Mario C. Chang

Metabolic imaging produces powerful visual assessments of organ function in vivo. Current techniques can be improved by safely increasing metabolic contrast. The gold standard, 2-[ ¹⁸ F]fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging, is limited by radioactive exposure and sparse assessment of metabolism beyond glucose uptake and retention. Deuterium magnetic resonance imaging (DMRI) with [6,6- ² H ₂ ]glucose is nonradioactive, achieves tumor metabolic contrast, but can be improved by enriched contrast from deuterated water (HDO) based imaging. Here, we developed a DMRI protocol employing [ ² H ₇ ]glucose. Imaging ² H-signal and measuring HDO production in tumor-bearing mice detected differential glucose utilization across baseline tumors, tumors treated with vehicle control or anti-glycolytic BRAFi and MEKi therapy, and contralateral healthy tissue. Control tumors generated the most ² H-signal and HDO. To our knowledge this is the first application of DMRI with [ ² H ₇ ]glucose for tumoral treatment monitoring. This approach demonstrates HDO as a marker of tumor glucose utilization and suggests translational capability in humans due to its safety, noninvasiveness, and suitability for serial monitoring.

Nonalcoholic fatty liver disease (NAFLD) prevalence is increasing annually and affects over a third of U.S. adults. NAFLD can progress to nonalcoholic steatohepatitis (NASH), characterized by severe inflammation and fibrosis. NASH is predicted to become the primary cause of liver transplant by 2030. Although the etiology of NAFLD/NASH is incompletely understood, dysregulated fatty acid oxidation is implicated in disease pathogenesis. Here, we developed a method for estimating hepatic β-oxidation from the metabolism of [D ₁₅ ]octanoate to deuterated water and detection with deuterium magnetic resonance methods. Application of this method to perfused liver from a mouse model of NAFLD revealed dysregulated hepatic β-oxidation, findings that we confirmed with in vivo imaging. The high-fat diet–induced NAFLD mouse studies indicated that decreased β-oxidative efficiency in the fatty liver could serve as a prognostic indicator of NAFLD progression. Furthermore, our method provides a clinically translatable imaging approach for determining hepatic β-oxidation efficiency.

The natural amino acid asparagine (Asn) is required by cells to sustain function and proliferation. Healthy cells can synthesize Asn through asparagine synthetase (ASNS) activity, whereas specific cancer and genetically diseased cells are forced to obtain asparagine from the extracellular environment. ASNS catalyzes the ATP-dependent synthesis of Asn from aspartate by consuming glutamine as a nitrogen source. Asparagine Synthetase Deficiency (ASNSD) is a disease that results from biallelic mutations in the ASNS gene and presents with congenital microcephaly, intractable seizures, and progressive brain atrophy. ASNSD often leads to premature death. Although clinical and cellular studies have reported that Asn deprivation contributes to the disease symptoms, the global metabolic effects of Asn deprivation on ASNSD-derived cells have not been studied. We analyzed two previously characterized cell culture models, lymphoblastoids and fibroblasts, each carrying unique ASNS mutations from families with ASNSD. Metabolomics analysis demonstrated that Asn deprivation in ASNS-deficient cells led to disruptions across a wide range of metabolites. Moreover, we observed significant decrements in TCA cycle intermediates and anaplerotic substrates in ASNS-deficient cells challenged with Asn deprivation. We have identified pantothenate, phenylalanine, and aspartate as possible biomarkers of Asn deprivation in normal and ASNSD-derived cells. This work implies the possibility of a novel ASNSD diagnostic via targeted biomarker analysis of a blood draw.

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.

