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4.4 Nitrite and nitrate
The measurement of NO in biological tissues has been difficult in the past because of the
short halfJife, the small amounts produced, and its lability in the presence of Oz.
By contrast nitrite and nitrate are stable metabolites whose presence in blood, urine and tissue
in humans was demonstrated many years ago (Mitchell, A. et al. 1916; Mitchell 1928;
Tannenbaum, Fett et al. 1928). The diazotization assay is the standard technique for
measuring inorganic nitrite and was described more than 150 years ago by Greiss (Greiss
1864; Greiss 1879). A two step assay is carried out with the reagents sulphanilic and N-(1-
naphthyl) ethylenediamine first mixed and then incubated with a nitrite containing sample
which interacts in a 1-l ratio generating a purple azo dye which can be monitored by
spectrophotometry at a wave length of 546nm (Greiss 1864; Green, Wagner et al. L982). This
remains the most common method of measuring nitrite and nitrate (Schmidt and Kelm 1996)
in fluid such as blood and urine (Wishnok, Tannenbaum et al. 1993; Baylis and Vallance
1998). Studies have suggested that it is circulating nitrite measurable in serum or plasma
rather than nitrate that more reflects the NO synthesis in both humans and animals (Bode-
Boger, Boger et al. 1999; Lauer, Preik et al. 2IN_I; Kleinbongard, Dejam et al. 2006).
However, the excretion rate in urine of nitrate is a more non-invasive method to measure
whole body NO synthesis and is particularly useful for obtaining baseline results and then
assessing responses to physical and pharmacological treatment (Kanno, Hirata et al. 1992;
Bode-Boger, Boger er al. 1994 Borgonio, Witte et al. 1999; Bode-Boger, Boger et d. 2000).
There are some difficulties in making these measurements in biological assays as NO
decomposes to nitrite and nitrate at different rates depending on the ambient conditions and
on the redox environment of the fluid being measured. As well, these two compounds are
present at different concentrations in urine, serum and/or blood therefore requiring the ability
to measure and calculate across a wide concentration range potentially from low nanomolar to
high micromolar levels (Schmidt and Kelm 1996). Although adaptation to enable this has
occurred as studies on the nitrogen compounds have progressed (Green, Wagner et al. 1982;
Corras and Wakid 1990; Ohta, Araki et al. 1994; Tsikas 2005). However even a very recent
comprehensive review of the topic states "Despite the chemical simplicity of nitrite and
nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids
as indicators of NO synthesis may be difficult" (Tsikas 2005). The Greiss reaction gives an
assay that is designed to measure single samples, although it can be modified to give online
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