A novel fluorescent lipid probe for dry eye: retrieval by tear lipocalin in humans

Abstract

Purpose: A fluorescent probe was used to identify mucin-depleted areas on the ocular surface and to 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 were 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 analyzed by gel electrophoresis. Immunoprecipitation verified FODE complexed to tear lipocalin in tears.

Results: FODE exhibits an isosbestic point at 473 nm, 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 produces visible staining at the contact sites of membranes, which correlates with removal of mucin. Despite the fact that tear lipocalin is reduced in dry eye patients, FODE removal follows similar rapid exponential decay functions for all subjects. FODE is bound to tear lipocalin in tears.

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.

Figure 1.

Mass spectrum (scan range m/z 100–600) of FODE after 6 months' storage in ethanol. Major peak at m/z 583.3 corresponds to the predicted mass of the MH⁻ chemical structure (inset) of FODE (C₃₈H₄₈O_5, molecular weight 584.78).

Figure 2.

(A) pH titration of absorbance for 0.74 μM FODE in 1% EtOH/1% Tween 80 (•) at 511 nm and fluorescein (○) at 490 nm of 0.005% fluorescein adapted with permission from Doughty MJ. pH dependent spectral properties of sodium fluorescein ophthalmic solutions revisited. Ophthalmic Physiol Opt. 2010;30:167–174. Copyright 2010 John Wiley and Sons. Inset: absorption spectra of 0.74 μM FODE/1% EtOH/1% Tween 80 in buffers from pH 3.5 to 12 show convergence at the isosbestic point at 473 nm. (B) Influence of Tween 80 on the spectra of FODE. A split cuvette separated FODE in buffer in one chamber and buffer with Tween in the second chamber (−) before mixing the two chambers and (– –) FODE/Tween 80 mixed. Peak absorbances shown in nm; 2 nm red shift of absorbance resulted from the mixture of FODE in Tween 80 solution.

Figure 3.

Photomicrograph of fluorescence image of impression cytology membrane taken from ocular surface 20 minutes after FODE instillation. (A) Green fluorescence of FODE: λ_ex = 483 ± 15 nm, λ_em = 535 ± 25 nm, and a dichroic filter (<506 nm cutoff). (B) Same area after DAPI staining, λ_ex = 365 ± 20 nm, λ_em = 445 ± 25 nm, and dichroic filter (<395 nm cutoff). (C) 3A and 3B merged. Original magnification ×200.

Figure 4.

Intensity of autofluorescence of impression cytology membranes. Bar graphs expressed as mean (error bars ± SD) intensity of autofluorescence nm computed from six independent photographs in (A) at λ_em = 535 and in (B) at λ_em = 445 nm. *P < 0.05.

Figure 5.

Time-dependent pattern of FODE removal from ocular surface as marked by impressions taken from different areas of the same patient at the following times: (A) 1 minute after FODE instillation, (B) 20 minutes, (C) 60 minutes. Original magnification: 50×.

Figure 6.

Fluorescence of tears versus time after FODE installation in dry eye (□) and control subjects (▪). Fluorescence intensity (FI) was measured at λ_ex = 490 nm and λ_em = 511 nm. Individual exponential regression curve was calculated. FI at 0 minutes was set as 100%, and FI of subsequent time points were also represented as percentage of FI at 0 minutes. Mean intensities were not significantly different between control and dry eye subjects (P = 0.30). Inset: fluorescent intensity of eluted fractions from immunoprecipitation on columns of protein A cross-linked agar bound with either anti-tear lipocalin immune serum or preimmune serum as shown. Two microliters of combined tears and FODE were incubated on the columns and eluted at pH 2.

Figure 7.

Size-exclusion gel filtration of pooled tear samples from dry eye subjects collected at 5 minutes. (•) Absorbance at 280 nm was corrected by linear regression analysis of absorbance of 280 vs. 310 nm. (○) Fluorescence intensity (λ_em = 511 nm). Inset left: Coomasssie-stained SDS tricine (4%–12%) acrylamide gradient gel. Lane 1, fractions 4 to 6. Lane 2, fractions 8 to 10. Lane 3, fractions 14 to 16. Lane 4, fractions 21 to 23. Lane 5, fractions 26 to 28. Lane M, molecular weight markers, size shown at the right. Lactoferrin (Lf), lysozyme (Ly), and tear lipocalin (TL) are indicated at left. Inset right: fluorescence of anion exchange chromatography performed on fractions 21 to 23 of control subjects collected at 10 to 20 minutes. 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.

Figure 8.

Absorbance spectra of extracts from various Schirmer strips in buffer. Spectra are displayed as mean values (n = 6) for extracts from each strip. Inset top: absorbance spectrum of human tears shows negligible absorbance >310 nm. Inset bottom: linear regression analysis was performed from graphs of absorbance at 280 vs. 310 nm from extracts of the Tear Flo strip.

Figure 9.

FODE staining of sites where Schirmer test II was performed (arrows). Photos taken after (A) 3 minutes and (B) 5 minutes. Photos were under a LED blue penlight (467 nm, LDP LLC) source, excitation band-pass filter (315–540 nm, 80% transmittance) (Beseler), emission yellow gel filter 312 (Rosco Laboratories) (cutoff <460 nm), and a 9× slit-lamp eyepiece (Leitz). Manual camera settings for photography were ISO 400, 1/4,” F2.9, VR, no flash, fine 13 m (Coolpix P6000; Nikon).

Figure 10.

Specificity of periodic acid-Schiff (PAS) staining of bovine sialomucin in various types of impression cytology membranes. Bar graphs show the difference in PAS intensity of areas of the membrane without mucin compared to those with mucin. Intensity calculations were made from reverse grayscale program provided by ImageJ software (NIH). Measurements represent the mean for six individual experiments (±SD). *P < 0.05. Photographs of representative membranes from one experiment are shown below the graph.

Figure 11.

Periodic acid-Schiff staining of surfactant-free mixed cellulose ester membrane. (A) Image of an area that contacted the ocular surface. (B) Image of control region of the same membrane not in contact with ocular surface. (C) Membrane alone. Manual camera settings for photography were ISO 64, 1/284,” F2.9, VR, no flash, fine 13 m (Coolpix P6000).

Figure 12.

Clinical image of SF-MCE membrane impression site. (A) Three minutes after FODE instillation. (B) Same area 25 minutes after instillation. Images were under a LED blue penlight (467 nm, LDP LLC) source, excitation band-pass filter (315–540 nm, 80% transmittance) (Beseler), emission yellow gel filter 312 (Rosco Laboratories) (cutoff <460 nm), and a 9× slit-lamp eyepiece (Leitz). Manual camera settings for photography were ISO 400, 1/4,” F2.9, VR, no flash, fine 13 m (Coolpix P6000).