A Preliminary Report on the Investigation of Prestin as a Biomarker for Idiopathic Sudden Sensorineural Hearing Loss

Abstract

To date, no specific biomarkers for idiopathic sudden sensorineural hearing loss (ISSHL) have been used. The aim of this study is to investigate whether prestin, the motor protein of cochlear outer hair cells, could be used as a biomarker candidate for the diagnosis and prognosis judgement of ISSHL. Blood samples of 14 ISSHL patients and 28 control patients without history of hearing loss were collected. Plasma prestin concentration was measured using Human Prestin (SLC26A5) ELISA Kit. The results showed that prestin was detectable in the plasma of all patients and the concentration of prestin was significantly higher in ISSHL patients with about half being above the average range of control patients. Moreover, in treatment responsive group, 6 of 10 patients had decreased prestin levels after treatment compared to those of before treatment, while the prestin levels of all the 4 patients in treatment unresponsive group increased in varying degrees. Our promising preliminary results suggest that prestin has the potential to serve as a biomarker to assist diagnosis and judge response to pharmacological treatments.

Keywords

prestin idiopathic sudden sensorineural hearing loss biomarker diagnosis

Introduction

Biomarkers help increase the accuracy of diagnosis, characterize disease, aid prognosis, predict response to treatment, and guide treatment. Currently, there are about 30 serum biomarkers that are routinely used in clinical oncology, for example, prostate-specific antigen for prostate cancer and cancer antigen (CA)-125 for ovarian cancer.¹ Unfortunately, there is no applicable serum biomarker that can be used for early diagnosis or management of inner ear disorders. In 2015, Parham hypothesized that outer hair cell (OHC)–specific protein, prestin, might be a biochemical marker for early detection of acquired sensorineural hearing loss.² He successfully verified his hypothesis using experimental animal models of noise-induced hearing loss and cisplatin ototoxicity.^3,4 However, so far there has been no data in human being to conform the theory. Idiophathic sudden sensorineural hearing loss (ISSHL) is a common inner ear disease characterized by hearing loss, and OHC damage is considered to be one of the possible mechanisms. Therefore, we hypothesized that the plasma concentration of prestin will increase in some of the patients with ISSHL at a certain stage after the onset of the disease. In the present study, we collected blood samples of patients with ISSHL and those of control patients without history of hearing loss. Human Prestin (SLC26A5) ELISA Kit was used to measure plasma concentration of prestin. The difference in prestin concentration between patients with ISSHL and control patients was compared, and the difference between responding patients and nonresponding patients after treatment was also analyzed.

Patients and Methods

This study was approved by the institutional review board of affiliated hospital of Jiangnan University and Wuxi People’s Hospital affiliated to Nanjing Medical University. Plasma concentration of prestin from 14 patients of ISSHL and 24 control patients without history of hearing loss was measured in this study. The age and sex ratio of the 2 groups were similar (Table 1). Plasma prestin concentrations of ISSHL were measured before and after treatment, respectively.

Table 1.

Clinical Characteristics of Patients With Sudden Sensorineural Hearing Loss and Normal Controls.

Patients with idiopathic sudden sensorineural hearing loss (n = 14)
Age, years	Mean (SD)	57.9 (15.4)
	Range	31-72
Sex	Male:female	7:8
Affected ears	Left:right	6:8
Interval between onset and treatment, days
	Mean (SD)	3.6 (1.4)
Normal controls (n = 24)
Age, years	Mean (SD)	54.0 (9.9)
	Range	33-76
Sex	Male:female	11:13

Abbreviations: SD, standard deviation.

The diagnostic criteria for ISSHL consisted of rapid onset of hearing loss greater than 30 dB in 3 consecutive frequencies occurring within 3 days. Audiometry, history taking, physical examination, and inner ear magnetic resonance imaging or computed tomography were performed to confirm the diagnosis of ISSHL. Patients with recognized causes of sensorineural hearing loss such as Meniere disease or autoimmune hearing loss were excluded. Additionally, pregnant patients and those who had received previous treatment were also excluded.

Patients with ISSHL were hospitalized and treated with the same strategy within 7 days after the onset of the disease. All patients received oral prednisolone 1 mg/kg for 5 days, intravenous ginkgo biloba extract, and intramuscular cobamamide every day during the whole course of treatment. Besides, batroxobin was administered intravenously once every other day according to patient’s coagulation function, which was monitored by blood test. The patients with ISSHL were separated into 2 groups according to pure tone audiometry performed at the end of treatment. According to Siegel criteria⁵, patients with complete recovery, partial recovery, and slight improvement were considered as the treatment responsive group. Patients with no recovery were considered as the treatment unresponsive group (Table 2).

