The focus of this review is the application of infrared (IR) spectroscopy to study human lens and tear lipids with minimal discussion of the role of lipids and the complex etiology of cataracts and dry eye. The power of IR spectroscopy to measure composition, conformation, structure, and environment of biomolecules was applied to diagnose cataracts and dry eye and follow the efficacy of treatment. Principal component analysis and other standard algorithms were used to analyze IR spectra.
A thin lipid layer on the surface of the tears is believed to contribute to tear stability (1,2). Most of the tear film lipid comes from the meibomian glands located in the eye lids (1–3). The major function of tears is to keep the underlying cornea clear and hydrated. Blinking spreads the tears across the surface of the cornea and the mechanical action of the blink expresses a lipid called meibum from the meibomian glands located in the eye lids on the lid margin (1,2). The meibum finds its way to the tear film surface where it forms a heterogeneous, duplex layer that is about 17 molecules thick (4). It is believed that the lipid layer decreases the surface tension of the tears, which allows them to spread, and lowers the evaporation rate of the underlying aqueous liquid. Shortly after blinking, if one does not blink again, the tear film "breaks up." In all cases of dry eye, the tear film is unstable and tear breakup times are shortened. Dry eye affects more than seven million people in the United States alone (5). The roles of lipids and the etiology of dry eye have been reviewed (1–3). This article reviews the application of IR spectroscopy to study meibum and determine if any relationships exist between tear film stability and meibum lipid composition, environment, and conformation (6–12).
ExperimentalMeibum and tear lipids can be collected a number of ways, such as by using a spatula, microcapillary pipettes, lipid absorbent tape, or paper strips (3). For most of the studies discussed below, meibum was collected by squeezing the eyelid between two cotton swabs. About 0.5 mg of meibum is expressed on the eyelid surface and collected using a sterile spatula. The meibum is then transferred to a silver chloride window and transmission IR spectra are measured. In the studies reviewed, IR spectra were measured using a Fourier transform IR (FT-IR) spectrometer equipped with a mercury cadmium telluride (MCT) detector (Nicolet 5000 Magna Series, Thermo Fisher Scientific, Inc.). The meibum on the AgCl window was placed in a temperature-controlled IR cell. The cell was jacketed by an insulated water coil connected to a circulating water bath (model R-134A, Neslab Instruments). The sample temperature was measured and controlled by a thermistor touching the sample cell window. The water bath unit was programmed to measure the temperature at the thermistor and to adjust the bath temperature so that the sample temperature could be set to the desired value. The rate of heating or cooling (1 °C/15 min) at the sample was also adjusted by the water bath unit. Temperatures were maintained within ±0.01 °C. IR data analysis was performed using GRAMS/386 software (Galactic Industries).
Cataracts and Lens Lipids
The lens is located in the front of the eye behind the cornea and functions to focus light on the retina at the back of the eye. The lipids of the lens do not turn over, and most of the lipids made at birth are present at death (13). Light passing through a human lens traverses through thousands of cellular membranes that serve as impermeable barriers to cations as well as a matrix for proteins. The phospholipid composition of human lens membranes is unusual. The human lens contains dihydrosphingomyelin, found in significant quantities only in the lens. Lens membranes are some of the most saturated membranes in the human body, and contain extremely high levels of cholesterol, particularly near the center of the lens (10:1 mole:mole cholesterol to phospholipid). The composition of the human lens changes dramatically with age and cataract. The roles of lipids and the etiology of cataracts have been reviewed (13). IR spectroscopy was used to study lens membrane lipid conformation in relation to compositional differences between species, and the aging and cataractous human lens (13–34).