Abstract
Background
COVID-19 has been associated with olfactory dysfunction in many infected patients. The rise of calcium levels in the nasal secretions plays an essential role in the olfaction process with a desensitization effect on the olfactory receptor neurons and a negative impact on the olfaction transmission. Ethylene diamine tetra acetic acid (EDTA) is a chelating agent that can bind free calcium in the nasal secretions, thereby reducing the adverse effects of calcium on olfactory function.
Objectives
The objective of this work is to demonstrate the effect of intranasal EDTA on improving olfactory dysfunction following COVID-19.
Methods
Fifty patients with a history of COVID-19 and olfactory dysfunction that persisted for more than 6 months were enrolled in the current prospective randomized clinical trial. Participants were randomized into 2 equal groups. Twenty-five patients were treated with olfactory training only, while the remaining 25 patients received treatment with olfactory training and a topical nasal spray of ethylene diamine tetra acetic acid. The olfactory function was assessed before treatment and 3 months later using the Sniffin’ Sticks test. Additionally, the determination of calcium level in the nasal secretions was performed using an ion-selective electrode before treatment and 3 months later.
Results
Eighty-eight percent of the patients treated with olfactory training in addition to EDTA exhibited clinical improvement, while 60% showed improvement in patients treated with olfactory training only. Furthermore, a significant decrease in the measured calcium level in the nasal secretions was demonstrated after the use of ethylene diamine tetra compared to patients treated with olfactory training only.
Conclusion
Ethylene diamine tetra acetic acid may be associated with an improvement of the olfactory function post-COVID-19.
Keywords: COVID-19, chelating agent, olfactory dysfunction, EDTA, sodium chloride, randomized, calcium, Sniffin’ sticks, anosmia, hyposmia
Introduction
Olfactory dysfunction is one of the most commonly identified disorders, affecting approximately 20% of adults. 1 Although olfactory dysfunction is universally present, the exact mechanism has not been fully confirmed. Additionally, the limited availability of an effective treatment protocol for olfactory dysfunction could indeed be attributed to the simultaneous presence of neurological and biochemical factors. 2
The new global coronavirus disease 2019, (COVID-19) is primarily caused by the severe acute respiratory syndrome coronavirus. 2 Coronaviruses have the capability to enter the brain through the cribriform plate, which is located near the olfactory bulb and epithelium. 3 The neurons in the olfactory region are at high risk of injury due to the significant viral load in the nasal cavity. 4 Sudden and complete loss of olfactory function has been reported in many COVID-19 patients. 5
Although ions comprise only 1% of nasal secretions, 6 the ionic microenvironment in the olfactory cleft plays a crucial role in the chemical-electrical transduction pathway, facilitating the transmitting of olfactory information from the nasal lumen to the central processing system. 7 The disturbance of ionic levels is associated with the presence or progression of many diseases. 8 Previous reports suggest the major role of calcium in olfactory receptor neurons and the mechanism of olfactory transmission. Calcium, in conjunction with calmodulin, regulates sensitivity to cyclic adenosine monophosphate (cAMP) by entering the cilium during the olfactory response. This leads to a decreased channel sensitivity of cyclic nucleotide-gated channels to cAMP. When the olfactory receptor cells are exposed to different odorants, it stimulates the influx of calcium through cyclic nucleotide-gated channels into the small volume within the cilia. Consequently, there is an increase in intra-ciliary calcium, which establishes negative feedback on various stages of the olfaction transmission mechanism.9,10 More specifically, an increase in nasal calcium levels may lead to desensitization of the olfactory receptor neurons. It is hypothesized that the changes in the olfaction sensitivity resulting from alterations in the nasal calcium levels can significantly affect the sensitivity of cyclic nucleotide-gated channels and, consequently, the excitability of receptor neurons in vivo. This, in turn, can contribute to an improvement in the olfaction process.11–13
Ethylene diamine tetra acetic acid (EDTA) was introduced as a chelating agent used for the removal of toxic heavy metal ions. The disodium salt of EDTA is a common ingredient in the formulation of various pharmaceutical preparations. 14 EDTA possesses the ability to bind divalent or trivalent metal ions, such as calcium and magnesium cations, through 4 carboxylate groups and 2 amines groups. 15 EDTA is capable of sequestering free calcium from nasal secretions, forming a stable complex product. This property of EDTA may have implications for the treatment of olfactory disorders.
