Insights into the molecular triggers of parosmia based on gas chromatography olfactometry.

Background: Parosmia is a debilitating condition in which familiar smells become distorted and disgusting, with consequences for diet and mental health. It is a feature of post-infectious olfactory loss, particularly resulting from COVID-19. There is currently little understanding of its pathophysiology, and the prevailing hypothesis for the underlying mechanism is aberrant growth of regenerating olfactory sensory neurons after damage.
Methods: We use gas-chromatograph olfactometry to individually present components of a complex olfactory mixture as a rapid screening tool for assessment of both quantitative and qualitative olfactory dysfunction in those with and without parosmia. This allows them to report the associated sensory effects and to identify those molecules which are altered or parosmic in nature.
Results: Here we show 15 different molecular triggers of this symptom. These trigger molecules are common to many in the parosmic volunteer group and share certain characteristics such as extremely low olfactory threshold and common molecular structure. Conclusions: We posit that specific highly odour-active molecules are the cause of the parosmic symptom in most cases and initiate the sense of disgust, suggesting that parosmia is, at least in part, a receptor-level phenomenon.

Introduction

Prior to the COVID-19 pandemic, olfactory dysfunction was largely unrecognised, and often underestimated by health care professionals. Since the spread of SARS-CoV-2, and the realisation that 50–65% of cases result in anosmia[1] (the loss of sense of smell), there is a greater awareness of the debilitating effect of olfactory disorders[2]. Typically, in cases post COVID-19, normal olfactory function returns within a few weeks, but one study estimates 12% of all cases result in long term smell dysfunction[3]. With >200 million confirmed cases of COVID-19 worldwide[4], this is a significant problem facing the global population today.

Parosmia often occurs in the early stages of recovery from anosmia, typically 2–3 months after onset[1], particularly in those whose anosmia was either acquired post-infection. It is characterised by episodes of triggered olfactory distortions in which familiar everyday smells become altered and unpleasant, to the extent that they become almost unrecognisable, and these distortions vary in strength and duration[5]. Note that throughout the paper, when we refer to parosmia triggers, we are referring to triggers of the episodes rather than triggers of disease onset. Those severely affected find their quality of life deteriorates as everyday activities such as eating, showering and social interactions become a challenge. They report being distressed and anxious about their future[5] and, with many food aromas being intolerable, they start to reject food, leading to significant changes in weight[6], a decline in mental health and, in severe cases, to clinical depression[7,8]. Although many mechanisms for parosmia have been proposed, there is very little fundamental understanding of its pathophysiology.

The aim of this work was to gain insight into the mechanisms involved in parosmia. In 2013, coffee and chocolate were found to elicit distorted olfactory experiences in parosmia[9] and more recently, coffee, meat, onion, garlic, egg, mint/toothpaste were identified in a thematic analysis of group posts on social media[5]. These foods contain aroma compounds with some of the lowest odour-thresholds known, and we suggest that these compounds may be involved in triggering episodes of parosmia. Our original hypothesis was based on that of Leopold[10] who proposed that parosmia was a result of incomplete characterisation of the odorant. As olfactory sensory neurons (OSN) regenerate from basal stem cells, selective detection of just the pungent highly odour-active compounds might result in an incomplete, and therefore distorted perception of certain foods and beverages. Whether this would be sufficient to cause the strong sense of disgust, often reported with parosmia, was not clear.

Our approach is to use GC-Olfactometry (GC-O) to determine which of the aroma compounds present in the headspace of coffee are responsible for distortions and the sense of disgust experienced by those with parosmia. Gas chromatography separates the hundreds of volatile components present in the sample headspace which, when coupled to an odour-port, allows subjects to sniff and describe each component as it elutes from the column and assess a variety of single aroma compounds in a short time.

In this paper we demonstrate that there are a small number of highly potent odorants responsible for the parosmia stimulus when smelt by those with parosmia. These odorants fall into four groups based on their physio-chemical characteristics which implies that only a small number of olfactory receptors are responsible for the sensation.

