Publications by authors named "Daniel A Tennant"

44 Publications

Proline metabolism and redox; maintaining a balance in health and disease.

Amino Acids 2021 Jul 22. Epub 2021 Jul 22.

Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK.

Proline is a non-essential amino acid with key roles in protein structure/function and maintenance of cellular redox homeostasis. It is available from dietary sources, generated de novo within cells, and released from protein structures; a noteworthy source being collagen. Its catabolism within cells can generate ATP and reactive oxygen species (ROS). Recent findings suggest that proline biosynthesis and catabolism are essential processes in disease; not only due to the role in new protein synthesis as part of pathogenic processes but also due to the impact of proline metabolism on the wider metabolic network through its significant role in redox homeostasis. This is particularly clear in cancer proliferation and metastatic outgrowth. Nevertheless, the precise identity of the drivers of cellular proline catabolism and biosynthesis, and the overall cost of maintaining appropriate balance is not currently known. In this review, we explore the major drivers of proline availability and consumption at a local and systemic level with a focus on cancer. Unraveling the main factors influencing proline metabolism in normal physiology and disease will shed light on new effective treatment strategies.
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http://dx.doi.org/10.1007/s00726-021-03051-2DOI Listing
July 2021

Prolyl-4-hydroxylase 3 maintains β-cell glucose metabolism during fatty acid excess in mice.

JCI Insight 2021 Jul 15. Epub 2021 Jul 15.

Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom.

The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a Hypoxia-Inducible Factor (HIF) target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. While PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about effects of this highly conserved enzyme in insulin-secreting β-cells in vivo. Here, we show that deletion of PHD3 specifically in β-cells (βPHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in βPHD3KO islets is associated with impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes and insulin secretion. Thus, PHD3 might be a pivotal component of the β-cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.
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http://dx.doi.org/10.1172/jci.insight.140288DOI Listing
July 2021

Loss of SDHB Promotes Dysregulated Iron Homeostasis, Oxidative Stress, and Sensitivity to Ascorbate.

Cancer Res 2021 Jul 14;81(13):3480-3494. Epub 2021 Jun 14.

PARCC, INSERM UMR970, Equipe Labellisée par la Ligue Contre le Cancer, Paris, France.

Succinate dehydrogenase is a key enzyme in the tricarboxylic acid cycle and the electron transport chain. All four subunits of succinate dehydrogenase are tumor suppressor genes predisposing to paraganglioma, but only mutations in the SDHB subunit are associated with increased risk of metastasis. Here we generated an knockout chromaffin cell line and compared it with deficient cells. Both cell types exhibited similar SDH loss of function, metabolic adaptation, and succinate accumulation. In contrast, cells showed hallmarks of mesenchymal transition associated with increased DNA hypermethylation and a stronger pseudo-hypoxic phenotype compared with cells. Loss of SDHB specifically led to increased oxidative stress associated with dysregulated iron and copper homeostasis in the absence of NRF2 activation. High-dose ascorbate exacerbated the increase in mitochondrial reactive oxygen species, leading to cell death in cells. These data establish a mechanism linking oxidative stress to iron homeostasis that specifically occurs in -deficient cells and may promote metastasis. They also highlight high-dose ascorbate as a promising therapeutic strategy for SDHB-related cancers. SIGNIFICANCE: Loss of different succinate dehydrogenase subunits can lead to different cell and tumor phenotypes, linking stronger 2-OG-dependent dioxygenases inhibition, iron overload, and ROS accumulation following SDHB mutation.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2936DOI Listing
July 2021

Metabolic adaptations to hypoxia in the neonatal mouse forebrain can occur independently of the transporters SLC7A5 and SLC3A2.

Sci Rep 2021 Apr 27;11(1):9092. Epub 2021 Apr 27.

University/British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK.

