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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 21  |  Issue : 4  |  Page : 248-253

Expression of 16.2kDa protein in elderly population: A quest for the detection of age related hearing impairment


1 Department of Biochemistry, National Institute of Miners' Health, JNARDDC Campus, Wadi, Nagpur, Maharashtra, India
2 Department of Botany, Cytogenetics, Genetics and Plant Breeding Section, University of Kalyani, Kalyani, West Bengal, India
3 School of Life Sciences, Jawaharlal Nehru Institute of Advanced Studies, Secunderabad, Telangana, India

Date of Web Publication16-Oct-2015

Correspondence Address:
Shubhangi Kailas Pingle
National Institute of Miners' Health, JNARDDC Campus, Wadi, Nagpur - 440 023, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0971-7749.167399

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  Abstract 

Introduction: Age related hearing impairment (ARHI) is the most common sensory deficit in the elderly population and has become a severe social health problem. Several mechanisms have been described for hearing impairment in elderly people. Probable cause of hearing impairment may result from damage in the inner and outer hair cell, loss of stria vascularis, thickening of basilar membrane, or loss of sensory elements in the basal end of the cochlea. The aim of present study is to identify the protein biomarkers associated with ARHI using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Materials and Methods: Total 96 study subjects (48 experimental and 48 controls) were selected for this study. Serum samples were used for biochemical analysis and one-dimensional electrophoresis. Statistical analysis was done by GraphPad software. Results: Glucose, serum creatinine, and urea levels were in normal range and mean urea and creatinine levels are insignificantly associated with ARHI while mean glucose level is significantly associated with ARHI. Smoking/alcohol consumption did not show any significant association with ARHI. The molecular weight of the differentially expressed protein was 16.2 kDa as calculated by gel image analyzer software. The differentially expressed protein may be cochlin tomoprotein (CTP) of 16 kDa which is an isomer of cochlin protein. CTP contains all the known mutation sites associated with deafness. Conclusion: The expression of differentially expressed protein was consistently observed among experimental subjects as compared to control. This protein may help in the prediagnosis of ARHI due to added advantage of enhanced expression in subjects of ARHI. Results revealed males were more prone to ARHI than female.

Keywords: Age related hearing impairment, Cochlin, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Serum protein


How to cite this article:
Thakkar LR, Pingle SK, Kumbhakar DV, Jawade AA, Tumane RG, Soni PN, Jain RK, Jamil K. Expression of 16.2kDa protein in elderly population: A quest for the detection of age related hearing impairment. Indian J Otol 2015;21:248-53

How to cite this URL:
Thakkar LR, Pingle SK, Kumbhakar DV, Jawade AA, Tumane RG, Soni PN, Jain RK, Jamil K. Expression of 16.2kDa protein in elderly population: A quest for the detection of age related hearing impairment. Indian J Otol [serial online] 2015 [cited 2018 Dec 11];21:248-53. Available from: http://www.indianjotol.org/text.asp?2015/21/4/248/167399


  Introduction Top


Age related hearing impairment (ARHI) or prebycusis is defined as a sensorineural hearing loss (SNHL) associated with advanced chronological age. Hearing loss is a gradual and progressive disease. It is the most common sensory deficit which has become a severe social health problem. Individuals aged 60 years and older are more prone to this health condition.[1] ARHI can get aggravated by other factors such as diabetes, reduced blood circulation, exposure to noise, and certain medications.[2] It is more common in males as compared to females. As per World Health Organization, in 2025, there will be 1.2 billion people over 60 years of age worldwide, with more than 500 million individuals who will suffer significant hearing loss from ARHI.[3]

