Analysis of 13 C and 14 C labeling in pyruvate and lactate in tumor and blood of lymphoma-bearing mice injected with 13 C- and 14 C-labeled pyruvate.

Measurements of hyperpolarized 13 C label exchange between injected [1-13 C]pyruvate and the endogenous tumor lactate pool can give an apparent first-order rate constant for the exchange. The determination of the isotope flux, however, requires an estimate of the labeled pyruvate concentration in the tumor. This was achieved here by measurement of the tumor uptake of [1-14 C]pyruvate, which showed that <2% of the injected pyruvate reached the tumor site. Multiplication of this estimated labeled pyruvate concentration in the tumor with the apparent first-order rate constant for hyperpolarized 13 C label exchange gave an isotope flux that showed good agreement with a flux determined directly by the injection of non-polarized [3-13 C]pyruvate, rapid excision of the tumor after 30 s and measurement of 13 C-labeled lactate concentrations in tumor extracts. The distribution of labeled lactate between intra- and extracellular compartments and the blood pool was investigated by imaging, by measurement of the labeled lactate concentration in blood and tumor, and by examination of the effects of a gadolinium contrast agent and a lactate transport inhibitor on the intensity of the hyperpolarized [1-13 C]lactate signal. These measurements showed that there was significant export of labeled lactate from the tumor, but that labeled lactate in the blood pool produced by the injection of hyperpolarized [1-13 C]pyruvate showed only relatively low levels of polarization. This study shows that measurements of hyperpolarized 13 C label exchange between pyruvate and lactate in a murine tumor model can provide an estimate of the true isotope flux if the concentration of labeled pyruvate that reaches the tumor can be determined.

Abstracts of the 24th international isotope society (UK group) symposium: synthesis and applications of labelled compounds 2015.

The 24th annual symposium of the International Isotope Society's United Kingdom Group took place at the Møller Centre, Churchill College, Cambridge, UK on Friday 6th November 2015. The meeting was attended by 77 delegates from academia and industry, the life sciences, chemical, radiochemical and scientific instrument suppliers. Delegates were welcomed by Dr Ken Lawrie (GlaxoSmithKline, UK, chair of the IIS UK group). The subsequent scientific programme consisted of oral presentations, short 'flash' presentations in association with particular posters and poster presentations. The scientific areas covered included isotopic synthesis, regulatory issues, applications of labelled compounds in imaging, isotopic separation and novel chemistry with potential implications for isotopic synthesis. Both short-lived and long-lived isotopes were represented, as were stable isotopes. The symposium was divided into a morning session chaired by Dr Rebekka Hueting (University of Oxford, UK) and afternoon sessions chaired by Dr Sofia Pascu (University of Bath, UK) and by Dr Alan Dowling (Syngenta, UK). The UK meeting concluded with remarks from Dr Ken Lawrie (GlaxoSmithKline, UK).

Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent.

The C2 domain of synaptotagmin I, which binds to anionic phospholipids in cell membranes, was shown to bind to the plasma membrane of apoptotic cells by both flow cytometry and confocal microscopy. Conjugation of the protein to superparamagnetic iron oxide nanoparticles allowed detection of this binding using magnetic resonance imaging. Detection of apoptotic cells, using this novel contrast agent, was demonstrated both in vitro, with isolated apoptotic tumor cells, and in vivo, in a tumor treated with chemotherapeutic drugs.

Detecting treatment response in a model of human breast adenocarcinoma using hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate.