Neonatal encephalopathy due to hypoxia-ischemia is associated with adverse neurodevelopmental effects. The involvement of branched chain amino acids (BCAAs) in this is largely unexplored. Transport of BCAAs at the plasma membrane is facilitated by SLC7A5/SLC3A2, which increase with hypoxia. We hypothesized that hypoxia would alter BCAA transport and metabolism in the neonatal brain. We investigated this using an organotypic forebrain slice culture model with, the SLC7A5/SLC3A2 inhibitor, 2-Amino-2-norbornanecarboxylic acid (BCH) under normoxic or hypoxic conditions. We subsequently analysed the metabolome and candidate gene expression. Hypoxia was associated with increased expression of SLC7A5 and SLC3A2 and an increased tissue abundance of BCAAs. Incubation of slices with C-leucine confirmed that this was due to increased cellular uptake. BCH had little effect on metabolite abundance under normoxic or hypoxic conditions. This suggests hypoxia drives increased cellular uptake of BCAAs in the neonatal mouse forebrain, and membrane mediated transport through SLC7A5 and SLC3A2 is not essential for this process. This indicates mechanisms exist to generate the compounds required to maintain essential metabolism in the absence of external nutrient supply. Moreover, excess BCAAs have been associated with developmental delay, providing an unexplored mechanism of hypoxia mediated pathogenesis in the developing forebrain.
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http://dx.doi.org/10.1038/s41598-021-88757-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079390PMC
April 2021

Systemic and adipocyte transcriptional and metabolic dysregulation in idiopathic intracranial hypertension.

JCI Insight 2021 May 24;6(10). Epub 2021 May 24.

Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

BACKGROUNDIdiopathic intracranial hypertension (IIH) is a condition predominantly affecting obese women of reproductive age. Recent evidence suggests that IIH is a disease of metabolic dysregulation, androgen excess, and an increased risk of cardiovascular morbidity. Here we evaluate systemic and adipose specific metabolic determinants of the IIH phenotype.METHODSIn fasted, matched IIH (n = 97) and control (n = 43) patients, we assessed glucose and insulin homeostasis and leptin levels. Body composition was assessed along with an interrogation of adipose tissue function via nuclear magnetic resonance metabolomics and RNA sequencing in paired omental and subcutaneous biopsies in a case-control study.RESULTSWe demonstrate an insulin- and leptin-resistant phenotype in IIH in excess of that driven by obesity. Adiposity in IIH is preferentially centripetal and is associated with increased disease activity and insulin resistance. IIH adipocytes appear transcriptionally and metabolically primed toward depot-specific lipogenesis.CONCLUSIONThese data show that IIH is a metabolic disorder in which adipose tissue dysfunction is a feature of the disease. Managing IIH as a metabolic disease could reduce disease morbidity and improve cardiovascular outcomes.FUNDINGThis study was supported by the UK NIHR (NIHR-CS-011-028), the UK Medical Research Council (MR/K015184/1), Diabetes UK, Wellcome Trust (104612/Z/14/Z), the Sir Jules Thorn Award, and the Midlands Neuroscience Teaching and Research Fund.
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http://dx.doi.org/10.1172/jci.insight.145346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262372PMC
May 2021

Tolerogenic effects of 1,25-dihydroxyvitamin D on dendritic cells involve induction of fatty acid synthesis.

J Steroid Biochem Mol Biol 2021 Jul 27;211:105891. Epub 2021 Mar 27.

Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, B15 2TT, United Kingdom. Electronic address:

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of immune function, promoting anti-inflammatory, tolerogenic T cell responses by modulating antigen presentation by dendritic cells (DC). Transcriptomic analyses indicate that DC responses to 1,25D involve changes in glycolysis, oxidative phosphorylation, electron transport and the TCA cycle. To determine the functional impact of 1,25D-mediated metabolic remodelling, human monocyte-derived DC were differentiated to immature (+vehicle, iDC), mature (+LPS, mDC), and immature tolerogenic DC (+1,25D, itolDC) and characterised for metabolic function. In contrast to mDC which showed no change in respiration, itolDC showed increased basal and ATP-linked respiration relative to iDC. Tracer metabolite analyses using C -labeled glucose showed increased lactate and TCA cycle metabolites. Analysis of lipophilic metabolites of C-glucose revealed significant incorporation of label in palmitate and palmitoleate, indicating that 1,25D promotes metabolic fatty acid synthesis in itolDC. Inhibition of fatty acid synthesis in itolDC altered itolDC morphology and suppressed expression of CD14 and IL-10 by these cells. These data indicate that the ability of 1,25D to induce tolerogenic DC involves metabolic remodelling leading to synthesis of fatty acids.
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http://dx.doi.org/10.1016/j.jsbmb.2021.105891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223499PMC
July 2021

A human pluripotent stem cell model for the analysis of metabolic dysfunction in hepatic steatosis.

iScience 2021 Jan 11;24(1):101931. Epub 2020 Dec 11.

