INTRODUCTION

Somatically, voice is not essential, but it is fundamental to man as an individual. It not only distinguishes man from other animals, but also helps him in his social activities, in the creation of his unique form of expression and in his artistic, scientific and moral manifestations.
Phonatory function changes constantly from youth to adulthood as a result of growth modifications, hormonal activity and structural deterioration of the mechanism. The alterations of larynx due to the normal aging process has been described by many authors (SEGRE, 197120; HIRANO, 1981 06; RAMING & RINGEL,1983 18; KAHÀNE,1987 12; ARRAIS, 19882; HIRANO, KURITA, SAKAGUCHI, 1989 07).

Among the structural changes of the larynx which are due to aging, thickening of deepest layer of the lamina propria of the vocal fold is of great importance. These changes in the vibratory tissue of the vocal cords make then less flexible resulting in a change of the phonatory function of old individuals. Therefore, the study of the collagen fibers of the vocal folds is of great importance in individuals who depend on their voices for their profession such as singers, teachers, salesmen and who face d ifficulties maintaining their phonatory characteristics.

Approximately one third of the protein found in the human body is collagen (MINOR,1980'S1. It is the largest macromolecule found in connective tissues. Its function is to provide resistance to tension in order to keep all tissues united. This is achieved through intermolecular bonds as well as bonds between collagen fibers and fibrils and chemical and/or physical links between collagen and other structural components of the extracellular matrix (MINOR, 1980'5). Collagen belongs to a group fo insoluble and conjugated proteins with a molecular weight of 30,000 daltons. It is characterized by its fibrillar organization and peculiar composition - aproximately 30% glycine, 10% proline and hydroxyproline, and a varyng amount of hydroxylysine and carbohydrates (MILLER & GAY, 1982) 14.

In tissues, collagen was mostly studied through biochemical, immunobiological, histochemical, and ultrastructural methods (JUNQUEIRA & MONTES, 1983) 11. The determination of the amount of hydroxyproline is the most utilized procedure for the study of tissue deposited collagen in the last 20 years (MILLER & GAY, 1982) 14.

So far there has been no biochemical proof of histologic changes of the vocal fold tunica mucosa as described by HIRANO, 19898.

The objective of this study is quantify the collagen found in the tunica mucosa of the human vocal folds and to evaluate the concentration of this protein individuals of both sexes and in different age groups through biochemical methods.

MATERIAL AND METHODS





Each larynx studied was removed from a white cadaver with less than 24 hours of postmortem. All 60 larynxes obtained at the Department of Pathology of the Escola Paulista de Medicina were microscopically selected and found to be structurally normal.

These larynxes were distribuited in 6 groups of 10, according to age and sex. Group I was composed of male children 6 months to two years old, Group II included female children in the same age range as group I, Group III included males ranging from 20 to 40 years of age and Group IV was composed of females in the same age range as Group III. Men 60 to 80 years of age were included in Group V and females of this age range were included in Group VI.

1. Preparation of the Material

To obtain the vocal folds, each larynx was divided into two halves, left and right, using the median sagittal plane. The internal perichondrium of the thyroid cartilage, from the anterior commissure to the plane of projection of the arytenoid cartilage's vocal process in the posterior part of the larynx, was removed along with the vocal cords.

Before right and left vocal folds of each larynx were wrapped ira aluminum foil and stored in a freezer at - 20° C for the biochemícal quantification of their collagen, the middle third of the tunica mucosa was removed. The weight of three portions varied form 40 to 130 miligrams.

2. Collagen Quantification

Collagen quantification was performed by hydroxyproline quantity determination according to the method of ROJKIND & GONZALES, 1974 19. After the assays, recuperatory tests proved the method to be reproducible (CHEHTER, 1987)^. The variation seen in between assays was less than 10%, in the same laboratory.

STATISTICAL ANALYSIS

The following non-parametric tests were used in the analysis of the results:

1. The Mann-Whitney test for two independent samples was used to compare the male and female values for the weight in milligrams of collagen per gram of vocal fold tunica mucosa.

2. The Kruskal-Wallis one-way analysis of variance by ranks (SIEGEL, 1978)2 was used to separately compare the values of collagen in milliliters per gram of vocal fold tunica mucosa of the men and women ira the three age groups studied in this work. When there was a significant difference, this analysis was supplemented with the test of multiple comparisons (HOLLANDER, 1973)9.

In all tests, 0.05 or 5% (>0.05) was considered the level of significance for the rejection of the null hypothesis. Significant values are marked with ara asterisk.

