Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.

Background: Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver diseases worldwide.There is a pressing clinical need to identify potential therapeutic targets for NASH treatment.Thioredoxin interacting protein (Txnip) is a stress responsive gene that has been implicated in the pathogenesis of NASH, but its exact role is not fully understood.Here, we investigated the liver-and gene-specific role of Txnip and its upstream/downstream signaling in the pathogenesis of NASH.Methods and Results: Using four independent NASH mouse models, we found that TXNIP protein abnormally accumulated in NASH mouse livers.A decrease in E3 ubiquitin ligase NEDD4L resulted in impaired TXNIP ubiquitination and its accumulation in the liver.TXNIP protein levels were positively correlated with that of CHOP, a major regulator of ER stress-mediated apoptosis, in NASH mouse liver.Moreover, gain-and loss-of-function studies showed that TXNIP increased protein not mRNA levels of Chop both in vitro and in vivo.Mechanistically, the C-terminus of TXNIP associated with the N-terminus of the α-helix domain of CHOP and decreased CHOP ubiquitination, thus increasing the stability of CHOP protein.Lastly, selective knockdown of Txnip by adenovirus-mediated shRNA (not targets Txnip antisense lncRNA) delivery in the livers of both young and aged NASH mice suppressed the expression of CHOP and its downstream apoptotic pathway, and ameliorated NASH by reducing hepatic apoptosis, inflammation, and fibrosis.Conclusions: Our study revealed a pathogenic role of hepatic TXNIP in NASH and identified a novel NEDD4L-TXNIP-CHOP axis in the pathogenesis of NASH.