University/BHF Centre for Cardiovascular Science, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh BioQuarter, 47 Little France Crescent, Edinburgh EH16 4TJ, UK.

Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term encompasses a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis, which have, to date, been challenging to model in the laboratory setting. Here, we present a human pluripotent stem cell (hPSC)-derived model of hepatic steatosis, which overcomes inherent challenges of current models and provides insights into the metabolic rewiring associated with steatosis. Following induction of macrovesicular steatosis in hepatocyte-like cells using lactate, pyruvate, and octanoate (LPO), respirometry and transcriptomic analyses revealed compromised electron transport chain activity. C isotopic tracing studies revealed enhanced TCA cycle anaplerosis, with concomitant development of a compensatory purine nucleotide cycle shunt leading to excess generation of fumarate. This model of hepatic steatosis is reproducible, scalable, and overcomes the challenges of studying mitochondrial metabolism in currently available models.
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http://dx.doi.org/10.1016/j.isci.2020.101931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773967PMC
January 2021

Intracellular sodium elevation reprograms cardiac metabolism.

Nat Commun 2020 08 28;11(1):4337. Epub 2020 Aug 28.

School of Cardiovascular and Medical Sciences, British Heart Foundation Centre of Research Excellence, King's College London, The Rayne Institute, St Thomas' Hospital, London, UK.

Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 μM ouabain 100 nM blebbistatin) or chronic myocardial Na load (PLM mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic 'fingerprint'. Control (PLM), transgenic (PLM), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by Na, P, C NMR followed by H-NMR metabolomic profiling. Elevated Na leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Na overload or inhibition of Na/Ca may be a new approach to ameliorate metabolic dysregulation in heart failure.
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http://dx.doi.org/10.1038/s41467-020-18160-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455741PMC
August 2020

Induction of the nicotinamide riboside kinase NAD salvage pathway in a model of sarcoplasmic reticulum dysfunction.

Skelet Muscle 2020 02 19;10(1). Epub 2020 Feb 19.

Institute of Metabolism and Systems Research, University of Birmingham, 2nd Floor IBR Tower, Edgbaston, Birmingham, B15 2TT, UK.

Background: Hexose-6-Phosphate Dehydrogenase (H6PD) is a generator of NADPH in the Endoplasmic/Sarcoplasmic Reticulum (ER/SR). Interaction of H6PD with 11β-hydroxysteroid dehydrogenase type 1 provides NADPH to support oxo-reduction of inactive to active glucocorticoids, but the wider understanding of H6PD in ER/SR NAD(P)(H) homeostasis is incomplete. Lack of H6PD results in a deteriorating skeletal myopathy, altered glucose homeostasis, ER stress and activation of the unfolded protein response. Here we further assess muscle responses to H6PD deficiency to delineate pathways that may underpin myopathy and link SR redox status to muscle wide metabolic adaptation.

Methods: We analysed skeletal muscle from H6PD knockout (H6PDKO), H6PD and NRK2 double knockout (DKO) and wild-type (WT) mice. H6PDKO mice were supplemented with the NAD precursor nicotinamide riboside. Skeletal muscle samples were subjected to biochemical analysis including NAD(H) measurement, LC-MS based metabolomics, Western blotting, and high resolution mitochondrial respirometry. Genetic and supplement models were assessed for degree of myopathy compared to H6PDKO.

Results: H6PDKO skeletal muscle showed adaptations in the routes regulating nicotinamide and NAD biosynthesis, with significant activation of the Nicotinamide Riboside Kinase 2 (NRK2) pathway. Associated with changes in NAD biosynthesis, H6PDKO muscle had impaired mitochondrial respiratory capacity with altered mitochondrial acylcarnitine and acetyl-CoA metabolism. Boosting NAD levels through the NRK2 pathway using the precursor nicotinamide riboside elevated NAD/NADH but had no effect to mitigate ER stress and dysfunctional mitochondrial respiratory capacity or acetyl-CoA metabolism. Similarly, H6PDKO/NRK2 double KO mice did not display an exaggerated timing or severity of myopathy or overt change in mitochondrial metabolism despite depression of NAD availability.

