Metabolic Analysis of Intracellular Pathogenic Bacteria Using NMR.

Pathogen proliferation and virulence depend on available nutrients, and these vary when the pathogen moves from outside of the host cell (extracellular) to the inside of the host cell (intracellular). Nuclear Magnetic Resonance (NMR) is a versatile analytical method, which lends itself for metabolic studies. In this chapter, we describe how 1H NMR can be combined with a cellular infection model to study the metabolic crosstalk between a bacterial pathogen and its host both in the extracellular and intracellular compartments. Central carbon metabolism is highlighted by using glucose labeled with the stable isotope 13C.

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Clarifying the appropriate application rates of N, P, and K fertilizers and the physiological mechanisms of wheat under water-saving recharge irrigation in the North China Plain would provide a theoretical basis for formulating reasonable fertilization plans for high-yield and high-efficiency wheat production. We established four treatments with different amounts of nitrogen (N), phosphorus (P2O5), and potassium (K2O) application: 0, 0, and 0 kg·hm-2 (F0), 180, 75, and 60 kg·hm-2 (F1), 225, 120, and 105 kg·hm-2 (F2), and 270, 165, and 150 kg·hm-2 (F3). During the jointing and anthesis stages of wheat, the relative water content of each treatment in the 0-40 cm soil layer was replenished to 70%, to investigate the differences in wheat flag leaf photosynthetic characteristics, distribution of 13C assimilates, grain starch accumulation, and fertilizer utilization. The results showed that the relative chlorophyll content of flag leaves, photosynthetic and chlorophyll fluorescence parameters, 13C assimilate allocation in each organ, enzyme activities involved in starch synthesis, and starch accumulation in the F1 treatment were significantly higher than that in F0 treatment, which was an important physiological basis for the 20.9% increase in grain yield. The above parameters and yield in the F2 and F3 treatments showed no significant increase compared to F1 treatment, while fertilizer productivity and agronomic efficiency of N, P, and K decreased by 17.5%-58.4% and 12.7%-50.7%, respectively. Therefore, F1 could promote flag leaf photosynthetic assimilate production and grain starch accumulation under water-saving supplementary irrigation conditions, resulting in higher grain yield and fertilizer utilization efficiency.

13C-Stable isotope resolved metabolomics uncovers dynamic biochemical landscape of gut microbiome-host organ communications in mice.

BACKGROUND: Gut microbiome metabolites are important modulators of host health and disease. However, the overall metabolic potential of the gut microbiome and interactions with the host organs have been underexplored. RESULTS: Using stable isotope resolved metabolomics (SIRM) in mice orally gavaged with 13C-inulin (a tracer), we first observed dynamic enrichment of 13C-metabolites in cecum contents in the amino acids and short-chain fatty acid metabolism pathways. 13C labeled metabolites were subsequently profiled comparatively in plasma, liver, brain, and skeletal muscle collected at 6, 12, and 24 h after the tracer administration. Organ-specific and time-dependent 13C metabolite enrichments were observed. Carbons from the gut microbiome were preferably incorporated into choline metabolism and the glutamine-glutamate/GABA cycle in the liver and brain, respectively. A sex difference in 13C-lactate enrichment was observed in skeletal muscle, which highlights the sex effect on the interplay between gut microbiome and host organs. Choline was identified as an interorgan metabolite derived from the gut microbiome and fed the lipogenesis of phosphatidylcholine and lysophosphatidylcholine in host organs. In vitro and in silico studies revealed the de novo synthesis of choline in the human gut microbiome via the ethanolamine pathway, and Enterococcus faecalis was identified as a major choline synthesis species. These results revealed a previously underappreciated role for gut microorganisms in choline biosynthesis. CONCLUSIONS: Multicompartmental SIRM analyses provided new insights into the current understanding of dynamic interorgan metabolite transport between the gut microbiome and host at the whole-body level in mice. Moreover, this study singled out microbiota-derived metabolites that are potentially involved in the gut-liver, gut-brain, and gut-skeletal muscle axes. Video Abstract.

