Nieto R, Calder A G, Anderson S E, Lobley G E
Rowett Research Institute, Bucksburn, Aberdeen, UK.
J Mass Spectrom. 1996 Mar;31(3):289-94. doi: 10.1002/(SICI)1096-9888(199603)31:3<289::AID-JMS299>3.0.CO;2-Z.
An alternative method for the determination of [15N]ammonia enrichment in biological fluids was developed. It is based on the use of glutamate dehydrogenase of bovine liver (EC 1.4.1.2.) with 2-oxopentanoic acid as substrate, to convert the ammonia present in the sample into norvaline, the enrichment of which can be measured by gas chromatography/mass spectrometry as its tertiary butyldimethylsilyl (TBDMS) derivative 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 product, norvaline, is not present in biological fluids and pre-processing of the sample is unnecessary. The procedure includes a pre-incubation stage which allows removal of contaminant ammonia present in the reagents used for the enzyme reaction. The contributions of other sources of nitrogen to norvaline production have been checked and quantified: these may provide limitations of the technique when samples for analysis are low in ammonia (e.g. arterial or hepatic venous blood). To reduce these contributions, short times of incubation are proposed. The results from two experiments in vivo in which two sheep were infused with [15N]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 [15N]ammonia infused.
开发了一种测定生物流体中[¹⁵N]氨富集度的替代方法。该方法基于使用牛肝谷氨酸脱氢酶（EC 1.4.1.2.），以2-氧代戊酸为底物，将样品中的氨转化为正缬氨酸，其富集度可在电子轰击选择性离子记录（SIR）条件下通过气相色谱/质谱法测定其三叔丁基二甲基硅烷基（TBDMS）衍生物。本方法的主要优点是比先前描述的方法更简单、更快，因为生物流体中不存在合成产物正缬氨酸，无需对样品进行预处理。该程序包括一个预孵育阶段，可去除酶反应所用试剂中存在的污染性氨。已检查并量化了其他氮源对正缬氨酸生成的贡献：当分析样品中的氨含量较低时（如动脉血或肝静脉血），这些可能会限制该技术的应用。为减少这些贡献，建议缩短孵育时间。给出了在两只绵羊肠系膜静脉内注入[¹⁵N]氯化铵的两项体内实验结果，并讨论了结果所产生的生物学意义。通过对注入的[¹⁵N]氨在肠系膜静脉和门静脉中的定量回收，证明了该程序的有效性。
