Climatic significance of isotope ratios

The hydrogen and oxygen isotope ratios of water, which can be measured by Isotope Ratio Mass Spectrometry (IRMS), exhibit climatic dependencies and are commonly exploited in hydrogeology. More generally, the overall carbon or hydrogen isotope ratios of plant organic matter, and in particular of tree-ring cellulose, have been frequently used for climatic reconstruction. However, since many physicochemical and biochemical fractionation phenomena are likely to contribute to the isotopic values, the interpretation of the climatic significance of isotopic parameters is not always straightforward. In the case of hydrogen and oxygen for instance, the climatic profile of the source meteoric water is not simply transferred to leaf water and many steps of the biosyntheses are accompanied by kinetic and thermodynamic isotope effects that depend on the individual mechanistic pathways. The information brought about by overall isotope ratios determined by IRMS is averaged over all fractionation effects undergone at the different molecular positions. In contrast, the NMR investigation of Site-specific Natural Isotope Fractionation (SNIF-NMR) gives simultaneous access to isotope ratios specific to individual positions in the molecule. Since the different atoms do not necessarily exhibit the same climatic dependency, the method provides complementary responses to the environmental conditions. In particular, the isotopic parameters of ethanol and water obtained by fermenting sugars in standardized conditions reflect climatic influences which took place at different periods of plant growth. As a consequence, statistical analyses based on multi-site isotopic variables provide powerful criteria for distinguishing geographical regions of cultivation characterized by different climatic features. Although the sensitivity to climatic variations is the most pronounced for plant water and for sugars formed at the first step of photosynthesis, other components such as lipids or minor metabolites also exhibit climatic dependencies. The combination of isotopic values pertaining to different atomic species and either averaged over the whole molecule (IRMS) or associated with different molecular sites (SNIF-NMR), provides complementary criteria, which can be exploited in terms of both climatic significance and mechanistic pathways of the individual atoms.     

Isotopic effects in the electronic spectra of tryptophan

Summary. No influence of isotopic substitution in deuterium-substituted tryptophan on the florescence excitation spectrum has previously been found out. Here, the isotopic effects of electronic excitation of deuterium-substituted tryptophan were experimentally and theoretically analyzed for first time. It was shown a short-wave shift of the UV-absorption maximum at 220 nm corresponding to the 360 cal/mol and short-wave shift for fluorescence spectrum corresponding to the 210 cal/mol. To account for this effect, the quantum chemical calculations of the geometric and electron structure, frequencies of normal vibrations and transition energies have been performed. The isotopic effects originate from the zero-point energies of ground and excited states. It was found that isotopic shifts depend on the position of isotope in the molecule and kind of transition. So, it can be utilized in the analysis of proteins structure and complexation.     

A novel strategy for the targeted analysis of protein and peptide metabolites

In many biological applications such as epitope discovery or drug metabolism studies, the detection of naturally processed exogenous proteins (e.g. vaccines or peptide therapeutics) and their metabolites is frequently complicated by the presence of a complex endogenous mixture of closely related or even identical compounds. We describe a method that incorporates stable isotope labelling of the protein of interest, allowing the selective screening of the intact molecule and all metabolites using a modified precursor ion scan. This method involves monitoring the low-molecular-weight fragment ions produced during MS/MS that distinguish isotopically labelled peptides from related endogenous compounds. All isotopically labelled peptides can be selected using this method. The technique makes no assumptions about the processed or post-translational state of the peptide, and hence can selectively screen out modified peptides that would otherwise be missed by single reaction monitoring approaches. This method does not replace single reaction monitoring or regular precursor scanning techniques; instead, it is a method that can be used when the assumptions required for the former two techniques cannot be predicted. The potential for this technique to be used in metabolism and pharmacokinetic experiments is discussed with specific examples looking at the metabolism of α-synuclein in serum and the brain.     