Oxygenation of Earth's atmosphere induced metabolic and ecologic transformations recorded in the Lomagundi-Jatuli carbon isotopic excursion.

The oxygenation of Earth's atmosphere represents the quintessential transformation of a planetary surface by microbial processes. In turn, atmospheric oxygenation transformed metabolic evolution; molecular clock models indicate the diversification and ecological expansion of respiratory metabolisms in the several hundred million years following atmospheric oxygenation. Across this same interval, the geological record preserves 13C enrichment in some carbonate rocks, called the Lomagundi-Jatuli excursion (LJE). By combining data from geologic and genomic records, a self-consistent metabolic evolution model emerges for the LJE. First, fermentation and methanogenesis were major processes remineralizing organic carbon before atmospheric oxygenation. Once an ozone layer formed, shallow water and exposed environments were shielded from UVB/C radiation, allowing the expansion of cyanobacterial primary productivity. High primary productivity and methanogenesis led to preferential removal of 12C into organic carbon and CH4. Extreme and variable 13C enrichments in carbonates were caused by 13C-depleted CH4 loss to the atmosphere. Through time, aerobic respiration diversified and became ecologically widespread, as did other new metabolisms. Respiration displaced fermentation and methanogenesis as the dominant organic matter remineralization processes. As CH4 loss slowed, dissolved inorganic carbon in shallow environments was no longer highly 13C enriched. Thus, the loss of extreme 13C enrichments in carbonates marks the establishment of a new microbial mat ecosystem structure, one dominated by respiratory processes distributed along steep redox gradients. These gradients allowed the exchange of metabolic by-products among metabolically diverse organisms, providing novel metabolic opportunities. Thus, the microbially induced oxygenation of Earth's atmosphere led to the transformation of microbial ecosystems, an archetypal example of planetary microbiology.IMPORTANCEThe oxygenation of Earth's atmosphere represents the most extensive known chemical transformation of a planetary surface by microbial processes. In turn, atmospheric oxygenation transformed metabolic evolution by providing oxidants independent of the sites of photosynthesis. Thus, the evolutionary changes during this interval and their effects on planetary-scale biogeochemical cycles are fundamental to our understanding of the interdependencies among genomes, organisms, ecosystems, elemental cycles, and Earth's surface chemistry through time.

Visualizing 13C-Labeled Metabolites in Maize Root Tips with Mass Spectrometry Imaging.

Tracing in vivo isotope-labeled metabolites has been used to study metabolic pathways or flux analysis. However, metabolic differences between the cells have been often ignored in these studies due to the limitation of solvent-based extraction. Here we demonstrate that the mass spectrometry imaging of in vivo isotope-labeled metabolites, referred to as MSIi, can provide important insights into metabolic dynamics with cellular resolution that may supplement the traditional metabolomics and flux analysis. Developing maize root tips are adopted as a model system for MSIi by supplementing 200 mM [U-13C]glucose in 0.1x Hoagland medium. MSIi data sets were acquired for longitudinal sections of newly grown maize root tips after growing 5 days in the medium. A total of 56 metabolite features were determined to have been 13C-labeled based on accurate mass and the number of carbon matching with the metabolite databases. Simple sugars and their derivatives were fully labeled, but some small metabolites were partially labeled with a significant amount of fully unlabeled metabolites still present, suggesting the recycling of "old" metabolites in the newly grown tissues. Some distinct localizations were found, including the low abundance of hexose and its derivatives in the meristem, the high abundance of amino acids in the meristem, and the localization to epidermal and endodermal cells for lipids and their intermediates. Fatty acids and lipids were slow in metabolic turnover and showed various isotopologue distributions with intermediate building blocks, which may provide flux information for their biosynthesis.

Comparison of δ13C and δ15N of ecologically relevant amino acids among beluga whale tissues.

