Lactate Metabolism in Human Lung Tumors.

Cancer cells consume glucose and secrete lactate in culture. It is unknown whether lactate contributes to energy metabolism in living tumors. We previously reported that human non-small-cell lung cancers (NSCLCs) oxidize glucose in the tricarboxylic acid (TCA) cycle. Here, we show that lactate is also a TCA cycle carbon source for NSCLC. In human NSCLC, evidence of lactate utilization was most apparent in tumors with high 18fluorodeoxyglucose uptake and aggressive oncological behavior. Infusing human NSCLC patients with 13C-lactate revealed extensive labeling of TCA cycle metabolites. In mice, deleting monocarboxylate transporter-1 (MCT1) from tumor cells eliminated lactate-dependent metabolite labeling, confirming tumor-cell-autonomous lactate uptake. Strikingly, directly comparing lactate and glucose metabolism in vivo indicated that lactate's contribution to the TCA cycle predominates. The data indicate that tumors, including bona fide human NSCLC, can use lactate as a fuel in vivo.

Metabolic Heterogeneity in Human Lung Tumors.

Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intraoperative (13)C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature.

Functional activation of pyruvate dehydrogenase in human brain using hyperpolarized [1-13 C]pyruvate.

PURPOSE: Pyruvate, produced from either glucose, glycogen, or lactate, is the dominant precursor of cerebral oxidative metabolism. Pyruvate dehydrogenase (PDH) flux is a direct measure of cerebral mitochondrial function and metabolism. Detection of [13 C]bicarbonate in the brain from hyperpolarized [1-13 C]pyruvate using carbon-13 (13 C) MRI provides a unique opportunity for assessing PDH flux in vivo. This study is to assess changes in cerebral PDH flux in response to visual stimuli using in vivo 13 C MRS with hyperpolarized [1-13 C]pyruvate. METHODS: From seven sedentary adults in good general health, time-resolved [13 C]bicarbonate production was measured in the brain using 90° flip angles with minimal perturbation of its precursors, [1-13 C]pyruvate and [1-13 C]lactate, to test the hypothesis that the appearance of [13 C]bicarbonate signals in the brain reflects the metabolic changes associated with neuronal activation. With a separate group of healthy participants (n = 3), the likelihood of the bolus-injected [1-13 C]pyruvate being converted to [1-13 C]lactate prior to decarboxylation was investigated by measuring [13 C]bicarbonate production with and without [1-13 C]lactate saturation. RESULTS: In the course of visual stimulation, the measured [13 C]bicarbonate signal normalized to the total 13 C signal in the visual cortex increased by 17.1% ± 15.9% (p = 0.017), whereas no significant change was detected in [1-13 C]lactate. Proton BOLD fMRI confirmed the regional activation in the visual cortex with the stimuli. Lactate saturation decreased bicarbonate-to-pyruvate ratio by 44.4% ± 9.3% (p < 0.01). CONCLUSION: We demonstrated the utility of 13 C MRS with hyperpolarized [1-13 C]pyruvate for assessing the activation of cerebral PDH flux via the detection of [13 C]bicarbonate production.

Isotopomer analyses with the tricarboxylic acid cycle intermediates and exchanging metabolites from the rat kidney.

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 C3 ]lactate instead of natural abundance lactate while the other group received [U-13 C3 ]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 C3 ]/[1,2,3-13 C3 ] 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 C3 ]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.

Recent progress in analysis of intermediary metabolism by ex vivo 13 C NMR.

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.

Oxidation of hyperpolarized [1-13 C]pyruvate in isolated rat kidneys.

Kidneys play a central role in numerous disorders but current imaging methods have limited utility to probe renal metabolism. Hyperpolarized (HP) 13 C magnetic resonance imaging is uniquely suited to provide metabolite-specific information about key biochemical pathways and it offers the further advantage that renal imaging is practical in humans. This study evaluated the feasibility of hyperpolarization examinations in a widely used model for analysis of renal physiology, the isolated kidney, which enables isolation of renal metabolism from the effects of other organs and validation of HP results by independent measurements. Isolated rat kidneys were supplied with either HP [1-13 C]pyruvate only or HP [1-13 C]pyruvate plus octanoate. Metabolic activity in both groups was confirmed by stable renal oxygen consumption. HP [1-13 C]pyruvate was readily metabolized to [13 C]bicarbonate, [1-13 C]lactate, and [1-13 C]alanine, detectable seconds after HP [1-13 C]pyruvate was injected. Octanoate suppressed but did not eliminate the production of HP [13 C]bicarbonate from [1-13 C]pyruvate. Steady-state flux analyses using non-HP 13 C substrates validated the utilization of HP [1-13 C]pyruvate, as observed by HP 13 C NMR. In the presence of octanoate, lactate is generated from a tricarboxylic acid cycle intermediate, oxaloacetate. The isolated rat kidney may serve as an excellent model for investigating and establishing new HP 13 C metabolic probes for future kidney imaging applications.

