Diagnosing sucrase-isomaltase deficiency: a comparison of a 13C-sucrose breath test and a duodenal enzyme assay.

BACKGROUND: Reduced activity of the sucrase-isomaltase (SI) enzyme can cause gastrointestinal symptoms. Biochemical measurement of SI activity in small intestinal biopsies is presently considered the gold standard for the diagnosis of SI deficiency, but this invasive test is not suitable as a routine diagnostic tool. AIM: To evaluate a 13C-sucrose-breath test (13CSBT) as a diagnostic tool for SI deficiency in an adult population. METHODS: 13CSBT results were compared to sucrase activity measured in duodenal biopsies. RESULTS: Forty patients with gastrointestinal symptoms were included in the study, 4 of whom had celiac disease and the rest (n = 36) had normal histological findings. Nine patients (22.5%) had low sucrase activity measured using duodenal biopsies. No correlation was observed between enzymatic sucrase activity and the 13CSBT results. The 13CSBT-curves for the celiac patients versus patients with normal duodenal histology demonstrated that the patients with celiac disease were within the lower range of the distribution. CONCLUSION: We observed a mismatch between the 13CSBT results and the biochemically measured sucrase activity, suggesting that SI activity is not uniformly distributed throughout the small intestines. This methodological discrepancy should be acknowledged when diagnosing SI deficiency.

Hydroponic Growth of [13C]-Labeled Tobacco for DNA Damage Studies in Cigarette Smokers.

Cigarette smoking is the acknowledged major cause of cancers of the lung and oral cavity and is an established important risk factor for multiple other cancers. DNA addition products (DNA adducts) caused by cigarette smoking are critical factors in its mechanism of carcinogenesis. However, most DNA adducts detected to date in humans cannot be specifically ascribed to smoking but rather have multiple exogenous and endogenous sources. In the study reported here, we prepared [13C]-labeled tobacco to address this problem. We report for the first time the successful growth from seeds to flowering under hydroponic conditions of highly [13C]-labeled tobacco in a controlled 13CO2 environment. The standard growth procedure with optimized conditions is described in detail. The [13C]-enrichment rate was assessed by quantifying nicotine and sugars and their [13C]-isotopologues in this tobacco using high-resolution mass spectrometry, reaching >94% in the tobacco leaves. The [13C]-labeled leaves after curing will be used to make cigarettes, allowing investigation of the specific contributions of tobacco smoke carcinogens to identified DNA adducts in smokers.

Hyperpolarized [2-13C, 3-2H3]Pyruvate Detects Hepatic Gluconeogenesis In Vivo.

The feasibility of hyperpolarized [2-13C, 3-2H3]pyruvate for probing gluconeogenesis in vivo was investigated in this study. Whereas hyperpolarized [1-13C]pyruvate has clear access to metabolic pathways that convert pyruvate to lactate, alanine, and bicarbonate, its utility for assessing pyruvate carboxylation and gluconeogenesis has been limited by technical challenges, including spectral overlap and an obscure enzymatic step that decarboxylates the labeled carbon. To achieve unambiguous detection of gluconeogenic products, the carbonyl carbon in pyruvate was labeled with 13C. To prolong the T1 relaxation time, [2-13C, 3-2H3]pyruvate was synthesized and dissolved with D2O after dynamic nuclear polarization. The T1 of [2-13C, 3-2H3]pyruvate in D2O could be improved by 76.9% (79.6 s at 1 T and 74.5 s at 3 T) as compared to [2-13C]pyruvate in water. Hyperpolarized [2-13C, 3-2H3]pyruvate with D2O dissolution was applied to rat livers in vivo under normal feeding and fasting conditions. A gluconeogenic product, [2-13C]phosphoenolpyruvate, was observed at 149.9 ppm from fasted rats only, highlighting the utility of [2-13C, 3-2H3]pyruvate in detecting key gluconeogenic enzyme activities such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase in vivo.

A comprehensive monitoring approach for a naturally anoxic aquifer beneath a controlled landfill.

