Aging Clin Exp Res DOI 10.1007/s40520-014-0282-3
ORIGINAL ARTICLE
Evaluation of serum antioxidants in age-related hearing loss Taye Jemilat Lasisi • Akeem Olawale Lasisi
Received: 3 March 2014 / Accepted: 28 October 2014 Ó Springer International Publishing Switzerland 2014
Abstract Background Age-related hearing loss (ARHL) has been linked to the shift in the pro-oxidant/antioxidant ratio. Our objectives were to assess serum levels of retinol and zinc among the elderly individuals and to correlate the levels with hearing threshold. Methods Prospective study of apparently healthy individuals aged C60 years of age. Participants had complete clinical history, physical examination and pure tone average conducted. Blood samples were collected for determination of serum levels of retinol and zinc. Mann– Whitney U test was used to compare retinol and zinc values. Pearson’s correlation test was used to determine the relationship between hearing threshold and serum levels of retinol and zinc. Results Among 126 elderly participants with mean age 67 ± 2.7 years; the mean pure tone average for air conduction was 29.3 ± 1.6 dBHL while the mean bone conduction was 36.5 ± 1.8 dBHL. The median values of serum retinol and zinc levels in the elderly participants who had hearing loss in the speech frequencies were 52 and 83.3 lg/L, respectively, while among participants with normal hearing threshold, values were 50 and 89.9 lg/L, respectively (p = 0.59 and 0.99, respectively). For the high frequencies, the median value of serum retinol and zinc levels among the elderly participants with normal hearing T. J. Lasisi (&) Departments of Physiology and Oral Pathology, College of Medicine, University of Ibadan, P. O. Box 22040, Ibadan, Nigeria e-mail: [email protected] A. O. Lasisi Department of Otorhinolaryngology, College of Medicine, University of Ibadan, Ibadan, Nigeria
threshold was 70.3 and 99.9 lg/L, while among those with hearing loss, it was 46.9 and 83.2 lg/L, respectively (p = 0.000 and 0.005, respectively). Conclusion Serum retinol and zinc levels were significantly lower among elderly with hearing loss involving the high frequencies. This is added evidence to extant literature on the possible role of antioxidants in the development of ARHL and suggests further study on the effect of antioxidants supplementation in the control of ARHL which is presently controversial and inconclusive. Keywords Age related hearing loss Elderly Antioxidants Retinol Zinc Hearing threshold
Introduction Age-related hearing loss (ARHL) or presbycusis is the most common sensory deficit in the elderly, and has become a great social and health challenge [1]. The progressive deterioration of hearing associated with aging, is the most common cause of hearing loss with reported prevalence of 23 % among people between ages 65 and 75, in the United States [2]. In Nigeria, the prevalence is 6.1 % [3]. Recently, the effects of reactive oxygen metabolites and their metabolites have been linked with ARHL among several forms of tissue damage [4, 5]. Reactive oxygen metabolites are generated in inflammatory responses, ischemic injuries to organs, and injuries resulting from the intracellular metabolism of chemicals and drugs. Reports from cadaveric and animal studies have shown that impaired blood flow and or oxygen delivery results in hair cell damage and strial atrophy, even in the absence of classic atherosclerotic changes [6–8]. Similarly, studies have also identified presbyacusis to be associated with
123
Aging Clin Exp Res
increased frequency of the mitochondria-DNA deletion in the cochleae and showed that treatment with antioxidants led to reduction in mitochondria-DNA deletions and improved auditory sensitivities in rats [9, 10]. Contrary to this, Sha et al. [11] recently reported that dietary manipulations can alter cochlear antioxidant capacity but do not ameliorate age-related sensorineural hearing loss in the CBA/J mouse. Antioxidants function by acting as scavengers of reactive oxygen metabolites and inhibitors of lipid peroxidation, reducing tissue damage and preventing hearing loss. Similarly, several epidemiological and clinical studies have revealed potential roles for dietary antioxidants in the ageassociated decline of immune function and the reduction of risk of morbidity and mortality from cancer and heart disease [12, 13]. However, the role of these antioxidants in the development and control of ARHL is still controversial. We hypothesized that low levels of serum antioxidants contribute to the development of ARHL. This study has chosen retinol and zinc because they also function as nutritional factors and their deficiencies have been previously reported [14]. Furthermore, this will serve as basis for the inclusion of other antioxidants and the role of intervention in future studies. The objectives of this study were to determine the serum levels of retinol and zinc among the elderly subjects with hearing loss and evaluate correlation, if any, with hearing loss and changes in the hearing threshold.
