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Metabolic dysfunction-associated steatotic liver disease (MASLD) prevalence is increasing annually and affects over a third of US adults. MASLD can progress to metabolic dysfunction-associated steatohepatitis (MASH), characterized by severe hepatocyte injury, inflammation, and eventual advanced fibrosis or cirrhosis. MASH is predicted to become the primary cause of liver transplant by 2030. Although the etiology of MASLD/MASH is incompletely understood, dysregulated fatty acid oxidation is implicated in disease pathogenesis. Here, we develop a method for estimating hepatic β-oxidation from the metabolism of [D15]octanoate to deuterated water and detection with deuterium magnetic resonance methods. Perfused livers from a mouse model of MASLD reveal dysregulated hepatic β-oxidation, findings that corroborate in vivoimaging. The high-fat-diet-induced MASLD mouse studies indicate that decreased β-oxidative efficiency in the fatty liver could serve as an indicator of MASLD progression. Furthermore, our method provides a clinically translatable imaging approach for determining hepatic β-oxidation efficiency.

OBJECTIVE--Fluxes through mitochondrial pathways are defective in insulin-resistant skeletal muscle, but it is unclear whether similar mitochondrial defects play a role in the liver during insulin resistance and/or diabetes. The [...]

Maintaining water homeostasis is fundamental for cellular function. Many diseases and drugs affect water balance and plasma osmolality. Water homeostasis studies in small animals require the use of invasive or terminal methods that make intracellular fluid volume and extracellular fluid volume (ECF) monitoring over time stressful and time consuming. We examined the feasibility of monitoring mouse ECF by a noninvasive method using time-domain nuclear magnetic resonance (TD-NMR). This technique allows differentiation of protons in a liquid environment (free fluid) from protons in soft tissues containing a majority of either small molecules (lean) or large molecules (fat). Moreover, this apparatus enables rapid, noninvasive, and repeated measurements on the same animal. We assessed the feasibility of coupling TD-NMR analysis to a longitudinal metabolic cage study by monitoring mice daily. We determined the effect of 24-h water deprivation on mouse body parameters and detected a sequential and overlapping decrease in free fluid and lean mass during water deprivation. Finally, we studied the effect of mineralocorticoids that are known to induce a transient increase in ECF but for which no direct measurements have been performed in mice. We showed, for the first time, that mineralocorticoids induced a transient ~15% increase in free fluid in conscious mice. TD-NMR is, therefore, the first method to allow direct measurement of discrete changes in ECF in conscious small animals. This method allows analysis of kinetic changes to stimuli before investigating with terminal methods and will allow further understanding of fluid disorders.

Hyperpolarized 89Y complexes are attractive NMR spectroscopy and MR imaging probes due to the exceptionally long spin–lattice relaxation time (T1≈ 10 min) of the 89Y nucleus. However, in vivoimaging of 89Y has not yet been realized because of the low NMR signal enhancement levels previously achieved for this ultra low-γnnucleus. Here, we report liquid-state 89Y NMR signal enhancements over 60 000 times the thermal signal at 298 K in a 9.4 T magnet, achieved after the dynamic nuclear polarization (DNP) of Y(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) samples at 3.35 T and 1.4 K. The 89Y DNP was shown to proceed by thermal mixing and the liquid state 89Y NMR signal enhancement was maximized by (i) establishing the optimal microwave irradiation frequency, (ii) optimizing the glassing matrix, (iii) choosing a radical with negligible inhomogeneous line broadening contribution to the ESR linewidth, and (iv) addition of an electron T1erelaxation agent. The highest enhancements were achieved using a trityl OX063 radical combined with a gadolinium relaxation agent in water-glycerol matrix. Co-polarization of 89YDOTA and sodium [1-13C]pyruvate showed that both 89Y and 13C nuclear species acquired the same spin temperature, consistent with thermal mixing theory of DNP. This methodology may be applicable for the optimization of DNP of other low-γnnuclei.

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

Hyperpolarized carbon-13 magnetic resonance (HP-MR) is a new metabolic imaging method the does not use ionizing radiation. Due to the inherent chemical specificity of MR, not only tracer uptake but also downstream metabolism of the agent is detected in a straightforward manner. HP [2-13C] dihydroxyacetone (DHA) is a promising new agent that directly interrogates hepatic glucose metabolism. DHA has three metabolic fates in the liver: glucose production, glycerol production and potential inclusion into triglycerides, and oxidation in the tricarboxylic acid cycle. Each pathway is regulated by flux through multiple enzymes. Using Duhamel’s formula, the kinetics of DHA metabolism is modeled, resulting in estimates of specific reaction rate constants. The multiple enzymatic steps that control DHA metabolism make more simplified methods for extracting kinetic data less than satisfactory. The described modeling paradigm effectively identifies changes in metabolism between gluconeogenic and glycogenolytic models of hepatic function.

Building an accurate lipid inventory relies on coordinated information from orthogonal analytical capabilities. Integrating the familiar workflow of liquid chromatography (LC), high-resolution mass spectrometry (HRMS), and tandem mass spectrometry (MS/MS) with proton nuclear magnetic resonance spectroscopy (1H NMR) would be ideal for building that inventory. For absolute lipid structural elucidation, LC–HRMS/MS can provide lower-level structural information with superior sensitivity, while 1H NMR can provide invaluable higher-order structural information for the disambiguation of isomers with absolute chemical specificity. Digitization of the LC eluent followed by splitting the microfractions into two flow paths in a defined ratio for HRMS/MS and NMR would be the ideal strategy to permit correlation of the MS and NMR data as a function of chromatographic retention time. Here, we report an active segmentation platform to transform analytical flow rate LC eluent into parallel microliter segmented flow queues for high confidence correlation of the MS, MS/MS, and NMR data. The practical details in implementing this strategy to achieve an integrated LC–MS–NMR platform are presented, including the development of an active segmentation technology using a four-port two-way valve to transform the LC eluent into parallel segmented flows for online MS analysis followed by offline segment-specific 1H NMR and optimization of the detector response toward segmented flow. To demonstrate the practicality of this novel platform, it was tested using lipid mixture samples.

Malignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Here, we show that cytoplasmic lipid droplet (LD) accumulation, indicative of a lipogenic phenotype, is a cellular adaption to extracellular acidity. LD marker PLIN2 is strongly associated with poor overall survival in breast cancer patients. Acid-induced LD accumulation is triggered by activation of the acid-sensing G-protein-coupled receptor (GPCR) OGR1, which is expressed highly in breast tumors. OGR1 depletion inhibits acid-induced lipid accumulation, while activation by a synthetic agonist triggers LD formation. Inhibition of OGR1 downstream signaling abrogates the lipogenic phenotype, which can be rescued with OGR1 ectopic expression. OGR1-depleted cells show growth inhibition under acidic growth conditions in vitroand tumor formation in vivo. Isotope tracing shows that the source of lipid precursors is primarily autophagy-derived ketogenic amino acids. OGR1-depleted cells are defective in endoplasmic reticulum stress response and autophagy and hence fail to accumulate LDs affecting survival under acidic stress.

Machine perfusion improves solid organ preservation for transplantation. We have demonstrated that antegrade perfusion preservation of hearts is superior to cold storage but may be limited by aortic valve incompetence. We hypothesized that retrograde perfusion (RP) through the coronary sinus may provide more reliable perfusate delivery to the heart. This study was designed to determine the optimal perfusion parameters and evaluate regional flow after RP of canine hearts. After donor cardiectomy, canine hearts (n 6) were established in a perfusion device (LifeCradle, Organ Transport Systems, Inc., Frisco, TX) through a coronary sinus catheter. Hearts were perfused at 5°C over flow rates from 10 to 35 ml/100 g myocardium/min for 20 minutes at each flow rate. Colored microspheres were used to quantify tissue perfusion. Oxygen consumption (MVO2) and perfusion parameters were measured. At end-perfusion, tissue was collected for proton magnetic resonance spectroscopy (1H MRS), microsphere analysis, and determination of myocardial edema. MVO2increased up to flow rates of 20 ml/100 g/min. Right ventricular (RV) perfusion was reduced at all flow rates. Increased lactate/alanine ratios by 1H MRS and reduced myocardial water content were noted in RV samples. RP results in excellent left ventricular (LV) perfusion. RV perfusion is reduced and oxidative metabolism in the right ventricle may not be maintained by RP. Further studies to evaluate effects of reduced RV perfusion by RP on functional recovery after transplantation are warranted.

GdDOTP5−is a highly charged, bone-seeking paramagnetic complex that could potentially detect bone lesions by magnetic resonance imaging (MRI). To date, its pharmacokinetics, effects on organ relaxivity, and interaction with hydroxyapatite (HA) has not been described.

¹³C NMR isotopomer analysis is a powerful method for measuring metabolic fluxes through pathways intersecting in the tricarboxylic acid cycle. However, the inherent insensitivity of ¹³C NMR spectroscopy makes application of isotopomer analysis to small tissue samples (mouse tissue, human biopsies, or cells grown in tissue culture) problematic. ¹H NMR is intrinsically more sensitive than ¹³C NMR and can potentially supply the same information via indirect detection of ¹³C providing that isotopomer information can be preserved. We report here the use of J-resolved HSQC (J-HSQC) for ¹³C isotopomer analysis of tissue samples. We show that J-HSQC reports isotopomer multiplet patterns identical to those reported by direct ¹³C detection but with improved sensitivity. Copyright 2001 Academic Press.

Beide Isotope von Silber, 107Ag und 109Ag, wurden mithilfe dynamischer Kernpolarisation (DNP) simultan polarisiert, was eine hohe Signalverstärkung und die NMR‐spektroskopische Charakterisierung von Ag‐Komplexen im mMKonzentrationen ermöglichte. Da beide Isotope lange Relaxationszeiten T1haben, wird das hyperpolarisierte NMR‐Signal des einen Isotops auch dann noch beobachtet, wenn die Magnetisierung des anderen Isotops durch Radiofrequenzpulse längst ausgelöscht wurde.

107Ag und109Aggehen eine simultane dynamische Kernpolarisation ein, wie Z. Kovacs et al. in der Zuschrift auf S. 540 ff.beschreiben. Die ausgeprägte NMR‐Signalverstärkung in Lösung ermöglicht die Ag‐NMR‐spektroskopische Charakterisierung von Ag+‐Komplexen im mM‐Bereich. Beide hyperpolarisierten Isotope haben lange T1‐Relaxationszeiten, sodass NMR‐Experimente möglich sind, bei denen das Signal des 107Ag‐Kerns nach der Zerstörung der 109Ag‐Magnetisierung durch Anregung mit Radiowellen detektiert werden kann.

107Ag and109Ag undergosimultaneous dynamic nuclear polarization as described by Z. Kovacs et al. in their Communication on page 525 ff.The large liquid‐state NMR enhancements achieved allow the Ag NMR spectroscopic characterization of Ag+complexes in the mMconcentration range. Both hyperpolarized isotopes have long T1relaxation times, enabling NMR experiments to be performed in which the signal from the 107Ag nuclei can be detected after destroying the 109Ag magnetization by radio‐frequency excitation.