10.14639/0392-100X-N2074
Objective. The goal of the study was to determine whether the level of OTOLIN-1, a protein whose expression is highly restricted to the inner ear,is increased in the body fluids of patients with inner ear disorders in comparison to healthy subjects. Methods. In the preliminary part of the study, OTOLIN-1 levels were measured in the serum, urine, and saliva of patients with an acute onset of Ménière´s disease and in healthy individuals. Subsequently, only serum OTOLIN-1 levels were taken into account and were compared between patients with acute onset of Ménière´s disease, sudden hearing loss, vestibular neuritis and healthy subjects. Results. The most reliable diagnostic parameter was OTOLIN-1 levels in serum. Serum samples of patients with Ménière’s disease and sudden hearing loss showed significantly higher OTOLIN-1 levels than those from healthy individuals. In addition, there was no significant difference between the serum concentration of OTOLIN-1 in patients with vestibular neuritis and the control group. Conclusions. Serum levels of OTOLIN-1 can potentially be used as a biomarker for acute onset of inner ear disorders due to its significant increase in patients with acute Meniere´s disease and sudden hearing loss in comparison to healthy individuals.
text/xml
1827-675X
2074
en
Kerstin  Willenborg
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
inner ear
A potential serological biomarker for inner ear pathologies: OTOLIN-1
Text.Serial.Journal
https://www.actaitalica.it/
42
Vestibology
2022-09-30
2022-03-17
2022-10-12
2022-10-12
364-371
1
Kerstin  Willenborg
2022/09/30
10.14639/0392-100X-N2074
364
Department of Otorhinolaryngology, Head and Neck Surgery, Hannover Medical School, Hannover, Germany
1827-675X
inner ear
en
371
A potential serological biomarker for inner ear pathologies: OTOLIN-1
https://www.actaitalica.it/article/download/2074/851
42
ACTA OTORHINOLARYNGOLOGICA ITALICA 2022;42:364-371; doi: 10.14639/0392-100X-N2074




Vestibology


A potential serological biomarker
for inner ear pathologies: OTOLIN-1
Un potenziale biomarcatore sierologico per le patologie dell’orecchio interno:
OTOLIN-1
Emilio Avallone1, Heike Schmitt1,2, Giorgio Lilli3, Athanasia Warnecke1,2, Anke Lesinski-Schiedat1, Thomas Lenarz1,2,
Kerstin Willenborg1
1
 Department of Otorhinolaryngology, Head and Neck Surgery, Hannover Medical School, Hannover, Germany; 2 Cluster of
Excellence “Hearing4all” of the German Research Foundation, Hannover Medical School, Hannover, Germany; 3 UOSD Audiologia,
Fondazione IRCCS Ca’ Granda Ospedale Maggiore, Milan, Italy


