Development of a novel 13C-labelled methionine breath test protocol for potential assessment of hepatic mitochondrial function in sheep using isotope-ratio mass spectrometry

Abstract The 13 C-labelled methionine breath test is a non-invasive test with the potential to evaluate metabolic liver mitochondrial dysfunction in vivo . This study aimed to develop an optimised protocol for breath collection in sheep and compare 13 C-methionine and methyl- 13 C-methionine as substrates for future breath test applications in ruminants. Two doses (1 mg/kg and 2 mg/kg) of 13 C-methionine or methyl- 13 C-methionine in saline were administered via intravenous injection to four South Australian Merino ewes aged three years with an average body weight of 65 kg. Exhaled breath samples were collected at baseline and every 10 min for 180 min after administration of the 13 C-labelled substrate, and analysed using an isotope ratio mass spectrometer. Dose recovery of 13 CO 2 derived from 13 C-methionine was higher at each time-point compared to that derived from methyl- 13 C-methionine, at both concentrations. 13 C-methionine dose evaluation indicated that intra-individual and inter-individual variability of percentage dose recovery was lower in animals dosed with 2 mg/kg 13 C-methionine compared to 1 mg/kg 13 C-methionine. Mean cumulative percentage dose recovery of animals administered 1 mg/kg and 2 mg/kg 13 C-methionine at 60 min, 120 min and 180 min was 5.34 ± 0.87%, 11.52 ± 1.29% and 16.30 ± 1.45%, and 12.30 ± 0.34%, 25.12 ± 0.59% and 34.45 ± 0.57%, respectively. We conclude that the 13 C-labelled methionine breath test can be reliably performed in sheep through intravenous administration of 2 mg/kg 13 C-methionine. This study also demonstrated the utility and advantage of 13 C-methionine compared to methyl- 13 C-methionine as substrates for future breath testing applications to evaluate hepatic mitochondrial function in sheep and other ruminants. Further development of the test could be employed to evaluate disease progression and efficacy of applied therapeutics in sheep models of human diseases characterised by hepatic dysfunction. In addition, the 13 C-methionine breath test could be utilised in animal production to assess hepatic metabolic function and the potential impact of nutritional mismanagement.