Development and application of isotopically-selective assays for nitric oxide metabolites.

The importance of nitric oxide (NO) in neurotransmission, immunological defense, and as a vasodilator in the regulation of blood pressure is well known, so there is a high interest in methodology for NOS turnover measurements, or methods that help to discover new metabolites of nitric oxide. Use of L-arginine with a [15]N stable label allows determination of the NO pathway throughput, but requires sensitive detection methods which are capable of isotopic discrimination. The Griess assay for nitrite has been combined with surface enhanced resonance Raman spectroscopy (SERRS) to allow more sensitive determination of nitrite, and also quantitative discrimination between [14]N and [15]N forms. The method was optimised for use with silver sol prepared from silver nitrate and citrate. The method was applied to the analysis of urine, serum and culture medium with recoveries of 96%, 85% and 95% respectively. Good reproducibility was achieved following pretreatment of samples by solid phase extraction. The limit of detection for nitrite was 5 nmol/l and the response was linear up to at least 10 mumol/l. In terms of isotopic discrimination between the [14]N and [15]N isotopes, [15]N nitrite was detectable at isotopic ratios of greater than 1:20 [15]N : [14]N. The time of analysis, excluding derivatisation by the Griess assay, was approximately 5 minutes. The method will be useful in metabolic tracer studies using stable labels. An alternative assay using ion chromatography - mass spectrometry allowed the detection of nitrite and nitrate without prior derivatisation, and showed good discrimination between nitrogen isotopes. Nitrite and nitrate were separated from each other and from matrix components by suppressed ion chromatography. Postcolumn oxidation and a chloride trap column were employed to improve senstivity and selectivity. The limit of detection for [14]N-nitrite and [14]N-nitrate was 200 nmol/l and the detection limit for [15]N-nitrite and [15]N-nitrate 50 nmol/l. Nitrate recovery was 93% from urine and 94% from serum. Recovery of nitrite was 96% from urine and 107% from serum.The IC-MS assay was used in a pilot study of primary pulmonary hypertension, in which NOS turnover was found to be significantly lower in the patient group than in the controls. The assay was also used in a study of NO donors where the donor compounds as well as nitrite and nitrate could be detected.