SUMMARY
Objective. The goal of the study was to determine whether the level of OTOLIN-1, a pro-
tein whose expression is highly restricted to the inner ear,is increased in the body fluids of
patients with inner ear disorders in comparison to healthy subjects.
Methods. In the preliminary part of the study, OTOLIN-1 levels were measured in the
serum, urine, and saliva of patients with an acute onset of Ménière´s disease and in healthy
individuals. Subsequently, only serum OTOLIN-1 levels were taken into account and were
compared between patients with acute onset of Ménière´s disease, sudden hearing loss,
vestibular neuritis and healthy subjects.
Results. The most reliable diagnostic parameter was OTOLIN-1 levels in serum. Serum
samples of patients with Ménière’s disease and sudden hearing loss showed significantly
higher OTOLIN-1 levels than those from healthy individuals. In addition, there was no
significant difference between the serum concentration of OTOLIN-1 in patients with ves-
tibular neuritis and the control group.
Conclusions. Serum levels of OTOLIN-1 can potentially be used as a biomarker for acute
onset of inner ear disorders due to its significant increase in patients with acute Meniere´s       Received: March 17, 2022
                                                                                                    Accepted: August 1, 2022
disease and sudden hearing loss in comparison to healthy individuals.
KEY WORDS: Ménière disease, OTOLIN-1, sudden hearing loss, biomarker, inner ear                     Correspondence
                                                                                                    Emilio Avallone
                                                                                                    Department of Otorhinolaryngology, Head and
RIASSUNTO                                                                                           Neck Surgery, Hannover Medical School, Carl
Obiettivo. Scopo principale dello studio è stato determinare se il livello di OTOLIN-1, una         Neuberg-Str. 1, 30625 Hannover, Germany.
proteina la cui espressione è altamente ristretta all’orecchio interno, aumenti nei fluidi          Tel. +49 5115323032
corporei di pazienti con diversi disturbi dell’orecchio interno, rispetto a soggetti sani.          E-mail: avallone.emilio@mh-hannover.de
Metodi. Nella parte preliminare dello studio, la concentrazione di OTOLIN-1 è stata
misurata nel siero, urina e saliva di pazienti con esordio acuto della malattia di Ménière            How to cite this article: Avallone E, Schmitt H,
e di soggetti sani. In seguito, solo i livelli di OTOLIN-1 nel siero sono stati considerati e         Lilli G, et al. A potential serological biomarker
paragonati in pazienti con malattia di Ménière, ipoacusia improvvisa, neurite vestibolare             for inner ear pathologies: OTOLIN-1. Acta
                                                                                                      Otorhinolaryngol Ital 2022;42:364-371. https://
e soggetti sani.                                                                                      doi.org/10.14639/0392-100X-N2074
Risultati. Il parametro diagnostico più affidabile è stato il livello sierico di OTOLIN-1. La
concentrazione sierica di OTOLIN-1 nei pazienti affetti da malattia di Ménière e ipoacusia          © Società Italiana di Otorinolaringoiatria
improvvisa era significativamente più alta rispetto agli individui sani. I livelli della proteina   e Chirurgia Cervico-Facciale
nel siero dei pazienti con neurite vestibolare e del gruppo di controllo non differivano
significativamente.                                                                                                OPEN ACCESS
Conclusioni. La concentrazione sierica di OTOLIN-1 può essere potenzialmente utilizzata             This is an open access article distributed in accordance with
come un biomarcatore per i disturbi dell’orecchio interno, dato l’incremento significativo          the CC-BY-NC-ND (Creative Commons Attribution-Non-
                                                                                                    Commercial-NoDerivatives 4.0 International) license. The
in pazienti con malattia di Ménière e ipoacusia improvvisa rispetto a individui sani.               article can be used by giving appropriate credit and mentio-
                                                                                                    ning the license, but only for non-commercial purposes and
PAROLE CHIAVE: malattia di Ménière, OTOLIN-1, ipoacusia improvvisa, biomarker,                      only in the original version. For further information: https://
orecchio interno                                                                                    creativecommons.org/licenses/by-nc-nd/4.0/deed.en




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                                                                                             OTOLIN-1 as an inner ear damage biomarker




