Non-Invasive Differentiation of M1 and M2 Activation in Macrophages Using Hyperpolarized 13C MRS of Pyruvate and DHA at 1.47 Tesla.
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ABSTRACT:
SUBMITTER: Qiao K
PROVIDER: S-EPMC8305442 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hyperpolarized (13)C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl-coenzyme A (acetyl-CoA). [1-(13)C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in (13)C-bicarbonate production after dichloroacetate (DCA) administration. With [1-(13)C]pyruvate, the (13)C label is released as (13 CO2 /(13)C-bicarbonate, and, hence, does not allow us to follow the fate of acetyl-CoA. Pyruvate labeled in the C2 position has been used to track the (13)C label into the TCA (tricarboxylic acid) cycle and measure [5-(13)C]glutamate as well as study changes in [1-(13)C]acetylcarnitine with DCA and dobutamine. This work investigates changes in the metabolic fate of acetyl-CoA in response to metabolic interventions of DCA-induced increased PDC flux in the fed and fasted state, and increased cardiac workload with dobutamine in vivo in rat heart at two different pyruvate doses. DCA led to a modest increase in the (13)C labeling of [5-(13)C]glutamate, and a considerable increase in [1-(13)C]acetylcarnitine and [1,3-(13)C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2-(13)C]lactate, [2-(13)C]alanine and [5-(13)C]glutamate. The change in glutamate with dobutamine was observed using a high pyruvate dose but not with a low dose. The relative changes in the different metabolic products provide information about the relationship between PDC-mediated oxidation of pyruvate and its subsequent incorporation into the TCA cycle compared with other metabolic pathways. Using a high dose of pyruvate may provide an improved ability to observe changes in glutamate.
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Project description:The differentiation of bacterial infection from other causes of inflammation is difficult in clinical practice and is critical where patient outcomes rely heavily on early interventions. In addition to physical exam and laboratory markers, several imaging modalities are frequently employed, but these techniques generally target the host immune response, rather than the living microorganisms themselves. Here, we describe a method to detect bacteria-specific metabolism using hyperpolarized (HP) 13C magnetic resonance spectroscopy. This technology allows visualization of the real-time conversion of enriched 13C substrates to their metabolic products, identified by their distinct chemical shifts. We have identified the rapid metabolism of HP [2-13C]pyruvate to [1-13C]acetate as a metabolic signature of common bacterial pathogens. We demonstrate this conversion in representative Gram-negative and Gram-positive bacteria, namely, Escherichia coli and Staphylococcus aureus, and its absence in key mammalian cell types. Furthermore, this conversion was successfully modulated in three mutant strains, corresponding to deletions of relevant enzymes.
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Project description:Renal cell carcinomas (RCC) are a heterogeneous group of tumors with a wide range of aggressiveness. Noninvasive methods to confidently predict the tumor biologic behavior and select appropriate treatment are lacking. Here, we investigate the dynamic metabolic flux in living RCC cells using hyperpolarized (13)C-pyruvate magnetic resonance spectroscopy (MRS) combined with a bioreactor platform and interrogated the biochemical basis of the MRS data with respect to cancer aggressiveness. RCC cells have significantly higher pyruvate-to-lactate flux than the normal renal tubule cells. Furthermore, a key feature distinguishing the localized from the metastatic RCC cells is the lactate efflux rate, mediated by the monocarboxylate transporter 4 (MCT4). The metastatic RCC cells have significantly higher MCT4 expression and corresponding higher lactate efflux, which is essential for maintaining a high rate of glycolysis. We show that such differential cellular transporter expression and associated metabolic phenotype can be noninvasively assessed via real-time monitoring of hyperpolarized (13)C-pyruvate-to-lactate flux.
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Project description:MRI using hyperpolarized (HP) carbon-13 pyruvate is being investigated in clinical trials to provide non-invasive measurements of metabolism for cancer and cardiac imaging. In this project, we applied HP [1-13 C]pyruvate dynamic MRI in prostate cancer to measure the conversion from pyruvate to lactate, which is expected to increase in aggressive cancers. The goal of this work was to develop and test analysis methods for improved quantification of this metabolic conversion. In this work, we compared specialized kinetic modeling methods to estimate the pyruvate-to-lactate conversion rate, kPL , as well as the lactate-to-pyruvate area-under-curve (AUC) ratio. The kinetic modeling included an "inputless" method requiring no assumptions regarding the input function, as well as a method incorporating bolus characteristics in the fitting. These were first evaluated with simulated data designed to match human prostate data, where we examined the expected sensitivity of metabolism quantification to variations in kPL , signal-to-noise ratio (SNR), bolus characteristics, relaxation rates, and B1 variability. They were then applied to 17 prostate cancer patient datasets. The simulations indicated that the inputless method with fixed relaxation rates provided high expected accuracy with no sensitivity to bolus characteristics. The AUC ratio showed an undesired strong sensitivity to bolus variations. Fitting the input function as well did not improve accuracy over the inputless method. In vivo results showed qualitatively accurate kPL maps with inputless fitting. The AUC ratio was sensitive to bolus delivery variations. Fitting with the input function showed high variability in parameter maps. Overall, we found the inputless kPL fitting method to be a simple, robust approach for quantification of metabolic conversion following HP [1-13 C]pyruvate injection in human prostate cancer studies. This study also provided initial ranges of HP [1-13 C]pyruvate parameters (SNR, kPL , bolus characteristics) in the human prostate.