The mechanism of hearing impairment has been studied by several researchers. Keles et al. reported that anatomy of the middle ear is affected much among the geriatric population; however, it doesn't affect the resonance frequency and mechanical conduction of sound. Briefly, hearing loss occurs as the stria vascularis is degenerated, which regulates the bioelectrical stability and metabolic health of the cochlea.[4] Moreover, damage in the inner or outer hair cell, thickening of basilar membrane or loss of sensory elements in the basal end of the cochlea is also a major cause of ARHI.[5] Martines et al. documented in his study that alteration in peripheral and central auditory systems may be one of the major causes of ARHI.[6] Studies in animal model done by Huang revealed that mutation in the cadherin 23 coding Ahl calcium-binding transmembrane protein and POU 4F3 gene can be responsible for ARHI.[7]

Research has been conducted in animal models to study the functions of protein coding genes of prestin, cochlin, connexin, otospiralin, cadherin 23, etc., of the ear. However, limited studies are available on protein biomarkers of human for early detection of ARHI. The study aims to identify complementary strategies of biomarkers studies, based on proteomic predictors of disease susceptibility to ARHI.


  Materials and Methods Top


Selection of study population

In this study, equally 48 subjects were selected from Dhamtari district of Chhattisgarh state as experimental (with hearing impairment) and control (with normal hearing acuity). The selection of experimental subjects was based on confirmed history of hearing disability. Subjects having age more than 60 years were included in this study. Those subjects having a history of diabetes and taking ototoxic drugs were excluded from the study. A standard questionnaire was used to record information on age, sex, height, weight, diet, habits, health history, and medication. The study was explained by the vernacular to the study subjects and written consent was obtained from them.

Collection of blood samples

Blood samples were collected in sodium fluoride (100 μl/ml of blood) tubes for blood glucose analysis. Remaining blood samples were allowed to clot and centrifuged at 1000 rpm for 5 min. Serum samples were stored at −40°C as per accepted procedure. Serum samples were used for biochemical parameters and electrophoresis studies.

Determination of biochemical parameters

The biochemical parameters that are, glucose, urea, and creatinine levels were determined by GOD-POD, Urea Berthelot, and alkaline picrate method using commercially available kits (i.e. beacon diagnostics and precision biotech), respectively. The absorbance was measured at 505, 600, and 510 nm, respectively, for glucose, urea, and creatinine. Each sample was tested in duplicate.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Biotech Yercaud) is a versatile technique widely used for protein separation based on their Mr and charges. The purpose of performing SDS-PAGE was to find a differential expression of proteins among experimental and control subjects. The standard protein markers ranging from 3.5 kDa to 205 kDa were used for molecular mass (Mr) determination (GeNei - Cat. No. 105975). SDS-PAGE was carried out on a discontinuous vertical thick layer with a resolving gel of 5% acrylamide in Tris-HCl buffer at pH 6.8 and a separating gel of 10% acrylamide in Tris-HCl buffer at pH 8.8. Electrophoresis was run in Tris-glycine electrode buffer at a voltage of 150 V. Serum sample and markers were mixed separately with sample buffer, containing 4% SDS, 2% 2-mercaptoethanol, glycerol, 0.02% bromophenol blue (tracking dye), and 0.01 M Tris-HCl buffer at pH 6.8. The mixtures were heated in a boiling water bath for 3 min to denature the proteins. Serum samples (40 µg per lane) were loaded in each well. After electrophoresis, the gel was kept in fixative solution for ½ h then stained with 0.05% Coomassie brilliant blue R-250 stain (Sigma, USA) for 2 h. and transferred into destaining solution until blue background color disappeared, and clear bands were visible. The relative migration distance (Rf) and Mr were determined using gel image analyzer software.

Statistical analysis

The mean and standard deviation was calculated by statistical software (GraphPad Software, Inc., 7825 Fay Avenue, Suite 230, La Jolla, CA 92037 USA). Chi-square test was used to evaluate the result of behavioral characteristics. Student's t-test was used to calculate the significance of biochemical parameter between experimental and control subjects. P < 0.05 was considered as statistically significant. Densitometric analysis was carried out by GelAnalyzer 2010 software.


