Volatomic analysis identifies compounds that can stratify non-alcoholic fatty liver disease

Background & aims: Analysis of volatile organic compounds (VOCs) in exhaled breath, 'volatomics', provides opportunities for non-invasive biomarker discovery and novel mechanistic insights into a variety of diseases. The purpose of this pilot study was to compare breath VOCs in an initial cohort of patients with non-alcoholic fatty liver disease (NAFLD) and healthy controls.

Methods: Breath samples were collected from 15 participants with Child-Pugh class A NAFLD cirrhosis, 14 with non-cirrhotic NAFLD, and 14 healthy volunteers. Exhaled breath samples were collected using an established methodology and VOC profiles were analysed by gas chromatography-mass spectrometry. The levels of 19 VOCs previously associated with cirrhosis were assessed. Peaks of the VOCs were confirmed and integrated using Xcalibur® software, normalised to an internal standard. Receiver-operating characteristic (ROC) curves were used to determine the diagnostic accuracy of the candidate VOCs.

Results: Terpinene, dimethyl sulfide, and D-limonene provided the highest predictive accuracy to discriminate between study groups. Combining dimethyl sulfide with D-limonene led to even better discrimination of patients with NAFLD cirrhosis from healthy volunteers (AUROC 0.98; 95% CI 0.93-1.00; p <0.001) and patients with NAFLD cirrhosis from those with non-cirrhotic NAFLD (AUROC 0.91; 95% CI 0.82-1.00; p <0.001). Breath terpinene concentrations discriminated between patients with non-cirrhotic NAFLD and healthy volunteers (AUROC 0.84; 95% CI 0.68-0.99; p = 0.002).

Conclusion: Breath terpinene, dimethyl sulfide, and D-limonene are potentially useful volatomic markers for stratifying NAFLD; in addition, a 2-stage approach enables the differentiation of patients with cirrhosis from those without. However, these observations require validation in a larger NAFLD population. (ClinicalTrials.gov Identifier: NCT02950610).

Lay summary: Breath malodour has been associated with a failing liver since the ancient Greeks. Analytical chemistry has provided us an insight into ubiquitous volatile organic compounds associated with liver (and other) diseases. This has vastly improved our understanding of the mechanistic processes of liver damage. Our study aims to identify volatile organic compounds which are specific to non-alcoholic fatty liver disease and that can be exploited for rapid diagnostics.

Keywords: ALT, alanine aminotransaminase; APRI, aminotransferase:platelet ratio index; ARFI, acoustic radiation force impulse; AST, aspartate aminotransferase; AUROC, area under the receiver-operating characteristics curve; BMI, body mass index; D-limonene; Dimethyl sulfide; GAVE, gastric antral vascular ectasia; GC-MS, gas chromatography mass spectrometry; GGT, gamma-glutamyltransferase; HA, hyaluronic acid; HOMA, homeostatic model assessment; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; Non-alcoholic fatty liver disease; PHG, portal hypertensive gastropathy; QC, quality control; T2DM, type 2 diabetes mellitus; TE, transient elastography; Terpinene; VOCs, volatile organic compounds; Volatile organic compounds; Volatomics.