The processes leading to high levels of arsenic (As), iron (Fe), and manganese (Mn) in groundwater, in a naturally reducing aquifer at a controlled municipal landfill site, are investigated. The challenge is to distinguish the natural water-rock interaction processes, that allow these substances to dissolve in groundwater, from direct pollution or enhanced dissolution of hydroxides as undesired consequences of the anthropic activities above. Ordinary groundwater monitoring of physical-chemical parameters and inorganic compounds (major and trace elements) was complemented by environmental isotopes of groundwater (tritium, deuterium, oxygen-18 and carbon-13) and dissolved gases (carbon-13 of methane and carbon dioxide and carbon-14 of methane). Pearson/Spearman correlation indices, as well as Principal Component Analysis (PCA), were used to determine the main correlations among variables. The concurrent presence of As, Fe and CH4, as reported in similar anoxic environments, suggests that anaerobic oxidation of methane could drive the reductive dissolution of As-rich Fe(III)(hydro)oxides. Manganese is more sensitive to carbon dioxide, possibly due to a decrease in pH which accelerates the dissolution of Mn-oxides. Finally, we found that tritium and deuterium, which have been used for decades as leachate tracer in groundwater, may be subject to false positives due to the reuse of water recovered from leachate treatment (which has the same isotopic signature of leachate) within the plants, to comply with the requirements of the circular economy. The integration of the environmental isotope analysis into the traditional monitoring approach can effectively support the comprehension of processes. However, this strategy needs to be complemented by a good conceptual hydrogeological model and expert evaluation to avoid misinterpretations.

Comparison of carbon, nitrogen, and oxygen stable isotope ratios and mercury concentrations in muscle tissues of five beaked whale species and sperm whales stranded in Hokkaido, Japan.

We studied δ13C, δ15N and δ18O values, and total mercury (THg) concentrations in muscle samples from deep-sea predators - five beaked whale species and sperm whales - stranded along the coast of Hokkaido, in the north of Japan in 2010 and 2019. The δ13C, δ15N and δ18O values, THg concentrations, and body length (BL) of Stejneger's beaked whales were similar to those of Hubbs' beaked whales, which belong to the same genus. In contrast, δ13C values, THg concentrations, and BL of Sato's beaked whales were markedly different from those of Baird's beaked whales, which belong to the same genus. Stejneger's and Hubbs' beaked whales living around Hokkaido may compete in their ecological niches, whereas Sato's and Baird's beaked whales may segregate their ecological niches. Although Cuvier's beaked whales and sperm whales belong to different genera and their BLs were significantly different, their δ13C and δ15N values were similar, probably because they can dive and stay in deeper waters than other beaked whale species. The δ13C values in combined samples from all whales increased with increasing BL, probably owing to the larger whale species' dietary preference for squid. The δ13C values in combined samples from all whales were positively correlated with THg concentrations, whereas the δ15N values in the combined samples were negatively correlated. The δ18O values in combined samples from most whales tended to be positively correlated with THg concentrations. These correlations may be explained by a higher THg load from deep-sea feeding than from pelagic feeding and by a feeding shift towards lower trophic levels.

MRI Application and Challenges of Hyperpolarized Carbon-13 Pyruvate in Translational and Clinical Cardiovascular Studies: A Literature Review.

Cardiovascular disease shows, or may even be caused by, changes in metabolism. Hyperpolarized magnetic resonance spectroscopy and imaging is a technique that could assess the role of different aspects of metabolism in heart disease, allowing real-time metabolic flux assessment in vivo. In this review, we introduce the main hyperpolarization techniques. Then, we summarize the use of dedicated radiofrequency 13C coils, and report a state of the art of 13C data acquisition. Finally, this review provides an overview of the pre-clinical and clinical studies on cardiac metabolism in the healthy and diseased heart. We furthermore show what advances have been made to translate this technique into the clinic in the near future and what technical challenges still remain, such as exploring other metabolic substrates.

Winter-ground microhabitat use by differently coloured phenotypes affects return rate in a long-distance migratory bird.

