HiSorb TD-GC-MS Innovation: Probing Liver Metabolism with Deuterated EVOCs Unveils Potential Novel Enzymatic Pathways and Biomarkers for Disease Diagnosis

Murgia A., Ahmed Y., Banda I., Manhota M., Fonseca . D. Gandelman O., Allsworth M., Boyle B., Rudman J., Clarke C., Martin A., Ferrandino G. 

1. Owlstone Medical, United Kingdom 

Poster PDF

Liver disease has a high prevalence in the modern world, with many different aetiologies. In most cases, liver disease is asymptomatic until it has progressed into the later stages, and diagnostic methods are typically invasive, preventing early screening. With the aim to develop a non-invasive breath test for early diagnosis of liver disease based on hepatic metabolic alterations, preclinical approaches are needed to detect the hepatic metabolisation of Exogenous Volatile Organic Compounds (EVOCs) substrates to detectable bio-products. This presentation unveils the findings of an in vitro study investigating the metabolism of deuterated EVOCs by Primary Human Hepatocytes (PHH). The study employs the HiSorb platform coupled with Thermal Desorption-Gas Chromatography-Mass Spectrometry technique (TD-GC-Orbitrap-MS) for precise analysis. 

Methods

1 x 10^6 PHH/mL were treated with five deuterated EVOCs (2-pentanone-d5, 2-butanol-d5, limonene-d5, nonanal-d18, and benzyl alcohol-d7) at a concentration of 10 ng/µL. EVOCs endpoint extraction was performed using HiSorb (37°C, 300 rpm, 2 hours). Control groups included vehicle treated PHH (Methanol: 0.02%) and spiked EVOCs in the absence of PHH. All samples were analyzed using targeted and untargeted qualitative analysis with TD-GC-Orbitrap-MS. Notably, the experiment was repeated to include the validation of Tolrestat and 4-Methylpyrazole, which inhibit aldo-ketoreductase 1B10 (AKR1B10) and alcohol dehydrogenase (ADH), respectively. Molecular features (MF) were obtained and the areas of compound ions were compared between experiment groups. The identity of significant (p < 0.05) compounds was established using mass-spectra annotation comparison and co-chromatography. 

Preliminary data

Targeted Analysis: The mass spectra fragment ions of EVOCs bioproducts were significantly higher in the presence of PHH compared to controls, with no detectable bioproduct production in the absence of PHH. The EVOCs substrates 2-butanol-d5, 2-pentanone-d5, and limonene-d5 showed no variation with or without PHH. However, nonanal-d18 and benzyl alcohol-d7 exhibited a decrease in the presence of PHH, suggesting these two EVOCs did not saturate the associated metabolic pathways. Enzyme inhibitor efficacy varied across EVOCs substrates, with the only notable/significant effect seen with 4-Methylpyrazole. Compared to non-inhibited PHH, with 4-Methylpyrazole we observed a lower reduction in benzyl alcohol-d7 (substrate) and lower production of benzaldehyde-d6 (bio-product). 

Untargeted Analysis: From a list of 2096 extracted MF, a total of 47 were found to be elevated in the presence of PHH compared to controls, suggesting these may be additional bioproducts. In agreement with this, elucidation of MF spectra identified 18 features corresponding to peak ions of expected bioproducts already detected as part of the targeted analysis. Additionally, 3 MF showed similar spectra to <6C deuterated organic acids and 3 deuterated terpenes. Interestingly, 1 MF was identified as non-deuterated 3-octanone, suggesting that this compound is generated by secondary metabolic alteration induced by the investigated compounds. Combined, all this data highlights that hepatic metabolism of target compounds can be evaluated in vitro using PHH. It also highlights how MS, when used for discriminating deuterated and non-deuterated spectra, is a critical modality for this approach. This approach could be applied to several aspects of medical research, from identifying pathological metabolic alterations as novel biomarkers for diagnostic purposes, to screening potential new drug targets and drug candidates for efficacy in affecting disease associated metabolic pathways. 

Novel aspect

Headspace analysis of PHH coupled with GC-MS is an advantageous approach for pre-clinical drug development and biomarker discovery.