Isotope Ratio Mass Spectrometer Research Articles

Dietary Phenylalanine and Tyrosine Requirements in Healthy Human Pregnancy

Abstract Objectives Phenylalanine (PHE), an indispensable amino acid, is required for protein synthesis and is fundamental for proper fetal development. PHE is the precursor for tyrosine (TYR), a conditionally indispensable amino acid that is crucial for synthesis of dopamine, norepinephrine and epinephrine. The current recommendation for PHE + TYR during pregnancy is 36 mg/kg/d, which is factorially determined and not based on experimental studies. Our objective was to experimentally determine PHE + TYR requirements during early (13–19 wks) and late (33–39 wks) gestation, accounting for potential changes in requirement since metabolism and rate of growth are not static throughout pregnancy. Methods 20 women (22–39 y) were studied using the non-invasive stable isotope based indicator amino acid oxidation technique (with L-[1–,13C]Leucine as indicator) for a total of 51 study days. A range of PHE intakes (5 to 100 mg/kg/d) were provided in early and late pregnancy as isonitrogenous and isocaloric meals on separate study days, in the absence of dietary tyrosine. Breath samples were collected at baseline and isotopic steady state. Samples were analyzed for ,13C enrichment on an Isotope Ratio Mass Spectrometer, and the PHE + TYR requirement was determined using two-phase linear regression crossover analysis to identify a breakpoint in ,13CO2 production (represents the minimum dietary requirement) in response to changes in PHE intake. Results Results suggest the PHE + TYR requirement is 43 and 55 mg/kg/d in early and late pregnancy respectively. Conclusions Our preliminary results suggest that PHE + TYR requirements in pregnancy are higher compared to current recommendations, and are different between gestational stages. These results are similar to our previous findings of increased requirements for dietary protein, lysine and PHE (in the presence of excess TYR) in late compared to early pregnancy. They also highlight the need to investigate the dietary requirement of the remaining indispensable amino acids during this life stage. Funding Sources Canadian Institute of Health Research.

Metabolic Availability of Lysine from Oats and Whole Milk Assessed in School-Aged Children

Abstract Objectives The objective of this study was to assess protein quality by determining the metabolic availability (MA) of lysine from commonly consumed plant and animal-based food sources in school-aged children, using the indicator amino acid oxidation (IAAO) method. Methods Using a repeated-measures design, six healthy school-aged children randomly received 10 lysine intakes from either crystalline L-lysine (3, 6, 9, 12 mg/kg/day), moist cooked oats (9, 12 mg/kg/day), oven-baked granola (12 mg/kg/day), or whole milk (6, 9, 12 mg/kg/day). All diets provided protein at 1.5 g/kg/day and calories at 1.7x the participant´s measured resting energy requirement. All test diets were isocaloric and isonitrogenous. Every study day, breath samples were collected at baseline and during isotopic steady state and the ,13C enrichment was measured using an isotope ratio mass spectrometer. MA of lysine from food sources was determined using slope ratio analysis of ,13C-phenylalanine oxidation response to feeding lysine from whole foods compared to L-lysine from a reference crystalline amino acid diet. Results Lysine from whole milk was highly available, (∼100%, similar to our crystalline-lysine reference). The MA of lysine from cooked oats was 92.7%. When the same oats were oven-baked the availability of lysine was reduced by ∼50%. This reduction may be due to lysine loss through the Maillard reaction. Conclusions Lysine is highly metabolically available from milk and from moist cooked oats. However, dry cooking of oats reduces lysine MA considerably. The protein quality of cereal- based diets is dependent in the lysine content and bioavailability of the individual foods. Therefore, it is imperative to study how the human body utilizes this nutrient, especially in growing children. These are the first studies that assess protein quality in commonly consumed foods directly in children. This data will help build a knowledge base that can be further used to develop accurate dietary combinations that fully meet amino acid needs. Funding Sources Supported by: Dairy Farmers of Canada and National Dairy Council.