Introduction                                                        The MD group included patients with probable or definite
                                                                    MD in its acute onset. According to the AAO-HNS crite-
The cellular and molecular structures of the inner ear are dif-
                                                                    ria 11, probable MD is diagnosed in presence of one defini-
ficult to assess due to its anatomy and delicacy. Currently, di-
                                                                    tive episode of vertigo lasting 20 minutes or longer, plus
agnoses of inner ear pathologies such as sudden hearing loss
                                                                    hearing loss, tinnitus, or aural fullness in the affected ear.
(SHL), Ménière’s disease (MD), or benign paroxysmal posi-
                                                                    Definite MD is diagnosed in presence of two or more spon-
tional vertigo (BPPV) is based on a classic diagnostic battery
                                                                    taneous episodes of vertigo lasting 20 minutes or longer,
that requires clinical assessment combined with audiological
                                                                    documented hearing loss and tinnitus or aural fullness. Ad-
examinations and imaging studies. In recent years, there is
                                                                    ditionally, these patients underwent electrocochleography
a growing interest in identifying inner ear biomarkers that
                                                                    test and/or glycerol dehydration test. Exclusion criteria
can potentially simplify the diagnosis of inner ear diseases 1.
                                                                    were a history of vestibular neuritis, ear operations and de-
Advances in the understanding of the inner ear biology have
                                                                    compensated hypertension.
led to the identification of several proteins that are specifi-
                                                                    For VN patients, the following inclusion criteria were
cally expressed in the cells of the cochlea and vestibule, and
                                                                    applied: sudden onset of severe dizziness, spontaneous
thereby represent promising inner ear biomarkers 1. Among
                                                                    horizontal-torsional nystagmus at the vestibular
these, OTOLIN-1 is a collagen-like protein that serves as
                                                                    examination, abnormal head impulse test, nausea,
an organic scaffold for the deposition of calcium carbonate
                                                                    vomiting, head shaking nystagmus, positive Romberg
into otoconia 2-4. It is expressed in the supporting cells of the
                                                                    test, positive Unterberger test and positive caloric test.
vestibular maculae and cristae, in the tectorial membrane, in
                                                                    Exclusion criteria were a history of MD, ear operations,
marginal cells of the stria vascularis, and in Claudius cells 2.
                                                                    presence of neurological symptoms and absence of acute
Recent studies have shown that OTOLIN-1 is detectable in
                                                                    hearing loss.
serum and that its level significantly increases in patients af-
fected by BPPV in comparison to healthy subjects 5,6, during
                                                                    Diagnostic procedures
aging 7, and after ear surgery 8. Therefore, it has the charac-
                                                                    All audiometry procedures were performed in soundproof
teristics of a biomarker, since it is specific for an organ, is
                                                                    rooms. Pure-tone audiometry was performed with a com-
easily accessible and measurable and can differentiate be-
tween healthy subjects and patients.                                puter audiometry system (Audio-DATA, CAS AD2117) at
The first aim of the study was to expand the range of body          frequencies from 0.125 to 8 kHz for air conduction hearing
fluids where OTOLIN-1 can reliably be detected by analyz-           thresholds and from 0.250 to 6 kHz for bone conduction
ing its presence in saliva, urine and serum and to choose           hearing thresholds.
the most appropriate fluid for the further analysis. The sec-       Electrocochleography (ECochG) was performed using a
ond aim was to determine whether additional pathologies,            needle electrode positioned at the promontory. The outer
such as SHL, MD and vestibular neuritis (VN), cause an              hair cells were stimulated with the use of clicks. The pro-
increase in OTOLIN-1 level in the fluid of choice in com-           cedure was performed using a Natus CareFusion Nicolet
parison to healthy controls and thus assess its specificity for     EDX device. The summating potential (SP) and the action
inner ear disorders.                                                potential of the auditory nerve (AP) were detected. A SP/
                                                                    AP limit was set to 0.30-0.40. Values exceeding this limit
                                                                    suggest endolymphatic hydrops 12-14.
Materials and methods                                               The glycerol dehydration test was performed by adminis-
Patients                                                            tering 1.5 mg/kg glycerol for body weight orally early in
The study consisted of four patient groups divided as fol-          the morning and after fasting. Three consecutive pure-tone
lows: 20 with acute onset of MD (mean age 51.2 years), 20           audiometry tests were performed after the administration.
with SHL (mean age 48.8 years), 18 with VN (mean age                The glycerol test was considered positive if the hearing
58.1 years) and 17 healthy controls (mean age 45.8 years).          threshold rose by at least 10 dB in three consecutive fre-
The healthy control group consisted of individuals with no          quencies.
previous history of MD, SHL, VN, BPPV, or ear operations.           Head shaking nystagmus test was performed using a chair
Inclusion criteria for SHL patients were pure-tone audiom-          with the patient sitting upright in front of the examiner. The
etry with an increase in the threshold of at least 30 dB in         clinician tilted the patient’s head down 30° and rotated on
three consecutive frequencies and with the onset of symp-           the horizontal plane in both directions 30 times. The pa-
toms within 72 hours 9,10. Exclusion criteria were the ab-          tient was asked to keep his eyes closed during the proce-
sence of an acute vertigo attack, previous history of MD            dure and was asked to open them at the end of the rotations.
and ear operations.                                                 Eye movements were observed with Frenzel’s glasses. The



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E. Avallone et al.




