Free-radical damage: A possible mechanism of laryngeal aging. (Original Article).

Abstract

We conducted a study of lipid peroxidation as a marker of age-related free-radical damage in the human larynx--the first study of its kind. A colorimetric assay for malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4HNE) was performed on extracts taken from thyroarytenoid muscle harvested from fresh cadaveric laryngeal specimens. Levels of MDA and 4-HNE were measured by spectrophotometry. Correlation studies were performed by linear regression analysis. We found that MDA levels in human thy roarytenoid muscle appeared to increase with age while 4-HNE levels showed a slight decrease with age. Our findings are consistent with those of previous studies of other organ systems and indicate that there is a need for further study of free-radical damage and the effects of aging on the human larynx and on voice production.

Introduction

The gradual deterioration of the human voice with age is a well-recognized phenomenon. Numerous acoustic and physiologic studies have characterized the qualitative changes in the aging voice, and recent studies have described the histopathologic abnormalities in the aging larynx, as well. However, the molecular processes that underlie these cellular and functional changes remain poorly understood.

Different theories have been postulated about the molecular mechanisms of senescence. The free-radical theory of aging is based on the hypothesis that the endogenous build-up of free-radical species results in oxidative molecular damage. Mecocci et al found age-dependent increases in markers of oxidative damage to DNA, protein, and lipids in human skeletal muscle. (1) evidence to support the free-radical theory includes observations that (1) oxidative damage to macromolecules accumulates with normal aging, (2) (2) variations in life spans among different species correlate inversely with rates of mitochondrial generation of oxidant species, (3) and (3) experimental interventions that extend life spans result in reductions in oxidative damage. (4)

To date, there has been no study of the role of free radicals and oxidative damage in the aging larynx. The goal of our study was to examine lipid peroxidation as a marker of free-radical damage in the aging larynx by assaying levels of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) in cadaveric thyroarytenoid muscle.

Materials and methods

Preparation of laryngeal tissue. Laryngeal specimens were obtained from 22 cadavers during autopsy within 24 hours of death and stored frozen at -20[degrees] C. At death, the ages of the subject donors ranged from 47 to 82 years. All specimens were taken from subjects who had had no history of laryngeal disease or trauma.

Larynges were thawed and sectioned, and exactly 500 mg of thyroarytenoid muscle was harvested from each larynx. Each sample was washed in ice-cold 0.9% sodium chloride and then minced and diluted to a 10% (by weight) solution with 20 mmol of ice-cold Tris-hydrochloric acid buffer (pH: 7.4) and butylated hydroxytoluene (BHT). Each sample was homogenized and centrifuged at 3,000 G at 4[degrees] C for 10 minutes. Supernatant was collected, and approximately 200 [micro]l was used for each assay.

Lipid peroxidation assay. A colorimetric assay for MDA and 4-HNE (Oxford Biomedical Research; Oxford, Mich.) was used in this study. This assay is based on a reaction between N-methyl-2-phenylindole and either MDA or 4-HNE. The reaction forms a stable chromophore that reaches maximal absorbance at 586 nm. (5) The addition of methanesulfonic acid is used to quantify the level of both MDA and 4-HNE (MDA + 4-HNE), and the addition of 37% hydrochloric acid makes possible the quantification of MDA alone. The actual concentration of 4-HNE alone is calculated as the difference between the two levels.

MDA + 4-HNE assay. A 200-[micro]l sample of supernatant was combined with 650 [micro]l of Nmethyl-2-phenylindole (10.3 mmol in acetonitrile and methanol). Then 150 [micro]1 of 15.4 M methanesulfonic acid and 10 [micro]1 of BHT were added to the sample and briefly vortexed. The mixture was incubated at 45[degrees] C for 40 minutes and then cooled on ice.

MDA assay. A 200-[micro]l sample of supernatant was combined with 650 [micro]l of N-methyl2-phenylindole (10.3 mmol in acetonitrile and methanol). Then 150 p1 of 37% hydrochloric acid and 10 [micro]l of BHT were added to the sample and briefly vortexed. Each sample was incubated at 45[degrees] C for 60 minutes and then cooled on ice.

Spectrophotometry. Spectrophotometry measuring absorbance at 586 nm of serial dilutions of MDA and 4-HNE standard solutions was used to create standard concentration curves for each assay. Standard curves were prepared fresh for each assay prior to spectrophotometry. Blank samples of 200 [micro]l of double-distilled water were used to zero the spectrophotometer prior to use.

Results

Sample concentrations ([micro]M/L) of MDA and 4-HNE were derived from standard concentration curves. MDA levels tended to increase with age and 4-HNE levels appeared to have an inverse correlation with age.

Regression analyses for MDA and 4-HNE were performed to derive lines of best fit (figure). The best-fit line for MDA was an upward slope with an [r.sup.2] value of 0.1095 (p = 0.123) and the 4-HNE line was a slightly downward slope with an [r.sup.2] value of 0.0469 (p = 0.321).