  Results Top


In the present study (n = 96) subjects of above 60 years age were enrolled and categorized into two groups, experimental and control. The study group comprised (n = 48) males and (n = 48) females. The average age of male and female among experimental subjects was 70.17 ± 7.38 and 70.60 ± 8.25 while in control average age of male and female was 71.1 ± 5.49 and 70.00 ± 6.75, respectively. All experimental n = 28 males (58.34%) and n = 20 females (41.66%) were having hearing impairment on the basis of their history.

[Table 1] provides the percent wise distribution of behavioral characteristics of male and female subjects in the study groups. The percentage of male smokers and alcohol consumers among experiment was insignificantly higher than control subjects. No history of smoking and alcohol consumption was found among females.
Table 1: Distribution of behavioral characteristics of male and female subjects

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[Table 2] represents the mean level of biochemical parameters in both the study groups. The random blood glucose, serum creatinine, and urea levels were found to be in normal range when comprised in regards to male and female. Statistically significant difference was observed in random glucose level while statistically insignificant variations were observed among the rest of the biochemical parameters in both the study subjects. When both groups categorized according to gender, all biochemical parameters were found to be under the normal range.
Table 2: Various biochemical parameters according to study groups

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The differentially expressed protein bands after SDS-PAGE were represented in [Figure 1]. The Mr of identified protein was determined by gel image analyzer software and compared with standard Mr markers.
Figure 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (full view)

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The molecular mass of differentially expressed protein was found to be 16.2 kDa by gel image analyzer. The results on the repeated performance of this experiment showed prominent expression of this protein in 42 (87.5%) subjects among experimental subjects, on the contrary less expression of this protein in 4 (8.33%) subjects were observed in control subjects as shown in [Figure 2]. The rest of the subjects did not show such type of protein expression.
Figure 2: Sodium dodecyl sulfate-.polyacrylamide gel electrophoresis showing expression of target protein in experimental samples which is absent in control group after Coomassie staining

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[Figure 3] shows densitometric analysis of gel. Each lane and bands were marked individually as depicted in window A. density of each expressed band of standard Mr marker is shown on window B while the Rf and Mr are depicted on window C. The densitometric analysis of the targeted protein (TP) band was performed and is depicted in [Figure 4]. It is evident that the average band intensity of the TP was found to be 164. It was absent in control, hence not detected by the software.
Figure 3: Densitometric analysis of gel

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Figure 4: Densitometric analysis of targeted protein band

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  Discussion Top


In this study 48 subjects were taken for each group that is, experimental and control. The mean age of both male and female were quite closer in both the study subjects.

Sousa et al. were reported that smoking and alcohol consumption do not have any correlation with ARHI.[8] Similar trend was observed in the present study. Scientists have raised the possibility of a connection between smoking and ARHI while others have not confirmed this correlation.[9] These habits were recorded quantitatively (yes or no) in the form of a questionnaire.

The mean level of glucose was found slightly higher among the experimental group as compared to control; however it falls under the normal range. A positive association between diabetes and ARHI is well-known and more prevalence of type II diabetes mellitus was reported in the geriatric population.[10] Frisina et al. reported that diabetes affects right ear more than the left ear.[11] Several studies have reported on the possible pathogenesis of diabetes associated with ARHI. Since in the present study no signs of diabetes were found, the incidence of ARHI may be due to different causes which may be clearer by further study.

The mean level of creatinine among the experimental group was slightly greater than control, but the reverse was observed in the case of mean urea level. However, both the readings were found in the normal range. Age-related decrease in both creatinine production and creatinine clearance explains that creatinine remains stable with increasing age. Glomerular filtration rate decreases with age, but this is not associated with an increase in serum creatinine.[12] The increase in plasma urea level was observed in the elderly people by some reported studies.[13],[14] Vural et al. reported that SNHL occurs more commonly in chronic kidney disease patients than the normal population.[15] In this study mean urea levels were in normal range, and it was insignificantly associated with ARHI. Enhanced level of serum urea and creatinine cannot predict the occurrence of hearing loss as reported by Meena et al.[16]

Scientists reported that more numbers of men were affected by hearing loss compared to women.[17] Pleis et al. were documented that men are more prone to hearing loss than women.[18] In connection with above findings, more expression of TP band was observed in male as compared to female subjects in the present study. It may be due to higher occupational exposure of male as compared to females.