Development of Minimally Invasive 13C-Glucose Breath Test to Examine Different Dietary Therapies in Patients with Glycogen Storage Disorders

Abstract Objectives The objectives of the current study were: 1) to establish the use of ,13C-Glucose Breath Test (,13C-GBT) and its oxidation to ,13CO2 as a minimally invasive technique to examine in vivo glucose oxidation in healthy adults, and 2) to measure the utilization of uncooked cornstarch (UCCS) and Glycosade® in patients with glycogen storage disease type Ia (GSD-Ia) and healthy controls using ,13C-GBT, based on the natural enrichment of ,13C in UCCS and Glycosade®. Methods Experiment 1- Ten healthy adults (22 – 33y) underwent ,13C-GBT protocols twice as a proof-of-principle, once without oral isotope dose (only D-glucose 75 g/d) and once with isotope (D-glucose 75 g/d + U-,13C-glucose 75 mg/d). Breath samples were collected at baseline and every 20 min for 240 min to measure ,13CO2. Experiment 2- Two patients (12 and 28y) with GSD-Ia and five healthy controls (10 – 32y) underwent ,13C-GBT protocols twice. Subjects received UCCS or Glycosade® orally (based on their current prescribed dose 42 – 100 g) after a 4 hour fast according to GSD-Ia fasting tolerance. Breath samples were collected at baseline and every 30 min for 240 min. ,13CO2 oxidation of glucose from UCCS and Glycosade® were measured using an isotope ratio mass spectrometer and compared using a paired t-test. Blood glucose was measured using a glucometer hourly to test hypoglycemia (≤3.4 mmol/L). Results Results 1- The mean rate of ,13CO2 in all healthy adults from D-glucose without U-,13C-glucose 0.435 ± 0.162 was significantly lower than the mean rate of ,13CO2 in the same group with U-,13C-glucose 3.518 ± 0.988. The peak occurred at 200 minutes in all healthy adults without and with U-,13C-glucose. Results 2- Glucose oxidation from Glycosade® was lower than glucose oxidation from UCCS in the 12y GSD-Ia. Glucose oxidation from UCCS and Glycosade® remained the same in the 28y GSD-Ia. However, oxidation of glucose by the 28y GSD-Ia for both starches displayed a higher rate, compared to age matched controls. Conclusions Our results show that the minimally invasive,13C-GBT test over 4 hours can be useful to examine glucose metabolism from various exogenous carbohydrate sources in GSD. Future studies are needed to use ,13C-GBT in response to different doses to determine optimal glucose management in GSD patients. Funding Sources Saudi Arabian Cultural Bureau, Ottawa and BC Children's Hospital Research Institute.

Real Ileal Digestibility of Sunflower Protein and Amino Acids in Humans

Abstract Objectives Sunflower protein has a relatively well-balanced amino acid profile but it is little used in human food. Nutritional quality of sunflower protein isolate has never been directly measured in humans. This study aims to determine the ileal digestibility of protein and amino acid of a sunflower isolate incorporated in biscuits, in healthy humans. Methods Healthy volunteers (n = 7) were intubated with a naso-ileal tube. They ingested every 30 min for 4 h, sunflower biscuits for a total of 156 g of biscuits and 25 g of 15N intrinsically labeled sunflower protein isolate. A non-absorbable marker was perfused into the ileum. Ileal samples were continuously collected during 8 h after the first meal. Blood was sampled every 30 min for 4 h and every hour after and total urine was collected every 2 h. To measure nitrogen ileal digestibility, the percentage of nitrogen and 15N enrichment in ileal contents were assessed by an elemental analyzer coupled with and isotope ratio mass spectrometer (EA-IRMS). Indispensable amino acid (IAA) ileal digestibility was determined by measuring amino acid 15N enrichment by GC-C-IRMS and their quantification by UHPLC. Metabolic losses of dietary nitrogen were assessed by measuring 15N in urine and plasma. The digestible indispensable amino acid score (DIAAS) and the net post-prandial utilization (NPPU) were calculated. Results Exogenous nitrogen appeared in the ileum 1 h after the first meal, its flow was stable between 4 h and 7 h. Cumulated dietary nitrogen in the ileum was 14.0 ± 4.0% over 8 h. Sunflower nitrogen real ileal digestibility was 86.0 ± 4.0% of ingested nitrogen. Mean IAA ileal digestibility was 87.0 ± 6.1% with values ranging from 84.4% for lysine and threonine to 89.3 ± 7.8% for methionine. DIAAS was 0.93 for lysine. After 8 h, 4.0 ± 1.0% of dietary nitrogen was incorporated in plasma protein. Cumulated exogenous nitrogen in urinary urea was 4.0 ± 1.4% and exogenous nitrogen in body urea after 8 h was 9.8 ± 2.5%. The NPPU was 72.2 ± 3.7% at 8 h. Conclusions Sunflower isolate ileal digestibility is relatively low compared to other protein isolate assessed in similar conditions (ileal tube and 15N labeling of protein). Despite a moderate deficiency in lysine, sunflower protein isolate has a good post-prandial metabolic utilization. Funding Sources French Research National Agency (ANR), financial support of SOFIPROTEOL under the FASO Project PRODIA.

