Gas Isotope Ratio Mass Spectrometry Research Articles

In situ simultaneous measuring method for the determination of key processes of soil organic carbon cycling: Soil microbial respiration using laser spectrometry.

Soil microbial heterotrophic C-CO2 respiration is important for C cycling. Soil CO2 differentiation and quantification are vital for understanding soil C cycling and CO2 emission mitigation. Presently, soil microbial respiration (SR) quantification models are based on native soil organic matter (SOM) and require consistent monitoring of δ13C and CO2. We present a new apparatus for achieving in situ soil static chamber incubation and simultaneous CO2 and δ13C monitoring by cavity ring-down spectroscopy (CRDS) coupled with a soil culture and gas introduction module (SCGIM) with multi-channel. After a meticulous five-point inter-calibration, the repeatability of CO2 and δ13C values by using CRDS-SCGIM were determined, and compared with those obtained using gas chromatography (GC) and isotope ratio mass spectrometry (IRMS), respectively. We examined the method regarding quantifying SR with various concentrations and enrichment of glucose and then applied it to investigate the responses of SR to the addition of different exogenous organic materials (glucose and rice residues) into paddy soils during a 21-day incubation. The CRDS-SCGIM CO2 and δ13C measurements were conducted with high precision (<1.0µmol/mol and 1‰, respectively). The optimal sampling interval and the amount added were not exceeded 4 h and 200mg C/100g dry soil in a 1 L incubation bottle, respectively; the 13C-enrichment of 3%-7% was appropriate. The total SR rates observed were 0.6-4.2µL/h/g and the exogenous organic materials induced -49%-28% of priming effects in native SOM mineralisation. Our results show that CRDS-SCGIM is a method suitable for the quantification of soil microbial CO2 respiration, requiring less extensive lab resources than GC/IRMS.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. II: Biohydrogenation and incorporation into bacterial lipids

To maintain membrane homeostasis, ruminal bacteria synthesize branched-chain fatty acids (BCFA) or their derivatives (vinyl ethers) that are recovered during methylation procedures as branched-chain aldehydes (BCALD). Many strains of cellulolytic bacteria require 1 or more branched-chain volatile fatty acid (BCVFA). Therefore, the objective of this study was to investigate BCVFA incorporation into bacterial lipids under different dietary conditions. The study was an incomplete block design with 8 continuous culture fermenters used in 4 periods with treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were high (HF) or low forage (LF, 67 or 33% forage, 33:67 alfalfa:orchardgrass), without or with supplemental corn oil (CO; 3% dry matter, 1.5% linoleic fatty acid), and without or with 2.15 mmol/d (5 mg/d 13C each of isovalerate, isobutyrate, and 2-methylbutyrate). After methylation of bacterial pellets collected from each fermenter's effluent, fatty acids and fatty aldehydes were separated before analysis by gas chromatography and isotope ratio mass spectrometry. Supplementation of BCVFA did not influence biohydrogenation extent. Label was only recovered in branched-chain lipids. Lower forage inclusion decreased BCFA in bacterial fatty acid profile from 9.45% with HF to 7.06% with LF and decreased BCALD in bacterial aldehyde profile from 55.4% with HF to 51.4% with LF. Supplemental CO tended to decrease iso even-chain BCFA and decreased iso even-chain BCALD in their bacterial lipid profiles. The main 18:1 isomer was cis-9 18:1, which increased (P < 0.01) by 25% from CO (data not shown). Dose recovery in bacterial lipids was 43.3% lower with LF than HF. Supplemental CO decreased recovery in the HF diet but increased recovery with LF (diet × CO interaction). Recovery from anteiso odd-chain BCFA and BCALD was the greatest; therefore, 2-methylbutyrate was the BCVFA primer most used for branched-chain lipid synthesis. Recovery in iso odd-chain fatty acids (isovalerate as primer) was greater than label recovery in iso even-chain fatty acids (isobutyrate as primer). Fatty aldehydes were less than 6% of total bacterial lipids, but 26.0% of 13C recovered in lipids were recovered in BCALD because greater than 50% of aldehydes were branched-chain. Because BCFA and BCALD are important in the function and growth of bacteria, especially cellulolytics, BCVFA supplementation can support the rumen microbial consortium, increasing fiber degradation and efficiency of microbial protein synthesis.

