technology has proved useful in detecting the
movement of NO, for example determining the specific parentage of L-arginine to NO to
methaemoglobin, determining the active site for NO or the binding site with other
compounds. Interestingly, despite the unpaired electron, the NO molecule is only detectable in
an excited state, while at a 'ground' state (unexcited state) the coupling of electron spin and
the orbital angular momentum makes it resonant silent on the spectroscope. It is possible to
identify signals at different frequencies which allow different molecules within compounds to
be identified. This is unless a wide band of frequency is occupied as is seen with the iron in
the haem proteins and from some of the other compounds used to trap NO such as metals or
thiols. The compounds used for trapping NO in this way are also sensitive to pH. At acidic pH
levels, the nitroso and nitrone compounds are unstable and can yield NO-type spectra even in
the absence of NO (Anoyo and Kohno 1991). At alkaline pH there can be inhibition of the
reaction between NO and haemoglobin (Feelisch, Kubitzek et al. 1996; Hakim, Sugimori et
al. 1996). Finally, the equipment required to perform electron paramagnetic resonance was,
and remains, expensive and considerable expertise is necessary (Archer 1993). In addition,
more direct methods of measuring exhalation were becoming available.
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