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. © 2022 John Wiley & Sons Ltd.

Cellular redox is intricately linked to energy production and normal cell function. Although the redox states of mitochondria and cytosol are connected by shuttle mechanisms, the redox state of mitochondria may differ from redox in the cytosol in response to stress. However, detecting these differences in functioning tissues is difficult. Here, we employed C-13 magnetic resonance spectroscopy (MRS) and co- polarized [1-C-13] pyruvate and [1,3- C-13(2)] acetoacetate ([1,3-C-13(2)]AcAc) to monitor production of hyperpolarized (HP) lactate and beta-hydroxybutyrate as indicators of cytosolic and mitochondrial redox, respectively. Isolated rat hearts were examined under normoxic conditions, during low-flow ischemia, and after pretreatment with either aminooxyacetate (AOA) or rotenone. All interventions were associated with an increase in [P-i]/[ATP] measured by P-31 NMR. In well-oxygenated untreated hearts, rapid conversion of HP [1-C-13]pyruvate to [1-C-13]lactate and [1,3-C-13(2)]AcAc to [1,3-C-13(2)]beta-hydroxybutyrate ([1,3-C-13(2)]beta-HB) was readily detected. A significant increase in HP [1,3-C-13(2)]beta-HB but not [1-C-13]lactate was observed in rotenone-treated and ischemic hearts, consistent with an increase in mitochondrial NADH but not cytosolic NADH. AOA treatments did not alter the productions of HP [1-C-13]lactate or [1,3-C-13(2)]beta-HB. This study demonstrates that biomarkers of mitochondrial and cytosolic redox may be detected simultaneously in functioning tissues using co-polarized [1-C-13]pyruvate and [1,3-C-13(2)]AcAc and C-13 MRS and that changes in mitochondrial redox may precede changes in cytosolic redox.

Purpose This study is to investigate time-resolved C-13 MR spectroscopy (MRS) as an alternative to imaging for assessing pyruvate metabolism using hyperpolarized (HP) [1-C-13]pyruvate in the human brain. Methods Time-resolved C-13 spectra were acquired from four axial brain slices of healthy human participants (n = 4) after a bolus injection of HP [1-C-13]pyruvate. C-13 MRS with low flip-angle excitations and a multichannel C-13/H-1 dual-frequency radiofrequency (RF) coil were exploited for reliable and unperturbed assessment of HP pyruvate metabolism. Slice-wise areas under the curve (AUCs) of C-13-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 C-13 reconstruction. Reproducibility of HP pyruvate exams was presented by performing two consecutive injections with a 45-minutes interval. Results [1-C-13]Lactate relative to the total C-13 signal (tC) was 0.21-0.24 in all slices. [C-13]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-C-13]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-C-13]pyruvate.

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 (C-13) MRI with hyperpolarized (HP) [1-C-13]-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-C-13]-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 C-13 data among the states.Results: At rest, HP [1-C-13]-lactate and [1-C-13]-alanine were detected in calf muscle, but [C-13]-bicarbonate was negligible. During moderate flexion-extension exercise, total HP C-13 signals (tC) increased 2.8-fold because of increased muscle perfusion (P = .005), and HP [1-C-13]-lactate-to-tC ratio increased 1.7-fold (P = .04). HP [C-13]-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 {C-13}]-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-C-13]-pyruvate MRI and the sensitivity of in vivo pyruvate metabolism to exercise states. (C) RSNA, 2021

Transient disruption of the blood-brain barrier (BBB) with focused ultrasound (FUS) is an emerging clinical method to facilitate targeted drug delivery to the brain. The focal noninvasive disruption of the BBB can be applied to promote the local delivery of hyperpolarized substrates. In this study, we investigated the effects of FUS on imaging brain metabolism using two hyperpolarized C-13-labeled substrates in rodents: [1-C-1(3)]-pyruvate and [1-C-1(3)]glycerate. The BBB is a rate-limiting factor for pyruvate delivery to the brain, and glycerate minimally passes through the BBB. First, cerebral imaging with hyperpolarized [1-C-1(3)]pyruvate resulted in an increase in total C-13 signals (p = 0.05) after disrupting the BBB with FUS. Significantly higher levels of both [1-C-1(3)]lactate (lactate/total C-13 signals, p = 0.01) and [C-13]bicarbonate (p = 0.008) were detected in the FUS-applied brain region as compared to the contralateral FUS-unaffected normal-appearing brain region. The application of FUS without opening the BBB in a separate group of rodents resulted in comparable lactate and bicarbonate productions between the FUS-applied and the contralateral brain regions. Second, C-13 imaging with hyperpolarized [1-C-13]glycerate after opening the BBB showed increased [1-C-1(3)]glycerate delivery to the FUS-applied region (p = 0.04) relative to the contralateral side, and [1-C-1(3)]lactate production was consistently detected from the FUS-applied region. Our findings suggest that FUS accelerates the delivery of hyperpolarized molecules across the BBB and provides enhanced sensitivity to detect metabolic products in the brain; therefore, hyperpolarized C-13 imaging with FUS may provide new opportunities to study cerebral metabolic pathways as well as various neurological pathologies.

