Abstract
Blood is the specimen of choice for most laboratory tests for diagnosis and disease monitoring. Sampling exhaled breath is a noninvasive alternative to phlebotomy and has the potential for real-time monitoring at the bedside. Improved instrumentation has advanced breath analysis for several gaseous compounds from humans. However, application to small animal models of diseases and physiology has been limited. To extend breath analysis to mice, we crafted a means for collecting nose-only breath samples from groups and individual animals who were awake. Samples were subjected to gas chromatography and mass spectrometry procedures developed for highly sensitive analysis of trace volatile organic compounds (VOCs) in the atmosphere. We evaluated the system with experimental systemic infections of severe combined immunodeficiency Mus musculus with the bacterium Borrelia hermsii. Infected mice developed bacterial densities of ∼107 per ml of blood by day 4 or 5 and in comparison to uninfected controls had hepatosplenomegaly and elevations of both inflammatory and anti-inflammatory cytokines. While 12 samples from individual infected mice on days 4 and 5 and 6 samples from uninfected mice did not significantly differ for 72 different VOCs, carbon monoxide (CO) was elevated in samples from infected mice, with a mean (95% confidence limits) effect size of 4.2 (2.8–5.6), when differences in CO2 in the breath were taken into account. Normalized CO values declined to the uninfected range after one day of treatment with the antibiotic ceftriaxone. Strongly correlated with CO in the breath were levels of heme oxygenase-1 protein in serum and HMOX1 transcripts in whole blood. These results (i) provide further evidence of the informativeness of CO concentration in the exhaled breath during systemic infection and inflammation, and (ii) encourage evaluation of this noninvasive analytic approach in other various other rodent models of infection and for utility in clinical management.
Highlights
Body temperature, respiratory rate, and pulse rhythm were known to the ancients as informative parameters for diagnosing, staging, and monitoring disease
After a survey for a large number of volatile organic compounds (VOC) in the exhaled breath of infected and non-infected mice, we identified the ratio of carbon monoxide (CO) to carbon dioxide (CO2) in the breath as an informative correlate of the state of infection of individual mice in this model and of the response to anti-infective therapy
BALB/c-scid mice were first accommodated over 7 d of training to restraint without distress for up to 10 min at nose-only ports of a manifold for breath collection (Figure 1 and Figure S1 of Supplementary Information)
Summary
Respiratory rate, and pulse rhythm were known to the ancients as informative parameters for diagnosing, staging, and monitoring disease. A lengthening list of laboratory-based assays of substances and cells of the blood, urine, and other specimens provide additional information on the bodily status. Assays of blood are seldom performed at the bedside, and it may be hours or days before results are known. For some conditions unaided olfaction of the practitioner was sufficiently accurate for clinical decision making. These examples inspired efforts to extend human olfaction by means of instrumentation that provided better sensitivity and discrimination. While the potential value of breath analysis for disease-specific diagnosis, such as in diabetes [2], cystic fibrosis [3], or cancer [4], is undoubted, our interest instead is in compounds in the breath that might provide a unique dimension for the assessment and monitoring of patients with a variety of conditions
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