2.1.1.1. Biological facts
In mammals, odorants are detected by the olfactory epithelium. Two others organs are thought to be involved in olfaction, they are the trigeminal nerves and the vomeronasal organ, see Figure 2.1. The role of the vomeronasal organ is however still unknown.
The olfactory epithelium is a 5 cm^ patch of yellow tissue located at the top of the nasal cavity approximately on a level with eye.^^ The mucosa is roughly 75 p,m thick and is host to between 1 0^ to 1 0* receptor cells distributed across the tissue Olfactory cells are bipolar neurons unique in the central neuron system due to their ability to regenerate every 30-45 days.*"^ From the dendrite end of each neuron, there are several hairlike appendages called cilia, which extend outward into the mucus that covers and protects the olfactory e p i t h e l i u m . T h e primary events of odor discrimination are thought to involve the interaction of odorous molecules with specific receptors on the cilia. The olfactory neurones also give rise to axons that are bundled to traverse the cruciform plate reaching the olfactory bulb in the brain. In the bulb these axons synapse onto secondary neurons known as mitra cells. This synapse, which is known as a glomerulus, is complicated and consists of a single mitral cell upon which 500 olfactory axons converge; from these signals are relayed to higher regions of the brain. The peripheral location of olfactory neurons, their remarkable capacity for post natal regeneration and their direct axon link to the brain sets olfactory neurons apart from other neurons of the central nervous system. Figure 2.2. is a schematic of the anatomy of the human olfactory. A detailed account of the anatomy of the human olfactory neuroepithelium is provided by Morrison and Moran.
In the mid-1980s, the biochemistry of olfaction was stimulated by the discovery that the level of cyclic adenosine monophosphate, cAMP, in isolated olfactory cilia increased rapidly when the cilia were exposed to certain odorants. It was subsequently demonstrated that odorants that have no effects on the level of cAMP induced a rapid change in the levels of inositol triphosphate, IP3.
Brain
Olfactory bulb Cribriform olate Tri seminal nerve
'Vomeronasal organ
Figure 2.1. Keys organs thought to be involved in the perception of odour
Epithelium' Mucus layer Olfactory nerve Mitral cell Glomeruli Cribriform plate Axons Olfactory bulb Receptor cells Cilia Odorant flow
Figure 2.2. Convergence of receptor cells signals on to glomeruli on the olfactory bulb.
It is well known that these two secondary messengers cause the opening of ion channels. Influx of positive ions into the cells initiates a decrease in voltage across the cell membrane, which ultimately results in the generation of a nerve impulse. The signal then runs to the olfactory bulb, which in turn, sends impulses to the primary olfactory cortex. From there, information is relayed both to higher cortical areas and to the limbic system, which controls emotion. The former processes the odor sensations so that subjects can recognize them, for a review see reference 17.
The above findings on the odor -induced cAMP and IP3 responses fit well with the discovery that guanine nucleotide binding proteins, G-proteins, were also involved in olfactory transduction. G-proteips mediate guanine triphosphate, GTP, dependent responses which act as intermediaries between ligand receptor and targets such as adenyl cyclase and phospholipase C, the intracellular enzymes responsible for the production of cAMP and IP3 respectively. Similar types of transduction process are linked to receptor proteins that are inserted into the cell membrane and th ^ cross the membrane in seven places, see Figure 2.3. Hence, it was postulated that odor receptors
1 18
might also belong to this seven-helix family of G-protein-coupled receptors. G- proteins are cell membrane proteins, which act as intermediaries between receptor activation and the subsequent activation of it second messenger. They have three different subunits. The portions that pass through the membrane exist as a-helices. These a-helices come together to form a cylinder and it is thought that the binding sites lie inside the cylinder. This is consistent with hormone receptors and with optical receptors in which the retinal-derived pigment is held in such a way.
In 1991, Buck and Axel^^ discovered the family of transmembrane proteins that u
were believed to be the odor receptors and some of the genes that encode them. They cloned and characterised eighteen different members of an extremely large multigene family that encodes the seven transmembrane proteins whose expression were restricted to the olfactory epithelium. The proteins found all contained the seven helical transmembrane structure and contained sequence similarity to other members of the ‘G- protein’ linked receptor family. It is now estimated that there are between 500-750 odorant receptor genes in humans.