Conclusions: These findings suggest a complex metabolic response to changes in muscle SR NADP(H) redox status that result in impaired mitochondrial energy metabolism and activation of cellular NAD salvage pathways. It is possible that SR can sense and signal perturbation in NAD(P)(H) that cannot be rectified in the absence of H6PD. Whether NRK2 pathway activation is a direct response to changes in SR NAD(P)(H) availability or adaptation to deficits in metabolic energy availability remains to be resolved.
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http://dx.doi.org/10.1186/s13395-019-0216-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031948PMC
February 2020

Succinate dehydrogenase deficiency in a chromaffin cell model retains metabolic fitness through the maintenance of mitochondrial NADH oxidoreductase function.

FASEB J 2020 01 22;34(1):303-315. Epub 2019 Nov 22.

Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Mutations in succinate dehydrogenase (SDH) lead to the development of tumors in a restricted subset of cell types, including chromaffin cells and paraganglia. The molecular basis for this specificity is currently unknown. We show that loss of SDH activity in a chromaffin cell model does not perturb complex I function, retaining the ability to oxidize NADH within the electron transport chain. This activity supports continued oxidation of substrates within the tricarboxylic acid (TCA) cycle. However, due to the block in the TCA cycle at SDH, the high glutamine oxidation activity is only maintained through an efflux of succinate. We also show that although the mitochondria of SDH-deficient cells are less active per se, their higher mass per cell results in an overall respiratory rate that is comparable with wild-type cells. Finally, we observed that when their mitochondria are uncoupled, SDH-deficient cells are unable to preserve their viability, suggesting that the mitochondrial metabolic network is unable to compensate when exposed to additional stress. We therefore show that in contrast to models of SDH deficiency based on epithelial cells, a chromaffin cell model retains aspects of metabolic "health," which could form the basis of cell specificity of this rare tumor type.
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http://dx.doi.org/10.1096/fj.201901456RDOI Listing
January 2020

New aspects of amino acid metabolism in cancer.

Br J Cancer 2020 01 10;122(2):150-156. Epub 2019 Dec 10.

Institute of Metabolism and Systems Research, University of Birmingham, Edgbaston, Birmingham, UK.

An abundant supply of amino acids is important for cancers to sustain their proliferative drive. Alongside their direct role as substrates for protein synthesis, they can have roles in energy generation, driving the synthesis of nucleosides and maintenance of cellular redox homoeostasis. As cancer cells exist within a complex and often nutrient-poor microenvironment, they sometimes exist as part of a metabolic community, forming relationships that can be both symbiotic and parasitic. Indeed, this is particularly evident in cancers that are auxotrophic for particular amino acids. This review discusses the stromal/cancer cell relationship, by using examples to illustrate a number of different ways in which cancer cells can rely on and contribute to their microenvironment - both as a stable network and in response to therapy. In addition, it examines situations when amino acid synthesis is driven through metabolic coupling to other reactions, and synthesis is in excess of the cancer cell's proliferative demand. Finally, it highlights the understudied area of non-proteinogenic amino acids in cancer metabolism and their potential role.
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http://dx.doi.org/10.1038/s41416-019-0620-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052246PMC
January 2020

Metabolic tracing reveals novel adaptations to skeletal muscle cell energy production pathways in response to NAD depletion.

Wellcome Open Res 2018 17;3:147. Epub 2019 Sep 17.

Institute of Metabolism and Systems Research, University of Birmingham, UK, Birmingham, B15 2TT, UK.

Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD homeostasis on these pathways is unknown. We utilised skeletal muscle myotube models to induce NAD depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U- C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. NAD excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells resulted in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity was increased in response to low NAD , which was rapidly reversed with repletion of the NAD pool using NR. NAD depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reversed these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD depletion remains unclear, and requires further investigation. These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD precursor usage in models of skeletal muscle physiology.
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http://dx.doi.org/10.12688/wellcomeopenres.14898.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305244PMC
September 2019

Nicotinamide Riboside Augments the Aged Human Skeletal Muscle NAD Metabolome and Induces Transcriptomic and Anti-inflammatory Signatures.

