High Throughput Screening of Serum γ-Glutamyl Dipeptides for Risk Assessment of Nonalcoholic Steatohepatitis with Impaired Glutathione Salvage Pathway.

Nonalcoholic fatty liver disease (NAFLD) is the most common preventable chronic liver disorder in developed countries, the prevalence of which is increasing worldwide due to its association with obesity and type 2 diabetes. However, the exact mechanisms of NAFLD pathophysiology remain poorly understood including its progression to the more severe nonalcoholic steatohepatitis (NASH). New advances for early detection and monitoring of NASH progression are limited due to the lack of specific blood biomarkers, thus requiring invasive liver biopsies for histopathology. Herein, multisegment injection-capillary electrophoresis-tandem mass spectrometry (MSI-CE-MS/MS) is validated as a high throughput, robust, and quantitative platform for targeted analysis of a panel of 16 serum γ-glutamyl dipeptides from a cohort of NASH adult patients from Japan (median age = 53 years, median BMI = 27 kg/m2, n = 116). Multiplexed separations based on MSI-CE-MS/MS enable the design of unique data workflows that rely on customizable serial sample injection formats for accurate determination of γ-glutamyl dipeptides with quality control. Also, the introduction of a liquid coolant device to the capillary outlet improves long-term migration time stability in CE. Unsupervised pattern recognition methods revealed two distinctive NASH subgroups based on their contrasting γ-glutamyl dipeptide status despite patients having similar clinical phenotypes and NASH activity scores (median NAS ≈ 6.0). There was an inverse correlation between serum γ-glutamyl dipeptide concentrations and γ-glutamyltransferease (GGT) enzyme activity (r = -0.46; p = 2.5 × 10-7), which was indicative of a low-risk (n = 64) as compared to a high-risk (n = 52) patient subgroup with impaired glutathione salvage pathway and likely poor clinical prognosis. Our findings highlight the key role of defects in the γ-glutamyl cycle for differentiation of NASH patients, which may enable better risk assessment of long-term survivorship as a complement to standard liver enzyme screens and histopathology.

Metabolomics Platform with Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Plasma Analysis.

Metabolome analysis using capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) has the potential to improve coverage of metabolite detection because of its high selectivity and sensitivity. Configuration of the interface between CE and HRMS to meet the ground connection is essential for enabling independent regulation of the electrical currents in the CE and electrospray field. In the present study, we applied an electrospray-ionization adapter equipped with a grounded nebulizer to CE-HRMS and tested the analytical performance for 34 charged compounds. The extracted-ion electropherograms, consisting of seven sets of isomers, showed reasonable peak shapes and separation for the annotation of each metabolite. The levels of 34 target analytes in a standard mixture were determined with a dynamic range of at least 102, maintaining linearity with r2 > 0.9. The repeatability and intermediate precision above the lower limit of quantification showed the relative standard deviation to be lower than 20%. In the spike-recovery experiment, 27 of the 34 metabolites in plasma extract were recovered at a rate of 80 to 120%, suggesting high accuracy. Furthermore, we assessed the feasibility of our platform in metabolome analysis using human-plasma extract. The results showed successful detection of 270 metabolites, indicating the potential of our platform to yield higher coverage of the metabolome. In addition, analysis of dilution integrity demonstrated the quantitative ability of metabolome analysis with CE-HRMS, although the existence of saturation or matrix effects were seen in the case of 33 of the metabolites. This study indicates that our platform has great potential for large-scale metabolome analysis of plasma for biological studies and clinical biomarker screening.