MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GC-MS-based metabolomics workflow that uses insoluble protein-derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion) and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol (ISO) treatment) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as LC or capillary electrophoresis.

中文翻译：

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改进的基于质谱的心脏代谢组学分析工作流程。

基于质谱的代谢组学方法是一种强大的技术，可绘制心脏复杂而相互关联的代谢途径。然而，将代谢物丰度标准化为心脏组织中的样本仍是一项技术挑战。在这里，我们描述了一种改进的基于GC-MS的代谢组学工作流程，该流程使用不溶性蛋白衍生的谷氨酸对每个样品中的代谢物进行归一化，并包括对蛋白衍生氨基酸的归一化以减少生物学变异并检测微小的代谢变化。此外，糖原在代谢组学工作流程中进行测量。我们通过首先比较两种不同的心脏摘除方法将这一工作流程应用于心脏代谢研究：Langendorff心脏法（主动脉反灌流）和采用改良的组织冷冻钳法原位冷冻小鼠心脏。然后，我们使用原位冷冻方法研究急性β-肾上腺素受体刺激（通过异丙肾上腺素（ISO）处理）对心脏代谢的影响。使用我们的工作流程并在几分钟之内，ISO降低了糖原代谢，糖酵解和克雷布斯循环所涉及的代谢物水平，但戊糖磷酸途径代谢物和许多游离氨基酸的水平保持不变。该观察结果与磷酸化的腺苷核苷酸丰度增加了6倍有关。这些结果支持以下观点：ISO会急剧加速葡萄糖的氧化代谢，以满足支持增加心率和心输出量所需的ATP需求。综上所述，