COMPOSITION OF LIGHT HYDROCARBONS IN JURASSIC TIGHT OILS IN THE CENTRAL SICHUAN BASIN, CHINA: ORIGIN AND SOURCE ROCK CORRELATION

Crude oil reserves in tight Middle and Lower Jurassic reservoirs are of increasing exploration interest in the central Sichuan Basin, SW China. However, the origin of these “tight oils” is poorly understood. In this study, sixteen samples of light oils/condensates from tight Middle and Lower Jurassic reservoir rocks were analysed using gas chromatography (GC) and isotope ratio mass spectrometry to investigate the oils’ origin and to classify them into genetic families. The tight oils can be divided into two families. Family I oils occur in the Gongshanmiao oilfield where reservoir units comprise the Da'anzhai Member of the Lower Jurassic Ziliujing Formation, the Lower Jurassic Lianggaoshan Formation, and the First Member of the Middle Jurassic Shaximiao Formation. Family I oils are characterized by relatively low values of the methylcyclohexane (MCH) and cyclohexane (CH) indexes, low values of Mango's parameter K2 for light hydrocarbon composition, and relatively negative δ13C values ranging from ‐30.8‰ to ‐28.9‰. Family I oils are inferred to be self‐sourced by lacustrine shales in the Da'anzhai Member and the Lianggaoshan Formation in the study area, both of which are rich in sapropelic organic matter. These source rocks also charged reservoirs in the First Member of the Shaximiao Formation. By contrast, the newly discovered Family II oils, which occur at the Jinhua oilfield and the as‐yet undeveloped Qiulin and Bajiaochang structures, are reservoired in the Second Member of Shaximiao Formation. Family II oils have higher values of the MCH index, CH index and Mango's K2 parameter, and δ13C values varying from ‐27.5‰ to ‐25.4‰. These oils have similar light hydrocarbon compositions and δ13C values to oils derived from source rocks in the Upper Triassic Xujiahe Formation which contain dominantly humic organic matter. Family II oils are therefore inferred to be derived from the coaly mudstones in the Xujiahe Formation.The different compositions of the tight oils in the First and Second Members of the Shaximiao Formation appear to be controlled by the distribution and thickness of source rocks in the study area. Thus, the Gongshanmiao oilfield where Family I oils occur in the First Member is close to the depocentre of source rocks in the Da'anzhai Member and Lianggaoshan Formation. These source rocks are inferred to have charged the First Member reservoirs which may also be present in nearby oil‐ and gas‐bearing structures such as Nanchong and Yingshan. By contrast, Family II oils occur in tight reservoirs in the Second Member in areas with thick successions of Upper Triassic Xujiahe Formation mudstone source rocks, such as the Jinhua oilfield. In areas where both source rocks are present such as the Zhongtaishan and Lianchi oilfields, Shaximiao Formation reservoirs appear to contain both Family I and Family II oils.

Determination of enantiomeric and stable isotope ratio fingerprints of active secondary metabolites in neroli (Citrus aurantium L.) essential oils for authentication by multidimensional gas chromatography and GC-C/P-IRMS

Neroli essential oil (EO), extracted from bitter orange blossoms, is one of the most expensive natural products on the market due to its poor yield and its use in fragrance compositions, such as cologne. Multiple adulterations of neroli EO are found on the market, and several authentication strategies, such as enantioselective gas chromatography (GC) and isotope ratio mass spectrometry (IRMS), have been developed in the last few years. However, neroli EO adulteration is becoming increasingly sophisticated, and analytical improvements are needed to increase precision. Enantiomeric and compound-specific isotopic profiling of numerous metabolites using multidimensional GC and GC-C/P-IRMS was carried out. These analyses proved to be efficient for geographical tracing, especially to distinguish neroli EO of Egyptian origin. In addition, δ2H values and enantioselective ratios can identify an addition of 10% of petitgrain EO. These results demonstrate that enantioselective and stable isotopic metabolite fingerprint determination is currently a necessity to control EOs.

Body fat of stock-type horses predicted by rump fat thickness and deuterium oxide dilution and validated by near-infrared spectroscopy of dissected tissues.