Isotopic fractionation during acid digestion of calcite: A combined ab initio quantum chemical simulation and experimental study.

Carbonate clumped isotope analysis involves the reaction of carbonate minerals with phosphoric acid to release CO2 for measurement in a gas-source isotope ratio mass spectrometer. Although the clumped isotope proxy is based on the temperature dependence of 13 C-18 O bonding preference in the mineral lattice, which is captured in the product CO2 , there is limited information on the phosphoric acid reaction mechanism and the magnitude of clumped isotopic fractionation (mass 63 in CO3 2- to mass 47 in CO2 ) during the acid digestion. We studied the reaction mechanism for the phosphoric acid digestion of calcite using first-principles density functional theory. We identified the transition state structures for each reaction involving different isotopologues and used the corresponding vibrational frequencies in reduced partition function theory to estimate the Δ47 acid fractionation. Experimental Δ47 data were acquired by processing the sample CO2 gas through the dual-inlet peripheral of a ThermoFinnigan MAT253 isotope ratio mass spectrometer. We showed that the acid digestion reaction, which results in the formation of CO2 enriched with 13 C-18 O bonds, began with the protonation of calcium carbonate in the presence of water. Our simulations yielded a relationship between the Δ47 acid fractionation and reaction temperature as Δ47 = -0.30175 + 0.57700 × (105 /T2 ) - 0.10791 × (105 /T2 )2 , with T varying between 298.15 and 383.15 K. We propose a reaction mechanism that shows a higher slope (Δ47 acid fractionation vs. 1/T2 curve) for the phosphoric acid digestion of calcite than in previous studies. The theoretical estimates from the present and earlier studies encapsulate experimental observations from both "sealed vessel" and "common acid bath" acid digestion methods.

Nitrogen isotopic analysis of nitrate in aquatic environment using cadmium-hydroxylamine hydrochloride reduction.

The nitrogen isotopic ratio of nitrate (δ15 N-NO3 - value) is a critical parameter for understanding nitrogen biogeochemical cycling in aquatic systems. Current approaches to the determination of δ15 N-NO3 - values involve time-intensive handling procedures, the use of toxic chemicals and complicated microbial incubation. A chemical reduction method for measuring the δ15 N-NO3 - values of aquatic samples was established. Nitrate was first quantitatively reduced to nitrite in a column filled with copper-coated cadmium granules, and the produced nitrite further reduced to nitrous oxide gas with hydroxylamine hydrochloride. The nitrogen isotope ratio of the produced nitrous oxide was measured using a continuous-flow isotope ratio mass spectrometer coupled with a purge and cryogenic trap system. The optimized experimental conditions were: solution acidity, H+ concentration of 0.46 M, pH = 0.34; dosage of hydroxylamine, molar ratio of NH2 OH to NO2 - of 4; reaction temperature, 45°C; and reaction time, 14-16 h. No salt effect was found for this method. The reproducibility of the δ15 N-NO3 - value for the laboratory standard was better than 0.3‰ for long-term measurements (20 nmol NO3 - requirement). This method provides a reliable approach for the determination of δ15 N-NO3 - values at natural abundance. It provides (1) high measurement accuracy, (2) ease of operation, (3) environmental-friendly procedure (less toxic regents used), and (4) suitability for both freshwater and saline water samples.

Effects of oxygen plasma ashing treatment on carbonate clumped isotopes.

For clumped isotope analysis (Δ47 ), hydrocarbon and organic molecules present an important contaminant that cannot always be removed by CO2 purification through a Porapak-Q trap. Low-temperature oxygen plasma ashing (OPA) is a quick and easy approach for treatment; however, the impact of this treatment on the original carbonate clumped isotope values has never been fully studied. We tested the isotopic impact of OPA using three natural samples with a large range of initial Δ47 values. Crushed and sieved (125 μm mesh) samples were placed into a Henniker Plasma HPT-100 plasma system and treated at a flow rate of 46 mL/min and a power of 100 W at a vacuum of 0.2 mbar for 10, 20, 30 and 60 min before clumped isotope analysis using two MAT 253 isotope ratio mass spectrometers modified to measure masses 44-49. OPA treatment for 30 min or more on calcite powder samples has the potential to alter the clumped isotopic composition of the samples beyond analytical error. A systematic positive offset is observed in all samples. The magnitude of this alteration translates to a temperature offset from known values ranging from 4°C to 13°C. We postulate that the observed positive offset in Δ47 occurs because the bonds within lighter isotopologues are preferentially broken by plasma treatment, leading to an artificial increase in the 'clumping' value of the sample. We recommend that any laboratory performing OPA treatments should reduce the runs to 10-20 min or carry out successive runs of 10 min followed by sample stirring, as this procedure showed no alteration in the initial Δ47 values. Our results validate the use of OPA for clumped isotope applications and will allow future research to use clumped isotopes for challenging samples such as oil-stained carbonates, bituminous shales or host rocks with very high organic carbon content.