PKM1 Exerts Critical Roles in Cardiac Remodeling Under Pressure Overload in the Heart.

BACKGROUND: Metabolic remodeling precedes most alterations during cardiac hypertrophic growth under hemodynamic stress. The elevation of glucose utilization has been recognized as a hallmark of metabolic remodeling. However, its role in cardiac hypertrophic growth and heart failure in response to pressure overload remains to be fully illustrated. Here, we aimed to dissect the role of cardiac PKM1 (pyruvate kinase muscle isozyme 1) in glucose metabolic regulation and cardiac response under pressure overload. METHODS: Cardiac-specific deletion of PKM1 was achieved by crossing the floxed PKM1 mouse model with the cardiomyocyte-specific Cre transgenic mouse. PKM1 transgenic mice were generated under the control of tetracycline response elements, and cardiac-specific overexpression of PKM1 was induced by doxycycline administration in adult mice. Pressure overload was triggered by transverse aortic constriction. Primary neonatal rat ventricular myocytes were used to dissect molecular mechanisms. Moreover, metabolomics and nuclear magnetic resonance spectroscopy analyses were conducted to determine cardiac metabolic flux in response to pressure overload. RESULTS: We found that PKM1 expression is reduced in failing human and mouse hearts. It is important to note that cardiomyocyte-specific deletion of PKM1 exacerbates cardiac dysfunction and fibrosis in response to pressure overload. Inducible overexpression of PKM1 in cardiomyocytes protects the heart against transverse aortic constriction-induced cardiomyopathy and heart failure. At the mechanistic level, PKM1 is required for the augmentation of glycolytic flux, mitochondrial respiration, and ATP production under pressure overload. Furthermore, deficiency of PKM1 causes a defect in cardiomyocyte growth and a decrease in pyruvate dehydrogenase complex activity at both in vitro and in vivo levels. CONCLUSIONS: These findings suggest that PKM1 plays an essential role in maintaining a homeostatic response in the heart under hemodynamic stress.

Dual-phase imaging of cardiac metabolism using hyperpolarized pyruvate.

PURPOSE: Previous cardiac imaging studies using hyperpolarized (HP) [1-13 C]pyruvate were acquired at end-diastole (ED). Little is known about the interaction between cardiac cycle and metabolite content in the myocardium. In this study, we compared images of HP pyruvate and products at end-systole (ES) and ED. METHODS: A dual-phase 13 C MRI sequence was implemented to acquire two sequential HP images within a single cardiac cycle at ES and ED during successive R-R intervals in an interleaved manner. Each healthy volunteer (N = 3) received two injections of HP [1-13 C]pyruvate for the dual-phase imaging on the short-axis and the vertical long-axis planes. Spatial distribution of HP 13 C metabolites at each cardiac phase was correlated to multiphase 1 H MRI to confirm the mechanical changes. Ratios of myocardial HP metabolites were compared between ES and ED. Segmental analysis was performed on the midcavity short-axis plane. RESULTS: In addition to mechanical changes, metabolic profiles of the heart detected by HP [1-13 C]pyruvate differed between ES and ED. The myocardial signal of [13 C]bicarbonate relative to [1-13 C]lactate was significantly smaller at ED than the ratio at ES (p < .05), particularly in mid-anterior and mid-inferoseptal segments. The distinct metabolic profiles in the myocardium likely reflect the technical aspects of the imaging approach such as the coronary flow in addition to the cyclical changes in metabolism. CONCLUSION: The study demonstrates that metabolic profiles of the heart, measured by HP [1-13 C]pyruvate, are affected by the cardiac cycle in which that the data are acquired.

Dynamic 13 C MR spectroscopy as an alternative to imaging for assessing cerebral metabolism using hyperpolarized pyruvate in humans.