Body condition score and percent body fat (BF; %) of horses are positively correlated with reproductive efficiency and are indicative of metabolic issues. However, BF in horses may be poorly predicted because current procedures are either subjective or dependent on one anatomical location. Therefore, the objectives of the current study were to compare 2 methods of predicting BF using rump fat thickness (RFT) and deuterium oxide (DO) dilution with actual tissue fat analysis by near-infrared spectroscopy (NIRS) in stock-type horses and to identify the relationship between BF and BCS. Twenty-four stock-type horses were selected to be humanely euthanized based on 3 primary criteria: geriatric, crippled, and/or unsafe. Approximately 20 h before slaughter, horses were weighed and BCS assessed to be 1 ( = 1; 433 kg), 2 ( = 1; 415 kg), 3 ( = 1; 376 kg), 4 ( = 7; 468 ± 13 kg), 5 ( = 10; 455 ± 11 kg), and 6 ( = 4; 493 ± 12 kg) and RFT was measured using ultrasonography. Blood samples were collected immediately before and 4 h after DO infusion (0.12 g/kg BW). Deuterium oxide concentration of plasma was determined by gas isotope ratio mass spectrometry. Horses were housed in a dry lot overnight before being individually sedated (1.1 mg xylazine/kg BW) and anesthetized using a jugular venipuncture (2.2 mg ketamine/kg BW), and potassium chloride (KCl) solution was administered to cease cardiac function before exsanguination. After euthanasia, horse carcasses were processed and dissected and tissues were collected for NIRS analysis. Body fat predicted by DO dilution was correlated with BF measured by NIRS analysis on various weight bases ( = 0.76 to 0.81, < 0.001), whereas no correlation was observed for BF predicted by RFT. A paired -test indicated that both DO dilution and RFT overestimated BF by 2.48 to 3.26% ( < 0.001) and 5.81 to 6.59% ( < 0.001), respectively, compared with NIRS analysis. Body condition scores affected BF measured by NIRS analysis ( ≤ 0.038) and predicted by DO dilution ( < 0.001) and RFT ( = 0.042). The current study provided evidence that DO dilution was a better prediction method for BF than RFT and that BCS may serve as a BF predictor in stock-type horses.

Gas chromatography and isotope ratio mass spectrometry of Pinot Noir wine volatile compounds (δ13C) and solid residues (δ13C, δ15N) for the reassessment of vineyard water-status

This paper describes a novel approach to reassess the water status in vineyards based on compound-specific isotope analysis (CSIA) of wine volatile organic compounds (δ13CVOC/VPDB) and bulk carbon and nitrogen isotopes, and the C/N molar ratios of the wine solid residues (δ13CSR/VPDB, δ15NSR/Air-N2). These analyses link gas chromatography/combustion and elemental analysis to isotope ratio mass spectrometry (GC/C/IRMS, EA/IRMS). Field-grown cultivars of Pinot Noir grapevines were exposed during six growing seasons (2009–2014) to controlled soil water availability, while maintaining identical the other environmental variables and agricultural techniques. Wines were produced from the grapes by the same oenological protocol. This permitted for the assessment of the effects in the biochemistry of wines solely induced by the changes in the plant-soil water status. This mimicked the more recurrent and prolonged periods of soil water deficiency due to climate changes. Water stress in grapevine was assessed by the measurement of the predawn leaf water potential (Ψpd) and the stable carbon isotope composition of the berry sugars during harvest (must sugars). For quantitation purposes and the normalization of the measured stable carbon isotope ratios of the VOCs, the wine samples were spiked with three standard compounds with known concentration and δ13CVPDB values. VOCs were extracted by liquid–liquid extraction and analyzed by gas chromatography/flame ionization detection (GC/FID), gas chromatography/mass spectrometry (GC/MS), and GC/C/IRMS. δ13C values were obtained for eighteen VOCs. The solid residues were obtained by freeze-drying wine aliquots and were analyzed for their C and N content and isotope composition by EA/IRMS. All the isotopic ratios (δ13CSR, δ15NSR, δ13CVOC) are highly correlated with the Ψpd values, indicating that the proposed gas chromatography and isotope ratio mass spectrometry approach is a useful tool to assess the changes in the water status of grapevine cultivars in different terroirs. The combined analytical approach was used for the first time to complement the assessment of soil water availability effects on the grapevine. The δ13C values of the volatile compounds helped confirm (or establish) their main source(s) and biosynthetic pathway(s). Importantly, we also show for the first time that the combination of C/N and δ15N values of freeze-dried wines have an unexplored potential for the study of nitrogen dynamics in soil/grape/wine systems.

Stable isotope analysis of dynamic lipidomics

Metabolic pathway flux is a fundamental element of biological activity, which can be quantified using a variety of mass spectrometric techniques to monitor incorporation of stable isotope-labelled substrates into metabolic products. This article contrasts developments in electrospray ionisation mass spectrometry (ESI-MS) for the measurement of lipid metabolism with more established gas chromatography mass spectrometry and isotope ratio mass spectrometry methodologies. ESI-MS combined with diagnostic tandem MS/MS scans permits the sensitive and specific analysis of stable isotope-labelled substrates into intact lipid molecular species without the requirement for lipid hydrolysis and derivatisation. Such dynamic lipidomic methodologies using non-toxic stable isotopes can be readily applied to quantify lipid metabolic fluxes in clinical and metabolic studies in vivo. However, a significant current limitation is the absence of appropriate software to generate kinetic models of substrate incorporation into multiple products in the time domain. Finally, we discuss the future potential of stable isotope-mass spectrometry imaging to quantify the location as well as the extent of lipid synthesis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