Isotopic discrimination between carrion and elytra clippings of lab-reared American burying beetles (Nicrophorus americanus): Implications for conservation and evaluation of feeding relationships in the wild.

Differences in stable isotope composition between an animal and its diet are quantified by experimentally derived diet-tissue discrimination factors. Appropriate discrimination factors between consumers and prey are essential for interpreting stable isotope patterns in ecological studies. While available for many taxa, these values are rarely estimated for organisms within the carrion food web. We used a controlled-diet stable isotope feeding trial to quantify isotopic diet-tissue discrimination factors of carbon (δ13 C values) and nitrogen (δ15 N values) from laboratory-reared Nicrophorus americanus raised on carrion. We used exoskeleton samples of beetle elytra (wing covers) to determine diet-tissue discrimination factors using a continuous flow isotope ratio mass spectrometer equipped with an elemental analyzer. We also measured the isotopic compositions of five species of co-occurring, wild-caught burying beetles and evaluated feeding relationships. We found differences in stable carbon discrimination between carrion sources (mammalian and avian) and lab-reared beetles, but no difference in stable nitrogen discrimination. Values for δ13 C did not differ among wild-caught burying beetle species, but values for δ15 N were significantly different for the three species with overlapping breeding seasons. Furthermore, wild-caught burying beetles within our study area do not appear to use avian carrion resources to rear their young. This study informs future interpretation of stable isotope data for insects within the carrion food web. In addition, these results provide insight into carrion resources used by co-occurring burying beetle species in situ. We also demonstrated that independent of adult food type, the larval food source has a significant impact on the isotopic signatures of adult beetles, which can be estimated using a minimally invasive elytra clipping.

A fluorination method for measuring the 13 C-13 C isotopologue of C2 molecules.

Doubly substituted isotope species ("clumped" isotopes) can provide insights into the biogeochemical history of a molecule, including its temperature of formation and/or its (bio)synthetic pathway. Here, we propose a new fluorination method for the measurement of 13 C-13 C species in C2 molecules using a conventional isotope ratio mass spectrometer. Target molecules include ethane, ethene and ethanol. 13 C-13 C isotope species in C2 molecules were measured as C2 F6 using a conventional isotope ratio mass spectrometer. Ethane and ethene are directly fluorinated to C2 F6 . Ethanol is measured after dehydration to ethene and subsequent fluorination of the latter. The method enables the measurement of the Δ13 C13 C values normalized against a reference working standard. The reproducibility of the whole protocol, including chemical modification steps and measurement of C2 F6 isotopologues, is better than ±0.14‰ for all the compounds. Ethane from natural gas samples and biologically derived ethanol show a narrow range of Δ13 C13 C values, varying from 0.72‰ to 0.90‰. In contrast, synthetic ethanol as well as putative abiotic ethane show Δ13 C13 C values significantly different from this range with values of 1.14‰ and 0.25‰, respectively. The method presented here provides alternative means of measuring 13 C-13 C species to that using high-resolution mass spectrometry. Preliminary data from natural and synthetic molecules re-emphasizes the potential of 13 C clumped isotope species as a (bio)marker.

Sniffing out cocoa bean traits that persist in chocolates by PTR-MS, ICP-MS and IR-MS

The cocoa botanical and geographical origin and the primary processing steps applied by cocoa farmers at the beginning of the supply chain influence the chemical compositional traits of the cocoa beans. These features are carried along the supply chain as intrinsic markers up to the final products. These intrinsic markers could be used for tracking and tracing purposes. In this study, we examined the retention and loss of compositional signatures from cocoa beans to chocolates. Volatile, elemental and stable isotope signatures of cocoa beans of 10 different origins and 11 corresponding chocolates were determined by high sensitivity-proton transfer reaction-mass spectrometry (HS-PTR-MS), inductively coupled plasma-MS (ICP-MS) and isotope ratio-MS (IR-MS), respectively. The volatile fingerprints provided mostly information on the origin and primary processing traits of the raw cocoa beans in the chocolates. Volatile compounds that are relevant markers include: acetic acid (m/z 61), benzene (m/z 79), pyridine (m/z 80), 2-phenylethanol (m/z 123), and maltol (m/z 127). On the other hand, the elemental and stable isotope characteristics are more indicative of the cocoa content and added ingredients. Possible elemental markers for cocoa origin include Fe, Cr, and Cd. VOCs appear to be the most robust markers carried from cocoa beans to chocolates of the groups examined. This provides the potential for track and trace of cocoa beans from farm to chocolates.