Olfactory training is considered a common therapeutic alternative for postviral olfactory loss and has a strong scientific foundation.16–18 This prospective study was conducted to test the use of intranasal EDTA in addition to olfactory training in order to decrease the rise of calcium in the nasal secretions and improve olfactory dysfunction following COVID-19. Furthermore, a comprehensive description of the comparison between patients treated with olfactory training alone with patients treated with olfactory training along with topical nasal spray EDTA was provided. This study represents the first published clinical trial evaluating the use of intranasal EDTA use as a topical treatment to improve olfactory dysfunction post-COVID-19 infection.
Materials and Methods
Study Design
A prospective randomized double clinical trial was conducted in the ENT Department of Damietta Faculty of Medicine, Al-Azhar University, Egypt. The study received approval from the Ethical Committee of Damietta Faculty of Medicine, Al Azhar University, Egypt (IRB,00012367-21-01-010). All methods were performed in accordance with the relevant guidelines and regulations.
Sample Size
Currently, there is no research available on the effects of EDTA on olfactory dysfunction related to COVID-19. Therefore, due to the lack of preliminary data or estimates of the effect size, the sample size for this study was determined based on feasibility. It is worth noting that reports have indicated that 79.5% of individuals with post-COVID-19 olfactory dysfunction may experience complete recovery within the first two months. 19 Additionally, a sample rejection rate of up to 10% was anticipated, and certain exclusion criteria were applied. In total, 300 patients were screened for eligibility, out of which 50 patients (31 females and 19 males) were randomly assigned to 2 groups. The enrolment period of all patients took place between March 2021 and December 2021, during which comprehensive characterization and examination were conducted. The study flowchart is depicted in Figure 1.
Figure 1.
The flow diagram of the proposed study.
Inclusion Criteria
To be enrolled in the study, patients had to meet the following inclusion criteria: being adults over 18 years of age, having a previous COVID-19 infection confirmed with a documented nasopharyngeal swab test, showing recovery from infection confirmed by a documented negative nasopharyngeal swab test, and exhibiting clinically confirmed signs of olfactory dysfunction persisting for more than six months.
Exclusion criteria included: (1) patients younger than 18 years; (2) patients with olfactory dysfunction less than 6 months after testing negative for SARS-CoV-2; (3) patients with a history of previous olfactory dysfunction related to trauma or surgery; (4) patients with congenital olfactory loss and neurodegenerative diseases; (5) patients with psychiatric diseases; (6) patients who were currently taking medication for olfactory dysfunction; (7) pregnant patients and (8) patients currently participating in other COVID-19 trials.
Entry into the Study
Before participating in the study, the patients were approached by one of the study team members who explained the study objective, potential benefits, and possible adverse effects. Subsequently, all patients signed an informed consent form. After obtaining informed consent, the patients received the proposed treatment.
Randomization Process
Patients were assigned to 2 equal groups using a randomization method known as unratified block randomization. The randomization was achieved using a computer-generated randomization plan, which provides an additional layer of objectivity and eliminates any potential for human bias. A block size of 4 was chosen, where for every 4 consecutive patients enrolled in the study, 2 would be assigned to each group. The block size was selected to maintain a balance between the groups and prevent any systematic imbalances in treatment assignment. The randomization plan was kept confidential and securely stored to maintain blinding and prevent any potential interference or manipulation. The study investigators followed the predetermined plan strictly, adhering to the randomization sequence as provided by the computer-generated list. This approach adds rigor and reliability to the study design, enhancing the validity of the results obtained.