PURPOSE: This study is to investigate time-resolved 13 C MR spectroscopy (MRS) as an alternative to imaging for assessing pyruvate metabolism using hyperpolarized (HP) [1-13 C]pyruvate in the human brain. METHODS: Time-resolved 13 C spectra were acquired from four axial brain slices of healthy human participants (n = 4) after a bolus injection of HP [1-13 C]pyruvate. 13 C MRS with low flip-angle excitations and a multichannel 13 C/1 H dual-frequency radiofrequency (RF) coil were exploited for reliable and unperturbed assessment of HP pyruvate metabolism. Slice-wise areas under the curve (AUCs) of 13 C-metabolites were measured and kinetic analysis was performed to estimate the production rates of lactate and HCO3- . Linear regression analysis between brain volumes and HP signals was performed. Region-focused pyruvate metabolism was estimated using coil-wise 13 C reconstruction. Reproducibility of HP pyruvate exams was presented by performing two consecutive injections with a 45-minutes interval. RESULTS: [1-13 C]Lactate relative to the total 13 C signal (tC) was 0.21-0.24 in all slices. [13 C] HCO3- /tC was 0.065-0.091. Apparent conversion rate constants from pyruvate to lactate and HCO3- were calculated as 0.014-0.018 s-1 and 0.0043-0.0056 s-1 , respectively. Pyruvate/tC and lactate/tC were in moderate linear relationships with fractional gray matter volume within each slice. White matter presented poor linear regression fit with HP signals, and moderate correlations of the fractional cerebrospinal fluid volume with pyruvate/tC and lactate/tC were measured. Measured HP signals were comparable between two consecutive exams with HP [1-13 C]pyruvate. CONCLUSIONS: Dynamic MRS in combination with multichannel RF coils is an affordable and reliable alternative to imaging methods in investigating cerebral metabolism using HP [1-13 C]pyruvate.

Hyperpolarized 13C MR Spectroscopy Depicts in Vivo Effect of Exercise on Pyruvate Metabolism in Human Skeletal Muscle.

Background Pyruvate dehydrogenase (PDH) and lactate dehydrogenase are essential for adenosine triphosphate production in skeletal muscle. At the onset of exercise, oxidation of glucose and glycogen is quickly enabled by dephosphorylation of PDH. However, direct measurement of PDH flux in exercising human muscle is daunting, and the net effect of covalent modification and other control mechanisms on PDH flux has not been assessed. Purpose To demonstrate the feasibility of assessing PDH activation and changes in pyruvate metabolism in human skeletal muscle after the onset of exercise using carbon 13 (13C) MRI with hyperpolarized (HP) [1-13C]-pyruvate. Materials and Methods For this prospective study, sedentary adults in good general health (mean age, 42 years ± 18 [standard deviation]; six men) were recruited from August 2019 to September 2020. Subgroups of the participants were injected with HP [1-13C]-pyruvate at resting, during plantar flexion exercise, or 5 minutes after exercise during recovery. In parallel, hydrogen 1 arterial spin labeling MRI was performed to estimate muscle tissue perfusion. An unpaired t test was used for comparing 13C data among the states. Results At rest, HP [1-13C]-lactate and [1-13C]-alanine were detected in calf muscle, but [13C]-bicarbonate was negligible. During moderate flexion-extension exercise, total HP 13C signals (tC) increased 2.8-fold because of increased muscle perfusion (P = .005), and HP [1-13C]-lactate-to-tC ratio increased 1.7-fold (P = .04). HP [13C]-bicarbonate-to-tC ratio increased 8.4-fold (P = .002) and returned to the resting level 5 minutes after exercise, whereas the lactate-to-tC ratio continued to increase to 2.3-fold as compared with resting (P = .008). Conclusion Lactate and bicarbonate production from hyperpolarized (HP) [1-carbon 13 {13C}]-pyruvate in skeletal muscle rapidly reflected the onset and the termination of exercise. These results demonstrate the feasibility of imaging skeletal muscle metabolism using HP [1-13C]-pyruvate MRI and the sensitivity of in vivo pyruvate metabolism to exercise states. © RSNA, 2021 Online supplemental material is available for this article.

Assessment of hepatic pyruvate carboxylase activity using hyperpolarized [1-13 C]-l-lactate.

