Odorant Metabolism Analysis by an Automated Ex Vivo Headspace Gas-Chromatography Method

Abstract

In the olfactory epithelium (OE), odorant metabolizing enzymes have the dual function of volatile component detoxification and active clearance of odorants from the perireceptor environment to respectively maintain the integrity of the tissues and the sensitivity of the detection. Although emphasized by recent studies, this enzymatic mechanism is poorly documented in mammals. Thus, olfactory metabolism has been characterized mainly in vitro and for a limited number of odorants. The automated ex vivo headspace gas-chromatography method that was developed here was validated to account for odorant olfactory metabolism. This method easily permits the measurement of the fate of an odorant in the OE environment, taking into account the odorant gaseous state and the cellular structure of the tissue, under experimental conditions close to physiological conditions and with a high reproducibility. We confirmed here our previous results showing that a high olfactory metabolizing activity of the mammary pheromone may be necessary to maintain a high level of sensitivity toward this molecule, which is critical for newborn rabbit survival. More generally, the method that is presented here may permit the screening of odorants metabolism alone or in mixture or studying the impact of aging, pathology, polymorphism or inhibitors on odorant metabolism.

Odorant-odorant metabolic interaction, a novel actor in olfactory perception and behavioral responsiveness

“…We previously developed a fast reliable and automated method to ex vivo measure endogenous global enzymatic odorant metabolism 23 , 24 . Briefly, in this validated method, a fresh total explant of newborn rabbit OE was put in a sealed 20 ml vial, in which a known concentration of gaseous odorant was injected.…”

“…The result that non-GST conjugated aldehydes, namely EA or vanillin, did not affect 2MB2 metabolism demonstrated that such an inhibition results from metabolic interaction toward GST. Our headspace gas-chromatography method for ex vivo measurement 24 allowed us to select 2MP2 as the best challenger. Moreover, and most importantly, it led us to show that in the vial headspace, a high level of residual/available 2MB2 (no metabolized) resulted from 2MB2-2MP2 interaction.…”

“…The aim of injecting a known gaseous odorant amount in the headspace above the olfactory mucosa made necessary the determination of the odorant gas-liquid partition coefficient. The latter was realized by using the Phase Ratio Variation (PRV) method 46 , as previously described by automatic equilibrium headspace - gas chromatography 24 . Briefly, aqueous solution of the 2MB2 or EA was prepared with the analyte concentration of 20 ppm and 100 ppm respectively.…”

“…The 2MB2 metabolism kinetic has been previously described and validated with this headspace gas chromatography method 24 . Here, the 2MB2 kinetic of metabolization was compared to those observed in presence of another odorant, i.e., a putative metabolic challenger or not.…”

“…Naturally contained in the rabbit milk effluvium, the mammary pheromone is a monomolecular signal, 2-methylbut-2-enal (2MB2) [the mammary pheromone being 2MB2, it will hereafter be termed 2MB2 for simplicity and clarity], involved in the interaction of rabbit neonates with the mother, their sucking behavior and early odor learning, and therefore their survival 17 – 21 . In the OE of newborn rabbits, it has been recently demonstrated that 2MB2 is metabolized by glutathione-S-transferase enzyme (GST), which belongs to the OME family, and that this metabolism is particularly active within the developmental time-window during which 2MB2 perception is vital for young rabbits (i.e., at birth compared to the weaning) 20 , 22 – 24 ; It is noteworthy that preferential substrates of GST are aldehydes, including 2MB2.…”