BACKGROUND: The recent introduction of a dynamic nuclear polarisation technique has permitted noninvasive imaging of tumour cell metabolism in vivo following intravenous administration of (13)C-labelled cell substrates. METHODS: Changes in hyperpolarised [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate metabolism were evaluated in both MDA-MB-231 cells and in implanted MDA-MB-231 tumours following doxorubicin treatment. RESULTS: Treatment of MDA-MB-231 cells resulted in the induction of apoptosis, which was accompanied by a decrease in hyperpolarised (13)C label flux between [1-(13)C]pyruvate and lactate, which was correlated with a decrease in the cellular NAD(H) coenzyme pool. There was also an increase in the rate of fumarate conversion to malate, which accompanied the onset of cellular necrosis. In vivo, the decrease in (13)C label exchange between pyruvate and lactate and the increased flux between fumarate and malate, following drug treatment, were shown to occur in the absence of any detectable change in tumour size. CONCLUSION: We show here that the early responses of a human breast adenocarcinoma tumour model to drug treatment can be followed by administration of both hyperpolarised [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate. These techniques could be used, therefore, in the clinic to detect the early responses of breast tumours to treatment.

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations.

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are "silent, " that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing "metabolic snapshots," can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY-an abbreviation for functional analysis by co-responses in yeast.

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 is essential for p53-null cancer cells.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4) controls metabolic flux through allosteric regulation of glycolysis. Here we show that p53 regulates the expression of PFKFB4 and that p53-deficient cancer cells are highly dependent on the function of this enzyme. We found that p53 downregulates PFKFB4 expression by binding to its promoter and mediating transcriptional repression via histone deacetylases. Depletion of PFKFB4 from p53-deficient cancer cells increased levels of the allosteric regulator fructose-2,6-bisphosphate, leading to increased glycolytic activity but decreased routing of metabolites through the oxidative arm of the pentose-phosphate pathway. PFKFB4 was also required to support the synthesis and regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) in p53-deficient cancer cells. Moreover, depletion of PFKFB4-attenuated cellular biosynthetic activity and resulted in the accumulation of reactive oxygen species and cell death in the absence of p53. Finally, silencing of PFKFB4-induced apoptosis in p53-deficient cancer cells in vivo and interfered with tumour growth. These results demonstrate that PFKFB4 is essential to support anabolic metabolism in p53-deficient cancer cells and suggest that inhibition of PFKFB4 could be an effective strategy for cancer treatment.

The susceptibility of tumors to the antivascular drug combretastatin A4 phosphate correlates with vascular permeability.

The acute effects of the antivascular drug, combretastatin A4 phosphate, on tumor energy status and perfusion were assessed using magnetic resonance imaging (MRI) and spectroscopy. Localized (31)P magnetic resonance spectroscopy showed that LoVo and RIF-1 tumors responded well to drug treatment, with significant increases in the P(i)/nucleoside triphosphate ratio within 3 h, whereas SaS, SaF, and HT29 tumors did not respond to the same extent. This variable response was also seen in MRI experiments in which tumor perfusion was assessed by monitoring the kinetics of inflow of the contrast agent, gadolinium diethylenetriaminepentaacetate. These data were analyzed to give the initial rate and time constant for inflow of contrast agent and the integral under the inflow curve. The differential susceptibility of the tumors to combretastatin A4 phosphate showed a positive correlation with prior MRI measurements of tumor vascular permeability, which was determined by measuring the inflow of a macromolecular contrast agent, BSA-gadolinium diethylenetriaminepentaacetate.

31P-NMR saturation transfer measurements of exchange between Pi and ATP in the reactions catalysed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase in vitro.

Recent 31P-NMR saturation transfer measurements of flux between Pi and ATP in the perfused rat heart (Kingsley-Hickman, P., Sako, E.Y., Andreone, P.A., St. Cyr, J.A., Michurski, S., Foker, J.E., From, A.H.L., Petein, M. and Ugurbil, K. (1986) FEBS Lett. 198, 159-163) have given a P/O ratio (mols ATP synthesised/atoms oxygen consumed) which was close to 6. This anomalously high value was attributed to exchange in the reaction catalysed by the mitochondrial F1F0-ATP synthase. We show here that this exchange could also be catalysed by the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. 31P-NMR saturation transfer measurements of the exchange catalysed by these enzymes in vitro, under conditions designed to mimic those present in the perfused rat heart, have shown that they could catalyse a quantitatively significant Pi-ATP exchange in vivo. A three-site exchange model is used to investigate the effects of Pi-ATP exchange on saturation transfer measurements of the reverse flux in the creatine kinase reaction. A discrepancy in the measured and forward and reverse fluxes in this reaction has been attributed previously to the participation of the gamma-phosphate of ATP in other exchange reactions.