Cell Rep 2019 08;28(7):1717-1728.e6

Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, UK; Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK; MRC-Arthritis Research UK Centre for Musculoskeletal Ageing Research, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK. Electronic address:

Nicotinamide adenine dinucleotide (NAD) is modulated by conditions of metabolic stress and has been reported to decline with aging in preclinical models, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD metabolome and if it can alter muscle mitochondrial bioenergetics. We supplemented 12 aged men with 1 g NR per day for 21 days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways, without altering mitochondrial bioenergetics. NR also depressed levels of circulating inflammatory cytokines. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR.
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http://dx.doi.org/10.1016/j.celrep.2019.07.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702140PMC
August 2019

Ex vivo metabolite profiling of paediatric central nervous system tumours reveals prognostic markers.

Sci Rep 2019 07 19;9(1):10473. Epub 2019 Jul 19.

Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom.

Brain tumours are the most common cause of cancer death in children. Molecular studies have greatly improved our understanding of these tumours but tumour metabolism is underexplored. Metabolites measured in vivo have been reported as prognostic biomarkers of these tumours but analysis of surgically resected tumour tissue allows a more extensive set of metabolites to be measured aiding biomarker discovery and providing validation of in vivo findings. In this study, metabolites were quantified across a range of paediatric brain tumours using H-High-Resolution Magic Angle Spinning nuclear magnetic resonance spectroscopy (HR-MAS) and their prognostic potential investigated. HR-MAS was performed on pre-treatment frozen tumour tissue from a single centre. Univariate and multivariate Cox regression was used to examine the ability of metabolites to predict survival. The models were cross validated using C-indices and further validated by splitting the cohort into two. Higher concentrations of glutamine were predictive of a longer overall survival, whilst higher concentrations of lipids were predictive of a shorter overall survival. These metabolites were predictive independent of diagnosis, as demonstrated in multivariate Cox regression models. Whilst accurate quantification of metabolites such as glutamine in vivo is challenging, metabolites show promise as prognostic markers due to development of optimised detection methods and increasing use of 3 T clinical scanners.
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http://dx.doi.org/10.1038/s41598-019-45900-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642141PMC
July 2019

Development and Validation of a Combined Hypoxia and Immune Prognostic Classifier for Head and Neck Cancer.

Clin Cancer Res 2019 09 10;25(17):5315-5328. Epub 2019 Jun 10.

Institute of Head and Neck Studies and Education, University of Birmingham, Birmingham, United Kingdom.

Purpose: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxia-immune classifier with potential application in patient prognostication and prediction of response to targeted therapy.

Experimental Design: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed using the The Cancer Genome Atlas (TCGA) HNC dataset ( = 275) and validated using two independent cohorts ( = 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining.

Results: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and survival profiles: hypoxia/immune, hypoxia/immune, and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively ( = 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxia/immune and hypoxia/immune tumors were overrepresented in "inflamed" and "immune-desert" microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3 T cells ( = -0.5464; = 0.0377), further corroborating the transcription-based classification.

Conclusions: We developed and validated a hypoxia-immune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3314DOI Listing
September 2019

Verteporfin selectively kills hypoxic glioma cells through iron-binding and increased production of reactive oxygen species.

Sci Rep 2018 09 25;8(1):14358. Epub 2018 Sep 25.

Institute of Metabolism and Systems Research, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

Gliomas are highly malignant brain tumours characterised by extensive areas of poor perfusion which subsequently leads to hypoxia and reduced survival. Therapies that address the hypoxic microenvironment are likely to significantly improve patient outcomes. Verteporfin, a benzoporphyrin-like drug, has been suggested to target the Yes-associated protein (YAP). Increased YAP expression and transcriptional activity has been proposed in other tumour types to promote malignant cell survival and thus YAP-inhibitor, verteporfin, may be predicted to impact glioma cell growth and viability. Due to the extensive hypoxic nature of gliomas, we investigated the effect of hypoxia on YAP expression and found that YAP transcription is increased under these conditions. Treatment of both primary and immortalised glioblastoma cell lines with verteporfin resulted in a significant decrease in viability but strikingly only under hypoxic conditions (1% O). We discovered that cell death occurs through a YAP-independent mechanism, predominately involving binding of free iron and likely through redox cycling, contributes to production of reactive oxygen species. This results in disruption of normal cellular processes and death in cells already under oxidative stress - such as those in hypoxia. We suggest that through repurposing verteporfin, it represents a novel means of treating highly therapy-resistant, hypoxic cells in glioma.
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http://dx.doi.org/10.1038/s41598-018-32727-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156578PMC
September 2018

Tissue metabolite profiles for the characterisation of paediatric cerebellar tumours.