Methods

Participants

This study (No 22/19) was approved by the University of Reading Research Ethics Committee. All participants received full information and gave their informed consent. All parosmic participants were recruited via Facebook support groups or local ENT consultants. The major inclusion criterion was those with post-infection olfactory loss (the aetiology most likely to result in parosmia[11]), whilst those with other aetiologies such as degenerative olfactory loss or traumatic brain injury were excluded from this study. Non-parosmic participants were recruited from within the Department of Food and Nutritional Sciences at the University of Reading, or through private Facebook pages. The initial study was carried out with pre-COVID-19 parosmic participants (N = 14) and non-parosmic participants (N = 15) between October 2019 and March 2020. This was supplemented with post-COVID-19 parosmic participants (N = 15) between July and September 2020. All volunteers completed a screening questionnaire (see Supplementary Information) before attending a study day in the Olfaction Laboratory at the University of Reading. Selection was based on the participants listing coffee as a key trigger, and answering “often” at least once to two key questions which have been reported to discriminate most efficiently between parosmic participants and those with quantitative olfactory disorders[12]:

Are odours that are pleasant to others, unpleasant to you? Never/rarely/often/always

Is the taste of food different to what you expect? Never/rarely/often/always

Olfactory function

The bilateral olfactory function of all participants was assessed at the beginning of the day using the well-established and validated orthonasal psychophysical Sniffin’ Sticks test (Burghart, Wedel, Germany)[13], based on the threshold of 2-phenylethanol (T), discrimination (D) and identification (I) tests. The resulting TDI scores, which range from 0 to 48, gives a measure of quantitative olfactory function.

Rationale for use of coffee

A cocktail of individual aroma compounds was initially considered for the GC-O study, but mixtures of compounds, especially those containing sulfur, are unstable, prone to oxidation, interact with each other and the solvent, and are onerous to prepare from fresh for each subject. This approach is also dependant on pre-selection of the likely trigger molecules from a base of thousands of volatile compounds, all of them potential triggers. The use of a foodstuff allowed screening of a range of volatile compounds, both triggers and non-triggers, in a more stable environment. Coffee was selected as it has been recognised on several occasions to be a major trigger of parosmia[5] and has the additional advantage of being widely consumed. However, coffee aroma is highly variable, and degrades over time. Our solution was to use catering sachets of pre-portioned, one mug instant coffee, to produce a material as consistent as possible, which would last the duration of the study. In effect, instant coffee was being used as a stable carrier for a wide range of potential trigger and non-trigger molecules.

Gas chromatography-mass spectrometry (GC-MS)

For standard coffee analysis, each sachet was made up of 300 ml of boiling water and the headspace extracted using solid phase microextraction (SPME). A concentrated sample prepared in 3 ml of water was also prepared to aid the identification of the aromas detected. Both standard and concentrated extracts were analysed by GC-MS using a typical program on a non-polar column and also on a polar column to confirm compound identities. Full details of the procedure are provided in the Supplementary Methods.

Gas chromatography-olfactometry (GC-O)

All volunteers assessed the standard coffee extract using GC-O on a non-polar column. In addition, three parosmic volunteers and two experts also assessed the coffee extract on a polar column to confirm compound identities. Full details of the chromatography are provided in the Supplementary Methods.