presence of three or more nystagmus beats indicated ves-          Results
tibular impairment.
                                                                  Quantification of OTOLIN-1 in body fluids
Romberg test was performed with the patient standing up,
                                                                  In the preliminary part of the study, the concentration of
with the feet together in parallel position, the arms along
                                                                  OTOLIN-1 was measured in the serum, urine and saliva of
the body and the eyes closed for one minute. If unsteadi-
                                                                  10 patients with acute MD, as an example of inner ear dis-
ness occurs, the test was positive.
                                                                  ease, and in 10 healthy individuals. In serum (Fig. 1A), the
For the Unterberger test, the patients marched in place with
                                                                  concentration of OTOLIN-1 ranged from 60.5 to 893.3 pg/
closed eyes, raising their knees up to approximately 45°,
                                                                  ml with a mean concentration of 502.2 ± 240.2 pg/ml in
performing 50 steps (one per second) with their arms ex-
                                                                  patients with MD (n = 9, one patient refused blood sam-
tended in front of them at 90°. Patients who showed a right
                                                                  pling), and from 26.7 to 367.9 pg/ml with a mean value
or left rotation equal or superior to 45° were classified as
                                                                  of 145.5 ± 109.2 pg/ml in control subjects (n = 10). Pa-
having a change in dynamic balance 15.
                                                                  tients with MD had a higher level of OTOLIN-1 in serum
Caloric nystagmus test was performed using a Hortmann
                                                                  in comparison to healthy subjects and the difference was
Video-CNG-Analyser videonystagmography system (Oto-
                                                                  statistically significant as shown by Mann Whitney test
metrics Natus Medical Denmark ApS, Taastrup, Denmark).            (p = 0.0007; p < 0.05). In saliva (Fig. 1B), the concentra-
Both ears were irrigated with 50 ml of water at 30 °C or          tion of OTOLIN-1 ranged from 17.2 to 1844.2 pg/ml with
44 °C for 30 seconds. During the procedure, patients re-          a mean value of 809.5 ± 547.2 pg/ml in patients with MD
mained supine with a 30° elevation of the upper body. Nys-        (n = 10), and from 611.9 to 1470.5 pg/ml with a mean value
tagmus was evaluated by the mean maximal slow phase               of 1046.0 ± 277.2 pg/ml in healthy individuals (n = 10).
velocity after warm and cool irrigation over 30 seconds.          Overall, in saliva OTOLIN-1 was more concentrated than
In our laboratory, caloric asymmetry higher than 20% after        in serum. In contrast, OTOLIN-1 was less concentrated in
warm and cool irrigation is defined as canal paresis.             urine (Fig. 1C). In patients with MD, it ranged from 0 to
                                                                  66.9 pg/ml, with the exception of one patient (1750 pg/
Biochemical analysis                                              ml), and in healthy subjects it ranged from 0 to 91.4 pg/
Blood, urine and saliva were collected from patients at           ml, with the exception of one individual (947.1 pg/ml). The
admission to the hospital. The blood was centrifuged at           average OTOLIN-1 level in urine of MD patients (n = 10)
1500 xg for 20 minutes and the supernatant (serum) was            and control subjects (n = 10) were 183.1 ± 550.9 pg/ml
stored at -20 °C, together with urine and saliva, until further   and 100.7 ± 299.5 pg/ml, respectively. In the saliva and
analysis. Human OTOLIN-1 was quantified by a highly               urine, there was no significant difference between patients
sensitive enzyme-linked immunosorbent assay (ELISA) kit           with MD and control subjects (p = 0.07; p > 0.05, p = 0.12;
(MyBiosource.com, San Diego, CA), following the manu-             p > 0.05 respectively). Due to the wide scatter of OTO-
facturer’s instructions. Serum samples were assayed in du-        LIN-1 levels in saliva and the low concentration of the pro-
plicate.                                                          tein in urine in both MD patients and control subjects, it
                                                                  was decided to monitor OTOLIN-1 only in serum samples
Statistical analysis                                              in further experiments.
Data storage and statistical analysis was performed
with Microsoft® Office Excel 2010 (Microsoft Corp.,               Quantification of OTOLIN-1 in serum of patients with MD,
Redmond, Wash.), GraphPad Prism 9.0.0 (121)                       SHL, VN and healthy controls
(GraphPad Software, San Diego, CA) and SigmaPlot                  In the second part of the study, OTOLIN-1 serum levels
14.5 (Systat Software, San Jose, CA) using a 5% criterion         in patients with two different inner ear disorders (MD and
for statistical significance. The Mann Whitney test was           SHL) and with retro-cochlear injury (VN) were compared
used for statistical comparison between two groups.               with healthy subjects (Fig. 2). The average concentration
A Spearman’s correlation test was applied to measure              of OTOLIN-1 was 439.1 ± 318.5 pg/ml in patients with
the correlation between OTOLIN-1 serum levels and                 MD (n = 20), 299.5 ± 224.1 pg/ml in patients with SHL
degree of hearing loss and SP/AP values. To quantify              (n = 20), 206.1 ± 136.3 pg/ml in patients with VN (n = 18)
the sensitivity and specificity of serum OTOLIN-1 in              and 171.6 ± 107.0 pg/ml in healthy individuals (n = 17).
distinguishing patients with MD or SHL from healthy               OTOLIN-1 was significantly higher in patients with MD
subjects, a Receiver-operating Characteristic (ROC)               in comparison to control subjects as shown by Mann
was performed. The level of significance was set at               Whitney test (p = 0.0003; p < 0.05), and to patients with
p < 0.05.                                                         VN (p = 0.001; p < 0.05). OTOLIN-1 was also significantly



366
                                                                                                                 OTOLIN-1 as an inner ear damage biomarker




  A                                                   B                                              C




 *** Indicate significant difference calculated by Mann Whitney test (p < 0.001)


Figure 1. OTOLIN-1 levels in serum (A), saliva (B) and urine (C) of Ménière’s disease patients (MD) and healthy controls. Columns and error bars represent mean
values ± SD. Individual data are shown with dots.


