High-precision Isotope Ratio Mass Spectrometry Research Articles

Natural Carbon Isotope Abundance of Plasma Metabolites and Liver Tissue Differs between Diabetic and Non-Diabetic Zucker Diabetic Fatty Rats

Background‘You are what you eat’ is an accurate summary for humans and animals when it comes to carbon isotope abundance. In biological material, natural13C/12C ratio is subject to minute variations due to diet composition (mainly from ingestion of C3 and C4 metabolism plants) and to the discrimination between ‘light’ and ‘heavy’ isotopes during biochemical reactions (isotope effects and isotopic fractionation).Methodology/Principal FindingsCarbon isotopic abundance was measured in ZDF (fa/+) and ZDF (fa/fa), (lean and obese-diabetic rats respectively) fed the same diet. By analysing plasma metabolites (glucose and non-esterified fatty acids), breath and liver tissue by high-precision isotope ratio mass spectrometry, we demonstrate for the first time statistically distinguishable metabolic carbon isotope abundance between ZDF (fa/+) and ZDF (fa/fa) rats based on plasma glucose, palmitic, oleic, linoleic, arachidonic acids and bulk analysis of liver tissue (P<0.005) resulting into clear isotopic fingerprints using principal component analysis. We studied the variation of isotopic abundance between both groups for each metabolite and through the metabolic pathways using the precursor/product approach. We confirmed that lipids were depleted in 13C compared to glucose in both genotypes. We found that isotopic abundance of linoleic acid (C18: 2n-6), even though both groups had the same feed, differed significantly between both groups. The likely reason for these changes between ZDF (fa/+) and ZDF (fa/fa) are metabolic dysregulation associated with various routing and fluxes of metabolites.Conclusion/SignificanceThis work provides evidence that measurement of natural abundance isotope ratio of both bulk tissue and individual metabolites can provide meaningful information about metabolic changes either associated to phenotype or to genetic effects; irrespective of concentration. In the future measuring the natural abundance δ13C of key metabolites could be used as endpoints for studying in vivo metabolism, especially with regards to metabolic dysregulation, and development and progression of metabolic diseases.

Effect of Artificial Reconstitution of the Interaction between the Plant Camptotheca acuminata and the Fungal Endophyte Fusarium solani on Camptothecin Biosynthesis

Fungal endophytes inhabit healthy tissues of all terrestrial plant taxa studied and occasionally produce host-specific compounds. We recently isolated an endophytic fungus, Fusarium solani, from Camptotheca acuminata, capable of biosynthesizing camptothecin (CPT, 1), but this capability substantially decreased on repeated subculturing. The endophyte with an impaired 1 biosynthetic capability was artificially inoculated into the living host plants and then recovered after colonization. Although the host-endophyte interaction could be reconstituted, biosynthesis of 1 could not be restored. Using a homology-based approach and high-precision isotope-ratio mass spectrometry (HP-IRMS), a cross-species biosynthetic pathway is proposed where the endophyte utilizes indigenous G10H (geraniol 10-hydroxylase), SLS (secologanin synthase), and TDC (tryptophan decarboxylase) to biosynthesize precursors of 1. However, the endophyte requires host STR (strictosidine synthase) in order to condense the nitrogen-containing moiety (tryptamine, 2) with the carbon-containing moiety (secologanin, 3) to form strictosidine (4) and complete the biosynthesis of 1. Biosynthetic genes of 1 in the seventh subculture generation of the endophyte revealed random and unpredictable nonsynonymous mutations. These random base substitutions led to dysfunction at the amino acid level. The controls, Top1 gene and rDNA, remained intact over subculturing, revealing that instability of biosynthetic genes of 1 was not reflected in the primary metabolic processes and functioning of the housekeeping genes. The present results reveal the causes of decreased production of 1 on subculturing, which could not be reversed by host-endophyte reassociation.

