HPLC analysis of closed, open, and reflex eye tear proteins

Changes in the closed, open and reflex eye tear proteins of normal subjects were compared and analysed. Tear proteins were resolved by high-performance liquid chromatography (HPLC) utilising both gel filtration (P-300 SW) and reverse-phase (C-18) columns and the HPLC fractions were further analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. The protein composition of the closed-eye tear was significantly different from that of the open and reflex-eye tear. Secretory IgA (sIgA) was the predominant protein in closed eye tears constituting 49% of the total protein compared to 11% in reflex tears, whereas lysozyme was the predominant protein (53%) in reflex tears. Levels of lactoferrin, lipocalin and lysozyme were relatively constant in both open and reflex tears. HPLC profiles of the closed-eye tears, upon continuous stimulation of lacrimal glands indicated that sIgA was significantly reduced whereas lactoferrin, lipocalin, and lysozyme were significantly increased. These results indicate that the tear composition upon waking attains that of the open eye within 4 to 5 minutes, and upon continuous stimulation this reflects the reflex-eye tear composition. It also indicates that mechanisms responsible for changes in concentration of constitutive and regulated tear protein with stimulus can be studied successfully using non-invasive methods to collect human tears.