PURPOSE: To evaluate the utility of hyperpolarized [1-13 C]-l-lactate to detect hepatic pyruvate carboxylase activity in vivo under fed and fasted conditions. METHODS: [1-13 C]-labeled sodium L-lactate was polarized using a dynamic nuclear polarizer. Polarization level and the T1 were measured in vitro in a 3 Telsa MR scanner. Two groups of healthy rats (fasted vs. fed) were prepared for in vivo studies. Each rat was anesthetized and intravenously injected with 60-mM hyperpolarized [1-13 C]-l-lactate, immediately followed by dynamic acquisition of 13 C (carbon-13) MR spectra from the liver at 3 Tesla. The dosage-dependence of the 13 C-products was also investigated by performing another injection of an equal volume of 30-mM hyperpolarized [1-13 C]-l-lactate. RESULTS: T1 and liquid polarization level of [1-13 C]-l-lactate were estimated as 67.8 s and 40.0%, respectively. [1-13 C]pyruvate and [1-13 C]alanine, [13 C]bicarbonate ( HCO3- ) and [1-13 C]aspartate were produced from hyperpolarized [1-13 C]-l-lactate in rat liver. Smaller HCO3- and larger aspartate were measured in the fed group compared to the fasted group. Pyruvate and alanine production were increased in proportion to the lactate concentration, whereas the amount of HCO3- and aspartate production was consistent between 30-mM and 60-mM lactate injections. CONCLUSION: This study demonstrates that a unique biomarker of pyruvate carboxylase flux, the appearance of [1-13 C]aspartate from [1-13 C]-l-lactate, is sensitive to nutritional state and may be monitored in vivo at 3 Tesla. Because [13 C] HCO3- is largely produced by pyruvate dehydrogenase flux, these results suggest that the ratio of [1-13 C]aspartate and [13 C] HCO3- (aspartate/ HCO3- ) reflects the saturable pyruvate carboxylase/pyruvate dehydrogenase enzyme activities.

Cardiac T2∗ measurement of hyperpolarized 13 C metabolites using metabolite-selective multi-echo spiral imaging.

PURPOSE: Noninvasive imaging with hyperpolarized (HP) pyruvate can capture in vivo cardiac metabolism. For proper quantification of the metabolites and optimization of imaging parameters, understanding MR characteristics such as T2∗ s of the HP signals is critical. This study is to measure in vivo cardiac T2∗ s of HP [1-13 C]pyruvate and the products in rodents and humans. METHODS: A dynamic 13 C multi-echo spiral imaging sequence that acquires [13 C]bicarbonate, [1-13 C]lactate, and [1-13 C]pyruvate images in an interleaved manner was implemented for a clinical 3 Tesla system. T2∗ of each metabolite was calculated from the multi-echo images by fitting the signal decay of each region of interest mono-exponentially. The performance of measuring T2∗ using the sequence was first validated using a 13 C phantom and then with rodents following a bolus injection of HP [1-13 C]pyruvate. In humans, T2∗ of each metabolite was calculated for left ventricle, right ventricle, and myocardium. RESULTS: Cardiac T2∗ s of HP [1-13 C]pyruvate, [1-13 C]lactate, and [13 C]bicarbonate in rodents were measured as 24.9 ± 5.0, 16.4 ± 4.7, and 16.9 ± 3.4 ms, respectively. In humans, T2∗ of [1-13 C]pyruvate was 108.7 ± 22.6 ms in left ventricle and 129.4 ± 8.9 ms in right ventricle. T2∗ of [1-13 C]lactate was 40.9 ± 8.3, 44.2 ± 5.5, and 43.7 ± 9.0 ms in left ventricle, right ventricle, and myocardium, respectively. T2∗ of [13 C]bicarbonate in myocardium was 64.4 ± 2.5 ms. The measurements were reproducible and consistent over time after the pyruvate injection. CONCLUSION: The proposed metabolite-selective multi-echo spiral imaging sequence reliably measures in vivo cardiac T2∗ s of HP [1-13 C]pyruvate and products.

Spectral fitting strategy to overcome the overlap between 2-hydroxyglutarate and lipid resonances at 2.25 ppm.

