Metabolism is a major driver of hydrogen isotope fractionation recorded in tree-ring glucose of Pinus nigra.
ABSTRACT: Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences. To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961-1995). We found fractionation signals (i.e. temporal variability in deuterium abundance) at glucose H1 and H2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO2 concentration beyond a response change point. They explain ≈ 60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth. We propose the signals are introduced at the leaf level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.
Project description:As the central carbon uptake pathway in photosynthetic cells, the Calvin-Benson cycle is among the most important biochemical cycles for life on Earth. A carbon flux of anaplerotic origin (i.e. through the chloroplast-localized oxidative branch of the pentose phosphate pathway) into the Calvin-Benson cycle was proposed recently. Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO<sub>2</sub> concentrations, C<sub>a</sub> . Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations. We report deuterium fractionation signals at H<sup>1</sup> and H<sup>2</sup> of starch glucose. Below a C<sub>a</sub> change point, these signals increase with decreasing C<sub>a</sub> consistent with modelled fractionations by anaplerotic flux. Under standard conditions (C<sub>a</sub> = 450 ppm corresponding to intercellular CO<sub>2</sub> concentrations, C<sub>i</sub> , of 328 ppm), we estimate negligible anaplerotic flux. At C<sub>a</sub> = 180 ppm (C<sub>i</sub> = 140 ppm), more than 10% of the glucose-6-phosphate entering the starch biosynthesis pathway is diverted into the anaplerotic pathway. In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin-Benson cycle in vivo. We propose the flux may help to: maintain high levels of ribulose 1,5-bisphosphate under source-limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build-up source strength; and counteract oxidative stress.
Project description:This study was designed to determine whether perdeuterated glucose experiences a kinetic isotope effect (KIE) as glucose passes through glycolysis and is further oxidized in the tricarboxylic acid (TCA) cycle. Metabolism of deuterated glucose was investigated in two groups of perfused rat hearts. The control group was supplied with a 1:1 mixture of [U-13C6]glucose and [1,6-13C2]glucose, while the experimental group received [U-13C6,U-2H7]glucose and [1,6-13C2]glucose. Tissue extracts were analyzed by 1H, 2H and proton-decoupled 13C NMR spectroscopy. Extensive 2H-13C scalar coupling plus chemical shift isotope effects were observed in the proton-decoupled 13C NMR spectra of lactate, alanine and glutamate. A small but measureable (?8%) difference in the rate of conversion of [U-13C6]glucose vs. [1,6-13C2]glucose to lactate, likely reflecting rates of CC bond breakage in the aldolase reaction, but conversion of [U-13C6]glucose versus [U-13C6,U-2H7]glucose to lactate did not differ. This shows that the presence of deuterium in glucose does not alter glycolytic flux. However, there were two distinct effects of deuteration on metabolism of glucose to alanine and oxidation of glucose in the TCA. First, alanine undergoes extensive exchange of methyl deuterons with solvent protons in the alanine amino transferase reaction. Second, there is a substantial kinetic isotope effect in metabolism of [U-13C6,U-2H7]glucose to alanine and glutamate. In the presence of [U-13C6,U-2H7]glucose, alanine and lactate are not in rapid exchange with the same pool of pyruvate. These studies indicate that the appearance of hyperpolarized 13C-lactate from hyperpolarized [U-13C6,U-2H7]glucose is not substantially influenced by a deuterium kinetic isotope effect.
