Abstract
Odorant-metabolizing enzymes are critically involved in the clearance of odorant molecules from the environment of the nasal neuro-olfactory tissue to maintain the sensitivity of olfactory detection. Odorant metabolism may also generate metabolites in situ, the characterization and function of which in olfaction remain largely unknown. Here, we engineered and validated an ex vivo method to measure odorant metabolism in real-time. Glassware containing an explant of rat olfactory mucosa was continuously flushed with an odorant flow and was coupled to a proton transfer reaction-mass spectrometer for volatile compound analysis. Focusing on carboxylic esters and diketone odorants, we recorded the metabolic uptake of odorants by the mucosa, concomitantly with the release of volatile odorant metabolites in the headspace. These results significantly change the picture of real-time in situ odorant metabolism and represent a new step forward in the investigation of the function of odorant metabolites in the peripheral olfactory process. Our method allows the systematic identification of odorant metabolites using a validated animal model and permits the screening of olfactory endogenously produced chemosensory molecules.
Highlights
Odorant-metabolizing enzymes are critically involved in the clearance of odorant molecules from the environment of the nasal neuro-olfactory tissue to maintain the sensitivity of olfactory detection
To investigate the metabolic capacity of ex vivo olfactory mucosa (OM) toward odorants in real-time, a continuous direct-injection mass spectrometry method was developed. It comprised the implementation of a 6-way valve creating a two-way circuit connected to a proton transfer reaction-mass spectrometry (PTR-MS) instrument that allowed direct odorant delivery independently above the biological material contained in a glassware in the first experimental branch, with the second branch serving as a control (Fig. 1)
Monitoring of the reaction with PTR-MS had been demonstrated to be possible by a previous study using discrete sampling of the headspace above the OM contained in vials[21]
Summary
Odorant-metabolizing enzymes are critically involved in the clearance of odorant molecules from the environment of the nasal neuro-olfactory tissue to maintain the sensitivity of olfactory detection. In a single study in humans, the presence of odorant metabolites has been demonstrated by an atmospheric pressure chemical ionization (APCI) ion source in exhaled breath after odorant inhalation[17] This direct-injection mass spectrometry technique is very suitable for real-time analysis of volatile molecules from biological environments[19]. Despite these advances, the significance of OMEs in the process of olfaction remains debatable because few aspects are known about the enzymatic mechanism and its ability to generate odorant metabolites, especially under experimental conditions directly focusing on the tissue involved: the neuroepithelium. Our results provide new insights into the in situ olfactory metabolism of odorants that update our understanding of nasal volatile metabolism that has been conceptualized in the olfactory physiologically based pharmacokinetic models[22,23] developed in nasal toxicology
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