PURPOSE: 1 H MRS provides a noninvasive tool for identifying mutations in isocitrate dehydrogenase (IDH). Quantification of the prominent 2-hydroxyglutarate (2HG) resonance at 2.25 ppm is often confounded by the lipid resonance at the same frequency in tumors with elevated lipids. We propose a new spectral fitting approach to separate these overlapped signals, therefore, improving 2HG evaluation. METHODS: TE 97 ms PRESS was acquired at 3T from 42 glioma patients. New lipid basis sets were created, in which the small lipid 2.25-ppm signal strength was preset with reference to the lipid signal at 0.9 ppm, incorporating published fat relaxation data. LCModel fitting using the new lipid bases (Fitting method 2) was conducted along with fitting using the LCModel built-in lipid basis set (Fitting method 1), in which the lipid 2.25-ppm signal is assessed with reference to the lipid 1.3-ppm signal. In-house basis spectra of low-molecular-weight metabolites were used in both fitting methods. RESULTS: Fitting method 2 showed marked improvement in identifying IDH mutational status compared with Fitting method 1. 2HG estimates from Fitting method 2 were overall smaller than those from Fitting method 1, which was because of differential assignment of the signal at 2.25 ppm to lipids. In receiver operating characteristic analysis, Fitting method 2 provided a complete distinction between IDH mutation and wild-type whereas Fitting method 1 did not. CONCLUSION: The data suggest that 1 H MR spectral fitting using the new lipid basis set provides a robust fitting strategy that improves 2HG evaluation in brain tumors with elevated lipids.

15 N-carnitine, a novel endogenous hyperpolarized MRI probe with long signal lifetime.

PURPOSE: The purpose of this study was to investigate hyperpolarization and in vivo imaging of [15 N]carnitine, a novel endogenous MRI probe with long signal lifetime. METHODS: L-[15 N]carnitine-d9 was hyperpolarized by the method of dynamic nuclear polarization followed by rapid dissolution. The T1 signal lifetimes were estimated in aqueous solution and in vivo following intravenous injection in rats, using a custom-built dual-tuned 15 N/1 H RF coil at 4.7 T. 15 N chemical shift imaging and 15 N fast spin-echo images of rat abdomen were acquired 3 minutes after [15 N]carnitine injection. RESULTS: Estimated T1 times of [15 N]carnitine at 4.7 T were 210 seconds (in H2 O) and 160 seconds (in vivo), with an estimated polarization level of 10%. Remarkably, the [15 N]carnitine coherence was detectable in rat abdomen for 5 minutes after injection for the nonlocalized acquisition. No downstream metabolites were detected on localized or nonlocalized 15 N spectra. Diffuse liver enhancement was detected on 15 N fast spin-echo imaging 3 minutes after injection, with mean hepatic SNR of 18 ± 5 at a spatial resolution of 4 × 4 mm. CONCLUSION: This study showed the feasibility of hyperpolarizing and imaging the biodistribution of HP [15 N]carnitine.

Characterization and compensation of f0 inhomogeneity artifact in spiral hyperpolarized 13 C imaging of the human heart.

PURPOSE: This study aimed to investigate the role of regional f0 inhomogeneity in spiral hyperpolarized 13 C image quality and to develop measures to alleviate these effects. METHODS: Field map correction of hyperpolarized 13 C cardiac imaging using spiral readouts was evaluated in healthy subjects. Spiral readouts with differing duration (26 and 45 ms) but similar resolution were compared with respect to off-resonance performance and image quality. An f0 map-based image correction based on the multifrequency interpolation (MFI) method was implemented and compared to correction using a global frequency shift alone. Estimation of an unknown frequency shift was performed by maximizing a sharpness objective based on the Sobel variance. The apparent full width half at maximum (FWHM) of the myocardial wall on [13 C]bicarbonate was used to estimate blur. RESULTS: Mean myocardial wall FWHM measurements were unchanged with the short readout pre-correction (14.1 ± 2.9 mm) and post-MFI correction (14.1 ± 3.4 mm), but significantly decreased in the long waveform (20.6 ± 6.6 mm uncorrected, 17.7 ± 7.0 corrected, P = .007). Bicarbonate signal-to-noise ratio (SNR) of the images acquired with the long waveform were increased by 1.4 ± 0.3 compared to those acquired with the short waveform (predicted 1.32). Improvement of image quality was observed for all metabolites with f0 correction. CONCLUSIONS: f0 -map correction reduced blur and recovered signal from dropouts, particularly along the posterior myocardial wall. The low image SNR of [13 C]bicarbonate can be compensated with longer duration readouts but at the expense of increased f0 artifacts, which can be partially corrected for with the proposed methods.