Methods
urinalysis tests were conducted and the participants were found to be free of any medical conditions. Consecutive eligible and consented elderly men and women C60 years of age were counseled. Each participant was taken through an already prepared questionnaire for that purpose. Specifically, questions aimed at eliciting otological and general medical conditions were asked and these included: otorrhoea, tinnitus, vertigo, otalgia, polyuria, polydipsia, significant weight loss, chronic cough and palpitation. Participants were also asked about history suggestive of allergy and use of such medications as: aminoglycosides, diuretics and 4-amino-quinolines antimalarial drugs. Individuals with history of living near noise such as in blacksmith shop, radio room/discotech and welder shop, for at least 2 h per day for at least 5 days a week were excluded. This was followed by ear, nose and throat examination and hearing test using pure tone average (PTA). Specifically, subjects were examined for evidence of arteriosclerosis by palpating the walls of the radial artery or locomotor brachialis—observing the pulsation of the brachial artery at the elbow. After the examination, collection of blood for estimation of serum levels of the biologic markers and pure tone average were conducted. The criteria for the diagnosis of the medical condition were based on simple definitions [15, 16]. The study was approved by the Oyo State Research Ethical Review Committee and the participants were recruited over a 9 month period. In this study, hearing loss was defined as pure tone average [30 dBHL and the control subjects were selected among elderly who had normal pure tone averages (0–30 dBHL).
Study design Blood sample collection and storage This was a prospective study of molecular and nutritional biology of hearing loss in apparently healthy elderly. The participants were drawn from the Ibadan community tagged to an out-reach research program involving IbadanLoyola University Genetics of Hypertension project organized for the detection and prevention of illnesses among the elderly in Ibadan, Oyo State, Nigeria. Included in the study were all elderly men and women C60 years of age who had no known medical condition while the exclusion criteria involved those with history of diabetes, stroke, hypertension, ear diseases, exposure to noise and ototoxic drugs such as aminoglycosides antibiotics, diuretics; ear infections, ear trauma or ear surgery. Participants recruitment Participants included were elderly who have been examined repeatedly by physicians in the outreach program. The blood pressure, random blood sugar and
123
Approximately, 5 ml of whole blood was collected using antecubital vein under aseptic condition. The samples were stored at -80 °C in batches for quantitative assay of retinol and zinc. Pure tone average The pure tone average was conducted using a computer audiometer BA 20 Kamplex with the subjects in sitting position in the sound-proof (acoustic) booth in the ENT clinic. The subjects were instructed to raise the hand if he heard the tone presented to the ears. The hearing acuity was measured in decibel hearing level (dBHL) at the frequencies 250—8,000 Hz. The average for the four frequencies 250, 500, 1,000, and 2,000 Hz was recorded as pure tone average for speech frequency while the average for the 3,000, 4,000, 6,000 and 8,000 Hz was recorded as the pure tone average for the high frequencies.
Aging Clin Exp Res
Quantification of serum retinol
Table 1 Distribution of serum zinc and retinol levels in the high frequency among the participants (n = 126)
Sample extraction and determination for retinol was done by measuring 0.125 lL of serum and diluting up to 500 lL volume with ultra-pure water. An antioxidant (10 g/dL ascorbic acid) was then added and the mixture was shaken for 15 min, followed by 5 min of sonication. Triton was added as detergent and an internal standard (400 lL of acetonitrile) was added and mixed properly. To this mixture 400 lL of n-hexane was added; the moisture was shaken for 5 min and centrifuged for 2 min at 8,000 revolutions per minute. The supernatant was collected and retinol determination was done using the high performance liquid chromatography (HPLC) method [14].
PTA (dBHL)
n
0–30
32
99.9
70.3
31–40
23
84.95
46.45
41–50
18
81.6
40.3
51–60
14
83.3
39.9
61–70
12
75.1
47.2
71–80
13
96.9
59.2
81–90 91–100
11 3
80.25 103.2
41.1 61.8
Determination of plasma zinc Serum was deproteinised in 1–9 mL of 10 % Trichloroacetic acid in 0.1 % lanthanum solution. Zinc levels were determined in the resultant supernatant using flame atomic absorption spectrophotometry (Model 205 Buck Scientific, East Norwalk CT, USA 06855). All reagents and materials used for the analysis were free of zinc contamination and the plastics used for the analysis were previously washed with Hydrochloric acid. The control and standard sera were included in the analysis at every sera assay to ensure reliability and quality of the procedure. An initial pilot study was conducted to test all instruments, this was followed by a preliminary statistical analysis to detect outliers and correct factors. Statistics Data were presented using descriptive statistics such as median and range for serum levels of retinol and zinc. Data was tested for normality using Kolmogorov–Smirnov test. Serum levels of retinol and zinc were not normally distributed, hence, the Mann–Whitney U test was used to compare average retinol and zinc values. Pearson’s correlation coefficient was used to determine the relationship between hearing threshold and serum levels of retinol and zinc. All analyses were done at 5 % level of significant. Data were analysed using the Stata software (version 7.0 for windows, College Station, TX, USA: stata Corp; 2001).