Procedure at the odour-port

Subjects were sat in front of the GC-O with their nose placed in, but not resting on, a glass cone. They were familiarised with the instrument, instructed to breathe normally during the run, and advised that they could stop at any time. As the aromas eluted from the column, three bits of information were requested from the subjects: (i) an odour description, (ii) an odour intensity, and (iii) an indication of whether the odour elicited a parosmic response. Since the description and identification of aromas in the absence of any other cues is difficult, all participants were presented with a flavour wheel before they started (Supplementary Fig. 1), which they could use as a reference during the GC-O run. It had been developed by two experts who sniffed samples of the same coffee (both at regular strength and concentrated) by GC-O. The words were categorised into food and non-food, and colour coded for quick reference. The flavour wheel was of more use to non-parosmic participants, as parosmic participants found it hard to describe many of the aromas, even with the help of the flavour wheel. Many resorted to using the terms “new coffee”, “that parosmia smell”, “trigger number 1” or “trigger number 2”. As each aroma eluted, parosmic participants were prompted to highlight anything that had a parosmic character or trigger. Intensity was scored on a general labelled magnitude scale (gLMS) with anchors at barely detectable, weak, medium, strong, very strong and strongest imaginable. This was chosen over the more common visual analogue scale to allow for instances where parosmic participants wanted to extend the range of scores upwards. All subjects carried out the GC-O of coffee twice. During the second run, the focus was on refining the descriptors with discussion between the researcher and the subject to help identify the compounds eluting.

Confirmation of identity of the trigger molecules

Supplementary Data 1 shows how the identification of each trigger was confirmed based on comparison of the mass spectrum, linear retention index and odour character with those of authentic standards. Three parosmic participants returned to assess coffee on a polar column to confirm the identity of trigger compounds. Once identified, selected trigger compounds diluted in mineral oil or propylene glycol at 10 mg L−1 were presented to two parosmic participants as described for the European test of olfactory capabilities[14]. They were asked to sniff the vial and indicate whether each compound released “that parosmia smell” which they had described previously.

Additional samples

Extracts of cocoa, meat, peanut butter, and red pepper were prepared for coffee with modifications described in Supplementary Methods. Human faecal samples were obtained with informed consent and kindly prepared under Class 2 conditions by members of the Food and Microbial Science Unit at the University of Reading with ethical approval from Reading Research Ethics Committee (number UREC 1520). The sample was mixed with an equal weight of water, and 3 g transferred to an SPME vial. Chromatography conditions were the same as for coffee.

Table 1

Summary of participant demographics.

Participant demographic data	No	Male	Female	Age (mean)	Age (range)	Age (SD)	CRS
All Participants	44	12	32	47	19–73	14	4
Pre-COVID-19 parosmics	14	3	11	56	33–73	9.6	1
Post-COVID-19 parosmics	15	3	12	37	19–60	12.2	1
Non-parosmics	15	6	9	49	33–71	13.2	2

CRS chronic rhinosinusitis.

Table 2

Compounds most frequently detected by parosmic participants.

Molecular triggers	Code	Odour threshold ug/L	Number times detected by parosmic	Number times reported as trigger
2-furanmethanethiol	T1	0.005[16]	24	20
2-ethyl-3,6-dimethylpyrazine	P1	0.01	18	15
2,3-diethyl-5-methylpyrazine	P2	0.05[16]	20	13
2-furanmethyl methyl disulfide	D1	0.04[20]	18	11
2-methyl-3-furanthiol	T2	0.0004[18]	19	10
2-methyl-3-furyl methyl disulfide	D2	0.004[18]	18	10
2-ethyl-3,5-dimethylpyrazine	P3	1[37]	17	10
3-methyl-2-butene-1-thiol	T3	0.01[19]	21	9
2-ethyl-3-methoxypyrazine	M1	0.4[21]	12	9
2-isobutyl-3-methoxypyrazine	M2	0.002[16]	17	7
3-mercapto-3-methylbutanol	T4		13	6
3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolone)	X1	0.5[16]	10	6
3-mercapto-3-methylbutyl formate	T5		15	5
2-methoxyphenol (guaiacol)	X2	12[16]	14	5
trimethylpyrazine	P4	9.6[21]	10	5
unknown LRI 981	X3		12	4
2-isopropyl-3-methoxyprazine	M3	0.001[16]	15	3
2,3-butanedione	X4	1[16]	15	2
4-ethylguaiacol	NT1	4[16]	10	0
(E)-β-damascenone	NT2	1[16]	9	0

T thiol, P pyrazine (trisubstituted), D disulfide, M methoxypyrazine, X unclassified, NT non-trigger.