Project description:Cancer cells undergo diverse metabolic adaptations to meet the energetic demands imposed by dysregulated growth and proliferation. Assessing metabolism in intact tumors allows the investigator to observe the combined metabolic effects of numerous cancer cell-intrinsic and -extrinsic factors that cannot be fully captured in culture models. We have developed methods to use stable isotope-labeled nutrients (e.g., [<sup>13</sup>C]glucose) to probe metabolic activity within intact tumors in vivo, in mice and humans. In these methods, the labeled nutrient is introduced to the circulation through an intravenous catheter prior to surgical resection of the tumor and adjacent nonmalignant tissue. Metabolism within these tissues during the infusion transfers the isotope label into metabolic intermediates from pathways supplied by the infused nutrient. Extracting metabolites from surgical specimens and analyzing their isotope labeling patterns provides information about metabolism in the tissue. We provide detailed information about this technique, from introduction of the labeled tracer through data analysis and interpretation, including streamlined approaches to quantify isotope labeling in informative metabolites extracted from tissue samples. We focus on infusions with [<sup>13</sup>C]glucose and the application of mass spectrometry to assess isotope labeling in intermediates from central metabolic pathways, including glycolysis, the tricarboxylic acid cycle and nonessential amino acid synthesis. We outline practical considerations to apply these methods to human subjects undergoing surgical resections of solid tumors. We also discuss the method's versatility and consider the relative advantages and limitations of alternative approaches to introduce the tracer, harvest the tissue and analyze the data.
Project description:The stable carbon isotope (13)C is used as a universal tracer in plant eco-physiology and studies of carbon exchange between vegetation and atmosphere. Photosynthesis fractionates against (13)CO(2) so that source sugars (photosynthates) are on average (13)C depleted by 20‰ compared with atmospheric CO(2). The carbon isotope distribution within sugars has been shown to be heterogeneous, with relatively (13)C-enriched and (13)C-depleted C-atom positions. The (13)C pattern within sugars is the cornerstone of (13)C distribution in plants, because all metabolites inherit the (13)C abundance in their specific precursor C-atom positions. However, the intramolecular isotope pattern in source leaf glucose and the isotope fractionation associated with key enzymes involved in sugar interconversions are currently unknown. To gain insight into these, we have analyzed the intramolecular isotope composition in source leaf transient starch, grain storage starch, and root storage sucrose and measured the site-specific isotope fractionation associated with the invertase (EC 188.8.131.52) and glucose isomerase (EC 184.108.40.206) reactions. When these data are integrated into a simple steady-state model of plant isotopic fluxes, the enzyme-dependent fractionations satisfactorily predict the observed intramolecular patterns. These results demonstrate that glucose and sucrose metabolism is the primary determinant of the (13)C abundance in source and sink tissue and is, therefore, of fundamental importance to the interpretation of plant isotopic signals.
Project description:Quantitative assessment of cerebral glucose consumption rate (CMR<sub>glc</sub>) and tricarboxylic acid cycle flux (V<sub>TCA</sub>) is crucial for understanding neuroenergetics under physiopathological conditions. In this study, we report a novel in vivo Deuterium (<sup>2</sup>H) MRS (DMRS) approach for simultaneously measuring and quantifying CMR<sub>glc</sub> and V<sub>TCA</sub> in rat brains at 16.4 Tesla. Following a brief infusion of deuterated glucose, dynamic changes of isotope-labeled glucose, glutamate/glutamine (Glx) and water contents in the brain can be robustly monitored from their well-resolved <sup>2</sup>H resonances. Dynamic DMRS glucose and Glx data were employed to determine CMR<sub>glc</sub> and V<sub>TCA</sub> concurrently. To test the sensitivity of this method in response to altered glucose metabolism, two brain conditions with different anesthetics were investigated. Increased CMR<sub>glc</sub> (0.46 vs. 0.28?µmol/g/min) and V<sub>TCA</sub> (0.96 vs. 0.6?µmol/g/min) were found in rats under morphine as compared to deeper anesthesia using 2% isoflurane. This study demonstrates the feasibility and new utility of the in vivo DMRS approach to assess cerebral glucose metabolic rates at high/ultrahigh field. It provides an alternative MRS tool for in vivo study of metabolic coupling relationship between aerobic and anaerobic glucose metabolisms in brain under physiopathological states.
