COMMON PATTERN IN ELECTROPHORETIC POLYPEPTIDE PROFILES OF TEAR FLUID OF HEALTHY SUBJECTS AND

CHANGES IN DRY EYE PATIENTS

Leonidas Traipe1, Luis Michea2, Remigio López3.

1 Fundación Oftalmológica Los Andes2 Facultad de Medicina, Universidad de Chile3 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

BackgroundBackground 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: 0.5 - 1 µl/min)

ObjectivesObjectives

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

MethodsMethodsSubjects: Forty nine healthy subjects (both sexes, age 21- 60 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), 312-318 (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), 970-976 (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 < 0.001 (Student’s T test)

ResultsResults

Tear Fluid Collection

1. Polyurethane minisponges are positioned for 3 minutes on the outerthird of the lower lid. Eyes can be either open or closed .

2. A series of micropipette tips are sectionedand organized to build up a sort of conicalfunnel (left). After tear absorption, theminisponge is located in the center of thefunnel-like construct which is thensupported on the opening of a 0.5 mlEppendorf centrifuge tube (right).

3. Tear fluid is recovered in the0.5 ml Eppendorf tube by centrifugation at 2000g x 3 min.

Tear samples in the 10-15 µl range

Tear protein quantification

Microliters

Representative tear samples (3-µl aliquots) from4 different subjects

b) After elution in methanol, the dyesolution corresponding to each spot isread in a spectrophotometer at 600 nm, and protein concentration is expressedin relation to the standard protein.

Representative readings

1.1770.43010

0.7680.2175

0.4290.1283

TEAR FLUIDBSA (1 mg/ml)VOLUME (µl)

1.1770.43010

0.7680.2175

0.4290.1283

TEAR FLUIDBSA (1 mg/ml)VOLUME (µl)a) Cellulose membranes after protein staining. Aliquots (1-5 µl) of A. Saliva, B. Tear fluid and C. Standard protein (BSA 1 mg/ml) were spotted on a cellulose membrane, fixed, stained with Coomassie blue and washed in 7% acetic acid until clear background.

Tear protein concentrationaround 4 mg/ml

Electrophorertic protein profiles of tear fluid from healthy subjects

Invariable (79, 18 and 14 kDa) and variable (66 kDa) polypeptides

Normal polypeptide pattern

Some healthy subjects (4%) with a markedly different polypeptide profile (arrow)

SDS-polyacrylamide gel electrophoresis (representative gels)

Lanes were loaded with 30 µg of tear protein per sample. After the fractionation, proteins were stained with Coomassie blue R-250.

A

FD E

CB

Top Bottom

Ban

d In

ten

sity

(a

.u.)

Densitometric tracings of electrophoretic polypeptide profiles oftear fluid from a single healthy subject

Band of 66 kDa (arrow in each tracing) corresponds to a highly variable band

For each tracing, the Y-axis represent band intensities. Tracings of protein migration in gelfractionation were performed from higher (left) to lower (right) molecular weights.

Both tear collection, electrophoretic fractionation and gel densitometry wereperformed in sextuplicate (A through F)

14

18

66

79

27

41

kDa

24

pH 10 NEPHGE pH 3

Bidimensional NEPHGE fractionation (size and charge) of tear polypeptides

Alb

Lf

TSPALys

pH 10 NEPHGE pH 3

kDa

79

41

66

2724

18

14

Lf, lactoferrin; Alb, Albumin; Lys, lysozyme; TSPA, lipocalin

Lupus C

LUPUS

TEAR POLYPEPTIDE PROFILES OF INDIVIDUAL PATIENTS WITH SEVERE DRY-EYE

ROSACEA SJÖGREN

*Tear fluid (6 μl) from both eyes were combined.

Loss of major normal bands

Increased intensity of 66 kDaband.

C, tear sample (7 μl) from a single healthy subject

Tear samples (7 μl) from the right and left eyes of a single subject.

All the normal electrophoreticbands are clearly more intense (increased evaporation?)

Tear samples (7 μl) from the right and left eyes of a single subject.

More intense bandsand some degree ofbilateral asymmetryare observed.

ConclusionsConclusions

In the normal tear fluid, a general electrophoretic polypeptide profile In the normal tear fluid, a general electrophoretic polypeptide profile consisting of both invariable and variable polypeptides can be consisting of both invariable and variable polypeptides can be identified. identified.

Lactoferrin, lipocalin and lysozyme are invariable polypeptides Lactoferrin, lipocalin and lysozyme are invariable polypeptides whereas albumin is a highly variable tear component.whereas albumin is a highly variable tear component.

Marked alterations in the tear polypeptide composition may occur Marked alterations in the tear polypeptide composition may occur among Dry-Eye patients, such as increased tear protein among Dry-Eye patients, such as increased tear protein concentration as well as selective changes in the presence of concentration as well as selective changes in the presence of invariable polypeptidesinvariable polypeptides