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Project description:In the heart, detection of hyperpolarized [(13)C]bicarbonate and (13)CO(2) by magnetic resonance (MR) after administration of hyperpolarized [1-(13)C]pyruvate is caused exclusively by oxidative decarboxylation of pyruvate via the pyruvate dehydrogenase complex (PDH). However, liver mitochondria possess alternative anabolic pathways accessible by [1-(13)C]pyruvate, which may allow a wider diagnostic range for hyperpolarized MR compared with other tissue. Metabolism of hyperpolarized [1-(13)C]pyruvate in the tricarboxylic acid (TCA) cycle was monitored in the isolated perfused liver from fed and fasted mice. Hyperpolarized [1-(13)C]pyruvate was rapidly converted to [1-(13)C]lactate, [1-(13)C]alanine, [1-(13)C]malate, [4-(13)C]malate, [1-(13)C]aspartate, [4-(13)C]aspartate, and [(13)C]bicarbonate. Livers from fasted animals had increased lactate:alanine, consistent with elevated NADH:NAD(+). The appearance of asymmetrically enriched malate and aspartate indicated high rates of anaplerotic pyruvate carboxylase activity and incomplete equilibration with fumarate. Hyperpolarized [(13)C]bicarbonate was also detected, consistent with multiple mechanisms, including cataplerotic decarboxylation of [4-(13)C]oxaloacetate via phosphoenolpyruvate carboxykinase (PEPCK), forward TCA cycle flux of [4-(13)C]oxaloacetate to generate (13)CO(2) at isocitrate dehydrogenase, or decarboxylation of [1-(13)C]pyruvate by PDH. Isotopomer analysis of liver glutamate confirmed that anaplerosis was sevenfold greater than flux through PDH. In addition, signal from [4-(13)C]malate and [4-(13)C]aspartate was markedly blunted and signal from [(13)C]bicarbonate was completely abolished in livers from PEPCK KO mice, indicating that the major pathway for entry of hyperpolarized [1-(13)C]pyruvate into the hepatic TCA cycle is via pyruvate carboxylase, and that cataplerotic flux through PEPCK is the primary source of [(13)C]bicarbonate. We conclude that MR detection of hyperpolarized TCA intermediates and bicarbonate is diagnostic of pyruvate carboxylase and PEPCK flux in the liver.
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Project description:This study was designed to determine the effects of deuteration in pyruvate on exchange reactions in alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and flux through pyruvate dehydrogenase (PDH). Although deuteration of a 13C enriched substrate is commonly used to increase the lifetime of a probe for hyperpolarization experiments, the potential impact of kinetic isotope effects on such substitutions has not been studied in detail. Metabolism of deuterated pyruvate was investigated in isolated rat hearts. Hearts were perfused with a 1:1 mixture of [U-13C3]pyruvate and [2-13C1]pyruvate or a 1:1 mixture of [U-13C3]pyruvate plus [2-13C1, U-2H3]pyruvate for 30?min before being freeze clamped. Another set of hearts received [2-13C1, U-2H3]pyruvate and was freeze-clamped at 3?min or 6?min. Tissue extracts were analyzed by 1H and 13C{1H} NMR spectroscopy. The chemical shift isotope effect of 2H was monitored in the 13C NMR spectra of the C2 resonance of lactate and alanine plus the C5 of glutamate. There was little kinetic isotope effect of 2H in pyruvate on flux through PDH, LDH or ALT as detected by the distribution of 13C, but the distribution of 2H differed markedly between alanine and lactate. At steady-state, alanine was a mixture of deuterated species, while lactate was largely perdeuterated. Consistent with results at steady-state, hearts freeze-clamped at 3?min or 6?min showed rapid removal of deuterium in alanine but not in lactate. Metabolism of hyperpolarized [1-13C1]pyruvate was compared to [1-13C1,U-2H3]pyruvate in isolated hearts. Consistent with the results from tissue extracts, there was little effect of deuteration on the kinetics of appearance of lactate, alanine or bicarbonate, but there was a small, time-dependent upfield chemical shift in the HP[1-13C1]alanine signal reflecting exchange of methyl deuterons with water protons. Together, these results demonstrate that (1) the kinetics of pyruvate metabolism in hearts detected by 13C NMR are not affected by replacement of the pyruvate methyl protons with deuterons and (2) that the loss of deuterium from the methyl position occurs rapidly during the conversion of pyruvate to alanine. The majority of the deuterium atoms are lost on the time-scale of a hyperpolarization experiment.
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