Treatment Regimen Procedures
Twenty-five patients were treated with olfactory training only using 4 standard odorants (phenyl ethyl alcohol [rose], eucalyptol [eucalyptus], citronellal [lemon], and eugenol [cloves]) for 3 months. 16 On the other hand, 25 patients were treated with olfactory training and 1% topical nasal spray of EDTA. The Department of Pharmaceutical Analytical Chemistry, Cairo Faculty of Pharmacy, Al-Azhar University, Egypt provided the appropriate standard procedures for formulating EDTA nasal spray solutions. Reports demonstrated that EDTA should be prepared less than 2% in pharmaceutical cosmetic preparations. Thus 1% EDTA was appropriate and safe to be used as a topical nasal solution. 19 Topical nasal spray EDTA, 1%, was prepared in phosphate buffer, pH 7.5. The medications were provided in nasal spray bottles which deliver a standardized volume of 0.1 ml. Patients were instructed and trained to instill the topical EDTA using a lying position with the head tilted back, which has been suggested to improve access to the upper nasal cavity. Variations in patient technique utilizing nasal spray delivery were controlled for in the study by providing detailed instructions to patients on the correct administration technique. Additionally, patients were observed and provided with feedback to ensure that they were complying with the recommended technique. Patients were also required to demonstrate their technique to the study staff and sign a compliance form to confirm their understanding of the correct technique.
Study Outcomes
Olfactory Function Assessment
The “Sniffin’ Sticks” test is a validated assessment tool for evaluating olfactory function. Odorants are administrated using felt-tip pens that carry a tampon soaked with different concentrations of liquid odorant (phenyl ethanol dissolved in propylene glycol). Sixteen odorant concentrations were created through a stepwise diluting process. The pen's tip is positioned in front of the patient nose and carefully moved from left to right nostril and backward. 20 The threshold score (T) was evaluated using three alternative forced choice paradigms where patients were repeatedly presented with triplets of pens and had to assign one pen containing an odorous solution from 2 blanks filled with the solvent. 21 A staircase paradigm was employed, starting with the lowest odor concentration. In this paradigm, correctly identifying the odorous pen twice in a row or providing one incorrect answer marked a turning point. A turning point led to a decrease or increase in concentration in the subsequent triplet of odors. The threshold score (T) was the mean of the last four turning points in the staircase, with the final score ranging between 1 and 16 points. The discrimination (D) score was evaluated by introducing three alternative forced choice paradigms where patients were repeatedly presented with 2 pens containing the same odorant, while the third pen smelled differently. Patients were asked to discriminate the single pen with a different smell. The score was the sum of correctly identified odors, between 0 and 16 points. The identification score (I) was evaluated by introducing single pens where patients were asked to identify and label the smell, using four alternative descriptors for each pen. The total score was the sum of correctly identified pens, thus subjects could score between 0 and 16 points. 22 The final TDI score was the sum of scores for threshold, discrimination, and identification scores, with a range between 1 and 48 points. A TDI score below 16.75 points was considered to represent anosmia, and a TDI score between 16.75 and 30.50 points was considered to represent hyposmia. A TDI score of 30.75 points or more signified normosmia.23,24
In the current study, the same tester of olfaction was used for all patients to ensure consistency and accuracy of the olfactory assessments. The tester was trained and experienced in conducting “Sniffin’ Sticks” test and was blinded to the treatment allocation of the patients. In addition, the time of day for smell tests was kept consistent among the patient population to control for any confounding effects or diurnal variation in olfactory function. All smell tests were administered in the morning between 9:00 am and 12:00 pm.
Determination of Calcium in the Nasal Secretions
Nasal secretions were collected from the patients, and the most accurate representation of the nasal fluid composition was obtained immediately after a sneeze due to the relatively large amount of secretion produced. Nasal fluid was usually collected using a small stainless steel (approximately 10 mm × 5 mm × 2 mm). The stainless steel was clamped onto the septum between the nostrils enhancing the fluid to drain into 1.5-ml tube. 25 The volume of nasal secretion obtained was transferred to a series of centrifugation tubes after adding 0.5 ml of phosphate buffer solution. To denature proteins, 3 ml of acetonitrile was added to the centrifugation tubes. The tubes were shaken for 1 min and centrifuged at 4000 r/min for 30 min. The protein-free supernatant was evaporated to dryness, and the residues were diluted with phosphate buffer solution in 10-ml volumetric flasks. The standard procedure for developing a screen-printed selective electrode 26 was developed by the Department of Pharmaceutical Analytical Chemistry, Cairo Faculty of Pharmacy, Al-Azhar University. It was used for the determination of calcium level in the nasal secretion samples from all patients before treatment and 3 months later.