Ecological applications of compound-specific stable isotope analysis (CSIA) of amino acids (AAs) include 1) tracking carbon pathways in food webs using essential AA (AAESS) δ13C values, and 2) estimating consumer trophic position (TP) by comparing relative differences of 'trophic' and 'source' AA δ15N values. Despite the significance of these applications, few studies have examined AA-specific SI patterns among tissues with different AA compositions and metabolism/turnover rates, which could cause differential drawdown of body AA pools and impart tissue-specific isotopic fractionation. To address this knowledge gap, especially in the absence of controlled diet studies examining this issue in captive marine mammals, we used a paired-sample design to compare δ13C and δ15N values of 11 AAs in commonly sampled tissues (skin, muscle, and dentine) from wild beluga whales (Delphinapterus leucas). δ13C of two AAs, glutamic acid/glutamine (Glx, a non-essential AA) and, notably, threonine (an essential AA), differed between skin and muscle. Furthermore, δ15N of three AAs (alanine, glycine, and proline) differed significantly among the three tissues, with glycine δ15N differences of approximately 10 ‰ among tissues supporting recent findings it is unsuitable as a source AA. Significant δ15N differences in AAs such as proline, a trophic AA used as an alternative to Glx in TP estimation, highlight tissue selection as a potential source of error in ecological applications of CSIA-AA. Amino acids that differed among tissues play key roles in metabolic pathways (e.g., ketogenic and gluconeogenic AAs), pointing to potential physiological applications of CSIA-AA in studies of free-ranging animals. These findings underscore the complexity of isotopic dynamics within tissues and emphasize the need for a nuanced approach when applying CSIA-AA in ecological research.

Quantifying genome-specific carbon fixation in a 750-meter deep subsurface hydrothermal microbial community.

Dissolved inorganic carbon has been hypothesized to stimulate microbial chemoautotrophic activity as a biological sink in the carbon cycle of deep subsurface environments. Here, we tested this hypothesis using quantitative DNA stable isotope probing of metagenome-assembled genomes (MAGs) at multiple 13C-labeled bicarbonate concentrations in hydrothermal fluids from a 750-m deep subsurface aquifer in the Biga Peninsula (Turkey). The diversity of microbial populations assimilating 13C-labeled bicarbonate was significantly different at higher bicarbonate concentrations, and could be linked to four separate carbon-fixation pathways encoded within 13C-labeled MAGs. Microbial populations encoding the Calvin-Benson-Bassham cycle had the highest contribution to carbon fixation across all bicarbonate concentrations tested, spanning 1-10 mM. However, out of all the active carbon-fixation pathways detected, MAGs affiliated with the phylum Aquificae encoding the reverse tricarboxylic acid (rTCA) pathway were the only microbial populations that exhibited an increased 13C-bicarbonate assimilation under increasing bicarbonate concentrations. Our study provides the first experimental data supporting predictions that increased bicarbonate concentrations may promote chemoautotrophy via the rTCA cycle and its biological sink for deep subsurface inorganic carbon.

Composition and metabolism of microbial communities in soil pores.

Delineation of microbial habitats within the soil matrix and characterization of their environments and metabolic processes are crucial to understand soil functioning, yet their experimental identification remains persistently limited. We combined single- and triple-energy X-ray computed microtomography with pore specific allocation of 13C labeled glucose and subsequent stable isotope probing to demonstrate how long-term disparities in vegetation history modify spatial distribution patterns of soil pore and particulate organic matter drivers of microbial habitats, and to probe bacterial communities populating such habitats. Here we show striking differences between large (30-150 µm Ø) and small (4-10 µm Ø) soil pores in (i) microbial diversity, composition, and life-strategies, (ii) responses to added substrate, (iii) metabolic pathways, and (iv) the processing and fate of labile C. We propose a microbial habitat classification concept based on biogeochemical mechanisms and localization of soil processes and also suggests interventions to mitigate the environmental consequences of agricultural management.