Characterization of tar-like material and producible oil in Misoa C2 and C3 reservoir sands of the Area 2 South (Ceuta Field, Venezuela)

Here we characterized asphaltic oil occurrences in the Misoa C2 and C3 reservoirs within Area 2 South of the Ceuta Field (Lake Maracaibo Basin, NW Venezuela). To perform the geochemical and petrological characterization of the C2 and C3 sub-units, which cover an interval in which tar-like material was detected, we analyzed core (18) and cutting (123) samples using the Rock-Eval 6 technique in reservoir mode. Samples were studied under fluorescence light and scanning electron microscopy (SEM), showing the presence of two charges of oils and the relationships within silica cements and clay minerals. To evaluate vertical inter-reservoir communication and lateral intra-reservoir continuity, we also studied 12 oil samples from wells in Area 2 South. Biomarkers and isotopic signatures were examined using gas chromatography-mass spectrometry (GC-MS) and isotope ratio mass spectrometry (IRMS). The results suggest that oil and core extracts belongs to a single family derived from two pulses of hydrocarbon generation from the La Luna source rock, which was deposited in a marine reducing environment. The oil samples are a mixture of a paleobiodegraded oil charge and a later fresh, more mature oil recharge. The geochemical analyses also evidence that the formation of heavy oil tar zones can be related to biodegradation and in-reservoir oil mixing, though other formation mechanisms for the tar are also feasible. Finally, the GC fingerprints of the oils are indicative of lateral reservoir continuities across the F-1 and F-2 faults and vertical communication between the Misoa C2 and C3 sub-units in Area 2 South, suggesting that tar occurrences do not act as permeability barriers within the two reservoirs studied.

LATE MESOLITHIC NARVA STAGE IN ESTONIA: POTTERY, SETTLEMENT TYPES AND CHRONOLOGY

This paper gives a systematized overview of different Narva stage sites in Estonia, describing their artefactual and archaeozoological material, and environmental conditions. We demonstrate the diversity of Narva stage settlement types (sites on coastal river estuaries, coast, coastal lagoons, inland river banks and shores of inland lakes) and economy (marine, terrestrial/inland aquatic and mixed subsistence) in the region. A further site-based description of Narva pottery is also provided in order to exemplify the similarities and differences of this earliest pottery type in the eastern Baltic. We also present a comprehensive list of all currently available Narva stage radiocarbon dates from Estonia and Ingermanland (north-western Russia) according to which the Narva-type pottery in the northern part of its distribution area dates to the period c. 5200-3900 cal BC. Additionally, the issues of dating food crust, especially the high risk of reservoir effect offsets, are emphasized. We carried out a comparative study dating contemporaneous food crusts and plant remains and conducting lipid residue analysis employing combined methods of gas chromatography-mass spectrometry (GC-MS) and isotope ratio mass spectrometry (GC-C-IRMS, EA-IRMS). The results demonstrate the implications and importance of characterizing lipid residues so that samples with reservoir correction can at least be identified. Introduction The beginning of pottery use is a substantial and important innovation in the history of humankind. It must have brought along a new concept not only for storage and cooking, but also changes in technology and production skills, transport and trading. The earliest pottery known to date is from eastern Asia (China, Japan and Russian Far East and eastern Siberia) and goes back to the end of the Pleistocene (e.g. Nakamura et al. 2001; Kuzmin 2006; Boaretto et al. 2009; Shevkomud & Yanshina 2012, 53). In the Baltic Sea region pottery first appears, depending on traditions of archaeological periodization, in the Early Neolithic (Finland, Russia, Latvia, Lithuania, Belarus) or Late Mesolithic period (Sweden, Denmark, Germany, Poland, Estonia) (e.g. Loze & Lijva 1989; Girininkas 2005, 105 f.; Kriiska 2009, fig. 5; Piezonka 2012, 24). Earlier in Estonia the introduction of pottery was assigned to the Neolithic period, as with other Baltic States (e.g. Jaanits et al. 1982, 61; Lang & Kriiska 2001, 89). Since the adoption of pottery generated no significant change in the local settlement pattern, subsistence economy nor, presumably, in social organization, the new periodization of the Stone Age in Estonia proceeds from the process of the introduction of domesticates. The latter occurred more than a thousand years after the adoption of pottery and resulted in major shifts in the society, settlement and economy in both Estonia and neighbouring areas (Kriiska 2009, 167; Nordqvist et al. 2015, 143). Two major pottery traditions, divided into many sub-variants, can be distinguished in the Baltic Sea region. One of the traditions embraces pottery types that comprise large pointed- or rounded-bottomed pots and, in places, also small and shallow saucer-shaped vessels (see e.g. Hallgren 2004, fig. 1). The know-how of making such vessels spread from the late 6th millennium through the early 5th millennium cal BC (e.g. Loze 1988, 101; Hallgren 2004, 136 f.; Piezonka 2008, 76; 2012, 42; Girininkas 2009, 127; Kriiska 2009, 161; Jennbert 2011, 99; Pesonen et al. 2012). Five types of early pottery are traditionally distinguished in the Baltic Sea region: (1) Ertebolle in southern Scandinavia, northern Germany and northern Poland (e.g. Hallgren 2004, 135; Jennbert 2011); (2) Early comb ware (or Sperrings) in southern Finland and north-western Russia (e.g. Luho 1957; German 2002); (3) Saraisniemi 1 in northern Finland and in Karelia, the Kola Peninsula in Russia and northern Sweden (e.g. Torvinen 2000; Haggren et al. …