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£o o hFigure 2.3. Schematic representation of a putative odorant receptor. The N-terminus is located extracellularly and the C-terminus intracellularly. The vertical cylinders delineate the seven putative a helices spanning the membrane.
The olfactory proteins are still poorly characterised and their binding affinity for odorants has yet to be demonstrated, although there is suggestive evidence that at least one of these receptors can actually respond to odor m o l e c u l e s . S t u d i e s based on molecular modelling have been carried out on the 0R 5 and OR 17 olfactory receptor g e n e s . F i r s t , it was pointed out that the 0R 5 gene product mediates the activation of G-protein-coupled IP3 production by odorants such as lilial and lyral, methoxypyrazines and carboxylic acids.^^ Furthermore, Singer and Shepherd tested docking of the odor molecule lyral in simulated docking experiments using molecular modeling.^^ The results pointed to specific ligand-binding residues distributed in helices 3 through to 7 that form a binding pocket of approximately 12 A from the extracellular surface of the receptor. This work supports the work of Axel and Buck*^ that helices 3, 4 and 5, which exhibit wide sequence diversity from one receptor to another, are involved in odorant binding. Generalisation of this finding to other olfactory receptors is supported by the recent observation by Lancet and Ben-Arie^"^ that the OR5 receptor shares homology with a wide range of olfactory receptors found in mammalian species, including rat, dog and human. Using a similar approach. Singer constructed a molecular model of the OR 17 receptor. Octanal was automatically docked in the m o d e l . T h e s e results
0^
predicted an odor-binding pocket of around lOA from the extracellular surface. However, the modelling of this important class of proteins is highly speculative because of insufficient knowledge of the olfactory mechanisms.
Currently the number of receptor types is estimated to be 500-750. This raises the question of how are these receptor are distributed in the olfactory epithelium. Are individual members of the receptor family expressed by every olfactory neuron or by only a subset of neurons? The latter is supported by evidence. Each sensory neuron expresses only one receptor and is therefore functionally distinct. Recent studies have shown that the olfactory epithelium is divided into broad zones according the types of receptors found in each zone, but that within each zone there is a random distribution of neurons expressing the same receptors and that members of each subset send their axons to only one or a few of some 2 0 0 0 glomeruli.^^
The olfactory mucus is roughly 10-30 |xm t h i c k . I n addition to protect the mucosa against damage by xenobiotics or microorganisms, the mucus has to provide a favourable milieu into which odorants can partition and gain access to and interact with receptors. The discovery in the mucus of small acidic proteins that accommodate hydrophobic molecules in aqueous environment provided new hopes in odorant transport process understanding. Odorant binding proteins, GBPs, belong to one of the most abundant class of proteins found in the olfactory apparatus. By their amino acid sequence, they belong to the family of lipocalins, generally involved in the transport of hydrophobic l i g a n d s . T h e i r concentration in mucus and respiratory epithelium of mammals is estimated to be high: 1% of soluble nasal proteins. GBP is a homodimer that has a central pocket lined with hydrophobic residues of which high proportion are aromatics. The central pocket has dimensions of 11*10*7 Â, i.e. 770 Â^with an opening size of 6*7 Â, although a much larger cavity of 1100*1300 Â has been suggested.^^
Assuming their involvement in the olfaction process, different physiological roles for GBPs have been proposed. Due to the hydrophobic nature of the odorant molecules and of the lipocalin pocket, it has been proposed that GBPs could carry the odorant molecules from the air to the olfactory receptors through the aqueous barrier of the mucus. It is also proposed that GBP-odorant complexes interact with the receptors. Subsequent to activation of the receptors, GBP may also be involved in removing odorants from the receptor sites to allow termination of the olfactory signal. Furthermore, based on the relatively weak affinity of GBPs for odorant molecules, Kdiss
value in the micromolar range, it has been proposed that OBPs would avoid the saturation of the olfactory receptor when odorant molecule are present at high concentration. It has also been demonstrated that OBPs bind with good affinity to 6-11 carbon alkylic aldehydes.^^
2.1.1.2. Theories of olfaction
Over the recent years a number of theories have been proposed in an attempt (5 explain how humans perceive odors. Here are reviewed the most prominent theories.
