Metabolic Insights Related to Sleep and Circadian Clocks from Mass Spectrometry-Based Analysis of Blood and Breath

Abstract

Most physiological processes in humans are synchronized with their environment by so-called circadian clocks. These molecular time-keeping machineries are present in almost every cell. While light is the most important external stimulus to reset the circadian clocks, they can also be entrained by other stimuli, such as feeding or rest/activity cycles. Sleep is both, one of the major outputs of circadian clocks and also an independently regulated recuperative neurobiological process. Both, circadian clocks and sleep are closely intertwined with metabolic regulation and their disruption is associated with adverse effects on metabolic health, such as type 2 diabetes, metabolic syndrome, obesity and cardiovascular diseases. Since sleep restriction and disruption of circadian clocks are common issues in a modern 24/7 society, their negative metabolic consequences constitute a major concern of public health. However, despite clear evidence for the association between sleep, circadian clocks and metabolism, many aspects of this relationship remain unclear. In this thesis, novel insights into metabolic processes related to sleep and circadian clocks were gained using cutting-edge high-resolution mass spectrometry (HRMS) techniques. The combination of high resolution and high mass accuracy enables the analysis of complex mixtures and delivers molecular information. HRMS therefore provides a powerful tool for metabolic profiling. Here, two HRMS approaches were used. Metabolites in exhaled breath were measured in real time by secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS), offering a non-invasive technique with a virtually unlimited sampling frequency for studying systemic metabolic processes. Moreover, metabolic profiling from blood samples was performed using electrospray ionization mass spectrometry coupled to ultra-high performance liquid chromatography. Thereby, chromatography adds an additional dimension of separation, which improves unambiguous compound identification. Within this thesis, a setup for the analysis of exhaled breath during sleep by SESI-HRMS was developed. This allowed an unprecedented monitoring of metabolome-wide regulation during sleep with a ten-second time resolution. Major metabolic pathways were found to undergo rapid and reversible changes upon sleep stage transitions. It seems likely that the relevance of this complex synchronization of metabolism and sleep architecture for human health and performance is higher than previously thought. The non-invasive fashion of SESI-HRMS and its capability for real-time information qualify this technique also as a promising tool for clinical diagnostics. Here, breath biomarkers for obstructive sleep apnea (OSA) were validated in a larger and broader cohort of patients confirming the previously stated association between breath levels of these metabolites and the severity of the disease. These findings suggest that breath analysis by SESI-HRMS may add a substantial objective value, especially for OSA screening, and bring SESI-HRMS a step closer to its clinical application. By analyzing breath, it was possible to unravel metabolic processes related to sleep architecture and obstructive sleep apnea, while the metabolic profiling of blood provided insights into the connection between circadian clocks and metabolism. An elongating effect on the circadian period length was observed for a range of metabolic factors in serum from metabolically unhealthy obese patients. The combination of the molecular information provided by HRMS and results from genome-wide association revealed insulin resistance as central aspect of this association. In contrast to humans, arctic species, such as Norwegian reindeer, have always been exposed to seasonally occurring conditions of constant light. Therefore, they might have developed strategies to cope with this reoccurring circadian disruption. In the scope of this thesis circadian regulation of metabolism in arctic reindeer was investigated in combination with their behavior across all seasons. In contrast their activity patterns, metabolism in reindeer was not synchronized with the experienced light schedule. While less rhythmic metabolites were found during constant light in summer, a surprisingly high number of metabolites displayed circadian rhythms in winter. These findings suggest that reindeer have developed mechanisms to decouple circadian regulation of metabolism from behavioral rhythmicity. In conclusion, this thesis demonstrates the value of breath analysis and the versatility of high-resolution mass spectrometry for the field of metabolomics. By using this technique, metabolic processes related to sleep and circadian clocks were unraveled and the diagnostic value of metabolic profiling in exhaled breath by SESI-HRMS was further validated.