Natural Abundance Isotopic Fractionation in the Fermentation Reaction: Influence of the Nature of the Yeast

Site-specific natural isotope fractionation studied by nuclear magnetic resonance (SNIF-NMR) provides isotopic criteria that characterize a biochemical transformation such as fermentation and enable isotopic ratios measured in end products to be correlated with those of their precursors. In principle, a given set of transfer coefficients applies only to bioconversions performed under strictly identical conditions, a situation that is hardly fulfilled in most usual fermentation processes. In particular, natural raw materials such as fruits frequently involve complex mixtures of various yeast strains present at different concentrations. Series of experiments performed with different yeasts, different concentrations of car- bohydrates, and different yields of the transformation have shown that, although glycolysis is associated with overall hydrogen fractionation effects that may exceed 40 ppm, the range of variation in the isotopic ratios of the fermentation products, ethanol and water, does not exceed a few parts per million. Provided that the yield in ethanol reaches values higher than 70%, the nature of the yeast strain has minimal influence on the isotopic ratio of the methyl site of ethanol (D/H)_I. In contrast, the isotope ratio of the methylene site, (D/H)_II, may exhibit significant enhancements, in particular when ethanol is left in contact for a long time with poorly alcohologenic yeasts. These behaviors are consistent with hydrogen transfers from the aqueous medium to the methylene site, and partly to the methyl site, occurring with relatively high kinetic isotope effects. Since water acts as an open pool of hydrogens, however, only small isotopic variations are produced in the course of the fermentation reaction. Moreover, the partial connection between hydrogens from the methyl site of ethanol and hydrogens from glucose operates with relatively small secondary isotopic effects. No significant changes in the percentages of intra- and inter-molecular transfers of hydrogen to the methyl site are observed as a function of the nature of the yeast. These results support the use of the methyl isotopic ratio of ethanol as a probe of the isotopic behavior of carbohydrate precursors, whatever the yeast strains present in natural fermentation media.     

Can heavy isotopes increase lifespan? Studies of relative abundance in various organisms reveal chemical perspectives on aging

Stable heavy isotopes co‐exist with their lighter counterparts in all elements commonly found in biology. These heavy isotopes represent a low natural abundance in isotopic composition but impose great retardation effects in chemical reactions because of kinetic isotopic effects (KIEs). Previous isotope analyses have recorded pervasive enrichment or depletion of heavy isotopes in various organisms, strongly supporting the capability of biological systems to distinguish different isotopes. This capability has recently been found to lead to general decline of heavy isotopes in metabolites during yeast aging. Conversely, supplementing heavy isotopes in growth medium promotes longevity. Whether this observation prevails in other organisms is not known, but it potentially bears promise in promoting human longevity.     

(12)C/(13)C fractionations in plant primary metabolism

Natural (13)C abundance is now an unavoidable tool to study ecosystem and plant carbon economies. A growing number of studies take advantage of isotopic fractionation between carbon pools or (13)C abundance in respiratory CO(2) to examine the carbon source of respiration, plant biomass production or organic matter sequestration in soils. (12)C/(13)C isotope effects associated with plant metabolism are thus essential to understand natural isotopic signals. However, isotope effects of enzymes do not influence metabolites separately, but combine to yield a (12)C/(13)C isotopologue redistribution orchestrated by metabolic flux patterns. In this review, we summarise key metabolic isotope effects and integrate them into the corpus of plant primary carbon metabolism.     

SIMS microscopy: a tool to measure the intracellular concentration of carbon 14-labelled molecules.

Monolayer cultures of human fibroblasts were incubated for 24 h with 14C-arginine and observed by means of SIMS microscopy (ion microscopy). Carbon 14 imaging showed the intracellular distribution of labelled arginine which featured high nuclear incorporation. The local concentration of this amino acid in different cells and intracellular structures was assessed through local isotopic 14C/12C ratio measurement. This relates the signal intensity of the labelling isotope carbon 14 to that of the corresponding natural isotope (carbon 12) of known tissular concentration. Using this method we were able to measure minor variations in the molecular concentration of arginine (expressed as mumol/g of tissue) between different fibroblasts. Results of this study indicate that SIMS microscopy is well adapted to carbon 14 detection and can provide quantitative maps of the cellular and subcellular distribution of 14C-labelled molecules.     