The Stereochemical Theorv of Odor: In 1946 Pauling indicated that a specific odor quality is due to the molecular shape and size of the chemical.^^ Similarly, he extended the idea of a ‘Steric Theory of Odor’ originally proposed by Moncrieff in 1949 that stated airborne chemical molecules are smelled when they fit into certain complimentary receptor sites on the olfactory receptor system.^® In 1952, Amoore postulated there is a limited number of receptor types, each of these recognizes a particular molecular shape and, when triggered, generates the signal for a primary odor.^^ He proposed primary odors, viz. ethereal, camphoraceous, musky, floral, minty, pungent and putrid. The molecular volume and shape similarity of typical odorants of each class and proposed shaped receptors for the first five and the generation of charged species for the last two, positive for pungent and negative for putrid. Amoore subsequently carried out ‘specific anosmia’ experiments in an attempt to prove the existence of primary odors and identify all them.^^ This work was inspired by Guillot’s suggestions that specific anosmia, the inability to detect one particular odor, was due to the affected person lacking one of the primary odor receptors. One of the main objections to the stereochemical theory is that there are many examples of substances that have a similar shape but different odors because of a difference in functional group.
The Shape Theorv: In 1957, Beets proposed the concept of modality instead of primary od6r and used a more statistical approach. He considered that a pure odorant is composed of identical molecules arriving in the vicinity of the receptor sites with different orientations and different conformations. These differently oriented molecules interact with a variety of sites to produce a set of interactions, finally generating a
pattern of information containing several modalities at various levels of intensity and corresponding to a given odor.^"^
The Penetrating and Puncturing Theorv: In 1967, Theimer and Davies proposed that odorant molecules must be absorbed into a thin-walled site on the olfactory nerve ending, penetrating it and leading to the formation of a small hole in the membrane. Accordingly, the theory stresses the primary importance of rates of desorption and molecular cross-sectional areas.
The Vibration Theorv: In 1938, Dyson suggested that the infrared resonance, which is a measurement of a molecules vibration, might be associated with odor.^^ This idea was popularised by Wright in the mid 1950’s as infrared spectrophotometers became generally available for such spectral measurements, which Wright was able to correlate with certain odorants.^^ By the mid-70’s it appeared that the theory of Wright had failed a critical test. The optical enantiomers of iwenthol and of (jarvone smelled distinctly different, although the corresponding infrared spectra were identical. And so this theory fell from favour.
Vibrational Induced Electron Tunelling Spectroscope Theorv: In 1996, Turin postulated the presence of an electric potential gap in a protein, with nicotinamide adenine dinucleotide and zinc ions providing the electrodes. Electrons cannot cross the gap unless an odorant molecule is placed between the so-called electrodes. To cross the gap the electron must lose energy and this it does by tunnelling through the orbitals of the odorant molecule and exciting vibrational modes in it as a result. Thus Turin has moved the search for correlations from the infrared to inelastic electron tunnelling spectra.^^
2.1.1.3. A combinatorial Process for odor interpretation
In 1999, Buck and co-workers appeared to have unravelled the mystery of how the nose can interpret abundance of different o d o r s . T h e y showed that the sense of smell in mammals is based on a combinatorial approach to recognising and processing odors. Instead of dedicating an individual odor receptor to a specific odor, the olfactory system uses an alphabet of receptors to create a specific smell response within the neurons of the brain. The olfactory system seems to use combinations of receptors to
greatly reduce the number of receptor types actually required to convey a broad range of odors. In the reported study, individual mouse neurons were exposed to a range of odorants. Using a technique called calcium imaging, the researchers detected which nerve cells were stimulated by a particular odor. Using this approach, it was shown that:
(i). Single receptors can recognise multiple odorants,
(ii). A single odorant is typically recognised by multiple receptors,
(iii). Different odorants are recognised by different combinations of receptors, thus indicating that the olfactory system uses a combinatorial coding scheme to encode the identities of odors.
