Isotope Tracer Experiments Research Articles

A timeline of stable isotopes and mass spectrometry in the life sciences.

This review retraces the role of stable isotopes and mass spectrometry in the life sciences. The timeline is divided into four segments covering the years 1920-1950, 1950-1980, 1980-2000, and 2000 until today. For each period methodic progress and typical applications are discussed. Application of stable isotopes is driven by improvements of mass spectrometry, chromatography, and related fields in sensitivity, mass accuracy, structural specificity, complex sample handling ability, data output, and data evaluation. We currently experience the vision of omics-type analyses, that is, the comprehensive identification and quantification of a complete compound class within one or a few analytical runs. This development is driven by stable isotopes without competition by radioisotopes. In metabolic studies as classic field of isotopic tracer experiments, stable isotopes and radioisotopes were competing solutions, with stable isotopes as the long-term junior partner. Since the 1990s the number of metabolic studies with radioisotopes decreases, whereas stable isotope studies retain their slow but stable upward tendency. Unique fields of stable isotopes are metabolic tests in newborns, metabolic experiments in healthy controls, newborn screening for inborn errors, quantification of drugs and drug metabolites in doping control, natural isotope fractionation in geology, ecology, food authentication, or doping control, and more recently the field of quantitative omics-type analyses. There, cells or whole organisms are systematically labeled with stable isotopes to study proteomic differences or specific responses to stimuli or genetic manipulation. The duo of stable isotopes and mass spectrometry will probably continue to grow in the life sciences, since it delivers reference-quality quantitative data with molecular specificity, often combined with informative isotope effects. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:58-85, 2017.

Coral mucus fuels the sponge loop in warm- and cold-water coral reef ecosystems.

Shallow warm-water and deep-sea cold-water corals engineer the coral reef framework and fertilize reef communities by releasing coral mucus, a source of reef dissolved organic matter (DOM). By transforming DOM into particulate detritus, sponges play a key role in transferring the energy and nutrients in DOM to higher trophic levels on Caribbean reefs via the so-called sponge loop. Coral mucus may be a major DOM source for the sponge loop, but mucus uptake by sponges has not been demonstrated. Here we used laboratory stable isotope tracer experiments to show the transfer of coral mucus into the bulk tissue and phospholipid fatty acids of the warm-water sponge Mycale fistulifera and cold-water sponge Hymedesmia coriacea, demonstrating a direct trophic link between corals and reef sponges. Furthermore, 21–40% of the mucus carbon and 32–39% of the nitrogen assimilated by the sponges was subsequently released as detritus, confirming a sponge loop on Red Sea warm-water and north Atlantic cold-water coral reefs. The presence of a sponge loop in two vastly different reef environments suggests it is a ubiquitous feature of reef ecosystems contributing to the high biogeochemical cycling that may enable coral reefs to thrive in nutrient-limited (warm-water) and energy-limited (cold-water) environments.

Elucidating the roles of stearoyl-CoA desaturase 1 in adipocyte fatty acid metabolism and cellular function