Stable carbon isotope analyses of nanogram quantities of particulate organic carbon (pollen) with laser ablation nano combustion gas chromatography/isotope ratio mass spectrometry.

RationaleAnalyses of stable carbon isotope ratios (δ 13C values) of organic and inorganic matter remains have been instrumental for much of our understanding of present and past environmental and biological processes. Until recently, the analytical window of such analyses has been limited to samples containing at least several μg of carbon.MethodsHere we present a setup combining laser ablation, nano combustion gas chromatography and isotope ratio mass spectrometry (LA/nC/GC/IRMS). A deep UV (193 nm) laser is used for optimal fragmentation of organic matter with minimum fractionation effects and an exceptionally small ablation chamber and combustion oven are used to reduce the minimum sample mass requirement compared with previous studies.ResultsAnalyses of the international IAEA CH‐7 polyethylene standard show optimal accuracy, and precision better than 0.5‰, when measuring at least 42 ng C. Application to untreated modern Eucalyptus globulus (C3 plant) and Zea mays (C4 plant) pollen grains shows a ~ 16‰ offset between these species. Within each single Z. mays pollen grain, replicate analyses show almost identical δ 13C values.ConclusionsIsotopic offsets between individual pollen grains exceed analytical uncertainties, therefore probably reflecting interspecimen variability of ~0.5–0.9‰. These promising results set the stage for investigating both δ 13C values and natural carbon isotopic variability between single specimens of a single population of all kinds of organic particles yielding tens of nanograms of carbon. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Assessing the fidelity of marine vertebrate microfossil δ18O signatures and their potential for palaeo-ecological and -climatic reconstructions

Conodont biogenic apatite has become a preferred analytical target for oxygen isotope studies investigating ocean temperature and palaeoclimate changes in the Palaeozoic. Despite the growing application in geochemically-based palaeoenvironmental reconstructions, the paucity or absence of conodont fossils in certain facies necessitates greater flexibility in selection of robust oxygen-bearing compounds for analysis. Vertebrate microfossils (teeth, dermal denticles, spines) offer a potential substitute for conodonts from the middle Palaeozoic. Vertebrate bioapatite is particularly advantageous given a fossil record extending to the present with representatives across freshwater to fully marine environments, thus widening the scope of oxygen isotope studies on bioapatite. However, significant tissue heterogeneity within vertebrates and differential susceptibility of these tissues to diagenetic alteration have been raised as potential problems affecting the reliability of the oxygen isotope ratios as palaeoclimatic proxies. Well-preserved vertebrate microfossils and co-occurring conodont fossils from the Upper Devonian and Lower Carboniferous of the Lennard Shelf, Canning Basin, Western Australia, were analysed using bulk (gas isotope ratio mass spectrometry, GIRMS) and in-situ (secondary ion mass spectrometry, SIMS) methodologies, with the latter technique allowing investigation of specific tissues within vertebrate elements. The δ18Oconodont results may be interpreted in terms of palaeolatitudinally and environmentally sensible palaeo-salinity and -temperature and provide a baseline standard for comparison against vertebrate microfossil δ18O values. Despite an absence of obvious diagenetic modification, GIRMS of vertebrate denticles yielded δ18O values depleted in 18O by 2–4‰ relative to co-occurring conodonts. SIMS analysis of dentine tissues exhibited significant heterogeneity, while hypermineralised tissues in both scales and teeth produced δ18O values comparable with those of associated conodonts. The susceptibility of permeable phosphatic fossil tissues to microbial activity, fluid interaction and introduction of mineral precipitates post-formation is demonstrated in the dentine of vertebrate microfossils, which showed significant heterogeneity and consistent depletion in 18O relative to conodonts. The hypermineralised tissues present in both teeth and scales appear resistant to many diagenetic processes and indicate potential for palaeoclimatic reconstructions and palaeoecological investigations.