Statistical Analysis
All statistical analyses were conducted using SPSS v23 statistical software (SPSS, Inc, Chicago, Illinois). The obtained results were tested for normality and parametric or nonparametric tests were applied accordingly. Unless specified otherwise, results were presented as mean ± standard deviation. Differences in frequencies were evaluated using Fisher's exact probability test. ANOVA test was employed for the assessment the repeated measures within subject factor and between subject factor. Unpaired t-test was utilized to compare and test the significance of the results of the two groups. Chi-squared test was employed wherever appropriate. Statistical significance was considered when p < .05.
Results
Fifty patients, who had documented previous COVID-19 and olfactory dysfunction persisted more than 6 months’ post-SARS-CoV-2 negative testing, were enrolled in this study. The patients ranged in age from 20 to 60 years. There were 31 females and 19 males. In total, 25 patients received only olfactory training, while the remaining 25 received olfactory training in addition to topical EDTA treatment. The complete characteristics of the patients were described in Table 1. Differences in frequencies for total comorbidities were assessed using Fisher's exact probability test. There was a nonsignificant difference in the frequency of smoking between the group of patients treated with olfactory training alone and the group treated with olfactory training in addition to topical EDTA (3/25 vs 5/25; p = .70). Also, there were no significance between the two groups for other comorbidities, as shown in Table 1. EDTA exhibited the ability to chelate calcium in the nasal secretions forming soluble complex products. The reaction pathway for the interaction between EDTA and calcium was proposed and illustrated in Figure 2.
Table 1.
Patient Demographics and Clinical Data.
| Character | Olfactory training | Olfactory training + EDTA | p (Fisher exact probability test) |
|---|---|---|---|
| Sample size, n | 25 | 25 | |
| Age (years), mean ± SD | 40.37 ± 8.58 | 41.25 ± 7.23 | |
| Days since symptoms to enrollment, mean ± SD | 180 ± 5.28 | 180 ± 6.32 | |
| Gender (male/female), n | 10/15 | 9/16 | |
| Smokers (current/never), n | 3/22 | 5/20 | 0.70 |
| Comorbidities, n | |||
| Asthma | 1 | 2 | 1.00 |
| Diabetes | 5 | 6 | 1.00 |
| Hypertension | 3 | 5 | 0.70 |
| Migraine | 0 | 1 | 1.00 |
| Current medication | Anti-histamine Metformin B-blocker ACE inhibitor Paracetamol |
Anti-histamine Glipizide Amlodipine ACE inhibitor Paracetamol |
Figure 2.
The schematic reaction pathway of EDTA with calcium.
EDTA, ethylene diamine tetra acetic acid.
The Sniffin’ Sticks test was used to assess olfactory function in all patients before treatment and 3 months later. Table 2 presents the mean pre- and post-treatment olfactory scores based on the treatment regimen. The change in the olfactory scores following treatment with olfactory training or olfactory training in addition to EDTA is depicted in Figure 3. In general, the change in TDI score was significantly greater in patients who received the olfactory training in addition to the EDTA group compared to the patients who received olfactory training (2.33 points, p = .008). More specifically in the patients who received olfactory training only, there was a significant improvement in the change of TDI scores but it did not reach clinical significance (5.2 points, p = .0004). As the patient was considered to be improved when the TDI score increased by 6 points. 27 On the other hand, in the patients who received the olfactory training in addition to EDTA, there was a significant improvement in the change of TDI scores which reached clinical significance (7.2 points, p = .00001). Furthermore, there was a trend toward improved T, D, and I scores (1.4 points, p = .091, 1.7 points, p = .005 and 2 points, p = .005, respectively) for the patients treated with olfactory training only. For the patients treated with olfactory training in addition to EDTA, there was a clinically significantly improved T, D, and I scores (2.4 points, p = .005, 2.9 points, p = .007, and 2.4 points, p = .004, respectively).
