Jones J G, Cottam G L, Miller B C, Sherry A D, Malloy C R
Mary Nell and Ralph B. Rogers Magnetic Resonance Center, University of Texas Southwestern Medical Center, Dallas 75235.
Anal Biochem. 1994 Feb 15;217(1):148-52. doi: 10.1006/abio.1994.1095.
13C NMR analysis of 13C-enriched glucose containing multiple isotopomers is hampered by chemical shift similarities of several carbon resonances and by the presence of two anomeric forms. A convenient and quantitative method of enzymatically oxidizing glucose to gluconate in tissue and perfusate extracts is presented. The six carbon resonances of the resulting 13C-enriched gluconate are fully resolved at high pH, thereby allowing a determination of the fractional population of each 13C isotopomer by 13C NMR. The utility of this method is demonstrated using the effluent from an isolated perfused liver containing 13C-enriched glucose produced by hepatic metabolism of sodium [1,2,3-13C3]propionate via the citric acid cycle and gluconeogenesis. An analysis of the gluconate C2 and C5 resonances in this sample showed that pentose phosphate activity was insignificant during this perfusion protocol. As demonstrated, this method provides a means of fully describing 13C isotopomer populations in enriched glucose samples where isotope may be derived from multiple metabolic pathways, thus expanding the scope of experimental design and enrichment strategies.
含有多种同位素异构体的13C富集葡萄糖的13C NMR分析受到几个碳共振的化学位移相似性以及两种异头物形式的存在的阻碍。本文提出了一种在组织和灌注液提取物中酶促将葡萄糖氧化为葡萄糖酸盐的简便定量方法。所得13C富集葡萄糖酸盐的六个碳共振在高pH下完全分辨，从而允许通过13C NMR测定每种13C同位素异构体的分数丰度。使用来自分离灌注肝脏的流出物证明了该方法的实用性，该流出物含有通过柠檬酸循环和糖异生由[1,2,3-13C3]丙酸钠的肝脏代谢产生的13C富集葡萄糖。对该样品中葡萄糖酸盐C2和C5共振的分析表明，在此灌注方案中磷酸戊糖活性微不足道。如所示，该方法提供了一种在富集葡萄糖样品中充分描述13C同位素异构体群体的方法，其中同位素可能来自多种代谢途径，从而扩大了实验设计和富集策略的范围。