                                                                                   higher in patients with SHL than in the healthy individuals
                                                                                   (p = 0.032; p < 0.05). There was no difference between pa-
                                                                                   tients with VN and the control subjects (p = 0.27; p > 0.05).
                                                                                   The data showed a significantly higher level of OTOLIN-1
                                                                                   in MD and SHL patients in comparison to control individu-
                                                                                   als. Additionally, to have a preliminary estimate of the po-
                                                                                   tential of serum OTOLIN-1 in distinguishing patients with
                                                                                   MD or SHL from healthy individuals, ROC analysis was
                                                                                   performed. The optimal cut-off value to discriminate MD
                                                                                   patients and control subjects was 243 pg/ml, which had a
                                                                                   sensitivity of 80.0%, a specificity of 76.5% and an area un-
                                                                                   der the curve of 0.82 (95% CI: 0.675-0.966). For the pre-
                                                                                   diction of SHL, the optimal cut-off value was 196 pg/ml,
                                                                                   which had a sensitivity of 60.0%, a specificity of 64.7%,
                                                                                   and an area under the curve of 0.68 (95% CI: 0.505-0.853).
                                                                                   To evaluate the potential of serum OTOLIN-1 as a common
                                                                                   biomarker for inner ear diseases, ROC analysis was per-
                                                                                   formed comparing the serum OTOLIN-1 values of both the
 ** Indicate significant difference calculated by Mann Whitney test (p < 0.05)     MD and SHL patients with healthy controls. The optimal
                                                                                   cut-off value to discriminate between patients with inner
Figure 2. OTOLIN-1 levels in the serum of Ménière’s disease patients (MD),         ear disorders and control subjects was 222 pg/ml, which
patients with sudden hearing loss (SHL), patients with vestibular neuritis (VN),
and healthy controls. Columns and error bars represent mean values ± SD.           had a sensitivity of 62.5%, a specificity of 70.6%, and an
Individual data are shown with dots.                                               area under the curve of 0.75 (95% CI: 0.625-0.875).




                                                                                                                                                         367
E. Avallone et al.




Relationship between OTOLIN-1 and audiological                                        described to increase with aging 7. To verify this
parameters in MD and SHL patients                                                     information in our cohort of subjects, OTOLIN-1 serum
It was investigated whether there was a correlation between the                       levels of the control group were matched with age. The
severity of hearing loss and OTOLIN-1 levels in MD and SHL                            control individuals were subdivided into three groups: 20-
patients. Hearing loss was evaluated based on pure tone audi-                         40 years old, 41-60 years old, > 60 years old (Fig. 3A).
ometry. For MD patients, the pure tone average was evaluated                          Mean OTOLIN-1 values were 137.1 ± 286.4 pg/ml for the
for lower frequencies, due to low-tone hearing loss caused by                         20-40 year group (n = 6), 202.9 ± 161.2 pg/ml in those
the disease (Tab. I). The frequencies considered were between                         41-60 year (n = 7), and 168.7 ± 312.1 pg/ml for controls
125 Hz and 1000 Hz, and the pure tone average for MD pa-                              > 60 years (n = 4). The youngest group showed a tendency
tients, referred to as low tone PTA (ltPTA) in this work, had a                       towards lower OTOLIN-1 levels in serum in comparison to
mean value of 44.29 dB. For SHL patients, the pure tone aver-                         the older groups. However, these differences were not sta-
age (PTA) was measured for frequencies between 500 Hz and                             tistically significant as determined by Mann Whitney test
2000 Hz (Tab. I) and had a mean value of 52.25 dB. The poten-                         between the groups 20-40 and 41-60 (p = 0.18; p > 0.05)
tial correlation between ltPTA and OTOLIN-1 in MD patients                            and between the group 20-40 and > 60 (p = 0.39; p > 0.05).
and PTA and OTOLIN-1 in SHL subjects was tested with a                                To investigate the relationship between OTOLIN-1 and age
Spearman’s correlation test that gave, respectively, rs = -0.016                      in patients with MD and SHL, that were shown (Fig. 2) to
(p = 0.94; p > 0.05) and rs = 0.27 (p = 0.24; p > 0.05), indicat-                     significantly differentiate from controls as far as OTOLIN-1
ing no association between the variables. Additionally, it was                        levels is concerned, patients were divided into the three
tested whether there was a relationship between the hydrops                           age groups described above. In MD subjects (Fig. 3B), the
index, SP/AP and OTOLIN-1 levels in MD patients. No cor-                              protein mean values were 484.6 ± 266.9 pg/ml for the 20-
relation could be found between protein levels and the SP/AP                          40 year group (n = 7), 298.6 ± 129.2 pg/ml for those 41-
coefficient, rs = -0.15 (p = 0.56; p > 0.05).                                         61 years (n = 6), and 514.1 ± 455.4 pg/ml for > 60 years
                                                                                      (n = 7). No significant differences were found between the
Relationship between OTOLIN-1 and age of subjects                                     younger and older patients as determined by Mann Whit-
OTOLIN-1 serum concentration has previously been                                      ney test between the groups 20-40 and > 60 (p = 0.40;