De novo lipogenesis (DNL) is disrupted in a wide range of human disease. Thus, quantification of DNL may provide insight into mechanisms and guide interventions if it can be performed rapidly and noninvasively. DNL flux is commonly measured by H-2 incorporation into fatty acids following deuterated water ((H2O)-H-2) administration. However, the sensitivity of this approach is limited by the natural abundance of C-13, which masks detection of H-2 by mass spectrometry. Here we report that high-resolution Orbitrap gas-chromatography mass-spectrometry resolves H-2 and C-13 fatty acid mass isotopomers, allowing DNL to be quantified using lower (H2O)-H-2 doses and shorter experimental periods than previously possible. Serial measurements over 24-hrs in mice detects the nocturnal activation of DNL and matches a H-3-water method in mice with genetic activation of DNL. Most importantly, DNL is detected in overnight-fasted humans in less than an hour and is responsive to feeding during a 4-h study. Thus, H-2 specific MS provides the ability to study DNL in settings that are currently impractical. Fat synthesis is necessary for normal physiology, but its dysregulation contributes to the pathology of many diseases. Here, the authors report a high-resolution mass spectrometry approach that quantifies fat synthesis flux in humans and mice following a brief and low dose of deuterated water.

Guanosine diphosphate mannose (GDP-Man) is the donor substrate required for mannosylation in the synthesis of glycoproteins, glycolipids and the newly discovered glycoRNA. Normal GDP-Man biosynthesis plays a crucial role in support of a variety of cellular functions, including cell recognition, cell communication and immune responses against viruses. Here, we report the detection of GDP-Man in human brain for the first time, using P-31 MRS at 7 T. The presence of GDP-Man is evidenced by the detection of a weak P-31 doublet at -10.7 ppm that can be assigned to the phosphomannosyl group (P beta) of the GDP-Man molecule. This weak but well-resolved signal lies 0.9 ppm upfield of UDP(G) P beta-multiplet from a mixture of UDP-Glc, UDP-Gal, UDP-GlcNAc and UDP-GalNAc. In reference to ATP (2.8 mM), the concentration of GDP-Man in human brain was estimated to be 0.02 +/- 0.01 mM, about 15-fold lower than the total concentration of UDP(G) (0.30 +/- 0.04, N = 17) and consistent with previous reports of UDP-Man in cells and brain tissue extracts measured by high-performance liquid chromatography. The reproducibility of the measured GDP-Man between test and 2-week retest was 21% +/- 15% compared with 5% +/- 4% for UDP(G) (N = 7). The measured concentrations of GDP-Man and UDP(G) are linearly correlated ([UDP(G)] = 4.3 [GDP-Man] + 0.02, with R = 0.66 and p = 0.0043), likely reflecting the effect of shared sugar precursors, which may vary among individuals in response to variation in nutritional intake and consumption. Given that GDP-Man has another set of doublet (P alpha) at -8.3 ppm that overlaps with NAD(H) and UDP(G)-P alpha signals, the amount of GDP-Man could potentially interfere with the deconvolution of these mixed signals in composition analysis. Importantly, this new finding may be useful in advancing our understanding of glycosylation and its role in the development of cancer, as well as infectious and neurodegenerative diseases.

Ketogenesis occurs in liver mitochondria where acetyl-CoA molecules, derived from lipid oxidation, are condensed into acetoacetate (AcAc) and reduced to beta-hydroxybutyrate (BHB). During carbohydrate scarcity, these two ketones are released into circulation at high rates and used as oxidative fuels in peripheral tissues. Despite their physiological relevance and emerging roles in a variety of diseases, endogenous ketone production is rarely measured in vivo using tracer approaches. Accurate determination of this flux requires a two-pool model, simultaneous BHB and AcAc tracers, and special consideration for the stability of the AcAc tracer and analyte. We describe the implementation of a two-pool model using a metabolic flux analysis (MFA) approach that simultaneously regresses liquid chromatography-tandem mass spectrometry (LC-MS/MS) ketone isotopologues and tracer infusion rates. Additionally, 1H NMR real-time reaction monitoring was used to evaluate AcAc tracer and analyte stability during infusion and sample analysis, which were critical for accurate flux calculations. The approach quantifies AcAc and BHB pool sizes and their rates of appearance, disposal, and exchange. Regression analysis provides confidence intervals and detects potential errors in experimental data. Complications for the physiological interpretation of individual ketone fluxes are discussed.

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 T-2*s of the HP signals is critical. This study is to measure in vivo cardiac T-2*s of HP [1-C-13]pyruvate and the products in rodents and humans.Methods: A dynamic C-13 multi-echo spiral imaging sequence that acquires [C-13]bicarbonate, [1-C-13]lactate, and [1-C-13]pyruvate images in an interleaved manner was implemented for a clinical 3 Tesla system. T-2* 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 T-2* using the sequence was first validated using a C-13 phantom and then with rodents following a bolus injection of HP [1-C-13]pyruvate. In humans, T-2* of each metabolite was calculated for left ventricle, right ventricle, and myocardium.Results: Cardiac T-2* s of HP [1-C-13]pyruvate, [1-C-13]lactate, and [C-13]bicarbonate in rodents were measured as 24.9 +/- 5.0, 16.4 +/- 4.7, and 16.9 +/- 3.4 ms, respectively. In humans, T-2* of [1-C-13]pyruvate was 108.7 +/- 22.6 ms in left ventricle and 129.4 +/- 8.9 ms in right ventricle. T-2* of [1-C-13]lactate was 40.9 +/- 8.3, 44.2 +/- 5.5, and 43.7 +/- 9.0 ms in left ventricle, right ventricle, and myocardium, respectively. T-2* of [C-13] 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 T-2* s of HP [1-C-13]pyruvate and products.

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 P-31-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.