Project description:The hydrogen isotope ratio of water cryogenically extracted from plant stem samples (δ<sup>2</sup>H<sub>stem_CVD</sub>) is routinely used to aid isotope applications that span hydrological, ecological, and paleoclimatological research. However, an increasing number of studies have shown that a key assumption of these applications-that δ<sup>2</sup>H<sub>stem_CVD</sub> is equal to the δ<sup>2</sup>H of plant source water (δ<sup>2</sup>H<sub>source</sub>)-is not necessarily met in plants from various habitats. To examine this assumption, we purposedly designed an experimental system to allow independent measurements of δ<sup>2</sup>H<sub>stem_CVD</sub>, δ<sup>2</sup>H<sub>source</sub>, and δ<sup>2</sup>H of water transported in xylem conduits (δ<sup>2</sup>H<sub>xylem</sub>) under controlled conditions. Our measurements performed on nine woody plant species from diverse habitats revealed a consistent and significant depletion in δ<sup>2</sup>H<sub>stem_CVD</sub> compared with both δ<sup>2</sup>H<sub>source</sub> and δ<sup>2</sup>H<sub>xylem</sub> Meanwhile, no significant discrepancy was observed between δ<sup>2</sup>H<sub>source</sub> and δ<sup>2</sup>H<sub>xylem</sub> in any of the plants investigated. These results cast significant doubt on the long-standing view that deuterium fractionation occurs during root water uptake and, alternatively, suggest that measurement bias inherent in the cryogenic extraction method is the root cause of δ<sup>2</sup>H<sub>stem_CVD</sub> depletion. We used a rehydration experiment to show that the stem water cryogenic extraction error could originate from a dynamic exchange between organically bound deuterium and liquid water during water extraction. In light of our finding, we suggest caution when partitioning plant water sources and reconstructing past climates using hydrogen isotopes, and carefully propose that the paradigm-shifting phenomenon of ecohydrological separation ("two water worlds") is underpinned by an extraction artifact.
Project description:<h4>Purpose</h4>The spatial resolution of <sup>18</sup>F-fluorodeoxyglucose PET does not allow the specific cellular origin of its signal to be determined, but it is commonly accepted that transport and trapping of <sup>18</sup>F-fluorodeoxyglucose reflects neuronal glucose metabolism. The main frameworks for the diagnosis of Alzheimer's disease suggest that hypometabolism measured with <sup>18</sup>F-fluorodeoxyglucose PET is a biomarker of neuronal injury and neurodegeneration. There is preclinical evidence to suggest that astrocytes contribute, at least partially, to the in vivo <sup>18</sup>F-fluorodeoxyglucose PET signal. However, due to a paucity of PET tracers for imaging astrocytic processes, the relationship between astrocyte function and glucose metabolism in human brain is not fully understood. The aim of this study was to investigate the longitudinal association between astrocyte function and glucose metabolism in Alzheimer's disease.<h4>Methods</h4>The current investigation combined longitudinal PET data from patients with autosomal dominant Alzheimer's disease, including data on astrocyte function (<sup>11</sup>C-deuterium-L-deprenyl binding) and glucose metabolism (<sup>18</sup>F-fluorodeoxyglucose uptake). Research participants included 7 presymptomatic and 4 symptomatic mutation carriers (age 44.9?±?9.8 years and 58.0?±?3.7 years, respectively) and 16 noncarriers (age 51.1?±?14.2 years). Eight carriers and eight noncarriers underwent longitudinal follow-up PET imaging at an average of 2.8?±?0.2 and 3.0?±?0.5 years from baseline, respectively.<h4>Results</h4>Longitudinal decline in astrocyte function as measured using <sup>11</sup>C-deuterium-L-deprenyl PET was significantly associated with progressive hypometabolism (<sup>18</sup>F-fluorodeoxyglucose uptake) in mutation carriers; no significant association was observed in noncarriers.<h4>Conclusion</h4>The emerging data shift the accepted wisdom that decreases in cerebral metabolism measured with <sup>18</sup>F-fluorodeoxyglucose solely reflect neuronal injury, and places astrocytes more centrally in the development of Alzheimer's disease.
