13C NMR 代谢组学评价氧葡萄糖剥夺后脑皮质切片即刻和延迟轻度低温。
13C NMR metabolomic evaluation of immediate and delayed mild hypothermia in cerebrocortical slices after oxygen-glucose deprivation.
机构信息
* Research Associate, ‖ Visiting Professor, ** Professor, The Department of Anesthesia and Perioperative Care, † Professor, The Department of Epidemiology and Biostatistics, ‡ Research Associate Professor, # Professor, The Department of Pharmaceutical Chemistry, The University of California San Francisco, San Francisco, California. § NMR Spectroscopist and Laboratory Manager, QB3 Physical Biosciences Division, Lawrence Berkeley National Laboratory, The University of California, Berkeley, California.
出版信息
Anesthesiology. 2013 Nov;119(5):1120-36. doi: 10.1097/ALN.0b013e31829c2d90.
BACKGROUND
Mild brain hypothermia (32°-34°C) after human neonatal asphyxia improves neurodevelopmental outcomes. Astrocytes but not neurons have pyruvate carboxylase and an acetate uptake transporter. C nuclear magnetic resonance spectroscopy of rodent brain extracts after administering [1-C]glucose and [1,2-C]acetate can distinguish metabolic differences between glia and neurons, and tricarboxylic acid cycle entry via pyruvate dehydrogenase and pyruvate carboxylase.
METHODS
Neonatal rat cerebrocortical slices receiving a C-acetate/glucose mixture underwent a 45-min asphyxia simulation via oxygen-glucose-deprivation followed by 6 h of recovery. Protocols in three groups of N=3 experiments were identical except for temperature management. The three temperature groups were: normothermia (37°C), hypothermia (32°C for 3.75 h beginning at oxygen--glucose deprivation start), and delayed hypothermia (32°C for 3.75 h, beginning 15 min after oxygen-glucose deprivation start). Multivariate analysis of nuclear magnetic resonance metabolite quantifications included principal component analyses and the L1-penalized regularized regression algorithm known as the least absolute shrinkage and selection operator.
RESULTS
The most significant metabolite difference (P<0.0056) was [2-C]glutamine's higher final/control ratio for the hypothermia group (1.75±0.12) compared with ratios for the delayed (1.12±0.12) and normothermia group (0.94±0.06), implying a higher pyruvate carboxylase/pyruvate dehydrogenase ratio for glutamine formation. Least Absolute Shrinkage and Selection Operator found the most important metabolites associated with adenosine triphosphate preservation: [3,4-C]glutamate-produced via pyruvate dehydrogenase entry, [2-C]taurine-an important osmolyte and antioxidant, and phosphocreatine. Final principal component analyses scores plots suggested separate cluster formation for the hypothermia group, but with insufficient data for statistical significance.
CONCLUSIONS
Starting mild hypothermia simultaneously with oxygen-glucose deprivation, compared with delayed starting or no hypothermia, has higher pyruvate carboxylase throughput, suggesting that better glial integrity is one important neuroprotection mechanism of earlier hypothermia.
背景
人类新生儿窒息后轻度脑低温（32°-34°C）可改善神经发育结局。星形胶质细胞而非神经元具有丙酮酸羧化酶和乙酸摄取转运体。给予[1-C]葡萄糖和[1,2-C]乙酸后，通过 C 磁共振波谱对啮齿动物脑组织提取物进行分析，可以区分神经胶质细胞和神经元之间的代谢差异，以及通过丙酮酸脱氢酶和丙酮酸羧化酶进入三羧酸循环。
方法
接受 C-乙酸盐/葡萄糖混合物的新生大鼠大脑皮质切片通过氧葡萄糖剥夺进行 45 分钟的窒息模拟，然后进行 6 小时的恢复。三组实验（每组 N=3 次实验）的方案完全相同，除了温度管理。三组温度分别为：正常体温（37°C）、低温（32°C，从氧葡萄糖剥夺开始 3.75 小时）和延迟低温（32°C，氧葡萄糖剥夺开始 15 分钟后开始 3.75 小时）。磁共振代谢产物定量的多变量分析包括主成分分析和最小绝对收缩和选择算子（L1 惩罚正则化回归算法）。
结果
最显著的代谢物差异（P<0.0056）是低温组[2-C]谷氨酰胺的最终/对照比值较高（1.75±0.12），而延迟组（1.12±0.12）和正常体温组（0.94±0.06），这意味着谷氨酸盐形成的丙酮酸羧化酶/丙酮酸脱氢酶比值更高。最小绝对收缩和选择算子发现与三磷酸腺苷保存相关的最重要代谢物：通过丙酮酸脱氢酶进入产生的[3,4-C]谷氨酸盐、作为重要渗透调节剂和抗氧化剂的[2-C]牛磺酸和磷酸肌酸。最终主成分分析得分图表明低温组形成了单独的聚类，但数据不足，无法进行统计学意义上的分析。
结论
与延迟开始或不开始低温相比，同时开始轻度低温与氧葡萄糖剥夺相比，丙酮酸羧化酶的通量更高，这表明更好的神经胶质细胞完整性是早期低温的一个重要神经保护机制。
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