This new combinatorial approach is illustrated in Table 2.1.
This explains how 500-750 receptors can describe many thousands of different odors. Buck and her colleagues also demonstrated that even slight changes in chemical structure activate different combinations of receptors. Thus, octanol smells like oranges, but the similar compound octanoic acid smells like sweat. Similarly, it was found that large amounts of a chemical bind to a wider variety of receptors than do small amounts of the same chemical. This explains why a large quantity of the chemical indole smells putrid, while a trace of the same chemical smells flowery.^^
2.1.1.4. Odor characterisation
Odor can be described using a number of different dimensions, viz. quality (floral woody), intensity (strong, moderate, weak) and threshold. Scientists studying human olfaction have developed physical, physiological and sensory techniques for two purposes: to study the human olfactory process and to quantify the sensory activity of chemicals.
Phvsical methods
Physical instruments have been designed to measure both odor quality and intensity. Electronic noses are the most prominent example. They are capable of identifying the aroma from different brands of coffee. This modem technique consists of an array of gas-sensitive semi-conductors, which are connected to a neural network. The signals recorded by the sensors produce a characteristic pattern for a given odor.
Table 2.1. Comparison of the Receptor Codes for Odorants that have Similar Structures but Different Odors^ SI S3 S6 S18 S19 S25 S41 S46 S50 S51 S79 S83 S85 S86
Hexanoic acid Rancid, sweaty,sour,goat-like, fatty Hexanol Sweet, herbal, woody, cognac, Scotch,
whiskey Heptanoic
acid 1 Rancid, sweaty sour, fatty
Heptanol Violet, sweet, woody, herbal, fresh, fatty Octanoic acid Rancid, sour, repulsive, sweaty, fatty Octanol Sweet, orange, rose, fatty, fresh, powerful,
waxy 1
Nonanoic acid Nonanol
m m i r m r m r m n
Waxy, cheese, nut-like, fattyFresh, rose, oily floral, odor of citronella oil, fatty
^Aliphatic acids and alcohols with the same same carbon chains were recognised by different combinations of Ors, thus providing a potential explanation for why they are perceived as having strikingly different odors. Taken from Ref.39. (Cr> Ocic/ ^
The electronic nose can be used not only for odor characterisation, but also for the quantitative determination of the concentrations of individual molecules in a complex environment. Although the concept of electronic nose has been developed to imitate the signal processing in the biological noses, in practice the elements of signal transduction, signal processing, identification of chemical patterns currently differ from those believed to be present in the biological system. Comparisons have been made between the human nose and electronic noses. In all cases, the human nose was more sensitive than its electronic counterpart. The response to concentration changes was also different for the two “noses”. The electronic nose was shown to respond linearly, whereas the human nose responds logarithmically. Neuner-Jehle and Etzweiler have reviewed theory and the prospect of development of electronic noses.
Phvsiological method
Measurable physiological responses to odor stimuli include changes of electrical potential at the olfactory bulb or olfactory receptor. Attempts have been made to correlate such successful electrical activity to odor perception. It has been shown that the intensity of an odorant stimulus is related to the amplitude of a DC-recorder electrical potential and to the frequencies of an AC-recorded electrical i m p u l s e . Mo s t of the studies have been carried out on animals. Electrical activity in the human brain has been developed to study the unpleasantness of odors. Results have been reviewed.
Sensorv methods
Another way to obtain a human’s perceptual response to an odor is to ask him for a verbal expression of the odor intensity and quality.
Quality
Can you measure the difference between one kind of smell and another ? The tests used to describe odor quality are known as odor profiling tests. These are the most complex of the sensory tests and, to ensure good quality, accurate and reproducible data are only carried out by highly trained and experienced sensory panellists."^^
Intensity
How strong is an odor? A number of different types of sensory scales are used to