Carbon sources and pathways for citrate secreted by human prostate cancer cells determined by NMR tracing and metabolic modeling.

Prostate epithelial cells have the unique capacity to secrete large amounts of citrate, but the carbon sources and metabolic pathways that maintain this production are not well known. We mapped potential pathways for citrate carbons in the human prostate cancer metastasis cell lines LNCaP and VCaP, for which we first established that they secrete citrate (For LNCaP 5.6 ± 0.9 nmol/h per 106 cells). Using 13C-labeled substrates, we traced the incorporation of 13C into citrate by NMR of extracellular fluid. Our results provide direct evidence that glucose is a main carbon source for secreted citrate. We also demonstrate that carbons from supplied glutamine flow via oxidative Krebs cycle and reductive carboxylation routes to positions in secreted citrate but likely do not contribute to its net synthesis. The potential anaplerotic carbon sources aspartate and asparagine did not contribute to citrate carbons. We developed a quantitative metabolic model employing the 13C distribution in extracellular citrate after 13C glucose and pyruvate application to assess intracellular pathways of carbons for secreted citrate. From this model, it was estimated that in LNCaP about 21% of pyruvate entering the Krebs cycle is converted via pyruvate carboxylase as an anaplerotic route at a rate more than sufficient to compensate carbon loss of this cycle by citrate secretion. This model provides an estimation of the fraction of molecules, including citrate, leaving the Krebs cycle at every turn. The measured ratios of 13C atoms at different positions in extracellular citrate may serve as biomarkers for (malignant) epithelial cell metabolism.

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.

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.

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.

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.

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.

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.

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.

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 [2-13C]pyruvate MR molecular imaging with whole brain coverage.

Hyperpolarized (HP) 13C Magnetic Resonance Imaging (MRI) was applied for the first time to image and quantify the uptake and metabolism of [2-13C]pyruvate in the human brain to provide new metabolic information on cerebral energy metabolism. HP [2-13C]pyruvate was injected intravenously and imaged in 5 healthy human volunteer exams with whole brain coverage in a 1-minute acquisition using a specialized spectral-spatial multi-slice echoplanar imaging (EPI) pulse sequence to acquire 13C-labeled volumetric and dynamic images of [2-13C]pyruvate and downstream metabolites [5-13C]glutamate and [2-13C]lactate. Metabolic ratios and apparent conversion rates of pyruvate-to-lactate (kPL) and pyruvate-to-glutamate (kPG) were quantified to investigate simultaneously glycolytic and oxidative metabolism in a single injection.

Quantitative and carbon isotope ratio analysis of fatty acids isolated from human brain hemispheres.

Our knowledge surrounding the overall fatty acid profile of the adult human brain has been largely limited to extrapolations from brain regions in which the distribution of fatty acids varies. This is especially problematic when modeling brain fatty acid metabolism, therefore, an updated estimate of whole-brain fatty acid concentration is necessitated. Here, we sought to conduct a comprehensive quantitative analysis of fatty acids from entire well-characterized human brain hemispheres (n = 6) provided by the Douglas-Bell Canada Brain Bank. Additionally, exploratory natural abundance carbon isotope ratio (CIR; δ13 C, 13 C/12 C) analysis was performed to assess the origin of brain fatty acids. Brain fatty acid methyl esters (FAMEs) were quantified by gas chromatography (GC)-flame ionization detection and minor n-6 and n-3 polyunsaturated fatty acid pentafluorobenzyl esters by GC-mass spectrometry. Carbon isotope ratio values of identifiable FAMEs were measured by GC-combustion-isotope ratio mass spectrometry. Overall, the most abundant fatty acid in the human brain was oleic acid, followed by stearic acid (STA), palmitic acid (PAM), docosahexaenoic acid (DHA), and arachidonic acid (ARA). Interestingly, cholesterol as well as saturates including PAM and STA were most enriched in 13 C, while PUFAs including DHA and ARA were most depleted in 13 C. These findings suggest a contribution of endogenous synthesis utilizing dietary sugar substrates rich in 13 C, and a combination of marine, animal, and terrestrial PUFA sources more depleted in 13 C, respectively. These results provide novel insights on cerebral fatty acid origin and concentration, the latter serving as a valuable resource for future modeling of fatty acid metabolism in the human brain.

Age-associated change in pyruvate metabolism investigated with hyperpolarized 13 C-MRI of the human brain.