31 P-MRS of the healthy human brain at 7 T detects multiple hexose derivatives of uridine diphosphate glucose.

Nucleotide sugars are required for the synthesis of glycoproteins and glycolipids, which play crucial roles in many cellular functions such as cell communication and immune responses. Uridine diphosphate-glucose (UDP-Glc) was previously believed to be the only nucleotide sugar detectable in brain by 31 P-MRS. Using spectra of high SNR and high resolution acquired at 7 T, we showed that multiple nucleotide sugars are coexistent in brain and can be measured simultaneously. In addition to UDP-Glc, these also include UDP-galactose (UDP-Gal), -N-acetyl-glucosamine (UDP-GlcNAc) and -N-acetyl-galactosamine (UDP-GalNAc), collectively denoted as UDP(G). Coexistence of these UDP(G) species is evident from a quartet-like multiplet at -9.8 ppm (M-9.8 ), which is a common feature seen across a wide age range (24-64 years). Lineshape fitting of M-9.8 allows an evaluation of all four UDP(G) components, which further aids in analysis of a mixed signal at -8.2 ppm (M-8.2 ) for deconvolution of NAD+ and NADH. For a group of seven young healthy volunteers, the concentrations of UDP(G) species were 0.04 ± 0.01 mM for UDP-Gal, 0.07 ± 0.03 mM for UDP-Glc, 0.06 ± 0.02 mM for UDP-GalNAc and 0.08 ± 0.03 mM for UDP-GlcNA, in reference to ATP (2.8 mM). The combined concentration of all UDP(G) species (average 0.26 ± 0.06 mM) was similar to the pooled concentration of NAD+ and NADH (average 0.27 ± 0.06 mM, with a NAD+ /NADH ratio of 6.7 ± 2.1), but slightly lower than previously found in an older cohort (0.31 mM). The in vivo NMR analysis of UDP-sugar composition is consistent with those from tissue extracts by other modalities in the literature. Given that glycosylation is dependent on the availability of nucleotide sugars, assaying multiple nucleotide sugars may provide valuable insights into potential aberrant glycosylation, which has been implicated in certain diseases such as cancer and Alzheimer's disease.

13 C NMR of glutamate for monitoring the pentose phosphate pathway in myocardium.

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 C2 ]glucose and the tissue extracts were analyzed. Metabolism of [2,3-13 C2 ]glucose yields [1,2-13 C2 ]pyruvate via glycolysis and [2,3-13 C2 ]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 C2 ]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 C2 ]lactate was minimal. Benfotiamine, known to induce the PPP, caused an increase in production of [4,5-13 C2 ]glutamate. In rats receiving [2,3-13 C2 ]glucose, brain extracts showed well-resolved signals from both [2,3-13 C2 ]lactate and [4,5-13 C2 ]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 C2 ]glucose.

Analysis of steady-state carbon tracer experiments using akaike information criteria.

INTRODUCTION: Carbon isotope tracers have been used to determine relative rates of tricarboxylic acid cycle (TCA) cycle pathways since the 1950s. Steady-state experimental data are typically fit to a single mathematical model of metabolism to determine metabolic fluxes. Whether the chosen model is appropriate for the biological system has generally not been evaluated systematically. An overly-simple model omits known pathways while an overly-complex model may produce incorrect results due to overfitting. OBJECTIVES: The objectives were to develop and study a method that systematically evaluates multiple TCA cycle mathematical models as part of the fitting process. METHODS: The problem of choosing overly-simple or overly-complex models was approached by developing software that automatically explores all possible combinations of flux through pyruvate dehydrogenase, pyruvate kinase, pyruvate carboxylase and anaplerosis at propionyl-CoA carboxylase, and equivalent pathways, all relative to TCA cycle flux. Typical TCA cycle metabolic tracer experiments that use 13C nuclear magnetic resonance for detection and quantification of 13C-enriched glutamate products were simulated and analyzed. By evaluating the multiple model fits with both the conventional sum-of-squares residual error (SSRE) and the Akaike Information Criterion (AIC), the software helps the investigator understand the interaction between model complexity and goodness of fit. RESULTS: When fitting alternative models of the TCA cycle metabolism, the SSRE may identify more than one model that fits the data well. Among those models, the AIC provides guidance as to which is the simplest of the candidate models is sufficient to describe the observed data. However under some conditions, AIC used alone inappropriately discriminates against necessary metabolic complexity. CONCLUSION: In combination, the SSRE and AIC help the investigator identify the model that best describes the metabolism of a biological system.

