BackgroundSystematic quantitative methodologies are needed to understand the heterogeneity of cell metabolism across cell types in normal physiology, disease, and treatment. Metabolic flux analysis (MFA) can be used to infer steady state fluxes, but it does not apply for transient dynamics. Kinetic flux profiling (KFP) can be used in the context of transient dynamics, and it is the current gold standard. However, KFP requires measurements at several time points, limiting its use in high-throughput applications.ResultsHere we propose transient MFA (tMFA) as a cost-effective methodology to quantify metabolic fluxes using metabolomics and isotope tracing. tMFA exploits the time scale separation between the dynamics of different metabolites to obtain mathematical equations relating metabolic fluxes to metabolite concentrations and isotope fractions. We show that the isotope fractions of serine and glycine are at steady state 8 h after addition of a tracer, while those of purines and glutathione are following a transient dynamics with an approximately constant turnover rate per unit of metabolite, supporting the application of tMFA to the analysis of folate metabolism. Using tMFA, we investigate the heterogeneity of folate metabolism and the response to the antifolate methotrexate in breast cancer cells. Our analysis indicates that methotrexate not only inhibits purine synthesis but also induces an increase in the AMP/ATP ratio, activation of AMP kinase (AMPK), and the inhibition of protein and glutathione synthesis. We also find that in some cancer cells, the generation of one-carbon units from serine exceeds the biosynthetic demand.ConclusionsThis work validates tMFA as a cost-effective methodology to investigate cell metabolism. Using tMFA, we have shown that the effects of treatment with the antifolate methotrexate extend beyond inhibition of purine synthesis and propagate to other pathways in central metabolism.

中文翻译：

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使用瞬时代谢通量分析定量叶酸代谢

背景需要系统的定量方法来了解正常生理、疾病和治疗中细胞类型之间细胞代谢的异质性。代谢通量分析 (MFA) 可用于推断稳态通量，但不适用于瞬态动力学。动力学通量分析 (KFP) 可用于瞬态动力学，它是当前的黄金标准。然而，KFP 需要在多个时间点进行测量，限制了其在高通量应用中的使用。结果在这里，我们建议将瞬态 MFA (tMFA) 作为一种经济有效的方法，使用代谢组学和同位素追踪来量化代谢通量。tMFA 利用不同代谢物动力学之间的时间尺度分离来获得将代谢通量与代谢物浓度和同位素分数相关联的数学方程。我们表明，在添加示踪剂后 8 小时，丝氨酸和甘氨酸的同位素分数处于稳态，而嘌呤和谷胱甘肽的同位素分数则遵循瞬态动力学，每单位代谢物的周转率近似恒定，支持将 tMFA 应用于叶酸代谢分析。使用 tMFA，我们研究了叶酸代谢的异质性和乳腺癌细胞对抗叶酸甲氨蝶呤的反应。我们的分析表明，甲氨蝶呤不仅抑制嘌呤合成，而且诱导 AMP/ATP 比率增加、AMP 激酶 (AMPK) 激活以及蛋白质和谷胱甘肽合成的抑制。我们还发现，在一些癌细胞中，丝氨酸产生的一碳单位超过了生物合成的需求。结论这项工作验证了 tMFA 作为一种经济有效的方法来研究细胞代谢。使用 tMFA，我们已经证明抗叶酸甲氨蝶呤治疗的效果超出了抑制嘌呤合成的范围，并传播到中枢代谢的其他途径。