Vacuolar compartmentation complicates the steady-state analysis of glucose metabolism and forces reappraisal of sucrose cycling in plants

Steady-state stable isotope labelling provides a method for generating flux maps of the compartmented network of central metabolism in heterotrophic plant tissues. Theoretical analysis of the contribution of the vacuole to the regeneration of glucose by endogenous processes shows that numerical fitting of isotopomeric data will only generate an accurate map of the fluxes involving intracellular glucose if information is available on the labelling of both the cytosolic and vacuolar glucose pools. In the absence of this information many of the calculated fluxes are at best unreliable or at worst indeterminate. This result suggests that the anomalously high rates of sucrose cycling and glucose resynthesis that have been reported in earlier steady-state analyses of tissues labelled with (13)C-glucose precursors may be an artefact of assuming that the labelling pattern of extracted glucose reflected the labelling of the cytosolic pool. The analysis emphasises that although subcellular information can sometimes be deduced from a steady-state analysis without recourse to subcellular fractionation, the success of this procedure depends critically on the structure of the metabolic network. It is concluded that methods need to be implemented that will allow measurement of the subcellular labelling pattern of glucose and other metabolites, as part of the routine analysis of the redistribution of label in steady-state stable isotope labelling experiments, if the true potential of network flux analysis for generating metabolic phenotypes is to be realized.     

Rapid-freeze preservation minimizes radioisotope leakage from zooplankton in feeding experiments

Preservation of labelled zooplankton by rapid freezing on dryice minimizes loss of ¹⁴C and ³²P. ¹⁴C retention in frozen samplesapproximates 100% even after 24 h storage. This is a major improvementover retention in chemical preservatives such as ethanol. Isotopeloss in ethanol is rapid (on a time scale of minutes) and extensive(up to 72%). ³²P appears to be more labile than ¹⁴C, and exposureof labelled animals to liquids must be minimized to effectivelyconserve this isotope. ³²P retention is then improved, but mayvary between 75 and 100%. We urge continued caution in feedingexperiments using ³²P, especially when dual labelling to measureselectivity.     

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as (15)N. By utilising hydroponic media that contain (15)N inorganic salts as the sole nitrogen source, near to 100% (15)N-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled (14)N- and (15)N-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of (14)N/(15)N peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the (14)N and (15)N peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct (14)N and (15)N peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2).     

A novel stable isotope labelling assisted workflow for improved untargeted LC–HRMS based metabolomics research

Many untargeted LC–ESI–HRMS based metabolomics studies are still hampered by the large proportion of non-biological sample derived signals included in the generated raw data. Here, a novel, powerful stable isotope labelling (SIL)-based metabolomics workflow is presented, which facilitates global metabolome extraction, improved metabolite annotation and metabolome wide internal standardisation (IS). The general concept is exemplified with two different cultivation variants, (1) co-cultivation of the plant pathogenic fungi Fusarium graminearum on non-labelled and highly ¹³C enriched culture medium and (2) experimental cultivation under native conditions and use of globally U-¹³C labelled biological reference samples as exemplified with maize and wheat. Subsequent to LC–HRMS analysis of mixtures of labelled and non-labelled samples, two-dimensional data filtering of SIL specific isotopic patterns is performed to better extract truly biological derived signals together with the corresponding number of carbon atoms of each metabolite ion. Finally, feature pairs are convoluted to feature groups each representing a single metabolite. Moreover, the correction of unequal matrix effects in different sample types and the improvement of relative metabolite quantification with metabolome wide IS are demonstrated for the F. graminearum experiment. Data processing employing the presented workflow revealed about 300 SIL derived feature pairs corresponding to 87–135 metabolites in F. graminearum samples and around 800 feature pairs corresponding to roughly 350 metabolites in wheat samples. SIL assisted IS, by the use of globally U-¹³C labelled biological samples, reduced the median CV value from 7.1 to 3.6 % for technical replicates and from 15.1 to 10.8 % for biological replicates in the respective F. graminearum samples.     