Results of SDS-PAGE revealed that more expression of the TP was observed among the experimental group as compared to control on repeated performance of the experiment. Densitometric analysis depicts the band intensity of TP was found to be 164 and same band was absent in control. The obtained protein having Mr of 16.2 kDa may be associated with ARHI. The Mr of differentially expressed protein resembles with Mr of 16 kDa, cochlin tomoprotein (CTP). CTP is widely studied in inner and middle ear functions. Among inner ear proteins, CTP constitutes a major part of the protein that is, 70%. CTP is an isoform of cochlin protein and is very specific to the perilymph. This protein plays a conjunctive role between brain and auditory nerves in the transmission of sound waves to the brain. Nanograms quantity of CTP in serum, perilymph and saliva was obtained by Ikezono et al.[19]

High level of cochlin proteins was detected in the inner ear by Western blot analysis as reported by Li et al.[20] Reason for the expression of protein bands only in the experimental subjects may be due to upregulation of COCH gene responsible for elevation of CTP protein concentration because of the hearing impairment due to age. Further, CTP contains all of the known mutation sites associated with an autosomal dominant, nonsyndromic, progressive SNHL (DFNA9).[21] Moreover, cochlin is a secretary protein in the extracellular matrix [22] and is highly expressed in the inner ear and also found in the spleen,[23] upon mutation it is not intracellular located, and follows the Golgi/ER pathway and further go through the proteolytic cleavage and glycosylation as reported by Robertson et al.[22] Further, Py et al. documented that due to inflammation, cochlin isoforms are released from the spleen into the blood stream after proteolytic cleavage. They used the serum as a sample to detect this protein by SDS-PAGE and Western blot analysis. They observed that, in gel electrophoresis, the cochlin isoform appeared as p8 (N-terminal fragment) and p18 forms (LCCL domain of cochlin), which strongly suggests that this protein is found in the serum.[23]

Another study conducted by Baek et al. to detect serum cochlin antibody titer by direct enzyme-linked immunosorbent assay using sera from hearing loss patients. They observed elevated levels of cochlin antibody titers in noise exposed and ARHI patients as compared to control.[24] This further confirms that cochlin antibody is secreted in the serum. The above observation is in line with the findings of the present study and is strongly supporting our findings. Although this finding is conflicting to the work carried out by Ikezono et al., 2004 and Ikezono et al., 2009 who reported that 16 kDa cochlin isoform named CTP corresponding to N-terminus of full-length cochlin and the LCCL domain did not detect in other body fluids except perilymph.[19],[25] From this study, it can be predicted that the protein bands which were prominently expressed in the experimental subjects but very less expressed in control subjects may be an isoform of cochlin protein that is, CTP.


  Conclusion Top


Results of these investigations revealed no significant correlation between smoking and alcohol consumption with ARHI. Males were more prone to ARHI than female in this study. Results of glucose were in the normal range which is inconsistent with the previously reported results in ARHI. Average serum creatinine and urea levels were in normal range and were insignificantly associated with ARHI. These findings pointed out some another cause of hearing impairment among experimental subjects which was tried to reveal by the SDS-PAGE studies. The Mr of the TP was 16.2 kDa which is close to 16 kDa CTP. CTP contains all of the known mutation sites associated with deafness. This protein can help in the prediagnosis of ARHI due to added advantage of the elevated level of (16.2 kDa) 16 kDa CTP in the earlier stage of the disease. Further research on a large number of samples using allied techniques such as audiometer, two-dimensional electrophoresis, LC-MS, etc., is needed to confirm the findings of the present study.

Acknowledgements

All authors would like to thank all the volunteers for participation in the study. We are grateful to Director, National Institute of Miners' Health for his constant support and encouragement.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
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