Carbon and nutrient accumulation in mangrove sediments affected by multiple environmental changes

In order to assess the impact of anthropogenic activities on carbon and nutrient accumulation, total organic carbon (TOC), nitrogen (TN), and phosphorus (TP) accumulation rates were examined in a 210Pb-dated mangrove sediment core from Sepetiba Bay, Brazil, a coastal region impacted by multiple environmental changes during the previous century. A 50-cm length sediment core was collected from a mangrove forest in Sepetiba Bay. Sediment subsamples were analyzed to measure TOC, TN, δ13C, and δ15N using an elemental analyzer attached to an isotope ratio mass spectrometer Thermo Finnigan Model Delta Plus XP, whereas colorimetric analysis were used to measure TP. For 210Pbex analyses, gamma-ray measurements were performed in a semiplanar intrinsic germanium high purity coaxial detector, coupled to a multichannel analyzer, whereas the sediment accumulation rate (SAR) was calculated according to the constant initial concentration (CIC) method. Also, carbon and nutrient fluxes were calculated using SAR and TOC, TN, and TP contents, whereas statistical differences were evaluated by ANOVA + Tukey HSD analysis with previous data normalization. The calculated sedimentation rate (~ 8.1 mm year−1) since the early 1900s was up to threefold higher than the global mean determined for mangrove forests (~ 2.8 mm year−1) and the regional sea level rise (~ 3.2 mm year−1). Significantly higher TOC, TN, and TP fluxes, up to nearly 1000, 90, and 15 g m−2 year−1, respectively, were observed after the water diversion from a nearby drainage basin in the 1950s and an increase in sewage effluent input, which increased in the early 1990s. After this period, lighter δ13C values (~ − 25‰) indicate an increased importance of the terrestrial organic matter source, while lower TOC:TN ratios (~ 11) and heavier δ15N values (~ + 9‰) suggest an increased influence of anthropogenic fertilization on inorganic nitrogen accumulation. The significantly higher accumulation rates during the last decades evidenced the role of mangrove sediments as sinks for anthropogenically enhanced inputs of carbon and nutrients. Also, studies on carbon and nutrient accumulation evidenced the need for further research in eutrophic coastal areas.

Impact of enteral arginine supplementation on lysine metabolism in humans: A proof-of-concept for lysine-related inborn errors of metabolism.

Patients with lysine-related inborn errors of metabolism (pyridoxine-dependent epilepsy [PDE] and glutaric aciduria type 1 [GA1]), follow a lysine-restricted diet with arginine-fortified lysine-free amino acid formula and additional oral arginine supplementation as a newer therapy for PDE. The rationale of arginine supplementation is based on arginine's ability to compete with lysine transport across cell membranes via shared transporter systems. Adequate doses of arginine required to competitively inhibit enteral lysine uptake has not been studied in humans This proof-of-concept study investigates the effect of incremental enteral arginine doses on whole-body lysine oxidation using an in vivo stable isotope tracer, L-[1-13 C] lysine, in healthy humans. Five healthy men completed six study days each consuming one dose of l-arginine HCl per study day; range = 50-600 mg/kg/d. Lysine intake was at DRI (30 mg/kg/d). Breath samples were analysed for L-[1-13 C] lysine oxidation to 13 CO2 using an isotope ratio mass spectrometer. Plasma amino acid concentrations were analysed using an amino acid analyser. Increasing doses of l-arginine HCl caused a linear decrease in whole-body lysine oxidation. Plasma arginine concentration increased, and plasma lysine concentration decreased below normal range with high arginine intakes. We provide the first empirical evidence of arginine-lysine antagonism in response to increasing oral arginine doses. Results suggest 300-600 mg/kg/d of l-arginine HCl and lysine intake restricted to DRI is needed to reduce enteral lysine uptake and systemic lysine oxidation. This could potentially lead to a recommended dose for arginine in lysine-related inborn errors of metabolism.

Refined protocol for δ13 C analysis of calcite and dolomite in carbonate mixture samples.