Metabolic coupling between soil aerobic methanotrophs and denitrifiers in rice paddy fields.

Paddy fields are hotspots of microbial denitrification, which is typically linked to the oxidation of electron donors such as methane (CH4) under anoxic and hypoxic conditions. While several anaerobic methanotrophs can facilitate denitrification intracellularly, whether and how aerobic CH4 oxidation couples with denitrification in hypoxic paddy fields remains virtually unknown. Here we combine a ~3300 km field study across main rice-producing areas of China and 13CH4-DNA-stable isotope probing (SIP) experiments to investigate the role of soil aerobic CH4 oxidation in supporting denitrification. Our results reveal positive relationships between CH4 oxidation and denitrification activities and genes across various climatic regions. Microcosm experiments confirm that CH4 and methanotroph addition promote gene expression involved in denitrification and increase nitrous oxide emissions. Moreover, 13CH4-DNA-SIP analyses identify over 70 phylotypes harboring genes associated with denitrification and assimilating 13C, which are mostly belonged to Rubrivivax, Magnetospirillum, and Bradyrhizobium. Combined analyses of 13C-metagenome-assembled genomes and 13C-metabolomics highlight the importance of intermediates such as acetate, propionate and lactate, released during aerobic CH4 oxidation, for the coupling of CH4 oxidation with denitrification. Our work identifies key microbial taxa and pathways driving coupled aerobic CH4 oxidation and denitrification, with important implications for nitrogen management and greenhouse gas regulation in agroecosystems.

Stable Isotope Labeling and Quantification of Photosynthetic Metabolites.

Stable isotope labeling with 13CO2 coupled with mass spectrometry allows monitoring the incorporation of 13C into photosynthetic intermediates and is a powerful technique for the investigation of the metabolic dynamics of photosynthesis. We describe here a protocol for 13CO2 labeling of large leaved plants and of Arabidopsis thaliana rosette, and a method for quantitative mass spectrometry analyses to uncover the labeling pattern of Calvin-Benson cycle sucrose, and starch synthesis as well as carbon-concentrating mechanism metabolites.

Amino acid-specific isotopes reveal changing five-dimensional niche segregation in Pacific seabirds over 50 years.

Hutchison's niche theory suggests that coexisting competing species occupy non-overlapping hypervolumes, which are theoretical spaces encompassing more than three dimensions, within an n-dimensional space. The analysis of multiple stable isotopes can be used to test these ideas where each isotope can be considered a dimension of niche space. These hypervolumes may change over time in response to variation in behaviour or habitat, within or among species, consequently changing the niche space itself. Here, we use isotopic values of carbon and nitrogen of ten amino acids, as well as sulphur isotopic values, to produce multi-isotope models to examine niche segregation among an assemblage of five coexisting seabird species (ancient murrelet Synthliboramphus antiquus, double-crested cormorant Phalacrocorax auritus, Leach's storm-petrel Oceanodrama leucorhoa, rhinoceros auklet Cerorhinca monocerata, pelagic cormorant Phalacrocorax pelagicus) that inhabit coastal British Columbia. When only one or two isotope dimensions were considered, the five species overlapped considerably, but segregation increased in more dimensions, but often in complex ways. Thus, each of the five species occupied their own isotopic hypervolume (niche), but that became apparent only when factoring the increased information from sulphur and amino acid specific isotope values, rather than just relying on proxies of δ15N and δ13C alone. For cormorants, there was reduction of niche size for both species consistent with a decline in their dominant prey, Pacific herring Clupea pallasii, from 1970 to 2006. Consistent with niche theory, cormorant species showed segregation across time, with the double-crested demonstrating a marked change in diet in response to prey shifts in a higher dimensional space. In brief, incorporating multiple isotopes (sulfur, PC1 of δ15N [baselines], PC2 of δ15N [trophic position], PC1 and PC2 of δ13C) metrics allowed us to infer changes and differences in food web topology that were not apparent from classic carbon-nitrogen biplots.