Table 2.
Results of the Measured Olfactory Scores and the Measured Nasal Calcium Concentration Levels Pre and Post Treatment.
| Olfactory training | Olfactory training + EDTA | |||
|---|---|---|---|---|
| Pre administration | Post administration | Pre administration | Post administration | |
| T score, mean ± SD | 2.30 ± 0.21 | 3.75 ± 1.04 | 1.99 ± 0.14 | 4.12 ± 0.92 |
| D score, mean ± SD | 6.23 ± 0.22 | 7.95 ± 1.23 | 6.26 ± 0.27 | 9.25 ± 1.09 |
| I score, mean ± SD | 5.22 ± 0.21 | 7.27 ± 1.36 | 5.28 ± 0.25 | 7.69 ± 1.27 |
| TDI score, mean ± SD | 13.76 ± 0.36 | 18.95 ± 3.58 | 13.54 ± 0.35 | 21.06 ± 3.05 |
| Nasal calcium concentration (mM), mean ± SD | 38.12 ± 1.64 | 28.88 ± 5.90 | 37.96 ± 1.51 | 23.88 ± 5.64 |
Figure 3.
Box and whisker plots showing the change of measured olfactory scores and the change of measured calcium concentration levels for group received olfactory training and group received olfactory training in addition to EDTA.
EDTA, ethylene diamine tetra acetic acid.
When examining individual patient scores, it was observed that olfactory function clinically improved in 60% of patients who received olfactory training alone. However, in those who received EDTA in addition to olfactory training, 88% of patients showed improvement. The proportion of patients who improved with the additional EDTA was statistically significantly higher than the training alone group (x2 = 7.06, df = 2, p = .03).
The concentration of calcium in the nasal secretions was assessed in all patients using screen-printed ion-selective electrodes. Electromotive force values were determined over a calcium concentration range of 100 to 0.001 mM. A standard calibration plot was constructed relating the electromotive force values to the calcium concentration. The designed electrode exhibited a near Nernstian slope of 29.23 mV/decade with a detection limit of 0.0001 mM in the dynamic range of 100 to 0.001 mM. The concentration of calcium in the nasal secretions was successfully determined using the developed electrode. The changes in the measured calcium level following treatment with olfactory training or olfactory training in addition to EDTA are depicted in Figure 3. The mean values of calcium concentration before treatment and 3 months later are presented in Table 2.
An ANOVA test was conducted to statistically test the repeated measures of the measured olfactory scores and the repeated measures of the measured calcium concentration values before and after treatment with olfactory training or olfactory training in addition to EDTA. A significant difference was obtained for the measures of the whole measures (Table 3). To assess whether the results of patients treated with olfactory training in addition to EDTA showed a statistical significance compared to patients treated with olfactory training only, the change of the olfactory scores and the change of the measured nasal calcium concentration were compared using an unpaired t-test. Results were presented in Table 4. Based on the findings, it can be concluded that patients who received olfactory training in addition to EDTA exhibited a statistically significant difference and a relevant clinical improvement in olfactory function. EDTA was generally well tolerated, with nasal discharge being the most commonly reported side effect. However, mild burning sensations in the nose or throat were also reported.
Table 3.
ANOVA Statistical Testing for the Measured Olfactory Score Values and the Measured Values of the Nasal Calcium Concentrations.
