Kauppinen R A, Pirttilä T R, Auriola S O, Williams S R
Department of Biochemistry and Biotechnology, A.I. Virtanen Institute, University of Kuopio, Finland.
Biochem J. 1994 Feb 15;298 ( Pt 1)(Pt 1):121-7. doi: 10.1042/bj2980121.
Incorporation of 13C label from either [1-13C]glucose to glutamate C-4 and lactate C-3 or from [2-13C]acetate to glutamate C-4 was monitored in situ in a superfused brain slice preparation by using 1H-detected/13C-edited (1H/13C) n.m.r. spectroscopy. The fractional enrichments of both metabolites were determined by this means in both brain slices and acid extracts of the preparations in order to assess their 1H-n.m.r. detectabilities. The 1H/13C satellite resonances from glutamate C-4 and lactate C-3 in brain tissue were followed from 4 min onwards in the presence of 5 mM [1-13C]glucose. Fractional enrichment of glutamate C-4 in the slice preparations was higher than in their acid extracts throughout the incubation of 100 min; at 30 min the enrichment was 15.9 +/- 0.6% in the slice preparations and 10.6 +/- 0.9% in extracts and at 100 min 24.5 +/- 1.7% compared with 19.7 +/- 0.4%, respectively. In contrast, lactate C-3 reached a steady-state fractional enrichment of approx. 43% by 15 min and there was no difference between the values determined in the slice preparations and the acid extracts. There was a significant difference between the glutamate C-4 fractional enrichments in the brain slices (7.4 +/- 0.6%) and extracts (5.1 +/- 0.3%) after 60 min of incubation with [2-13C]acetate. Thus 13C label from both glucose and exogenous acetate enters a pool of glutamate that is more amenable to 1H n.m.r. detection than total acid-extracted brain biochemical glutamate, whereas lactate is labelled with full 1H n.m.r. visibility. The results are discussed in the light of the biochemical factors that affect glutamate 1H-n.m.r. susceptibility and thus its n.m.r. visibility.
通过使用氢检测/碳编辑（1H/13C）核磁共振波谱法，在灌流脑片制备物中对13C标记从[1-13C]葡萄糖掺入谷氨酸C-4和乳酸C-3或者从[2-13C]乙酸盐掺入谷氨酸C-4的过程进行了原位监测。通过这种方法测定了脑片和制备物酸提取物中这两种代谢物的分数富集，以评估它们的1H核磁共振检测能力。在存在5 mM [1-13C]葡萄糖的情况下，从4分钟起跟踪脑组织中谷氨酸C-4和乳酸C-3的1H/13C卫星共振。在100分钟的孵育过程中，脑片制备物中谷氨酸C-4的分数富集始终高于其酸提取物中的分数富集；在30分钟时，脑片制备物中的富集为15.9±0.6%，提取物中的为10.6±0.9%，在100分钟时分别为24.5±1.7%和19.7±0.4%。相比之下，乳酸C-3在15分钟时达到约43%的稳态分数富集，并且在脑片制备物和酸提取物中测定的值之间没有差异。在用[2-13C]乙酸盐孵育60分钟后，脑片（7.4±0.6%）和提取物（5.1±0.3%）中谷氨酸C-4的分数富集存在显著差异。因此，来自葡萄糖和外源性乙酸盐的13C标记进入了一个比总酸提取脑生化谷氨酸更易于进行1H核磁共振检测的谷氨酸池，而乳酸则具有完全的1H核磁共振可见性。根据影响谷氨酸1H核磁共振敏感性及其核磁共振可见性的生化因素对结果进行了讨论。