Measurements of exchange in the reaction catalysed by creatine kinase using 14C and 15N isotope labels and the NMR technique of saturation transfer.

31P-NMR measurements of saturation transfer have been used to measure exchange between the gamma-phosphate of ATP and phosphocreatine and between the beta-phosphate of ATP and the beta-phosphate of ADP in the reaction catalysed by creatine kinase in vitro. The calculated exchange fluxes have been compared with measurements of 15N label exchange between creatine and phosphocreatine and 14C label exchange between ATP and ADP. At pH 8.0 the fluxes between phosphocreatine and the gamma-phosphate of ATP and between the beta-phosphates of ATP and ADP, measured by saturation transfer, were the same and equal, within experimental error, to the fluxes between creatine and phosphocreatine, measured by 15N label exchange, and between ADP and ATP, measured by 14C label exchange. At pH 7.0 the flux between phosphocreatine and the gamma-phosphate of ATP, measured by saturation transfer, was equal, within experimental error, to the flux between creatine and phosphocreatine, measured by 15N label exchange. However, at low ADP concentrations (less than 0.2 mM), the flux between ATP and ADP measured by saturation transfer was significantly less than that between phosphocreatine and ATP and, more importantly, less than the ADP-ATP exchange flux measured by 14C label exchange. The saturation transfer and isotope exchange measurements at pH 7.0 have shown that it is valid to equate saturation transfer measurements of exchange between phosphocreatine and ATP in vivo with the potential for net chemical flux through the reaction. The observed discrepancy at pH 7.0 between the 14C and saturation transfer measurements of ATP----ADP exchange can be explained if there is significant loss of saturation in an intermediate in the exchange reaction. Under these conditions analysis of the exchange according to two-site exchange model is invalid. In magnetisation transfer measurements of exchange in other enzyme catalysed reactions, the possible presence of a kinetically significant intermediate and therefore the validity of data analysis using a two-site exchange model should be considered.

A comparison of 31P-NMR saturation transfer and isotope-exchange measurements of creatine kinase kinetics in vitro.

31P-NMR measurements of saturation transfer have been used to measure the exchange of phosphate between phosphocreatine and the gamma-phosphate of ATP in the reaction catalysed by creatine kinase in vitro. The similarity of the calculated exchange flux with the flux estimated from an isotope-exchange experiment, in which exchange of 15N label between creatine and phosphocreatine was measured, showed that the two-site-exchange model, normally used in the analysis of saturation-transfer data, is valid in this case. 15N label exchange was monitored using a heteronuclear 31P/15N spin-echo NMR experiment in which the incorporation of 15N label into phosphocreatine was detected by following the phase modulation of the spin-spin coupled 31P resonance. The isotope-exchange experiment should prove to be useful in studies of creatine kinase in systems where the low concentration of the enzyme precludes saturation-transfer measurements, for example in muscle mitochondria preparations.

Centrifugal analysis of undiluted packed human erythrocyte lysates. Studies of the association of glyceraldehyde-phosphate dehydrogenase with the membrane fraction.

Concentrated human erythrocyte lysates (greater than 99% initial haematocrit) were subjected to high centrifugal fields. This caused the membrane fraction to separate from the cytoplasmic portion, due to the lower density of the former. Enzyme distribution data indicated that glyceraldehyde-phosphate dehydrogenase was predominantly in the cytoplasmic fraction.

Determination of buffering capacity of rat myocardium during ischemia.