Sci Rep 2018 08 10;8(1):11992. Epub 2018 Aug 10.

Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.

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.
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http://dx.doi.org/10.1038/s41598-018-30342-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086878PMC
August 2018

Oncogenic IDH1 Mutations Promote Enhanced Proline Synthesis through PYCR1 to Support the Maintenance of Mitochondrial Redox Homeostasis.

Cell Rep 2018 03;22(12):3107-3114

Institute of Metabolism and Systems Research, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Electronic address:

Since the discovery of mutations in isocitrate dehydrogenase 1 (IDH1) in gliomas and other tumors, significant efforts have been made to gain a deeper understanding of the consequences of this oncogenic mutation. One aspect of the neomorphic function of the IDH1 R132H enzyme that has received less attention is the perturbation of cellular redox homeostasis. Here, we describe a biosynthetic pathway exhibited by cells expressing mutant IDH1. By virtue of a change in cellular redox homeostasis, IDH1-mutated cells synthesize excess glutamine-derived proline through enhanced activity of pyrroline 5-carboxylate reductase 1 (PYCR1), coupled to NADH oxidation. Enhanced proline biosynthesis partially uncouples the electron transport chain from tricarboxylic acid (TCA) cycle activity through the maintenance of a lower NADH/NAD ratio and subsequent reduction in oxygen consumption. Thus, we have uncovered a mechanism by which tumor cell survival may be promoted in conditions associated with perturbed redox homeostasis, as occurs in IDH1-mutated glioma.
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http://dx.doi.org/10.1016/j.celrep.2018.02.084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5883319PMC
March 2018

High-Speed Tracer Analysis of Metabolism (HS-TrAM).

Wellcome Open Res 2018 12;3. Epub 2018 Jan 12.

Institute of Metabolism and Systems Research, University of Birmingham, West Midlands, UK.

Tracing the fate of stable isotopically-enriched nutrients is a sophisticated method of describing and quantifying the activity of metabolic pathways. Nuclear Magnetic Resonance (NMR) offers high resolution data, yet is under-utilised due to length of time required to collect the data, quantification requiring multiple samples and complicated analysis. Here we present two techniques, quantitative spectral filters and enhancement of the splitting due to J-coupling in H, C-HSQC NMR spectra, which allow the rapid collection of NMR data in a quantitative manner on a single sample. The reduced duration of HSQC spectra data acquisition opens up the possibility of real-time tracing of metabolism including the study of metabolic pathways . We show how these novel techniques can be used to trace the fate of labelled nutrients in a whole organ model of kidney preservation prior to transplantation using a porcine kidney as a model organ, and also show how the use of multiple nutrients, differentially labelled with C and N, can be used to provide additional information with which to profile metabolic pathways.
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http://dx.doi.org/10.12688/wellcomeopenres.13387.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811808PMC
January 2018

Metabolic implications of hypoxia and pseudohypoxia in pheochromocytoma and paraganglioma.

Cell Tissue Res 2018 05 15;372(2):367-378. Epub 2018 Feb 15.

Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

Hypoxia is a critical driver of cancer pathogenesis, directly inducing malignant phenotypes such as epithelial-mesenchymal transition, stem cell-like characteristics and metabolic transformation. However, hypoxia-associated phenotypes are often observed in cancer in the absence of hypoxia, a phenotype known as pseudohypoxia, which is very well documented in specific tumour types, including in paraganglioma/pheochromocytoma (PPGL). Approximately 40% of the PPGL tumours carry a germ line mutation in one of a number of susceptibility genes of which those that are found in succinate dehydrogenase (SDH) or in von Hippel-Lindau (VHL) genes manifest a strong pseudohypoxic phenotype. Mutations in SDH are oncogenic, forming tumours in a select subset of tissues, but the cause for this remains elusive. Although elevated succinate levels lead to increase in hypoxia-like signalling, there are other phenotypes that are being increasingly recognised in SDH-mutated PPGL, such as DNA hypermethylation. Further, recently unveiled changes in metabolic re-wiring of SDH-deficient cells might help to decipher cancer related roles of SDH in the future. In this review, we will discuss the various implications that the malfunctioning SDH can have and its impact on cancer development.
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http://dx.doi.org/10.1007/s00441-018-2801-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915505PMC
May 2018

Combined Analysis of NMR and MS Spectra (CANMS).

