June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
FODE Highlights Impression Based Mucin Defects on the Ocular Surface in Dry Eye Patients: Kinetics and Retrieval by Tear Lipocalin
Author Affiliations & Notes
  • Po-Ting Yeh
    Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
    Ophthalmology, Jules Stein Eye Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA
  • Ben Glasgow
    Ophthalmology, Jules Stein Eye Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA
    Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA
  • Richard Casey
    Ophthalmology, Jules Stein Eye Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 932. doi:
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      Po-Ting Yeh, Ben Glasgow, Richard Casey; FODE Highlights Impression Based Mucin Defects on the Ocular Surface in Dry Eye Patients: Kinetics and Retrieval by Tear Lipocalin. Invest. Ophthalmol. Vis. Sci. 2013;54(15):932.

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Abstract
 
Purpose
 

A fluorescent probe was used to identify mucin depleted areas on the ocular surface and test the hypothesis that tear lipocalin, retrieves lipids from the eyes of normal and dry eye subjects.

 
Methods
 

Fluorescein labeled octadecyl ester, FODE, was characterized by mass spectrometry and absorbance spectrophotometry. The use of FODE to define mucin defects was studied with impression membranes under conditions that selectively deplete mucin. The kinetics of FODE removal from the ocular surface was analyzed by sampling tears from control and dry eye patients at various times. The tear protein-FODE complexes were isolated by gel filtration and ion exchange chromatographies, monitored with absorption and fluorescent spectroscopies and analysed by gel electrophoresis. Immunoprecipitation verified FODE complexed to tear lipocalin.

 
Results
 

FODE exhibits an isosbestic point at 473nm, pKa of 7.5, and red shift relative to fluorescein. The low solubility of FODE in buffer is enhanced with 1% Tween 80 and ethanol. FODE adheres to the ocular surface of dry eye patients. FODE produced visible staining at the contact sites of membranes, which correlated with removal of mucin. Despite the fact that tear lipocalin was reduced in dry eye patients, FODE removal followed similar rapid exponential decay functions for all subjects. The majority of FODE was eluted bound to tear lipocalin.

 
Conclusions
 

Tear lipocalin retrieves lipid rapidly from the human ocular surface in mild to moderate dry eye disease and controls. With improvements in solubility, FODE may have potential as a fluorescent probe to identify mucin depleted areas.

 
 
FPLC of tear samples from dry eye subjects. (●) Absorbance at 280 nm (○) Fluorescence intensity (λem=511 nm). Inset left, coomasssie-stained tricine-SDS-PAGE. Lane 1, fx 4-6. Lane 2, fx 8-10. Lane 3, fx 14-16. Lane 4, fx 21-23. Lane 5, fx 26-28. Lane M, molecular weight markers. Lactoferrin (Lf), lysozyme (Ly) and tear lipocalin (TL) are indicated at left. Inset right, fluorescence of anion exchange chromatography performed on fractions 21-23 of control subjects. Fluorescence was seen only in the elution buffer (high salt) fractions. Gel filtration chromatography of control sample was nearly identical to that of dry eye.
 
FPLC of tear samples from dry eye subjects. (●) Absorbance at 280 nm (○) Fluorescence intensity (λem=511 nm). Inset left, coomasssie-stained tricine-SDS-PAGE. Lane 1, fx 4-6. Lane 2, fx 8-10. Lane 3, fx 14-16. Lane 4, fx 21-23. Lane 5, fx 26-28. Lane M, molecular weight markers. Lactoferrin (Lf), lysozyme (Ly) and tear lipocalin (TL) are indicated at left. Inset right, fluorescence of anion exchange chromatography performed on fractions 21-23 of control subjects. Fluorescence was seen only in the elution buffer (high salt) fractions. Gel filtration chromatography of control sample was nearly identical to that of dry eye.
 
 
FODE staining of sites where Schirmer test II was performed (arrows). Photos taken after: (A) 3 minutes (B) 5 minutes.
 
FODE staining of sites where Schirmer test II was performed (arrows). Photos taken after: (A) 3 minutes (B) 5 minutes.
 
Keywords: 480 cornea: basic science • 583 lipids  
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