Table I. Audiologic diagnostic values in Ménière’s disease patients (MD) and patients with sudden hearing loss (SHL).
 MD patients         Age             Sex            ltPTA 1        ECohG 2      Glycerol Test SHL patients           Age     Sex         PTA 3
    1                      F                 41              42                  /       +            1             55        M           90
    2                      F                 70              55                0.56      /            2             19        M          6.67
    3                      F                 68              70                  /       +            3             62        M         116.67
    4                      F                 32              50                0.32      -            4             50        M          8.33
    5                      M                 23              42                0.55      +            5             41        F          115
    6                      M                 55              47                0.71      +            6             76        F         113.33
    7                      F                 65               8                0.26      +            7             57        M         23.33
    8                      F                 49              63                0.23      +            8             81        F           30
    9                      F                 78             36.25              0.42      -            9             22        M           50
    10                     F                 84             42.50              0.59      /           10             62        M          120
    11                     F                 33             67.50              0.63      -           11             38        M         11.67
    12                     F                 27             22.50              0.53      +           12             41        F         11.68
    13                     F                 71             52.50              0.39      +           13             36        M         28.33
    14                     M                 33             42.50              0.55      +           14             67        F         21.67
    15                     F                 33             38.75                /       +           15             56        F           45
    16                     F                 55              35                0.62      /           16             56        F         41.67
    17                     M                 20             22.50              0.57      -           17             19        M           30
    18                     M                 49             38.75              0.51      +           18             19        F         63.33
    19                     F                 58             28.75              0.34      -           19             51        F         63.33
    20                     M                 66             81.25              0.68      +           20             68        M           30
1
    Low tone pure tone average; 2 Electrocochleography; 3 Pure tone average.




368
                                                                                                             OTOLIN-1 as an inner ear damage biomarker




   A                                                 B                                                   C




Figure 3. OTOLIN-1 levels in healthy subjects (A), MD (B) and SHL (C) divided in different age groups.


p > 0.05) and between the groups 41-60 and > 60 (p = 0.22;                       damaged glomerular membrane as a consequence of diabetic
p > 0.05). In SHL patients (Fig. 3C), mean OTOLIN-1                              nephropathy 16. Even though we showed that OTOLIN-1 is
values were 458.5 ± 330.2 pg/ml for the 20-40 year group                         detectable in saliva and urine, the sampling drawbacks of
(n = 4), 278.5 ± 212.7 pg/ml for those 41-61 years (n = 10),                     these two body fluids lead us to conclude that OTOLIN-1 in
and 368.5 ± 129.5 pg/ml for > 60 years (n = 6). Although                         serum is the most reliable diagnostic parameter.
younger SHL patients had, in tendency, higher values of                          We demonstrated that the serum concentration of OTO-
OTOLIN-1, no significant differences were found, as de-                          LIN-1 in MD and SHL patients was significantly higher
termines by Mann Whitney test between the groups 20-40                           than in the control group. In particular, OTOLIN-1 serum
and > 60 (p = 0.08; p > 0.05) and between the groups 41-60                       levels were highest in MD patients. On the other hand,
and > 60 (p = 0.18; p > 0.05).                                                   OTOLIN-1 levels in VN patients was not significantly dif-
                                                                                 ferent from controls. Taking into account that OTOLIN-1
Discussion                                                                       has been shown to increase as a consequence of BPPV 5,6
                                                                                 and after mastoidectomy 8, we suggest that OTOLIN-1 lev-
One of the characteristics of an effective biomarker is easy                     els correlate positively with isolated damage to the inner
sampling. As shown previously 5-8, OTOLIN-1 is detectable                        ear.
in serum and requires a blood withdrawal. In contrast, the                       Pathologies involving mainly retro-cochlear structures,
collections of saliva and urine are easier to obtain and are                     such as the VN, may not further alter the serum level of the
not invasive. In our study, we tested the possibility to detect
                                                                                 protein. From our results, we propose a new hypothesis on
OTOLIN-1 in these two additional body fluids. OTOLIN-1
                                                                                 the pathophysiological construct of the inner ear, in which
levels in saliva of healthy subjects and MD patients showed
                                                                                 a pathological event resulting in inner ear damage releases
high heterogeneity. This observation can be explained by
                                                                                 a large amount of OTOLIN-1 into the bloodstream. The ex-
the different volume of saliva produced by individuals, for
                                                                                 act mechanism of OTOLIN-1 release in the bloodstream is
example, dehydrated subjects have less saliva leading to
                                                                                 yet to be elucidated. A possible explanation could be the in-
a more concentrated protein in the sample. On the other
                                                                                 crease of the physiological filtration of OTOLIN-1 through
hand, the concentration of OTOLIN-1 in urine was very
                                                                                 the blood-labyrinthine barrier that also occurs in healthy
low in all groups, with the exception of two individuals. The
                                                                                 subjects. Although serum OTOLIN-1 levels increased in
protein has a molecular weight of 70 kDa that exceeds the
                                                                                 MD and SHL patients, we did not find a positive correlation
renal glomerular filtration capacity, and for this reason it is
                                                                                 between this level and severity of hearing loss. We also did
retained in blood and is barely detectable in urine. The two
                                                                                 not find a correlation between the extent of endolymphatic
individuals with high OTOLIN-1 urine levels may have a
                                                                                 hydrops, measured by the SP/AP ratio, and serum level of