Worldwide obesity rates have risen to epidemic proportions, with over 600 million obese individuals across the globe. These individuals are prone to obesity-related health complications, including type 2 diabetes, hypertension, cancer, and cardiovascular disease. Obesity also coincides with the expansion of adipose tissue, which has an important role storing excess calories in the form of triacylglycerol (TAG) within adipocyte lipid droplets. The predominant fatty acids (FAs), comprising adipocyte TAGs, are monounsaturated FAs, which are produced by stearoyl-CoA desaturase 1 (SCD1). Specifically, SCD1 converts saturated FAs palmitate (PA) and stearate (SA) into palmitoleate and oleate, respectively. Interestingly, whole-body SCD1-deficiency is associated with reduced lipogenesis and adiposity. This places SCD1 as a potential target for obesity therapies; however, our understanding of the mechanisms linking SCD1 with changes in adipocyte function remains unclear. This thesis provides important new insights into how SCD1 impacts adipocyte FA metabolism and cellular function. Using a specific SCD1 inhibitor, changes in lipid metabolism, global gene expression, inflammation, cellular stress, and basal insulin signaling were assessed in 3T3-L1 adipocytes. Results demonstrated that SCD1 inhibition caused a reduction in TAGs and phospholipids, which coincided with the downregulation of genes associated with the biosynthesis of these lipid fractions. Cellular diacylglycerols were increased with SCD1 inhibition and insulin signaling was partially impaired. In contrast, markers of cellular stress were unaltered. Furthermore, the FA composition of each lipid fraction was dramatically modified, with SCD1-inhibited adipocytes specifically upregulating the elongation of PA to SA. Stable isotope tracer experiments revealed that this elongation was occurring via elongase 6. Additionally, reduced SCD1 activity in adipocytes exacerbated the effects of exogenous SA on markers of inflammation. Taken together, SCD1 activity has many indirect influences on adipocyte metabolism in addition to its role in FA desaturation. Importantly, SCD1 facilitates the storage of FAs in TAGs and the ability of adipocytes to handle exogenous FAs. SCD1 also prevents saturated FA and DAG accumulation, and preserves insulin signaling in adipocytes. Ultimately this thesis highlights the importance of SCD1 in the maintenance of adipocyte cellular function, and emphasizes the wide-ranging impact of SCD1 on adipocyte FA metabolism.

Gas Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Determination of Isotopologue and Tandem Mass Isotopomer Fractions of Primary Metabolites for (13)C-Metabolic Flux Analysis.

For the first time an analytical work flow based on accurate mass gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOFMS) with chemical ionization for analysis providing a comprehensive picture of (13)C distribution along the primary metabolism is elaborated. The method provides a powerful new toolbox for (13)C-based metabolic flux analysis, which is an emerging strategy in metabolic engineering. In this field, stable isotope tracer experiments based on, for example, (13)C are central for providing characteristic patterns of labeled metabolites, which in turn give insights into the regulation of metabolic pathway kinetics. The new method enables the analysis of isotopologue fractions of 42 free intracellular metabolites within biotechnological samples, while tandem mass isotopomer information is also accessible for a large number of analytes. Hence, the method outperforms previous approaches in terms of metabolite coverage, while also providing rich isotopomer information for a significant number of key metabolites. Moreover, the established work flow includes novel evaluation routines correcting for isotope interference of naturally distributed elements, which is crucial following derivatization of metabolites. Method validation in terms of trueness, precision, and limits of detection was performed, showing excellent analytical figures of merit with an overall maximum bias of 5.8%, very high precision for isotopologue and tandem mass isotopomer fractions representing >10% of total abundance, and absolute limits of detection in the femtomole range. The suitability of the developed method is demonstrated on a flux experiment of Pichia pastoris employing two different tracers, i.e., 1,6(13)C2-glucose and uniformly labeled (13)C-glucose.

Anaerobic ammonium oxidation and its contribution to nitrogen removal in China's coastal wetlands.

Over the past several decades, human activities have caused substantial enrichment of reactive nitrogen in China’s coastal wetlands. Although anaerobic ammonium oxidation (anammox), the process of oxidizing ammonium into dinitrogen gas through the reduction of nitrite, is identified as an important process for removing reactive nitrogen, little is known about the dynamics of anammox and its contribution to nitrogen removal in nitrogen-enriched environments. Here, we examine potential rates of anammox and associate them with bacterial diversity and abundance across the coastal wetlands of China using molecular and isotope tracing techniques. High anammox bacterial diversity was detected in China’s coastal wetlands and included Candidatus Scalindua, Kuenenia, Brocadia, and Jettenia. Potential anammox rates were more closely associated with the abundance of anammox bacteria than to their diversity. Among all measured environmental variables, temperature was a key environmental factor, causing a latitudinal distribution of the anammox bacterial community composition, biodiversity and activity along the coastal wetlands of China. Based on nitrogen isotope tracing experiments, anammox was estimated to account for approximately 3.8–10.7% of the total reactive nitrogen removal in the study area. Combined with denitrification, anammox can remove 20.7% of the total external terrigenous inorganic nitrogen annually transported into China’s coastal wetland ecosystems.