Table 2.

Siegel Criteria and Grouping of ISSHL Patients According to Treatment Results.

	Recovery From Hearing Loss (Siegel Classification)	Explanation
Treatment responsive	Complete recovery	Final hearing level^a better than 25 dB, regardless of the amount of gain.
	Partial recovery	Gained more than 15 dB in hearing and had a final hearing level between 25 and 45 dB.
	Slight improvement	15 dB gain and a final hearing level poorer than 45 dB.
Treatment unresponsive	No improvement	Gain less than 15 dB

Abbreviations: ISSHL, idiopathic sudden sensorineural hearing loss.

^aFinal hearing level: arithmetic mean value of 500, 1000, 2000, and 4000 Hz according to Committee on Hearing and Equilibrium of the American Academy of Otolaryngology—Head and Neck Surgery.

Blood samples were collected using EDTA as an anticoagulant and centrifuged for 15 minutes at 1000×g within 30 minutes of collection. The samples were stored in a –80°C refrigerator until time of assay. Prestin concentration was measured using Human Prestin (SLC26A5) ELISA Kit (MyBioSource.com, San Diego, California) as described in the manufacturer’s instruction manual. A 1:50 dilution was prepared, and the optical density in the wells of the enzyme-linked immunosorbent assay (ELISA) microplate was measured at 450 nm using a KHB ST-360 plate reader (Kehua Bio-engineering Co., Ltd, Shanghai, China).

Results

Prestin was detected in the plasma samples of all patients. Figure 1 shows the distribution of plasma prestin concentration for the 2 groups. In control patients, prestin levels ranged from 85.4 to 1628.25 pg/mL, with an average of 840.24 ± 496.22 (mean [SD]) pg/mL. Among patients with ISSHL, prestin levels before treatment ranged from 295.25 to 9648.80 pg/mL, with an average concentration of 1955.98 ± 2501.48 (mean [SD]) pg/mL, which is significantly higher than that of control patients (Figure 2). Approximately half of patients with ISSHL had levels above the average level of control patients.

Figure 1.

Plasma prestin concentration of patients with idiopathic sudden sensorineural hearing loss (ISSHL) and control patients.

Figure 2.

Comparison of mean plasma prestin levels. Error bars represent SD. ISSHL indicates idiopathic sudden sensorineural hearing loss, SD, standard deviation. ***P < .001 compared to control.

Of the 14 patients with ISSHL, 10 patients were evaluated as treatment responsive and 4 patients as treatment unresponsive (Table 3). Correlation of initial plasma prestin level before treatment with treatment outcome was calculated by Spearman rank sum test. The result showed that plasma prestin levels before treatment in patients with ISSHL did not correlate with treatment outcomes according to the Siegel classification (r = −.327, P = .254). Prestin levels after treatment ranged from 0 to 7610.45 pg/mL, with an average concentration of 1653.26 ± 1967.60 (mean [SD]) pg/mL. In the treatment responsive group, the prestin levels of 6 patients decreased after treatment compared to those of pretreatment, while the prestin levels of all the 4 patients in treatment unresponsive group increased in varying degrees.

Table 3.

Audiologic Data and Plasma Prestin Levels in ISSHL Patients.

Patient Number	PTA Before Treatment, dB	PTA After Treatment, dB	Treatment Outcome (Siegel Criteria)	Sample Time After Onset, Days	Prestin Level Before Treatment, pg/mL	Sample Time After Onset, Days	Prestin Level After Treatment, pg/mL
1	77.5	21.25	Complete recovery	5	347.70	12	0.00
2	77.5	50	Slight improvement	3	2848.42	11	1862.55
3	83.75	51.25	Slight improvement	7	531.10	13	6.65
4	80	35	Partial recovery	5	3831.05	10	7610.45
5	63.75	28.75	Partial recovery	6	9648.80	12	242.85
6	66.25	72.5	No recovery	3	190.35	10	321.50
7	97.5	88.75	No recovery	4	714.35	10	3108.00
8	116.25	117.5	No recovery	2	1471.85	12	2719.60
9	87.5	60	Slight improvement	5	426.35	9	1289.25
10	36.25	20	Complete recovery	4	1080.40	9	1550.05
11	42.5	41.25	No recovery	4	740.55	8	949.75
12	68.75	50	Slight improvement	4	2408.35	15	1769.60
13	105	83.75	Slight improvement	6	295.25	15	400.10
14	80	27.5	Partial recovery	5	2849.15	10	1315.35

Abbreviations: ISSHL, idiopathic sudden sensorineural hearing loss; PTA, pure-tone average.