| Group | Threshold (T) statistical assessment results | ||||
|---|---|---|---|---|---|
| Source of variation | df | Sum of squares | Mean square | p | |
| Olfactory training | Between group | 1 | 26.06 | 26.06 | 1.40 × 10−8 |
| Within group | 48 | 27.05 | 0.56 | ||
| Olfactory training + EDTA | Between group | 1 | 57.03 | 57.03 | 2.66 × 10−15 |
| Within group | 48 | 20.94 | 0.44 | ||
| Discrimination (D) statistical assessment results | |||||
| Olfactory training | Between group | 1 | 36.98 | 36.98 | 1.20 × 10−8 |
| Within group | 48 | 37.59 | 0.78 | ||
| Olfactory training + EDTA | Between group | 1 | 111.60 | 111.60 | 1.16 × 10−17 |
| Within group | 48 | 30.48 | 0.635 | ||
| Identification (I) statistical assessment results | |||||
| Olfactory training | Between group | 1 | 51.61 | 51.61 | 2.1 × 10−9 |
| Within group | 48 | 45.66 | 0.95 | ||
| Olfactory training + EDTA | Between group | 1 | 72.24 | 72.24 | 2.49 × 10−12 |
| Within group | 48 | 40 | 0.83 | ||
| Nasal calcium concentration statistical assessment results | |||||
| Olfactory training | Between group | 1 | 1067.22 | 1076.22 | 1.39 × 10−9 |
| Within group | 48 | 917.28 | 19.11 | ||
| Olfactory training + EDTA | Between group | 1 | 2487 | 2487 | 4.05 × 10−16 |
| Within group | 48 | 819 | 17.07 | ||
Statistical significance is indicated by bold.
Table 4.
Unpaired t Results for Statistical Comparing the Change of the Measured Olfactory Scores and the Change of Nasal Calcium Concentrations Between the Two Groups.
| Parameter | Change of T score | Change of D score | Change of I score | Change of TDI score | Change of calcium level |
|---|---|---|---|---|---|
| t | 1.17 | 1.52 | 1.58 | 1.61 | 1.67 |
| p | 0.014 | 0.001 | 0.002 | 0.008 | 0.002 |
Discussion
Olfactory dysfunction has become a common symptom associated with many cases of coronavirus.28,29 Several reports have indicated an increase in calcium levels in the nasal secretions which can have negative effects on the olfactory mechanism. The shift in calcium concentration holds physiological significance and correlates with the olfaction management process.11–13
The current study was specifically designed to investigate the effectiveness of EDTA in improving olfactory dysfunction post-COVID-19. As olfactory training is commonly used as therapy for postviral olfactory loss, a prospective randomized clinical trial was conducted to assess the effect of intranasal EDTA in addition to olfactory training for olfactory dysfunction treatment. EDTA is a widely used chelating agent that has the ability to form strong water-soluble metal complexes with divalent and trivalent cations. These cations are incorporated into a ring-like structure resulting in the formation of complex products between the selected ion and EDTA. The chelation process is primarily influenced by pH and the presence of other competing metal ions. 30 Specifically, at a pH of 7.5, EDTA selectively forms a calcium–EDTA complex even in the presence of sodium, potassium, or magnesium cations. As a result of this mechanism, the use of EDTA leads to a decrease in calcium levels in nasal secretions. The findings of the current study suggest that reducing calcium levels in nasal secretions through EDTA may be associated with an improvement in olfactory function.
It is very important to use precise methods to assess the olfactory function. The Sniffin’ Sticks test is a common assessment tool for evaluating human olfactory function. This test consists of three subtests, odor threshold (T), odor discrimination (D), and odor identification (I), each with a potential score of up to 16 points. A composite “TDI” score is calculated by summing the scores from the three subtests. The obtained results demonstrated that patients who received EDTA in addition to olfactory training exhibited significant improvement in the olfactory function, as evidenced by improved overall olfactory test scores.
It is also recommended to quantify the calcium level in the nasal secretions. Potentiometric determination of the calcium using a screen-printed selective electrode offers the advantage of being suitable for small sample volumes, which is ideal for the current study on nasal secretions. 31 The electrode was designed and utilized to determine the calcium concentration before and after the described treatment. The observed sharp decrease in calcium levels can be attributed to the chelation of the calcium and the formation of the calcium EDTA complex product. While our findings suggest the potential benefits of EDTA in improving olfactory dysfunction, it is essential to expand this research to larger and more diverse populations considering the limitations posed by our small sample size.
This study had several limitations. The primary limitation is the small sample size, which resulted in an underpowered analysis. Further studies are needed to validate the association between changes in the nasal calcium concentration and the olfactory function. It is important to expand this study to include larger and more diverse populations. Additionally, investigating the use of EDTA for olfactory dysfunction caused by factors other than COVID-19 infection should be considered.