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the protein in MD subjects. We conclude that OTOLIN-1             as monitoring inner ear damage in patients undergoing
serum concentrations can be used in diagnosis of inner ear        chemotherapy or ototoxic antibiotic therapy. Additionally,
diseases, but that it does not provide information about the      it could be used as a supplementary diagnostic aid in the
severity of disease.                                              evaluation of patients with specific audio-vestibular symp-
Additionally, the relationship between OTOLIN-1 levels            toms following head trauma to assess damage to the laby-
and age was investigated. OTOLIN-1 serum levels have              rinth. Likewise, it could be a marker of specific injury after
been reported to increase with increasing age of healthy          otologic surgery. It would be interesting to investigate its
subjects 7. Our data showed that in tendency, OTOLIN-1            possible prognostic value by examining the serum levels
levels were higher in the serum of older healthy subjects         of the protein during follow-up of patients with inner ear
in comparison to younger ones, but statistical significance       diseases and correlate these with the clinical picture; in par-
was not reached. We have also investigated the relation-          ticular, this would allow to evaluate the effects of therapies.
ship between OTOLIN-1 levels and age in our cohort of             A limitation of the study is the small number of individuals
patients. In contrast to healthy subjects, in SHL younger         enrolled, which allowed only preliminary evaluation of the
patients OTOLIN-1 levels tended to be higher than in older        potential of OTOLIN-1 as a biomarker for inner ear dis-
subjects, whereas no tendency and no significant differ-          orders. Furthermore, it was not possible to know whether
ences could be found in individuals with MD. While aging          some of our patients had an acoustic neuroma instead of
might increase OTOLIN-1 levels in control subjects, it does
                                                                  only an inner ear disorder, because it was not always possi-
not seem to play the same role in patients with inner ear
                                                                  ble to obtain the results of the suggested MRI from patients
disorders or, alternatively, the aging effect is masked by the
                                                                  and we had no information from the local ENT physicians.
pathology.
                                                                  However, our study is the first in which OTOLIN-1 has
To properly define the potential clinical applications of
                                                                  been detected in other body fluids other than serum. In ad-
OTOLIN-1, it is necessary to determine age-specific
                                                                  dition, we showed that the serum level increases in MD and
thresholds of protein serum levels. Although our study is a
small-scale preliminary investigation, we sketched poten-         SHL in comparison to healthy subjects and VN patients.
tial serum OTOLIN-1 cut-off values that can be used to dif-       Preliminary cut-off values were sketched out. A further as-
ferentiate MD or SHL affected subjects from healthy indi-         pect of our study that has never been previously considered
viduals. The cut-off value for diagnosing MD appears to be        is the investigation of the correlations between OTOLIN-1
particularly reliable, having high sensitivity and specificity.   and the entity of hearing loss, as well as between the pro-
The threshold to differentiate SHL patients from healthy          tein and hydrops. We hope that the growing interest in this
subjects has a good compromise of sensitivity and speci-          area of otology will lead to further studies in order to in-
ficity. As the serum OTOLIN-1 levels of MD patients are           crease the sample size, thus enabling more accurate cut-off
much higher than for SHL patients, it is clear that a higher      values to be calculated.
cut-off value is needed to discriminate MD patients.
In our work, we evaluated OTOLIN-1 as a common marker             Conclusions
for inner ear diseases. We estimated a cut-off value that
provides a preliminary indication of the potential of OTO-        Our study provides evidence that OTOLIN-1 can be used
LIN-1 in distinguishing healthy and inner ear disorders af-       as a serum marker for inner ear damage. In the foreseeable
fected subjects. Further studies with a higher number of          future, OTOLIN-1 can potentially be applied as an addi-
controls and patients will be necessary to calculate more         tional examination tool to improve the specificity of the di-
reliable cut-off ranges of the protein in healthy subjects and    agnostic process in inner ear diseases, for example in MD.
the thresholds to be applied for different inner ear patholo-     Furthermore, it could be implemented to assess damage to
gies, especially in relation to the age of the patient.           the inner ear following procedures such as ear surgeries,
Our results showed that OTOLIN-1 appears to be a prom-            use of ototoxic drugs, or a traumatic event resulting in a
ising biomarker for the diagnosis of MD. This opens up            labyrinthine concussion.
a number of opportunities for differential diagnosis, for
example in patients with acute vertigo, allowing subjects         Acknowledgements
with MD to be differentiated from individuals with VN or          We thank Chiara Baccolini for valuable advice in analysing
neurological conditions (e.g. multiple sclerosis, vestibular      the data and writing the manuscript.
migraine or stroke).
The potential use of OTOLIN-1 as a biomarker for inner            Conflict of interest statement
ear disorders led us to envisage several applications, such       The authors declare no conflict of interest.