The carbon isotope composition of carbonates is widely used in petrology and paleoenvironments as a proxy for carbon sources and environmental parameters during mineral formation or late diagenesis. However, most carbonate minerals in nature are complicated mixtures (i.e., coexisting calcite and dolomite). Precise separation of individual signals in mixed carbonate minerals is essential to obtain information regarding these indexes because of the diverse isotopic behavior of different minerals. The conditions for pretreating calcite-dolomite mixtures to determine each of their δ13 C values were optimized with a series of experiments varying the purging and reaction conditions using an isotope ratio mass spectrometer equipped with an online GasBench II interface. Sequential phosphoric acid reactions of pure calcite and dolomite were performed to measure the characteristics of single minerals under different temperatures and reaction times. Artificial mixtures with varied proportions of calcite and dolomite were used to verify the precision of the proposed method. The optimized pretreatment conditions for δ13 C analysis of calcite in the mixture involved purging the sample bottle using helium and then reacting it with 99% H3 PO4 for 100 min in a cold water bath at 2°C. The optimized pretreatment conditions for δ13 C analysis of dolomite in the mixture involved purging the sample bottle using helium gas after δ13 Ccal measurement and reacting it with H3 PO4 on a heating plate for 10 h at 45°C. This study introduced a workflow with ultimate precision for the selective δ13 C measurements of calcite-dolomite mixtures combining a GasBench II with a continuous flow isotope ratio mass spectrometer.

Laser based technique for monitoring heavy water leaks in nuclear reactors: performance validation with conventional techniques

In a pressurized heavy water reactor (PHWR), monitoring leakages of heavy water from coolant and moderator systems is very important for reactor safety as well as health of operating personnel. In view of this, heavy water leak monitoring system based on off-axis integrated cavity output spectroscopy (OA-ICOS) technique is developed. The developed system estimates the amount of heavy water in the samples by measuring the HDO concentration with the detection sensitivity of ±1.2 ppm. The performance of this system is studied by measuring various samples obtained from the Dhruva research reactor at Bhabha Atomic Research Centre. The validation of the data obtained using this system is carried out using tritium activity measurements of same samples. The results are also compared with D/H ratio measurements obtained by isotope ratio mass spectrometer (IRMS) . The measured HDO concentration of various samples using the developed system shows a good correlation with the measurements using IRMS and tritium activity measurement techniques.

Stable isotope analysis of non-exchangeable hydrogen in carbohydrates derivatised with N-methyl-bis-trifluoroacetamide by gas chromatography – Chromium silver reduction/High temperature Conversion-isotope ratio mass spectrometry (GC-CrAg/HTC-IRMS)

A novel procedure for the rapid isotope analysis of the carbon-bound non-exchangeable (CBNE) hydrogen in mono and disaccharides has been developed to demonstrate the feasibility of detecting undeclared addition of exogenous sugar products in foods and beverages susceptible to economically motivated adulteration. The procedure utilizes a simple one-step reaction, with the derivatising agent N-methyl-bis-trifluoroacetamide, to substitute the exchangeable hydroxyl-hydrogens with trifluoroacetate derivatives that are sufficiently volatile to be separated and measured by a gas chromatograph coupled to an isotope ratio mass spectrometer. The conversion of the derivatised sugars into the measuring gas is achieved using a high temperature chromium-silver reactor that retains carbon, oxygen and fluorine whilst releasing hydrogen gas for stable isotope measurement. The new procedure has advantages over existing methods in terms of ease of use, analysis time and compound-specific information. Sugars from fruit juice and honey have been measured to demonstrate the feasibility of using this technique.

An ideal solution? Optimising pretreatment methods for artificially mummified ancient Egyptian tissues.

RationaleAlthough the analysis of skeletal remains dominates the study of ancient dietary stable isotopes, mummified bodies also allow short‐term diet to be studied through the analysis of soft tissues. The application of resins, waxes and oils during mummification can affect the results obtained. This study assesses a range of methods for removing such substances from mummified tissue.MethodsAn experimental mummification model following ancient Egyptian methods was created using a modern pig leg. Sub‐samples of skin, muscle and bone were removed and coated with a range of substances used in Egyptian mummification. Four methods were used to clean these samples before the measurement of the carbon and nitrogen stable isotope ratios of their gelatinised collagen content using a ThermoFinnigan Flash Elemental analyser coupled to a DeltaPlus XL isotope ratio mass spectrometer via a ConFlo III interface.ResultsThe results showed that embalming materials can significantly affect dietary stable isotope ratios, and that these substances are most effectively removed using a mixture of polar and non‐polar solvents. Results indicate that bone samples demineralised with HCl and skin samples produce more accurate results than bone samples demineralised with EDTA or muscle samples.ConclusionsThe choice of tissue and the preparation methods used can have a significant effect on the accuracy of stable isotope data obtained from mummified tissue, particularly when embalming materials are also present. A mixture of solvents appears to be a more effective cleaning agent than a single solvent. Demineralisation with HCl is preferable for well‐preserved bone, as used in this study, but whether this is the case for more fragile, less well‐preserved bone requires further study. Skin samples produce more consistent data than muscle, but visually distinguishing between these tissues is not simple on ancient mummies.