Free radical mechanism investigation of the side-chain alkylation of toluene with methanol on basic zeolites X

The side-chain alkylation of toluene represents a novel, environmentally friendly, and low cost route for the production of styrene. However, the yield of styrene produced in this way is currently low, and the mechanism responsible for the side-chain alkylation of toluene is poorly understood. Furthermore, the reason for the higher catalytic efficiency of CsX over NaX and KX remains unclear. In this work, the free radical mechanism of the side-chain alkylation of toluene over basic zeolite X has been elucidated using quantum chemical calculations, together with isotope tracing experiments and the reaction between p-nitrotoluene and methanol. The adsorption isotherm of methanol showed that Cs+ ions could block methanol from accessing the β-cage, which is where the side-chain alkylation reaction occurred. Furthermore, the H–D exchange results between toluene and deuterated toluene (C6D5CD3) showed that CsX was more efficient as a catalyst than KX for the conversion of toluene to the corresponding benzyl radical (C6H5CH2•). These two results therefore explain the higher catalytic activity of CsX towards side-chain alkylation than KX. Based on the free radical mechanism, the selectivity of styrene could be increased from 17.4% to 59.4% using CO2 as carrier gas instead of N2.

ICT: isotope correction toolbox.

Isotope tracer experiments are an invaluable technique to analyze and study the metabolism of biological systems. However, isotope labeling experiments are often affected by naturally abundant isotopes especially in cases where mass spectrometric methods make use of derivatization. The correction of these additive interferences--in particular for complex isotopic systems--is numerically challenging and still an emerging field of research. When positional information is generated via collision-induced dissociation, even more complex calculations for isotopic interference correction are necessary. So far, no freely available tools can handle tandem mass spectrometry data. We present isotope correction toolbox, a program that corrects tandem mass isotopomer data from tandem mass spectrometry experiments. Isotope correction toolbox is written in the multi-platform programming language Perl and, therefore, can be used on all commonly available computer platforms. Source code and documentation can be freely obtained under the Artistic License or the GNU General Public License from: https://github.com/jungreuc/isotope_correction_toolbox/ {christian.jungreuthmayer@boku.ac.at,juergen.zanghellini@boku.ac.at} Supplementary data are available at Bioinformatics online.

GLYCOLATE OXIDASE3, a Glycolate Oxidase Homolog of Yeast l-Lactate Cytochrome c Oxidoreductase, Supports l-Lactate Oxidation in Roots of Arabidopsis.

In roots of Arabidopsis (Arabidopsis thaliana), l-lactate is generated by the reduction of pyruvate via l-lactate dehydrogenase, but this enzyme does not efficiently catalyze the reverse reaction. Here, we identify the Arabidopsis glycolate oxidase (GOX) paralogs GOX1, GOX2, and GOX3 as putative l-lactate-metabolizing enzymes based on their homology to CYB2, the l-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses l-lactate with a similar efficiency to glycolate; in contrast, the photorespiratory isoforms GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than l-lactate. The key factor making GOX3 more efficient with l-lactate than GOX1 and GOX2 is a 5- to 10-fold lower Km for the substrate. Consequently, only GOX3 can efficiently metabolize l-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-of-function and overexpressor plants indicate that l-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on l-lactate as a sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicating that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss of function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes l-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of l-lactate after its formation under normoxia.

Sauna, sweat and science – quantifying the proportion of condensation water versus sweat using a stable water isotope (2H/1H and 18O/16O) tracer experiment

Most visitors of a sauna appreciate the heat pulse that is perceived when water is poured on the stones of a sauna stove. However, probably only few bathers are aware that this pleasant heat pulse is caused by latent heat being released onto our skin due to condensation of water vapour. In order to quantify the proportion of condensation water versus sweat to dripping water of test persons we conducted sauna experiments using isotopically labelled (δ18O and δ2H) thrown water as tracer. This allows differentiating between ‘pure sweat’ and ‘condensation water’. Two ways of isotope mass balance calculations were applied and yielded similar results for both water isotopes. Accordingly, condensation contributed considerably to dripping water with mean proportions of 52 ± 12 and 54 ± 7 % in a sauna experiment in winter semester 2011/12 and 30 ± 13 and 33 ± 6 % in a sauna experiment in winter semester 2012/13, respectively, depending on the way of calculating the isotope mass balance. It can be concluded from the results of our dual isotope labelling sauna experiment that it is not all about sweat in the sauna.

