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.

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.