A comprehensive understanding of carbon cycling pathways in the soil-plant system is needed to develop models that accurately predict global carbon reservoir responses to anthropogenic perturbations. Honey is a carbon-rich natural food produced by wild and managed pollinating insects all over the world; the composition of a single sample is a function of millions of pollinator-plant interactions. We studied the 13C/12C and Δ14C of 121 honey samples sourced from the United States, and found a significant old carbon contribution. The effect is observed from 25 to 45° latitude, not correlated with 13C/12C, and consistent with a previously published study on European honeys. In specific cases, the measured values were up to 20 ‰ (Δ14C) higher than the expected atmospheric 14CO2 value for the given year, which shows a significant old carbon contribution. We hypothesize that the older carbon is from plant liquids derived in part from soil carbon or stored nonstructural carbohydrates from plants, which shifts the calibrated age of the sample by 5 years or more. Our work is the first to describe the widespread occurrence of old carbon in honey and shows that radiocarbon measurements can be a powerful tool to trace carbon allocations in terrestrial food webs and detect the atmosphere-soil-plant carbon cycle contributions.

OBJECTIVE: Metformin is a widely prescribed first line drug for the treatment of type 2 diabetes mellitus (DM). Studies have shown that the use of metformin is often associated with a decrease in vitamin B12 (B12) levels in patients with DM. Few studies have shown that this effect could be mitigated with calcium supplementation. In the present study, we quantified the effect of metformin, and metformin co-administered with calcium on B12 absorption using a novel stable isotope [13C] cyanocobalamin tracer.
METHODS: A pilot crossover study was conducted to estimate the bioavailability of B12 in healthy subjects, using [13C] cyanocobalamin as a tracer. In the study, [13C] cyanocobalamin was administered orally to the participants followed by hourly venous sampling to measure the concentration of the tracer and estimate bioavailability. This protocol was followed for three experiment days, each separated by a one month wash out period. As part of the study, all participants received the tracer alone for the control day (C), metformin 850 mg along with the tracer for the metformin day (M) and metformin 850 mg with calcium 500 mg and the tracer for the metformin calcium day (MC).
RESULTS: Seven participants completed all three experiment days. The mean B12 bioavailability (±SD, n = 7) was 42.6 ± 10.2% for the control day (C), 30.8 ± 15.3% for the metformin day (M) and 46.4 ± 8.6% for the metformin-calcium day (MC). Repeated measures ANOVA was done and the pairwise comparison showed a significant difference in the B12 bioavailability between control and metformin day (C vs M p = 0.010), and between the metformin and metformin with calcium day (M vs MC p = 0.003).
CONCLUSIONS: B12 bioavailability reduced significantly from baseline (C) when metformin (M) was administered and this reduction was reversed when calcium was co-administered (MC) in healthy participants. In patients using metformin, calcium supplementation as a strategy to prevent B12 deficiency needs to be further studied.

Stable carbon isotopes are a powerful tool to study photosynthesis. Initial applications consisted of determining isotope ratios of plant biomass using mass spectrometry. Subsequently, theoretical models relating C isotope values to gas exchange characteristics were introduced and tested against instantaneous online measurements of 13C photosynthetic discrimination. Beginning in the twenty-first century, laser absorption spectroscopes with sufficient precision for determining isotope mixing ratios became commercially available. This has allowed collection of large data sets at lower cost and with unprecedented temporal resolution. More data and accompanying knowledge have permitted refinement of 13C discrimination model equations, but often at the expense of increased model complexity and difficult parametrization. This chapter describes instantaneous online measurements of 13C photosynthetic discrimination, provides recommendations for experimental setup, and presents a thorough compilation of equations available to researchers. We update our previous 2018 version of this chapter by including recently improved descriptions of (photo)respiratory processes and associated fractionations. We discuss the capabilities and limitations of the diverse 13C discrimination model equations and provide guidance for selecting the model complexity needed for different applications.

