Natural Isotope Abundance in Metabolites: Techniques and Kinetic Isotope Effect Measurement in Plant, Animal, and Human Tissues

The natural isotope abundance in bulk organic matter or tissues is not a sufficient base to investigate physiological properties, biosynthetic mechanisms, and nutrition sources of biological systems. In fact, isotope effects in metabolism lead to a heterogeneous distribution of 2H, 18O, 13C, and 15N isotopes in metabolites. Therefore, compound-specific isotopic analysis (CSIA) is crucial to biological and medical applications of stable isotopes. Here, we review methods to implement CSIA for 15N and 13C from plant, animal, and human samples and discuss technical solutions that have been used for the conversion to CO2 and N2 for IRMS analysis, derivatization and isotope effect measurements. It appears that despite the flexibility of instruments used for CSIA, there is no universal method simply because the chemical nature of metabolites of interest varies considerably. Also, CSIA methods are often limited by isotope effects in sample preparation or the addition of atoms from the derivatizing reagents, and this implies that corrections must be made to calculate a proper δ-value. Therefore, CSIA has an enormous potential for biomedical applications, but its utilization requires precautions for its successful application. © 2017 Elsevier Inc.The authors thank the support of the initiative PLAISIR (Pays de la Loire Association for International Structure on “Isotopomic Research”) funded by the French Regional International Strategy Grant Pays de la Loire, and the Australian Research Council through a Future Fellowship grant (under contract FT140100645)

Intramolecular C-13 pattern in hexoses from autotrophic and heterotrophic C-3 plant tissues

The stable carbon isotope 13C is used as a universal tracer in plant eco-physiology and studies of carbon exchange between vegetation and atmosphere. Photosynthesis fractionates against 13CO2 so that source sugars (photosynthates) are on average 13C depleted by 20% compared with atmospheric CO2. The carbon isotope distribution within sugars has been shown to be heterogeneous, with relatively 13C-enriched and 13C-depleted C-atom positions. The 13C pattern within sugars is the cornerstone of 13C distribution in plants, because all metabolites inherit the 13C 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 3.2.1.26) and glucose isomerase (EC 5.3.1.5) 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 13C abundance in source and sink tissue and is, therefore, of fundamental importance to the interpretation of plant isotopic signals

Carbon allocation to major metabolites in illuminated leaves is not just proportional to photosynthesis when gaseous conditions (CO2 and O2) vary

In gas-exchange experiments, manipulating CO2 and O2 is commonly used to change the balance between carboxylation and oxygenation. Downstream metabolism (utilization of photosynthetic and photorespiratory products) may also be affected by gaseous conditions but this is not well documented. Here, we took advantage of sunflower as a model species, which accumulates chlorogenate in addition to sugars and amino acids (glutamate, alanine, glycine and serine). We performed isotopic labelling with 13CO2 under different CO2/O2 conditions, and determined 13C contents to compute 13C-allocation patterns and build-up rates. The 13C content in major metabolites was not found to be a constant proportion of net fixed carbon but, rather, changed dramatically with CO2 and O2. Alanine typically accumulated at low O2 (hypoxic response) while photorespiratory intermediates accumulated under ambient conditions and at high photorespiration, glycerate accumulation exceeding serine and glycine build-up. Chlorogenate synthesis was relatively more important under normal conditions and at high CO2 and its synthesis was driven by phosphoenolpyruvate de novo synthesis. These findings demonstrate that carbon allocation to metabolites other than photosynthetic end products is affected by gaseous conditions and therefore the photosynthetic yield of net nitrogen assimilation varies, being minimal at high CO2 and maximal at high O2.We thank the Australian Research Council for its support via a Future Fellowship awarded to G.T. under contract FT140100645

PhenoMeter: A Metabolome Database Search Tool Using Statistical Similarity Matching of Metabolic Phenotypes for High-Confidence Detection of Functional Links

