METHOD FOR THE DETERMINATION OF (NH3)-N-15 ENRICHMENT IN BIOLOGICAL SAMPLES BY GAS CHROMATOGRAPHY ELECTRON IMPACT IONIZATION MASS-SPECTROMETRY/

An alternative method for the determination of [N-15]ammonia enrichmen t in biological fluids was developed. It is based on the use of glutam ate dehydrogenase of bovine liver (EC 1.4.1.2.) with 2-oxopentanoic ac id as substrate, to convert the ammonia present in the sample into nor valine, the enrichment of which can be measured by gas chromatography/ mass spectrometry as its tertiary butyldimethylsilyl (TBDMS) derivativ e under electron impact selective ion recording (SIR) conditions. The principal advantage of the present approach is that it is simpler and quicker than the previously described methods, because the synthetic p roduct, norvaline, is not present in biological fluids and pre-process ing of the sample is unnecessary. The procedure includes a pre-incubat ion stage which allows removal of contaminant ammonia present in the r eagents used for the enzyme reaction. The contributions of other sourc es of nitrogen to norvaline production have been checked and quantifie d: these may provide limitations of the technique when samples for ana lysis are low in ammonia (e.g. arterial or hepatic venous blood). To r educe these contributions, short times of incubation are proposed. The results from two experiments in vivo in which two sheep were infused with [N-15]ammonium chloride in the mesenteric vein are presented and the biological implications which arise from the results are discussed . The validity of the procedures was demonstrated by the quantitative recovery from the mesenteric and portal veins of [N-15] ammonia infuse d.