Rapid mixing between old and new C pools in the canopy of mature forest trees

Stable C isotope signals in plant tissues became a key tool in explaining growth responses to the environment. The technique is based on the fundamental assumption that the isotopic composition of a given unit of tissue (e.g. a tree ring) reflects the specific C uptake conditions in the leaf at a given time. Beyond the methodological implications of any deviation from this assumption, it is of physiological interest whether new C is transferred directly from sources (a photosynthesizing leaf) to structural sinks (e.g. adjacent stem tissue), or inherently passes through existing (mobile) C pools, which may be of variable (older) age. Here, we explore the fate of (13)C-labelled photosynthates in the crowns of a 30-35 m tall, mixed forest using a canopy crane. In all nine study species labelled C reached woody tissue within 2-9 h after labelling. Four months later, very small signals were left in branch wood of Tilia suggesting that low mixing of new, labelled C with old C had taken place. In contrast, signals in Fagus and Quercus had increased, indicating more intense mixing. This species-specific mixing of new with old C pools is likely to mask year- or season-specific linkages between tree ring formation and climate and has considerable implications for climate reconstruction using stable isotopes as proxies for past climatic conditions.     

Microbeam analysis of Ca exchange and uptake in the fine roots of spruce: influence of pH and aluminum

Summary A novel stable isotope labelling procedure for microbeam analysis was developed to monitor exchange and uptake of nutrients, primarily Mg, K and Ca, by root tips at the cellular level. Initially root samples were analysed from 2-year-old spruce trees, originating both from a nursery and from a polluted forest site, (1) for the cortex cell wall accessibility and nutrient binding properties, (2) for the influence of low pH and elevated aluminum concentrations on Ca binding to cortex cell walls, and (3) for long-range transport into the secondary xylem, proximal to the labelled root tip. In nursery control plants, Ca is localized mainly in the apoplast of the cortex. Exchange of Mg, K, Ca in the cell wall of the cortex and the primary xylem with label in incubation solutions is almost completed to equilibration within 30 min. In the secondary xylem we could detect Mg, K, and Ca from labelling solutions in minute amounts after 30 min, and as a major fraction after 48 h. This indicates that stable isotope labelling can be used to study both ion-exchange properties of the apoplast and long-range transport. Slight acidification of the labelling incubation media to pH 4.5 reduced Ca binding to the cortex cell walls slightly, but acidification to the extreme value of pH 2.3 reduced binding 41%. A combination of pH 4.5 and increased free aluminum reduced the binding by 83%. In a preliminary attempt to analyse the nutrient binding capability of the root-tip apoplast from pollution affected trees, we exposed fine roots of 2-year-old spruce from an acidified and polluted site showing typical low levels of Ca and Mg in the cortical cell walls to Ca-enriched media. Under these conditions the Ca content of cortex cell walls doubled upon incubation at pH 4.7, reaching 40% of the total binding capacity of our nursey control plants.     

Kinetic isotope effects significantly influence intracellular metabolite 13C labeling patterns and flux determination

Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from ¹³C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of ¹³C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the ¹³C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the ¹³C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in ¹³C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in ¹³C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.     

