Centre for Integrative Bioinformatics, VU University Amsterdam, 1081HV Amsterdam, The Netherlands.
Bioinformatics. 2010 Mar 1;26(5):653-60. doi: 10.1093/bioinformatics/btq018. Epub 2010 Jan 22.
Quantitative determination of metabolic fluxes in single tissue biopsies is difficult. We report a novel analysis approach and software package for in vivo flux quantification using stable isotope labeling.
We developed a protocol based on brief, timed infusion of (13)C isotope-enriched substrates for the tricarboxylic acid (TCA) cycle followed by quick freezing of tissue biopsies. NMR measurements of tissue extracts were used for flux estimation based on a computational model of carbon transitions between TCA cycle metabolites and related amino acids. To this end, we developed a computational framework in which metabolic systems can be flexibly assembled, simulated and analyzed. Flux parameters were quantified from NMR multiplets by a partial grid search followed by repeated Nelder-Mead optimizations implemented on a computer grid. We implemented a model of the TCA cycle and showed by extensive simulations that the timed infusion protocol reliably quantitates multiple fluxes. Experimental validation of the method was done in vivo on hearts of anesthetized pigs under two different conditions: basal state (n = 7) and cardiac stress caused by infusion of dobutamine (n = 7). About nine tissue samples (40-200 mg dry-weight) were taken per heart. TCA cycle flux was 6.11 +/- 0.28 (SEM) micromol/min x gdw at baseline versus 9.29 +/- 1.03 micromol/min x gdw for dobutamine stress. Oxygen consumption calculated from the TCA cycle flux and from 'gold standard' blood gas-based measurements were close, correlating with r=0.88 (P < 10(-4)). Spatial heterogeneity in metabolic fluxes is detectable amongst the small samples. We propose that our novel isotope snapshot methodology is suitable for flux measurements in biopsies in vivo.
Non-profit organizations will, upon request, be granted a non-exclusive license to use the software for internal research and teaching purposes at no charge. A web interface for using the software on our computer grid is available under http://www.ibi.vu.nl/programs/
定量测定单个组织活检中的代谢通量较为困难。我们报告了一种使用稳定同位素标记进行体内通量定量分析的新分析方法和软件包。
我们开发了一种基于短暂、定时输注（13）C 同位素标记的三羧酸（TCA）循环底物的方案，随后快速冷冻组织活检。通过基于 TCA 循环代谢物和相关氨基酸之间的碳转换的计算模型，对组织提取物的 NMR 测量值进行通量估计。为此，我们开发了一种计算框架，可以灵活组装、模拟和分析代谢系统。通过在计算机网格上执行的局部网格搜索和重复的 Nelder-Mead 优化，从 NMR 多重峰中定量通量参数。我们实现了 TCA 循环的模型，并通过广泛的模拟表明，定时输注方案可可靠地定量多种通量。在两种不同条件下（基础状态，n = 7；输注多巴酚丁胺引起的心脏应激，n = 7）对麻醉猪的心脏进行了体内方法验证。每个心脏取约 9 个组织样本（40-200mg 干重）。TCA 循环通量在基础状态下为 6.11 +/- 0.28（SEM）µmol/min x gdw，而在多巴酚丁胺应激下为 9.29 +/- 1.03µmol/min x gdw。从 TCA 循环通量和基于“黄金标准”血气的测量值计算的耗氧量接近，相关系数为 r=0.88（P < 10(-4)）。在小样本中可检测到代谢通量的空间异质性。我们提出，我们的新型同位素快照方法适合用于体内活检中的通量测量。
非营利组织将根据要求获得非专有的软件许可证，以便在内部研究和教学中免费使用。可通过以下网址使用我们的计算机网格上的软件的网络界面：http://www.ibi.vu.nl/programs/