Thermal oxidation does not fractionate soil organic carbon with differing biological stabilities

AbstractThermal analysis techniques have been used to differentiate soil organic carbon (SOC) pools with differing thermal stability. A correlation between thermal and biological stability has been indicated in some studies, while others reported inconsistent relationships. Despite these controversial findings and no standardized method, several recently published studies used thermal analysis techniques to determine the biological stability and quality of SOC in mineral soils. This study examined whether thermal oxidation at temperature levels between 200°C and 400°C, combined with evolving gas analysis and isotope ratio mass spectrometry, is capable of identifying SOC pools with differing biological stability in mineral soils. Soil samples from three sites being under Miscanthus (C4‐plant) cultivation for more than 17 years following former agricultural cropland (only C3‐plant) cultivation were used. Due to natural shifts in 13C content, young and labile Miscanthus‐derived SOC could be distinguished from stable and old C3‐plant‐derived SOC. The proportion of Miscanthus‐derived SOC increased significantly with increasing temperatures up to 350°C in bulk soil samples, indicating increasing oxidation of labile and young SOC with increasing temperatures. Use of density fractions to validate the thermally oxidized SOC from bulk soil samples revealed that the thermal oxidation patterns did not reflect the biological stability of SOC. The suggested biologically labile particulate organic carbon (light fraction from density fractionation) was clearly enriched in Miscanthus‐derived young SOC. The thermal oxidation patterns, however, revealed preferential oxidation of these biologically labile fractions not at low temperatures, but rather at higher temperatures. The reverse was found for the biologically stable mineral‐associated density fraction (heavy fraction). Based on different soil types, it was concluded that the thermal stability of SOC between 200°C and 400°C is not a suitable indicator of the biological stability of SOC and, thus, thermal oxidation is not capable of fractionating SOC pools with differing biological stability.

Demonstration of a Novel Platform For Measuring Gut Microbiome Metabolic Dynamics

Evaluation of bacterial populations inhabiting the gut microbiome may offer insights to disease development. Further, dietary intake of carbohydrates and a Western diet may shape the metabolic dynamics of the gut microbiome. However, the ability to quantify metabolic dynamics in these bacterial populations is lacking. We developed a stable isotope tracer platform to quantify metabolic pathways based on their ability to produce 13CO2 from various substrates. A model system was first established using Lactobacillus rhamnosus added to a 10 ml Exetainer tube. 13C substates in solution were then added. Samples were heated at 37°C for 4 to 12 hrs. The headspace in each tube was sampled and analyzed by gas isotope ratio mass spectrometry. We demonstrated that 13CO2 production from isotopically enriched solutions of glucose, fructose, acetate, pyruvate, octanoate, oxalate, and urea were able to be quantitated. Samples were tested under both aerobic and anaerobic conditions. Anaerobic conditions produced more 13CO2 gas. L. rhamnosus had limited ability to metabolize amino acids phenylalanine and methionine and other sugars (sorbitol, xylose and lyxose). We then used this technique to quantitate metabolic dynamics in fecal samples. After collection of fecal samples from a healthy individual, approximately 1 ml of diluted fecal samples was added to solutions of 13C‐oxalate and 13C‐urea. Fecal samples were heated at 37°C for 4 to 12 hrs and tested under anaerobic conditions. We demonstrated 13CO2 production from these substrates. When these same fecal samples were left at 22°C for 5 days, the samples continued to show enzymatic conversion to 13CO2. In conclusion, we have demonstrated a simple platform for measuring metabolic dynamics of the gut microbiome in fecal samples.