Rethinking 13C-metabolic flux analysis - The Bayesian way of flux inference.

Metabolic reaction rates (fluxes) play a crucial role in comprehending cellular phenotypes and are essential in areas such as metabolic engineering, biotechnology, and biomedical research. The state-of-the-art technique for estimating fluxes is metabolic flux analysis using isotopic labelling (13C-MFA), which uses a dataset-model combination to determine the fluxes. Bayesian statistical methods are gaining popularity in the field of life sciences, but the use of 13C-MFA is still dominated by conventional best-fit approaches. The slow take-up of Bayesian approaches is, at least partly, due to the unfamiliarity of Bayesian methods to metabolic engineering researchers. To address this unfamiliarity, we here outline similarities and differences between the two approaches and highlight particular advantages of the Bayesian way of flux analysis. With a real-life example, re-analysing a moderately informative labelling dataset of E. coli, we identify situations in which Bayesian methods are advantageous and more informative, pointing to potential pitfalls of current 13C-MFA evaluation approaches. We propose the use of Bayesian model averaging (BMA) for flux inference as a means of overcoming the problem of model uncertainty through its tendency to assign low probabilities to both, models that are unsupported by data, and models that are overly complex. In this capacity, BMA resembles a tempered Ockham's razor. With the tempered razor as a guide, BMA-based 13C-MFA alleviates the problem of model selection uncertainty and is thereby capable of becoming a game changer for metabolic engineering by uncovering new insights and inspiring novel approaches.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling.

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Current methods for hyperpolarized [1-13C]pyruvate MRI human studies.

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of HP agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate-by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods and Equipment study groups. It further aims to provide a comprehensive reference for future consensus, building as the field continues to advance human studies with this metabolic imaging modality.

Constraining activity and growth substrate of fungal decomposers via assimilation patterns of inorganic carbon and water into lipid biomarkers.

Fungi are among the few organisms on the planet that can metabolize recalcitrant carbon (C) but are also known to access recently produced plant photosynthate. Therefore, improved quantification of growth and substrate utilization by different fungal ecotypes will help to define the rates and controls of fungal production, the cycling of soil organic matter, and thus the C storage and CO2 buffering capacity in soil ecosystems. This pure-culture study of fungal isolates combined a dual stable isotope probing (SIP) approach, together with rapid analysis by tandem pyrolysis-gas chromatography-isotope ratio mass spectrometry to determine the patterns of water-derived hydrogen (H) and inorganic C assimilated into lipid biomarkers of heterotrophic fungi as a function of C substrate. The water H assimilation factor (αW) and the inorganic C assimilation into C18:2 fatty acid isolated from five fungal species growing on glucose was lower (0.62% ± 0.01% and 4.7% ± 1.6%, respectively) than for species grown on glutamic acid (0.90% ± 0.02% and 7.4% ± 3.7%, respectively). Furthermore, the assimilation ratio (RIC/αW) for growth on glucose and glutamic acid can distinguish between these two metabolic modes. This dual-SIP assay thus delivers estimates of fungal activity and may help to delineate the predominant substrates that are respired among a matrix of compounds found in natural environments.IMPORTANCEFungal decomposers play important roles in food webs and nutrient cycling because they can feed on both labile and more recalcitrant forms of carbon. This study developed and applied a dual stable isotope assay (13C-dissolved inorganic carbon/2H) to improve the investigation of fungal activity in the environment. By determining the incorporation patterns of hydrogen and carbon into fungal lipids, this assay delivers estimates of fungal activity and the different metabolic pathways that they employ in ecological and environmental systems.

Implications of dietary carbon incorporation in fish carbonates for the global carbon cycle.