Conclusion
This article demonstrated the effect of intranasal EDTA in reducing the elevated nasal calcium level and improving dysfunction post-COVID-19. Following the use of EDTA in addition to olfactory training, a significant improvement from anosmia to hyposmia was observed, accompanied by a notable decrease in nasal secretions calcium level. Further research is recommended to validate the efficacy of EDTA in treating olfactory dysfunction and its association with decreased calcium levels in nasal secretions.
Acknowledgments
The author(s) would like to thank the Deanship of scientific research at Umm Al-Qura University for supporting this work by grant code (23UQU4290565DSR57).
Footnotes
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article. This work was supported by the Deanship of scientific research at Umm Al-Qura University (grant number 23UQU4290565DSR57).
ORCID iDs: Ahmed H. Abdelazim https://orcid.org/0000-0002-8907-3497
References
- 1.Brämerson A, Johansson L, Ek L, et al. Prevalence of olfactory dysfunction: the Skövde population-based study. Laryngoscope. 2004;114(4):733–737. [DOI] [PubMed] [Google Scholar]
- 2.Kurahashi T, Menini A. Mechanism of odorant adaptation in the olfactory receptor cell. Nature. 1997;385(6618):725–729. [DOI] [PubMed] [Google Scholar]
- 3.Felsenstein S, Herbert JA, McNamara PS, et al. COVID-19: immunology and treatment options. Clin Immunol. 2020;215:108448. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Han AY, Mukdad L, Long JL, et al. Anosmia in COVID-19: mechanisms and significance. Chem Senses. 2020;45(6):423–428. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Eliezer M, Hautefort C, Hamel A-L, et al. Sudden and complete olfactory loss of function as a possible symptom of COVID-19. JAMA otolaryngology–Head & Neck Surgery. 2020;146(7):674–675. [DOI] [PubMed] [Google Scholar]
- 6.Ramvikas M, Arumugam M, Chakrabarti S, et al. Nasal vaccine delivery. In: Micro and nanotechnology in vaccine development. Elsevier; 2017:279–301. [Google Scholar]
- 7.Lam K, Conley DB, Liu K, et al. Effect of ionic compositions in nasal irrigations on human olfactory thresholds. Laryngoscope. 2015;125(2):E50–E56. [DOI] [PubMed] [Google Scholar]
- 8.Sauer G, Richter C-P, Klinke R. Sodium, potassium, chloride and calcium concentrations measured in pigeon perilymph and endolymph. Hear Res. 1999;129(1-2):1–6. [DOI] [PubMed] [Google Scholar]
- 9.Menini A. Calcium signalling and regulation in olfactory neurons. Curr Opin Neurobiol. 1999;9(4):419–426. [DOI] [PubMed] [Google Scholar]
- 10.Panagiotopoulos G, Naxakis S, Papavasiliou A, et al. Decreasing nasal mucus Ca improves hyposmia. Rhinology. 2005;43(2):130–134. [PubMed] [Google Scholar]
- 11.Whitcroft K, Merkonidis C, Cuevas M, et al. Intranasal sodium citrate solution improves olfaction in post-viral hyposmia. Rhinology. 2016;54(4):368–374. [DOI] [PubMed] [Google Scholar]
- 12.Whitcroft K, Ezzat M, Cuevas M, et al. The effect of intranasal sodium citrate on olfaction in post-infectious loss: results from a prospective, placebo-controlled trial in 49 patients. Clin Otolaryngol. 2017;42(3):557–563. [DOI] [PubMed] [Google Scholar]
- 13.Whitcroft K, Gunder N, Cuevas M, et al. Intranasal sodium citrate in quantitative and qualitative olfactory dysfunction: results from a prospective, controlled trial of prolonged use in 60 patients. Eur Arch Oto-Rhino-Laryngol. 2021;278(8):1–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Hart JR. Ethylenediaminetetraacetic acid and related chelating agents. Ullmann's Encyclopedia of Industrial Chemistry. 2000;13:573–578. [Google Scholar]