Angew Chem Int Ed Engl 2017 04 8;56(15):4140-4144. Epub 2017 Mar 8.

Institute of Metabolism and Systems Research, University of Birmingham, IBR West Tower, Birmingham, B15 2TT, UK.

Cellular metabolism in mammalian cells represents a challenge for analytical chemistry in the context of current biomedical research. Mass spectrometry and NMR spectroscopy together with computational tools have been used to study metabolism in cells. Compartmentalization of metabolism complicates the interpretation of stable isotope patterns in mammalian cells owing to the superimposition of different pathways contributing to the same pool of analytes. This indicates a need for a model-free approach to interpret such data. Mass spectrometry and NMR spectroscopy provide complementary analytical information on metabolites. Herein an approach that simulates C multiplets in NMR spectra and utilizes mass increments to obtain long-range information is presented. The combined information is then utilized to derive isotopomer distributions. This is a first rigorous analytical and computational approach for a model-free analysis of metabolic data applicable to mammalian cells.
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http://dx.doi.org/10.1002/anie.201611634DOI Listing
April 2017

Metabolic differences between cold stored and machine perfused porcine kidneys: A H NMR based study.

Cryobiology 2017 02 3;74:115-120. Epub 2016 Dec 3.

Department of Renal Surgery, University Hospitals Birmingham, Birmingham, UK.

Hypothermic machine perfusion (HMP) and static cold storage (SCS) are the two methods used to preserve deceased donor kidneys prior to transplant. This study seeks to characterise the metabolic profile of HMP and SCS porcine kidneys in a cardiac death donor model. Twenty kidneys were cold flushed and stored for two hours following retrieval. Paired kidneys then underwent 24 h of HMP or SCS or served as time zero controls. Metabolite quantification in both storage fluid and kidney tissue was performed using one dimensional H NMR spectroscopy. For each metabolite, the net gain for each storage modality was determined by comparing the total amount in each closed system (i.e. total amount in storage fluid and kidney combined) compared with controls. 26 metabolites were included for analysis. Total system metabolite quantities following HMP or SCS were greater for 14 compared with controls (all p < 0.05). In addition to metabolic differences with control kidneys, the net metabolic gain during HMP was greater than SCS for 8 metabolites (all p < 0.05). These included metabolites related to central metabolism (lactate, glutamate, aspartate, fumarate and acetate). The metabolic environments of both perfusion fluid and the kidney tissue are strikingly different between SCS and HMP systems in this animal model. The total amount of central metabolites such as lactate and glutamate observed in the HMP kidney system suggests a greater degree of de novo metabolic activity than in the SCS system. Maintenance of central metabolic pathways may contribute to the clinical benefits of HMP.
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http://dx.doi.org/10.1016/j.cryobiol.2016.11.006DOI Listing
February 2017

Isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), fumarate hydratase (FH): three players for one phenotype in cancer?

Biochem Soc Trans 2016 08;44(4):1111-6

Institute of Metabolism and Systems Research, University of Birmingham, Birmingham B15 2TT, U.K.

In the early 1920s Otto Warburg observed that cancer cells have altered metabolism and from this, posited that mitochondrial dysfunction underpinned the aetiology of cancers. The more recent identification of mutations of mitochondrial metabolic enzymes in a wide range of human cancers has now provided a direct link between metabolic alterations and cancer. In this review we discuss the consequences of dysfunction of three metabolic enzymes involved in or associated with the tricarboxylic acid (TCA) cycle: succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) focusing on the similarity between the phenotypes of cancers harbouring these mutations.
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http://dx.doi.org/10.1042/BST20160099DOI Listing
August 2016

(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.