In this study, hyperpolarized 13 C MRI (HP-13 C MRI) was used to investigate changes in the uptake and metabolism of pyruvate with age. Hyperpolarized 13 C-pyruvate was administered to healthy aging individuals (N = 35, ages 21-77) and whole-brain spatial distributions of 13 C-lactate and 13 C-bicarbonate production were measured. Linear mixed-effects regressions were performed to compute the regional percentage change per decade, showing a significant reduction in both normalized 13 C-lactate and normalized 13 C-bicarbonate production with age: - 7 % ± 2 % per decade for 13 C-lactate and - 9 % ± 4 % per decade for 13 C-bicarbonate. Certain regions, such as the right medial precentral gyrus, showed greater rates of change while the left caudate nucleus had a flat 13 C-lactate versus age and a slightly increasing 13 C-bicarbonate versus age. The results show that both the production of lactate (visible as 13 C-lactate signal) as well as the consumption of monocarboxylates to make acetyl-CoA (visible as 13 C-bicarbonate signal) decrease with age and that the rate of change varies by brain region.

Investigating cerebral perfusion with high resolution hyperpolarized [1-13 C]pyruvate MRI.

PURPOSE: To investigate high-resolution hyperpolarized (HP) 13 C pyruvate MRI for measuring cerebral perfusion in the human brain. METHODS: HP [1-13 C]pyruvate MRI was acquired in five healthy volunteers with a multi-resolution EPI sequence with 7.5 × 7.5 mm2 resolution for pyruvate. Perfusion parameters were calculated from pyruvate MRI using block-circulant singular value decomposition and compared to relative cerebral blood flow calculated from arterial spin labeling (ASL). To examine regional perfusion patterns, correlations between pyruvate and ASL perfusion were performed for whole brain, gray matter, and white matter voxels. RESULTS: High resolution 7.5 × 7.5 mm2 pyruvate images were used to obtain relative cerebral blood flow (rCBF) values that were significantly positively correlated with ASL rCBF values (r = 0.48, 0.20, 0.28 for whole brain, gray matter, and white matter voxels respectively). Whole brain voxels exhibited the highest correlation between pyruvate and ASL perfusion, and there were distinct regional patterns of relatively high ASL and low pyruvate normalized rCBF found across subjects. CONCLUSION: Acquiring HP 13 C pyruvate metabolic images at higher resolution allows for finer spatial delineation of brain structures and can be used to obtain cerebral perfusion parameters. Pyruvate perfusion parameters were positively correlated to proton ASL perfusion values, indicating a relationship between the two perfusion measures. This HP 13 C study demonstrated that hyperpolarized pyruvate MRI can assess cerebral metabolism and perfusion within the same study.

A user independent denoising method for x-nuclei MRI and MRS.

PURPOSE: X-nuclei (also called non-proton MRI) MRI and spectroscopy are limited by the intrinsic low SNR as compared to conventional proton imaging. Clinical translation of x-nuclei examination warrants the need of a robust and versatile tool improving image quality for diagnostic use. In this work, we compare a novel denoising method with fewer inputs to the current state-of-the-art denoising method. METHODS: Denoising approaches were compared on human acquisitions of sodium (23 Na) brain, deuterium (2 H) brain, carbon (13 C) heart and brain, and simulated dynamic hyperpolarized 13 C brain scans, with and without additional noise. The current state-of-the-art denoising method Global-local higher order singular value decomposition (GL-HOSVD) was compared to the few-input method tensor Marchenko-Pastur principal component analysis (tMPPCA). Noise-removal was quantified by residual distributions, and statistical analyses evaluated the differences in mean-square-error and Bland-Altman analysis to quantify agreement between original and denoised results of noise-added data. RESULTS: GL-HOSVD and tMPPCA showed similar performance for the variety of x-nuclei data analyzed in this work, with tMPPCA removing ˜5% more noise on average over GL-HOSVD. The mean ratio between noise-added and denoising reproducibility coefficients of the Bland-Altman analysis when compared to the original are also similar for the two methods with 3.09 ± 1.03 and 2.83 ± 0.79 for GL-HOSVD and tMPPCA, respectively. CONCLUSION: The strength of tMPPCA lies in the few-input approach, which generalizes well to different data sources. This makes the use of tMPPCA denoising a robust and versatile tool in x-nuclei imaging improvements and the preferred denoising method.