BACKGROUND: Diagnosing lactose malabsorption is usually based on hydrogen excretion in breath after a lactose challenge. However, a proportion of subjects with lactose malabsorption will not present a rise in hydrogen. Measuring excretion of methane or stable isotope labeled 13CO2 after ingestion of 13C-lactose has been proposed to mitigate this problem.
OBJECTIVE: The aim of the study was to assess the performance of measuring methane and 13CO2 in individuals with normal hydrogen excretion compared to a genetic lactase non-persistence test.
METHODS: Individuals referred for lactose breath testing and healthy controls were included. Participants received 13C-enriched lactose, performed breath testing, and underwent genotyping for a marker of lactase non-persistence (13910C*T). Using genotype as gold standard, the performance of measuring methane and 13CO2 excretion was assessed.
RESULTS: 151 subjects participated in the study, 50 of which presented a lactase non-persistent genotype. Of these, 72% were correctly diagnosed through hydrogen excretion of ≥ 20 ppm above baseline. In subjects with normal hydrogen excretion, cumulative 13C excretion had an area under the curve (AUC) of the receiver operating characteristics (ROC) curve of 0.852. Sensitivity was 93% and specificity was 51% for the current cutoff of 14.5%. The optimal cutoff was 12.65% (sensitivity 93%, specificity 70%). The ROC curve of peak methane had an AUC of 0.542 (sensitivity of 14%, specificity of 91% for cutoff ≥ 10 ppm).
CONCLUSIONS: In individuals with genetically demonstrated lactase non-persistence and negative hydrogen breath test, the use of 13C-lactose with measurement of 13CO2 excretion and hydrogen is a well-performing test to detect the lactose malabsorption and performs better than methane in our cohort.

OBJECTIVE: To investigate the safety and value of hyperpolarized (HP) MRI of [1-13C]pyruvate in healthy volunteers using deuterium oxide (D2O) as a solvent.
METHODS: Healthy volunteers (n = 5), were injected with HP [1-13C]pyruvate dissolved in D2O and imaged with a metabolite-specific 3D dual-echo dynamic EPI sequence at 3T at one site (Site 1). Volunteers were monitored following the procedure to assess safety. Image characteristics, including SNR, were compared to data acquired in a separate cohort using water as a solvent (n = 5) at another site (Site 2). The apparent spin-lattice relaxation time (T1) of [1-13C]pyruvate was determined both in vitro and in vivo from a mono-exponential fit to the image intensity at each time point of our dynamic data.
RESULTS: All volunteers completed the study safely and reported no adverse effects. The use of D2O increased the T1 of [1-13C]pyruvate from 66.5 ± 1.6 s to 92.1 ± 5.1 s in vitro, which resulted in an increase in signal by a factor of 1.46 ± 0.03 at the time of injection (90 s after dissolution). The use of D2O also increased the apparent relaxation time of [1-13C]pyruvate by a factor of 1.4 ± 0.2 in vivo. After adjusting for inter-site SNR differences, the use of D2O was shown to increase image SNR by a factor of 2.6 ± 0.2 in humans.
CONCLUSIONS: HP [1-13C]pyruvate in D2O is safe for human imaging and provides an increase in T1 and SNR that may improve image quality.

Different tissues are used for stable isotope analysis in cetacean investigations. However, variation in the isotopic composition of tissues with different turnover rates has been reported for cetaceans. To better understand stable carbon and nitrogen isotopes (δ13C and δ15N) in skin compared to other tissues, this study assessed the isotopic variation among the liver, muscle, and skin of Guiana dolphins (Sotalia guianensis), as well as the influence of sex on these variations. No differences were found in δ13C among male tissues, but females showed lower values in the liver compared to muscle and skin. Differences in δ15N were observed among all tissues, with different variation patterns for males and females. Four females were distinguished from males and other females by their 13C depletion in all tissues and δ15N variation pattern. We conclude that skin and muscle may be equivalent in δ13C values for Guiana dolphins. The multiple-tissue analysis brings new insights into their feeding ecology and provides background for stable isotope analysis using non-destructive sampling techniques in small cetaceans.

Pesticide degradation in wetland systems intercepting agricultural runoff is often overlooked and mixed with other dissipation processes when assessing pesticide concentrations alone. This study focused on the potential of compound-specific isotope analysis (CSIA) to estimate pesticide degradation in a stormwater wetland receiving pesticide runoff from a vineyard catchment. The fungicide dimethomorph (DIM), with diastereoisomers E and Z, was the prevalent pesticide in the runoff entering the wetland from June to September 2020. DIM Z, the most commonly detected isomer, exhibited a significant change (Δ(13C) > 3 ‰) in its carbon isotopic composition in the wetland water compared to the runoff and commercial formulation, which indicated degradation. Laboratory DIM degradation assays, including photodegradation and biodegradation in oxic wetland water with and without aquatic plants and in anoxic sediments, indicated that DIM degradation mainly occurred in the wetland sediments. The rapid degradation of both DIM isomers (E:t1/2 = 1.2 ± 0.6, Z: t1/2 = 1.5 ± 0.8 days) in the wetland sediment led to significant carbon isotopic fractionation (εDIM-E = -3.0 ± 0.6 ‰, εDIM-Z = -2.0 ± 0.2 ‰). In contrast, no significant isotope fractionation occurred during DIM photodegradation, despite the rapid isomerization of the E isomer to the Z isomer and a half-life of 15.3 ± 2.2 days for both isomers. DIM degradation was slow (E: t1/2 = 56-62 days, Z: t1/2 = 82-103 days) in oxic water with plants, while DIM persisted (120 days) in water without plants. DIM CSIA was thus used to evaluate the in situ biodegradation of DIM Z in the wetland. The DIM Z degradation estimates based on a classical concentration mass balance (86-94 %) were slightly higher than estimates based on the isotopic mass balance (61-68 %). Altogether, this study shows the potential of CSIA to conservatively evaluate pesticide degradation in wetland systems, offering a reliable alternative to classical labor-intensive mass balance approaches.).