Results The subjects included 126 elderly (59 males and 67 females) and the age ranged from 60 to 98 years with mean age of 67 ± 2.7 years. The mean PTA for air conduction (AC) was 29.3 ± 1.6 dBHL while the mean for the bone conduction (BC) was 36.5 ± 1.8 dBHL. The median of the
Zinc (lg/L)
Retinol (lg/L)
PTA pure tone average, dBHL decibel hearing loss
Table 2 Distribution of serum zinc and retinol levels in the speech frequency among the participants (n = 126) PTA (dBHL)
n
Zinc (lg/L)
Retinol (lg/L)
0–30
88
83.3
50
31–40
13
84.2
50.2
41–50
9
75.1
52
51–60
10
106.7
59.2
61–70
3
56
23
71–80
3
103.2
61.8
PTA pure tone average, dBHL decibel hearing loss
PTA for speech frequencies was 25 dBHL, while for the high frequencies; it was 45 dBHL. Using the 30 dBHL as the cut-off, hearing loss in the speech (250–2,000 Hz) frequencies was seen in 38 (30.2 %) participants and normal hearing range was seen in 88 (69.8 %) participants. In contrast, high frequency (3,000–8,000 Hz) hearing loss was seen in 94 (74.6 %) participants while hearing was within normal range in 32 (25.4 %) participants; and combined speech and high frequency hearing loss accounted for 16 (12.7 %). Tables 1 and 2 show the frequency distribution of the serum levels of retinol and zinc respectively, among the subjects according to the hearing threshold in decibel hearing level (dBHL). The serum levels of retinol was between 21 and 80.2 lg/L, (median 50.65 lg/L), while the serum levels of Zinc was between 56 and 117.9 lg/L (median 84.15 lg/L). The median values of serum levels of retinol in the elderly subjects who had hearing loss in the speech frequencies (250–2,000 Hz) was 52 lg/L, while among participants with normal hearing threshold, it was 50 lg/L. For the high frequencies, the median values of serum levels of retinol among the elderly subjects with normal hearing threshold was 70.3 lg/L, while among those with hearing loss, it was 46.9 lg/L.
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Aging Clin Exp Res Table 3 Comparison of serum zinc and retinol between elderly with and without hearing loss in the speech frequency PTA (dBHL)
n
Minimum
Maximum
Median
p value 0.99
Zinc (lg/L)
B30
88
56
118
83.3
[30
38
56
116
89.9
Retinol (lg/L)
B30 [30
88 38
23 21
80.2 72
50 52
0.59
PTA pure tone average, dBHL decibel hearing loss
Table 4 Comparison of serum zinc and retinol between elderly with and without hearing loss in the high frequency PTA (dBHL)
n
Minimum
Maximum
Median
p value 0.005
Zinc (lg/L)
B30
32
56
118
99.9
[30
94
56
118
83.2
Retinol (lg/L)
B30
32
23
80
70.3
[30
94
21
72
46.9
0.000
PTA pure tone average, dBHL decibel hearing loss
On the other hand, in the speech frequencies (250–2,000 Hz), the median value of serum Zinc levels among the elderly subjects with normal hearing threshold was 83.3 lg/L, while among those with hearing loss the median was 89.9 lg/L. However, for high frequencies, the median serum level of Zinc among the elderly subjects with normal hearing threshold was 99.9 lg/L, while among those with hearing loss the median was 83.2 lg/L. Tables 3 and 4 compare the serum levels of retinol and zinc and the hearing threshold between subjects with and without hearing loss. Serum levels of retinol and zinc were significantly lower in the elderly with hearing loss in the high frequency (p = 0.005 and p = 0.000, respectively). Although not significant, there was negative correlation between serum retinol, zinc and hearing loss in the speech (r = -0.08, p = 0.42; r = -0.1, p = 0.31, respectively) and high frequencies (r = -0.16, p = 0.09; r = -0.14, p = 0.15, respectively).
Discussion Overall, our results suggest that low serum levels of retinol and zinc are associated with hearing loss involving high frequencies among the elderly. These findings may indicate that low levels of retinol and zinc may have a role in the development of ARHL and this may suggest potential use in the control of presbyacusis, although this is still subject to findings from more studies.