RESULTS

Table I contains the values found for the weight of collagen (mg.) in one gram of vocal fold tunica mucosa in the different age groups.

DISCUSSION

According to SEGRE, 197120, the symptoms due to aging are the following: vocal weakness, hoarseness, reduction of the vocal phonatory intensity due to decreasing expiratory volume and vocal fold vibration amplitude, trembling voice resulting from the loss of the subglottic pressure and air - column pressure which passes through the glottis, monotonous voice, opaque and harmonic-lacking voice because of the reduced actin of the supraglottic resonators and lack of coordination due to the loss of mucosa sensitivity, muscular atrophy and alterations ín the fundamental frequency. These symptoms are initially seen in singing voice which represents a refinement of speaking voice.

All these changes are responsible for the alterations in voice during aging, characterized mainly by discrete hoarseness, harsness and tremor. These alterations were also investigated by PTACEK & SANDER, 1966 17. They specified the characteristics which differentiate the voices of young adults from voices of the elderly. The frequency and intensity of voice is smaller ira the elderly. The elderly present more hoarseness and a greater occurrence of sonority breaks.

In another study by PTACEK et ai, 1966", a significant reduction in vocal ressitura, intensity, maximum phonatory time and infra-oral respiratory pressure and vital capacity was observed in old individuals.

PERELLÓ,1962 16, compared the movements of the tunica mucosa during the passage of an air stream from below the vocal folds to the wavelike undulations of a rug. The tunica must be very flexible, humidified and reasonably free from the deeper layers of muscles for correct phonation.

For RAMING & RINGER, 1983 18, shimmer, which is the variation of intensity around the fundamental frequency, was the only acoustic value which varied significantly when young adults were compared to the elderly. Significant alterations in the average fundamental frequency, jitter (the variation in fundamental frequency height) and vocal range were not seen. We believe the increase in vocal fold tunica mucosa collagen decreases its elasticity and therefore promotes variations in height and intensity around the fundamental frequency of voice with an increase in jitter and shimmer values.

Ourwork is in agreement to the work of ALARCOS,BEHLAU & TOSI, 1983 1. They observed that senile male voices become more lower in picht and female voices become more higher in picht when compared to young adults. They also found a smaller control in the sustained emission expressed in greater jitter values.

In our opinion, the alterations which occur in the Structure of the vocal folds greatly influence the symptoms of phonatory aging. Changes which are related to collagen and alter vocal fold elasticity, are the most important. Alterations of voice due to an increase in vocal fold vibratory tissue rigidity are a direct result of this tissue's histochemical characteristics.

As we dosed the collagen in the vocal fold tunica mucosa of males and females belonging to three different age groups, we observed an increase ín the amount of collagen as the individual grows old.

Comparing the values found for both sexes, only the mate group representing old individuals (60 - 80 years) presented a significantly greater amount collagen. KAHANE, 1987 12, also observed significantly greater differences in the aging process of the male larynx. The male larynx presents histological changes after the theed decade while its female counterpart changes much later, after the fifth decade of life. HIRANO et al, 19898, stated female individuals show smaller fibrotic tissue changes than males.

We believe the increase in vocal fold tunica mucosa collagen will decrease elasticity of this tissue because of collagen's rigidity and bonding characteristics. KAHANE´s works, 1988 3 are in agreement to the findings in this present research and state that the collagen fibers of the deep layer of the tunica mucosa run anterior - posteriorly and parallel to the adjacent vocal muscle.

A decrease in the elasticity of the tunica mucosa as a result of the in crease in the amount of fibrous component sin the connective tissue of the vocal folds contributes to alterations observed in phonation, specially of the fundamental frequency of voice, as the individual gets old. This agrees with the works of SEGRE, (1971) 20, HOLLIEN & SHIPP (1972) 10, KAHANE (1987)12, BEHLAY et al (1988) 3. These authors described alterations in the fundamental frequency of the voice of elderly patients.
In our opinion, tone elevation seen in the elderly individual occurs because of a increase in rigidity which predominates over changes which lead to flaccidity like the decrease in muscular force caused by the atrophy of the vocal muscle observed by FERRERI (1959)5 and ARRAIS (1988)2. Female tone of voice tends to diminish with age since collagen increase is not as great as it is in males.