Project description:The persistent organic pollutant DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) is still indispensable in the fight against malaria, although DDT and related compounds pose toxicological hazards. Technical DDT contains the dichloro congener DDD (1-chloro-4-[2,2-dichloro-1-(4-chlorophenyl)ethyl]benzene) as by-product, but DDD is also formed by reductive degradation of DDT in the environment. To differentiate between DDD formation pathways, we applied deuterium NMR spectroscopy to measure intramolecular deuterium distributions (2H isotopomer abundances) of DDT and DDD. DDD formed in the technical DDT synthesis was strongly deuterium-enriched at one intramolecular position, which we traced back to 2H/1H fractionation of a chlorination step in the technical synthesis. In contrast, DDD formed by reductive degradation was strongly depleted at the same position, which was due to the incorporation of 2H-depleted hydride equivalents during reductive degradation. Thus, intramolecular isotope distributions give mechanistic information on reaction pathways, and explain a puzzling difference in the whole-molecule 2H/1H ratio between DDT and DDD. In general, our results highlight that intramolecular isotope distributions are essential to interpret whole-molecule isotope ratios. Intramolecular isotope information allows distinguishing pathways of DDD formation, which is important to identify polluters or to assess DDT turnover in the environment. Because intramolecular isotope data directly reflect isotope fractionation of individual chemical reactions, they are broadly applicable to elucidate transformation pathways of small bioactive molecules in chemistry, physiology and environmental science.
Project description:We apply stable isotope tracing, mass-spectrometry-based untargeted metabolomics, to reveal the biochemical space labeled by <sup>13</sup>C-substrates in bone-marrow-derived macrophages. At the pathway level, classically (lipopolysaccharide [LPS]-polarized, M1) and alternatively (interleukin [IL]-4-polarized, M2) polarized macrophages were <sup>13</sup>C-labeled with surprising concordance. Total pools of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an intermediate in the hexosamine biosynthetic pathway, were equally abundant in LPS- and IL-4-polarized macrophages. Informatic scrutiny of <sup>13</sup>C-isotopologues revealed that LPS-polarized macrophages leverage the pentose phosphate pathway to generate UDP-GlcNAc, whereas IL-4-polarized macrophages rely on intact glucose and mitochondrial metabolism of glucose carbon. Labeling from [<sup>13</sup>C]glucose is competed by unlabeled fatty acids and acetoacetate, underscoring the broad roles for substrate metabolism beyond energy conversion. Finally, the LPS-polarized macrophage metabolite itaconate is imported into IL-4-polarized macrophages, in which it reprograms [<sup>13</sup>C]glucose metabolism. Thus, use of fully unsupervised isotope tracing metabolomics in macrophages reveals polarization-state-specific metabolic pathway connectivity, substrate competition, and metabolite allocation among cellular compartments.
Project description:Deuterium Magnetic Resonance Spectroscopy (DMRS) is a non-invasive technique that allows the detection of deuterated compounds in vivo. DMRS has a large potential to analyze uptake, perfusion, washout or metabolism, since deuterium is a stable isotope and therefore does not decay during biologic processing of a deuterium labelled substance. Moreover, DMRS allows the distinction between different deuterated substances. In this work, we performed DMRS of deuterated 3-O-Methylglucose (OMG). OMG is a non-metabolizable glucose analog which is transported similar to D-glucose. DMRS of OMG was performed in phantom and in vivo measurements using a preclinical 7 Tesla MRI system. The chemical shift (3.51 ± 0.1 ppm) and relaxation times were determined. OMG was injected intravenously and spectra were acquired over a period of one hour to monitor the time evolution of the deuterium signal in tumor-bearing rats. The increase and washout of OMG could be observed. Three different exponential functions were compared in terms of how well they describe the OMG washout. A mono-exponential model with offset seems to describe the observed time course best with a time constant of 1910 ± 770 s and an offset of 2.5 ± 1.2 mmol/l (mean ± std, N = 3). Chemical shift imaging could be performed with a voxel size of 7.1 mm x 7.1 mm x 7.9 mm. The feasibility of DMRS with deuterium labelled OMG could be demonstrated. These data might serve as basis for future studies that aim to characterize glucose transport using DMRS.