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Funding                                                                        4
                                                                                    Murayama E, Takagi Y, Nagasawa H. Immunohistochemical localiza-
                                                                                    tion of two otolith matrix proteins in the otolith and inner ear of the
This research was funded by the Cluster of Excellence                               rainbow trout, Oncorhynchus mykiss: comparative aspects between
of the German Research Foundation (DFG; “Deutsche                                   the adult inner ear and embryonic otocysts. Histochem Cell Biol
Forschungsgemeinschaft”) EXC 2177/1 “Hearing4all”,                                  2004;121:155-166. https://doi.org/10.1007/s00418-003-0605-5
and the Hannover Medical School, Hannover, Germany.
                                                                               5
                                                                                    Parham K, Sacks D, Bixby C, et al. Inner ear protein as a biomarker
                                                                                    in circulation? Otolaryngol Head Neck Surg 2014;151:1038-1040.
                                                                                    https://doi.org/10.1177/0194599814551127
Author contributions                                                           6
                                                                                    Sacks D, Parham K. Preliminary report on the investigation of
EA conceived and designed the study, carried out the sam-                           the association between BPPV and osteoporosis using biomark-
pling, the data analysis and wrote the manuscript. HS per-                          ers. Otol Neurotol 2015;36:1532-1536. https://doi.org/10.1097/
                                                                                    MAO.0000000000000853
formed the biochemical analysis and contributed with the                       7
                                                                                    Tabtabai R, Haynes L, Kuchel GA, et al. Age-related increase in blood
data analysis. GL helped with sample collection and the                             levels of Otolin-1 in humans. Otol Neurotol 2017;38:865-869. https://
design of the study. AW and AL-S gave a valuable contribu-                          doi.org/10.1097/MAO.0000000000001426
tion in reviewing the manuscript. TL and KW designed the                       8
                                                                                    Dogan M, Sahin M, Kurtulmus Y. Otolin-1, as a potential marker for
                                                                                    inner ear trauma after mastoidectomy. J Int Adv Otol 2019;15:200-
study and reviewed the manuscript.                                                  203. https://doi.org/10.5152/iao.2019.5155
                                                                               9
                                                                                    Gurgel RK, Jackler RK, Dobie RA, et al. A new standardized format
Ethical consideration                                                               for reporting hearing outcome in clinical trials. Otolaryngol Head Neck
The ethics approval was given by the Hannover Medical                               Surg 2012;147:803-807. https://doi.org/10.1177/0194599812458401
School Ethics Committee (approval no. 3231-2016 and no.
                                                                               10
                                                                                    Chandrasekhar SS, Tsai Do BS, Schwartz SR, et al. Clinical practice
                                                                                    guideline: sudden hearing loss (update). Otolaryngol Head Neck Surg
8199-2018).                                                                         2019;161:S1-S45. https://doi.org/10.1177/0194599812436449
The research was conducted ethically, with all study pro-                      11
                                                                                    Committee on Hearing and Equilibrium Guidelines for the Diag-
cedures being performed in accordance with the require-                             nosis and Evaluation of Therapy in Meniere’s Disease. Otolaryn-
                                                                                    gol Head Neck Surg 1995;113:181-185. https://doi.org/10.1016/
ments of the World Medical Association’s Declaration of                             S0194-5998(95)70102-8
Helsinki.                                                                      12
                                                                                    Moon IJ, Park GY, Choi J, et al. Predictive value of electroco-
Written informed consent was obtained from each partici-                            chleography for determining hearing outcomes in Meniere’s dis-
pant/patient for study participation and data publication.                          ease. Otol Neurotol 2012;33:204-210. https://doi.org/10.1097/
                                                                                    MAO.0b013e318241b88c
                                                                               13
                                                                                    Gibson WP, Prasher DK, Kilkenny GP. Diagnostic signifi-
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