(13)C glucose labelling studies using 2D NMR are a useful tool for determining ex vivo whole organ metabolism during hypothermic machine perfusion of kidneys.
(13)C glucose labelling studies using 2D NMR are a useful tool for determining ex vivo whole organ metabolism during hypothermic machine perfusion of kidneys.
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DOI：
10.1186/s13737-016-0037-0
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Ludwig C
中科院分区：
文献类型：
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作者：
Nath J;Smith T;Hollis A;Ebbs S;Canbilen SW;Tennant DA;Ready AR;Ludwig C
本研究的目的是确定使用核磁共振 (NMR) 示踪剂研究（富含 13C 的葡萄糖）来检测低温机器灌注 (HMP) 期间猪肾灌注液和皮质组织中离体从头代谢的可行性。使用器官恢复系统 LifePort 肾脏灌注装置对猪肾脏 (n = 6) 进行 24 小时的 HMP 处理。将均匀富含稳定同位素 13C（[U-13C] 葡萄糖）的葡萄糖以 10 mM 的浓度掺入 KPS-1 样灌注液中。使用 1D 1H 和 2D 1H,13C 异核单量子相干 (HSQC) NMR 光谱对灌注液进行分析。然后通过量化灌注液和组织样本中含有 13 C 的关键代谢物的比例来研究代谢活性。灌注液和组织提取物中存在的许多中心代谢物中 13C 显着富集，其中乳酸和丙氨酸最为明显。在 HMP 期间，灌注液中的富集乳酸总量（每个样品）有所增加（6 小时时为 31.1±12.2 nmol，24 小时时为 93.4±25.6 nmol，p< 0.01）。富集丙氨酸的总量以类似的方式增加（6 小时时为 1.73±0.89 nmol，24 小时时为 6.80±2.56 nmol，p<0.05）。此外，一些样品中还明显存在少量富集的乙酸盐和谷氨酸。这项研究最终证明了 HMP 期间发生了从头代谢，并强调了这种低温、缺氧环境中的活跃代谢途径。虽然大部分富含 13C 的葡萄糖被代谢为糖酵解终点代谢物（例如乳酸），但非糖酵解途径衍生物的存在表明 HMP 期间的代谢比之前想象的更为复杂。使用 2D NMR 进行同位素标记的离体器官灌注研究是可行且信息丰富的。
The aim of this study is to determine the feasibility of using nuclear magnetic resonance (NMR) tracer studies (13C-enriched glucose) to detect ex vivo de novo metabolism in the perfusion fluid and cortical tissue of porcine kidneys during hypothermic machine perfusion (HMP). Porcine kidneys (n = 6) were subjected to 24 h of HMP using the Organ Recovery Systems LifePort Kidney perfusion device. Glucose, uniformly enriched with the stable isotope 13C ([U-13C] glucose), was incorporated into KPS-1-like perfusion fluid at a concentration of 10 mM. Analysis of perfusate was performed using both 1D 1H and 2D 1H,13C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The metabolic activity was then studied by quantifying the proportion of key metabolites containing 13C in both perfusate and tissue samples. There was significant enrichment of 13C in a number of central metabolites present in both the perfusate and tissue extracts and was most pronounced for lactate and alanine. The total amount of enriched lactate (per sample) in perfusion fluid increased during HMP (31.1 ± 12.2 nmol at 6 h vs 93.4 ± 25.6 nmol at 24 h p < 0.01). The total amount of enriched alanine increased in a similar fashion (1.73 ± 0.89 nmol at 6 h vs 6.80 ± 2.56 nmol at 24 h p < 0.05). In addition, small amounts of enriched acetate and glutamic acid were evident in some samples. This study conclusively demonstrates that de novo metabolism occurs during HMP and highlights active metabolic pathways in this hypothermic, hypoxic environment. Whilst the majority of the 13C-enriched glucose is metabolised into glycolytic endpoint metabolites such as lactate, the presence of non-glycolytic pathway derivatives suggests that metabolism during HMP is more complex than previously thought. Isotopic labelled ex vivo organ perfusion studies using 2D NMR are feasible and informative.