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. 2020 Jun 15;2(5):100137.
doi: 10.1016/j.jhepr.2020.100137. eCollection 2020 Oct.

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

Rohit Sinha  1 , Khalida A Lockman  2 , Natalie Z M Homer  3 , Edward Bower  3 , Paul Brinkman  4 , Hugo H Knobel  5 , Jonathan A Fallowfield  6 , Alan J Jaap  7 , Peter C Hayes  1 , John N Plevris  1
Affiliations

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

Rohit Sinha et al. JHEP Rep. .

Abstract

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.

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Conflict of interest statement

The authors have no conflicts of interest pertaining to this study to declare. Please refer to the accompanying ICMJE disclosure forms for further details.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Levels of volatile organic compounds in exhaled breath in study participants. Healthy (healthy controls; n = 14), NC NAFLD (n = 14) and NAFLD C (n = 15). Levels were adjusted for a unit toluene-d8 (internal standard reference) and BMI. Data expressed as mean with standard error of mean. A one-way ANOVA was performed with post-test Bonferroni correction. Significance is denoted as ∗p <0.05, ∗∗p <0.01, and ∗∗∗p <0.001. BMI, body mass index; NAFLD C, non-alcoholic fatty liver disease with cirrhosis; NC NAFLD, non-cirrhotic non-alcoholic fatty liver disease.
Fig. 2
Fig. 2
AUROC curve for breath terpinene in classifying patients with non-cirrhotic NAFLD (n = 14) vs. healthy participants (n = 14). Discrimination used for internal model validation - ranking patient with non-cirrhotic NAFLD was measured by AUROC. AUROC, area under the receiver operating curve; NAFLD, non-alcoholic fatty liver disease.
Fig. 3
Fig. 3
AUROC curve for a combination of D-limonene and dimethyl sulfide in classifying patients with NAFLD cirrhosis (n = 15) vs. healthy participants (n = 14). Discrimination used for internal model validation - ranking patient with NAFLD cirrhosis was measured by AUROC. AUROC, area under the receiver operating curve; NAFLD, non-alcoholic fatty liver disease.
Fig. 4
Fig. 4
AUROC curve for a combination of D-limonene and dimethyl sulfide in classifying patients with NAFLD cirrhosis (n = 15) vs. non-cirrhotic NAFLD (n = 14). AUROC, area under the receiver operating curve; NAFLD, non-alcoholic fatty liver disease.
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