Discussion

Lack of serum biomarkers has been an impediment to early diagnosis and treatment of inner ear disorders. Recently, Parham et al demonstrated that otolin-1 may be able to act as a serum biomarker for vestibular disease.⁶ Inspired by this finding, he suggested that prestin, the motor protein of cochlear OHCs, may serve as a biomarker for OHC damage based on the following reasons: First, prestin is exclusively expressed in the lateral membrane of the OHCs. Second, after degeneration of OHCs, prestin can be found in phagosomes in the supporting cells which will end up in circulation. Third, prestin is 80 kDa in size which is small enough to cross the blood-labyrinthine barrier and enter circulation. Finally, because of extreme sensitivity of the ELISA, picogram quantities of prestin released into circulation can be detected.²

Idiopathic sudden sensorineural hearing loss is a disease with no known exact cause, generally defined as idiopathic and thought to occur as a result of multifactorial causes. Although the pathophysiology of ISSHL remains unknown, damage of OHCs is thought to be one of the important events that lead to ISSHL, especially in patients with high-frequency hearing loss.⁷ The result of present study showed that prestin can be detected and quantified using ELISA technique in plasma of both patients with ISSHL and control patients without history of hearing loss. Furthermore, the average concentration in ISSHL is much higher than those of control patients. These results can be explanatory. According to the theory of Parham, serum prestin in control patients results in normal homeostatic turnover in the membrane of healthy OHCs. Shortly after OHCs damage, prestin concentration will rise and reach on a peak on some day within 1 week.⁸ However, specificity is an important criterion for an ideal biomarker. Although the average prestin concentration in the ISSHL group was higher than that in the control group, the prestin concentrations in ISSHL group significantly overlapped with those in normal control group. Only half of the patients had higher concentration than the average level of the control group. These results suggested that OHCs damage happened only in part of patients with ISSHL, and prestin may serve as a potential biomarker for adjuvant diagnosis in these patients.

The result of Spearman rank sum test showed that there was no correlation between the prestin values before treatment and treatment outcomes. Considering the small sample size, it is unreasonable to draw a conclusion, and further analysis based on large sample size is needed. Interestingly, we found that the change in prestin plasma concentration after treatment is indicative of treatment response. In the treatment responsive group, 6 of 10 patients’ prestin levels decreased after treatment compared to those of after treatment, while in all the 4 patients with no hearing recovery, prestin levels significantly increased. We hypothesized that when the treatment is effective and pathogenic factors of OHC damage cease, prestin concentration is expected to decrease, while as the treatment is ineffective and pathogenic mechanisms persist, prestin concentration is expected to increase. Similar phenomenon was observed in the study of James Naples who suggested that prestin may serve as a biomarker of cochlear injury that is sensitive to therapeutic interventions in cisplatin ototoxicity.⁹ Therefore, we suggested that changes in plasma prestin level may be a potential indicator of the prognosis of ISSHL.

There are some limitations in this study. First, the sample size was small. Further studies are necessary to observe the reference values in more control patients and changes in prestin level in larger number of patients with ISSHL. Relationships between prestin level and clinical variables of patients with ISSHL also need to be investigated. Second, the plasma prestin concentration and treatment results of patients with ISSHL should be observed for a longer period of time, at least 4 weeks after the end of treatment. Third, because of the different time of treatment after onset, the time of prestin measurement in patients with ISSHL is different. Finally, each sample was measured only once, and few samples had concentrations below the detection limit (7.8 pg/mL) specified in the manufacturer’s instruction manual. Despite all these limitations, the results of our study still indicate prestin may be a potential biomarker for the diagnosis and treatment of ISSHL.

Footnotes

Authors’ Note

Changling Sun and Xiaolin Xuan contributed equally to this study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by the Natural Science Foundation of Jiangsu Province [BK20160195]; Wuxi Young Medical Talents [QNRC054].

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