Marine bony fish are important participants in Earth's carbon cycle through their contributions to the biological pump and the marine inorganic carbon cycle. However, uncertainties in the composition and magnitude of fish contributions preclude their integration into fully coupled carbon-climate models. Here, we consider recent upwards revisions to global fish biomass estimates (2.7-9.5×) and provide new stable carbon isotope measurements that show marine fish are prodigious producers of carbonate with unique composition. Assuming the median increase (4.17×) in fish biomass estimates is linearly reflected in fish carbonate (ichthyocarbonate) production rate, marine fish are estimated to produce between 1.43 and 3.99 Pg CaCO3 yr-1, but potentially as much as 9.03 Pg CaCO3 yr-1. Thus, marine fish carbonate production is equivalent to or potentially higher than contributions by coccolithophores or pelagic foraminifera. New stable carbon isotope analyses indicate that a significant proportion of ichthyocarbonate is derived from dietary carbon, rather than seawater dissolved inorganic carbon. Using a statistical mixing model to derive source contributions, we estimate ichthyocarbonate contains up to 81 % dietary carbon, with average compositions of 28-56 %, standing in contrast to contents <10 % in other biogenic carbonate minerals. Results also indicate ichthyocarbonate contains 5.5-40.4 % total organic carbon. When scaled to the median revised global production of ichthyocarbonate, an additional 0.08 to 1.61 Pg C yr-1 can potentially be added to estimates of fish contributions to the biological pump, significantly increasing marine fish contributions to total surface carbon export. Our integration of geochemical and physiological analyses identifies an overlooked link between carbonate production and the biological pump. Since ichthyocarbonate production is anticipated to increase with climate change scenarios, due to ocean warming and acidification, these results emphasize the importance of quantitative understanding of the multifaceted role of marine fish in the global carbon cycle.

Hyperpolarized 13 C metabolic imaging of the human abdomen with spatiotemporal denoising.

PURPOSE: Improving the quality and maintaining the fidelity of large coverage abdominal hyperpolarized (HP) 13 C MRI studies with a patch based global-local higher-order singular value decomposition (GL-HOVSD) spatiotemporal denoising approach. METHODS: Denoising performance was first evaluated using the simulated [1-13 C]pyruvate dynamics at different noise levels to determine optimal kglobal and klocal parameters. The GL-HOSVD spatiotemporal denoising method with the optimized parameters was then applied to two HP [1-13 C]pyruvate EPI abdominal human cohorts (n = 7 healthy volunteers and n = 8 pancreatic cancer patients). RESULTS: The parameterization of kglobal = 0.2 and klocal = 0.9 denoises abdominal HP data while retaining image fidelity when evaluated by RMSE. The kPX (conversion rate of pyruvate-to-metabolite, X = lactate or alanine) difference was shown to be <20% with respect to ground-truth metabolic conversion rates when there is adequate SNR (SNRAUC > 5) for downstream metabolites. In both human cohorts, there was a greater than nine-fold gain in peak [1-13 C]pyruvate, [1-13 C]lactate, and [1-13 C]alanine apparent SNRAUC . The improvement in metabolite SNR enabled a more robust quantification of kPL and kPA . After denoising, we observed a 2.1 ± 0.4 and 4.8 ± 2.5-fold increase in the number of voxels reliably fit across abdominal FOVs for kPL and kPA quantification maps. CONCLUSION: Spatiotemporal denoising greatly improves visualization of low SNR metabolites particularly [1-13 C]alanine and quantification of [1-13 C]pyruvate metabolism in large FOV HP 13 C MRI studies of the human abdomen.

Functional activation of pyruvate dehydrogenase in human brain using hyperpolarized [1-13 C]pyruvate.