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In the work of Seidman et al. [9] on prospective randomized study of the effect of antioxidants, nutritional supplementation, and dietary restriction on age-related hearing loss in rats, they concluded that nutrient supplementation might reduce the progression of age-related hearing loss. They proposed that the mechanism of development of presbyacusis involves the generation of reactive oxygen metabolites and resultant damage to mitochondria. This study has chosen retinol and zinc because they function as nutritional factors and their deficiencies have been studied and reported [14] under disease conditions. Furthermore, this will serve as basis for the inclusion of other antioxidants and the role of intervention in future studies. Although further studies may need to include a number of other antioxidants, the findings from this study also add to the increasing body of evidence implicating reduction in antioxidant activity in the damage to hearing loss associated with aging. There is support in the literature for the protective effects of antioxidants such as tocopherols as scavengers of reactive oxygen metabolite and inhibitors of lipid peroxidation [7, 8]. In the aging cochlea, reduction of blood supply and the ongoing need for energy generation through oxidative phosphorylation have been identified as processes which encourage the generation of reactive oxygen molecules within the cochlea [4, 5]. Studies have also demonstrated markedly decreased flow within the circulatory system in the elderly population [17, 18]. Similarly, changes in whole blood viscosity and red blood cell rigidity have been correlated with high frequency hearing loss in elderly humans [18]. Human cadaveric study have also shown significant evidence supporting the concept that hypoperfusion and/or oxygen delivery results in hair cell damage, atrophy of striae and gradual loss of capillaries in the spiral ligament of the scala vestibuli with an increase in intravascular strands and avascular channels [19, 20]. Prazma et al. [18] and others [21–23] have observed that diminished flow in capillaries of the basal turn of the cochlea, and increased blood viscosity and red blood cell rigidity appear to correlate with high-frequency hearing loss in elderly human subjects thus suggesting a relationship between vascular atrophy and presbyacusis [23, 24]. In addition, studies have demonstrated a significant reduction in erythrocyte velocity, increased vascular permeability, reduced capillary diameters, and a decrease in auditory sensitivity with aging [24, 25]. Dietary antioxidants have been reported to decrease the interaction and activation of immune, endothelial, and smooth muscle cells of vessel walls in the early stage of atherogenesis [25, 26]. In our methodology, simple definitions using history, physical examination, basic hematological investigation and urinalysis were applied as criteria for the possible health conditions which might predispose to ARHL among
Aging Clin Exp Res
the participants. This was to ensure that the contributory factors to hearing loss were excluded and the subjects would include only those with presbyacusis. We recognize the poor specificity of our screening method though it is believed that such was adequate for an initial study of ARHL in our environment. In addition, these subjects had been involved in our previous researches and their health status was known and documented. A negative correlation was not found between the hearing threshold and the serum levels of these two antioxidants although not significant. This could be explained by the multiplicity of factors and the complex interactions of the factors which could affect the hearing threshold and levels of these antioxidants. The hearing frequencies were classified into high and low frequencies to identify the particular frequencies involved, although it is known that age-related hearing loss usually starts from the high frequencies with involvement of the speech frequencies later.
Conclusion The findings from this study have shown that serum retinol and zinc levels were significantly lower among the elderly participants with hearing loss involving the high frequencies. This is added evidence to extant literature on the possible role of antioxidants in the development of ARHL and suggests further study on the effect of antioxidants supplementation in the control of ARHL which is presently controversial and inconclusive. Acknowledgments The authors thank Prof. Babatunde L. Salako of the Ibadan-Loyola University Genetics of Hypertension project and Prof. Oye Gureje of the Ibadan Study of Ageing project for their contributions to this work. Conflict of interest
Authors declare no conflict of interest.
References 1. Huang Q, Tang J (2010) Age related hearing loss or presbycusis. Eur Arch Otorhinolaryngol 267(8):1179–1191 2. Gates GA, Mills JH (2005) Presbycusis. Lancet 366:1111–1120 3. Lasisi AO, Abiona T, Gureje O (2010) The prevalence and correlates of self-reported hearing impairment from the Ibadan Study of Ageing. Trans R Soc Trop Med Hyg 104:518–523 4. Seidman MD, Ahmad N, Joshi D, Seidman J, Thawani S, Quirk WS (2004) Age-related hearing loss and its association with reactive oxygen species and mitochondrial DNA damage. Acta Otolaryngol Suppl 552:16–24 5. Nadol JB Jr, Merchant SN (2001) Histopathology and molecular genetics of hearing loss in the human. Int J Pediatr Otorhinolaryngol 61:1–15