Migratory bird populations are declining globally at alarming rates. Non-breeding site conditions affect breeding populations, but generalising non-breeding habitat conditions over large spatial regions cannot address potential fine-scale differences across landscapes or local populations. Plumage characteristics can mediate the effects of environmental conditions on individual fitness. However, whether different phenotypes use distinctive non-breeding sites, and whether they respond to non-breeding site conditions differently remains largely unknown. Stable isotopes (δ13C, δ15N, δ2H) of inert tissues are useful to infer habitat characteristics and geographic origins where those tissues were grown. We collected winter-grown feathers from pied flycatchers (Ficedula hypoleuca) on their breeding grounds over several years from males whose dorsal plumage colouration ranged continuously from brown to black and assessed their stable isotope values as proxies of local habitat conditions. Based on feather δ2H profiles we found that browner males spent their non-breeding season in drier habitats than black males. Assignment to origin analysis shows potential regional non-breeding ground separation between differently coloured males. High within-individual repeatability of both δ13C and δ15N indicate the pied flycatcher males return yearly to similar areas. Blacker males were more likely to return to the breeding grounds after dry years compared with brown males. The opposite was found in wet years. Our study demonstrates that different phenotypes are exposed to different non-breeding site conditions which can differentially affect individual survivorship. This has important ramifications for population dynamics under predicted climate change scenarios where especially brown phenotype pied flycatcher males may be under a risk of decreasing.

Hyperpolarized [1-13C]-pyruvate MRS evaluates immune potential and predicts response to radiotherapy in cervical cancer.

BACKGROUND: Monitoring pyruvate metabolism in the spleen is important for assessing immune activity and achieving successful radiotherapy for cervical cancer due to the significance of the abscopal effect. We aimed to explore the feasibility of utilizing hyperpolarized (HP) [1-13C]-pyruvate magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) to evaluate pyruvate metabolism in the human spleen, with the aim of identifying potential candidates for radiotherapy in cervical cancer. METHODS: This prospective study recruited six female patients with cervical cancer (median age 55 years; range 39-60) evaluated using HP [1-13C]-pyruvate MRI/MRS at baseline and 2 weeks after radiotherapy. Proton (1H) diffusion-weighted MRI was performed in parallel to estimate splenic cellularity. The primary outcome was defined as tumor response to radiotherapy. The Student t-test was used for comparing 13C data between the groups. RESULTS: The splenic HP [1-13C]-lactate-to-total carbon (tC) ratio was 5.6-fold lower in the responders than in the non-responders at baseline (p = 0.009). The splenic [1-13C]-lactate-to-tC ratio revealed a 1.7-fold increase (p = 0.415) and the splenic [1-13C]-alanine-to-tC ratio revealed a 1.8-fold increase after radiotherapy (p = 0.482). The blood leukocyte differential count revealed an increased proportion of neutrophils two weeks following treatment, indicating enhanced immune activity (p = 0.013). The splenic apparent diffusion coefficient values between the groups were not significantly different. CONCLUSIONS: This exploratory study revealed the feasibility of HP [1-13C]-pyruvate MRS of the spleen for evaluating baseline immune potential, which was associated with clinical outcomes of cervical cancer after radiotherapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT04951921 , registered 7 July 2021. RELEVANCE STATEMENT: This prospective study revealed the feasibility of using HP 13C MRI/MRS for assessing pyruvate metabolism of the spleen to evaluate the patients' immune potential that is associated with radiotherapeutic clinical outcomes in cervical cancer. KEY POINTS: • Effective radiotherapy induces abscopal effect via altering immune metabolism. • Hyperpolarized 13C MRS evaluates patients' immune potential non-invasively. • Pyruvate-to-lactate conversion in the spleen is elevated following radiotherapy.

Carbon 14 and Carbon 13 Syntheses of Velagliflozin.

Velagliflozin is the active ingredient of the first oral liquid medication approved by the Food and Drug Administration for the treatment of diabetes in cats. This compound belongs to the known class of sodium-glucose cotransporter 2 inhibitors approved to treat diabetes in human. Here, we report the detailed synthesis of velagliflozin labeled with carbon 14 and carbon 13.

Hyperpolarized Carbon-13 Magnetic Resonance Imaging: Technical Considerations and Clinical Applications.

Hyperpolarized (HP) carbon-13 (13C) MRI represents an innovative approach for noninvasive, real-time assessment of dynamic metabolic flux, with potential integration into routine clinical MRI. The use of [1-13C]pyruvate as a probe and its conversion to [1-13C]lactate constitute an extensively explored metabolic pathway. This review comprehensively outlines the establishment of HP 13C-MRI, covering multidisciplinary team collaboration, hardware prerequisites, probe preparation, hyperpolarization techniques, imaging acquisition, and data analysis. This article discusses the clinical applications of HP 13C-MRI across various anatomical domains, including the brain, heart, skeletal muscle, breast, liver, kidney, pancreas, and prostate. Each section highlights the specific applications and findings pertinent to these regions, emphasizing the potential versatility of HP 13C-MRI in diverse clinical contexts. This review serves as a comprehensive update, bridging technical aspects with clinical applications and offering insights into the ongoing advancements in HP 13C-MRI.