Physiological Daily Inhalation Rates for Health Risk Assessment in Overweight/Obese Children, Adults, and Elderly

Physiological daily inhalation rates reported in our previous study for normal-weight subjects 2.6-96 years old were compared to inhalation data determined in free-living overweight/obese individuals (n = 661) aged 5-96 years. Inhalation rates were also calculated in normal-weight (n = 408), overweight (n = 225), and obese classes 1, 2, and 3 adults (n = 134) aged 20-96 years. These inhalation values were based on published indirect calorimetry measurements (n = 1,069) and disappearance rates of oral doses of water isotopes (i.e., (2)H2 O and H2 (18)O) monitored by gas isotope ratio mass spectrometry usually in urine samples for an aggregate period of over 16,000 days. Ventilatory equivalents for overweight/obese subjects at rest and during their aggregate daytime activities (28.99 ± 6.03 L to 34.82 ± 8.22 L of air inhaled/L of oxygen consumed; mean ± SD) were determined and used for calculations of inhalation rates. The interindividual variability factor calculated as the ratio of the highest 99th percentile to the lowest 1st percentile of daily inhalation rates is higher for absolute data expressed in m3 /day (26.7) compared to those of data in m3/kg-day (12.2) and m3/m2-day (5.9). Higher absolute rates generally found in overweight/obese individuals compared to their normal-weight counterparts suggest higher intakes of air pollutants (in μg/day) for the former compared to the latter during identical exposure concentrations and conditions. Highest absolute mean (24.57 m3/day) and 99th percentile (55.55 m3 /day) values were found in obese class 2 adults. They inhale on average 8.21 m3 more air per day than normal-weight adults.

Metabolic fate of lactate after anoxia at 20°C in the Western painted turtle

Western painted turtles (Chrysemys picta bellii) can survive anoxia for 120 days at 3°C and tolerate plasma lactate concentrations approaching 200 mM. The metabolic fate of lactate accumulated over 6 hours of anoxia and 39 hours of recovery at 20°C was investigated with stable isotope tracer methodology. A bolus infusion of [U‐13C]‐lactate tracer was given via an intra‐arterial catheter 2 hours into anoxia. 13C enrichment of metabolites and CO2 from expired air, blood and bone were measured using gas chromatography and isotope ratio mass spectrometry. The tracer completely equilibrated with the lactate pool by the end of anoxia. M3‐glucose enrichment slowly increased throughout the recovery period, and liver glycogen accounted for 26–33% of the label at the end of recovery. CO2 was the fate for ~44% of the lactate carbon, and CO2 exchange with the bone accounted for ~18% of the label, indicating that the shell is an important sink for metabolically produced CO2. The plasma concentration and enrichment of beta‐hydroxybutyrate also increased. Our results demonstrate that painted turtles are more like mammals in that oxidation accounts for the largest fraction of the labeled carbon. Turtles primarily utilize some other substrate to replenish muscle glycogen stores, most likely glucose derived from liver. This research was funded by Saint Louis University and the National Institutes of Health.

Assessment of body fat in the pony: Part II. Validation of the deuterium oxide dilution technique for the measurement of body fat

Excessive accumulations or depletions of body fat have been associated with increased morbidity and mortality in horses and ponies. An objective, minimally-invasive method to accurately quantify body fat in living animals is required to aid nutritional management and define welfare/performance limits. To compare deuterium oxide (D(2) O) dilution-derived estimates of total body water (TBW) and body fat with values obtained by 'gold standard' proximate analysis and cadaver dissection. D(2) O dilution offers a valid method for the determination of TBW and body fat in equids. Seven mature (mean ± s.e. 13 ± 3 years, 212 ± 14 kg, body condition scores 1.25-7/9), healthy, Welsh Mountain pony mares, destined for euthanasia (for nonresearch purposes) were used. Blood samples were collected before and 4 h after D(2) O (0.11-0.13 g/kg bwt, 99.8 atom percent excess) administration. Plasma was analysed by gas isotope ratio mass spectrometry following filtration and zinc reduction. After euthanasia, white adipose tissue (WAT) mass was recorded before all body tissues were analysed by proximate chemical analyses. D(2) O-derived estimates of TBW and body fat were strongly associated with proximate analysis- and dissection-derived values (all r(2) >0.97, P≤0.0001). Bland-Altman analyses demonstrated good agreements between methods. D(2) O dilution slightly overestimated TBW (0.79%, limits of agreement (LoA) -3.75-2.17%) and underestimated total body lipid (1.78%, LoA -0.59-4.15%) and dissected WAT (0.72%, LoA -2.77-4.21%). This study provides the first validation of the D(2) O dilution method for the minimally-invasive, accurate, repeatable and objective measurement of body water and fat in living equids.