6. Gourevitch B, Edeline J (2011) Age related changes in the guinea pig auditory cortex relationship with brainstem changes and comparison with tone induced hearing loss. Eur J Neurosci 34:1953–1965 7. Derin A, Agirdir B, Derin N, Dinc O, Gu¨ney K, Ozcaglar H et al (2004) The effects of L-carnitine on presbyacusis in the rat model. Clin Otolaryngol Allied Sci 29:238–241 8. Seidman MD, Khan MJ, Bai U, Shirwany N, Quirk WS (2000) Biologic activity of mitochondrial metabolites on aging and agerelated hearing loss. Am J Otol 21:161–167 9. Takumida M, Anniko M (2002) Nitric oxide in the inner ear. Curr Opin Neurol 15:11–15 10. Seidman MD (2000) Effects of dietary restriction and antioxidants on presbyacusis. Laryngoscope 110:727–738 11. Sha S, Kanicki A, Halsey K, Anne Wearne K, Schacht J (2012) Antioxidant enriched diet does not delay the progression of age related hearing loss. Neurobiol Aging 33(5):15–16 12. Byers T, Bowman B (1993) Vitamin E supplements and coronary heart disease. Nutr Rev 51:333–336 13. Meydani M (2002) The Boyd Orr lecture. Nutrition interventions in aging and age associated diseases. Proc Nutr Soc 61(2):165–171 14. Lasisi OA (2009) The role of retinol in the aetiology and outcome of suppurative otitis media. Eur Arch Oto-Rhino-Laryngol 266:647–652 15. Lasisi OA, Ayodele JK, Ijaduola GTA (2005) Challenges in the management of childhood sensorineural hearing loss in Subsaharan Africa, Nigeria. Int J Paed Otorhinolaryngol 70:625–629 16. Lasisi OA, Sulaiman OA, Afolabi OA (2007) Socio-economic status and hearing loss in chronic suppurative otitis media in Nigeria. Ann Trop Paed 27:291–296 17. Nelson EG, Hinojosa R (2006) Presbycusis: a human temporal bone Study of individuals with downward sloping audiometric patterns of hearing loss and review of the literature. Laryngoscope 116(Suppl. 112):1–12 18. Prazma J, Butler B, Waters G, Anderson TL, Pillsbury HC (1987) Proceedings of the 71st Annual Meeting of the Federation of American Societies for Experimental Biology, vol 46. Washington, DC, p 1538 (abstract 1538) 19. Gatehouse S, Lowe GD (1990) Whole blood viscosity and red cell filterability as factors in sensorineural hearing impairment in the elderly. Acta Otolaryngol Suppl (Stockh) 476:37–43 20. Pujol R, Guy R, Puel J, Lenoir M, Eybalin M, Recasens M (1990) Glutamate neurotoxicity in the cochlea: a possible consequence of ischaemic or anoxic conditions occurring in aging. Acta Otolaryngol Suppl (Stockh) 476:32–36 21. Stephane FM, Hajime U, Liberman MC (2013) Efferent feedback minimizes cochlea neuropathy from moderate noise exposure. J Neurosci 33:5542–5552 22. Nakashima T, Nagamawa S, Sone M, Tomonaga M, Hayashi H (2003) Disorders of cochlear blood flow. Brain Res Rev 42:17–28 23. Ratnam DV, Ankola DD, Bhardwaj V, Sahana DK, Kumar MN, Raevi V (2006) Role of antioxidants in prophylaxis and therapy; a pharmaceutical perspective. J Control Release 113:189–209 24. Gopinath B, Flood VM, McMahon CM, Burlutsky G, Spankovich C, Hood LJ (2011) Dietary antioxidants intake is associated with the prevalence but not incidence of age related hearing loss. J Nutr Health Aging 15:896–900 25. Kopke R, Bielefeld E, Liu J, Zheng J, Jackson R et al (2005) Prevention of impulse noise induced hearing loss with antioxidants. Acta Otolaryngological 135(3):235–243 26. Blomhoff R (2005) Dietary antioxidants and cardiovascular diseases. Curr Opin Lipidol 16:47–54
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ORIGINAL ARTICLE
Evaluation of serum antioxidants in age-related hearing loss Taye Jemilat Lasisi • Akeem Olawale Lasisi
Received: 3 March 2014 / Accepted: 28 October 2014 Ó Springer International Publishing Switzerland 2014
Abstract Background Age-related hearing loss (ARHL) has been linked to the shift in the pro-oxidant/antioxidant ratio. Our objectives were to assess serum levels of retinol and zinc among the elderly individuals and to correlate the levels with hearing threshold. Methods Prospective study of apparently healthy individuals aged C60 years of age. Participants had complete clinical history, physical examination and pure tone average conducted. Blood samples were collected for determination of serum levels of retinol and zinc. Mann– Whitney U test was used to compare retinol and zinc values. Pearson’s correlation test was used to determine the relationship between hearing threshold and serum levels of retinol and zinc. Results Among 126 elderly participants with mean age 67 ± 2.7 years; the mean pure tone average for air conduction was 29.3 ± 1.6 dBHL while the mean bone conduction was 36.5 ± 1.8 dBHL. The median values of serum retinol and zinc levels in the elderly participants who had hearing loss in the speech frequencies were 52 and 83.3 lg/L, respectively, while among participants with normal hearing threshold, values were 50 and 89.9 lg/L, respectively (p = 0.59 and 0.99, respectively). For the high frequencies, the median value of serum retinol and zinc levels among the elderly participants with normal hearing T. J. Lasisi (&) Departments of Physiology and Oral Pathology, College of Medicine, University of Ibadan, P. O. Box 22040, Ibadan, Nigeria e-mail: [email protected] A. O. Lasisi Department of Otorhinolaryngology, College of Medicine, University of Ibadan, Ibadan, Nigeria
threshold was 70.3 and 99.9 lg/L, while among those with hearing loss, it was 46.9 and 83.2 lg/L, respectively (p = 0.000 and 0.005, respectively). Conclusion Serum retinol and zinc levels were significantly lower among elderly with hearing loss involving the high frequencies. This is added evidence to extant literature on the possible role of antioxidants in the development of ARHL and suggests further study on the effect of antioxidants supplementation in the control of ARHL which is presently controversial and inconclusive. Keywords Age related hearing loss Elderly Antioxidants Retinol Zinc Hearing threshold