Current methods for hyperpolarized [1-13C]pyruvate MRI human studies.

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of HP agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate-by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods and Equipment study groups. It further aims to provide a comprehensive reference for future consensus, building as the field continues to advance human studies with this metabolic imaging modality.

Anaerobic dihydrogen consumption of nutrient-limited aquifer sediment microbial communities examined by stable isotope analysis.

The biogeochemical consequences of dihydrogen (H2) underground storage in porous aquifers are poorly understood. Here, the effects of nutrient limitations on anaerobic H2 oxidation of an aquifer microbial community in sediment microcosms were determined in order to evaluate possible responses to high H2 partial pressures. Hydrogen isotope analyses of H2 yielded isotope depletion in all biotic setups indicating microbial H2 consumption. Carbon isotope analyses of carbon dioxide (CO2) showed isotope enrichment in all H2-supplemented biotic setups indicating H2-dependent consumption of CO2 by methanogens or homoacetogens. Homoacetogenesis was indicated by the detection of acetate and formate. Consumption of CO2 and H2 varied along the differently nutrient-amended setups, as did the onset of methane production. Plotting carbon against hydrogen isotope signatures of CH4 indicated that CH4 was produced hydrogenotrophically and fermentatively. The putative hydrogenotrophic Methanobacterium sp. was the dominant methanogen. Most abundant phylotypes belonged to typical ferric iron reducers, indicating that besides CO2, Fe(III) was an important electron acceptor. In summary, our study provides evidence for the adaptability of subsurface microbial communities under different nutrient-deficient conditions to elevated H2 partial pressures.

Effects of lipid extraction on stable isotope ratios of carbon and nitrogen in muscles of freshwater fish.

The extraction of lipids by the Folch method from the muscles of all the fish studied led to statistically significant differences in the values of δ15N. At the same time, lipid extraction led to a statistically significant increase in δ13C in pike and roach, and to a statistically insignificant decrease in δ13C in perch and bream. Thus, lipid extraction cannot serve as a universal method of sample preparation for the analysis of the isotopic composition of carbon (13C/12C) and nitrogen (15N/14N) in fish muscles. The differences between the δ13C values in the samples before and after lipid extraction were statistically investigated by different models. It is shown that mathematical correction method models can be used, but the results are depending on the fish types.

A continuous flow bioreactor system for high-throughput hyperpolarized metabolic flux analysis.

Hyperpolarized carbon-13 labeled compounds are increasingly being used in medical MR imaging (MRI) and MR imaging (MRI) and spectroscopy (MRS) research, due to its ability to monitor tissue and cell metabolism in real-time. Although radiological biomarkers are increasingly being considered as clinical indicators, biopsies are still considered the gold standard for a large variety of indications. Bioreactor systems can play an important role in biopsy examinations because of their ability to provide a physiochemical environment that is conducive for therapeutic response monitoring ex vivo. We demonstrate here a proof-of-concept bioreactor and microcoil receive array setup that allows for ex vivo preservation and metabolic NMR spectroscopy on up to three biopsy samples simultaneously, creating an easy-to-use and robust way to simultaneously run multisample carbon-13 hyperpolarization experiments. Experiments using hyperpolarized [1-13C]pyruvate on ML-1 leukemic cells in the bioreactor setup were performed and the kinetic pyruvate-to-lactate rate constants ( k PL ) extracted. The coefficient of variation of the experimentally found k PL s for five repeated experiments was C V = 35 % . With this statistical power, treatment effects of 30%-40% change in lactate production could be easily differentiable with only a few hyperpolarization dissolutions on this setup. Furthermore, longitudinal experiments showed preservation of ML-1 cells in the bioreactor setup for at least 6 h. Rat brain tissue slices were also seen to be preserved within the bioreactor for at least 1 h. This validation serves as the basis for further optimization and upscaling of the setup, which undoubtedly has huge potential in high-throughput studies with various biomarkers and tissue types.

