BackgroundThe burden of liver disease in the UK has risen dramatically and there is a need for improved diagnostics.AimsTo determine which breath volatiles are associated with the cirrhotic liver and hence diagnostically useful.MethodsA two-stage biomarker discovery procedure was used. Alveolar breath samples of 31 patients with cirrhosis and 30 healthy controls were mass spectrometrically analysed and compared (stage 1). 12 of these patients had their breath analysed after liver transplant (stage 2). Five patients were followed longitudinally as in-patients in the post-transplant period.ResultsSeven volatiles were elevated in the breath of patients versus controls. Of these, five showed statistically significant decrease post-transplant: limonene, methanol, 2-pentanone, 2-butanone and carbon disulfide. On an individual basis limonene has the best diagnostic capability (the area under a receiver operating characteristic curve (AUROC) is 0.91), but this is improved by combining methanol, 2-pentanone and limonene (AUROC curve 0.95). Following transplant, limonene shows wash-out characteristics.ConclusionsLimonene, methanol and 2-pentanone are breath markers for a cirrhotic liver. This study raises the potential to investigate these volatiles as markers for early-stage liver disease. By monitoring the wash-out of limonene following transplant, graft liver function can be non-invasively assessed.

Breath samples were taken from 31 patients with liver disease and 30 controls in a clinical setting and proton transfer reaction quadrupole mass spectrometry (PTR-Quad-MS) used to measure the concentration of volatile organic compounds (VOCs). All patients had cirrhosis of various etiologies, with some also suffering from hepatocellular cancer (HCC) and/or hepatic encephalopathy (HE). Breath limonene was higher in patients with No-HCC than with HCC, median (lower/upper quartile) 14.2 (7.2/60.1) versus 3.6 (2.0/13.7) and 1.5 (1.1/2.3) nmol mol−1 in controls. This may reflect disease severity, as those with No-HCC had significantly higher UKELD (United Kingdom model for End stage Liver Disease) scores. Patients with HE were categorized as having HE symptoms presently, having a history but no current symptoms and having neither history nor current symptoms. Breath limonene in these groups was median (lower/upper quartile) 46.0 (14.0/103), 4.2 (2.6/6.4) and 7.2 (2.0/19.1) nmol mol−1, respectively. The higher concentration of limonene in those with current symptoms of HE than with a history but no current symptoms cannot be explained by disease severity as their UKELD scores were not significantly different. Longitudinal data from two patients admitted to hospital with HE show a large intra-subject variation in breath limonene, median (range) 18 (10–44) and 42 (32–58) nmol mol−1.

Isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether), C3H2ClF5O, is a commonly used inhalation anaesthetic. Using a proton transfer reaction mass spectrometer (PTR-MS) we have detected isoflurane in the breath of patients several weeks following major surgery. That isoflurane is detected in the breath of patients so long after being anaesthetised raises questions about when cognitive function has fully returned to a patient. Temporal profiles of isoflurane concentrations in breath are presented for five patients (F/M 3/2, mean age 50 years, min–max 36–58 years) who had undergone liver transplant surgery. In addition, results from a headspace analysis of isoflurane are presented so that the product ions resulting from the reactions of H3O+ with isoflurane in PTR-MS could be easily identified in the absence of the complex chemical environment of breath. Six product ions were identified. In order of increasing m/z (using the 35Cl isotope where appropriate) these are (m/z 51), CHFCl+ (m/z 67), CF3CHCl+ (m/z 117), C3F4OCl+ (m/z 163), C3H2F4OCl+ (m/z 165), and C3F4OCl+ H2O (m/z 183). No protonated parent was detected. For the headspace study both clean air and CO2 enriched clean air (4% CO2) were used as buffer gases in the drift tube of the PTR-MS. The CO2 enriched air was used to determine if exhaled breath would affect the product ion branching ratios. Importantly no significant differences were observed, and therefore for isoflurane the product ion distributions determined in a normal air mixture can be used for breath analysis. Given that PTR-MS can be operated under different reduced electric fields (E/N), the dependence of the product ion branching percentages for isoflurane on E/N (96–138 Td) are reported.