13C glucose labelling studies using 2D NMR are a useful tool for determining ex vivo whole organ metabolism during hypothermic machine perfusion of kidneys

BACKGROUND 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). METHODS 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. RESULTS 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. CONCLUSIONS 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.