Hyperpolarized 13 C metabolic imaging of the human abdomen with spatiotemporal denoising.

PURPOSE: Improving the quality and maintaining the fidelity of large coverage abdominal hyperpolarized (HP) 13 C MRI studies with a patch based global-local higher-order singular value decomposition (GL-HOVSD) spatiotemporal denoising approach. METHODS: Denoising performance was first evaluated using the simulated [1-13 C]pyruvate dynamics at different noise levels to determine optimal kglobal and klocal parameters. The GL-HOSVD spatiotemporal denoising method with the optimized parameters was then applied to two HP [1-13 C]pyruvate EPI abdominal human cohorts (n = 7 healthy volunteers and n = 8 pancreatic cancer patients). RESULTS: The parameterization of kglobal = 0.2 and klocal = 0.9 denoises abdominal HP data while retaining image fidelity when evaluated by RMSE. The kPX (conversion rate of pyruvate-to-metabolite, X = lactate or alanine) difference was shown to be <20% with respect to ground-truth metabolic conversion rates when there is adequate SNR (SNRAUC > 5) for downstream metabolites. In both human cohorts, there was a greater than nine-fold gain in peak [1-13 C]pyruvate, [1-13 C]lactate, and [1-13 C]alanine apparent SNRAUC . The improvement in metabolite SNR enabled a more robust quantification of kPL and kPA . After denoising, we observed a 2.1 ± 0.4 and 4.8 ± 2.5-fold increase in the number of voxels reliably fit across abdominal FOVs for kPL and kPA quantification maps. CONCLUSION: Spatiotemporal denoising greatly improves visualization of low SNR metabolites particularly [1-13 C]alanine and quantification of [1-13 C]pyruvate metabolism in large FOV HP 13 C MRI studies of the human abdomen.

Distinguishing metabolic signals of liver tumors from surrounding liver cells using hyperpolarized 13 C MRI and gadoxetate.

PURPOSE: To use the hepatocyte-specific gadolinium-based contrast agent gadoxetate combined with hyperpolarized (HP) [1-13 C]pyruvate MRI to selectively suppress metabolic signals from normal hepatocytes while preserving the signals arising from tumors. METHODS: Simulations were performed to determine the expected changes in HP 13 C MR signal in liver and tumor under the influence of gadoxetate. CC531 colon cancer cells were implanted into the livers of five Wag/Rij rats. Liver and tumor metabolism were imaged at 3 T using HP [1-13 C] pyruvate chemical shift imaging before and 15 min after injection of gadoxetate. Area under the curve for pyruvate and lactate were measured from voxels containing at least 75% of normal-appearing liver or tumor. RESULTS: Numerical simulations predicted a 36% decrease in lactate-to-pyruvate (L/P) ratio in liver and 16% decrease in tumor. In vivo, baseline L/P ratio was 0.44 ± 0.25 in tumors versus 0.21 ± 0.08 in liver (p = 0.09). Following administration of gadoxetate, mean L/P ratio decreased by an average of 0.11 ± 0.06 (p < 0.01) in normal-appearing liver. In tumors, mean L/P ratio post-gadoxetate did not show a statistically significant change from baseline. Compared to baseline levels, the relative decrease in L/P ratio was significantly greater in liver than in tumors (-0.52 ± 0.16 vs. -0.19 ± 0.25, p < 0.05). CONCLUSIONS: The intracellular hepatobiliary contrast agent showed a greater effect suppressing HP 13 C MRI metabolic signals (through T1 shortening) in normal-appearing liver when compared to tumors. The combined use of HP MRI with selective gadolinium contrast agents may allow more selective imaging in HP 13 C MRI.

Advanced Hyperpolarized 13C Metabolic Imaging Protocol for Patients with Gliomas: A Comprehensive Multimodal MRI Approach.