PURPOSE: Pyruvate, produced from either glucose, glycogen, or lactate, is the dominant precursor of cerebral oxidative metabolism. Pyruvate dehydrogenase (PDH) flux is a direct measure of cerebral mitochondrial function and metabolism. Detection of [13 C]bicarbonate in the brain from hyperpolarized [1-13 C]pyruvate using carbon-13 (13 C) MRI provides a unique opportunity for assessing PDH flux in vivo. This study is to assess changes in cerebral PDH flux in response to visual stimuli using in vivo 13 C MRS with hyperpolarized [1-13 C]pyruvate. METHODS: From seven sedentary adults in good general health, time-resolved [13 C]bicarbonate production was measured in the brain using 90° flip angles with minimal perturbation of its precursors, [1-13 C]pyruvate and [1-13 C]lactate, to test the hypothesis that the appearance of [13 C]bicarbonate signals in the brain reflects the metabolic changes associated with neuronal activation. With a separate group of healthy participants (n = 3), the likelihood of the bolus-injected [1-13 C]pyruvate being converted to [1-13 C]lactate prior to decarboxylation was investigated by measuring [13 C]bicarbonate production with and without [1-13 C]lactate saturation. RESULTS: In the course of visual stimulation, the measured [13 C]bicarbonate signal normalized to the total 13 C signal in the visual cortex increased by 17.1% ± 15.9% (p = 0.017), whereas no significant change was detected in [1-13 C]lactate. Proton BOLD fMRI confirmed the regional activation in the visual cortex with the stimuli. Lactate saturation decreased bicarbonate-to-pyruvate ratio by 44.4% ± 9.3% (p < 0.01). CONCLUSION: We demonstrated the utility of 13 C MRS with hyperpolarized [1-13 C]pyruvate for assessing the activation of cerebral PDH flux via the detection of [13 C]bicarbonate production.

Simultaneous fMRI and metabolic MRS of hyperpolarized [1-13C]pyruvate during nicotine stimulus in rat.

Functional MRI (fMRI) and MRS (fMRS) can be used to noninvasively map cerebral activation and metabolism. Recently, hyperpolarized 13C spectroscopy and metabolic imaging have provided an alternative approach to assess metabolism. In this study, we combined 1H fMRI and hyperpolarized [1-13C]pyruvate MRS to compare cerebral blood oxygenation level-dependent (BOLD) response and real-time cerebral metabolism, as assessed with lactate and bicarbonate labelling, during nicotine stimulation. Simultaneous 1H fMRI (multislice gradient echo echo-planar imaging) and 13C spectroscopic (single slice pulse-acquire) data were collected in urethane-anaesthetized female Sprague-Dawley rats (n = 12) at 9.4 T. Animals received an intravenous (i.v.) injection of either nicotine (stimulus; 88 µg/kg, n = 7, or 300 µg/kg, n = 5) or 0.9% saline (matching volume), followed by hyperpolarized [1-13C]pyruvate injection 60 s later. Three hours later, a second injection was administered: the animals that had previously received saline were injected with nicotine and vice versa, both followed by another hyperpolarized [1-13C]pyruvate i.v. injection 60 s later. The low-dose (88 µg/kg) nicotine injection led to a 12% ± 4% (n = 7, t-test, p ~ 0.0006 (t-value -5.8, degrees of freedom 6), Wilcoxon p ~ 0.0078 (test statistic 0)) increase in BOLD signal. At the same time, an increase in 13C-bicarbonate signal was seen in four out of six animals. Bicarbonate-to-total carbon ratios were 0.010 ± 0.004 and 0.018 ± 0.010 (n = 6, t-test, p ~ 0.03 (t-value -2.3, degrees of freedom 5), Wilcoxon p ~ 0.08 (test statistic 3)) for saline and nicotine experiments, respectively. No increase in the lactate signal was seen; lactate-to-total carbon was 0.16 ± 0.02 after both injections. The high (300 µg/kg) nicotine dose (n = 5) caused highly variable BOLD and metabolic responses, possibly due to the apparent respiratory distress. Simultaneous detection of 1H fMRI and hyperpolarized 13C-MRS is feasible. A comparison of metabolic response between control and stimulated states showed differences in bicarbonate signal, implying that the hyperpolarization technique could offer complimentary information on brain activation.