REFERÊNCIA BIBLIOGRÁFICA

1. ALARCOS, A.; BEHLAU, M. S.; TOSI, O. - Computer acoustical analysis of senile voices. ProceedingsXIX IALPCongms, Edimburgh, 1983.
2. ARRAIS, A. -Aspectos morfológicos e1 istonrétricos das pregas vocais humanas em crianças, adultos e idosos. São Paulo, 1988. Tese - Doutorado - Escola Paulista de Medicina.
3. BEHLAU, M. S.; PONTES, P. A. L.; TOSI, O.; GANANÇA, M. M. - Prebifonia: tratamento da deterioração vocal inerente à idade. Acta AWHO, 7: 50 - 5, 1988.
4. CHEHTER, L. - Redução da fibrme hepática induzida pelo tetracloreto de carbono, pela administração pnfvia de vitamina A. São Paulo, 1987. Tese - Mestrado - Escola Paulista de Medicina.
5. FERRERI, G. - Senescence of the larynx. Ital. Gen. Rev. Otorhino-laringol., 1: 640 -709,1959.
6. HIRANO, M. -Structure of the vocal folds in normal and disease stats. Anatomical and physical studies. In: CONFERENCE ON THE ASSESSMENT OF VOCAL PATHOLOGY,1, Rockville, 1981. Proceediugs. Rockville, Bethesda, ASHA reports 11, 1981, p. 11-27.
7. HIRANO, M.; KURITA, S.; NAKASHIMA, T. - Growth, development and aging of human vocal folds. In: BLESS, D. M. & ABBS, J. H. Vocalfoldsphysiology. San Diego, College Hif, p. 22 -43,1983.
8. HIRANO, M.; KURITA, S.; SAKAGUCHI, S. - Aging of vibratory tissue of human vocal folds. Acta Otolaiyugol. (Stockh), 107: 428 - 33, 1989.
9. HOLLANDER, M. & WOLFE, D. A. -Nonparametricstatical methods. New York, Wiley, 503p, 1973.
10. HOLLIEN, H. &SHIPP, T.-Speaking fundamentalfrequency and chronological age in males. J. Speech Hear. Res., 15:155 -9,1972. 11. JUNQUEIRA, L. C. V. & MONTES, G. S. - Biology of collagen-proteoglican interation. Arch. Histol. Jap., 46: 589 - 629, 1983.
12. KAHANE, J. C. - Connective tissue changes in the larynx and their effects on voice. J. Voice, l: 23 - 30, 1987.
13. KARANE, J. C. - Histologic Structure and properties of human vocal folds. Ear, Nose and Throat. J., 67: 322 - 31, 1988.
14. MILLER, E. J. M. & GAY, S. - Collagen: An overview. In: COLOWICK, S. P. & KAPLAN, N. O. -Methods in enziomology. New York, Academie, 1982. p.3 - 33.
15. MINOR, R. - Collagen metabolism: a comparison of diseases of collagen and diseases affecting collagen. Am. J. Pathol., 98:22778,1980.
16. PERELLÓ, I. - La théorie muco-ondulatoire de Ia phonation. Ann. Otolaryngol, 79: 722 - 5, 1962.
17. PTACEK, P. H. & SANDER, E. K. - Age recognition from voice. J. Speech Hear. Res., 9: 273 - 77, 1966.
18. RAMIG, L. A. & RINGEL, R. L. - Effects of physiological aging on releeted acoustic characteristics of voice. J. Speech Hear. Res., 26: 22 - 30, 1983.
19. ROJKIND, M. & GONZALES, E. -An improved method for determiningspecificradioactivitisofproline "C andhydroxyproline "C in collagen and in non-collagen proteins. Anal. Biochem. 57. 17, 1974.
20. SEGRE, R. - Senescence of the voice. Ear, Nose and ThroatJ., 50:223 -7,1971.
21. SIEGEL, S. -Estatística no parametrica. Mexico, Trillas, 1975. 346

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*Aluno Pós-graduado do Curso de Pós-graduação em Otorrinolaringologia e Cirurgia de Cabeça e Pescoço, nível doutorado, da Escola Paulista de Medicina.

** Professor Titular do Departamento de Otorrinolaringologia e Distúrbios da Comunicação Humana da Escola Paulista de Medicina.

*** Professor Adjunto da Disciplina de Gastroenterologia do Departamento de Medicina Interna da Escola Paulista de Medicina.

Escola Paulista de Medicina - Rua Botucatu, 740 - VI. Clementino - São Paulo - Fone: 549-7041 - CEP: 04023-900.

Tese de Mestrado apresentada ao Curso de Pós-graduação em Otorrinolaringologia da Escola Paulista de Medicina. Ano 1991. Trabalho realizado com recursos do CAPES

Artigo recebido em 10 de janeiro de 1993.
Artigo aceito em 21 de janeiro de 1993.