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Paediatric brain tumors are becoming well characterized due to large genomic and epigenomic studies. Metabolomics is a powerful analytical approach aiding in the characterization of tumors. This study shows that common cerebellar tumors have metabolite profiles sufficiently different to build accurate, robust diagnostic classifiers, and that the metabolite profiles can be used to assess differences in metabolism between the tumors. Tissue metabolite profiles were obtained from cerebellar ependymoma (n = 18), medulloblastoma (n = 36), pilocytic astrocytoma (n = 24) and atypical teratoid/rhabdoid tumors (n = 5) samples using HR-MAS. Quantified metabolites accurately discriminated the tumors; classification accuracies were 94% for ependymoma and medulloblastoma and 92% for pilocytic astrocytoma. Using current intraoperative examination the diagnostic accuracy was 72% for ependymoma, 90% for medulloblastoma and 89% for pilocytic astrocytoma. Elevated myo-inositol was characteristic of ependymoma whilst high taurine, phosphocholine and glycine distinguished medulloblastoma. Glutamine, hypotaurine and N-acetylaspartate (NAA) were increased in pilocytic astrocytoma. High lipids, phosphocholine and glutathione were important for separating ATRTs from medulloblastomas. This study demonstrates the ability of metabolic profiling by HR-MAS on small biopsy tissue samples to characterize these tumors. Analysis of tissue metabolite profiles has advantages in terms of minimal tissue pre-processing, short data acquisition time giving the potential to be used as part of a rapid diagnostic work-up.

Altered cellular metabolism is now accepted to be at the core of many diseases including cancer. Over the past 20 years, NMR has become a core technology to study these metabolic perturbations in detail. This chapter reviews current NMR-based methods for steady-state metabolism and, in particular, the use of non-radioactive stable isotope-enriched tracers. Opportunities and challenges for each method, such as 1D (1)H NMR spectroscopy and (13)C carbon-based NMR spectroscopic methods, are discussed. Ultimately, the combination of NMR and mass spectra as orthogonal technologies are required to compensate for the drawbacks of each technique when used singly are discussed.

Background: The metabolic processes occurring within the preserved kidney during hypothermic machine perfusion (HMP) are not well characterized. The aim of this study was to use nuclear magnetic resonance (NMR) spectroscopy to examine the metabolomic profile of HMP perfusate from human cadaveric kidneys awaiting transplantation and to identify possible discriminators between the profiles of kidneys with delayed graft function (DGF) and immediate graft function (IGF)., Methods: Perfusates from HMP kidneys were sampled at 45 min and 4 hr of preservation with the LifePort Kidney Transporter 1.0 (Organ Recovery Systems, Chicago, IL) using KPS-1. Prepared samples underwent 1-D Proton-NMR spectroscopy, and resultant spectra were analyzed. Clinical parameters were collected prospectively., Results: Perfusate of 26 transplanted cadaveric kidneys was analyzed; 19(73%) with IGF and 7(27%) with DGF. Glucose concentrations were significantly lower in DGF kidneys compared to those with IGF at both 45 min (7.772 vs. 9.459 mM, P = 0.006) and 4 hr (8.202 vs. 10.235 mM, P = 0.003). Concentrations of inosine and leucine were significantly different between DGF and IGF kidneys at 45 min (0.002 vs. 0.013 mM, P = 0.009 and 0.011 vs. 0.006 mM, P = 0.036), and gluconate levels were also significantly different between DGF and IGF kidneys at 4 hr (49.099 vs. 59.513 mM, P = 0.009)., Conclusion: Significant metabolic activity may be occurring in kidneys during HMP. The NMR spectroscopy of the perfusate can identify differences in the metabolomic profiles of DGF and IGF kidneys that might have a predictive role in viability assessment. Modification of harmful metabolic processes may improve outcomes for HMP kidneys.

Introduction: Hypothermic machine perfusion offers great promise in kidney transplantation and experimental studies are needed to establish the optimal conditions for this to occur. Pig kidneys are considered to be a good model for this purpose and share many properties with human organs. However it is not established whether the metabolism of pig kidneys in such hypothermic hypoxic conditions is comparable to human organs., Methods: Standard criteria human (n = 12) and porcine (n = 10) kidneys underwent HMP using the LifePort Kidney Transporter 1.0 (Organ Recovery Systems) using KPS-1 solution. Perfusate was sampled at 45 minutes and 4 hours of perfusion and metabolomic analysis performed using 1-D 1H-NMR spectroscopy., Results: There was no inter-species difference in the number of metabolites identified. Of the 30 metabolites analysed, 16 (53.3%) were present in comparable concentrations in the pig and human kidney perfusates. The rate of change of concentration for 3-Hydroxybutyrate was greater for human kidneys (p<0.001). For the other 29 metabolites (96.7%), there was no difference in the rate of change of concentration between pig and human samples., Conclusions: Whilst there are some differences between pig and human kidneys during HMP they appear to be metabolically similar and the pig seems to be a valid model for human studies.

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.