Rapid cycling of recently fixed carbon in a Spartina alterniflora system: a stable isotope tracer experiment

Carbon dynamics in vegetated ecosystems are influenced by plants, belowground bacteria, and their interactions. Consequently, quantifying the fate of new plant production, identifying bacterial carbon sources, and evaluating plant–microbe interactions can provide insight to carbon cycling and storage. To follow short-term carbon transformations in a Spartina alterniflora—soil system, we applied 13C-labeled CO2 to aboveground leaves and chased it belowground into roots and bacterial lipids. Plant mesocosms were exposed to 13CO2 for 0, 1, 3, or 6 h. Incorporation of 13CO2 by plants and soil microbes was measured immediately after the incubation (Day 0) and 24 h later (Day 1). During a 24 h period, 41–64 % of the 13CO2 fixed by S. alterniflora was retained in leaves, 2.7–6.4 % was transferred to roots, and 30–55 % was lost via respiration. Small fractions of 13C assimilated by aboveground leaves were detected belowground in bacterial lipids on Day 1. Enrichment of lipids specific to sulfate reducing bacteria (10-methyl C16:0, cy-C17:0) indicated tight coupling between aboveground plant production and belowground anaerobic metabolisms. Overall, we found that a substantial fraction of new production was returned to the atmosphere within 24 h and that belowground bacteria were tightly coupled to plant dynamics.

Study of Si kinetics in interfacial SiO2 scavenging in HfO2 gate stacks

In this study, what occurs at the SiO2/substrate interface and how Si atoms in SiO2 behave in interfacial SiO2 scavenging in a HfO2 gate stack have been investigated. Since the same scavenging occurs on both Si and SiC substrates, the SiC substrate is used to study the role of the substrate. The characterizations of the SiO2/SiC interface show no Si growth on the substrate and no consumption of the substrate in the SiO2 scavenging. Isotope tracing experiments for a HfO2/SiO2/Si stack further demonstrate that atomic Si is generated in scavenging and diffuses out through the HfO2 layer. On the basis of these findings, the reaction at the SiO2/substrate interface is thermodynamically discussed.

The mechanism of borosilicate glass corrosion revisited

Currently accepted mechanistic models describing aqueous corrosion of borosilicate glasses are based on diffusion-controlled hydrolysis, hydration, ion exchange reactions, and subsequent re-condensation of the hydrolyzed glass network, leaving behind a residual hydrated glass or gel layer. Here, we report results of novel oxygen and silicon isotope tracer experiments with ternary Na borosilicate glasses that can be better explained by a process that involves the congruent dissolution of the glass, which is spatially and temporally coupled to the precipitation and growth of an amorphous silica layer at an inwardly moving reaction interface. Such a process is thermodynamically driven by the solubility difference between the glass and amorphous silica, and kinetically controlled by glass dissolution reactions at the reaction front, which, in turn, are controlled by the transport of water and solute elements through the growing corrosion zone. Understanding the coupling of these reactions is the key to understand the formation of laminar or more complex structural and chemical patterns observed in natural corrosion zones of ancient glasses. We suggest that these coupled processes also have to be considered to realistically model the long-term performance of silicate glasses in aqueous environments.

Development of a stable MnCo2O4 cocatalyst for photocatalytic CO2 reduction with visible light.