To determine the buffering capacity of ischemic rat myocardium, lactate production was altered by glycogen depletion prior to total global ischemia. Lactate production was monitored by 1H-NMR spectroscopy in perfused rat hearts and determined by enzymatic assay of freeze-clamped tissue extracts. Intracellular pH was measured by 31P-NMR spectroscopy. The relationship between total lactate produced and pH varied considerably, depending on the final pH reached. At pH greater than 6.4 this relationship is linear with a total buffering capacity (delta lactate/delta pH) of 25 mumol H+/g wet weight per pH unit. At lower pH values (pH less than 6.4), the total buffering capacity increases progressively. Since ischemia is invariably accompanied by ATP and phosphocreatine (PCr) hydrolysis, the proton production/consumption during high-energy phosphate hydrolysis must be considered when evaluating the intrinsic buffering capacity of the myocardium against proton loads produced by lactate production from glucose and glycogen. Schemes are presented which allow an estimation of the contribution of ATP and PCr hydrolysis and the buffering by the CO2/HCO3- system during ischemia. At pH greater than 6.4, the majority (about 60%) of buffering is due to hydrolysis of adenosine triphosphate, phosphocreatine in the heart, and neutralization of sodium bicarbonate in the perfusate. At pH less than 6.4 an increasing proportion of cardiac buffering is from intrinsic cardiac buffers, most likely from intracellular proteins. After correction for these contributions to the observed total cardiac buffering capacity, the intrinsic buffering capacity of the myocardium can be accounted for by a high capacity (170 mumol/g wet weight) but low pKa (5.2) buffering system.

The inhibition of erythrocyte glyceraldehyde-3-phosphate dehydrogenase. In situ PMR studies.

The kinetics of inhibition of human erythrocyte glyceraldehyde-3-phosphate dehydrogenase by iodoacetate were studied in the intact cell and in vitro. The kinetics were determined using 1H-NMR to follow solvent exchange of 1H and 2H at the C-2 position of lactate. The exchange occurs via a series of enzyme-catalysed reactions, including that catalysed by glyceraldehyde-3-phosphate dehydrogenase. A direct assay with quenching of the inhibition was also used to check the results. Iodoacetate was shown to act as an active site-directed inhibitor of the dehydrogenase. The enzyme inhibition patterns, which are characterised by a binding step and a kinetic step, are similar in situ and in vitro. Membrane binding, however, was found to alter the inhibition pattern for the enzyme in vitro.

31P-NMR studies of glucose and glutamine metabolism in cultured mammalian cells.

31P-NMR measurements of the concentrations of phosphorus-containing metabolites in mammalian cells immobilised and perifused with glucose and glutamine as sole carbon source have shown that the intracellular Pi concentration is significantly higher in cells perifused with glutamine than with glucose. The data are consistent with the proposal that the rate of glutamine utilisation may be controlled by the activity of phosphate-activated glutaminase.

Spin ECHO proton NMR studies of the metabolism of malate and fumarate in human erythrocytes. Dependence on free NAD levels.

The NAD-dependent conversion of malate to lactate in human erythrocytes was studied by spin echo proton NMR. A pathway involving the decarboxylation of oxaloacetate catalysed by haemoglobin is proposed to account for the observed reaction. NADP-dependent reaction was negligible. The rate of the reaction was measured in intact erythrocytes under controlled conditions. This rate correlates with that obtained with lysates at 30 microM free NAD and that obtained with purified human erythrocyte enzymes at about 15 microM NAD. The total extractable NAD in the intact cells was 70-90 microM. Experiments with cells containing elevated NAD levels could be explained by a significant fraction of the NAD being weakly bound (Kd about 1 mM) to haemoglobin.

Uncoupling protein 2 from carp and zebrafish, ectothermic vertebrates.