Stable Isotope Analysis of Intact Oxyanions Using Electrospray Quadrupole-Orbitrap Mass Spectrometry.

The stable isotopes of sulfate, nitrate, and phosphate are frequently used to study geobiological processes of the atmosphere, ocean, as well as land. Conventionally, the isotopes of these and other oxyanions are measured by isotope-ratio sector mass spectrometers after conversion into gases. Such methods are prone to various limitations on sensitivity, sample throughput, or precision. In addition, there is no general tool that can analyze several oxyanions or all the chemical elements they contain. Here, we describe a new approach that can potentially overcome some of these limitations based on electrospray hyphenated with Quadrupole Orbitrap mass spectrometry. This technique yields an average accuracy of 1-2‰ for sulfate δ34S and δ18O and nitrate δ15N and δ18O, based on in-house and international standards. Less abundant variants such as δ17O, δ33S, and δ36S, and the 34S-18O "clumped" sulfate can be quantified simultaneously. The observed precision of isotope ratios is limited by the number of ions counted. The counting of rare ions can be accelerated by removing abundant ions with the quadrupole mass filter. Electrospray mass spectrometry (ESMS) exhibits high-throughput and sufficient sensitivity. For example, less than 1 nmol sulfate is required to determine 18O/34S ratios with 0.2‰ precision within minutes. A purification step is recommended for environmental samples as our proposed technique is susceptible to matrix effects. Building upon these initial provisions, new features of the isotopic anatomy of mineral ions can now be explored with ESMS instruments that are increasingly available to bioanalytical laboratories.

Dietary phenylalanine requirements during early and late gestation in healthy pregnant women

Phenylalanine is an indispensable amino acid and, via tyrosine, is the precursor for the neurotransmitters dopamine, norepinephrine, and epinephrine. Currently, dietary requirements for phenylalanine during pregnancy are unknown. This study's aim was to determine phenylalanine requirements (in the presence of excess tyrosine) during early and late gestation using direct amino acid oxidation (DAAO; with l-[1-13C]phenylalanine) and indicator amino acid oxidation (IAAO; with l-[1-13C]leucine). Twenty-three healthy women (age: 30.4±3.1y, mean ± SD) were studied at a range of phenylalanine intakes (5.5-30.5 mg · kg-1 · d-1 in early and late pregnancy using DAAO, and 2.5-30.5 mg · kg-1 · d-1 in late pregnancy using IAAO) for a total of 76 study days. Test intakes were provided as 8 isocaloric and isonitrogenous meals with 1.5 g · kg-1 · d-1 protein and energy at 1.7 times the measured resting energy expenditure. Breath samples were analyzed on an isotope ratio mass spectrometer for 13C enrichment. Phenylalanine requirement was determined using a 2-phase linear regression crossover model to identify a breakpoint in 13CO2 production (representing the mean requirement) in response to phenylalanine intakes. Phenylalanine requirement during early pregnancy was determined to be 15 mg · kg-1 · d-1 (95% CI: 10.4, 19.9 mg · kg-1 · d-1); during late pregnancy, it was determined to be 21 mg · kg-1 · d-1 by DAAO (95% CI: 17.4, 24.7 mg · kg-1 · d-1) and IAAO (95% CI: 10.5, 32.2 mg · kg-1 · d-1). Our results suggest a higher requirement (40%) for phenylalanine during late pregnancy than during early pregnancy. Moreover, the early pregnancy requirements are higher than the previous adult male requirement (9.1 mg · kg-1 · d-1; 95% CI: 4.6, 13.6 mg · kg-1 · d-1), although the 95% CIs overlap. Both DAAO and IAAO methods provided similar breakpoints in late pregnancy, showing that the DAAO method was appropriate even though low phenylalanine intakes could not be tested. These results have potential implications for gestation stage-specific dietary phenylalanine recommendations in future.This trial was registered at clinicaltrials.gov as NCT02669381.