Introduction Age-related hearing loss (ARHL) or presbycusis is the most common sensory deficit in the elderly, and has become a great social and health challenge [1]. The progressive deterioration of hearing associated with aging, is the most common cause of hearing loss with reported prevalence of 23 % among people between ages 65 and 75, in the United States [2]. In Nigeria, the prevalence is 6.1 % [3]. Recently, the effects of reactive oxygen metabolites and their metabolites have been linked with ARHL among several forms of tissue damage [4, 5]. Reactive oxygen metabolites are generated in inflammatory responses, ischemic injuries to organs, and injuries resulting from the intracellular metabolism of chemicals and drugs. Reports from cadaveric and animal studies have shown that impaired blood flow and or oxygen delivery results in hair cell damage and strial atrophy, even in the absence of classic atherosclerotic changes [6–8]. Similarly, studies have also identified presbyacusis to be associated with
123
Aging Clin Exp Res
increased frequency of the mitochondria-DNA deletion in the cochleae and showed that treatment with antioxidants led to reduction in mitochondria-DNA deletions and improved auditory sensitivities in rats [9, 10]. Contrary to this, Sha et al. [11] recently reported that dietary manipulations can alter cochlear antioxidant capacity but do not ameliorate age-related sensorineural hearing loss in the CBA/J mouse. Antioxidants function by acting as scavengers of reactive oxygen metabolites and inhibitors of lipid peroxidation, reducing tissue damage and preventing hearing loss. Similarly, several epidemiological and clinical studies have revealed potential roles for dietary antioxidants in the ageassociated decline of immune function and the reduction of risk of morbidity and mortality from cancer and heart disease [12, 13]. However, the role of these antioxidants in the development and control of ARHL is still controversial. We hypothesized that low levels of serum antioxidants contribute to the development of ARHL. This study has chosen retinol and zinc because they also function as nutritional factors and their deficiencies have been previously reported [14]. Furthermore, this will serve as basis for the inclusion of other antioxidants and the role of intervention in future studies. The objectives of this study were to determine the serum levels of retinol and zinc among the elderly subjects with hearing loss and evaluate correlation, if any, with hearing loss and changes in the hearing threshold.
Methods
urinalysis tests were conducted and the participants were found to be free of any medical conditions. Consecutive eligible and consented elderly men and women C60 years of age were counseled. Each participant was taken through an already prepared questionnaire for that purpose. Specifically, questions aimed at eliciting otological and general medical conditions were asked and these included: otorrhoea, tinnitus, vertigo, otalgia, polyuria, polydipsia, significant weight loss, chronic cough and palpitation. Participants were also asked about history suggestive of allergy and use of such medications as: aminoglycosides, diuretics and 4-amino-quinolines antimalarial drugs. Individuals with history of living near noise such as in blacksmith shop, radio room/discotech and welder shop, for at least 2 h per day for at least 5 days a week were excluded. This was followed by ear, nose and throat examination and hearing test using pure tone average (PTA). Specifically, subjects were examined for evidence of arteriosclerosis by palpating the walls of the radial artery or locomotor brachialis—observing the pulsation of the brachial artery at the elbow. After the examination, collection of blood for estimation of serum levels of the biologic markers and pure tone average were conducted. The criteria for the diagnosis of the medical condition were based on simple definitions [15, 16]. The study was approved by the Oyo State Research Ethical Review Committee and the participants were recruited over a 9 month period. In this study, hearing loss was defined as pure tone average [30 dBHL and the control subjects were selected among elderly who had normal pure tone averages (0–30 dBHL).
Study design Blood sample collection and storage This was a prospective study of molecular and nutritional biology of hearing loss in apparently healthy elderly. The participants were drawn from the Ibadan community tagged to an out-reach research program involving IbadanLoyola University Genetics of Hypertension project organized for the detection and prevention of illnesses among the elderly in Ibadan, Oyo State, Nigeria. Included in the study were all elderly men and women C60 years of age who had no known medical condition while the exclusion criteria involved those with history of diabetes, stroke, hypertension, ear diseases, exposure to noise and ototoxic drugs such as aminoglycosides antibiotics, diuretics; ear infections, ear trauma or ear surgery. Participants recruitment Participants included were elderly who have been examined repeatedly by physicians in the outreach program. The blood pressure, random blood sugar and
123
Approximately, 5 ml of whole blood was collected using antecubital vein under aseptic condition. The samples were stored at -80 °C in batches for quantitative assay of retinol and zinc. Pure tone average The pure tone average was conducted using a computer audiometer BA 20 Kamplex with the subjects in sitting position in the sound-proof (acoustic) booth in the ENT clinic. The subjects were instructed to raise the hand if he heard the tone presented to the ears. The hearing acuity was measured in decibel hearing level (dBHL) at the frequencies 250—8,000 Hz. The average for the four frequencies 250, 500, 1,000, and 2,000 Hz was recorded as pure tone average for speech frequency while the average for the 3,000, 4,000, 6,000 and 8,000 Hz was recorded as the pure tone average for the high frequencies.
Aging Clin Exp Res
Quantification of serum retinol
Table 1 Distribution of serum zinc and retinol levels in the high frequency among the participants (n = 126)
Sample extraction and determination for retinol was done by measuring 0.125 lL of serum and diluting up to 500 lL volume with ultra-pure water. An antioxidant (10 g/dL ascorbic acid) was then added and the mixture was shaken for 15 min, followed by 5 min of sonication. Triton was added as detergent and an internal standard (400 lL of acetonitrile) was added and mixed properly. To this mixture 400 lL of n-hexane was added; the moisture was shaken for 5 min and centrifuged for 2 min at 8,000 revolutions per minute. The supernatant was collected and retinol determination was done using the high performance liquid chromatography (HPLC) method [14].
PTA (dBHL)
n
0–30
32
99.9
70.3
31–40
23
84.95
46.45
41–50
18
81.6
40.3
51–60
14
83.3
39.9
61–70
12
75.1
47.2
71–80
13
96.9
59.2
81–90 91–100
11 3
80.25 103.2
41.1 61.8
Determination of plasma zinc Serum was deproteinised in 1–9 mL of 10 % Trichloroacetic acid in 0.1 % lanthanum solution. Zinc levels were determined in the resultant supernatant using flame atomic absorption spectrophotometry (Model 205 Buck Scientific, East Norwalk CT, USA 06855). All reagents and materials used for the analysis were free of zinc contamination and the plastics used for the analysis were previously washed with Hydrochloric acid. The control and standard sera were included in the analysis at every sera assay to ensure reliability and quality of the procedure. An initial pilot study was conducted to test all instruments, this was followed by a preliminary statistical analysis to detect outliers and correct factors. Statistics Data were presented using descriptive statistics such as median and range for serum levels of retinol and zinc. Data was tested for normality using Kolmogorov–Smirnov test. Serum levels of retinol and zinc were not normally distributed, hence, the Mann–Whitney U test was used to compare average retinol and zinc values. Pearson’s correlation coefficient was used to determine the relationship between hearing threshold and serum levels of retinol and zinc. All analyses were done at 5 % level of significant. Data were analysed using the Stata software (version 7.0 for windows, College Station, TX, USA: stata Corp; 2001).
Results The subjects included 126 elderly (59 males and 67 females) and the age ranged from 60 to 98 years with mean age of 67 ± 2.7 years. The mean PTA for air conduction (AC) was 29.3 ± 1.6 dBHL while the mean for the bone conduction (BC) was 36.5 ± 1.8 dBHL. The median of the
Zinc (lg/L)
Retinol (lg/L)
PTA pure tone average, dBHL decibel hearing loss
Table 2 Distribution of serum zinc and retinol levels in the speech frequency among the participants (n = 126) PTA (dBHL)
n
Zinc (lg/L)
Retinol (lg/L)
0–30
88
83.3
50
31–40
13
84.2
50.2
41–50
9
75.1
52
51–60
10
106.7
59.2
61–70
3
56
23
71–80
3
103.2
61.8
PTA pure tone average, dBHL decibel hearing loss
PTA for speech frequencies was 25 dBHL, while for the high frequencies; it was 45 dBHL. Using the 30 dBHL as the cut-off, hearing loss in the speech (250–2,000 Hz) frequencies was seen in 38 (30.2 %) participants and normal hearing range was seen in 88 (69.8 %) participants. In contrast, high frequency (3,000–8,000 Hz) hearing loss was seen in 94 (74.6 %) participants while hearing was within normal range in 32 (25.4 %) participants; and combined speech and high frequency hearing loss accounted for 16 (12.7 %). Tables 1 and 2 show the frequency distribution of the serum levels of retinol and zinc respectively, among the subjects according to the hearing threshold in decibel hearing level (dBHL). The serum levels of retinol was between 21 and 80.2 lg/L, (median 50.65 lg/L), while the serum levels of Zinc was between 56 and 117.9 lg/L (median 84.15 lg/L). The median values of serum levels of retinol in the elderly subjects who had hearing loss in the speech frequencies (250–2,000 Hz) was 52 lg/L, while among participants with normal hearing threshold, it was 50 lg/L. For the high frequencies, the median values of serum levels of retinol among the elderly subjects with normal hearing threshold was 70.3 lg/L, while among those with hearing loss, it was 46.9 lg/L.