Mixed‐mode chromatography/isotope ratio mass spectrometry

Liquid chromatography coupled to molecular mass spectrometry (LC/MS) has been a standard technique since the early 1970s but liquid chromatography coupled to high-precision isotope ratio mass spectrometry (LC/IRMS) has only been available commercially since 2004. This development has, for the first time, enabled natural abundance and low enrichment delta(13)C measurements to be applied to individual analytes in aqueous mixtures creating new opportunities for IRMS applications, particularly for the isotopic study of biological molecules. A growing number of applications have been published in a range of areas including amino acid metabolism, carbohydrates studies, quantification of cellular and plasma metabolites, dietary tracer and nucleic acid studies. There is strong potential to extend these to new compounds and complex matrices but several challenges face the development of LC/IRMS methods. To achieve accurate isotopic measurements, HPLC separations must provide baseline-resolution between analyte peaks; however, the design of current liquid interfaces places severe restrictions on compatible flow rates and in particular mobile phase compositions. These create a significant challenge on which reports associated with LC/IRMS have not previously focused. Accordingly, this paper will address aspects of chromatography in the context of LC/IRMS, in particular focusing on mixed-mode separations and their benefits in light of these restrictions. It aims to provide an overview of mixed-mode stationary phases and of ways to improve high aqueous separations through manipulation of parameters such as column length, temperature and mobile phase pH. The results of several practical experiments are given using proteogenic amino acids and nucleosides both of which are of noted importance in the LC/IRMS literature. This communication aims to demonstrate that mixed-mode stationary phases provide a flexible approach given the constraints of LC/IRMS interface design and acts as a practical guide for the development of new chromatographic methods compatible with LC/IRMS applications.

Analysis of quantization error in high-precision continuous-flow isotope ratio mass spectrometry

High-precision isotope ratio mass spectrometry (IRMS) systems are equipped with digitizers that deliver effective maximum digitization depths of 16 to 24 bits; however, there are no analyses of the proper board depth required to retain high precision in continuous-flow techniques. We report an experimental and theoretical evaluation of quantization error in continuous-flow IRMS (CF-IRMS). CO 2 samples (100 pmol–30 nmol) were injected into a gas chromatography combustion IRMS system (GC–CIRMS). The analog signal was digitized by high precision, 24-bit ADC boards at 10 Hz, and was post-processed to simulate 12, 14, and 16-bit data sets. δ 13 C pdb values were calculated for all data sets by the conventional “summation” method or by curve-fitting the chromatographic peaks to the exponentially modified Gaussian (EMG) function. Benchmarks of S.D.( δ 13 C pdb ) = 0.3, 0.6, and 1.0‰ were considered to assess precision. In the presence of significant quantization noise, curve-fitting required several-fold less CO 2 than the summation method to reach a given benchmark. We derived an equation to describe the theoretical limitations of precision for the summation method as a function of CO 2 admitted to the source and the step size of the boards. Theory was in close agreement with the observed lower limit of precision for the simulated 16-bit data set. Curve-fitting achieved a precision of S.D. <0.3‰ for injections 20-fold smaller than summation for CO 2 samples collected on an IRMS with 16-bit resolution. By mitigating the impact of quantization noise, curve-fitting expands the dynamic range within a single run to include lower analyte levels, and effectively reduces the need for high pumping capacities and high precision ADC boards.

High-Precision Isotope Ratio Mass Spectrometry and Stable Isotope Precursors for Tracer Studies in Cell Culture

The use of stable isotope-labeled tracers is demonstrated in an in vitro system with analysis by high-precision isotope ratio mass spectrometry (IRMS), using n-3 long-chain polyunsaturated fatty acid (LCP) biosynthesis from [U-13C]18:3n-3 (18:3n-3*) in Y79 human retinoblastoma cells as a model system. The cells were cultured as a suspension in RPMI 1640 medium supplemented with 15% fetal calf serum at 37°C with 5% CO2 in air. They were harvested by sedimentation and cell lipids were extracted to determine the presence of 18:3n-3* metabolites using gas chromatography–combustion (GCC)–IRMS. As the dose of 18:3n-3* was systematically increased from treatment to treatment, the atom percent excess and the amounts of biosynthesized LCP* increased, while the percentage dose in each n-3 LCP* remained constant. Cultures incubated with 0.5 μmol (10 μM) of albumin-bound 18:3n-3, composed of 18:3n-3* diluted 1/60 or 1/100 with natural abundance 18:3n-3, yielded products with enrichments about 1.5 at.% excess (δ13CPDB < 1500‰), which is optimal for high-precision measurements. Kinetics in Y79 cells incubated with 18:3n-3* showed that n-3 LCP* incorporation increased over time; 18:3n-3*, 20:5n-3*, 22:5n-3*, and 22:6n-3* were detected at all time points with the 1/60 dilution. These data document experimental parameters for optimal stable isotope use and IRMS detection for in vitro tracer methodology.

