Forbes Martin, Geisberger Sabrina, Pietzke Matthias, Mastrobuoni Guido, Kempa Stefan
Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin Institute for Medical Systems Biology, Berlin, Germany.
Handb Exp Pharmacol. 2023;277:165-180. doi: 10.1007/164_2022_621.
The understanding of biochemical processes of metabolism is gained through the measurement of the concentration of intermediates and the rate of metabolite conversion. However, the measurement of metabolite concentrations does not give a full representation of this dynamic system. To understand the kinetics of metabolism, the system must be described and quantified in terms of metabolite flow as a function of time. In order to measure the metabolite flow, or more precisely the metabolic flux through a biological system, substrates of the cell are labelled with stable isotopes. The usage of these substrates by the cell leads to the incorporation of the isotopes into downstream intermediates.The most important metabolic pathways are encompassed in the central carbon metabolism (CCM). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the central carbon metabolism "is the most basic aspect of life". It includes all metabolites and enzymatic reactions within: glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), amino acids and nucleotide metabolic pathways. Some molecules are at the crossroad of metabolic pathways, interconnecting diverse metabolic and therefore functional outcomes. Labelling these nodal metabolites and analysing their isotopic composition allows the precise determination of the metabolic flow within the biochemical networks that they are in.Application of stable isotope labelled substrates allows the measurement of metabolic flux through a biochemical pathway. The rapid turnover of metabolites in pathways requires pulse-feeding cells with a labelled substrate. This method allows for the determination of different cell states. For example, the action of a drug from immediate impact until the compensatory response of the metabolic system (cell, organs, organisms). Pulsed labelling is an elegant way to analyse the action of small molecules and drugs and enables the analysis of regulatory metabolic processes in short time scales.
对代谢生化过程的理解是通过测量中间产物的浓度和代谢物转化速率来实现的。然而，代谢物浓度的测量并不能完全反映这个动态系统。为了理解代谢动力学，必须根据代谢物随时间变化的流量来描述和量化这个系统。为了测量代谢物流量，或者更准确地说是通过生物系统的代谢通量，细胞的底物用稳定同位素进行标记。细胞对这些底物的利用会导致同位素掺入下游中间产物中。最重要的代谢途径包含在中心碳代谢（CCM）中。根据京都基因与基因组百科全书（KEGG），中心碳代谢“是生命最基本的方面”。它包括以下所有代谢物和酶促反应：糖酵解和糖异生、磷酸戊糖途径（PPP）、三羧酸（TCA）循环、氧化磷酸化（OXPHOS）、氨基酸和核苷酸代谢途径。一些分子处于代谢途径的交叉点，连接着不同的代谢过程以及因此产生的功能结果。标记这些节点代谢物并分析它们的同位素组成，可以精确确定它们所处生化网络中的代谢流量。应用稳定同位素标记的底物可以测量通过生化途径的代谢通量。途径中代谢物的快速周转需要用标记底物对细胞进行脉冲式投喂。这种方法可以确定不同的细胞状态。例如，一种药物从即时作用直到代谢系统（细胞、器官、生物体）的代偿反应。脉冲标记是分析小分子和药物作用的一种巧妙方法，能够在短时间尺度上分析调节性代谢过程。
