COMMON PATTERN IN ELECTROPHORETIC POLYPEPTIDE PROFILES OF TEAR FLUID OF HEALTHY SUBJECTS AND CHANGES IN DRY EYE PATIENTS Leonidas Traipe 1, Luis Michea 2, Remigio López 3. 1 Fundación Oftalmológica Los Andes 2 Facultad de Medicina, Universidad de Chile 3 ICBM (Biología Celular y Molecular), Facultad de Medicina, Universidad de Chile No finantial relationships between the authors and any company or person exist in regard to the present study

Background Significant quantitative changes in the production of tear fluid may occur in Dry-Eye patients (low scores in Schirmer test). Frequently, those alterations are associated with a lower stability of the tear film (low Break-up times) what would suggest qualitative changes in tear macromolecular components. Direct biochemical analysis of tear is rare quite probably because of the low abundance of this fluid (5-15 µl/eye; tear flow: µl/min)

Objectives General: To contribute to the characterization of normal human tear fluid by the quantitative and qualitative determination of proteins Specific: To assess constancy and variability of tear protein composition among Dry-Eye patients

Methods Subjects : Forty nine healthy subjects (both sexes, age y.o. (36.4 ± 10.7) who were not displaying blepharitis, allergy, Dry-Eye or had not experienced ocular surgery were included. These subjects were no comsumers of medication, alcohol or cigarrettes and were not contact-lens wearers. Also, fifteen Dry Eye patients (AAO criteria) of identical age and sexes were included. Some of these patients had been diagnosed with either rheumatologic or dermatologic diseases. In all cases, informed consents were obtained. Tear Collection : Absorption by positioning polyurethane minisponges on the outer third of the margin of the lower lid (see Figure 1). Details in Cornea 25 (3), (2006). Protein Quantification : Tear aliquots (1-3 microliters) were spotted on cellulose membranes, fixed and stained with an alcoholic solution of Coomassie blue. After several washes of the membrane in 7% acetic acid, the dye was eluted in 1 ml of ethanol- ammonia and the absorbance of the eluate was determined in a spectrophotometer. Bovine serum albumin was used as standard. Details are given in Cornea 26 (8), (2007). Osmolarity. Definite volumes of tear fluid (Vm) were diluted with a definite volume of double-distilled water (Vw). Osmolarity of both water (Ow) and the diluted tear sample (Om) were measured in a osmometer. Tear osmolarity (Ot) was calculated by Ot = (200 x Om) - (Vw x Ow) Vm Protein electrophoresis. One dimensional SDS-PAGE was performed according to the conventional procedure of Laemmli and bidimensional electrophopresis (NEPHGE) according to O’Farrrell. Proteomics. MALDI MS and MALDI MS/MS.

TEAR OSMOLARITY (mOsm/L) HEALTHY CONTROLS 294 ± 19 (N = 21) DRY-EYE 332 ± 13 (N = 9) p < (Student’s T test) Results

Conclusions In the normal tear fluid, a general electrophoretic polypeptide profile consisting of both invariable and variable polypeptides can be identified. In the normal tear fluid, a general electrophoretic polypeptide profile consisting of both invariable and variable polypeptides can be identified. Lactoferrin, lipocalin and lysozyme are invariable polypeptides whereas albumin is a highly variable tear component. Lactoferrin, lipocalin and lysozyme are invariable polypeptides whereas albumin is a highly variable tear component. Marked alterations in the tear polypeptide composition may occur among Dry-Eye patients, such as increased tear protein concentration as well as selective changes in the presence of invariable polypeptides Marked alterations in the tear polypeptide composition may occur among Dry-Eye patients, such as increased tear protein concentration as well as selective changes in the presence of invariable polypeptides