Isotope analysis method for the herbicide bromoxynil and its application to study photo-degradation processes

Bromoxynil is an increasingly applied nitrile herbicide used for post-emergent control of annual broadleaved weeds. Compound-specific isotope analysis (CSIA) of the compound is of interest for studying its environmental fate, yet is challenging following its polar nature. We present a CSIA method for bromoxynil that includes offline thin-layer chromatography purification followed by an elemental analyzer isotope ratio mass spectrometer (EA-IRMS). This method was shown to be accurate and precise for δ13C and δ15N analysis of the compound (standard deviation of replicate standards <0.5‰). The method was applied to photodegraded samples, either radiated under laboratory condition with a UV lamp, or exposed to sunlight under environmental conditions. Dominating degradation products were similar in both cases. Nevertheless, isotope effects differed, presenting a strong inverse carbon isotope effect (εC = 4.74 ± 0.82‰) and a weak inverse nitrogen isotope effect (εN = 0.76 ± 0.12‰) for the laboratory experiment, and an insignificant carbon isotope effect (εC = 0.34 ± 0.44‰) and a normal nitrogen isotope effect (εN = −3.70 ± 0.30‰) for the natural conditions experiment. The differences in δ13C vs. δ15N enrichment trends suggest different mechanism for the two processes. Finally, the obtained dual isotope trend for natural conditions provide the basis for studying the dominance of photodegradation as a degradation route in the environment.

Metabolic breath signature of 13C-enriched wheat bran consumption related to gut fermentation in humans: a Fiber-TAG study

AbstractIntroductionDietary fibers (DF) have been classified mainly according to their physico-chemical and fermentability properties but it remains unclear whether such classification is relevant when addressing their health effects. Indeed, the nature of physiological effects induced by DF, particularly through their interaction with gut microbiota, remains poorly known due to their diversity, to gut microbiota inter-subjects variability and to the lack of validated non-invasive biomarkers to characterize DF-gut microbiota interaction. The aim of this pilot study was 1) to follow the metabolic fate of 13C-labeled DF through the assessment of 13C-labelled gut-derived metabolites in excreted breath and 2) to evaluate novel non-invasive breath-derived biomarkers of DF-gut microbiota interactions.Materials and methodsSix healthy women (29.7 ± 1.7 years old, BMI: 23.2 ± 0.9kg/m2, fiber intake: 23 ± 1g/d) consumed in research settings a controlled breakfast containing eight 13C-labelled wheat bran biscuits (50 g of labelled wheat bran, 3.0At%13C). 13C-labelled wheat bran was obtained from wheat cultivated under 13CO2 enriched atmosphere. Samples of expired gases were collected during 24 h after ingestion in order to measure H2 and CH4 by gas chromatography (GC) with piezoelectric detection and 13CO2 and 13CH4 by gas chromatography coupled with an isotope ratio mass spectrometer (GC-IRMS). Apart test breakfast, subjects only consumed standardized meals without fibers.ResultsThe analysis of H2 and CH4 24h-kinetic measurements distinguished 2 groups in terms of fermentation related gas excretion: the high-CH4 producers with high baseline CH4 concentrations (42.1 ± 13.7ppm) and low baseline H2 concentrations (7.3 ± 5.8ppm) and the low-CH4 producers with low baseline CH4 concentrations (6.5 ± 3.6ppm) and high baseline H2 concentrations (20.8 ± 16.0ppm). Following the 13C-wheat bran biscuits’ ingestion, postprandial H2 and CH4 concentrations increased more significantly in the high-CH4 producer subjects. 13C enrichment was detectable in expired gases in all subjects. 13CO2 kinetics were similar for all subjects and correspond to the oxidation of the digestible part of the bran. The appearance of 13CH4 was significantly enhanced and prolonged after 180 min in high-CH4 producers compared to low-CH4 producers, suggesting distinct fiber fermentation profile.DiscussionThis pilot study allowed to consider novel procedures for development of non-invasive breath biomarkers of fiber-gut microbiota interactions. Assessment of expired gas excretion following 13C-labelled fiber ingestion allowed deciphering distinct fermentation profiles: high-CH4 producers vs low-CH4 producers and accordingly provide a related non-invasive breath metabolic signature of the fiber fermentation for each profile. Further gut microbiota and 13C-metabolites analysis will permit to relate the gut bacteria composition with breath gas excretion kinetics according to fiber fermentation profile.