Arnoldi L, Valsecchi G, Magni F, Monti L D, Piatti P M, Costa S, Kienle M G
IRCCS San Raffaele, Milan, Italy.
J Mass Spectrom. 1998 May;33(5):444-52. doi: 10.1002/(SICI)1096-9888(199805)33:5<444::AID-JMS651>3.0.CO;2-B.
Tritiated water and radioactive tracers have been used to monitor glucose production by primary cultures of hepatocytes. More recently, 3H2O has been replaced for by 2H2O in 'in vivo' studies addressed at the evaluation of the relative contribution of gluconeogenesis to total glucose production. In this work, the possibility of using 2H2O to determine the ratio between the glucogenic flux and the overall flux through glucose 6-phosphate in isolated liver cells in vitro was evaluated. For this purpose, hepatocytes from either fasted or fed rats were incubated with a medium containing 6, 12 and 25% of 2H2O in the presence of either 2 or 20 mM pyruvate. Isotopomer analysis of six different mass clusters (m/z 328, 314, 242, 212, 187 and 145) was carried out by gas chromatography/mass spectrometry (GC/MS) of glucose aldonitrile pentaacetate. For each cluster, ions at m/z +1, +2, +3 and +4 were monitored. From the combination of different clusters the enrichment at C-6 and C-2 of glucose was computed and the C-6/C-2 ratio was considered to represent the contribution of gluconeogenesis to total glucose production, as suggested previously. Based on the results obtained, conditions selected to be optimum for the use of the method in studies on the modulation of gluconeogenesis were as follows: incubation of hepatocytes with 20 mM pyruvate in 12% 2H2O followed GC/electron ionization MS analysis of the clusters of ions at m/z 328, 314 and 187 of the glucose derivative to calculate enrichment at the C-2 and C-6 positions of glucose.
氚水和放射性示踪剂已被用于监测原代肝细胞培养物中的葡萄糖生成。最近，在评估糖异生对总葡萄糖生成的相对贡献的“体内”研究中，2H2O已取代3H2O。在这项工作中，评估了在体外分离的肝细胞中使用2H2O来确定糖异生通量与通过葡萄糖6-磷酸的总通量之间比例的可能性。为此，将禁食或喂食大鼠的肝细胞与含有6%、12%和25% 2H2O的培养基在2 mM或20 mM丙酮酸存在下孵育。通过葡萄糖醛腈五乙酸酯的气相色谱/质谱（GC/MS）对六个不同质量簇（m/z 328、314、242、212、187和145）进行同位素异构体分析。对于每个簇，监测m/z +1、+2、+3和+4处的离子。如先前所示，通过不同簇的组合计算葡萄糖在C-6和C-2处的富集，并将C-6/C-2比例视为代表糖异生对总葡萄糖生成的贡献。根据获得的结果，在糖异生调节研究中选择该方法使用的最佳条件如下：将肝细胞与20 mM丙酮酸在12% 2H2O中孵育，随后对葡萄糖衍生物的m/z 328、314和187处的离子簇进行GC/电子电离质谱分析，以计算葡萄糖在C-2和C-6位置的富集。