Human asparagine synthetase (ASNS) catalyzes the conversion of aspartate to asparagine in an ATP-dependent reaction that utilizes glutamine as a nitrogen source while generating glutamate, AMP, and pyrophosphate as additional products. Asparagine Synthetase Deficiency (ASNSD) is an inborn error of metabolism in which children present with homozygous or compound heterozygous mutations in the ASNS gene. These mutations result in ASNS variant protein expression. It is believed that these variant ASNS proteins have reduced enzymatic activity or stability resulting in a lack of sufficient asparagine production for cell function. Reduced asparagine production by ASNS appears to severely hinder fetal brain development. Although a variety of approaches for assaying ASNS activity have been reported, we present here a straightforward method for the in vitro enzymatic analysis by detection of AMP production. Our method overcomes limitations in technical feasibility, signal detection, and reproducibility experienced by prior methods like high-performance liquid chromatography, ninhydrin staining, and radioactive tracing. After purification of FLAG-tagged R49Q, G289A, and T337I ASNS variants from stably expressing HEK 293T cells, this method revealed a reduction in activity of 90, 36, and 96%, respectively. Thus, ASNS protein expression and purification, followed by enzymatic activity analysis, has provided a relatively simple protocol to evaluate structure–function relationships for ASNS variants reported for ASNSD patients.

The compound β-lapachone, a naturally derived naphthoquinone, has been utilized as a potent medicinal nutrient to improve health. Over the last twelve years, numerous reports have demonstrated distinct associations of β-lapachone and NAD(P)H: quinone oxidoreductase 1 (NQO1) protein in the amelioration of various diseases. Comprehensive research of NQO1 bioactivity has clearly confirmed the tumoricidal effects of β-lapachone action through NAD⁺-keresis, in which severe DNA damage from reactive oxygen species (ROS) production triggers a poly-ADP-ribose polymerase-I (PARP1) hyperactivation cascade, culminating in NAD⁺/ATP depletion. Here, we report a novel combination strategy with aminooxyacetic acid (AOA), an aspartate aminotransferase inhibitor that blocks the malate-aspartate shuttle (MAS) and synergistically enhances the efficacy of β-lapachone metabolic perturbation in NQO1⁺ breast cancer. We evaluated metabolic turnover in MDA-MB-231 NQO1⁺, MDA-MB-231 NQO1⁻, MDA-MB-468, and T47D cancer cells by measuring the isotopic labeling of metabolites from a [U-¹³C]glucose tracer. We show that β-lapachone treatment significantly hampers lactate secretion by ~85% in NQO1⁺ cells. Our data demonstrate that combinatorial treatment decreases citrate, glutamate, and succinate enrichment by ~14%, ~50%, and ~65%, respectively. Differences in citrate, glutamate, and succinate fractional enrichments indicate synergistic effects on central metabolism based on the coefficient of drug interaction. Metabolic modeling suggests that increased glutamine anaplerosis is protective in the case of MAS inhibition.

Treatment of cancers with β-lapachone causes NAD(P)H: quinone oxidoreductase 1 (NQO1) to generate an unstable hydroquinone that regenerates itself in a futile cycle while producing reactive oxygen species (ROS) in the form of superoxide and subsequently hydrogen peroxide. Rapid accumulation of ROS damages DNA, hyperactivates poly-ADP-ribose polymerase-I, causes massive depletion of NAD⁺/ATP, and hampers glycolysis. Cells overexpressing NQO1 subsequently die rapidly through an NAD⁺-keresis mechanism. Assessing changes in glycolytic rates caused by NQO1 bioactivation would provide a means of assessing treatment efficacy, potentially lowering the chemotherapeutic dosage, and reducing off-target toxicities. NQO1-mediated changes in glycolytic flux were readily detected in A549 (lung), MiaPaCa2 (pancreatic), and HCT-116 (colon) cancer cell lines by ²H-NMR after administration of [²H₇]glucose. The deuterated metabolic products ²H-lactate and HDO were quantified, and linear relationships with glucose consumption for both products were observed. The higher concentration of HDO compared to ²H-lactate allows for more sensitive measurement of the glycolytic flux in cancer. Gas chromatography-mass spectrometry analysis agreed with the NMR results and confirmed downregulated energy metabolism in NQO1⁺ cells after β-lapachone treatment. The demonstrated method is ideal for measuring glycolytic rates, the effects of chemotherapeutics that target glycolysis, and has the potential for in vivo translation.