HF-W Chronometry and Inner Solar System Accretion Rates

Models for the mechanisms of accretion of the terrestrial planets are re-examined using the experimental technique of high-precision isotope ratio mass spectrometry of tungsten (W). The decay of 182Hf to 182W (via 182Ta) provides a new kind of radiometric chronometer of planet formation processes. Hafnium and W, the parent and daughter trace elements, are highly refractory; however, Hf is lithophile and strongly partitioned into the silicate portion of a planet, whereas W is moderately siderophile and preferentially partitioned into a coexisting metallic phase. More than 90% of terrestrial W has gone into the Earth’s core during its formation. The residual silicate portion, the Earth’s primitive mantle, has a Hf/W ratio in the range 10–40, an order of magnitude higher than chondritic (~1.3). Tungsten isotopic data for the Earth and the Moon suggest that we can date a major event of planet formation: The Moon formed about 50 Myrs after the start of the solar system, providing strong support for the Giant Impact Theory of lunar origin. Recent simulations of this event imply that the Earth was probably only half formed at the time. From this we can deduce the planetary accretion rate. Tungsten isotope data for Mars provide evidence of a much shorter accretion interval, perhaps as little as 10 Myrs, but the rates for the Earth over the same time interval could have been comparable. The large W isotopic heterogeneities on Mars could only have been produced within the first 30 Myrs of the solar system. Large-scale mixing, e.g. from convective overturn, as is thought to drive the Earth’s plates, must be absent from Mars.

12] Assessing metabolism of β-[13C]carotene using high-precision isotope ratio mass spectrometry

This chapter describes a stable isotope tracer method based on the use of highly enriched β-[U-13C]carotene and high-precision isotope ratio mass spectrometry (IRMS). Biological applications of IRMS are reviewed in the chapter. In gas chromatography combustion (GCC)–IRMS, combusted analytes are detected as the CO2+ produced in a high-sensitivity tight ion source. Each carbon atom of the analyte molecule has an independent chance of ionization and therefore detection limits are related to moles of analyte carbon, rather than to moles of analyte as in gas chromatography/mass chromatography (GC/MS). The ionization probability and molecular ion stability of CO2 is constant for all analytes. For these reasons, compounds of low ionization efficiency, low stability, and high molecular weight tend to be detected with high sensitivity by GCC–IRMS compared to conventional organic GC/MS. GCC–IRMS permits use of low doses typical of daily β-carotene intake, which do not perturb endogenous pool sizes of β-carotene or retinol. β-[13C]carotene is clearly evident in plasma by 3 hr after a dose of 2 mg β-[U-13C]carotene. In contrast, no detectable increase in concentration of labeled βC could be observed prior to 5 hr postdose with 40 mg β-carotene-d8 uring organic mass spectrometry.

Quantitative evaluation of carbon isotopic fractionation during reversed-phase high-performance liquid chromatography

The fractionation of 13C during low-performance preparative LC and high-performance LC is reported quantitatively for methyl palmitate and using high-precision isotope ratio mass spectrometry (IR-MS). For both preparative and high-performance analytical columns, 13C enrichment is about 7‰ greater than the parent starting material, drops sharply in the first section of the peak and then settles to a value about 1‰ below that of the starting material. Recycling over a single HPLC column did not induce greater fractionation. These results emphasize the importance of quantitative peak collection for high-precision IR-MS studies, particularly the first part of the peak where the isotope ratio changes rapidly.

The Absolute Isotopic Composition and Atomic Weight of Terrestrial Nickel.

Twenty-nine samples of high-purity nickel metals, reagent salts and minerals, collected from worldwide sources, have been examined by high-precision isotope ratio mass spectrometry for their nickel isotopic composition. These materials were compared directly with SRM 986, certified isotopie standard for nickel, using identical measurement techniques and the same instrumentation. This survey shows no statistically significant variations among the samples investigated, indicating that the certified atomic weight and associated uncertainty for SRM 986 is applicable to terrestrial nickel samples. The atomic weight calculated for SRM 986 is 58.69335±0.00015 [2]. The currently recommended IUPAC value for terrestrial nickel is 58.69±0.01.

Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor

Solvent exchange of 18O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectrometry. Buried water is labeled by equilibration of the protein in 18O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18O label eluting with the protein in 10-300 s is determined by an H2O-CO2 equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH greater than or equal to 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 18O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18O labeling of carboxyl groups at low pH. In addition to 18O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent (static accessibility greater than 0) and hydrogen-bonded main chain O, and their pH min is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate.