The Laryngoscope Lippincott Williams & Wilkins, Inc. © 2007 The American Laryngological, Rhinological and Otological Society, Inc. Concentration and Distribution of Hyaluronic Acid in Human Vocal Folds Mariana Dantas Aumond Lebl, MD, MSc; Joa˜ o Roberto Maciel Martins, MD, PhD; Helena Bonciani Nader, PhD; Manuel de Jesus Simo˜es, MSc, PhD; Noemi De Biase, MD, MSc, PhD Objective/Hypothesis: To evaluate the concentra- tion and distribution of hyaluronic acid (HA) in hu- man adult vocal folds. Study Design: Experimental study. Methods: Histologic samples of the anterior, middle, and posterior regions of the vocal folds were studied to determine the tissue distribution of HA. The HA was traced with hyaluronan-binding proteins (HAPB) from bovine cartilage. Chemical bonds were detected through the avidin-biotin immunoperoxidase method. A control with the spe- cific Streptomyces hyalurolyticus hyaluronidase was conducted to avoid false-positive results. The concen- tration levels of HA were determined through a fluorescence-based assay method with the same HAPB and europium-labeled streptavidin. Results: Women presented higher HA concentrations than men in every studied region of the vocal folds and overall HA concentration levels twice as higher. In regard to the distribution of HA, the macula flavae and the vocal fold intermediate and deep layers pre- sented marked staining, with moderate staining around the vessels, on the gland capsules, and the connective tissue surrounding the vocal fold muscu- lar fibers. Conclusions: HA is diffusely distributed along the vocal folds; the profuse amounts of HA ob- served in the female vocal folds may be related to impact absorption, acting as a protective factor against the characteristic high vibration frequency. Key Words: Hyaluronan, vocal folds, fluorassay, histo- chemical method, bovine hyaluronidase. Laryngoscope, 117:595–599, 2007 INTRODUCTION The hyaluronic acid (HA) is a glycosaminoglycan (GAG) produced by fibroblasts and one of the main com- ponents of the lamina propria (LP) of human vocal folds (VF). 1,2 Many physiologic effects are attributed to the HA, such as maintenance of tissue stability, viscoelasticity, lubrication, osmotic pressure, resistance to flow, and bar- rier against infectious agents and macromolecules. HA also takes part in many cellular functions such as control of morphogenesis and differentiation, cell locomotion, tis- sue regeneration, and inflammatory response. It has also been associated with the mitotic process and tumoral growth, as well as with protection against degradation of proteoglycans. 3–5 Particularly significant to the VF vibra- tion is the viscoelasticity conferred by the HA. 3 Disrup- tions in the structure and distribution of HA on the VF may generate alterations on the mucosal wave with an impact in voice production. HA is a polymer of great promise as a therapeutic agent to reestablish the viscoelastic properties of VF with alterations of the cover layer, such as sulci and scars, either as injected 6–8 or by stimulated production, thus increasing its concentration levels in the LP. 9 Despite the well-known functions of HA in the biomechanical proper- ties of biological tissues, there are few morphologic studies describing the concentration and disposition of this mac- romolecule in the VF. The objective of this work is to evaluate the concentration and distribution of HA in hu- man adult VF. MATERIALS AND METHODS Sample Thirty-two VF were obtained from the autopsies of 16 ca- davers (8 men and 8 women), ranging from 38 to 68 years of age, within a period of less than 8 hours postmortem and without laryngeal lesion or pathology associated with the cause of death. The collected material was immediately immersed in physiologic saline solution (0.9% NaCl) during a maximum period of 1 hour and evaluated under surgical microscope. The exclusion criterion was presence of any lesion or tissue modification observable at this stage. Left and right VF of each subject were alternately selected for histology and HA quantification. HA Quantification A fluoroassay method for determination of HA in biological fluids was developed using hyaluronan-binding protein (HABP). 4 HABP is isolated from the bovine cartilage and comprises the From the Department of Otorhinolaryngology (M.D.A.L., N.D.B.), the Department of Clinical Endocrinology (J.R.M.M.), the Department of Molec- ular Biology (H.B.N.), and the Department of Morphology: Histology and Structural Biology (M.D.J.S.), Universidade Federal de Sa˜o Paulo, Sao Paulo, SP, Brazil; and the Department of Speech Pathology (N.D.B.), Pon- tifı ´cia Universidade Cato´lica de Sa˜o Paulo, Sao Paulo, SP, Brazil. Editor’s Note: This Manuscript was accepted for publication Novem- ber 27, 2006. Source of financial support: CAPES (Coordenac¸a˜o de Aperfeic¸oa- mento de Pessoal de Nı ´vel Superior). Send correspondence to R. Madre Rita Amada de Jesus, 106, 04721- 050, Sao Paulo, Brazil. E-mail: nbiase@terra.com.br DOI: 10.1097/MLG.0b013e31802ffe17 Laryngoscope 117: April 2007 Lebl et al.: Hyaluronic Acid in Human Vocal Folds 595