Quantitative measurement of redox state in human brain by 31 P MRS at 7T with spectral simplification and inclusion of multiple nucleotide sugar components in data analysis.

PURPOSE: To develop a simplified method for quantitative measurement of NAD+ /NADH (nicotinamide adenine dinucleotides) levels in human brain by 31 P MRS without interference from the α-ATP signal and with inclusion of multiple UDP-sugar components. METHODS: Simple pulse-acquire 31 P MR spectra were collected at 7T with and without a frequency-selective inversion pulse to remove the dominant α-ATP signal from the underlying NAD(H) signal. Careful inspection of the 31 P signal at -9.8 ppm previously assigned to UDP-glucose revealed multiple UDP-sugar components that must also be considered when deconvoluting the NAD(H) signal to quantify NAD+ and NADH. Finally, the overlapping NAD(H) and UDP(G) resonances were deconvoluted into individual components using Voigt lineshape analysis and UDP(G) modeling. RESULTS: The inversion-based spectral editing method enabled clean separation of the NAD(H) signal from the otherwise dominant α-ATP signal. In addition, the upfield signal near -9.8 ppm appears more "quartet-like" than a simple doublet consistent with contributions from other nucleotide sugars such as UDP-galactose, UDP-N-acetyl-galactosamine, and UDP-N-acetyl-glucosamine in addition to UDP-glucose. Deconvolution of the combined NAD(H) and UDP(G) signals showed that the measured NAD+ /NAD ratio was heavily influenced by UDP(G) modeling (7.5 ± 1.8 when the UDP(G) signal was fitted as multiple doublets versus 5.3 ± 0.6 when a simplified pseudo doublet model was used). In a test/re-test experiments separated by 2 weeks, consistent NAD+ /NADH ratios were measured in the brain of seven human subjects. CONCLUSIONS: The NAD+ /NADH ratio in human brain can be measured using 31 P MR spectra simplified by spectral editing and with inclusion of multiple UDP-sugar components in the data analysis.

Remodeling of substrate consumption in the murine sTAC model of heart failure.

BACKGROUND: Energy metabolism and substrate selection are key aspects of correct myocardial mechanical function. Myocardial preference for oxidizable substrates changes in both hypertrophy and in overt failure. Previous work has shown that glucose oxidation is upregulated in overpressure hypertrophy, but its fate in overt failure is less clear. Anaplerotic flux of pyruvate into the tricarboxylic acid cycle (TCA) has been posited as a secondary fate of glycolysis, aside from pyruvate oxidation or lactate production. METHODS AND RESULTS: A model of heart failure that emulates both valvular and hypertensive heart disease, the severe transaortic constriction (sTAC) mouse, was assayed for changes in substrate preference using metabolomic and carbon-13 flux measurements. Quantitative measures of O2 consumption in the Langendorff perfused mouse heart were paired with 13C isotopomer analysis to assess TCA cycle turnover. Since the heart accommodates oxidation of all physiological energy sources, the utilization of carbohydrates, fatty acids, and ketones were measured simultaneously using a triple-tracer NMR method. The fractional contribution of glucose to acetyl-CoA production was upregulated in heart failure, while other sources were not significantly different. A model that includes both pyruvate carboxylation and anaplerosis through succinyl-CoA produced superior fits to the data compared to a model using only pyruvate carboxylation. In the sTAC heart, anaplerosis through succinyl-CoA is elevated, while pyruvate carboxylation was not. Metabolomic data showed depleted TCA cycle intermediate pool sizes versus the control, in agreement with previous results. CONCLUSION: In the sTAC heart failure model, the glucose contribution to acetyl-CoA production was significantly higher, with compensatory changes in fatty acid and ketone oxidation not reaching a significant level. Anaplerosis through succinyl-CoA is also upregulated, and is likely used to preserve TCA cycle intermediate pool sizes. The triple tracer method used here is new, and can be used to assess sources of acetyl-CoA production in any oxidative tissue.