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Renal metabolism is essential for kidney functions and energy homeostasis in the body. The TCA cycle is the hub of metabolism, but the metabolic activities of the cycle in the kidney have rarely been investigated. This study is to assess metabolic processes at the level of the TCA cycle in the kidney based on isotopomer distributions in multiple metabolites. Isolated rat kidneys were perfused with media containing common substrates including lactate and alanine for an hour. One group of kidneys received [U- 13 C 3 ]lactate instead of natural abundance lactate while the other group received [U- 13 C 3 ]alanine instead of natural abundance alanine. Perfused kidneys and effluent were prepared for analysis using NMR spectroscopy. 13 C-labeling patterns in glutamate, fumarate, aspartate and succinate from the kidney extracts showed that pyruvate carboxylase and oxidative metabolism through the TCA cycle were comparably very active, but pyruvate cycling and pyruvate dehydrogenase were relatively less active. Isotopomer analyses with fumarate and malate from effluent, however, indicated that pyruvate carboxylase was much more active than the TCA cycle and other metabolic processes. The reverse equilibrium of oxaloacetate with four-carbon intermediates of the cycle was nearly complete (92%), based on the ratio of [2,3,4- 13 C 3 ]/[1,2,3- 13 C 3 ] in aspartate or malate. 13 C enrichment in glucose with 13 C-lactate supply was higher than that with 13 C-alanine. Isotopomer analyses with multiple metabolites (i.e., glutamate, fumarate, aspartate, succinate and malate) allowed us to assess relative metabolic processes in the TCA cycle in the kidney supplied with [U- 13 C 3 ]lactate. Data from the analytes were generally consistent, indicating highly active pyruvate carboxylase and oxidative metabolism through the TCA cycle. Different 13 C-labeling patterns in analytes from the kidney extracts versus effluent suggested metabolic compartmentalization., (© 2023 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Advanced imaging technologies, large-scale metabolomics, and the measurement of gene transcripts or enzyme expression all enable investigations of intermediary metabolism in human patients. Complementary information about fluxes in individual metabolic pathways may be obtained by ex vivo 13 C NMR of blood or tissue biopsies. Simple molecules such as 13 C-labeled glucose are readily administered to patients prior to surgical biopsies, and 13 C-labeled glycerol is easily administered orally to outpatients. Here, we review recent progress in practical applications of 13 C NMR to study cancer biology, the response to oxidative stress, gluconeogenesis, triglyceride synthesis in patients, as well as new insights into compartmentation of metabolism in the cytosol. The technical aspects of obtaining the sample, preparing material for analysis, and acquiring the spectra are relatively simple. This approach enables convenient, valuable, and quantitative insights into intermediary metabolism in patients., (© 2022 John Wiley & Sons, Ltd.)

After administration of 13 C-labeled glucose, the activity of the pentose phosphate pathway (PPP) is often assessed by the distribution of 13 C in lactate. However, in some tissues, such as the well-oxygenated heart, the concentration of lactate may be too low for convenient analysis by NMR. Here, we examined 13 C-labeled glutamate as an alternative biomarker of the PPP in the heart. Isolated rat hearts were perfused with media containing [2,3- 13 C 2 ]glucose and the tissue extracts were analyzed. Metabolism of [2,3- 13 C 2 ]glucose yields [1,2- 13 C 2 ]pyruvate via glycolysis and [2,3- 13 C 2 ]pyruvate via the PPP. Pyruvate is in exchange with lactate or is further metabolized to glutamate through pyruvate dehydrogenase and the TCA cycle. A doublet from [4,5- 13 C 2 ]glutamate, indicating flux through the PPP, was readily detected in 13 C NMR of heart extracts even when the corresponding doublet from [2,3- 13 C 2 ]lactate was minimal. Benfotiamine, known to induce the PPP, caused an increase in production of [4,5- 13 C 2 ]glutamate. In rats receiving [2,3- 13 C 2 ]glucose, brain extracts showed well-resolved signals from both [2,3- 13 C 2 ]lactate and [4,5- 13 C 2 ]glutamate in 13 C NMR spectra. Assessment of the PPP in the brain based on glutamate had a strong linear correlation with lactate-based assessment. In summary, 13 C NMR analysis of glutamate enabled detection of the low PPP activity in isolated hearts. This analyte is an alternative to lactate for monitoring the PPP with the use of [2,3- 13 C 2 ]glucose., (© 2021 John Wiley & Sons, Ltd.)

The pentose phosphate pathway (PPP) is essential for reductive biosynthesis, antioxidant processes and nucleotide production. Common tracers such as [1,2- 13 C 2 ]glucose rely on detection of 13 C in lactate and require assumptions to correct natural 13 C abundance. Here, we introduce a novel and specific tracer of the PPP, [2,3- 13 C 2 ]glucose. 13 C NMR analysis of the resulting isotopomers is informative because [1,2- 13 C 2 ]lactate arises from glycolysis and [2,3- 13 C 2 ]lactate arises exclusively through the PPP. A correction for natural abundance is unnecessary. In rats receiving [2,3- 13 C 2 ]glucose, the PPP was more active in the fed versus fasted state in the liver and the heart, consistent with increased expression of key enzymes in the PPP. Both the PPP and glycolysis were substantially increased in hepatoma compared with liver. In summary, [2,3- 13 C 2 ]glucose and 13 C NMR simplify assessment of the PPP., (© 2019 John Wiley & Sons, Ltd.)

The source of hyperpolarized (HP) [(13)C]bicarbonate in the liver during metabolism of HP [1-(13)C]pyruvate is uncertain and likely changes with physiology. Multiple processes including decarboxylation through pyruvate dehydrogenase or pyruvate carboxylase followed by subsequent decarboxylation via phosphoenolpyruvate carboxykinase (gluconeogenesis) could play a role. Here we tested which metabolic fate of pyruvate contributed to the appearance of HP [(13)C]bicarbonate during metabolism of HP [1-(13)C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The (13)C NMR of HP [(13)C]bicarbonate was observed in the liver of fed rats, but not in fasted rats where pyruvate carboxylation and gluconeogenesis was active. To further explore the relative fluxes through pyruvate carboxylase versus pyruvate dehydrogenase in the liver under typical conditions of hyperpolarization studies, separate parallel experiments were performed with rats given non-hyperpolarized [2,3-(13)C]pyruvate. (13)C NMR analysis of glutamate isolated from the liver of rats revealed that flux from injected pyruvate through pyruvate dehydrogenase was dominant under fed conditions whereas flux through pyruvate carboxylase dominated under fasted conditions. The NMR signal of HP [(13)C]bicarbonate does not parallel pyruvate carboxylase activity followed by subsequent decarboxylation reaction leading to glucose production. In the liver of healthy well-fed rats, the appearance of HP [(13)C]bicarbonate exclusively reflects decarboxylation of HP [1-(13)C]pyruvate via pyruvate dehydrogenase., (© 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)