Transplant Res 2016 5;5. Epub 2016 Aug 5.

Institute of Metabolism and Systems Research (IMSR), College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

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.
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http://dx.doi.org/10.1186/s13737-016-0037-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974776PMC
August 2016

Probing Cancer Cell Metabolism Using NMR Spectroscopy.

Adv Exp Med Biol 2016 ;899:89-111

Institute of Metabolism and Systems Research, University of Birmingham, IBR, Edgbaston, Birmingham, B15 2TT, UK.

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.
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http://dx.doi.org/10.1007/978-3-319-26666-4_6DOI Listing
June 2017

Mitochondrial metabolic remodeling in response to genetic and environmental perturbations.

Wiley Interdiscip Rev Syst Biol Med 2016 07 19;8(4):272-85. Epub 2016 May 19.

Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Mitochondria are metabolic hubs within mammalian cells and demonstrate significant metabolic plasticity. In oxygenated environments with ample carbohydrate, amino acid, and lipid sources, they are able to use the tricarboxylic acid cycle for the production of anabolic metabolites and ATP. However, in conditions where oxygen becomes limiting for oxidative phosphorylation, they can rapidly signal to increase cytosolic glycolytic ATP production, while awaiting hypoxia-induced changes in the proteome mediated by the activity of transcription factors such as hypoxia-inducible factor 1. Hypoxia is a well-described phenotype of most cancers, driving many aspects of malignancy. Improving our understanding of how mitochondria change their metabolism in response to this stimulus may therefore elicit the design of new selective therapies. Many of the recent advances in our understanding of mitochondrial metabolic plasticity have been acquired through investigations of cancer-associated mutations in metabolic enzymes, including succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase. This review will describe how metabolic perturbations induced by hypoxia and mutations in these enzymes have informed our knowledge in the control of mitochondrial metabolism, and will examine what this may mean for the biology of the cancers in which these mutations are observed. WIREs Syst Biol Med 2016, 8:272-285. doi: 10.1002/wsbm.1334 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wsbm.1334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982039PMC
July 2016

Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism.

Nat Commun 2015 Nov 2;6:8784. Epub 2015 Nov 2.

Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.

The tricarboxylic acid (TCA) cycle is a central metabolic pathway responsible for supplying reducing potential for oxidative phosphorylation and anabolic substrates for cell growth, repair and proliferation. As such it thought to be essential for cell proliferation and tissue homeostasis. However, since the initial report of an inactivating mutation in the TCA cycle enzyme complex, succinate dehydrogenase (SDH) in paraganglioma (PGL), it has become clear that some cells and tissues are not only able to survive with a truncated TCA cycle, but that they are also able of supporting proliferative phenotype observed in tumours. Here, we show that loss of SDH activity leads to changes in the metabolism of non-essential amino acids. In particular, we demonstrate that pyruvate carboxylase is essential to re-supply the depleted pool of aspartate in SDH-deficient cells. Our results demonstrate that the loss of SDH reduces the metabolic plasticity of cells, suggesting vulnerabilities that can be targeted therapeutically.
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http://dx.doi.org/10.1038/ncomms9784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632646PMC
November 2015

A roadmap for interpreting (13)C metabolite labeling patterns from cells.

Curr Opin Biotechnol 2015 Aug 28;34:189-201. Epub 2015 Feb 28.

School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK.

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments.
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http://dx.doi.org/10.1016/j.copbio.2015.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552607PMC
August 2015

Metabolomic perfusate analysis during kidney machine perfusion: the pig provides an appropriate model for human studies.

PLoS One 2014 12;9(12):e114818. Epub 2014 Dec 12.

Department of Renal Surgery, University Hospitals Birmingham, Birmingham, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114818PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4264773PMC
January 2016

Metabolomic analysis of perfusate during hypothermic machine perfusion of human cadaveric kidneys.

Transplantation 2015 Apr;99(4):754-9

1 Department of Renal Surgery, University Hospitals Birmingham, Birmingham, United Kingdom. 2 Department of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom. 3 Biomolecular NMR Spectroscopy, University of Birmingham, Birmingham, United Kingdom. 4 School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom.

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.
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http://dx.doi.org/10.1097/TP.0000000000000398DOI Listing
April 2015
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