Abstract: 585 LIPIDOMICS CAN BE USED IN PERSONALIZING CHOLESTEROL SYNTHESIS AND ABSORPTION INHIBITOR TREATMENTS

Metabolic pathway flux is a fundamental element of biological activity, which can be quantified using a variety of mass spectrometric techniques to monitor incorporation of stable isotope-labelled substrates into metabolic products. This article contrasts developments in electrospray ionisation mass spectrometry (ESI-MS) for the measurement of lipid metabolism with more established gas chromatography mass spectrometry and isotope ratio mass spectrometry methodologies. ESI-MS combined with diagnostic tandem MS/MS scans permits the sensitive and specific analysis of stable isotope-labelled substrates into intact lipid molecular species without the requirement for lipid hydrolysis and derivatisation. Such dynamic lipidomic methodologies using non-toxic stable isotopes can be readily applied to quantify lipid metabolic fluxes in clinical and metabolic studies in vivo. However, a significant current limitation is the absence of appropriate software to generate kinetic models of substrate incorporation into multiple products in the time domain. Finally, we discuss the future potential of stable isotope-mass spectrometry imaging to quantify the location as well as the extent of lipid synthesis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

Isotopic metrology of carbon dioxide. II. Effects of ion source materials, conductance, emission, and accelerating voltage on dual-inlet cross contamination.

We report high-precision isotopic carbon dioxide measurements, made before and after ion source modification to gas isotope ratio mass spectrometry (IRMS) instruments. Measurement protocols were designed to explore the effects of ion source material substitution, source conductance, inlet pressure, electron emission, acceleration potential, and inlet changeover equilibration time. After modification of the IRMS instruments at the National Institute of Standards and Technology (NIST) and the Max-Planck-Institute for Chemistry (MPI-Mainz), immediate changes were observed. At NIST, measurements were no longer sensitive to inlet equilibration times greater than 15 s, and different settings of ion source conductance resulted in delta(13)C shifts of about 0.04 per thousand per 10 per thousand measurement difference between sample and reference, a five-fold improvement. No significant changes in machine performance were observed after a month of use. After a year, performance had degraded slightly, but was controlled by ion source cleaning and the use of low-energy ion acceleration to minimize sputtering. At MPI-Mainz, results were very similar. We report cross-contamination coefficients measured since 1996, and discuss the role of adsorption, ion implantation, and sputtering on cross contamination in mass spectrometry systems. We recommend that users of high-precision IRMS instruments test for and minimize the effects described.

Estimation of Neonatal Body Composition: Isotope Dilution versus Total-Body Electrical Conductivity

The objective of the study is to evaluate neonatal body composition determined by the isotope dilution method compared with the total-body electrical conductivity (TOBEC) method. An oral dose of ¹⁸O- and ²H-labeled water was given to 40 healthy term newborns, and 26 infants successfully completed the protocol. The isotope concentrations in urine samples were measured by gas-isotope ratio mass spectrometry. Fat and percent body fat (%F) estimated by isotope dilution were correlated with fat and %F estimated. Total body water measured by isotope dilution of ¹⁸O and ²H was 2.44 ± 0.36 and 2.49 ± 0.35 kg, respectively (p < 0.001). Fat and %F estimated by ¹⁸O, ²H, and TOBEC were 0.48 ± 0.20 kg and 13.7 ± 5.1%, 0.48 ± 0.19 kg and 13.7 ± 4.6%, and 0.38 ± 0.21 kg and 10.9 ± 5.0%, respectively. Neither fat nor %F estimates by isotope dilution showed good correlation with the TOBEC estimates (r = 0.22–0.57). Because of the wide variation of water content in individuals, the isotope dilution method may not be the optimal way of assessing body composition, and specifically body fat, in newborns.

Validation of equilibration and chromium reduction methods for deuterium measurements of fluid volumes.

Determinations of fluid volumes are of importance for correct treatment of patients subjected to shock and trauma. Gas isotope ratio mass spectrometry (GIRMS) is an advanced method for analysis of stable isotopes. These can be used as tracers for measurement of various fluid volumes. In the current in vitro study, deuterium was used to determine different volumes of water simulating a range of body fluid volumes from neonates to adults. A high-precision scale gave control weights (i.e., volumes), and two methods, equilibration (EQ) and chromium reduction (CR), were compared by use of a GIRMS. The coefficient of variation was <1% when using both EQ (0.45%) and CR (0.79%). The variability was greater at small volumes, and, when regression equations for the relation between measured and calculated volumes were used as formulas, the deviation was 0.4% using EQ and 2.8% using CR at the volume of 1,000 ml. At larger volumes, the deviation when using CR approached 1%. These variations are better than previously published data using other methods. It was concluded that GIRMS is a suitable technique for fluid volume determinations in neonates as well as in adult patients, using deuterium as a tracer. EQ and CR methods were both regarded to give acceptable variabilities in this in vitro study. GIRMS may in the future increasingly be used clinically for accurate measurements of body fluid volumes.