内蒙古巴彦淖尔市纳林套海汉墓出土人骨的稳定同位素分析

稳定同位素分析技术近年来发展为复原古代民族食物结构、社会经济模式的有效手段。本文应用该技术对内蒙古自治区纳林套海汉代墓葬出土人骨中的C、N同位素比值进行了测定。结果表明, 纳林套海汉代居民日常饮食结构中保持着非常高的动物性食物摄入, 植物类食物的摄入中以C4类植物为主。结合其他相关研究结果, 我们认为西汉王朝通过实施移民屯垦和属国等政策, 大力推动了河套地区的农业和畜牧业生产, 改变了河套地区原有的经济模式和人们的饮食结构。本文的研究结果可以为复原汉代北部边疆的经济模式研究提供有益的线索和证据。     

Synthesis of stable isotope labelled internal standards for drug-drug interaction (DDI) studies

The syntheses of stable isotope labelled internal standards of important CYP-isoform selective probes, like testosterone 1, diclofenac 3, midazolam 5, and dextromethorphan 7, as well as their corresponding hydroxylated metabolites 6β-hydroxytestosterone 2, 4'-hydroxydiclofenac 4, 1'-hydroxymidazolam 6 and dextrorphan 8 are reported. Microwave-enhanced H/D-exchange reactions applying either acid, base, or homogeneous and heterogeneous transition metal catalysis, or combinations thereof proved to be highly efficient for direct deuterium labelling of the above mentioned probes. Compared to conventional stepwise synthetic approaches, the combination of H/D exchange and biotransformation provides the potential for considerable time- and cost savings, in particular for the synthesis of the stable isotope labelled internal standards of 4'-hydroxydiclofenac 4 and 1'-hydroxymidazolam 6.     

Strategies for metabolic flux analysis in plants using isotope labelling

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.     

Relationship between large and small tumor-binding cells in the spleen and bone marrow.

By employing a distinctive feature of natural killer (NK) cells, i.e., spontaneous target cell binding, the present study aimed to follow a relatively large cohort of target-binding cells (TBC), subdivided according to size and the presence of the radioautographically labelled nucleotide, tritiated thymidine, incorporated during a 1- or 6-hour exposure period. Labelling among all TBC in the spleen was 5% by 1 h after a single injection of the isotope and this value did not change significantly after 6 h of isotope exposure. There was an insignificant increase in the labelling index of spleen-localized, labelled, large TBC throughout and beyond the isotope exposure period for at least 48 h, concomitant with a significant increase in the labelling index of spleen-localized, small TBC during the same period. In the bone marrow, small TBC showed only 2% labelling by 1 h after a single injection of isotope, while 21% of large TBC in that organ were synthesizing DNA during the same period. The results are consistent with a precursor-product relationship between large and small TBC within the bone marrow but not the spleen, and with a bone marrow-to-spleen migration of small (+/- large) TBC. Moreover, a minor population of large TBC was detected in the spleen with kinetic characteristics distinct from those of the bone marrow.     

15N stable isotope labelling of slugs (Gastropoda: Pulmonata)

Stable isotope tracers are a promising tool for investigating the ecology of terrestrial slugs, including predator‐prey relationships, migration behaviour, nutrient turnover and dietary routing. The objective of the present feasibility study was to label two economically important slug groups, Deroceras reticulatum and keeled slugs (families Limacidae and Milacidae, respectively), with the stable isotope ¹⁵N under controlled laboratory conditions. Significant isotopic enrichment in slug tissue was detected after 4 days and persisted for at least 10 days after slugs had been fed on ¹⁵N enriched food for a period of 15 days. The time course of ¹⁵N uptake into slug tissues and its relation to food consumption were well described mathematically. Estimated mean ¹⁵N assimilation efficiencies from labelled maize mixed with unlabelled wheat bran were 30% and 38%, respectively, for the species groups studied. These findings suggest that slugs can be readily and efficiently labelled and that it is feasible to devise protocols for producing large numbers of isotopically labelled slugs for use in ecological studies. A simple method is described for the collection and analysis of cutaneous mucus from individual slugs which can be used to test uniformity of isotopic labelling.