- 15.Pribil R. Analytical Applications of EDTA and Related Compounds: International Series of Monographs in Analytical Chemistry. Elsevier; 2013. [Google Scholar]
- 16.Hummel T, Rissom K, Reden J, et al. Effects of olfactory training in patients with olfactory loss. Laryngoscope. 2009;119(3):496–499. [DOI] [PubMed] [Google Scholar]
- 17.Haehner A, Tosch C, Wolz M, et al. Olfactory training in patients with Parkinson's disease. PloS One. 2013;8(4):e61680. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Damm M, Pikart LK, Reimann H, et al. Olfactory training is helpful in postinfectious olfactory loss: A randomized, controlled, multicenter study. Laryngoscope. 2014;124(4):826–831. [DOI] [PubMed] [Google Scholar]
- 19.Lanigan RS, Yamarik TA. Final report on the safety assessment of EDTA, calcium disodium EDTA, diammonium EDTA, dipotassium EDTA, disodium EDTA, TEA-EDTA, tetrasodium EDTA, tripotassium EDTA, trisodium EDTA, HEDTA, and trisodium HEDTA. Int J Toxicol. 2002;21:95–142. [DOI] [PubMed] [Google Scholar]
- 20.Hummel T, Sekinger B, Wolf SR, et al. Sniffin’sticks’: olfactory performance assessed by the combined testing of odor identification, odor discrimination and olfactory threshold. Chem Senses. 1997;22(1):39–52. [DOI] [PubMed] [Google Scholar]
- 21.Croy I, Lange K, Krone F, et al. Comparison between odor thresholds for phenyl ethyl alcohol and butanol. Chem Senses. 2009;34(6):523–527. [DOI] [PubMed] [Google Scholar]
- 22.Oleszkiewicz A, Schriever V, Croy I, et al. Updated Sniffin’Sticks normative data based on an extended sample of 9139 subjects. Eur Arch Oto-Rhino-Laryngol. 2019;276(3):719–728. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Reden J, Lill K, Zahnert T, et al. Olfactory function in patients with postinfectious and posttraumatic smell disorders before and after treatment with vitamin A: a double-blind, placebo-controlled, randomized clinical trial. Laryngoscope. 2012;122(9):1906–1909. [DOI] [PubMed] [Google Scholar]
- 24.Hummel T, Kobal G, Gudziol H, et al. Normative data for the “Sniffin’Sticks” including tests of odor identification, odor discrimination, and olfactory thresholds: an upgrade based on a group of more than 3,000 subjects. Eur Arch Oto-Rhino-Laryngol. 2007;264(3):237–243. [DOI] [PubMed] [Google Scholar]
- 25.Burke W. The ionic composition of nasal fluid and its function. Health. 2014;6(8):720–728. [Google Scholar]
- 26.Li M, Li Y-T, Li D-W, et al. Recent developments and applications of screen-printed electrodes in environmental assays—A review. Anal Chim Acta. 2012;734:31–44. [DOI] [PubMed] [Google Scholar]
- 27.Rumeau C, Nguyen D, Jankowski R. How to assess olfactory performance with the Sniffin’Sticks test®. Eur Ann Otorhinolaryngol Head Neck Dis. 2016;133(3):203–206. [DOI] [PubMed] [Google Scholar]
- 28.Boscolo-Rizzo P, Borsetto D, Fabbris C, et al. Evolution of altered sense of smell or taste in patients with mildly symptomatic COVID-19. JAMA Otolaryngology–Head & Neck Surgery. 2020;146(8):729–732. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Whitcroft KL, Hummel T. Olfactory dysfunction in COVID-19: diagnosis and management. JAMA. 2020;323(24):2512–2514. [DOI] [PubMed] [Google Scholar]
- 30.Schwarzenbach G. The general, selective, and specific formation of complexes by metallic cations. In: Advances in Inorganic Chemistry and Radiochemistry. Elsevier; 1961:257–285. [Google Scholar]
- 31.Dimeski G, Badrick T, St John A. Ion selective electrodes (ISEs) and interferences—a review. Clin Chim Acta. 2010;411(5-6):309–317. [DOI] [PubMed] [Google Scholar]