A theoretical analysis of free Gibbs Energy and NMR 1H 13C chemical shifts of the effect of introduce methyl groups on diphenyl rings, to produce different isomers of (E)-1-(α,Ꞵ-dimethylbenzylidene)-2,2-diphenylhydrazine, is presented. IR vibrational frequencies, Mulliken charges, molecular electrostatic potential (MEP), Gibbs free energy (G) and 1H- and 13C-NMR chemical shifts were obtained by theoretical calculations. In this analysis it was found that the position of the methyl group affects the values of the 1H- and 13C-NMR chemical shifts and the ∆G and ∆H thermodynamic properties of formation and reaction, these properties vary with the same trend, for the isomers studied. Gibbs free energy calculations show that the theoretical (E)-1-(3,4-Dimethylbenzylidene)-2,2-diphenylhydrazine isomer is the most stable, which explains the success of the experimental synthesis of this compound among the other isomers. For this molecule, the C of the HC=N group is the most nucleophilic and the H is the least acidic. The 1H-NMR chemical shifts of protons show a strong correlation with the C=N distance. It was also observed that methyl affects the ν(C=N) frequencies, the C=N distance increases when the inductive effect of the methyl groups is in the structure.

Molecular stable isotope ratios are a novel type of dietary biomarker with high sensitivity and specificity for certain foods. Among these, fatty acid carbon isotope ratios (CIRs) have strong potential but have not been investigated as dietary biomarkers.
We evaluated whether fatty acid CIRs and mass proportions were associated with meat, fish, and sugar-sweetened beverage (SSB) intake.
Thirty-two men [aged 46.2 ± 10.5 y; BMI (kg/m2): 27.2 ± 4.0] underwent a 12-wk inpatient dietary intervention at the National Institute of Diabetes and Digestive and Kidney Diseases in Phoenix, Arizona. Men were randomly assigned to 1 of 8 dietary treatments varying the presence/absence of dietary meat, fish, and SSBs in all combinations. Fatty acid CIRs and mass proportions were measured in fasting blood samples and adipose tissue biopsies that were collected pre- and postintervention. Dietary effects were analyzed using multivariable regression and receiver operating characteristic AUCs were calculated using logistic regression.
CIRs of the several abundant SFAs, MUFAs and arachidonic acid (20:4n-6) in plasma were strongly associated with meat, as were a subset of these fatty acids in RBCs. Effect sizes in plasma ranged from 1.01‰ to 1.93‰ and were similar but attenuated in RBCs. Mass proportions of those fatty acids were not associated with diet. CIRs of plasma dihomo-γ-linolenic acid (20:3n-6) and adipose palmitic acid (16:0) were weakly associated with SSBs. Mass proportions of plasma odd-chain fatty acids were associated with meat, and mass proportions of plasma EPA and DHA (20:5n-3 and 22:6n-3) were associated with fish.
CIRs of plasma and RBC fatty acids show promise as sensitive and specific measures of dietary meat. These provide different information from that provided by fatty acid mass proportions, and are informative where mass proportion is not. This trial is registered at www.
gov as NCT01237093.

Glutamine is under scrutiny regarding its metabolic deregulation linked to energetic reprogramming in cancer cells. Many analytical techniques have been used to better understand the impact of the metabolism of amino acids on biological processes, however only a few are suited to work with complex samples. Here, we report the use of a general dissolution dynamic nuclear polarization (D-DNP) formulation using an unexpensive radical as a multipurpose tool to study glutamine, with insights from enzymatic modelling to complex metabolic networks and fast imaging. First, hyperpolarized [5-13 C] glutamine is used as molecular probe to study the kinetic action of two enzymes: L-asparaginase that has been used as an anti-metabolic treatment for cancer, and glutaminase. These results are also compared with those acquired with another hyperpolarized amino acid, [1,4-13 C] asparagine. Second, we explored the use of hyperpolarized (HP) substrates to probe metabolic pathways by monitoring metabolic profiles arising from hyperpolarized glutamine in E. coli extracts. Finally, a highly concentrated sample formulation is proposed for the purpose of fast imaging applications. We think that this approach can be extended to formulate other amino acids as well as other metabolites and provide complementary insights into the analysis of metabolic networks.