Nath Jay, Smith Tom, Hollis Alex, Ebbs Sam, Canbilen Sefa W, Tennant Daniel A, Ready Andrew R, Ludwig Christian
Department of Renal Surgery, University Hospitals Birmingham, Birmingham, UK ; Institute of Metabolism and Systems Research (IMSR), College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
Institute of Metabolism and Systems Research (IMSR), College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
Transplant Res. 2016 Aug 5;5:7. doi: 10.1186/s13737-016-0037-0. eCollection 2016.
The aim of this study is to determine the feasibility of using nuclear magnetic resonance (NMR) tracer studies ((13)C-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 (13)C ([U-(13)C] glucose), was incorporated into KPS-1-like perfusion fluid at a concentration of 10 mM. Analysis of perfusate was performed using both 1D (1)H and 2D (1)H,(13)C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The metabolic activity was then studied by quantifying the proportion of key metabolites containing (13)C in both perfusate and tissue samples.
There was significant enrichment of (13)C 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 (13)C-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.
本研究的目的是确定使用核磁共振（NMR）示踪研究（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。
使用器官回收系统LifePort肾脏灌注装置对猪肾（n = 6）进行24小时的HMP。将均匀富含稳定同位素¹³C的葡萄糖（[U-¹³C]葡萄糖）以10 mM的浓度加入到类似KPS-1的灌注液中。使用一维¹H和二维¹H、¹³C异核单量子相干（HSQC）核磁共振波谱对灌注液进行分析。然后通过量化灌注液和组织样品中含¹³C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多种中心代谢物中¹³C有显著富集，其中乳酸和丙氨酸最为明显。HMP期间灌注液中富集乳酸的总量（每个样品）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方式增加（6小时时为1.73±o89 nmol，24小时时为6.80±2.56 nmol，p < 0.05）。此外，在一些样品中还明显存在少量富集的乙酸盐和谷氨酸。
本研究确凿地证明了HMP期间发生了从头代谢，并突出了这种低温、低氧环境中的活跃代谢途径。虽然大部分富含¹³C的葡萄糖代谢为糖酵解终点代谢物如乳酸，但非糖酵解途径衍生物的存在表明HMP期间的代谢比以前认为的更复杂。使用二维NMR的同位素标记离体器官灌注研究是可行且信息丰富的。
Zotero 插件
