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4.2
The use of L-arginine as a substrate for NO production is a relatively specific reaction. This
allows the production of NO to be correlated to the loss of L-arginine and the gain of L-
citrulline, and it is possible to measure both of these in a laboratory setting (Senshu, Sato et al.
1992). However the baseline concentrations of L-arginine need to be known, which is difficult
in vivo. Also, while the formation of L-citrulline can be measured (Hecker, Sessa et al. 1990,
Senshu, 1992 #898; Bredt and Schmidt 1996), this is made more difficult in vivo where
certain cells such as vascular endothelial cells and macrophages (and likely others) are able to
regenerate L-citrulline back to L-arginine at a variable rate to act as further substrate (see
Chapter 3.4.3 'Control of the nitric oxide synthase isoenzymes'). This recycling clearly
precludes the use of measurement of the levels of these compounds to accurately reflect the
production of NO.
The NOS enzymes stimulate soluble guanylate cylase (sGC) with subsequent accumulation of
cyclic guanosine monophosphate (cGMP). Again, it is possible to measure the levels of this
protein, however, this requires access to, or the releasing of, the protein from its usual
intracellular site. This is also non specific for NO as both sGC and sGMP operate in many
cellular enzyme pathways (Schultz, Bohme et al. 1969; White and Aurbach 1969; Archer
L993; Craven andIgnarro 1996).
4.3
Methaemoglobin
NO can be measured by the transformation of reduced haemoglobin (Fe 2) as it is oxidised to
methaemoglobin (Fe 3*; which can then measured by spectrophotometry (detection threshold
= I nmol). The reaction is rapid and will be almost stoichometric under most experimental
conditions, with the only interfering compound likely to be superoxide anion production
(Sutton, Roberts et al. 1976). The advantages are that spectrophotometers are widely
available, there is no need to acidify the sample and that methaemoglobulin remains relatively
stable. Accumulation of oxyhaemoglobin is not a problem due to the much higher affinity of
haemoglobin for NO than Oz. The disadvantages are that it is more useful for biological
samples other than exhalate, considerable expertise is required and the assay will detect other
nitrosyl grcups in any given sample (Noack, Kubitzek et al. lgg2; Murphy and Noack 1994;
Feelisch, Kubitzek et al. 1996).
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