Targeted Breath Biopsy® Profiling Of Induced Biomarkers Unveils A Metabolic Adaptation In Cirrhosis Toward Alcohol Production

Giuseppe Ferrandino 1, Federico Ricciardi 1, Antonio Murgia 1, Menisha Manhota 1, Daniela Fonseca 1, Louise Nicholson-Scott 1, Chloe Fitzpatrick 1, Olga Gandelman 1, Max Allsworth 1, Billy Boyle 1, Agnieszka Smolinska 1 2, Carmen A Ginesta Frings 3 4, Jorge Contreras 3, Victor Gabrielli3, Claudia Asenjo-Lobos 5, Francisca Bascur5, Viviana Barrientos 4, Nataly Clavo 4, Melissa Jerez 6, Luis Méndez 3 4 

1. Owlstone Medical, 183 Cambridge Science Park, Milton Road, Cambridge CB4 0GJ, UK. 
2. Department of Pharmacology and Toxicology, School for Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands. 
3. Unidad de Gastroenterología y Endoscopía, Clínica Alemana, Facultad de Medicina Clínica Alemana, Universidad de Desarrollo, Santiago 7650568, Chile. 
4. Unidad de Endoscopia, Hospital Padre Hurtado, Santiago 8880465, Chile. 
5. Centro de Estudios Clínicos, Instituto de Ciencias e Innovación en Medicina (ICIM), Facultad de Medicina Clínica Alemana, Universidad del Desarrollo, Santiago 7610315, Chile. 
6. Nursing School, Universidad de Las Américas, Santiago 8242125, Chile. 

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Background and Aims

Subjects with chronic liver disease have shown higher circulating ethanol levels after fasting or carbohydrates ingestion. However, clinical functional data about the capability of the injured liver to cope with alcohols remain limited. In this study we used exhaled breath analysis to explore the conversion of an alcohol to the corresponding ketone, and a ketone to the corresponding alcohol, to establish the suitability of potential metabolic alterations and identify subjects with cirrhosis using a non-invasive breath test. 

Method

2-butanol, and 2-pentanone (the substrates) were selected as an alcohol and a ketone based on their safe profile for human consumption, their reported hepatic metabolism, and their presence in breath. Hepatic metabolism was confirmed by simultaneous treatment of human suspension hepatocytes with 10 ng/µl of 2-butanol, and 2-pentanone to measure their conversion to respectively 2-butanone, and 2-pentanol (the bioproducts) by using headspace analysis coupled with gas-chromatography mass-spectrometry (GC-MS). Clinical metabolism of these compounds was evaluated by measurement of the breath profile in 14 subjects with cirrhosis and 14 controls before and at different timepoints (10 - 120 minutes) after simultaneous ingestion of 2-butanol (100 mg) and 2-pentanone (100 mg) using Breath Biopsy® OMNI. 

Results

The bioproducts were present in the head space of primary hepatocytes treated with the substrates and absent in control conditions omitting primary hepatocytes or substrates. After an overnight fasting all the participants showed breath levels of investigated compounds with no differences between healthy and cirrhosis groups (p > 0.05). After ingestion of the substrates, all the subjects showed a > 100-fold increase of investigated compounds within 10 minutes. Subjects with cirrhosis showed higher levels of 2-butanol and 2-pentanol (p < 0.05) compared to healthy controls between 20- and 90-minutes post-administration. An exploratory classification model built on these breath-induced biomarkers at 20 minutes showed an area under the ROC curve of 0.92.  

Conclusion

2-butanol and 2-pentanone are metabolized to the corresponding ketone and alcohol in primary hepatocytes, supporting previous findings reporting hepatic metabolism. Subjects with cirrhosis showed higher breath levels of secondary alcohols independently from administration of the alcohol or the corresponding ketone, suggesting that the hepatic function is more balanced toward the alcohol production compared to healthy controls. This metabolic adaptation may contribute to higher circulating ethanol levels observed in subjects with chronic liver disease and be exploited in a breath test for cirrhosis detection.