2232 Agarose gel electrophoresis of human tears: No polymeric mucin detected

Purpose: The established methods for observing the morghnl-og;i nF the teer film a=~ based on ;nierfrrenc~ techniques. Since interference patterns are visible only in e pathologically thickened tear film, these techniques are not useful for the physiological tear film morphology. Methods: A ne;.approach is Brewster Angle Microscopy n-which allows the studv of ultra-thin films. In ophthalmology it is applied +or the in vitro observation of spread Meibomian gla d secretion. When p-polarized He-Ne-laserlight is locident at an interface of two refracting media (n ,n), no visible liqht is reflected if the anole'of'incidence is the Etrewster angle. The Brewster-angle for an air-water-interface is 53.1. If a laver of amphiohilic molecules (e.g. Meibomian giand secretibn) is spread on a uater subphase, light is reflected that can be used to image the spread lipid layer. Results: Non-pathological Meibomian gland secretion forms homogeneous films including phases of higher and lower reflectivity. The mobility of these phases varies according to the compression of the films. The secretion is characterized by a rapid spreading after touching the water surface. Conclusion: The BAM technique allows new insights on Meibomian lipid films in vitro. Using isotropic Fresnel formulae the thickness of different phases of the films can be determined. Purpose It has long been assumed that polymers conjunctwal mucins are dissolved in the aqueous component of the precomeal tear film and arc respmwble for the viscoelastic properties of tears observed m vitro This study investigated whether polymeric mucins could be detected in onstimulated tears Methods Unstimulated tear samples (0.5-5~1) .were collected from the inferior meniscus of healthy volunteers of both sexes using glass caplllaly tubes. These were pooled and run on agarose gel (1% w/v in Tris-Acetate-EDTA buffer) in a flat-bed apparatus together with secretory IgA, rabbit conjunctival mucin, and human conjunctival mucm as markers. The gel was vacuum-blotted onto nitmcellulose paper and stamed using a biotinylated lectin (Wheat Germ Agglutinin) and then the avidin-peroxidase-substnlte (Diaminobenzidine) system. Results No polymeric mucins could be detected in the pooled unstimulated human tears. Polymeric mucins from human cqunctiva and rabblt conjunctiva did mignte on the gel and were easily detected at a concentration of 1.25mgiml. Conclu.sion Polymeric mucins may not play as large a role as has been previously thought m maintaining the stabihty of the aqueous component of the precorneal tear film. Other high molecular weight molecules, or then interactions, may be responsible for the rheological properbes of tears I …