Uncoupling protein 1 (UCP1) is of demonstrated importance in mammalian thermogenesis, and early hypotheses regarding the functions of the newly discovered UCP homologues, UCP2, UCP3 and others, have focused largely on their potential roles in thermogenesis. Here we report the amino acid sequences of two new UCPs from ectothermic vertebrates. UCPs from two fish species, the zebrafish (Danio rerio) and carp (Cyprinus carpio), were identified in expressed sequence tag databases at the European Molecular Biology Laboratory. cDNAs from a C. carpio 'peritoneal exudate cell' cDNA library and from a D. rerio 'day 0 fin regeneration' cDNA library were obtained and fully sequenced. Each cDNA encodes a 310 amino acid protein with an average 82% sequence identity to mammalian UCP2s. The fish UCP2s are about 70% identical to mammalian UCP3s, and 60% identical to mammalian UCP1s. Carp and zebrafish are ectotherms--they do not raise their body temperatures above ambient by producing excess heat. The presence of UCP2 in these fish thus suggests the protein may have function(s) not related to thermogenesis.

A multinuclear NMR study of 2,3-bisphosphoglycerate metabolism in the human erythrocyte.

Resonances from 13C, 31P and 1H have been detected simultaneously in suspensions of human erythrocytes using a modified NMR spectrometer equipped with a probe tuned to four different frequencies. The utility of multinuclear NMR in the study of cellular metabolism is demonstrated with an investigation of 13C label flux through the 2,3-bisphosphoglycerate bypass in human erythrocytes. In a single experiment, the respective contributions of this bypass and the pentose-phosphate shunt were found to be 27 and 10% of the total glycolytic rate.

Changes in phosphatidylethanolamine metabolism in regenerating rat liver as measured by 31P-NMR.

31P-NMR spectra of regenerating rat liver in vivo show increases in resonance intensities in the phosphomonoester (PME) region and decreases in the phosphodiester (PDE) region as early as 12 h post partial hepatectomy, which return to normal by 8 days. The compounds primarily responsible for these changes have been identified in perchloric acid extracts as the phosphomonoester phosphoethanolamine and the phosphodiester glycerophosphoethanolamine (GPE), indicating altered phosphatidylethanolamine metabolism. A corresponding increase in diacylglycerol (DAG) levels during regeneration indicates a possible role for a phosphatidylethanolamine-specific phospholipase C in cellular proliferation. These results suggest that changes in phospholipid metabolites previously associated with neoplastic tissue can also be induced by normal tissue undergoing rapid cellular proliferation. The spectral changes observed in the regenerating rat liver are similar to changes seen in spectra from the livers of human patients in several disease states, indicating that 31P-NMR may allow non-invasive study of cell turnover in liver disease.

31P-NMR saturation transfer studies of aerobic Escherichia coli cells.

31P-NMR measurements of saturation transfer have been used to measure the flux between Pi and ATP in Escherichia coli cells respiring on an endogenous carbon source. Measurements were made in the wild type and in cells genetically modified to give a 5-fold higher concentration of the F1F0-ATP synthase. The flux in the two cell types was not significantly different. This, together with studies using inhibitors specific for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase and the ATP synthase, suggests that the observed flux arises predominantly from glycolytic rather than ATP synthase activity. Although this conclusion is in disagreement with previous experiments on E. coli, it is in agreement with recent experiments on yeast.

Endoplasmic reticulum: the major contributor to the PDE peak in hepatic 31P-NMR spectra at low magnetic field strengths.

31P-NMR spectra of liver in vivo, subcellular fractions and model systems were acquired in order to characterise further the hepatic phosphodiester peak seen at low magnetic field strengths previously shown to be predominantly due to phospholipid bilayers. The data obtained in this study in vitro suggested that the phospholipid membranes of the endoplasmic reticulum provide the dominant contribution to this phosphodiester peak. Support for this hypothesis was provided by experiments on rats. Phenobarbitone, which is known to induce proliferation of the endoplasmic reticulum produced a considerable increase in intensity of the phosphodiester peak in liver spectra in vivo.