The synthesis of uniform MnCo2O4 microspheres and their cooperation with a visible light harvester to achieve efficient photocatalytic CO2 reduction under ambient conditions are reported here. The MnCo2O4 materials were prepared by a facile two-step solvothermal-calcination method and were characterized by XRD, SEM, TEM, EDX, XPS, elemental mapping, and N2 adsorption measurements. By using the MnCo2O4 microspheres as a heterogeneous cocatalyst, the photocatalytic performance of the CO2-to-CO conversion catalysis was remarkably enhanced, and no decrease in the promotional effect of the cocatalyst was observed after repeatedly operating the reaction for six cycles. (13)CO2 isotope tracer experiments verified that the CO product originated from the CO2 reactant. The effect of synthetic conditions and various reaction parameters on the photocatalytic activity of the system were investigated and optimized. The stability of the MnCo2O4 cocatalyst in the CO2 reduction system was confirmed by several techniques. Moreover, a possible mechanism for MnCo2O4-cocatalyzed CO2 photoreduction catalysis is proposed.

Analysis of Cell Metabolism Using LC-MS and Isotope Tracers.

Here we discuss our methods to analyze small polar compounds involved in central carbon metabolism using LC-MS. Methods described include sample extraction procedures for cells and medium, as well as for plasma/serum, urine, CSF, and tissue samples. Different extraction solvents are assessed. Our methods for using (13)C stable isotope tracers to examine the kinetics and distributions of mass isotopologues of many metabolites are discussed. Quantification methods are described for (13)C stable isotope tracer experiments as well as for unlabeled experiments. These methods were applied in a fumarate hydratase deficient cell model to show how isotope tracing can demonstrate shifts in metabolic pathways and, together with metabolite exchange rates, can be used to gain insights into changes in cell metabolism.

Metal-free synthesis of ortho-CHO diaryl ethers by a three-component sequential coupling.

A practical, metal-free, and highly chemoselective approach was developed for the synthesis of ortho-CHO diaryl ethers by a three-component sequential coupling of arynes, N,N-dimethylformamide (DMF), and diaryliodonium salts. Diverse functional groups including halo, nitryl, and bulky substituents and heteroaromatics are well tolerated. Mechanistically, isotopic tracer experiments reveal that the diaryliodonium salt serves as an electrophile to trap the transient intermediates generated from the [2 + 2] cyclization of an aryne and DMF.

Molecular evidence of digestion and absorption of epibiotic bacterial community by deep-sea crab Shinkaia crosnieri.

The hydrothermal vent crab Shinkaia crosnieri is considered to obtain nutrition from the epibiotic bacteria found on the setae, but previous studies have not shown how nutrients can be transferred from the epibionts to the host. In this study, microscopic observations of S. crosnieri intestinal components detected autofluorescent setae fragments and pigmentation derived from the digestion of epibionts in a dye-stained epibiont tracer experiment. An in vitro digestion experiment with epibiotic populations using an intestinal extract demonstrated the degradation of epibiotic cells by digestive enzymes. A phylogenetic analysis showed that many of the bacterial 16S ribosomal RNA gene sequences obtained from the intestine were closely related to the sequences of the epibionts, thus they were probably derived from the epibionts. A stable isotope tracer experiment also indicated that (13)C assimilated by the epibionts provided a carbon (nutrition) source for the host. Both activity measurements and isotope studies showed that chemosynthetic metabolism by the gut microbial components were inactive. Together with the feeding behaviour of living S. crosnieri, these results indicate that S. crosnieri ingests the epibionts using maxillipeds and assimilates them via its digestive organs as a nutrient source. The results of this study elucidate the mechanism of nutritional transfer in ectosymbiosis between chemosynthetic bacteria and deep-sea invertebrates.

You are not always what we think you eat: selective assimilation across multiple whole‐stream isotopic tracer studies

Analyses of 21 15N stable isotope tracer experiments, designed to examine food web dynamics in streams around the world, indicated that the isotopic composition of food resources assimilated by primary consumers (mostly invertebrates) poorly reflected the presumed food sources. Modeling indicated that consumers assimilated only 33–50% of the N available in sampled food sources such as decomposing leaves, epilithon, and fine particulate detritus over feeding periods of weeks or more. Thus, common methods of sampling food sources consumed by animals in streams do not sufficiently reflect the pool of N they assimilate. Isotope tracer studies, combined with modeling and food separation techniques, can improve estimation of N pools in food sources that are assimilated by consumers. Food web studies that use putative food samples composed of actively cycling (more readily assimilable) and refractory (less assimilable) N fractions may draw erroneous conclusions about diets, N turnover, and trophic linkages of consumers. By extension, food web studies using stoichiometric or natural abundance approaches that rely on an accurate description of food‐source composition could result in errors when an actively cycling pool that is only a fraction of the N pool in sampled food resources is not accounted for.