This article describes PhenoMeter, a new type of metabolomics database search that accepts metabolite response patterns as queries and searches the MetaPhen database of reference patterns for responses that are statistically significantly similar or inverse for the purposes of detecting functional links. To identify a similarity measure that would detect functional links as reliably as possible, we compared the performance of four statistics in correctly top-matching metabolic phenotypes of Arabidopsis thaliana metabolism mutants affected in different steps of the photorespiration metabolic pathway to reference phenotypes of mutants affected in the same enzymes by independent mutations. The best performing statistic, the PhenoMeter Score (PM Score), was a function of both Pearson correlation and Fisher’s Exact Test of directional overlap. This statistic outperformed Pearson correlation, biweight midcorrelation and Fisher’s Exact Test used alone. To demonstrate general applicability, we show that the PhenoMeter reliably retrieved the most closely functionally-linked response in the database when queried with responses to a wide variety of environmental and genetic perturbations. Attempts to match metabolic phenotypes between independent studies were met with varying success and possible reasons for this are discussed. Overall, our results suggest that integration of pattern-based search tools into metabolomics databases will aid functional annotation of newly recorded metabolic phenotypes analogously to the way sequence similarity search algorithms have aided the functional annotation of genes and proteins. PhenoMeter is freely available at MetabolomeExpress (https://www.metabolome-express.org/phenometer.php)

Experimental evidence for a hydride transfer mechanism in plant glycolate oxidase catalysis

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C3 and C4 plant species, we analyzed kinetic parameters of purified recombinant C3 (Arabidopsis thaliana) and C4 (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The 12C/13C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the 13C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C3 and C4 enzymes

Rubisco is not really so bad

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most widespread carboxylating enzyme in autotrophic organisms. Its kinetic and structural properties have been intensively studied for more than half a century. Yet important aspects of the catalytic mechanism remain poorly understood, especially the oxygenase reaction. Because of its relatively modest turnover rate (a few catalytic events per second) and the competitive inhibition by oxygen, Rubisco is often viewed as an inefficient catalyst for CO2 fixation. Considerable efforts have been devoted to improving its catalytic efficiency, so far without success. In this review, we re-examine Rubisco's catalytic performance by comparison with other chemically related enzymes. We find that Rubisco is not especially slow. Furthermore, considering both the nature and the complexity of the chemical reaction, its kinetic properties are unremarkable. Although not unique to Rubisco, oxygenation is not systematically observed in enolate and enamine forming enzymes and cannot be considered as an inevitable consequence of the mechanism. It is more likely the result of a compromise between chemical and metabolic imperatives. We argue that a better description of Rubisco mechanism is still required to better understand the link between CO2 and O2 reactivity and the rationale of Rubisco diversification and evolution.C. B. and G. D. F. acknowledge funding by the Australian Government through the Australian Research Council Centre of Excellence for Translational Photosynthesis (Project CE140100015), and G. T. thanks the Australian Research Council for its support via a Fellowship under contract FT140100645

Covariation between oxygen and hydrogen stable isotopes declines along the path from xylem water to wood cellulose across an aridity gradient

Oxygen and hydrogen isotopes of cellulose in plant biology are commonly used to infer environmental conditions, often from time series measurements of tree rings. However, the covariation (or the lack thereof) between δ18O and δ2H in plant cellulose is still poorly understood. We compared plant water, and leaf and branch cellulose from dominant tree species across an aridity gradient in Northern Australia, to examine how δ18O and δ2H relate to each other and to mean annual precipitation (MAP). We identified a decline in covariation from xylem to leaf water, and onwards from leaf to branch wood cellulose. Covariation in leaf water isotopic enrichment (Δ) was partially preserved in leaf cellulose but not branch wood cellulose. Furthermore, whilst δ2H was well-correlated between leaf and branch, there was an offset in δ18O between organs that increased with decreasing MAP. Our findings strongly suggest that postphotosynthetic isotope exchange with water is more apparent for oxygen isotopes, whereas variable kinetic and nonequilibrium isotope effects add complexity to interpreting metabolic-induced δ2H patterns. Varying oxygen isotope exchange in wood and leaf cellulose must be accounted for when δ18O is used to reconstruct climatic scenarios. Conversely, comparing δ2H and δ18O patterns may reveal environmentally induced shifts in metabolism