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Aging Clin Exp Res Table 3 Comparison of serum zinc and retinol between elderly with and without hearing loss in the speech frequency PTA (dBHL)
n
Minimum
Maximum
Median
p value 0.99
Zinc (lg/L)
B30
88
56
118
83.3
[30
38
56
116
89.9
Retinol (lg/L)
B30 [30
88 38
23 21
80.2 72
50 52
0.59
PTA pure tone average, dBHL decibel hearing loss
Table 4 Comparison of serum zinc and retinol between elderly with and without hearing loss in the high frequency PTA (dBHL)
n
Minimum
Maximum
Median
p value 0.005
Zinc (lg/L)
B30
32
56
118
99.9
[30
94
56
118
83.2
Retinol (lg/L)
B30
32
23
80
70.3
[30
94
21
72
46.9
0.000
PTA pure tone average, dBHL decibel hearing loss
On the other hand, in the speech frequencies (250–2,000 Hz), the median value of serum Zinc levels among the elderly subjects with normal hearing threshold was 83.3 lg/L, while among those with hearing loss the median was 89.9 lg/L. However, for high frequencies, the median serum level of Zinc among the elderly subjects with normal hearing threshold was 99.9 lg/L, while among those with hearing loss the median was 83.2 lg/L. Tables 3 and 4 compare the serum levels of retinol and zinc and the hearing threshold between subjects with and without hearing loss. Serum levels of retinol and zinc were significantly lower in the elderly with hearing loss in the high frequency (p = 0.005 and p = 0.000, respectively). Although not significant, there was negative correlation between serum retinol, zinc and hearing loss in the speech (r = -0.08, p = 0.42; r = -0.1, p = 0.31, respectively) and high frequencies (r = -0.16, p = 0.09; r = -0.14, p = 0.15, respectively).
Discussion Overall, our results suggest that low serum levels of retinol and zinc are associated with hearing loss involving high frequencies among the elderly. These findings may indicate that low levels of retinol and zinc may have a role in the development of ARHL and this may suggest potential use in the control of presbyacusis, although this is still subject to findings from more studies.
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In the work of Seidman et al. [9] on prospective randomized study of the effect of antioxidants, nutritional supplementation, and dietary restriction on age-related hearing loss in rats, they concluded that nutrient supplementation might reduce the progression of age-related hearing loss. They proposed that the mechanism of development of presbyacusis involves the generation of reactive oxygen metabolites and resultant damage to mitochondria. This study has chosen retinol and zinc because they function as nutritional factors and their deficiencies have been studied and reported [14] under disease conditions. Furthermore, this will serve as basis for the inclusion of other antioxidants and the role of intervention in future studies. Although further studies may need to include a number of other antioxidants, the findings from this study also add to the increasing body of evidence implicating reduction in antioxidant activity in the damage to hearing loss associated with aging. There is support in the literature for the protective effects of antioxidants such as tocopherols as scavengers of reactive oxygen metabolite and inhibitors of lipid peroxidation [7, 8]. In the aging cochlea, reduction of blood supply and the ongoing need for energy generation through oxidative phosphorylation have been identified as processes which encourage the generation of reactive oxygen molecules within the cochlea [4, 5]. Studies have also demonstrated markedly decreased flow within the circulatory system in the elderly population [17, 18]. Similarly, changes in whole blood viscosity and red blood cell rigidity have been correlated with high frequency hearing loss in elderly humans [18]. Human cadaveric study have also shown significant evidence supporting the concept that hypoperfusion and/or oxygen delivery results in hair cell damage, atrophy of striae and gradual loss of capillaries in the spiral ligament of the scala vestibuli with an increase in intravascular strands and avascular channels [19, 20]. Prazma et al. [18] and others [21–23] have observed that diminished flow in capillaries of the basal turn of the cochlea, and increased blood viscosity and red blood cell rigidity appear to correlate with high-frequency hearing loss in elderly human subjects thus suggesting a relationship between vascular atrophy and presbyacusis [23, 24]. In addition, studies have demonstrated a significant reduction in erythrocyte velocity, increased vascular permeability, reduced capillary diameters, and a decrease in auditory sensitivity with aging [24, 25]. Dietary antioxidants have been reported to decrease the interaction and activation of immune, endothelial, and smooth muscle cells of vessel walls in the early stage of atherogenesis [25, 26]. In our methodology, simple definitions using history, physical examination, basic hematological investigation and urinalysis were applied as criteria for the possible health conditions which might predispose to ARHL among
Aging Clin Exp Res
the participants. This was to ensure that the contributory factors to hearing loss were excluded and the subjects would include only those with presbyacusis. We recognize the poor specificity of our screening method though it is believed that such was adequate for an initial study of ARHL in our environment. In addition, these subjects had been involved in our previous researches and their health status was known and documented. A negative correlation was not found between the hearing threshold and the serum levels of these two antioxidants although not significant. This could be explained by the multiplicity of factors and the complex interactions of the factors which could affect the hearing threshold and levels of these antioxidants. The hearing frequencies were classified into high and low frequencies to identify the particular frequencies involved, although it is known that age-related hearing loss usually starts from the high frequencies with involvement of the speech frequencies later.
Conclusion The findings from this study have shown that serum retinol and zinc levels were significantly lower among the elderly participants with hearing loss involving the high frequencies. This is added evidence to extant literature on the possible role of antioxidants in the development of ARHL and suggests further study on the effect of antioxidants supplementation in the control of ARHL which is presently controversial and inconclusive. Acknowledgments The authors thank Prof. Babatunde L. Salako of the Ibadan-Loyola University Genetics of Hypertension project and Prof. Oye Gureje of the Ibadan Study of Ageing project for their contributions to this work. Conflict of interest
Authors declare no conflict of interest.
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