Considering whole-body metabolism in hyperpolarized MRI through 13 C breath analysis-An alternative way to quantification and normalization?

PURPOSE: Hyperpolarized [1-13 C]pyruvate MRI is an emerging clinical tool for metabolic imaging. It has the potential for absolute quantitative metabolic imaging. However, the method itself is not quantitative, limiting comparison of images across both time and between individuals. Here, we propose a simple signal normalization to the whole-body oxidative metabolism to overcome this limitation. THEORY AND METHODS: A simple extension of the model-free ratiometric analysis of hyperpolarized [1-13 C]pyruvate MRI is presented, using the expired 13 CO2 in breath for normalization. The proposed framework was investigated in two porcine cohorts (N = 11) subjected to local renal hypoperfusion defects and subsequent [1-13 C]pyruvate MRI. A breath sample was taken before the [1-13 C]pyruvate injection and 5 min after. The raw MR signal from both the healthy and intervened kidney in the two cohorts was normalized using the 13 CO2 in the expired air. RESULTS: 13 CO2 content in the expired air was significantly different between the two cohorts. Normalization to this reduced the coefficients of variance in the aerobic metabolic sensitive pathways by 25% for the alanine/pyruvate ratio, and numerical changes were observed in the bicarbonate/pyruvate ratio. The lactate/pyruvate ratio was largely unaltered (<2%). CONCLUSION: Our results indicate that normalizing the hyperpolarized 13 C-signal ratios by the 13 CO2 content in expired air can reduce variation as well as improve specificity of the method by normalizing the metabolic readout to the overall metabolic status of the individual. The method is a simple and cheap extension to the hyperpolarized 13 C exam.

Hyperpolarized 13 C Metabolic MRI of Patients with Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related death in the United States. However, early response assessment using the current approach of measuring changes in tumor size on computed tomography (CT) or MRI is challenging. PURPOSE: To investigate the feasibility of hyperpolarized (HP) [1-13 C]pyruvate MRI to quantify metabolism in the normal appearing pancreas and PDA, and to assess changes in PDA metabolism following systemic chemotherapy. STUDY TYPE: Prospective. SUBJECTS: Six patients (65.0 ± 7.6 years, 2 females) with locally advanced or metastatic PDA enrolled prior to starting a new line of systemic chemotherapy. FIELD STRENGTH/SEQUENCE: 3-T, T1-weighted gradient echo, metabolite-selective 13 C echoplanar imaging. ASSESSMENT: Time-resolved HP [1-13 C]pyruvate data were acquired before (N = 6) and 4-weeks after (N = 3) treatment initiation. Pyruvate metabolism, as quantified by pharmacokinetic modeling and metabolite area-under-the-curve ratios, was assessed in manually segmented PDA and normal appearing pancreas ROIs (N = 5). The change in tumor metabolism before and 4-weeks after treatment initiation was assessed in primary PDA (N = 2) and liver metastases (N = 1), and was compared to objective tumor response defined by response evaluation criteria in solid tumors (RECIST) on subsequent CTs. STATISTICAL TESTS: Descriptive tests (mean ± standard deviation), model fit error for pharmacokinetic rate constants. RESULTS: Primary PDA showed reduced alanine-to-lactate ratios when compared to normal pancreas, due to increased lactate-to-pyruvate or reduced alanine-to-pyruvate ratios. Of the three patients who received HP [1-13 C]pyruvate MRI before and 4-weeks after treatment initiation, one patient had a primary tumor with early metabolic response (increase in alanine-to-lactate) and subsequent partial response according to RECIST, one patient had a primary tumor with relatively stable metabolism and subsequent stable disease by RECIST, and one patient had metastatic PDA with increase in lactate-to-pyruvate of the liver metastases and corresponding progressive disease according to RECIST. DATA CONCLUSION: Altered pyruvate metabolism with increased lactate or reduced alanine was observed in the primary tumor. Early metabolic response assessed at 4-weeks after treatment initiation correlated with subsequent objective tumor response assessed using RECIST. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

Do foraging ecology and contaminants interactively predict parenting hormone levels in common eider?