Fe(II)-catalyzed recrystallization of goethite revisited.

Results from enriched (57)Fe isotope tracer experiments have shown that atom exchange can occur between structural Fe in Fe(III) oxides and aqueous Fe(II) with no formation of secondary minerals or change in particle size or shape. Here we derive a mass balance model to quantify the extent of Fe atom exchange between goethite and aqueous Fe(II) that accounts for different Fe pool sizes. We use this model to reinterpret our previous work and to quantify the influence of particle size and pH on extent of goethite exchange with aqueous Fe(II). Consistent with our previous interpretation, substantial exchange of goethite occurred at pH 7.5 (≈ 90%) and we observed little effect of particle size between nanogoethite (average size of 81 × 11 nm; ≈ 110 m(2)/g) and microgoethite (average size of 590 × 42 nm; ≈ 40 m(2)/g). Despite ≈ 90% of the bulk goethite exchanging at pH 7.5, we found no change in mineral phase, average particle size, crystallinity, or reactivity after reaction with aqueous Fe(II). At a lower pH of 5.0, no net sorption of Fe(II) was observed and significantly less exchange occurred accounting for less than the estimated proportion of surface Fe atoms in the particles. Particle size appears to influence the amount of exchange at pH 5.0 and we suggest that aggregation and surface area may play a role. Results from sequential chemical extractions indicate that (57)Fe accumulates in extracted Fe(III) goethite components. Isotopic compositions of the extracts indicate that a gradient of (57)Fe develops within the goethite with more accumulation of (57)Fe occurring in the more easily extracted Fe(III) that may be nearer to the surface.

Imaging of trace elements in tissues: with a focus on laser ablation inductively coupled plasma mass spectrometry.

Elemental imaging techniques are capable of showing the spatial distribution of elements in a sample. Their application in biomedical sciences is promising, but they are not yet widely employed. The review gives a short overview about techniques available and then focuses on the advantages of using laser ablation inductively coupled plasma mass spectrometry for elemental bioimaging. Current examples for the use of elemental imaging with medical context are given to illustrate the potential of this type of analysis for clinical applications. Recently, synchrotron-based techniques and laser ablation inductively coupled plasma mass spectrometry have been successfully applied to analyse the spatial distribution of elements in biological samples of medical relevance. Elemental bioimaging methods have a great potential for medical applications. They are complementary to molecular imaging and histological staining and are especially attractive when used in combination with stable isotope tracer experiments.

Hydrogen Storage Materials Comprising Conjugated Hydrocarbon Polymers with LiH: Comparison of Cyclic Durability between LiH–Polyacetylene, −Poly(p-phenylene), and −Poly(diphenylacetylene) and Mechanistic Investigation upon LiH–Poly(p-phenylene)

Hydrogen storage and release properties of composites were compared: lithium hydride with conjugated macromolecules of polyacetylene (PA), poly(p-phenylene) (PPP), and poly(diphenylacetylene) (PDPA). Cyclic hydrogen absorption and desorption experiments showed that LiH–PPP has better durability than either LiH–PA or LiH–PDPA, probably because the higher structural stability of PPP prevented unfavorable side reactions that might inactivate LiH. Mechanistic investigations of hydrogen release reactions on LiH–PPP were conducted using isotopic tracer experiments, electrical conductivity measurements, and IR and NMR spectroscopy. Those results indicate that the hydrogen release reaction proceeded via electron transfer from hydride ion to conjugation systems of polymer molecules. This proposed mechanism is identical to those for the LiH–PA composite.