New methodology for VOC analysis from intubated mouse models 

INTRODUCTION

New methodology developed by experts in breath research to streamline sample collection from animal models

Exhaled breath contains volatile organic compounds (VOCs) that originate from biological processes in the body, and therefore have the potential to be utilized as biomarkers for clinical use. As breath biomarkers have been identified across a broad range of disease areas they are particularly promising for the broader adoption of next-generation non-invasive diagnostic and monitoring tools. However, the translation of breath tests to clinical use remains limited, which is partly due to the lack of consistent methodologies and quality controls across the breath research literature.

To address the challenges in clinical translation and expand the range of breath-based tests, a reliable animal model breath analysis method for pre-clinical studies is key. Establishing a method using inbred mice in controlled laboratory settings as a substitute for human breath can reduce the variability and challenges, allow for a better understanding of breath as a sampling medium using an animal model, and ultimately expediting the identification and validation of breath biomarkers for clinical use.

Several papers have previously aimed to study the breath of mouse models. In order to ensure that VOCs analyzed are truly exclusively from the breath, and to improve the signal-to-noise ratio, collection and analysis of breath from intubated mice can provide complementary data to advance the understanding of the mouse breath matrix. 

In a recent study, we developed a method for accurately characterizing the VOCs in the breath of healthy intubated mice using GC-MS, capable of superior control over the signal-to-noise ratios than is currently possible for human, and mouse breath. Finally, as a proof-of-principle and to assess the progress in data analysis quality, we compared the on-breath VOCs identified from intubated mouse models to the on-breath VOCs in human exhaled breath.  

Flexivent system

VOC “on-breath” classification

The mouse breath sampling system was developed by modifying the commercial flexiVent® FX1 small animal ventilator.

In order to conduct an initial assessment of the relative background of the flexiVent system, 500ml of air was collected from each of the 5 different scenarios of blank samples (Control – an unused sorbent tube, A- ambient air in the room, B- filtered ambient air, C- unfiltered air in the flexiVent system, D- filtered air in the flexiVent system) while the system was in operation. The data presented in the figure above demonstrates the contamination originating from the flexiVent can be removed by the presence of a second filter. Subsequent experiments showed that optimal performance can be maintained by replacing the filter paper at the start of each test day.

The majority of compounds identified as in common between mouse and human breath with the greatest fold change in breath to blank were associated with the gut microbiome or were plant-derived. Examples of these include trimethylamine (TMA) and dimethyl sulfone.