Global climate change is causing abiotic shifts such as higher air and ocean temperatures, and disappearing sea ice in Arctic ecosystems. These changes influence Arctic-breeding seabird foraging ecology by altering prey availability and selection, affecting individual body condition, reproductive success, and exposure to contaminants such as mercury (Hg). The cumulative effects of alterations to foraging ecology and Hg exposure may interactively alter the secretion of key reproductive hormones such as prolactin (PRL), important for parental attachment to eggs and offspring and overall reproductive success. However, more research is needed to investigate the relationships between these potential links. Using data collected from 106 incubating female common eiders (Somateria mollissima) at six Arctic and sub-Arctic colonies, we examined whether the relationship between individual foraging ecology (assessed using δ13C, δ15N) and total Hg (THg) exposure predicted PRL levels. We found a significant, complex interaction between δ13C, δ15N and THg on PRL, suggesting that individuals cumulatively foraging at lower trophic levels, in phytoplankton-dominant environments, and with the highest THg levels had the most constant significant relationship PRL levels. Cumulatively, these three interactive variables resulted in lowered PRL. Overall, results demonstrate the potential downstream and cumulative implications of environmentally induced changes in foraging ecology, in combination with THg exposure, on hormones known to influence reproductive success in seabirds. These findings are notable in the context of continuing environmental and food web changes in Arctic systems, which may make seabird populations more susceptible to ongoing stressors.

Mechanistic inference of the metabolic rates underlying [Formula: see text]C breath test curves.

Carbon stable isotope breath tests offer new opportunities to better understand gastrointestinal function in health and disease. However, it is often not clear how to isolate information about a gastrointestinal or metabolic process of interest from a breath test curve, and it is generally unknown how well summary statistics from empirical curve fitting correlate with underlying biological rates. We developed a framework that can be used to make mechanistic inference about the metabolic rates underlying a 13C breath test curve, and we applied it to a pilot study of 13C-sucrose breath test in 20 healthy adults. Starting from a standard conceptual model of sucrose metabolism, we determined the structural and practical identifiability of the model, using algebra and profile likelihoods, respectively, and we used these results to develop a reduced, identifiable model as a function of a gamma-distributed process; a slower, rate-limiting process; and a scaling term related to the fraction of the substrate that is exhaled as opposed to sequestered or excreted through urine. We demonstrated how the identifiable model parameters impacted curve dynamics and how these parameters correlated with commonly used breath test summary measures. Our work develops a better understanding of how the underlying biological processes impact different aspect of 13C breath test curves, enhancing the clinical and research potential of these 13C breath tests.

Carbon-13 labeling of ibrutinib for human microdosing.

Ibrutinib is an oral medication for the treatment of B cell malignancies. During its clinical development, a stable isotopologue of ibrutinib was required for the assessment of the drug's absolute oral bioavailability via intravenous microdosing. The following work describes a 10-step, gram-scale production of carbon-13 labeled ibrutinib from [13 C6 ]phenol (13 C6 , 99%) in 31% overall yield with >99% chemical purity and >99% enantiomeric excess (ee), suitable for intravenous microdosing in humans.

Stable isotope synthesis of glycine transporter 1 inhibitor Iclepertin (BI 425809) and its major metabolites.

Stable isotope labeled Iclepertin (BI 425809, 1) and its major metabolites are needed as internal standards in bioanalytical studies. BI 425809 consists of two main building blocks, 5-methylsulfonyl-2-[(1R)-2,2,2-trifluoro-1-methyl-ethoxy]benzoic acid (2) and 3-[(1R,5R)-3-azabicyclo[3.1.0]hexan-5-yl]-5-(trifluoromethyl)isoxazole (3) linked to each other via an amide bond. We used fluoro[13 C6 ]benzene as the starting material in the preparation of [13 C6 ]-2. This intermediate was then employed to access carbon 13 labeled Iclepertin ([13 C6 ]-1) and other metabolites. The major metabolite BI 761036 (6), which resulted from cytochrome P450 oxidation and amide hydrolysis of BI 425809, was prepared labeled with carbon 13 and nitrogen 15 via two synthetic routes. In the first route, diethyl [13 C3 ]malonate, [13 C]methyl iodide, and hydroxyl[15 N]amine were used to provide [13 C4 ,15 N]-BI 761036 ([13 C4 ,15 N]-6a) in 13 steps in 6% overall yield, whereas in the second route, [13 C3 ]propargyl alcohol, potassium [13 C]cyanide, and [15 N]ammonia were used to furnish [13 C4 ,15 N]-BI 761036 ([13 C4 ,15 N]-6b) in 11 steps in 1% overall yield. The detailed stable isotope synthesis of 1 and its major metabolites is described.