Using Breath Biopsy to measure limonene as a marker of cirrhosis

Detecting liver disease is challenging but breath limonene could hold the solution

Publication information: G. Ferrandino et al. Breath Biopsy Assessment of Liver Disease Using an Exogenous Volatile Organic Compound—Toward Improved Detection of Liver Impairment, Clinical and Translational Gastroenterology 2020; 11:e00239. DOI: 10.14309/ctg.0000000000000239

Disease Area: Liver disease (cirrhosis, NAFLD, NASH)

Application: Early detection, progression monitoring

Sample medium: Breath

Products: ReCIVA® Breath SamplerCASPER®Portable Air SupplyBreath Biopsy®EVOC® Probes

Analysis approach: GC-MS (Q Exactive GC Hybrid Quadrupole-Orbitrap Mass Spectrometer)


  • Detecting liver disease is challenging and relies on expensive and invasive methods.
  • Limonene, a naturally occurring chemical found in food and drink, has previously been linked to cirrhosis.
  • Levels of limonene on breath differentiate between healthy and cirrhotic individuals with 75% accuracy.


Globally, levels of liver disease are rising rapidly, with as many as 3 in 10 adults and 1 in 10 children affected, in some countries.1 Unfortunately, despite their increasing incidence, detecting liver diseases, such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) as well as more advanced cirrhosis, remains challenging.

Detection currently is expensive and relies heavily upon invasive methods such as blood and tissue biopsy. Further, these tests largely reflect liver damage and not current liver function. A non-invasive method suitable for screening would enable earlier detection and would be hugely beneficial – unlocking cheaper and more effective treatment pathways. Such a test would also be useful for reducing the cost of recruitment of patients and non-invasive monitoring of the effects of investigational drugs in pharmaceutical clinical trials.

Two prior publications have established the relevance of breath limonene for detection of liver disease. Fernandez del Rio et al.2 initially linked limonene with liver diseases, by comparing levels of limonene on the breath of liver cirrhosis patients pre- and post-liver transplant. In a follow-up paper (O’Hara M. E. et al)3, the same group demonstrated that limonene levels moderately increased in patients with hepatocellular carcinoma (HCC) with underlying cirrhosis.

delRio Limonene plots

Figure 1. Results from Fernandez del Rio et al.


Validating Limonene as a Biomarker for Liver Disease

Wanting to validate these earlier findings, Ferrandino et al., collected breath samples with the Breath Biopsy® Collection Station before performing targeted analysis via gas chromatography mass spectrometry (GC-MS) in the Breath Biopsy Platform.4 The study compared the levels of limonene on breath of healthy volunteers to that of patients with cirrhosis or cirrhotic hepatocellular-carcinoma.

Limonene Boxplots

Figure 2: Boxplots demonstrating the levels of limonene on exhaled breath from patient’s in different groups (healthy, cirrhosis and cirrhosis with hepatocellular carcinoma (HCC)) (left). Boxplots of levels of exhaled limonene for cirrhosis patients by Child-Pugh classification of disease severity.

This study confirmed that levels of limonene in samples increase in cirrhosis patients, and the increase correlates with disease severity as measured by Child-Pugh classification. Based on a limonene threshold of 10.2 ng/L the area under the receiver operating characteristic curve was 0.78 with a 73% sensitivity and 77% specificity for separating cirrhosis patients from healthy controls.

Limonene correlations

Figure 3: A predictive model based on limonene abundance discriminates cirrhosis from healthy with 73% sensitivity and 77% specificity (left). Exhaled limonene correlates with clearance (bilirubin) and protein synthesis capacity (albumin, INR) of the liver but not with markers of liver damage (ALT).

GIF Limonene Animation

The study also examined limonene on breath in comparison to existing blood biomarkers. Limonene correlated with three liver function markers (bilirubin, albumin and international normalized ratio (INR)). Significantly however, there was no correlation with alanine amino transferase (ALT) a measure of liver damage. Limonene analysis therefore could allow the early detection of liver diseases, where liver function is affected but liver damage has not yet occurred.

What do you think about limonene as a biomarker for liver disease? Visit the Breath Biopsy Community and have your say.

In this study the amount of limonene each subject consumed was not standardized. While this accurately reflects the varying levels of limonene consumption in the average person’s diet, further work will need to examine whether limonene dose can be standardized to generate a more reliable diagnostic model.

The Breath Biopsy OMNI Assay enables simultaneous targeted and untargeted analysis of the same breath samples. The results of the untargeted analysis will be available soon. This may reveal additional biomarkers of liver function that can further enhance these results and provide an even stronger predictive model of liver disease.


You can hear more about our work on limonene in our webinar and poster. Our EVOC® Probe approach allows for high-contrast, targeted VOC analysis informed by biological evidence. If you are interested in breath analysis for liver diseases or would like to find out more about the EVOC Probe approach, contact our team.



  1. Definition & Facts of NAFLD & NASH:
  2. Fernandez de Rio et al. (2015) Volatile Biomarkers in Breath Associated With Liver Cirrhosis — Comparisons of Pre- and Post-liver Transplant Breath Samples EBioMedicine 2(9), p1243-1250, DOI: 10.1016/j.ebiom.2015.07.027 
  3.  M E O’Hara et al. (2016) Limonene in exhaled breath is elevated in hepatic encephalopathy J. Breath Res. DOI: 10.1088/1752-7155/10/4/046010
  4. Ferrandino et al. (2020) Breath Biopsy Assessment of Liver Disease Using an Exogenous Volatile Organic Compound—Toward Improved Detection of Liver Impairment Clinical and Translational Gastroenterology 11:e00239. DOI: 10.14309/ctg.0000000000000239

Using Breath Biopsy to identify biomarkers of liver function