Discussion

Different theories have been proposed to explain the cellular and molecular mechanisms that underlie mammalian senescence. One of these theories holds that the primary cause of age-related loss of function might be attributable to somatic DNA mutations. Although few chromosomal mutations have been associated with aging, many specific mitochondrial DNA mutations do accumulate in various tissues (e.g., the brain, heart, and skeletal muscle) as individuals age. (6) Because mitochondria are the primary sites of oxidative phosphorylation, the accumulation of mitochondrial DNA mutations might lead to a progressive decrease in the body's ability to produce energy in affected cells. Age-related mitochondrial DNA mutations have recently been demonstrated in the human larynx. (7)

Proponents of the theory examined in our study espouse that free radicals are a primary causative factor in aging. The human body continuously produces free-radical oxygen species. A gradual deterioration of the body's ability to scavenge free radicals allows them to accumulate. The accumulation of free radicals leads to oxidative damage to DNA, protein, and lipids. Commonly studied byproducts of oxidative damage are 8-hydroxy-2-deoxyguanosine (DNA damage), MDA (lipid peroxidation), and protein carbonyls (protein oxidation). (1)

With regard to lipid peroxidation, oxidative aldehydes appear to arise as a result of beta cleavage reactions of lipid hydroperoxides, although the exact mechanism of this process has not been fully delineated. (8) Like MDA, 4-HNE is an oxidative aldehyde and a well-studied byproduct of lipid peroxidation. Increased levels of 4-HNE have been identified in many pathologic processes, including Alzheimer's disease, amyotrophic lateral sclerosis, and cerebral hypoxia. (9-11)

Although numerous studies have confirmed the presence of increased MDA levels in various aging tissues, 4-HNE levels have not previously been shown to be anything more than a byproduct of pathologic processes. Moreover, no study to date has shown that there is an association between 4-HNE and aging. 4-HNE immunoreactivity has been found to be higher in the brains of postnatal rats than in those of adult rats, indicating that 4-HNE might have a role in early mammalian development. (12)

Our findings are consistent with those of studies of lipid peroxidation in other organ systems. In our study, MDA levels appeared to increase in thyroarytenoid muscle as a function of age. In contrast, 4-HNE levels did not increase but displayed a slight negative correlation with age. The regression line for MDA approached but did not reach full statistical significance (p = 0.123). Additional studies of larger populations might eventually allow for statistical confirmation of the association between aging and increased MDA levels in the larynx.

One limitation of our study was the fact that we could not account for the influence of different confounding factors such as smoking, lifelong vocal habits, and the general health of our donors. Moreover, the use of cadaveric laryngeal tissue was complicated by the passage of time between death and the harvest of laryngeal tissue. Finally, the tissues we examined might have been subject to stresses underlying the primary cause of death and therefore could conceivably have skewed our results. However, these limitations were unavoidable. Unlike studies of aging and other tissues (e.g., skeletal muscle and skin), in which samples can be removed from living persons with minimal morbidity, vocal fold tissue cannot be removed without causing significant functional complications. Perhaps studying animal models is one way to overcome some of these limitations.

In conclusion, this is the first published study that attempted to investigate free-radical damage within the aging human larynx. Our preliminary findings suggest that MDA levels increase with age in human thyroarytenoid muscle. It also appears that 4-HNE levels do not increase with age. Although additional studies are needed to confirm these findings, our study provides some support for the theory that free-radical damage has a role in the aging of the larynx and in the associated decline of the human voice.

[FIGURE OMITTED]

References

(1.) Mecocci P, Fano G, Fulle S, et al. Age-dependent increases in oxidative damage to DNA, lipids, and proteins in human skeletal muscle. Free Radic Biol Med 1999;26:303-8.

(2.) Muscari C, Giaccari A, Giordano E, et al. Role of reactive oxygen species in cardiovascular aging. Mol Cell Biochem 1996;160-161:159-66.

(3.) Sohal RS, Svensson I, Sohal BH, Brunk UT. Superoxide anion radical production in different animal species. Mech Ageing Dev 1989;49 129-35.

(4.) Chipalkatti S, Dc AK, Aiyar AS. Effect of diet restriction on some biochemical parameters related to aging in mice. J Nutr 1983;113:944-50.

(5.) Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: Malonaldehyde and 4-hydroxynonenal. Methods Enzymol 1990;186:407-21.

(6.) Kowald A. The mitochondrial theory of aging. Biol Signals Recept 2001;10: 162-75.

(7.) Manaligod JM, Milam M, Hill SA, Ct al. Age-related mitochondrial DNA mutations in the human larynx. Laryngoscope 2000; 110:2123-7.

(8.) Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 1991;11:81-128.

(9.) McGrath LT, McGleenon BM, Brennan S, et al. Increased oxidative stress in Alzheimer's disease as assessed with 4-hydroxynonenal but not malondialdehyde. QJM 2001;94:485-90.

(10.) Pedersen WA, Fu W, Keller JN, et al. Protein modification by the lipid peroxidation product 4-hydroxynonenal in the spinal cords of amyotrophic lateral sclerosis patients. Ann Neurol 1998;44:819-24.

(11.) McKracken E, Graham DI, Nilsen M, et al. 4-Hydroxynonenal immunoreactivity is increased in human hippocampus after global ischemia. Brain Pathol 2001;11:414-21.

(12.) Cao Q, Ong WY, Halliwell B. Lipid peroxidation in the postnatal rat brain. Formation of 4-hydroxynonenal in the supraventricular corpus callosum of postnatal rats. Exp Brain Res 2001;137:205-13.

From the Department of Otolaryngology--Head and Neck Surgery, University of Iowa Hospital and Clinics, Iowa City.

Reprint requests: Jose M. Manaligod, MD, Assistant Professor. Department of Otolaryngology--Head and Neck Surgery, University of Iowa Hospital and Clinics, 200 Hawkins Dr., Iowa City, IA 52242. Phone: [phone]; fax: [phone]; e-mail: [e-mail]