This study aimed to implement a multimodal 1H/HP-13C imaging protocol to augment the serial monitoring of patients with glioma, while simultaneously pursuing methods for improving the robustness of HP-13C metabolic data. A total of 100 1H/HP [1-13C]-pyruvate MR examinations (104 HP-13C datasets) were acquired from 42 patients according to the comprehensive multimodal glioma imaging protocol. Serial data coverage, accuracy of frequency reference, and acquisition delay were evaluated using a mixed-effects model to account for multiple exams per patient. Serial atlas-based HP-13C MRI demonstrated consistency in volumetric coverage measured by inter-exam dice coefficients (0.977 ± 0.008, mean ± SD; four patients/11 exams). The atlas-derived prescription provided significantly improved data quality compared to manually prescribed acquisitions (n = 26/78; p = 0.04). The water-based method for referencing [1-13C]-pyruvate center frequency significantly reduced off-resonance excitation relative to the coil-embedded [13C]-urea phantom (4.1 ± 3.7 Hz vs. 9.9 ± 10.7 Hz; p = 0.0007). Significantly improved capture of tracer inflow was achieved with the 2-s versus 5-s HP-13C MRI acquisition delay (p = 0.007). This study demonstrated the implementation of a comprehensive multimodal 1H/HP-13C MR protocol emphasizing the monitoring of steady-state/dynamic metabolism in patients with glioma.

Chemical and C and N stable isotope compositions of three species of epiphytic Tillandsia in a Caribbean coastal zone: air pollution sources and biomonitoring implications.

We characterized the elemental and C and N stable isotope compositions of Tillandsia fasciculata Sw., Tillandsia balbisiana Schult. & Schult.f. and Tillandsia recurvata (L.) L. samples collected in Cienfuegos (Cuba). Results showed high enrichment factors for S, Hg, Cd, Pb, P, Zn, Cu, Mo, Sb and Ca in all Tillandsia species, indicating inputs from local anthropogenic activities (road traffic, industries and cement production). Carbon concentrations and δ13C varied from 38.3-47.7 % and -20.4 to -13.4 ‰ within the three species, respectively. δ13C showed seasonal dependence with the dry and wet periods and more 13C-depleted values in urban/industrial areas, coherent with the input of anthropogenic emissions. Nitrogen concentrations (0.4-1.3 %) and δ15N values (-9.9-4.4 ‰) exhibit larger variations and are positively correlated in the three species. The most positive δ15N in T. recurvata (-0.2-4.4 ‰) are attributed to contributions from industrial activities and road traffic. In fact, both δ15N and total nitrogen (TN) values increase in sites with higher road traffic and show significant correlations with typical road traffic and industrial tracers. Finally, we calculate an average total nitrogen deposition rate of 4.4 ± 2.3 kg ha-1 a-1 from N content in T. recurvata, similar to the existing values determined in the region by field measurements, but higher than the global terrestrial average.

Preparation of [1'-13 C]citric acid as a probe in a breath test to evaluate tricarboxylic acid cycle flux.

[1'-13 C]Citric acid (1) was efficiently prepared from dimethyl 1,3-acetonedicarboxylate in two steps as a probe for a breath test. The synthetic method was selected because of the yield and reproducibility. Compound 1 was orally administrated to rats, and the time course of the increase of 13 CO2 /12 CO2 ratios (Δ13 CO2 ) in their breath was successfully followed, indicating the metabolism of 1. Thus, the 13 C-breath test using 1 is a promising method to evaluate tricarboxylic acid (TCA) cycle flux.

Radiocarbon integrity of dissolved inorganic carbon (DIC) samples stored in plastic and glass bottles: implications for reliable groundwater age dating.

Various approaches based on the natural variations of carbon isotopes (14C and 13C) in dissolved inorganic carbon (DIC) are routinely used to study groundwater dynamics and to estimate recharge rates by deriving groundwater ages. However, differences in 14C activities in groundwater samples collected repeatedly from the same wells and discordantly young 14C groundwater ages compared to noble gases led some authors to question the validity of radiocarbon dating. Poor sampling protocols and storage effects (14C contamination) for radiocarbon analysis are a critical factor in explaining age determination discrepancies. We evaluated the impact of storage protocols on carbon isotope exchange with atmospheric carbon dioxide by comparing glass versus standard plastic field sampling bottles for various storage times before radiocarbon and 13C analyses. The 14C bias after 12 months in pre-evacuated glass vials was minimal and within analytical precision. However, storage of DIC samples in plastic sampling bottles led to marked changes in 14C and 13C contents (up to ∼15 pmC and ∼ 5 ‰, respectively, after 12 months), meaning contamination led to younger groundwater age estimations than it should have been. Protocols for sampling and storing DIC samples for radiocarbon using pre-evacuated glass bottles help avoid atmospheric 14CO2 contamination and microbial activity.